Letter
`
`pubs.acs.org/acsmedchemlett
`
`†
`
`∥
`
`‡
`
`§
`
`Discovery of the First Potent Inhibitors of Mutant IDH1 That Lower
`Tumor 2‑HG in Vivo
`Janeta Popovici-Muller,*,†
`Shunqi Yan,
`Jeremy M. Travins,
`Francesco G. Salituro,
`Jeffrey O. Saunders,
`†
`†
`†
`†
`†
`⊥
`Fang Zhao,
`Stefan Gross,
`Lenny Dang,
`Katharine E. Yen,
`Hua Yang,
`Kimberly S. Straley,
`†
`†
`∇
`#
`#
`#
`Shengfang Jin,
`Kaiko Kunii,
`Valeria R. Fantin,
`Shunan Zhang,
`Qiongqun Pan,
`Derek Shi,
`†
`†
`Scott A. Biller,
`and Shinsan M. Su
`†
`Agios Pharmaceuticals, 38 Sidney Street, Cambridge, Massachusetts 02139, United States
`‡
`Ember Therapeutics, 855 Boylston Street, 11th Floor, Suite B, Boston, Massachusetts 02116, United States
`§Sage Therapeutics, 215 First Street, Cambridge, Massachusetts 02141, United States
`∥
`Schrödinger, Inc., 120 West 45th Street, New York, New York 10036, United States
`⊥
`Sundia MediTech Company, Ltd., Building 8, 388 Jialilue Road, Zhangjiang High-Tech Park, Shanghai 201203, China
`∇
`Oncology Research Unit, Pfizer Worldwide Research and Development, La Jolla Laboratories, San Diego, California 92121, United
`States
`#Shanghai ChemPartner Co., LTD, 998 Halei Road, Zhangjiang Hi-tech Park, Pudong New Area, Shanghai 201203, China
`*S Supporting Information
`
`ABSTRACT: Optimization of a series of R132H IDH1
`inhibitors from a high throughput screen led to the first
`potent molecules that show robust tumor 2-HG inhibition in a
`xenograft model. Compound 35 shows good potency in the
`U87 R132H cell based assay and ∼90% tumor 2-HG inhibition
`in the corresponding mouse xenograft model following BID
`dosing. The magnitude and duration of tumor 2-HG inhibition
`correlates with free plasma concentration.
`
`KEYWORDS: Mutant IDH1, tumor 2-HG, R132H IDH1 inhibitors
`
`T he family of isocitrate dehydrogenases (IDHs) includes
`
`two NADP dependent isoforms IDH1 and IDH2, which
`catalyze the oxidative decarboxylation of isocitrate to produce
`carbon dioxide, α-ketoglutarate (α-KG), and NADPH.1,2,14
`The implication of a role for IDH in cancer was revealed after
`somatic mutations in IDH1 were identified through a genome
`wide mutation analysis in glioblastoma.3 This landmark study
`was followed by high throughput sequencing, which revealed
`the presence of mutations in IDH1 in more than 70% of grade
`II−III gliomas and secondary glioblastomas,4 as well as in
`approximately 10−15% of patients with acute myeloid leukemia
`(AML).5 These somatic mutations were found at a key arginine
`residue belonging to the catalytic triad found in the enzyme’s
`active site (R132 for IDH1). This active site mutation results in
`loss-of-function for the oxidative decarboxylation of isocitrate
`and confers a novel gain-of-function for the production of the
`oncometabolite D-2-hydroxyglutarate (2-HG).6 Further charac-
`terization of
`the mutation showed that overexpression of
`mutant IDH1 in U87-MG, a human glioblastoma cell
`line,
`resulted in 100-fold elevated levels of 2-HG relative to the same
`cells expressing vector alone (data not shown).6 Recently, it
`was demonstrated that 2-HG is a competitive inhibitor of
`multiple α-KG-dependent dioxygenases, including histone and
`
`DNA demethylases,7,8 and several studies have shown that 2-
`HG producing IDH mutants are involved in global histone and
`DNA methylation alterations which may contribute to
`tumorigenesis through epigenetic rewiring.9,10 Taken together,
`these findings implicate mutant IDH1 as an oncogene and a
`compelling drug target for new therapies for glioma and AML
`patients.
`In order to identify small molecule inhibitors of IDH1,11,12
`we conducted a high-throughput screening (HTS) campaign
`against R132H IDH1 mutant protein homodimer. Library
`screen followed by confirmation of the active hits provided
`phenyl-glycine inhibitor 1. Detailed kinetic mechanism-of-
`action studies showed compound 1 binding to be reversible and
`behaving as competitive inhibitor with respect to α-KG and
`uncompetitive with respect to NADPH (data not shown).
