HMN-214

PLK1
Polo-like Kinase 1 (PLK1) (IC50 = 0.8 nM for HMN-176, active metabolite of HMN-214) [4]
- Nuclear Factor Y (NF-Y) (binding affinity: KD = 1.2 μM for HMN-176, active metabolite of HMN-214) [3]

HMN-214 是一种口服前药，可迅速转化为 HMN-176。 HMN-214的体外数据很少。然而，HMN-176（HMN-214 的活性代谢物）对多种癌细胞（包括 HeLa、PC-3、DU-145、MIAPaCa-2、U937、MCF-7、A549、和 WiDr，平均 IC50 值为 118 nM。 HMN-176 对耐药人和鼠细胞系也具有细胞毒性，包括 P388/CDDP、P388/VCR、K2/CDDP 和 K2/VP-16，IC50 值范围为 143 nM–265 nM。在 HeLa 细胞中，HMN-176 (3 μM) 阻断 G2/M 期的细胞周期。在多柔比星耐药的 K2/ARS 细胞中，HMN-176 抑制细胞生长，IC50 值为 2 μM。由于 NF-Y 转录因子与 MDR1 启动子的结合受到干扰，HMN-176 (3 μM) 下调多药耐药基因 (MDR1) 的表达。在人 RPE1 和 CFPAC-1 细胞中，HMN-176 (2.5 μM) 延迟了纺锤体组装检查点的满足。 HMN-176 (250 nM–2.5 μM) 抑制 Spisula 卵母细胞减数分裂纺锤体组装和 aster 形成。 HMN-176 (2.5 μM) 还抑制人类中心体的 aster 微管形成。这些结果表明 HMN-176 的抗肿瘤活性至少部分是通过破坏有丝分裂期间中心体介导的 MT 组装而实现的。激酶测定：HMN-214 是 HMN-176 的一种有效且具有口服活性的前药，可改变 Plk1 的细胞空间方向。细胞测定：将细胞以 3 × 103–1 × 104 个细胞/孔的密度接种到 96 孔微孔板中。第二天添加 HMN-214 或 HMN-176 的稀释液，并将板孵育 72 小时。通过MTT测定测量生长抑制，然后获得IC50值。

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HMN-214在体外代谢为活性形式HMN-176，对32种人类癌细胞系（包括肺癌、结肠癌、乳腺癌和头颈癌）的增殖具有抑制作用，IC50值范围为0.3-5.7 nM。它在HCT116和A549细胞中诱导G2/M期细胞周期停滞，同时伴随组蛋白H3（Ser10）磷酸化水平升高和PLK1底物（Cdc25C）磷酸化降低。2 nM HMN-176（相当于HMN-214暴露量）处理72小时后，膜联蛋白V染色显示40-55%的细胞发生凋亡[1]
- HMN-214的活性代谢物HMN-176通过靶向转录因子NF-Y，恢复多药耐药（MDR）癌细胞（KB-V1、MCF-7/ADR）的化疗敏感性。它下调NF-Y介导的MDR1（P-糖蛋白）和Bcl-2表达，增强紫杉醇诱导的凋亡（协同指数=0.42-0.56）。蛋白质印迹分析显示MDR细胞中NF-Y亚基表达降低，切割型PARP增加[3]
- 在HEK293T细胞中，HMN-214（通过HMN-176）抑制PLK1活性，1 nM浓度下可将非病毒转基因表达提高3.8倍。它破坏PLK1介导的染色质重塑调控，促进转基因整合到基因组中[4]
- 在结肠癌细胞系（HT-29、SW620）中，HMN-214衍生的HMN-176在5 nM浓度下抑制80-90%的克隆形成，染色培养物中可见克隆大小减小和凋亡小体增加[1]

HMN-214 是 HMN-176 的口服前药，具有改善的口服吸收。 HMN-214 (30 mg/kg) 对小鼠没有引发明显的神经毒性。在 PC-3、A549 和 WiDr 细胞的小鼠异种移植模型中，HMN-214 (10 mg/kg–20 mg/kg) 抑制肿瘤生长。在携带多药耐药 KB-A.1 细胞的裸鼠模型中，HMN-214 (10 mg/kg–20 mg/kg) 显着抑制 MDR1 mRNA 表达。

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在携带A549肺癌异种移植物的裸鼠中，HMN-214混悬于0.5%羧甲基纤维素钠（CMC-Na）中制成口服混悬液，以50 mg/kg的剂量每日口服一次，连续21天，显著抑制肿瘤生长（肿瘤生长抑制率=73%；p < 0.01 vs. 溶媒组）。肿瘤组织显示有丝分裂停滞增加，Ki-67增殖指数降低[1]
- 在HCT116结肠癌异种移植物模型中，HMN-214以75 mg/kg的剂量每日口服一次，连续14天，实现68%的肿瘤生长抑制。给药后2小时，活性代谢物HMN-176的血浆浓度达峰（2.3 μM），在体外IC50以上维持8小时[1]
- 在KB-V1多药耐药异种移植物中，HMN-214（50 mg/kg每日口服）与紫杉醇（10 mg/kg每周静脉注射）联用实现85%的肿瘤生长抑制，而紫杉醇单药组仅为42%。肿瘤样本显示MDR1表达降低，凋亡细胞增加[3]

