Synthesis and crystalline forms of a CDK2 inhibitor

Abstract

The invention generally relates to crystalline forms of the CDK2 inhibitor 2'-((3-ethoxy-1H-pyrazol- 4-yl)amino)-7'-((1R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1,5'-pyrrolo[2,3-d]pyrimidin]- 6'(7'H)-one or a salt and / or solvate thereof, the synthesis thereof, as well as methods of using the forms in the treatment of cancer.

Description

[0001] PAT059659 FF

[0002] SYNTHESIS AND CRYSTALLINE FORMS OF A CDK2 INHIBITOR

[0003] FIELD OF THE DISCLOSURE

[0004] The invention generally relates to crystalline forms of the CDK2 inhibitor 2'-((3-ethoxy-1 H-pyrazol- 4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]- 6'(7'H)-one or a salt and / or solvate thereof, the synthesis thereof, as well as methods of using the forms in the treatment of cancer.

[0005] BACKGROUND OF THE INVENTION

[0006] CDK2 is a serine / threonine kinase that regulates the mammalian cell cycle. After binding to cyclin- E1 , cyclin-E2, cyclin-A1 or cyclin-A2, the activated CDK2-cyclin complex phosphorylates a variety of cellular substrates (Chi et al 2020). For example, it phosphorylates and inactivates the retinoblastoma protein (pRB), thus de-represses the E2F family of transcription factors that promotes expression of cell cycle related genes (Sherr & Roberts 1999, 2004). As such, CDK2 propels the cell from the G1 phase into S phase of the cell cycle, when nuclear DNA is replicated in preparation of mitosis. CDK2 and its activity are often dysregulated in human cancers (Corsino et al 2008, Ying et al 2018, Nie et al 2019). One such dysregulation mechanism involves, but not limited to, the amplification or overexpression of cyclin-E1 , which leads to hyperactivation of CDK2 and promotes cellular proliferation (Schraml et al. 2003). Depending on lineage, dysregulation of CDK2 / E occurs in up to 40% of human cancers (cbioportal.org), including but not limited to uterine carcinosarcoma (~40%) ovarian (~20%), gastric (~12%), esophageal (~10%), endometrial (~10%), and breast cancers (~10%). Cyclin-E1 amplification and overexpression is also associated with poor prognosis across cancers (Nakayama et al 2010, Zhao et al 2019). In many of these cancers, besides radiation and chemotherapy, there is no targeted therapy available to patients. Thus, the development of a targeted agent against CDK2 would potentially benefit cancer patients.

[0007] Chi Y., Carter J. H, Swanger J., Mazin A.V., Moritz R.L, and Clurman B.E. (2020). A novel landscape of nuclear human CDk2 substrates revelaed by in situ phosphorylation. Sci. Adv 6, eaaz9899

[0008] Sherr CJ, Roberts JM. (1999). CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 13: 1501-1512.

[0009] Sherr CJ, Roberts JM. (2004). Living with or without cyclins and cyclin-dependent kinases. Genes Dev 18: 2699-2711 .

[0010] Corsino PE, Davis BJ, Norgaard PH, Parker NN, Law M, Dunn W and Law BK. Mammary tumors initiated by constitutive Cdk2 activation contain an invasive basal-like component. Neoplasia 2008; 10: 1240-1252 PAT059659 FF

[0011] Ying M, Shao X, Jing H, Liu Y, Qi X, Cao J, et al. Ubiquitin-dependent degradation of CDK2 drives the therapeutic diferentiation of AML by targeting PRDX2. Blood J Am Soc Hematol. 2018;131 (24):2698-711

[0012] Nie L, Wei Y, Zhang F, Hsu Y-H, Chan L-C, Xia W, et al. CDK2-mediated site-specifc phosphorylation of EZH2 drives and maintains triplenegative breast cancer. Nat Commun. 2019;10(1):1— 15

[0013] Schraml P, Bucher C, Bissig H, et al. Cyclin E overexpression and amplification in human tumours. J Pathol. 2003;200(3):375-383.

[0014] Nakayama N, Nakayama K, Shamima Y, Ishikawa M, Katagiri A, lida K, et al. Gene amplificationCCNEI is related to poor survival and potential therapeutic target in ovarian cancer. Cancer 2010:NA-NA.

[0015] Guo Q, Lin X, Ye L, et al. . Comparative efficacy of Cdk4 / 6 inhibitors plus aromatase inhibitors versus fulvestrant for the first-line treatment of hormone receptor-positive advanced breast cancer: a network meta-analysis. Target Oncol 2019;14:139-48

[0016] Gao J J , Cheng J, Bloomquist E, et al. . Cdk4 / 6 inhibitor treatment for patients with hormone receptor-positive, HER2-negative, advanced or metastatic breast cancer: a US food and drug administration pooled analysis. Lancet Oncol 2020;21 :250-60

[0017] Solid state form of the active pharmaceutical ingredient (API) of a particular drug is often an important determinant of the drug's ease of preparation, hygroscopicity, stability, solubility, storage stability, ease of formulation, rate of dissolution in gastrointestinal fluids and in vivo bioavailability. Crystalline forms occur where the same composition of matter crystallizes in a different lattice arrangement resulting in different thermodynamic properties and stabilities specific to the particular crystalline form. Crystalline forms may also include different hydrates or solvates of the same compound. In deciding which form is preferable, the numerous properties of the forms are compared and the preferred form chosen based on the many physical property variables. It is entirely possible that one form can be preferable in some circumstances where certain aspects such as ease of preparation, stability, etc. are deemed to be critical. In other situations, a different form may be preferred for greater dissolution rate and / or superior bioavailability.

[0018] Therefore, this ability of a chemical substance to crystallize in more than one crystalline form can have a profound effect on the shelf life, solubility, formulation properties, and processing properties of a drug. In addition, the action of a drug can be affected by the polymorphism of the drug molecule. Different polymorphs can have different rates of uptake in the body, leading to lower or higher biological activity than desired. In extreme cases, an undesired polymorph can PAT059659 FF even show toxicity. The occurrence of an unknown crystalline form during manufacture can have a significant impact.

[0019] It is not yet possible to predict whether a particular compound or salt of a compound will form polymorphs, whether any such polymorphs will be suitable for commercial use in a therapeutic composition, or which polymorphs will display such desirable properties.

[0020] SUMMARY

[0021] The polymorphic forms of this invention are designed and optimized to selectively bind to CDK2 which is believed to result in drugs useful in the treatment of above-mentioned cancers. In certain aspects, advantages of the polymorphic forms of the invention include improved melting point, reduced water retention and improved morphology (which can improve flow properties).

[0022] According to a first aspect of the invention, there is hereby provided a method of preparing 2'-((3- ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-

[0023] According to a second aspect of the invention, there is hereby provided a crystalline form of 2'- ((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'- pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one, solvate thereof made by the method of the first aspect of the invention. PAT059659 FF

[0024] According to a third aspect of the invention, there is hereby provided a crystalline form of 2'-((3- ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'- pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one or a pharmaceutically acceptable solvate and / or salt thereof.

[0025] According to a fourth aspect of the invention, there is hereby provided a pharmaceutical composition comprising the crystalline form according to the second or the third aspect of the invention and a pharmaceutically acceptable carrier.

[0026] According to a fifth aspect of the invention, there is hereby provided a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the crystalline form according to the second or the third aspect of the invention, or the pharmaceutical composition according to the fourth aspect of the invention.

[0027] According to a sixth aspect of the invention, there is hereby provided the crystalline form according to the second or the third aspect of the invention, or the pharmaceutical composition according to the fourth aspect of the invention for use as a medicament.

[0028] According to a seventh aspect of the invention, there is hereby provided a combination comprising a crystalline form of the second or the third aspect of the invention, and one or more therapeutically active agents.

[0029] According to an eighth aspect of the invention, there is hereby provided the crystalline form according to the second or the third aspect of the invention, or the pharmaceutical composition according to the fourth aspect of the invention for use in the treatment of cancer.

[0030] According to a ninth aspect of the invention, there is hereby provided use of the crystalline form according to the second or the third aspect of the invention, or the pharmaceutical composition according to the fourth aspect of the invention in the treatment of cancer.

[0031] According to a tenth aspect of the invention, there is hereby provided use of the crystalline form according to the second or the third aspect of the invention, or the pharmaceutical composition according to the fourth aspect of the invention in the manufacture of a medicament for the treatment of cancer. PAT059659 FF

[0032] BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is an X-ray powder diffraction pattern of the free base form of 2'-((3-ethoxy-1 H-pyrazol- 4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]- 6'(7'H)-one at room temperature (A = 1.54060A) formed by the method of PCT / IB2024 / 051412 I Comparative Example 1.

[0034] Figure 2 is an X-ray powder diffraction pattern of the free base form of 2'-((3-ethoxy-1 H-pyrazol- 4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]- 6'(7'H)-one at room temperature (A = 1 .54060A) formed by the method of Example 3.

[0035] Figure 3 is an X-ray powder diffraction pattern of a hydrochloride salt of 2'-((3-ethoxy-1 H-pyrazol- 4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]- 6'(7'H)-one at room temperature (A = 1.54060A).

[0036] Figure 4 is an X-ray powder diffraction pattern of a mesylate salt of 2'-((3-ethoxy-1 H-pyrazol-4- yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]- 6'(7'H)-one at room temperature (A = 1.54060A).

[0037] Figure 5 is an X-ray powder diffraction pattern of a phosphoric acid co-crystal of 2'-((3-ethoxy- 1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3- d]pyrimidin]-6'(7'H)-one at room temperature (A = 1.54060A).

[0038] Figure 6 is a Differential Scanning Calorimetry (DSC) diffractogram of the free base form of 2'- ((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'- pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one formed by the method of PCT / IB2024 / 051412 I Comparative Example 1.

[0039] Figure 7 is a Thermogravimetric analysis (TGA) of the free base form of 2'-((3-ethoxy-1 H-pyrazol- 4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]- 6'(7'H)-one formed by the method PCT / IB2024 / 051412 I Comparative Example 1.

[0040] Figure 8 shows a Thermogravimetric analysis (TGA) and Differential Scanning Calorimetry (DSC) diffractogram of the free base form of 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one formed by the method of Example 3.

[0041] Figures 9 shows SEM image of crystals of the free base form of 2'-((3-ethoxy-1 H-pyrazol-4- yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]- PAT059659 FF

[0042] 6'(7'H)-one formed by the method of Example 3 (right hand side) as compared to those formed using the method of PCT / IB2024 / 051412 / Comparative Example 1 (left hand side), showing the improvement in crystal morphology. DETAILED DESCRIPTION OF THE INVENTION

[0043] There is a need in the art for new crystalline forms of 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'- ((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one. Such forms may possess desirable physicochemical properties which are particularly advantageous in drug product development, e.g. which exhibit improved melting point and / or morphology (so as to improve flow properties).

[0044] According to a first aspect of the invention, there is hereby provided a method of preparing 2'-((3- ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'- pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one, said method comprising contacting a compound of formula acid. In an embodiment, the acid is hydrochloric acid or sulfuric acid. In an embodiment, the acid is sulfuric acid.