`Given its attractive chemical
`structure and well-defined
`inhibitory properties, we selected this compound as a starting
`point for further optimization.
`
`Received: August 3, 2012
`Accepted: September 1, 2012
`Published: September 17, 2012
`
`© 2012 American Chemical Society
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`Figure 1. HTS hit 1 and phenyl-glycine scaffold synthesis.
`
`Figure 2. Key structural elements that influence the binding affinity of the phenyl-glycine scaffold.
`
`Table 1. C-Terminus R1 SAR
`
`aThe IC50 values for the R132H homodimer are the mean of at least two determinations performed as described in the Supporting Information.
`
`We report herein that optimization of 1 led to the
`identification of 35, the first reported R132H IDH1 inhibitor
`to show robust in vivo reduction of 2-HG levels in a tumor
`xenograft model.
`The phenyl-glycine scaffold was readily assembled via four
`component Ugi reaction,13 as depicted retrosynthetically in
`Figure 1. Compound 1 was synthesized using cyclopentyl
`isocyanide, o-methyl benzaldehyde, m-fluoroaniline, and (2-
`thiophen-2-yl) acetic acid as starting materials. If 2-chloroacetic
`acid is used for the Ugi acid component, intermediate 2 (R4 =
`Cl) is
`readily obtained and can be used for
`further
`functionalization at R4 through nucleophilic displacement of
`the chlorine.
`Upon identification of 1 as a screening hit, we set out to
`understand the key structural elements that were responsible
`for the binding affinity of this compound to the R132H IDH1
`
`protein (Figure 2). The molecule displays mostly hydrophobic
`features, with three aromatic rings positioned around two
`amide carbonyl groups, with a rather high clogP (5.6). Starting
`from a closely related analog 3 (IC50 = 0.08 μM), we first
`initiated a substitution pattern investigation of the phenyl-
`glycine backbone. The eutomer/distomer relationship of the α-
`carbon stereocenter was established by chiral synthesis of
`analog 3 starting from D-and L-mandelic acid,14 which provided
`4 (S) and 5 (R) enantiomers, respectively, with compound 4
`(IC50 = 0.06 μM) possessing essentially all of the activity found
`in the racemate. The enantiospecificity of
`this enzyme
`inhibition held true in many analogs subsequently investigated
`(data not shown).
`rapid exploration of structure−activity relationships
`For
`(SARs), all subsequent compounds were profiled in their
`racemic form. Geminal substitution at the α-carbon as depicted
`
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`Table 2. N-Terminus R4 SAR
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`Letter
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`aThe IC50 values for R132H homodimer are the mean of at least two determinations performed as described in the Supporting Information.
`
`for 6 incurred an 18-fold potency loss compared to the case of
`3. Next, alkylation of the secondary amide nitrogen as shown
`for 7 caused a 45-fold loss in potency compared to the case of
`3, while replacement of either the C-terminus or N-terminus
`carbonyl groups (compounds 8 and 9) with a CH2 moiety
`resulted in significant loss of biochemical activity, highlighting
`the importance of both amide moieties for binding affinity.
`
`We then started a systematic investigation of SAR for the N-
`and C-terminus regions of the scaffold, as well as the central
`aromatic moieties, with a key objective to improve properties,
`including decreasing the lipophilicity of the initial hit 1, while
`improving biochemical potency.
`R1 functional group exploration (Table 1) revealed that
`carbocycles were well tolerated, with cyclohexyl 10 slightly
`better (IC50 = 0.05 μM) than the starting HTS hit 1. As the
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`Table 3. Selectivity and Cell Based Profiling of Potent Phenyl-Glycine Analogs
`
`HT1080
`U87
`IDH1wt
`HT1080
`R132C
`U87
`R132H
`IC50 (μM)b
`GI50 (μM)
`IC50 (μM)
`IC50 (μM)a
`GI50 (μM)
`IC50 (μM)
`IC50(μM)a
`cLogP
`compd
`7.7 (32%)
`>20
`0.39
`0.05
`>20
`0.58
`0.09
`5.6
`1
`2.9 (22%)
`>20
`0.15
`0.03
`>20
`0.19
`0.05
`6.2
`10
`19.7 (32%)
`>20
`0.22
`0.06
`>20
`0.29
`0.10
`4.9
`18
`19.3 (34%)
`>20
`0.26
`0.05
`>20
`0.21
`0.05
`5.0
`19
`6.3 (39%)
`>3
`0.09
`0.03
`>20
`0.36
`0.06
`6.5
`20
`>100
`>20
`0.48
`0.16
`>20
`0.07
`0.07
`4.7
`35
`>100
`>20
`0.11
`0.04
`>20
`0.24
`0.08
`6.3
`36
`>100
`>20
`0.17
`0.04
`>20
`0.37
`0.07
`7.1
`37
`aThe IC50 values for R132H and R132C and wt homodimers are the mean of at least two determinations performed as described in the Supporting
`Information. bFor compounds with less than 100% enzyme inhibition, the maximum inhibition achieved is shown.
