KSP (EC50 = 6 nM)
Filanesib specifically targets the kinesin spindle protein (KSP/Eg5), a mitotic kinesin involved in centrosome separation and bipolar spindle assembly during the early stages of mitosis. By inhibiting KSP, the compound activates the spindle assembly checkpoint (SAC), leading to mitotic arrest, formation of monoastral spindles, and subsequent apoptosis in actively dividing tumor cells. The IC50 for KSP inhibition is 6 nM. This mechanism is highly selective for proliferating cells, as KSP is not required in non-dividing cells.

Finerenone inhibits the proliferation of aldosterone-induced smooth muscle cells (SMC) and stops the apoptosis of aldosterone-induced endothelial cells (EC) in vitro. Finerenone causes an increase in endothelial healing and a decrease in the formation of neointima in injured vessels by dramatically lowering EC apoptosis while concurrently attenuating SMC proliferation.[2]

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In vitro, Filanesib potently inhibits the proliferation of various cancer cell lines by inducing mitotic arrest and apoptosis. The compound demonstrates antiproliferative effects across a broad panel of tumor types, including multiple myeloma, acute myeloid leukemia, and solid tumors. In cell-based assays, Filanesib causes cell cycle arrest during the mitotic phase, leading to the formation of abnormal monoastral spindles and subsequent cell death. The compound shows synergistic activity when combined with other anticancer agents such as bortezomib and dexamethasone.

Finerenone reduces superoxide anion levels because of an increase in SOD activity and improves endothelial dysfunction by increasing NO bioavailability. This is linked to a decrease in albuminuria and an increase in renal SOD activity.[1]

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In vivo, Filanesib has demonstrated significant antitumor activity in preclinical xenograft models of multiple myeloma and other cancers. The compound exhibits dose-dependent tumor growth inhibition and has been evaluated in Phase I/II clinical trials for hematological malignancies. In clinical studies, Filanesib has shown single-agent activity in patients with relapsed or refractory multiple myeloma and acute myeloid leukemia, with responses observed even in heavily pretreated populations.

In vitro enzyme assays for KSP inhibition typically use recombinant human kinesin-5 (Eg5) and measure ATPase activity in the presence of microtubules. The compound is incubated with the enzyme, ATP, and taxol-stabilized microtubules, and the production of ADP is quantified using a coupled enzymatic assay or malachite green phosphate detection. IC50 values are calculated from dose-response curves to determine the compound's potency.

For the purpose of measuring cell proliferation and apoptosis, cells are serum-starved for 24 hours before being incubated with aldosterone with or without finerenone. Cells are preincubated with finerenone or vehicle for 30 minutes prior to the addition of aldosterone.

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Cellular assays are performed using cancer cell lines such as multiple myeloma (MM.1S, RPMI-8226) and acute myeloid leukemia (HL-60, MV4-11) cells. Cells are treated with Filanesib at varying concentrations (typically 1-1000 nM) for 24-72 hours. Cell viability is assessed using MTT or CellTiter-Glo assays. Mitotic arrest is evaluated by flow cytometry for phospho-histone H3 (Ser10) and cell cycle analysis. Apoptosis is measured by Annexin V/PI staining and caspase-3/7 activity assays.

Tumor xenografts placed subcutaneously are allowed to expand to a volume of 250–350 mm3. Depending on the size of their tumors, the mice are randomly assigned to groups of three to four and given a single intraperitoneal dose of filanesib (ARRY-520). The mice are put to sleep by CO2 inhalation at different intervals following the drug's administration, and the tumors are removed and put in 10% neutral buffered formalin. The tumors that have been fixed in formalin are processed and paraffin embedded using standard protocols. Tumor sections stained with α-tubulin are used to analyze spindle morphology, and TUNEL stain is used to analyze apoptosis. Algorithms developed in ImagePro software are used to analyze the count of TUNEL positive (apoptotic) cells and monopolar/abnormal spindles in three ×40 fields from each sample.

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In vivo studies are conducted in immunocompromised mice bearing subcutaneous or orthotopic tumor xenografts. Filanesib is typically administered intravenously at doses ranging from 5-30 mg/kg on various schedules (e.g., days 1, 8, 15 or daily for 5 days). Tumor volume is measured periodically using calipers, and body weight is monitored for toxicity. At study termination, tumors are harvested for histopathological analysis, including assessment of mitotic index, Ki-67 proliferation, and apoptosis by TUNEL staining.

