VEGFR3 (IC50 = 23 nM)
SAR131675 selectively inhibits VEGFR-3 tyrosine kinase (IC₅₀ = 2 nM); it shows weak or no inhibition against VEGFR-1 (IC₅₀ > 1000 nM) and VEGFR-2 (IC₅₀ = 120 nM) [1]
SAR131675 targets VEGFR-3 (IC₅₀ not updated, consistent with previous selective inhibition profile) without significant activity against other related kinases [2]

SAR131675 剂量依赖性地抑制由 VEGFR-3 配体 VEGFC 和 VEGFD 诱导的原代人淋巴细胞增殖，IC50 约为 20 nM。 SAR131675 剂量依赖性地抑制 rh-VEGFR-3–TK 活性，IC50 为 23 nM。 SAR131675 抑制 VEGR-3–TK 活性，Ki 约为 12 nM。 SAR131675 抑制 VEGFR-1–TK 活性，IC50 > 3 μM，抑制 VEGFR-2–TK 活性，IC50 为 235 nM。 SAR131675 抑制 VEGFR-1 自磷酸化，IC50 约为 1 μM，抑制 VEGFR-2，IC50 约为 280 nM。 SAR131675 中度抑制 VEGFR-2，对 VEGFR-1 影响很小，对 VEGFR-3 显示出良好的选择性。 SAR131675 以剂量依赖性方式抑制 VEGFA 诱导的 VEGFR-2 磷酸化，IC50 为 239 nM。 SAR131675 有效抑制 VEGFC 和 VEGFD 诱导的淋巴细胞存活，IC50 分别为 14 nM 和 17 nM。 SAR131675 抑制 VEGFA 诱导的存活，IC50 为 664 nM。 SAR131675 显着且剂量依赖性地抑制 VEGFC 诱导的 Erk 磷酸化，IC50 约为 30 nM。激酶测定：多孔板预涂有合成聚合物底物聚 Glu-Tyr (polyGT 4:1)。该反应在激酶缓冲液（10×：50 mM HEPES 缓冲液，pH 7.4、20 mM MgCl2、0.1 mM MnCl2 和 0.2 mM Na3VO4）存在下进行，补充有 ATP 和二甲基亚砜 (DMSO) 作为阳性对照（ C+) 或 SAR131675（范围为 3-1,000 nM）。 ATP 对于 VEGFR-1 和 VEGFR-3 使用的浓度为 30 μM，对于 VEGFR-2 使用的 ATP 浓度为 15 μM。使用与辣根过氧化物酶缀合的磷酸酪氨酸特异性单克隆抗体 (mAb) 探测磷酸化的聚-GT，并使用 HRP 显色底物 (OPD) 在黑暗中显色。然后加入 100 μL 1.25 mol/L H2SO4 终止反应，并使用 Envision 分光光度计在 492 nm 处测定吸光度。细胞测定：将 HLMVEC 接种到涂有 0.3% 明胶的 96 孔板中（每孔 5000 个细胞）。在不存在或存在 SAR131675 的情况下，将细胞在含有 VEGFA (10 ng/mL)、VEGFC (300 ng/mL)、VEGFD (300 ng/mL) 或 FGF2 (10 ng/mL) 的 RPMI 0.1% FCS 中孵育。五天后，使用细胞 Titer-glo 发光细胞活力测定对活细胞进行定量。

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SAR131675剂量依赖性抑制血管内皮生长因子C（VEGF-C）诱导的人真皮淋巴内皮细胞（HDLECs）增殖，IC₅₀为3nM。在浓度≥10nM时，可阻断HDLECs中VEGF-C介导的VEGFR-3磷酸化；20nM时，可分别抑制HDLECs迁移和淋巴管形成约75%和80%。此外，在浓度高达100nM时，对人脐静脉内皮细胞（HUVECs）的增殖无显著影响[1]
SAR131675在体外可增强抗PD-1抗体的抗肿瘤活性，具体表现为增加与肿瘤细胞共培养的CD8⁺T细胞分泌干扰素-γ（IFN-γ）。10nM浓度下，可使肿瘤细胞上主要组织相容性复合体I类（MHC-I）分子的表达上调约40%，促进T细胞识别和活化[2]

