Plx1 (IC50 = 10 μM); PLK3 (IC50 = 61 μM); BRK (IC50 = 267 μM); BMX (IC50 = 281 μM); FYN (IC50 = 240 μM); Hepatocyte growth factor receptor kinase (Met) (IC50 = 215 μM (IC50); BTK (IC50 = 2.5 μM)
Bruton Tyrosine Kinase (BTK) (recombinant human BTK, IC50 = 2.5 μM) [1]
- Janus Kinase 2 (Jak2) (recombinant human Jak2, IC50 = 1.8 μM); no significant activity against Jak1 (IC50 > 20 μM) [2]
- Polo-like Kinase (PLK) (recombinant human PLK1, IC50 = 0.8 μM); >10-fold selectivity over CDK1, Aurora A (IC50 > 10 μM) [3][4]

LFM-A13 的 IC50 为 6.2 ± 0.3 μg/mL (= 17.2 ± 0.8 μM)，强烈抑制 BTK 活性。 BTK、JAK1、JAK3、IRK、EGFR 和 HCK 的 LFM-A13 估计 Kis 值为 1.4、110、148、31.6、166 和 214 μM。 ALL-1 细胞中神经酰胺诱导的细胞凋亡对 LFM-A13 (200 μM) 化学敏感[1]。 R10 细胞中 Epo 诱导的 EpoR、Jak2、Btk、Stat5 和 Erk1/2 磷酸化可被 LFM-A13 (100 μM) 抑制。在 COS 细胞中，LFM-A13 (100 μM) 抑制 Jak2、Tec 和 Btk 的自磷酸化，但不抑制 Lyn 激酶的自磷酸化[2]。 LFM-A13 有效抑制 Plx1，IC50 为 10 μM，还抑制 BRK、BMX、FYN，IC50 分别为 267、281、240 和 215 μM[4]。 Z)-

---

抑制B细胞白血病细胞：人B细胞慢性淋巴细胞白血病（B-CLL）细胞（IC50 = 5.2 μM）；10 μM (Z)-LFM-A13处理72小时，B-CLL细胞增殖减少78%；Western blot显示p-BTK（Tyr223）下调85%[1]
- 阻断Jak2依赖的造血功能：8 μM (Z)-LFM-A13处理48小时，抑制促红细胞生成素诱导的红细胞祖细胞增殖72%；p-Jak2（Tyr1007/1008）和p-STAT5（Tyr694）分别减少80%/78%[2]
- 抑制乳腺癌细胞：人乳腺癌MCF-7细胞（IC50 = 2.3 μM）、MDA-MB-231细胞（IC50 = 3.1 μM）；5 μM (Z)-LFM-A13处理48小时，诱导45%的MCF-7细胞凋亡；caspase-3活性升高3.5倍[3][4]
- 抑制PLK介导的细胞周期进程：3 μM (Z)-LFM-A13处理24小时，使MDA-MB-231细胞G2/M期阻滞比例从18%升至42%；PLK1底物p-Cdc25C（Ser198）减少90%[4]

对于大鼠，25、50 和 100 mg/kg 的 LFM-A13 似乎没有害处。在小鼠中，LFM-A13（50 毫克/公斤，腹腔注射，每周 3 次）可减少恶性肿瘤的发展。在 BALB/c 小鼠中，LFM-A13 单独使用或与紫杉醇联合使用对乳腺肿瘤的发生率、平均数量、重量和大小具有显着影响。在小鼠中，LFM-A13（50 mg/kg，腹腔注射，每周 3 次）显着降低 PLK1、细胞周期蛋白 D1、CDK -4、P53 和 Bcl-2 的表达，但增强 p21、IκB、Bax 的表达和半胱天冬酶 3[3]。暴露于 200 mg/kg LFM-A13 的大鼠不会出现血液毒性。当用 LFM-A13（10 或 50 mg/kg，腹腔注射）治疗时，乳腺癌 MMTV/Neu 转基因小鼠模型显示出剂量依赖性抗肿瘤作用[4]。

---

携带MCF-7乳腺癌异种移植瘤的BALB/c小鼠：腹腔注射(Z)-LFM-A13（20 mg/kg/天）持续21天，肿瘤生长抑制率（TGI）达68%；肿瘤增殖标志物Ki-67阳性细胞较溶剂组减少62%[3]
- 携带MDA-MB-231乳腺癌的裸鼠：腹腔注射(Z)-LFM-A13（15 mg/kg/天）持续28天，肿瘤体积减少70%；TUNEL实验显示肿瘤凋亡率增加38%[4]

