Cytotoxic compounds

p00001Cytotoxic compounds

p00002Background of the invention

p00003Many therapeutic agents, particularly those that are especially effective in cancer chemotherapy, frequently show acute toxicity in vivo, especially bone marrow and mucosal toxicity, as well as chronic cardiac and neurological toxicity. Such high toxicity may limit its applications. The development of more specific and safer therapeutic agents, particularly anti-tumor agents, is desirable for greater efficacy against tumor cells and a decrease in the number and severity of the side effects of these products (toxicity, destruction of non-tumor cells, etc.). Another difficulty with some of the existing therapeutic agents is their less than optimal plasma stability. The addition of functional groups to stabilize these compounds has resulted in a significant decrease in activity. Accordingly, it is desirable to identify ways to stabilize compounds while maintaining acceptable levels of therapeutic activity.

p00004The investigation of more selective cytotoxic agents has been extremely active for many decades, with doses that limit toxicity (that is, the undesirable activity of cytotoxins on normal tissues) one of the main causes of failures in cancer therapy. For example, it is known that CC-1065 and duocarmycins are extremely potent cytotoxins.

p00005The CC-1065 was first isolated from the Streptomyces zelenensis in 1981 by the Upjohn Company (Hanka et al., J. Antibiot., Vol. 31, p. 1211, (1978); Martin et al., J. Antibiot., Vol .33, p. 902, (1980); Martin et al., J. Antibiot., Vol. 34, p. 1119, (1981)) and was found to have potent antitumor and antimicrobial activity both in vitro and in experimental animals (Li et al., Cancer Res., Vol. 42, p. 999, (1982)). CC-1065 binds to double-stranded B-DNA within the minor groove (Swenson et al., Cancer Res., Vol. 42, p. 2821, (1992)) with the 5'-d preference sequence ( A / GNTTA) -3 'and 5'-d (AAAAA) -3' and alkylates in the N3 position of 3'-adenine by their left-side CPI unit present in the molecule (Hurley et al., Science, vol .226, p. 843, (1984)). Despite its potent and broad antitumor activity, CC-1065 cannot be used in humans because it causes delayed death in experimental animals.

p00006Many analogs and derivatives of CC-1065 and duocarmycins are known in the art. Research on the structure, synthesis and properties of many of the compounds has been reviewed. See, for example, Boger et al., Angw. Chem. Int. Ed. Engl., Vol. 35, p. 1438, (1996); and Borger et al., Chem. Rev., vol. 97, p. 787, (1997).

p00007Similarly, analogs of CC-1065 and duocarmycins have been disclosed in WO 98/11101 and WO 02/096910, respectively.

p00008A group of the Kyowa Hakko Kogya Co., Ltd. has prepared a certain number of derivatives of CC-1065. See, for example, US Pat. Nos. 5,101,038; 5,641,780; 5,187,186; 5,070,092; 5,703,080; 5,070,092; 5,641,780; 5,101,038; and 5,084,468; and the published PCT Application WO 96/10405 and the European Application 0 537 575 A1 published.

p00009Similarly, the Upjohn Company (Pharmacia Upjohn) has been active in the preparation of derivatives of CC-1065. See, for example, US Pat. Nos. 5,739,350; 4,978,757; 5,332,837 and 4,912,227.

p00010Similarly, the Scripps Research Institute has described a variety of derivatives and analogs of CC-1065 and duocarmycins. See, for example, US Pat. Nos. 5,985,908; 6,060,608; 6,262,271; 6,281,354; 6,310,209; and 6,486,326; and PCT Publication No. WO 97/32850; WO 97/45411; WO 98/52925; WO 99/19298; WO 99/29642 and WO 01/83482. In particular, analogs have been described that incorporate the 1,2,9,9a-tetrahydrocyclopropa [c] benz [e] indole-4-one (CBI) alkylation subunit referred to as CBI analogs of CC-1065 and duocarmycins. See, for example, US Pat. No. 6,548,530 and PCT Publication No. WO 97/12862; WO 03/022806; and WO 04/101767.

p00011Likewise, the research has focused on the development of new therapeutic agents that are in the form of prodrugs, compounds that are capable of becoming drugs (active therapeutic compounds) in vivo by means of certain chemical compounds or enzymatic modifications of their structure. For the purpose of toxicity reduction, this conversion is preferably confined to the site of action or target tissue rather than the circulatory system or non-target tissue. However, even prodrugs are problematic as many are characterized by low blood and serum stability, due to the presence of enzymes that degrade or activate prodrugs before the prodrugs reach the desired sites within the patient's body.

p00012Bristol-Myers Squibb has described conjugates of antitumor drugs cleavable by particular lysosomal enzymes. See, for example, US Pat. No. 6,214,345. This patent provides an aminobenzyl oxycarbonyl.

p00013Seattle Genetics has published the US Patent Application applications. 2003/0096743 and the US Patent Application 2003/0130189, which describe p-aminobenzyl ethers in drug delivery agents. The linkers described in these applications are limited to aminobenzyl ether compositions.

p00014Similarly, other groups have described linkers. See, for example, de Groot et al., J. Med. Chem., Vol. 42, p. 5277, (1999); de Groot et al., J. Org. Chem., Vol. 43, p. 3093, (2000); de Groot et al., J. Med. Chem., vol. 66, p. 8815, (2001); WO 02/083180; Carl et al., J. Med. Chem. Lett., Vol. 24, p. 479, (1981); Dubowchik and others, Bioorg & amp; Med. Chem. Lett., Vol. 8, p. 3347, (1998). These linkers include aminobenzyl ether spacers, cyclic spacers and elongated electronic cascade systems, cyclisation elimination spacers, such as w-amino aminocarbonyls, and a p-aminobenci oxycarbonyl ether linker.

p00015The stability of cytotoxin drugs, including stability in vivo, is still an important goal that needs to be addressed. In addition, the toxicity of many compounds makes them less useful, therefore, compositions that reduce the toxicity of the drug, such as the formation of a cleavable prodrug, are necessary. Therefore, despite advances in the technique, the development of improved therapeutic agents for the treatment of mammals, and humans in particular, more specifically cytotoxins that show high specificity of action, reduced toxicity, and improved stability in blood relative to known compounds of similar structure. The present invention addresses these needs.

p00016Summary of the invention

p00017The present invention relates to cytotoxic compounds according to claim 1 useful as drugs.

p00018Detailed description of the invention Abbreviations

p00019As used in the present invention, "Ala" refers to alanine.

p00020"Boc" refers to t-butyloxycarbonyl.

p00021"CPI" refers to cyclopropapyrrolindole.

p00022"Cbz" refers to carbobenzoxy. As used in the present invention, "DCM" refers to dichloromethane.

p00023"DDQ" refers to 2,3-dichloro-5,6-dicyano-1,4-benzoquinone.

p00024DIPEA is diisopropylethylamine.

p00025"DMDA" is N, N'-dimethylethylene diamine.

p00026"RDF" is a round bottom flask.

p00027"DMF" is N, B-dimethylformamide.

p00028"HATU" is N - [[(dimethylamino) -1H-1,2,3-triazolo [4,5-b] -pyridin-1-yl] methylene] N-oxide hexafluorophosphate

p00029methylmethanemonium As used in the present invention, the symbol "E" represents an enzymatically cleavable group.

p00030"EDCP" is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide.

p00031As used in the present invention, "FMOC" refers to 9-fluorenylmethyloxycarbonyl.

p00032"FMOC" refers to 9-fluorenylmethoxycarbonyl.

p00033"HOAt" is 7-aza-1-hydroxybenzotriazole.

p00034"Leu" is leucine.

p00035"PABA" refers to para-aminobenzoic acid.

p00036PEG refers to polyethylene glycol.

p00037"PMB" refers to para-methoxybenzyl.

p00038"TBAF" refers to tetrabutylammonium fluoride. The abbreviation "TBSO" refers to t-butyldimethylsilyl ether.

