Research paper
Synthesis and biological evaluation of quinazoline derivatives – A SAR study of novel inhibitors of ABCG2

Presented variations of the moieties based on the basic quinazoline scaffold should contribute to a better understanding of the SAR regarding quinazoline derivatives as inhibitors of ABCG2.
https://doi.org/10.1016/j.ejmech.2018.10.026Get rights and content

Highlights

  • SAR of structural requirements for high potency of quinazoline-type ABCG2 inhibitors.

  • Enhanced activity against ABCG2.

  • Type of interaction studies with substrate Hoechst 33342.

  • Investigation of conformational changes caused by inhibitor binding.

Abstract

Multidrug resistance (MDR) is a major obstacle for effective chemotherapeutic treatment of cancer frequently leading to failure of the therapy. MDR is often associated with the overexpression of ABC transport proteins like ABCB1 or ABCG2 which efflux harmful substances out of cells at the cost of ATP hydrolysis. One way to overcome MDR is to apply potent inhibitors of ABC transporters to restore the sensitivity of the cells toward cytostatic agents. This study focusses on the synthesis and evaluation of novel 2,4-disubstituted quinazoline derivatives regarding the structure-activity-relationship (SAR), their ability to reverse MDR and their mode of interaction with ABCG2. Hence, the inhibitory potency and selectivity toward ABCG2 was determined. Moreover, the intrinsic cytotoxicity and the reversal of MDR were investigated. Interaction type studies with the substrate Hoechst 33342 and conformational analyses of ABCG2 with 5D3 monoclonal antibody were performed for a better understanding of the underlying mechanisms. In our study we could further enhance the inhibitory effect against ABCG2 (compound 31, IC50: 55 nM) and identify the structural features that are crucial for inhibitory potency, the impact on transport activity and binding to the protein.

Introduction

ATP-Binding cassette (ABC) transport proteins are found among eukaryotic and prokaryotic organisms [1]. In eukaryota they are expressed in different tissues where they provide membrane transport of nutrients and are responsible for an active efflux of different molecules upon ATP hydrolysis, protecting the cells from potentially harmful compounds [2,3]. Human ABC transport proteins form a superfamily of 48 members, classified into seven subfamilies ABC A to ABC G [[4], [5], [6]]. The Breast Cancer Resistance Protein (BCRP, ABCG2) for instance is expressed amongst others in tissues like the gut, bile, canaliculi, placenta, blood-testis, and blood-brain barriers excreting and limiting absorption of potentially harmful substrate molecules [7,8]. ABCG2 is one of three major members of the ABC transport protein family besides P-glycoprotein (P-gp, ABCB1) and multidrug resistance associated protein 1 (MRP1, ABCC1), which are often associated with the occurrence of multidrug resistance (MDR) in cancer [4,[9], [10], [11]]. Breast Cancer Resistance Protein was discovered in 1998 by Doyle et al. when they selected a human breast cancer cell line for doxorubicin resistance in the presence of the ABCB1 inhibitor verapamil [12,13]. More cytostatic drugs like the anthracene derivative mitoxantrone (MX) or the active metabolite of irinotecan, namely SN-38, were identified as substrates of ABCG2 [14,15]. Therefore, a chemotherapeutic therapy might lack effectiveness due to overexpression of ABCG2, leading to a low intracellular concentration of the cytostatic. High expression rates of ABCG2 were for instance found in solid tumors and hematopoietic tumors [16]. A possible way to resensitize such cancer cells could be the co-administration of potent inhibitors of ABCG2.

The first potent inhibitor was discovered and isolated from Aspergillus fumigatus, and named fumitremorgin C (FTC). Despite its high potency, FTC never found clinical application owing to neurotoxic side effects [17]. More promising was the second generation inhibitor Ko143, exhibiting a high potency together with selectivity in the submicromolar range toward ABCG2 [18,19]. The tyrosine kinase inhibitor gefitinib is used in clinical application for the treatment of non-small cell lung carcinoma (NSCLC). It was found that co-administration with topoisomerase I inhibitors was an effective approach to overcome the resistance of ABCG2 overexpressing cells [20]. However, only few potent and selective inhibitors of ABCG2 have been described yet [21].

