Synthesis and biological evaluation of flavones and benzoflavones as inhibitors of BCRP/ABCG2

doi:10.1016/j.ejmech.2013.06.035

European Journal of Medicinal Chemistry

Volume 67, September 2013, Pages 115-126

https://doi.org/10.1016/j.ejmech.2013.06.035 Get rights and content

Highlights

•
Investigation of flavones, 7,8- and 5,6-benzoflavones for BCRP inhibition.
•
7,8-Benzoflavones are potent inhibitors of BCRP.
•
Selective BCRP inhibitors and one broad spectrum inhibitor found.
•
Little cytotoxicity of selected compounds.

Abstract

Multidrug resistance (MDR) often leads to a failure of cancer chemotherapy. Breast Cancer Resistance Protein (BCRP/ABCG2), a member of the superfamily of ATP binding cassette proteins has been found to confer MDR in cancer cells by transporting molecules with amphiphilic character out of the cells using energy from ATP hydrolysis. Inhibiting BCRP can be a solution to overcome MDR. We synthesized a series of flavones, 7,8-benzoflavones and 5,6-benzoflavones with varying substituents at positions 3, 3′ and 4′ of the (benzo)flavone structure. All synthesized compounds were tested for BCRP inhibition in Hoechst 33342 and pheophorbide A accumulation assays using MDCK cells expressing BCRP. All the compounds were further screened for their P-glycoprotein (P-gp) and Multidrug resistance-associated protein 1 (MRP1) inhibitory activity by calcein AM accumulation assay to check the selectivity towards BCRP. In addition most active compounds were investigated for their cytotoxicity. It was observed that in most cases 7,8-benzoflavones are more potent in comparison to the 5,6-benzoflavones. In general it was found that presence of a 3-OCH₃ substituent leads to increase in activity in comparison to presence of OH or no substitution at position 3. Also, it was found that presence of 3′,4′-OCH₃ on phenyl ring lead to increase in activity as compared to other substituents. Compound 24, a 7,8-benzoflavone derivative was found to be most potent being 50 times selective for BCRP and showing very low cytotoxicity at higher concentrations.

Graphical abstract

Introduction

Cancer is the main cause of millions of deaths worldwide. Since decades chemotherapy has been a major form of the treatment for different types of cancers. Unfortunately, the majority of cancers are either resistant to chemotherapy or acquire multidrug resistance (MDR) during treatment. MDR is an acquired drug resistance by tumour cells that consists of simultaneous emergence of cellular resistance to toxic action of chemotherapeutic drugs originally used and to other chemicals having different chemical structure and mechanism of action. As a result of MDR, chemotherapeutic agents fail to target the tumour cells and cancer becomes untreatable by chemotherapy [1], [2].

One of the mechanisms by which tumours develop multidrug resistance is over expression of efflux transport proteins, especially certain ATP-binding cassette (ABC) transporters in the plasma membrane of cancer cells. ABC transporters utilize energy obtained from hydrolysis of ATP to efflux chemically unrelated compounds [3], [4], [5]. Until now 48 ABC transporters have been identified in humans.

P-glycoprotein (P-gp) and the multidrug resistance associated protein 1 (MRP1) belonging to subfamily ABCC have been shown to confer resistance to a broad spectrum of chemotherapeutic agents. More recently BCRP (breast cancer resistance protein) which is also known as ABCG2 has been discovered and proved to cause resistance in tumour cells too.

Although BCRP was first cloned in doxorubicin resistant MCF-7 breast cancer cells [6], later it has been also found to confer resistance to mitoxantrone (hence named MXR) [7] and subsequently it was found in the placenta (hence named ABCP) [8]. BCRP is an ABC transporter protein consisting of 655 amino acids and having a molecular weight of 72 kDa. It is a half transporter containing a single NH₂-terminal nucleotide binding domain (NBD) followed by six transmembrane domains (TMD). It has been proposed that, to achieve its functionality, BCRP needs to be homo- or heterodimerized [6], [7]. BCRP has been found to be present in many normal tissues other than in tumour cells, including apical membrane of placental syncytiotrophoblasts, endocrine cells of the pancreas, epithelial cells in the small intestine and colon, liver caniculi, blood–brain and blood–testis barrier, gallbladder epithelium and stem cells [8], [9], [10]. In normal tissues BCRP protects the body from various endogenous and exogenous toxins.

