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Discovery of AG-120 (Ivosidenib): A First-in-Class Mutant IDH1 Inhibitor for the Treatment of IDH1 Mutant Cancers

Somatic point mutations at a key arginine residue (R132) within the active site of the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) confer a novel gain of function in cancer cells, resulting in the production of d-2-hydroxyglutarate (2-HG), an oncometabolite. Elevated 2-HG levels are implicated in epigenetic alterations and impaired cellular differentiation. IDH1 mutations have been described in an array of hematologic malignancies and solid tumors. Here, we report the discovery of AG-120 (ivosidenib), an inhibitor of the IDH1 mutant enzyme that exhibits profound 2-HG lowering in tumor models and the ability to effect differentiation of primary patient AML samples ex vivo. Preliminary data from phase 1 clinical trials enrolling patients with cancers harboring an IDH1 mutation indicate that AG-120 has an acceptable safety profile and clinical activity.


Inhibitory Behavior of AG-120
In vitro, AG-120 showed no time-dependence in IC50 values between 1 and 16 hours of incubation for the mIDH1-R132 homodimer. AG-120 showed non-competitive inhibitory behavior with respect to the α-KG substrate ( Figure S1) and appears to be non-competitive with respect to the NADPH cofactor by standard equilibrium kinetic analysis ( Figure S2).
These observations are consistent with AG-120 being a rapid equilibrium inhibitor of the mIDH1-R132H homodimer in the presence or absence of substrate and cofactor. However, we cannot confirm this mode of action because NADPH is found to be tightly pre-bound in recombinant enzyme preparations. 1 Against the IDH1-WT homodimer, AG-120 showed uncompetitive inhibition with respect to the NADP cofactor ( Figure S3). In the presence of increasing concentrations of AG-120, the observed rate constant (kobs) was a hyperbolic function of inhibitor concentration. Along with the observed time-dependence in IC50 values between 1 and 16 hours, these data are consistent with AG-120 being a slow-binding inhibitor of the IDH1-WT homodimer ( Figure S4).
With respect to the inhibition of the IDH1-WT enzyme, it is worth noting that mice with complete knock-out of Idh1 showed completely normal development and fertility, although they were more susceptible to lethal doses of lipopolysaccharide than WT litter-mates, 2 suggesting that inhibition of IDH1 under homeostasis should be tolerable. Overall, preclinical profiling of AG-120 fully supports further clinical studies in the context of diseases associated with mutation of IDH1. Tables   Table S1. In vitro ADME and in vivo PK properties of AGI-14100

Supplementary Figures and
In vitro ADME   . AG-120 showed non-competitive inhibition with respect to alpha-ketoglutarate (α-  AG-120 is a non-competitive inhibitor of the mIDH1-R132H homodimer with respect to NADPH. Linear regression of the reciprocal plots for each titration series of varying cofactor versus fixed concentration of inhibitor intersects at a negative value on the x-axis, indicative of a non-competitive mode of inhibition. However, it is not conclusive that AG-120 can bind to the apo-form of the enzyme given that recombinantly expressed mIDH1-R132 enzymes exhibit low nM Km values for NADPH and already contain pre-bound co-factor (observed in crystallography studies, data not shown).

Figure S3
. AG-120 is an uncompetitive inhibitor of the IDH1-WT homodimer with respect to NADP  In the presence of increasing concentrations of AG-120, the observed rate constant (kobs) is a hyperbolic function of inhibitor concentration, indicative of AG-120 being a slow-binding inhibitor of the IDH1-WT homodimer.   Enhanced ability of progenitor cells to form differentiated colonies, consistent with the onset of cellular differentiation, was observed for mIDH1, but not IDH1-WT, patient myeloblasts when grown for 12 days in methylcellulose in the presence of AG-120 (5 μM) compared with vehicle control (DMSO, Figure S5A). Maturation of AML blasts was also evaluated by flow cytometry analysis for changes in cell-surface markers associated with myeloid differentiation (CD15, CD24, CD11b, and CD14). Treatment of mIDH1 AML cells with AG-120 always induced increases in one or more of these differentiation markers, although there was significant heterogeneity in the marker(s) induced, as well as in the kinetics of their appearance, with both features varying from one patient sample to another. This is illustrated