`
`Figure 3. Tumor 2-HG inhibition following one and three BID doses of 150 mg/kg of 35 via IP route in the U87 R132H tumor xenograft model.
`
`ring size decreased from cyclohexyl 10 to cyclopropyl 13,
`potency decreased gradually to low micromolar values.
`Replacement of cyclohexyl with aromatic rings (o-tolyl, benzyl)
`as shown for 14 and 15 led to a 10−30-fold decrease in
`biochemical potency compared to the case of 10. In an attempt
`to improve the properties of these compounds by decreasing
`the clogP through heteroatom substitution in the cyclohexyl
`ring, we found that pyran 16 was 10-fold less potent than 10,
`while piperidine 17 suffered a nearly 100-fold loss of
`biochemical potency.
`Evaluation of R2 substituents revealed that for the α-aromatic
`ring ortho-substitution was most favored, while replacement of
`the phenyl group with heterocycles or carbocycles afforded only
`low micromolar potency analogs.14 A preliminary survey of R3
`pointed to meta-substituted aromatic groups being most
`favorable, while aliphatic moieties, acyclic or cyclic, or
`heteroatom containing carbocycles provided analogs with a
`significant drop in biochemical potency.14 These observations
`coupled with the C-terminus amide SAR results shown in Table
`1 suggested that the phenyl-glycine scaffold was binding in a
`highly lipophilic region of the enzyme.
`Having elucidated SAR on three areas of the scaffold, we
`continued our exploration on the N-terminus R4 substituents in
`an additional approach to improve the compound physical
`chemical properties and decrease the overall lipophilicity of the
`original hit. Synthetic chemistry readily amenable to parallel
`arrays allowed us to rapidly explore a variety of functional
`groups at the N-terminus (Table 2).
`
`Replacement of thiophene in compound 1 by other carbon-
`linked heterocycles, such as thiazole 18, 4-pyridyl 19, or 3-
`indole 20 analogs, provided similar biochemical potency to 1 in
`the 0.05−0.1 μM range. Modification of the pyridine 19 to
`pyrimidine 21 caused a 22-fold drop in potency. Our
`investigation next focused on nitrogen linked systems directly
`prepared from intermediate 2 (R4 = Cl) via chlorine
`displacement (Figure 1). Replacement of chlorine in compound
`22 with amino group in analog 23 caused a potency drop from
`0.9 to 7.8 μM. A small set of cycloalkyl amines with an
`adjustment of ring size led to diminishing biochemical potency
`from cyclopropyl 24 (0.19 μM) to cyclohexyl 26 (1.27 μM).
`Interestingly, when the cyclohexyl group in 26 was replaced by
`a phenyl ring in compound 27, biochemical potency was
`substantially enhanced from 1.27 μM to 0.06 μM. Alkylation of
`the aniline nitrogen as shown in analog 28 maintained the
`potency at 0.05 μM. Use of aromatic rings for R4 did improve
`the potency compared to the aliphatic analogs (24−26);
`however, clogP increased as well,
`retaining their highly
`hydrophobic character. Encouraged by the good potency of
`aromatic tertiary amine 28, we next tested a small set of tertiary
`aliphatic amines, with the aim of decreasing the lipophilicity of
`the scaffold by introduction of basic solubilizing groups. Among
`the examples that were evaluated, morpholine 30 displayed the
`best biochemical potency (0.24 μM), while pyrrolidine 29 and
`piperazine 31 afforded weakly active single digit micromolar
`analogs. Addition of a fused phenyl ring to pyrrolidine as shown
`in 32 improved the potency by 10-fold at the expense of an
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`increased clogP value. We then continued exploration of
`nitrogen heterocycles from intermediate 2. Replacing the
`pyrrolidine ring in compound 29 with pyrazole 33 improved
`biochemical potency by 4-fold, while triazole 34 offered a slight
`improvement in binding affinity. Gratifyingly, N-(2-methyl)-
`imidazole 35 restored the biochemical potency to 0.07 μM,
`while fused nitrogen linked heterocycles (benzimidazole 26,
`indole 37) maintained the same potency at the expense of
`increased clogP values. Overall, R4-substitution allowed
`introduction of a diverse set of substituents that were well
`tolerated, possibly indicating that this part of the phenyl glycine
`scaffold may be binding in a solvent exposed area of the
`protein.
