NO334841B1 - Pyrimidine derivatives, processes for their preparation, pharmaceutical compositions comprising such and such compounds for the treatment of abnormal cell growth - Google Patents

Abstract

The present invention relates to a compound of formula (I) wherein R 1 -R 4 are as defined herein. Such novel pyrimidine derivatives are useful in the treatment of abnormal cell growth, such as cancer, in mammals. The present invention also relates to a method of using such compounds in the treatment of abnormal cell growth in mammals, in particular humans and pharmaceutical compositions containing such compounds.

Description

Background for the invention

The present invention relates to pyrimidine derivatives, a method for their preparation, pharmaceutical preparations comprising such and such compounds for the treatment of abnormal cell growth.

It is known that a cell can become cancerous by the transformation of part of its DNA into an oncogene (ie a gene which, when activated, leads to the formation of malignant tumor cells). Many oncogenes encode proteins that are aberrant tyrosine kinases capable of causing cell transformation. Alternatively, the overexpression of a normal proto-oncogene tyrosine kinase can also result in proliferative disorders, sometimes resulting in a malignant phenotype.

Receptor tyrosine kinases are enzymes that span the cell membrane and have an extracellular binding domain for growth factors such as epidermal growth factor, a transmembrane domain and an intracellular part that functions as a kinase to phosphorylate specific tyrosine residues in proteins and thus influence cell proliferation. Other receptor tyrosine kinases include c-erbB-2, c-met, tie-2, PDGFr, FGFr and VEGFR. It is known that such kinases are often aberrantly expressed in common human cancers such as breast cancer, gastrointestinal cancer such as colon, rectal or stomach cancer, leukemia and ovarian, bronchial or pancreatic cancer. It has also been shown that epidermal growth factor receptor (EGFR), which has tyrosine kinase activity, is mutated and/or overexpressed in many human cancers such as brain, lung, squamous cell, bladder, stomach, breast, head and neck, oesophageal, gynecological and thyroid tumours.

Accordingly, it has been known that inhibitors of receptor tyrosine kinases are useful as selective inhibitors of the growth of mammalian cancer cells. For example, erbstatin, a tyrosine kinase inhibitor, selectively impairs growth in athymic nude mice of a transplanted human breast carcinoma expressing epidermal growth factor receptor tyrosine kinase (EGFR), but is without effect on growth of another carcinoma that does not express EGF receptor . Thus, selective inhibitors of certain receptor tyrosine kinases are useful in the treatment of abnormal cell growth, especially cancer, in mammals. In addition to receptor tyrosine kinases, selective inhibitors of certain non-receptor tyrosine kinases, such as FAK (focal adhesion kinase), lek, src, abl or serine/threonine kinases (eg: cyclin-dependent kinases , useful in the treatment of abnormal cell growth, especially cancer, in mammals.FAK is also known as protein tyrosine kinase 2, PTK2.

Compelling evidence indicates that FAK, a cytoplasmic, non-receptor tyrosine kinase, plays an essential role in cell-matrix signal transduction pathways (Clark and Brugge 1995, Science 268: 233-239) and that its aberrant activation is associated with an increase in metastatic potential of tumors (Owens et al. 1995, Cancer Research 55: 2752-2755). FAK was originally identified as a 125 kDa protein highly tyrosine-phosphorylated in cells transformed by v-Src. FAK was subsequently found to be a tyrosine kinase that localizes to focal adhesions, which are points of contact between cultured cells and their underlying substratum and sites of intense tyrosine phosphorylation. FAK is phosphorylated and, thus, activated in response to extracellular matrix (ECM) binding to integrins. Recently, studies have demonstrated that an increase in FAK mRNA levels accompanied by invasive transformation of tumors and attenuation of expression of FAK (through the use of antisense oligonucleotides) induces apoptosis in tumor cells (Xu et al. 1996, Cell Growth and Piff. 7: 413 -418). In addition to being expressed in most tissue types, FAK is found at elevated levels in most human cancers, especially in highly invasive metastases.

Various compounds, such as styrene derivatives, have also been shown to have tyrosine kinase inhibitory properties. Five European patent publications, ie EP 0 566 226 A1 (published 20 October 1993), EP 0 602 851 A1 (published 22 June 1994), EP 0 635 507 A1 (published 25 January 1995), EP 0 635 498 A1 (published January 25, 1995) and EP 0 520 722 A1 (published December 30, 1992), refer to certain bicyclic derivatives, especially quinazoline derivatives, which have anti-cancer properties resulting from their tyrosine kinase inhibitory properties.

WO patent application WO 92/20642 (published November 26, 1992) also discloses certain bis-mono- and bicyclic aryl and heteroaryl compounds as tyrosine kinase inhibitors useful in inhibiting abnormal cell proliferation. WO Patent Applications WO96/16960 (published June 6, 1996), WO 96/09294 (published March 6, 1996), WO 97/30034 (published August 21, 1997), WO 98/02434 (published January 22, 1998), WO 98/02437 (published January 22, 1998) and WO 98/02438 (published January 22, 1998) also refer to substituted bicyclic heteroaromatic derivatives as tyrosine kinase inhibitors useful for the same purpose.

U.S. Patent Application No. 60/435,670, filed December 20, 2002 (Attorney Docket No. PC25339) relates to a broad class of novel pyrimidine derivatives that are selective inhibitors of FAK. As such, these compounds are useful in the treatment of abnormal cell growth.

Accordingly, a need exists for additional selective inhibitors of certain receptor and non-receptor tyrosine kinases useful in the treatment of abnormal cell growth, such as cancer, in mammals. The present invention provides pyrimidine derivatives, a method for their preparation, pharmaceutical preparations comprising such as well as such compounds for the treatment of abnormal cell growth.

Summary of the invention

The present invention relates to a compound of formula 1

where

R<1> and R2 are each a hydrogen atom, or R<1> and R<2> taken together with the carbon atom to which they are attached form a 2-hydroxy-indan-1-yl ring;

R3 is selected from

(a) a hydrogen atom when R<1> and R<2> together form an indanyl ring as indicated above, (b) -(C6-C10)aryl optionally substituted with from one to three groups selected from

-SO2(Ci-C6)alkyl, SO2NH2, -N((Ci-C6)alkyl)(SO2(Ci-C6)alkyl) and halogen,

(c) a heteroaryl group selected from chromanyl or furanyl, each optionally substituted with -(C 1 -C 6 )alkyl, (d) -(C 3 -C 10 )cycloalkyl or -(C 3 -C 10 )cycloalkenyl, both optionally substituted with hydroxy, (e ) morpholinyl, pyrrolidinyl or pyridinyl, each optionally substituted with

-SO2(CrC6)alkyl or -N((CrC6)alkyl)(SO2(CrC6)alkyl, or

(f) (C 1 -C 6 )alkyl substituted with a group selected from -N((C 1 -C 6 )alkyl)(SO 2 (C 1 -C 6 )alkyl) and O(C 1 -C 6 )alkyl,

n is an integer from 1 to 3.

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Preferred is a compound with formula 1 above where R<1> and R<1> are hydrogen and n is 1.

Further preferred is the compound of formula 1 above where R3 is -(C6-Cio)aryl, optionally substituted with one to three groups independently selected from the group consisting of -SO2(Ci-C6)alkyl, -SO2NH2,-N((Ci. C 6 )alkyl)(SO 2 (C 1 -C 6 )alkyl) and halogen; or a heteroaryl group selected from chromanyl or furanyl, each optionally substituted with -(C 1 -C 6 )alkyl.

Further preferred is compound of formula 1 above, where R3 is selected from the group consisting of

(d) -(C 3 -C 10 )cycloalkyl or -(C 3 -C 10 )cycloalkenyl, both optionally substituted with hydroxy, (e) morpholinyl, pyrrolidinyl or pyridinyl, each optionally substituted with - SO 2 (C 1 -C 6 )alkyl or -N( (C 1 -C 6 )alkyl)(SO 2 (C 1 -C 6 )alkyl, and (f) (C 1 -C 6 )alkyl substituted with a group selected from -N((C 1 -C 6 )alkyl)(SO 2 (C 1 -C 6 )alkyl) and O(C 1 -C 6 )alkyl.

Also further preferred is compound of formula 1 above, where R<3> is -(Ci-Ce)alkyl substituted with one group selected from the group consisting of

-N((C 1 -C 6 )alkyl)(SO 2 (C 1 -C 6 )alkyl) and -O(C 1 -C 6 )alkyl.

