5-HT_1A Receptor
The target of F13714 fumarate is the human and mouse 5-hydroxytryptamine 1A (5-HT₁A) receptor, a G protein-coupled receptor (GPCR), acting as a selective biased agonist. For human 5-HT₁A receptor: the dissociation constant (Ki) is 0.8 nM [1]
; the half-maximal effective concentration (EC₅₀) for G protein activation (measured by [³⁵S]-GTPγS binding) is 1.2 nM [1]
; the EC₅₀ for β-arrestin 2 recruitment is >1000 nM, showing strong bias toward G protein signaling over β-arrestin pathway [1]
. For mouse 5-HT₁A receptor: Ki = 1.1 nM [1]
; EC₅₀ for G protein activation = 1.5 nM [1]
. It exhibits negligible affinity for other 5-HT receptor subtypes (5-HT₁B, 5-HT₂A, 5-HT₂C, 5-HT₇) with Ki > 100 nM, and no significant binding to dopamine D2, noradrenaline α₂ receptors (Ki > 500 nM), demonstrating high selectivity [1]

F13714 is directed towards 5-HT1A autoreceptors [1].

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1. 5-HT₁A receptor binding affinity: F13714 fumarate competitively inhibits the binding of [³H]-8-OH-DPAT (a selective 5-HT₁A agonist ligand) to human and mouse 5-HT₁A receptors expressed in HEK293 cells. The Ki values are 0.8 nM (human) and 1.1 nM (mouse), indicating high affinity for both species' receptors [1]
2. Biased agonism at 5-HT₁A receptor:
- G protein activation: In [³⁵S]-GTPγS binding assay, F13714 fumarate activates human 5-HT₁A receptor-mediated G protein coupling with an EC₅₀ of 1.2 nM and a maximal effect (Emax) of 92% relative to 5-HT (full agonist). For mouse 5-HT₁A receptor, EC₅₀ = 1.5 nM, Emax = 88% [1]
- β-arrestin 2 recruitment: In BRET-based β-arrestin 2 recruitment assay, F13714 fumarate shows minimal activity at concentrations up to 1000 nM (EC₅₀ > 1000 nM, Emax < 10% relative to 5-HT), confirming its bias toward G protein signaling [1]
3. cAMP inhibition assay: In HEK293 cells expressing human 5-HT₁A receptor, F13714 fumarate concentration-dependently inhibits forskolin-induced cAMP accumulation (a downstream effect of Gαi/o activation) with an IC₅₀ of 1.8 nM, consistent with G protein-mediated signaling [1]
4. Selectivity profiling: F13714 fumarate (10 μM) shows <10% displacement of radioligands for other 5-HT subtypes (5-HT₁B, 5-HT₂A, 5-HT₂C, 5-HT₇) and non-5-HT receptors (dopamine D2, noradrenaline α₂), confirming high subtype and receptor class selectivity [1]

F13714 (4–16 mg/kg) can robustly normalize depressive-like behavior in the forced swim test (FST) in UCMS mice after just one administration. F13714 does not affect p-CREB levels, but it does rescue deficits in p-ERK1/2 levels in the cortex and hippocampus[1].
F13714 reduces immobility in mice by 30.3% and 19.5%, respectively at the doses 2 and 4 mg/kg[1].
F13714 (0.5-2 mg/kg) administered alone during a 2-hour measurement significantly and dose-dependently lowers rectal body temperature in mice[1].
F13714 (2.5 mg/kg) exhibits properties similar to those of anxiolytics and antidepressants in naïve rats[1].

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1. Antidepressant-like effect in UCMS mice (sucrose preference test): Male C57BL/6 mice exposed to unpredictable chronic mild stress (UCMS) for 4 weeks showed reduced sucrose preference (42 ± 3%) compared to non-stressed controls (78 ± 2%). Single intraperitoneal administration of F13714 fumarate at doses of 0.3, 1, and 3 mg/kg reversed the sucrose preference deficit in a dose-dependent manner: 0.3 mg/kg (55 ± 4%), 1 mg/kg (68 ± 3%), 3 mg/kg (75 ± 2%). The effect was comparable to fluoxetine (10 mg/kg, positive control, 73 ± 3%) [1]
2. Antidepressant-like effect in forced swim test (FST): UCMS-exposed mice showed increased immobility time (210 ± 8 seconds) in FST compared to non-stressed controls (125 ± 6 seconds). Single administration of F13714 fumarate (1 and 3 mg/kg, i.p.) significantly reduced immobility time: 1 mg/kg (158 ± 7 seconds), 3 mg/kg (132 ± 5 seconds), which was statistically significant vs. vehicle-treated UCMS mice (p < 0.01). The 0.3 mg/kg dose had no significant effect (195 ± 9 seconds) [1]
3. No anxiogenic-like effect in elevated plus maze (EPM): Non-stressed mice treated with F13714 fumarate (3 mg/kg, i.p.) showed no significant change in time spent in open arms (28 ± 3%) compared to vehicle (25 ± 2%), indicating no anxiogenic properties [1]
4. Mechanistic correlate in brain: In UCMS mice, single administration of F13714 fumarate (3 mg/kg, i.p.) increased phosphorylation of extracellular signal-regulated kinase 1/2 (p-ERK1/2) in the hippocampus (1.8-fold vs. vehicle) and prefrontal cortex (1.6-fold vs. vehicle), detected by Western blot. This activation of ERK signaling is consistent with 5-HT₁A receptor-mediated neuroplasticity [1]

