(BMS-986142): A Reversible Inhibitor of Bruton's ... - ACS Publications

Article pubs.acs.org/jmc

Discovery of 6‑Fluoro-5‑(R)‑(3‑(S)‑(8-fluoro-1-methyl-2,4-dioxo-1,2dihydroquinazolin-3(4H)‑yl)-2-methylphenyl)-2‑(S)‑(2hydroxypropan-2-yl)-2,3,4,9-tetrahydro‑1H‑carbazole-8carboxamide (BMS-986142): A Reversible Inhibitor of Bruton’s Tyrosine Kinase (BTK) Conformationally Constrained by Two Locked Atropisomers Scott H. Watterson,* George V. De Lucca, Qing Shi, Charles M. Langevine, Qingjie Liu, Douglas G. Batt, Myra Beaudoin Bertrand, Hua Gong, Jun Dai, Shiuhang Yip, Peng Li, Dawn Sun, Dauh-Rurng Wu, Chunlei Wang, Yingru Zhang, Sarah C. Traeger, Mark A. Pattoli, Stacey Skala, Lihong Cheng, Mary T. Obermeier, Rodney Vickery, Lorell N. Discenza, Celia J. D’Arienzo, Yifan Zhang, Elizabeth Heimrich, Kathleen M. Gillooly, Tracy L. Taylor, Claudine Pulicicchio, Kim W. McIntyre, Michael A. Galella, Andy J. Tebben, Jodi K. Muckelbauer, ChiehYing Chang, Richard Rampulla, Arvind Mathur, Luisa Salter-Cid, Joel C. Barrish, Percy H. Carter, Aberra Fura, James R. Burke, and Joseph A. Tino Bristol-Myers Squibb Research and Development, P.O. Box 4000, Princeton, New Jersey 08543, United States S Supporting Information *

ABSTRACT: Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase, is a member of the Tec family of kinases. BTK plays an essential role in B cell receptor (BCR)-mediated signaling as well as Fcγ receptor signaling in monocytes and Fcε receptor signaling in mast cells and basophils, all of which have been implicated in the pathophysiology of autoimmune disease. As a result, inhibition of BTK is anticipated to provide an effective strategy for the clinical treatment of autoimmune diseases such as lupus and rheumatoid arthritis. This article details the structure−activity relationships (SAR) leading to a novel series of highly potent and selective carbazole and tetrahydrocarbazole based, reversible inhibitors of BTK. Of particular interest is that two atropisomeric centers were rotationally locked to provide a single, stable atropisomer, resulting in enhanced potency and selectivity as well as a reduction in safety liabilities. With significantly enhanced potency and selectivity, excellent in vivo properties and efficacy, and a very desirable tolerability and safety profile, 14f (BMS-986142) was advanced into clinical studies.

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INTRODUCTION Bruton’s tyrosine kinase (BTK) is a nonreceptor tyrosine kinase expressed in all hematopoietic cells with the exception of T cells and differentiated plasma cells. BTK is a member of the Tec family of kinases which includes TEC, BTK, ITK, TXK, and BMX. In B cells, BTK plays an essential role in B cell receptor (BCR)-mediated activation and proliferation, as well as T cell costimulation.1 Specifically, when an antigen binds to the B cell receptor, BTK is recruited to a macro-molecular complex where it is phosphorylated on two key tyrosine residues.1 Upon activation, BTK subsequently phosphorylates PLCγ.1 Further downstream signaling leads to the activation of both the NFAT and NFκB pathways,1 resulting in cell proliferation, antibody and cytokine production, and costimulatory molecule expression (e.g., CD80, CD86, and CD69). In © 2016 American Chemical Society

addition to its role in BCR signaling, BTK is involved in myeloid cell functions where it has been shown to be a crucial component in low affinity activating Fcγ receptor signaling (e.g., FcγRIIa and FcγRIIIa) in monocytes and FcεRI signaling in mast cells and basophils.2 When BTK is activated through the Fcγ and Fcε receptors, downstream signaling results in the expression of pro-inflammatory cytokines, chemokines, and cell adhesion molecules.2 Finally, BTK-dependent signaling is required for RANK-L controlled osteoclastogenesis in monocytic precursors.3 Rituximab, an anti-CD20 B cell depleting antibody approved for the clinical treatment of rheumatoid arthritis, clearly Received: July 21, 2016 Published: September 1, 2016 9173

DOI: 10.1021/acs.jmedchem.6b01088 J. Med. Chem. 2016, 59, 9173−9200

Journal of Medicinal Chemistry

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Figure 1. Identification of lead BMS-935177 (2).

Figure 2. Compound 2 has four interconverting atropisomers which were separated by chiral SFC.

clinical therapeutic agents. With an appropriately positioned noncatalytic cysteine residue (Cys481) in the kinase domain,10 there has been particular interest in identifying inhibitors that covalently bind to Cys481 as potential agents for both oncological and autoimmune diseases. One such drug, ibrutinib, was approved for the treatment of non-Hodgkin’s lymphoma and chronic lymphocytic leukemia.11 Several additional covalent inhibitors have advanced into clinical trials for the treatment of rheumatoid arthritis.12 In addition to covalent inhibitors, there has been significant interest in developing reversible inhibitors of BTK,13 including our own efforts.14−16 As part of our effort to identify reversible inhibitors of BTK, we previously disclosed the discovery of a novel carbazole series of inhibitors, providing 1 as an initial lead.15a Recently, we disclosed the structure−activity relationships (SAR) to further optimize this series leading to carbazole 2.15b Compound 2 (Figure 1) provided potent inhibition of BTK with an IC50 of 3 nM and a reasonable human whole blood IC50 of 550 nM. With a desirable pharmacokinetic profile, 2 was further evaluated in vivo for both efficacy and tolerability. In animal models of human rheumatoid arthritis, a mouse collagen-induced arthritis (CIA) model and a Fcγ dependent, B cell independent mouse collagen antibody-induced arthritis (CAIA) model, 2 demonstrated significant efficacy at oral doses of 20 and 30 mg/kg q.d.15b Unfortunately, in tolerability studies, 2 provided a less than desirable therapeutic margin (99%). The absolute stereochemistry of 7a was established by single crystal X-ray analysis to be the (S)quinazolinedione atropisomer, but with a mixture of atropisomers at carbazole C4, as seen in Figure 6. The addition of the second carbonyl would allow for the bicyclic dione to orient in either of two possible conformations and still retain the interaction with the conserved water, but interestingly, the more potent (S)-configuration 7a would be expected to bind in the same orientation as that observed in the X-ray cocrystal structure of 4. With the enhanced whole blood potency of 7a, it is reasonable to anticipate that the additional carbonyl might be 9178



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Scheme 9. Preparation of Boronic Ester Intermediatesa

Reagents and conditions: (a) Δ, xylene, 90%; (b) CDI, THF, 90%; (c) bis(pinacolato)diboron, dichloro 1,1′-bis(diphenylphosphino)ferrocenepalladium(II)−CH2Cl2 adduct, KOAc, toluene, 90%; (d) SFC chiral separation; (e) MeI, Cs2CO3, DMF, 90%.

a

Scheme 10. Preparation of Aniline 39a

a

Reagents and conditions: (a) pyr2IBF4, CH2Cl2, 63%; (b) Zn(CN)2, Pd(PPh3)4, DMF, 83%; (c) NaOH, EtOH/H2O, 91%.

potentially enhance both the selectivity profile and the overall tolerability profile of the series by eliminating any off-target liabilities associated with the less target relevant atropisomer, as recently supported in the literature.18 In further support of this, there are well established instances where the desired pharmacological activity of a chiral drug is associated with one enantiomer while off-target liabilities are linked to the other enantiomer.19 Since a single, stable atropisomer should behave more like a traditional chiral center, this strategy was expected to also minimize the complications associated with progressing a mixture of interconverting atropisomers.18d Fully racemic compound 8 (Figure 9, mixture of 4 atropisomers) with a chloro substituent at carbazole C3 was prepared and was found to have similar activity against BTK as both 2 and 7a (IC50 = 4 nM). When prepared with the (S)quinazolinedione atropisomer, the resulting mixture of carbazole C4 atropisomers was separated by chiral SFC to give 9a and 9b as single, stable atropisomers. It is worth noting that the chiral prioritization changed with the addition of the chloro at carbazole C3, with the bioactive conformation now derived from the (R)-atropisomer. As expected, 9a (R-isomer with the linker methyl oriented down) was substantially more potent than 9b (S-isomer, linker methyl up), as seen in Figure 9

Figure 4. Co-crystal structure (1.9 Å) of compound 4 bound to the kinase domain of BTK (PDB ID: 5JRS).

binding to the kinase domain by addition of small substituents at carbazole C3, as depicted in Figure 8. We rationalized that this strategy would provide improvements in activity, particularly human whole blood potency. Additionally, we anticipated that a locked, predefined conformation could 9179



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Figure 5. Quinazoline-diones 5 and 6, a route to locking one of the atropisomeric centers.

Table 2. In Vitro Potency of Carbazoles 9a and 12 vs Carbazoles 2 and 7a

in vitro activity cmpd

R

BTK IC50 (nM)

2 7a 9a 12a 12b 12c

NA H Cl Me CN F

3.0 ± 0.10 2.3 ± 1.2 1.0 ± 0.5 0.65 (n = 2) 0.27 ± 0.04 0.45 ± 0.12

a

mouse PK (10 mg/kg)

JAK2/BTK selectivity

Ramos IC50 (nM)

94× 510× 1,900× 1,200× 7,100× 4,400×

26 ± 15 19 ± 9 11 ± 5 17 (n = 2) 6.0 ± 0.4 11 ± 9

a

hWB IC50 (nM) 550 ± 100 190 ± 30 90 ± 41 240 (n = 2) 120 (n = 1) 90 ± 60

a

CMax (μM)

AUC (μM·h)

% formation of 13

8.9 2.2b 4.7 6.3 0.12 5.5

80 25b 41 48 0.31 28

NA 4b 66 (10) 0.5 ND 15

a

IC50 values are shown as mean values of at least three determinations unless specified otherwise. bMouse PK at 5 mg/kg; NA = not applicable; ND = not determined.

(BTK IC50 = 1.0 nM vs 130 nM, respectively). Computational modeling of both isomers 9a and 9b based on the cocrystal structure of 4 bound in the kinase domain of BTK suggested that the less active isomer 9b orients the quinazolinedione moiety into solvent, thus minimizing van der Waal interactions that may be favorable for binding (Figure 10). Additionally, the linker methyl group is projected up into the P-loop of the kinase domain. These combined effects apparently disrupt the hydrogen bond interactions with Met477 in the hinge region of the pocket, likely contributing to the observed loss in potency. As predicted, locking the carbazole C4 atropisomer, providing a single, stable atropisomer (9a), resulted in a 6-

fold improvement in the human whole blood activity relative to 2 (IC50 = 90 nM vs 550 nm) (Table 2). Although the benefits of the locked, predefined conformation on whole blood potency are reasonably clear, it is less clear as to what role the C3 chloro may play in modulating the electronic nature of the core through inductive effects. Interestingly, compound 9a was 1,900-fold selective for BTK over JAK2 (Janus kinase 2), demonstrating significantly improved selectivity relative to both 2 (JAK2/BTK: 94×) and 7a (JAK2/BTK: 550×). In stability studies in methanol and plasma at 37 °C, both locked atropisomers 9a and 9b were stable for greater than 5 days. With a significantly improved human whole blood potency and 9180



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Figure 6. X-ray crystal structures of 7a with the locked (S)-quinazolinedione atropisomer revealing a mixture of two interconverting C4 atropisomers (CCDC # 1501158).

Figure 7. Distribution of carbazole C4 atropisomers (7a) in multispecies plasma studies at 37 °C over 4 h.

Figure 9. Compound 9a, a single, stable atropisomer.

With the enhanced human whole blood potency and selectivity observed with a single atropisomer (9a), we continued to explore carbazole C3 substitution, as outlined in Table 2. Interestingly, replacement of the chloro with a more neutral methyl (12a) maintained activity against BTK as well as the improved selectivity observed for 9a. Surprisingly, 12a was less potent in the human whole blood assay with an IC50 of 240 nM, consistent with the human whole blood potency observed for 7a. This could be attributed to the similarity in the electronic nature of the cores. In a mouse PK study, 12a showed similar if not better exposure when compared to 9a, but with less than 1% formation of the quinazolinedione des-methyl metabolite 13a. Substitution with an electronically withdrawing nitrile group (12b) significantly improved the BTK binding with an IC50 of 0.27 nM (9-fold over 7a), suggesting that the hydrogen bonding interactions of the carbazole NH and perhaps the carboxamide to the hinge are likely strengthened

Figure 8. Medicinal chemistry strategy: Lock the C4 atropisomer into the preferred conformation for optimal binding to BTK.

an overall improved selectivity profile (data not shown) relative to 2, compound 9a was advanced into multispecies PK studies. Unfortunately, substantial demethylation of the quinazolinedione was observed in mouse (66%) and rat (80%) to give metabolite 10 (Figure 11). However, very little formation of 10 was seen in dogs (10 0.003 0.41b 3.8b

± 0.005 ± 0.001 ± 0.003 ± 0.0.003

0.090 ± 0.040 0.14 ± 0.08 0.089 ± 0.046

2 IC50 (μM)a 0.026 ± 0.015 0.008b 0.040 ± 0.030 >10 0.014b

0.550 ± 0.100 2.1 ± 0.2 ND

a IC50 values are shown as mean values of at least three determinations unless specified otherwise. bIC50 values as single determinations; PBMC = peripheral blood mononuclear cells; ND = not determined.

