Benzothiazoles and thiazolopyridines as sirtuin modulators

ES 2 396 913 T3

DESCRIPTION

Sirtuin modulating compounds.

Background

The Silent Information Regulatory (SIR) gene family represents a group of highly conserved genes in the genomes of organisms ranging from archaebacteria to a variety of eukaryotes (Frye, 2000). The encoded SIR proteins are involved in various processes ranging from gene silencing regulation to DNA repair. Proteins encoded by members of the SIR gene family exhibit high sequence conservation in a 250 amino acid core domain. A well-characterized gene in this family is S. cerevisiae SIR2, involved in the silencing of HM loci that contain information specifying the yeast sexual type, the effects of telomere position, and cell aging (Guarente, 1999; Kaeberlein et al. al., 1999; Shore, 2000). Yeast Sir2 protein belongs to the family of histone deacetylases (reviewed in Guarente, 2000; Shore, 2000). The Sir2 homolog, CobB, in Salmonella typhimurium, functions as a NAD (nicotinamide adenine dinucleotide) -dependent ADP-ribosyl transferase (Tsang and Escalante-Semerena, 1998).

The Sir2 protein is a class III deacetylase that uses NAD as a co-substrate (Imai et al., 2000; Moazed, 2001; Smith et al., 2000; Tanner et al., 2000; Tanny and Moazed, 2001). Unlike other deacetylases, many of which are involved in gene silencing, Sir2 is insensitive to class I and II histone deacetylase inhibitors such as trichostatin A (TSA) (Imai et al., 2000; Landry et al. , 2000a; Smith et al., 2000).

The deacetylation of acetyl-lysine by Sir2 is firmly linked to the hydrolysis of NAD, producing nicotinamide and the new compound acetyl-ADP ribose (Tanner et al., 2000; Landry et al., 2000b; Tanny and Moazed, 2001). The NAD-dependent deacetylase activity of Sir2 is essential, since its functions can connect its biological role with cellular metabolism in yeast (Guarente, 2000; Imai et al., 2000; Lin et al., 2000; Smith et al. ., 2000). Mammalian Sir2 homologs have NAD-dependent histone deacetylase activity (Imai et al., 2000; Smith et al., 2000). Most of the information on Sir2-mediated functions comes from studies in yeast (Gartenberg, 2000; Gottschling, 2000).

Biochemical studies have shown that Sir2 can easily deacetylate the amino termini of histones H3 and H4, resulting in the formation of 1-O-acetyl-ADP-ribose and nicotinamide. Strains with additional copies of SIR2 exhibit greater rDNA silencing and a 30% longer life span. Additional copies of the SIR2 C. elegans homolog, sir-2.1, and the D. melanogaster dSir2 gene have recently been shown to greatly extend life in these organisms. This implies that the SIR2-dependent regulatory pathway for aging arose early in evolution and has been well conserved. Today, the Sir2 genes are believed to have evolved to enhance an organism's health and stress resistance to increase its chances of surviving adversity.

SIRT3 is a homologue of SIRT1 conserved in prokaryotes and eukaryotes (P. Onyango et al., Proc. Natl. Acad. Sci. USA 99: 13653-13658 (2002)). The SIRT3 protein is targeted to mitochondrial crests by a unique domain located at the N terminus. SIRT3 possesses NAD + -dependent deacetylase activity and is ubiquitously expressed, particularly in metabolically active tissues. Upon transfer to mitochondria, SIRT3 is believed to be cleaved into a smaller active form by mitochondrial matrix processing peptidase (MPP) (B. Schwer et al., J. Cell Biol. 158: 647-657 (2002) ).

It has been known for more than 70 years that caloric restriction improves the health and extends the life of mammals (Masoro, 2000). The life of yeast, like that of metazoans, is extended by interventions that resemble caloric restriction, such as lowering glucose. The discovery that both yeast and flies lacking the SIR2 gene do not live longer when calorie restricted provides evidence that SIR2 genes mediate beneficial health effects of this diet (Anderson et al., 2003; Helfand and Rogina, 2004). Likewise, mutations that reduce the activity of the yeast glucose-sensitive cAMP (adenosine 3 ', 5'-monophosphate) -dependent pathway extend life in wild-type cells in mutant sir2 strains, demonstrating that SIR2 it is probably a key 3 'component of the caloric restriction pathway (Lin et al., 2001).

Porcu et al (Trends in Pharmacological Sciences, Elsevier, Haywarth, GB, vol 26, no. 2, 2005, pp. 94-103) describe a series of compounds that are described as modulators of sirtuin.

JP07291976 describes a series of imidazo [2,1-b] thiazole derivatives. WO2006122011 describes thiazole-based HCV inhibitors.

Summary

New sirtuin modulator compounds are provided in the present invention,

In one aspect, the invention provides sirtuin A modulator compounds of Structural Formula (XVIII):

ES 2 396 913 T3

<img file="ES2396913T3_D0001.tif" />

or its pharmaceutically acceptable salt, in which

R¹⁹ each Z14, Z15 and each Z10, Z11 are selected,

<img file="ES2396913T3_D0002.tif" />

1 ', in which:

in which:

zero to two of Z10, Z11, Z12 or Z13 are N;

at least one of Z14, Z15 and Z- |₆ is N, NR1 ', S or O;

zero to one of Z14, Z15 and Z- |₆ is S or O;

zero to two of Z14, Z15 and Z16 are N or NR1 ';

zero to an R²⁰ it is a solubilizing group; and zero to one R1 'is optionally substituted linear or branched C1-C3 alkyl;

each R²⁰ is independently selected from H or a solubilizing group;

R²¹ is selected from -NR1'-C (O) -, -NR1'-S (O) 2-, -NRT-C (O) -NR1-, -NR1'-C (S) -NR1'-, -NR1 '-C (S) -NR1'-CR1'R1'-, NR1'-C (O) -CR1'R1'-NR1'-, -NR1^,-C (= NR1^,) -NR1'-, -C (O) -NR-i'-, -C (O) -NR-i'-S (O) 2-, -NR1'-, -CR-fR · / -, - NRJ-C (O) CR1 '= CR1'-, -NR1'-S (O) 2-NR1'-, -NR1'-C (O) -NR1'-S (O) 2-, -NR1'- CR1'R1'-C (O) -NR1'-, -CR1'R1'-C (O) -NRJ-, -NR1 'C (O) -CR1' = CR1'-CR1'R1'-, -NR1 '-C (= N-CN) -NR1'-, -NR1'-C (O) -CR1'R1'-O-, -NR1'-C (O) -CR1'R1'-CR1'R1'- O-, -NR1 'S (O) 2-CR1'R1'-, -NR1'-S (O) 2-CR1'R1'- CR1'R1'-, -NR1'-C (O) -CR1' R1'-; -NR1'-C (O) -CR1'R1'-CR1'R1'-, -NR1'-C (S) -NR1 'CR1'R1'-CR1'R1'-, -NR1'-C (O) -OR-,

<img file="ES2396913T3_D0003.tif" />

wherein each R1 'is independently selected from H or optionally substituted linear or branched C1-C3 alkyl; Y

R³¹ is selected from an optionally substituted monocyclic or bicyclic aryl, or an optionally substituted monocyclic or bicyclic heteroaryl, with the exception that when R²¹ is -NR1'-C (O) -, R³¹ not 4-cyanophenyl or

ES 2 396 913 T3 and when R²¹ is -NRi'-S (O) 2-, R³¹

<img file="ES2396913T3_D0004.tif" />

In another aspect, the invention provides sirtuin B modulator compounds of Structural Formula (XX):

<img file="ES2396913T3_D0005.tif" />

or its pharmaceutically acceptable salt, in which

R¹⁹ is selected from:

in which:

each Z10, Z11, Z12 and Z13

<img file="ES2396913T3_D0006.tif" />

each Z₁₄, Z₁₅ and Z₁₆ is independently selected from N, NR₁', S, O, CR²⁰ or CR₁', in which:

zero to two of Z₁₀, Z₁₁, Z₁₂ or Z₁₃ are N;

at least one of Z₁₄, Z₁₅ and Z₁₆ is N, NR₁', O or S;

zero to one of Z₁₄, Z₁₅ and Z₁₆ is S or O;

zero to two of Z₁₄, Z₁₅ and Z₁₆ are N or NR₁';

zero to an R²⁰ it is a solubilizing group; and zero to an R₁'is C alkyl₁-C₃ optionally substituted linear or branched;

each R²⁰ is independently selected from H or a solubilizing group;

R^{20 a} is independently selected from H or a solubilizing group;

R²¹ is selected from -NRAC (O) -, -NRf-S (O) 2-, -NR1'-C (O) -NRA, -NRAC (S) -NRA, -NR1^,-C (S) -NR1^,-CR1^,R1'-, NR₁'-C (O) -CR₁'R₁'-NR₁'-, -NR₁'-C (= NR₁') -NR₁'-, -C (O) -NRA, -C (O) -NRAS (O) 2-, -CR-fRA, -NR1^,-C (O) -CR1^,= CR1'-, -NR1'-S (O) 2-NR1'-, -NR1'-C (O) -NR1'-S (O) 2-, -NR1^,-CR1^,R1^,-C (O) -NR1'-, -CR1^,R1^,-C (O) -NR1'-, -NR1^,-C (O) -CR1^,= CR1'CRt'RA, -NR1'-C (= N-CN) -NR1'-, -NR1^,-C (O) -CR1^,R1^,-O-, -NR1^,-C (O) -CR1^,R1^,-CR1^,R1^,-O-, -NR1^,-S (O) 2-CR1^,R1'-, NR1^,-S (O) 2-CR1^,R1'- CR1'R1'-, -NR1^,-C (O) -CR1^,R1'-; -NR1^,-C (O) -CR1^,R1^,-CR1^,R1'-, -NR1^,-C (S) -NR1^,-CR1^,R1^,-CR1^,R1'-, -NR1'-C (O) -O-,

ES 2 396 913 T3

<img file="ES2396913T3_D0007.tif" />

where each R₁'is independently selected from H or C alkyl₁-C₃ optionally substituted linear or branched; Y

R is selected from optionally substituted monocyclic or bicyclic aryl, or optionally substituted monocyclic or bicyclic heteroaryl, where when R¹⁹ it is

<img file="ES2396913T3_D0008.tif" />

20a and Z₁₀, Z11, Z₁₂ and Z₁₃ are each CH, R is a solubilizing group.

In yet another aspect, the invention provides sirtuin B modulator compounds of Structural Formula (XXI):

<img file="ES2396913T3_D0009.tif" />

or its pharmaceutically acceptable salt, in which

R²¹ is selected from -NRr-C (O) -, -NR1'-S (O) 2-, -NR1'-C (O) -NR1'-, -NRr-C (S) -NR1'-, -NRr -C (S) -NRr-CRrRr-, NR1'-C (O) -CR1'R1'-NR1'-, -NRr-C ^ NRO-NRr-, -C (O) -NR; -, -C (O) -NR1'-S (O)₂-, -NR1'-, -CR / R1'-, -NRr-C (O) CR1 '= CR1'-, -NR1'-S (O) 2-NR1'-, -NR1'-C (O) -NR1'-S (O) 2-, -NR1'-CR1'R1'-C (O) -NR1'-, -CR1'R1'-C (O) -NR1'-, -NR1'C (O ) -CR1 '= CR1'-CR1'R1'-, -NR1'-C (= N-CN) -NR1'-, -NR1'-C (O) -CR1'R1'-O-, -NR1' -C (O) -CR1'R1'-CR1'R1'-O-, -NR1'S (O) 2-CR1'R1'-, -NRr-S (O) 2-CR1'Rr- CRrRr-, -NR1 '-C (O) -CRrRr-; -NRr-C (O) -CR1^,Rr-CR1^,Rr-, -NR1'-C (S) -NRr-

<img file="ES2396913T3_D0010.tif" />

wherein each R1 'is independently selected from H or optionally substituted linear or branched C1-C3 alkyl; Y

R is an optionally substituted monocyclic or bicyclic heteroaryl, or an optionally substituted bicyclic aryl, where:

when R²¹ is -NH-C (O) -CH2-, R³² it is not thien-2-yl unsubstituted;

when R²¹ is -NH-C (O) -, R³² it is not furan-2-yl, 5-bromofuran-2-yl or 2-phenyl-4-methylthiazol-5-yl;

32 when R is -NH-S (O)₂-, R is not unsubstituted naphthyl or 5-chlorothien-2-yl.

ES 2 396 913 T3

In another aspect, the invention provides sirtuin D) modulator compounds of Structural Formula (XXII):

<img file="ES2396913T3_D0011.tif" />

or a pharmaceutically acceptable salt thereof, where:

R²¹ is selected from -NRÍ-C (O) -, -NRT-S (O)₂-, -NRr-C (O) -NRÍ-, -NRr-C (S) -NRÍ-, -NRr-C (S) -NRT-CRÍRT-, NR1'-C (O) -CR1'R1'- NR1'-, -NR1'-C (= NR1 ') - NR1'-, -C (O) -NR1'-, -C (O) -NR1'-S (O) 2-, -NR1'-, -CR1'R1'-, -NR1'-C (O) CRT = CRT-, -NRr-S (O) 2-NRT-, -NRT-C (O) -NRr-S (O)₂-, -NRT-CRTRr-C (O) -NRÍ-, -CRÍRr-C (O) -NRT-, -NR1'C (O) -CR1 '= CR1'-CR1'R1'-, -NR1'- C (= N-CN) -NR1'-, -NR1'-C (O) -CR1'R1'-O-, -NR1'-C (O) -CR1'R1'-CR1'R1'-O- , -NR1'S (O) 2-CRTRT-, -NR1^,-S (O) 2-CR1^,R1'- CRÍRT-, -NR1^,-C (O) -CR1^,R1'-; -NRr-C (O) -CRTRT-CRÍRr-, -NRÍ-C (S) -NRT-

<img file="ES2396913T3_D0012.tif" />

wherein each Rf is independently selected from H or optionally substituted linear or branched C1-C3 alkyl; Y

R³³ is an optionally substituted phenyl, where:

when R²¹ is -NRi'-C (O) -, R₁'is not H;

when R²¹ is -NH-C (O) -CH₂ or -NH-C (O) -CH₂-O-, R³³ it is not unsubstituted phenyl or 4-halophenyl; Y

33 when R is -NH-S (O)₂-, R is unsubstituted phenyl, 2,4- or 3,4-dimethylphenyl, 2,4-dimethyl-5-methoxyphenyl, 2-methoxy3,4-dichlorophenyl, 2-methoxy-5-bromophenyl-3,4-dioxyethylenephenyl , 3,4-dimethoxyphenyl, 3,4-dichlorophenyl, 3,4-dimethylphenyl, 3- or 4-methylphenyl, 4-alkoxyphenyl, 4-phenoxyphenyl, 4-halophenyl, 4-biphenyl or 4-acetylaminophenyl.

In one aspect, the invention provides sirtuin E) modulator compounds of Structural Formula (XXII):

<img file="ES2396913T3_D0013.tif" />

or its pharmaceutically acceptable salt, where:

R²¹ is selected from -NH-C (O) - or -NH-C (O) -CH₂-; Y

R³³ is phenyl substituted with

a) a -N (CH3) group₂ ;

b) a CN group at position 3;

c) a -S (CH3) group; or

<img file="ES2396913T3_D0014.tif" />

ES 2 396 913 T3 bridging positions 3 and 4.

In any of the options A), B), C) or D), where Ri 'is substituted, Ri' is substituted with one or more of -OH, halogen, -OR^to, -O-COR^to, -COR^to, -C (O) R^to, -CN, -NO2, - COOH, -COOR^to, -OCO2R^to, -C (O) NR^toR^b, -OC (O) NR^toR^b, SO3H, -NH2, -NHR^to, - N (R^toR^b), -COOR^to, -CHO, -CONH2, -CONHR^to, -CON (R^toR^b), -NHCOR^to, -NRCOR^to, - NHCONH2, -NHCONR^toH, -NHCON (R^toR^b), -NR^cCONH2, -NR^cCONR^toH, -NR^cWITH (R^toR^b), -C (= NH) -NH2, -C (= NH) -NHR^to, -C (= NH) N (R^toR^b), -C (= NR^c) -NH2, -C (= NR^c) -NHR^to, -C (= NR^c) -N (R^toR^b), -NH-C (= NH) -NH2, -NH-C (= NH) -NHR^to -NH-C (= NH) N (R^toR^b), -NH-C (= NR^c) -NH2, -NH-C (= NR^c) -NHR^to, -NH-C (= NR^c) -N (R^toR^b), -NR^dHC (= NH) -NH2, -NR^d-C (= NH) -NHR^to, NR^d-C (= NH) -N (R^toR^b), -NR^d-C (= NR^c) -NH2, -NR^d-C (= NR^c) -NHR^to, -NR^d-C (= NR^c) -N (R^toR^b), -NHNH2, -NHNHR^to, NHR^toR^b, -SO2NH2, -SO2NHRa, -SO2NR^toR^b, -CH = CHR^to, -CH = CR^toR^b, -CR^c= CR^toR^b, CR^c= CHR^to, -CR^c= CR^toR^b, -CCR^to, -SH, -SOkR^to, -S (O) kOR^to and -NH-C (= NH) -NH2, where k is 0, 1 or 2

R^to-R^d are each independently an aliphatic, substituted aliphatic, benzyl, substituted benzyl, aromatic, or substituted aromatic group; Y

-NR^toR^btaken together, they can also be in the form of a non-aromatic, substituted or unsubstituted heterocyclic group;

where a non-aromatic heterocyclic group, benzyl group or aryl group may also have an aliphatic or substituted aliphatic group as a substituent; a substituted aliphatic group may also have a non-aromatic heterocyclic ring, a substituted non-aromatic heterocyclic ring, benzyl, substituted benzyl, aryl or substituted aryl as a substituent; and a non-aromatic, aliphatic, substituted, substituted aryl or substituted benzyl heterocyclic group may have more than one substituent.

Also provided are pharmaceutical compositions comprising one or more compounds of Formulas XVIII XXII, or their salts.

In another aspect, the application considers methods for using sirtuin modulator compounds, or compositions comprising sirtuin modulator compounds. In certain embodiments, sirtuin modulator compounds that can increase the level and / or activity of a sirtuin protein can be used for a variety of therapeutic applications including, for example, increasing the life of a cell, and treating and / or o prevent a wide variety of diseases and disorders including, for example, diseases or disorders related to aging or stress, diabetes, obesity, neurodegenerative diseases, chemotherapeutic substance-induced neuropathy, neuropathy associated with an ischemic episode, eye diseases and / or disorders, cardiovascular disease, blood clotting disorders, inflammation and / or hot flashes, etc. Sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can also be used to treat a disease or disorder in a subject that would benefit from increased mitochondrial activity, to enhance muscle performance, to increase ATP levels in muscles or to treat or prevent tissue and muscle damage associated with hypoxia or ischemia. In other embodiments, sirtuin modulator compounds that decrease the level and / or activity of a sirtuin protein can be used for a variety of therapeutic applications including, for example, increasing cellular sensitivity to stress, increasing apoptosis, treating cancer, stimulate appetite and / or stimulate weight gain, etc. As will be described in more detail below, the methods comprise administering to a subject in need thereof a pharmaceutically effective amount of a sirtuin modulator compound.

In certain aspects, the sirtuin modulating compounds can be administered alone or in combination with other compounds, including other sirtuin modulating compounds or other therapeutic agents.

Brief description of the figures

Figure 1 shows a schematic view of the Cellular ATP Assay described in Example 5.

Figure 2 shows a dose response curve for ATP levels in cells after resveratrol tr eatment.

Detailed description

1. Definitions

As used herein, the following terms and expressions will have the meanings set forth below. Unless otherwise defined, all scientific and technological terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art.

The singular forms un, an, and el / la include reference to plurals unless the context clearly indicates otherwise.

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The term agent is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, an antibody, a protein or its portion, eg, a peptide), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) tissues or cells. The activity of such agents can render them suitable as therapeutic agents, that is, biologically, physiologically or pharmacologically active substances that act locally or systemically in a subject.

The term "bioavailable" when referring to a compound is recognized in the art and refers to a form of a compound that allows the same, or a portion or amount thereof, to be administered, absorbed by, incorporated into, or physiologically available to a subject or patient to whom it is administered.

"Biologically active" portion of a sirtuin refers to a portion of a sirtuin protein that has a biological activity, such as the ability to deacetylate. Biologically active portions of a sirtuin can comprise the central domain of sirtuins. The biologically active portions of SIRT1 having nos. GenBank accession NP_036370 spanning the NAD + binding domain and the substrate binding domain, for example, may include, without limitation, amino acids 62-293 of no. GenBank accession NP_036370, encoded by nucleotides 237 to 932 of nos. GenBank accession NM_012238. Consequently, this region is sometimes called the central domain. Other biologically active portions of SIRT1, also sometimes referred to as core domains, include amino acids 261 to 447 of nos. GenBank accession NP_036370, encoded by nucleotides 834 to 1394 with nos. GenBank accession NM_012238; amino acids 242 to 493 with no. GenBank accession NP_036370, encoded by nucleotides 777 to 1532 with nos. GenBank accession NM_012238; or amino acids 254 to 495 with no. GenBank accession NP_036370, encoded by nucleotides 813 to 1538 with nos. GenBank accession NM_012238.

The term "companion animals" refers to dogs and cats. As used herein, the term "dog (s)" denotes any member of the Canis familiaris species, of which there are a large number of different breeds. The term "cat (s)" refers to a feline animal, including domestic cats and other members of the Felidae family, genus Felis.

The term comprising and the term comprises / n are used in the open and inclusive sense indicating that additional elements may be included.

The term "conserved residue" refers to an amino acid that is a member of a group of amino acids that have certain properties in common. The term "conservative amino acid substitution" refers to the substitution (conceptually or otherwise) of an amino acid from said group with a different amino acid from the same group. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins from homologous organisms (Schulz, GE and RH Schirmer., Principles of Protein Structure, Springer-Verlag). According to such analyzes, the amino acid groups in which the amino acids within a group preferentially exchange with each other, and therefore resemble each other, can be defined, mainly in their impact on the structure of the protein in general. (Schulz, GE and RH Schirmer, Principles of Protein Structure, Springer-Verlag). An example of a set of amino acids defined in this mode includes: (i) a charged group, consisting of Glu and Asp, Lys, Arg, and His, (ii) a positively charged group, consisting of Lys, Arg, and His, (iii) a negatively charged group, consisting of Glu and Asp, (iv) an aromatic group, consisting of Phe, Tyr, and Trp, (v) a nitrogen ring group, consisting of His and Trp, (vi) a large non-polar aliphatic group, consisting of Val, Leu and Ile, (vii) a slightly polar group, consisting of Met and Cys, (viii) a group of small residues, consisting of Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gln and Pro, (ix) an aliphatic group, consisting of Val, Leu, Ile, Met and Cys, and (x) a small hydroxyl group, consisting of Ser and Thr.

Diabetes refers to hyperglycemia or ketoacidosis, as well as general and chronic metabolic abnormalities that arise from a prolonged state of hyperglycemia or a reduction in glucose tolerance. Diabetes encompasses both Type I and Type II (Non-Insulin Dependent Diabetes Mellitus or NIDDM) forms of the disease. Risk factors for diabetes include the following: waistline greater than 40 inches in men or 35 inches in women, blood pressure 130/85 mmHg or greater, triglycerides above 150 mg / dl, fasting glucose greater than 100 mg / dl or high-density lipoprotein less than 40 mg / dl in men or 50 mg / dl in women.

A direct activator of a sirtuin is a molecule that activates a sirtuin by binding to it. A sirtuin inhibitor is a molecule that inhibits a sirtuin by binding to it.

The term ED50 is known in the art. In certain embodiments, ED50 means the dose of a drug that produces 50% of its maximum response or effect, or alternatively, the dose that produces a predetermined response in 50% of test subjects or preparations. The term LD50 is known in the art. In certain embodiments, LD50 means the dose of a drug that is fatal in 50% of test subjects. The term "therapeutic index" is an art recognized term that refers to the therapeutic index of a drug, defined as LD50 / ED50.

ES 2 396 913 T3

The term hyperinsulinemia refers to a state in an individual in which the level of insulin in the blood is higher than normal.

The term including is used to express that it includes, but is not limited to. including and including, but not limited to, are used interchangeably.

The term "insulin resistance" refers to a state in which a normal amount of insulin produces a subnormal biological response relative to the biological response in a subject who does not experience insulin resistance.

An insulin resistance disorder, as discussed herein, refers to any disease or condition that is caused by or where insulin resistance contributes to insulin resistance. Examples include: diabetes, obesity, metabolic syndrome, insulin resistance syndromes, syndrome X, insulin resistance, high blood pressure, hypertension, cholesterol, dyslipidemia, hyperlipidemia, dyslipidemia, atherosclerotic disease including stroke, coronary artery disease or myocardial infarction, hyperglycaemia, hyperinsulinaemia and / or hyperproinsulinaemia, glucose intolerance, delayed insulin release, Diabetic complications including coronary heart disease, angina pectoris, congestive heart failure, stroke, cognitive functions in dementia, retinopathy, peripheral neuropathy, nephropathy, glomerulonephritis, glomerulosclerosis, nephrotic syndrome, hypertensive nephrosclerosis, some cancers (such as endometrial, breast, prostate and colon), pregnancy complications, poor female reproductive health (such as menstrual irregularities, infertility, irregular ovulation, polycystic ovary syndrome (PCOS)), lipodystrophy, cholesterol-related disorders such as gallstones, cholecystitis and cholelithiasis, gout, obstructive sleep apnea and respiratory problems, osteoarthritis, and prevention and treatment of bone loss, p. eg, osteoporosis.

The term cattle refers to domesticated quadrupeds, including those raised for meat and various derivatives, e.g., a bovine animal, including cows and other members of the genus Bos, a pig animal including domestic pigs and other members of the genus Sus , a sheep animal that includes sheep and other members of the genus Ovis, domestic goats, and other members of the genus Capra; domesticated quadrupeds bred for specialized tasks such as use as a beast of burden, e.g. eg, an equine, including domestic horses and other members of the family Equidae, genus Equus.

The term mammal is known in the art, and examples of mammals include humans, primates, livestock (including bovines, swine, etc.), companion animals (eg, canines, felines, etc.), and rodents ( eg, mice and rats).

The term "natural form" when referring to a compound means a compound that is in a form, e.g. eg, a composition, in which it can be found naturally. For example, since resveratrol can be found in red wine, it is present in red wine in a natural form. A compound is not found in a natural form if, e.g. For example, the compound has been purified and separated from at least some of the other molecules found with the compound naturally. A natural compound refers to a compound that can be found in nature, that is, a compound that has not been designed by man. A natural compound may have been made by man or by nature.

A natural compound refers to a compound that can be found in nature, that is, a compound that has not been designed by man. A natural compound may have been made by man or by nature. For example, resveratrol is a natural compound. A non-natural compound is a compound that is not known to exist in nature or does not occur in nature.

Obese individuals or individuals suffering from obesity are generally individuals who have a body mass index (BMI) of at least 25 or more. Obesity may or may not be associated with insulin resistance.

The terms parenteral administration and parenterally administered are known in the art and refer to modes of administration other than enteral and topical administration, usually by injection, and include without limitation intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital injection and infusion. , intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrasternal.

"Patient, subject, individual, or host" refers to either a human or a non-human animal.

The term "percent identical" refers to a sequence identity between two amino acid sequences or between two nucleotide sequences. Identity can be determined by comparing a position in each sequence that can be aligned for comparison purposes. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical in that position; when the equivalent site is occupied by the same or similar amino acid residue (eg, similar in steric and / or electronic nature), then the molecules can be considered homologous (similar) at that position. The term "percent homology, similarity or identity" refers to a function of the number of identical or similar amino acids in the positions shared by the compared sequences. The expression as a percentage

ES 2 396 913 T3 "Homology, similarity or identity" refers to a function of the number of identical or similar amino acids in the positions shared by the compared sequences. Various alignment algorithms and / or programs can be used, including FASTA, BLAST, or ENTREZ. FASTA and BLAST, available as part of the GCG Sequence Analysis Package (University of Wisconsin, Madison, Wis.), And can be used with, e.g. eg, default settings. ENTREZ is available through the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD. In one embodiment, the percent identity of two sequences can be determined with the GCG program with a gap weight of 1, p. For example, each amino acid gap is weighed as if it were a single amino acid or uneven nucleotide between the two sequences.

Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace &amp; Co., San Diego, California, USA Preferably, an alignment program is used that allows gaps in the sequence to align the sequences. Smith-Waterman is a type of algorithm that allows gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). The GAP program using the Needleman and Wunsch alignment method can also be used to align sequences. An alternative search strategy uses the MPSRCH software, which runs on a MASPAR computer. MPSRCH uses a Smith-Waterman algorithm to rate sequences on a massively parallel computer. This approach improves the ability to choose distantly related pairs, and tolerates especially small gaps and errors in nucleotide sequences. Nucleic acid encoded amino acid sequences can be used to search both protein and DNA databases.

The term "pharmaceutically acceptable carrier" is known in the art and refers to a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting any composition or its components. . Each vehicle must be acceptable in the sense of being compatible with the composition in question and its components, and must not be detrimental to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and wax suppositories; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffers; and (21) other compatible non-toxic substances used in pharmaceutical formulations.

The term polynucleotide and the term nucleic acid are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogues. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci defined from binding analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications can be imparted to the structure of the polynucleotide before or after polymer assembly. The nucleotide sequence can be interrupted by non-nucleotide components. A polynucleotide can further be modified, such as by conjugation with a tagging component. The term "recombinant polynucleotide" means a polynucleotide of genomic, cDNA, semisynthetic or synthetic origin, which either does not occur in nature or is linked to another polynucleotide in an unnatural arrangement.

The term "prophylactic or therapeutic treatment" is known in the art and refers to the administration of a drug to a host. If administered before the clinical manifestation of the unwanted condition (eg. g., disease or unwanted condition of the host animal), then the treatment is prophylactic, that is, it protects the host against the development of the unwanted condition, whereas if it is administered after the manifestation of the unwanted condition, the Treatment is therapeutic (that is, it is intended to reduce, alleviate or maintain the existing unwanted condition or the side effects derived from it).

The term "protecting group" is known in the art and refers to temporary substituents that protect a potentially reactive functional group from unwanted chemical transformations. Examples of such protecting groups include carboxylic acid esters, silyl ethers of alcohols and acetals and ketals of aldehydes and ketones, respectively. The field of protecting group chemistry has been reviewed by Greene and Wuts in Protective Groups in Organic Synthesis (2^to ed., Wiley: New York, 1991).

The term "pyrogen-free", with reference to a composition, refers to a composition that does not contain a pyrogen in an amount that would lead to an adverse effect (eg, irritation, fever, inflammation, diarrhea,

ES 2 396 913 T3 respiratory problems, endotoxic shock, etc.) in a subject to whom the composition has been administered. For example, the term is intended to encompass compositions that are free, or substantially free, of an endotoxin such as, for example, a lipopolysaccharide (LPS).

Replicative life of a cell refers to the number of daughter cells produced by an individual stem cell. Chronological age or chronological life, on the other hand, refers to the length of time that a population of undivided cells remains viable when nutrients are depleted. Increasing the life of a cell or extension of the life of a cell, applied to cells or organisms, refers to increasing the number of daughter cells produced by a cell; Increase the ability of cells or organisms to deal with stress and fight damage, e.g. eg, to DNA, proteins; and / or increase the ability of cells or organisms to survive and exist in a living state for longer under a particular condition, e.g. eg, stress (eg, heat shock, osmotic stress, high energy radiation, chemically induced stress, DNA damage, inadequate salt level, inadequate nitrogen level, or inadequate nutrient level). Life can be increased by at least 20%, 30%, 40%, 50%, 60% or between 20% and 70%, 30% and 60%, 40% and 60% or more, using the methods described in the present memory.

"Sirtuin activating compound" refers to a compound that increases the level of a sirtuin protein and / or increases at least one activity of a sirtuin protein. In an illustrative embodiment, a sirtuin activating compound can increase at least one biological activity of a sirtuin protein by at least about 10%, 25%, 50%, 75%, 100% or more. Illustrative biological activities of sirtuin proteins include deacetylation, e.g. eg, from histones and p53; life extension; increased genomic stability; transcription silencing; and control of the secretion of oxidized proteins between stem and daughter cells.

"Sirtuin inhibitory compound" refers to a compound that reduces the level of a sirtuin protein and / or reduces at least one activity of a sirtuin protein. In an illustrative embodiment, a sirtuin inhibitor compound can reduce at least one biological activity of a sirtuin protein by at least about 10%, 25%, 50%, 75%, 100% or more. Illustrative biological activities of sirtuin proteins include deacetylation, e.g. eg, from histones and p53; life extension; increased genomic stability; transcription silencing; and control of the secretion of oxidized proteins between stem and daughter cells.

"Sirtuin modulator compound" refers to a compound of Formulas XVIII-XXII as described herein. In illustrative embodiments, a sirtuin modulator compound can either increase (eg, activate or stimulate), reduce (eg, inhibit or suppress), or otherwise change the functional property or biological activity of a sirtuin protein. Sirtuin modulator compounds can act to modulate a sirtuin protein either directly or indirectly. In certain embodiments, a sirtuin modulator compound can be a sirtuin activator compound or a sirtuin inhibitor compound.

"Sirtuin protein" refers to a member of the sirtuin deacetylase family of proteins, or preferably the sir2 family, which include the yeast proteins Sir2 (GenBank Accession No. P53685), C. elegans Sir-2.1 (Accession No. in GenBank NP_501912) and human SIRT1 (GenBank Accession No. NM_012238 and NP_036370 (or AF083106)) and SIRT2 (GenBank Accession No. NM_012237, NM_030593, NP_036369, NP_085096) and AF08310796). Other members of the family include additional Sir2 yeast-like genes called HST genes (Sir two homologues) HST1, HST2, HST3, and HST4, and the five human homologs hSIRT3, hSIRT4, hSIRT5, hSIRT6, and hSIRT7 (Brachmann et al. (1995 ) Genes Dev. 9: 2888 and Frye et al. (1999) BBRC 260: 273). Preferred sirtuins are those with greater similarities to SIRT1, i.e. hSIRT1, and / or Sir2 than to SIRT2, such as those members that have at least part of the N-terminal sequence present in SIRT1 and absent in SIRT2, such as SIRT3 .

SIRT1 protein refers to a member of the sir2 family of sirtuin deacetylases. In one embodiment, a SIRT1 protein includes the yeast proteins Sir2 (GenBank Accession No. P53685), C. elegans Sir-2.1 (GenBank Accession No. NP_501912), human SIRT1 (GenBank Accession No. NM_012238, or NP_036370 (or AF083106)) and human SIRT2 (GenBank Accession No. NM_012237, NM_030593, NP_036369, NP_085096 or AF083107) and their equivalents and fragments. In another embodiment, a SIRT1 protein includes a polypeptide comprising a sequence consisting of, or consisting essentially of, the amino acid sequence set forth in nos. GenBank accession NP_036370, NP_501912, NP_085096, NP_036369 or P53685. SIRT1 proteins include polypeptides that comprise all or a portion of the amino acid sequence set forth in nos. GenBank accession NP_036370, NP_501912, NP_085096, NP_036369 or P53685; the amino acid sequence set forth in nos. GenBank accession NP_036370, NP_501912, NP_085096, NP_036369 or P53685 with 1 to about 2, 3, 5, 7, 10, 15, 20, 30, 50, 75 or more conservative amino acid substitutions; an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to nos. GenBank accession NP_036370, NP_501912, NP_085096, NP_036369 or P53685, and their functional fragments. Polypeptides of the invention also include homologues (eg, orthologs and paralogs), variants, or fragments, of nos. GenBank accession NP_036370, NP_501912, NP_085096, NP_036369 or P53685.

SIRT3 protein refers to a member of the sirtuin deacetylase family of proteins and / or a homologue of a SIRT1 protein. In one embodiment, a SIRT3 protein includes human SIRT3 proteins (accession no.

ES 2 396 913 T3

GenBank AAH01042, NP_036371 or NP_001017524) and mouse SIRT3 (GenBank accession number NP_071878), and their equivalents and fragments. In another embodiment, a SIRT3 protein includes a polypeptide comprising a sequence consisting of, or consisting essentially of, the amino acid sequence set forth in nos. GenBank accession AAH01042, NP_036371, NP_001017524 or NP_071878. SIRT3 proteins include polypeptides that comprise all or a portion of the amino acid sequence set forth in nos. GenBank accession AAH01042, NP_036371, NP_001017524 or NP_071878; the amino acid sequence set forth in nos. Access in GenBank AAH01042, NP_036371, NP_001 017524 or NP_071878 with 1 to about 2, 3, 5, 7, 10, 15, 20, 30, 50, 75 or more conservative amino acid substitutions; an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to nos. GenBank accession AAH01042, NP_036371, NP_001017524 or NP_071878, and their functional fragments. The polypeptides of the invention also include homologues (e.g. g., orthologs and paralogs), variants or fragments, of no. GenBank accession AAH01042, NP_036371, NP_001017524 or NP_071878. In one embodiment, a SIRT3 protein includes a fragment of the SIRT3 protein that is produced by cleavage with a mitochondrial matrix processing peptidase (MPP) and / or a mitochondrial intermediate peptidase (MIP).

The term "substantially homologous" when used in relation to amino acid sequences, refers to sequences that are substantially identical or similar in sequence to one another, giving rise to conformational homology and therefore retention, to a useful degree, of one or more biological (including immunological) activities. The expression is not intended to imply a common evolution of the sequences.

The term synthetic is known in the art and refers to production by chemical or enzymatic synthesis in vitro.

The terms systemic administration, systemically administered, peripheral administration, and peripherally administered are known in the art and refer to the administration of a composition, therapeutic substance, or other material other than directly to the central nervous system, such that it enters the central nervous system. of the patient and, therefore, is subject to metabolism and other similar processes.

The term "therapeutic agent" is known in the art and refers to any chemical moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject. The term also means any substance for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the improvement of physical or mental development and / or conditions in an animal or a human being.

The term "therapeutic effect" is known in the art and refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance. The phrase "therapeutically effective amount" means that the amount of said substance produces a certain desired local or systemic effect with a reasonable benefit / risk ratio applicable to any treatment. The therapeutically effective amount of said substance will vary depending on the subject and the disease being treated, the weight and age of the subject, the severity of the disease, the mode of administration and the like, which can be easily determined by the person skilled in the art. . For example, certain compositions described herein can be administered in an amount sufficient to produce a desired effect with a reasonable benefit / risk ratio applicable to such treatment.

Transcriptional regulatory sequence is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, that induce or control the transcription of the protein-encoding sequences to which they are operably linked. In preferred embodiments, the transcription of one of the recombinant genes is under the control of a promoter sequence (or other transcriptional regulatory sequence) that controls the expression of the recombinant gene in a cell type intended for expression. It is also to be understood that the recombinant gene may be under the control of transcriptional regulatory sequences that are the same or different from those sequences that control the transcription of the natural forms of the genes described in the present invention.

Treating a condition or disease refers to curing as well as alleviating at least one symptom of the condition or disease.

A vector is a self-replicating nucleic acid molecule that transfers an inserted nucleic acid molecule into and / or between host cells. The term includes vectors that function primarily for insertion of a nucleic acid molecule into a cell, replication of vectors that function primarily for nucleic acid replication, and expression vectors that function for transcription and / or translation of DNA or RNA. Also included are vectors that provide more than one of the aforementioned functions. As used herein, expression vectors are defined as polynucleotides that, when introduced into an appropriate host cell, can be transcribed and translated into polypeptide (s). An expression system usually connotes a suitable host cell comprised of an expression vector that can function to produce a desired expression product.

The term vision impairment refers to decreased vision, which is often only partially reversed or irreversible after treatment (eg, surgery). Particularly severe vision impairment is

ES 2 396 913 T3 calls blindness or loss of vision, which refers to a total loss of vision, vision worse than

20/200 and cannot be improved with corrective lenses, or to a visual field of less than 20 degrees in diameter (10 degree radius).

2. Sirtuin modulators

In one aspect, the invention provides novel sirtuin modulator compounds for treating and / or preventing a wide variety of diseases and disorders, including, for example, diseases or disorders related to aging or stress, diabetes, obesity, neurodegenerative diseases. , eye diseases and disorders, cardiovascular disease, blood clotting disorders, inflammation, cancer and / or hot flashes, etc. Sirtuin modulatory compounds that increase the level and / or activity of a sirtuin protein can also be used to treat a disease or disorder in a subject who would benefit from increased mitochondrial activity, to enhance muscle performance, to increase the muscle ATP levels or to treat or prevent damage to muscle tissue associated with hypoxia and ischemia. Other compounds described herein may be suitable for use in a pharmaceutical composition and / or one or more methods described herein.

In certain embodiments, the compounds of Structural Formula (XVIII) have the formula:

<img file="ES2396913T3_D0015.tif" />

or its pharmaceutically acceptable salt, where

R is selected from H or a solubilizing group;

R²¹ is selected from -NH-C (O) - or -NH-C (O) -CH₂-; Y

R is selected from an optionally substituted monocyclic or bicyclic aryl, or an optionally substituted monocyclic or bicyclic heteroaryl.

Typically, R¹⁹ in the compounds of Structural Formula (XVIII) it is selected from phenyl, pyridyl, thienyl or furyl, particularly optionally substituted phenyl.

<img file="ES2396913T3_D0016.tif" />

ES 2 396 913 T3

<img file="ES2396913T3_D0017.tif" />

<img file="ES2396913T3_D0018.tif" />

Typically, R³¹ is selected from phenyl, pyrazolyl, furyl, pyridyl, pyrimidinyl, thienyl, naphthyl, benzopyrazolyl, benzofuryl, quinolinyl, quinoxalinyl or benzothienyl and where R³¹ is optionally substituted.

Typically, R²¹ is selected from -NH-C (O) - or -NH-C (O) -CH2-;

Typically, R¹⁹ in the compounds of Structural Formula (XX) it is selected from phenyl, pyridyl, thienyl or furyl, particularly optionally substituted phenyl. Typically, R^{20 a} is selected from H, -CH2-N (CH3) 2,

<img file="ES2396913T3_D0019.tif" />

ES 2 396 913 T3

<img file="ES2396913T3_D0020.tif" />

: yl, benzopyrazolyl,

Typically, R²¹ is selected from -NH-C (O) - or -NH-C (O) -CH2-;

In certain embodiments of the compounds of Structural Formula (XXI), R³² is selected from pyrrolyl, pyrazolyl, pyrazinyl, furyl, pyridyl, pyrimidinyl, or thienyl, and R³² it is optionally substituted and optionally benzofused.

In certain embodiments of the compounds of Structural Formula (XXI), R³² is selected from benzofuryl, methylfuryl, benzothienyl, pyridyl, pyrazinyl, pyrimidinyl, pyrazolyl, wherein said methylfuryl, pyridyl, pyrazinyl, pyrimidinyl or pyrazolyl is optionally benzofused and where R³² it is optionally substituted or additionally substituted.

In the compounds of Structural Formula (XXII), preferably R is selected from -NH-C (O) - or -NH-C (O) CH2-.

The compounds of the invention, including the novel compounds of the invention, can also be used in the methods described herein.

The compounds and their salts described in the present invention also include their corresponding hydrates (eg, hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrate) and solvates. Suitable solvents for the preparation of solvates and hydrates can generally be selected by the person skilled in the art.

The compounds and their salts can be present in amorphous or crystalline forms (including cocrystalline and polymorphic forms).

In the compounds described above, the bivalent groups described as possible values ​​for variables can have any orientation, as long as said orientation results in a stable molecule. Preferably, however, the left side of a bivalent group (eg, -NR / -C (O) -) is attached to a bivalent arylene or heteroarylene group (eg,

31

R) and the right side of a bivalent group is attached to a monovalent aryl group (eg, R).

The sirtuin modulator compounds of the invention having hydroxyl substituents, unless otherwise indicated, can also form the related secondary metabolites, such as phosphate, sulfate, acyl derivatives (eg, acetyl, acid acyl fatty) and sugar (eg, glucurondate, glucose) (eg, from hydroxyl groups), particularly those derived from sulfate, acyl and sugar. In other words, the -OH substituent groups also include -OSO3 · M⁺, where M⁺ is a suitable cation (preferably H⁺, NH₄⁺ or an alkali metal ion such as Na⁺ okay⁺) and sugars such as

<img file="ES2396913T3_D0021.tif" />

These groups are generally cleavable to -OH by hydrolysis or by metabolic (eg, enzymatic) cleavage.

In certain embodiments, the compounds of the invention exclude one or more of the species described in Table 4.

ES 2 396 913 T3

The sirtuin modulator compounds of the invention advantageously modulate the level and / or activity of a sirtuin protein, particularly the deacetylase activity of the sirtuin protein.

Separately or in addition to the properties just mentioned, certain sirtuin modulator compounds of the invention do not substantially have one or more of the following activities: PI3-kinase inhibition, aldorreductase inhibition, tyrosine kinase inhibition, EGFR tyrosine kinase transactivation , coronary dilatation or spasmolytic activity, at concentrations of the compound that are effective to modulate the deacetylation activity of a sirtuin protein (e.g. g., such as a SIRT1 and / or SIRT3 protein).

An alkyl group is a non-aromatic, straight-chain, branched, or cyclic hydrocarbon that is fully saturated. Typically, a straight or branched chain alkyl group has between 1 and about 20 carbon atoms, preferably between 1 and about 10, and a cyclic alkyl group has between 3 and about 10 carbon atoms, preferably between 3 and about 8. Examples of straight chain and branched alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl, and octyl. A C1-C4 branched or straight chain alkyl group is also called a lower alkyl group.

An alkenyl group is a straight-chain, branched, or cyclic non-aromatic hydrocarbon containing one or more double bonds. Typically, the double bonds are not located at the end of the alkenyl group, such that the double bond is not adjacent to another functional group.

An alkynyl group is a straight-chain, branched, or cyclic non-aromatic hydrocarbon containing one or more triple bonds. Typically, the triple bonds are not located at the end of the alkynyl group, such that the triple bond is not adjacent to another functional group.

A ring (eg, a 5- to 7-membered ring) or a cyclic group includes carbocyclic and heterocyclic rings. Said rings can be saturated or unsaturated, including aromatic ones. Heterocyclic rings typically contain 1 to 4 heteroatoms, although the oxygen and sulfur atoms cannot be adjacent to each other.

Aromatic (aryl) groups include carbocyclic aromatic groups such as phenyl, naphthyl, and anthracyl, and heteroaryl groups such as imidazolyl, thienyl, furyl, pyridyl, pyrimidyl, pyranyl, pyrazolyl, pyrrolyl, pyrazinyl, thiazolyl, oxazolyl, and tetrazolyl.

Aromatic groups also include fused, polycyclic aromatic ring systems in which a carbocyclic aromatic ring or a heteroaryl ring is fused to one or more heteroaryl rings. Examples include benzothienyl, benzofuryl, indolyl, quinolinyl, benzothiazole, benzoxazole, benzimidazole, quinolinyl, isoquinolinyl, and isoindolyl.

Non-aromatic heterocyclic rings are non-aromatic carbocyclic rings that include one or more hetero atoms such as nitrogen, oxygen, or sulfur in the ring. The ring can have five, six, seven or eight members. Examples include tetrahydrofuryl, tetrahydrothiophenyl, morpholino, thiomorpholino, pyrrolidinyl, piperazinyl, piperidinyl, and thiazolidinyl, along with the cyclic form of sugars.

A ring fused to a second ring shares at least one bond in common.

Suitable substituents on a non-aromatic heterocyclic (carbocyclic and heteroaryl) alkyl, alkenyl, alkynyl, aryl, or aryl group are those that do not substantially interfere with the ability of the disclosed compounds to have one or more of the properties described herein. A substituent substantially interferes with the properties of a compound when the magnitude of the property is reduced by more than about 50% in a compound with the substituent compared to a compound without the substituent. Examples of suitable substituents include -OH, halogen (-Br, -Cl, -I and -F), -OR^to, -O-COR^to, -COR^to, -C (O) R^to, CN, -NO², -COOH, -COOR^to, -OCO2R^to, - C (O) NR^toR^b, -OC (O) NR^toR^b, -SO3H, -NH2, -NHR^to, -N (R^toR^b), -COOR^to, -CHO, - CONH2, -CONHR^to, -CON (R^toR^b), -NHCOR^to, -NRCOR^to, -NHCONH2, -NHCONR^toH, - NHCON (R^toR^b), -NR^cCONH2, NR^cCONR^toH, -NR^cWITH (R^toR^b), -C (= NH) -NH2, - C (= NH) -NHR^to, -C (= NH) -N (R^toR^b), -C (= NR^c) -NH2, -C (= NR^c) -NHR^to, C (= NR^c) -N (R^toR^b), -NH-C (= NH) -NH2, -NH-C (= NH) -NHR^to, -NH-C (= NH) -N (R^toR^b), -NH-C (= NR^c) -NH2, -NH-C (= NR^c) NHR^to, -NH-C (= NR^c) -N (R^toR^b), -NR^dHC (= NH) -NH2, - NR^d-C (= NH) -NHR^to, -NR^d-C (= NH) -N (R^toR^b), -NR^d-C (= NR^c) -NH2, -NR^d-C (= NR^c) -NHR^to, -NR^d-C (= NR^c) -N (R^toR^b), -NHNH2, -NHNHR^to, -NHR^toR^b, -SO2NH2, -SO2NHRa, -SO2NR^toR^b, CH = CHR^to, -CH = CR^toR^b, -CR^c= CR^toR^b, CR^c= CHR^to, -CR^c= CR^toR^b, -CCR^to, -SH, -SOkR^to (k is 0, 1 or 2), -S (O) kOR^to (k is 0, 1 or 2) and -NH-C (= NH) -NH2. R^to-R^d they are each independently an aliphatic, substituted aliphatic, benzyl, substituted benzyl, aromatic or substituted aromatic group, preferably an alkyl, benzyl or aryl group. Also, NR^toR^bTaken together, they can also be in the form of a substituted or unsubstituted non-aromatic heterocyclic group. A non-aromatic heterocyclic group, benzyl group or aryl group may also have an aliphatic or substituted aliphatic group as a substituent. A substituted aliphatic group can also have a non-aromatic heterocyclic ring, a substituted non-aromatic heterocyclic ring, benzyl, substituted benzyl, aryl or substituted aryl as a substituent. A non-aromatic, aliphatic, substituted, substituted aryl, or substituted benzyl heterocyclic group can have more than one substituent.

