WO2021119236A1 - Preparation of a chk1 inhibitor compound - Google Patents

Abstract

The invention provides a novel synthetic route for the preparation of the Chk-1 inhibitor compound; also provided by the invention is a novel process for the preparation of the synthetic intermediate of formula (11); as well as novel process intermediates per se.

Description

PREPARATION OF A CHK1 INHIBITOR COMPOUND

This application claims priority from US provisional patent application number 62/946,296 filed on 10 December 2019, the contents of which are incorporated herein by reference in their entirely.

This invention relates to processes for preparing the Chk-1 inhibiitor compound 5-[[5- [4-(4-Fluoro-1-methyl-4-piperidyl)-2-methoxy-phenyl]-1H-pyrazol-3-yl]amino]pyrazine- 2-carbonitrile, processes for preparing synthetic intermediates, and to novel chemical intermediates for use in the processes.

Background of the Invention

Chk-1 is a serine/threonine kinase involved in the induction of cell cycle checkpoints in response to DNA damage and replicative stress [Clin. Can. Res. 2007; 13(7)]. Cell cycle checkpoints are regulatory pathways that control the order and timing of cell cycle transitions. Most cancer cells have impaired G1 checkpoint activation due to a defective p53 tumor suppressor protein. Hahn et al., “Rules for making human tumor cells” N. Engl. J. Med. 2002; 347: 1593-603 and Hollstein et al., “p53 mutations in human cancers” Science 1991; 253: 49-53) have reported that tumours are associated with mutations in the p53 gene, a tumour suppressor gene found in about 50% of all human cancers.

Chk-1 inhibition abrogates the intra S and G2/M checkpoints and has been shown to selectively sensitise tumour cells to well known DNA damaging agents. Examples of DNA damaging agents where this sensitising effect has been demonstrated include Gemcitabine, Pemetrexed, Cytarabine, Irinotecan, Camptothecin, Cisplatin, Carboplatin [Clin. Cancer Res. 2010, 16, 376], Temozolomide [Journal of Neurosurgery 2004, 100, 1060], Doxorubicin [Bioorg. Med. Chem. Lett. 2006;16:421- 6], Paclitaxel [WO2010149394], Hydroxy urea [Nat. Cell. Biol. 2005 Feb;7(2): 195-20], the nitroimidazole hypoxia-targetted drug TH-302 (Meng et al., AACR, 2013 Abstract No. 2389) and ionising radiation [Clin. Cancer Res. 2010, 16, 2076]. See also the review article by McNeely, S., et al., “CHEK again: Revisiting the development of CHK1 inhibitors for cancer therapy, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/ j.pharmthera.2013.10.005.

Recently published data have also shown that Chk-1 inhibitors may act synergistically with PARP inhibitors [Cancer Res:, 66: (16)], Mek inhibitors [Blood. 2008 September 15; 112(6): 2439-2449], Farnesyltransferase inhibitors [Blood. 2005 Feb 15; 105(4): 1706- 16], Rapamycin [Mol. Cancer Ther. 2005 Mar;4(3):457-70], Src inhibitors [Blood. 2011 Feb 10; 117(6): 1947-57] and WEE1 inhibitors (Chaudhuri et al., Haematologica, 2013.093187).

Resistance to chemotherapy and radiotherapy, a clinical problem for conventional therapy, has been associated with activation of the DNA damage response in which Chk-1 has been implicated (Chk-1 activation is associated with radioresistence in glioblastoma [Nature ; 2006; 444(7):756-760] and the inhibition of Chk-1 sensitises lung cancer brain metastases to radiotherapy [Biochem. Biophys. Res. Commun. 2011 March 4;406(1):53-8]).

It is also envisaged that Chk-1 inhibitors, either as single agents or in combination, may be useful in treating tumour cells in which constitutive activation of DNA damage and checkpoint pathways drive genomic instability. This phenotype is associated with complex karyotypes in samples from patients with acute myeloid leukemia (AML) [Cancer Research 2009, 89, 8652], In vitro antagonisation of the Chk-1 kinase with a small molecule inhibitor or by RNA interference strongly reduces the clonogenic properties of high-DNA damage level AML samples. In contrast Chk-1 inhibition has no effect on normal hematopoietic progenitors. Furthermore, recent studies have shown that the tumour microenvironment drives genetic instability [Nature ;

2008; (8): 180- 192] and loss of Chk-1 sensitises cells to hypoxia/reoxygenation [Cell Cycle ; 2010; 9(13):2502], In neuroblastoma, a kinome RNA interference screen demonstrated that loss of Chk-1 inhibited the growth of eight neuroblastoma cell lines. Tumour cells deficient in Fanconi anemia DNA repair have shown sensitivity to Chk-1 inhibition [Molecular Cancer 2009, 8:24], It has been shown that the Chk-1 specific inhibitor PF-00477736 inhibits the growth of thirty ovarian cancer cell lines [Bukczynska et al, 23^rd Lome Cancer Conference] and triple negative negative breast cancer cells [Cancer Science 2011, 102, 882], Also, PF-00477736 has displayed selective single agent activity in a MYC oncogene driven murine spontaneous cancer model [Ferrao et al, Oncogene (15 August 2011)]. Chk-1 inhibition, by either RNA interference or selective small molecule inhibitors, results in apoptosis of MYC- overexpressing cells both in vitro and in an in vivo mouse model of B-cell lymphoma [Hoglund et al., Clinical Cancer Research, Online First September 20, 2011], The latter data suggest that Chk-1 inhibitors would have utility for the treatment of MYC- driven malignancies such as B-cell lymphoma/leukemia, neuroblastoma and some breast and lung cancers. Ewing sarcoma cell lines have also been reported to be sensitive to Chk kinase inhibitors (McCalla et al., Kinase Targets in Ewing's Sarcoma Cell Lines using RNAi-based & Investigational Agents Screening Approaches, Molecular Targets 2013, Boston, USA).

It has also been reported that mutations that reduce the activity of DNA repair pathways can result in synthetically lethal interactions with Chk1 inhibition. For example, mutations that disrupt the RAD50 complex and ATM signaling increase responsiveness to Chk1 inhibition [Al-Ahmadie et al. , Synthetic lethality in ATM- deficient RAD50-mutant tumors underlie outlier response to cancer therapy]. Likewise, deficiencies in the Fanconi anemia homologous DNA repair pathway lead to sensitivity to Chk1 inhibition [Chen et al., Chk1 inhibition asd a strategy for targeting fanconi anemia (FA) DNA repair pathway deficient tumors. Mol. Cancer 2009 8:24, Duan et al., Fanconi anemia repair pathway dysfunction, a potential therapeutic target in lung cancer. Frontiers in Oncology 20144:1]. Also, human cells that have loss of function in the Rad 17 gene product are sensitive to Chk1 suppression [Shen et al., Synthetic lethal interaction between tumor suppressor RAD17 and Chk1 kinase in human cancer cells. 2014 SACNAS National Conference Abstract].

Various attempts have been made to develop inhibitors of Chk-1 kinase. For example, WO 03/10444 and WO 2005/072733 (both in the name of Millennium) disclose aryl/heteroaryl urea compounds as Chk-1 kinase inhibitors. US2005/215556 (Abbott) discloses macrocyclic ureas as kinase inhibitors. WO 02/070494, WO2006014359 and WO2006021002 (all in the name of lcos) disclose aryl and heteroaryl ureas as Chk-1 inhibitors. WO/2011/141716 and WO/2013/072502 both disclose substituted pyrazinyl- phenyl ureas as Chk-1 kinase inhibitors. WO2005/009435 (Pfizer) and WO2010/077758 (Eli Lilly) disclose aminopyrazoles as Chk-1 kinase inhibitors.

WO2015/120390 discloses a class of substituted phenyl-pyrazolyl-amines as Chk-1 kinase inhibitors. One of the compounds disclosed is the compound 5-[[5-[4-(4-fluoro- 1-methyl-4-piperidyl)-2-methoxy-phenyl]-1H-pyrazol-3-yl]amino]pyrazine-2-carbonitrile, the synthesis of which is described in Example 64 and Synthetic Method L in WO2015/120390, as illustrated in Figure 46 of the present application.

The Chk-1 kinase inhibitor compound 5-[[5-[4-(4-fluoro-1-methyl-4-piperidyl)-2- methoxy-phenyl]-1H-pyrazol-3-yl]amino]pyrazine-2-carbonitrile is useful in the treatment of cancers as disclosed in WO2015/120390.