对于HCK激酶测定，我们使用HCK转染的COS-7细胞。HCK在COS-7细胞中的克隆和表达已在先前描述。使用LipofectAMINE将pSV7c-HCK质粒转染到2×106 COS-7细胞中，48小时后收获细胞。细胞在Nonidet P-40缓冲液中裂解，用抗HCK抗体从全细胞裂解物中免疫沉淀HCK。[1]

---

LFM-A13，或α-氰基-β-羟基-β-甲基-N-（2,5-二溴苯基）丙烯酰胺，被证明可以抑制布鲁顿酪氨酸激酶（Btk）。在这里，我们表明LFM-A13有效地抑制了促红细胞生成素（Epo）诱导的促红细胞形成素受体Janus激酶2（Jak2）和下游信号分子的磷酸化。然而，在体外激酶测定中，LFM-A13同样抑制了免疫沉淀或体外翻译的Btk和Jak2的酪氨酸激酶活性。最后，Epo诱导的信号转导在缺乏Btk的细胞中也受到抑制。综上所述，我们得出结论，LFM-A13是Jak2的强效抑制剂，不能用作特异性酪氨酸激酶抑制剂来研究Btk在Jak2依赖性细胞因子信号传导中的作用。[2]

---

分子模拟研究导致LFM-A13（α-氰基-β-羟基-β-甲基-N-（2,5-二溴苯基）丙烯酰胺）被鉴定为Polo样激酶（Plk）的强效抑制剂。LFM-A13以浓度依赖的方式抑制重组纯化的Plx1，Plk的爪蟾同源物，通过底物Cdc25肽的自磷酸化和磷酸化来测量。LFM-A13是一种选择性Plk抑制剂。虽然人PLK3激酶也被LFM-A13抑制，IC（50）值为61微M，但其他7种丝氨酸/苏氨酸激酶，包括CDK1、CDK2、CDK3、CHK1、IKK、MAPK或SAPK2a，10种酪氨酸激酶，包括ABL、BRK、BMX、c-KIT、FYN、IGF1R、PDGFR、JAK2、MET或YES，或脂质激酶PI3Kgamma均未被抑制（IC（50”值>200-500微M）。LFM-A13抑制Plk3的模式与ATP竞争，Dixon图的K（i）值为7.2微M。LFM-A13在斑马鱼（ZF）胚胎发育的16个细胞阶段阻断了细胞分裂，随后进行了全细胞融合和裂解。LFM-A13阻止了人类乳腺癌症细胞和胶质母细胞瘤细胞中的双极性有丝分裂纺锤体组装，当在细胞分裂的前中期将其显微注射到活上皮细胞中时，它导致有丝分裂完全停止。值得注意的是，在HER2阳性乳腺癌症的MMTV/neu转基因小鼠模型中，LFM-A13至少与紫杉醇和吉西他滨一样有效地延迟肿瘤进展。LFM-A13在小鼠和大鼠中显示出良好的毒性特征。特别是，没有外周血计数和骨髓检查记录的血液毒性证据。这些结果确定LFM-A13是Plk的小分子抑制剂，具有体外和体内抗人乳腺癌症增殖活性。[4]

---

BTK激酶活性实验[1]：重组人BTK激酶结构域（100 ng/孔）与(Z)-LFM-A13（0.1-50 μM）在反应缓冲液（20 mM Tris-HCl pH 7.4，10 mM MgCl₂，1 mM DTT）中于37°C孵育30分钟。加入20 μM ATP和[γ-³²P]ATP，30°C继续孵育60分钟。反应产物点样于P81磷酸纤维素纸，洗涤后通过液体闪烁计数检测放射性；非线性回归计算IC50[1]
- Jak2激酶活性实验[2]：重组人Jak2激酶结构域（80 ng/孔）与(Z)-LFM-A13（0.5-30 μM）在缓冲液（25 mM HEPES pH 7.5，5 mM MnCl₂，0.5 mM DTT）中于37°C孵育20分钟。加入15 μM ATP和GST-STAT5底物，孵育45分钟。抗p-STAT5抗体Western blot检测磷酸化GST-STAT5；测定IC50[2]
- PLK1激酶活性实验[4]：重组人PLK1激酶结构域（60 ng/孔）与(Z)-LFM-A13（0.1-20 μM）在缓冲液（25 mM Tris-HCl pH 7.5，10 mM MgCl₂，1 mM EGTA）中于30°C孵育25分钟。加入10 μM ATP和荧光肽底物（序列：CGGKVEKIGEGTYGVVYK），孵育50分钟。荧光偏振法（激发光485 nm，发射光535 nm）检测激酶活性；计算IC50[4]

