Discovery and Optimization of Quinolinone Derivatives as ......IDH1 is highly polar and bound to...

Discovery and Optimization of Quinolinone Derivatives as Potent,Selective, and Orally Bioavailable Mutant Isocitrate Dehydrogenase1 (mIDH1) InhibitorsJian 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 invarious human cancers. Inhibition of mutant IDH1 (mIDH1) with small molecules has been clinically validated as a promisingtherapeutic treatment for acute myeloid leukemia and multiple solid tumors. Herein, we report the discovery and optimizationof a series of quinolinones to provide potent and orally bioavailable mIDH1 inhibitors with selectivity over wild-type IDH1. TheX-ray structure of an early lead 24 in complex with mIDH1-R132H shows that the inhibitor unexpectedly binds to an allostericsite. Efforts to improve the in vitro and in vivo absorption, distribution, metabolism, and excretion (ADME) properties of 24yielded a preclinical candidate 63. The detailed preclinical ADME and pharmacology studies of 63 support further developmentof quinolinone-based mIDH1 inhibitors as therapeutic agents in human trials.

■ INTRODUCTION

The human cytoplasmic isocitrate dehydrogenases (IDH) are aclass of nicotinamide adenine dinucleotide phosphate(NADP+)-dependent enzymes that catalyze the oxidativedecarboxylation of isocitrate to α-ketoglutarate (α-KG), whichis one of the key reactions in the Krebs (citric acid) cycle.1,2

There are three isoforms in this family. IDH1 resides in thecytoplasm, whereas IDH2 and IDH3 localize in the mitochon-dria. Heterozygous mutations of IDH1 at Arg132, a residue thatis critical for the substrate recognition, have been frequentlyidentified in multiple cancer types,2−4 including grade II−IIIgliomas and secondary glioblastomas (GBMs) (where theyoccur at a rate of 70−90%),5,6 acute myeloid leukemia (AML,10−17%),7,8 intrahepatic cholangiocarcinoma (7−20%),9 andcentral and periosteal chondrosarcomas (46−52%).10,11 Themost commonly observed IDH1mutations in these cancer typesinclude R132H, R132C, R132G, R132S, and R132L, whereasIDH2mutations R140Q or R172K have been identified in AML

and less frequently in solid tumors. These mutant enzymes losetheir wild-type enzyme activity, acquiring a new function toreduce α-ketoglutarate (α-KG) to D-2-hydroxyglutarate (2-HG).7,12 As a result, human cancer cells harboring mutant IDH1(mIDH1) show aberrantly elevated 2-HG levels.7 2-HG hasbeen described as an “oncometabolite” since it inhibits the classof α-KG-dependent enzymes involved in epigenetic regulation,cell signaling, and collagen synthesis. In particular, 2-HG impairsDNA demethylation through inhibition of ten-eleven trans-location-2 (TET2), which in turn results in the hyper-methylation of DNA CpG islands and impairs hematopoieticcell differentiation.13 2-HG also impairs histone demethylationthrough inhibition of a number of lysine demethylases.14,15 Twomutant IDH1 (mIDH1) inhibitors, AGI-519816 and GSK321,17

have demonstrated the reduction of intratumoral 2-HG levels

Received: March 7, 2019Published: June 14, 2019

Article

pubs.acs.org/jmcCite This: J. Med. Chem. 2019, 62, 6575−6596

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and result in tumor regression in mIDH1-containing tumorxenograft mouse models. These studies suggest that mIDH1inhibitors can be used as therapeutic agents to induce thedifferentiation of proliferating cancer cells.Three classes of mIDH1 inhibitors based on their distinct

binding sites have been reported (Chart 1).16−31 Class-Iinhibitors, such as SYC-43520,21 and thiohydantoin-16,22 bind

directly to the active site of the protein. Since the active site ofIDH1 is highly polar and bound to co-factor nicotinamideadenine dinucleotide phosphate hydrogen (NADPH), it ischallenging 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 anallosteric site, reducing the enzymatic activity of IDH1 through a

Chart 1. Structures of the Known Mutant IDH1 Inhibitors

Journal of Medicinal Chemistry Article

DOI: 10.1021/acs.jmedchem.9b00362J. Med. Chem. 2019, 62, 6575−6596

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conformational change of several critical amino acid residues(Arg100, Arg109, and His132). X-ray structures of compoundsGSK321,17 Bay1436032,23,24 IDH889,25 IDH305,26 Sanofibisimidazole 3,27 and CRUK-MI 20a28 revealed that thesecompounds are all class-II inhibitors. AG-881, a dual inhibitor ofmIDH1 and mIDH2, is the only example of a class-III inhibitorto date. The X-ray structures of AG-881, solved by Yun et al.,revealed that it binds mIDH1 and mIDH2 in a commonallosteric pocket located at the dimer interface.29 The sterichindrance in the substrate binding site caused by the bent anddisplaced α10 helixes due to AG-881 binding in the allostericpocket may account for the inhibitory effect of this compound.Several other inhibitors, such as AGI-5198, AG-120,30 andBRD2879,31 have been reported, but their X-ray structures havenot been published. AG-120 (ivosidenib, a selective mIDH1inhibitor) has recently been approved by the Food and DrugAdministration for the treatment of relapsed/refractory AMLpatients harboring mIDH1. The clinical data demonstrated thativosidenib (AG-120) normalized the plasma 2-HG levels inAML patients to the level observed in healthy volunteers andrestored the proper differentiation of hematopoietic stem cellsand reduced leukemic burden.18 These clinical observationsvalidate the role of mIDH1 inhibitors in AML tumorgenesis andthe potential clinical application of selective mIDH inhibitors inAML patients bearing IDH1 mutations. However, AG-120 isreported to demonstrate a high efflux ratio (ER > 350) with verylow apical to basal transport in Madin−Darby canine kidney(MDCK) cells transfected with the multidrug transporter P-glycoprotein (P-gp).19 The likely inability of AG-120 to crossthe blood−brain barrier could limit its clinical utility in low-grade glioma and secondary GBM. Hence, there is still an urgentneed to develop novel mIDH1 inhibitors that penetrate the

blood−brain barrier for the treatment of mIDH1 harboringbrain cancers.Herein, we report the discovery of a series of quinolinone

derivatives as potent IDH1-R132H inhibitors and theidentification of candidate 63 for preclinical studies in oncology.In vitro absorption, distribution, metabolism, and excretion(ADME) data also indicate that candidate 63 may cross theblood−brain barrier, suggesting that the quinolinone-basedmIDH1 inhibitors may be further optimized for the treatment ofmIDH1-containing low-grade glioma and secondary GBM.

■ RESULTS AND DISCUSSION

Discovery of Quinolinone Hit from High-ThroughputScreening (HTS). To identify mIDH1-R132H selective hits, ahigh-throughput screening (HTS) campaign against a set of over400 000 compounds was conducted using a diaphorase assay tomeasure the consumption of NADPH, a co-substrate of mIDH1.The initial HTS actives were triaged and tested in an eight-pointdose titration for IC50 determination. The selected HTS hitswere confirmed by thermal shift assay, surface plasmonresonance assay, and an orthogonal biochemical inhibitionassay using RapidFire high-throughout-mass spectrometry(Agilent) to determine the 2-HG level. The confirmed hitswere then tested for the inhibition of mutant IDH1-R132C andselectivity against wild-type IDH1. The selective hits wereclassified/clustered as different chemical series and singletonsbased on their structural similarity. Among five chemical seriesidentified from the HTS campaign, the quinolinone scaffold ofhit 1 emerged as an attractive chemical series due to itsreasonably good ligand efficiency (LE) and physical properties(molecular weight (MW), Log P/LogD). In a mechanism ofaction study, substrate competition assays indicated that early

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 MLM, left % 30 min solubility (μM)

1 Me Me H 0.704 17.6 27.9 0.042 Me H H 0.238 30.8 42.3 <0.013 H Me H 0.303 9.6 17.8 <0.014 CI H H 0.267 93.9 89.9 <0.015 H H H 3.32 88.0 69.4 0.936 CI H Me >25 63.7 22.0 <0.01

aHLM: human liver microsome; MLM: mouse liver microsome.



