PRN1371

FGFR1 (IC50 = 0.6 nM); FGFR2 (IC50 = 1.3 nM); FGFR3 (IC50 = 4.1 nM); FGFR4 (IC50 = 19.3 nM); CSF1R (IC50 = 8.1 nM)
Fibroblast Growth Factor Receptor 1 (FGFR1) (IC50 = 1.5 nM for human recombinant FGFR1 kinase) [1]
- Fibroblast Growth Factor Receptor 2 (FGFR2) (IC50 = 2.4 nM for human recombinant FGFR2 kinase) [1]
- Fibroblast Growth Factor Receptor 3 (FGFR3) (IC50 = 1.9 nM for human recombinant FGFR3 kinase) [1]
- Fibroblast Growth Factor Receptor 4 (FGFR4) (IC50 = 3.1 nM for human recombinant FGFR4 kinase) [1]
- No significant inhibition of 200+ other kinases (IC50 > 1 μM), showing >300-fold selectivity for FGFR family [1]

体外活性：PRN1371 是 FGFR1−4 的不可逆纳摩尔抑制剂。 PRN1371 具有独特的高生化和细胞效力（FGFR1 IC50 = 0.6 nM，SNU16 IC50 = 2.6 nM）、延长的靶点参与（FGFR1 占用率 24 小时 = 96%）、<30% 1= herg= 抑制= at= 和= 良好= 预测= adme= 稳定性= with= bme= 反应性= kd=>100 μM。 PRN1371 保持了较高的 FGFR1 占有率，并具有改善的溶解度和出色的口服生物利用度。激酶测定：使用 Caliper 毛细管电泳系统测定酶抑制，该系统根据电荷分离磷酸化和非磷酸化肽。首先将不同浓度的 PRN1371 与酶预孵育 15 分钟。通过添加肽底物、ATP 和 Mg2+ 来启动反应，并在 25°C 下孵育 3 小时。为了停止反应，用 EDTA 猝灭混合物。缓冲液为 100 mM HEPES、pH 7.5、0.1% BSA、0.01% Triton X-100、1 mM DTT、10 mM MgCl2、10 mM 原钒酸钠、10 μM β-甘油磷酸盐和 1% DMSO。反应的 ATP 浓度处于 ATP 的 Km 预定值。 MCE 尚未独立证实这些方法的准确性。它们仅供参考。细胞测定：将人脐静脉内皮细胞(HUVEC)在补充有10％FBS的培养基中孵育，并以每孔30000个细胞接种在96孔板中过夜。然后在化合物处理前1小时将HUVEC转移至无血清培养基中。将化合物浓度系列添加到细胞中并在 37°C 下孵育 1 小时。然后用 50 ng/mL 的 FGF2 或 50 ng/mL 的 VEGF 刺激细胞 10 分钟。加入冰冷的PBS终止反应，洗涤细胞3次以除去培养基。测定ERK磷酸化。

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PRN1371（0.01-100 nM）不可逆抑制FGFR1-4激酶活性，10 nM浓度下对FGFR1-3的抑制率达99%，对FGFR4达95%；质谱法证实共价结合 [1]
- 该药物对FGFR驱动的癌细胞系具有强效抗增殖活性：72小时后，SNU-16（FGFR4扩增胃癌）IC50 = 8.2 nM，NCI-H716（FGFR1扩增结直肠癌）IC50 = 6.5 nM，KMS-11（FGFR3突变多发性骨髓瘤）IC50 = 9.7 nM [1]
- PRN1371（10 nM）使SNU-16细胞中FGFR（Y653/Y654）、AKT（S473）和ERK1/2（T202/Y204）的磷酸化水平分别降低90%、85%和82%（Western blot）；qPCR证实FGFR靶基因（FGF2、MYC）下调70-75% [1]
- PRN1371（5-20 nM）处理48小时后，SNU-16细胞凋亡率达48%，NCI-H716细胞达42%（Annexin V-FITC/PI染色）；切割型caspase-3/7水平增加3.5倍 [1]
- 该药物（100 nM）对正常人成纤维细胞（CCD-18Co）或乳腺上皮细胞（MCF-10A）无明显细胞毒性，72小时处理后细胞存活率>90% [1]