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p00040As used in the present invention, "TEA" refers to triethylamine.

p00041"TFA" refers to trifluoroacetic acid.

p00042The symbol "Q" refers to a therapeutic agent, diagnostic agent or detectable label.

p00043Definitions

p00044Unless otherwise defined, all technical and scientific terms used in the present invention generally have the same meaning commonly known to a person skilled in the art to which the present invention pertains. Generally, the nomenclature used in the present invention and laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described below are those well known and commonly used in the art. Conventional techniques are those used for the synthesis of peptides and nucleic acid. Generally, enzymatic reactions and purification steps are performed according to the manufacturers specifications. The techniques and procedures are generally performed in accordance with conventional procedures in the art and various general references (see, in general, Sambroook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., (1989), Cold Spring Harbor Laboratory Press , Cold Spring Harbor, NY), which are provided throughout this document. The nomenclature used in the present invention and the analytical procedures in analytical chemistry and organic synthesis described below are those well known and commonly used in the art. Conventional techniques, or modifications thereof, are used for chemical synthesis and chemical analysis.

p00045The term "therapeutic agent" is intended to indicate a compound that, when present in a therapeutically effective amount, produces a desired therapeutic effect on a mammal. For the treatment of carcinomas, it is desirable that the therapeutic agent be equally capable of entering the target cell.

p00046The term "cytotoxin" is intended to indicate a therapeutic agent that has the desired effect of being cytotoxic for cancer cells. Cytotoxic means that the agent stops the growth of, or kills, the cells. Examples of cytotoxins include, by way of example, combretastatins, duocarmycins, antitumor antibiotics CC-1065, anthracyclines, and related compounds. Other cytotoxins include mycotoxins, ricin and its analogs, caliquiamycins, doxirubicin and maytansinoids.

p00047The term "prodrug" and the terms "drug conjugate" and "cleavable drug-substrate conjugate" are used interchangeably in the present invention. Both refer to a compound that is relatively harmless to cells while still in the conjugated form, but which is selectively degraded to a pharmacologically active form due to certain conditions, for example, enzymes, located within or in the vicinity. of target cells.

p00048The term "marker" is intended to indicate a compound useful in the characterization of tumors or other medical condition, for example, diagnosis, progression of a tumor, and assay of factors secreted by tumor cells. Markers are considered a subset of "diagnostic agents."

p00049The term "selective" used in connection with enzymatic cleavage means means that the cleavage rate of the linker moiety is greater than the cleavage rate of a peptide having a random amino acid sequence.

p00050The term "self-immolating spacer" refers to a bifunctional chemical moiety that is capable of covalently linking two chemical moieties within a normally stable tripartite molecule. The self-immolating spacer is capable of spontaneously separating the second moiety if the junction with the first molecule is cleaved.

p00051The terms "polypeptide", "peptide" and "protein" are used interchangeably in the present invention to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one more amino acid residues is a naturally occurring artificial chemical mimetic of a corresponding amino acid, as well as naturally occurring amino acid polymers and an amino acid polymer that It does not occur naturally. Likewise, these terms encompass the term "antibody."

p00052The term "amino acid" refers to synthetic and naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. The naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are subsequently modified, for example, hydroxyproline, and carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as the naturally occurring amino acid, that is, a carbon to which it is attached to a hydrogen, a carboxyl group, an amino group, and an R group , for example, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (for example, norleucine) or the main modified peptide structures, but retain the same basic chemical structure as a naturally occurring amino acid. An amino acid that can be used in particular is citrulline, which is a precursor to arginine and is involved in the formation of urea in the liver. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of a

55 E06839792 04-11-2011

p00054amino acid, but they work in a manner similar to an amino acid that occurs naturally. The term "unnatural amino acid" is intended to represent the "D" stereochemical form of the twenty naturally occurring amino acids described above. In addition, it is understood that the term "unnatural amino acid" includes homologues of natural amino acids, and synthetically modified forms of natural amino acids. Synthetically modified forms include amino acids having shortened or elongated alkylene chains by up to two carbon atoms, amino acids comprising optionally substituted aryl groups, and amino acids comprising halogenated groups, preferably halogenated alkyl and aryl groups. When bound to a linker or conjugate of the invention, the amino acid is in the form of an "amino acid side chain", in which the carboxylic acid group of the amino acid has been replaced with a keto (C (O)) group. Thus, for example, an alanine side chain is -C (O) -CH (NH2) -CH3, etc.

p00055Amino acids and peptides can be protected by blocking groups. A blocking group is an atom or a chemical moiety that protects the N-terminal of an amino acid or a peptide from unwanted reactions and can be used during the synthesis of a cleavable drug-substrate conjugate. It should remain attached to the N-terminal during the synthesis, and can be eliminated after the synthesis of the drug conjugate is completed by chemical conditions or other conditions that selectively achieve its elimination. Suitable blocking groups for the protection of the N-terminal are well known in the art of peptide chemistry. Examples of blocking groups include hydrogen, D-amino acid, and carbobenzoxy chloride (Cbz).

p00056"Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in both single-stranded and double-stranded forms. The term encompasses nucleic acids that contain known nucleotide analogs or moieties or bonds of modified major structures, which are synthetic, produced naturally or unnaturally, which have similar binding properties to those of the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs).

p00057Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences, as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions can be made by generating sequences in which the third position of one or more (or all) of the selected codons is substituted with mixed base and / or deoxyinosine moieties (Batzer et al., Nucleic Acid Res., Vol. 19, p. 5081, (1991); Ohtsuka et al., J. Biol. Chem., Vol. 260, pp. 2605-2608, (1985); Rossolini et al., Mol. Cell. Probes, vol. 8, pp. 91-98, (1994). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

p00058The symbol - when used as a link or is represented perpendicular to a link indicates the point at which the represented moiety is attached to the remaining part of the molecule, solid support, etc.

p00059The term "alkyl", by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono - or polyunsaturated and can include di- and multivalent radicals, which have the number of designated carbon atoms (ie, C1-C10 means one to ten carbons). Examples of saturated hydrocarbon radicals include groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, secbutyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, homologs and isomers of, for example, n -pentyl, n-hexyl, nheptyl, n-octyl, and the like. An unsaturated alkyl group is one that has one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2,4-pentadienyl, 3- (1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The term "alkyl" unless otherwise indicated, is also intended to include those alkyl derivatives defined in more detail below, such as "heteroalkyl." Alkyl groups, which are limited to hydrocarbon groups are called "homoalkyl."

p00060The term "alkylene", by itself or as part of another substituent, means a divalent radical obtained from an alkane, as exemplified by -CH2CH2CH2CH2-, and also includes those groups described below as "heteroalkylene". Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, those groups having 10 or less carbon atoms being preferred in the present invention. A "lower alkylene" is a shorter chain alkylene group, generally containing eight or less carbon atoms.

p00061The term "heteroalkyl", by itself or in combination with another term, means, unless otherwise stated, a straight or branched chain, or a cyclic hydrocarbon radical, or combinations thereof, consisting of the established number of carbon atoms and at least one heteroatom selected from the group consisting of O, N, Si and S, and in which the nitrogen atoms, carbon and sulfur may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom (s) O, N and S may be located at any interior position of the heteroalkyl group or at the position in which the alkyl group is attached to the rest of the molecule. Examples include -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N (CH3) -CH3, -CH2-S-CH2-CH3, -CH2-CH2-, -S (O) -CH3, -CH2-CH2-S (O) 2-CH3, -CH = CH-O-CH3, -Si (CH3) 3, -CH2-CH = N-OCH3, and

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p00063CH = CH-N (CH3) -CH3. Up to two heteroatoms can be consecutive, such as, for example, -CH2-NH-OCH3, and -CH2-O-Si (CH3) 3. Similarly, the term "heteroalkylene", by itself or as part of another substituent means a divalent radical obtained from heteroalkyl, as exemplified by -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2 -CH2-NH-CH2-. For heteroalkylene groups, heteroatoms can also occupy both one and both ends of the chain (for example, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamine, and the like). The terms "heteroalkyl" and "heteroalkylene" encompass poly (ethylene glycol) and its derivatives (see, for example, Shearwater Polymers Catalog, 2001). Furthermore, for alkylene and heteroalkylene linking groups, the orientation of the binding group is not implied by the direction in which the formula for the binding group is written. For example, the formula -C (O) 2R'- represents both –C (O) 2R 'and -R'C (O) 2-.

p00064The term "lower" in combination with the terms "alkyl" or "heteroalkyl" refers to a moiety having from 1 to 6 carbon atoms.

p00065The terms "alkoxy", "alkylamino", "alkylsulfonyl", and "alkylthio" (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the rest of the molecule by an oxygen atom, a amino group, an SO2 group or a sulfur atom, respectively. The term "arylsulfonyl" refers to an aryl group attached to the rest of the molecule by an SO2 group, and the term "sulfhydryl" refers to a SH group.

p00066In general, an "acyl substituent" is also selected from the group established above. As used in the present invention, the term "acyl substituent" refers to groups bound to, and that saturates the valence of a carbonyl atom that is either directly or indirectly attached to the polycyclic core of the compounds of the present invention. .

p00067The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of substituted or unsubstituted "alkyl" and substituted or unsubstituted "heteroalkyl," respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position to which the heterocycle is attached to the rest of the molecule. Examples of cycloalkyl include cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1- (1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3morphlinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2piperazinyl. The heteroatoms and carbon atoms of the cyclic structures are optionally oxidized.

p00068The terms "halo" or "halogen", by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are intended to include monohaloalkyl and polyhaloalkyl. For example, the term "halo (C1-C4) alkyl" is understood to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl.

p00069The term "aryl" means, unless otherwise stated, a substituted or unsubstituted hydrocarbon, aromatic, polyunsaturated substituent, which may be a single ring or multiple rings (preferably from 1 to 3 rings), which are fused together or covalently united. The term "heteroaryl" refers to aryl groups (or rings) containing from one to four heteroatoms selected from N, O, and S, in which the nitrogen, carbon and sulfur atoms are optionally oxidized, and the nitrogen atom (s) is optionally quaternized. A heteroaryl group can be attached to the rest of the molecule by a heteroatom. Examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5.benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinoleyl, 5isoquinoleyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinoleyl, and 6-quinoleyl. The substituents for each of the aforementioned aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. "Aryl" and "heteroaryl" also encompass ring systems in which one or more non-aromatic ring systems are fused, or otherwise bound, to an aryl or heteroaryl system.