ES 2 396 913 T3

The combinations of substituents and variables contemplated by the present invention are only those that result in the formation of stable compounds. As used herein, the term "stable" refers to compounds that possess sufficient stability to allow manufacture, and that maintain the integrity of the compound for a period of time sufficient to be useful for the purposes detailed in the specification. present memory.

A hydrogen bond donor group is a functional group that has a positively charged hydrogen atom (eg, -OH, -NH2, -SH) or a group (eg, an ester) that is metabolized to a group capable of donating a hydrogen bond.

As used herein, a "solubilizing group" is a moiety that has sufficient hydrophilic character to improve or increase the water solubility of the compound in which it is included, as compared to an analogous compound that does not include the group. Hydrophilicity can be achieved by any means, such as the inclusion of functional groups that ionize under the conditions of use to form charged moieties (e.g. g. carboxylic acids, sulfonic acids, phosphoric acids, amines, etc.); groups that include permanent charges (eg, quaternary ammonium groups); and / or heteroatoms or heteroatomic groups (eg, O, S, N, NH, N- (CH2) and R^to, N (CH2) and C (O) R^to, N- (CH2) and C (O) OR^to, N- (CH2) and S (O) 2R^to-, N- (CH2) and S (O) 2OR^to, N- (CH2) and C (O) NR^toR^to, etc., where R^to is selected from hydrogen, lower alkyl, lower cycloalkyl, (C6-C14) aryl, phenyl, naphthyl, arylalkyl (C7-C20) and benzyl, where R^to is optionally substituted; yy is an integer between 0 and 6), optionally substituted heterocyclic groups (e.g., - (CH2) nR^b, - (CH2) nC (O) -R^b, - (CH2) nO- (CH2) n-Rb, where R^b is selected from an optionally substituted saturated monocyclic heterocycle, an optionally substituted saturated bicyclic fused heterocycle, an optionally substituted saturated spirobicyclic heterocycle, an optionally substituted heteroaryl, and an optionally substituted and partially substituted non-aryl heterocycle; and n is an integer between 0 and 2). It is to be understood that the substituents present in R^to or R^b they must not improve or increase water solubility compared to their unsubstituted counterparts to be within the scope of this definition. Such substituents are required not to significantly reverse the advance in water solubility provided by the R moiety.^to or R^b not replaced.

In one embodiment, the solubilizing group increases the water solubility of the corresponding compound lacking the solubilizing group by at least 5 times, preferably at least 10 times, more preferably at least 20 times, and most preferably at least 50 times.

100 101 101

In a preferred embodiment, the solubilizing group is a moiety of the formula: - (CH2) nR -N (R) (R), where:

n is selected from 0, 1 or 2;

R¹⁰⁰ is selected from a bond, -C (O) - or -O (CH2) n; and each R¹⁰¹ is independently selected from:

to. hydrogen;

b. linear or branched C1-C4 alkyl, wherein said alkyl is optionally substituted with halo, CN, OH, O (linear or branched C1-C4 alkyl), N (RT) (RT) or = O;

c.

<img file="ES2396913T3_D0022.tif" />

d.

<img file="ES2396913T3_D0023.tif" />

and.

ES 2 396 913 T3

<img file="ES2396913T3_D0024.tif" />

or

F. both remains R¹⁰¹ are taken together with the nitrogen atom to which they are attached to form a ring of the structure

<img file="ES2396913T3_D0025.tif" />

<img file="ES2396913T3_D0026.tif" />

or

g. both remains R¹⁰¹ are taken together with the nitrogen atom to which they are attached to form a 5-membered heteroaryl ring containing 1 to 3 additional N atoms, wherein said heteroaryl ring is optionally substituted with R1 ';

in which:

each Z is independently selected from -O-, -S-, -NR1'-, or -C (R⁵⁰) (R⁵⁰)-, where:

at least three of Z20, Z21, Z22, and Z23 are -C (R⁵⁰) (R⁵⁰)-;

at least three of Z24, Z25, Z26, Z27, and Z28 are -C (R⁵⁰) (R⁵⁰)-;

at least four of Z30, Z31, Z32, and Z33 are -C (R⁵⁰) (R⁵⁰) -; and at least four of Z34, Z35, Z36, Z37, and Z38 are -C (R⁵⁰) (R⁵⁰)-;

each R1 is independently selected from hydrogen or a linear or branched C1-C3 alkyl optionally substituted with one or more substituents selected from halo, -CN, -OH, -OCH3, -NH2, NH (CH3), -N (CH3) 2 o = O;

each R⁵⁰ is independently selected from R1 ', halo, CN, OH, O- (linear or branched C1-C4 alkyl), N (R1') (R1 '), = CR1', SR1 ', = NR1', = NOR1 'or = O;

two R⁵⁰ any suitable non-cyclics are optionally linked together directly or via a C1 to C2 alkylene, alkenylene or alkanedylidene bridge to produce a fused or spiro bicyclic ring; Y

ES 2 396 913 T3

<img file="ES2396913T3_D0027.tif" />

the ring structure is optionally benzo-fused or fused to a monocyclic heteroaryl to produce a bicyclic ring.

For the sake of clarity, the term alkylene, alkenylene or alkanedylidene bridge C₁ to C₂ means the multivalent structures -CH₂-, -CH₂-CH₂-, -CH =, = CH-, -CH = CH- or = CH-CH =. The two remains R⁵⁰ that are optionally bonded to each other can either be on the same carbon atom or on different carbon atoms. The first option produces a spiro bicyclic ring, while the second produces a condensed bicyclic ring. It will be obvious to those skilled in the art that when two R⁵⁰ are linked together to form a ring (either directly or through one of the aforementioned bridges), one or more terminal hydrogen atoms will be lost in each R⁵⁰ . Therefore, a remainder R⁵⁰ suitable non-cyclic available to form a ring is an R moiety⁵⁰ non-cyclic comprising at least one terminal hydrogen atom.

101 101

In another preferred embodiment, the solubilizing group is a moiety of the formula: - (CH₂)_n-OR, where n and R are as defined above.

In another preferred embodiment, the solubilizing group is a moiety of the formula: - (CH₂)_n-C (O) -R₁', where n and R₁'are as defined above.

102 102

In a preferred embodiment, a solubilizing group is selected from - (CH₂)_n-R, where n is 0, 1 or 2; and R is selected from

<img file="ES2396913T3_D0028.tif" />

<img file="ES2396913T3_D0029.tif" />

<img file="ES2396913T3_D0030.tif" />

<img file="ES2396913T3_D0031.tif" />

ES 2 396 913 T3

<img file="ES2396913T3_D0032.tif" />

ES 2 396 913 T3 where R1 'are as defined above.

In an even more preferred embodiment, a solubilizing group is selected from 2-dimethylaminoethylcarbamoyl, piperazin-1-ylcarbonyl, piperazinylmethyl, dimethylaminomethyl, 4-methylpiperazin-1-ylmethyl, 4-aminopiperidin-1-yl-methyl, 4-fluoropiperidin-1 -yl-methyl, morpholinomethyl, pyrrolidin-1-ylmethyl, 2-oxo-4-benzylpiperazin-1-ylmethyl, 4-benzylpiperazin-1-ylmethyl, 3-oxopiperazin-1-ylmethyl, piperidin-1-ylmethyl, piperazin-1-ylethyl , 2,3-dioxopropylaminomethyl, thiazolidin-3-ylmethyl, 4-acetylpiperazin-1-ylmethyl, 4-acetylpiperazin-1-yl, morpholino, 3,3-difluoroazetidin-1-ylmethyl, 2H-tetrazol-5-ylmethyl, thiomorpholin-4-ylmethyl, 1-oxothiomorpholin-4-ylmethyl, 1, 1-dioxothiomorpholin-4-ylmethyl, 1H-imidazol-1-ylmethyl, 3,5-dimethylpiperazin-1ylmethyl, 4-hydroxypiperidin-1-ylmethyl, M-methyl (1-acetylpiperidin-4-yl) -aminomethyl, M-methylquinuclidin-3ylaminomethyl , 1 H-1,2,4-triazol-1-ylmethyl, 1-methylpiperidin-3-yl-oxymethyl or 4-fluoropiperidin-1-yl.

To the extent that it does not fall within any of the definitions set forth above, the term "solubilizing group" also includes residues described as attached to the 7-position of 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3 acid. -carboxylic (ciprofloxacin) and its derivatives, as described in PCT publications WO 2005026165, WO 2005049602 and wO 2005033108, and in European patent publications EP 0343524, EP 0688772, EP 0153163, EP 0159174; as well as water solubilizing groups described in US Patent Publication 2006/0035891. The description of each of these patent publications is incorporated herein by reference.

The indicated double bonds in a structure like:

are intended to include both the (E) and (Z) settings. Preferably, the double bonds are in the (E) configuration.

A sugar is an aldehyde or ketone derivative of a straight chain polyhydric alcohol, containing at least three carbon atoms. A sugar can exist as a linear molecule or, preferably, as a cyclic molecule (eg, in the form of pyranose or furanose). Preferably, a sugar is a monosaccharide such as glucose or glucuronic acid. In embodiments of the invention where, for example, prolonged residence of a compound derived with a sugar is desired, the sugar is preferably a non-natural sugar. For example, one or more hydroxyl groups are substituted with another group, such as a halogen (eg, chlorine). The stereochemical configuration at one or more carbon atoms can also be altered, compared to a natural sugar. An example of a suitable non-natural sugar is sucralose.

A fatty acid is a carboxylic acid that has a long chain hydrocarbon moiety. Typically, a fatty acid has an even number of carbon atoms in the range of 12 to 24, often 14 to 20. Fatty acids can be saturated or unsaturated and substituted or unsubstituted, but are typically unsubstituted. Fatty acids can be natural or unnatural. In embodiments of the invention where, for example, prolonged residence of a compound having a fatty acid moiety is desired, the fatty acid is preferably a non-natural sugar. The acyl group of a fatty acid consists of the hydrocarbon moiety and the carbonyl moiety of the carboxylic acid functionality, but excludes the -OH moiety associated with the carboxylic acid functionality.

Also included in the present invention are salts, particularly the pharmaceutically acceptable salts, of the sirtuin modulator compounds described herein. Compounds of the present invention that possess sufficient acidic character, sufficient base character, or both functional groups, can react with any of a number of inorganic bases, and inorganic and organic acids to form a salt. Alternatively, compounds that are inherently charged, such as those with a quaternary nitrogen, can form a salt with an appropriate counter ion (eg, a halide such as bromide, chloride or fluoride, particularly bromide).

Acids commonly used to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid and the like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenyl sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of such salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate propriolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexene-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gammahydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like.

ES 2 396 913 T3

Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases useful for preparing the salts of the present invention therefore include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like.

In accordance with another embodiment, the present invention provides methods for producing the above defined sirtuin modulator compounds. Compounds can be synthesized using standard techniques. Advantageously, these compounds are conveniently synthesized from readily available starting materials.

Synthetic chemistry methodologies and transformations useful for synthesizing the sirtuin modulator compounds described in the present invention are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations (1989); TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, 2^to Ed. (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis (1995).

In an illustrative embodiment, a sirtuin modulator compound can cross the cytoplasmic membrane of a cell. For example, a compound can have a cell permeability of at least about 20%, 50%, 75%, 80%, 90%, or 95%.

The sirtuin modulator compounds described herein also have one or more of the following characteristics: the compound can be essentially non-toxic to a cell or subject; the sirtuin modulator compound can be an organic molecule or a small molecule of 2000 amu or less, 1000 amu or less; a compound can have a half-life under normal atmospheric conditions of at least about 30 days, 60 days, 120 days, 6 months, or 1 year; the compound can have a half-life in solution of at least about 30 days, 60 days, 120 days, 6 months, or 1 year; a sirtuin modulator compound can be more stable in solution than resveratrol by at least a factor of about 50%, 2 times, 5 times, 10 times, 30 times, 50 times, or 100 times; a sirtuin modulator compound can promote deacetylation of DNA repair factor Ku70; a sirtuin modulator compound can promote deacetylation of Rc1A / p65; a compound can increase the turnover rate and enhance the sensitivity of cells to TNF-induced apoptosis.

In certain embodiments, a sirtuin modulator compound does not have any substantial ability to inhibit a class I histone deacetylase (HDAC), class II HDAC, or HDAC I and II, at concentrations (eg, in vivo) effective to modulate sirtuin deacetylase activity. For example, in preferred embodiments, the sirtuin modulator compound is a sirtuin activating compound and is chosen to have an EC50 value to activate sirtuin deacetylase activity of at least 5 times less than the EC50 value for inhibition of a HDAC I and / or HDAC II, and even more preferably at least 10 times, 100 times or even 1000 times less. Methods for assaying HDAC I and / or HDAC II activity are known in the art, and kits for performing such assays are commercially available. See p. eg, BioVision, Inc. (Mountain View, CA; world wide web at biovision.com) and Thomas Scientific (Swedesboro, NJ; world wide web tomassci.com).

In certain embodiments, a sirtuin modulator compound does not have any substantial ability to modulate sirtuin homologs. In one embodiment, a human sirtuin protein activator may not have any substantial ability to activate a sirtuin protein from lower eukaryotes, particularly yeast or human pathogens, at concentrations (eg, in vivo) effective to activate activity. of human sirtuin deacetylase. For example, one can choose a sirtuin activating compound that has an EC50 value to activate a human sirtuin, such as SIRT1 and / or SIRT3, the deacetylase activity that is at least 5 times less than the EC50 value to activate a sirtuin of yeast, such as Sir2 (such as Candida, S. cerevisiae, etc.), and even more preferably at least 10 times, 100 times or even 1000 times less. In another embodiment, an inhibitor of a sirtuin protein from lower eukaryotes, particularly human or yeast pathogens, has no substantial ability to inhibit a human sirtuin protein at concentrations (eg, in vivo) effective to inhibit the deacetylase activity of a lower eukaryotic sirtuin protein. For example, one can choose a sirtuin inhibitor compound that has an IC50 value to inhibit a human sirtuin, such as SIRT1 and / or SIRT3, the deacylase activity that is at least 5 times less than the IC50 value to inhibit a sirtuin of yeast, such as Sir2 (such as Candida, S. cerevisiae, etc.), and even more preferably at least 10 times, 100 times or even 1000 times less.

In certain embodiments, a sirtuin modulator compound may have the ability to modulate one or more homologs of the sirtuin protein, such as, for example, one or more human SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7. In one embodiment, a sirtuin modulator compound has the ability to modulate both a SIRT1 and a SIRT3 protein.

In other embodiments, a SIRT1 modulator does not have any substantial ability to modulate other homologs of the sirtuin protein, such as, for example, one or more of human SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7, at concentrations (e.g. ., in vivo) effective to modulate human SIRT1 deacetylase activity. For example, a sirtuin modulator compound having an ED50 value can be chosen to modulate the activity.

ES 2 396 913 human SIRT1 deacetylase T3 that is at least 5 times lower than the ED50 value to modulate one or more

Human SIRT2, SIRT3, SIRT4, SIRT5, SIRT6 or SIRT7, and even more preferably at least 10 times, 100 times or even 1000 times less. In one embodiment, a SIRT1 modulator does not have any substantial ability to modulate a SIRT3 protein.

In other embodiments, a SIRT1 modulator does not have any substantial ability to modulate other sirtuin protein homologs, such as, for example, one or more of human sIrT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7, at concentrations (e.g. ., in vivo) effective to modulate human SIRT3 deacetylase activity. For example, a sirtuin modulator compound having an ED50 value to modulate human SIRT3 deacetylase activity that is at least 5 times lower than the ED50 value can be chosen to modulate one or more SIRT1, SIRT2, SIRT4, SIRT5, SIRT6 or human SIRT7, and even more preferably at least 10 times, 100 times or even 1000 times less. In one embodiment, a SIRT3 modulator does not have any substantial ability to modulate a SIRT1 protein.

In certain embodiments, a sirtuin modulator compound may have a binding affinity for a sirtuin protein of about 10M, 10M, 10M, 10M, or less. A sirtuin modulator compound can reduce (activator) or increase (inhibitor) the apparent Km value of a sirtuin protein for its substrate or NAD + (or other cofactor) by a factor of at least about 2, 3, 4, 5, 10 , 20, 30, 50 or 100. In certain embodiments, Km values ​​are determined using the mass spectrometry assay described herein. Preferred activator compounds reduce the Km value of a sirtuin for its substrate or cofactor to a greater degree than that caused by resveratrol at a similar concentration, or reduce the Km value of a sirtuin for its substrate or cofactor similar to that caused by resveratrol in a lower concentration. A sirtuin modulator compound can increase the Vmax value of a sirtuin protein by a factor of at least about 2, 3, 4, 5, 10, 20, 30, 50, or 100. A sirtuin modulator compound may have an ED50 value to modulate the deacetylase activity of a SIRT1 and / or SIRT3 protein of less than about 1 nM, less than about 10 nM, less than about 100 nM, less than about 1 pM, less of about 10 pM, less than about 100 pM, or between about 1-10 nM, between about 10-100 nM, between about 0.1-1 pM, between about 1-10 pM, or between about 10-100 pM. A sirtuin modulator compound can modulate the deacetylase activity of a SIRT1 and / or SIRT3 protein by a factor of at least about 5, 10, 20, 30, 50, or 100, as measured in a cellular assay or a cell-based assay. A sirtuin activating compound can cause at least approximately 10%, 30%, 50%, 80%, 2-fold, 5-fold, 10-fold, 50-fold, or 100-fold greater induction of deacetylase activity of a sirtuin protein relative to with the same concentration of resveratrol. A sirtuin modulator compound may have an ED50 value to modulate SIRT5 at least about 10 times, 20 times, 30 times, 50 times greater than that to modulate SIRT1 and / or SIRT3.

3. Illustrative uses

In certain aspects, the application considers methods for modulating the level and / or activity of a sirtuin protein and methods for its use.

In certain embodiments, the invention contemplates methods for use of sirtuin modulator compounds wherein the sirtuin modulator compounds activate a sirtuin protein, e.g. For example, they increase the level and / or activity of a sirtuin protein. Sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can be useful for a variety of therapeutic applications including, for example, increasing the life of a cell and treating and / or preventing a wide range of diseases. and disorders including, for example, diseases or disorders related to aging or stress, diabetes, obesity, neurodegenerative diseases, cardiovascular disease, blood clotting disorders, inflammation, cancer and / or hot flashes, etc. The methods comprise administering to a subject in need thereof a pharmaceutically effective amount of a sirtuin modulator compound, e.g. eg, a sirtuin activating compound.

In other embodiments, the application considers methods for use of sirtuin modulator compounds in which the sirtuin modulator compounds reduce sirtuin activity, e.g. For example, they reduce the level and / or activity of a sirtuin protein. Sirtuin modulating compounds that reduce the level and / or activity of a sirtuin protein may be useful for a variety of therapeutic applications including, for example, increasing cellular sensitivity to stress (including increasing radiosensitivity and / or chemosensitivity). , increase the amount and / or rate of apoptosis, cancer treatment (optionally in combination with another chemotherapeutic agent), stimulation of appetite and / or stimulation of weight gain, etc. The methods comprise administering to a subject in need thereof a pharmaceutically effective amount of a sirtuin modulator compound, e.g. eg, a sirtuin inhibitor compound.

While Applicants do not wish to be swayed by theory, it is believed that the activators and inhibitors of the present invention may interact with a sirtuin at the same site within the sirtuin protein (eg, active site or site that affects the Km or Vmax value of the active site). This is believed to be the reason why certain classes of sirtuin activators and inhibitors may have substantial structural similarity.

ES 2 396 913 T3

In certain embodiments, the sirtuin modulator compounds described herein can be taken alone or in combination with other compounds. In one embodiment, a mixture of two or more sirtuin modulator compounds can be administered to a subject in need thereof. In another embodiment, a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein can be administered with one or more of the following compounds: resveratrol, butein, fisetin, piceatannol, or quercetin. In an illustrative embodiment, a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein can be administered in combination with nicotinic acid. In another embodiment, a sirtuin modulator compound that reduces the level and / or activity of a sirtuin protein can be administered with one or more of the following compounds: nicotinamide (NAM), suranim; NF023 (a G protein antagonist); NF279 (a purinergic receptor antagonist); Trolox (6-hydroxy-2,5,7,8, tetramethylchroman-2-carboxylic acid); (-) - epigallocatechin (hydroxy at sites 3,5,7,3 ', 4', 5 '); (-) - epigallocatechin gallate (Hydroxy 5,7,3 ', 4', 5 'and gallate ester sites at 3); cyanidin chloride (3,5,7,3 ', 4'-pentahydroxyflavilium chloride); delphinidin chloride (3,5,7,3 ', 4', 5'-hexahydroxyflavilium chloride); miricetin (cannabiscetin; 3,5,7,3 ', 4', 5'-hexahydroxyflavone); 3,7,3 ', 4', 5'-pentahydroxyflavone; gossypetin (3,5,7,8,3 ', 4'hexahydroxyflavone), sirtinol; and splithomycin (see eg, Howitz et al. (2003) Nature 425: 191; Grozinger et al. (2001) J. Biol. Chem. 276: 38837; Dedalov et al. (2001) PNAS 98: 15113; and Hirao et al. (2003) J. Biol. Chem 278: 52773). In yet another embodiment, one or more sirtuin modulating compounds can be administered with one or more therapeutic agents for the treatment or prevention of various diseases, including, for example, cancer, diabetes, neurodegenerative diseases, cardiovascular disease, blood clotting, inflammation. , hot flashes, obesity, aging, stress, etc. In various embodiments, combination therapies comprising a sirtuin modulator compound can refer to (1) pharmaceutical compositions comprising one or more sirtuin modulator compounds in combination with one or more therapeutic agents (e.g. g., one or more therapeutic agents described herein); and (2) co-administration of one or more sirtuin modulating compounds with one or more therapeutic agents where the sirtuin modulating compound and the therapeutic agent have not been formulated in the same compositions (but may be present within the same kit or package. , such as a blister pack or other multi-chamber package; connected, separately sealed containers (eg. g., aluminum bags) that can be separated by the user; or a kit in which the sirtuin modulator compound (s) and the other therapeutic agent (s) are in separate containers). When separate formulations are used, the sirtuin modulator compound can be administered concurrently, intermittently, staggered, before, after, or combinations thereof, with the administration of another therapeutic agent.

In certain embodiments, methods for reducing, preventing, or treating diseases or disorders using a sirtuin modulator compound may also comprise increasing the level of a sirtuin protein, such as human SIRT1, SIRT2, and / or SIRT3, or their counterparts. Increasing protein levels can be achieved by introducing one or more copies of a nucleic acid encoding a sirtuin into a cell. For example, the level of a sirtuin can be increased in a mammalian cell by introducing into the mammalian cell a nucleic acid encoding sirtuin, e.g. eg, increasing the level of SIRT1 by introducing a nucleic acid that encodes the amino acid sequence set forth in no. access code in GenBank NP_036370 and / or increase the level of SIRT3 by introducing a nucleic acid that encodes the amino acid sequence set forth in no. GenBank accession AAH01042. The nucleic acid can be under the control of a promoter that regulates the expression of the SIRT1 and / or SIRT3 nucleic acid. Alternatively, the nucleic acid can be introduced into the cell at a site in the genome that is downstream of a promoter. Methods for increasing the level of a protein using these methods are known in the art.

A nucleic acid that is introduced into a cell to increase the protein level of a sirtuin can encode a protein that is at least about 80%, 85%, 90%, 95%, 98%, or 99% identical to the sequence of a sirtuin, p. eg, SIRT1 protein (GenBank accession number NP_036370) and / or SIRT3 (GenBank accession number AAH01042). For example, the nucleic acid encoding the protein may be at least about 80%, 85%, 90%, 95%, 98%, or 99% identical to a nucleic acid encoding a SIRT1 protein (eg, No. GenBank accession number NM_012238) and / or SIRT3 (eg, GenBank accession number BC001042). The nucleic acid can also be a nucleic acid that hybridizes, preferably under stringent hybridization conditions, to a nucleic acid encoding a wild-type sirtuin, e.g. g., the SIRT1 protein (GenBank accession number NMM_012238) and / or SIRT3 (eg, GenBank accession number BC001042). Stringent hybridization conditions can include hybridization and a wash in 0.2 x SSC at 65 ° C. When using a nucleic acid encoding a protein that is different from a wild-type sirtuin protein, such as a protein that is a fragment of a wild-type sirtuin, the protein is preferably biologically active, e.g. For example, it is capable of deacetylation. It is only necessary to express in a cell a portion of sirtuin that is biologically active. For example, a protein that differs from wild-type SIRT1, which has no. GenBank accession NP_036370, preferably its core structure. The core structure is sometimes referred to amino acids 62-293 of no. GenBank accession NP_036370, which are encoded by nucleotides 237 to 932 of nos. GenBank accession NM_012238, which covers NAD binding as well as substrate binding domains. The core domain of SIRT1 can also refer to about amino acids 261 to 447 of nos. GenBank accession NP_036370, encoded by nucleotides 834 to 1394 with nos. GenBank accession NM_012238; at about amino acids 242 to 493 with no. GenBank accession NP_036370, encoded by nucleotides 777 to 1532 with nos. GenBank accession NM_012238; or up to about amino acids 254 to 495 with no. GenBank accession NP_036370, encoded by nucleotides 813 to

ES 2 396 913 T3

1538 with no. GenBank accession NM_012238. Whether or not a protein retains a biological function, e.g. eg, deacetylation ability, can be determined according to methods known in the art.

In certain embodiments, methods of reducing, preventing, or treating diseases or disorders using a sirtuin modulator compound may also comprise reducing the level of a sirtuin protein, such as human SIRT1, SIRT2, and / or SIRT3, or their counterparts. Reducing the level of a sirtuin protein can be achieved according to methods known in the art. For example, a siRNA, antisense nucleic acid, or ribozyme targeting sirtuin can be expressed in the cell. A dominant negative sirtuin mutant can also be used, e.g. eg, a mutant that is not capable of deacetylation. For example, the SIRT1 mutant H363Y, described, eg, can be used. eg, in Luo et al. (2001) Cell 107: 137. Alternatively, agents that inhibit transcription can be used.

Methods for modulating sirtuin protein levels also include methods for modulating the transcription of genes encoding sirtuins, methods for stabilizing / destabilizing the corresponding mRNAs, and other methods known in the art.

Aging / Stress

In one embodiment, the application considers a method that extends the life of a cell, extends the proliferative capacity of a cell, delays aging of a cell, promotes survival of a cell, delays cellular senescence in a cell, mimics the effects from caloric restriction, increases a cell's resistance to stress or prevents apoptosis of a cell, contacting the cell with a sirtuin modulator compound of the invention that increases the level and / or activity of a sirtuin protein. In an illustrative embodiment, the methods comprise contacting the cell with a sirtuin activating compound.

The methods described herein can be used to increase the amount of time that cells, particularly primary cells (i.e., cells obtained from an organism, eg, a human), can be kept alive in culture. mobile. Embryonic stem cells (ES) and pluripotent cells, as well as the differentiated cells derived from them, can also be treated with a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein to maintain cells, or its offspring, in culture for longer periods of time. Said cells can also be used for transplantation into a subject, e.g. eg, after ex vivo modification.

In one embodiment, cells that are intended to be preserved for long periods of time can be treated with a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein. Cells can be in suspension (eg, blood cells, serum, biological growth media, etc.) or in tissues or organs. For example, blood taken from an individual for transfusion purposes can be treated with a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein to preserve blood cells for longer periods of time. Furthermore, blood to be used for forensic purposes can also be preserved by employing a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein. Other cells that can be treated to extend their life or to protect against apoptosis include cells for consumption, e.g. eg, non-human mammalian cells (such as meat) or plant cells (such as vegetables).

Sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can also be applied during phases of development and growth in mammals, plants, insects or microorganisms, in order to, e.g. eg, alter, delay or accelerate development and / or growth processes.

In another embodiment, sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can be used to treat cells useful for cell transplantation or therapy, including for example solid tissue grafts, organ transplants, cell suspensions, cells stem, bone marrow cells, etc. The cells or tissue may consist of an autograft, an allograft, an isograft, or a xenograft. Cells or tissue can be treated with the sirtuin modulator compound prior to administration / implantation, concurrent with administration / implantation and / or post-administration / implantation in a subject. Cells or tissue can be treated prior to donor cell removal, ex vivo after donor cell or tissue removal post-implantation into the recipient. For example, the donor or recipient may be treated systemically with a sirtuin modulator compound or may have a subset of cells / tissue treated locally with a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein. In certain embodiments, the cells or tissue (or donors / recipients) may further be treated with another therapeutic agent useful to prolong graft survival, such as, for example, an immunosuppressive agent, a cytokine, an angiogenic factor, etc.

In still other embodiments, cells can be treated with a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein in vivo, e.g. eg, to increase its life or prevent apoptosis. For example, the skin can be protected from aging (eg, development of wrinkles, loss of elasticity, etc.) by treating the skin or epithelial cells with a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein. In an illustrative embodiment, the skin is contacted with a composition

ES 2 396 913 T3 pharmaceutical or cosmetic comprising a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein. Exemplary skin conditions or skin conditions that can be treated in accordance with the methods described herein include disorders or diseases associated with or caused by inflammation, sun damage, or natural aging. For example, the compositions find utility in the prevention or treatment of contact dermatitis (including irritant contact dermatitis and allergic contact dermatitis), atopic dermatitis (also known as allergic eczema), actinic keratosis, keratinization disorders (including eczema), epidermolysis bullosa diseases (including pemphigus), exfoliative dermatitis, seborrheic dermatitis, erythema (including erythema multiforme and erythema nodosum), damage caused by the sun or other light sources, discoid lupus erythematosus, dermatomyositis, psoriasis, skin cancer and the effects of natural aging. In another embodiment, sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can be used for the treatment of wounds and / or burns to promote healing, including for example first, second or third degree burns and / or thermal, chemical or electrical burns. The formulations can be administered topically, to the skin or mucosal tissue, as an ointment, lotion, cream, microemulsion, gel, solution or the like, as will be described in more detail herein, within the context of an effective administration regimen to achieve the desired result.

Topical formulations comprising one or more sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can also be used as preventive compositions, e.g. eg, chemopreventive. When used in a chemopreventive method, susceptible skin is treated prior to any visible conditions in a particular individual.

The sirtuin modulator compounds can be administered locally or systemically to a subject. In one embodiment, a sirtuin modulator compound is administered locally to the tissue or organ of a subject by injection, topical formulation, etc.

In another embodiment, a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein can be used to treat or prevent a cell senescence-induced disease or condition in a subject; methods for reducing the rate of senescence of a subject, eg. eg, after the onset of senescence; methods to extend the life of a subject; methods to treat or prevent a life-related disease or condition; methods for treating or preventing a disease or condition related to the proliferative capacity of cells; and methods of treating or preventing a disease or condition resulting from cell damage or death. In certain embodiments, the method does not work to reduce the rate of occurrence of life-shortening diseases of a subject. In certain embodiments, a method does not work to reduce mortality caused by a disease, such as cancer.

In yet another embodiment, a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein can be administered to a subject in order to generally increase the life of their cells and to protect their cells against stress and / or against apoptosis. Treating a subject with a compound described herein is believed to be similar to subjecting the subject to hormesis, ie, mild stress that is beneficial to organisms and can extend life.

Sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can be administered to a subject to prevent aging and the consequences related to aging, such as stroke, heart disease, heart failure, arthritis, high blood pressure, and disease. Alzheimer's. Other conditions that can be treated include eye disorders, e.g. g., associated with the aging of the eye, such as cataracts, glaucoma and macular degeneration. Sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can also be administered to subjects for the treatment of diseases, e.g. g., chronic diseases, associated with cell death, in order to protect cells from cell death. Illustrative diseases include those associated with neuronal cell death, neuronal dysfunction, or muscle cell death or dysfunction, such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, amniotropic lateral sclerosis, and muscle dystrophy; AIDS; fulminant hepatitis; diseases associated with degeneration of the brain, such as Creutzfeld-Jakob disease, retinitis pigmentosa, and cerebellar degeneration; myelodysplasia such as aplastic anemia; ischemic diseases such as myocardial infarction and stroke; liver diseases such as alcoholic hepatitis, hepatitis B, and hepatitis C; joint diseases such as osteoarthritis; atherosclerosis; alopecia; skin damage caused by UV light; lichen planus; skin atrophy; waterfalls; and graft rejection. Cell death can also be caused by surgery, drug therapy, chemical exposure, or radiation exposure.

Sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can also be administered to a subject suffering from an acute illness, e.g. eg damage to an organ or tissue, eg. eg, a subject suffering from a stroke or myocardial infarction or a subject suffering from a spinal cord injury. Sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can also be used to repair an alcoholic liver.

Cardiovascular disease

ES 2 396 913 T3

In another embodiment, the application considers a method of treating and / or preventing cardiovascular disease, administering to a subject in need thereof a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein.

Cardiovascular diseases that can be treated or prevented using sirtuin modulating compounds that increase the level and / or activity of a sirt protein include cardiomyopathy or myocarditis; such as idiopathic cardiomyopathy, metabolic cardiomyopathy, alcoholic cardiomyopathy, drug-induced cardiomyopathy, ischemic cardiomyopathy and hypertensive cardiomyopathy. Furthermore, treatable or preventable with the use of the compounds and methods described herein are atheromatous disorders of the major blood vessels (macrovascular disease) such as the aorta, coronary arteries, carotid arteries, cerebrovascular arteries, renal arteries. , the iliac arteries, the femoral arteries, and the popliteal arteries. Other vascular diseases that can be treated or prevented include those related to platelet aggregation, retinal arterioles, glomerular arterioles, nerve vessels, cardiac arterioles, and associated capillary beds of the eye, kidney, heart, and central nervous systems, and peripheral. Sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can also be used to increase an individual's plasma HDL levels.

Still other disorders that can be treated with sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein include restenosis, e.g. g., that follows a coronary intervention, and disorders related to an abnormal level of high-density or low-density cholesterol.

In one embodiment, a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein can be administered as part of a combination therapy with a cardiovascular agent including, for example, an antiarrhythmic agent, an antihypertensive agent, a blood glucose blocker. calcium channels, a cardioplegic solution, a cardiotonic agent, a fibrinolytic agent, a sclerosing solution, a vasoconstrictor, a vasodilator, a nitric oxide donor, a potassium channel blocker, a sodium channel blocker, statins, or a naturiuretic agent.

In one embodiment, a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein can be administered as part of a combination therapy with an anti-arrhythmia agent. Antiarrhythmic agents are often organized into four main groups according to their mechanism of action: type I, sodium channel blockade; type II, beta-adrenergic blockade; type III, prolonged repolarization; and type IV, calcium channel blockade. Type I anti-arrhythmic agents include lidocaine, moricizine, mexiletine, tocainide, procainamide, encainide, phlecanide, tocainide, phenytoin, propafenone, quinidine, disopyramide, and flecainide. Type II anti-arrhythmic agents include propranolol and esmolol. Type III agents include agents that act by prolonging the duration of the action potential, such as amiodarone, artilide, bretilium, clofilium, isobutilide, sotalol, azimilide, dofetilide, dronedarone, ersentilide, ibutilide, tedisamil, and threcetylide. Type IV anti-arrhythmic agents include verapamil, diltaizem, digitalis, adenosine, nickel chloride, and magnesium ions.

In another embodiment, a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein can be administered as part of a combination therapy with another cardiovascular agent. Examples of cardiovascular agents include vasodilators, eg, hydralazine; angiotensin converting enzyme inhibitors, eg captopril; anti-angina agents, for example isosorbide nitrate, glyceryl trinitrate and pentaerythritol tetranitrate; anti-arrhythmic agents, eg quinidine, procainaltide and lignocaine; cardioglycosides, eg, digoxin and digitoxin; calcium antagonists, eg, verapamil and nifedipine; diuretics, such as thiazides and related compounds, for example bendrofluazide, chlorothiazide, chlorotalidone, hydrochlorothiazide and other diuretics, for example fursemide and triamterene, and sedatives, for example nitrazepam, flurazepam and diazepam.

Other exemplary cardiovascular agents include, for example, a cyclooxygenase inhibitor such as aspirin or indomethacin, a platelet aggregation inhibitor such as clopidogrel, ticlopidene, or aspirin, fibrinogen antagonists, or a diuretic such as chlorothiazide, hydrochlorothiazide, flumethiazide, hydroflumethiazide, bendlumethiazide. , methylchlorthiazide, trichloromethiazide, polythiazide or benzthiazide as well as ethacrynic acid trichrinephen, chlorthalidone, furosemide, musolimine, bumetanide, triamterene, amiloride, and spironolactone and salts of these compounds, angiotensin-converting enzyme inhibitors such as captopril, zofenopril, fosinopril, enalapril, ceranopril, cilazopril, delapril, pentopril, quinapril, ramipril antagonists, and salts of lisinopril said angiotensin II such as losartan, irbesartan or valsartan, thrombolytic agents such as tissue plasminogen activator (tPA), recombinant tPA, streptokinase, urokinase, prourokinase and anisoylated streptokinase plasminogen activated complex (APSAC, Eminase, Beecham Laboratories), or animal salivary gland plasminogen activators, calcium channel blockers such as verapamil, nifedipine, or diltiazem, thromboxane receptor antagonists such as ifetroban , prostacyclin mimetics or phosphodiesterase inhibitors. Such combination products, if formulated as a fixed dose, employ the compounds of the present invention within the dosage range described above and the other pharmaceutically active agent within its approved dosage range.

Still other illustrative cardiovascular agents include, for example, vasodilators, e.g. eg, benziclan, cinnarizine, citicoline, cycladelate, cyclonicate, ebumamonin, fenoxezil, flunarizine, ibudilast, ifenprodil, lomerizine, naflole, nikamate, nosergoline, nimodipine, papaverine, pentifylline, nofizpomin, vincacetina

ES 2 396 913 T3 pentoxifylline, prostacyclin derivatives (such as prostaglandin E1 and prostaglandin I2), an endothelin receptor blocking drug (such as bosentan), diltiazem, nicorandil and nitroglycerin. Examples of the brain protective drug include radical scavengers (such as edaravone, vitamin E and vitamin C), glutamate antagonists, AMPA antagonists, kainate antagonists, NMDA antagonists, GABA agonists, growth factors, opioid antagonists, phosphatidylcholine precursors, serotonin agonists, Na channel inhibitors⁺/AC²⁺ and K channel opening drugs⁺ . Examples of brain metabolic stimulants include amantadine, tiapride, and gamma-aminobutyric acid. Examples of the anticoagulant include heparins (such as heparin sodium, heparin potassium, dalteparin sodium, dalteparin calcium, heparin calcium, parnaparin sodium, and danaparoid sodium), warfarin, enoxaparin, argatroban, batroxobin, and sodium citrate. Examples of antiplatelet drugs include ticlopidine hydrochloride, dipyridamole, cilostazol, ethyl icosapentate, sarpogrelate hydrochloride, dilazep hydrochloride, trapidil, a non-steroidal anti-inflammatory agent (such as aspirin), beraprostsodium, ilofrost, and indobuterol. Examples of thrombolytic drugs include urokinase, tissue-type plasminogen activators (such as alteplase, tisokinase, nateplase, pamiteplase, monteplase, and rateplase), and nasaruplase. Examples of the antihypertensive drug include angiotensin converting enzyme inhibitors (such as captopril, alacepril, lisinopril, imidapril, quinapril, temocapril, delapril, benazepril, cilazapril, trandolapril, enalapril, ceronapril, spinalpril, peril, imidapril, mobipirapril, ramindopril, immigrant and randolapril), angiotensin II antagonists (such as losartan, candesartan, valsartan, eprosartan, and irbesartan), calcium channel blockers (such as aranidipine, efonidipine, nicardipine, bamidipine, benidipine, manidipine, cilnidipine, nisoldipine, nitrendipine, nifedipine, nilvadipine, felodipine, amlodipine, diltiazem, bepridil, clentiazem, fendilin, galopamil, mibeiadipine, teridilotine, prenidipine, mibeidylamine, ellidylotine, prenidipine, miberadilotine, lercanidipine, nimodipine, cinnarizine, flunarizine, lidoflazine, lomerizine, benziclane, etafenone and perhexilina), β-adrenaline receptor blockers (propranolol, pindolol, indenolol, carteolol, bunitrolol, atenolol, acebutolol, metoprolol, timolol, nipradilol, penbutolol, nadolol, tilisolol, carvedilol, bisoprolol, betaxolol, celiprolol, bopindolol, bevantolol, labetalol, alprenolol, buolfotunfeolol, buolfotun and amosulal butylidine, butofilolol, carazolol, cetamolol, chloranolol, dilevalol, epanolol, levobunolol, mepindolol, metipranolol, moprolol, nadoxolol, nevibolol, oxprenolol, practol, pronetalol, sotalol, sufinalol, talindolol, talindolol tertalol, toliprolol, xybenolol, and esmolol), α-receptor blockers (such as amosulalol, prazosin, terazosin, doxazosin, bunazosin, urapidil, phentolamine, arotinolol, dapiprazole, fenspiride, indoramin, labetalol, naphtopinoline, tampazolin, nicotinol, naphtopinol, and taminozulosin yohimbine), sympathetic nerve inhibitors (such as clonidine, guanfacine, guanabenz, methyldopa, and reserpine), hydralazine, todralazine, budralazine, and cadralazine. Examples of the antianginal drug include nitrate drugs (such as amyl nitrate, nitroglycerin, and isosorbide), β-adrenaline receptor blockers (such as propranolol, pindolol, indenolol, carteolol, bunitrolol, atenolol, acebutolol, metoprolol, timolol, nipradilol, tilisolol, penbutol, , carvedilol, bisoprolol, betaxolol, celiprolol, bopindolol, bevantolol, labetalol, alprenolol, amosulalol, arotinolol, befunolol, bucumolol, bufetolol, buferalol, buprandolol, butylidine, butofilolol, carazolol, cetamolol, chloranolol, dilevalol, epanolol, levobunolol, mepindolol, metipranolol, moprolol, nadoxolol, nevibolol, oxprenolol, practol, pronetalol, sotalol, sufinalol, talindole channels, calcium channel blockers, andxibolol (tertalol) such as aranidipine, efonidipine, nicardipine, bamidipine, benidipine, manidipine, cilnidipine, nisoldipine, nitrendipine, nifedipine, nilvadipine, felodipine, amlodipine, diltiazem, bepridil, clentiazem, fendiline galopamil, mibefradil, prenylamine, semotiadil, terodiline, verapamil, cilnidipine, elgodipine, isradipine, lacidipine, lercanidipine, nimodipine, cinnarizine, flunarizine, lidoflazine, lomerizine, trapsol, dilazidyl, and perimethrimetamoline, ethazidylphenide, and perimehetamoline enoxaparin and aspirin. Examples of diuretics include thiazide diuretics (such as hydrochlorothiazide, methicothiazide, trichlormethiazide, benzylhydrochlorothiazide, and penflutizide), loop diuretics (such as furosemide, ethacrynic acid, bumetanide, pyretanide, azosemide, and torasemide diuretics).⁺ (spironolactone, triamterene, and potassium canrenoate), osmotic diuretics (such as isosorbide, D-mannitol, and glycerin), nontiazide diuretics (such as methicran, trypamide, chlorthalidone, and mefruside), and acetazolamide. Examples of the cardiotonic include digitalis formulations (such as digitoxin, digoxin, methyldigoxin, deslanoside, vesnarinone, lanatoside C, and proscylaridine), xanthine formulations (such as aminophylline, choline theophylline, diprophylline, and proxifylline), catecholamine formulations (such as dopacarmine, dobpautamine, dobpautamine) ), PDE III inhibitors (such as amrinone, olprinone, and milrinone), denopamine, ubidecarenone, pimobendan, levosimendan, aminoethylsulfonic acid, vesnarinone, carperitida and colforsin daropat. Examples of antiarrhythmic drugs include ajmaline, pyrmenol, procainamide, cybenzoline, disopyramide, quinidine, aprindine, mexyletin, lidocaine, phenyloin, pilsicainide, propafenone, flecainide, atenolol, acebutolol, sotalol, propranolioindol, methoprolol, propranolioindole, methoprolol, nipranzelantonekaol, pyltindol bepridil and verapamil. Examples of the antihyperlipidemic drug include atorvastatin, simvastatin, pravastatin sodium, fluvastatin sodium, clinofibrate, clofibrate, simfibrate, fenofibrate, bezafibrate, cholestimidc, and cholestyramine. Examples of the immunosuppressant include azathioprine, mizoribine, cyclosporine, tacrolimus, gusperimus, and methotrexate.

Cell death / Cancer

Sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can be administered to subjects who have recently received or are likely to receive a dose of radiation or toxin. In one embodiment, the radiation or toxin dose is received as part of a work-related or medical procedure, e.g. eg, work in a nuclear power plant, flying in an airplane, X-rays, CT scans, or the administration of a radioactive dye for medical imaging; In such an embodiment, the compound is administered as a prophylactic measure. In another embodiment, the radiation or toxin is received unintentionally, e.g. For example, as a consequence of an industrial accident, inhabiting a site of natural radiation, terrorist act or act of war that

ES 2 396 913 T3 implies radioactive or toxic material. In such a case, the compound is preferably administered as soon as possible after exposure to inhibit apoptosis and the subsequent development of acute radiation syndrome.

Sirtuin modulator compounds can also be used to treat and / or prevent cancer. In certain embodiments, sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can be used to treat and / or prevent cancer. Caloric restriction has been associated with a reduction in the incidence of age-related disorders, including cancer (see eg, Bordone and Guarente, Nat. Rev. Mol. Cell Biol. (2005 epub); Guarente and Picard, Cell 120: 473-82 (2005); Berrigan, et al., Carcinogenesis 23: 817-822 (2002); and Heilbronn and Ravussin, Am. J. Clin. Nutr. 78: 361-369 (2003)). In addition, the yeast Sir2 protein has been shown to be required for life extension by glucose restriction (see eg, Lin et al., Science 289: 2126-2128 (2000); Anderson et al., Nature 423: 181-185 (2003)), a yeast model for caloric restriction. Accordingly, an increase in the level and / or activity of a sirtuin protein may be useful in treating and / or preventing the incidence of age-related disorders, such as cancer. In other embodiments, sirtuin modulator compounds that reduce the level and / or activity of a sirtuin protein can be used to treat and / or prevent cancer. For example, inhibitory compounds can be used to stimulate acetylation of substrates such as p53 and therefore increase apoptosis, as well as reduce the life of cells and organisms, make them more sensitive to stress, and / or increase radiosensitivity and / or or chemosensitivity of a cell or organism. Therefore, inhibitory compounds can be used, e.g. eg, to treat cancer. Illustrative cancers that can be treated using a sirtuin modulator compound are those of the brain and kidney; hormone-dependent cancer, including breast, prostate, testicular, and ovarian cancer; lymphomas and leukemias. In cancer associated with solid tumors, a modulator compound can be administered directly to the tumor. Cancer of blood cells, p. eg, leukemia, can be treated by administering a modulator compound to the bloodstream or bone marrow. The development of benign cells can also be treated, e.g. eg, warts. Other diseases that can be treated include autoimmune diseases, e.g. eg, systemic lupus erythematosus, scleroderma, and arthritis, where autoimmune cells should be killed. Viral infections such as herpes, HIV, adenovirus, and malignant and benign disorders associated with HTLV-1 can also be treated with the administration of a sirtuin modulator compound. Alternatively, cells can be obtained from a subject, treated ex vivo to kill certain undesirable cells, e.g. eg, cancer cells, and administered again to the same subject or to a different subject.

Other chemotherapeutic agents that can be co-administered with the modulator compounds described herein as having anti-cancer activity (e.g. (g., compounds that induce apoptosis, compounds that reduce life, or compounds that make cells sensitive to stress) include: aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cytofosphamine, darabiphuncarbatrolin, daziphondrine, dazoterrinecarbatrolin, dazorphosphamine diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, methotrexate, murethane, methothane, methothane, methothane, methothane, methothanol mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristiae, vindesine, and vinorelbine.