Even more preferred is the compound described above with formula 2

where A is selected from the group consisting of: where m is an integer from 0-3 and R<13>is a substituent selected from the group consisting of (b) -SO2(CrC6)alkyl, SO2NH2, -N((CrC6) alkyl)(SO 2 (C 1 -C 6 )alkyl) and halogen when A is -(C 6 -C 10 )aryl;

(c) -(C 1 -C 6 )alkyl when A is a heteroaryl group; and

(e) -SO 2 (C 1 -C 6 )alkyl or -N((C 1 -C 6 )alkyl)(SO 2 (C 1 -C 6 )alkyl) when A is a pyridyl group. Also even more preferred is the compound described above with formula 3

where B is selected from the group consisting of:

where m is an integer from 0-6 and R<13>is a hydroxy group.

Also even more preferred is the compound described above with formula 4

where D is selected from the group consisting of:

where q is an integer from 1-2, m is an integer from 0-5 and R<13>s

(c) -(C 1 -C 6 >alkyl when D is a heteroaryl group,

(d) hydroxy when D is -(C 3 -C 10 )cycloalkyl or -(C 3 -C 10 )cycloalkenyl, (e) -SO 2 (C 1 -C 6 )alkyl or -N((C 1 -C 6 )alkyl)(SO 2 (C 1 -C 6 )alkyl) C6)alkyl when D is morpholinyl, pyrrolidinyl or pyridinyl.

Even more preferred is compound of formula 1, selected from the group consisting of: 5-[4-(3-methanesulfonyl-benzylamino)-5-trifluoromethyl-pyrimidin-2-ylamino]-1,3-dihydro-indol-2-one ;

Ethanesulfonic acid methyl-{3-[2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-propyl}-amide;

5-{4-[(isocroman-1-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one;

5-{4-[2-(pyridin-3-yloxy)-propylamino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one;

3-{[2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-methyl}-benzenesulfonamide;

5-{4-[(1-methanesulfonyl-piperidin-3-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one;

N-(3-{[2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-methyl}-phenyl)-methanesulfonamide;

N-methyl-N-{2-[2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-ethyl}-methanesulfonamide;

5-{4-[(4-methanesulfonyl-morpholin-2-ylmethyl)-amino]-5-trifluoromethyl pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one;

5-[4-(3-methanesulfonylmethyl-benzylamino)-5-trifluoromethyl-pyrimidin-2-ylamino]-1,3-dihydro-indol-2-one;

5-{4-[(1-methanesulfonyl-pyrrolidin-3-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one;

N-methyl-N-{3-[2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-propyl}-methanesulfonamide;

5-{4-[2-(1-methanesulfonyl-piperidin-2-yl)-ethylamino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one;

5-{4-[(4-methanesulfonyl-pyridin-2-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one;

5-[4-(3-isopropoxy-propylamino)-5-trifluoromethyl-pyrimidin-2-ylamino]-1,3-dihydro-indol-2-one;

5-{4-[(5-methyl-furan-2-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one;

5-{4-[(bicyclo[2,2,1]hept-5-en-2-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2- on;

N-(4-fluoro-3-{[2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-methyl}-phenyl)- N-methyl-methanesulfonamide;

5-{4-[(1-methanesulfonyl-piperidin-3-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one;

5-{4-[(6-methanesulfonyl-pyridin-2-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one;

5-{4-[(5-methanesulfonyl-pyridin-3-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one;

5-[4-(2-methanesulfonyl-benzylamino)-5-trifluoromethyl-pyrimidin-2-ylamino]-1,3-dihydro-indol-2-one;

5-{4-[(1-pyrimidin-2-yl-piperidin-3-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one;

5-{4-[2-(1-methanesulfonyl-piperidin-2-yl)-ethylamino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one;

5-{4-[2-(1-methanesulfonyl-piperidin-2-yl)-ethylamino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one;

N-(2-{[2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-methyl}-phenyl)-methanesulfonamide;

5-{4-[(1-methanesulfonyl-pyrrolidin-2-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one;

N-methyl-N-(2-{[2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-methyl}-phenyl)- methanesulfonamide;

N-methyl-N-(2-methyl-6-{[2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-methyl} -phenyl)-methanesulfonamide;

5-[4-(2-hydroxy-indan-1-ylamino)-5-trifluoromethyl-pyrimidin-2-ylamino]-1,3-dihydro-indol-2-one;

5-{4-[(1-hydroxy-cyclopentylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one;

5-{4-[2-hydroxy-2-(1-methanesulfonyl-piperidin-2-yl)-ethylamino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one; and

N-methyl-N-(3-{[2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-methyl}-pyridin-2 -yl)-methanesulfonamide

Also covered is the method for producing the compound of formula 1

where

R<1> and R<2> are each a hydrogen atom, or R<1> and R2 taken together with the carbon atom to which they are attached form a 2-hydroxy-indan-1-yl ring;

R3 is selected from

(a) a hydrogen atom when R<1> and R<2> together form an indanyl ring as indicated above, (b) -(C 6 -C 10 )aryl optionally substituted with from one to three groups selected from -SO 2 (C 1 -C 6 ) alkyl, SO 2 NH 2 , -N((C 1 -C 6 )alkyl)(SO 2 (C 1 -C 6 )alkyl) and halogen, (c) a heteroaryl group selected from chromanyl or furanyl, each optionally substituted with -(C 1 -C 6 )alkyl, ( d) -(C3-C10)cycloalkyl or -(C3-C10)cycloalkenyl, both optionally substituted with hydroxy, (e) morpholinyl, pyrrolidinyl or pyridinyl, each optionally substituted with

-SO2(CrC6)alkyl or -N((CrC6)alkyl)(SO2(CrC6)alkyl, or

(f) (C 1 -C 6 )alkyl substituted with a group selected from -N((d -C 6 )alkyl)(SO 2 (C 1 -C 6 )alkyl) and O(C 1 -C 6 )alkyl,

n is an integer from 1 to 3.

or a pharmaceutically acceptable salt, solvate or hydrate thereof;

where the method comprises the steps

(a) react 5-amino-oxindole, 2 with dichloropyrimidine 3 in the presence of a Lewis acid and a suitable base to prepare a 2-substituted pyrimidine 4; (b) react pyrimidine 4 with NH2-(CR<1>R2)nR<3>In the presence of a suitable base to give a compound of formula 1.

The invention also relates to the compound according to claim 1, for the treatment of abnormal cell growth in a mammal.

The invention further relates to a pharmaceutical preparation for the treatment of abnormal cell growth in a mammal comprising an amount of a compound according to claim 1 which is effective for the treatment of abnormal cell growth, and a pharmaceutically acceptable carrier.

The abnormal cell growth can be cancer, including lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tube, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland , soft tissue sarcoma, cancer of the urethra, cancer of the penis, cancer of the prostate, chronic or acute leukemia, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or urethra, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, spinal cord tumours, brainstem glioma, pituitary adenoma or a combination of one or more of the preceding cancers.

The abnormal cell growth can be solid cancer tumors such as breast, lung, colon, brain, prostate, stomach, pancreas, ovary, skin (melanoma), endocrine, uterine, testicular and bladder cancer.

The abnormal cell growth can also be a benign proliferative disease, including psoriasis, benign prostatic hypertrophy or restinosis.

Anti-angiogenesis agents, such as MMP-2 (matrix metalloproteinase 2) inhibitors, MMP-9 (matrix metalloproteinase 9) inhibitors, and COX-II

(cyclooxygenase II) inhibitors, can be used together with a compound of formula 1 in the methods and pharmaceutical preparations described here. Examples of useful COX-II inhibitors include CELEBREX™

(alecoxib), valdecoxib and rofecoxib. Examples of useful matrix metalloproteinase inhibitors are described in WO 96/33172 (published October 24, 1996), WO 96/27583 (published March 7, 1996), European Patent Application No. 97304971.1 (filed July 8, 1997), European Patent Application No. 99308617.2 (filed October 29, 1999), WO 98/07697 (published February 26, 1998), WO 98/03516 (published January 29, 1998), WO 98/34918 (published August 13, 1998), WO 98/34915 (published August 13, 1998), WO 98/33768 (published August 6, 1998), WO 98/30566 (published July 16, 1998), European Patent Publication 606,046 (published July 13, 1994), European Patent Publication 931,788 ( published 28 July 1999), WO 90/05719 (published 31 May 1990), WO 99/52910 (published 21 October 1999), WO 99/52889 (published 21 October 1999), WO 99/29667 (published 17 . June 1999), PCT International Application No. PCT/IB98/01113 (filed 21 July 1998), European Patent Application No. 99302232.1 (filed 25 March 1999), UK Patent Application No. 9912961.1 (in filed June 3, 1999), US Provisional Application No. 60/148,464 (filed August 12, 1999), US Patent 5,863,949 (issued January 26, 1999), US Patent 5,861,510 (issued January 19, 1999) and European Patent Publication 780,386 (published 25 June 1997). Preferred MMP-2 and MMP-9 inhibitors are those that have little or no activity for inhibiting MMP-1. More preferred are those that selectively inhibit MMP-2 and/or MMP-9 in relation to the other matrix metalloproteinases (ie MMP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7 , MMP-8, MMP-10, MMP-11, MMP-12 and MMP-13).