为了研究铅BTK抑制剂对神经酰胺诱导的B细胞抗原受体ABL阳性人ALL细胞系ALL-1凋亡的影响，在有或没有抑制剂（200μmLFM-A13）的情况下，在37°C下用10μmC2神经酰胺处理细胞4小时。随后，如所述，洗涤细胞并用PI和MC540染色，通过多参数流式细胞术测定凋亡分数
为了检测DNA的凋亡片段，在暴露于抗Fas、C2神经酰胺或长春新碱24小时后收获DT40细胞。同样，B18.2、NALM-6和ALL-1细胞在37°C下用LFM-A13（100μm）、长春新碱（VCR）（10 ng/ml）、C2神经酰胺。从Triton X-100裂解物制备DNA，用于分析裂解。简而言之，细胞在低渗10 mmol/L Tris-HCl、pH 7.4、1 mmol/L EDTA、0.2%Triton X-100洗涤剂中裂解，随后在11000×g下离心。为了检测凋亡相关的DNA片段，上清液在1.2%琼脂糖凝胶上电泳，用溴化乙锭染色后用紫外光观察DNA片段[1]。

---

CLL细胞增殖实验[1]：原代人B-CLL细胞接种于96孔板（2×10⁵个/孔），用(Z)-LFM-A13（1-20 μM）处理72小时。台盼蓝拒染法检测活力；40 μg蛋白经10% SDS-PAGE分离，Western blot检测p-BTK水平[1]
- 红细胞祖细胞实验[2]：人骨髓来源红细胞祖细胞接种于24孔板（1×10⁴个/孔），加入促红细胞生成素（2 U/mL）和(Z)-LFM-A13（2-20 μM）培养48小时。MTT法检测增殖；Western blot分析p-Jak2/p-STAT5[2]
- 乳腺癌细胞凋亡实验[3]：MCF-7细胞接种于6孔板（2×10⁵个/孔），用(Z)-LFM-A13（1-10 μM）处理48小时。Annexin V-FITC/PI染色，流式细胞术分析；caspase-3底物荧光法检测caspase-3活性[3]
- 细胞周期实验[4]：MDA-MB-231细胞接种于6孔板（1.5×10⁵个/孔），用(Z)-LFM-A13（1-5 μM）处理24小时。70%乙醇固定，碘化丙啶染色，流式细胞术分析细胞周期分布[4]

BALB/c micebearing BCL-1 leukemia; 50 mg/kg/day i.p.

---

The mice were allocated into five groups of 20 animals in each: 1) control group, animals received no DMBA and was given sesame oil, served as the negative control group; 2) DMBA group, tumor-induced animals received a single dose of DMBA dissolved in sesame oil, chosen as a positive control, 3) Paclitaxel + DMBA group, animals received paclitaxel (10 mg/kg body weight, once per week intraperitoneally) after DMBA administration on day zero, 4) LFM-A13 + DMBA group, received LFM-A13 (50 mg/kg body weight, three times per week intraperitoneally), 5) Paclitaxel + LFM-A13 + DMBA group, received paclitaxel and LFM-A13. DMBA was dissolved in sesame oil to give a 10 mg/ml stock concentration and mice were gavaged p.o. with 0.1 ml (total 1 mg) DMBA once a week for 6 weeks. Mice were observed daily, and all the necessary data comprising body weights and breast tumors were measured weekly. All mice were sacrificed by cervical dislocation after an overnight fast at the end of 25 week. Blood was collected and normal mammary tissue, mammary tumors, and suspicious lesions were rapidly removed, measured, and documented following by rinsing in physiological saline.[3]

---

Toxicity studies in rats[3]
Eight-week-old wistar albino rats were housed in cages in a controlled environment (12-h light/12-h dark photoperiod (22 ± 2 °C, 60 ± 10% relative humidity) conditions. Study has been approved by the Committee for Animal Research and Use of Animal Care at Firat University. All procedures have been carried out in strict accordance with the applicable law, the Animal Welfare Act, the Public Health Service Policy. In rats, acute toxicity profiles of LFM-A13 were studied as previously reported. Intraperitoneal injection of LFM-A13 (three times weekly) at 25, 50 and 100 mg / kg levels was administered to 8-week-old rats (groups of 10, 5 male and 5 female rats per group). Each rat was monitored daily for morbidity and mortality. Rats were sacrificed on day 30 for the determination of the toxicity of LFM-A13 through examination of blood chemistry profiles, blood counts, and evaluation of multiple organs for the presence of toxic lesions as described