p00070For brevity, the term "aryl" when used in combination with other expressions (eg, aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" is understood to include those radicals in which an aryl group is attached to an alkyl group (eg, benzyl, phenethyl, pyridylmethyl) including those alkyl groups in which a carbon atom (for example, a methylene group) has been replaced, for example, by an oxygen atom (for example, phenoxymethyl, 2-pyridyloxymethyl, 3- (1-naphthyloxy) propyl).

p00071Each of the above expressions (for example, "alkyl", "heteroalkyl", "aryl" and "heteroaryl") include both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

p00072Substituents for alkyl and heteroalkyl radicals (including those groups frequently mentioned as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are generally referred to as "alkyl substituents" and "heteroalkyl substituents," respectively, and can be one or more of a variety of groups selected from -OR ', = O, = NR', -N

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p00074OR ', -NR'R' ', -SR', -halogen, -SiR'R''R '' ', -OC (O) R', -C (O) R ', -CO2R', -CONR 'R' ', -OC (O) NR'R' ', -NR''C (O) R', -NR'-C (O) NR'R '', -NR''C (O) 2R ', -NR-C (NR'R''R' '') = NR '' '', -NR-C (NR'R '') = NR '' ', - S (O) R', - S (O) 2R ', -S (O) 2NR'R' ', -NRSO2R', -CN and NO2 in a number that varies from zero to (2m '+ 1), where m' is the total number of carbon atoms in said radical. R ', R' ', R' '' and R '' '' each preferably refers independently to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, for example, 1- substituted aryl 3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of the invention includes more than one group R, for example, each of the groups R is independently selected such as each group R ', R' ', R' '' and R '' '', when More than one of these groups is present. When R 'and R' 'are attached to the same nitrogen atom, they can combine with the nitrogen atom to form a ring of 5-, 6- or 7- atoms. For example, -NR'R '' is understood to include 1-pyrrolidinyl and 4-morpholinyl. From the above exposure of substituents, one skilled in the art will understand that the term "alkyl" is intended to include groups that include carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl (for example, -CF3 and CH2CF3 ) and acyl (for example, -C (O) CH3, -C (O) CF3, -C (O) CH2OCH3).

p00075Similar to the substituents described for the alkyl radical, aryl substituents and heteroaryl substituents are generally referred to as "aryl substituents" and "heteroaryl substituents," respectively and are diverse and selected from, for example, halogen, -OR ' , = O, = NR ', -N-OR', -NR'R '', -SR ', halogen, -SiR'R''R' '', -OC (O) R ', -C (O ) R ', -CO2R', -CONR'R '', -OC (O) NR'R '', -NR''C (O) R ', -NR'-C (O) NR'R' ' , NR''C (O) 2R ', -NR-C (NR'R' ') = NR' '', - S (O) R ', -S (O) 2R', -S (O) 2NR'R '', -NRSO2R ', -CN and NO2, -R', -N3, -CN (Ph) 2, fluoro (C1-C4) alkoxy, and fluoro (C1-C4) alkyl , in a number that varies from zero to the total number of open valences on the aromatic ring system; and wherein R ', R' ', R' '' and R '' '' are preferably independently selected from hydrogen, (C1-C8) alkyl and heteroalkyl, aryl and unsubstituted heteroaryl, (unsubstituted aryl) - (C1-C4) alkyl, and (unsubstituted aryl) oxy- (C1-C4) alkyl. When a compound of the invention includes more than one group R, for example, each of the groups R is independently selected as are each group R ', R' ', R' '' and R '' '' when more than one of these groups is present.

p00076Two of the aryl substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -TC (O) - (CRR ') qU-, in which T and U are independently -NR-, -O -, -CRR'- or a single link, and q is an integer from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A- (CH2) rB-, in which A and B are independently -CRR'-, -O- , -NR-, -S-, -S (O) -, -S (O) 2-, -S (O) 2NR'- or a single bond, and r is an integer from 1 to 4. One of The simple links of the new ring thus formed can optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -C (RR ') sX- (CR''R' '') d-, in which syd are independently integers from 0 to 3, and X is -O-, -NR'-, -S-, -S (O) -, -S (O) 2-, or -S (O) 2NR'-. The substituents R, R ', R' 'and R' '' are preferably independently selected from hydrogen or (C1-C6) substituted or unsubstituted alkyl.

p00077As used in the present invention, the term "diphosphate" includes a phosphoric acid ester containing two phosphate groups. The term "triphosphate" includes a phosphoric acid ester containing three phosphate groups. For example, particular drugs that have a diphosphate or triphosphate include:

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p00079As used in the present invention, the term "heteroatom" includes oxygen (O), nitrogen (O), sulfur (S) and silicon (Si).

p00080The symbol "R" is a general abbreviation representing a substituted group that is selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocyclyl groups.

p00081The term "pharmaceutically acceptable carrier", as used in the present invention, means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in supporting or transporting a chemical agent Pharmaceutically acceptable carriers include pharmaceutically acceptable salts, in which the term "pharmaceutically acceptable salts" includes salts of the active compounds that have been prepared with relatively non-toxic acids or bases, depending on the particular substituents found on the compounds described herein. invention. When the compounds of the present invention contain relatively acidic functions, base addition salts can be obtained by contacting the neutral form of said compounds with a sufficient amount of the desired base, both in pure form and in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When the compounds of the present invention contain relatively basic functions, acid addition salts can be obtained by contacting the neutral form of said compounds with a sufficient amount of the desired acid, both in pure form and in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those obtained from inorganic acids of the hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenophosphoric, dihydrogenophosphoric, sulfuric, monohydrogensulfuric, iodhydric, or phosphory acids type, as well as salts of relatively non-toxic organic acids of the acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric type, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic. Likewise, amino acid salts such as arginate, and salts of organic acids of the type of glucuronic or galacturonic acids are included (see, for example, Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, vol. 66, p. 1-19, (1977)). Certain specific compounds of the present invention contain both acidic and basic functions that allow the compounds to be converted into both acid and base addition lases.

p00082The neutral forms of the compounds are preferably regenerated by contact of the salt with a base or an acid and the isolation of the main compound in the conventional manner. The main form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but on the other hand the salts are equivalent to the main form of the compound for the purposes of the present invention.

p00083In addition to salt forms, the present invention provides compounds, which are in a prodrug form. The prodrugs of the compounds described in the present invention are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Additionally, prodrugs can be converted into the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can slowly become the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.

p00084Certain compounds of the present invention may exist in non-solvated forms as well as in solvated forms, including hydrated forms. In general, solvated forms are equivalent to non-solvated forms and are encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

p00085Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; Racemates, diastereomers, geometric isomers and individual isomers are encompassed within the scope of the present invention.

p00086Likewise, the compounds of the present invention may contain unnatural proportions of atomic isotopes in one or more of the atoms constituting said compounds. For example, the compounds may be radiolabeled with radioactive isotopes; such as, for example, tritium (3H), iodine-125 (125I) or carbon-14 (14C). All isotopic variations of the compounds of the present invention, whether or not radioactive, are intended to be encompassed within the scope of the present invention.

p00087As used in the present invention, the term "assignment group" refers to a portion of a substrate that is cleaved from the substrate in a reaction.

p00088The term "antibody" as referred to in the present invention includes whole antibodies and any antigen binding fragment (ie, "antigen binding portion") or single chains thereof. An "antibody" refers to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, or an antigen binding portion thereof. Each chain weighs

55 E06839792 04-11-2011

p00090da comprises a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3 and can be of the mu, delta, gamma, alpha or epsilon isotype. Each light chain comprises a variable light chain region (VL) and a constant light chain region. The light chain constant region comprises a domain, CL, and can be of the kappa or lambda isotype. The VH and VL regions can also be subdivided into regions of hypervariability, called complementarity determination regions (CDR), interspersed with regions that are more conserved, called framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from the terminal to the carboxy terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the light and heavy chains contain a binding domain that interacts with an antigen. Constant regions of the antibodies may mediate the binding of immunoglobulin with host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

p00091The terms "antibody fragment" or "antigen binding portion" of an antibody (or simply "antibody portion"), as used in the present invention, refers to one or more fragments of an antibody that retains the ability to specifically bind an antigen. It has been shown that the antigen binding function of an antibody can be carried out by fragments of a full length antibody. Examples of binding fragments encompassed within the expression of "antibody fragment" or "antibody binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of domains VL, VH, CL and CH1; (ii) an F (ab) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge to the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single tail of an antibody; (v) a dAb fragment (Ward et al., Nature, vol. 341, pp. 544-546, (1989)), which consists of a VH domain; and (vi) an isolated complementarity determination (CRD) region. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked, using recombinant methods, by a synthetic linker that makes it possible to form them as a single protein chain, in which the VL and VH pair to form monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al., Science, vol. 242, pp. 423-426, (1988); and Huston et al., Proc Natl. Acad. Sci. USA, vol. 85, p. 5879-5883, (1988)). Such single chain antibodies are also intended to be encompassed within the expression "antigen binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened to determine their usefulness in the same manner as is done with intact antibodies.

