Discovery and Optimization of Quinolinone Derivatives as Potent

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Discovery and Optimization of Quinolinone Derivatives as Potent, Selective, and Orally Bioavailable Mutant Isocitrate Dehydrogenase 1 (mIDH1) Inhibitors Jian Lin,*,† Wei Lu,‡ Justin A. Caravella,∥ Ann Marie Campbell, R. Bruce Diebold, Anna Ericsson, Edward Fritzen, Gary R. Gustafson, David R. Lancia, Jr., Tatiana Shelekhin, Zhongguo Wang, Jennifer Castro, Andrea Clarke, Deepali Gotur, Helen R. Josephine, Marie Katz, Hien Diep, Mark Kershaw, Lili Yao, Goss Kauffman, Stephen E. Hubbs, George P. Luke, Angela V. Toms, Liann Wang, Kenneth W. Bair, Kenneth J. Barr, Christopher Dinsmore,* Duncan Walker, and Susan Ashwell§ Forma Therapeutics, Inc., 500 Arsenal Street, Suite 100, Watertown, Massachusetts 02472, United States S Supporting Information *

ABSTRACT: Mutations at the arginine residue (R132) in isocitrate dehydrogenase 1 (IDH1) are frequently identified in various human cancers. Inhibition of mutant IDH1 (mIDH1) with small molecules has been clinically validated as a promising therapeutic treatment for acute myeloid leukemia and multiple solid tumors. Herein, we report the discovery and optimization of a series of quinolinones to provide potent and orally bioavailable mIDH1 inhibitors with selectivity over wild-type IDH1. The X-ray structure of an early lead 24 in complex with mIDH1-R132H shows that the inhibitor unexpectedly binds to an allosteric site. Efforts to improve the in vitro and in vivo absorption, distribution, metabolism, and excretion (ADME) properties of 24 yielded a preclinical candidate 63. The detailed preclinical ADME and pharmacology studies of 63 support further development of quinolinone-based mIDH1 inhibitors as therapeutic agents in human trials.

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INTRODUCTION The human cytoplasmic isocitrate dehydrogenases (IDH) are a class of nicotinamide adenine dinucleotide phosphate (NADP+)-dependent enzymes that catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG), which is one of the key reactions in the Krebs (citric acid) cycle.1,2 There are three isoforms in this family. IDH1 resides in the cytoplasm, whereas IDH2 and IDH3 localize in the mitochondria. Heterozygous mutations of IDH1 at Arg132, a residue that is critical for the substrate recognition, have been frequently identified in multiple cancer types,2−4 including grade II−III gliomas and secondary glioblastomas (GBMs) (where they occur at a rate of 70−90%),5,6 acute myeloid leukemia (AML, 10−17%),7,8 intrahepatic cholangiocarcinoma (7−20%),9 and central and periosteal chondrosarcomas (46−52%).10,11 The most commonly observed IDH1 mutations in these cancer types include R132H, R132C, R132G, R132S, and R132L, whereas IDH2 mutations R140Q or R172K have been identified in AML © XXXX American Chemical Society

and less frequently in solid tumors. These mutant enzymes lose their wild-type enzyme activity, acquiring a new function to reduce α-ketoglutarate (α-KG) to D-2-hydroxyglutarate (2HG).7,12 As a result, human cancer cells harboring mutant IDH1 (mIDH1) show aberrantly elevated 2-HG levels.7 2-HG has been described as an “oncometabolite” since it inhibits the class of α-KG-dependent enzymes involved in epigenetic regulation, cell signaling, and collagen synthesis. In particular, 2-HG impairs DNA demethylation through inhibition of ten-eleven translocation-2 (TET2), which in turn results in the hypermethylation of DNA CpG islands and impairs hematopoietic cell differentiation.13 2-HG also impairs histone demethylation through inhibition of a number of lysine demethylases.14,15 Two mutant IDH1 (mIDH1) inhibitors, AGI-519816 and GSK321,17 have demonstrated the reduction of intratumoral 2-HG levels Received: March 7, 2019 Published: June 14, 2019 A

DOI: 10.1021/acs.jmedchem.9b00362 J. Med. Chem. XXXX, XXX, XXX−XXX

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Chart 1. Structures of the Known Mutant IDH1 Inhibitors

directly to the active site of the protein. Since the active site of IDH1 is highly polar and bound to co-factor nicotinamide adenine dinucleotide phosphate hydrogen (NADPH), it is challenging to develop a potent substrate-like (α-KG-like) inhibitor with favorable druglike properties and good blood− brain barrier (BBB) permeability. Class-II inhibitors occupy an allosteric site, reducing the enzymatic activity of IDH1 through a

and result in tumor regression in mIDH1-containing tumor xenograft mouse models. These studies suggest that mIDH1 inhibitors can be used as therapeutic agents to induce the differentiation of proliferating cancer cells. Three classes of mIDH1 inhibitors based on their distinct binding sites have been reported (Chart 1).16−31 Class-I inhibitors, such as SYC-43520,21 and thiohydantoin-16,22 bind B

DOI: 10.1021/acs.jmedchem.9b00362 J. Med. Chem. XXXX, XXX, XXX−XXX

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Figure 1. Structure and biochemical activity of HTS hit 1 and potential opportunities for SAR exploration.