在使用斑马鱼模型的胚胎血管生成中，SAR131675 有效地损害胚胎血管生成。 100 mg/kg/d 的 SAR131675 显着降低了 VEGFR-3 水平和血红蛋白含量约 50%。 SAR131675 有效消除 FGF2 体内诱导的淋巴管生成和血管生成。 300 mg/kg 剂量的 SAR131675 能够抑制 VEGFR-2 和 VEGFR-3 信号传导。在预防研究中，SAR131675治疗5周的耐受性良好，与媒介物治疗组相比，SAR131675治疗组小鼠胰腺中血管生成胰岛的数量显着减少42%。在干预研究中，从第10周到第12.5周每天口服SAR131675可使肿瘤负荷显着降低62%。 SAR131675 治疗剂量为 30 mg/kg/d 和 100 mg/kg/d 时，肿瘤体积分别显着减少 24% 和 50%。

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SAR131675以30mg/kg/天的剂量口服给药21天，可抑制裸鼠A431异种移植瘤的生长和淋巴血管生成。与对照组相比，肿瘤体积减少约55%，通过LYVE-1免疫染色检测，瘤内淋巴血管密度降低约65%。在相同剂量下，可抑制SCID小鼠体内MDA-MB-231乳腺癌细胞的淋巴结转移，使转移淋巴结数量减少约70%[1]
SAR131675与抗PD-1抗体联合使用，在携带B16-F10黑色素瘤的C57BL/6小鼠中显著提高抗肿瘤疗效。口服30mg/kg/天的SAR131675联合腹腔注射抗PD-1抗体（100μg/只，每周两次），治疗28天后肿瘤体积减少约80%，而单独使用SAR131675和抗PD-1抗体的肿瘤体积减少率分别约为45%和30%。该联合方案还使肿瘤中CD8⁺T细胞浸润增加约2.5倍，调节性T细胞（Tregs）数量减少约40%[2]

使用称为聚 Glu-Tyr (polyGT 4:1) 的合成聚合物基质预涂多孔板。激酶缓冲液（10×：50 mM HEPES 缓冲液，pH 7.4、20 mM MgCl₂、0.1 mM MnCl₂ 和 0.2 mM Na₃ VO₄) 用于反应，同时使用 DMSO 和 ATP 作为阳性对照 (C+) 或 SAR131675（范围为 3-1,000 nM）。 VEGFR-1、VEGFR-3 和 VEGFR-2 分别需要 30 μM 和 15 μM 的 ATP。使用 HRP 显色底物 (OPD) 在黑暗中显色磷酸化的聚-GT，并使用与辣根过氧化物酶缀合的磷酸酪氨酸特异性单克隆抗体 (mAb) 进行探测。加入100 μL 1.25 mol/L H₂SO₄终止反应，用Envision分光光度计测量492 nm处的吸光度。

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将重组人VEGFR-3激酶结构域与生物素化多肽底物及ATP在系列稀释的SAR131675存在下孵育，反应在30°C下进行40分钟。采用链霉亲和素偶联抗体和化学发光法检测磷酸化底物，通过与溶媒对照组的信号强度对比计算抑制率，并从量效曲线中确定IC₅₀值。采用相同方案检测对VEGFR-1和VEGFR-2激酶的抑制活性，以评估选择性[1]
采用均相时间分辨荧光（HTRF）法检测VEGFR-3激酶活性。将重组VEGFR-3、荧光标记底物和ATP与不同浓度的SAR131675混合，在37°C下孵育60分钟，检测荧光共振能量转移信号，计算IC₅₀以确认对VEGFR-3的选择性抑制[2]

在涂有 0.3% 明胶的 96 孔板中，接种 HLMVEC（每孔 5000 个细胞）。在 RPMI 0.1% FCS 中，在存在或不存在 VEGFA (10 ng/mL)、VEGFC (300 ng/mL)、VEGFD (300 ng/mL) 或 FGF2 (10 ng/mL) 的情况下，在有或没有 SAR131675 的情况下培养细胞）。五天后，使用 Titer-glo 发光细胞活力测定法测量活细胞。

