Indian Journal of Chemistry Vol. 55B, June 2016, pp. 724-733

Study of solvatochromic behavior and antimicrobial activities of some newly synthesized bis-azo-dapsone congeners Jyotirmaya Sahoo* & Paidesetty Sudhir Kumar Department of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Siksha‘O’Anusandhan University, Bhubaneswar 751 003, India E-mail: [email protected] Received 14 January 2015; accepted (revised) 3 April 2016 In the present study, a series of new bisazo dyes derived from dapsone have been synthesized in one step, using diazotized coupling reaction and evaluated for their in-vitro antimicrobial activity. Ampicillin and fluconazole have been taken as reference antibiotics (RA). The structure of synthesized compounds are confirmed by different spectral techniques viz. elemental analysis, 1H NMR, UV-Vis, FT-IR and mass spectrometry. The solvatochromic behavior of the synthesized compounds are also studied by UV-Vis spectrometry. The compound 4b has been observed with significant antibacterial activity against Shigella flexneri, Escherichia coli, Vibrio cholera and Streptococcus mitis in comparison to standard drug whereas all the compounds except 4f show significant antifungal activity against Aspergillus niger. The results have been statistically interpreted by one way analysis of variance (ANOVA) followed by Dunnett’s Post Hoc test. Exploitation of dapsone molecule by the attachment of different nucleophiles may be responsible for the significant increase of antimicrobial activity. However, the 8-hydroxy quinoline linked bisazo dapsone showed highest significant antimicrobial activity than the other newly synthesized bisazo dapsone analogues in comparison to RA. Keywords: Azocoupling, antibacterial, antifungal, dapsone, solvatochromic

The manifestation of skin related microbial infections still remain a challenging clinical problem. Some crucial factors include an increasing multidrug resistance of microbial pathogens towards antibiotics and more incidences of multiple serious adverse effects. Microbial resistance towards antibiotics makes the work of researchers searching for new organic compounds having potential effect against pathogenic bacteria and fungus more challenging. Dapsone is a chemically derived sulphone antimycobacterial agent and the most commonly used in conjugation with rifampicin and clofazimine dye for treatment of the mycobacterial leprae infection1, as an anti-infective agent. It is also used for treatment of malaria and recently pneumocysticcarnii pneumonia (PCP) in HIV patient2. The mode of antibacterial action of a dapsone inhibits synthesis of bacterial nutrient dihydro folic acid via competition with para amino benzoate for active site of dihydro folic acid ester synthetase. It has also anti-inflammatory and immunomodulatory effect3. The azo linked sulphonamide prodrug such as prontosil (sulfonamidochryosoidine) was the first effective chemotherapeutic agents that could be used systemically for streptococcal infection in humans4.

The preparation of diazo-coupled ligands such as dithizone and hydroxyl quinoline coupled to benzidinecarboxy methyl cellulose were applied towards the recovery of trace metals from sea water5. Azo compounds are versatile compounds that have various applications in industry as well as photosensitive species in photographic system6. In addition to these, heterocyclics bearing azo moiety also have a wide range of pharmacological activities including antibacterial7, antitumor8, 9 antioxidant and urease inhibitor10. Furthermore, 4-hydroxy coumarin and their derivatives have shown their diverse biological activities including antibacterial11, antioxidant12, antiviral13, anti14 15 inflammatory and antitumor . Similarly, 8-hydroxy quinoline (oxine) and their metal complexes have shown activity over a broad pharmacological spectrum including antiseptic16, antibacterial17, anticancer18 and anti amoebic19 activities. Some other bio-active organic compounds having good coupling properties are with thiobarbituric acid, salicylic acid, thymol and β-napthol. Literature survey revealed that the diazotization of dapsone can be coupled with α-naphthol in the presence of sodium carbonate20. Therefore it was thought of interest to

SAHOO & KUMAR: BIS-AZO-DAPSONE CONGENERS

incorporate bisazo group from diazotized dapsone at reactive active sites of different potent coupling components by azo coupling reaction and to investigate their synergistic antimicrobial effect. Some of the effective biologically active drugs containing azo linkage are phenazopyridine, basalazide and prontosil for the treatment of urinary tract, analgesic, anti-inflammatory and antistreptococcal activities respectively. Results and Discussion Chemistry A series of seven bisdiazo compounds derived from dapsone 4a-g were synthesized by coupling of diazotized dapsone with different nucleophiles in presence of nitrosyl chloride under mild conditions (Scheme I). The excess of nitrous acid in the reaction was neutralized by addition of urea crystals. The nucleophiles are β-napthol, 4-hydroxycoumarin, salicylaldehyde, salicylic acid, 8-hydroxyquinoline, thiobarbituric acid and thymol. The coupling agents such as thiobarbituric ring were synthesized by