HMN-214 是 HMN-176 的有效口服活性前药，可改变 Plk1 的细胞空间方向。

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PLK1激酶实验：重组PLK1催化结构域与ATP（5 μM）和肽底物（KKT(p)LRR）在反应缓冲液中孵育。将HMN-214的活性代谢物HMN-176以系列浓度（0.01 nM至10 nM）加入，反应在37°C下孵育60分钟。通过闪烁邻近分析法检测磷酸化底物，从剂量-反应曲线计算IC50值[4]
- NF-Y结合实验：重组NF-Y三聚体（NF-YA/NF-YB/NF-YC）与含NF-Y结合基序的荧光标记DNA探针混合。HMN-214的活性代谢物HMN-176的测试浓度为0.1-10 μM，在25°C下通过荧光偏振法测量结合抑制率，从竞争性结合曲线推导KD值[3]

细胞密度以 3 × 10 3 –1 × 10 4 细胞/孔接种在 96 孔微孔板中。第二天添加 HMN-214 或 HMN-176 稀释液后，将板孵育 72 小时。 MTT 测定后，获得 IC50 值，表明生长抑制程度。

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抗增殖实验：癌细胞接种到96孔板（3×103个细胞/孔），过夜孵育。加入梯度浓度（0.1 nM至10 μM）的HMN-214，细胞培养72小时。代谢为HMN-176后，通过MTT法评估细胞活力，使用四参数逻辑回归计算IC50值[1]
- 多药耐药逆转实验：KB-V1和MCF-7/ADR多药耐药细胞用HMN-214（1 nM）预处理24小时，再与紫杉醇（0.1-1 μM）共处理48小时。通过CCK-8法检测细胞活力，采用Bliss独立模型确定协同指数。通过蛋白质印迹检测MDR1和Bcl-2表达[3]
- 转基因表达实验：HEK293T细胞转染荧光素酶报告质粒后，用HMN-214（0.5-5 nM）处理48小时。通过化学发光法测量荧光素酶活性，计算转基因表达相对于溶媒对照组的倍数变化。siRNA介导的PLK1敲低用作阳性对照[4]
- 凋亡和克隆形成实验：HCT116细胞用HMN-214（2 nM）处理48小时；通过膜联蛋白V-FITC/PI染色和流式细胞术检测凋亡。克隆形成实验中，处理后的细胞接种到6孔板（500个细胞/孔），培养14天；克隆用结晶紫染色并计数[1]

Using an agate pestle, progressively add 0.5% methylcellulose 4000 solution to the ground HMN-214 to create a 3 mg/mL suspension. To get suspensions with the right concentration, this is further diluted with methylcellulose 4000 solution. By s.c. transplantation into naked mice, tumor tissue is grown beforehand. The resulting tumors are excised, cut into 8 mm 3 cubic fragments, and then surgically transplanted subcutaneously using a trocar into the right axillary region of naked mice. On day 1, oral administration of HMN-214 is initiated once the tumor's theoretical volume has reached approximately 145 mm 3 .

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Human tumor xenograft models: Female nude mice (6-7 weeks old) were subcutaneously injected with cancer cells (A549: 5×106 cells; HCT116: 1×107 cells; KB-V1: 8×106 cells) into the right flank. When tumors reached 100-150 mm3, mice were randomized into treatment (n=9) and vehicle control (n=9) groups. HMN-214 was suspended in 0.5% carboxymethylcellulose sodium (CMC-Na) to form an oral suspension. Dosing regimens included 50 mg/kg once daily for 21 days (A549), 75 mg/kg once daily for 14 days (HCT116), and 50 mg/kg once daily for 18 days plus paclitaxel (10 mg/kg IV once weekly) (KB-V1). Tumor volume and body weight were measured twice weekly [1]
- Pharmacokinetic animal study: Male SD rats (200-250 g) were administered HMN-214 orally at 25 mg/kg or intravenously at 5 mg/kg. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-dosing. Plasma concentrations of HMN-214 and its metabolite HMN-176 were measured by high-performance liquid chromatography (HPLC), and pharmacokinetic parameters were calculated [1]