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hit 1 is competitive against α-KG and noncompetitive againstNADPH (data is not shown here). As depicted in Figure 1, HTShit 1 was divided into three regions: the left-hand side (LHS),the linker, and the right-hand side (RHS). Defining the criticalcomponents in each region was the focus of subsequentstructure−activity relationship (SAR) studies.Early SAR and Lead Generation. Initial SAR studies on

HTS hit 1 focused on investigating which portions of themolecule are critical for its activity against mIDH1-R132H. Wefirst investigated the SAR of the substituents R1, R2, and R3 whileholding the rest of the molecule constant (Table 1). Compound1 hasmethyl groups at the 6- and 7-positions, and the data showsthat the removal of either methyl group (analogs 2 and 3) resultsin a 2−3-fold increase in potency. Analogs 1−3 all have poorstability after incubation with either mouse or human livermicrosomes (MLM or HLM, respectively). Each compound has31% or less remaining after a 30 min incubation with HLM.Replacement of the 6-Me group with chlorine (analog 4) notonly maintained potency but also improved the metabolicstability significantly (HLM: 94% remaining after 30 min;MLM: 90% remaining after 30 min). The removal of bothmethyl groups from R1 and R2 in compound 5 not only incurreda 5-fold loss in potency but also improved the microsomalstability. Compound 6, in which the quinolinone nitrogen of 4 ismethylated, had no measurable inhibitory activity. This suggeststhat the quinoline NH is making a key interaction with theprotein. In general, all these analogs suffered from poorsolubility.To further understand if the tetrazole ring, the methoxy

group, and the COgroup of the quinolinone core are involvedin interactions with the protein, several key singletons wereprepared and studied. As shown in Table 2, the removal of the

entire tetrazole ring of 1 reduced the activity of 7 by 73-fold. Thereplacement of the OMe group of 1 with hydrogen (analog 8)caused a 4-fold loss in potency. The carbonyl group of thequinolinone core appears to be essential for inhibition, as thereplacement of quinolinone with the quinoline ring in 9abolished all activities. Replacing the tetrazole in 1with a pyrrolering (compound 10) caused a dramatic loss in potency,suggesting that one or more of the tetrazole nitrogens aremaking a critical interaction.Exploration of SAR in the RHS/LHS regions was carried out

using a combinatorial reductive amination library consisting offour LHS cores and various primary or secondary amines andanilines (Figure 2). Initially, we hypothesized that hit 1 binds in

the active site near NADPH, and the tetrazole ring may makehydrogen bonds with the key residues, as the substrate α-KGdoes. Therefore, we selected anilines containing hydrogen bonddonors and/or acceptors for incorporation in the library.Quinoline core (D) yielded no active compounds, again

suggesting that the carbonyl and/or NH in the quinolinone coreare critical for the activity against mIDH1-R132H. The otherthree LHS cores showed a similar trend in SAR. The 6-chloro-quinolinone core (B) gave the most active compounds,including many with good physical properties. Most of thecompounds made with aliphatic primary amines (e.g., 11) andall compounds derived from secondary aliphatic amines andanilines are inactive (representative data is shown in Table 3).The table also shows that the replacement of the tetrazole with acyano group (e.g., 12 and 14−16) resulted in submicromolaractivity for mIDH1-R132H, but repositioning the cyano groupto the meta position (13) or extending the linker with amethylene group (11) resulted in the complete loss of activity.Interestingly, the incorporation of a nicotinonitrile as in both 14and 16 resulted in a small increase in potency when comparedwith their counterpart benzonitriles (12 and 15). One couldhypothesize that the nitrogen atom of the nicotinonitrilemakes akey interaction with the protein, since compound 21 retainssome activity (5.2 μM) even though it lacks a 4-CN group. Thereplacement of the tetrazole ring (17) with other heterocyclicrings like imidazole (18), thiadiazole (19), or morpholine (20)gave different degrees of decrease in potency (1.7, 45, or 100μM, respectively), suggesting that the position of nitrogen(s) inthe heterocyclic ring is important. It was hypothesized that thenitrile or tetrazole nitrogen plays a role as a H-bond acceptor.The last two examples (22 and 23) may indicate that the proteinrequires a para-substituted H-bond acceptor that is co-planarwith the phenyl ring.

Exploration of RHS Region of Compounds 12 and 14.The early SAR studies described above suggested that the CNgroup in 12 or 14 is a good replacement for the tetrazole ring of

Table 2. SAR of Critical Components

Figure 2. Library design for LHS and RHS SAR exploration.



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1. These compounds lack the methoxy group found on 1, whichis 4-fold more potent than the corresponding compound 8 thatlacks it, as described earlier. We reasoned that the potency of 12and 14 could, therefore, be further improved by the installationof a methoxy group or other lipophilic substituents of the similarsize on the 4-nitrile aryl ring in the RHS region. As shown inTable 4, this effort led to the identification of two more potentmIDH1 inhibitors 24 and 32. The addition of a hydrophobicgroup such as o-OMe (24), m-OMe (25), o-OEt (27), o-Me

(15), 2,6-di-OMe (28), o-OCF3 (29), and o-CF3 (31) to thephenyl ring had a positive impact on the activity compared withthe unsubstituted analog 12. Interestingly, the 2-methylnicoti-nonitrile analog 32 had excellent potency (0.072 μM), whereasthe 4-methylnicotinonitrile (16) was 5-fold less potent (0.344μM). An o-F (30), which is substantially smaller and lesshydrophobic than OMe, had no significant impact on theactivity compared to 12. Installation of an electron-withdrawinggroup (EWG) such as CF3 (33) or Cl (34) reduced the activity

Table 3. SAR of RHS Region



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dramatically, most likely due to the reduction of the basicity ofthe pyridine nitrogen. The two most potent compounds 24 and

32 showed reasonably good HLM and MLM stability, but lowsolubility was still a concern.

Exploration of LHS Region of Compound 24. Com-pounds 35−41 (shown in Table 5), with different R substituentsin the quinolinone core, were designed to explore the topologyand nature of the area around the 6-position. Large substituentssuch as Br (35), tert-butyl (36), and CF3 (37) are all wellaccommodated in this apparently hydrophobic site, thusresulting in excellent potency. A relatively small group such asamethyl (38), F (40), orH (41) gave a 7−17-fold loss in activitycompared with 35. It was observed that all electron-withdrawinggroups (Cl, Br, CF3, and F) improve the metabolic stability bothin human and mouse, as exemplified by in compounds 24, 35,37, and 40. Considering potency, metabolic stability, andsolubility, the 6-Cl-substituted compound 24 was considered tohave the best-balanced profile.

In Vivo Pharmacokinetics (PK) and Cell BiologyProfiles of Leads. Although mIDH1-R132H was the primarytarget of this effort, IDH1-R132C is another IDH1 mutationfrequently identified in AML and solid tumors. To assess theselectivity profile of our leads, wemeasured the activity of 24 and32 against IDH1-R132C mutant and wild-type IDH1 enzymesusing a NADPH fluorescence-based biochemical assay. Thecellular activity of compounds 24 and 32 was also evaluated incell-based assays including a glioblastoma cell line (U87) and acolorectal carcinoma cell line (HCT116). These cell linesoverexpress either mIDH1-R132H or mIDH1-R132C andproduce high levels of 2-HG compared to the cells transfectedwith the vector alone. After the treatment of these cell lines withinhibitors for 48 h, the levels of 2-HG in the media weremeasured by liquid chromatography−mass spectrometry (LC−MS) to generate IC50 values. As shown in Table 6, compounds24 and 32 are highly selective over wild-type IDH1 (both IC50 >75 μM). Both compounds showed submicromolar cell potencyfor mIDH1-R132H (200−300 nM range) and low micromolarpotency for mIDH1-R132C in the HCT116 cell line.Compound 24 showed about 4-fold selectivity of R132H overR132C in U87 cell assay, whereas 32 is equally potent for bothIDH1 mutants R132C and R132H.By virtue of their good cellular potency and selectivity, as well

as moderate in vitro metabolic stability in mice, 24 and 32 wereevaluated in PK experiments (PO, 5 mpk). After oraladministration in female BALB/c mice at a dose of 5 mg/kg,blood levels were detected up to 24 h. The maximum totalconcentration of 24 (1.06 μM) was reached after 1 h; total

Table 4. Optimization of RHS (Ar) of Compounds 12 and 14

Table 5. Optimization of LHS (R) of Compound 24

cpd no. R IDH1-R132H, IC50 (μM) HLM/MLM, left % 30 min solubility (μM)

24 Cl 0.127 81/42 0.4335 Br 0.054 89/39 0.1836 tBu 0.076 6/3.9 <0.1

37 CF3 0.053 76/89 <0.138 Me 0.132 1.2/0.49 1.3339 OMe 0.140 21.6/10.5 0.6740 F 0.138 81/46 3.3241 H 0.913 35/4.6 6.13



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exposure area under the curve (AUC)0−8 h was 2.45 μM h.Compound 24 has a plasma half-life (T1/2) of 1.27 h, and its totalplasma concentration was 0.1 μM at 4 h. The second leadcompound 32 has a very similar PK profile, as shown in Table 7.

