The present invention relates to novel 2,6-diaminopyridine derivatives that inhibit cyclin-dependent kinases. These compounds and their pharmaceutically acceptable salts and esters have antiproliferative activity and are useful, inter alia, in the treatment or control of cancer, in particular solid tumors. This invention also relates to pharmaceutical compositions containing such compounds and to methods of treating or controlling cancer, most particularly the treatment or control of breast, lung, colon and prostate tumors. Finally, this invention is also directed to novel intermediate compounds useful in the preparation of the novel diaminopyridines herein disclosed.

Uncontrolled cell proliferation is the hallmark of cancer. Cancerous tumor cells typically have some form of damage to the genes that directly or indirectly regulate the cell-division cycle.

The progression of cells through the various phases of the cell cycle is regulated by a series of multienzyme complexes consisting of a regulatory protein, a cyclin, and a kinase. These kinases are called cyclin-dependent kinases (Cdks). The Cdks are expressed throughout the cell cycle, while the levels of the cyclins vary depending on the stage of the cell cycle.

The four primary phases of cell cycle control are generally describes as G-i, S, G2, and M. Some essential enzymes for cell cycle control appear to be cyclin D/Cdk4, cyclin D/Cdk6, cyclin E/Cdk2, cyclin A/Cdk2, and cyclin B/Cdk1 (also known as Cdc2/cyclin B). Cyclin D/Cdk4, cyclin D/Cdk6, and cyclin E/Cdk2 control passage through the Grphase and the Gr to S-phase transition by phosphorylation of the retinoblastoma phosphoprotein, pRb. Cyclin A/Cdk2 regulates passage through the S-phase, and cyclin B/Cdk1 controls the G2 checkpoint and regulates entry into M (mitosis) phase.

The cell cycle progression is regulated by Cdk1 (cdc2) and Cdk2 beyond early Gi when cells are committed to cytokinesis. Therefore, drug inhibition of these Cdks is likely not only to arrest cell proliferation, but also to trigger apoptotic cell death. Once the cells pass the G1 restriction point and are committed to S phase, they become independent of growth factor stimulation for continued cell cycle progression.

Following completion of DNA replication, cells enter the G2 phase of the cell cycle in preparation for M phase and cytokinesis. Cdk1 has been shown to regulate passage of cells through these later phases of the cell cycle in association with both cyclins A and B. Complete activation of Cdk1 requires both cyclin binding and specific phosphorylation (Morgan, D. O., De Bondt, H. L, Curr. Opin. Cell. Biol. 1994, 6, 239-246). Once activated, Cdk1/cyclin complexes prepare the cell for division during M phase.

The transition from Gi phase into S phase as stated above is regulated by the complex of Cdk4 with cyclin D and Cdk2. with cyclin E. These complexes phosphorylate the tumor suppressor protein Retinoblastoma (pRb), releasing the transcription factor E2F and allowing the expression of genes required in S phase (Nevins, J. R. Science 1992, 258, 424-429; Lavia, P. BioEssays 1999, 21, 221-230). Blocking the activity of the Cdk4/cyclin D and Cdk2/cyclin E complexes arrests the cell cycle in Gi phase. For example, the proteins of the 1NK4 family, including p16INK4a, which block the kinase activity of the Cdk4/cyclin D complex, cause arrest in Gi (Sherr, C. J. Science 1996, 274, 1672-1677). The specific block has been reviewed (Vidal, A. Gene 2000, 247, 1-15).

Recent experiments show that the complex of Cdk4 with cyclin D3 also plays a role in cell cycle progression through G2 phase. Inhibition of this complex, either by p16 or using a dominant negative Cdk4, results in arrest in G2 phase in cells that do not express pRb (Gabrielli B. G. et al. J. Biol. Chem. 1999, 274, 13961-13969).

Numerous defects in the pRb pathway have been shown to be involved in various cancers. For example, overexpression of Cdk4 has been observed in cases of hereditary melanoma (Webster, K. R. Exp. Opin. Invest. Drugs 1998, 7, 865-887); cyclin D is overexpressed in many human cancers (Sherr, C. J. Science 1996, 274, 1672-1677); p16 is mutated or deleted in many tumors (Webster, K. R. Exp. Opin. Invest. Drugs 1998, 7, 865-887); and pRb function is lost through mutation or deletion in many human cancers (Weinberg, R. A. Cell 1995, 81, 323-330). Defects in this pathway have also been shown to have an effect on prognosis. For example, loss of p16 is correlated with poor prognosis in non-small-cell lung carcinoma (NSCLC) and malignant melanoma (Tsihlias, J. et al. Annu. Rev. Med. 1999, 50, 401-423). Abnormalities of cyclin D1 and/or pRb at the gene and/or expression level were present in more than 90% of a series of non-small cell lung cancer specimens, indicating that cyclin D1 and/or pRb represent an important step in lung tumorigenesis (Marchetti, A. et al. Int. J. Cancer 1998, 75, 573-582). In 49 out of 50 pancreatic carcinomas (98%), the pRb/p16 pathway was abrogated exclusively through inactivation of the p16 gene and cyclin D connected (Schutte, M. et al. Cancer Res. 1998, 57, 3126-3134). For a review on the relation between expression of pRb and the cyclin/cyclin dependent kinases in a number of tissues see Teicher, B.A.. Cancer Chemother. Pharmacol. 2000, 46, 293-304.

Because of the involvement of the Cdk4/cyclin D/pRb pathway in human cancer through its role in regulating progression of the cell cycle from Gi to S phase, and the potential therapeutic benefit from modulating this pathway, there has been considerable interest in agents that inhibit or promote elements of this pathway. For example, effects on cancer cells have been shown using antibodies, antisense oligonucleotides and overexpression or addition of proteins involved in the pathway. See, e.g., Lukas, J. et al. Nature 1995, 79, 573-582; Nevins, J. R. Science 1992, 258, 424-429; Lim, I. K. et al. Molecular Carcinogenesis 1998, 23, 25-35; Tam, S. W. et al. Oncogene 1994, 9, 2663-2674; Driscoll, B. et al. Am. J. Physiol. 1997, 273 (Lung Cell. MoI. Physiol.), L941-L949; and Sang, J. et al. Chin. Sci. Bull. 1999, 44, 541-544).

The role of cdks in the regulation of cellular proliferation is thus well established. For example, as shown above, there is an extensive body of literature validating the use of compounds inhibiting targets in the Cdk4 , Cdk2 and Cdk1 pathways as anti-proliferative therapeutic agents. Inhibitors of cellular proliferation thus act as reversible cytostatic agents that are useful in the treatment of disease processes which feature abnormal cellular growth, such as cancers and other cell proliferative disorders including, for example inflammation (e.g. benign prostate hyperplasia, familial adenomauosis, polyposis, neuro¬ fibromatosis, atherosclerosis, pulmonary fibrosis, arthritis, psoriasis, inflammatory bowel disease, transplantation rejections infections), viral infections (including, but not limited to herpervirus, poxvirus, Epstein-Barr virus), autoimmune disease (e.g. lupus, rheumatoid arthritis, psoriasis, inflammatory bowel disease), neurodegenerative disorders (including but not limited to Alzheimer's disease), and neurodegenerative diseases (e.g. Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy, and cerebral degeneration).

Several distinct classes of small molecules have been identified as inhibitors of Cdks: olomoucine and other purine analogs, flavopiridol, staurosporine, UCN-01 and other indolocarbazoles, 9-hydroxyellipticine, indirubin, paullones, diaryl ureas, quinazolines, indopyrazoles, [2, 3-d] pyridopyrimidines, fascaplysin, aminothiazoles, diaminothiazoles, p-teridinones, and pyrazoles or example (Carlson et. al., Cancer Res.. 1996, 56, 2973-2978: De Azevedo et al., Eur. J. Biochem., 1997, 243, 518-526; Bridges, A.J., Exp. Opin. Ther. Patents. 1995, 5, 12451257; Reinhold et al., J. Biol. Chem. 1998, 278, 3803-3807; Kakeya, H. et. al., Cancer Res.. 1998, 58, 704-710; Harper, J.W., Cancer Surveys 1997, 29, 91-107; Harrington, E.A., et al., Proc. Natl. Acad. Sci. USA 1998, 95, 11945-11950; Meijer, L., et al., Eur. J. Biochem.. 2000, 267, 1-13; Garrett, M. D. et. al., Current Opin. Genetics Develop. 1999, 9, 104-111; Mgbonyebi, O. P. et al., Cancer Res.. 1999, 59, 1903-1910; Hoessel et al., Nature Cell Biology. 1999, 1, 60-67; Zaherevitz et al., Cancer Res., 1999, 59, 2566-2569; Honma, T., et al., 221st National ACS Meeting.. 2001: Medi 136; Sielecki, T.M., et al., Bioorg. Med. Chem. Lett. 2001, 11, 1157-1160; Nugiel, D. A., et al., J. Med. Chem., 2001, 44, 1334-1336; Fry, D. W. et al., J. Biol. Chem. 2001 , 276, 16617-15523; Soni, R., et al., Biochem. Biophys. Res. Commun. 2000, 275, 877; Ryu, C-K. et al., Bioorg. Med. Chem. Lett, 2000, 10, 461; Jeong, H-W., et al., Bioorg. Med. Chem. Lett. 2000, 10, 1819; Toogood et al., J. Med. Chem., 2000, 43, 4606-4616; Chong, W., Fischer, Curr. Opin. in Drug Discov. and Develop., 2001, 4, 623-634, WO0009921845, Toogood. P., WO0119825, Toogood P., WO0138315, Reich S.H., WO0179198, Webster, K. US 6,262,096.

For reviews of compounds inhibiting the Cdk4/cyclin D pathway see: Harris, W. and Wilkinson, S., Emerging Drugs.. 2000, 5, 287-297; Dumas, J., Exp. Opin. Ther. Patents. 2001 , 11, 405-429; Sielecki T., et. al., J. Med. Chem.. 2000, 43, 1-18. The present invention relates to novel diaminopyridines of the

formula

wherein

R1 is selected from the group consiting of

heterocycle and lower alkyl-heterocycle, wherein the

heterocycle moiety in both instances optionally may be substituted by up

to four substituents independently selected from the group consisting of:

by oxo, OR12, CO2R12,NR5R6, S(O)nR15, aryl

R2 is selected from the group consisting of

aryl, heteroaryl, cycloalkyl and heterocycle, wherein each

may be substituted by up to four substituents independently

selected from the group consisting of:

(a) lower alkyl, (b) lower alkyl substituted by halogen or OR10, (c) halogen, (d) OR1* (e) NO2,

(f) CN '5 6 (g) NR5R6, (h) S(O)n-R9, and (i) SO2-NR16R17; R5 and R6 are each independently selected from the group consisting of

(a) H,

(b) lower alkyl,

(c) lower alkyl substituted by oxo, CO2R12, OR12, NR13R14, C(O)NR13R14 , SO2R15, NSO2R12, heteroaryl, heterocycle, or heterocycle substituted by oxo, (d) cycloalkyl,

(e) cycloalkyl substituted by CO2R12, OR12, NR13R14 , C(O)NR13R14 or SO2R15, (f) aryl,

(g) aryl substituted by NR13R14, OR12, CO2R12, C(O)NR13R14 , SO2R15, halogen, lower alkyl, or lower alkyl substituted by halogen, OR12, oxo, CO2R12, C(O)NR13R14 or NR13R14,

(h) SO2R15,

(i) CO2R12,

(j) COR12, and

(k) OR12

P

or alternatively, the group -NR5R6 can form a ring having a total of

from 3 to 7 ring atoms, said ring atoms comprising in addition to the

nitrogen to which R5 and R6 are bonded, carbon ring atoms, said carbon

ring atoms optionally being replaced by one or more additional N or O ring

atoms or the group SO2, and said ring atoms optionally being substituted

by OH, oxo, NR13R14, lower alkyl and lower alkyl substituted by OR12;

R7 is selected from the group consisting of (a) H,

(b) lower alkyl,

(C) lower alkyl substituted by OR12, CO2R12, NR5R6, or C(O)NR5R6, (d) halogen, (e) OXO, (f) aryl, (g) aryl substituted by up to three substituents independently selected from lower alkyl, halogen and NR5R6, (h) cycloalkyl, (i) cycloalkyl substituted by OH, oxo, or NH2, (D SO2R15, and (k) COR12 ;

