Dipeptidyl peptidase inhibitors

[0001] The invention relates to compounds that can be used to inhibit dipeptidyl peptidases as well as compositions of substances and kits containing these compounds. The present invention also relates to methods of inhibiting dipeptidyl peptidases and methods of treatment using compounds of the invention.

[0002] Dipeptidyl peptidase IV (EC.3.4.14.5, according to the IUBMB enzyme nomenclature) is a type II membrane protein which is referred to in the literature by many different names, including DPP4, DP4, DAPIV, FAPp, adenosine deaminase complexing protein 2 2, adenosine deaminase (ADAbp) binding protein, dipeptidylaminopeptidase IV; Xaa-Pro-dipeptidylaminopeptidase; Gly-Pro naphthylamidase; postproline dipeptidylaminopeptidase IV; CD26 lymphocyte antigen; GP110 glycoprotein; dipeptidyl peptidase IV; glycinoproline aminopeptidase; glycoprotein aminopeptidase; X-prolyl dipeptidylaminopeptidase; pep X; CD26 leukocyte antigen; glycylprilyl dipeptidylaminopeptidase; dipeptidyl-peptydohydrolaza; glycoprolyl aminopeptidase; dipeptidyl-aminopeptidase IV; DPP IV / CD26; aminoacylprilyl dipeptidylaminopeptidase; T cell-releasing Tp103 molecule; X-pdapA. In this specification, dipeptidyl peptidase IV is referred to as "DPP-IV."

[0003] DPP-IV is a non-classical serine aminodipeptidase that removes Xaa-Pro dipeptides from the amino terminus (N-terminus) of polypeptides and proteins. DPP-IV dependent slow release of X-Gly or X-Ser dipeptides has also been reported for some naturally occurring peptides.

[0004] Expression of DPP-IV occurs constitutively on epithelial and endothelial cells of various tissues (intestines, liver, lungs, kidneys and placenta); DPP-IV is also found in body fluids. Expression of DPP-IV also occurs on circulating T cells; cell surface antigen, CD-26 has been shown to be synonymous with DPP-IV. DPP-IV is involved in many disease states, some of which are discussed below.

[0005] DPP-IV is responsible for the metabolic cleavage of certain endogenous peptides (GLP1 (7-36), glucagon) in vivo and has proteolytic activity against various other peptides (GHRH, NPY, GLP-2, VIP) in vitro.

[0006] GLP-1 (7-36) is a 29-amino acid peptide produced by post-translational processing of proglucagon in the small intestine. GLP-1 (7-36) has many in vivo activities, including stimulation of insulin secretion, inhibition of glucagon secretion, induction of satiety, and delaying gastric emptying. Based on the physiological profile of GLP-1 (7-36), it is thought to be beneficial in the prevention and treatment of type II diabetes and potentially obesity. For example, exogenous administration of GLP-1 (7-36) (continuous infusion) has been found to be effective in diabetic patients in this patient population. Unfortunately, GLP-1 (736) degrades rapidly in vivo and has been found to have a short half-life in vivo (t_V2 = 1.5 minutes). [0007] Based on studies on genetically cultured DPP-IV knock out mice and in vivo and in vitro studies with selective DPP-IV inhibitors, DPP-IV has been shown to be the primary degrading enzyme for GLP-1 (7-36) in vivo . GLP-1 (7-36) is effectively degraded by DPP-IV to GLP-1 (9-36), which is believed to act as a physiological antagonist for GLP-1 (7-36). In vivo inhibition of DPP-IV is therefore considered useful for the potential for endogenous levels of GLP-1 (7-36) and for enhancing the formation of its GLP-1 antagonist (9-36). Therefore, DPP-IV inhibitors are believed to be useful agents for preventing, delaying the progression and / or treatment of conditions mediated by DPP-IV, in particular diabetes, and more specifically type 2 diabetes, diabetic dyslipidemia, and disorders.

EP 1 586 571 B3 glucose tolerance (IGT), conditions of impaired fasting plasma glucose (IFG), metabolic acidosis, ketoacidosis, for appetite regulation and obesity.

[0008] DPP-IV expression in T cells is increased after mitogenic or antigenic stimulation (Mattern, T., et al., Scand J. Immunol., 1991,33, 737). DPP-IV inhibitors and DPP-IV antibodies have been reported to suppress mitogen stimulated and antigen stimulated T cells in a dose dependent manner (Schon, E., et al., Biol. Chem., 1991,372, 305). Various other T-cell functions, such as cytokine production, IL-2-mediated cell proliferation and B helper cell activity have been shown to be dependent on DPP-IV activity (Schon, E., et al., Scand. J. Immunol., 1989 , 29, 127). DPR-IV inhibitors based on boroProline (Flentke, GR, et al., Proc. Nat. Acad. Sci. USA, 1991,88, 1556), although unstable, were effective in inhibiting antigen-induced proliferation of lymphocytes and IL-2 production in rodent CD4 + T helper cells. Such boronic acid inhibitors have been shown to have an in vivo effect in mice, suppressing the formation of antibodies induced by antigenic challenge (Kubota, T. et al., Clin. Exp. Immun., 1992, 89, 192). The role of DPP-IV in regulating T cell activation may also be partly attributed to its cell surface binding to transmembrane phosphatase, CD45. Inhibitors of DPPIV or inactive site ligands may likely disrupt the CD45-DPP-IV junction. It is known that CD45 is an integral component of the T cell signaling apparatus. DPP-IV has been reported to be essential for the penetration and infectivity of HIV-1 and HIV-2 viruses in CD4 + T cells (Wakselman, M., Nguyen, C., Mazaleyrat , J.-P., Callebaut, C., Krust, B., Hovanessian, AG, Inhibition of HIV-1 infection of CD 26+ but not CD 26-cells by a potent cyklopeptidic inhibitor of the DPP-IV activity of CD 26. Abstract P, 44 of the 24.sup.th European Peptide Symposium 1996). In addition, DPP-IV has been shown to bind to the enzyme adenosine deaminase (ADA) on the surface of T cells (Kameoka, J., et al., Science, 193, 26 466). ADA deficiency causes severe complex immunodeficiency (SCID) in humans. This ADA-CD26 interaction may be the key to SCID pathophysiology. It follows that DPP-IV inhibitors may be useful immunosuppressants (or drugs that suppress cytokine release) for the treatment of, among others: organ transplant rejection; autoimmune diseases such as inflammatory bowel disease, multiple sclerosis and rheumatoid arthritis, and AIDS treatment.

[0009] DPP-IV lung endothelial cells have been shown to be an adhesive molecule for metastatic breast cancer in rats and prostate cancer cells (Johnson, RC, et al., J., Cell. Biol., 1993, 121, 1423). It is known that DPP-IV binds to fibronectin and that some metastatic tumor cells carry large amounts of fibronectin on their surface. Strong DPP-IV inhibitors may be useful as drugs to prevent metastases of, for example, breast and prostate tumors into the lungs.

[0010] High levels of DPP-IV expression have also been found in human skin fibroblast cells of patients with psoriasis, rheumatoid arthritis (RA) and lichen planus (Raynaud, F., et al., J. Cell. Physiol., 1992, 151, 378) . Thus DPP-IV inhibitors may be useful as agents for the treatment of dermatological diseases such as psoriasis and lichen planus.

[0011] High DPP-IV activity was found in tissue homogenates from patients with benign prostate hyperplasia and in prostatosomes. These are prostate-derived organelles, important for increasing sperm motility (Vanhoof, G., et al., Eur. J. Clin. Chem. Clin. Biochem., 1992, 30, 333). DPPIV inhibitors may also act to suppress sperm motility and thus act as male contraceptives. Conversely, DPP-IV inhibitors are involved in the treatment of infertility, especially female infertility caused by polycystic ovary syndrome (PCOS, Stein-Leventhal syndrome), which is stable in

EP 1 586 571 B3 is characterized by thinning of the ovarian capsule and the formation of numerous follicular cysts. It causes infertility and galactorrhoea.

[0012] DPP-IV is thought to play a role in the cleavage of various cytokines (stimulation of hematopoietic cells), growth factors and neuropeptides.

[0013] Stimulated hematopoietic cells are useful in the treatment of disorders that are characterized by a reduced number of hematopoietic cells or their precursors in vivo. These conditions often occur in patients who are immunosuppressed, for example as a result of chemotherapy and / or radiation therapy for cancer. Type IV dipeptidyl peptidase inhibitors have been found to be useful to stimulate the growth and differentiation of hematopoietic cells in the absence of exogenously added cytokines or other growth factors or stromal cells. This finding contradicts the dogma in the field of hematopoietic cell stimulation, which states that the addition of cytokines or cytokine producing cells (stromal cells) is an essential element for maintaining and stimulating the growth of hematopoietic cells in culture. (See, e.g. International Application PCT / US93 / 017173 published as WO 94/03055).

[0014] DPP-IV in human plasma has been shown to cleave N-terminal Tyr-Ala from a growth hormone releasing factor and causes inactivation of this hormone. Thus DPP-IV inhibitors may be useful in the treatment of short stature due to growth hormone deficiency (dwarfism) and to promote GH-dependent tissue growth or regrowth.

[0015] DPP-IV can also cleave neuropeptides and it has been shown to modulate the activity of neuroactive peptides of substance P, neuropeptide Y and CLIP (Mentlein, R., Dahms, P., Grandt, D., Kruger, R., Proteolytic processing of neuropeptide Y and peptide YY by dipeptidyl peptidase IV, Regul. Pept., 49, 133,

1993; Wetzel, W., Wagner, T., Vogel, D., Demuth, H.-U., Balschun, D., Effects of the CLIP fragment ACTH 20-24 on the duration of REM sleep episodes, Neuropeptides, 31.41 , 1997). Thus DPP-IV inhibitors may also be useful as agents for regulating or normalizing neurological diseases.

[0016] DPP-IV has been shown to be inhibited by a number of compounds. However, there is still a need for new DPP-IV inhibitors that have beneficial potency, stability, selectivity, toxicity and / or pharmacological properties. Accordingly, the present invention provides a new class of DPP-IV inhibitors.

Summary of the Invention [0017] The present invention relates to compounds having DPP-IV inhibitory activity. It is noted that these compounds may also have inhibitory activity on other S9 proteases and can therefore be used against both these other S9 proteases and against DPP-IV. The present invention also provides compositions, industrial products and kits containing these compounds.

[0018] In one embodiment, a pharmaceutical composition is provided that comprises the DPP-IV inhibitor of the present invention as an active ingredient. The pharmaceutical compositions of the invention may optionally contain 0.001% -100% of one or more of one of the DPP-IV inhibitors of the invention. These pharmaceutical compositions can be administered or coadministered by various routes, including, for example, orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, liposomally, inhaled, vaginally, intraocularly, by topical delivery (e.g. stent), subcutaneous, adipose tissue, intra-articular or intratracheal. The compositions may also be administered or co-administered in sustained release dosage forms. [0019] The invention also relates to kits and other industrial products for the treatment of disease states associated with DPP-IV.

[0020] In one embodiment, a kit is provided that comprises a composition comprising at least one DPP-IV inhibitor of the present invention in combination with instructions. The instructions may indicate the disease state for which the composition is to be administered, storage information, dosage information and / or instructions on how to administer the composition. The kit may also contain packaging materials. The packaging material may be a container for storing the composition. The kit may also contain optional additional components, such as syringes for administration of the compounds. The kit may contain the composition in single administration or multiple administration forms.

