US009 199997B2

(12) United States Patent (10) Patent No.: US 9,199,997 B2 Yamamoto et al. (45) Date of Patent: *Dec. 1, 2015

(54) PURINONE DERIVATIVE HYDROCHLORIDE WO O3,O37890 5, 2003 WO 2005/O11597 2, 2005 WO 2007 142755 12/2007 (71) Applicant: ONO PHARMACEUTICAL CO., WO 2008/060301 5, 2008 LTD., Osaka (JP) WO 2008/121742 10, 2008 WO 2010/009342 1, 2010 (72) Inventors: Shingo Yamamoto, Osaka (JP); Toshio WO 2011, 152351 12/2011 Yoshizawa, Osaka (JP) WO 2013/081016 6, 2013

(73) Assignee: ONO PHARMACEUTICAL CO., OTHER PUBLICATIONS LTD., Osaka (JP) International Preliminary Report on Patentability and Written Opin (*) Notice: Subject to any disclaimer, the term of this ion issued Jun. 3, 2014 in International (PCT) Application No. PCT/ patent is extended or adjusted under 35 JP2012/080769. U.S.C. 154(b) by 0 days. International Search Report issued Jan. 29, 2013 in International This patent is Subject to a terminal dis (PCT) Application No. PCT/JP2012/080769. claimer. International Search Report issued Aug. 16, 2011 in International (PCT) Application No. PCT/JP2011/062377. (21) Appl. No.: 14/360,725 Vetrie, David, et al., “The gene involved in X-linked agam maglobulinaemia is a member of the Src family of protein-tyrosine (22) PCT Filed: Nov. 28, 2012 kinases”. Nature, vol. 361, Jan. 21, 1993, pp. 226-233. Uckun, Fatih M. et al., “Bruton's Tyrosine Kinase as a New Thera (86). PCT No.: PCT/UP2012/080769 peutic Target'. Anti-Cancer Agents in Medicinal Chemistry, vol. 7. 2007, pp. 624-632. S371 (c)(1), Supplementary European Search Report issued Sep. 19, 2013 in (2) Date: May 27, 2014 European Application No. 11789754. (87) PCT Pub. No.: WO2013/081016 Anderson, Chem. And Biol., vol. 10, 2003, pp. 787-797. CAS RN 1222785810, entered STN May 13, 2010. PCT Pub. Date: Jun. 6, 2013 Yasuhiro et al., “ONO-4059, a novel oral Bruton's tyrosine kinase (Btk) inhibitor that demonstrates potent pharmacodynamic activity (65) Prior Publication Data through Phosphorylated Btk (P-Btk) inhibition, in addition to effec US 2014/033OO15 A1 Nov. 6, 2014 tive anti-tumour activity in a TMD-8 (DLBCL) xenograft model”, American Association for Cancer Research Annual Meeting 2013, (30) Foreign Application Priority Data Abstract No. 2452. Rule et al., “A Phase I Study Of The Oral Btk Inhibitor ONO-4059 In Nov. 29, 2011 (JP) ...... 2011-259.662 Patients With Relapsed/Refractory B-Cell Lymphoma”, 55' Ameri can Society of Hematology, session: 624, program No. 4397, 2013. (51) Int. Cl. Dyer et al., “The Bruton's tyrosine kinase (BTK) inhibitor ONO CO7D 473/00 (2006.01) 4059: Single-agent activity in patients with relapsed and refractory A6 IK3I/522 (2006.01) non-GCB-DLBCL”, 2014 ASCO Annual Meeting, Abstract No. CO7D 473/34 (2006.01) 8553. (52) U.S. Cl. Extended European Search Report issued Apr. 1, 2015 in correspond CPC ...... C07D473/00 (2013.01); A61K3I/522 ing European Application No. 12852725.6. (2013.01); C07D473/34 (2013.01) (Continued) (58) Field of Classification Search CPC ...... A61K31/522; C07D 473/00 USPC ...... 514/262:544/276 Primary Examiner — Taofiq A Solola See application file for complete search history. (74) Attorney, Agent, or Firm — Wenderoth, Lind & Ponack, (56) References Cited LLP. U.S. PATENT DOCUMENTS (57) ABSTRACT 7,071,199 B1 7/2006 Hirst et al. 8,557.803 B2 10/2013 Yamamoto et al. The purinone derivative 6-amino-9-(3R)-1-(2-butynoyl)-3- 8,940,725 B2 1/2015 Yamamoto et al. pyrrolidinyl-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin 2003/0171360 A1 9, 2003 Gross et al. 2008/0076921 A1 3/2008 Honigberg et al. 8-one hydrochloride has Btk-selective inhibitory activity and, 2013, OO793.27 A1 3/2013 Yamamoto et al. in addition to having excellent metabolic stability, it is a 2013,0217880 A1 8/2013 Yamamoto et al. compound that exhibits a high level of solubility and absorp 2014/0330015 A1 11/2014 Yamamoto et al. tion with respect to the free base and can be crystallized, hence it can serve as atherapeutic agent for diseases involving FOREIGN PATENT DOCUMENTS B cells and mast cells. JP 2003-509427 3, 2003 JP 2010-504324 2, 2010 WO 01, 19828 3, 2001 1 Claim, 3 Drawing Sheets U.S. Patent Dec. 1, 2015 Sheet 1 of 3 US 9,199,997 B2

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40 60 80 100 120 140 160 180 200 220 °C US 9, 199,997 B2 1. 2 PURNONE DERVATIVE HYDROCHLORIDE from alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; Z' represents CO, OCO, NHCO, or CS: R'' TECHNICAL FIELD and R' each independently represent H, unsubstituted C-C alkyl, Substituted C-C alkyl, unsubstituted C-C het The present invention relates to 6-amino-9-(3R)-1-(2-bu eroalkyl, substituted C-C heteroalkyl, unsubstituted C-C, tynoyl)-3-pyrrolidinyl-7-(4-phenoxyphenyl)-7,9-dihydro cycloalkyl, Substituted C-C cycloalkyl, unsubstituted 8H-purin-8-one hydrochloride (hereinafter, referred to as the C-C heterocycloalkyl, and substituted C-C heterocy compound of the present invention), which has Btkinhibitory cloalkyl: or R'' and R together form a bond; and R' activity and is useful as a therapeutic agent for autoimmune represents H, substituted or unsubstituted C-C alkyl, sub diseases, cancer, and the like; crystals thereof, and a pharma 10 stituted or unsubstituted C-C heteroalkyl, C-C alkoxy ceutical composition containing the same. alkyl, C-C alkylaminoalkyl, Substituted or unsubstituted C-C cycloalkyl, or substituted or unsubstituted aryl (the BACKGROUND ART definitions of these groups have been excerpted)) (refer to Patent Documents 1, 2, and 3). Bruton's tyrosine kinase (abbreviated below as “Btk”) 15 On the other hand, for example, compounds represented by belongs to the Tec family of kinases, which are non-receptor general formula (B) tyrosine kinases, and is selectively expressed in the B cell and myelocyte lines. Btk plays an important role in signal trans duction in B cells and is a factor that contributes to the C2 survival, differentiation, proliferation, and activation of B cells. Signaling in B cells via the B cell antigen receptor R4B (B) (BCR) induces a broad range of biological responses, and N A abnormal signal transduction here causes abnormal B cell s (CR2PR3P) activation and the formation of pathogenic autoantibodies. B N N / Btk is believed to form a link in the BCR-mediated signal 25 ck / 1 O transduction pathways into B cells. Thus, X-linked agamma Q2B Q 2NN globulinemia (XLA) is known to be caused by a defect in the V human Btk gene that results in the induction of abnormal B RIB R5B cell differentiation and a drastic decline in immunoglobulin production (refer to Non-patent Document 1). The symptoms 30 of this disease include a substantial decline in B cells in the (in the formula, Q' and Q are independently