Several substrates of BCRP have been identified, which include anticancer drugs such as mitoxantrone [11], podophyllotoxins etoposid and teniposid, flavopiridol [12], topotecan, irinotecan [13] and its active metabolite SN-38. To overcome MDR acquired by over expression of BCRP several efforts have been made by investigating inhibitors of BCRP. These structurally unrelated compounds include naturally occurring flavonoids [14], [15], [16], [17], broad spectrum inhibitors like tariquidar [18] and elacridar, chromons [19] and methoxy-stilbenes [20]. Several research groups have investigated chalcones (precursor of flavonoids) for their inhibitory effect [21], [22], [23], [24]. Fumitremorgin C was found to be a very potent inhibitor of BCRP [13], but its use was limited due to neurotoxicity. Its non-toxic analogue Ko143 is the most potent and selective inhibitor of BCRP known today.

Recently a flavonoid, the 7,8-benzoflavone was identified as very potent inhibitor of BCRP [15], [25]. To further investigate benzoflavones for their potential to inhibit BCRP, we synthesized several 7,8- and 5,6-benzoflavones bearing varying substitutions at positions 3, 3′ and 4′. Also several substituted synthetic flavones were investigated to allow comparison of their inhibitory potential with that of the benzoflavones. These compounds were checked for their inhibitory effect on BCRP in Hoechst 33342 and pheophorbide A accumulation assays. The compounds were also investigated for their effect on P-gp and MRP1. Compound 24, a 7,8-benzoflavone bearing three methoxy substituents at positions 3, 3′,4′ was found to be most potent having an inhibitory concentration only about 2 fold less than Ko143 which is the most potent specific BCRP inhibitor [26], while being almost 3 fold more potent than the unsubstituted 7,8-benzoflavone.

Section snippets

Chemistry

In the current study we synthesized and investigated several flavones (1–13), 7,8-benzoflavones (14–25) and 5,6-benzoflavones (26–34). Flavones (1–9) and both type of benzoflavones were synthesized from their precursor chalcones and benzochalcones. The precursors were synthesized by Claisen–Schmidt condensation using substituted acetophenones and benzaldehydes in presence of NaOH or LiOH as base in ethanol as reported earlier [23]. The synthesis of the studied compounds is depicted in Scheme 1

Conclusion

Naturally occurring as well as synthetic flavonoids have been shown to inhibit BCRP and P-gp in several studies. In the current study we synthesized and investigated several 7,8-benzoflavones, 5,6-benzoflavones along with comparison to multisubstituted flavones for their BCRP inhibition potential. All the compounds were evaluated using two different substrates of BCRP. From the activity data obtained in the Hoechst 33342 and pheophorbide A assays we were able to propose the effect of

Chemistry

The general synthetic route to target flavones and benzoflavones is depicted in Scheme 1 and Scheme 2. Syntheses and characterization of precursor chalcones and benzochalcones have been described earlier [23]. Synthesis of final flavones and benzoflavones was carried out as reported earlier with small modifications [27], [29].

All chemicals were purchased from Acros Organics, Alfa Aesar or Sigma–Aldrich. During the synthesis reaction progress was monitored using analytical thin layer

Acknowledgements

We would like to thank Dr. A. H. Schinkel (The Netherlands Cancer Institute, Amsterdam, The Netherlands) for kindly providing the MDCK BCRP cell line. K.J. thanks to DAAD (Germany) for doctoral fellowship.