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condensation of thiourea and sodium salt of diethyl malonate in hydrochloric acid. The IR spectrum of precursor 4-HC showed strong absorption band at 3417 cm−1 corresponding to –OH group and band at 1698 cm−1 with respect to lactone carbonyl of 4-hydroxycoumarin. Similarly, 8-hydroxyquinoline was made from 2-aminophenol and glycerol in presence of H2SO4 and oxidized by nitrobenzene. The generated bisphenyldiazonium salt from dapsone has the highly electrophilic nitrogen which undergoes simple azo-coupling reaction with appropriate nucleophiles21. Progress of the reaction was monitored by TLC using suitable solvent system. The majority of the synthesized compounds were colored and their physical characteristics are reported in Table I. The infrared spectra of the all the prepared compounds showed sharp absorption bands due to presence of sulphonyl SO2str., OHstr., Ar-CHstr, and –N=N-str. The Ar-CHstr. signal in the infrared spectrum of the prepared compound 4b shows two strong broad absorption bands at 3140 and 3460 cm−1 due to stretching vibration of hydroxyl group of 8-HQ

Scheme I

INDIAN J. CHEM., SEC B, JUNE 2016

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Table I ― Physical characteristic data of newly synthesized bis azo dapsone analogues 4a-g Compd

R- (coupling component)

Mol. formula

m/z

Rf

m.p. (°C)

Colour

Yield (%)

4a

4-hydroxy

C26H18N4O6S

594.99

0.6

310-20

Yellow

85

C30H20N6O4S

560.13

0.8

210-20

Dark Red

85

C26H18N4O8S

514.09

0.8

230-40

Brick Red

92

C32H22N4O4S

558.13

0.7

270-80

Cherry Red

85

C26H18N4O8S

545.03

0.6

220-30

Brick Red

78

C20H14N8O6S3

558.57

0.8

270-80

Brick Red

80

C32H34N4O4S

517.13

0.8

130-40

Brown

73

Ο

Ο

ΟΗ

coumarin

8- hydroxy

4b

Ν

ΟΗ

quinoline 4c

Ο salicylaldehyde ΗΟ

4d

ΟΗ β-napthol

ΟΗ

4e

Ο salicylic acid 4f

ΗΟ

Thiobarbituric acid

Ο

ΗΝ Ν Η

S 4g

Ο

Thymol

CH3

OH H3C

CH3

and due to intermolecular and intramolecular hydrogen bonding between hydroxyl group and nitrogen of quinoline nucleus. So the absorption band is broad22. The compound 4a shows three strong absorption bands at 1745, 1398 and 1146 cm−1 assigned to stretching vibration of lactone carbonyl of

coumarin and asym/sym stretching of sulphone group of dapsone respectively. The medium frequency bands appeared at 1508 and 1293 cm−1 which were assigned to (-N=N-)str. and C-Ostr. respectively. Instead of functional thiol (SH) group, the compound 4f was in the thionic form indicated by the absorption band in

SAHOO & KUMAR: BIS-AZO-DAPSONE CONGENERS

the region around 1348 cm−1. The FT-IR spectrum of compound 4f showed two strong absorption bands at 1690 and 1654 cm−1 due to presence of the dicarbonyl group in the structure. The infrared spectrum of the compound 4a showed a strong broad absorption band at 3460 cm−1 corresponding to –OH group due to intra molecular hydrogen bonding between enolic –OH and azo nitrogen and the sharp band at 1745 cm−1 with respect to lactone carbonyl of coumarin and further chemically present enolic hydroxyl confirmed by treatment with aqueous solution of ferric chloride. In salicylaldehyde analogue 4c, the carbonyl stretching of aldehyde and hydroxyl group showed absorption bands at 1656 and 3451 cm−1 respectively. The reported LC-MS spectra of all synthesized compounds 4a-g revealed molecular ion peaks which strongly confirmed their predicted molecular formula. The compound 4f at Rt 1.568 min having percentage of area 96.78% and molecular ion peak at m/z 556 strongly supports the predicted molecular formula C20H14N8O6S3. The 1H NMR spectrum of aromatic protons of compound 4a appeared at δ 7.92-7.89 doublet, 7.42-7.36 multiplet, 7.65-7.55 multiplet and 7.59 -7.42 doublet and among these aromatic protons, H-6 and H-8 protons were more shielded23. 1H NMR of the synthesized compound 4f suggested that unsubstituted active methylene of thiobarbituric acid showed singlet peak at δ 4.05 for two protons not

