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Design and synthesis of novel tamoxifen analogues that avoid CYP2D6 metabolism

https://doi.org/10.1016/j.ejmech.2016.02.026Get rights and content

Highlights

  • Design and synthesis of Tamoxifen analogues bearing ester groups and different basic terminal moieties.

  • Esterified analogues avoids CYP2D6 metabolism ensuring equal clinical outcomes for all patients.

  • Novel analogues showed higher growth inhibition potency than TAM against MCF-7 cell lines.

  • Novel analogues showed a broad spectrum activity over a panel of human cancer cell lines.

  • Novel analogues are more selective towards ER-α than ER-β.

Abstract

Tamoxifen (TAM) is a widely used drug in the prophylaxis and treatment of breast cancer. TAM is metabolized to the more active 4-hydroxytamoxifen (4-OH-TAM) and endoxifen by cytochrome P450 (CYP) mainly CYP2D6 and CYP3A4 enzymes. Due to the genetic polymorphisms in CYP2D6 genes, high variation in the clinical outcomes of TAM treatment is observed among women of different populations. To address this issue, novel TAM analogues with possible altered activation pathways were synthesized. These analogues were tested for their antiproliferative action on MCF-7 breast cancer cell lines as well as their binding affinity for estrogen receptor (ER) ER-α and ER-β receptors. These entire novel compounds showed better antiproliferative activity than did TAM on the MCF-7 cells. Moreover, compound 10 exhibited a half maximal growth inhibition (GI50) that was 1000 times more potent than that of TAM (GI50 < 0.005 μM vs 1.58 μM, respectively). Along with a broad spectrum activity on various cancer cell lines, all the TAM analogues showed considerable activity on the ER-negative breast cancer cell line. For further study, compound 10 was incubated in human liver microsomes (HLM), human hepatocytes (hHEP) and CYP2D6 supersomes. The active hydroxyl metabolite was detected after incubation in HLM and hHEP, implicating the involvement of other enzymes in its metabolism. These results prove that this novel series of TAM analogues might provide improved clinical outcomes for poor 2D6 metabolizers.

Introduction

Breast cancer is considered to be the most common cancer among women worldwide, with 1.7 million women diagnosed with the disease in 2012 [1]. The majority of breast cancer cases (almost 80%) are classified as hormone-dependent cancer, since estrogen, acting via estrogen receptoralpha (ER-α) or estrogen receptor beta (ER-β), is the major inducer of the development and growth of the tumor. These are also called ER-positive breast cancers. The remaining cases are not induced by estrogen and are classified as hormone-independent, or ER-negative, cancers. Since the growth of hormone-dependent cancer cells can be down-regulated by the oppositely active hormones, several endocrine therapies that limit the actions of estrogen have been developed over the past years. These endocrine therapies have played an important part in treating and improving the outcomes of postmenopausal women with all stages of the disease [2]. Selective estrogen receptor modulators (SERMs) have also been studied for their anti-cancer activity. These are chemically diverse compounds that lack the steroidal structure of estrogens yet can bind to ERs [3]. In addition, SERMs exhibit partial agonist and antagonist properties in different tissues and organs [4], [5].

Tamoxifen (TAM) was the first SERM to be utilized in the treatment of metastatic breast cancer and is considered to be among the most effective drug in treating ER-positive breast cancers, either alone or better in combination with aromatase inhibitors. Moreover, the drug reduces the risk of recurrence and death when used as adjuvant therapy in early stage or in metastatic cancer [6]. The efficacy of TAM is derived from its two clinically active metabolites, 4-hydroxytamoxifen (4-OH-TAM) and endoxifen, both of which have a greater affinity towards ER-α and a much higher antiestrogenic potency in breast cancer cells than does the parent drug (Fig. 1).

TAM metabolism and activation to the more active endoxifen is mediated mainly via cytochrome P450 (CYP) enzymes, specifically the CYP2D6 and CYP3A4 isoforms. Other non CYP as UGT and SULT genes are contributing to the clearance of TAM. All these enzymes are polymorphic with many well-characterized variants [7]. CYP2D6 is known to have highly variable enzymatic activity as a result of polymorphisms in the genes that encode the enzyme. Such genetic polymorphisms can lead to the formation of proteins that lack enzymatic activity or to enzymes with reduced activity [5], [8].

The aim of this study was to synthesize new TAM analogues that maintain the main pharmacophoric features of TAM yet are metabolized via a metabolic pathway that does not involve the CYP2D6 enzyme, thus avoiding the genetic polymorphisms of this enzyme and giving more equal clinical benefits to patients. During the course of this work, several structural modifications to TAM were investigated, including substitution of a methyl in place of an ethyl group on the triphenylethylene (TPE) backbone. The effects of such a change on binding affinity to ER-α receptors, growth inhibition of MCF-7 cancerous cell line and selectivity were investigated. In addition, the effect of blocking the metabolic para-hydroxylation on ring C was studied by placing a hydroxyl group or an ester group, namely acetate, butanoate or decanoate. Furthermore, the dimethylaminoethoxy side chain at ring B of TAM that plays a crucial role in TAM antagonistic action [9] was modified to a pyrrolidinylethoxy or a piperidinylethoxy group. The effect of cyclization, size of the cyclic structure and alteration of the basicity of nitrogen were all extensively investigated (Fig. 2). The novel TAM analogues were prepared as depicted in (Scheme 1).

Compounds 1 was synthesized using standard McMurry coupling reaction of 4,4′- dihydroxybenzophenone with acetophenone using titanium tetrachloride/zinc as catalyst to give the triphenyl ethylene backbone in yield 87% [10], [11].

Compound 1 was then treated with the appropriate base hydrochloride salt in the presence of potassium carbonate to form monoalkylated and dialkylated ether derivatives. Products were purified using column chromatography to provide the monoalkylated derivative 24 with yield 45%. The esterification of the monoalkylated compounds was then achieved via using commercially available acid chlorides in a basic medium. Compounds 213 were obtained as 1:1 mixture of E/Z isomers. Attempts to isolate the E/Z isomers using column chromatography as well as preparative HPLC were not successful.

13C NMR confirmed the formation of isomers since most of the signals were duplicated. Such duplication of signal has been previously reported by Bedford and Richardson [12].

Section snippets

Results and discussion

All novel TAM analogues were tested for their antiproliferative effect on MCF-7 cell lines and for their binding affinity to ER-α. Compound 8, the esterified analogue with the highest ER-α binding affinity (half maximal inhibitor concentration [IC50] = 0.0077 μM), and compound 3, its hydroxyl congener (IC50 = 0.0008 μM), were additionally tested for their binding affinity to ER-β. The differential selectivities between ER-α and ER-β were calculated for these two compounds (Table 1).

All of the

Conclusion

We designed and synthesized a series of TAM analogues that are metabolized via activation pathways that do not involve the CYP2D6 enzyme, thus avoiding the variability in response caused by genetic polymorphism of this enzyme. All these novel compounds have a better antiproliferative effect on MCF-7 cells than does TAM. Moreover, these novel, rigid analogues show improved activity and high selectivity towards ER-α receptors compared to TAM. It is worth to mention that for compounds 3, 8 and

Acknowledgements

The authors are deeply grateful to the authority of the National Cancer Institute, USA, for the antitumor screening. This project was supported financially by the Science and Technology Development Fund (STDF), Egypt, Grant No: 5386.

The authors are deeply grateful to Dr. Bianca Liederer, Dr. Peter Fan and Ms. Teresa Mulder, Drug Metabolism & Pharmacokinetics, Genentech, South San Francisco, CA for their support in the metabolite ID study.

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