[0021] In another embodiment, an industrial article is provided that comprises a composition comprising at least one DPP-IV inhibitor of the present invention in combination with packaging materials. The packaging material may be a container for storing the composition. The container may optionally contain a label indicating the disease state for which the composition is to be administered, storage information, dosage information and / or instructions on how to administer the composition. The kit may also contain optional additional components, such as syringes for administration of the compounds. The kit may contain the composition in single administration or multiple administration forms.

[0022] Also provided are methods for making the compounds, compositions and kits of the present invention. For example, a number of synthetic schemes are provided for synthesizing compounds of the present invention.

[0023] Also provided are methods of using the compounds, compositions and kits of the present invention.

[0024] In one embodiment, the compounds, compositions, kits and industrial products are used to inhibit DPP-IV.

[0025] In another embodiment, a method of using a compound of the present invention is provided for the manufacture of a medicament for use in the treatment of a disease state that is known to be mediator of DPP-IV or known to be treated with DPP- inhibitors IV.

[0026] Examples of diseases that can be treated by administering the compounds and compositions of the present invention include, but are not limited to, conditions in which the mediator is DPP-IV, in particular diabetes, more specifically type 2 diabetes, diabetic dyslipidemia, disturbed conditions glucose tolerance (IGT), conditions of fasting plasma glucose (IFG), metabolic acidosis, ketoacidosis, appetite regulation, obesity, immunosuppressants or regulation of cytokine release, autoimmune diseases such as bowel inflammation, multiple sclerosis and rheumatoid arthritis, AIDS, cancers (prevention of metastasis, e.g. breast or prostate cancer), dermatological diseases such as psoriasis and lichen planus, infertility treatment female contraception and neurological disorders.

[0027] It is noted with respect to all the above embodiments of the invention that the present invention includes in its intentions all pharmaceutically acceptable ionized forms (e.g. salts) and solvates (e.g. hydrates) of the compounds, regardless of whether such ionized forms and solvates are in detail advisable because it is well known in the art to administer pharmaceutical agents in ionized or solvated form. It is also noted that if no particular stereochemistry is specified, the indication of the compound includes in its intentions all possible stereoisomers (e.g. enantiomers or diastereomers, depending on the number of chiral centers), regardless of whether the compound is present as a single isomer or as a mixture of isomers . In addition, unless indicated otherwise, the indication of the compound includes in swo4

EP 1 586 571 B3 intends all possible resonance forms and tautomers. With reference to the claims, the term "compound represented by the formula" includes the compound and all pharmaceutically acceptable ionized forms and solvates, all possible stereoisomers, and all possible resonant forms and tautomers, unless otherwise specified in the specific claim.

BRIEF DESCRIPTION OF THE FIGURE [0028] Figure 1 illustrates a schematic ribbon diagram of the DPP-IV structure, with highlighted secondary structural structural elements.

DEFINITIONS [0029] Unless otherwise indicated, the following terms used in the specification and claims will have the following meanings for the purpose of this application.

[0030] "Amino" means a -NH group₂.

[0031] "Animal" includes humans, non-human mammals (e.g. dogs, cats, rabbits, cattle, horses, sheep, goats, pigs, deer, and the like) and non-mammals (e.g. birds, and the like ).

[0032] "Aromatic" means a moiety in which its atoms form an unsaturated ring system, all atoms in the ring system have hybridization² and the total number of electrons pi is equal to 4n + 2. The aromatic ring may be such that the ring atoms are only carbon atoms or may contain carbon atoms and non-carbon atoms (see heteroaryl).

[0033] "Aryl" means a monocyclic or polycyclic ring assembly in which each ring is aromatic or forms an aromatic ring assembly when fused to one or more rings. Typically, Cx aryl and Cx-y aryl are typically used in which X and Y indicate the number of ring carbon atoms.

[0034] "Disease" means any unhealthy condition of an animal or part thereof, and includes an unhealthy condition that can be caused by or associated with a medical or veterinary therapy applied to the animal, i.e., "side effects" of such therapy.

[0035] As used herein, the term "fused ring" refers to a ring that is bonded to another ring to form a compound having a bicyclic structure in which the ring atoms common to both rings are bonded directly to each other. Non-exclusive examples of typical fused rings are decalin, naphthalene, anthracene, phenanthrene, indole, furan, benzofuran, quinoline, and the like. Compounds having fused ring systems may be saturated, partially saturated, carbocyclic, heterocyclic, aromatic, heteroaromatic, and the like.

[0036] "Halo" means fluorine, chlorine, bromine or iodine.

[0037] The term "isomers" means any compound having the same molecular formula but differing in the type or sequence of binding of atoms or the arrangement of atoms in space. Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers". Stereoisomers that are not mirror images of one another are referred to as "diastereomers" and stereoisomers that are non-superimposable mirror images are referred to as enantiomers or sometimes "optical isomers". The carbon atom bound to four different substituents is referred to as the "chiral center." A compound with one chiral center has two enantiomeric forms of opposite chirality. A mixture of two enantiomeric forms is referred to as a "racemic mixture." A compound with more than one chiral center has 2ⁿ"¹ enantiomeric pairs, where n is the number of chiral centers. Relationships with more than one center

EP 1 586 571 B3 can exist as a single diastereomer or as a mixture of diastereomers, referred to as a "diastereomeric mixture." When one chiral center is present, the stereoisomer can be characterized by the absolute configuration of this chiral center. Absolute configuration refers to the arrangement in space of substituents attached to the chiral center. Enantiomers are characterized by the absolute configuration of their chiral centers and described by the precedence rules of R- and S Cahn, Ingold and Prelog. The conventions of stereochemical nomenclature, methods for determining stereochemistry, and separation of stereoisomers are well known in the art (e.g., see "Advanced Organic Chemistry", 4th edition, March, Jerry, John Wiley & Sons, New York, 1992).

[0038] "Pharmaceutically acceptable" means what is useful in the manufacture of a pharmaceutical composition that is generally safe, non-toxic and is not biologically or otherwise undesirable, and includes what is acceptable for veterinary use and use pharmaceutical for people.

[0039] The term "pharmaceutically acceptable salts" means salts of the inhibitors of the present invention which are pharmaceutically acceptable as defined above and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as acetic acid, propionic acid, hexanoic acid, heptane acid, cyclopentanopionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric, benzoic acid, o- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2.2.2] oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4'-methylenebis acid (3- hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, mucous acid and the like.

[0040] Pharmaceutically acceptable salts also include base addition salts, which can be formed when acidic protons are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like.

[0041] The term "therapeutically effective amount" means that amount which, when administered to an animal to treat a disease, is sufficient to treat that disease.

[0042] The term "treatment" means any administration of a compound of the present invention and includes:

(1) preventing the occurrence of a disease in an animal that may be predisposed to the disease but not yet experiencing or showing the pathology and / or symptomology of the disease, (2) inhibiting the disease in an animal that is experiencing or exhibiting pathologies or symptommology of the disease (i.e. inhibiting further progression of pathology and / or symptomology), or (3) ameliorating the disease in an animal, who experience or exhibit pathologies or symptommologies of the disease (i.e., reversal of pathology and / or symptomology).

DETAILED DESCRIPTION OF THE INVENTION

EP 1 586 571 B3 [0043] The present invention relates to compounds, compositions, kits and industrial products that can be used to inhibit dipeptidyl IV peptidases (hereinafter referred to as DPP-IV).

[0044] DPP-IV (EC.3.4.14.5 also known as DPP4, DP4, DAP-IV, adenosine deaminase 2 complexing protein 2, adenosine deaminase binding protein (ADAbp) or CD26) is a 240kDa protein with 766 residues, which is highly specific, membrane-bound non-classical serine aminodipeptidase. DPPIV has a serine type of protease activity, cleaving dipeptides from the amino terminus of peptides with proline or alanine in the penultimate position. In addition, slow release X-Gly or X-Ser dipeptides have been reported for some naturally occurring peptides. DPP-IV is constitutively expressed on epithelial and endothelial cells of various tissues (intestines, liver, lungs and placenta), and is also found in body fluids. DPP-IV is also expressed on circulating T lymphocytes and has been shown to be synonymous with CD-26 cell surface antigen. The full-length wild-type form of DPP-IV is described in GenBank Accession Number NM_001935 ("Dipeptidyl peptidase IV (CD 26) gene expression in enterocyte-like colon cancer cell lines HT-29 and Caco-2. Cloning of the complete human coding sequence and changes of dipeptidyl peptidase IV mRNA levels during cell differentiation ", Darmoul, D., Lacasa, M., Haricault, L., Marguet, D., Sapin, C., Trotot, P., Barbat, A. and Trugnan, G ., J. Biol. Chem., 267 (7), 4824-4833, 1992).

[0045] DPP-IV belongs to the S9 serine protease family, more specifically the S9B family. Other members of the S9 family include, but are not limited to:

S9A subfamily: Dipeptidyl peptidase; Oligopeptidase B (EC 3.4.21.83);

Oligopeptidase B; Prolylooligopeptidase (EC 3.4.21.26);

S9B subfamily: Dipeptidylaminopeptidase A; Dipeptidylaminopeptidase B Dipeptidylpeptidase IV (EC 3.4.14.5); Dipeptidyl peptidase V alpha subunit of fibroblast activation protein; Seprase S9C Subfamily: Acylaminoacylpeptidase (EC 3.4.19.1) [0046] It is noted that the compounds of the present invention may also have inhibitory activity against other members of the S9 family and may therefore be used in disease states associated with these other family members.

1. CRYSTALLINE STRUCTURE DPP-IV [0047] At Syrrx, Inc. (San Diego, California) DPP-IV crystal structure has recently been solved. Cognition of the crystal structure was used to direct the design of the present DPP-IV inhibitors. [0048] Figure 1 illustrates a ribbon diagram of the DPP-IV structure, with highlighted secondary structural elements of the protein. DPP-IV is a cylindrical particle with an approximate height of 70 A and a diameter of 60 A. In the center of the Figure, the catalytic triad DPP-IV (Ser642, Asp720 and His752) is illustrated by a "ball and stick" presentation. This triad of amino acids is located in the peptidase domain or DPP-IV catalytic domain. The catalytic domain is catalytically linked to the β propeller domain. The DPP-IV catalytic domain includes residues 1-67 and 511-778. The DPP-IV catalytic domain adopts the characteristic α / β hydrolase fold. The core of this domain contains an 8-thread β sheet with all but one parallel thread. The α sheet is significantly twisted and is flanked by three α helices on one side and five α helices on the other. Topology of β strands is 1,2, -1x, 2x and (1x) (JS Richardson: The anatomy and taxonomy of protein structure; (1981) Adv. Protein Chem. 269, 15076-15084.). Many have been identified

EP 1 586 571 B3 residues that contribute to the shape and charge characteristics of the active site. Understanding these residues was an important contribution to the design of DPP-IV inhibitors of the present invention.

