Mutant isocitrate dehydrogenase 1 (IDH1)
- IDH1 R132H: IC50 = 8 nM [3]
- IDH1 R132C: IC50 = 11 nM [3]
- Wild-type IDH1: IC50 = 180 nM [3]
The compound does not inhibit IDH2 R140Q, IDH2 R172Q, or wild-type IDH2 (IC50 > 10,000 nM) [3].

- DS-1001b treatment (1 and 10 µM for 72 hours) markedly decreased intracellular 2-HG levels in IDH1-mutant chondrosarcoma cell lines (JJ012 with IDH1 R132G and L835 with IDH1 R132C), but had no effect on IDH2-mutant (SW1353) or IDH wild-type (OUMS27, NDC5-1) cell lines. Intracellular D-2-HG levels were 100-fold higher than L-2-HG and were completely blocked by 1 µM DS-1001b in both IDH1-mutant cell lines. [2]
- DS-1001b impaired proliferation in JJ012 and L835 cells in a dose-dependent manner. The GI50 values were 81 nM (day 14) for JJ012 and 77 nM (6 weeks) for L835 cells. The drug had little effect on the proliferation of IDH wild-type cell lines (OUMS27 and NDC5-1; GI50 > 10 µM). [2]
- Under 3D spheroid and hypoxic (1% O2) conditions, DS-1001b decreased cell viability in a dose-dependent manner. GI50 values for JJ012 cells at 7 days were 289.6 nM (monolayer), 1,444 nM (spheroids), and 1,363 nM (1% O2). For L835 cells at 3 weeks, GI50 values were 160.9 nM (monolayer) and 2,878 nM (1% O2). [2]
- Treatment with DS-1001b decreased intracellular 2-HG levels in a dose-dependent manner in JJ012 and L835 cells, with complete blockage of 2-HG production at 1 µM for 72 hours. [2]
- In L835 cells, DS-1001b (10 µM for 6 weeks) upregulated chondrocyte differentiation pathway genes (SOX9, RUNX2, COL2A1, COL10A1, ACAN) at the mRNA and protein (SOX9) levels, as measured by RT-PCR and western blot. [2]
- In JJ012 cells, DS-1001b (1 µM for 7 days) induced G1 phase arrest (5.4% increase in G0/G1, 4.4% decrease in S phase), upregulated CDKN1C (p57) mRNA and protein expression, as shown by RT-PCR and western blot. [2]
- DS-1001b treatment (1 µM for 7 days) significantly reduced the levels of H3K4me3 and H3K9me3 in JJ012 and L835 cells, while H3K27me3 levels remained unchanged, as determined by western blot analysis. [2]
- Chromatin immunoprecipitation (ChIP)-qPCR showed that DS-1001b treatment (1 µM for 7 days) markedly reduced H3K9me3 levels at the SOX9 locus in L835 cells and at the CDKN1C locus in JJ012 cells. [2]
- DS-1001b treatment (100 nM for 2 weeks) in 3D pellet cultures of L835 cells increased lacunar spaces, decreased nuclear size and hyperchromasia, increased nuclear SOX9 expression, and significantly increased the Alcian blue-positive chondrocyte matrix area. [2]
- DS-1001b treatment inhibited production of 2-HG from cells with IDH1 R132H or IDH1 R132C at 20–50 nM. [3]

- In a xenograft mouse model with subcutaneous JJ012 (IDH1 R132G) tumors, continuous oral administration of DS-1001b (mixed in sterilized pellet food) significantly impaired subcutaneous tumor growth compared to the control diet group. Tumor weight was also markedly decreased in the treatment group at the final follow-up. [2]
- Intratumoral 2-HG levels were markedly lower in DS-1001b -treated mice than in untreated mice (P < 0.001). [2]
- DS-1001b suppressed the increase of plasma 2-HG levels in the mouse model, whereas levels gradually increased in untreated mice (P = 0.006). [2]
- RT-PCR confirmed that DS-1001b treatment significantly upregulated CDKN1C in xenograft tumors, consistent with in vitro results. Immunohistochemical analysis showed nuclear CDKN1C expression in some DS-1001b -treated JJ012 cells, which was not observed in untreated cells. [2]
- In a glioblastoma patient-derived xenograft (PDX) model with heterozygous IDH1 R132H, administration of DS-1001b showed a great reduction of 2-HG in the tumor and clear anti-tumor effects against subcutaneous A1074 PDX model. [3]

- DS-1001b ’s inhibitory activity on mutant and wild-type IDH enzymes was tested using in vitro enzyme assays. The IC50 values for IDH1 R132H, IDH1 R132C, and wild-type IDH1 were determined to be 8 nM, 11 nM, and 180 nM, respectively. The compound did not inhibit IDH2 R140Q, IDH2 R172Q, or wild-type IDH2 (IC50 values > 10,000 nM). [3]
- X-ray crystallography was performed on the ternary complex of IDH1 R132C, NADPH, and compound A (a DS-1001b derivative). The analysis revealed that compound A is located in the allosteric pocket at the dimer surface, and IDH1 R132C is in an "open" inactive form. [3]