R9 is selected from the group consisting of

(a) H, and (b) lower alkyl;

(a) lower alkyl, (b) aryl, and (c) aryl substituted by halogen or NR5R6;

R11 is selected from the group consisting of

(a) H, (b) lower alkyl, and (c) lower alkyl substituted by oxo and halogen;

R12 is selected from the group consisting of

(a) H, (b) lower alkyl, and (c) lower alkyl substituted by NR5R6 or OR11; R13 and R14 are each independently selected from the group consisting of

(a) H,

(b) lower alkyl,

(c) lower alkyl substituted by CO2R12, OR12, NR5R6, C(O)NR5R6, SO2R15, NSO2R12, heteroaryl, heterocycle, or heterocycle substituted by oxo,

(d) cycloalkyl,

(e) cycloalkyl substituted by CO2R12, OR12, NR5R6, CONR5R6 or SO2R15, (f) aryl, (g) aryl substituted by NR5R6, OR12, CO2R12, CONR5R6 , SO2R15, halogen, lower alkyl, and lower alkyl substituted by halogen, OR12, oxo, CO2R12, C(O)NR5R6 or NR5R6; (h) SO2R15,

(O CO2R12,

(j) COR12, and

(k)

OR12

Rl1°\ K^°

or alternatively, the group -NR13R14 can form a ring having a total of from 3

to 7 ring atoms, said ring atoms comprising in addition to the nitrogen to which

R13 and R14 are bonded, carbon ring atoms, said carbon ring atoms optionally

being replaced by one or more additional N or O ring atoms, and said ring atoms

optionally being substituted by OH, oxo, NR5R6, lower alkyl and lower alkyl

substituted by OR12;

R15 is selected from the group consisting of

(a) aryl,

(b) aryl substituted by the group halogen, CO2R12, SO2R10, COR12, lower alkyl and lower alkyl substituted by halogen, OR12, oxo, CO2R12, C(O)NR5R6 or NR5R6, (c) heteroaryl, (d) heteroaryl substituted by the group halogen, CO2R12, SO2R10, COR12, lower alkyl and lower alkyl substituted by halogen, OR12, 0X0, CO2R12, C(O)NR5R6 or NR5R6, (e) NR5R6,

(f ) lower alkyl,

( (gg)) l loowweerr a allkkyyll substituted by halogen, OR12, oxo, CO2R12, C C((OO))NNRR55RR66 or NR5R6, (h) heterocycle, and

(i) heterocycle substituted by the group CO2R12, COR12, SO2R12, lower alkyl, C(O)NR5R6 or NR5R6;

R16 and R17 are each independently selected from the group consisting of

(a) H, and

(b) lower alkyl,

or, alternatively, the group -NR16R17 can form a ring having a total of from

3 to 7 ring atoms, said ring atoms comprising in addition to the nitrogen to which

R16 and R17 are bonded, carbon ring atoms, said carbon ring atoms optionally

being replaced by one or more additional N or O ring atoms, and said ring atoms

optionally being substituted by lower alkyl, OH, oxo and NH2; and

n is O, 1 or 2;

or the pharmaceutically acceptable salts or esters thereof.

These compounds inhibit cyclin-dependent kinases. These compounds

and their pharmaceutically acceptable salts and esters have antiproliferative

activity and are useful in the treatment or control of cancer, in particular solid

tumors.

The present invention also relates to pharmaceutical compositions

comprising one or more compounds of the invention, or a pharmaceutically

acceptable salt or ester thereof, and a pharmaceutically acceptable carrier or

excipient. The present invention further relates to a method for treating or controlling cancer, more particularly the treatment or control of a solid tumor, most particularly to the treatment or control of breast, lung and colon and prostate tumors by administering to a patient in need of such therapy a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt or ester thereof.

Finally, this invention also relates to novel intermediate compounds useful in the preparation of a compound of formula I.

As used herein, the following terms shall have the following definitions.

"Aryl" means a monovalent, monocyclic or bicyclic, aromatic carbocyclic hydrocarbon radical, preferably a 6-10 membered aromatic aromatic ring system . Preferred aryl groups include, but are not limited to, phenyl, naphthyl, tolyl and xylyl.

"Carbonyl" means the radical C=O.

"Cycloalkyl" means a non-aromatic, partially or completely saturated monovalent cyclic hydrocarbon radical containing 3 to 8 atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

"Effective amount" means an amount that is effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.

"Halogen" means fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.

"Hetero atom" means an atom selected from N1 O and S. "Heteroaryl" means an aromatic heterocyclic ring system containing up to two rings. Preferred heteroaryl groups include, but are not limited to, thienyl, furyl, indolyl, pyrrolyl, pyridinyl, pyridine, pyrazinyl, oxazolyl, thiaxolyl, quinolinyl, pyrimidinyl, imidazole, benzofuran and tetrazolyl.

"Heterocycle" or "heterocyclyl" means a saturated or partially unsaturated, non- aromatic cyclic radical of 3 to 8 ring atoms in which from one to 3 ring atoms are hetero atoms selected from nitrogen, oxygen, S(O)n (where n is an integer from 0 to 2), or a combination thereof, the remaining ring atoms being C. Examples of preferred heterocycles are piperidine, piperazine, pyrrolidine, morpholine, indoline, tetrahydropyranyl, thiomorpholino, pentamethylene sulfide, and pentamethylene sulfone.

Ki" refers to a measure of the thermodynamic binding of the ligand/inhibitor (that is, a compound according to the invention) to the target protein. Kj can be measured, inter alia, as is described in Example 53, infra.

"Lower alkyl" alone or in conjunction with another term, e.g. lower alkyl- heterocycle, denotes a straight-chain or branched saturated aliphatic hydrocarbon having 1 to 6, preferably 1 to 4, carbon atoms. Typical lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, t-butyl, 2-butyl, pentyl, hexyl and the like.

"Oxo" means =0.

"Pharmaceutically acceptable ester" refers to a conventionally esterified compound of formula I having a carboxyl group, which esters retain the biological effectiveness and properties of the compounds of formula I and are cleaved in vivo (in the organism) to the corresponding active carboxylic acid. Examples of ester groups which are cleaved (in this case hydrolyzed) in vivo to the corresponding carboxylic acids (R40C(=O)OH) are lower alkyl esters which may be substituted with NR41R42 where R41 and R42 are lower alkyl, or where NR41R42 taken together form a monocyclic aliphatic heterocycle, such as pyrrolidine, piperidine, morpholine, N-methylpiperazine, etc.; acyloxyalkyl esters of the formula R40C(=O)OCHR43OC(=O)R44 where R43 is hydrogen or methyl, and R44 is lower alkyl or cycloalkyl; carbonate esters of the formula R40C(=O)OCHR43OC(=O)OR45 where R43 is hydrogen or methyl, and R45 is lower alkyl or cycloalkyl; or aminocarbonylmethyl esters of the formula R40C(=O)OCH2C(=O)NR41R42 where R41 and R42 are hydrogen or lower alkyl, or where NR41R42 taken together form a monocyclic aliphatic heterocycle, such as pyrrolidine, piperidine, morpholine, N-methylpiperazine, etc. As used herein, R40 has the same definition as R1.

Examples of lower alkyl esters are the methyl, ethyl, and n-propyl esters, and the like. Examples of lower alkyl esters substituted with NR41R42 are the diethylaminoethyl, 2-(4-morpholinyl)ethyl, 2-(4-methylpiperazin-1-yl)ethyl esters, and the like. Examples of acyloxyalkyl esters are the pivaloxymethyl, 1-acetoxyethyl, and acetoxymethyl esters. Examples of carbonate esters are the 1-(ethoxycarbonyloxy)ethyl and 1- (cyclohexyloxycarbonyloxy)ethyl esters. Examples of aminocarbonylmethyl esters are the N,N-dimethylcarbamoylmethyl and carbamoylmethyl esters.

Further information concerning examples of and the use of esters for the delivery of pharmaceutical compounds is available in Design of Prodrugs. Bundgaard H ed. (Elsevier, 1985). See also, H. Ansel et. al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) at pp. 108-109; Krogsgaard-Larsen, et. al., Textbook of Drug Design and Development (2d Ed. 1996) at pp. 152-191.

"Pharmaceutically acceptable salt" refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of formula I and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Sample acid-addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluene sulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Sample base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethylammonium hydroxide. The chemical modification of a pharmaceutical compound (i.e. drug) into a salt is a technique well known to pharmaceutical chemists to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds. See, e.g., H. Ansel et. al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) at pp. 196 and 1456-1457.

"Pharmaceutically acceptable," such as pharmaceutically acceptable carrier, excipient, etc., means pharmacologically acceptable and substantially non-toxic to the subject to which the particular compound is administered.

"Substituted," as in substituted alkyl, means that the substitution can occur at one or more positions and, unless otherwise indicated, that the substituents at each substitution site are independently selected from the specified options.

"Therapeutically effective amount" means an amount of at least one compound of Formula I1 or a pharmaceutically acceptable salt or ester thereof, that significantly inhibits proliferation and/or prevents differentiation of a human tumor cell, including human tumor cell lines.

In one embodiment, the present invention relates to compounds of formula I

or the pharmaceutically acceptable salts or esters thereof, wherein R1 and R2 are as defined above.

In a preferred embodiment of the compounds of formula I, R2 is phenyl, preferably phenyl substituted by halogen, most preferably F, OR12 wherein R12 is lower alkyl or lower alkyl. In a most preferred embodiment, R2 is phenyl substituted by one or two F molecules and one OR12 group wherein R12 is lower alkyl, preferably methyl.

In another preferred embodiment of the compounds of formula I, R2 is as defined above and R1 is selected from the group

wherein

R3 is selected from the group H, lower alkyl, lower alkyl substituted by oxo, OR12, CO2R12, NR5R6, SO2R15, aryl or C(O)NR5R6,

CO2R7, COR12, C(O)NR5R6, and SO2R15;

R4 is selected from the group H, OR11, lower alkyl, NR5R6, NO2, 0X0 CN, and halogen;

R5 and R6 are each independently selected from the group

H, lower alkyl,

lower alkyl substituted by oxo, CO2R12, OR12, NR13R14, C(O)NR13R14 , SO2R15, NSO2R12, heteroaryl, heterocycle, or heterocycle substituted by oxo,

cycloalkyl,

cycloalkyl substituted by CO2R12, OR12, NR13R14, C(O)NR13R14 or SO2R15,

aryl,

aryl substituted by NR13R14, OR12, CO2R12, CONR13R14, SO2R15, halogen, lower alkyl, and lower alkyl substituted by halogen, OR12, oxo, CO2R12, CONR13R14 or NR13R14;

SO2R15, CO2R12,

COR12, and

OR12

R11O O \ p p <- ^

or alternatively, the group -NR5R6 can form a ring having a total of

from 3 to 7 ring atoms, said ring atoms comprising in addition to the

nitrogen to which R5 and R6 are bonded, carbon ring atoms, said

carbon ring atoms optionally being replaced by one or more

additional N or O ring atoms or the group SO2, and said ring atoms

optionally being substituted by OH, oxo, N13R14, lower alkyl and

lower alkyl substituted by OR12;

R7 is selected from the group H, lower alkyl, lower alkyl substituted by OR12, CO2R12, NR5R6, or CONR5R6, halogen, oxo, aryl, aryl substituted by up to three substituents independently selected from lower alkyl, halogen, or NR5R6, . cycloalkyl, cycloalkyl substituted by OH, oxo, or NH2, SO2R15, and COR12 ;

R10 is selected from the group lower alkyl, aryl, and aryl substituted by halogen or NR5R6;

R11 is selected from the group H, lower alkyl, and lower alkyl substituted by oxo and halogen;

R12 is selected from the group H lower alkyl, and lower alkyl substituted by halogen, oxo, NR5R6Or OR11;

R13 and R14 are independently selected from H, lower alkyl, lower alkyl substituted by CO2R12, OR12, NR5R6, C(O)NR5R6, SO2R15, NSO2R12, heteroaryl, heterocycle, or heterocycle substituted by oxo,

cycloalkyl,

cycloalkyl substituted by CO2R12, OR12, NR5R6, C(O)NR5R6 or SO2R15,

aryl,

aryl substituted by NR5R6, OR12, CO2R12, C(O)NR5R6, SO2R15, halogen, lower alkyl, and lower alkyl substituted by halogen, OR12, oxo, CO2R12, C(O)NR5R6 and NR5R6;

SO2R15,

CO2R12,

COR12, and

OR12

R11O. O S

or alternatively, the group -NR13R14 can form a ring having a total of

from 3 to 7 ring atoms, said ring atoms comprising in addition to the

nitrogen to which R13 and R14 are bonded, carbon ring atoms, said

carbon ring atoms optionally being replaced by one or more

additional N or O ring atoms, and said ring atoms optionally being

substituted by OH, oxo, NR5R6, lower alkyl and lower alkyl

substituted by OR12;

R15 is selected from the group

aryl,

aryl substituted by the group halogen, CO2R12, SO2R10, COR12 , lower alkyl and lower alkyl substituted by halogen, OR12, oxo, CO2R12, C(O)NR5R6 or NR5R6,

heteroaryl, heteroaryl substituted by the group halogen, CO2R12, SO2R10, COR12, lower alkyl and lower alkyl substituted by halogen, OR12, oxo, CO2R12, C(O)NR5R6 or NR5R6,

NR5R6,

lower alkyl,

lower alkyl substituted by the group halogen, OR12, oxo, CO2R12, C(O)NR5R6 or NR5R6,

heterocycle, and

heterocycle substituted by the group CO2R12, COR12,

SO2R12, lower alkyl, C(O)NR5R6 or NR5R6;

X is selected from the group

S,

SO,

SO2, O; and

n is O, 1 or 2,

or the pharmaceutically acceptable salts or esters thereof.