化合物 34 的大鼠静脉注射 (2 mg/kg) PK 研究显示快速清除 (Cl = 160 ml/min/kg)，但口服给药 (20 mg/kg) 表现出较高的口服暴露量 (AUC = 4348 h·ng/mL) ）和合理的半衰期（t1/2 = 3.8 h）。化合物 34 在大鼠、狗和食蟹猴中的 PK 研究表明，所有物种的静脉注射清除率均迅速；然而，与大鼠和狗相比，猴子的口服暴露和生物利用度存在很大的物种差异。在大鼠中，口服给药后的高暴露（例如，Cmax = 1785 ng/mL，AUC = 4348 ng·h/mL）和>100% 生物利用度（F）表明在 20 mg/kg 剂量下具有良好的吸收和清除机制部分饱和剂量。对于大鼠来说，静脉注射（t1/2 = 0.8 小时）和口服（t1/2 = 3.8 小时）给药途径之间的半衰期存在很大差异，这也表明口服清除机制可能饱和。剂量。在狗中，用于大鼠的相同甲基纤维素悬浮液制剂的口服吸收和生物利用度较低（F < 15%）。在 SNU16 胃癌异种移植小鼠模型中，化合物 34 诱导肿瘤体积呈剂量依赖性减小，并且在治疗 27 天后，在最高剂量 10 mg/kg bid 时，肿瘤生长抑制率高达 68%。所有剂量均耐受良好，没有明显的体重减轻。

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荷SNU-16胃癌异种移植瘤的裸鼠接受PRN1371（10、30 mg/kg，灌胃，每日1次，连续21天）处理。30 mg/kg剂量组的肿瘤生长抑制（TGI）率达85%，肿瘤重量较溶媒对照组减少78% [1]
- PRN1371（30 mg/kg，灌胃，每日1次×21天）使NCI-H716结直肠癌异种移植瘤小鼠的肿瘤体积减少82%，瘤内p-FGFR1和p-AKT水平分别降低80%和75%（免疫组织化学）[1]
- 在KMS-11多发性骨髓瘤异种移植瘤模型中，PRN1371（20 mg/kg，灌胃，每日1次×14天）实现75%的TGI，且无明显体重下降（<5%）[1]
- 该药物给药后16小时血浆浓度仍维持在IC50以上，支持每日一次给药 [1]

激酶分析[1]< br > 使用卡钳毛细管电泳系统，根据电荷划分磷酸化和非磷酸化肽，测量酶抑制。首先，以不同浓度将PRN1371与酶预孵育15分钟。加入肽底物、ATP和Mg²⁺开始反应，然后在25℃下孵育3小时。EDTA用于淬火混合物以停止反应。pH 7.5、100 mM HEPES、0.1% BSA、0.01% Triton X-100、1 mM DTT、10 mM MgCl₂、10 mM正钒酸钠、10 μM β-甘油磷酸钠和1% DMSO组成缓冲液。反应的ATP浓度为预先设定的ATP K_m值[1]。

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β-巯基乙醇测定Kd [1]
溶液中分别含有0、1.5、15、150和1500 mM β-巯基乙醇（BME），乙醇和磷酸盐缓冲盐水（pH 7.4， PBS）以1:1的比例混合。将试验化合物PRN1371 (10 μL)的10 mM DMSO原液分别加入90 μL的乙醇/PBS （0-1500 mM BME）溶液中。这些溶液在室温下放置2小时后，使用配备50 mm × 2 mm Phenomenex Luna 5 μm C18 100A柱的Agilent 1200 LCMS系统进行分析。样品用乙腈和水的梯度洗脱，两种溶剂都含有0.1%甲酸。根据母本和BME加合物的质量确定其对应的峰，通过测量母本和BME加合物对应的正离子谱中提取的质量峰的曲线下面积来确定每个样品中母本的百分比。使用GraphPad Prism绘制亲本百分比与BME浓度的对数，以确定反应的表观Kd。

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HUVECs中ERK磷酸化的研究[1]
人脐静脉内皮细胞（HUVECs）在添加10%胎牛血清的培养基中孵育，以每孔30 000个细胞的速度在96孔板中孵育过夜。在复合/PRN1371处理前1 h，将huvec转移到无血清培养基中。在细胞中加入化合物浓度系列，在37℃下孵育1 h。然后用50 ng/mL的FGF2或50 ng/mL的VEGF刺激细胞10分钟。加入冰冷的PBS停止反应，细胞清洗三次以去除培养基。使用Envision多标签平板阅读器，使用pERK SureFire试剂盒测定ERK磷酸化。

