Accepted Manuscript Synthesis and cytotoxic evaluation of novel dihydrobenzo[h]cinnoline-5,6-diones Tuyet Anh Dang Thi, Lena Decuyper, Hoang Thi Phuong, Doan Vu Ngoc, Ha Thanh Nguyen, Tra Thanh Nguyen, Thanh Do Huy, Hung Huy Nguyen, Matthias D’hooghe, Tuyen Van Nguyen PII: DOI: Reference: S0040-4039(15)30038-1 http://dx.doi.org/10.1016/j.tetlet.2015.08.084 TETL 46668 To appear in: Tetrahedron Letters Received Date: Revised Date: Accepted Date: 16 June 2015 21 August 2015 31 August 2015 Please cite this article as: Thi, T.A.D., Decuyper, L., Phuong, H.T., Ngoc, D.V., Nguyen, H.T., Nguyen, T.T., Huy, T.D., Nguyen, H.H., D’hooghe, M., Nguyen, T.V., Synthesis and cytotoxic evaluation of novel dihydrobenzo[h]cinnoline-5,6-diones, Tetrahedron Letters (2015), doi: http://dx.doi.org/10.1016/j.tetlet 2015.08.084 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain Graphical abstract Synthesis and cytotoxic evaluation of novel dihydrobenzo[h]cinnoline-5,6-diones Synthesis and cytotoxic evaluation of novel dihydrobenzo[h]cinnoline-5,6-diones Tuyet Anh Dang Thi,a Lena Decuyper,b Hoang Thi Phuong,a Doan Vu Ngoc,a Ha Thanh Nguyen,a Tra Thanh Nguyen,a Thanh Do Huy,a Hung Huy Nguyen,c Matthias D’hooghe,b,* Tuyen Van Nguyena, a Institute of Chemistry, Vietnam Academy of Science and Technology, 18-Hoang Quoc Viet, CauGiay, Hanoi, Vietnam b SynBioC Research Group, Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium c Hanoi University of Science, 19-Le Thanh Tong str., Hoan Kiem, Hanoi, Vietnam Abstract A convenient one-pot multicomponent synthetic approach was developed en route to novel functionalized dihydrobenzo[h]cinnoline-5,6-diones using 2-hydroxy-1,4-naphthoquinone, methylhydrazine and a variety of aromatic aldehydes Sixteen new derivatives were thus prepared and subsequently evaluated in terms of their cytotoxicity profile, revealing a promising anticancer activity of nine of the compounds against KB and Hep-G2 human tumor cell lines Keywords: Dihydrobenzo[h]cinnoline-5,6-diones; Domino reactions; Naphthoquinones; Cytotoxicity The quinone skeleton is ubiquitously present in nature as a constituent of biologically active molecules in living organisms.1 Nowadays, quinones represent the second largest class of antitumor agents approved by the FDA for clinical use.2 Their structural features make them particularly appropriate to take part in several biological oxidation processes due to the redox properties linked to the fully aromatic system.3 In this way, either one- or two-electron reduction provokes cellular damage through direct alkylation of proteins and nucleic acids or oxidative stress to crucial cellular macromolecules such as DNA, lipids and proteins, caused by the production of highly reactive oxygen species.4 Accordingly, this class of compounds has attracted considerable attention in the field of organic and medicinal chemistry within the pursuit of new bioactive agents Among these, the naphthoquinones, often substituted with heterocyclic groups, represent a valuable class of compounds showing pronounced biological activities associated with, inter alia, antibacterial,5 antifungal,5 antiviral,6 antimalarial,7 trypanocidal,4a,5b,8 insecticidal,9 antiangiogenic10 and antiinflammatory11 properties Furthermore, numerous examples of naphthoquinone compounds are known for the treatment of skin diseases and different cancer types.3a,3b,5e Both 1,4- and, more rarely, 1,2-naphthoquinones occur naturally as toxic secondary metabolites of naphthalene, a principal aromatic hydrocarbon present in ambient air However, direct comparisons between the two isomers have indicated that their reduction potentials are similar, while selectivity differences exist in terms of their electrophilic reactions, which is translated into different biological targets and associated activities.12 In continuation of our previous research on functionalized heterocyclic naphthoquinones,13 we present herein the synthesis of novel dihydrobenzo[h]cinnoline-5,6-diones as cytotoxic agents, starting from 2-hydroxy-1,4-naphthoquinones using a one-pot multicomponent domino reaction (MDR) A domino reaction has been defined as a reaction involving two or more bond-forming transformations, based on functionalities induced