In another embodiment R1 is a heterocycle that is substituted with H, lower

alkyl, S(O)nR15, CO2R7, COR12, C(O)NR13R14.

In another embodiment R1 is a heterocycle that is substituted by lower

alkyl that is substituted by aryl.

In another preferred embodiment, the invention relates to

compounds of formula

wherein R3 and R4 are as defined above and R18, R19, R20 and R21 are each independently selected from lower alkyl, halogen and OR12. The preferred halogens are F and Cl.

Preferably, R3 is selected from the group CO2R7, COR12, SO2R15, C(O)NR5R6, lower alkyl and lower alkyl substituted by aryl, preferably phenyl. Most preferably, R3 is SO2R15. Preferably, R15 is lower alkyl or NR5R6.

Preferred R4 groups include H, OR11 and lower alkyl.

Preferred R5 and R6 groups are those wherein each is idependently selected from H and lower alkyl, or the group -NR5R6 can form a ring having a total of from 3 to 7 ring atoms, said ring atoms comprising in addition to the nitrogen to which R5 and R6 are bonded, carbon ring atoms, said carbon ring atoms optionally being replaced by one or more additional N or O ring atoms and said ring atoms optionally being substituted by OH, oxo and NH2, lower alkyl or lower alkyl substituted by OR12.

The preferred R12 is lower alkyl.

In still another preferred embodiment there are provided the compounds of formula l(a), wherein R represents hydrogen; benzyl; (Ci-Cβ)alkyl; -S(O)2-(C1-C6)alkyl; -C(O)-(Ci-C6)alkyl; -C(O)2-(C1-C6)alkyl; and -C(O)NH-(CrC6)alkyl;

R4 represents hydrogen; and

R18, R19, R20 and R21 independently from each other represent (CrC^alkyl; -O-(Ci-C6)alkyl; and halogen.

Examples of compounds of formula l(a) include:

[2-Amino-6-(1-methanesulfonyl-piperidin-4-ylamino)-pyridi n-3-yl]-(2-methoxy- phenyl)-methanone (Example 6);

[2-Amino-6-(1-methanesulfonyl-piperidin-4-ylamino)-pyridi n-3-yl]-(2-fluoro- phenyl)-methanone (Example 10);

4-[6-Amino-5-(2-fluoro-benzoyl)-pyridin-2-ylamino]-piperi dine-1-carboxylic acid ethyl ester (Example 11);

[2-Amino-6-(1-ethanesulfonyl-piperidin-4-ylamino)-pyridin -3-yl]-(2-fluoro-phenyl)- methanone (Example 12);

1-{4-[6-Amino-5-(2-fluoro-benzoyl)-pyridin-2-ylamino]-pip eridin-1-yl}-ethanone (Example 13); 1-{4-[6-Amino-5-(2-fluoro-benzoyl)-pyridin-2-ylamino]-piperi din-1-yl}-propan-1- one (Example 14);

4-[6-Amino-5-(2-fluoro-benzoyl)-pyridin-2-ylannino]-piper idine-1-carboxylic acid ethylamide (Example 15);

4-[6-Amino-5-(2-fluoro-benzoyl)-pyridin-2-ylamino]-piperi dine-1-carboxylic acid methylamide (Example 16);

[2-Amino-6-(1-methanesulfonyl-piperidin-4-ylamino)-pyridi n-3-yl]-(3-methoxy- phenyl)-methanone (Example 18);

4-[6-Amino-5-(5-fluoro-2-methoxy-benzoyl)-pyridin-2-ylami no]-piperidine-1- carboxylic acid ethyl ester (Example 20);

[2-Amino-6-(1-methanesulfonyl-piperidin-4-ylamino)-pyridi n-3-yl]-(5-fluoro-2- methoxy-phenyl)-methanone (Example 23);

[2-Amino-6-(1-ethanesulfonyl-piperidin-4-ylamino)-pyridin -3-yl]-(5-fluoro-2- methoxy-phenyl)-methanone (Example 24);

{2-Amino-6-[1-(propane-1-sulfonyl)-piperidin-4-ylamino]-p yridin-3-yl}-(5-fluoro-2- methoxy-phenyl)-methanone (Example 25);

{2-Amino-6-[1-(propane-2-sulfonyl)-piperidin-4-ylamino]-p yridin-3-yl}-(5-fluoro-2- methoxy-phenyl)-methanone (Example 26);

4-[6-Amino-5-(5-fluoro-2-methoxy-benzoyl)-pyridin-2-ylami no]-piperidine-1- carboxylic acid methyl ester (Example 27);

4-[6-Amino-5-(5-fluoro-2-methoxy-benzoyl)-pyridin-2-ylami no]-piperidine-1- carboxylic acid propyl ester (Example 28); [2-Amino-6-(1-benzyl-piperidin-4-ylamino)-pyridin-3-yl]-(5-f luoro-2-methoxy- phenyl)-methanone (Example 29);

1 -{^[δ-Annino-S-CS-fluoro^-metho'xy-benzoyO-pyridin^-ylamino l-pipericlin-i -yl}- ethanone (Example 30);

1-{4-[6-Amino-5-(5-fluoro-2-methoxy-benzoyl)-pyridin-2-yl amino]-piperidin-1-yl}- propan-1-one (Example 31);

4-[6-Amino-5-(5-fluoro-2-methoxy-benzoyl)-pyridin-2-ylami no]-piperidine-1- carboxylic acid methylamide (Example 32);

4-[6-Amino-5-(5-fluoro-2-methoxy-benzoyl)-pyridin-2-ylami no]-piperidine-1- carboxylic acid ethylamide (Example 33);

[2-Amino-6-(1-methyl-piperidin-4-ylamino)-pyridin-3-yl]-( 5-fIuoro-2-methoxy- phenyl)-methanone (Example 34);

[2-Amino-6-(1-methanesulfonyl-piperidin-4-ylamino)-pyridi n-3-yl]-(4-methoxy- phenyl)-methanone (Example 36);

[2-Amino-6-(1-methanesulfonyl-piperidin-4-ylamino)-pyridi n-3-yl]-(4-fluoro- phenyl)-methanone (Example 38);

[2-Amino-6-(1-methanesulfonyl-piperidin-4-ylamino)-pyridi n-3-yl]-(2,3-difluoro-6- methoxy-phenyl)-methanone (Example 40);

4-[6-Amino-5-(2,3-difluoro-6-methoxy-benzoyl)-pyridin-2-y lamino]-piperidine-1- carboxylic acid ethyl ester (Example 41); 1-{4-[6-Amino-5-(2,3-difluoro-6-methoxy-benzoyl)-pyridin-2-y lamino]-piperidin-1- yl}-ethanone (Example 42);

1-{4-[6-Amino-5-(5-fluoro-2-methoxy-4-methyl-benzoyl)-pyr idin-2-ylamino]- piperidin-1-yl}-ethanone (Example 44);

[2-Amino-6-(1-methanesulfonyl-piperidin-4-ylamino)-pyridi n-3-yl]-(5-fluoro-2- methoxy-4-methyl-phenyl)-methanone (Example 45);

4-[6-Amino-5-(5-fluoro-2-methoxy-4-methyl-benzoyl)-pyridi n-2-ylamino]- piperidine-1-carboxylic acid ethyl ester (Example 46);

1-{4-[6-Amino-5-(4-chloro-5-fluoro-2-methoxy-benzoyl)-pyr idin-2-ylamino]- piperidin-1-yl}-ethanone (Example 48);

[2-Amino-6-(1-methanesulfonyl-piperidin-4-ylamino)-pyridi n-3-yl]-(4-chloro-5- fluoro-2-methoxy-phenyl)-methanone (Example 49);

4-[6-Amino-5-(5-fluoro-2-methoxy-4-chloro-benzoyl)-pyridi n-2-ylamino]- piperidine-1-carboxylic acid ethyl ester (Example 50); and

[2-Amino-6-(1-methanesulfonyl-piperidin-4-ylamino)-pyridi n-3-yl]-(2,6-difluoro- phenyl)-methanone (Example 52).

The compounds disclosed herein and covered by formula I above may exhibit tautomerism or structural isomerism. It is intended that the invention encompasses any tautomeric or structural isomeric form of these compounds, or mixtures of such forms, and is not limited to any one tautomeric or structural isomeric form depicted in the formula above.

The compounds of the present invention can be prepared by any conventional means, known to the skilled artisan. Suitable processes for synthesizing these compounds are provided in the examples. Generally, compounds of formula I can be prepared

according to the synthetic route described in scheme 1 , wherein unless explicitly

otherwise stated, R1 and R2 are as defined above.

The commercially available starting material, 2,6-dichloronicotinic acid (from

Aldrich. In Examples 1 and 2), can be regio-selectively converted to 2-amino-6-chloro-

N-methoxy-N-methylnicotinamide (Example 3) via 2-amino-6-chloronicotinic acid or 2,6-

dichloro-N-methoxy-N-methylnicotinamide as the intermediates (F. Mutterer and C.

Weis, Helvetica Chimica Acta, 59, 1976, 222-229). The R2 group is introduced by

reaction with organolithium reagents that are either commercially available or, readily

prepared from commercially available halides or substituted benzene. The final

compounds of the invention are then obtained by displacement of the 6-chloro group

with a variety of commercially available amines (R1NH2). When not commercially

available, these reactants are prepared or cited in the individual examples.

step e

scheme 1

Consequently as a further embodiment of the present invention, there is provided the process for the production of a compound of formula I, whereby

a) a compound of the formula

is converted into a compound of the formula

b) said compound of formula (B) is further reacted in the presence of an organolithium compound of the formula

R2-Li (C)

to give the compound of formula

c) said compound of formula (D) is further reacted in the presence of an amine of formula

R1-NH2 (E)

to give the corresponding compound of formula I, which can

d) be separated from the reaction mixture and, if desired, be turned into a pharmaceutically acceptable salt or ester; wherein

R1 and R2 have the meanings given above.

Separating a mixture of stereoisomers into the optically pure stereoisomers (when compound of formula I is chiral)

The optional separation of isomeric structures of formula I can be carried out according to known methods such as for example resolution or chiral high pressure liquid chromatography (also known as chiral HPLC). Resolution methods are well known, and are summarized in "Enantiomers, Racemates, and Resolutions" (Jacques, J. et al. John Wiley and Sons, NY, 1981). Methods for chiral HPLC are also well known, and are summarized in "Separation of Enantiomers by Liquid Chromatographic Methods" (Pirkle, W. H. and Finn, J. in "Asymmetric Synthesis", Vol. 1 , Morrison, J. D., Ed., Academic Press, Inc., NY 1983, pp. 87-124).

