Eg5 (IC50 = 200 nM)
The therapeutic target is mitotic Kinesin Eg5 (also known as KIF11)[1]
The therapeutic target is mitotic Kinesin Eg5, which exerts effects by allosterically inhibiting the ATPase activity of the motor domain of Eg5[2]
The therapeutic target is mitotic Kinesin Eg5[3]

Dimethylenastron (1.0 µmol) causes a less severe scarring, but it does not considerably extend the bleb's life in comparison to the control group. In the rabbits treated with glaucoma filtration surgery, dimethylenastron (1.0 µmol) shows a significantly reduced ratio of intraocular pressure and a milder, though not significantly reduced, subconjunctival fibrotic reaction[3].

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Glaucoma filtration surgery model (Chinchilla rabbits)
1. Administration regimen and ocular response: Glaucoma filtration surgery (mimicking clinical practice) was performed on 37 female chinchilla rabbits (ChBBCH). Different regimens of subconjunctival injection of Dimethylenastron were given: single-dose groups (unilateral injection of 1.0 μmol or 3.0 μmol at baseline), multiple-dose groups (injection of 1.0 μmol or 3.0 μmol at baseline + 3 days + 7 days postoperatively), and control groups (surgery only, vehicle (DMSO) administration)[3]
2. Fibrosis and bleb scarring: Bleb scarring occurred in the surgery-only group, vehicle group, and 3.0 μmol Dimethylenastron group; the 1.0 μmol group showed milder subconjunctival fibrotic reaction, but the bleb survival time was not significantly prolonged (Kaplan-Meier log rank test, p=0.053)[3]
3. Intraocular pressure (IOP) changes: IOP in all groups was correlated with the fibrotic process and returned to normal levels within 14 days after surgery; the 1.0 μmol Dimethylenastron group had a significantly reduced IOP ratio, but did not sufficiently improve surgical outcomes[3]
4. Histological verification: Immunohistochemistry (smooth muscle actin (SMA), CD31) and histological analysis showed that the 1.0 μmol Dimethylenastron group had milder subconjunctival fibrotic reaction, but did not achieve a significant improvement in surgical efficacy[3]

Dimethylenastron has an IC50 of 200 nM, making it a strong Eg5 inhibitor. Five other kinesin subfamilies (kinesin 1/4/7/10 and one ungrouped, originating from four different organisms) are not inhibited by dimethylenastron. In HeLa cells, dimethylenastron (0.5, 1 μM) induces cell accumulation in the G2/M phase.

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1. Detection of ADP release rate of Eg5 motor domain: MANT-ADP reagent was used to detect the effect of Dimethylenastron on the ADP release rate of Eg5 motor domain. Experimental groups with/without microtubules were set up respectively, and the changes in ADP release rate after treatment with different concentrations of the drug were quantitatively determined to verify the allosteric inhibitory effect of the drug on Eg5 ATPase activity; the experiment was repeated 3 times, results were expressed as mean ± SD, and statistical tests were used to analyze differences (P<0.01)[2]

Transwell assays measure the invasion of cells in response to dimethylenastron. Matrigel or fibronectin is applied to the transwell filters' upper surface. After adding cells suspended in 200 μL serum-free media to the chamber, the chamber is put in a 24-well plate with the entire medium inside. The matrigel on the upper surface of the filters is removed with cotton swabs once the filters have been gently removed from their 24-hour incubation at 37°C. The cells on the underside of transwell filters are photographed after being fixed for 30 minutes in 4% paraformaldehyde and stained for 10 minutes in 0.1% crystal violet. The number of invading cells is counted in five random fields per filter for quantitative evaluation. The number of invasive cells in the drug-treatment group divided by the number of invasive cells in the control group is used to calculate the extent of cell invasion[2].

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Pancreatic cancer cell migration assay (scratch wound healing method)
1. Experimental procedure: PANC1 pancreatic cancer cells were seeded in serum-free medium and cultured to confluence. Scratches were made on the cell monolayer with a pipette tip, and medium containing 0, 3, 10 μmol/L Dimethylenastron with 10% or 5% serum was added respectively. After 24 hours of culture, images of the scratch area were taken, and the number of cells migrating to the scratch area was counted to quantify migration ability; the experiment was repeated 3 times, results were expressed as mean ± SD, and statistical tests were used to analyze differences from the control group (P<0.05 or P<0.01)[2]
### Pancreatic cancer cell invasion assay (Transwell method)
1. Experimental procedure: The upper side of the Transwell chamber filter was coated with matrigel or fibronectin respectively. PANC1 cells treated with 0, 3, 10 μmol/L Dimethylenastron were seeded into the upper chamber of the Transwell, and medium containing serum was added to the lower chamber. After 24 hours of culture, cells on the underside of the filter were fixed and stained, and the number of invasive cells was counted after imaging; the experiment was repeated 3 times, results were expressed as mean ± SD, and statistical tests were used to analyze differences from the control group (P<0.05 or P<0.01)[2]
### Pancreatic cancer cell proliferation assay
1. Sulforhodamine B (SRB) assay: PANC1 cells were seeded in culture plates and treated with gradient concentrations of Dimethylenastron for 24 hours or 72 hours respectively. Cells were fixed, stained with SRB solution, unbound dye was eluted, and absorbance values were detected with a spectrophotometer to calculate the percentage of cell proliferation; the experiment was repeated 3 times, results were expressed as mean ± SD[2]
2. MTT assay: The operational procedure was consistent with the SRB assay, replacing with MTT reagent to detect cell viability and calculate proliferation percentage; SRB and MTT data were compared to verify the inhibitory effect of the drug on proliferation (P<0.05)[2]
### Detection of Eg5 expression in pancreatic cancer cells (immunofluorescence method)
1. Experimental procedure: Immunofluorescence staining was performed on pancreatic cancer cell lines (PANC1, EPP85, BxPC3, CFPAC1, AsPAC1) and normal pancreatic epithelial cells. Primary antibody targeted Eg5 (red fluorescence), DAPI stained nuclei (blue fluorescence). After imaging, the expression level and nuclear distribution of Eg5 were quantitatively analyzed; the experiment was repeated 3 times, and results showed that Eg5 expression in pancreatic cancer cells was 9-16 times higher than that in normal cells (P<0.01)[2]