Table 1. Structure−Activity Relationship of Substituents R1, R2, and R3a

cpd ID

R1

R2

R3

IDH1-R132H, IC50 (μM)

HLM, left % 30 min

1 2 3 4 5 6

Me Me H CI H CI

Me H Me H H H

H H H H H Me

0.704 0.238 0.303 0.267 3.32 >25

17.6 30.8 9.6 93.9 88.0 63.7

MLM, left % 30 min 27.9 42.3 17.8 89.9 69.4 22.0

solubility (μM) 0.04 50% inhibitionc 3.2

a

The studies were conducted in Pharmaron Inc. bThe study was conducted by Cerep. cDetails is shown in the Supporting information. PatchClamp data was obtained from ChanTest.

d

N

DOI: 10.1021/acs.jmedchem.9b00362 J. Med. Chem. XXXX, XXX, XXX−XXX

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Scheme 1. Synthesis of Quinolinone Derivatives 2−41a

Reagents and conditions: (a) NaBH(OAc)3, AcOH, 1,2-dichloroethane (DCE), room temperature (rt) to 60 °C.

a

Scheme 2. Synthesis of Compound 44a

a Reagents and conditions: (a) DMF, POCl3, 80 °C, overnight; (b) 12 M HCl, reflux, 24 h; (c) HBr, 115 °C, 4 days; (d) 4-amino-2methoxybenzonitrile, NaBH(OAc)3, AcOH, rt, overnight; (e) pyridin-2-yl methanol, diethyl azodicarboxylate (DEAD), PPh3, tetrahydrofuran (THF), rt to 50 °C, overnight.

Scheme 3. Library Synthesis of Compounds 46−52a

a

Reagents and conditions: (a) ROH, DEAD, PPh3, THF; (b) RBr, K2CO3, CH3CN, reflux, 24 h.

compounds 45−52 were prepared via the Mitsunobu reaction or alkylation of 44e or 45g with an appropriate alcohol or alkyl halide in either singleton or library fashion. Intermediate 45g, the 8-hydroxy quinolinone analog, was prepared by a different route as described in the Supporting Information. We have developed an effective synthetic route to prepare (S)-3-(1-aminoethyl)-6-chloroquinolin-2(1H)-one hydrochloride 71 with high optical purity (Scheme 4).32−34 In this route, Ellman’s chiral sulfinamide ((R)-2-methylpropane-2-sulfinamide) was used to stereoselectively introduce the benzylic methyl group.36,37 After condensation of Ellman’s sulfinamide

As described in Scheme 2, the synthesis of compound 44 began with the cyclization of N-(4-chloro-3-methoxyphenyl)acetamide (44a) with the Vilsmeier−Haack reagent35 (generated in situ from dimethylformamide (DMF)/POCl3) at 80 °C. The resulting 2-chloro-3-formyl quinoline (44b) was hydrolyzed to yield the 2-quinolone (44c). Cleavage of the methyl ether moiety with HBr at 115 °C gave the free phenol (44d). Reductive amination of 44d with 4-amino-2-methoxybenzonitrile and NaBH(OAc)3 afforded intermediate (44e). Mitsunobu reaction of 44e with pyridin-2-yl methanol then provided the final product 44. As shown in Scheme 3, O

DOI: 10.1021/acs.jmedchem.9b00362 J. Med. Chem. XXXX, XXX, XXX−XXX

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Scheme 4. Synthesis of Intermediate 71a

Reagents and conditions: (a) (R)-2-methylpropane-2-sulfinamide, CuSO4, 55 °C, DCE, overnight, 81%; (b) MeMgBr, dichloromethane (DCM), −50 to −60 °C, 3 h, 63%; (c) 1 N HCl, dioxane, reflux overnight, >98%. a

Scheme 5. Synthesis of Compounds 56 and 59−64a

Reagents and conditions: (a) N,N-diisopropyl ethylamine (DIEA), DMSO, 110 °C; (b) DIEA, EtOH, microwave, 140−150 °C; (c) Pd2(dba)3, dppf, Zn(CN)2, DMF, 120 °C, overnight.

a

Scheme 6. Synthesis of Compounds 57 and 58a

Reagents and conditions: (a) ethyl 2-cyanoacetate, piperidine, EtOH, rt, 30 min, reflux, 2 h; (b) Ti(OiPr)4, MeMgBr, −78 °C to rt; (c) Ti(OiPr)4, EtMgBr, BF3·OEt2, −78 °C to rt; (d) 6-fluoro-2-methylnicotinonitrile, DIEA, DMSO, 130 °C.

a

The syntheses of compounds 57 and 58 required the preparation of an intermediate bearing a 3-nitrile functional group on the quinolinone core (Scheme 6). Friedlander condensation of 2-amino-5-chlorobenzaldehyde with ethyl 2cyanoacetate in refluxing ethanol provided 6-chloro-2-oxo-1,2dihydroquinoline-3-carbonitrile (72) in good yield. Compound 57 was then prepared by the conversion of nitrile 72 to the α,αdimethyl-substituted carbinamine 73 with methylmagnesium bromide in the presence Ti(OiPr)4,38 followed by nucleophilic displacement of fluoride from 6-fluoro-2-methylnicotinonitrile. Compound 58 required the preparation of a cylclopropanated intermediate, which was prepared using the procedure developed by Bertus et al.39 The treatment of the quinolinone nitrile 72 with 1.1 equiv of Ti(OiPr)4 and 2.2 equiv of EtMgBr

with aldehyde 71a, the resulting imine 71b was treated with methylmagnesium bromide to provide the sulfinamide 71c. The pure (R,S)-diastereomer was easily isolated by column chromatography in high yield. Cleavage of the chiral auxiliary and simultaneous hydrolysis of the 2-chloroquinoline moiety under mildly acidic conditions (1 N HCl in dioxane) gave the amine 71 in quantitative yield as the hydrochloride salt. The same methodology was employed to prepare the opposite enantiomer, (R)-3-(1-aminoethyl)-6-chloroquinolin-2(1H)one hydrochloride. With enantiomerically pure (S)-3-(1-aminoethyl)-6-chloroquinolin-2(1H)-one 71 in hand, we were able to prepare analogs 56 and 59−64 via nucleophilic substitution with the appropriate aryl halides at elevated temperatures (Scheme 5). P