Some specific examples of MMP inhibitors useful in combination with the compounds above are AG-3340, RO 32-3555, RS 13-0830 and the compounds indicated in the following list: 3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl ]-(1-hydroxycarbamoyl-cyclopentyl)-amino]-propionic acid;

3- exo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylic acid-hydroxyamide;

(2R,3R)-1-[4-(2-chloro-4-fluoro-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylic acid hydroxyamide;

4-[4-(4-Fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylic acid hydroxyamide;

3-[[4-(4-Fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclobutyl)-amino]-propionic acid;

4-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylic acid hydroxyamide;

3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-3-carboxylic acid hydroxyamide;

(2R,3R)-1-[4-(4-fluoro-2-methyl-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylic acid hydroxyamide;

3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-1-methyl-ethyl)-amino]-propionic acid;

3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(4-hydroxycarbamoyl-tetrahydro-pyran-4-yl)-amino]-propionic acid;

3-exo-3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylic acid-hydroxyamide;

3-endo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3,2,1]octane-3-carboxylic acid hydroxyamide; and

3-[4-(4-Fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-furan-3-carboxylic acid hydroxyamide;

and pharmaceutically acceptable salts, solvates and prodrugs of said compounds.

The compounds of formula 1 and the pharmaceutically acceptable salts, solvates and prodrugs thereof can also be used in combination with signal transduction inhibitors, such as agents that can inhibit EGFR (epidermal growth factor receptor) responses, such as EGFR antibodies, EGF antibodies and molecules that are EGFR inhibitors; VEGF (vascular endothelial growth factor) inhibitors; and erbB2 receptors or inhibitors, such as organic molecules or antibodies that bind to the erbB2 receptor, such as HERCEPTIN™ (Genentech, Inc. of South San Francisco, California, USA).

EGFR inhibitors are described in, for example, WO 95/19970 (published July 27, 1995), WO 98/14451 (published April 9, 1998), WO 98/02434 (published January 22, 1998) and US Patent 5,747,498 (assigned 5 May 1998). EGFR inhibitory agents include CI-1033 (Pfizer Inc.), the monoclonal antibodies C225 and anti-EGFR 22Mab (ImClone Systems Incorporated of New York, New York, USA), compounds ZD-1839 (AstraZeneca), BIBX-1382 (Boehringer Ingelheim), MDX-447 (Medarex Inc. of Annandale, New Jersey, USA) and OLX-103 (Merck & Co. Whitehouse Station, New Jersey, USA), VRCTC-310 (Ventech Research) and EGF fusion toxin (Seragen Inc . in Hopkinton, Massachusettes).

VEGF inhibitors, for example CP-547,632 and AG-13736 (Pfizer, Inc.), SU-5416 and SU-6668 (Sugen Inc. of South San Francisco, California, USA), can also be combined with a compound of formula 1 .VEGF inhibitors are described in, for example, WO 99/24440 (published May 20, 1999), PCT International Application PCT/IB99/00797 (filed May 3, 1999), in WO 95/21613 (published August 17, 1995), WO 99/61422 (published December 2, 1999), US Patent 5,834,504 (issued November 10, 1998), WO 98/50356 (published November 12, 1998), US Patent 5,883,113 (issued March 16, 1999), US Patent 5,886,020 (issued March 23, 1999), US Patent 5,792,783 (issued August 11, 1998), WO 99/10349 (published March 4, 1999), WO 97/32856 (published September 12, 1997), WO 97/22596 (published June 26, 1997), WO 98/54093 (published December 3, 1998), WO 98/02438 (published January 22, 1998), WO 99/16755 (published April 8, 1999) and WO 98/02437 (published 22 January 1998). Other examples of some specific VEGF inhibitors are IM862 (Cytran Inc. of Kirkland, Washington, USA); anti-VEGF monoclonal antibody from Genentech, Inc. of South San Francisco, California; and angiozyme, a synthetic ribozyme from Ribozyme (Boulder, Colorado) and Chiron (Emeryville, California).

ErbB2 receptor inhibitors, such as CP-724,714 (Pfizer, Inc.), GW-282974 (Glaxo Wellcome plc) and the monoclonal antibodies AR-209 (Aronex Pharmaceuticals Inc. of The Woodlands, Texas, USA) and 2B-1 (Chiron), can be administered in combination with a compound of formula 1. Such erbB2 inhibitors include those described in WO 98/02434 (published January 22, 1998), WO 99/35146 (published July 15, 1999), WO 99/ 35132 (published July 15, 1999), WO 98/02437 (published January 22, 1998), WO 97/13760 (published April 17, 1997), WO 95/19970 (published July 27, 1995), US Patent 5,587,458 ( granted December 24, 1996) and US Patent 5,877,305 (granted March 2, 1999). ErbB2 receptors or inhibitors applicable to the present invention are also described in US Provisional Application No. 60/117,341, filed on January 27, 1999 and in US Provisional Application No. 60/117,346, filed on January 27, 1999.

Other antiproliferative agents that can be used with the compounds above include inhibitors of HDI (CI-994, Pfizer Inc.), MEK (CI-1040, Pfizer Inc.), the enzyme farnesyl protein transferase and the receptor tyrosine kinase PDGFr, including the compounds described and claimed in the following US patent applications: 09/221946 (filed Dec. 28, 1998); 09/454058 (filed Dec. 2, 1999); 09/501163 (filed Feb. 9, 2000); 09/539930 (filed Mar. 31, 2000); 09/202796 (filed May 22, 1997); 09/384339 (filed Aug. 26, 1999); and 09/383755 (filed Aug. 26, 1999); and the compounds described and claimed in the following US provisional patent applications: 60/168207 (filed November 30, 1999); 60/170119 (filed Dec. 10, 1999); 60/177718 (filed Jan. 21, 2000); 60/168217 (filed 30 November 1999) and 60/200834 (filed 1 May 2000). The compounds according to the invention can also be used in combination with inhibitors of topoisomerase I, e.g. irinotecan (Camptosar®) and edotecarin. A compound of formula 1 may also be used with other agents useful for the treatment of abnormal cell growth or cancer, including agents capable of enhancing antitumor immune responses, such as CTLA4 (cytotoxic lymphocyte antigen 4) antibodies and other agents capable of to block CTLA4; and anti-proliferative agents such as other farnesyl protein transferase inhibitors, for example the farnesyl protein transferase inhibitors described in the references listed in the "Background" section, supra. Specific CTLA4 antibodies that can be used in the present invention include those described in US Provisional application 60/113,647 (filed December 23, 1998).

"Abnormal cell growth", as used herein, unless otherwise indicated, refers to cell growth that is independent of normal regulatory mechanisms (eg, loss of contact inhibition). This includes abnormal growth of: (1) tumor cells (tumors) that proliferate by expression of a mutated tyrosine kinase or overexpression of a receptor tyrosine kinase; (2) benign and malignant cells in other proliferative diseases where aberrant tyrosine kinase activation occurs; (4) any tumors that proliferate by receptor tyrosine kinases; (5) any tumors that proliferate by aberrant serine/threonine kinase activation; and (6) benign and malignant cells in other proliferative diseases where aberrant serine/threonine kinase activation occurs.

The compounds above are powerful inhibitors of FAK protein tyrosine kinases and are thus all suitable for therapeutic use as antiproliferative agents (e.g. anticancer agents), antitumor agents (e.g. effective against solid tumors), antiangiogenesis agents (e.g. .for stopping or preventing the proliferation of blood vessels) in mammals, especially in humans. In particular, the compounds above are useful for the prevention and treatment of a number of human hyperproliferative disorders such as malignant and benign tumors of the liver, kidney, bladder, breast, stomach, ovary, colorectum, prostate, pancreas, lung, vulva, thyroid, hepatic carcinomas, sarcomas , glioblastomas, head and neck and other hyperplastic disorders such as benign hyperplasia of the skin (eg psoriasis) and benign hyperplasia of the prostate (eg BPH). It is also expected that a compound as defined above may have activity against a number of leukemias and lymphoid malignancies.

