Synthesis, characterization and in vitro biological screening of 4-hydroxy naphthalen-1-yl, naphtho[1,2-b]furan, benzo[h]chromene and 5,6-dihydropyridazine derivatives containing sulfonamide moiety

In this study, a series of 4-((4-hydroxynaphthalen-1-yl)diazenyl)benzenesulfonamides have been prepared by subsequent diazotization of sulfonamide derivatives and coupling with 1-naphthol in alkaline medium. Cyclization of 4-((4-hydroxynaphthalen-1-yl)diazenyl)benzenesulfonamides with cinnamic acid in the presence of a basic catalyst afforded the novel naphtho[1,2-b]furans. Also, 4-((4-hydroxynaphthalen-1-yl)diazenyl)benzenesulfonamides can be cyclized with α‐cyanocinnamonitriles to afford 2-amino-3-cyano-4-phenyl-4H-benzo[h]chromenes. 4-(4-amino-3,5dicyano-6-iminopyridazin-1(6H)-yl)benzenesulfonamides were obtained at room temperature by treatment of 2-amino-1,1,3-tricyanopropene with a diazonium salt of sulfonamide derivatives. The structures of newly synthesized compounds were confirmed by analytical data and spectroscopic techniques. The antimicrobial activity of the obtained compounds was assessed in vitro by qualitative and quantitative (minimum inhibitory concentration) (MIC) assays.

Chromene derivatives are very an important class of heterocyclic compounds, widely distributed in natural products.Chromene and its derivatives have also been recognized as one type of 'privileged medicinal scaffolds' due to their unique pharmacological and biological activities 23 .Dong et al. 24,25 designed and prepared a series of 4-amino-2H-benzo[h]chromen-2-one and 4-amino-7,8,9,10-tetrahydro-2Hbenzo[h]chromen-2-one derivatives based on the potent anticancer agents neo-tanshinlactone and its 4-ethyl analogue 26 .
Sulfonamides are another important compounds family for the medicinal industry, and they are now extensively used drugs for the treatment or conservation of different illnesses 27 .In clinical medicine, they have been used as anticancer 28 , antimicrobial 29 , antiobesity 30 , carbonic anhydrase 31 and acetylcholinesterase inhibitor agents for Alzheimer's disease 32 .

Syntheses and characterizations of the compounds
A series of 4-((4-hydroxynaphthalen-1yl)diazenyl)benzenesulfonamides 3a-e were synthesized by coupling of diazonium salt of sulfonamide derivatives 1a-e with 1-naphthol 2 in presence of 10% sodium hydroxide (Scheme 1).Diazotization was carried out in the presence of nitrosyl chloride at 0-5 o C. The structure of compounds 3a-e was determined by their elemental analysis and spectral data.Elementary analysis indicated that sulfur was present.The infrared spectra of all isolated compounds were consistent with the assumed structures.The infrared spectra of compounds 3a-e showed the presence of absorption band at 3357-3448 cm -1 which is characteristic of the hydroxyl group beside two absorptions bands for azo and sulfone groups.The representative 1 HNMR spectrum of compound 3a (DMSO-d6) shown 7.1, 7.75, 8.35, 8.80 (4d, 4H, naphtho-H), 7.47, 7.58 (2m, 2H, naphtho-H), 7.94, 8.24 (2d, 4H, AB-system), 8.10 (s, 2H, NH2 exchangeable with D2O), 12.36 (br, 1H, OH exchangeable with D2O).The molecular ion peak of compound 3c was observed at m/z 410 (42.83%) corresponding to the molecular formula C19H14N4O3S2, and the base peak was found in the spectrum at m/z 65.The enolic-OH groups of all the compounds were chemically detected by the treatment with a FeCl3 solution, which gives characteristic color.

