Tetrahydroindazoles as Interleukin-2 Inducible T-Cell Kinase Inhibitors

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Tetrahydroindazoles as Interleukin‑2 Inducible T‑Cell Kinase Inhibitors. Part II. Second-Generation Analogues with Enhanced Potency, Selectivity, and Pharmacodynamic Modulation in Vivo Jason D. Burch,† Kathy Barrett,† Yuan Chen,† Jason DeVoss,† Charles Eigenbrot,† Richard Goldsmith,† M. Hicham A. Ismaili,† Kevin Lau,† Zhonghua Lin,† Daniel F. Ortwine,† Ali A. Zarrin,† Paul A. McEwan,‡ John J. Barker,‡ Claire Ellebrandt,§ Daniel Kordt,§ Daniel B. Stein,§ Xiaolu Wang,§ Yong Chen,∥ Baihua Hu,∥ Xiaofeng Xu,∥ Po-Wai Yuen,∥ Yamin Zhang,∥ and Zhonghua Pei*,† J. Med. Chem. 2015.58:3806-3816. Downloaded from pubs.acs.org by UNIV OF WINNIPEG on 08/20/18. For personal use only.

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Genentech Inc., 1 DNA Way, South San Francisco, California 94080, United States Evotec (U.K.) Ltd., 114 Milton Park, Abingdon, Oxfordshire OX14 4RZ, United Kingdom § Evotec AG, Manfred Eigen Campus, Essener Bogen 7, D-22419 Hamburg, Germany ∥ Pharmaron Beijing Ltd. Co., No. 6 TaiHe Road BDA, 100176 Beijing, P. R. China ‡

S Supporting Information *

ABSTRACT: The medicinal chemistry community has directed considerable efforts toward the discovery of selective inhibitors of interleukin-2 inducible T-cell kinase (ITK), given its role in T-cell signaling downstream of the T-cell receptor (TCR) and the implications of this target for inflammatory disorders such as asthma. We have previously disclosed a structureand property-guided lead optimization effort which resulted in the discovery of a new series of tetrahydroindazole-containing selective ITK inhibitors. Herein we disclose further optimization of this series that resulted in further potency improvements, reduced off-target receptor binding liabilities, and reduced cytotoxicity. Specifically, we have identified a correlation between the basicity of solubilizing elements in the ITK inhibitors and off-target antiproliferative effects, which was exploited to reduce cytotoxicity while maintaining kinase selectivity. Optimized analogues were shown to reduce IL-2 and IL-13 production in vivo following oral or intraperitoneal dosing in mice.

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one example5f and the reduction of IL-4 production and lung inflammation in another.5b We have previously disclosed the evolution of an indazole series of ITK inhibitors discovered from an HTS campaign5m using property- and structure-based methods to a new series of tetrahydroindazole (THI) inhibitors, ultimately resulting in the potent and selective inhibitor GNE-9822 (1, Figure 1).5q As we continued to profile this lead compound toward in vivo efficacy studies, we uncovered off-target receptor binding and undesirable antiproliferative effects in hepatocytes and Jurkat cells (vide infra). Herein we disclose further optimization efforts that simultaneously eliminated these liabilities while further improving ITK inhibitory potency and ultimately resulted in inhibitors capable of modulating T-cell receptor signaling in vivo.

INTRODUCTION The Tec family kinases include IL-2 inducible T cell kinase (ITK), resting lymphocyte kinase (RLK), Bruton’s tyrosine kinase (Btk), tyrosine kinase expressed in hepatocellular carcinoma (Tec), and bone marrow expressed kinase (Bmx). ITK is a tyrosine kinase that is involved in T cell development, differentiation, and effector function.1 While ITK, RLK, and Tec are all expressed in T cells, the prominent role for ITK and RLK in T cell receptor signaling (TCR), phospholipase γ-1 (PLCγ1) phosphorylation and subsequent Ca2+ mobilization has been established through murine knockout studies.2 Several groups have shown that ITK deficiency results in selective impairment of T helper 2 (Th2) cell function relative to other T cell subtypes.3 Furthermore, following ovalbumin challenge, ITK −/− mice demonstrate reduced lung inflammation, eosinophil infiltration, and mucous production.4a Studies using an ITK kinase-dead transgenic mouse model suggest that ITK kinase activity is required for the control of Th2 responses and the development of allergic asthma.4b The wealth of preclinical evidence supporting the role of ITK in allergic asthma and other inflammatory disorders has prompted intense research effort from the pharmaceutical community toward the discovery of selective inhibitors of this kinase.5 Preclinical in vivo activity using ITK inhibitors has been reported, resulting in the reduction of IL-2 production in © 2015 American Chemical Society

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CHEMISTRY Compounds were prepared in a manner analogous to that disclosed previously (Scheme 1).5q Racemic nitropyrazoles 2i− ix were reduced to aminopyrazoles 3i−ix with palladium on carbon under an atmosphere of hydrogen, then the requisite inhibitors 17−30 were obtained by HATU-mediated coupling Received: December 23, 2014 Published: April 6, 2015 3806

DOI: 10.1021/jm501998m J. Med. Chem. 2015, 58, 3806−3816

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Journal of Medicinal Chemistry

Scheme 2. Synthesis of Tetrahydroindazole Carboxylic Acid 6a

Reagents and conditions: (a) TMSCF3, NaI, THF, 65 °C; (b) pMeC6H4SO3H, acetone/water, 50 °C; (c) diethyl oxalate, t-BuOK, THF, −70 °C; (d) hydrazine, AcOH, 120 °C; (e) NaOH, EtOH, water, 50 °C. a

Figure 1. Previously disclosed indazole and tetrahydroindazole series of ITK inhibitors.

Scheme 1. General Synthesis of Inhibitors 17−30a

butoxide7 provided α-keto ester 10, which was subsequently converted to carboxylic acid 6 via cyclization with hydrazine followed by ester hydrolysis. While the synthesis of aminopyrazoles 3i−iii was presented in our previous publication,5q preparation of nitropyrazoles 2iv−ix is shown in Scheme 3. Addition of phenyl magnesium Scheme 3. Synthesis of Nitropyrazoles 2iv−ixa

a

a

with tetrahydroindazole carboxylic acids 4−6. Single stereoisomers were obtained by supercritical fluid chromatography (SFC) on chiral solid supports when necessary. The synthesis of tetrahydroindazole carboxylic acids 4 and 5 has been disclosed previously;5q synthesis of carboxylic acid 6 is shown in Scheme 2. Difluorocyclopropanation6 of protected cyclohexanone 7 yielded cyclopropane 8, which was deprotected under acidic conditions to provide ketone 9. Claisen condensation with diethyl oxalate mediated by potassium tert-

bromide to aldehydes 11v−vii provided benzylic alcohols 12v− vii, which were subsequently converted to nitropyrazole sulfones by a Mitsunobu reaction8 with nitropyrazole followed by mCPBA-mediated oxidation. Nitropyrazole 2ix at the sulfoxide oxidation state could be obtained by careful control of the equivalents of the oxidizing agent. Finally, the lithium anion of tetrahydrothiopyran sulfoxide (13) was added to

Reagents and conditions: (a) PhMgBr, THF, 0 °C → rt; (b) 4nitropyrazole, PPh3, DIAD, THF, 0 °C → rt; (c) mCPBA, DCM, 0 °C (excess for 2v−viii; 1.0 equiv for 2ix); (d) n-BuLi, THF, −78 °C then benzaldehyde, −78 → −20 °C.