Structure-Based Guided Optimization of 24 and 32.Xu et al. have previously shown that the a-KG substrate bindsnear the NADPH site, where the substrate is coordinated by thearginine triad residues (Arg100, Arg109, and His132).1

Compound 1 is a competitive inhibitor with respect to α-KGbut noncompetitive with respect to NADPH binding. Weattempted to dock compound 1 in the α-KG/NADPH bindingsite of mIDH1-R132H (PBD: 3INM);12 however, we wereunable to find a predicted binding mode for compound 1 orcompound 24 that was consistent with the SAR data andmechanism of inhibition. It seemed possible that either theinhibitors bind in an allosteric site or the inhibitor-boundstructures differ significantly from the publicly available X-raycrystal structures of mIDH1-R132H.To understand the binding modes and guide further

optimization of these leads, we obtained X-ray crystal structuresof mIDH1-R132H in complex with 24 and 32. The X-ray crystalstructure of 24 complexed with mIDH1-R132H homodimer inthe presence of NADPH was solved at a resolution of 2.8 Å. Thestructure shows the expected dimeric structure of mIDH1, withone compound bound per monomer (Figure 3A). Unexpect-edly, the compound binds in an induced-fit pocket approx-imately 15 Å away from the NADPH binding site (Figure 3B).Binding of compound 24 causes the central α-helix to shift andpartially occupy the substrate binding site. The Arg triadresidues rearrange so they are directed toward the inducedallosteric site near compound 24, as shown in Figure 3C. Theinhibitor, therefore, appears to be competitive with respect tothe substrate binding, even though the inhibitor binding sitedoes not overlap with the substrate site. The compound makesseveral key interactions with the protein, as shown in Figure 3D.All crucial components of 24 as identified from our initial SARstudies can be interpreted by interactions with the protein, asseen in the crystal structure. The cyanomoiety forms a hydrogenbond with the backbone amide NH of Leu120, whereas thelinker benzyl amine (NH) makes a H-bond with the carbonyl ofIle128. The 2-methoxy group of the benzonitrile ring sits wellinto a small hydrophobic cleft formed by Trp124 and Ile113.Both the carbonyl (CO) and the NH of the quinolinone formhydrogen bonds with Arg109 and Asp279, respectively. The 6-

chloro substituent of the quinolinone ring fills in a hydrophobicpocket formed by residues Trp267, Ala258, Ile130, and Ile128.As shown in Figure 3E, analog 32 binds with mIDH-R132Hprotein in a similar mode, except that the pyridine nitrogen inthe RHS region makes a water-mediated hydrogen bond withthe backbone NH of Ile218. This may explain why anicotinonitrile RHS can enhance potency. The incorporationof a pyridine nitrogen at this position does not appear to havemuch effect on potency, but the pyridine compounds aremodestly less lipophilic than the phenyl analogs. These co-crystal structures of 24 and 32 enabled a structure-based designstrategy to be used in further optimization.As indicated in Figure 3C, the 7- and 8-positions of the

quinolinone scaffold are oriented so that the additionalsubstitution at these positions could further increase thepotency. In addition, this region of the binding pocket ispartially exposed, so substitution at these positions afforded anopportunity to add polar functional groups with the goal ofmodulating the physiochemical properties of the compounds.Table 8 shows several compounds including a more hydrophilicgroup (or a solubilizing group) at either the 7- or 8-position.This approach was partially successful in that very high potencyand selectivity for mIDH1-R132H were achieved by theaddition of 7-substituents (for example, compounds 42, 44,46, 48−53), yet significant challenges remained to be overcometo improve druglike characteristics, including solubility, micro-somal stability, and oral bioavailability. Compared to 7-substituted analogs like 42, 44, and 46, 8-substitutedcompounds (such as 43, 45, and 47) retained some potencyfor mIDH1-R132H, but activity for mIDH1-R132C wasimpaired.We next turned our attention to the modification of the

benzylic linker. This linker adopts a similar conformation in thecrystal structures with both 24 and 32, as shown in Figure 4. Wehypothesized that the incorporation of an appropriatesubstituent on the α-position of the benzylic amine linkercould constrain the linker to adopt the bioactive conformationand, therefore, reduce the entropic cost of ligand binding. Wedefine the torsion angles φ1 and φ2 as the torsion angles shownin Figure 5A. The crystal structure of 24 shows that the boundligand has φ1 and φ2 values of approximately 305 and 160°,respectively. This linker conformation positions the 2-methoxybenzonitrile ring so that it is nearly perpendicular tothe quinolinone ring (Figure 4). We performed an ab initiotorsion scan of a fragment of compound 24 with anunsubstituted CH2N linker, and the same fragment having amethyl group on this linker. The results of the torsion scan areshown in Figure 5. Figure 5A illustrates that compounds with anunsubstituted linker havemultiple energyminima, including oneenergy minimum near the conformation seen in the IDH1 co-crystal structure. Figure 5B shows that the methyl-substitutedlinker with (S) stereochemistry is more conformationallyconstrained, and the bioactive conformation is still near anenergy minimum. This extra constraint on the linkerconformation would be expected to modestly improve the

Table 6. Biochemical and Cell Biology Profiles of Leads

biochem assays cell assays

cpdno.

IDH1-R132H,IC50 (μM)

IDH1-R132C,IC50 (μM)

IDH1 WT,IC50 (μM)

HCT116 2HG IDH1-R132H, IC50 (μM)

HCT116 2HG IDH1-R132C, IC50 (μM)

U87 2HG IDH1-R132H, IC50 (μM)

U87 2HG IDH1-R132C, IC50 (μM)

24 0.127 2.25 100 0.266 1.92 0.316 1.2532 0.072 0.82 75 0.219 3.21 0.274 0.258

Table 7. In Vitro and In VivoMouse PK Parameters of 24 and32

mouse PK (PO, 5 mpk)

cpdno.

IDH1-R132H,

IC50 (μM)MLM,

left % 30 minsolubility(μM)

half-lifeT1/2(h)

Cmax(μM)

AUC0−8 h(μM h)

24 0.127 42 0.43 1.27 1.06 2.4532 0.072 69 0.64 3.57 0.48 2.66



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Figure 3. (A) Overview of the X-ray crystal structure of mIDH1-R132H dimer and inhibitor 24. IDH1 monomers are shown in magenta and orangeribbons. Inhibitor 24 is shown with green carbons and NADPH molecules are shown with yellow carbons. (B) View of a single IDH1 monomer,rotated 90° relative to (A). Inhibitor 24 binds in a site that is toward the right side of the picture. (C) Close-up view of the allosteric binding site ofmIDH1-R132H and inhibitor 24. Positions 7 and 8 of the quinolinone ring are the only portion of compound 24 that appears to be exposed. (D) Viewof the binding site of compound 24 (PDB: 6O2Y). (E) X-ray crystal structure of mIDH1-R132H and inhibitor 32 (PDB: 6O2Z).



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potency. By contrast, the (R) enantiomer is expected to besubstantially less potent, because it has an energy minimum thatdoes not resemble the bioactive conformation observed in thecrystal structure. For the (R) enantiomer to adopt the bioactive

conformation, there would be a steric clash between the methylgroup and the carbonyl O of the quinolinone.Methyl substitution could also block the metabolism at the α-

position of the benzylic group. As X-ray structure of 24 suggeststhat the protein can only accommodate a small group in thevicinity of the benzylic linker, we selected and investigatedseveral small R groups, such as α-methyl, gem-dimethyl, andspirocyclopropyl (Table 9). The introduction of a racemic α-methyl group in the benzylic linker of 54 maintained goodpotency and microsomal stability in both human and mouse. Itwas also observed that the solubility was improved 19-fold over32, probably due to the disruption of molecular planarity by theα-methyl group. The two pure enantiomers 55 (R) and 56 (S)were synthesized via independent routes to determine the chiralpreference for ligand binding. As hypothesized, the (S)-methylenantiomer (56) was favored (IC50: 0.020 μM for R132H; 0.183μM for R132C), whereas the (R)-methyl enantiomer (55) lostmost of its activity (IC50: 19 μM for R132H). The (S)-methylenantiomer (56) also demonstrated excellent HLM and MLMstability and improved solubility. The gem-dimethyl substitu-tion, compound 57, showed a 3.9-fold loss in activity versus theunsubstituted linker, and the spirocyclopropyl substitution (58)showed a minimal loss in potency.

