Patent Grant
7615634
The present invention relates to novel 4-aminopyrimidine-5-one derivatives that inhibit cyclin-dependent kinases, most particularly cyclin-dependent kinase 4 (Cdk4). 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 diaminopyrimidines 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 G1, 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 G1-phase and the G1- 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 G1 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 G1 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 G1 phase. For example, the proteins of the INK4 family, including p16INK4a, which block the kinase activity of the Cdk4/cyclin D complex, cause arrest in G1 (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 G1 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. Mol. Physiol.), L941-L949; and Sang, J. et al. Chin. Sci. Bul. 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, pteridinones, 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., 221stNational 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(5), 623-634, WO099/21845, Toogood. P., WO0119825, Toogood P., WO0138315, Reich S. H., WO0179198, Webster, K. U.S. Pat. No. 6,262,096.
The class of diaminopyridimines is represented by compounds of formula
stated to inhibit Cdk4 and FAK3. See WO0012485 (Astra Zeneca).
WO9118887 (Smith Kline Beecham) relates to diaminopyrimidines of formula
that are stated to inhibit gastric secretion.
WO0039101 (Astra Zeneca) relates to pyrimidine compounds of formula
stated to act as anti-cancer agents.
WO0164653 (Astra Zeneca) relates to pyrimidine compounds of the formula
described to act as Cdk inhibitors and FAK inhibitors.
WO0164654 (Astra Zeneca) relates to pyrimidine compounds of formula
described to act as Cdk inhibitors and FAK inhibitors.
Additionally, WO0164656 (Astra Zeneca) relates to pyrimidine compounds of formula
also described as Cdk inhibitors and FAK inhibitors.
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 diaminopyrimidines of the formula
wherein
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;
selected from lower alkyl, halogen and NR5R6,
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;
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
These compounds inhibit cyclin-dependent kinases, most particularly Cdk4. 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 salt or ester thereof.
Finally, this invention also relates to novel intermediate compounds useful in the preparation of a compound of formula I.
Definitions
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 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 N, 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.
“IC50” refers to the concentration of a particular compound according to the invention required to inhibit 50% of a specific measured activity. IC50 can be measured, inter alia, as is described in Example390A, infra.
“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. Ki can be measured, inter alia, as is described in Example 390B, 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 ═O.
“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 (R4° C(═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.
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 I, 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, or OR12 wherein R12 is 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
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;
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;
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. Preferably, R3 is selected from the group CO2R7, COR12 and SO2R15 Most preferably R3 is SO2R15 and R15 is lower alkyl or NR5R6. Preferred R4 groups include H, OR11 and lower alkyl. Preferred R5 and R6groups are those wherein 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.
Examples of compounds of formula I(a) include:
Another preferred embodiment of the invention relates to compounds of formula
wherein R4, R5, R6, R18, R19, R20 and R21 are as defined above.
Examples of such compounds include:
Preferred R5 and R6 groups are independently selected from H, COR12 and SO2R15.
Another preferred embodiment of the invention relates to compounds of formula
wherein R4′, R5, R6, R18, R19, R20 and R21 are as defined above. Examples of such compounds include:
Preferred R4′ groups include H, OR11 and lower alkyl. Preferred R5 and R6 groups are those wherein the group —NR5R6 forms a ring having 3 to 7 ring atoms, said ring optionally including one or more additional N or O ring atoms and optionally being substituted by, OH, oxo, NH2, lower alkyl or lower alkyl substituted by OR12.
The following compounds are also preferred embodiments according to the instant invention:
[4-Amino-2-(4-amino-cyclohexylamino)-pyrimidin-5-yl]-(2, 3-difluoro-6-methoxy-phenyl)-methanone
[4-Amino-2-( 1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2-fluoro-6-methoxy-phenyl)-methanone
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. Suitable processes for synthesizing these compounds are provided in the examples. Generally, compounds of formula I can be prepared according to one of the below described synthetic routes.
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 degrees 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 degrees and about room temperature, preferably at about 0 degrees.
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 1.
As mentioned above, the compounds of the present invention, including the compounds of formula I, 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.
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 II 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: thymidilaate 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 Sci., 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:
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.
To a solution of 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid (1.00 g, 5.40 mmol, Sigma) in dimethylformamide were added O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate (3.06 g, 8.1 mmol, Aldrich), 1-hydroxy-benzotriazole hydrate (1.09 g, 8.1 mmol, Aldrich) followed by diisopropylethylamine (3.94 g, 29.7 mmol) at 0° C. After stirring for 10-15 minutes, N,O-dimethyl-hydroxylamine. HCl (790 mg, 8.1 mmol, Aldrich) was added. The reaction was stirred at 0° C. to room temperature for 2 hours and then diluted with ethyl acetate and water. The aqueous layer was separated 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 1:1 hexane/ethyl acetate to give 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide as a white solid (1.189 g, 98% yield). HRMS, observed: 242.0836; Calcd for M+: 242.0837
A) Preparation of Aryllithium
To a solution of 1-fluoro-3-iodobenzene (11.0 g, 4.975 mmol, Aldrich) in anhydrous tetrahydrofuran (8 mL) at −78° C., was added slowly a solution of n-butyllithium in hexane (1.6 M, 3.42 mL, 5.47 mmol, Aldrich) over 20 minutes. The reaction was stirred at −78° C. for another 30 minutes to give a clear solution which was used in the next step.
B) 4-Amino-2-ethylsulfanylpyrimidine-5-carboxylic acid methoxymethylamide (200 mg, 0.8292 mmol, Example 1) was dissolved in anhydrous tetrahydrofuran (4 mL) and cooled to −78° C. A solution of freshly prepared 3-fluorophenyl lithium (˜3 equiv, from Step A) was added and the orange colored reaction mixture was stirred at −78° C. for 1 to 2 hours until the complete consumption of the starting material. The resulting mixture was quenched with aqueous ammonium chloride solution, extracted with ethyl acetate (3×20 mL), washed with brine (2×10 mL), dried over sodium sulfate and evaporated in vacuo. The residue was purified on silica gel with 80/20→60/40 of hexane/ethyl acetate to give (4-amino-2-ethyl-sulfanylpyrimidin-5-yl)-(3-fluorophenyl)methanone as a light yellow solid (159 mg, 71 % yield). MS (APCI): 278 (M+H).
A solution of (4-amino-2-ethyl-sulfanyl-pyrimidin-5-yl)-(3-fluorophenyl)methanone (141.5 mg, 0.5102 mmol, Example 2) in chloroform (15 mL) was cooled to 0° C. and treated with 3-chloroperoxybenzoic acid (381.6 mg, ˜1.55 mmol, ˜70% purity from Aldrich) and the reaction was stirred at the same temperature for 1.5 hours. The reaction mixture was diluted with methylene chloride (50 mL) and washed with 10% aqueous sodium thiosulfate (2×10 mL), brine, dried and concentrated. The crude product was purified on silica gel with 60:40 of hexane/ethyl acetate to give (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(3-fluoro-phenyl)-methanone as a white solid (118.2 mg, 75% yield). HRMS, observed: 309.0582; Calcd for M+: 309.0583
A suspension of (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(3-fluoro-phenyl)-methanone (20.1 mg, 0.0650 mmol, Example 3), 4-(4-methylpiperazino)aniline (15.8 mg, 0.0826 mmol, Appolo Chemical) and p-toluenesulfonic acid hydrate (15 mg, 0.0777 mmol, Aldrich) in isopropanol (2.5 mL) was placed in a sealed tube and heated at 100 to 110° C. under microwave conditions for 1 hour. The resulting mixture was evaporated in vacuo and the crude product was purified on silica gel with 95:5 of dichloromethane/methanol to give [4-amino-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-pyrimidin-5-yl]-(3-fluoro-phenyl)-methanone as a light yellow solid (16.4 mg, 68% yield). HRMS, observed: 407.1994; Calcd for (M+H)+: 407.1990.
4-Amino-2-ethylsulfanylpyrimidine-5-carboxylic acid methoxymethylamide (200 mg, 0.830 mmol, Example 1) was dissolved in anhydrous tetrahydrofuran (3 mL) and cooled to −78° C. A solution of 2-methoxyphenyl lithium (˜3 equiv, freshly prepared following the same procedure as Step A of Example 2) was added. The reaction was stirred at −78° C. for 1.5 hours and quenched with aqueous ammonium chloride solution. The resulting mixture was extracted with ethyl acetate (3×20 mL), washed with brine (2×10 mL), dried over sodium sulfate and evaporated in vacuo. The residue was purified on silica gel with 60:40 of hexane/ethyl acetate to give (4-amino-2-ethylsulfanylpyrimidin-5-yl)-(2-methoxy-phenyl)-methanone as a white solid (224.1 mg, 94% yield). HRMS, observed: 290.0961; Calcd for (M+H)+: 290.0958.
A solution of (4-amino-2-ethylsulfanylpyrimidin-5-yl)-(2-methoxy-phenyl)-methanone (1.25 g, 4.32 mmol, Example 5) in chloroform (80 mL) was cooled to 0° C. and treated with 3-chloroperoxybenzoic acid (2.8 g, ˜12.5 mmol, ˜77% purity from Aldrich) and the reaction was stirred at the same temperature for 1.5 hours. The reaction mixture was diluted with methylene chloride (150 mL) and washed with 10% aqueous sodium thiosulfate (2×20 mL), brine, dried and concentrated. The crude product was purified on silica gel with 60:40 of hexane/ethyl acetate to give (4-amino-2-ethanesulfonylpyrimidin-5-yl)-(2-methoxyphenyl)-methanone as a white solid (1.06 g, 76% yield). HRMS, observed: 344.0679; Calcd for (M+Na)+: 344.0675.
A suspension of (4-amino-2-ethanesulfonylpyrimidin-5-yl)-(2-methoxyphenyl)-methanone (20.0 mg, 0.062 mmol, Example 6), 4-(4-methylpiperazino)aniline (16.6 mg, 0.087 mmol, Appollo Chemical) and p-toluenesulfonic acid hydrate (15 mg, 0.0777 mmol, Aldrich) in isopropanol (2.5 mL) was placed in a sealed tube and heated at 100° C. under microwave conditions for 1 hour. The resulting mixture was concentrated in vacuo and the crude product was purified on silica gel with 95:5 of dichloromethane/methanol to give [4-amino-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-pyrimidin-5-yl]-(2-methoxyphenyl)-methanone as a light yellow solid (10.6 mg). HRMS, observed: 419.2190; Calcd for (M+H)+: 419.2195.
A suspension of (4-amino-2-ethanesulfonylpyrimidin-5-yl)-(2-methoxyphenyl)-methanone (26.6 mg, 0.0828 mmol, Example 6) and 4-(4-isopropylpiperazin-1-yl)phenylamine (19.5 mg, 0.0889 mmol) in isopropanol (2.5 mL) was placed in a sealed tube and heated at 110° C. under microwave conditions for 1 hour. The resulting mixture was concentrated and the crude product was purified on silica gel with 95:5 of dichloromethane/methanol to give [4-amino-2-[4-(4-isopropyl-piperazin-1-yl)-phenylamino]-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone as a light yellow solid (15.6 mg, 42% yield). HRMS, observed: 447.2507; Calcd for (M+H)+: 447.2503.
The 4-(4-isopropylpiperazin-1-yl)phenylamine was prepared as follows:
A mixture of 1-(4-nitrophenyl)piperazine (2.00 g, 9.7 mmol) (Acros Organics), powdered potassium carbonate (2.7 g, 19.3 mmol), 2-bromopropane (0.96 mL, 10.1 mmol), potassium iodide (50 mg) and a catalytic amount of 18-crown-6 in acetonitrile (15 mL) was heated at reflux overnight. The mixture was filtered, and the filter cake was washed with acetonitrile. The solvent was evaporated from the combined filtrates and the residue was partitioned between water and ethyl acetate. The layers were separated and the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried (MgSO4), filtered, evaporated and chromatographed (0-66% acetone/dichloromethane) to give 1-(1-methylethyl)-4-(4-nitrophenylpiperazine).
A mixture of 1-(1-methylethyl)-4-(4-nitrophenylpiperazine) (1.7 g, 6.8 mmol) (prepared as above) and 10% palladium-on-charcoal in ethanol (30 mL) was hydrogenated at room temperature and atmospheric pressure overnight. The catalyst was filtered off and the filter cake washed thoroughly with ethanol. The mixture was evaporated under reduced pressure to give 4-(4-isopropylpiperazin-1-yl)phenylamine as a dark brown oil. CDK4 IC50=0.171 μM; CDK1 IC50=3.564 μM; CDK2 IC50=10.000 μM; HCT 116 IC90=20.890 μM.
A suspension of (4-amino-2-ethanesulfonylpyrimidin-5-yl)-(2-methoxyphenyl)-methanone (20.0 mg, 0.0650 mmol, Example 6) and ethyl 4-amino-1-piperidinecarboxylate (15.1 mg, 0.0876 mmol, Aldrich) in isopropanol (2.5 mL) was placed in a sealed tube and heated at ˜100-110° C. under microwave conditions for ˜1 hour and the resulting mixture was evaporated in vacuo. The crude product was purified on silica gel with 95:5 of dichloromethane/methanol to give 4-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid ethyl ester as a light yellow solid (22.1 mg, 89% yield). HRMS, observed: 400.1984; Calcd for (M+H)+: 400.1980.
The same procedure was used as described in Example 9 starting from (4-amino-2-ethanesulfonylpyrimidin-5-yl)-(2-methoxyphenyl)-methanone (125.4 mg, 0.3902 mmol, Example 6) and 4-amino-1-Boc-piperidine (101.0 mg, 0.484 mmol, Astatech) to give 4-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid tert-butyl ester (158.2 mg, 95% yield). HRMS, observed: 428.2296; Calcd for (M+H)+: 428.2293.
4-[4-Amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid tert-butyl ester (1.96 g, Example 10) was dissolved in dichloromethane (25 mL), cooled to 0° C. and treated with trifluoroacetic acid (12.5 mL). After stirring 1 hour at 0° C., the reaction mixture was concentrated in vacuo to give 4.29 g of [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone as the trifluoroacetic acid salt. A portion of the crude product was purified on HPLC and then neutralized with sodium carbonate, washed with brine and dried to give [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone free base. -. HRMS, observed: 328.1771; Calcd for (M+H)+: 328.1768.
The same procedure was used as described in Example 2, starting from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methylamide (200 mg, 0.830 mmol, Example 1) and a solution of 2,6-difluorophenyl lithium (˜3 equiv, freshly prepared from 1-bromo-2,6-difluorobenzene (Aldrich), as in Example 2A), to give (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2,6-difluoro-phenyl)-methanone as a white solid (170 mg, 70% yield). HRMS, observed:296.0667; Calcd for (M+H)+: 296.0664.
The same procedure as described in Example 3, starting from (4-amino-2-ethyl-sulfanyl-pyrimidin-5-yl)-(2,6-difluoro-phenyl)-methanone, Example 12, was used to give (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(2,6-difluoro-phenyl)-methanone as a white solid (78% yield). HRMS, observed: 350.0385; Calcd for (M+Na)+: 350.0381.
The same procedure as described in Example 7 was used, starting from (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(2,6-difluoro-phenyl)-methanone (18.4 mg, 0.0565 mmol, Example 13) to give [4-amino-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-pyrimidin-5-yl]-(2,6-difluoro-phenyl)-methanone. H RMS, observed: 425.1901; Calcd for (M+H)+: 425.1896.
The same procedure as described in Example 8, starting from (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(2,6-difluoro-phenyl)-methanone, Example 13, was used to give [4-amino-2-[4-(4-isopropyl-piperazin-1-yl)-phenylamino]-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone. HRMS, observed: 453.2213; Calcd for (M+H)+: 453.2209.
The same procedure as described in Example 9 was used, starting from (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(2,6-difluoro-phenyl)-methanone, Example 13, to give 4-[4-amino-5-(2,6-difluoro-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid ethyl ester (158.2 mg, 95% yield). HRMS, observed: 406.1690; Calcd for (M+H)+: 406.1685.
To a solution of [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone trifluoroacetic acid salt (80.9 mg, Example 11) in methylene chloride (4 mL) were added triethylamine (0.095 mL, 0.681 mmol, Aldrich) and methyl chloroformate (9.0 mg, 0.0868 mmol, Aldrich) at 0° C. The mixture was stirred for 1 hour at 0° C., the reaction mixture was concentrated in vacuo and crude product was purified on silica gel with 95:5 of dichloromethane/methanol to give 4-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid methyl ester (27.0 mg). HRMS, observed: 386.1827; Calcd for (M+H)+: 386.1823.
The same procedure as described in Example 17 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 11, and propylchloroformate (Aldrich), to afford 4-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid propyl ester. HRMS, observed: 414.2140; Calcd for (M+H)+: 414.2136.
The same procedure as described in Example 17 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 11, and isobutyl chloroformate (Aldrich), to afford 4-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid iso-butyl ester. HRMS, observed: 428.2295; Calcd for (M+H)+: 428.2293
To a solution of [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone trifluoroacetic acid salt, (31 mg, Example 11) in dimethylformamide (2 mL) were added triethylamine (19 mg, 0.193 mmol, Aldrich), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (6.6 mg, 0.0344 mmol, Aldrich), 1-hydroxybenzotriazole hydrate (5.0 mg, 0.037 mmol, Aldrich), followed by acetic acid (2.3 mg, 0.034 mmol). The reaction mixture was stirred overnight at room temperature, taken up in ethyl acetate (15 mL), washed with brine and evaporated. The crude product was purified on silica gel with 95:5 of dichloromethane/methanol to give 1-[4-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-ethanone (11.2 mg). HRMS, observed: 370.1875; Calcd for (M+H)+: 370.1874.
The same procedure as described in Example 20 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 11, and propionic acid (Aldrich), to give 1-[4-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-propan-1-one. H RMS, observed: 384.2034; Calcd for (M+H)+: 384.2030.
The same procedure as described in Example 20 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone trifluoroacetic acid salt Example 11, and butyric acid (Aldrich), to give 1-[4-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-butan-1-one. HRMS, observed: 398.2189; Calcd for (M+H)+: 398.2187.
The same procedure as described in Example 20 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone trifluoroacetic acid salt Example 11 and isobutyric acid (Aldrich), to give 1-[4-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-3-methyl-butan-1-one. HRMS, observed: 412.2350; Calcd for (M+H)+: 412.2343.
The same procedure as described in Example 20 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 11, and diethylaminopropionic acid hydrochloride (Aldrich), to give 1-[4-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-3-diethylamino-propan-1-one. HRMS, observed: 455.2769; Calcd for (M+H)+: 455.2765.
To a solution of [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone trifluoroacetic acid salt (92.4 mg, Example 11) in methylene chloride (3 mL) were added triethylamine (0.12 mL, 0.861 mmol, Aldrich) and methyl isocyanate (6.2 mg, 0.107 mmol, Fluka) at 0° C . After stirring for 1 hour at 0° C., the reaction mixture was concentrated in vacuo and crude product was purified on silica gel with 95:5 of dichloromethane/methanol to give 4-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid methylamide (32.8 mg). HRMS, observed: 385.1985; Calcd for (M+H)+: 385.1983.
The same procedure as described in Example 25 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 11, and ethyl isocyanate (Aldrich), to give 4-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid ethylamide. HRMS, observed: 399.2143; Calcd for (M+H)+: 399.2139.
The same procedure as described in Example 25 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 11, and propyl isocyanate (Aldrich), to give 4-[4-Amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid propylamide. HRMS, observed:413.2299; Calcd for (M+H)+: 413,2296.
To a solution of [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone trifluoroacetic acid salt (66.3 mg, Example 11) in methylene chloride (3 mL) were added triethylamine (0.09 mL, 0.617 mmol, Aldrich) and methanesulfonyl chloride (8.9 mg, 0.0774 mmol, Aldrich) at 0° C. After stirring for 0.5 to 1 hour at 0° C., the reaction mixture was concentrated in vacuo and crude product was purified on silica gel with 95:5 of dichloromethane/methanol to give [4-Amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone (19.2 mg). HRMS, observed: 406.1546; Calcd for (M+H)+: 406.1544.
The same procedure as described in Example 28 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 11, and ethanesulfonyl chloride (Aldrich), to give [4-amino-2-(1-ethanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone. HRMS, observed: 420.1704; Calcd for (M+H)+: 420.1700.
The same procedure as described in Example 28 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 11, and propanesulfonyl chloride (Aldrich), to give [4-amino-2-[1-(propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone. HRMS, observed: 434.1860; Calcd for (M+H)+: 434.1857.
The same procedure was used as described in Example 2, starting from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide (from Example 1) and a solution of 2-tolyllithium (˜3 equiv, freshly prepared from 2-iodotoluene, as in Example 2A), to give (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-o-tolyl-methanone as a white solid. MS (M+H)+: 274.
The same procedure as described in Example 3 was used, starting from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-o-tolyl-methanone, Example 31, to give (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-o-tolyl-methanone as a white solid. HRMS, observed: 306.0910; Calcd for (M+H)+: 306.0907.