2. INHIBITORYDPP-IV [0049] DPP-IV inhibitors of the present invention are compounds selected from the group consisting of: 2- {6- [3-aminopiperidin-1-yl] -3-methyl-2,4-dioxo-3,4 dihydro-2H-pyrimidin-1-ylmethyl} -benzonitrile; and 2- [6- (3-aminopiperidin-1-yl) -3-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-ylmethyl] -4-fluorobenzonitrile.

[0050] Specific examples of DPP-IV inhibitors according to the present invention further include:

2- {6- [3 (R) -aminopiperydyn-1-yl] -3-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-ylmethyl} -benzonitrile;

and

2- [6- (3 (R) -aminopiperydyn-1-yl) -3-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-ylmethyl] -4fluorobenzonitryl.

[0051] In another embodiment, the present invention provides the compounds in the form of a pharmaceutically acceptable salt.

[0052] In yet another embodiment, the present invention provides compounds present in a mixture of stereoisomers. In yet another embodiment, the present invention provides the compounds as a single stereoisomer.

[0053] In yet another embodiment, the present invention provides pharmaceutical compositions containing the compound as an active ingredient. In yet another embodiment, the present invention provides pharmaceutical compositions, which composition is a solid formulation adapted for oral administration. In yet another particular variation, the present invention provides a pharmaceutical composition, which composition is a tablet. In another particular embodiment, the present invention provides a pharmaceutical composition, which composition is a liquid formulation adapted for oral administration. In yet another particular variation, the present invention provides a pharmaceutical composition, which composition is a liquid formulation adapted for parenteral administration.

[0054] In yet another particular embodiment, the present invention provides a pharmaceutical composition, which composition is adapted for administration by a route selected from oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, transbuccal, intranasal, liposome, inhalation, vaginal, intraocular, by topical (e.g. catheter or stent), subcutaneous, fat, intra-articular and intra-articular administration.

[0055] In another embodiment, the present invention provides a kit comprising a compound of the present invention and instructions that contain one or more information selected from the group consisting of an indication of the disease state in which the compound is to be administered, compound storage information, information dosing and instructions on how to administer the compound. In another embodiment, the present invention provides a kit that contains the compound in multi-dose form.

[0056] In another embodiment, the present invention provides an industrial article comprising a compound of the present invention and packaging materials. In another embodiment, the packaging material comprises a container for storing the compound. In yet another embodiment, the invention provides an article of manufacture comprising a marking indicating one or more members of a group consisting of the disease state in which the compound is to be administered, storage information, dosage information and instructions on how to administer the compound.

[0057] In another embodiment, the invention provides an industrial article, which industrial article comprises the compound in a multi-dose form.

[0058] In another embodiment, the present invention provides a compound of the present invention for use as a medicament.

[0059] In another embodiment, the present invention provides the use of a compound of the present invention in the manufacture of a medicament for treating cancer in a patient in need of such treatment.

[0060] In another embodiment, the present invention provides the use of a compound of the present invention in the manufacture of a medicament for the treatment of a disease in which the disease is type I diabetes or type II diabetes. [0061] In another embodiment, the present invention provides the use of a compound of the present invention in the manufacture of a medicament for the treatment of autoimmune disorders such as rheumatoid arthritis, psoriasis and multiple sclerosis, but not limited to a patient in need of such treatment [0062] in another embodiment, the present invention provides the use of a compound of the present invention in the manufacture of a medicament for the treatment of cancer, wherein the cancer being treated is colon, prostate, breast, thyroid, skin, lung, or head and neck cancer.

[0063] In another embodiment, the present invention provides the use of a compound of the present invention in the manufacture of a medicament for the treatment of a condition characterized by inadequate activation or concentration of lymphocytes or hematopoietic cells in a patient in need of such treatment.

[0064] In another embodiment, the present invention provides the use of a compound of the present invention in the manufacture of a medicament for treating an infection in a patient in need of such treatment.

[0065] In another embodiment, the present invention provides the use of a compound of the present invention in the manufacture of a medicament for the treatment of a condition characterized by symptoms of immune deficiency in a patient in need of such treatment.

[0066] In other of its embodiments, the present invention provides a method for producing pyrimidinedione of formula

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including:

(i) mixing 6-chloro-1H-pyrimidine-2,4-dione with an aryl halide of formula

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wherein Hal is Br, Cl or I, under conditions sufficient to produce a compound of formula

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EP 1 586 571 B3 (ii) alkylation of the subsequent product with a methyl allograft under sufficient conditions to form a compound in wawran

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(iii) kagkogkzwaniep the above product in terms of the earth about waver νη₂ • 2HCl

W [0067] In this case, the wickerwork of Wckwkckic, hence the creation of Uncnwwiwku Unlnj wbnjjujn towrenkin, together with the department. In jnOkk en zayagoólkcya wOmick, including a student from the department of bnkaggzck.

[0068] In the following, zowzwbin, oircmiOckwOiwknm jnzt: io 2- {6- [3 (R) -cmikWoionrcOck-1-clw] -3-mntclw-2,4-Oiwkzw-3,4-OiacOrw-2H-oircmiOck-1 -clwmntclw} bnkzwkitrcl.

[0069] ZcuwcSc Zia wOkWśkin wzzcztkiza wckwkcń and wzznlkiza Oclzzcza wckwkcń, wOmick or owjnOckyzcya woizcwckcza herein or zcztrznockcza compounds Sn wzzcztkin tckin wżawkckic, wOmickc and / or owjnOckzzn compounds wbnjmują IZH wzznlkin fwrmc Owouzzyzclkcya fcrmcynutczzkin zwli, zcrówkw in owztczi owjnOckzznow ztnrnwizwmnru or minzzckikc ztnrnwizwmnrów. in Ockcm zdzoznoólkc orzcok about the cinemas of wzkczckw ikcyznj.

PwOwbkin, kinOc in którcmkwlwink of wckwkcń, wOmick or owjnOckyzcya woizcwckcya herein or the compounds zcztrznockcya wbnykn jnzt jnOkw or więynj ynktrów owtnkyjclkin yhirclkcya, t iktnkyją jnzt wbjęyin wbu mwSliwcya ynktrów yhirclkcya, jnśli in Ockcm zzyznoólkcm orzcocOku cinemas wzkczckw ikcyznj.

A. Salts and hydrates of DPP-IV inhibitors [0070] NclnSc pay attention to, Sn compounds inside kikinjzznow in the liver are present in fwmin and sheep, in particular in the context of the compound. Nc orzckłcO, zckrnz kikinjzznow wżkclcaku wbnjmujn orznkzztcłynkin compounds wnOłuo kikinjzznow wżkclcaku and ztwzwwckin iya in fwrmin iya Owouzzyzclkcya fcrmcynutcyzkin zwli owyawOząycya wO róSkcyh kwczów and zczcO wrockiyzkcya and kinwrockiyzkcya zowOkin of orwynOurcmi Owbrzn zkckcmi in zztuyn.

[0071] The compounds of the KinOc wnOłuo kikinjzznow wżkclcaku mcją fwrmę wwlknj zczcOc compounds mwoą bcc wżtwwrzwkn in owztcyi Owouzzyzclknj fcrmcynutcyzkin zwli cOOcycjknj of kwcznm orznz rnckyję fwrmc wwlknj zczcOc therefore Owouzzyzclkcm fcrmcynutcyzkin kwcznm kinwrockiyzkcm or wrockiyzkcm, ko. aclwonkwwwOwrków, tckiya jck yalwrwwwOwrnk, brwmwwwOwrnk, jwOwwwOwrnk; ikkn kwczc miknrclkn and iya wowwicOcyjyn zwnnyn, tckin jck zicryzck, thursday, fwzfwrck, nty .; and clkilw- and mwkwcrclwzulfwkickc, tckin jck ntckwzulfwkick, twlun30 kwzulfwkick and bnkznkwzulfwkick; and ikkw wczwoccy wrockiyzkn i iw WowwOOwyn zwnnn, tckin jck wytck, wikick, mclnikick, burzztykick, yctrckick, bnkzwnzck, zcliyclck and czkwrbikick. Dclzzn zwln cOOcycjkn of kwczcmi wnOłuo kikinjzznow wżkclcaku wbnjmują, cln cinemas ult Ow kiya worckiyzwkn: cOcoikick, cloikick, croikick, czocrcoikick, wwOwrwzicryzck, wwOwrwzicryzck, brwmnk, mcślck, kcmfwrck, kcmfwrwzulfwkick, kcorclck, yalwrnk, yalwrwbnkzwnzck, ycklwonktckworwoiwkick, Oiolukwkick, OiwwOwrwfwzfwrck, Oikitrwbnkzwnzck , OwOnyclw10

EP 1 586 571 B3 sulfate, fumarate, galactan (from mucous acid), galacturonate, glucoheptanoate, gluconate, glutamate, glycerophosphate, hemisuccinate, hemisulfate, heptanoate, hexanoate, hippuran, hydrochloride, hydrobromide, iodide, 2-hydroxyethane sulfate isobutyrate, lactate, lactobionate, malate, malonate, mandelate, metaphosphate, methanesulfonate, methylbenzoate, monohydrogen phosphate, 2-naphthalene sulfonate, nicotinate, nitrate, oxalate, oleate, pamoesate, pectinate, persulfate, phenylacetate, 3-phenylpropionate, phosphate, phosphonate and phthalate. It should be taken into account that the forms of the free bases will usually differ slightly in physical properties from the forms of their respective salts, such as solubility in polar solvents, but in other respects the salts are equivalent to the forms of the corresponding free bases for the purposes of the present invention.

[0072] When the compounds of the present invention are in the form of the free acid, a pharmaceutically acceptable salt with the base can be prepared by reacting the free acid form with a pharmaceutically acceptable inorganic or organic base. Examples of such bases are alkali metal hydroxides, including potassium, sodium and lithium hydroxides; alkaline earth metal hydroxides such as barium and calcium hydroxides; alkali metal alkoxides, e.g. potassium ethoxide and sodium propoxide; and various organic bases such as ammonium hydroxide, piperidine, diethanolamine and N-methylglutamine. Aluminum salts of the compounds of the present invention are also included. Further base salts of the present invention include, but are not limited to: copper, ferric, ferrous, lithium, magnesium, manganese, manganese, potassium, sodium and zinc salts. Salts of organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, e.g. salts of arginine, betaine, caffeine, chloroprocaine, choline, N, N'-dibenzylethylenediamine (benzathine), dicyclohexylamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, Netamine, glucamine, Netamine , iso-propylamine, lidocaine, lysine, meglumine, N-methyl-D-glucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethanolamine, triethylamine, trimethylamine, tripropylamine and tris- (hydroxymethyl) methylamine (tromethamine). It should be taken into account that the forms of the free acid will usually differ slightly in physical properties from those of their respective salts, such as solubility in polar solvents, but in other respects the salts are equivalent to those of the corresponding free acids for the purposes of the present invention.

[0073] Protected derivatives of the compounds of the present invention can also be prepared. Examples of techniques applicable to the creation and removal of protecting groups can be found in TW Greene, Protecting Groups in Organic Synthesis, 3rd edition, John Wiley &amp; Sons, Inc. 1999.

[0074] The compounds of the present invention may also conveniently be prepared or formed in the process of the invention as solvates (e.g. hydrates). Hydrates of compounds of the present invention may conveniently be prepared by recrystallization from a water / organic solvent mixture using organic solvents such as dioxane, tetrahydrofuran or methanol.

