Selective FAK inhibitors

专利摘要:
A compound of the formula (I) where R
公开号:AU2012216894A1
申请号:U2012216894
申请日:2012-02-17
公开日:2013-09-05
发明作者:Ylva BERGMAN；Neil Choi；Richard Charles FOITZIK；Danny GANAME；Catherine Fae HEMLEY；Ian Peter Holmes；Romina LESSENE；Gillian Elizabeth LUNNISS；Marica NIKAC；Scott Raymond WALKER
申请人:Cancer Therapeutics CRC Pty Ltd；
IPC主号:C07D239-42

专利说明:
WO 2012/110774 PCT/GB2012/000176 1 SELECTIVE FAK INHIBITORS This invention relates to 2,4,5-substituted pyrimidines that inhibit Focal Adhesion Kinase (FAK), also known as protein tyrosine kinase 2 (PTK2), and to pharmaceutical 5 compositions containing such compounds. This invention also relates to a method of using such compounds for the prevention and/or treatment of proliferative diseases, such as cancer. Background 10 Directional cell migration is important in many physiological and pathological processes including embryonic development, wound healing, angiogenesis, tumour invasion and metastasis. Transduction of extracellular signals, that stimulate cells to move directionally, may be induced by a number of processes including trans membrane integrins binding to extra cellular matrix proteins and the action of growth 15 factors (for example EGF, lGF and VEGF) on the extracellular domains of their cognate receptors. FAK is a non receptor tyrosine kinase that mediates signals from both trans membrane integrins and growth factor receptors. FAK has been reported to play a 20 central role in coordinating these diverse extra cellular signals, integrating them in a fashion that results in directional movement of cells through their external environment (Tomar and Schlaepfer. Current Opinion in Cell Biology: 2009, 21, 676 683). 25 Integrin clustering or the activation of a growth factor receptor (for example EGFR, IGF-1 R, Her2 and VEGFR) promotes FAK autophosphorylation at Y397. Phosphorylated Y397 FAK then binds to c-Src (referred to as Src herein) and Src mediated phosphorylation of FAK at Y576 and Y577 occurs to give rise to an active FAK-Src complex. Active FAK-Src then facilitates signaling via a number of 30 biochemical pathways which influence processes such as cell adhesion, migration, invasion, cell survival, proliferation, acquisition of chemotherapy resistance and metastasis (Brunton and Frame. Current Opinion in Pharmacology: 2008, 8, 437-432 and Chatzizacharias et al. Expert Opinion in Therapeutic Targets: 2007, 11(10), 1315-1328). 35 WO 2012/110774 PCT/GB2012/000176 2 Cell adhesion Functional studies addressing the role of FAK in cell adhesion suggest that it contributes to both focal adhesion assembly (Richardson and Parsons. Nature: 1996, 380, 538-540) and focal adhesion turnover (Fincham et al. Oncogene: 1995, 10(11), 5 2247-2252). Inhibition of FAK by RNAi in both human and mouse cell lines, resulting in decreased FAK protein levels, has been shown to reduce cell adhesion to a fibronectin/laminin-coated plate in vitro (Tsutsumi et al. International Journal of Oncology: 2008, 33(1), 215-224). 10 Cell miQration There is strong evidence that FAK is a key regulator of cell migration (Angelucci and Bologna. Current Pharmaceutical Design: 2007, 13, 2129-2145 and Mitra et al. Nature Reviews Molecular Cell Biology: 2005, 6, 56-68). Cells derived from FAK -/ mouse embryos exhibit reduced migration as a result of impaired adhesion turnover 15 (6i et al. Nature: 1995, 377, 539-544). Moreover, displacement of FAK from focal adhesions reduces cell migration (Gilmore and Romer. Molecular Biology of the Cell: 1996, 7(8), 1209-1224), whilst over-expression in CHO cells stimulates migration (Cary et al. Journal of Cell Science: 1996, 7, 1787-1794). In addition, inhibition of FAK by RNAi in both human and mouse cell lines, resulting in decreased FAK protein 20 levels, has been shown to reduce cell migration in an in vitro haptotactic migration assay (Tsutsumi et al. International Journal of Oncology: 2008, 33(1), 215-224). Cell invasion FAK activation has been shown to enhance matrix degrading invasive behaviour. 25 FAK-Src signaling through cellular apoptosis susceptibility protein (CAS) (Liao et al. Journal of Experimental and Clinical Cancer Research: 2008, 27:15) leads to the expression of matrix metalloproteases (MMPs) including MMP2 and MMP9. FAK-Src activation also promotes cell surface expression of MMP14 via phosphorylation of endophilin A2. MMP14 then activates MMP2 by cleavage of pro-MMP2 to its active 30 form (Siesser and Hanks. Clinical Cancer Research: 2006, 12(11), 3233-3237). Highly invasive cancer cells form specialized actin-rich extra cellular matrix degrading membrane protrusions known as invadopodia which are rich in matrix-degrading proteases such as MMPs. Both FAK and Src have been shown to be instrumental in the formation of invadopodia (Chan et al. Journal of Chemical Biology: 2009, 185(2), 35 357-370).
WO 2012/110774 PCT/GB2012/000176 3 Cell survival FAK has been shown to play an important role in cell survival. Activation of FAK has been shown to result in suppression of anoikis (apopotosis in response to an 5 inappropriate extra cellular matrix environment) (Frisch et al Journal of Cell Biology. 1996, 134(3), 793-799 and Xu et al Cell Growth and Differentiation. 1996, 7(4), 413 418). Studies have demonstrated that FAK activates multiple downstream pathways to suppress anoikis in both fibroblasts and epithelial cells (Zouq et al. Journal of Cell Science: 2008, 122, 357-367). In human intestinal crypt cells signalling via the 10 association of FAK with 01 integrin and subsequent binding with Src up regulates expression of the anti-apoptotic proteins BcI-XL and Mcl-i via P13-K/Akt-1 signalling. P13-K/Akt-1 signalling also down regulates expression of the pro-apoptotic activators Bax and Bak, causes phosphorylation of the pro-apoptotic sensitizer Bad and antagonizes p38p activation. Dissociation of FAK/Src results in a 15 sustained/enhanced activation of p38p which is an apoptosis/anoikis driver (Bouchard et al. Apoptosis: 2008, 13, 531-542). Cell proliferation Reduction in the expression of either FAK or P1 integrin and hence disruption of the 20 P1-FAK signalling axis results in decreased initial proliferation of micro-metastatic cells distributed in the lung. Using 3D cultured D2 cells a strong correlation was observed between FAK Y397 and Y861 phosphorylation and proliferative ability (Shibue and Weinberg. PNAS 2009, 106(25), 10290-10295). HL-60 Cells, transfected to over express FAK, have been shown to double at a rate 1.5 times 25 faster than control HL-60 cells. Studies revealed a marked induction of cyclin D3 expression and CDK activity in the cells over expressing FAK Activation of Pj3 K/Akt-1 signalling, a process associated with FAK activation in a number of studies, was identified as a probable cause of the cyclin expression/activation (Yamamoto et al. Cellular Signaling: 2003, 15. 575-583). 30 Acquisition of chemotherapy resistance Exposure of the cisplatin sensitive ovarian cancer cell line OAW42 to repeated cycles of cisplatin treatment and subsequent recovery resulted in the formation of chemo resistant OAW42-R cells. Studies aimed at identifying the cause of this chemo 35 resistance revealed that FAK was constituently active in both the sensitive and WO 2012/110774 PCT/GB2012/000176 4 chemo-resistant cells. However, inhibition of phosphorylation of Y397 FAK was induced by treatment with cisplatin in OAW42 cells but not in OAW42-R cells (Poulain and co-workers. Gynaecologic oncology: 2006, 101, 507-519). The effects of FAK inhibition on chemo-resistance has also been studied in vitro and in vivo using 5 the FAK inhibitor TAE226, alone and in combination with docetaxel, in taxane sensitive (SKOV3ip1 and HeyA8) and taxane-resistant (HeyA8-MDR) ovarian cancer cell lines. TAE226 has the structure: 0 NN- N'l CIq H H N1 N H and is described in WO 2004/080980 and WO 2005/016894. in vitro, TAE226 10 inhibited the phosphorylation of FAK at both Y397 and Y661 sites, inhibited cell growth in a time- and dose-dependent manner, and enhanced docetaxel-mediated growth inhibition by 10- and 20-fold in the taxane-sensitive and taxane-resistant cell lines, respectively. in vivo, FAK inhibition by TAE226 significantly reduced tumour burden in the HeyA8, SKOV3ip1, and HeyA8-MDR models (46-64%) compared with 15 vehicle-treated controls. However, the greatest efficacy was observed with concomitant administration of TAE226 and docetaxel in all three models (85-97% reduction). In addition, TAE226 in combination with docetaxel significantly prolonged survival in tumour-bearing mice (Halder et al. Cancer Res: 2007, 67(22), 10976 10983). 20 Metastatic potential Several studies have examined the role of FAK protein levels and it's relation to tumor progression in animal models. In a mouse skin carcinogenesis model using FAK +/- mice, reduced FAK protein expression correlated with decreased papilloma 25 formation (46%), compared with FAK +/+ wild-type control mice (McLean et al. Cancer Research: 2001, 61, 8385-8389). Using human breast carcinoma cells, researchers showed that FAK siRNA treated cells were inhibited from metastasizing to the lung after orthotopic implantation in nude mice (Benlimame et al. Journal of Cell Biology: 2005, 171, 505-516). Similar experiments using short hairpin RNA 30 (shRNA) against FAK in 4T1 mouse breast carcinoma cells resulted in an inhibition of metastasis to the lungs after orthotopic implantation in mammary pads (Mitra et al.
WO 2012/110774 PCT/GB2012/000176 5 Oncogene: 2006, 25, 4429-4440). Inhibition of FAK by dominant negative expression in 4T1 mouse breast carcinoma cells reduced tumour growth and angiogenesis in mice (Mitra et al. Oncogene: 2006, 25, 5969-5984). Use of a Cre/loxP recombination system to disrupt FAK function in the mammary epithelium of a transgenic model of 5 breast cancer has demonstrated that FAK expression is required for the transition of premalignant hyperplasias to carcinomas and their subsequent metastases. The observed decrease in tumor progression was further correlated with impaired mammary epithelial proliferation suggesting that FAK plays a critical role in mammary tumor progression (Lahlou et al. PNAS USA: 2007, 104(51), 20302-20307). 10 In accordance with the above observations over expression of FAK mRNA and/or protein has been reported in numerous human cancers including colorectal cancer (de Heer. European Journal of Surgical Oncology: 2008, 34(11), 1253-1261), prostate cancer (Tremblay, L., W. Hauck, et al. International Journal of Cancer: 1996, 15 68(2), 164-171), breast cancer (Watermann et al. British Journal of Cancer 2005, 93(6), 694-698) and melanomas (Hess et al. Cancer Research: 2005, 65(21), 9851 60). Furthermore FAK over expression is frequently correlated with more aggressive phenotypes of these cancers. 20 Thus, there is strong evidence to suggest that a FAK inhibitor would have application for the reduction of cell adhesion, cell migration, cell invasion, cell proliferation and chemo-resistance. Furthermore, a FAK inhibitor would have applicability to induce apoptosis for cells in inappropriate extra cellular matrix environments and reduce angiogenesis. 25 A compound which is a seltective FAK inhibitor would enable the targeting of specific biological pathways, without any potential issues caused by the inhibition of any targets, such as other protein kinases. 30 Accordingly, compounds that selectively inhibit FAK would be useful for the treatment of proliferative diseases, such as cancer. Two compounds reported to inhibit FAK are PF-562,271 and PF-573,228.
WO 2012/110774 PCT/GB2012/000176 6 0 H NH ~ F 0 N CF 0= I a..-.- N I O N N N N N N N F ' H H H H1 PF-562,271 PF-573,228 PF-562,271 is described in W02004/056786, W02004/056807, W02005/023780, WO2007/063384 and Roberts et al. Cancer Res 2008, 68(6), 1935-1944. 5 PF-573,228 is described in Slack-Davis et al. J. Biol. Chem. 2007, 282(20), 14845 14852. In addition to these specifically described compounds, further classes of FAK 10 inhibitors are disclosed in W02008/129380, W02008/115369, W02009/105498, US2010/113475, W02009/143389, W02009/071535, W02010/055117, W02010/058030, W02010/058032, W02007/140222, and W02009/024332. Summary of the invention 15 The present inventors have discovered a particular class of compounds which are effective as selective FAK inhibitors. These compounds may exhibit selectivity for FAK over kinases such as IGF-1R (insulin-like growth factor 1 receptor), IR (insulin receptor) and CDKs (cyclin-dependent kinases), as well as over other kinases, such as VEGFR 1, VEGFR2 and VEGFR3. Additionally, the compounds of the invention 20 may have enhanced selectivity for the inhibition of cytochrome p 4 50 enzymes, specifically the 2C9 and 3A4 isoforms. Furthermore, the compounds of the invention may be less prone to the formation of adducts with glutathione. In a first aspect, the present invention provides compounds of the following formula 25 (1): R 2 R N R N N R
R
3
H
WO 2012/110774 PCT/GB2012/000176 7 wherein: R' is selected from: H and
RN
3 NI N N4 wherein: 5 RNI is selected from H, C 1 3 alkyl and C(=0)Me;
RN
2 is selected from H, C1.3 alkyl and C(=0)Me;
RN
3 is selected from H, C13 alkyl and C(=O)Me;
RN
4 is selected from H and CH 3 ;
R
2 is selected from H and N5 N6 N N,, N 10 wherein:
RN
5 is selected from H, C1.3 alkyl and C(=0)Me; RN6 is selected from H, C13 alkyl and C(=0)Me; and wherein only one of R' and R 2 is H; 15 R 3 is selected from 0-C2 alkyl, C2 alkyl, halo, cyano, where the C 1
.
2 alkyl group may be substituted by one or more fluoro groups;
R
4 is selected from CF 3 , halo, CF 2 H and CN; and
R
5 is selected from groups of the following formulae: 70 H R N R6 R (R ) (R ) 20 wherein:
R
6 is selected from H, (CHRc1), 1 C(O)N(R)N6)Z and (CH 2 )n2C(O)OZ 2 ; wherein: n1 is 1; Re1 is H or Me;
RN
6 is H or CH 3 ; 25 Z' is H, CH 3 or OCH 3 ; n2 is 1; and WO 2012/110774 PCT/GB2012/000176 8
Z
2 is CH 3 ; and where only one of RN 6 and Z' can be CH 3 ,
R
7 is selected from H, and (CH 2 )mIC(O)N(R m )Y', wherein: ml is 0 or 1; 5 RM1 is H; and Y' is H, Me or OCHa; and only one of R 6 and R 7 is H; and
R
8 is H or, when R 7 is C(=O)NH 2 , R 8 is selected from H and C 1
-
2 alkyl. 10 A second aspect of the present invention provides a composition comprising a compound of the first aspect and a pharmaceutically acceptable carrier or diluent. A third aspect of the invention provides a compound of the first aspect for use in a method of therapy. 15 A fourth aspect of the invention provides for the use of a compound of the first aspect in the preparation of a medicament for treating a disease ameliorated by the inhibition of FAK. The fourth aspect of the invention also provides a compound of the first aspect for use in the method of treatment of a disease ameliorated by the 20 inhibition of FAK. A further aspect of the invention provides an active compound as described herein for use in a method of treatment of the human or animal body, preferably in the form of a pharmaceutical composition. 25 Another aspect of the invention provides a method of inhibiting FAK in vitro or in vivo, comprising contacting a cell with an effective amount of an active compound as described herein. 30 Each of the groups R' to R" will be discussed in more detail below.
WO 2012/110774 PCT/GB2012/000176 9 R1 R' may have one of the following structures: N3 N1 N2 R H N N N (R a) (R1b) (RC) (R id) N4 (Rle) When R' is H, R 2 (discussed below) is not H. 5 Each of RN1, RN 2 and RN 3 is independently selected from H, C 1
.
3 alkyl (i.e. methyl, ethyl, prop-1-yl and prop-2-yl) and C(=O)Me and RN 4 is selected from either H or methyl. 10 R2 R2 may have one of the following structures: NS N6 H N N R N (R2A) (R 2 B) (R 2 c) 15 When R 2 is H, R' (discussed above) is not H.
RN
5 and RN 6 are independently is selected from H, C 1
.
3 alkyl (i.e. methyl, ethyl, prop 1-yl and prop-2-yl) and C(=0)Me. 20 R3
R
3 is selected from O-C2 alkyl, C 1
.
2 alkyl, halo, cyano, where the C1.2 alkyl group may be substituted by one or more fluoro groups. Thus, R 3 may be: WO 2012/110774 PCT/GB2012/000176 10 a) halo: F, Cl, Br, 1; b) cyano: CN c) C 1
.
2 alkyl, optionally substituted by one or more fluoro groups: CH 3 , CH 2 F, CHF 2 ,
CF
3 , CH 2
CH
3 , CH 2
CH
2 F, CH 2
CHF
2 , CH 2
CF
3 , CHFCH 3 , CHFCH 2 F, CHFCHF 2 , 5 CHFCF 3 , CF 2
CH
3 , CF 2
CH
2 F, CF 2
CHF
2 , CF 2
CF
3 ; d) 0-C1.2 alkyl, wherein the C12 alkyl group is optionally substituted by one or more fluoro groups: 0-CH 3 , 0-CH 2 F, 0-CHF 2 , 0-CF 3 , O-CH 2
CH
3 , 0-CH 2
CH
2 F, 0-CH 2
CHF
2 , 0-CH 2
CF
3 , 0-CHFCH 3 , O-CHFCH 2 F, O-CHFCHF 2 , 0-CHFCF 3 , 0-CF 2
CH
3 , O-CF 2
CH
2 F, O-CF 2
CHF
2 , O-CF 2
CF
3 . 10 R4
R
4 is selected from CF 3 , halo (i.e. F, C, Br, 1), CF 2 H and CN. In some embodiments, the halo group is either Cl or Br. 15 R5
R
5 is selected from groups of the following formulae: 7 0 H R N R& R * (R ) (R ) 20 R 6
R
6 is selected from H, (CHRc1), 1 C(O)N(RN Z and (CH 2 )1 2
C(O)OZ
2 ; wherein: n1 is 1; Rd' is H or Me; RN6 is H or CH 3 ; 25 Z' is H, CH 3 or OCH 3 ; n2 is 1; and
Z
2 is CH 3 ; wherein only one of RN 6 and Z' may be CH 3 . 30 When R6 is H, R 7 (discussed below) is not H.
WO 2012/110774 PCT/GB2012/000176 11 If R 6 is (CHRc'),,C(O)N(RN 6 )Z1, it may be selected from: CH 2
C(O)NH
2 ,
CH
2 C(O)NHCH3, CH 2
C(O)NHOCH
3 , CH 2
C(O)NCH
3 0CH 3 , CHCH 3
C(O)NH
2 ,
CHCH
3
C(O)NHCH
3 , CHCH 3
C(O)NHOCH
3 ,, and CHCH 3
C(O)NCH
3 0CH 3 . 5 If R 6 is (CH 2 )n 2
C(O)OZ
2 , it is CH 2
C(O)OCH
3 .
R
7 H, and (CH 2 )m1C(O)N(R m )Y', wherein: 10 ml is 0 or 1; RMl is H; and Y' is H, Me or OCH 3 ; When R7 is H, R' (discussed above) is not H. 15 When R' is (CH 2 )m1C(O)N(R
M
')Y
1 , it may be selected from C(O)NH 2 , C(O)NHCH 3 ,
C(O)NHOCH
3 , CH 2
C(O)NH
2 , CH 2 C(O)NHCHaand CH 2
C(O)NHOCH
3 .
R
8 20 RB is H, except for when R 7 is C(=O)NH 2 , it may alternatively be C 1
.
2 alkyl. i.e. methyl or ethyl. Includes Other Forms Included in the above are the well known ionic, salt, solvate, and protected forms of 25 these substituents. For example, a reference to carboxylic acid (-COOH) also includes the anionic (carboxylate) form (-COO-), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (-N*HR'R 2 ), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. 30 Similarly, a reference to a hydroxyl group also includes the anionic form (-0-), a salt or solvate thereof, as well as conventional protected forms of a hydroxyl group. Isomers, Salts, Solvates, Protected Forms, and Prodrugs Certain compounds may exist in one or more particular geometric, optical, 35 enantiomeric, diasteriomeric, epimeric, stereoisomeric, tautomeric, conformational, or WO 2012/110774 PCT/GB2012/000176 12 anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d and I-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; a- and P-forms; axial and equatorial forms; boat-, 5 chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms"). Note that, except as discussed below for tautomeric forms, specifically excluded from the term "isomers", as used herein, are structural (or constitutional) isomers (i.e. 10 isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, -OCH 3 , is not to be construed as a reference to its structural isomer, a hydroxymethyl group, -CH 2 OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a 15 class of structures may well include structurally isomeric forms falling within that class (e.g., C 1
.
7 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl). The above exclusion does not pertain to tautomeric forms, for example, keto-, enol-, 20 and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro. H 0 OH H+ , O O / 0* -C-C .C=C C=C H+ keto enol enolate Note that specifically included in the term "isomer" are compounds with one or more 25 isotopic substitutions. For example, H may be in any isotopic form, including 'H, 2 H (D), and 3 H (T); C may be in any isotopic form, including 12C, "C and 14 C; 0 may be in any isotopic form, including 60 and "0; and the like. Unless otherwise specified, a reference to a particular compound includes all such 30 isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g., fractional crystallisation and chromatographic means) of such isomeric forms are WO 2012/110774 PCT/GB2012/000176 13 either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner. Unless otherwise specified, a reference to a particular compound also includes ionic, 5 salt, solvate, and protected forms of thereof, for example, as discussed below. It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the active compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge et al. J. 10 Pharm. Sci., 66, 1-19 (1977). For example, if the compound is anionic, or has a functional group which may be anionic (e.g., -COOH may be -COO~), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali 15 metal ions such as Na* and K*, alkaline earth cations such as Ca2* and Mg 2 +, and other cations such as Af*. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH 44 ) and substituted ammonium ions (e.g., NHR*,
NH
2
R
2 +, NHR 3 *, NR 4 *). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, 20 butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH 3
)
4 *. 25 If the compound is cationic, or has a functional group which may be cationic (e.g., -NH 2 may be -NH 3 +), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulphuric, sulphurous, nitric, nitrous, phosphoric, and phosphorous. Examples of suitable 30 organic anions include, but are not limited to, those derived from the following organic acids: acetic, propionic, succinic, glycolic, stearic, palmitic, lactic, malic, pamoic, tartaric, citric, gluconic, ascorbic, maleic, hydroxymaleic, phenylacetic, glutamic, aspartic, benzoic, cinnamic, pyruvic, salicyclic, sulfanilic, 2 acetyoxybenzoic, fumaric, phenylsulfonic, toluenesulfonic, methanesulfonic, 35 ethanesulfonic, ethane disulfonic, oxalic, pantothenic, isethionic, valeric, lactobionic, WO 2012/110774 PCT/GB2012/000176 14 and gluconic. Examples of suitable polymeric anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose. 5 It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the active compound. The term "solvate" is used herein in the conventional sense to refer to a complex of solute (e.g. active compound, salt of active compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc. 10 It may be convenient or desirable to prepare, purify, and/or handle the active compound in a chemically protected form. The term "chemically protected form", as used herein, pertains to a compound in which one or more reactive functional groups are protected from undesirable chemical reactions, that is, are in the form of a protected or protecting group (also known as a masked or masking group or a 15 blocked or blocking group). By protecting a reactive functional group, reactions involving other unprotected reactive functional groups can be performed, without affecting the protected group; the protecting group may be removed, usually in a subsequent step, without substantially affecting the remainder of the molecule. See, for example, Protective Groups in Orqanic Synthesis (T. Green and P. Wuts, Wiley, 20 1999). For example, a hydroxy group may be protected as an ether (-OR) or an ester (-OC(=O)R), for example, as: a t-butyl ether; a benzyl, benzhydryl (diphenylmethyl), or trityl (triphenylmethyl) ether; a trimethylsilyl or t-butyldimethylsily ether; or an 25 acetyl ester (-OC(=0)CH 3 , -OAc). For example, an aldehyde or ketone group may be protected as an acetal or ketal, respectively, in which the carbonyl group (>C=O) is converted to a diether (>C(OR) 2 ), by reaction with, for example, a primary alcohol. The aldehyde or ketone group is 30 readily regenerated by hydrolysis using a large excess of water in the presence of acid. For example, an amine group may be protected, for example, as an amide or a urethane, for example, as: a methyl amide (-NHCO-CH3); a benzyloxy amide ( 35 NHCO-OCH 2 C3H 5 , -NH-Cbz); as a t-butoxy amide (-NHCO-OC(CH 3
)
3 , -NH-Boc); a 2- WO 2012/110774 PCT/GB2012/000176 15 biphenyl-2-propoxy amide (-NHCO-OC(CH 3
)
2
CH
4
C
6
H
5 , -NH-Bpoc), as a 9 fluorenylmethoxy amide (-NH-Fmoc), as a 6-nitroveratryloxy amide (-NH-Nvoc), as a 2-trimethylsilylethyloxy amide (-NH-Teoc), as a 2,2,2-trichloroethyloxy amide (-NH Troc), as an allyloxy amide (-NH-Alloc), as a 2(-phenylsulphonyl)ethyloxy amide ( 5 NH-Psec); or, in suitable cases, as an N-oxide (>NO-). For example, a carboxylic acid group may be protected as an ester for example, as: an C1-7 alkyl ester (e.g. a methyl ester; a t-butyl ester); a C1.7 haloalkyl ester (e.g., a C1.7 trihaloalkyl ester); a triCO 1 7 alkylsilyl-C.
7 alkyl ester; or a C5.20 aryl-C 1
.
7 alkyl ester 10 (e.g. a benzyl ester; a nitrobenzyl ester); or as an amide, for example, as a methyl amide. For example, a thiol group may be protected as a thioether (-SR), for example, as: a benzyl thioether; an acetamidomethyl ether (-S-CH 2
NHC(=O)CH
3 ). 15 It may be convenient or desirable to prepare, purify, and/or handle the active compound in the form of a prodrug. The term "prodrug", as used herein, pertains to a compound which, when metabolised (e.g. in vivo), yields the desired active compound. Typically, the prodrug is inactive, or less active than the active compound, but may provide advantageous handling, administration, or metabolic 20 properties. For example, some prodrugs are esters of the active compound (e.g. a physiologically acceptable metabolically labile ester). During metabolism, the ester group (-C(=0)OR) is cleaved to yield the active drug. Such esters may be formed by esterification, for example, of any of the carboxylic acid groups (-C(=O)OH) in the parent compound, with, where appropriate, prior protection of any other reactive 25 groups present in the parent compound, followed by deprotection if required. Examples of such metabolically labile esters include those wherein R is C1-7 alkyl (e.g. -Me, -Et); C1.7 aminoalkyl (e.g. aminoethyl; 2-(N,N-diethylamino)ethyl; 2-(4 morpholino)ethyl); and acyloxy-C 1
.
7 alkyl (e.g. acyloxymethyl; acyloxyethyl; e.g. pivaloyloxymethyl; acetoxymethyl; 1-acetoxyethyl; 1-(1-methoxy-1-methyl)ethyl 30 carbonxyloxyethyl; 1-(benzoyloxy)ethyl; isopropoxy-carbonyloxymethyl; 1 isopropoxy-carbonyloxyethyl; cyclohexyl-carbonyloxymethyl; 1-cyclohexyl carbonyloxyethyl; cyclohexyloxy-carbonyloxymethyl; 1-cyclohexyloxy carbonyloxyethyl; (4-tetrahydropyranyloxy) carbonyloxymethyl; 1-(4 tetrahydropyranyloxy)carbonyloxyethyl; (4-tetrahydropyranyl)carbonyloxymethyl; and 35 1-(4-tetrahydropyranyl)carbonyloxyethyl).
WO 2012/110774 PCT/GB2012/000176 16 Also, some prodrugs are activated enzymatically to yield the active compound, or a compound which, upon further chemical reaction, yields the active compound. For example, the prodrug may be a sugar derivative or other glycoside conjugate, or may 5 be an amino acid ester derivative. Selectivity The selectivity of the compounds for inhibiting FAK over other kinases such as VEGFR1, VEGFR2 and/or VEGFR3, IGF-1R, IR and CDKs can be demonstrated by 10 biochemical assay results (see, for example, the FAK kinase assay and VEGFR3 assays described below). The selectivity of the compounds for FAK over the inhibition of cytochrome p450 enzymes, specifically the 2C9 and 3A4 isoforms may be determined using standard 15 inhibition assays. How prone the compounds of the invention may be to the formation of adducts with glutathione may be determined by the protocol described in Walker, et al. Biorg. Med. Chem. Letts. 2008, 18, 6071-6077. 20 The selectivity of compounds for inhibiting FAK over other enzymes may be expressed as ratio of other enzyme's inhibition (IC 50 ) to the FAK inhibition (IC 5 o). For example, to determine the selectivity of a compound for inihibitng FAK over VEGFR3, the compound's IC 5 o for VEGFR3 is divided by the compound's IC 5 o for FAK to give a 25 ratio. The higher the ratio, the more selective the compound is. Further Embodiments The following embodiments and preferences may be combined with one another as appropriate. 30 In some embodiments, R 2 is H and R 1 is: R NN N , wherein RN1 is selected from H, C13 alkyl (i.e. methyl, ethyl, prop-1 yl and prop-2-yl) and C(=O)Me. In some of these embodiments, it may be preferred WO 2012/110774 PCT/GB2012/000176 17 that RN1 is C(=O)Me. In others of these embodiments, it may be preferred that RN1 is H, methyl or ethyl. In further of these embodiments, it may be preferred that RN1 is H. In other embodiments, R 2 is H and R 1 is: N2 R N2 5 , wherein R is selected from H and C 1
.
3 alkyl (i.e. methyl, ethyl, prop-1-yl and prop-2-yl). In these embodiments, it may be preferred that RN 2 is selected from H and methyl. In these embodiments, it may also be preferred that RN 2 is ethyl. 10 In other embodiments, R 2 is H and R' is: RN I N * , wherein RN 3 is selected from H and C 1
.
3 alkyl (i.e. methyl, ethyl, prop-1-yl and prop-2-yl). In these embodiments, it may be preferred that RN 3 is selected from H and methyl. 15 In other embodiments, R 2 is H and R' is: N4 , wherein RN4 is selected from H and methyl. In these embodiments, it may be preferred that RN 4 is H. In some embodiments, R 1 is H and R 2 is: N5 R N N 20 * , where RN 5 is selected from H and C1.3 alkyl (i.e. methyl, ethyl, prop 1-yl and prop-2-yl). In these embodiments, it may be preferred that RN5 is selected from H and methyl.