- Cells (JJ012, L835, SW1353, OUMS27, NDC5-1) were treated with various concentrations of DS-1001b. For IC50/GI50 determination, cells were treated for varying durations (e.g., 7 days for JJ012, 3-6 weeks for L835). Cell proliferation was assessed by counting cumulative cell numbers. Cell viability under normoxic, hypoxic (1% O2), and 3D spheroid cultures was evaluated. [2]
- Intracellular levels of total 2-HG and enantiomers (D-2-HG and L-2-HG) were measured by LC-MS/MS and LC-TOFMS after 72 hours of DS-1001b treatment. [2]
- For RNA-seq analysis, L835 cells were treated with DS-1001b at 10 µM for 6 weeks, and JJ012 cells were treated with 1 µM for 7 days. Gene expression changes were analyzed by pathway analysis and confirmed by qRT-PCR. [2]
- Western blot analysis was performed on L835 cells treated with DS-1001b (0, 1, 10 µM for 6 weeks) and JJ012 cells (1 µM for 7 days) to assess protein levels of SOX9, CDKN1C, H3K4me3, H3K9me3, and H3K27me3. [2]
- Cell cycle progression in JJ012 cells treated with DS-1001b (1 µM for 7 days) was analyzed by flow cytometry using BrdU and 7-AAD staining. [2]
- For 3D pellet culture, L835 cells were cultured in chondrogenic medium to form pellets. After 4 weeks, pellets were treated with DS-1001b at 100 nM for an additional 2 weeks. Histological analysis included H&E, SOX9, Collagen II immunohistochemistry, and Alcian blue staining. [2]
- Chromatin immunoprecipitation (ChIP)-qPCR was performed on L835 and JJ012 cells treated with DS-1001b (1 µM for 7 days) using antibodies specific for H3K4me3, H3K9me3, and H3K27me3 at the SOX9 and CDKN1C loci. [2]

- For the in vivo efficacy study in chondrosarcoma model, NOD-SCID mice were subcutaneously transplanted with JJ012-Luc+ cells. Starting 3 weeks after transplantation (following confirmation of tumor engraftment by bioluminescence imaging), DS-1001b was administered continuously to mice (n=7) mixed with sterilized pellet food. The control group (n=5) received control diet. Tumor volume was measured, and bioluminescence imaging was performed to monitor tumor burden. Plasma 2-HG levels were measured every 2 or 3 weeks. At the final follow-up, intratumoral 2-HG levels, tumor weight, and CDKN1C expression (by qRT-PCR and immunohistochemistry) were evaluated. [2]
- In a glioblastoma patient-derived xenograft (PDX) model with IDH1 R132H, DS-1001b was administered to show anti-tumor effects and reduction of 2-HG in the tumor. The specific dosing regimen is not detailed in the provided text, but the compound was effective against the subcutaneous A1074 PDX model. [3]

- DS-1001b is an orally bioavailable compound. [2]
- Brain exposure of the compound was tested in mice using radioactivity of [14C]-labeled DS-1001a . The results suggested that DS-1001a (the active moiety of DS-1001b ) penetrates the blood-brain barrier (BBB). [3]
- The plasma concentration of DS-1001b in the mouse xenograft model was in the range of 2.4-10.4 µM. [2]

- In the mouse xenograft model, continuous administration of DS-1001b for up to 6 weeks did not cause body weight loss or other serious side effects, suggesting long-term continuous administration is clinically feasible. [2]

[1]. Isoxazole derivative as mutated isocitrate dehydrogenase 1 inhibitor. WO2016052697A1.

[2]. Selective inhibition of mutant IDH1 by DS-1001b ameliorates aberrant histone modifications and impairs tumor activity in chondrosarcoma. Oncogene. 2019 Oct;38(42):6835-6849.

[3]. Characterization of a novel BBB-permeable mutant IDH1 inhibitor, DS-1001b. Annals of Oncology, 2019, 30: v145-v146.

- DS-1001b is a clinical investigational drug. A Phase I clinical trial for treating glioma patients with IDH1 mutations is underway (ClinicalTrials.gov identifier: NCT03030066). [2][3]
- The patent application for DS-1001b is published under the Patent Cooperation Treaty with publication number WO2016/052697 A1. [2]
- The compound is a tert-butylamine salt of DS-1001a . [3]
- The compound is highly selective for mutant IDH1 over wild-type IDH1 and both mutant and wild-type IDH2. The IC50 for wild-type IDH1 is 180 nM, and >10,000 nM for all IDH2 forms tested. [3]

C29H29CL3FN3O4

608.91566824913

607.12

1898207-64-1

Safusidenib;1898206-17-1

139600317

White to off-white solid powder

2

7

5

40

859

0

ClC1C=C(C=C(C=1C1C(C(N2C=C(C)C3C(/C=C/C(=O)O)=CC=CC2=3)=O)=C(C(C)(C)F)ON=1)Cl)Cl.NC(C)(C)C

UPPAAWQBZQBNIE-USRGLUTNSA-N

InChI=1S/C25H18Cl3FN2O4.C4H11N/c1-12-11-31(17-6-4-5-13(19(12)17)7-8-18(32)33)24(34)21-22(30-35-23(21)25(2,3)29)20-15(27)9-14(26)10-16(20)281-4(2,3)5/h4-11H,1-3H3,(H,32,33)5H2,1-3H3/b8-7+

t-Butylamine (E)-3-(1-(5-(2-fluoropropan-2-yl)-3-(2,4,6-trichlorophenyl)isoxazole-4-carbonyl)-3-methyl-1H-indol-4-yl)acrylate

DS-1001B DS 1001B DS1001B DS-1001 DS 1001 DS1001.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~29 mg/mL (~47.63 mM)

Solubility in Formulation 1: ≥ 2.42 mg/mL (3.97 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 24.2 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.42 mg/mL (3.97 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 24.2 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)