Copper mediated one-pot synthesis of quinazolinones and exploration of piperazine linked quinazoline derivatives as anti-mycobacterial agents

A facile method was developed for the synthesis of quinazolinone derivatives in a one-pot condensation reaction via in situ amine generation using ammonia as the amine source and with the formation of four new C–N bonds in good to excellent yields. With the optimised method, we synthesized a library of piperazine linked quinazoline derivatives and the synthesized compounds were evaluated for their inhibitory activity against Mycobacterium tuberculosis. The compounds 8b, 8e, 8f, 8m, 8n and 8v showed potent anti-mycobacterial activity with MIC values of 2–16 μg mL−1. All the synthesized compounds follow Lipinski's rules for drug likeness.


Introduction
Nitrogen-containing heterocycles are present in a wide range of bioactive natural products and synthetic drug candidates. 1 Among them, quinazolines and their derivatives represent medicinally important structural cores present in a number of drug candidates. 2 They possess a wide range of biological activities including anticancer, 3 antiviral, 4 antitubercular 5 and antibacterial 6 properties.
Recently, ammonia has attracted wide attention as a costeffective and efficient nitrogen source. 7 A number of homogeneous transition-metal catalysed reactions for the synthesis of organic amines using gaseous or liquid ammonia are reported. 8,9 Owing to its safety and ease of handling, aqueous ammonia is even more attractive as a substrate.
In view of the medicinal and pharmacological importance of quinazolinones, several methods on the synthesis of this class of compounds have been reported. Zhan and co-workers 10 in 2013 reported an interesting approach by condensation of substituted anthranilamides with different aldehydes in presence of copper oxide for the synthesis of substituted quinazolinone derivatives. In 2014, Hung and co-workers 11 reported synthesis of quinazolinone by using 2-bromobenzoic acid and substituted amidines as starting materials. Abe et al. 12 reported the synthesis of quinazolinones by using 2-amino benzoic acid with substituted nitriles (Scheme 1).
Tuberculosis is a transmissible disease caused by Mycobacterium tuberculosis (Mtb) complex and recognized to have high mortality rate globally 13 According to WHO reports, 10 million cases were reported in the year 2017, where India is the leading country with the highest burden of TB. 14 Emergence of drug-   In the present method, we have developed a copper mediated oxidative coupling of different aldehydes and 2-bromobenzoic acid, using aq. ammonia as a less expensive nitrogen source. We explored the double amination of aryl halides to the corresponding amines and also acids to amides at the same substrate. With the established method, we could successfully synthesize 4-substituted piperazine/piperidine linked C2-aryl/ heteroaryl quinazolines. The synthesized compounds were evaluated for their in vitro inhibitory activity against Mycobacterium tuberculosis H37Rv. clog P values were determined using SwissADME.