selected from peripheral blood and an increased susceptibility to bacterial CX', CX’, and nitrogen; Q represents N or CH; X' and infections. Btk is also known to participate in mast cell acti X’ are independently selected from the group consisting of Vation and in the physiological functions of platelets. Due to hydrogen, (C-C) alkyl, cyano, halogen, and so forth; R is this, compounds that have a Btkinhibitory activity are effec 35 selected from the group consisting of hydrogen and (C-C) tive for the treatment of diseases in which B cells or mast cells alkyl; yB represents 0 or an integer from 1 to 3: RandR participate, for example, allergic diseases, autoimmune dis are independently selected from hydrogen and (C-C) alkyl; eases, inflammatory diseases, thromboembolic diseases, and R" is selected from the group consisting of alkyl, heterocy cancers (refer to Non-patent Document 2). clyl, aryl, heteroaryl, and so forth; and R is selected from The following compounds are known as prior art for the 40 the group consisting of alkyl, heterocyclyl, and Substituted compounds of the present invention. heterocyclyl (the definitions of these groups have been Compounds represented by general formula (A) are known excerpted)) (refer to Patent Document 4) are known as com as compounds that have a Btk inhibitory activity pounds that have a purinone skeleton. Compounds represented by general formula (C) are also 45 known C 1 (A) -Ar" (C3) 50 4C (C) QS Q2C / NH2 XC N N 55 l s - QC N1 N R2C 4. N 4. N^Y, ( )nC W QC YNZA R6-A 60 (in the formula, X is selected from the group consisting of nitrogen and CR: R is selected from the group consisting R8-4 R7-4 of hydrogen, halogen, Substituted or unsubstituted alkyl, and soforth; Q' is selected from the group consisting of O, S, and (in the formula, L, represents CH, O, NH, or S. Ar' repre 65 so forth; Z is selected from the group consisting of oxygen, sents substituted or unsubstituted aryl or substituted or unsub sulfur, and NY;Y is selected from the group consisting of stituted heteroaryl; Y' represents any substituent selected hydrogen, Substituted or unsubstituted alkyl, and so forth; US 9, 199,997 B2 3 4 Q’, Q, and Q are independently selected from the group 19.74, 20.42, 21.05, 22.57, 23.21, 23.85, and 24.70 degrees consisting of hydrogen, Substituted or unsubstituted alkyl, in a powder X-ray diffraction spectrum; substituted or unsubstituted aryl, and so forth; R is selected 6. The crystal as in any of 2 to 5 above which is charac from the group consisting of hydrogen and Substituted or terized by the powder X-ray diffraction spectral chart in unsubstituted alkyl; and nC represents 0, 1, 2, 3, or 4 (the 5 FIG.3: definitions of these groups have been excerpted)) (refer to 7. The crystal as in any of 2 to 6 above having an endot Patent Document 5). hermic peakata peak temperature of 21.6°C. in differential In addition, Patent Document 6 discloses a compound hav scanning calorimetry; ing a purinone backbone as Formula 20 (see paragraph 8. The crystal as in any of 2 to 7 above which is charac 0028). 10 terized by the differential scanning calorimetry chart in The present invention relates to 6-amino-9-(3R)-1-(2-bu FIG. 4; tynoyl)-3-pyrrolidinyl-7-(4-phenoxyphenyl)-7,9-dihydro 9. A pharmaceutical composition comprising 6-amino-9- (3R)-1-(2-butynoyl)-3-pyrrolidinyl-7-(4-phenoxyphe 8H-purin-8-one hydrochloride, which has Btk-selective nyl)-7,9-dihydro-8H-purin-8-one hydrochloride; inhibitory activity, and in addition to excellent metabolic 15 10. The pharmaceutical composition as in 9 above that is a stability, has greater solubility and absorption than the free Btkinhibitor; base; Such matters are neither described nor Suggested by the 11 The pharmaceutical composition as in 10 above that is prior art documents. an agent for the prevention and/or treatment of a Btk Patent Document 1: Japanese Translation of PCT Application related disease; No. 2010-504324 12 The pharmaceutical composition as in 11 above Patent Document 2: WO 2008/121742 wherein the Btk-related disease is an allergic disease, Patent Document 3: WO 2010/009342 autoimmune disease, inflammatory disease, thromboem Patent Document 4: WO 2008/060301 bolic disease, bone-related disease, or cancer; and Patent Document 5: WO 2007/142755 13. The pharmaceutical composition as in 12 above Patent Document 6: Japanese Translation of PCT Application 25 wherein the cancer is non-Hodgkin’s lymphoma, etc. No. 2003-509427 The compound of the present invention has Btk-selective Non-Patent Document 1: Nature, Vol. 361, pages 226-233, inhibitory activity, and in addition to having excellent meta 1993 bolic stability, is a compound with greater solubility and Non-Patent Document 2: Anticancer Agents in Medicinal absorption than the free base; therefore, it is useful as an Chemistry, Vol. 7, No. 6, pages 624-632, 2007 30 outstandingly safe therapeutic agent for a disease involving B cells and mast cells such as non-Hodgkin’s lymphoma. DISCLOSURE OF THE INVENTION BRIEF DESCRIPTION OF THE DRAWINGS The problem to be solved by the present invention is the development of a compound that has Btk-selective inhibitory 35 FIG. 1 shows a powder X-ray diffraction spectral chart of a activity, and in addition to excellent metabolic stability, has crystal of 6-amino-9-(3R)-1-(2-butynoyl)-3-pyrrolidinyl greater solubility and absorption than the free base in order to 7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one (in FIG. provide a very stable agent for the treatment of a disease 1, the vertical axis represents intensity (counts), and the hori involving B cells and mast cells. In addition, a compound that Zontal axis represents 20 (degrees)); has excellent stability as the active ingredient of a pharma 40 FIG. 2 shows a differential scanning calorimetry (DSC) ceutical product and that can be crystallized to enable long chart of a crystal of 6-amino-9-(3R)-1-(2-butynoyl)-3-pyr term storage is preferred. rolidinyl-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8- As the result of their diligent and incisive research to solve One: the aforementioned problem, the inventors discovered that FIG.3 shows a powder X-ray diffraction spectral chart of a the compound of the present invention has Btk-selective 45 crystal of 6-amino-9-(3R)-1-(2-butynoyl)-3-pyrrolidinyl inhibitory activity, and in addition to excellent metabolic 7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one hydro stability, has greater solubility and absorption than the free chloride (in FIG. 3, the vertical axis represents intensity base, and can be crystallized, thereby completing the present (counts), and the horizontal axis represents 20 (degrees)); and invention. FIG. 4 shows