Mitochondrial pyruvate carrier [2]
Monocarboxylate transporters [1]

Compound 19 (7ACC2), which suppresses SiHa cell proliferation in lactate-containing media, has an EC50 of 0.22 μM. It works for 72 hours. The high affinity MCT1 transporter is the main factor influencing lactate uptake in SiHa cells[1]. Compound 19, or 7ACC2, exhibits remarkable chemical stability in simulated gastric (SGF) and intestinal (SIF) fluids, a good apparent permeability coefficient (Papp) through the Caco-2 monolayer, and a high level of metabolic stability on human hepatocytes, mouse liver microsomes, and human liver microsomes [1]. 7ACC2 is a strong inhibitor of mitochondrial pyruvate transport that increases intracellular pyruvate buildup, hence blocking the uptake of extracellular lactate[2].

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1. Effects on cancer cell metabolism and growth: 7ACC2 (10 µM) treatment of SiHa cells showed differential effects on cell growth and oxygen consumption rate (OCR) depending on glucose/lactate availability in the medium; it did not inhibit MCT1 activity (assessed by ¹⁴C-lactate uptake and intracellular H⁺ concentration measurements in MCT1-expressing Xenopus oocytes), nor did it affect MCT4 activity in MCT4-expressing Xenopus oocytes; in SiHa cells, it increased glycolytic flux (elevated extracellular acidification rate, ECAR), reduced mitochondrial respiration (decreased OCR), promoted intracellular pyruvate accumulation, blocked extracellular lactate uptake, increased glucose consumption, altered the lactate/glucose ratio, and modified the abundance of glycolysis-derived pyruvate/lactate, TCA cycle intermediates, and serine synthesis pathway metabolites; in isolated SiHa cell mitochondria, it inhibited pyruvate-dependent OCR and [2−¹⁴C]pyruvate uptake in a dose- and time-dependent manner, and affected BCECF fluorescence (reflecting mitochondrial pH changes) in a dose-dependent way; in FaDu tumor spheroids, 20 µM 7ACC2 induced cytotoxic effects (in contrast to cytostatic effects of MCT1 inhibitor AR-C155858), stimulated glycolysis, caused uncompensated inhibition of mitochondrial respiration, increased cellular debris accumulation, altered 2-deoxyglucose-IRDye (2DG-IR) distributioccumulation (especially in the spheroid core), and modified GLUT-1 staining distribution at different spheroid depths; knockdown of MPC1 in FaDu cells mimicked the effect of 7ACC2 on 2DG-IR accumulation in the spheroid core [2]

Treatment with 7ACC2 (3 mg/kg; intraperitoneal administration; daily; for 5 days or 10 days) dramatically reduces the growth of tumors in mice. 7ACC2 decreases hypoxia in vivo, which radiosensitizes tumor cells[2]. When mice are given 7ACC2 (compound 19; 3 mg/kg) intraperitoneally, they quickly reach a Cmax of 1246 ng/ml (4 μM) (Tmax=10 min), which is accompanied by a 4.5-hour plasma half-life[1].

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1. Metabolic effects in tumor xenografts: In SiHa tumor xenografts in nude mice, administration of 3 mg/kg 7ACC2 (2 h treatment) altered in vivo ¹³C-MRS spectra after hyperpolarized ¹³C-pyruvate injection, reduced the relative ¹³C-lactate NMR signal and the ¹³C lactate/pyruvate ratio [2]
2. Antitumor and radiosensitizing effects: 3 mg/kg 7ACC2 treatment for 9 days reduced SiHa xenograft tumor volume in nude mice; daily administration of 3 mg/kg 7ACC2 (for 10 or 5 days) sensitized SiHa xenografts to radiotherapy (single 16 Gy irradiation or 5 fractions of 4 Gy), reducing tumor growth; it also reduced tumor hypoxia (assessed by CA9/pimonidazole staining in FaDu spheroids and tumor pO₂ measurements by EPR oximetry in SiHa xenografts) [2]

1. Mitochondrial pyruvate transport assay: Isolated mitochondria from SiHa cells were treated with different doses of 7ACC2 for varying durations, then [2−¹⁴C]pyruvate uptake was measured to evaluate the inhibitory effect of 7ACC2 on mitochondrial pyruvate transport; BCECF fluorescence was used to detect pH changes in isolated SiHa mitochondria treated with increasing doses of 7ACC2 to reflect the impact on pyruvate transport; OCR measurements were performed on isolated SiHa mitochondria exposed to pyruvate/malate (with/without methylpyruvate) after 10 µM 7ACC2 treatment to assess mitochondrial respiration inhibition [2]
2. MCT activity assay: MCT1-expressing Xenopus oocytes were treated with increasing doses of 7ACC2, and ¹⁴C-lactate uptake and intracellular H⁺ concentration were measured to evaluate MCT1 inhibition; the same assays were conducted on MCT4-expressing Xenopus oocytes to assess MCT4 activity [2]