Reagents and conditions: (a) H2 (1 atm), 10% Pd/C, EtOH, rt; (b) 4−6, HATU, iPr2NEt, DMF, rt.

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phosphorylation of PLCγ-1 was measured. When comparing Jurkat cell proliferation versus inhibition of PLCγ phosphorylation (Figure 2), we were encouraged by the lack of

benzaldehyde to provide benzyl alcohol 14 as a mixture of diastereomers.9 Mitsunobu reaction and oxidation provided nitropyrazole 2viii. For inhibitor 18, a slightly modified protocol (Scheme 4) was necessary due to issues with elimination of the requisite Scheme 4. Synthesis of Inhibitor 18a

a

Reagents and conditions: (a) 4-nitropyrazole, PPh3, DIAD, THF, 0 °C → rt; (b) 10% Pd/C, H2, MeOH, rt; (c) 4b, HATU, iPr2NEt, DMF, rt; (d) mCPBA, DCM, 0 °C; (e) TFA, iPr3SiH, DCM, rt. Figure 2. Inhibition of Jurkat cell proliferation versus inhibition of phosphorylation of PLCγ-1 for all THI inhibitors color-coded by calculated most basic pKa (7.5, red). The potencies are expressed as −log10(IC50), where IC50 values are in nM unit.

nitropyrazole. 2-(Methylthio)-1-phenylethan-1-ol (15) was converted to the aminopyrazole 16 via a Mitsonobu reaction without oxidation to the sulfone followed by reduction of the nitro group. Amide bond formation with SEM-protected indazole acid 4b,5q oxidation to the sulfone with mCPBA, and then SEM deprotection provided 18.

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correlation between cellular ITK inhibition and antiproliferative effects (cytotoxicity), which suggests that the cytotoxicity is not caused by ITK inhibition. We then invested significant effort to identify off-target(s) responsible for this cytotoxicity. After extensive kinase profiling of a number of compounds, we were unable to identify an offtarget kinase or combination of kinases, inhibition of which correlated with observed cytotoxicity in Jurkat cells.12 Instead, a plot of amine basicity within the THI series showed that reduced basicity (calculated most basic pKa 50% at 0.1 μM). This promising in vitro profile prompted further profiling of this compound, during which undesirable in vitro toxicity liabilities were uncovered. When subjected to the CEREP receptor panel, 1 bound strongly (96%) to the 5-HT2A receptor, dopamine, and 5-HT transporters (102%) and the Ca2+ channel (88%), and also exhibited strong inhibition (88%) of the hERG channel (patch clamp assay) at 10 μM.10 Compound 1 exhibited antiproliferative effects in hepatocytes (IC50 = 15 μM), where ITK is not expressed. In retrospect, receptor promiscuity might have been expected, given that the structure of 1 shares the pharmacophore features of binding to hERG and other receptors: strongly basic amine (calculated pKa ∼ 9) tethered to a lipophilic, polyaromatic core.11 On the other hand, the undesired broad cellular cytotoxicity was somewhat surprising, given that kinase selectivity had improved dramatically relative to our earlier chemical matter. This cytotoxicity would make 1 unsuitable for asthma indication. We thus chose to explore the reduction of cytotoxicity associated with this series of compounds. We developed and implemented a proliferation assay of Jurkat cells with no TCR stimulation by the measurement of ATP production, which allowed us to gauge the off-target cytotoxicity of the compounds. Jurkat cells were selected for this investigation as this was the same cell type used for determination of cellular ITK activity, where inhibition of 3808

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Journal of Medicinal Chemistry Table 1. SAR of THI Analogues Containing Nonbasic Benzylic Side Chainsa

a

All values are the mean of two or more independent assays. bStereochemistry of 1 was assigned by X-ray crystallography (Figure 3). Stereochemistry of the benzylic carbon of the rest of the compounds was assigned by analogy: the more active enantiomer is presumed to have the same stereochemistry as in 1. The relative stereochemistry of 20−23 is unknown, and only the two most potent diastereomers of the four possible are shown. cInhibition of PLC-γ phosphorylation in Jurkat cells with TCR stimulation. dAntiproliferation effect on Jurkat cells without TCR stimulation. eSolubility in aqueous phosphate buffer solution (pH 7.4).

plasma protein was present) to the extent of below the cytotoxicity IC50 compared to compound 1, solubility in the Jurkat proliferation assay buffer conditions, where 10% FBS was present, was higher than the observed proliferation IC50s in all cases (data not shown). Having identified chemical matter with improved cytotoxicity profiles, we were interested in further improving ITK inhibitory potency. We postulated that further optimization of the interactions between the ligand and the lipophilic pocket near Phe435 (cf. Figure 3) could be a source of improving potency, and Table 2 highlights our effort in this regard. Replacement of the gem-dimethyl substituent present in earlier analogues with a cyclopropyl methyl moiety (25) resulted in a significant improvement in cellular potency relative to the series progenitor 24.5q An additional order of magnitude improvement was achieved by replacement of the methylene moiety in the cyclopropane ring by a difluoromethylene (26). Combining the pertinent discoveries summarized in Tables 1 and 2, we were able to discover two additional noteworthy compounds, 27 (GNE-6688) and 28 (GNE-4997), with 28 achieving cellular potency of 4 nM (Figure 4). These compounds, in addition to 20, were selected for in-depth profiling, and the results are summarized in Table 3 in comparison to our earlier lead 1 (GNE-9822). All compounds showed improved potency relative to 1, with comparable kinase selectivity. Compound 28 is especially noteworthy in this

Figure 3. Co-crystal structure of 1 with the kinase domain of ITK.5q Favorable interactions between protein−ligand are denoted by dashed lines with cylinders. The solvent accessible surface of the protein is color coded by lipophilic potential (pink is polar; green is hydrophobic). Figure was created with MOE (www.chemcomp.com).

regard, demonstrating a 10-fold improvement in ITK enzymatic and cellular potency while improving magnitude of kinase selectivity at 0.1 μM (a concentration that is >1000-fold over ITK Ki) compared to compound 1. The in vitro cytotoxicity window was improved from 85-fold for 1 to 3000-fold for 28. 3809

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Journal of Medicinal Chemistry Table 2. Optimization of the Tetrahydroindazole Subunita

Table 3. Profile of Inhibitors 1, 20, 27, and 28a compd

1

20

27

28

ITK Ki PLCg IC50b Jurkat prolif IC50 cytotox windowc LCK Ki select foldd kinase sele solubility log D7.4 TPSA MDCK permf hPPB (%) HHep Clg CEREP panelh hERG %inhi hepatotox IC50

0.0007 0.045 4.7 85× 0.34 470× 6/286 68 2.8 78 4.6 93 5.0 5/40 88 6.2

0.0002 0.028 30 1070× 1.6 8000× 10/285 45 3.6 92 1.9 94 13 0/40 3.6 83

0.0002 0.039 29 740× 0.26 1600× 9/225 6.5 3.5 109 4.4 97 13 0/40 34 >100

0.00009 0.004 12 3000× 0.46 5000× 7/225 3.9 2.8 109 1.5 97 5.6 1/40 6.8 >100

a

Ki, IC50, and solubility have the unit of micormolar (μM). bInhibition of PLC-γ phosphorylation in Jurkat cells with TCR stimulation. c Cytotoxicity window was calculated by dividing Jurkat prolif IC50 by PLCg IC50. dSelectivity fold was calculated by Lck Ki divided by ITK Ki. eKinase selectivity is gauged by the number of kinases inhibited >70% at 0.1 μM out of the total number of kinases in the kinase panel at Invitrogen. fPermeability in MDCK cell line in the unit of 10−6 cm/ s. gClearance in human hepatocytes in the unit of mL/min/kg. h Number of receptors in the CEREP receptor panel showing >70% binding at 10 μM. iPercentage of hERG inhibition at a compound concentration of 10 μM.

a

All values are the mean of two or more independent assays. Absolute stereochemistry of the more potent isomers of 25 and 26 was inferred from the X-ray crystal structure of 30 bound to ITK (Figure 5). b

Figure 4. Structures and potencies of compounds 27 and 28.