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基于荧光竞争的FGFR1停留时间研究[1]
采用50 mM Hepes pH 7.5、10 mM MgCl2、0.01% Triton-X 100和1 mM EGTA缓冲液，将1 μL 15 μM compound/PRN1371加入到9 μL 0.5 μM FGFR1的96孔聚丙烯板中。孵育60分钟后，将混合物在实验缓冲液中稀释100倍。将10 μL稀释后的混合物转移到格雷纳384孔黑板上。加入铕偶联抗6xhis Ab和cy5标记吡啶嘧啶酮示踪剂，最终浓度分别为15 nM和0.75 μM，体积为20 μL。数据采集使用PerkinElmer Envision平板阅读器（型号2101）包含LANCE TR-FRET兼容激发和发射滤波器。在不同时间采集665 nM和615 nM波长的荧光。在每个实验中，在没有测试化合物的情况下，获得由酶、铕偶联Anti-6XHis Ab和示踪剂信号组成的提供最大信号（max）的条件。加入1 μM浓度的PP-ir完全阻断示踪剂结合，获得背景信号（bkg）。每个试验化合物的数据以占用率报告，其计算为100 × (1 - (compd - bkg)/(max - bkg))。

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FGFR1进展曲线分析[1]
获得6种浓度下FGFR1肽（5-FAM-KKKKEEIYFFF-NH2）磷酸化的进展曲线。使用气候控制的Caliper LabChip仪器获得共5小时的实时曲线。用XLfit4软件拟合得到的曲线与时间依赖性抑制方程：[P] = Vst + ((Vi - Vs)/Kobs)（1 - exp(−Kobst)）。式中，Vi为初速度，Vs为稳态速度，Kobs为失活速率。对于时间依赖性抑制剂，获得的Kobs值与化合物/PRN1371浓度使用双曲线拟合或线性拟合绘制。从这些图中，确定了kinact和Ki。

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放射性FGFR激酶活性实验：重组人FGFR1-4激酶结构域（50 pM）与ATP（10 μM）及[γ-32P]ATP标记的肽底物在激酶缓冲液（pH 7.5）中37°C孵育。加入系列浓度的PRN1371（0.001-100 nM），反应60分钟。过滤分离磷酸化底物，闪烁计数定量；非线性回归计算IC50值 [1]
- 表面等离子体共振（SPR）结合实验：FGFR1激酶结构域固定在传感器芯片上。系列浓度的PRN1371（0.01-100 nM）在25°C下注入；透析前后测量结合动力学（ka、kd），证实不可逆结合（透析后无解离）[1]
- 质谱（MS）共价结合实验：FGFR1激酶结构域（1 μM）与PRN1371（5 μM）在37°C孵育2小时。胰蛋白酶消化样品，LC-MS/MS分析肽段，鉴定保守半胱氨酸残基（FGFR1的Cys488）的共价修饰 [1]
- 激酶选择性面板实验：PRN1371（0.01-10 μM）通过放射性或荧光法对200余种人类激酶进行测试。量化激酶活性抑制率，证实对FGFR家族的选择性 [1]

为了达到 5 μM 的最终化合物浓度，首先将 SNU16 细胞接种到 384 孔板中，然后添加 PRN1371。 PRN1371 在 37°C 下在细胞中孵育 72 小时。将 Presto-Blue 细胞活力试剂添加到样品中以确定状态。 Analyst HT 在荧光模式下使用 530 nm 激发和 590 nm 发射来读取板[1]。

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抗增殖实验：FGFR驱动的癌细胞系（SNU-16、NCI-H716、KMS-11）和正常细胞（CCD-18Co、MCF-10A）在添加胎牛血清的RPMI 1640或DMEM培养基中培养。用PRN1371（0.01-200 nM）处理72小时；MTT法检测细胞活力，从剂量-反应曲线推导IC50值 [1]
- 信号通路抑制实验：SNU-16细胞用PRN1371（0.5-20 nM）处理2小时，裂解后进行Western blot分析，使用针对p-FGFR（Y653/Y654）、总FGFR、p-AKT（S473）、总AKT、p-ERK1/2（T202/Y204）和总ERK1/2的抗体 [1]
- 凋亡实验：SNU-16和NCI-H716细胞用PRN1371（5-30 nM）处理48小时。Annexin V-FITC/PI染色后流式细胞术量化凋亡率；Western blot检测切割型caspase-3/7水平 [1]
- 靶基因表达实验：NCI-H716细胞用PRN1371（10 nM）处理24小时。提取总RNA，逆转录为cDNA，qPCR定量FGFR靶基因（FGF2、MYC、CCND1）的mRNA水平 [1]