WO 2012/110774 PCT/GB2012/000176 18 it may be preferred that R1 is selected from: N1 N2 R N N N N~ , and In some embodiments, R 1 is H and R 2 is: N6 R NN 5 * , where RN 6 is selected from H and C 1 .3 alkyl (i.e. methyl, ethyl, prop 1-yl and prop-2-yl). In these embodiments, it may be preferred that RN 6 is selected from H and methyl. It may be further preferred that R 1 is H and R 2 is: Me, N N 10 In some embodiments, R 3 is selected from F, Me, Et, OMe and OCF 3 . In some of these embodiments, R 3 is OMe. 15 In some embodiments, R 4 is selected from CF 3 , Cl, Br, CF 2 H, and CN. In further embodiments, R 4 is selected from CF 3 , Cl and CF 2 H. In further embodiments, R 4 is selected from CF 3 and Cl. It may be preferred that R 4 is CF 3 . 20 In some embodiments, it may be preferred that R 5 is a group of the following formulae: R7 R 6R 8
(R
5 a) In some embodiments, R 7 is H and R3 is (CHRc1),,C(O)N(RN6)Z.
WO 2012/110774 PCT/GB2012/000176 19 In further embodiments, R 7 is H and R' is selected from CH 2
C(O)NH
2 ,
CH
2
C(O)NHCH
3 , CHCH 3
C(O)NH
2 and CHCH 3
C(O)NHCH
3 . It may be preferred that R 7 is H and R 6 is selected from CH 2
C(O)NH
2 , 5 CHCH 3
C(O)NH
2 and CH 2
C(O)NHCH
3 , and more preferably from CH 2
C(O)NH
2 , and
CHCH
3
C(O)NH
2 , Most preferably R' is CH 2
C(O)NH
2 . In some embodiments, R8 is H and R 7 is (CH 2 )m1C(O)N(Rm')Y'. 10 In further embodiments, R 6 is H and R 7 is selected from C(O)NH 2 , C(O)NHCH 3 ,
CH
2
C(O)NH
2 and CH 2
C(O)NHCH
3 . It may be preferred that R 6 is H and R7 is C(O)NH 2 . 15 In some embodiments where R6 is H and R 7 is C(O)NH 2 , R" is methyl. In some embodiments, it may be preferred that R5 is a group of the following formula: H N 0 (R b) In selected embodiments of the invention, the compounds may of formula la: 0 Ril a CF3 N N H 20 wherein RO is selected from: HN N HN N N WO 2012/110774 PCT/GB2012/000176 20 and R 3 a is selected from Me, Et, OMe, OCF 3 , and F. In selected embodiments of the invention, the compounds may of formula lb: Na R 3bCF R ' I(Ib) N N Rb H wherein R b is selected from OMe and OCF 3 , and Rs' is selected from: 0
NH
2 N ~' NH 2 5 and Embodiments of the inventions are compounds of the examples, including compounds 1 to 13. Embodiments of particular interest include compounds 3, 6 and 10 11. General synthesis methods The compounds of the invention can be prepared employing the following general methods and using procedures described in detail in the experimental section. The 15 reaction conditions referred to are illustrative and non-limiting. Compounds of formula I, as described above, can be prepared by synthetic strategies outlined below, wherein the definitions above apply: 20 Scheme A R
R
3 L ' N LR NH 2 H R "Y1 + I I N 4 R R R 4 F1 F2 F3 WO 2012/110774 PCT/GB2012/000176 21 Compounds of formula F1 may be reacted with substituted synthetic anilines of formula F2 (as prepared in scheme C, D , E, F, G and H) to form intermediates of formula F3 where L' and L 2 may be the same or different and include Cl, Br, I, SMe,
SO
2 Me and R 4 = CF 3 , halogen, CF 2 H or CN. 5 Compounds of the formula F1 may be prepared where L' and L 2 are different (see scheme B) to allow regioselective substitution or when L'=L 2 suitable reaction conditions can be employed (choice of solvent, reaction temperature, addition of a Lewis acid, for example ZnCl 2 in diethyl ether) to allow L' to be selectively displaced 10 over L 2 . Where regiochemical mixtures and di-substitution are obtained the regioisomers may be separated by chromatography. Compounds of the formula F1 where L'=L 2 are either commercially available, for example 2,4-dichloro-5-(trifluoromethyl)pyrimidine, 2,4-dichloro-5-fluoropyrimidine, 15 2,4,5-trichloropyrimidine, 2,4-dichloro-5-bromopyrimidine, 2,4-dichloro-5 iodopyrimidine, 2,4-dichloro-5-cyanopyrimidine or may be prepared readily from commercial starting materials. Where R 4 = CF 3 and differentiation of L' and L 2 is desirable, the method outlined in scheme B may be employed. 20 Scheme B CI N Cl SMe N Cl SMe N I MeN N KIN
CF
3 ZnCI 2 CF HI CF 3 THF GI G2 G3 Commercially available 2,4-dichloro-5-(trifluoromethyl)pyrimidine (G1) can be selectively reacted with sodium thiomethoxide in the presence of zinc(II) chloride to give 2-thiomethyl-4-chloro-5-(trifluoromethyl)pyrimidine (G2). 2-Thiomethyl-4-chloro 25 5-(trifluoromethyl)pyrimidine (G2) can be further reacted, for example by conversion to 2-thiomethyl-4-iodo-5-(trifluoromethyl)pyrimidine (G3) under Finkelstein conditions and/or by oxidation with mCPBA to give the corresponding sulfone if further differentiation of the 2 and 4-position is required or if additional activation is desirable. 30 WO 2012/110774 PCT/GB2012/000176 22 Scheme C 3 NBoc NBoc NBocR N R N HNJ + I- Ii-- -j - I 0,NDa Pd/C 0 2 N H 2 N G5 F4 F5 F6 Commercially available tert-butyl piperazine-1-carboxylate (G5) and 4-fluoro-2 substituted-1-nitrobenzenes of the formula F4 can be reacted in an SNAr reaction to 5 give tert-butyl 4-(3-substituted-4-nitrophenyl)piperazine-1-carboxylates of the formula F5. Subsequent reduction via hydrogenation in the presence of a catalyst, for example palladium on charcoal or platinum oxide when R 3 is Cl, Br or 1, gives the corresponding anilines of the formula F6. 10 Scheme D OH CF B O= 'OH O SON 1~ 0 2 //CF 3 0 0. F7 d/ -CF 3 P BocN LDA BocN O 3 P G6 G7 NBoc NBoc
H
2 02N Pd/C H2N R3R F8 F9 The corresponding 4-piperidine analogues of F6 can be prepared by a sequence of reactions starting with the conversion of commercially available tert-butyl 4 oxopiperidine-1-carboxylate (G6) to vinyl triflate G7. Coupling of G7 in a Suzuki type 15 reaction with aryl boronic acids, or analogous boronic esters, of the formula F7 gives tetrahydropyridines of the formula F8. Subsequent reduction via hydrogenation in the presence of a catalyst, for example palladium on charcoal, gives anilino-piperidines of the formula F9. Alternatively, protected aniline analogues of compounds of the WO 2012/110774 PCT/GB2012/000176 23 formula F7, for example Boc or CBz protected anilines, can be employed in the Suzuki coupling step. This negates the need for a later reduction step and may be beneficial when R 3 is halo. 5 Scheme E -L3 Pd + HO'B .N : 0 2 N
R
3 O
O
2 N FI G8 F11
H
2
R
3 NH R NBoc Pt0 2 Boc20
H
2 N H2N F12 F13 3-Piperidine analogues can be prepared by reaction of commercially available compounds of the formula F1 0, where L 3 =I or Br, with pyridin-3-ylboronic acid (G8) in a Suzuki type reaction to form intermediates of the formula F11. Reduction of 10 compounds of the formula F11 with hydrogen in the presence of a catalyst, for example platinum oxide, gives intermediates of the formula F12 which may be protected using Boc anhydride to give compounds of the formula F13. Scheme F L Pd + +HO, NR 0 2 N 2 3 OH 0 2 N R3 F14 G9 F15 H2 R R3 : R / N NBoc PtO 2 H Boc 2 O
H
2 N N
H
2 N 15 F16 F17 WO 2012/110774 PCT/GB2012/000176 24 2-Piperidine analogues can be prepared by reaction of commercially available compounds of the formula F14, where L'=l or Br, with pyridin-2-ylboronic acid (G9) in a Suzuki type reaction to form intermediates of the formula F15. Reduction of compounds of the formula F15 with hydrogen in the presence of a catalyst, for 5 example platinum oxide, gives intermediates of the formula F16 which may be protected using Boc anhydride to give compounds of the formula F17. Scheme G R3 NBoc Pd R H 2
R
3 O HN
O
2 N N Pd/C H 2 N N 1 2 N NBoc NBoc P18 G5 F19 F20 10 Compounds of the formula F20 can be prepared by coupling of commercially available tert-butyl piperazine-1-carboxylate (G5) and compounds of the formula F18, where L 3 =1 or Br, in a Buchwald type reaction to give intermediates of the formula F19. Compounds of the formula F19 can be reduced with hydrogen in the presence of a catalyst, for example palladium on charcoal or platinum oxide, to give anilines of 15 the formula F20. Scheme H R X X R' F23 Y, Y 0 RS CO 2 H R 08 R Rs O R F21 F22 F24 H Y RR F25 WO 2012/110774 PCT/GB2012/000176 25 Compounds of the formula F21 may be reacted to form esters of the formula F22 where X=Br or I, R 8 =H or Me and Y is selected from a single bond, -CH and -CHCH 3 -. When Rol= t-Bu, Boc anhydride may be employed or where Ro'=Me 5 methanol in the presence of an acid, for example sulfuric acid, may be used to form the desired ester. Esters of the formula F22 can be reacted with terminal acetylenes of the formula F23 in a Sonagashira type coupling to give acetylenes of the formula F24 where R 9 =TMS, TES or (CH 3
)
2 C*OH. R 9 may then be removed to generate compounds of the formula F25. When R 9 =TMS or TES potassium carbonate or tetra 10 n-butyl ammonium fluoride may be employed to induce this transformation. When
RS=(CH
3
)
2 C*OH, sodium hydride in refluxed toluene may be used. Scheme I H RR R H N N Y L, Pd/Cu(I) R~ NN -N 0 R . R ,- R 4 0 , NI R 2H R ONR S0 R R 2 3 225 F26 H y H y N Nl NNY N) 0N R .- N R 0 R" N, R4 2 R 2 F28 F27 15 Pyrimidines of the formula F3 may be reacted with terminal acetylenes of the formula F25 to give acetylenes of the formula F26 in a Sonagashira type coupling. The acetylene in compounds of the formula F26 may be reduced to an alkane of the formula F27 using hydrogen gas in the presence of a transition metal catalyst. The 20 exact choice of catalyst and conditions employed is dependent on the nature of R 4 . For example, where R 4
=CF
3 , 10% Pd/C may be used, where R 4 CI, platinum oxide is employed. Esters of the formula F27 may then be deprotected to give carboxylic acids of the formula F28. Where RO'=Me or Et, lithium hydroxide solutions may be employed. Where R 01 =t-Bu, acidic solutions, for example trifluoroacetic acid in WO 2012/110774 PCT/GB2012/000176 26 dichloromethane may be used. It will be appreciated that under acidic conditions Boc protecting groups in R' and R 2 will also be cleaved. Scheme J H
R
3 R 11 R H y R 1.N, R N N OH N, / r, 4 O HATU R R
R
2 F28 R H y N NN I j-NR R R'N R0 O
R
2 5 F29 Carboxylic acids of the formula F28 can be converted to amides of the formula F29 using a suitable amine or ammonia salt in the presence of a peptide coupling agent, for example HATU. 10 Scheme K H
R
3 N R H R R N N
R
2 F27 R H N N 4 NRR 11 R R R 2 F29 Alternatively, when RO'=Me, esters of the formula F27 may be directly converted to amides of the formula F29 by reaction with an amine at elevated temperatures. 15 WO 2012/110774 PCT/GB2012/000176 27 Scheme L Re R' H X 0 x o F23 O 0 NH NH | NH F30 F31 F32 Where molecules with lactams fused to the right hand side aromatic ring are required compounds of the formula F30 can be reacted with terminal acetylenes of the 5 formula F23 in a Sonagashira type coupling to give acetylenes of the formula F31 where R 9 =TMS, TES or (CH 3
)
2 C*OH. R 9 may then be removed to generate compounds of the formula F32. When R9=TMS or TES, potassium carbonate or tetra-n-butyl ammonium fluoride may be employed to induce this transformation. When R 9
=(CH
3
)
2 C*OH, sodium hydride in refluxed toluene may be used. 10 Compounds of the formula F32 can then be coupled to compounds of the formula F3 and further elaborated as described above and below. Scheme M R 'R R R R N N F FR 3 Compounds of the formula F29, or analogues containing lactams, with Boc protecting groups present in R' or R 2 (in the place of RN1, RN 2 , RN 3 , RN 4 an NS) a hnb deprotected under acidic conditions, for example using trifluoroacetic acid in dichloromethane solutions, to give the corresponding parent piperazine or piperidine 20 compounds of the formula F33. Scheme N Y11 N R ~~NR R R NP R N 0 ' 15F29 F 33 WO 2012/110774 PCT/GB2012/000176 28 Compounds of the formula F33 may then be further modified by derivitisation of the amine functionality. For example, compounds of the formula F34 where RN1, RN 2 , RN 3
RN
4 , RNSor RNS = Me may be prepared by reductive alkylation with formaldehyde in the presence of sodium triacetoxyborohydride. Derivatives were RN N 2 , RN 3 R or 5 RN 6 = Et may be prepared by reductive alkylation with acetaldehyde in the presence of sodium triacetoxyborohydride. Compounds of the formula F34 where RN1, RN 2 , RNS, RNS or RN6 = acetyl may be prepared by reaction of compounds of the formula F33 with a suitable acylating agent, for example acetic anhydride. 10 An alternate strategy for the formation of compounds of the formula F27, where
R
3
=CF
3 and R 7 =H, is to prepare compounds of the formula F37, as outlined in scheme N. Scheme N H N' cF SMe N I Pd S' N MCPBA O cF 0 N1 C1F 3 Y R8 O R O R F25 G3 F35 N CF, H 2 N CF, S N )-I'S N ' // \ I 1 Y -010 0 0 N 0 0 O 0 1 0 0 O RR 15 F36 F37 Coupling of esters of the formula F25, where R 8 =H, with 4-iodo-2-(methylthio)-5 (trifluorornethyl)pyrimidine (G3) under Sonagashira conditions gives acetylenes of the formula F35. Oxidation, using MCPBA, gives sulfones of the formula F36. Reduction of the acetylene using hydrogen, in the presence of a catalyst, for example 10% 20 palladium on charcoal, gives compounds of the formula F37. Compounds of the formla F37 can be reacted with anilines of the formula F2 under acidic conditions, for example in the presence of trifluoro acetic acid to give WO 2012/110774 PCT/GB2012/000176 29 compounds of the formula F27 which can then be further elaborated as described above. Use of Compounds of the Invention 5 The present invention provides active compounds, specifically, active 2,4,5 substituted pyrimidines . The term "active", as used herein, pertains to compounds which are capable of inhibiting FAK activity, and specifically includes both compounds with intrinsic activity 10 (drugs) as well as prodrugs of such compounds, which prodrugs may themselves exhibit little or no intrinsic activity. Assays which may be used in order to assess the FAK inhibition offered by a particular compound are described in the examples below. 15 The present invention further provides a method of inhibiting FAK inhibition in a cell, comprising contacting said cell with an effective amount of an active compound, preferably in the form of a pharmaceutically acceptable composition. Such a method may be practised in vitro or in vivo. 20 The present invention further provides active compounds which inhibit FAK activity as well as methods of methods of inhibiting FAK activity comprising contacting a cell with an effective amount of an active compound, whether in vitro or in vivo. 25 Active compounds may also be used as part of an in vitro assay, for example, in order to determine whether a candidate host is likely to benefit from treatment with the compound in question. The invention further provides active compounds for use in a method of treatment of 30 the human or animal body. Such a method may comprise administering to such a subject a therapeutically-effective amount of an active compound, preferably in the form of a pharmaceutical composition. The term "treatment", as used herein in the context of treating a condition, pertains 35 generally to treatment and therapy, whether of a human or an animal (e-g. in WO 2012/110774 PCT/GB2012/000176 30 veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e. prophylaxis) is also 5 included. The term "therapeutically-effective amount" as used herein, pertains to that amount of an active compound, or a material, composition or dosage from comprising an active compound, which is effective for producing some desired therapeutic effect, 10 commensurate with a reasonable benefit/risk ratio. Cancer The present invention provides active compounds which are anticancer agents. One of ordinary skill in the art is readily able to determine whether or not a candidate 15 compound treats a cancerous condition for any particular cell type, either alone or in combination. Examples of cancers include, but are not limited to, bone cancer, brain stem glioma, breast Cancer, cancer of the adrenal gland, cancer of the anal region, cancer of the 20 bladder. cancer of the endocrine system, cancer of the oesophagus, cancer of the head or neck, cancer of the kidney or ureter, cancer of the liver, cancer of the parathyroid gland, cancer of the penis, cancer of the small intestine, cancer of the thyroid gland, cancer of the urethra, carcinoma of the cervix, carcinoma of the endometrium, carcinoma of the fallopian tubes, carcinoma of the renal pelvis, 25 carcinoma of the vagina, carcinoma of the vulva, chronic or acute leukemia, colon cancer, cutaneous or intraocular melanoma, haemetological malignancies, Hodgkin's disease, lung cancer, lymphocytic lymphomas, neoplasms of the central nervous system (CNS), ovarian cancer, pancreatic cancer, pituitary adenoma, primary CNS lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, 30 sarcoma of soft tissue, skin cancer, spinal axis tumors, stomach cancer and uterine cancer. Any type of cell may be treated, including but not limited to, lung, gastrointestinal (including, e.g., bowel, colon), breast (mammary), ovarian, prostate, liver (hepatic), 35 kidney (renal), bladder, pancreas, brain, and skin.
WO 2012/110774 PCT/GB2012/000176 31 The anti cancer treatment defined hereinbefore may be applied as a sole therapy or may involve, in addition to the compound of the invention, conventional surgery or radiotherapy or chemotherapy. Such chemotherapy may include one or more of the 5 following categories of anti-tumour agents: (i) other antiproliferative/antineoplastic drugs and combinations thereof, as used in medical oncology, such as alkylating agents (for example cisplatin, oxaliplatin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulphan, temozolamide and nitrosoureas); antimetabolites (for example 10 gemcitabine and antifolates such as fluoropyrimidines like 5 fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside, and hydroxyurea); antitumour antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic agents (for example vinca alkaloids like vincristine, vinblastine, vindesine 15 and vinorelbine and taxoids like taxol and docetaxel (Taxotere) and polokinase inhibitors); and topoisomerase inhibitors (for example epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan and camptothecin); (ii) cytostatic agents such as antioestrogens (for example tamoxifen, fulvestrant, toremifene, raloxifene, droloxifene and iodoxyfene), antiandrogens (for example 20 bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH antagonists or LHRH agonists (for example goserelin, leuprorelin and buserelin), progestogens (for example megestrol acetate), aromatase inhibitors (for example as anastrozole, letrozole, vorazole and exemestane) and inhibitors of 5*-reductase such as finasteride; 25 (iii) anti-invasion agents (for example c-Src kinase family inhibitors like 4-(6 chloro-2,3-methylenedioxyanilino)-7-[2-(4-methylpiperazin-1 -yl)ethoxy)-5 tetrahydropyran-4-yloxyquinazoline (AZD0530; International Patent Application WO 01/94341), N-(2-chloro-6-methylphenyl)-2-{6-[4-(2-hydroxyethyl)piperazin-1-yl]-2 methylpyrirmidin-4-ylamino}thiazole-5-carboxamide (dasatinib BMS-354825; J. Med. 30 Chem., 2004, 47, 6658-6661 and and 4-((2,4-dichloro-5-methoxyphenyl)amino)-6 methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinoline-3-carbonitrile (bosutinib, SKI-606; Cancer research (2003), 63(2), 375-81), and metalloproteinase inhibitors like marimastat, inhibitors of urokinase plasminogen activator receptor function or antibodies to Heparanase); WO 2012/110774 PCT/GB2012/000176 32 (iv) inhibitors of growth factor function: for example such inhibitors include growth factor antibodies and growth factor receptor antibodies (for example the anti erbB2 antibody trastuzumab [HerceptinT], the anti-EGFR antibody panitumumab, the anti erbBl antibody cetuximab [Erbitux, C225] and any growth factor or growth factor 5 receptor antibodies disclosed by Stern et al. Critical reviews in oncology/haematology, 2005, Vol. 54, pp11-29); such inhibitors also include tyrosine kinase inhibitors, for example inhibitors of the epidermal growth factor family (for example EGFR family tyrosine kinase inhibitors such as N-(3-chloro-4-fluorophenyl) 7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine (gefitinib, ZD1839), N-(3 10 ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (erlotinib, OSI 774) and 6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)-quinazolin-4 amine (Cl 1033), erbB2 tyrosine kinase inhibitors such as lapatinib, inhibitors of the hepatocyte growth factor family, inhibitors of the platelet-derived growth factor family such as imatinib, inhibitors of serine/threonine kinases (for example RaslRaf 15 signalling inhibitors such as farnesyl transferase inhibitors, for example sorafenib (BAY 43-9006)), inhibitors of cell signalling through MEK and/orAKT kinases, inhibitors of the hepatocyte growth factor family, c-kit inhibitors, abl kinase inhibitors, IGF receptor (insulin-like growth factor) kinase inhibitors; aurora kinase inhibitors (for example AZD11 52, PH739358, VX-680, MLN8054, R763, MP235, MP529, VX-528 20 AND AX39459) and cyclin dependent kinase inhibitors such as CDK2 and/or CDK4 inhibitors; (v) antiangiogenic agents such as those which inhibit the effects of vascular endothelial growth factor, [for example the anti vascular endothelial cell growth factor antibody bevacizumab (AvastinT) and VEGF receptor tyrosine kinase inhibitors such 25 as 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1 -methylpiperidin-4 ylmethoxy)quinazoline (ZD6474; Example 2 within WO 01/32651), 4-(4-fluoro-2 methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline (AZD2171; Example 240 within WO 00/47212), vatalanib (PTK787; WO 98/35985) and SU11248 (sunitinib; WO 01/60814), compounds such as those disclosed in International Patent 30 Applications W097/22596, WO 97/30035, WO 97/32856 and WO 98/13354 and compounds that work by other mechanisms (for example linomide, inhibitors of integrin avb3 function and angiostatin)]; (vi) vascular damaging agents such as Combretastatin A4 and compounds disclosed in International Patent Applications WO 99/02166, WO 00/40529, WO 35 00/41669, WO 01/92224, WO 02/04434 and WO 02/08213; WO 2012/110774 PCT/GB2012/000176 33 (vii) antisense therapies, for example those which are directed to the targets listed above, such as ISIS 2503, an anti-ras antisense; (viii) gene therapy approaches, including for example approaches to replace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2, GDEPT (gene 5 directed enzyme pro drug therapy) approaches such as those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme and approaches to increase patient tolerance to chemotherapy or radiotherapy such as multi drug resistance gene therapy; and (ix) immunotherapy approaches, including for example ex vivo and in vivo 10 approaches to increase the immunogenicity of patient tumour cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte macrophage colony stimulating factor, approaches to decrease T cell anergy, approaches using transfected immune cells such as cytokine transfected dendritic cells, approaches using cytokine transfected tumour cell lines and approaches using 15 anti idiotypic antibodies A combination of particular interest is with docetaxel. Other possible combinations of interest include with gemcitabine, cisplatin and the camptothecin prodrug irinotecan. 20 Administration The active compound or pharmaceutical composition comprising the active compound may be administered to a subject by any convenient route of administration, whether systemically/ peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. 25 transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, 30 intraarticular, subarachnoid, and intrasternal; by implant of a depot, for example, subcutaneously or intramuscularly. The subject may be a eukaryote, an animal, a vertebrate animal, a mammal, a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine (e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. a horse), a primate, simian (e.g. a monkey or ape), a monkey (e.g. marmoset, baboon), an ape 35 (e.g. gorilla, chimpanzee, orang-utan, gibbon), or a human.
WO 2012/110774 PCT/GB2012/000176 34 Formulations While it is possible for the active compound to be administered alone, it is preferable to present it as a pharmaceutical composition (e.g. formulation) comprising at least 5 one active compound, as defined above, together with one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilisers, preservatives, lubricants, or other materials well known to those skilled in the art and optionally other therapeutic or prophylactic agents. 10 Thus, the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition comprising admixing at least one active compound, as defined above, together with one or more pharmaceutically acceptable carriers, excipients, buffers, adjuvants, stabilisers, or other materials, as described herein. 15 The term "pharmaceutically acceptable" as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or 20 complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for 25 example, Reminqton's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include 30 the step of bringing into association the active compound with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. 35 WO 2012/110774 PCT/GB2012/000176 35 Formulations may be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, tablets, losenges, granules, powders, capsules, cachets, pills, ampoules, suppositories, pessaries, ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses, electuaries, or aerosols. 5 Formulations suitable for oral administration (e.g. by ingestion) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion 10 or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste. A tablet may be made by conventional means, e.g., compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free 15 flowing form such as a powder or granules, optionally mixed with one or more binders (e.g. povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers or diluents (e.g. lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc, silica); disintegrants (e.g. sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl 20 cellulose); surface-active or dispersing or wetting agents (e.g. sodium lauryl sulfate); and preservatives (e.g. methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid). Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or 25 controlled release of the active compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach. 30 Formulations suitable for topical administration (e.g. transdermal, intranasal, ocular, buccal, and sublingual) may be formulated as an ointment, cream, suspension, lotion, powder, solution, past, gel, spray, aerosol, or oil. Alternatively, a formulation may comprise a patch or a dressing such as a bandage or adhesive plaster impregnated with active compounds and optionally one or more excipients or 35 diluents.
WO 2012/110774 PCT/GB2012/000176 36 Formulations suitable for topical administration in the mouth include losenges comprising the active compound in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active compound in an inert basis such as gelatin 5 and glycerin, or sucrose and acacia; and mouthwashes comprising the active compound in a suitable liquid carrier. Formulations suitable for topical administration to the eye also include eye drops wherein the active compound is dissolved or suspended in a suitable carrier, 10 especially an aqueous solvent for the active compound. Formulations suitable for nasal administration, wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid 15 inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid for administration as, for example, nasal spray, nasal drops, or by aerosol administration by nebuliser, include aqueous or oily solutions of the active compound. 20 Formulations suitable for administration by inhalation include those presented as an aerosol spray from a pressurised pack, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichoro-tetrafluoroethane, carbon dioxide, or other suitable gases. 25 Formulations suitable for topical administration via the skin include ointments, creams, and emulsions. When formulated in an ointment, the active compound may optionally be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active compounds may be formulated in a cream with an oil-in water cream base. If desired, the aqueous phase of the cream base may include, for 30 example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane- 1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the active compound through the skin or other affected areas. Examples of such dermal 35 penetration enhancers include dimethylsulfoxide and related analogues.