Results and discussion
Direct employment of ammonia as a reagent in transition-metal catalysis is generally a challenging task. In the current optimised method, copper mediated oxidative coupling is developed between aldehydes and 2-bromobenzoic acid, using aq. ammonia as a nitrogen source. Various conditions for the conversion of 1 to 3 are studied and the results are discussed in Table 1. We initiated our studies by using copper oxide and NMP as solvent in the absence of oxygen at 80 C for 24 h ( Table  1, entry 1). The reaction did not proceed. However, we observed the formation of the product in small quantities when the Scheme 1 Methodologies for the synthesis of quinazolinones.
Scheme 2 Reaction scope. oxide and CuI as catalysts in presence of oxygen, NMP and DMSO as the solvent, the reaction proceeded smoothly. DMSO as a solvent was found to be more favourable (Table 1, entry 9-10). The reaction was studied with different solvents viz., ACN, toluene, DMF and the reaction proceeded with altered yields (Table 1, entry [11][12][13][14]. When the catalyst was changed to copper diacetate and coppertriate the reaction was sluggish and resulted in low yields (Table 1, entry 15-17). The reaction was found to proceed with optimal yields with CuI as catalyst and DMSO as the solvent (entry 10). Aer optimizing the reaction conditions, we focussed on expanding the substrate scope of this transformation and the results are summarized in (Scheme 2). It is observed that electron-donating substituents such as methoxy, amino, and methyl on 2-phenyl were well tolerated under the optimal reaction conditions, with 70-78% yields (3b, 3c, 3g and 3h). Similarly, halogen substituents like 2,4-dichloro, 2,6-dichloro  This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 43533-43538 | 43535 and 3-nitro are also tolerated, yielding the desired products (3j, 3i and 3l) in good to high yields (75-79%). Heterocycles like pyridyl, furan, thiophene and isoxazole at C-2 position (3d, 3e, 3f and 3m) are also well tolerated with good to moderate yields (78-82%). With 4-bromo and 4-cyano (3o and 3n) substituents, the reactions proceeded smoothly (Scheme 2).
We performed the gram scale synthesis with 1.5 gm of 2bromo benzoic acid and benzaldehyde as the starting materials which resulted in 1.2 g of the nal product (Scheme 3). As the reaction with TEMPO did not show the product formation, the free radical mediated mechanism is observed and conventional mechanism in one-pot protocol is proposed (Scheme 3). Based on the control experiments, a plausible reaction mechanism is proposed and depicted in Scheme 4. Our mechanistic investigation was supported by ESI-QTOF-MS technique and collected the mass data at different time intervals with <5 ppm error. Initially, under copper catalysis substrate 1 gets converted to intermediate I. on oxidation gives the desired products 3a-o in good to moderate yields, for the corresponding product the peak was observed with [M + H] at m/z of 223.0896. The product formation was observed aer 6 h but not completely, further preceding the reaction for 10-12 h to get the complete conversion (Fig. 2).
A series of 2-arylquinazoline derivatives were synthesized with the optimized method as described in Scheme 2. The obtained quinazolinones (3a-f) were further chlorinated using POCl 3 and N,N-diethyl aniline to provide the corresponding 2aryl chloroquinazoline intermediates 4a-f. The chlorinated intermediates 4a-f were treated with piperazine 5 to yield 2-aryl-4-(piperazin-1-yl)quinazoline 6a-f. Coupling of 6a-f with a number of carboxylic acids 7a-i using HATU as coupling reagent afforded the corresponding amide derivatives 8a-z in moderate to excellent yields. Structures of all the newly synthesized compounds were conrmed by 1 H NMR, 13 C NMR and HRMS (ESI) spectroscopic techniques (Scheme 5).
The synthesized derivatives were evaluated for their antimicrobial activity against ESKAP pathogen panel (results included in the ESI †) and Mycobacterium tuberculosis H37Rv strain. [18][19][20] While, the compounds were found to be inactive against ESKAP pathogenic panel, some of the compounds were found to exhibit promising inhibitory activity against M. tuberculosis H37Rv strain (Table 2). A perusal of the results indicated that among the amide derivatives, compounds 8f exhibited potent anti-mycobacterial activity with MIC of 2 mg mL À1 and 8n showed an MIC of 4 mg mL À1 . Further, the results indicated that four compounds 8b, 8e, 8m and 8v showed MIC of 16 mg mL À1 whereas other molecules were found to be moderately active or inactive. Presence of methoxy group on 2-phenyl moiety as in 8k and 8m resulted in moderate activity with MIC of 16-64 mg mL À1 . Replacement of C2-phenyl with 4-pyridyl was found to be a favourable lead. Substitution of R 2 position with electron withdrawing groups like 2-chloro-5-bromo phenyl 8t showed inhibitory activity with MIC of 32 mg mL À1 . Compounds having 2-phenyl with electron donating groups like 3,5-dimethoxy and 3,4,5-trimethoxy 8s and 8q were found to be inactive but 4-N,Ndimethylphenyl was found to be good lead with MIC of 2 mg mL À1 . Compounds with halogen containing groups like 5bromo-2-chlorophenyl 8y exhibited moderate activity with MIC of 32 mg mL À1 whereas 4-iodo 8r was inactive and unfortunately rest of the molecules were devoid of activity (Table 2 and Fig. 3).

Conclusions
In conclusion, we have developed an operationally simple, onepot and cost-efficient method for the preparation of quinazolinones with diverse substituents. This method uses mild catalytic system which enables effective construction of four C-N bonds in one pot operation through in situ amine generation, conrmed through the ESI-MS technique. By using the optimised method, we generated a library of new piperazine linked 2-aryl/hetero-aryl-quinazoline derivatives which were evaluated for their anti-microbial activity against ESKAP pathogen panel and also against M. tuberculosis. Among the tested compounds, 8f exhibited selective and potent anti-mycobacterial activity with MIC value 2 mg mL À1 . Compounds 8b, 8e, 8m and 8v exhibited moderate anti-mycobacterial activity with MIC value 16 mg mL À1 . All the synthesized compounds obey the Lipinski rule of clog P values.

Conflicts of interest
The authors declare no conict of interest.