DOI: 10.1021/acs.jmedchem.9b00362 J. Med. Chem. XXXX, XXX, XXX−XXX

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Scheme 7. General Synthetic Route for Compound 67 and Analogsa

a Reagents and conditions: (a) R′OH, DEAD, PPh3, THF; (b) R′Br, K2CO3, CH3CN, reflux, 24 h; (c) Ac2O, DIEA, ethyl acetate (EtOAc); (d) DMF, POCl3, 80 °C, overnight; (e) NaOMe, MeOH, THF; (f) MeMgBr, DCM, −78 °C to rt; (g) Dess−Martin periodinane, DCM; (h) (R)-2methylpropane-2-sulfinamide, Ti(OiPr)4, THF; (i) L-selectride, THF; (j) 1 N HCl, dioxane; (k) 2-chloro-4-methoxypyrimidine-5-carbonitrile, DIEA, DMSO, 110 °C.

followed by the addition of a Lewis acid (BF3·OEt2) provided the desired cylcopropanated intermediate 74. Nucleophilic substitution then afforded the final compound 58. The syntheses of the 7-substituted quinolinones 65−70 (Scheme 7) began with the acetylation of the requisite anilines to provide acetanilides (65a−70a), which were then cyclized with in situ-generated Vilsmeier−Haack reagent to afford the various 7-substituted quinolinone intermediates (65b−70b). Subsequent transformations (steps e−k) were achieved using procedures and conditions detailed in our previous publications to obtain the final compounds.34−36

(ESI)). High-performance liquid chromatograph (HPLC) analyses were obtained using a XBridge Phenyl or C18 column (5 μm, 50 × 4.6 mm2, 150 × 4.6 mm2 or 250 × 4.6 mm2) with UV detection (Waters 996 PDA) at 254 nm or 223 nm using a standard solvent gradient program (methods 1−3 shown in the Supporting Information). Racemic mixtures of final compounds were separated into individual enantiomers by chiral supercritical fluid chromatography (SFC) under the indicated conditions. Chemical and chiral (where applicable) purities were >95% for all final compounds, as assessed by LC−MS and chiral SFC analysis, respectively. Further details on the analytical conditions used for individual compounds may be found in the Supporting Information. High-resolution mass spectrometry (HRMS) data were collected on a Waters Time of Flight (Waters Acquity I Class UPLC with Xevo G2-XS Q Tof HRMS and PDM-UPLC-HRMS-1) instrument using electrospray ionization. Materials. HCT116-IDH1-R132H/+ and HCT116-IDH1R132C/+ were licensed from Horizon (HD-104-021 and HD-104013). HT1080 and U87MG cells were commercially available from ATCC. Various human IDH1-R132 mutant cDNA ORF clones were purchased from Origene (R132H: RC400096; R132C: RC400097, R132L: RC400098; R132G: RC400099; R132S: RC400100). IDH1 mutant expressing U87MG cells were generated by transfecting U87MG parent cells with mutant IDH1-R132 mutant cDNA and selecting under G418. Synthesis and Characterization of Compounds 2−70. Details of compound synthesis and characterization can be either found below or in the Experimental Section, Supporting Information. 4-(((6-Chloro-2-oxo-1,2-dihydroquinolin-3-yl)methyl)amino)-2methoxybenzonitrile (24). To a 100 mL round-bottom flask were added 6-chloro-2-oxo-1,2-dihydroquinoline-3-carbaldehyde (200 mg, 0.963 mmol), 4-amino-2-methoxybenzonitrile (150 mg, 1.01 mmol), and AcOH (0.276 mL, 4.82 mmol) in DCE (15 mL). Finally, sodium triacetoxyborohydride (364 mg, 1.93 mmol) was added, and the mixture was stirred at room temperature overnight. LC−MS indicated only about 50% conversion. The reaction mixture was diluted with EtOAc (60 mL) and washed with water (2×) and brine. The organic phase was dried over Na2SO4, filtered, and concentrated to yield a crude. The crude was dissolved in 3 mL of DMSO and purified by preparative HPLC to yield the desired product 24 (34 mg, 10.4% yield). 1 H NMR (300 MHz, CDCl3) δ ppm 11.11 (br s, 1H), 7.58 (s, 1H), 7.49−7.43 (m, 1H), 7.42−7.33 (m, 1H), 7.27−7.19 (m, 2H), 6.14 (dd, J = 8.50, 2.05 Hz, 1H), 6.06 (d, J = 1.76 Hz, 1H), 4.37 (s, 2H), 3.80− 3.72 (m, 3H). LC−MS (ESI) m/z calcd for C18H15ClN3O2 [M + H]+, 340.09; found, 340.00, Rt = 2.34 min (method 1), >99% purity. 6-(((6-Chloro-2-oxo-1,2-dihydroquinolin-3-yl)methyl)amino)-2methylnicotinonitrile (32). A suspension of 6-chloro-2-oxo-1,2-

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CONCLUSIONS In summary, a novel quinolinone HTS hit 1 was identified and optimized via singleton and combinatorial library syntheses to generate early leads 24 and 32. X-ray structures unexpectedly revealed that both 24 and 32 bind in an allosteric, induced-fit pocket in the mIDH1-R132H protein. Structure-based rational design guided the optimization of potency and druglike properties, leading to the identification of compound 63 which proved suitable for exploring the effect of inhibition of the production of 2-HG by IDH1-R132H/IDH1-R132C in preclinical in vivo PK/PD xenograft models. Compound 63 has good overall selectivity vs the wild-type IDH protein and potently inhibits IDH1 mutants R132H, R132C, R132G, and R132L, suggesting broad utility across the various known R132 mutations. Compound 63 demonstrated excellent cell permeability, oral bioavailability, and ADME/PK properties. Oral dosing of 63 in the mouse mIDH1 tumor xenograft model shows a robust reduction of the tumor-derived 2-HG level, which is a PD biomarker of mIDH1 activity. The preclinical profile of compound 63 suggests it may have potential as a treatment of AML, GBM, or other forms of mIDH1-driven cancer.