in the previous step, taking place without changing the reaction conditions or adding catalysts and/or additional reagents A multicomponent reaction Tel.: +84 917683979 (TVN), +32 92649394 (MD), E-mail: ngvtuyen@hotmail.com (TVN), matthias.dhooghe@UGent.be (MD) involves at least three substrates and is considered to be a subgroup of domino reactions 14 The applicability of this green approach in organic synthesis is increasing continuously and is widely accepted This can be attributed to the high synthetic efficiency created by decreasing the number of time-consuming reaction steps and laboratory operations required, the amount of chemicals and solvents used and the waste produced.14,15 The synthesis of a considerable series of drug-like compounds with high molecular complexity and structural diversity, including naphthoquinone-fused heterocycles, is based on the aforementioned domino strategy These have recently been described in the literature and demonstrate the relevance of this topic in modern organic chemistry 16 The synthesis of the dihydrobenzo[h]cinnoline-5,6-dione skeleton has only been described once before, starting from 2,3-epoxy-1,4-naphthoquinones The substrates were treated with active methylene compounds in basic medium followed by subsequent reaction with hydrazine under acid catalysis.17 In addition, their antioxidant and cytotoxic evaluation was performed In line with our previous research related to functionalized heterocyclic naphthoquinones,13g the synthesis of the novel naphthoquinone derivatives was pursued in the present work by using a different approach To that end, a one-pot multicomponent domino strategy was employed starting from simple and readily available substrates, namely 2-hydroxy-1,4-naphthoquinone 1, methylhydrazine and aromatic aldehydes Thus, a solution of 2-hydroxy-1,4-naphthoquinone and equivalents of aromatic aldehydes 3a-o in t-BuOH was heated under reflux for 30-60 min, after which a solution of methylhydrazine in t-BuOH was added The resulting mixture was then further heated under reflux for 2-3 h In this way, 15 new fused naphthoquinones 4a-o were selectively obtained in 43-60% yield after purification by silica gel column chromatography (Scheme 1, Table 1).18 The proposed molecular structures of the functionalized naphthoquinones 4a-o were assigned by means of 1H NMR, 13C NMR and IR analysis techniques The mass spectra displayed the molecular ion signals at m/z (M+) values pertaining to the molecular formulae In addition, a single crystal X-ray analysis of compound 4f, as depicted in Figure 1, provided irrefutable evidence for the formation of this uncommon dihydrobenzo[h]cinnoline-5,6-dione heterocyclic skeleton Scheme Synthesis of naphthoquinones 4a-o A possible mechanistic interpretation of this multicomponent reaction begins with the formation of 1,2,4-naphtalenetriones by Knoevenagel condensation of 2-hydroxy-1,4-naphthoquinone with the aromatic aldehydes 3, followed by dehydration Besides, the nucleophilic addition of methylhydrazine to a second equivalent of aromatic aldehydes results in the formation of 1arylmethylidene-2-methylhydrazines 8, after elimination of water Compounds behave as Michael acceptors for the addition of the in situ prepared hydrazones (route a).19 The adducts undergo tautomerization and intramolecular cyclization affording the fused naphthoquinone derivatives 11 Keto-enol tautomerization and elimination of water finally lead to the title compounds (Scheme 2) However, other mechanistic proposals, such as for example condensation of hydrazone with trione at C4 (route b) to furnish an intermediate 12 prone to undergo electrocyclic cyclization toward tricycle 13, followed by proton abstraction, should not be ruled out The mechanism could be further investigated by prestirring methylhydrazine with an aromatic aldehyde (e.g benzaldehyde 3a) and 2-hydroxy-1,4-naphthoquinone with a different aldehyde (e.g 4-methoxybenzaldehyde 3c) in order to explore the possibility of having two different Ar-groups in the final product and the fidelity of their incorporation, which could be an indication of reversibility Scheme Proposed mechanisms for the formation of compounds Table Synthesis of dihydrobenzo[h]cinnoline-5,6-diones 