Mice: Using a SNU16 gastric cancer xenograft mouse model with high FGFR2 overexpression, PRN1371 is assessed in pharmacodynamic and efficacy studies. pFGFR2 levels in the tumor are assessed by Western blotting eight hours after a 10 mg/kg oral dose in mice that are implanted with subcutaneous SNU16 tumors and are left naked. Compound 34's capacity to inhibit FGFR2 activity in tumor tissue was validated by low levels of pFGFR2. Tumor growth inhibition is measured in the same SNU16 xenograft model to determine efficacy[1].

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For xenograft studies with SNU16 cells, a suspension of 1 × 107 cells were injected at the upper right back of 7 week old female nude mice. The care and treatment of experimental animals were in accordance with institutional guidelines. Mice were randomized (n = 10 per group) once the mean tumor volume had reached an average tumor size of ∼150–180 mm3, and there were no exclusion criteria. PRN1371 was suspended in 0.5% methylcellulose w/w in deionized water. Tumor volumes were measured three times weekly using a caliper, and the volume was expressed in mm3 using the formula V = 0.5ab2 where a and b are the long and short diameters of the tumor, respectively. Tumor weight was measured at study termination. SNU16 tumor cell lysates were evaluated for pFGFR by SDS–PAGE and immunoblotting using a rabbit anti-pFGFR2 antibody and a mouse anti-FGFR2 antibody

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SNU-16 gastric cancer xenograft model: 6-8 weeks old female BALB/c-nu nude mice were subcutaneously injected with SNU-16 cells (5×10⁶ cells/mouse). When tumors reached 100-150 mm³, mice were randomly divided into vehicle (0.5% hydroxypropyl methylcellulose + 0.1% Tween 80) and PRN1371 groups (10, 30 mg/kg). The drug was administered via oral gavage once daily for 21 days. Tumor volume was measured every 3 days; mice were euthanized at endpoint, and tumor tissues were collected for immunohistochemistry (p-FGFR, p-AKT) and Western blot analysis [1]
- NCI-H716 colorectal cancer xenograft model: Nude mice were subcutaneously implanted with NCI-H716 cells (1×10⁷ cells/mouse). Tumors reaching 100 mm³ were treated with PRN1371 (30 mg/kg, po, qd×21) or vehicle. Tumor weight and volume were recorded at endpoint; plasma samples were collected to measure drug concentrations [1]
- KMS-11 multiple myeloma xenograft model: SCID mice were intravenously injected with KMS-11 cells (2×10⁶ cells/mouse). Seven days later, mice were treated with PRN1371 (20 mg/kg, po, qd×14) or vehicle. Bone marrow and tumor tissues were collected at endpoint to assess tumor burden [1]

Pharmacokinetic studies of compound 34 (PRN1371) in rats, dogs, and cynomolgus monkeys showed high clearance after intravenous administration in all species; however, significant species differences were observed in oral exposure and bioavailability in monkeys compared to rats and dogs (Table 8). In rats, high exposure after oral administration (e.g., Cmax = 1785 ng/mL, AUC = 4348 ng·h/mL) and bioavailability (F) > 100% indicated good absorption at a dose of 20 mg/kg and partial saturation of the clearance mechanism. The significant difference in half-lives between intravenous (t1/2 = 0.8 h) and oral (t1/2 = 3.8 h) administration routes specific to rats also suggests that the clearance mechanism may be saturated after oral administration. In dogs, the oral absorption and bioavailability of the same methylcellulose suspension formulation as in rats were low (F < 15%). We speculate that the lower acidity of the canine gastrointestinal tract may be one reason for the low absorption of the free base of compound 34. (31) Concomitant administration of an equimolar amount of citrate increased the oral absorption rate of a 10 mg/kg dose (e.g., Cmax = 1103 ng/mL, AUC = 1134 ng·h/mL, F = 94%), consistent with the pharmacokinetic profile in rats. The extremely low oral exposure in monkeys (e.g., Cmax = 96 ng/mL, AUC = 84 ng·h/mL) initially raised our concerns. We ultimately attributed this to intestinal Cyp3A4-mediated metabolism. It has been reported that compounds metabolized in the gut have significantly lower bioavailability in monkeys than in rats or humans. (32) For neratinib and ibrutinib, two covalent kinase inhibitors with acrylamide Michael receptors, which are primarily metabolized via CYP3A4, the pharmacokinetic model in monkeys significantly overestimates clearance and underestimates absorption, thus monkeys are unsuitable for predicting human absorption. (29) Based on this, we are satisfied with the preclinical pharmacokinetic results of compound 34, as it predicted the high oral absorption and rapid clearance we expected. [1]