Converting a compound of formula I that bears a basic nitrogen into a pharmaceutically acceptable acid addition salt The optional conversion of a compound of formula I that bears a basic nitrogen into a pharmaceutically acceptable acid addition salt can be effected by conventional means. For example, the compound can be treated with an inorganic acid such as for example hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, or with an appropriate organic acid such as acetic acid, citric acid, tartaric acid, methanesulfonic acid, p-toluene sulfonic acid, or the like.

Converting a compound of formula I that bears a carboxylic acid group into a pharmaceutically acceptable alkali metal salt

The optional conversion of a compound of formula I that bears a carboxylic acid group into a pharmaceutically acceptable alkali metal salt can be effected by conventional means. For example, the compound can be treated with an inorganic base such as lithium hydroxide, sodium hydroxide, potassium hydroxide, or the like.

Converting a compound of formula I that bears a carboxylic acid group into a pharmaceutically acceptable ester

The optional conversion of a compound of formula I that bears a carboxylic acid group into a pharmaceutically acceptable ester can be effected by conventional means. The conditions for the formation of the ester will depend on the stability of the other functional groups in the molecule to the reaction conditions. If the other moieties in the molecule are stable to acidic conditions, the ester may be conveniently prepared by heating in a solution of a mineral acid (e.g., sulfuric acid) in an alcohol. Other methods of preparing the ester, which may be convenient if the molecule is not stable to acidic conditions include treating the compound with an alcohol in the presence of a coupling agent and in the optional presence of additional agents that may accelerate the reaction. Many such coupling agents are known to one skilled in the art of organic chemistry. Two examples are dicyclohexylcarbodiimide and triphenylphosphine /diethyl azodicarboxylate. In the case where dicyclohexylcarbodiimide is used as the coupling agent, the reaction is conveniently carried out by treating the acid with the alcohol, dicyclohexylcarbodiimide, and the optional presence of a catalytic amount (0-10 mole%) of N,N-dimethylaminopyridine, in an inert solvent such as a halogenated hydrocarbon (e.g., dichloromethane) at a temperature between about 0 0C and about room temperature, preferably at about room temperature. In the case where triphenylphosphine/diethyl azodicarboxylate is used as the coupling agent, the reaction is conveniently carried out by treating the acid with the alcohol, triphenylphosphine and diethyl azodicarboxylate, in an inert solvent such as an ether (e.g., tetrahydrofuran) or an aromatic hydrocarbon (e.g., benzene) at a temperature between about 0 0C and about room temperature, preferably at about 0 0C.

In an alternative embodiment, the present invention includes pharmaceutical compositions comprising at least one compound of formula I , or a pharmaceutically acceptable salt or ester thereof and an a pharmaceutically acceptable excipient and/or carrier.

These pharmaceutical compositions can be administered orally, for example in the form of tablets, coated tablets, dragees, hard or soft gelatin capsules, solutions, emulsions or suspensions. They can also be administered rectally, for example, in the form of suppositories, or parenterally, for example, in the form of injection solutions.

The pharmaceutical compositions of the present invention comprising compounds of formula I, and/or the salts or esters thereof, may be manufactured in a manner that is known in the art, e.g. by means of conventional mixing, encapsulating, dissolving, granulating, emulsifying, entrapping, dragee-making, or lyophilizing processes. These pharmaceutical preparations can be formulated with therapeutically inert, inorganic or organic carriers. Lactose, corn starch or derivatives thereof, talc, steric acid or its salts can be used as such carriers for tablets, coated tablets, dragees and hard gelatin capsules. Suitable carriers for soft gelatin capsules include vegetable oils, waxes and fats. Depending on the nature of the active substance, no carriers are generally required in the case of soft gelatin capsules. Suitable carriers for the manufacture of solutions and syrups are water, polyols, saccharose, invert sugar and glucose. Suitable carriers for injection are water, alcohols, polyols, glycerine, vegetable oils, phospholipids and surfactants. Suitable carriers for suppositories are natural or hardened oils, waxes, fats and semi-liquid polyols. The pharmaceutical preparations can also contain preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifying agents, sweetening agents, coloring agents, flavoring agents, salts for varying the osmotic pressure, buffers, coating agents or antioxidants. They can also contain other therapeutically valuable substances, including additional active ingredients other than those of formula I.

As mentioned above, the compounds of the present invention, including the compounds of formula I1 are useful in the treatment or control of cell proliferative disorders, including chemoprevention of cancer. Chemoprevention is defined as inhibiting the development of invasive cancer by either blocking the initiating mutagenic event or by blocking the progression of pre-malignant cells that have already suffered an insult of inhibiting tumor relapse.

These compounds and formulations containing said compounds are particularly useful in the treatment or control of solid tumors, such as, for example, breast, colon, lung and prostate tumors. Furthermore the compounds according to the present invention are useful for the manufacture of medicaments for the treatment of cancer, especially of solid tumors, and more preferably of breast, lung, colon and prostate tumors.

A therapeutically effective amount of a compound in accordance with this invention means an amount of compound that is effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is within the skill in the art.

The therapeutically effective amount or dosage of a compound according to this invention can vary within wide limits and may be determined in a manner known in the art. Such dosage will be adjusted to the individual requirements in each particular case including the specific compound(s) being administered, the route of administration, the condition being treated, as well as the patient being treated. In general, in the case of oral or parenteral administration to adult humans weighing approximately 70 Kg, a daily dosage of about 10 mg to about 10,000 mg, preferably from about 200 mg to about 1 ,000 mg, should be appropriate, although the upper limit may be exceeded when indicated. The daily dosage can be administered as a single dose or in divided doses, or for parenteral administration, it may be given as continuous infusion.

The compounds of this invention may be used in combination (administered in combination or sequentially) with known anti-cancer treatments such as radiation therapy or with cytostatic or cytotoxic agents, such as for example, but not limited to, DNA interactive agents, such as cisplatin or doxorubicin; topoisomerase Il inhibitors such as etoposide: topoisomerase I inhibitors such as CPT-11 or topotecan; tublin interacting agents, such as paclitaxel, docetaxel or epothilones; hormonal agents such as tamoxifen: thymidilate synthaes inhibitors, such as 5-fluorouracil; and anti-metabolites such as methotrexate. Compounds of formula I may also be useful in combination with modulators of p53 transactivation.

If formulated as a fixed dose, the above-described combination products include the compounds of this invention within the dosage range described above and the other pharmaceutically active agent or treatment within its approved dose range. For example, an early cdk1 inhibitor olomucine has been found to act synergistically with well known cytotoxic agents in inducing apoptosis. (J. Cell ScL, 1995, 108, 2897-2904). Compounds of formula I may also be administered sequentially with known anticancer or cytoxic agents when concommitant administration or a combination is inappropriate. This invention is not limited in the sequence of administration: compounds of formula I may be administered either prior to or after administration of the known anticancer or cytotoxic agent. For example, the cytotoxic activity of the cdk inhibitor flavopiridol is affected by the sequence of administration with anticancer agents. (Cancer Research, 1997, 57, 3375).

In another embodiment, the present invention also relates to novel intermediates useful in the preparation of compounds of formula I. These novel intermediates include the following compounds: 2,6-Dichloro-N-methoxy-N-methyl-nicotinamide (Example 1);

2-Amino-6-chloro-nicotinic acid (Example 2);

2-Amino-6-chloro-N-methoxy-N-methyl-nicotinamide (Example 3);

(2,6-Dichloro-pyridin-3-yl)-(2-methoxy-phenyl)-methanone (Example 4);

(2-Amino-6-chloro-pyridin-3-yl)-(2-methoxy-phenyl)-methan one (Example 5);

(2-Amino-6-chloro-pyridin-3-yl)-(2-fluoro-phenyl)-methano ne (Example 7);

4-[6-Amino-5-(2-fluoro-benzoyl)-pyridin-2-ylamino]-piperi dine-1-carboxylic acid tert-butyl ester (Example 8);

[2-Amino-6-( piperidin-4-ylamino)-pyridin-3-yl]-(2-fluoro-phenyl)-methano ne (Example 9);

(2-Amino-6-chloro-pyridin-3-yl)-(3-methoxy-phenyl)-methan one (Example 17);

(2-Amino-6-chloro-pyridin-3-yl)-(5-fluoro-2-methoxy-pheny l)-methanone (Example 19);

4-[6-Amino-5-(5-fluoro-2-methoxy-benzoyl)-pyridin-2-ylami no]-piperidine-1- carboxylic acid tert-butyl ester (Example 21);

[4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluor o-2-methoxy-phenyl)- methanone trifluoroacetic acid salt (Example 22);

(2-Amino-6-chloro-pyridin-3-yl)-(4-methoxy-phenyl)-methan one (Example 35);

(2-Amino-6-chloro-pyridin-3-yl)-(4-fluoro-phenyl)-methano ne (Example 37); (2-Amino-6-chloro-pyridin-3-yl)-(2,3-difluoro-6-methoxy-phen yl)-methanone (Example 39);

(2-Amino-6-chloro-pyridin-3-yl)-(5-fluoro-2-methoxy-4-met hyl-phenyl)-methanone (Example 43);

(2-Amino-6-chloro-pyridin-3-yl)-(4-chloro-5-fluoro-2-meth oxy-phenyl)-methanone (Example 47); and

(2-Amino-6-chloro-pyridin-3-yl)-(2,6-difluoro-phenyl)-met hanone (Example 51 ).

The following examples illustrate preferred methods for synthesizing and using the compounds and formulations of the present invention. These examples and preparations are illustrative and are not intended to be limiting. It should be understood that there may be other embodiments which fall within the spirit and scope of the invention as defined by the claims appended hereto. Examples:

Example 1

2,6-Dichloro-N-methoxy-N-methyl-nicotinamide

MW 192.00 MW 235.07 C6H3CI2NO2 C8H8CI2N2O2

To a solution of 2,6-dichloronicotinic acid (500 mg, 2.63 mmol, Aldrich 90%) in anhydrous N,N-dimethylformamide (5 ml_) were added O-benzotriazol-1-yl-N,N,N',N'- tetramethyluronium hexafluorophosphate (1.28 g, 3.37 mmol, Aldrich), 1- hydroxybenzotriazole hydrate (456 mg, 3.37 mmol, Aldrich) followed by N1N- diisopropyiethylamine (1.3 g, 10.0 mmol) and N,O-dimethylhydroxylamine hydrochloride (306 mg, 3.15 mmol, Aldrich) at 0 0C. The reaction was stirred for 1~2 hrs, treated with water and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried and concentrated. The crude product was purified on silica gel with hexanes/ethyl acetate to give 2,6-Dichloro-N-methoxy-N-methyl-nicotinamide as a white solid (304.5 mg, 50% yield). HRMS, observed: 233.9968, Calcd for M+: 233.9963 Example 2

2-Amino-6-chloro-nicotinic acid

MW 192.00 MW 172.57 C6H3CI2NO2 C6H5CIN2O2

A solution of 2,6-dichloronicotinic acid (10 g, 46.9 mmol, Aldrich 90%) was dissolved in concentrated ammonium hydroxide (100 ml_, 29.4%, Fisher) and heated at 130-1600C in a pressure bottle for 1-2 days before solvent was removed in vacuo. The residue was treated with water and the pH was lowered to ~8 with concentrated hydrochloric acid. The resulting mixture was extracted with ethyl acetate and the combined organic extracts were washed with brine, dried, concentrated and the crude product was recrystallized from ethyl acetate to give 2-Amino-6-chloro-nicotinic acid as white crystalline (2.74g, 34 % yield). HRMS, observed: 172.0042, Calcd for M+: 172.0040 Example 3

2-Amino-6-chloro-N-methoxy-N-methyl-nicotinamide

Method 1

MW 235.07 MW 215.64 C8H8CI2N2O2 C 'n8K1 MnOCINX 3W)2

2,6-Dichloro-N-methoxy-N-methyl-nicotinamide (202.4 rhg, 0.8610 mmol, Example 1) and concentrated ammonium hydroxide (1.8 mL, 29.4%, Fisher) in tetrahydrofuran (2.5 mL) were heated to 155 ° C in the microwave for 2 hr. The reaction mixture was concentrated and the residue was purified on silica gel with hexanes/ethyl acetate to give 2-Amino-6-chloro-N-methoxy-N-methyl-nicotinamide (14.3 mg, 8% yield). HRMS, observed: 215.0463, Calcd for M+: 215.0462.