Our workgroup decided to modify the structure of gefitinib, to increase its relatively low inhibitory potency toward ABCG2. First step was to start with the underlying quinazoline scaffold introducing a phenyl moiety at position 2 und to introduce various substituents at the aniline linker at position 4. First results from the Hoechst 33342 accumulation assay with ABCG2 overexpressing cells were promising, yielding several compounds with higher potencies than Ko143, one of the most potent inhibitor of ABCG2 known to date [[22], [23], [24], [25], [26]]. In our most recent study we investigated a modification of the quinazoline scaffold using a pyrido[2,3-d]pyrimidine structure [27]. We found an enantiotopic relation of the inhibitory activities between those scaffolds leading to several highly potent derivatives. Also, the intrinsic cytotoxicities of these new compounds were low.

In the present study, we decided to investigate different modifications of our basic 4-anilino-2-phenylquinazoline scaffold. We replaced the aromatic residue at position 2 by five-membered heteroaromatic moieties as well as hydrogen to determine the impact of the substitution at this position on the inhibitory activity toward ABCG2. Also, we altered the 4-amino linker by replacing it by oxygen or sulphur. Furthermore, the linker was replaced by an amido group. Based on the results from the oxygen and sulphur linkers we methylated the amino linker to investigate the importance of a H-donor function on inhibitory activity.

With these new insights into the structure-activity relationship (SAR) of this novel class of inhibitors new tailored inhibitors of ABCG2 can be designed.

Section snippets

Chemistry

A brief description of the synthetic route for all compounds is presented in Scheme 1.

Quinazolinone derivatives containing a five-membered aromatic ring at position 2 were synthesized via cyclic condensation from anthranilamide together with thiophene-2-carbaldehyde or 1H-pyrrole-2-carbaldehyde to yield the corresponding precursor 1 or 2. Chlorination of the carbonyl function at position 4 was achieved by reaction with POCl3 to yield 3 and 4. The 2-substituted-4-chloroquinazoline derivatives

Conclusions

In this study several modifications of the quinazoline scaffold were carried out. The first series based on scaffold A contained the five-membered heterocycles thiophene and pyrrole at position 2. For both heterocycles the highest inhibitory activity in the Hoechst 33342 accumulation assay was obtained with a 3-cyano substituent of the anilino linker at position 4 (see compound 7; IC50: 178 nM and 11; IC50: 156 nM), revealing even higher potencies toward ABCG2 than standard inhibitor Ko143 (IC50

Materials

Chemicals were purchased from Acros Organics (Geel, Belgium), Alfa Aesar (Karlsruhe, Germany), Sigma Aldrich (Steinheim, Germany) or Merck (Darmstadt, Germany) and used without further purification. Reactions in the microwave were performed with 50 mL vials using with a CEM Discover SP (CEM GmbH, Kamp-Lintfort, Germany). Reaction progress was monitored using thin layer chromatography (TLC) with an aluminium plate coated with silica gel 60 F254 (Merck Millipore, Billerica, MA, USA). As eluent a

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      Starting materials 2a-d were reacted with substituted 4-nitrophenol or 4-nitrophenylamine in PhCl to afford various quinoline derivatives 3a-b and 3d-i (Scheme 1). Intermediate 3b was methylated by the use of CH3I and NaH to yield 3c [42]. Reduction of the nitro groups of 3a-i via iron powder and HOAc in 90% EtOH provided the amino precursors 4a-i. Subsequently, the amines 4a-i were acidylated with phenyl chloroformate in the presence of pyridine to facilitate 5a-i which were suffered hydrazinolysis reactions with 80% hydrazine hydrate with vigorous agitation.

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