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showed in 1H NMR spectra. Two amide protons and highly deshielded NH/OH proton appeared at δ 11.25, 11.85 and 14.55 respectively10. Eight aromatic protons and shielded aryl protons of 4,4'-bisazothiobarbituric acid of dapsone appeared at δ 7.72-8.03. Singlet proton of –CH-N=N(diazomethine) appeared at δ 3.45-3.55. For compound 4c it was observed that aldehydic-H and hydroxyl group showed singlet peak at δ 8.85 and 10.56 respectively. Solvatochromic effect on synthetic bis-azo dapsone congeners, 4a-g The UV-Vis absorption spectra of compounds 4a-g were recorded over the range of λ max between 300-600 nm using different solvents. The absorption spectra of the dyes 4a-g were recorded in eight different solvents at a concentration of 10−5 to 10−6 M and results are reported in Table II. Seven prepared dyes 4a-g showed the solvatochromic effect for different coupling components attached with dapsone using eight different solvents such as acetone, methanol, ethanol, isopropanol, tetrahydrofuran, 1,4dioxane, dimethyl sulfoxide and dimethyl formamide. According to UV-Vis data in Table II, for the compound 4d, introduction of two moles of β-napthol substituent to dapsone, coupled with diazo functional groups at the C-4 and C-4′ position gave a large bathochromic shift in most of the solvents with respect to λmax. In compound 4f, insertion of two

Table II ― Electronic absorption spectral data (λmax) nm of newly synthesized bis azo dapsone analogues 4a-g Compd

λmax, nm λmax, nm (ν, cm−1) λmax, nm (ν, cm−1) λmax, nm (ν, cm−1) λmax, nm (ν, cm−1) λmax, nm (ν, cm−1) λmax, nm (ν, cm−1) λmax, nm (ν, cm−1) −1 (ν, cm ) (Acetone)

4a

( Ethanol)

420 296(33783.8), (-23696.7) 394(25380.7)

(Methanol)

(Propan-2-ol)

297(33670), 394(25380.7)

−

(DMF)

(Dioxane)

(DMSO)

(THF)

292(34482.8), 295(33898.3), 271(36900.4), 409(24449.9) 419(23866.3) 414(24154.6)

423 (-23640.7)

4b

−

394(25380.7), 242(29691.2), 242(30450.7), 454(22026.4) 393(25432.3) 396(25252.5)

387(25839.8)

294,395 (-25316.5)

402 (-24838.5)

397 (-25188.9)

4c

−

256(39062.5), 257(38910.5), 250(43859.6), 389(25706.9), 371(26954.2) 362(27624.3) 359(27855.2) 488(20491.8)

362 (-27624.3)

379 (-26385.2)

−

4d

−

483 (-20686.8)

292(34989.5), 483(20695.4)

−

488 (-20491.8)

297(33670), 485 -20618.6)

4e

−

267(37369.2), 379(26385.2)

270(37037), 375(26666.7)

−

406 (-24618.4)

363 (-27548.2)

406 (-24630.5)

−

4f

410 293(34129.7), 290(34482.8), (-24390.2) 397(25188.9) 402(24875.6)

−

414 (-24154.6)

414 (-24154.6)

415 (-24038.5)

415 (-24096.4)

4g

378 (59.24) 384 (264041.7)

378 (26455)

387 (25839.8)

−

382 (26178)

387 (25389.8) 386 (25906.7)

304(32894.7), 303(32938.1), 487(20500.2) 485(20593.1)

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INDIAN J. CHEM., SEC B, JUNE 2016

moles of thiobarbituric acid substituents attached to bis-azo dapsone at the C-4 and C-4' position resulted in the second largest bathochromic shift in three different apolar solvents with respect to the λ max compared to other synthetic dyes. Thermal analysis To study the thermal behavior of the bisazo analogues DSC was performed. The heat capacity (CP) verses temperature (T) curve yields peak temperature of (237.25°C) with onset temperature of 191.32°C and endset temperature of 266.02°C followed by left balance limit of 184.94°C and right balance limit of 275.27°C. The exothermic peak obtained for compound 4c corresponds to its melting point observed by open capillary method at 230-240°C. The exothermic peak curve represents the crystallization phase of the synthesized compound24. In vitro anti microbiological activity Majority of the synthesized bis azo dapsone analogues showed effective antimicrobial activity. The mean zone of inhibition among the different compounds was found to be significantly different with p value of 0.00. The maximum zone of inhibition (ZI) in mm observed by newly synthesized bis azo dapsone analogues are 4b (23±1.1), 4a (18.17±2.48), 4e (13±0.63), 4b (15±2.83), 4e (14±1.27), 4c (15.33±1.37), 4b (17.17±1.17), 4b (11.17±1.94), 4b (13.5±1.76), 4c (15±1.41), 4g (10.17±0.41), 4b (22.67±1.37), 4f (21±1.79) and 4b (17 ± 1.41) against Bacillus circulans, Shigella flexneri, Escherichia coli, Pseudomonas aeruginosa, Micrococcus luteus, Klebsiella pneumoniae, Bacillus subtilis, Pectobacterium carotovorum, Salmonella enterica paratyphi, Salmonella enterica ser. typhi, Salmonella enterica typhimurium, Vibrio cholera, Staphylococcus aureus and Streptococcus mitis respectively (mean ± SD). A perusal of Table III shows that the pair-wise comparison of each of the newly synthesized compounds with standard is expressed in mean difference ± SEM, and revealed that compound 4b showed highest significant antibacterial activity among all the synthesized bis azo dapsone analogues when compared with ampicillin (p< 0.05) against Bacillus circulans, Shigella flexneri, Escherichia coli, Vibrio cholera and Streptococcus mitis whereas compound 4a, 4c and 4e showed significant antibacterial activity than RA (p< 0.05) against