The same procedure as described in Example 7 was used, starting from (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-o-tolyl-methanone, Example 32, to give [4-amino-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-pyrimidin-5-yl]-o-tolyl-methanone. MS (M+H)+, 403.
The same procedure as described in Example 8 was used, starting from (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-o-tolyl-methanone, Example 32, to give [4-amino-2-[4-(4-isopropyl-piperazin-1-yl)-phenylamino]-pyrimidin-5-yl]-o-tolyl-methanone. HRMS, observed: 431.2557; Calcd for (M+H)+: 431.2554.
The same procedure as described in Example 9 was used, starting from (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-o-tolyl-methanone, Example 32, to give 4-[4-amino-5-(2-methyl-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid ethyl ester as a white solid. HRMS, observed:384.2035; Calcd for (M+H)+: 384.2030.
The same procedure was used as described in Example 2, starting from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methylamide (from Example 1) and a solution of 2-fluorophenylilithium (˜3 equiv, freshly prepared from 1-fluoro-2-iodobenzene as in Example 2A), to give (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2-fluoro-phenyl)-methanone. MS (M+H)+: 278.
The same procedure as described in Example 3 was used, starting from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2-fluoro-phenyl)-methanone, Example 36, to give (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(2-fluoro-phenyl)-methanone as a white solid. MS (M+H)+: 310.
The same procedure as described in Example 7 was used, starting from (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(2-fluoro-phenyl)-methanone, Example 37, to give [4-amino-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-pyrimidin-5-yl]-(2-fluoro-phenyl)-methanone. MS (M+H)+407.
The same procedure as described in Example 8 was used, starting from (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(2-fluoro-phenyl)-methanone, Example 37, to give [4-amino-2-[4-(4-isopropyl-piperazin-1-yl)-phenylamino]-pyrimidin-5-yl]-(2-fluoro-phenyl)-methanone. MS (M+H)+, 435.
The same procedure as described in Example 9 was used, starting from (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(2-fluoro-phenyl)-methanone, Example 37, to give 4-[4-amino-5-(2-fluoro-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid ethyl ester as a white solid. MS (M+H)+, 388.
[4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2-fluoro-phenyl)-methanone, Example 151, was treated as described in Example 28 to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2-fluoro-phenyl)-methanone. MS (M+H)+, 394.
The same procedure as described in Example 29 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2-fluoro-phenyl)-methanone, Example 151, to give [4-amino-2-(1-ethanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2-fluoro-phenyl)-methanone. MS (M+H)+, 408.
The same procedure was used as described in Example 2, starting from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methylamide (from Example 1) and a solution of 2-trifluoromethylphenylilithium (˜3 equiv, freshly prepared from 1-iodo-2-trifluoromethylbenzene as in Example 2A), to give (4-Amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2-trifluoromethyl-phenyl)-methanone. MS (M+H)+: 328.
The same procedure as described in Example 3 was used, starting from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2-trifluoromethyl-phenyl)-methanone, Example 43, to give (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(2-trifluoromethyl-phenyl)-methanone as a white solid. MS (M+H)+: 360.
The same procedure as described in Example 7 was used, starting from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2-trifluoromethyl-phenyl)-methanone, Example 43, to give [4-amino-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-pyrimidin-5-yl]-(2-trifluoromethyl-phenyl)-methanone. MS (M+H)+, 457.
The same procedure as described in Example 8 was used, starting from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2-trifluoromethyl-phenyl)-methanone, Example 43, to give [4-amino-2-[4-(4-isopropyl-piperazin-1-yl)-phenylamino]-pyrimidin-5-yl]-(2-trifluoromethyl-phenyl)-methanone. MS (M+H)+, 485
A solution of 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methylamide (400 mg, 1.65 mmol, Example 1) was dissolved in anhydrous tetrahydrofuran (6 mL) and cooled to −78° C. A solution of the freshly prepared 2-methoxyl-5-fluorophenyl lithium (˜5 equiv, prepared following the same procedure as described in Step A of Example 2) was added over 10-30 minutes and the orange colored reaction mixture was stirred at −78 to 0° C. for 1-3 hours until the complete consumption of the starting material. The resulting mixture was quenched with aqueous ammonium chloride solution, extracted with ethyl acetate (3×20 mL), washed with brine (2×10 mL), dried over sodium sulfate and evaporated in vacuo. The residue was purified on silica gel with 80/20→60/40 of hexane/ethyl acetate to give (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-phenyl)-methanone as a white solid (277 mg). MS (M+H)+, 308.
The same procedure as described in Example 3 was used, starting with (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-phenyl)-methanone, Example 47, to give (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-phenyl)-methanone as a white solid. MS (M+H)+: 340.
The same procedure as described in Example 7 was used, starting with (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-phenyl)-methanone, Example 47, to give [4-amino-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone. MS (M+H)+, 437.
The same procedure as described in Example 8 was used, starting with (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-phenyl)-methanone, Example 47, to give [4-amino-2-[4-(4-isopropyl-piperazin-1-yl)-phenylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone. MS (M+H)+: 465.
The same procedure as described in Example 9 was used, starting with (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-phenyl)-methanone, Example 47, to give 4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid ethyl ester as a white solid. MS (M+H)+, 418.
The same procedure was used as described in Example 9 starting from (4-amino-2-ethanesulfonylpyrimidin-5-yl)-(2-methoxyphenyl)-methanone, Example 6, and 3-amino-piperidine-1-carboxylic acid tert-butyl ester (Astatech) to give 3-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid tert-butyl ester. MS (M+H)+, 428.
3-[4-Amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid tert-butyl ester (76 mg, Example 52) was dissolved in dichloromethane (4 mL), cooled to 0° C. and treated with trifluoroacetic acid (1 mL). After stirred 30 minutes at 0° C., the reaction mixture was concentrated in vacuo to give [4-amino-2-(piperidin-3-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone as a trifluoroacetic acid salt. A portion of this material was partitioned between ethyl acetate and aqueous sodium carbonate to give the free base. (129.5 mg). MS (M+H)+, 328.
To a solution of [4-amino-2-(piperidin-3-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone trifluoroacetic acid salt (42 mg, Example 53) in dichloromethane (3 mL) were added triethylamine (48 mg, Aldrich) and methyl chloroformate (6.1 mg, Aldrich) at 0° C. After stirring for 1 hour at 0° C., the reaction mixture was concentrated in vacuo and crude product was purified on silica gel with 95:5 of dichloromethane/methanol to give 3-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid methyl ester (20.5 mg). MS (M+H)+, 386. CDK4 IC50=0.459 μM; CDK1 IC50=0.469 μM; CDK2 IC50=0.713 μM; HCT 116 IC90=6.437 μM.
The same procedure as described in Example 54 was used, starting with [4-amino-2-(piperidin-3-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 53, and ethyl chloroformate (Aldrich) to give 3-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid ethyl ester. MS (M+H)+, 400.
The same procedure as described in Example 20 was used, starting with [4-amino-2-(piperidin-3-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 53, to give 1-[3-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-ethanone. MS (M+H)+, 370.
The same procedure as described in Example 25 was used, starting with [4-amino-2-(piperidin-3-ylamino)-pyrimidin-5-yl]-(2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 53, and dimethylcarbamyl chloride (Aldrich) to give 4-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid dimethylamide. MS (M+H)+, 399.
The same procedure was used as described in Example 10 starting from (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-phenyl)-methanone (1.01 g, 2.979 mmol, Exam pie 48), to give 4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid tert-butyl ester (921.7 mg) (which is the same compound that is prepared according to the process of Example 164). MS (M+H)+, 446.
4-[4-Amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid tert-butyl ester (839.6 mg, Example 58) was dissolved in dichloromethane (11 mL), cooled to 0° C. and treated with trifluoroacetic acid (5.5 mL). After stirring ˜1 minute at 0° C., the reaction mixture was concentrated in vacuo to give the product as a trifluoroacetic acid salt (1.76 g). A portion of this salt (1.00 g) was dissolved in ethyl acetate, neutralized with excess aqueous sodium carbonate solution, washed with brine, dried and concentrated in vacuo to give [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (423.7 mg). MS (M+H)+, 346.
To a solution of [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone as a trifluoroacetic acid salt (57.1 mg, Example 59) in dichloromethane (2 mL) were added triethylamine (58 mg, 8 equiv, Aldrich) and methyl chloroformate (6.7 mg, 1 equiv, Aldrich) at 0° C. After stirring for ˜2 hours at 0° C., the reaction mixture was concentrated in vacuo and crude product was purified on silica gel with 95:5 of dichloromethane/methanol to give 4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid methyl ester (23.7 mg). MS (M+H)+, 404.
The same procedure as described in Example 60 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 59, and propyl chloroformate (Aldrich) to give 4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid propyl ester. MS (M+H)+, 432.
The same procedure as described in Example 60 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 59, and acetyl chloride (Aldrich) to give 1-[4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-ethanone. MS (M+H)+, 388.
The same procedure as described in Example 60 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 59, and propionyl chloride (Aldrich) to give 1-[4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-propan-1-one. MS (M+H)+, 402.
The same procedure as described in Example 60 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 59, and butyryl chloride (Aldrich) to give 1-[4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-butan-1-one. MS (M+H)+, 416.
The same procedure as described in Example 60 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 59, and methanesulfonyl chloride (Aldrich), to give [4-amino-2-( 1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone. MS (M+H)+, 424.
The same procedure as described in Example 60 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 59, and ethanesulfonyl chloride (Aldrich), to give [4-amino-2-( 1-ethanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone. MS (M+H)+, 438.
The same procedure as described in Example 60 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 59, and 1-propanesulfonyl chloride (Aldrich), to give [4-amino-2-[ 1-(propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-methoxy-phenyl)-methanone. MS (M+H)+, 452.
The same procedure as described in Example 60 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 59, and trifluoromethanesulfonyl chloride (Aldrich), to give [4-amino-2-(1-trifluoromethanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone. MS (M+H)+, 478.
The same procedure was used as described in Example 9 starting from (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-phenyl)-methanone (400 mg, 1.18 mmol, Example 48) and (R)-3-amino-pyrrolidine-1-carboxylic acid tert-butyl ester (233 mg,1.25 mmol, Astatech), to give (R)-3-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-pyrrolidine-1-carboxylic acid tert-butyl ester (447 mg). MS (M+H)+, 432.
The same procedure as described in Example 59 was used, starting from (R)-3-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-pyrrolidine-1-carboxylic acid tert-butyl ester, Example 69, to give (R)-[4-amino-2-(pyrrolidin-3-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone as a trifluoroacetic acid salt, and also as the free base. MS (M+H)+, 332.
The same procedure as described in Example 60 was used starting from (R)-[4-amino-2-(pyrrolidin-3-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 70, to give (R)-3-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-pyrrolidine-1-carboxylic acid methyl ester. MS (M+H)+, 390.
The same procedure as described in Example 60 was used, starting from (R)-[4-amino-2-(pyrrolidin-3-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 70, and ethyl chloroformate (Aldrich) to give (R)-3-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-pyrrolidine-1-carboxylic acid ethyl ester. MS (M+H)+, 404.
The same procedure as described in Example 62 was used starting from (R)-[4-amino-2-(pyrrolidin-3-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 70, to give (R)-1-[3-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-pyrrolidin-1-yl]-ethanone. MS (M+H)+, 374
The same procedure as described in Example 63 was used starting from (R)-[4-amino-2-(pyrrolidin-3-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 70, to give (R)-1-[3-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-pyrrolidin-1-yl]-propan-1-one. MS (M+H)+, 388.
The same procedure as described in Example 65 was used, starting from (R)-[4-amino-2-(pyrrolidin-3-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 70, to give (R)-[4-amino-2-(1-methanesulfonyl-pyrrolidin-3-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone. MS (M+H)+, 410.
The same procedure as described in Example 66 was used, starting from (R)-[4-amino-2-(pyrrolidin-3-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 70, to give (R)-[4-amino-2-(1-ethanesulfonyl-pyrrolidin-3-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone. MS (M+H)+, 424.
The same procedure was used as described in Example 9 starting from (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-phenyl)-methanone, Example 48, and (S)-3-amino-pyrrolidine-1-carboxylic acid tert-butyl ester (Astatech), to give (S)-3-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-pyrrolidine-1-carboxylic acid tert-butyl ester. MS (M+H)+, 432.
The same procedure as described in Example 59 was used, starting from (S)-3-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-pyrrolidine-1-carboxylic acid tert-butyl ester, Example 77, to give (S)-[4-amino-2-(pyrrolidin-3-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone as a trifluoroacetic acid salt and also as the free base. MS (M+H)+, 332.
The same procedure as described in Example 75 was used, starting from (S)-[4-amino-2-(pyrrolidin-3-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 78, to give (S)-[4-amino-2-(1-methanesulfonyl-pyrrolidin-3-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone. MS (M+H)+, 410.
The same procedure as described in Example 76 was used, starting from (S)-[4-amino-2-(pyrrolidin-3-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 78, to give (S)-[4-amino-2-(1-ethanesulfonyl-pyrrolidin-3-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone. MS (M+H)+, 424.
To a solution of N,N-dimethylaminoacetic acid (9.8 mg, 0.095 mmol, Aldrich) in dimethylformamide (2 mL) were added 1-hydroxybenzotriazole hydrate (16.5 mg, 0.108 mmol, Aldrich), O-benzotriazol-1-yl-N,N, N′,N′-tetramethyluronium hexafluorophosphate (40.8 mg, 0.108 mmol, Aldrich). After it was stirred for 20 minutes, a solution of [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt (47 mg, Example 59) and triethylamine (47 mg, 0.469 mmol, Aldrich) in dimethylformamide (2 mL) were added. The mixture was stirred overnight at room temperature before it was concentrated, taken up in ethyl acetate, washed with brine and evaporated. The crude product was purified on HPLC to give 1-[4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-2-dimethylamino-ethanone (12.5 mg). MS (M+H)+: 431.
The same procedure as described in Example 81 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 59, and N,N-diethylaminoacetic acid (Aldrich) to give 1-[4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-2-diethylamino-ethanone. MS (M+H)+: 459.
The same procedure as described in Example 81 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 59, and morpholin-4-yl-acetic acid (Aldrich) to give 1-[4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-2-morpholin-4-yl-ethanone. MS (M+H)+: 473.
The same procedure as described in Example 81 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 59, and dimethylaminopropionic acid (TCI-US) to give 1-[4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-3-dimethylamino-propan-1-one. MS (M+H)+: 445.
The same procedure as described in Example 81 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 59, and diethylaminopropionic acid hydrochloride (Aldrich) to give 1-[4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-3-diethylamino-propan-1-one. MS (M+H)+: 473.
The same procedure as described in Example 81 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 59, and 1-piperidine propionic acid (Aldrich) to give 1-[4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-3-piperidin-1-yl-propan-1-one. MS (M+H)+: 485.
The same procedure as described in Example 81 was used, starting with 3-morpholin-4-yl-propionic acid (prepared by the method of Kempf, Dale J., et al., J. Med. Chem.; 36:3, 1993, pp 320-330), to give 1-[4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-3-morpholin4-yl-propan-1-one. MS (M+H)+: 487.
The same procedure as described in Example 47 was used, starting from 4-amino-2-ethylsulfanylpyrimidine-5-carboxylic acid, Example 1, and 2-bromo-4,6-difluoroanisole (Astatech), to give (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2,6-difluoro-3-methoxy-phenyl)-methanone as a white solid. MS (M+H)+, 326.
The same procedure as described in Example 3 was used, starting with (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2,6-difluoro-3-methoxy-phenyl)-methanone, Example 88, to give (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(2,6-difluoro-3-methoxy-phenyl)-methanone as a white solid. MS (M+H)+: 358.
A suspension of (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(2,6-difluoro-3-methoxy-phenyl)-methanone (20.0 mg, 0.056 mmol, Example 89) and 1-(4-amino-piperidin-1-yl)-ethanone (21.1 mg, 0.148 mmol, prepared as described in U.S. Pat. No. 5,817,828) in isopropyl alcohol (2.5 mL) was heated at 120° C. in a sealed tube under microwave conditions for 0.3 to 1 hour. The resulting reaction mixture was evaporated in vacuo and crude product was purified on silica gel with 95:5 of dichloromethane/methanol to give 1-[4-[4-amino-5-(2,6-difluoro-3-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-ethanone as a white solid (18.3 mg). MS (M+H)+, 406.
The same procedure as described in Example 28 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2,6-difluoro-3-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 93, to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2,6-difluoro-3-methoxy-phenyl)-methanone. MS (M+H)+, 442.
The same procedure was used as described in Example 90 starting from (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(2,6-difluoro-3-methoxy-phenyl)-methanone, Example 89, and 4-amino-1-Boc-piperidine (Astatech) to give 4-[4-amino-5-(2,6-difluoro-3-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid tert-butyl ester. MS (M+H)+, 464.
The same procedure was used as described in Example 59 starting from 4-[4-amino-5-(2,6-difluoro-3-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid tert-butyl ester, Example 92, to give [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2,6-difluoro-3-methoxy-phenyl)-methanone as a trifluoroacetic acid salt, and also as the free base. MS (M+H)+, 364.
The same procedure was used as described in Example 60, starting with [4-amino-2-( piperidin-4-ylamino)-pyrimidin-5-yl]-(2,6-difluoro-3-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 93, to give 4-[4-amino-5-(2,6-difluoro-3-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid methyl ester. MS (M+H)+, 422
The product of the reaction of 2-iodophenol (Aldrich) and ethyl iodide in hot acetone containing potassium carbonate was treated with n-butyl lithium as described in Example 2A and subsequently reacted with 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide Example 1, using the procedure of Example 47 to give (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2-ethoxy-phenyl)-methanone as a white solid. MS (M+H)+, 304
The same procedure as described in Example 3 was used, starting from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2-ethoxy-phenyl)-methanone, Example 95, to give (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(2-ethoxy-phenyl)-methanone as a white solid. MS (M+H)+: 336.
The same procedure as described in Example 90 was used, starting with (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(2-ethoxy-phenyl)-methanone, Example 96, to give 1-[4-[4-amino-5-(2-ethoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin- 1-yl]-ethanone as a white solid. MS (M+H)+: 384.
A solution of 2-bromo4,5-difluoro-anisole (1.95 g, 8.75 mmol, Astatech) in a mixture of pentane/tetrahydrofuran (5 mL/17 mL) was cooled to −72° C. and treated with n-butyl lithium (2.5 M in hexane, 3.6 mL, 9.0 mmol). After stirring for 10 minutes, a solution of 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide (0.78 g, 3.21 mmol, Example 1) in tetrahydrofuran (3 mL) was added. The temperature was slowly raised to −35° C. for ˜2 hours. The reaction was quenched at −35° C. with 5% aqueous ammonium chloride solution and the mixture was diluted with ethyl acetate/hexane (100 mL). The organic phase was separated, washed with 1:1 water/saturated aqueous sodium chloride solution and dried (Na2SO4). The residue after solvent removal was purified by HPLC chromatography (Waters Prep 500 eluting with 35% ethyl acetate/hexane) to give, in approximately 1:1 ratio, the two positional isomers (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2,3-difluoro-6-methoxy-phenyl)-methanone identical to material prepared in Example 102, and (4-Amino-2-ethylsulfanyl-pyrimidin-5-yl)-(4,5-difluoro-2-methoxy-phenyl)-methanone as a white solid after evaporation. HRMS, observed: 326.0772; Calcd for M+: 326.0770.
The same procedure as described in Example 3 was used, starting with (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(4,5-difluoro-2-methoxy-phenyl)-methanone, Example 98, to give (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(4,5-difluoro-2-methoxy-phenyl)-methanone as a white solid. MS (M+H)+: 358.
The same procedure as described in Example 90 was used, starting with (4-amino-2-ethanesulfonyl -pyrimidin-5-yl)-(4,5-difluoro-2-methoxy-phenyl)-methanone, Example 99, to give 1-[4-[4-amino-5-(4,5-difluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-ethanone as a white solid. MS (M+H)+: 406.
The same procedure as described in Example 90 was used, starting with (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(4,5-difluoro-2-methoxy-phenyl)-methanone, Example 99, and 1-methanesulfonyl-piperidin-4-ylamine (compound with trifluoro-acetic acid), Example 162, to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(4,5-difluoro-2-methoxy-phenyl)-methanone as a white solid. MS (M+H)+: 442.
The same procedure as described in Example 47 was used, starting from 4-amino-2-ethylsulfanylpyrimidine-5-carboxylic acid methoxymethylamide, Example 1, and 2-bromo4,5-difluoro-anisole (Astatech), to give (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2,3-difluoro-6-methoxy-phenyl)-methanone as a white solid. MS (M+H)+, 326.
The same procedure as described in Example 3 was used, starting with (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2,3-difluoro-6-methoxy-phenyl)-methanone, Example 102, to give (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(2,3-difluoro-6-methoxy-phenyl)-methanone as a white solid. MS (M+H)+: 358.
The same procedure as described in Example 90 was used, starting from (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(2,3-difluoro-6-methoxy-phenyl)-methanone, Example 103, to give 1-[4-[4-amino-5-(2,3-difluoro-6-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-ethanone as a white solid. MS (M+H)+: 406.