Table 5. Partial in Vitro Profiling Data for 14f parameter

result

protein binding (bound)

99.1% human 98.5% mouse 98.3% rat 99.1% dog 98.8% monkey Ames negative IC50 = 5.5 μM 10/13% @ 10 μM (1 and 4 Hz) 40% @ 10 μM >40 μM 1A2, 2B6, 2D6 2.6 μM 2C8 17.1 μM 2C9 32.2 μM 2C19 8.5 μM 3A4 673/725 nm/s (pH 5.5/7.4) ND due to insufficient recovery 99% ie; LCMS (ESI) m/z calcd for C32H27FN4O4 [M + H]+ 551.2. Found: (M + H − H2O)+ 533.2. 1H NMR (400 MHz, DMSO-d6) δ 11.39 (s, 1H), 8.15 (br. s., 1H), 8.02−7.93 (m, 2H), 7.83 (s, 1H), 7.72 (dd, J = 14.4, 8.3 Hz, 1H), 7.52−7.41 (m, 3H), 7.37− 7.30 (m, 2H), 7.10−7.03 (m, 1H), 7.03−6.97 (m, 2H), 4.98 (d, J = 1.1 Hz, 1H), 3.74 (t, J = 8.4 Hz, 3H), 1.74 (s, 3H), and 1.46 (d, J = 4.4 Hz, 6H). 3-Chloro-4-(3-(8-fluoro-1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-7-(2-hydroxypropan-2-yl)-9H-carbazole-1-carboxamide (8).16a A mixture of ethyl 5-bromo-8-carbamoyl9H-carbazole-2-carboxylate (16, 0.100 g, 0.277 mmol) and Nchlorosuccinimide (recrystallized from toluene; 0.037 g, 0.277 mmol) in carbon tetrachloride (10 mL) and dimethylformamide (2 mL) was stirred at room temperature for 112 h. The mixture was filtered, and the collected precipitate was washed with carbon tetrachloride and dried overnight under reduced pressure. The residue was purified by column chromatography on silica gel (40 g), eluting with hexanes followed by a mixture of ethyl acetate and hexanes (30%, then 50%), to give ethyl 5-bromo-8-carbamoyl-6-chloro-9H-carbazole2-carboxylate as a fluffy white solid (18a, 0.071g, 0.187 mmol, 67% yield). LCMS (ESI) m/z calcd for C16H12BrClN2O3 [M + H]+ 395.0. Found: 395.0 and 397.0. 1H NMR (400 MHz, DMSO-d6) δ 12.13 (s, 1H), 8.77 (d, J = 8.6 Hz, 1H), 8.53 (d, J = 1.1 Hz, 1H), 8.36 (br. s., 1H), 8.29 (s, 1H), 7.89 (dd, J = 8.4, 1.5 Hz, 1H), 7.74 (br. s., 1H), 4.38 (q, J = 7.0 Hz, 2H), and 1.38 (t, J = 7.0 Hz, 3H). Alternative Procedure. To a mixture of ethyl 5-bromo-8-carbamoyl9H-carbazole-2-carboxylate (90 g, 249 mmol), carbon tetrachloride (2.9 L) and N-methyl-2-pyrrolidone (0.60 L) was added Nchlorosuccinimide (36.1 g, 271 mmol). The reaction mixture was stirred at 45 °C for 2 h. After cooling to room temperature, the solid was collected by vacuum filtration. The solid was stirred in methanol (1.0 L) at 60 °C for 2 h and then cooled to room temperature. The solid was collected and dried to give ethyl 5-bromo-8-carbamoyl-6chloro-9H-carbazole-2-carboxylate (18a, 70 g, 167 mmol, 67% yield) (95% purity). The filtrate was concentrated under reduced pressure to remove carbon tetrachloride. To the N-methyl-2-pyrrolidone residue was added water (2.0 L). The resulting precipitate was collected and dried to give an additional 14 g of product (25% yield, 75% purity). A solution of ethyl 5-bromo-8-carbamoyl-6-chloro-9H-carbazole-2carboxylate (4.14 g, 10.5 mmol) in tetrahydrofuran (200 mL), cooled in a dry ice−acetone bath, was treated portionwise over 30 min with 1.6 M methyllithium in hexanes (45.8 mL, 73.2 mmol). The mixture was stirred at −78 °C for 60 min and then was treated portionwise with a saturated aqueous solution of ammonium chloride. Water was added, and the mixture was extracted with ethyl acetate (2×). The combined organic layers were washed with water (2×), and the aqueous phases were combined and extracted with dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was crystallized from ethyl acetate, the precipitate was collected by vacuum filtration, and the filtrate was concentrated under reduced pressure and purified by silica gel chromatography, eluting with a mixture of ethyl acetate and hexanes (0−100%), to give an additional solid. The two solids were combined to give 4-bromo-3-chloro-7-(2-hydroxypropan-2-yl)-9Hcarbazole-1-carboxamide as a light yellow solid (3.13 g, 8.20 mmol, 9188



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11.77 (br. s., 1H), 11.50 (s, 1H), 8.28 (br. s., 1H), 8.14 (s, 1H), 7.87− 7.80 (m, 2H), 7.65 (ddd, J = 10.0, 8.8, 1.0 Hz, 1H), 7.59 (br. s., 1H), 7.55−7.49 (m, 3H), 7.28 (dd, J = 6.4, 2.6 Hz, 1H), 7.23 (td, J = 8.0, 4.6 Hz, 1H), 7.05 (dd, J = 8.4, 1.5 Hz, 1H), 6.70 (d, J = 8.6 Hz, 1H), 5.00 (s, 1H), 1.72 (s, 3H), and 1.45 (d, J = 2.9 Hz, 6H). 3-Chloro-4-(R)-(3-(R)-(8-fluoro-2,4-dioxo-1,2-dihydroquinazolin3(4H)-yl)-2-methylphenyl)-7-(2-hydroxypropan-2-yl)-9H-carbazole1-carboxamide (11).16a A mixture of 4-bromo-3-chloro-7-(2-hydroxypropan-2-yl)-9H-carbazole-1-carboxamide (18a, 0.360 g, 0.943 mmol), 8-fluoro-3-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)quinazoline-2,4(1H,3H)-dione (34,0.392 g, 0.990 mmol), 1,1′-bis(diphenylphosphino)ferrocenepalladium(II) dichloride−dichloromethane adduct (0.039 g, 0.047 mmol) and cesium carbonate (0.615 g, 1.89 mmol) in dioxane (10 mL) and water (2.5 mL) was heated at 100 °C overnight. The cooled mixture was diluted with ethyl acetate, washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel, eluting with ethyl acetate and hexanes (0−50%−70%, containing 1% methanol) to provide 3-chloro-4-(3-(8-fluoro-2,4-dioxo-1,2-dihydroquinazolin3(4H)-yl)-2-methylphenyl)-7-(2-hydroxypropan-2-yl)-9H-carbazole-1carboxamide (mixture of four atropisomers) as a white solid (0.361 g, 0.632 mmol, 67% yield). LCMS (ESI) m/z calcd for C31H24ClFN4O4 [M + H]+ 571.2. Found: (M + H − H2O) 553.3. 1H NMR (500 MHz, CD3OD) δ 8.09 (d, J = 1.1 Hz, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.81− 7.69 (m, 1H), 7.64−7.54 (m, 2H), 7.48 (dd, J = 7.8, 1.1 Hz, 1H), 7.43−7.29 (m, 1H), 7.31−7.24 (m, 1H), 7.18 (dd, J = 8.3, 1.7 Hz, 1H), 7.16−7.07 (m, 1H), 6.92 (dd, J = 8.6, 5.3 Hz, 1H), 1.94−1.71 (m, 3H), 1.65−1.50 (m, 6H). A sample of the mixture of four atropisomers of 3-chloro-4-(3-(8fluoro-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-7(2-hydroxypropan-2-yl)-9H-carbazole-1-carboxamide (0.250 g, 0.438 mmol) was separated by chiral SFC as follows: column, Chiralpak ADH (5 × 25 cm, 5 μm); mobile phase, CO2−IPA (60:40) at 220 mL/ min, 35 °C, 100 bar; sample preparation, 21 mg/mL in MeOH; injection, 3.0 mL. The third peak eluting from the column provided 11 as a single, stable atropisomer (0.024 g, 0.042 mmol, 10% yield). HPLC purity: 96%; tr = 3.39 min (Method D). Chiral purity: >98%; LCMS (ESI) m/z calcd for C31H24ClFN4O4 [M + H]+ 571.2. Found: (M + H − H2O)+ 553.2. 1H NMR (400 MHz, MeOH-d4) δ 8.09 (s, 1H), 7.95 (d, J = 7.9 Hz, 1H), 7.74 (d, J = 1.1 Hz, 1H), 7.60−7.54 (m, 2H), 7.54−7.43 (m, 1H), 7.37 (d, J = 7.5 Hz, 1H), 7.28 (d, J = 4.6 Hz, 1H), 7.11 (dd, J = 8.6, 1.5 Hz, 1H), 6.91 (d, J = 8.6 Hz, 1H), 2.00− 1.76 (m, 3H), 1.69−1.52 (m, 6H) 4-(R)-(3-(S)-(8-Fluoro-1-methyl-2,4-dioxo-1,2-dihydroquinazolin3(4H)-yl)-2-methylphenyl)-7-(2-hydroxypropan-2-yl)-3-methyl-9Hcarbazole-1-carboxamide (12a).16a A dark, homogeneous solution of 3-bromo-4-methylaniline (36, 5.00 g, 26.9 mmol) and N-iodosuccinimide (4.53 g, 20.16 mmol) and bis(pyridine)iodonium tetraborofluorate (2.70g, 7.26 mmol) in dichloromethane (100 mL) was stirred at room temperature overnight. The reaction mixture was diluted with dichloromethane, washed with a saturated aqueous solution of sodium bisulfite, washed with water, and dried over anhydrous sodium sulfate. Concentration under reduced pressure followed by purification by flash silica gel chromatography using a mixture of ethyl acetate in hexane (1%−2%−3%) afforded 5-bromo-2-iodo-4-methylaniline (37, 5.27 g, 16.9 mmol, 63% yield) as a yellow solid. HPLC tr = 2.03 min (Method C); LCMS (ESI) m/z calcd for C7H7BrIN [M + H]+ 212.0. Found: mass spectrum m/z (M + H)+ = 212.0 and 214.0. 1H NMR (500 MHz, CDCl3) δ 7.49 (d, J = 0.6 Hz, 1H), 6.94 (s, 1H), 4.17− 3.91 (br.s, 2H), and 2.25 (s, 3H). A mixture of 5-bromo-2-iodo-4-methylaniline (37, 5.25 g, 16.8 mmol) and zinc cyanide (0.988 g, 8.41 mmol) in N,Ndimethylformamide (80 mL) was degassed well with vacuum and n itr og e n ( 3× ) . T o t h e m i x t u r e w a s a d d e d t e t r a k i s (triphenylphosphine)palladium (0) (0.972 g, 0.841 mmol) with degassing, and the yellow, homogeneous solution was immersed in an oil bath at 90 °C and stirred overnight. The reaction mixture was diluted with ethyl acetate, washed with a 10% lithium chloride solution (2×), and washed with brine. The organic layer was collected, and the

SFC chromatography to give two single, stable atropisomers as follows: column, AS-H 3 × 25 cm; 5 μm; BPR pressure, 100 bar; temperature, 40 °C; flow rate, 140 mL/min; mobile phase, CO2/ MeOH (60/40); detector wavelength, 280 nm; injection, 1.15 mL; sample preparation, 3.56 mg/25 mL MeOH/DMSO (5:1), 30 mg/ mL. The second peak eluting from the column provided 9a as a single, stable atropisomer and as a white solid. HPLC purity: 98.8%; tr = 9.13 min (Method A); 98.3%; tr = 9.62 min (Method B). Chiral purity: >99.2%; LCMS (ESI) m/z calcd for C32H26ClFN4O4 [M + H]+ 585.2. Found: (M + H − H2O)+ 567.1. 1H NMR (400 MHz, CDCl3) δ 10.44 (s, 1H), 8.11 (d, J = 7.9 Hz, 1H), 7.73 (s, 1H), 7.67 (d, J = 1.1 Hz, 1H), 7.57−7.52 (m, 1H), 7.46 (ddd, J = 13.9, 8.1, 1.5 Hz, 1H), 7.41− 7.37 (m, 1H), 7.36 (d, J = 7.5 Hz, 1H), 7.25−7.23 (m, 1H), 7.23−7.21 (m, 1H), 6.97 (d, J = 8.4 Hz, 1H), 3.90 (d, J = 8.1 Hz, 3H), 3.50 (d, J = 5.7 Hz, 3H), 1.86 (s, 3H), and 1.64 (s, 6H). 3-Chloro-4-(R)-(3-(S)-(8-fluoro-2,4-dioxo-1,2-dihydroquinazolin3(4H)-yl)-2-methylphenyl)-7-(2-hydroxypropan-2-yl)-9H-carbazole1-carboxamide (10). A mixture of 4-bromo-3-chloro-7-(2-hydroxypropan-2-yl)-9H-carbazole-1-carboxamide (18a, 1.00 g, 2.62 mmol), 8-fluoro-3-(S)-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)phenyl)quinazoline-2,4(1H,3H)-dione (33, 1.25 g, 3.14 mmol), tripotassium phosphate (2 M in water) (3.93 mL, 7.86 mmol), and tetrahydrofuran (12 mL) was degassed (3×). 1,1′-Bis(di-tertbutylphosphino)ferrocene palladium dichloride (0.085 g, 0.131 mmol) was added, and the reaction mixture was degassed (2×). The reaction mixture was stirred at room temperature overnight. Analysis confirmed that the reaction was ∼50% complete. Additional catalyst was added, and the reaction mixture was degassed and stirred at 50 °C for 7 h and then at room temperature over the weekend. The reaction mixture was diluted with ethyl acetate, washed with water, washed with brine, and dried over anhydrous sodium sulfate. Concentration under reduce pressure followed by purification by flash silica gel chromatography using a mixture of ethyl acetate and hexane (50%− 62%−75%−85%) afforded 3-chloro-4-(3-(S)-(8-fluoro-2,4-dioxo-1,2dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-7-(2-hydroxypropan-2yl)-9H-carbazole-1-carboxamide (1.20 g, 2.10 mmol, 80% yield) as an off-white solid. HPLC tr = 2.63 min (Method C); LCMS (ESI) m/z calcd for C31H24ClFN4O4 [M + H]+ 571.2. Found: (M + H − H2O) 553.2. A sample of 3-chloro-4-(3-(S)-(8-fluoro-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-7-(2-hydroxypropan-2-yl)-9H-carbazole-1-carboxamide (mixture of 2 atropisomers) was separated by chiral SFC to give two single, stable atropisomers using the following conditions: column, OJ-H 3 × 25 cm; 5 μm; BPR pressure, 100 bar; temperature, 40 °C; flow rate, 150 mL/min; mobile phase, CO2/ MeOH (60/40); detector wavelength, 220 nm; injection, 1.2 mL; sample preparation, 1.19 mg/25 mL MeOH/DCM (2:1), 48 mg/mL. The second peak eluting from the column provided 10 as a single, stable atropisomer and as an off-white solid. HPLC purity: 99.5%; tr = 9.67 min (Method A); 99.0%; tr = 9.47 min (Method B). Chiral purity: >99.5%; LCMS (ESI) m/z calcd for C31H24ClFN4O4 [M + H]+ 571.2. Found: (M + H − H2O)+ 553.2. 1H NMR (400 MHz, DMSO-d6) δ 9189