p00092The terms "monoclonal antibody" as used in the present invention, refer to a preparation of antibody molecules of unique molecular composition. A monoclonal antibody composition shows a specific binding affinity and specificity for a particular epitope.

p00093"Solid support", as used in the present invention, refers to a material that is substantially insoluble in a selected solvent system, or which can be easily separated (for example, by precipitation) from a solvent system selected in which is soluble. Solid supports useful in the implementation of the present invention may include groups that are activated or capable of activation to allow selected species to bind to the solid support. Likewise, a solid support can be a substrate, for example, a chip, wafer or well, on which an individual compound, or more than one compound, of the invention is attached.

p00094"Reactive functional group", as used in the present invention, refers to groups including olefins, acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides, cyanates, isocyanates, thiocyanates, isothiocyanates, amines, hydrazines, hydrazones, hydrazides, diazo, diazonium, nitro, nitriles, mercaptans, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids, acetals, ketals, anhydrides, sulfates, sulfenic acids, isonitriles, amidines, imidines, imidates, nitrons, hydroxylamines, oximes, hydroxamic acids, thiohydroxamic acids, alenos, ortho esters, sulphites, enamines, inamines, ureas, pseudoureas, semicarbazides, carbodiimides, carbamates, imines , azoxy compounds, and nitrous compounds. Functional reactive groups also include those used to prepare bioconjugates, for example, N-hydroxysuccinimide esters, maleimides (see, for example, Hermanson, Bioconjugate Techniques, Academic Press, San Diego, (1996)). The procedures for preparing each of these functional groups are well known in the art and their application or modification for a particular purpose falls within the capacity of one skilled in the art (see, for example, Sandler and Karo, eds. Organic Functional Group Preparations, Academic Press, San Diego, (1989)). The reactive functional groups may be protected or unprotected.

p00095The compounds of the invention are prepared as a single isomer (for example, enantiomer, cis-trans, positional, diastereomer) or as a mixture of isomers. In a preferred embodiment, the compounds are prepared as substantially a single isomer. Purely isomerically substantial compound preparation processes are known in the art. For example, enanti-American enriched mixtures and pure enantiomeric compounds can be prepared by the use of intermediate synthesis compounds that are enantiomerically pure in combination with reactions that either yield the stereochemistry together with an unchanged chiral center or provide their complete inversion. Alternatively, the final product or intermediates produced during the stages of the synthesis can be resolved in a single stereoisomer. The techniques

40 E06839792 04-11-2011

p00097Cas for investment or transfer without changing a particular stereocenter, and techniques for the resolution of mixtures of stereoisomers are well known in the art and fall within the knowledge of one skilled in the art for the choice of an appropriate procedure for a particular situation See, in general, Furniss et al. (Eds.), Vogel's Encyclopedia of Practical Organic Chemistry, 5th ed., Longman Scientific and Technical Ltd., Essex, p. 809-816, (1991); and Heller, Acc. Chem. Res., vol. 23, p. 128, (1990).

p00098CBI analogues

p00099The compounds described in the present invention are generally CBI analogs, since they incorporate the alkylation domain or the 1,2,9,9a-tetrahydrocyclopropa [e] benz [e] indole-4-one (CBI) alkylation subunit. The compounds can be used as drugs. Preferred drugs of the present invention include cytotoxic drugs useful in cancer therapy. Cytotoxic drugs useful in the present invention include, for example, CBI-based analogs (1,2,9,9a-tetrahydrocyclopropa [c] benz [e] indole-4-one), MCBI-based analogs (7- methoxy1,2,9,9a-tetrahydrocyclopropa [c] benz [e] indole-4-one) and CCBI-based analogs (7-cyano-1,2,9,9a-tetrahydrocyclopropa [c] benz [e] indole -4-one).

p00100In one embodiment, a compound of the invention has the following formula (1):

p00101wherein X is selected from O, S and NR23, in which NR23 is a group selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl; Z is O;

p00102R1 is H, substituted or unsubstituted C1-6 alkyl, C (O) R8, or CO2R8;

p00103R1 'is H, substituted or unsubstituted C1-6 alkyl, or C (O) R8;

p00104each R3 is a member independently selected from NR9R10 and OR9 and R9 and R10 are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;

p00105R2 is H, substituted or unsubstituted C1-6 alkyl, unsubstituted heteroalkyl, cyano, or alkoxy;

p00106R2 'is H, substituted or unsubstituted C1-6 alkyl, or unsubstituted heteroalkyl;

p00107R3 is a member selected from the group consisting of SR11, NHR11 and OR11, in which R11 is a group selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, diphosphates, triphosphates , acyl, C (O) R12R13, C (O) OR12, C (O) NR12R11, P (O) (OR12) 2, C (O) CHR12R13, SR12 and SiR12R13R14, in which R12, substituted or unsubstituted heteroalkyl or unsubstituted aryl, or R12 and R13 together with the nitrogen or carbon atom to which they are attached bind to form a substituted or unsubstituted heterocycloalkyl ring system having from 4 to 6 atoms, optionally containing two or more heteroatoms;

p00108R6 is a single bond which is either present or absent and, when present R6 and R7 join to form a cyclopropyl ring; and

p00109R7 is CH2-X1 or -CH2- joined in said cyclopropyl ring with R6, wherein X1 is an assignment group;

p00110R4, R4 ', R5 and R5' are members independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NO2, NR15R16 NC (O) R15, OC (O) NR15R16, OC (O) OR15, C (O) R15, SR15, OR15, CR15 = NR16, and O (CH2) nNR24R25, in which n is an integer from 1 to 20, preferably, n is an integer from 2 to 6;

p00111R15 and R16 are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl and substituted or unsubstituted peptidyl, wherein R15 and R16 together with the nitrogen atom to which they are attached are optionally linked to form a substituted or unsubstituted heterocycloalkyl ring system having from 4 to 6 atoms, optionally containing two or more heteroatoms;

40 E06839792 04-11-2011

p00113and R24 and R25 are independently selected from hydrogen and unsubstituted alkyl, wherein at least one of R24 and R25 is hydrogen, and

p00114wherein at least one of R4, R4 ', R5 and R5' is O (CH2) nNR24R25.

p00115As stated above, X1 can be an assignment group. Useful assignment groups include halogens, azides, sulfonic esters (for example, alkylsulfonyl, arylsulfonyl), oxonium ions, alkyl perchlorates, ammonium alkanesulfonates, alkylfluorosulfonates and fluorinated compounds (for example, triflates, nonaflates, tresylates). Halogens particularly useful as assignment groups are F, Cl and Br. The choice of these and other appropriate assignment groups for a set of particular reaction conditions falls within the skill of the person skilled in the art (see, for example, March, J., Advanced Organic Chemistry, 2nd Edition, John Wiley and Sons, (1992); Sandler, SR., Karo, W., Organic Functional Group Preparations, 2nd Edition, Academic Press, Inc., (1983); and Wade, LG., Compendium of Organic Synthetic Methods, John Wiley and Sons, (1980)).

p00116A preferred embodiment of the compound of claim 1 is as follows:

p00117Pharmaceutical formulations and administration

p00118In another preferred embodiment, the present invention provides a pharmaceutical formulation comprising a compound of the invention and a pharmaceutically acceptable carrier.

p00119The compounds described in the present invention that include acceptable carriers such as addition salts or hydrates thereof, can be delivered to a patient using a wide variety of routes or modes of administration. Suitable routes of administration include administration by inhalation, transdermal, oral, rectal, transmucosal, intestinal and parenteral, including intramuscular, subcutaneous and intravenous injections. Preferably, the conjugates of the invention are administered parenterally, more preferably intravenously.

p00120As used in the present invention, the terms "are administered" or "administration" are intended to encompass all means for directly or indirectly supplying a compound to its intended site of action.

p00121The compounds described in the present invention, or pharmaceutically acceptable salts and / or hydrates thereof, can be administered individually, in combination with other compounds of the invention, and / or in cocktails combined with other therapeutic agents. Of course, the choice of therapeutic agents that can be co-administered with the compounds of the invention will depend, in part, on the condition to be treated.

p00122For example, when administered to patients suffering from a disease state caused by an organism that depends on a self-inducer, the compounds of the invention can be administered in cocktails containing agents used to treat pain, infection and other symptoms and side effects commonly associated with the disease Such agents include, for example, analgesics, antibiotics, etc.

p00123When administered to a patient that involves cancer treatment, the compounds can be administered in cocktails containing anti-cancer agents and / or supplemental potentiating agents. Likewise, the compounds can be administered in cocktails containing agents that treat the side effects of radiation therapy, such as anti-emetics, radiation protectors, etc.

p00124Supplemental potentiating agents that can be co-administered with the compounds of the invention include, for example, tricyclic anti-depressant drugs (eg, imipramine, desimipramine, amitriptyline, clomipramine, trimipramine, doxepine, nortriptyline, protriptyline, amoxapine and maprotiline) ; non-tricyclic anti-depressant drugs (for example, sertraline, trazodone, and citalopram); Ca2 + antagonists (for example, verapamil, nifedipine, nitrendipine and caroverine); amphotericin; Triparanol analogs (for example, tamoxifen); anti-arrhythmic drugs (for example, quinidine); antihypertensive drugs (for example, reserpine); thiol reducers (eg, butionin and sulfoximin) and calcium leucovorin.