1. SiHa cell metabolic assay: SiHa cells were treated with 10 µM 7ACC2 for 24 h in media with different glucose/lactate combinations; cell growth (72 h) and OCR were measured to evaluate metabolic effects; ¹³C-glucose labeling was used to analyze intracellular metabolite abundance (glycolysis-derived pyruvate/lactate, TCA cycle intermediates, serine synthesis pathway metabolites), glucose consumption, and lactate/glucose ratio; ECAR was measured to assess glycolytic activity; lactate consumption/secretion was quantified after 24 h incubation in lactate/glucose-containing media [2]
2. FaDu spheroid assay: FaDu spheroids were treated with 20 µM 7ACC2 for up to 9 days; spheroid growth, cellular density, and cellular debris accumulation were monitored over time; 2DG-IR (3 h exposure) distributioccumulation (periphery to core, core region) was assessed by immunofluorescence; GLUT-1 staining at different spheroid depths was quantified by immunofluorescence; MPC1 knockdown in FaDu cells was achieved via shRNA, and 2DG-IR accumulation in the spheroid core was evaluated [2]

Animal/Disease Models: 7-week- old female NMRI nude mice with radiotherapy administered[2]
Doses: 3 mg/kg
Route of Administration: intraperitoneal (ip)administration; daily; for 5 days or 10days
Experimental Results: A significant increase in tumor growth delay was observed.

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1. Tumor xenograft metabolic assessment: Nude mice bearing SiHa tumor xenografts were administered 3 mg/kg 7ACC2; 2 h after administration, hyperpolarized ¹³C-pyruvate was injected, and in vivo ¹³C-MRS spectra were acquired to measure ¹³C-lactate/pyruvate ratios [2]
2. Antitumor efficacy assay: Nude mice with SiHa xenografts were treated with 3 mg/kg 7ACC2 daily for 9 days, and tumor volume was monitored; for radiosensitization experiments, mice received daily 3 mg/kg 7ACC2 for 10 days (single 16 Gy irradiation) or 5 days (5 fractions of 4 Gy), with 7ACC2 formulated in DMSO (10-day treatment) or 5% DMA/50% HPβCD/45% sodium phosphate buffer (5-day treatment); tumor pO₂ was measured by EPR oximetry at different time points after 3 mg/kg 7ACC2 administration [2]

[1]. Synthesis and pharmacological evaluation of carboxycoumarins as a new antitumor treatment targeting lactate transport in cancer cells. Bioorg Med Chem. 2013 Nov 15;21(22):7107-17.

[2]. Interruption of lactate uptake by inhibiting mitochondrial pyruvate transport unravels direct antitumor and radiosensitizing effects. Nat Commun. 2018 Mar 23;9(1):1208.

1. Mechanism of action: 7ACC2 inhibits mitochondrial pyruvate transport (MPC), leading to intracellular pyruvate accumulation and thus blocking the uptake of extracellular lactate by cancer cells; it stimulates glycolysis, leading to uncompensated inhibition of mitochondrial respiration in tumor cells; its inhibitory effect on MPC can reduce tumor hypoxia, thereby enhancing the radiosensitivity of tumor xenografts [2] 2. Therapeutic potential: 7ACC2 is a novel anticancer drug targeting lactate metabolism, with direct antitumor effects and radiosensitizing properties; its mechanism of action is different from that of MCT1 inhibitors (AR-C155858), inducing cytotoxicity rather than cell inhibition in tumor spheroids [2] 3. Structural background: 7ACC2 belongs to carboxycoumarin derivatives, which are a class of compounds developed as potential anti-tumor drugs that target lactate transport in cancer cells; palladium-catalyzed Buchwald-Hartwig coupling reaction is an effective method for synthesizing aminocarboxycoumarin derivatives (7ACC2 class compounds) [1]

C18H15NO4

309.32

309.1

C, 69.89; H, 4.89; N, 4.53; O, 20.69

1472624-85-3

72696735

Light yellow to yellow solid powder

1.4±0.1 g/cm3

548.9±50.0 °C at 760 mmHg

285.8±30.1 °C

0.0±1.6 mmHg at 25°C

1.672

4.17

1

5

4

23

495

0

O1C(C(C(=O)O[H])=C([H])C2C([H])=C([H])C(=C([H])C1=2)N(C([H])([H])[H])C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])=O

XTKDQPFUOFAMRL-UHFFFAOYSA-N

InChI=1S/C18H15NO4/c1-19(11-12-5-3-2-4-6-12)14-8-7-13-9-15(17(20)21)18(22)23-16(13)10-14/h2-10H,11H2,1H3,(H,20,21)

7-[benzyl(methyl)amino]-2-oxochromene-3-carboxylic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

Solubility in Formulation 1: 2.5 mg/mL (8.08 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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.

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