Numidargistat (CB-1158 B, INCB-01158) is a novel, potent and orally bioavailable immunomodulating agent acting as an arginase inhibitor with anticancer effects. It exhibits IC50s of 86 nM and 296 nM for inhibitng recombinant human arginase 1 and recombinant human arginase 2, respectively. CB-1158 blocked myeloid cell-mediated suppression of T cell proliferation in vitro and reduced tumor growth in multiple mouse models of cancer, as a single agent and in combination with checkpoint blockade, adoptive T cell therapy, adoptive NK cell therapy, and the chemotherapy agent gemcitabine. CB-1158 increased tumor-infiltrating CD8+ T cells and NK cells, inflammatory cytokines, and expression of interferon-inducible genes. CB-1158 may be potentially useful in renal cell cancer, breast cancer, non-small cell lung cancer, acute myeloid leukemia, and other tumor types where arginase-secreting MDSCs are known to play an immunosuppressive role.

Physicochemical Properties

Molecular Formula	C11H22BN3O5
Molecular Weight	287.1204829216
Exact Mass	287.165
CAS #	2095732-06-0
Related CAS #	Numidargistat dihydrochloride;Numidargistat 2HCl (CB1158, INCB01158)
PubChem CID	131801114
Appearance	Off-white to light yellow solid powder
Hydrogen Bond Donor Count	5
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	6
Heavy Atom Count	20
Complexity	381
Defined Atom Stereocenter Count	3
SMILES	OC([C@@]1(CN(C([C@H](C)N)=O)C[C@@H]1CCCB(O)O)N)=O
InChi Key	ZZJLMZYUGLJBSO-LAEOZQHASA-N
InChi Code	InChI=1S/C11H22BN3O5/c1-7(13)9(16)15-5-8(3-2-4-12(19)20)11(14,6-15)10(17)18/h7-8,19-20H,2-6,13-14H2,1H3,(H,17,18)/t7-,8-,11-/m0/s1
Chemical Name	(3R,4S)-1-(L-alanyl)-3-amino-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid
Synonyms	CB-1158 INCB01158,CB1158 INCB 01158,CB 1158 INCB-01158, Numidargistat
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	IC50: 86 nM (Arginase 1); 296 nM (Arginase 2)[1]
ln Vitro	Numidargistat is a strong and orally accessible arginase inhibitor with IC50 values of 86 and 296 nM for recombinant human arginase 1 and 2, respectively. Numidargistat inhibits native rginase 1 (Arg1) in human granulocyte, erythrocyte, and hepatocyte lysate at IC50s of 178 nM, 116 nM, and 158 nM, respectively, and inhibits Arg1 in cancer patient plasma (IC50, 122 nM). Numidargistat effectively inhibits arginase in human HepG2, K562 cell lines, and primary human hepatocytes with IC50 values of 32, 139, and 210 μM, respectively. Numidargistat has no effect on NOS. Furthermore, Numidargistat is not directly cytotoxic to murine cancer cell lines [1].
ln Vivo	In mice, imidargistat (100 mg/kg, po, twice daily) reduces myeloid cells and increases the number of cytotoxic cells that infiltrate tumors. In mice containing CT26 cancer cells, the combination of fumarate plus gemcitabine or PD-L1 inhibition slows the formation of tumors[1].
Cell Assay	Intracellular arginase activity is determined for the arginase-expressing HepG2 and K-562 cell lines as follows. HepG2 cells are seeded at 100,000 cells per well one day prior to treatment with CB-1158. K-562 cells are seeded at 200,000 cells per well on the day of CB-1158 treatment. Cells are treated with a dose-titration of CB-1158 in SILAC RPMI-1640 media containing 5% heat-inactivated and dialyzed FBS, antibiotics/anti-mycotic, 10 mM L-arginine, 0.27 mM L-lysine, and 2 mM L-glutamine. The medium is harvested after 24 h and urea generated is determined. Wells containing media without cells are used as background controls. For assessing the effect of CB-1158 on Arg1 in primary hepatocytes, frozen human hepatocytes are thawed, allowed to adhere onto collagen-coated wells for 4 h, and then incubated for 48 h in SILAC-RPMI containing 10 mM L-ornithine, no L-arginine, and a dose-titration of CB-1158, at which time the media are analyzed for urea[1].
References	[1]. Inhibition of arginase by CB-1158 blocks myeloid cell-mediated immune suppression in the tumor microenvironment. J Immunother Cancer. 2017 Dec 19;5(1):101.
Additional Infomation	Numidargistat is an aminopyrrolidine that is pyrrolidine substituted by (2S)-2-aminopropanoyl, amino, carboxy, and 3-boronopropyl groups at positions 1, 3, 3, and 4 (the 3R,4S-stereoisomer). It is a selective arginase inhibitor being developed by Calithera Biosciences for the treatment of solid tumours. It has a role as an EC 3.5.3.1 (arginase) inhibitor, an antineoplastic agent and an immunomodulator. It is a member of boronic acids, a pyrrolidinecarboxylic acid, a N-acylpyrrolidine, an aminopyrrolidine, an amino monocarboxylic acid and a primary amino compound. CB-1158 is under investigation in clinical trial NCT03910530 (A Study of INCMGA00012, INCB001158, and the Combination in Japanese Participants With Advanced Solid Tumors). Numidargistat is an orally available inhibitor of arginase, a manganese-dependent enzyme that hydrolyzes the amino acid arginine to form ornithine and urea, with potential immunomodulating and antineoplastic activities. Upon administration, numidargistat inhibits the breakdown of arginine by arginase, which is produced by myeloid cells, and restores arginine levels. This allows arginine to stimulate the synthesis of nitric oxide and the secretion of pro-inflammatory cytokines and chemokines, which induces the proliferation and activation of T-cells. Therefore, this agent may prevent the immunosuppressive effects of tumor-infiltrating myeloid cells and promote lymphocyte-mediated immune responses against tumor cells. Arginase is produced by neutrophils, macrophages and myeloid-derived suppressor cells (MDSC) and plays a role in inflammation-associated immunosuppression.

Solubility Data

Solubility (In Vitro)

DMSO : ~100 mg/mL (~348.29 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.71 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 25.0 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: ≥ 2.5 mg/mL (8.71 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 3: ≥ 2.5 mg/mL (8.71 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 25.0 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.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.4829 mL	17.4143 mL	34.8286 mL
	5 mM	0.6966 mL	3.4829 mL	6.9657 mL
	10 mM	0.3483 mL	1.7414 mL	3.4829 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.