All compounds also exhibited reduced receptor binding, hERG inhibition, and hepatotoxicity (Table 3 and Table S1 in Supporting Information). Compounds 20, 27, and 28 do, however, have lower solubility than compound 1. We were able to obtain an X-ray crystal structure of 28 bound to ITK (Figure 5). The binding mode is consistent with that of 1 (Figure 3).5q The inhibitor amide NH engages the carbonyl of Met438 and the pyrazole participates in a donor− acceptor pair with the NH of Met438 and the carbonyl of Glu436, thus anchoring the inhibitor to the hinge region. The cyclic sulfone serves to orient the benzyl phenyl ring for an edge-to-face interaction with Phe437. The difluoromethylene group of the cyclopropane occupies a lipophilic pocket adjacent to Phe435 (the “gatekeeper” residue). The observed increase in potency for these difluorocyclopropane inhibitors is likely due to more efficient space-filling of this pocket. Having demonstrated improved cellular potency and improved in vitro safety, we tested the compounds in an in vivo pharmacodynamics (PD) model. Cross-linking of the T cell receptor (TCR) with an antibody against CD3 (145-2C11)

Figure 5. Co-crystal structure of 28 complexed with the kinase domain of ITK (PDB code 4rfm). The two fluorine atoms occupy a hydrophobic pocket within ITK adjacent to the gatekeeper Phe435. Interactions and surface depictions are the same as in Figure 3

resulted in the release of interleukin-2 (IL-2) and other cytokines in a dose- and time-dependent fashion (data not shown). Mice were first dosed with ITK inhibitors at different dose levels. One hour later, anti-CD3 was administered (10 μg, IV) and serum IL-2 and IL-13 levels were measured by Meso Scale Discovery (MSD) after 1.5 h. We chose to measure IL-2 and IL-13 levels as these two cytokines are affected by ITKmediated TCR signaling.1 As shown in Figure 6, IL-2 levels were decreased in a dose-dependent manner, reaching a maximum 98% reduction at the 200 mg/kg of compound 20. We observed a similar trend with regard to IL-13 levels, reaching 100% reduction at the 200 mg/kg dose. Compound 3810

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Varian Prostar instrument, using a Phenomenex Gemini-NX C-18 (0.3 cm × 5 cm; 5 μm particle size) stationary phase, with 0.1% aqueous formic acid/acetonitrile (for acidic or neutral compounds) or 0.1% aqueous ammonium hydroxide/acetonitrile (for basic compounds) gradients as the mobile phase (typically 5−85% acetonitrile over 10 min) with a flow rate of 60 mL/min. Preparative SFC separations were performed on a PIC Solutions instrument, with conditions indicated in the Experimental Section. Proton NMR spectra were measured on Bruker 300 or 400 MHz spectrometers, and chemical shifts are reported in ppm downfield from TMS (0 ppm). High-resolution mass spectrometry of final compounds were performed on a Thermo UHPLC/QE with a Thermo-Q Exactive mass spectrometry detector using ESI ionization, after elution on a Acquity BEH C18 (2.1 mm × 50 mm; 1.7 μm particle size) stationary phase using a gradient of water/acetonitrile (3−97% over 7 min; 0.1% formic acid in both phases). The purity of final compounds 14−32 was verified by HPLC on an Agilent 1200 instrument, with an Agilent SB C-18 (2.1 mm × 30 mm; 1.8 μm particle size) stationary phase and a gradient of water/ acetonitrile (5−95% over 6 min; 0.05% TFA in both phases) at a flow rate of 0.4 mL/min. Quantification of target and impurities was done by UV detection at 254 nm, and is >95% in all cases. Compounds 17, 24, 25, and 25′ were reported as examples 30, 17, and 26, respectively, in the previous paper.5q 6,6-Dimethyl-N-(1-(2-(methylsulfonyl)-1-phenylethyl)-1H-pyrazol-4-yl)-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (18). To a solution of 2-methylsulfanyl-1-phenyl-ethanone (452 mg, 2.72 mmol, 452 mg) in methanol (5 mL, 5 mL) and THF (5 mL) was added sodium borohydride (1.5 equiv, 4.07 mmol, 157.4 mg), and the mixture was stirred at rt for 90 min. Excess hydride was quenched with ∼10 mL satd NH4Cl(aq), then the mixture was diluted with 50 mL of brine and extracted with 50 mL of EtOAc. The organic extracts were dried (Na 2 SO 4 ) and concentrated in vacuo to provide 2methylsulfanyl-1-phenyl-ethanol (447 mg, 97.7% yield) of sufficient purity to be used directly in the next step. To a solution of 2-methylsulfanyl-1-phenyl-ethanol (447 mg, 2.65 mmol, 447 mg) and 4-nitro-1H-pyrazole (1.1 equiv, 2.92 mmol, 330.4 mg) in THF (10 mL) was added triphenylphosphine (1.1 equiv, 2.92 mmol, 766.5 mg) and diisopropyl azodicarboxylate (1.1 equiv, 2.92 mmol, 0.605 mL). The mixture was stirred overnight at rt. After in vacuo concentration, the residue was purification by CombiFlash (40 g; 100:0 to 50:50 heptane:EtOAc over 20 min) to provide provide 1(2-methylsulfanyl-1-phenyl-ethyl)-4-nitro-pyrazole (666 mg, 2.53 mmol, 95.2% yield) containing small amounts of other impurities by NMR, but which was used without further purification. To a solution of 1-(2-methylsulfanyl-1-phenyl-ethyl)-4-nitropyrazole (666 mg, 2.53 mmol) in methanol (10 mL) was added palladium on carbon (0.1 equiv, 0.253 mmol, 538.4 mg), and the mixture was stirred under an atmosphere of H2 (balloon) for 2 h. LCMS shows complete consumption of starting material. The catalyst was deactivated by the addition of CH2Cl2, then the mixture was filtered through Celite (CH2Cl2 and MeOH rinse). The filtrate was concentrated in vacuo to provide 1-(2-methylsulfanyl-1-phenylethyl)pyrazol-4-amine of sufficient purity to be used directly. General Procedure A: Amide Coupling. To a solution of 1-(2methylsulfanyl-1-phenyl-ethyl)pyrazol-4-amine (2.53 mmol, 590 mg) and 6,6-dimethyl-1-(2-trimethylsilylethoxymethyl)-5,7-dihydro-4H-indazole-3-carboxylic acid (1.0 equiv, 2.53 mmol, 821 mg) in dimethylformamide (10 mL) was added HATU (1.0 equiv, 2.53 mmol, 992 mg) and N,N′-diisopropylethylamine (1.5 equiv, 3.80 mmol, 0.668 mL), and the mixture was stirred overnight at rt. The mixture was diluted with 100 mL of EtOAc and washed with 100 mL of satd NaHCO3(aq) and 2 × 100 mL of 1:1 H2O:brine. The organic extracts were dried (Na2SO4) and concentrated in vacuo. The product was purification by CombiFlash (40 g; dry load; 100:0 to 50:50 heptane:EtOAc over 20 min) to give 6,6-dimethyl-N-[1-(2-methylsulfanyl-1-phenyl-ethyl)pyrazol-4-yl]-1-(2-trimethylsilylethoxymethyl)-5,7-dihydro-4H-indazole-3-carboxamide (1.31 g, 2.43 mmol, 96% yield). To a solution of 6,6-dimethyl-N-[1-(2-methylsulfanyl-1-phenylethyl)pyrazol-4-yl]-1-(2-trimethylsilylethoxymethyl)-5,7-dihydro-4H-