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EXPERIMENTAL SECTION

General. Unless otherwise noted, reagents and solvents were used as received from commercial suppliers. Proton nuclear magnetic resonance (NMR) spectra were obtained on either the Bruker or Varian spectrometer at 300 MHz. Spectra are given in ppm (δ), and coupling constant, J, is reported in hertz. Tetramethylsilane was used as an internal standard. Mass spectra were collected using a Waters ZQ Single Quad Mass Spectrometer (ion trap electrospray ionization Q

DOI: 10.1021/acs.jmedchem.9b00362 J. Med. Chem. XXXX, XXX, XXX−XXX

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The material was chromatographed by Biotage MPLC (10 g of silica gel column, 0−50% EtOAc in hexanes) to provide the title compound 56 as a solid (51.5 mg, 0.145 mmol, 76% yield, HPLC purity >95% at 220 nm). 1H NMR (300 MHz, DMSO-d6): δ ppm 11.99 (s, 1H), 7.91 (d, J = 7.30 Hz, 1H), 7.72−7.80 (m, 2H), 7.62 (d, J = 8.80 Hz, 1H), 7.45− 7.53 (m, 1H), 7.30 (d, J = 8.79 Hz, 1H), 6.35−6.55 (m, 1H), 5.12−5.34 (m, 1H), 2.36 (s, 3H), 1.42 (d, J = 6.70 Hz, 3H). LC−MS (ESI) m/z: [M + H]+, 339.00; Rt = 2.40 min (method 1), >99% purity. HRMS (ESI) calcd for C18H16ClN4O [M + H]+, 339.1013; found 339.1011. (S)-4-((1-(6-Chloro-2-oxo-1,2-dihydroquinolin-3-yl)ethyl)amino)2-methoxybenzonitrile (59). A solution of (S)-3-(1-aminoethyl)-6chloroquinolin-2(1H)-one hydrochloride 71 (201 mg, 0.776 mmol) and 4-fluoro-2-methoxybenzonitrile (236 mg, 1.56 mmol) in DMSO (5 mL) was treated with DIEA (400 μL, 2.29 mmol) and stirred at 110 °C for 3 days. The sample was diluted with water (75 mL) and extracted with DCM (2 × 50 mL), dried, and filtered. Silica gel was added, and the solvent was evaporated under reduced pressure. The material was chromatographed by Biotage MPLC (silica gel, 0−70% EtOAc in hexanes, with isocratic elution when peaks came off) to provide a gum. The material was dissolved in DCM (10 mL), washed with water (2 × 10 mL), dried (Na2SO4), filtered, and evaporated to provide 76 mg of yellow powder. The sample was mixed with MeCN (4 mL) and water (2 mL), frozen on a dry ice/acetone bath, and lyophilized to give the title compound 59 as a solid (71.1 mg, 0.193 mmol, 24.93% yield, HPLC purity 96.3% at 220 nm). 1H NMR (300 MHz, DMSO-d6): δ ppm 12.07 (s, 1H), 7.77 (d, J = 2.35 Hz, 1H), 7.74 (s, 1H), 7.50 (dd, J = 8.65, 1.91 Hz, 1H), 7.35−7.20 (m, 3H), 6.27 (s, 1H), 6.06 (d, J = 7.90 Hz, 1H), 4.79−4.65 (m, 1H), 3.75 (s, 3H), 1.43 (d, J = 6.45 Hz, 3H). LC−MS (ESI) m/z: [M + H]+, 354.00; Rt = 2.37 min (method 1), 96% purity. HRMS (ESI) calcd for C19H17ClN3O2 [M + H]+, 354.1009; found 354.1007. (S)-6-((1-(6-Chloro-2-oxo-1,2-dihydroquinolin-3-yl)ethyl)amino)4-methoxynicotinonitrile (60). In an 80 mL microwave vessel were combined 6-chloro-4-methoxynicotinonitrile (1 g, 60 mmol), (S)-3-(1aminoethyl)-6-chloroquinolin-2(1H)-one hydrochloride 71 (1.34 g, 53 mmol), and DIEA (1.98 mL, 11.4 mmol) in 21 mL of EtOH (200 proof). The reaction mixture was microwaved at 140 °C for 4.5 h, cooled to room temperature, and concentrated to dryness under reduced pressure. The material was purified twice by ISCO using a 40 g of “gold” column with a gradient elution of EtOAc in DCM to provide the title compound 60 (478 mg, 24% yield). 1H NMR (300 MHz, DMSO-d6): δ ppm 11.99 (br s 1H), 8.16 (s, 1H), 7.90 (d, J = 7.41 Hz, 1H), 7.75 (d, J = 2.46 Hz, 1H), 7.72 (s, 1H), 7.48 (dd, J1 = 8.52 Hz, J2 = 2.46 Hz, 1H), 7.29 (d, J = 8.52 Hz, 1H), 6.25 (br s, 1H), 5.22 (br s, 1H), 3.85 (s, 3H), 1.41 (d, J = 6.6 Hz, 3H). LC−MS (ESI) m/z: [M + H]+, 355.10; Rt = 4.38 min (method 2), >99% purity. HRMS (ESI) calcd for C18H16ClN4O2 [M + H]+, 355.0962; found 355.0957. mp: 248−249 °C. (S)-6-((1-(6-Chloro-2-oxo-1,2-dihydroquinolin-3-yl)ethyl)amino)2-methoxynicotinonitrile (61). A solution of (S)-3-(1-aminoethyl)-6chloroquinolin-2(1H)-one hydrochloride 71 (69.7 mg, 0.269 mmol) and 6-fluoro-2-methoxynicotinonitrile (45.2 mg, 0.297 mmol) in DMSO (1.5 mL) was treated with DIEA (141 μL, 0.807 mmol) and stirred at 110 °C for 1 h. LC−MS at 45 min showed that the reaction had gone to completion. The sample was pipetted onto water (20 mL), resulting in the formation of a white precipitate. The precipitate was extracted with EtOAc (2 × 15 mL), dried (Na2SO4), and filtered. Silica gel was added, and the solvent was evaporated under reduced pressure. The material was chromatographed by Biotage MPLC (10 g of silica gel column) with 0−75% EtOAc in hexanes, with isocratic elution when peaks came off to provide the title compound 61 as a white solid (68.8 mg, 0.194 mmol, 72.1% yield, HPLC purity 100% at 220 nm). 1H NMR (300 MHz, DMSO-d6) δ ppm 11.97 (br s, 1H), 8.13 (br s, 1H), 7.77 (d, J = 2.35 Hz, 1H), 7.73 (s, 1H), 7.60 (d, J = 8.50 Hz, 1H), 7.48 (dd, J = 8.79, 2.35 Hz, 1H), 7.29 (d, J = 9.09 Hz, 1H), 6.26 (br s, 1H), 5.20 (br s, 1H), 3.72 (br s, 3H), 1.44 (d, J = 7.04 Hz, 3H). LC−MS (ESI) m/z calcd for C18H16ClN4O2 [M + H]+, 355.10; found, 355.00; Rt = 2.38 min (method 1), >99% purity. (S)-5-((1-(6-Chloro-2-oxo-1,2-dihydroquinolin-3-yl)ethyl)amino)6-methoxypicolinonitrile (62). To a solution of 5-fluoro-6-methox-