WO 2012/110774 PCT/GB2012/000176 37 When formulated as a topical emulsion, the oily phase may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. 5 Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabiliser(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream 10 formulations. Suitable emulgents and emulsion stabilisers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulphate. The choice of suitable oils or fats for the formulation is based on achieving the desired 15 cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations may be very low. Thus the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, 20 propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. 25 Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used. Formulations suitable for rectal administration may be presented as a suppository 30 with a suitable base comprising, for example, cocoa butter or a salicylate. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active compound, such carriers as are known in the art to be appropriate. 35 WO 2012/110774 PCT/GB2012/000176 38 Formulations suitable for parenteral administration (e.g. by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal), include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, and 5 solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. Examples of suitable isotonic vehicles for use in such formulations include 10 Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the active compound in the solution is from about 1 ng/ml to about 10 pg/ml, for example from about 10 ng/ml to about 1 pg/ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition 15 requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Formulations may be in the form of liposomes or other microparticulate systems which are designed to target the active compound to blood components or one or more organs. 20 Dosage It will be appreciated that appropriate dosages of the active compounds, and compositions comprising the active compounds, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of 25 therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in 30 combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects. 35 WO 2012/110774 PCT/GB2012/000176 39 Administration in vivo can be effected in one dose, continuously or intermittently (e.g. in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for 5 therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. In general, a suitable dose of the active compound is in the range of about 100 pg to 10 about 250 mg per kilogram body weight of the subject per day. Where the active compound is a salt, an ester, prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately. 15 EXAMPLES The following are examples are provided solely to illustrate the present invention and are not intended to limit the scope of the invention, as described herein. Acronyms 20 For convenience, many chemical moieties are represented using well known abbreviations, including but not limited to, methyl (Me), ethyl (Et), n-propyl (nPr), iso propyl (iPr), n-butyl (nBu), tert-butyl (tBu), n-hexyl (nHex), cyclohexyl (cHex), phenyl (Ph), biphenyl (biPh), benzyl (Bn), naphthyl (naph), methoxy (MeO), ethoxy (EtO), benzoyl (8z), and acetyl (Ac). 25 For convenience, many chemical compounds are represented using well known abbreviations, including but not limited to, methanol (MeOH), ethanol (EtOH), iso propanol (i-PrOH), methyl ethyl ketone (MEK), ether or diethyl ether (Et2O), acetic acid (AcOH), dichloromethane (methylene chloride, DCM), trifluoroacetic acid (TFA), 30 dimethylformamide (DMF), tetrahydrofuran (THF), dimethylsulfoxide (DMSO), meta chloroperoxybenzoic acid (mCPBA), tert-butyloxycarbonyl (Boc), trimethylsilyl (TMS), triethylsilyl (TES), 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), diphenylphosphoryl azide (DPPA), 1,8 diazabicyclo[5.4.0]undec-7-ene (DBU), 1-ethyl-3-(3-dimethylaminopropyl) 35 carbodiimide (EDCI), 4-dimethylaminopyridine (DMAP), tetra-n-butylammonium WO 2012/110774 PCT/GB2012/000176 40 fluoride (TBAF), N,N-Diisopropylethylamine (DIPEA), 1-hydroxybenzotriazole (HOBt), and 1,2-dichloroethene (DOE). General Experimental Details 5 Unless otherwise stated the following generalisations apply. 1 NMR spectra were recorded on either a Bruker Avance DRX300 (300 MHz), a Bruker Ultrasheild plus (400 MHz) or a Varian Unity Inova 600 (600 MHz) spectrometer. The multiplicity of a signal is designated by the following 10 abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; br, broad; m, multiplet. All observed coupling constants, J, are reported in Hertz. "C NMR were recorded on a Bruker Avance DRX300 (75 MHz), a Bruker Ultrasheild plus (100 MHz) or a Varian Unity Inova 600 (150 MHz) spectrometer in a broad band decoupled mode. 15 LC/MS data was generated using either a Finnigan LCQ Advantage Max (LCMS-A), a Waters 70 3100 system (LCMS-B) or an Agilent 6100 Series Single Quad LC/MS (LCMS-C). LCMS Method A (LCMS-A) 20 instrument: Finnigan LCQ Advantage Max Pump: Finnigan Surveyor LC Pump Finnigan Surveyor Autosampler Finnigan Surveyor PDA Detector 25 LC conditions: Reverse Phase HPLC analysis Column: Gemini 3p C18 20x4.Omm 110A Injection Volume 1OpL Solvent A: Water 0.1% Formic Acid 30 Solvent B: Acetonitrile 0.1% Formic Acid Gradient: 10-100% B over 10min Detection: 100-600nm MS conditions: 35 Ion Source: Ion trap WO 2012/110774 PCT/GB2012/000176 41 Ion Mode: ES positive Temp: 300*C Capillary V- 25 Detection: Ion counting 5 Scan Range: 80-1OOOA mu Scan Time: 0.2 sec Acquisition time: 10min LCMS Method B (LCMS-B) 10 Instrument: Waters ZQ 3100 -Mass Detector Waters 2545-Pump Waters SFO System Fluidics Organizer Waters 2996 Diode Array Detector Waters 2767 Sample Manager 15 LC conditions: Reverse Phase HPLC analysis Column: XBridge TM C18 5pm 4.6x100mm Injection Volume 10pL 20 Solvent A: Water 0.1% Formic Acid Solvent B: Acetonitrile 0.1% Formic Acid Gradient: 10-100% B over 10min Flow rate: 1.5 mL/min Detection: 100-600nm 25 MS conditions: Ion Source: Single-quadrupole Ion Mode: ES positive Source Temp: 150*C 30 Desolvation Temp: 350*C Detection: Ion counting Cpillary (KV)-3.00 Cone (V): 30 Extractor (V):3 35 RF Lens (V): 0.1 WO 2012/110774 PCT/GB2012/000176 42 Scan Range: 100-1000 Amu Scan Time: 0,5 sec Acquisition time: 10min Gas Flow 5 Desolvation L/hr-650 LCMS Method C (LCMS-C) Instrument: Agilent 6100 Series Single Quad LC/MS Agilent 1200 Series HPLC 10 Pump: 1200 Series G1311A Quaternary pump Autosampler: 1200 Series G1329A Thermostatted Autosampler Detector: 1200 Series G1314B Variable Wavelength Detector LC conditions: 15 Reverse Phase HPLC analysis Column: Luna C8(2) 5p 50X 4.6mm 100A Column temperature: 30*C Injection Volume: 5pL Solvent A: Water 0.1% Formic Acid 20 Solvent B: Acetonitrile 0.1% Formic Acid Gradient: 5-100% B over 10min Detection: 254 nm or 214 nm MS conditions: 25 Ion Source: Quadrupole Ion Mode: Multimode-ES Drying gas temp: 300"C Vaporizer temperature: 200"C Capillary voltage (V): 2000 (positive) 30 Capillary voltage (V): 4000 (negative) Scan Range: 100-1000 Step size: 0.1 sec Acquisition time: 10min WO 2012/110774 PCT/GB2012/000176 43 Analytical thin-layer chromatography was performed on Merck silica gel 60F254 aluminium-backed plates which were visualised using fluorescence quenching under UV light or acidic anisaldehyde or a basic potassium permanganate dip. Flash chromatography was performed using either a Teledyne Isco CombiFlash Rf purifi 5 cation system using standard RediSep@ cartridges or a Biotage Isolera purification system using either Grace or Biotage silica cartridges. Where necessary, anhydrous solvents were prepared using a Braun purification system or purchased from Sigma-Aldrich. 10 Example 1: 2-(2-(2-(2-((2-Methoxy-4-(piperazin-1-yl)phenyl)amino)-5 (trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetamide (1) O 0 O NH 0
NO
2 + B NH
NO
2 N NH 2 F N F BOCN BOC N 11 12 0 0
N
0 H 0 N N CI00 ON N BOC N ON NNCF, BOCN 13 14 15 0 O U 0
N
0 H ~ 0 H N N N N N CF 3 N CF, BOC'NN BOCN 16 17
H
2 N 0 H 2 N 0 O O H I0H N N -. N N N N N N CF 3 Nf N CF 3 BOC'N HN 18 1 WO 2012/110774 PCT/GB2012/000176 44 (a) tert-Butyl 4-(3-methoxy-4-nitrophenyl)piperazine-1-carboxylate (/1) A mixture of 5-fluoro-2-nitroanisole (3.00 g, 17.5 mmol), tert-butyl piperazine-1 carboxylate (4.57 g, 24.5 mmol) and potassium carbonate (3.63 g, 26.3 mmol,) in dimethyl sulfoxide (20 mL) was heated at 90 *C for 1.5 hours under a nitrogen 5 atmosphere. The reaction mixture was cooled and diluted with water (100 mL). The resulting precipitate was filtered and dried to give a bright yellow solid. The solid was suspended in a 5:1 solution of petroleum ether/diethyl ether (50 mL), sonicated for 2 minutes, filtered and dried to give the title compound (/1) (3.95 g, 66%) as a bright yellow solid; 'H NMR (300 MHz, de-DMSO) 6 1.42 (s, 9 H), 3.46 (brs, 8 H), 3.90 (s, 3 10 H), 6.52 (s, 1 H), 6.57 (d, 1 H, J = 9.5 Hz) and 7.89 (d, 1 H, J = 9.3 Hz). LCMS Method B: rt 7.45 min; m/z 338.2 [M+H]+. (b) tert-Butyl 4-(4-amino-3-methoxyphenyl)piperazine-1-carboxylate (12) To a solution of tert-butyl 4-(3-methoxy-4-nitrophenyl)piperazine-1-carboxylate (/1) 15 (1.90 g, 5.63 mmol) in methanol (20 mL) and ethyl acetate (80 mL) was added 10% Pd/C (0.500 g). The mixture was evacuated and backfilled three times with hydrogen gas then stirred under a hydrogen atmosphere for 18 hours. The crude reaction mixture was filtered through a plug of Celite, washing with ethyl acetate and the filtrate evaporated in vacuo then dried under high vacuum to give a dark red viscous 20 oil. The oil was suspended in a 2:1 solution of diethyl ether and petroleum ether (15 mL) and sonicated for 3 minutes. The resulting mixture was evaporated in vacuo and dried under high vacuum to give the title compound (12) (1.55 g, 89%) as a dark red solid; 1 H NMR (300 MHz, d 6 -DMSO) 6 1.47 (s, 9 H), 2.97 (t, 4 H, J = 4.7 Hz), 3.57 (t, 4 H, J = 4.7 Hz), 3.83 (s, 3 H), 6.40 (dd, I H, J = 8.3, 2.1 Hz), 6.50 (d, I H, J = 2.0 25 Hz) and 6.64 (d, 1 H, J = 8.3 Hz). LCMS Method B: rt 5.02 min; m/z 308.3 [M+H]*. (c) telt-Butyl 4-(4-((4-chloro-5-(trifiuoromethyl)pyrimidin-2-yl)amino)-3 methoxyphenyl)piperazine-1-carboxylate (13) To a stirred chilled solution (0 *C) of 2,4-dichloro-5-(trifluromethyl)pyrimidine (0.800 g, 30 3.68 mmol) under a stream of dry N 2 gas in 1:1 dichloroethane/tert-butanol (50 mL) was added a solution of zinc chloride (1 M in diethyl ether; 4.42 mL, 4.42 mmol). After 1 hour of stirring at 0 *C, a solution of tert-butyl 4-(4-amino-3 methoxyphenyl)piperazine-1-carboxylate (12) in 1:1 dichloroethane/tert-butanol (15 mL) (1.13 g, 3.68 mmol, 1 eq.) was added drop wise over 30 minutes followed by the 35 drop wise addition of a solution of triethylamine (0.565 mL, 0.410 mmol, 1.1 eq.) in WO 2012/110774 PCT/GB2012/000176 45 1:1 dichloroethane/tert-butanol (5 mL) over 30 minutes, The resulting mixture was then stirred vigorously at room temperature for 18 hours. The volatiles were evaporated under reduced pressure and the residue then dried under high vacuum. Ethyl acetate and water were added and the aqueous layer was extracted with ethyl 5 acetate (3x30 mL). The combined organic extracts were washed with water, brine, dried (magnesium sulfate), filtered and evaporated to give the crude product as a dark yellow foam. The crude product was purified by flash column chromatography on silica gel (0-20% ethyl acetate/petroleum ether) to yield the title compound (13) (1.25 g, 69%) as a yellow solid; 'H NMR (300 MHz, CDCl 3 ) 6 1.48 (s, 9 H), 3.11 (t, 4 10 H, J = 5.2 Hz), 3.59 (t, 4 H, J = 5.0 Hz), 3.88 (s, 3 H), 6.54 (s, 1 H), 6.57 (d, 1 H, J= 9.4 Hz), 7.83 (s, 1 H), 8.17 (d, 1 H, J = 8.5 Hz) and 8.53 (s, 1 H). LCMS Method B: rt 9.24 min; m/z 488.3 [M+HY. (d) tert-Butyl 4-(3-methoxy-4-((4-((2-(2-methoxy-2-oxoethyl)phenyl)ethynyl)-5 15 (trifluoromethyi)pyrimidin-2-y)amino)phenyl)piperazine-1-carboxylate (15) To a solution of tert-butyl 4-(4-((4-chloro-5-(trifluoromethyl)pyrimidin-2-yl)amino)-3 methoxyphenyl)piperazine-1-carboxylate (172) (0.350 g, 0.717 mmol) in dimethylformamide (2.5 mL) and triethylamine (0.450 mL) was added methyl 2-(2 ethynylphenyl)acetate (14: prepared according to the procedure of Peng, C. et al.; 20 Adv. Synth. Catal. 2008, 350, 2359 - 2364 or as described below) (0.137 g, 0.789 mmol), trans-dichlorobis(triphenylphosphine) palladium(II) (0.025 g, 0.036 mmol), Cul (0.014 g, 0.072 mmol) and triphenylphosphine (0.019 g, 0.072 mmol). The reaction mixture was heated under microwave irradiation at 120 0C whilst stirring for 15 minutes. The reaction mixture was diluted with ethyl acetate and water then the 25 resulting mixture was extracted with ethyl acetate (3x1 5 mL). The combined organic extracts were washed with water, brine, dried (magnesium sulfate), filtered and evaporated to give a brown solid. The solid was purified by flash column chromatography on silica gel (10-50% ethyl acetate/petroleum ether) to give the title compound (15) (0.360 g, 80%) as an orange crystalline solid; 1 H NMR (300 MHz, 30 CDC 3 ) 6 1.49 (s, 9 H), 3.11 (s, 4 H), 3.60 (s, 4 H), 3.71 (d, 3 H, J = 1.0 Hz), 3.89 (s, 3 H), 3.96 (s, 2 H), 6.55 (s, 1 H), 6.59 (s, 1 H), 7.26-7.45 (m, 3 H), 7.68 (d, 1 H, J= 7.6 Hz), 7.83 (s, 1 H), 8.27 (d, 1 H, J = 7.3 Hz), and 8.60 (s, 1 H). LCMS Method B: rt 9.55 min; m/z 626.4 [M+Hf.
WO 2012/110774 PCT/GB2012/000176 46 (e) tert-Butyl 4-(3-methoxy-4-((4-(2-(2-methoxy-2-oxoethy)phenethy)-5 (trifluoromethyl)pyrimidin-2-yl)amino)phenyl)piperazine-1-carboxylate (16) To a solution of tert-butyl 4-(3-methoxy-4-((4-((2-(2-methoxy-2 oxoethyl)phenyl)ethynyl)-5-(trifluoromethyl)pyrimidin-2-yl)amino)phenyl)piperazine- 1 5 carboxylate (5) (0.358 g, 0.572 mmol) in ethyl acetate (25 mL) and dimethylformamide (5 mL) was added 10% Pd/C (0.200 g). The mixture was evacuated and backfilled three times with hydrogen gas then stirred under a hydrogen atmosphere for 18 hours. The reaction mixture was filtered through a plug of Celite, washing with ethyl acetate then the filtrate was evaporated in vacuo and 10 dried under high vacuum to give the title compound (16) (0.360 g, 100%) as a viscous yellow oil; 1 H NMR (300 MHz, CDC1 3 ) 5 1.48 (s, 9 H), 3.00-3.20 (m, 8 H), 3.59 (t, 4 H, J= 4.8 Hz), 3.67 (s, 3 H), 3.75 (s, 2 H), 3.90 (s, 3 H), 6.55-6.57 (m, 2 H), 7.19-7.26 (m, 4 H), 7.75 (s, 1 H), 8.26 (d, 1 H, J = 9.4 Hz) and 8.50 (s, 1 H). LCMS Method B: rt 6.55 min; m/z 630.5 [M+H]*. 15 (f) Lithium 2-(2-(2-(2-((4-(4-(tert-butoxycarbonyl)piperazin-1-y)-2 methoxyphenyl)amino)-5-(trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetate (17) To a solution of tert-butyl 4-(3-methoxy-4-((4-(2-(2-methoxy-2-oxoethyl)phenethyl)-5 (trifluoromethyl)pyrimidin-2-yl)amino)phenyl)piperazine-1-carboxylate (/6) (0.360 g, 20 0,572 mmol) in tetrahydrofuran (17 mL), methanol (3 mL) and water (5 mL) was added lithium hydroxide (0.042 g, 1.77 mmol) and the resulting mixture was stirred at room temperature for 20 hours. The reaction volatiles were evaporated in vacuo and the resulting residue was diluted with ethyl acetate and saturated aqueous sodium hydrogen carbonate. The aqueous phase was extracted with ethyl acetate (3x20 mL) 25 and the combined organic extracts were washed with saturated aqueous sodium hydrogen carbonate (20 mL), dried (magnesium sulfate), filtered, evaporated in vacuo and dried to give the title compound (/7) (0.356 g, 100%) as a yellow foam; 'H NMR (300 MHz, CDC 3 ) 5 1.48 (s, 9 H), 2.96-3.06 (m, 8 H) 3.56-3.62 (m, 6 H), 3.77 (s, 3 H), 6.47-6.51 (m, 2 H), 7.00-7.11 (m, 2 H), 8.00 (brs, 2 H) and 8.43 (s, 1 H). 30 LCMS Method B: rt 9.00 min, m/z 616.4 [M+H}*. (g) tert-Butyl 4-(4-((4-(2-(2-amino-2-oxoethy)phenethyl)-5-(trifluoromethyl)pyrimidin 2-y)amino)-3-methoxyphenyl)piperazine-1-carboxylate (18) To a stirred solution of lithium 2-(2-(2-(2-((4-(4-(tert-butoxycarbonyi)piperazin-1-yl)-2 35 methoxyphenyl)amino)-5-(trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetate ((7) WO 2012/110774 PCT/GB2012/000176 47 (0.352 g, 0.672 mmol), 1-hydroxybenzotriazole (0.093 g, 0.686 mmol) and 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide) hydrochloride (0.121 g, 0.629 mmol) in dry tetrahydrofuran (16 mL) and dimethylformamide (7 mL) was added N,N diisopropylethylamine (0.498 mL, 2.86 mmol) under a nitrogen atmosphere. The 5 reaction mixture was stirred for 10 minutes at room temperature then ammonium carbonate (0.263 g, 2.859 mmol) was added and the resulting suspension was stirred at room temperature for 18 hours. The reaction volatiles were evaporated in vacuo and the resulting residue was diluted with ethyl acetate (15 mL) and water (10 mL). The layers were separated and the aqueous layer was extracted with ethyl acetate 10 (2x15 mL). The combined organic extracts were washed with water, brine, dried (magnesium sulfate) and concentrated in vacuo to give a yellow foam which was purified by flash column chromatography on silica gel (55-85% ethyl acetate/petroleum ether) to give the title compound (/8) (0.276 g, 87%) as a yellow foam; 1 H NMR (300 MHz, CDC1 3 ) 5 1.48 (d, 9 H, J= 1.1 Hz), 3.09 (d, 8 H, J = 3.9 15 Hz), 3.58 (brs, 4 H), 3.66 (s, 2 H), 3.88 (d, 3 H, J = 1.0 Hz), 5.48 (brs, 1 H), 5.78 (brs, 1 H), 6.54 (d, 1 H, J = 1.3 Hz), 6.57 (s, 1 H), 7.23-7.25 (brs, 4 H), 7.72 (s, 1 H), 8.13 (d, 1 H, J = 8.6 Hz) and 8.48 (s, 1 H). LCMS Method B: rt 8.52 min; m/z 615.4 [M+H]*. 20 (f) 2-(2-(2-(2-((2-methoxy-4-(piperazin-1-yl)phenyl)amino)-5 (trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetamide (1) To a solution of tert-butyl 4-(4-((4-(2-(2-amino-2-oxoethyl)phenethyl)-5 (trifluoromethyl)pyrimidin-2-y)amino)-3-methoxyphenyl)piperazine-1-carboxylate (18) (0.276 g, 0.449 mmol) in dichloromethane (8 mL), was added trifluoroacetic acid 25 (0.669 mL, 8.98 mmol) and the resulting mixture was stirred under a nitrogen atmosphere for 18 hours. The solvents were evaporated in vacuo to give a yellow residue which was dissolved in 3:1 chloroform/2-propanol and 1 M aqueous sodium hydroxide solution. The aqueous layer was extracted with 3:1 chloroform,2-propanol (3x1 5 mL) and the combined organic extracts were washed with water, brine, dried 30 (magnesium sulfate), filtered and evaporated to give the title compound (1) (0.229 g, 99%) as a yellow foam; 1 H NMR (300 MHz, d 6 -DMSO) 6 2.87-3.10 (m, 12 H), 3.46 (s, 2 H), 3.78 (s, 3 H), 4.32 (d, 1 H, J = 4.2 Hz), 6.48 (dd, 1 H, J = 2.2, 8.7 Hz), 6.61 (d, 1 H, J = 2.1 Hz), 6.88 (brs, 1 H), 7.15-7.30 (m, 4 H), 7.38 (brs, 1 H), 7.46 (brs, 1 H), 8.51 (s, 1 H) and 8.81 (s, 1 H). LCMS Method B: rt 5.65 min; m/z 515.4 [M+H)*. 35 WO 2012/110774 PCT/GB2012/000176 48 Synthesis of Intermediate 14: Methyl 2-(2-Ethynylphenyl)acetate (14). 0 0 0 0 OH 01 19 110 14 (a) Methyl 2-(2-iodophenyl)acetate (/9) 5 2-(2-lodophenyl)acetic acid (5.00 g, 19.1 mmol) was placed into a reaction flask and dissolved in MeOH (150 mL). Sulfuric acid (250 pL) was added and reaction mixture was stirred and heated at 80 *C under nitrogen for 16 hours. The resulting mixture was cooled to room temperature and the volatiles removed by evaporation under reduced pressure. The residue was taken up in ethyl acetate (100 mL), washed with 10 10% NaHCO 3 (100 mL), dried (MgSO 4 ) and evaporated under reduced pressure to give the title compound (/9) (5.20g, 99%) as a clear liquid; 'H NMR (400 MHz, CDC 3 ) 6 7,85 (dd, J = 7.9, 1.0 Hz, 1H), 7.35 - 7.27 (m, 2H), 6.97 (ddd, J = 7.9, 7,0, 2.1 Hz, 1H), 3.81 (s, 2H), 3.72 (s, 3H). 15 (b) Methyl 2-(2-((trirnethylsilyl)ethynyl)phenyl)acetate ((10) Methyl 2-(2-iodophenyl)acetate (19) (4.65 g, 16.8 mmol), PdCl 2 (PPh 3
)
2 (295 mg, 421 pmol) and Cu(I)l (80.0 mg, 421 ppmol) were placed into an oven dried reaction flask under nitrogen. (Trimethylsilyl)acetylene (2.80 mL, 20.2 mmol), dry degassed THF (20 mL) and triethylamine (20 mL) were added and the reaction mixture was stirred 20 at room temperature for 16 hours. The volatiles were removed under reduced pressure to give a black residue which was adsorbed onto silica then chromatographed on silica gel (0-5% ethyl acetate/petroleum benzine 40-60 *C) to give the title compound (110) (4.63 g, 99%) as a light brown liquid; 'H NMR (400 MHz, CDCI 3 ) 6 7.48 (dd, J = 7.5, 0.8 Hz, 1H), 7.32 - 7.14 (m, 3H), 3.84 (s, 2H), 3.71 25 (s, 3H), 0.26 (s, 9H). LCMS Method C: rt 6.64 min. (c) Methyl 2-(2-ethynylphenyl)acetate (14) Methyl 2-(2-((trinethylsilyl)ethynyl)phenyl)acetate (/10) (4.63 g, 19.0 mmol) was dissolved in DCM (200 mL) and TBAF (1.0 M in THF) (28.5 mL, 28.5 mmol, 1.5 eq) 30 was added at 0 *C. The resulting solution was stirred at room temperature for 1 hour before washing with 10% NaHCO 3 (100 mL). The organic layer was dried (MgSO 4 ) then evaporated under reduced pressure to give a dark brown/black residue. The WO 2012/110774 PCT/GB2012/000176 49 residue was adsorbed onto silica and then chromatographed on silica gel (0-10% ethyl acetate/petroleum benzine 40-60 *C) to give the title compound (14) (2.76 g, 83%) as a red liquid; 'H NMR (400 MHz, CDC1 3 ) 6 7.52 (dd, J = 7.6, 1.1 Hz, 1H), 7.43 - 7.16 (m, 3H), 3,88 (d, J = 9.6 Hz, 2H), 3.77 - 3.52 (m, 3H), 3.28 (s, 1H). 5 Synthesis of Intermediate 115: Methyl 2-(2-(2-(2-(methylsulfonyl)-5 (trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetate (115) 0 C1 N C S N C1 ,S N I CFN CF N CF+ 111 112 14 N C 3 N 0 0 113 114 115 (a) 4-Chloro-2-(methylthio)-5-(trifluoromethyl)pyrimidine (111) 10 To a solution of the 2,4-dichloro-5-(trifluoromethyl)pyrimidine (2.50 g, 11.5 mmol) in THF (50 mL) in an ice bath under nitrogen was added zinc(II) chloride (1.0 M in ether, 13.8 mL, 13.8 mmol) dropwise. The mixture was stirred in the ice bath for two hours, then sodium methanethiolate (0.888 g, 12.7 mmol) was added. The mixture was stirred overnight, allowing the reaction to slowly come to room temperature. After 18 15 hours the reaction was quenched with 2 M HCl (15 mL), and the organics removed by evaporation under reduced pressure. The aqueous residue was diluted with brine (15 mL), and extracted with DCM (3x30 mL). The combined organic phases were dried (phase separator) and carefully evaporated to give a pale yellow oil. Chromatography (2x40g silica cartridge, 0-20% DCM/n-hexane) followed by carefully 20 evaporation of solvent (40 *C @ 400 mmHg then room temperature @ 200 mmHg) gave the title compound (/11) (2.149 g, 82% yield) as a colourless oil; 1 H NMR (600 MHz, CDCI 3 ) 6 8.66 (s, 1 H), 2.61 (s, 3H). LCMS Method C: rt: 7.95 min; m/z 229.1[M+H]+. Note: /11 is volatile. 25 (b) 4-/odo-2-(methylthio)-5-(trifluoromnethy/)pyrinidine (112) 4-Chloro-2-(methylthio)-5-(trifluoromethyl)pyrimidine (111) (5.00 g, 21.9 mmol) was placed into a reaction flask then sodium iodide (9,80 g, 65.6 mmol) and hydroiodic WO 2012/110774 PCT/GB2012/000176 50 acid (58%) (70 mL) were added. The reaction mixture was stirred for 48 hours in darkness then diluted with water (200 mL) where upon a colourless solid precipitated. The precipitate was collected by filtration and was washed with 10% NaHCO 3 solution until neutral. The resulting solid was washed with water (100 mL) then 5 suction dried for 2 hours to give the title compound (112) (3.956 g, 57%) as a pale yellow solid; 'H NMR (400 MHz, CDCl 3 ) 6 8.42 (s, 1H), 2.58 (s, 3H). LCMS Method C: rt 6.30 min; m/z 321.0 [M+H]*. (c) Methyl 2-(2-((2-(methylthio)-5-(trifluoromethyl)pyrimidin-4 10 yl)ethynyl)phenyl)acetate (113) 4-lodo-2-(methylthio)-5-(trifluoromethyl)pyrimidine (/12) (2.00 g, 6.24 mmol), PdCl 2 (PPh 3
)
2 (438 mg, 625 pmol), Cul (119 mg, 625 pmol) and triphenylphosphine (164 mg, 625 pmol) were placed into an oven dried microwave reaction vial under nitrogen. Methy) 2-(2-ethynylphenyl)acetate (14) (1.31 g, 7.49 mmol), THF (20 mL) 15 and triethylamine (10 mL) were added and the resulting mixture was stirred at 100 *C under microwave irradiation for 10 min. The volatiles were evaporated under reduced pressure then the residue was adsorbed onto silica from DCM. The pre-adsorbed material was chromatographed on silica gel (0-25% ethyl acetate/petroleum benzine 40-60 *C) to give the title compound (/13) (1.571 g, 69%) as an orange solid; 1 H 20 NMR (400 MHz, CDC13) 6 8.71 (d, J= 0.8 Hz, 1H), 7.68 (dd, J= 7.7, 1.1 Hz, 1H), 7.50 - 7.29 (m, 3H), 3.93 (s, 2H), 3.71 (d, J = 3.4 Hz, 3H), 2.62 (d, J = 3.4 Hz, 3H). (d) Methyl 2-(2-((2-(methylsufony)-5-(trifluoromethyl)pyrimidin-4 yl)ethynyl)phenyl)acetate (114) 25 Methyl 2-(2-((2-(methylthio)-5-(trifluoromethyl)pyrimidin-4-yl)ethynyl)phenyl)acetate (113) (3.14 g, 8.57 mmol) was dissolved in DCM (150 mL) and the resulting solution cooled to 0 *C. mCPBA (70%; 4.65 g, 18.9 mmol) was added then the reaction mixture was allowed to warm to room temperature, at which, stirring was continued overnight. The crude mixture was washed with 10% NaHCO 3 (200 mL) and the 30 layers were separated. The organics were dried (MgSO 4 ) then evaporated under reduced pressure to give a light yellow solid. The solid was adsorbed onto silica then chromatographed on silica gel (0-50% ethyl acetate/ petroleum benzine 40-60 *C) to give the title compound (114) (2.876 g, 84%) as a yellow solid; 'H NMR (400 MHz, CDCla) 6 9.13 (d, J = 0.7 Hz, 1H), 7.73 (dd, J = 7.6, 0.9 Hz, 1H), 7.54 - 7.46 (m, 1H), WO 2012/110774 PCT/GB2012/000176 51 7.44 - 7.32 (m, 2H), 3.94 (s, 2H), 3.77 - 3.67 (m, 3H), 3.43 (s, 3H). LCMS Method C: rt 5.90 min; m/z 421.0 (M+Na), 399.1 (M+1), 367.0 (M-OMe), 339.1 (M-COOMe). (e) Methyl 2-(2-(2-(2-(methyisulfonyl)-5-(trifluoromethyl)pyrimidin-4 5 yl)ethy)pheny)acetate (115) Methyl 2-(2-((2-(methylsulfonyl)-5-(trifluoromethyl)pyrimidin-4 yl)ethynyl)phenyl)acetate (114) (1.50 g, 3.76 mmol) was taken up in DMF (30 mL) then 10% Pd/C (750 mg) was added. The resulting suspension was stirred under H 2 (1 atm) for 16 hours at room temperature. The crude reaction mixture was filtered 10 through Celite, washing with MeOH. The filtrate was evaporated under reduced pressure to give a yellow liquid which was adsorbing onto silica. The silica adsorbed material was chromatographed on silica gel (0-100% ethyl acetate/petroleum benzine 40-60 *C) to give the title compound (115) (1.38 g, 91%) as a yellow solid; 'H NMR (400 MHz, CDCI 3 ) 6 9.07 (d, J = 0.7 Hz, 1 H), 7.30 - 7.12 (m, 4H), 3.72 (s, 2H), 3.68 15 (s, 3H), 3.41 - 3.35 (m, 2H), 3.35 (s, 3H), 3.20 (dd, J= 9.6, 6.3 Hz, 2H). LCMS Method C: rt 5.92 min; m/z 425.1 (M+Na), 403.1 (M+1), 401.1 (M-1), 371.1 (M-OMe), 343.1 (M-COOMe).
WO 2012/110774 PCT/GB2012/000176 52 Example 2: 2-(2-(2-(2-((2-Methoxy-4-(piperidin-4-yl)phenyl)amino)-5 (trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetamide (2) 0 N OT 0 N O Fa 0 0 116 0 NH2- 0 N2H H H 0 NO NBB N OO 0 11911 208 21 22~ 51 (116 H HN 0 ~BOCC CF, ''- N CF, 01 0,, 0,, 119 Hs5 120 SOC't40 CF,. CF,' N, 0 ~ N N 1 N N N N NHI 0, 0, 121 122 BOCpN w HN (3.0 ____N CF 3 _ NH, NN NH, NJ NZ_,F N N N ,H I .H 123 2 (a) tert-Butyl 4-(((trifluoromethyl)sulfonyl)oxy) -5, 6-dihydropyridine- 1(2H)-carboxylate 5 (116) Lithium diisopropylamide (2 M in heptane/THF/ethylbenzene; 15.1 mL, 30.1 mmol) was added drop wise to a solution of tert-butyl 4-oxopiperidine-1-carboxylate (3.00 g, 15.1 mmol) in THF (50 mL) at -78 *C and the mixture left to stir for 30 minutes. A solution of N-phenyl-bis(trifluoromethanesulfonimide) (6.46 g, 18.1 mmol) in THF (60 10 mL) was then added dropwise over 30 minutes to the reaction and mixture left to stir for 30 minutes at -78 *C. The resulting mixture was then allowed to warm to room temperature and was stirred for 24 hours. The solvent was partially removed (ca 80 mL) and the reaction mixture quenched with saturated NaHCO 3 solution (50 mL). DCM (50 mL) was added to the solution and the layers separated. The aqueous layer WO 2012/110774 PCT/GB2012/000176 53 was then extracted with DCM (2 x 50 mL). The organic layers were combined and washed with 0.2 M citric acid solution (50 mL), 1 M NaOH (50 mL), brine (50 mL) and dried over Na 2
SO
4 .The solvent was removed under reduced pressure to give a brown oil which was purified by column chromatography on silica gel (0-10% diethyl 5 ether in petroleum benzine 40-60 *C) to afford the title compound (116) (2.48 g, 50%) as an orange oil which crystallized on cooling to -18 *C; 1 H NMR (400 MHz, CDCl 3 ) 5 5.76 (s, 1H), 4.05 - 4,04 (in, 2H), 3.63 (t, J = 5.6 Hz, 2H), 2.46 - 2.43 (m, 2H), 1.47 (s, 9H). 10 (b) tert-Butyl (2-methoxy-4-(4,4,5, 5-tetramethyl-1,3,2-dioxaborolan-2 yl)phenyl)carbamate (117) 2-Methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (1.00 g, 4.01 mmol), Boc anhydride (1.75 g, 8.02 mmol) and toluene (10 mL) were stirred under nitrogen at 110 0C for 20 hours. The volatiles were evaporated under reduced pressure then 15 the residue adsorbed onto silica gel. Chromatography (SiO 2 , 0-20% ethyl acetate/petroleum benzine 40-80 *C) gave the title compound (/17) (1 409, 100%) as a colourless solid; 1 H NMR (400 MHz, CDCl) 6 8.11 (d, J = 7.9 Hz, 1H), 7.43 (dd, J = 8.0, 0.8 Hz, 1 H), 7.26 (dd, J = 7.8, 6.9 Hz, 1 H), 3.92 (s, 3H), 1.53 (s, 9H), 1.35 (s, 12H). LCMS Method C: rt 6.85 min. 20 (c) tert-Butyl 4-(4-((tert-butoxycarbonyl)amino)-3-methoxyphenyl)-5,6 dihydropyridine-1(2H)-carboxylate (118) To a solution of tert-butyl 4-(((trifluoromethyl)sulfonyl)oxy)-5,6-dihydropyridine-1 (2H) carboxylate (116) (474 mg, 1.43 mmol), tett-butyl (2-methoxy-4-(4,4,5,5-tetramethyl 25 1,3,2-dioxaborolan-2-yl)phenyl)carbamate (/17) (500 mg, 1.43 mmol) and Pd(PPh3)4 (165 mg, 0.143 mmol) in DME (15 mL) was added a solution of 3.5 M aqueous NaHCO 3 (2.00 mL, 5.0 eq) and the resulting suspension was heated at 80 0C for 16 hours. On cooling to room temperature ethyl acetate (70 mL) was added and the resulting solution was washed with water (50 mL). The organic layer was dried 30 (MgSO 4 ) and evaporated to dryness. The residue was chromatographed (SiO 2 , 0 20% ethyl acetate/petroleum benzine 40-60 *C) to give the title compound (118) (520 mg, 90%) as a pale yellow liquid; 'H NMR (400 MHz, CDCl 3 ) 6 8.03 (d, J = 8.1 Hz, 1H), 7.09 (s, 1H), 6.96 (dd, J = 8.4, 1.3 Hz, 1H), 6.88 (d. J = 1.7 Hz, 1H), 5.98 (s, 1H), 4.08 (d, J = 1.9 Hz, 2H), 3.89 (s, 3H), 3.64 (t, J = 5.6 Hz, 2H), 2.52 (s, 2H), 1.54 35 (s, 9H), 1.51 (s, 9H). LCMS Method C: rt 6.87 min; m/z 349.1 [M-t-Bu+2*.