References (35)

L.M. Chan et al.
The ABCs of drug transport in intestine and liver: efflux proteins limiting drug absorption and bioavailability
Eur. J. Pharm. Sci.
(2004)
F. Staud et al.
Breast cancer resistance protein (BCRP/ABCG2)
Int. J. Biochem. Cell Biol.
(2005)
S. Zhang et al.
Structure activity relationships and quantitative structure activity relationships for the flavonoid-mediated inhibition of breast cancer resistance protein
Biochem. Pharmacol.
(2005)
A. Pick et al.
Structure-activity relationships of flavonoids as inhibitors of breast cancer resistance protein (BCRP)
Bioorg. Med. Chem.
(2011)
J. Yuan et al.
Synthesis of methylated quercetin derivatives and their reversal activities on P-gp- and BCRP-mediated multidrug resistance tumour cells
Eur. J. Med. Chem.
(2012)
Y. Han et al.
Modulation of breast cancer resistance protein (BCRP/ABCG2) by non-basic chalcone analogues
Eur. J. Pharm. Sci.
(2008)
K. Juvale et al.
Investigation of chalcones and benzochalcones as inhibitors of breast cancer resistance protein
Bioorg. Med. Chem.
(2012)
M. Bennett et al.
Aspects of the Algar–Flynn–Oyamada (AFO) reaction
Tetrahedron
(1996)
G. Casano et al.
Anti-HIV and antiplasmodial activity of original flavonoid derivatives
Bioorg. Med. Chem.
(2010)
H. Muller et al.
New functional assay of P-glycoprotein activity using Hoechst 33342
Bioorg. Med. Chem.
(2007)

A. Pick et al.

Structure–activity relationships of new inhibitors of breast cancer resistance protein (ABCG2)

Bioorg. Med. Chem.

(2008)

H. Mueller et al.

Comparison of the usefulness of the MTT, ATP, and calcein assays to predict the potency of cytotoxic agents in various human cancer cell lines

J. Biomol. Screen.

(2004)

A.T. Fojo et al.

Expression of a multidrug-resistance gene in human tumors and tissues

Proc. Natl. Acad. Sci. U S A

(1987)

W.S. Dalton et al.

Drug-resistance in multiple myeloma and non-Hodgkin's lymphoma: detection of P-glycoprotein and potential circumvention by addition of verapamil to chemotherapy

J. Clin. Oncol.

(1989)

P. Krishnamurthy et al.

Role of ABCG2/BCRP in biology and medicine

Annu. Rev. Pharmacol. Toxicol.

(2006)

O. Polgar et al.

ABCG2: structure, function and role in drug response

Expert Opin. Drug Metab. Toxicol.

(2008)

G. Schmitz et al.

ABC transporters and cholesterol metabolism

Front Biosci.

(2001)

Cited by (81)