The Invention

The present invention provides improved processes for making the Chk-1 kinase inhibitor compound 5-[[5-[4-(4-fluoro-1-methyl-4-piperidyl)-2-methoxy-phenyl]-1H- pyrazol-3-yl]amino]pyrazine-2-carbonitrile (referred to herein also as the compound of formula (I), compound (13) or the Chk-1 inhibitor.

In one general aspect, the improved process of the present invention is represented by the sequence of reactions set out in Scheme 1 below (and in Figure 44).

Scheme 1

The synthetic route shown in Scheme 1 and Figure 44 has a number of advantages over the synthetic route described in WO2015/120390. For example, the route depicted in Scheme 1 is significantly shorter in terms of both longest linear sequence (6 vs 10 steps) and total steps (7 vs 10). The new route will also provide enhanced yields of product due to the shorter sequence as well as the avoidance of the low yielding cryogenic chemistry with n-butyl lithium described in the WO2015/120390 process. Finally, the improved process of the present invention uses readily available and stable building blocks and the subsequent intermediates derived from the process are readily isolable crystalline solids.

The improved synthetic route makes use of the same two final steps (removal of the Boc protecting group followed by reductive methylation) as the synthetic route described in WO 2015/120390 but the synthesis of the Boc-protected intermediate 11 in the present route differs from the synthesis of intermediate (11) in WO2015/120390. Accordingly, in one aspect (Embodiment 1.1) the invention provides a process for the preparation of a compound of the formula (11):

which process comprises the reaction of a compound of the formula (A) with a compound of the formula (B):

where LG¹ is a leaving group (for example chlorine, bromine, iodine or trifluoromethansulfonate), and BG is a B(OH)₂ or a boronate ester group such as

in the presence of a palladium catalyst and a base. The palladium catalyst typically comprises one or more phosphine ligands such as 2- dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (XPhos), triphenylphosphine, 2- dicyclohexylphosphino-2',6'-dimethoxybiphenyl and 4,5-Bis(diphenylphosphino)-9,9- dimethylxanthene (XantPhos), one preferred ligand being XPhos.

The base can be a carbonate base such as potassium carbonate or caesium carbonate and the reaction may be carried out in a polar solvent such as dimethyl formamide (DMF) or dioxane (e.g. aqueous dioxane). The reaction mixture is typically subjected to heating, for example to a temperature of about 100°C.

Thus, in particular embodiments, the invention provides:

1.2 A process according to Embodiment 1.1 wherein the leaving group LG¹ is bromine. 1.3 A process according to Embodiment 1.1 or Embodiment 1.2 wherein the group

BG is

or B(OH)₂.

1.4 A process according to Embodiment 1.3 wherein the group BG is

1.5 A process according to Embodiment 1.4 wherein the group BG is B(OH)₂.

1.6 A process according to any one of Embodiments 1.1 to 1.5 wherein the base is a carbonate base.

1.7 A process according to Embodiment 1.6 wherein the carbonate base is potassium carbonate.

1.8 A process according to any one of Embodiments 1.1 to 1.7 wherein the palladium catalyst comprises a phosphine ligand selected from 2- dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (XPhos), triphenylphosphine, 2- dicyclohexylphosphino-2',6'-dimethoxybiphenyl and 4,5-Bis(diphenylphosphino)-9,9- dimethylxanthene (XantPhos).

1.9 A process according to Embodiment 1.7 wherein the phosphine ligand is XPhos. 1.10 A process according to any one of Embodiments 1.1 to 1.7 wherein the palladium catalyst is selected from Pd₂dba₃/XPhos, Pd₂dba₃/SPhos and Pd G2 Pt-Bu₃.

1.11 A process according to Embodiment 1.10 wherein the palladium catalyst is Pd₂dba₃/XPhos. In a further aspect (Embodiment 2.1), the invention provides a process for the preparation of a compound of the formula (A):

which process comprises the reaction of a compound of the formula (C) with a compound of the formula (D):

where LG² is a leaving group (e.g. bromine or chlorine) in the presence of a base.

The polar solvent can be a non-aqueous aprotic solvent such as dimethylformamide.

The base is typically a carbonate base such as potassium carbonate.

The reaction is typically carried out with heating to a non-extreme temperature, for example a temperature in the range from 60 °C to °80 C, e.g. about °70 C.

Accordingly, in further embodiments, the invention provides: 2.2 A process according to Embodiment 2.1 wherein the leaving group LG² is chlorine.

2.3 A process according to Embodiment 2.1 or Embodiment 2.2 wherein the base is potassium carbonate. 2.4 A process according to any one of Embodiments 2.1 to 2.3 wherein the reaction is carried out in a non-aqueous aprotic solvent such as dimethylformamide.

2.5 A process according to any one of Embodiments 2.1 to 2.4 wherein the process is carried out at a temperature in the range from 60 °C to °80 C, e.g. about 70 °C. In a further aspect (Embodiment 3.1), the invention provides a process for the preparation of a compound of the formula (B):

which process comprises the reaction of a compound of the formula (E):

where LG³ is a group capable of being displaced by a boronate or boronic acid group by reaction in the presence of a palladium catalyst and a base with a compound of the formula R^xR^yB-BR^x'R^y' where Rx, Ry, Rx' and Ry' are all hydrogen, or R^xR^yB and BR^x'R^y' each form boronate ester groups such

The group LG³ can be a halogen such as chlorine or bromine, or a trifluoromethanesulfonate group, a particular example being chlorine.

The palladium catalyst can be, for example Pd₂dba₃/ XPhos, or Pd₂dba₃/SPhos where (Pd₂(dba)₃) is tris(dibenzylideneacetone)dipalladium(0), XPhos is 2-dicyclohexyl- phosphino-2',4',6'-triisopropylbiphenyl and SPhos is 2-dicyclohexylphosphino-2',6'- dimethoxybiphenyl, with one preferred ligand being XPhos.

The base can be a carbonate base such as an alkali metal carbonate (e.g. sodium or potassium carbonate), or an alkoxide or alkanoate base such as an alkaline metal alkoxide or alkanoate, examples being potassium acetate and sodium tert- butoxide and mixtures thereof. The reaction is typically carried out in a polar solvent, for example ethanol or dioxane, at a moderately elevated temperature, for example to a temperature in the range from about 65 °C to about 105 °C, e.g. about 100 °C.

Accordingly, in further embodiments, the invention provides:

3.2 A process according to Embodiment 3.1 wherein LG³ is selected from chlorine, bromine and a trifluoromethanesulfonate group.

3.3 A process according to Embodiment 3.2 wherein LG³ is chlorine.

3.4 A process according to any one of Embodiments 3.1 to 3.3 wherein the palladium catalyst is Pd₂dba₃/ XPhos (e.g. in a ratio of approximately 1:2).

3.5 A process according to any one of Embodiments 3.1 to 3.4 wherein the compound of the formula R^xR^yB-BR^x'R^y' is selected from bis(pinacolato)diboron and tetrahydroxydi boron.

3.6 A process according to Embodiment 3.5 wherein the compound of the formula R^xR^yB-BR^x'R^y' is bis(pinacolato)diboron.

3.7 A process according to Embodiment 3.5 wherein the compound of the formula R^xR^yB-BR^x'R^y' is tetrahydroxydiboron. 3.8 A process according to any one of Embodiments 3.1 to 3.7 wherein the base is an alkali metal acetate.

3.9 A process according to Embodiment 3.7 wherein the base is potassium acetate.

3.10 A process according to any one of Embodiments 3.1 to 3.9 wherein the reaction is carried out in a polar solvent such as ethanol or dioxane.

3.11 A process according to Embodiment 3.9 wherein the polar solvent is dioxane.

In a further aspect (Embodiment 4.1), the invention provides a process for the preparation of a compound of the formula (E) by the reaction of a compound of the formula (F):

with a fluorinating agent capable of replacing the hydroxyl group with fluorine.

In further embodiments, the invention provides:

4.2 A process according to Embodiment 4.1 wherein the fluorinating agent is selected from diethylaminosulfur trifluoride (DAST), diethylaminodifluorosulfinium tetrafluoroborate (XtalFluor-E) and morpholinodifluorosulfinium tetrafluoroborate (XtalFluor-M).

4.3 A process according to Embodiment 4.2 wherein the fluorinating agent is diethylaminodifluorosulfinium tetrafluoroborate.

The compound of formula (F) can be prepared by reacting a compound of the formula (5) with a compound of the formula (G) in the presence of magnesium under Grignard reaction conditions:

The reaction is usually carried out in an ether solvent, for example 2-methyl- tetrahydrofuran, typically in the presence of a catalytic amount of iodine.