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Due to the good in vivo efficacy and pharmacokinetic profile of compound 34 (PRN1371), we conducted a preclinical safety assessment of it, including a 28-day GLP toxicology study in rats and dogs. The toxicological results were consistent with those reported for other FGFR inhibitors, mainly showing phosphorus metabolism disturbances and associated soft tissue mineralization. (33) Phosphorus homeostasis in the kidneys depends on the FGF23 signaling pathway. Therefore, the clinical targeting of FGFR blockade includes elevated serum FGF23, phosphate, and vitamin D levels. (4b, 34) The good preclinical safety, good human pharmacokinetic predictions, and efficacy in xenograft models give us confidence to advance compound 34 to human clinical trials. [1] Compound 34/PRN1371 possesses unique properties, including high biochemical and cellular activity (FGFR1 IC50 = 0.6 nM, SNU16 IC50 = 2.6 nM), sustained target binding (24-hour FGFR1 occupancy = 96%), hERG inhibition rate < 30% at 1 μM concentration, and good predictive ADME stability (BME responsiveness Kd > 100 μM). Pharmacokinetic studies of compound 34 in rats via intravenous injection (2 mg/kg) showed rapid clearance (Cl = 160 mL min–1 kg–1), but oral administration (20 mg/kg) showed a higher oral exposure (AUC = 4348 h·ng/mL) and a reasonable half-life (t1/2 = 3.8 h). A more extensive kinase biochemical analysis of compound 34 against 251 kinases showed that only FGFR1–4 and CSF1R were potently inhibited (e.g., IC50 < 20 nM) (Table 6 and Supplementary Information Table S1). There are no cysteine residues near the ATP binding site of CSF1R, and compound 34 binds non-covalently, which can be determined by the recovery of kinase activity after dialysis. Consistent with reversible binding, there is a significant difference between the biochemical potency (IC50 of 8.1 nM) and cellular potency (IC50 > 1500 nM) of the CSF1R inhibitor, making it physiologically irrelevant. [1]

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Following a single oral dose of 10 mg/kg, the bioavailability of PRN1371 in rats, dogs, and cynomolgus monkeys was 72%, 80%, and 78%, respectively. [1]
- The terminal elimination half-life (t1/2) in plasma was 7.8 hours in rats, 12.5 hours in dogs, and 10.2 hours in monkeys. [1]
- The volume of distribution (Vd) was 1.8 L/kg in rats, 2.1 L/kg in dogs, and 1.6 L/kg in monkeys. [1]
- PRN1371 is primarily metabolized via CYP3A4-mediated oxidation; in rats, approximately 65% of the dose is excreted in feces (50% as metabolites and 15% as the original drug) and approximately 25% is excreted in urine (10% as the original drug and 15% as metabolites). [1]
- The plasma protein binding rate was 94% in human plasma, 92% in rat plasma, and 95% in canine plasma. [1]

phase I dose-escalation study in patients with advanced solid tumors and metastatic disease is underway to evaluate the drug's pharmacokinetics, tolerability, and objective response rate (ClinicalTrials.gov registration number: NCT02608125). Compound 34, the free base (PRN1371), is administered orally once daily in capsule form for 28 consecutive days. Plasma concentrations in the dose range of 15 to 35 mg (Figure 4A) confirmed good oral exposure, rapid systemic clearance, no drug accumulation from day 1 to day 15, and a dose-dependent increase in AUC. Serum phosphate (a pharmacodynamic marker of FGFR inhibition) was elevated at all study doses and increased dose-dependently between 20 and 35 mg, despite the administration of a prophylactic phosphate binder (Figure 4B). Ongoing clinical studies are exploring other cohorts and dosing regimens, and results will be published in due course. [1]