a differential scanning calorimetry (DSC) More specifically, the present invention relates to: 50 chart of a crystal of 6-amino-9-(3R)-1-(2-butynoyl)-3-pyr 1 6-amino-9-(3R)-1-(2-butynoyl)-3-pyrrolidinyl-7-(4- rolidinyl-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8- phenoxyphenyl)-7,9-dihydro-8H-purin-8-one hydrochlo one hydrochloride. ride; 2 A crystal of 6-amino-9-(3R)-1-(2-butynoyl)-3-pyrrolidi BEST MODE FOR CARRYING OUT THE nyl-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one 55 INVENTION hydrochloride; 3 The crystal as in 2 above having at least 2 or more peaks The present invention is described in greater detail below. at angle 20 selected from approximately 8.11, 8.43, 11.57. The term “Btk-selective inhibitory activity” means Btk 12.73, 13.85, 14.20, 14.67, 14.91, 15.94, 16.64, 18.06, selective inhibitory activity with regard to tyrosine kinases 19.74, 20.42, 21.05, 22.57, 23.21, 23.85, and 24.70 degrees 60 other than Btk, particularly Lck, Fyn, and Lyn A. Due to this in a powder X-ray diffraction spectrum; property, unexpected adverse reactions caused by inhibiting 4 The crystal as in 2 or 3 above having peaks at angle 20 other tyrosine kinases can be avoided. of approximately 8.11, 8.43, 14.20, 14.67, 14.91 and 23.21 In the present invention 6-amino-9-(3R)-1-(2-butynoyl)- degrees in a powder X-ray diffraction spectrum; 3-pyrrolidinyl-7-(4-phenoxyphenyl)-7,9-dihydro-8H-pu 5 The crystal as in any of 2 to 4 above having peaks at 65 rin-8-one (hereinafter, abbreviated as Compound A) means angle 20 selected from approximately 8.11, 8.43, 11.57. the compound represented by the following structural for 12.73, 13.85, 14.20, 14.67, 14.91, 15.94, 16.64, 18.06, mula US 9, 199,997 B2 5 6 Moreover, Table 1 below shows the diffraction angle 20 and Chemical Formula 4 the relative intensity in the powder X-ray diffraction spec trum.

5 TABLE 1 O Diffraction angle 20 (degrees) Relative intensity (%) 5.88 1OO 10.31 6.3 NH 10 1O.S6 8.4 11.47 7.8 11.84 9.7 N N N 12.63 S.1 1360 21.4 l 2 X=o 15.55 45.8 N 17.11 57 N 15 1844 14.9 19.74 11.7 20.40 41.2 N 21.03 7.4 21.76 10.1 22.39 14.3 - 2O 22.77 10.1 O 23.48 3.5 24.00 4.5 (in the formula, the symbol Moreover, as shown in FIG. 2, the crystals of Compound A 25 showed endothermic peaks corresponding to melting repre Chemical Formula 5 sented by an onset temperature of approximately 169° C. and a peak temperature of approximately 172°C. As shown in Table 2 below, the aforementioned screening represents the B position). 30 was performed using 18 species of acidic counter-ions. Study of Acid Addition Salts of Compound A Counter screening of acid addition salts of Compound A TABLE 2 was performed by the following method using Compound A Acidic counter-ion Step (1) Step (2) prepared in Example 8 below and various acidic counterions. Hydrochloric acid Amorphous Oil Amorphous powders were obtained by mixing a molar as Sulfuric acid Amorphous Oil amount of Compound A with equivalent amount of each Acetic acid Amorphous Crystals Citric acid Amorphous Crystals acidic counterion; as step (I) methyl tert-butyl ether (MTBE) (+)-tartaric acid Oil Crystals was added to the mixture and the precipitate was scraped up Phosphoric acid Amorphous Crystals with a micro-spatula, or as step (2), if no crystals precipitated Fumaric acid Amorphous Crystals Lactic acid Oil Crystals in aforementioned step (1), methanol was added to the mix- 40 Succinic acid Oil Crystals ture, and it was allowed to dry naturally. When crystals were Methanesulfonic acid Amorphous Oil obtained by this screening method, the physical property data p-toluenesulfonic acid Amorphous Oil Benzenesulfonic acid Amorphous Oil was measured under the conditions shown below. The physi (-)-camphorsulfonic acid Amorphous Oil cal property data for Compound A was acquired beforehand, (+)-camphorsulfonic acid Amorphous Oil and that was compared with the physical property data of the 2-naphthalenesulfonic acid Oil Oil crystals obtained from the aforementioned counter screening 1-hydroxy-2-naphthoic acid Oil Crystals Benzoic acid Oil Crystals process. Nicotinic acid Nicotinic acid Crystals 1 Powder X-Ray Diffraction Spectrum crystals receptor antagonist, thromboxane Syn yngitis, mastitis, meningitis, myelitis, myocarditis, myositis, thetase inhibitors, steroids and the like. nephritis, oophoritis, orchitis, osteitis, pancreatitis, parotitis, Other drugs that Supplement and/or enhance the protective pericarditis, peritonitis, pharyngitis, pleuritis, phlebitis, and/or therapeutic efficacy of the compound of the present pneumonia, proctitis, prostatitis, pyelonephritis, rhinitis, sal invention against autoimmune diseases include, for example, pingitis, sinusitis, stomatitis, Synovitis, tendinitis, tonsillitis, immunosuppressants, steroids, disease-modifying antirheu uveitis, vaginitis, vasculitis, Vulvitis, and the like. 25 matic drugs, elastase inhibitors, cannabinoid-2 receptor ago Examples of a thromboembolic disease in the present nists, prostaglandins, prostaglandin synthetase inhibitors, invention include myocardial infarction, angina pectoris, phosphodiesterase inhibitors, metalloproteinase inhibitors, reocclusion after angioplasty, restenosis after angioplasty, adhesion molecule inhibitors, anti-cytokine protein prepara reocclusion after aortocoronary bypass, restenosis after aor tions such as anti-TNF-C. preparations, anti-IL-1 prepara tocoronary bypass, cerebral infarction, transient ischemia, 30 tions, and anti-IL-6 preparations, and cytokine inhibitors, peripheral vascular occlusive disease, pulmonary embolism, non-steroidal anti-inflammatory drugs, anti-CD20 antibod deep vein thrombosis, and the like. ies, and the like. Examples of a bone-related disease in the present invention Other drugs that Supplement and/or enhance the protective include osteoporosis, periodontitis, metastasis of cancer to and/or therapeutic efficacy of the compound of the present bone, osteoarthritis, hypercalcemia, bone fractures, Behcet’s 35 invention against inflammatory diseases include, for disease, and the like. example, Steroids, elastase inhibitors, cannabinoid-2 receptor Examples of cancer in the present invention include non agonists, prostaglandins, prostaglandin synthetase inhibitors, Hodgkin’s lymphomas, and among those B cell non phosphodiesterase inhibitors, metalloproteinase inhibitors, Hodgkin’s lymphoma is most applicable, for example, Bur adhesion molecule inhibitors, anti-leukotriene agents, anti kitt's lymphoma, AIDS-related lymphoma, marginal Zone 40 cholinergic agents, thromboxane A2 receptor antagonists, B-cell lymphoma (nodal marginal Zone B cell lymphoma, thromboxane synthase inhibitors, Xanthine derivatives, extranodal