Reactivity of Biomass-Derived Olefins with Elemental Sulfur: Mechanistic Insight
2024, European Journal of Organic Chemistry
Lignocellulosic biomass remains underutilized despite its annual production in gigaton quantities. Sulfur is another vastly underutilized waste product of fossil fuel refining. New mechanistic insight into the reactions of sulfur unveiled since 2020 suggest a rich and hitherto unexplored chemistry between biomass‐derived olefins and elemental sulfur. In this study, four biomass‐derived olefins (eugenol (1), 4‐allyl‐2,6‐dimethoxyphenol (2), o‐eugenol (3), and 2‐allyl‐6‐methylphenol (4)) were reacted with elemental sulfur to elucidate the S−C bond‐forming and other reactivity of these compounds. Each of the compounds was reacted with elemental sulfur in three sulfur : organic reactant ratios (2 : 1, 4 : 1 and 9 : 1) and at two temperatures (180 °C or 230 °C). Product mixtures were characterized using ¹H NMR spectrometry and GC‐MS analysis. Products resulting from a range of mechanisms were unveiled, including inverse vulcanization, S−C_{allylic/benzylic} bond formation, S−C_aryl bond formation, intramolecular cyclization, C−C σ‐bond scission, and C−O σ‐bond scission. It is anticipated that the insights from this study will support further synergy between the critical sustainability goals of biomass and sulfur utilization.
Evaluation of oxindole derivatives as a potential anticancer agent against breast carcinoma cells: In vitro, in silico, and molecular docking study
2023, Toxicology in Vitro
In this study we have performed the in vitro anticancer activity of spiro oxindole derivatives against MCF-7 (human Adreno carcinoma) and MDA-MB-231 (triple negative breast cancer) cell lines to propose a possible role of these derivatives in the treatment of cancer. Compound 6, which has an N-benzyl substitution with a chloro group on the indolin-2-one scaffold, had the most potent activity against MCF-7 (3.55 ± 0.49 μM) and MDA-MB-231 (4.40 ± 0.468 μM) of all the synthesized molecules. A normal mouse embryonic fibroblast (NIH/3 T3) cell line was used to test the cellular toxicity of these derivatives. The results showed that none of the compounds were cytotoxic to normal cells. In addition, pharmacokinetic (ADME) and toxicity study profiles were predicted in silico. All the synthesized derivatives (1 to 7) demonstrated the necessary physicochemical properties for bioavailability. Finally, in vitro results of promising compound 6 were validated using molecular docking and dynamic simulation studies, which revealed their binding affinities and conformational stability in the binding cavity. Thus, these derivatives may serve as lead structures for a new generation of anticancer agents.
Current perspectives on benzoflavone analogues with potent biological activities: A review
2022, Arabian Journal of Chemistry
Medicinal chemistry investigators have isolated and synthesized benzoflavone analogues with diverse bioactivities including enzyme inhibitory activity, central nervous system (CNS) activity, anti-inflammatory activity, anti-microorganism activity, hypoglycemic activity, and receptor modulating potential. SAR (Structure-Activity Relationship) studies have been conducted extensively and plenty of benzoflavone analogues have been prepared for potential targets. Herein, we review the pharmacology properties and SAR for benzoflavone analogues, and provide a brief summarization of synthetic strategies, wishing to give perspective on the research and development of benzoflavone derivatives.
Recent advances in the synthesis of 4H-chromen-4-ones (2012 − 2021)
2022, Advances in Heterocyclic Chemistry
Chromones is a family of oxygen heterocyclic compounds whose synthetic versatility and broad spectrum of bioactive properties has been widely reported over the past decade. The increasing number of publications related to the synthesis of this heterocyclic system highlights diverse synthetic approaches, using different starting materials, with novel and efficient synthetic details, applying alternative heating conditions, that provides a huge number of polyfunctionalized 4H-chromen-4-one derivatives. The purpose of this chapter is to present a comprehensive survey of the methodologies developed for the synthesis of 2,3-unsubstituted, 2- and 3-substituted and 2,3-disubstituted 4H-chromen-4-ones, focusing on the literature since 2012, in nearly 450 publications.
Structure-based designing and synthesis of 2-phenylchromone derivatives as potent tyrosinase inhibitors: In vitro and in silico studies
2021, Bioorganic and Medicinal Chemistry
The present study describes the discovery of novel inhibitors of mushroom tyrosinase enzyme. For that purpose, a series of varyingly substituted 2-phenylchromone analogues 1–28 were synthesized and characterized in detail by various spectroscopic techniques (UV–Vis, FTIR, ¹H NMR, ¹³C NMR) and mass spectrometry. All the derivatives (1–28) were screened in vitro for their inhibitory potential against mushroom tyrosinase enzyme. Interestingly, all the synthetic compounds displayed good to excellent inhibitory activity with IC₅₀ values ranging from 0.093 ± 0.003 μg/ml to 23.58 ± 0.94 μg/ml for brominated 3-hydroxy-2-phenylchromones and 0.22 ± 0.017 μg/ml to 22.22 ± 1.1 μg/ml for brominated 2-phenylchromones against tyrosinase in comparison to the standard kojic acid (IC₅₀ = 1.79 ± 0.64 μg/ml). Remarkably, the bromine atoms attached on ring A attribute to increases the inhibitory potential of 2-phenylchromone moiety and anti-tyrosinase assay demonstrated that compound 10 (IC₅₀ = 0.093 ± 0.003 µg/ml) was found almost nineteenfold, 11 (IC₅₀ = 0.126 ± 0.015 µg/ml) fourteenfold and 26 (IC₅₀ = 0.22 ± 0.017 µg/ml) about eightfold more active than the positive control. Notably, among the already literature reported tyrosinase inhibitors, these analogues have been found the most active inhibitors of mushroom tyrosinase with the lowest possible IC₅₀ values. To design and develop novel tyrosinase inhibitors using 2-phenylchromone as a structural motif in the future, a limited structure-activity relationship was established. Moreover, in silico studies were carried out to rationalize the binding mode of interactions of all the targeted compounds (1–28) with the active site of enzymes. The experimental and theoretical results are in parallel with one another. In addition, molecular description was performed with the drug-likeness and bioactivity scores. Computational analysis predicted that few compounds are in a linear correlation with Lipinski’s RO5 indicating superb drug-likeness and bioactivity score for drug targets.
Rational drug design of 6-substituted 4-anilino-2-phenylpyrimidines for exploration of novel ABCG2 binding site
2021, European Journal of Medicinal Chemistry
Citation Excerpt :
Starting from the above stated compounds, our workgroup was able to develop several new potent inhibitors of ABC transporters in general, and of ABCG2 in particular. Until today, compound 5 with its quinazoline scaffold is one of the most potent inhibitors of ABCG2 [26]; Compound 6 with its quinoline scaffold is one of the 20 most potent multitarget ABCB1, ABCC1, and ABCG2 inhibitors [11,15,27–36]; the consequent downsizing of the main scaffold to a pyrimidine led to the discovery of compound 7 as a novel ABCG2 inhibitor [37]; and subsequent development of small-molecule pyrimidines led to one of the most potent reversers of ABCG2-mediated MDR, compound 8 [17]. Fig. 2 shows the stated compounds 5–8.
In the search for novel, highly potent, and nontoxic adjuvant chemotherapeutics to resolve the major issue of ABC transporter-mediated multidrug resistance (MDR), pyrimidines were discovered as a promising compound class of modern ABCG2 inhibitors. As ABCG2-mediated MDR is a major obstacle in leukemia, pancreatic carcinoma, and breast cancer chemotherapy, adjuvant chemotherapeutics are highly desired for future clinical oncology. Very recently, docking studies of one of the most potent reversers of ABCG2-mediated MDR were reported and revealed a putative second binding pocket of ABCG2. Based on this (sub)pocket, a series of 16 differently 6-substituted 4-anilino-2-phenylpyrimidines was designed and synthesized to explore the potential increase in inhibitory activity of these ABCG2 inhibitors. The compounds were assessed for their influence on the ABCG2-mediated pheophorbide A transport, as well as the ABCB1- and ABCC1-mediated transport of calcein AM. They were additionally evaluated in MDR reversal assays to determine their half-maximal reversal concentration (EC₅₀). The 6-substitution did not only show increased toxicity against ABCG2-overexpressing cells in combination with SN-38 but also a negative influence on cell viability in general. Nevertheless, several candidates had EC₅₀ values in the low double-digit nanomolar concentration range, qualifying them as some of the most potent reversers of ABCG2-mediated MDR. In addition, five novel multitarget ABCB1, ABCC1, and ABCG2 inhibitors were discovered, four of them exerting their inhibitory power against the three stated transporters at least in the single-digit micromolar concentration range.

