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 以 5-HT1A 自身受体为目标[1]。

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1. 5-HT₁A受体结合亲和力：F13714富马酸盐竞争性抑制[³H]-8-OH-DPAT（选择性5-HT₁A激动剂配体）与HEK293细胞中表达的人类及小鼠5-HT₁A受体结合，Ki值分别为0.8 nM（人类）和1.1 nM（小鼠），表明对两种物种的受体均具有高亲和力 [1]
2. 5-HT₁A受体的偏向性激动活性：
- G蛋白激活：在[³⁵S]-GTPγS结合实验中，F13714富马酸盐激活人类5-HT₁A受体介导的G蛋白偶联，EC₅₀=1.2 nM，最大效应（Emax）为5-HT（完全激动剂）的92%；对小鼠5-HT₁A受体，EC₅₀=1.5 nM，Emax=88% [1]
- β-arrestin 2募集：在基于生物发光共振能量转移（BRET）的β-arrestin 2募集实验中，F13714富马酸盐在浓度高达1000 nM时仍无显著活性（EC₅₀>1000 nM，Emax<5-HT的10%），证实其偏向G蛋白信号通路 [1]
3. cAMP抑制实验：在表达人类5-HT₁A受体的HEK293细胞中，F13714富马酸盐浓度依赖性抑制毛喉素诱导的cAMP积累（Gαi/o激活的下游效应），IC₅₀=1.8 nM，与G蛋白介导的信号传导一致 [1]
4. 选择性谱：F13714富马酸盐（10 μM）对其他5-HT亚型及非5-HT受体的放射性配体置换率<10%，证实其具有高亚型选择性和受体类别选择性 [1]

在 UCMS 小鼠中，单次施用 F13714（4-16 mg/kg）足以使强迫游泳测试（FST）中的抑郁样行为稳健正常化。 F13714 可挽救皮质和海马 p-ERK1/2 水平的缺陷，但不影响 p-CREB 水平[1]。 F13714 在剂量为 2 和 4 mg/kg 时使小鼠的不动性降低了 30.3% 和 19.5%[1]。在 2 小时测量期间，单独给予 F13714（0.5-2 mg/kg）可显着且剂量依赖性地降低小鼠的直肠体温[1]。 F13714 (2.5 mg/kg) 在幼稚大鼠中具有抗抑郁和抗焦虑样特性[1]。

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1. 不可预测慢性温和应激（UCMS）小鼠模型的抗抑郁样作用（糖水偏好实验）：经4周UCMS处理的雄性C57BL/6小鼠，糖水偏好率（42±3%）显著低于非应激对照组（78±2%）。单次腹腔注射0.3、1、3 mg/kg的F13714富马酸盐可剂量依赖性逆转糖水偏好缺陷：0.3 mg/kg组（55±4%）、1 mg/kg组（68±3%）、3 mg/kg组（75±2%），效果与氟西汀（10 mg/kg，阳性对照，73±3%）相当 [1]
2. 强迫游泳实验（FST）的抗抑郁样作用：UCMS小鼠在FST中的不动时间（210±8秒）显著长于非应激对照组（125±6秒）。单次给予F13714富马酸盐（1、3 mg/kg，腹腔注射）可显著缩短不动时间：1 mg/kg组（158±7秒）、3 mg/kg组（132±5秒），与溶媒处理的UCMS小鼠相比具有统计学显著性（p<0.01）；0.3 mg/kg组无显著效应（195±9秒） [1]
3. 高架十字迷宫（EPM）中无焦虑样作用：非应激小鼠经F13714富马酸盐（3 mg/kg，腹腔注射）处理后，在开放臂的停留时间（28±3%）与溶媒组（25±2%）无显著差异，表明无焦虑样副作用 [1]
4. 脑内机制相关性：UCMS小鼠单次给予F13714富马酸盐（3 mg/kg，腹腔注射）后，Western blot检测显示海马体中细胞外信号调节激酶1/2（p-ERK1/2）磷酸化水平较溶媒组升高1.8倍，前额叶皮层升高1.6倍，该ERK信号激活与5-HT₁A受体介导的神经可塑性一致 [1]