marginal Zone B-cell lymphoma, splenic marginal expectorants, antibacterial agents, antihistamines, anti-cy Zone B-cell lymphoma), diffuse large B-cell lymphoma, pri tokine protein preparations, cytokine inhibitors, forskolin mary effusion lymphoma, lymphoma-like granulomatous preparations, mediator release inhibitors, non-steroidal anti disease, follicular lymphoma, B-cell chronic lymphocytic 45 inflammatory drugs, and the like. leukemia, B cell prolymphocytic leukemia, lymphoplasma Other drugs that Supplement and/or enhance the protective cytic leukemia/Waldenstrom's macroglobulinemia, plasma and/or therapeutic efficacy of the compound of the present cytoma, mantle cell lymphoma, mediastinal large B-cell lym invention against thromboembolic diseases include, for phoma, intravascular large B-cell lymphoma, and hairy cell example, thrombolytic agents, heparin, heparinoids, low leukemia. Moreover, examples of cancer in the present inven 50 molecular weight heparin, warfarin, thrombin inhibitors, fac tion include cancers other than non-Hodgkin’s lymphoma tor Xa inhibitors, ADP receptor antagonists, cyclooxygenase Such as pancreatic endocrine tumors and multiple myeloma. inhibitors, and the like. Examples of pancreatic endocrine tumors include insuli Other drugs that Supplement and/or enhance the protective noma, gastrinoma, glucagonoma, Somatostatinoma, VIP-pro and/or therapeutic efficacy of the compound of the present ducing tumor (VIPoma), PP-producing tumor (PPoma), 55 invention against bone-related diseases include, for example, GRF-producing tumor, and the like. bisphosphonates, prostaglandins, vitamin D preparations, The compound of the present invention can be adminis calcium preparations, estrogen preparations, calcitonin tered together with another drug as a concomitant medication preparations, ipriflavone preparations, protein anabolic Ste tO: roids, Vitamin K preparations, cathepsin K inhibitors, par (1) Supplement and/or enhance the preventive and/or thera 60 athyroid hormones, growth factors, caspase-1 inhibitors, peutic effect of the said compound; PTHrP derivatives, metalloproteinase inhibitors, farnesoid X (2) improve the kinetics/absorption, or reduce the dose of the receptor agonists, anti-androgen agents, selective estrogen said compound; and/or receptor modulators (SERMs), progesterone agonists, cal (3) mitigate the side effects of the said compound. cium receptor antagonists (calcylitics), strontium prepara The concomitant medication that contains the other drug, 65 tions, C-calcitonin gene-related peptide preparations, osteo and the compound of the present invention can be adminis genetic protein preparations, anti-RANKL antibodies, anti tered as a formulation that combines both components therein TNF-C. antibodies, anti-IL-6 antibodies, and the like. US 9, 199,997 B2 11 12 Other drugs that Supplement and/or enhance the protective dine, hydroxychloroquine, bucillamine, methotrexate, and/or therapeutic efficacy of the compound of the present leflunomide, lobenzarit Sodium, aurothioglucose, Sodium invention against non-Hodgkin’s lymphoma include, for aurothiomalate, and the like. example, alkylating agents, antimetabolites, antitumor anti Examples of elastase inhibitors include ONO-5046, ONO biotics, plant alkaloids, hormone drugs, platinum com 6818, MR-889, PBI-1101, EPI-HNE-4, R-665, ZD-0892, pounds, anti-CD20 antibody, other anti-cancer agents and the ZD-8321, GW-3 11616, DMP-777, L-659286, L-680833, like. L-683845, AE-3763, and the like. Examples of antihistamines include azelastine hydrochlo Examples of prostaglandins (hereinafter, abbreviated as ride, ebastine, epinastine hydrochloride, emedastine fuma PG) include PGE1 drugs (e.g. alprostadil alfadex, alprostadil, rate, auranofin, oxatomide, olopatadine hydrochloride, dil 10 etc.), PGI2 drugs (e.g. beraprost sodium, etc.), PG receptor chlorpheniramine maleate, clemastine fumarate, ketotifen agonists, PG receptor antagonists, and the like. Examples of fumarate, cimetidine, dimenhyrinate, diphenhydramine PG receptors include PGE receptors (EP1, EP2, EP3, EP4), hydrochloride, cyproheptadine hydrochloride, cetirizine PGD receptors (DP, CRTH2), PGF receptors (FP), PGI2 hydrochloride, desloratadine, terfenadine, famotidine, fex receptors (IP), TX receptors (TP), and the like. ofenadine hydrochloride, bepotastine, bepotastine besilate, 15 Examples of prostaglandin synthetase inhibitors include mizolastine, meduitazine, mometaSone furoate, ranitidine, salaZoSulfapyridine, mesalazine, olsalazine, 4-aminosali ranitidine hydrochloride, loratadine, promethazine hydro cylic acid, JTE-522, auranofin, carprofen, diphenpyramide, chloride, homochlorcyclizine hydrochloride, etc. flunoxaprofen, flurbiprofen, indometacin, ketoprofen, lor Examples of leukotriene antagonists include pranlukast noxicam, loxoprofen, meloxicam, oxaprozin, parsalmide, hydrate, montelukast Sodium, Zafirlukast, ablukast, pobi piproxen, piroXicam, piroXicam cinnamate, Zaltoprofen, pra lukast, Sulukast, iralukast sodium, Verlukast, ritolukast, noprofen, and the like. cinalukast, pirodomast, tomelukast, doqualast, and the like. Examples of phosphodiesterase inhibitors include rollip Examples of anti-allergy drugs include amlexanoX, azelas ram, cilomilast, Bay 19-8004, NIK-616, roflumilast (BY tine hydrochloride, israpafant, ibudilast, imitrodast Sodium, 25 217), cipamfylline (BRL-61063), atizoram (CP-80633), ebastine, epinastine hydrochloride, emedastine fumarate, ONO-6126, SCH-351591, YM-976, V-11294A, PD-168787, oXatomide, oZagrel hydrochloride, olopatadine hydrochlo D-4396, IC-485 and the like. ride, cromoglicic acid, Sodium cromoglicate, ketotifen fuma Examples of adhesion molecule inhibitors include alpha-4 rate, seratrodast, cetirizine hydrochloride, Suplatast tosilate, integrin antagonists and the like. taZanolast, terfenadine, domitroban calcium hydrate, tra 30 Examples of anti-TNF-C. preparations include anti-TNF-C. nilast, nedocromil, fexofenadine, fexofenadine hydrochlo antibodies, soluble TNF-C. receptors, anti-TNF-C. receptor ride, pemirolast potassium, meduitazine, ramatroban, repiri antibodies, soluble TNF-C. binding proteins, and the like, and nast, loratadine, and the like. particularly infliximab and etanercept. Examples of thromboxane A2 receptor antagonists include 35 Examples of anti-IL-1 preparations include anti-IL-1 anti seratrodast, domitroban calcium hydrate, and ramatroban. bodies, soluble IL-1 receptors, anti-IL-1Ra antibodies and/or The thromboxane synthase inhibitors can be exemplified anti-IL-1 receptor antibodies, and the like, particularly anak by imitrodast sodium and oZagrel hydrochloride. inra. Examples of Steroids include amcinonide, hydrocortisone