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(2.10 g, 9.26 mmol) in tetrahydrofuran (45 mL) was heated at 60 °C for 60 min. After cooling to room temperature, the mixture was diluted with ethyl acetate (∼70 mL), stirred for 15 min, and filtered under reduced pressure. The solid was washed with ethyl acetate and dried. The filtrate was concentrated, triturated with methanol, filtered under reduced pressure, and washed with methanol. The solids were combined to give 4-bromo-7-(ethoxycarbonyl)-3-methyl-9H-carbazole-1-carboxylic acid (1.40 g, 3.72 mmol, 88% yield) as a pale yellow solid. HPLC tr = 3.39 min (Method C); LCMS (ESI) m/z calcd for C17H14BrNO4 [M + H]+ 376.0. Found: 376.2 and 378.2. A heterogeneous mixture of 4-bromo-7-(ethoxycarbonyl)-3-methyl9H-carbazole-1-carboxylic acid (1.40 g, 3.72 mmol), 1-ethyl-3-(3(dimethylamino)propyl)carbodiimide, hydrochloride (1.070 g, 5.58 mmol), and 1H-benzo[d][1,2,3]triazol-1-ol, monohydrate (0.855 g, 5.58 mmol) in a mixture of tetrahydrofuran (30 mL) and dichloromethane (5 mL) was stirred at room temperature for 60 min. Ammonium hydroxide (0.217 mL, 5.58 mmol) was added, and the heterogeneous reaction mixture was stirred at room temperature overnight. The mixture was diluted with ethyl acetate and washed with a saturated aqueous solution of sodium bicarbonate. Unfortunately, the mixture remained heterogeneous, resulting in a difficult separation. Consequently, the mixture was filtered, and the cake was washed with water followed by ethyl acetate, resulting in an off-white solid which was determined to be the product. The solid was triturated with methanol and dried to give ethyl 5-bromo-8-carbamoyl-6-methyl-9Hcarbazole-2-carboxylate (0.908 g, 2.42 mmol, 65% yield) as an offwhite solid HPLC tr = 3.15 min (Method C); LCMS (ESI) m/z calcd for C17H15BrN2O3 [M + H]+ 375.0. Found: 375.1 and 377.1. 1H NMR (400 MHz, DMSO-d6) δ 11.88 (s, 1H), 8.77 (d, J = 8.6 Hz, 1H), 8.48 (d, J = 0.9 Hz, 1H), 8.20 (br. s., 1H), 7.84 (dd, J = 8.5, 1.4 Hz, 1H), 7.59 (br. s., 1H), 4.37 (q, J = 7.0 Hz, 2H), 2.55 (s, 3H), and 1.37 (t, J = 7.0 Hz, 3H). The ethyl acetate/water filtrated was separated, and the organic layer was washed with brine. The organic layer was collected and dried over anhydrous sodium sulfate. Concentration under reduced pressure followed by trituration with methanol with sonication afforded additional ethyl 5-bromo-8-carbamoyl-6-methyl9H-carbazole-2-carboxylate (0.366 g, 0.975 mmol, 26% yield) as a yellow solid. Total product: 1.27 g (91%). To a solution of ethyl 5-bromo-8-carbamoyl-6-methyl-9H-carbazole-2-carboxylate (0.500 g, 1.33 mmol) in tetrahydrofuran (12 mL) at −78 °C was added methyllithium (1.6 M in ether) (3.0 equiv; 2.50 mL, 4.00 mmol) dropwise over 10 min. The reaction mixture was stirred at −78 °C for 30 min. Additional methyllithium (2.0 equiv; 1.6 M in ether) (1.67 mL, 2.67 mmol) was added, and the heterogeneous reaction mixture was stirred at −78 °C for 30 min. HPLC analysis indicated that there was still starting material remaining. An additional 2.0 equiv of methyllithium was added, and the reaction was stirred for 45 min. The reaction mixture was quenched at −78 °C with a saturated aqueous solution of ammonium chloride. After warming to room temperature, the mixture was diluted with ethyl acetate, washed with water, and washed with brine. The organic layer was collected, the aqueous layers were sequentially extracted with ethyl acetate, and the combined organic layers were dried over anhydrous sodium sulfate. Concentration under reduce pressure afforded a quantitative yield of the product as an off-white solid. The solid was triturated with methanol with sonication and filtered to give 4-bromo-7-(2hydroxypropan-2-yl)-3-methyl-9H-carbazole-1-carboxamide (0.336 g, 0.930 mmol, 70% yield) as a tan solid. HPLC tr = 2.77 min (Method C); LCMS (ESI) m/z calcd for C17H17BrN2O2 [M + H]+ 361.1. Found: (M + H − H2O) 343.2 and 345.2. 1H NMR (500 MHz, DMSO-d6) δ 11.42 (s, 1H), 8.56 (d, J = 8.6 Hz, 1H), 8.12 (br. s., 1H), 7.91−7.89 (m, 2H), 7.48 (br. s., 1H), 7.36 (dd, J = 8.3, 1.7 Hz, 1H), 5.06 (s, 1H), 2.53 (s, 3H), and 1.51 (s, 6H). The filtrate was purified by reverse-phase preparative HPLC, and the desired fractions were immediately neutralized with a saturated aqueous solution of sodium bicarbonate and concentrated under reduced pressure. The residue was dissolved in ethyl acetate and water, and the organic layer was collected and washed with brine. The aqueous layers were sequentially extracted with ethyl acetate, and the organic layers were combined and dried over anhydrous sodium sulfate. Concentration under reduced

aqueous layers were sequentially extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate and concentrated to give the crude product as a brown solid which was purified by flash silica gel chromatography using a mixture of ethyl acetate and hexane (1%−2.5%−5%−50%) to give a brown solid. The compound was further purified by trituration with methanol to give 2amino-4-bromo-5-methylbenzonitrile (3 crops) (38, 2.68 g, 12.7 mmol, 75% yield) as a tan solid. HPLC tr = 1.70 min (Method C); LCMS (ESI) m/z calcd for C8H7BrN2 [M + H]+ 211.0. Found: 211.1 and 213.1. The filtrate was purified by flash silica gel chromatography using a mixture of ethyl acetate in hexane (5%−10%) to give additional 2-amino-4-bromo-5-methylbenzonitrile (0.272 g, 1.29 mmol, 8% yield) as a tan solid. Total: 2.95 g (83% yield). A heterogeneous mixture of 2-amino-4-bromo-5-methylbenzonitrile (38, 2.95 g, 14.0 mmol) in a mixture of ethanol (21 mL) and sodium hydroxide (2 M in water) (34.9 mL, 69.9 mmol) was heated at reflux overnight. Within 10 min, the solution became homogeneous. The solvent was removed under reduced pressure, and the aqueous residue was diluted with water, the pH was adjusted to ∼5 with concentrated hydrochloric acid, and stirred for 30 min to ensure pH stability. The solid was collected by vacuum filtration, washed well with water, and dried to give the product as a pale yellow solid. The wet solid was dissolved in ethyl acetate, washed with brine, and dried over anhydrous sodium sulfate. Concentration under reduced pressure afforded 2amino-4-bromo-5-methylbenzoic acid (39, 2.92 g, 12.7 mmol, 91% yield) as a pale yellow solid. HPLC tr = 1.52 min (Method C); LCMS (ESI) m/z calcd for C8H8BrNO2 [M + H]+ 230.0. Found: 230.1 and 232.1. To a suspension of 2-amino-4-bromo-5-methylbenzoic acid (39, 2.92 g, 12.7 mmol) in a mixture of 37% hydrochloric acid (12.7 mL) and water (4.3 mL) cooled with a salt−ice bath was added a 3 M solution of sodium nitrite (0.963 g, 14.0 mmol) in water (4.5 mL) dropwise slowly via syringe. After the addition was complete, the creamy solution was stirred for 45 min (ice-salt bath). A 7 M solution of tin(II) chloride dihydrate (8.59 g, 38.1 mmol) in hydrochloric acid (37%, 8.2 mL) was added dropwise slowly via syringe. The ice-bath was removed, and the reaction mixture was stirred at room temperature for 60 min. The thick, off-white colored reaction mixture was filtered under reduced pressure, and the solid was washed well with water and dried overnight. The solid was transferred to a roundbottomed flask and dispersed in methanol with sonication. The suspension was concentrated under reduced pressure, and the solid was triturated with dichloromethane with sonication. The solid was collected by vacuum filtration and washed with dichloromethane to give 4-bromo-2-hydrazinyl-5-methylbenzoic acid hydrochloride (2.17 g, 7.71 mmol, 61% yield) as a white solid. HPLC tr = 0.798 min (Method C); LCMS (ESI) m/z calcd for C8H9BrN2O2 [M + H]+ 245.0. Found: 245.1 and 247.1. A mixture of 4-bromo-2-hydrazinyl-5-methylbenzoic acid, hydrochloride (2.17 g, 7.71 mmol), ethyl 3-oxocyclohexanecarboxylate (1.44 g, 8.48 mmol), and acetic acid (1.32 mL, 23.1 mmol) in toluene (40 mL) was heated in an oil bath at 117 °C for 5 h. The solvent was removed under reduced pressure, and the residue (dried well) was diluted with toluene (18 mL) and trifluoroacetic acid (4.5 mL). The reaction mixture was stirred in an oil bath at 90 °C overnight. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (with sonication), and the precipitate was collected by vacuum filtration and washed with ethyl acetate to give a yellow solid. The filtrate was concentrated under reduced pressure, and the residue was suspended in ethyl acetate with sonication. The solid was collected to give additional product which was combined with the previous batch to give 5-bromo-2-(ethoxycarbonyl)-6-methyl-2,3,4,9-tetrahydro-1H-carbazole-8-carboxylic acid (2.04 g, 4.77 mmol, 62% yield) as a yellow solid. The solid was triturated with methanol to give 5-bromo2-(ethoxycarbonyl)-6-methyl-2,3,4,9-tetrahydro-1H-carbazole-8-carboxylic acid (1.60 g, 4.21 mmol, 55% yield) as a pale yellow solid. HPLC tr = 3.22 min (Method C); LCMS (ESI) m/z calcd for C17H18BrNO4 [M + H]+ 380.1. Found: 380.2 and 382.2. A solution of 5-bromo-2-(ethoxycarbonyl)-6-methyl-2,3,4,9-tetrahydro-1H-carbazole-8-carboxylic acid (1.60 g, 4.21 mmol) and DDQ 9190



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g, 0.644 mmol) in N,N-dimethylformamide (7 mL) was degassed well with vacuum and nitrogen (3×). To the mixture was added tetrakis(triphenylphosphine)palladium(0) (0.074 g, 0.064 mmol) with degassing, and the yellow, homogeneous solution was immersed in an oil bath at 95 °C and stirred overnight. The reaction mixture was diluted with ethyl acetate, washed with a 10% lithium chloride solution (2×), and washed with brine. The organic layer was collected, and the aqueous layers were sequentially extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate and concentrated to give the crude product as a yellow solid. The solid was triturated with dichloromethane with sonication to give 4-bromo-3cyano-7-(2-hydroxypropan-2-yl)-9H-carbazole-1-carboxamide (18b, 0.400 g, 1.08 mmol, 83% yield) as a pale yellow solid. LCMS (ESI) m/z calcd for C17H14BrN3O2 [M + H]+ 372.0. Found: (M + H − H2O)+ = 356.0. 1H NMR (500 MHz, DMSO-d6) δ 12.12 (s, 1H), 8.55 (d, J = 8.6 Hz, 1H), 8.38 (s, 1H), 8.31 (br. s., 1H), 8.03 (s, 1H), 7.74 (br. s., 1H), 7.49 (dd, J = 8.6, 1.1 Hz, 1H), 5.15 (s, 1H), and 1.52 (s, 6H). A mixture of 4-bromo-3-cyano-7-(2-hydroxypropan-2-yl)-9H-carbazole-1-carboxamide (18b, 0.400 g, 1.08 mmol), 8-fluoro-1-methyl-3(S)-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)quinazoline-2,4(1H,3H)-dione (19, 0.573 g, 1.40 mmol), cesium carbonate (0.700 g, 2.15 mmol), and dioxane (5 mL) was degassed with vacuum and nitrogen (3×). 1,1′-Bis(diphenylphosphino)ferrocenepalladium(II) dichloride−dichloromethane adduct (0.053 g, 0.064 mmol) was added, and the reaction mixture was degassed (2×). The reaction mixture was immersed in an oil bath at 88 °C and stirred for 2 days. The mixture was diluted with ethyl acetate, washed with water, washed with brine, and dried over anhydrous sodium sulfate. Concentration under reduce pressure followed by purification by flash silica gel chromatography using a mixture of ethyl acetate in hexane (50%−75%−85%−100%) afforded the product (0.350 g, 0.608 mmol, 57% yield) as a pale yellow solid. The compound was further purified by reverse phase preparative HPLC to give 3-cyano-4-(3-(8-fluoro-1methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-7(2-hydroxypropan-2-yl)-9H-carbazole-1-carboxamide (0.200 g, 0.347 mmol, 32% yield) as a white sold. HPLC purity: 98.2%; tr = 9.01 min (Method A); 98.2%; tr = 8.81 min (Method B); LCMS (ESI) m/z calcd for C33H26FN5O4 [M + H]+ 576.2. Found: 576.1. 1H NMR (400 MHz, DMSO-d6) δ 11.96 (s, 1H), 8.45 (s, 1H), 8.35 (br. s., 1H), 8.00−7.94 (m, 1H), 7.92 (s, 1H), 7.77−7.68 (m, 2H), 7.60−7.52 (m, 2H), 7.41 (dd, J = 6.6, 2.2 Hz, 1H), 7.38−7.29 (m, 1H), 7.13 (t, J = 10.1 Hz, 1H), 6.85 (t, J = 8.1 Hz, 1H), 5.05 (d, J = 2.0 Hz, 1H), 3.74 (dd, J = 8.1, 2.6 Hz, 3H), 1.73 (s, 3H), and 1.48−1.43 (m, 6H). A sample of 3-cyano-4-(3-(8-fluoro-1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-7-(2-hydroxypropan-2-yl)-9Hcarbazole-1-carboxamide (mixture of 4 atropisomers) was separated by chiral SFC chromatography using the following preparative conditions to give four single, stable atropisomers: column, ChiralPak AD-H 25 × 3.0 cm; 5 μm; temperature, 45 °C; flow rate, 150 mL/min; mobile phase, CO2/ iPA (65/35); detector wavelength, 220 nm. The forth peak eluting from the column provided 12b as a single, stable atropisomer and as a pale yellow solid. HPLC purity: 99.9%; tr = 9.08 min (Method A); 99.7%; tr = 8.82 min (Method B). Chiral Purity: 98.9% ie. LCMS (ESI) m/z calcd for C33H26FN5O4 [M + H]+ 576.2. Found: 576.1. 3-Fluoro-4-(R)-(3-(S)-(8-fluoro-1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-7-(2-hydroxypropan-2-yl)9H-carbazole-1-carboxamide (12c).16a To a suspension of 2-amino4-bromo-5-fluorobenzoic acid (10.0 g, 42.7 mmol) in a mixture of hydrochloric acid (37%, 42.7 mL) and water (14.3 mL) cooled with a salt-ice bath was added a 3 M solution of sodium nitrite (3.24 g, 47.0 mmol) in water (15.7 mL) dropwise slowly via syringe. After the addition was complete, the creamy solution was stirred for 30 min (ice-salt bath). A 7 M solution of tin(II) chloride dihydrate (28.9 g, 128 mmol) in hydrochloric acid, 37% (27.5 mL), was added dropwise slowly via syringe. The ice-bath was removed, and the reaction mixture was stirred at room temperature for 45 min. The thick, cream colored reaction mixture was filtered under reduced pressure, and the solid was washed well with water and dried overnight under reduced pressure.