In another general aspect, the invention provides a process comprising a sequence of process steps correspondingly generally to the process steps disclosed in synthetic route L in WO2015/120390 but with modifications to improve yields, product quality and suitability for scale-up. The modified process is represented by the sequence of reaction steps shown in Scheme 2 (and Figure 45).

Differences between the process of the invention as illustrated by Scheme 2 above and the process described in synthetic route L in WO2015/120390 include the following:

• The preparation of compound 17 in the process of Scheme 2 uses n-hexyl lithium in methyl-tetrahydrofuran whereas the corresponding step in Route L in WO201 5/120390 uses n-butyl lithium in THF. In the step of fluorinating compound 17 to give compound 18 in the process of Scheme 2, the DAST fluorinating agent (also used in the process of Scheme L in WO2015/120390) can be replaced by diethylaminodifluorosulfinium tetrafluoroborate (XtalFluor-E) or morpholinodifluorosulfinium tetrafluoroborate (XtalFluor-M)

The conversion of compound 18 to compound 19 in the process of Scheme 2 uses acetonitrile in THF in the presence of the base potassium tert- butoxide (KOf-Bu) whereas the corresponding step in Route L in WO2015/120390 involves a Grignard reaction and uses magnesium, and bromo-acetonitrile in THF.

The reductive methylation of the final intermediate 24 to give the Chk-1 inhibitor compound (compound 13) is carried out using sodium cyanoborohydride (NaCNBH₃) as the reducing agent whereas the corresponding step in the process in Scheme L in WO2015/120390 uses sodium triacetoxy borohydride (Na(OAc)₃BH) as the reducing agent.

Accordingly, in further embodiments, the invention provides:

5.1 A process for the preparation of a compound of the formula 17, which process comprises the reaction of a compound of the formula 15 with a compound of the formula 16 in the presence of hexyl lithium:

5.2 A process for the preparation of a compound of the formula 18, which process comprises the reaction of a compound of the formula 17:

with a fluorinating agent selected from diethylaminodifluorosulfinium tetrafluoroborate (XtalFluor-E) and morpholinodifluorosulfinium tetrafluoroborate (XtalFluor-M), preferably diethylaminodifluorosulfinium tetrafluoroborate (XtalFluor-E).

5.3 A process for the preparation of a compound of the formula 19, which process comprises the reaction of a compound of the formula 18 with acetonitrile in the presence of a strong base (e.g. potassium tert- butoxide):

5.4 A process for the preparation of a compound of the formula 13 by the reductive methylation of a compound of the formula 24 using formaldehyde (e.g. in the form of paraformaldehyde) and sodium cyanoborohydride:

5.5 A process for the preparation of a compound of the formula 13 by the reductive methylation of a compound of the formula 24 using paraformaldehyde and a reducing agent selected from sodium cyanoborohydride and sodium triacetoxyborohydride:

5.6 A process for the preparation of compound 13, which process comprises the process of any one of Embodiments 1.1 to 1.11 followed by removal of the protecting group PG and reductive methylation. 5.7 A process according to Embodiment 5.6 wherein the reductive methylation is as defined in either of Embodiments 5.4 and 5.5.

5.8 A process for the preparation of a compound of formula 24:

which process comprises the process of any one of Embodiments 1.1 to 1.11 followed by removal of the protecting group PG, for example using an acid such as hydrochloric acid.

In a further aspect, the invention provides novel intermediates for use in the processes of the invention.

Accordingly, in further embodiments, the invention provides: 6.1 A compound of the formula (A):

wherein LG¹ is as defined in any one of the preceding embodiments. 6.2 A compound of the formula (B):

wherein BG is as defined in any one of the preceding embodiments.

Further aspects and embodiments of the invention will be apparent from the Examples provided below.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a ¹H NMR spectrum of Compound 3 formed by the reaction of Compound 1 with Compound 2 as shown in Scheme 1 and described in Example 1A.

Figure 2 is a ¹H NMR spectrum of Compound 6 formed by the reaction of Compound 4 with Compound 5 as shown in Scheme 1 and described in Example 1B.

Figure 3 is a ¹H NMR spectrum of Compound 7 formed by the reaction of Compound 6 with the fluorinating agent XtalFluor-E as shown in Scheme 1 and described in Example 1C.

Figure 4 is is a mass spectrum of Compound 7 showing an [M+Na] peak at 366.1 (Expected [M+Na]: 366.12) Figure 5 is is a mass spectrum of Compound 8a formed by the reaction of Compound 7 with B₂(OH)₄ in the presence of a palladium catalyst as described in Example 1D.

Figure 6 is a ¹H NMR spectrum of Compound 8a.

Figure 7 is the mass spectrum of Compound 11 formed by the reaction of Compound 3 and Compound 8 as shown in Scheme 1 and described in Example 1F.

Figure 8 is the in-process control (IPC) profile obtained by HPLC of the reaction of compound 1 with sodium methoxide to give 4-bromo-2-methoxy-benzaldehyde as described in Example 2A.

Figure 9 is the HPLC profile of the product of Example 2A after purification.

Figure 10 is the ¹H NMR spectrum of the product of Example 2A.

Figure 11 is the ¹³C NMR spectrum of the product of Example 2A.

Figure 12 is the IPC of the reaction described in Example 2B (preparation of compound 15)

Figure 13 is the HPLC profile of the product of the reaction described in Example 2B after workup and solvent removal.

Figure 14 is the HPLC profile obtained using the process for preparing compound 15 described in WO2015/120390.

Figure 15 is the ¹H NMR spectrum for the compound 15.

Figure 16 is the ¹³C NMR spectrum for the compound 15.

Figure 17 is an HPLC trace of the reaction mixture obtained after 1.5 hours and before ketone addition in the process step described in Example 2C.

Figure 18 is the HPLC trace of the reaction mixture obtained after warming to room temperature for 1 hour after ketone addition in the process step described in Example 2C.

Figure 19 is the HPLC trace after work up and solvent removal in the process step described in Example 2C.

Figure 20 is the ¹H NMR spectrum for compound 17.

Figure 21 is the ¹³C NMR spectrum for compound 17.

Figure 22 is the IPC for the fluorination of compound 17 to give compound 18 as described in Exampe 2D. Figure 23 is the IPC obtained 1 hour after p-TSA addition in the process of Example 2D.

Figure 24 is the IPC obtained for the final purified solid in the process of Example 2D

Figure 25 is the ¹H NMR spectrum for the product of the process of Example 2D (compound 18).

Figure 26 is the ¹³C NMR spectrum for the product of the process of Example 2D (compound 18).

Figure 27 is the HPLC profile for the product of Example 2E (Compound 19)

Figure 28 is the 1H NMR spectrum for compound 19.

Figure 29 is the HPLC profile for the product of Example 2F (compound 20)

Figure 30 is the ¹H NMR spectrum for compound 20.

Figure 31 is the HPLC profile for the product of Example 2G (compound 21)

Figure 32 is the HPLC profile compound 21 when produced by the process described in WO2015/120390.

Figure 33 is the ¹H NMR spectrum of compound 21.

Figure 34 is the ¹H NMR spectrum of compound 21 with the addition of D₂0.

Figure 35 is an HPLC profile of the reaction product of Example 2H.

Figure 36 is is the ¹H NMR spectrum of compound 23 formed by the process of Example 2H.

Figure 37 is the ¹H NMR spectrum of compound 23 with the addition of D₂0.

Figure 38 is the HPLC profile of the product of the reaction described in Example 3.

Figure 39 is ¹H NMR spectrum of compound 24 formed by the process of Example 3.

Figure 40 is the HPLC profile obtained in Run 2 in the process of Example 3.

Figure 41 is the HPLC profile of the product of the reaction described in Example 4 (preparation of compound 13 - the Chk-1 inhibitor).

Figure 42 is the ¹H NMR spectrum for the Chk-1 inhibitor compound.

Figure 43 is the HPLC profile of the product obtained from Run 2 of the process described in Example 4.

Figure 44 is reaction Scheme 1 showing the novel reaction of the first aspect of the invention. Figure 45 is a reaction scheme showing an improved version of the synthetic route described in WO2015/120390.

Figure 46 is shows Synthetic Method L from WO2015/120390.

Figure 47 is the mass spectrum of compound 8. Figure 48 is the ¹H NMR specrum of compound 8.

EXAMPLES

The invention will now be illustrated, but not limited, by reference to the specific embodiments described in the following examples.

