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)

At an IC50 of 6.2 ± 0.3 μg/mL (= 17.2 ± 0.8 μM), LFM-A13 strongly suppresses BTK activity. The LFM-A13 estimated Kis values for BTK, JAK1, JAK3, IRK, EGFR, and HCK are 1.4, 110, 148, 31.6, 166, and 214 μM. Ceramide-induced apoptosis in ALL-1 cells is chemosensitive to LFM-A13 (200 μM)[1]. Epo-induced phosphorylation of EpoR, Jak2, Btk, Stat5, and Erk1/2 in R10 cells is suppressed by LFM-A13 (100 μM). In COS cells, LFM-A13 (100 μM) suppresses auto-phosphorylation of Jak2, Tec, and Btk, but not Lyn kinase auto-phosphorylation[2]. Potently inhibiting Plx1 at an IC50 of 10 μM, LFM-A13 also inhibits BRK, BMX, FYN, and has IC50s of 267, 281, 240, and 215 μM[4]. Z)-

For rats, LFM-A13 at 25, 50, and 100 mg/kg does not appear to be harmful. In mice, LFM-A13 (50 mg/kg, i.p., three times a week) reduces the development of malignant tumors. In BALB/c mice, LFM-A13 either by itself or in conjunction with paclitaxel exhibits a significant impact on the incidence, mean number, weight, and size of breast tumors. In mice, LFM-A13 (50 mg/kg, i.p., three times a week) dramatically reduces the expression of PLK1, cyclin D1, CDK -4, P53, and Bcl-2, but enhances the expression of p21, IκB, Bax, and caspase 3[3]. Rats exposed to 200 mg/kg of LFM-A13 do not experience hematologic toxicity. The MMTV/Neu transgenic mouse model of breast cancer shows dose-dependent anti-tumor effects when treated with LFM-A13 (10 or 50 mg/kg, ip)[4].

For HCK kinase assays, we used HCK-transfected COS-7 cells. The cloning and expression of HCK in COS-7 cells has been described previously. The pSV7c-HCK plasmid was transfected into 2 × 106 COS-7 cells using LipofectAMINE, and the cells were harvested 48 h later. The cells were lysed in Nonidet P-40 buffer, and HCK was immunoprecipitated from the whole cell lysates with an anti-HCK antibody.[1]

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LFM-A13, or alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)propenamide, was shown to inhibit Bruton's tyrosine kinase (Btk). Here we show that LFM-A13 efficiently inhibits erythropoietin (Epo)-induced phosphorylation of the erythropoietin receptor, Janus kinase 2 (Jak2) and downstream signalling molecules. However, the tyrosine kinase activity of immunoprecipitated or in vitro translated Btk and Jak2 was equally inhibited by LFM-A13 in in vitro kinase assays. Finally, Epo-induced signal transduction was also inhibited in cells lacking Btk. Taken together, we conclude that LFM-A13 is a potent inhibitor of Jak2 and cannot be used as a specific tyrosine kinase inhibitor to study the role of Btk in Jak2-dependent cytokine signalling.[2]

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Molecular modeling studies led to the identification of LFM-A13 (alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)propenamide) as a potent inhibitor of Polo-like kinase (Plk). LFM-A13 inhibited recombinant purified Plx1, the Xenopus homolog of Plk, in a concentration-dependent fashion, as measured by autophosphorylation and phosphorylation of a substrate Cdc25 peptide. LFM-A13 was a selective Plk inhibitor. While the human PLK3 kinase was also inhibited by LFM-A13 with an IC(50) value of 61 microM, none of the 7 other serine/threonine kinases, including CDK1, CDK2, CDK3, CHK1, IKK, MAPK1 or SAPK2a, none of the 10 tyrosine kinases, including ABL, BRK, BMX, c-KIT, FYN, IGF1R, PDGFR, JAK2, MET, or YES, or the lipid kinase PI3Kgamma were inhibited (IC(50) values >200-500 microM). The mode of Plk3 inhibition by LFM-A13 was competitive with respect to ATP with a K(i) value of 7.2 microM from Dixon plots. LFM-A13 blocked the cell division in a zebrafish (ZF) embryo model at the 16-cell stage of the embryonic development followed by total cell fusion and lysis. LFM-A13 prevented bipolar mitotic spindle assembly in human breast cancer cells and glioblastoma cells and when microinjected into living epithelial cells at the prometaphase stage of cell division, it caused a total mitotic arrest. Notably, LFM-A13-delayed tumor progression in the MMTV/neu transgenic mouse model of HER2 positive breast cancer at least as effectively as paclitaxel and gemcitabine. LFM-A13 showed a favorable toxicity profile in mice and rats. In particular there was no evidence of hematologic toxicity as documented by peripheral blood counts and bone marrow examinations. These results establish LFM-A13 as a small molecule inhibitor of Plk with in vitro and in vivo anti-proliferative activity against human breast cancer.[3]

In order to examine the effects of the lead BTK inhibitor on ceramide-induced apoptosis in B cell antigen receptor-ABL positive human ALL cell line ALL-1, cells were treated for 4 h at 37 °C with 10 μmC2-ceramide in the presence or absence of the inhibitor (200 μm LFM-A13 ). Subsequently, cells were washed and stained with PI and MC540, and the apoptotic fractions were determined by multiparameter flow cytometry, as described.

To detect apoptotic fragmentation of DNA, DT40 cells were harvested 24 h after exposure to anti-Fas, C2-ceramide, or vincristine. Similarly, B18.2, NALM-6, and ALL-1 cells were treated with LFM-A13 (100 μm), vincristine (VCR) (10 ng/ml), C2-ceramide (C2-CER) (10 μm), LFM-A13 (100 μm) + VCR (10 ng/ml), and LFM-A13 (100 μm) + C2-CER (10 μm) for 24 h at 37 °C. DNA was prepared from Triton X-100 lysates for analysis of fragmentation. In brief, cells were lysed in hypotonic 10 mmol/liter Tris-HCl, pH 7.4, 1 mmol/liter EDTA, 0.2% Triton X-100 detergent and subsequently centrifuged at 11,000 × g. To detect apoptosis-associated DNA fragmentation, supernatants were electrophoresed on a 1.2% agarose gel, and the DNA fragments were visualized by ultraviolet light after staining with ethidium bromide[1].

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

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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]

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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

[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]

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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]

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

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.94 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (6.94 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.94 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)