untitled European Journal of Chemistry 3 (1) (2012) 44‐50 European Journal of Chemistry ISSN 2153‐2249 (Print) / ISSN 2153‐2257 (Online)  2012 EURJCHEM DOI:10.5155/eurjchem.3.1.44‐50.517 European Journal of Chemistry Journal homepage: www.eurjchem.com Synthesis, antimicrobial activity and absorption studies of some novel heterocyclic dyes based on 4‐hexylbenzene‐1,3‐diol Hitendra Mangubhai Patel Department of Chemistry, Vithalbhai Patel and Rajratna Purushotamdas Tulsibhai Patel Science College, Sardar Patel University, Vallabh Vidyanagar, Gujarat, 388120, India *Corresponding author at: Department of Chemistry, Vithalbhai Patel and Rajratna Purushotamdas Tulsibhai Patel Science College, Sardar Patel University, Vallabh Vidyanagar, Gujarat, 388120, India. Tel.: +91.982.4334922; fax: +91.982.4334922. E‐mail address: shreeniketan71@yahoo.in (H.M. Patel). ARTICLE INFORMATION ABSTRACT Received: 31 August 2011 Received in revised form: 12 October 2011 Accepted: 15 November 2011 Online: 31 March 2012 KEYWORDS Aniline derivatives were diazotized and coupled with 3‐aminocrotononitrile to give the corresponding 2‐arylhydrazono‐3‐ketiminobutyronitriles. Cyclization of these arylhydrazono derivatives with hydrazine monohydrate afforded 5‐amino‐4‐arylazo‐3‐methyl‐1H‐pyrazoles which were subsequently diazotized and coupled with malononitrile to yield a series of pyrazolylhydrazonomalononitriles. These compounds were then reacted with hydrazine monohydrate to provide, heterocyclic dyes, which were further diazotized and coupled with 4‐hexylbenzene‐1,3‐diol to produce novel heterocyclic tetraazo dyes which were characterized by elemental analysis and spectral methods. The antimicrobial activity and absorption characteristics of the dyes were also examined in detail. Pyrazole Solvatochromism Heterocyclic dyes Antimicrobial activity 3‐Aminocrotononitrile 4‐Hexylbenzene‐1,3‐diol 1. Introduction The growing interest in the pyrazole chemistry lies in designing new synthetic approach, theoretical calculations and applications of newer spectroscopic techniques. The usage of many pyrazole derivatives has undoubtedly created considerable attention in developing many different synthetic procedures in pharmaceuticals, agrochemicals, dyestuff. The recent developments in the synthetic routes and the chemistry of pyrazoles have been thoroughly reviewed [1‐5]. The condensation of β‐enaminonitriles and β‐ketoesters with hydrazines continues to be the most widely used method for constructing the aminopyrazoles and pyrazolones, respectively [6,7]. The amino derivatives of pyrazoles belong to important compounds used for preparation of other functional derivatives mainly for the synthesis of condensed heterocyclic systems [8‐ 12]. Also, fused pyrazoles are important compounds that have many derivatives with a wide range of interesting properties, such as antihyperglycemic, analgestic, anti‐inflammatory, anti‐ pyretic, anti‐bacterial, hypoglycaemic and sedative‐hypnotic activities. Recently, some pyrazoles were reported to have non‐ nucleoside HIV‐1 reverse transcriptase inhibitory activity [13‐ 15]. Some azopyrazole derivatives also find application in dyes, biological and pharmacological studies and complexes [16‐18]. The use of heterocyclic intermediates in the synthesis of azo disperse dyes is well established and the resultant dyes exhibit good tinctorial strength and brighter dyeing than those derived from aniline‐based diazo components. For instance, amino‐ substituted thiazole, benzothiazole [19,20] and benzoiso‐ thiazole [21] compounds afford highly electronegative diazo components and consequently, provide a pronounced batho‐ chromic effect compared to the corresponding benzoid compounds. Moreover, azo disperse dyes containing 3‐methyl‐ 1H‐pyrazole‐5‐one and 4‐hexylbenzene‐1,3‐diol as coupling component have also been described as having red‐violet colours in the literature [22‐25]. We report here the synthesis of a series of new heterocyclic tetraazo dyes based on 4‐hexylbenzene‐1,3‐diol. Antimicrobial activity and absorption ability of these dyes substituted with electron‐withdrawing and electron‐donating groups at their o‐, m‐ and p‐position were also examined in detail. 