Research paper
Discovery of novel CA-4 analogs as dual inhibitors of tubulin polymerization and PD-1/PD-L1 interaction for cancer treatment

https://doi.org/10.1016/j.ejmech.2020.113058Get rights and content

Highlights

  • A series of novel CA-4 analogs as dual inhibitors of tubulin polymerization and PD-1/PD-L1 were discovered.

  • TP5 exhibited strong antiproliferative effects against five cancer cell lines with an IC50 of 800 nM in HepG2 cells.

  • TP5 inhibited tubulin polymerization, suppressed HepG2 cell migration, arrested cell at G2/M phase, and induced apoptosis.

  • TP5 exhibited moderate anti-PD-1/PD-L1 activity with IC50 of 48.76 μM in an HTRF assay.

  • TP5 suppressed tumor growth in an immune checkpoint humanized mouse model with a TGI of 57.9% at 100 mg/kg.

Abstract

A series of novel CA-4 analogs as dual inhibitors of tubulin polymerization and PD-1/PD-L1 were designed, synthesized and bio-evaluated. Among them, compound TP5 exhibited strongest inhibitory effects against five cancer cell lines with an IC50 value of 800 nM in HepG2 cells. In addition, mechanism studies revealed that TP5 could effectively inhibit tubulin polymerization, suppress HepG2 cells migration and colony formation, and cause cell arrest at G2/M phase and induce apoptosis. Furthermore, TP5 exhibited moderate anti-PD-1/PD-L1 activity with IC50 values of 48.76 μM in a homogenous time-resolved fluorescence (HTRF) assay. In vivo efficacy studies indicated that TP5 could significantly suppress tumor growth in an immune checkpoint humanized mouse model with a Tumor Growth Suppression (TGI) of 57.9% at 100 mg/kg without causing significant toxicity. Moreover, TP5 did not cause in vivo cardiotoxicity in BALB/c mice. These results suggest that the novel CA-4 analogs may serve as a starting point for developing more potent dual inhibitors of tubulin polymerization and PD-1/PD-L1.

Introduction

Cancer has become the second leading cause of death and a major public health problem globally [1]. Current molecularly targeted therapies aim to bind selectively to a single biological entity (e.g. a protein) to avoid unwanted off-target effects. However, cancer is a complex disease with multiple signaling pathways dysregulated, therefore, single-target drugs cannot adequately achieve therapeutic effects. This provoked the search for therapeutics with multi-targeting capabilities [2]. Compared with single-target drugs, multi-target therapeutics can act on more than one target, thus achieve greater efficacy and are less vulnerable to drug resistance [3,4].

Recent studies showed that the combination of anti-PD-1/PD-L1 antibodies and cytotoxic agents (e.g. tubulin inhibitors: paclitaxel and BNC105) can achieve synergistic effects and better antitumor efficacy, and that the combination therapy could be safer than chemotherapeutic agents alone [[5], [6], [7], [8]]. However, combination therapy has several drawbacks including unpredictable pharmacokinetics (PK) and pharmacodynamics (PD) with a mixture of two or more drugs. An alternative approach is to design a single molecule with the capability to act on multiple validated cancer drug targets. It is relatively easy to predict the PK and PD of a single molecule. In addition, single molecules are more amenable to structural modifications which offers the added advantage of performing structure−activity relationship studies more rapidly.

We have previously described the discovery of several classes of Combretastatin A-4 (CA-4)-based tubulin inhibitors [9,10] and resorcinol dibenzyl ether-based PD-1/PD-L1 inhibitors [11,12], which showed potent anticancer activities in vitro and in vivo. Microtubules play a key role in the division and proliferation of tumor cells, and have been targeted by many anti-tumor drugs such as taxanes and vinca alkoloids [13]. CA-4, as a representative tubulin inhibitor binding at the colchicine site, exhibits potent antitumor activities in various cancer cell lines [14]. However, the further development of CA-4 was hindered by the poor water solubility, toxic side effects and P-gp mediated multi-drug resistance (MDR) [15]. As for cancer immunotherapy, it is one of the most actively pursued areas in anticancer drug discovery because of its high selectivity and low toxicity [16]. Currently, PD-1/PD-L1 is one of the most promising targets in the field of immuno-oncology [17]. In the past decades, numerous PD-1/PD-L1 inhibitors have been discovered, including small molecules, peptidomimetics, and monoclonal antibodies [18,19].

