Synthesis of hexahydrofuro[3,2-c]quinoline, a martinelline type analogue and investigation of its biological activity

Background Candida susceptibility commonly occurs in breast cancer patients. Of which, Candida albicans is considered as a common pathogen causing candidiasis. Martinella iquitosensis (Bignoniaceae) is one of the species belonged to Martinella, distributed widely in Amazon basin. Its root extract yielded two complex substituted tetrahydroquinolines, Martinelline and Martinellic acid which were the first natural non-peptide bradykinin receptor antagonists identified. Findings In this study, a novel martinelline type analogue, named 2,3,3a,4,5,9b-hexahydro-8-phenoxy-4-(pyridin-2-yl)furo[3,2-c]quinoline, was synthesized and its preliminary anticancer activity and antifungal potential were investigated. This compound showed potential anticancer activity against MDAMB-231 breast cancer cells. Meanwhile it could enhance the fungistatic activity of miconazole against Candida albicans. Conclusions These findings provide an implication for the continue investigation and development of martinelline type analogues as therapeutic agents in the future.


Background
Breast cancer patients are commonly susceptible to candidiasis. Candida albicans (C. albicans) is one of the opportunistic fungi especially observed in immunocompromised patients (Calderone and Fonzi 2001), such as cancer patients. Previous studies have investigated the prevalence of candidiasis in cancer patients. Among 845 women with multiple lymph node positive or metastatic breast carcinoma receiving high-dose chemotherapy and autologous bone marrow transplantation at Duke University Medical Center during 1992-1997, 29 of them (3.4 %) developed candidemia. Of which, 23 % of them were found to be infected with C. albicans. The mortality was highest for the women who were infected by C. albicans (71 %) (Gottfredsson et al. 2003). In 2005, 400 adult patients with the hematological malignancy, head neck or solid tumor (including breast cancer) were recruited randomly into the study on admission to the regional cancer center of the Norfolk and Norwich University Hospital. There were 56.8 % (227 of 400) of all cancer patients and 18.9 % (43 of 227) of those who had clinical and microbiological evidence of oral candidiasis. Among 269 yeast isolates recovered from 227 patients, C. albicans was the most common yeast (74 %) causing colonization and infection (Schelenz et al. 2011).
Martinella can be used as indigenous medicine for eye disease caused by bacteria in various ethnolinguistic groups of some South American countries (Witherup et al. 1995). Martinella iquitosensis (Bignoniaceae), one of the species belonged to Martinella, is a tropical plant with dark purple corolla in South American and distributed widely in Amazon basin (Zuntini and Lohmann 2014). Its root extract yielded two complex substituted tetrahydroquinolines, Martinelline and Martinellic acid ( Fig. 1a) which were the first natural non-peptide bradykinin receptor antagonists identified by Witherup et al. (1995). Afterwards, the studies on the synthesis of new compounds with this interesting core have been increased. For instance, hexahydro-2H-pyrano[3,2-c] quinolines were found to be a potent agent against pathogenic gram-negative bacteria and Magesh et al. discovered that one of the synthesized compounds exhibits good bacteriolytic activity against Virbio vulnificus and Vibrio parahaemolticus (Magesh et al. 2004). Another research group (Kantevari et al. 2011) also synthesized a series of hexahydro-2H-pyrano[3,2-c]quinolines and studied their activity against the Mycobacrtium tuberculosis H37Rv. Three of the synthesized compounds showed a comparable activity as ethambutol. In addition, it was reported that hexahydro-2H-pyrano[3,2-c]quinolines could be used as selective σ 1 receptor ligand for the treatment of pain (Diaz et al. 2013).
Heterocyclic compounds are important candidates in the development of new class of structural entities for medicinal applications. Quinoline is a heterocyclic aromatic nitrogen containing compound characterized by a double-ring structure that has a benzene ring fused to pyridine at two adjacent carbon atoms (Keri and Patil 2014). Among these structures, tetrahydroquinoline derivatives demonstrated extensive biological activities. They included anticancer (Subramanian et al. 2011), antioxidant (Dorey et al. 2000 and antifungal (Vargas Méndez et al. 2010) activity. The construction and stereochemistry of the tricyclic ring system have also been recently reported (Calleja et al. 2014). In the previous years, we have prepared and studied some simple 2-subsituted terahydroquinoline alkaloid analogues as antitumor agents possessing notable cytotoxicity towards human Hep3B heptocellular carcinoma cells (Lam et al. 2013). In addition, we have reported the antimicrobial activity of some novel synthesized quinoline analogues (Lam et al. 2014;Chung et al. 2015). Inspired by unique core moiety of Martinelline and the reported promising bioactivity of tetrahydroquinolines and their related natural product analogues, we have designed compound 1 (Fig. 1b) and its potential biological activity was tested on MDAMB-231 breast cancer cells and C. albicans.
Strategically, we aimed to develop a simple and mild reaction pathway for the synthesis of compound 1 using one-pot multicomponent reaction with substituted aniline, aldehyde and alkene. Various catalysts, such as NbCl 5 (da Silva et al. 2014), Fe 2 (SO 4 )·xH 2 O (Khan et al. 2011;Das et al. 2014), In(OTf ) 3 (Priestley et al. 2013) and BiCl 3 (Kouznetsov et al. 2011) were reported to be effective for this reaction. Particularly, bismuth and its compounds are recognized as safe and green Lewis acid catalysts (Leonard et al. 2002;Mohan 2010). With increasing concern on the advancement of "green reaction" in last decade, the application of bismuth(III) compounds and their important roles in organic synthesis have been clearly addressed in recent researches (Gaspard-Iloughmane and Le Roux 2004;Bothwell et al. 2011;Ollevier 2013). Herein, we report a bismuth(III)-catalyzed synthesis of substituted tetrahydroquionlines.

Determination of minimum inhibitory concentration (MIC) and sensitization assay
Candida albicans was obtained from American Type of Culture Collection. The MIC values of synthesized compound 1 and miconazole nitrate were determined by the broth dilution method. Various concentrations of compound 1 and miconazole were loaded from a starting concentration of 50 µM containing 0.1 % dimethyl sulfoxide (DMSO) as vehicle and they were diluted serially. DMSO (0.1 %) was used as a vehicle control. The fungal samples were then incubated at 37 °C for 48 h. The minimum concentrations of compound 1 and miconazole that induced a complete growth inhibition would be determined as their MIC values. For sensitization study, with compound 1 at 50 µM, miconazole was added at a starting concentration of 1.56, 0.78 and 0.39 µM respectively. After incubation, the fungal samples were treated with MTS/PMS as above (Lam et al. 2015a).

Synthesis of compound 1
We first screened different metal salts as catalyst for this reaction (Table 1). It was found that only metal(III) salts (Table 1, Entry 5-7) gave our desired compound 1 and, in particular, bismuth(III) nitrate pentahydrate provided the highest yield among all the selected catalysts. With such preliminary screening, we used Bi(NO 3 ) 3 ·5H 2 O as catalyst for further optimization of the reaction under various conditions aiming to enhance the product yield (Table 2). It is noted that addition of catalyst in 0.2 mol equivalent and the reaction carried in ethanol (Table 2, Entry 7) provided the best yield. With the present work, further investigation is ongoing to develop a greener and more effective reaction for the synthesis of compound 1.

Anticancer activity of compound 1
Compound 1 at 50 µM (~17 µg/ml) could readily induce cell death on MDAMB-231 cells with significant cellular morphological changes when compared with the untreated control (Fig. 2a) such as cell rounding and shrinkage (Fig. 2b) which were similar to those from the positive reference, doxorubicin (Fig. 2c) at 8 µM after 24 h. As shown in Fig. 2d, a dose dependent cytotoxicity of compound 1 on MDAMB-231 breast cancer cells was observed after a 48 h of incubation.

MIC of compound 1
The MIC value of miconazole on C. albicans was found to be 3 µM. Compound 1, however, did not exhibit antifungal activity on C. albicans of up to 50 µM. However, we found that compound 1 could enhance the antifungal activity of miconazole on C. albicans. In the subsequent tests, compound 1 was added simultaneously with different concentrations of miconazole. As shown in Fig. 3, compound 1 could significantly potentiate the antifungal action of miconazole. Recently, we have shown that corilagin could sensitize Hep3B hepatoma cells to cisplatin and doxorubicin (Gambari et al. 2014). Here we suggest that compound 1 at 50 µM could significantly improve the fungistatic property of miconazole against C. albicans.

Conclusions
In this work, the synthesis and preliminary in vitro biological application of a novel martinelline type analogue, named 2, 3,3a,4,5,9b-hexahydro-8-phenoxy-4-(pyridin-2-yl)furo [3,2-c]quinoline, were described. Interestingly, this compound showed potential anticancer activity against MDAMB-231 breast cancer cells and it could simultaneously potentiate the fungistatic activity of miconazole against a common human pathogenic fungus, C. albicans. As the obtained compound 1 consists of three chiral centres, there should be eight stereoisomers exist. Further work will be carried out to isolate each isomer and investigate their individual potential biological activity in order to elucidate if chirality is important in this group of compounds from the pharmaceutical point of view. Authors' contributions PYC carried out the chemistry experiments and drafted the manuscript. JCO participated in the chemistry experiments. CHC participated in the antifungal test. ZXB participated in the anticancer study. WYW participated in the chemistry experiments. KHL conceived of the study, participated in the design of the study and carried out the chemistry experiments. CHC conceived of the study, and participated in its design of the study, carried out the anticancer and antifungal studies and helped to draft the manuscript. All authors read and approved the final manuscript.