Structural Characterization and Antimicrobial Activities of 7H-Benzo[h]chromeno[2,3-d]pyrimidine and 14H-Benzo[h]chromeno[3,2-e][1,2,4]triazolo[1,5-c] pyrimidine Derivatives

Three new series of chromene molecules have been synthesized in order to explore their antimicrobial activity. The series encompass 2-substituted 14-(4-halophenyl)-12-methoxy-14H-benzo[h]chromeno[3,2-e][1,2,4]-triazolo[1,5-c]pyrimidines 7a–o, 9-benzylideneamino-7-(4-halo-phenyl)-5-methoxy-8-imino-7H-benzo-[h]chromeno[2,3-d]pyrimidines 8a–b and 3-ethoxycarbonyl-14-(4-halophenyl)-12-methoxy-14H-benzo-[h]chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine-2-one derivatives 12a–b. The structure of these novel compounds were confirmed using IR, 1H- and 13C-NMR as well as MS spectroscopy. The new compounds were evaluated in vitro for their antimicrobial activity and it was demonstrated that 7H-benzochromenopyrimidine and derivatives of 14H-benzochromenotriazolopyrimidine exhibited the most promising antibacterial activities compared to the reference antimicrobial agents. The structure activity relationship (SAR) studies of the target compounds agreed with the in vitro essays and confirmed higher potent antimicrobial activity against some of the tested microorganisms.


Introduction
The development of antimicrobial agents is an area of great activity due to the emergence of multidrug resistance in common pathogens and the appearance of new infections. Infectious diseases are considered the second-leading cause of death worldwide; consequently, tremendous efforts have been made to develop new antimicrobial agents that are active especially against the drug resistant strains.
The majority of antimicrobial agents are diverse five-and six-membered heterocyclic molecules that play a crucial role in the metabolism of all living cells [1]. Moreover, a great deal of interest has been directed toward condensed ring systems due to their various types of physiological activities and the success in utilizing them as privileged medicinal scaffolds. In particular, fused chromene and benzochromene molecules have become some of the best potential candidates for pharmacological purposes due to their antimicrobial [2,3], antileishmanial [4,5], anticancer [6,7], antiproliferative [8],  [9,10], hypertensive [11], antitumor [12][13][14][15] effects and activities, as well as for the treatment of Alzheimer's disease [16] and schizophrenia disorders [17].

Antibacterial Evaluation
The target compounds 7a-o, 8a-c and 12a-c were tested in vitro for their antimicrobial activities by the agar diffusion method using Mueller-Hinton agar medium for bacteria and Sabouraud's agar medium for fungi [32,33]. The tested microorganisms were obtained from the Regional Center for Mycology & Biotechnology (RCMP), Al-Azhar University.

Antibacterial Evaluation
The target compounds 7a-o, 8a-c and 12a-c were tested in vitro for their antimicrobial activities by the agar diffusion method using Mueller-Hinton agar medium for bacteria and Sabouraud's agar medium for fungi [32,33]. The tested microorganisms were obtained from the Regional Center for Mycology & Biotechnology (RCMP), Al-Azhar University.

Antibacterial Evaluation
The target compounds 7a-o, 8a-c and 12a-c were tested in vitro for their antimicrobial activities by the agar diffusion method using Mueller-Hinton agar medium for bacteria and Sabouraud's agar medium for fungi [32,33]. The tested microorganisms were obtained from the Regional Center for Mycology & Biotechnology (RCMP), Al-Azhar University.

General Information
Commercial-grade solvents and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. Melting points were measured with a Stuart Scientific (Stone, Staffordshire, UK) apparatus and are uncorrected. IR spectra were determined as KBr pellets on a Jasco FT/IR 460 plus spectrophotometer (Jasco, Tokoyo, Japan). 1 H-NMR (500 MHz) and 13 C-NMR (125 MHz) spectra were recorded using a Bruker AV 500 MHz spectrometer (Bruker, Billerica, MA, USA). Chemical shifts (δ) are expressed in parts per million (ppm). The 1 H-NMR and 13 C-NMR spectra of the compounds are provided in the Supplementary Material. The MS were measured using a Shimadzu GC/MS-QP5050A spectrometer (Shimadzu, Tokoyo, Japan). Elemental analyses were carried out at the Regional Centre for Mycology & Biotechnology (RCMP, Al-Azhar University, Cairo, Egypt) and the results were within ±0.3% of calculated values. Analytical thin layer chromatography (TLC) on silica gel precoated F 254 plates (Merck, Billerica, MA, USA) was used to check the purity of the final compounds and intermediates.  [34][35][36][37], [29][30][31] and [29][30][31], respectively).

General Procedure for the Synthesis of Compounds 7a-c
A mixture of aminoimino compound 6a-c (3.88, 4.04, or 4.49 g, respectively, 0.01 mol) and formic acid or methyl formate (0.46 g or 0.6 g, 0.01 mol) in dry benzene (30 mL) was refluxed for 5 h. The solvent was extracted and the resulting products were recrystallized from ethanol/1,4-dioxane to give 7a-c. The physical data of the compounds 7a-c are as follows:

General Procedure for the Preparation of Compounds 7d-f
A mixture of aminoimino compounds 6a-c (3.88, 4.04, or 4.49 g, respectively, 0.01 mol) and acetyl chloride or acetic anhydride (30 mL) was refluxed for 2 h. The solvent was extracted and the resulting product was recrystallized from 1,4-dioxan to give 7d-f. The physical data of the compounds 7d-f are as follows: Method B: A mixture of compounds 8a-c (4.76, 4.92 or 5.37 g, respectively, 0.01 mol), dioxane (20 mL) and piperidine (0.5 mL) was refluxed for 2 h. The solvent was extracted and the resulting product was recrystallized from ethanol/1,4-dioxane to give 7m-o in 60% yield (as verified by m.p., mixed m.p., identical IR and MS spectrum).
The physical data of the compounds 7m-o are as follows: