Research paperSynthesis and biological investigation of 2,4-substituted quinazolines as highly potent inhibitors of breast cancer resistance protein (ABCG2)
Graphical abstract
Introduction
ATP-binding cassette (ABC) transport proteins comprise a large superfamily of membrane proteins that are present in all living organisms from bacteria, to fungi, plants and animals [1]. While in prokaryotes importers and exporters exist, in eukaryotes only export proteins are present. In humans 48 members are expressed that are classified according to their phylogenetic similarities in seven subfamilies ABCA to ABCG [2], [3], [4]. ABC export transporters utilize the energy from hydrolysis of ATP to efflux their substrates out of the cell, commonly against a concentration gradient. While most ABC transporters have distinct substrates, a few are multyspecific recognizing a wide variety of structurally and mechanistically unrelated molecules [5]. In this context, overexpression of multispecific ABC transporters was found to play a major role in the resistance of tumors against chemotherapeutic drugs, which lead to insufficient concentrations of the drug inside the cancer cell, resulting in failure of the therapy [6]. Because structurally unrelated drugs are affected by this type of resistance it is referred to as multidrug resistance (MDR). Three ABC transport proteins, namely P-glycoprotein (P-gp, ABCB1), multidrug resistance-associated protein 1 (MRP1, ABCC1) and breast cancer resistance protein (BCRP, ABCG2) are most commonly associated with MDR [1], [7], [8], [9]. ABCG2 was the last multyspecific ABC transporter to be discovered in 1998 by Doyle et al. and found to be highly overexpressed in drug-resistant solid and hematopoietic tumors [2], [10]. ABCG2 is a half transporter consisting of 655 amino acids and possessing only one cytosolic nucleotide-binding domain (NBD) and one transmembrane domain (TMD) [11]. To function it is assumed to form dimers or tetramers unlike ABCB1 and ABCC1, which form fully functional monomers (full transporters) [12], [13], [14], [15]. ABCG2 differs in the topological organization from ABCB1 and ABCC1 as it contains a N-terminal nucleotide binding domain (NBD), while in full transporters the NBD is located C-terminal after the corresponding alpha-helical transmembrane domain.
ABCG2 recognizes and transports numerous anticancer drugs including conventional chemotherapeutic and targeted small therapeutic molecules [16]. The cytostatic agent mitoxantrone for instance, was found to be a substrate of ABCG2 [17], [18]. Several Other chemotherapeutics were identified as substrates of ABCG2 too, like the camptothecin derivatives topotecan, irinotecan and its active metabolite SN-38 [19], [20]. Moreover, a variety of photosensitizers like pheophorbide A, protoporphyrin IX and other related compounds are also substrates of ABCG2, suggesting that ABCG2 is a possible cause for cellular resistance to photodynamic therapy [1]. Regarding inhibitors of ABCG2, potent and selective compounds are still rather limited [21]. Most notably, the diketopiperazin Ko143 was found to be selective toward ABCG2 at a concentration of less than 1 μM and considerably less toxic than its precursor fumitremorgin C (FTC) [22], [23]. Some potent inhibitors of ABCG2 have also been reported by the workgroup of Di Pietro: in one of their first studies they investigated methoxy stilbenes [24] and quinoxaline-substituted chalcones [25] as inhibitors of ABCG2. More recently nontoxic and potent inhibitors based on a chromone scaffold [26], as well as an indeno [1,2-b]indole scaffold [27] have been reported. Here, some of the chromone derivatives showed activities similar to Ko143.
Moreover, a notable overlap of substrates as well as inhibitors between the transport proteins ABCG2 and ABCB1 was observed in earlier studies [28]. Thus, when treating tumors with anticancer-agents that are substrates of both transport proteins, a strong influence on the pharmacokinetic can be expected. A possible way to overcome MDR is by co-administration with multispecific inhibitors like the tyrosine kinase inhibitor gefitinib, which is able to inhibit both transporters and reduce the efflux of chemotherapeutics from the central nervous system (CNS) as reported by Zhuang et al. [29] In the same context, GF120918 (Elacridar) was found to be a potent inhibitor of both, ABCG2 and ABCB1 and was therefore investigated regarding its ability to overcome natural barriers like CNS and BBB [30], [31].
Previous studies of our working group investigated compounds containing a quinazoline scaffold like the TKI gefitinib. Quinazoline derivatives with a phenyl substitution at position 2 and a substituted aniline moiety at position 4 were found to be potent inhibitors of ABCG2 [32], [33], [34]. Highest inhibitory potencies were obtained with nitro, hydroxy and cyano substituents in meta and para position of the anilino residue. Except for the disubstitution with 3,4-dimethoxy, considerably high selectivity toward ABCG2 was observed in comparison to inhibition of ABCB1 and ABCC1 in cells overexpressing these transporters. Moreover, MDR related to both cytostatic drugs SN-38 and mitoxantrone could be reversed by the most potent compounds. Very recently we presented 4-substituted quinazoline derivatives containing a pyridyl substitution at position 2 [35]. They showed a considerably higher inhibitory potency against ABCG2 compared to Ko143 and also a very low intrinsic cytotoxicity resulting in high therapeutic ratios.
This study investigates substitution at the aniline linker at position 4 together with the phenyl group at position 2. Additional modifications at both aromatic residues are explored to gain more insights in structure activity relationships and to guide the synthesis of more potent inhibitors of ABCG2.
Section snippets
Chemistry
A brief description of the synthetic route for all compounds is presented in Scheme 1. Intermediate 2-substituted quinazolinones were synthesized by a cyclic condensation reaction of anthranilamide with the corresponding substituted aldehyde to yield the derivatives 1–7. Subsequently, the quinazolinone derivatives were refluxed with POCl3 to obtain the 2-substituted-4-chloroquinazoline derivatives 8–14. Nucleophilic aromatic substitution of the 4-chloroquinazoline derivatives with different
Conclusions
The current study investigates the inhibitory potency of substituted 2-phenyl-4-anilinoquinazolines toward ABCG2. Modifications were carried out on the aromatic systems at position 2 and 4 of the quinazoline scaffold. Due to findings in earlier studies only meta and para substitutions were investigated apart from compound 40 with an ortho substitution [29]. First, the importance of the aromatic system in position 4 was confirmed by replacing the aromatic ring of the aniline residue (15) with a
Materials
Chemicals were purchased from Acros Organics (Geel, Belgium), Alfa Aesar (Karlsruhe, Germany) or Sigma Aldrich (Steinheim, Germany) or Merck (Darmstadt, Germany) and used without further purification. For the microwave, 50 mL vials were used with a CEM Discover SP (CEM GmbH, Kamp-Lintfort, Germany). Reaction progress was monitored by thin layer chromatography (TLC) with an aluminium plate coated with silica gel 60 F254 (Merck Millipore, Billerica, MA, USA). TLCs were carried out with a mixture
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