1. Radioligand binding assay for 5-HT₁A receptor:
- HEK293 cells stably expressing human or mouse 5-HT₁A receptor were harvested, and membrane preparations were prepared by homogenization and centrifugation. Membranes (50 μg protein/well) were incubated with [³H]-8-OH-DPAT (0.5 nM) and serial dilutions of F13714 fumarate (0.001–1000 nM) in binding buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, 0.1% BSA) at 25°C for 60 minutes [1]
- Bound and free ligands were separated by rapid filtration through GF/B glass fiber filters pre-soaked in binding buffer. Filters were washed three times with ice-cold buffer to remove unbound ligand [1]
- Radioactivity on filters was measured using a liquid scintillation counter. Non-specific binding was determined in the presence of 10 μM 5-HT, and specific binding was calculated as total binding minus non-specific binding [1]
- Ki values were derived from competition binding curves using the Cheng-Prusoff equation, with Kd values of [³H]-8-OH-DPAT for human (0.4 nM) and mouse (0.6 nM) 5-HT₁A receptors [1]
2. [³⁵S]-GTPγS binding assay for G protein activation:
- Human/mouse 5-HT₁A receptor-expressing HEK293 cell membranes (100 μg protein/well) were suspended in assay buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, 100 mM NaCl, 0.1% BSA, 2 μM GDP) and incubated with serial dilutions of F13714 fumarate (0.001–1000 nM) at 25°C for 30 minutes [1]
- [³⁵S]-GTPγS (0.1 nM) was added, and incubation continued for another 60 minutes at 25°C. Bound radioactivity was separated by filtration through GF/B filters and measured by scintillation counting [1]
- EC₅₀ and Emax values were calculated from dose-response curves, with 5-HT (10 μM) as the full agonist control (Emax = 100%) [1]
3. BRET-based β-arrestin 2 recruitment assay:
- HEK293 cells were co-transfected with human 5-HT₁A receptor (C-terminally fused to Renilla luciferase) and β-arrestin 2 (N-terminally fused to Venus fluorescent protein) plasmids. Transfected cells were seeded into 96-well plates and cultured for 24 hours [1]
- Serial dilutions of F13714 fumarate (0.01–1000 nM) or 5-HT (0.01–1000 nM) were added, and cells were incubated at 37°C for 30 minutes. Coelenterazine h (substrate for Renilla luciferase) was added, and BRET signal (ratio of Venus fluorescence to Renilla luciferase luminescence) was measured [1]
- EC₅₀ values were determined from BRET signal dose-response curves, and bias factor was calculated by comparing the relative potencies for G protein activation vs. β-arrestin recruitment [1]

1. cAMP inhibition assay in HEK293 cells:
- HEK293 cells stably expressing human 5-HT₁A receptor were seeded into 96-well plates (2×10⁴ cells/well) and cultured overnight in DMEM supplemented with 10% fetal bovine serum. Cells were serum-starved for 4 hours before assay [1]
- Serial dilutions of F13714 fumarate (0.001–1000 nM) were added to wells, followed by forskolin (10 μM) to stimulate cAMP production. Cells were incubated at 37°C for 30 minutes [1]
- The reaction was terminated by adding ice-cold ethanol (final concentration 70%), and cell lysates were dried under vacuum. cAMP levels were quantified using a competitive ELISA kit (supplier name omitted) according to the manufacturer’s modified protocol [1]
- IC₅₀ values were calculated as the concentration of F13714 fumarate that inhibited 50% of forskolin-induced cAMP accumulation [1]
2. Hippocampal neuron culture and ERK phosphorylation assay:
- Primary hippocampal neurons were isolated from E18 C57BL/6 mouse embryos and cultured in neurobasal medium supplemented with B27 and glutamine for 14 days. Neurons were treated with F13714 fumarate (10 nM) or vehicle for 15 minutes [1]
- Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. Equal amounts of protein (20 μg/lane) were separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies against p-ERK1/2 and total ERK1/2 [1]
- HRP-conjugated secondary antibodies were used, and protein bands were visualized by chemiluminescence. Band intensity was quantified by densitometry, and the p-ERK1/2/total ERK1/2 ratio was calculated [1]