dihydroquinoline-3-carbaldehyde (150 mg, 0.722 mmol) and 6-amino2-methylnicotinonitrile (115 mg, 0.867 mmol) in DCE (20 mL) was treated with AcOH (0.124 mL, 2.167 mmol) and stirred for 20 min. Sodium triacetoxyborohydride (459 mg, 2.167 mmol) was added. The mixture was placed under nitrogen and stirred at room temperature. After 30 min, the suspension went into the solution. The brown solution was stirred at ambient temperature over the weekend, during which time a material precipitated. The mixture was diluted with EtOAc (50 mL), washed with water (2 × 50 mL) and brine (50 mL), dried (Na2SO4), filtered, and evaporated. The residue (∼0.13 g) was dissolved in methanol, treated with silica gel, and evaporated. The crude material was chromatographed by Biotage MPLC (25 g silica gel column) with 0−10% MeOH/DCM to yield the title compound 32 (40 mg, 17%). 1H NMR (300 MHz, DMSO-d6): δ 12.02 (br, 1H), 7.72− 7.79 (m, 3H), 7.47 (dd, J1 = 2.34 Hz, J2 = 8.79 Hz, 1H), 7.29 (d, J = 8.79 Hz, 1H), 6.75 (d, J = 8.8 Hz, 1H), 4.31 (sd, J = 5.57 Hz, 1H), 4.11 (s, 1H), 3.14 (d, J = 5.27 Hz, 1H), 2.36 (s, 3H). LC−MS (ESI) m/z calcd for C17H14ClN4O [M + H]+, 325.09; found, 325.00, Rt = 1.31 min (method 3), >99% purity. Synthesis of (S)-3-(1-Aminoethyl)-6-chloroquinolin-2(1H)-one Hydrochloride (Intermediate 71). Step 1 (Scheme 4): (R,E)-N-((2,6Dichloroquinolin-3-yl)methylene)-2-methylpropane-2-sulfinamide (71b). To a mixture of 2,6-dichloroquinoline-3-carbaldehyde (15.0 g, 66.37 mmol) and (R)-2-methylpropane-2-sulfinamide (8.85 g, 73.14 mmol) in DCE (150 mL) was added CuSO4 (16.0 g, 100.25 mmol). The resulting mixture was stirred at 55 °C overnight. After thin-layer chromatography (TLC) and MS showed complete disappearance of starting materials, the mixture was cooled to room temperature and filtered through a pad of Celite. The Celite was then rinsed with DCM. The filtrate was evaporated to dryness in vacuo and purified by SiO2 column chromatography (0−25% hexanes/EtOAc) to afford the title compound 71b, as a yellow solid (17.7 g, 81% yield). Step 2 (Scheme 4): (R)-N-((S)-1-(2,6-Dichloroquinolin-3-yl)ethyl)2-methylpropane-2-sulfinamide (71c). To a solution of (R,E)-N((2,6-dichloroquinolin-3-yl)methylene)-2-methylpropane-2-sulfinamide (8.85 g, 26.88 mmol) in anhydrous DCM (200 mL) at −60 °C was added dropwise MeMgBr (3 M solution in diethyl ether, 13.5 mL, 40.54 mmol). The resulting reaction mixture was stirred at about −60 to −50 °C for 3 h and then stirred at −20 °C overnight under an atmosphere of N2. After TLC and MS showed complete disappearance of starting materials, saturated NH4Cl (163 mL) was added at −20 °C, and the resulting mixture was stirred for 10 min. The aqueous phase was extracted with DCM (100 mL × 3), dried over anhydrous Na2SO4, filtered, and evaporated. The residue was purified by column chromatography on an ISCO chromatography system (SiO2: gold column; gradient; hexanes to 100% EtOAc) to provide the title compound 71c as a yellow solid (5.8 g, 63% yield). Step 3 (Scheme 4): (S)-3-(1-Aminoethyl)-6-chloroquinolin-2(1H)one Hydrochloride (71). A mixture of (R)-N-((S)-1-(2,6-dichloroquinolin-3-yl)ethyl)-2-methylpropane-2-sulfinamide (6.6 g, 19.13 mmol) in 1,4-dioxane (41 mL) and 1 N HCl (41 mL) was heated at reflux overnight. The solvents were evaporated in vacuo, and the resulting residue was dissolved in hot water and lyophilized. The crude product was triturated with diethyl ether to afford the title compound 71 as a yellow solid (4.9 g, 98.8% yield, enantiomeric excess: 98.4%). 1H NMR (300 MHz, DMSO-d6): δ ppm 12.4 (br s, 1H), 8.32 (br s, 2H), 8.07 (s, 1H), 7.85 (d, J = 2.2 Hz, 1H), 7.63 (dd, J1 = 8.8 Hz, J2 = 2.5 Hz, 1H), 7.40 (d, J = 8.8 Hz, 1H), 4.40−4.45 (m, 1H), 1.53 (d, J = 8.5 Hz, 3H). LC−MS (ESI) m/z calcd for C11H12ClN2O [M + H]+, 223.07; found, 223.10, Rt = 3.42 min (method 2). Syntheses of Compounds 56 and 59−64 (Scheme 5). (S)-6-(1-(6Chloro-2-oxo-1,2-dihydroquinolin-3-yl)ethylamino)-2-methylnicotinonitrile (56). A mixture of 6-fluoro-2-methylnicotinonitrile (28.6 mg, 0.210 mmol) and (S)-3-(1-aminoethyl)-6-chloroquinolin-2(1H)one hydrochloride (intermediate 71, 49.6 mg, 0.191 mmol) was treated with DMSO (1.4 mL) and DIEA (0.10 mL, 0.573 mmol). The solution was stirred at 110 °C for 2 h. LC−MS indicated that the reaction had gone to completion. The sample was mixed with water (20 mL) and extracted with DCM (3 × 15 mL). The extracts were dried (Na2SO4), filtered, treated with silica gel, and evaporated under reduced pressure. R