View all citing articles on Scopus

View full text

Original articleSynthesis and biological evaluation of flavones and benzoflavones as inhibitors of BCRP/ABCG2

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Chemistry

Conclusion

Chemistry

Acknowledgements

Eur. J. Pharm. Sci.

Int. J. Biochem. Cell Biol.

Biochem. Pharmacol.

Bioorg. Med. Chem.

Eur. J. Med. Chem.

Eur. J. Pharm. Sci.

Bioorg. Med. Chem.

Tetrahedron

Bioorg. Med. Chem.

Bioorg. Med. Chem.

Bioorg. Med. Chem.

J. Biomol. Screen.

Expression of a multidrug-resistance gene in human tumors and tissues

Proc. Natl. Acad. Sci. U S A

Drug-resistance in multiple myeloma and non-Hodgkin's lymphoma: detection of P-glycoprotein and potential circumvention by addition of verapamil to chemotherapy

J. Clin. Oncol.

Role of ABCG2/BCRP in biology and medicine

Annu. Rev. Pharmacol. Toxicol.

ABCG2: structure, function and role in drug response

Expert Opin. Drug Metab. Toxicol.

ABC transporters and cholesterol metabolism

Front Biosci.

Original article
Synthesis and biological evaluation of flavones and benzoflavones as inhibitors of BCRP/ABCG2