WO 2012/110774 PCT/GB2012/000176 54 (d) tert-Butyl 4-(4-((tert-butoxycarbonyl)amino)-3-methoxyphenyl)piperidine-1 carboxylate (119) A suspension of tert-butyl 4-(4-((tert-butoxycarbonyl)amino)-3-methoxyphenyl)-5,6 5 dihydropyridine-1(2H)-carboxylate (/18) (500 mg, 1.23 mmol) and 10% Pd/C (50 mg) in MeOH (30 mL) was stirred under an atmosphere of hydrogen for 16 hours. The resulting mixture was filtered through celite, washing with ethyl acetate (70 mL) then the filtrate evaporated to dryness. The residue was chromatographed (SiOz, 0-20% ethyl acetate/petroleum benzine 40-60 *C) to give the title compound (119) (411 mg, 10 82%) as a pale yellow liquid; 'H NMR (400 MHz, CDCl 3 ) 5 7.94 (d, J = 7.4 Hz, 1 H), 6.99 (s, 1 H), 6.76 (d, J = 8.3 Hz, 1 H), 6.67 (s, 1 H), 4.21 (s, 2H), 3.84 (s, 3H), 2.77 (t, J = 12.2 Hz, 2H), 2.57 (ddd, J = 12.1, 9.0, 3.3 Hz, 1H), 1.79 (d, J = 12.6 Hz, 2H), 1.68 - 1.54 (m, 2H), 1.50 (s, 9H), 1.47 (s, 9H). 15 (e) Methyl 2-(2-(2-(2-((2-methoxy-4-(piperidin-4-yl)phenyl)amino)-5 (trifluoromethyl)pyrimidin-4-y)ethyl)pheryl)acetate (120) Methyl 2-(2-(2-(2-(methylsulfonyl)-5-(trifluoromethyl)pyrimidin-4 yl)ethyl)phenyl)acetate (/15) (200 mg, 0.497 mmol) and tert-butyl 4-(4-((tert butoxycarbonyl)amino)-3-methoxyphenyl)piperidine-1 -carboxylate (/19) (303 mg, 20 0.745 mmol) were dissolved in trifluoroethanol (4 mL). Trifluoroacetic acid (200 plL) was added and the resulting mixture was heated at 100 'C under microwave irradiation for 1 hour. The crude reaction mixture was adsorbed onto silica gel and separated by silica gel chromatography (0-20% MeOH/DCM) to give the title compound (/20) (215 mg, 82%) as a light brown liquid; 'H NMR (400 MHz, d 6 25 Acetone) 6 8.64 (s, IH), 8.37 (dd, J = 8.3, 3.7 Hz, 1H), 7.29 (ddd, J = 13.2, 6.9, 1.7 Hz, 3H), 7.22 (ddd, J = 8.9, 6.1, 1.7 Hz, IH), 7.10 (d, J= 1.5 Hz, 1H), 6.97 (dd, J = 8.3, 1.5 Hz, 1H), 3.98 (d, J = 7.4 Hz, 3H), 3.82 (s, 2H), 3.74 - 3.60 (m, 5H), 3.27 (t, J = 11.7 Hz, 2H), 3.22 - 3.06 (m, 4H), 3.00 (s, 1H), 2.34 - 2.17 (m, 2H), 2.12 (dd, J= 4.0, 3.5 Hz, 2H), 2.08 (dt, J = 6.6, 2.2 Hz, 3H). LOMS Method C: rt 5.23 min; m/z 30 529.1 [M+1]*. (f) tert-Butyl 4-(3-methoxy-4-((4-(2-(2-methoxy-2-oxoethyl)phenethyl)-5 (triftuoromethy/)pyrimidin-2-y)amino)pheny)piperidine-1-carboxylate (121) A solution of methyl 2-(2-(2-(2-((2-methoxy-4-(piperidin-4-yl)phenyl)amino)-5 35 (trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetate (120) (300 mg, 0.567 mmol) and WO 2012/110774 PCT/GB2012/000176 55 Boc anhydride (247 mg, 1.13 mmol) in DCM (10 mL) was stirred at room temperature under nitrogen for 20 hours. The volatiles were evaporated under reduced pressure to give a brown liquid which was adsorbed onto silica gel. Chromatography (SiO 2 , 0 15% MeOH/DCM) gave the title compound (/21) (210 mg, 59% yield) as a brown 5 liquid; 'H NMR (400 MHz, CDCl 3 ) 6 8.56 (s, 1H), 8.40 (d, J = 8.3 Hz, 1H), 7.95 (s, 1H), 7.31 - 7.18 (m, 4H), 6.87 (dd, J = 8.4, 1.7 Hz, 1H), 6.79 (d, J = 1.7 Hz, 1H), 4.28 (d, J = 6.5 Hz, 2H), 3.95 (d, J = 4.0 Hz, 3H), 3.78 (s, 2H), 3.70 (s, 3H), 3.26 - 3.02 (m, 4H), 2.84 (t, J = 12.2 Hz, 2H), 2.66 (t, J = 3.4 Hz, 1H), 1.87 (d, J = 12.7 Hz, 2H), 1.66 (dd, J = 12.9, 3.4 Hz, 2H), 1.52 (s, 9H). LCMS Method C: rt 7.24 min; m/z 629.2 10 [M+1]*, 627.0 [M-1]-. (g) Lithium 2-(2-(2-(2-((4-(1-(tert-butoxycarbonyl)piperidin-4-y)-2 methoxypheny)amino)-5-(trifluoromethy)pyrimidin-4-yl)ethyl)phenyl)acetate (122) A solution of tert-butyl 4-(3-methoxy-4-((4-(2-(2-methoxy-2-oxoethyl)phenethyl)-5 15 (trifluoromethyl)pyrimidin-2-yl)amino)phenyl)piperidine-1-carboxylate (121) (210 mg, 0.334 mmol) and LiOH.H 2 0 (42 mg, 1.0 mmol) in THF (10 mL), water (2 mL) and MeOH (1 mL) was stirred at room temperature for 20 hours. The volatiles were evaporated under reduced pressure to give the title compound (122) as a light yellow solid, 300 mg, excess mass due to inorganic salts present. LCMS Method C: rt 7.31 20 min; m/z 615.1 [M+1]*, 613.2 [M-1]~. (h) tert-Butyl 4-(4-((4-(2-(2-amino-2-oxoethyl)phenethyl)-5-(trifluoromethyl)pyrimidin 2-yl)amino)-3-methoxyphenyl)piperidine-1-carboxylate (123) A solution of lithium 2-(2-(2-(2-((4-(1-(tert-butoxycarbony)piperidin-4-y)-2 25 methoxyphenyl)amino)-5-(trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetate (/22) (210 mg, 0.338 mmol), HATU (257 mg, 0.676 mmol), ammonium chloride (362 mg, 6.76 mmol) and DIPEA (115 pL) in dry DMF (4 mL) was stirred at room temperature overnight. The volatiles were evaporated under reduced pressure and the residue diluted with ethyl acetate. The resulting solution was washed with 10% NaHCO 3 then 30 the organic layer dried (MgSO4). The volatiles were removed under reduced pressure and the residue chromatographed (SiO 2 , 0-100% ethyl acetate/petroleum benzine 40-60 *C) to give the title compound (/23) (185 mg, 89%) as a colourless solid; 1 H NMR (400 MHz, CDCl 3 ) 6 8.54 (s, 1 H), 8.26 (d, J = 8.3 Hz, 1 H), 7.88 (s, 1 H), 7.25 (dd, J = 4.4, 2.4 Hz, 2H), 6.86 (dd, J= 8.3, 1.7 Hz, 1 H), 6.76 (d, J= 1.7 Hz, 1 H), 5.49 35 (d, J = 39.2 Hz, 2H), 4.24 (s, 2H), 3.92 (s, 3H), 3.70 (s, 2H), 3.20 - 3.02 (m, 4H), 2.80 WO 2012/110774 PCT/GB2012/000176 56 (m, 2H), 2.64 (s, 2H), 1.84 (d, J = 12.8 Hz, 2H), 1.62 (dd, J = 12.3, 3.5 Hz, 2H), 1.49 (s, 9H). LCMS Method C: rt 6.64 min; m/z 636.2 [M+Na]*, 614.1 [M+1]*, 612.2 [M-1], 558.2 [M-t-Bu+2]*. 5 (i) 2-(2-(2-(2-((2-Methoxy-4-(piperidn-4-yl)phenyl)amnino)-5-(trifluoromethyl)pyrimidin 4-y)ethyI)phenyl)acetamide (2) Trifluoroacetic acid (1 mL) was added to a stirred solution of tert-butyl 4-(4-((4-(2-(2 amino-2-oxoethyl)phenethyl)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-3 methoxyphenyl)piperidine-1-carboxylate (123) (185 mg, 0.301 mmol) in DCM (10 mL) 10 and the resulting solution was stirred at room temperature for 2 hours. DCM (20 mL) and 10% NaHCO 3 solution (10 mL) were added then the organic layer was dried (MgSO 4 ) and evaporated to dryness under reduced pressure to give the title compound (2) (125 mg, 81%) as a colourless solid; 'H NMR (400 MHz, CD 2 Cl 2 ) 6 8.49 (s, 1H), 8.31 (d, J = 8.3 Hz, 1H), 7.91 (s, 1H), 7.28 - 7.12 (m, 3H), 6.94 (bs, 2H), 15 6.85 (d, J = 8.2 Hz, 1H), 6.78 (dd, J = 11.8, 3.1 Hz, 1H), 6.35 (s, 1H), 5.94 (s, 1H), 3.88 (s, 3H), 3.65 (s, 2H), 3.42 (d, J = 12,3 Hz, 2H), 3.24 - 3.02 (m, 4H), 2.91 (t, J 11.3 Hz, 2H), 2.75 - 2.61 (m, 1H), 2.10 - 1.83 (m, 4H). LCMS Method C: rt 4.92 min; m/z 514.1 [M+1]*, 521.1 [M-1]~. 20 Example 3: 2-(2-(2-(2-((2-Methoxy-4-(1-methylpiperidin-4-yl)phenyl)amino)-5 (trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetamide (3)
GF
3 NN CF3S H H N N N N NNH 0 H 'No' 2 3 Formaldehyde solution (37% aq.; 32 pL, 0.39 mmol) was added to a stirred solution of 2-(2-(2-(2-((2-methoxy-4-(piperidin-4-yl)pheny)amino)-5-(trifluoromethyl)pyrimidin 25 4-yl)ethyl)phenyl)acetamide (2) (40 mg, 78 pmol) in dry MeOH (2 mL). Sodium triacetoxyborohydride (83 mg, 0.39 mmol) was added under nitrogen and the resulting mixture was stirred at room temperature for 2 hours. The crude mixture was diluted with ethyl acetate and adsorbed onto silica gel. Chromatography (SiO 2 , 0 20% MeOH/DCM) gave the title compound (3) (25 mg, 61%) as a solid; 1 H NMR (400 30 MHz, CD 2
CI
2 ) 6 8.53 (s, 1H), 8.34 (d, J = 8.3 Hz, 1H), 7.94 (s, 1H), 7.29 - 7.15 (m, 4H), 6.92 (dd, J= 8.3, 1.4 Hz, 1H), 6.82 (d, J = 1.7 Hz, 1H), 6.12 (bs, IH), 5.84 (bs, WO 2012/110774 PCT/GB2012/000176 57 1H), 3.91 (s, 3H), 3.68 (s, 2H), 3.60 (d, J = 10.6 Hz, 2H), 3.24 - 3.03 (m, 4H), 2.80 (s, 3H), 2.79 (m, 2H), 2.71 (m, 1H), 2.42 - 2.20 (m, 2H), 2.01 (m, 2H). LCMS Method C: rt 4.92 min; m/z 528.1 [M+1]*, 526.1 [M-1]-. 5 Example 3A: Alternative synthesis of 2-(2-(2-(2-((2-Methoxy-4-(piperidin-4 yl)phenyl)amino)-5-(trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetamide (2) and 2-(2-(2-(2-((2-Methoxy-4-(1 -methylpiperid in-4-yl)phenyl)amino)-5 (trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetamide (3) 011 F0 Br O Br BOC N NO 2 0 2 NN 124 125 ~ H 2 H C BOC'N BOC 126 127 BOCN 0 BOCN N O 0,0 128 121 BOC'N H0 NH2 HN 0NH CF3 NN"NNH N N C2 H INI N 23 2 0 N CF 3
NH
2 N N 0,H 3 10 (a) 4-Bromo-2-methoxy-1-nitrobenzene (124) Sodium metal (3.14 g, 136 mmol) was added portion-wise to methanol (300 mL) under a nitrogen atmosphere. Once a homogeneous solution was obtained 4-bromo 2-fluoro-1-nitrobenzene (20.0 g, 91 mmol) was added and the resulting mixture stirred at 60 0C for 1 h. The volatiles were evaporated and the solid residue WO 2012/110774 PCT/GB2012/000176 58 suspended in water (400 mL). The resulting suspension was filtered washing with water (2x50 mL). The filter cake was air dried to give the title compound (124) (20.1 g, 95% yield) as a pale yellow solid; 'H NMR (400 MHz, CDCI3) 6 7.75 (d, J = 8.6 Hz, 1H), 7.24 (d, J = 1.9 Hz, 1H), 7.18 (dd, J = 8.6, 1.9 Hz, 1H), 3.97 (s, 3H). LCMS 5 Method C: rt 5.85 min. (b) tert-Butyl 4-(3-methoxy-4-nitrophenyl)-5,6-dihydropyridine-1(2HI)-carboxylate (125) A mixture of 4-bromo-2-methoxy-1-nitrobenzene (124) (2.364 g, 10.2 mmol) tert-butyl 10 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1 (2H)-carboxylate (3.00 g, 9.70 mmol), potassium carbonate (4.02 g, 29.1 mmol) and DMF (60 mL) was degassed with three vacuum/nitrogen cycles then PdCl 2 (dppf)-DCM solvate (450 mg, 6 mol%) added under nitrogen in a Schlenk tube. A second tube was prepared in the same manner (ie. a total of 6.00 g starting boronate), and both tubes were heated to 15 85 *C under nitrogen. After 17 hours both tubes were cooled under nitrogen and added to 5% w/v aqueous lithium chloride (600 mL). The resulting mixture was extracted with ether (300 mL) and ethyl acetate (3x300 mL). The combined organic extracts were washed with water (600 mL) and brine (600 mL), dried (sodium sulphate) then evaporated to dryness. The residue was chromatographed (120 g 20 silica cartridge, 0-60% ethyl acetate/petroleum benzine 40-60 *C) to give the title compound (125) (5.863 g, 91% yield) as a yellow solid; 1 H NMR (400 MHz, CDC 3 ) 6 7.86 (d, J = 8.5 Hz, 1H), 7.04 - 6.97 (m, 2H), 6.17 (s, 1H), 4.12 (d, J = 2.5 Hz, 2H), 3.98 (s, 3H), 3.65 (t, J = 5.6 Hz, 2H), 2.52 (s, 2H), 1.49 (s, 9H). LCMS Method C: rt 6.27 min; m/z 279.0 [M-tBu+2H)*, 235.1 [M-Boc+2H]*. 25 (c) tert-Butyl 4-(4-amino-3-methoxypheny1)piperidine-1-carboxylate (126) tert-Butyl 4-(3-methoxy-4-nitrophenyl)-5,6-dihydropyrid ine-1 (2H)-carboxylate (125) (5.609 g, 16.78 mmol) was dissolved in 1:1 ethanol: ethyl acetate (500 mL) and Pd/C (2.50 g) was added. The mixture was stirred vigorously under hydrogen for five hours 30 then filtered through celite. The celite was washed with ethyl acetate (500 mL) and the combined filtrates evaporated to give the title compound (126) (4.93 g, 96% yield) as an off-white solid; 'H NMR (400 MHz, CDC 3 ) 6 6.69 - 6.60 (m, 3H), 4.22 (s, 2H). 3.84 (s, 3H), 2.78 (t, J = 12.4 Hz, 2H), 2.54 (tt, J = 121, 3.5 Hz, 1H), 1.80 (d, J = 13.1 Hz, 2H), 1.65 - 1.53 (m, 2H), 1.48 (s, 9H). LCMS Method C: rt 4.91 min; m/z 251.1 35 [M-tBu+2H]*, 207.2 (M-Boc+2H]*.
WO 2012/110774 PCT/GB2012/000176 59 (d) tert-Butyl 4-(4-((4-chloro-5-(trifluoromethy)pyrimidin-2-yI)amino)-3 methoxyphenyl)piperidine-1-carboxylate (127) A solution of 2,4-dichloro-5-(trifluoromethyl)pyrimidine (981 mg, 4.52 mmol) in 1:1 5 tert-butanol: dichloroethane (40 mL) was cooled to 0 'C under nitrogen then 1.0 M zinc(II) chloride in ether (4.52 mL, 4.52 mmol) was added dropwise. After one hour a solution of tert-butyl 4-(4-amino-3-methoxyphenyl)piperidine-1-carboxylate (/26) (1.26 g, 4,11 mmol) and triethylamine (0.860 mL, 6.17 mmol) in 1:1 tert-butanol: dichloroethane (50 mL) was added dropwise at -10 'C then the mixture was stirred 10 for 16 hours allowing the temperature to rise to room temperature. The mixture was concentrated, evaporated onto silica and chromatographed (120 g silica cartridge, 0 10% ethyl acetate/petroleum benzine 40-60 *C) to give the title compound (127) (1.277 g. 64% yield) as a white solid; 'H NMR (400 MHz, CDC13) 6 8.57 (s, 1H), 8.28 (d, J = 8.3 Hz, 1H), 8.01 (s, 1H), 6.86 (dd, J = 8.3, 1.8 Hz, 1H), 6.76 (d, J = 1.8 Hz, 15 1 H), 4.25 (br s, 2H), 3.91 (s, 3H), 2.87 - 2.73 (m, 2H), 2.64 (tt, J = 12.1, 3.6 Hz, 1 H), 1.83 (d, J = 12.5 Hz, 2H), 1.69 - 1.54 (m, overlaps with water), 1.49 (s, 9H). LCMS Method C: rt 7.12 min; m/z 431.0 [M-tBu+2H]*, 387.1 [M-Boc+2H]*; m/z 485.1 [M-H)~. (e) tert-Butyl 4-(3-methoxy-4-((4-((2-(2-methoxy-2-oxoethyl)phenyl)ethyny)-5 20 (trifluoromethyl)pyrirnidin-2-yl)amino)phenyl)piperidine-1-carboxylate (128) A mixture of methyl 2-(2-ethynylphenyl)acetate (14) (0.472 g, 2.71 mmol), tert-butyl 4 (4-((4-chloro-5-(trifluoromethyl)pyrimidin-2-yl)amino)-3-methoxyphenyl)piperidine-1 carboxylate (/27) (1.10 g, 2.26 mmol), triphenylphosphine (59 mg, 10 mol%), copper(l) iodide (43 mg, 10 mol%), bis(triphenylphosphine)palladium(11) chloride (79 25 mg, 5 mol%). DMF (12 mL) and triethylamine (1.26 mL, 9.04 mmol) was degassed with nitrogen in a microwave tube then heated under microwave irradiation at 120"C for 15 minutes. Three additional tubes were prepared and heated as described above (i.e. a total of 4.40 g of /27). The cooled mixtures were combined, and poured into water (600 mL). The resulting mixture was extracted with dichloromethane (3x 250 30 mL) then the combined organic phases were washed with water (2x 300 mL), brine (300 mL), dried over sodium sulfate and evaporated to dryness. The residue was chromatographed (120 g silica cartridge, 0-50% ethyl acetate/petroleum benzine 40 60 "C) to give the title compound (128) (5.52 g, 98% yield) as a yellow solid; 'H NMR (400 MHz, CDCl 3 ) 6 8.63 (s, 1H), 8.38 (d, J = 8.3 Hz, 1H), 8.00 (s, 1H), 7.69 (dd, J = 35 7.7, 0.9 Hz, 1H), 7.43 (td, J = 7.6, 1.4 Hz, 1H), 7.36 (d, J = 7.9 Hz, 1H), 7.32 (dd, J = WO 2012/110774 PCT/GB2012/000176 60 7.5, 1.4 Hz, 1 H), 6.87 (dd, J = 8.4, 1.8 Hz, 1H), 6.76 (d, J = 1.8 Hz, 1H), 4.26 (s, 2H), 3.97 (s, 2H), 3.91 (s, 3H), 3.71 (s, 3H), 2.81 (t, J = 12.6 Hz, 2H), 2.64 (tt, J = 12.2, 3.6 Hz, 1 H), 1.84 (d, J = 12.6 Hz, 2H), 1.69 - 1.55 (m, overlaps with water), 1.49 (s, 9H). LCMS Method C: rt 7.05 min; m/z (625.1 [M+H]*, 569.1 [M-tBu+2H]*; m/z 623.2 [M 5 H]i. (f) tert-Butyl 4-(3-methoxy-4-((4-(2-(2-methoxy-2-oxoethyi)phenethyl)-5 (trifluoromethyl)pyrimidin-2-yl)amino)phenyl)piperidine-1-carboxylate (121) tert-Butyl 4-(3-methoxy-4--((4-((2-(2-methoxy-2-oxoethyl)phenyl)ethynyl)-5 10 (trifluoromethyl)pyrimidin-2-yl)amino)phenyl)piperidine-1 -carboxylate (/26) (5.52 g, 8.84 mmol) was dissolved in DMF (250 mL) then triethylamine (1 mL) and Pd/C (2.50 g) were added and the mixture stirred at 30 *C under hydrogen. After 16 hours the mixture was filtered through celite and the celite washed with DMF (250 mL). Triethylamine (1 mL) and Pd/C (2.50 g) were added to the combined filtrates and the 15 mixture stirred at 30 'C under hydrogen. After 18 hours the resulting mixture was filtered through celite washing with ethyl acetate (300 mL). The filtrate was divided into two lots; each was poured into water (800 mL) and the resulting mixture extracted with ethyl acetate (3x 300 mL). The ethyl acetate extracts from each lot were combined, washed with water (2x 500 mL) and brine, dried (sodium sulfate) and 20 evaporated. The two workups were combined and chromatographed (120 g silica cartridge, 0-20% ethyl acetate/petroleum benzine 40-60 *C) to give the title compound (121) (3.620 g, 65% yield) as a white solid; 'H NMR (400 MHz, CDCI 3 ) 6 8.55 (s, 1H), 8.37 (d, J = 8.3 Hz, 1H), 7.91 (s, 1H), 7.29 - 7.18 (m, overlaps with
CHC
3 ), 6.85 (dd, J = 8.3, 1.8 Hz, 1H), 6.77 (d, J 1.8 Hz, 1H), 4.26 (s, 2H), 3.93 (S, 25 3H), 3.68 (s, 3H), 3.19 - 3.01 (m, 4H), 2.81 (t, J 12.7 Hz, 2H), 2.64 (tt, J = 12.0, 3.5 Hz, 1 H), 1.85 (d, J = 13.3 Hz, 2H), 1.70 - 1.57 (m, overlaps with water), 1.49 (s, 9H). LCMS Method C: rt 7.14 min; m/z 629.2 [M+H]*, 573.1 [M-tBu+2H]*; m/z 627.2 [M-H]. (g) tert-Butyl 4-(4-((4-(2-(2-amino-2-oxoethyl)phenethyl)-5-(trifluorornethyl)pyrimidin 30 2-y)amino)-3-methoxyphenyl)piperidine-1-carboxylate (123) tert-Butyl 4-(3-methoxy-4-((4-(2-(2-methoxy-2-oxoethyl)phenethyl)-5 (trifluoromethyl)pyrimidin-2-yl)amino)phenyl)piperidine-1-carboxylate (121) (3.62 g, 5.76 mmol) was dissolved in THF (200 mL) then a solution of lithium hydroxide monohydrate (1.21 g, 28.8 mmol) in water (100 mL) was added. The resulting 35 mixture was stirred for 18 hours at room temperature then concentrated. The residue WO 2012/110774 PCT/GB2012/000176 61 was poured into saturated sodium bicarbonate (200 mL) and water (300 mL). The resulting mixture was extracted with ethyl acetate (3x300 mL) and the combined organic phases were washed with brine, dried over sodium sulfate and evaporated. The residue was evaporated from toluene then dissolved in THF (70 mL) and DMF 5 (12 mL) at 30 *C under nitrogen. The mixture was stirred vigorously while HOBT (1.012 g, 7.49 mmol), EDCI.HCI (1.330 g, 7.49 mmol) and DIPEA (5.02 mL, 28.8 mmol) were added. After five minutes ammonium carbonate (2.77 g, 28.8 mmol) was added and stirring continued for 16 hours. The resulting mixture was poured into saturated sodium bicarbonate (300 mL) and extracted with ethyl acetate (3x300 mL). 10 The combined organic phases were washed with 1:1 saturated brine: water (2x300 mL), brine (300 mL), dried over sodium sulfate and evaporated. The residue was chromatographed (120 g silica cartridge, 0-80% ethyl acetate/petroleum benzine 40 60 *C) to give the title compound ((23) (2.698 g, 76% over two steps) as a white solid; 'H NMR (400 MHz, CDCl 3 ) 6 8.54 (s, 1H), 8.28 (d, J = 8.3 Hz, 1H), 7.86 (s, 15 1H), 7.29 - 7.22 (m, overlaps with residual CHCl 3 ), 6.86 (dd, J = 8.3, 1.9 Hz, 1H), 6.76 (d, J = 1.9 Hz, 1H), 5.37 (d, J 15.9 Hz, 2H), 4.25 (s, 2H), 3.93 (s, 3H), 3.71 (s, 2H), 3.18 - 3.04 (m, 4H), 2.81 (t, J 12.5 Hz, 2H), 2.64 (tt, J = 12.0, 3.4 Hz, 1H), 1.84 (d, J = 12.9 Hz, 2H), 1.69 - 1.55 (m, overlaps with water), 1.49 (s, 9H). LCMS Method C: rt 6.59 min; m/z 614.2 [M+HI*, 558.1 [M-tBu+2H]*; m/z 612.2 [M-H]-. 20 (h) 2-(2-(2-(2-((2-Methoxy-4-(piperidin-4-yl)phenyl)amino)-5 (trifluorornethyl)pyrimidin-4-y)ethyl)phenyl)acetamide (2) A solution of tert-butyl 4-(4-((4-(2-(2-amino-2-oxoethyl)phenethyl)-5 (trifluoromethyl)pyrimidin-2-yl)amino)-3-methoxyphenyl)piperidine-1 -carboxylate (123) 25 (2.694 g, 4.39 mmol) was dissolved in dichloromethane (150 mL), and TFA (15 mL) was added. The resulting mixture was stirred for 16 hours then concentrated. The residue was suspended in 10% sodium hydroxide (200 mL) then extracted with ethyl acetate (5x200 mL). The combined organic phases were washed with brine (300 mL), dried over sodium sulfate and evaporated to give the title compound (2) (2.252 30 9, 100% yield) as a white solid; 'H NMR (400 MHz, CDC 3 ) 6 8.53 (s, 1H), 8.26 (d, J = 8.3 Hz, 1 H), 7.85 (s, 1 H), 7.29 - 7.21 (m, overlaps with CHCl 3 ), 6.87 (d, J = 8.3 Hz, 1H), 6.80 (s, 1H), 5.43 (d, J = 10.1 Hz, 2H), 3.92 (d, J = 1.3 Hz, 3H), 3.70 (s, 2H), 3.20 (d, J = 12.7 Hz, 2H), 3.15 - 3.05 (m, 4H), 2.75 (t, J = 12.2 Hz, 2H), 2.61 (tt, J 12.0, 3.5 Hz, 1 H), 1.89 - 1.76 (m, overlaps with water), 1.67 (qd, J = 12.7, 3.9 Hz, WO 2012/110774 PCT/GB2012/000176 62 2H). LCMS Method C: rt 4.92 min; m/z 514.1 [M+H]*, 536.1 [M+Na]*; m/z 512.2 [M H] (i) 2-(2-(2-(2-((2-Methoxy-4-(1-methylpiperidin-4-yl)pheny)amino)-5 5 (trifluoromethyl)pyrimidin-4-yl)ethy)phenyl)acetamide (3) 2-(2-(2-(2-((2-Methoxy-4-(piperidin-4-yl)phenyl)amino)-5-(trifluoromethyl)pyrimidin-4 yl)ethyl)phenyl)acetamide (2) (2.249 g, 4.38 mmol) was dissolved in methanol (220 mL) and 37% formaldehyde solution (0.483 mL, 17.5 mmol) was added. After five minutes sodium tris(acetoxy)borohydride (4.641 g, 21.9 mmol) was added and 10 stirring continued at room temperature for two hours. The volatiles were evaporated and the residue suspended in 5% aqueous sodium hydroxide (200 mL). The resulting mixture was extracted with ethyl acetate (5x200 mL) then the combined organic phases were washed with brine (500 mL), dried over sodium sulfate and evaporated. The residue was evaporated from ether and the solvent traces removed under high 15 vacuum to give the title compound (3) (2.196 g, 95% yield) as a white solid; 1 H NMR (400 MHz, CDC 3 ) 6 8.53 (s, 1H), 8.24 (d, J = 8.2 Hz, 1H), 7.85 (s, IH), 7.28 - 7.22 (m, overlaps with CHCI 3 ), 6.88 (dd, J = 8.3, 1.9 Hz, 1H), 6.81 (d, J = 1.8 Hz, 1H), 5.41 (s, 2H), 3.90 (s, 3H), 3.70 (s, 2H), 3.16 - 3.06 (m, 4H), 2.99 (d, J = 12.1 Hz, 2H), 2.48 (tt, J = 10.5, 5.9 Hz, 1H), 2.34 (s, 3H), 2.07 (td, J = 11.1, 4.1 Hz, 2H), 1.89 20 1.77 (m, overlaps with water). LCMS Method C: rt 4.94 min; m/z 5281 [M+H]*, 550.1 [M+Na]*; m/z 526.2 [M-H] Example 4: 2-(2-(2-(2-((4-(1 -Ethylpi peridin-4-yl)-2-methoxyphenyl)amino)-5 (trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetamide (4) HN 0 -N N' N ONN O NANHN N NNH 0H 0 H 25 2 4 Aceteldehyde (18 mg, 0.39 mmol) was added to a stirred solution of 2-(2-(2-(2-((2 methoxy-4-(piperidin-4-yl)phenyl)amino)-5-(trifluoromethyl)pyrimidin-4 yl)ethyl)phenyl)acetamide (2) (40 mg, 78 pmol) in dry MeOH (2 mL). Sodium triacetoxyborohydride (83 mg, 0.39 mmol) was added under nitrogen and the 30 resulting mixture was stirred at room temperature for 2 hours. The crude mixture was diluted with ethyl acetate and adsorbed onto silica gel. Chromatography (SiO 2 , 0- WO 2012/110774 PCT/GB2012/000176 63 20% MeOH/DCM) gave the title compound (4) (10 mg, 24%) as a light coloured solid; 'H NMR (400 MHz, CD 2
CI
2 ) 6 8.52 (s, 1H), 8.34 (d, J= 8.3 Hz, 1H), 7.92 (s, 1H), 7.26 - 7.14 (m, 4H), 6.92 (d, J = 8.3 Hz, 1H), 6.82 (d, J = 1.7 Hz, 1H), 5.91 (bs, IH), 5.87 (bs, 1H), 3.91 (s, 3H), 3.66 (s, 2H), 3.63 (m, 2H), 3.20 - 3.01 (m, 6H), 2.73 (d, J 5 = 11.7 Hz, 2H), 2.42 - 2.21 (m, 3H), 2.02 (d, J = 18.0 Hz, 4H), 1.37 (t, J 7.3 Hz, 3H). LCMS Method C: rt 4.96 min; m/z 542.2 {M+1]*, 540.1 M-1]-. Example 5: 3-(2-(2-((2-Methoxy-4-(piperidin-4-yi)phenyl)amino)-5 (trifluoromethyl)pyrimidin-4-yl)ethyl)benzamide (5) OH - NH 2 ------ NH 2 N H, NHz o TMS 0 129 130 131 BOC B 'NHH
NH
2
NH
2 BOCN I Ho', D 8 CNO 118 132 133 NHO 0 H N
C
F3 NI N NH 2 N.0 - g I& N Y I yy :: BOC' N, " ~H.c- , 126 134 135
N
0 H_ 2' 0 1 CFa N H BOC, N HNG-N 10 136 5 (a) 3-lodobenzamide (129) To a stirred solution of 3-iodobenozic acid (2.00 g, 8.06 mmol) and DIPEA (5.62 mL, 32.3 mmol) in MeCN (100 mL) at room temperature was added HOBT (1.63 g, 12.1 mmol) and EDCI.HCI (2.32 g, 12.1 mmol). After stirring for 10 minutes, ammonium 15 carbonate (4.65 g, 46.4 mmol) was added and the resulting solution was stirred overnight. The volatiles were removed in vacuo to yield a crude solid which was suspended in water (100 mL). The resulting suspension was sonicated for 10 minutes then the solid collected by filtration. The filter cake was washed with water (20 mL) and dried to yield the title compound (129) (1 65 g 83%) as a brown solid; 'H WO 2012/110774 PCT/GB2012/000176 64 NMR (400 MHz, CDCI 3 ) 5 8.13 (t, J = 1.6 Hz, 1H), 7.83 (ddd, J = 7.9,1.7, 1.1 Hz, 1H), 7.73 (ddd, J = 7.8, 1.7, 1.1 Hz, 1H), 7.16 (t, J = 7.8 Hz, 1H), 6.18 - 5.49 (m, J 137.3 Hz, 2H). LCMS Method C: rt = 5.04 min; m/z = 248 [M+1]*. 5 (b) 3-((Trimethylsilyl)ethynyl)benzamide (130) A solution of 3-iodobenzamide (129) (1.65 g, 6.66 mmol), Cu(I)l (127 mg, 0.666 mmol) and triphenylphosphine (524 mg, 2.00 mmol) in THF (100 mL) and triethylamine (4.64 mL, 33.3 mmol) was sonicated for 10 minutes under a nitrogen atmosphere. PdCl 2 (PPh 3
)
2 (642 mg, 0.67 mmol) and TMS acetylene (1.88 mL, 13.3 10 mmol) were then added and the reaction stirred at room temperature for 3 days. The volatiles were evaporated in vacuo and the residue chromatographed on silica gel (0 50% ethyl acetate/petroleum benzine 40-60 *C) to give the title compound (130) (1.53 g); 'H NMR (400 MHz, CDCl 3 ) 5 7.78 (t, J = 1.5 Hz, 1H), 7.67 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 7.53 - 7.48 (m, 1H), 7.29 (td, J = 7.8, 0.5 Hz, 1H), 0.15 (s, 9H). 15 (c) 3-Ethynylbenzamide (131) Potassium carbonate (1.84 g, 13.3 mmol) was added to a stirred solution of 3 ((trimethylsilyl)ethynyl)benzamide (/30) (1.45 g, 6.66 mmol) in MeOH (15 mL) at room temperature. The resulting suspension was stirred for 30 minutes then water (100 20 mL) and EtOAc (100 mL) were added. The resulting precipitate was collected via filtration and after air drying this was suspended in a 1:1 mix of acetone and methanol (15 mL). After sonication the resulting suspension was filtered to give the title compound (131) (335 mg, 35%) which was used without additional purification. 25 (d) 2-Methoxy-4-(1,2,3,6-tetrahydropyridin-4-yl)aniline (132) To a solution of tert-butyl 4-(4-((tert-butoxycarbonyl)amino)-3-methoxyphenyl)-5,6 dihydropyridine-1(2H)-carboxylate (/18) (6.7 g, 0.016 mol) in DCM (60 mL) was added TFA (6.0 mL, 0.081 mol) and the resulting solution was stirred at room temperature for 14 hours. An additional 5 mL of TFA was added and the reaction was 30 stirred for a further 6 hours. The volatiles were removed in vacuo and the residue taken up in CH 3
CN/H
2 0 and lyophilised to give the title compound (/32) as the TFA salt (6.38 g, 91%) as a brown oil; 'H NMR (400 MHz, d 4 -MeOH) 6 7.31 (dd, J = 7.3, 2.8 Hz, 1H), 7.21 (t, J = 2.0 Hz, 1H), 7.13 - 7.09 (m, 1H), 6.20 (tt, J = 3.4, 1.6 Hz, 1H), 3.83 (dd, J = 5.7, 2.4 Hz, 2H), 3.43 (t, J = 6.1 Hz, 2H), 2.77 (ddd, J = 8.0, 3.9, 35 1.9 Hz, 2H). LCMS Method C: rt 1.38 min; m/z 201.1 [M+H]*.
WO 2012/110774 PCT/GB2012/000176 65 (e) tert-Butyl 4-(4-amino-3-methoxyphenyl)-5,6-dihydropyridine-1(2H)-carboxylate (133) To a stirred solution of 2-methoxy-4-(1,2,3,6-tetrahydropyridin-4-yl)aniline (132) 5 (6.38 g, 14.8 mmol) in MeOH (50 mL) at room temperature was added a solution of (Boc) 2 0 (3.27 g, 15.0 mmol) in MeOH (20 mL) drop-wise over 5 minutes and the resultant solution was stirred for 1 hour. The mixture was concentrated in vacuo and the residue was taken up in 2 M NaOH (50 mL) solution. The aqueous layer was extracted with CH 2
CI
2 (4x50 mL) and the combined organic extracts were washed 10 with brine (50 mL) then dried (MgSO 4 ). The solvent was removed in vacuo to give the title compound (/33) (1.97 g, 43%) as a brown oil; 'H NMR (400 MHz, CDCl 3 ) 6 6.85 - 6.80 (m, IH), 6.67 (d, J = 8.1 Hz, 1H), 5.90 (s, 1H), 4.05 (d, J = 2.7 Hz, 1H), 3.87 (s, 3H), 3.62 (t, J = 5.7 Hz, 1H), 2.49 (s, 2H), 1.49 (s, 9H). 15 (f) tert-Butyl 4-(4-amino-3-methoxyphenyl)piperidine-1-carboxylate (126) To a suspension of 10% Pd/C (0.068 g) in DMF (2 mL) under nitrogen was added a solution of tert-butyl 4-(4-amino-3-methoxyphenyl)-5,6-dihydropyridine-1 (2H) carboxylate (133) (2.0 g, 6.5 mmol) in EtOH (50 mL) and the resulting suspension was stirred under an atmosphere of hydrogen at room temperature for 24 hours. The 20 reaction mixture was filtered through celite, washing with ethyl acetate (100 mL). The filtrate was evaporated to dryness to give the title compound (126) (1.42 g, 72%) as a light brown solid; 'H NMR (400 MHz, CDCI 3 ) 6 6.66 - 6.58 (m, 3H), 4.21 (s, 2H), 3.76 - 3.66 (m, 4H), 2.53 (tt, J = 12.1, 3.5 Hz, 1H), 1.84 - 1.75 (m, 2H), 1.55 (tdd, J = 11.5, 6.4, 3.1 Hz, 1H), 1.47 (s, 9H). 25 (g) tert-Butyl 4-(4-((4-chloro-5-(trifluoromethyl)pyrimidin-2-yl)amino)-3 methoxyphenyl)piperidine-1-carboxylate (134) Zinc chloride (1.0 M in Et 2 0) (8.56 mL, 8.56 mmol) was added to a solution of 2,4 dichloro-5-(trifluoromethyl)pyriimidine (1.06 mL, 7.85 mmol) in 1:1 DCE/t-BuOH 30 (10 mL) at 0 'C under nitrogen. The mixture was stirred for 1 hour at 0 "C and then tert-butyl 4-(4-amino-3-methoxyphenyl)piperidine-1-carboxylate (126) (1.97 g, 7.13 mmol) in 1:1 DCE/tBuOH (30 mL) was added. A solution of triethylamine (600 pL, 0.432 mmol, 1.1 eq) in 1:1 DCE/t-BuOH (10 mL) was next added drop-wise at 0 0C The reaction mixture was vigorously stirred for a further 30 minutes at 0 *C after the 35 final addition and then at room temperature for 24 hours. The solvent was removed in WO 2012/110774 PCT/GB2012/000176 66 vacuo to afford a brown oily residue which was purified by column chromatography on silica gel (0-45% EtOAc in petroleum benzine 40-60 *C) to give a pale yellow oil which solidified upon standing. This was triturated sequentially with water, 1:1 water/MeOH and MeOH and the precipitate was filtered to afford a white solid. The 5 solid was recrystallised from water to give the title compound (134) (0.92 9, 30%) as a white solid; 1 H NMR (400 MHz, CDCi 3 ) 6 8.57 (s, 1H), 8.28 (d, J = 8.3 Hz, 1H), 8.01 (s, 1 H), 6.86 (dd, J = 8.4, 1.7 Hz, 1 H), 6.76 (d, J = 1.8 Hz, 1 H), 4.25 (s, 2H), 3.91 (s, 3H), 2.81 (t, J = 11.8 Hz, 2H), 2.64 (tt, J = 12.1, 3.6 Hz, 1H), 1.83 (d, J = 13.5 Hz, 2H), 1.69 - 1.57 (m, 2H), 1.49 (s, 9H). LCMS Method C: rt 7.00 min; m/z = 487.0 10 [M+H]*, 431.0 [M-tButyl+H}+, 485.0 [M-H]-. (h) tert-Butyl 4-(4-((4-((3-carbamoylpheny)ethynyl)-5-(trifluoromethy)pyrimidin-2 yl)amino)-3-methoxyphenyl)piperidine-1-carboxylate (135) To a mixture of tert-butyl 4-(4-((4-chloro-5-(trifluoromethyl)pyrimidin-2-yl)amino)-3 15 methoxyphenyl)piperidine-1-carboxylate (/34) (0.098 g, 0.20 mmol), 3 ethynylbenzamide (131) (0.038 g, 0.26 mmol), PdCl 2 (PPh 3
)
2 (9 mg, 0.01 mmol), triphenylphosphine (0.010 g, 0.04 mmol) and copper(l) iodide (0.009 g, 0.05 mmol) in DMF (2 mL), that had been degassed with nitrogen for 10 minutes, was added triethylamine (0.090 mL, 0.65 mmol). The mixture was degassed with nitrogen then 20 heated to 120 0C for 25 minutes under microwave irradiation. On cooling to room temperature the resulting mixture was left overnight, then an additional portion of 3 ethynylbenzamide (126) (0.047 g, 0.32 mmol) was added. After degassing with nitrogen the reaction was heated at 120 "C for 25 minutes under microwave irradiation. The volatiles were evaporated under reduced pressure and the residue 25 purified using silica gel column chromatography (20-70% EtOAc/petroleum benzine 40-60 0C) to give the title compound (135) (0.073 g, 61%) as a yellow solid; 'H NMR (400 MHz, CDCl3) 6 8.66 (s, 1 H), 8.35 (d, J = 8.3 Hz, 1 H), 8.04 (dd, J = 1.4, 1.4 Hz, 1H), 8.02 (s, 1H), 7.93 (ddd, J = 7.9, 1.8, 1.2 Hz, 1H), 7.80 (m, 1H), 7.53 (m, 1H), 6.88 (dd, J = 8.3, 1.6 Hz, 1H), 6.76 (d, J = 1.8 Hz, 1H), 4.27 (m, 2H), 3.91 (s, 3H), 30 2.81 (m, J = 12.2 Hz, 2H), 2.64 (tt, J = 12.2, 3.4 Hz, 1H), 1.84 (m, 2H), 1.65 (m, 2H), 1.49 (s, 9H). LCMS Method C: rt 6.59 min; m/z 496.1 [(M-Boc)+H]', 540.0 [(M-t Bu)+H]*.