pressure afforded additional 4-bromo-7-(2-hydroxypropan-2-yl)-3methyl-9H-carbazole-1-carboxamide (0.073 g, 0.202 mmol, 15% yield) as a pale yellow solid. Total product: 0.409 g (85% yield). A mixture of 4-bromo-7-(2-hydroxypropan-2-yl)-3-methyl-9Hcarbazole-1-carboxamide (0.091 g, 0.252 mmol), 8-fluoro-1-methyl-3(S)-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)quinazoline-2,4(1H,3H)-dione (19, 0.134 g, 0.327 mmol), tripotassium phosphate (2 M in water) (0.378 mL, 0.756 mmol), and tetrahydrofuran (2.0 mL) was degassed with vacuum and nitrogen (3×). 1,1′-Bis(ditert-butylphosphino)ferrocene palladium dichloride (8.21 mg, 0.013 mmol) was added, and the reaction mixture was degassed (2×). The reaction mixture was stirred at room temperature overnight. The dark reaction mixture was diluted with ethyl acetate, washed with water, washed with brine, and dried over anhydrous sodium sulfate. Concentration under reduce pressure followed by purification by flash silica gel chromatography using a mixture of ethyl acetate and hexane (50%−62%−75%−85%) afforded 4-(3-(S)-(8fluoro-1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-7-(2-hydroxypropan-2-yl)-3-methyl-9H-carbazole-1-carboxamide (mixture of 2 atropisomers at carbazole C4, 0.087 g, 0.149 mmol, 59% yield) as a pale yellow solid. HPLC purity: 97.0%; tr = 10.69 min (Method A); 97.3%; tr = 10.45 min (Method B); LCMS (ESI) m/z calcd for C33H29FN4O4 [M + H]+ 565.2. Found: (M + H − H2O) 547.4. 1H NMR (500 MHz, DMSO-d6) δ 11.20 (s, 1H), 8.10 (br. s., 1H), 7.97 (ddd, J = 7.9, 7.2, 1.0 Hz, 1H), 7.90 (s, 1H), 7.78 (s, 1H), 7.76−7.68 (m, 1H), 7.53−7.48 (m, 1H), 7.44 (dd, J = 7.9, 1.2 Hz, 1H), 7.40 (br. s., 1H), 7.36−7.30 (m, 1H), 7.23 (dd, J = 7.4, 1.2 Hz, 1H), 6.98 (ddd, J = 12.0, 8.4, 1.5 Hz, 1H), 6.68 (t, J = 7.9 Hz, 1H), 4.94 (d, J = 3.1 Hz, 1H), 3.73 (dd, J = 8.2, 3.5 Hz, 3H), 2.17 (s, 3H), 1.66 (s, 3H), and 1.48−1.41 (m, 6H). A sample of 4-(3-(S)-(8-fluoro-1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-7-(2-hydroxypropan-2-yl)-3-methyl-9H-carbazole-1-carboxamide (mixture of 2 atropisomers) was separated by SFC chromatography as follows: column, ChiralPak AD-H 25 × 3.0 cm; 5 μm; temperature, 40 °C; flow rate, 150 mL/min; mobile phase, CO2/MeOH (65/35); detector wavelength, 220 nm. The second peak eluting from the column provided 12a as single, stable atropisomer and as a white solid. HPLC purity: 100%; tr = 10.75 min (Method A); 99.8%; tr = 10.49 min (Method B). Chiral Purity: >99.5% ie; LCMS (ESI) m/z calcd for C33H29FN4O4 [M + H]+ 565.2. Found: (M + H − H2O) 547.4. 1H NMR (500 MHz, DMSO-d6) δ 11.19 (s, 1H), 8.10 (br. s., 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.90 (s, 1H), 7.78 (s, 1H), 7.76−7.68 (m, 1H), 7.53−7.48 (m, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.40 (br. s., 1H), 7.34 (td, J = 8.0, 4.0 Hz, 1H), 7.23 (d, J = 7.5 Hz, 1H), 6.96 (dd, J = 8.5, 1.2 Hz, 1H), 6.68 (d, J = 8.3 Hz, 1H), 4.93 (s, 1H), 3.73 (d, J = 8.0 Hz, 3H), 2.18 (s, 3H), 1.66 (s, 3H), and 1.44 (d, J = 5.5 Hz, 6H). 3-Cyano-4-(R)-(3-(S)-(8-fluoro-1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-7-(2-hydroxypropan-2-yl)9H-carbazole-1-carboxamide (12b).16a A mixture of 4-bromo-7-(2hydroxypropan-2-yl)-9H-carbazole-1-carboxamide (16, 2.00 g, 5.76 mmol),15b,c N-iodosuccinimide (1.69 g, 7.49 mmol), and pyridine (1.86 mL, 23.0 mmol) in N,N-dimethylformamide (20 mL) was heated in an oil bath at 65 °C for 2 days. The reaction mixture was cooled to room temperature, diluted with ethyl acetate, washed with a 10% aqueous solution of lithium chloride (2×), and washed with brine. The organic layer was collected, and the aqueous layers were sequentially extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate. Concentration followed by purification by flash silica gel chromatography using a mixture of ethyl acetate in hexane (50%−65%) afforded 4-bromo-7-(2-hydroxypropan2-yl)-3-iodo-9H-carbazole-1-carboxamide (23, 0.609 g, 1.29 mmol, 23% yield) as a yellow solid. LCMS (ESI) m/z calcd for C16H14BrIN2O2 [M + H]+ 472.9. Found: (M + H − H2O)+ = 456.9. 1H NMR (500 MHz, DMSO-d6) δ 11.66 (s, 1H), 8.49 (d, J = 8.6 Hz, 1H), 8.38 (s, 1H), 8.26 (br. s., 1H), 7.94 (d, J = 1.1 Hz, 1H), 7.58 (br. s., 1H), 7.40 (dd, J = 8.6, 1.4 Hz, 1H), 5.09 (s, 1H), and 1.51 (s, 6H). A mixture of 4-bromo-7-(2-hydroxypropan-2-yl)-3-iodo-9H-carbazole-1-carboxamide (23, 0.609 g, 1.29 mmol) and zinc cyanide (0.076 9191



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The solid was transferred to a round-bottomed flask and triturated with methanol with sonication. The suspension was filtered under reduced pressure, and the solid was washed with methanol and dried. Since a significant amount of the product was in the filtrate, the solution was concentrated, and the residue was triturated with dichloromethane. The product was collected by vacuum filtration and dried well. The solids were combined to give 4-bromo-5-fluoro-2hydrazinylbenzoic acid hydrochloride (5.37 g, 18.6 mmol, 44% yield) as a white solid. LCMS (ESI) m/z calcd for C7H6BrFN2O2 [M + H]+ 249.0. Found: 249.1 and 251.1. A heterogeneous mixture of 4-bromo-5-fluoro-2-hydrazinylbenzoic acid hydrochloride (5.37 g, 18.8 mmol), ethyl 3-oxocyclohexanecarboxylate (3.52 g, 20.7 mmol), and acetic acid (3.23 mL, 56.4 mmol) in toluene (90 mL) was heated in an oil bath at 110 °C for 20 h. The solvent was removed under reduced pressure, and the residue was diluted with toluene (43 mL) and trifluoroacetic acid (11 mL). The homogeneous reaction mixture was stirred overnight in an oil bath heated to 90−94 °C. The reaction mixture was diluted with ethyl acetate, sonicated, and filtered under reduced pressure to give the product. The filtrate was concentrated and resuspended in ethyl acetate with sonication. The solid was collected and washed with ethyl acetate. The combined solids contained a significant amount of 5bromo-6-fluoro-2,3,4,9-tetrahydro-1H-carbazole-2,8-dicarboxylic acid. The solid was triturated with methanol to give 3.22 g of the product (HPLC purity: 79%). The solid was retriturated with methanol to give 5-bromo-2-(ethoxycarbonyl)-6-fluoro-2,3,4,9-tetrahydro-1H-carbazole8-carboxylic acid (2.87 g, 7.47 mmol, 40% yield) as a pale yellow solid. LCMS (ESI) m/z calcd for C16H15BrFNO4 [M + H]+ 284.0. Found: 384.1 and 386.1. The methanol filtrates were combined and concentrated. Trituration with methanol and filtration provided additional 5-bromo-2-(ethoxycarbonyl)-6-fluoro-2,3,4,9-tetrahydro1H-carbazole-8-carboxylic acid (0.513 g, 1.34 mmol) as a light tan solid. A solution of 5-bromo-2-(ethoxycarbonyl)-6-fluoro-2,3,4,9-tetrahydro-1H-carbazole-8-carboxylic acid (2.87 g, 7.47 mmol) and DDQ (3.73 g, 16.4 mmol) in tetrahydrofuran (45 mL) was heated at 60 °C for 90 min. After cooling to room temperature, the homogeneous mixture was diluted with ethyl acetate (∼50 mL) and stirred for 60 min. The resulting precipitate was collected by vacuum filtration, and the solid was washed with ethyl acetate and dried. The filtrate was concentrated, triturated with methanol with sonication, filtered, and washed with methanol. The solids were combined to give 4-bromo-7(ethoxycarbonyl)-3-fluoro-9H-carbazole-1-carboxylic acid (2.39 g, 6.29 mmol, 84% yield) as a pale yellow solid. LCMS (ESI) m/z calcd for C16H11BrFNO4 [M + H]+ 380.0. Found: 380.0 and 382.0. A heterogeneous mixture of 4-bromo-7-(ethoxycarbonyl)-3-fluoro9H-carbazole-1-carboxylic acid (2.39 g, 6.29 mmol), 1-ethyl-3-(3(dimethylamino)propyl)carbodiimide hydrochloride (1.81 g, 9.43 mmol), and 1H-benzo[d][1,2,3]triazol-1-ol, monohydrate (1.44 g, 9.43 mmol) in a mixture of tetrahydrofuran (30 mL) and dichloromethane (5 mL) was stirred at room temperature for 20 min. Ammonium hydroxide (0.367 mL, 9.43 mmol) was added, and the heterogeneous reaction mixture was stirred at room temperature for 4 h. The mixture was diluted with ethyl acetate, washed with a saturated aqueous solution of sodium bicarbonate (2×), and washed with brine. The organic layer was collected, and the aqueous layers were sequentially extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate. Concentration under reduced pressure followed by trituration with methanol with sonication afforded ethyl 5-bromo-8-carbamoyl-6-fluoro-9H-carbazole-2-carboxylate (2.26 g, 5.96 mmol, 95% yield) as a pale yellow solid. LCMS (ESI) m/z calcd for C16H12BrFN2O3 [M + H]+ 279.0. Found: 379.1 and 381.1. 1H NMR (500 MHz, DMSO-d6) δ 12.02 (s, 1H), 8.70 (d, J = 8.3 Hz, 1H), 8.51 (d, J = 1.1 Hz, 1H), 8.29 (br. S., 1H), 8.10 (d, J = 10.3 Hz, 1H), 7.87 (dd, J = 8.5, 1.5 Hz, 1H), 7.74 (br. S., 1H), 4.37 (q, J = 6.9 Hz, 2H), and 1.37 (t, J = 7.1 Hz, 3H). To a solution of ethyl 5-bromo-8-carbamoyl-6-fluoro-9H-carbazole2-carboxylate (0.500 g, 1.32 mmol) in tetrahydrofuran (9.0 mL) at −78 °C was added methyllithium (1.6 M in ether) (3.0 equiv; 2.47 mL, 3.96 mmol) dropwise over 10 min. The reaction mixture was

stirred at −78 °C for 30 min. Additional methyllithium (2.0 equiv; 1.6 M in ether) (1.65 mL, 2.64 mmol) was added, and the reaction mixture was stirred at −78 °C for 45 min. The homogeneous reaction was quenched at −78 °C with a saturated aqueous solution of ammonium chloride. After warming to room temperature, the mixture was diluted with ethyl acetate, washed with water, and washed with brine. The organic layer was collected, and the aqueous layers were sequentially extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate. Concentration under reduce pressure afforded a pale yellow solid which was purified by reverse phase preparative HPLC. The desired fractions were immediately neutralized with a saturated aqueous solution of sodium bicarbonate and concentrated under reduced pressure. The residue was dissolved in a mixture of ethyl acetate and water, and the organic layer was collected and washed with brine. The aqueous layers were sequentially extracted with ethyl acetate, and the combined organic layers were dried over anhydrous sodium sulfate. Concentration under reduced pressure afforded 4-bromo-3-fluoro-7-(2-hydroxypropan-2yl)-9H-carbazole-1-carboxamide (0.240 g, 0.657 mmol, 50% yield) as a pale yellow solid. LCMS (ESI) m/z calcd for C16H14BrFN2O2 [M + H]+ 365.0. Found: (M + H − H2O)+ 347.1 and 349.1. 1H NMR (500 MHz, DMSO-d6) δ 11.58 (s, 1H), 8.50 (d, J = 8.6 Hz, 1H), 8.22 (br. S., 1H), 7.96 (d, J = 10.3 Hz, 1H), 7.94 (d, J = 1.1 Hz, 1H), 7.65 (br. S., 1H), 7.39 (dd, J = 8.5, 1.5 Hz, 1H), 5.09 (s, 1H), and 1.51 (s, 6H). A mixture of 4-bromo-3-fluoro-7-(2-hydroxypropan-2-yl)-9H-carbazole-1-carboxamide (0.100 g, 0.274 mmol), 8-fluoro-1-methyl-3-(S)(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)quinazoline-2,4(1H,3H)-dione (19, 0.146 g, 0.356 mmol), tripotassium phosphate (2 M in water) (0.411 mL, 0.821 mmol), and tetrahydrofuran (2.0 mL) was degassed with vacuum and nitrogen (3×). 1,1′-Bis(ditert-butylphosphino)ferrocene palladium dichloride (8.92 mg, 0.014 mmol) was added, and the reaction mixture was degassed (2×). The reaction mixture was stirred at room temperature overnight. The dark reaction mixture was diluted with ethyl acetate, washed with water, washed with brine, and dried over anhydrous sodium sulfate. Concentration under reduce pressure followed by purification by flash silica gel chromatography using a mixture of ethyl acetate and hexane (50%−62%−75%−85%) provided 3-fluoro-4-(3(S)-(8-fluoro-1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2methylphenyl)-7-(2-hydroxypropan-2-yl)-9H-carbazole-1-carboxamide (0.084 g, 0.146 mmol, 53% yield) as an off-white solid. HPLC purity: 98.3%; tr = 10.87 min (Method A); 98.0%; tr = 10.64 min (Method B); LCMS (ESI) m/z calcd for C32H26F2N4O4 [M + H]+ 569.2. Found: (M + H − H2O)+ 551.4. 1H NMR (500 MHz, DMSO-d6) δ 11.39 (s, 1H), 8.21 (br. s., 1H), 7.99−7.92 (m, 2H), 7.84 (s, 1H), 7.73 (ddt, J = 14.4, 8.0, 1.4 Hz, 1H), 7.59 (br. s., 1H), 7.54−7.47 (m, 2H), 7.40 (dd, J = 7.2, 1.7 Hz, 1H), 7.34 (tt, J = 7.9, 4.0 Hz, 1H), 7.04 (ddd, J = 11.5, 8.6, 1.5 Hz, 1H), 6.88 (t, J = 8.0 Hz, 1H), 4.99 (d, J = 2.2 Hz, 1H), 3.74 (dd, J = 8.2, 1.0 Hz, 3H), 1.77 (s, 3H), and 1.49−1.42 (m, 6H). A sample of 3-fluoro-4-(3-(S)-(8-fluoro-1-methyl-2,4-dioxo-1,2dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-7-(2-hydroxypropan-2yl)-9H-carbazole-1-carboxamide (mixture of 2 atropisomers) was separated by chiral SFC to give two single, stable atropisomers using the following conditions: column, AS-H 3 × 25 cm; 5 μm; BPR pressure, 100 bar; temperature, 40 °C; flow rate, 120 mL/min; mobile phase, CO2/ MeOH (70/30); detector wavelength, 220 nm; injection, 2 mL; sample preparation, 18 mg/5 mL MeOH, ∼3.6 mg/mL. The first peak eluting from the column provided 12c as a single, stable atropisomer and as a white solid. HPLC purity: 99.3%; tr = 10.70 min (Method A); 99.4%; tr = 10.45 min (Method B). Chiral purity: >99.4% ie; LCMS (ESI) m/z calcd for C32H26F2N4O4 [M + H]+ 569.2. Found: (M + H − H2O)+ 551.3.0. 1H NMR (500 MHz, DMSO-d6) δ 11.39 (s, 1H), 8.21 (br. s., 1H), 8.00−7.92 (m, 2H), 7.84 (s, 1H), 7.73 (ddd, J = 14.4, 8.0, 1.4 Hz, 1H), 7.59 (br. s., 1H), 7.55−7.47 (m, 2H), 7.40 (dd, J = 7.2, 1.4 Hz, 1H), 7.33 (td, J = 8.0, 4.0 Hz, 1H), 7.05 (dd, J = 8.3, 1.4 Hz, 1H), 6.89 (d, J = 8.3 Hz, 1H), 4.99 (s, 1H), 3.74 (d, J = 8.0 Hz, 3H), 3.17 (d, J = 5.3 Hz, 3H), 1.77 (s, 3H), and 1.46 (d, J = 4.2 Hz, 6H). 5-(3-(S)-(8-Fluoro-1-methyl-2,4-dioxo-1,2-dihydroquinazolin3(4H)-yl)-2-methylphenyl)-2-(R)-(2-hydroxypropan-2-yl)-2,3,4,9-tet9192