`As
`the SAR investigation revealed functional group
`modifications that provided potent inhibitors in the R132H
`enzymatic assay, we selected a focused set of analogs for
`evaluation against R132C IDH1 mutant15 and wild-type IDH1
`enzymes. Additionally, compounds were profiled in the
`glioblastoma U87 cells
`that overexpress mutant R132H
`IDH1, as well as the HT1080 chondrosarcoma cell
`line,
`which expresses the endogenous R132C IDH1 mutant.16 These
`cell lines produce significant levels of 2-HG compared to vector
`cells alone. Upon treatment with inhibitor for 48 h, the levels of
`2-HG were measured in the media by LCMS, to generate IC50
`values.14 Within the same experiment, 50% growth inhibition
`(GI50) was determined by measuring total cellular ATP after 72
`h of compound treatment.
`As shown in Table 3, the majority of compounds showed
`similar biochemical potency against the R132C IDH1 mutant
`and displayed cellular IC50 values less than 0.5 μM in both U87
`and HT1080 cell lines, with a 3−5-fold shift in enzyme to cell
`potency in most cases. Exquisite selectivity for R132H and
`R132C IDH1 mutant isoforms was demonstrated by the poor
`biochemical activity against the wild-type IDH1 and the lack of
`induction of nonspecific cell death (GI50 > 20 μM).
`Compound 35, equipotent in both enzyme R132H and U87
`cellular assays, was selected for additional in vivo profiling in the
`U87 R132H tumor xenograft mouse model (Figure 3). In vitro
`and in vivo DMPK studies were conducted for compound 35.
`This analog showed rapid turnover
`in human and rat
`microsomal incubations with an estimated hepatic extraction
`ratio of 0.93 and 0.85, respectively. Plasma protein binding was
`95.7% in mouse using the equilibrium dialysis method.
`Reasonable plasma exposure was achieved via intraperitoneal
`dosing at 50 mg/kg (AUC0−24h = 20800 h·ng/mL), enabling
`the use of inhibitor 35 for further in vivo studies. Female nude
`mice bearing U87 R132H tumor xenografts14 were dosed via IP
`route with 150 mg/kg of 35 formulated in 0.5% MC and 0.2%
`Tween 80, and then they were compared to the vehicle control
`animals. Blood and tumor samples were taken at different time
`points following compound administration. The plasma and
`inhibitor 35, as well as
`tumor concentrations of
`the
`corresponding tumor 2-HG concentrations were determined
`using sensitive and specific LC/MS/MS methods. The
`unbound plasma concentration of 35 was calculated using the
`total plasma concentration of 35 and free fraction of 35 in
`mouse plasma (4.3%).
`Following a single dose of 35, the estimated plasma free
`concentration of 35 was higher than the in vitro cellular IC50
`value (0.07 μM) for over 10 h. The magnitude and duration of
`tumor 2-HG inhibition correlated well with the free plasma
`concentration of 35. Compared to a single dose, a repeat dose
`of 35 provided longer exposure coverage time (drug exposure >
`
`Letter
`
`IC50) while the Cmax of 35 was similar following single and BID
`dosing. Better tumor 2-HG inhibition was achieved following
`BID dosing compared to a single dose, where the maximum
`tumor 2-HG inhibition was 89.4% and 69%, respectively. These
`results demonstrated that tumor 2-HG inhibition correlated
`with the duration of drug exposure and that robust tumor 2-HG
`inhibition is achievable with adequate and sustainable drug
`exposure.
`In conclusion, we have discovered the first class of potent
`IDH1 mutant inhibitors through optimization of HTS hits.
`Compound 35 is a potent inhibitor of 2-HG production in U87
`R132H cells and shows ∼90% tumor 2-HG inhibition in vivo
`following three BID doses. As high levels of 2-HG have been
`shown to alter the epigenetic state and biology of cells,9,10,17 the
`utility of this molecule will be important to assess the biological
`consequences of IDH mutations and the potential of IDH
`inhibitors for treating IDH mutant tumors.
`
`■ ASSOCIATED CONTENT
`*S Supporting Information
`in vivo studies,
`Experimental procedures for assay protocols,
`and synthesis and characterization of compounds. This material
`is available free of charge via the Internet at http://pubs.acs.org.
`
`■ AUTHOR INFORMATION
`Corresponding Author
`*Tel: (617) 649-8604. Fax: (617) 649-8618. E-mail: janeta.
`popovici-muller@agios.com.
`Notes
`The authors declare no competing financial interest.
`
`■ ACKNOWLEDGMENTS
`
`We thank Dr. Nageshwara Rao KV and Dr. Sarma BVNBS at
`SAI Advantium for their contribution to the synthesis of
`compound 8.
`
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`656−659.
`
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`View publication statsView publication stats
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`855
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`dx.doi.org/10.1021/ml300225h | ACS Med. Chem. Lett. 2012, 3, 850−855
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`Rigel Exhibit 1011
`Page 6 of 6
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