WO 2012/110774 PCT/GB2012/000176 67 (i) tert-Butyl 4-(4-((4-(3-carbamoylphenethyl)-5-(trifluoromethy)pyrimidin-2-yl)amino) 3-methoxyphenyl)piperidine-1-carboxylate (136) A mixture of tert-butyl 4-(4-((4-((3-carbamoylphenyl)ethynyl)-5 (trifluoromethyl)pyrimidin-2-yl)amino)-3-methoxyphenyl)piperidine-1 -carboxylate (135) 5 (0.073 g, 0.12 mmol) and 10% palladium on activated carbon (0.042 g) in DMF (3 mL) was stirred under a hydrogen atmosphere for 20 hours. The resulting mixture was filtered and filtrate evaporated to dryness under reduced pressure. The residue was chromatographed on silica gel (20-100% acetone/petroleum benzine 40-60 *C) to give the title compound (136) (0.070 g, 95%). 10 (j) 3-(2-(2-((2-Methoxy-4-(piperidin-4-yl)phenyl)amino)-5-(trifluoromethyl)pyrimidin-4 yl)ethyl)fbenzamide (5) A mixture of tert-butyl 4-(4-((4-(3-carbamoylphenethyl)-5-(trifluoromethyl)pyrimidin-2 yl)amino)-3-methoxyphenyl)piperidine-1-carboxylate (136) (0.070 g, 0.12 mmol) and 15 TFA (0.200 mL, 2.61 mmol) in THF (3 mL) was stirred 16 hours at room temperature. The mixture was concentrated under reduced pressure and azeotroped with toluene. The residue was then purified using a SCX cartridge (MeOH, 0.5% NH/MeOH). The methanolic ammonia eluent was concentrated then dissolved in hot acetonitrile which was allowed to cool to room temperature. The cooled acetonitrile solution was filtered 20 and the filtrate was concentrated, taken up in hot acetonitrile (2 mL) and water (1 mL) then freeze dried to give the title compound (5) (49.4 mg, 85%) as a white solid. A portion of this material was further purified by mass directed auto preparative HPLC to give the title compound (5) (4.5 mg) as a while solid; 'H NMR (400 MHz, d 6 DMSO) 6 8.90 (m, 1H), 8.57 (s. 1H), 8.41 (s, 1H), 7.93 (s, 1H), 7.77 (s, 1H), 7.71 (m, 25 1H), 7.62 (dd, J = 7.5, 7.5 Hz, 1H), 7.34 (m, 2H), 6.94 (dd, J = 7.3, 1.6 Hz, 1H), 6.82 (ddd, J = 7.9, 3.4, 1.5 Hz, 1H), 3.82 (s, 3H), 3.12 (m, 2H), 3.05 (m, 4H), 2.89 (m, 1H), 2.68 (m, 2H), 2.20 (m, 1H), 1.78 (m, 2H), 1.64 (m, 2H). LCMS Method C: rt 4.89 min; m/z 500.1[M+H]. 30 WO 2012/110774 PCT/GB2012/000176 68 Example 6: 3-(2-(2-((2-Methoxy-4-(1-methylpiperidin-4-yl)phenyl)amino)-5 (trifluoromethyl)pyrimidin-4-yl)ethyl)benzamide (6) HN NN N NH 2 N N NH 2 I~ NI~
CF
2 CF 2 HNN 5 6 To a solution of 3-(2-(2-((2-methoxy-4-(piperidin-4-yl)phenyl)amino)-5 5 (trifluoromethyl)pyrimidin-4-yl)ethyl)benzamide (5) (0.021 g, 0.042 mmol) in anhydrous methanol (2.00 mL) was added 37% aqueous formaldehyde (0.012 mL, 0.16 mmol) and sodium triacetoxyborohydride (0.044 g, 0.21 mmol) under a N 2 atmosphere. The mixture was then stirred for 1.5 hours. The resulting mixture was concentrated under reduced pressure and diluted with EtOAc (10 mL) and washed 10 with sat. aqueous NaHCO 3 (10 mL). The Aqueous was further extracted with EtOAc (10 mL) and the combined organic layers were washed with brine (10 mL) and water (10 mL) then dried using a phase separation cartridge. The organics were concentrated under reduced pressure and purified using silica gel column chromatography (0-30% MeOH/EtOAc + 1% 2 M ethanolic NH 3 ). The product was 15 then taken up in minimal hot acetonitrile and water and freeze dried to give the title compound (6) (0.016 g, 74%) as a white solid; 'H NMR (400 MHz, d-Acetone) 6 8.62 (s, 1H), 8.27 (dd, J = 8.3, 4.1 Hz, 1H), 8.15 (s, 1H), 7.90 (s, 1H), 7.78 (d, J = 7.5 Hz, 1 H), 7.41 (m, 3H), 6.99 (d, J = 1.7 Hz, 1 H), 6.89 (dd, J = 8.2, 1.6 Hz, 1 H), 6.55 (s, 1H), 3.98 (s, 3H), 3.20 (in, 4H), 2.90 (m, 2H), 2.57 (m, 1H), 2.22 (s, 3H), 1.98 (td, J 20 11.2, 3.8 Hz, 2H), 1,78 (m, 4H). LCMS Method C: rt 4.97 min; m/z 514.2 [M+H]*. Example 7: 3-(2-(2-((4-(1 -Ethylpiperidin-4-yl)-2-methoxyphenyl)amino)-5 (trifluoromethyl)pyrimidin-4-yl)ethyl)benzamide (7) H I10 H HN N N "iNH 2 NN N
NH
2 _ ,- N - 0 ..- Y N NN -. ~ N 5 7 25 To a solution of 3-(2-(2-((2-methoxy-4-(piperidin-4-yl)phenyl)anino)-5 (trifluoromethyl)pyrimidin-4-yl)ethyl)benzamide (5) (0.021 g, 0.04 mmol) in methanol (2.00 mL) was added acetaldehyde (0.010 mL, 0.18 mmol) followed by sodium triacetoxyborohydride (0.044 g, 0.21 mmol) under a N 2 atmosphere. The mixture was WO 2012/110774 PCT/GB2012/000176 69 then stirred at room temperature for 5 hours. The resulting mixture was concentrated under reduced pressure and purified using silica gel column chromatography (0-30% MeOH/EtOAc + 1% 2 M ethanolic NH 3 ). The product was further purified using mass directed auto-preparative HPLC to give the title compound (7) (3.5 mg, 16%); 1 H 5 NMR (400 MHz, d-Acetone) 6 8.62 (s, 1 H), 8.24 (m, 2H), 7.91 (s, 1 H), 7.78 (ddd, J = 7.5, 1.4, 1.4 Hz, 1H), 7.41 (m, 3H), 7.01 (d, J = 1.7 Hz, 1H), 6.89 (dd, J = 8.3, 1.7 Hz, IH), 6.60 (m, 1H), 3.97 (s, 3H), 3.21 (m, 4H), 3.12 (m, 2H), 2.55 (m, 3H), 2.16 (m, 2H), 1.85 (m, 4H), 1.11 (t, J = 7.2 Hz, 3H). LCMS Method C: rt 4.98 min; n/z 528.2 [M+H]*. 10 WO 2012/110774 PCTIGB2012/000176 70 Example 8: 2-(2-(2-(2-((2-Ethyl-4-(piperidin-4-yl)phenyl)amino)-5 (triflIuo rom ethyl) pyri mid in-4-yI)ethyl)phenyl)acetam ide (8) 0 00 0
CF
3 - ---- 0 116 137 0
H
2 N N 0----~-N -6 0-y" B +- +N Br B .K 138 137 H _ N 0N Nq 00 0~ 139 140 H7 N. N CI H N CF 3 0,N 0 00 -6CF 3 0 ON N 0 Y0~ 141 14 142 H H N Nr ' N. N. NrCF 3 N - , CF 3 YNH No0 0 0 0 143 144 N N N.',P N N_, CF, - NH 2 8 5 WO 2012/110774 PCT/GB2012/000176 71 (a) tert-Butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine 1(2H)-carboxylate (137) Bis(pinacolato)diboron (0.511 g, 2.01 mmol), potassium acetate (0.592 g, 6.04 mmol), dppf (56 mg, 5 mol%), and PdC 2 (dppf) dichloromethane solvate (83 mg, 5 5 mol%) were loaded into a Schlenk tube, and purged with nitrogen. A solution tert butyl 4-(((trifluoromethyl)sulfonyl)oxy)-5,6-dihydropy ridine-1 (2H)-carboxylate (116) (1.00 g, 3.02) in dioxane (5 mL) was added, the mixture degassed with 3x vacuum/nitrogen cycles then brought to 80 "C under nitrogen. After 16 hours the mixture was cooled, and added to water (100 mL). The resulting mixture was 10 extracted with dichloromethane (3x50 mL), and the combined DCM extracts washed with brine (50 mL), dried over sodium sulfate and evaporated. Chromatography (40 g silica cartridge, 0-100% ethyl acetate/petroleum benzine 40-60 *C) gave the title compound (/37) (383 mg, 62% yield) as a crystalline white solid; 'H NMR (400 MHz,
CDCI
3 ) 5 6.45 (s, 1H), 3.94 (d, J = 2.7 Hz, 2H), 3.43 (t, J = 5.5 Hz, 2H), 2.22 (s, 2H), 15 1.45 (s, 9H), 1.26 (s). LCMS Method C: rt 6.48 min; m/z 254.2 [M-tBu+2H]*, 210.2 JM-Boc+2H]'. (b) Benzyl (4-bromo-2-ethylphenyf)carbamate (38) 4-Bromo-2-ethylaniline (500 mg, 2.50 mmol) was dissolved in toluene (25 mL), 20 sodium carbonate (397 mg, 3.75 mmol) and benzyl chloroformate (0.428 mL, 3.00 mmol) were added and the mixture stirred under nitrogen at room temperature. After 20 hours water (25 mL) was added, the aqueous phase separated and washed with ethyl acetate (2x25 mL). The combined organic extracts were washed with brine, dried over sodium sulfate and evaporated. Chromatography (40 g silica cartridge, 0 25 100% ethyl acetate/petroleum benzine 40-60 *C) gave the title compound (/38) (708 mg, 85%) as a pink solid; 'H NMR (400 MHz, CDCl 3 ) 5 7.72 (s, 1H), 7.44 - 7.28 (m, 7H), 6.43 (s, 1H), 5.20 (s, 2H), 2.54 (q, J = 7.6 Hz, 2H), 1.21 (t, J = 7.6 Hz, 3H). LCMS Method C: rt 6.46 min; m/z 258.0 [M-PhCH 2
O+CH
3 OH]. 30 (c) tert-Butyl 4-(4-(((benzy/oxy)carbony)amino)-3-ethylpheny)-5 6-dihydropyridine 1(2H)-carboxylate (139) Potassium carbonate (215 mg, 1.55 mmol) the tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2 dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate (37) (160 mg, 0.517 mmol), benzyl (4-bromo-2-ethylphenyl)carbamate (134) (182 mg, 0.543 mmol), 35 PdCl 2 (dppf)-DCM solvate (22 mg, 5 mol%) and DMF (5 mL) were loaded into a WO 2012/110774 PCT/GB2012/000176 72 Schienk tube, and degassed with 3x vacuum/nitrogen cycles. The mixture was brought to 80 *C under nitrogen then after 18 hours cooled and poured into water (100 mL). DCM (75 mL) and brine (50 mL) were added, the aqueous phase was washed with further DCM (2x75 mL), and the combined DCM extracts washed with 5 brine, dried and evaporated. Chromatography (40 g silica cartridge, 0-80% ethyl acetate/petroleum benzine 40-60 0C) gave the title compound (139) (129 mg, 57% yield) as a colourless oil; 'H NMR (400 MHz, CDC 3 ) 6 7.77 (s, 1H), 7,45 - 7.31 (m, 5H), 7.22 (dd, J = 8.5, 2.1 Hz, 1H), 7.18 (d, J = 2.1 Hz, 1H), 6.48 (s, 1H), 5.99 (s, 1H), 5.21 (s, 2H), 4.06 (d, J = 2.5 Hz, 2H), 3.63 (t, J = 5.7 Hz, 2H), 2.58 (q, J = 7.6 10 Hz, 2H), 2.50 (s, 2H), 1.49 (s, 9H), 1.22 (t, J = 7.6 Hz, 3H). LCMS Method C: rt 6.67 min; m/z 337.1 {M-Boc+2H)*, 381.1 {M-tBu+2H)*. (d) tert-Butyi 4-(4-amino-3-ethylpheny)piperidine-1-carboxylate (140) fert-Butyi 4-(4-(((benzyloxy)carbonyl)amino)-3-ethylphenyl)-5,6-dihyd ropy ridine 15 1(2H)-carboxylate (139) (0.500 g, 1.14 mmol) was dissolved in ethanol (20 mL), and a slurry of 10% Pd/C (0.25 g) in ethanol (2 mL) was added. The mixture was stirred under hydrogen for 18 hours, then filtered through celite, washing the celite with ethanol (30 mL). The combined filtrates were evaporated, and chromatography (12 g silica cartridge, 0-80% ethyl acetate/petroleum benzine 40-60 *C) gave the title 20 compound (140) (0.212 g, 61% yield) as a pink syrup; 1 H NMR (400 MHz, CDC13) 6 6.90 (d, J = 2.0 Hz, 1H), 6.87 (dd, J = 8.0, 2.1 Hz, IH), 6.63 (d, J = 8.0 Hz, 1H), 4.21 (s, 2H), 3.55 (s, 2H), 2.78 (t, J = 12.2 Hz, 2H), 2.59 - 2.46 (m, 3H), 1.78 (d, J = 13.2 Hz, 2H), 1.66 - 1.53 (m, overlaps with water), 1.48 (s, 9H), 1.25 (t, J = 7.5 Hz, 3H). LCMS Method C: rt 5.33 min; m/z 249.2 [M-tBu+2H]*, 205.2 [M-Boc+2H]*. 25 (e) tert-Butyl 4-(4-((4-chloro-5-(trifluoromethy)pyrimidin-2-yl)amino)-3 ethyiphenyl)piperidine- 1-carboxy/ate (141) A solution of 2,4-dichloro-5-(trifluromethyl)pyrimidine (166 mg, 0.766 mmcl) in 1:1 DCE: t-BuOH (5 mL) was stirred at 0 *C under nitrogen. 1.0 M Zinc(II) chloride in 30 ether (0.77 mL, 0.77 mmol) was added and the mixture stirred for one hour. tert-Buty 4-(4-amino-3-ethylphenyl)piperidine-1-carboxylate (/40) (212 mg, 0.70 minol) in 1:1 DCE: t-BuOH (5 mL) was added dropwise, and after 30 minutes triethylamine (146 pL, 1.05 mmol) in 1:1 DCE: t-BuOH (5 mL) was added and the mixture allowed to slowly come to room temperature. After 18 hours the mixture was concentrated, and 35 chromatography (12 g silica cartridge, 0-50% ethyl acetate/cyclohexane) gave the WO 2012/110774 PCT/GB2012/000176 73 title compound (141) (283 mg, 84% yield) as a pale pink solid; 'H NMR (400 MHz,
CDC
3 ) 6 8.51 (d, J = 0.6 Hz, 1H), 7.60 (d, J = 8.3 Hz, 1H), 7.15 - 7.07 (m, 3H), 4.25 (s, 2H), 2.80 (t, J = 12.1 Hz, 2H), 2.69 - 2.59 (m, 3H), 1.83 (d, J = 12.9 Hz, 2H), 1.69 - 1.59 (m, 2H), 1.49 (s, 9H), 1.23 (t, J = 7.6 Hz, 3H). LCMS Method C: rt 6.94 min; 5 m/z 429.1 [M-tBu+2H]*, 385.1 [M-Boc+2H]*; m/z 483.1 [M-H-. (0 tert-Butyl 4-(3-ethyl-4-((4-((2-(2-methoxy-2-oxoethyl)phenyt)ethyny)-5 (trifluoromethyl)pyrimidin-2-yl)amino)phenyl)piperidine-1-carboxylate (142) A mixture of tert-butyl 4-(4-((4-chloro-5-(trifluoromethyl)pyrimidin-2-yl)amino)-3 10 ethylphenyl)piperidine-1-carboxylate (/41) (283 mg, 0.584 mmol), methyl 2-(2 ethynylphenyl)acetate (14) (122 mg, 0.700 mmol), copper(l) iodide (17 mg, 15 mol%), triphenylphosphine (23 mg, 15 mol%), bis(triphenylphosphine)palladium(ll) chloride (41 mg, 10 mol%), triethylamine (0.33 mL, 2.3 mmol) and DMF (3 mL) was degassed with a nitrogen then heated under microwave irradiation (120 *C/15 min). The cooled 15 mixture was poured into water (30 mL) and extracted with ethyl acetate (3x25 mL). The combined organic extracts were washed with brine (2x50 mL), dried over sodium sulfate and evaporated. Chromatography (12 g silica cartridge, 0-80% ethyl acetate/petroleum benzine 40-60 *C) gave the title compound (142) (311 mg, 86% yield) as a brown oil; 'H NMR (400 MHz, CDC1 3 ) 6 8.57 (s, 1H), 7.73 - 7.65 (m, 2H), 20 7.46 - 7.28 (m), 7.15 - 7.08 (m), 4.25 (s, 2H), 3.95 (s, J = 9.6 Hz, 2H), 3.70 (s, 3H), 2.80 (t, J = 12.1 Hz, 2H), 2.70 - 2.59 (m, 3H), 1.84 (d, J = 12.9 Hz, 2H), 1.65 (td, J = 12.8, 4.1 Hz, 2H), 1.49 (s, 9H), 1.25 (t, J = 7.6 Hz, 3H). LCMS: rt 7.01 min; m/z 623.1 [M+H1*, 567.1 [M-tBu+2H]*, 523.1 [M-Boc+2H]*; m/z 621.2 [M-H]. 25 (g) tert-Butyl 4-(3-ethyl-4-((4-(2-(2-methoxy-2-oxoethyl)phenethyl)-5 (trifluoromethyf)pyrimidin-2-yl)amino)phenyl)piperidine-1-carboxylate (143) tert-Butyl 4-(3-ethyl-4-((4-((2-(2-methoxy-2-oxoethyl)phenyl)ethynyl)-5 (trifluoromethyl)pyrimidin-2-yl)amino)phenyl)piperidine-1 -carboxylate (142) (311 mg, 0.499 mmol) was dissolved in ethanol (20 mL), and a slurry of Pd/C (150 mg) in 30 ethanol (2 mL) was added. The mixture was stirred under hydrogen for 18 hours, filtered through celite, washing the celite with ethanol (20 mL) and the filtrate concentrated. The mixture was taken up in ethanol (20 mL), and a slurry of Pd/C (150 mg) in ethanol (2 mL) was added followed by triethylamine (20 PL). The mixture was stirred under hydrogen for 18 hours, filtered through celite, the celite washed 35 with ethanol (10 mL) and the combined filtrates evaporated. The residue was taken WO 2012/110774 PCT/GB2012/000176 74 up in DMF (10 mL), a slurry of Pd/C (150 mg) in DMF (2 mL) was added and the mixture stirred under hydrogen. After 16 hours the mixture was filtered through celite, and the celite washed with ethyl acetate (100 mL). The combined filtrate was evaporated to give a pale green oil. Chromatography (12 g silica cartridge, 0-60% 5 ethyl acetate/petroleum benzine 40-60 *C) gave the title compound (143) (177.1 mg, 57% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl 3 ) 6 8.49 (d, J = 0.5 Hz, 1H), 7.73 (d, J = 8.1 Hz, IH), 7.25 - 7.16 (m, 4H), 7.12 - 7.07 (m, 2H), 7.06 (s, 1H), 4.25 (br s, 2H), 3,72 (s, 2H), 3.67 (s, 3H), 3.14 - 3.01 (m, 4H), 2.81 (t, J = 12.2 Hz, 2H), 2.72 - 2.59 (m, 3H), 1.84 (d, J 13.0 Hz, 2H), 1.69 - 1.56 (m, overlaps with water in 10 solvent), 1.49 (s, 9H), 1.26 (t, J 7.1 Hz, 3H). LCMS: rt 7.16 min; m/z 627.2 [M+H]*; 571.1 [M-tBu+2H]'; m/z 625.2 [M-H]-. (h) tert-Butyl 4-(4-((4-(2-(2-amino-2-oxoethyl)phenethyl)-5-(trifluoromethyl)pyrimidin 2-yl)amino)-3-ethyiphenyl)piperidine-1-carboxylate (144) 15 tert-Butyl 4-(3-ethyl-4-((4-(2-(2-methoxy-2-oxoethyl) phenethyl)-5 (trifluoromethyl)pyrimidin-2-yl)amino)phenyl)piperidine-1-carboxylate ((43) (177 mg, 0.282 mmol) was dissolved in THF (10 mL), and a solution of lithium hydroxide monohydrate (59.0 mg, 1.41 mmol) in water (2 mL) was added. The mixture was stirred for 18 hours then concentrated. The residue was suspended in saturated 20 sodium bicarbonate (20 mL), and extracted with ethyl acetate (3x20 mL). The combined ethyl acetate phases were washed with brine, dried (sodium sulphate) and evaporated. The residue was dissolved in THF (10 mL) and DMF (1 mL) at 30 *C and HOBt (50 mg, 0.37 mmol), EDCI (65 mg, 0.37 mmol) and DIPEA (0.246 mL, 1.41 mmol) were added. After ten minutes ammonium carbonate (135 mg, 1.41 mmol) 25 was added and the mixture was stirred for 18 hours at 30 "C. The mixture was concentrated, the residue diluted with saturated sodium bicarbonate (25 mL) and extracted with ethyl acetate (3x 25 mL). The combined ethyl acetate phases were washed with brine (25 mL), dried (sodium sulfate) and evaporated. Chromatography (12 g silica cartridge, 0-100% ethyl acetate/petroleum benzine 40-60 "C) gave the 30 title compound (/44) (122 mg, 71% over two steps) as a white solid; 'H NMR (400 MHz, CDCl 3 ) 6 8.49 (s, 1H), 7.56 (d, J = 8.1 Hz, 1 H), 7.28 - 7.22 (m, overlaps with CHCI), 7.22 - 7.15 (m, 1H), 7.13 - 7.05 (m, 3H), 5.38 (s, 1H), 5.15 (s, IH), 4.25 (s, 2H), 3.64 (s, 2H), 3.11 - 2.99 (m, 4H), 2.80 (t, J = 12.1 Hz, 2H), 2.69 - 2.60 (m, 3H), 1.84 (d, J = 12.3 Hz, 2H), 1.69 - 1.60 (m, 2H), 1.49 (s, 9H), 1.23 (t, J = 7.6 Hz, 3H). 35 LCMS: rt 6.65 min; n/z 612.2 [M+H)*, 556.1 [M-tBu+2H]*; m/z 610.2 [M-H)-.
WO 2012/110774 PCT/GB2012/000176 75 (i) 2-(2-(2-(2-((2-Ethyl-4-(piperidin-4-yl)pheny)amino)-5-(trifluoromethyl)pyrimidin-4 yI)ethyI)phenyI)acetamide (8) tert-Butyl 4-(4-((4-(2-(2-amino-2-oxoethyi)phenethyl)-5-(trifluoromethyl)pyrimidin-2 5 yl)amino)-3-ethylphenyl)piperidine-1-carboxylate (144) (120 mg, 0.20 mmol) was dissolved in DCM (20 mL), TFA (2 mL) was added and the mixture stirred for 16 hours at room temperature. The mixture was concentrated and the residue suspended in 10% sodium hydroxide (10 mL) and brine (10 mL). The mixture was extracted with ethyl acetate (4x20 mL), the combined extracts washed with brine, 10 dried (sodium sulphate) evaporated and the residue evaporated for ether to give the title compound (8) (93 mg, 93% yield) as a white solid; 'H NMR (400 MHz, d6-DMSO) 6 9.47 (s, 1H), 8.49 (s, 1H), 7.37 (s, 1H), 7.26 - 7.18 (m, 2H), 7.17 - 7.09 (m, 4H), 7.05 (dd, J = 8.2, 1.9 Hz, 1H), 6.89 (s, 1H), 3.44 (s, 2H), 3.09 - 2.88 (m, 6H), 2.62 2.54 (m, overlaps with DMSO), 1.70 (d, J = 11.4 Hz, 2H), 1.52 (qd, J = 12.3, 3.7 Hz, 15 2H), 1.10 (t, J = 7.5 Hz, 3H). LCMS Method C: 4.96 min; m/z 512.2 [M+H]*, 534.2 [M+Na]*; m/z 510.2 IM-HT. Example 9: 2-(2-(2-(2-((2-Ethyl-4-(I-methylpiperidin-4-yl)phenyl)amino)-5 (trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetamide (9) HH N NN CF, NH2 CFa NHz HN NO 20 8 9 2-(2-(2-(2-((2-Ethyl-4-(piperidin-4-yl)phenyl)amino)-5-(trifluoromethyl)pyrimidin-4 yl)ethyl)phenyl)acetamide (8) (84 mg, 0.16 mmol) was dissolved in methanol (8 mL), 37% formaldehyde solution (53 pL, 0.66 mmol) was added and the mixture stirred for ten minutes at room temperature. Sodium tris(acetoxy)borohydride (174 mg, 0.821 25 mmol) was added, and after two hours the mixture was concentrated, The residue was diluted with 10% sodium hydroxide (15 mL) and brine (15 mL), and the mixture extracted with ethyl acetate (4x25 mL). The combined ethyl acetate phases were washed with brine, dried over sodium sulfate, evaporated and the residue evaporated from DCM to give the title compound (9) (72 mg, 84% yield) as a white solid; 'H NMR 30 (400 MHz, d 6 -DMSO) 5 9.46 (s, 1H), 8.49 (s, 1H), 7.36 (s, 1H), 7.21 (t, J = 4.6 Hz, 2H), 7.18 - 7.10 (m, 4H), 7.06 (dd J = 8.2, 1.6 Hz, IH), 6.88 (s, 1H), 3.44 (s, 2H), WO 2012/110774 PCT/GB2012/000176 76 3.07 - 2.90 (m, 4H), 2.86 (d, J = 11.3 Hz, 2H), 2.57 (q, J = 7.5 Hz, 2H), 2.48 - 2.37 (m, 1H), 2.19 (s, 3H), 2.02 - 1.89 (m, 2H), 1.79 - 1.59 (m, 4H), 1.09 (t, J = 7.5 Hz, 3H). LCMS Method C: rt 5.04 min; m/z 526.2 [M+H]*; m/z 524.2 [M-H]'. 5 Example 10: 2-(2-(2-(2-((4-(Piperidin-4-yi)-2-(trifIuoromethoxy)phenyI)amino)-5 (trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetamide (10) 0 0 NH2 14 145 .CF ,CF N N~ +0 O 0 Br NH2 Br O O N O 146 137 O.CF, 0
'CF
3 O
ONNH
2 N N NH, CF, + N 147 148 145
H
2 N 0 HN 0
.CF
3 0 ' CF 3 0 N H H N N H N CF,
CF
3 W49 150 HN 0 .CF, HN N F 10 10 WO 2012/110774 PCT/GB2012/000176 77 (a) 2-(2-Ethynylpheny/)acetamide (145) To a stirred solution of methyl 2-(2-ethynylphenyl)acetate (/4) (0.200 g, 1.15 mmol) in methanol (2 mL) at 0 0C in a thick-walled Schlenk tube was added magnesium nitride (0.290 g, 2.87 mmol) in a single portion. The tube was sealed immediately and 5 allowed to warm to room temperature in a water bath over 1 hour and then heated at 80 *C for 22 hours. After cooling to room temperature the resulting mixture was diluted with EtOAc (100 mL) and saturated aqueous NaHCO 3 (80 mL). The layers were separated and the aqueous layer was extracted with EtOAc (80 mL), neutralised with 2 M aq. HCI and extracted with EtOAc (2 x 80 mL). The organic 10 layers were combined, washed with brine (100 mL), dried (MgSO4), filtered and concentrated in vecuo to give a yellow solid. Silica gel chromatography (40 g Si Cartridge, 0-50% EtOAc in dichloromethane) gave the title compound (145) (0.093 g, 51% yield) as a white solid; 'H NMR (400 MHz, de-DMSO) 5 7.44 (dd, J = 7.6, 1.0 Hz, 1 H), 7.40 - 7.28 (m, 3H), 7.24 (td, J = 7.4, 1.7 Hz, 1 H), 6.93 (s, 1 H), 4.32 (s, 1 H), 15 3.59 (s, 2H). LCMS Method C: rt 4.68 min; m/z 160.2 [M+H]*. (b) Benzyl (4-bromo-2-(trifluoromethoxy)phenyl)carbamate (146) 4-Bromo-2-(trifluoromethoxy)aniline (1.0 g, 3.9 mmol) was dissolved in dry toluene (25 mL), sodium carbonate (0.621 g, 5.86 mmol) and benzyl chloroformate (0.669 20 mL, 4.69 mmol) was added and the mixture stirred under nitrogen at room temperature for 22 hours. The reaction was then heated to 80 0C and stirred at this temperature for 17 hours and then heated further to reflux and stirred for 22 hours. Water (100 mL) was added to the coiled mixture, the aqueous phase separated and washed with ethyl acetate (2x100 mL). The combined organic extracts were washed 25 with 0.5 M aq. citric acid (70 mL), water (70 mL), brine (70 mL), dried (MgSO4), filtered and concentrated in vacuo to give a pink solid. The crude material was purified by silica gel chromatography (40 g Si cartridge, 0-20% EtOAc in 40-60 'C petroleum benzine) to give the title compound (146) (1.153 g, 76% yield) as a white solid; 'H NMR (400 MHz, CDCl 3 ) 5 8.15 (d, J = 8.7 Hz, 1H), 7.44 - 7.36 (m, 7H), 6-95 30 (s, 1H), 5.22 (s, 2H). LCMS Method C: rt 6.67 min; m/z 389.9 [M-H]. (c) tert-Butyl 4-(4-amino-3-(trifluoromethoxy)phenyl)piperidine- 1 -carboxylate (147) A solution of 2 M aq. Na 2
CO
3 (1.85 mL, 3.70 mmol) was added to a mixture of tert butyl 4-(4,4,5,5-tetramethyl-1, 3,2-dioxaborolan-2-y)-5,6-dihydropyridine-1(2H) 35 carboxylate (137) (-50% pure, 0.915 g, 1.480 mmol), benzyl (4-bromo-2- WO 2012/110774 PCT/GB2012/000176 78 (trifluoromethoxy)phenyl)carbamate (146) (0.635 g, 1.63 mmol), LiCI (0.125 g, 2.96 mmol), Pd(PPh) 2
CI
2 (0.052 g, 0.074 mmol) and TBAB (0.048 g, 0.15 mmol) in dry 1,4-dioxane (15 mL). The reaction mixture was stirred at 80 *C for 17 hours, then filtered through celite, which was washed with EtOAc and MeOH and the filtrate was 5 concentrated in vacuo to give a pale yellow gum. The crude material was purified by silica gel chromatography (40 g Si Cartridge, 0-15% EtOAc in petroleum benzine 40 60 *C) to give a pale yellow gum (0.515 g). To a solution of the intermediate in EtOAc (20 mL) was added 10% Pd/C (80 mg) in EtOAc (5 mL). The reaction was then stirred at room temperature for 18 hours under an atmosphere of hydrogen and then 10 filtered through a pad of celite, which was washed with EtOAc (100 mL). The solvent was removed in vacuo to give crude product which was purified by silica gel chromatography (40 g Si Cartridge, 0-30% EtOAc in petroleum benzine 40-60 *C) to give the title compound (147) (80% purity, 0.220 g, 33% yield over 2 steps) as a pale yellow gum; 'H NMR (400 MHz, CDCI 3 ) 5 6.98 - 6.95 (m, 1H), 6.92 (dd, J = 8.2, 1.8 15 Hz, 1H), 6.74 (d, J= 8.2 Hz, IH), 4.30 - 4.13 (m, 2H), 3.77 (s, 2H), 2.77 (t, J= 12.5 Hz, 2H), 2.54 (tt, J = 12.1, 3.5 Hz, 1H), 1.78 (d, J = 13.1 Hz, 2H), 1,57 - 1.50 (m, 2H, obscured by water signal), 1.48 (s, 9H). (d) tert-Butyl 4-(4-((4-chloro-5-(triffuoromethyl)pyrimidin-2-yl)amino)-3 20 (trifluoromethoxy)phenyl)piperidine-1-carboxylate (148) 2,4-Dichloro-5-(trifluoromethyl)pyrimidine (0-110 g, 0.506 mmol) was stirred in a 1:1 t BuOH:1,2-dichloroethane mixture (10 mL) at 0 "C. A 1.0 M ZnC1 2 solution in diethyl ether (0.578 mL, 0.578 mmol) was added cautiously, after addition the reaction was left stirring at 0 *C for 30 minutes. A solution of tert-butyl 4-(4-amino-3 25 (trifluoromethoxy) phenyl)piperidine-1-carboxylate (147) (impure, -80% pure, 0.217 g, 0.482 mmol) in 1:1 t-BuOH:1,2-dichloroethane (5 mL) was added drop-wise at 0 "C followed by a solution of NEt 3 (0.081 mL, 0.58 mmol) in 1:1 t-BuOH:1,2 dichloroethane (5 mL) and the reaction was allowed to warm to room temperature and was stirred for 18 hours, then at 60 *C for 24 hours. The organic solvents were 30 evaporated in vacuo and the crude gum was purified by silica gel chromatography (40 g Si cartridge, 0-40% EtOAc in petroleum benzine 40-60 *C) to give the title compound (148) (0.085 g, 33% yield) as a pale yellow oily solid; 'H NMR (400 MHz, d 6 -DMSO) 6 10.38 (s, 1H), 8.72 (s, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.33 - 7.29 (m, 2H), 4.13 - 4.04 (m, 2H), 2.88 - 2.72 (m, 3H), 1.79 (d, J = 12.1 Hz, 2H), 1.56 - 1.44 (m, 35 2H), 1.42 (s, 9H). LCMS Method C: rt 7.02 min; m/z 539.0, 541.0 [M-H].
WO 2012/110774 PCT/GB2012/000176 79 (e) tert-Butyl 4-(4-((4-((2-(2-amino-2-oxoethyl)phenyl)ethynyl)-5 (trifluoromethyl)pyrimidin-2-yl)amino)-3-(trifluoromethoxy)phenyl)piperidine- 1 carboxylate (149) 5 To a nitrogen de-gassed solution of 2-(2-ethynylphenyl)acetamide (/41) (0.029 g, 0.18 mmol) and tert-butyl 4-(4-((4-chloro-5-(trifluoromethyl)pyrimidin-2-yl)amino)-3 (trifluoromethoxy)phenyl)piperidine-1-carboxylate (148) (0.082 g, 0.15 mmol) in dry DMF (4 mL) were added triethylamine (0.085 mL, 0.61 mmol), triphenylphosphine (6.0 mg, 0.023 mmol), trans-dichlorobis (triphenylphosphine)palladium (II) (0.011 g, 10 0.015 mmol) and Cu(I)l (4.0 mg, 0.023 mmol). The reaction mixture was heated under microwave irradiation at 120 *C for 20 minutes, concentrated to dryness in vacuo and purified by silica gel chromatography (12 g Si cartridge, 0-100% EtOAc in petroleum benzine 40-60 *C) to give the title compound (/49) (0.073 g, 73% yield) as a pale yellow solid; 'H NMR (400 MHz, d 6 -DMSO) 6 10.11 (s, IH), 8.74 (s, 1H), 7.58 15 (dd, J = 7.7, 6.2 Hz, 2H), 7.53 - 7.47 (m, 1H), 7.42 - 7.27 (m, 5H), 6.99 (s, 1H), 4.14 - 4.03 (m, 2H), 3.67 (s, 2H), 2.87 - 2.72 (m, 3H), 1.79 (d, J = 12.8 Hz, 2H), 1.50 (qd, J = 12.5, 4.1 Hz, 2H), 1.42 (s, 9H). LCMS Method C: rt 6.63 min; m/z 564.0 [M Boc+2H*. 20 (f tert-Butyl 4-(4-((4-(2-(2-amino-2-oxoethyl)phenethyl)-5-(trifluoromethy)pyrimidin-2 yl)amino)-3-(trifhioromethoxy)pheny/)piperidine-1-carboxy/ate (150) tert-Butyl 4-(4-((4-((2-(2-amino-2-oxoethyl)phenyl)ethynyl)-5-(trifluoromethyl) pyrimidin-2-yl)amino)-3-(trifluoromethoxy)phenyl)piperidine-1-carboxylate (/49) (72.0 mg, 0.108 mmol) was dissolved in dry DMF (7 mL) under an atmosphere of nitrogen, 25 and a slurry of 10% Pd(OH) 2 /C (0.050 g) in EtOAc (2 mL) was added. The mixture then was stirred vigorously under hydrogen for 24 hours. Upon completion, the reaction was filtered through a pad of celite, which was washed with EtOAc (40 mL). The combined filtrates were concentrated in vacuo to give a pale yellow oil. The crude product was purified by silica gel chromatography (12 g Si Cartridge, 0-100% 30 EtOAc in petroleum benzine 40-60 *C) to give the title compound (150) (0.064 g, 88% yield) as an off-white solid; 1 H NMR (400 MHz, d 6 -DMSO) 6 9.80 (s, 1H), 8.60 (s, 1 H), 7.61 (d, J = 8.1 Hz, I H), 7.39 (s, 1 H), 7.32 - 7.26 (m, 2H), 7.21 (dt, J = 6.7, 3.4 Hz, IH), 7.18 - 7.08 (m, 3H), 6.90 (s, 1H), 4.09 (d, J = 12.1 Hz, 2H), 3.45 (s, 2H), 3.09 - 2.94 (m, 4H), 2.88 - 2.72 (m, 3H) 1.79 (d, J = 12.3 Hz, 2H), 1.59 - 1.38 (m, 35 11H). LCMS Method C: rt 6.75 min, m/z 668.1 [M+H]*.
WO 2012/110774 PCT/GB2012/000176 80 (g) 2-(2-(2-(2-((4-(Piperidin-4-yl)-2-(trifluoromethoxy)phenyl)amino)-5-(trifluoromethyl) pyrimidin-4-yl)ethyl)phenyl)acetamide (10) tert-Butyl 4-(4-((4-(2-(2-amino-2-oxoethyl)phenethyl)-5-(trifluoromethyl)pyrimidin-2 5 yl)amino)-3-(trifluoromethoxy)phenyl)piperidine-1 -carboxylate (150) (0.062 g, 0.093 mmol) was dissolved in dry DCM (6 mL) under an atmosphere of nitrogen. Trifluoroacetic acid (0.142 mL, 1.86 mmol) was added to the solution and the reaction was stirred at room temperature for 20 hours. Volatiles were removed in vacuo, EtOAc (50 mL) and 2 M aq. NaOH (50 mL) were added to the residue and the 10 layers were separated. The aqueous layer was extracted with EtOAc (2x70 mL), the combined organics were washed with water (50 mL), brine (50 mL), dried (MgSO 4 ), filtered and concentrated in vacuo to give an off-white solid. The crude material was purified by silica gel chromatography (12 g Si Cartridge, 0-100% EtOAc in petroleum benzine 40-60 *C, then 0-100% methanol in EtOAc, then 1% ammonia in methanol), 15 to give the title compound (10) (0.042 g, 80% yield) as a white solid; 'H NMR (400 MHz, d 6 -DMSO) 6 9.78 (s, 1H), 8.59 (s, 1H), 7.59 (d, J = 8.3 Hz, 1H), 7.39 (s, 1H), 7.29 - 7.17 (m, 3H), 7.18 - 7.06 (m, 3H), 6.90 (s, 1 H), 3.45 (s, 2H), 3.09 - 2.92 (m, 6H), 2.71 - 2.53 (m, 3H), 1.71 (d, J = 11.7 Hz, 2H), 1.49 (qd, J = 12.1, 3.7 Hz, 2H). LCMS Method C: rt 5,07 min; m/z 568.1 [M+H)*. 20 Example Ii: 2-(2-(2-(2-((4-(1-Methylpiperidin-4-yI)-2-(trifluoromethoxy) phenyl)amino)-5-(trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetamide (ii)
H
2 N 0 H 2 N 0 oCF,
.CF
3 N N N N N9 N / CF, N.. CF 3 HN 10 11 25 To a suspension of 2-(2-(2-(2-((4-(piperidin-4-yl)-2-(trifluoromethoxy)phenyl)amino)-5 (trifluoromethyl) pyrimidin-4-yl)ethyl)phenyl)acetamide (10) (0.039 g, 0.069 mmol) in anhydrous methanol (4 mL) was added a 37% aq. solution of formaldehyde (0.020 mL, 0.28 mmol) under an atmosphere of nitrogen followed by sodium triacetoxyborohydride (0.073 g, 0.34 mmol). The reaction was stirred at room 30 temperature for 3 hours. The volatiles were then removed in vacuo and the residue WO 2012/110774 PCT/GB2012/000176 81 was diluted with EtOAc (50 mL) and sat. aq. NaHCO 3 (30 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2x30 mL), the combined organic layers were washed with brine (40 mL), dried (MgSO 4 ), filtered and concentrated in vacuo to give a gum which was taken up in DCM (- 10 mL) and 5 methanol (- 1 mL) and concentrated in vacuo. The process was repeated with only DCM twice. The resulting gum was then suspended in diethyl ether (5 mL) and the solvent was removed in vacuo. The procedure was repeated to give the title compound (11) (0.037 g, 93% yield) as a white fluffy solid: 'H NMR (400 MHz, d 6 DMSO) 6 9.79 (s, 1H), 8.59 (s, 1H), 7.60 (d, J = 8.3 Hz, 1H), 7.39 (s, 1H), 7.28 (dd, J 10 = 8.4, 1.9 Hz, 1H), 7.26 - 7.23 (m, 1H), 7.23 - 7.19 (m, 1H), 7.18 - 7.08 (m, 3H), 6.89 (s, 1H), 3.45 (s, 2H), 3.08 - 2.93 (m, 4H), 2.90 - 2.83 (m, 2H), 2.57 - 2.52 (m, 1H, obscured by residual solvent signal), 2.19 (s, 3H), 1.96 (td, J= 11.5, 2.0 Hz, 2H), 1.80 - 1.72 (m, 2H), 1.65 (ddd, J = 24.6, 12.4, 3.7 Hz, 2H). LCMS Method C: rt 5.11 min; m/z 582.1 [M+H]*. 15 Example 12: 2-(2-(2-(2-((2-methyl-4-(1-methylpiperidin-4-yl)phenyl)amino)-5 (trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetamide (12) N H' H O o XY - 0 BB OO'B N OT N OI 0 ~ .
0 151 137 152
H