In one preferred embodiment of the invention, cancer is selected from lung cancer, bone cancer, pancreatic cancer, gastric cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, gynecological, rectal cancer, cancer in the anal region, stomach cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tube, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, cancer of the oesophagus, cancer of the small intestine, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, squamous cell, prostate cancer, chronic or acute leukaemia, lymphocytic lymphomas, cancer of the bladder, cancer of the kidneys or urethra, renal cell carcinoma, carcinoma of the kidney pelvis, central nervous system (CNS) neoplasms, primary CNS lymphoma, spinal cord tumors, brain, pituitary adenoma or a combination of one or more of the preceding cancers.

In a more preferred embodiment, the cancer is selected from a solid tumor, such as in the breast, lung, colon, brain, prostate, stomach, pancreas, ovary, skin (melanoma), endocrine system, uterus, testicle and bladder.

The compounds above may also be applicable in the treatment of further disorders where aberrant expression of ligand/receptor interactions or activation or signaling events related to different protein tyrosine kinases are involved. Such disorders may include those of a neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic nature where aberrant function, expression, activation or signaling of erbB tyrosine kinases is involved. In addition, the compounds above may have therapeutic utility in inflammatory, angiogenic and immunological disorders involving both identified and hitherto unidentified tyrosine kinases which are inhibited by the compounds according to the present invention.

The term "treatment of", as used herein, unless otherwise indicated, means the reversal, alleviation, inhibition of the development of or prevention of the disorder or condition for which this term is used or one or more symptoms of such disorder or condition. The term "treatment", as used herein, unless otherwise indicated, denotes the effect of treatment as "treatment of" is defined immediately above.

The present invention also provides a pharmaceutical preparation comprising a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, as defined above, together with a pharmaceutically acceptable adjuvant, diluent or carrier.

The invention further provides a method for producing a pharmaceutical preparation according to the invention which comprises mixing a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, as defined above, with a pharmaceutically acceptable adjuvant, diluent or carrier.

For the above therapeutic uses, the dose administered will of course vary with the compound used, the method of administration, the treatment desired and the disorder indicated. The daily dose of the compound of formula (1)/salt/solvate (active ingredient) may be in the range from 1 mg to 1 gram, preferably 1 mg to 250 mg, more preferably 10 mg to 100 mg.

Detailed description of the invention

The compounds with formula 1 can be prepared using the synthetic route described in Scheme 1. The substituents in Scheme 1 have the same meaning as the substituents defined for formula 1. R<4> is hydrogen.

Compounds of formula 1 can be prepared by starting from 5-amino-oxindole (2) and pyrimidine (3). Combination of 3 with an equimolar amount of a Lewis acid at temperatures ranging from -15 to 45°C for a period of 10-60 minutes in an inert solvent (or solvent mixture) followed by addition of 2 and a suitable base provides after a period of 1-24 hours the intermediate 4-chloropyrimidine (4) in high yields. Examples of inert solvents include THF, 1,4-dioxane, n-BuOH, i-PrOH, dichloromethane and 1,2-dichloroethane. Examples of suitable bases used may include (i) non-nucleophilic organic bases, for example triethylamine or diisopropylethylamine (ii) inorganic bases such as potassium carbonate or cesium carbonate or (iii) resin-bound bases such as MP carbonate.

Examples of Lewis acids include halide salts of magnesium, copper, zinc, tin or titanium. In the next reaction, intermediate 4 is reacted with an amine of formula 5 either neat or in the presence of an inert solvent (or solvent mixture) at temperatures in the range from 0 to 150°C to give the compounds of formula 1. Optionally, this reaction can be carried out in presence of a suitable base. Examples of suitable solvents for this reaction include THF, 1,4-dioxane, DMF, N-methyl-pyrrolidinone, EtOH, n-BuOH, i-PrOH, dichloromethane, 1,2-dichloroethane, DMSO or acetonitrile. Suitable bases are as described above.

Compounds according to the present invention can be synthetically converted into other compounds according to the invention by techniques known to those skilled in the art. For illustrative purposes and without limitation, such methods include: a) removal of a protecting group by methods described in T.W. Greene and P.G.M. Wuts, "Protective Groups in Organic Synthesis", Second Edition, John Wiley and Sons, New York, 1991; e.g. removal of a BOC protecting group with an acid source such as HCl or trifluoroacetic acid.

b) displacement of a leaving group (halide, mesylate, tosylate, etc) with functional groups such as a primary or secondary amine, thiol

or alcohol to form a secondary or tertiary amine, thioether or ether, respectively.

c) treatment of phenyl (or substituted phenyl) carbamates with primary or secondary amines to form the corresponding ureas as in

Thavonekham, B et. eel. Synthesis (1997), 10, p.1189;

d) reduction of propargyl or homopropargyl alcohols or N-BOC-protected primary amines to the corresponding E-allylic or E-homoallylic

derivatives by treatment with sodium bis(2-methoxyethoxy)aluminum hydride (Red-AI) as in Denmark, S. E.; Jones, T. K. J. Org. Chem. (1982) 47, 4595-4597 or van Benthem, R.A.T.M.; Michels, J.J.; Speckamp, W. N. Synlett

(1994), 368-370; e) reduction of alkynes to the corresponding Z-alkene derivatives by treatment with hydrogen gas and a Pd catalyst as in Tomassy, B. et. eel.

Synth. Commun. (1998), 28, p. 1201

f) treatment of primary and secondary amines with an isocyanate, acid chloride (or other activated carboxylic acid derivative), alkyl/aryl chloroformate or

sulfonyl chloride to give the corresponding urea, amide, carbamate or sulfonamide; g) reductive amination of a primary or secondary amine using an aldehyde or ketone and a suitable reducing reagent. h) treating alcohols with an isocyanate, acid chloride (or other activated carboxylic acid derivative), alkyl/aryl chloroformate or sulfonyl chloride to give it

corresponding carbamate, ester, carbonate or sulphonic acid ester.

Amines of formula 5 can be obtained and used directly or alternatively prepared by those skilled in the art using common chemical transformations. For example, arylalkylamines or heteroarylalkylamines can be prepared from the corresponding nitrile by catalytic hydrogenation using catalysts such as Pd/C or Raney nickel or by lithium aluminum hydride reduction, (see Rylander, Catalytic Hydrogenation in Organic Synthesis, Academic Press, 1979).

The nitrile starting materials can either be purchased or prepared from the corresponding aryl/heteroaryl bromide, iodide or triflate and Zn(CN) 2 using Pd coupling conditions found in Tschaen, D. M., et. al Synthetic Communications (1994), 24, 6, pp. 887-890.

Alternatively, benzylamines or heteroarylmethylamines can be prepared by reacting the appropriate arylalkyl or heteroarylalkyl halide and the potassium salt of (BOC^NH (reference) and subsequent removal of the Boc groups with acid.

Amines, protected forms of amines, precursors of amines and precursors of the protected forms of amines of formula 5 can be prepared by combining the appropriate alkyne or alkenyl stannane, alkenyl borane, alkenyl boronic acid, boronic acid ester with the appropriate aryl or heteroaryl bromide , -iodide or -triflate using Pd coupling conditions as found in Tsuji, J.; Palladium Reagents and Catalysis, John Wiley and Sons 1999 and references cited therein.

Appropriately protected amines of formula 5 can be converted to various amines of formula 5 according to methods known to those skilled in the art such as: (a) oxidation of a thioether to sulfoxide or sulfone. (b) N-alkylation of a sulfanilide can be achieved under phase transfer using conditions described by Brehme, R. "Synthesis", (1976), pp. 113-114.

As understood by those skilled in the art, chemical conversion to convert an aryl halide or triflate or heteroaryl halide or triflate to an aromatic or heteroaromatic amine can be performed using conditions currently described in the literature, see Hartwig, J.F.: "Angew. Chem. Int . Oath." (1998), 37, pp. 2046-2067, Wolfe, J.P.; Wagaw, S.; Marcoux, J.F.; Buchwald, S. L.; "Acc. Chem. Res.", (1998), 31, pp. 805-818, Wolfe, J.P.; Buchwald, S. L.; "J. Org. Chem.", (2000), 65, pp. 1144-1157, Muci, A.R.; Buchwald, S. L; "Topics in Current Chemistry" (2002), pp. 131-209 and references therein. Furthermore, as understood by those skilled in the art, these same chemical aryl or heteroaryl amination transformations can alternatively be performed on nitrile (or primary amide) precursors to yield amines of formula 5 after nitrile (or amide) reduction. Protected amines of formula 5 can further be converted into various amines of formula 5 according to methods known to those skilled in the art.