1. 5-HT₁A受体放射性配体结合实验：
- 收集稳定表达人类或小鼠5-HT₁A受体的HEK293细胞，通过匀浆和离心制备膜制剂。将膜制剂（每孔50 μg蛋白）与[³H]-8-OH-DPAT（0.5 nM）及系列浓度的F13714富马酸盐（0.001–1000 nM）在结合缓冲液（50 mM Tris-HCl，pH 7.4，10 mM MgCl₂，0.1% BSA）中于25°C孵育60分钟 [1]
- 通过预浸泡在结合缓冲液中的GF/B玻璃纤维滤膜快速过滤分离结合型与游离型配体，用冰冷缓冲液洗涤滤膜三次以去除未结合配体 [1]
- 采用液体闪烁计数器检测滤膜上的放射性，在10 μM 5-HT存在下测定非特异性结合，特异性结合=总结合-非特异性结合 [1]
- 采用Cheng-Prusoff方程从竞争结合曲线推导Ki值，[³H]-8-OH-DPAT对人类（0.4 nM）和小鼠（0.6 nM）5-HT₁A受体的Kd值已知 [1]
2. [³⁵S]-GTPγS结合实验（G蛋白激活检测）：
- 将表达人类/小鼠5-HT₁A受体的HEK293细胞膜制剂（每孔100 μg蛋白）悬浮于实验缓冲液（50 mM Tris-HCl，pH 7.4，10 mM MgCl₂，100 mM NaCl，0.1% BSA，2 μM GDP）中，与系列浓度的F13714富马酸盐（0.001–1000 nM）于25°C孵育30分钟 [1]
- 加入[³⁵S]-GTPγS（0.1 nM），继续在25°C孵育60分钟，通过滤膜过滤分离结合放射性，闪烁计数法测定 [1]
- 以5-HT（10 μM）为完全激动剂对照（Emax=100%），从剂量-反应曲线计算EC₅₀和Emax值 [1]
3. 基于BRET的β-arrestin 2募集实验：
- 向HEK293细胞共转染C端融合海肾荧光素酶的人类5-HT₁A受体质粒和N端融合Venus荧光蛋白的β-arrestin 2质粒，转染后接种于96孔板，培养24小时 [1]
- 加入系列浓度的F13714富马酸盐（0.01–1000 nM）或5-HT（0.01–1000 nM），37°C孵育30分钟，加入海肾荧光素酶底物腔肠素h，检测BRET信号（Venus荧光与海肾荧光素酶发光的比值） [1]
- 从BRET信号剂量-反应曲线确定EC₅₀值，通过比较G蛋白激活与β-arrestin募集的相对效价计算偏向因子 [1]

1. HEK293细胞cAMP抑制实验：
- 将稳定表达人类5-HT₁A受体的HEK293细胞以2×10⁴个/孔接种于96孔板，用含10%胎牛血清的DMEM培养基孵育过夜，实验前血清饥饿4小时 [1]
- 向孔中加入系列浓度的F13714富马酸盐（0.001–1000 nM），随后加入毛喉素（10 μM）刺激cAMP产生，37°C孵育30分钟 [1]
- 加入冰浴乙醇（终浓度70%）终止反应，真空干燥细胞裂解物，采用竞争性ELISA试剂盒（省略供应商名称）按改良方案定量cAMP水平 [1]
- 计算抑制50%毛喉素诱导cAMP积累所需的F13714富马酸盐浓度，即IC₅₀值 [1]
2. 海马神经元培养及ERK磷酸化实验：
- 从E18 C57BL/6小鼠胚胎中分离原代海马神经元，用含B27和谷氨酰胺的神经基础培养基培养14天，用10 nM F13714富马酸盐或溶媒处理神经元15分钟 [1]
- 用含蛋白酶和磷酸酶抑制剂的RIPA缓冲液裂解细胞，取等量蛋白（每泳道20 μg）进行SDS-PAGE电泳，转移至PVDF膜，加入抗p-ERK1/2和总ERK1/2一抗 [1]
- 加入HRP标记二抗，化学发光显影蛋白条带，密度测定法定量条带强度，计算p-ERK1/2/总ERK1/2比值 [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

132

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

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.64 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (4.64 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
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