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calcd for C17H15ClN5O [M + H]+, 340.10; found, 340.96; Rt = 2.35 min (method 1), 97% purity. (S)-2-((1-(6-Chloro-7-methoxy-2-oxo-1,2-dihydroquinolin-3-yl)ethyl)amino)-4-methoxypyrimidine-5-carbonitrile (67). A mixture of 2-chloro-4-methoxypyrimidine-5-carbonitrile (45.9 mg, 0.271 mmol) and (S)-3-(1-aminoethyl)-6-chloro-7-methoxyquinolin-2(1H)-one hydrochloride 75 (see the Supporting Information, 70.1 mg, 0.242 mmol) was dissolved in DMSO (1.6 mL) and DIEA (127 μL, 0.727 mmol). The solution was stirred at 110 °C for 45 min. Water (20 mL) was added, and the reaction mixture was extracted with DCM (2 × 15 mL). The extracts were dried (Na2SO4) and filtered. Silica gel was then added, and the solvent was evaporated under reduced pressure. The material was purified by column chromatography on a Biotage MPLC chromatography system (10 g of silica gel column, eluted with 0−100% EtOAc in hexanes, with isocratic elution when peaks eluted). The product fractions were washed with water (40 mL), dried (Na2SO4), filtered, and evaporated under reduced pressure. The material was treated with MeCN (0.8 mL) and water (0.4 mL) to provide a thick slurry. The mixture was frozen on a dry ice/acetone bath, then lyophilized to provide the title compound 67 as a white solid (70.3 mg, 0.182 mmol, 75% yield, HPLC purity 100% at 220 nm). 1H NMR (300 MHz, DMSO-d6): δ ppm 11.83 (s, 1H), 8.50−8.74 (m, 1H), 8.48 (d, J = 1.76 Hz, 1H), 7.77 (s, 1H), 7.68 (d, J = 7.04 Hz, 1H), 6.94 (s, 1H), 5.15−5.29 (m, 1H), 3.78−4.00 (m, 3H), 3.88 (s, 3H), 1.36−1.46 (m, 3H). LC−MS (ESI) m/z [M + H]+, 386.10; Rt = 10.10 min (method 2), >99% purity. HRMS (ESI) calcd for C18H17ClN5O3 [M + H]+, 386.1020; found 386.1019. IDH1-R132H and IDH1-R132C Enzymatic Assay. Assays were performed in a 384-well black plate. An aliquot of 250 nL of compound was incubated with 10 μL of 30 nM IDH1-R132H or 10 nM IDH1R132C recombinant protein in assay buffer (50 mM Tris pH = 7.5, 150 mM NaCl, 5 mM MgCl2, 0.1% (w/v) bovine serum albumin, and 0.01% Triton X-100) in each well at 25 °C for 15 min. After the plate was centrifuged briefly, an aliquot of 10 μL of 2 mM α-ketoglutarate and 20 μM NADPH solution prepared in assay buffer was then added to each well, and the reaction was maintained at 25 °C for 45 min. An aliquot of 10 μL of diaphorase solution (0.15 U/mL of diaphorase and 30 μM resazurin in assay buffer) was added to each well. The plate was maintained at 25 °C for 15 min and then read on a plate reader with excitation and emission wavelengths at 535 and 590 nm, respectively. The IC50 of a given compound was calculated by fitting the dose− response curve of inhibition of NADPH consumption at a given concentration with the four-parameter logistic equation. Each compound was assayed in triplicate. The IC50 value is the mean ± standard deviation of multiple determinations. Cellular 2-HG Assay Using HCT116 Mutant IDH1 Cells. HCT116 isogenic IDH1-R132H and IDH1-R132C mutant cells were cultured in growth media (McCoy’s 5A, 10% fetal bovine serum, 1× antibiotic− antimycotic solution and 0.3 mg/mL G418) in 5% CO2 in an incubator at 37 °C. To prepare the assay, cells were trypsinized and resuspended in assay media (McCoy’s 5A with no L-glutamine, 10% fetal bovine serum, 1× antibiotic−antimycotic solution and 0.3 mg/mL of G418). An aliquot of 10 000 cells/100 μL was transferred to each well of a clear 96-well tissue culture plate. The cells were incubated in 5% CO2 at 37 °C in an incubator overnight to allow for proper cell attachment. An aliquot of 50 μL of compound containing assay media was then added to each well, and the assay plate was kept in 5% CO2 at 37 °C in an incubator for 24 h. The media were then removed from each well, and 150 μL of a methanol/water mixture (80/20 v/v) was added to each well. The plates were kept at −80 °C freezer overnight to allow for complete cell lysis. An aliquot of 125 μL of extracted supernatant was analyzed by RapidFire high-throughout-mass spectrometry (Agilent) to determine the cellular 2-HG level. The IC50 of a given compound was calculated by fitting the dose−response curve of cellular 2-HG inhibition at a given concentration with the four-parameter logistic equation. In Vivo PKPD Studies Using HCT116-IDH1-R132H/+ or HCT116R132C/+ Xenograft Mouse Model. An aliquot of 5X10E6 HCT116IDH1-R132H/+ or HCT116-IDH1-R132C/+ cells in 100 μL phosphate-buffered saline was injected subcutaneously under the