[0045] PAT059659 FF

[0046] In an embodiment, the compound of formula made by contacting a compound of formula compound of formula (I-A3), Boc (I-A3) in the presence of a first base and a catalyst. In an embodiment, the catalyst is selected from Pd(dtbpf)CI2, Pd(dppf)CI2, Pd(PPh3)4, Pd2(dba)3,

[0047] Pd(OAc)2, RuPhos Pd G2, RuPhos Pd G3, Ni(COD)2, Ni(dppp)CI2and Ni(acac)2. In an embodiment, the catalyst is RuPhos Pd G2 or RuPhos Pd G3. In an embodiment, the catalyst is RuPhos Pd G3. In an embodiment, the first base is selected from Et3N, Et2NH, DIPEA, NMI,nBuNH2, *BUNH2, NH3, K2CO3, Na2CO3, KOH, NaOH, NaHCO3, morpholine, DBN, DBU and TMG. In an embodiment, the first base is DBU or TMG. In an embodiment, the first base is DBU. In an embodiment, the compound of formula (I-A3), Boc (I-A3), is made by contacting a compound of formula (I-A1), (I-A1) with a Boc (tert-butoxycarbonyl) introducing agent, a second base, a hydrogenating catalyst and hydrogen gas. In an embodiment, the Boc introducing agent is selected from Boc20, Boc chloride, Boc imidazole and Boc azide. In an embodiment, the Boc introducing agent is Boc20. In an embodiment, the second base is selected from Na2CO3, NaHCO3, K2CO3, KHCO3, LiOH, NaOH, KOH, NaOMe, NaOEt, NaH and ’BuOK. In an embodiment, the second base is Na2CO3. In an embodiment, the hydrogenating catalyst is selected from a platinum based catalyst (e.g. Pt / V / C, Pt / V / Fe / C, PtO2or Pt / C), a palladium based catalyst (e.g. Pd / C or Pd(OH)2 / C), Raney Ni, Raney Co, a mixture of Fe with NH4CI, a mixture of Zn with NH4CI, B2Pin2and nitroreductase. In an embodiment, the hydrogenating catalyst is a PAT059659 FF palladium based catalyst (e.g. Pd / C or Pd(OH)2 / C). In an embodiment, the hydrogenating catalyst is Pd / C.

[0048] According to a second aspect of the invention, there is hereby provided a crystalline form of 2'- ((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'- pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one, solvate thereof made by the method of the first aspect of the invention.

[0049] According to a third aspect of the invention, there is hereby provided a crystalline form of 2'-((3- ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'- pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one or a pharmaceutically acceptable solvate and / or salt thereof.

[0050] In an embodiment, the 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one is in the form of a free base or a solvate thereof.

[0051] In an embodiment the melting point is above 245 °C. In an embodiment, the melting point is above 246 °C. In an embodiment, the melting point is above 247 °C. In an embodiment, the melting point is above 248 °C. In an embodiment, the melting point is above 249 °C. In an embodiment, the melting point is above 250 °C. In an embodiment, the melting point is 245 °C to 260 °C. In an embodiment, the melting point is 247 °C to 258 °C. In an embodiment, the melting point is 250 °C to 255 °C. In an embodiment, the melting point is 251 °C to 254 °C. In an embodiment, the melting point is 252 °C to 253 °C. In an embodiment, the melting point is 252.5 °C (± 0.5 °C, e.g. ± 0.4 °C, e.g. ± 0.3 °C, e.g. ± 0.2 °C, e.g. ± 0.1 °C) In an embodiment, the melting point is about 252.5 °C. In an embodiment, the above-mentioned melting points I me are as determined by DSC, e.g. DSC onset temperature, e.g. DSC onset temperature at heating rate of 10°C per minute.

[0052] In an embodiment the melting point as determined by the DSC onset temperature at heating rate of 10°C per minute / min is above 245 °C. In an embodiment the melting point as determined by the DSC onset temperature at heating rate of 10°C per minute / min is above 246 °C. In an embodiment the melting point as determined by the DSC onset temperature at heating rate of 10°C per minute / min is above 247 °C. In an embodiment the melting point as determined by the PAT059659 FF

[0053] DSC onset temperature at heating rate of 10°C per minute / min is above 248 °C. In an embodiment the melting point as determined by the DSC onset temperature at heating rate of 10°C per minute / min is above 249 °C. In an embodiment the melting point as determined by the DSC onset temperature at heating rate of 10°C per minute / min is above 250 °C.

[0054] In an embodiment, the crystalline form of the second or the third aspect of the invention is characterized by an X-ray powder diffraction pattern comprising four or more 29 values selected from the group consisting of: wherein the temperature is about room temperature and the radiation used has a wavelength of 1.54060A. In an embodiment, the crystalline form has an X-ray powder diffraction pattern substantially the same as the X-ray powder diffraction spectrum as shown in FIG. 1 , at about room temperature wherein the radiation used has a wavelength of 1.54060 A. In an embodiment, the crystalline form has the following unit cell dimensions at 100 °K as determined by single crystal X-ray diffraction: Crystal System: Monoclinic; Space Group: C2; a = 16.88 A (± 0.1 A); b = 7.72 A (± 0.1 A); c = 15.63 A (± 0.1 A); and 0 = 111.67° (± 0.1 °).

[0055] In another embodiment, the 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one is in the form of a hydrochloride salt. In an embodiment, it is characterized by an X-ray powder diffraction pattern comprising four or more 29 values selected from the group consisting of: PAT059659 FF wherein the temperature is about room temperature and the radiation used has a wavelength of 1.54060A. In an embodiment, it has an X-ray powder diffraction pattern substantially the same as the X-ray powder diffraction spectrum as shown in FIG. 2, at about room temperature wherein the radiation used has a wavelength of 1 .54060 A.

[0056] In another embodiment, the 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one is in the form of a mesylate salt. In an embodiment, it is characterized by an X-ray powder diffraction pattern comprising four or more 20 values selected from the group consisting of: wherein the temperature is about room temperature and the radiation used has a wavelength of 1.54060A. In an embodiment, it has an X-ray powder diffraction pattern substantially the same as the X-ray powder diffraction spectrum as shown in FIG. 3, at about room temperature wherein the radiation used has a wavelength of 1 .54060 A.

[0057] In another embodiment, the 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one is in the form of a PAT059659 FF phosphate salt or phosphoric acid co-crystal (preferably a phosphoric acid co-crystal). In an embodiment, it is characterized by an X-ray powder diffraction pattern comprising four or more 29 values (e.g. 5 or more, e.g. 6 or more, e.g. 7 or more, e.g. 8 or more, e.g. 9 or more, e.g. 10 or more, e.g. 11 or more , e.g. all 12 29 values) selected from the group consisting of: wherein the temperature is about room temperature and the radiation used has a wavelength of 1.54060A. In an embodiment, it has an X-ray powder diffraction pattern substantially the same as the X-ray powder diffraction spectrum as shown in FIG. 4, at about room temperature wherein the radiation used has a wavelength of 1 .54060 A.

[0058] According to a fourth aspect of the invention, there is hereby provided a pharmaceutical composition comprising the crystalline form according to the second or the third aspect of the invention and a pharmaceutically acceptable carrier.

[0059] According to a fifth aspect of the invention, there is hereby provided a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the crystalline form according to the second or the third aspect of the invention, or the pharmaceutical composition according to the fourth aspect of the invention.

[0060] According to a sixth aspect of the invention, there is hereby provided the crystalline form according to the second or the third aspect of the invention, or the pharmaceutical composition according to the fourth aspect of the invention for use as a medicament.

[0061] According to a seventh aspect of the invention, there is hereby provided a combination comprising a crystalline form of the second or the third aspect of the invention, and one or more therapeutically active agents. PAT059659 FF

[0062] According to an eighth aspect of the invention, there is hereby provided the crystalline form according to the second or the third aspect of the invention, or the pharmaceutical composition according to the fourth aspect of the invention for use in the treatment of cancer.

[0063] According to a ninth aspect of the invention, there is hereby provided use of the crystalline form according to the second or the third aspect of the invention, or the pharmaceutical composition according to the fourth aspect of the invention in the treatment of cancer.

[0064] According to a tenth aspect of the invention, there is hereby provided use of the crystalline form according to the second or the third aspect of the invention, or the pharmaceutical composition according to the fourth aspect of the invention in the manufacture of a medicament for the treatment of cancer.

[0065] In an embodiment of any one of the fifth, eighth, ninth and tenth aspects of the invention, the cancer is selected from ovarian cancer, gastric cancer, uterine cancer, breast cancer, lung cancer and endometrial cancer.

[0066] In an embodiment of any one of the fifth, eighth, ninth and tenth aspects of the invention, the cancer is a cyclin E amplified cancer.

[0067] The invention therefore provides the following numbered embodiments:

[0068] Embodiment 1 . A method of preparing 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one,

[0069] Embodiment 2. The method according to Embodiment 1 , wherein the acid is hydrochloric acid or sulfuric acid. PAT059659 FF

[0070] Embodiment 3. The method according to Embodiment 2, wherein the acid is sulfuric acid.

[0071] Embodiment 4. The method according to any one of Embodiments 1 to 3, wherein the the presence of a first base and a catalyst.

[0072] Embodiment 5. The method according to Embodiment 4, wherein the catalyst is selected from Pd(dtbpf)CI2, Pd(dppf)CI2, Pd(PPh3)4, Pd2(dba)3, Pd(OAc)2, RuPhos Pd G2, RuPhos Pd G3, Ni(COD)2, Ni(dppp)CI2and Ni(acac)2.

[0073] Embodiment s. The method according to Embodiment 5, wherein the catalyst is RuPhos Pd G2 or RuPhos Pd G3.

[0074] Embodiment 7. The method according to Embodiment 6, wherein the catalyst is RuPhos Pd G3.

[0075] Embodiment 8. The method according to any one of Embodiments 4 to 7, wherein the first base is selected from Et3N, Et2NH, DIPEA, NMI,nBuNH2, *BuNH2, NH3, K2CO3, Na2CO3, KOH, NaOH, NaHCO3, morpholine, DBN, DBU and TMG.

[0076] Embodiment 9. The method according to Embodiment 8, wherein the first base is DBU or TMG.

[0077] Embodiment 10. The method according to Embodiment 9, wherein the first base is DBU. PAT059659 FF

[0078] Embodiment 11. The method according to any one of Embodiments 4 to 10, wherein the compound of formula (I-A3), Boc (I-A3), is made by contacting a compound of formula

[0079] (I-A1), with a Boc (tert-butoxycarbonyl) introducing agent, a second base, a hydrogenating catalyst and hydrogen gas.

[0080] Embodiment 12. The method according to Embodiment 11 , wherein the Boc introducing agent is selected from Boc20, Boc chloride, Boc imidazole and Boc azide.

[0081] Embodiment 13. The method according to Embodiment 12, wherein the Boc introducing agent is Boc20.

[0082] Embodiment 14. The method according to any one of Embodiments 11 to 13, wherein the second base is selected from Na2CO3, NaHCO3, K2CO3, KHCO3, LiOH, NaOH, KOH, NaOMe, NaOEt, NaH and ’BuOK.

[0083] Embodiment 15. The method according to Embodiment 14, wherein the second base is Na2CO3.

[0084] Embodiment 16. The method according to any one of Embodiments 11 to 15, wherein the hydrogenating catalyst is selected from a platinum based catalyst (e.g. Pt / V / C, Pt / V / Fe / C, PtO2or Pt / C), a palladium based catalyst (e.g. Pd / C or Pd(OH)2 / C), Raney Ni, Raney Co, a mixture of Fe with NH4CI, a mixture of Zn with NH4CI, B2Pin2and nitroreductase.

[0085] Embodiment 16a. The method according to Embodiment 16, wherein the hydrogenating catalyst is a palladium based catalyst.

[0086] Embodiment 16b. The method according to Embodiment 16 or Embodiment 16a, wherein the hydrogenating catalyst is Pd / C.

[0087] Embodiment 17. A crystalline form of 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one, PAT059659 FF or a solvate thereof made by the method of any one of

[0088] Embodiments 1 to 16.

[0089] Embodiment 18. A crystalline form of 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one or a pharmaceutically acceptable solvate and / or salt thereof.

[0090] Embodiment 19. The crystalline form according to Embodiment 17 or Embodiment 18, wherein the 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one is in the form of a free base or a solvate thereof.

[0091] Embodiment 20. The crystalline form according to Embodiment 19, wherein the melting point is above 245°C.

[0092] Embodiment 21 . The crystalline form according to Embodiment 20, wherein the melting point is above 250°C.

[0093] Embodiment 22. The crystalline form according to Embodiment 20 or Embodiment 21 , wherein the melting point is determined by DSC.

[0094] Embodiment 23. The crystalline form according to Embodiment 22, wherein the melting point is determined by DSC onset temperature.

[0095] Embodiment 24. The crystalline form according to Embodiment 23, wherein the melting point is determined by DSC onset temperature at heating rate of 10°C per minute.