Examples of anti-IL-6 preparations include anti-IL-6 anti Sodium Succinate, prednisolone sodium Succinate, methyl 40 bodies, soluble IL-6 receptors, anti-IL-6 receptor antibodies, prednisolone sodium Succinate, ciclesonide, difluprednate, and the like, particularly tocilizumab. betamethasone propionate, dexamethasone, deflazacort, tri Examples of cytokine inhibitors include Suplatast tosylate, amcinolone, triamcinolone acetonide, halcinonide, dexam T-614, SR-31747, sonatimod, and the like. ethasone palmitate, hydrocortisone, flumetasone pivalate, Examples of anticholinergic agents include trihex prednisolone butylacetate, budesonide, prasterone Sulfate, 45 yphenidyl, trihexyphenidyl hydrochloride, biperiden, mometasonem furoate, fluocinonide, fluocinolone acetonide, biperiden hydrochloride, and the like. fludroxycortide, flunisolide, prednisolone, alclometaSone Examples of Xanthine derivatives include aminophylline, propionate, clobetasol propionate, dexamethasone propi theophylline, doxofylline, sipamphylline, diprophylline, and onate, deprodone propionate, fluticasone propionate, the like. beclometaSone propionate, betamethasone, methylpredniso 50 Examples of expectorants include foeniculated ammonia lone, methylprednisolone Suleptanate, methylprednisolone spirit, Sodium bicarbonate, bromhexine hydrochloride, car Sodium Succinate, dexamethasone sodium phosphate, hydro bocysteine, ambroxol hydrochloride, methylcysteine hydro cortisone sodium phosphate, prednisolone sodium phos chloride, acetylcysteine, L-cysteine ethyl ester hydrochlo phate, diflucortolone Valerate, dexamethasone Valerate, ride, tyloxapol, and the like. betamethasone Valerate, prednisolone Valerate acetate, corti 55 Sone acetate, diflorasone acetate, dexamethasone acetate, tri Examples of antibacterials include Sodium cefuroxime, amcinolone acetate, paramethasone acetate, halopredone meropenem trihydrate, netilmicin Sulfate, Sisomicin Sulfate, acetate, fludrocortisone acetate, prednisolone acetate, meth ceftibuten, PA-1806, IB-367, tobramycin, PA-1420, doxoru ylprednisolone acetate, clobetasone butyrate, hydrocortisone bicin, astromicin sulfate, cefetamet pivoxil hydrochloride, butyrate, hydrocortisone butyrate propionate, betamethasone 60 and the like. butyrate propionate, and the like. Examples of mediator release agents include tranilast, Examples of immunosuppressants include azathioprine, Sodium cromoglicate, amlexanox, repirinast, ibudilast, daza ascomycin, everolimus, Salazosulfapyridine, cyclosporine, nolast, pemirolast potassium, and the like. cyclophosphamide, sirolimus, tacrolimus, bucillamine, Examples of thrombolytic agents include alteplase, uroki methotrexate, leflunomide, and the like. 65 nase, tisokinase, nasaruplase, nateplase, t-PA, pamiteplase, Examples of disease-modifying anti-rheumatic drugs monteplase, prourokinase, streptokinase, and the like. include D-penicillamine, actarit, auranofin, Salazosulfapyri An example of a heparinoid is fondaparinux. US 9, 199,997 B2 13 14 Examples of low molecular weight heparins include dan Examples of strontium preparations include strontium aparoid sodium, enoxaparin (sodium), nadroparin calcium, ranelate and the like. bemiparin (sodium), reviparin (Sodium), tinzaparin (sodium), Examples of anti-RANKL antibodies include denosumab and the like. (AMG 162) and the like. Examples of thrombin inhibitors include argatroban, Examples of osteogenetic protein preparations includeYM Ximelagatran, melagatran, dabigatran, bivalirudin, lepirudin, 484 and the like. hirudin, desirudin, and the like. Examples of alkylating agents include nitrogen mustard Examples of ADP receptor antagonists include ticlopidine N-oxide hydrochloride, cyclophosphamide, Ifosfamide, mel hydrochloride, clopidogrel sulfate, and the like. phalan, thiotepa, carboquone, buSulfan, nimustine hydro Examples of cyclooxygenase inhibitors include aspirin and 10 chloride, dacarbazine, ranimustine, and the like. the like. Examples of antimetabolites include methotrexate, mer Examples of bisphosphonate preparations include alendr captopurine, 6-mercaptopurine riboside, fluorouracil, onate Sodium hydrate, ibandronic acid, incadronate diso tegafur, tegafur uracil, carmofur, doxifluridine, cytarabine, dium, etidronate disodium, olpadronate, clodronate sodium 15 enocitabine, tegafur gimestat otastat potassium, gemcitabine hydrate, Zoledronic acid, tiludronate disodium, neridronate, hydrochloride, cytarabine ocfosfate, procarbazine hydro pamidronate disodium, piridironate, minodronic acid hydrate, chloride, hydroxycarbamide, and the like. sodium risedronate hydrate, YM 175 and the like. Examples of anticancer antibiotics include actinomycin D, Examples of vitamin D preparations include alfacalcidol, mitomycin C, daunorubicin hydrochloride, doxorubicin falecalcitriol, calcitriol, 1C.,25-dihydroxy cholecalciferol, hydrochloride, aclarubicin hydrochloride, neocarzinostatin, dihydrotachysterol, ST-630, KDR, ED-71, rocaltrol, tacal pirarubicin hydrochloride, epirubicin (hydrochloride), idaru ciol, maxacalcitol and the like. bicin hydrochloride, chromomycin A3, bleomycin (hydro Examples of calcium preparations include calcium chlo chloride), peplomycin Sulfate, therarubicin, Zinostatin Sti ride, calcium gluconate, calcium glycerophosphate, calcium malamer, and the like. lactate, calcium L-aspartate, calcium hydrogen phosphate 25 Examples of plant preparations include vinblastine Sulfate, and the like. Vincristine Sulfate, vindesine Sulfate, irinotecan hydrochlo Examples of estrogen preparations include estradiol, estra ride, etoposide, flutamide, vinorelbine tartrate, docetaxel diol benzoate, estradiol cypionate, estradiol dipropionate, hydrate, paclitaxel, and the like. estradiol enanthate, estradiol hexahydrobenzoate, estradiol Examples of hormones include estramustine phosphate phenylpropionate, estradiol undecanoate, estradiol Valerate, 30 Sodium, mepitioStane, epitioStanol, goserelin acetate, fos estrone, ethynyl estradiol, mestranol and the like. Examples of calcitonin preparations include calcitonin, festrol (diethylstilbestrol phosphate), tamoxifen citrate, salmon calcitonin, chicken calcitonin, Secalciferol, elcatonin, toremifene citrate, fadrozole hydrochloride hydrate, medrox TJN-135 and the like. yprogesterone acetate, bicalutamide, leuprorelin acetate, Examples of ipriflavone preparations include ipriflavone 35 anastroZole, exemestane, and the like. and the like. Examples of platinum compounds include carboplatin, cis Examples of protein anabolic steroids include platin, medaplatin, and the like. oxymetholone, stanozolol, nandrolone decanoate, nan Examples of anti-CD20 antibodies include rituximab, ibri drolone phenylpropionate, nandrolone