The same procedure as described in Example 90 was used, starting from (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(2,3-difluoro-6-methoxy-phenyl)-methanone, Example 103, and 1-methanesulfonyl-piperidin-4-ylamine (with trifluoro-acetic acid), Example 162, to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone. MS (M+H)+, 442.
The same procedure as described in Example 92 was used, starting from (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(2,3-difluoro-6-methoxy-phenyl)-methanone, Example 103, to give 4-[4-amino-5-(2,3-difluoro-6-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid tert-butyl ester identical to material prepared in Example 166. MS (M+H)+, 464.
The same procedure was used as described in Example 59, using material from Example 106, to give [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone as a trifluoroacetic acid salt, and also as the free base. MS (M+H)+, 364.
The same procedure as described in Example 60 was used, starting from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone trifluoroacetic acid salt, Example 107, to give 4-[4-amino-5-(2,3-difluoro-6-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid methyl ester. MS (M+H)+, 422.
The same procedure as described in Example 47 was used, starting from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide, Example 1, and 2-bromo4-chloroanisole (Aldrich), to give (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(5-chloro-2-methoxy-phenyl)-methanone as a white solid. MS (M+H)+, 324.
The same procedure as described in Example 3 was used, starting from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(5-chloro-2-methoxy-phenyl)-methanone, Example 109, to give (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(5-chloro-2-methoxy-phenyl)-methanone as a white solid. MS (M+H)+: 356.
The same procedure as described in Example 90 was used, starting with (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(5-chloro-2-methoxy-phenyl)-methanone, Example 110, to give 1-[4-[4-amino-5-(5-chloro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-ethanone as a white solid. MS (M+H)+: 404.
The same procedure as described in Example 90 was used, starting with (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(5-chloro-2-methoxy-phenyl)-methanone, Example 110, and 1-methanesulfonyl-piperidin-4-ylamine (with trifluoro-acetic acid), Example 162, to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(5-chloro-2-methoxy-phenyl)-methanone. MS (M+H)+, 404.
(4-Amino-cyclohexyl)-carbamic acid tert-butyl ester (Astatech, 872 mg, 4.0 mmol) was added to a stirred solution of sulfone, (1.03 g, 3.30 mmol, Example 48) in dimethylformamide (10 mL). The mixture was stirred at 90° C. for 1 hour and then cooled to room temperature and poured into water. The solid was filtered and dried and passed through a short pad of silica gel column (eluted with 2% methanol/dichloromethane) to give a pale yellow solid. 1.42 g, 94%. MS (M+H)+=460.
Trans-[4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-cyclohexyl]-carbamic acid tert-butyl ester (1.32 g) obtained from Example 113 was dissolved in 50% trifluoroacetic acid/dichloromethane (30 mL) and the solution was stirred at room temperature for 30 minutes. The solvent was removed and the residue was treated with saturated aqueous sodium bicarbonate solution. The mixture was extracted three times with ethyl acetate/tetrahydrofuran (3:1, 30 mL). The extracts were combined, dried (Na2SO4) and concentrated under reduced pressure to a solid (980 mg). Yield, 95%. MS(ES) (M+H)+=360.
[4-Amino-2-(4-amino-cyclohexylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (250 mg, 0.70 mmol, Example 114) was dissolved in tetrahydrofuran (8 mL) and the solution was cooled to 0° C. To the stirred solution, triethylamine (81 mg, 0.80 mmol) and methanesulfonylchloride (Aldrich, 81 mg, 0.80 mmol) were added successively. The reaction was quenched 5 minutes later with saturated aqueous sodium bicarbonate solution (15 mL) and the mixture was extracted with methylene chloride (3×15 mL). The extracts were dried (Na2SO4) and concentrated to give a solid, which was chromatographed on silica gel (2.5% methanol in dichloromethane) to give a white solid. 195.08 mg. 64%. MS(ES) (M+H)+=438.
The title compound was made by a similar procedure as for Example 115 using [4-amino-2-(4-amino-cyclohexylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone, Example 114, and ethanesulfonyl chloride (Aldrich). MS(ES) (M+H)+=452.
The title compound was made by a similar procedure as for Example 115 using [4-amino-2-(4-amino-cyclohexylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone, Example 114, and ethylchloroformate (Aldrich). MS(ES) (M+H)+=432.
The title compound was made by a similar procedure as for Example 115 using [4-amino-2-(4-amino-cyclohexylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone, Example 114, and 2-methoxyethylchloroformate (TCI-US). MS(ES) (M+H)+=462.
The title compound was made by a similar procedure as for Example 115 using [4-amino-2-(4-amino-cyclohexylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone, Example 114, and acetic anhydride (Aldrich). MS(ES) (M+H)+=402.
The title compound was prepared as in Example 113 and Example 114 using (4-amino-cyclohexyl)-carbamic acid tert-butyl ester (Altech) and (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(2,3-difluoro-6-methoxy-phenyl)-methanone Example 103. MS (M+H)+=378.
The title compound was made by a similar procedure as for Example 119 using trans-[4-amino-2-(4-amino-cyclohexylamino)-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone Example 120, and acetic anhydride (Aldrich). MS(ES) (M+H)+=420.
A. (4-Methanesulfonylamino-cyclohexyl)-carbamic acid tert-butyl ester
To a stirred solution of (4-amino-cyclohexyl)-carbamic acid tert-butyl ester (Altech, 1.24 g, 5.79 mmol) in methylene chloride (50 mL) at 0° C., triethylamine (202 mg, 6.95 mmol) and methansulfonylchloride (Aldrich, 796 mg, 6.95 mmol) were added successively and the mixture was stirred for 40 minutes. The solvent was removed and the residue was washed with water to give a white solid, which was directly used for the next step.
B. N-(4-Amino-cyclohexyl)-methanesulfonamide; compound with trifluoro-acetic acid
The compound obtained from step A was dissolved in 50% trifluoroacetic acid/dichloromethane and the mixture was stirred at room temperature for 30 minutes. The excess acid was removed under reduced pressure and the residue was triturated with ether to give a white powder, 1.51 g, 90% for two steps. MS(ES) (M+H)+=193.
To a stirred solution of trans-1,4-diaminocyclohexane (5.12 g, 44.87 mmol, Aldrich) in tetrahydrofuran (200 mL) at 0° C., ethanesulfonylchloride (4.48 mL, 47.11 mmol) was added dropwise and the mixture was stirred for 3 hours. The solid was filtered and washed with tetrahydrofuran (3×50 mL) and dried to give a white solid which was dissolved in saturated aqueous sodium bicarbonate solution and the mixture was extracted with ethyl acetate (3×30 mL). The extracts were combined and dried with sodium sulfate and the solvent was removed to give a white solid. 2.8 g, MS(ES) (M+H)+=207.
To a stirred solution of N-(4-amino-cyclohexyl)-methanesulfonamide, Example 122, (211 mg, 0.69 mmol) in dimethylformamide, triethylamine (40 μL, 2.87 mmol) and the compound of Example 103 (200mg, 0.57 mmol) were added and the mixture was stirred at 90° C. for 1 hour. Then the mixture was poured into water and extracted with ethyl acetate, dried (sodium sulfate) and concentrated. The residue was chromatographed to give the product. 185 mg, 71%. MS(ES) (M+H)+=456.
To a stirred solution of trans-4-amino-cyclohexanol hydrochloride (108 mg, 0.71 mmol, Aldrich) in dimethylformamide, were added sodium bicarbonate(98 mg, 0.92 mmol) and (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-phenyl)-methanone (201 mg, 0.59 mmol, Example 48) and the mixture was stirred at 90° C. for 1 hour. The mixture was poured into water and extracted with ethyl acetate. The extracts were dried with sodium sulfate and concentrated. The residue was chromatographed (2.5% methanol in dichloromethane) to give a white solid. 172 mg, 81%. MS(ES) (M+H)+=361.
To a stirred solution of sulfoxide (300 mg, 0.93 mmol, Example 163), tetrahydro-thiopyran-4-ylamine (170 mg, 1.45 mmol, made by the procedure of K. Ramahingam et al. J. Org. Chem., 1981, 46(22), 4376), was added and the mixture was stirred at 80° C. for 2 hours. The solvent was removed under reduced pressure and the residue was chromatographed (ethyl acetate/hexane, 20% then 40%) to give a light yellow solid. 155 mg, 46%. MS (M+H)+=363.
The thiopyran obtained from Example 126 (85 mg, 0.23 mmol) was dissolved in methylene chloride (10 mL) and cooled to 0° C. To the stirred solution, meta-chloroperoxybenzoic acid (Aldrich, 77%, 80 mg, 0.35 mmol) was added and the mixture was stirred for 90 minutes. The reaction was quenched with 10% aqueous sodium thiosulfate solution and the organic layer was separated and washed with aqueous sodium carbonate solution (10%). The solution was dried with sodium sulfate and the solvent was removed on a rotary evaporator. The residue was chromatographed (50% ethyl acetate/hexanes) to give the product. 31 mg, 34%. MS(ES) (M+H)+=395.
The title compound was made by a similar procedure as for Example 113, using 4-(1-ethyl-piperidin-3-yloxy)-phenylamine and the compound of Example 48. MS(ES) (M+H)+=466.
The 4-(1-ethyl-piperidin-3-yloxy)-phenylamine was prepared as follows:
A. 1-Ethyl-3-(4-nitro-phenoxy)-pyrrolidine
B. 4-(1-Ethyl-pyrrolidin-3-yloxy)-phenylamine
The title compound was made by a similar procedure as for Example 113, using 4-(1-ethyl-pyrrolidin-3-yloxy)-phenylamine (prepared as described above in Example 128) and the compound of Example 48. MS(ES) (M+H)+=452.
The title compound was made by a similar procedure as for Example 113, using 4-(2-dimethylamino-2-methyl-propoxy)-phenylamine and the compound of Example 48. MS(ES) (M+H)+=454.
The 4-(2-dimethylamino-2-methyl-propoxy)-phenylamine was prepared as follows:
A. [1,1-Dimethyl-2-(4-nitro-phenoxy)-ethyl]-dimethyl-amine
B. 4-(2-Dimethylamino-2-methyl-propoxy)-phenylamine
The title compound was made by a similar procedure as for Example 113, using R-4-(2-pyrrolidin-1-yl-propoxy)-phenylamine and the compound of Example 48. MS(ES) (M+H)+=466.
The R-4-(2-pyrrolidin-1-yl-propoxy)-phenylamine was prepared as follows:
A. (R)-1-[1-Methyl-2-(4-nitro-phenoxy)-ethyl]-pyrrolidine
The title compound was made by a similar procedure as for Example 113, using [3-(4-amino-phenyl)-2-hydroxy-propyl]-ethyl-carbamic acid tert-butyl ester and the compound of Example 48. MS(ES) (M+H)+=540.
The [3-(4-amino-phenyl)-2-hydroxy-propyl]-ethyl-carbamic acid tert-butyl ester was prepared by the following procedure: To a stirred solution of 1-allyl4-nitrobenzene (2.85 g, 17.47 mmol; prepared by the procedure of EP 1013636 A, 1999) in dichloromethane (100 mL) at room temperature was added 3-chlorperoxybenzoic acid (6.03 g, 57-86%, Aldrich) and the mixture stirred overnight. The solution was washed with excess aqueous sodium thiosulfate, then with aqueous sodium bicarbonate solution and the organic solution was dried (magnesium sulfate), evaporated, and chromatographed on silica gel giving 1.94 g (62%) of 24-nitorbenzyloxirane as a yellow oil. This oil (0.92 g 5.13 mmol) in methanol was stirred overnight at room temperature with aqueous ethylamine (2 mL, 70% in water, Aldrich). The methanol was removed by evaporation and the residue was dissolved in 1N aqueous hydrochloric acid. The aqueous solution was washed with ether and the aqueous layer was made basic with 4N aqueous sodium hydroxide. Tetrahydrofuran was added to dissolve the solids and di-tert-butyl dicarbonate (1.35 g, 6.16 mmol, Aldrich) was added and the mixture stirred at room temperature for 2 hours. The reaction was extracted with dichloromethane, dried (magnesium sulfate), evaporated and chromatographed on silica gel to give ethyl-[2-hydroxy-3-(4-nitro-phenyl)-propyl]carbamic acid tert-butyl ester as a yellow oil, 1.23 g, 74%. MS(ES) (M+H)+=325. This nitro compound (1.06 g, 3.26 mmol) in ethanol (4 mL) and 10% Palladium on Carbon was hydrogenated at 35 psi for 1.5 hours. The mixture was filtered and evaporated to give [3-(4-amino-phenyl)-2-hydroxy-propyl]-ethyl-carbamic acid tert-butyl ester as a yellow gum, 0.94 g (98%) MS(ES) (M+H)+=295.
The title compound was made by a similar procedure as for Example 113 using 3-(4-amino-phenoxy)-pyrrolidine-1-carboxylic acid tert-butyl ester and the compound of Example 48. MS(ES) (M+H)+=524.
The 3-(4-amino-phenoxy)-pyrrolidine-1-carboxylic acid tert-butyl ester was prepared as follows:
A. 3-(4-Nitro-phenoxy)-pyrrolidine-1-carboxylic acid tert-butyl ester
B. 3-(4-Amino-phenoxy)-pyrrolidine-1-carboxylic acid tert-butyl ester
The title compound was made by a similar procedure as for Example 113, using 4-dimethylamino-aniline (Aldrich) and the compound of Example 48. MS(ES) (M+H)+=382.
The title compound was made by a similar procedure as for Example 113, using 3-aminoethyl-indole (Aldrich) and the compound of Example 48. MS(ES) (M+H)+=406.
The title compound was made by a similar procedure as for Example 113, using N-(3-aminopropyl)-pyridone (Aldrich) and the compound of Example 48. MS(ES) (M+H)+=388.
The title compound was made by a similar procedure as for Example 113, using (4-amino-butyl)-carbamic acid tert-butyl ester (Aldrich) and the compound of Example 48. MS(ES) (M+H)+=334.
The title compound was made by a similar procedure as for Example 113, using (3-amino-propyl)-carbamic acid tert-butyl ester (Aldrich) and the compound of Example 48. MS(ES) (M+H)+=420.
3-(4-Amino-phenoxy)-piperidine-1-carboxylic acid tert-butyl ester (see below) and the compound of Example 48 were treated as in Example 113, the resulting product was then treated as in Example 114, and then as in Example 115 to give [4-amino-2-[4-(1-methanesulfonyl-piperidin-3-yloxy)-phenylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone. MS(ES) (M+H)+=516.
The 3-(4-amino-phenoxy)-piperidine-1-carboxylic acid tert-butyl ester was prepared as follows:
A. 3-(4-Nitro-phenoxy)-piperidine-1-carboxylic acid tert-butyl ester
B. 3-(4-Amino-phenoxy)-piperidine-1-carboxylic acid tert-butyl ester
3-(4-Amino-phenoxy)-piperidine-1-carboxylic acid tert-butyl ester ((prepared as described above in Example 139) and the compound of Example 48 were treated as in Example 113, the resulting product was then treated as in Example 114, and then as in Example 115 to give 3-[4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-phenoxy]-piperidine-1-carboxylic acid ethyl ester. MS(ES) (M+H)+=510.
The title compound was made by a similar procedure as for Example 115 with methanesulfonylchloride (Aldrich) and the compound of Example 137. MS(ES) (M+H)+=412.
The title compound was made by a similar procedure as for Example 115, using 4-fluorophenyl-sulfonyl chloride (Aldrich) and the compound of Example 137. MS(ES) (M+H)+=492.
The title compound was made by a similar procedure as for Example 115 using phenyl-sulfonylchloride (Aldrich) and [4-amino-2-(3-amino-propylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone, which was made from the condensation of (3-amino-propyl)-carbamic acid tert-butyl ester (Aldrich) and the compound of Example 48 following the procedure of Example 113 and Example 114.MS(ES) (M+H)+=460.
To a stirred suspension of the compound of Example 59 in the form of its trifluoroacetic acid salt (45 mg), triethylamine (150 μL, 1.08 mmol) was added followed by 4-dimethylamino-phenyl-isocyanate (Aldrich, 33 mg, 0.20 mmol), and the solution was stirred at room temperature for 1.5 hours. The solution was then concentrated and the residue was purified by reverse phase HPLC to give a solid, (34 mg). MS(ES) (M+H)+=508.
The title compound was made by a similar procedure as used in Example 144 using cyclohexyl isocyanate (Aldrich) and the compound of Example 59 to give 4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid cyclohexylamide. MS(ES) (M+H)+=471.
The title compound was made by a similar procedure as used in Example 144, using diethoxyphosphinyl isocyanate (Aldrich) and the compound of Example 59 to give [4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carbonyl]-phosphoramidic acid diethyl ester. MS(ES) (M+H)+=525.
The title compound was made by a similar procedure as used in Example 144, using ethyl isocyanatoacetate (Aldrich) and the compound of Example 59, to give [4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carbonyl]-amino)-acetic acid ethyl ester. MS(ES) (M+H)+=475.
The title compound was made by a similar procedure as used in Example 115, starting with isopropylsulfonyl chloride (Aldrich) and the compound of Example 59, to give [4-amino-2-[1-(propane-2-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone MS(ES) (M+H)+=452.
The title compound was made by a similar procedure as used in Example 117, using Isopropyl chloroformate (Aldrich) and the compound of Example 113. MS(ES) (M+H)+=446.
The same procedure as described in Example 10 was used, starting from (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(2-fluoro-phenyl)-methanone, Example 37, to give 4-[4-amino-5-(2-fluoro-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid tert-butyl ester. MS (M+H)+, 416.
The same procedure as described in Example 11 was used, starting from 4-[4-amino-5-(2-fluoro-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid tert-butyl ester, Example 150, to give [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2-fluoro-phenyl)-methanone. MS (M+H)+, 315.
To a solution of [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (50 mg, 0.145 mmol, Example 59) and diisopropylethylamine (Aldrich, 330 μL, 1.894 mmol) in anhydrous tetrahydrofuran (15 mL) cooled to 5° C., was added a solution of 2,5-dimethyl-3-thiophenesulfonyl chloride (Maybridge, 45.3 mg, 0.218 mmol) in methylene chloride (3 mL). The reaction mixture was stirred at room temperature overnight and then diluted with 50 mL of methylene chloride. The resulting solution was washed with saturated aqueous sodium bicarbonate (2×10 mL), dried over anhydrous magnesium sulfate and concentrated in vacuo. Purification of the crude residue by flash chromatography (Biotage system, KP-Sil™ 32-63 μm, 60 Å silica gel) eluting with 25% ethyl acetate in hexane yielded [4-amino-2-[1-(2,5-dimethyl-thiophene-3-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (69 mg, 92%) as white solids. HR-MS (ES, m/z) calculated for C23H26FN5O4S2 [(M+H)+] 520.1483, observed 520.1494.
The title compound was prepared from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone, Example 59, and 3-thiophenesulfonyl chloride (Maybridge) by the procedure described in Example 152. HR-MS (ES, m/z) calculated for C21H23FN5O4S2 [(M+H)+] 492.1170, observed 492.1175.
The title compound was prepared from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone, Example 59, and benzo[b]thiophene-3-sulfonyl chloride (Maybridge) by the procedure described in Example 152. HR-MS (ES, m/z) calculated for C25H25FN5O4S2 [(M+H)+] 542.1327, observed 542.1331.
The title compound was prepared from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone, Example 59, and 1,3,5-trimethyl-1H-pyrazole-4-sulfonyl chloride (Maybridge) by the procedure described in Example 152. HR-MS (ES, m/z) calculated for C23H28FN7O4S [(M+H)+] 518.1981, observed 518.1986.
This compound was prepared from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone, Example 59, and 2-carbomethoxy-3-thiophenesulfonyl chloride (Maybridge) by the procedure described in Example 152. HR-MS (ES, m/z) calculated for C23H25FN5O6S2 [(M+H)+]550.1225, observed 550.1232.
A solution of 4-amino-2-methylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide (Example 168) in tetrahydrofuran was cooled to −78° C. and treated as in Example 47. The resulting (4-amino-2-methanesulfanyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-phenyl)-methanone was treated with meta-chloroperoxybenzoic acid (Aldrich) as in Example 165 to give (4-amino-2-methanesulfinyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-phenyl)-methanone (LR-ES) (M+H)+: 310.
A solution of (4-amino-2-methanesulfinyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-phenyl)-methanone (235 mg, 0.73 mmol, Example 157) and tetrahydro-pyran-4-ylamine (Combi-Blocks) (260 mg, 2.3 mmol) in N-methylpyrrolidinone (15 ml) was heated for 30 minutes at 100° C. The solution was cooled, poured into water and extracted into ethyl acetate (2×). The combined organic extracts were washed with 5% sodium bicarbonate and saturated sodium chloride. The solution was dried (Na2SO4) and solvent was removed under vacuum to give a crude solid. Purification was by silica gel chromatography to give [4-amino-2-(tetrahydro-pyran-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone as a white solid. HRMS, observed: 346.1445; Calcd for M+: 346.1441.