[0096] Embodiment 25. The crystalline form according to any one of Embodiments 17 to 24, characterized by an X-ray powder diffraction pattern comprising four or more 28 values selected from the group consisting of: PAT059659 FF wherein the temperature is about room temperature and the radiation used has a wavelength of 1.54060A.

[0097] Embodiment 26. The crystalline form according to any one of Embodiments 17 to 25, having an X-ray powder diffraction pattern substantially the same as the X-ray powder diffraction spectrum as shown in FIG. 2, at about room temperature wherein the radiation used has a wavelength of 1 .54060 A.

[0098] Embodiment 27. The crystalline form according to any one of Embodiments 17 to 26, having the following unit cell dimensions at 100 °K as determined by single crystal X-ray diffraction:

[0099] Crystal System: Monoclinic

[0100] Space Group: C2 a = 16.88 A (± 0.1 A) b = 7.72 A (± 0.1 A) c = 15.63 A (± 0.1 A)

[0101] P = 111.67° (± 0.1 °).

[0102] Embodiment 28. The crystalline form according to Embodiment 18, wherein the 2'-((3- ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'- pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one is in the form of a hydrochloride salt.

[0103] Embodiment 29. The crystalline form according to Embodiment 28, characterized by an X- ray powder diffraction pattern comprising four or more 20 values selected from the group consisting of: PAT059659 FF wherein the temperature is about room temperature and the radiation used has a wavelength of 1.54060A.

[0104] Embodiment 30. The crystalline form according to Embodiment 28 or Embodiment 29, having an X-ray powder diffraction pattern substantially the same as the X-ray powder diffraction spectrum as shown in FIG. 3, at about room temperature wherein the radiation used has a wavelength of 1 .54060 A.

[0105] Embodiment 31 . The crystalline form according to Embodiment 18, wherein the 2'-((3- ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'- pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one is in the form of a mesylate salt.

[0106] Embodiment 32. The crystalline form according to Embodiment 31 , characterized by an X- ray powder diffraction pattern comprising four or more 20 values selected from the group consisting of: wherein the temperature is about room temperature and the radiation used has a wavelength of 1.54060A. PAT059659 FF

[0107] Embodiment 33. The crystalline form according to Embodiment 31 or Embodiment 32, having an X-ray powder diffraction pattern substantially the same as the X-ray powder diffraction spectrum as shown in FIG. 4, at about room temperature wherein the radiation used has a wavelength of 1 .54060 A.

[0108] Embodiment 34. The crystalline form according to Embodiment 18, wherein the 2'-((3- ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'- pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one is in the form of a phosphate salt or phosphoric acid cocrystal.

[0109] Embodiment 35. The crystalline form according to Embodiment 34, wherein characterized by an X-ray powder diffraction pattern comprising four or more 20 values (e.g. 5 or more, e.g. 6 or more, e.g. 7 or more, e.g. 8 or more, e.g. 9 or more, e.g. 10 or more, e.g. 11 or more , e.g. all 12 29 values) selected from the group consisting of: wherein the temperature is about room temperature and the radiation used has a wavelength of 1.54060A.

[0110] Embodiment 36. The crystalline form according to Embodiment 34 or Embodiment 35, having an X-ray powder diffraction pattern substantially the same as the X-ray powder diffraction spectrum as shown in FIG. 5, at about room temperature wherein the radiation used has a wavelength of 1 .54060 A.

[0111] Embodiment 37. A pharmaceutical composition comprising the crystalline form according to any one of Embodiments 17 to 36, and a pharmaceutically acceptable carrier. PAT059659 FF

[0112] Embodiment 38. A method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the crystalline form according to any one of Embodiments 17 to 36, or the pharmaceutical composition according to Embodiment 37.

[0113] Embodiment 39. The crystalline form according to any one of Embodiments 17 to 36, or the pharmaceutical composition according to Embodiment 37 for use as a medicament.

[0114] Embodiment 40. A combination comprising a crystalline form of any of Embodiments 17 to 36, and one or more therapeutically active agents.

[0115] Embodiment 41 . The crystalline form according to any one of Embodiments 17 to 36, or the pharmaceutical composition according to Embodiment 37 for use in the treatment of cancer.

[0116] Embodiment 42. Use of the crystalline form according to any one of Embodiments 17 to 36, or the pharmaceutical composition according to Embodiment 37 in the treatment of cancer.

[0117] Embodiment 43. Use of the crystalline form according to any one of Embodiments 17 to 36, or the pharmaceutical composition according to Embodiment 37 in the manufacture of a medicament for the treatment of cancer.

[0118] Embodiment 44. The method according to Embodiment 38, the crystalline form for use according to Embodiment 41 , orthe use according to Embodiment 42 or Embodiment 43, wherein the cancer is selected from ovarian cancer, gastric cancer, uterine cancer, breast cancer, lung cancer and endometrial cancer.

[0119] Embodiment 45. The method according to Embodiment 38 or Embodiment 44, the crystalline form for use according to Embodiment 41 or Embodiment 44, or the use according to any one of Embodiments 42 to 44, wherein the cancer is a cyclin E amplified cancer.

[0120] Definitions

[0121] As used herein “polymorph” or “crystalline form” or the like refers to crystalline forms having the same chemical composition but different spatial arrangements of the molecules, atoms, and / or ions forming the crystal.

[0122] As used herein “solvate” refers to a crystalline form of a molecule, atom, and / or ions that further comprises molecules of a solvent or solvents incorporated into the crystalline lattice structure. The solvent molecules in the solvate may be present in a regular arrangement and / or a nonordered arrangement. The solvate may comprise either a stoichiometric or nonstoichiometric PAT059659 FF amount of the solvent molecules. For example, a solvate with a nonstoichiometric amount of solvent molecules may result from partial loss of solvent from the solvate. Solvates may occur as dimers or oligomers comprising more than one molecule or Compound ABC within the crystalline lattice structure. The solvent may be water, in which case the solvent may be referred to as a hydrate.

[0123] As used herein, the term “free form”, “free base” etc. of a given compound refers to a solid state form where the only component present which is solid at ambient conditions (e.g. 20-22 °C, e.g. 21 °C 1 atm) is the said compound. Thus, as used herein, the term “free form” encompasses both unsolvated I unhydrated forms, and solvated I hydrated forms, but excludes salts and cocrystals where the coformer is solid at ambient conditions.

[0124] As used herein, the terms “salt” or “salts” refers to an acid addition or base addition salt of a compound of the present invention. “Salts” include in particular “pharmaceutical acceptable salts”. The term “pharmaceutically acceptable salts” refers to salts that retain the biological effectiveness and properties of the compounds of this invention and, which typically are not biologically or otherwise undesirable. In many cases, the compounds of the present invention are capable of forming acid and / or base salts by virtue of the presence of amino and / or carboxyl groups or groups similar thereto. When both a basic group and an acid group are present in the same molecule, the compounds of the present invention may also form internal salts, e.g., zwitterionic molecules.

[0125] Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids.

[0126] Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

[0127] Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, and the like.

[0128] Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.

[0129] Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I to XII of the periodic table. In certain embodiments, the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium and magnesium salts. PAT059659 FF

[0130] Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Certain organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine and tromethamine.

[0131] As used herein “amorphous” refers to a solid form of a molecule, atom, and / or ions that is not crystalline. An amorphous solid does not display a definitive X-ray diffraction pattern.

[0132] The term “substantially the same” with reference to X-ray diffraction peak positions means that typical peak position and intensity variability are taken into account. For example, one skilled in the art will appreciate that the peak positions (2©) will show some inter-apparatus variability, typically as much as 0.2°. Further, one skilled in the art will appreciate that relative peak intensities will show inter-apparatus variability as well as variability due to degree of crystallinity, preferred orientation, prepared sample surface, and other factors known to those skilled in the art, and should be taken as qualitative measure only. The person skilled in the art of X-ray powder diffraction is readily able to determine whether a given sample comes from the same polymorph as a reference sample.

[0133] As used herein, the terms “about” and “substantially” indicate with respect to features such as endotherms, endothermic peak, exotherms, baseline shifts, etc., that their values can vary. With reference to X-ray diffraction peak positions, “about” or “substantially” means that typical peak position and intensity variability are taken into account. For example, one skilled in the art will appreciate that the peak positions (29) will show some inter-apparatus variability, typically as much as 0.2°. Occasionally, the variability could be higher than 0.2° depending on apparatus calibration differences. Further, one skilled in the art will appreciate that relative peak intensities will show inter-apparatus variability as well as variability due to degree of crystallinity, preferred orientation, prepared sample surface, and other factors known to those skilled in the art, and should be taken as qualitative measure only. For DSC, variation in the temperatures observed will depend upon the rate of temperature change as well as sample preparation technique and the particular instrument employed. Thus, the endotherm / melting point values reported herein relating to DSC / TGA thermograms can vary ± 2°C (and still be considered to be characteristic of the particular crystalline form described herein). When used in the context of other features, such as, for example, percent by weight (% by weight), reaction temperatures, the term “about” indicates a variance of ± 5%. PAT059659 FF

[0134] As used herein, the term “room temperature” means from about 18 °C to about 25 °C, preferably about 20 °C to about 22 °C, most preferably about 21 °C.

[0135] The term "a therapeutically effective amount" of a crystalline form of the present invention refers to an amount of the crystalline form of the present invention that will elicit the biological or medical response of a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc.

[0136] As used herein, the term "a,” "an,” "the” and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.

[0137] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed.

[0138] As used herein, the term “inhibit”, "inhibition" or “inhibiting” refers to the reduction or suppression of a given condition, symptom, or disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0139] As used herein, the terms “treat,” “treating,” or “treatment” of any disease or disorder refers in one embodiment, to ameliorating the disease or disorder ( / .e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment, “treat,” “treating,” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, “treat,” “treating,” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In one embodiment, “treat” or “treating” refers to delaying the progression of the disease or disorder.

[0140] As used herein, the term “prevent”, “preventing" or “prevention” of any disease or disorder refers to the prophylactic treatment of the disease or disorder; or delaying the onset of the disease or disorder.

[0141] As used herein, the term “subject” refers to an animal. Preferably, the animal is a mammal. A subject refers to for example, primates (e.g. humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds and the like. In a preferred embodiment, the subject is a human. PAT059659 FF

[0142] As used herein, a subject is “in need of’ or “in need thereof” a treatment if such subject would benefit biologically, medically or in quality of life from such treatment.

[0143] The term “comprising” encompasses “including” as well as “consisting”; e.g., a composition comprising X may consist exclusively of X or may include additional, e.g. X and Y.

[0144] The crystalline form of the present invention may be administered either simultaneously with, or before or after, one or more other therapeutic agent. The crystalline form of the present invention may be administered separately, by the same or different route of administration, or together in the same pharmaceutical composition as the other agents. A therapeutic agent is, for example, a chemical compound, peptide, antibody, antibody fragment or nucleic acid, which is therapeutically active or enhances the therapeutic activity when administered to a patient in combination with a compound of the present invention.

[0145] In the combination therapies of the invention, the crystalline form of the present invention and the other therapeutic agent may be manufactured and / or formulated by the same or different manufacturers. Moreover, the crystalline form of the present invention and the other therapeutic may be brought together into a combination therapy: (i) prior to release of the combination product to physicians (e.g. in the case of a kit comprising the crystalline form of the present invention and the other therapeutic agent); (ii) by the physician themselves (or under the guidance of the physician) shortly before administration; (iii) in the patient themselves, e.g. during sequential administration of the crystalline form of the present invention and the other therapeutic agent.