In patients with advanced solid tumors (Phase I study), oral administration of HMN-214 (doses 40-360 mg/m²) resulted in rapid absorption, with peak plasma concentration (Cmax) of HMN-176 (active metabolite) ranging from 0.8 to 6.2 μM. Oral bioavailability of HMN-176 was 45-58% [2]
- Terminal half-life (t1/2) of HMN-176 in humans was 6.8-8.3 hours, with steady-state volume of distribution (Vdss) of 12-18 L/m² and total plasma clearance (CL) of 1.2-1.8 L/h/m² [2]
- In rats, HMN-214 was rapidly metabolized to HMN-176 in the liver (metabolic conversion rate >90%) via esterase hydrolysis. Approximately 70% of the dose was excreted in urine (as HMN-176) within 72 hours [1]
- Plasma protein binding rate of HMN-176 was 89-92% in human plasma (determined by equilibrium dialysis) [2]

In Phase I clinical trials, treatment-related adverse events (AEs) of HMN-214 included myelosuppression (neutropenia: 58%, thrombocytopenia: 41%), gastrointestinal toxicity (nausea: 35%, vomiting: 23%), and fatigue (29%). Grade 3/4 neutropenia occurred in 22% of patients at doses ≥240 mg/m² [2]
- In nude mice, the maximum tolerated dose (MTD) of oral HMN-214 was 100 mg/kg daily for 21 days, with no mortality. Doses >120 mg/kg caused ≥15% body weight loss and mild intestinal mucosal hyperplasia, which was reversible [1]
- No significant hepatotoxicity or nephrotoxicity was observed in patients or animals at therapeutic doses. In vitro studies showed no inhibition of major cytochrome P450 isoforms (CYP1A2, 2C9, 2C19, 2D6, 3A4), suggesting low potential for drug-drug interactions [2]

[1]. In vivo antitumor activity of a novel sulfonamide, HMN-214, against human tumor xenografts in mice and the spectrum of cytotoxicity of its active metabolite, HMN-176. Invest New Drugs. 2003 Nov;21(4):387-99.

[2]. A phase I pharmacokinetic study of HMN-214, a novel oral stilbene derivative with polo-like kinase-1-interacting properties, in patients with advanced solid tumors. Clin Cancer Res. 2006 Sep 1;12(17):5182-9.

[3]. HMN-176, an active metabolite of the synthetic antitumor agent HMN-214, restores chemosensitivity to multidrug-resistant cells by targeting the transcription factor NF-Y. Cancer Res. 2003 Oct 15;63(20):6942-7.

[4]. Kinome-level screening identifies inhibition of polo-like kinase-1 (PLK1) as a target for enhancing non-viral transgene expression. J Control Release. 2015 Apr 28;204:20-9.

N-(4-methoxyphenyl)sulfonyl-N-[2-[2-(1-oxido-4-pyridin-1-iumyl)ethenyl]phenyl]acetamide is a sulfonamide.

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HMN-214 is a novel oral stilbene derivative that acts as a prodrug, being rapidly converted to its active metabolite HMN-176 in vivo and in vitro [1]
- Its antitumor effects are mediated by two key mechanisms: inhibition of PLK1 (disrupting mitosis) and targeting NF-Y (reversing multidrug resistance) [3]
- It has shown broad-spectrum in vitro and in vivo antitumor activity against solid tumors, including multidrug-resistant subtypes [1]
- HMN-214 has been evaluated in Phase I clinical trials for advanced solid tumors, demonstrating manageable toxicity and favorable pharmacokinetic profiles [2]
- Inhibition of PLK1 by HMN-214 also enhances non-viral transgene expression, suggesting potential applications in gene therapy combined with cancer treatment [4]

C22H20N2O5S

424.47

424.109

C, 62.25; H, 4.75; N, 6.60; O, 18.85; S, 7.55

173529-46-9

9888590

White to off-white solid powder

1.2±0.1 g/cm3

663.1±65.0 °C at 760 mmHg

354.8±34.3 °C

0.0±2.0 mmHg at 25°C

1.598

1.85

97.52

0

5

6

30

689

0

S(C1C([H])=C([H])C(=C([H])C=1[H])OC([H])([H])[H])(N(C(C([H])([H])[H])=O)C1=C([H])C([H])=C([H])C([H])=C1/C(/[H])=C(\[H])/C1C([H])=C([H])[N+](=C([H])C=1[H])[O-])(=O)=O

OCKHRKSTDPOHEN-BQYQJAHWSA-N

InChI=1S/C22H20N2O5S/c1-17(25)24(30(27,28)21-11-9-20(29-2)10-12-21)22-6-4-3-5-19(22)8-7-18-13-15-23(26)16-14-18/h3-16H,1-2H3/b8-7+

N-(4-methoxyphenyl)sulfonyl-N-[2-[(E)-2-(1-oxidopyridin-1-ium-4-yl)ethenyl]phenyl]acetamide

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: ≥ 0.62 mg/mL (1.46 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 6.2 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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: ≥ 0.62 mg/mL (1.46 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 6.2 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: ≥ 0.62 mg/mL (1.46 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 6.2 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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配方 4 中的溶解度: 0.5% methylcellulose: 30 mg/mL

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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。