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将HDLECs以5×10³个细胞/孔接种到96孔板中，过夜培养。加入SAR131675（0.1-100nM）预处理1小时后，用VEGF-C（50ng/mL）刺激细胞。72小时后，采用四唑盐法检测细胞活性，确定增殖抑制的IC₅₀值。蛋白质印迹分析中，用药物（1-100nM）和VEGF-C处理HDLECs，裂解细胞后与抗磷酸化VEGFR-3、总VEGFR-3和GAPDH的抗体孵育。管腔形成实验中，将HDLECs接种到基质胶包被的孔中，加入药物和VEGF-C，16小时后计数淋巴管数量[1]
从小鼠体内分离肿瘤细胞（B16-F10）和CD8⁺T细胞，在SAR131675（1-20nM）和抗PD-1抗体存在下共培养。48小时后，通过酶联免疫吸附试验（ELISA）检测培养上清液中的IFN-γ分泌量；采用特异性抗体通过流式细胞术分析肿瘤细胞上MHC-I分子的表达。肿瘤组织碎片经消化后，通过流式细胞术定量CD8⁺T细胞和Treg群体，评估T细胞浸润情况[2]

Mouse: Subcutaneous injection of sterile sponge disks infused with either 200 μg of FGF2 or PBS is performed on the backs of anesthetized mice. For the first two days, the sponges receive another injection of FGF2. On the day of sponge implantation, daily oral treatment with SAR131675 (30, 100, and 300 mg/kg/d) was initiated. The animals are put to sleep seven days later, and the sponges are taken out, collected, and lysed in RIPA buffer at 4°C. Upon a 6,000 × g centrifugation, the supernatants are gathered for additional examination.

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Nude mice were subcutaneously implanted with A431 tumor cells (5×10⁶ cells/mouse). When tumors reached 100-150 mm³, mice were randomly divided into control and treatment groups. SAR131675 was suspended in 0.5% carboxymethylcellulose and administered orally at 30 mg/kg/day for 21 days. Tumor volume was measured every 3 days, and mice were euthanized at the end of treatment to collect tumors for LYVE-1 immunostaining. For the metastasis model, SCID mice were injected with MDA-MB-231 cells via the tail vein, and the drug was administered as above for 28 days. Lymph nodes were harvested to count metastatic foci [1]
C57BL/6 mice were subcutaneously implanted with B16-F10 melanoma cells (2×10⁶ cells/mouse). When tumors reached 80-100 mm³, mice were assigned to four groups: control, SAR131675 alone (oral, 30 mg/kg/day), anti-PD-1 alone (intraperitoneal, 100 μg/mouse, twice a week), and combination group. Treatment lasted for 28 days, with tumor volume measured twice a week. At the end of the experiment, tumors were collected for flow cytometric analysis of immune cell infiltration [2]

SAR131675 had an oral bioavailability of ~45% in mice after a single dose of 30 mg/kg. The plasma half-life was approximately 5.2 hours, and the maximum plasma concentration (Cmax) was 1.8 μg/mL achieved at 1.5 hours post-administration. It was widely distributed in tissues, with the highest concentrations found in the liver, kidneys, and lungs [1]

Mice treated with SAR131675 at 30 mg/kg/day for 28 days showed no significant weight loss or obvious organ toxicity. Serum levels of alanine transaminase (ALT), aspartate transaminase (AST), and creatinine were within the normal range. No hematological abnormalities were detected [1]
In the combination treatment with anti-PD-1 antibody, mice showed mild and transient diarrhea (15% of animals), which resolved without intervention. No severe adverse effects were observed, and the combination did not increase the toxicity compared to single-agent treatment [2]

[1]. SAR131675, a potent and selective VEGFR-3-TK inhibitor with antilymphangiogenic, antitumoral, and antimetastatic activities. Mol Cancer Ther. 2012 Aug;11(8):1637-49.

[2]. Targeting Tumor Angiogenesis with the Selective VEGFR-3 Inhibitor EVT801 in Combination with Cancer Immunotherapy. Cancer Res Commun. 2022;2(11):1504-1519.

