Elsevier

European Journal of Medicinal Chemistry

Volume 143, 1 January 2018, Pages 1919-1930
European Journal of Medicinal Chemistry

Research paper
Design and synthesis of novel thiobarbituric acid derivatives targeting both wild-type and BRAF-mutated melanoma cells

https://doi.org/10.1016/j.ejmech.2017.11.006Get rights and content

Highlights

  • A series of novel thio- and seleno-barbituric acid derivatives were synthesized.

  • The cytotoxicity was evaluated against wild-type and BRAF-mutated melanoma cells.

  • 2b and 8b most effectively inhibited viability of melanoma cells and induced apoptotic cell death.

  • 2b and 8b were effective towards melanoma cells with both wild-type and mutant BRAF.

  • 2b, 2d, and 8b also effectively inhibited viability of other tumor cell lines.

Abstract

A series of novel thio- and seleno-barbituric acid derivatives were synthesized by varying the substituents at N1 and N3 (ethyl, methyl, allyl, and phenyl), and C5 tethered with dienyl and trienyl moieties attached to substituents such as phenyl, 2-furanyl, 2-thiophenyl, 1-naphthyl, and 3-pyridyl. The cytotoxic potential of these derivatives was evaluated by using MTT assay against melanoma cell lines expressing either wild-type (CHL-1) or mutant (UACC 903) BRAF gene. Among all, 2b and 8b were identified as the most potent compounds. Both 2b and 8b inhibited viability of various melanoma cells and induced cell death as evidenced by Live and Dead assay. Western blot analysis showed that they induce PARP cleavage and inhibit anti-apoptotic Bcl-2, Bcl-xL and Survivin in a dose-dependent manner within 24 h of the treatment. Novel thiobarbituric acid analogs also inhibited viability of various other solid tumor cell lines, such as pancreatic, breast, and colon. Overall, 2b, 2d, and 8b emerged as the most effective compounds and make good leads for the development of future therapeutic agents.

Introduction

Cancer is one of the leading causes of death worldwide and accounted for 8.2 million deaths (nearly 14.6% of all deaths) in 2012 [1]. Deaths from cancer worldwide are projected to continue to rise to over 13 million annually within next two decades. In the United States alone, an estimated 1,685,210 new cancer cases are diagnosed with estimated deaths of 595,690 in 2016 [2]. Among all cancers, melanoma is one of the most deadly form of skin cancer with 2 million new cases annually in the United States [2]. If diagnosed and removed while it is still thin and limited to the outermost skin layer, it is almost 100% curable. However, no effective therapy is available for treatment once the cancer advances and metastasizes to other parts of the body. Commonly used drugs, dacarbazine and temozolomide, in melanoma patients provide unsatisfactory outcomes. The BRAFV600E inhibitor PLX4032 (vemurafenib) [3] (Fig. 1) has proved to be a remarkable drug but develops resistance in about 7 months, and also works only on tumors with BRAFV600E mutations. Most patients require multiple lines of therapy as cancer cells acquire resistance against the chemotherapeutic agents by different mechanisms. The combination therapies with BRAF and MEK inhibitors such as Cobimetinib or Trametinib (Fig. 1) also did not show much improvement [4]. Management of patients with resistance continues to present an extensive confront to researchers and oncologists. Many clinically effective anti-cancer drugs, including those for melanoma used today, suffer from serious drawbacks as majority of patients with metastatic solid tumors die after chemotherapeutic regimens failed to induce objective response or when resistance developed [5]. Therefore, to combat this resistance, still there is an impetus to identify and develop more potent therapeutic agents with fewer side effects in cancer therapy.

In recent years, thiobarbituric acids have been reported to exhibit a broad spectrum of biological properties such as anti-convulsant [6], anti-inflammatory [7], [8], anti-bacterial [9], anti-HIV [10], anti-hypnotic [11], anti-parkinsonian [12], and anti-cancer activities [13], [14], [15], including anti-melanoma properties [16], [17]. Among them some thiobarbituric acid derivatives, such as merbarone [18], merocyanine 540 [19] and its selenium analog selenomerocyanine 56 [20] (Fig. 1) have been identified as possible modulators of apoptosis in several cancer cells, including glioblastoma multiforme (GBM), melanoma, leukemia, and cancers of breast, lung, prostate, and cervix; albeit not particularly for cancers that are resistant to current therapy. An effective lead thiobaribituic acid analog 2a (Scheme 1) was recently identified in our laboratory from a library of 50,000 chemicals, originally screened to identify compounds that could target temozolomide (TMZ) resistant brain tumor cells. The lead compound 2a was effective on both TMZ resistant and sensitive glioma cells [21]. Interestingly, our preliminary studies showed 2a to also possess activity towards human melanoma cells. Therefore, we hypothesized that the structure of 2a can be modulated by varying substitutions at N1, N3, and C5 positions to create a potent compound for melanoma treatment (Fig. 2). Therefore, in the present study, we conducted a structure-activity relationship (SAR) study to further optimize 2a structure to develop an efficacious novel thio-/seleno-barbituric acid compound for melanoma and possibly other solid tumors. In addition, 2a was considered as a valuable lead compound owing to its selective cytotoxicity to various cancer cells including melanoma, while sparing the normal cells. We synthesized 24 novel thio- and selenobarbituric derivatives by varying the substituents at N1, N3, and C5 tethered with dienyl and trienyl moieties attached with different substituents like phenyl, 2-furanyl, 2-thiophenyl, 1-naphthyl and 3-pyridyl (Scheme 1, Scheme 2, Scheme 3). The efficacy of novel analogs was evaluated in a diverse panel of cancer cell lines and the two best identified compounds 2b and 8b, were further characterized in both wild-type and BRAFV600E mutant melanoma cells to get an insight into their mechanism of action.

Section snippets

Design

The lead thiobarbituric acid analog 2a was found to be effective on both BRAF-mutant and wild-type melanoma cells. We hypothesized that careful manipulation of 2a could lead to more effective analogs for a broader range of melanomas as well as other cancers. In this regard, we modified various functionalities by manipulating substitutions at N1-, N3- and C5-positions of 2a as well as by replacing S by Se (Fig. 2). While choosing substitutions, we also made sure that the resulting modified

Conclusion

We synthesized a series of thio- and seleno-barbituric acid analogs with a variety of substitutions at N1, N3, and C5 positions. Many of these compounds inhibited viability of both BRAF wild-type (CHL-1) and mutant (UACC903) human melanoma cells. Based on the SAR study compounds 2b, 2d, and 8b were found to be the most effective analogs. Live/Dead assay showed compound 2b to be more effective than 8b in inducing cell death; causing nearly 65–70% cell death at 25 μM concentration in human

General

Melting points were recorded on a Fischer-Johns melting point apparatus and are uncorrected. NMR spectra were recorded using a Bruker Avance 500 MHz spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) downfield from the internal standard. The signals are quoted as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublet) and dt (doublet of triplet). HRMS were determined at the Chemistry Instrumentation Center, State University of New York

Acknowledgement

The authors thank the Department of Pharmacology, Penn State College of Medicine, and Penn State Cancer Institute for financial support. This study was also supported partially by NIH's NCI Grant R21 CA167406-02 & Elsa U. Pardee Foundation Grant (SYL). The authors thank Dr. Jyh-Ming Lin, Solution Phase NMR Facility at Core Research Facilities of the Penn State College of Medicine for recording NMR spectra.

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    1

    Current address. Department of Biomedical Sciences, Cooper Medical School of Rowan University, 401 S Broadway, Camden, NJ 08103.

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