cyclohexylpropi tumomab, ocrelizumab, and the like. onate, metenolone acetate, mestanolone, ethylestrenol, calus 40 Examples of other anticancer agents include L-asparagi terone and the like. nase, octreotide acetate, porfimer Sodium, mitoxantrone Examples of Vitamin K preparations include menate acetate, and the like. trenone, phytonadione and the like. The concomitant medication used together with the com Examples of cathepsin K inhibitors include ONO-5334, pound of the present invention can include not only drugs that AAE 581, SB 462795, and odanacatib, and the like. 45 have been discovered to date, but also drugs that may be Examples of parathyroid hormone (PTH) include dried discovered in the future. thyroid, levothyroxine Sodium, liothyronine Sodium, propy The compound of the present invention is generally admin lthiouracil, thiamazole, teriparatide acetate and the like. istered systemically or locally, and as an oral or parenteral Examples of growth factors include fibroblast growth fac form. Examples of oral formulations include liquids for oral tor (FGF), vascular endothelial growth factor (VEGF), hepa 50 administration (e.g. elixirs, syrups, pharmaceutically accept tocyte growth factor (HGF), insulin-like growth factor (IGF) able water-based formulations, Suspensions, and emulsions) and the like. and Solids for oral administration (e.g. tablets (including Sub Examples of caspase-1 inhibitors include nitroflubiprofen, lingual tablets and orally disintegrating tablets), pills, cap pralnacasan and the like. Sules (including hard capsules, soft capsules, gelatin cap Examples of PTHrP derivatives include hPTHrP. 55 Sules, and microcapsules), powders, granules, and lozenges), RS-66271 and the like. and the like. Examples of parenteral formulations include Examples of the farnesoid X receptor agonists include Solutions (e.g. injectables (such as Subcutaneous injectables, SR-45023A and the like. intravenous injectables, intramuscular injectables, intraperi Examples of anti-androgen agents include osateronacetate toneal injectables, and drip formulations), eye drops (e.g. and the like. 60 aqueous eye drops (such as aqueous eye drops, aqueous eye Examples of selective estrogen receptor modulators drop Suspensions, viscous eye drops, solubilized eye drops, (SERMs) include TSE-424, WJ-713/MPA, lasofoxifene tar etc.) and nonaqueous eye drops (such as nonaqueous eye trate, raloxifene hydrochloride, tamoxifen citrate and the like. drops and nonaqueous eye drop Suspensions, etc.)), topical Examples of progesterone agonist include trimegestone formulations (e.g. ointments (such as ophthalmic ointments, and the like. 65 etc.)), ear drops), and the like. These preparations can be Examples of calcium receptor antagonists (calcilytics) controlled release formulations such as rapid release formu include NPS-423557 and the like. lations, Sustained release formulations, and the like. These US 9, 199,997 B2 15 16 preparations can be produced by publicly known methods The dose of the compound of the present invention can be such as the methods described in The Japanese Pharmaco selected appropriately depending on the condition, age, type poeia. of formulation, and the like, and in the case of an oral prepa AS agents for oral administration, the liquid preparations ration preferably 1 to 100 mg. or more preferably 5 to 30 mg for oral administration can be produced, for example, by can be administered 1 to several times a day (e.g. 1 to 3 times). dissolving, Suspending, or emulsifying the compound of the Moreover, the compound of the present invention can be present invention in a commonly used diluent (e.g. purified administered parenterally 1 to several times a day in a range water, ethanol, or a mixture thereof, etc.). These liquid prepa of 50 g to 500 mg per dose, or can be continuously admin rations may also contain a wetting agent, Suspending agent, istered intravenously in a range from 1 to 24 hours per day. emulsifier, Sweetener, flavoring, fragrance, preservative, 10 Of course, as noted above, the dose will depend upon buffer, and the like. various conditions and, as a result, cases will occur wherein As a solid for oral administration, the Solid oral prepara an amount less than the above dosage will be sufficient or tions can be prepared by mixing the compound of the present cases will occur wherein those ranges must be exceeded. invention with, an excipient (e.g. lactose, mannitol, glucose, 15 microcrystalline cellulose, starch, etc.), a binder (e.g. hydrox EXAMPLES ypropyl cellulose, polyvinyl pyrrolidone, magnesium meta silicate aluminate, etc.), a disintegrant (e.g. cellulose calcium The present invention is described in detail below through glycolate, etc.), a lubricant (e.g. magnesium Stearate, etc.), a examples, but is by no means limited thereto. stabilizer, a solubilizer (e.g. glutamic acid, aspartic acid, etc.), The solvents in parentheses shown in the sections on chro and the like, and formulating according to conventional meth matographic separation and TLC indicate the elution solvent ods. As needed, coating can be carried out with a coating or development solvent that was used, and the ratio represents agent (e.g. Sugar, gelatin, hydroxypropyl cellulose, hydrox the ratio by volume. ypropyl methylcellulose phthalate, etc.) and two or more Unless otherwise stated, the NMR data is 'H-NMR data. layers can be applied. 25 The items in parentheses shown in the NMR sections rep As parenteral preparations, topical preparations can be resent the solvents used in measurement. produced using a publicly known method and a commonly The compound names used in this Description are gener used formulation. For example, an ointment can be prepared ally names generated based on IUPAC nomenclature or gen by incorporating or dissolving the compound of the present erated using ACD/Name(R), a computer program from invention into a base. The ointment base can be selected from 30 Advanced Chemistry Development, Inc., that performs nam publicly known ointment bases or a commonly used ointment ing based on IUPAC rules. base. For example, one item alone or a mixture of two or more items selected from the following can be used: higher fatty Example 1 acids and higher fatty acid esters (e.g. adipic acid, myristic acid, palmitic acid, Stearic acid, oleic acid, adipate esters, 35 N,N-dibenzyl-6-chloro-5-nitropyrimidine-4-amine myristate esters, palmitate esters, Stearate esters, oleate esters, etc.), waxes (e.g. beeswax, spermaceti, ceresin, etc.), Surfactants (e.g. polyoxyethylene alkyl ether phosphate A solution of dibenzylamine (10.2 g) in dichloromethane esters, etc.), higher alcohols (e.g. cetanol, Stearyl alcohol, (30 mL) was