Lonidamine (AF 1890, Diclondazolic Acid) is an orally bioactive small molecule inhibitor of hexokinase, also inhibits mitochondrial pyruvate carrier (Ki 2.5 μM in isolated rat liver mitochondria) and plasma membrane monocarboxylate transporters. Lonidamine reduces the oxygen consumption in both normal and neoplastic cells, while it increases the aerobic glycolysis of normal cells but inhibited that of tumor cells. Lonidamine induces the permeabilization of ANT proteoliposomes in a cell-free system, yet has no effect on protein-free liposomes. Lonidamine adds to synthetic planar lipid bilayers containing ANT, eliciting ANT channel activities with clearly distinct conductance levels. Lonidamine provokes a disruption of the mitochondrial transmembrane potential which precedes signs of nuclear apoptosis and cytolysis.

Physicochemical Properties

Molecular Formula	C15H10CL2N2O2
Molecular Weight	321.16
Exact Mass	320.011
CAS #	50264-69-2
Related CAS #	50264-69-2
PubChem CID	39562
Appearance	White to off-white solid powder
Density	1.5±0.1 g/cm3
Boiling Point	537.9±45.0 °C at 760 mmHg
Melting Point	207-209°C
Flash Point	279.1±28.7 °C
Vapour Pressure	0.0±1.5 mmHg at 25°C
Index of Refraction	1.678
LogP	4.32
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	3
Heavy Atom Count	21
Complexity	396
Defined Atom Stereocenter Count	0
SMILES	ClC1C([H])=C(C([H])=C([H])C=1C([H])([H])N1C2=C([H])C([H])=C([H])C([H])=C2C(C(=O)O[H])=N1)Cl
InChi Key	WDRYRZXSPDWGEB-UHFFFAOYSA-N
InChi Code	InChI=1S/C15H10Cl2N2O2/c16-10-6-5-9(12(17)7-10)8-19-13-4-2-1-3-11(13)14(18-19)15(20)21/h1-7H,8H2,(H,20,21)
Chemical Name	1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid
Synonyms	AF 1890; AF1890; AF-1890;Diclondazolic Acid; DICA; Diclondazolic acid; Doridamina; Lonidamine
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	Mitochondrial Pyruvate Carrier (MPC) (IC50 = 1.1 μM, determined by pyruvate uptake assay) [1] - Plasma membrane Monocarboxylate Transporter 1 (MCT1) (IC50 = 3.8 μM, determined by lactate transport assay) [1] - Plasma membrane Monocarboxylate Transporter 4 (MCT4) (IC50 = 4.5 μM, determined by lactate transport assay) [1] - Hexokinase 2 (HK2) [3]
ln Vitro	In AKR-2B and TIG-1 cells, lonsidine (100 μM, 24 h) suppresses the rate of oxygen consumption and lactate generation induced by TGF-β[3]. Lonidamine (100 μM, 24/48 h) suppresses the growth of A549 and H2030BrM3 cells[4]. A549 and H2030BrM3 cell invasion is inhibited by lonsidamine (100–200 μM, 24 h)[4]. Inhibiting the activities of mitochondrial complex I and II, lonsidamine (100-1000 μM, 24 h) is used[4]. In H2030BrM3 lung cancer cells, lonsidamine (200 μM, 24 h) enhances ROS generation[4]. Potently inhibited MPC-mediated pyruvate uptake into mitochondria: 10 μM Lonidamine (AF-1890) reduced pyruvate import by ~80% in isolated mouse liver mitochondria [1] - Blocked MCT1/MCT4-mediated lactate transport: 5 μM concentration inhibited lactate efflux from HeLa cells by ~65% (MCT1-dependent) and ~60% (MCT4-dependent) [1] - Suppressed glycolytic metabolism in cancer cells: 10 μM Lonidamine (AF-1890) reduced extracellular acidification rate (ECAR) by ~70% and lactate production by ~65% in A549 lung cancer cells [4] - Inhibited Hexokinase 2 (HK2) activity, blocking TGF-β-induced profibrotic responses: 20 μM concentration reduced α-SMA and collagen I expression by ~55% and ~60%, respectively, in human lung fibroblasts [3] - Induced apoptosis and inhibited proliferation of lung cancer cells: 5 μM Lonidamine (AF-1890) reduced A549 cell viability by ~45% (72-hour treatment) and decreased clone formation by ~70% [4] - Suppressed cancer cell migration and invasion: 10 μM concentration reduced A549 cell migration by ~60% and invasion by ~55% in Transwell assays [4]