SAHOO & KUMAR: BIS-AZO-DAPSONE CONGENERS

Shigella flexneri, Salmonella enterica ser. typhi and Escherichia coli respectively. Among all the compounds 4b is the only compound which showed highest zone of inhibitions (35±1.27), (25±1.79), (18±0.63) and (23±1.27) against Aspergillus niger, Trychophyton rubrum, Candida albicans and Candida glabrata respectively and 4g with zone of inhibition (29±1.27) against Cryptococcus neoformans (mean ± SD). With reference to Table IV, the pair-wise comparison of each of the newly synthesized compounds with fluconazole is expressed in mean difference ± SEM, and revealed that all the compounds except 4f and 4g showed significant antifungal activity than fluconazole (p< 0.05) against Aspergillus niger whereas against Trychophyton rubrum all the newly synthesized bis azo dapsone analogues tremendously showed significant antifungal activity (p< 0.05). Only compound 4b showed significant antifungal activity against Candida glabrata. The inhibitory properties of different dapsone derived compounds was determined in terms of

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MIC (µgmL−1) within a concentration range of 500-31.25 µgmL−1. The MIC values of the test analogues against different bacterial strains are mentioned in Table V. The compound 4b showed excellent antibacterial activity against all the organisms, at a MIC level of 31.25 µgmL−1 except E. coli, P. carotovorom, S. enterica ser. typhi and S. enterica typhimurium. The compound 4e has the second highest MIC in the series, which can inhibit the growth of seven organisms at a minimum inhibitory concentration level of 31.25 µgmL−1. P. carotovorom is resistant to all the compounds except 4a and 4b at MIC 500 µgmL−1. However, the majority of compounds can effectively inhibit the growth of Bacillus circulans, Shigella flexneri, Salmonella enterica ser. typhi and Staphylococcus aureus at 31.25 µgmL.−1 The azo based dapsone derivative, 4f is the only compound, which can inhibit the growth of most of the organisms at a minimum inhibitory concentration level not less than 500 µgmL−1. The reference antibiotic (ampicillin) is able to exhibit its MIC against all the bacterial strains at 31.25 µgmL−1.

Table IV ― Zone of inhibition (mm) of newly synthesized bis azo dapsone analogues 4a-g against bacterial strains (Mean difference± S.E.M.) Compd

An

Tr *

*

1.33 ± 0.47 16±0.47* 3±0.47* 15±0.45* 11±0.47* 1±0.47 -1±0.47

4a 4b 4c 4d 4e 4f 4g

3.83±0.68 8.83±0.68* 6±0.68* 6.83±0.65* 1.83±0.68* 3.83±0.68* 4±0.68*

Ca

Cg

Cn

-16±0.53 -8±0.53 -13±0.53 -14±0.51 -8±0.53 -17±0.53 -17±0.53

-5±0.64 4±0.64* -4±0.64 -7±0.61 -8.17±0.64 -7±0.64 -6.83±0.64

-4.67±0.66 -2.67±0.66 -7.67±0.66 -4.67±0.64 -5.67±0.66 -9.67±0.66 1.33±0.66

Results expressed in mean ± S.E.M. (n=6), (statistical significance at *p500 − 31.25 4a 500 125 125 31.25 31.25 31.25 31.25 31.25 31.25 31.25 31.25 125 31.25 31.25 4b − 31.25 125 31.25 31.25 125 − 31.25 125 31.25 125 − 31.25 4c − 500 250 62.5 250 125 31.25 − 250 250 125 31.25 − 31.25 4d − 31.25 31.25 >500 125 31.25 31.25 125 500 31.25 31.25 31.25 − − 4e − 500 250 >500 >500 >500 − 500 125 >500 500 − 31.25 >500 4f − >500 31.25 250 250 500 − − 125 − 31.25 125 >500 31.25 4g − Ampicillin 31.25 31.25 31.25 31.25 31.25 31.25 31.25 31.25 31.25 31.25 31.25 31.25 31.25 31.25 MIC-Minimum Inhibitory Concentration, - No zone of inhibition, Bc- Bacillus circulans, Sf- Shigella flexneri, Ec- Escherichia coli, Pa- Pseudomonas aeruginosa , Ml- Micrococcus luteus, Kp- Klebsiella pneumoniae, Bs- Bacillus subtilis, , Pc- Pectobacterium carotovorum, Sp- Salmonella enterica paratyphi, Se- Salmonella enterica ser.typhi, St-Salmonella enterica typhimurium, Vc-Vibrio cholera, Sa-Staphylococcus aureus, Sm- Streptococcus mitis. RA- Reference Antibiotic (Ampicillin).