Article

rahydro-1H-carbazole-8-carboxamide (14a). A sample of 5-bromo2-(2-hydroxypropan-2-yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide16b (320 mg, 0.914 mmol) was separated by chiral SFC using the following conditions: column, Chiral OD-H 3 × 25 cm; 5 μm; flow rate, 85 mL/min; mobile phase, CO2/MeOH (65/35); injection, 3 mL; sample preparation, 320 mg/50 mL MeOH/MeCN (1:1). The second peak eluting from the column provided (R)-5-bromo-2-(2hydroxypropan-2-yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide as a white solid (165 mg, 0.471 mmol, 50% yield). Chiral purity: >99.5% ie; LCMS (ESI) m/z calcd for C16H19BrN2O2 [M + H]+ 351.1. Found: 351.0 and 353.0. 1H NMR (400 MHz, CD3OD) δ ppm 7.38 (d, J = 8.14 Hz, 1H), 7.16 (d, J = 8.14 Hz, 1H), 3.37−3.45 (m, 1H), 2.90−2.97 (m, 1H), 2.79−2.89 (m, 1H), 2.54−2.64 (m, 1H), 2.20−2.28 (m, 1H), 1.80−1.90 (m, J = 11.94, 11.94, 4.95, 2.20 Hz, 1H), 1.41−1.53 (m, J = 12.43, 12.43, 12.32, 5.28 Hz, 1H), and 1.30 (s, 6H). A mixture of 8-fluoro-1-methyl-3-(S)-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)quinazoline-2,4(1H,3H)-dione (19, 49.2 mg, 0.120 mmol), 5-(R)-bromo-2-(2-hydroxypropan-2-yl)2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (40 mg, 0.114 mmol), cesium carbonate (74.4 mg, 0.228 mmol), and 1,1′-bis(diphenylphosphino)ferrocenepalladium(II) dichloride−dichloromethane adduct (4.66 mg, 5.71 μmol) in tetrahydrofuran (2 mL) and water (0.50 mL) was heated at 40 °C for 3 h. The reaction mixture was diluted with dichloromethane, washed with water, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO, 20−100% ethyl acetate/hexane, 40 g silica gel column) to give 14a as a white solid. HPLC purity: 97.2%; tr = 8.56 min (Method B); LCMS (ESI) m/z calcd for C32H31FN4O4 [M + H]+ 555.2. Found: 555.2. 1H NMR (400 MHz, CDCl3) δ 10.04 (s, 1H), 8.13 (dd, J = 7.2, 3.9 Hz, 1H), 7.52−7.34 (m, 4H), 7.28−7.21 (m, 2H), 6.97 (dd, J = 7.7, 6.2 Hz, 1H), 3.89 (d, J = 7.7 Hz, 3H), 2.92 (d, J = 16.5 Hz, 1H), 2.71−2.58 (m, 1H), 2.27−2.19 (m, 1H), 2.17−2.09 (m, 1H), 2.03 (d, J = 12.3 Hz, 1H), 1.93−1.76 (m, 4H), and 1.29 (s, 6H). 5-(3-(S)-(8-Fluoro-1-methyl-2,4-dioxo-1,2-dihydroquinazolin3(4H)-yl)-2-methylphenyl)-2-(S)-(2-hydroxypropan-2-yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (14b). A sample of 5-bromo2-(2-hydroxypropan-2-yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide16b (320 mg, 0.914 mmol) was separated by chiral SFC using the following conditions: column, Chiral OD-H 3 × 25 cm; 5 μm; flow rate, 85 mL/min; mobile phase, CO2/MeOH (65/35); injection, 3 mL; sample preparation, 320 mg/50 mL MeOH/MeCN (1:1). The first peak eluting from the column provided 5-bromo-2-(S)-(2hydroxypropan-2-yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide as a single, stable atropisomer as a white solid (164 mg, 0.468 mmol, 50% yield). Chiral purity: >99.5% ie; LCMS (ESI) m/z calcd for C16H19BrN2O2 [M + H]+ 351.1. Found: 351.0 and 353.0. 1H NMR (400 MHz, CD3OD) δ ppm 7.38 (d, J = 8.14 Hz, 1H), 7.16 (d, J = 8.14 Hz, 1H), 3.37−3.45 (m, 1H), 2.90−2.97 (m, 1H), 2.79−2.89 (m, 1H), 2.54−2.64 (m, 1H), 2.20−2.28 (m, 1H), 1.80−1.90 (m, J = 11.94, 11.94, 4.95, 2.20 Hz, 1H), 1.41−1.53 (m, J = 12.43, 12.43, 12.32, 5.28 Hz, 1H), and 1.30 (s, 6H). A mixture of 8-fluoro-1-methyl-3-(S)-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)quinazoline-2,4(1H,3H)-dione (19, 49.2 mg, 0.120 mmol), 5-bromo-2-(S)-(2-hydroxypropan-2-yl)2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (40 mg, 0.114 mmol), cesium carbonate (74.4 mg, 0.228 mmol), and 1,1′-bis(diphenylphosphino)ferrocenepalladium(II) dichloride−dichloromethane adduct (9.33 mg, 0.011 mmol) in tetrahydrofuran (2 mL) and water (0.500 mL) was heated at 40 °C for 3 h. The reaction mixture was diluted with dichloromethane, washed with water, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography using a mixture of ethyl acetate and hexane (20%−100%) to give 14b (0.038 g, 0.066 mmol, 58% yield) as a pale yellow solid. HPLC purity: 95.9%; tr = 8.54 min (Method A); 98.0%; tr = 8.92 min (Method B); LCMS (ESI) m/z calcd for C32H31FN4O4 [M + H]+ 555.2. Found: 555.2. 1H NMR (400 MHz, CDCl3) δ 10.04 (s, 1H), 8.12 (d, J = 7.7 Hz, 1H), 7.51−7.45 (m, 1H), 7.43−7.34 (m, 3H), 7.26−7.21 (m, 2H), 6.97 (dd, J = 7.6, 3.9 Hz,

1H), 3.92−3.87 (m, 3H), 2.92 (d, J = 12.1 Hz, 1H), 2.70−2.57 (m, 1H), 2.29−2.18 (m, 1H), 2.13 (d, J = 14.3 Hz, 1H), 2.03 (br. s., 1H), 1.92−1.77 (m, 4H), and 1.28 (d, J = 2.4 Hz, 6H). The absolute stereochemistry of 14b was determined by single crystal X-ray analysis (CCDC # 1501160).

6-Chloro-5-(R)-(3-(S)-(8-fluoro-1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-2-(R)-(2-hydroxypropan-2yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (14c).16a A solution of sodium nitrite (3.03 g, 43.9 mmol) in water (14.8 mL) was added dropwise to a cooled (−10 °C, sodium chloride−ice bath) suspension of 2-amino-4-bromo-5-chlorobenzoic acid (10.0 g, 39.9 mmol) in hydrochloric acid (37%, 39.9 mL) and water (13.3 mL) at such a rate that the temperature did not exceed 0 °C. The resulting suspension was stirred at 0 °C for 15 min and was then treated with a solution of tin(II) chloride hydrate (22.7 g, 120 mmol) in hydrochloric acid (37%, 17 mL). The resulting mixture was warmed to room temperature and stirred for 60 min. The precipitate was collected by vacuum filtration, washed with water, and air-dried overnight to give 4bromo-5-chloro-2-hydrazinylbenzoic acid hydrochloride as an offwhite solid (12.9 g, 37.5 mmol, 96% yield). LCMS (ESI) m/z calcd for C7H6BrClN2O4 [M + H]+ 264.9. Found: 265.0 and 267.0. 1H NMR (400 MHz, DMSO-d6) δ 9.05 (br. s., 1H), 7.95 (s, 1H), and 7.55 (s, 1H). A suspension of 4-bromo-5-chloro-2-hydrazinylbenzoic acid hydrochloride (12.9 g, 37.6 mmol), ethyl 3-oxocyclohexanecarboxylate (7.03 g, 41.3 mmol), and acetic acid (6.45 mL, 113 mmol) in toluene (188 mL) was heated at 105 °C overnight. After 16 h, additional acetic acid (6 mL) and ethyl 3-oxocyclohexanecarboxylate (2.00 g) were added, and the reaction mixture was heated at 110 °C for 4.5 h. The mixture was concentrated, and the residue was diluted with toluene (100 mL) and trifluoroacetic acid (20 mL), and the suspension was heated at 90 °C overnight. The cooled mixture was concentrated, and the residue was suspended in ethyl acetate. The resulting solid was collected by vacuum filtration, washed with ethyl acetate, and air-dried to give 5bromo-6-chloro-2-(ethoxycarbonyl)-2,3,4,9-tetrahydro-1H-carbazole8-carboxylic acid as a yellow solid (11.0 g, 27.6 mmol, 73% yield). LCMS (ESI) m/z calcd for C16H15BrClNO4 [M + H]+ 400.0. Found: 400.2 and 402.2. 1H NMR (400 MHz, DMSO-d6) δ 13.44 (br. s., 1H), 11.24 (s, 1H), 7.69 (s, 1H), 4.12 (qd, J = 7.1, 2.3 Hz, 2H), 3.23−2.81 (m, 5H), 2.23−2.09 (m, 1H), 1.91−1.75 (m, 1H), and 1.22 (t, J = 7.0 Hz, 3H). Following the procedure used to prepare 14e, 5-bromo-6-chloro-2(ethoxycarbonyl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxylic acid was converted into ethyl 5-bromo-8-carbamoyl-6-chloro-2,3,4,9tetrahydro-1H-carbazole-2-carboxylate (light brown solid, 8.54 g, 78% yield). LCMS (ESI) m/z calcd for C16H16BrClN2O3 [M + H]+ 399.0. Found: 399.1 and 401.1. A solution of ethyl 5-bromo-8-carbamoyl-6-chloro-2,3,4,9-tetrahydro-1H-carbazole-2-carboxylate (7.03 g, 17.6 mmol) in tetrahydrofuran (200 mL) was cooled in a dry ice−acetone bath and treated portionwise over 40 min with 1.6 M methyllithium in tetrahydrofuran (66.0 mL, 106 mmol). After 60 min, the mixture was treated slowly at −78 °C with a saturated aqueous solution of ammonium chloride and stirred for 10 min while warming to room temperature. The mixture was extracted with dichloromethane (3×), and the combined organic phases were washed sequentially with water and brine, dried over 9193



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cm, 5 μm); mobile phase, CO2/MeOH (50:50) at 124 mL/min, 100 bar, 45 °C; sample preparation, 39 mg/mL in MeOH/DMSO (4:1); injection, 2.33 mL. The second peak eluting from the column provided the (S) isomer, 5-bromo-6-chloro-2-(S)-(2-hydroxypropan-2-yl)2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide, as an off-white solid (0.923 g, 2.39 mmol, 39% yield). LCMS (ESI) m/z calcd for C16H18BrClN2O2 [M + H]+ 385.0. Found: 385.2 and 387.2. 1H NMR (500 MHz, DMSO-d6) δ 11.06 (s, 1H), 8.12 (br. s., 1H), 7.74 (s, 1H), 7.49 (br. s., 1H), 4.23 (s, 1H), 3.27 (d, J = 5.0 Hz, 1H), 2.93 (dd, J = 17.1, 4.6 Hz, 1H), 2.72 (t, J = 11.8 Hz, 1H), 2.48−2.37 (m, 1H), 2.12 (d, J = 9.2 Hz, 1H), 1.69−1.59 (m, 1H), 1.38−1.24 (m, 1H), and 1.14 (s, 6H). A mixture of 5-bromo-6-chloro-2-(S)-(2-hydroxypropan-2-yl)2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (100 mg, 0.259 mmol), 8-fluoro-1-methyl-3-(S)-(2-methyl-3-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)phenyl)quinazoline-2,4(1H,3H)-dione (19, 112 mg, 0.272 mmol), cesium carbonate (169 mg, 0.519 mmol), and 1,1′-bis(diphenylphosphino)ferrocenepalladium(II) dichloride− dichloromethane adduct (16.9 mg, 0.021 mmol) in tetrahydrofuran (4.3 mL) and water (1.1 mL) was heated at 50 °C overnight. The reaction mixture was diluted with ethyl acetate, washed with water, washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography using a mixture of ethyl acetate and hexane (50−80%), followed further purification by reverse-phase, preparative HPLC to afford 6-chloro-5-(3-(S)-(8-fluoro-1-methyl-2,4dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-2-(S)-(2-hydroxypropan-2-yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (0.059 g, 0.129 mmol, 50% yield) as a pale yellow solid and as a mixture of two atropisomers. HPLC purity: 97.8%; tr = 9.08 min (Method A); 98.9%; tr = 9.72 min (Method B). Chiral purity: >99.5% ie; LCMS (ESI) m/z calcd for C32H30ClFN4O4 [M + H]+ 589.2. Found: 589.2. 1H NMR (500 MHz, DMSO-d6) δ 10.89 (s, 1H), 8.13 (br. s., 1H), 7.94 (d, J = 7.9 Hz, 1H), 7.76−7.68 (m, 2H), 7.46 (br. s., 1H), 7.40−7.28 (m, 3H), 7.22−7.16 (m, 1H), 4.21−4.13 (m, 1H), 3.77−3.64 (m, 3H), 2.95−2.82 (m, 1H), 2.40 (d, J = 16.3 Hz, 1H), 1.94−1.70 (m, 3H), 1.70−1.61 (m, 3H), 1.58−1.48 (m, 1H), and 1.16−1.02 (m, 7H). A sample of 6-chloro-5-(3-(S)-(8-fluoro-1-methyl-2,4-dioxo-1,2dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-2-(S)-(2-hydroxypropan-2-yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (mixture of two atropisomers, 0.071 g, 0.121 mmol) was separated by chiral SFC to give two single, stable atropisomers as follows: column, OD-H (3 × 25 cm, 5 μm); mobile phase, CO2−MeOH (70:30) at 180 mL/min, 35 °C, 100 bar; injection, 3 mL (430 s cycle time); sample preparation, 0.071 mg/mL in MeOH/CH2Cl2 (4:1), ∼8.4 mg/mL. The first peak eluting from the column provided 14d (0.034, 0.056 mmol, 47% yield) as a single, stable atropisomer and as a white solid. HPLC purity: 95.2%; tr = 9.09 min (Method A); 96.1%; tr = 9.73 min (Method B). Chiral purity: >99.5% ie; LCMS (ESI) m/z calcd for C32H30ClFN4O4 [M + H]+ 589.2. Found: 589.4. 1H NMR (400 MHz, CD3OD) δ 8.06−7.99 (m, 1H), 7.69 (s, 1H), 7.60 (ddd, J = 14.3, 8.1, 1.4 Hz, 1H), 7.40 (d, J = 7.5 Hz, 1H), 7.34−7.27 (m, 2H), 7.24 (dd, J = 7.5, 1.1 Hz, 1H), 3.85 (d, J = 7.9 Hz, 3H), 2.90 (dd, J = 16.5, 5.1 Hz, 1H), 2.56 (dd, J = 16.4, 12.0 Hz, 1H), 2.10−2.02 (m, 1H), 2.00−1.84 (m, 2H), 1.77 (s, 3H), 1.75−1.68 (m, 1H), 1.28 (d, J = 3.1 Hz, 1H), and 1.22 (d, J = 2.2 Hz, 6H). The absolute stereochemistry of 14d was determined by single crystal X-ray analysis (CCDC # 1501161). 6-Fluoro-5-(R)-(3-(S)-(8-fluoro-1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-2-(R)-(2-hydroxypropan-2yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (14e).16a A heterogeneous mixture of 5-bromo-2-(ethoxycarbonyl)-6-fluoro-2,3,4,9tetrahydro-1H-carbazole-8-carboxylic acid (0.513 g, 1.34 mmol), 1ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (0.384 g, 2.00 mmol), and 1H-benzo[d][1,2,3]triazol-1-ol, monohydrate (0.307 g, 2.00 mmol) in a mixture of tetrahydrofuran (10 mL) and dichloromethane (1.65 mL) was stirred at room temperature for 20 min. Ammonium hydroxide (0.078 mL, 2.00 mmol) was added, and the heterogeneous reaction mixture was stirred at room temperature

anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with a mixture of ethyl acetate and hexanes (0−100%) to give 5-bromo-6-chloro-2-(2-hydroxypropan-2-yl)-2,3,4,9-tetrahydro-1Hcarbazole-8-carboxamide as a yellow solid (4.66 g, 15.2 mmol, 86% yield). LCMS (ESI) m/z calcd for C16H18BrClN2O2 [M + H]+ 385.0. Found: 385.2 and 387.2. A sample of racemic 5-bromo-6-chloro-2-(2-hydroxypropan-2-yl)2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (2.35 g, 6.09 mmol) was separated by chiral SFC as follows: column, Chiralpak IA (3 × 25 cm, 5 μm); mobile phase, CO2/MeOH (50:50) at 124 mL/min, 100 bar, 45 °C; sample preparation, 39 mg/mL in MeOH/DMSO (4:1); injection, 2.33 mL. The first peak eluting from the column provided the (R)-isomer, 5-bromo-6-chloro-2-(R)-(2-hydroxypropan-2-yl)2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide as a yellow solid (1.15 g, 2.98 mmol, 49% yield). LCMS (ESI) m/z calcd for C16H18BrClN2O2 [M + H]+ 385.0. Found: 385.2 and 387.2. 1H NMR (500 MHz, DMSO-d6) δ 11.07 (s, 1H), 8.12 (br. s., 1H), 7.75 (s, 1H), 7.57−7.45 (m, 1H), 4.23 (s, 1H), 3.27 (d, J = 4.7 Hz, 1H), 2.93 (dd, J = 17.2, 4.7 Hz, 1H), 2.78−2.67 (m, 1H), 2.48−2.39 (m, 1H), 2.16−2.08 (m, 1H), 1.69−1.59 (m, 1H), 1.37−1.26 (m, 1H), 1.14 (s, 6H). A mixture of 8-fluoro-1-methyl-3-(S)-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)quinazoline-2,4(1H,3H)-dione (19, 134 mg, 0.327 mmol), 5-bromo-6-chloro-2-(R)-(2-hydroxypropan-2-yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (120 mg, 0.311 mmol), cesium carbonate (203 mg, 0.622 mmol), and 1,1′bis(diphenylphosphino)ferrocenepalladium(II) dichloride−dichloromethane adduct (20.3 mg, 0.025 mmol) in tetrahydrofuran (5 mL) and water (1.3 mL) was heated at 50 °C overnight. The reaction mixture was diluted with ethyl acetate, washed with water, washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography using a mixture of ethyl acetate in hexanes (50−70−80%) to give 136 mg of the product. The compound was further purified by reversephase, preparative HPLC to give 6-chloro-5-(3-(S)-(8-fluoro-1-methyl2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-2-(R)-(2hydroxypropan-2-yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (0.088 g, 0.149 mmol, 48% yield) as a solid and as a mixture of two atropisomers. HPLC purity: 99%; tr = 1.77 min (Method D); LCMS (ESI) m/z calcd for C32H30ClFN4O4 [M + H]+ 589.2. Found: 589.4. 1 H NMR (500 MHz, DMSO-d6) δ 10.90 (s, 1H), 8.14 (br. s., 1H), 7.99−7.93 (m, 1H), 7.77−7.68 (m, 2H), 7.46 (br. s., 1H), 7.41−7.29 (m, 3H), 7.22−7.17 (m, 1H), 4.25−4.11 (m, 1H), 3.77−3.66 (m, 3H), 2.95−2.83 (m, 1H), 2.47−2.34 (m, 1H), 1.97−1.72 (m, 3H), 1.70− 1.63 (m, 3H), 1.54 (dd, J = 11.4, 3.5 Hz, 1H), 1.17−0.98 (m, 7H). A sample of 6-chloro-5-(3-(S)-(8-fluoro-1-methyl-2,4-dioxo-1,2dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-2-(R)-(2-hydroxypropan-2-yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (mixture of two atropisomers, 0.085 g, 0.144 mmol) was separated by chiral SFC to give two single stable atropisomers as follows: column, OD-H (3 × 25 cm, 5 μm); mobile phase, CO2−MeOH (70:30) at 180 mL/min, 35 °C, 100 bar; injection, 3 mL (430 s cycle time); sample preparation, 0.071 mg/mL in MeOH/CH2Cl2 (4:1), ∼8.4 mg/mL. The second peak eluting from the column provided 14c (0.049, 0.082 mmol, 57% yield) as single, stable atropisomer and as an off-white solid. HPLC purity: 97.8%; tr = 9.08 min (Method A); 98.9%; tr = 9.72 min (Method B). Chiral purity: >99.5% ie; LCMS (ESI) m/z calcd for C32H30ClFN4O4 [M + H]+ 589.2. Found: 589.4. 1H NMR (400 MHz, DMSO-d6) δ 10.86 (s, 1H), 8.12 (br. s., 1H), 7.98−7.93 (m, 1H), 7.76−7.66 (m, 2H), 7.49−7.35 (m, 2H), 7.34−7.28 (m, 2H), 7.20 (d, J = 6.2 Hz, 1H), 4.17 (br. s., 1H), 3.72 (d, J = 7.9 Hz, 3H), 2.88 (d, J = 12.8 Hz, 1H), 2.44−2.34 (m, 1H), 1.99−1.71 (m, 3H), 1.64 (s, 3H), 1.60−1.48 (m, 1H), and 1.08 (d, J = 7.3 Hz, 7H). 6-Chloro-5-(R)-(3-(S)-(8-fluoro-1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-2-(S)-(2-hydroxypropan-2yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (14d).16a A sample of racemic 5-bromo-6-chloro-2-(2-hydroxypropan-2-yl)2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (2.35 g, 6.09 mmol) was separated by chiral SFC as follows: column, Chiralpak IA (3 × 25 9194



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chloromethane adduct (6.90 mg, 8.45 μmol) was added, and the reaction mixture was degassed (2×). The mixture was immersed in an oil bath at 52 °C and stirred overnight. The reaction mixture was diluted with ethyl acetate, washed with water, washed with brine, and dried over anhydrous sodium sulfate. Concentration under reduce pressure afforded the crude product mixture which was purified by flash silica gel chromatography using a mixture of ethyl acetate and hexane (50%−62%−75%) to afford 6-fluoro-5-(3-(S)-(8-fluoro-1methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-2(R)-(2-hydroxypropan-2-yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (0.050 g, 0.086 mmol, 61% yield) as a white solid. HPLC purity: 99.2%; tr = 10.67 min (Method A); 98.9%; tr = 10.35 min (Method B); LCMS (ESI) m/z calcd for C32H30F2N4O4 [M + H]+ 573.2. Found: 573.2. 1H NMR (500 MHz, DMSO-d6) δ 10.79−10.74 (m, 1H), 8.05 (br. s., 1H), 7.98−7.93 (m, 1H), 7.76−7.69 (m, 1H), 7.57−7.51 (m, 1H), 7.43 (br. s., 1H), 7.40−7.26 (m, 4H), 4.19−4.13 (m, 1H), 3.74− 3.68 (m, 3H), 2.94−2.84 (m, 1H), 2.49−2.35 (m, 2H), 1.92−1.80 (m, 3H), 1.76−1.68 (m, 3H), 1.62−1.52 (m, 1H), and 1.12−1.06 (m, 6H). A sample of 6-fluoro-5-(3-(S)-(8-fluoro-1-methyl-2,4-dioxo-1,2dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-2-(R)-(2-hydroxypropan-2-yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (mixture of 2 atropisomers) was separated by chiral SFC to give two single, stable atropisomers using the following conditions: column, AS-H 3 × 25 cm; 5 μm; BPR pressure, 100 bar; temperature, 35 °C; flow rate, 120 mL/min; mobile phase, CO2/MeOH (70/30); detector wavelength, 220 nm; injection, 1.7 mL; sample preparation, 45 mg/5 mL MeOH and ∼9 mg/mL. The first peak eluting from the column provided 14e as a single, stable atropisomer and as a white solid. HPLC purity: 98.2%; tr = 10.66 min (Method A); 98.6%; tr = 10.33 min (Method B). Chiral purity: >99.5% ie; LCMS (ESI) m/z calcd for C32H30F2N4O4 [M + H]+ 573.2. Found: 573.2. 1H NMR (500 MHz, DMSO-d6) δ 10.79−10.74 (m, 1H), 8.05 (br. s., 1H), 7.98−7.93 (m, 1H), 7.76− 7.69 (m, 1H), 7.57−7.51 (m, 1H), 7.43 (br. s., 1H), 7.40−7.26 (m, 4H), 4.19−4.13 (m, 1H), 3.74−3.68 (m, 3H), 2.94−2.84 (m, 1H), 2.49−2.35 (m, 2H), 1.92−1.80 (m, 3H), 1.76−1.68 (m, 3H), 1.62− 1.52 (m, 1H), and 1.12−1.06 (m, 6H). 6-Fluoro-5-(R)-(3-(S)-(8-fluoro-1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-2-(S)-(2-hydroxypropan-2yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (14f).16a A sample of 5-bromo-6-fluoro-2-(2-hydroxypropan-2-yl)-2,3,4,9-tetrahydro1H-carbazole-8-carboxamide (97 g, 264 mmol, see procedure for 14e) was separated by chiral SFC as follows: column, Chiralpak OD-H (3 × 25 cm, 5 μm); BPR pressure, 100 bar; temperature, 40 °C; flow rate, 150 mL/min; mobile phase, CO2/MeOH (70:30); detector wavelength, 240 nm; injection, 1.33 mL (3.5 min cycle time); sample preparation, 97 g/2590 mL MeOH/CH2Cl2 (1:1), ∼38 mg/mL. The first peak eluting from the column provided, after a methanol trituration, 5-bromo-6-fluoro-2-(S)-(2-hydroxypropan-2-yl)-2,3,4,9tetrahydro-1H-carbazole-8-carboxamide (37 g, 102 mmol, 71% yield) as an off-white solid. HPLC purity: >99%; tr = 2.67 min (Method C). Chiral purity: 99.5% ie; LCMS (ESI) m/z calcd for C16H18BrFN2O2 [M + H]+ 369.1. Found: 369.1 and 371.1. 1H NMR (500 MHz, DMSO-d6) δ 10.96 (s, 1H), 8.07 (br. s., 1H), 7.55 (d, J = 10.3 Hz, 1H), 7.50 (br. s., 1H), 4.24 (s, 1H), 3.26 (dd, J = 15.8, 4.4 Hz, 1H), 2.93 (dd, J = 17.1, 4.6 Hz, 1H), 2.72 (t, J = 11.7 Hz, 1H), 2.48−2.40 (m, 1H), 2.12 (d, J = 9.2 Hz, 1H), 1.70−1.62 (m, 1H), 1.32 (qd, J = 12.4, 5.3 Hz, 1H), and 1.14 (s, 6H). A mixture of 5-bromo-6-fluoro-2-(S)-(2-hydroxypropan-2-yl)2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (6.00 g, 16.25 mmol), 8-fluoro-1-methyl-3-(S)-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)quinazoline-2,4(1H,3H)-dione (19, 8.00 g, 19.50 mmol), tripotassium phosphate (2 M in water) (24.4 mL, 48.8 mmol), and tetrahydrofuran (72 mL) was degassed with vacuum and nitrogen (3×). 1,1′-Bis(di-tert-butylphosphino)ferrocene palladium dichloride (0.530 g, 0.813 mmol) was added, and the reaction mixture was degassed (2×). The reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate, washed with water, washed with brine, and dried over anhydrous sodium sulfate. Concentration under reduce pressure followed by purification by flash silica gel chromatography using a mixture of ethyl acetate and

for 60 min (homogeneous). The mixture was diluted with ethyl acetate, washed with a saturated aqueous solution of sodium bicarbonate (2×), and washed with brine. The organic layer was collected, and the aqueous layers were sequentially extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate. Concentration followed by trituration with methanol with sonication afforded ethyl 5-bromo-8-carbamoyl-6-fluoro-2,3,4,9tetrahydro-1H-carbazole-2-carboxylate (0.432 g, 1.12 mmol, 84% yield) as a pale yellow solid. HPLC purity: >99%; tr = 2.89 min (Method C); LCMS (ESI) m/z calcd for C16H16BrFN2O3 [M + H]+ 383.0. Found: 383.1 and 385.1. To a solution of ethyl 5-bromo-8-carbamoyl-6-fluoro-2,3,4,9tetrahydro-1H-carbazole-2-carboxylate (10.0 g, 26.1 mmol) in tetrahydrofuran (200 mL) at −78 °C was added methyllithium (1.6 M in ether) (3 equiv; 49 mL, 78 mmol) dropwise over 30 min The reaction mixture was stirred at −78 °C for 45 min. An additional 2 equiv of methyllithium (33 mL) was added over 25 min, and the reaction mixture was stirred at −78 °C for an additional 1.5 h. The reaction was quenched at −78 °C with a saturated aqueous solution of ammonium chloride was warmed to room temperature. The mixture was diluted with ethyl acetate, washed with water, and washed with brine. The organic layer was collected, and the aqueous layers were sequentially extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was dissolved in ∼100 mL of ethyl acetate and filtered through a pad of Celite topped with a pad of silica gel in a 600 mL fritted funnel using ethyl acetate (∼1 L). Concentration under reduced pressure afforded 5-bromo-6-fluoro-2(2-hydroxypropan-2-yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (9.24 g, 25.03 mmol, 96% yield) as a pale yellow solid. LCMS (ESI) m/z calcd for C16H18BrFN2O2 [M + H]+ 369.1. Found: 369.2 and 371.2. A sample of racemic 5-bromo-6-fluoro-2-(2-hydroxypropan-2-yl)2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide was separated by chiral SFC as follows: column, Chiralpak AD-H (3 × 25 cm, 5 μm); temperature, 45 °C; flow rate, 120 mL/min; mobile phase, CO2/ MeOH (50:50); detector wavelength, 220 nm; injection, 1 mL (20 μg/mL). The first peak eluting from the column provided the (R)isomer, 5-bromo-6-fluoro-2-(R)-(2-hydroxypropan-2-yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide, as a white solid. HPLC purity: >99%; tr = 2.73 min (Method C); LCMS (ESI) m/z calcd for C16H18BrFN2O2 [M + H]+ 369.1. Found: 369.2 and 371.2. 1H NMR (500 MHz, DMSO-d6) δ 10.96 (s, 1H), 8.07 (br. s., 1H), 7.55 (d, J = 10.3 Hz, 1H), 7.50 (br. s., 1H), 4.24 (s, 1H), 3.26 (dd, J = 15.8, 4.4 Hz, 1H), 2.93 (dd, J = 17.1, 4.6 Hz, 1H), 2.72 (t, J = 11.7 Hz, 1H), 2.48− 2.40 (m, 1H), 2.12 (d, J = 9.2 Hz, 1H), 1.70−1.62 (m, 1H), 1.32 (qd, J = 12.4, 5.3 Hz, 1H), and 1.14 (s, 6H). A mixture of 5-bromo-6-fluoro-2-(R)-(2-hydroxypropan-2-yl)2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (0.052 g, 0.141 mmol), 8-fluoro-1-methyl-3-(S)-(2-methyl-3-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)phenyl)quinazoline-2,4(1H,3H)-dione (19, 0.075 g, 0.183 mmol), cesium carbonate (0.092 g, 0.282 mmol), and dioxane (0.8 mL) was degassed with a vacuum and nitrogen (3×). 1,1′-Bis(diphenylphosphino)ferrocenepalladium(II) dichloride−di9195