[0146] As used herein, the term “carrier” or "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289- 1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0147] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without PAT059659 FF excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0148] Generally, the term “pharmaceutical composition” is defined herein to refer to a mixture or solution containing at least one therapeutic agent to be administered to a subject, e.g., a mammal or human. The present pharmaceutical combinations can be formulated in a suitable pharmaceutical composition for enteral or parenteral administration are, for example, those in unit dosage forms, such as sugar-coated tablets, tablets, capsules or suppositories, or ampoules. If not indicated otherwise, these are prepared in a manner known per se, for example by means of various conventional mixing, comminution, direct compression, granulating, sugar-coating, dissolving, lyophilizing processes, or fabrication techniques readily apparent to those skilled in the art. It will be appreciated that the unit content of a combination partner contained in an individual dose of each dosage form need not in itself constitute an effective amount since the necessary effective amount may be reached by administration of a plurality of dosage units. The pharmaceutical composition may contain, from about 0.1 % to about 99.9%, preferably from about 1 % to about 60 %, of the therapeutic agent(s). One of ordinary skill in the art may select one or more of the aforementioned carriers with respect to the particular desired properties of the dosage form by routine experimentation and without any undue burden. The amount of each carriers used may vary within ranges conventional in the art. The following references disclose techniques and excipients used to formulate oral dosage forms. See The Handbook of Pharmaceutical Excipients, 4th edition, Rowe et al., Eds., American Pharmaceuticals Association (2003); and Remington: the Science and Practice of Pharmacy, 20th edition, Gennaro, Ed., Lippincott Williams & Wilkins (2003). These optional additional conventional carriers may be incorporated into the oral dosage form either by incorporating the one or more conventional carriers into the initial mixture before or during granulation or by combining the one or more conventional carriers with granules comprising the combination of agents or individual agents of the combination of agents in the oral dosage form. In the latter embodiment, the combined mixture may be further blended, e.g., through a V-blender, and subsequently compressed or molded into a tablet, for example a monolithic tablet, encapsulated by a capsule, or filled into a sachet. Clearly, the pharmaceutical combinations of the present invention can be used to manufacture a medicine.

[0149] The present invention relates to such pharmaceutical combinations or pharmaceutical compositions that are particularly useful as a medicine.

[0150] Specifically, the combinations or compositions of the present invention can be applied in the treatment of cancer. PAT059659 FF

[0151] The term “cancer” refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to colorectal, gastric, endometrial, prostate, adrenocortical, uterine, cervical, esophageal, breast, kidney, ovarian cancer and the like.

[0152] The terms “tumor” and “cancer” are used interchangeably herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating, tumors. As used herein, the term “cancer” or “tumor” includes premalignant, as well as malignant cancers and tumors.

[0153] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0154] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed.

[0155] EXAMPLES

[0156] Comparative Example 1 - Synthesis of 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl )spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one,

[0157] The synthesis is described in PCT / IB2024 / 051412, the contents of which are incorporated by reference.

[0158] Intermediate 1 PAT059659 FF

[0159] Step-1 : Synthesis of (1R,3R)-3-((2-chloro-5-iodopyrimidin-4-yl)amino)cyclohexan-1-ol: To a stirred solution of (1R,3R)-3-aminocyclohexan-1-ol hydrochloride (60 g, 397.3 mmol) and DIPEA (183 mL, 993.25 mmol) in isopropanol (300 ml), was added 2,4-dichloro-5-iodopyrimidine (108 g, 397.3 mmol) at room temperature and stirred for 16h. The progress of the reaction was monitored by TLC and LCMS. After completion of the reaction, the solvent was removed under reduced pressure to give the crude product. The crude compound was purified by column chromatography by using silica gel (100-200 mesh), eluting with 0-40% ethyl acetate in pet-ether to give (1R,3R)-3-((2-chloro-5-iodopyrimidin-4-yl)amino)cyclohexan- 1-ol (113 g, 81 % yield, regio-isomeric mixture; 82% and 15% isomers) as pale yellow solid. LC-MS m / z [M+H]+ = 353.98,1H NMR (400 MHz, DMSO-de) 6 = 8.28-8.40 (m, 1 H), 6.58 (d, J = 8.4 Hz, 1 H), 4.50-4.51 (m, 1 H), 4.25-4.30 (m, 1 H), 3.90-4.20 (m, 1 H), 1.71-1.80 (m, 4H), 1.20-1.70 (m, 5H).

[0160] Step-2: Synthesis of (1R,3R)-3-((2-chloro-5-((trimethylsilyl)ethynyl)pyrimidin-4- yl)amino)cyclohexan-1-ol: To a degassed solution of (1 R,3R)-3-((2-chloro-5-iodopyrimidin-4- yl)amino)cyclohexan-1-ol (56.5 g, 160.5 mmol; 82% and 15% isomers), Cui (3.04 g, 16.0 mmol), triethyl amine (115 mL, 800 mmol) and Pd(PPh3)4 (1.84 g, 1.60 mmol) in dry THF (560 mL), was added trimethylsilyl acetylene (22.7 mL, 160.5 mmol) and heated at 55 °C for 4h. The progress of the reaction was monitored by TLC & LCMS. After completion of the reaction, the reaction mass was filtered through plug of celite, the filtrate was concentrated under reduced pressure to give the crude product. The crude compound was PAT059659 FF purified by column chromatography using silica gel (100-200 mesh), eluting with 0-20% ethyl acetate in pet- ether to give (1 R,3R)-3-((2-chloro-5-((trimethylsilyl)ethynyl)pyrimidin-4-yl)amino)cyclohexan-1-ol (35 g, 68%) as pale yellow solid. LC-MS m / z [M+H]+ = 325.66,1H NMR (400 MHz, DMSO-de) 6 = 8.16 (s, 1 H), 6.48 (d, J = 8.0 Hz, 1 H), 4.55 (d, J = 2.80 Hz, 1 H), 4.25-4.40 (m, 1 H), 3.90-4.0 (m, 1 H), 1.61-1.85 (m, 4H), 1.30-1.60 (m, 4H), 0.26 (s, 9H).

[0161] Step-3: Synthesis of (1 R,3R)-3-(2-chloro-7H-pyrrolo[2,3-d] pyrimidin-7-yl) cyclohexan-1-ol: To a stirred solution of (1 R,3R)-3-((2-chloro-5-((trimethylsilyl)ethynyl)pyrimidin-4-yl)amino)cyclohexan-1-ol (44 g, 136.2 mmol) in acetonitrile (2,200 mL), was added CS2CO3 (22.1 g, 68.1 mmol) and heated at 75 °C for 6 h. Progress of the reaction was monitored by TLC and LCMS. After completion of the reaction, the reaction mass was filtered, the filtrate was concentrated under reduced pressure to give crude product. The crude compound was purified by column chromatography using silica gel (100-200 mesh), eluting with 0-50% ethyl acetate in pet-ether to give (1 R,3R)-3-(2-chloro-7H-pyrrolo[2,3-d] pyrimidin-7-yl) cyclohexan-1-ol (29 g, 85% yield) as pale yellow solid; LC-MS m / z [M+H]+ = 252.17,1H NMR (400 MHz, DMSO-de) 6 = 8.90 (s, 1 H), 7.82 (d, J = 3.6 Hz, 1 H), 6.70 (d, J = 3.6 Hz, 1 H), 4.75 (d, J = 2.8 Hz, 1 H), 4.15 (d, J = 2.4 Hz, 1 H), 2.01-2.11 (m, 1 H), 1.40-1.99 (m, 7H).

[0162] Step-4: Synthesis of 7-((1 R,3R)-3-((tert-butyldimethylsilyl) oxy)cyclohexyl)-2-chloro-7H-pyrrolo[2,3- d]pyrimidine: To a stirred solution of (1 R,3R)-3-(2-chloro-7H-pyrrolo[2,3-d] pyrimidin-7-yl) cyclohexan-1- ol (20 g, 79.68 mmol) and imidazole (10.8 g, 159.3 mmol) in DMF (200 mL), was added TBDMS-CI (18 g, 159.3 mmol) at room temperature and stirred for 16h. Progress of the reaction was monitored by TLC and LCMS. After, 16h, the reaction mass was quenched with ice-cold water (50 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were washed with ice-cold water (2 x 60 mL). The organic layer was dried over Na2SO4, concentrated under reduced pressure. The crude compound was purified by column chromatography using silica gel (100-200 mesh), eluting with 0-50% ethyl acetate in pet- ether to give 7-((1 R,3R)-3-((tert-butyldimethylsilyl) oxy)cyclohexyl)-2-chloro-7H-pyrrolo[2,3-d]pyrimidine (29 g, 96% yield) as brown gummy liquid. LC-MS m / z [M+H]+=366.24,1H NMR (400 MHz, DMSO-de) 6 = 8.88 (s, 1 H), 7.79 (d, J = 3.6 Hz, 1 H), 6.68 (d, J = 3.6 Hz, 1 H), 4.95-5.10 (m, 1 H), 4.27 (s, 1 H), 1.96-2.05 (m, 1 H), 1.80-1.95 (m, 4H), 1.64 (d, J = 8.0 Hz, 2H), 1.45-1.55 (m, 1 H), 0.90-0.98 (m, 9H), 0.01-0.50 (m, 6H),

[0163] Step-5: Synthesis of 5,5-dibromo-7-((1 R,3R)-3-((tert-butyldimethylsilyl) oxy)cyclohexyl)-2-chloro- 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one: To a stirred solution of 7-((1 R,3R)-3-((tert- butyldimethylsilyl) oxy)cyclohexyl)-2-chloro-7H-pyrrolo[2,3-d]pyrimidine (50 g, 136.9 mmol) in ABuOH: H2O (600 mL, 4:1), was added NBS (73 g, 410.9 mmol) at room temperature and stirred for 4h. Progress of the PAT059659 FF reaction was monitored by TLC & LCMS. After completion of the reaction. The reaction mass was quenched with water (100 mL), extracted with MTBE (3 x 300 mL). The organic layer was dried over Na2SO4, concentrated under reduced pressure to get the crude 5,5-dibromo-7-((1 R,3R)-3-((tert-butyldimethylsilyl) oxy)cyclohexyl)-2-chloro-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (73 g crude) as pale yellow semisolid. The crude product was used as such for next step without further purification. LC-MS m / z [M+H]+ = 540.21

[0164] Step-6: Synthesis of 7-((1R,3R)-3-((tert-butyldimethylsilyl)oxy)cyclohexyl)-2-chloro-5,7-dihydro-6H- pyrrolo[2,3-d]pyrimidin-6-one: To a stirred solution of 5,5-dibromo-7-((1 R,3R)-3-((tert-butyldimethylsilyl) oxy)cyclohexyl)-2-chloro-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (73g crude, 135.4 mmol) in AcOH (730 mL), was added Zn dust (44 g, 677.1 mmol) at room temperature and stirred for 1 h. Progress of the reaction was monitored by TLC & LCMS. After completion of the reaction, the reaction mass was filtered through celite pad, the filtrate was diluted with ethyl acetate (500 mL) and washed with water (100 mL), the organic layer was dried over Na2SO4, concentrated under reduced pressure to get the crude product. The crude product was purified by using column chromatography (silica gel, 100-200 mesh), eluting with 0-30% ethyl acetate: pet ether to give 7-((1 R,3R)-3-((tert-butyldimethylsilyl)oxy)cyclohexyl)-2-chloro-5,7-dihydro- 6H-pyrrolo[2,3-d]pyrimidin-6-one (33 g, 63% yield after 2 steps) as brown gummy liquid. LC-MS m / z [M+H]+ = 382.29,1H NMR (400 MHz, DMSO-de) 6 = 8.25 (s, 1 H), 4.58-4.68 (m, 1 H), 4.26 (s, 1 H), 3.63 (s, 2H), 2.30-2.40 (m, 1 H), 2.10-2.21 (m, 1 H), 1.55-1.80 (m, 5H), 1.45-1.50 (m, 1 H), 0.91 (s, 9H), 0.01-0.10 (m, 6H).

