Synthesis and Antimicrobial Evaluation of Some Tricyclic Substituted Benzo [ h ] quinazolines , Benzo [ h ] quinolines and Naphthaleno [ d ] thiazoles

Objectives: The notable advancing in the medicinal chemistry arena against infectious diseases has resulted in the discovery of numerous valuable antimicrobial drugs that saved millions of lives throughout the last few decades. Nevertheless, the continuous reporting of multi-drug resistant strains of a number of diverse germs makes the need for novel broad-spectrum anti-infective agents still exists. Methods: Three series of ring-equivalent tricyclic benzo[h]quinazolines (compounds IIIa,b, IVa,b and Va,b), benzo[h]quinolines (compounds VIa-g, VIIa-c and VIIIa-c), and naphthaleno[d]thiazoles analogs (compounds X & XIa-e ) were designed as bioisosteres and synthesized. Benzo[h]quinazolines were respectively obtained via treating chalcones IIa,b with urea, thiourea and guanidine carbonate in basic medium The one-pot reactions of 6-methoxy-1-tetralone I with different aromatic aldehydes and active methylenecontaining agents namely ethylcyanoacetate, malononitrile and thiocyanoacetamide in the presence of excess ammonium acetate afforded benzo[h]quinolines compounds VIa-g, VIIa-c and VIIIa-c respectively. Naphthaleno[d]thiazoles X & XIa-e were yielded upon cyclocondensation of the bromotetralone IX with thiourea and aldehydethiosemicarbazones. The chemical structures of the synthesized agents were elucidated based on their spectral data and elemental analyses. Results: The antimicrobial activity of some of the synthesized compounds was screened and only 2-bromo derivative IX and aminothiazole compound X exhibited broad antimicrobial effectiveness. The antifungal MIC value of X was1.85 mg/mL. Conclusion: The new synthesized compounds, designed as bioisosteres, showed excellent potency against the tested gram (+) bacterial and fungal strains compared to reference drugs.


INTRODUCTION
Life threatening systemic infections continue to be a remarkably significant problem in healthcare.The microbial self-defense everlasting genetic mutations resulted in the elevated levels of multi-drug resistant strains of variable virulent pathogens and emerging of new infectious diseases.This issue is especially pronounced in immune-compromised and hospitalized patients and those under immune-suppressing therapy in organ transplantation and cancer treatment 1,2 .
In view of the drawbacks of many of the marketed antimicrobial agents and the high incidence of bacterial resistance to their activities, there is an urgently high demand for discovery of safer and more effective broad-spectrum agents.Among the substantial armories in the medicinal chemistry arsenal are the design and development of novel chemical entities of diverse mechanisms of actions to surmount any potential crossresistance of the current medications 3 .Quinazoline nucleus is one of fused heterocyclic ring systems that possesses a wide array of biological activities.These pharmacological activities include DNA binding 4 , antitumor [5][6][7] , benzodiazepine and GABA receptor ligand activity 8 , antiviral 9,10 , antibacterial 11 , antituberculosis 12,13 and cellular phosphorylation and tyrosine kinase inhibition 14 .Many marketed drugs comprise quinazoline nucleus in their structures such as; the α1-blocker alfuzocin, which is used in the treatment of BPH (benign prostate hyperplasia), the oral hypoglycemic balaglitazone, dacomitinibthat is used in the treatment of non-small-cell lung carcinoma (in phase III clinical trials) and the broad-spectrum antifungal albaconazole 15 (Figure 1).
Similarly, quinoline condensed ring system has been found in a considerable number of pharmacologically active natural products and in clinically active drugs [16][17][18] .The broad diversity of pharmacological activities of quinoline-containing compounds includes antipsychotic 19 , anti-inflammatory 20,21 , antioxidant 22 , anti-HIV 23 , antifungal 24 , treatment of lupus 25 , treatment of neurodegenerative diseases 26 , antitubercular 27,28 , antiprotozoal 29,30 and anticancer 31,32 .Among the marketed drugs carrying quinoline nucleus is the fluoroquinolone antibacterial ciprofloxacin.Most of the quinolone family members of drugs are the antimalarial chloroquine, the anti-retroviral saquinavir, the HMG-CoA reductase inhibitor pitavastatin and the farnesyltransferase inhibitor tipifarnibusedfor the treatment of leukemia 33 (Figure 2).