Table 8. 7- and 8-Substitution SAR in LHS Region

Figure 4. Overlapped bioactive conformation of 24 (purple) and 32(green) seen in the X-ray crystal structures.



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Taken together, the data showed that an (S)-methyl-substituted benzylic linker (56) was the optimal choice, notonly providing very potent inhibitory activity against the R132Hand R132C mutants but also conferring excellent metabolicstability and enhanced solubility (Table 9). In particular, theaddition of this (S)-methyl substituent on the benzylic linker of24 gave the most potent inhibitor in the series thus far,compound 59 (biochemical IC50: 14 nM; cell HCT116 IC50: 7nM). This compound also achieved a 10-fold enhancement insolubility (4 μM) and a modest improvement in MLM stability.Subsequent efforts were focused on finding the bestcombination of this “magic” (S)-Me linker and the optimalRHS pieces. We chose and designed various phenyl, pyridine,and pyrimidine rings containing a para-nitrile and a meta-OMeor meta-Me groups as RHS pieces (highlighted in blue in Table10). The incorporation of an (S)-methyl group at the benzyliclinker resulted in compounds (59−64) with very potentbiochemical and cellular activities against both mIDH1-R132H and mIDH1-R132C. Compound 63, bearing the 4-

methoxypyrimidine-5-carbonitrile group in the RHS region,yielded the best overall potency and property profile. The designrationale for this pyrimidine carbonitrile was based on theanalysis of the two overlapped ligands 24 and 32 in their X-raystructures, as depicted in Figure 4. The benefits of pyrimidinereplacement of the phenyl ring are 2-fold: one nitrogen can serveas an acceptor to form a water-mediated hydrogen bond with theprotein as 32 does, whereas the other nitrogen can lower theenergy for the optimal conformation. These specific interactionsmay be compensating for the expected increase in desolvationenergy for a more polar pyrimidine ring (vs a phenyl ring as inthe case of 59). Furthermore, a pyrimidine ring increasespolarity and lowers LogD, thus potentially improving solubilityand giving more favorable physicochemical properties.We next revisited the optimization of the quinolinone core,

with a focus on fine-tuning the LHS of compound 63 (Table11). A small R group-like F (65) at the 7-position had nofavorable effect on potency or solubility. Larger groups such ascyclopropyl, methoxy, isopropoxy, cyclopropylmethoxy, and 2-pyridinemethoxy (66−70) all showed an improvement inpotency for both the biochemical assay (IC50 range: 4−17 nM)and the HCT116 cell-based assays (IC50 range: 4−49 nM).Interestingly, compounds 67−70 also demonstrated a 5−10-fold increase in potency against mIDH1-R132C compared with63−66. However, most of the compounds (65−70) sufferedfrom poor solubility. Considering a combination of potency,selectivity, and druglike properties, compounds 63 and 67 wereselected as preclinical candidates for further exploration.

In Vitro and in Vivo Biology and PharmacologyProfiling of 63 and 67. Profiling in various mIDH1-expressingU87 cells revealed that compound 63 inhibited 2-HGproduction by several IDH1-R132 mutants (R132C, R132G,R132L) with similar potency as that observed for R132H (Table12), although it is a weaker inhibitor for the R132S mutant.Interestingly, compound 67, which has only a small structuralchange (7-OMe vs 7-H), showed a 10-fold improvement incellular potency across all mutants. High passive cellularpermeability of compounds 63 and 67 was seen both in an

Figure 5. Energy vs conformation for fragments of compound 24. Torsion angles φ1 and φ2, given in degrees, are the dihedral angles around the firsttwo bonds in the linker from the quinolinone (C−C and C−N bonds, respectively). Energies determined by ab initio calculations (B3LYP/6-311G**+) are contoured at an interval of 2 kcal/mol. The conformation of the linker seen in the IDH1 co-crystal structure of 24 is marked with a red“X”. (A) results for the unsubstituted linker. (B) results for the (S)-methyl linker. In some configurations (particularly withφ1∼ 60°), themethyl grouphas a steric clash with the carbonyl, causing these configurations to be disfavored.

Table 9. Exploration of Linker SAR



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Table 10. Combination of the “Magic” (S)-Me Linker and Various RHS

biochem assays cell assays

cpdno.

IDH1-R132H, IC50(μM)

IDH1-R132C, IC50(μM)

HCT116 2HG IDH1-R132H,IC50 (μM)

HCT116 2HG IDH1-R132C,IC50 (μM)

HLM/MLM,left % 30 min

LogD(7.4)

solubility(μM)

56 0.020 0.183 0.234 0.269 98/94 3.74 5.759 0.014 0.111 0.007 0.213 75/71 3.67 4.160 0.017 0.178 0.051 0.411 100/100 3.38 15.661 0.024 0.190 0.152 0.092 82/nd 3.47 0.0862 0.044 1.015 0.1851 0.338 100/60 2.50 4.863 0.018 0.130 0.045 0.233 100/100 2.68 10.564 0.040 0.443 0.293 0.293 97/98 3.11 22.4

Table 11. Optimization of LHS of 63



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artificial membrane [parallel artificial membrane permeationassay (PAMPA)] and a cancer coli-2 (Caco-2) cell system(Table 11). Both compounds have good cell permeability andlow efflux ratio (ER: 1.00 for 63; 1.65 for 67), which indicate alow probability of P-glycoprotein driven efflux. As the MDR1-MDCK assay has been widely used as a surrogate model for BBBpenetration to further understand if compound 63 is a P-gpsubstrate or is a likely brain-penetrant CNS drug candidate, wemeasured the permeability of 63 in both wild-type and MDR1-MDCK assays. As shown in Table 13, compound 63 has highcell permeability and a low efflux ratio. Its net efflux ratio(ERMDR1‑MDCK/ERMDCK = 1.89) indicates that it is not a P-gpsubstrate. Together with low efflux ratios in Caco-2 and MDR1-MDCK artificial membranes, high permeability (MDCK Papp),and good microsomal stability, compound 63 has the potentialto cross the blood−brain barrier and may have potentialtherapeutic utility against mt-IDH1-harboring brain tumors.In vitro and in vivo pharmacokinetic profiling of 63 and 67

was performed across multiple species (rat, mouse, and dog).Low clearance (CL) in vitro was observed in all species for bothinhibitors (Table 14). The plasma protein binding (PPB) of 63is slightly lower than the PPB of 67 in human, mouse, rat, anddog probably due to its lower LogD. The data in Table 14 also

revealed that both 63 and 67 have low clearance in humanhepatocytes with half-lives of 4.3 and 3.1 h, respectively.The PK profiles of 63 and 67 were assessed in several

preclinical species (mouse, rat, and dog). The results fromintravenous (IV) and oral (PO) administration are summarizedbelow (Table 15). Both compounds showed low in vivoclearance and high oral exposure in rat, mouse, and dog. The PKprofile of 63 is clearly superior to that of 67 in rat and mouse. Ifplasma protein binding values (PPB%) of 63 and 67 are used todetermine unbound plasma AUCinf and Cmax for each species,inhibitor 63 has a 10-fold higher free drug concentration in bothrat (AUCunb: 0.459 vs 0.043 μMh) and mouse (AUCunb: 2.06 vs0.178 μMh). In the dog intravenous PK study, although 67 has alonger half-life and lower clearance than 63, both compoundshave nearly identical unbound plasma concentrations. Giventhat compound 63 has superior potency and PK propertiesoverall, it was selected as the candidate for further preclinicalstudies.

In Vivo Pharmacology Study of 63. To assess theactivities of 63 in vivo, PK−pharmacodynamics (PD) experi-ments in a mouse xenograft tumor model were used todetermine the degree of exposure required to suppress 2-HGlevels. Compound 63 was administered to HCT116-IDH1-

Table 12. Cellular Activity (IC50: μM) in Inhibiting 2-HG in Various mt-IDH1 Expressing U87 Cells

IDH1 mutation (IC50, μM) bidirectional permeability in Caco-2 cell line

cpd no. R132H R132C R132G R132L R132S PAMPAa 10−6 cm/s mean Papp (B−A) (10−6, cm/s) Papp (B−A) (10−6, cm/s) efflux ratio

63 0.018 0.13 0.12 0.06 1.49 18.0 8.66 8.64 1.0067 0.001 0.004 0.003 0.005 0.129 12.7 5.73 9.47 1.65

aPAMPA: parallel artificial membrane permeation assay.