In vitro activity of the compounds of formula 1 can be determined by the following procedure. More particularly, the following experiments provide a method for determining whether compounds of formula 1 inhibit tyrosine kinase activity of the catalytic construct FAK(410-689). The experiment is an ELISA-based format, which measures inhibition of poly-glu-tyr phosphorylation by FAK(410-689).

The trial protocol has three parts:

I. Purification and cleavage of His-FAK(410-689)

II. FAK410-689- (also known as FAKcd) activation

III. FAKcd kinase ELISA

Materials:

-Ni-NTA agarose (Qiagen)

-XK-16 column (Amersham-Pharmacia)

-300 mM Imidizole

-Superdex 200 HiLoad 16/60 prep quality column (Amersham Biotech.) -Antibody: Anti-phosphotyrosine HRP-conjugated Py20 (Transduction labs).

-FAKcd: Cleaned and activated internally

-TMB Microwell Peroxidase Substrate (Oncogene Research Products #CL07)

-BSA: Sigma #A3294

-Tween-20: Sigma #P1379

-DMSO: Sigma #D-5879

-D-PBS: Gibco #14190-037.

Reagents for purification:

-Buffer A: 50 mM HEPES pH 7.0,

500 mM NaCl,

0.1 mMTCEP

Complete TM Protease Inhibitor Cocktail Tablets (Roche)

-Buffer B: 25 mM HEPES pH 7.0,

400 mM NaCl

0.1 mM TCEP.

-Buffer C: 10 mM HEPES pH 7.5,

200 mM ammonium sulfate

0.1 mM TCEP.

Reagents for activation

-FAK(410-689): 3 tubes of frozen aliquots with 150ul/tube total 450ul with 1.48mg/ml (660ug) -His-Src(249-524): -0.74mg/ml stock in 10mM HEPES, 200 mM (NH4)2SO4-Src reaction buffer (Upstate Biotech):

100 mM Tris-HCl pH7.2,

125 mM MgCl2,

25 mM MnCl2,

2 mM EDTA,

250 µM Na3V04,

2 mM DTT

-Mn2+/ATP cocktail (Upstate Biotech)

75 mM MnCl 2

500 µM ATP

20 mM MOPS pH 7.2

1 mM Na 3 VO 4

25 mM -glycerol phosphate

5 mM EGTA

1 mM DTT

-ATP: 150 mM stock

-MgCl2: 1 M stock

-DTT: 1M stock

Reagents for FAKcd kinase ELISA

-Phosphorylation buffer:

50 mM HEPES, pH 7.5,

125 mM NaCl,

48 mM MgCl 2

-Wash buffer: TBS + 0.1% Tween-20.

-Block Buffer:

Tris buffer saline solution,

3% BSA,

0.05% Tween-20, filtered.

-Plate coating buffer:

50 mg/ml Poly-Glu-Tyr (Sigma #P0275) in phosphate-buffered saline

(DPBS).

-ATP: 0.1 M ATP in H2O or HEPES, pH 7.

Note: ATP trial buffer:

Prepare as 75 µM ATP in PBS, so that 80 µl i

120 ul reaction volume=50uM final ATP concentration.

I. Purification of His-FAKcd(410-689)

1. Resuspend 130 g of baculovirus cell paste containing the overexpressed His-FAKcd410-689 recombinant protein in 3 volumes (400 ml) of Buffer A,

2. Light cells with one pass on a microfluidizer

3. Remove cell debris by centrifugation at 40C for 35 minutes at 14,000 rpm in a Sorval SLA-1500 rotor. 4. Transfer the supernatant to a clean tube and add 6.0 ml of Ni-NTA agarose (Qiagen) 5. Incubate the suspension with gentle rocking at 40C for 1 hour 6. Centrifuge the suspension at 700 x g in a swinging bucket rotor. 7. Discard the supernatant and resuspend the agarose beads in 20.0 ml of Buffer A 8. Transfer the beads to an XK-16 column (Amersham-Pharmacia) connected to FPLCTM. 9. Wash the agarose beads with 5 column volumes of Buffer A and elute the column with a step gradient of Buffer A containing 300 mM Imidizole. 10. Perform a buffer exchange of the eluted fractions into Buffer B 11. After buffer exchange, pool the fractions and add thrombin at a 1:300 (w/w) ratio and incubate overnight at 13°C to remove the N -terminal His tag (His-FAK410-698 FAK410-689 (also known as FAKcd)). 12. Add the reaction mixture back onto the Ni-NTA column equilibrated with Buffer A and collect the flow-through. 13. Concentrate the flow-through down to 1.7 ml and load directly onto a Superdex 200 HiLoad 16/60 prep quality column equilibrated with Buffer C. The desired protein elutes between 85 - 95 ml.

14. Aliquot the FAKcd protein and store frozen at -80°C

II. FAK activation

1. To 450 ul of FAK(410-689) at 1.48 mg/ml (660ug) add the following:

30 µl of 0.037 mg/ml (1 µM) His-Src(249-524)

30 µl of 7.5 mM ATP

12 µl of 20 mM MgCl 2

10ul Mn2+/ATP cocktail (UpState Biotech.)

4 µl of 6.7 mM DTT

60ul Src reaction buffer (UpState Biotech.)

2. Incubate the reaction for at least 3 hours at room temperature

At time two, almost all FAK(410-689) is singly phosphorylated. The second phosphorylation is slow. At 120 (t = 120 minutes), add 10 µl of 150 mM

ATP.

Two = (Start) 90% single phosphorylated FAK(410-689) (1 P04)

T43= (43 min) 65% single phosphorylated (1 P04), 35% double phosphorylated

(2 P04)

T90= (90 min) 45% 1 P04, 55% 2 P04

T150= 15% 1 P04, 85% 2 P04

T210= <10% 1 P04, >90% 2 P04 desalted sample

3. Add 180 µl aliquots of the desalted material to NiNTA spin column and incubate on spin column 4. Spin at 10k rpm (microfuge), for 5 min to isolate and collect the flow-through (Activated FAK(410-689)) and remove His -Src

(captured on the column)

III. FAKcd kinase ELISA

1. Coat 96-well Nunc MaxiSorp plates with poly-glu-tyr (pGT) at 10 µg/well: Prepare 10 µg/ml pGT in PBS and aliquot 100 µl/well. Incubate the plates at 37°C overnight, aspirate the supernatant, wash the plates 3 times with wash buffer and pat dry before storing at 4°C. 2. Prepare compound stock solutions of 2.5 mM in 100% DMSO. Stocks are then diluted to 60X the final concentration in 100% DMSO and diluted 1:5 in kinase phosphorylation buffer. 3. Prepare a 75 uM working ATP solution in kinase phosphorylation buffer. Add 80 µl to each well for a final ATP concentration of 50 µM. 4. Transfer 10 µl of the diluted compounds (0.5 log serial dilutions) to each well of the pGT assay plate, running each compound in triplicate on the same plate. 5. On ice, dilute FAKcd protein to 1:1000 in kinase phosphorylation buffer. Add 30 ul per well. 6. Note: Linearity and the appropriate dilution must be pre-determined for each portion of protein. The enzyme concentration chosen must be such that quantification of the experimental signal will be approximately 0.8-1.0 at OD450 and in the linear range of the reaction rate. 7. Prepare both a No ATP Control (Noise) and a No Compound Control (Signal): 8. (Noise) One blank row of wells receives 10 µl of 1:5 diluted compounds in DMSO, 80 µl of phosphorylation buffer (minus ATP) and 30 µl FAKcd solution. 9. (Siganl) Control wells receive 10 µl of 1:5 diluted DMSO (minus compound) in kinase phosphorylation buffer, 80 µl of 75 µM ATP and 30 µl of 1:1000 FAKcd enzyme. 10. Incubate the reaction at room temperature for 15 minutes with gentle shaking on a plate shaker. 11. End the reaction by aspirating the reaction mixture and washing 3 times with washing buffer. 12. Dilute phospho-tyrosine HRP-conjugated (pY20HRP) antibody to 0.250ug/ml (1:1000 of stock) in blocking buffer. Add 100 ul per well and incubate with shaking for 30 min. at room temperature 13. Aspirate the supernatant and wash the plate 3 times with washing buffer. 14. Add 100 ul per well of room temperature TMB solution to initiate color development. Color development is finished after approximately 15-30 seconds. by adding 100ul of 0.09M H2SO4pr. well. 15. The signal is quantified by measuring absorbance at 450 nm on a BioRad microplate reader or a microplate reader capable of reading at OD450. 16. Inhibition of tyrosine kinase activity would result in a reduced absorbance signal. The signal is typically 0.8-1.0 OD units. The values are given as IC50, uM concentration.

