Research paper
Extending the structure−activity relationship study of marine natural ningalin B analogues as P-glycoprotein inhibitors

https://doi.org/10.1016/j.ejmech.2016.09.070Get rights and content

Highlights

  • A series of novel ningalin B analogues were synthesized.

  • A ring and its two methoxy groups are important pharmacophores.

  • Compound 23 shows most potent P-gp modulator in reversing paclitaxel resistance.

  • Compound 23 restores intracellular antitumor drug accumulation by inhibiting P-gp.

Abstract

In the present study, a total of 25 novel ningalin B analogues were synthesized and evaluated for their P-gp modulating activity in a P-gp overexpressed breast cancer cell line LCC6MDR. Preliminary structure-activity study shows that A ring and its two methoxy groups are important pharmacophores for P-gp inhibiting activity. Among all derivatives, 23 is the most potent P-gp modulator with EC50 of 120–165 nM in reversing paclitaxel, DOX, vinblastine and vincristine resistance. It is relatively safe to use with selective index at least greater than 606 compared to verapamil. Mechanistic study demonstrates that compound 23 reverses P-gp mediated drug resistance by inhibiting transport activity of P-gp, thereby restoring intracellular drug accumulation. In summary, our study demonstrates that ningalin B analogue 23 is a non-cytotoxic and effective P-gp chemosensitizer that can be used in the future for reversing P-gp mediated clinical cancer drug resistance.

Introduction

P-glycoprotein (P-gp or ABCB1) belongs to the ATP-Binding Cassette (ABC) super-family of proteins [1]. Overexpression of P-gp in cancer patients has been correlated with chemoresistance and poor prognosis. It can pump various anticancer drugs out of cancer cells and reducing intracellular drug concentration below their therapeutic levels [2], [3], [4], [5]. P-gp has also been found cancer stem cells that are highly drug resistant [6]. Considerable effort has been spent on developing P-gp inhibitors [7], [8], [9], [10], [11], [12], [13], [14]. However, only a few non-toxic and specific P-gp inhibitors have been found [15], [16], [17], [18], [19], [20] and none of these inhibitors can be used clinically. Therefore, searching for novel P-gp inhibitors with low toxicity and high potency remains an important approach to reverse clinical multidrug resistance (MDR).

Although P-gp was the most characterized ABC family member in terms of its structure and mechanism of action [3], [21], [22], its exact binding sites of inhibitors remains elusive. Recently, non-toxic natural products including curcumin [23], kaempferol [24], quercetin [25], epigallocatechin gallate [26], lamallarine K, lamallarine I, lamallarine O [27] and their derivatives or analogues such as quercetin pentamethyl ether [28], permethyl lamallarine D, permethyl ningalin D and permethyl ningalin B (shown in Fig. 1) [29], [30], [31] have been discovered as a novel source for providing new P-gp modulators [10]. We also have synthesized analogues of natural marine product ningalin B and characterized their P-gp inhibitory activity. We found that four ningalin B analogues had potent P-gp inhibitory activity, were safe to use and specific towards P-gp (compounds 14 in Fig. 1) [32], [33], [34]. Structure-activity relationship study revealed that substituent at C-ring and the linkers between N atom of pyrroledione and C-ring were important. Compounds 3 and 4 were the most potent and non-cytotoxic lead compounds. In this report, we will study the effects of substituent in rings A and C on P-gp modulating activity.

Section snippets

Chemistry

As shown in Scheme 1, three N-substituted phthalimide derivatives and two N-substituted 3-arylpyrroledione derivatives were synthesized. Phthalimide was reacted with 3,4,5-trimethoxybenzylmethanesulfonate,2-bromo-1-(4-methoxyphenyl)ethanone, and 2-bromo-1-(4-benzoloxy-3-methoxyphenyl)ethanone in the presence of K2CO3 in DMF to afford compounds 6, 7, and 8 respectively. Synthetic compound 9 [35] was coupled with 2-bromo-1-(4-benzoloxy-3-methoxyphenyl)ethanone in the presence of K2CO3 in DMF to

Conclusion

In the present study, a total of 25 novel ningalin B analogues were synthesized and characterized for their P-gp modulating activity in a P-gp overexpressed breast cancer cell line LCC6MDR. Several important pharmacophores for modulating P-gp were found including (1) phenyl rings A and B, (2) di-methoxylation at rings A and (3) tri-substitution at ring C with ortho-bromo, meta-methoxy and para-trimethoxybenzyloxy groups. Among all ningalin B derivatives, 23 with dimethoxy groups at rings A and

General

All non-aqueous reactions were carried out under nitrogen atmosphere in freshly distilled anhydrous solvents. Starting materials and reagents were reagent-grade and were used without further purification unless otherwise stated. Solvents were dried according to standard procedures. Analytical thin-layer chromatography (TLC) was performed on pre-coated plates (silica gel 60 F254) purchased from Merck KGaA and they were visualized under short (254 nm) and long (365 nm) UV light. Column

Materials for biological studies

DMSO, verapamil, cyclosporine A, doxorubicin (DOX), paclitaxel (PTX), vinblastine, vincristine and rhodamine 123 were purchased from Sigma-Aldrich. Dulbecco's modified Eagle's medium (DMEM), trypsin-ethylenediaminetetracetic acid (EDTA), and penicillin/streptomycin were from Gibco BRL. Fetal bovine serum (FBS) was from Hyclone Laboratories. 2-(4,5-Dimethylthiazol-2-yl-)-5-[3-(carboxymethoxy)phenyl]- 2-(4-sulfophenyl)-2H-tetrazolium (MTS) and phenazine methosulfate (PMS) were purchased from

Acknowledgment

This project was funded by NSFC-Shandong Joint Fund for Marine Science Research Centers (U1406402), National Natural Science of China (NSFC 81172926), Project of Strategic Importance (1-ZE22) of the Hong Kong Polytechnic University and the General Research Fund (PolyU 5606/12 M) of the Research Grant Council of Hong Kong.

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    These two authors contribute equally to this work.

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