[0075] The term "pharmaceutically acceptable salt" as used herein is intended to include all compounds of the present invention that are used in the form of a salt, especially when the salt confers improved pharmacokinetic properties on the compound compared to the free form of the compound or other compound salt. The form of a pharmaceutically acceptable salt may also confer on the compound desirable pharmacokinetic properties that it previously did not possess, and may even positively affect the pharmacodynamics of the compound with respect to its therapeutic activity in the body. An example of pharmacokinetic properties, on

A beneficial effect that can be had is the way the compound is transported across cell membranes, which in turn can directly and positively affect the absorption, distribution, biotransformation and excretion of the compound. Although the route of administration of the pharmaceutical composition is important and bioavailability may be critically affected by various anatomical, physiological and pathological factors, the solubility of the compound is usually dependent on the nature of the particular form of its salt used. One skilled in the art will know that an aqueous solution of a compound will provide the fastest absorption of the compound in the body of the subject being treated, while lipid solutions and suspensions as well as solid dosage forms will result in less rapid absorption of the compound.

3. INDICATIONS FOR THE USE OF DPP-IV INHIBITORS [0076] DPP-IV is believed to contribute to the pathology and / or symptomology of many different diseases, therefore the reduction of DPP-IV activity in the subject by inhibition can be used for therapeutic management in these disease states. Examples of various diseases that can be treated using DPP-IV inhibitors of the present invention are described herein. It is noted that additional diseases in addition to those disclosed herein may be identified at a later time because the biological roles that DPP-IV plays in various pathways are becoming more fully understood.

[0077] One set of indications in which the DPP-IV inhibitors of the present invention may be used is the prevention and treatment of diabetes and obesity, in particular type 2 diabetes, diabetic dyslipidemia, impaired glucose tolerance (IGT) conditions, impaired glucose levels fasting (IFG), metabolic acidosis, ketoacidosis, appetite regulation and obesity.

[0078] DPP-IV inhibitors of the present invention may also be used as immunosuppressants (or drugs that suppress cytokine release) for the treatment of, inter alia: organ transplant rejection; autoimmune diseases such as inflammatory bowel disease, multiple sclerosis and rheumatoid arthritis; and in the treatment of AIDS.

[0079] DPP-IV inhibitors of the present invention may also be used to treat various cancers, including breast cancer, lung cancer and prostate cancer.

[0080] The DPP-IV inhibitors of the present invention can also be used to treat dermatological diseases such as psoriasis, rheumatoid arthritis (RA) and lichen planus.

[0081] DPP-IV inhibitors of the present invention may also be used to treat infertility and amenorrhea.

[0082] DPP-IV inhibitors of the present invention can also be used to modulate the cleavage of various cytokines (stimulation of hematopoietic cells), growth factors and neuropeptides. For example, such conditions often occur in patients in immunosuppressive states, for example as a consequence of chemotherapy and / or radiation therapy for cancer.

[0083] DPP-IV inhibitors of the present invention may also be used to prevent or reduce cleavage of N-terminal Tyr-Ala from growth hormone releasing factor. Accordingly, these inhibitors can be used to treat short stature associated with growth hormone deficiency (dwarfism) and to promote GH-dependent tissue growth or regrowth.

[0084] The DPP-IV inhibitors of the present invention may also be used to treat various disease states associated with neuropeptide cleavage and may thus be useful for regulating or normalizing neurological disorders.

EP 1 586 571 B3 [0085] For oncological indications, DPP-IV inhibitors of the present invention may be used in combination with other agents to inhibit unwanted and uncontrolled cell proliferation. Examples of other antiproliferative agents that may be used in combination with the DPPIV inhibitors of the present invention include, but are not limited to, retinoic acid and its derivatives,

TM TM

2-methoxyestradiol, ANGIOSTATIN protein, ENDOSTATIN protein, suramin, squalamine, metalloproteinase-1 tissue inhibitor, metalloproteinase-2 tissue inhibitor, plasminogen 1 activator inhibitor, plasminogen 2 activator inhibitor, cartilage derived inhibitor, paclitaxel, platelet factor 4, sulfate factor 4 ), sulfonated chitin derivatives (made from king crab shells), sulfonated polysaccharide - peptidoglycan complex (sp-pg), staurosporine, modulators of matrix metabolism, including, for example, proline analogues ((1-azetidine-2-carboxylic acid (LACA)), cishydroxyproline, d, 1-3,4 -dehyde-proline, thiaproline, beta.-aminopropionitrile fumarate, 4-propyl-5- ( 4-pyridinyl) -2 (3H) -oxazolone, methotrexate, mitoxantrone, heparin, interferons, macroglobulin 2 serum, chimp-3, chymostatin, beta.-cyclodextrin tetradecasulfate, eponemycin; fumagillin, sodium gold thiomalate, d-penicillamine (CDPT), beta-1-anti-collagenase, alpha, 2-antiplasmin, bisantrene, disodium lobenzarite, n-2-carboxyphenyl-4-chloroantranilic or "CCA" disodium salt, thalidomide; angiostatic steroid, carboxyamine imidazole; metalloproteinase inhibitors such as BB94. Other anti-angiogenesis agents that can be used include antibodies, preferably monoclonal antibodies against these angiogenic growth factors: bFGF, aFGF, FGF-5, VEGF, VEGF-C, HGF / SF and Ang-1 / Ang-2 isoforms. Ferrara N. and Alitalo, K. "Clinical application of angiogenic growth factors and their inhibitors" (1999) Nature Medicine 5: 13591364.

4. ENHANCING COMPOSITIONS AND DPP-IV HIBITORS [0086] A variety of compositions and methods of administration may be used in combination with the DPP-IV inhibitors of the present invention. Such compositions in addition to the DPP-IV inhibitors of the present invention may contain typical pharmaceutical excipients and other conventional pharmaceutically inactive agents. In addition, the compositions may contain active ingredients in addition to the DPP-IV inhibitors of the present invention. These additional active ingredients may include additional compounds of the invention, and / or one or more other pharmaceutically active agents.

[0087] The compositions may be in gaseous, liquid, semi-liquid or solid form, formulated in a manner suitable for the route of administration to be used. Capsules and tablets are typically used for oral administration. For parenteral administration, reconstitution of a lyophilized powder prepared as described herein is typically used.

[0088] Compositions containing DPP-IV inhibitors of the present invention may be administered or co-administered orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbucally, intranasally, liposomally, inhaled, vaginally, intraocularly. topical administration (e.g. via catheter or stent), subcutaneous, adipose tissue, intra-articular or intrasternal administration. The compounds and / or compositions of the invention may also be administered or co-administered in slow release dosage forms.

[0089] DPP-IV inhibitors and compositions containing them may be administered or co-administered in any conventional dosage form. Co-administration in the context of the invention is intended to mean the administration of more than one therapeutic agent, one of which contains a DPP-IV inhibitor, during coordinated treatment13

EP 1 586 571 B3 to achieve an improved clinical result. Such co-administration may also be coexisting, i.e. occurring in overlapping periods of time.

[0090] Solutions or suspensions used for parenteral, intradermal, subcutaneous or topical administration may optionally contain one or more of the following components: a sterile diluent such as water for injection, saline solution, non-volatile oil of plant or animal origin, polyethylene glycol, glycerin, propylene glycol or other synthetic solvent; bactericides such as benzyl alcohol and methyl parabens; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates and phosphates; tonicity adjusting agents such as sodium chloride or dextrose, and agents for controlling the acidity or alkalinity of the composition, such as alkaline or acidifying agents or buffers such as carbonates, bicarbonates, phosphates, hydrochloric acid and organic acids such as acetic and citric acid. Parenteral preparations may optionally be enclosed in ampoules, disposable syringes or single or multiple dose vials made of glass, plastic or other suitable material.

[0091] When the DPP-IV inhibitors of the present invention show insufficient solubility, methods for solubilizing compounds can be used. Such methods are known to those skilled in the art and include, but are not limited to, the use of cosolvents such as dimethyl sulfoxide (DMSO), the use of surfactants such as TWEEN, or dissolution in aqueous sodium bicarbonate. Compounds such as prodrugs can also be used to formulate effective pharmaceutical compositions.

[0092] A solution, suspension, emulsion or the like may form in the composition when the DPP-IV inhibitors of the present invention are mixed or added. The form of the resulting composition will depend on many factors, including the intended route of administration and the solubility of the compound in the chosen carrier or vehicle. The effective concentration needed to alleviate the disease being treated can be empirically determined.

[0093] The compositions of the present invention may be provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, dry inhalable powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and emulsions oil-in-water containing appropriate amounts of compounds, especially their pharmaceutically acceptable salts, preferably sodium salts. Pharmaceutically and therapeutically active compounds and their derivatives are typically formulated and administered in unit dosage forms or multi-dose forms. Unit dosage forms herein refer to physically discrete units suitable for human and animal subjects and individually packaged, as is known in the art. Each dosage unit contains a specific amount of the therapeutically active compound suitable to produce the desired therapeutic effect in association with the required carrier, vehicle or pharmaceutical diluent. Examples of unit dosage forms include ampoules and syringes, individually packaged tablets or capsules. Unit dosage forms may be administered in parts or multiples thereof. Multi-dose forms are many identical unit dosage forms packaged in a single container for administration in separate unit dosage forms. Examples of multi-dose forms include vials, tablet or capsule bottles, or pint or gallon bottles. Thus, the multi-dose form is a plurality of unit dosage forms not separated during packaging.

[0094] In addition to one or more DPP-IV inhibitors of the present invention, the composition may include: a diluent such as lactose, sucrose, dicalcium phosphate or carboxymethyl cellulose; a lubricant such as magnesium stearate, calcium stearate and talc; a binder such as starch, natural gums such as acacia and gelatin, molasses, polyvinylpyrrolidone, celluloses and their derivatives, povidone, crospovidones and other such binders known to those skilled in the art. Liquid compositions suitable for administration as pharmaceuticals can for example be prepared by dissolving, dispersing or otherwise mixing the active compound as defined above and optional pharmaceutical auxiliaries in a carrier such as, for example, water, saline, aqueous dextrose, glycerol, glycols , ethanol, and the like to form a solution or suspension. If desired, the pharmaceutical composition for administration may also contain small amounts of auxiliary substances such as wetting agents, emulsifying or solubilizing agents, pH buffering agents and the like, for example sodium acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, sodium acetate triethanolamine, triethanolamine oleate, and other such agents. Actual methods for making such dosage forms are known in the art or will be apparent to those skilled in the art; see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975. The compositions or formulations for administration will in each case contain the DPP-IV inhibitor of the present invention in an amount sufficient to reduce DPP-IV activity in vivo, thus treating the subject's disease state.

[0095] Dosage forms or compositions may optionally contain one or more DPP-IV inhibitors of the present invention in the range of 0.005% to 100% (w / w), and the balance will be supplemented with additional substances as described herein. For oral administration, the pharmaceutically acceptable composition may optionally contain any one or more of the commonly used inactive ingredients, such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, talc, cellulose derivatives, croscarmellose sodium, glucose, sucrose, magnesium carbonate. sodium saccharin, talc. Such compositions include solutions, suspensions, tablets, capsules, powders, dry inhalable powders, and sustained release formulations, such as, but not limited to, implants and micro-encapsulated delivery systems, and biodegradable, biocompatible polymers such as collagen, acetate ethylene vinyl, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others. Methods for making these formulations are known to those skilled in the art. The compositions may optionally contain 0.01% -100% (w / w) of one or more DPP-IV inhibitors, optionally 0.1-95%, and optionally 1-95%.

