Free Fatty Acid Receptor 4 (FFA4/GPR120): AH-7614 is a negative allosteric modulator of FFA4. It inhibits signaling induced by both endogenous and synthetic agonists. Functional inhibition potencies are reported. [2]
Selectivity: AH-7614 did not antagonize activation of the related free fatty acid receptor 1 (FFA1/GPR40) by the agonist TUG-770 in calcium mobilization assays. [2]

In U2OS cells expressing FFA4, AH-7614 (Compound 39) (0.063-1 μM) inhibits the intracellular Ca2+ response brought on by linoleic acid and FFAR4 agonist [1]. In NCI-H716 cells, the increasing impact of GSK137647A on glucose-stimulated insulin production was eliminated by AH-7614 (100 μM) [1]. TUG-891-mediated internalization of FFA4 from the cell surface is blocked by AH-7614 (0.001-10 μM; 15 minutes) (pIC50=7.70) [2]. AH-7614 (10 μM; 30 minutes) inhibits intracellular myo-inositol monophosphate and FFA4 phosphorylation increases brought on by agonists [2].

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Inhibition of Calcium Mobilization (hFFA4): In Flp-In T-REx 293 cells inductively expressing human FFA4-eYFP, AH-7614 potently inhibited calcium mobilization induced by EC80 concentrations of agonists. pIC50 values: against α-linolenic acid (50 μM) = 7.51 ± 0.08; against TUG-891 (500 nM) = 8.13 ± 0.08. [2]
Inhibition of β-Arrestin-2 Recruitment (hFFA4): In HEK293T cells co-expressing hFFA4-eYFP and β-arrestin-2-Renilla luciferase, AH-7614 inhibited agonist-induced BRET signals. pIC50 values: against αLA = 7.66 ± 0.05; against TUG-891 = 7.55 ± 0.07. [2]
Activity at Murine FFA4: AH-7614 also potently inhibited β-arrestin-2 recruitment to murine FFA4 induced by αLA (pIC50 = 8.05 ± 0.08) and TUG-891 (pIC50 = 7.93 ± 0.06). [2]
Selectivity against FFA1: In 1321N1 cells stably expressing human FFA1, AH-7614 (up to 10 μM) had no effect on TUG-770-induced calcium mobilization, demonstrating selectivity for FFA4 over FFA1. [2]
Inhibition of Receptor Internalization: In Flp-In T-REx 293 cells expressing hFFA4-mVenus, AH-7614 inhibited TUG-891-induced receptor internalization with pIC50 = 7.70 ± 0.10. Concentration-response studies showed that AH-7614 primarily decreased the maximal response to TUG-891 in a saturable manner, consistent with negative allosteric modulation. [2]
Mechanism as Negative Allosteric Modulator: Detailed analysis with four structurally distinct FFA4 agonists (TUG-891, TUG-1197, GSK137647A, Cpd A) showed that AH-7614 reduced both potency and maximal response in a saturable manner. Operational model fitting yielded estimates for log α (effect on affinity) and log β (effect on efficacy) for each agonist pair (see Table 1 in the paper). Some probe dependence was observed, with GSK137647A showing a significantly lower log β compared to other agonists. [2]
Inhibition of Inositol Monophosphate Accumulation: In cells expressing hFFA4-mVenus, AH-7614 (10 μM) blocked TUG-891-induced accumulation of inositol monophosphates. [2]
Inhibition of Receptor Phosphorylation: In cells expressing mFFA4-eYFP, AH-7614 pretreatment (1-10 μM) concentration-dependently inhibited TUG-891 (10 μM)-induced phosphorylation of Thr347 and Ser350 residues in the receptor C-terminus, detected using phospho-specific antisera. [2]
Effect on Adipocyte Differentiation: In murine C3H10T1/2 mesenchymal stem cells, treatment with AH-7614 (10 μM) during differentiation induction (IID medium: insulin, IBMX, dexamethasone) significantly reduced Oil Red O staining (triglyceride deposits), indicating inhibition of adipogenesis. AH-7614 also limited IID-induced PPARγ mRNA upregulation but did not affect Runx2 downregulation. [2]

In mice, AH7614 (50 μg administered intraperitoneally every 4 μd for 20 days) can inhibit the formation of tumors [3]. ? When paired with epirubicin, AH7614 (50 μg; intratumoral injection given one day prior to the latter) can increase the susceptibility of cancer cells to chemotherapy by blocking GPR120 signaling and preventing tumor growth [3].