A solution of [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (60 mg, 0.17 mmol, Example 59) and bromomethylacetate (60 mg, 0.39 mmol, Aldrich) in dimethylformamide (1.5 mL) was stirred at room temperature for 6 hours. The solution was poured into water and extracted into methylene chloride (2×). 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 silica gel chromatography to give [4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin- 1-yl]-acetic acid methyl ester as a white solid. HRMS, observed: 418.1888; Calcd for M+: 418.1885.
A solution of [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (300 mg, 0.87 mmol, Example 59) and diisopropylethylamine (2 mL) in tetrahydrofuran (80 mL) was stirred at +5° C. To this was added dropwise dimethylsulfamoyl chloride (320 mg, 2.23 mmol, Aldrich) in methylene chloride (15 mL). The reaction was stirred at room temperature for 2 hours, poured into water and extracted into methylene chloride (2×50 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 silica gel chromatography to give 4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-sulfonic acid dimethylamide as a white solid. HRMS, observed: 453.1720; Calcd for M+: 453.1715.
A solution of [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (75 mg, 0.22 mmol, Example 59) and diisopropylethylamine (2 mL) in tetrahydrofuran (40 mL) was stirred at +5° C. To this was added dropwise 3,5-dimethyl-isoxazole-4-sulfonyl chloride (100 mg, 0.51 mmol, Maybridge). The reaction was stirred at room temperature for 2 hours, poured into water and extracted into methylene chloride (2×50 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 silica gel chromatography to give [4-amino-2-[1-(3,5-dimethyl-isoxazole-4-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone as a white solid. HRMS, observed: 505.1669; Calcd for M+: 505.1664.
A) 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° C. 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 (2×50 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
B) A suspension of (1-methanesulfonyl-piperidin-4-yl)-carbamic acid tert-butyl ester (1.14 g, 4.1 mmol, from Example 162, Step A) 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 1.20 g of 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoro-acetic acid (100% yield). HRMS, observed: 177.0692; Calcd for M+: 177.0698
To a stirred solution of (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-phenyl)-methanone (11.1 g, 36 mmol, Example 47) in dichloromethane (300 mL) chilled to 0° C. was added portionwise over about 30 minutes 74% meta-chloroperoxybenzoic acid (10.0 g, 43 mmol). The reaction was followed by thin layer chromatography and when the starting material was consumed (˜45 minutes) the reaction mixture was quenched by the addition of 10% aqueous sodium thiosulfate (20 mL). The organic layer was removed and washed once with 10% aqueous sodium thiosulfate, twice with saturated aqueous sodium carbonate, and once with brine. The organic layer was dried (Na2SO4) and evaporated to give a quantitative yield of a mixture of 9:1 (by NMR) of oxidized products. A small portion of this material was chromatographed on silica gel. Eluting with 1:1 ethyl acetate/hexane provided the minor component, the same as in Example 59, pure (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-phenyl)-methanone as a pale yellow solid, MP 174-175. Mass spectrum (LR-APCI) M+: 339.
Eluting with 9:1 ethyl acetate/methanol provided the major component, (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-phenyl)-methanone as a pale yellow solid, mp 150-152. Mass spectrum (LR-APCI) M+: 323.
A solution composed of a 9:1 mixture of (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-phenyl)-methanone and (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-phenyl)-methanone (11.6 g, 36 mmol, Example 163) and 4-amino-1N-Boc-piperidine (Astatech Inc.) (10.8 g, 54 mmol) in 1-methyl-2-pyrrolidinone (50 mL, Aldrich) was heated at 70° C. for 30 minutes. The reaction was diluted with water and extracted with ethyl acetate. The dried (Na2SO4) organic solution was evaporated and the residue was triturated with ethyl ether/hexane to give 95% yield of 4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid tert-butyl ester as a colorless solid, mp 122-124 dec. Mass Spectrum (LR-ES) M+: 445.
A stirred solution of (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2,3-difluoro-6-methoxy-phenyl)-methanone (7.4 g, 23 mmol, Example 102) in dichloromethane (150 mL) at 0° C. was treated portionwise with 74% meta-chloroperoxybenzoic acid (6 g, 26 mmol) over about 30 minutes. The cold reaction mixture was quenched with 25 ml of 10% aqueous sodium thiosulfate solution and the layers separated. The organic layer was washed once with 10% aqueous sodium thiosulfate solution, twice with saturated aqueous sodium carbonate solution, once with brine, dried (Na2SO4) and evaporated. Trituration of the residue with hexane provided a quantitative yield of (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(2,3-difluoro-6-methoxy-phenyl)-methanone as a colorless solid, mp 165 dec. Mass Spectrum (LR-ES) (M+H)+: 342.
A solution of (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(2,3-difluoro-6-methoxy-phenyl)-methanone (7.7 g, 23 mmol, Example 165) and 4-amino-1N-Boc-piperidine (6.8 g, 34 mmol, Astatech) in 30 mL of 1-methyl-2-pyrrolidinone was heated at 70° C. for 30 minutes. The reaction mixture was diluted with water and extracted with ethyl acetate. The dried (Na2SO4) organic solution was evaporated and the residue was chromatographed on silica gel. Elution with 3:2 ethyl acetate/hexane provided 80% yield of 4-[4-amino-5-(2,3-difluoro-6-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid tert-butyl ester as a colorless solid, mp 114 dec., identical to material prepared in Example 106. Mass Spectrum (LR-ES) M+: 463.
To a stirred solution of 4-amino-2-methylsulfanyl-pyrimidine-5-carboxylic acid ethyl ester (24.0 g, 112.5 mmol, Salor) in ethanol (400 mL) and water (200 mL) was added lithium hydroxide (9.536 g, 225.0 mmol). The suspension was stirred at room temperature for 3 hours during which all the solids went into solution. The ethanol was removed in vacuo and additional water was added to make a total volume of 300 mL. The resulting solution was washed with 2×100 mL of ether which was discarded. To the aqueous solution was added concentrated hydrochloric acid (20 mL) to bring the pH of the aqueous solution to about pH 3 during which the desired product precipitated out. The product was filtered, washed with 25 mL of water, suction dried and then dried in a vacuum oven at 50° C. to give 20.6 g (98.8%) of the product as a white solid.
To a stirred solution of 4-amino-2-methylsulfanyl-pyrimidine-5-carboxylic acid (20.6 g, 111.2 mmol, Example 167) in dimethylformamide (200 mL) was added O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate (63.91 g, 166.8 mmol, Aldrich) and 1-hydroxybenzotriazole hydrate (22.77 g, 166.8 mmol, Aldrich) and the resulting mixture was cooled to 0° C. (ice-water bath). Diisopropylethylamine (108 mL, Aldrich) was added. followed by N,O-dimethylhydroxylamine hydrochloride (16.61 g, 166.8 mmol, Aldrich) and the resulting mixture was stirred at 0° C. for 2 hours. The reaction mixture was diluted with 1 L of ethyl acetate and washed with 800 mL of water. The aqueous phase was removed and extracted with 3×500 mL of ethyl acetate. The combined organic extracts were washed with 500 mL of water, dried over anhydrous magnesium sulfate, filtered and concentrated to give crude product. The crude product was loaded on the top of a 3 inch wide×4 inch long silica-gel column (230400 mesh) and eluted with 20%, 50%, 75% ethyl acetate in hexane and 250 mL fractions were collected. Appropriate fractions containing the product were combined and concentrated to give 21.0 g of product. This was taken in 50 ml ethyl acetate to give a slurry and, while stirring, 200 mL of hexane was added. The product was filtered, washed with 50 mL of hexane, suction dried and then dried in the oven at 40° C. to give 16.3 g (64.2 %) of 4-amino-2-methylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide.
Alternatively, a suspension of 4-amino-2-methylsulfanyl-pyrimidine-5-carboxylic acid (1.01 g, 5.45 mmol, Example 167), N,O-dimethylhydroxylamine hydrochloride (0.59 g, 6.0 mmol, Aldrich), triethylamine (0.84 mL, 6.0 mmol) in dichloromethane (25 mL) was treated with 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (1.25 g, 6.52 mmol, Aldrich). The mixture was stirred at room temperature overnight resulting in a clear solution. The reaction was diluted with ethyl acetate (100 mL), washed with water (2×25 mL), brine, and dried over magnesium sulfate. The dried organic solution was filtered through a plug of silica gel and eluted with ethyl acetate to give, after evaporation, 1.05 g, (85%) of 4-amino-2-methylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide. MS(ES) (M+H)+=229.
n-Butyl lithium (46 mL, 115 mmol, 2.5 M solution in hexanes, Aldrich) was added to a solution of 3,4-difluoroanisole (12.98 mL, 109.5 mmol, Aldrich) in anhydrous tetrahydrofuran (100 mL) cooled to −78° C. using an addition funnel. The resulting solution was warmed to −40 C for 15 min then cooled back to −78° C. before a solution of Example 168 (5 g, 21.9 mmol) in anhydrous tetrahydrofuran (25 mL) was added slowly via syringe. The reaction mixture turned yellow. It was stirred at −78° C. for 30 minutes and then warmed up to −50° C. After stirring at −50° C. for 1 hour, the cooling bath was removed and the reaction mixture was allowed to warm up to −30° C. where it was quenched with a saturated aqueous solution of ammonium chloride. Water was added and the solution was extracted with ethyl acetate, the organic layers were washed with brine, dried (anhydrous sodium sulfate) and concentrated. The residue was triturated in hexanes, and the solids were filtered off. Purification of the solids by flash chromatography (silica gel, eluting with 30%, 40%, 50% ethyl acetate in hexanes) gave the product. The product was recrystallized with ethyl acetate and hexanes to give ˜3.6 g of off-white solids. The filtrate was concentrated, and the residue was purified by flash column chromatography (silica gel, eluting with 100% methylene chloride, then 10% ethyl acetate in methylene chloride) to give an additional amount of product. Total yield: 4.3561 g (64%), off-white solids. MS (M+H)+: 312.
4-Amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide (300 mg, 1.238 mmol, Example 1) in tetrahydrofuran (1 mL) was added slowly via syringe to a stirred, −78° C. solution of 3 equiv. of alkyl lithium, prepared as in Example 2A from 3-fluoroanisole (Aldrich), and the reaction mixture was stirred for 1.5 hours at −78° C. and then 30 minutes at 0° C. The reaction was quenched with saturated aqueous sodium bicarbonate and extracted with ethyl ether. The organic layers were washed with brine, dried and concentrated. Purification was accomplished by chromatography on a Biotage flash column eluting with 20-40% ethyl acetate in hexane to give the product as a yellow oil.
The title compound was prepared in a similar manner as described in Example 163 from the compound of Example 170 and the crude product was used without further purification.
A mixture of 4-Amino-2-ethanesulfinyl-pyrimidin-5-yl)-2-fluoro-6-methoxy-phenyl)-methanone (53.38 mg, 0.165 mmol, Example 171), 1-actyl-4-aminopiperidine (35.19 mg, 0.248 mmol), diisopropylethylamine (0.1437 mL, 0.825 mmol, Aldrich) and ethanol (5 mL) was refluxed for 4 hours and evaporated. The resulting residue was chromatographed eluting with 5% methanol in ethyl acetate containing 0.1% of triethylamine to give the product as a yellow solid. HR-MS (ES) obs. 388.1784, calcd. (M+H)+388.1780.
In a similar manner as that described in Example 170, the title compound was prepared from the compound of Example 1 and the alkyl lithium reagent prepared from the treatment of 2-bromo-5-fluorophenol (Crescent Chem.) with methyl iodide and potassium carbonate in hot acetone followed by treatment of this product with n-butyl lithium as in Example 2A, to give a white solid. LR-MS: (M+H)+308.
The title compound was prepared in a similar manner as described in Example 163 from the compound of Example 173 and the crude product was used without further purification.
The title compound was prepared in a similar manner as described in Example 172 from the material prepared in Example 174 (136 mg, 0.421 mmol) and 4-amino-1-Boc-piperidine (126.5 mg, 0.631 mmol, Astatech) to give, after chromatography, 70 mg (38%) of product as a white glass. LR-MS: (M+H)+446
The compound of Example 175 (70 mg) in dichloromethane (2 mL) and trifluoroacetic acid (2 mL) was stirred for 2 hours at room temperature and evaporated to give the title compound (72 mg) as the trifluoroacetic acid salt.
The trifluoroacetic acid salt of [4-amino-2-(piperidin-4-ylamino)pyrimidin-5-yl-(4-fluoro-2-methoxy-phenyl)-methanone (72.18 mg, 0.157 mmol, Example 176) in dichloromethane (5 mL) and triethylamine (0.2188 mL, 1.570 mmol) was chilled and methanesulfonyl chloride (0.017 mL, 0.22 mmol, Aldrich) was added and the mixture was stirred at room temperature for several hours. Chromatography provided 42 mg (63%) of the title compound as a white solid. HR-ES(+), observed: 424.1453; Calcd for (M+H)+: 424.1450.
The title compound was prepared in a similar manner as described in Example 170 from 1-fluoro-3,5-dimethoxybenzene (Aldrich) to give the product as a yellow oil.
The title compound was prepared in a similar manner as described in Example 173 from Example 177 and the crude product was obtained in 100% yield and used without further purification.
The title compound was prepared in a similar manner as described in Example 175 from material prepared in Example 179 and the product was chromatographed on silica gel using 80% ethyl acetate/hexane as the eluting solvent to give product in 86% yield.
The compound of Example 180 was treated with 1:1 dichloromethane and trifluoroacetic acid as in Example 176 and evaporated to give the title compound as the trifluoroacetic acid salt.
In a manner similar to that used in Example 177, the trifluoroacetic acid salt of [4-Amino-2-(piperidin-4-ylamino)pyrimidin-5-yl-(2-fluoro-4,6-dimethoxy-phenyl)-methanone (84.5 mg, 0.173 mmol, Example 182) was treated with methanesulfonylchloride (0.026 mL, 0.346 mmol, Aldrich) and the product chromatographed to give 46% of yellow solids. HR-MS (ES): obs. 454.1558, calcd. 454.1555.
In a similar manner as used in Example 177, the trifluoroacetic acid salt of [4-amino-2-(piperidin-4-ylamino)pyrimidin-5-yl-(2-fluoro-4,6-dimethoxy-phenyl)-methanone (84.5 mg, 0.173 mmol, Example 182) was treated with acetic anhydride (0.0327 mL, 0.346 mmol, Fisher) to yield the title compound and the product chromatographed to give 66% of yellow solids. HR-MS (ES): obs. 418.1890, calcd. 418.1885.
The compound was prepared from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide, Example 1, and 3,4,5-trifluoroanisole (Aldrich) in an analogous manner as described in Example 169.
The compound was prepared from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide, Example 1, and 2-bromo-6-methoxy-pyridine (Aldrich) in an analogous manner as described in Example 169.
The compound was prepared from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide, Example 1, and 2,3,4-trifluoroanisole (Matrix) in an analogous manner as described in Example 169.
The compound was prepared from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide, Example 1, and 2-bromo-3-methyl-thiophene (Aldrich) in an analogous manner as described in Example 169.
The compound was prepared from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide, Example 1, and 2-bromo4-fluorotoluene (Aldrich) in an analogous manner as described in Example 169.
The compound was prepared from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide, Example 1, and 2-bromo-4-fluoro-1-trifluoromethyl-benzene (Aldrich) in an analogous manner as described in Example 169.
The compound was prepared from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide, Example 1, and 2-bromo4-fluoro-1-isopropoxy-benzene (prepared from 2-bromo-4-fluoro-phenol, Aldrich) in an analogous manner as described in Example 169.
The compound was prepared from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide, Example 1, and 2-bromo-1-ethoxy-4-fluoro-benzene (prepared from 2-bromo-4-fluoro-phenol, Aldrich) in an analogous manner as described in Example 169.
The compound was prepared from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide, Example 1, and 2-bromo-1-ethyl-4-fluoro-benzene (prepared from 2-bromo-4-fluoroacetophenone, WO0015634) in an analogous manner as described in Example 169.
The compound was prepared from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide, Example 1, and 1-bromo-2-methoxy-4-trifluoromethyl-benzene (prepared from 3-(trifluoromethyl)-anisole, Aldrich) in an analogous manner as described in Example 169.
The compound was prepared from 4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2,3,4-trifluoro-6-methoxy-phenyl)-methanone (Example 184) in an analogous manner as described in Example 163. The crude product was used without further purification.
The compound was prepared from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(3-methoxy-pyridin-2-yl)-methanone (Example 185) in an analogous manner as described in Example 163. The crude product was used without further purification.
The compound was prepared from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(3,4,5-trifluoro-2-methoxy-phenyl)-methanone (Example 186) in an analogous manner as described in Example 163. The crude product was used without further purification.
The compound was prepared from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(3-methyl-thiophen-2-yl)-methanone (Example 187) in an analogous manner as described in Example 163. The crude product was used without further purification.
The compound was prepared from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(5-fluoro-2-methyl-phenyl)-methanone (Example 188) in an analogous manner as described in Example 163. The crude product was used without further purification.
The compound was prepared from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(5-fluoro-2-trifluoromethyl-phenyl)-methanone (Example 189) in an analogous manner as described in Example 163. The crude product was used without further purification.
The compound was prepared from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(5-fluoro-2-isopropoxy-phenyl)-methanone (Example 190) in an analogous manner as described in Example 163. The crude product was used without further purification.
The compound was prepared from (4-amino-2-methylsulfanyl-pyrimidin-5-yl)-(2-ethoxy-5-fluoro-phenyl)-methanone (Example 191) in an analogous manner as described in Example 163. The crude product was used without further purification.
The compound was prepared from (4-amino-2-methylsulfanyl-pyrimidin-5-yl)-(2-ethyl-5-fluoro-phenyl)-methanone (Example 192) in an analogous manner as described in Example 163. The crude product was used without further purification.
The compound was prepared from (4-amino-2-methylsulfanyl-pyrimidin-5-yl)-(2-methoxy-4-trifluoromethyl-phenyl)-methanone (Example 193) in an analogous manner as described in Example 163. The crude product was used without further purification.
The compound was prepared from (4-amino-2-ethylsulfinyl-pyrimidin-5-yl)-(2,3,4-trifluoro-6-methoxy-phenyl)-methanone (Example 194) in an analogous manner as described in Example 172. HR-MS (ES, m/z) calculated for C19H21N5O3F3 [(M+H)+] 424.1591, observed 424.1593. CDK4 IC50=0.01 μM; CDK1 IC50=0.128; CDK2 IC50=0.051; HCT 116 IC90=2.4 μM.
The compound was prepared from (4-amino-2-ethylsulfinyl-pyrimidin-5-yl)-(3-methoxy-pyridin-2-yl)-methanone (Example 195) in an analogous manner as described in Example 172. HR-MS (ES, m/z) calculated for C18H23N6O3 [(M+H)+] 371.1826, observed 371.1829.
The compound was prepared from (4-amino-2-ethylsulfinyl-pyrimidin-5-yl)-(3,4,5-trifluoro-2-methoxy-phenyl)-methanone (Example 196) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) in an analogous manner as described in Example 172. HR-MS (ES, m/z) calculated for C18H21N5O4SF3 [(M+H)+] 460.1261, observed 460.1267.
The compound was prepared from (4-amino-2-ethylsulfinyl-pyrimidin-5-yl)-(2,3,4-trifluoro-6-methoxy-phenyl)-methanone (Example 194) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) in an analogous manner as described in Example 172. HR-MS (ES, m/z) calculated for C18H21N5O4SF3[(M+H)+] 460.1261, observed 460.1267. CDK4 IC50=0.001; CDK1 IC50=0.005; CDK2 IC50=0.002; HCT 116 IC90=0.630 μM.
The compound was prepared from (4-amino-2-ethylsulfinyl-pyrimidin-5-yl)-(3-methoxy-pyridin-2-yl)-methanone (Example 195) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) in an analogous manner as described in Example 172. HR-MS (ES, m/z) calculated for C17H23N6O4S [(M+H)+] 407.1496, observed 407.1501.
To a mixture of [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (100 mg, 0.290 mmol, Example 59) and triethylamine (243 uL, 1.740 mmol) in tetrahydrofuran (3 mL) was added methyl vinyl sulfone (76 uL, 0.870 mmol, Aldrich). The reaction mixture was stirred at room temperature for 2 days and concentrated. The residue was partitioned between ethyl acetate (20 mL) and water (20 mL). The layers were separated and the aqueous layer was extracted with ethyl acetate (10 mL). The combined organic layers were washed with brine and dried over anhydrous magnesium sulfate. The solids were filtered off, and the filtrate was concentrated in vacuo to give a yellow solid (160 mg). Recrystallization from ethanol give [4-amino-2-[1-(2-methanesulfonyl-ethyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone as white solids (63.1 mg, 48% yield). HR-MS (ES, m/z) calculated for C20H27N5O4SF [(M+H)+] 452.1763, observed 452.1766.
The compound was prepared from (4-amino-2-ethylsulfinyl-pyrimidin-5-yl)-(3-methyl-thiophen-2-yl)-methanone (Example 197) in an analogous manner as described in Example 172. HR-MS (ES, m/z) calculated for C17H22N5O2S [(M+H)+] 360.1489, observed 360.1492.