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INDIAN J. CHEM., SEC B, JUNE 2016

Experimental Section The chemicals used for present research studies were of synthetic grade obtained from Merck Specialities Pvt. Ltd., Mumbai, India and Hi-Media Laboratories Pvt. Ltd, Mumbai, India. The prepared products were analyzed by FT-IR (JASCO FT-IR 4100 Spectrophotometer) using KBr pellets. LC-MS were recorded on Shimadzu mass spectrophotometer.The 1H NMR spectra were recorded on a Bruker H1 NMR 400 MHz instrument using tetramethylsilane as an internal standard and the chemical shifts are reported in δ (ppm). UV-Vis (Jasco V-630 Spectrophotometer) and elemental analysis for C,H,N and S were performed on PerkinElmer model 2400 CHNS/O analyzer. Differential scanning calorimetry was carried out on Mettler Toledo STARe system using aluminium cans calibrated with indium to ensure accuracy of the calorimetric scale. The melting points were determined by open capillary method (Elico). Synthesis of 1,1'-(1E,1'Z)-4,4'-sulfonyl-bis-(4,1phenylene)-bis-(diazene-2,1-diyl) dinaphthalen-2-ol, 4d Dapsone (2.309 g, 9.3 mmol) was dissolved in aqueous hydrochloric acid; the solution was cooled with stirring to 0-5°C and sodium nitrite (0.413 g, 6 mmol) was added to it. The mixture contained excess nitrous acid which was destroyed by the addition of urea. The clear diazonium salt solution was slowly poured into a solution of 2-naphthol (1.0 g, 6.9 mmol) in 10% NaOH (10 mL) at 0-5°C with constant stirring for about 30 min and pH of the solution maintained between 5-6. The deep cherry red product was filtered off, washed with water, dried and purified by recrystallization from ethanol. General method of synthesis of compounds 5,5'(1E)-4,4′-sulfonyl-bis-(1,4-phenylene)-bis-(diazene2,1-diyl)-bis-(coupling component) 4b, 4c, 4e and 4f; 3,3'-(1E,1'E)-4,4'-sulfonyl-bis-(4,1-phenylene)bis-(diazene-2,1-diyl)-bis-(4-hydroxy-2H-chromen2-one) 4a; and 4,4'-(1E,1'E)-4,4'-sulfonyl-bis-(4,1phenylene)-bis-(diazene-2,1-diyl)-bis-(2-isopropyl5-methylphenol)25, 4g An ice-cold solution of sodium nitrite (0.207 g, 3 mmol) was added drop-wise to the solution of dapsone (3 mmol) with conc. H2SO4 (8-9 mmol) and water (5 mL) on an ice bath. The temperature of the reaction was maintained at 5°C. When addition was completed, the solution was kept for 30 min