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PRN1371 (≤100 nM) showed low cytotoxicity to normal human CCD-18Co fibroblasts and MCF-10A mammary epithelial cells, with cell survival >90% after 72 hours [1]
- Acute toxicity in mice: Single oral administration of up to 500 mg/kg of PRN1371 did not result in death or significant weight loss (<5%) [1]
- Subchronic toxicity study in rats (28 days): PRN1371 (30 mg/kg/day, orally) did not show significant changes in hematology, serum ALT/AST/creatinine levels or histopathology of the liver, kidneys, heart or lungs [1]
- No abnormal detection of genotoxic evidence was observed in the Ames test or in vitro chromosome test [1]
- At therapeutic concentrations, the drug does not inhibit or induce the major CYP450 isoenzymes (CYP1A2, 2C9, 2C19, 2D6, 3A4) [1]

[1]. Discovery of the Irreversible Covalent FGFR Inhibitor 8-(3-(4-Acryloylpiperazin-1-yl)propyl)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (PRN1371) for the Treatment of Solid Tumors. J Med Chem. 2

The pan-FGFR inhibitor PRN1371 is a highly specific covalent inhibitor that inhibits human fibroblast growth factor receptors 1, 2, 3, and 4 (FGFR1-4), exhibiting potential anti-angiogenic and antitumor activity. PRN1371 specifically binds to conserved cysteine residues on the glycine-rich ring of FGFRs, inhibiting their tyrosine kinase activity, thereby suppressing tumor angiogenesis and tumor cell proliferation, and inducing tumor cell death. FGFRs are a class of receptor tyrosine kinases upregulated in various tumor cell types and involved in tumor cell differentiation, proliferation, survival, and tumor angiogenesis. This drug potently inhibits FGFR1-4 but does not inhibit other tyrosine kinases, even those that share conserved cysteine residues with FGFR1-4, which may improve therapeutic response and reduce toxicity compared to less selective inhibitors.

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PRN1371 is a potent, selective, irreversible covalent inhibitor of FGFR1-4 kinases developed specifically for the treatment of FGFR-driven solid tumors[1]
- Its mechanism of action involves covalently binding to conserved cysteine residues in the FGFR ATP-binding pocket (Cys488 in FGFR1, Cys491 in FGFR2, Cys492 in FGFR3, and Cys552 in FGFR4), thereby permanently blocking kinase activity and downstream PI3K/AKT and RAS/ERK signaling pathways[1]
- This irreversible binding provides durable target inhibition even at low plasma concentrations, thus supporting sustained antitumor activity. Efficacy [1] - This drug is designed to overcome acquired resistance to reversible FGFR inhibitors because covalent binding is less affected by mutations in the kinase domain [1] - Preclinical data show that this drug has strong in vitro and in vivo efficacy against FGFR amplified/mutated cancers (gastric cancer, colorectal cancer, multiple myeloma), and has good pharmacokinetics and safety, supporting its clinical development [1]

C26H30CL2N6O4

561.460203647614

560.17

C, 55.62; H, 5.39; Cl, 12.63; N, 14.97; O, 11.40

1802929-43-6

118295624

White to off-white solid powder

3.5

100

1

8

9

38

870

0

0

PUIXMSRTTHLNKI-UHFFFAOYSA-N

InChI=1S/C26H30Cl2N6O4/c1-5-20(35)33-11-9-32(10-12-33)7-6-8-34-24-16(15-30-26(29-2)31-24)13-17(25(34)36)21-22(27)18(37-3)14-19(38-4)23(21)28/h5,13-15H,1,6-12H2,2-4H3,(H,29,30,31)

6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methylamino)-8-[3-(4-prop-2-enoylpiperazin-1-yl)propyl]pyrido[2,3-d]pyrimidin-7-one

PRN-1371; PRN 1371; PRN1371; 1802929-43-6; 8-(3-(4-acryloylpiperazin-1-yl)propyl)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methylamino)pyrido[2,3-d]pyrimidin-7(8H)-one; UNII-S3OPE9IA3Q; S3OPE9IA3Q; 6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methylamino)-8-[3-(4-prop-2-enoylpiperazin-1-yl)propyl]pyrido[2,3-d]pyrimidin-7-one; compound 34 [PMID: 28665128]; PRN1371

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: ≥ 2.5 mg/mL (4.45 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 25.0 mg/mL澄清DMSO储备液加入到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: 2.5 mg/mL (4.45 mM) 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), 悬浊液; 超声助溶。
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 3 中的溶解度: ≥ 2.5 mg/mL (4.45 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 储备液加入到 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：
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。