Table 13. Permeability of 63 in MDCK Cell Line

bidirectional permeability in MDCK cell linea bidirectional permeability in MDR1-MDCK cell lineb

cpd no. Papp (A−B) (10−6, cm/s) Papp (B−A) (10−6, cm/s) efflux ratio Papp (A−B) (10−6, cm/s) Papp (B−A) (10−6, cm/s) efflux ratio net efflux ratio

63 12.61 7.97 0.63 8.35 9.93 1.19 1.89aMadin−Darby canine kidney (MDCK) epithelial cells. bMDR1-MDCK cells originate from the transfection of MDCK cells with the MDR1 gene,the gene encoding for the efflux protein, P-glycoprotein (P-gp).

Table 14. ADME Profiles of Selected Preclinical Candidates 63 and 67a

human hepatocytes

cpdno.

human PPB(%)

mouse PPB(%)

rat PPB(%)

dog PPB(%)

media PPB(%)

LogD(7.4)

RLM/HLM/MLM/DLM Clint(mL/(min mg))

T1/2(h)

Clint (total)(mL/(min kg))

63 98.10 98.07 97.97 96.31 76.04 2.68 7.0/7.0/7.0/7.0 4.32 13.6567 98.56 99.17 99.55 97.72 65.38 2.74 10.1/7.2/7.0/7.0 3.07 19.14

aPPB (%) is the percentage of plasma protein binding; RLM is rat liver microsome; HLM is human liver microsome; MLM is mouse livermicrosome; and DLM is dog liver microsome.

Table 15. PK Profiles of Selected Preclinical Candidates 63 and 67 in Preclinical Species

rat PK (IV_1 mg/kg; PO_5 mg/kg) mouse PK (IV_1 mg/kg; PO_5 mg/kg) dog PK (IV, 0.5 mg/kg)

cpdno.

t1/2(h)

Cmax(μM)total

AUC inf(μM h) total

(free)

CL(mL/(minkg)) F%

t1/2(h)

Cmax(μM)total

AUC inf (μMh) total (free)

CL(mL/(min kg)) F%

t1/2(h)

AUC inf (μMh) total (free)

CL(mL/(min kg))

63 2.2 3.8 22.6 (0.459) 8.6 81 10.0 5.9 106.7 (2.06) 3.3 130 5.2 3.3 (0.123) 7.767 2.6 1.9 10.7 (0.043) 16.4 75 3.5 5.2 21.5 (0.178) 8.5 85 6.2 5.8 (0.132) 4.3



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R132H/+ or HCT116-IDH1-R132C/+ xenograft bearingfemale BALB/c Nude mice at three doses (5, 12.5, 25 mg/kg), administered at 12 h intervals. Plasma and xenograft tumor

samples were collected at 4, 12, and 24 h post last dose todetermine the exposure of 63 in plasma and tumor, as well as tomeasure the inhibition of mIDH1 activity (as measured by a

Figure 6. (a−d) Compound 63 PK/PD study in HCT116-IDH1-R132H/+ xenograft model. (a) Free concentration of 63 in plasma after three-doseoral administration (5, 12.5, and 25 mg/kg) with a 12 h dosing interval in a mouse HCT116-IDH1-R132H/+ xenograft model; (b) free concentrationof 63 in the tumor after three-dose oral administration (5, 12.5, and 25 mg/kg) with a 12 h dosing interval in a mouse HCT116-IDH1-R132H/+xenograft model; (c) percent 2-HG inhibition in tumors with PO doses of 5, 12.5, and 25 mpk at three different time points (4, 12, 24 h). (d) In vivoactivity (2-HG % inhibition) of 63 vs free compound concentration in the tumor.

Figure 7. (a−d) Compound 63 PK/PD study in HCT116-IDH1-R132C/+ xenograft model. (a) Free concentration of 63 in plasma after three-doseoral administration (5, 12.5, and 25mg/kg) with a 12 h dosing interval in mouse HCT116-IDH1-R132C/+ xenograft model; (b) free concentration of63 in the tumor after three-dose oral administration (5, 12.5, and 25 mg/kg) with a 12 h dosing interval in a mouse HCT116-IDH1-R132C/+xenograft model; (c) Percent 2-HG inhibition in tumors with PO doses of 5, 12.5, and 25 mpk at three different time points (4, 12, 24 h). (d) In vivoactivity (2-HG % inhibition) of 63 vs free compound concentration in the tumor.



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reduction in levels of 2-HG) in the tumor. Compound 63 is98.1% mouse plasma protein bound, hence the fractionunbound (fu) is 0.019. We used this factor to convert the totalcompound concentration to free (unbound) concentration inboth plasma and tumor.In both IDH1 mutant models, the free concentration of 63

was comparable in plasma and xenograft tumors, and exposureswere dose-dependent (Figures 6 and 7). In comparison to thevehicle-treated group, 63 showed a time- and dose-dependentinhibition of 2-HG levels in plasma (Figures 6a and 7a) and inthe tumor (Figures 6b and 7b). At the highest dose tested inthese studies (25 mg/kg), treatment with 63 inhibited 2-HGlevels in the tumor by >90% for up to 24 h after the last dose inthe HCT116-IDH1-R132H/+ xenograft model (Figure 6c) andto similar levels for at least 12 h in theHCT116-IDH1-R132C/+model (Figure 7c). Calculations based upon the percentage ofsuppression of 2-HG concentration in the tumor versus the freedrug concentration in the tumor gave in vivo IC50 values of 49and 46 nM in the HCT116-IDH1-R132H or HCT116-IDH1-R132C models, respectively (Figures 6d and 7d). Whencorrected for unbound levels of 63, there is an excellentcorrelation in potency among the biochemical assay, cellularassay, and in vivo studies (Table 16).

As shown in Figure 6c, the PK/PD studies of 63 in theHCT116-IDH1-R132H/+ xenograft model suggested that a

12.5 mpk BID (twice daily) or 25 mpk QD (once daily)schedule is required to maintain continuous >90% 2-HGinhibition in the tumor, whereas results from the HCT116-IDH1-R132C/+ xenograft model suggest that a 25 mpk BIDdose schedule is needed to maintain unbound drug levels aboveIC90 (>90% 2-HG inhibition) in the tumor (Figure 7c). Inaddition to the investigation of the PK/PD properties of 63, wefurther explored its suitability as a clinical candidate, includingan assessment of physical properties and ADME/safety profile.As summarized in Table 17, compound 63 is a potent mIDH1inhibitor with a favorable druglike property profile. Compound63 demonstrated excellent in vitro liver microsome and plasmastability across species and most likely has limited potential fordrug−drug interactions as it showed low inhibitory activityagainst a panel of cytochrome P450 (CYP450) enzymes (e.g.,15% inhibition for CYP3A4 at 10 μM). Reversible and time-dependent inhibition (TDI) of CYP3A4 by compound 63 wasalso investigated. The data suggested that compound 63 isneither a time-dependent nor time-independent inhibitor ofCYP3A4 below 5 μM (shown in the Supporting Information).Although compound 63 has moderate human ether-a-go-go-related gene (hERG) activity (3.2 μM), it showed a potentialsafety window of 200-fold with respect to potency for IDH1-R132H. We envision that the reduction of the lipophilicity of 63(lower LogD7.4) could be the way to further decrease its hERGactivity.

