Design, Synthesis and Anticancer activity of novel Triazole substituted Quinazoline Hybrids

Paduri Karunakar1,2, Swetha Gujjewar2, Somesh Sharma2, Srinivasu Pothukanuri2, Krubakaran Muthusamy2, Premkumar Arumugam2, Chebolu Naga Sesha Sai Pavan Kumar*1 1Department of Sciences and Humanities, Chemistry Division, Vignan’s Foundation for Science, Technology & Research, Vadlamudi, Guntur, Andhra Pradesh-522 213, India 2GVK Biosciences Private Limited, IDA Nacharam, Hyderabad, Telangana-500 076, India


INTRODUCTION
Quinazoline motif has substantiated to be a powerful and adaptable constituent for progress of diverse pharmacological entities (Hameed et al., 2018).
Quinazolines also display biological roles like cellular phosphorylation inhibitors, ligands for benzodiazepine and GABA receptors in the central ner-vous system (Bertelli et al., 2000) and as DNA binding motifs (Lewerenz et al., 2003;Malecki et al., 2004). They have also shown to possess effectual α-adrenergic blocking activity. Intriguing this, and in continuance of our program on the synthesis of diverse novel heterocyclic systems showing signi icant biological activities (Kumar et al., 2011(Kumar et al., , 2013Sriramoju et al., 2019;Santhoshi et al., 2018) here in we designed and synthesized 13 structurally different quinazoline derivatives and assessed for in vitro anticancer activity against HepG2 (liver carcinoma) & MCF-7 (breast cancer) cancer cell lines. Some of the compounds displayed substantial anticancer activity in micro molar (µM) concentration out of various synthesized hybrids.

MATERIALS AND METHODS
Chemicals & reagents were procured from Sigma, Merck and are directly used. Melting Points are ascertained in sulfuric acid bath using narrow capillary tubes. TLC was sprinted on silica gel glass plates containing 60 F-254 and visualization was carried with UV light or with iodine. Perkin-Elmer 1000 instrument was used for recording IR Spectra with KBr pellets. 1 H (Proton) NMR was recorded in CDCl 3 /CD 3 OD/DMSO-d 6 solvents using internal standard tetramethylsilane at 400 MHz / 500 MHz operating frequency, 13 C (Carbon 13) NMR spectra were recorded on 100 & 125 MHz frequency instruments. Agilent-LCMS instrument was used for recording mass spectra.
Compound 2 on treatment with aq. ammonia at ambient temperature afforded corresponding amide (3), which on re luxing with aq. 3N NaOH furnished quinazoline-4-one (4) product. Quinazolinone 4 was reacted with POCl 3 to attain chloro quinazoline (5). Chloroquinazoline 5 on further treatment with hydrazine in THF produced 6-bromo-2-cyclopropyl-4-hydrazinylquinazoline (6). Subsequent cyclization of compound 6 was achieved by heating in Ac 2 O & AcOH for 2 h to obtain the desired triazoloquinazoline 7. Triazoloquinazoline 7 on reaction with diverse range of aryl boronic acids by Suzuki coupling reaction (Miyaura et al., 1979;Miyaura and Suzuki, 1995) with the aid of Pd(OAc) 2 catalyst & S-Phos ligand in K 2 CO 3 and 1,4-dioxane attained disubstituted triazoloquinazolines (8A-G). Structures of all new hybrids were envisaged and con irmed on the source of IR, 1 HNMR, 13 CNMR & mass spectral data. Primarily, when the Suzuki coupling reaction is carried out at re lux conditions for longer time (16 h), interestingly, the side chain cyclopropyl ring moiety due to relatively less stability it undergoes transformation to vinyl moiety and produces the corresponding quinazolines (8A & 8B). In order to rectify this problem, the coupling reaction time is reduced to 2 h and the corresponding products are obtained in good yields (8C-G).
By fascinating this, we also used benzoyl chloride (9) for the introduction of phenyl ring at the 2 nd position and for the synthesis of various phenyl substituted triazoloquinazoline derivatives. For this, 5-bromoanthranilicacid (1) was treated with benzoyl chloride and prepared subsequent benzoxazinone (10) and the same synthetic sequence was adopted for the synthesis of bromo triazoloquinazoline (15). Furthermore, Suzuki coupling reaction is successfully employed on compound 15 with various boronic acids with PdCl 2 (dppf).DCM complex in 1,4-dioxane for 12 h afforded requisite triazolo quinazolines (16H-16L) in reasonable to good yields depicted in Scheme 2. Thus synthesized quinazolines (Scheme 3) were screened against two cancer cell lines for evaluation of in vitro anticancer activity and considerable results are obtained for some of the compounds (Tables 1 and 2).
The source of the cell lines used in the present study and the culture media are elaborated in reference (Ravichandran and Manoj, 2014). IC 50 in vitro growth inhibitory values of the compounds are assessed compared with the reference drug Puromycin. The cytotoxic activities of the compounds were tested using a CellTiter-Glo Luminescence based cellular cytotoxicity assay against HepG2 (ATCC Cat No HB-8065) & MCF7 . The titled cell lines were irstly cultured using Dulbeccos Modi ied Eagle Medium (DMEM), and supplemented through 10% heatinactivated Fetal Bovine Serum; in humidi ied 5% CO 2 atmosphere. When cells reached to 80-90% con luency, were separated from the lasks by trypsinization, neutralized, tallied and then seeded 5000 cells/well in a 96 well clear bottom tissue culture plates. After 24 h, the cells were treated with mentioned compounds & incubated for 72 h at 37 o C in 5% CO 2 incubator. Following incubation, 100µL of CellTiter-Glo ® reagent was added and mixed well for 10 min. The luminescence was calculated using an Envision 2104 multimode plate reader. The IC 50 (50% inhibitory concentration) were estimated from the conspired absorbance data for the dose response curves (Figures 1 and 2). The cytotoxicity (IC 50 values in µM) of the triazolo quinazolines was determined by analyzing the data using Graphpad Prism statistical tool and values are tabulated in Tables 1 and 2. Out of the 13 compounds under study, the compounds 8A, 16H and 16K displayed potent activity (14-49 µM) on both HepG2 (liver carcinoma) and MCF-7 (breast cancer) cell lines. Compounds 8B, 8F, 16L, and 15 displayed substantial activity against HepG2 cancer cell line (34-65 µM). Moreover, compounds 8 C-E, 8G, 16 I-J did not show any activity on the tested cell lines. These results indicate that introduction of vinyl and phenyl moiety is more important rather than cyclopropyl ring for cytotoxicity.

CONCLUSIONS
In the study, a variety of triazolo[4,3-c] quinazolines were synthesized in an ef icient manner and the compounds were well characterized by spectroscopic methods. The titled synthesized hybrids were screened for possible anticancer activity with two cancer cell lines and some of the compounds (8A, 16H, 16K) displayed substantial activity. SAR studies and synthesis of other substituted compounds and its library is underway in our laboratory for further biological activity will be reported in due course.