| Vascular disrupting agents have a novel class of antitumor drugs targeting at tubulin and some candidates have been in clinical trials.In our prior studies,a new kind of leads with scaffold of N-(4-Quinazoline)-3,4-dihydroquinoxaline-2-onewas discovered as a novel kind of VDAswith very high potency of anti-proliferative activity in cellular with GI50 values at low nanomolar level.As a continue study,we focused on modifications of the 2-position substituents on the quinazoline ring with a strong purposiveness to get new high potent compounds withimproved drug-like properties as new potential VDA drug candidates.In the path of modification,considering the structure–activity relationship in known compounds and drawing on the experience of our past studies,we kept the quinazoline ring,lactam ring and the 7-position methoxy group which enhance the activity.In the next step,the reactant must be changed and the synthesis should be redesigned from the very beginning,since we decided to replace the methoxy on 2-position on quinazoline ring with chlorine.In the new design,the most tough problem was to reduce the nitro group into amino group,as the original method could not give pure product.Acetic acid/Zinc powder was finally chosen as the new reaction system,providing a stable,high yield,mild reaction condition and reducing the difficulty of the whole path.According to the docking,we assumed that a pocket setting for compound 12 c should exist.Therefore,we chose some large groups to verifyour theory after we got the key intermediate:4-(2-chloroquinazolin-4-yl)-7-methoxy-3,4-dihydroquinoxalin-2(1H)-one,such as n-butylamine,cyclopropylamine and 3,3-difluoropyrrolidine.Then we received a brilliant result: all of the compound showed a great anti-proliferative activity in the first set of cellular assay.Furthermore,as shown in the results,the drug-like properties of most compounds,particularly the aqueous solubility,were better than that of the lead compound,especially the 16 e : 81 μg/m L,which was a good point to verify our assumption while the Log P of all the compound except 16 d was between 1 to 3,resulting a better intestinal absorption.We picked a number of high anti-proliferative activity compounds for tubulin assembly and colchicine binding assays(16a-g,16 i,16j,16q),taking the CA-4 as the positive control.Consistent with our previous results,compounds 16 c,16d,16 g,16j,and 16 q were highly active in the tubulin assembly assay with low to submicromolar inhibitory IC50 values(0.42 to 0.77 μM),comparable with those of 12c(0.77 μM)and CA-4(0.54 μM)in the same assay.The molecular simulation with high-resolution stathmin-4 protein crystal structure was used to seek the binding site.Compound4-(2-(3-hydroxypropylamino)quinazolin-4-yl)-7-methoxy-3,4-dihydroq uinoxalin-2(1H)-one(16e)and lead compound 12 c were docked at the colchicine binding site with the tubulin crystal structure(PDB code: 5LYJ).It showed that molecules 16 e and 12 c overlapped well with the original ligand CA-4 in the binding site,while the outspread C2-side chain in 16 e formed two hydrogen bonds with Tyr 202 and Asp 251.These findings supported the postulate that,similar to CA-4,this compound series targeted the tubulin-colchicine binding site.As a kind of cytotoxicity compounds,this series was born with limitations and faults.Wide anti-tumor spectrum also means low specificity,and high activity sometimes equals toxic.We walked on the glass while there was some good news: the reversibility of the binding between VDA and tubulin led to lower toxicity of compounds and the drug-like properties were better than ever,though specificity was still to be solved.Complementation between this series and paclitaxel may occur,or these compounds replace paclitaxel,owing to the progress of resisting recurrent malignant glioma in clinical trials. |
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