These chemotherapeutic agents can be categorized by their mechanism of action into, for example, the following groups: anti-metabolite / anti-cancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabiae) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); antiproliferative / antimitotic agents, including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones and navelbine, epidipodophyllotoxins, tenipodophylotoxin agents DNA (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, hexamethylmelaminoxaliplatin, ifosfamide, melphalan, merchlorethamide, mitomycin, mitoxantrone, nitrosourea, paclitaxel, plicamycin, procarbazine, teniposide, etophosfoethylene) (VP16), triophosphophoside (VP) antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase that systemically metabolizes L-asparagine and blocks cells that do not have the ability to synthesize their own asparagine); antiplatelet agents; antiproliferative / antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide, and analogs, melphalan, chlorambucil), ethyleneimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmzoustine, analogs) dacarbazinine (DTIC); antiproliferative / antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone inhibitor analogs (estrogen, tamoxifen, goserrelin, bicalutamide, nilutamide) and aromatase (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts, and other thrombin inhibitors); fibrinotic agents (such as activator of the

ES 2 396 913 T3 tissue plasminogen, streptokinase and urokinase), aspirin, COX-2 inhibitors, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigration agents; antisegregating agents (breveldin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (TNP-470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF), fibroblast growth factor inhibitor (FGF), epidermal growth factor (EGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, 10 daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, corticosteroids (corticosteroids), irinotecan) and mitoxantrone, corticosteroids (irinotecan) hydrocortisone, methylpednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors; inducers of mitochondrial dysfunction and activators of caspase; chromatin disruptors.

These chemotherapeutic agents can be used by themselves with a sirtuin modulator compound described herein by inducing cell death or reducing life or increasing sensitivity to stress and / or in combination with other chemotherapeutic agents. Many combinatorial therapies have been developed, including, but not limited to, those listed in Table 1.

Table 1: Illustrative Combinatorial Therapies for Cancer Treatment.

<td>Denomination</td><td>Therapeutic agents</td>

<td>ABV</td><td>Doxorubicin, Bleomycin, Vinblastine</td>

<td>ABVD</td><td>Doxorubicin, Bleomycin, Vinblastine, Dacarbazine</td>

<td>AC (Mom)</td><td>Doxorubicin, Cyclophosphamide</td>

<td>AC (Sarcoma)</td><td>Doxorubicin, Cisplatin</td>

<td>AC (Neuroblastoma)</td><td>Cyclophosphamide, Doxorubicin</td>

<td>ace</td><td>Cyclophosphamide, Doxorubicin, Etoposide</td>

<td>ace</td><td>Cyclophosphamide, Doxorubicin</td>

<td>AD</td><td>Doxorubicin, Dacarbazine</td>

<td>AP</td><td>Doxorubicin, Cisplatin</td>

<td>arac-dnr</td><td>Cytarabine, Daunorubicin</td>

<td>B-CAVe</td><td>Bleomycin, Lomustine, Doxorubicin, Vinblastine</td>

<td>bcvpp</td><td>Carmustine, Cyclophosphamide, Vinblastine, Procarbazine, Prednisone</td>

<td>beacopp</td><td>Bleomycin, Etoposide, Doxorubicin, Cyclophosphamide, Vincristine, Procarbazine, Prednisone, Filgrastim</td>

<td>BEP</td><td>Bleomycin, Etoposide, Cisplatin</td>

<td>BEEP</td><td>Bleomycin, Cisplatin, Ifosfamide, Mesna</td>

<td>bomp</td><td>Bleomycin, Vincristine, Cisplatin, Mitomycin</td>

<td>AC</td><td>Cytarabine, Asparaginase</td>

<td>cape</td><td>Cisplatin, Methotrexate, Bleomycin, Vincristine</td>

<td>coffee</td><td>Cyclophosphamide, Doxorubicin, Fluorouracil</td>

<td>cal-g</td><td>Cyclophosphamide, Daunorubicin, Vincristine, Prednisone, Asparaginase</td>

ES 2 396 913 T3

<td>Denomination</td><td>Therapeutic agents</td>

<td>CAMP</td><td>Cyclophosphamide, Doxorubicin, Methotrexate, Procarbazine</td>

<td>CHAP</td><td>Cyclophosphamide, Doxorubicin, Cisplatin</td>

<td>CaT</td><td>Carboplatin, Paclitaxel</td>

<td>CAV</td><td>Cyclophosphamide, Doxorubicin, Vincristine</td>

<td>CAVE ADD</td><td>CAV and Etoposide</td>

<td>CA-VP16</td><td>Cyclophosphamide, Doxorubicin, Etoposide</td>

<td>DC</td><td>Cyclophosphamide, Carboplatin</td>

<td>CDDP / VP-16</td><td>Cisplatin, Etoposide</td>

<td>CEF</td><td>Cyclophosphamide, Epirubicin, Fluorouracil</td>

<td>CEPP (B)</td><td>Cyclophosphamide, Etoposide, Prednisone, with or without / Bleomycin</td>

<td>CEV</td><td>Cyclophosphamide, Etoposide, Vincristine</td>

<td>CF</td><td>Cisplatin, Fluorouracil, or Carboplatin Fluorouracil</td>

<td>CHAP</td><td>Cyclophosphamide or Cyclophosphamide, Altretamine, Doxorubicin, Cisplatin</td>

<td>Ch1VPP</td><td>Chlorambucil, Vinblastine, Procarbazine, Prednisone</td>

<td>CHOP</td><td>Cyclophosphamide, Doxorubicin, Vincristine, Prednisone</td>

<td>CHOP-BLEO</td><td>Add Bleomycin to CHOP</td>

<td>CISCA</td><td>Cyclophosphamide, Doxorubicin, Cisplatin</td>

<td>CLD-BOMP</td><td>Bleomycin, Cisplatin, Vincristine, Mitomycin</td>

<td>CMF</td><td>Methotrexate, Fluorouracil, Cyclophosphamide</td>

<td>CMFP</td><td>Cyclophosphamide, Methotrexate, Fluorouracil, Prednisone</td>

<td>CMFVP</td><td>Cyclophosphamide, Methotrexate, Fluorouracil, Vincristine, Prednisone</td>

<td>CMV</td><td>Cisplatin, Methotrexate, Vinblastine</td>

<td>CNF</td><td>Cyclophosphamide, Mitoxantrone, Fluorouracil</td>

<td>CNOP</td><td>Cyclophosphamide, Mitoxantrone, Vincristine, Prednisone</td>

<td>COB</td><td>Cisplatin, Vincristine, Bleomycin</td>

<td>CODE</td><td>Cisplatin, Vincristine, Doxorubicin, Etoposide</td>

<td>COMLA</td><td>Cyclophosphamide, Vincristine, Methotrexate, Leucovorin, Cytarabine</td>

<td>COMP</td><td>Cyclophosphamide, Vincristine, Methotrexate, Prednisone</td>

<td>Copper regime</td><td>Cyclophosphamide, Methotrexate, Fluorouracil, Vincristine, Prednisone</td>

<td>COP</td><td>Cyclophosphamide, Vincristine, Prednisone</td>

ES 2 396 913 T3

<td>Denomination</td><td>Therapeutic agents</td>

<td>COPE</td><td>Cyclophosphamide, Vincristine, Cisplatin, Etoposide</td>

<td>COPP</td><td>Cyclophosphamide, Vincristine, Procarbazine, Prednisone</td>

<td>CP (lymphocytic leukemia chronicle)</td><td>Chlorambucil, Prednisone</td>

<td>CP (ovarian cancer)</td><td>Cyclophosphamide, Cisplatin</td>

<td>CT</td><td>Cisplatin, Paclitaxel</td>

<td>CVD</td><td>Cisplatin, Vinblastine, Dacarbazine</td>

<td>CVI</td><td>Carboplatin, Etoposide, Ifosfamide, Mesna</td>

<td>CVP</td><td>Cyclophosphamide, Vincristine, Prednisone</td>

<td>CVPP</td><td>Lomustine, Procarbazine, Prednisone</td>

<td>CYVADIC</td><td>Cyclophosphamide, Vincristine, Doxorubicin, Dacarbazine</td>

<td>GIVES</td><td>Daunorubicin, cytarabine</td>

<td>DAT</td><td>Daunorubicin, Cytarabine, Thioguanine</td>

<td>DAV</td><td>Daunorubicin, Cytarabine, Etoposide</td>

<td>DCT</td><td>Daunorubicin, Cytarabine, Thioguanine</td>

<td>DHAP</td><td>Cisplatin, Cytarabine, Dexamethasone</td>

<td>GAVE</td><td>Doxorubicin, Ifosfamide</td>

<td>DTIC / Tamoxifen</td><td>Dacarbazine, Tamoxifen</td>

<td>DVP</td><td>Daunorubicin, Vincristine, Prednisone</td>

<td>EAP</td><td>Etoposide, Doxorubicin, Cisplatin</td>

<td>EC</td><td>Etoposide, Carboplatin</td>

<td>EFP</td><td>Etoposide, Fluorouracil, Cisplatin</td>

<td>Elf</td><td>Etoposide, Leucovorin, Fluorouracil</td>

<td>EMA 86</td><td>Mitoxantrone, Etoposide, Cytarabine</td>

<td>EP</td><td>Etoposide, Cisplatin</td>

<td>EVE</td><td>Etoposide, Vinblastine</td>

<td>FAC</td><td>Fluorouracil, Doxorubicin, Cyclophosphamide</td>

<td>FAM</td><td>Fluorouracil, Doxorubicin, Mitomycin</td>

<td>FAMTX</td><td>Methotrexate, Leucovorin, Doxorubicin</td>

<td>FAP</td><td>Fluorouracil, Doxorubicin, Cisplatin</td>

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<td>Denomination</td><td>Therapeutic agents</td>

<td>F-CL</td><td>Fluorouracil, Leucovorin</td>

<td>FEC</td><td>Fluorouracil, Cyclophosphamide, Epirubicin</td>

<td>EDF</td><td>Fluorouracil, Etoposide, Cisplatin</td>

<td>FL</td><td>Flutamide, Leuprolide</td>

<td>FZ</td><td>Flutamide, goserelin acetate implant</td>

<td>HDMTX</td><td>Methotrexate, Leucovorin</td>

<td>Hexa-CAF</td><td>Altretamine, Cyclophosphamide, Methotrexate, Fluorouracil</td>

<td>ICE-T</td><td>Ifosfamide, Carboplatin, Etoposide, Paclitaxel, Mesna</td>

<td>IDMTX / 6-MP</td><td>Methotrexate, Mercaptopurine, Leucovorin</td>

<td>IE</td><td>Ifosfamide, Etoposide, Mesna</td>

<td>IfoVP</td><td>Ifosfamide, Etoposide, Mesna</td>

<td>IPA</td><td>Ifosfamide, Cisplatin, Doxorubicin</td>

<td>M-2</td><td>Vincristine, Carmustine, Cyclophosphamide, Prednisone, Melphalan</td>

<td>MAC-III</td><td>Methotrexate, Leucovorin, Dactinomycin, Cyclophosphamide</td>

<td>MACC</td><td>Methotrexate, Doxorubicin, Cyclophosphamide, Lomustine</td>

<td>MACOP-B</td><td>Methotrexate, Leucovorin, Doxorubicin, Cyclophosphamide, Vincristine, Bleomycin, Prednisone</td>

<td>MAID</td><td>Mesna, Doxorubicin, Ifosfamide, Dacarbazine</td>

<td>m-BACOD</td><td>Bleomycin, Doxorubicin, Cyclophosphamide, Vincristine, Dexamethasone, Methotrexate, Leucovorin</td>

<td>MBC</td><td>Methotrexate, Bleomycin, Cisplatin</td>

<td>MC</td><td>Mitoxantrone, Cytarabine</td>

<td>MF</td><td>Methotrexate, Fluorouracil, Leucovorin</td>

<td>MICE</td><td>Ifosfamide, Carboplatin, Etoposide, Mesna</td>

<td>MINE</td><td>Mesna, Ifosfamide, Mitoxantrone, Etoposide</td>

<td>mini-BEAM</td><td>Carmustine, Etoposide, Cytarabine, Melphalan</td>

<td>MOBP</td><td>Bleomycin, Vincristine, Cisplatin, Mitomycin</td>

<td>MOP</td><td>Mechlorethamine, Vincristine, Procarbazine</td>

<td>MOPP</td><td>Mechlorethamine, Vincristine, Procarbazine, Prednisone</td>

<td>MOPP / ABV</td><td>Mechlorethamine, Vincristine, Procarbazine, Prednisone, Doxorubicin, Bleomycin, Vinblastine</td>

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<td>Denomination</td><td>Therapeutic agents</td>

<td>MP (multiple myeloma)</td><td>Melphalan, Prednisone</td>

<td>MP (prostate cancer)</td><td>Mitoxantrone, Prednisone</td>

<td>MTX / 6-MO</td><td>Methotrexate, Mercaptopurine</td>

<td>MTX / 6-MP / VP</td><td>Methotrexate, Mercaptopurine, Vincristine, Prednisone</td>

<td>MTX-CDDPAdr</td><td>Methotrexate, Leucovorin, Cisplatin, Doxorubicin</td>

<td>MV (breast cancer)</td><td>Mitomycin, Vinblastine</td>

<td>MV (acute myeloid leukemia)</td><td>Mitoxantrone, Etoposide</td>

<td>M-VAC Methotrexate</td><td>Vinblastine, Doxorubicin, Cisplatin</td>

<td>MVP Mitomycin</td><td>Vinblastine, Cisplatin</td>

<td>MVPP</td><td>Mechlorethamine, Vinblastine, Procarbazine, Prednisone</td>

<td>NFL</td><td>Mitoxantrone, Fluorouracil, Leucovorin</td>

<td>NOVP</td><td>Mitoxantrone, Vinblastine, Vincristine</td>

<td>Takeover bid</td><td>Vincristine, Prednisone, Doxorubicin</td>

<td>OPPA</td><td>Add Procarbazine to OPA.</td>

<td>PAC</td><td>Cisplatin, doxorubicin</td>

<td>PAC-I</td><td>Cisplatin, Doxorubicin, Cyclophosphamide</td>

<td>PA-CI</td><td>Cisplatin, doxorubicin</td>

<td>Pc</td><td>Paclitaxel, Carboplatin or Paclitaxel, Cisplatin</td>

<td>PCV</td><td>Lomustine, Procarbazine, Vincristine</td>

<td>PE</td><td>Paclitaxel, Estramustine</td>

<td>PFL</td><td>Cisplatin, Fluorouracil, Leucovorin</td>

<td>POC</td><td>Prednisone, Vincristine, Lomustine</td>

<td>ProMACE</td><td>Prednisone, Methotrexate, Leucovorin, Doxorubicin, Cyclophosphamide, Etoposide</td>

<td>ProMACE / cytaBOM</td><td>Prednisone, Doxorubicin, Cyclophosphamide, Etoposide, Cytarabine, Bleomycin, Vincristine, Methotrexate, Leucovorin, Cotrimoxazole</td>

<td>PRoMACE / MOPP</td><td>Prednisone, Doxorubicin, Cyclophosphamide, Etoposide, Mechlorethamine, Vincristine, Procarbazine, Methotrexate, Leucovorin</td>

<td>Pt / VM</td><td>Cisplatin, Teniposide</td>

<td>PVA</td><td>Prednisone, Vincristine, Asparaginase</td>

<td>PVB</td><td>Cisplatin, Vinblastine, Bleomycin</td>

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<td>Denomination</td><td>Therapeutic agents</td>

<td>PVDA</td><td>Prednisone, Vincristine, Daunorubicin, Asparaginase</td>

<td>SMF</td><td>Streptozocin, Mitomycin, Fluorouracil</td>

<td>TAD</td><td>Mechlorethamine, Doxorubicin, Vinblastine, Vincristine, Bleomycin, Etoposide, Prednisone</td>

<td>TCF</td><td>Paclitaxel, Cisplatin, Fluorouracil</td>

<td>TIP</td><td>Paclitaxel, Ifosfamide, Mesna, Cisplatin</td>

<td>TTT</td><td>Methotrexate, Cytarabine, Hydrocortisone</td>

<td>Topo / CTX</td><td>Cyclophosphamide, Topotecan, Mesna</td>

<td>VAB-6</td><td>Cyclophosphamide, Dactinomycin, Vinblastine, Cisplatin, Bleomycin</td>

<td>VAC</td><td>Vincristine, Dactinomycin, Cyclophosphamide</td>

<td>VACAdr</td><td>Vincristine, Cyclophosphamide, Doxorubicin, Dactinomycin, Vincristine</td>

<td>VAD</td><td>Vincristine, Doxorubicin, Dexamethasone</td>

<td>VATH</td><td>Vinblastine, Doxorubicin, Thiotepa, Flouximesterone</td>

<td>VBAP</td><td>Vincristine, Carmustine, Doxorubicin, Prednisone</td>

<td>VBCMP</td><td>Vincristine, Carmustine, Melphalan, Cyclophosphamide, Prednisone</td>

<td>VC</td><td>Vitiorcibine, Cisplatin</td>

<td>VCAP</td><td>Vincristine, Cyclophosphamide, Doxorubicin, Prednisone</td>

<td>YOU</td><td>Viilorcibiae, Doxorubicin</td>

<td>VelP</td><td>Vinblastine, Cisplatin, Ifosfamide, Mesna</td>

<td>Vip</td><td>Etoposide, Cisplatin, Ifosfamide, Mesna</td>

<td>VM</td><td>Mitomycin, Vinblastine</td>

<td>VMCP</td><td>Vincristine, Melphalan, Cyclophosphamide, Prednisone</td>

<td>VP</td><td>Etoposide, Cisplatin</td>

<td>V-TAD</td><td>Etoposide, Thioguanine, Daunorubicin, Cytarabine</td>

<td> 5+2</td><td>Cytarabine, Daunorubicin, Mitoxantrone</td>

<td> 7 + 3</td><td>Cytarabine with /, Daunorubicin or Idarubicin or Mitoxantrone</td>

<td>8 in 1</td><td>Methylprednisolone, Vincristine, Lomustine, Procarbazine, Hydroxyurea, Cisplatin, Cytarabine, Dacarbazine</td>

In addition to conventional chemotherapeutic agents, the sirtuin modulator compounds described herein, as capable of inducing cell death or reducing life, can also be used with RNAi or antisense RNA, or with other polynucleotides to inhibit the expression of the components. cells that contribute to unwanted cell proliferation that are the target of conventional chemotherapy. Such targets are, by way of illustration only, growth factors, growth factor receptors, cell cycle regulatory proteins, transcription factors, or signal transduction kinases.

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Combination therapies comprising the sirtuin modulating compounds and a conventional chemotherapeutic agent may be advantageous over combination therapies known in the art, as the combination allows the conventional chemotherapeutic agent to exert a greater effect at a lower dose. In a preferred embodiment, the effective dose (ED50) for a chemotherapeutic agent, or the combination of conventional chemotherapeutic agents, when used in combination with a sirtuin modulator compound, is at least 2 times less than the ED50 of the chemotherapeutic agent alone. , and even more preferably 5 times, 10 times or even 25 times. Conversely, the therapeutic index (TI) for said chemotherapeutic agent or combination of said chemotherapeutic agent used in combination with the sirtuin modulator compound described herein can be at least 2 times higher than the TI of a conventional chemotherapeutic regimen. alone, and even more preferably 5 times, 10 times or even 25 times greater

Neuronal disorders / diseases

In certain aspects, sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can be used to treat patients suffering from neurodegenerative diseases, and traumatic or mechanical injury to the central nervous system (CNS), to the spinal cord. or the peripheral nervous system (PNS). Neurodegenerative disease typically involves reductions in the mass and volume of the human brain, which may be due to the atrophy and / or death of brain cells, which are much deeper than those in a healthy person where they are attributed to aging. Neurodegenerative diseases can progress gradually, after a prolonged period of normal brain function, due to progressive degeneration (eg. g., nerve cell dysfunction and death) of specific brain regions. Alternatively, neurodegenerative diseases can have a rapid onset, such as those associated with trauma or toxins. The actual onset of brain degeneration can precede clinical expression by many years. Examples of neurodegenerative diseases include, but are not limited to, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), Diffuse Lewy, chorea-acantocytosis, primary lateral sclerosis, eye diseases (ocular neuritis), chemotherapy-induced neuropathies (eg. g., by vincristine, paclitaxel, bortezomib), neuropathies induced by diabetes and Friedreich's ataxia. Sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can be used to treat these disorders and others that will be described below.

AD is a chronic, incurable, and cannot be stopped CNS disorder. It occurs gradually, producing amnesia, unusual behavior, personality changes, and a decline in reasoning skills. These losses are associated with the death of specific types of brain cells, and the breakdown of connections and their supporting networks (eg, glial cells) between them. AD has been described in reverse in childhood development. In most people with AD, symptoms appear by age 60. The earliest symptoms include recent memory loss, poor judgment, and personality changes. Later in the illness, people with AD may forget how to perform simple tasks, such as washing their hands. Eventually, people with AD lose all reasoning ability and become dependent on other people for their daily care. Eventually, the illness becomes so debilitating that patients are confined to bed and typically develop coexisting illnesses.

PD is a chronic, incurable, and cannot be stopped CNS disorder. It occurs gradually and produces uncontrolled body movements, stiffness, tremors, and dyskinesia. These motor system problems are related to the death of brain cells in an area of ​​the brain that produces dopamine, a chemical that helps control muscle activity. In most people with PD, symptoms appear by age 50. The initial symptoms of PD consist of a pronounced tremor affecting the extremities, notably in the hands or lips. The subsequent characteristic symptoms of PD are stiffness or retardation of movement, a sluggish gait, stooped posture, and impaired balance. There is a wide range of secondary symptoms such as amnesia, dementia, depression, emotional changes, swallowing difficulties, abnormal speech, sexual dysfunction, and bladder and bowel problems. These symptoms will begin to interfere with routine activities, such as holding a fork or reading a newspaper. Eventually, people with PD become so disabled that they are often confined to bed.

ALS (motor neuronal disease) is a chronic, incurable CNS disorder that cannot be stopped. It attacks motor neurons, the components of the CNS that connect the brain to skeletal muscles. In ALS, motor neurons deteriorate and eventually die, and while a person's brain normally remains fully functioning and alert, the command to move the nerve reaches the muscles. Most people with ALS are between the ages of 40 and 70. The first motor neurons to weaken are those that control the arms and legs. People with ALS may have trouble walking, they may drop things, fall, have muddled speech, and laugh or cry uncontrollably. Eventually, the muscles in the extremities begin to atrophy from disuse. This muscle weakness becomes debilitating and the person needs a wheelchair or cannot even function out of bed.

The causes of these neurological diseases have long been unknown. Conventionally they are defined as different diseases, even they clearly show similarities in the basic processes and

ES 2 396 913 T3 commonly demonstrate overlapping symptoms much greater than would be expected by chance alone.

Current disease definitions cannot adequately address the overlap problem and a new classification of neurodegenerative disorders has been generated.

HD is another neurodegenerative disease that stems from genetically programmed degeneration of neurons in certain areas of the brain. This degeneration causes uncontrolled movements, loss of intellectual faculties and emotional disturbances. HD is a familial disease, passed from father to son through a dominant mutation in the wild-type gene. Some early symptoms of HD are mood swings, depression, irritability or difficulty driving, learning new things, remembering an event, or making a decision. As the disease progresses, concentration on intellectual tasks becomes increasingly difficult and the patient has difficulty feeding himself and swallowing.

Tay-Sachs disease and Sandhoff disease are glycolipid storage diseases caused by a lack of liosomal β-hexosaminidase (Gravel et al., In The Metabolic Basis of Inherited Disease, eds. Scriver et al., McGraw-Hill , New York, pp. 2839-2879, 1995). In both disorders, ganglioside GM2 and glycolipid-related substrates for β-hexosaminidase accumulate in the nervous system and trigger acute neurodegeneration. In the most severe forms, the onset of symptoms occurs in early childhood. A precipitous neurodegenerative course then follows, with affected children presenting with motor dysfunction, seizures, visual loss, and deafness. Death usually occurs between 2-5 years of age. Neuronal loss through an apoptotic mechanism has been demonstrated (Huang et al., Hum. Mol. Genet. 6: 1879-1885, 1997).

Apoptosis is known to play a role in the pathogenesis of AIDS in the immune system. However, HIV-1 also induces neurological disease. Shi et al. (J. Clin. Invest. 98: 1979-1990, 1996) examined the apoptosis induced by HIV-1 infection in the CNS in an in vitro model and in brain tissue of AIDS patients, and discovered HIV infection -1 of apoptosis induced in primary brain cultures in neurons and astrocytes in vitro. Neuron and astrocyte apoptosis was also detected in brain tissue of 10/11 AIDS patients, including 5/5 HIV-1 patients with dementia and 4/5 without dementia.

There are four main peripheral neuropathies associated with HIV, namely sensory neuropathy, AIDP / CIPD, drug-induced neuropathy, and CMV-related neuropathy.

The most common type of AIDS-associated neuropathy is distal symmetric polyneuropathy (DSPN). This syndrome is the consequence of nerve degeneration and is characterized by numbness and a prickling and needling sensation. DSPN causes some serious abnormalities and mainly causes numbness or tingling in the feet and slower reflexes in the ankles. In general it occurs with more intense immunosuppression and is gradually progressive. Tricyclic antidepressant treatment relieves symptoms but does not affect underlying nerve damage.

A less common but more severe type of neuropathy is known as acute or chronic inflammatory demyelinating polyneuropathy (AIDP / CIDP). In AIDP / CIDP, there is damage to the fatty membrane that covers the nerve impulses. This class of neuropathy involves inflammation and resembles the muscle breakdown often identified with long-term use of AZT. It can be the first manifestation of HIV infection, where the patient may not report pain, but cannot respond to conventional reflex tests. This class of neuropathy can be associated with seroconversion, in which case it can sometimes resolve spontaneously. It can serve as a sign of HIV infection and indicate that it might be time to consider antiviral therapy. AIDP / CIDP can be of autoimmune origin.

Drug-induced or toxic neuropathies can be very painful. Antiviral drugs commonly cause peripheral neuropathy, as do other drugs, eg. eg, vincristine, dilantin (an anti-seizure drug), high-dose vitamins, isoniazid, and folic acid antagonists. Peripheral neuropathy is often used in clinical trials for antivirals as a dose-limiting side effect, meaning that more drugs should not be given. In addition, the use of such drugs can exacerbate otherwise mild neuropathies. Usually, these drug-induced neuropathies are reversible with drug discontinuation.

CMV causes several neurological syndromes in AIDS, including encephalitis, myelitis, and polyradiculopathy.

Neuronal loss is also a salient feature of earlier diseases, such as CreutzfeldtJakob disease in humans, BSE in cattle (mad cow disease), Scrapie disease in sheep and goats, and feline spongiform encephalopathy (FSE) in cats. Sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein may be useful in treating or preventing neuronal loss due to these foregoing diseases.

In another embodiment, a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein can be used to treat or prevent any disease or disorder involving axonopathy. Distal axonopathy is a type of peripheral neuropathy that arises from some metabolic or toxic alteration of the neurons of the peripheral nervous system (PNS). It is the most frequent response of the nerves to alterations 37

ES 2 396 913 T3 metabolic or toxic, and as such can be caused by metabolic diseases such as diabetes, kidney failure, deficiency syndromes such as malnutrition and alcoholism, or the effects of toxins or drugs. The most common cause of distal axonopathy is diabetes, and the most common cause of distal axonopathy is diabetic neuropathy. The most distal portions of the axons are usually the first to degenerate, and the axonal atropy progresses slowly into the nerve cell body. If the noxious stimulus is removed, regeneration is possible, although the prognosis is reduced depending on the duration and intensity of the stimulus. People with distal axonopathies usually experience glove and sock motor and sensory disturbances. The functions of the deep tendons and the autonomic nervous system (SnA) are also lost or diminished in affected areas.

Diabetic neuropathies are neuropathic disorders associated with diabetes mellitus. These conditions usually result from diabetic microvascular lesions that involve the small blood vessels that supply the nerves (vasa nervorum). Relatively common conditions that can be associated with diabetic neuropathy include third nerve palsy; mononeuropathy; mononeuritis multiplex; diabetic amyotrophy; painful polyneuropathy; ; and thoracoabdominal neuropathy. The clinical manifestations of diabetic neuropathy include, for example, sensorimotor polyneuropathy such as numbness, sensory loss, dysesthesia, and night pain; autonomic neuropathy such as delayed gastric emptying or gastroparesis; and cranial neuropathy such as oculomotor neuropathies (3rd) or mononeuropathies of the thoracic or lumbar spinal nerves.

Peripheral neuropathy is the medical term for damage to the nerves of the peripheral nervous system, which can be caused either by diseases of the nerve or by side effects of systemic disease. Peripheral neuropathies vary in their presentation and origin, and can affect the nerve or neuromuscular joint. The main causes of peripheral neuropathy include seizures, nutritional deficiencies, and HIV, although diabetes is the most likely cause. The mechanical pressure of staying in one position for a long time, a tumor, intraneural bleeding, exposure of the body to extreme conditions such as radiation, cold temperatures or toxic substances can also cause peripheral neuropathy.

In an illustrative embodiment, a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein can be used to treat or prevent multiple sclerosis (MS), including relapsing MS and monosympathetic MS, and other demyelinating conditions, such as , for example, chronic inflammatory demyelinating polyneuropathy (CIDP), or symptoms associated with these.

MS is a chronic, often disabling disease of the central nervous system. Several convergent lines of evidence indicate the possibility that the disease is caused by an alteration in immune function, although the cause of this alteration has not been established. The alteration allows cells of the immune system to attack myelin, the fat that contains the insulating sheath that surrounds nerve axons located in the central nervous system (CNS). When myelin is damaged, electrical pulses cannot travel quickly or normally through nerve fiber pathways in the brain and spinal cord. This causes the breakdown of normal electrical conductivity within the axons, fatigue, and disturbances in vision, strength, coordination, balance, sensation, and functioning of the bladder and intestines.

As such, MS is now a common and well-known neurological disorder characterized by episodes of inflammation and demyelination that occur in some part of the CNS. However, practically always without compromise of the associated peripheral nerves. Demyelination produces a situation analogous to that which results from cracks or tears in an insulation that surrounds an electrical cable. That is, when the insulating sheath breaks, the circuit is short-circuited and the associated electrical appliance will operate intermittently or not at all. This loss of myelin surrounding nerve fibers causes short circuits in the nerves that run through the brain and spinal cord, resulting in symptoms of MS. This demyelination has also been observed to occur in areas, as opposed to the entire central CNS. Also, such demyelination can be intermittent. Therefore, such plaques are spread both in time and space.

The pathogenesis is believed to involve a local breakdown of the blood-brain barrier causing a localized and inflammatory immune response, with consequent damage to myelin and neurons.

Clinically, MS exists in both sexes and can occur at any age. However, its most common presentation is in the relatively young adult, usually with a simple focal lesion such as optic nerve damage, an area of ​​anesthesia (loss of sensation) or paresthesia (location of loss of sensation), or muscle weakness. . In turn, vertigo, double vision, localized pain, incontinence, and pain in the arms and legs can occur when flexing the neck, as well as a wide variety of less frequent symptoms.

An initial attack of MS is usually temporary, and it may take weeks, months, or years for a new attack. Some people can enjoy a stable, relatively episode-free life for a large number of years, while others less fortunate experience a downhill course to complete paralysis. Commonly there are a series of remissions and relapses, in which each relapse leaves the patient somewhat worse than before. Relapses can be triggered by episodes of stress, viral infections, or toxins. High body temperature, ie a fever, will worsen the condition, or a reduction in temperature, for example, from a cold bath, could improve the condition.

ES 2 396 913 T3

In yet another embodiment, a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein can be used to treat trauma to nerves, including trauma due to disease, injury (including surgical intervention), or environmental trauma (e.g. eg, neurotoxins, alcoholism, etc.).

Sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein may also be useful in preventing, treating, and alleviating symptoms of various PNS disorders, such as those described below. The PNS is made up of the nerves that lead to or branch from the spinal cord and the CNS. Peripheral nerves manipulate a diverse range of body functions, including sensory, motor, and autonomic functions. When a person has peripheral neuropathy, the nerves in the PNS have been damaged. Nerve damage can arise from a number of causes, such as illness, physical injury, poisoning, or malnutrition. These agents can affect the afferent or efferent nerves. Depending on the cause of the damage, the nerve cell axon, its protective myelin sheath, or both can be damaged or destroyed.

The term peripheral neuropathy encompasses a wide range of disorders in which nerves outside the brain and spinal cord, the peripheral nerves, have been damaged. Peripheral neuropathy can also be called peripheral neuritis, or if many nerves are involved, the terms polyneuropathy or polyneuritis can be used.

Peripheral neuropathy is a widespread disorder that has many underlying causes. Some of these causes are common, such as diabetes, and others are extremely rare, such as acrylamide poisoning and certain inherited disorders. The most common cause of peripheral neuropathy worldwide is leprosy. Leprosy is caused by the bacterium Mycobacterium leprae, which attacks the peripheral nerves of affected people.

Leprosy is extremely rare in the United States, where diabetes is the most common cause of peripheral neuropathy. It has been estimated that more than 17 million people in the United States and Europe have diabetes-related polyneuropathy. Many neuropathies are idiopathic; of unknown cause. The most common of the inherited neuropathies in the United States is Charcot-Marie-Tooth disease, which affects approximately 125,000 people.

Another of the better known peripheral neuropathies is Guillain-Barré syndrome, which arises from complications associated with viral diseases, such as cytomegalovirus, Epstein-Barr virus and human immunodeficiency virus (HIV), or bacterial infection including Campylobacter disease jejuni and Lyme. The worldwide incidence rate is approximately 1.7 cases per 100,000 people per year. Other known causes of peripheral neuropathies include chronic alcoholism, varicella-zoster virus infection, botulism, and polio. Peripheral neuropathy can develop as a primary symptom, or it can be due to another disease. For example, peripheral neuropathy is only a symptom of diseases such as amyloid neuropathy, certain types of cancer, or inherited neurological disorders. These diseases can affect the PNS and CNS, as well as other tissues in the body.

Other diseases of the PNS treatable with sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein include: brachial plexus neuropathies (diseases of the cervical and first thoracic roots, nerve trunk, and peripheral nerve components of the brachial plexus. Clinical manifestations include regional pain, paresthesia, muscle weakness, and reduced sensation in the upper extremities. These disorders can be associated with trauma, including congenital injuries; thoracic outlet syndrome; neoplasms, neuritis, radiation therapy; and other ailments. See Adams et al., Principles of Neurology, 6^to ed, page 1351-2); diabetic neuropathies (peripheral, autonomic disorders of the cranial nerves associated with diabetes mellitus). These conditions usually result from diabetic microvascular lesions that involve the small blood vessels that supply the nerves (vasa nervorum). Relatively common conditions that can be associated with diabetic neuropathy include third nerve palsy; mononeuropathy; mononeuritis multiplex; diabetic amyotrophy; painful polyneuropathy; ; and thoracoabdominal neuropathy (see Adams et al., Principles of Neurology, 6th ed, p1325); mononeuropathies (disease or trauma involving a single isolated peripheral nerve, or out of proportion to indicate diffuse peripheral nerve dysfunction). Mononeuritis multiplex refers to a condition characterized by multiple isolated nerve lesions. Mononeuropathies can stem from a wide variety of causes including ischemia; traumatic injury; compression; connective tissue diseases; cumulative trauma disorders; and other ailments; neuralgia (severe pain that occurs in conjunction with the course or distribution of a peripheral or cranial nerve); neoplasms of the peripheral nervous system (neoplasms arising from the peripheral nervous tissue). This includes neurofibromas; Schwannomas; granular cell tumors; malignant peripheral nerve sheath tumors (see DeVita Jr et al., Cancer: Principles and Practice of Oncology, 5th ed, p1750-1); and nerve compression syndromes (mechanical compression of the nerves or nerve roots from internal or external causes). This can lead to a conduction block to nerve impulses due to, for example, myelin sheath dysfunction or axonal loss. Nerve and nerve sheath injuries can be caused by ischemia; inflammation; or a direct mechanical effect; neuritis (a general term indicating inflammation of a peripheral or cranial nerve). Clinical manifestations can include pain; paresthesia; paresis or hyperesthesia; polyneuropathies (diseases of multiple peripheral nerves). The various forms are categorized by the type of nerve affected (eg. g., sensory, motor, or autonomic), by the distribution of the nerve lesion (eg, distal vs. proximal), by the primarily affected nervous component (eg, demyelinating vs. axonal), by etiology, or by hereditary pattern.

ES 2 396 913 T3

In another embodiment, a sirtuin activating compound can be used to treat or prevent chemotherapeutic agent-induced neuropathy. The sirtuin modulator compounds can be administered prior to the administration of the chemotherapeutic agent, simultaneously with the administration of the chemotherapeutic drug and / or after the initiation of the administration of the chemotherapeutic drug. If the sirtuin activating compound is administered after the initiation of chemotherapeutic drug administration, it is desirable that the sirtuin activating compound is administered before, or with the first signs, of chemotherapeutic agent-induced neuropathy.

Chemotherapy drugs can damage any part of the nervous system. Fortunately, encephalopathy and myelopathy are very rare. Nerve nerve damage is much more common and can be a side effect of treatment experienced by people with cancer, such as lymphoma. Most neuropathies affect the sensory nerves more than the motor nerves. Therefore, common symptoms are tingling, numbness, or loss of balance. The longest nerves in the body appear to be the most sensitive, which is why most patients experience numbness or pricking in the hands and feet.

The chemotherapy drugs most commonly associated with neuropathy are vinca-alkaloids (anticancer drugs originally derived from a member of the periwinkle plant) and a platinum-containing drug called cisplatin. Vinca alkaloids include the drugs vinblastine, vincristine, and vindesine. Many combination chemotherapy treatments for lymphoma, for example CHOP and CVP, contain vincristine, which is the drug most often known to cause this problem. In fact, it is the risk of neuropathy that limits the dose of vincristine that can be administered.

Studies have shown that most patients will lose some reflexes in their legs as a result of vincristine treatment, and many will experience some degree of tingling (paresthesia) in the fingers and toes. Neuropathy usually does not manifest itself at the beginning of treatment, but usually occurs after a few weeks. Stopping the drug at the onset of symptoms is not essential, but if neuropathy progresses, it may be necessary. It is very important that patients report such symptoms to physicians, since nerve damage is largely reversible if the drug is discontinued. Most doctors will usually reduce the dose of vincristine or switch to another form of vinca-alkaloid such as vinblastine or vindesine if symptoms are mild. Sometimes the nerves that supply the intestines are affected, causing abdominal pain and constipation.

In another embodiment, a sirtuin activator compound can be used to treat or prevent a polyglutamine disease. Huntington's disease (HD) and spinocerebellar ataxia type 1 (SCA1) are just two examples of a class of genetic diseases caused by dynamic mutations that involve the expansion of repeats of triplet sequences. In reference to this common mechanism, these disorders are called trinucleotide repeat diseases. At least 14 such diseases are known to affect humans. Nine of them, including SCA1 and Huntington's disease, have CAG as the repeat sequence (see Table 2 below). Since CAG encodes an amino acid called glutamine, these nine trinucleotide repeat disorders are collectively known as polyglutamine diseases.

While the genes involved in various polyglutamine diseases have little in common, the disorders they cause follow a strikingly similar course. Each disease is characterized by a progressive degeneration of a different group of nerve cells. The main symptoms of these diseases are similar, although not identical, and usually affect middle-aged people. Given the similarities in symptoms, it is hypothesized that polyglutamine diseases progress through common cellular mechanisms. In recent years, scientists have made great strides to reveal what those mechanisms are.

Above a certain threshold, the greater the number of glutamine repeats in a protein, the earlier the onset of the disease and the more severe the symptoms. This indicates that the abnormally long glutamine pathways render its host protein toxic to nerve cells.

To test this hypothesis, scientists have generated genetically modified mice that express proteins with long polyglutamine pathways. Regardless of whether the mice express full-length proteins or only those portions of the proteins that contain the glutamine pathways, they develop symptoms of polyglutamine diseases. This indicates that a long polyglutamine pathway is itself damaging cells and does not have to be part of a functional protein to cause damage.

For example, it is believed that SCA1 symptoms are not caused directly by loss of normal ataxin-1 function but instead by the interaction between ataxin-1 and another protein called LANP. The LANP protein is necessary for nerve cells to communicate with each other, and therefore for survival. When the mutant ataxin-1 protein accumulates within nerve cells, it traps the LANP protein, interfering with its normal function. After a while, the non-functioning of LANP seems to cause nerve cells to malfunction.

ES 2 396 913 T3

Table 2. Summary of polyglutamine diseases.

<td>Disease</td><td>Gene name</td><td>Chromosomal location</td><td>Hereditary pattern</td><td>Protein</td><td>Normal repeat length</td><td>Repeat length with disease</td>

<td>Spinobulbar muscular atrophy (Kennedy disease)</td><td>AR</td><td>Xq13-21</td><td>recessive linked to X</td><td>androgen receptor (AR)</td><td> 9-36</td><td> 38-62</td>

<td>Huntington's disease</td><td>HD</td><td>4p16.3</td><td>autosomal dominant</td><td>huntingtin</td><td> 6-35</td><td> 36-121</td>

<td>Dentatorubral-pallidolusian atrophy (Haw River syndrome)</td><td>DRPLA</td><td>12p13.31</td><td>autosomal dominant</td><td>atrophin-1</td><td> 6-35</td><td> 49-88</td>

<td>Spinocerebellar ataxia type 1</td><td>SCA1</td><td>6p23</td><td>autosomal dominant</td><td>ataxin-1</td><td> 6-44</td><td> 39-82</td>

<td>Spinocerebellar ataxia type 2</td><td>SCA2</td><td>12q24.1</td><td>autosomal dominant</td><td>ataxin-2</td><td> 15-31</td><td> 36-63</td>

<td>Spinocerebellar ataxia type 3 (Machado-</td><td>SCA3</td><td>14q32.1</td><td>autosomal dominant</td><td>ataxin-3</td><td> 12-40</td><td> 55-84</td>

<td>Joseph's disease)</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Spinocerebellar ataxia type 6</td><td>SCA6</td><td>19p13</td><td>autosomal dominant</td><td>voltage-gated calcium channel subunit o1a</td><td> 4-18</td><td> 21-33</td>

<td>Spinocerebellar ataxia type 7</td><td>SCA7</td><td>3p12-13</td><td>autosomal dominant</td><td>ataxin-7</td><td> 4-35</td><td> 37-306</td>

<td>Spinocerebellar ataxia type 17</td><td>SCA17</td><td>6q27</td><td>autosomal dominant</td><td>binding protein</td><td> 25-42</td><td> 45-63</td>

TATA

Many transcription factors have also been found in the neuronal inclusions of various diseases. These transcription factors may interact with polyglutamine-containing proteins and then become entrapped in neuronal inclusions. This in turn could prevent transcription factors from turning genes into intermittent genes as needed by cells. Another observation is the hypoacetylation of histones in affected cells. This has led to the hypothesis that Histone Deacetylase Class I / II (HDAC I / II) inhibitors, which are known to increase histone acetylation, may be a new therapy for polyglutamine diseases (US patent application 10 / 476,627; Method of treating neurodegenerative, psychiatric, and other disorders with deacetylase inhibitors).

In yet another embodiment, the application provides a method of treating or preventing a neuropathy related to ischemic injuries or diseases, such as coronary heart disease (including congestive heart failure and myocardial infarctions), stroke, emphysema, hemorrhagic shock, peripheral vascular disease ( upper and lower extremities) and transplant-related injuries.

In certain embodiments, the application provides a method of treating a cell of the central nervous system to prevent damage in response to a decrease in blood flow to the cell. Typically, the intensity of damage that can be prevented will largely depend on the degree of reduction in the bloodstream and the duration of the reduction. As an example, the normal amount of perfusion to brain gray matter in humans is approximately 60 to 70 mL / 100 g of brain tissue / min. Central nervous system cell death typically occurs when blood flow decreases below approximately 810 mL / 100 g of brain tissue / min, while at slightly higher levels (i.e., 20-35 mL / 100 g of

ES 2 396 913 T3 brain tissue / min) the tissue remains alive but is unable to function. In one embodiment, apoptotic or necrotic cell death can be prevented. In yet another embodiment, ischemia-mediated damage, such as cytotoxic edema or anoxemia of central nervous system tissue, can be prevented. In each embodiment, the central nervous system cell can be a spinal cell or a brain cell.

Another aspect encompasses administering a sirtuin activating compound to a subject to treat an ischemic condition of the central nervous system. A variety of central nervous system ischemic conditions can be treated with the sirtuin activating compounds described herein. In one embodiment, the ischemic picture is a stroke that produces any type of ischemic damage to the central nervous system, such as apoptotic or necrotic cell death, cytotoxic edema, or anoxia of central nervous system tissue. Stroke can impact any area of ​​the brain or be caused by any cause known to commonly cause a stroke. In an alternative to this embodiment, the stroke is a brain stem stroke. Generally speaking, brainstem strokes affect the brainstem, which involuntarily controls life-sustaining functions such as breathing, blood pressure, and heartbeat. In another alternative to this embodiment, the stroke is a cerebellar stroke. Typically, cerebellar strokes impact the cerebellum area of ​​the brain, which controls balance and coordination. In still another embodiment, the stroke is an embolic stroke. Generally speaking, embolic strokes can impact any region of the brain and typically cause an artery to be blocked by vasocclusion. In even another alternative, the stroke may be a hemorrhagic stroke. Like ischemic strokes, hemorrhagic strokes can impact any region of the brain and usually result in the rupture of a blood vessel characterized by hemorrhage (bleeding) within the brain or in the surrounding areas. In another embodiment, the stroke is a thrombotic stroke. Typically, thrombotic strokes result from the blockage of a blood vessel by accumulated deposits.

In another embodiment, the ischemic condition may stem from a disorder that occurs in a part of the subject's body outside of the central nervous system, but causes a reduction in blood flow to the central nervous system. These disorders may include, but are not limited to, a peripheral vascular disorder, venous thrombosis, pulmonary embolism, arrhythmia (eg. eg, atrial fibrillation, a transient ischemic attack, unstable angina, or sickle cell anemia. In turn, the ischemic picture of the central nervous system can occur as a consequence of a surgical procedure. By way of example, the subject may undergo heart surgery, lung surgery, spinal surgery, brain surgery, vascular surgery, abdominal surgery, or organ transplant surgery. Organ transplant surgery may include heart, lung, pancreas, kidney, or liver transplant surgery. In turn, the ischemic picture of the central nervous system can occur as a consequence of trauma or injury to a part of the subject's body outside the central nervous system. By way of example, trauma or injury can cause a degree of bleeding that significantly reduces the total volume of blood in the subject's body. Due to this reduction in total volume, the amount of blood flow to the central nervous system is concomitantly reduced. As another example, trauma or injury can also cause the formation of a vasocclusion that restricts blood flow to the central nervous system.

Of course, it is contemplated that sirtuin activating compounds can be used to treat central nervous system ischemic disease regardless of the cause of the condition. In one embodiment, the ischemic condition stems from a vasocclusion. Vasocclusion can be any type of occlusion, but is typically a thrombosis or stroke. In another embodiment, the ischemic condition can be caused by bleeding. The hemorrhage can be any type of hemorrhage, but is generally a cerebral hemorrhage or a subarachnoid hemorrhage. In still another embodiment, the ischemic picture can be caused by a narrowing of a vessel. Generally speaking, the vessel may narrow as a result of vasoconstriction, as occurs during vasospasms, or due to arteriosclerosis. In still another embodiment, the ischemic condition stems from injury to the brain or spine.

In another aspect, a sirtuin activating compound can be administered to reduce the size of the ischemic nucleus infarct following an ischemic central nervous system picture. Likewise, the sirtuin activating compound can also be beneficially administered to reduce the size of the ischemic penumbra or the transitional zone that follows an ischemic central nervous system picture.

In one embodiment, a combination drug regimen can include drugs or compounds for the treatment or prevention of neurodegenerative disorders or secondary conditions associated with these conditions. Therefore, a combination drug regimen can include one or more sirtuin activators and one or more antineurodegeneration agents. For example, one or more sirtuin activator compounds can be combined with an effective amount of one or more of: L-DOPA; a dopamine agonist; an adenosine A2A receptor antagonist; a COMT inhibitor; an MAO inhibitor; an N-NOS inhibitor; a sodium channel antagonist; an antagonist selective N-methyl D-aspartate (NMDA) receptor antagonist; an AMPA / kainate receptor antagonist; a calcium channel antagonist; a GABA-A receptor agonist; an acetyl choline esterase inhibitor; a matrix metalloprotease inhibitor; a PARP inhibitor; an inhibitor of p38 MAP kinase or c-junN-terminal kinases; TPA; NDA antagonists; beta-interferons; growth factors; glutamate inhibitors; and / or as part of a cell therapy.