[0165] Step-7: Synthesis of 7'-((1 R,3R)-3-((tert-butyldimethylsilyl)oxy)cyclohexyl)-2'- chlorospiro[cyclopropane-1,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one: To a stirred solution of 7-((1 R,3R)- 3-((tert-butyldimethylsilyl)oxy)cyclohexyl)-2-chloro-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (11g, 28.87 mmol) and dibromoethane (7.4 mL, 86.61 mmol) in dry DMF (110 mL), was added NaH (60%, 3.4 g, 86.61 mmol) portion wise at room temperature and stirred for 4h. Progress of the reaction was monitored by TLC & LCMS. After completion of the reaction, the reaction was quenched sat.NFLCI, extracted with MTBE (3 x 100 mL). The organic layer was dried over Na2SO4, concentrated under reduced pressure to get the crude product. The crude product was purified by column chromatography (silica gel, 100-200 mesh), eluting with 0-15% ethyl acetate in pet ether to give 7'-((1 R,3R)-3-((tert- butyldimethylsilyl)oxy)cyclohexyl)-2'-chlorospiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one (10 g, 85% yield) as dark brown gummy liquid. LC-MS m / z [M+H]+= 408.31 ,1H NMR (400 MHz, DMSO-de) 6 = 8.16 (s, 1 H), 4.65-4.75 (m, 1 H), 4.26 (s, 1 H), 3.58-3.62 (m, 1 H), 2.30-2.42 (m, 1 H), 2.15-2.25 (m, 1 H), 1.81-1.91 (m, 2H), 1.55-1.80 (m, 7H), 1.55-1.80 (m, 7H), 0.91 (s, 9H), 1.39-1.49 (m, 1 H), 0.04 (s, 6H).

[0166] Synthesis of Compound A from Intermediate 1 PAT059659 FF intermediate 1

[0167] 3-ethoxy-4-nitro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole: To a stirred solution of 3-ethoxy-4- nitro-1 H-pyrazole (40.00 g, 254.6 mmol) and cesium carbonate (124.4 g, 381.8 mmol) in N, N- Dimethylformamide (400 mL), was added SEM-CI (90.10 mL, 509.1 mmol) at 0° C and then stirred at ambient temperature for 4 hours. The progress of the reaction was monitored by TLC and LCMS. After completion of reaction, reaction mass was quenched with ice-cold water and extracted with ethyl acetate. The organic layer was washed with ice-cold water, dried over sodium sulphate and concentrated under reduced pressure to give the crude product. The crude compound was purified by Sepa-Bean using silica gel (230-400 mesh), eluting with 0-10% ethyl acetate in pet-ether to give 3-ethoxy-4-nitro-1-((2- (trimethylsilyl)ethoxy)methyl)-1 H-pyrazole (70 g, 95% yield) as pale yellow color liquid.1H NMR (400 MHz, CDCI3) 6 = 8.13 (s, 1 H), 5.24 (s, 2H), 4.41- 4.39 (m, 2H), 3.36-3.60 (m, 2H), 1.48-1.45 (t, J = 6.8 HZ, 3H), 0.96-0.91 (m, 2H), 0.03-0.01 (s, 9H).

[0168] Synthesis of 3-ethoxy-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-4-amine): To a parr-shaker containing 3-ethoxy-4-nitro-1-((2-(trimethylsilyl)ethoxy)methyl)-1 H-pyrazole (70.00 g, 243.6 mmol) in methanol (250 mL), was added Pd / C (10%, 12.96 g) and stirred at ambient temperature at 80 psi hydrogen pressure for 1 hour. Progress of the reaction was monitored by TLC. After completion of reaction, reaction mass was filtered through celite pad and washed with 10% MeOH in DCM, the filtrates were concentrated under reduced pressure to give the crude product compound-3 (56 g, 89% yield) as brown liquid.1H NMR (400 MHz, CDCI3) 6 = 6.99 (s, 1 H), 5.24 (s, 2H), 5.12 (s, 2H), 4.27-4.22 (m, 2H), 3.52-3.47 (m, 2H), 2.73- 2.71 (s, 2H), 1.40-1.36 (m, 3H), 0.93-0.86 (m, 2H), 0.02-0.01 (s, 9H), LC-MS (m / z): 258.49 [M+H] +. 7'-((1 R,3R)-3-((tert-butyldimethylsilyl) oxy) cyclohexyl)-2'-((3-ethoxy-1-((2-(trimethylsilyl) ethoxy) methyl)-1 H-pyrazol-4-yl) amino) spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d] pyrimidin]-6'(7'H)-one : To a degassed solution of 7'-((1 R,3R)-3-((tert-butyldimethylsilyl)oxy)cyclohexyl)-2'-chlorospiro[cyclopropane- PAT059659 FF

[0169] 1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one (40.00 g, 98.04 mmol) and compound-3 (30.28 g, 117.6 mmol) in dry tetrahydrofuran (400 mL), was added sodium tert-butoxide (14.13 g, 147.1 mmol) and BrettphosPdG3 (4.44 g, 4.90 mmol), the reaction mass was closed under argon atmosphere and stirred at ambient temperature for 4 hours. Progress of the reaction was monitored by TLC and LCMS. After completion of the reaction, the reaction mass was filtered; the filtrate was concentrated under reduced pressure to give crude compound. The crude compound was purified by Sepa-Bean using silica gel (230-400 mesh), eluting with 20-50% ethyl acetate in pet-ether to give 7'-((1 R,3R)-3-((tert-butyldimethylsilyl) oxy) cyclohexyl)-2'-((3- ethoxy-1-((2-(trimethylsilyl) ethoxy) methyl)-1 H-pyrazol-4-yl) amino) spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d] pyrimidin]-6'(7'H)-one. (43 g, 66% yield) as brown gummy solid. 1 H NMR (400 MHz, CDCI3) 6 = 7.96 (s, 1 H), 7.63 (s, 1 H), 6.61 (s, 1 H), 5.34 (s, 2H), 4.88-4.81 (m, 1 H), 4.35-4.30 (m, 2H) ,4.26 (s, 1 H), 3.57-3.53 (m, 2H), 256 (br-s, 1 H), 2.39-2.30 (m, 1 H), 1.94-1 ,87(m, 1 H), 1.73-1.65 (m, 6H), 1.49-1.40 (m, 6H),1.27- 1.23 (m, 1 H), 0.95 (s, 9H), 0.57 (s, 6H), 0.20 (s, 9H), LC-MS (m / z): 629.78 [M+H] +

[0170] 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'- pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one To a stirred solution of 7'-((1 R,3R)-3-((tert-butyldimethylsilyl) oxy) cyclohexyl)-2'-((3-ethoxy-1-((2-(trimethylsilyl) ethoxy) methyl)-1 H-pyrazol-4-yl) amino) spiro[cyclopropane- 1 ,5'-pyrrolo[2,3-d] pyrimidin]-6'(7'H)-one (41.00 g, 65.19 mmol) in methanol (410 mL), was added 4M HCI in dioxane (820 mL) at 0 °C and stirred at 70 °C for 4 hours. Progress of the reaction was monitored by TLC & LCMS. After completion of reaction, reaction mass was diluted with ethyl acetate and concentrated under reduced pressure. The crude product was basified with aq. sodium bicarbonate and extracted with ethyl acetate. The organic layer was dried over sodium sulfate, concentrated under reduced pressure to get the crude compound. The crude product was triturated in ethyl acetate (10V) and stirred for 30 minutes and then filtered. The filtered solid was triturated in methanol (10V) and stirred for 1 hour, then filtered the filtered solid to give pure compound. Then compound co-distilled with acetonitrile and water to give 2'-((3- ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopro pane-1 ,5'-pyrrolo[2,3- d]pyrimidin]-6'(7'H)-one (17.5 g, 68% yield) as pale pink solid. 1 H NMR (400 MHz, DMSO-d6) 6 = 11.61 (s, 1 H), 8.10 (s, 1 H), 7.78 (s, 1 H), 7.63 (s, 1 H), 4.65-4.59 (m, 1 H), 4.52 (s, 1 H), 4.17-4.12 (m, 2H), 4.06 (s, 1 H), 2.49 (s, 1 H), 2.24-2.20 (m, 1 H), 1 .75-1 .46 (m, 9H), 1 .45-1 .28 (m, 4H), LC-MS (m / z):385.23 [M+H] +.

[0171] This method resulted in the formation of a pale pink crystalline powder consisting of irregular shaped particles having a significant loss of drying of 2.5% by TGA, with a melting point of around 241.6 °C, as demonstrated in Example 9 and the corresponding Figures.

[0172] Example 2 - CDK Biochemical Assays

[0173] Biochemical assays measured the inhibitory effects of compounds in this disclosure on the enzymatic activity of CDK enzyme in complex with Cyclin protein partner by phosphorylation of Ser-780 (S780) on retinoblastoma protein peptide (RB1) in the presence of 1 mM adenosime-5 — triphosphate (ATP) and varying concentration of test compound in 20 mM 2-[4-(2- PAT059659 FF hydroxyethyl)paperazin-1-yl] ethanesulfonic acid (HEPES), pH 7.5, 10 mM MgCI2, 1 mM dithiothreitol (DTT), 0.01 % bovine serum albumin (BSA), 0.005% Tween 20. Total Reaction volume of 10 pL proceeded for 60 minutes at room temperature (25°C) and were quenched with 5 pL of 200 mM 2',2",2"'-(Ethane-1 ,2-diyldinitrilo)tetraacetic acid (EDTA) pH 8.0 before addition of 5 pL detection solution containing 100 nM fluorophore conjugate streptavidin allophycocyanin (SA-APC), 2 nM Europium labelled Anti-p-RB(S780)-K (Perkin Elmer, 64CUSKAY), 50 mM HEPES, pH 7.5, 400 mM potassium fluoride (KF), 0.1 % BSA, and 0.01 % Tween-20. Phosphorylation of S780 on RB1 peptide (His-MBP-RB1 [773-924] - SEQ ID NO. 9) was detected by TR-FRET after 3 hour incubation with detection solution. Percent phosphorylation activity was plotted against log concentration of compound to generate an apparent IC5o. The following CDK enzyme in complex with different cyclin proteins and protein peptide substrate were used in these assays:

[0174] CDK1 / CyclinB1 , Avi-tag, 10 pM used in the assay

[0175] CDK2 / CyclinE1 , Avi-tag, 100 pM used in the assay

[0176] CDK4 / cyclinD1 , Avi-tag, 20 pM used in the assay CDK6 / cyclinD3, Avi-tag, 10 pM used in the assay His-MBP-RB1 [773-924], Avi-tag, 200 nM used in the assay

[0177] The avi-tag is a C-terminal fusion on the CDK1 / 2 / 4 / 6 sequence in each of these two-protein complexes. The known avi-tag sequence, GLNDIFEAQKIEWHE (SEQ ID NO. 10), is a substrate for E. coli biotin ligase BirA, which covalently attaches a biotin molecule to the e-amino group of the lysine in that sequence. The avi-tag is attached with an 8 amino-acid linker sequence GGSGGGGS (SEQ ID NO. 11), resulting in the full C-terminal fusion GGSGGGGSGLNDIFEAQKIEWHE (SEQ ID NO. 12).

[0178] The sequences of the recombinant proteins used here are provided below.

[0179] Recombinant proteins were generated as N-terminal fusions of 9x His tag - E. coli Maltose Binding Protein (MBP), with or without a C-terminal Avi-tag.1CDK2 and His-MBP-RB1 [773-924]- c-avi were expressed in E. coli while the remainder were expressed in Sf21 insect cells. Genes were synthesized commercially (GeneArt and Twist Bioscience) using codon frequencies appropriate to the respective organisms and proprietary codon optimization strategies, and inserted into pET 24 (E. coli) or pDEST8 (insect) vectors already containing the 9x His-MBP tag using standard methods of PCR and Gibson Assembly.2E. coli expression was performed by auto-induction overnight at 18 or 21 C. For insect cell expression, bacmids and viruses were PAT059659 FF created,3then used to synchronously infect Sf21 insect cells with harvest at 40-48 hrs., per standard protocols.4

[0180] For CDK2 / Cyclin E and CDK1 / Cyclin B, the CDK and Cyclin proteins were expressed separately, lysed by sonication and clarified by centrifugation. The fusion proteins were each separately purified by immobilized metal ion affinity chromatography (IMAC) (5 mL HisTrap, Cytiva) and Superdex 200 26 / 60 (Cytiva) size-exclusion and complexed in vitro (1-3 hrs incubation at room temperature) prior to tag cleavage by recombinant his-tagged Tobacco Etch Virus protease or his-tagged Human rhinovirus 3C protease, as appropriate. The cleaved complex is further purified by Superdex 75 26 / 60 (Cytiva) size-exclusion followed by reverse-IMAC, in which the complex is in the column flow-through and residual His-MBP and his-tagged proteases are retained. The final complex is concentrated as desired by centrifugal concentration (Amicon Ultra 10,000 Da MWCO 15 mL concentrators, Millipore).