FAK-inducible cell-based ELISA: Final protocol

Materials:

Reacti-Bind Goat Anti-Rabbit Plates 96-well (Pierce Product#15135ZZ @$115.00)

FAKpY397 rabbit polyclonal antibody (Biosource #44624 @315.00

USD)

ChromePure Rabbit IgG, whole molecule (Jackson Laboratories #001-000-003 @60/25 mg USD)

UBI aFAK Clone 2A7 Mouse Monoclonal Antibody (Upstate #05-182 @ $289.00)

Peroxidase-conjugated AffiniPure goat anti-mouse IgG (Jackson Labs #115-035-146 @95/1.5 mL USD)

SuperBlock TBS (Pierce Product #37535ZZ @99 USD)

Bovine Serum Albumin (Sigma #A-9647 @117.95/100g USD)

TMB Peroxidase Substrate (Oncogene Research Products #CL07-100 ml @$40.00)

Na3V04Sodium Orthovanadate (Sigma #S6508 @43.95/50g USD) MTT Substrate (Sigma # M-2128 @25.95/500mg USD)

Growth media: DMEM+10%FBS, P/S, Glu, 750 ug/ml Zeocin and 50 ug/ml Hygromycin (Zeocin InVitrogen #R250-05 @ $725 and Hygromycon InVitrogen #R220-05 @ $150)

Mifepristone InVitrogen # H110-01 @ $125

Complete TM EDTA-Free Protease Inhibitor Pellet, Boehringer Mannheim #1873580

FAK cell-based protocol for selectivity of kinase-dependent phosphoFAKY397

Procedure:

An inducible FAK cell-based assay in ELISA format for chemical screening to identify tyrosine kinase-specific inhibitors was developed. The cell-based experiment utilizes the mechanism of the GeneSwitchTM system (InVitrogen) to exogenously control expression and phosphorylation of FAK and the kinase-dependent autophosphorylation site at residue Y397.

Inhibition of kinase-dependent autophosphorylation at Y397 results in a reduced absorbance signal at OD450. The signal is typically 0.9 to 1.5 OD450 units with noise falling in the range of 0.08 to 0.1 OD450 units. The values are given as IC50, uM concentration.

On day 1, grow A431 «FAKwt in T175 flasks. On the day before running FAK cell experiments, seed A431«FAKwt cells in growth media on 96-well U-bottom plates. Allow cells to stand at 37°C, 5% CO 2 in 6 to 8 hr before FAK induction. Prepare Mifepristone stock solution of 10 µM in 100% ethanol. The stock solution is then diluted to 10 X the final concentration in the growth media. Transfer 10 µl of this dilution (final concentration of 0.1 nM Mifepristone) to each well. Allow cells to stand at 37°C, 5% CO2 overnight (12 to 16 hours). Also prepare control wells without Mifepristone induction of FAK expression and phosphorylation.

On day 2, coat Goat Anti-Rabbit plate(s) with 3.5 µg/ml phospho-specific FAKpY397 polyclonal antibody prepared in SuperBlock TBS buffer and allow plate(s) to shake on a plate shaker at room temperature for 2 hours. Optionally, control wells can be coated with 3.5 ug/ml control Capture antibody (Whole Rabbit IgG molecules) prepared in SuperBlock TBS. Wash away excess FAKpY397 antibody 3 times using buffer. Block Anti-FAKpY397 coated plate(s) with 200 µl per well of 3%BSA/0.5%Tween blocking buffer for 1 hour at room temperature on the plate rack. While the plate(s) are being blocked, prepare compound stock solutions of 5 mM in 100% DMSO. The stock solutions are then serially diluted to 100X of the final concentration in 100% DMSO. Prepare a 1:10 dilution using 100X solution in growth media and transfer 10 µl of the appropriate compound dilutions to each well containing either FAK-induced or uninduced control A431 cells for 30 min at 37°C, 5% CO2- Prepare RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF and one complete TM EDTA- free protease inhibitor pellet per 50 ml solution). At the end of the 30 minute compound treatment, wash away compound 3 times using TBS-T wash buffer. Lyse cells with 100 µl/well of RIPA buffer.

On the coated plate, remove blocking buffer and wash 3 times using TBS-T wash buffer. Using a 96-well automated microdispenser, transfer 100 µl of whole cell lysate (from step 6) to the Goat Anti-Rabbit FAKpY397 coated plate(s) to capture phosphoFAKY397 proteins. Shake at room temperature for 2 hours. Wash away unbound proteins 3 times using TBS-T wash buffer. Prepare 0.5 µg/ml (1:2000 dilution) of UBI aFAK detection antibody in 3%BSA/0.5% Tween blocking buffer. Add 100 ul of UBI aFAK solution per well and shake for 30 minutes at room temperature. Wash away excess UBI aFAK antibody 3 times using TBS-T wash buffer. Prepare 0.08 µg/ml (1:5000 dilution) of secondary Anti-Mouse Peroxidase (Anti-2MHRP) conjugated antibody. Add 100 ul per well of the Anti-2MHRP solution and shake for 30 minutes at room temperature. Wash away excess Anti-2MHRP antibody 3 times using TBS-T wash buffer. Add 100 ul per well of room temperature TMB substrate solution to allow color development. End TMB reaction with 100 ul per well of TMB stop solution (0.09M H2SO4) and quantify the signal by measuring absorbance at 450 nm on a BioRad microplate reader.

In a preferred embodiment, the compounds have an in vivo activity as determined by a kinase assay, e.g. as described herein, at less than 100 nM. Preferably, the compounds have an IC 50 of less than 25 nM in the kinase assay and more preferably less than 10 nM. In a further preferred embodiment, the compounds show an IC50 in a FAK cell-based assay, e.g. as described herein, of less than 1 M, more preferably less than 100 nM and most preferably less than 25 nM.

Administration of the compounds according to the present invention (below the "active compound(s)") can be carried out by any method that entails delivery of the compounds to the site of action. These methods include oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion), topical and rectal administration.

The amount of the active compound administered will depend on the individual being treated, the severity of the disorder or condition, the frequency of administration, the disposition of the compound and the discretion of the prescribing physician. However, an effective dose is in the range of approx. 0.001 to approx. 100 mg per kg body weight per day, preferably approx. 1 to approx. 35 mg/kg/day, in single or divided doses. For a person of 70 kg, this will give an amount of approx. 0.05 to approx. 7 g/day, preferably approx. 0.2 to approx. 2.5 g/day. In some cases, dose levels below the lower limit of the above range may be more than sufficient, while in other cases, even larger doses may be used without causing any harmful side effect, provided that such larger doses are first divided into many small doses for administration during of the day.

The active compound may be used as sole therapy or may involve one or more other anti-tumor substances, for example those selected from, for example, mitotic inhibitors, for example vinblastine; alkylating agents, such as cis-platin, carboplatin and cyclophosphamide; anti-metabolites, for example 5-fluorouracil, cytosine-arabinoside and hydroxyurea, or for example one of the preferred anti-metabolites described in European Patent Application No. 239362 such as N-(5-[N-(3,4-dihydro- 2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl)-L-glutamic acid; growth factor inhibitors; cell cycle inhibitors; intercalating antibiotics, such as adriamycin and bleomycin; enzymes, such as interferon; and anti-hormones, for example anti-estrogens such as Nolvadex<®> (tamoxifen) or, for example anti-androgens such as Casodex<®> (4'-cyano-3-(4-fluorophenylsulfonyl)-2-hydroxy- 2-methyl-3'-(trifluoromethyl)propionanilide). Such combined treatment can be achieved by simultaneous, sequential or separate dosing of the individual components of the treatment.

The pharmaceutical preparation may for example be in a form suitable for oral administration such as a tablet, capsule, pill, powder, extended release formulations, solution, suspension, for parenteral injection as a sterile solution, suspension or emulsion, for topical administration as an ointment or cream or for rectal administration as a suppository. The pharmaceutical preparation may be in unit dosage forms suitable for easy administration of precise doses. The pharmaceutical preparation will comprise a conventional pharmaceutical carrier or additive and a compound according to the invention as active ingredient. In addition, it may include other medicinal or pharmaceutical agents, carriers, adjuvants, etc.