ES 2 396 913 T3

Illustrative N-NOS inhibitors include 4- (6-amino-pyridin-2-yl) -3-methoxyphenol 6- [4- (2-dimethylamino-ethoxy) -2-methoxy-phenyl] -pyridin-2-yl-amine , 6- [4- (2-dimethylamino-ethoxy) -2,3-dimethyl-phenyl] -pyridin-2-yl-amine, 6- [4- (2-pyrrolidinyl-ethoxy) 2,3-dimethyl-phenyl ] -pyridin-2-yl-amine, 6- [4- (4- (n-methyl) piperidinyloxy) -2,3-dimethyl-phenyl] -pyridin-2-yl-amine, 6- [4- (2-dimethylamino -ethoxy) -3-methoxy-phenyl] -pyridin-2-yl-amine, 6- [4- (2-pyrrolidinyl-ethoxy) -3-methoxy-phenyl] -pyridin-2-yl-amine, 6- {4- [2 (6,7-dimethoxy-3,4-dihydro-1h-isoquinolin-2-yl) -ethoxy] -3-methoxy-phenyl} -pyridin-2-yl-amine, 6- { 3-methoxy-4- [2- (4-phenethyl-piperazin-1-yl) -ethoxy] -phenyl} -pyridin-2-yl-amine, 6- {3-methoxy-4- [2- (4- methyl-piperazin-1-yl) -ethoxy] -phenyl} -pyridin-2-yl-amine, 6- {4- [2 (4-dimethylamin-o-piperidin-1-yl) -ethoxy] -3-methoxy -phenyl} -pyridin-2-yl-amine, 6- [4- (2-dimethylamino-ethoxy) -3-ethoxy-phenyl] -pyridin-2-yl-amine, 6- [4- (2-pyrrolidinyl-ethoxy) -3-ethoxy-phenyl] -pyridin-2-yl-amine, 6- [4- (2-dimethylamino-ethoxy) -2-isopropyl-phenyl] -pyridin-2-yl-amine, 4- (6-amino-pyridin-yl) -3-cyclopropyl-phenol 6- [2-cyclopropyl- 4- (2-dimethylamino-ethoxy) -phenyl] -pyridin-2-yl-amine, 6 [2-cyclopropyl-4- (2-pyrrolidin-1-yl-ethoxy) -phenyl] -pyridin-2-yl- amine, 3- [3- (6-amino-pyridin-2-yl) -4-cyclopropyl-phenoxy] -pyrrolidine-1-carboxylic acid tert-butyl ester, 6- [2-cyclopropyl-4- (1-methyl-pyrrolidine- 3-yl-oxy) -phenyl] -pyridin-2-yl-amine, 4- (6-amino-pyridin-2-yl) -3-cyclobutyl-phenol 6- [2-cyclobutyl-4- (2-dimethylamino-ethoxy) -phenyl] -pyridin-2-yl-amine, 6- [2- Cyclobutyl-4- (2-pyrrolid-in-1-yl-ethoxy) -phenyl] -pyridin-2-yl-amine, 6- [2-cyclobutyl-4- (1-methyl-pyrrolidin-3-yl-oxy) - Phenyl] -pyridin-2-yl-amine, 4- (6-amino-pyridin-2-yl) -3-cyclopentyl-phenol 6- [2-cyclopentyl-4- (2-dimethylamino-ethoxy) -phenyl] -pyrid- in-2-yl-amine, 6- [2-cyclopentyl4- (2-pyrrolidin-yl-ethoxy) -phenyl] -pyridin-2-yl-amine, 3- [4- (6-amino-pyridin-2-yl) -3-methoxy-phenoxy] pyrrolidine-1-carboxylic acid tert-butyl ester 6- [4- (1-methyl-pyrrolidin-3-yl-oxy) -2-methoxy-phenyl] -pyridin-2-yl-amine, 4 [4- (6-amino-pyridin-2-yl) -3-methoxy-phenoxy -] - piperidine-1-carboxylic acid 6 - [2-methoxy-4- (1-methyl-piperidin-4-yl-oxy) -phenyl] -pyridin-2-yl-amine, 6- [4- (allyloxy) -2-methoxy-phenyl] -pyridin-2 -ylamine, 4- (6-amino-pyridin-2-yl) -3-methoxy-6-allylphenol 12 and 4- (6-aminopyridin-2-yl) -3-methoxy-2-allyl-phenol 13 4- (6- amino-pyridin-2-yl) -3-methoxy-6-propyl-phenol 6- [4- (2-dimethylamino-ethoxy) -2-methoxy-5-propyl-phenyl] -pyridin-yl-amine, 6- [2 -isopropyl-4- (pyrrolidin-3-yl-oxy) -phenyl] -pyridin-2-yl-amine, 6- [2-isopropyl-4 (piperidin-3-yl-oxy) -phenyl] -pyridin-2 -ylamine, 6- [2-isopropyl-4- (1-methyl-azetidin-3-yl-oxy) -phenyl] -pyridin-2-yl-amine, 6- [2-isopropyl-4- (1-methyl -piperidin-4-yl-oxy) -phenyl] -pyridin-2-yl-amine, 6- [2-isopropyl-4- (1-methyl-pyrrolidin-3-yl-oxy) -phenyl] -pyridin-2-yl-amine- 6- [2-isopropyl-4- (1-methyl-pyrrolidin-3- yl-oxy) -phenyl] -pyridin-2-yl-amine, 6- [2-isopropyl-4- (2-methyl-2-azabicyclo [2.2.1] hept-5-yl-oxy) -phenyl] - pyridin-2-yl-amine, 6- [4- (2-dimethylamino-ethoxy) -2-methoxy-phenyl] -pyridin-2-yl-amine, 6- {4- [2 (benzyl-methyl-amino) -ethoxy] -2-methoxy-phenyl} -pyridin-2-yl-amine, 6- [2-methoxy-4- (2-pyrrolidin-1-yl-ethoxy) -phenyl] -pyridin-2-ylamine, 2- (6-amino-pyridin-2-yl) -5- (2-dimethylamino-ethoxy) -phenol 2- [4- (6-amino-pyridin-2-yl) -3-methoxy-phenoxy] -acetamide 6 [4- (2-amino-ethoxy) -2-methoxy-phenyl] -pyridin-2-yl-amine, 6- {4- [2- (3,4-dihydro-1h-isoquinolin-2-yl) -ethoxy] -2-methoxy-phenyl} -pyridin-2-yl-amine, 2- [4- (6-amino-pyridin-2-yl) -3-methoxy-phenoxy] -ethanol 6- {2-methoxy -4- [2- (2,2,6,6-tetramethyl-piperidin-1-yl) ethoxy] -phenyl} -pyridin-2-yl-amine, 6- {4- [2- (2,5- dimethyl-pyrrolidin-1-yl) -ethoxy] -2-methoxy-phenyl} -pyridin-2-yl-amine, 6- {4- [2- (2,5-dimethyl-pyrrolidin-1-yl) -ethoxy] -2-methoxy-phenyl} -pyridin-2-yl-amine, 2- [4- (6-amino-pyridin- 2-yl) -3-methoxy-phenoxy] -1- (2,2,6,6-tetramethyl-piperidin-1-yl) -ethanone 6- [2-methoxy-4- (1-methyl-pyrrolidin-2-yl -methoxy) -phenyl] -pyridin-2-yl-amine, 6- [4- (2-dimethylamino-ethoxy) -2-propoxy-phenyl] -pyridin-2-yl-amine, 6- {4- [2- ( benzyl-methyl-amino) -ethoxy] -2-propoxy-phenyl} -pyridin-2-ylamine 6- [4- (2-ethoxy-ethoxy) -2-methoxy-phenyl] -pyridin-2-yl-amine, 6- [4- (2-dimethylamino-ethoxy) -2-isopropoxy-phenyl] -pyridin-2-ylamine, 6- [4- (2-ethoxy-ethoxy) -2-isopropoxy-phenyl] -pyridin-2- yl-amine, 6- [2-methoxy-4- (3-methyl-butoxy) -phenyl] -pyridin-2-yl-amine, 6- [4- (2-dimethylamino-ethoxy) -2-ethoxy-phenyl ] -pyridin-2-yl-amine, 6- {4- [2- (benzyl-methyl-amino) -ethoxy-2-ethoxy-phenyl} -pyridin-2-ylamine, 6- [2-ethoxy-4- (3-methyl-butoxy) -phenyl] -pyridin-2-yl-amine, 1- (6-amino-3-aza-bicyclo [3.1.0] hex-3-yl) -2- [4- (6-aminopyridin-2-yl) -3-ethoxy-phenoxy] -ethanone 6- [ 2-ethoxy-4- (2-pyrrolidin-1-yl-ethoxy) -phenyl] -pyridin-2-yl-amine, 3- {2- [4- (6-amino-pyridin-2-yl) -3-ethoxy -phenoxy] -ethyl} -3-aza-bicyclo [3.1.0] hex-6-yl-amine, 1- (6-amino-3-aza-bicyclo [3.1.0] hex-3-yl) -2 - [4- (6-aminopyridin-2-yl) -3-methoxy-phenoxy] -ethanone 3- {2- [4- (6-amino-pyridin-2-yl) -3-methoxy-phenoxy] -ethyl } -3-aza-bicyclo [3.-1.0] hex-6-ylamine, 6- [2-isopropoxy-4- (2-pyrrolidin-1-yl-ethoxy) -phenyl] -pi-ridin-2-yl -amine, 6- {4- [2- (Benzyl-methyl-amino) -ethoxy] -2-isopropoxy-phenyl -} - pyridin-2-yl-amine, 6- [4- (2-dimethylamino-ethoxy) -2-methoxy- 5-propyl-phenyl] -pyridin-2-yl-amine, 6- [5-allyl-4- (2-dimethylamino-ethoxy) -2-methoxy-phenyl] -pyridin-2-yl-amine, 6- [5- allyl-2-methoxy-4- (2-pyrrolidin-1-yl-ethoxy) -phenyl] -pyridin-2-yl-amine, 6- [3-allyl-4- (2-dimethylamino-ethoxy) -2- methoxy-phenyl] -pyridin-2-yl-amine, 6- [2-methoxy-4- (pyrrolidin-3-yl-oxy) -phenyl] -pyridin-2-ylamine, 6- [2-methoxy-4- (1-methylpyrrolidin-3-yl-oxy) -phenyl] -pyridin-2-yl-amine, 6- [2-ethoxy-4- (pyrrolidin-3-yl-oxy) -phenyl] -pyridin-2-ylamine, 6- [2-isopropoxy-4- (pyrrolidin-3-yl-oxy) -phenyl] - pyridin-2-yl-amine, 6- [2-methoxy-4- (piperidin-4-yl-oxy) -phenyl] -pyridin-2-ylamine, 6- [2-methoxy-4- (2,2, 6,6-tetramethyl-piperidin-4-yl-oxy) -phenyl] -pyridin-2-yl-amine, 6- [2-isopropoxy-4- (pyrrolidin-3-yl-oxy) phenyl] -pyridin-2 -ylamine, 3- [4- (6-amino-pyridin-2-yl) -3-methoxy-phenoxy] -azetidine-1-carboxylic acid tert-butyl ester 6- [4- (azetidine-3- yl-oxy) -2-methoxy-phenyl] -pyridin-2-yl-amine, 6- [2-methoxy-4- (1-methyl-azetidin-3-yl-oxy) -phenyl] -pyridin-2-yl-1amine, 6- [2-isopropoxy-4- (pyrrolidin-3-yl- oxy) -phenyl] -pyridin-2-yl-amine, 6- [2-isopropoxy-4- (pyrrolidin-3-yl-oxy) -phenyl] -pyridin-2-yl-amine, 6- [2-methoxy-4 - (pyrrolidin-3-yl-oxy) -phenyl] -pyridin-2-yl-amine, 6- [2-methoxy-4- (1-methyl-pyrrolidin-3-yl-oxy) -phenyl] -pyridin2- yl-amine, 6- [2-methoxy-4- (1-methyl-pyrrolidin-3-yl-oxy) -phenyl] -pyridin-2-yl-amine, 6- [2-methoxy-4- (2- methyl-2-azabicyclo [2.2.1] hept-5-yl-oxy) -phenyl] -pyrid-in-2-yl-amine, 6- [2-methoxy-4- (1-methyl-piperidin-4-yl-oxy) -phenyl] -pyridin-2-yl-amine, 6 [4- (1-ethyl-piperidin-4-yl-oxy ) -2-methoxy-phenyl] -pyridin-2-yl-amine, 6- [5-allyl-2-methoxy-4- (1-methylpyrrolidin-3-yl-oxy) -phenyl] -pyr-idin-2-yl -amine, 6- [4- (2-dimethylamino-ethoxy) -2,6-dimethyl-phenyl] -pyridin-2-yl-amine, 6- [2,6-dimethyl-4- (3-piperidin-1 -yl-propoxy) phenyl] -pyridin-2-yl-amine, 6- [2,6-dimethyl-4- (2-pyrrolidin-1-yl-ethoxy) -phenyl] -pyridin-2-yl-1- amine, 6- {2,6-dimethyl-4- [3- (4-methylpiperazin-1-yl) -propoxy] -phenyl} -pyridin-2-yl-amine, 6- [2,6-dimethyl-4- ( 2-morpholin-4-yl-ethoxy) -phenyl] -pyridin-2-yl-amine, 6- {4- [2 (benzyl-methyl-amino) -ethoxy] -2,6-dimethyl-phenyl} -pyridine -2-yl-amine, 2- [4- (6-amino-pyridin-2-yl) -3,5-dimethyl-phenoxy] -acetamide 6- [4- (2-amino-ethoxy) -2.6 -dimethyl-phenyl] -pyridin-2-yl-amine, 6- [2-isopropyl-4- (2-pyrrolidin-1-yl-ethoxy) -phenyl] -pyridin-2-yl-amine, 2- (2,5-dimethyl-pyrrolidin-1-yl) -6- [2-isopropyl-4- (2-pyrrolidin-1-yl-ethoxy) -phenyl] -pyridine 6- {4- [2- ( 3,5-dimethyl-piperidin-1-yl) ethoxy] -2-isopropyl-phenyl} -pyridin-2-yl-amine, 6- [4- (2-dimethylamino-ethoxy) -2-isopropyl-phenyl] - pyridin-2-yl-amine, 6- [2-tert-butyl4- (2-dimethylamino-ethoxy) -phenyl] -pyridin-2-yl-amine, 6- [2-tert-butyl-4- ( 2-pyrrolidin-1-yl-ethoxy) -phenyl -] - pyridin-2-yl-amine, 6- [4 (2-pyrrolidinyl-ethoxy) -2,5-dimethylphenyl] -pyr-idin-2-yl- amine, 6- [4- (2-dimethylamino-ethoxy) -2,5-dimethyl-phenyl] -pyridin-2-yl-amine, 6- [4- (2- (4-phenethylpiperazin-1-yl) -ethoxy) -2,5-dimethyl-phenyl] -pyridin-2-yl-amine, 6- [2-cyclopropyl-4- (2-dimethylamino-1-methylethoxy) -phenyl] -pyridin-2-yl-amine, 6- [cyclobutyl-4- (2-dimethylamino-1-methyl-ethoxy) -phenyl] -pyridin-2-yl-amine, 6- [4- (allyloxy) -2-cyclobutyl-phenyl] -pyridi-n-2ylamine, 2-allyl-4- (6-amino-pyridin-2-yl) -3-cyclobutyl-phenol and 2-allyl-4- (6-amino-pyridin-2-yl) -5-cyclobutyl-phenol 4- (6- amino-pyridin-2-yl) -5-cyclobutyl-2-propyl-phenol 4- (6-amino-pyridin-2-yl) -3-cyclobutyl-2-propyl-phenol 6- [243

ES 2 396 913 T3 cyclobutyl-4- (2-dimethylamino-1-methyl-ethoxy) -5-propyl-phenyl] -pyridin-2-yl-amine, 6- [2-cyclobutyl-4- (2- dimethylamino-1-methylethoxy) -3-propyl-1-phenyl] -pyridin-2-yl-amine, 6- [2-cyclobutyl-4- (2-dimethylamino-ethoxy) -5-propyl-phenyl] -pyridine- 2-yl-amine, 6- [2-cyclobutyl-4- (2-dimethylamino-ethoxy) -3-propyl-phenyl] -pyridin-2-yl-amine, 6- [2-cyclobutyl-4- (1-methyl- pyrroli-din-3-yl-oxy) -5-propylphenyl] -pyridin-2-yl-amine, 6- [cyclobutyl-4- (1-methyl-1-pyrrolidin-3-yl-oxy) -3-propyl-phenyl] -pyridin-2-yl-amine, 2- (4-benzyloxy-5-hydroxy-2-methoxy -phenyl) -6- (2,5-dimethyl-pyrrol-1-yl) -pyridine 6- [4- (2-dimethylamino-ethoxy) -5-ethoxy-2-methoxy-phenyl] -pyridin-2-ylamine , 6- [5-ethyl-2-methoxy-4- (1-methyl-piperidin-4-yl-oxy) -phenyl] -pyr-idin-2-yl-amine, 6- [5-ethyl-2- methoxy-4- (piperidin-4-yl-oxy) phenyl] -pyridin-2-yl-amine, 6- [2,5-dimethoxy-4- (1-methyl-pyrrolidin-3-yl-oxy) -phenyl ] -pyr-idin-2-yl-amine, 6- [4- (2-dimethylaminoethoxy) -5-ethyl-2-methoxy-phenyl] -pyridin-2-yl-amine.

Illustrative NMDA receptor antagonists include (+) - (1S, 2S) -1- (4-hydroxyphenyl) -2- (4-hydroxy-4-phenylpiperidino) -1-pro-panol, (1S, 2S) -1- (4-hydroxy-3-methoxyphenyl) -2- (4-hydroxy-4-phenylpiperidino) -1-propanol, (3R, 4S) -3 (4- (4-fluorophenyl) -4-hydroxypiperidin-1- yl -) - chroman-4,7-diol, (1R *, 2R *) - 1- (4-hydroxy-3-methylphenyl) -2- (4- (4-fluoro-phenyl) -4-hydroxypiperidin-1-yl ) -propan-1-ol-mesylate or its pharmaceutically acceptable acid addition salt.

Illustrative dopamine agonists include ropininol; L-dopa decarboxylase inhibitors such as carbidopa or benserazide, bromocriptine, dihydroergocriptine, etisulergine, AF-14, alaptide, pergolide, piribedil; dopamine D1 receptor agonists such as A-68939, A-77636, dihydrexin, and SKF-38393; dopamine D2 receptor agonists such as carbergoline, lisuride, N-0434, naxagolide, pD-118440, pramipexole, quinpirol, and ropinirole; dopamine / e-adrenergic receptor agonists such as DPDMS and dopexamine; dopamine / 5-HT / 5-HT-1 A uptake inhibitor agonists such as roxindole; dopamine / opiate receptor agonists such as NIH-10494; α2-adrenergic antagonist / dopamine agonists such as terguride; a2-adrenergic antagonist / dopamine D2 agonists such as ergolines and talipexole; dopamine uptake inhibitors such as GBR-12909, GBR-13069, GYKI-52895, and NS-2141; Monoamine oxidase-B inhibitors, such as selegiline, N- (2-butyl) -N-methylpropargylamine, N-methyl-N- (2-pentyl) propargylamine, AGN-1133, ergot derivatives, lazabemide, LU-53439, MD-280040 and mofegiline; and COMT inhibitors such as CGP-28014.

Illustrative acetyl cholinesterase inhibitors include donepizil, 1- (2-methyl-1H-benzimide-zol-5-yl) -3- [1 (phenylmethyl) -4-piperidinyl] -1-propanone; 1- (2-phenyl-1H-benzimidazol-5-yl) -3- [1- (phenylmethyl) -4-piperidinyl] -1-propanone; 1 (1-Ethyl-2-methyl-1H-benzimidazol-5-yl) -3- [1- (phenylmethyl) -4-p-iperidinyl] -1-propanone; 1- (2-methyl-6-benzothiazolyl) -3- [1 (phenylmethyl) -4-piperidinyl] -1-propanone; 1- (2-methyl-6-benzothiazolyl) -3- [1 - [(2-methyl-4-thiazolyl) methyl] -4-piperidinyl] -1propanone; 1- (5-methyl-benzo [b] thienyl-2-yl) -3- [1- (phenylmethyl) 4-piperidinyl] -1-propanone; 1- (6-methyl-benzo [b] thien-2-yl) -3- [1 (phenylmethyl) -4-piperidinyl] -1-prop-anone; 1- (3,5-dimethyl-benzo [b] thien-2-yl) -3- [1- (phenylmethyl) -4-piperidin-yl] -1-propanone; 1 (benzo [b] thien-2-yl) -3- [1- (phenylmethyl) -4-piperidinyl] -1-propanone; 1- (benzofuran-2-yl) -3- [1- (phenylmethyl) -4-piperidinyl] -1-propanone; 1- (1-phenylsulfonyl-6-methyl-indol-2-yl) -3- [1- (phenylmethyl) -4-pip-eridinyl] -1-propanone; 1- (6-methyl-indol-2-yl) -3- [1 (phenylmethyl) -4-piper-idinyl] -1-propanone; 1- (1-phenylsulfonyl-5-amino-indol-2-yl) -3- [1- (phenylm-ethyl) -4-piperidinyl] -1propanone; 1- (5-amino-indol-2-yl) -3- [1- (phenylmethyl) -4-piperidinyl] -1-propanone; and 1- (5-acetylamino-indol-2-yl) -3- [1 (phenylmethyl) -4-piperidinyl] -1-propanone. 1- (6-quinolyl) -3- [1- (phenylmethyl) -4-piperidinyl] -1-propanone; 1- (5-indolyl) -3- [1 (phenylmethyl) -4-piperidinyl] -1-propanone; 1- (5-benzthienyl) -3- [1- (phenylmethyl) -4-piperidinyl] -1-pro-panone; 1- (6-quinazolyl) -3 [1- (phenylmethyl) -4-piperidinyl] -1-propanone; 1- (6-benzoxazolyl) -3- [1- (phenylmethyl) -4-piperidinyl] -1-propanone; 1- (5benzofuryl) -3- [1- (phenylmethyl) -4-piperidinyl] -1-propanone; 1- (5-methyl-benzimidazol-2-yl) -3- [1- (phenylmethyl) -4-piperidinyl] -1propanone; 1- (6-methyl-benzimidazol-2-yl) -3- [1- (phenylmethyl) -4-piperidinyl] -1-propanone; 1- (5-chloro-benzo [b] thien-2-yl) -3- [1 (phenylmethyl) -4-piperidin-yl] -1-propanone; 1- (5-azaindol-2-yl) -3- [1- (phenylmethyl) 4-piperidinyl] -1-propanone; 1- (6azabenzo [b] thien-2-yl) -3- [1- (phenylmethyl) -4-piperidinyl] -1-propanone; 1- (1H-2-oxo-pyrrolo [2 ', 3', 5,6] benzo [b] thieno-2-yl) -3- [1 (phenylmethyl) -4-piperidinyl] -1-propanone; 1- (6-methyl-benzothiazol-2-yl) -3- [1- (phenylmethyl) -4-piperidinyl] -1-propanone; 1- (6-methoxy-indol-2-yl) -3- [1- (phenylmethyl) -4-piperldinyl] -1-propanone; 1- (6-methoxy-benzo [b] thien-2-yl) -3- [1- (phenylmethyl) -4piperidinyl] -1-propanone 1- (6-acetylamino-benzo [b] thien-2-yl) -3- [1- (phenylmethyl) -4-piperid-ynyl] -1-propanone; 1- (5-acetylamino-benzo [b] thien-2-yl) -3- [1- (phenylmethyl -) - 4-piperidinyl] -1-propanone; 6-hydroxy-3- [2- [1- (phenylmethyl) -4-piperidinyl] ethyl] -1,2-benzisoxazole; 5-methyl-3- [2- [1- (phenylmethyl) -4-piperidinyl-] ethyl] -1,2-benzisoxazole; 6-methoxy-3 [2- [1 (phenylmethyl) -4piperidinyl] ethyl] -1,2-benzisoxazole; 6-acetamide-3- [2- [1- (phenylmethyl) -4-piperidinyl] -ethyl] -1,2-benzisoxazole; 6-amino-3- [2- [1 (phenylmethyl) -4-piperidinyl] ethi-1] -1,2-benzisoxazole; 6- (4-morpholinyl) -3- [2- [1- (phenylmethyl) -4-piperidin-yl] ethyl] -1,2-benzisoxazole;

5.7-dihydro-3- [2- [1- (phenylmethyl) -4-piperidi-nyl] ethyl] -6H-pyrrolo [4,5-f] -1,2-benzisoxazol-6-one; 3- [2- [1- (phenylmethyl) -4-piperidinyl] ethyl] -1,2-benzisothiazole; 3- [2- [1- (phenylmethyl) -4-piperidinyl] ethenyl] -1,2-benzisoxazole; 6-phenylamino-3- [2- [1 (phenylmethyl) -4-piperidinyl] ethyl] -1,2, -benzisoxazole; 6- (2-thiazolyl) -3- [2- [1- (phenylmethyl) -4-piperidinyl] ethyl] -1,2-benzis-oxazole; 6 (2-oxazolyl) -3- [2- [1- (phenylmethyl) -4-piperidinyl] ethyl] -1,2-benzisoxazole; 6-pyrrolidinyl-3- [2- [1- (phenylmethyl) -4-piperidinyl] ethyl] -1, -2-benzisoxazole; 5,7-dihydro-5,5-dimethyl-3- [2- [1- (phenylmethyl) -4-piperid-ynyl] ethyl] -6H-pyrrolo [4,5-f] -1,2-benzisoxazole- 6-one;

6.8- dihydro-3- [2- [1- (phenylmethyl) -4-piperidinyl] ethyl] -7H-pyrrolo [5,4-g] -1,2-benzisoxazol-7-one; 3- [2- [1- (phenylmethyl) -4-piperidinyl] ethyl] -5,6, -8-trihydro-7H-isoxazolo [4,5-g] -quinolin-7-one; 1-benzyl-4 - ((5,6-dimethoxy-1-indanon) -2yl) methylpiperidine, 1-benzyl-4 - ((5,6-dimethoxy-1-indanon) -2-ylidenyl) methylpiperidine, 1- benzyl-4 - ((5-methoxy-1-indanon) -2-yl) methylp-iperidine, 1-benzyl-4 - ((5,6-diethoxy-1-indanon) -2-yl) methylpiperidine, 1-benzyl- 4 - ((5,6-methylenedioxy-1-indanon) -2-yl) methylpiperidine, 1- (m-nitrobenzyl) -4 - ((5,6-dimethoxy-1-indanon) -2-yl) methylpiperidine, 1- cyclohexymethyl-4 - ((5,6-dimethoxy-1indanon) -2-yl) methylpiperidine, 1- (m-florobenzyl) -4 - ((5,6-dimethoxy-1-indanon) -2-yl) methylpiperidine, 1-benzyl-4 - ((5,6-dimethoxy-1-indanon) -2-yl) propylpiperidine and 1-benzyl-4 - ((5-isopropoxy-6-methoxy-1-indanon) -2-yl) methylpiperidine.

Illustrative calcium channel antagonists include diltiazem, omega-conotoxin GVIA, methoxyiverapamil, amlodipine, felodipine, lacidipine, and mibefradil.

ES 2 396 913 T3

Illustrative GABA-A receptor modulators include clomethiazole; IDDB; gaboxadol (4,5,6,7tetrahydroisoxazolo [5,4-c] pyridin-3-ol); ganaxolone (3a-hydroxy-3p-methyl-5a-pregnan-20-one); fengabine (2 [(butylimino) - (2-chlorophenyl) methyl] -4-chlorophenol); 2- (4-methoxyphenyl) -2,5,6,7,8,9-hexahydro-pyrazolo [4,3-c] cinolin-3-one; 7-cyclobutyl-6- (2-methyl-2H-1,2,4-triazol-3-ylmethoxy) -3-phenyl-1,2,4-triazolo [4,3-b] pyridazine; (3-fluoro-4-methy) phenyl) -N - ({- 1 - [(2-methylphenyl) methyl] -benzimidazol-2-yl} methyl) -N-pentylcarboxamide; and 3- (aminomethyl) -5-methylhexanoic acid.

Illustrative potassium channel openers include diazoxide, flupirtine, pinacidil, levcromakalim, rilmakalim, cromakalim, PCO-400, and SKP-450 (2- [2 (1,3-dioxolone) -2-methyl] -4- (2 '-oxo-1'-pyrrolidinyl) -6-nitro-2H-1-benzopyran).

Exemplary AMPA / kainate receptor antagonists include 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX); 6-nitro-7-sulfamoylbenzo [f] quinoxaline-2,3-dione (NBQX); 6,7-dinitroquinoxaline-2,3-dione (DNQX); 1- (4-aminophenyl) -4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride; and 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo- [f] quinoxaline.

Illustrative sodium channel antagonists include ajmaline, procainamide, flecainide, and riluzole.

Illustrative matrix metalloprotease inhibitors include 4- [4- (4-fluorophenoxy) benzenesulfonylamino] tetrahydropyran-4-carboxylic acid hydroxyamide; 5-Methyl-5- (4- (4'-fluorophenoxy) -phenoxy) -pyrimidine-2,4,6-trione; 5-n-Butyl-5- (4- (4'-fluorophenoxy) -phenoxy) -pyrimidine-2,4,6-trione and prinomistat.

Poly (ADP ribose) polymerase (PARP) is an abundant nuclear enzyme activated by individual DNA strand breaks to synthesize poly (ADP ribose) from NAD. Under normal conditions, PARP is involved in oxidative stress base cleavage repair by activating and recruiting DNA repair enzymes into the nucleus. Therefore, PARP plays a role in cell necrosis and DNA repair. PARP is also involved in regulating the expression of cytokines that mediate inflammation. Under conditions where DNA damage is excessive (such as from acute overexposure to pathological injury), PARP becomes excessively activated resulting in cell-based energy insufficiency that is characterized by NAD depletion and leads to ATP consumption, necrosis cell, tissue injury and organ damage / failure. PARP is believed to contribute to neurodegeneration, depleting nicotinamide adenine dinucleotide (NAD +) which then reduces adenosine triphosphate (ATP; Cosi and Marien, Ann. NY Acad. Sci., 890: 227, 1999) contributing to cell death that can be prevented with PARP inhibitors. Illustrative PARP inhibitors can be found in Southan and Szabo, Current Medicinal Chemistry, 10: 321, 2003.

Illustrative inhibitors of p38 MAP kinase and c-jun-N-terminal kinases include pyridyl imidazoles, such as PD 169316, isomeric PD 169316, SB 203580, SB 202190, SB 220026, and RWJ 67657. Others are described in US Patent 6,288. 089, and are incorporated by reference herein.

In an illustrative embodiment, a combination therapy to treat or prevent MS comprises a therapeutically effective amount of one or more sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein and one or more of Avonex® (interferon beta -1a), Tysabri® (natalizumab) or Fumaderm® (BG12 / Oral fumarate).

In another embodiment, a combination therapy for treating or preventing neuropathy or conditions associated with diabetes comprises a therapeutically effective amount of one or more sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein and one or more tricyclic antidepressants. (TCA) (including, for example, imipramine, amitriptyline, desipramine, and nortriptyline), serotonin reuptake inhibitors (SSRIs) (including, for example, fluoxetine, paroxetiae, sertralen and citalopram) and antiepileptic drugs (AEDs) (including for example gabapentin, carbamazepine and topimirate).

In another embodiment, the application provides a method for treating or preventing a polyglutamine disease using a combination comprising at least one sirtuin activating compound and at least one HDAC I / II inhibitor. Examples of HDAC I / II inhibitors include hydroxamic acids, cyclic peptides, short chain fatty acids, and depudecin.

Examples of hydroxamic acids and hydroxamic acid derivatives include, but are not limited to, trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), oxamflatin, suberic bishydroxamic acid (SBHA), m-carboxy-cinnamic acid-bishhydroxamic acid ( CBHA), valproic acid and pyroxamide. TSA was isolated as an antifungal antibiotic (Tsuji et al. (1976) J. Antibiot (Tokyo) 29: 1-6) and was found to be a potent inhibitor of mammalian HDAC (Yoshida et al. (1990) J. Biol. Chem . 265: 17174-17179). The finding that TSA resistant cell lines have altered HDAC demonstrates that this enzyme is an important target for TSA. Other hydroxamic acid-based HDAC inhibitors, SAHA, SBHA, and CBHA are synthetic compounds capable of inhibiting HDAC at or below micromolar concentration in vitro or in vivo. Glick et al. (1999) Cancer Res. 59: 43924399. These hydroxamic acid-based HDAC inhibitors all possess an essential structural feature: a polar hydroxamic terminal attached through a hydrophobic methylene spacer (eg, 6 carbons in length) to another polar site that is attached to a hydrophobic moiety. terminal (eg, benzene ring). Developed compounds having such essential characteristics are also within the scope of hydroxamic acids that can be used as HDAC inhibitors.

ES 2 396 913 T3

The cyclic peptides used with HDAC inhibitors are primarily cyclic tetrapeptides. Examples of cyclic peptides include, but are not limited to, trapoxin A, apicidin, and depsipeptide. Trapoxin A is a cyclic tetrapeptide that contains a 2-amino-8-oxo-9,10-epoxy-decanoyl (AOE) residue. Kijima et al. (1993) J. Biol. Chem. 268: 22429-22435. Apicidin is a fungal metabolite that exhibits potent broad-spectrum antiprotozoal activity and inhibits HDAC activity at nanomolar concentrations. Darkin-Rattray et al. (1996) Proc. Natl. Acad. Sci. USA 93; 13143-13147. The depsipeptide is isolated from Chromobacterium violaceum, and has been shown to inhibit HDAC activity at micromolar concentrations.

Examples of benzamides include, but are not limited to, MS-27-275. Saito et al. (1990) Proc. Natl. Acad. Sci. USA 96: 4592-4597. Examples of short chain fatty acids include, but are not limited to, butyrates (eg, butyric acid, arginine butyrate, and phenylbutyrate (PB)). Newmark et al. (1994) Cancer Lett. 78: 1-5; and Carducci et al. (1997) Anticancer Res. 17: 3972-3973. Furthermore, depudecin, which has been shown to inhibit HDAC at micromolar concentrations (Kwon et al. (1998) Proc. Natl. Acad. Sci. USA 95: 3356-3361), also falls within the scope of the histone deacetylase inhibitor, as described herein.

Blood clotting disorders

In other aspects, sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can be used to treat or prevent blood clotting disorders (or hemostatic disorders). As used interchangeably herein, the term haemostasis and the term blood coagulation refer to the control of bleeding, including the physiological properties of vasoconstriction and coagulation. Blood clotting helps maintain the integrity of the mammalian circulation after injury, inflammation, disease, birth defect, dysfunction, or other disturbance. After the initiation of clotting, blood clotting proceeds through the sequential activation of certain proenzymes in plasma to their enzyme form (see, for example, Coleman, RW et al. (Eds.) Haemostasis and Thrombosis, Second edition, (1987)). These plasma glycoproteins, including Factor XII, Factor XI, Factor IX, Factor X, Factor VII, and prothrombin, are zymogens of serine proteases. Most of these blood clotting enzymes are effective on a physiological scale only when they are assembled in complexes on membrane surfaces with protein cofactors such as Factor VIII and Factor V. Other blood factors modulate and localize the formation of clots, or dissolve blood clots. Activated protein C is a specific enzyme that inactivates procoagulant components. Calcium ions are involved in many of the reactions to the components. Blood coagulation follows either the intrinsic pathway, where all protein components are present in the blood, or the extrinsic pathway, where the cell membrane protein tissue factor plays a critical role. Clot formation occurs when fibrinogen is cleaved by thrombin to form fibrin. Blood clots are made up of activated platelets and fibrin.

Furthermore, the formation of blood clots not only limits bleeding in the event of injury (hemostasis), but can lead to severe organ damage and death in the context of atherosclerotic diseases due to occlusion of a major artery or vein. Thrombosis is therefore the formation of blood clots at the wrong time and place. It involves a cascade of complicated and regulated biochemical reactions between circulating blood proteins (clotting factors), blood cells (particularly platelets), and elements of the wall of a damaged vessel.

Accordingly, the present application provides anticoagulation and antithrombotic treatments that aim to inhibit the formation of blood clots in order to prevent or treat blood clotting disorders, such as myocardial infarction, stroke, loss of a limb due to peripheral artery disease or pulmonary embolism.

As used interchangeably herein, modulating or modulating hemostasis and regulating or regulating hemostasis include induction (eg, stimulation or enhancement) of hemostasis, as well as inhibition (eg, reduction or decrease). ) of hemostasis.

In one aspect, the application provides a method of reducing or inhibiting hemostasis in a subject by administering a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein. The compositions and methods described herein are useful for the treatment or prevention of thrombotic diseases. As used herein, the term "thrombotic disorder" includes any disorder or condition characterized by excessive or unwanted coagulation or hemostatic activity, or a hypercoagulable state. Thrombotic disorders include diseases or disorders involving platelet adhesion and thrombus formation, and may manifest as an increased propensity for thrombosis formation, e.g. eg, increased numbers of thrombi, thrombosis at an early age, a familial tendency to thrombosis, and thrombosis at unusual sites. Examples of thrombotic disorders include, but are not limited to, thromboembolism, deep vein thrombosis, pulmonary embolism, stroke, myocardial infarction, miscarriage, thrombophilia associated with antithrombin III deficiency, protein C deficiency, protein S deficiency, resistance to activated protein C, dysfibrinogenemia, fibrinolytic disorders, homocystinuria, pregnancy, inflammatory disorders, myeloproliferative disorders, arteriosclerosis, angina, p. g., unstable angina, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, cancer metastasis, sickle cell disease, glomerular nephritis, and drug-induced thrombocytopenia (including, for example, heparin-induced thrombocytopenia). What's more,

ES 2 396 913 T3 sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can be administered to prevent thrombotic events or to prevent re-occlusion during or after therapeutic coagulation lysis or procedures such as angioplasty or surgery .

In another embodiment, a combination drug regimen may include drugs or compounds for the treatment or prevention of blood clotting disorders or secondary conditions associated with these conditions. Therefore, a combination drug regimen may include one or more sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein and one or more anticoagulation or antithrombosis agents. For example, one or more sirtuin modulator compounds can be combined with an effective amount of one or more of: Oral aspirin, heparin and Warfarin which inhibits Vit K-dependent factors, low molecular weight heparins which inhibit factors X and II, thrombin inhibitors, GP IIbIIIa platelet receptor inhibitors, tissue factor (TF) inhibitors, inhibitors of the human von Willebrand factor, inhibitors of one or more factors involved in hemostasis (in particular in the coagulation cascade). In addition, sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can be combined with thrombolytic agents, such as t-PA, streptokinase, reptylase, TNK-t-PA, and staphylokinase.

Weight control

In another aspect, sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can be used to treat or prevent weight gain or obesity in a subject. For example, sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can be used, for example, to treat or prevent hereditary obesity, dietary obesity, hormone-related obesity, drug-related obesity. , to reduce the weight of a subject, or to reduce or prevent weight gain in a subject. A subject in need of such treatment may be an obese subject, prone to becoming obese, overweight, or prone to being overweight. Subjects prone to becoming obese or overweight can be identified, for example, on the basis of family history, diet, activity level, medication intake, or various combinations thereof.

In still other embodiments, sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can be administered to subjects suffering from a variety of other diseases and conditions that can be treated or prevented by promoting weight loss in the subject. Such diseases include, for example, high blood pressure, hypertension, high blood cholesterol levels, dyslipidemia, type 2 diabetes, insulin resistance, glucose intolerance, hyperinsulinemia, coronary artery disease, angina pectoris, congestive heart failure, stroke, gallstones, cholecystitis and cholelithiasis, gout, osteoarthritis, obstructive sleep apnea and respiratory problems, some types of cancer (such as endometrial, breast, prostate and colon), pregnancy complications, poor female reproductive health (such as menstrual irregularities, infertility, irregular ovulation), bladder control problems (such as stress urinary incontinence); uric acid nephrolithiasis; psychological disorders (such as depression, eating disorders, distorted body image, and low self-esteem). Stunkard AJ, Wadden TA. (Editors) Obesity: theory and therapy, Second Edition. New York: Raven Press, 1993. Finally, AIDS patients may develop lipodystrophy or insulin resistance in response to combination therapies for AIDS.

In another embodiment, sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can be used to inhibit adipogenesis or fat cell differentiation, either in vitro or in vivo. In particular, high circulating levels of insulin and / or insulin-like growth factor (IGF) 1 will be prevented by recruiting preadipocytes to differentiate into adipocytes. Such methods can be used to treat or prevent obesity.

In other embodiments, sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can be used to reduce appetite and / or increase satiety, thereby causing weight loss or preventing weight gain. A subject in need of such treatment can be an overweight, obese subject, or a subject prone to being overweight or becoming obese. The method may comprise administering daily or every other day, or once a week, a dose, e.g. eg, in the form of a pill, to a subject. The dose may be an appetite reducing dose.

In other embodiments, a sirtuin modulator compound that reduces the level and / or activity of a sirtuin protein can be used to stimulate appetite and / or weight gain. A method can comprise administering to a subject, such as a subject who you need it, a pharmaceutically effective amount of a sirtuin modulating agent that reduces the level and / or activity of a sirtuin protein, such as SIRT1 and / or SIRT3. A subject in need of such treatment may be a subject suffering from cachexia or may be prone to cachexia. A combination of agents can also be administered. A method may further comprise monitoring the subject for disease status or sirtuin activation, for example in adipose tissue.

Methods to stimulate fat accumulation in cells can be used in vitro, to establish cellular models of weight gain, which can be employed, e.g. For example, to identify other drugs that prevent weight gain.

ES 2 396 913 T3

Methods for modulating adipogenesis or fat cell differentiation, either in vitro or in vivo, are also provided. In particular, high circulating levels of insulin and / or insulin-like growth factor (IGF) 1 will be prevented by recruiting preadipocytes to differentiate into adipocytes. Such methods can be used to modulate obesity. One method of stimulating adipogenesis may comprise contacting a cell with a sirtuin modulating agent that lowers the level and / or activity of a sirtuin protein.

In another embodiment, the application provides methods for reducing fat or lipid metabolism in a subject by administering a sirtuin modulator compound that reduces the level and / or activity of a sirtuin protein. The method includes administering to a subject an amount of a sirtuin modulator compound, e.g. eg, in an amount effective to reduce fat mobilization into the blood by WAT cells and / or to reduce fat burning by BAT cells.

Methods for promoting appetite and / or weight gain may include, for example, first identifying a subject as in need of reduced fat or lipid metabolism, e.g. eg, by weighing the subject, determining their BMI, or evaluating the subject's fat content or sirtuin activity in the subject's cells. The method may also include monitoring the subject, e.g. g., during and / or after administration of a sirtuin modulator compound. Administration can include one or more doses, e.g. g., given as a bolus or continuously. Monitoring can include evaluating a hormone or metabolite. Illustrative hormones include leptin, adiponectin, resistin, and insulin. Illustrative metabolites include triglycerides, cholesterol, and fatty acids.

In one embodiment, a sirtuin modulator compound that reduces the level and / or activity of a sirtuin protein can be used to modulate (eg, increase) the amount of subcutaneous fat in a tissue, e.g. eg, in facial tissue or other tissue associated with the surface of the neck, hand, leg, or lips. The sirtuin modulator compound can be used to increase the stiffness, water retention, or support properties of the tissue. For example, the sirtuin modulator compound can be applied topically, e.g. eg, in association with another agent, eg. eg, for treating tissue associated with the surface. The sirtuin modulator compound can also be injected subcutaneously, e.g. eg, within the region where subcutaneous fat alteration is desired.

A method of modulating weight may further comprise monitoring the subject's weight and / or the level of modulation of sirtuins, for example, in adipose tissue.

In an illustrative embodiment, a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein can be administered as a combination therapy to treat or prevent weight gain or obesity in a subject. For example, one or more sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can be administered in combination with one or more anti-obesity agents. Illustrative antiobesity agents include, for example, phenylpropanolamine, ephedrine, pseudoephedrine, phentermine, a cholecystokinin A agonist, a monoamine reuptake inhibitor (such as sibutramine), a sympathomimetic agent, a serotonergic agent (such as dexfenfluramine, or a fenflonistamine). dopamine (as bromocriptine), an agonist or mimetic of melanocyte-stimulating hormone receptors, an analog of melanocyte-stimulating hormone, a cannabinoid receptor antagonist, a melanin-concentrating hormone antagonist, the OB (leptin) protein, a leptin analog, a leptin receptor agonist, a galanin antagonist, or a GI lipase inhibitor or reducer (such as orlistat). Other anorexic agents include bombesin agonists, dehydroepiandrosterone or its analogs, glucocorticoid receptor agonists and antagonists, orexin receptor antagonists, urocortin-binding protein antagonists, glucagon-like peptide 1 receptor agonists such as Exendin and ciliary neurotrophic factors such as Axokine.

In another embodiment, sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can be administered to reduce drug-induced weight gain. For example, a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein can be administered as a combination therapy with drugs that can stimulate appetite or cause weight gain, in particular weight gain due to factors that other than fluid retention. Examples of medications that can cause weight gain include, for example, diabetes treatments, including for example sulfonylureas (such as glipizide and glyburide), thiazolidinediones (such as pioglitazone and rosiglitazone), meglitinides, nateglinide, repaglinide, sulfonylurea medications, and insulin; antidepressants, including for example tricyclic antidepressants (such as amitriptyline and imipramine), irreversible monoamine oxidase inhibitors (MaOI), selective serotonin reuptake inhibitors (SSRIs), bupropion, paroxetine and mirtazapine; steroids, such as for example prednisone; hormonal therapy; lithium carbonate; valproic acid; carbamazepine; chlorpromazine; thiothixene; beta blockers (such as propranolo); alpha blockers (such as clonidine, prazosin, and terazosin); and contraceptives, including oral contraceptives (birth control pills) or other contraceptives that contain estrogen and / or progesterone (Depo-Provera, Norplant, Ortho), testosterone, or Megestrol. In another illustrative embodiment, sirtuin modulator compounds that increase the level and / or activity of a sirtuin may be administered as part of a smoking cessation program, to prevent weight gain, or to reduce the gain already achieved.

Diabetes / metabolic disorders

ES 2 396 913 T3

In another aspect, sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can be used to treat or prevent a metabolic disorder, such as insulin resistance, a prediabetic state, type II diabetes, and / or its agents. complications. Administration of sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can increase insulin sensitivity and / or reduce insulin levels in a subject. A subject in need of such treatment may be a subject who has insulin resistance or another precursor symptom of type II diabetes, who has type II diabetes, or who is prone to any of these conditions. For example, the subject can be a subject having insulin resistance, e.g. g., who has high circulating levels of insulin and / or associated conditions, such as hyperlipidemia, dyslipogenesis, hypercholesterolemia, glucose intolerance, high blood glucose levels, other manifestations of syndrome X, hypertension, atherosclerosis and lipodystrophy.

In an illustrative embodiment, a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein can be administered as a combination therapy to treat or prevent a metabolic disorder. For example, one or more sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can be administered in combination with one or more antidiabetic agents. Illustrative antidiabetic agents include, for example, an aldose reductase inhibitor, a glycogen phosphorylase inhibitor, a sorbitol dehydrogenase inhibitor, a protein tyrosine phosphatase 1B inhibitor, a dipeptidyl protease inhibitor, insulin (including orally bioavailable insulin preparations ), a mimetic of insuilin, metformin, acarbose, a ligand of peroxisome proliferator activated receptor γ (PPAR-γ) such as troglitazone, rosaglitazone, pioglitazone or GW-1929, a sulfonylurea, glipazide, glyburide, or chloropropamide, where the amounts of the first and second compounds result in a therapeutic effect. Other antidiabetic agents include a glucosidase inhibitor, a glucagon-like peptide 1 (GLP-1), insulin, a PPARα / γ dual agonist, a meglithimide, and an aP2 inhibitor. In an illustrative embodiment, an antidiabetic agent may be a dipeptidyl peptidase IV (DP-IV or DPP-IV) inhibitor such as, for example, LAF237 from Novartis (NVP DPP728; 1 ​​- [[[2 - [(5-cyanopyridin-2 -yl) amino] ethyl] amino] acetyl] -2-cyano- (S) -pyrrolidine) or MK-04301 from Merck (see eg, Hughes et al., Biochemistry 38: 11597-603 (1999)) .

Inflammatory diseases

In other aspects, sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can be used to treat or prevent a disease or disorder associated with inflammation. Sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can be administered before the onset, during or after the onset of inflammation. When used prophylactically, the compounds are preferably provided in advance of any inflammatory response or symptoms. Administration of the compounds can prevent or attenuate inflammatory responses or symptoms.

Illustrative inflammatory conditions include, for example, multiple sclerosis, rheumatoid arthritis, psoriatic arthritis, degenerative joint disease, sponduloarthropathies, gouty arthritis, systemic lupus erythematosus, juvenile arthritis, rheumatoid arthritis, osteoarthritis, osteoporosis, diabetes (e.g. g., insulin-dependent diabetes mellitus or juvenile-onset diabetes), menstrual spasms, cystic fibrosis, inflammatory bowel disease, irritable bowel syndrome, Crohn's disease, mucous colitis, ulcerative colitis, gastritis, esophagitis, pancreatitis, peritonitis, Alzheimer's disease, shock, ankylosing spondylitis, gastritis, conjunctivitis, pancreatitis (acute or chronic), multiple organ injury syndrome (eg. g., secondary to septicemia or trauma), myocardial infarction, atherosclerosis, stroke, reperfusion injury (eg, due to cardiopulmonary bypass or kidney dialysis), acute glomerulonephritis, vasculitis, thermal injury (ie, sunburn) , necrotizing enterocolitis, granulocyte transfusion associated syndrome and / or Sjogren's syndrome. Illustrative inflammatory conditions of the skin include, for example, eczema, atopic dermatitis, contact dermatitis, urticaria, scleroderma, psoriasis, and dermatoses with acute inflammatory components.

In another embodiment, sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can be used to treat or prevent allergies and respiratory conditions, including asthma, bronchitis, pulmonary fibrosis, allergic rhinitis, oxygen toxicity, emphysema. , chronic bronchitis, acute respiratory distress syndrome and any other chronic obstructive pulmonary disease (COPD). The compounds can be used to treat chronic hepatitis, including hepatitis B and hepatitis C.

