IACS-10759 HCl (IACS10759; IACS-010759) was identified as a potent and orally bioavailable inhibitor of complex I of oxidative phosphorylation (OXPHOS) with anticancer activity. In isolated mitochondria or permeabilized cells, ATP production or oxygen consumption was inhibited at single digit nM concentrations in the presence of malate/glutamate, but not succinate. More directly, IACS-10759 inhibited the conversion of NADH to NAD+ in an immunoprecipitated complex I assay at low nM concentrations. Using genetic and pharmacological approaches, the specific complex I subunit inhibited by IACS-10759 has been identified and the mechanism of complex I inhibition is being investigated. Importantly, IACS-10759 is orally bioavailable with excellent physicochemical properties in preclinical species and achieved significant in vivo efficacy with daily oral dosing of 10-25 mg/kg. Specifically, there was a >50 day extension of median survival in an orthotopic AML cell line xenograft and robust regression in DLBCL and GBM xenograft models. In light of these results, as well as its drug like profile IACS-10759 has entered IND enabling studies with first-in-human studies targeted for third quarter of 2015.

Physicochemical Properties

Molecular Formula	C25H26CLF3N6O4S
Molecular Weight	599.024953365326
Exact Mass	598.137
Elemental Analysis	C, 50.13; H, 4.38; Cl, 5.92; F, 9.51; N, 14.03; O, 10.68; S, 5.35
CAS #	1807523-99-4
Related CAS #	IACS-010759; 1570496-34-2; 1570496-34-2 (HCl); 1807524-00-0 (besylate); 1807524-05-5; 1807524-01-1 (mesylate)
PubChem CID	91864637
Appearance	Typically exists as white to off-white solids at room temperature
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	12
Rotatable Bond Count	7
Heavy Atom Count	40
Complexity	909
Defined Atom Stereocenter Count	0
InChi Key	LUSCFOVOISLXTM-UHFFFAOYSA-N
InChi Code	InChI=1S/C25H25F3N6O4S.ClH/c1-16-29-23(24-30-22(32-38-24)18-6-8-20(9-7-18)37-25(26,27)28)31-34(16)15-17-4-3-5-19(14-17)33-12-10-21(11-13-33)39(2,35)36;/h3-9,14,21H,10-13,15H2,1-2H3;1H
Chemical Name	5-(5-methyl-1-(3-(4-(methylsulfonyl)piperidin-1-yl)benzyl)-1H-1,2,4-triazol-3-yl)-3-(4-(trifluoromethoxy)phenyl) -1,2,4-oxadiazolehydrochloride
Synonyms	IACS010759 hydrochloride; IACS-010759; IACS 010759;IACS-10759 HCl; IACS 10759; IACS10759 HCl
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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.
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	complex I of oxidative phosphorylation (OXPHOS) Mitochondrial electron transport chain complex I (no specific IC50, Ki, or EC50 values provided) [2] Oxidative phosphorylation complex I (inhibited ATP production and oxygen consumption at single-digit nM concentrations in the presence of malate/glutamate; inhibited the conversion of NADH to NAD+ at low nM concentrations in an immunoprecipitated complex I assay) [1]
ln Vitro	IACS-010759 hydrochloride (10, 30, 100 nM; for 4 or 5 days) lowers the viability of primary AML and promotes apoptosis [1]. IACS-010759 hydrochloride (0.001, 0.01, 0.1, 1, 10, 100, 1000 nM; 72 h) substantially suppresses OCR and galactose-dependent H460 cell survival with practically the same IC50 value of 1.4 nM[1]. IACS-010759 hydrochloride has similar action in mouse (average IC50=5.6 nM), rat (IC50=12.2 nM) and cynomolgus monkey (IC50=8.7 nM) cell lines [1]. Treatment with IACS-010759 robustly inhibited proliferation and induced apoptosis in brain cancer and acute myeloid leukemia (AML) cell models reliant on oxidative phosphorylation (OXPHOS). The inhibitory effects are likely attributed to energy depletion and reduced aspartate production, which further impairs nucleotide biosynthesis [2] In isolated mitochondria or permeabilized cells, IACS-010759 inhibited ATP production and oxygen consumption at single-digit nM concentrations when malate/glutamate was present, but showed no inhibitory effect in the presence of succinate. Additionally, in an immunoprecipitated complex I assay, the compound inhibited the conversion of NADH to NAD+ at low nM concentrations [1]