Figure 6. Reduction of serum IL-2 and IL-13 following IP dosing of ITK inhibitor 20 in mice.

concentration measured at 2.5 h of the study increased as dose increased, reaching a plasma concentration of ∼80 μM at the highest dose.14

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CONCLUSION In conclusion, we have further optimized a tetrahydroindazole series of ITK inhibitors, discovering second generation analogues with increased potency and decreased off-target in vitro toxicity. Specifically, we have demonstrated an interesting relationship between cellular toxicity and compound basicity in the absence of significant differences in kinase selectivity. Taking advantage of this observation, we were able to design compounds with decreased cytotoxicity. Furthermore, we have now demonstrated in vivo pathway modulation with our optimized inhibitors.

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EXPERIMENTAL SECTION

Chemistry. General. All commercially available reagents and solvents were used as received without further purification or drying unless otherwise stated. Reactions using air- or moisture-sensitive reagents were performed under an atmosphere of nitrogen using freshly opened EMD DriSolv solvents. Reaction progress was monitored by TLC and/or HPLC. Flash chromatography was performed with Isco CombiFlash Companion systems using prepacked silica gel columns (40−60 μm particle size RediSep or 20−40 μm spherical silica gel RediSep Gold columns, or similar columns from other vendors). Preparative HPLC purifications were performed on a 3811

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Journal of Medicinal Chemistry

N-(1-((1,1-Dioxidotetrahydro-2H-thiopyran-4-yl)(phenyl)methyl)1H-pyrazol-4-yl)-6,6-dimethyl-4,5,6,7-tetrahydro-1H-indazole-3carboxamide (19). Prepared by following general procedures A and B. Chiral HPLC (ChiralPak IB-3 (4.6 mm × 50 mm, 3 μm particle size); eluent = Hex:EtOH 60:40; 1.0 mL/min, 3.5 MPA, 25 °C) purification gave 19 as the fast-eluting isomer. 1H NMR (CDCl3, 300 MHz): δ 8.62 (s, 1H), 8.14 (s, 1H), 7.56 (s, 1H), 7.43−7.30 (m, 5H), 4.83 (d, J = 10.5 Hz, 2H), 3.03−2.91 (m, 4H), 2.86−2.73 (m, 3H), 2.41 (s, 2H), 1.95−1.79 (m, 4H), 1.56 (t, J = 6.3 Hz, 2H), 1.01 (s, 6H). MS: m/z = 482 (M + H). HPLC retention time: 1.97 min. 4-((4-Amino-1H-pyrazol-1-yl)(phenyl)methyl)tetrahydro-2H-thiopyran 1-oxide. A solution of mCPBA (570 mg, 3.30 mmol, 1.00 equiv) in AcOEt (5 mL) was added dropwise to a stirred solution of 4nitro-1-[phenyl(tetrahydro-2H-thiopyran-4-yl)methyl]-1H-pyrazole (1.0 g, 3.30 mmol, 1.00 equiv) in dichloromethane (50 mL) at 0 °C. After 30 min, the resulting solution was diluted with 250 mL of AcOEt and washed with 3 × 150 mL of saturated solution of sodium carbonate and 3 × 150 mL of brine. The organic was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied onto a silica gel column with ethyl acetate/ petroleum ether (1/2 to 2/1). This resulted in 500 mg (47%) and 400 mg (38%) of the two diastereomers (stereochemistry unassigned) of 4-nitro-1-[phenyl(1-oxo-tetrahydro-2H-thiopyran-4-yl)methyl]-1Hpyrazole. General Procedure C: Reduction of Nitro Group. Each diastereomer was then reduced individually: Hydrogen gas was introduced into a mixture of 4-nitro-1-[phenyl(1-oxo-tetrahydro-2Hthiopyran-4-yl)methyl]-1H-pyrazole (500 mg, 1.57 mmol, 1.00 equiv) and palladium on carbon (500 mg) in methanol (50 mL). After 30 min at room temperature, the solids were filtered out. The filtrate was concentrated under vacuum. This resulted in 425 mg (94%) of 4amino-1-[phenyl(1-oxo-tetrahydro-2H-thiopyran-4-yl)methyl]-1Hpyrazole as a light-yellow solid. 6,6-Dimethyl-N-(1-((1-oxidotetrahydro-2H-thiopyran-4-yl)(phenyl)methyl)-1H-pyrazol-4-yl)-4,5,6,7-tetrahydro-1H-indazole-3carboxamide (20 and 20′). Prepared by following general procedure A. Each diastereomer was reacted independently, then resolved into their constituent stereoisomers by chiral LCMS yielding the four diastereomeric products. Chiral HPLC conditions: ChiralPak IA-3 (4.6 mm × 50 mm, 3 μm particle size); eluent = Hex (0.1% Et3N):EtOH 50:50; 1.0 mL/min, 5.8 MPA, 25 °C. 20: 1H NMR (300 MHz, CDCl3) δ 8.85 (s, 1H), 8.14 (s, 1H), 7.56 (s, 1H), 7.41 (d, J = 1.5 Hz, 2H), 7.39−7.26 (m, 3H), 4.96 (d, J = 10.8 Hz, 1H), 3.03−2.94 (m, 2H), 2.85 (t, J = 6.3 Hz, 2H), 2.72−2.60 (m, 1H), 2.52−2.34 (m, 4H), 2.27−1.97 (m, 2H), 1.56 (t, J = 6.4 Hz, 2H), 1.49−1.38 (m, 2H), 1.00 (s, 6H). MS: m/z = 466 (M + H). HPLC retention time: 2.71 min. 20′: 1H NMR (300 MHz, CDCl3) δ 8.84 (s, 1H), 8.14 (s, 1H), 7.56 (s, 1H), 7.41 (d, J = 1.5 Hz, 2H), 7.39−7.26 (m, 3H), 4.96 (d, J = 10.8 Hz, 1H), 3.03−2.93 (m, 2H), 2.85 (t, J = 6.3 Hz, 2H), 2.72−2.60 (m, 1H), 2.51−2.34 (m, 4H), 2.27−1.97 (m, 2H), 1.56 (t, J = 6.3 Hz, 2H), 1.48−1.38 (m, 2H), 1.00 (s, 6H). MS: m/z = 466 (M + H). HPLC retention time: 5.89 min. Chiral HPLC conditions for the other two less potent isomers: ChiralPak IB (4.6 mm × 250 mm, 5 μm particle size); eluent = Hex (0.1% Et3N):EtOH 70:30; 1.0 mL/min, 5.9 MPA, 25 °C. HPLC retention time: 9.74 and 15.63 min, respectively. N-(1-((1,1-Dioxidotetrahydrothiophen-3-yl)(phenyl)methyl)-1Hpyrazol-4-yl)-6,6-dimethyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (21 and 21′). Prepared in an analogous manner to compound 19. The stereoisomers were separated by preparative chiral HPLC instead of SFC (chiral HPLC conditions: Lux Cellulose-4 (4.6 mm × 150 mm, 3 μm particle size); eluent = Hex:EtOH 60:40; 1.0 mL/min, 4.2 MPA, 25 °C). 21: 1H NMR (300 MHz, CDCl3) δ 8.62 (s, 1H), 8.14 (s, 1H), 7.57 (s, 1H), 7.43−7.31 (m, 5H), 5.06 (d, J = 10.5 Hz, 1H), 3.69−3.66 (m, 1H), 3.24−2.98 (m, 3H), 2.86−2.82 (t, J = 6.3 Hz, 2H), 2.78−2.70 (m, 1H), 2.43 (s, 2H), 2.24−2.22 (m, 1H), 2.01−1.94 (m, 1H), 1.59−1.55 (t, J = 6.3 Hz, 2H), 1.02 (s, 6H). MS: m/z = 468 (M + H); HPLC retention time: 12.3 min. 21′: 1H NMR (300 MHz, CDCl3) δ 8.68 (s, 1H), 8.10 (s, 1H), 7.57 (s, 1H), 7.40− 7.32 (m, 5H), 5.04 (d, J = 10.5 Hz, 1H), 3.67−3.65 (m, 1H), 3.32−