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Breast Cancer Xenograft Model: BALB/c nude mice bearing MCF-7/ADM (epirubicin-resistant) tumor xenografts were treated with intratumoral injections of AH-7614 (50 μg) once every 4 days, alone or in combination with intraperitoneal epirubicin (4 mg/kg). Treatment groups included vehicle, epirubicin alone, AH-7614 alone, GPR120-siRNA alone, AH-7614+epirubicin, and GPR120-siRNA+epirubicin. [3]
Tumor Growth Inhibition: Combination therapy with AH-7614 and epirubicin significantly reduced tumor growth compared to either treatment alone. At day 20, tumor volumes and tumor weights were significantly lower in the AH-7614+epirubicin group compared to epirubicin alone. [3]
Molecular Effects in Tumors: Western blot analysis of tumor tissues showed that AH-7614 treatment decreased ABCG2 and FASN expression, consistent with its mechanism of action. [3]

Cell Lines: HEK293T, Flp-In T-REx 293 cells (with inducible FFA4 expression), 1321N1 cells (with stable FFA1 expression), and C3H10T1/2 murine mesenchymal stem cells were used. [2]
Calcium Mobilization Assay: Cells were seeded in poly-D-lysine-coated 96-well plates. FFA4 expression was induced with doxycycline where applicable. Cells were loaded with Fura2-AM (3 μM) for 45 minutes, washed, and equilibrated in HBSS. Fluorescence emission at 510 nm was measured after excitation at 340/380 nm using a Flexstation plate reader. For antagonism, cells were preincubated with AH-7614 for 15 minutes before agonist addition. [2]
β-Arrestin-2 BRET Assay: HEK293T cells were co-transfected with eYFP-tagged FFA4 and β-arrestin-2-Renilla luciferase plasmids. Cells were preincubated with AH-7614 for 15 minutes, then incubated with coelenterazine h (2.5 μM) for 10 minutes at 37°C, followed by agonist stimulation for 5 minutes. BRET was measured as the ratio of emission at 535 nm to 475 nm. [2]
Receptor Internalization Assay (High-Content Imaging): Flp-In T-REx 293 cells expressing hFFA4-mVenus were plated in 96-well plates, induced with doxycycline overnight, then treated with AH-7614 and TUG-891 for 30 minutes at 37°C. Cells were fixed with 4% paraformaldehyde, nuclei stained with Hoechst33342, and imaged using a Cellomics ArrayScan II. Internalized mVenus was quantified and normalized to cell number. [2]
Inositol Monophosphate Assay: Cells were incubated with agonists for 1 hour in the presence of 10 mM LiCl. IP1 accumulation was measured using an HTRF-based kit according to manufacturer's instructions. [2]
Western Blot for Receptor Phosphorylation: Cells expressing mFFA4-eYFP were pretreated with AH-7614 for 30 minutes, then stimulated with TUG-891 (10 μM) for 5 minutes. Cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with phospho-specific antiserum recognizing phosphorylated Thr347 and Ser350. Detection used LI-COR Odyssey CLx Imager. [2]
Adipocyte Differentiation Assay: C3H10T1/2 cells were differentiated for 5 days in IID medium (100 nM insulin, 500 μM IBMX, 10 nM dexamethasone) with or without AH-7614 (10 μM). Oil Red O staining visualized triglyceride deposits. Staining was quantified by dissolving in isopropanol and measuring absorbance at 405 nm. RT-qPCR assessed PPARγ, Runx2, and mFFA4 mRNA levels normalized to cyclophilin. [2]

Animals:** Four-week-old female BALB/c nude mice were housed under specific pathogen-free conditions. All procedures were approved by the Institutional Animal Care and Use Committee. [3]
* **Tumor Establishment:** Mice received subcutaneous estrogen pellets (0.72 mg 17β-estradiol, 60-day release) three days before cell injection. MCF-7/ADM cells (5 × 10⁶) in 100 μL PBS were injected subcutaneously into mouse flanks. [3]
* **Treatment Regimen:** When tumors reached 150-200 mm³, mice were randomized to six groups (n=6 per group): (1) vehicle; (2) epirubicin (4 mg/kg, i.p., once every 4 days); (3) GPR120-siRNA (10 nmol, intratumoral, once every 4 days); (4) AH-7614 (50 μg, intratumoral, once every 4 days); (5) GPR120-siRNA + epirubicin (siRNA one day prior to epirubicin); (6) AH-7614 + epirubicin (AH-7614 one day prior to epirubicin). [3]
* **Tumor Measurement:** Tumor volume was calculated as length × width²/2. After 20 days of treatment, mice were euthanized, tumors were removed, weighed, and processed for Western blot analysis. [3]