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temperature, 40 °C; flow rate, 150 mL/min; mobile phase, CO2/ MeOH (70:30); detector wavelength, 220 nm; injection, 1.8 mL (4.6 min cycle time); sample preparation, MeOH/CH2Cl2 (1:1). The second peak eluting from the column provided 14g as a single, stable atropisomer and as a white solid. HPLC purity: 99.9%; tr = 10.62 min (Method A); 99.9%; tr = 10.28 min (Method B). Chiral purity: 97.7% ie; LCMS (ESI) m/z calcd for C32H30F2N4O4 [M + H]+ 573.2. Found: 573.2. 1H NMR (500 MHz, DMSO-d6) d 10.77 (s, 1H), 8.05 (br. s., 1H), 7.94 (dd, J = 7.9, 1.0 Hz, 1H), 7.72 (ddd, J = 14.4, 8.0, 1.4 Hz, 1H), 7.55 (d, J = 10.5 Hz, 1H), 7.43 (br. s., 1H), 7.40−7.35 (m, 1H), 7.34−7.28 (m, 3H), 4.17 (s, 1H), 3.72 (d, J = 8.0 Hz, 3H), 2.90 (d, J = 4.7 Hz, 1H), 2.87 (d, J = 4.7 Hz, 1H), 2.45−2.35 (m, 1H), 2.00−1.91 (m, 1H), 1.86 (dd, J = 15.3, 5.5 Hz, 2H), 1.71 (s, 3H), 1.57 (td, J = 11.9, 3.3 Hz, 1H), 1.10 (s, 3H), and 1.08 (s, 3H). 8-Fluoro-1-methyl-3-(R,S)-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)phenyl)quinazoline-2,4(1H,3H)-dione (17). A solution of 3-(3-bromo-2-methylphenyl)-8-fluoroquinazoline-2,4(1H,3H)-dione (4.80 g, 13.8 mmol) in dimethylformamide (25 mL) was treated with cesium carbonate (13.4 g, 41.2 mmol). The suspension was stirred at room temperature and treated dropwise, but quickly, with iodomethane (4.30 mL, 68.7 mmol) and then stirred rapidly at room temperature for 1 h. The mixture was diluted with ethyl acetate and water (200 mL). The organic phase was separated and washed sequentially with water and brine and then was dried and concentrated to provide 3-(3-bromo-2-methylphenyl)-8-fluoro-1methylquinazoline-2,4(1H,3H)-dione as a tan solid (4.80 g, 13.3 mmol, 96% yield). LCMS (ESI) m/z calcd for C16H12BrFN2O2 [M + H]+ 363.0. Found: 363.0 and 365.0. A mixture of 3-(3-bromo-2-methylphenyl)-8-fluoro-1-methylquinazoline-2,4(1H,3H)-dione (4.80 g, 13.2 mmol), 4,4,4′,4′,5,5,5′,5′octamethyl-2,2′-bi(1,3,2-dioxaborolane) (4.36 g, 17.2 mmol), potassium acetate (3.89 g, 39.6 mmol), and 1,1′-bis(diphenylphosphino)ferrocenepalladium(II) dichloride−dichloromethane adduct (0.540 g, 0.661 mmol) in dioxane (65 mL) was heated at reflux for 2 h. After cooling to room temperature, the mixture was filtered through Celite, and the solids were rinsed with ethyl acetate. The filtrate was diluted with ethyl acetate, washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was subjected to column chromatography on silica gel (80 g), eluting with ethyl acetate and hexanes (20−50%), to provide 17 (4.61 g, 11.2 mmol, 85% yield). LCMS (ESI) m/z calcd for C22H24BFN2O4 [M + H]+ 411.2. Found: 411. 1H NMR (400 MHz, CDCl3) δ 8.14−8.08 (m, 1H), 7.93 (dd, J = 7.5, 1.3 Hz, 1H), 7.48 (ddd, J = 14.0, 8.0, 1.5 Hz, 1H), 7.34 (t, J = 7.6 Hz, 1H), 7.27−7.20 (m, 2H), 3.88 (d, J = 7.9 Hz, 3H), 2.36 (s, 3H), and 1.36 (s, 12H). 8-Fluoro-1-methyl-3-(S)-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl) phenyl)quinazoline-2,4(1H,3H)-dione (19) and 8-Fluoro-1-methyl-3-(R)-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)quinazoline-2,4(1H,3H)-dione (35).16 A sample of racemic 8-fluoro-1-methyl-3-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)quinazoline-2,4(1H,3H)-dione (17) was separated by chiral SFC as follows: column, (R,R)-Whelk-O1 (3 × 25 cm, 5 μm); mobile phase, CO2−MeOH (70:30) at 200 mL/min, 100 bar, 30 °C; sample preparation, 97.3 mg/mL in MeOH/DCM (1:1); injection, 4 mL. The first peak eluting from the column provided the (S)-isomer, 8-fluoro-1-methyl-3-(S)-(2-methyl-3-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)quinazoline-2,4(1H,3H)dione (19), as a single, stable atropisomer and as a white solid. LCMS (ESI) m/z calcd for C22H24BFN2O4 [M + H]+ 411.2. Found: 411. 1H NMR (400 MHz, CDCl3) δ 8.14−8.08 (m, 1H), 7.93 (dd, J = 7.5, 1.3 Hz, 1H), 7.48 (ddd, J = 14.0, 8.0, 1.5 Hz, 1H), 7.34 (t, J = 7.6 Hz, 1H), 7.27−7.20 (m, 2H), 3.88 (d, J = 7.9 Hz, 3H), 2.36 (s, 3H), and 1.36 (s, 12H). The absolute stereochemistry of 19 as (S) was determined by single crystal X-ray analysis of carbazole 7a. The second peak eluting from the column provided the (R)-isomer, 8-fluoro-1-methyl-3-(R)-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl) quinazoline-2,4(1H,3H)-dione (35), as a white solid and a single, stable atropisomer. LCMS (ESI) m/z calcd for C22H24BFN2O4 [M + H]+ 411.2. Found: 411. 1H NMR (400 MHz, CDCl3) δ 8.13−8.08 (m, 1H), 7.93 (dd, J = 7.5, 1.3 Hz, 1H), 7.48

hexane (50%−62%−75%−85%) afforded 6-fluoro-5-(3-(S)-(8-fluoro1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-methylphenyl)2-(S)-(2-hydroxypropan-2-yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (8.7 g, 15.19 mmol, 93% yield) as an off-white solid. HPLC purity: 99%; tr = 2.80 min (Method C); LCMS (ESI) m/z calcd for C32H30F2N4O4 [M + H]+ 573.2. Found: 573.2. A sample of 6-fluoro-5-(3-(S)-(8-fluoro-1-methyl-2,4-dioxo-1,2dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-2-(S)-(2-hydroxypropan-2-yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (23.7 g, 41.3 mmol) was separated by chiral SFC to give two single, stable atropisomers as follows: column, AS-H (3 × 25 cm, 5 μm); BPR pressure, 100 bar; temperature, 40 °C; flow rate, 150 mL/min; mobile phase, CO2/MeOH (70:30); detector wavelength, 220 nm; injection, 1.8 mL (4.6 min cycle time); sample preparation, MeOH/CH2Cl2 (1:1). The first peak eluting from the column provided 6-fluoro-5-(R)(3-(S)-(8-fluoro-1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)2-methylphenyl)-2-(S)-(2-hydroxypropan-2-yl)-2,3,4,9-tetrahydro-1Hcarbazole-8-carboxamide (12.7 g, 22.2 mmol, 54% yield) as a single, stable atropisomer and as an off-white solid. The compound was suspended in methanol with sonication, and the solid was collected by vacuum filtration, washed with methanol, and dried well to give 14f (11.2 g, 19.5 mmol, 88% yield) as a white solid. HPLC purity: 99.9%; tr = 11.05 min (Method A); 99.9%; tr = 10.72 min (Method B). Chiral purity: 99.8% ie; Optical rotation: [α]D20 (c = 2.10, CHCl3) = +63.8°; LCMS (ESI) m/z calcd for C32H30F2N4O4 [M + H]+ 573.2. Found: 573.5. Anal. calcd for C32H30F2N4O4, 0.72% H2O: C 65.56, H 5.42, N 9.55. Found: C 65.69, H 5.40, N 9.52. 1H NMR (500 MHz, DMSO-d6) δ 10.78 (s, 1H), 8.07 (br. s., 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.72 (dd, J = 14.2, 8.0 Hz, 1H), 7.56 (d, J = 10.8 Hz, 1H), 7.45 (br. s., 1H), 7.42−7.36 (m, 1H), 7.34 (d, J = 6.9 Hz, 1H), 7.34−7.31 (m, 1H), 7.29 (dd, J = 7.5, 1.3 Hz, 1H), 4.17 (s, 1H), 3.73 (d, J = 8.0 Hz, 3H), 2.91 (dd, J = 16.8, 4.4 Hz, 1H), 2.48−2.37 (m, 1H), 1.98−1.89 (m, 2H), 1.87 (d, J = 11.0 Hz, 1H), 1.76 (s, 3H), 1.59 (td, J = 11.5, 4.1 Hz, 1H), 1.20−1.12 (m, 1H), and 1.11 (s, 6H). 13 C NMR (126 MHz, DMSO-d6) δ 168.2 (d, J = 1.8 Hz, 1C), 160.1 (d, J = 3.6 Hz, 1C), 151.9 (d, J = 228.9 Hz, 1C), 150.5 (d, J = 41.8 Hz, 1C), 148.7 (d, J = 205.3 Hz, 1C), 139.2, 135.1, 135.0, 134.8, 131.4, 130.6, 130.0 (d, J = 7.3 Hz, 1C), 128.5, 127.1 (d, J = 4.5 Hz, 1C), 125.7, 124.3 (d, J = 2.7 Hz, 1C), 123.6 (d, J = 8.2 Hz, 1C), 123.0 (d, J = 23.6 Hz, 1C), 120.8 (d, J = 20.0 Hz, 1C), 118.4, 115.3 (d, J = 7.3 Hz, 1C), 108.8 (d, J = 5.4 Hz, 1C), 106.7 (d, J = 28.2 Hz, 1C), 70.4, 45.4, 34.3 (d, J = 14.5 Hz, 1C), 27.1, 26.8, 24.8, 24.7, 22.1, and 14.5. 19FNMR (470 MHz, DMSO-d6) δ −121.49 (dt, J = 22.9, 11.4 Hz, 1F), and −129.56 (d, J = 11.4 Hz, 1F). The absolute configuration of 14f was confirmed by single crystal Xray analysis (CCDC # 1501162).

6-Fluoro-5-(S)-(3-(S)-(8-fluoro-1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-2-(S)-(2-hydroxypropan-2yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (14g). A sample of 6-fluoro-5-(3-(S)-(8-fluoro-1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-2-(S)-(2-hydroxypropan-2-yl)-2,3,4,9tetrahydro-1H-carbazole-8-carboxamide (23.7 g, 41.3 mmol) was separated by chiral SFC to give two single, stable atropisomers as follows: column, AS-H (3 × 25 cm, 5 μm); BPR pressure, 100 bar; 9196