dripped into a solution of 4,6-dichloro-5-nitro cetostearyl alcohol, etc.), silicone oils (e.g. dimethyl polysi 40 pyrimidine (10g) in dichloromethane (70 mL) on an ice bath. loxane, etc.), hydrocarbons (e.g. hydrophilic petrolatum, Then triethylamine (14.4 mL) was added, and the mixture white petrolatum, purified lanolin, liquid paraffin, etc.), gly was stirred for 1 hour. Water was added to the reaction mix cols (e.g. ethylene glycol, diethylene glycol, propylene gly ture, the organic layer was washed with a saturated aqueous col, polyethylene glycol, macrogol, etc.), plant oils (e.g. cas Sodium chloride solution and dried over anhydrous sodium tor oil, olive oil, Sesame oil, turpentine oil, etc.), animal oils 45 Sulfate, and the solvent was concentrated under reduced pres (e.g. mink oil, egg yolk oil, squalane, squalene, etc.), water, sure to obtain the title compound (19.2 g) with the physical absorption promoters, and anti-irritants. A humectant, preser property value shown below. Vative, stabilizer, antioxidant, fragrance, and the like may also TLC: Rf 0.50 (hexane:ethyl acetate=7:1). be included therein. As parenteral preparations, injectables include Solutions, 50 Example 2 Suspensions, and emulsions as well as injectables in Solid form to be used after dissolution or Suspension in a solvent at tert-butyl (3R)-3-(6-(dibenzylamino)-5-nitropyrimi the time of use. For example, an injectable can be used by din-4-yl)amino)pyrrolidine-1-carboxylate dissolving, Suspending, or emulsifying the compound of the present invention in a solvent. Examples of the Solvent 55 The compound prepared in Example 1 (19 g) and tert-butyl include distilled water for injection, physiological saline (3R)-3-aminopyrrolidine-1-carboxylate (10.5 g) were dis Solution, vegetable oil, propylene glycol, polyethylene gly solved in dioxane (58 mL). Triethylamine (8.1 mL) was col, an alcohol Such as ethanol, or a combination thereof. The added, and the mixture was stirred for 5 hours at 50° C. The injectable can also contain a stabilizer, a solubilizer (e.g. reaction mixture was returned to room temperature, the Sol glutamic acid, aspartic acid, PolySorbate 80R, etc.), a sus 60 vent was distilled off, water was added, and extraction was pending agent, an emulsifier, a soothing agent, a buffer, a performed with ethyl acetate. The organic layer was washed preservative, and the like. The injectable can be sterilized in with Saturated aqueous sodium chloride Solution, then dried the final process or can be manufactured using aseptic pro over anhydrous sodium sulfate, and the solvent was distilled cessing methods. The injectable can also be manufactured as off. The residue was purified by silica gel column chroma a sterile solid form, for example, a freeze-dried product, and 65 tography to obtain the title compound (27.0 g) with the physi can be used after dissolution in distilled water for injection or cal property value shown below. another solvent that is either sterile or sterilized prior to use. TLC: Rf 0.29 (hexane:ethyl acetate=4:1) US 9, 199,997 B2 17 18 Example 3 Example 7 tert-butyl (3R)-3-((5-amino-6-(dibenzylamino)pyri midin-4-yl)aminopyrrolidine-1-carboxylate (3R)-6-amino-9-pyrrolidin-3-yl-7-(4-phenoxyphe 5 nyl)-7,9-dihydro-8H-purin-8-one dihydrochloride An ethyl acetate (360 mL) solution of the compound pre pared in Example 2 (17.5 g) was dripped into a mixture of zinc At room temperature 4 NHCl/dioxane (13 mL) was added (23.3 g) and a 3.0 M aqueous ammonium chloride Solution to a methanol (13 mL) Suspension of the compound prepared (11.4 g) on an ice bath, and the temperature was immediately in Example 6 (1.3 g 2.76 mmol. 1.0 equivalent), and the raised to room temperature. After stirring for 2 hours, the 10 mixture was stirred for 1 hour. The solvent was then distilled reaction mixture was filtered through CeliteTM and the solvent off to obtain the title compound (1.5 g) with the physical was distilled off. The residue was purified by silica gel col property value shown below. umn chromatography to obtain the title compound (12.4 g) TLC: Rf 0.50 (dichloromethane:methanol:28% ammonia with the physical property value shown below. 15 water=9:1:0.1) TLC: Rf 0.69 (hexane:ethyl acetate=1:1) Example 4 Example 8 tert-butyl (3R)-3-6-(dibenzylamino)-8-oxo-7,8-di 6-amino-9-(3R)-1-(2-butynoyl)-3-pyrrolidinyl-7- hydro-9H-purin-9-ylpyrrolidin-1-carboxylate (4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one (Compound A) The compound prepared in Example 3 (8.4 g) and 1.1'- carbonyl diimidazole (5.9 g) were dissolved in tetrahydrofu ran (120 mL) and the solution was stirred for 15 hours at 60° 25 C. The solvent was distilled off from the reaction mixture, Chemical Formula 8 water was added, and extraction with ethyl acetate was per formed. The organic layer was washed with Saturated aque ous sodium chloride Solution, then dried over anhydrous O sodium sulfate, and the solvent was distilled off. The residue 30 was purified by silica gel column chromatography to obtain the title compound (7.8 g) with the physical property value shown below. NH2 TLC: Rf 0.28 (hexane:ethyl acetate=2:1) 35 N1S-N Example 5 l 2 X= O N N tert-butyl (3R)-3-(6-amino-8-oxo-7,8-dihydro-9H purin-9-yl)pyrrolidine-1-carboxylate 40 The compound prepared in Example 4 (7.8 g) was dis solved in methanol (240 mL) and ethyl acetate (50 mL), 20% Pearlman's catalyst (Pd(OH)/C) (8.0 g, 100 wt %) was added, hydrogen gas replacement was carried out, and stir 45 ring was performed for 7.5 hours at 60° C. The reaction mixture was filtered through CeliteTM and the solvent was After 2-butylnoic acid (34 mg), 1-ethyl-3-(3-dimethylami distilled off to obtain the title compound (5.0 g) with the nopropyl) carbodiimide hydrochloride (EDC) (78 mg), 1-hy physical property value indicated below. droxybenzotriazole (HOBt) (62 mg), and triethylamine (114 TLC: Rf 0.50 (ethyl acetate) 50 LL) were added to a solution of the compound prepared in Example 7 (100 mg) in dimethyl formamide (3 mL), the Example 6 mixture was stirred at room temperature for 3 hours. Water was added to the reaction mixture and extraction with ethyl tert-butyl (3R)-3-6-amino-8-oxo-7-(4-phenoxyphe 55 acetate was performed. The organic layer was washed with nyl)-7,8-dihydro-9H-purin-9-ylpyrrolidine-1-car saturated Sodium carbonate solution and Saturated aqueous boxylate Sodium chloride Solution, then dried over anhydrous sodium sulfate, and the solvent was distilled off. The residue was At room temperature p-phenoxy phenylboronic acid (2.1 purified by thin layer chromatography (dichloromethane: g), copper(II) acetate (1.48 g), molecular sieve 4A (2.5 g), and 60 methanol:28% ammonia water-90:10:1) to obtain the title pyridine (0.82 mL) were added to a dichloromethane suspen compound (75 mg) with the physical property values shown sion (200 mL) of the compound prepared in Example 5 (2.5 g), followed by stirring for 21 hours. The reaction mixture below. was filtered through CeliteTM and the residue was purified by TLC: Rf 0.68 (ethyl acetate:methanol=9:1); silica gel column chromatography to obtain the title com 65 1H-NMR (CDC1): 8 1.94-2.03, 2.23-2.39, 2.80-3.01, pound (1.3 g) with the physical property value shown below. 