Method 2:

MW 172.5720 MW 215.6408 C6H5CIN2O2 C8H10CIN3O2 To a solution of 2-Amino-6-chloro-nicotinic acid (4.99g, 28.965 mmol, Example 2) in anhydrous N,N-dimethylformamide (5OmL) were added 1-hydroxybenzotriazole hydrate (6.21 g, 40.57 mmol, Advanced ChemTech) and O-benzotriazol-1-yl-N, N1NT1N'- tetramethyluroniumhexafluorophosphate (15.38 g, 40.5648 mmol, Aldrich) at O0C. A solution of N.O-dimethylhydroxylamine hydrochloride (306 mg, 3.15 mmol, Aldrich) in anhydrous N,N-dimethylformamide (15ml_) was treated with N,N-diisopropylethylamine (8.28 g, 64.1 mmol) and immediately added to the reaction which was stirred at O0C for ~10 minutes then at room temperature for 4-5 hrs. The reaction was diluted with ethyl acetate (15OmL) and water (100 mL) and the aqueous layer was extracted with ethyl acetate (3 x 150 mL). The combined organic layers were washed with water (3x) and saturated sodium chloride, dried and concentrated. The crude product was purified on silica gel with 90/10→70/30 of hexanes/ethyl acetate to give 2-Amino-6-chloro-N- methoxy-N-methyl-nicotinamide (4.72 g, 21.92 mmol, 75.7 % yield). HRMS, observed: 215.0463, Calcd for M+: 215.0462.

Example 4

(2,6-Dichloro-pyridin-3-yl)-(2-methoxy-phenyl)-methanone

MW235.07 MW234.0376 MW 282.1282 C8H8Cl2N2O2 C7H7IO C13H9Cl2NO2

To a solution of 2-iodoanisole (4.97g, 21.24 mmol, Aldrich 98%) in anhydrous tetrahydrofuran (33 mL) at -78 0C, was added a solution of n-butyllithium in hexane (2.5 M, 8.5 mL, 21.25 mmol, Aldrich). The reaction was stirred at -78 0C for 0.5~1 hr to give a solution of the 2-methoxyphenyl lithium for the following reaction. To a solution of 2,6-Dichloro-N-methoxy-N-methyl-nicotinamide (503.3 mg, 2.141 mmol, Example 1) in anhydrous tetrahydrofuran (8 ml_) was added a solution of freshly prepared 2-methoxyphenyl lithium (3~5 equiv, from above) and the reaction was stirred at -78 0C for 30-60 mins until the complete consumption of starting material. The resulting mixture was quenched with aqueous ammonium chloride solution, extracted with ethyl acetate, washed with saturated sodium chloride, dried over sodium sulfate and evaporated in vacuo. The residue was purified on silica gel with hexanes/ethyl acetate to give (2,6-Dichloro-pyridin-3-yl)-(2-methoxy-phenyl)-methanone as a light brown waxy solid (314.3 mg, 52% yield). HRMS, observed: 281.0023, Calcd for M+: 281.0010.

Example 5

(2-Amino-6-chloro-pyridin-3-yl)-(2-methoxy-phenyl)-methan one

MW 282.1282 MW 262.6978 C13H9CI2NO2 C13H11ClN2O2

A solution of (2,6-Dichloro-pyridin-3-yl)-(2-methoxy-phenyl)-methanone (50.3 mg, 0.178 mmol, Example 4) in 1,4-dioxane (2 ml_) was treated with concentrated ammonium hydroxide (0.24 mL, 29.4%) and heated to 150° C in the microwave for 3 hrs. The resulting mixture was treated with water and ethyl acetate, washed with saturated sodium chloride, dried over sodium sulfate and evaporated in vacuo. The residue was purified on silica gel with hexanes/ethyl acetate to give (2-Amino-6-chloro- pyridin-3-yl)-(2-methoxy-phenyl)-methanone as a off-white solid (23.7 mg, 50.7% yield). HRMS, observed: 262.0502, Calcd for M+: 262.0509.

Example 6

[2-Amino-6-(1-methanesulfonyl-piperidin-4-ylamino)-pyridi n-3-yl]-(2-methoxy-phenyl)- methanone

A) Preparation of methanesulfonyl-piperidin-4-yIamine with trifluoro-acetic acid

MW 178.25 + 114.02 C6H14N2O2S . C2HF3O2

A solution of piperidin-4-yl-carbamic acid tert-butyl ester (1.0 g, 5.0 mmol, Astratech, Inc.) and diisopropylethylamine (4 ml_) in tetrahydrofuran (40 mL) was stirred at +5 0C. To this was added methanesulfonyl chloride (1.0 g, 8.8 mmol) in a bolus. The reaction was brought to room temperature for 1 hour, poured into water and extracted into methylene chloride (2x50 mL). The combined organic extracts were washed with 5% aqueous sodium bicarbonate. The organic solution was dried (Na2SO4) and solvent was removed under vacuum to give a crude solid. Purification was by trituration with ether/hexane to give (1 -methanesulfonyl-piperidin-4-yl)-carbamic acid tert-butyl ester as a white solid. Mass spectrum (ES) H+: 278

A suspension of (1-methanesulfonyl-piperidin-4-yl)-carbamic acid tert-butyl ester (1.14 g, 4.1 mmol, from above) in methylene chloride (15 mL) was treated at room temperature with trifluoroacetic acid (5.3 mL). After stirring for 2 hours, all solvent was removed and the residue was triturated with ether. This was filtered, washed with ether and dried in vacuum to give 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoro-acetic acid (1.20 g, 100% yield). HRMS, observed: 177.0692; Calcd for M+: 177.0698.

B) Preparation of [2-Amino-6-(1-methanesulfonyl-piperidin-4-ylamino)-pyridin-3 -yl]-(2- methoxy-phenyl)-methanone

MW 262.69 MW 178.25 + 114.02 MW 404.49 C13H11CIN2O2 C6H14N2O2S . C2HF3O2 C19H24N4O4S

A mixture of (2-Amino-6-chloro-pyridin-3-yl)-(2-methoxy-phenyl)-methanone (24.9 mg, 0.0948 mmol, Example 5), 1-methanesulfonyl-piperidin-4-ylamine (50.6 mg, 0.284 mmol, from Step A above ), N,N-diisopropylethylamine (38mg, 0.294 mmol) and ethanol (2.7 mL) were heated at 160-180 ° C in a sealed tube under microwave conditions for 0.5-4 hrs. The resulting reaction mixture was concentrated in vacuo and the residue was purified on silica gel with methylene chloride/methanol to give [2- Amino-6-(1-methanesulfonyl-piperidin-4-ylamino)-pyridin-3-yI ]-(2-methoxy-phenyl)- methanone as a white solid (22.5 mg, 58% yield). HRMS, observed: 405.1594, calcd for (M+H)+: 405.1591. Example 7

(2-Amino-6-chloro-pyridin-3-yl)-(2-fluoro-phenyl)-methano ne

MW 215.6408 MW 222.0016 MW 250.66 C8H10CIN3O2 C6H4FI C12H8CIFN2O

2-Amino-6-chloro-N-methoxy-N-methyl-nicotinamide (500 mg, 2.32 mmol, Example 3) was dissolved in anhydrous tetrahydrofuran (14 ml_) and cooled to -78 0C. A solution of 2-fluorophenyl lithium (4-6 equiv, freshly prepared following the same procedure as in Example 4) was added. The reaction was stirred at -78 0C for 1~3 hrs and quenched with aqueous ammonium chloride solution. The resulting mixture was extracted with ethyl acetate, washed with saturated sodium chloride, dried over sodium sulfate and evaporated in vacuo. The product was then purified on silica gel to give (2-Amino-6-chloro-pyridin-3-yl)-(2-fluoro-phenyl)-methanone as a white solid (445.5 mg, 77 % yield). HRMS, observed: 250.0306, Calcd for M+: 250.0309. Example 8

4-[6-Amino-5-(2-fluoro-benzoyl)-pyridin-2-ylamino]-piperi dine-1 -carboxylic acid tert-butyl ester

MW 250.66 MW 200.28 MW 414.48 C12H8CIFN2O C10H20N2O2 C22H27FN4O3

The title compound was prepared from (2-Amino-6-chlprp-pyridin-3-yl)-(2-fluoro-

phenyl)-methanone (Example 7) and 4-amino-1-N-Boc-piperidine (Astatech, >96%)

using the procedure described in Step B , Example 6. HRMS, observed: 415.2143,

Calcd for (M+H)+: 415.2140.

Example 9

[2-Amino-6-( piperidin-4-ylamino)-pyridin-3-yl]-(2-fluoro-phenyl)-methano ne

MW 414.48 MW 314.37 C22H27FN4O3 C17H19FN4O 4-[6-Amino-5-(2-fluoro-benzoyl)-pyridin-2-ylamino]-piperidin e-1-carboxylic acid tert-butyl ester (269.6 mg, 0.6504 mmol, Example 8) was dissolved in dichloromethane (5 ml_), cooled to 0 0C and treated with trifluoroacetic acid (2.5 ml_). After stirring -30 minutes, the reaction mixture was concentrated in vacuo. The salt was then neutralized with saturated sodium carbonate (~3 ml_), extracted with ethyl acetate/methylene chloride (~50ml_), washed with saturated sodium chloride (~3 ml_), dried over sodium sulfate and evaporated in vacuo to give [2-Amino-6-( piperidin-4-ylamino)-pyridin-3-yl]- (2-fluoro-phenyl)-methanone as an off-white solid (200 mg, 0.636 mmol, 98 % yield).

Example 10

[2-Amino-6-(1-methanesulfonyl-piperidin-4-ylamino)-pyridi n-3-yl]-(2-fluoro-phenyl)- methanone

MW 250.66 MW 178.25 MW 392.46 C12H8CIFN2O C6H14N2O2S C18H21FN4O3S

The title compound was prepared from (2-Amino-6-chloro-pyridin-3-yl)-(2-fluoro- phenyl)-methanone (Example 7) and 1-methanesulfonyl-piperidin-4-ylamine (Step A, Example 6) using the procedure described in Step B, Example 6. HRMS, observed: 392.1329, calcd for M+: 392.1318. Example 11

4-[6-Amino-5-(2-fluoro-benzoyl)-pyridin-2-ylamino]-piperi dine-1 -carboxylic acid ethyl

ester

MW 250.66 MW 172.23 MW 386.43 C12H8CIFN2O C8H16N2O2 C20H23FN4O3

The title compound was prepared from (2-Amino-6-chloro-pyridin-3-yl)-(2-fluoro-

phenyl)-methanone (Example 7) and ethyl-4-amino-1-piperidine carboxylate (Aldrich)

using the procedure described in Step B, Example 6. HRMS, observed: 386.1762,

calcd for lvT: 386.1754.

Example 12

[2-Amino-6-(1-ethanesulfonyl-piperidin-4-ylamino)-pyridin -3-yl]-(2-fluoro-phenyl)-

methanone

MW 314.37 MW 406.48 C17H19FN4O Ci9H23FN4O3S To a solution of [2-Amino-6-( piperidin-4-ylamino)-pyridin-3-yl]-(2-fluoro-phenyl)- methanone (19.2 mg, 0.0611 mmol, Example 9) in methylene chloride (1.5 ml_) were added triethyl amine (12.6 mg, 0.124 mmol, Aldrich) in methylene chloride (0.2 ml_) and a solution of ethanesulfonyl chloride (8.66 mg, 0.066 mmol, Aldrich 99+%) in methylene chloride (0.2 ml_) at room temperature. After ~30 mins the reaction mixture was concentrated and purified by reversed phase HPLC as described in Example 11 to give [2-Amino-6-(1-ethanesulfonyl-piperidin-4-ylamino)-pyridin-3- yl]-(2-fluoro-phenyl)- methanone as a white solid (15.1 mg, 60 % yield). HRMS, observed: 407.1533, calcd for (M+H)+: 407.1548.