Furthermore, sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can be used to treat autoimmune diseases and / or inflammation associated with autoimmune diseases such as autoimmune diseases of organs and tissues (e.g. g., Raynaud's syndrome), scleroderma, myasthenia gravis, transplant rejection, endotoxic shock, septicemia, psoriasis, eczema, dermatitis, multiple sclerosis, autoimmune thyroiditis, systemic lupus erythematosus, Addison's disease, autoimmune polyglandular disease (also known as autoimmune polyglandular disease). polyglandular autoimmune) and Grave's disease.

In certain embodiments, one or more sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can be taken alone or in combination with other compounds useful to treat or prevent inflammation. Illustrative anti-inflammatory agents include, for example, steroids (eg, cortisol, cortisone, fludrocortisone, prednisone, 6α-methylprednisone, triamcinolone, betamethasone, or dexamethasone), drugs

ES 2 396 913 T3 non-steroidal anti-inflammatory drugs (NSAIDs (eg, aspirin, acetaminophen, tolmetin, ibuprofen, mefenamic acid, piroxicam, nabumetone, rofecoxib, celecoxib, etodolac, or nimesulide). In another embodiment, the other therapeutic agent is a antibiotic (eg, vancomycin, penicillin, amoxicillin, ampicillin, cefotaxime, ceftriaxone, cefixime, rifampinmetronidazole, doxycycline, or streptomycin) In another embodiment, the other therapeutic agent is a PDE4 inhibitor (eg. g., roflumilast or rolipram). In another embodiment, the other therapeutic agent is an antihistamine (eg, cyclizine, hydroxyzine, promethazine, or diphenhydramine). In another embodiment, the other therapeutic agent is an antimalarial agent (eg, artemisinin, artemether, artsunate, chloroquine phosphate, mefloquine hydrochloride, doxycycline hydrate, proguanil hydrochloride, atovaquone, or halofantrine). In one embodiment, the other therapeutic agent is drotrecogin alfa.

Other examples of anti-inflammatory agents include, for example, aceclofenac, acemetacin, eacetamidocaproic acid, acetaminophen, acetaminosalol, acetanilide, acetylsalicylic acid, S-adenosylmethionine, alclofenac, alclomethasone, alfentanil, algestone, allylprofenic acid, almino-salicylic acid aluminum, amcinonide, amfenac, aminochloroxazin, 3-amino-4-hydroxybutyric acid, 2-amino-4-picoline, aminopropylon, aminopyrine, amixethrin, ammonium salicylate, ampiroxicam, amtolmetin guacil, anileridine, antipyrine, anthraphenine, apazone, beclomethasone, bendazac, benorylate, benoxaprofen, benzpiperilon, benzydamine, benzylmorphine, bermoprofen, betamethasone, betamethasone-17-bisatabolic acid, betamethasone-17-valentabolide, p-acetaminophen, betamethasone-17-acetaminophen acid, p-acetaminophen 5-bromosalicylic, bromosaligenin, bucetin, buclóxic acid, bucoloma, budesonide, bufexamac, bumadizon, buprenorphine, butacetin, butibufen, butorphanol, carbamazepine, carbifen, carprofen, carsalam, chlorobutanol, chloroprednisone, chlortenoxazin, choline salicylate, cincofen, cinmetacin, ciramadol, clidanac, clobetasol, clocortolone, clomethacin, clonitazene, clonixin, clopirac, cloprednol, clove, codeine, codeine methyl bromide, codeine phosphate, cortisol codeine phosphate, codeine phosphate , cortivazole, cropropamide, crotetamide, cyclazociae, deflazacort, dehydrotestosterone, desomophin, desonide, deoxymethasone, dexamethasone, dexamethasone-21-isonicotinate, dexoxadrol, dextromoramide, dextropropoxyphene, deoxycorticosterone, dezocine, diampromide, diamorphone, diclofenac, difenamizole, difenpiramide, diflorasone, diflucortolone, diflunisal, difluprednate, dihydrocodeine, enol dihydrocodeinone acetate, dihydromorphine, acetylsalicylate dihydroxyaluminum, dimenoxadol, dimepheptanol, dimethylthiambutene, butyrate dioxaphetyl, dipipanone, diprocetil, dipyrone, ditazole, droxicam, emorphazone, enfenamic acid, enoxolone, epirizole, eptazocin, etersalate, ethenzamide, ethoheptazine, ethoxazene, ethylmethylthiambutene, ethylmorphine, etodolac, etofenamate, etonitazene, eugenol, felbinac, fenbufen, phenclózic acid, fendosal, fenoprofen, fentanyl, fentiazac, fepradinol, feprazone, floctaonitazene, fluxxazene, flusonofolide, fluazinolidene, flusonopholidamic acid fluocinolone acetonide, fluocinonide, fluocinolone acetonide, fluocortin butyl, fluocortolone, fluoresone, fluorometholone, fluperolone, flupirtine, fluprednidene, fluprednisolone, fluproquazone, flurandrenolide, flurbiprofen, fluticasone, formocortal, phosphosal, gentisic acid, glafenin, glucamethacin, salicylate glycol, guaiazulene, halcinonide, halobetasol, halomethasone, haloprednone, heroin, hydrocodone, hydrocortamate, hydrocortonehydrocortiscortisone hydrocodone, hydrocortonaucine hydroxide, hydrocortiscortone hydrochloride hydrocortisone 21-lysinate, hydrocortisone cypionate, hydromorphone, hydroxypetidine, ibufenac, ibuprofen, ibuproxam, imidazole salicylate, indomethacin, indoprofen, isofezolac, isoflupredone, isoflupredone acetate, isoladol, isomethadone, isonixin, isoxepac, isoxicam, ketobemidone, ketoprofen, cetorolac, p-lactofenetide, lefetamine, levalorphan, levorphanol, lysomethadone, lysicofenazyl-lophenol, lysofenazyl-lysichane , mazipredone, meclofenamic acid, medrisone, mefenamic acid, meloxicam, meperidine, meprednisone, meptazinol, mesalamine, metazocine, methadone, metotrimeprazine, methylprednisolone, Methylprednisolone acetate, methylprednisolone sodium succinate, methylprednisolone sulepnate, methiazinic acid, metofolin, metopon, mofebutazone, mofezolac, mometasone, morazone, morphine, morphine hydrochloride, morphine sulfate, morphine, napholine, myrbuphine, 1-morphine, salicylate naphthyl salicylate, naproxen, narcein, nefopam, nicomorphine, nifenazone, niflumic acid, nimesulide, 5, -nitro-2, -propoxyacetanilide, norlevorphanol, normethadone, normorphine, norpipanone, olsalazine, opium, oxaceprole, oxamethacin, oxaprozin, oxycodone, oxymorphone, oxyphenbutazone, papaveretum, paramethasone, paranylin, parsalmide, pentazocine, perisoxal, phenacetin, fenadoxone, phenazocin, phenytoxyl saline, phenytoxinylchloride, phenytoxinylchloride, phenytoxinylchloride, phenytoxinyl sodium , phenyl salicylate, feniramidol, picetoprofen, piminodine, pipebuzone, piperilone, pyrazolac, pyritramide, piroxicam, pirprofen, pranoprofen, prednicarbate, prednisolone, prednisone, prednival, prednylidene, proglumethacin, proheptazine, promedol, propacetamol, properidin, propyram, propoxyphene, propiphenazone, procuazone, protizinic acid, proxazole, ramifenazone, remifentanil, rimazolidin methyl sulfate, salicylamide, salicylamide, salicylamide or salicylamide, salicylamide or salicylamide acid, salicylamide or salicylamide acid, salicylamide or salicylamide acid salicylic, salicylsulfuric acid, salsalate, salverine, symmetride, sufentanil, sulfasalazine, sulindac, superoxide dismutase, suprofen, suxibuzone, talniflumate, tenidap, tenoxicam, terofenamate, tetrandrine, thiazolinobutazone, thiaprofenic acid, thiaramide, tilidine, tinoridine, thixocortol, tolfenamic acid, tolmetin, tramadol, triamcinolone, triamcinolone acetonide, tropesin, viminol, xenbucin, xpiracimoprofen, and zomethine.

In an illustrative embodiment, a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein can be administered with a selective COX-2 inhibitor to treat or prevent inflammation. Illustrative selective COX-2 inhibitors include, for example, deracoxib, parecoxib, celecoxib, valdecoxib, rofecoxib, etoricoxib, lumiracoxib, 2- (3,5-difluorophenyl) -3-- [4- (methylsulfonyl) phenyl] -2 -cyclopenten-1-one, (S) -6,8-dichloro-2- (trifluoromethyl) -2H-1-benzopyran-3-carboxylic acid, 2- (3,4-difluorophenyl) -4- (3-hydroxy -3-methyl-1-butoxy) -5- [4 (methylsulfonyl) phenyl] -3- (2H) -pyridazinone, 4- [5- (4-fluorophenyl) -3- (trifluoromethyl) -1H-pyrazole-1 -yl] benzenesulfonamide, tert-butyl benzyl-4 - [(4-oxopiperidin-1-yl} sulfonyl] piperidine-4-carboxylate, 4- [5- (phenyl) -3- (trifluoromethyl) -1H-pyrazol-1-yl] benzenesulfonamide, its salts and their prodrugs.

ES 2 396 913 T3

Hot flushes

In another aspect, sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can be used to reduce the incidence or intensity of hot flashes that are symptoms of a disorder. For example, the method in question includes the use of sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein, alone or in combination with other agents, to reduce the incidence or intensity of hot flashes in cancer patients. In other embodiments, the method provides for the use of sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein to reduce the incidence or intensity of hot flashes in menopausal and post-menopausal women.

In another aspect, sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can be used as therapy to reduce the incidence or intensity of hot flashes that are side effects of other drug therapy, e.g. eg, drug-induced hot flashes. In certain embodiments, a method of treating and / or preventing drug-induced hot flashes comprises administering to a patient in need thereof a formulation comprising at least one hot flash-inducing compound and at least one modulator compound of sirtuin that increases the level and / or the activity of a sirtuin protein. In other embodiments, a method of treating drug-induced hot flashes comprises separately administering one or more hot flash-inducing compounds and one or more sirtuin modulating compounds, e.g. eg, where the sirtuin modulator compound and the hot flash agent have not been formulated in the same compositions. When using separate formulations, the sirtuin modulator compound can be administered (1) at the same time as the administration of the hot flash agent, (2) intermittently with the hot flash agent, (3) staggered relative to the administration of the agent. hot flash inducer, (4) prior to administration of the hot flash inducing agent, (5) subsequent to administration of the hot flash inducing agent, and (6) various combinations thereof. Illustrative hot flash inducing agents include, for example, niacin, phaloxifene, antidepressants, antipsychotics, chemotherapeutics, calcium channel blockers, and antibiotics.

In one embodiment, sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can be used to reduce the side effects of hot flashes of a vasodilator or antilipemic agent (including anticolesterolemic agents and lipotropic agents). In an illustrative embodiment, a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein can be used to reduce hot flashes associated with niacin administration.

Nicotinic acid, 3-pyridinecarboxylic acid or niacin, is an antilipidemic agent marketed, for example, under the names Nicolar®, SloNiacin®, Nicobid® and Time Release Niacin®. Nicotinic acid has been used for many years in the treatment of lipidemic disorders such as hyperlipidemia, hypercholesterolemia, and atherosclerosis. This compound has long been known to exhibit beneficial effects of reducing total cholesterol, low-density lipoproteins or LDL cholesterol, triglycerides, and apolipoprotein in (Lp (a)) in the human body, while increasing the desirable high-density lipoprotein or HDL cholesterol.

Typical doses range from about 1 gram to about 3 grams daily. Nicotinic acid is normally administered two to four times after meals, depending on the dosage form selected. Nicotinic acid is currently marketed in two dosage forms. One consists of an immediate or rapid-release tablet to be administered three or four times a day. Immediate release (IR) nicotinic acid formulations generally release virtually all of the nicotinic acid within about 30 to 60 minutes after ingestion. The other dosage form is a sustained release form that is suitable to administer between two and four times per day. In contrast to IR formulations, sustained release (SR) nicotinic acid formulations are designed to release significant amounts of drug for absorption into the bloodstream during specific time intervals in order to maintain therapeutic levels of nicotinic acid over a period of time. extended such as 12 or 24 hours after ingestion.

As used herein, the term "nicotinic acid" is intended to encompass nicotinic acid or a compound other than nicotinic acid itself, which the body metabolizes to nicotinic acid, thus producing essentially the same effect as nicotinic acid. Illustrative compounds that produce an effect similar to that of nicotinic acid include, for example, nicotinyl alcohol tartrate, dglucitol hexanicotinate, aluminum nicotinate, niceritrol, and d, 1-alpha-tocopheryl nicotinate. Each such compound will be collectively referred to herein as nicotinic acid.

In another embodiment, the application provides a method of treating and / or preventing hyperlipidemia with reduced hot flash side effects. The method comprises the steps of administering to a subject in need thereof a therapeutically effective amount of nicotinic acid and a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein in an amount sufficient to reduce hot flashes. In an illustrative embodiment, the nicotinic acid and / or the sirtuin modulator compound can be administered at night.

In another representative embodiment, the method involves the use of sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein to reduce the hot flash side effects of raloxifene. Raloxifene acts like estrogen in certain parts of the body, but it is not a hormone. Helps prevent

ES 2 396 913 T3 osteoporosis in women who have reached menopause. Osteoporosis causes bones to gradually thin, brittle, and more likely to break. Evista slows the loss of bone mass that occurs with menopause, reducing the risk of spinal fractures due to osteoporosis. A common side effect of raloxifene is hot flashes (sweating and hot flashes). This can be uncomfortable for women who are already experiencing hot flashes due to menopause.

In another representative embodiment, the method involves the use of sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein to reduce the side effects of hot flashes by antidepressant or antipsychotic agents. For example, sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can be used in conjunction with (administered separately or together) a serotonin reuptake inhibitor, a 5HT2 receptor antagonist, an anticonvulsant, an inhibitor. of norepinephrine reuptake, an α-adrenoreceptor antagonist, an NK-3 antagonist, an NK1 receptor antagonist, a PDE4 inhibitor, a neuropeptide Y5 receptor antagonist, a D4 receptor antagonist, a 5HT1A receptor antagonist, a 5HT1D receptor antagonist, a CRF antagonist, a monoamine oxidase inhibitor, or a sedative-hypnotic drug.

In certain embodiments, sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can be used as part of a serotonin reuptake inhibitor (SRI) treatment to reduce hot flashes. In certain preferred embodiments, the SRI is a selective serotonin reuptake inhibitor (SSRI), such as a fluoxetinoid (fluoxetine, norfluoxetine) or a nefazodonoid (nefazodone, hydroxynefazodone, oxonefazodone). Other illustrative SSRIs include duloxetine, venlafaxine, milnacipran, citalopram, fluvoxamine, paroxetine, and sertraline. The sirtuin modulator compound that increases the level and / or activity of a sirtuin protein can also be used as part of a sedative-hypnotic treatment, such as those selected from the group consisting of a benzodiazepine (such as alprazolam, chlordiazepoxide, clonazepam, clorazepate , clobazam, diazepam, halazepam, lorazepam, oxazepam, and prazepam), zolpidem, and barbiturates. In still other embodiments, a sirtuin modulator compound that increases the level and / or activity of a sirtuin protein can be used as part of treatment with a 5-HT1A receptor partial agonist, such as those selected from the group consisting of buspirone, flesinoxan, gepirone, and ipsapirone. Sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can also be used as part of a treatment with a norepinephrine reuptake inhibitor, such as those selected from tertiary amine tricyclics and secondary amine tricyclics. Illustrative tertiary amine tricyclics include amitriptyline, clomipramine, doxepin, imipramine, and trimipramine. Illustrative secondary amine tricycles include amoxapine, desipramine, maprotiline, nortriptyline, and protriptyline. In certain embodiments, sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can be used as part of a treatment with a monoamine oxidase inhibitor, such as those selected from the group consisting of isocarboxazid, phenelzine, tranylcypromine , selegiline and moclobemide.

In yet another representative embodiment , sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can be used to reduce the side effects of hot flashes caused by chemotherapeutic agents, such as cyclophosphamide, tamoxifen.

In another embodiment, sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can be used to reduce the side effects of hot flashes caused by calcium channel blockers, such as amlodipine.

In another embodiment, sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can be used to reduce the side effects of hot flashes caused by antibiotics. For example, sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can be used in combination with levofloxacin. Levofloxacin is used to treat infections of the sinuses, skin, lungs, ears, airways, bones, and joints caused by susceptible bacteria. Levofloxacin is also frequently used to treat urinary infections, including those resistant to other antibiotics, as well as prostatitis. Levofloxacin is effective in treating infectious diarrhea caused by the bacteria E. coli, campylobacter jejuni, and shigella. Levofloxacin is also used to treat various obstetric infections, including mastitis.

Eye disorders

One aspect of the present application is a method of inhibiting, reducing or in some way treating vision impairment, by administering to a patient a therapeutic dose of a sirtuin modulator selected from a compound described herein or its pharmaceutically acceptable salt.

In certain respects, vision impairment is caused by damage to the optic nerve or the central nervous system. In particular embodiments, the damage to the optic nerve is caused by high intraocular pressure, such as that created by glaucoma. In other particular embodiments, the damage to the optic nerve is caused by inflammation of the nerve, which is often associated with an infection or an immune (eg, autoimmune) response such as optic neuritis.

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Glaucoma describes a group of disorders that are associated with a visual field defect, excavation of the optic disc, and damage to the optic nerve. These disorders are commonly called glaucomatous optic neuropathies. Most glaucomas are generally, but not always, associated with elevated intraocular pressure. Illustrative forms of glaucoma include glaucoma and penetrating keratoplasty, acute angle closure, angle closure closure, chronic open angle, angle recession, drug-induced hyphema, aphakic and pseudoaphakic glaucoma, intraocular tumors, juvenile glaucoma, lens particle, of low tension, malignant, neovascular, phacolytic, phacomorphic, pigmentary, plateau iris, primary congenital, primary open angle, pseudoexfoliation, secondary congenital, suspicion in the adult, unilateral, uveitic, ocular hypertension, ocular hypotonia, Posner-Schlossman syndrome and scleral expansion procedure in ocular hypertension and primary open angle glaucoma.

Intraocular pressure can also be increased with various surgical procedures, such as phacoemulsification (ie, cataract surgery) and implantation of structures such as artificial lenses. In addition, spinal surgeries in particular, or any surgery in which the patient is prone to spending an extended period of time, can increase interocular pressure.

Optic neuritis (ON) is inflammation of the optic nerve and causes acute vision loss. It is highly associated with multiple sclerosis (MS), since 15-25% of MS patients initially present with ON, and 50-75% of ON patients are diagnosed with MS. ON is also associated with infection (eg, viral infection, meningitis, syphilis), inflammation (eg, from a vaccine), infiltration, and ischemia.

Another condition that leads to optic nerve damage is ischemic optic neuropathy (AION). There are two types of AION. Arteritic AION due to giant cell arteritis (vasculitis), leading to acute vision loss. Non-arteritic AION encompasses all cases of ischemic optic neuropathy that are not due to giant cell arteritis. The pathophysiology of AION is not entirely clear, although it appears to incorporate both inflammatory and ischemic mechanisms.

Other optic nerve damage is typically associated with demyelination, inflammation, ischemia, toxins, or trauma to the optic nerve. Illustrative conditions in which the optic nerve is damaged include demyelinating optic neuropathy (optic neuritis, retrobulbar optic neuritis), optic nerve sheath meningioma, adult optic neuritis, childhood optic neuritis, anterior ischemic optic neuropathy, posterior ischemic optic neuropathy , compressive optic neuropathy, papillary edema, pseudo papillary edema and toxic / nutritional optic neuropathy.

Other neurological conditions associated with vision loss, although not directly related to optic nerve damage, include amblyopia, Bell's palsy, chronic progressive external ophthalmoplegia, multiple sclerosis, pseudotumor cerebri, and trigeminal neuralgia.

In certain respects, vision impairment is caused by retinal damage. In particular embodiments, the retinal damage is caused by disturbances in the bloodstream to the eye (eg, arteriosclerosis, vasculitis). In particular embodiments, the retinal damage is caused by rupture of the macula (eg, exudative or non-exudative macular degeneration).

Illustrative retinal diseases include age-related exudative macular degeneration, age-related non-exudative macular degeneration, age-related macular degeneration from electronic retinal prosthesis and RPE transplantation, acute multifocal placoid pigmentary epitheliopathy, acute retinal necrosis, Best's disease, occlusion of the arterial branch of the retina, occlusion of the venous branch of the retina, Autoimmune retinopathies associated with cancer, central retinal artery occlusion, central retinal vein occlusion, central serous chorioretinopathy, Eales disease, epimacular membrane, reticulum degeneration, macroaneurysm, diabetic macular edema, Irvine macular edema- Gas, macular orifice, subretinal neovascular membranes, subacute diffuse neuroretinitis, non-pseudophasic cystoid macular edema, syndrome of presumed ocular histoplasmosis, exudative retinal detachment, postoperative retinal detachment, proliferative retinal detachment, rhegmatogenous retinal detachment, tractional retinal detachment, retinitis pigmentosa, CMV retinitis, retinoblastoma, premature retinopathy, shotgun retinopathy, background diabetic retinopathy, proliferative diabetic retinopathy, retinopathy due to hemoglobinopathies, Purtscher retinopathy, Valsalva retinopathy, juvenile retinoschisis, senile retinoschisis, Terson syndrome and white spot syndrome.

Other illustrative diseases include bacterial eye infections (eg, conjunctivitis, keratitis, tuberculosis, syphilis, gonorrhea), viral infections (eg, ocular herpes simplex virus, varicella zoster virus, cytomegalovirus retinitis, virus human immunodeficiency (HIV)) as well as progressive external retinal necrosis secondary to HIV or other eye diseases associated with immunodeficiency or HIV. In turn, eye diseases include fungal infections (eg, Candida choroiditis, histoplasmosis), protozoal infections (eg, toxoplasmosis), and others such as ocular toxocariasis and sarcoidosis.

One aspect of the application is a method of inhibiting, reducing, or treating vision impairment in a subject undergoing treatment with a chemotherapeutic drug (eg, a neurotoxic drug, a drug that

ES 2 396 913 T3 raises intraocular pressure such as a steroid), by administering to the subject in need of such treatment a therapeutic dose of a sirtuin modulator described herein.

Another aspect of the application is a method of inhibiting, reducing or treating vision impairment in a subject undergoing surgery, including eye surgeries or other surgeries performed in a prone position, as in spinal cord surgery, by administering to the subject in need of such treatment a therapeutic dose of a sirtuin modulator described herein. Eye surgeries include cataract surgery, iridiotomy, and lens replacement. Another aspect of the invention is the treatment, including inhibition and prophylactic treatment, of aging-related eye diseases, including cataracts, dry eyes, damage to the retina and the like, by administering to the subject in need of such treatment a therapeutic dose of a sirtuin modulator described in the present invention.

Cataract formation is associated with several biochemical changes in the lens of the eye, such as a reduction in the levels of antioxidants, ascorbic acid and glutathione, increased lipids, oxidation of amino acids and proteins, increased calcium and sodium, loss of amino acids and reduction of lens metabolism. The lens, lacking blood vessels, is suspended in extracellular fluids in the front of the eye. Nutrients such as ascorbic acid, glutathione, vitamin E, selenium, bioflavonoids, and carotenoids are required to maintain the transparency of the lens. Reduced levels of selenium salts produce an increase in free radical-inducing hydrogen peroxide, which is neutralized by the selenium-dependent antioxidant enzyme glutathione peroxidase. The lens-protective glutathione peroxidase is also dependent on the amino acids methionine, cysteine, glycine, and glutamic acid.

Cataracts can also appear due to an inability to properly metabolize the galactose found in lactose-containing dairy products, a disaccharide made up of the monosaccharide galactose and glucose. Cataracts can be prevented, delayed, and possibly even reversed, if caught early and corrected metabolically.

Damage to the retina is attributed, among others, to reactions initiated by free radicals in glaucoma, diabetic retinopathy, and age-related macular degeneration (AMD). The eye is a part of the central nervous system and has a limited regenerative capacity. The retina is made up of numerous nerve cells that contain the highest concentration of polyunsaturated fatty acids (PFAs) and is subject to oxidation. Free radicals are generated by UV light entering the eye and mitochondria in rods and cones, which generate the energy needed to transform light into visual impulses. Free radicals cause peroxidation of PFA by hydroxyl or superoxide radicals which in turn spread additional free radicals. Free radicals cause temporary or permanent damage to retinal tissue.

Glaucoma is generally viewed as a disorder that causes elevated intraocular pressure (IOP), which causes permanent damage to the nerve fibers in the retina, but one sixth of all glaucomas do not cause elevated IOP. This disorder is now perceived as one of reduced vascular perfusion and an increase in neurotoxic factors. Recent studies have implicated elevated levels of glutamate, nitric oxide, and peroxynitrite in the eye as the causes of the death of retinal ganglion cells. Neuroprotective agents may be the future of glaucoma care. For example, nitric oxide synthase inhibitors block the formation of peroxynitrite from nitric oxide and superoxide. In a recent study, animals treated with aminoguanidine, a nitric oxide synthase inhibitor, had a reduction in retinal ganglion cell loss. It was concluded that nitric oxide in the eye caused cytotoxicity in many tissues and neurotoxicity in the central nervous system.

Diabetic retinopathy occurs when the underlying blood vessels generate microvascular abnormalities consisting mainly of microaneurysms and intraretinal hemorrhages. Oxidative metabolites are directly involved in the pathogenesis of diabetic retinopathy, and free radicals increase the generation of growth factors leading to increased proliferative activity. Nitric oxide produced by the endothelial cells of the vessels can also cause the smooth muscle cells to relax and cause vasodilation of vessel segments. Retinal ischemia and hypoxia occur after arterial basement membrane thickening, endothelial proliferation, and loss of pericytes. Inadequate oxygenation causes capillary obliteration or non-perfusion, arteriovenous shunts, slow blood flow, and impaired ability of red blood cells to release oxygen. Lipid peroxidation of retinal tissue also occurs as a consequence of free radical damage.

The macula is responsible for our sharp central vision and is made up of light-sensitive cells (cones) while the underlying retinal pigment epithelium (RPE) and choroid nourish and help remove waste materials. The RPE nourishes the cones with the vitamin A substrate for photosensitive pigments and digests the outer tips of the detachment of the cones. RPE is exposed to high levels of UV radiation, and secretes factors that inhibit angiogenesis. The choroid contains a dense vascular network that provides nutrients and removes waste materials.

In AMD, the tips of the detached cones become indigestible by RPE, where the cells expand and die after collecting too much undigested material. Waste material accumulations do not

ES 2 396 913 digested T3, called drusen, form under the RPE. Photoxic damage also causes lipofuscin accumulation in RPE cells. Intracellular lipofuscin and the accumulation of drusen in Bruch's membrane interfere with the transport of oxygen and nutrients to the retinal tissues, ultimately leading to RPE and photoreceptor dysfunction. In exudative AMD, blood vessels expand from the choriocapillaries through defects in Bruch's membrane and can grow under the RPE, dislodging it from the choroid, and leaking fluid or bleeding.

Macular pigment, one of the protective factors that prevents sunlight from damaging the retina, is formed by the accumulation of nutritionally derived carotenoids such as lutein, the fatty yellow pigment that serves as a delivery vehicle for other important nutrients and zeaxanthin. Antioxidants, such as vitamins C and E, beta-carotene, and lutein, plus zinc, selenium, and copper, are all found in healthy macula. In addition to providing nutrition, these antioxidants protect against free radical damage that initiates macular degeneration.

Another aspect of the application is the prevention or treatment of damage to the eye caused by stress, chemical attack or radiation, by administering to the subject in need of such treatment a dose of a sirtuin modulator described herein. Radiation damage or electromagnetic damage to the eye can include that caused by CRT or exposure to sunlight or UV.

In one embodiment, a combination drug regimen can include drugs or compounds for the treatment or prevention of ocular disorders or secondary conditions associated with these conditions. Accordingly, a combination drug regimen can include one or more sirtuin activators and one or more therapeutic agents for the treatment of an eye disorder. For example, one or more sirtuin activating compounds can be combined with an effective amount of one or more of: an agent that reduces intraocular pressure, an agent for treating glaucoma, an agent for treating optic neuritis, an agent for treating CMV retinopathy , an agent for treating multiple sclerosis and / or an antibiotic, etc.

In one embodiment, a sirtuin modulator can be administered in conjunction with a therapy to reduce intraocular pressure. One group of therapies involves blocking water production. For example, topical beta-adrenergic antagonists (timolol and betaxolol) decrease aqueous production. Topical timolol causes IOP to drop in 30 minutes with maximum effects in 1-2 hours. A reasonable regimen is Timoptic 0.5%, in one drop every 30 minutes for 2 doses. The carbonic anhydrase inhibitor, acetazolamide, also reduces water production and must be administered in conjunction with topical beta-antagonists. A starting dose of 500 mg is given followed by 250 mg every 6 hours. This medicine can be administered orally, intramuscularly, or intravenously. In addition, alpha 2 agonists (eg, Apraclonidine) work by reducing water production. Its effects are additive to topically administered beta-blockers. They have been approved for use in the management of acute elevation of pressure after anterior chamber laser procedures, but have been described as effective in treating acute angle-closure glaucoma. A reasonable regimen is one drop every 30 minutes for 2 doses.

A second group of therapies to reduce intraocular pressure involves reducing the vitreous volume. Hyperosmotic agents can be used to treat an acute attack. These agents draw water out of the balloon, making the blood hyperosmolar. Oral glycerol is often used at a dose of 1 mL / kg in a cold 50% solution (mixed with lemon juice for a more pleasant taste). Glycerol is converted to glucose in the liver; people with diabetes may need additional insulin if they become hypoglycemic after receiving glycerol. Oral isosorbide is an inert alcohol that can also be used as an osmotic agent for patients with acute angle-closure glaucoma. The usual dose is 100 g taken po (220 ml of a 45% solution). This inert alcohol should not be confused with isosorbide dinitrate, a nitrate-based heart medication used for angina and congestive heart failure. Intravenous mannitol at a dose of 1.0-1.5 mg / kg is also effective and well tolerated in patients with nausea and vomiting. These hyperosmotic agents should be used with caution in any patient with a history of congestive heart failure.

A third group of therapies involves facilitating the watery flow from the eye. Miotic agents pull the iris from the iridocorneal angle and can help relieve obstruction of the trabecular meshwork by the peripheral iris. Pilocarpine 2% (blue eyes) -4% (brown eyes) can be given every 15 minutes for the first 1-2 hours. More frequent administration or higher doses can precipitate a systemic cholinergic crisis. NSAIDs are sometimes used to reduce inflammation.

Illustrative therapeutic agents for reducing intraocular pressure include ALPHAGAN® P (Allergan) (brimonidine tartrate ophthalmic solution), AZOPT® (Alcon) (brinzolamide ophthalmic suspension), BETAGAN® (Allergan) (levobunolol hydrochloride ophthalmic solution, uSp), BETIMOL® (Vistakon) (timolol ophthalmic solution), BETOPTIC S® (Alcon) (betaxolol HCl), BRIMONIDINE TARTRATE (Bausch &amp; Lomb), CARTEOLOL HYDROCHLORIDE (Bausch &amp; Lomb), COSOpT® (Merck) (dorzolamide hydrochloride-timolol maleate ophthalmic solution), LuMiGAN® (Allergan) (bimatoprost ophthalmic solution), OPTIPRANOLOL® (Bausch &amp; Lomb) (metipranolol ophthalmic solution), TIMOLOL GFS (Falcon) timolol maleate ophthalmic gel-forming solution), TIMOPTIC® (Merck) (timolol maleate ophthalmic solution), TRAVATAN® (Alcon) (travoprost ophthalmic solution), TRUSOPT® (Merck) (dorzolamide hydrochloride ophthalmic solution) and XALATAN® (Pharmacia &amp; Upjohn) (latanoprost ophthalmic solution).

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In one embodiment, a sirtuin modulator can be administered in conjunction with therapy to treat and / or prevent glaucoma. An example of a glaucoma drug is DARANIDe® tablets (Merck) (Dichlorphenamide).

In one embodiment, a sirtuin modulator can be administered in conjunction with therapy to treat and / or prevent optic neuritis. Examples of drugs for optic neuritis include DECADRON® phosphate injection (Merck) (Dexamethasone sodium phosphate), DEPO-MEDROL® (Pharmacia &amp; Upjohn) (methylprednisolone acetate), HYDROCORTONE® tablets (Merck) (Hydrocortisone), ORAPRED® ( Biomarin) (prednisolone sodium phosphate oral solution) and PEDIApReD® (Celltech) (prednisolone sodium phosphate, uSp).

In one embodiment, a sirtuin modulator can be administered in conjunction with therapy to treat and / or prevent CMV retinopathy. Treatments for CMV retinopathy include CYTOVENE® (ganciclovir capsules) and VALCYTE® (Roche Laboratories) (valganciclovir hydrochloride tablets).

In one embodiment, a sirtuin modulator can be administered in conjunction with a therapy to treat and / or prevent multiple sclerosis. Examples of such drugs include DANTRIUM® (Procter &amp; Gamble Pharmaceuticals) (dantrolene sodium), NoVaNTRONE® (Serono) (mitoxantrone), AVONEX® (Biogen Idec) (Interferon beta-la), BETASERON® (Berlex) (Interferon beta- 1b), CoPaXoNE® (Teva Neuroscience) (glatiramer acetate injection) and REBIF® (Pfizer) (interferon beta-1a).

In turn, macrolides and / or mycophenolic acid, which has multiple activities, can be co-administered with a sirtuin modulator. Macrolide antibiotics include tacrolimus, cyclosporin, sirolimus, everolimus, ascomycin, erythromycin, azithromycin, clarithromycin, clindamycin, lincomycin, dirithromycin, josamycin, spiramycin, diacetyl-midecamycin, tylosin, leincithromycin-77, and leincithromycin3, leincithromycin, and lycosin, rhoxithromycin-77

Diseases and disorders associated with mitochondria

In certain embodiments, the invention contemplates methods of treating diseases or disorders that would benefit from increased mitochondrial activity. The methods comprise administering to a subject in need thereof a therapeutically effective amount of a sirtuin activating compound. An increase in mitochondrial activity refers to increasing the activity of the mitochondria while maintaining the overall numbers of mitochondria (p. g., mitochondrial mass), increasing the numbers of mitochondria, thus increasing mitochondrial activity (eg, stimulating mitochondrial biogenesis), or combinations thereof. In certain embodiments, diseases and disorders that would benefit from mitochondrial activity include diseases or disorders associated with mitochondrial dysfunction.

In certain embodiments, methods of treating diseases and disorders that would benefit from increased mitochondrial activity may comprise identifying a subject suffering from mitochondrial dysfunction. Methods for diagnosing mitochondrial dysfunction may involve molecular, pathological, and / or biochemical genetic analyzes, which are summarized in Cohen and Gold, Cleveland Clinic Journal of Medicine, 68: 625-642 (2001). One method of diagnosing mitochondrial dysfunction is the Thor-Byrne-ier scale (see, eg, Cohen and Gold, supra; Collin S. et al., Eur Neurol. 36: 260-267 (1996)). Other methods for determining mitochondrial number and function include, for example, enzymatic assays (e.g. g., a mitochondrial enzyme or an ATP biosynthesis factor such as an ETC enzyme or a Krebs cycle enzyme), determination of mitochondrial mass, mitochondrial volume and / or mitochondrial number, quantification of mitochondrial DNA, monitoring of intracellular calcium homeostasis and / or cellular responses to disturbances of this homeostasis, evaluation of the response to an apoptogenic stimulus, determination of the production of free radicals. Such methods are known in the art and are described, for example, in US Patent Publication No. 2002/0049176 and references cited there.

Mitochondria are critical to the survival and proper functioning of almost all eukaryotic cell types. Mitochondria in virtually any cell type can have birth or acquired defects that affect their function. Therefore, the clinically important signs and symptoms of mitochondrial defects that affect respiratory chain function are heterogeneous and variable, depending on the distribution of defective mitochondria among cells and the severity of their deficits, and on physiological demands. of affected cells. Non-dividing tissues with high energy requirements, eg. For example, nervous tissue, skeletal muscle, and cardiac muscle are particularly susceptible to respiratory chain dysfunction, but any organ can be affected.

Diseases and disorders associated with mitochondrial dysfunction include diseases and disorders in which deficits in the activity of the mitochondrial respiratory chain contribute to the development of such diseases or disorders in a mammal. This includes 1) congenital genetic deficiencies in activity of one or more components of the mitochondrial respiratory chain; and 2) acquired deficiencies in the activity of one or more components of the mitochondrial respiratory chain, where said deficiencies are caused by a) oxidative damage during aging; b) elevation of intracellular calcium; c) exposure of affected cells to nitric oxide; d) hypoxia or ischemia; e) deficits associated with microtubules in axonal transport of mitochondria, or f) expression of mitochondrial uncoupling proteins.

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Diseases or disorders that would benefit from increased mitochondrial activity in general include, for example, diseases in which free radical-mediated oxidative injury leads to tissue degradation, diseases in which cells inappropriately undergo apoptosis and diseases in which cells cannot undergo apoptosis. Illustrative diseases or disorders that would benefit from increased mitochondrial activity include, for example, AD (Alzheimer's disease), ADPD (Alzheimer's disease and Parkinson's disease), AMDF (Ataxia, Myoclonus and deafness), autoimmune diseases, cancer, CIPO (chronic intestinal pseudo-obstruction with myopathy and ophthalmoplegia), congenital muscular dystrophy, CPEO (chronic progressive external ophthalmoplegia), DEAF (maternal inherited deafness or aminolicoside-induced deafness), DEMCHO (dementia and chorea), diabetes mellitus (Type I or Type II), DIDMOAD (Diabetes Insipidus, Diabetes Mellitus, optic atrophy, deafness), DMDF (Diabetes Mellitus and deafness) , dystonia, exercise intolerance, ESOC (epilepsy, strokes, optic atrophy and cognitive impairment), FBSN (familial bilateral striatal necrosis), FICP (fatal infantile cardiomyopathy plus MELAS associated cardiomyopathy), GER (gastrointestinal reflux), HD (Huntington's disease), KSS (Kearns Sayre syndrome), late-onset myopathy, LDYT (Leber's hereditary optic neuropathy and dystonia), Leigh's syndrome, LHON (Leber's hereditary optic neuropathy), LIMM (fatal infantile mitochondrial myopathy), MDM (myopathy and diabetes mellitus), MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes), MEPR (myoclonic epilepsy and psychomotor regression), MERME (MERRF / MELAS superimposed disease), MERRF (myoclonic epilepsy and ragged red muscle fibers), MHCM (maternal inherited hypertrophic cardiomyopathy), MICM (maternal inherited cardiomyopathy), MILS (maternal inherited Leigh syndrome), mitochondrial encephalocardiomyopathy, mitochondrial encephalomyopathy, encephalomyopathy MM (mitochondrial myopathy), MMC (maternal myopathy and cardiomyopathy), MNGIE (myopathy and external ophthalmoplegia, neuropathy, gastrointestinal encephalopathy, encephalopathy), multisystem mitochondrial disorder (myopathy, encephalopathy, blindness, hearing loss, peripheral neuropathy), NARP (neurogenic muscle weakness, ataxia, and retinitis pigmentosa; Alternative phenotype at this locus is described as Leigh's disease, PD (Parkinson's disease), Pearson's syndrome, PEM (progressive encephalopathy), PEO (progressive external ophthalmoplegia), PME (progressive myoclonic epilepsy), PMPS (Pearson's medullary syndrome). pancreas), psoriasis, RTT (Ret syndrome), schizophrenia, SIDS (sudden infant death), SNHL (sensory-neural hearing loss), Varied familial presentation (clinical manifestations range from spastic paraparesis and progressive multisystemic disorder, and fatal cardiomyopathy up to truncal ataxia, dysarthria, severe hearing loss, mental regression, ptosis, ophthalmoparesis, distal cyclones, and diabetes mellitus), or Wolfram syndrome.

Other diseases and disorders that would benefit from increased mitochondrial activity include, for example, Friedreich's ataxia and other ataxias, amyotrophic lateral sclerosis (ALS) and other neuromotor diseases, macular degeneration, epilepsy, Alpers syndrome, elimination syndrome. Multiple mitochondrial DNA, MtDNA deletion syndrome, Complex I deficiency, Complex II deficiency (SDH), Complex III deficiency, cytochrome oxidase (COX, Complex IV) deficiency, Complex V deficiency, adenine nucleotide translocator (ANT) deficiency, pyruvate dehydrogenase (PDH) deficiency, ethylmalonic aciduria with lactic acidemia, 3-methyl glutaconic aciduria with lactic acidemia, epilepsy refractory with declines during an infection, Asperger's syndrome with declines during an infection, autism with declines during an infection, attention deficit hyperactivity disorder (ADHD), declining cerebral palsy during infection, declining dyslexia during infection, maternal inherited thrombocytopenia and leukemia syndrome, MARIAHS syndrome (mitochondrial ataxia, recurrent infections, aphasia, hypouricemia / hypomyelination, seizures and dicarboxylic aciduria), dystonia ND6, syndrome of cyclical vomiting with declines during infection, 3-hydroxy isobutyric aciduria with lactic acidemia, diabetes mellitus with lactic acidemia, Uridine-sensitive neurological syndrome (URNS), dilated cardiomyopathy, splenic lymphoma, and renal tubular acidosis / diabetes / ataxis syndrome.

In other embodiments, the invention contemplates methods of treating a subject suffering from mitochondrial disorders arising from, but not limited to, post-traumatic head injury and brain edema, stroke (methods of the invention useful for preventing reperfusion injury ), Lewy body dementia, hepatorenal syndrome, acute liver failure, NASH (nonalcoholic steatohepatitis), antimetastasis / prodifferentiation cancer therapy, Idiopathic congestive heart failure, atrial fibrillation (nonvalvular), Wolff-Parkinson-White syndrome, idiopathic heart block, prevention of reperfusion injury in acute myocardial infarctions, familial migraines, irritable bowel syndrome, secondary prevention of myocardial infarctions no Q waves, premenstrual syndrome, prevention of renal failure in hepatorenal syndrome, anti-phospholipid antibody syndrome, eclampsia / pre-eclampsia, sterility due to poor ovarian response, ischemic heart disease / angina, and Shy-Drager and lack of autonomy syndromes not classified.

In yet another embodiment, methods for treating mitochondrial disorders associated with drug-related side effects are contemplated. The types of pharmaceutical agents associated with mitochondrial disorders include reverse transcriptase inhibitors, protease inhibitors, DHOD inhibitors, and the like. Examples of reverse transcriptase inhibitors include, for example, Azidothymidine (AZT), Stavudine (D4T), Zalcitabine (ddC), Didanosine (DDI), Fluoroiodoarauracil (FIAU), Lamivudine (3TC), Abacavir, and the like. Examples of protease inhibitors include, for example, Ritonavir, Indinavir, Saquinavir, Nelfinavir, and the like. Examples of dihydro-orthoate dehydrogenase (DHOD) inhibitors include, for example, Leflunomide, Brequinar, and the like.

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Reverse transcriptase inhibitors not only inhibit reverse transcriptase, but also gamma polymerase that is required for mitochondrial function. Inhibition of gamma polymerase activity (eg, with the reverse transcriptase inhibitor) then leads to mitochondrial dysfunction and / or a reduction in mitochondrial mass manifesting in the patient as hyperlactatemia. This type of condition can benefit from an increase in the number of mitochondria and / or an advance in mitochondrial functioning, eg. g., by administration of a sirtuin activating compound.

Common symptoms of mitochondrial diseases include cardiomyopathy, muscle weakness and atrophy, developmental delays (motor, language, cognitive, or executive), ataxia, epilepsy, renal tubular acidosis, peripheral neuropathy, optic neuropathy, autonomic neuropathy, neurogenic bowel dysfunction, Sensorineural deafness, neurogenic bladder dysfunction, dilated cardiomyopathy, migraine, liver failure, lactic acidemia, and diabetes mellitus.

In certain embodiments, the invention contemplates methods of treating a disease or disorder that would benefit from increased mitochondrial activity, which involves administering to a subject in need one or more sirtuin activating compounds in combination with another therapeutic agent such as, for example, an agent useful to treat mitochondrial dysfunction (such as antioxidants, vitamins or cofactors of the respiratory chain), an agent useful for reducing a symptom associated with a disease or disorder involving mitochondrial dysfunction (such as an anticonvulsant agent, an agent useful for relieving neuropathic pain, an agent for treating cardiac dysfunction), a cardiovascular agent (as described below in more detail), a chemotherapeutic agent (as described in more detail below) or an anti-neurodegeneration agent (as described in more detail below). In an illustrative embodiment, the invention provides methods for treating a disease or disorder that would benefit from increased mitochondrial activity, which involves administering to a subject in need, one or more sirtuin activating compounds in combination with one or more of the following: coenzyme Q10, L-carnitine, thiamine, riboflavin, niacinamide, folate, vitamin E, selenium, lipoic acid, or prednisone. Compositions comprising such combinations are also provided in the present invention.

In illustrative embodiments, the invention contemplates methods of treating diseases or disorders that would benefit from increased mitochondrial activity, by administering to a subject in need thereof, a therapeutically effective amount of a sirtuin activating compound. Illustrative diseases or disorders include, for example, neuromuscular disorders (eg, Friedreich's ataxia, muscular dystrophy, multiple sclerosis, etc.), neuronal instability disorders (eg. g., seizure disorders, migraine, etc.), developmental delay, neurodegenerative disorders (eg. g., Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, etc.), ischemia, renal tubular acidosis, neurodegeneration related to aging and cognitive impairment, chemotherapy fatigue, menopause or irregularities in the menstrual cycle or ovulation related to age or chemotherapy-induced, mitochondrial myopathies, mitochondrial damage (eg. g., calcium accumulation, excitotoxicity, exposure to nitric acid, hypoxia, etc.), and mitochondrial dysregulation.

A gene defect underlying Friedreich's ataxia (FA), the most common inherited ataxia, was recently identified and is called frataxin. In AF, after a period of normal development, coordination deficits develop that progress to paralysis and death, typically between the ages of 30 and 40. The most severely affected tissues are the spinal cord, peripheral nerves, myocardium, and pancreas. Patients typically lose motor control and are confined to wheelchairs, and typically suffer from heart failure and diabetes. The genetic basis of FA involves GAA trinucleotide repeats in an intron region of the gene encoding frataxin. The presence of these repeats produces a reduction in the transcription and expression of the gene. Frataxin is involved in the regulation of mitochondrial iron content. When cellular frataxin content is subnormal, excess iron accumulates in the mitochondria, promoting oxidative damage and consequent mitochondrial degeneration and dysfunction. When intermediate numbers of GAA repeats are present in the intron of the frataxin gene, the severe clinical phenotype of ataxia cannot develop. However, these intermediate-length trinucleotide extensions are found in 25-30% of patients with non-insulin-dependent diabetes mellitus, compared with approximately 5% of the non-diabetic population. In certain embodiments, sirtuin activating compounds can be used to treat patients with disorders related to frataxin deficiencies or defects, including Friedreich's ataxia, myocardial dysfunction, diabetes mellitus, and complications of diabetes such as peripheral neuropathy.

Muscular dystrophy refers to a family of diseases that involve impaired neuromuscular structure and function, often resulting in musculoskeletal atrophy and myocardial dysfunction. In the case of Duchenne muscular dystrophy, mutations or deficits in a specific protein are implicated in its etiology. Mice with their dystrophin genes inactivated exhibit some characteristics of muscular dystrophy and have approximately 50% deficits in mitochondrial respiratory chain activity. A final common pathway for neuromuscular degeneration in most cases is calcium-mediated impairment of mitochondrial function. In certain embodiments, sirtuin activating compounds can be used to reduce the rate of impairment in muscle functional abilities and to improve muscle functional status in patients with muscular dystrophy.

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Multiple sclerosis (MS) is a neuromuscular disease characterized by focal inflammatory and autoimmune degeneration of brain white matter. Periodic exacerbations or attacks are significantly correlated with upper respiratory tract and other infections, both bacterial and viral, indicating that mitochondrial dysfunction plays a role in MS. Depression of neuronal mitochondrial respiratory chain activity caused by nitric oxide (produced by astrocytes and other cells involved in inflammation) is implicated as a molecular mechanism that contributes to SD. In certain embodiments, sirtuin activating compounds can be used for the treatment of patients with multiple sclerosis, both prophylactically and during disease exacerbation episodes.

Epilepsy is frequently present in patients with mitochondrial cytopathies, involving a range of seizure frequency and intensity, e.g. eg, absence, tonic, atonic, myoclonic, and status epilepticus, occurring in isolated episodes or many times a day. In certain embodiments, sirtuin activating compounds can be used to treat patients with seizures secondary to mitochondrial dysfunction, including reducing the intensity and frequency of seizure activity.

Metabolic studies in patients with recurrent migraines indicate that deficits in mitochondrial activity are frequently associated with this disorder, manifesting as impaired oxidative phosphorylation and excess lactate production. These deficits are not necessarily due to genetic defects in mitochondrial DNA. People with migraines are hypersensitive to nitric oxide, an endogenous cytochrome c oxidase inhibitor. Furthermore, patients with mitochondrial cytopathies, e.g. eg, MELAS, often suffer from recurrent migraines. In certain embodiments, sirtuin activating compounds can be used to treat patients with recurrent migraines, including headaches resistant to ergot compounds or serotonin receptor antagonists.