2 N N O '" 0 HN 0 0NCF 0 0 NN. 0 "II - -9 153 115 154 0 N NH H IHI 155 12 20 WO 2012/110774 PCT/GB2012/000176 82 (a) Benzyf (4-bromo-2-methylphenyl)carbamate (151) 2-Bromo-4-methylaniline (5.00 g, 26.9 mmol), benzyl chloroformate (5.75 mL, 40.3 mmol), Na 2
CO
3 (4.27 g, 40.3 mmol) and toluene (100 mL) were stirred under nitrogen at room temperature for 20 hours. The resulting mixture was diluted with 5 ethyl acetate and washed with water (100 mL). The organic layer was dried (MgSO 4 ) and the volatiles removed by evaporation under reduced pressure. Petroleum benzene 40-60 *C was added and the resulting precipitate collected by filtration to give the title compound (151) as colourless needles (8.50 g, 99%); 'H NMR (400 MHz, CDC 3 ) 6 7.80 - 7.67 (m, 1H), 7.44 - 7.28 (m, 6H), 6.40 (s, 1H), 5.20 (s, 2H), 10 2.21 (s, 3H). LCMS Method C: rt 6.36 min; m/z 320 [M+1]*. (b) tert-Buty 4-(4-(((benzyloxy)carbonyt)amino)-3-methylphenyl)-5,6-dihydropyridine 1(2H)-carboxylate (152) A suspension of benzyl (4-bromo-2-methylphenyl)carbamate (151) (1.00 g, 3.12 15 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine 1(2H)-carboxylate (37) (1.16 g, 3.74 mmol), PdCl 2 (dppf)-DCM complex (310 mg, 0.374 mmol) and potassium carbonate (1.29 g, 9.36 mmol) in DMF (30 mL) was stirred under nitrogen at 80 "C for 16 hours. The volatiles were evaporated under reduced pressure and the residue adsorbed onto silica gel. Chromatography on silica 20 gel (0-30% ethyl acetate/petroleum benzine 40-60 *C) gave the title compound (152) (949 mg, 72%) as a yellow liquid; 'H NMR (400 MHz, CDC 3 ) 6 7.83 - 7.70 (m, 1H), 7.41 - 7.34 (m, 5H), 7.21 (dd, J = 8.5, 1.9 Hz, 1H), 7.16 (d, J = 1.8 Hz, 1H), 6.56 (s, 1H), 5.97 (s, 1H), 5.20 (s, 2H), 4.05 (d, J = 2.9 Hz, 2H), 3.61 (t, J = 5.7 Hz, 2H), 2.48 (d, J = 1.5 Hz, 2H), 2.23 (s, 3H), 1.50 (d, J = 2.6 Hz, 9H). 25 (c) tert-Buty 4-(4-amino-3-methylphenyl)piperidine-1-carboxylate (153) A suspension of ted-butyl 4-(4-(((benzyloxy)carbonyl)aiiino)-3-niethylphenyl)-5,6 dihydropyridine-1(2H)-carboxylate (152) (798 mg, 2.49 mmol) and 10% Pd/C (250 mg) in MeOH (50 mL) was stirred under hydrogen (1 atm) for 16 hours at room 30 temperature. The resulting mixture was filtered through celite, washing with MeOH, then the volatiles were removed by evaporation under reduced pressure to give the the title compound (153) (550 mg, 76%) as a purple/brown liquid; 1 H NMR (400 MHz,
CDC
3 ) 6 6.95 (d, J = 6.7 Hz, 3H), 4.22 (s, 2H), 2.77 (s, 2H), 2.54 (s, 1H), 2.29 (s, 3H), 1.77 (d, J = 12.7 Hz, 2H), 1.56 (dd, J = 12.6, 3.7 Hz, 9H), 1.47 (s, 9H). LCMS 35 Method C rt 5.09 min; m/z 235.1 [M-tBu+2f, 191.2 [M-Boc+2.
WO 2012/110774 PCTIGB2012/000176 83 (d) Methyl 2-(2-(2-(2-((2-methyl-4-(piperidin-4-y)pheny)amino)-5 (trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetate (154) A solution of methyl 2-(2-(2-(2-(methylsulfonyl)-5-(trifluoromethyl)pyrimidin-4 5 yl)ethyl)phenyl)acetate (115) (450 mg, 1.11 mmol), tert-butyl 4-(4-amino-3 methylphenyl)piperidine-1-carboxylate (153) (390 mg, 1.34 mmol) and TFA (0.25 mL) in 2,2,2-TFE (3 mL) was heated at 120 *C under microwave irradiation for 30 minutes. The resulting mixture was adsorbed onto silica gel and chromatographed (0-50% MeOH/DCM) to give the title compound (154) (478 mg, 83%) as a viscous 10 liquid; 1 H NMR (400 MHz, CDCl 3 ) 6 9.14 (bs, 1H), 8.76 (bs, 1H), 8.51 (s, 1H), 7.58 (d, J = 8.2 Hz, 1 H), 7.21 (ddt, J = 10.7, 7.4, 4.3 Hz, 4H), 7.15 - 7.06 (m, 1 H), 3.65 (s, 2H), 3.60 (d, J = 12.6 Hz, 2H), 3.49 (s, 3H), 3.15 - 3.02 (m, 6H), 2.87 - 2.71 (m, 1 H), 2.32 (s, 3H), 2.07 (d, J = 7.8 Hz, 4H). LCMS Method C: rt 5.24 min; m/z 513.2 [M+1]*. 15 (e) Methyl 2-(2-(2-(2-((2-methy-4-(1-methylpiperidin-4-yl)phenyl)amino)-5 (trifluoromethyl)pyrimidin-4-yl)ethy)phenyl)acetate (/55) Methyl 2-(2-(2-(2-((2-methyl-4-(piperidin-4-yl)phenyl)amino)-5 (trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetate (54) (1.10 g, 2.15 mmol) was 20 dissolved in dry MeOH (20 mL) and formaldehyde solution (37% aq; 348 pL, 4.29 mmol) was added. Sodium triacetoxyborohydride (2.27 g, 10.7 mmol) was added under nitrogen and the resultant mixture was stirred at room temperature for 16 hours. Ethyl acetate (50 mL) was added and the mixture was washed with 10% NaHCO 3 solution (20 mL). The organic layer was separated, dried (MgSO 4 ) then the 25 volatiles removed by evaporation under reduced pressure. Chromatography (Si0 2 , 0 50% MeOH/DCM) gave the title compound (155) (480 mg, 42%) as a cream solid; 'H NMR (400 MHz, CDC3l) 6 8.54 (s, 1H), 7.83 - 7.73 (m, 1H), 7.35 (s, 1H), 7.32 - 7.19 (m, 3H), 7.19 - 7.09 (m, 2H), 7.07 - 6.97 (m, 1H), 3.76 (s. 2H), 3.70 (s, 3H), 3.23 (d, J = 11.5 Hz, 2H), 3.17 - 3.04 (m, 4H), 2.61 - 2.51 (m, IH), 2.50 (s, 3H), 2.35 (s, 3H), 30 2.33 - 2.24 (m, 2H), 2.02 - 1.84 (m, 4H). LCMS Method C: rt 5.25 min; m/z 527.2 [M+1].
WO 2012/110774 PCT/GB2012/000176 84 (f) 2-(2-(2-(2-((2-Methyl-4-(1-methylpiperidin-4-y/)phenyl)amino)-5 (trifluoromethyl)pyrimidin-4-yi)ethy)phenyl)acetamide (12) Methyl 2-(2-(2-(2-((2-methyl-4-(I-methylpiperidin-4-yl)phenyl)amino)-5 (trifluoromethyi)pyrimidin-4-yl)ethyl)phenyl)acetate (155) (476 mg, 0.906 mmol) and 5 LiOH.H 2 0 (113 mg, 2.71 mmol) in a mixture of THF (20 mL), water (4 mL) and MeOH (2 mL) were stirred at room temperature for 24 hours. The volatiles were evaporated under reduced pressure to give a light yellow solid which was dissolved in dry DMF (10 mL) and dry THF (10 mL). HOBT (171 mg, 1.26 mmol), EDCl (196 mg, 1.26 mmol), ammonium carbonate (458 mg, 4.87 mmol) and DIPEA (829 pL, 4.87 mmol) 10 were added and the resulting mixture was stirred at room temperature for 16 hours. The volatiles were evaporated under reduced pressure and the residue taken up in ethyl acetate. The resulting solution was washed with 10% NaHCO 3 , the layers separated and the organic layer was dried (MgSO 4 ). The volatiles were evaporated under reduced pressure and the residue chromatographed (SiO 2 , 0-100% 15 MeOH/DCM) to give the title compound (12) (358 mg, 52%) as a cream solid; 'H NMR (400 MHz, CDC1 3 ) 6 8.51 (s, I H), 7.58 (d, J = 8.9 Hz, 1H), 7.34 (s, 1H), 7.28 7.19 (m, 4H), 7.14 - 7.09 (m, 2H), 5.66 (s, 1H), 5.49 (s, 1H), 3.65 (s, 2H), 3.13 - 3.04 (m, 4H), 3.04 - 2.97 (m, 2H), 2.54 - 2.41 (m, 1 H), 2.35 (s, 3H), 2.30 (s, 3H), 2.08 (td, J = 11.3, 3.8 Hz, 2H), 1.88 - 1.80 (m, 4H). LCMS Method C: rt 4.91 min; m/z 512.2 20 [M+1]*, 510.2 [M-1J.
WO 2012/110774 PCT/GB2012/000176 85 Example 13: 2-(2-(2-(2-((2-Fluoro-4-(1-methylpiperidin-4-yl)phenyl)amino)-5 (trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetamide (13) FCT F H j~ NH2 F HN 'BO0C O'B BOC + L) N Ise j1g 157 F F N + N O 0o z oQC No-.b"c 58 15 169 F H F HN N N NH 162 1 9 FH F ,- N.~H
N.
CF F H NHNH, HN6 NH1NH (a) tert-Butyl (2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2 5 yl)phenyl)carbamate (156) A mixture of 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (1.00 g, 4.22 mmol) and Boc 2 0 (1.14 g, 5.21 mmol) in toluene (15 mL) was heated at reflux for 16 hours. A further portion of Boc 2 O (1.04 g, 4.8 mmol) was added and the mixture heated for a further 20 hours then another portion of Boc 2 O (1.4 g, 6.4 mmol) 10 was added. After heating at reflux for a further 24 hours a catalytic amount of DMAP was added and the mixture heated at reflux for 30 minutes before concentrating under reduced pressure to give the title compound (56) (1.42 g, 99%); H NMR (400 MHz, CDC 3 ) 6 7.52 (m, 2H), 7.14 (dd, J = 7.5, 7.5 Hz, 1H), 1.40 (s, 12H), 1.35 (s, 9H). 15 WO 2012/110774 PCT/GB2012/000176 86 (b) tert-Butyl 4-(4-((tert-butoxycarbony)amino)-3-fluorophenyl)-5,6-dihydropyridine 1(2H)-carboxylate (157) To a mixture of tert-butyl 4-(((trifluoromethyl)sulfonyl)oxy)-5,6-dihydropyridine-1 (2H) carboxylate (116) (0.504 g, 1.52 mmol), tert-butyl (2-fluoro-4-(4,4,5,5-tetramethyl 5 1,3,2-dioxaborolan-2-yl)phenyl)carbamate (162) (0.496 g, 1.47 mmol), and PdCl 2 (PPh 3
)
2 (0.055 g, 0.078 mmol) under a N 2 atmosphere was added dioxane (15 mL) and the mixture was bubbled with N 2 for 5 minutes before addition of 3.0 M aqueous sodium carbonate (1.5 mL, 4.5 mmol). The reaction was heated at reflux for 6 hours then concentrated under reduced pressure. The residue was 10 chromatographed on silica gel (0-25% EtOAc/petroleum benzine 40-60 *C) to give the title compound (/57) (0.409 g, 71%); 1 H NMR (400 MHz, CDC1 3 ) 5 7.12 (m, 3H), 6.09 (s, 1 H), 4.08 (m, J = 2.8 Hz, 2H), 3.63 (dd, J = 5.6, 5.6 Hz, 2H), 2.49 (m, 2H), 1.42 (s, 18H). 15 (c) tert-Butyl 4-(4-((tert-butoxycarbonyt)amino)-3-fluoropheny)piperidine- 1 carboxylate (154) A mixture of tert-butyl 4-(4-((tert-butoxycarbonyl)amino)-3-fluorophenyl)-5,6 dihydropyridine-1(2H)-carboxylate (/53) (0.409 g, 1.04 mmol) and 10% Pd/C (0.043 g) in EtOAc (20 mL) was stirred for 16 hours at room temperature under a 20 H 2 atmosphere. The mixture was filtered through celite and the filtrate concentrated under reduced pressure to give the title compound (154) (0.376 g, 92%) as a white foam; 'H NMR (400 MHz, CDC 3 ) 6 7.09 (dd, J = 8.1, 8.1 Hz, 1H), 6.95 (m, 2H), 4.25 (m, 2H), 2.79 (dd, J = 12.0, 12.0 Hz, 2H), 2.65 (m, IH), 1.82 (m, 2H), 1.43 (s, 18H). 25 (d) Methyl 2-(2-(2-(2-((2-fioro-4-(piperidin-4-yl)phenyl)amino)-5 (trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetate (158) To a mixture of tert-butyl 4-(4-((tert-butoxycarbonyl)amino)-3-fluorophenyl)piperidine 1-carboxylate (/57) (0.376 g, 0.954 mmol) and methyl 2-(2-(2-(2-(methylsulfonyl)-5 (trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetate (/15) (0.268 g, 0.667 mmol) in 30 2,2,2-trifluoroethanol (15 mL) was added TFA (0.8 mL). The resulting mixture was heated under microwave irradiation at 100 *C for 20 minutes then 15 minutes then concentrated under reduced pressure to give a crude sample of the title compound (/58) (0.344 g) which was used without purification.
WO 2012/110774 PCT/GB2012/000176 87 (e) tert-Butyl 4-(3-fluoro-4-((4-(2-(2-methoxy-2-oxoethy)phenethyl)-5 (trifluoromethyl)pyrimidin-2-yl)amino)phenyl)piperidine-1-carboxylate (160) A mixture of crude methyl 2-(2-(2-(2-((2-fluoro-4-(piperidin-4-yl)phenyl)amino)-5 (trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetate (159) (0.344 g), Boc 2 0 (0.214 g, 5 0.98 mmol) and a catalytic amount of DMAP in DCM (15 mL) was stirred for 20 hours. Triethylamine (0.10 mL, 0.72 mmol) was then added and the mixture stirred for 20 hours at 40 *C. Further portions of triethylamine (1.50 mL, 10.8 mmol) and Boc 2 0 (0.260 g, 1.19 mmol) were added and stirring continued at 40 "C for 4 hours. The mixture was concentrated under reduced pressure and chromatographed on 10 silica gel (0-75% EtOAc/petroleum benzine 40-60 *C) to give the title compound (160) (0.206 g, 50%) in 79% purity by LCMS, this material was used subsequently without further purification; 'H NMR (400 MHz, CDCI 3 ) 6 8.57 (s, 1 H), 8.28 (dd, J = 8.6, 8.6 Hz, 1H), 7.48 (d, J = 2.4 Hz, 1H), 7.22 (m, 4H), 7.00 (m, 2H), 4.25 (m, 2H), 3.75 (s, 2H), 3.68 (s, 3H), 3.11 (m, 4H), 2.81 (m, 2H), 2.64 (m, IH), 1.84 (m, 2H), 1.62 (dd, J 15 = 12.6, 4.5 Hz, 2H), 1.49 (s, 9H); LCMS Method C: rt 7.05 min; m/z 617.2 [M+H]*, 561.1 [(M-t-Bu)+H]*. (f) tert-Butyl 4-(4-((4-(2-(2-amino-2-oxoethyl)phenethyl)-5-(trifluoromethyl)pyrimidin-2 yl)amino)-3-fluorophenyl)piperidine-1-carboxylate (161) 20 To a solution of tert-butyl 4-(3-fluoro-4-((4-(2-(2-nethoxy-2-oxoethyl)phenethyl)-5 (trifluoromethyl)pyri midin-2-yl)amino)phenyl)piperidine-1 -carboxylate (160) (0.206 g) in THF (5 mL) was added aqueous 2 M LiOH (0.500 mL, 1.00 mmol) and water (0.5 mL) and the resulting solution was stirred for 16 hours at room temperature. Aqueous LiCH (1.5 M; 0.5 mL) was added and the reaction stirred for a further 20 hours at 25 room temperature. The mixture was then heated to 50 *C and methanol (1 mL) added before stirring for 20 hours. The volatiles were evaporated under reduced pressure and the residue azeotroped with toluene twice before addition of THF (4 mL), solid lithium hydroxide (0.021 g, 0.87 mmol), water (1 mL) and methanol (1 mL). The resulting mixture was stirred at room temperature for 5 hours and then heated to 30 50 *C for 16 hours. The volatiles were evaporated under reduced pressure and the residue diluted with water. The aqueous solution was extracted with EtOAc (2x20 mL) before acidifying to a pH of 3 with aqueous HCI. The resulting precipitate was collected as a pellet by centrifugation then dissolved in methanol and concentrated under reduced pressure. The residue was azeotroped with toluene then taken up in 35 DMF (6 mL) to which EDCl (0.080 g, 0.42 mmol), HOBT (0.061 g, 0.45 mmol) and WO 2012/110774 PCT/GB2012/000176 88 DIPEA (0.29 mL, 1.67 mmol) were added. The mixture was stirred at room temperature for 30 minutes under nitrogen before addition of ammonium carbonate (0.129 g, 1.66 mmol). The resulting mixture was stirred at 30 'C for 20 hours before the volatiles were evaporated under reduced pressure. Water (20 mL) was added 5 and the resulting suspension was extracted with EtOAc (3x20 mL). The combined organic extracts were concentrated under reduced pressure and the residue chromatographed on silica gel (0-100% EtOAc/petroleum benzine 40-60 *C then 70 100% EtOAc/petroleum benzine 40-60 *C) to give the title compound (161) (0.049 g, 24%); 'H NMR (400 MHz, d 6 -Acetone) 6 8.71 (s, 1H), 8.60 (s, IH), 7.98 (dd, J = 8.4, 10 8.4 Hz, 1H), 7.29 (m, 1H), 7.17 (m, 5H), 6.66 (s, 1H), 6.22 (s, 11H), 4.21 (m, IH), 3.63 (s, 2H), 3.16 (m, 4H), 2.09 (m, 2H), 1.87 (m, 3H), 1.59 (qd, J = 12.7, 4.4 Hz, 2H), 1.46 (s, 9H); LCMS Method C: rt 6.46 min; m/z 602.1 [M+H]*, 624.1 [M+Na]*, 546.1 [(M- t-Bu)+H)*, 502.1 [(M-Boc)+H]*. 15 (g) 2-(2-(2-(2-((2-Fluoro-4-(piperidin-4-yI)phenyl)amino)-5-(trifluoromethy)pyrimidin 4-y)ethy)phenyl)acetamide (162) To a solution of tert-butyl 4-(4-((4-(2-(2-amino-2-oxoethyl)phenethyl)-5 (trifluoromethyl)pyrimidin-2-yI)amino)-3-fluorophenyl)piperidine-1 -carboxylate (161) (0.049 g, 0.081 mmol) in THF (3 mL) was added TFA (0.20 mL, 2.6 mmol). The 20 resulting mixture was then stirred at room temperature for 20 hours then 40 *C for 24 hours. The volatiles were removed by evaporation under reduced and DCM (2 mL) and TFA (1.00 mL, 13.1 mmol) were added. After stirring at 30 *C for 18 hours the mixture was concentrated under reduced pressure and the residue azeotroped twice with toluene (10 mL) to give the title compound (/62) (0.037 g, 91%). LCMS Method 25 C: rt 4.84 min; rn/z 502.1[M+H)* (h) 2-(2-(2-(2-((2-Fivoro-4-(1-methylpiperidin-4-yl)phenyl)amino)-5 (trifluoromethyl)pyrimidin-4-y)ethy)phenyl)acetamide (13) To a solution of 2-(2-(2-(2-((2-fluoro-4-(piperidin-4-yl)phenyl)amino)-5 30 (trifluoromethyl)pyrimidin-4-yl)ethyl)phenyl)acetamide (/62) (0.037 g, 0.074 mmol) in anhydrous methanol (3 mL) was added 37% aqueous formaldehyde (0.022 mL, 0.30 mmol) and sodium triacetoxyborohydride (0.081 g, 0.38 mmol) under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 1 hour. The volatiles were evaporated under reduced pressure and the residue taken up 35 saturated aqueous NaHCO 3 solution (25 mL). The resulting mixture was extracted WO 2012/110774 PCT/GB2012/000176 89 with EtOAc (2x25 mL) then the combined organic layers were washed with brine (25 mL), dried (phase separation cartridge) and evaporated to dryness under reduced pressure. The residue was purified using silica gel column chromatography (0-50% MeOH/EtOAc with 1% 2 M ethanolic ammonia) to give the title compound (13) (0.020 5 g, 52%); 1 H NMR (400 MHz, d 6 -Acetone + d 4 -MeOH) 6 8.54 (s, 1 H), 7.88 (dd, J = 8.5 Hz, 1H), 7.16 (m, 6H), 3.80 (s, 2H), 3.63 (s, 2H), 3.10 (m, 4H), 2.95 (m, 2H), 2.54 (m, 1H), 2.27 (s, 3H), 1.79 (rn, 4H). LCMS Method C: rt 4.93 min; m/z 516.1 [M+H]*. Biological Assays 10 The activity of compounds of the invention can be profiled using biochemical and cellular assays. Primary potency at FAK can be assessed using an Alpha Screen
T
M technology biochemical assay. 15 The kinetics of this binding may be further studied using a surface plasmon resonance (SPR) technology assay using a BiacoreTM S51 sensor to establish Ka, kd and consequently KD. When off rates from the protein greatly exceed on rates, as may occur for highly potent compounds, Ko gives an accurate measure of protein 20 ligand binding affinity. The ability of compounds of the invention to inhibit FAK within cells can be assessed with an ELISA -type assay performed using a Meso Scale Discovery SECTOR Imager 6000 instrument. In this assay the ability of compounds of the invention to 25 inhibit phosphorylation of Y397-FAK is determined. The effect of compounds of the invention on inhibition of cellular proliferation resulting from non-FAK activity may be assessed using a 2D proliferation assay using a suitable cell line. This gives an indication of off-target activities and potential toxicity 30 arising from them. Therefore, comparing inhibition of phosphorylation of Y397-FAK and 2D proliferation gives a measure of FAK specific mediated effects and also of potential toxicity resulting from off-target activity. Primary potency at VEGFR3 can be assessed using an Alpha ScreenTM technology 35 biochemical assay.
WO 2012/110774 PCT/GB2012/000176 90 FAK biochemical Alpha Screen assay A biotin labeled peptide is used as substrate (amino acid sequence: Biotin-Glu-Gly Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH 2 ). FAK enzyme 5 was expressed in insect cells as catalytic domain (amino acids 411-686) N-terminally tagged with six histidine amino acids and a Tobacco Etch Virus (TeV) cleavage sequence. After lysing the cells by sonication, the kinase was purified by Ni Immobilised Metal Affinity Chromatography chromatography, TeV cleavage leaving a N-terminal glycine, and gel filtration. The 15 pi assay reactions are run in Greiner 10 brand white 384-well low volume plates. All reactions contained 10 mM HEPES pH 7.4, 25 mM NaCI, 10 mM MgCl 2 , 0.01 % (v/v) Tween-20, 50 pM Na 3
VO
4 , 0.01% (w/v) albumin from chicken egg white, 111 nM peptide substrate, 80 pM ATP, and 4 ng/reaction FAK enzyme, with the enzyme being omitted from negative control reactions. Compounds were added in a volume of 100 nl from dilution series made 15 up in DMSO, positive and negative control reactions receiving the same volume DMSO without compounds. The plates were sealed with adhesive seals and incubated for 90 minutes at 30'C. The reactions were stopped with the detection reagents added at the same time. Product formation was quantified as amplified luminescence between PerkinElmer AlphaScreen T M beads, using Streptavidin-coated 20 donor and anti-phosphotyrosine (P-Tyr-100) acceptor beads. To each reaction, 5 pl containing 10 mM HEPES pH 7.4, 25 mM NaCl, 100 mM EDTA, 0.01 % (vlv) Tween 20, and 6.25 pjg/ml of each bead type were added. Plates were incubated for 6 hours before being read on a PerkinElmer EnVision TM plate reader in HTS Alphascreen TM mode. IC 5 0 values were obtained by calculating percent inhibition (%l) for each 25 reaction relative to controls on the same plate (%=(l-CN)/(CP-CN) where CN/ CP are the averages of the negative/ positive reactions, respectively), then fitting the %I data vs. compound concentration [1] to %l=(A+((B-A)/(1+((C/[l])^D)))) where A is the lower asymptote, B is the upper asymptote, C is the IC 5 0 value, and D is the slope factor. 30 Results Compound IC 5 o (nM) 1 1.9 2 7.1 3 2.2 4 3.6 WO 2012/110774 PCT/GB2012/000176 91 5 7.2 6 2.1 7 9.4 8 5.2 9 3.6 10 2.7 11 2.2 12 3.6 13 5.6 FAK BiacorenM SPR assay Binding parameters of compounds were determined using a BiacoreTM S51 sensor. An anti-GST antibody was immobilized onto a CM5 chip by primary amine-coupling 5 in accordance with the manufacturer's recommendations. In running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% Surfactant P20, 10 mM MgCl 2 , and 1% DMSO) N-terminally GST-fused purified FAK enzyme was captured on both spot I and 2. Spot 1 was subsequently blocked by loading with 10 30 nM PF-562,271 at the beginning of each cycle. Concentration series' of the test compounds were injected over the spots at 25*C. The specific binding was calculated as difference between spot 2 and 1 signals followed by solvent correction. Fitting to a one site binding model yielded the kinetic rate constants kd and ka and the equilibrium binding constant KD=kdlka. 15 For compounds with an expected K < 5 nM N-terminally GST-fused purified FAK enzyme was captured on spot 2 of the anti-GST antibody coated chip only. After the injection cycle of a compound the chip surface was regenerated with 10 mM glycine HCl, pH2.2 before capturing the enzyme again. The binding sensorgrams were 20 analysed as described before. Results Compound K 0 (nM) 1 0.29 2 0.74 WO 2012/110774 PCT/GB2012/000176 92 3 0.21* 4 1.5 6 2.9 8 0.81 9 1.5 10 0.79 11 1.7 * This result was measured using an extended washing step of 30 minutes. P397Y-FAK Inhibition MSD platform cellular biomarker assay 5 Compounds of the invention may be tested for in vitro activity in the following assay: 96-well plates (cat#MA6000, Meso Scale Discovery) are coated with 30pL/well of mouse monoclonal FAK antibody (63D5] (cat#ab72140, Abcam) pre-diluted in PBS to a concentration of 1mg/mL. The plates are sealed with adhesive film and incubated 10 for 16 hours at 40C. The antibody is then flicked out of the plates and 150pL of 3% {w/v) Blocker A (cat#R93AA-1, Meso Scale Discovery) is added. The plates are resealed with adhesive film and incubated at room temperature on a shaker set at medium speed for 2 hours. The plates are then washed three times with a solution containing 50mM Tris-HCI pH 7.5, 0.15M NaCl and 0.02% Tween-20, before cell 15 lysate addition described below. Cells are split 1:2 into T1 50 cell culture flasks 2 days prior to compound treatment. On the day prior to compound treatment, 200pL media containing 20,000 cells is seeded into all wells of white, clear-bottom, TC treated, pclear, 96-well microtitre 20 plates (cat#655098, Greiner Bio-One), and the plates are incubated at 370C and 5%
CO
2 for 36 hours. 1 pL/well of compound is then added from dilution series prepared in DMSO. Negative control wells receive the same volume of DMSO without compounds, and positive control wells receive 2pM of a control compound in the same volume of DMSO. Cells are treated for 1 hour at 37*C and 5% CO 2 . The 25 media/compounds are then flicked off and 55pL/well of ice-cold complete lysis buffer is added. Complete lysis buffer is prepared by adding 1 tablet PhosSTOP complete phosphatase inhibitor (cat#04906837001, Roche) and 1 tablet Complete, Mini, EDTA-free, protease inhibitor (cat#04693159001, Roche) per I OmL of incomplete WO 2012/110774 PCT/GB2012/000176 93 lysis buffer (150mM NaCI, 20mM Tris-HCI pH 7.5, 1mM EDTA, 1mM EGTA, 1% Triton-X 100). Plates are incubated on ice for 30 minutes, with 30 seconds high speed plate shaking every 5 minutes. 40pL/well of cell lysate is transferred to the coated, blocked and washed 96-well microtitre plates described above. The 96-well 5 plates are sealed with adhesive film and incubated for 16 hours at 4"C. The plates are then washed three times with a solution containing 50mM Tris-HCI pH 7.5, 0.15M NaCl and 0.02% Tween-20 and tapped dry. 25pL/well of detection solution (1% [wIv] Blocker A (cat#R93AA-1, Meso Scale Discovery) in 50mM Tris-HCI pH 7.5, 0.15M NaCl and 0.02% Tween-20, with 1:600 rabbit polyclonal FAK phospho Y397 antibody 10 (cat#ab39967, Abcam), 1:1000 anti-rabbit sulfo-tag antibody (cat#R32AB-1 Meso Scale Discovery) and 1:40 reconstituted Blocker D-M (cat#D609-0100, Rockland Immunochemicals for Research)) is added, and the plates resealed with adhesive film and incubated for 1 hour at room temperature on a plate shaker set to medium speed. Plates are then washed three times with a solution containing 50mM Tris-HCI 15 pH 7.5, 0.15M NaCl and 0.02% Tween-20 and tapped dry. 150pL/well of Read Buffer T + Surfactant (cat#R92TC-1, Meso Scale Discovery) is then added, and pFAK-397 levels quantified using a Meso Scale Discovery SECTOR Imager 6000 instrument. IC5o values are determined by first calculating percent inhibition (%I) for each lysate 20 relative to controls on the same plate (%I=(S-CP)/(CN-CP)) where S is the sample result, CN is the average result of DMSO only treated negative controls, and CP is the average result of 2pM treated positive controls. %I is plotted against compound concentration (l] and the data fitted using the following equation, %I=(A+((B A)/(1+((C/[I])AD)))), where A is the lower asymptote, B is the upper asymptote, C is 25 the IC50 value, and D is the slope factor. Results for MDA-231-LNA cells % response of control at 2 pM 1 9 122 2 12 91 3 7 102 4 13 108 6 264 80 9 59 112 WO 2012/110774 PCT/GB2012/000176 94 10 114 116 11 16 117 12 390 77 13 14 90 2D Cellular proliferation assay Cells are split 1:4 into T75 cell culture flasks two days prior to cell seeding. A variety of cancer cell lines can be utilized in this assay. 5 On the day of cell seeding 1OOpL/well of media containing 1000-5000 cells are added to 96-well microtitre plates (Cat.#655 180, greiner bio-one) except wells G1 2 and H12 to which 100pi of media is added. In a second plate, a single row of cells is seeded at the same concentration. This second plate is known as the t=0 plate and is 10 used to calculate the relative cell number prior to addition of test agent. The plates containing cells are incubated for 24 hours at 37*C/ 5%CO2. 0.5pLIwell of compound is then added from dilution series prepared in DMSO. A compound with known potency is included for each set of plates in order to assess assay performance. Negative control wells receive the same volume of DMSO without compounds. 15 Background signal is determined from wells containing media alone. The t=0 plate is read using addition of a resazurin-based reagent (see below) on the day that other plates have compound added to them. Plates containing cells to which compound has been added are then incubated for 3 days at 37*C and 5% CO 2 . 20 After 3 days of incubation, cell proliferation is quantified by addition of 20 pl/well of a resazurin-based reagent with a typical composition as follows: Resazurin, Sigma# R7017-1G, 0.015% w/v; methylene blue, Sigma# MB-1(25g), 0.0025% w/v; potassium hexacyanoferrate (1ll), Sigma# P8131-100G, 0.033 w/v; potassium hexacyanoferrate (11) trihydrate, Sigma# P9387-1 OG, 0.042% w/v; in PBS buffer. 25 Plates are incubated with resazurin-based reagent for 1-4 hours (37*C, 5% C02) prior to the determination of fluorescence at, or near (5 79 Ex/58 4 Em). Percentage inhibition of proliferation (%I) for each treated well relative to controls on the same plate is calculated using the equation %I=(S-B)-(T 0
-B)/(CN-B)-(T
0 -B) where 30 S is the sample result B is the background fluorescence, To is the t=0 value and CN is the average result of DMSO only treated negative controls. For IC50 WO 2012/110774 PCT/GB2012/000176 95 determination, %I is plotted against compound concentration [1] and the data fitted using the following equation, %l=(A+((B-A)/(1 +((C/[1])^D)))), where A is the lower asymptote, B is the upper asymptote, C is the IC 50 value, and D is the slope factor. 5 Results for MDA-231-LNA cells Compound IC 50 (pM) 1 1.43 2 2.77 3 6.62 4 1.91 5 3.06 6 2.24 7 4.93 8 2.48 9 2.90 10 1.69 11 2.58 12 4.83 13 2.76 VEGFR3 Biochemical asay Compounds of the invention may be tested for in vitro activity in the following assay: 10 A biotin labeled peptide is used as substrate (amino acid sequence: Biotin-Glu-Gly Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH 2 ). VEGFR3 cytoplasmic domain (amino acids 798-1298) was purchased as N-terminal GST fusion protein ("the enzyme"). The 15 pl assay reactions are run in Greiner brand white 384-well low volume plates. All reactions contained 10 mM HEPES pH 7.4, 15 10 mM MgCl 2 , 0.01 % (vlv) Tween-20, 50 pM Na 3
VO
4 , 0.01% (w/v) albumin from chicken egg white, 1 mM Dithiothreitol, 111 nM peptide substrate, 500 pM ATP, and 3.8 ng/reaction enzyme, with the enzyme being omitted from negative control reactions. Compounds were added in a volume of 100 nl from dilution series prepared in DMSO, positive and negative control reactions receiving the same 20 volume DMSO without compound. The plates were sealed with adhesive seals and incubated for 90 minutes at 30 degree Celsius. The reactions were stopped with the WO 2012/110774 PCT/GB2012/000176 96 detection reagents added at the same time as follows: Product formation was quantified as amplified luminescence between PerkinElmerAlphaScreen TM beads, using Streptavidin-coated donor and anti-phosphotyrosine (P-Tyr-100) acceptor beads. To each reaction, 5 pl containing 10 mM HEPES pH 7.4, 25 mM NaCI, 5 100 mM EDTA, 0.01 % (v/v) Tween-20, and 6.25 pg/ml of each bead type were added. Plates were incubated for 6 hours before being read on a PerkinElmer EnVision TM plate reader in HTS AlphascreenTM mode. ICso values were obtained by calculating percent inhibition (%I) for each reaction relative to controls on the same plate (%I=(1-CN)/(CP-CN) where CN/ CP are the averages of the negative/ positive 10 reactions, respectively), then fitting the %l data vs. compound concentration [1] to %I=(A+((B-A)/(1+((C/[1])AD)))) where A is the lower asymptote, B is the upper asymptote, C is the IC50 value, and D is the slope factor. The above assay was also run in a slightly modified form in some cases (indicated 15 below with *). In these cases, VEGFR3 cytoplasmic domain (amino acids 818-1177, lacking 949-1002 of UniProt accession number P35916) was expressed and purified as N-terminal Hexa-His-fusion protein ("the enzyme"), rather than using the N terminal GST-fusion protein. 20 Results Compound IC 50 (nM) 1 275* 2 3685* 3 538 5 5750* 6 10440 7 13260* 8 1176 9 1010 10 >66000 11 59763 12 240* 13 205