The compound was prepared from (4-amino-2-ethylsulfinyl-pyrimidin-5-yl)-(5-fluoro-2-methyl-phenyl)-methanone (Example 198) in an analogous manner as described in Example 172. HR-MS (ES, m/z) calculated for C19H23N5O2F [(M+H)+] 372.1831, observed 372.1834.
The compound was prepared from (4-amino-2-ethylsulfinyl-pyrimidin-5-yl)-(3-methyl-thiophen-2-yl)-methanone (Example 197) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) in an analogous manner as described in Example 172. HR-MS (ES, m/z) calculated for C16H22N5O3S2 [(M+H)+] 396.1159, observed 396.1162.
The compound was prepared from (4-amino-2-ethylsulfinyl-pyrimidin-5-yl)-(5-fluoro-2-methyl-phenyl)-methanone (Example 198) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) in an analogous manner as described in Example 172. HR-MS (ES, m/z) calculated for C18H23N5O3SF [(M+H)+] 408.1500, observed 408.1504.
The compound was prepared from (4-amino-2-ethylsulfinyl-pyrimidin-5-yl)-(5-fluoro-2-trifluoromethyl-phenyl)-methanone (Example 199) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) in an analogous manner as described in Example 172. HR-MS (ES, m/z) calculated for C18H20N5O3SF4 [(M+H)+] 462.1218, observed 462.1222.
The compound was prepared from (4-amino-2-ethylsulfinyl-pyrimidin-5-yl)-(5-fluoro-2-isopropoxy-phenyl)-methanone (Example 200) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) in an analogous manner as described in Example 172. HR-MS (ES, m/z) calculated for C20H27N5O4SF [(M+H)+] 452.1766, observed 452.1766.
The compound was prepared from (4-amino-2-ethylsulfinyl-pyrimidin-5-yl)-(2-ethoxy-5-fluoro-phenyl)-methanone (Example 201) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) in an analogous manner as described in Example 172. HR-MS (ES, m/z) calculated for C19H25N5O4SF [(M+H)+] 438.1606, observed 438.1610.
The compound was prepared from (4-amino-2-ethylsulfinyl-pyrimidin-5-yl)-(2-ethyl-5-fluoro-phenyl)-methanone (Example 202) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) in an analogous manner as described in Example 172. HR-MS (ES, m/z) calculated for C19H25N5O3SF [(M+H)+] 422.1660, observed 422.1657.
The compound was prepared from (4-amino-2-ethylsulfinyl-pyrimidin-5-yl)-(2-methoxy-4-trifluoromethyl-phenyl)-methanone (Example 203) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) in an analogous manner as described in Example 172. HR-MS (ES, m/z) calculated for C19H23N5O4SF3 [(M+H)+] 474.1418, observed 474.1418.
To a solution of 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide (600 mg, 2.476 mmol, Example 167) in tetrahydrofuran (20 mL) cooled to −20° C. was added thiophen-2-yl-magnesium bromide (9.9 mL, 9.9 mmol, 1 M in tetrahydrofuran, Aldrich) over a period of 15 minutes. The reaction mixture was stirred at −20° C. for 1 hour and at 0° C. for another hour then the ice bath was removed to allow the temperature to go up to room temperature. After standing at room temperature for 12 hours, saturated solution of sodium bicarbonate (1 mL), water (100 mL) and diethyl ether (20 mL) were each added. The mixture was filtered and the layers were separated. The product was extracted with diethyl ether (2×20 mL). The combined organic layers were washed with brine and dried over anhydrous magnesium sulfate. The solids were filtered off, and the filtrate was concentrated in vacuo. Purification of the crude residue by flash chromatography (Biotage system, 60 A silica gel, eluting with 1.4% ethyl acetate and 0.1 % triethylamine in methylene chloride) yield 4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-thiophen-2-yl-methanone as off-white solids (465 mg, 71%).
The compound was prepared from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-thiophen-2-yl-methanone (Example 219) in an analogous manner as described in Example 163. The crude product was used without further purification.
The compound was prepared from (4-amino-2-ethylsulfinyl-pyrimidin-5-yl)-(3-methyl-thiophen-2-yl)-methanone (Example 220) in an analogous manner as described in Example 172. HR-MS (ES, m/z) calculated for C17H22N5O2S [(M+H)+] 360.1489, observed 360.1492.
The compound was prepared from (4-amino-2-methylsulfinyl-pyrimidin-5-yl)-(2-fluoro-6-methoxy-phenyl)-methanone (Example 171) in an analogous manner as described in Example 175. HR-MS (ES, m/z) calculated for C22H29N5O4F [(M+H)+] 446.2198, observed 446.2203.
The compound was prepared from 4-[4-amino-5-(2-fluoro-6-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid tert-butyl ester (Example 222) in an analogous manner as described in Example 176.
The compound was prepared from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2-fluoro-6-methoxy-phenyl)-methanone (Example 223) in an analogous manner as described in Example 177.
The compound was isolated as a side product from the preparation of [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2-fluoro-6-methoxy-phenyl)-methanone (Example 223). HR-MS (ES, m/z) calculated for C19H20N5O3F4 [(M+H)+] 442.1497, observed 442.1501.
The compound was prepared from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (Example 107) and chloropropanesulfonyl chloride (Aldrich) in an analogous manner as described in Example 177. HR-MS (ES, m/z) calculated for C20H25N5O4SF2Cl [(M+H)+] 504.1279, observed 504.1284.
The reaction mixture of [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (100 mg, 0.198 mmol, Example 226), dl-2-amino-1-propanol (158 uL, 1.980 mmol, TCl) and sodium iodide (30 mg, 0.198 mmol) in tetrahydrofuran (4 mL) was heated at 150° C. for 1 hour using a Smith Creator microwave synthesizer. Upon cooling to room temperature, the solvent was removed and acetonitrile (3 mL) was added. The mixture was filtered and trifluoroacetic acid (0.25 mL) was added. Purification by reverse phase HPLC (Cl8, eluting with acetonitrile and water) gave (4-amino-2-[1-[3-(2-hydroxy-1-methyl-ethylamino)-propane-1-sulfonyl]-piperidin-4-ylamino]-pyrimidin-5-yl)-(2,3-difluoro-6-methoxy-phenyl)-methanone (70 mg, 65% yield) as off-white solids. HR-MS (ES, m/z) calculated for C23H33N6O5SF2[(M+H)+] 543.2196, observed 543.2202.
The compound was prepared from [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (Example 226) and 1-methylpiperazine (Fluka) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C25H36N7O4SF2[(M+H)+] 568.2512, observed 568.2519. CDK4 IC50=0.001; CDK1 IC50=0.039; CDK2 IC50=0.017; IC90=0.274 μM.
The compound was prepared from [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (Example 226) and (R)-2-amino-1-butanol (Aldrich) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C24H35N6O5SF2 [(M+H)+] 557.2352, observed 557.2360.
[4-Amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (Example 226) was reacted with sodium acetate in an analogous manner as described in Example 227 to give acetic acid 3-[4-[4-amino-5-(2,3-difluoro-6-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-sulfonyl]-propyl ester. The crude acetic acid 3-[4-[4-amino-5-(2,3-difluoro-6-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-sulfonyl]-propyl ester was hydrolyzed with 5% potassium hydroxide to give [4-amino-2-[1-(3-hydroxy-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone. HR-MS (ES, m/z) calculated for C20H26N5O5SF2 [(M+H)+] 486.1617, observed 486.1623.
The compound was prepared from [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (Example 226) and pyrrolidine (Aldrich) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C24H33N6O4SF2 [(M+H)+] 539.2247, observed 539.2252.
The compound was prepared from [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (Example 226) and morpholine (Aldrich) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C24H33N605SF2 [(M+H)+] 555.2196, observed 555.2199.
The compound was prepared from [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (Example 226) and 1-(2-hydroxyethyl)piperazine (Aldrich) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C26H38N7O5SF2 [(M+H)+] 598.2618, observed 598.2624.
The compound was prepared from [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (Example 226) and N-(2-methoxyethyl)methylamine (TCI America) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C24H35N6O5SF2[(M+H)+] 557.2352, observed 557.2357.
The compound was prepared from [4-amino−2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (Example 226) and 2-amino-2-methyl-1-propanol (Aldrich) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C24H35N6O5SF2 [(M+H)+] 557.2352, observed 557.2359.
The compound was prepared from [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (Example 226) and 1-amino-2-propanol (Eastman Kodak) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C23H33N6O5SF2 [(M+H)+] 543.2196, observed 543.2200.
The compound was prepared from [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (Example 226) and (S)-(+)-2-amino-1-propanol (Aldrich) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C23H33N6O5SF2[(M+H)+] 543.2196, observed 543.2200.
The compound was prepared from [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (Example 226) and (R)-2-amino-3-methyl-1-butanol (Aldrich) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C25H37N6O5SF2[(M+H)+] 571.2509, observed 571.2514.
The compound was prepared from [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (Example 226) and (R)-2-amino-1-propanol (Aldrich) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C23H33N6O5SF2[(M+H)+] 543.2196, observed 543.2201.
The compound was prepared from [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (Example 226) and (S)-2-amino-1-butanol (Aldrich) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C24H35N6O5SF2[(M+H)+] 557.2352, observed 557.2356.
The compound was prepared from [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (Example 226) and 3-amino-pentane-1,5-diol (prepared from the methyl oxime of diethyl 1,3-acetonedicarboxylate, Aldrich, by reduction with lithium aluminum hydride in diethyl ether) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C25H37N6O6SF2[(M+H)+] 587.2458, observed 587.2464.
The compound was prepared from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (Example 59) and chloropropanesulfonyl chloride (Aldrich) in an analogous manner as described in Example 177. HR-MS (ES, m/z) calculated for C20H26N5O4SFCl[(M+H)+] 486.1373, observed 486.1375.
The compound was prepared from [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (Example 242) and N-(2-methoxyethyl)methylamine (TCI America) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C24H36N6O5SF [(M+H)+] 539.2447, observed 539.2449.
The compound was prepared from [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (Example 242) and 1-(2-hydroxyethyl)piperazine (Aldrich) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C26H39N7O5SF [(M+H)+] 580.2712, observed 580.2716.
The compound was prepared from [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (Example 242) and (S)-2-amino-1-propanol (Aldrich) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C23H34N6O5SF [(M+H)+] 525.2290, observed 525.2294.
The compound was prepared from [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (Example 242) and (R)-2-amino-1-butanol (Aldrich) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C24H36N6O5SF [(M+H)+]539.2450, observed 539.2447.
The compound was prepared from [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (Example 242) and 1-amino-2-propanol (Eastman Kodak) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C23H34N6O5SF [(M+H)+]525.2290, observed 525.2295.
The compound was prepared from [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (Example 242) and (R)-2-amino-3-methyl-1-butanol (Aldrich) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C25H38N6O5SF [(M+H)+]553.2603, observed 553.2600.
The compound was prepared from [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (Example 242) and 2-amino-1-methoxy-propane (Aldrich) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C24H36N6O5SF [(M+H)+]539.2447, observed 539.2450.
The compound was prepared from [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (Example 242) and 2-amino-2-methyl-1-propanol (Aldrich) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C24H36N6O5SF [(M+H)+]539.2447, observed 539.2449.
The compound was prepared from [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (Example 242) and (R)-2-amino-1-propanol (Aldrich) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C23H34N6O5SF [(M+H)+]525.2290, observed 525.2294.
The compound was prepared from [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (Example 242) and (S)-2-amino-1-butanol (Aldrich) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C24H36N6O5SF [(M+H)+]539.2447, observed 539.2449.
The mixture of [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (100 mg, 0.290 mmol, Example 59), 1,4-butane sultone (81 uL, 0.870 mmol, Aldrich) and triethylamine (41 uL, 0.290 mmol) in tetrahydrofuran (3 mL) was heated at 150° C. for 1 hour using the Smith Creator microwave synthesizer. Upon cooling to room temperature, the reaction mixture was concentrated. Purification of the crude residue by reverse phase HPLC (C18, eluting with water and acetonitrile) gave [4-amino-2-[1-(4-hydroxy-butane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone as white solids (120 mg, 86%). HR-MS (ES, m/z) calculated for C21H29N5O5SF [(M+H)+]482.Example 221, observed 482.1874.
To a mixture of 1,4-butane sultone (5 g, 36.72 mmol, Aldrich) and thionyl chloride (2.92 mL, 39.66 mmol, Aldrich) was added dimethylformamide (0.2 mL). The reaction mixture was heated at reflux for 4 hours and then concentrated to give 4-chloro-butane-1-sulfonyl chloride as yellow oil. The crude product was used without further purification.
The compound was prepared from [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (Example 59) and 4-chloro-butane-1-sulfonyl chloride (Example 254) in an analogous manner as described in Example 177. HR-MS (ES, m/z) calculated for C21H27N5O4SFCI [(M+H)+]500.1532, observed 500.1529.
The compound was prepared from [4-amino-2-[1-(4-chloro-butane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (Example 255) and 1-methyl-piperazine (Aldrich) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C26H39N7O4SF [(M+H)+]564.2767, observed 564.2763.
The compound was prepared from [4-amino-2-[1-(4-chloro-butane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (Example 255) and pyrrolidine (Aldrich) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C25H36N6O4SF [(M+H)+]535.2500, observed 535.2498.
The compound was prepared from [4-amino-2-[1-(4-chloro-butane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (Example 255) and 1-amino-2-propanol (Eastman Kodak) in an analogous manner as described in Example 227. HR-MS (ES, m/z) calculated for C24H35N6O5SF [(M+H)+]539.2451, observed 539.2447.
The compound was prepared from 4-amino-2-methylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide (Example 168) and 1,2-difluoro-4,5-dimethoxy-benzene (Aldrich) in an analogous manner as described in Example 169. HR-MS (ES, m/z) calculated for C14H14N3O3SF2 (M+) 341.0646, observed 341.0644.
The compound was prepared from (4-amino-2-methylsulfanyl-pyrimidin-5-yl)-(2,3-difluoro-5,6-dimethoxy-phenyl)-methanone (Example 259) in an analogous manner as described in Example 163. The crude product was used without further purification.
The compound was prepared from (4-amino-2-methylsulfinyl-pyrimidin-5-yl)-(2,3-difluoro-5,6-dimethoxy-phenyl)-methanone (Example 260) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) in an analogous manner as described in Example 172. HR-MS (ES, m/z) calculated for C19H24N5O5SF [(M+H)+]472.1465, observed 472.1461.
To a solution of [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2,3-difluoro-5,6-dimethoxy-phenyl)-methanone (30 mg, 0.0636 mmol, Example 261) in methylene chloride (20 mL) cooled to 5° C. was added aluminum chloride (129 mg, 0.954 mmol, Fluka). The reaction mixture was stirred at room temperature for 24 hours and quenched with water. Tetrahydrofuran (40 mL) was added and the layers were separated. The organic layer was washed with water (1×5 mL), brine (1×5 mL) and dried over anhydrous sodium sulfate. The solids were filtered off and the filtrate was concentrated in vacuo. Purification of the crude residue by flash chromatography (Biotage system, 60 A silica gel, eluting with ethyl acetate) yielded [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2,3-difluoro-6-hydroxy-5-methoxy-phenyl)-methanone as yellow solids (15 mg, 52%). HR-MS (ES, m/z) calculated for C18H22N5O5SF2 [(M+H)+]458.1307, observed 458.1304.
To a solution of [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2,3-difluoro-5,6-dimethoxy-phenyl)-methanone (100 mg, 0.212 mmol, Example 261) in methylene chloride (50 mL) cooled to −78° C. was added boron tribromide (2.19 mL, 2.190 mmol, 1 M solution in methylene chloride, Aldrich). The reaction mixture was warmed slowly to 0° C. over a period of 1 hour. After being stirred at 0° C. for 1 hour, the reaction was quenched with a solution of aqueous sodium bicarbonate. The aqueous layer was extracted with methylene chloride. The combined layers were washed with water and dried over anhydrous sodium sulfate. The solids were filtered off and the filtrate was concentrated in vacuo. Purification of the crude residue by reversed phase HPLC (C18, eluting with water and acetonitrile) yielded [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2,3-difluoro-5,6-dihydroxy-phenyl)-methanone as yellow solids (50 mg, 52%). HR-MS (ES, m/z) calculated for C17H20N5O5SF2 [(M+H)+]444.1151, observed 444.1148.
To a solution of 1,2-difluoro-4,5-dimethoxybenzene (5 g, 28.71 mmol, Aldrich) in methylene chloride (300 mL) cooled to 0° C. was added aluminum chloride (14.16 g, 106.2 mmol, Fluka) in several portions. At the end of the addition, the ice bath was removed and the reaction mixture was stirred at room temperature for 12 hours. Water was added to quench the reaction. Diethyl ether (200 mL) was added and the layers were separated. The aqueous layer was extracted with diethyl ether (50 mL). The combined organic layers were washed with water, brine and dried over anhydrous magnesium sulfate. The solids were filtered off and the filtrate was concentrated in vacuo. Purification of the crude residue by flash chromatography (Biotage system, 60 Å silica gel, eluting with 10% ethyl acetate in hexanes) yielded 4,5-difluoro-2-methoxy-phenol as dark oil (3.75 g, 82%).
To a solution of 4,5-difluoro-2-methoxy-phenol (3.75 g, 23.42 mmol, Example 264) in methylene chloride (60 mL) cooled to 0° C. were added N,N-diisopropylethylamine (4.895 mL, 28.10 mmol), tert-butyldimethylsilyl chloride (4.367 g, 28.10 mmol, Aldrich), and dimethylaminopyridine (286 mg, 2.342 mmol, Aldrich). At the end of addition, the ice bath was removed and the reaction mixture was stirred at room temperature for 90 minutes. Water (30 mL) was added and the layers were separated. The aqueous layer was extracted with methylene chloride (20 mL). The combined organic layers were washed with brine and dried over anhydrous magnesium sulfate. The solids were filtered off and the filtrate was concentrated in vacuo. Purification of the crude residue by flash chromatography (Biotage system, 60 Å silica gel, eluting with 3% ethyl acetate in hexanes) yielded tert-butyl-(4,5-difluoro-2-methoxy-phenoxy)-dimethyl-silane as a clear oil (5.22 g, 81%).
To a solution of tert-butyl-(4,5-difluoro-2-methoxy-phenoxy)-dimethyl-silane (5.2 g, 18.95 mmol) in tetrahydrofuran (60 mL) cooled to −78° C. was added n-butyllithium (9.096 mL, 22.74 mmol) dropwise over a period of 15 minutes. At the end of the addition, the reaction mixture was stirred at −78° C. for 1 hour. Dimethylformamide (1.737 mL, 22.74 mmol) was added and the reaction mixture was stirred at −78° C. for 2 hours. Saturated solution of ammonium chloride (˜5 mL) was added, and then the mixture was diluted with 100 mL of water. The aqueous layer was extracted with ethyl acetate (2×75 mL). The combined organic layers were washed with brine and dried over anhydrous magnesium sulfate. The solids were filtered off and the filtrate was concentrated in vacuo. Purification of the crude residue by flash chromatography (Biotage system, 60 Å silica gel, eluting with 4% ethyl acetate in hexanes) yielded 3-(tert-butyl-dimethyl-silanyloxy)-5,6-difluoro-2-methoxy-benzaldehyde as clear oil (3.96 g, 69%). LR-MS (M+) 302.2.
To a solution of 5-bromo-2,4-dichloropyrimidine (753.6 mg, 3.307 mmol, Matrix Chemicals) in tetrahydrofuran (20 mL) cooled to −30° C. was added dropwise isopropylmagnesium chloride. The temperature was maintained below −25° C. throughout the addition. At the end of addition, the reaction mixture was stirred at −30° C. for 20 minutes and a solution of 3-(tert-butyl-dimethyl-silanyloxy)-5,6-difluoro-2-methoxy-benzaldehyde (1 g, 3.307 mmol, Example 266) in tetrahydrofuran was added dropwise. The temperature was maintained below −25° C. throughout the addition. The reaction mixture was allowed to warm up to 0° C. after the addition was completed. After being stirred at 0° C. for 45 minutes, saturated solution of ammonium chloride (˜3 mL) was added. The mixture was taken up in water and ethyl acetate. The aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with brine and dried over anhydrous magnesium sulfate. The solids were filtered off and the filtrate was concentrated in vacuo. Purification of the crude residue by flash chromatography (Biotage system, 60 Å silica gel, eluting with 5-10% ethyl acetate in hexanes) yielded [3-(tert-butyl-dimethyl-silanyloxy)-5,6-difluoro-2-methoxy-phenyl]-(2,4-dichloro-pyrimidin-5-yl)-methanol as white solids (965 mg, 65%). LR-MS (M+) 451.1.