Neuropsychological or neurodevelopmental delays are often seen in children with mitochondrial diseases. The development and remodeling of neural connections require intensive biosynthetic activity, particularly involving synthesis of neuronal membranes and myelin, both of which require pyrimidine nucleotides as cofactors. Uridine nucleotides are involved in the inactivation and transfer of sugars to glycolipids and glycoproteins. Cytidine nucleotides are derived from uridine nucleotides, and are crucial for the synthesis of important membrane phospholipid constituents such as phosphatidylcholine, which receives its choline residue from cytidine diphosphocholine. In the case of mitochondrial dysfunction (due either to defects in mitichondrial DNA or to any of the acquired or conditional deficits such as nitric oxide-mediated or exicytoxic mitochondrial dysfunction) or other conditions that result in impaired pyrimidine synthesis, cell proliferation and axonal extension are impaired at crucial stages in the development of neural interconnections and circuits, which produces a delay or delay in the development of neuropsychological functions such as language, motor function, executive, and cognitive abilities. In autism, for example, magnetic resonance spectroscopic measurements of brain phosphate compounds indicate that there is a global subsynthesis of membranes and membrane precursors indicated by reduced levels of uridine diphospho-sugars, and cytidine nucleotide derivatives involved in the synthesis of the membranes. Disorders characterized by developmental delay include Rett syndrome, generalized developmental delay (or PDD-NOS unspecified generalized developmental delay to distinguish it from specific subcategories such as autism), autism, Asperger syndrome, and attention deficit / hyperactivity disorder (ADHD), which is being recognized as a delay in the development of the activity of the neural circuit underlying executive functions. In certain embodiments, sirtuin activating compounds may be useful for treating patients with neurodevelopmental delays (eg, motor, language, executive function, and cognitive skills), or other neurodevelopmental and neuropsychological delays in the brain. central nervous system and somatic development in non-neural tissues such as muscle and endocrine glands.

The two most significant severe neurodegenerative diseases associated with aging, Alzheimer's disease (AD) and Parkinson's disease (PD), involve mitochondrial dysfunction in their pathogenesis. Complex I deficiencies in particular are frequently found not only in nigrostriatal neurons that degenerate in Parkinson's disease, but also in peripheral tissues and in cells such as muscle and platelets of patients suffering from Parkinson's disease. In Alzheimer's disease, the activity of the mitochondrial respiratory chain is often depressed, especially that of Complex IV (Cytochrome c Oxidase). In addition, mitochondrial respiratory function is depressed as a result of aging, further amplifying the deleterious sequelae of additional molecular damage that affect respiratory chain function. Factors other than primary mitochondrial dysfunction underlie neurodegeneration in AD, PD, and related disorders. Excitotoxic stimulation and nitric oxide are implicated in both diseases, the factors of which exacerbate deficits in the mitochondrial respiratory chain and whose damaging actions are exaggerated in a history of respiratory chain dysfunction. Huntington's disease also involves mitochondrial dysfunction in affected regions of the brain, with cooperative interactions of excitotoxic stimulation and mitochondrial dysfunction, contributing to neuronal degeneration. In certain embodiments, sirtuin activator compounds may be useful for treating and attenuating the progression of aging-related neurodegenerative diseases, including AD and PD.

One of the main genetic defects in amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) is a mutation or deficiency in copper-zinc superoxide dismutase (SOD 1), an antioxidant enzyme. 59

ES 2 396 913 T3 mitochondria produce and are primary targets for reactive oxygen species. Inefficient transfer of electrons to oxygen in mitochondria is the most significant physiological source of free radicals in mammalian systems. Deficiencies of antioxidants or antioxidant enzymes can result in or exacerbate mitochondrial degeneration. Mice transgenic for mutated SOD1 develop symptoms and pathology similar to those in human ALS. The development of the disease in these animals has been shown to involve oxidative destruction of mitochondria, followed by functional impairment of motor neurons and the onset of clinical symptoms. The skeletal muscle of patients with ALS shows little activity of the mitochondrial Complex I. In certain embodiments, sirtuin activating compounds may be useful to treat ALS, to reverse or delay the progression of clinical symptoms.

Oxygen deficiency produces direct inhibition of the activity of the mitochondrial respiratory chain, depriving cells of a terminal electron acceptor for reoxidation of cytochrome c in Complex IV, and indirectly, especially in the nervous system, through secondary post-anoxic excitotoxicity and nitric oxide formation. In conditions such as cerebral anoxia, angina, or sickle cell crisis, the tissues are relatively hypoxic. In such cases, compounds that can increase mitochondrial activity protect tissues from the deleterious effects of hypoxia, attenuate delayed secondary cell death, and accelerate recovery from stress and hypoxic tissue injury. In certain embodiments, sirtuin activating compounds may be useful in preventing delayed cell death (apoptosis in regions such as the hippocampus or cortex, which occurs approximately 2 to 5 days after an episode of cerebral ischemia) after ischemic or hypoxic attack. to the brain.

Acidosis due to renal dysfunction is frequently seen in patients with mitochondrial disease, whether the underlying respiratory chain dysfunction is congenital or induced by ischemia or cytotoxic agents such as cisplatin. Renal tubular acidosis often requires exogenous sodium bicarbonate to maintain tissue and blood pH. In certain embodiments, sirtuin activating compounds may be useful for treating renal tubular acidosis and other forms of renal dysfunction caused by deficits in the mitochondrial respiratory chain.

During normal aging, there is a progressive deterioration in the functioning of the mitochondrial respiratory chain. Beginning at approximately age 40, there is an exponential increase in the accumulation of mitochondrial DNA defects in humans, and a concurrent impairment in the nuclear regulated elements of mitochondrial respiratory activity. Many mitochondrial DNA lesions have a selection advantage during mitochondrial turnover, especially in postmitotic cells. The proposed mechanism is that mitochondria with a defective respiratory chain produce less oxidative damage to themselves than mitochondria with intact functional respiratory chains (mitochondrial respiration is the primary source of free radicals in the body). Therefore, normally functioning mitochondria accumulate oxidative damage to membrane lipids more rapidly than defective mitochondria, and are therefore tagged for degradation by liposomes. Since mitochondria within cells have a half-life of approximately 10 days, a selection advantage may result in rapid replacement of functional mitochondria with those with decreased respiratory activity, especially in slowly dividing cells. The net result is that once a mutation occurs in a gene for a mitochondrial protein that reduces oxidative damage to mitochondria, those defective mitochondria will rapidly populate the cell, diminishing or eliminating its respiratory capabilities. The accumulation of these cells produces aging or degenerative disease in the body. This is consistent with the progressive mosaic appearance of cells with defective electron transport activity in muscle, where cells lack Cytochrome oxidase c (COX) activity intertwined between cells with normal activity, and a higher incidence of COX cells. -negative in biopsies of older subjects. The organism, during aging, or in a variety of mitochondrial diseases, is therefore faced with a situation in which irreplaceable postmitotic cells (e.g. neurons, skeletal and cardiac muscle) must be preserved and their function maintained until a significant degree, in view of an inexorable progressive deterioration in the functioning of the mitochondrial respiratory chain. Neurons with dysfunctional mitochondria become progressively more sensitive to attacks, such as excitotoxic injury. Mitochondrial failure contributes to most of the degenerative diseases (especially neurodegeneration) that accompany aging. Congenital mitochondrial diseases often involve early-onset neurodegeneration similar in fundamental mechanism to disorders that occur during aging in people born with normal mitochondria. In certain embodiments, sirtuin activator compounds may be useful for treating or attenuating cognitive decline and other degenerative consequences of aging.

Damage to mitochondrial DNA is more extensive and persists longer than damage to nuclear DNA in cells subjected to oxidative stress or cancer chemotherapeutic agents, such as cisplatin, due to greater vulnerability and less efficient repair of mitochondrial DNA. Although mitochondrial DNA may be more sensitive to damage than nuclear DNA, it is relatively resistant, in some situations, to mutagenesis by chemical carcinogens. This is because mitochondria respond to some types of mitochondrial DNA damage by destroying their faulty genomes rather than trying to repair them. This produces global mitochondrial dysfunction for a period after cytotoxic chemotherapy. Clinical use of chemotherapeutic agents such as cisplatin, mitomycin, and cytoxan is often accompanied by fatigue from debilitating chemotherapy, periods

ES 2 396 913 T3 of prolonged weakness and intolerance to exercise, which can persist even after recovery from hematological and gastrointestinal toxicities of the toxicities of said agents. In certain embodiments, sirtuin activating compounds may be useful for the treatment and prevention of mitochondrial dysfunction-related cancer chemotherapy side effects.

A crucial function of the ovary is to maintain the integrity of the mitochondrial genome in oocytes, since the mitochondria that pass to a fetus are all derived from those present in the oocytes at the time of conception. Deletions in mitochondrial DNA become detectable around the age of menopause, and are also associated with abnormal menstrual cycles. Since cells cannot directly detect and respond to defects in mitochondrial DNA, but can only detect secondary defects that affect the cytoplasm, such as difficulty in breathing, oxy-induction states or deficits in the synthesis of pyrimidine, these products of mitochondrial function are involved as a signal for oocyte selection and follicular atresia, ultimately triggering menopause when maintenance of genomic fidelity and functional mitochondrial activity can no longer be guaranteed. This is analogous to apoptosis in DNA-damaged cells, which undergo an active process of cell suicide when genomic fidelity can no longer be achieved with repair processes. Women with mitochondrial cytopathies affecting the gonads often experience premature menopause or exhibit abnormalities in the primary cycle. Cytotoxic chemotherapy for cancer often induces premature menopause, with a consequent increased risk of osteoporosis. Chemotherapy-induced amenorrhea is generally due to ovarian failure. The incidence of chemotherapy-induced amenorrhea increases as a function of age in premenopausal women receiving chemotherapy, indicating a mitochondrial compromise. Inhibitors of mitochondrial respiration or protein synthesis inhibit hormone-induced ovulation and further inhibit the production of ovarian steroid hormones in response to pituitary gonadotropins. Women with Down syndrome typically experience menopause prematurely, and are also subject to an early onset of dementia of the Alzheimer's type. Reduced cytochrome oxidase activity is consistently found in tissues from patients with Down syndrome and late-onset Alzheimer's disease. Proper support of mitochondrial function or compensation for mitochondrial dysfunction is therefore useful to protect against aging-related or chemotherapy-induced menstrual cycle or ovulation irregularities or menopause. In certain embodiments, sirtuin activating compounds may be useful for treating and preventing amenorrhea, irregular ovulation, menopause, or secondary consequences of menopause.

In certain embodiments, sirtuin activator compounds may be useful for the treatment of mitochondrial myopathies. Mitochondrial myopathies range from mild, slowly progressive extraocular muscle weakness to fatal, severe childhood myopathies and multisystemic encephalomyopathies. Some syndromes have been defined, with some overlap between them. Established syndromes affecting the muscles include progressive external ophthalmoplegia, Kearns-Sayre syndrome (with ophthalmoplegia, pigmentary retinopathy, cardiac conduction defects, cerebellar ataxia, and sensorineural deafness), MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) , MERFF syndrome (myoclonic and ragged red fiber epilepsy), limb-waist distribution weakness and infantile myopathy (benign or severe and fatal). Muscle biopsy specimens stained with modified Gomori trichrome stain show ragged red fibers due to excessive accumulation of mitochondria. Biochemical defects in substrate transport and utilization, the Krebs cycle, oxidative phosphorylation, or the respiratory chain can be detected. Numerous mutations and point deletions of mitochondrial DNA have been described, transmitted in a maternal, non-Mendelian, inherited pattern. Mutations occur in nuclear-encoded mitochondrial enzymes.

In certain embodiments, sirtuin activating compounds may be useful in treating patients suffering from toxic damage to the mitochondria, such as toxic damage due to calcium accumulation, excitotoxicity, nitric acid exposure, drug-induced toxic damage, or hypoxia.

A fundamental mechanism of cell injury, especially in excitable tissues, involves the excessive influx of calcium into cells, as a consequence of either a leak through the plasma membrane or defects in the mechanisms for handling intracellular calcium. Mitochondria are important sites of calcium sequestration, and preferably use energy from the respiratory chain to absorb calcium, rather than ATP synthesis, resulting in a downward spiral of mitochondrial failure, as calcium absorption in the cells mitochondria produces decreased energy transduction capacity.

Excessive stimulation of neurons with excitatory amino acids is a common mechanism for the death or injury of cells of the central nervous system. Activation of glutamate receptors, especially the subtype designated NMDA receptors, produces mitochondrial dysfunction, in part through elevation of intracellular calcium during excitotoxic stimulation. Conversely, deficits in mitochondrial respiration and oxidative phosphorylation sensitize cells to excitotoxic stimuli, leading to cell death or injury during exposure to levels of excitotoxic neurotransmitters or toxins that would be harmless to normal cells.

Nitric oxide (approximately 1 micromolar) inhibits cytochrome oxidase (Complex IV) and consequently mitochondrial respiration; furthermore, prolonged exposure to nitric oxide (NO) irreversibly reduces the activity of Complex I. Physiological or pathophysiological concentrations of NO thus inhibit pyrimidine biosynthesis. The

ES 2 396 913 T3 Nitric oxide is involved in a variety of neurodegenerative disorders including inflammatory and autoimmune diseases of the central nervous system, and is involved in mediating excitotoxic and post-hypoxic damage to neurons.

Oxygen is the terminal electron acceptor in the respiratory chain. Oxygen deficiency hampers the activity of the electron transport chain, producing less pyrimidine synthesis as well as less ATP synthesis by oxidative phosphorylation. Human cells proliferate and retain viability under practically anaerobic conditions, if uridine and pyruvate (or a similar agent effective in oxidizing NADH to optimize glycolytic ATP production) are provided.

In certain embodiments, sirtuin activator compounds may be useful for treating diseases or disorders associated with mitochondrial dysregulation.

The transcription of mitochondrial DNA encoding the components of the respiratory chain requires nuclear factors. In neuronal axons, mitochondria must travel to and from the nucleus in order to maintain respiratory chain activity. If axonal transport is hampered by hypoxia or by drugs such as taxol that affect microtubule stability, mitochondria distant from the nucleus suffer loss of cytochrome oxidase activity. Therefore, treatment with a sirtuin activator compound may be useful in promoting nuclear mitochondrial interactions.

Mitochondria are the primary source of free radicals and reactive oxygen species, due to overflow from the mitochondrial respiratory chain, especially when defects in one or more components of the chain impede the orderly transfer of electrons from metabolic intermediates to molecular oxygen. To reduce oxidative damage, cells compensate by expressing mitochondrial uncoupling proteins (UCP), of which several have been identified. UCP-2 is transcribed in response to oxidative damage, inflammatory cytokines, or excess lipid loads, e.g. eg, fatty liver and steatohepatitis. UCPs reduce the overflow of reactive oxygen species from the mitochondria, discharging proton gradients across the inner mitochondrial membrane, in effect wasting the energy produced by metabolism and rendering cells vulnerable to energy stress in exchange for reduced oxidative injury.

Muscle performance

In other embodiments, the invention contemplates methods of improving muscle performance by administering an effective amount of a sirtuin activating compound. For example, sirtuin activating compounds may be useful for improving physical endurance (e.g. g., the ability to perform a physical task such as exercise, physical work, sports activities, etc.), inhibiting or delaying physical fatigue, improving blood oxygen levels, improving energy in healthy people, improving capacity work and endurance, reducing muscle fatigue, reducing stress, increasing cardiac and cardiovascular function, improving sexual capacity, increasing muscle ATP levels and / or reducing lactic acid in the blood. In certain embodiments , the methods involve administering an amount of a sirtuin activating compound that increases mitochondrial activity, increases mitochondrial biogenesis, and / or increases mitochondrial mass.

Sports performance refers to the capacity of the athlete's muscles when participating in sports activities. Increased athletic performance, strength, speed and endurance are measured by an increase in the force of muscle contraction, an increase in the amplitude of the muscle contraction, a shortening in the reaction time of the muscle between stimulation and contraction. Athlete refers to an individual who participates in sports at any level and who seeks to achieve a better level of strength, speed, and endurance in their performance, such as bodybuilders, cyclists, long-distance runners, short-distance runners, etc. An athlete may train hard, that is, do intense sports activities more than three days a week or for competitions. An athlete can also be a gym enthusiast, looking to improve general health and well-being, improve energy levels, train for about 1-2 hours about 3 times a week. The increase in sports performance is manifested by the ability to overcome muscle fatigue, maintain activity for longer periods of time and have a more effective training.

In the field of muscular performance of an athlete, it is desired to create conditions that allow competition or training at higher levels of resistance for a prolonged period of time. However, acute and intense anaerobic use of skeletal muscles often results in impaired athletic performance, with loss of strength and energy, increased onset of muscle fatigue, inflammation, and dysfunction. It is now recognized that even a single session of exhaustive exercise, or any acute trauma to the body such as muscle injury, resistance or exhaustive muscular exercise, or elective surgery, is characterized by disturbed metabolism that affects muscle performance in both the short-term phase. as long term. Both the metabolic / enzymatic activity of the muscle and the gene expression are affected. For example, breakdown of skeletal muscle nitrogen metabolism as well as reduction of metabolic energy sources occur during extensive muscle activity. Amino acids, including branched chain amino acids, are released from muscles followed by deamination to elevate serum ammonia and local oxidation as a source of muscle fuel, increasing metabolic acidosis. In addition, there is a deterioration in the catalytic efficiency of muscle contraction events, as well as an alteration in the enzymatic activities of the muscle.

ES 2 396 913 T3 nitrogen and energy metabolism. Likewise, protein catabolism is initiated if the rate of protein synthesis is reduced coupled with an increase in non-contractable protein degradation. These metabolic processes are also accompanied by the generation of free radicals, which also damage muscle cells.

Recovery from fatigue during acute and extended exercise requires the reversal of metabolic and non-metabolic fatigue factors. Known factors involved in human muscle fatigue, such as lactate, ammonia, hydrogen ions, etc., provide an incomplete and unsatisfactory explanation of the fatigue / recovery process, and other unknown agents are likely to be involved (Baker et al. , J. Appl. Physiol. 74: 22942300, 1993; Bazzarre et al., J Am. Coll. Nutr. 11: 505-511, 1992; Dohm et al., Fed. Proc. 44: 348-352, 1985; Edwards in: Biochemistry of Exercise, Proceedings of the Fifth International Symposium on the Biochemistry of Exercise (Kutrgen, Vogel, Poormans, eds.), 1983; MacDougall et al., Acta Physiol. Scand. 146: 403-404, 1992; Walser et al., Kidney Int. 32: 123-128, 1987). Several studies have also looked at the effects of nutritional supplements and herbal supplements to increase muscle performance.

Apart from muscular performance during endurance exercise, free radicals and oxidative stress parameters are affected in pathophysiological states. A substantial body of data now indicates that oxidative stress contributes to progressive muscle atrophy in pathophysiological states (reviewed in Clarkson, PM Antioxidants and physical performance. Crit. Rev. Food Sci. Nutr. 35: 31-41; 1995; Powers, SK; Lennon, SL Analysis of cellular responses to free radicals: Focus on exercise and skeletal muscle. Proc. Nutr. Soc. 58: 10251033; 1999). For example, with regard to muscle disorders in which both endurance and muscle function are compensated, the role of nitric oxide (NO) has not been implicated. In muscular dystrophies, especially those due to defects in proteins that make up the dystrophin-glycoprotein complex (DGC), it has been associated with the enzyme that synthesizes NO, nitric oxide synthase (NOS). Recent studies of dystrophies related to DGC defects indicate that a mechanism of cellular injury is functional ischemia related to alterations in cellular NOS and disruption of a normal protective action of NO. This protective action is the prevention of local ischemia during contraction-induced increases in sympathetic vasoconstriction. Rando (Microsc Res Tech 55 (4): 223-35, 2001), has shown that oxidative injury precedes pathological changes and that muscle cells with defects in DGC are more susceptible to oxidative challenges. Excessive lipid peroxidation due to free radicals was also shown to be a factor in myopathic diseases such as McArdle's disease (Russo et al., Med Hypotheses. 39 (2): 147-51, 1992). Likewise, mitochondrial dysfunction is known to correlate with progressive aging-related muscle atrophy (sarcopenia) and free radical damage has been suggested, although little investigated, as a contributing factor (reviewed in Navarro, A .; Lopez-Cepero, JM; Sanchez del Pino, ML Front. Biosci. 6: D26-44; 2001). Other indications include acute sarcopenia, for example muscle atrophy and / or cachexia associated with burns, bed rest, immobilization of a limb, or major thoracic, abdominal and / or orthopedic surgery. It is contemplated that the methods of the present invention will also be effective in treating muscle-related conditions.

In certain embodiments, the invention provides new dietary compositions comprising modulators of sirtuin, a method for their preparation, and a method for using the compositions to enhance athletic performance. Accordingly, therapeutic compositions, foods and beverages are provided which have actions that enhance physical endurance and / or inhibit physical fatigue of those engaged in broadly defined exercises, including sports that require endurance, and physical work that requires muscular effort. Said dietary compositions can comprise electrolytes, caffeine, vitamins, carbohydrates, etc.

Other uses

Sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can be used to treat or prevent viral infections (such as influenza, herpes, or papilloma virus infections) or as antifungal agents. In certain embodiments, sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can be administered as part of a combination drug therapy with another therapeutic agent for the treatment of viral diseases, including for example acyclovir, ganciclovir and zidovudine. In another embodiment, sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can be administered as part of a combination drug therapy with another antifungal agent, including for example topical antifungals such as ciclopirox, clotrimazole, econazole. , miconazole, nystatin, oxiconazole, terconazole and tolnaftate, or systemic antifungals such as fluconazole (Diflucan), itraconazole (Sporanox), ketoconazole (Nizoral) and miconazole (Monistat IV).

Subjects that can be treated as described herein include eukaryotes, such as mammals, e.g. eg, humans, sheep, horses, pigs, canines, felines, non-human primates, mice, and rats. Cells that can be treated include eukaryotic cells, e.g. g., from a subject described above, or plant cells, yeast cells, and prokaryotic cells, e.g. eg, bacterial cells. For example, modulator compounds can be administered to farm animals to improve their ability to tolerate farm conditions longer.

ES 2 396 913 T3

Sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can also be used to increase life, stress resistance and resistance to apoptosis in plants. In one embodiment, a compound is applied to plants, e.g. eg, on a periodic basis, or to mushrooms. In another embodiment, the plants are genetically modified to produce a compound. In another embodiment, the plants and fruits are treated with a compound prior to harvesting and shipping to increase resistance to damage during shipping. Plant seeds can also be contacted with the compounds described herein, e.g. For example, to keep them.

In other embodiments, sirtuin modulator compounds that increase the level and / or activity of a sirtuin protein can be used to modulate life in yeast cells. Situations where it may be desirable to extend the life of yeast cells include any process where yeast is used, e.g. eg brewing beer, yogurt, and bakery items, eg. eg, bread. Using yeast with an extended life may result in less yeast usage or may make yeast more active for longer periods of time. Yeast or other mammalian cells used to produce proteins can also be treated as described herein.

Sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can also be used to increase life, stress resistance and resistance to apoptosis in insects. In this embodiment, the compounds would be applied to useful insects, e.g. eg, bees and other insects that are involved in the pollination of plants. In a specific embodiment, a compound would be applied to bees involved in the production of honey. In general, the methods described herein can be applied to any organism, e.g. eg, eukaryote, which may be of commercial importance. For example, they can be applied to fish (aquaculture) and birds (eg, chicken and poultry).

Higher doses of sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can also be used as a pesticide, interfering with the regulation of silenced genes and with the regulation of apoptosis during development. In this embodiment, a compound can be applied to plants using a method known in the art that ensures that the compound is bioavailable to insect larvae and not to plants.

At least in view of the connection between reproduction and longevity (Longo and Finch, Science, 2002), sirtuin modulating compounds that increase the level and / or activity of a sirtuin protein can be applied to effect the reproduction of organisms. such as insects, animals and microorganisms.

essays

Still other methods contemplated herein include screening methods to identify compounds or agents that modulate sirtuins. An agent can be a nucleic acid, such as an aptamer. Assays can be performed in a cell-based or cell-free format. For example, an assay may comprise incubating (or contacting) a sirtuin with a test agent under conditions in which a sirtuin can be modulated by an agent known to modulate sirtuin, and monitoring or determining the level of modulation of sirtuin in presence of the test agent relative to the absence of the test agent. The level of modulation of a sirtuin can be determined by determining its ability to deacetylate a substrate. Illustrative substrates are acetylated peptides available from BiOMOL (Plymouth Meeting, PA). Preferred substrates include p53 peptides, such as those comprising an acetylated K382. A particularly preferred substrate is Fluor de Lys-SIRT1 (BIOMOL), that is, the acetylated peptide Arg-His-Lys-Lys. Other substrates are peptides of human histones H3 and H4 or an acetylated amino acid. Substrates can be fluorogenic. The sirtuin can be SIRT1, Sir2, SIRT3, or portions thereof. For example, recombinant SIRT1 can be obtained from BIOMOL. The reaction can be carried out for about 30 minutes and stopped, e.g. eg with nicotinamide. The HDAC Fluorescent Activity Assay / Drug Discovery Kit (AK-500, BiOmOl Research Laboratories) can be used to determine the level of acetylation. Similar trials are described in Bitterman et al. (2002) J. Biol. Chem. 277: 45099. The level of modulation of sirtuin in an assay can be compared to the level of modulation of sirtuin in the presence of one or more compounds (separately or simultaneously) described in the present invention, which can serve as positive or negative controls. The sirtuins for use in the assays can be full length sirtuin proteins or portions thereof. Since it has been shown herein that activator compounds appear to interact with the N-terminus of SIRT1, proteins for use in the assays include N-terminal portions of sirtuins, e.g. eg, about 1-176 or 1255 amino acids of SIRT1; about 1-174 or 1-252 amino acids of Sir2.

In one embodiment, a screening assay comprises (i) contacting a sirtuin with a test agent and an acetylated substrate under conditions appropriate for the sirtuin to deacetylate the substrate in the absence of the test agent; and (ii) determining the level of acetylation of the substrate, where a lower level of acetylation of the substrate in the presence of the test agent relative to the absence of the test agent indicates that the test agent stimulates deacetylation by sirtuin, whereas a higher level of acetylation of the substrate in the presence of the test agent relative to the absence of the test agent indicates that the test agent inhibits deacetylation by sirtuin.

ES 2 396 913 T3

Methods for identifying a modulating agent, e.g. For example, stimulate or inhibit, sirtuins in vivo may comprise (i) contacting a cell with a test agent and a substrate capable of entering a cell in the presence of a class I and class II HDAC inhibitor under conditions appropriate to that sirtuin deacetylates the substrate in the absence of the test agent; and (ii) determining the level of acetylation of the substrate, where a lower level of acetylation of the substrate in the presence of the test agent relative to the absence of the test agent indicates that the test agent stimulates deacetylation by sirtuin, while that a higher level of acetylation of the substrate in the presence of the test agent relative to the absence of the test agent indicates that the test agent inhibits deacetylation by sirtuin. A preferred substrate is an acetylated peptide, which is also preferably fluorogenic, as described in more detail herein. The method further comprises lysing the cells to determine the level of acetylation of the substrate. Substrates can be added to cells in a concentration ranging from about 1 pM to about 10mM, preferably from about 10pM to 1mM, even more preferably from about 100pM to 1mM, such as about 200pM. A preferred substrate is an acetylated lysine, e.g. eg, ε-acetyl lysine (Fluor de Lys, FdL) or Fluor de Lys-SIRT1. A preferred class I and class II HDAC inhibitor is trichostatin A (TSA), which can be used at concentrations ranging from about 0.01 to 100pM, preferably from about 0.1 to 10pM, such as 1pM. Incubation of cells with the test compound and the substrate can be carried out for about 10 minutes to 5 hours, preferably for about 1-3 hours. Since TSA inhibits all class I and class II HDACs, and that certain substrates, e.g. g., Fluor de Lys, are deficient for SIRT2 and even worse for SIRT3-7, such an assay can be used to identify modulators of SIRT1 in vivo.

5. Pharmaceutical compositions

The sirtuin modulator compounds described herein can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. For example, sirtuin modulator compounds and their physiologically acceptable salts and solvates can be formulated for administration, for example, by injection (e.g. g., SubQ, IM, IP), inhalation or insufflation (either by mouth or nose) or oral, buccal, sublingual, transdermal, nasal, parenteral or rectal administration. In one embodiment, a sirtuin modulator compound can be administered locally, at the site where the target cells are present, ie, in a specific tissue, organ, or fluid (eg, blood, cerebrospinal fluid, etc.).

Sirtuin modulator compounds can be formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations in general can be found in Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA. For parenteral administration, an injection is preferred, which can be intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the compounds can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the compounds can be formulated in solid form and redissolved or suspended immediately prior to use. Also included are lyophilized forms.

For oral administration, the pharmaceutical compositions may take the form, for example, of tablets, lozenges or capsules prepared by conventional means with pharmaceutically acceptable excipients, such as binding agents (for example, pregelatinized cornstarch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); bulking agents (eg, lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (eg, magnesium stearate, talc, or silica); disintegrants (eg potato starch or sodium starch glycolate); or wetting agents (eg, sodium lauryl sulfate). Tablets can be coated according to methods well known in the art. Liquid preparations for oral administration may take the form, for example, of solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives, such as suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (eg lectin or acacia); non-aqueous vehicles (eg, almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (eg, methyl- or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, color and sweetening agents as appropriate. Preparations for oral administration can be suitably formulated to give controlled release of the active compound.

For administration by inhalation (eg, pulmonary administration), the sirtuin modulating compounds may conveniently be administered in the form of an aerosol spray presentation from pressurized containers or from a nebulizer, with the use of a suitable propellant, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.

ES 2 396 913 T3

The sirtuin modulator compounds can be formulated for parenteral administration by injection, e.g. eg, rapid intravenous injection or continuous infusion. Formulations for injections may be presented in unit dosage form, eg, in ampoules, or in multidose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg sterile pyrogen-free water, prior to use.

The sirtuin modulator compounds can also be formulated in rectal compositions such as suppositories or retention enemas, containing for example conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations previously described, the sirtuin modulator compounds can also be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (eg, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, sirtuin modulating compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or with ion exchange resins, or as very poorly soluble derivatives, for example as a highly soluble salt. slightly soluble. The controlled-release formula also includes patches.

In certain embodiments, the compounds described herein can be formulated for administration to the central nervous system (CNS) (reviewed in Begley, Pharmacology &amp; Therapeutics 104: 29-45 (2004)). Conventional approaches to CNS drug delivery include: neurosurgical strategies (eg, intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (p. g., production of a chimeric fusion protein comprising a transport peptide that has affinity for an endothelial cell surface molecule in combination with an agent that is itself capable of crossing the BBB) in an attempt to exploit one of the endogenous transport routes of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (eg. g., conjugation of water soluble agents to cholesterol or lipid carriers); and the transient disruption of BBB integrity by hyperosmotic rupture (resulting from infusion of mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide).

One possibility to achieve sustained release kinetics is to embed or encapsulate the active compound in nanoparticles. The nanoparticles can be administered as a powder, as a mixture of powder with added excipients, or as suspensions. Colloidal nanoparticle suspensions can be easily administered through a small diameter cannula.

Nanoparticles are particles with a diameter from about 5 nm to about 1000 nm. The term nanoparticles, as used hereinafter, refers to particles formed by a polymeric matrix in which the active compound is dispersed, also known as nanospheres, and also refers to nanoparticles that are composed of a core containing the active compound, surrounded by a polymeric membrane, also known as nanocapsules. In certain embodiments, nanoparticles having a diameter of from about 50 nm to about 500 nm, in particular from about 100 nm to about 200 nm, are preferred.

Nanoparticles can be prepared by in situ polymerization of dispersed monomers or by using preformed polymers. Since polymers that are prepared in situ are often not biodegradable and / or contain highly toxic by-products, preformed polymers are preferred. Preformed polymer nanoparticles can be prepared by different techniques, e.g. eg, by emulsion evaporation, solvent displacement, salt removal, mechanical grinding, microprecipitation, and emulsification diffusion.

With the methods described above, nanoparticles can be formed with various types of polymers. For use in the method of the present invention, nanoparticles made from biocompatible polymers are preferred. The term "biocompatible" refers to material that after introduction into a biological environment has no significant effects on the biological environment. Of the biocompatible polymers, those polymers which are also biodegradable are especially preferred. The term "biodegradable" refers to material that after introduction into a biological environment degrades enzymatically or chemically into smaller molecules, which can subsequently be removed. Examples are hydroxycarboxylic acid polyesters such as poly (lactic acid) (PLA), poly (glycolic acid) (PGA), polycaprolactone (PCL), copolymers of lactic acid and glycolic acid (PLGA), copolymers of lactic acid and caprolactone, polyepsilon caprolactone, polyhydroxy butyric acid and poly (ortho) esters, polyurethanes, polyanhydrides, polyacetals, polydihydropyrans, polycyanoacrylates, natural polymers such as alginate and other polysaccharides, including dextran and cellulose, collagen and albumin.

Suitable surface modifiers can preferably be selected from known organic and inorganic pharmaceutical excipients. Such excipients include various polymers, low molecular weight oligomers, natural products, and surfactants. Preferred surface modifiers include nonionic and ionic surfactants. Representative examples of surface modifiers include gelatin, casein, lecithin

ES 2 396 913 T3 (phosphatides), gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glycerol monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers, e.g. eg, macrogol esters such as ketomacrogol 1000, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, e.g. For example, the commercially available TweensTM polyethylene glycols, polyoxyethylene stearates, colloidal silicon dioxide, phosphates, sodium dodecyl sulfate, calcium carboxymethylcellulose, sodium carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose hydroxypropylmethylcellulose, hydroxypropylcellulose crystalline phosphate and aluminum trioxypropylcellulose, magnesium hydroxypropylcellulosephthalate, aluminum hydroxypropylcellulosephthalate , polyvinyl alcohol and polyvinylpyrrolidone (PVP). Most of these surface modifiers are known as pharmaceutical excipients and are described in detail in the Handbook of Pharmaceutical Excipients, jointly published with the American Pharmaceutical Association and The Pharmaceutical Society of Great Britain, the Pharmaceutical Press, 1986.

Another description of how to prepare nanoparticles can be found, for example, in US Patent No. 6,264,922.

Liposomes are another drug delivery system that is easily injectable. Thus, in the method of the invention the active compounds can also be administered in the form of a liposome delivery system. Liposomes are known to those skilled in the art. Liposomes can be formed from a variety of phospholipids such as cholesterol, stearylamine, or phosphatidylcholines. The liposomes usable for the method of the invention encompass all types of liposomes, including without limitation, small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles.

Liposomes are used for a variety of therapeutic purposes and, in particular, to transport therapeutic agents to target cells. Advantageously, liposome drug formulations offer the potential for improved drug delivery properties, including, for example, controlled drug release. An extension of the circulation time is often required for liposomes to reach a target region, cell or site. In particular, this is necessary when the target region, cell or site is not located close to the site of administration. For example, when liposomes are administered systemically, it is desirable to coat the liposomes with a hydrophilic agent, eg, a coating of hydrophilic polymer chains such as polyethylene glycol (PEG) to extend the blood circulation time of the liposomes. Such surface modified liposomes are commonly referred to as sterically stabilized or long circulating liposomes.

A surface modification to a liposome is the attachment of PEG chains, typically having a molecular weight of from about 1000 daltons (Da) to about 5000 Da, and up to about 5 mole percent (%) of the lipids that make up the liposomes (see, for example, Stealth Liposomes, CRC Press, Lasic, D. and Martin, F., eds., Boca Raton, Fla., (1995)), and references cited therein. The pharmacokinetics exhibited by such liposomes are characterized by a dose-dependent reduction in the absorption of the liposomes by the liver and spleen by the mononuclear phagocyte system (MPS), and a significantly prolonged circulation time in the blood, in comparison with liposomes without surface modification, which tend to be rapidly cleared from the blood and accumulate in the liver and spleen.

In certain embodiments, the complex is protected to increase the circulatory half-life of the complex, or it is protected to increase the resistance of nucleic acid to degradation, eg, degradation by nucleases.

As used herein, protect and its cognates, such as protected, refers to the ability of the protective moieties to reduce the non-specific interaction of the complexes described herein with serum complement or with other species present in serum. in vitro or in vivo. Protective moieties can reduce interaction or binding to these species through one or more mechanisms, eg, non-specific electronic or steric non-specific interactions. Examples of such interactions include non-specific electrostatic interactions, charge interactions, Van der Waals interactions, steric hindrance, and the like. For a moiety to act as a protective moiety, the mechanism (s) by which it can reduce interaction, association, or binding with serum complement or other species does not have to be identified. One can determine whether a moiety can act as a protective moiety, by determining whether or to what extent a complex binds to serum species.

It should be noted that protective remains can be multifunctional. For example, a protective moiety can function as, for example, a target factor. A protective moiety can also be called multifunctional with respect to the mechanism (s) by which it protects the complex. Without wishing to be limited by proposed theory or mechanism, examples of such a multifunctional protective moiety are synthetic pH-sensitive endosomal membrane-disrupting polymers, such as PPAA or PEAA. Certain poly (alkylacrylic acids) have been shown to disrupt endosomal membranes, while leaving the outer cell surface membrane intact (Stayton et al. (2000) J. Controll. Release 65: 203-220; Murthy et al. (1999 ) J. Controll. Release 61: 137-143; WO 99/34831), thus increasing cellular bioavailability and functioning as a target factor. However, PPAA reduces the binding of serum complement to the complexes in which it is incorporated, thus functioning as a protective moiety.

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Another way of producing a formulation, particularly a solution, of a sirtuin modulator such as resveratrol or its derivative, is through the use of cyclodextrin. By cyclodextrin is meant α-, β-, or γ-cyclodextrin. Cyclodextrins are described in detail in Pitha et al., US Patent No. 4,727,064, which is incorporated herein by reference. Cyclodextrins are cyclic oligomers of glucose; these compounds form inclusion complexes with any drug whose molecule can fit into the lipophilic-seeking cavities of the cyclodextrin molecule.

The cyclodextrin of the compositions according to the invention can be α-, β-, or γ-cyclodextrin. Α-Cyclodextrin contains six glucopyranose units; Β-cyclodextrin contains seven glucopyranose units; and γ-cyclodextrin contains eight glucopyranose units. The molecule is believed to form a truncated cone having a core aperture of 4.7-5.3 angstroms, 6.0-6.5 angstroms, and 7.5-8.3 angstroms at α-, β- or γcyclodextrin, respectively. The composition according to the invention can comprise a mixture of two or more of the α-, β- or γ-cyclodextrins. Typically, however, the composition according to the invention will comprise only one of the α-, β- or γ-cyclodextrins.

The most preferred cyclodextrins in compositions according to the invention are compounds of amorphous cyclodextrins. By amorphous cyclodextrin is meant non-crystalline mixtures of cyclodextrins where the mixture is prepared from α-, β- or γ-cyclodextrin. In general, amorphous cyclodextrin is prepared by non-selective alkylation of the desired cyclodextrin species. Suitable alkylating agents for this purpose include, but are not limited to, propylene oxide, glycidol, iodoacetamide, chloroacetate, and 2-diethylaminoethylchloride. The reactions are carried out to produce mixtures containing a plurality of components, thus preventing the crystallization of the cyclodextrin. Various alkylated cyclodextrins can be prepared and will, of course, vary depending on the starter species of the cyclodextrin and the alkylating agent that is employed. Among the amorphous cyclodextrins suitable for compositions according to the invention are hydroxypropyl, hydroxyethyl, glucosyl, maltosyl and maltotriosyl derivatives of β-cyclodextrin, carboxyamidomethyl-β-cyclodextrin, carboxymethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin and diethylamino- β-cyclodextrin.

An example of resveratrol dissolved in the presence of a cyclodextrin is provided in Marier et al., J. Pharmacol. Exp. Therap. 302: 369-373 (2002), where a 6 mg / mL resveratrol solution was prepared using 0.9% saline containing 20% ​​hydroxylpropyl-β-cyclodextrin.

As mentioned above, the compositions of the invention comprise an aqueous preparation of preferably substituted amorphous cyclodextrin and one or more modulators of sirtuin. The relative amounts of sirtuin and cyclodextrin modulators will vary depending on the relative amount of each of the sirtuin modulators and the effect of cyclodextrin on the compound. In general, the weight ratio of the sirtuin modulator compound to the weight of the cyclodextrin compound will range from 1: 1 to 1: 100. A weight to weight ratio in a range of 1: 5 to 1:50 and more preferably in a range of 1:10 to 1:20 of the compound selected from the sirtuin to cyclodextrin modulators is believed to be the most effective. to increase the circulating availability of the sirtuin modulator.

It should be noted that if the aqueous solution comprising the sirtuin modulators and a cyclodextrin is to be administered parenterally, especially via the intravenous route, a cyclodextrin will be substantially free of pyrogenic contaminants. Various forms of cyclodextrin, such as forms of amorphous cyclodextrin, are available from a number of vendors, including Sigma-Aldrich, Inc. (St. Louis, Mo., USA). A method for the production of hydroxypropyl-β-cyclodextrin is described in Pitha et al., US Patent No. 4,727,064.

Further description of the use of cyclodextrin to solubilize compounds can be found in US 2005/0026849.

Rapidly disintegrating or dissolving dosage forms are useful for rapid absorption, particularly buccal and sublingual absorption of pharmaceutically active agents. Rapidly dissolving dosage forms are beneficial for patients, such as elderly patients or pediatric patients, who have difficulty swallowing typical solid dosage forms, such as tablets and oblong tablets. In addition, fast-dissolving dosage forms overcome disadvantages associated with, for example, chewable dosage forms, where the length of time an active agent remains in the patient's mouth plays an important role in determining the amount of masking of the patient. taste and the degree to which the patient can object roughness in the throat of the active agent.

To overcome these problems, manufacturers have developed a series of fast dissolving solid dose oral formulations. They are available from manufacturers including Cima Labs, Fuisz Technologies Ltd., Prographarm, RP Scherer, Yamanouchi-Shaklee, and McNeil-PPC, Inc. All of these manufacturers market different types of fast dissolving solid dosage forms. See p. eg, Cima Labs patents and publications such as US Patent Nos. 5,607,697, 5,503,846, 5,223,264, 5,401,513, 5,219,574 and 5,178,878, WO 98/46215, WO 98/14179; patents for Fuisz Technologies, now part of BioVail, such as US Patent Nos. 5,871,781, 5,869,098, 5,866,163, 5,851,553, 5,622,719, 5,567,439, and 5,587,172; US Patent No.

ES 2 396 913 T3

5,464,632 to Prographarm; patents for RP Scherer such as US Patent Nos.

4,642,903, 5,188,825, 5,631,023, and 5,827,541; patents for Yamanouchi-Shaklee such as US Patent Nos. 5,576,014 and 5,446,464; patents for Janssen such as US Patent Nos.

5,807,576, 5,635,210, 5,595,761, 5,587,180 and 5,776,491; US Patent Nos.

5,639,475 and 5,709,886 for Eurand America, Inc .; US Patent Nos. 5,807,578 and 5,807,577 for LAB Pharmaceutical Research; patents for Schering Corporation such as US Patent Nos. 5,112,616 and 5,073,374; US Patent No. 4,616,047 to Laboratoire L. LaFon; US Patent No. 5,501,861 to Takeda Chemicals Inc., Ltd .; and US Patent No. 6,316,029 for Elan.

In an example of fast dissolving tablet preparation, fast dissolving tablet granules prepared by spray drying or pre-compaction processes are mixed with excipients and compressed into tablets using conventional tabletting machinery. The granules can be combined with a variety of carriers, including low density, high moldability saccharides, low moldability saccharides, polyol blends, and then directly compressed into a tablet that exhibits a better dissolution and disintegration profile.

Tablets in accordance with the present invention typically have a hardness of from about 2 to about 6 Strong-Cobb units (scu). Tablets within this hardness range rapidly disintegrate or dissolve when chewed. Additionally, tablets disintegrate rapidly in water. On average, a typical 1.1 to 1.5 gram tablet disintegrates in 1-3 minutes without shaking. This rapid disintegration facilitates the administration of the active material.

The granules used to prepare tablets can be, for example, mixtures of carbohydrates or low density alkaline earth metal salts. For example, a mixture of alkaline earth metal salts includes a combination of calcium carbonate and magnesium hydroxide. Similarly, a fast dissolving tablet can be prepared according to the methods of the present invention, incorporating the use of A) spray dried extra light calcium carbonate / maltodextrin, B) magnesium hydroxide and C) a combination of polyols Eutectic that includes Sorbitol Instant, Xylitol and Mannitol. These materials have been combined to produce a low density tablet that dissolves very quickly and promotes rapid disintegration of the active ingredient. Furthermore, pre-compacted and spray-dried granules can be combined in the same tablet.

For preparation of a fast dissolving tablet, a sirtuin modulator useful in the present invention may be in particulate, granular, crystalline, oily or dissolution form. The sirtuin modulator for use in the present invention may be a spray dried product or an adsorbate that has been pre-compacted to a harder granular form that reduces the drug taste. A pharmaceutical active ingredient for use in the present invention can be spray dried with a carrier that prevents the active ingredient from being easily extracted from the tablet by chewing.

In addition to being added directly to the tablets of the present invention, the drug itself can be processed through the pre-compaction process to achieve a higher density before being incorporated into the formulation.

The pre-compaction process used in the present invention can be used to administer poorly soluble pharmaceutical materials, in such a way as to improve the release of said pharmaceutical materials compared to traditional dosage forms. This could allow the use of lower dosage levels to deliver equivalent bioavailable levels of drug and thus reduce the toxicity levels of both new and commercialized chemical entities. Poorly soluble pharmaceutical materials can be used in the form of nanoparticles, which are nanometer-sized particles.

In addition to the active ingredient and granules prepared from low density alkaline earth metal salts and / or water soluble carbohydrates, fast dissolving tablets can be formulated using conventional carriers or excipients and established pharmaceutical techniques. Conventional carriers or excipients include, but are not limited to, diluents, binders, adhesives (i.e. cellulose derivatives and acrylic derivatives), lubricants (i.e. magnesium or calcium stearate, vegetable oils, polyethylene glycols, talc, sodium lauryl sulfate , polyoxyethylene monostearate), disintegrants, colorants, flavors, preservatives, sweeteners and various materials such as buffers and adsorbents.

A further description of the preparation of fast dissolving tablets can be found, for example, in US Pat. 5,939,091.

Pharmaceutical compositions (including osmotic preparations) may comprise between about 0.00001 and 100%, such as between 0.001 and 10% or between 0.1% and 5% by weight of one or more of the sirtuin modulator compounds described in the present memory.

In one embodiment, a sirtuin modulator compound described herein is incorporated into a topical formulation that contains a topical carrier that is generally suitable for topical drug administration and that comprises any such materials known in the art. The topical vehicle can be selected so as to provide the composition in the desired form, e.g. e.g. as an ointment, lotion, cream, microemulsion, gel, 69

ES 2 396 913 T3 oil, solution or the like, and can be comprised of a material of natural or synthetic origin. It is preferable that the selected carrier does not adversely affect the active agent or other components of the topical formulation.

Examples of suitable topical carriers include water, alcohols, and other non-toxic organic solvents, glycerin, mineral oil, silicone, petroleum jelly, lanolin, fatty acids, vegetable oils, waxes, and the like.

The formulations can be colorless and odorless ointments, lotions, creams, microemulsions and gels.

The sirtuin modulating compounds can be incorporated into ointments, which in general are semi-solid preparations typically based on petrolatum or petrolatum derivatives. The specific ointment base to use, as will be appreciated by those skilled in the art, is one that provides optimal drug delivery and, preferably, also provides other desired characteristics, e.g. eg, emolliency or the like. As with other vehicles, an ointment base must be inert, stable, non-irritating, and non-sensitizing. As explained in Remington's (supra) ointment bases can be grouped into four classes: oil bases; emulsifying bases; emulsion bases; and water-soluble bases. Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum. Emulsifying ointment bases, also known as absorbent ointment bases, contain little or no water and include, for example, hydroxystearine sulfate, anhydrous lanolin, and hydrophilic petrolatum. Emulsion ointment bases are either water-in-oil (W / O) emulsions or oil-in-water (O / W) emulsions and include, for example, cetyl alcohol, glyceryl monostearate, lanolin, and stearic acid. Illustrative water-soluble ointment bases are prepared from polyethylene glycols (PEGs) of varying molecular weight; again, see Remington's, supra, for more information.

Sirtuin activator compounds can be incorporated into lotions, which are generally frictionless preparations to be applied to the surface of the skin, and are typically liquid or semi-liquid preparations in which the solid particles, including the active agent, are present in a base. of water or alcohol. Lotions are usually suspensions of solids, and may comprise a liquid oily emulsion of the oil-in-water type. Lotions are preferred formulations for treating large areas of the body, due to the ease of application of a more fluid composition. In general it is necessary that the insoluble matter in a lotion be finely divided. Lotions will typically contain suspending agents to produce better dispersions as well as compounds useful for locating and holding the active agent in contact with the skin, e.g. eg, methyl cellulose, sodium carboxymethyl cellulose, or the like. An illustrative lotion for use in conjunction with the present method contains propylene glycol mixed with a hydrophilic petrolatum such as that available under the brand name Aquaphor.^RTM from Beiersdorf, Inc. (Norwalk, Conn.).