indazole-3-carboxamide (300 mg, 0.556 mmol) in tetrahydrofuran (5 mL) was added 3-chloroperbenzoic acid (2.2 equiv, 1.22 mmol, 274 mg), and the mixture was stirred for 60 min at rt. The mixture was diluted with 50 mL of EtOAc and washed with 50 mL of satd aq NaHCO3 and 50 mL of brine. The organic extracts were dried (Na2SO4) and concentrated in vacuo. Purification by CombiFlash (24 g; dry load; 70:30 to 30:70 heptane: EtOAc over 20 min) provided 6,6-dimethyl-N-[1-(2-methylsulfonyl-1- phenyl-ethyl)pyrazol-4-yl]-1(2-trimethylsilylethoxymethyl)-5,7-dihydro-4H-indazole-3-carboxamide (90 mg, 0.157 mmol, 28% yield). General Procedure B: Removal of SEM Group. To a solution of 6,6-dimethyl-N-[1-(2-methylsulfonyl-1-phenyl-ethyl)pyrazol-4-yl]-1(2-trimethylsilylethoxymethyl)-5,7-dihydro-4H-indazole-3-carboxamide (90.0 mg, 0.157 mmol) in trifluoroacetic acid (2 mL) was added triisopropylsilane (5 equiv, 0.787 mmol, 0.163 mL), and the mixture was stirred for 90 min at rt. After in vacuo concentration, the residue was purified by reverse-phase HPLC, followed by SFC on a chiral stationary phase to provide the title compounds as single enantiomers. SFC conditions: Lux Cellulose-3 (4.6 mm × 50 mm, 5 μm particle size) at 30% methanol w/0.1% NH4OH; 5 mL/min, 120 bar, 40 °C. The fast eluting isomer is the more potent inhibitor of ITK, and the stereochemistry was assigned by analogy. 1H NMR (400 MHz, DMSO-d6) δ 12.85−12.62 (s, 1H), 10.22−9.97 (s, 1H), 8.29−8.24 (s, 1H), 7.75−7.70 (s, 1H), 7.46−7.40 (m, 2H), 7.40−7.27 (m, 3H), 6.02−5.95 (dd, J = 9.7, 3.9 Hz, 1H), 4.58−4.47 (dd, J = 14.9, 9.7 Hz, 1H), 3.94−3.83 (dd, J = 14.9, 4.0 Hz, 1H), 2.71−2.62 (m, 5H), 2.40− 2.36 (s, 2H), 1.52−1.43 (t, J = 6.4 Hz, 2H), 0.99−0.92 (s, 6H). MS: m/z = 442 (M + H). SFC retention time: 0.47 min. 4-((4-Amino-1H-pyrazol-1-yl)(phenyl)methyl)tetrahydro-2H-thiopyran-1,1-dioxide. Into a 2 L three-necked round-bottom flask purged and maintained with a nitrogen atmosphere, was placed tetrahydro2H-thiopyran-4-carbaldehyde (65.0 g, 499.2 mmol, 1.00 equiv) and tetrahydrofuran (300 mL). PhMgBr (1M, 750 mL, 1.50 equiv) was added dropwise to the stirred solution at 0 °C. The resulting solution was then stirred for 12 h at room temperature. The reaction progress was monitored by TLC with PE/DCM = 2/1. The reaction was then quenched by the addition of 500 mL of saturated NH4Cl and extracted with 3 × 500 mL of ethyl acetate. The combined organic was washed with 3 × 500 mL of brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel column chromatography (1/20 of acetate/petroleum ether) to provide 27.0 g (26%) of phenyl(tetrahydro-2H-thiopyran-4-yl)methanol as a yellow oil. DIAD (18.9 g, 93.47 mmol, 1.50 equiv) was added to the solution of phenyl(tetrahydro-2H-thiopyran-4-yl)methanol (13.0 g, 62.4 mmol, 1.00 equiv), 4-nitro-1H-pyrazole (8.5 g, 75.17 mmol, 1.20 equiv), and PPh3 (24.6 g, 93.8 mmol, 1.50 equiv) in tetrahydrofuran (300 mL) dropwise at 0 °C and stirred for 12 h at room temperature under nitrogen. The reaction solution was concentrated under vacuum. The residue was purified by a silica gel column eluting with ethyl acetate/ petroleum ether (1/10) to afford 4-nitro-1-[phenyl(tetrahydro-2Hthiopyran-4-yl)methyl]-1H-pyrazole (13.0 g, 69%) as a white solid. A solution of mCPBA (23.90 g, 138.4 mmol, 3.00 equiv) in AcOEt (50 mL) was added to the solution of 4-nitro-1-[phenyl(tetrahydro2H-thiopyran-4-yl)methyl]-1H-pyrazole (14.0 g, 46.15 mmol, 1.00 equiv) in dichloromethane (200 mL) dropwise at 5 °C and stirred for 30 min at 5 °C. The resulting solution was diluted with 300 mL of DCM, washed with 3 × 300 mL of saturated solution of Na2CO3, dried over anhydrous Na2SO4, and concentrated under vacuum to give 4-((4-nitro-1H-pyrazol-1-yl)(phenyl)methyl)tetrahydro-2H-thiopyran1,1-dioxide (13.0 g, 84%) as a white solid. A mixture of 4-((4-nitro-1H-pyrazol-1-yl)(phenyl)methyl)tetrahydro-2H-thiopyran 1,1-dioxide (8.00 g, 23.8 mmol, 1.00 equiv), methanol (500 mL), AcOEt (800 mL), and Raney Ni (6.0 g) was stirred for 12 h at room temperature under an atmosphere of hydrogen. The solids were filtered out. The filtrate was concentrated under vacuum to afford 4-((4-amino-1H-pyrazol-1-yl)(phenyl)methyl)tetrahydro-2H-thiopyran-1,1-dioxide (6.5 g, 89%) as a lightbrown solid. This material was used in the next step without purification. 3812

