Synthesis and biological evaluation of 7-methoxy-1-(3,4,5-trimethoxyphenyl)-4,5-dihydro-2H-benzo[e]indazoles as new colchicine site inhibitors
Graphical abstract
Introduction
Microtubule-targeting agents (MTAs) could not only treat cancers by inhibition of mitosis, but also acting on interphase cells to affect traffic on the microtubule, cell migration and invasiveness, immunomodulatory, vascular disruption, and so on.1, 2, 3, 4, 5, 6 This is why MTAs was described as ‘targeted chemotherapy’ by some researchers, and this research field kept active.7, 8 MTAs include inhibitors interacting with four binding sites: the laulimalide, taxane/epothilone, vinca alkaloid, and colchicine sites. In recent years, a colchicine site inhibitor (CSI) combretastatin A-4 (CA-4) (Fig. 1A) showed promising prospects and several analogs have entered clinical trials.9, 10, 11, 12 However, none of them has been approved so far, prompting the development of new CSIs to treat cancers.13, 14, 15, 16, 17
The basic pharmacophores of the CSIs consist of two hydrophobic rings A (often trimethoxyphenyl) and B linked with a bridge double bond in the cis-configuration (Fig. 1A). They might undergo cis–trans isomerization of the double bond to generate the isomers with significantly reduced biological activity.3, 9, 16 To eliminate this problem of instability, a novel type of CSI with the scaffold 1-phenyl-4,5-dihydro-2H-benzo[e]indazole, represented by compound A1 (Fig. 1B), was found in our previous study. The bioactive configuration of these compounds was locked by cyclization between the bridge double bond and ring A, and their binding mode to tubulin was revealed by the X-ray co-crystal structure. This type of CSI showed good antitumor activity in vitro and in vivo.16 It encouraged us to study the new CSIs represented by compound C1, of which the bioactive configuration was locked by cyclization between the bridge double bond and ring B (Fig. 1B). The antitumor activity, primary structure-activity relationship (SAR) and docking binding mode of these new CSIs were explored.
Compound C1 was synthesized by the method showed in Scheme 1. Key intermediate 1 was synthesized from 2,6-dimethoxynaphthalene by the method we reported before.17, 18 Then it was reacted with 3,4,5-trimethoxybenzoyl chloride (2) under the condition of LDA in anhydrous THF to generate 1,3-diketone intermediate 3. It could undergo cyclization reaction by stirring with hydrazine hydrate in EtOH to produce title compound. Compounds C2–C5 could be produced by the similar method using different substituted hydrazines (4) to replace hydrazine hydrate in the cyclization reaction. The position of the R substitutes of these compounds was confirmed to be the nitrogen adjacent to the A ring on pyrazole in our previous study.16
The inhibitory activity against tubulin polymerization of presentative compound C1 was evaluated firstly, which showed good activity and better than the templet compound A1 slightly (Table 1). Considered that the template compound showed good inhibitory activity against human colon cancer cell lines, the inhibitory activities against COLO205 of the compound C1 were tested (Table 1). It also showed better activity obviously than compound A1. Then, the primary exploration of the SAR of this type of CSI was carried out, which focused on the substitutes on the pyrazole ring. Introduction of methyl on the pyrazole ring of compound C1 obtained compound C2, which showed improved inhibitory activity against tubulin polymerization slightly (Table 1). In addition, introduction of polar ethoxyl, aromatic phenyl and aliphatic ethyl acetate groups on the pyrazole ring obtained compounds C3, C4 and C5. These three compounds had much weaker activity against tubulin polymerization than compound C1. The inhibitory activities against human colon cancer cell lines COLO205 and HCT-116 of the title compounds were evaluated. They displayed similar activity change corresponding with the inhibitory activity against tubulin polymerization. Compounds C1 and C2 showed best inhibitory activity with IC50 of low nanomolar concentrations.
The SAR of this type of CSI was different with that of templet compounds represented by A1, of which any substitutes on the pyrazole ring would reduce the activity because of the interruption of the formation of the important water-bridged hydrogen bonds.16 In order to understand it better, the predictive binding mode of compound C1 was obtained by molecular docking using crystal structure of the tubulin-compound A1 complex as template (PDB code 5Z4P).16 As displayed in Fig. 2, compound C1 was buried in the binding site deeply than compound A1 slightly, which consistent with its better inhibitory activity against tubulin polymerization. Notably, the pyrazole ring of compound C1 bound to the inside of the pocket contrary to that of compound A1. There is limited room in the pocket left for the substitutes on it, which might be the reason why only the derivative substituted with small group methyl on the pyrazole ring of compound C1 could keep good activity.
Furthermore, compound C1 was sent to undertaken NCI-60 Human Tumor Cell Lines Screen. The screen utilizes 60 different human tumor cell lines, representing leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney cancers, to identify and characterize novel compounds with growth inhibition or killing of tumor cell lines.19, 20 Compound C1 displayed good activity with GI50 < 10 nM against 57 cell lines (Fig. 3), and showed special good activity with TGI < 10 nM against 9 cell lines, such as colon cancer COLO 205, CNS cancer SF-539 and melanoma SK-MEL-5 (full report see Supplementary data). It indicated that this new CSI is a valuable broad-spectrum antitumor compound for further study.
In summary, on the basis of our previous study, new CSIs which the bioactive configuration was locked by cyclization between the bridge double bond and ring B were found. It eliminated the cis-trans isomerization problem, and showed better activity than templet compound. Compounds C1 and C2 displayed the best activity, with tubulin polymerization IC50 of 3.4 and 1.5 μM, and growth IC50 of low nanomolar concentrations against human colon cancer cell lines. The primary SAR was explained by the docking binding mode study. In addition, compound C1 showed excellent broad-spectrum antitumor activity in the NCI-60 Human Tumor Cell Lines Screen. This study provides new promising CSIs for the further development as antitumor agents.
Section snippets
Acknowledgment
We thank the National Natural Science Foundation of China (21572266) and the Shanghai Rising-Star Program of China (16QA1404800) for financial support.
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These authors contributed equally.