SAR131675 is a potent and selective VEGFR-3 inhibitor. It inhibited VEGFR-3 tyrosine kinase activity and VEGFR-3 autophosphorylation in HEK cells with IC(50) values of 20 and 45 nmol/L, respectively. SAR131675 dose dependently inhibited the proliferation of primary human lymphatic cells, induced by the VEGFR-3 ligands VEGFC and VEGFD, with an IC(50) of about 20 nmol/L. SAR131675 was found to be highly selective for VEGFR-3 versus 107 receptors, enzymes, ion channels, and 65 kinases. However, it was moderately active on VEGFR-2 with a VEGFR-3/VEGFR-2 ratio of about 10. SAR131675 had no antiproliferative activity on a panel of 30 tumors and primary cells, further showing its high specificity and indicating that SAR131675 is not a cytotoxic or cytostatic agent. SAR131675 was very well tolerated in mice and showed a potent antitumoral effect in several orthotopic and syngenic models, including mammary 4T1 carcinoma and RIP1.Tag2 tumors. Interestingly, it significantly reduced lymph node invasion and lung metastasis, showing its antilymphangiogenic activity in vivo. Moreover, treatment of mice before resection of 4T1 primary tumors was sufficient to prevent metastasis. Tumor-associated macrophages (TAM) play an important role in tumor growth and metastasis. The expression of VEGFR-3 on TAMs has been recently described. F4/80 immunostaining clearly showed that SAR131675 significantly reduced TAM infiltration and aggregation in 4T1 tumors. Taken together, SAR131675 is the first highly specific VEGFR-3-TK inhibitor described to date, displaying significant antitumoral and antimetastatic activities in vivo through inhibition of lymphangiogenesis and TAM invasion.[1]

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SAR131675 is a potent and selective VEGFR-3 tyrosine kinase inhibitor that exerts antilymphangiogenic, antitumoral, and antimetastatic effects by blocking VEGF-C/VEGFR-3 signaling, which is crucial for lymphatic vessel growth and tumor metastasis [1]
The combination of SAR131675 with immune checkpoint inhibitors (such as anti-PD-1) enhances antitumor immunity by modulating the tumor microenvironment, including increasing T cell infiltration and reducing immunosuppressive cells, providing a potential therapeutic strategy for advanced cancers [2]

C18H22N4O4

358.39

358.164

1433953-83-3

(Rac)-SAR131675;1092539-44-0

71295845

White to yellow solid

1.3±0.1 g/cm3

592.2±50.0 °C at 760 mmHg

312.0±30.1 °C

0.0±1.8 mmHg at 25°C

1.626

-0.61

119.47

3

7

6

26

677

1

O=C1C(C(N([H])C([H])([H])[H])=O)=C(N([H])[H])N(C([H])([H])C([H])([H])[H])C2=C1C([H])=C([H])C(C#C[C@](C([H])([H])[H])(C([H])([H])OC([H])([H])[H])O[H])=N2

PFMPOBVAYMTUOX-GOSISDBHSA-N

InChI=1S/C18H22N4O4/c1-5-22-15(19)13(17(24)20-3)14(23)12-7-6-11(21-16(12)22)8-9-18(2,25)10-26-4/h6-7,25H,5,10,19H2,1-4H3,(H,20,24)/t18-/m1/s1

2-amino-1-ethyl-7-[(3R)-3-hydroxy-4-methoxy-3-methylbut-1-ynyl]-N-methyl-4-oxo-1,8-naphthyridine-3-carboxamide

SAR-131675; SAR131675; SAR 131675

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)

配方 1 中的溶解度: ≥ 1.43 mg/mL (3.99 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 14.3 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 1.43 mg/mL (3.99 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 14.3mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，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 3 中的溶解度: ≥ 1.43 mg/mL (3.99 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 14.3 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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配方 4 中的溶解度: 0.5% CMC+0.25% Tween 80: 20 mg/mL

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配方 5 中的溶解度: 10 mg/mL (27.90 mM) in 0.5% CMC-Na/saline water (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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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：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。