with occasional stirring to complete the diazotization. Then it was poured into an ice-cold solution of different coupling components such as 8-hydroxy quinoline, salicylaldehyde, 4-hydroxycoumarin, salicylic acid, thiobarbituric acid and thymol (0.543 g, 3 mmol) in 10 mL of acetate buffer solution (pH 5) in ethanol. The resultant mixtures were stirred at 0-5°C and allowed to stand in an ice bath for 1 h. The coloured products obtained were filtered and washed with water. The obtained products were dried and purified by recrystallization from ethanol. 1,1'-(1E,1'Z)-4,4'-Sulfonyl-bis-(1,4-phenylene)-bis(diazene-2,1-diyl)-dinaphthalen-2-ol, 4d The product was prepared by diazotized dapsone followed by coupling with β-napthol. Cherry red color powder. Yield 85%. Rf: 0.7. m.p.270-80°C. UV-Vis (ethanol): 483 nm; (T%, ethanol): 20686.8; IR (KBr): 3372, (O-Hstr.), 1591, 1621, (C=Cstr. napthyl), 1502 (-N=N-),1254 (O-H bend), 1393, 1142, (SO2str. of sulfone ), 1101 cm−1 (C-N str.); 1H NMR (DMSO-d6, 300 MHz): δ 8.11-8.36 (m, 8H, dapsone diaryl-H), 7.15 (dd, napthyl H-3, J = 8.4 Hz), 8.03 (dd, napthyl H-4, J = 9.6 Hz), 8.04 (dd, napthyl H-5), 7.44 (m, napthyl H-7), 8.07(dd, napthyl H-8); LC-MS (RT,% area): 3.043, 91; m/z 556.7 (M-1). Anal. Calcd for C32H22N4O4S: C, 62.80; H, 3.96; N, 10.03; S, 5.74. Found: C, 62.84; H, 3.92; N, 10.01; S, 5.73%. 3,3'-(1E,1'E)-4,4'-Sulfonyl-bis-(1,4-phenylene)-bis(diazene-2,1-diyl)-bis-(4-hydroxy-2H-chromen-2-one, 4a 4-Hydroxy coumarin was synthesized by the Claisen condensation of 2-hydroxy acetophenone with diethyl carbonate in NaH as previously reported26. The product was prepared by diazotized dapsone and followed by coupling with 4-hydroxy coumarin. Yellow colored powder. Yield 85%. Rf: 0.6; m.p.310-20°C. UV-Vis (ethanol): 394 nm; (T%, ethanol): 25380.7; IR (KBr): 3460 (O-Hstr.), 1745 (C=Ostr. of lactone carbonyl), 1398,1146 (SO2str. of sulfone), 1627 (C=Cstr. of pyrone), 1508 (-N=N-), 1293 (C-Ostr.), 1103 cm−1 (C-Nstr.); 1H NMR (DMSO-d6, 300 MHz): δ 7.72-8.03 (m, 8H, dapsone diaryl-H), 7.89 (d, coumarin H-5, J = 7.2 Hz), 7.42 (m, coumarin H-6), 7.69 (m, coumarin H-7), 7.59 (d, coumarin H-8, J = 7.2 Hz); LC-MS (RT,% area): 2.346, 90.35; m/z 595. Anal. Calcd for C26H18N4O6S: C, 60.60; H, 3.05; N, 9.42;S, 5.39. Found: C, 60.57; H, 3.01; N, 9.39; S, 5.37%.

SAHOO & KUMAR: BIS-AZO-DAPSONE CONGENERS

5,5'-(1E)-4,4′-Sulfonyl-bis-(1,4-phenylene)-bis(diazene-2,1-diyl) diquinolin-8-ol, 4b The product was prepared by diazotized dapsone followed by coupling with 8-hydroxy quinoline. Dark red color powder. Yield 85%. Rf: 0.8; m.p. 210-20°C. UV-Vis (ethanol): 454 nm; (T%, ethanol): 22026.4; IR (KBr): 3460, 3140 (O-Hstr.), 1589 (C=Cstr. Quinoline/phenyl), 1637 (C=Nstr. Quinoline), 1504 (-N=N-), 1255 (O-H bend), 1395, 1140 (SO2str. of sulfone), 1012 cm−1 (C-Nstr.); 1H NMR (DMSO-d6, 300 MHz): δ 9.91 (s, 1H, OH), 8.15-8.17 (m, 8H, dapsone diaryl-H ), 8.79 (d, quinoline H-2, J = 9.0 Hz), 7.65 (m, quinoline H-3, J = 9.0 Hz), 8.17 (d, quinoline H-4), 7.67 (d, quinoline H-6, J = 8.0 Hz), 7.26 (d, quinoline H-7, J = 8.0 Hz); LC-MS (RT,% area): 2.759, 80.47; m/z 559.23 (M-1). Anal. Calcd for C30H20N6O4S: C, 64.28; H, 3.60; N, 14.99; S, 5.72. Found: C, 64.30; H, 3.65; N, 14.98; S, 5.69%. 5,5'-(1E,1'E)-4,4'-Sulfonyl-bis-(1,4-phenylene)-bis(diazene-2,1-diyl)-bis-(2-hydroxy benzaldehyde, 4c The product was prepared by diazotized dapsone and followed by coupling with salicyaldehyde. Brick red colored powder. Yield 92%. Rf: 0.8; m.p.230-40°C. UV-Vis (ethanol) 371 nm; (T%, ethanol): 26954.2; IR (KBr): 3451 (O-Hstr.), 2923, 2848 (-CHstr. of aldehyde), 1656 (C=Ostr.), 1515 (-N=N-), 1251(O-H bend), 1395, 1140 (SO2str. of sulfone), 1009 cm−1 (C-Nstr.); 1H NMR (DMSO-d6, 300 MHz): δ 10.56 (s, 1H, aldehydic- H), 8.17-8.43 (m, 8H, dapsone diaryl-H), 8.85 (s, 1H, aldehydic-H), 7.36 (dd, salicylaldehyde H-3, J = 8.84 Hz ), 7.82 (dd, salicylaldehyde H-4, J = 8.84 Hz ), 8.03 (s, 1H, salicylaldehyde H-6); LC-MS (RT,% area): 3.259, 73.47; m/z 513.19 (M-1). Anal. Calcd for C26H18N4O8S: C, 60.69; H, 3.53; N, 10.89; S, 6.23. Found: C, 60.66; H, 3.51; N, 10.85; S, 6.19%. 5,5'-(1E,1'E)-4,4'-Sulfonyl-bis-(1,4-phenylene)-bis(diazene-2,1-diyl)-bis-(2-hydroxybenzoic acid), 4e The product was prepared by diazotized dapsone and followed by coupling with salicylic acid. Brick red colored powder. Yield 78%. Rf: 0.6. m.p.220-30°C. UV-Vis (ethanol): 379 nm; (T%, ethanol): 26385.2; IR (KBr): 3373 (O-Hstr.), 1668 (C=Ostr. of carboxylic acid), 1590 (C=Cstr. of salicylic acid), 1512 (-N=N-), 1250 (O-H bend.), 1395, 1144 (SO2str. of sulfone), 1009 cm−1 (C-Nstr.); 1H NMR (CDCl3, 300 MHz): δ 11.99 (s, 1H, COOH), 11.78 (s, 1H, OH), 8.18-8.36 (m, 8H, dapsone diaryl-H), 7.42 (dd, salicylic H-3, J = 7.5 Hz), 8.07 (dd, salicylic