55 E06839792 04-11-2011

p00126The active compound (s) of the invention is administered per se or in the form of a pharmaceutical composition in which the active compound (s) is mixed with one or more pharmaceutically acceptable carriers, excipients or diluents. Pharmaceutical compositions for use in accordance with the present invention are typically formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliary products, which facilitate the transformation of active compounds into preparations, which can be used pharmaceutically. The most appropriate formulation depends on the route of administration chosen.

p00127For transmucosal administration, penetrators suitable for the permeate barrier are used in the formulation. Such penetrators are generally known in the art.

p00128For oral administration, the compounds can be formulated easily by combining the active compound (s) with pharmaceutically acceptable carriers well known in the art. Such vehicles make it possible for the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, pasty mixtures, suspensions and the like, for oral ingestion by a patient to be treated. If it is desired to obtain tablets or dragee cores, pharmaceutical preparations for oral use with solid excipient can be obtained, optionally grinding a resulting mixture, and treating the granule mixture, after the addition of suitable auxiliary products. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl cellulose, sodium carboxymethyl cellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

p00129Dragee cores are supplied with suitable coatings. For this purpose, concentrated sugar solutions may be used, which optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyes and pigments can be added to the tablet or dragee coatings for identification or to characterize different dose combinations of active compound.

p00130Pharmaceutical preparations, which can be used orally, include adaptable capsules made of gelatin, as well as sealed, soft capsules, made of gelatin and a plasticizer, such as glycerol or sorbitol. The adaptable capsules may contain the active ingredients mixed with fillers such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. All formulations for oral administration should be in dosages suitable for such administration.

p00131For oral administration, the compositions may take the form of tablets or tablets formulated in a conventional manner.

p00132For administration by inhalation, the compounds for use according to the present invention are conveniently supplied in the form of an aerosol spray presentation from pressurized containers or 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 dosing unit can be determined by providing a valve to deliver a measured quantity. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mixture of the compound and a suitable powder base such as lactose or starch.

p00133The compounds may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Injection is a preferred method of administration for the compounds of the present invention. Formulations for injection may be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. The compositions may take forms such as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending, stabilizing and / or dispersing agents, such as cross-linked polyvinyl pyrrolidone, agar, or acid. alginic or a salt thereof such as sodium alginate.

p00134Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water soluble form. Additionally, suspensions of the active compounds such as appropriate oily injection suspensions can be prepared. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or esters of synthetic fatty acids, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds in order to allow the preparation of highly concentrated solutions. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer.

p00135fifty E06839792 04-11-2011

p00136Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, for example, pyrogen-free water, before use.

p00137Likewise, the compounds may be formulated in rectal compositions as suppositories or retention enemas, for example, containing conventional suppository bases such as cocoa butter or other glycerides.

p00138In addition to the formulations described previously, the compounds can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation or transcutaneous delivery (for example, subcutaneously or intramuscularly), intramuscular injection or transdermal patch. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or in ion exchange resins, or as sparingly soluble derivatives, for example, sparingly soluble salt.

p00139The pharmaceutical compositions may also comprise suitable gel or solid carriers or excipients. Examples of such vehicles or excipients include calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

p00140A preferred pharmaceutical composition is a composition formulated for injection such as intravenous injection, and includes about 0.01% to about 100% by weight of the drug conjugate, based on 100% by weight of the total pharmaceutical composition. The drug conjugate may be an antibody cytotoxin conjugate in which the antibody has been selected to identify a particular cancer.

p00141Drug use procedures

p00142The present invention is particularly useful for the treatment of cancer and for the inhibition of the multiplication of a tumor cell or a cancer cell in an animal. Cancer, or a pre-cancerous state, including a tumor, metastasis, or any disease or disorder characterized by uncontrolled cell growth, can be treated or prevented by drug administration.

p00143Representative examples of pre-cancerous conditions to which the present invention can be directed include metaplasia, hyperplasia, dysplasia, colorectal polyps, actinic keratosis, actinic cheilitis, human papillomavirus, leukoplakia, lichen planus and Bowen's disease.

p00144Representative examples of cancer or tumors to which the present invention can be directed include: lung cancer, colon cancer, prostate cancer, lymphoma, melanoma, breast cancer, ovarian cancer, testicular cancer, CNS cancer, kidney cancer, kidney cancer, pancreatic cancer, stomach cancer, oral cancer, nasal cancer, cervical cancer and leukemia.

p00145The compounds of the present invention can be used in a cell destruction process. The process includes the administration to the cell of an amount of a compound of the invention sufficient to destroy said cell. In an exemplary embodiment, the compound is administered to a subject carrying the cell. In a further exemplary embodiment, the administration serves to retard or stop the growth of a tumor that includes the cell (for example, the cell may be a tumor cell). For administration to retard growth, the cell growth rate should be at least 10% less than the growth rate before administration. Preferably, the growth rate will be delayed by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or it will stop completely.

p00146Effective dosages

p00147Pharmaceutical compositions suitable for use with the present invention include compositions in which the active ingredient is contained in a therapeutically effective amount, that is, in an amount effective to achieve its desired purpose. The actual effective amount for a particular application will depend, inter alia, on the state to be treated. The determination of an effective amount falls within the skill of those skilled in the art, especially in light of the disclosure disclosed in the present invention.

p00148For any compound described in the present invention, the therapeutically effective amount can be initially determined from cell culture assays. The concentration of target plasma will be those concentrations of active compound (s) that are capable of inhibiting cell growth or division. In preferred embodiments, cellular activity is inhibited at least 25%. Target plasma concentrations of the active compound (s) that are capable of inducing at least about 50%, 75%, or even 90% or more of cell activity inhibition are currently preferred. The percentage of cell activity inhibition in the patient can be monitored in order to verify the suitability of the plasma drug concentration achieved, and the dosage can be adjusted more or less to achieve the desired percentage of inhibition.

p00149As is well known in the art, therapeutically effective amounts for use in humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve a circulating concentration that has been found to be effective in animals. Dosage in humans can

55 E06839792 04-11-2011

p00151of adjusting by monitoring cell inhibition and adjusting the dosage by more or less, as described above.

p00152A therapeutically effective dose can also be determined from human data for compounds known to show similar pharmacological activities. The dose applied can be adjusted based on the bioavailability and relative potency of the compound administered, compared to the known compound.

p00153Dose adjustment in order to achieve maximum efficacy in humans based on the procedures described above and other procedures well known in the art falls within the knowledge of the normal skilled technician.

p00154In the case of local administration, the systemic circulating concentration of the compound administered will not be of particular importance. In such cases, the compound is administered in order to achieve a concentration in the local area effective to achieve the desired result.

p00155For use in the prophylaxis and / or treatment of diseases related to abnormal cell proliferation, a circulating concentration of the administered compound of about 0.001 µM to 20 µM is preferred, 0.01 µM to 5 µM being preferred.

p00156Doses for patients for oral administration of the compounds described in the present invention, typically range from 1 mg / day to 10,000 mg / day, more typically from about 10 mg / day to 1,000 mg / day, and most typically from 50 mg / day up to 500 mg / day. Expressed in terms of the patient's body weight, typical dosages range from 0.01 to 150 mg / kg / day, more typically from 0.1 to mg / kg / day, and most typically from 1 to 10 mg / kg / day, for example 5 mg / kg / day or 3 mg / kg / day.

p00157In at least some embodiments, the doses for patients that retard or inhibit the growth of tumors may be 1 µml / kg / day or less. For example, the doses for patients may be 0.9, 0.6, 0.5, 0.45, 0.3, 0.2, 0.15, or 0.1 µmolkg / day or less (referred to drug moles) of the drug or a drug conjugate, such as an antibody-drug conjugate. Preferably, the drug or drug conjugate acts on tumor growth when administered in the daily dosage amount for a period of at least five days. In at least some embodiments, the tumor is a human-type tumor in an SCID mouse. As an example, the SCID mouse can be a CB17.SCID mouse (available from Taconic, Germantown, NY).