DOI: 10.1021/jm501998m J. Med. Chem. 2015, 58, 3806−3816

Article

Journal of Medicinal Chemistry

CDCl3) δ 8.68 (s, 1H), 8.12 (s, 1H), 7.62−7.57 (m, 3H), 7.37−7.30 (m, 3H), 5.55 (d, J = 9.6 Hz, 1H), 4.13−4.05 (m, 2H), 3.06−2.82 (m, 4H), 2.43 (s, 2H), 2.04−2.02 (m, 2H), 1.89−1.70 (m, 3H), 1.59−1.50 (m, 3H), 1.01 (s, 6H). MS: m/z = 482 (M + H). HPLC retention time: 1.4 min. 23′: 1H NMR (300 MHz, CDCl3) δ 8.91 (s, 1H), 8.06 (s, 1H), 7.63 (s, 1H), 7.44−7.41 (m, 2H), 7.32−7.26 (m, 3H), 5.71 (d, J = 8.7 Hz, 1H), 4.43−4.35 (m, 1H), 3.11−2.95 (m, 4H), 2.82−2.70 (m, 2H), 2.40 (s, 2H), 2.03−1.98 (m, 2H), 1.84−1.70 (m, 2H), 1.64− 1.47 (m, 4H), 0.99 (s, 6H). MS: m/z = 482 (M + H). HPLC retention time: 5.7 min. 7-Methyl-1,4-dioxaspiro[4.5]dec-7-ene (7). A solution of 1methoxy-3-methylbenzene (11 g, 90.04 mmol, 1.00 equiv) in ether (60 mL) was added dropwise to liquid ammonia (150 mL) at −78 °C. t-Butyl alcohol (60 mL) was added dropwise to the above solution at −78 °C, then sodium (5.20 g, 226 mmol, 2.50 equiv) was added in portions. The resulting solution was warmed to −35 °C and stirred at −35 °C for 2 h. The resulting solution was diluted with 200 mL of pentane, quenched with 100 mL of water (carefully and very slowly), extracted with 2 × 100 mL of pentane, dried over anhydrous sodium sulfate, and concentrated under vacuum to give 9.2 g (82%) of 1methoxy-5-methylcyclohexa-1,4-diene as colorless oil. A solution of 1-methoxy-5-methylcyclohexa-1,4-diene (4.6 g, 37.04 mmol, 1.00 equiv), dichloromethane (100 mL), ethane-1,2-diol (11.5 g, 185 mmol, 5.00 equiv), and 4-methylbenzene-1-sulfonic acid (277 mg, 1.61 mmol, 0.05 equiv) was stirred at room temperature overnight. The reaction mixture was washed with 2 × 50 mL of saturated sodium bicarbonate solution and 3 × 50 mL of water. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by silica gel column chromatography, eluting with petroleum ether to give 2.8 g (49%) of 7-methyl-1,4-dioxaspiro[4.5]dec-7-ene as a colorless oil. 7,7-Difluoro-1-methylbicyclo[4.1.0]heptan-3-one (8). NaI (680 mg, 4.53 mmol, 0.50 equiv), tetrahydrofuran (28 mL), 7-methyl-1,4dioxaspiro[4.5]dec-7-ene (1.4 g, 9.08 mmol, 1.00 equiv), and TMSCF3 (3.23 g, 22.75 mmol, 2.51 equiv) were weighed into a 100 mL sealed tube. The tube was purged and maintained with an inert atmosphere of nitrogen. The reaction was stirred at 65 °C for 12 h and then quenched with 20 mL of water. The resulting solution was extracted with ethyl acetate. The combined organic layer was washed with saturated Na2S2O3 and brine, dried over anhydrous sodium sulfate, and then concentrated under vacuum. The residue was purified by silica gel column chromatography eluting with ethyl acetate/petroleum ether (1/100) to give 1.6 g (86%) of 7,7-difluoro-1-methylspiro[bicyclo[4.1.0]heptane-3,2-[1,3]dioxolane] as a colorless oil. A solution of 7,7-difluoro-1-methylspiro[bicyclo[4.1.0]heptane-3,2[1,3]dioxolane] (1.60 g, 7.83 mmol, 1.00 equiv) and PTSA (135 mg, 0.78 mmol, 0.10 equiv) in acetone (25 mL)/water (5 mL) was stirred for 12 h at 50 °C. The reaction mixture was diluted with 300 mL of diethyl ether. The organic layer was washed with saturated sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to give 1.10 g (88%) of 7,7-difluoro1- methylbicyclo[4.1.0]heptan-3-one as a light-yellow oil. 5,5-Difluoro-5a-methyl-1,4,4a,5,5a,6-hexahydrocyclopropa[f ]indazole-3-carboxylic Acid (6). Under nitrogen t-BuOK (3.1 mL, 1 M in THF, 1.00 equiv) was added dropwise into a solution of 7,7difluoro-1-methylbicyclo[4.1.0]heptan-3-one (500 mg, 3.12 mmol, 1.00 equiv) and diethyl oxalate (456 mg, 3.12 mmol, 1.00 equiv) in tetrahydrofuran (10 mL) at −70 °C. The reaction mixture was stirred for 12 h at −70 °C, quenched by 5 mL of saturated NH4Cl, and then extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. This resulted in 620 mg (76%) of ethyl 2-[7,7-difluoro-6-methyl-4oxobicyclo[4.1.0]heptan-3-yl]-2-oxoacetate as a brown oil. A solution of hydrazine hydrate (763 mg, 15.24 mmol, 6.40 equiv) and ethyl 2-[7,7-difluoro-6-methyl-4-oxobicyclo[4.1.0]heptan-3-yl]-2oxoacetate (620 mg, 2.38 mmol, 1.00 equiv) in acetic acid (15 mL) was stirred for 12 h at 120 °C. The reaction was cooled to room temperature, and the pH of the solution was adjusted to 8−9 with saturated sodium bicarbonate solution. The resulting solution was extracted with ethyl acetate. The organic extract was washed with