Example 13

1-{4-[6-Amino-5-(2-fluoro-benzoyl)-pyridin-2-ylamino]-pip eridin-1-yl}-ethanone

MW 314.37 MW 356.40 C17H19FN4O C19H21FN4O2

The title compound was prepared from [2-Amino-6-(piperidin-4-ylamino)-pyridin- 3-yl]-(2-fluoro-phenyl)-methanone (Example 9) and acetyl chloride (Aldrich 98.5 %) using the procedure described in Example 12. HRMS, observed: 356.1655, Calcd for M+: 356.1649. Example 14

1-{4-[6-Amino-5-(2-fluoro-benzoyl)-pyridin-2-ylamino]-pip eridin-1-yl}-propan-1-one

MW 314.37 MW 370.43 C17H19FN4O C20H23FN4O2 The title compound was prepared from [2-Amino-6-(piperidin-4-ylamino)-pyridin- 3-yl]-(2-fluoro-phenyl)-methanone (Example 9) and propionyl chloride (Aldrich 98 %) using the procedure described in Example 12. HRMS, observed: 370.1807, Calcd for M+: 370.1805.

Example 15

4-[6-Amino-5-(2-fluoro-benzoyl)-pyridin-2-ylamino]-piperi dine-1-carboxylic acid ethylamide

MW 314.37 MW 385.44 C17H19FN4O ^20^24 FN,O, The title compound was prepared from [2-Amino-6-(piperidin-4-ylamino)-pyridin- 3-yl]-(2-fluoro-phenyl)-methanone (Example 9) and ethyl isocyanate (Aldrich 98%) using the procedure described in Example 12. HRMS, observed: 385.1917, calcd for M+: 385.1914.

Example 16

4-[6-Amino-5-(2-fluoro-benzoyl)-pyridin-2-ylamino]-piperi dine-1-carboxylic acid methylamide

MW 314.37 MW 371.42 C17H19FN4O C19H22FN5O2

The title compound was prepared from [2-Amino-6-(piperidin-4-ylamino)-pyridin- 3-yl]-(2-fluoro-phenyl)-methanone (Example 9) and methyl isocyanate (Aldrich 98 %) using the procedure described in Example 12. HRMS, observed: 371.1756, calcd for M+: 371.1758. Example 17

(2-Amino-6-chloro-pyridin-3-yl)-(3-methoxy-phenyl)-methan one

MW 215.6408 MW 234.0376 MW 262.70 C8H10CIN3O2 C7H7IO C13H11CINo 2Ov->2

The title compound was prepared from 2-Amino-6-chloro-N-methoxy-N-methyl- nicotinamide (Example 3) and 3-iodoanisole (Aldrich) using the procedure described in Example 7. HRMS, observed: 262.0514, Calcd for M+: 262.0509.

Example 18

[2-Amino-6-(1-methanesulfonyl-piperidin-4-ylamino)-pyridi n-3-yl]-(3-methoxy-phenyl)- methanone

MW 262.70 MW 178.25 MW 404.49 C13H11CIN2O2 C6H14N2O2S C19H24N4O4S The title compound was prepared from (2-Amino-6-chloro-pyridin-3-yl)-(3- methoxy-phenyl)-methanone (Example 17) and 1-methanesulfonyl-piperidin-4-ylamine (Step A, Example 6) using the procedure described in Step B, Example 6. HRMS, 405.1595 observed:, Calcd for (M+H)+: 405.1591.

Example 19

(2-Amino-6-chloro-pyridin-3-yl)-(5-fluoro-2-methoxy-pheny l)-methanone

MW 215.6408 MW 205.0277 MW 280.6882 C8H10CIN3O2 C7H6BrFO α,H,nCIFNoO,

The title compound was prepared from 2-Amino-6-chloro-N-methoxy-N-methyl- nicotinamide (Example 3) and 2-bromo-4-fluoroanisole (Aldrich) using the procedure described in Example 7. HRMS, observed: 280.0417, Calcd for M+: 280.0415. Example 20

4-[6-Amino-5-(5-fluoro-2-methoxy-benzoyl)-pyridin-2-ylami no]-piperidine-1-carboxylic acid ethyl ester

MW 280.6882 MW 172.23 MW 416.4562 C13H10CIFN2O2 C8H16N2O2 C21H25FN4O4

The title compound was prepared from (2-Amino-6-chloro-pyridin-3-yl)-(5-fluoro- 2-methoxy-phenyl)-methanone (Example 19) and ethyl-4-amino-1-piperidine carboxylate (Aldrich 96) using the procedure described in Step B, Example_6. HRMS, observed: 417.1938, Calcd for (M+H)+: 417.1933.

Example 21

4-[6-Amino-5-(5-fluoro-2-methoxy-benzoyl)-pyridin-2-ylami no]-piperidine-1-carboxylic acid tert-butyl ester

MW 280.6882 MW 200.2831 MW 444.5104 C13H10CIFN2O2 C10H20N2O2 C23H29FN4O4 The title compound was prepared from (2-amino-6-chloropyridin-3-yl)-(5-fluoro-2- methoxyphenyl)methanone (Example 19) and 4-amino-i-N-Bocpiperidine (Astatech, >96%) using the procedure described in Step B, Example 6. HRMS, observed: 445.2252, Calcd for (M+H)+: 445.2246.

Example 22

[4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluor o-2-methoxy-phenyl)-methanone trifluoroacetic acid salt..

MW 444.5104 MW 344.3921 + C23H29FN4O4 C18H21FN4O2 - XC2HF3O2

A solution of 4-[6-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyridin-2-ylamino] - piperidine-1-carboχylic acid tert-butyl ester (256.1 mg, 0.5761 mmol, Example 21) in dichloromethane (5 ml_) was cooled to 00C and treated with trifluoroacetic acid (2.5 ml_). After stirring 30 mins, the reaction mixture was concentrated in vacuo to give [4-amino- 2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy- phenyl)-methanone as the trifluoroacetic acid salt (519.8 mg). MS (M+H)+: 345. Example 23

[2-Amino-6-(1-methanesulfonyl-piperidin-4-ylamino)-pyridi n-3-yl]-(5-fluoro-2-methoxy- phenyl)-methanone

MW 280.6882 MW 178.25 MW422.4820 C13H10CIFN2O2 C6H14N2O2S C19H23FN4O4S

The title compound was prepared from (2-amino-6-chloropyridin-3-yl)-(5-fluoro-2- methoxyphenyl)methanone (Example 19) and 1-methanesulfonylpiperidin-4-ylamine (Step A, Example 6) using the procedure described in Step B, Example 6. HRMS, observed: 423.1502, Calcd for (M+H)+: 423.1497. Ki for cdk4 = 0.040 μM, cdk2 = 0.098 μM, and IC50 for HCT116 cell line = 9 μM.

Example 24

[2-Amino-6-(1-ethanesulfonyl-piperidin-4-ylamino)-pyridin -3-yl]-(5-fluoro-2-methoxy- phenyl)-methanone

MW 344.3921 + MW 436.5091 C20H25FN4O4S

To a solution of [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2 - methoxy-phenyl)-methanone trifluoroacetic acid salt (0.0589 mmol, Example 22) in methylene chloride (1.25 mL) were added triethylamine (0.065 ml_, 0.466 mmol, Aldrich) and a solution of ethanesulfonyl chloride (8.1 mg, 0.062 mmol, Aldrich 99+%) in methylene chloride (0.33 mL) at 0 0C. After 5 minutes the reaction mixture was allowed to stir at room temperature for 1 hr before it was concentrated in vacuo. The residue was absorbed on silica gel and purified with 99.5/0.5->95/5 of methylene chloride/methanol to give [2-amino-6-(1-ethanesulfonyl-piperidin-4-ylamino)-pyridin-3- yl]-(5-fluoro-2-methoxy-phenyl)-methanone as a white solid (16.0 mg, 63% yield). HRMS, observed: 437.1660, Calcd for (M+H)+: 437.1654.

Example 25

{2-Amino-6-[1-(propane-1-sulfonyl)-piperidin-4-ylamino]-p yridin-3-yl}-(5-fluoro-2- methoxy-phenyl)-methanone

MW 344.3921 + MW 450.54 C18H21 FN4O2.xC2HF 3,OW2 C21H27FN4O4S

The title compound was prepared from [4-amino-2-(piperidin-4-yIamino)- pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt (Example 22) and 1-propanesulfonyl chloride (Aldrich 97%) using the procedure described in Example 24. HRMS, observed 451.1817, Calcd for (M+H)+: 451.1810. Example 26

{2-Amino-6-[1-(propane-2-sulfonyl)-piperidin-4-ylamino]-p yridin-3-yl}-(5-fluoro-2- methoxy-phenyl)-methanone

MW 344.3921 + MW 450.54 C18H21FN4O2-XC2HF3O2 C21H27FN4O4S

The title compound was prepared from [4-amino-2-(piperidin-4-ylamino)- pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt (Example 22) and isopropyl sulfonyl chloride (Aldrich 97%) using the procedure described in Example 24. HRMS, observed: 451.1814, Calcd for (M+H)+: 451.1810. Example 27

4-[6-Amino-5-(5-fluoro-2-methoxy-benzoyl)-pyridin-2-ylami no]-pipendine-1-carboxylic acid methyl ester

MW 344.3921 + MW 402.4291 C18H21FN4O21XC2HF3O2 C20H23FN4O4

The title compound was prepared from [4-amino-2-(piperidin-4-ylamino)- pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt (Example 22) and methylchloroformate (Aldrich 99%) using the procedure described in Example 24. HRMS, observed: 403.1781 , Calcd for (M+H)+: 403.1776. Example 28

4-[6-Amino-5-(5-fluoro-2-methoxy-ben2oyl)-pyridin-2-ylami no]-piperidine-1-carboxylic acid propyl ester

MW 344.3921 + MW 430.4833 C J«18H' '2,<1F' U 1 N4,OW,2.xG,HF 3,OU.2 C22H27FN4O4

The title compound was prepared from [4-amino-2-(piperidin-4-ylamino)- pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyI)-methanone trifluoroacetic acid salt (Example 22) and 1 -propyl chloroformate (Aldrich 98%) using the procedure described in Example 24. HRMS, observed: 431.2095, Calcd for (M+H)+: 431.2089.

Example 29

[2-Amino-6-(1-benzyl-piperidin-4-ylamino)-pyridin-3-yl]-( 5-fluoro-2-methoxy-phenyl)- methanone

MW 280.6882 MW 190.2907 MW 434.5179 C13H10CIFN2O2 ^12^18^2 C25H27FN4O2

The title compound was prepared from (2-amino-6-chloropyridin-3-yl)-(5-fluoro-2- methoxyphenyl)methanone (Example 19) and 4-amino-1-benzylpiperidine (Ardrich) using the procedure described in Step B, Example 6. HRMS, observed: 435.2196, Calcd for (M+H)+: 435.2191. Example 30

1 -{4-[6-Amino-5-(5-fluoro-2-methoxy-benzoyl)-pyridin-2-ylamin o]-piperidin-1 -yl}- ethanone

MW 344.3921 + MW 386.43 C18H21FN4O2.xC2HF3O2 C20H23FN4O3

The title compound was prepared from [4-amino-2-(piperidin-4-ylamino)- pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt (Example 22) and acetyl chloride (Aldrich 98.5 %) using the procedure described in Example 24. HRMS, observed: 387.1833, Calcd for (M+H)+: 387.1827. Example 31

i^-tθ-Amino-S-Cδ-fluoro^-methoxy-benzoyO-pyπdin-Z-ylam inol-piperidin-i-y^-propan-

1-one

MW 344.3921 + MW 400.46 C '11n8H' '2o11FN M4OW2o-XC9HFo 3OW2 C2 1 H2 5 FN4 O3

The title compound was prepared from [4-amino-2-(piperidin-4-ylamino)-

pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt

(Example 22) and propionyl chloride (Aldrich 98%) using the procedure described in

Example 24. HRMS, observed: 401.1988, Calcd for (M+H)+: 401.1984 .