A G -1 2 0 i s a n u n -c o m p e t i t i v e i n h i b i t o r o f I D H 1 -W T w i t h r e s p e c t t o N
in Figure S5B, which shows that treatment of mIDH1 blasts (from Patients #2 and #4) in liquid culture with AG-120 for 6 days induced an increase in the mean fluorescence intensities for CD15 and CD24 (the most uniform features of the AG-120-induced differentiation effect across AML samples), and in the side scatter (SSC, granularity) and forward scatter (FSC, relative size) parameters. Treatment of IDH1-WT AML cells with AG-120 never induced increases in the expression level of any of these markers (e.g. Patient #1 in Figure 5B), indicating that AG-120 is effective solely on mIDH1 cells. These results were confirmed by cytology as a decrease in the number of blast cells and an increase in the number of more mature myeloid cells (myelocytes and metamyelocytes) in the mIDH1 patient samples following 6 days of treatment with AG-120 (0.5 μM, 1 μM, or 5 μM) compared with DMSO control ( Figure S5C). These data show that AG-120 induces myeloid differentiation in mIDH1-R132H and -R132C, but not IDH1-WT, primary human AML BM samples.

Scheme S1. Synthesis of AG-120
Starting from the commercially available 3,3-difluorocyclobutan-1-amine hydrochloride 12, isocyanide 14 was easily obtained following procedures reported in the literature (see Supporting Information references) and used without further purification in the next reaction.

Expression and purification of WT and mutant IDH enzymes
WT and mutant IDH1 and IDH2 enzymes were expressed and purified as previously reported. 4 Determination of compound inhibition potency against the mIDH1-R132H enzyme reaction using a diaphorase/resazurin coupled system In the primary reaction, the reduction of α-KG acid to D-2-hydroxyglutarate ( Florescence was read on a Spectramax plate reader at Ex 544 Em 590. Recombinant protein was expressed and purified as previously described. 5 Compounds or compound dilutions were prepared in 100% dimethyl sulfoxide (DMSO) concentration and diluted 1:100 into the final reaction. mIDH1-R132C was assayed under similar conditions, with the exception that the 1X Buffer was 50 mM K2HPO4 pH 6.5, 40 mM NaHCO3, 5 mM MgCl2, 10% glycerol,

Assay of the IDH1-WT enzyme reaction for determination of inhibitor potency
IDH1-WT enzyme was assayed in a modified version of the assay used for mIDH1-R132H.
Since this enzyme converts NADP to NADPH stoichiometrically with the conversion of isocitrate to α-KG, NADPH product can be continuously assayed by direct coupling to the diaphorase/resazurin system and reading resorufin production at Ex 544 Em 590. Assays

Assay of the IDH2-WT enzyme reaction for determination of inhibitor potency
Inhibitory potency of compounds against the IDH2-WT enzyme was determined in a coupled assay to diaphorase. In this assay, production of NADPH by IDH2-WT was linked to a concomitant reduction of resazurin to the highly fluorescent resorufin. Enzyme was diluted to 0.06 g/mL in 40 L 1X Assay Buffer (150 mM NaCl, 50 mM potassium phosphate pH 7, 10 mM MgCl2, 10% glycerol, 2 mM B-ME, 0.03% BSA), to which 1 L of compound was added in DMSO. The mixture was incubated for 16 hours at room temperature (RT Buffer), incubated for 5 minutes at 25°C, and read as described above. mIDH2-R172K was assayed as for mIDH2-R140Q with the following modifications: 1.25 g/mL of protein was used, the Substrate Mix contained 50 M NADPH and 6.4 M α-KG, and the compound was incubated for 1 hour before starting the reaction.

Cell assays
Cells were seeded in their respective growth media at a density of 5000 (U87MG, HCCC-9810, COR-L 105) or 2500 (HT1080) cells/well into 96-well microtiter plates and incubated overnight at 37°C and 5% CO2. The next day, AG-120 was prepared in 100% DMSO as a 10 mM stock and then diluted in media for a final concentration of 0.1% DMSO. Highest concentration dose was 3 µM. Medium was removed from the cell plates and 200 µL of the compound dilutions were added to each well. For neurospheres, compounds and cells (40,000/well) were plated together at the same time. After 48 hours of incubation with compound at 37°C, 100 µL of media was removed from each well and analyzed as described below. The cell plates were then allowed to incubate another 24 hours. At 72 hours post compound addition, a 10 mL/plate of Promega Cell Titer Glo reagent was thawed and mixed.
The cell plate was removed from the incubator and allowed to equilibrate to RT. Then 100 µL of reagent was added to each well of media. The cell plate was placed on an orbital shaker for 10 minutes and then allowed to sit at RT for 20 minutes. The plate was then read for luminescence with an integration time of 500 ms to determine any compound effects on growth inhibition (half maximal inhibition of cell proliferation, GI50).
The concentrations of 2-HG in media were determined using a non-validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method.
The following protein precipitation method was used for sample preparation: value >0.98 in linear regression. The peak area ratios of the analyte relative to the internal standard were used for 2-HG quantitation. Linearity was achieved in the 2-HG concentration range from 10 to 30,000 ng/mL for cell pellets.