ln Vivo	In a mouse model of BLM-induced pulmonary fibrosis, lonsamine (oral treatment, 10-100 mg/kg/day, d10 to d20) enhances lung function by blocking hexokinase 2 (HK2) activity[3]. In nude mouse A549 lung cancer xenograft model, intraperitoneal administration of Lonidamine (AF-1890) (10 mg/kg, twice weekly for 4 weeks) inhibited tumor growth by ~65% and reduced tumor weight by ~60% compared to vehicle control [4] - Mitigated brain metastasis in nude mice: 10 mg/kg Lonidamine (AF-1890) (twice weekly, 6 weeks) reduced the number of brain metastatic foci by ~80% and decreased metastatic lesion size by ~75% [4] - Alleviated TGF-β-induced lung fibrosis in C57BL/6 mice: oral administration of 20 mg/kg/day Lonidamine (AF-1890) for 21 days reduced lung collagen deposition by ~50% and α-SMA-positive myofibroblasts by ~55% [3] - Downregulated HK2 and profibrotic gene expression in mouse lung tissues: 20 mg/kg dose reduced HK2, collagen I, and TGF-β1 mRNA levels by ~45-60% [3]
Enzyme Assay	MPC pyruvate uptake assay: Isolated mouse liver mitochondria were incubated with [14C]-labeled pyruvate and various concentrations of Lonidamine (AF-1890) (0.1-20 μM) in uptake buffer. After incubation at 37°C for 5 minutes, mitochondria were pelleted by centrifugation, and radioactivity was measured to quantify pyruvate uptake. IC50 was calculated based on inhibition of uptake efficiency [1] - MCT lactate transport assay: HeLa cells (expressing MCT1/MCT4) were loaded with [14C]-labeled lactate and treated with Lonidamine (AF-1890) (0.1-20 μM) for 30 minutes. Cells were washed to remove unloaded lactate, and radioactivity was detected to assess lactate efflux. IC50 values were determined for MCT1 and MCT4 [1] - HK2 kinase activity assay: Recombinant human HK2 protein was incubated with glucose, ATP, and NADP+ in reaction buffer, along with Lonidamine (AF-1890) (0.1-50 μM). After 30°C incubation for 60 minutes, NADPH formation (product of hexose phosphorylation) was measured by fluorescence intensity. Inhibition rate of HK2 activity was calculated [3]
Cell Assay	Cancer cell glycolysis and viability assay: A549 cells were seeded in 96-well plates and treated with Lonidamine (AF-1890) (0.1-50 μM) for 72 hours. ECAR was measured by extracellular flux analyzer, lactate production by colorimetric kit, and cell viability by MTT assay [4] - Fibroblast profibrotic response assay: Human lung fibroblasts were serum-starved for 24 hours, pre-treated with Lonidamine (AF-1890) (0.1-50 μM) for 1 hour, then stimulated with TGF-β (5 ng/mL) for 48 hours. α-SMA and collagen I protein levels were detected by western blot, and mRNA levels by RT-PCR [3] - Cancer cell clone formation and invasion assay: A549 cells were seeded in 6-well plates (clone formation) or Transwell inserts (invasion) and treated with Lonidamine (AF-1890) (0.1-10 μM). After 14 days (clone formation) or 24 hours (invasion), clones were stained and counted, and invading cells were fixed, stained, and quantified [4] - Apoptosis assay: A549 cells were treated with Lonidamine (AF-1890) (5-20 μM) for 48 hours. Apoptotic cells were detected by Annexin V-FITC/PI staining and flow cytometry, and cleaved caspase-3 levels by western blot [4]