We carefully analyzed the pharmacophores of the small molecule PD-1/PD-L1 inhibitor BMS-200 and the tubulin inhibitor CA-4, with four pharmacophoric points (core group, linker, aryl group and tail group) identified for the PD-1/PD-L1 inhibitors (Fig. 1) [11,12,20,21]. We found that CA-4 and the core group (red colored structural moieties in Fig. 1) of BMS-200 have similar physicochemical properties (e.g. Log P and tPSA), and that large substitutions on the hydroxyl group of CA-4 is tolerable based on the docking analysis [10], this inspired us to design dual inhibitors of tubulin and PD-1/PD-L1 through a hybridization strategy. With this in mind, we designed, synthesized and bio-evaluated 20 novel CA-4 analogs as dual-acting tubulin and PD-1/PD-L1 inhibitors. Among them, compound TP5 displayed high anti-proliferative potency against a panel of cancer cell lines as well as moderate anti-PD-1/PD-L1 activity, making it a promising lead compound for future development.

Section snippets

Chemistry

The synthesis of CA-4 analogs TP1–20 is outlined in Scheme 1. Briefly, the hydroxyl group of 4-(hydroxymethyl)benzaldehyde compound 1 was brominated by tribromoborane to give 4-(bromomethyl)benzaldehyde intermediate M1. Then the benzyl bromide moiety of intermediate M1 was reacted with the hydroxyl group of CA-4 to yield the benzylated CA-4 analog M2. Finally, the NaBH3CN-mediated reductive amination was applied to convert M2 and various amines to the desired compounds TP1–20 according to a

Conclusions

In summary, novel CA-4 analogs based on PD-1/PD-L1 inhibitor and CA-4 were synthesized as dual inhibitors of tubulin and PD-1/PD-L1. Among them, compound TP5 showed strongest inhibitory effects against five cancer cell lines with an IC50 value of 0.8 μM in HepG2 cells and negligible cytotoxicity to normal cells (IC50 > 40 μM for HEK-293 and NCM460). In addition, mechanism of action studies suggest that TP5 exerted its effects by inhibiting tubulin polymerization, suppressing HepG2 cell

Experimental section

General methods. Reagents and solvents were obtained from commercial sources and used without further purification. CA-4 was purchased from InvivoChem (Libertyville, IL 60048, USA) and BMS-202 was purchased from Targetmol. All the reactions were monitored by TLC using silica gel GF/UV 254. All melting points were measured using a X-5 micro melting point apparatus. Flash chromatography was performed using silica gel (300–400 mesh). The 1H and 13C NMR spectra were recorded on Bruker AV-400

Synthesis of compounds TP1–20

(Z)-1-(4-((2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)methyl)benzyl)piperidine-2-carboxylic acid (TP1). The title compound was obtained as light yellow solid (3.9 mg, 15% yield); mp: 166.9–168.2 °C; 1H NMR (400 MHz, DMSO) δ 7.32 (d, J = 8.1 Hz, 2H), 7.27 (d, J = 8.0 Hz, 2H), 6.97–6.91 (m, 2H), 6.87 (dd, J = 8.4, 1.5 Hz, 1H), 6.56 (s, 2H), 6.52–6.44 (m, 2H), 4.83 (s, 2H), 3.86 (d, J = 13.4 Hz, 1H), 3.75 (s, 3H), 3.63 (d, J = 2.5 Hz, 9H), 3.50 (d, J = 14.0 Hz, 1H), 3.08 (dd, J = 7.6, 4.0 Hz,

Supporting information

1H NMR, 13C NMR, HRMS, and HPLC spectra.

Notes

The authors declare no competing financial interest.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by scientific research project of high level talents (No. C1051008) in Southern Medical University of China; and International Science and Technology Cooperation Projects of Guangdong Province (No. G819310411).

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