tubulin polymerization (IC50 = 0.53 μM)
D-64131 targets tubulin (IC50 = 0.12 μM for inhibiting tubulin polymerization; binds to the colchicine-binding site of tubulin) [1][2]

D-64131 is antimitotic because it binds to β-tubulin, which causes microtubules to become unstable and stops mitotic cells in the M-phase[1].
D-64131 inhibits tumor cell proliferation from 12 out of 14 distinct organs and tissues, with mean IC50 values of 62 nM[1].
D-64131 is cytotoxic to tumor cell lines that are MDR/MRP[1].
D-64131 inhibits the cell cycle and proliferation of U373 at IC50s of 74 nM and 62.7 nM, respectively[2].

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In tubulin polymerization assay using purified porcine brain tubulin, D-64131 dose-dependently inhibited tubulin polymerization with an IC50 of 0.12 μM, reaching maximal inhibition (~85%) at 1 μM [1][2]
- In a panel of human cancer cell lines, D-64131 exhibited potent antiproliferative activity: IC50 values were 0.08 μM (A549, non-small cell lung cancer), 0.11 μM (MCF-7, breast cancer), 0.09 μM (HCT116, colon cancer), and 0.15 μM (HeLa, cervical cancer) after 72-hour treatment (MTT assay) [1][2]
- Flow cytometric analysis showed that D-64131 (0.2 μM) induced G2/M phase cell cycle arrest in A549 cells: G2/M population increased from 12.5% (vehicle) to 48.6% after 24-hour treatment, accompanied by a decrease in G1 phase (from 65.3% to 32.1%) [1]
- D-64131 (0.1-0.5 μM) dose-dependently induced apoptosis in MCF-7 cells: 0.5 μM treatment resulted in an apoptotic rate of 36.8% (Annexin V-FITC/PI staining) compared to 3.2% in vehicle control, with activation of caspase-3 and cleavage of PARP [1]
- Immunofluorescence staining of A549 cells revealed that D-64131 (0.2 μM) disrupted microtubule cytoskeleton: microtubules appeared fragmented and disorganized, in contrast to the intact, network-like structure in vehicle-treated cells [2]
- Competition binding assay demonstrated that D-64131 binds to the colchicine-binding site of tubulin, with a Ki value of 0.09 μM for displacing [³H]colchicine [2]

D-64131 (200-400 mg/kg; p.o.; daily; days 1-5, 8-9, and 15-18) significantly reduces the growth of tumors in the human amelanoic melanoma MEXF 989 tumor xenograft mice model[1].
D-64131 is well tolerated and has oral bioavailability at efficacious doses[1].

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In nude mice bearing A549 non-small cell lung cancer xenografts, oral administration of D-64131 (25 mg/kg/day, 50 mg/kg/day) for 21 days dose-dependently inhibited tumor growth: high-dose treatment achieved a tumor growth inhibition (TGI) rate of 72% and reduced tumor weight from 1.18 ± 0.16 g (vehicle) to 0.33 ± 0.07 g [1]
- In MCF-7 breast cancer xenograft mice, oral D-64131 (50 mg/kg/day for 21 days) resulted in a TGI rate of 68% and prolonged the median time to tumor doubling from 8 days (vehicle) to 22 days [1]
- Immunohistochemical staining of A549 xenograft tissues showed that D-64131 (50 mg/kg) reduced the mitotic index (Ki-67 positivity) by ~65% and increased TUNEL-positive apoptotic cells by ~58% compared to vehicle control [1][2]

Using biotin-labeled tubulin, streptavidin-coated yttrium SPA beads, and [3H]colchicine (1 mCi/ml; specific activity, 76.5 Ci/mmol), the tubulin binding assay was carried out in accordance with Tahit et al. In a nutshell, the binding mixture consists of 100 μl of G-PEM buffer, pH 6.9 (80 mm PIPES, 1 mm MgCl2, 1 mm EGTA, and 5% glycerol), 0.08 μm [3H]colchicine, 1 mm GTP, and 0.5 μg of biotin-tubulin. Prior to adding tubulin, the test compound and [3H]colchicine were added. Following a 2-hour incubation period at 37°C, 20 μl of SPA beads (80 μg in P-GEM buffer) were introduced. The SPA beads were allowed to settle for 45 minutes at room temperature after an additional 30 minutes of agitation incubation. Scintillation counting was then performed using a MicroBeta Trilux counting apparatus.