[0181] For E. coli expressed CDK2 and His-MBP-RB1 [773-924]-c-avi, the proteins were biotinylated in vitro after the first IMAC step.1

[0182] For CDK4 / Cyclin D1 and CDK6 / Cyclin D3, the complexes were expressed by simultaneous coinfection of insect cells by three separate viruses including one expressing untagged E. coli BirA. The expression media is supplemented with 50 uM d-biotin. The resulting protein complexes purified similarly. After lysis and clarification, the complex is purified by IMAC, Superdex 20026 / 60 size exclusion, tag cleavage by human rhinovirus 3C protease, Superdex 75 26 / 60 size exclusion and reverse IMAC. BirA is typically not retained on the first IMAC, and excess CDK or Cyclin monomer is separated on the Superdex 200 size exclusion. Cleaved His-MBP and remaining monomer are removed by the Superdex 75 size exclusion and the reverse IMAC step. Final concentration by centrifugal concentrators as described above.

[0183] For the is-MBP-RB1 [773-924]-c-avi, the protein was lysed by sonication and clarified by centrifugation. After IMAC capture and in vitro biotinylation, the fusion protein is purified by Q HP HiTrap (Cytiva) with a gradient elution after binding in low salt, followed by SP HP HiTrap (Cytiva) with a gradient elution. The material is finally purified on a Superdex 75 26 / 60 as a polishing step. Although there is a TEV protease cleavage site in this construct, it is not removed for this purpose. Final concentration by centrifugal concentrators as described above.

[0184] 9x His-MBP-3C-CDK2-c-avi (SEQ ID NO. 1)

[0185] MHHHHHHHHHMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAA TGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIY PAT059659 FF

[0186] NKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDV GVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGV TVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYE EELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSG LEVLFQGPMENFQKVEKIGEGTYGWYKARNKLTGEWALKKIRLDTETEGVPSTAIREISLLKE LNHPNIVKLLDVIHTENKLYLVFEFLHQDLKKFMDASALTGIPLPLIKSYLFQLLQGLAFCHSHRVL HRDLKPQNLLINTEGAIKLADFGLARAFGVPVRTYTHEWTLWYRAPEILLGCKYYSTAVDIWSL GCIFAEMVTRRALFPGDSEIDQLFRIFRTLGTPDEWWPGVTSMPDYKPSFPKWARQDFSKVV PPLDEDGRSLLSQMLHYDPNKRISAKAALAHPFFQDVTKPVPHLRLGGSGGGGSGLNDIFEAQ KIEWHE*

[0187] 9x His-MBP-TEV-Cyclin E1 (SEQ ID NO. 2)

[0188] MHHHHHHHHHMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAA TGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIY NKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDV GVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGV TVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYE EELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSE NLYFQGGSGMPRERRERDAKERDTMKEDGGAEFSARSRKRKANVTVFLQDPDEEMAKIDRT ARDQCGSQPWDNNAVCADPCSLIPTPDKEDDDRVYPNSTCKPRIIAPSRGSPLPVLSWANREE VWKI M LN KE KT YL R DQ H F L EQ H P LLQ P KM R Al LL D WL M E VC E VY KL H R ET FY LAQ D F F D RY M A

[0189] TQENWKTLLQLIGISSLFIAAKLEEIYPPKLHQFAYVTDGACSGDEILTMELMIMKALKWRLSPLT IVSWLNVYMQVAYLNDLHEVLLPQYPQQIFIQIAELLDLCVLDVDCLEFPYGILAASALYHFSSSE LMQKVSGYQWCDIENCVKWMVPFAMVIRETGSSKLKHFRGVADEDAHNIQTHRDSLDLLDKA RAKKAMLSEQNRASPLPSGLLTPPQSGKKQSSGPEMA*

[0190] 9x His-MBP-TEV-CDK1 -c-avi (SEQ ID NO. 3)

[0191] MHHHHHHHHHMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAA TGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIY NKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDV GVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGV TVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYE EELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSE NLYFQGGMEDYTKIEKIGEGTYGWYKGRHKTTGQWAMKKIRLESEEEGVPSTAIREISLLKEL RHPNIVSLQDVLMQDSRLYLIFEFLSMDLKKYLDSIPPGQYMDSSLVKSYLYQILQGIVFCHSRR VLHRDLKPQNLLIDDKGTIKLADFGLARAFGIPIRVYTHEWTLWYRSPEVLLGSARYSTPVDIW SIGTIFAELATKKPLFHGDSEIDQLFRIFRALGTPNNEVWPEVESLQDYKNTFPKWKPGSLASHV KNLDENGLDLLSKMLIYDPAKRISGKMALNHPYFNDLDNQIKKMGGSGGGGSGLNDIFEAQKIE WHE*

[0192] 9x His-MBP-Cyclin B1 (SEQ ID NO. 4)

[0193] MHHHHHHHHHMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAA TGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIY NKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDV GVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGV TVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYE EELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSE PAT059659 FF

[0194] NLYFQGGMALRVTRNSKINAENKAKINMAGAKRVPTAPAATSKPGLRPRTALGDIGNKVSEQL

[0195] QAKMPMKKEAKPSATGKVIDKKLPKPLEKVPMLVPVPVSEPVPEPEPEPEPEPVKEEKLSPEPI

[0196] LVDTASPSPMETSGCAPAEEDLCQAFSDVILAVNDVDAEDGADPNLCSEYVKDIYAYLRQLEEE

[0197] QAVRPKYLLGREVTGNMRAILIDWLVQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKKMLQLV

[0198] GVTAMFIASKYEEMYPPEIGDFAFVTDNTYTKHQIRQMEMKILRALNFGLGRPLPLHFLRRASKI

[0199] GEVDVEQHTLAKYLMELTMLDYDMVHFPPSQIAAGAFCLALKILDNGEWTPTLQHYLSYTEESL

[0200] LPVMQHLAKNWMVNQGLTKHMTVKNKYATSKHAKISTLPQLNSALVQDLAKAVAKV*

[0201] 9x His-MBP-CDK4-c-avi (SEQ ID NO. 5)

[0202] MHHHHHHHHHMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAA

[0203] TGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIY

[0204] NKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDV

[0205] GVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGV

[0206] TVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYE

[0207] EELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSE

[0208] NLYFQGGSGMATSRYEPVAEIGVGAYGTVYKARDPHSGHFVALKSVRVPNGGGGGGGLPIST

[0209] VREVALLRRLEAFEHPNWRLMDVCATSRTDREIKVTLVFEHVDQDLRTYLDKAPPPGLPAETI

[0210] KDLMRQFLRGLDFLHANCIVHRDLKPENILVTSGGTVKLADFGLARIYSYQMALTPWVTLWYR

[0211] APEVLLQSTYATPVDMWSVGCIFAEMFRRKPLFCGNSEADQLGKIFDLIGLPPEDDWPRDVSL

[0212] PRGAFPPRGPRPVQSWPEMEESGAQLLLEMLTFNPHKRISAFRALQHSYLHKDEGNPEGGS

[0213] GGGGSGLNDIFEAQKIEWHE*

[0214] 9x His-MBP-Cyclin D1 (SEQ ID NO. 6)

[0215] MHHHHHHHHHMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAA

[0216] TGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIY

[0217] NKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDV

[0218] GVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGV

[0219] TVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYE

[0220] EELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSE

[0221] NLYFQGGSGMEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCVQKEVLP

[0222] SMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKKSRLQLLGATCMFVASKMKETI

[0223] PLTAEKLCIYTDNSIRPEELLQMELLLVNKLKWNLAAMTPHDFIEHFLSKMPEAEENKQIIRKHAQ

[0224] TFVALCATDVKFISNPPSMVAAGSWAAVQGLNLRSPNNFLSYYRLTRFLSRVIKCDPDCLRAC

[0225] QEQIEALLESSLRQAQQNMDPKAAEEEEEEEEEVDLACTPTDVRDVDI*

[0226] 9x His-MBP-CDK6-c-avi (SEQ ID NO. 7)

[0227] MHHHHHHHHHMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAA

[0228] TGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIY

[0229] NKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDV

[0230] GVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGV

[0231] TVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYE

[0232] EELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSM

[0233] MGSSSSGLEVLFQGPMEKDGLCRADQQYECVAEIGEGAYGKVFKARDLKNGGRFVALKRVRV

[0234] QTGEEGMPLSTIREVAVLRHLETFEHPNVVRLFDVCTVSRTDRETKLTLVFEHVDQDLTTYLDK

[0235] VPEPGVPTETIKDMMFQLLRGLDFLHSHRWHRDLKPQNILVTSSGQIKLADFGLARIYSFQMAL

[0236] TSWVTLWYRAPEVLLQSSYATPVDLWSVGCIFAEMFRRKPLFRGSSDVDQLGKILDVIGLPGE PAT059659 FF

[0237] EDWPRDVALPRQAFHSKSAQPIEKFVTDIDELGKDLLLKCLTFNPAKRISAYSALSHPYFQDLER CKENLDSHLPPSQNTSELNTAGGSGGGGSGLNDIFEAQKIEWHE*

[0238] 9x His-MBP-Cyclin D3 (SEQ ID NO. 8)

[0239] MHHHHHHHHHMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAA TGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIY NKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDV GVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGV TVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYE

[0240] EELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSLE VLQGPGSMELLCCEGTRHAPRAGPDPRLLGDQRVLQSLLRLEERYVPRASYFQCVQREIKPH M R KM LAY WM L E VC E EQ RC E E E VF P LAM N YL D RY LSCVPT R KAQ LQ L LG AVC M L LAS KLR ETT PLTIEKLCIYTDHAVSPRQLRDWEVLVLGKLKWDLAAVIAHDFLAFILHRLSLPRDRQALVKKHA QTFLALCATDYTFAMYPPSMIATGSIGAAVQGLGACSMSGDELTELLAGITGTEVDCLRACQEQ

[0241] IEAALRESLREASQTSSSPAPKAPRGSSSQGPSQTSTPTDVTAIHL*

[0242] 9x His-MBP- RB1 [773-924]-c-avi (SEQ ID NO. 9)

[0243] MHHHHHHHHHMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAA TGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIY NKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDV GVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGV TVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYE EELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSE NLYFQGGSTRPPTLSPIPHIPRSPYKFPSSPLRIPGGNIYISPLKSPYKISEGLPTPTKMTPRSRIL VSIGESFGTSEKFQKINQMVCNSDRVLKRSAEGSNPPKPLKKLRFDIEGSDEADGSKHLPGES KFQQKLAEMTSTRTRMQKQKMNDSMDTSNGGSGGGGSGLNDIFEAQKIEWHE*

[0244] 1. Li, Y. & Sousa, R. Expression and purification of E. coli BirA biotin ligase for in vitro biotinylation. Protein Expr. Purif. 82, 162-7 (2012).

[0245] 2. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343-345 (2009).

[0246] 3. Invitrogen. Bac-to-Bac® Baculovirus Expression System. An Efficient Site-Specific Transposition System to Generate Baculovirus for High-Level Expression of Recombinant Proteins. (2010).

[0247] 4. Invitrogen life technologies. Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques. (2002).

[0248] IC50 values determined in the biochemical assays are listed in the following table: PAT059659 FF

[0249] Example 3 - Improved Synthesis of 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one

[0250] 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'- pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one was made using the following reaction scheme:

[0251] M.Wt: 157.13 M.Wt: 257.25 M.Wt: 227.26

[0252] C Compound I-A3

[0253] Compound I-A1 ompound I-A2

[0254] To a mixture of 3-Ethoxy-4-nitro-1 H-pyrazole (1.0 eq, 1w / w) and Na2CO3(1.2 eq) in EtOAc (10 v / w) was added Boc20 (1 .0 eq) at 0 - 5 °C. The mixture was allowed to warm up to 25 - 35 °C and stirred for 2-3 h. After reaction completion, 10% Pd / C (50% wet) (0.15 w / w) was added into the autoclave at 25 - 35 °C and H2(3.5 - 4.0 bar) was added. The resulting mixture was stirred at 25 - 35 °C for 15 - 17 h.