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H-4, J = 7.7 Hz), 8.36 (s, 1H, salicylic H-6); LC-MS (RT,% area); 2.301, 92.08; m/z 545.08 (M+1). Anal. Calcd for C26H18N4O8S: C, 57.14; H, 3.32; N, 10.25; S, 5.87. Found: C, 57.11; H, 3.30; N, 10.21; S,5.86%. 5,5'-(1E)-4,4'-Sulfonyl-bis-(1,4-phenylene)-bis(diazene-2,1-diyl)-bis-(2-thiobarbituric acid), 4f The product was prepared by diazotized dapsone and followed by coupling with thiobarbituric acid. Dark red color powder. Yield 85%. Rf: 0.8. m.p.175-85°C. UV-Vis (ethanol): 397 nm; (T%, ethanol): 24875.6; IR (KBr): 3373, 3466 (OH/NHstr.), 1690, 1654 (C=Ostr.), 1348(C=Sstr.), 1512 (-N=N-), 1348, 1141 (SO2str. of sulfone), 1004 (C-Nstr.) cm−1; 1 H NMR (DMSO-d6, 300 MHz): δ 7.72-8.03 (m, 8H, dapsone diaryl-H), 11.25 (s, 1H, thiobarbituric acid NH), 11.85 (s, 1H, thiobarbituric acid NH), 14.55 (s, 1H, thiobarbituric acid OH), 3.45-3.55 (s, 1H, CH-N=N-); LC-MS (RT,% area); 1.568, 96.78; m/z; 556.08 (M-1). Anal. Calcd for C20H14N8O6S3: C, 43.03; H, 2.53; N, 20.06; S, 17.22. Found: C, 43.05; H, 2.52; N, 20.04; S, 17.21%. 4,4'-(1E,1′E)-4,4'-Sulfonyl-bis-(1,4-phenylene)bis-(diazene-2,1-diyl)-bis-(2-isopropyl-5methylphenol), 4g The product was prepared by diazotized dapsone and followed by coupling with thymol. Dark red color powder. Yield 85%. Rf: 0.8. m.p.75-185°C. UV-Vis (ethanol): 384 nm; (T%, ethanol): 26041.7; IR (KBr): 3362, 3237 (OHstr.), 1630, 1590 (C=Cstr.), 1496 (-N=N-), 1280 (-OH bend), 1140 cm−1 (SO2str. of sulfone); 1 H NMR (DMSO-d6, 300MHz): δ 8.17-8.39 (d, 8H, dapsone diaryl-H), 9.69 (s,1H, thymol OH), 2.34 (s, 3H, CH3 of thymol), 3.25 (s, 1H, -CH(CH3)2), 1.25 (s, 6H, -CH(CH3)2, 7.09 (s, 1H, aryl thymol H), 7.64 (s, 1H, aryl thymol H); LC-MS (RT,% area); 2.839, 63.47; m/z 516.71 (M-1). Anal. Calcd for C32H34N4O4S: C, 67.35; H, 6.00; N, 9.82; S, 5.62. Found: C, 67.32; H, 5.55; N, 9.84; S, 5.61%. Microbiology In vitro antimicrobial activity The above newly synthesized bis-azo dye derivatives of dapsone were investigated over different microbial strains viz. Escherichia coli (MTCC 614), S. enterica ser typhi (MTCC 773), S. enterica typhimurium (MTCC 98), S. enterica paratyphi (MTCC 3220), Shigella flexneri (MTCC 1457), Pseudomonas aeruginosa (MTCC 1035),