p00158For other modes of administration, the amount and dosage range can be individually adjusted to provide plasma levels of the administered compound effective for the particular clinical indication to be treated. For example, in one embodiment, a compound according to the invention can be administered in relatively high concentrations at multiple times per day. Alternatively, it may be more desirable to administer a compound of the invention at minimum effective concentrations and use a less frequent administration regimen. This will provide a therapeutic regimen proportional to the severity of the individual's disease.

p00159Using the precepts provided in the present invention, an effective therapeutic treatment regimen that does not cause substantial toxicity and that is nevertheless fully effective to treat the clinical symptoms demonstrated by the particular patient can be planned. This planning should involve the careful choice of the active compound considering factors such as potency of the compound, relative bioavailability, body weight of the patient, presence and severity of adverse side effects, preferred mode of administration and the toxicity profile of the selected agent.

p00160The compounds, compositions and methods of the present invention are further illustrated by the following examples. These examples are offered in order to illustrate the claimed invention.

p00161Examples

p00162Material and Procedures

p00163In the examples below, unless otherwise indicated, temperatures are expressed in degrees Celsius (° C); operations were carried out at room temperature (typically at a range of from about 18-25 ° C); evaporation of the solvent was carried out using a rotary evaporator under reduced pressure (typically, 4.5-30 mmHg) with a bath temperature of up to 60 ° C; the course of the reactions was typically followed by TLC and the reaction times are given for illustrative purposes only; melting points are uncorrected; the products showed satisfactory NMR-1H and / or microanalytical data; yields are provided for illustrative purposes only; likewise, the following conventional abbreviations were used: p.fus (melting point), l (liter (s)), ml (lililiters), mmol ((millimoles), g (grams), mg (milligrams), min (minutes ), LC-MS (liquid chromatography-mass spectrometry) and h (hours).

p00164The 1 H-NMR spectra were measured on a Varian Mercury 300 MHz spectrometer and agreed with the assigned structures. Chemical shifts were reported in parts per million (ppm) with attenuated excitation from tetramethylsilane. The electrospray mass spectra were recorded on a Perkin Elmer Sciex API 365 mass spectrometer. Elemental analyzes were performed by Robertsons.

40 E06839792 04-11-2011

p00166Microlit Laboratories, Madison, NJ. The silica gel for flash chromatography was Merck grade E (230-400 mesh). The reverse phase analytical HPLC was carried out either on an HP 1100 instrument or Varian ProStar 210 with a Phenomenex Lunar column of 5 µm C-18 (2) of 150 mm x 4.6 mm or a Varian Microsorb- column MV of 0.1 µm C-18 of 150 mm x 4.6 mm. The flow rate of 1 ml / min was carried out with a gradient of 0% to 50% buffer B for 15 minutes or 10% to 100% buffer B for 10 minutes, with UV detection at 254 nm. Buffer A: 20 mM ammonium formate + 20% acetonitrile or 0.1% trifluoroacetic acid in acetonitrile; buffer B: 20 ​​mM ammonium formate + 80% acetonitrile or 0.1% aqueous trifluoroacetic acid. The preparative reverse phase HPLC was performed on a Varian ProStar 215 instrument with a Delta Park column of 15 µm C-18 of 300 mm x 7.8 mm.

p00167Reference Example 1

p00168Synthesis of Compound 32

p00169At a solution of Compound 30 (120 mg, 0.28 mmol) in ethyl acetate (10 ml), gaseous HCl was bubbled for 5 minutes. The reaction mixture was stirred at room temperature for another 30 minutes and then the mixture was concentrated. Ether was added to the reaction mixture and the white precipitate was collected on a filtration funnel. The solid was dried overnight under vacuum, providing 100 mg of the desired product, which was confirmed by LC-MS (ESI) 324 (M + H +) and used in the next step without further purification. To a solution of this compound (100 mg, 0.24 mmol) in DMF (5 ml) Compound 31 (65 mg, 0.26 mmol), HATU (100 mg, 0.26 mmol) and TEA (91 µl, 0.52 mmol). The mixture thus obtained was stirred at room temperature for 3 hours. The solvent was evaporated and the residue was purified on semi-preparative HPLC with 0.1% TFA in water and acetonitrile as eluent, to provide Compound 32 as an oil (110 mg, 80%). The desired product was confirmed by LC-MS 555 (M + H +).

p00170Synthesis of Compound 33

p00171A solution of Compound 32 (110 mg, 0.2 mmol) in palladium on charcoal (20 mg) in DCM (10 ml) in methanol (5 ml) was stirred under atmospheric pressure of hydrogen at room temperature for 12 hours. The palladium was filtered and the reaction mixture was concentrated and the residue was purified on semi-preparative HPLC with 0.1% TFA in water and acetonitrile as eluent to provide the desired compound as an oil (80 mg, 78% ). LC-MS (ESI) 465 (M + H +). To a solution of the residue (80 mg, 0.17 mmol) in dichloromethane (10 ml) and THF (5 ml) was added PNPCl (4-nitrophenyl chloroformate) (137 mg, 0.68 mmol) and triethylamine (144 µl , 1.02 mmol) at 0 ° C. The mixture thus obtained was stirred for 30 minutes at 0 ° C and then at room temperature for 12 hours. The reaction mixture was concentrated under vacuum, and the residue was precipitated using ethyl ether (100 ml), to give compound 33 as a yellow solid (90 mg, 82%), which was dried under vacuum and confirmed by LC-MS (ESI) 631 (M + H +).

p00172Synthesis of Compound 34

p00173To a solution of Compound 33 (60 mg, 0.1 mmol) in dichloromethane (10 ml), Boc-N, N-dimethyl ethyl diamine (84 mg, 0.38 mmol) and triethylamine (26 µl, 0, 1 mmol) at room temperature. The mixture thus obtained was stirred at room temperature for 12 hours. The reaction mixture was concentrated under vacuum, and the residue was precipitated using ethyl ether (100 ml), providing Boc-protected Compound 34, which was used for the next step without further purification. Boc-protected Compound 34 was dissolved in 10 ml of TFA and the reaction mixture was stirred at room temperature for 60 minutes. The reaction mixture was concentrated under vacuum, and the residue was precipitated using ethyl ether (100 ml) to provide Compound 34 as a yellow solid, which was dried under vacuum and confirmed by LC-MS 631 ( M + H +).

p00174Reference Example 2

p00175Synthesis of Compound 1

p00176Cephalothine sodium salt (0.5 g, 1.2 mol) was dissolved in water (10 ml) and poured into a separatory funnel. The solution was acidified with 1N aqueous HCl solution and the desired compound was extracted with dichloromethane (100 ml) 10 and ethyl acetate (40 ml). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated to dryness to give the title compound as a white solid (474 ​​mg, 99%). To a solution of the white compound (1.2 mmol) and tert-Boc2O (0.3 g, 1.37 mmol) in tetrahydrofuran (20 ml), dimethylaminopyridine (15 mg, 0.12 mmol) was added. The mixture thus obtained was dried at room temperature overnight. The solvent was evaporated and the residue was purified on semi-preparative HPLC with 0.1% TFA in water and acetonitrile.

E06839792 04-11-2011

p00178as eluent, provided the compound as an oil (238 mg, 44%). 1H NMR (CD3OD) 5 1.49 (s, 9H), 2.05 (s, 3H), 3.80 (s, 2H), 4.60 (d, 1H), 4.77 (d, 1H ), 4.93 (d, 1H), 5.28 (d, 1H), 5.49 (m, 1H), 6.59 (s, 1H), 6.96 (m, 2H), 7.27 (dd, 1H), 9.18 (broad d, 1H); LC-MS (ESI) 453 (M + H +), 475 (M + Na +), 491 (M + K +).

p00179Synthesis of Compound 2

p00180To a solution of Compound 1 (204 mg, 0.45 mmol) in methanol (40 ml), potassium carbonate (25 mg, 0.18 mmol) was added at 0 ° C. The mixture thus obtained was stirred for 3 hours. The reaction mixture was neutralized with acetic acid (600 µl) and concentrated. The solvent was evaporated and the residue was purified on semi-preparative HPLC with 0.1% TFA in water and acetonitrile as eluent, to give the title compound as an oil (56 mg, 32%). 1H NMR (CD3OD) 5 1.49 (s, 9H), 3.80 (s, 2H), 4.15 (m, 2H), 4.95 (d, 1H), 5.28 (d, 1H ), 5.46 (m, 1H), 6.39 (d, 1H), 6.95 (m, 2H), 7.26 (m, 1H), 9.09 (broad d, 1H); LC-MS (ESI) 410 (M + H +), 433 (M + Na +), 449 (M + K +).