■ CHEMISTRYThe syntheses of quinolinones 2−41 were carried out from thecommercially available 6- or 7-substituted 2-oxo-1,2-dihydro-quinoline-3-carbaldehydes (2a−41a) through reductive amina-tion using suitable amines or anilines, as shown in Scheme 1.Detailed procedures and reaction conditions for the preparationof 2−41 have been previously described.32−34

Table 16. Biochemical, in Vitro Cellular Potency (Unbound),and in Vivo Potency (Unbound) for 63

HCT116_mIDH1-R132H HCT116_mIDH1-R132C

enzyme(nM)

cell 2-HG(nM)

in vivo 2-HG(nM)

enzyme(nM)

cell 2-HG(nM)

in vivo 2-HG (nM)

18 45 49 130 52 46

Table 17. Summary of Key Properties and Activities of 63

on-target efficacy

mIDH1-R132H IC50 (μM) 0.018mIDH1-R132C IC50 (μM) 0.130cell HCT116-R132H IC50 (μM) 0.045cell HCT116-R132C IC50 (μM) 0.233PK(ub)-PD HCT116-R132H 2HG IC50 (μM) 0.049PK(ub)-PD HCT116-R132C 2HG IC50 (μM) 0.046

target-related selectivity

wild-type IDH1 IC50 (μM) 35.0mIDH2-R172K IC50 (μM) 33.8mIDH2-R140Q IC50 (μM) 76.6

physical properties

MW/cLogD (7.4)/PSA 356/2.68/100kinetic solubility, pH = 7.4 (μM) 9.4CaCo-2 Papp A−B/B−A (cm−6/s) (efflux ratio) 8.66/8.64 (ER: 1.00)MDR1-MDCK Papp A−B/B−A (cm−6/s) (efflux ratio) 8.35/9.93 (ER: 1.19)

ADME and safety profiling

CYP3A4/2D6/2C9/2C19/1A2 (% inhibition @ 10 μM) 15.4/10.2/13.6/22.7/35.4CYP3A4 TDI IC50 0/30 min ± NADPH (μM)a no IC50 shiftPXR HepG2 @10 μM (% rel rifampicin)a 15liver microsome stability MLM/RLM/DML/HLM (% remain 30 min) 106/100/94/10844 kinase panel profileb two receptors >50% inhibitionc

hERG PatchClamp IC50 (μM)d 3.2

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



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http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.9b00362/suppl_file/jm9b00362_si_001.pdf


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

compounds 45−52 were prepared via the Mitsunobu reactionor alkylation of 44e or 45g with an appropriate alcohol or alkylhalide in either singleton or library fashion. Intermediate 45g,the 8-hydroxy quinolinone analog, was prepared by a differentroute as described in the Supporting Information.We have developed an effective synthetic route to prepare

(S)-3-(1-aminoethyl)-6-chloroquinolin-2(1H)-one hydrochlor-ide 71 with high optical purity (Scheme 4).32−34 In this route,Ellman’s chiral sulfinamide ((R)-2-methylpropane-2-sulfina-mide) was used to stereoselectively introduce the benzylicmethyl group.36,37 After condensation of Ellman’s sulfinamide

Scheme 1. Synthesis of Quinolinone Derivatives 2−41a

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

Scheme 2. Synthesis of Compound 44a

aReagents and conditions: (a) DMF, POCl3, 80 °C, overnight; (b) 12 M HCl, reflux, 24 h; (c) HBr, 115 °C, 4 days; (d) 4-amino-2-methoxybenzonitrile, 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

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



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with aldehyde 71a, the resulting imine 71b was treated withmethylmagnesium bromide to provide the sulfinamide 71c. Thepure (R,S)-diastereomer was easily isolated by columnchromatography in high yield. Cleavage of the chiral auxiliaryand simultaneous hydrolysis of the 2-chloroquinoline moietyunder mildly acidic conditions (1 N HCl in dioxane) gave theamine 71 in quantitative yield as the hydrochloride salt. Thesame methodology was employed to prepare the oppositeenantiomer, (R)-3-(1-aminoethyl)-6-chloroquinolin-2(1H)-one hydrochloride.With enantiomerically pure (S)-3-(1-aminoethyl)-6-chloro-

quinolin-2(1H)-one 71 in hand, we were able to prepare analogs56 and 59−64 via nucleophilic substitution with the appropriatearyl halides at elevated temperatures (Scheme 5).

The syntheses of compounds 57 and 58 required thepreparation of an intermediate bearing a 3-nitrile functionalgroup on the quinolinone core (Scheme 6). Friedlandercondensation of 2-amino-5-chlorobenzaldehyde with ethyl 2-cyanoacetate in refluxing ethanol provided 6-chloro-2-oxo-1,2-dihydroquinoline-3-carbonitrile (72) in good yield. Compound57 was then prepared by the conversion of nitrile 72 to the α,α-dimethyl-substituted carbinamine 73 with methylmagnesiumbromide in the presence Ti(OiPr)4,

38 followed by nucleophilicdisplacement of fluoride from 6-fluoro-2-methylnicotinonitrile.Compound 58 required the preparation of a cylclopropanatedintermediate, which was prepared using the proceduredeveloped by Bertus et al.39 The treatment of the quinolinonenitrile 72 with 1.1 equiv of Ti(OiPr)4 and 2.2 equiv of EtMgBr

Scheme 4. Synthesis of Intermediate 71a

aReagents 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%.

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

aReagents 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.

Scheme 6. Synthesis of Compounds 57 and 58a

aReagents 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.



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followed by the addition of a Lewis acid (BF3·OEt2) providedthe desired cylcopropanated intermediate 74. Nucleophilicsubstitution then afforded the final compound 58.The syntheses of the 7-substituted quinolinones 65−70

(Scheme 7) began with the acetylation of the requisite anilinesto provide acetanilides (65a−70a), which were then cyclizedwith in situ-generated Vilsmeier−Haack reagent to afford thevarious 7-substituted quinolinone intermediates (65b−70b).Subsequent transformations (steps e−k) were achieved usingprocedures and conditions detailed in our previous publicationsto obtain the final compounds.34−36

■ CONCLUSIONSIn summary, a novel quinolinone HTS hit 1 was identified andoptimized via singleton and combinatorial library syntheses togenerate early leads 24 and 32. X-ray structures unexpectedlyrevealed that both 24 and 32 bind in an allosteric, induced-fitpocket in the mIDH1-R132H protein. Structure-based rationaldesign guided the optimization of potency and druglikeproperties, leading to the identification of compound 63which proved suitable for exploring the effect of inhibition ofthe production of 2-HG by IDH1-R132H/IDH1-R132C inpreclinical in vivo PK/PD xenograft models. Compound 63 hasgood overall selectivity vs the wild-type IDH protein andpotently inhibits IDH1 mutants R132H, R132C, R132G, andR132L, suggesting broad utility across the various known R132mutations. Compound 63 demonstrated excellent cell perme-ability, oral bioavailability, and ADME/PK properties. Oraldosing of 63 in themousemIDH1 tumor xenograft model showsa robust reduction of the tumor-derived 2-HG level, which is aPD biomarker of mIDH1 activity. The preclinical profile ofcompound 63 suggests it may have potential as a treatment ofAML, GBM, or other forms of mIDH1-driven cancer.

■ EXPERIMENTAL SECTIONGeneral.Unless otherwise noted, reagents and solvents were used as

received from commercial suppliers. Proton nuclear magneticresonance (NMR) spectra were obtained on either the Bruker orVarian spectrometer at 300 MHz. Spectra are given in ppm (δ), andcoupling constant, J, is reported in hertz. Tetramethylsilane was used asan internal standard. Mass spectra were collected using a Waters ZQSingle Quad Mass Spectrometer (ion trap electrospray ionization

(ESI)). High-performance liquid chromatograph (HPLC) analyseswere obtained using a XBridge Phenyl or C18 column (5 μm, 50 × 4.6mm2, 150 × 4.6 mm2 or 250 × 4.6 mm2) with UV detection (Waters996 PDA) at 254 nm or 223 nm using a standard solvent gradientprogram (methods 1−3 shown in the Supporting Information).Racemic mixtures of final compounds were separated into individualenantiomers by chiral supercritical fluid chromatography (SFC) underthe indicated conditions. Chemical and chiral (where applicable)purities were >95% for all final compounds, as assessed by LC−MS andchiral SFC analysis, respectively. Further details on the analyticalconditions used for individual compounds may be found in theSupporting Information. High-resolution mass spectrometry (HRMS)data were collected on a Waters Time of Flight (Waters Acquity I ClassUPLC with Xevo G2-XS Q Tof HRMS and PDM-UPLC-HRMS-1)instrument using electrospray ionization.

Materials. HCT116-IDH1-R132H/+ and HCT116-IDH1-R132C/+ were licensed from Horizon (HD-104-021 and HD-104-013). HT1080 and U87MG cells were commercially available fromATCC. Various human IDH1-R132 mutant cDNA ORF clones werepurchased from Origene (R132H: RC400096; R132C: RC400097,R132L: RC400098; R132G: RC400099; R132S: RC400100). IDH1mutant expressing U87MG cells were generated by transfectingU87MG parent cells with mutant IDH1-R132 mutant cDNA andselecting under G418.

Synthesis and Characterization of Compounds 2−70. Detailsof compound synthesis and characterization can be either found belowor in the Experimental Section, Supporting Information.