Generation of HT1080 mIDH1-R132C xenografts
All animal studies were approved by the Institutional Animal Care and Use Committee and conducted in compliance with all national and local guidelines and regulations.

IDH mutational status
Genomic DNA was purified from diagnostic PB and/or BM specimens using the Qiagen DNeasy extraction kit following the manufacturer's instructions. IDH1/2 mutational status was analyzed by PCR amplification of IDH genes (exon 4 genomic regions, containing the mutational hotspot codon mIDH1-R132 and codons mIDH2-R140 and -R172) and direct sequencing, as reported. [6][7] Ex vivo AG-120 treatment

General Synthetic Procedures and Experimental Notes
In the following examples, the chemical reagents were purchased from commercial sources (such as Alfa, Acros, Sigma Aldrich, TCI, and Shanghai Chemical Reagent Company) and used without further purification. Flash chromatography was performed on an Isolera One General procedure for the Ugi reaction [8][9][10][11][12][13][14][15] A mixture of aldehyde (3.5 mmol) and aniline (3.5 mmol) in MeOH (8 mL) was stirred at RT for 30 minutes. Then, acid (3.5 mmol) was added and the reaction mixture was stirred for another 30 minutes. Isocyanide (3.5 mmol) was then added. The resulting mixture was then stirred at RT overnight and quenched by addition of H2O. The resulting mixture was partitioned between ethyl acetate (EtOAc) and H2O. The organic layer was separated, washed with brine, dried over anhydrous Na2SO4, and then concentrated. The residue was purified by silica gel column chromatography using dichloromethane (DCM)/MeOH as eluent to afford the desired product.

Synthetic Details and Characterization of Compounds
Compound 2 was prepared according to the following scheme, using the following protocol: Step A: N-(3,3-difluorocyclobutyl)formamide (13). A 2-L sealed reactor was charged with 3,3-difluorocyclobutanamine hydrochloride ( The residual solid was dissolved in EtOAc (600 mL) and filtered. The filtrate was concentrated under reduced pressure. The residue was triturated with n-hexane (400 mL) and filtered. The solid was collected and dried to afford 74 g desired product (13) as off-white solid (86% yield). (
Step  Compound 3 was prepared according to the following scheme, using the following protocol: Step A: (S)-1-(methoxycarbonyl)pyrrolidine-2-carboxylic acid. Methyl carbonochloridate (1.65 g, 17.4 mmol) was added to a solution of (S)-pyrrolidine-2-carboxylic acid (22) (1 g, 8.7 mmol) in a solution of THF (10 mL) and saturated NaHCO3 (10 mL) at 0°C and stirred at RT overnight. The mixture was extracted with EtOAc and the organic layer was concentrated to afford the desired product 23 (1.2 g, 80% yield) as colorless oil.
Step B: Compound 3. The product was synthesized via the general procedure for the Ugi reaction set forth above. 1  Compound 4 was prepared according to the following scheme, using the following protocol:
Step  Compound 9 was prepared according to the following scheme, using the following protocol: Step A:
Evaporation of the solvent gave the desired product 29 (4.8 g, 92.3% yield) as colorless oil.
Step E: The same as general procedure for Buchwald reaction set forth above.
Step B: 3-Fluoro-5-nitrobenzene-1-sulfonyl chloride (36). Benzyl(3-fluoro-5nitrophenyl)sulfane 35 (3.0 g) was dissolved in DCM (30 mL). To this solution, deionized H2O (30 mL) was added. Then chlorine was bubbled slowly into the stirred reaction mixture until the complete consumption of the starting material as monitored by TLC. The organic layer was separated, washed with saturated aqueous Na2S2O3 solution, dried and concentrated to afford the crude product 36, which was used for the next step without further purification.
Step E: The same as general procedures for Ugi reaction set forth above.
Step F: The same as general procedures for Buchwald reaction set forth above.
The residue was purified by prep-TLC to afford 11 (34 mg, 46% yield). 1  Compound AG-120 was prepared according to the following scheme, using the following protocol: Step
Step B: Compound AG-120. Pd2(dba)3 (17 g, 18.6 mmol)  and stirred at 100C for 3 hours. The mixture was filtered and the filtrate was concentrated, then the diastereomeric residue was separated by column chromatography on silica gel and eluted with DCM/EtOAc (100:1 to 4:1) to give the desired isomer, which was then further purified by crystallization from EtOAc to get the desired product AG-120 (95 g, 22% yield, 99.7 %de) as a white solid. 1