[0096] Salts of DPP-IV inhibitors, preferably sodium salts, can be prepared with carriers that protect the compound against rapid removal from the body, such as formulations or coatings controlling release over time. The formulations may further contain other active compounds to obtain the desired combinations or properties.

A. Formulations for oral administration [0097] Dosage forms for oral administration may be solid, gel or liquid forms. Examples of solid dosage forms include, but are not limited to, tablets, capsules, granules and loose powders. More specific examples of oral tablets include compressed pastilles and chewable tablets which may be coated with enteric coatings, sugar coatings or film coatings. Examples of capsules are soft and hard gelatin capsules. Granules and powders can be supplied in non-effervescent or effervescent forms. Each of them can be combined with other ingredients known to those skilled in the art.

In some embodiments, the DPP-IV inhibitors of the present invention are provided as solid dosage forms, preferably capsules or tablets. Tablets, pills, capsules, cachets and the like may optionally contain one or more of the following ingredients, or compounds of a similar type: binder; a diluent; disintegrant; lubricant; glidant; sweetener; and flavoring.

[0099] Examples of binders that may be used include, but are not limited to, microcrystalline cellulose, gum tragacanth, glucose solution, acacia, gelatin solution, sucrose and starch paste.

[0100] Examples of lubricants that may be used include, but are not limited to, talc, starch, magnesium or calcium stearate, lycopodium and stearic acid.

[0101] Examples of diluents that may be used include, but are not limited to, lactose, sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate.

[0102] Examples of glidants that may be used include, but are not limited to, colloidal silicon dioxide.

[0103] Examples of disintegrants that may be used include, but are not limited to, croscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methyl cellulose, agar and carboxymethyl cellulose.

[0104] Examples of coloring agents that may be used include, but are not limited to, any of the water-soluble FD and C dyes approved for use, mixtures thereof; and water-insoluble FD and C dyes suspended on alumina hydrate.

[0105] Examples of sweeteners that can be used include, but are not limited to, sucrose, lactose, mannitol and artificial sweeteners such as sodium cyclamate and saccharin, and many spray dried flavors.

[0106] Examples of flavors that may be used include, but are not limited to, natural flavors extracted from plants, such as fruit and synthetic blends of compounds that produce a pleasant impression such as, but not limited to restrictions on them, peppermint oil and methyl salicylate.

[0107] Examples of wetting agents that may be used include, but are not limited to, propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether.

[0108] Examples of antimetemetic coatings that may be used include, but are not limited to, fatty acids, fats, waxes, shellac, ammoniated shellac, and cellulose acetate phthalate.

[0109] Examples of film-forming coatings that can be used include, but are not limited to, hydroxyethyl cellulose, sodium carboxymethyl cellulose, polyethylene glycol 4000, and cellulose acetate phthalate^froms.

[0110] If oral administration is desired, a salt of the compound may optionally be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition may be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition may also be formulated in combination with antacids or other such ingredients.

[0111] When the capsule dosage form is, it may optionally additionally contain a liquid carrier such as oil. In addition, unit dosage forms may optionally additionally comprise 16

A variety of other materials that modify the physical form of the dosage unit, for example, sugar coatings and other enteric agents can be used.

[0112] The compounds of the present invention may also be administered as a component of an elixir, suspension, syrup, wafer, spray formulation, chewing gum or the like. The syrup may optionally contain, in addition to the active compounds, sucrose as a sweetening agent, and certain preservatives, dyes and flavors.

[0113] DPP-IV inhibitors of the present invention may also be mixed with other active substances that do not interfere with the desired effect, such as antacida, H2 blockers and diuretics. For example, if a compound is used to treat asthma or hypertension, it may be used with other bronchodilators and antihypertensive agents, respectively.

[0114] Examples of pharmaceutically acceptable carriers that may contain tablets containing the DPP-IV inhibitors of the present invention include, but are not limited to, binders, lubricants, diluents, disintegrants, coloring agents, flavors and wetting agents . Enteric-coated tablets, due to the enteric coating, resist gastric acid and dissolve or disintegrate in a neutral or alkaline environment in the intestine. Sugar-coated tablets may be compressed tablets that have been coated with various layers of pharmaceutically acceptable substances. Film-coated tablets may be compressed tablets that have been coated with polymers or other suitable coating. Repeatedly compressed tablets may be compressed tablets made by more than one compression cycle using the pharmaceutically acceptable substances previously mentioned. Coloring agents may also be used in tablets. Flavors and sweeteners can also be used in tablets, and they are particularly useful in the production of pastilles and chewable tablets.

[0115] Examples of liquid oral forms that can be used include, but are not limited to, aqueous solutions, emulsions, suspensions, solutions and / or suspensions reconstituted from effervescent granules.

[0116] Examples of aqueous solutions that can be used include, but are not limited to, elixirs and syrups. The term elixirs used herein refers to clear, sweetened hydroalcoholic preparations. Examples of pharmaceutically acceptable carriers that can be used in elixirs include, but are not limited to, solvents. Particular examples of solvents that can be used include glycerin, sorbitol, ethyl alcohol and syrup. The term syrup as used herein refers to concentrated aqueous sugar solutions, for example, sucrose. The syrups may optionally additionally contain a preservative.

[0117] The term emulsions refers to two-phase systems in which one liquid is dispersed in the form of small grains in another liquid. The emulsions may be oil-in-water or water-in-oil emulsions. Examples of pharmaceutically acceptable agents that can be used in emulsions include, but are not limited to, non-aqueous liquids, emulsifying agents and preservatives.

[0118] Examples of pharmaceutically acceptable substances that can be used in non-effervescent granules intended to be reconstituted into a liquid oral dosage form include diluents, sweeteners and wetting agents.

[0119] Examples of pharmaceutically acceptable substances that can be used in effervescent granules to be reconstituted into a liquid oral dosage form include organic acids and a source of carbon dioxide.

EP 1 586 571 B3 [0120] Coloring and flavoring agents may optionally be used in all of the above dosage forms.

[0121] Particular examples of preservatives that can be used include glycerin, methyl and propylparaben, benzoic acid, sodium benzoate and alcohol.

[0122] Particular examples of liquid non-aqueous agents that can be used in emulsions include mineral oil and cottonseed oil.

[0123] Particular examples of emulsifying agents that can be used include gelatin, acacia, tragacanth, bentonite and surfactants such as polyoxyethylene sorbitan monooleate.

[0124] Particular examples of suspending agents that may be used include sodium carboxymethyl cellulose, pectin, tragacanth, Veegum and acacia. Diluents include lactose and sucrose. Sweeteners include sucrose, syrups, glycerin and artificial sweeteners such as sodium cyclamate and saccharin.

[0125] Particular examples of wetting agents that may be used include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether.

[0126] Particular examples of organic acids that can be used include citric acid and tartaric acid.

[0127] Sources of carbon dioxide that can be used in effervescent compositions include sodium bicarbonate and sodium carbonate. Coloring agents include all FD and C water-soluble dyes approved for use, and mixtures thereof.

[0128] Particular examples of flavors that can be used include natural flavors extracted from plants, and synthetic blends of compounds that produce a pleasant taste sensation.

[0129] For a solid dosage form, the solution or suspension, for example in propylene carbonate, vegetable oils or triglycerides, is preferably encapsulated in a gelatin capsule. Such solutions, their preparation and encapsulation are disclosed in US Patent Nos. 4,328,245; 4 409 239; and 4,410,545.

For such a liquid dosage form, the solution, for example in polyethylene glycol, may be diluted with a sufficient amount of a pharmaceutically acceptable liquid carrier, e.g. water, so that it is easily measurable for administration.

[0130] Alternatively, liquid or semi-solid oral preparations can be prepared by dissolving or dispersing the active compound or salts thereof in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate) and other such carriers, and encapsulating these solutions or suspensions in hard or soft gelatin capsules. Other useful formulations include those set out in US Patent Nos. Re 28,819 and 4,358,603.

B. Single injection solutions [0131] The present invention also relates to compositions designed for the administration of DPP-IV inhibitors of the present invention by parenteral administration, generally characterized by injection, subcutaneous, intramuscular or intravenous administration. Injectable preparations may be prepared in any conventional form, for example, as liquid solutions or suspensions, solid forms suitable for dissolution or suspension in a liquid prior to injection, or as emulsions.

EP 1 586 571 B3 [0132] Examples of inactive agents that can be used in conjunction with the injection formulations of the present invention include, but are not limited to, water, saline, dextrose, glycerol or ethanol. Injectable compositions may also optionally contain small amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubilizers and other such agents such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrin. Implantation of a slow or sustained release system is also contemplated here such that a constant dosage level is maintained (see e.g. U.S. Patent No. 3,710,795). The percentage of active compound in such parenteral compositions is highly dependent on its specific type, and on the activity of the compound and the needs of the subject.

[0133] Parenteral administration of the formulation includes intravenous, subcutaneous and intramuscular administration. Preparations for parenteral administration include sterile injectable solutions, sterile dry soluble products such as the lyophilized powders described herein ready to be reconstituted with the solvent immediately before use, including hypodermic tablets, sterile injectable suspensions, sterile dry soluble products ready for combination from the vehicle immediately before use, and sterile emulsions. The solutions may be aqueous or non-aqueous.

[0134] Examples of suitable carriers for intravenous administration include, but are not limited to, physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol and polypropylene glycol, and mixtures thereof. [0135] Examples of pharmaceutically acceptable carriers that may optionally be used in parenteral preparations include, but are not limited to, aqueous vehicle, non-aqueous vehicle, antimicrobials, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances.

[0136] Examples of aqueous vehicles that may optionally be used include sodium chloride for injection, Ringer's injection, isotonic dextrose for injection, sterile water for injection, dextrose and lactated Ringer's injection.

[0137] Examples of non-aqueous parenteral vehicles that may optionally be used include vegetable oils, cottonseed oil, sesame oil, corn oil and peanut oil.

[0138] Antimicrobials may be added to parenteral formulations at bacteriostatic or fungistatic concentrations, especially when the formulations are packaged in multi-dose containers and designed so that multiples of parts can be stored and withdrawn. Examples of antimicrobials that may be used include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl esters of p-hydroxybenzoic acid, thimerosal, benzalkonium chloride and benzethonium chloride.

[0139] Examples of isotonic agents that may be used include sodium chloride and dextrose. Examples of buffering agents that can be used include phosphate and citrate. Examples of antioxidants that can be used include sodium bisulfite. Examples of local anesthetics that can be used include procaine hydrochloride. Examples of suspending and dispersing agents that may be used include sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose and polyvinylpyrrolidone. Examples of emulsifying agents that can be used include polysorbate 80 (TWEEN 80). Ion sequestering or chelating agents include EDTA.

[0140] Pharmaceutical carriers may also optionally include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

[0141] The concentration of DPP-IV inhibitor in the parenteral preparation can be adjusted such that the injection provides a pharmaceutically effective amount to exert the desired pharmacological effect. As is known in the art, the exact concentration and / or dosage used of a DPP-IV inhibitor will ultimately depend on the age and condition of the patient or animal.