4-(((6-Chloro-2-oxo-1,2-dihydroquinolin-3-yl)methyl)amino)-2-methoxybenzonitrile (24). To a 100 mL round-bottom flask wereadded 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, sodiumtriacetoxyborohydride (364 mg, 1.93 mmol) was added, and themixture was stirred at room temperature overnight. LC−MS indicatedonly about 50% conversion. The reaction mixture was diluted withEtOAc (60 mL) and washed with water (2×) and brine. The organicphase was dried over Na2SO4, filtered, and concentrated to yield acrude. The crude was dissolved in 3 mL of DMSO and purified bypreparativeHPLC to yield the desired product 24 (34mg, 10.4% yield).1H 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)-2-methylnicotinonitrile (32). A suspension of 6-chloro-2-oxo-1,2-

Scheme 7. General Synthetic Route for Compound 67 and Analogsa

aReagents 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)-2-methylpropane-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.



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dihydroquinoline-3-carbaldehyde (150 mg, 0.722 mmol) and 6-amino-2-methylnicotinonitrile (115 mg, 0.867 mmol) in DCE (20 mL) wastreated with AcOH (0.124 mL, 2.167 mmol) and stirred for 20 min.Sodium triacetoxyborohydride (459 mg, 2.167 mmol) was added. Themixture was placed under nitrogen and stirred at room temperature.After 30 min, the suspension went into the solution. The brownsolution was stirred at ambient temperature over the weekend, duringwhich time amaterial precipitated. Themixture 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) wasdissolved inmethanol, treated with silica gel, and evaporated. The crudematerial was chromatographed by Biotage MPLC (25 g silica gelcolumn)with 0−10%MeOH/DCM to yield the title compound 32 (40mg, 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 = 8.79Hz, 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 calcdfor 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-((2,6-Dichloroquinolin-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, 73.14mmol) in DCE (150 mL) was added CuSO4 (16.0 g, 100.25 mmol).The resulting mixture was stirred at 55 °C overnight. After thin-layerchromatography (TLC) and MS showed complete disappearance ofstarting materials, the mixture was cooled to room temperature andfiltered through a pad of Celite. The Celite was then rinsed with DCM.The filtrate was evaporated to dryness in vacuo and purified by SiO2column chromatography (0−25% hexanes/EtOAc) to afford the titlecompound 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-sulfina-mide (8.85 g, 26.88 mmol) in anhydrous DCM (200 mL) at −60 °Cwas added dropwise MeMgBr (3 M solution in diethyl ether, 13.5 mL,40.54 mmol). The resulting reaction mixture was stirred at about −60to −50 °C for 3 h and then stirred at −20 °C overnight under anatmosphere of N2. After TLC and MS showed complete disappearanceof starting materials, saturated NH4Cl (163 mL) was added at −20 °C,and the resulting mixture was stirred for 10min. The aqueous phase wasextracted with DCM (100 mL × 3), dried over anhydrous Na2SO4,filtered, and evaporated. The residue was purified by columnchromatography on an ISCO chromatography system (SiO2: goldcolumn; gradient; hexanes to 100% EtOAc) to provide the titlecompound 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-dichlor-oquinolin-3-yl)ethyl)-2-methylpropane-2-sulfinamide (6.6 g, 19.13mmol) in 1,4-dioxane (41 mL) and 1 N HCl (41 mL) was heated atreflux overnight. The solvents were evaporated in vacuo, and theresulting residue was dissolved in hot water and lyophilized. The crudeproduct was triturated with diethyl ether to afford the title compound71 as a yellow solid (4.9 g, 98.8% yield, enantiomeric excess: 98.4%). 1HNMR (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-(6-

Chloro-2-oxo-1,2-dihydroquinolin-3-yl)ethylamino)-2-methylnico-tinonitrile (56). A mixture of 6-fluoro-2-methylnicotinonitrile (28.6mg, 0.210 mmol) and (S)-3-(1-aminoethyl)-6-chloroquinolin-2(1H)-one hydrochloride (intermediate 71, 49.6 mg, 0.191 mmol) was treatedwith DMSO (1.4 mL) and DIEA (0.10 mL, 0.573 mmol). The solutionwas stirred at 110 °C for 2 h. LC−MS indicated that the reaction hadgone to completion. The sample was mixed with water (20 mL) andextracted with DCM (3 × 15 mL). The extracts were dried (Na2SO4),filtered, treated with silica gel, and evaporated under reduced pressure.

The material was chromatographed by BiotageMPLC (10 g of silica gelcolumn, 0−50% EtOAc in hexanes) to provide the title compound 56as a solid (51.5 mg, 0.145 mmol, 76% yield, HPLC purity >95% at 220nm). 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)-6-chloroquinolin-2(1H)-one hydrochloride 71 (201 mg, 0.776 mmol)and 4-fluoro-2-methoxybenzonitrile (236 mg, 1.56 mmol) in DMSO (5mL) was treated with DIEA (400 μL, 2.29 mmol) and stirred at 110 °Cfor 3 days. The sample was diluted with water (75 mL) and extractedwith DCM (2 × 50 mL), dried, and filtered. Silica gel was added, andthe solvent was evaporated under reduced pressure. The material waschromatographed by Biotage MPLC (silica gel, 0−70% EtOAc inhexanes, 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 ofyellow powder. The sample was mixed with MeCN (4 mL) and water(2 mL), frozen on a dry ice/acetone bath, and lyophilized to give thetitle 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): δppm 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 = 7.90Hz, 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 werecombined 6-chloro-4-methoxynicotinonitrile (1 g, 60 mmol), (S)-3-(1-aminoethyl)-6-chloroquinolin-2(1H)-one hydrochloride 71 (1.34 g, 53mmol), and DIEA (1.98 mL, 11.4 mmol) in 21 mL of EtOH (200proof). The reaction mixture was microwaved at 140 °C for 4.5 h,cooled to room temperature, and concentrated to dryness underreduced pressure. The material was purified twice by ISCO using a 40 gof “gold” column with a gradient elution of EtOAc in DCM to providethe 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 =2.46Hz, 1H), 7.29 (d, J = 8.52Hz, 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 forC18H16ClN4O2 [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)-6-chloroquinolin-2(1H)-one hydrochloride 71 (69.7 mg, 0.269 mmol)and 6-fluoro-2-methoxynicotinonitrile (45.2 mg, 0.297 mmol) inDMSO (1.5 mL) was treated with DIEA (141 μL, 0.807 mmol) andstirred at 110 °C for 1 h. LC−MS at 45 min showed that the reactionhad gone to completion. The sample was pipetted onto water (20 mL),resulting in the formation of a white precipitate. The precipitate wasextracted with EtOAc (2 × 15 mL), dried (Na2SO4), and filtered. Silicagel was added, and the solvent was evaporated under reduced pressure.The material was chromatographed by BiotageMPLC (10 g of silica gelcolumn) with 0−75% EtOAc in hexanes, with isocratic elution whenpeaks came off to provide the title compound 61 as a white solid (68.8mg, 0.194 mmol, 72.1% yield, HPLC purity 100% at 220 nm). 1HNMR(300MHz, 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, 2.35Hz, 1H), 7.29 (d, J = 9.09Hz, 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/zcalcd for C18H16ClN4O2 [M + H]+, 355.10; found, 355.00; Rt = 2.38min (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-