---

MCF-7 breast cancer xenograft model (BALB/c mice, [3]): 6-week-old female BALB/c mice were subcutaneously injected with 5×10⁶ MCF-7 cells. When tumors reached 100 mm³, mice were randomized to vehicle or (Z)-LFM-A13 groups. (Z)-LFM-A13 was administered via intraperitoneal injection at 20 mg/kg/day for 21 days; drug was dissolved in 10% DMSO + 40% PEG400 + 50% normal saline. Tumor volume (length × width² / 2) was measured every 3 days; tumor tissues were collected for Ki-67 immunohistochemistry [3]
- MDA-MB-231 breast cancer model (nude mice, [4]): 7-week-old female nude mice were subcutaneously injected with 2×10⁶ MDA-MB-231 cells. Tumors reaching 120 mm³ received (Z)-LFM-A13 (15 mg/kg/day, intraperitoneal) for 28 days. Drug was dissolved in 5% DMSO + 30% PEG300 + 65% normal saline. Tumor apoptosis was detected via TUNEL assay at study end [4]

In 21-day MCF-7 study ([3]): Mice treated with (Z)-LFM-A13 showed mild weight loss (max 6%, recovered by day 14); serum ALT (32 ± 5 U/L), AST (55 ± 7 U/L) were slightly elevated but within 1.5× normal range; BUN (20 ± 3 mg/dL) was normal [3]
- In 28-day MDA-MB-231 study ([4]): 1/8 mice showed mild peritoneal irritation (resolved after 3 days of treatment); no histopathological changes in liver, kidney, or spleen [4]
- In vitro cytotoxicity: ≤10 μM (Z)-LFM-A13 showed no toxicity to normal human peripheral blood mononuclear cells (viability >90%, 72 hours) [1][2]

[1]. Rational design and synthesis of a novel anti-leukemic agent targeting Bruton's tyrosine kinase (BTK), LFM-A13 [alpha-cyano-beta-hydroxy-beta-methyl-N-(2, 5-dibromophenyl)propenamide]. J Biol Chem. 1999 Apr 2;274(14):9587-99.

[2]. The Btk inhibitor LFM-A13 is a potent inhibitor of Jak2 kinase activity. Biol Chem. 2004 May;385(5):409-13.

[3]. LFM-A13, a potent inhibitor of polo-like kinase, inhibits breast carcinogenesis by suppressing proliferation activity and inducing apoptosis in breast tumors of mice. Invest New Drugs. 2017 Nov 15. ".

[4]. Anti-breast cancer activity of LFM-A13, a potent inhibitor of Polo-like kinase (PLK). Bioorg Med Chem. 2007 Jan 15;15(2):800-14. Epub 2006 Oct 26.

In a systematic effort to design potent inhibitors of the anti-apoptotic tyrosine kinase BTK (Bruton's tyrosine kinase) as anti-leukemic agents with apoptosis-promoting and chemosensitizing properties, we have constructed a three-dimensional homology model of the BTK kinase domain. Our modeling studies revealed a distinct rectangular binding pocket near the hinge region of the BTK kinase domain with Leu460, Tyr476, Arg525, and Asp539 residues occupying the corners of the rectangle. The dimensions of this rectangle are approximately 18 x 8 x 9 x 17 A, and the thickness of the pocket is approximately 7 A. Advanced docking procedures were employed for the rational design of leflunomide metabolite (LFM) analogs with a high likelihood to bind favorably to the catalytic site within the kinase domain of BTK. The lead compound LFM-A13, for which we calculated a Ki value of 1.4 microM, inhibited human BTK in vitro with an IC50 value of 17.2 +/- 0.8 microM. Similarly, LFM-A13 inhibited recombinant BTK expressed in a baculovirus expression vector system with an IC50 value of 2.5 microM. The energetically favorable position of LFM-A13 in the binding pocket is such that its aromatic ring is close to Tyr476, and its substituent group is sandwiched between residues Arg525 and Asp539. In addition, LFM-A13 is capable of favorable hydrogen bonding interactions with BTK via Asp539 and Arg525 residues. Besides its remarkable potency in BTK kinase assays, LFM-A13 was also discovered to be a highly specific inhibitor of BTK. Even at concentrations as high as 100 micrograms/ml (approximately 278 microM), this novel inhibitor did not affect the enzymatic activity of other protein tyrosine kinases, including JAK1, JAK3, HCK, epidermal growth factor receptor kinase, and insulin receptor kinase. In accordance with the anti-apoptotic function of BTK, treatment of BTK+ B-lineage leukemic cells with LFM-A13 enhanced their sensitivity to ceramide- or vincristine-induced apoptosis. To our knowledge, LFM-A13 is the first BTK-specific tyrosine kinase inhibitor and the first anti-leukemic agent targeting BTK.[11]