Examples of parenteral administration forms include solutions or suspensions of active compounds in sterile aqueous solutions, for example aqueous propylene glycol or dextrose solutions. Such dosage forms can, if desired, be suitably buffered.

Suitable pharmaceutical carriers include inert diluents or fillers, water and various organic solvents. The pharmaceutical preparations may, if desired, contain additional ingredients such as flavourings, binders, additives and the like. Thus, for oral administration, tablets containing various additives such as citric acid can be used together with various disintegrants such as starch, alginic acid and certain complex silicates and with binders such as sucrose, gelatin and acacia. In addition, smoothing agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tableting purposes. Solid preparations of a similar type can also be used in soft and hard filled gelatin capsules. Preferred materials for this include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral administration, the active compound may be combined with various sweetening or flavoring agents, dyes or coloring agents and, if desired, emulsifying or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin or combinations thereof.

Methods for the preparation of various pharmaceutical preparations with a specific amount of active compound are known or will be clear to those skilled in the art. For examples, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easter, Pa., 15th Edition (1975).

The Examples and Preparations given below further illustrate and exemplify the compounds according to the present invention and methods for producing such compounds. In the following examples, molecules with a single chiral center, unless otherwise indicated, may exist as a racemic mixture. Molecules with two or more chiral centers, unless otherwise indicated, exist as a racemic mixture of the diastereomers. Simple enantiomers/diastereomers can be obtained by methods known to those skilled in the art.

When HPLC chromatography is referred to in the Preparations and Examples below, the general conditions used, unless otherwise indicated, are as follows. The column used is a ZORBAX® RXC18 column (manufactured by Hewlett Packard) with 150 mm length and 4.6 mm internal diameter. The tests have been run on a Hewlett Packard-1100 system. A gradient-solvent method is used running 100 percent ammonium acetate/acetic acid buffer (0.2 M) to 100 percent acetonitrile over 10 minutes. The system then runs on a wash cycle with 100 percent acetonitrile for 1.5 minutes and then 100 percent buffer solution for 3 minutes. The flow rate over this period is a constant 3 ml/minute.

In the following examples and formulations, "Et" means ethyl, "Ac" means acetyl, "Me" means methyl and "Bu" means butyl.

Examples

General methods:

Preparation of 5-nitrooxindole:

Fuming nitric acid (8.4 ml) was added dropwise to a solution of oxindole (26 g) in 100 ml of concentrated sulfuric acid at -15°C. Care was taken to keep the reaction temperature at -15°C. After the addition was complete, the reaction mixture was stirred for 30 minutes and then poured into ice water. A yellow precipitate formed which was isolated by filtration to give 34 grams (98%) of 5-nitro-oxindole.

Preparation of 5-amino-oxindole (2):

To a solution of 5-nitrooxindole (25 g) in 120 ml of dimethylacetamide in a Parr flask was added 10% Pd/C (0.5 g). The mixture was hydrogenated (2.8 kg/cm<2>H 2 ) for 16 hours. The catalyst was removed by filtration and the filtrate was diluted with ether (2L) to give 5-amino-oxindole (10.5 g; 50%).

Preparation of 2,4-dichloro-5-trifluoromethylpyrimidine (3): 5-trifluoromethyluracil (250 g, 1.39 mol) and phosphorus oxychloride (655 mL, 6.94 mol, 5 equiv) were charged to a 3L 4-necked flask equipped with overhead stirrers, a reflux cooler, an addition funnel and an internal thermocouple. The content was

maintained under a nitrogen atmosphere as concentrated phosphoric acid (85 wt%, 9.5 mL, 0.1 eq.) was added in one portion to the slurry, resulting in a moderately exothermic reaction. Diisopropylethylamine (245 mL, 1.39 mol, 1 eq.) was then added dropwise over 15 min at such a rate that the internal

temperature of the reaction mixture reached 85-90°C at the end of the addition. At the end of the amine addition, the reaction mixture became a homogeneous light-orange solution. Heating was initiated and the orange solution was held at 100°C for 20 hours, at which time HPLC analysis of the reaction mixture showed that the starting material was consumed. External heating was removed and the contents of the flask were cooled to 40°C and then added dropwise to a cooled mixture of 3N HCl (5 L, 10 equiv) and diethyl ether (2L) while the temperature in the cooling vessel was maintained between 10 and 15°C . The layers were separated and the aqueous layer was extracted once with ether (1L). The combined organic layers were collected, washed with water until the washes were neutral (5 x 1.5L washes), dried with MgSO\4 and concentrated to give 288 g (95% yield) of a bright yellow-orange oil with 96% purity (HPLC). This material can be further purified by distillation (bp 109°C at 79 mmHg).

Preparation of 5-(4-chloro-5-trifluoromethyl-pyrimidin-2-vlamino)-1,3-dihydro-indol-2-one (4): To a solution of 5-trifluoromethyl-2,4-dichloropyrimidine (214 .8 g; 0.921 mol) in 1:1 DCE/tBuOH (1.240 L) was added zinc chloride 1M solution in ether (1 eq; 0.921 L). After 0.5 h, 5-amino-oxindole (124 g; 0.837 mol) was added followed by triethylamine (129.4 mL; 0.921 mol) while maintaining the temperature at 25°C. The reaction mixture was stirred at room temperature overnight, then concentrated and the product triturated from methanol as a yellow solid (224.3 g; 82%).<1>H NMR (DMSO-de, 400 MHz) δ 3.29 ( s, 2H), 6.76 (d, J = 7.9 Hz, 2H), 7.39 (d, J = 8.3 Hz), 7.51 (br s, 1H), 8.71 (s , 1H), 10.33 (s, 1H), 10.49 (s, 1H). <13>C NMR (DMSO-de, 100 MHz) δ 177.0, 161.3, 158.7 (br), 140.7, 132.8, 126.9, 123.7 (q, J = 268 Hz), 121.0, 118.7, 111.2 (q, J = 32 Hz), 109.6, 36.7; HPLC ret. hour: 5.759 min. LRMS (M+) 329.1, 331.1. Example 1 5-[4-(R-1-phenyl-ethylamino)-5-trifluoromethyl-pyrimidin-2-ylamino]-1,3-dihydro-indol-2-one

To a solution of 1:1 DCE/t-BuOH alcohol (1:1 ratio, 4 mL) and 5-(4-chloro-5-trifluoromethyl-pyrimdin-2-ylamino)-1,3-dihydro-indol-2 -one (0.15 g; 0.456 mmol) was added (R)(+) alpha-phenethylamine (0.071 mL; 0.547 mmol) and diisopropylethylamine (0.081 mL, 0.456 mmol). The resulting solution was stirred under nitrogen and heated to 80°C for 16 hours. The reaction mixture was cooled to room temperature, diluted with -10 ml of a 1:1 mixture of dichloromethane and methanol followed by the addition of 0.5 g of MP carbonate. The resulting mixture was stirred, filtered, concentrated and purified by silica gel chromatography (97:2.8:0.3 ratio of chloroform/methanol/concentrated ammonium hydroxide). The desired title compounds were obtained as a white solid (0.021 g; 11%). HPLC ret. time: 6.46 min. LRMS (M+) 413.4

The following compounds according to the invention were prepared by heating chloropyrimidine (4) with a suitable amine as in Example 1. Amines used in these reactions were either obtained commercially and used as received or alternatively they were prepared by usual synthesis methods for amines known for professionals in the field. Unless otherwise indicated, compounds having chiral centers were prepared as racemic mixtures.