In the examples, the following abbreviations are used. ACN acetonitrile AC₂0 acetic anhydride AcOH acetic acid aq aqueous Boc tert-butoxycarbonyl DAST diethylaminosulfur trifluoride DCM dichloromethane DIPEA diisopropylethylamine DMF dimethylformamide DMSO dimethylsulfoxide NEt₃ triethylamine Et₂0 diethyl ether EtOAc ethyl acetate EtOH ethanol HPLC high performance liquid chromatography IPC In-process control iPrOAc isopropyl acetate KOAc potassium acetate LCMS liquid chromatography-mass spectrometry LiHMDS lithium bis(trimethylsilyl)amide MeCN acetonitrile MeOH methanol 2-MeTHF 2-methyl-tetrahydrofuran MTBE methyl tert-butyl ether NaBH(OAc)₃ sodium triacetoxyborohydride n-HexLi n-hexyl lithium NaOtBu sodium tertiary butoxide NMP N-methyl-2-pyrrolidone NMR nuclear magnetic resonance

PdCI₂(dppf).DCM [1,1'-Bis(diphenylphosphino)ferrocene]dichloropalladium(ll), complex with dichloromethane

PdG2 Pt-Bu₃ chloro[(tri-tert-butylphosphine)-2-(2-aminobiphenyl)] palladium(ll) pTSA para-toluene sulfonic acid RBF round bottom flask rotovap rotary evaporator SM starting material

SPhos 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl t-BuOK sodium tertiary butoxide THF tetrahydrofuran TFA trifluoroacetic acid XPhos 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl

Proton magnetic resonance (¹H NMR) spectra were recorded on a Bruker 400 instrument operating at 400 MHz, in the solvent indicated at 27°C, unless otherwise stated and are reported as follows: chemical shift d/ppm (multiplicity where s=singlet, d=doublet, dd, double doublet, t=triplet, q=quartet, m=multiplet, br=broad, number of protons).

Liquid chromatography and mass spectroscopy analyses were carried out using the system and operating conditions set out below. Where atoms with different isotopes are present and a single mass quoted, the mass quoted for the compound is the monoisotopic mass (i.e. ³⁵CI; ⁷⁹Br etc.)

HPLC Method Details:

Unless otherwise noted, the processes described below used the following HPLC method to track both reaction conversion and intermediate purity. Alternative methods exist for compounds 12 and CHK1 Inhibitor compound 13 and those method details are also listed below.

Procedure: 1.1. Preparation of Mobile Phase

1.1.1. Mobile Phase A (10mM Ammonium Acetate in Water)

To 1L of water, add 0.77g of Ammonium Acetate, mix well. Different volumes may be used provided the ratio of Ammonium Acetate to water remains unchanged. 1.1.2. Mobile Phase B - 100% ACN

1.2. Blank - 50:50 MPA/MPB

1.3. Sample Diluent: 50:50 MPA/MPB

1.4. Retention Time Marker Preparation - Weigh out approximately 10mg of retention time marker into a flask. Dissolve with 10mL of sample diluent. 1.5. Purity Sample Preparation - Weigh out approximately 10mg of material into a flask. Dissolve with 10mL of sample diluent.

1.6. Reaction Sample Preparation - Dilute 10uL of reaction solution into 0.5mL of diluent, and filter if necessary.

Chromatographic Conditions

Procedure:

2.1. Preparation of Mobile Phase

2.1.1. Mobile Phase A (10mM Ammonium Acetate in Water)

To 1L of water, add 0.77g of ammonium acetate, mix well. Different volumes may be used provided the ratio of ammonium acetate to water remains unchanged.

2.1.2. Mobile Phase B - 3:1 ACN/MeOH

Combine 750mL ACN with 250mL MeOH, mix well. Different volumes may be used provided the ratio of ACN to MeOH remains unchanged.

2.2. Blank - 50:50 MeOH/ACN 2.3. Sample Diluent: 50:50 MeOH/ACN

2.4. Retention Time Marker Preparation - Weigh out approximately 10mg of retention time marker into a flask. Dissolve with 10mL of sample diluent.

2.5. Purity Sample Preparation - Weigh out approximately 10mg of material into a flask. Dissolve with 10mL of sample diluent. 2.6. Reaction Sample Preparation - Dilute 10uL of reaction solution into 0.5mL of diluent, and filter if necessary.

Chromatographic Conditions

HPLC Method for Purity Determination of Intermediates 12 and CHK1 Inhibitor

1.0 PREPARATION OF MOBILE PHASE

1.1.1. Mobile Phase A (0.1% TFA in Water) To 1 L of water, add 1 mL of TFA, mix well. Different volumes may be used provided the ratio of TFA to water remains unchanged.

1.1.2. Mobile Phase B (3:1 ACN/MeOH)

To 750 mL of AON, add 250 mL of MeOH, mix well. Different volumes may be used provided the ratio of MeOH to AON remains unchanged. 1.2. Blank - 100% MeOH

1.3. Sample Diluent: 100% MeOH 1.4. Retention Time Marker Preparation - Weigh out approximately 5mg of retention time marker into a vial. Dissolve with 20mL of sample diluent.

1.5. Sample Preparation - Weigh out material and dilute with a sufficient quantity of Diluent to result in a 0.25 mg/mL sample solution. Chromatographic Conditions

Example 1

1 A. 5-((5-bromo-1H-pyrazol-3-yl)amino)pyrazine-2-carbonitrile

Aryl bromide (1.0 g, 1 equiv), aryl chloride (1.29 g, 1.5 equiv), K₂CO₃ (1.719 g, 2.0 equiv) and a magnetic stir bar were added to a 20 mL vial with 5 mL (5.0 V) DMF and heated to 70 °C. The reaction was stirred for 20 hours. Upon completion monitored by HPLC, the reaction was cooled to rt, precipitated with 20 mL (20 V) water and stirred for 30 minutes. The slurry was filtered and washed with 5 mL (5 V) water then 10 mL (10 V) /- PrOAc. HPLC analysis of the product showed a major peak at 5.326.

Mass Spectrum: Expected [M+H]: 264.98 + 266.98 (⁸¹Br) - Observed [M+H]: 265.0 and 267.0 (⁸¹ Br) The NMR spectrum of the product is shown in Figure 1.

1 B. tert-Butyl 4-(4-chloro-3-methoxyphenyl)-4-hvdroxypiperidine-1-carboxylate

Magnesium (1.10 g, 1.0 equiv), iodine (0.06 g, 0.005 equiv), and a magnetic stir bar were added to a 300 mL round bottom flask and put under a nitrogen atmosphere. 60 mL (6.0 V) of 2-MeTHF was added to the flask and heated to 50 °C. Aryl bromide was slowly added over 15 minutes as a 2.0 V 2-MeTHF solution. The reaction was stirred at 50 °C for 1 hour. Upon completion of the metalation, the reaction was cooled to 0 °C and the ketone was added to the reaction as a 2.0 V solution in 2-MeTHF. The reaction was stirred for 30 minutes then warmed to room temperature and stirred for 1 hour. The reaction was quenched with a saturated ammonium chloride solution. The organic layer was washed with water. The solvent was removed by rotovap to give a yellow oil. The oil was dissolved in 100 mL (10.0 V) heptane and heated to reflux. The solution was then allowed to cool to rt to precipitate a white solid. The solid was filtered and washed with 50 mL (5.0 V) of heptane to give a white solid (9.57 g). The ¹H NMR of the product is shown in Figure 2.

1C. tert-Butyl 4-(4-chloro-3-methoxyphenyl)-4-fluoropiperidine-1-carboxylate

Alcohol 6 (8.54 g) was added to a 200 mL round bottom flask with a magnetic stir bar and dissolved in 40 ml (5.0 V) anhydrous DCM. NEt₃-3HF (4.88 ml, 1.2 equiv) was added to the solution. The vessel was cooled to 0 °C. Xtal-Fluor-E (6.87 g, 1.2 equiv) was slowly added. The reaction was stirred for 1 hour then allowed to warm to room temperature and stirred for 1 hour. The reaction was quenched with saturated sodium bicarbonate. The organic layer was removed, and the DCM was removed by rotovap to give an orange oil. The product was purified by column chromatography (4:1 heptane: EtOAc) to give a white solid after the solvent is removed (5.66 g, 66% yield).

HPLC analysis showed the major peak at 12.142.

The ¹H NMR is shown in Figure 3 and the mass spectrum is shown in Figure 4 (Expected [M+Na]: 366.12 - Observed [M+Na]: 366.1).