2.91 (m, 4H), 2.86 (t, J = 6 Hz, 2H), 2.44 (s, 2H), 2.02−1.86 (m, 2H), 1.59−1.55 (t, J = 6.3 Hz, 2H), 1.02 (s, 6H). MS: m/z = 468 (M + H). HPLC retention time: 15.1 min. N-(1-((1,1-Dioxidotetrahydro-2H-thiopyran-3-yl)(phenyl)methyl)1H-pyrazol-4-yl)-6,6-dimethyl-4,5,6,7-tetrahydro-1H-indazole-3carboxamide (22 and 22′). Prepared in an analogous manner to compound 19. The stereoisomers were separated by preparative chiral HPLC instead of SFC (chiral HPLC conditions: ChiralPak IA (4.6 mm × 250 mm, 3 μm particle size); eluent = (Hex + 0.1% Et3N):EtOH 50:50; 1.0 mL/min, 4.2 MPA, 25 °C). 22: 1H NMR (300 MHz, CDCl3) δ 9.00 (s, 1H), 8.14 (s, 1H), 7.61 (s, 1H), 7.44− 7.29 (m, 5H), 4.99 (d, J = 9.3 Hz, 1H), 3.35 (d, J = 4.5 Hz, 1H), 3.08− 3.02 (m, 1H), 2.93−2.84 (m, 4H), 2.72−2.58 (m, 1H), 2.58 (s, 2H), 2.13−2.05 (m, 2H), 2.01 (s, 1H), 1.72−1.67 (m, 1H), 1.59 (t, J = 7.5 Hz, 2H), 1.32−1.26 (m, 1H), 1.03 (s, 6H). MS: m/z = 482 (M + H). HPLC retention time: 15.5 min. 22′: 1H NMR (300 MHz, CDCl3) δ 8.92 (s, 1H), 8.11 (s, 1H), 7.68 (s, 1H), 7.39−7.29 (m, 5H), 5.07 (d, J = 9.3 Hz, 1H), 3.27−3.21 (m, 1H), 3.06−2.84 (m, 6H), 2.72 (s, 2H), 2.11−2.03 (m, 2H), 1.77−1.72 (m, 1H), 1.55 (t, J = 7.5 Hz, 2H), 1.25−1.20 (m, 1H), 1.03 (s, 6H). MS: m/z = 482 (M + H). HPLC retention time: 13.2 min. 2-((4-Amino-1H-pyrazol-1-yl)(phenyl)methyl)tetrahydro-2H-thiopyran-1,1-dioxide. Butyllithium (84.2 mL, 1.20 equiv) was added to the solution of tetrahydro-2H-thiopyran-1-oxide (20.7 g, 175.13 mmol, 1.00 equiv) in tetrahydrofuran (200 mL) dropwise at −78 °C and stirred for 1 h at −78 °C under nitrogen. To this was added benzaldehyde (18.6 g, 175.27 mmol, 1.00 equiv) dropwise at −78 °C and stirred for 6 h at −78 °C. The reaction was then quenched by 500 mL of saturated NH4Cl, extracted with 3 × 500 mL of ethyl acetate, and concentrated under vacuum. The residue was purified by a silica gel column eluting with DCM:MeOH (20/1) to afford 2-(hydroxy(phenyl)methyl)tetrahydro-2H-thiopyran 1-oxide (30.0 g, 76%) as a light-yellow solid. DIAD (39.6 g, 196.0 mmol, 1.50 equiv) was added to the solution of 2-(hydroxy(phenyl)methyl)tetrahydro-2H-thiopyran-1-oxide (29.3 g, 130.6 mmol, 1.00 equiv), 4-nitro-1H-pyrazole (17.7 g, 156.53 mmol, 1.20 equiv), and PPh3 (51.4 g, 196.18 mmol, 1.50 equiv) in tetrahydrofuran (700 mL) dropwise at 0 °C and stirred overnight at 25 °C under nitrogen. The reaction was then quenched by 1 L of water, extracted with 3 × 1000 mL of ethyl acetate, and concentrated under vacuum. The residue was purified by a silica gel column eluting with ethyl acetate/petroleum ether (1/1) to afford 2-((4-nitro-1Hpyrazol-1-yl)(phenyl)methyl)-tetrahydro-2H-thiopyran 1-oxide (40 g, crude) as a yellow oil. mCPBA was added to the solution of 2-((4-nitro-1H-pyrazol-1yl)(phenyl)methyl)tetrahydro-2H-thiopyran-1-oxide (62.4 g, 195.4 mmol, 1.00 equiv) in dichloromethane (700 mL) at 0 °C under stirred for 4 h at 25 °C. The reaction was then quenched by the addition of 1000 mL of sodium carbonate/H2O, washed with 2 × 500 mL water and 2 × 500 mL of brine, and concentrated under vacuum. The residue was purified by a silica gel column eluting with ethyl acetate/petroleum ether (1/3) to afford 2-((4-nitro-1H-pyrazol-1yl)(phenyl)methyl)tetrahydro-2H-thiopyran-1,1-dioxide (22 g, crude) as a yellow solid. A mixture of 2-((4-nitro-1H-pyrazol-1-yl)(phenyl)methyl)tetrahydro-2H-thiopyran-1,1-dioxide (12 g, 35.78 mmol, 1.00 equiv), 10% palladium on carbon (6.0 g), methanol (300 mL), and ethyl acetate (300 mL) was stirred for 3 h at room temperature under hydrogen. The solids were filtered out. The filtrate was concentrated under vacuum to afford 2-((4-amino-1H-pyrazol-1-yl)(phenyl)methyl)tetrahydro-2H-thiopyran 1,1-dioxide (11.0 g, crude) as a light-red solid. N-(1-((1,1-Dioxidotetrahydro-2H-thiopyran-2-yl)(phenyl)methyl)1H-pyrazol-4-yl)-6,6-dimethyl-4,5,6,7-tetrahydro-1H-indazole-3carboxamide (23 and 23′). Prepared in an analogous manner to compound 19 starting with 2-(hydroxy(phenyl)methyl)tetrahydro2H-thiopyran-1-oxide. The stereoisomers were separated by preparative chiral HPLC instead of SFC (chiral HPLC conditions: ChiralPak IB-3 (4.6 mm × 150 mm, 3 μm particle size); eluent = Hex:EtOH 80:20; 1.0 mL/min, 4.2 MPA, 25 °C). 23: 1H NMR (300 MHz, 3813

DOI: 10.1021/jm501998m J. Med. Chem. 2015, 58, 3806−3816

Article

Journal of Medicinal Chemistry

buffered saline (pH 7.4). Then 1.5 h after αCD3 antibody administration, animals were euthanized by CO2 inhalation and plasma was collected for PK measurements and serum was collected for Meso Scale Discovery (MSD) measurement of both IL-2 and IL13. Jurkat Viability Assay. Cell viability assay was performed by using ATPlite kit (PerkinElmer) according to the vendor’s protocol with some modifications described below: Jurkat T-cells (10,000 cells/well) were suspended in assay medium RPMI 1640 (Sigma) containing 10% FCS and 2 mM L-glutamine, seeded in 384-well plate, and added with compounds that were prediluted in assay medium (omitting FCS and L-glutamine) and that were assayed at seven concentrations in triplicate. Plates were then incubated at 37 °C with 5% CO2 for 48 h. And afterward, plates were added with ATPlite substrate reagent to each well in a ratio of 1:1 (v/v), incubated at room temperature with shaking at 700 rpm for 2 min using an orbital microplate shaker, and then subjected for luminescence measurement with Envision.

brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel column chromatography eluting with ethyl acetate/petroleum ether (1/2) to give 300 mg (49%) of ethyl 5,5-difluoro-5a-methyl-1,4,4a,5,5a,6hexahydrocyclopropa[f ]indazole-3-carboxylate (10). A solution of ethyl 5,5-difluoro-5a-methyl-1,4,4a,5,5a,6-hexahydrocyclopropa-[f ]indazole-3-carboxylate (300 mg, 1.17 mmol, 1.00 equiv), ethanol (12 mL), water (2.4 mL), and sodium hydroxide (469 mg, 11.72 mmol, 10.02 equiv) was stirred for 2 h at 50 °C. The reaction mixture was concentrated under vacuum, and the residue was dissolved in 50 mL of water. The pH value of the solution was adjusted to 4 to 5 with 1 N of hydrogen chloride. The solid was collected by filtration and dried under vacuum to provide 250 mg (94%) of 5,5-difluoro-5a-methyl-1,4,4a,5,5a,6-hexahydrocyclopropa[f ]indazole-3-carboxylic acid (6). 1H NMR (400 MHz, DDMSO-d6) δ 12.99 (s, 2H), 3.01−2.98 (m, 3H), 2.79−2.74 (dd, J = 17.2 Hz, J = 3.2 Hz, 1H), 1.76 (d, J = 15.6 Hz, 1H), 1.33 (s, 3H). N-( 1-Benzyl-1 H-pyrazol-4 -yl)-5,5-di fluor o-5a-methyl1,4,4a,5,5a,6-hexahydrocyclopropa[f ]indazole-3-carboxamide (26 and 26′). Prepared by following general procedures A and C. The stereoisomers were separated by preparative chiral HPLC instead of SFC (chiral HPLC conditions: ChiralPak IA-3 (4.6 mm × 50 mm, 3 μm particle size); eluent = (Hex + 0.1% Et2NH):EtOH 50:50; 1.0 mL/min, 4.2 MPA, 25 °C). 26′: 1H NMR (300 MHz, CDCl3) δ 8.75 (s, 1H), 8.03 (s, 1H), 7.60 (s, 1H), 7.38−7.22 (m, 5H), 5.29 (s, 2H), 3.37−3.02 (m, 3H), 2.88−2.74 (m, 1H), 1.66−1.59 (m, 1H), 1.41 (s, 3H). MS: m/z = 384 (M + H). HPLC retention time: 1.4 min. 26: 1H NMR (300 MHz, CDCl3) δ 8.67 (s, 1H), 8.03 (s, 1H), 7.61 (s, 1H), 7.37−7.24 (m, 5H), 5.29 (s, 2H), 3.34−3.03 (m, 3H), 2.80−2.71 (m, 1H), 1.66−1.58 (m, 1H), 1.40 (s, 3H). MS: m/z = 384 (M + H). HPLC retention time: 2.8 min. N-(1-((1,1-Dioxidotetrahydro-2H-thiopyran-4-yl)(phenyl)methyl)1H-pyrazol-4-yl)-5a-methyl-1,4,4a,5,5a,6-hexahydrocyclopropa[f ]indazole-3-carboxamide (27). Prepared by following general procedures A and C. The stereoisomers were separated by preparative chiral HPLC instead of SFC (chiral HPLC conditions: ChiralPak IB-3 (4.6 mm × 50 mm, 3 μm particle size); eluent = Hex (0.1% Et3N):EtOH 60:40; 1.0 mL/min, 3.0 MPA, 25 °C). 27: 1H NMR (300 MHz, CDCl3) δ 8.70 (s, 1H), 8.13 (s, 1H), 7.57 (s, 1H), 7.40−7.43 (m, 2H), 7.29−7.36 (m, 3H), 4.84 (d, 1H, J = 10.8 Hz), 3.38 (d, 1H, J = 16.8 Hz), 2.86−3.05 (m, 6H), 2.68−2.79 (m, 2H), 1.80−1.89 (m, 4H), 1.24 (s,3H), 1.06−1.12 (m, 1H), 0.41−0.42 (m, 1H), 0.13−0.23 (m, 1H). MS: m/z = 480 (M + H). HPLC retention time: 2.40 min. N-(1-((1,1-Dioxidotetrahydro-2H-thiopyran-2-yl)(phenyl)methyl)1H-pyrazol-4-yl)-5,5-difluoro-5a-methyl-1,4,4a,5,5a,6hexahydrocyclopropa[f ]indazole-3-carboxamide (28). Prepared by following general procedures A. The stereoisomers were separated by preparative chiral HPLC instead of SFC (SFC conditions: ChiralCel OJ-3 (4.6 mm × 100 mm, 3 μm particle size) at 5−40% (MeOH + 0.1% DEA); 5 mL/min, 100 bar, 40 °C). 28: 1H NMR (300 MHz, CDCl3) δ 8.94 (s, 1H), 7.91 (s, 1H), 7.67 (s, 1H), 7.44 (d, J = 3.0 Hz, 2H), 7.34−7.30 (m, 3H), 5.68 (d, J = 4.5 Hz, 1H), 4.42−4.35 (m, 1H), 3.50 (s, 2H), 3.30−2.94 (m, 6H), 2.69 (d, J = 9.0 Hz, 2H), 2.02 (s, 2H), 1.94−1.57 (m, 4H), 1.51−1.49 (m, 1H), 1.39 (s, 3H). MS: m/z = 516 (M + H). SFC retention time: 3.4 min. Biochemical and cellular assays were reported in the previous paper.5q Animal Studies. Six−eight-week-old female C57/Bl6 animals were purchased from Jackson Laboratories (Bar Harbor, ME). All animals used in this study were housed and maintained at Genentech in accordance with American Association of Laboratory Animal Care guidelines. All experimental studies conducted under protocols were approved by the Institutional Animal Care and Use Committee of Genentech Lab Animal Research in an AAALACi-accredited facility in accordance with the Guide for the Care and Use of Laboratory Animals and applicable laws and regulations. For PKPD studies, animals were dosed with small molecules by intraperitoneal injection in 200 μL of 5% cremophor with 50 mM citrate buffer. One h later, animals were given 10 μg of anti-CD3 antibody by intravenous tail vein injection in 200 μL of phosphate

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ASSOCIATED CONTENT

* Supporting Information S

X-ray data collection and refinement for compound 28; statistics of X-ray crystal structure of 28 bound to ITK; percentage of inhibition of various receptors by compound 1 at 10 μM; correlation between calculated and measured pKa of THI analogues; plasma-free concentration of compound 20 at t = 2.5 h in the Pk-PD study. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 650-467-1754. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the analytical and purification group at Genentech for NMR, kinetic solubility, log D measurement, and compound purification.

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ABBREVIATIONS USED ACN, acetonitrile; ATP, adenosinetriphosphate; Cl, clearance; %F, bioavailability (fraction absorbed); DIAD, diisopropyl azodicarboxylate; DMF, N,N-dimethylformamide; HATU, (1[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate); HPLC, high-pressure liquid chromatography; HTS, high-throughput screen; IL, interleukin; ITK, interleukin-2 inducible T-cell kinase; LE, ligand efficiency; mCPBA, meta-chloroperbenzoic acid; MDCK, Madin−Darby canine kidney; MOE, Molecular Operating Environment; PBS, phosphate buffer solution; PLCγ, phosphoinositide phospholipase C gamma; PPB, plasma protein binding; Pyr, pyridine; rt, room temperature; SAR, structure− activity relationships; SEM, 2-(trimethylsilyl)ethoxymethyl; SFC, supercritical fluid chromatography; T1/2, half-life; TCR, T-cell receptor; TFA, trifluoroacetic acid; Th2, T-helper cell, type 2; THI, tetrahydroindazole; THF, tetrahydrofuran; TMS, tetramethylsilane; TPSA, topological polar surface area

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REFERENCES

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