---

The goals of the present study were to define the anticancer activity of LFM-A13 (α-cyano-β-hydroxy-β-methyl-N-(2,5-dibromophenyl)-propenamide), a potent inhibitor of Polo-like kinase (PLK), in a mouse mammary cancer model induced by 7,12-dimethylbenz(a)anthracene (DMBA) in vivo and explore its anticancer mechanism(s). We also examined whether the inhibition of PLK by LFM-A13 would improve the efficiency of paclitaxel in breast cancer growth in vivo. To do this, female BALB/c mice received 1 mg of DMBA once a week for 6 weeks with oral gavage. LFM-A13 (50 mg/kg body weight) was administered intraperitoneally with DMBA administration and continued for 25 weeks. We found that LFM-A13, paclitaxel, and their combination have a significant effect on the DMBA-induced breast tumor incidence, mean tumor numbers, average tumor weight, and size. At the molecular level, the administration of LFM-A13 hindered mammary gland carcinoma development by regulating the expression of PLK1, cell cycle-regulating proteins cyclin D1, cyclin dependent kinase-4 (CDK-4), and the CDK inhibitor, p21. Moreover, LFM-A13 treatment upregulated the levels of IκB, the pro-apoptotic proteins Bax, and caspase-3, and down-regulated p53 and the antiapoptotic protein Bcl-2 in mammary tumors. The combination of LFM-A13 with paclitaxel was found to be more effective compared with either agent alone. Collectively, these results suggest that LFM-A13 has an anti-proliferative activity against breast cancer in vivo and that LFM-A13 and paclitaxel combination could be a strategy for the treatment of breast cancer.[3]

---

(Z)-LFM-A13 is a multi-targeted kinase inhibitor, initially identified as a BTK inhibitor for B-cell leukemias, later found to inhibit Jak2 and PLK1, expanding its potential to hematologic disorders and breast cancer [1][2][3][4]
- Its antitumor mechanism varies by target: inhibiting BTK-mediated B-cell activation (leukemia), blocking Jak2-STAT5 signaling (hematopoiesis), and suppressing PLK1-dependent cell cycle progression (breast cancer) [1][2][4]
- Preclinical data support its efficacy in breast cancer, but its multi-target profile may increase off-target risks compared to selective inhibitors [3][4]

C11H8BR2N2O2

360.001420974731

357.895

C, 36.70; H, 2.24; Br, 44.39; N, 7.78; O, 8.89

244240-24-2

LFM-A13;62004-35-7

54676905

Light yellow to yellow solid powder

1.9±0.1 g/cm3

487.9±45.0 °C at 760 mmHg

248.9±28.7 °C

0.0±1.3 mmHg at 25°C

1.677

3.42

73.12

2

3

2

17

386

0

C/C(=C(\C#N)/C(=O)NC1=C(C=CC(=C1)Br)Br)/O

UVSVTDVJQAJIFG-VURMDHGXSA-N

InChI=1S/C11H8Br2N2O2/c1-6(16)8(5-14)11(17)15-10-4-7(12)2-3-9(10)13/h2-4,16H,1H3,(H,15,17)/b8-6-

2-Cyano-N-(2,5-dibromophenyl)-3-hydroxy-2-butenamide

LFM-A13; LFM A13; lfm-a13; 244240-24-2; (Z)-2-cyano-N-(2,5-dibromophenyl)-3-hydroxybut-2-enamide; CHEMBL228043; 62004-35-7; SMR001230714; alpha-Cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)propenamide; SR-01000075965; LFM A13

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)

DMSO: 72 mg/mL (200.0 mM) Water:<1 mg/mL Ethanol:<1 mg/mL

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.94 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。

---

配方 2 中的溶解度: 2.5 mg/mL (6.94 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。

---

View More

配方 3 中的溶解度: ≥ 2.5 mg/mL (6.94 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。

---

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。