1D. tert- Butyl 4-fluoro-4-(3-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)phenyl)piperidine-1-carboxylate (8)

Pd₂dba₃(7 mg, 0.01 equiv), KOAc (0.2 g, 2.5 equiv), XPhos (16 mg, 0.04 equiv), B₂Pin₂ (0.517 g, 2.5 equiv), and a magnetic stir bar were added to a 20 ml vial and put under N₂. A 10.0 V dioxane stock solution (2.8 ml) of arylchloride 7 (0.28 g, 1 equiv) was made under N₂ and added to the vial. The reaction mixture was heated to 100 °C and stirred for 4 hours. Upon completion, the reaction mixture was diluted with EtOAc washed with water, dried over sodium sulfate, filtered and the solvent evaporated to give a yellow oil. The product was purified via flash chromatography (0 -> 20 -> 100 Heptane: EtOAc) to give 8 as a yellow oil (0.195 g) (55%). Mass spectrum: Expected [M+Na]: 458.25, Observed [M+Na]: 458.3

The mass spectrum is shown in Figure 47.

The ¹H NMR spectrum is shown in Figure 48.

Further reactions were carried out using Pd₂dba₃/XPhos (1:2), Pd₂dba₃/SPhos (1:2), PdG2 Pt-Bu3 and Pd(dppf)Cl₂ as the catalysts. In each case, the catalyst was used in an amount of 5 mol%, and KOAc was present in an amount corresponding to 3 equivalents and bis(pinacolato)diboron was present in an amount corresponding to 3 equivalents. The reactions were carried out in dioxane and the reaction mixtures were heated for 22 hours.

The yields and amounts of residual starting materia in each of these reactions are set out in the table below.

1E. (4-(1-(tert-butoxycarbonyl)-4-fluoropiperidin-4-yl)-2-methoxyphenyl)boronic acid

(8a)

XPhos, Pd XPhos G2, KOAc, B₂(OH)₄, and a magnetic stir bar were added to a 4 ml vial and put under N₂. Compound 7 (1eq) was added to the reaction in a 10V solution in EtOH and heated to 70 °C overnight (white slurry to reddish to brown slurry).

The reaction was cooled to rt and 15V of water was added to precipitate brown and black solids. The reaction was purified by column chromatography to give a white solid.

The mass spectrum of the product is shown in Figure 5 (Expected [M+Na]: 458.25 - Observed [M+Na]: 458.3) and the ¹H NMR spectrum is shown in Figure 6.

1F. tert-butyl 4-(4-(3-((5-cvanopyrazin-2-yl)amino)-1H-pyrazol-5-yl)-3-methoxyphenyl)-

4-fluoropiperidine-1-carboxylate

Pd, ligand, base, bromide, and a magnetic stir bar were added to a 4 ml vial and put under N₂. A stock solution of boron reagent (20.0 V) was made under N₂. The boron reagent was added to the reaction vessel followed by 10 V of H₂O and heated to 100 °C. The reactions were washed with water and diluted with EtOAc and analyzed by HPLC. The HPLC trace showed a major peak at 10.801.

The mass spectrum of the product is shown in Figure 7 (Expected [M+H]: 494.22 - Observed [M+H]: 494.3). Example 2

Improvements in the Synthetic Route Described in Example 64 and Synthetic Method

L in WO2015/120390

The conditions used to prepare the CHK1 inhibitor (Compound 13) were optimized and made amenable to scale-up. Several steps were optimized to improve yield, quality, and to ensure that the operations for each step are suitable to large scale implementation. The improved route is shown in Figure 45.

In this section, references to the “original process” or “original procedure” or “previous process” and like terms refer to the process described in Synthetic Method L as disclosed in WO2015/120390. 2A. 4-Bromo-2-methoxy-benzaldehyde

Aldehyde 13 (500 g) was charged to a 12 L reactor. 6.6 V MeOH (3.3 L) was added to the reactor and cooled to 0 - 5 °C. NaOMe solution (25 wt% in MeOH, 1597 g, 1690 ml) was added (exotherm 0 -> 20 °C). The reaction was agitated under reflux (65 - 70 °C) for 1 hour (light yellow slurry). The reaction was cooled to 0 - 5 °C and 15.0V (7.5 L) of water was added over 2 minutes (exotherm 0 -> 20 °C). A white slurry resulted. The reactor was cooled to 10 °C. The solid was filtered and washed with 5.0 V water (2.5 L) to give a white solid (450 g, 83% yield). The solid was dissolved in 7.0 V of toluene (3.5 L) and washed with 5.0 V water (2.5 L) (to remove trace basic salts which interfere with the next reaction). The toluene solution of 14 was used as is in the next step.

The IPC at 2 hours is shown in Figure 8 and the data obtained are shown in the table below.

The HPLC trace after purification is shown in Figure 9 and the data for the HPLC trace are as set out in the table below.

The ¹H NMR spectrum of the product is shown in Figure 10 and the ¹³C NMR spectrum in Figure 11. The ¹H and ¹³C NMR data are as follows:

¹H NMR (400 MHz, Chloroform-d) δ 10.38 (d, J = 0.8 Hz, 1H), 7.67 (d, J = 8.2 Hz, 1H), 7.17 (ddd, J= 8.2, 1.7, 0.8 Hz, 1H), 7.15 (d, J= 1.7 Hz, 1H), 3.92 (s, 3H).

¹³C NMR (101 MHz, Chloroform-d) δ 188.87, 162.05, 130.68, 129.81, 124.32, 123.83, 115.50, 56.13. 2B. 2-(4-bromo-2-methoxyphenyl)-1,3-dioxolane (15)

A 7.0 V toluene (3200 ml) solution of 14 from the previous step was transferred to a 12 L 4-neck RBF equipped with mechanical stirring and a Dean-Stark apparatus. pTSA- H₂O (38.1 g) and ethylene glycol (672 ml) were added to the flask. The solution was heated to reflux in a heating mantle over 2 hours while collecting water plus ethylene glycol in a Dean-Stark apparatus (internal temperature 110-115 °C; internal temperature increased throughout reaction). To sample the reaction for HPLC, one drop of NEt was added to an HPLC vial with 2 ml MeCN and three drops of the reaction solution were added to the basic MeCN. This neutralization was critical to prevent deprotection under the HPLC conditions.

Upon completion (<2% SM), the reaction was quenched with 0.5 V NEt (230 ml) (exotherm 21 °C to 23 °C). The reaction was transferred to an 11 L reactor and washed with 5.0 V of saturated sodium bicarbonate (2300 ml) (exotherm 23 °C to 26 °C). The aqueous layer was removed, and the organic layer was washed with 5.0 V water (2300 ml). The solvent was removed by rotovap. 4.0 V of 2-MeTHF (2 L) was added and the solvent was removed again to give 15 as an orange to red oil (523 g crude, 483.3 g compound 15 by qNMR (trimethoxybenzene internal standard), 93% yield)

The IPC after two hours (old HPLC method) is shown in Figure 12 and the data are set out in the table below.

The HPLC trace after workup and solvent removal (new HPLC method) is shown in Figure 13 and the data are set out in the table below.

For comparison purposes, the HPLC purity obtained by the previous process (the corresponding process step described in Synthetic Method L in WO2015/120390) is shown in Figure 14. The ¹H and ¹³C NMR data for the compound 15 are shown in Figures 15 and 16 and the data are set out below.

¹H NMR (400 MHz, Chloroform-d) δ 10.38 (d, J = 0.8 Hz, 1H), 7.67 (d, J = 8.2 Hz, 1 H), 7.17 (ddd, J= 8.2, 1.7, 0.8 Hz, 1H), 7.15 (d, J= 1.7 Hz, 1H), 3.92 (s, 3H).

¹³C NMR (101 MHz, Chloroform-d) δ 158.34, 128.15, 125.17, 123.76, 123.57, 114.39, 98.95, 65.34, 55.97.

2C. Tert-butyl 4-(4-(1,3-dioxolan-2-yl)-3-methoxyphenyl)-4-hydroxypiperidine-1- carboxylate (17)

Aryl bromide 15 (460 g) was dissolved in 4.0 V 2-MeTHF (1850 ml) and added to a 12L 4-neck RBF with a mechanical stirrer. The vessel was put under N₂ and cooled to -78 °C. n-HexLi in hexane (2.3 M) (925 ml) was added dropwise to the reaction via an addition funnel over 30 minutes keeping the reaction below -60 °C (some precipitate forms on the side then a brown suspension is formed by the end of the addition) and stirred for 1 additional hour at -78 °C. N-Boc piperidone 16 (441 g) was dissolved in 2.0 V 2-MeTHF (920 ml) and filtered to remove insoluble solids that clog addition funnel. The solution was added dropwise to the reaction over 1 hour via an addition funnel keeping the temperature below -60 °C (turns to a brown solution). The reaction was warmed to rt over 1 hour and stirred for 1 hour at rt. 5.0 V of saturated NH4CI (2300 ml) was slowly added to the reaction over 1 minute (marginal exotherm). The aqueous layer was removed, and the organic layer was washed with 5.0 V water (2300 ml). The solvent was removed by rotovap and chased with 2 L of 2-MeTHF. 2L of DCM was added and removed by rotovap to give 17 as an orange oil (991 g crude, 302.7 g, NMR, 45% yield).