Figure. 2. Representative examples of marketed quinoline-containing Drugs
Moreover, benzothiazole fused heterocyclic scaffold has been reported to have a relatively variable pharmacological effects such as antimicrobial 34 , anticancer 35,36 , anti-inflammatory 37 , β-2 adrenoceptor agonist and antidepressant activities 38 .Figure 3 depicts some of benzothiazole-containing drugs like; the sulfonamide diuretic ethoxozolmide, the antiviral and immuno-suppressive frentizole, the glutamate receptor antagonist riluzole used in treatment of amyotrophic lateral sclerosis, the anti-diabetic zopolrestat and the amyloid imaging agent thioflavin T 38 .
Driven by the urgent need to develop novel antimicrobial agents of broader spectrum of activity, more potency and less toxicity and in the shade of the aforementioned facts, we were interested in the synthesis of some structurally related congener tricycles of benzo[h]quinazolines, benzo[h]quinolines and naphthaleno [d]thiazoles.The designed isosteric tricycles were synthesized to rummage their potential synergistic antimicrobial activities.

Chemistry
Melting points were determined on STUART scientific melting point apparatus and were uncorrected.The reactions times were determined using the thin layer chromatography (TLC) technique which was performed using plates of aluminum oxide coated with silica gel F 254 (Merck) neutral type and the eluent was chloroform/methanol (9:1).The organic solvents and extracts were dried with anhydrous Na2SO4 or anhydrous MgSO4.The infrared (IR) spectra were recorded on Bruker FT-IR spectrophotometer as potassium bromide discs.Mass spectra (MS) were performed at 70 EV with Shimadzu GCMS, QP1000 EX using the Electron Ionization Technique (EI).The proton nuclear magnetic resonance ( 1 H-NMR) spectra were recorded in (DMSO-d6 and CDCl3 on Varian Gemini spectrophotometer at 200 MH and Varian Mercury spectrophotometer at 300 MHz, using tetramethylsilan (TMS) as an internal reference and the chemical shift values (δ) are given in part per million (ppm).Elemental analysis were carried out at the Micro analytical Center, Cairo University.6-Methoxy-1-tetralone I is commercially available.

General procedure for the synthesis of quinazolinesIIIa&b, IVa&b and Va&b
An alcoholic solution of urea, thiourea or guanidine carbonate (0.01mol) in absolute ethanol (10 mL) was added to a mixture of the appropriate chalcones IIa and/or IIb (0.01mol), potassium hydroxide (1.12g, 0.02 mol) in absolute ethanol (30 mL).The reaction mixture was allowed to reflux for 6 h and the solvent was removed under reduced pressure.The separated residue was washed with water and crystallized from ethanol.

Materials
All microorganisms used were obtained from the culture collection of the department of Microbiology and Immunology, Faculty of Pharmacy, Helwan University.The compounds were tested against E. coli and S. aureusinnutient broth at pH 7 and against B. subtilis in bacto brain heart infusion broth at pH 7 and C. albicans in broth containing 1 % neopeptone and 2 % dextrose at pH 5.7 Media for disc sensitivity tests were nutrient agar and Muller Hinton agar (MHA) purchased from Difco.The disc diameter was 6 mm.Non sterile powder of the tested compounds were dissolved in sterile DMSO to yield 5 mg/ ml passed through 0.2 μm membrane filters (Millipore corp.Bedford, Mass).

Disc diffusion test
20 mLof Muller-Hinton agar (MHA) at 55 o C, inoculated with 1mL of the microbial culture (10 6 CFU/Ml), was poured in sterile Petri dish and left to solidify.A sterile filter paper disc impregnated with solution of the compound under testing(100μg/mL in DMF) was placed on the surface of agar , and the plate was incubated overnight at 37 o C. The diameter of the zone of inhibition was measured and compared with the standard zone produced by amoxicillin.

Tube dilution technique
Nine dilutions of the tested compounds were made in Mueller-Hinton broth to obtain test concentrations ranging from 50 to 0.2 μg/mL.The concentrations of the microorganism were adjusted as to give an innoculum of 106 CFU in each tube.The tubes were incubated overnight at 37 o C.The inhibitory concentration was determined and compared to that of Amphotricin B, Clotrimazole and Ketoconazole.