[0142] Dosage units of parenteral preparations can be packaged in ampoules, vials or syringes with a needle. According to the knowledge and practice of art, all preparations for parenteral administration should be sterile.

[0143] Injectable preparations may be designed for topical and systemic administration. Typically, a therapeutically effective dose is formulated to contain a concentration of at least about 0.1% w / w to about 90% w / w or more, preferably above 1% w / w DPP-IV inhibitor to the treated tissue / tissues. The DPP-IV inhibitor may be administered at once, or divided into several smaller doses to be administered at intervals of time. It should be understood that the exact dosage and duration of treatment will depend on the site where the composition is topically administered, the vehicle and other variables that can be determined empirically using known test protocols or by extrapolating in vivo or in vitro test data. It should be noted that concentration and dose values ​​may also vary depending on the age of the subject being treated. Next, it should be understood that for each particular subject, it may be necessary to adjust the assessment of a particular dosage regime over time, depending on individual needs and the professional person administering or supervising the administration of the formulation. Thus, the concentration ranges presented herein are intended as examples and are not intended to limit the scope or practical implementation of the claimed formulations.

[0144] The DPP-IV inhibitor may optionally be suspended in micronized or other suitable form or may be derivatized to obtain a more soluble active product or to produce a prodrug. The form of the resulting mixture depends on many factors, including the intended route of administration and the solubility of the compound in the selected vehicle or vehicle. The effective concentration is the concentration sufficient to alleviate the symptoms of the disease state and can be determined empirically.

C. Anilized Powders [0145] DPP-IV inhibitors of the present invention may also be prepared as lyophilized powders that can be reconstituted for administration as solutions, emulsions and other mixtures. Lyophilized powders can also be formulated as solids or gels.

[0146] Sterile, lyophilized powder may be prepared by dissolving the compound in a sodium phosphate buffer solution containing dextrose or other suitable excipient. The desired formulation is obtained by subsequent sterile filtration followed by lyophilization under standard conditions known to those skilled in the art. Briefly, a lyophilized powder can optionally be prepared by dissolving dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, sucrose or other suitable agent, about 1-20%, preferably about 5 to 15%, in a suitable buffer, such as citrate , sodium or potassium phosphate or other such buffer known to those skilled in the art, typically at approximately neutral pH. The DPP-IV inhibitor is then added to the resulting mixture, preferably above room temperature, more preferably about 30-35 ° C, and stirred until dissolved. The resulting mixture is diluted for

EP 1 586 571 B3 adding more buffer to the desired concentration. The resulting mixture is sterilized by filtration or treated to remove particles and to ensure sterility, and lyophilized after being portioned into vials. Each vial may contain a single dose or multiple doses of DPP-IV inhibitor.

D. Topical administration [0147] DPP-IV inhibitors of the present invention may also be administered as topical mixtures. Topical mixtures can be used for topical or systemic administration. The resulting mixture may be a solution, suspension, emulsion or the like and they are formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigation, sprays, suppositories, bandages, skin patches and other formulations suitable for topical administration.

[0148] DPP-IV inhibitors can be formulated as sprays for topical administration, such as by inhalation (see US Patent Nos. 4 044 126, 4 414 209 and 4 364 923, which describe sprays for providing a steroid useful in the treatment of inflammatory diseases, particularly asthma). These formulations for administration to the respiratory tract may be in the form of an aerosol or nebulizer solution, or micronized blowing powder, alone or in combination with an inert carrier such as lactose. In this case, the formulation particles will typically have diameters below 50 microns, preferably below 10 microns.

[0149] DPP-IV inhibitors may also be formulated for topical administration, such as topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams and lotions, and for application to the eye or to the reservoir or spinal cord application . Topical administration is contemplated for transdermal delivery and administration to the eyes or mucosa or for inhalation therapy. Nasal solutions of the DPP-IV inhibitor may also be administered alone or in combination with other pharmaceutically acceptable excipients.

E. Formulations for and enrc. Respiratory administration [0150] Depending on the condition being treated, other routes of administration may also be used, such as topical application, transdermal patches, and rectal administration. For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic effect. Rectal suppositories are used herein in the sense of solid bodies for insertion into the rectum that melt or soften at body temperature, releasing one or more pharmacologically or therapeutically active ingredients. Pharmaceutically acceptable substances used in rectal suppositories are media or vehicle and agents to increase the melting point. Examples of substrates include cocoa butter (theobromic oil), mixtures of glycerin and gelatin, carbowax, (polyoxyethylene glycol) and suitable mixtures of mono-, di- and triglycerides of fatty acids. Combinations of different substrates can be used. Agents for increasing the melting point include olbrot and wax. Rectal suppositories can be made by compression or molding. The typical weight of a rectal suppository is about 2 to 3 g. Tablets and capsules for rectal administration may be prepared using the same pharmaceutically acceptable substances and the same methods as for formulations for oral administration.

F. PosaPayment of forms

EP 1 586 571 B3 [0151] Below are specific examples of oral, intravenous and tablet formulations that can optionally be used with the compounds of the present invention. It is noted that these formulations may vary depending on the particular compound used and the indication for which the formulation is to be used.

ORAL FORMULATION

Compound of the invention Citric acid monohydrate

Sodium hydroxide

Flavor

Water

10-100 mg 105 mg 18 mg

qs up to 100 ml

INTRAVENIC FORMULATION

Compound of the invention Dextrose Monohydrate Citric acid monohydrate

Sodium hydroxide

Water for injections

TABLET FORMULATION

0.1-10 mg

qs to give isotonicity 1.05 mg 0.18 mg qs to 1.0 ml

Compound of the invention 1%

73% microcrystalline cellulose

25% stearic acid

Colloidal silica 1%

5. KITS CONTAINING DPP-IV INHIBITORS [0155] The invention also relates to kits and other industrial products for the treatment of diseases associated with DPP-IV. It is noted that diseases include all conditions for which DPP-IV has activity contributing to the pathology and / or symptomology of the condition.

and [0156] In one embodiment, a kit is provided that comprises a composition comprising at least one DPP-IV inhibitor of the present invention in combination with instructions. The instructions may indicate the disease state in which the composition is to be administered, storage information, dosage information and / or instructions on how to administer the composition. The kit may also contain packaging materials. The packaging material may be a container for holding the composition. The i5 kit may also optionally contain an additional component, such as a syringe, for administration of the composition.

The kit may contain the composition in unit or multi-dose dosage forms. [0157] In another embodiment, the present invention provides an industrial article comprising a composition comprising at least one DPP-IV inhibitor of the present invention in combination with packaging materials. The packaging material may be a container for storing the compound. The container may optionally contain a label indicating the disease state in which the composition is to be administered, storage information, dosage information and instructions on how to administer

EP 1 586 571 B3 composition. The kit may also optionally contain additional components, such as syringes for administration of the composition. The kit may contain the composition in unit or multi-dose dosage forms.

[0158] It is noted that the packaging materials used in the industrial kits and articles of the present invention can form a plurality of divided containers, such as a divided bottle or a divided foil packet. The container may be in any conventional shape or form, as is known in the art, made from a pharmaceutically acceptable material, for example a paper or cardboard box, a glass or plastic bottle or jar, a resealable pouch (for example to hold a "supply" of tablets) to be placed in another container) or blister pack with single doses to be squeezed out of the pack in accordance with the therapeutic schedule. The type of container used will depend on the exact dosage form, for example, a conventional cardboard box will generally not be used to hold a liquid suspension. It is possible that more than one container will be used in a single package to sell a single dosage form. For example, tablets may be contained in a bottle, which in turn is contained in a box. Typically, the kit includes instructions for administering separate ingredients. The kit form is particularly advantageous when the separate components are administered in different dosage forms (e.g., oral, topical, transdermal and parenteral), are administered at different time intervals, or when it is desirable to select the dosage of the individual components by the prescribing physician.

[0159] One particular example of a kit according to the present invention is the so-called blister pack. Blister packs are well known in the packaging industry and are widely used for packaging pharmaceutical dosage units (tablets, capsules and the like). Blister packs generally consist of a sheet of relatively rigid material covered with a film of plastic material, preferably transparent. During the packaging process, recesses are formed in the plastic film. The recesses have the size and shape of individual tablets or capsules to be packaged, or have the size and shape to accommodate the multiple tablets and / or capsules to be packaged. Then, tablets or capsules are placed in the cavities and a sheet of relatively rigid material is heat sealed with a plastic film on the side of the foil that is opposite to the direction in which the cavities were formed. As a result, tablets or capsules are sealed individually or collectively as needed in the cavities between the plastic film and the sheet. Preferably, the strength of the sheet is such that tablets or capsules can be removed from the blister pack by manually pressing on the depressions and thus forming an opening in the sheet at the location of the depression. The tablet or capsule can then be removed from the sheet through said opening.

[0160] Another particular embodiment of the kit may be a dispenser designed to dispense daily doses one at a time during their intended use. Preferably, the dispenser is equipped with a memory aid device so as to further facilitate compliance with the regime. An example of such a memory support device is a mechanical counter that indicates the number of daily doses that have been issued. Another example of such a memory enhancement device is battery-powered microchip memory coupled to a liquid crystal screen or booster sound that, for example, gives the date when the last daily dose was taken and / or reminds you when to take the next dose.

EP 1 586 571 B3

EXAMPLES

1. Preparation of DPP-IV inhibitors [0161] Various methods can be developed to synthesize compounds of the present invention. Representative methods for synthesizing these compounds are provided in the examples. It is noted, however, that the compounds of the present invention may also be synthesized by other synthetic routes that others may design.

[0162] It is easy to understand that some of the compounds of the present invention have atoms with connections to other atoms that give the compound particular stereochemistry (e.g., chiral centers). It is known that during the synthesis of the compounds of the present invention, mixtures of different stereoisomers (enantiomers, diastereomers) may be formed. Unless specific stereochemistry is specified, listing of the compound is intended to include all of the various possible stereoisomers.

[0163] Various methods are known in the art for separating mixtures of different stereoisomers. For example, a racemic mixture of a compound may be reacted with an optically active resolving agent to give a pair of diastereomeric compounds. Then the diastereomers can be separated to recover optically pure enantiomers. Dissociable complexes (e.g. crystalline diastereomeric salts) can also be used to separate enantiomers. Diastereomers will typically have physical properties (e.g., melting points, boiling points, solubilities, reactivity, etc.) sufficiently different enough to be able to be easily separated using these differences. For example, diastereomers can typically be separated by chromatography or by separation / separation techniques based on differences in solubility. A more detailed description of the techniques that can be used to separate stereoisomers of compounds from their racemic mixtures can be found in Jean Jacques Andre Collet, Samuel H. Wilen, Enantiomers, Racemates and Resolutions, John Wiley &amp; Sons, Inc. (1981).

[0164] The compounds of the present invention may also be prepared as pharmaceutically acceptable acid addition salts by reacting the free base compound with a pharmaceutically acceptable inorganic or organic acid. Alternatively, a pharmaceutically acceptable base addition salt may be prepared by reacting the compound in the form of the free acid with a pharmaceutically acceptable inorganic or organic base. Inorganic and organic acids and bases suitable for the preparation of pharmaceutically acceptable salts of the compounds are set out in the definition part of this application. Alternatively, the salt compounds can be prepared using the salts of the substrates or intermediates.