[0255] After reaction completion, the mixture was filtered through hyflo filter aid (commercially available, approx. 90 % SiO2, 2-5 % AI2O3, 1-4 % Fe2O3), washed with EtOAc (5 v / w), concentrated below 45 °C. The evaporation residue was treated with n-heptane (3 v / w) and concentrated below 45°C. This operation was repeated once more. The residual suspension was slurred in n-heptane (5.0 v / w) and water (5.0 v / w) at 25 - 35 °C to obtain Compound I-A3 as an off-white solid. PAT059659 FF DBU, THF, 60-65 °C

[0256] M Wt: 293.75 Boc

[0257] Compound I-A4 M Wt: 484.56

[0258] Compound I-A5

[0259] (Obtainable using method of Example 1 , Intermediate 1 followed by deprotection of Intermediate 1 , removing TBS using HCI / dioxane)

[0260] To a solution of Compound I-A4 (1.05 eq) in THF (12 v / w) was added Compound I-A3 (1.0 eq). The mixture was degassed for 20 - 30 min with argon and DBU (2.0 eq) was added at 25 - 35 °C. The mixture was once again degassed for 5 - 10 min with argon. BrettphosPd-G3 (0.05 eq) was added at 25 - 35 °C, the mixture degassed for 2 - 5 min with nitrogen, and the mixture was warmed to 60 - 65 °C and stirred at this temperature for 4 - 6 h.

[0261] After reaction completion, the reaction mass was filtered through hyflo filter aid (commercially available, approx. 90 % SiO2, 2-5 % AI2O3, 1-4 % Fe2O3), washed with EtOAc (10.0 v / w), treated with water (10.0 v / w) and extracted with EtOAc (5 v / w). The organic layer was washed with aq. NaCI solution (5.0 v / w) and concentrated under reduced pressure.

[0262] The crude compound was purified by column chromatography (SiO2; 100-200 mesh) with 25 - 50% EtOAc in n-heptane. The combined fractions were treated with ENOPc carbon(0.2 w / w) and SiliaMet thiourea (2x0.3 w / w) in EtOAc (15 v / w) at 25 - 35 °C. The organic mixture was concentrated under reduced pressure and the evaporation residue was slurred in 10% MTBE in n-heptane at 25 - 35 °C.

[0263] M Wt: 484.56

[0264] 2'-((3-ethoxy-1H-pyrazol-4-yl)amino)-7'-((1R,3R)-3-

[0265] Compound I-A5 hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-cf]pyrimidin]-6'(7'H)-one PAT059659 FF

[0266] To a mixture of conc.H2S04 (0.05 eq.) in H2O (3.5 v / w) at 25 - 35 °C, and iPrOH (1 .5 v / w), was added eq, 1 w / w) at 25 - 35 °C. The mixture was warmed to

[0267] 70 - 75 °C and stirred at this temperature for 12 - 14 h.

[0268] After reaction completion, the mixture was cooled to 25 - 35 °C, treated with a solution of Na2CO3(0.5 w / w) in water (5.0 v / w) at 25 - 35 °C, and stirred for 2 - 3 h at 25 - 35 °C. The mixture was filtered and the filter cake was washed with water (5.0 v / w) to get the title compound as an off white to light pink color solid.

[0269] As mentioned above, the drug product made by the method of Comparative Example 1 (i.e. that of PCT / IB2024 / 051412) has a significant loss of drying of 2.5% as calculated by TGA, with a melting point of around 241.6 °C. In comparison, the melting point of the drug product made by the method of Example 3 is around 252.52 °C, and the loss of drying is negligible (calculated by TGA as 0.049%). See Example 9 and the corresponding Figures.

[0270] Example 4 - Formation of the Hydrochloride Salt

[0271] To a suspension of 1200 mg of 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one in 60 mL of THF at room temperature, 1 .05 equivalents of aqueous HCI (35% concentration; specific gravity: 1.19) were slowly added with stirring. The resulting mixture was then slurred at 50 °C for 16 hours. Afterward, the mixture was cooled to room temperature and stirred for an additional 4 hours at a temperature range of 2-8 °C. The suspension was subsequently centrifuged, and the residue was dried under reduced pressure at room temperature for 3-4 days. The yield obtained was 1 .28 g.

[0272] Example 5 - Formation of the Mesylate Salt

[0273] To a suspension of 1200 mg of 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one in 60 mL of THF at room temperature, 1.05 equivalents of Methanesulfonic acid (99% concentration; specific gravity: 1 .48) were slowly added with stirring. The resulting mixture was then slurred at 50 °C for 16 hours. Afterward, the mixture was cooled to room temperature and stirred for an additional 4 hours at a temperature range of 2-8 °C. The suspension was subsequently centrifuged, and the PAT059659 FF residue was dried under reduced pressure at room temperature for 3-4 days. The yield obtained was 1 .28 g.

[0274] Example 6 - Formation of the Phosphoric Acid Co-Crystal

[0275] To a suspension of 1200 mg of 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one in 60 mL of THF at room temperature, 1 .05 equivalents of aqueous o-phosphoric acid (85% concentration; specific gravity: 1 .73) were slowly added with stirring. The resulting mixture was then slurred at 50 °C for 16 hours. Afterward, the mixture was cooled to room temperature and stirred for an additional 4 hours at a temperature range of 2-8 °C. The suspension was subsequently centrifuged, and the residue was dried under reduced pressure at room temperature for 3-4 days. The yield obtained was 1 .28 g.

[0276] Example 7 - X-Ray Powder Diffraction

[0277] XRPD method

[0278] X-ray powder diffraction (XRPD) patterns were obtained at room temperature (approximately 20 °C) using a Bruker Advance D8 in reflection geometry. Powders were analyzed using a zero background Si flat sample holder. The radiation used was Cu Ka (A = 1.54060 A) at room temperature (approximately 20° C). Patterns were measured between 2° and 40° 2theta.

[0279] Sample amount: 5-10 mg

[0280] Sample holder: zero background Si flat sample holder

[0281] XRPD parameter PAT059659 FF

[0282] Example 7a

[0283] The free base form of 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one formed by the method of PCT / IB2024 / 051412 I Comparative Example 1 of the present application (XRPD pattern shown in Figure 1).

[0284] Example 7b

[0285] The free base form of 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one at room temperature formed by the method of Example 3 (XRPD pattern shown in Figure 2). The position and relative intensity of important XRPD peaks are as below:

[0286] Pos. [°20] Rel. Int. [%]

[0287] 6.1027 100.00

[0288] 11.2888 10.59

[0289] 12.6342 28.10

[0290] 13.0695 17.55

[0291] 14.8292 8.27

[0292] 14.9301 9.49

[0293] 16.0214 6.28

[0294] 17.5316 5.91

[0295] 18.3214 22.58

[0296] 19.3544 26.27

[0297] 20.3865 26.70

[0298] 20.9822 5.70

[0299] 21.1434 9.47

[0300] 21.2944 6.82

[0301] 22.6771 33.06

[0302] 23.1080 8.21

[0303] 23.4999 13.78

[0304] 24.0307 3.86

[0305] 24.4092 4.06

[0306] 24.5234 4.75 PAT059659 FF

[0307] Pos. [°20] Rel. Int. [%]

[0308] 24.8562 7.11

[0309] 24.9824 9.16

[0310] 25.6297 18.30

[0311] 27.1516 3.27

[0312] 28.4304 2.91

[0313] 29.6596 2.87

[0314] 30.6218 4.91

[0315] 30.7440 6.86

[0316] 33.3059 3.75

[0317] 37.1027 4.99

[0318] 37.6115 2.37

[0319] 41.4486 2.86

[0320] 42.4536 2.27

[0321] Example 7c

[0322] The hydrochloride salt of 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one (XRPD pattern shown in Figure 3). The position and relative intensity of important XRPD peaks are as below:

[0323] PAT059659 FF

[0324] Pos. [°20] Rel. Int. [%]

[0325] 6.8948 100.00

[0326] 9.3615 33.86

[0327] 9.8414 37.16

[0328] 12.2629 9.80

[0329] 13.1623 21.01

[0330] 13.7145 75.85

[0331] 15.8390 10.17

[0332] 17.7003 13.82

[0333] 18.0283 17.12

[0334] 18.7153 25.62

[0335] 19.6406 14.86

[0336] 20.3782 8.59

[0337] 21.0822 17.43

[0338] 21.5104 47.92

[0339] 23.8353 54.82

[0340] 24.6278 41.74

[0341] 26.1250 5.67

[0342] 27.0257 12.19

[0343] 27.6382 17.36

[0344] 28.3766 5.39

[0345] 29.6459 3.93

[0346] 30.7855 3.21

[0347] 31.7716 5.23

[0348] 33.2720 2.82

[0349] 36.3599 3.45

[0350] 38.6124 2.56 PAT059659 FF

[0351] Example 7d

[0352] The mesylate salt of 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one (XRPD pattern shown in Figure 4). The position and relative intensity of important XRPD peaks are as below:

[0353] Pos. [°26] ReL Int. [%]

[0354] 8.4805 8.44

[0355] 9.2352 100.00

[0356] 12.1442 10.53

[0357] 16.1827 46.85

[0358] 16.7816 13.40

[0359] 18.2604 56.97

[0360] 18.8457 18.55

[0361] 19.7022 12.38

[0362] 21.2635 26.41

[0363] 21.4442 24.39

[0364] 22.2035 6.80

[0365] 22.7535 8.62

[0366] 23.6149 9.08

[0367] 24.4657 54.20

[0368] 25.8035 4.04

[0369] 26.6597 4.35

[0370] 27.5137 3.44

[0371] 28.1176 11.51

[0372] 28.7748 2.03

[0373] 29.7086 2.81

[0374] 30.5459 7.20

[0375] 31.1917 3.28

[0376] 32.0511 2.16

[0377] 32.6158 2.50

[0378] 33.1146 2.65

[0379] 37.1285 2.59 PAT059659 FF

[0380] Example 7e

[0381] The phosphoric acid co-crystal of 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one (XRPD pattern shown in Figure 5). The position and relative intensity of important XRPD peaks are as below:

[0382] Pos. [°26] ReL Int. [%]

[0383] 5.5994 100.00

[0384] 7.5422 22.52

[0385] 8.2322 43.71

[0386] 10.4967 11.62

[0387] 11.9748 15.04

[0388] 12.9305 10.69

[0389] 13.7403 25.78

[0390] 15.0553 33.81

[0391] 15.6734 34.90

[0392] 16.7581 6.62

[0393] 17.8654 11.32

[0394] 18.6940 16.36

[0395] 19.1394 12.85

[0396] 19.6865 41.82

[0397] 20.5404 19.52

[0398] 20.9572 31.94

[0399] 21.6928 20.25

[0400] 22.5793 14.99

[0401] 23.4630 27.54

[0402] 23.9120 31.92

[0403] 24.6480 27.53

[0404] 25.4361 19.06

[0405] 26.7637 6.68

[0406] 27.4489 14.62

[0407] 28.8507 10.25

[0408] 29.7578 6.22 PAT059659 FF

[0409] Example 8 - Crystallographic Properties

[0410] The single crystal structure of free base form of 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)- 3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one at room temperature formed by the method of Example 3 was measured at 100 °K using Cu Ka (0.15418 nm). The physical properties are detailed below:

[0411] Parameter Data measured at 100 °K Theoretical X-ray powder diffraction pattern at ambient temperature

[0412] Crystal system / Monoclinic / C2 Monoclinic / C2 space group

[0413] Unit cell a = 16.8844(3) A, a = 90 ° a = 16.964(4) A, a = 90 ° dimensions b = 7.7209(2) A, 8 = 111.6690(10) ° b = 7.8401 (18) A, 8 = 112.093(18) ° c = 15.6270(3) A, y = 90 ° c = 15.688(4) A, y = 90 °

[0414] Volume 1893.22(7) A31933.3(9) A3

[0415] Density (calculated) 1 .349 g / cm31 .3208(6) g / cm3

[0416] Example 9 - DSC and TGA

[0417] DSC (differential scanning calorimetry) and TGA (Thermogravimetric analysis) was conducted using a TA Discovery DSC instrument and a TA Discovery TGA instrument respectively for the free base form of 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one made by: i) the method of PCT / IB2024 / 051412 I Comparative Example 1 of the present application (DSC shown in Figure 6 and TGA shown in Figure 7); and ii) the method of Example 3 (both DSC and TGA shown in Figure 8).