Claims (12)

1. Compound of formula 1 where R<1> and R<2> are each a hydrogen atom, or R<1> and R<2> taken together with the carbon atom to which they are attached form a 2-hydroxy-indan-1-yl ring; R 3 is selected from (a) a hydrogen atom when R<1> and R 2 together form an indanyl ring as indicated above, (b) -(C 6 -C 10 )aryl optionally substituted with from one to three groups selected from -SO 2 (C 1 -C 6 )alkyl, SO 2 NH 2 , -N((C 1 -C 6 )alkyl)(SO 2 (C 1 -C 6 )alkyl) and halogen, (c) a heteroaryl group selected from chromanyl or furanyl, each optionally substituted with -(C 1 -C 6 )alkyl, (d ) -(C3-C10)cycloalkyl or -(C3-C10)cycloalkenyl, both optionally substituted with hydroxy, (e) morpholinyl, pyrrolidinyl or pyridinyl, each optionally substituted with -SO2(CrC6)alkyl or -N((CrC6)alkyl )(SO 2 (C 1 -C 6 )alkyl, or (f) (C 1 -C 6 )alkyl substituted with a group selected from -N((d -C 6 )alkyl)(SO 2 (C 1 -C 6 )alkyl) and O(C 1 -C 6 )alkyl, n is an integer from 1 to 3. or a pharmaceutically acceptable salt, solvate or hydrate thereof. 2. Compound according to claim 1 where R<1> and R<1> are hydrogen and n is 1. 3. A compound according to any one of the preceding claims wherein R 3 is -(C 6 -C 10 )aryl, optionally substituted with one to three groups independently selected from the group consisting of -SO 2 (C 1 -C 6 )alkyl, -SO 2 NH 2 , -N( (C 1 -C 6 )alkyl)(SO 2 (C 1 -C 6 )alkyl) and halogen; or a heteroaryl group selected from chromanyl or furanyl, each optionally substituted with -(C 1 -C 6 )alkyl. 4. Compound according to claim 1 or 2, where R<3> is selected from the group consisting of (d) -(C3-Ci0)cycloalkyl or -(C3-Ci0)cycloalkenyl, both optionally substituted with hydroxy, (e) morpholinyl, pyrrolidinyl or pyridinyl, each optionally substituted with - 802(O!-C6)alkyl or -N((C1-C6)alkyl)(SO2(C1-C6)alkyl, and (f) (C1-C6)alkyl substituted with a group selected from -N((C 1 -C 6 )alkyl)(SO 2 (C 1 -C 6 )alkyl) and O(C 1 -C 6 )alkyl. 5. Compound according to claim 1 or 2, where R<3> is -(Ci-Ce)alkyl substituted with one group selected from the group consisting of -N((CrC6)alkyl)(SO2(CrC6)alkyl) and -O( C 1 -C 6 )alkyl. 6. Compound according to any one of the preceding claims of formula 2 where A is selected from the group consisting of: where m is an integer from 0-3 and R<13>is a substituent selected from the group consisting of (b) -SO2(Ci-C6)alkyl, SO2NH2, -N((Ci-C6)alkyl)(SO2( C 1 -C 6 )alkyl) and halogen when A is -(C 1 -C 10 )aryl; (c) -(C 1 -C 6 )alkyl when A is a heteroaryl group; and (e) -SO 2 (C 1 -C 6 )alkyl or -N((C 1 -C 6 )alkyl)(SO 2 (C 1 -C 6 )alkyl) when A is a pyridyl group. 7. Compound according to any of the preceding claims of formula 3 where B is selected from the group consisting of: where m is an integer from 0-6 and R<13>is a hydroxy group. 8. Compound according to any one of the preceding claims of formula 4 where D is selected from the group consisting of: where q is an integer from 1-2, m is an integer from 0-5 and R<13> is (c) -(CrC6)alkyl when D is a heteroaryl group, (d) hydroxy when D is-(C3- Cio)cycloalkyl or -(C3-Cio)cycloalkenyl, (e) -SO2(C1-C6)alkyl or -N((C1-C6)alkyl)(SO2(C1-C6)alkyl when D is morpholinyl, pyrrolidinyl or pyridinyl. 9. A compound according to claim 1, selected from the group consisting of: 5-[4-(3-methanesulfonyl-benzylamino)-5-trifluoromethyl-pyrimidin-2-ylamino]-1,3-dihydro-indol-2-one; Ethanesulfonic acid methyl-{3-[2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-propyl}-amide; 5-{4-[(isocroman-1-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one; 5-{4-[2-(pyridin-3-yloxy)-propylamino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one; 3-{[2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-methyl}-benzenesulfonamide; 5-{4-[(1-methanesulfonyl-piperidin-3-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one; N-(3-{[2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-methyl}-phenyl)-methanesulfonamide; N-methyl-N-{2-[2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-ethyl}-methanesulfonamide; 5-{4-[(4-methanesulfonyl-morpholin-2-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one; 5-[4-(3-methanesulfonylmethyl-benzylamino)-5-trifluoromethyl-pyrimidin-2-ylamino]-1,3-dihydro-indol-2-one; 5-{4-[(1-methanesulfonyl-pyrrolidin-3-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one; N-methyl-N-{3-[2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-propyl}-methanesulfonamide; 5-{4-[2-(1-methanesulfonyl-piperidin-2-yl)-ethylamino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one; 5-{4-[(4-methanesulfonyl-pyridin-2-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one; 5-[4-(3-isopropoxy-propylamino)-5-trifluoromethyl-pyrimidin-2-ylamino]-1,3-dihydro-indol-2-one; 5-{4-[(5-methyl-furan-2-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one; 5-{4-[(bicyclo[2,2,1]hept-5-en-2-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2- on; N-(4-fluoro-3-{[2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-methyl}-phenyl)- N-methyl-methanesulfonamide; 5-{4-[(1-methanesulfonyl-piperidin-3-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one; 5-{4-[(6-methanesulfonyl-pyridin-2-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one; 5-{4-[(5-methanesulfonyl-pyridin-3-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one; 5-[4-(2-methanesulfonyl-benzylamino)-5-trifluoromethyl-pyrimidin-2-ylamino]-1,3-dihydro-indol-2-one; 5-{4-[(1-pyrimidin-2-yl-piperidin-3-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one; 5-{4-[2-(1-methanesulfonyl-piperidin-2-yl)-ethylamino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one; 5-{4-[2-(1-methanesulfonyl-piperidin-2-yl)-ethylamino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one; N-(2-{[2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-methyl}-phenyl)-methanesulfonamide; 5-{4-[(1-methanesulfonyl-pyrrolidin-2-ylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one; N-methyl-N-(2-{[2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-methyl}-phenyl)- methanesulfonamide; N-methyl-N-(2-methyl-6-{[2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-methyl} -phenyl)-methanesulfonamide; 5-[4-(2-hydroxy-indan-1-ylamino)-5-trifluoromethyl-pyrimidin-2-ylamine 1,3-dihydro-indol-2-one; 5-{4-[(1-hydroxy-cyclopentylmethyl)-amino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one; 5-{4-[2-hydroxy-2-(1-methanesulfonyl-piperidin-2-yl)-ethylamino]-5-trifluoromethyl-pyrimidin-2-ylamino}-1,3-dihydro-indol-2-one; and N-methyl-N-(3-{[2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-methyl}-pyridin- 2-yl)-methanesulfonam 10. Process for the preparation of compound of formula 1 where R<1>and R2 are each a hydrogen atom, or R<1>and R2 taken together with the carbon atom to which they are attached form a 2-hydroxy-indan-1-yl ring; R3 is selected from (a) a hydrogen atom when R<1>and R<2> together form an indanyl ring as indicated above, (b) -(C6-C10)aryl optionally substituted with from one to three groups selected from -SO2( CrC6)alkyl, SO2NH2, -N((CrC6)alkyl)(SO2(CrC6)alkyl) and halogen, (c) a heteroaryl group selected from chromanyl or furanyl, each optionally substituted with -(C1-C6)alkyl, (d) -(C3-Cio)cycloalkyl or -(C3-Cio)cycloalkenyl, both optionally substituted with hydroxy, (e) morpholinyl, pyrrolidinyl or pyridinyl, each optionally substituted with -SO2(Ci-C6)alkyl or -N((Ci- C 6 )alkyl)(SO 2 (C 1 -C 6 )alkyl, or (f) (C 1 -C 6 )alkyl substituted with a group selected from -N((d -C 6 )alkyl)(SO 2 (C 1 -C 6 )alkyl) and O(C 1 -C 6 )alkyl , n is an integer from 1 to 3. or a pharmaceutically acceptable salt, solvate or hydrate thereof; wherein the process comprises the steps of (a) reacting 5-amino-oxindole, 2 with dichloropyrimidine 3 in the presence of a Lewis acid and a suitable base to prepare a 2-substituted pyrimidine 4; (b) react pyrimidine 4 with NH2-(CR<1>R2)nR<3>In the presence of a suitable base to give a compound of formula 1. 11. Compound according to claim 1, for the treatment of abnormal cell growth in a mammal. 12. A pharmaceutical preparation for the treatment of abnormal cell growth in a mammal comprising an amount of a compound according to claim 1 which is effective for the treatment of abnormal cell growth, and a pharmaceutically acceptable carrier.