The sirtuin modulating compounds can be incorporated into creams, which are generally viscous liquid or semi-solid emulsions, or oil-in-water or water-in-oil. Cream bases are washable in water and contain an oil phase, an emulsifier and a water phase. The oily phase in general is composed of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase by volume, and generally contains a humectant. The emulsifier in a cream formulation, as discussed in Remington's, supra, is generally a nonionic, anionic, cationic or amphoteric surfactant.

Sirtuin modulating compounds can be incorporated into microemulsions, which are generally thermodynamically stable, isotropically transparent dispersions of two immiscible liquids, such as oil and water, stabilized by an interfacial film of surfactant molecules (Encyclopedia of Pharmaceutical Technology (New York: Marcel Dekker, 1992), volume 9). For the preparation of microemulsions, the surfactant (emulsifier), cosurfactant (co-emulsifier), an oil phase and an aqueous phase are necessary. Suitable surfactants include any surfactant useful in the preparation of emulsions, e.g. eg, emulsifiers typically used in the preparation of creams. The co-surfactant (or co-emulsifier) ​​is generally selected from the group of polyglycerol derivatives, glycerol derivatives and fatty alcohols. Preferred emulsifier / co-emulsifier combinations are generally, but not necessarily, selected from the group consisting of: glyceryl monostearate and polyoxyethylene stearate; polyethylene glycol and ethylene glycol palmitostearate; and caprylic and capric triglycerics and oil macrogolglycerides. The aqueous phase includes not only water, but typically also buffers, glucose, propylene glycol, polyethylene glycols, preferably lower molecular weight polyethylene glycols (e.g. g., PEG 300 and PEG 400), and / or glycerol and the like, while the oil phase in general will comprise, for example, fatty acid esters, modified vegetable oils, silicone oils, mixtures of mono- and triglycerides , mono- and di-esters of PEG (eg, oleoyl macrogol glycerides), etc.

Sirtuin modulating compounds can be incorporated into gel formulations that are generally semi-solid systems consisting of suspensions composed of small inorganic particles (biphasic systems) or large organic molecules substantially uniformly distributed throughout a carrier (monophasic gels). Monophasic gels can be prepared, for example, by combining the active agent, a liquid carrier, and a suitable gelling agent such as tragacanth (at 2-5%), sodium alginate (at 2-10%), gelatin (at 2-15%). %), methylcellulose (at 3-5%), sodium carboxymethylcellulose (at 2-5%), carbomer (at 0.3-5%) or polyvinyl alcohol (at 10-20%) together and mixing until a characteristic semi-solid product is produced. Other suitable gelling agents include methylhydroxycellulose, polyoxyethylene-polyoxypropylene, hydroxyethylcellulose, and gelatin. While gels commonly employ liquid aqueous vehicles, alcohols and oils can also be used as the liquid vehicle.

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Various additives, known to those skilled in the art, can be included in formulations, e.g. eg, topical formulations. Examples of additives include, but are not limited to, solubilizers, skin permeation enhancers, opacifiers, preservatives (e.g. g., antioxidants), gelling agents, buffering agents, surfactants (particularly nonionic and amphoteric surfactants), emulsifiers, emollients, thickening agents, stabilizers, humectants, colorants, fragrance, and the like. The inclusion of solubilizers and / or skin permeation enhancers is particularly preferred, along with emulsifiers, emollients, and preservatives. An optimal topical formulation comprises about: 2% by weight to 60% by weight, preferably 2% by weight to 50% by weight of solubilizer and / or skin permeation enhancer; 2% by weight to 50% by weight, preferably 2% by weight to 20% by weight of emulsifiers; 2% by weight to 20% of emollients; and 0.01 to 0.2% by weight of preservatives, where the active agent and vehicle (eg, water) make up the remainder of the formulation.

A skin permeation enhancer serves to facilitate the passage of therapeutic levels of the active agent through a reasonably sized area of ​​unbroken skin. Suitable enhancers are known in the art and include, for example: lower alcohols such as methanol ethanol and 2-propanol; alkylmethyl sulfoxides such as dimethylsulfoxide (DMSO), decylmethylsulfoxide (C10 MSO), and tetradecylmethyl sulfoxide; pyrrolidones such as 2-pyrrolidone, N-methyl-2-pyrrolidone and N - (- hydroxyethyl) pyrrolidone; urea; N, N-diethyl-m-toluamide; C2 -C6 alkanediols; various solvents such as dimethyl formamide (DMF), N, N-dimethylacetamide (DMA), tetrahydrofurfuryl alcohol; and the 1-substituted azacycloheptan-2-ones, particularly 1-n-dodecylcyclazacycloheptan-2-one (laurocapram; available under the brand name Azone^RTM from Whitby Research Incorporated, Richmond, Va.).

Examples of solubilizers include, but are not limited to, the following: hydrophilic ethers such as diethylene glycol monoethyl ether (ethoxydiglycol, available as Transcutol^RTM) and ethylene glycol monoethyl ether oleate (available as Softcutol^RTM); polyethylene castor oil derivatives such as polyoxy 35 castor oil, polyoxy 40 hydrogenated castor oil, etc .; polyethylene glycol, particularly low molecular weight polyethylene glycols such as PEG 300 and pEg 400, and polyethylene glycol derivatives such as caprylic / capric glycerides PEG-8 (sold as Labrasol^RTM); alkylmethylsulfoxides such as DMSO; pyrrolidones such as 2-pyrrolidone and N-methyl-2-pyrrolidone; and DMA. Many solubilizers can also act as absorption enhancers. A single solubilizer can be incorporated into the formulation, or a mixture of solubilizers.

Suitable emulsifiers and co-emulsifiers include, without limitation, those emulsifiers and co-emulsifiers described with respect to microemulsion formulations. Emollients include, for example, propylene glycol, glycerol, isopropyl myristate, polypropylene glycol-2 (PPG-2) myristyl ether propionate, and the like.

Other active agents can also be included in the formulations, e.g. g., other anti-inflammatory agents, analgesics, antimicrobial agents, antifungal agents, antibiotics, vitamins, antioxidants, and sun blocking agents commonly found in sunscreen formulations, including without limitation, anthranlates, benzophenones (particularly benzophenone-3), camphor derivatives, cinnamates (eg octyl methoxycinnamate), dibenzoylmethanes (eg. g., butyl methoxydibenzoylmethane), p-aminobenzoic acid (PABA) and its derivatives, and salicylates (eg, octyl salicylate).

In certain topical formulations, the active agent is present in an amount in the range of about 0.25% by weight to 75% by weight of the formulation, preferably in the range of about 0.25% by weight to 30% by weight. of the formulation, more preferably in the range of about 0.5% by weight to 15% by weight of the formulation, and most preferably in the range of about 1.0% by weight to 10% by weight of the formulation.

The topical skin treatment compositions can be packaged in a suitable container that corresponds to their viscosity and consumer end use. For example, a lotion or cream can be packaged in a bottle or ball applicator, or in an aerosol device with a propellant, or in a container equipped with a pump suitable for finger operation. If the composition is a cream, it can simply be stored in a non-deformable hand-squeeze bottle or container, such as a tube or lidded bottle. The composition can also be included in capsules such as those described in US Patent No. 5,063,507. Accordingly, closed containers are also provided containing a cosmetically acceptable composition as defined herein.

In an alternative embodiment, a pharmaceutical formulation for oral or parenteral administration is provided, in which case the formulation comprises a microemulsion comprising a modulator compound as described above, but may contain carriers, additives, etc. alternatives that are pharmaceutically acceptable, particularly suitable for oral or parenteral drug administration. Alternatively, a microemulsion containing a modulator compound can be administered orally or parenterally substantially as described above, without modification.

Phospholipid complexes, e.g. g., resveratrol-phospholipid complexes, and their preparation, are described in US Patent Application Publication No. 2004/116386. Methods for stabilizing active components using polyol / polymer microcapsules, and their preparation, are described in US20040108608. Procedures for dissolving lipophilic compounds in aqueous solution with amphiphilic block copolymers are described in WO 04/035013.

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Eye conditions can be treated or prevented, eg. g., with systemic, topical, intraocular injection of a sirtuin modulator compound, or by insertion of a sustained release device that releases a sirtuin modulator compound. A sirtuin modulator compound that increases or decreases the level and / or activity of a sirtuin protein can be administered in a pharmaceutically acceptable ophthalmic vehicle such that the compound remains in contact with the ocular surface for a period of time sufficient to allow the compound to penetrate the corneal and internal regions of the eye, such as the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris / ciliary groove, lens, choroid / retina and sclera. The pharmaceutically acceptable ophthalmic vehicle can be, for example, an ointment, vegetable oil, or an encapsulating material. Alternatively, the compounds of the invention can be injected directly into the aqueous or vitreous humor. In another alternative, the compounds can be administered systemically, such as by injection or intravenous infusion, for treatment of the eye.

The sirtuin modulator compounds described herein can be preserved in an oxygen-free environment in accordance with methods in the art. For example, resveratrol or its analog can be prepared in a sealed capsule for oral administration, such as Capsugel from Pfizer, Inc.

Cells, p. g., treated ex vivo with a sirtuin modulator compound, can be administered according to the methods for administering a graft to a subject, which can be accompanied, e.g. g., from the administration of an immunosuppressive drug, e.g. eg, cyclosporin A. For general principles of medicinal formulations, the reader may refer to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn &amp; W. Sheridan eds, Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy, ED Ball, J. Lister &amp; P. ​​Law, Churchill Livingstone, 2000.

The toxicity and therapeutic efficacy of sirtuin modulator compounds can be determined by standard pharmaceutical procedures in cell culture or experimental animals. LD50 is the fatal dose for 50% of the population. The ED50 is the therapeutically effective dose in 50% of the population. The dose relationship between toxic and therapeutic effects (LD50 / ED50) is the therapeutic index. Sirtuin modulating compounds that exhibit high therapeutic indices are preferred. While sirtuin modulating compounds that exhibit toxic side effects can be used, precautions should be taken to design a delivery system that targets such compounds to the affected tissue site in order to minimize potential damage to uninfected cells and thus reduce the side effects.

Data obtained from cell culture assays and animal studies can be used to formulate a range of dosage for use in humans. The dose of such compounds may lie within a range of circulating concentrations that include the ED50 with little or no toxicity. The dose can vary within this range, depending on the form used and the route of administration used. For any compound, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a range of concentration in circulating plasma that includes the IC50 (ie, the concentration of the test compound that achieves maximum mean inhibition of symptoms) as determined in cell culture. Such information can be used to more precisely determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

6. Kits

Also provided herein are kits, e.g. eg, kits for therapeutic purposes or kits to modulate cell life or modulate apoptosis. A kit can comprise one or more sirtuin modulator compounds, e.g. eg, in pre-measured doses. A kit may optionally comprise devices for contacting cells with the compounds and instructions for use. Devices include syringes, stents, and other devices for introducing a sirtuin modulator compound into a subject (eg, a subject's blood vessel) or for applying it to a subject's pi .

Another type of kit contemplated by the invention consists of kits for identifying sirtuin modulating compounds. Such kits contain (1) a sirtuin or a sirtuin-containing material and (2) a sirtuin modulator compound of the invention, which are in separate containers. Such kits can be used, for example, to perform a competition-type assay to test other compounds (typically provided by the user) for sirtuin modulating activity. In certain embodiments, these kits also comprise means for determining sirtuin activity (eg, a peptide with an appropriate reporter, such as those described in the Examples).

In yet another embodiment, the invention provides a composition comprising a sirtuin modulator of the present invention and another therapeutic agent [the same used in combination therapies and combination compositions] in separate dosage forms, but associated with each other. The term associated with each other, as used herein, means that the separate dosage forms are packaged together or are in some way linked together in such a way that it is readily visible that the separate dosage forms are intended to be marketed. and administered as part of the same regimen. The agent and the sirtuin modulator are preferably packaged together in a blister pack or other multi-chamber pack,

ES 2 396 913 T3 separately sealed containers (such as foil bags or the like) that the user can separate (eg by tearing at cut lines between the two packages).

In yet another embodiment, the invention provides a kit comprising, in separate containers, a) a sirtuin modulator of the present invention; and b) another therapeutic agent such as those described herein.

The practice of the present methods will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the art. These techniques are explained in detail in the bibliography. See, for example, Molecular Cloning A Laboratory Manual, 2^to Ed., Ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (DN Glover ed., 1985); Oligonucleotide Synthesis (MJ Gait ed., 1984); Mullis et al. US Patent No. 4,683,195; Nucleic Acid Hybridization (BD Hames &amp; SJ Higgins eds. 1984); Transcription And Translation (BD Hames &amp; SJ Higgins eds. 1984); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., NY); Gene Transfer Vectors For Mammalian Cells (JH Miller and MP Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vol. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes 1-IV (DM Weir and CC Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

Examples

The invention will now be described generally and more readily understood with reference to the following examples which are included solely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention in any way.

Example 1: Synthesis and characterization of sirtuin modulators

Experimental section

Abbreviations used in the experimental section:

HATU = O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate

NMM = 4-methylmorpholine

DIEA = N, N-diisopropylethylamine

DMF = N, N-dimethylformamide

CH2Cl2 = sichloromethane

EtOAc = ethyl acetate

MeOH = methanol

Na2SO4 = sodium sulfate

PPA = polyphosphoric acid

Et3N = triethylamine rt = room temperature

Preparation of 2-imidazo [2,1-b] thiazol-6-yl-phenylamine:

<img file="ES2396913T3_D0033.tif" />

In a typical preparation, 123 mg of 2-aminothiazole (1.23 mmol) and 2-bromo-2'-nitroacetophenone (300 mg, 1.23 mmol) were mixed with 15 mL of methyl ethyl acetone, and stirred under reflux. for 18 hours. Then it was cooled to room temperature and filtered. The filtrate was concentrated. The resulting solids were mixed with 20 mL of EtOH and 5 drops of concentrated HBr were added. The reaction mixture was stirred under reflux for 6 hours. Everything dissolved at this point, and LC / MS indicated the formation of the desired nitro intermediate (MS, M⁺+ H = 246). The mix of 73

ES 2 396 913 T3 reaction was concentrated and mixed with 20 mL of dilute aqueous NaHCO3. The resulting solids were collected by filtration and dried to provide 300 mg of the nitro intermediate. This material was mixed with 15 mL of MeOH and 3 mL of water, along with 6 eq of sodium hydrosulfide hydrate. The reaction mixture was stirred under reflux for 8 hours. Then it was cooled to room temperature and concentrated. The aqueous layer was extracted with CH2Cl2. The combined organic layers were dried (Na2SO4) and concentrated to provide 260 mg of 2-imidazo [2,1b] thiazol-6-yl-phenylamine.

Preparation of Compound 203:

<img file="ES2396913T3_D0034.tif" />

2-Imidazo [2,1-b] thiazol-6-yl-phenylamine (64 mg, 0.30 mmol) was mixed with 1 mL of pyridine, together with 60 mg of 3,4-dimethoxybenzoyl chloride (0.30 mmol). The reaction mixture was reacted in the Biotage microwave reactor at 160 ° C for 10 min. The reaction mixture was cooled to room temperature and concentrated. The resulting residue was purified by chromatography (Isco, gradient elution, CH₂Cl₂ up to 9: 1 CH₂Cl₂/ MeOH) to provide the desired product (MS, M⁺ + H = 380).

Preparation of Compound 204:

The same procedure was employed as in the preparation of Compound 203, using the appropriate acid chloride.

Preparation of Compounds 707, 739 and 740:

The same procedure was employed as in the preparation of Compound 203 except with 2-amino-4-methylthiazole at the beginning of the synthetic sequence and the appropriate acid chloride in the last amide formation step.

Preparation of 6- (2-nitro-phenyl) -imidazo [2,1-b] thiazole-3-carboxylic acid ethyl ester:

<img file="ES2396913T3_D0035.tif" />

In a typical preparation, 2.1 g of ethyl 2-aminothiazole-4-carboxylate (Combi-Blocks, 0.0123 mol) were mixed with 25 mL of methyl ethyl ketone, together with 2-bromo-2'-nitroacetophenone ( 3.0 g, 0.0123 mol). The reaction mixture was stirred under reflux for 18 hours. It was then cooled to room temperature and filtered to remove some of the solids. The filtrate was concentrated to provide 3.10 g of 6- (2-nitro-phenyl) -imidazo [2,1-b] thiazole-3-carboxylic acid ethyl ester (MS, M⁺ + H = 318).

Preparation of [6- (2-nitro-phenyl) -imidazo [2,1-b] thiazol-3-yl] -methanol:

<img file="ES2396913T3_D0036.tif" />

ES 2 396 913 T3

Acid ethyl ester was mixed

6- (2-Nitro-phenyl) -imidazo [2,1-b] thiazole-3-carboxylic acid (14.50 g, 0.0458 mol) with 100 mL of THF and 100 mL of water containing 7.3 g NaOH (4 eq). The reaction mixture was stirred at room temperature for 18 hours. Then he concentrated. The aqueous layer was washed once with CH2Cl2 and then acidified with 6N HCl. The solids were collected by filtration and dried to provide 7.4 mg of the acidic intermediate. This material (7.4 g, 0.0256 mol) was mixed with 200 mL of anhydrous THF together with NMM (2.8 mL, 0.0256 mol) and cooled to 0 ° C. Isobutyl chloroformate (3.35 mL, 0.0256 mol) was added and the reaction mixture was stirred in an ice bath for 3 hours. NaBH4 (0.97 g, 0.0256 mol) was added as a solution in 30 mL of water. The reaction mixture was stirred at 0 ° C for 45 min, then warmed to room temperature and concentrated. The aqueous layer was extracted with CH2Cl2. The combined organic layers were dried (Na2SO4) and concentrated to provide the crude product. Purification by chromatography (Isco, using a pentane / EtOAc mixture) provided 5.20 g of [6- (2-nitro-phenyl) -imidazo [2.1b] thiazol-3-yl] -methanol (74% of performance).

Preparation of 4- [6- (2-amino-phenyl) -imidazo [2,1-b] thiazol-3-ylmethyl] -piperazine-1-carboxylic acid tert-butyl ester:

<img file="ES2396913T3_D0037.tif" />

[6- (2-Nitro-phenyl) -imidazo [2,1-b] thiazol-3-yl] -methanol (1.0 g, 3.64 mmol) was dissolved in 100 mL of CH2Cl2 along with 1 eq of Et3N (0.51 mL). Methanesulfonyl chloride (1 eq, 0.28 mL) was added and the reaction mixture was warmed to room temperature and stirred for 15 min. It was then quenched with brine and extracted with CH2Cl2. The combined organic layers were dried (Na2SO4) and concentrated to provide the mesylate intermediate. This material was mixed with 4 mL of CH₃CN together with 0.51 mL of Et₃N and 680 mg of Boc-piperazine (3.64 mmol) and stirred at room temperature for 1 day. The reaction mixture was concentrated and the resulting residue was partitioned between CH2Cl2 and water. The organic layer was dried (Na2SO4) and concentrated to provide an essentially quantitative yield of the product. This material was mixed with 6 mL of MeOH and 1 mL of water, along with 200 mg of sodium hydrosulfide hydrate. The resulting reaction mixture was stirred under reflux for 24 hours. Then it was cooled to room temperature and concentrated. The resulting residue was diluted with 2 mL of water and extracted with CH2Cl2. The combined organic layers were dried (Na2SO4) and concentrated to provide 0.90 g of 4- [6- (2-aminophenyl) -imidazo [2,1-b] thiazol-3-ylmethyl] - tert-butyl acid ester piperazine-1-carboxylic.

Preparation of Compound 207:

<img file="ES2396913T3_D0038.tif" />

4- [6- (2-amino-phenyl) -imidazo [2,1-b] thiazol-3-ylmethyl] -piperazine-1-carboxylic acid tert-butyl ester (0.3 mmol) was mixed with 1 mL of pyridine together with 1 eq (60 mg) of 3,4-dimethoxybenzoyl chloride. The reaction mixture was reacted in a Biotage microwave reactor at 160 ° C for 10 min. Then it was cooled to room temperature and concentrated. The resulting crude product was purified by chromatography (Isco, gradient elution, CH2Cl2 to 95% CH2Cl2, 4% MeOH and 1% Et3N). The purified product was then treated with 2 mL of 25% TFA in CH2Cl2 for 2 hours. It was then concentrated and the resulting residue was triturated with Et2O to provide the desired product as the TFA salt (MS, M⁺ + H = 478).

Preparation of Compounds 208, 326, 327, 328, 329, 330, 337, 338, 440, 441, 442, 443, 444, 445, 446, 447, 448, 510, 511, 512, 543, 544, 708, 709, 710, 733, 735, 736, 737, 738, 743 and 744:

ES 2 396 913 T3

The same procedure was employed as in the preparation of Compound 207, using the appropriate acid chloride or sulfonyl chloride. Compounds 623, 624, 625, 644, 645, 692, 695, 697 and 698 were prepared according to the procedure used for the preparation of Compound 207, using the appropriate acid chloride. Acid chlorides were commercially available or were prepared from carboxylic acids as follows: Carboxylic acid (1.0 mmol), thionyl chloride (2.0 mmol), and a catalytic amount of N, N-dimethylformamide (DMF) (2 drops) were refluxed in toluene (2 mL) for 1 hour. The reaction was cooled to room temperature and concentrated in vacuo to provide the desired acid chlorides.

Preparation of 2- (3-Dimethylaminomethyl-imidazo [2,1-b] thiazol-6-yl) -phenylamine:

<img file="ES2396913T3_D0039.tif" />

[6- (2-Nitro-phenyl) -imidazo [2,1-b] thiazol-3-yl] -methanol (435 mg, 1.58 mmol) was dissolved in 25 mL of CH₂Cl₂ along with 1 eq of Et3N (0.330 mL). Methanesulfonyl chloride (1 eq, 0.12 mL) was added and the reaction mixture was warmed to room temperature and stirred for 15 min. It was then quenched with brine and extracted with CH2Cl2. The combined organic layers were dried (Na2SO4) and concentrated to provide the mesylate intermediate. This material was mixed with 4 mL of THF together with 4 mL of a 2N dimethylamine solution in THF and stirred at room temperature for 3 hours. The reaction mixture was concentrated and the resulting residue was partitioned between CH2Cl2 and water. The organic layer was dried (Na2SO4) and concentrated to provide an essentially quantitative yield of the product. This material was mixed with 6 mL of MeOH and 1 mL of water, along with 200 mg of sodium hydrosulfide hydrate. The resulting reaction mixture was stirred under reflux for 6 hours. It was then cooled to room temperature, diluted with 100 mL of absolute EtOH, and concentrated. The resulting residue was mixed with 20 mL of 9: 1 CH2Cl2 / MeOH and filtered. The filtrate was concentrated to provide 2- (3-dimethylaminomethyl-imidazo [2,1b] thiazol-6-yl) -phenylamine.

Preparation of Compound 205:

<img file="ES2396913T3_D0040.tif" />

h₂n

5 = \ \ / N— '/ -

2- (3-Dimethylaminomethyl-imidazo [2,1-b] thiazol-6-yl) -phenylamine (0.3 mmol) was mixed with 1 mL of pyridine together with 1 eq (60 mg) of 3,4 chloride -dimethoxybenzoyl. The reaction mixture was reacted in a Biotage microwave reactor at 160 ° C for 10 min. Then it was cooled to room temperature and concentrated. The resulting crude product was purified by chromatography (Isco, gradient elution, CH₂Cl₂ up to 95% CH₂Cl₂, 4% MeOH and 1% Et3N) to provide the desired product as a light yellow solid (MS, M⁺ + H = 437).

Preparation of Compound 206:

The same procedure was employed as in the preparation of the appropriate Compound 205.

using acid chloride

Preparation of 2- (3-morpholin-4-ylmethyl-imidazo [2,1-b] thiazol-6-yl) -phenylamine:

<img file="ES2396913T3_D0041.tif" />

ES 2 396 913 T3

[6- (2-Nitro-phenyl) -imidazo [2,1-b] thiazol-3-yl] -methanol (435 mg, 1.58 mmol) was dissolved in 25 mL of CH2Cl2 along with 1 eq of Et3N ( 0.330 mL). Methanesulfonyl chloride (1 eq, 0.12 mL) was added and the reaction mixture was warmed to room temperature and stirred for 15 min. It was then quenched with brine and extracted with CH2Cl2. The combined organic layers were dried (Na2SO4) and concentrated to provide the mesylate intermediate. This material was mixed with 6 mL of CH₃CN together with 0.33 mL of Et₃N and 0.14 mL of morpholine. The reaction mixture was stirred at room temperature for 18 hours. The next day, it was concentrated and the resulting residue was partitioned between CH2Cl2 and water. The organic layer was dried (Na₃SO4) and concentrated to provide an essentially quantitative yield of the product. This material was mixed with 6 mL of MeOH and 1 mL of water, along with 200 mg of sodium hydrosulfide hydrate. The resulting reaction mixture was stirred under reflux for 6 hours. It was then cooled to room temperature, diluted with 100 mL of absolute EtOH, and concentrated. The resulting residue was mixed with 20 mL of 9: 1 CH2Ch / MeOH and filtered. The filtrate was concentrated to provide 2- (3-morpholin-4-ylmethyl-imidazo [2,1b] thiazol-6-yl) -phenylamine.

Preparation of Compound 209:

<img file="ES2396913T3_D0042.tif" />

2- (3-Morpholin-4-ylmethyl-imidazo [2,1-b] thiazol-6-yl) -phenylamine (0.3 mmol) was mixed with 1 mL of pyridine together with 1 eq (60 mg) of chloride of 3,4-dimethoxybenzoyl. The reaction mixture was reacted in a Biotage microwave reactor at 160 ° C for 10 min. Then it was cooled to room temperature and concentrated. The resulting crude product was purified by chromatography (Isco, gradient elution, CH2O2 up to 95% CH2O2, 4% MeOH and 1% Et₃N) to provide the desired product as a light yellow solid (MS, M⁺ + H = 479).

Preparation of Compound 210:

The same procedure was employed as in the preparation of Compound 209, using the appropriate acid chloride.

Preparation of 6- (2-nitro-phenyl) -imidazo [2,1-b] thiazole-2-carboxylic acid ethyl ester:

<img file="ES2396913T3_D0043.tif" />

In a typical preparation, 1.0 g of ethyl 2-aminothiazole-5-carboxylate (Astatech, 5.81 mmol) was mixed with 50 mL of acetone together with 1.42 g of 2-bromo-2'-nitroacetophenone and stirred under reflux for 18 hours. Then it leaked. The filtrate was concentrated to provide the intermediate amide (MS, M⁺ + H = 336). This material was mixed with 20 mL of EtOH together with 6 drops of concentrated HBr and stirred under reflux for 4 hours. The reaction mixture was cooled to room temperature and concentrated. The resulting residue was diluted with NaHCO₃ dilute, watery. The solids were collected by filtration and dried to provide 6- (2-nitro-phenyl) -imidazo [2,1-b] thiazole-2-carboxylic acid ethyl ester (MS, M⁺ + H = 318).

Preparation of [6- (2-nitro-phenyl) -imidazo [2,1-b] thiazol-2-yl] -methanol:

ES 2 396 913 T3

<img file="ES2396913T3_D0044.tif" />

6- (2-Nitro-phenyl) -imidazo [2,1-b] thiazole-2-carboxylic acid ethyl ester (660 mg, 2.08 mmol) was dissolved in 12 mL of THF and NaOH (4 eq ) as a solution in 10 mL of water. The reaction mixture was stirred at 50 ° C for 12 hours. Then it was cooled to room temperature and concentrated. The aqueous layer was acidified with 6N HCl to pH 5. The solids were collected by filtration and dried to provide an essentially quantitative yield of the acid. This material (2.08 mmol) was mixed with 20 mL of anhydrous THF along with NMM (0.23 mL, 2.08 mmol) and cooled in an ice bath. Isobutyl chloroformate (0.27 mL, 2.08 mmol) was added and the reaction mixture was stirred at 0 ° C for 30 min. NaBH was added₄ (80 mg, 2.08 mmol) as a solution in 5 mL of water. The reaction mixture was stirred at 0 ° C for 30 minutes and then concentrated. The aqueous layer was extracted with CH2CE. The combined organic layers were dried (Na2SO4) and concentrated. Purification by chromatography (Isco, gradient elution using a mixture of CH2Cl2 and MeOH) provided 190 mg of [6- (2-nitrophenyl) -imidazo [2,1-b] thiazol-2-yl] -methanol.

Preparation of 2- (2-Dimethylaminomethyl-imidazo [2,1-b] thiazol-6-yl) -phenylamine:

<img file="ES2396913T3_D0045.tif" />

Essentially the same imidazo [2,1-b] thiazol-6-yl) -phenylamine procedure was employed except starting material.

used during the preparation of 2- (3-dimethylaminomethyl used [6- (2-nitro-phenyl) -imidazo [2,1-b] thiazol-2-yl] -methanol as

Preparation of Compound 178:

<img file="ES2396913T3_D0046.tif" />

2- (2-Dimethylaminomethyl-imidazo [2,1-b] thiazol-6-yl) -phenylamine (0.3 mmol) was mixed with 1 mL of pyridine together with 1 eq (60 mg) of 3,4 chloride -dimethoxybenzoyl. The reaction mixture was reacted in a Biotage microwave reactor at 160 ° C for 10 min. Then it was cooled to room temperature and concentrated. The resulting crude product was purified by chromatography (Isco, gradient elution, CH2O2 to 95% CH2O2, 4% MeOH, and 1% Et3N) to provide the desired product as a light yellow solid (MS, M⁺ + H = 437).

Preparation of Compound 179:

The same procedure was employed as in the preparation of Compound 178, using the appropriate acid chloride.

Preparation of 4- [6- (2-amino-phenyl) -imidazo [2,1-b] thiazol-2-ylmethyl] -piperazine-1-carboxylic acid tert-butyl ester:

ES 2 396 913 T3

<img file="ES2396913T3_D0047.tif" />

except that [6- (2-nitro-phenyl) was used. Essentially the same procedure used during the preparation of 4 [6- (2-aminophenyl) -imidazo [2,1-b] thiazole acid tert-butyl ester was used. -3-ylmethyl] -piperazine-1-carboxylic, imidazo [2,1-b] thiazol-2-yl] -methanol as starting material.

Preparation of Compound 270:

<img file="ES2396913T3_D0048.tif" />

4- [6- (2-amino-phenyl) -imidazo [2,1-b] thiazol-2-ylmethyl] -piperazine-1-carboxylic acid tert-butyl ester (0.2 mmol) was mixed with 1 mL of pyridine together with 1 eq (40 mg) of 3,4-dimethoxybenzoyl chloride. The reaction mixture was reacted in a Biotage microwave reactor at 160 ° C for 10 min. Then it was cooled to room temperature and concentrated. The resulting crude product was purified by chromatography (Isco, gradient elution, CH2O2 to 95% CH2O2, 4% MeOH and 1% Et3N). The purified product was then treated with 2 mL of 25% TFA in CH2Cl2 for 2 hours. It was then concentrated and the resulting residue was triturated with Et2O to provide the desired product as the TFA salt (MS, M⁺ + H = 478).

Preparation of Compound 271 and Compound 513:

The same procedure was employed as in the preparation of Compound 270, using the appropriate acid chloride.

Preparation of a 1: 1 mixture of 6- (2-Chloro-pyridin-3-yl) -imidazo [2,1-b] thiazole-3-carboxylic acid ethyl ester and 6- (2-bromo) acid ethyl ester -pyridin-3-yl) -imidazo [2,1-b] thiazole-3-carboxylic:

<img file="ES2396913T3_D0049.tif" />

A 1: 1 mixture of 2-bromo-1- (2-chloro-pyridin-3-yl) -ethanone and 2-bromo-1- (2-bromo-pyridin-3-yl) -ethanone was prepared according to the procedure outlined in WO 2005/061476. This mixture (5.6 g, approximately 0.0240 mol) was mixed with 150 mL of methyl ethyl ketone together with 2-amino-thiazole-4-carboxylic acid ethyl ester (4.6 g) and stirred under reflux for 18 hours. The reaction mixture was concentrated. The resulting residue was mixed with 150 mL of: CH2Cl2 and filtered. The filtered solids were 2-amino-thiazole-4-carboxylic acid ethyl ester without

ES 2 396 913 T3 react. The filtrate was concentrated to provide an essentially pure 1: 1 mixture of 6 (2-Chloro-pyridin-3-yl) -imidazo [2,1-b] thiazole-3-carboxylic acid ethyl ester and 6-acid ethyl ester. - (2-bromo-pyridin-3-yl) -imidazo [2.1b] thiazole-3-carboxylic acid (3.0 g total).

Preparation of a 1: 1 mixture of 6- (2-Chloro-pyridin-3-yl) -3-morpholin-4-ylmethyl-imidazo [2,1-b] thiazole and 6- (2-bromo-pyridin-3-yl ) -3-morpholin-4-ylmethyl-imidazo [2,1-b] thiazole:

<img file="ES2396913T3_D0050.tif" />

The 1: 1 mixture of 6- (2-Chloro-pyridin-3-yl) -imidazo [2,1-b] thiazole-3-carboxylic acid ethyl ester and 6- (2-bromo-pyridine) -3-yl) -imidazo [2,1-b] thiazole-3-carboxylic acid (3.0 g) was mixed with 100 mL of THF together with 25 mL of water containing 3 g of NaOH. The reaction mixture was stirred at 50 ° C for 3 hours. Then it was cooled to room temperature and concentrated. The aqueous layer was acidified to pH 5 with 6N HCl, and the resulting mixture was filtered. The solids were collected to provide 2.14 g of the intermediate acid.

This 1: 1 mixture of acid (2.14 g) was mixed with 250 mL of anhydrous THF together with NMM (0.85 mL, mmol) and cooled in an ice bath. Isobutyl chloroformate (1.0 ml) was added and the reaction mixture was warmed to room temperature and stirred for 3 hours. The reaction mixture was cooled in an ice bath and NaBH was added.₄ (0.29 g) as a solution in 20 mL of water. The reaction mixture was stirred for 30 minutes and then warmed to room temperature. It was concentrated and subsequently extracted with CH₂Cl₂. The combined organic layers were dried (Na₂SW₄) and concentrated to provide 1.5 g of the intermediate alcohol.

This 1: 1 mixture of intermediate alcohol (1.5 g) was mixed with 100 mL of CH₂Cl₂ together with Et₃N (0.80 mL) and cooled in an ice bath. Methanesulfonyl chloride (0.44 ml) was added and the reaction mixture was warmed to room temperature. The reaction mixture was quenched with brine and the two layers were separated. The organic layer was dried (Na₂SW₄) and concentrated to provide the intermediate mesylate. This material was immediately mixed with 30 mL of CH3CN along with 0.80 mL of Et₃N and 0.5 mL of morpholine. The reaction mixture was stirred at 50 ° C for 3 hours. The reaction mixture was concentrated under reduced pressure and the resulting residue was partitioned between CH₂Cl₂ and brine. The organic layer was separated, dried (Na₂SW₄) and concentrated to supply the gross product. Chromatographic purification (Isco, gradient elution, CH2Cl2 to 95% CH2Cl2, 4% MeOH and 1% Et3N) provided 720 mg of a 1: 1 mixture of 6- (2-Chloro-pyridin-3-yl) -3 -morpholin-4-ylmethyl-imidazo [2,1-b] thiazole and 6- (2-bromo-pyridin-3-yl) -3-morpholin-4-ylmethyl-imidazo [2,1-b] thiazole.

Preparation of (4-methoxy-benzyl) - [3- (3-morpholin-4-ylmethyl-imidazo [2,1-b] thiazol-6-yl) -pyridin-2-yl] -amine:

<img file="ES2396913T3_D0051.tif" />

The 1: 1 mixture of 6- (2-Chloro-pyridin-3-yl) -3-morpholin-4-ylmethyl-imidazo [2,1-b] thiazole and 6- (2-bromo-pyridin-3-yl ) -3-morpholin-4ylmethyl-imidazo [2,1-b] thiazole (600 mg) was mixed with 15 mL of toluene together with 0.47 mL of 4-methoxybenzylamine and stirred under reflux for 5 days. The reaction mixture was cooled to room temperature and partitioned between CH₂Cl₂ and brine. The organic layer was separated, dried (Na₂SW₄) and concentrated to supply the gross product. Purification by chromatography (Isco, gradient elution, CH2Cl2 to 95% CH2Cl2, 4% MeOH and 1% Et3N) provided 200 mg of (4-methoxy-benzyl) - [3- (3-morpholin-4-ylmethyl-imidazo [2,1-b] thiazol-6-yl) -pyridin-2-yl] -amine.

Preparation of 3- (3-morpholin-4-ylmethyl-imidazo [2,1-b] thiazol-6-yl) -pyridin-2-ylamine:

ES 2 396 913 T3

<img file="ES2396913T3_D0052.tif" />

(4-Methoxy-benzyl) - [3- (3-morpholin-4-ylmethyl-imidazo [2,1-b] thiazol-6-yl) -pyridin-2-yl] -amine (100 mg, 0 , 23 mmol) with 2 mL of CH₂Cl₂ together with triethylsilane (0.11 mL, 2 eq). Trifluoroacetic acid (1 mL) was added and the reaction mixture was stirred at room temperature for 18 hours. The next day, the reaction mixture was concentrated. The resulting residue was triturated with Et2O to give an essentially quantitative yield of 3- (3-morpholin-4-ylmethyl-imidazo [2,1-b] thiazol-6-yl) -pyridin-2-ylamine as the TFA salt. .

Preparation of Compound 621:

<img file="ES2396913T3_D0053.tif" />

The TFA salt of 3- (3-morpholin-4-ylmethyl-imidazo [2,1-b] thiazol-6-yl) -pyridin-2-ylamine (0.1 mmol) was mixed with 1 mL of pyridine together with 0.1 mmol of 2-quinoxaloyl chloride. The reaction mixture was reacted in a microwave reactor at 160 ° C for 10 min. Then it was cooled to room temperature and concentrated to provide the crude product. Purification by preparative HPLC, using a mixture of aqueous CH3CN that had been buffered with 0.1% TFA, provided 18 mg of the desired product as the TFA salt (MS, M⁺ + H = 472).

Preparation of 5- (2-nitro-phenyl) -thiazol-2-ylamine:

<img file="ES2396913T3_D0054.tif" />

In a typical preparation, 2-amino-5-bromothiazole monohydrobromide (Aldrich, 5.00g, 0.0192 mol) was mixed with 40 mL of toluene, 40 mL of ethanol and 20 mL of water. 2-Nitrophenyl boronic acid (3.2 g, 0.0192 mol) was added, along with 2.35 g of [1,1'-bis (diphenylphosphino) ferrocene] dichloro-palladium (II) complex with CH₂Cl₂ (1: 1) and 6.10 g of anhydrous sodium carbonate. The reaction mixture was stirred at 90 ° C for 18 hours. Then it was cooled to room temperature and concentrated. The resulting residue was mixed with 500 ml of EtOAc and washed with water (3x50 ml). The organic layer was filtered to remove the black precipitate. The filtrate was extracted with dilute 1N HCl. The combined aqueous layers were concentrated to near dryness. The resulting residue was purified by preparative HPLC, using an aqueous acetonitrile mixture that had been buffered with 0.1% TFA to provide 108 mg of 5- (2-nitrophenyl) -thiazol-2ylamine (MS, M⁺ + H = 222).

Preparation of 2- (2-nitro-phenyl) -imidazo [2,1-b] thiazole-6-carboxylic acid ethyl ester:

ES 2 396 913 T3

<img file="ES2396913T3_D0055.tif" />

Got mixed up

5- (2-nitro-phenyl) -thiazol-2-ylamine (100 mg, 0.452 mmol) with 10 mL of methyl ethyl ketone together with 1.5 equivalents of ethyl ethyl bromopyruvate. The reaction mixture was stirred under reflux for 5 hours. The reaction mixture was cooled to room temperature and concentrated. The crude product was purified by chromatography (Isco, gradient elution, CH₂Cl₂ at 9: 1 CH₂Cl₂/ MeOH) to provide 60 mg of 2- (2-nitro-phenyl) -imidazo [2,1-b] thiazole-6-carboxylic acid ethyl ester (MS, M⁺ + H = 318).

Preparation of 2- (2-amino-phenyl) -imidazo [2,1-b] thiazole-6-carboxylic acid methyl ester:

<img file="ES2396913T3_D0056.tif" />

2- (2-Nitro-phenyl) -imidazo [2,1-b] thiazole-6-carboxylic acid ethyl ester (60 mg, 0.189 mmol) was mixed with 3 mL of MeOH together with sodium hydrosulfide hydrate (32 mg, 0.567 mmol) in 1 mL of water. The reaction mixture was stirred under reflux for 1 hour and monitored by LC / MS. The reduction of the nitro group was completed at this point and the ethyl ester group had been exchanged with the corresponding methyl derivative. The reaction mixture was cooled to room temperature and concentrated. The aqueous layer was extracted with CH2Cl2. The combined organic layers were dried (Na₂SO-0 and concentrated to provide an essentially quantitative yield of 2- (2-aminophenyl) -imidazo [2,1-b] thiazole-6-carboxylic acid methyl ester (MS, M⁺ + H = 274).

Preparation of Compound 703:

<img file="ES2396913T3_D0057.tif" />

2- (2-Amino-phenyl) -imidazo [2,1-b] thiazole-6-carboxylic acid methyl ester (27 mg, 0.095 mmol) was mixed with 1 mL of pyridine together with 22 mg of 3,4 chloride , 5-trimethoxybenzoyl. The reaction mixture was reacted in a microwave reactor at 160 ° C for 10 minutes. Then it was cooled to room temperature and concentrated. The resulting crude product was purified by preparative HPLC using an aqueous acetonitrile mixture that had been buffered with 0.1% TFA to provide 108 mg of 2- [2- (3,4,5-trimethoxybenzoylamino) phenyl] acid methyl ester. -imidazo [2,1-b] thiazole-6-carboxylic (MS, M⁺ + H = 468).

Preparation of Compound 704:

<img file="ES2396913T3_D0058.tif" />

ES 2 396 913 T3

2- [2- (3,4,5-Trimethoxybenzoylamino) -phenyl] -imidazo [2,1-b] thiazole-6-carboxylic acid methyl ester (6 mg) was mixed with 1 mL of THF. Sodium hydroxide (10 mg) was added as a solution in 1 mL of water. The reaction mixture was stirred at room temperature for 4 hours and then concentrated. The resulting crude product was purified by preparative HPLC using an aqueous acetonitrile mixture that had been buffered with 0.1% TFA to provide 108 mg of 2- [2- (3,4,5-trimethoxy-benzoylamino) acid methyl ester). -phenyl] -imidazo [2,1-b] thiazole-6-carboxylic acid (MS, M⁺ + H = 454).

Preparation of 3- (chloromethyl) -6- (2-nitrophenyl) imidazo [2,1-b] thiazole:

<img file="ES2396913T3_D0059.tif" />

(3-Nitro-5-thiazolo [5,4-c] pyridin-2-yl-phenyl) -methanol (1.375 g, 5 mmol) was suspended in 25 mL of CH2Cl2 and cooled with an ice bath. Thionyl chloride (3.6 ml, 10 eq) was added dropwise and the reaction mixture was slowly warmed to room temperature. After stirring overnight, 100 mL of ether was added to the reaction mixture, and the resulting suspension was filtered to collect 1.55 g of the desired product (MS, M⁺ + H = 293.1).

Preparation of 4-benzyl-1 - ((6- (2-nitrophenyl) imidazo [2,1-b] thiazol-3-yl) methyl) piperazin-2-one:

<img file="ES2396913T3_D0060.tif" />

3- (chloromethyl) -6- (2-nitrophenyl) imidazo [2,1-b] thiazole (292 mg, 1 mmol), 4-benzylpiperazin-2-one (380 mg, 2 mmol) and NaH (88 mg, 2.2 eq) in 4 mL of dry DMF. The reaction was heated to 100 ° C overnight. After cooling to room temperature, the reaction mixture was partitioned between ethyl acetate and water. The organic layer was collected, dried and evaporated to provide the crude product, which was further purified by reverse HPLC to give 211 mg of the desired product 4-benzyl-1 - ((6- (2-nitrophenyl) imidazo [2, 1-b] thiazol-3-yl) methyl) piperazin-2one (MS, M⁺ + H = 448.1).

Preparation of 1 - ((6- (2-aminophenyl) imidazo [2,1-b] thiazol-3-yl) methyl) -4-benzylpiperazin-2-one:

<img file="ES2396913T3_D0061.tif" />

To a suspension of 200 mg of 1 - ((6- (2-nitrophenyl) imidazo [2,1-b] thiazol-3-yl) methyl) -4-benzylpiperazin-2-one in 5 mL of MeOH was added 250 mg of sodium hydrosulfide hydrate. The reaction mixture was heated to 135 ° C for 30 minutes (MW). After cooling to room temperature, the mixture was diluted with 50 ml of water and extracted with CH2Cl2. The combined organic layers were dried (Na2SO4) and concentrated to provide the crude product, which can be further purified by reverse phase HPLC to provide 135 mg of the target product 1 - ((6- (2-aminophenyl) imidazo [2, 1-b] thiazol-3-yl) methyl) -4-benzylpiperazin-2-one (MS, M⁺ + H = 418.1).

Preparation of Compound 628:

ES 2 396 913 T3

<img file="ES2396913T3_D0062.tif" />

A mixture of 1 - ((6- (2-aminophenyl) imidazo [2,1-b] thiazol-3-yl) methyl) -4-benzylpiperazin-2-one (44 mg, 0.1 mmol) and 2-quinoxalloyl chloride (21 mg, 1.1 eq) in 2.5 mL of pyridine for 20 minutes at 160 ° C (MW). After cooling to room temperature, the pyridine was removed and the crude reaction product was redissolved in methanol and purified by reverse phase HPLC to provide 22 mg of the desired product (MS, M⁺ + H = 574.1).

Preparation of 617, 618, 647, 648, 676, 677, 678, 679, 699, 741,742 and 711:

These compounds were prepared analogously to Compound 628. Products were purified by reverse phase HPLC.

Preparation of 2- (3-pyrrolidin-1-ylmethyl-imidazo [2,1-b] thiazol-6-yl) -phenylamine:

<img file="ES2396913T3_D0063.tif" />

[6- (2-Nitro-phenyl) -imidazo [2,1-b] thiazol-3-yl] -methanol (110 mg, 0.4 mmol) was cooled in CH₂Cl₂ (5 ml) with triethylamine (56 ul, 1 eq) at 0 ° C. Methylsulfonyl chloride (31 ul, 1 eq) was added dropwise, stirred at 0 ° C for 10 min, warmed to room temperature and stirred for 15 min. The reaction was quenched by the addition of brine, and the mesylate was extracted with CH₂Cl₂, dried over Na₂SW₄ and concentrated. The residue was dissolved in acetonitrile (3 ml), and triethylamine (31 ul, 1 eq) was added, followed by pyrrolidine (66 ul, 2 eq). The reaction mixture was stirred for 2 hours, concentrated, and treated with pentane. Column chromatography in CH₂Cl₂ (0 to 4% MeOH gradient) provided 6- (2-Nitro-phenyl) -3-pyrrolidin-1-ylmethyl-imidazo [2,1-b] thiazole. This material was dissolved in methanol (16 ml) and a solution of sodium hydrogen sulfide (112 mg, 5 eq) in water (4 ml) was added. The reaction mixture was refluxed for 2 days with additional charges of NaHS (2 x 112 mg). The reaction was concentrated to remove methanol, and the aqueous solution was extracted with CH2Cl2 (3 x 40 mL). The organic layer was dried over Na₂SW₄, and concentrated to obtain the product as a yellow film, 109 mg. (MS, M⁺ + H = 299.1)

Preparation of Compound 620:

<img file="ES2396913T3_D0064.tif" />

2- (3-Pyrrolidin-1-ylmethyl-imidazo [2,1-b] thiazol-6-yl) -phenylamine (200 umol) in 2 ml of pyridine was stirred with 3,4,5-trimethoxybenzoyl chloride (200 umol, 46 mg). The solution was stirred for 3 hours, concentrated to dryness, treated with Methanol, and purified by preparative HPLC. The fractions were lyophilized to obtain 36 mg of the product as a TFA salt. (MS, M⁺ + H = 493.1.)

Compound 619 was prepared in a manner analogous to Compound 620, using the appropriate acid chlorides. 84

ES 2 396 913 T3

Preparation of 6- (2-nitro-phenyl) -imidazo [2,1-b] thiazole-3-carboxylic acid ethyl ester:

<img file="ES2396913T3_D0065.tif" />

In a typical preparation, (2-Amino-thiazol-4-yl) -acetic acid methyl ester (1.0 g, 5.8 mmol) was mixed with 30 mL of methyl ethyl ketone together with 2-bromo-2 ' -nitroacetophenone (1.42 g, 5.8 mmol). The reaction mixture was refluxed for 1 hour and stirred at 90 ° C overnight. It was then cooled to room temperature and concentrated to a red oil. Attempts to precipitate the product by dissolving in methanol and adding water resulted in an emulsion. Methanol was removed by rotary evaporation, and the aqueous emulsion was charged to a separatory funnel. The pH was adjusted to 9 with NaHCO3, and the mixture was extracted with CH₂Cl₂ (3 x 50 ml). The combined organic layer was dried over Na2SO4, concentrated to a red oil, and purified by silica gel chromatography (CH2Cl2 with a 0 to 5% MeOH gradient). Obtained as a red solid (0.59 g, 32% yield). (MS, M⁺ + H = 318.0.)