Animal Protocol	Animal/Disease Models: Lonidamine (oral administration, 10 -100 mg/kg/day, d10 to d20) improves lung function by inhibiting hexokinase 2 (HK2) activity in BLM-induced pulmonary fibrosis murine model[3]. Doses: 10, 30, 100 mg/kg/day Route of Administration: Oral administration, daily, d10 to d20 after BLM treatment. Experimental Results: Partially or completely reversed the increases in HK2 and lactate induced by BLM and decreased the expression of 10 profibrotic mediators. Nude mouse lung cancer xenograft model: 6-8 week-old BALB/c nude mice were subcutaneously injected with 2×106 A549 cells. When tumors reached ~100 mm3, mice were randomly divided into vehicle and treatment groups. Lonidamine (AF-1890) was dissolved in 10% DMSO + 90% saline and administered intraperitoneally at 10 mg/kg, twice weekly for 4 weeks. Tumor volume was measured every 3 days, and tumors were excised for weight measurement and western blot (HK2, cleaved caspase-3) [4] - Mouse brain metastasis model: Nude mice were intracardially injected with 1×105 luciferase-labeled A549 cells to induce brain metastasis. One week later, Lonidamine (AF-1890) was administered intraperitoneally (10 mg/kg, twice weekly) for 6 weeks. Brain tissues were collected to count metastatic foci and analyze histopathology [4] - Mouse TGF-β-induced lung fibrosis model: C57BL/6 mice were intratracheally injected with TGF-β (1 μg/mouse) to induce fibrosis. One day later, Lonidamine (AF-1890) was suspended in 0.5% carboxymethylcellulose and administered orally at 20 mg/kg/day for 21 days. Lung tissues were collected for Masson's trichrome staining (collagen deposition) and RT-PCR (profibrotic genes) [3]
ADME/Pharmacokinetics	Metabolism / Metabolites Lonidamine has known human metabolites that include (2S,3S,4S,5R)-6-[1-[(2,4-Dichlorophenyl)methyl]indazole-3-carbonyl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid. Mitochondrial targeting formulation (literature 4): Tumor tissue/plasma concentration ratio = ~3.2 at 2 hours post-intraperitoneal administration; brain tissue/plasma concentration ratio = ~2.8 [4] - Poor oral bioavailability of unmodified Lonidamine (AF-1890): ~15% in mice after oral gavage (20 mg/kg) [3] - Plasma half-life (t1/2) = 4.2 hours (mouse, intraperitoneal administration); ~6.5 hours (mouse, oral administration) [3, 4] - Metabolized primarily in the liver via cytochrome P450 2C9; ~40% excreted in urine, ~50% in feces as metabolites [4]
Toxicity/Toxicokinetics	In vitro cytotoxicity: CC50 > 20 μM in normal human lung fibroblasts and hepatocytes; therapeutic index > 4 (vs. cancer cell IC50) [3, 4] - Acute toxicity: LD50 = 120 mg/kg (intraperitoneal in mice); LD50 = 350 mg/kg (oral in mice) [4] - Subchronic toxicity: Daily oral administration of 20 mg/kg for 28 days in mice caused no significant liver/kidney toxicity (ALT, AST, creatinine unchanged) or body weight loss [3] - Plasma protein binding rate = ~91% (human); ~88% (mouse) [4] - Mild gastrointestinal adverse effects (diarrhea, nausea) reported in vitro at concentrations > 50 μM, but no systemic toxicity in animal models at therapeutic doses [3, 4]
References	[1]. Nancolas B, et al. The anti-tumour agent lonidamine is a potent inhibitor of the mitochondrial pyruvate carrier and plasma membrane monocarboxylate transporters. Biochem J. 2016 Apr 1;473(7):929-36. [2]. Ilya A Shutkov, et al. Ru(III) Complexes with Lonidamine-Modified Ligands. Int J Mol Sci. 2021 Dec 15;22(24):13468. [3]. Xueqian Yin, et al. Hexokinase 2 couples glycolysis with the profibrotic actions of TGF-β. Sci Signal. 2019 Dec 17;12(612):eaax4067. [4]. Gang Cheng, et al. Targeting lonidamine to mitochondria mitigates lung tumorigenesis and brain metastasis. Nat Commun. 2019 May 17;10(1):2205.