3.50-3.63, 3.67-3.80, 3.86-4.02, 4.03-4.18, 4.23-4.33, 4.42 TLC: Rf 0.18 (hexane:ethyl acetate=1:1) 4.51, 5.11-5.25, 7.04-7.23, 7.34-7.45, 8.20-8.23 US 9, 199,997 B2 19 20 Example 9 First 5uL/well of either a solution prepared by diluting the test compound in dimethyl sulfoxide (DMSO) or DMSO alone was added to the wells of a 96-well assay plate together 6-amino-9-(3R)-1-(2-butynoyl)-3-pyrrolidinyl-7- with 10 uL/well of substrate/enzyme mixture and allowed to (4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one react for 20 minutes at 30°C. The substrate/enzyme mixture hydrochloride solutions were prepared by dilution in kinase buffer (DL dithiothreitol (DTT; 2.7 mM) and 1.33x kinase buffer) so that the final concentration of Tyr-1 peptide would be 4 uM, and the final concentration of Btk would be 5 nM. Next 5uL/well Chemical Formula 9 10 of adenosine triphosphate was added (ATP; final concentra tion 36 uM) and reacted for 1 hour at 30°C. After the reaction was completed, 10 uL of development solution prepared by O diluting development reagent B in development buffer 128 fold was added and reacted for an additional 1 hour at 30° C. 15 Then the enzyme reaction was stopped by adding 10 uL of stop solution. The fluorescence intensity in each well was NH2 measured using a fluorescence plate reader (Fusion Universal HCI Microplate Analyzer, PerkinElmer, Inc.) at 520 nm and 445 N1 N N nm. In accordance with the instructions accompanying the kit, the phosphorylation rate was determined by the ratio of l 2 X= O the emission at 445 nm (coumalin emission) in relation to N N emission at 520 nm (fluorescein emission). The inhibition (%) was calculated from the following for mula. 25 Phosphorylation inhibition rate (%)=1-(A-A)/ (Ac-A)x100 Mathematical Formula 1 A: Phosphorylation rate when test compound was added A: Phosphorylation rate without ATP (blank) 30 A: Phosphorylation rate with DMSO alone (control) The compound prepared in Example 8 (3.0 g) was placed in The 50% inhibition rate of the test compound (ICso value) a 300 mL 3-neck pear-shaped flask, ethyl acetate (30 mL) and was calculated from an inhibition curve based on the rate of 1-propanol (4.5 mL) were added, and the external tempera inhibition at each concentration of the test compound. ture was set at 70° C. (internal temperature 61° C.). After it Measurements of the inhibitory activity on other kinases was confirmed that the compound prepared in Example 8 had 35 were made in the same manner as described above using dissolved completely, 10% HC1/methanol (3.5 mL) was various kinases such as Lck, Fyn, Lyn A (Invitrogen Corpo added, and after precipitation of crystals was confirmed, the ration) in place of Btk. crystals were ripened by the following sequence: external Results revealed that the ICso value of the compound pre temperature 70° C. for 30 min, external temperature 60° C. pared in Example 9 was 0.0021 uM. for 30 min, external temperature 50 C for 60 min, external In addition, Btk-selective inhibitory activity rates of the temperature 40C for 30 min, room temperature for 30 min, 40 compound prepared in Example 9 toward other kinases, par and on an ice bath for 30 min. The resulting crystals were ticularly Lck, Fyn, and LynA, were calculated on the basis of filtered, washed with ethyl acetate (6 mL), and dried under the ICso values for each kinase, and these are shown in Table vacuum at 50° C. to obtain white crystals of the title com 4 below. pound (2.76 g) with the physical property values shown 45 below. TABLE 4 TLC: Rf 0.55 (dichloromethane:methanol=9:1); LckICso 1H-NMR (CDOD): 8 1.97-2.07, 2.38-2.52, 2.63-2.80, BtkICso Fyn ICsoBtkICso LynAICso BtkICso 3.51-3.63, 3.77-3.94, 4.00-4.19, 4.27-4.35, 5.26-5.38, 7.08 Example 9 375 1057 1662 7.23, 7.38-7.52, 8.44-8.47 50 Pharmacological Test Examples Results show that the compound of the present invention not only has Btkinhibitory activity, but also has Btk-selective inhibitory activity toward other kinases. Biological Example 1 55 Biological Example 2 Measurement of Btk Inhibitory Activity and Blood Kinetics in Dogs Selectivity Toward Btk (In Vitro) 60 The kinetic profiles of compound A and the salt thereof (the The measurement of Btk enzyme inhibitory activity was compound prepared in Example 9) in blood were evaluated in performed using the following reagents: Z-LYTETM Kinase male beagle dogs under fasting conditions. Intravenous Assay Kit-Tyr 1 (containing Tyr 1 peptide, Thy 1 phospho administration, as well as both oral liquid and oral Suspension peptide, 5x kinase buffer, ATP, development reagent B, devel administrations were carried out for Compound A prepared in opment buffer, and stop reagent), Tyr 1 peptide (Invitro 65 Example 8, and oral administration by capsule was carried genTM), and Btk (InvitrogenTM) according to the instructions out for the compound prepared in Example 9. For the intra accompanying the kit. venous and oral Solution administrations, a solubilized solu US 9, 199,997 B2 21 22 tion of Compound A dissolved in WellSolve (Celeste Inc.) Solution test I liquid, Japanese Pharmacopoeia dissolution heated to 60° C. was used. Using the solubilized solutions, test II liquid, diluted McIlvaine buffer (pH 4.0, pH 7.4), doses of 1 mg/1 mL/kg were rapidly administered intrave purified water, and artificial intestinal fluids (FaSSIF, FeS nously with a syringe via a forelimb cephalic vein, and doses SIF). With stirring at 700 rpm with a magnetic stirrer, of 1 mg/5 mL/kg were administered by gavage using a cath approximately 1 mL samples were taken from the test Sus eter. Approximately 300 uL blood samples were drawn from pension at 30 min and 24 h after the start of the test, and after the jugular vein as follows: for intravenous administration filtering with a 0.2 um filter, the solubility of Compound A before administration, 2, 5, 15, and 30 minutes after admin was measured by HPLC under the conditions shown below. istration, and 1, 2, 4, 6, 8, and 24 hours after administration; Table 6 below compares the solubility of Compound A and for oral administration-before administration, 5, 15, and 30 10 the compound prepared in Example 9. minutes after administration, and 1, 2, 4, 6, 8, and 24 hours