*For HPLC IPC analysis quench 5 drops of reaction solution into 1 ml water then add 1 ml MeCN (important to add MeCN second or IPC will not be accurate)

The HPLC trace obtained after 1.5 hours and before ketone addition (old column) is shown in Figure 17 and the HPLC data are set out below.

The HPLC trace obtained after warming to room temperature for 1 hour after ketone addition (old column) is shown in Figure 18 and the data from the HPLC are shown below.

The HPLC trace after work up and solvent removal (fresh column) is shown in Figure

19 and the HPLC data are set out below.

The ¹H and ¹³C NMR spectra for compound 17 are shown in Figures 20 and 21 and the NMR data are set out below.

¹H NMR (400 MHz, Chloroform-d) δ 7.42 (d, J= 8.0 Hz, 1H), 7.02 (d, J= 1.7 Hz, 1H), 6.96 (dd, J= 8.0, 1.6 Hz, 1H), 6.06 (s, 1H), 4.08-3.93 (m, 4H), 3.90 (d, J= 13.7 Hz,

1 H), 3.80 (s, 3H), 3.14 (t, J= 12.8 Hz, 2H), 2.71 (s, 1H), 1.89 (td, J= 13.2, 4.7 Hz, 2H), 1.72-1.53 (m, 2H), 1.43 (s, 9H). ¹³C NMR (101 MHz, Chloroform-d) δ 157.74, 154.94, 151.21, 126.70, 124.34, 116.50, 107.41 , 99.15, 79.60, 71.36, 65.27, 55.62, 39.77, 37.98, 28.47.

2D. tert-butyl 4-fluoro-4-(4-formyl-3-methoxyphenyl)piperidine-1-carboxylate (18)

The alcohol 17 (231 g crude from previous step, 66 g) was added to a 1 L 3-neck RBF equipped with a magnetic stir bar. 5.0 V anhydrous DCM (330 ml) was added to the flask and put under N₂. The solution was cooled to 0 °C. NEt₃-3HF (34 ml) was added in one portion to the reaction mixture. XtalFluor-E (47.8 g) was added slowly over 15 minutes (exotherm 0 °C -> 10 °C). The reaction was stirred at 0 °C for 1 hour. The reaction was warmed to rt and stirred for 1 additional hour at rt (turns from red orange to dark). The reaction was then slowly added to a 15.0 V solution saturated NaHCO₃ solution (1000 ml) over 10 minutes at rt with significant gas evolution. The organic layer was separated, and the solvent was removed by rotovap to give an orange oil. The crude oil was dissolved in 5.0 V 2-MeTHF (330 ml) and added to a 1000 ml RBF with a magnetic stir bar. 2 V H₂O (125 ml) was added to the vessel along with p-TSA- H₂O (3 g). The reaction was stirred for 1 hour at rt, then filtered.

5.0 V (330 ml) of brine was added to the solution. The aqueous layer was removed, and the solvent was removed by rotovap to give a dark oil. The dark oil was dissolved in 2 V /-PrOH (125 ml). A solution of 50 wt%/wt% NaHSO₃ with respect to SM (33 g) in 1.0 V water (66 ml) was added to the solution. The mixture was stirred for 15 minutes with significant solid precipitation. The solid was filtered and washed with 5 V MTBE (330 ml) to give a white solid. The wet cake was added to a 1000 ml Erlenmeyer flask. 5 V EtOAc (330 ml) and 5 V 5% Na₂CO₃ (330 ml) was added to the white solid. This mixture was stirred for 1 hour as the solids dissolved. The aqueous layer was removed. The organic layer was washed with 5.0 V water (this separation is a little slow ~30 min to settle). The solvent was removed by rotovap to give a yellow solid or oil (60 g). 10V heptane (660 ml) was added to precipitate a solid and stirred for 1 hour. The solid was filtered and washed with 5 V heptane (330 ml) to isolate the product 18 as a pale yellow to white solid (29.03 g, 50% yield)

Compared to the previous process, this updated process with a more stable fluorinating agent provides higher yield (50 vs 40%) and also similar overall quality (~96.8% Purity) while the filtration is significantly faster with the updated solvent system isopropanol.

After warming for 1 hour, the IPC was as shown in Figure 22 and the IPC data are as shown below.

The IPC 1 hour after p-TSA addition is shown in Figure 23 and the IPC data are shown below.

The IPC data for the final purified solid are shown in Figure 24 and the table of data below.

The ¹H and ¹³C NMR spectra are shown in Figures 25 and 26 and the NMR data are set out below.

¹H NMR (400 MHz, Chloroform-d) δ 10.42 (s, 1H), 7.80 (dd, J = 8.1, 1.0 Hz, 1H), 7.04 (d, J = 1.5 Hz, 1 H), 6.92 (dd, J = 8.2, 1.5 Hz, 1 H), 4.21 - 4.00 (m, 2H), 3.94 (s, 3H), 3.24 - 2.90 (m, 2H), 2.11 - 1.82 (m, 4H), 1.48 (s, 9H).

¹³C NMR (101 MHz, Chloroform-d) δ 189.33, 162.05, 154.80, 152.36 (d, J= 20.8 Hz), 128.90, 124.18, 116.31 (d, J = 8.0 Hz), 107.35 (d, J= 12.5 Hz), 95.45, 93.68, 80.02, 55.83, 39.65, 36.38 (d, J= 22.8 Hz), 28.54.

2E. tert-Butyl 4-(4-(2-cyano-1-hydroxyethyl)-3-methoxyphenyl)-4-fluoropiperidine-1- carboxylate (19)

t-BuOK (1.00g, Aldrich 156671, Lot#MKCF5835) was added to Easymax 100 mL reactor which was purged with nitrogen. THF 15 mL (anhydrous, Sigma Aldrich 401757 lot # SHBK1580) was then added. The jacket temperature was lowered to - 25°C and then MeCN (1.54 mL, anhydrous, Sigma Aldrich 271004 lot # SHBK3750) was added slowly over ~2 min and kept stirring for 30 min. The jacket temperature was lowered to -30 °C. Compound 18 (2.00 g, Lot# BJG-174) dissolved in 5 mL THF was slowly charged to reaction mixture over 5 min and stir for 4 h with jacket temperature at -30 °C. IPC by HPLC showed the completion of the reaction. 20 mL of IPAc and 20 mL water were added and stirred for 5 min. The mixture was then transferred to a separatory funnel and allowed for phase separation. The organic phase was washed with water 20mL x 2. The pH of the second aqueous wash is 6. The organic phase is dried over Na₂SO₄ and filtered and the Na₂SO₄ was washed with THF (5 mL). The organic solution was concentrated by rotatory evaporator about 8m L. The product crystallized. Heptane (6 mL) was added and kept at 4 °C for 2 hours. The white solid was collected by vacuum filtration. The solid was washed with heptane (2 mL × 2) and dried in vacuum oven for 2 hours at 21 °C and 2.038 g (yield 90.9%) of 19 as a white solid was obtained with the HPLC purity of 97%.

The workup and isolation of this process was simplified from the original procedure and this new process provides comparable yield and purity to the original process. The HPLC trace for the product is shown in Figure 27 and the data are set out below.

MS (m/z) [M + Na]+ found 401.2

The 1H NMR spectrum is shown in Figure 28 and the NMR data are set out below.