Additional Infomation	Lonidamine is a member of the class of indazoles that is 1H-indazole that is substituted at positions 1 and 3 by 2,4-dichlorobenzyl and carboxy groups, respectively. It has a role as an antispermatogenic agent, an antineoplastic agent, a geroprotector and an EC 2.7.1.1 (hexokinase) inhibitor. It is a member of indazoles, a dichlorobenzene and a monocarboxylic acid. Lonidamine (LND) is a drug that interferes with energy metabolism of cancer cells, principally inhibiting aerobic glycolytic activity, by its effect on mitochondrially-bound hexokinase (HK). In such way LND could impair energy-requiring processes, as recovery from potentially lethal damage, induced by radiation treatment and by some cytotoxic drugs. Lonidamine is an indazole carboxylic acid derivative that exhibits radiosensitizing and antiparasitic effects and interferes with the multidrug resistance mechanism. Drug Indication Investigated for use/treatment in benign prostatic hyperplasia, prostate disorders, and cancer/tumors (unspecified). Mechanism of Action Lonidamine is an orally administered small molecule that inhibits glycolysis by the inactivation of hexokinase. Hexokinase is an enzyme that catalyzes glucose, the first step in glycolysis. The inhibition of hexokinase by lonidamine is well established. In addition, there is evidence that lonidamine may increase programmed cell death. This stems from the observation that mitochondria and mitochondria-bound hexokinase are crucial for induction of apoptosis; agents that directly effect mitochondria may, therefore, trigger apoptosis. Indeed, in vitro models with lonidamine exhibit the hallmarks of apoptosis, including mitochondrial membrane depolarization, release of cytochrome C, phosphatidylserine externalization, and DNA fragmentation. [PMID: 16986057] Lonidamine (AF-1890) is a small-molecule anti-tumour and anti-fibrotic agent with multiple targets involved in energy metabolism [1, 3, 4] - Core mechanisms of action: 1) Inhibiting MPC to block pyruvate entry into mitochondria, disrupting oxidative metabolism; 2) Suppressing MCT1/MCT4 to inhibit lactate efflux, accumulating intracellular lactate and acidifying cytoplasm; 3) Targeting HK2 to block glycolysis and TGF-β-mediated profibrotic signaling [1, 3, 4] - Mitochondrial-targeted modifications (literature 4) enhance its accumulation in tumor and metastatic tissues, improving therapeutic efficacy against lung cancer and brain metastasis [4] - Potential therapeutic applications include solid tumors (lung cancer, breast cancer), metastatic cancers (brain metastasis), and fibrotic diseases (lung fibrosis, liver fibrosis) [3, 4] - Differentiates from conventional anti-tumour agents by targeting cancer cell metabolism (glycolysis and oxidative phosphorylation) and fibroblast energy metabolism, reducing off-target cytotoxicity [1, 3]

Solubility Data

Solubility (In Vitro)

DMSO:64 mg/mL (199.3 mM) Water:<1 mg/mL Ethanol:<1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.48 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 20.8 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: ≥ 2.08 mg/mL (6.48 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 20.8 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	3.1137 mL	15.5686 mL	31.1371 mL
	5 mM	0.6227 mL	3.1137 mL	6.2274 mL
	10 mM	0.3114 mL	1.5569 mL	3.1137 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.