权利要求:
Claims (26)
[1] 1. A compound of the formula (1): R 2 R N R N N R 5 R 3 H wherein: 5 R' is selected from: H and RN 3 N1 N 2 N4 R N N R '-NaNNR wherein: RN1 is selected from H, C13 alkyl and C(=O)Me; RN 2 is selected from H, C-3 alkyl and C(=O)Me; 10 RN 3 is selected from H, C 1 3 alkyl and C(=O)Me; RN 4 is selected from H and CH 3 ; R2 is selected from H and N5 N R N5N N R N a wherein: 15 RN5 is selected from H, C1-3 alkyl and C(=O)Me; RN6 is selected from H, C 1 - 3 alkyl and C(=0)Me; and wherein only one of R' and R 2 is H; R 3 is selected from 0-C2 alkyl, C12 alkyl, halo, cyano, where the C12 alkyl group may be substituted by one or more fluoro groups; 20 R 4 is selected from CF 3 , halo, CF 2 H and CN; and R' is selected from groups of the following formulae: WO 2012/110774 PCT/GB2012/000176 98 0 H R N R 6 R (R") (R ) wherein: R 6 is selected from H, (CHRc1)r 1 C(O)N(RN6 )Z and (CH 2 )n 2 C(O)OZ 2 ; wherein: n1 is 1; 5 Rc 1 is H or Me; RN 6 is H or CH 3 ; Z' is H, CH 3 or OCH 3 ; n2 is 1; and Z 2 is CH 3 ; 10 and where only one of RN 6 and Z' can be CH 3 , R' is selected from H, and (CH 2 )mI1C(O)N(RM1)Y1, wherein: ml is 0 or 1; RM' is H; and Y' is H, Me or OCH 3 ; 15 and only one of R' and R 7 is H; and R3 is H or, when R' is C(=O)NH 2 , R3 is selected from H and C1-2 alkyl.
[2] 2. A compound according to claim 1, wherein R 2 is H and R' is: N1 R s wherein RN1 is C(=O)Me. 20
[3] 3. A compound according to claim 1, wherein R 2 is H and R' is: N1 R 11N N , wherein RN is H, methyl or ethyl.
[4] 4. A compound according to claim 1, wherein R 2 is H and R 1 is: N2 R Nwr 25 *, wherein RN 2 is selected from H, methyl and ethyl. WO 2012/110774 PCT/GB2012/000176 99
[5] 5. A compound according to claim 1, wherein R 2 is H and R' is: RN 3 N , wherein RN 3 is selected from H and methyl. 5
[6] 6. A compound according to claim 1, wherein R 2 is H and R' is: N4 , wherein RN 4 is selected from H and methyl.
[7] 7. A compound according to claim 1, wherein R 1 is H and R 2 is: N5 R NN Ns , where RN 5 is selected from H and methyl. 10
[8] 8. A compound according to claim 1, wherein R 1 is H and R 2 is: R NN , where RN6 is selected from H and methyl. 15
[9] 9. A compound according to any one of claims 1 to 8, wherein R 3 is selected from F, Me, Et, OMe and OCF 3 .
[10] 10. A compound according to any one of claims 1 to 9, wherein R 3 is OMe. 20
[11] 11. A compound according to any one of claims 1 to 10, wherein R 4 is selected from CF 3 , Cl and CF 2 H .
[12] 12. A compound according to claim 11, wherein R' is CF 3 . 25
[13] 13. A compound according to any one of claims 1 to 11, wherein R 5 is a group of the following formula: WO 2012/110774 PCT/GB2012/000176 100 R 6 R R7 * (Rsa)
[14] 14. A compound according to claim 13, wherein R 7 is H and R 6 is selected from CH 2 C(O)NH 2 , CH 2 C(O)NHCH 3 , CHCH 3 C(O)NH 2 and CHCH 3 C(O)NHCH 3 . 5
[15] 15. A compound according to claim 14, wherein R 7 is H and R 6 is selected from CH 2 C(O)NH 2 , CHCH 3 C(O)NH 2 and CH 2 C(O)NHCH 3 .
[16] 16. A compound according to claim 15, wherein R 7 is H and R 6 is selected from 10 CH 2 C(O)NH 2 , and CHCH3C(O)NH 2 .
[17] 17. A compound according to claim 13, wherein R 6 is H and R7 is selected from C(O)NH 2 , C(O)NHCH 3 , CHqC(O)NH 2 and CH 2 C(O)NHCH 3 . 15
[18] 18. A compound according to claim 17, wherein R 6 is H and R 7 is C(O)NH 2 .
[19] 19. A compound according to claim 18, wherein R 8 is methyl.
[20] 20. A compound according to any one of claims 1 to 11, wherein R' is a group of 20 the following formula: H N 0 (R 5 b)
[21] 21. A composition comprising a compound according to any one of claims 1 to 20, and a pharmaceutically acceptable carrier or diluent WO 2012/110774 PCT/GB2012/000176 101
[22] 22. A compound a compound according to any one of claims 1 to 20 for use in a method of therapy.
[23] 23. The use of a compound according to any one of claims 1 to 20 in the 5 preparation of a medicament for treating a disease ameliorated by the inhibition of FAK.
[24] 24. A compound according to any one of claims 1 to 20 for use in the method of treatment of a disease ameliorated by the inhibition of FAK. 10
[25] 25. A compound according to any one of claims 1 to 20 for use in a method of treatment of the human or animal body.
[26] 26. A method of inhibiting FAK in vitro or in vivo, comprising contacting a cell with 15 an effective amount of a compound according to any one of claims 1 to 20.