4a-o Entry 10 11 12 13 14 15 a Ar H 3-BrC6H4 4-MeOC6H4 3-MeOC6H4 Naphth-2-yl 4-BrC6H4 4-ClC6H4 3,4-OCH2O-C6H3 2-MeOC6H4 4-NO2C6H4 4-(MeSO2)C6H4 4-NMe2C6H4 4-HOC6H4 3-MeO-4-OH-C6H3 2-NO2-5-OH-C6H3 Compound (yield)a a (50%) b (51%) c (53%) d (47%) e (47%) f (45%) g (43%) h (53%) i (60%) j (51%) k (48%) l (45%) m (45%) n (43%) o (47%) After purification by column chromatography (SiO2) Figure Molecular structure of tricyclic compound 4f In order to broaden the reaction scope, the above-described reaction employing aliphatic aldehydes was examined in the next part For this purpose, cyclohexane carboxaldehyde and isobutyraldehyde were used in combination with 2-hydroxy-1,4-naphthoquinone and methylhydrazine as the starting materials The reactions were followed via TLC analysis, however, only giving rise to highly complex reaction mixtures Apparently, an electrophilic benzylic position is required for this MCR to proceed, resulting in reaction failure in the case of aliphatic aldehydes To evaluate their cytotoxic potential, the newly synthesized 1,2-naphthoquinone derivatives 4a-o were subjected to in vitro biological assessment against two human cancer cell lines, KB and HepG2.20,21 The results of the cytotoxicity evaluation, as compared to the anticancer reference compound ellipticine, are summarized in Table As evidenced by these results, the majority of the derivatives exhibit at least moderate cytotoxic activity against the KB and Hep-G2 cell lines Nine of the new dihydrobenzo[h]cinnoline-5,6-diones (4a, 4b, 4d, 4f, 4g, 4h, 4j, 4k, 4m) even display a considerable activity profile with IC50-values below µM against both cell lines, being only slightly higher than those of the anticancer drug ellipticine In particular, nitro compound 4j can be identified as the most promising agent with IC50-values of 0.56 and 0.77 µM against the KB and Hep-G2 cell lines, respectively These results clearly suggest the relevance of this interesting new class of dihydrobenzo[h]cinnoline-5,6-diones in the framework of cancer therapy research and medicinal chemistry Further optimization of the core structures toward potent cytotoxic agents should definitely be considered in future research Both electron-donating and electron-withdrawing phenyl substituents were assessed in this study in order to evaluate their influence on the reaction outcome and the subsequent biological testing However, no clear effect of the substitution pattern was observed on the yields (43-60%) (Table 1) as well as on the IC50-values obtained during the cytotoxicity analyses (Table 2), pointing to the need for further elaboration of the pharmacophore for the development of suitable structure-activity relationships and the design of more potent dihydrobenzo[h]cinnoline-5,6-dione scaffolds Table Cytotoxicity evaluation of the prepared naphthoquinone derivatives 4a-o Entry Compound 10 11 12 13 14 15 16 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4n 4o Ellipticine IC50 (µM) KB 3.70 3.43 23.86 4.58 7.42 2.29 3.60 2.87 >292 0.56 1.33 5.81 2.02 12.61 135.73 1.26 IC50 (µM) Hep-G2 3.63 3.22 20.69 3.56 15.40 2.93 2.14 3.64 >292 0.77 2.71 16.85 4.46 10.93 145.90 1.42 In conclusion, the efficient and straightforward preparation of a series of functionalized dihydrobenzo[h]cinnoline-5,6-diones was described, using a one-pot multicomponent approach employing 2-hydroxy-1,4-naphthoquinone, methylhydrazine and a variety of aromatic aldehydes Subsequent biological assessment pointed out the relevance of a number of these novel scaffolds in terms of their cytotoxic activity, implying their potential for further studies in the field of anticancer research Acknowledgements The authors are indebted to the Vietnamese National Foundation for Science and Technology Development (NAFOSTED, code: 104.01-2013.27) and to Ghent University – Belgium (BOF) for financial support References and notes Thomson, R H Naturally occurring quinones; 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(l) Khanna, G.; Chaudhary, A.; Khurana, J M Tetrahedron Lett 2014, 55, 6652-6654; (m) Mahajan, S.; Khullar, S.; Mandal, S K.; Singh, I P Chem Commun 2014, 50, 10078-10081; (n) Brahmachari, G.; 17 18 19 20 21 Banerjee, B ACS Sustainable Chem Eng 2014, 2, 411-422; (o) Prasanna, P.; Balamurugan, K.; Perumal S.; Menéndez, J C Green Chem 2011, 13, 2123-2129 Berghot, M A.; Kandeel, E M.; Abdel-Rahman, A H.; Marwa, A.