Following the closure of the conjunctival suture, one of the following agents is injected using a metallic needle (30 G) that is placed into the subconjunctival space at the nasal border of the superior rectus muscle: Following surgery, the rabbits in the control group receive no adjuvant, the other groups receive one unilateral subconjunctival injection of Dimethylenastron (1.0 µmol, 3.0 µmol) or the vehicle (DMSO, 99.9%, 10 mg/mL) alone at baseline. This means that the injection is given immediately after surgery, and in two additional groups, it is given twice more at days 3 and 7 after (1.0 µmol, 3.0 µmol)[3].

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Animal experiment of glaucoma filtration surgery (Chinchilla rabbits)
1. Experimental animals: 37 female chinchilla rabbits (ChBBCH)[3]
2. Surgical operation: Glaucoma filtration surgery simulating clinical practice was performed[3]
3. Administration regimen:
- Single-dose group: Unilateral subconjunctival injection of 1.0 μmol or 3.0 μmol Dimethylenastron at surgical baseline, or vehicle injection only[3]
- Multiple-dose group: Unilateral subconjunctival injection of 1.0 μmol or 3.0 μmol Dimethylenastron at surgical baseline, 3 days and 7 days postoperatively respectively[3]
4. Observation period and endpoint: Clinical indicators such as bleb scarring and IOP changes were recorded for 14 days postoperatively; animals were sacrificed on day 14, and ocular tissues were collected for histological and immunohistochemical analysis (SMA, CD31 staining)[3]

1. Ocular toxicity: Two patients experienced transient fibrinogenic reactions after subconjunctival injection of 3.0 μmol dimethylsetron; no other adverse reactions, such as inflammation or optical turbidity, were observed; the excipient (DMSO) was well tolerated [3].

[1]. Development and biological evaluation of potent and specific inhibitors of mitotic Kinesin Eg5. Chembiochem. 2005 Jul;6(7):1173-7.

[2]. Dimethylenastron suppresses human pancreatic cancer cell migration and invasion in vitro via allosteric inhibition of mitotic kinesin Eg5. Acta Pharmacol Sin. 2011 Dec;32(12):1543-8.

[3]. The effect of adjuvant dimethylenastron, a mitotic Kinesin Eg5 inhibitor, in experimental glaucoma filtration surgery. Curr Eye Res. 2010 Dec;35(12):1090-8.

1. Drug Classification: Dimethylsetron is a quinazoline/thione compound that specifically inhibits the mitotic kinesin Eg5 [1][2][3] 2. Mechanism of Action: Dimethylsetron binds to the motor domain of Eg5 through allosteric action, reducing the release rate of ADP and inhibiting the ATPase activity of Eg5; its inhibitory effect on pancreatic cancer cell migration and invasion is not related to its inhibition of proliferation [2] 3. Research and Development Background: Eg5 plays a key role in the assembly of the bipolar spindle, and its inhibitors have shown significant anticancer activity in preclinical studies; Eg5 is highly expressed in pancreatic cancer cells (9-16 times higher than in normal cells), suggesting that it can be used as a therapeutic target for pancreatic cancer [2] 4. Application potential: Dimethylsetron can inhibit the migration and invasion of pancreatic cancer cells, providing a new mechanism for pancreatic cancer chemotherapy; it can slightly reduce subconjunctival fibrosis during glaucoma filtration surgery, but the current concentration has not been sufficient to improve the surgical effect [2][3]

C16H18N2O2S

302.39

302.108

C, 63.55; H, 6.00; N, 9.26; O, 10.58; S, 10.60

863774-58-7

11609157

White to off-white solid powder

1.3±0.1 g/cm3

475.6±55.0 °C at 760 mmHg

241.4±31.5 °C

0.0±1.2 mmHg at 25°C

1.674

2.47

3

3

1

21

512

0

O=C1CC(C)(C)CC2NC(NC(C1=2)C1C=C(O)C=CC=1)=S

RUOOPLOUUAYNPY-UHFFFAOYSA-N

InChI=1S/C16H18N2O2S/c1-16(2)7-11-13(12(20)8-16)14(18-15(21)17-11)9-4-3-5-10(19)6-9/h3-6,14,19H,7-8H2,1-2H3,(H2,17,18,21)

4-(3-hydroxyphenyl)-7,7-dimethyl-2-sulfanylidene-3,4,6,8-tetrahydro-1H-quinazolin-5-one

Dimethylenastron

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 (8.27 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 (8.27 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.)