ypicolinonitrile (47.6 mg, 0.313 mmol) and (S)-3-(1-aminoethyl)-6chloroquinolin-2(1H)-one hydrochloride 71 (74.5 mg, 0.287 mmol) in DMSO (2 mL) was added DIEA (153 μL, 0.876 mmol). The solution was stirred at 110 °C for 12 h. Once LC−MS analysis indicated that most starting material was consumed, the mixture was diluted with water (30 mL) in DCM (30 mL). The organic extract was dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude material was purified by column chromatography on Biotage chromatography system (eluted with 0−50% EtOAc in hexanes) to provide the title compound 62 (42.7 mg, 42% yield). 1H NMR (300 MHz, DMSO-d6): δ ppm 12.07 (s, 1H), 7.72−7.79 (m, 2H), 7.50 (dd, J = 8.79, 2.35 Hz, 1H), 7.33 (m, 2H), 6.65 (d, J = 7.62 Hz, 1H), 6.48 (d, J = 7.92 Hz, 1H), 4.72 (quin, J = 6.82 Hz, 1H), 3.97 (s, 3H), 1.50 (d, J = 6.74 Hz, 3H). LC−MS (ESI) m/z calcd for C18H16ClN4O2 [M + H]+, 355.10; found, 355.06; Rt = 1.44 min (method 1), >99% purity. (S)-2-((1-(6-Chloro-2-oxo-1,2-dihydroquinolin-3-yl)ethyl)amino)4-methoxypyrimidine-5-carbonitrile (63). To a solution of 2-chloro4-methoxypyrimidine-5-carbonitrile (65.4 mg, 0.386 mmol) and (S)-3(1-aminoethyl)-6-chloroquinolin-2(1H)-one hydrochloride 71 (100 mg, 0.386 mmol) in DMSO (0.60 mL) was added DIEA (0.135 mL, 0.772 mmol). The solution was stirred at 110 °C for 4 h. Once LC−MS analysis indicated that most starting material was consumed, the mixture was diluted with DCM and washed with water (2×) and brine (1×). The organic extract was dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude material was purified by column chromatography on Biotage chromatography system (eluted with 0−80% EtOAc in hexanes) to provide the title compound 63 (34.1 mg, >95% HPLC pure @ 220 nm, 26% yield). 1H NMR (300 MHz, DMSO-d6, at 120 °C): δ ppm 11.65 (br s, 1H), 8.42 (s, 1H), 8.20 (br s, 1H), 7.79 (s, 1H), 7.68 (s, 1H), 7.45 (d, J = 8.8 Hz, 1H), 7.33 (d, J = 8.8 Hz, 1H), 5.32 (m, 1H), 3.94 (s, 3H), 1.50 (d, J = 6.3 Hz, 3H). LC−MS (ESI) m/z: [M + H]+, 356.00; Rt = 2.31 min (method 1), >99% purity. HRMS (ESI) calcd for C17H15ClN5O2 [M + H]+, 356.0914; found 356.0910. (S)-2-((1-(6-Chloro-2-oxo-1,2-dihydroquinolin-3-yl)ethyl)amino)4-methylpyrimidine-5-carbonitrile (64). Step 1 (Scheme 5): (S)-3-(1((5-Bromo-4-methylpyrimidin-2-yl)amino)ethyl)-6-chloroquinolin2(1H)-one. A mixture of 5-bromo-2-chloro-4-methylpyrimidine (440 mg, 2.122 mmol) and (S)-3-(1-aminoethyl)-6-chloroquinolin-2(1H)one hydrochloride 71 (500 mg, 1.930 mmol) was dissolved in DMSO (3 mL) and DIEA (5.79 mmol, 748 mg, 1 mL), and the solution was stirred at 110 °C for 12 h. Once LC−MS indicated that most of the starting material was consumed, the mixture was cooled to room temperature and stirred for 2 days. The solution was then diluted with water and extracted with DCM (2×). The extracts were dried (Na2SO4), filtered, and evaporated under reduced pressure. The crude material was purified by silica gel chromatography on a Biotage chromatography system (50 g column, eluted with 0−80% EtOAc/ hexanes) to afford the title compound (635 mg, 84% yield). 1H NMR (300 MHz, DMSO-d6): δ ppm 11.94 (s, 1H), 8.13−8.29 (m, 1H), 7.66−7.88 (m, 2H), 7.46 (dd, J = 8.79, 2.35 Hz, 1H), 7.20−7.32 (m, 1H), 5.08 (br s, 1H), 2.17−2.37 (m, 3H), 1.25−1.46 (m, 3H). LC−MS (ESI) m/z calcd for C16H15BrClN4O [M + H]+, 393.01; found, 395.84; Rt = 2.59 min (method 1). Step 2 (Scheme 5): (S)-2-((1-(6-Chloro-2-oxo-1,2-dihydroquinolin-3-yl)ethyl)amino)-4-methylpyrimidine-5-carbonitrile (64). A mixture of Pd2(dba)3 (11.63 mg, 0.013 mmol), dppf (14.0 mg, 0.025 mmol), (S)-3-(1-((5-bromo-4-methylpyrimidin-2-yl)amino)ethyl)-6chloroquinolin-2(1H)-one (635 mg, 1.613 mmol), and dicyanozinc (379 mg, 3.23 mmol) in DMF (30 mL) was purged with nitrogen for 10 min. The mixture was then heated at 120 °C overnight. Once LC−MS showed 50% conversion, the volatiles were removed under vacuum. Water was added to the resulting residue, and solids were removed by filtration. The crude material was purified by silica gel chromatography on a Biotage chromatography system (25 g column eluted with 10− 100% EtOAc/hexanes) to afford the title compound 64 (517 mg, 94% yield) 1H NMR (300 MHz, DMSO-d6): δ ppm 11.98 (s, 1H), 8.46− 8.74 (m, 2H), 7.67−7.89 (m, 2H), 7.18−7.53 (m, 2H), 5.08−5.42 (m, 1H), 2.14−2.45 (m, 3H), 1.25−1.50 (m, 3H). LC−MS (ESI) m/z S