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Animals: Four-week-old female BALB/c nude mice were housed under specific pathogen-free conditions. All procedures were approved by the Institutional Animal Care and Use Committee. [3]
Tumor Establishment: Mice received subcutaneous estrogen pellets (0.72 mg 17β-estradiol, 60-day release) three days before cell injection. MCF-7/ADM cells (5 × 10⁶) in 100 μL PBS were injected subcutaneously into mouse flanks. [3]
Treatment Regimen: When tumors reached 150-200 mm³, mice were randomized to six groups (n=6 per group): (1) vehicle; (2) epirubicin (4 mg/kg, i.p., once every 4 days); (3) GPR120-siRNA (10 nmol, intratumoral, once every 4 days); (4) AH-7614 (50 μg, intratumoral, once every 4 days); (5) GPR120-siRNA + epirubicin (siRNA one day prior to epirubicin); (6) AH-7614 + epirubicin (AH-7614 one day prior to epirubicin). [3]
Tumor Measurement: Tumor volume was calculated as length × width²/2. After 20 days of treatment, mice were euthanized, tumors were removed, weighed, and processed for Western blot analysis. [3]

[1]. Identification of diarylsulfonamides as agonists of the free fatty acid receptor 4 (FFA4/GPR120). Bioorg Med Chem Lett. 2014 Jul 15;24(14):3100-3.

[2]. Probe-Dependent Negative Allosteric Modulators of the Long-Chain Free Fatty Acid Receptor FFA4. Mol Pharmacol. 2017 Jun;91(6):630-641.

[3]. Fatty acid receptor GPR120 promotes breast cancer chemoresistance by upregulating ABC transporters expression and fatty acid synthesis. EBioMedicine. 2019 Feb;40:251-262.

Background: AH-7614 (originally designated compound 39) was first described by Sparks et al. (2014) as an FFA4 antagonist. It is a xanthene derivative of a diarylsulfonamide-based FFA4 agonist. This study provides the first detailed characterization of its mechanism as a negative allosteric modulator. [2]
Chemical Synthesis: AH-7614 was synthesized from xanthone via reduction with sodium borohydride followed by reaction with p-toluenesulfonamide in acetic acid. Purity was confirmed as 99.7% by HPLC. Structural identity was confirmed by ¹H NMR, ¹³C NMR, and ESI-HRMS. [2]
Mechanism of Action: AH-7614 is a negative allosteric modulator of FFA4, not a competitive antagonist. Evidence includes: (1) saturable inhibition (maximal effect reached at high concentrations with residual agonist response); (2) reduction of both potency and maximal response depending on agonist; (3) probe-dependent effects across different agonist chemotypes. [2]
Probe Dependence: AH-7614 showed subtle probe dependence in its modulation, with significantly lower log β values (effect on efficacy) for the sulfonamide-based agonist GSK137647A compared to carboxylate-based agonists. [2]
Utility as a Tool Compound: The study demonstrates that AH-7614, when used in combination with the inactive structural analog TUG-1387, can define FFA4-specific biologic functions, such as its role in adipocyte differentiation of mesenchymal stem cells. [2]
Structural Derivatives: Two key derivatives were synthesized and tested: TUG-1387 (amide replacement, inactive at FFA4) and TUG-1506 (thioxanthene replacement, retained NAM activity with altered properties). [2]

C20H17NO3S

351.42

351.093

C, 68.36; H, 4.88; N, 3.99; O, 13.66; S, 9.12

6326-06-3

233085

White to off-white solid powder

1.37g/cm3

505.9ºC at 760mmHg

259.8ºC

1.685

5.64

1

4

3

25

528

0

OZCQEUZTOAAWDK-UHFFFAOYSA-N

InChI=1S/C20H17NO3S/c1-14-10-12-15(13-11-14)25(22,23)21-20-16-6-2-4-8-18(16)24-19-9-5-3-7-17(19)20/h2-13,20-21H,1H3

4-methyl-N-(9H-xanthen-9-yl)benzenesulfonamide

AH-7614 AH7614 AH 7614

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 : ~100 mg/mL (~284.56 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.11 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.

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