ln Vivo	In mice with NB-1 (PGD null) subcutaneous xenografts, IACS-010759 hydrochloride (5, 10, 25 mg/kg/day; oral; for 21 days) at dosages of 5 or 10 mg/kg caused tumor reduction, negligible weight loss, and intolerance to the 25 mg/kg dose [1]. Less frequent dosing regimens (Q2D or Q3D) may somewhat enhance survival, but IACS-010759 hydrochloride (10?mg/kg; oral; QD (daily) or QD×5 (5 d on/2 d off); for 35 d) prolonged median survival from 28 days to over 60 days [1]. With a high volume of distribution and low plasma clearance, IACS-010759 hydrochloride (0.3 mg/kg for intravenous; 1 mg/kg for oral) has a prolonged terminal half-life (>24 hours) [1]. In in vivo models of brain cancer and AML, IACS-010759 potently inhibited tumor growth at well-tolerated doses [2] In an orthotopic AML cell line xenograft model, daily oral administration of IACS-010759 at doses ranging from 10 mg/kg to 25 mg/kg resulted in a >50-day extension of median survival. In diffuse large B-cell lymphoma (DLBCL) and glioblastoma multiforme (GBM) xenograft models, the same dosing regimen led to robust tumor regression [1]
Enzyme Assay	Isolated mouse complex I assay[1] Complex I was isolated from mouse heart mitochondria using an adaptation of the method of Sharpley and colleagues. The concentration of IACS-010759 in Fig. 1f was 60 nM. The NADH:decylubiquinone assay is described in Sharpley et al, and the APAD+ and H2O2 assays are described in Birrell et al. An immunoprecipitated complex I assay was performed to assess the inhibitory effect of IACS-010759 on complex I activity. The key reaction monitored in this assay was the conversion of NADH to NAD+. Different concentrations of the compound were tested, and it was found that IACS-010759 could inhibit this conversion reaction at low nM concentrations [1]
Cell Assay	Generation of clonal cell lines resistant to IACS-010759[1] H292 cells (1 × 106 cells/plate) were seeded in 15-cm dishes in galactose growth medium and treated with 1 nM IACS-010759 (IC65) for 3 weeks, followed by exposure to 8 nM IACS-010759 (IC95) until resistant clones emerged. Twenty-six resistant clones were isolated from four independent experiments and were seeded at 5 × 103 cells/well in 96-well plates in 100 µl galactose growth medium. After cells became fully attached, IACS-010759 or rotenone was added to a final concentration of 370 nM to 18 pM for 3 d. Plates were scanned in the IncuCyte live-cell analysis system before analysis via Hoechst and PI. Subsequently, both Hoechst and PI using an Operetta high-content imaging system. RNA-seq was conducted on the parental line and 12 resistant clones, uncovering a single nonsynonymous, heteroplasmic (35–50%), recurrent mutation in the mitochondrial-encoded gene MT-ND1 in 9 of the 12 resistant clones that conferred the L55F (T3469C) amino acid change. Paired-end reads were initially aligned to transcript sequences of complex I genes with Bowtie 2 (ref.53), and the aligned fragments were probabilistically assigned to transcripts using eXpress54. Variants from the reference genome were called using the ‘mpileup’ command in SAMtools. MutPred55 analysis of the L55F variant classifies the alteration as potentially pathogenic (MutPred score = 0.8); this alteration is found at a very low frequency in mtDNA sequences in Genebank (1:30,589 based on full-length mitochondrial genomes deposited in Genebank before 28 October 2015), suggesting it is unlikely to be a polymorphism. The mutation was confirmed in four of the resistant clones by cloning the MT-ND1 gene sequence and analyzing purified plasmid DNA via Sanger sequencing using the following primers: Forward: 5′-GTAAAACGACGGCCAGT-3′ and Reverse: 5′-AACAGCTATGACCATG-3′. Brain cancer and AML cell lines that are reliant on OXPHOS were selected as the in vitro cell models. These cells were treated with IACS-010759, and subsequent assessments were conducted to measure cell proliferation and apoptosis. The results showed that the compound significantly inhibited cell proliferation and induced obvious apoptosis in these cell models [2] Isolated mitochondria or permeabilized cells were used for the experiment. The cells or mitochondria were exposed to IACS-010759 under two different conditions: one with malate/glutamate present and the other with succinate present. After treatment, the levels of ATP production and oxygen consumption were detected to evaluate the impact of the compound on mitochondrial function and OXPHOS activity [1]