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right flank of 6−8 weeks old female BALB/c nude mice. The tumor size was calipered and calculated by (a × b2/2), where “b” is the smallest diameter and “a” is the largest diameter. Once the tumor size reached ∼300 mm3, the animals were randomized to various groups (N = 9/ group) for compound treatment. Compound 63 was formulated with 10% ethanol, and 90% poly(ethylene glycol)400 was administrated orally three times with 12 h dosing interval. The dosing volume was 10 mL/kg animal weight. The animals were euthanized at 4, 12, 24 h post last dose. Plasma samples were collected for the measurement of compound 63 concentration in plasma. The tumor samples were harvested for the tumoral 2-HG level measurement by LC−MS. Protein Expression and Purification for Crystallography for 24 and 32. Protein expression and purification, crystallization and structure determination, data collection and refinement statistics tables for 24 (PDB: 6O2Y) and 32 (PDB: 6O2Z). X-ray structures are shown in the Supporting Information.

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compounds 24 and 32; CrownBio for PK/PD studies in HCT116-IDH1-R132H/+ and R132C/+ xenograft models; Drs. Rob Sarisky, Paul Ehrlich, Hesham Mohamed, and Frances Duffy-Warren for very helpful discussions.

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ABBREVIATIONS ADME, absorption, distribution, metabolism, and excretion; Arg, arginine; AUC, area under the curve; BBB, blood−brain barrier; Brine, a saturated aqueous solution of sodium chloride; Caco-2, cancer coli-2; CL, clearance; Cmax, maximum concentration; CYP, cytochrome P450; DCM, dichloromethane; DCE, 1,2-dichloroethane; DIEA, N,N-diisopropyl ethylamine; DMSO, dimethyl sulfoxide; EtOAc, ethyl acetate; EtOH, ethanol; Ex, example; F, oral bioavailability; h, hour; His, histidine; hERG, human ether-a-go-go-related gene; HPLC, high-performance liquid chromatography; HRMS, high-resolution mass spectrometry; IV, intravenous; IP, intraperitoneal; Ki, inhibition constant; LC−MS, liquid chromatography−mass spectrometry; Log D7.4, log of partition coefficient between octanol and pH7.4 aqueous buffer; MDCK, Madin−Darby canine kidney; MeOH, methanol; min, minute; NADPH, nicotinamide adenine dinucleotide phosphate hydrogen; PAMPA, parallel artificial membrane permeation assay; P-gp, P-glycoprotein; Papp, apparent permeability; PK, pharmacokinetics; PK/PD, pharmacokinetic−pharmacodynamic; PO, by mouth; PPB, plasma protein binding; THF, tetrahydrofuran; PSA, polar surface area

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.9b00362. Protein expression and purification for crystallography (Section S2); crystallization and structure determination of 24 and 32 (Section S3); crystal parameters, data collection and refinement statistics for 24 and 32 (Section S4); details of the synthetic procedures and analytical data for intermediates 44a−70f, and compounds 24−70 (Sections S4−S26); kinase profiling (Cerep Kinase panel) of 63 (Section S27); PXR HepG2 assay and CYP3A4 TDI assay data for 63 (Section S29); hERG PatchClamp assay for 63 (S32); cell lines and cellular assays (Sections S32−S33) (PDF) Molecular formula strings are available separately in comma-separated values file format (CSV)

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Accession Codes

Atomic coordinates and experimental data for the co-crystal structures of 24 (PDB: 6O2Y) and 32 (PDB: 6O2Z) in complex with mIDH-R132H will be released upon article publication.

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J.L.). *E-mail: [email protected] (C.D.). ORCID

Jian Lin: 0000-0001-8428-7958 Present Addresses †

Casma Therapeutics, Cambridge, Massachusetts 02139, United States (J.L.). ‡ KSQ Therapeutics, Cambridge, Massachusetts 02139, United States (W.L.). ∥ Camp4 Therapeutics, Cambridge, Massachusetts 02139, United States (J.C.). § Ra Pharmaceuticals, Inc., Cambridge, Massachusetts 02140, United States (S.A.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Anu Mahadevan and the chemists at Organix Inc. for their contribution to the syntheses of intermediates and compounds; Drs. Adam J. Stein, Andre White and colleagues from Xtal BioStructures, Inc. for solving X-ray structures of T

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