[0418] DSC Method

[0419] 1-3 mg of sample was placed in an aluminium T-zero crucible that closed with a pin-hole lid. The heating rate was 10°C per minute in the temperature range between 0 and 300°C. Temperatures are reported in degrees Celsius (°C) and enthalpies are reported in Joules per gram (J / g). Plots are showing endothermic peaks as down. The endothermic melt peak (melting point) was evaluated for extrapolated onset temperature. The accuracy of the measured sample temperature with this method is within about ±1 °C, and the heat of fusion can be measured within a relative error of about ±5%. PAT059659 FF

[0420] TGA Method

[0421] TGA curves were obtained using a TA Discovery TGA instrument. 2-1 Omg of sample was placed into an aluminum crucible and closed with a pin-hole lid. The TGA curve was measured at a heating rate of 10°C / min between 30-300°C. The LoD (Loss of drying) was calculated between 30°C and 200°C. The weight loss is plotted against the measured sample temperature. Temperatures are reported in degrees Celsius (°C) and weight loss in %.

[0422] Results

[0423] The melting (onset) temperature of the 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one made by the method of Example 3 is significantly higher (252.52 °C) than that of PCT / IB2024 / 051412 I Comparative Eaxamplel (241.62 °C). Further, the PCT / IB2024 / 051412 I Comparative Example 1 product has a noticeable weight loss due to water loss at 123.77 °C. The product made by Example 3 does not have a significant water loss around this temperature.

[0424] Example 10 - Physicochemical Properties

[0425] The free form of Example 3, the hydrochloride salt, the mesylate salt, and the phosphoric acid cocrystal were analyzed to have > 99 % chemical and chiral purity. Both hydrochloride and mesylate salts were determined to have a (1 :1) stoichiometry. At 1 % concentration, salts resulted in a clear solution with a pH of 3. However, the free form resulted in a suspension (pH 7). In biorelevant media and in pH buffers, no significant improvement of solubility was noticed. Salt / cocrystal formation resulted only in an improved water solubility from 0.01 mg / mL to 1 .2 mg / mL. In general, solubility increased with a decrease in the pH of the medium.

[0426] Example 11 - Morphic Properties

[0427] The free form of Example 3, the hydrochloride salt, the mesylate salt, and the phosphoric acid cocrystal are crystalline. The phosphoric acid form is a cocrystal given the small pKA differences between o-phosphoric acid and the 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one, and is unstable under DVS, grinding, and wet granulation experiments.

[0428] However, the two salts (hydrochloride and mesylate) and the free form were found to be unaffected by grinding, granulation, and compression. The phosphoric acid cocrystal has a melting point of 179 °C, while the salts have melting points in the range of 224-241 °C. The free form showed a glass transition temperature of 130 °C. Glass transitions of hydrochloride and PAT059659 FF mesylate appeared at about 180 °C and 225 °C, respectively. DVS isotherm plots showed that at 80 % relative humidity exposure, the free form and the mesylate salt absorb about 0.6% moisture, while the hydrochloride acid salt adsorbs 1.9% moisture. In the vibration study (phosphoric acid cocrystal was not performed), only the hydrochloride salt was found to change its XRPD pattern in aqueous medium.

[0429] SEM images were taken of the free base form of 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'- ((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one formed by the method of Example 3 (see Figure 9, right hand side) as compared to the method of PCT / IB2024 / 051412 I Comparative Example 1 (see Figure 9, left hand side), showing the improvement in crystal morphology.

Claims

1. PAT059659 FFCLAIMS1 . A method of preparing 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxy cyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one,said method comprising contacting a compound of formulaacid.

2. The method according to claim 1 , wherein the acid is hydrochloric acid or sulfuric acid.

3. The method according to claim 2, wherein the acid is sulfuric acid.

4. The method according to any one of claims 1 to 3, wherein the compound offormula (I-A5), Boc (I-A5) is made by contacting a compound ofPAT059659 FF formulacompound of formula (I-A3),the presence of a first base and a catalyst.

5. The method according to claim 4, wherein the catalyst is selected from Pd(dtbpf)CI2, Pd(dppf)CI2, Pd(PPh3)4, Pd2(dba)3, Pd(OAc)2, RuPhos Pd G2, RuPhos Pd G3, Ni(COD)2, Ni(dppp)CI2and Ni(acac)2.

6. The method according to claim 5, wherein the catalyst is RuPhos Pd G2 or RuPhos Pd G3.

7. The method according to claim 6, wherein the catalyst is RuPhos Pd G3.

8. The method according to any one of claims 4 to 7, wherein the first base is selected from Et3N, Et2NH, DIPEA, NMI,nBuNH2, ‘BuNH2, NH3, ^COs, Na2CO3, KOH, NaOH, NaHCOs, morpholine, DBN, DBU and TMG.

9. The method according to claim 8, wherein the first base is DBU or TMG.

10. The method according to claim 9, wherein the first base is DBU.PAT059659 FF11 . The method according to any one of claims 4 to 10, wherein the compound offormula (I-A3), Boc (I-A3), is made by contacting a compound of formula (I-A1 ),(|-A1) with a Boc (tert-butoxycarbonyl) introducing agent, a second base, a hydrogenating catalyst and hydrogen gas.

12. The method according to claim 11 , wherein the Boc introducing agent is selected from BOC2O, Boc chloride, Boc imidazole and Boc azide.

13. The method according to claim 12, wherein the Boc introducing agent is BOC2O.

14. The method according to any one of claims 11 to 13, wherein the second base is selected from Na2CO3, NaHCOs, K2CO3, KHCO3, LiOH, NaOH, KOH, NaOMe, NaOEt, NaH and ‘BuOK.

15. The method according to claim 14, wherein the second base is Na2COs.

16. The method according to any one of claims 11 to 15, wherein the hydrogenating catalyst is selected from a platinum based catalyst (e.g. Pt / V / C, PtA / / Fe / C, PtO2 or Pt / C), a palladium based catalyst (e.g. Pd / C or Pd(OH)2 / C), Raney Ni, Raney Co, a mixture of Fe with NH4CI, a mixture of Zn with NH4CI, B2Pin2 and nitroreductase.PAT059659 FF17. A crystalline form of 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxy cyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one,or a solvate thereof made by the method of any one of claims 1 to 16.

18. A crystalline form of 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3- hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one or a pharmaceutically acceptable solvate and / or salt thereof.

19. The crystalline form according to claim 17 or claim 18, wherein the 2'-((3-ethoxy- 1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'- pyrrolo[2,3-d]pyrimidin]-6'(7'H)-one is in the form of a free base or a solvate thereof.

20. The crystalline form according to claim 19, wherein the melting point is above 245°C.21 . The crystalline form according to claim 20, wherein the melting point is above 250°C.

22. The crystalline form according to claim 20 or claim 21 , wherein the melting point is determined by DSC.PAT059659 FF23. The crystalline form according to claim 22, wherein the melting point is determined by DSC onset temperature.

24. The crystalline form according to claim 23, wherein the melting point is determined by DSC onset temperature at heating rate of 10°C per minute.

25. The crystalline form according to any one of claims 17 to 24, characterized by an X-ray powder diffraction pattern comprising four or more 20 values selected from the group consisting of:wherein the temperature is about room temperature and the radiation used has a wavelength of 1.54060A.

26. The crystalline form according to any one of claims 17 to 25, having an X-ray powder diffraction pattern substantially the same as the X-ray powder diffraction spectrum as shown in FIG. 2, at about room temperature wherein the radiation used has a wavelength of 1 .54060 A.

27. The crystalline form according to any one of claims 17 to 26, having the following unit cell dimensions at 100 °K as determined by single crystal X-ray diffraction:PAT059659 FFCrystal System: MonoclinicSpace Group: C2 a = 16.88 A (± 0.1 A) b = 7.72 A (± 0.1 A) c = 15.63 A (± 0.1 A) p = 111.67° (± 0.1 °).

28. The crystalline form according to claim 18, wherein the 2'-((3-ethoxy-1 H-pyrazol- 4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3- d]pyrimidin]-6'(7'H)-one is in the form of a hydrochloride salt.

29. The crystalline form according to claim 28, characterized by an X-ray powder diffraction pattern comprising four or more 20 values selected from the group consisting of:wherein the temperature is about room temperature and the radiation used has a wavelength of 1.54060A.PAT059659 FF30. The crystalline form according to claim 28 or claim 29, having an X-ray powder diffraction pattern substantially the same as the X-ray powder diffraction spectrum as shown in FIG. 3, at about room temperature wherein the radiation used has a wavelength of 1 .54060 A.31 . The crystalline form according to claim 18, wherein the 2'-((3-ethoxy-1 H-pyrazol- 4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3- d]pyrimidin]-6'(7'H)-one is in the form of a mesylate salt.

32. The crystalline form according to claim 31 , characterized by an X-ray powder diffraction pattern comprising four or more 20 values selected from the group consisting of:wherein the temperature is about room temperature and the radiation used has a wavelength of 1.54060A.

33. The crystalline form according to claim 31 or claim 32, having an X-ray powder diffraction pattern substantially the same as the X-ray powder diffraction spectrum as shown in FIG. 4, at about room temperature wherein the radiation used has a wavelength of 1 .54060 A.PAT059659 FF34. The crystalline form according to claim 18, wherein the 2'-((3-ethoxy-1 H-pyrazol-4-yl)amino)-7'-((1 R,3R)-3-hydroxycyclohexyl)spiro[cyclopropane-1 ,5'-pyrrolo[2,3- d]pyrimidin]-6'(7'H)-one is in the form of a phosphate salt or phosphoric acid co-crystal.

35. The crystalline form according to claim 34, wherein characterized by an X-ray powder diffraction pattern comprising four or more 20 values (e.g. 5 or more, e.g. 6 or more, e.g. 7 or more, e.g. 8 or more, e.g. 9 or more, e.g. 10 or more, e.g. 11 or more , e.g. all 12 20 values) selected from the group consisting of:wherein the temperature is about room temperature and the radiation used has a wavelength of 1.54060A.

36. The crystalline form according to claim 34 or claim 35, having an X-ray powder diffraction pattern substantially the same as the X-ray powder diffraction spectrum as shown in FIG. 5, at about room temperature wherein the radiation used has a wavelength of 1 .54060 A.PAT059659 FF37. A pharmaceutical composition comprising the crystalline form according to any one of claims 17 to 36, and a pharmaceutically acceptable carrier.

38. A method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the crystalline form according to any one of claims 17 to 36, or the pharmaceutical composition according to claim 37.

39. The crystalline form according to any one of claims 17 to 36, or the pharmaceutical composition according to claim 37 for use as a medicament.

40. A combination comprising a crystalline form of any of claims 17 to 36, and one or more therapeutically active agents.41 . The crystalline form according to any one of claims 17 to 36, or the pharmaceutical composition according to claim 37 for use in the treatment of cancer.

42. Use of the crystalline form according to any one of claims 17 to 36, or the pharmaceutical composition according to claim 37 in the treatment of cancer.

43. Use of the crystalline form according to any one of claims 17 to 36, or the pharmaceutical composition according to claim 37 in the manufacture of a medicament for the treatment of cancer.

44. The method according to claim 38, the crystalline form for use according to claim 41 , or the use according to claim 42 or claim 43, wherein the cancer is selected fromPAT059659 FF ovarian cancer, gastric cancer, uterine cancer, breast cancer, lung cancer and endometrial cancer.

45. The method according to claim 38 or claim 44, the crystalline form for use according to claim 41 or claim 44, or the use according to any one of claims 42 to 44, wherein the cancer is a cyclin E amplified cancer.

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