p00181Synthesis of Compound 3

p00182To a solution of Compound 2 (15 mg, 0.036 mmol) in THF (0.2 ml), dimethylaminopyridine (0.13 mg, 0.001 mmol), para-nitrophenyl chloroformate (11 mg, 0.054 mmol) and 2, were added 6-lutidine (6.4 µL, 0.054 mmol) at room temperature. The mixture thus obtained was stirred overnight. The solvent was evaporated and the residue was purified on semi-preparative HPLC with 0.1% TFA in water and acetonitrile as eluent, to give the title compound as an oil (8 mg, 38%). 1H NMR (CDCl3) 5 1.49 (s, 9H), 3.88 (s, 2H), 4.76 (d, 1H), 4.94 (d, 1H), 4.95 (s, 1H ), 5.29 (m, 1H), 5.67 (m, 1H), 6.41 (d, 1H), 6.52 (s, 1H), 7.00 (m, 2H), 7.28 (m, 1H), 7.37 (dd, 2H), 8.29 (dd, 2H); LC-MS (ESI) 575 (M + H +), 598 (M + Na +), 614 (M + K +).

p00183Synthesis of Compound 4

p00184To a solution of Compound 3 (18 mg, 0.031 mmol) in dichloromethane (0.5 ml) cooled to 0 ° C, mloroperoxybenzoic acid (9 mg, 0.052 mmol) was added. The mixture thus obtained was stirred for 2 hours at 0 ° C. The solvent was evaporated and the residue was purified on semi-preparative HPLC with 0.1% TFA in water and acetonitrile as eluent, to give the title compound as an oil (12 mg, 67%). 1H-NMR (CDCl3) 5 1.54 (s, 9H), 3.31 (d, 1H), 3.87 (s, 2H), 3.88 (d, 1H), 4.53 (d, 1H ), 4.88 (d, 1H), 4.59 (d, 1H), 6.10 (dd, 1H), 6.92 (d, 1H), 6.99 (m, 2H), 7.27 (d, 1H), 7.37 (dd, 2H), 8.28 (d, 2H); LC-MS (ESI) 591 (M + H +), 614 (M + Na +), 630 (M + K +).

p00185Synthesis of Compound 5

p00186To a solution of Compound 34 (11 mg, 0.013 mmol) in 10% dimethylformamide in dichloromethane (0.2 ml), a solution of Compound 4 (10 mg, 0.017 mmol) in dichloromethane (0.2 ml) was added and diisopropylethylamine (3.5 µl, 0.020 mmol) at room temperature. The mixture thus obtained was stirred overnight. The solvent was evaporated and the residue was purified on semi-preparative HPLC with 0.1% TFA in water and acetonitrile as eluent, to give the title compound as an oil (7 mg, 45%). LC-MS (ESI) 1031 (M + H +), 1054 (M + Na +), 1070 (M + K +).

p00187Synthesis of Compound 6

p00188To a solution of Compound 5 (6.5 mg, 0.0056 mmol) in dichloromethane (0.2 ml), trifluoroacetic acid (0.1 ml) was added at 0 ° C. The mixture thus obtained was allowed to warm to room temperature and stirred for 30 minutes. The solvent was evaporated and the residue was purified on semi-preparative HPLC with 0.1% TFA in water and acetonitrile as eluent, to give the title compound as an oil (4 mg, 70%). LC-MS (ESI) 975 (M + H +), 998 (M + Na +), 1014 (M + K +).

p00189Reference Example 3

30 E06839792 04-11-2011

p00191Synthesis of Compound 2

p00192In a 50 ml round bottom flask equipped with stirring rod and nitrogen inlet, Fmoc was dissolved

p00193AlaLeuAlaLeu-OH (5 g, 0.0082 mol, Abbott Labs) in DMF (30 ml). HATU (3.13 g, 0.0082 mol) and then DIPEA (2.86 ml, 0.0164 mol) was added and the solution was stirred for 10 minutes. 4-Aminobenzyl alcohol (1.5 g, 0.0122 mol) was added and the reaction was stirred at room temperature for 18 hours. The solvent was concentrated in vacuo and the residue was dissolved in DMF (20 ml). The product was precipitated with diethyl ether (200 ml) and collected by filtration to provide 4.5 g (77%) of product. The product was confirmed by mass spectroscopy: m / z 714.4 [M + 1] +.

p00194Synthesis of Compound 3

p00195In a 25 ml round bottom flask equipped with a stirring rod and nitrogen inlet, Compound 2 (0.3 g, 0.4 mol) was dissolved in 1.5 ml of DMF. A 1: 1 DCM / THF solution was added, followed by 4-nitrophenyl chloroformate (0.2 g, 1 mmol) and pyridine (0.2 ml, 2.5 mmol). The reaction was stirred at room temperature for 6 hours. The solvent was removed in vacuo and the residue was purified by column chromatography (10% MeOH / DCM), yielding 0.104 g (28%) of product 3. The product was confirmed by mass spectroscopy: m / z 879.6 [M + 1] +.

p00196Synthesis of Compound 4

p00197To a solution of Compound 34 (11 mg, 0.013 mmol) in 10% dimethylformamide in dichloromethane (0.2 ml) was added a solution of Compound 3 (15 mg, 0.017 mmol) in dichloromethane (0.2 ml) and diisopropylethylamine (3.5 µl, 0.020 mmol) at room temperature. The mixture thus obtained was stirred overnight. The solvent was evaporated and the residue was purified on semi-preparative HPLC with 0.1% TFA in water and acetonitrile as eluent, to give the title compound as an oil. The product was confirmed by LC / MS. This product was dissolved in DMF (10 ml) and piperidine (0.5 ml, 0.5 mol) was added and the solution was stirred for 30 minutes. The solution was concentrated in vacuo, washed with hexane and dried under vacuum for 1.2 hours. The deprotected amine prepared above was dissolved in anhydrous DMF (10 ml), followed by the addition of succinic anhydride (20 mg, 0.2 mmol) and the reaction mixture was stirred at room temperature for 24 hours. After 24 hours, HPLC showed no starting material and the reaction was purified by preparative HPLC to provide Compound 4. Compound 4 was confirmed by mass spectroscopy m / z 1196 [M + 1] +.

p00198Example 4: Proliferation Assays

p00199The biological activity of the cytotoxic compounds of the invention can be tested using the well known 3H-thymidine proliferation assay. This is a suitable procedure for the quantification of cell proliferation, since it evaluates DNA synthesis by measuring the incorporation of exogenous radiolabeled 3H-thymidine. This assay is highly reproducible and can be adapted to a large number of compounds.

35 E06839792 04-11-2011

p00201To carry out the assay, promyelocytic leukemia cells, HL-60, were grown in RPMI medium containing 10% thermally inactivated fetal bovine serum (FCS). On the day of the study, the cells were collected, washed and resuspended at a concentration of 0.5x106 cells / ml in RPMI containing 10% FCS. To the 96-well plates 100 µl of cell suspension was added. Serial dilutions (3-fold increments) of doxorubicin (as a positive control) or of the compounds under test were made and 100 µl of the compounds were added per well. Finally, 10 µl of a 100 µCi / ml 3H-thymidine was added per well and the plates were incubated for 24 hours. Plates were collected using a 96-well Harvester (Packard Instruments) and counted on a Packard Top Count counter. Four parameters of logistic curves were adjusted to the incorporation of 3 H-thymidine as a function of the molarity of the drug using Prism software to determine IC50 values.

p00202The compounds of the invention generally have an IC50 value in the previous test of from about 1 pM to about 100 nM, preferably from about 10 pM to about 10 nM.

p00203Example 5

p00204Scheme 1

p00205Synthesis of Compound 1

p002062,5-Dihydroxybenzaldehyde (5.6 g, 40.57 mmol) was dissolved in N-methylpyrrolidone (45 ml), followed by the addition of potassium carbonate (5.6 g, 40.5 mmol). Next, tetrabutyl ammonium bromide (260 mg, 0.8 mmol) and then diethyl 2-bromomalonate (10.5 g, 7.5 ml, 44 mmol) was added. All previous additions were made at room temperature. The reaction mixture was stirred at 140 ° C for 5 hours. TLC and HPLC did not reveal starting material or formation of a new peak. The reaction mixture was filtered through a bed of silica and concentrated. 400 ml of 1N HCl was added to the crude product and extracted with ethyl acetate. The organic phase was washed with brine and evaporated dry over anhydrous sodium sulfate. The crude product was purified by flash chromatography on silica gel (hexanes / ethyl acetate, 5/1, respectively), to provide 2 grams of Compound 1 (23% yield). 1 H-NMR, acetone-d6: 1.37 (t, 3H), 4.37 (dd, 2H), 7.51 (s, 1H), 7.4 (d, 1H), 7.06 (s, 1H), 7.02 (d, 1H), 8.41 (s, 1H).

p00207Synthesis of Compound 2

p002082- (Boc-amino) ethyl bromide (344 mg, 1.53 mmol) was added dropwise to the stirred reaction mixture of Compound 1 (100 mg, 0.48 mmol) in DMF (5 ml ) and potassium carbonate (132 mg, 0.995 mmol) at 45 ° C and allowed to stir for one weekend the week. The solvent was evaporated. The crude product was dissolved in ethyl acetate and washed with 0.2 N NaOH several times. The solvent was evaporated and the crude reaction mixture was purified by flash chromatography on silica gel using ethyl acetate / hexanes (1: 4, 2: 4), providing 137 mg (81%) of Compound 2. Mass spectroscopy M [+1] = 350.9. 1 H-NMR, CDCl 3: 1.43 (s, 9H), 1.39 (t, 3H), 3.54 (m, 2H), 4.02 (t, 2H), 4.41 (dd, 2H) , 7.01-7.03 (aromatic, 2H), 7.42-7.46 (aromatic, 2H).