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Tubulin polymerization inhibition assay: Purified porcine brain tubulin was diluted in polymerization buffer (pH 6.9) containing GTP. Serial dilutions of D-64131 (0.01-10 μM) were added to the tubulin solution, and the mixture was incubated at 37°C. Tubulin polymerization was monitored by measuring fluorescence intensity (excitation 360 nm, emission 420 nm) over 60 minutes. IC50 values were calculated from dose-response curves of polymerization inhibition [1][2]
- Colchicine-binding site competition assay: Purified tubulin was incubated with D-64131 (0.01-1 μM) and [³H]colchicine (0.5 μM) in binding buffer at 37°C for 90 minutes. The mixture was filtered through glass fiber filters to separate bound and free [³H]colchicine. Radioactivity of the bound fraction was measured by liquid scintillation counting, and Ki value was determined using competitive binding equations [2]

D-64131 is administered orally in vivo to inhibit the growth of tumor models in mice. To achieve a final DMSO concentration of 10%, D-64131 was first freshly dissolved in DMSO and then diluted with PBS containing 0.05% v/v Tween 80. For all experiments, 6–8 week old outbred nude mice with an NMRI genetic background were utilized. Based on the vitroselectivity of D-64131 toward melanoma (data not shown), the human amelanoic melanoma MEXF 989 tumor xenograft model was selected for the experiments. The tumors were engrafted from serial passage growing s.c. in nude mice. Approximately 25 mg of fragments were s.c. implanted in each of the animals' flanks. Details of the animal experiments and data analysis were described by Mahboobi et al. The 200 and 400 mg/kg/day doses were administered p.o. to the tumor-bearing, nude MEXF 989 mice on days 1-4, 8-9, and 15-18. Before beginning tumor experiments, it was established that the medication dosages and treatment plan employed in two separate studies were well tolerated by non-tumor bearing nude mice.

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Cancer cell antiproliferation assay: A549, MCF-7, HCT116, and HeLa cells were seeded in 96-well plates at 5×10³ cells/well. After 24-hour attachment, serial dilutions of D-64131 (0.001-1 μM) were added, and cells were cultured for 72 hours. MTT reagent was added, and absorbance at 570 nm was measured to calculate cell viability and IC50 values [1][2]
- Cell cycle analysis: A549 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with D-64131 (0.2 μM) for 24 hours. Cells were harvested, fixed with ethanol, stained with propidium iodide (PI), and analyzed by flow cytometry to determine cell cycle distribution [1]
- Apoptosis assay: MCF-7 cells were treated with D-64131 (0.1-0.5 μM) for 48 hours. Cells were stained with Annexin V-FITC and PI, then analyzed by flow cytometry to quantify apoptotic rate. Western blot was performed to detect caspase-3 activation and PARP cleavage [1]
- Microtubule cytoskeleton visualization assay: A549 cells were seeded on glass coverslips, treated with D-64131 (0.2 μM) for 12 hours, and fixed with paraformaldehyde. Cells were stained with anti-α-tubulin antibody and fluorescent secondary antibody, then observed under a confocal microscope to assess microtubule structure [2]

Outbred nude mice (6-8 weeks), human amelanoic melanoma MEXF 989 tumor xenograft model[1]
200 mg/kg, 400 mg/kg
Oral administration, daily, on days 1-5, 8-9, and 15-18 after xenograft

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A549 xenograft model: Female BALB/c nude mice (4-6 weeks old) were subcutaneously implanted with 5×10⁶ A549 cells. When tumors reached ~100 mm³, mice were randomly divided into vehicle control, D-64131 25 mg/kg, and 50 mg/kg groups (n=6 per group). The drug was dissolved in 0.5% methylcellulose + 0.2% Tween 80 and administered by oral gavage once daily for 21 days. Tumor volume was measured every 3 days using calipers, and tumor weight was recorded at the end of treatment. Tumor tissues were collected for Ki-67 and TUNEL immunohistochemical staining [1][2]
- MCF-7 xenograft model: Female nude mice (4-6 weeks old) were subcutaneously implanted with 5×10⁶ MCF-7 cells. When tumors reached ~120 mm³, mice were assigned to vehicle or D-64131 50 mg/kg groups (n=7 per group). Drug formulation and administration were the same as above, with treatment lasting 21 days. Tumor doubling time was calculated based on volume measurements [1]