Scheme 3. Fragmentation pattern of naphtho[1,2-b]furan 6c
The formation of 6 from the reaction of 3 with 4 is assumed to proceed via initial addition naphtholate anion (C-2) in 3 to the activated double bond in 4 to yield the non-isolable intermediate Michael adduct (A) followed by intramolecular cyclization and subsequent decarboxylation to afford the naphthofurans 6 (Scheme 4).
The reaction of compound 3a with αcyanocinnamonitriles was investigated.Thus, the reaction of compound 3a with α-cyanocinnamonitriles 7 in refluxing DMF in the presence of piperidine afforded 2-amino-3-cyano-4-phenyl-4Hbenzo[h]chromenes 8a,b.The structure of 8 was supported by elemental analysis and spectral data.The infrared spectra of compounds 8a, b displayed absorption bands for NH2, C≡N, N=N and SO2 functions.The mass spectrum of compound 8a showed a molecular ion peak at m/z 481 (1.64%) corresponding to the molecular formula C26H19N5O3S.The formation of 8 from the reaction of 3a with 7 is assumed to proceed via initial addition of naphtholate anion (C-2) in 3a to the activated double bond in 7 to yield the non-isolable intermediate Michael adduct (B) followed by intramolecular cyclization through nucleophilic addition of the hydroxyl group to the cyano group and tautomerization 38 to afford benzochromene 8 (Scheme 5) .

Antimicrobial activity and minimal inhibition concentration
The newly synthesized compounds were evaluated for their in-vitro antibacterial activity against Staphylococcus aureus, Bacillus subtilis as examples of Gram-positive bacteria, Proteus vulgarisand Escherichia coli as examples of Gram-negative bacteria, using two standard antibiotics, Ampicillin, and Gentamycin as reference drugs and antifungal potential against a representative panel of fungal strains i.e.Aspergillus fumigatus (filamentous fungi), and Candida albicans (yeast), using one standard antibiotic, Amphotericin B as reference drug.The compounds were tested for their activity at a concentration of 10 mg/mL using inhibition zone diameter in mm as a criterion for the antimicrobial activity, and the results are shown in (Table 1).Based on the results, the newly synthesized compounds tested displayed variable invitro antimicrobial activities under these screening conditions.Interestingly, the tested compounds exhibited significant antifungal activities against the filamentous fungus (Aspergillus fumigatus) and unicellular yeast (Candida albicans).The highest antifungal activity was detected for compound 3b followed by 3a, 3e, 3d, 3c, respectively.However, compound 3e exhibited the highest activity against Gram-positive bacteria, Staphylococcus aureus as compared with the standard antibiotic, Ampicillin, followed by 3b, 3a, 8a and 3d, respectively.On the other hand, compound 3b exhibited the highest activity against Gram-positive bacteria, Bacillus subtilis followed by 3a, 3e, 3d and 6a, respectively.Moreover, the tested Gram-negative bacteria; Proteus vulgaris was highly susceptible to compound 3e followed by, 3d, 8a, 3b, 3a, 6a and 6b as compared with the standard antibiotic, Gentamycin.The order of activity against Escherichia coli was 3e >3b >3a >3d > 6a > 8a >3c >6c >6b >8b.Table 1.In-vitro antimicrobial activities of the synthesized compounds tested at 10 mg/mL by well diffusion agar assay and expressed as inhibition zone diameter (mm) in the form of mean ± standard deviation.Amphotericin B, ampicillin and gentamycin were used as standard drugs against the tested fungi, Grampositive and Gram-negative bacteria, respectively.

Experimental
All analyses were done at the Microanalytical Center, Cairo University, Cairo (Egypt).Melting points (uncorrected) were determined in open capillaries on a Gallenkamp melting point apparatus (Sanyo Gallenkamp, Southborough, UK).IR spectra (KBr discs) were recorded using a Shimadzu FT-IR 8400S spectrophotometer (Shimadzu, Kyoto, Japan), Infrared (IR) Spectra were recorded as KBr disks.NMR Spectra were recorded on a Bruker spectrophotometer (Bruker, Karlsruhe, Germany). 1 H spectrum was run at 400 MHz in deuterated dimethylsulfoxide (DMSO-d6).Chemical shifts are expresses in values (ppm) relative to TMS as an internal standard.Mass spectral data were given by a GCMS-QP1000 EX-spectrometer (Shimadzu, Kyoto, Japan) at 70 eV.All reagents used were of the Analytical grade.Compounds α-cyanocinnamonitriles 7 41 and 2amino-1,1,3-tricyanopropene 9 42 have been synthesized as previously reported.