To a solution of [3-(tert-butyl-dimethyl-silanyloxy)-5,6-difluoro-2-methoxy-phenyl]-(2,4-dichloro-pyrimidin-5-yl)-methanol (955 mg, 2.116 mmol, Example 267) in methylene chloride (6 mL) and water (1 mL) were added sodium bicarbonate (80 mg, 0.952 mmol), tetrabutylammonium bromide (21 mg, 0.0635 mmol), TEMPO (3.3 mg, 0.0212 mmol, Aldrich). The mixture was cooled to 0° C. and sodium hypochlorite (2.548 mL, 2.539 mmol, Clorox™) was added. The reaction mixture was stirred at 0° C. for 30 minutes and more sodium hypochlorite (0.5 mL) was added. Thin layer chromatography (silica gel, 10% ethyl acetate in hexanes) still showed the alcohol starting material. More sodium hypochlorite (3×0.5 mL) was added until no alcohol was seen. The reaction mixture was diluted with methylene chloride (30 mL) and water (20 mL). The layers were separated, and the organic phase was washed with water (2×10 mL), brine (1×10 mL) and dried over anhydrous magnesium sulfate. The solids were filtered off and the filtrate was concentrated in vacuo to give [3-(tert-butyl-dimethyl-silanyloxy)-5,6-difluoro-2-methoxy-phenyl]-(2,4-dichloro-pyrimidin-5-yl)-methanone as pale yellow oil (910 mg, 96%). The crude product was used without further purification.
Ammonia gas was bubbled through a solution of [3-(tert-butyl-dimethyl-silanyloxy)-5,6-difluoro-2-methoxy-phenyl]-(2,4-dichloro-pyrimidin-5-yl)-methanone (900 mg, 2.003 mmol, Example 268) in toluene (10 mL) for about 30 minutes. The reaction mixture was concentrated to dryness to give (4-amino-2-chloro-pyrimidin-5-yl)-[3-(tert-butyl-dimethyl-silanyloxy)-5,6-difluoro-2-methoxy-phenyl]-methanone (1.02 g, yellow gum). The crude product was used without further purification.
To a solution of (4-amino-2-chloro-pyrimidin-5-yl)-[3-(tert-butyl-dimethyl-silanyloxy)-5,6-difluoro-2-methoxy-phenyl]-methanone (860 mg, 2.0 mmol, Example 269) in ethanol (12 mL) was added 1-methanesulfonyl-piperidin-4-ylamine trifluoroacetate salt (613.8 mg, 2.1 mmol, Example 162) and diisopropylethylamine (1.2 mL, 6.889 mmol). The mixture was heated at reflux for 4 hours and then concentrated to dryness. The residue was dissolved in ethyl acetate (20 mL). The solution was washed with brine (1×10 mL) and dried over anhydrous magnesium sulfate. The solids were filtered off and the filtrate was concentrated in vacuo. Purification of the crude residue by flash chromatography (Biotage system, 60 Å silica gel, eluting with 45-60% ethyl acetate in hexanes) yielded [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-[3-(tert-butyl-dimethyl-silanyloxy)-5,6-difluoro-2-methoxy-phenyl]-methanone as white glass (684 mg, 60%). LR-MS (M+) 572.1.
To a solution of [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-[3-(tert-butyl-dimethyl-silanyloxy)-5,6-difluoro-2-methoxy-phenyl]-methanone (675 mg, 1.181 mmol, Example 270) in tetrahydrofuran (10 mL) was added tetrabutylammonium fluoride (1.240 mL, 1.24 mmol, 1 M solution in tetrahydrofuran, Aldrich). The mixture was stirred at room temperature for 15 minutes and then concentrated to dryness. The residue was taken up in methylene chloride and washed with saturated solution of sodium bicarbonate. The organic layer was dried over anhydrous magnesium sulfate. The solids were filtered off and the filtrate was concentrated in vacuo. Purification of the crude residue by flash chromatography (Biotage system, 60 A silica gel, eluting with 65-80% ethyl acetate in hexanes then 5% methanol in ethyl acetate) yielded [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2,3-difluoro-5-hydroxy-6-methoxy-phenyl)-methanone as white solids (315 mg, 58%). HR-MS (ES, m/z) calculated for C18H22N5O5SF2 [(M+H)+]458.1308, observed 458.1304.
A solution of [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (290 mg, 0.72 mmol, Example 59 and 3-chloropropanesulfonyl chloride (165 mg, 0.93 mmol, Aldrich) was treated with diisopropylethyl amine (102 mg, 102 mmol, Aldrich) in methylene chloride (200 mL). This was stirred for 1 hour at 5° C. The reaction was washed with 5% aqueous sodium bicarbonate, dried (MgSO4), and the solvent concentrated in vacuum. This solid was dissolved in tetrahydrofuran, filtered through celite, reduced in volume and triturated with ether to give 300 mg (82%) of [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone as a white solid. HRMS, observed: 486.1375; Calcd for (M+H)+: 486.1373
A suspension of [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (25 mg, 0.051 mmol, Example 272), potassium iodide (30 mg, 0.18 mmol), and pyrollidine (400 mg, 5.6 mmol) in dioxane (8 mL) was heated at 90° C. under nitrogen for 14 hours. The resulting solution was concentrated to ˜1 mL and triturated with water. Solids were filtered and dissolved in methylene chloride. After washing with water, the organic layer was separated and dried (Na2SO4). The product was purified by trituration with tetrahydrofuran/ethyl ether and filtered to give [4-amino-2-[1-(3-pyrrolidin-1-yl-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone as a white solid (12 mg, 40% yield). HRMS, observed: 521.2343; Calcd for (M+H)+: 521.2341
A suspension of [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (80 mg, 0.16 mmol, Example 273), potassium iodide (100 mg, 0.61 mmol), and potassium acetate (120 mg, 1.22 mmol) in dimethylformamide (6 mL) was heated at 65-85° C. under nitrogen for 14 hours. The resulting solution was diluted with 50 mL of cold water plus 20 mL of saturated aqueous sodium chloride. Solids were filtered, collected and dissolved in methylene chloride. After washing with water, the organic layer was separated and dried (Na2SO4). The residue was purified by silica gel chromatography (ethyl acetate/hexane) and crystallized from methylene chloride/ethyl ether to give acetic acid 3-[4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-sulfonyl]-propyl ester as a white solid (42 mg, 51% yield). HRMS, observed: 510.1822; Calcd for (M+H)+: 510.1817
A suspension of acetic acid 3-[4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-sulfonyl]-propyl ester (25 mg, 0.049 mmol, Example 274) in a solution of potassium hydroxide (50 mg, 0.9 mmol), water (1 mL) and ethanol (6 mL) was stirred at 25° C. for 1 hour. The solution was cooled, acidified with acetic acid and reduced in volume to 2 mL. This was diluted with water and the product extracted into methylene chloride. After washing with water, the organic layer was separated and dried (Na2SO4). The residue was purified by silica gel chromatography (ethyl acetate) and crystallized from methylene chloride/hexane to give [4-amino-2-[1-(3-hydroxy-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone as a white solid (12 mg, 50% yield). HRMS, observed: 468.1716; Calcd for (M+H)+: 468.1712
A suspension of [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (80 mg, 0.16 mmol, Example 272), potassium iodide (200 mg, 1.2 mmol), morpholine (200 mg, 4.3 mmol) in dioxane (12 mL) was heated at reflux under nitrogen for 48 hours. The resulting solution was diluted with 20 mL of cold water plus 20 mL of saturated aqueous sodium chloride. Solids were filtered and dissolved in methylene chloride. The organic layer was separated and dried (Na2SO4). The residue was purified by silica gel chromatography (triethylamine/methanol/methylene chloride 1:10:90) and crystallized from methylene chloride/hexane to give [4-amino-2-[1-(3-morpholin-4-yl-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone as a white solid (15 mg, 17% yield). HRMS, observed: 537.2296; Calcd for (M+H)+: 537.2290
A suspension of [4-amino-2-[l -(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (210 mg, 0.43 mmol, Example 272), potassium iodide (200 mg, 1.2 mmol) and ammonia (1.5 mL, 7M in methanol, Aldrich) was heated at 100-110° C. in a sealed tube with stirring for 6 hours. Solvent was removed and the crude amine product was dissolved in tetrahydrofuran (12 mL). The resulting solution was cooled to 0-5° C., treated with diisopropylethylamine (200 mg, 2.0 mmol, Aldrich) and then with a bolus of methanesulfonyl chloride (100 mg, 0.88 mmol, Aldrich). After stirring at ambient temperature for 3 hours, the solution was washed with 10% NaHCO3. The organic layer was separated and dried (Na2SO4). The residue was purified by silica gel chromatography (5% methanol/methylene chloride) and precipitated from tetrahydrofuran/ethyl ether to give N-(3-[4-[4-amino-5-(5-fluoro-2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-sulfonyl]-propyl)-methanesulfonamide as a white solid (60 mg, 26% yield). HRMS, observed: 445.1652; Calcd for (M+H)+: 445.1647
A suspension of [4-amino-2-[1-(3-chloro-propane-1-sulfonyl)-piperidin-4-ylamino]-pyrimidin-5-yl]-(5-fluoro-2-methoxy-phenyl)-methanone (32 mg, 0.066 mmol, Example 272), potassium iodide (50 mg, 0.3 mmol), N-methylpiperazine (500 mg, 5.0 mmol, Aldrich) in dioxane (15 mL) was heated at reflux under nitrogen for 16 hours. The reaction was diluted with methylene chloride (40 mL) and washed (2×) with water. The organic layer was separated and dried (Na2SO4). The residue was purified by silica gel chromatography (methanol/methylene chloride) and triturated from ethyl ether and filtered to give (4-amino-2-[1-[3-(4-methyl-piperazin-1-yl)-propane-1-sulfonyl]-piperidin-4-ylamino]-pyrimidin-5-yl)-(5-fluoro-2-methoxy-phenyl)-methanone as a white solid (12 mg, 33% yield). HRMS, observed: 550.2612; Calcd for (M+H)+: 550.2607
A suspension of [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (200 mg, 0.45 mmol, Example 105) in methylene chloride (40 mL) was treated at 0-5° C. with aluminum chloride (620 mg, 4.7 mmol). The solution was stirred at ˜7° C. for 2.5 hours and treated with ice water (20 mL). The mixture was treated with 10% NaHCO3 (15 mL) and extracted with a mixture of tetrahydrofuran and methylene chloride (2×), dried (Na2SO4), and filtered. Removal of solvent gave an oil that crystallized upon trituration with methylene chloride. This crude solid was purified by silica gel chromatography (5-10% methanol/methylene chloride to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2,3-difluoro-6-hydroxy-phenyl)-methanone as a white solid (75 mg, 39%) after solvent removal. HRMS, observed: 428.1203; Calcd for (M+H)+: 428.1199
A) 2-Bromo-1-(2,2-diethoxy-ethoxy)-4-fluoro-benzene
Bromo-4-fluorophenol (50 g, 0.26 mol, Lancaster) was combined with 2-bromodiethylacetal (52 g, 0.26 mol, Aldrich) and potassium carbonate (37 g, 0.266 mol) in dimethylformamide (500 mL) and was heated at 125° C. internally for 14 hours. The reaction was cooled, filtered (cake washed with methylene chloride) and the organics diluted with 1.5 L of water. The mixture was extracted with 20% ether/hexane (2×), washed with water (2×) and dried (Na2SO4). Removal of solvent afforded ˜60 g of 2-bromo-1-(2,2-diethoxy-ethoxy)4-fluoro-benzene (˜70%) as a decomposing oil and taken to the next step.
B) 7-Bromo-5-fluoro-benzofuran
2-Bromo-1-(2,2-diethoxy-ethoxy)-4-fluoro-benzene (60 g, 0.22 mol, Step A) was combined with Amberlyst-15 (20 g, Aldrich) in chlorobenzene (500 mL, Aldrich) and stirred with a paddle stirrer while the temperature was maintained at very gentle reflux. At the same time an azeotrope containing ethanol and chlorobenzene was removed via downward distillation. After 2 hours, more chlorobenzene was added (200 mL) and additional azeotrope was collected. The content of the flask was filtered and excess chlorobenzene was distilled (45° C., at ˜1 mm). The residue was purified by silica gel chromatography (5-10% ethyl ether/hexane) to give 7-bromo-5-fluoro-benzofuran (14 g, ˜33%) and taken to the next step. H1NMR (300 MHz, DMSOd6), ppm: 8.20 (d), 1H, 7.52(m), 2 H, 7.10(d), 1H.
C) 5-Fluoro-benzofuran-7-carbaldehyde
7-Bromo-5-fluoro-benzofuran (7 g, 32 mmol, Step B) was dissolved in a mixture of tetrahydrofuran (350 mL) and pentane (100 mL), cooled to −95 to −100° C., and treated with n-butyl lithium (14 mL, 35 mmol, 2.5M in hexane, Aldrich). After stirring for 5 minutes, a solution of dimethylformamide (5 mL) in tetrahydrofuran (15 mL) was added dropwise and the reaction temperature raised to −20° C. This was treated with aqueous ammonium chloride (50 mL) and stirred for 0.5 hour at ambient temperature. The reaction mixture was concentrated in vacuo and diluted with hexane. The organic fraction was washed with water and dried (Na2SO4). Removal of solvent afforded crude 5-fluoro-benzofuran-7-carbaldehyde (˜30%) as an oil. Purification was by silica gel chromatography (5-10% ethyl ether/hexane). HRMS, observed: 163.0201; Calcd for (M+H)+: 163.0195
D) (2,4-Dichloro-pyrimidin-5-yl)-(5-fluoro-benzofuran-7-yl)-methanol
A solution of 5-bromo-2,4-dichloro-pyrimidine (1.0 g, 4.4 mmol, Aldrich) was dissolved in tetrahydrofuran (50 mL), cooled to −30° C., and treated slowly with isopropylmagnesium chloride (2.2 mL, 4.4 mmol, 2M in tetrahydrofuran, Aldrich). After stirring 0.5 hour between −30° C. and −25° C., the reaction was treated with a solution of 5-fluoro-benzofuran-7-carbaldehyde (0.65 g, 0.40 mmol, Step C) in tetrahydrofuran (10 mL). The temperature was slowly raised to −10° C. and then the reaction was quenched with aqueous 20% ammonium chloride. After 0.5 hours of stirring at ambient temperature, the mixture was diluted with hexane (100 mL), washed with water and dried (Na2SO4). Removal of solvent afforded crude (2,4-dichloro-pyrimidin-5-yl)-(5-fluoro-benzofuran-7-yl)-methanol as an oil. Purification was by silica gel chromatography (50% ethyl acetate/hexane) to give 930 mg of oil (˜70%). HRMS, observed: 311.9863; Calcd for (M+H)+: 311.9869
E) (2,4-Dichloro-pyrimidin-5-yl)-(5-fluoro-benzofuran-7-yl)-methanone
At 20° C., a solution of (2,4-dichloro-pyrimidin-5-yl)-(5-fluoro-benzofuran-7-yl)-methanol (0.9 g, 0.29 mmol, Step D) in ethylacetate (110 mL) was treated with a slurry of manganese oxide (9.0 g, Aldrich) in ethyl acetate (110 mL). After stirring for 3.5 hours, the reaction was filtered through celite and solvent removed in vacuo to give 0.82 g of (2,4-dichloro-pyrimidin-5-yl)-(5-fluoro-benzofuran-7-yl)-methanone as a foam and taken to the next step.
F) (4-Amino-2-chloro-pyrimidin-5-yl)-(5-fluoro-benzofuran-7-yl)-methanone
At 20° C., a stirred suspension of (2,4-dichloro-pyrimidin-5-yl)-(5-fluoro-benzofuran-7-yl)-methanone (0.80 g, 2.6 mmol, Step E) in toluene (70 mL) was treated with a slow stream of ammonia gas (Matheson) for 1 hour. The suspension was cooled, filtered and the filter cake washed with water. The filter cake was dissolved in acetonitrile, filtered through celite and the solvent removed to give a solid. This was crystallized from acetonitrile to give 0.4 g of (4-amino-2-chloro-pyrimidin-5-yl)-(5-fluoro-benzofuran-7-yl)-methanone (52%) as a white solid. HRMS, observed: 291.0208; Calcd for (M+H)+: 291.0211
G) [4-Amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2,3-difluoro-6-hydroxy-phenyl)-methanone
Methanesulfonyl-piperidin-4-ylamine.trifluoroacetic acid salt (0.68 g, 2.3 mmol, Example 162), (4-amino-2-chloro-pyrimidin-5-yl)-(5-fluoro-benzofuran-7-yl)-methanone (0.5 g, 1.7 mmol, Step F), and diisopropylethylamine (1 mL, Aldrich) were combined with ethanol (20 mL) and refluxed for 12 hours. The reaction was cooled and diluted with ice water (˜60 mL). After 2 hours, the suspension was filtered and dried at 50° C. and high vacuum to give 0.35 g (57%) of [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2,3-difluoro-6-hydroxy-phenyl)-methanone as a white solid. HRMS, observed: 434.1297; Calcd for (M+H)+: 434.1293. CDK4 IC50=0.030 μM; CDK1 IC50=0.058 μM; CDK2 IC50=0.002 μM; HCT 116 IC90=3.300 μM.
A solution of 4-amino-2-methylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide (456 mg, 3 mmol, Example 168) in tetrahydrofuran (3 mL) chilled to 0° C. and added phenylmagnesium chloride (4 mL, 2M in tetrahydrofuran, Aldrich). The mixture was stirred cold for 0.5 hours and at room temperature for 2 hours and then quenched with aqueous ammonium chloride. The product was extracted with ethyl acetate, washed with aqueous sodium bicarbonate, brine, and dried (MgSO4). Removal of solvent and chromatography on silica gel (3:1 hexane/ethyl acetate) gave, after crystallization from dichloromethane/hexane, 310 mg of 4-amino-2-methylsulfanyl-pyrimidin-5-yl)-phenyl-methanone, MP 137-138° C. MS(ES) MH+246
The title compound was prepared in a similar manner as described in Example 163 using material prepared in Example 281 and the crude product used without purification.
Treatment of the material prepared in Example 282 with material prepared in Example 162 in ethanol containing triethylamine at reflux for one hour, gave, after chilling and filtering, [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-phenyl-methanone, as a colorless solid, MP 249-250° C. MS(ES) MH+376
Treatment of [4-amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (Example 107) in tetrahydrofuran and diisopropylethyl amine with 98% formic acid gave the title compound after extraction and chromatography on silica gel using methanol/triethylamine/dichloromethane (2:2:96) and crystallization from dichloromethane/hexane, MP 263° C. (dec). MS(ES) MH+392
[4-Amino-2-(piperidin-4-ylamino)-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (308 mg, 0.848 mmol, Example 107) in dioxane (3 mL) and sulfamide (814 mg, 8.48 mmol, Aldrich) refluxed overnight. The mixture was diluted with water and sodium bicarbonate and extracted with dichloromethane. The organic solution was washed with water, dried (MgSO4), filtered and evaporated. Chromatography on silica gel (methanol/triethylamine/ethyl acetate 5:2:95) gave, after crystallization from ethanol/ethyl ether, 255 mg of colorless solid, MP 217-218° C. MS(ES) MH+443
4-[4-Amino-5-(2,3-difluoro-6-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-sulfonic acid amide (50 mg, Example 285) in dichloromethane (10 mL), N-methyl morpholine (0.1 mL, Aldrich) and dimethylaminopyridine (1 mg, Aldrich) was chilled in ice and added acetic anhydride (5 drops). The mixture was stirred cold for two hours and quenched with methanol. The mixture was evaporated to dryness and chromatographed on silica gel (dichloromethane, then ethyl acetate). Fractions corresponding to product were combined and crystallized from dichloromethane/ethyl ether to give 45 mg of colorless material, MP 153° C. (dec). MS(ES) MH+485
A solution of 5-bromo-2,4-dichloropyrimidine (18.8 g, 82.5 mmol, Aldrich) in tetrahydrofuran (50 mL) was chilled to −30° C. and isopropylmagnesium chloride (41.25 mL, 82.5 mmol, 2M in tetrahydrofuran, Aldrich) was added. The mixture was stirred at −30° C. for 20 minutes and a solution of 2,3-difluoro-6-methoxybenzaldehyde (12.91 g, 75 mmol, Apollo) in tetrahydrofuran (40 mL) was added slowly at this temperature. The mixture was then stirred at 0° C. for one hour and quenched with aqueous ammonium chloride. Extraction with ethyl acetate and crystallization from ethyl ether/hexanes gave 20.5 g of rac-(2,4-dichloro-pyrimidin-5-yl)-(2,3-difluoro-6-methoxy-phenyl)-methanol as a light yellow solid, MP 99-101° C. MS(ES) MH+322
A mixture of rac-(2,4-dichloro-pyrimidin-5-yl)-(2,3-difluoro-6-methoxy-phenyl)-methanol (20.5 g, 63.8 mmol, Example 287) in dichloromethane (180 mL) was chilled with stirring to 0° C. and added water (20 mL), sodium bicarbonate powder (2.42 g, 28.73 mmol, Aldrich), tetrabutylammonium bromide (0.617 g, 1.915 mmol, Aldrich), and 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical (0.1 g, 0.638 mmol, TEMPO, Aldrich). Sodium hypochlorite solution (6.15%, Aldrich) was added portionwaise until all starting material was oxidized (TLC ethyl acetate/hexanes 3:1). The cold reaction mixture was then washed with water (2×), dried (MgSO4) and concentrated adding hexanes. Filtration of the cold mixture gave 17.4 g of (2,4-dichloro-pyrimidin-5-yl)-(2,3-difluoro-6-methoxy-phenyl)-methanone, MP 137-139° C.