Example 32

4-[6-Amino-5-(5-fluoro-2-methoxy-benzoyl)-pyridin-2-ylami no]-piperidine-1-carboxylic

acid methylamide

MW 344.3921 + MW 401.44 C '18H' '2O11FN M4CW2 XC9HFO 3OW.2 C20H24FN5O3 The title compound was prepared from [4-amino-2-(piperidin-4-ylamino)- pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt (Example 22) and methyl isocyanate (Aldrich, 98%) using the procedure described in Example 24. HRMS, observed: 402.1940, Calcd for (M+H)+: 402.1936.

Example 33

4-[6-Amino-5-(5-fluoro-2-methoxy-benzoyl)-pyridin-2-ylami no]-piperidine-1-carboxylic acid ethylamide

MW 344.3921 + MW 415.47 C18H21FN4O2-XC2HF3O2 C21H26FN5O3

The title compound was prepared from [4-amino-2-(piperidin-4-ylamino)- pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt (Example 22) and ethyl isocyanate (Aldrich 98 %) using the procedure described in Example 24. HRMS, observed: 416.2097, Calcd for (M+H)+: 416.2093. Example 34

[2-Amino-6-(1-methyl-piperidin-4-ylamino)-pyridin-3-yl]-( 5-fluoro-2-methoxy-phenyl)- methanone

MW 344.3921 + MW 358.42 C18H21 FN4O2-XC2HF3O2 C19H23FN4O2

To a solution of [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2 - methoxy-phenyl)-methanone trifluoroacetic acid salt (0.0575 mmol, Example 22) in N,N-dimethylformamide (1.5 mL) were added potassium carbonate (55.6 mg, 0.4024 mmol, Aldrich) and iodomethane (14.8 mg, 0.104 mmol, Aldrich 99.5%) in N1N- dimethylformamide (0.35 mL). The reaction was stirred at 0 0C for 4 hrs. The resulting mixture was diluted with ethyl acetate (30 mL), washed with water, brine, dried over sodium sulfate and evaporated in vacuo. The residue was purified on reversed phase HPLC to give [2-Amino-6-(1-methyl-piperidin-4-ylamino)-pyridin-3-yl]-(5-f luoro-2- methoxy-phenyl)-methanone as a white solid (4.8 mg, 24 % yield). HRMS, observed: 358.1810, Calcd for M+: 358.1805. Example 35

(2-Amino-6-chloro-pyridin-3-yl)-(4-methoxy-phenyl)-methan one

MW 215.64 MW 234.04 MW 262.70 C8H10CIN3O2 C7H7IO C13H11CIN2O2 The title compound was prepared from 2-Amino-6-chloro-N-methoxy-N-methyl- nicotinamide (Example 3) and 4-methoxyphenyllithium freshly prepared from 4- iodoanisole (Aldrich) using the procedure described in Example 7. HRMS, observed: 262.0512, Calcd for M+: 262.0509

Example 36

[2-Amino-6-(1-methanesulfonyl-piperidin-4-ylamino)-pyridi n-3-yl]-(4-methoxy-phenyl)- methanone

MW 262.70 MW 178.25 MW 404.49 C13H11CIN2O2 C6H14N2O2S C19H24N4O4S The title compound was prepared from (2-Amino-6-chloro-pyridin-3-yl)-(4- methoxy-phenyl)-methanone (Example 35) and 1-methanesulfonyl-piperidin-4-ylamine (Step A, Example 6) using the procedure described in Step B, Example 6. HRMS, observed: 405.1595, calcd for (M+H)+: 405.1591.

Example 37

(2-Amino-6-chloro-pyridin-3-yl)-(4-fluoro-phenyl)-methano ne

MW 215.64 MW 222.00 MW 250.66 C8H10CIN3O2 C6H4FI C12H8CIFN2O

The title compound was prepared from 2-Amino-6-chloro-N-methoxy-N-methyl- nicotinamide (Example 3) and 1-fluoro-4-iodobenzene (Aldrich 99%) using the procedure described in Example 7. HRMS, observed: 250.0314, Calcd for M+: 250.0309. Example 38 [2-Amino-6-(1-methanesulfonyl-piperidin-4-ylamino)-pyridin-3 -yl]-(4-fluoro-phenyl)- methanone

MW 250.66 MW 178.25 MW 392.46 C12H8CIFN2O C6H14N2O2S C18H21FN4O3S The title compound was prepared from (2-amino-6-chloro-pyridin-3-yl)-(4- fluorophenyl)methanone (Example 37) and 1-methanesulfonyl-piperidin-4-ylamine (Step A, Example 6) using the procedure described in Step B, Example 6. HRMS, observed: 393.1391 , Calcd for (M+H)+: 393.1391. Example 39 (2-Amino-6-chloro-pyridin-3-yl)-(2,3-difluoro-6-methoxy-phen yl)-methanone

MW 215.64 MW 223.02 MW 298.68 C8H10CIN3O2 C7H5BrF2O C13H9CIF2N2O2 The title compound was prepared from 2-Amino-6-chloro-N-methoxy-N-methyl- nicotinamide (Example 3) and 2-bromo-4,5-difluoroanisole (Apollo) using the procedure described in Example 7. HRMS, observed: 298.0318, Calcd for M+: 298.0321.

Example 40

[2-Amino-6-(1-methanesulfonyl-piperidin-4-ylamino)-pyridi n-3-yl]-(2,3-difluoro-6- methoxy-phenyl)-methanone

MW 298.68 MW 178.25 MW440.47 C J13H' '09C' IF5N5O. C6H14N2O2S C1QH,,F,NXLS

The title compound was prepared from (2-Amino-6-chloro-pyridin-3-yl)-(2,3- difluoro-6-methoxy-phenyl)-methanone (Example 39) and 1-methanesulfonyl-piperidin- 4-ylamine (Step A, Example 6) using the procedure described in Step B, Example 6. HRMS, observed: 441.1407, Calcd for (M+H)+: 441.1403. Ki for cdk4 = 0.017 μM, cdk1 = 0.060 μM, cdk2 = 0.054 and IC50 for HCT116 cell line = 2.6 μM. Example 41

4-[6-Amino-5-(2,3-difluoro-6-methoxy-benzoyl)-pyridin-2-y lamino]-piperidine-1-

carboxylic acid ethyl ester

MW 298.68 MW 172.23 MW 434.45 C13H9CIF2N2O2 C8H16N2O2 C21H24F2N4O4

The title compound was prepared from (2-Amino-6-chloro-pyridin-3-yl)-(2,3-

difluoro-6-methoxy-phenyl)-methanone (Example 39) and ethyl-4-amino-1-piperidine

carboxylate (Aldrich 96%) using the procedure described in Step B, Example 6. HRMS,

observed: 435.1841 , Calcd for (M+H)+: 435.1839.

Example 42

1_{4-[6-Amino-5-(2,3-difluoro-6-methoxy-benzoyl)-pyridin- 2-ylamino]-piperidin-1-yl}-

ethanone

MW 298.68 MW 142.20 MW 404.42 C '«13H' '«9CIF, 2Nι i,2Ow.2 C7H14N2O C20H22F2N4O3 The title compound was prepared from (2-Amino-6-chloro-pyridin-3-yl)-(2,3- difluoro-6-methoxy-phenyl)-methanone (Example 39) and 1-(4-amino-piperidin-1-yl)- ethanone (prepared as described in Manetti et ai, Biorg. Med. Chem. Lett., 13 (2003), 2303-2306) using the procedure described in Step B, Example 6. HRMS, observed: 405.1734, Calcd for (M+H)+: 405.1733.

Example 43

(2-Amino-6-chloro-pyridin-3-yl)-(5-fluoro-2-methoxy-4-met hyl-phenyl)-methanone

MW 215.64 MW 140.16 MW 294.72 C8H10CIN3O2 C8H9FO C14H12CIFN2O2

To a solution of 4-fluoro-3-methylanisole (0.75 mL, 802 mg, 5.55 mmol, Aldrich 97%) in anhydrous tetrahydrofuran (5 mL) at -78 0C, was added slowly a solution of n- butyllithium in hexanes (2.5 M1 2.22 mL, 5.55 mmol, Aldrich). The reaction was stirred at -78 0C for 10 mins before it was allowed to warm to -25 ~0°C and stirred for 1.5 hour. The resulting aryllithium reagent was cooled back to -78 0C and reacted with 2-Amino-6- chloro-N-methoxy-N-methyl-nicotinamide as described in Example 7 to give (2-Amino- 6-chloro-pyridin-3-yl)-(5-fluoro-2-methoxy-4-methyl-phenyl)- methanone as an off-white solid. HRMS, observed: 295.0645, Calcd for (M+H)+: 295.0644. Example 44

i^-Iβ-Amino-S-Cδ-fluoro^-methoxy^-methyl-benzoyO-pyricl in-Z-ylaminoj-pipericlin-i- yl}-ethanone

MW 294.72 MW 142.20 MW 400.46 C14H12CIFN2O2 C7H14N2O C 'o211H 25FN4O3 The title compound was prepared from (2-Amino-6-chIoro-pyridin-3-yl)-(5-fluoro- 2-methoxy-4-methyl-phenyl)-methanone (Example 43) and 1-(4-amino-piperidin-1-yl)- ethanone (prepared as described in D. Manetti et al, Biorg. Med. Chem. Lett., 13 (2003), 2303-2306) using the procedure described in Step B, Example 6.. HRMS, observed: 401.1084, Calcd for (M+H)+: 401.1084.

Example 45

[2-Amino-6-(1 -methanesulfonyl-piperidin-4-ylamino)-pyridin-3-yl]-(5-fluor o-2-methoxy-4- methyl-phenyl)-methanone

MW 294.72 MW 178.25 MW 436.51 C -Η1ΛH1 '.12,CIFN920^,2 C6H14N2O2S C20H25FN4O4S The title compound was prepared from (2-Amino-6-chloro-pyridin-3-yl)-(5-fIuoro- 2-methoxy-4-methyl-phenyl)-methanone (Example 43) and 1-methanesulfonyI- piperidin-4-ylamine (Step A, Example 6) using the procedure described in Step B, Example 6. HRMS, observed 437.1653, Calcd for (M+H)+: 437.1654.

Example 46

4-[6-Amino-5-(5-fluoro-2-methoxy-4-methyl-benzoyl)-pyridi n-2-ylamino]-piperidine-1- carboxylic acid ethyl ester

MW 294.72 MW 172.23 MW 430.48 C14H12CIFN2O2 C8H16N2O2 C22H27FN4O4

The title compound was prepared from (2-Amino-6-chloro-pyridin-3-yl)-(5-fluoro- 2-methoxy-4-methyl-phenyl)-methanone (Example 43) and ethyl-4-amino-1-piperidine carboxylate (Aldrich 96%) using the procedure described in Step B, Example 6. HRMS, observed: 431.2090, Calcd for (M+H)+: 431.2089. Example 47

(2-Amino-6-chIoro-pyridin-3-yl)-(4-chloro-5-fluoro-2-meth oxy-phenyl)-methanone

A) Preparation of 5-Chloro-4-fluoro-2-iodoanisole

C7H5CIFIO C7H5CIFIO

To a solution of 3-chloro-4-fluoroanisoIe (4.69 g, Lancaster) in chloroform (250 ml_) was added silver trifluoroacetate (23.2 g, Aldrich) followed by iodine (15.8 g, Aldrich) in several portions. The reaction mixture was stirred for 2 hours and filtered through Celite. The filtrate was washed with water, brine, dried and concentrated. The crude product was purified by crystallization from ether/petroleum ether to give 5-chloro-4-fluoro-2-iodoanisole (5.0 g). MS (M+H)+, 285.

B) Preparation of (2-Amino-6-chloro-pyridin-3-yl)-(4-chloro-5-fluoro-2-methoxy -phenyl)- methanone

MW 215.64 MW 286.47 MW 315.13 C8H10CIN3O2 C7H5CIFlO C13H9CI2FN2O2 The title compound was prepared from 2-Amino-6-chloro-N-methoxy-N-methyl- nicotinamide (Example 3) and 5-chloro-4-fluoro-2-iodoanisole (prepared as described in Step A above) using the procedure described in Example 7. HRMS, observed: 312.9943, Calcd for (M-H)+: 312.9947.

