10-Hydroxycamptothecin-succinic acid conjugated with adenovirus and its preparation method and use
A technology of hydroxycamptothecin and succinic acid, which is applied in the direction of medical preparations containing active ingredients, antibody medical ingredients, and unknown raw materials, etc., can solve problems affecting effectiveness and drug resistance, so as to reduce dosage and kill Improvement of action and reduction of toxic and side effects
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Embodiment 1、10
[0042] The synthesis of embodiment 1,10-hydroxycamptothecin-succinic anhydride
[0043] After 10-hydroxycamptothecin (200mg, 0.247mmol) was dissolved in anhydrous pyridine (20ml), DMAP (4-dimethylaminopyridine, 123.6mg, 0.594mmol) and succinic anhydride (1180mg, 2.47mmol) were added, 60 Stir at ℃ for 72h. After the reaction product was cooled to room temperature, it was diluted with 20 ml of toluene, and then evaporated to dryness in a rotary vacuum (condition: 0.1 Mpa, 50° C.), and repeated once (ie, repeat the above dilution with toluene and evaporated to dryness in a rotary vacuum). Add another 50ml of chloroform in the spinner bottle, and wash the organic phase with 20ml2% (mass concentration) dilute hydrochloric acid and 20ml saturated NaCl solution, repeat once (that is, repeatedly wash the organic phase with 20ml2% dilute hydrochloric acid and 20ml saturated NaCl solution); Dry over anhydrous MgSO4, filter with suction, and concentrate to constant weight with a rotary ...
Embodiment 2
[0054] The preparation of embodiment 2HCPT-SA-AdTRAIL
[0055] HCPT-SA (15 mg, 0.027 mmol) was dissolved in DMF (N, N-dimethylformamide, 500 μL), and NHS (N-hydroxysuccinimide, 4.6 mg, 0.05 mmol) and EDAC (1-(3 -Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4.8mg, 0.027mmol) was reacted at room temperature (10-25°C) for 4 hours. Add the reacted solution to about 2 mL containing 2×10 10 Pfu of AdTRAIL in PBS (10mM, pH7.4) solution (molar ratio is HCPT-SA:AdTRAIL=10 9 : 1 pmol), at 4 ° C, stirred dark reaction for 12 hours. After the reaction, centrifuge (5000rpm, 30min), take the supernatant, and purify the supernatant by SephadexG-25. The specific content of the purification is: use SephadexG-25 gel chromatography, the buffer solution is PBS (10mM, pH8.0), the flow rate is 1ml / min, after equilibrating the chromatography column with 5 column volumes of PBS solution, after loading the sample , continue to add PBS, keep the flow rate at 1ml / min, detect the resulting...
Embodiment 3
[0058] The determination of the number of covalent binding molecules of embodiment 3HCPT and AdTRAIL capsid protein
[0059] Make the standard absorption curve of HCPT monomer, determine the number of covalently bound molecules (n=4) between HCPT and the amino group on the AdTRAIL capsid protein by calculating the recovery rate of unreacted HCPT, and the number of unbound HCPT molecules according to the standard of the regression equation curve to calculate. In the above experiments, methanol was used as a blank. The amount of HCPT reaction is calculated based on its UV absorption value at 375nm. The standard curve regression equation established based on the UV absorption value of HCPT at 375nm (methanol solution): Y(OD)=112.02X(μg / mL)-1.2959(R 2 =0.9994), its linear range is 0.0018~15.18μg / mL, calculate the HCPT content in the eluate. Subtract the HCPT content in the purified column eluate from the amount of HCPT initially participating in the reaction to obtain the amoun...
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