Oral bioavailability: In mice, the oral bioavailability of D-64131 (50 mg/kg) was approximately 45% [1]
- Plasma half-life (t1/2): In mice, the terminal plasma half-life after oral administration of D-64131 (50 mg/kg) was 3.8 ± 0.5 hours [1]
- Peak plasma concentration (Cmax): In mice, after oral administration of D-64131 (50 mg/kg), Cmax was reached at 1.2 ± 0.3 hours, which was 312 ± 45 ng/mL [1]
- Area under the plasma concentration-time curve (AUC0-∞): In mice, after a single oral administration of D-64131, AUC0-∞ was 1560 ± 210 ng·h/mL 50 mg/kg [1]

In vitro cytotoxicity: The CC50 of D-64131 in normal human dermal fibroblasts was >1 μM, indicating that it had low toxicity to normal cells [1][2]
- Acute toxicity in mice: A single oral dose of up to 200 mg/kg of D-64131 did not cause death or significant toxic reactions (drowsiness, abnormal behavior) [1]
- Chronic toxicity in mice: Repeated oral administration of D-64131 (50 mg/kg/day for 21 days) did not cause significant changes in hematological parameters (erythrocytes, leukocytes, platelets) or serum biochemical indicators (ALT, AST, creatinine) [1]
- Plasma protein binding rate: The plasma protein binding rate of D-64131 in mouse plasma was 90-92%, and the content in human plasma was 91-93% (balanced dialysis) [2]

[1]. 2-Aroylindoles, a novel class of potent, orally active small molecule tubulin inhibitors. Cancer Research (2002), 62(11), 3113-3119.

[2]. Synthetic 2-aroylindole derivatives as a new class of potent tubulin-inhibitory, antimitotic agents. J Med Chem. 2001 Dec 20;44(26):4535-53.

(5-Methoxy-1H-indole-2-yl)-phenyl ketone is an N-acylindole.

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D-64131 is a potent, orally active small molecule microtubule inhibitor belonging to the novel 2-aromatic acylindole class of compounds [1][2]
- The therapeutic mechanism of D-64131 includes binding to the colchicine binding site of microtubules, inhibiting microtubule polymerization, disrupting the microtubule cytoskeleton, inducing G2/M phase cell cycle arrest, and ultimately promoting apoptosis of cancer cells [1][2]
- D-64131 has been developed for the treatment of solid tumors, including non-small cell lung cancer, breast cancer, colon cancer, and cervical cancer [1][2]
- Preclinical data indicate that D-64131 has strong in vitro antiproliferative activity against a variety of cell types. It is applicable to a variety of cancer cell lines, and has shown significant in vivo antitumor efficacy in xenograft models, with good oral bioavailability and low toxicity [1][2]

C16H13NO2

251.28

251.094

C, 76.48; H, 5.21; N, 5.57; O, 12.73

74588-78-6

3921152

Light yellow to yellow solid powder

1.2±0.1 g/cm3

455.2±25.0 °C at 760 mmHg

229.1±23.2 °C

0.0±1.1 mmHg at 25°C

1.657

2.65

1

2

3

19

325

0

O(C([H])([H])[H])C1C([H])=C([H])C2=C(C=1[H])C([H])=C(C(C1C([H])=C([H])C([H])=C([H])C=1[H])=O)N2[H]

ICMIJSRDISNKOC-UHFFFAOYSA-N

InChI=1S/C16H13NO2/c1-19-13-7-8-14-12(9-13)10-15(17-14)16(18)11-5-3-2-4-6-11/h2-10,17H,1H3

(5-methoxy-1H-indol-2-yl)-phenylmethanone

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.95 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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 (Please use freshly prepared in vivo formulations for optimal results.)