[0165] The free acid or free base forms of the compounds can be prepared from the corresponding base addition salt or acid addition salt. For example, an acid addition salt compound can be converted to the corresponding free base by treatment with a suitable base (e.g., ammonium hydroxide, sodium hydroxide, and the like). The compound in the form of a base addition salt can be converted to the corresponding free acid by treatment with a suitable acid (e.g. hydrochloric acid, etc).

[0166] Protected derivatives of the compounds can be prepared by methods known to those skilled in the art. A detailed description of the techniques applicable to the creation and removal of protecting groups can be found in TW Greene, Protecting Groups in Organic Synthesis, 3rd edition, John Wiley &amp; Sons, Inc. 1999.

[0167] The compounds of the present invention may be conveniently prepared or obtained when carrying out the method of the invention in the form of solvates (e.g. hydrates). Hydrates of compounds according to

EP 1 586 571 B3 of the present invention can conveniently be prepared by recrystallization from a water / organic solvent mixture, using organic solvents such as dioxane, tetrahydrofuran or methanol.

[0168] The compounds of the present invention may also be prepared as their individual stereoisomes by reacting a racemic mixture of an active resolving agent to form a pair of diastereomeric compounds, separating the diastereomers and recovering the optically pure enantiomer. Although the separation of enantiomers can be carried out using covalent diasteromeric derivatives of compounds, dissociable complexes (e.g. crystalline diastereomeric salts). Diastereomers have different physical properties (e.g. melting points, boiling points, solubilities, reactivity, etc.) and can be easily separated by taking advantage of these differences.

Diastereomers can be separated by chromatography or by means of separation / separation techniques based on differences in solubility. Then the optically pure enantiomer can be recovered, along with the resolving agent, by any practical non-racemizing agent. A more detailed description of the techniques that can be used to separate stereoisomers of compounds from their i5 mixtures can be found in Jean Jacques Andre Collet, Samuei H, Wllen, Enantiomers, Racematics and Resolutions, John Wiley &amp; Sons, Inc. (1981).

[0169] The symbols and conventions used in the present description for methods, schemes and examples are consistent with those used in modern scientific literature, for example in the Journal of the American Chemical Society or the Journal of Biological Chemistry. Standard single-letter or ^ r ^ liit ^ rc ^ w ^ e ^ k: rć) typh, which, unless otherwise indicated, are used in the L configuration for the determination of amino acid residues. Unless otherwise indicated, all substrates were obtained from commercial suppliers and used without further purification. In particular, the following abbreviations may be used in the examples and description:

<td>g (grams);</td><td>mg (milligrams);</td>

<td>l (liters);</td><td>ml (milliliters);</td>

<td>μΙ (microliters);</td><td>psi (pounds per square inch);</td>

<td>M (molar);</td><td>mM (millimolar);</td>

<td>iv (intravenously);</td><td>Hz (hertz);</td>

<td>MHz (megahertz);</td><td>mole (moles);</td>

<td>mmol (millimoles);</td><td>RT (ambient temperature);</td>

<td>min (minutes); h (hours);</td><td></td>

<td>Tt (melting point);</td><td>TLC (thin layer chromatography);</td>

<td>Tr (retention time);</td><td>RP (reverse phase system);</td>

<td>MeOH (methanol);</td><td>i-PrOH (isopropanol);</td>

<td>TEA (triethylamine);</td><td>TFA (trifluoroacetic acid);</td>

<td>TFAA (trifluoroacetic anhydride);</td><td>THF (tetrahydrofuran);</td>

<td>DMSO (dimethyl sulfoxide);</td><td>EtOAc (ethyl acetate);</td>

EP 1 586 571 B3

<td>DME (1,2-dimethoxyethane);</td><td>DCM (dichloromethane);</td>

<td>DCE (dichloroethane);</td><td>DMF (N, N-dimethylformamide);</td>

<td>DMPU (N, N'-dimethylpropylene urea);</td><td>CDI (1,1-carbonyl diimidazole);</td>

<td>IBCF (isobutyl chloroformate);</td><td>HOAc (acetic acid);</td>

<td>HOSu (N-hydroxysuccinimide);</td><td>HOBT (1-hydroxybenzotriazole);</td>

<td>et₂O (diethyl ether);</td><td>EDCI (ethylcarbodiimide hydrochloride);</td>

<td>BOC (tert-butyloxycarbonyl);</td><td>FMOC (9-fluorenylmethoxycarbonyl);</td>

<td>DCC (dicyclohexylcarbodiimino);</td><td>CBZ (benzyloxycarbonyl);</td>

<td>Ac (acetyl);</td><td>atm (atmosphere);</td>

<td>TMSE (2- (trimethylsilyl) ethyl);</td><td>TMS (trimethylsilyl);</td>

<td>TIPS (triisopropylsilyl);</td><td>TBS (t-butyldimethylsilyl);</td>

<td>DMAP (4-dimethylaminopyridine);</td><td>Me (methyl);</td>

<td>OMe (methoxy);</td><td>Et (ethyl);</td>

<td>Et (ethyl);</td><td>tBu (tert-butyl);</td>

<td colspan="2">HPLC (high pressure liquid chromatography);</td>

<td colspan="2">BOP (bis (2-oxo-3-oxazolidinyl) phosphinyl chloride);</td>

<td colspan="2">TBAF (tetra-n-butylammonium fluoride);</td>

<td colspan="2">mCPBA (meta-chloroperbenzoic acid.</td>

[0170] All references to ether or Et₂O refer to diethyl ether; brine refers to a saturated NaCl solution. Unless otherwise indicated, all temperatures are expressed in ° C (degrees Celsius). Unless otherwise indicated, all reactions were carried out under an inert atmosphere at RT.

[0171] Spectra ¹H NMR was recorded on a Bruker Avance 400 apparatus. Chemical shifts are expressed in parts per million (ppm). Coupling constants are given in hertz (Hz). The fission patterns describe visible multiplets and are designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad).

[0172] Low resolution mass spectra (MS) and compound purity data were obtained on a Waters ZQ LC / MS single quadrupole system equipped with an ion source (ESI) based on ionization by electric field spraying, a UV detector (220 and 254 nm), and scattered light laser detector (ELSD). Thin layer chromatography was performed on 0.25 mm E silica gel plates. Merck (60F-254), visualized with UV light, 5% ethanolic phosphomolybdic acid solution, ninhydrin or p-anisaldehyde solution. Column chromatography was performed on silica gel plates (230-400 mesh, Merck).

2. Scheme of synthesis of DPP-IV inhibitors according to the present invention

EP 1 586 571 B3 [0173] DPP-IV inhibitors of the present invention can be synthesized according to various reaction schemes. Certain reaction schemes are provided for illustration in the examples. Other reaction schemes can be easily designed by those skilled in the art.

[0174] In the reactions described hereinafter, it may be necessary to protect reactive functional groups, for example hydroxyl, amino, imino, thio or carboxy groups, when they are desired in the final product, to avoid their unwanted participation in the reactions. Typical protecting groups may be used in accordance with standard practice, see for example TW Greene and PGM Wuts in "Protective Groups in Organic Chemistry" John Wiley and Sons, 1991.

[0175] In each of the above reaction schemes, the various substituents may be selected from the various substituents described herein.

[0176] Descriptions of the syntheses of particular compounds of the present invention based on the above reaction schemes are provided herein.

3. Examples of DPP-IV inhibitors [0177] The present invention is further illustrated in detail by the following examples describing the synthesis of specific compounds of the invention, but not limiting the scope of the invention.

Experimental methods [0178]

<img file="PL1586571T6_D0005.tif" />

2- (6-Chrrr-2,4-yirksr-3,4-yihkyrr-2H-pirkmiykh-1-klrmetklr) behzrhitrkl (2). To a solution of 6-chlorouracil (20 g, 122 mmol) in a mixture of DMF-DMSO (6: 1, 600 mL) under nitrogen at 0 ° C, sodium hydride (60%, 5.5 g, 137 mmol) was added portionwise. After 0.5h, lithium bromide (8 g, 96 mmol) was added to the mixture and stirred for 15 min at 0 ° C. A solution of α-bromo-o-tolunitrile (25.1 g, 128 mmol) in DMF (30 ml) was added dropwise, and stirred at this temperature for 1 h, and then overnight at RT. It will be understood that the alkylation of the amine may be carried out under standard conditions known in the art, including using a base such as NaH, LiH or the like, in an organic solvent or mixture of solvents. Solvents may include DMSO, THF, DMF and the like, or mixtures thereof. In addition, additives, including LiBr, LiI, NaI and the like can be used. The mixture was evaporated and co-evaporated with water in vacuo to remove most of the DMF, and then poured into ice water (1 L). The precipitate was collected by filtration. The crude product was suspended in hot AcOEt-CHCl3 and sonicated for 5 min, allowed to stand at 0 ° C for 1h, and then filtered to give a white solid of the title compound (19 g) in 54% yield. It will also be understood that purification can be carried out using various methods known in the art, including washing with an aqueous / organic solvent or mixture of solvents, recrystallization and / or column chromatography. Nie30

Restrictive examples of organic solvents and solvent mixtures include ethyl acetate, isopropyl acetate, acetone, THF and the like.

¹H-NMR (400MH DMSO): δ 11^,82 (s, 1H), 7^,87 (d, 1H, J = 7^,6 H^from) 7^,71 (t, 1H, J = 7^,6 Hz), 7^,51 (t, 1H, J = 7.6 Hz), 7.37 (d, 1H, J = 8 Hz), 6.06 (s, 1H), 5.31 (s, 2H).

MS (ES) [m + H] calcd for C12HgClN3O2, 262.0; found 262.0.

[0180] 2- (6-Chloro-3-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-ylmethyl) benzonitrile (3). To a cold (0 ° C) solution of benzylated 6-chlorouracil 2 (10 g, 38 mmol) in DMF-THF (1: 1,300 ml) under nitrogen was added NaH (60%, 1.6 g, 39.9 mmol) in portions, then LiBr (2 g) was added. The mixture was stirred at rt for 20 min. Iodomethane (5.4 mL, 76 mmol) was added, then the flask was sealed and stirred at this temperature for 10 min, at rt for 2h, and at 35 ° C overnight, and then concentrated in vacuo. It will be understood that the alkylation of the amine may be carried out under standard conditions known in the art, including using a base such as NaH, LiH or the like in an organic solvent or mixture of solvents. Solvents may include DMSO, THF, DMF and the like, or mixtures thereof. In addition, additives, including LiBr, Lii, Nal and the like can be used. For example, alkylation can be carried out using methyl iodide and K2CO3 in acetone. The reaction may be carried out at a temperature of about 15-45 ° C, preferably at a temperature of about 20-43 ° C, and more preferably at a temperature of about 35-41 ° C, until the reaction is complete. The residue was dissolved in CHCl₃ and washed with water and brine, dried (Na2SO4), and filtered and then concentrated in vacuo. The crude product was crystallized from THF-hexanes to give 7.6 g (72%) of the title compound 3. It will be understood by those skilled in the art that benzonitrile can be purified in various organic solvents or solvent mixtures. For example, benzonitrile can be purified by the addition of a mixture of dichloromethane and heptane. Optionally, the benzonitrile may be further purified in an organic solvent or mixture of solvents such as dichloromethane, chloroform, acetonitrile, THF, ethyl acetate, isopropyl acetate and the like. Preferably, the product is purified and washed with ethyl acetate.