General Procedure for Synthesis of 4-((4hydroxynaphthalen-1-yl)diazenyl)benzenesulfonamides 3a-e:
Sulfonamide (0.01 mole) was suspended in water (50 ml).Hydrochloric acid (10 ml, 36%) was added dropwise to this well stirred.The mixture was gradually heated up to 70 °C till clear solution obtained.The solution was cooled to 0-5 °C in an ice bath.A solution of NaNO2 (0.5 mg) in water (5ml) previously cooled to 0 °C, was then added over a period 5 minutes with stirring.1-naphthol (0.01 mole) was dissolved in 10% NaOH (10 ml) and then put ice to cool to 5 o C.Then, diazonium salt solution was added occasionally stirring very slowly to the 1-naphthol solution.The reaction mixture was left to complete for 15 min and occasional stirring; then the formed precipitate was filtered and dried in air and then recrystallized from proper solvent to give 3.

General Procedure for Synthesis of naphtho[1,2b]furans 6a-c.
To a mixture of compound 3 (0.01 mole) and cinnamic acid 4 (0.01 mole) in DMF (10 ml), a few drops piperidine was added.The reaction mixture was refluxed for 2 h.After cooling, the precipitate was filtered and recrystallized from proper solvent to give 6.

Procedure for Synthesis of benzo[h]chromenes 8a,b.
To a mixture of compound 3 (0.01 mole) and α-cyanocinnamonitrile 7(0.01 mole) in DMF (10 ml), a few drops triethylamine was added.The reaction mixture was refluxed for 1 h.After cooling, the precipitate was filtered and recrystallized from proper solvent to give 8.

General Procedure for Synthesis of 4-(4-amino-3,5dicyano-6-iminopyridazin-1(6H)-yl)benzenesulfonamides 11a-e:
Sulfonamide (0.01 mole) was suspended in water (50 ml).Hydrochloric acid (10 ml, 36%) was added drop wise to this well stirred.The mixture was gradually heated up to 70 °C till clear solution obtained.The solution was cooled to 0-5 °C in an ice bath.A solution of NaNO2 (0.5 gm) in water (5ml) previously cooled to 0 °C, was then added over a period 5 minutes with stirring.2-Amino-1,1,3-tricyanopropene 9 (0.01 mole) was dissolved in ethanol (10 ml) in the presence of sodium acetate (1 gram) and then put ice to cool to 5 o C.Then, diazonium salt solution was added occasionally stirring very slowly to the 2-Amino-1,1,3tricyanopropene solution.The reaction mixture was left to complete for 3h and occasional stirring; then the formed precipitate was filtered and dried in air and then recrystallized from proper solvent to give 11.

Biological evaluation
All microbial strains were provided from the culture collection of the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt.The antimicrobial activity was investigated on a dozen of newly synthesized compounds in order to increase the selectivity of these derivatives towards test microorganisms using the agar diffusion method using Mueller-Hinton agar medium for bacteria and Sabouraud's agar medium for fungi 43,44 .Briefly, 100 μl of the test bacteria/fungi were grown in 10 mL of fresh media until they reached a count of approximately 10 8 cells/ml for bacteria or 10 5 cells/mL for fungi.All the newly synthesized compounds were weighed and dissolved in dimethyl sulfoxide to prepare extract stock solution.
One hundred µL of each sample at 5 mg/mL was added to each well (10 mm diameter holes cut in the agar gel).The plates were incubated for 24-48 h at 37 °C (for bacteria and yeast) and 48 h at 28 °C (for filamentous fungi).After incubation, the microorganism's growth was observed.Ampicillin and Gentamycin were used as standard antibacterial drugs while amphotericin B was used as a standard antifungal drug.The resulting inhibition zone diameters were measured in millimeters and used as a criterion for the antimicrobial activity.If an organism is placed on the agar, it will not grow in the area around the well if it is susceptible to the chemical.This area of no growth around the disc is known as a Zone of inhibition.The size of the clear zone is proportional to the inhibitory action of the compound under investigation.Solvent controls (DMSO) were included in every experiment as negative controls.DMSO was used for dissolving the tested compounds and showed no inhibition zones, confirming that it does not influence the growth of the tested microorganisms.The active compounds were further investigated to determine their antimicrobial activity expressed regarding minimum inhibitory concentration (MIC) using the modified agar well diffusion method that mentioned above.Different concentrations of each active compound were tested and compared with standard drugs.The MIC was then determined as the lowest concentration inhibiting the growth of the organism after 24-48 h 43,45 .

Table 2 .
The antibacterial activities of the synthesized compounds expressed as minimum inhibitory concentration (µg/ml).