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

GB9624482D0|1995-12-18|1997-01-15|Zeneca Phaema S A|Chemical compounds|

---

AU719434B2|1996-02-13|2000-05-11|Astrazeneca Ab|Quinazoline derivatives as VEGF inhibitors|

---

EP0885198B1|1996-03-05|2001-12-19|AstraZeneca AB|4-anilinoquinazoline derivatives|

---

GB9718972D0|1996-09-25|1997-11-12|Zeneca Ltd|Chemical compounds|

---

WO1998035985A1|1997-02-12|1998-08-20|The Regents Of The University Of Michigan|Protein markers for lung cancer and use thereof|

---

GB9714249D0|1997-07-08|1997-09-10|Angiogene Pharm Ltd|Vascular damaging agents|

---

ES2274634T3|1998-08-29|2007-05-16|Astrazeneca Ab|PIRIMIDINE COMPOUNDS.|

---

GB9828511D0|1998-12-24|1999-02-17|Zeneca Ltd|Chemical compounds|

---

GB9900334D0|1999-01-07|1999-02-24|Angiogene Pharm Ltd|Tricylic vascular damaging agents|

---

GB9900752D0|1999-01-15|1999-03-03|Angiogene Pharm Ltd|Benzimidazole vascular damaging agents|

---

CN100360505C|1999-02-10|2008-01-09|阿斯特拉曾尼卡有限公司|Quinazoline derivatives as angiogenesis inhibitors|

---

GB9905075D0|1999-03-06|1999-04-28|Zeneca Ltd|Chemical compounds|

---

DE60029007T2|1999-11-05|2007-01-11|Astrazeneca Ab|QUINAZOLINE DERIVATIVES AS VEGF HEMMER|

---

UA73976C2|2000-02-15|2005-10-17|Суджен, Інк.|Pyrrole substituted 2-indolinone compounds, a pharmaceutical composition, a method for modulation of catalytic activity of protein kinase and a method for the treatment of disease related to protein kinase|

---

GB0004886D0|2000-03-01|2000-04-19|Astrazeneca Uk Ltd|Chemical compounds|

---

GB0004890D0|2000-03-01|2000-04-19|Astrazeneca Uk Ltd|Chemical compounds|

---

GB0004887D0|2000-03-01|2000-04-19|Astrazeneca Uk Ltd|Chemical compounds|

---

BR0111230A|2000-05-31|2003-06-10|Astrazeneca Ab|Compound, and, use and process for the preparation thereof|

---

SI1292594T1|2000-06-06|2005-02-28|Astrazeneca Ab|Quinazoline derivatives for the treatment of tumours|

---

BR0112225A|2000-07-07|2003-05-06|Angiogene Pharm Ltd|Compound, pharmaceutical composition, use of a compound, and process for preparing a compound|

---

CA2411160A1|2000-07-07|2002-01-17|Angiogene Pharmaceuticals Limited|Colchinol derivatives as vascular damaging agents|

---

DK1438053T3|2001-10-17|2008-12-08|Boehringer Ingelheim Pharma|Pyrimidine derivatives, drug containing these compounds, their use and methods for their preparation|

---

GB0206215D0|2002-03-15|2002-05-01|Novartis Ag|Organic compounds|

---

UA80767C2|2002-12-20|2007-10-25|Pfizer Prod Inc|Pyrimidine derivatives for the treatment of abnormal cell growth|

---

US7109337B2|2002-12-20|2006-09-19|Pfizer Inc|Pyrimidine derivatives for the treatment of abnormal cell growth|

---

RS20060097A|2002-12-20|2008-11-28|Pfizer Products Inc.,|Pyrimidine derivatives for the treatment of abnormal cell growth|

---

GB0305929D0|2003-03-14|2003-04-23|Novartis Ag|Organic compounds|

---

ES2380206T3|2003-08-15|2012-05-09|Novartis Ag|2,4-Pyrimidinediamines useful for the treatment of neoplastic diseases, inflammatory and immune system disorders|

---

KR100694732B1|2003-09-05|2007-03-14|화이자 프로덕츠 인크.|Selective synthesis of cf3-substituted pyrimidines|

---

BRPI0511132A|2004-05-14|2007-11-27|Pfizer Prod Inc|pyrimidine derivatives and pharmaceutical composition comprising the same|

---

JP2007537230A|2004-05-14|2007-12-20|ファイザー・プロダクツ・インク|Pyrimidine derivatives for the treatment of abnormal cell proliferation|

---

GB0419160D0|2004-08-27|2004-09-29|Novartis Ag|Organic compounds|

---

GB0419161D0|2004-08-27|2004-09-29|Novartis Ag|Organic compounds|

---

WO2006076442A2|2005-01-14|2006-07-20|Janssen Pharmaceutica N.V.|Triazolopyrimidine derivatives|

---

CA2632149C|2005-12-01|2011-11-15|Pfizer Products Inc.|Pyrimidine derivatives for the treatment of abnormal cell growth|

---

JO3235B1|2006-05-26|2018-03-08|Astex Therapeutics Ltd|Pyrrolopyrimidine compounds and their uses|

---

CN101679264A|2007-03-16|2010-03-24|斯克里普斯研究学院|inhibitors of focal adhesion kinase|

---

CN101801401A|2007-03-16|2010-08-11|佛罗里达大学研究基金会|Kinase protein binding inhibitors|

---

DK2146779T3|2007-04-18|2016-11-28|Pfizer Prod Inc|Sulfonylamid derivatives to treat abnormal cell growth.|

---

EP2065380A1|2007-08-22|2009-06-03|F.Hoffmann-La Roche Ag|Pyridoneamide derivatives as focal adhesion kinase inhibitors and their use for the treatment of cancer|

---

US8461147B2|2007-12-03|2013-06-11|Boehringer Ingelheim International Gmbh|Diaminopyridines for the treatment of diseases which are characterised by excessive or anomal cell proliferation|

---

US20100317663A1|2008-02-19|2010-12-16|Jerry Leroy Adams|Anilinopyridines as inhibitors of fak|

---

CA2723961C|2008-05-21|2017-03-21|Ariad Pharmaceuticals, Inc.|Phosphorous derivatives as kinase inhibitors|

---

WO2009153589A1|2008-06-17|2009-12-23|Astrazeneca Ab|Pyridine compounds|

---

JO3067B1|2008-10-27|2017-03-15|Glaxosmithkline Llc|Pyrazolylaminopyridines as inhibitors of fak|

---

UY32240A|2008-11-14|2010-06-30|Boeringer Ingelheim Kg|NEW 2,4-DIAMINOPIRIMIDINAS, ITS PHARMACEUTICALLY ACCEPTABLE SALTS, COMPOSITIONS CONTAINING THEM AND APPLICATIONS.|

---

AR074209A1|2008-11-24|2010-12-29|Boehringer Ingelheim Int|USEFUL PYRIMIDINE DERIVATIVES FOR CANCER TREATMENT|

---

AR074210A1|2008-11-24|2010-12-29|Boehringer Ingelheim Int|PIRIMIDINE DERIVATIVES AS INHIBITORS OF PTK2-QUINASA|

---

US20110071158A1|2009-03-18|2011-03-24|Boehringer Ingelheim International Gmbh|New compounds|

---

WO2010126922A1|2009-04-30|2010-11-04|Glaxosmithkline Llc|Benzimidazolecarboxamides as inhibitors of fak|

---

US8410126B2|2009-05-29|2013-04-02|Boehringer Ingelheim International Gmbh|Pyrimidine inhibitors of PKTK2|

---

TW201100441A|2009-06-01|2011-01-01|Osi Pharm Inc|Amino pyrimidine anticancer compounds|

---

WO2010141796A2|2009-06-05|2010-12-09|Cephalon, Inc.|PREPARATION AND USES OF 1,2,4-TRIAZOLO [1,5a] PYRIDINE DERIVATIVES|

---

US20120196858A1|2009-08-12|2012-08-02|Poniard Pharmaceuticals ,Inc.|Method of promoting apoptosis and inhibiting metastasis|

---

US8466155B2|2009-10-02|2013-06-18|Boehringer Ingelheim International Gmbh|Pyrimidines|

---

KR101147550B1|2009-10-22|2012-05-17|한국과학기술연구원|2,7-Substituted thieno[3,2-d]pyrimidine compounds as protein kinase inhibitors|

---

MX340819B|2010-06-29|2016-07-26|Poniard Pharmaceuticals Inc |Oral formulation of kinase inhibitors.|

---

NZ604801A|2010-06-30|2015-03-27|Verastem Inc|Synthesis and use of focal adhesion kinase inhibitors|

---

DE102010034699A1|2010-08-18|2012-02-23|Merck Patent Gmbh|pyrimidine derivatives|

---

US20120244141A1|2010-09-28|2012-09-27|Boehringer Ingelheim International Gmbh|Stratification of cancer patients for susceptibility to therapy with PTK2 inhibitors|

---

WO2012045194A1|2010-10-09|2012-04-12|Abbott Laboratories|Benzodiazepinones as fak inhibitors for treatment of cancer|

---

WO2012045195A1|2010-10-09|2012-04-12|Abbott Laboratories|Pyrrolopyrimidines as fak and alk inhibiters for treatment of cancers and other diseases|

---

EP2675794B1|2011-02-17|2019-02-13|Cancer Therapeutics Crc Pty Limited|Selective fak inhibitors|DE102010034699A1|2010-08-18|2012-02-23|Merck Patent Gmbh|pyrimidine derivatives|

---

EP2675793B1|2011-02-17|2018-08-08|Cancer Therapeutics Crc Pty Limited|Fak inhibitors|

---

EP2675794B1|2011-02-17|2019-02-13|Cancer Therapeutics Crc Pty Limited|Selective fak inhibitors|

---

WO2014027199A1|2012-08-14|2014-02-20|Cancer Therapeutics Crc Pty Ltd|Fak and flt3 inhibitors|

---

AU2013302320A1|2012-08-17|2015-02-26|Cancer Therapeutics Crc Pty Limited|VEGFR3 inhibitors|

---

CN104837835A|2012-08-17|2015-08-12|癌症治疗合作研究中心有限公司|VEGFR3 inhibitors|

---

US10106834B2|2013-10-09|2018-10-23|The General Hospital Corporation|Methods of diagnosing and treating B cell acute lymphoblastic leukemia|

---

CN107235931B|2017-07-11|2019-09-24|大连医科大学|New pyrimidine anti-tumor compounds and preparation method thereof and purposes|

---

CN109503398B|2018-12-19|2021-07-02|江苏智远科创科技有限公司|Preparation method of N-methyl-4-methoxyaniline|

---

CN113646303A|2019-03-28|2021-11-12|安普利亚治疗有限公司|Salts and crystalline forms of FAK inhibitors|

---

WO2021237273A1|2020-05-28|2021-12-02|Amplia Therapeutics Pty Ltd|Methods of treating pulmonary fibrosis|

法律状态:
2016-11-10| FGA| Letters patent sealed or granted (standard patent)|

优先权:

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申请号 | 申请日 | 专利标题

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US201161443773P| true| 2011-02-17|2011-02-17||

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US61/443,773||2011-02-17||

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US201161523489P| true| 2011-08-15|2011-08-15||

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US61/523,489||2011-08-15||

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US201161579729P| true| 2011-12-23|2011-12-23||

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US61/579,729||2011-12-23||

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PCT/GB2012/000176|WO2012110774A1|2011-02-17|2012-02-17|Selective fak inhibitors|

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