A solution of (2,4-dichloro-pyrimidin-5-yl)-(2,3-difluoro-6-methoxy-phenyl)-methanone (13.7 g, Example 288) in toluene (200 mL) was stirred at room temperature and ammonia gas was bubbled in for 30 minutes. Water (200 mL) was then added and the mixture was stirred for 15 minutes. Hexane (200 mL) was added and the solids filtered and washed with water and hexane. The solids were dissolved in hot acetonitrile (˜700 mL), filtered hot, concentrated to about 300 mL and chilled. Filtration gave 10.6 g of (4-amino-2-chloro-pyrimidin-5-yl)-(2,3-difluoro-6-methoxy-phenyl)-methanone, MP 238-239° C. (dec). MS(ES) MH+300
To a stirred solution of trans-[4-amino-2-(4-amino-cyclohexylamino)-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (100 mg, 0.27 mmol, Example 120) and triethylamine (200 uL) in tetrahydrofuran (3 mL), 2-bromoethanol (20 uL, 0.28 mmol, Aldrich) was added and the mixture was stirred at reflux for 5 hours. The reaction was quenched with water and the mixture was extracted with ethyl acetate (3×10 mL) and the extracts were dried with sodium sulfate. Removal of solvent gave a yellow solid which was purified by reverse phase HPLC to give a pale yellow solid. 86 mg, 75%. MS (ES) MH+=422. CDK4 IC50=0.020 μM; CDK1 IC50=0.018 μM; CDK2 IC50=0.005 μM; HCT 116 IC90=0.179 μM.
To a stirred solution of trans-[4-amino-2-[4-(2-hydroxy-ethylamino)-cyclohexylamino]-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (106 mg, 0.25 mmol, Example 120) in 1,4-dioxane (3 mL), 2-bromoethanol (80 uL, 1.12 mmol, Aldrich) and sodium bicarbonate (50 mg, 0.60 mmol) were added and the mixture was heated at 113° C. for 24 hours. The reaction was quenched with water and the mixture was extracted with ethyl acetate (3×10 mL) and the extracts were dried with sodium sulfate. The solvent was removed and the residue was chromatographed (8% 9N ammonia in methanol/dichloromethane) to give a pale yellow solid. 97 mg, 84%. MS (ES) MH+=466.
To a stirred solution of trans-[4-amino-2-(4-amino-cyclohexylamino)-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (100 mg, 0.27 mmol, Example 120) and triethylamine (125 mg, 0.33 mmol) in tetrahydrofuran (5 mL), succinic anhydride (35 mg, 0.35 mmol, Aldrich,) was added and the mixture was refluxed for 2 hours. The mixture was cooled and the solid was filtered and dried to yield a white powder. 130 mg, 100%. MS (ES) MH+=478.
To a stirred solution of trans-[4-amino-2-(4-amino-cyclohexylamino)-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (565 mg, 1.5 mmol, Example 120) and triethylamine (167 mg, 0.25 mL, 1.69 mmol) in tetrahydrofuran (20 mL) at 0° C., 3-chloroproane sulfonyl chloride (0.20 mL, 1.65 mmol, Aldrich) was added. The mixture was stirred for 10 minutes and the reaction was quenched with water. The mixture was extracted with ethyl acetate and the extracts were combined and dried with sodium sulfate. The solvent was removed and the residue was chromatographed (4% methanol/dichloromethane) to give 600 mg of yellow solid. Yield, 77%. MS (ES) MH+=518.
To a stirred solution of 3-chloro-propane-1-sulfonic acid [4-[4-amino-5-(2,3-difluoro-6-methoxy-benzoyl)-pyrimidin-2-ylamino]-cyclohexyl]-amide (104 mg, 0.20 mmol, Example 293) in tetrahydrofuran (5 mL), morpholine (100 mg, 1.15 mmol, Aldrich) was added and the mixture was heated at reflux for 24 hours. The reaction was quenched with water and the mixture was extracted with ethyl acetate (3×5 mL). The extracts were combined and dried with sodium sulfate and concentrated. The residue was chromatographed (8% 9N ammonia in methanol/dichloromethane) to give a pale yellow solid. 57 mg, 50%. MS (ES) MH+=569. CDK4 IC50=0.015 μM; CDK1 IC50=0.008 μM; CDK2 IC50=0.005 μM; HCT 116 IC90=0.374 μM.
To a stirred solution of 3-chloro-propane-1-sulfonic acid [4-[4-amino-5-(2,3-difluoro-6-methoxy-benzoyl)-pyrimidin-2-ylamino]-cyclohexyl]-amide (104 mg, 0.20 mmol, Example 293) in tetrahydrofuran (5 mL), N-methylpiperizine (100 mg, 1 mmol, Aldrich,) was added and the mixture was heated at reflux for 24 hours. The reaction was quenched with water and the mixture was extracted with ethyl acetate (3×5 mL). The extracts were combined and dried with sodium sulfate and concentrated. The residue was chromatographed (8% 9N ammonia in methanol/dichloromethane) to give a pale yellow solid. 85 mg, 73%. MS (ES) MH+=582.
This compound was made with Example 293 and pyrolidine (Aldrich) by a similar procedure to the synthesis of Example 295. MS (ES) MH+=553.
The chloride (80 mg, 0.18 mmol, Example 293) was dissolved in ethanol (1 mL). To it was added potassium acetate (86.5 mg, 0.88 mmol) and potassium iodide (8 mg, 0.05 mmol). The mixture was heated in a pressure tube in a microwave for 30 minutes. The mixture was filtered and the solid was washed with ethanol (4 mL). The filtrate (about 5 mL) was treated with 5% potassium hydroxide (2 mL) and the solution was stirred at room temperature for 30 minutes. The solution was neutralized with trifluoroacetic acid and concentrated. The residue was purified by reversed phase HPLC to give a pale yellow solid. 25 mg, 28%. MS (ES) MH+=500.
The chloride (530 mg, 1.02 mmol, Example 293) was dissolved in tetrahydrofuran (8 mL). To it was added potassium t-butoxide (395 mg, 3.53 mmol, Aldrich) and potassium iodide (10 mg). The mixture was heated at reflux for 3 hours and the reaction was quenched with 0.5N hydrochloric acid (1 mL). The mixture was extracted with ethyl acetate (3×10 mL) and the extracts were combined and dried with sodium sulfate. Removal of solvent gave the crude which was chromatographed (5% methanol/dichloromethane) to give a pale yellow solid. 412 mg, 85%. MS (ES) MH+=482. CDK4 IC50=0.005 μM; CDK1 IC50=0.028 μM; CDK2 IC50=0.008 μM; HCT 116 IC90=0.320 μM.
To a stirred solution of trans-[4-amino-2-(4-amino-cyclohexylamino)-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (105 mg, 0.28 mmol, Example 120) and sodium bicarbonate (84 mg, 1 mmol) in 1,4-dioxane (3 mL), bis-2-chloroethyl-methylamine hydrochloride (58 mg, 0.30 mmol, Aldrich) was added and the mixture was stirred at reflux overnight. An additional 20 mg of bis-2-chloroethyl-methylamine hydrochloride (Aldrich) was added and the reaction was refluxed for another 12 hours. The reaction was quenched with water and the mixture was extracted with ethyl acetate (3×10 mL) and the extracts were dried with sodium sulfate. Removal of solvent gave a yellow solid which was chromatographed (8% 9N ammonia in methanol/dichloromethane) to give a pale yellow solid. 16 mg, 12%. MS (ES) MH+=461.
To a stirred solution of trans-[4-amino-2-(4-amino-cyclohexylamino)-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (64 mg, 0.16 mmol, Example 120) and sodium carbonate (0.49 mmol, 52 mg) in ethanol (3 mL), 1,4-dibromobutane (22 uL, 0.16 mmol, Aldrich) was added and the mixture was stirred at reflux for 3 days. The solvent was removed under reduced pressure and the residue was purified by reversed phase HPLC to give a white solid. 30 mg, 43%. MS (ES) MH+=432.
To a stirred solution of trans-[4-amino-2-(4-amino-cyclohexylamino)-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (60 mg, 0.16 mmol, Example 120) in ethylene glycol dimethyl ether, formaldehyde (37% in water, 36 uL, 0.477 mmol, Aldrich) and formic acid (30 uL, 0.785 mmol, Aldrich) were added and the mixture was stirred at reflux for 2 hours. The solvent was removed under reduced pressure and the residue was purified by reversed phase HPLC to give a white solid. 27 mg, 67%. MS (ES) MH+=406.
To a stirred solution of 1,2,3,6-tetrahydropyridine (1.5 g, 18.04 mmol, Aldrich) and triethylamine (36.12 mmol, 5.05 mL) in methylene chloride (60 mL) at 0° C., methane sulfonyl chloride (1.82 mL, 23.45 mmol, Aldrich) was added and the mixture was stirred for 2.5 hours. The reaction was quenched with water and the mixture was extracted with methylene chloride. The extracts were dried with MgSO4 and the solvent was removed under reduced pressure to give the desired product as an off-white solid. 2.20 g, 76%. MS (ES) MH+=162.
To a stirred solution of N-methanesulfony-1,2,3,4-tetrahydropyridine (2.18 g, 13.52 mmol, Example 302) in methylene chloride (30 mL) at 0° C., meta-chloroperoxybenzoic acid (3.94 g, 77%, 17.5 mmol, Aldrich) in 20 mL of methylene chloride was added and the mixture was stirred for 30 minutes. An additional 600 mg of meta-chloroperoxybenzoic acid was added and the mixture was stirred for 6 hours. The reaction was quenched with 30% sodium thiosulfate solution and the mixture was extracted with methylene chloride. The extracts were combined, washed with 5% sodium carbonate and dried with MgSO4. The solvent was removed to give the desired product as a white solid. 2.40 g, 100%. The compound was used directly for the next step.
To a stirred solution of rac-3-methanesulfonyl-7-oxa-3-aza-bicyclo[4.1.0]heptane (0.92 g, 5.19 mmol, Example 303) in dimethylformamide (20 mL), sodium azide (550 mg, 8.46 mmol) was added and the mixture was stirred at 80° C. for 6 hours. The mixture was cooled and poured into water and extracted with ethyl acetate (3×10 mL). The extracts were combined, washed with water, brine and dried with MgSO4. The solvent was removed to give the desired product as colorless oil. 304 mg, 30.4%. The compound was used directly for the next step.
The azide (300 mg, 1.36 mmol, Example 304) was dissolved in ethanol and hydrogenated at 45 psi under the catalyst of 10% palladium on carbon (30 mg) for 1 hour. The mixture was filtered and the filtrate was concentrated to give a white solid. 245 mg, 93%. The compound was used directly for the next step.
The amine (44 mg, 0.22 mmol, Example 305) and chloropyrimidine (55 mg, 0.18 mmol, Example 289) were dissolved in ethanol (5 mL). To the stirred mixture at reflux, diisopropylethylamine (64 uL, Aldrich) was added and the mixture was refluxed for 20 hours. The solvent was removed and the residue was purified by HPLC (10-60%, acetonitrile/water) to give a white solid, 25 mg, 30%. MS (ES) MH+=458.
To a stirred solution of the epoxide (177 mg, 1 mmol, Example 303) in tetrahydrofuran (8 mL), lithium bromide (543 mg, 6.25 mmol, Aldrich) and copper(II)bromide (725 mg, 3.25 mmol, Aldrich) were added and the mixture was stirred at room temperature overnight. The mixture was poured into 0.1N hydrochloric acid (33 mL) and extracted with ethyl acetate (3×10 mL). The extracts were combined, washed with water, brine and dried with MgSO4. The solvent was removed to give the desired product as colorless oil after passing through an aluminum column (eluted with 5% methanol/dichloromethane). 237 mg, 92%. H1NMR (300 MHz, CDCl3), ppm: 2.00-2.20 (m), 1 H; 2.38-2.52 (m), 1 H; 2.70 (br), 1 H (OH); 2.99 (s), 3H; 3.0-3.20 (m), 2 H; 3.50-3.51 (m), 1 H; 3.80-3.97 (m), 2 H; 4.0-4.10 (m), 1 H. The compound was used directly for the next step.
To a stirred solution of the bromide (70 mg, 0.27 mmol, Example 307) in dimethylformamide (3 mL), sodium azide (80 mg, 1.23 mmol) was added and the mixture was stirred at 84° C. overnight. The solvent was removed and the residue was chromatographed (50% ethyl acetate/hexanej to give the desired product as a colorless oil. 51 mg, 86%. H1NMR (300MHz, CDCl3), ppm: 1.60-1.70 (m), 1 H; 2.05-2.20(m), 1 H; 2.80-3.05 (m), 2 H; 2.83 (s), 3H; 3.10 (br), 1 H (OH); 3.40-3.51 (m), 1 H; 3.51-3.62 (m), 1 H. 3.65-3.80 (m), 2 H.
This cis-amino alcohol was made by a similar procedure to the synthesis of Example 305 using material from Example 308 and the compound was used directly for the next step.
The compound was made with Example 309 and Example 289 by a similar procedure to the synthesis of Example 306. MS (ES) MH+=458.
To the stirred solution of the azide (500 mg, 2.27 mmol, Example 304) in tetrahydrofuran (8 mL) at 0° C., sodium hydride (60% in oil dispersion, 180 mg, 4.54 mmol, Aldrich) was added slowly and the mixture was stirred for 30 minutes. Methyl iodide (184 uL, 2.95 mmol, Aldrich) was then added and the mixture was stirred at room temperature overnight. The reaction was quenched with saturated ammonium chloride solution and the mixture was extracted with ethyl acetate. The extract was washed with brine and dried with sodium sulfate. The solvent was removed and the residue was purified by chromatography (15% ethyl acetate/hexanes first and then 35%) to give a yellow oil. 280 mg, 53%.
The azide (270 mg, 1.15 mmol, Example 311) in ethanol was hydrogenated at 40 psi under the catalysis of palladium on carbon for 1 hour. The mixture was filtered and the filtrate was concentrated to give an off-white solid. 230 mg, 96%. It was used directly for the next step.
The compound was made with Example 312 and Example 289 by a similar procedure to the synthesis of Example 306. MS (ES) MH+=472.
To a stirred solution of alcohol (65 mg, 0.14 mmol, Example 306) in acetone (4 mL), Jones reagent (made by adding 0.5 mL of concentrated sulfuric acid to a solution of chromium(VI) oxide, 0.67 g, Aldrich, in 2 mL of water) was slowly added until the orange color persisted for 20 minutes. The mixture was stirred for 1 hour and 2 mL of isopropanol was added. The mixture was again stirred for 30 minutes followed by slow addition of saturated sodium bicarbonate solution until pH=7. The mixture was extracted with ethyl acetate/tetrahydrofuran (2:1, 3×10 mL) and the extracts were combined and dried with sodium sulfate. The solvent was removed and the residue was chromatographed on HPLC (20-70%) to give a white solid, 4.3 mg, 7%. MS (ES) MH+=456.
1-(Diphenylmethyl)-3-hydroxyazetidine (300 mg, 1.26 mmol, Oakwood,) was dissolved in ethyl acetate (10 mL). To it 10% palladium on carbon (100 mg) and di-tert-butyl dicarbonate (329 mg, 1.51 mmol, Aldrich) were added and the mixture was hydrogenated at 50 psi overnight. Filtration and removal of solvent gave the desired product. 218 mg, 100%. MS (ES) MH+=174.
The alcohol (1.0 g, 5.77 mmol, Example 315) was dissolved in methylene chloride (20 mL). To it triethylamine (1.60 mL, 11.54 mmol) and methane sulfonyl chloride (536 uL, 5.77 mmol, Aldrich) were added and the mixture was stirred at 0° C. for 3 hours. The solvent was removed and the residue was partitioned between ethyl acetate and water. The organic layer was separated and dried with sodium sulfate and concentrated to give a white solid. 1.51 g, 100%. The compound was used directly for the next step.
To a stirred solution of methane sulfonate (930 mg, 3.70 mmol, Example 316) in dimethylformamide (20 mL), sodium azide (962 mg, 14.8 mmol, Aldrich) was added and the mixture was stirred at 90° C. overnight. The mixture was poured into water and extracted with ethyl acetate (5×10 mL) and the extracts were combined, washed with water, brine and dried with sodium sulfate. The solvent was removed to give a clear oil. 710 mg, 97%. The compound was directly used for the next step.
To a stirred solution of the azide (1.15 g, 5.80 mmol, Example 317) in ethanol (20 mL), 10% palladium on carbon (93 mg) was added and the mixture was hydrogenated for 1 hour at 48 psi. The mixture was filtered through a pad of celite and the solvent was removed to a colorless oil. 940 mg, 94%. The compound was directly used for the next step.
To a stirred suspension of chloropyrimidine (700 mg, 2.34 mmol, Example 289) in methylene chloride (30 mL), amine (525 mg, 5.64 mmol, Example 318) in ethanol (30 mL) was added and the mixture heated at 80° C. for 4.5 hours. The solvent was removed and the residue was dissolved in ethyl acetate and the mixture was washed successively with 0.5N hydrochloric acid and water. The organic layer was dried with sodium sulfate and concentrated to give a white solid. 1.0 g, 98%. MS (ES) MH+=436.
To a stirred suspension of the protected amine (940 mg, 2.16 mmol, Example 319) in methylene chloride (30 mL), trifluoroacetic acid (20 mL) was added at 0° C. and the mixture stirred for 2 hours. The solvent was removed and the residue was partitioned between ethyl acetate and 5% sodium carbonate solution. The aqueous layer was extracted with ethyl acetate/tetrahydrofuran (1:1, 3×10 ml). The extracts were combined and the solution was dried with sodium sulfate. Removal of solvent gave a yellow solid. 711 mg, 98%. MS (ES) MH+=336.
To a stirred solution of amine (80 mg, 0.239 mmol, Example 320) in tetrahydrofuran (30 mL), triethylamine (67 uL, 0.48 mmol) and methane sulfonic anhydride (50 mg, 0.29 mmol) were added successively and the mixture was stirred for 2 hours. Ice water was added and the mixture was extracted with ethyl acetate (3×10 mL). The extracts were combined and dried with sodium sulfate and the solvent was removed to give a yellow solid, which was purified on HPLC (10-80%, acetonitrile/water. 61 mg; 62%. MS (ES) MH+=414.
This compound was made with Example 320 and ethyl sulfonyl chloride (Aldrich) by a similar procedure to the synthesis of Example 321. MS (ES) MH+=428.
This compound was made with Example 320 and isopropyl sulfonyl chloride (Aldrich) by a similar procedure to the synthesis of Example 321. MS (ES) MH+=442.
The same procedure as described in Example 47 was used, starting from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide, Example 1, and 2-bromo-4-methylanisole (Aldrich), to give (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2-methoxy-5-methyl-phenyl)-methanone as a white solid. MS (M+H)+, 304.
A solution of (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2-methoxy-5-methyl-phenyl)-methanone, 571 mg, 1.885 mmol, Example 324) in chloroform (16 mL) was cooled to −15° C. and treated with 3-chloroperoxybenzoic acid (503.7 mg, ˜2.25 mmol, ˜70% purity from Aldrich) and the reaction was stirred at the same temperature for 1 hour. The reaction mixture was diluted with methylene chloride (20 mL) and washed with 10% aqueous sodium thiosulfate (2×5 mL), 10% aqueous sodium carbonate (2×5 mL), brine, dried and concentrated to give (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(2-methoxy-5-methyl-phenyl)-methanone as a white solid. MS (M+H)+: 320.
A suspension of (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(2-methoxy-5-methyl-phenyl)-methanone (22.3 mg, 0.0698 mmol, Example 325) and 1-(4-amino-piperidin-1-yl)-ethanone (14.9 mg, 0.105 mmol, prepared as described in U.S. Pat. No. 5,817,828) in isopropyl alcohol (2.5 mL) was heated at 120° C. in a sealed tube under microwave conditions for 0.3 to 1 hour. The resulting reaction mixture was evaporated in vacuo and crude product was purified on silica gel with 95:5 of dichloromethane/methanol to give 1-[4-[4-amino-5-(2-methoxy-5-methyl-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-ethanone as a white solid (23.1 mg). MS (M+H)+, 384.
The same procedure as described in Example 326 was used, starting with (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(2-methoxy-5-methyl-phenyl)-methanone (Example 325) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2-methoxy-5-methyl-phenyl)-methanone. MS (M+H)+, 420
The same procedure as described in Example 47 was used, starting from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide, (Example 1), and 2-bromo-4-methoxyanisole (Aldrich), to give (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2,5-dimethoxy-phenyl)-methanone as a white solid. MS (M+H)+, 320.
The same procedure as described in Example 325 was used, starting from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2,5-dimethoxy-phenyl)-methanone (Example 328) to give (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(2,5-dimethoxyphenyl)-methanone as a white solid. MS (M+H)+: 336
The same procedure as described in Example 326 was used, starting with (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(2,5-dimethoxyphenyl)-methanone (Example 329) and 1-(4-amino-piperidin-1-yl)-ethanone to give 1-[4-[4-amino-5-(2,5-dimethoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-ethanone as a white solid. MS (M+H)+, 400.