¹H NMR (400 MHz, Chloroform-c/) d 7.47 (d), 7.03 (m), 5.83 (d), 5.08 (dt), 3.99 (d), 3.83 (s), 2.78 (m), 1.99 (m), 1.43 (s)

2F. tert-Butyl 4-(4-(2-cyanoacetyl)-3-methoxyphenyl)-4-fluoropiperidine-1-carboxylate

(20)

Alcohol 19 (14.00 g) was charged to 500 mL reactor with overhead stirrer. DCM 280 mL (anhydrous, contains 40-150 ppm amylene as stabilizer, Sigma Aldrich 270997 lot #SHBK5890) was then charged. It formed a clear solution. The flask was purged with nitrogen and cooled to -12.5 °C (jacket temperature, reaction T was -9.0 °C) while stirring at 200r/m. Dess Martin (24.08 g, 1.5 eq., Sigma Aldrich 274623 Lot MKCG4097) was then added in one portion. The reaction mixture temperature slightly increased to -8.7 °C. And then slowly down to -9.9 °C. 6 hours later, IPC by HPLC showed 49% conversion.7.84 g of Dess Martin and water 0.40 mL were then added and stirred at -9.8 °C overnight at stirring rate 200r/m. IPC by HPLC showed the reaction was 99.3% conversion. The reaction was quenched with a solution of KHCO₃ (8.4 g)/Na₂S₂0₃ (42 g, Aldrich 217263 Lot#mkch9633) /water (140 mL) while stirring rate at 500r/m. The temperature raised to ~12 °C. The jacket temperature was then set to 15 °C. Stirring was stopped and allowed for phase splitting. iPrOAc (200 mL) was added and the organic layer was separated. The organic phase was washed with a solution of KHCO₃ (7.50 g)/ Na₂S₂O₃ (37.5 g) /water (125 mL) again. The organic phase was dried over Na₂SO₄ and concentrated (30 °C/75torr) and left in 4 °C refrigerator overnight. The product was collected by vacuum filtration and was washed with iPrOAc 20 mL heptane 10 mL. 11.22 g (80.6% yield) (HPLC purity 94.1% @ 235nm) of ketone 20 was obtained after drying in vacuum oven at 28 °C overnight.

The workup and isolation of this process was simplified from the original procedure and this new process provides comparable yield and purity to the original process. A summary of changes is shown in the table below.

The HPLC trace for the product is shown in Figure 29 and the data are set out below.

The ¹H NMR spectrum for compound 20 is shown in Figure 30 and the NMR data are set out below.

¹H NMR (400 MHz, Chloroform-d) δ 7.73 (d), 7.34 (s), 7.16 (m), 5.75 (s), 4.86 (m), 4.49 (s), 3.99 (m), 3.32 (s), 3.05 (s), 1.99 (m), 1.43 (s), 1.17 (d)

2G. tert-Butyl 4-(4-(3-amino-1 H-pyrazol-5-yl)-3-methoxyphenyl)-4-fluoropiperidine-1- carboxylate (21)

Compound 20 (18.8 g) was added to a round bottom flask. EtOH (150 mL , Sigma Aldrich 459844 lot #SHBK8246) and NH₂NH₂CH₃-HCO₂H (5.75g, 1.25 eq. Aldrich 259748 lot#MKCH3748) were added and heated to 65 °C for 4 hours. IPC by HPLC showed the reaction was 99.1% completion. The reaction mixture was filtered to remove insoluble impurities. The filtrate was concentrated to 1/3 of the volume and then was added i-PrOAc (300 mL ). It was washed with 1) water 200 mL ; 2) KHCO₃ (7 g)/water 200 mL ; 3) water 200 mL ; dried over Na₂SO₄ and filtered. The product was in organic layer. Organic layer was concentrated to a residue using a rotary evaporator with a 32 °C water bath. During the concentration, solid came out of solution. MTBE 100 mL was then added and kept at room temperature for 30 minutes. The product was collected by vacuum filtration. The solid was washed with 10 mL of MTBE and dried in vacuum oven at room temperature overnight. 16.15 g (87.0% yield) of product 21 was obtained as an off white solid. MS(m/z) [M + H]+ found 391.2

Significant improvements were made to this process compared to the original procedure. The hydrazine source was optimized which led to a significantly shorter rxn time (3 vs 24h) and higher yield (87% vs 79%) and higher purity (97.9% vs 84.4%).

The HPLC trade for the reaction product is shown in Figure 31 and the data are set out below.

For comparison purposes, the HPLC profile for the previous process is shown in Figure 32.

The ¹H NMR spectrum of compound 21 and the ¹H NMR spectrum with D₂0 are shown in Figures 33 and 34 respectively, and the NMR data are set out below.

¹H NMR (400 MHz, Chloroform-d) δ 11.57 (s), 7.62 (s), 7.05 (m), 5.88 (s), 4.79 (m), 3.89 (s), 3.33 (s), 3.06 (s), 2.00 (m), 1.44 (s).

With D₂0 - ¹H NMR (400 MHz, Chloroform-d) δ 7.61 (s), 7.03 (m), 5.89 (s), 3.99 (s), 3.87 (s), 3.05 (s), 1.97 (m), 1.41 (s).

2H. tert-Butyl 4-(4-(3-((5-cyanopyrazin-2-yl)amino)-1H-pyrazol-5-yl)-3-methoxyphenyl)-

4-fluoropiperidine-1-carboxylate

Compound 21 (15.0 g), NMP (30 mL , Sigma-Aldrich 328634 lot SHBJ0436 anhydrous) were added to a round bottom flask. 2-Chloro-5-cyanopyrazine 22 (5.63 g, 1.05 eq., Chem-lmpex cat#26290 lot#001554-0158299-13111) and DIPEA (7.70 mL , 1.15 eq., Sigma Aldrich 387649 Lot#SHBJ2645) was added. The reaction was heated to 50 °C for 22 hours. IPC by HPLC showed 95.4% conversion with 4.2 % isomer. 2 mL DI PEA (0.30 eq) was added and heated to 60 °C overnight. IPC by HPLC showed the conversion was 99.2% conversion with 4.2% isomer. To the reaction was added iPrOAc (150 mL) and water (200 ml) and was left at room temperature for 2 hours. The solid product was collected by vacuum filtration and washed with water (30 mL) and iPrOAc (30 mL). It was dried in vacuum oven at 60 °C overnight. 17.95g (yield: 94.69%) of product 23 was obtained with the HPLC purity 98.6%. The HPLC profle is shown in Figure 35 and the HPLC data are set out below.

Mass spectrum: MS(m/z) [M + H]+ found 494.2

The ¹H NMR and ¹H NMR with D₂0 added are shown in Figures 36 and 37 and the chemical shift values are as set out below.

¹H NMR (400 MHz, Chloroform-d) δ 12.65 (d), 10.74 (s), 8.66 (d), 8.53 (s), 7.67 (d), 7.10 (m), 4.85 (s), 3.94 (s), 3.32 (s), 3.07 (s), 1.96 (m), 1.43 (s), 1.17 (d) With D₂0 - ¹H NMR (400 MHz, Chloroform-d) δ 8.62 (d), 8.51 (s), 7.65 (d), 7.09 (m), 6.93 (s), 3.91 (s), 3.05 (s), 1.96 (m), 1.41 (s)

Example 3

Hydrolysis of tert-butyl 4-[4-[3-[(5-cyanopyrazin-2-yl)aminol-1H-pyrazol-5-yl]-3- methoxy-phenyl]-4-fluoro-piperidine-1-carboxylate (23) to give 5-[[5-[4-(4-Fluoro-4- piperidyl)-2-methoxy-phenyl]-1H-pyrazol-3-yl]amino]pyrazine-2-carbonitrile (24)

Intermediate 23 (16.00 g) and 1,4-dioxane (320 mL , Sigma Aldrich 296309, Lot#SHBD8744V) were added to a round bottom flask and heated to 85 °C to dissolve the solid. It was concentrated using a rotatory evaporator. To it was added DCM (80 mL , Sigma Aldrich 270997, Lot#SHBK5890) under the protection of nitrogen and then cooled to 0 °C. 4M HCI/1,4-dioxane (80 mL , Sigma Aldrich 345547 Lot#SHBK6535) was added and stirred at 0 °C for 1.5 hour. IPC by HPLC showed 44% conversion. Ice bath was removed and the reaction was stirred for 1.5 hours. The reaction was deemed complete by HPLC. The reaction was slowly added to a solution of KHCO₃ (50 g, 1.25 eq) /water (200 mL). The quenched reaction was kept at 0 °C overnight. The product was collected by vacuum filtration and washed with water (50 mL). The solid was dried in vacuum oven at 40 °C overnight. Product 24 (12.48 g) was obtained with the HPLC purity 99.0% and water 13.66% by Karl Fischer. The HPLC profile is shown in Figure 38 and the HPLC data are set out below.

Mass spectrum of compound 24: MS(m/z) [M + H]+ found 394.2

The ¹H NMR spectrum of compound 24 is shown in Figure 39 and the chemical shift data are set out below. ¹H NMR (400 MHz, Chloroform-d) δ 12.66 (s), 10.51 (d), 8.60 (d), 7.70 (d), 7.05 (m), 3.94 (s), 3.00 (m), 2.50 (t), 2.06 (m)

Run 2:

Intermediate 23 (166.00 g) and DCM (2.0L) were added to a jacketed reactor at 5 °C to dissolve the solid. To this solution as charged 4M HCI/1,4-dioxane (80 mL , Sigma Aldrich 345547 Lot#SHBK6535) was added and stirred at 0 °C for 25 hour. HPLC analysis showed full conversion. The reaction solution was concentrated to approximately 5vol and to it was charged 5vol of heptane to induce precipitation of the product as the HCI salt. Product 24 (169 g) was obtained with the HPLC purity 98.1%.