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ypicolinonitrile (47.6 mg, 0.313 mmol) and (S)-3-(1-aminoethyl)-6-chloroquinolin-2(1H)-one hydrochloride 71 (74.5 mg, 0.287 mmol) inDMSO (2 mL) was added DIEA (153 μL, 0.876 mmol). The solutionwas stirred at 110 °C for 12 h. Once LC−MS analysis indicated thatmost starting material was consumed, the mixture was diluted withwater (30 mL) in DCM (30 mL). The organic extract was dried overNa2SO4, filtered, and evaporated under reduced pressure. The crudematerial was purified by column chromatography on Biotagechromatography system (eluted with 0−50% EtOAc in hexanes) toprovide the title compound 62 (42.7 mg, 42% yield). 1H NMR (300MHz, 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-chloro-4-methoxypyrimidine-5-carbonitrile (65.4 mg, 0.386 mmol) and (S)-3-(1-aminoethyl)-6-chloroquinolin-2(1H)-one hydrochloride 71 (100mg, 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−MSanalysis indicated that most starting material was consumed, themixture was diluted with DCM and washed with water (2×) and brine(1×). The organic extract was dried over Na2SO4, filtered, andevaporated under reduced pressure. The crude material was purified bycolumn chromatography on Biotage chromatography system (elutedwith 0−80% EtOAc in hexanes) to provide the title compound 63 (34.1mg, >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 = 8.8Hz, 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; found356.0910.(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-chloroquinolin-2(1H)-one. A mixture of 5-bromo-2-chloro-4-methylpyrimidine (440mg, 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 wasstirred at 110 °C for 12 h. Once LC−MS indicated that most of thestarting material was consumed, the mixture was cooled to roomtemperature and stirred for 2 days. The solution was then diluted withwater and extracted with DCM (2×). The extracts were dried(Na2SO4), filtered, and evaporated under reduced pressure. Thecrude material was purified by silica gel chromatography on a Biotagechromatography 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-dihydroquino-

lin-3-yl)ethyl)amino)-4-methylpyrimidine-5-carbonitrile (64). Amixture of Pd2(dba)3 (11.63 mg, 0.013 mmol), dppf (14.0 mg, 0.025mmol), (S)-3-(1-((5-bromo-4-methylpyrimidin-2-yl)amino)ethyl)-6-chloroquinolin-2(1H)-one (635 mg, 1.613 mmol), and dicyanozinc(379mg, 3.23mmol) in DMF (30mL) was purged with nitrogen for 10min. The mixture was then heated at 120 °C overnight. Once LC−MSshowed 50% conversion, the volatiles were removed under vacuum.Water was added to the resulting residue, and solids were removed byfiltration. The crude material was purified by silica gel chromatographyon 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

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). Amixture of2-chloro-4-methoxypyrimidine-5-carbonitrile (45.9 mg, 0.271 mmol)and (S)-3-(1-aminoethyl)-6-chloro-7-methoxyquinolin-2(1H)-one hy-drochloride 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) wasadded, and the reaction mixture was extracted with DCM (2 × 15 mL).The extracts were dried (Na2SO4) and filtered. Silica gel was thenadded, and the solvent was evaporated under reduced pressure. Thematerial was purified by column chromatography on a Biotage MPLCchromatography system (10 g of silica gel column, eluted with 0−100%EtOAc in hexanes, with isocratic elution when peaks eluted). Theproduct fractions were washed with water (40 mL), dried (Na2SO4),filtered, and evaporated under reduced pressure. The material wastreated with MeCN (0.8 mL) and water (0.4 mL) to provide a thickslurry. The mixture was frozen on a dry ice/acetone bath, thenlyophilized 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 (300MHz, 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 (method2), >99% purity. HRMS (ESI) calcd for C18H17ClN5O3 [M + H]+,386.1020; found 386.1019.

IDH1-R132H and IDH1-R132C Enzymatic Assay. Assays wereperformed in a 384-well black plate. An aliquot of 250 nL of compoundwas incubated with 10 μL of 30 nM IDH1-R132H or 10 nM IDH1-R132C recombinant protein in assay buffer (50 mMTris pH = 7.5, 150mMNaCl, 5 mMMgCl2, 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 wascentrifuged briefly, an aliquot of 10 μL of 2 mM α-ketoglutarate and 20μM NADPH solution prepared in assay buffer was then added to eachwell, and the reaction was maintained at 25 °C for 45 min. An aliquot of10 μL of diaphorase solution (0.15 U/mL of diaphorase and 30 μMresazurin in assay buffer) was added to each well. The plate wasmaintained at 25 °C for 15 min and then read on a plate reader withexcitation 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 givenconcentration with the four-parameter logistic equation. Eachcompound was assayed in triplicate. The IC50 value is the mean ±standard deviation of multiple determinations.

Cellular 2-HG Assay Using HCT116 Mutant IDH1 Cells. HCT116isogenic IDH1-R132H and IDH1-R132Cmutant cells were cultured ingrowth media (McCoy’s 5A, 10% fetal bovine serum, 1× antibiotic−antimycotic solution and 0.3 mg/mL G418) in 5% CO2 in an incubatorat 37 °C. To prepare the assay, cells were trypsinized and resuspendedin assay media (McCoy’s 5A with no L-glutamine, 10% fetal bovineserum, 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 clear96-well tissue culture plate. The cells were incubated in 5% CO2 at 37°C in an incubator overnight to allow for proper cell attachment. Analiquot of 50 μL of compound containing assay media was then addedto each well, and the assay plate was kept in 5% CO2 at 37 °C in anincubator for 24 h. The media were then removed from each well, and150 μL of a methanol/water mixture (80/20 v/v) was added to eachwell. The plates were kept at −80 °C freezer overnight to allow forcomplete cell lysis. An aliquot of 125 μL of extracted supernatant wasanalyzed by RapidFire high-throughout-mass spectrometry (Agilent) todetermine the cellular 2-HG level. The IC50 of a given compound wascalculated by fitting the dose−response curve of cellular 2-HGinhibition at a given concentration with the four-parameter logisticequation.

In Vivo PKPD Studies Using HCT116-IDH1-R132H/+ or HCT116-R132C/+ Xenograft Mouse Model. An aliquot of 5X10E6 HCT116-IDH1-R132H/+ or HCT116-IDH1-R132C/+ cells in 100 μLphosphate-buffered saline was injected subcutaneously under the



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right flank of 6−8 weeks old female BALB/c nude mice. The tumor sizewas calipered and calculated by (a × b2/2), where “b” is the smallestdiameter 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 with10% ethanol, and 90% poly(ethylene glycol)400 was administratedorally three times with 12 h dosing interval. The dosing volume was 10mL/kg animal weight. The animals were euthanized at 4, 12, 24 h postlast dose. Plasma samples were collected for the measurement ofcompound 63 concentration in plasma. The tumor samples wereharvested 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 andstructure determination, data collection and refinement statistics tablesfor 24 (PDB: 6O2Y) and 32 (PDB: 6O2Z). X-ray structures are shownin the Supporting Information.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.jmed-chem.9b00362.

Protein expression and purification for crystallography(Section S2); crystallization and structure determinationof 24 and 32 (Section S3); crystal parameters, datacollection and refinement statistics for 24 and 32 (SectionS4); details of the synthetic procedures and analytical datafor intermediates 44a−70f, and compounds 24−70(Sections S4−S26); kinase profiling (Cerep Kinasepanel) of 63 (Section S27); PXR HepG2 assay andCYP3A4 TDI assay data for 63 (Section S29); hERGPatchClamp assay for 63 (S32); cell lines and cellularassays (Sections S32−S33) (PDF)Molecular formula strings are available separately incomma-separated values file format (CSV)

Accession CodesAtomic coordinates and experimental data for the co-crystalstructures of 24 (PDB: 6O2Y) and 32 (PDB: 6O2Z) in complexwith mIDH-R132H will be released upon article publication.

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: [email protected] (J.L.).*E-mail: [email protected] (C.D.).ORCIDJian Lin: 0000-0001-8428-7958Present Addresses†Casma Therapeutics, Cambridge, Massachusetts 02139,United States (J.L.).‡KSQ Therapeutics, Cambridge, Massachusetts 02139, UnitedStates (W.L.).∥Camp4 Therapeutics, Cambridge, Massachusetts 02139,United States (J.C.).§Ra Pharmaceuticals, Inc., Cambridge, Massachusetts 02140,United States (S.A.).NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank Dr. Anu Mahadevan and the chemists at Organix Inc.for their contribution to the syntheses of intermediates andcompounds; Drs. Adam J. Stein, Andre White and colleaguesfrom Xtal BioStructures, Inc. for solving X-ray structures of

compounds 24 and 32; CrownBio for PK/PD studies inHCT116-IDH1-R132H/+ and R132C/+ xenograft models;Drs. Rob Sarisky, Paul Ehrlich, HeshamMohamed, and FrancesDuffy-Warren for very helpful discussions.

■ ABBREVIATIONSADME, absorption, distribution, metabolism, and excretion;Arg, arginine; AUC, area under the curve; BBB, blood−brainbarrier; Brine, a saturated aqueous solution of sodium chloride;Caco-2, cancer coli-2; CL, clearance; Cmax, maximum concen-tration; 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-reso-lution mass spectrometry; IV, intravenous; IP, intraperitoneal;Ki, inhibition constant; LC−MS, liquid chromatography−massspectrometry; LogD7.4, log of partition coefficient betweenoctanol and pH7.4 aqueous buffer; MDCK, Madin−Darbycanine 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, pharmacoki-netics; PK/PD, pharmacokinetic−pharmacodynamic; PO, bymouth; PPB, plasma protein binding; THF, tetrahydrofuran;PSA, polar surface area

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