Preparation of [6- (2-Nitro-phenyl) -imidazo [2,1-b] thiazol-3-yl] -acetic acid:

<img file="ES2396913T3_D0066.tif" />

6- (2-Nitro-phenyl) -imidazo [2,1-b] thiazole-3-carboxylic acid ethyl ester (466 mg, 1.47 mmol) in 2: 1 THF / Water was combined with 4 eq of NaOH (234 mg). The reaction mixture was heated at 50 ° C for 3 hours. The reaction mixture was concentrated to dryness, the residue was dissolved in water (20 ml), washed with CH2Cl2, and the aqueous layer was adjusted to pH = 3 with 4N HCl. The solids were collected by filtration, washed with water and dried to obtain the acidic product as a brown solid (442 mg, 99% yield) (MS, M⁺ + H = 304.0)

Preparation of Compound 649:

<img file="ES2396913T3_D0067.tif" />

[6- (2-Nitro-phenyl) -imidazo [2,1-b] thiazol-3-yl] -acetic acid (30 mg, 100 uMol), N-methylpiperazine (10 mg, 1, 0 eq) and N, N-Diisopropylethylamine (52 uL, 3.0 eq) in CH₂Cl₂. HOAT (16 mg, 1.2 eq) was added to the reaction mixture followed by EDCI (29 mg, 1.5 eq). The reaction stirred overnight. After adding 50% saturated NaHCO3 (2 mL) and extracting with CH2Cl2 (3 x 3 mL), the organic layer was dried over Na2SO4 and concentrated. Trituration with pentane provided the amide product as a brown solid.

The above amide was dissolved in methanol (4 ml) with NaHS (34 mg, 6 eq) and microwaved at 150 ° C for 30 min. MgSO was loaded₄ to the reaction mixture and after filtering, concentrating and treating with CH₂Cl₂ (2x) the desired aniline was obtained in the form of a red film.

The aniline from above was dissolved in pyridine (2 ml) and 2-quinoxalloyl chloride (38 mg, 2.0 eq) was charged as a solid. After stirring overnight, the reaction was concentrated to dryness and purified on HPLC.

Preparative ES 2 396 913 T3 to obtain the title compound as an orange solid (32.2 mg, 44% yield in stages). (MS, M⁺ + H = 512.2)

Preparation of Compound 650:

<img file="ES2396913T3_D0068.tif" />

To a vial was added 4- [6- (2-amino-phenyl) -imidazo [2,1-b] thiazol-3-ylmethyl] -piperazine-1-carboxylic acid tert-butyl ester (82 mg, 0.2 mmol), triethylamine (56 ul, 2 eq) and CH₂Cl₂ anhydrous (3 ml). 2-Quinoxaloyl (40 mg, 1.0 eq) was added as a solid. The reaction mixture was stirred for 18 hours, concentrated, and treated with CH2Cl2. Purification on silica gel with a CH eluent₂Cl₂ (with gradient 95: 4: 1 CH₂Cl₂: MeOH: Et₃N) provided Compound 650 as a yellow solid. (MS, M⁺ + H = 570.2)

Preparation of Compound 651:

<img file="ES2396913T3_D0069.tif" />

Compound 650 (105 mg, 0.185 mmol) was treated with 30% TFA in CH₂Cl₂ (4 ml) for 2 hours, treated with CH₂Cl₂ (3x) and ether (3x), to obtain crude Compound 441. Half of this material (92 umol) was dissolved in CH₂Cl₂ (5 ml), together with Et₃N (70 ul) and cooled to 0 ° C. Acetic anhydride (10 ul, 1 eq) was added and the reaction mixture was warmed to room temperature for 1 hour. The reaction was quenched with the addition of methanol and water, and concentrated to dryness. Reverse phase preparative HPLC purification and lyophilization provided Compound 651 as a TFA salt. (MS, M⁺ + H = 512.2)

Preparation of 2- [6- (2-Nitro-phenyl) -imidazo [2,1-b] thiazol-3-yl] -ethanol

<img file="ES2396913T3_D0070.tif" />

[6- (2-Nitro-phenyl) -imidazo [2,1-b] thiazol-3-yl] -acetic acid (300 mg, 1.0 mmol) was suspended in THF (20 ml) and stirred with NMM (110 ul, 1 eq) at room temperature for 1 hour. The reaction mixture was cooled to 0 ° C and isobutyl chloroformate (131 ul, 1 eq) was charged, and the reaction mixture was stirred at 0 ° C for 2 hours, at which time the mixed anhydride formation was complete. . A mixture of NaBH was added₄ (38 mg) in water (5 ml) dropwise at 0 ° C and warmed to room temperature and stirred for 1 hour. The reaction did not proceed to completion with 1 eq .; therefore, the addition of NaBH was repeated₄ with 3 eq of NaBH₄. The reaction was not complete after 1 hour, therefore the reaction mixture was concentrated to dryness, charged with fresh THF, followed by NaBH₄ (1 eq) and stirred overnight. LC-MS indicated that the reaction was complete, therefore the mixture was concentrated to dryness and CH was added.₂Cl₂ (50 ml) and water (20 ml). The layers parted, and the 86

ES 2 396 913 T3 aqueous layer was extracted with CH2Cl2 (3 x 50 mL). The combined organic layers were dried over Na2SO4 and concentrated to dryness. The product was purified on silica gel (Pentane with a gradient from 15% to 100%

EtOAc), concentrated and lyophilized from CH₃CN: H2O (160 mg, 55% yield). (MS, M⁺ + H = 290.0)

Preparation of 4- {2- [6- (2-nitro-phenyl) -imidazo [2,1-b] thiazol-3-yl] -ethyl} -piperazine-1-carboxylic acid tert-butyl ester:

<img file="ES2396913T3_D0071.tif" />

2- [6- (2-Nitro-phenyl) -imidazo [2,1-b] thiazol-3-yl] -ethanol (40 mg, 0.14 mmol) was dissolved in anhydrous CH2Cl2 and cooled to 0 ° C . Triethylamine (19ul, 1eq) was added followed by methanesulfonyl chloride (11ul, 1eq). The reaction mixture was warmed to room temperature and stirred for 30 min. LC-MS indicated that the reaction was incomplete, therefore the addition of Triethylamine (19 ul, 1eq) and methanesulfonyl chloride (11 ul, 1 eq) was repeated. The reaction mixture was quenched with the addition of 2 ml of brine, extracted with CH2Cl2 (2x2 ml), dried over Na2SO4 and concentrated to the mesylate as a yellow film.

The mesylate was dissolved in anhydrous acetonitrile (2 ml), Triethylamine (38 ul, 2 eq), and stirred with N-Boc-piperazine (26 mg, 2 eq) overnight. The reaction mixture was still exclusively the mesylate. The reaction mixture was charged with sodium iodide (41 mg) and stirred for 6 days, then purified by reverse phase preparative HPLC. Fractions were made alkaline with NaHCO₃ (sat), concentrated to remove CH3CN, and the aqueous layer was extracted with CH2Cl2. The organic layer was dried and concentrated to obtain the product as a yellow film. (MS, M⁺ + H = 458.2)

Preparation of Compound 680:

<img file="ES2396913T3_D0072.tif" />

4- {2- [6- (2-Nitro-phenyl) -imidazo [2,1-b] thiazol-3-yl] -ethyl} piperazine-1- carboxylic (24 mg, 0.05 mmol), NaHS (30 mg, 10 eq) and 5 ml of methanol. The reaction mixture was heated in the microwave at 150 ° C for 30 min. The reaction proceeded, but was not completed. An additional 30 mg of NaHS was loaded, and the reaction mixture was microwaved at 150 ° C for 30 minutes. Again, 15 mg of NaHS was loaded and microwaved at 160 ° C for 20 minutes. The solids were removed by filtration. The solution was dried over MgSO4 and concentrated to obtain the amine intermediate. This amine (0.05 mmol) was mixed with pyridine (3 ml), with 2-quinoxaloyl chloride (20 mg, 2 eq) and heated in the microwave at 160 ° C for 10 min. The reaction was partially complete, therefore after charging another two equivalents of 2-quinoxalloyl chloride (20 mg) the reaction was heated in the microwave for 20 minutes at 160 ° C. The reaction mixture was concentrated to dryness and purified by silica gel chromatography (eluent CH₂Cl₂, gradient 0 to 5% MeOH). The residue was treated with 25% TFA / CH₂Cl₂ for 3 hours, concentrated and purified on reverse phase preparative HPLC. (MS, M⁺ + H = 484.2).

ES 2 396 913 T3

Preparation of 3-Chloromethyl-6- (2-nitro-phenyl) -imidazo [2,1-b] thiazole:

<img file="ES2396913T3_D0073.tif" />

A solution of [6- (2-Nitro-phenyl) -imidazo [2,1-b] thiazol-3-yl] -methanol (1.0 g, 3.63 mmol) in anhydrous dichloromethane (15 ml) was Thionyl chloride (2 ml, 7.5 eq) was added slowly. The solution became homogeneous, followed by the development of a yellow precipitate. After 5 minutes, a catalytic amount of DMF (1 drop) was added and the mixture was stirred for 1 hour, concentrated to dryness, treated with CH2Cl2 (2x) then ether (1x) and dried under reduced pressure. 1.53 g of a yellow solid was obtained and was assumed to be a quantitative yield. (MS, M⁺ + H = 294.0)

Preparation of Compound 700:

<img file="ES2396913T3_D0074.tif" />

Displacement: 3-Chloromethyl-6- (2-nitro-phenyl) -imidazo [2,1-b] thiazole (126 mg, 0.300 mmol) in 2 ml of 1-methylpiperazine was heated in microwave at 110 ° C for 30 minutes . The reaction mixture was concentrated to dryness, and treated with methanol to obtain crude 3- (4-Methyl-piperazin-1-ylmethyl) -6- (2-nitro-phenyl) -imidazo [2,1-b] thiazole.

Nitro-reduction: The above residue was dissolved in ethanol (20 ml) and 10% palladium on carbon was added with stirring. The atmosphere was evacuated and refilled with nitrogen (3x) and ballooned with H2 (1 atm) for 18 hours. The reaction mixture was filtered through Celite, concentrated to dryness, and treated with CH₂Cl₂ and pentane to obtain the amine in the form of a red oil.

Amide formation: The above amine was dissolved in pyridine (3 ml), added to a microwave tube containing 2-quinoxalyl chloride (64 mg, 1.1 eq) and microwaved for 30 minutes at 160 ° C. Only 50% of the reaction was complete, therefore another portion of 2-quinoxalyl chloride was charged and heating continued for 30 minutes at 160 ° C. The reaction mixture was concentrated to dryness and purified by reverse phase preparative HPLC. (MS, M⁺ + H = 512.2)

Compounds 714, 715, 716 and 717 were prepared in a manner analogous to Compound 700, using the appropriate amines. (Boc protecting groups were removed by treatment with 25% TFA in CH2Cl2 for 3 hours, before purification).

Preparation of Compound 718:

<img file="ES2396913T3_D0075.tif" />

6- (2-Nitro-phenyl) -imidazo [2,1-b] thiazole-3-carboxylic acid ethyl ester (0.342 g, 1 mmol) was dissolved in 3: 1 Ethanol: THF (80 ml). 10% Pd / C (30 mg) was added to the reaction mixture and the reaction mixture was stirred under

ES 2 396 913 T3 a balloon of H2 (1 atm) for 7 days with periodic changes of additional catalyst. The reaction mixture was filtered through Celite, concentrated to dryness and treated with pentane to obtain the aniline as an orange solid, 289 mg.

A portion of the above aniline (56 mg, 200 umol) was dissolved in pyridine (4 ml) and stirred with 2-quinoxalyl chloride (46 mg 1.2 eq) overnight at room temperature. The reaction mixture was quenched with ethanol, concentrated to dryness, and treated with CH₂Cl₂/ pentane. The residue was dissolved in CH₂Cl₂ and washed with 50% NaHCO₃ saturated, aqueous, dried over Na₂SW₄. The product was purified on silica gel (CH₂Cl₂ with a gradient from 0 to 5% methanol). (MS, M⁺ + H = 444.1)

Compound 720 was prepared in an identical fashion to Compound 718, using [6- (2-Nitrophenyl) -imidazo [2,1-b] thiazol-3-yl] -acetic acid methyl ester as the starting material.

Preparation of Compound 719:

<img file="ES2396913T3_D0076.tif" />

6- {2 - [(Quinoxaline-2-carbonyl) -amino] -phenyl} -imidazo [2,1-b] thiazole-3-carboxylic acid ethyl ester was dissolved in 1:10 THF: Methanol (33 ml) and stirred with 1M aqueous NaOH (4 mL) overnight. The reaction was completed by LCMS. The reaction mixture was concentrated to remove organics and charged with water (20 mL). The aqueous alkaline (pH = 13) layer was washed with CH₂Cl₂ (2 x 20 ml). The aqueous layer was acidified (pH = 2) with 4M HCl and extracted with CH₂Cl₂ (3 x 20 ml). The combined organic layers were dried over Na₂SW₄, filtered and concentrated with pentane to obtain the desired product as an orange solid. (MS, M⁺ + H = 416.0)

Compound 721 was prepared in an identical fashion to Compound 719, using the analog methyl ester starting material.

Preparation of Compound 745:

<img file="ES2396913T3_D0077.tif" />

Displacement: 3-Chloromethyl-6- (2-nitro-phenyl) -imidazo [2,1-b] thiazole (0.200 mmol), imidazole (68 mg, 5 eq) and triethylamine (140 ul) were heated in acetonitrile (3 ml) at 110 ° C for 30 minutes in a microwave. The reaction mixture was concentrated to dryness.

Nitro-reduction: The residue from above was dissolved in methanol (6 ml), and a mixture of NaHS (67 mg, 6 eq) in water (1 ml) was added, and the reaction was stirred at 60 ° C overnight. The next morning, another portion of NaHS (67 mg, 6 eq) was loaded and the reaction was heated at 85 ° C for 3 hours. The reaction was cooled, concentrated to dryness, diluted with CH2Cl2 and water, and extracted with CH2Cl2 (2 x 40 mL). The combined organic layers were dried (Na2SO4) and concentrated.

Amide formation: The above amine was dissolved in pyridine (3 ml) and stirred with 2-quinoxalyl chloride (46 mg, 1.2 eq) at room temperature for 3 hours. The reaction mixture was concentrated to dryness and purified by reverse phase preparative HPLC. And it was lyophilized with HCl to obtain the HCl salt. (MS, M⁺ + H = 452.1)

ES 2 396 913 T3

Example 2: Identification of sirtuin modulators

A fluorescence polarization-based assay or mass spectrometry was used to identify modulators of SIRT1 activity. The same assay was used to identify modulators of any sirtuin protein. Fluorescence polarization assays use one or two different peptides based on a fragment of p53, a known sirtuin deacetylation target. Compounds 1-18 were tested using a substrate containing peptide 1, having 14 amino acid residues as follows: GQSTSSHSK (Ac) NleSTEG (SeC ID NO: 1) where K (Ac) is an acetylated lysine residue and Nle is a norleucine. The peptide is labeled with the MR121 fluorophore (635nm excitation / 680nm emission) at the C-terminus and with biotin at the N-terminus. The peptide substrate sequence is based on p53 with various modifications. In particular, all arginine and leucine residues other than acetylated lysine have been replaced with serine, so that the peptide is not susceptible to trypsin cleavage in the absence of deacetylation. Furthermore, the naturally present methionine residue in the sequence has been replaced with norleucine, since methionine can be susceptible to oxidation during synthesis and purification. Compounds 19-56 were tested using a substrate containing peptide 2, having 20 amino acid residues as follows: EE-K (biotin) GQSTSSHSK (Ac) NleSTEG-K (MR121) -EE-NH2 (SEQ ID NO: 2) where K (biotin) is a biotinylated lysine residue, K (Ac) is an acetylated lysine residue, Nle is norleucine and K (MR121) is a lysine residue modified by a MR121 fluorophore. This peptide is labeled with the MR121 fluorophore (635nm excitation / 680nm emission) at the C-terminus and with biotin at the N-terminus. The sequence of the peptide substrates are based on p53 with various modifications. In particular, all arginine and leucine residues other than acetylated lysine residues have been replaced with serine, so that the peptides are not susceptible to trypsin cleavage in the absence of deacetylation. Furthermore, the naturally present methionine residues in the sequences have been replaced with norleucine, since methionine can be susceptible to oxidation during synthesis and purification. As an alternative substrate in the assay, the following peptide 3 has also been used to test Compounds 19 to 56: Ac-EE-K (biotin) GQSTSSHSK (Ac) NleSTEG-K (5TMR) -EE-NH2 (SEQ ID NO. : 3) where K (Ac) is an acetylated lysine residue and Nle is a norleucine. The peptide is labeled with the 5TMR fluorophore (540nm excitation / 580nm emission) at the C-terminus. The peptide substrate sequence is also based on p53 with various modifications. Furthermore, the naturally occurring methionine residue in the sequence was replaced with norleucine, since methionine can be susceptible to oxidation during synthesis and purification.

Peptide substrates were exposed to a sirtuin protein in the presence of NAD⁺ to allow deacetylation of the substrate and make it sensitive to trypsin cleavage. Then trypsin was added and the reaction was run to completion (ie, the deacetylated substrate was cleaved) releasing the MR121 or 5TMR fragment. Streptavidin is then added to the reaction, where it can bind both the uncleaved substrate (ie, any remaining acetylated substrate) and the non-fluorescent portion of the cleaved peptide substrate (ie, the biotin-containing fragment). The fluorescence polarization signal observed for the streptavidin-bound full-length peptide substrates was higher than the fluorescence polarization signal observed for the released MR121 or 5TMR C-terminal fragment. In this way, the fluorescence polarization obtained is inversely proportional to the level of deacetylation (eg, the signal is inversely proportional to the activity of the sirtuin protein). The results were read on a microplate fluorescence polarization reader (Molecular Devices Spectramax MD) with suitable excitation and emission filters.

Fluorescence polarization assays using peptide 1 were carried out as follows: 0.5 pM peptide substrate and pNAD are incubated⁺ 150 pM with 0.1 pg / mL of SIRT1 for 60 minutes at 37 ° C in a reaction buffer (Tris-acetate 25, mM, pH8, 137 mM Na-Ac, 2.7 mM K-Ac, 1 mM Mg -Ac, 0.05% Tween-20, 0.1% Pluronic F127, 10 mM CaCl2, 5 mM DTT, 0.025% BSA, 0.15 mM nicotinamide). Test compounds 1-18 were solubilized in DMSO and added to the reaction at 11 concentrations ranging from 0.7 pM to 100 pM.

Fluorescence polarization assays using peptide 2 can be carried out as follows: 0.5 pM peptide substrate and pNAD were incubated⁺ 120 pM with 3 nM SIRT1 for 20 minutes at 25 ° C in a reaction buffer (25 mM Tris-acetate, pH8, 137 mM Na-Ac, 2.7 mM K-Ac, 1 mM Mg-Ac, 0.05 % Tween-20, 0.1% Pluronic F127, 10 mM CaCl2, 5 mM DTT, 0.025% BSA). Test compounds 19-56 were solubilized in DMSO and added to the reaction at 10 concentrations ranging from 300 pM to 0.15 pM in triple dilutions.

After incubation with SIRT1, nicotinamide was added to the reaction to a final concentration of 3 mM to stop the deacetylation reaction and 0.5 pg / mL of trypsin was added to cleave the deacetylated substrate. The reaction was incubated for 30 minutes at 37 ° C in the presence of 1 pM streptavidin. Fluorescent polarization was determined at excitation wavelengths (650 nm) and emissions (680 nm). The level of activity of the sirtuin protein in the presence of various concentrations of the test compound is then determined and can be compared with the level of activity of the sirtuin protein in the absence of the test compound, and / or with the level of activity of the proteins. sirtuin in the negative control (eg, inhibition level) and in the positive control (eg, activation level) described above.

For fluorescence polarization assays, a control for inhibition of sirtuin activity is carried out by adding 1 pL of 500 mM nicotinamide as a negative control at the beginning of the reaction (e.g., allows determination of maximum sirtuin inhibition ). A control was carried out for activation of the activity of

ES 2 396 913 T3 sirtuin using 3 nM sirtuin protein, with 1 pL DMSO in place of compound, to achieve initial substrate deacetylation (eg, to determine normalized sirtuin activity).

The mass spectrometry assay uses a peptide having 20 amino acid residues as follows: Ac-EE-K (biotin) -GQSTSSHSK (Ac) NleSTEG-K (5TMR) -EE-NH2 (SEQ ID NO: 3) where K (Ac) is an acetylated lysine residue and Nle is a norleucine. The peptide is labeled with the 5TMR fluorophore (excitation 540nm / emission 580nm) at the C-terminus. The peptide substrate sequence is based on p53 with various modifications. Furthermore, the naturally occurring methionine residue in the sequence was replaced with norleucine, since methionine can be susceptible to oxidation during synthesis and purification.

The mass spectrometric assay is carried out as follows: 0.5 pM peptide substrate and βNAD are incubated⁺ 120 pM with 10 nM SIRT1 for 25 minutes at 25 ° C in reaction buffer (50 mM Tris-acetate, pH 8, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCL, 5 mM DTT, 0.05% BSA). Test compounds can be added to the reaction as described above. The SirT1 gene is cloned into a vector containing the T7 promoter and transformed into BL21 (DE3). After a 25 minute incubation with SIRT1, 10 pL of 10% formic acid is added to stop the reaction. Reactions are sealed and frozen for later mass spectrometric analysis. Determination of the mass of the substrate peptide allows the precise determination of the degree of acetylation (ie, the starting material) compared to the deacetylated peptide (product).

For the mass spectrometry-based assay, a control for inhibition of sirtuin activity is carried out by adding 1 pL of 500 mM nicotinamide as a negative control at the beginning of the reaction (e.g., allows determination of sirtuin inhibition maximum). A control for activation of sirtuin activity is performed using 10 nM sirtuin protein, with 1 pL DMSO in place of the compound, to determine the amount of deacetylation of the substrate at a given time point within the linear range of the assay. This time point is the same as that used for the test compounds and, within the linear range, the time point represents a change in rate.

For each of the preceding assays, the SIRT1 protein was expressed and purified in the following manner. The SirT1 gene was cloned into a vector containing the T7 promoter and transformed into BL21 (DE3). The protein was expressed by induction with 1 mM IPTG as an N-terminal His-tagged fusion protein at 18 ° C overnight, and harvested at 30,000 x g. Cells were lysed with lysozyme in lysis buffer (50 mM Tris-HCl, 2 mM Tris [2-carboxyethyl] phosphine (TCEP), 10 pM ZnCl2, 200 mM NaCl) and further sonicated for 10 min for complete lysis. . The protein was purified on a Ni-NTA column (Amersham) and the fractions containing the pure protein were pooled, concentrated and run through a column (Sephadex S200 26/60 global). The soluble protein containing the peak was collected and passed through an ion exchange column (MonoQ). Gradient elution (200 mM - 500 mM NaCl) produced the pure protein. This protein was concentrated and dialyzed against dialysis buffer (20 mM Tris-HCl, 2 mM TCEP) overnight. The protein was aliquoted and frozen at -80 ° C until further use.

The sirtuin modulator compounds that activated SIRT1 were identified using the assay described above and are listed in Table 4. The ED50 values ​​for the activator compounds in the fluorescence polarization (FP) or mass spectrometry (MS) assay ) are represented by A '(ED50 = &lt;5pM), A (ED50 = 5-50 pM), B (ED50 = 51-100 pM), C (ED50 = 101-150 pM) and D (ED50 =&gt; 150 pM ). NT means that the compound was not tested using the indicated assay. NA means that the compound was not active in the indicated assay. Number of times of activation, as determined in the MS is represented by A (number of times of activation&gt; 250%), B (number of times of activation &lt;250%) or C (no number of times of activation). The resveratrol ED50 value for SIRT1 activation is 16 pM and the number of resveratrol activation times for SIRT1 in the MS assay is approximately 200%. Similarly, IC50 values ​​for inhibitory compounds are represented by A (IC50 = &lt;50 pM), B (IC50 = 51-100 pM), C (IC50 = 101-150 pM) and D (IC50 =&gt; 150 pM ).

ES 2 396 913 T3

Table 4. Sirt1 activators

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. TIMES OF ACT. MS</td>

<td> 178</td><td> 437</td><td>\ —OO HN \</td><td></td><td>to</td><td>to</td>

<td> 179</td><td> 467</td><td>\ —0 0 HN \</td><td></td><td>TO'</td><td>TO</td>

<td> 203</td><td> 380</td><td>\ —0 0 HN GO^-OR</td><td></td><td>TO'</td><td>B</td>

<td> 204</td><td> 410</td><td>\ -o ° HN ΌαΟ</td><td></td><td>TO'</td><td>TO</td>

ES 2 396 913 T3

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. TIMES OF ACT. MS</td>

<td> 205</td><td> 437</td><td>\ —Oo HN ^ = \ N— ' /</td><td></td><td>TO</td><td>TO</td>

<td> 206</td><td> 467</td><td>\ —0 O HN S ^ N ^ = \ N— ' /</td><td></td><td>TO</td><td>TO</td>

<td> 207</td><td> 478</td><td>\ —OO H. HN^S'rsA HN ^ ___ '</td><td></td><td>TO</td><td>TO</td>

<td> 208</td><td> 508</td><td>\ —0 0 ^ = 0 HN s- ^ n ^ = \ HN ^ __ / N— '</td><td></td><td>TO</td><td>TO</td>

ES 2 396 913 T3

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. TIMES OF ACT. MS</td>

<td> 209</td><td> 479</td><td>\ —O 0 0. HN __ N— '</td><td></td><td>TO</td><td>TO</td>

<td> 210</td><td> 509</td><td>\ —OO HN S - ^ - N ^ = \ __ / N— '</td><td></td><td>TO</td><td>B</td>

<td> 270</td><td> 478</td><td>\ —OO To HN or N— ' H</td><td></td><td>TO</td><td>TO</td>

<td> 271</td><td> 508</td><td>\ —OO HN or N—^FH</td><td></td><td>TO'</td><td>TO</td>

ES 2 396 913 T3

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. TIMES OF ACT. MS</td>

<td> 326</td><td> 478</td><td>-OR k HN S- ^ N) = \ HN \ N— '</td><td></td><td>TO</td><td>B</td>

<td> 327</td><td> 461</td><td>\ C HN HN N— '</td><td></td><td>TO</td><td>TO</td>

<td> 328</td><td> 418</td><td>k HN HN ^ __ N— '</td><td></td><td>B</td><td>TO</td>

<td> 329</td><td> 462</td><td>O ^ O » HN s- ^ ν X = \ HN ^ __ N—</td><td></td><td>TO</td><td>TO</td>

ES 2 396 913 T3

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. TIMES OF ACT. MS</td>

<td> 330</td><td> 443</td><td>.N rf HN V = \ HN \ N— '</td><td></td><td>TO</td><td>TO</td>

<td> 338</td><td> 458</td><td>o ^ and Vy / ° HN S - ^ - N ^ = \ HN \ __ / N— '</td><td></td><td>TO</td><td>TO</td>

<td> 440</td><td> 468</td><td>or / ° HN S - ^ - N} = \ yi-J ^ w HN ^ __ N— '</td><td></td><td>TO'</td><td>TO</td>

ES 2 396 913 T3

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. TIMES OF ACT. MS</td>

<td> 441</td><td> 470</td><td>Q HN S- ^ N / = \ HN ^ __ Nt</td><td></td><td>TO'</td><td>TO</td>

<td> 442</td><td> 472</td><td> ° / ⁰HN S - ^^ N /^&gt;= \ HN N— '</td><td></td><td>TO'</td><td>TO</td>

<td> 443</td><td> 436</td><td>Λθ HN s - ^^ n ^ = \ yL / vJ HN ^ __ / N— '</td><td></td><td>TO</td><td>TO</td>

<td> 444</td><td> 464</td><td>—S H. HN s_n) = \ VLZv? HN ^ __ N— '</td><td></td><td>TO</td><td>TO</td>

ES 2 396 913 T3

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. TIMES OF ACT. MS</td>

<td> 445</td><td> 432</td><td>/ ° HN ^ = \ yJw HN N— '</td><td></td><td>TO</td><td>B</td>

<td> 446</td><td> 424</td><td>HN γΐλν / HN \ __ N-!</td><td></td><td>TO</td><td>B</td>

<td> 510</td><td> 457</td><td>H HN s ^ n HN ^ __ / N— '</td><td></td><td>TO'</td><td>TO</td>

<td> 512</td><td> 484</td><td>^HNVC ^ / ⁰HN HN \ __ N— '</td><td></td><td>TO'</td><td>TO</td>

ES 2 396 913 T3

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. TIMES OF ACT. MS</td>

<td> 513</td><td> 470</td><td>Q HN OR N— ' H</td><td></td><td>TO'</td><td>TO</td>

<td> 543</td><td> 420</td><td>/ = \ N. N HN^S^ r ^^N\ HN \ __ ^ N— '</td><td></td><td>TO'</td><td>TO</td>

<td> 544</td><td> 474</td><td>^s / ⁰HN s „n HN ^ N— '</td><td></td><td>TO</td><td>TO</td>

ES 2 396 913 T3

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. TIMES OF ACT. MS</td>

<td> 617</td><td> 471,1</td><td>p NN // \ n HN 0 N— '</td><td></td><td>TO'</td><td>TO</td>

<td> 618</td><td> 469,1</td><td>Q HN S- ^ N p = \ CH</td><td></td><td>TO'</td><td>B</td>

<td> 619</td><td> 455</td><td>HN } = \ VnJ *^-\ j Qr</td><td></td><td>TO'</td><td>B</td>

100

ES 2 396 913 T3

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. TIMES OF ACT. MS</td>

<td> 620</td><td> 493,1</td><td>/ \ 0 0 HN \ = \ UJnJ Qr</td><td></td><td>TO'</td><td>B</td>

<td> 621</td><td> 472</td><td>or USA ¹ zX TO 0</td><td></td><td>TO'</td><td>TO</td>

<td> 623</td><td> 437</td><td>HN N ^ __ ^ N— '</td><td></td><td>ND</td><td>C</td>

<td> 624</td><td> 458</td><td>HN S '- ^ N / = \ HN ^ __ N — í</td><td></td><td>TO'</td><td>TO</td>

101

ES 2 396 913 T3

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. SOMETIMES OF ACT. MS</td>

<td> 625</td><td> 496</td><td>HN 5 = \ HN N— '</td><td></td><td>TO'</td><td>TO</td>

<td> 628</td><td> 574,1</td><td>Q Ho N S ^ N&gt; = \ Q «UJHU * N— 'm: OR</td><td></td><td>TO'</td><td>B</td>

<td> 644</td><td> 434</td><td>h^N&gt; or N S- ^ N 5 = x Vn __N— '</td><td></td><td>TO</td><td>TO</td>

<td> 645</td><td> 469</td><td>vz &gt; = ° N S ^^ N 5 = \ , _ ^ N ^ A N \ __</td><td></td><td>TO'</td><td>TO</td>

102

ES 2 396 913 T3

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. TIMES OF ACT. MS</td>

<td> 647</td><td> 473,1</td><td>N ) = \ —N 0— '</td><td></td><td>TO'</td><td>TO</td>

<td> 648</td><td> 402,1</td><td>N or-'</td><td></td><td>TO'</td><td>B</td>

<td> 649</td><td> 512,2</td><td>Q Ή HN _orCn ^ ~ O or</td><td></td><td>TO'</td><td>TO</td>

103

ES 2 396 913 T3

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. SOMETIMES OF ACT. MS</td>

<td> 650</td><td> 570,2</td><td>OR / XNN HN vO V^or</td><td></td><td>ND</td><td></td>

<td> 651</td><td> 512,2</td><td>Q NN HN me</td><td></td><td>TO'</td><td>TO</td>

<td> 676</td><td></td><td>N S ^ N / = \ Y or-⁷</td><td></td><td>TO'</td><td>TO</td>

104

ES 2 396 913 T3

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. SOMETIMES OF ACT. MS</td>

<td> 677</td><td></td><td>Q A · N NN— ' 0</td><td></td><td>TO'</td><td>TO</td>

<td> 678</td><td></td><td> \ %/^N'N HN S - ^ N 5 = \ XNz? A / r \ J^HNX /</td><td></td><td>TO</td><td>B</td>

<td> 679</td><td></td><td>F _hnH ^ ° \ S- ^ N) = \ V //^hnx 7</td><td></td><td>TO</td><td>B</td>

105

ES 2 396 913 T3

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. SOMETIMES OF ACT. MS</td>

<td> 680</td><td> 484,2</td><td>0 TO N or</td><td></td><td>TO'</td><td>TO</td>

<td> 692</td><td> 469</td><td>CH HN S ^ N 5 = \ HN ^ __ '</td><td></td><td>C</td><td>B</td>

<td> 695</td><td> 454</td><td>F HN S „N y * = \ HN ^ __ ^ N— '</td><td></td><td>TO'</td><td>TO</td>

106

ES 2 396 913 T3

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. SOMETIMES OF ACT. MS</td>

<td> 697</td><td> 596</td><td> /^OhHO — B X: HN S-'tísN&gt; = \₀ UHJ \ ^ -NN— ' Τ-⁰</td><td></td><td>nd</td><td>c</td>

<td> 698</td><td> 502</td><td>FF^F_X / = \ HN S- ^ NX = \ HN N— ' \ ___ /</td><td></td><td>TO'</td><td>TO</td>

<td> 699</td><td></td><td>Q TO N S- ^ sN 5 = \ Y or-^J</td><td></td><td>TO</td><td>TO</td>

107

ES 2 396 913 T3

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. TIMES OF ACT. MS</td>

<td> 700</td><td> 512,2</td><td>P N_v .N N s ^ ny = \</td><td></td><td>TO'</td><td>TO</td>

<td> 703</td><td> 468</td><td>\ —0 0 'Λ HN \</td><td></td><td>ND</td><td>C</td>

<td> 704</td><td> 454</td><td>\ —O 0 'T. HN or ^N- ^ r ^^s ) ^ \ JHJuZ-V?</td><td></td><td>ND</td><td>C</td>

108

ES 2 396 913 T3

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. TIMES OF ACT. MS</td>

<td> 707</td><td> 386,1</td><td>Q Ho HN 5 = \ h₃c</td><td></td><td>TO'</td><td>TO</td>

<td> 708</td><td> 494,2</td><td>V- / y = or HN S ^ N) = \ HN ^ ___ '</td><td></td><td>TO'</td><td>TO</td>

<td> 709</td><td> 494,1</td><td>HN^s^ ny = \ HN \ __ ^ N— '</td><td></td><td>ND</td><td>C</td>

<td> 710</td><td> 494,1</td><td>ΛΑ / = ° HN s- ^ ny = \ HN ^ __ N— '</td><td></td><td>TO'</td><td>TO</td>

109

ES 2 396 913 T3

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. TIMES OF ACT. MS</td>

<td> 711</td><td></td><td>Q N \ ArV-H » -TO- ON— ' or</td><td></td><td>TO</td><td>B</td>

<td> 714</td><td></td><td>TO. N s- ^ nx = \_F&lt;and</td><td></td><td>TO</td><td>B</td>

<td> 715</td><td></td><td>or N_k N N S'q ^ N, A \ S ^_N-&gt; l_V</td><td></td><td>TO'</td><td>B</td>

<td> 716</td><td></td><td>Q N S ^ N ,./Y</td><td></td><td>TO'</td><td>TO</td>

110

ES 2 396 913 T3

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. TIMES OF ACT. MS</td>

<td> 717</td><td></td><td>Q N</td><td></td><td>TO</td><td>B</td>

<td> 718</td><td> 444,1</td><td>Q N_K N N oA __ / 0</td><td></td><td>ND</td><td>C</td>

<td> 719</td><td> 416</td><td>Q TO N S - ^ - N 5 = \ o-4&gt; or</td><td></td><td>TO</td><td>TO</td>

<td> 720</td><td></td><td>or NN 5 = or N S- ^ N) = \ V _-or</td><td></td><td>ND</td><td>C</td>

111

ES 2 396 913 T3

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. TIMES OF ACT. MS</td>

<td> 721</td><td></td><td>Q AN S „N V 0</td><td></td><td>TO</td><td>TO</td>

<td> 733</td><td> 484,2</td><td>N '\ O zZ _ HV? Vy^N~ F^N'^ O</td><td></td><td>TO'</td><td>TO</td>

<td> 735</td><td> 514,2</td><td>9 h \\ n. &lt;^S&gt; N ^N_V V / Ζ ~~ λ Q OMe</td><td></td><td>TO'</td><td>TO</td>

<td> 736</td><td> 514,2</td><td>0 ^ X ^ -OMe í '^S') = N OR^{&gt; n}Aj</td><td></td><td>TO'</td><td>TO</td>

<td> 737</td><td> 500,1</td><td>OR _s .F Λ^Ν· ΖχΖ</td><td></td><td>TO'</td><td>TO</td>

<td> 738</td><td> 448,1</td><td>V ^^0MeA = t ¿rO V / -⁷ Ex</td><td></td><td>TO</td><td>TO</td>

112

ES 2 396 913 T3

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. TIMES OF ACT. MS</td>

<td> 739</td><td> 424,1</td><td>CmjCx ^oMe^h₃^eat</td><td></td><td>TO'</td><td>TO</td>

<td> 740</td><td> 377,1</td><td>H, C 9 _ZL_CHH_SC</td><td></td><td>TO'</td><td>B</td>

<td> 741</td><td> 498,1</td><td>Q N^S^ -r ^^N / = \ - NN— ' M or</td><td></td><td>TO'</td><td>TO</td>

<td> 742</td><td> 487,1</td><td>Q NN ^ = 0 N S „N} = \ p- /</td><td></td><td>TO'</td><td>B</td>

113

ES 2 396 913 T3

<td>COMPOUND NO.</td><td>[M + H] +</td><td>STRUCTURE</td><td>ED50 FP TEST</td><td>ED50 MS TEST</td><td>NO. TIMES OF ACT. MS</td>

<td> 743</td><td> 466,1</td><td>or-^S'-^N X = \ ^F</td><td></td><td>TO</td><td>TO</td>

<td> 744</td><td> 437,1</td><td>F OR_K ) = N hnHJ} - \</td><td></td><td>B</td><td>TO</td>

<td> 745</td><td> 452,1</td><td>0 N. N N &gt; = \ U = /</td><td></td><td>TO'</td><td>TO</td>

Example 3: Identification of sirtuin modulators using SIRT3

A fluorescence polarization assay was used to identify modulators of SIRT3 activity. The same assay was used to identify modulators of any sirtuin protein. The assay uses a 5-peptide substrate based on a Histone H4 fragment, a known sirtuin deacetylation target. The substrate contains a peptide having 14 amino acid residues as follows: Biotin-GASSHSK (Ac) VLK (MR121) (SEQ ID NO: 4) where K (Ac) is an acetylated lysine residue. The peptide is labeled with the MR121 fluorophore (635nm excitation / 680nm emission) at the C-terminus and with biotin at the N-terminus.

The peptide substrate was exposed to a sirtuin protein in the presence of NAD⁺ to allow deacetylation of the substrate and make it sensitive to trypsin cleavage. Trypsin is then added and the reaction is carried out to completion (ie, the deacetylated substrate is cleaved) releasing the MR121 or TMR fragment. Streptavidin is then added to the reaction, where it can bind both the uncleaved substrate (ie, any remaining acetylated substrate) and the non-fluorescent portion of the cleaved peptide substrate (ie, the biotin-containing fragment). The fluorescence polarization signal observed for the streptavidin-bound full-length peptide substrate is higher than the fluorescence polarization signal observed for the released MR121 or TMR terminal C-fragment. Therefore, the fluorescence polarization obtained is inversely proportional to the level of deacetylation (e.g., the signal is inversely proportional to the activity of the protein

114

ES 2 396 913 T3 sirtuin). The results are read on a microplate fluorescence polarization reader (Molecular Devices

Spectramax MD) with suitable excitation and emission filters.

Fluorescence polarization assays can be performed as follows: 0.5 pM peptide substrate and βNAD are incubated⁺ 50 pM with 2 nM SIRT3 for 60 minutes at 37 ° C in a reaction buffer (25 mM Trisacetate, pH8, 137 mM Na-Ac, 2.7 mM K-Ac, 1 mM Mg-Ac, 0.1% Pluronic F127, 10 mM CaCl2, 1 mM TCEP, 0.025% BSA). Test compounds are solubilized in DMSO and added to the reaction at 11 concentrations ranging from 0.7 pM to 100 pM. The SIRT3 protein used in the assays corresponded to amino acid residues 102-399 of N-terminal His-tagged human SIRT3. The protein was overexpressed in E. coli and purified on a nickel chelate column using standard techniques. After a 60 minute incubation with SIRT3, nicotinamide is added to the reaction to a final concentration of 3 mM to stop the deacetylation reaction and 0.5 pg / mL of trypsin is added to cleave the deacetylated substrate. The reaction is incubated for 30 minutes at 37 ° C in the presence of 1 mM streptavidin. Fluorescent polarization is determined at excitation wavelengths (650 nm) and emissions (680 nm). The level of activity of the sirtuin protein in the presence of various concentrations of the test compound is then determined and can be compared with the level of activity of the sirtuin protein in the absence of the test compound, and / or with the level of activity of the proteins. sirtuin in the negative control (e.g. inhibition level) and positive control (e.g. inhibition level). g., activation level) previously described.

A control for inhibition of sirtuin activity is carried out, adding 30 mM nicotinamide at the beginning of the reaction (eg, allows the determination of the maximum inhibition of sirtuin). A control for activation of a sirtuin activity is performed using 0.5 pg / mL sirtuin protein to achieve initial deacetylation of the substrate (eg, to determine normalized sirtuin activity).

EXAMPLE 4: Cell-based sirtuin activity assays

Fat mobilization test. 3T3 L1 cells are plated with 2 ml of 30,000 cells / ml in Dulbecco's Modified Eagle's Medium (DMEM) / 10% newborn calf serum in 24-well plates. Individual wells are allowed to differentiate by addition of 100 nM Rosiglitazone. Undifferentiated control cells are kept in DMEM / 10% new newborn calf serum throughout the assay. At 48 hours (2 days), adipogenesis is initiated by the addition of DMEM / 10% fetal calf serum / 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) / 1 pM dexamethasone. At 96 hours (4 days), adipogenesis is allowed to advance by removing the media and adding 2 ml of DMEM / 10% fetal calf serum along with 10 pg / ml of insulin or 100 nM of Rosiglitazone. At 144 hours (6 days) and 192 hours (8 days), all wells are switched to DMEM / 10% fetal calf serum.

At 240 hours (10 days after original plating of cells), test compounds in a range of concentrations are added to individual wells in triplicate, along with 100 nM Rosiglitazone. Three wells of undifferentiated cells are kept in DMEM / 10% newborn calf serum and three wells of differentiated control cells are kept in fresh DMEM / 10% newborn calf serum medium with 100 nM Rosiglitazone. As a positive control for fat mobilization, resveratrol (an activator of SIRT1) is used at concentrations ranging in triple dilutions from 100 pM to 0.4 pM.

At 312 hours (13 days), the medium is removed and the cells are washed twice with PBS. 0.5 mL of Solution Red O is added to the oil (provided in the Adipogenesis Assay Kit, Cat. No. ECM950, Chemicon International, Temecula, CA) per well, including wells that do not have cells as a background control. The plates are incubated for 15 minutes at room temperature and then the red dye solution O is extracted into the oil and the wells are washed 3 times with 1 mL of wash solution (Adipogenesis Assay Kit). After the last wash is removed, the stained plates are viewed, scanned, or photographed. The dye is removed (Adipogenesis Assay Kit) and quantitated on a plate reader at 520 nM. The quantitative and visual results are shown in Figure 16.

Primary root ganglion cell (DRG) protection assay. Test compounds are tested in an axon protection assay as described (Araki et al. (2004) Science 305 (5686): 1010-3). Briefly, mouse DRG explants of E12.5 embryos are grown in the presence of 1 nM nerve growth factor. Non-neuronal cells are removed from the cultures, adding 5-fluorouracil to the culture medium. Test compounds are added 12 to 24 hours prior to axon transections. Neurite transection is performed at 10-20 days in vitro (DIV) using an 18 gauge needle to remove the neuronal cell bodies.

EXAMPLE 5: Cell-based ATP assay

This example describes the effect of the SIRT1 activator, resveratrol on cellular ATP levels in NCI-H358 cells. Cellular ATP levels are indirectly measured by cellular metabolic rates and, by extension, mitochondrial function. Since SIRT1 activation has been linked to increased mitochondrial biogenesis in vivo, this study is designed to determine whether resveratrol increases mitochondrial function, using cellular ATP levels as the reading. The ATP assay is combined with the cell viability assay so that cellular ATP levels can be normalized to viable cells. ATP levels

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Cellular ES 2 396 913 T3 were measured using the ATPLite 1Step kit (PerkinElmer), and cell viability was measured using the AlamarBlue ™ cell permeable stain.

The cellular ATP assay is a multiplexing assay that measures both the ATP levels and the viability of a given cell sample. This assay is performed in a 96-well assay plate and data is reported as [ATP] / viability for each well of the assay plate.

The ATPLite 1Step ™ Kit is a luminescent cell-based assay for the detection of ATP. The kit contains a lyophilized substrate mixture, comprised of D-luciferin and the firefly enzyme (Photinus piralis) luciferase. The kit also contains a detergent-based reconstitution buffer that induces lysis of cell membranes. Luciferase in the assay mixture catalyzes a reaction between free cellular ATP and D-luciferin to produce bioluminescence according to the schematic reaction outlined below. The amount of light produced is proportional to the concentration of cellular ATP.

The AlamarBlue ™ Assay is a single-step assay that uses a soluble, non-toxic cell-permeable dye that is added to the growth medium of the cells. This dye undergoes electron reduction in viable cells but not in dead cells. The reduced dye product provides a fluorescent signal that can be monitored with a fluorescence plate reader (545 nm excitation and 575 nm emission). The amount of fluorescence generated in a given well is proportional to the number of viable cells. The viability signal generated by this assay is used to normalize the ATP signal from ATPLite 1Step ™ assay results.

Preparation of a test substance for the cellular ATP assay: Resveratrol was weighed and placed in a brown vial. The material was dissolved in 100% vehicle (DMSO) to provide a final concentration of 10 mM (stock solution). The stock solution was serially diluted with 100% DMSO as described in SOP 7.10. Final resveratrol concentrations in the compound plate were 0.008, 0.023, 0.069, 0.206, 0.617, 1.852, 5.556, 16.67, 50, and 150 pM.

The effect of resveratrol on cellular ATP levels in NCI-H358 cells (100 pL) was examined using the described cellular ATP assay. The experimental design is summarized in Figure 1. In this assay, NCI-H358 cells (obtained from American Tissue Culture Collection, ATCC) are seeded into 96-well microplates (10⁴ cells / well). The NCI-H358 growing culture medium consists of RPMI 1640 medium enriched with 10% FBS, 100 mg / mL streptomycin, and 100 units / mL penicillin. Cell microplates were treated in triplicate with 15 pL of 10 concentrations of resveratrol (0.008, 0.023, 0.069, 0.206, 0.617, 1.852, 5.556, 16,667, 50 and 150 pM) or 15 pL of vehicle (DMSO; final concentration of 0, 5%; 12 replicates per plate). After 48 hours of compound treatment under cell growth conditions, the plates were removed from the incubator and 15 µl of AlamarBlue ™ dye was added to each well. Cell microplates were incubated with dye for 2 hours under growth conditions, and then fluorescence was measured using a plate reader. The medium containing AlamarBlue ™ was removed and the plates were washed in 100 µl of PBS per well. This wash was removed and 200 µl of 1x ATPLite 1Step reagent was added to each well. Luminescence was then measured using a plate reader. The ATP signal for each well, measured by luminescence scan, was normalized to its corresponding cell viability value, measured by fluorescence scan, to generate the average ATP level per viable cell unit (ATP / vCell). The ATP / vCell for each treatment was then normalized to the average ATP / vCell vehicle for its respective cell chip, yielding the normalized ATP / vCell (norm. ATP / vCell). Finally the normalized ATP / vCell for each single treatment was averaged over the plaque replicas, generating the average normalized ATP / vCell. Resveratrol doses that increase cellular ATP levels have normalized ATP / vCell values ​​greater than 1.0. The concentration of resveratrol producing 50% of the maximum increase in normalized ATP / vCell (EC50 ATP) was determined by best fit curve analysis using a sigmoid dose response curve model.

The ATP levels of cells treated with 10 concentrations of resveratrol or vehicle alone were measured. Each of these ATP levels was normalized to cell viability in the corresponding treatment well, generating the ATP / vCell value. Each ATP / vCell value was subsequently normalized to its average ATP / vCell vehicle values ​​for its respective cell microplate.

Data is represented as normalized ATP / vCell (arbitrary units). Figure 2 indicates the best fit sigmoid dose and response curve for the 10 resveratrol concentrations plotted against the corresponding normalized ATP / vCell values. These values ​​represent an average of the three replicates of the plates. Resveratrol increases ATP levels in NCI-H358 cells in a dose-dependent manner. The maximum increase in cellular ATP levels was 3.0 times and occurred with the 50 pM resveratrol treatment. The EC50 value of ATP for resveratrol was determined at 29 pM.

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