¹1 H NMR ⁽400 MH ^ DMSO^): δ 7^,87 (d, 1H, J = 7^,6 H^from) 7^,70 (t, 1H, J = 7^,6 H^from) 7^,51 (t, 1H, J = 7^,6 Hz), 7^,40 (d, 1H, J = 8 Hz), 6.21 (s, 1H), 5.38 (s, 2H), 3.28 (s, 3H).

MS (ES) [m + H] calculated for C 13 HnClN₃O, 276.1; found 276.1.

[0181] 2- {6- [3 (R) -Aminopiperidin-1-yl] -3-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-ylmethyl} benzonitrile (4). 2- (6-Chloro-3-methyl-2,4-dioxo-3,4-dihydro-2-H-pyrimidin-1-ylmethyl) benzonitrile (330 mg, 1.08 mmol), (R) -3 dihydrochloride -aminopiperidine (246 mg, 1.4 mmol) and sodium bicarbonate (500 mg, 5.4 mmol) were mixed with 200 mg of activated molecular sieves (4A) in dry MeOH (5 ml) at 100 ° C for 2 h. The reaction mixture was filtered through Celite, concentrated in vacuo, and then diluted with CHCl₃, and washed with water. The aqueous phase was extracted with CHCl₃ and the combined organic phases were washed with water, dried (Na₃SO4), and filtered. TFA (1 mL) was added to the solution, and then concentrated in vacuo. The residue was dissolved in a small amount of MeOH, and ETO was added to induce precipitation. The mixture was left at RT overnight. It will be understood by those skilled in the art that condensation of the amine or the hydrochloride of the amine can be carried out in an organic solvent or a mixture of solvents with a base, such as potassium carbonate, sodium bicarbonate and the like, or mixtures thereof. The solvent can be both protic and aprotic solvents and mixtures thereof. For example, the solvent may be a mixture of isopropyl alcohol and water. The reaction mixture may be further heated to about 30-100 ° C, preferably about 35-55 ° C, and more preferably about 4550 ° C, until the reaction is complete. The solvents were decanted and the precipitate was washed twice with Et₃ABOUT,

EP 1 586 571 B3 to obtain 270 mg of product in the form of an off-white powder. It should be understood that the product can be further purified by washing with an organic solvent or a mixture of solvents. Non-limiting examples of solvents or solvent mixtures include isopropyl acetate, ethyl acetate, dichloromethane, heptane, and the like. Further, the product can be optionally purified by column chromatography.

[0182] The benzonitrile product may be isolated in the form of the free base, if desired, but preferably it can be further converted into the corresponding acid addition salt such as the benzoic acid salt. Preferably, the benzonitrile product is treated with benzoic acid to give 2- [6- (3-aminopiperidin-1-yl) -3-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-ylmethyl benzoate. ] benzonitrile (4).

io Preparation and isolation of the benzoate salt can be carried out by conventional methods for the formation of acid addition salts.

[0183] In each of the above steps, the isolation and / or purification of intermediates can be avoided if the intermediates are obtained from the reaction mixture as relatively pure compounds and intermediates or impurities from the reaction mixture do not interfere with subsequent reaction steps.

Where possible, one or more isolation steps can be eliminated to obtain shorter processing times, and the elimination of further processing can also result in higher reaction yields.

<img file="PL1586571T6_D0006.tif" />

[0185] 4-Fluoro-2-methylbenzonitrile (31). A mixture of 2-bromo-5-fluorotoluene (3.5 g, 18.5 mmol) and CuCN (2 g, 22 mmol) in DMF (100 mL) was refluxed for 24 hours. The reaction mixture was diluted with water and extracted with hexane. The organics were dried over MgSO4 and the solvent removed to give product 31 (60% yield).

¹H^-NMR ⁽400 MH ^ CDClA δ 7^,60 (dd, J = 5.6, 8^,8 Hz, 1H), 6^,93^-7^,06 (m, 2H), 2^,55 (s, 3H).

[0186] 2-Bromomethyl-4-fluorobenzonitrile (32). A mixture of 4-fluoro-2-methylbenzonitrile (2 g, 14.8 mmol), NBS (2.64 g, 15 mmol) and AIBN (100 mg) in CCl4 was refluxed under nitrogen for 2 hours. The reaction mixture was cooled to room temperature. The precipitate was removed by filtration. The organic solution was concentrated to give the crude product as an oil, which was used in the next step without further purification.

3° ¹H^-NMR ⁽400 MH ^ CDClA δ 7^,68 J = 5A 8^,4 H ^ 1H) 7^,28 J = 2A 8^,8 H ^ 1H) 7^,12 1H) 4^,6 fo 2H).

2- (6-Chloro-3-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-ylmethyl) -4-fluorobenzonitrile (33). Mixture of crude 3-methyl-6-chlorouracil (0.6 g, 3.8 mmol), 2-bromomethyl-4-fluorobenzonitrile (0.86 g, 4 mmol) and K2CO3 (0.5 g, 4 mmol) in DMSO (10 ml) was stirred at 60 ° C for 2 hours.

EP 1 586 571 B3

The reaction mixture was diluted with water and extracted with EtOAc. The organics were dried over MgSO₄ and the solvent removed. The residue was purified by column chromatography. 0.66 g of product was obtained (yield: 60%).

¹H-NMR ⁽400 MH ^ CDCl₃^): δ 7^,73 (dd, J = 7.2, 8.4 Hz, 1H^), 7^,26 (d, J-4.0 Hz, 1H^), 7^,11^-7^,17 (m, 1H^), 6^,94 (dd, J = 2.0, 9.0 Hz, 1H), 6.034 (s, 2H), 3.39 (s, 3H).

MS (ES) [m + H] calcd for C13H9CFN3O2, 293.68; found 293.68.

[0188] 2- [6- (3-Aminopiperidin-1-yl) -3-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-ylmethyl] -4-fluorobenzonitrile (34). 2- (6-Chloro-3-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-ylmethyl) -4-fluorobenzonitrile (300 mg, 1.0 mmol), (R) -3- dihydrochloride aminopiperidine (266 mg, 1.5 mmol) and sodium bicarbonate (500 mg, 5.4 mmol) were mixed in a sealed tube in EtOH (3 mL) at 100 ° C for 2 h. After HPLC purification, the final compound was obtained as a salt TFA.

¹H^-NMR ⁽400 MH ^ CD₃FROM^): δ 7^,77^-7^,84 1H) 7^,16^-7^,27 (m, 2H) 5^,46 (s, 1H), 5^,17^-5^,34 (ABq, 2H, J =

35.2, 15.6 Hz), 3.33-3.47 (m, 2H), 3.22 (s, 3H), 2.98-3.08 (m, 1H), 2.67-2 , 92 (m, 2H), 2.07-2.17 (m, 1H), 1.82-1.92 (m, 1H), 1.51-1.79 (m, 2H).

MS (ES) [m + H] calcd for C18H20FN5O2, 357.38; found 357.38.

4. In vitro suppression in vitro [0189] Protease inhibiting activity by DPP-IV inhibitors can easily be determined by methods known to those skilled in the art, since appropriate in vitro assays for measuring protease activity and its inhibition by test compounds are known . Examples of test agents that can be used to measure the activity and selectivity of protease inhibition are provided below.

DPP-IV assay [0190] Solutions of compounds in dimethyl sulfoxide (DMSO) were prepared at various concentrations (final concentration &lt;10mM) and then diluted with assay buffer containing: 20mM Tris, pH 7.4; 20mM KCI; and 0.1 mg / ml BSA. Human DPP-IV (0.1 nM final concentration) was added to the dilutions and pre-incubated for 10 minutes at ambient temperature, followed by reaction with AP-7-amido-4-trifluoromethyl coumarin (AP-AFC; 10 μΜ final concentration). The total reaction volume was 10-100 μΙ, depending on the test formats used (384 or 96 well plates). The reaction kinetics (excitation λ = 400 nm; emission λ = 505 nm) was monitored for 5-10 minutes or the end point was measured after 10 minutes. Braking constants (IC₅₀) was calculated from the enzyme curves using standard mathematical models.

FAPc assay [0191] Solutions of compounds in dimethyl sulfoxide (DMSO) were prepared at various concentrations (final concentration &lt;10mM) and then diluted with assay buffer containing: 20mM Tris, pH 7.4; 20mM KCl; and 0.1 mg / ml BSA. Human FAPa (final concentration 2 nM) was added to the dilutions and pre-incubated for 10 minutes at ambient temperature, followed by reaction with AP-7-amido-4-trifluoromethyl coumarin (AP-AFC; final concentration 40 μM). The total reaction volume was 10-100 μΙ, depending on the test formats used (384 or 96 well plates). The reaction kinetics (excitation λ = 400 nm; emission λ = 505 nm) was monitored for 5-10 minutes or the end point was measured after 10 minutes. Braking constants (IC₅₀) was calculated from the enzyme curves using standard mathematical models.

EP 1 586 571 B3

PREP test [0192] Solutions of compounds in dimethyl sulfoxide (DMSO) were prepared at various concentrations (final concentration &lt;10mM) and then diluted with assay buffer containing: 20mM sodium phosphate, pH 7.4; 0.5mM EDTA; 0.5mM DTT; and 0.1mg / ml BSA. PREP (EC3.4.21.26 from Flavobacterium meningosepticum; final concentration 0.2 nM) was added to the dilutions. PREP and the compound were preincubated for 10 minutes at ambient temperature, after which the reaction was initiated with ZGP-AMC (final concentration 10 μΜ). The total reaction volume was 10-100 μΙ, depending on the test formats used (384 or 96 well plates). The reaction kinetics (excitation λ = 375 nm; emission λ = 460 nm) was monitored for 5-10 minutes or the end point was measured after 10 minutes. Braking constants (IC5₀) was calculated from the enzyme curves using standard mathematical models.

Tryptase test [0193] Solutions of compounds in dimethyl sulfoxide (DMSO) were prepared at various concentrations (final concentration &lt;10mM) and then diluted with assay buffer containing: 100mM Hepes, pH 7.4; 0.01% Brij35; and 10 °% glycerol. Tryptase (rhLung beta; final concentration 0.1 nM) was added to the dilutions and pre-incubated with the compound for 10 minutes at ambient temperature. The enzymatic reaction was initiated with 25 μΜ Z-lys-SBzl and 400 μΜ DTNB. The total reaction volume was 100 μΙ in 96 well Costar A / 2 96 well plates. The reaction was monitored colorimetrically (λ = 405 nm) for 10 minutes. Inhibition constants (IC50) were calculated from enzyme curves using standard mathematical models.

[0194] The compounds of the invention were tested in accordance with the above-described protease inhibition assays and were observed to exhibit selective DPP-IV inhibitory activity. For example, the compounds of the invention have been found to inhibit DPP-IV activity at concentrations at least 50 times lower than those required to achieve equally active inhibition for FAPa. The apparent inhibition constants (K) for compounds of the invention against DPP-IV ranged from about 10^-9M to about 10^-5M.