Chemistry
The starting material 6-methoxy-1-tetralone I, comprises a cyclic buterophenone which is suitable to furnish the versatile intermediateα,β-unsaturated ketones, the chalcones IIa-f.These chalcones were synthesized via Claisen-Schemidt condensation, a type of aldol condensation of aromatic aldehydes with the corresponding substituted acetophenone in the presence of sodium, potassium or barium hydroxide [39,40] as illustrated in Scheme 1.
The reaction of I with different aromatic aldehydes namely, p-chlorobenzaldehyde, p-dimethylaminobenzaldehyde, 3,4-dimethoxybenzaldehyde, cinnamaldehyde, p-nitrobenzaldhyde and m-methoxybenzaldehye, in the presence of 10% potassium hydroxide uneventfully afforded the corresponding 2-[(substituted phenyl)methylene]-6-methoxy-3,4-dihydro1(2H)-naphthalenes IIa-f in 65-90% yields.The structures of compounds IIa-f were confirmed by microanalytical analyses and spectral data (IR, 1 H-NMR and mass spectra).The IR spectra showed the characteristic carbonyl stretching bands at 1637-1663 cm -1 in addition to other bands correlating with their structures.The 1 H-NMR spectrum of IIa, as an example, revealed two triplets at  2.91, 3.14 ppm referring to the C3 and C4 alicyclic methylene protons, two singlet signals at  3.93, 6.71 ppm attributed to OCH3, and the vinylic proton respectively, alongside multiplet signals at  6.80-8.22ppm attributed to the seven aromatic protons of the compound.The mass spectrum of the same compound showed two isotopic molecular ion peaks at m/z 297 and 299 which is in agreement with its molecular formula.In addition, the mass spectra of the chalcone derivatives IIb-f exhibited the molecular ion peaks of the compounds confirming their structural formulae These chalcones are considered to be useful intermediates in several cyclization reactions to produce different types of heterocyclic compounds according to the used reactants and the reaction's conditions 40,41 .Thus, in the present investigation, the target quinazolines IIIa,b, VIa,b and Va,b were obtained by condensation of the prepared chalcones IIa,b with urea , thiourea and guanidine salt respectively in refluxing ethanol under alkaline medium conditions.The appearance of the absorption bands of the NH and C=O groups at 3344and 1668 cm -1 , respectively in the IR spectrum of compound IIIb agreed with the proposed structure.In addition, the 1 H-NMR spectrum of compound IIIa revealed the disappearance of the vinylic proton singlet assigned for IIa, and the presence of the alicyclic and methoxy protons at  2.91, 3.14 and 3.93 ppm respectively in addition to multiplet signals at  6.90-7.96ppm for the seven aromatic protons.
Furthermore, the mass spectra of IIIa and IIIb displayed the molecular ion peaks which were in agreement with the proposed structures in addition to a common peak at m/z 229 assigned to a molecular fragment ion with side chain p-substituted phenyl missing moiety.
The quinoline analogs VI, VII and VIII could have been synthesized as cited in the literature through a base catalyzed cyclocondensation reaction of ,unsaturated ketones with ethylcyanoacetate in the presence of ammonium acetate [42].This method is time consuming and suffers from some difficulties especially with propenones bearing heterocyclic moieties.Another approach involves the condensation of an active acetyl function and the appropriate arylidinecyanoacetate, arylidinethiocyanoacetamide or arylidinemalononitrile in the presence of ammonium acetate as a source of ammonia [43].The one-step multi-component reaction approach was effectively employed since it is easy to perform and gives better yields in less reaction times.
The IR spectra for compounds VIa-g showed stretching bands at 3462-3281, 2212-2219 and 1635-1715 cm -1 corresponding to NH, CN and C=O groups, respectively.The 1 H-NMR spectrum of compound VIa displayed a broad D2O exchangeable singlet signal at 12.5 ppm attributed to the lactam proton, alongside multiple signals at  6.9-8.1 ppm for the seven aromatic protons.The MS spectrum of compound VIIIa displayed a base peak at m/z 361 attributed for its molecular ion.The sensitivity of the microorganisms towards the tested compounds is identified in the following manner: * significant.** Highly significant.