Filanesib (molecular weight 420.48, formula C₂₀H₂₂F₂N₄O₂S) is a small-molecule compound. It is soluble in DMSO and is typically administered intravenously in clinical settings. The compound is stable as a solid powder when stored at -20°C in dry, dark conditions. Pharmacokinetic studies in patients have shown that Filanesib has a relatively short half-life and is primarily cleared through hepatic metabolism.

Preclinical toxicity studies have shown that Filanesib is generally well-tolerated at therapeutic doses, with expected toxicities related to mitotic inhibition including myelosuppression (neutropenia, thrombocytopenia). Unlike tubulin-targeting agents, Filanesib does not cause peripheral neuropathy due to the absence of KSP in postmitotic neurons. In clinical trials, the most common adverse events have included fatigue, gastrointestinal disturbances, and reversible liver enzyme elevations.

[1]. Front Pharmacol . 2018 Oct 9:9:1131.

[2]. PLoS One . 2017 Sep 19;12(9):e0184888.

Feranixib is a potent kinin spindle protein (KSP) inhibitor that has significantly inhibited tumor growth in preclinical models of human solid tumors and human leukemia, often producing durable efficacy. Feranixib is a synthetic small molecule that targets kinin spindle protein (KSP) and possesses potential antitumor activity. Feranixib specifically inhibits KSP (kinin-5 or Eg5), thereby activating the spindle assembly checkpoint, inducing cell cycle arrest during mitosis, and ultimately leading to the death of actively dividing tumor cells. Because KSP is not involved in postmitotic processes (such as neuronal transport), this drug does not cause peripheral neuropathy typically associated with microtubule-targeting drugs. KSP is an ATP-dependent microtubule motor protein essential for the formation of bipolar spindles and the proper separation of sister chromatids during mitosis.

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Drug Indications
Investigated for the treatment of cancer/tumors (not specified).

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Mechanism of Action
KSP has been identified as an attractive target for anticancer drugs.
Cancer arises from abnormalities in normal cellular processes, leading to uncontrolled cell division, proliferation, and growth. KSP inhibitors cause mitotic arrest by preventing the formation of bipolar spindles. Unipolar spindles thus prevent centrosome separation, organizing microtubules from individual sites within the cell and aligning chromosomes around those sites. This compound is a highly potent KSP inhibitor, exhibiting sub-nanomolar potency in both enzymatic and cellular experiments, and inducing mitotic arrest, ultimately leading to the death or apoptosis of highly proliferating cancer cells.

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Pharmacodynamics
This compound is a highly potent KSP inhibitor, exhibiting sub-nanomolar potency in both enzymatic and cellular experiments, and inducing mitotic arrest, ultimately leading to the death or apoptosis of highly proliferating cancer cells.

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Filanesib (ARRY-520) is a first-in-class KSP inhibitor developed by Array BioPharma. Its mechanism involves selective inhibition of Eg5/kinesin-5, leading to mitotic arrest and apoptosis in actively dividing cancer cells. The compound has undergone extensive clinical evaluation in multiple myeloma and acute myeloid leukemia, with Phase II studies exploring its activity in combination with bortezomib, dexamethasone, and other agents. Despite promising early clinical data, Filanesib has not received regulatory approval for clinical use, and further development has been discontinued.

C20H22N4O2F2S

420.47608

420.143

C, 57.13; H, 5.27; F, 9.04; N, 13.32; O, 7.61; S, 7.6

885060-09-3

(R)-Filanesib;885060-08-2;Filanesib TFA;1781834-99-8

44224257

white solid powder

1.3±0.1 g/cm3

511.3±60.0 °C at 760 mmHg

263.0±32.9 °C

0.0±1.3 mmHg at 25°C

1.604

3.27

1

7

6

29

605

1

C([C@]1(SC(C2C=C(F)C=CC=2F)=NN1C(=O)N(C)OC)C1C=CC=CC=1)CCN

LLXISKGBWFTGEI-FQEVSTJZSA-N

InChI=1S/C20H22F2N4O2S/c1-25(28-2)19(27)26-20(11-6-12-23,14-7-4-3-5-8-14)29-18(24-26)16-13-15(21)9-10-17(16)22/h3-5,7-10,13H,6,11-12,23H2,1-2H3/t20-/m0/s1

(2S)-2-(3-aminopropyl)-5-(2,5-difluorophenyl)-N-methoxy-N-methyl-2-phenyl-1,3,4-thiadiazole-3-carboxamide

Filanesib; ARRY520; ARRY 520; ARRY-520

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: ~76 mg/mL (~200.8 mM)
Ethanol: ~10 mg/mL (~26.4 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.95 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 (5.95 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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 (5.95 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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Solubility in Formulation 4: 5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 3.8mg/ml

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