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Vibrio cholera (MTCC 3906), Klebsiella pneumonia (MTCC 109), Micrococcus luteus (MTCC 1809), Bacillus circulans (MTCC 490), Streptococcus mitis (MTCC 2695), Pectobacterium carotovorum (MTCC 1428), Aspergillus niger (MTCC 9933) and Candida albicans (MTCC 3017) were procured from the Institute of Microbial Technology and Gene Bank (IMTECH), Chandigarh, India. Few of the strains were gifted by University Department of Pharmaceutical Sciences, Utkal University, Bhubaneswar, Odisha, India and Staphylococcus aureus and Bacillus subtilis strain hswx88 (Ref 27) were isolated in the Pharmaceutical Biotechnology Division of the University Department of Pharmaceutical Sciences, Utkal University and also incorporated for evaluation of antimicrobial activity. Standard drugs used for antimicrobial assay were ampicillin and fluconazole (Hi-Media) used for bacterial and fungal strains respectively. The antimicrobial activity of the novel bis-azo dye derivatives of dapsone was performed using cup and plate method. The antimicrobial diffusion test was performed using a cell suspension of about 1.5 × 106 CFU mL−1 employing a McFarland turbidity standard No. 0.5 (Ref 28). Sterile petriplates were selected for performing antimicrobial sensitivity test of the said synthesized compounds (4a-g). Each plate was filled with sterile molten nutrient agar for antibacterial activity and sabouraud dextrose agar medium for antifungal activity with appropriate volume29. Freshly cultured, 24 h old, 100 µL of inoculums were evenly distributed over the sterile solid medium. Cups of 6 mm diameter were made using sterile borer. Stock solution of above synthesized compounds and reference antibiotics were made using DMF at a concentration level of 1 µgµL−1. Each cup was filled with a definite volume of above mentioned solutions (25 µL, containing 25 µg) and incubated 24 h for bacterial strains and 72 h for fungal strains at 37°C. The diameter of zone of inhibition was measured using the Hi-Antibiotic Zone Scale (Hi-Media). Determination of minimum inhibitory concentration (MIC)30 1 mgmL−1 stock solution of synthesized compounds as well as reference compounds was prepared using 10% DMF solution (distilled water). Further, five different concentrations of (500 − 31.25 µg mL−1) were prepared by two fold serial dilution method and poured into the cups and incubated at 37°C for 18-24 h. After incubation MIC was determined31.

Statistical analysis The observed data on zone of inhibition for different synthesized compounds on different microbial strains were subjected to one way analysis of variance (ANOVA) for comparison of mean. The mean zone of inhibition for each compound on each strain was compared with the reference compound (RA) through Dunnett Post Hoc test. The test of significance was done at 5% level of type one error. The null hypothesis zone of inhibition for test compound was higher than the reference compound. Sample size determination The minimum sample size determination was done for one way analysis of variance with the help of the on line software https://www.statstodo.com/ SSizAOV_Pgm.php under the guidance of a statistician. A minimum sample size of five was calculated taking probability of type 1 error (d) = 0.05, power (1-β) =0.8, number of groups 13 within group SD=2. However a sample size of six has been taken in the study for each compound against each strain. Conclusion It can be concluded from the results obtained from antimicrobial screening that the bisazodapsone analogues, particularly 8-hydroxyquinoline coupled compound 4b showed significant antibacterial activity against Shigella flexneri, Escherichia coli, Vibrio cholera and Streptococcus mitis. 4-Hydroxycoumarin, salicylaldehyde and salicylic acid coupled bis azo dapsone exhibited significant antibacterial activity against Shigella flexneri, Salmonella enterica ser.typhi and Escherichia coli respectively. Majority of the compounds showed significant antifungal activity against Aspergillus niger and Trychophyton rubrum. Corroborating our findings, exploitation of dapsone molecule by the attachment of different nucleophiles may be responsible for the significant increase of antimicrobial activity among which the significant antimicrobial activity of compound 4b may be due to coupling of 8-hydroxyquinoline with dapsone. Hence, further investigations may be continued to justify the above bis azo dapsones as therapeutic agents for the treatment of various microbial infections. Acknowledgement The authors are thankful to the Dean, School of Pharmaceutical Sciences, Siksha ‘O’ Anusandhan

SAHOO & KUMAR: BIS-AZO-DAPSONE CONGENERS

University, HOD, UDPS, Utkal University, Director NISER, Director IMMT, Bhubaneswar, India, Director Piramal Healthcare, Ahmadabad, India for spectral analysis and The Institute of Microbial Technology and Gene bank (IMTECH), Chandigarh, India for providing microorganisms. The authors also express their heartiest gratitude to Dr. B. B. Nanda, Dy. Director, Regional Institute of Planning, Applied Economics and Statistics, Bhubaneswar, India for his sincere guidance during the course of statistical interpretation.

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