The same procedure as described in Example 326 was used, starting with 1-[4-[4-amino-5-(2,5-dimethoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-ethanone (Example 330) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2,5-dimethoxy-phenyl)-methanone. MS (M+H)+, 436
The same procedure as described in Example 47 was used, starting from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide (Example 1) and 2-bromo-3-methoxyanisole (Aldrich), to give (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2,6-dimethoxy-phenyl)-methanone as a white solid. MS (M+H)+, 320.
The same procedure as described in Example 325 was used, starting from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2,6-dimethoxy-phenyl)-methanone Example 328 to give (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(2,6-dimethoxyphenyl)-methanone as a white solid. MS (M+H)+: 336
The same procedure as described in Example 326 was used, starting with (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(2,6-dimethoxyphenyl)-methanone (Example 333) and 1-(4-amino-piperidin-1-yl)-ethanone to give 1-[4-[4-amino-5-(2,6-dimethoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-ethanone as a white solid. MS (M+H)+, 400.
The same procedure as described in Example 326 was used, starting with 1-[4-[4-amino-5-(2,6-dimethoxy-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-ethanone (Example 333) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2,6-dimethoxy-phenyl)-methanone. MS (M+H)+, 436
The same procedure as described in Example 326 was used, starting with (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(2,5-dimethoxyphenyl)-methanone (Example 325) and (trans-4-amino-cyclohexyl)-carbamic acid tert-butyl ester (Astatech) to give [4-[4-amino-5-(2-methoxy-5-methyl-benzoyl)-pyrimidin-2-ylamino]-cyclohexyl]-carbamic acid tert-butyl ester as a white solid. MS (M+H)+, 456
The same procedure was used as described in Example 59, using material from Example 336, to give [4-amino-2-(4-amino-cyclohexylamino)-pyrimidin-5-yl]-(2-methoxy-5-methyl-phenyl)-methanone as the free base. MS (M+H)+, 356.
The similar procedure as described in Example 60 was used, starting from [4-amino-2-(4-amino-cyclohexylamino)-pyrimidin-5-yl]-(2-methoxy-5-methyl-phenyl)-methanone (Example 337) and acetyl chloride (Aldrich) to give N-[4-[4-amino-5-(2-methoxy-5-methyl-benzoyl)-pyrimidin-2-ylamino]-cyclohexyl]-acetamide. MS (M+H)+, 398.
A similar procedure as described in Example 60 was used, starting from [4-amino-2-(4-amino-cyclohexylamino)-pyrimidin-5-yl]-(2-methoxy-5-methyl-phenyl)-methanone (Example 337) and methanesulfonyl chloride (Aldrich) to give N-[4-[4-amino-5-(2-methoxy-5-methyl-benzoyl)-pyrimidin-2-ylamino]-cyclohexyl]-methanesulfonamide. MS (M+H)+, 398.
The same procedure as described in Example 170 was used, starting from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide (Example 1) and 4-fluoro-3-methylanisole (Aldrich), to give (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-4-methyl-phenyl)-methanone as a white solid, MS (M+H)+, 322 and (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(3-fluoro-6-methoxy-2-methyl-phenyl)-methanone as a white solid, MS (M+H)+, 322
The procedure as described in Example 325 was used, starting from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-4-methyl-phenyl)-methanone (Example 340) to give (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-4-methyl-phenyl)-methanone as a white solid. MS (M+H)+: 338
The same procedure as described in Example 326 was used, starting with (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-4-methyl-phenyl)-methanone (Example 341) and 1-(4-amino-piperidin-1-yl)-ethanone to give 1-[4-[4-amino-5-(5-fluoro-2-methoxy-4-methyl-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-ethanone as a white solid. MS (M+H)+, 402.
The same procedure as described in Example 326 was used, starting with 1-[4-[4-amino-5-(5-fluoro-2-methoxy-4-methyl-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-ethanone (Example 341) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-4-methyl-phenyl)-methanone. MS (M+H)+, 436
The same procedure as described in Example 326 was used, starting with (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-4-methyl-phenyl)-methanone (Example 341) and (trans4-amino-cyclohexyl)-carbamic acid tert-butyl ester (Astatech) to give [4-[4-amino-5-(5-fluoro-2-methoxy-4-methyl-benzoyl)-pyrimidin-2-ylamino]-cyclohexyl]-carbamic acid tert-butyl ester as a white solid. MS (M+H)+, 474
The same procedure was used as described in Example 59, using material from Example 344, to give [4-amino-2-(4-amino-cyclohexylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-4-methyl-phenyl)-methanone as the free base. MS (M+H)+, 374.
The similar procedure as described in Example 60 was used, starting from [4-amino-2-(4-amino-cyclohexylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-4-methyl-phenyl)-methanone (Example 345) and acetyl chloride (Aldrich) to give N-[4-[4-amino-5-(5-fluoro-2-methoxy-4-methyl-benzoyl)-pyrimidin-2-ylamino]-cyclohexyl]-acetamide. MS (M+H)+, 416.
The similar procedure as described in Example 60 was used, starting from [4-amino-2-(4-amino-cyclohexylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-4-methyl-phenyl)-methanone (Example 345) and methanesulfonyl chloride (Aldrich) to give N-[4-[4-amino-5-(5-fluoro-2-methoxy-4-methyl-benzoyl)-pyrimidin-2-ylamino]-cyclohexyl]-methanesulfonamide. MS (M+H)+, 452.
A solution of [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2,3-difluoro-6-methoxy-phenyl)-methanone (49.7 mmol, 0.112 mmol, Example 105) in methanol (4 mL) was treated with sodium methoxide (25 wt % in methanol, 0.29 mL, Aldrich) and heated at 130° C. in microwave for 3 hours. The reaction was concentrated and the crude product was purified on silica gel (95:5 methylene chloride/methanol) to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(3-fluoro-2,6-dimethoxy-phenyl)-methanone as a white solid. MS (M+H)+, 454.
In a similar procedure as described in Example 348, sodium ethoxide in ethanol was used to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2-ethoxy-3-fluoro-6-methoxy-phenyl)-methanone as a slightly yellow solid. MS (M+H)+, 468.
The procedure as described in Example 325 was used, starting from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(3-fluoro-6-methoxy-2-methyl-phenyl)-methanone (Example 340) to give (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(3-fluoro-6-methoxy-2-methyl-phenyl)-methanone. MS (M+H)+: 338
The same procedure as described in Example 326 was used, starting with (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(3-fluoro-6-methoxy-2-methyl-phenyl)-methanone (Example 350) and 1-(4-amino-piperidin-1-yl)-ethanone to give 1-[4-[4-amino-5-(3-fluoro-6-methoxy-2-methyl-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-ethanone as a white solid. MS (M+H)+, 402.
The same procedure as described in Example 326 was used, starting with (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(3-fluoro-6-methoxy-2-methyl-phenyl)-methanone (Example 350) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(3-fluoro-6-methoxy-2-methyl-phenyl)-methanone. MS (M+H)+, 438
To a solution of 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide (500 mg, 2.06 mmol, Example 1) in dry tetrahydrofuran (6 mL) at −30˜−40° C., was added 4-tolylmagnesium bromide (10.5 mL, Aldrich). The reaction was stirred at the same temperature for 2 hours before quenching with aqueous ammonium chloride. Work-up and purification as in Example 47 gave (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-p-tolyl-methanone as a white solid. MS (M+H)+, 274.
The same procedure as described in Example 325 was used, starting from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-p-tolyl-methanone (Example 353) to give (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-p-tolyl-methanone as a white solid. MS (M+H)+: 290
The same procedure as described in Example 326 was used, starting with (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-p-tolyl-methanone (Example 354) and 1-(4-amino-piperidin-1-yl)-ethanone to give 1-[4-[4-amino-5-(4-methyl-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-ethanone as a white solid. MS (M+H)+, 354.
The same procedure as described in Example 326 was used, starting with (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-p-tolyl-methanone (Example 354) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-p-tolyl-methanone. MS (M+H)+, 390
The same procedure as described in Example 353 was used, starting from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide (Example 1) and 4-methoxyphenylmagnesium bromide (Aldrich) to give (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-4-methoxy-phenyl-methanone. MS (M+H)+, 290.
The same procedure as described in Example 325 was used, starting from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-4-methoxy-phenyl-methanone (Example 357) to give (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(4-methoxy-phenyl)-methanone as a white solid. MS (M+H)+: 306
The same procedure as described in Example 326 was used, starting with (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(4-methoxy-phenyl)-methanone (Example 358) and 1-(4-amino-piperidin-1-yl)-ethanone to give 1-[4-[4-amino-5-(4-methoxyl-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-ethanone as a white solid. MS (M+H)+, 370.
The same procedure as described in Example 326 was used, starting with (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(4-methoxy-phenyl)-methanone (Example 358) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(4-methoxy-phenyl)-methanone. MS (M+H)+, 406
The same procedure as described in Example 353 was used, starting from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide (Example 1) and 4-chlorophenyl magnesium bromide (Aldrich) to give (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(4-chloro-phenyl)-methanone. MS (M+H)+, 294.
The same procedure as described in Example 325 was used, starting from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(4-chloro-phenyl)-methanone (Example 361) to give (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(4-chloro-phenyl)-methanone as a white solid. MS (M+H)+: 310
The same procedure as described in Example 326 was used, starting with (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(4-chloro-phenyl)-methanone (Example 362) and 1-(4-amino-piperidin-1-yl)-ethanone to give 1-[4-[4-amino-5-(4-chloro-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-ethanone as a white solid. MS (M+H)+, 374.
The same procedure as described in Example 326 was used, starting with (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(4-chloro-phenyl)-methanone (Example 362) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(4-chloro-phenyl)-methanone. MS (M+H)+, 410
The same procedure as described in Example 353 was used, starting from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide (Example 1) and 4-fluorophenyl magnesium bromide (Aldrich) to give (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(4-fluoro-phenyl)-methanone. MS (M+H)+, 278.
The same procedure as described in Example 325 was used, starting from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(4-fluoro-phenyl)-methanone (Example 365) to give (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(4-fluoro-phenyl)-methanone as a white solid. MS (M+H)+: 294
The same procedure as described in Example 326 was used, starting with (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(4-fluoro-phenyl)-methanone (Example 366) and 1-(4-amino-piperidin-1-yl)-ethanone to give 1-[4-[4-amino-5-(4-fluoro-benzoyl)-pyrimidin-2-ylamino]-piperidin-1-yl]-ethanone as a white solid. MS (M+H)+, 358.
The same procedure as described in Example 326 was used, starting with (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(4-fluoro-phenyl)-methanone (Example 366) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(4-fluoro-phenyl)-methanone. MS (M+H)+, 394
The same procedure as described in Example 348 was used to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2,4-dimethoxy-phenyl)-methanone as a white solid. MS (M+H)+, 454.
The same procedure as described in Example 349 was used to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2,4-dimethoxy-phenyl)-methanone as a white solid. MS (M+H)+, 468.
The same procedure as described in Example 47 was used, starting from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide (Example 1) and 4-bromo-2-fluoroanisole (Aldrich), to give (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(3-fluoro-4-methoxy-phenyl)-methanone as a white solid. MS (M+H)+, 308.
The same procedure as described in Example 3 was used, starting with (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-phenyl)-methanone (Example 371) to give (4-amino-2-ethanesulfonyl-pyrimidin-5-yl)-(5-fluoro-4-methoxy-phenyl)-methanone as a white solid. MS (M+H)+: 340.
The same procedure as described in Example 90 was used starting with material from Example 372 and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(3-fluoro-4-methoxy-phenyl)-methanone as a white solid. MS (M+H)+: 424.
The same procedure as described in Example 326 was used, starting with (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-4-methyl-phenyl)-methanone (Example 341) and 4-amino-1-Boc-piperidine (Astatech) to give 4-[4-amino-5-(5-fluoro-2-methoxy-4-methyl-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid tert-butyl ester as a white solid. MS (M+H)+, 460.
To a solution of 3-chloro-4-fluoroanisole (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-chloro4-fluoro-2-iodoanisole (5.0 g). MS (M+H)+, 285.
To a solution of 5-chloro-4-fluoro-2-iodoanisole (1.0 g, Example 375) was added isopropylmagnesium chloride (1.92 mL, 2M in tetrahydrofuran, Aldrich) and the reaction was stirred at −40 to −35° C. for 40 minutes before 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide (170 mg, Example 1) in tetrahydrofuran (4 mL) was added. The resulting reaction was quenched with aqueous ammonium chloride. Work-up and purification as in Exam pie 47 gave (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(4-chloro-5-fluoro-2-methoxy-phenyl)-methanone. MS (M+H)+, 342.
The same procedure as described in Example 325 was used, starting from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(4-chloro-5-fluoro-2-methoxy-phenyl)-methanone (Example 376) to give (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(4-chloro-5-fluoro-2-methoxy-phenyl)-methanone as a white solid. MS (M+H)+: 358
The same procedure as described in Example 326 was used, starting with (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(4-chloro-5-fluoro-2-methoxy-phenyl)-methanone (Example 377) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(4-chloro-5-fluoro-2-methoxy-phenyl)-methanone as a white solid. MS (M+H)+, 458.
The same procedure as described in Example 376 was used, starting with 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide (Example 1) and 2-bromo-4,6-difluoroanisole (Metrix) to give (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(3,5-difluoro-2-methoxy-phenyl)-methanone. MS (M+H)+, 326.
The same procedure as described in Example 325 was used, starting from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(3,5-difluoro-2-methoxy-phenyl)-methanone (Example 379) to give (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(3,5-difluoro-2-methoxy-phenyl)-methanone. MS (M+H)+: 342
The same procedure as described in Example 326 was used, starting with (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(3,5-difluoro-2-methoxy-phenyl)-methanone (Example 380) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(3,5-difluoro-2-methoxy-phenyl)-methanone as a white solid. MS (M+H)+, 442.
The same procedure as described in Example 375 was used, starting from 4-fluoro-2-methylanisole (Aldrich), to give 1-fluoro-2-iodo-4-methoxy-5-methyl-benzene, MS (M+H)+, 267 and 1-fluoro-3-iodo-4-methoxy-5-methyl-benzene, MS (M+H)+, 267.
The same procedure as described in Example 376 was used, starting from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide (Example 1) and 1-fluoro-2-iodo-4-methoxy-5-methyl-benzene (Example 382) to give (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2-fluoro-5-methoxy-4-methyl-phenyl)-methanone. MS (M+H)+, 322.
The same procedure as described in Example 325 was used, starting from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(2-fluoro-5-methoxy-4-methyl-phenyl)-methanone (Example 383) to give (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(2-fluoro-5-methoxy4-methyl-phenyl)-methanone as a white solid. MS (M+H)+: 338
The same procedure as described in Example 326 was used, starting with (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(2-fluoro-5-methoxy-4-methyl-phenyl)-methanone (Example 384) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(2-fluoro-5-methoxy-4-methyl-phenyl)-methanone as a white solid. MS (M+H)+, 438.
The same procedure as described in Example 376 was used, starting from 4-amino-2-ethylsulfanyl-pyrimidine-5-carboxylic acid methoxy-methyl-amide (Example 1) and 1-fluoro-3-iodo-4-methoxy-5-methyl-benzene (Example 382) to give (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-3-methyl-phenyl)-methanone. MS (M+H)+, 322.
The same procedure as described in Example 325 was used, starting from (4-amino-2-ethylsulfanyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-3-methyl-phenyl)-methanone (Example 386) to give (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-3-methyl-phenyl)-methanone as a white solid. MS (M+H)+: 338
The same procedure as described in Example 326 was used, starting with (4-amino-2-ethanesulfinyl-pyrimidin-5-yl)-(5-fluoro-2-methoxy-3-methyl-phenyl)-methanone (Example 387) and 1-methanesulfonyl-piperidin-4-ylamine; compound with trifluoroacetic acid (Example 162) to give [4-amino-2-(1-methanesulfonyl-piperidin-4-ylamino)-pyrimidin-5-yl]-(5-fluoro-2-methoxy-3-methyl-phenyl)-methanone as a white solid. MS (M+H)+, 438.
A solution of (4-amino-2-chloro-pyrimidin-5-yl)-(2,3-difluoro-6-methoxy-phenyl)-methanone (120 mg, 0.40 mmol, Example 289) and 4-amino-benzenesulfonamide (260 mg, 1.51 mmol, Aldrich) in isopropyl alcohol (30 mL) was stirred for 2 hours at 150° C. in a sealed pressure bottle. The reaction was cooled and poured into water (˜60 mL) and the resulting precipitate was filtered and air dried. This residue was purified by silica gel chromatography (2% triethylamine/ethyl acetate) to give 4-[4-amino-5-(2,3-difluoro-6-methoxy-benzoyl)-pyrimidin-2-ylamino]-benzenesulfonamide as a white solid. MS: M+H; 436
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 IC50 values and Ki values of less than 1.0 μM. Additionally, the antiproliferative potency of some compounds of the invention was tested in the human colon tumor cell line HCT116 with IC90 values reported from an MTT assay of less than 30 μM, preferably less than 5 μM.
A: IC50 Measurement
To determine inhibition of Cdk4, Cdk2 and Cdk1 activity, kinase assays were conducted using FlashPlate™ assays (NEN™-Life Science Products). FlashPlate assays were performed 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, Tex. Proteins were co-expressed in High Five™ insect cells and the complex was purified on glutathione Sepharose resin (Pharmacia, Piscataway, N.J.) 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 62 Kd protein was under the control of an IPTG inducible promoter in an M15 E. coli 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 NaCl, and 1 mM DTT. Purified Rb fusion protein stocks were quantitated for protein concentration, aliquoted, and stored at −70° C.
For all three kinase assays reported herein, 96-well FlashPlates were coated with Rb protein at 10 μg/ml, using 100 ul per well. Plates were incubated at 4° C. overnight or at room temperature for 3 hours on a shaker. To control for nonspecific phosphorylation, one row of wells was coated with 100 μl/well coating buffer (20 mM HEPES, 0.2 M NaCl). Plates were then washed twice with wash buffer (0.01% Tween 20 in phosphate-buffered saline). Compounds to be tested (“test compounds”) were added to the wells at 5× final concentration. Reactions were initiated by immediate addition of 40 μl reaction mix (25 mM HEPES, 20 mM MgCl2, 0.002% Tween 20, 2 mM DTT, 1 μM ATP, 4 nM 33P-ATP) and a sufficient amount of enzyme to give counts that were at least 10-fold above background. Plates were incubated at room temperature on a shaker for 30 minutes. Plates were washed four times with the wash buffer, sealed, and counted on the TopCount scintillation counter (Packard Instrument Co., Downers Grove, Ill.]. The percent inhibition of Rb phosphorylation, which is a measure of the inhibition of CDK activity, was determined according to the following formula:
where “test compound” refers to the average counts per minute of the test duplicates, “nonspecific” refers to the average counts per minute when no CyclinD/Cdk4, etc., was added, and “total” refers to the average counts per minute when no compound was added. The IC50 value is the concentration of test compound that reduces by 50% the protein-kinase induced incorporation of the radiolabel under the test conditions described.
B: KI Measurement
Alternatively, inhibition activity may be measured using Ki. Using the protein constructs described above in Example 390 A 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 3× their respective Kms for ATP.
In the CDK4 assay, test compounds were diluted to 3× their final concentrations in 25 mM Hepes, pH 7.0, 6.25 mM MgCl2, 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, 6.25 mM MgCl2, 0.003% Tween-20, 0.3 mg/ml 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 1.6 uM 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-His6 (PerkinElmer Life Sciences) in 25 mM Hepes, pH 7.0, 0.5 mg/ml BSA were added. Following a one hour incubation at 37 deg 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 665 nm, normalized for europium readings at 615 nm. 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 and Km refers to the Michaelis-Menten constant.
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 10 mM MgCl2. For CDK1 and CDK2, the respective reagent ATP concentrations were 162 uM and 90 uM. CDK1 at a reagent concentration of 0.15 ng/ul and CDK2 at a reagent concentration of 0.06 ng/ul were used. Reagent concentrations of detection reagents were adjusted between 3-12 nM Eu-Ab and 60-90 nM APC-antiHis 6 to give signal to background ratios of at least 10 to 1.
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° C. 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 37° C. 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)×100). The concentration at which 90% inhibition of cell proliferation is obtained (the lC90) is determined from the linear regression of a plot of the logarithm of the concentration versus percent inhibition.
Manufacturing Procedure:
Manufacturing Procedure:
Manufacturing Procedure:
Manufacturing Procedure:
While 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.
This application is a division of U.S. application Ser. No. 10/770,379, filed Feb. 2, 2004, now U.S. Pat. No. 7,157,455, This application claims the benefit of Provisional Applications Ser. No. 60/514,926, filed Oct. 28, 2003, and Ser. No. 60/446,273, filed Feb. 10, 2003.
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| Number | Date | Country | |
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| 20060229330 A1 | Oct 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60514926 | Oct 2003 | US | |
| 60446273 | Feb 2003 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10770379 | Feb 2004 | US |
| Child | 11471001 | US |