-M Med Chem 2014, 4, 381-388 General procedure for the synthesis of dihydrobenzo[h]cinnoline-5,6-diones 4: A mixture of 2-hydroxy1,4-naphthoquinone (1 equiv.) and aromatic aldehyde (2 equiv.) in t-BuOH (5 mL/50 mg of 1) was heated under reflux for 30-60 min, after which methylhydrazine (1 equiv.) in t-BuOH was added The reaction was heated under reflux for another 2-3 h, followed by the addition of water and extraction with EtOAc (three times) The combined organic phases were washed with water and dried (MgSO 4) Finally, the solvent was removed in vacuo to afford the crude product, which was then purified by column chromatography on silica gel (EtOAc/hexane, 3/7) 3,4-Bis(4-bromophenyl)-1-methyl-1,4dihydrobenzo[h]cinnoline-5,6-dione 4f: violet crystals, 45% yield Mp 179-180°C IR (KBr): 3443; -1 3048; 2928; 1687; 1624; 1529; 1480; 1372; 1254; 1067; 1007; 939; 818; 773 cm H NMR (CDCl3, 500 MHz): 8.09 (1H, dd, J = 1.0, 7.5 Hz); 7.80 (1H, d, J = 7.5 Hz); 7.72 (2H, d, J = 8.5 Hz); 7.63 (1H, dt, J = 1.5, 8.0 Hz); 7.51 (1H, t, J = 8.0 Hz); 7.49 (2H, d, J = 8.5 Hz); 7.36 (2H, d, J = 8.5 Hz); 7.17 (2H, d, J = 8.5 13 Hz); 5.62 (1H, s); 4.10 (3H, s) C NMR (CDCl3, 125 MHz): 179.9; 176.6; 149.4; 147.1; 139.3; 133.7; 132.8; 132.2; 131.9; 131.5; 130.6; 130.4; 130.2; 129.1; 128.6; 126.8; 125.4; 121.4; 109.2; 46.4; 34.8 + HRMS (ESI) [M+H] : Calcd for C25H16Br2N2O2: 533.9579, Found: 533.9567 and 535.9553 (a) Mantenuto, S.; Mantellini, F.; Favi, G.; Attanasi, O A Org Lett 2015, 17, 2014-2017; (b) Serrano, I.; Monge, D.; Álvarez, E.; Fernández, R.; Lassaletta, J M Chem Commun 2015, 51, 4077-4080; (c) Yang, H.-B.; Zhao, Y.-Z.; Sang, R.; Shi, M J Org Chem 2014, 79, 3519-3528; (d) Wu, W.; Yuan, X.; Hu, J.; Wu, X.; Wei, Y.; Liu, Z.; Lu, J Org Lett 2013, 15, 4524-4527 Cell culture and cell viability assay Two human cancer cell lines (epidermoid carcinoma cell line (KB) and hepatoma carcinoma cell line (HepG2)), obtained from the American Type Culture Collection (USA) ATCC, were used for cytotoxic evaluation The cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in a humidified atmosphere (95% air and 5% CO2) The exponentially growing cells were used throughout the experiments The inhibitory effects of the compounds on the growth of the human cancer cell lines were determined by measuring the metabolic activity using a 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay.21 Briefly, human cancer cell lines (1×105 cells/mL) were treated for days with a series of concentrations of the compounds (in DMSO): 0.125, 0.5, 2.0, 8.0, 32.0, and 128.0 µg/mL After incubation, 0.1 mg MTT solution (50 µL of a mg/mL solution) was added to each well, and the cells were then incubated at 37°C for h The plates were centrifuged at 1000 rpm for 10 at room temperature, and the media were then carefully aspirated Dimethylsulfoxide (150 µL) was added to each well to dissolve the formazan crystals The plates were read immediately at 540 nm on a microplate reader (TECAN GENIOUS) All the experiments were performed three times, and the mean absorbance values were calculated The results are expressed as the percentage of inhibition that produced a reduction in the absorbance by the treatment of the compounds compared to the untreated controls A dose-response curve was generated, and the inhibitory concentration of 50% (IC50) was determined for each compound as well as each cell line Carmichael, J.; DeGraff, W G.; Gazdar, A F.; Minna, J D.; Mitchell, J B Cancer Res 1987, 47, 936-942 ...Graphical abstract Synthesis and cytotoxic evaluation of novel dihydrobenzo[h ]cinnoline-5,6-diones Synthesis and cytotoxic evaluation of novel dihydrobenzo[h ]cinnoline-5,6-diones Tuyet... evaluated in terms of their cytotoxicity profile, revealing a promising anticancer activity of nine of the compounds against KB and Hep-G2 human tumor cell lines Keywords: Dihydrobenzo[h ]cinnoline-5,6-diones; ... elaboration of the pharmacophore for the development of suitable structure-activity relationships and the design of more potent dihydrobenzo[h]cinnoline-5,6-dione scaffolds Table Cytotoxicity evaluation