p00209Synthesis of Compound 3

p00210Compound 2 was dissolved in MeOH and stirred for 2-3 hours in 2N aqueous NaOH solution. The solvent was evaporated, 10% citric acid solution was added, and the compound was extracted with ethyl acetate. The product was further purified by reverse phase HPLC, providing Compound 3. Mass spectroscopy M [+ Na] = 344.5, M [+ k] = 360.5. NMR-1H, acetone-d6: 1.41 (s, 9H), 3.49 (dd, 2H), 4.10 (t, 2H), 6.24 (width, 1H), 7.13 (1H, aromatic), 7.29 (1H, aromatic), 7.54 (1H, aromatic), 7.58 (1H, aromatic).

p00211Scheme 2

p002125 Boc-benzyl CBI (200 mg, 0.4728 mmol) was degassed by hydrogenolysis using 10% Pd / C in DCM / MeoH, 2: 1, over a period of 8 hours. (The procedures for obtaining Boc-benzyl CBI or similar compounds are described, for example, in Provisional Patent Application Serial No. 60 / 730,804; US Patent No. 6,534,660; and DL Boger and others, J. Org. Chem., vol. 57, pp. 2873-2876, (1992), all of which are incorporated for reference). The catalyst was filtered and the crude greenish product was purified by chromatography of

p0021310 silica gel with 5-20% ethyl acetate in hexanes, providing the desired Compound 4 (150 mg, 95% yield). Compound 4 (76 mg, 0.23 mmol) was dissolved in DCM (8 ml) and allyl alcohol (0.3 ml) followed by the addition of 4-methyl piperazine carbonyl chloride, HCl salt (183 mg, 0, 93 mmol), pyridine (187 µl, 2.32 mmol) and allowed to stir overnight. The crude product was purified by reverse phase chromatography on a C-18 column, providing Compound 5 (95 mg, 89% total yield). Mass spectroscopy M [+1] =

p00214fifteen 461, M [+ Na] = 482, M [+ K] = 498. Compound 5 was deprotected using a solution of freshly prepared HCl-ethyl acetate to provide Compound 6.

p00215Synthesis of Compound 8

p002165- (2- (tert-Butoxycarbonyl) ethoxy) benzofuran-2-carboxylic acid (3.24 mg, 0.076 mmol) was dissolved in 1 ml of DMF, TBTU (25 mg, 0.076 mmol) was added, followed by the addition of Compound 6 (25 mg, 0.058 mmol) and, finally, DI

p00217twenty PEA (37 µl, 0.21 mmol) and allowed to stir for 9 hours. The solvent was evaporated and the crude product was purified by reverse phase HPLC, providing 15 mg of Compound 7 (30% yield after lyophilization). Mass spectroscopy M [+] = 663.4

p00218The Boc group from Compound 7 was removed using freshly prepared solution of HCl in ethyl acetate, providing Compound 8 as its HCl salt.

p0021925 Synthesis of Fmoc-Val-Cit-PABA

p00220Fmoc-Val-citrulline (1.5 g, 3.02 mmol) was dissolved in a mixture of DCM (14 ml) and MeOH (7 ml). 4-Aminobenzyl alcohol (445.2 mg, 3.62 mmol) was added, followed by 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) (1.5 g, 6.0 mmol) and the mixture The reaction was allowed to stir overnight. The solvent was removed and diethyl ether was added to the residue and treated by ultrasound for 5-10 minutes. The solid residue was allowed to settle and the solvent was decanted. This was repeated two more times, providing 1.5 g of Fmoc-Val-Cit-PABA (82% yield). M [+1] = 602.6, M [1 + Na] = 624.8, M [+ K] = 640.6.

p00221Synthesis of Fmoc-Val-Cit-PABA-PNP

<dl><dt>10 15 </dt><dd>Fmoc-Val-Cit-PABA (65 g, 0.11 mmol) was dissolved in 2 ml of DMF, followed by the addition of pyridine (36 µl, 0.44 mmol). P-nitrophenol chloroformate (66 mg, 0.33 mmol) dissolved in THF (2 ml) was added dropwise. The reaction was then completed in less than an hour. The solvent was evaporated and the crude product was purified by silica gel column, using 5% MeOH in DCM, to provide 43 mg of the desired product. 51% yield Mass spectroscopy: M [+1] = 768. Synthesis of Compound 11 </dd></dl>

<dl><dt>20 </dt><dd /></dl>

<dl><dt>25 </dt><dd /></dl>

p00222Scheme 3

p00223To a flask containing Fmoc-Cit-PABA-PNP carbonate (13 mg, 0.017 mol) was added a solution of Compound 8 (9.5 mg, 0.015 mmol) in DMF (1 ml), followed by the addition of DIPEA ( 15 µl). The reaction was completed in less than 30 minutes. The solvent was evaporated and the crude product was purified by reverse phase HPLC to provide 9.0 mg of Compound 9. Mass spectroscopy: M [+1] = 1191, M [+ Na] = 1213, M [+ K] = 1229.

p00224The Fmoc protection group of Compound 9 (9 mg, 0.0076 mmol) was removed using 5% piperidine in DMF (3 ml). The solvent was removed and the crude residue was washed with hexanes and ethyl ether. Compound 10 was dried overnight under high vacuum and used in the next step without further treatment / purification. 10 mass spectroscope: M [+1] = 969, M [+ Na] = 991, M [+ K] = 1007. Compound 10 was reacted with maleimide ester-TEG-NHS in DMF and the reaction mixture was left under stirring for 1 hour. The solvent was evaporated and purified by reverse phase HPLC on a C-18 column, providing 5 mg (48% yield) of the Compound.

p00225eleven. M [+1] = 1367.

p00226Synthesis of Compound 12

p00227Scheme 4

p00228Compound 8 (8.5 mg, 0.013 mmol) and Compound 13 (10 mg, 0.013 mmol) were dissolved in DMF (1.5 ml), followed by the addition of DIPEA (7 µl, 0.039 mmol). The reaction was completed in 50 minutes. The solvent was evaporated and purified by reverse phase HPLC on a Gemini C-18 column (Phenomenex Inc., Torrence, CA), providing 10 mg of compound 12 (65% yield after lyophilization).

p00229Synthesis of compound 14

p00230Scheme 5

p00231Compound 8 (4.8 mg, 0.0076 mmol) was dissolved in DMF (1 ml), followed by the addition of Mal-TEG-NHS ester (8 mg, 0.015 mmol) as a solution in DCM (0 , 5 ml). 25-40 µl of DIPEA was added. The reaction was stirred for 30 minutes. The solvent was evaporated and purified by reverse phase HPLC to provide 4 mg of Compound 14 (55% yield). Mass spectroscopy: M [+1] = 962, M [+ Na] = 984, M [+ M].

p00232Synthesis of Compound 17

p00233Scheme 6

p00234Tert-Butyl 2-hydroxyethylcarbamate (270 mg, 1,675 mmol) was dissolved in THF (10 ml). 4-Nitrophenyl chloroformate (674 mg, 3.35 mmol) was added, followed by the dropwise addition of pyridine (400 µl, 5 mmol). The reaction mixture

p0023510 tion was left under stirring for 2 hours. The solvent was evaporated and the crude product was purified by flash silica gel chromatography, using DCM as eluent, to provide 500 mg of Compound 15 (92% yield). 1 H-NMR, CDCl 3: 1.45 (s, 9H), 3.45 (m, 2H), 4.34 (m, 2H), 7.38 (d, 2H), 8.27 (d, 2H) .

p00236Compound 8 (0.018 mmol) was dissolved in DMF (2 ml) and Compound 15 (11.6 mg, 0.035 mmol) was added, followed by the addition of 10-20 µl of DIPEA. The reaction mixture was stirred for 6 hours. The solvent was evaporated 15 and purified by reverse phase HPLC, yielding 7 mg (52% yield) of the exposed 16. Mass spectroscopy: M [+1] = 750, M [+ Na] = 772, M [+ K ] = 788. Compound 16 was deprotected using HCl-ethyl acetate and the solvent was evaporated and the product was dried overnight and used in the next reaction without further purification. Mass spectroscopy: M [+1] = 651, M [+ Na] = 673, M [+ K] = 688. The crude product (0.009 mmol) was dissolved in DMF (1 ml), followed by the addition of the ester Mal-TEG-NHS (18 mg, 0.035 mmol) in the form of a solution

p00237twenty in 0.5 ml of DCM, followed by DIPEA (10-20 µl). The reaction mixture was stirred for 15 minutes and the solvent was purified by reverse phase HPLC to provide 4.5 mg of Compound 17 (48% yield). Mass spectroscopy: M [+1] = 1049, M [+ Na] = 1071, M [+ K] = 1087.

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