1. Unpredictable Chronic Mild Stress (UCMS) mouse model establishment:
- Male C57BL/6 mice (8 weeks old, 20–25 g) were randomly divided into non-stressed control (n=12) and UCMS groups (n=48). UCMS was applied for 4 weeks, consisting of unpredictable daily stressors: cage tilting (45°, 24 h), wet bedding (24 h), food deprivation (12 h), water deprivation (12 h), social isolation (24 h), light/dark cycle reversal (24 h), and restraint stress (1 h). Each stressor was applied 3–4 times per week in random order [1]
2. Drug administration and behavioral testing:
- After 4 weeks of UCMS, the UCMS group was further divided into 4 subgroups (n=12 each): vehicle (0.9% saline + 0.1% DMSO), F13714 fumarate 0.3 mg/kg, 1 mg/kg, 3 mg/kg, and fluoxetine 10 mg/kg (positive control). All drugs were administered as a single intraperitoneal injection (volume 10 μL/g body weight) [1]
- Sucrose preference test (SPT): Conducted 24 hours after drug administration. Mice were individually housed with two bottles: one containing 1% sucrose solution and the other containing tap water. After 24 hours, bottle weights were measured, and sucrose preference was calculated as (sucrose intake / total fluid intake) × 100% [1]
- Forced swim test (FST): Conducted 48 hours after drug administration. Mice were placed in transparent cylinders (20 cm diameter, 30 cm height) filled with 25°C water (20 cm depth) for 6 minutes. Immobility time (time spent floating without active swimming or climbing) was recorded during the last 4 minutes [1]
- Elevated plus maze (EPM): Conducted 72 hours after drug administration. The maze consisted of two open arms (30×5 cm) and two closed arms (30×5×15 cm) elevated 50 cm above the floor. Mice were placed in the center facing an open arm, and time spent in open arms was recorded for 5 minutes [1]
3. Brain tissue collection and Western blot:
- Mice were euthanized 1 hour after FST (49 hours post-drug administration). Hippocampus and prefrontal cortex were dissected on ice, snap-frozen in liquid nitrogen, and stored at -80°C. Tissues were homogenized in RIPA buffer with protease/phosphatase inhibitors, and protein lysates were used for Western blot analysis of p-ERK1/2 and total ERK1/2 [1]

[1]. The selective 5-HT 1A receptor biased agonists, F15599 and F13714, show antidepressant-like properties after a single administration in the mouse model of unpredictable chronic mild stress. Psychopharmacology (Berl). 2021 May 10.

1. Fumaric acid F13714 is a selective, G protein-biased 5-HT₁A receptor agonist developed for the treatment of major depressive disorder (MDD)[1]
2. Mechanism of action: Fumaric acid F13714 selectively binds to 5-HT₁A receptors and preferentially activates G protein-mediated signal transduction (Gαi/o) rather than the recruitment of β-arrestin 2. This biased signaling can inhibit cAMP production, activate the ERK1/2 pathway, and promote neuroplasticity in brain regions associated with depression (hippocampus, prefrontal cortex) without inducing β-arrestin-mediated receptor desensitization [1]. 3. Preclinical therapeutic advantages: Compared with non-biased 5-HT₁A receptor agonists (e.g., 8-OH-DPAT) or selective serotonin reuptake inhibitors (e.g., fluoxetine), fumarate F13714 exerts a rapid antidepressant-like effect after a single dose in the UCMS model (a validated preclinical model of depression), avoiding the problem of delayed onset of traditional antidepressants [1]. 4. Selectivity: The high selectivity of 5-HT₁A receptors relative to other 5-HT subtypes and non-5-HT receptors suggests a lower risk of off-target side effects (e.g., hypotension, sedation), which is usually associated with non-selective 5-HT ligands [1].

C25H29CLF2N4O5

538.9768

538.18

C, 55.71; H, 5.42; Cl, 6.58; F, 7.05; N, 10.40; O, 14.84

208109-39-1

F 13714; 208109-38-0

9958888

Solid powder

4

10

8

37

666

0

CC1=C(N=C(C=C1)CNCC2(CCN(CC2)C(=O)C3=CC(=C(C=C3)F)Cl)F)NC.C(=C/C(=O)O)\C(=O)O

HZJWIYFEDQNBEU-WLHGVMLRSA-N

InChI=1S/C21H25ClF2N4O.C4H4O4/c1-14-3-5-16(27-19(14)25-2)12-26-13-21(24)7-9-28(10-8-21)20(29)15-4-6-18(23)17(22)11-15;5-3(6)1-2-4(7)8/h3-6,11,26H,7-10,12-13H2,1-2H3,(H,25,27);1-2H,(H,5,6)(H,7,8)/b;2-1+

(E)-but-2-enedioic acid;(3-chloro-4-fluorophenyl)-[4-fluoro-4-[[[5-methyl-6-(methylamino)pyridin-2-yl]methylamino]methyl]piperidin-1-yl]methanone

F-13714; F13714; F-14679; F 14679; F14679; F13714 fumarate; F 13714

2934.99.9001

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.

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

DMSO: ~250 mg/mL (~463.9 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.64 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.64 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.)