Example 48

1-{4-[6-Amino-5-(4-chloro-5-fluoro-2-methoxy-benzoyl)-pyr idin-2-ylamino]-piperidin-1- yl}-ethanone

MW 315.13 MW 142.20 MW 420.87 C13H9CI2FN2O2 C7H14N2O C20H22CIFN4O3

The title compound was prepared from (2-Amino-6-chloro-pyridin-3-yl)-(4-chloro- 5-fluoro-2-methoxy-phenyl)-methanone (Example 47) and 1-(4-amino-piperidin-1~yl)- ethanone (prepared as described in D. Manetti et al, Biorg. Med. Chem. Lett, 13 (2003), 2303-2306) using the procedure described in Step B, Example 6. HRMS, observed: 421.1439, Calcd for (M+H)+: 421.1437. Example 49

[2-Amino-6-(1-methanesulfonyl-piperidin-4-ylamino)-pyridi n-3-yl]-(4-chloro-5-fluoro-2- methoxy-phenyl)-methanone

MW 315.13 MW 178.25 MW 456.93 C13H9CI2FN2O2 C6H14N2O2S C19H22CIFN4O4S The title compound was prepared from (2-Amino-6-chloro-pyridin-3-yl)-(4-chloro- 5-fluoro-2-methoxy-phenyl)-methanone (Example 47) and 1-methanesulfonyl-piperidin- 4-ylamine (Step A, Example 6) using the procedure described in Step B, Example 6. HRMS, observed: 457.1106, Calcd for (M+H)+: 457.1107. Ki for cdk4 = 0.008 μM, cdk1 = 0.154 μM, cdk2 = 0.039 μM, and IC50 for HCT116 cell line = 3.0 μM.

Example 50

4-[6-Amino-5-(5-fluoro-2-methoxy-4-chloro-benzoyi)-pyridi n-2-ylamino]-piperidine-1- carboxylic acid ethyl ester

MW 315.13 MW 172.23 MW 450.90 C13H9CI2FN2O2 C8H16N2O2 C21H24CIFN4O4 The title compound was prepared from (2-Amino-6-chloro-pyridin-3-yl)-(4-chloro- 5-fluoro-2-methoxy-phenyl)-methanone (Example 47) and ethyl-4-amino-1-piperidine carboxylate (Aldrich 96%) using the procedure described in Step B, Example 6. HRMS, observed: 451.1545, Calcd for (M+H)+: 451.1543.

Example 51

(2-Amino-6-chloro-pyridin-3-yl)-(2,6-difluoro-phenyl)-met hanone

MW 215.64 MW 192.99 MW 268.65 C8H10CIN3O2 C6H3BrF2 C12H7CIF2N2O

The title compound was prepared from 2-Amino-6-chloro-N-methoxy-N-methyl- nicotinamide (Example 3) and 2-bromo-1 ,3-difluorobenzene (Aldrich) using the procedure described in Example 7. HRMS, observed: 268.0217, Calcd for M+: 268.0215. Example 52

[2-Amino-6-(1-methanesulfonyl-pipeπclin-4-ylamino)-pyrid in-3-yl]-(2,6-difluoro-phenyl)- methanone

MW 268.65 MW 178.25 MW 410.45 C12H7CIF2N2O C6H14N2O2S ci8H20F2N4O3S

The title compound was prepared from (2-Amino-6-chloro-pyridin-3-yl)-(2,6- difluoro-phenyl)-methanone (Example 51) and 1-methanesulfonyl-piperidin-4-ylamine (Step A, Example 6) using the procedure described in Step B, Example 6. HRMS, observed: 411.1298, Calcd for (M+H)+: 411.1297.

The pharmacological properties of the compounds of this invention may be confirmed by a number of pharmacological assays. The exemplified pharmacological assays which follow have been carried out with the compounds according to the invention and their salts. The compounds of the invention exhibited cdk4/cyclin D activity with Ki values of less than 3.0 μM; cdk1 and cdk2 activities with Ki values of less than 8.0 μM. Additionally, the antiproliferative potency of some compounds of the invention was tested in the human colon tumor cell line HCT116 with IC50 values reported from an MTT assay of less than 35 μM, preferably less than 5 μM. Example 53

Kinase Assays

Ki: Measurement

Using recombinant human cyclin B-CDK1 , human cyclin E-CDK2 or human cyclin D1-CDK4 complexes. GST-cyclinE (GST-cycE), CDK2, GST-cyclinB (GST-cycB), CDK1 , GST-CDK4 and cyclin D1 (cycD1) cDNA clones in baculovirus vectors were provided by Dr. W. Harper at the Baylor College of Medicine, Houston, TX. Proteins were co-expressed in High Five™ insect cells and the complex was purified on glutathione Sepharose resin (Pharmacia, Piscataway, NJ) as previously described (Harper, J. W. et al. Cell 1993, 75, 805-816). A 6x-Histidine tagged truncated form of retinoblastoma (Rb) protein (amino acid 386-928) was used as the substrate for the cycD1-CDK4, cycB-CDK1 and the cycE-CDK2 assays (the expression plasmid was provided by Dr. Veronica Sullivan, Department of Molecular Virology, Roche Research Centre, Welwyn Garden City, United Kingdom). The Rb protein is a natural substrate for phosphorylation by CDK4, CDK2 and CDK1 (see Herwig and Strauss Eur. J. Biochem. Vol. 246 (1997) pp.581-601 and the references cited therein).

The expression of the 62Kd protein was under the control of an IPTG inducible promoter in an M15 E. coll strain. Cells were lysed by sonication and purification was carried out by binding lysates at pH 8.0 to a Ni-chelated agarose column pretreated with 1 mM imidazole. The resin was then washed several times with incrementally decreasing pH buffers to pH 6.0, and eluted with 500 mM imidazole. Eluted protein was dialysed against 20 mM HEPES pH 7.5, 30% glycerol, 200 mM NaCI, and 1 mM DTT. Purified Rb fusion protein stocks were quantitated for protein concentration, aliquoted, and stored at -70° C. Using the protein constructs described above, CDK1 , CDK2, and CDK4 HTRF assays were set up. These were done in 96-well format and read in 384-well plate format. The assays were run at 3x their respective Kms for ATP.

In the CDK4 assay, test compounds were diluted to 3x their final concentrations in 25 mM Hepes, pH 7.0, 6.25mM MgCI2, 1.5 mM DTT, 135 μM ATP. The DMSO concentration was no greater than 4.76%. Twenty microliters were added to the wells of a 96-well plate. The kinase reaction was initiated by the addition of 40 μl /well of a solution containing 0.185 μM Rb and 2.25 μg/ml CDK4 in 25 mM Hepes, pH 7.0, 6.25mM MgCI2, 0.003% Tween-20, 0.3mg/m! BSA, 1.5 mM DTT. Blank wells without CDK4 were included. The plates were incubated at 37°C for 30 minutes with shaking. The kinase reaction was terminated by the addition of 15 μl/well of 1.6uM anti-phospho- Rb (Ser 780) antibody (Cell Signaling Inc.) in 25 mM Hepes, pH 7.0 , 24 mM EDTA, 0.2 mg/ml BSA. After 30 minutes at 37°C, 15 μl/well of 3 nM Lance-Eu-W1024 labeled anti- rabbit IgG and 60 nM Allophycocyanin conjugated anti-Hisδ (PerkinElmer Life Sciences) in 25 mM Hepes, pH 7.0, 0.5 mg/ml BSA were added. Following a one hour incubation at 37° C, 35 μl of each well, in duplicate, were transferred to 384-well black plates. The plates were read using either ViewLux or Victor V readers (PerkinElmer Life Sciences) using an excitation wavelength of 340 nm and dual emission wavelengths of 615 nm and 665 nm. IC50 values (the concentration of test compounds reducing the assay control fluorescence read-out by 50%) were first calculated from net readings at 665nm, normalized for europium readings at 615nm. For ATP competitive inhibitors, the Ki values were calculated according to the following equation:

Ki = IC50/(1 + S/Km) where S refers to the substrate concentration (ATP) and Km refers to the Michaelis-Menten constant for the ATP.

The CDK1 and CDK2 assays were similarly run except for small differences in reagent and protein concentrations: The compound and enzyme buffers for both assays contained 1OmM MgCI2. For CDK1 and CDK2, the respective reagent ATP concentrations were 162uM and 9OuM. CDK1 at a reagent concentration of 0.15ng/ul and CDK2 at a reagent concentration of 0.06ng/ul were used. Reagent concentrations of detection reagents were adjusted between 3-12nM Eu-Ab and 60-9OnM APC-antiHis 6 to give signal to background ratios of at least 10 to 1.

Example 54

Cell Based Assays (Tetrazolium dye proliferation assav)("MTT Assay")

Proliferation was evaluated by the tetrazolium dye assay according to the procedure of Denizot and Lang (Denizot, F. and Lang, R. J Immunol Methods 1986, 89, 271-277). The cell line used was HCT116, a colorectal carcinoma cell line obtained from the American Type Cell Culture Collection (ATCC; Rockville, MD). The cells were grown in McCoy's 5A medium supplemented with 10% FCS and L-glutamine.

Cells were plated at the appropriate seeding density to give logarithmic growth over the course of the assay in a 96-well tissue culture plate. Plates were incubated overnight at 37 0C in a humidified incubator with 5% CO2. The next day, test compounds were serially diluted to four times the final concentration in the appropriate medium containing 1.2% DMSO. One-fourth final volume of each dilution was added in duplicate to the plates containing cells. The same volume of 1.2% DMSO in medium was added to a row of "control wells" such that the final concentration of DMSO in each well was 0.3%. Wells to which no cells were added served as the "blank." Wells to which no inhibitor was added served as "no inhibitor control." The plates were returned to the incubator, and at set time points (determined by their growth curves) plates were analyzed as described below.

3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (thiazolyl blue; MTT; Sigma) was added to each well to yield a final concentration of 1 mg/ml. Plates were returned to the incubator for 2.5-3 hours at 370C . The MTT-containing medium was removed and the resulting formazan metabolite was solubilized in 100% ethanol with shaking for 15 minutes at room temperature. Absorbance readings were taken in a microtiter plate reader (Dynatech and Molecular Devices plate readers were used interchangeably) at a wavelength of 570 nm with a 650 nm reference. Percent inhibition (% INH) is calculated by subtracting the absorbance of the blank well from all wells, then subtracting the ratio of the average absorbance of each test duplicate (SAVE) by the average of the controls (CAVE) from 1.00. The final number is then multiplied by 100 (% INH = (1.00 - SAVE/CAVE) x 100). The concentration at which 90% inhibition of cell proliferation is obtained (the ICgo) is determined from the linear regression of a plot of the logarithm of the concentration versus percent inhibition.

Example 55

Tablet Formulation

Manufacturing Procedure:

1. Mix Items 1 , 2 and 3 in a suitable mixer for 15 minutes. 2. Granulate the powder mix from Step 1 with 20% Povidone K30 Solution (Item 4). 3. Dry the granulation from Step 2 at 50°C. 4. Pass the granulation from Step 3 through a suitable milling equipment. 5. Add the Item 5 to the milled granulation Step 4 and mix for 3 minutes. 6. Compress the granulation from Step 5 on a suitable press. Example 56

Capsule Formulation

Manufacturing Procedure:

1. Mix Items 1 , 2 and 3 in a suitable mixer for 15 minutes. 2. Add Items 4 & 5 and mix for 3 minutes. 3. Fill into a suitable capsule.

Example 57

Injection Solution/Emulsion Preparation

Manufacturing Procedure:

1. Dissolve item 1 in item 2. 2. Add items 3, 4 and 5 to item 6 and mix until dispersed, then homogenize. 3. Add the solution from step 1 to the mixture from step 2 and homogenize until the dispersion is translucent. 4. Sterile filter through a 0.2 μm filter and fill into vials.

Example 58

Injection Solution/Emulsion Preparation

Manufacturing Procedure:

1. Dissolve item 1 in item 2. 2. Add items 3, 4 and 5 to item 6 and mix until dispersed, then homogenize. 3. Add the solution from step 1 to the mixture from step 2 and homogenize until the dispersion is translucent. 4. Sterile filter through a 0.2 μm filter and fill into vials.

While the invention has been illustrated by reference to specific and preferred embodiments, those skilled in the art will understand that variations and modifications may be made through routine experimentation and practice of the invention. Thus, the invention is intended not to be limited by the foregoing description, but to be defined by the appended claims and their equivalents.