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(ddd, J = 13.9, 8.1, 1.5 Hz, 1H), 7.37−7.31 (m, 1H), 7.27−7.20 (m, 2H), 3.88 (d, J = 7.9 Hz, 3H), 2.36 (s, 3H), and 1.36 (s, 12H). Alternative Preparation of 19. A solution of 8-fluoro-3-(2-methyl3-(S)-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)quinazoline-2,4(1H,3H)-dione (33, 40 g, 101 mmol) in tetrahydrofuran (400 mL) was treated with cesium carbonate (99 g, 303 mmol) and iodomethane (12.6 mL, 202 mmol). The resulting cloudy solution was stirred at room temperature overnight. Water (300 mL) was added, and the reaction mixture was extracted with ethyl acetate (3 × 150 mL). The combined organic phases were washed sequentially with brine and water, and dried and concentrated. The residue was purified by recrystallization from ethyl acetate to provide 8-fluoro-1-methyl-3(S)-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)quinazoline-2,4(1H,3H)-dione as a white solid (19, 38 g, 92.6 mmol, 92% yield). 3-(3-Bromo-2-methylphenyl)-8-fluoroquinazoline-2,4(1H,3H)dione (31).16 A suspension of 8-fluoro-1H-benzo[d][1,3]oxazine-2,4dione (3.00 g, 16.6 mmol) in xylenes (50 mL) was treated with 3bromo-2-methylaniline (3.08 g, 16.6 mmol) and heated to reflux. After 6 h, the mixture was allowed to cool to room temperature overnight. The resulting suspension was diluted with hexanes, and the precipitate was collected by filtration, rinsed with hexanes, and air-dried to provide 2-amino-N-(3-bromo-2-methylphenyl)-3-fluorobenzamide as a white solid (30, 4.50 g, 84% yield). LCMS (ESI) m/z calcd for C14H12BrFN2O [M + H]+ 323.0. Found: 323.0 and 325.0. 1H NMR (400 MHz, CDCl3) δ 7.69 (d, J = 7.9 Hz, 1H), 7.65 (br. s., 1H), 7.50− 7.46 (m, 1H), 7.32 (d, J = 8.1 Hz, 1H), 7.19−7.11 (m, 2H), 6.73−6.64 (m, 1H), 5.69 (br. s., 2H), and 2.44 (s, 3H). A solution of 2-amino-N-(3-bromo-2-methylphenyl)-3-fluorobenzamide (30, 5.70 g, 17.6 mmol) in tetrahydrofuran (100 mL) was treated with bis(trichloromethyl)carbonate (triphosgene) (6.28 g, 21.2 mmol) at room temperature and stirred for 15 min. The mixture was diluted with ethyl acetate, carefully treated with a saturated aqueous solution of sodium bicarbonate, and stirred at room temperature until gas evolution stopped. The separated organic phase was washed sequentially with a saturated aqueous solution of sodium bicarbonate, water, and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was triturated with ether to provide 3-(3-bromo-2-methylphenyl)-8-fluoroquinazoline-2,4(1H,3H)-dione (31, 6.00 g, 17.2 mmol, 97% yield) as an off white solid. LCMS (ESI) m/z calcd for C15H10BrFN2O2 [M + H]+ 349.0. Found: 349.0 and 351.0. 1H NMR (400 MHz, CDCl3) δ 8.59 (d, J = 17.6 Hz, 1H), 7.99 (d, J = 8.1 Hz, 1H), 7.70 (dd, J = 7.8, 1.2 Hz, 1H), 7.54−7.43 (m, 1H), 7.28−7.21 (m, 2H), 7.21−7.17 (m, 1H), and 2.28 (s, 3H). 8-Fluoro-3-(R,S)-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) phenyl)quinazoline-2,4(1H,3H)-dione (32).16 A stirred mixture of 3-(3-bromo-2-methylphenyl)-8-fluoroquinazoline-2,4(1H,3H)-dione (0.349 g, 1.00 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl2,2′-bi(1,3,2-dioxaborolane) (0.305 g, 1.20 mmol), 1,1′-bis(diphenylphosphino)ferrocenepalladium(II) dichloride−dichloromethane adduct (0.041 g, 0.050 mmol), and potassium acetate (0.245 g, 2.50 mmol) in dioxane (20 mL) and dimethyl sulfoxide (4 mL) was bubbled with nitrogen for 5 min, then heated at 90 °C overnight. The cooled mixture was partitioned between ethyl acetate and water. The organic phase was washed sequentially with a saturated aqueous solution of sodium bicarbonate, washed with water, washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with a mixture of ethyl acetate and hexanes (20:80) to give 32 (0.326 g, 82% yield) as a white solid. LCMS (ESI) m/z calcd for C21H22BFN2O4 [M + H]+ 397.2. Found: 397.2. 1H NMR (500 MHz, DMSO-d6) δ 11.78 (s, 1H), 7.80 (d, J = 7.2 Hz, 1H), 7.72 (dd, J = 7.4, 1.5 Hz, 1H), 7.71−7.56 (m, 1H), 7.45−7.35 (m, 1H), 7.35−7.29 (m, 1H), 7.29−7.16 (m, 1H), 2.22 (s, 3H), and 1.33 (s, 12H). 8-Fluoro-3-(R)-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) phenyl)quinazoline-2,4(1H,3H)-dione (34) and 8-Fluoro-3(S)-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)quinazoline-2,4(1H,3H)-dione (33). A sample of 8-fluoro-3(R,S)-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-

phenyl)quinazoline-2,4(1H,3H)-dione (32) was separated by chiral SFC to give two single, stable atropisomers as follows: column, Chiralcel OD-H (5 × 25 cm, 5 μm); mobile phase, CO2-MeOH (70:30) at 300 mL/min, 100 bar, 40 °C; sample preparation, 103 mg/ mL in DCM−MeOH (44:56); injection, 5.0 mL. The first peak eluting from the column provided the (R)-atropisomer, 8-fluoro-3-(R)-(2methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)quinazoline-2,4(1H,3H)-dione (34), as a white solid. The second peak eluting from the column provided the (S)-enantiomer 34 as a white solid. The mass spectrum and 1H NMR for each enantiomeric atropisomer were the same as those for intermediate 32. Human Recombinant BTK Enzyme Assay. To V-bottom 384well plates were added test compounds, human recombinant BTK (1 nM, Invitrogen Corporation), fluoresceinated peptide (1.5 μM), ATP (20 μM), and assay buffer (20 mM HEPES at pH 7.4, 10 mM MgCl2, 0.015% Brij 35 surfactant, and 4 mM DTT in 1.6% DMSO), with a final volume of 30 μL. After incubating at room temperature for 60 min, the reaction was terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LabChip 3000 (Caliper, Hopkinton, MA) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to control reactions with no enzyme (for 100% inhibition) and controls with no inhibitor (for 0% inhibition). Dose−response curves were generated to determine the concentration required for inhibiting 50% of BTK activity (IC50). Compounds were dissolved at 10 mM in DMSO and evaluated at 11 concentrations. BCR-Stimulated Calcium Flux in Ramos B Cells. Human Ramos (RA1) B cells (ATCC CRL-1596) at a density of 2 × 106 cells/mL in RPMI minus phenol red (Invitrogen 11835-030) and 50 mM HEPES (Invitrogen 15630-130) containing 0.1% BSA (Sigma A8577) were added to one-half volume of calcium loading buffer (BD bulk kit for probenecid sensitive assays, # 640177) and incubated at room temperature in the dark for 1 h. Dye-loaded cells were pelleted (Beckmann GS-CKR, 1200 rpm, room temperature, 5 min) and resuspended at room temperature in RPMI minus phenol red with 50 mM HEPES and 10% FBS to a density of 1 × 106 cells/mL. 150 μL aliquots (150,000 cells/well) were plated into 96 well poly-D-lysine coated assay plates (BD 35 4640) and briefly centrifuged (Beckmann GS-CKR 800 rpm, 5 min, without brake). Next, 50 μL compound dilutions in 0.4% DMSO/RPMI minus phenol red +50 mM HEPES + 10% FBS were added to the wells, and the plate was incubated at room temperature in the dark for 1 h. The assay plate was briefly centrifuged as above prior to measuring calcium levels. Using the FLIPR1 (Molecular Devices), cells were stimulated by adding goat antihuman IgM (Invitrogen AHI0601) to 2.5 μg/mL. Changes in intracellular calcium concentrations were measured for 180 s, and percent inhibition was determined relative to peak calcium levels seen in the presence of stimulation only. Whole Blood Assays of BCR-Stimulated CD69 Expression on B Cells. To measure BCR-stimulated B cells, heparanized human whole blood was added with various concentrations of test compound and stimulated with 30 μg/mL AffiniPure F(ab′)2 fragment goat anti human IgM (Jackson 109-006-1299-endotoxin cleared) and 10 ng/mL human IL-4 (Peprotech 200-04) for 18 h at 37 °C with agitation. The cells were stained with FITC-conjugated mouse antihuman CD20 (BD Pharmingen 555622) and PE-conjugated mouse antihuman CD69 monoclonal antibody (BD Pharmingen 555531), lysed and fixed, then washed. The amount of CD69 expression was quantitated by the mean fluorescence intensity (MFI) after gating on the CD20-positive B cell population as measured by FACS analysis. B cells in mouse whole blood were stimulated in an similar way, using AffinPure F(ab′)2 Fragment goat anti mouse IgG + IgM (Jackson Cat#115-006-068) at 100 μg/mL to stimulate and staining with allophycocyanin (APC) rat antimouse CD19 antibody (BD Biosciences 550992) to identify the B cells and CD69 quantitation with FITC-conjugated antimouse CD69 monoclonal antibody (BD Biosciences 553236). JAK2 Tyrosine Kinase Assay. The assays were performed in Vbottomed 384-well plates. The final assay volume was 30 μL prepared from 15 μL additions of enzyme and substrates (fluoresceinated 9197



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the mean score of treatment groups with P < 0.0001. One-sided Dunnett’s test was used to determine significance of inhibition of each treatment group compared to the vehicle control group. Collagen Antibody-Induced Arthritis in Mice. Female BALB/c mice (8−10 weeks of age; Harlan) were injected IP with a mixture of four monoclonal antimouse type II collagen antibodies (1 mg of each). Daily oral dosing was immediately started with vehicle (EtOH/TPGS/ PEG300; 5:5:90), compound 6 (10 or 30 mg/kg) or dexamethasone (1 mg/kg). Three days later, the mice were injected IP with 1.25 mg/ kg LPS (E. coli O111:B4; Sigma). Thereafter, mice were monitored 3×/week for the development and severity of paw inflammation. Each paw was visually scored by the following scheme: +0 = normal; +1 = one (or more) joints inflamed on digits; +2 = mild-moderate inflammation of plantar surface of paw and paw thickness modestly increased; +3 = moderate-severe inflammation of plantar surface of paw and paw thickness significantly increased; +4 = ankylosis of ankle joint (significantly reduced hock joint motion on flexion/extension). Clinical paw scores for all four paws were summed for each mouse, and mean ± SEM was calculated for each treatment group. Single Crystal X-ray Diffraction Data Collection. Single crystal X-ray diffraction data were collected on a Bruker-AXS APEX2 CCD system using Cu Kα radiation (λ = 1.5418 Å). Indexing and processing of the measured intensity data were carried out with the APEX2 software package/program suite.21 When indicated, crystals were cooled in the cold stream of an Oxford Cryosystem22 during data collection. The structures were solved by direct methods and refined on the basis of observed reflections using the crystallographic package SHELXTL.21 The derived atomic parameters (coordinates and temperature factors) were refined through full matrix least-squares. The function minimized in the refinements was Σw(|Fo| − |Fc|).22 R is defined as Σ∥Fo| − |Fc∥/Σ|Fo|, while Rw = [Σw(|Fo| − |Fc|)2/Σw|Fo|2]1/2 where w is an appropriate weighting function based on errors in the observed intensities. Difference maps were examined at all stages of refinement. Hydrogens were introduced in idealized positions with isotropic temperature factors, but no hydrogen parameters were varied. X-ray Crystallography of 14f Bound to the Kinase Domain of BTK. A baculovirus construct of His-TEV-hBTK (E396-S659) was used to generate protein for X-ray crystallography as previously reported.14 For protein/compound complex formation, 2 μL of compound 14f (50 mM DMSO stock) was added to 1.3 mL of hBTK at 0.38 mg/mL and incubated at room temperature for 3 h and then concentrated to 9.1 mg/mL prior to set up drops. Crystals were grown at room temperature using the hanging drop vapor diffusion method. The drop consisted of 1.5 μL protein solution and 1 μL reservoir solution containing 3% (w/v) methyl ether PEG 5000, 25% (w/v) PEG 8000 and 0.2 M Tris-HCl, pH 7.0. Macroseeding was performed to initiate crystal growth. The crystals appeared within a few days and continued to grow for 2−3 weeks. Crystals were flash-cooled in liquid nitrogen for data collection with 25% glycerol and 75% reservoir solution as cryoprotectant. Diffraction data were collected by Shamrock Structures, Inc. at Canadian Light Source, beamline 08ID. hBTK/compound 14f cocrystals belonged to the space group p212121: a = 46.6 Å, b = 69.8 Å, c = 89.0 Å, α = β = γ = 90.0°. The 1.50 Å resolution structure was determined by molecular replacement using a previously determined in-house BTK structure (unpublished results). The structure of hBTK + compound 14f has been deposited to RCSB with PDB ID: 5T18.

peptide and ATP) and test compounds in assay buffer (100 mM HEPES at pH 7.4, 10 mM MgCl2, 25 mM beta-glycerolphosphate, 0.015% Brij 35 surfactant, and 4 mM DTT). The reaction was initiated by the combination of Jak2 tyrosine kinase with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LabChip 3000 by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicleonly reactions for 0% inhibition. The final concentration of reagents in the assays is ATP, 30 μM; Jak2 fluorescent peptide, 1.5 μM; Jak2, 1 nM; and DMSO, 1.6%. Dose−response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in DMSO and evaluated at 11 concentrations, each in duplicate. IC50 values were derived by nonlinear regression analysis. NZB/W Lupus-Prone Mice. Baseline body weight, proteinuria, and serum dsDNA titers were determined for female NZB/WF1 mice (Jackson Laboratories) at 24 weeks of age prior to their randomization into treatment groups, each with n = 12. Mice were dosed by oral gavage, QD, for 16 weeks and included the following treatment groups: 14f at 30 mg/kg in vehicle (80:20 PEG400/water) and prednisolone (Sigma P6004) at 10 mg/kg. Mice were routinely monitored for overall health, and body weight, proteinuria, and dsDNA titers were measured every 3 weeks. Proteinuria was measured using a colorimetric assay for albumin (Siemens Albustix Reagent Strips for Urinalysis). Anti-dsDNA antibody titers were measured by applying serial dilutions of serum samples to DNA-coated ELISA plates. Bound antibody was detected with HRP-conjugated polyclonal goat-antimouse IgG. The individual titers were expressed as relative to that of a pooled serum standard. At the end of the study, final body weight and proteinuria were measured. Terminal blood samples were drawn for PK and anti-dsDNA titers. The amount of BTK inactivation in blood from these mice, 24 h after their last dose, was determined as described above. At the end of the study, kidneys (n = 5−6 per group) were collected in 10% Neutral Buffered Formalin for histological evaluation. Fixed kidney tissues were routinely processed and paraffin embedded. Kidney sections were stained with periodic acid Schiff and hematoxylin (PASH) and hematoxylin and eosin (H&E) for the evaluation of nephritis severity. Blinded to treatment group, severity of nephritis was evaluated using the following criteria. For glomerular damage: 1, mesangial matrix thickening and/or mesangial cell proliferation; 2, crescent formation, cellular deposits/casts in Bowman’s space; 3, cellular infiltration, composed of mononuclear cells in glomerular tufts; and 4, fibrosis of Bowman’s capsule. For tubular damage: 1, infiltration of mononuclear cells; 2, severity of tubular epithelial cell damage; and 3, protein casts. For tubule-interstitial damage: 1, fibrosis; and 2, infiltration of mononuclear cells. Each subcategory was assigned a score from 0 to 4. The total score for each mouse was the sum of the above nine subcategories with the highest possible score of 36. To evaluate IC deposition in the glomeruli, immunohistochemistry was performed using biotinylated goat antimouse IgG, (Vector Laboratories, Burlingame, CA) with 45 μg/ mL normal goat serum (Jackson Immunoresearch Laboratories, Inc.) as the negative control. Sections were conjugated to horse radish peroxidase using Vectastain ABC ELITE reagent (Vector Laboratories) and developed with Deep Space Black chromogen (Biocare Medical) for visualization of IgG. IC deposition was evaluated under 100× magnification by dividing the kidney into three sections and randomly selecting 10 glomeruli from each section. Each glomerulus was subdivided into four quadrants, and each quadrant was assigned a score from 0 to 4 based on the severity of IgG deposition, defined as 0, unremarkable; 1, minimal linear deposition and/or focal lesion; 2, mild linear deposition or multifocal lesions; 3, moderate/segmental lesions; 4, marked/global or diffuse deposition. The total score of each glomerulus was calculated, as was the total score of each kidney, and the group average score was determined. Statistical analysis was performed using a one-way ANOVA model after a square root transformation of the data to establish significant overall difference in

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01088. Single crystal X-ray experimental details for 7a, 9a, 14b, 14d, and 14f (PDF) PDB coordinates for the computational models of 9a (PDB) 9198



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PDB coordinates for the computational models of 9b (PDB) Accession Codes

PDB ID code for 14f: 5T18.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 609-252-6778. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED BTK, Bruton’s tyrosine kinase; NFAT, nuclear factor of activated T cells; NF-κB, nuclear factor kappa-light chain enhancer of activated B cells; PK, pharmacokinetic; SAR, structure−activity relationship; SFC, super critical fluid chromatography; hERG, human ether-a-go-go-related gene

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REFERENCES

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