Animal Protocol	10-25 mg/kg; oral; IACS-010759 was formulated in a 0.5% methylcellulose suspension and dosed by oral gavage according to described schedules. [1] Mice with DLBCL and GBM xenograft models Three types of xenograft models were established, including orthotopic AML cell line xenografts, DLBCL xenografts, and GBM xenografts. IACS-010759 was administered to the animals via oral gavage, with a dosing frequency of once daily. The dosage range of the compound was set from 10 mg/kg to 25 mg/kg [1]
ADME/Pharmacokinetics	IACS-010759 has oral bioavailability and exhibits excellent physicochemical properties in preclinical species [1]
Toxicity/Toxicokinetics	IACS-010759 was well-tolerated at the doses used in the in vivo studies [2]
References	[1]. Protopopova M. IACS-10759: A novel OXPHOS inhibitor which selectively kill tumors with metabolic vulnerabilities. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4380. doi:10.1158/1538-7445.AM2015-4380. [2]. An inhibitor of oxidative phosphorylation exploits cancer vulnerability. Nat Med. 2018 Jul;24(7):1036-1046.
Additional Infomation	Tumor cells normally depend on both glycolysis and oxidative phosphorylation (OXPHOS) to provide the energy and macromolecule building blocks needed to enable continued tumor cell growth. Genetic or epigenetic inactivation of one of these two redundant pathways represents a metabolic vulnerability that should be susceptible to an inhibitor of the other pathway. Through an extensive medicinal chemistry campaign, IACS-10759 was identified as a potent inhibitor of complex I of oxidative phosphorylation. In isolated mitochondria or permeabilized cells, ATP production or oxygen consumption was inhibited at single digit nM concentrations in the presence of malate/glutamate, but not succinate. More directly, IACS-10759 inhibited the conversion of NADH to NAD+ in an immunoprecipitated complex I assay at low nM concentrations. Using genetic and pharmacological approaches, the specific complex I subunit inhibited by IACS-10759 has been identified and the mechanism of complex I inhibition is being investigated. Importantly, IACS-10759 is orally bioavailable with excellent physicochemical properties in preclinical species and achieved significant in vivo efficacy with daily oral dosing of 10-25 mg/kg. Specifically, there was a >50 day extension of median survival in an orthotopic AML cell line xenograft and robust regression in DLBCL and GBM xenograft models. In light of these results, as well as its drug like profile IACS-10759 has entered IND enabling studies with first-in-human studies targeted for third quarter of 2015. IACS-010759 is a clinical-grade small-molecule inhibitor targeting mitochondrial electron transport chain complex I. Metabolic reprogramming is an emerging hallmark of tumor biology, and although extensive research has been conducted on the therapeutic targeting of glycolysis, the development of drugs targeting mitochondrial OXPHOS has remained largely unexplored, partly due to the incomplete understanding of tumor contexts where OXPHOS is essential. This compound exploits the metabolic vulnerability of tumors dependent on OXPHOS, making it a potential therapeutic agent for cancers such as brain cancer and AML [2] IACS-010759 was identified as a potent inhibitor of OXPHOS complex I through an extensive medicinal chemistry campaign. Genetic and pharmacological approaches have been employed to identify the specific complex I subunit inhibited by the compound, and investigations into the mechanism of complex I inhibition are ongoing. Due to its favorable in vivo efficacy and drug-like properties, IACS-010759 has entered Investigational New Drug (IND)-enabling studies, with first-in-human trials targeted for the third quarter of 2015 [1] IACS-010759 is currently being evaluated in phase 1 clinical trials for relapsed/refractory AML and solid tumors [2]

Solubility Data

Solubility (In Vitro)

DMSO: >60 mg/mL Water:N/A Ethanol:N/A

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.17 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	1.6694 mL	8.3468 mL	16.6937 mL
	5 mM	0.3339 mL	1.6694 mL	3.3387 mL
	10 mM	0.1669 mL	0.8347 mL	1.6694 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.