The HPLC profile is shown in Figure 40. Example 4

Reductive alkylation of 5-[[5-[4-(4-Fluoro-4-piperidyl)-2-methoxy-phenyl]-1H-pyrazol-3-yl]aminolpyrazine-2-carbonitrile (24) to give 5-[[5-[4-(4-Fluoro-4-piperidyl)-2- methoxy-phenyl]-1H-pyrazol-3-yl]aminolpyrazine-2-carbonitrile

Run 1:

Intermediate 24 (5.00 g) and dioxane (100 mL , Aldrich 296309 lot#SHBD8744V) were added to a 200mL flask. Under the protection of nitrogen, paraformaldehyde (1.15 g, 3 eq., Aldrich 158127 Lot#STBH8870) and NaBH(OAc)₃ (8.09 g, 3 eq., Aldrich 316393 Lot#SHBJ5376) were added and stirred at room temperature. It was slightly exothermc and the temperature slowly increased from 18.6 to 20.9 °C. A water bath was then used to control the temperature at ~19 °C. The reaction was slow based on LC analysis with 85.0% 24; 12.0% CHK1 Inhibitor and a 2.9% impurity. Paraformaldehyde (0.4 g) and NaBH(OAc)₃ (5.4 g) were added and stirred at rt overnight. The reaction was still slow with 60.7% SM; 29.6% product and three impurities at 2.6%, 6.1% and 1.0%. Water (0.25 mL ) was added to the reaction and stirred at rt overnight. The reaction was almost complete with 0.72% Compound , 95.0% CHK1 inhibitor, and two impurities at 3.3% and 1.0% level. To the reaction was added to i-PrOAc 200 mL (Sigma- Aldrich 537462 Lot#MKCH2786), water 200 mL and KHCO₃ 45 g (Fisher P184-500 lot 144811). There was off gas. After stirring for 20 min the phases were separated. The organic layer was washed with water (100 mL ). 37% HCI (3.0 mL , Sigma-Aldrich 258148 -2.5L Lot#SHBK0374) in 200 mL water was added to the organic solution after a polish filtration of the organic layer. After shaking and phase separation in a separatory funnel, the organic layer was washed with water (20mL ). The combined aqueous layer (pH=1 by pH paper) was added i-PrOAc (200 mL ) and K₂CO₃ (8.0 g, Fisher P208-500 loti 45088). After shaking in a separatory funnel and phase separation, the aqueous layer was washed with i-PrOAc (100 mL ). The organic solution was then washed with water 30 mL and dried over Na₂SO₄ and filtered. The organic solution was concentrated by rotary evaporator at 35 °C down to ~10 mL of residual solvent. MTBE (40 mL, Fisher E127-4, lot# 167039) was added and left in refrigerator at 4 °C overnight. The product, which was a light yellow solid, was collected by vacuum filtration and was washed with MTBE (10 mL). It was dried in vacuum oven at 30 °C overnight. 4.35 g (89% yield) of CHK1 inhibitor was obtained with HPLC purity at 95.1%

Notes:

1. Quenching the reaction by KHCO₃ might not be necessary. The reaction may be quenched by HCI solution directly

2. In a 500 mg scale reaction using more wet Compound 24 as the starting material, the reaction was faster and generated less impurities. The purity of the final product was 97.4%. This suggested that the reaction needs small amount of water to proceed well, likely because small amount of water increases the solubility of Compound 23 significantly in dioxane.

The HPLC profile of the Chk-1 inhibitor compound is shown in Figure 41 and the HPLC data are set out below.

Mass spectrum: MS(m/z) [M + H]+ found 408.1

The ¹H NMR spectrum for the Chk-1 inhibitor compound is shown in Figure 42 and the chemical shift data are set out below.

¹H NMR (400 MHz, Chloroform-d) δ 12.64 (s), 10.74 (s), 8.66 (m), 7.67 (d), 7.10 (m), 4.86 (dt), 3.93 (s), 3.56 (s), 3.33 (s), 2.73 (m), 2.12 (m), 1.17 (d)

Run 2:

To a jacketed reactor under nitrogen atmosphere was charged intermediate 24 (123 g) followed by MeOH (1.24L). To this solution was charged DIPEA (122.2g, 3eq) and the solution was aged at 30 °C for 2h. To this solution was then charged formalin (39g, 1 ,5eq) and aged for 2h prior to charging NaBH(OAc)3 (148g, 2.2eq). The reaction was aged for 18h at which time HPLC analysis indicated complete conversion. To the reaction solution was charged aqueous 25% ammonia solution (250g). Stir the resulting suspension at 20 °C for NLT 3h. The slurry was filtered and washed with water (3X 450mL ). The wet cake was dried at 50 °C for 20h. After drying the final CHK1 Inhibitor was collected (105g, 82% yield) with a purity of 96.6%.

The HPLC profile of the product is shown in Figure 43.

Equivalents The foregoing examples are presented for the purpose of illustrating the invention and should not be construed as imposing any limitation on the scope of the invention. It will readily be apparent that numerous modifications and alterations may be made to the specific embodiments of the invention described above and illustrated in the examples without departing from the principles underlying the invention. All such modifications and alterations are intended to be embraced by this application.

Claims

1. A process for the preparation of a compound of the formula (11):

which process comprises the reaction of a compound of the formula (A) with a compound of the formula (B):

where LG¹ is a leaving group (for example chlorine, bromine, iodine or trifluoromethansulfonate), and BG is a B(OH)2 or a boronate ester group such as in the presence of a palladium catalyst and a base.

2. A process according to claim 1 wherein the leaving group LG¹ is bromine.

3. A process according to claim 1 or claim 2 wherein the group BG is

or B(OH)₂.

4. A process according to any one of claims 1 to 3 wherein the palladium catalyst is selected from Pd₂dba₃/XPhos, Pd₂dba₃/SPhos and Pd G2 Pt-Bu₃.

5. A process according to claim 4 wherein the palladium catalyst is Pd₂dba₃/XPhos.

6. A process for the preparation of a compound of the formula (A):

which process comprises the reaction of a compound of the formula (C) with a compound of the formula (D):

where LG² is a leaving group (e.g. bromine or chlorine) in the presence of a base.

7. A process for the preparation of a compound of the formula (B):

which process comprises the reaction of a compound of the formula (E):

where BG is a boronic acid or boronate ester group and LG³ is a group capable of being displaced by a boronate or boronic acid group, by reaction in the presence of a palladium catalyst and a base, with a compound of the formula R^xR^yB-BR^x'R^y' where Rx, Ry, Rx' and Ry' are all hydrogen, or R^xR^yB and BR^x'R^y' each form boronate ester groups such as

8. A process for the preparation of a compound of the formula (E) by the reaction of a compound of the formula (F):

with a fluorinating agent capable of replacing the hydroxyl group with fluorine, wherein LG3 is as defined in claim 7.

9. A process for the preparation of a compound of the formula 13 by the reductive methylation of a compound of the formula 24 using formaldehyde (e.g. in the form of paraformaldehyde) and sodium cyanoborohydride:

10. A process for the preparation of a compound of the formula 13 by the reductive methylation of a compound of the formula 24 using paraformaldehyde and a reducing agent selected from sodium cyanoborohydride and sodium triacetoxyborohydride:

11. A process for the preparation of compound 13:

which process comprises the process of any one of claims 1 to 5 followed by removal of the protecting group PG and reductive methylation.

12. A process according to claim 11 wherein the reductive methylation is as defined in either of claims 10 and 11.

13. A process for the preparation of a compound of formula 24:

which process comprises the process of any one of claims 1 to 5 followed by removal of the protecting group PG, for example using an acid such as hydrochloric acid.

13. A compound of the formula (A):

wherein LG¹ is as defined in any one of the preceding claims.

14. A compound of the formula (B):

wherein BG is as defined in any one of the preceding claims.

15. An invention as defined in any one of Embodiments 1.1 to 1.11 1 to 2.5, 3.1 to 3.11 , 4.1 to 4.3, 5.1 to 5.7 and 6.1 to 6.2 herein.

16. A process for preparing a compound of the formula 13 sustantially as shown in Figure 44 or Figure 45 herein.