2. Experimental 2.1 Synthesis All the chemicals used for the synthesis of the compounds were obtained from various companies (commercial grade) and were further purified by crystallization. The solvents used were of spectroscopic grade. IR spectra were determined using a Perkin‐elmer Spectrum GX FT‐IR model, on a KBr disc. Nuclear magnetic resonance (1H NMR) spectra were recorded on a Hitachi R‐1500 in deuterated dimethylsulphoxide (DMSO‐d6) using tetramethylsilane (TMS) as the internal reference; chemical shifts were (δ) given in ppm. Ultraviolet‐ visible (UV‐vis) absorption spectra were recorded on a Carl Zeiss UV/VIS Specord spectrometer at the wavelength of maximum absorption (λmax) in a range of solvents, i.e. DMSO, DMF, acetonitrile, methanol, acetic acid and chloroform at the various concentrations (1x10‐6 ‐ 1x10‐8 M). Melting points were determined by open capillary method and are uncorrected. Elemental analysis was done on a Perkin Elmer CHNS/O Analyzer 2400 Series II were recorded on Agilent 1100 MSD. 2.1.1. Synthesis of 2‐arylhydrazono‐3‐ketiminobutyronitriles (1a‐1j) and 5‐amino‐4‐arylazo‐3‐methyl‐1H‐pyrazoles (2a‐ 2j) Patel / European Journal of Chemistry 3 (1) (2012) 44‐50 45 Scheme 1 Scheme 2 2‐Arylhydrazone‐3‐ketiminobutyronitriles (1a‐1j) and 5‐ amino‐4‐arylazo‐3‐methyl‐1H‐pyrazoles (2a‐2j) were prepared according to the literature procedures [1,2]. The general route for the synthesis of 2‐arylhydrazono‐3‐ ketiminobutyronitriles and 5‐amino‐4‐arylazo‐3‐methyl‐1H‐ pyrazoles is shown in (Scheme 1). 2.1.2. Synthesis of pyrazolylhydrazonomalononitriles 3a‐3j 5‐Amino‐4‐arylazo‐3‐methyl‐1H‐pyrazoles (0.01 mol) were dissolved in a mixture of glacial acetic acid and concentrated hydrochloric acid (20 mL, ratio 1:1) and the solution was then cooled to 273.15‐278.15 K. Sodium nitrite (0.69 g, 0.01 mol) in water (10 mL) was then added to this solution drop wise with vigorous stirring, during about 1 h, while cooling at 273.15 ‐ 278.15 K. Then the resulting diazonium solution was added in portions over 30 min to a vigorously stirred solution of malononitrile (0.66 g, 0.01 mol) in pyridine (10 mL) at between 273.15 and 278.15 K, maintaining the pH at 7‐8 by simultaneous addition of sodium acetate solutions. The mixture was then stirred for 2 h. at between 273.15 and 278.15 K. The precipitated product separated upon dilution with water (50 mL) was filtered off, washed with water several times, dried and crystallized from DMF‐H2O. 2.1.3. Synthesis of heterocyclic disazo dyes 4a‐4j Equimolar amounts (0.005 mol) of 3a‐3j and hydrazine monohydrate in ethanol (30 mL) were heated, under reflux, for 4 h. The reaction mixture was concentrated in vacuo and then triturated with water whereby the resulting solid product was collected by filtration and crystallized from DMF‐H2O. The general route for the synthesis of disazo dyes 4a‐4j is shown in (Scheme 2). 2.1.4. Synthesis of heterocyclic tetraazo dyes 5a‐5j Diazotization of 4a‐4j and coupling with 4‐hexylbenzene‐ 1,3‐diol were prepared according to the literature procedure [26]. The general route for the synthesis of heterocyclic tetraazo dyes 5a‐5j is shown in (Scheme 3). 4‐(3’‐Methyl‐4’‐phenylazo‐1’H‐pyrazole‐5’‐ylazo)‐3,5‐[4,4’‐ dihexyl‐1,1’,3,3’‐tetrahydroxy‐diphenylazo]‐1H‐pyrazole (5a): yellowish Orange. Yield: 77%. M.p.: 186‐187 oC. FT‐IR (KBr, cm‐ 1): 3460, 3449 (2 OH), 3280, 3203 (2 NH), 3086 (Ar‐H), 2991 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 0.90 (t, 3H, CH3 ),1.31‐1.59 (m, 6H, ‐CH2 ), 2.45 (s, 3H, CH3), 2.62 (s, 2H, ‐CH2), 6.45 (s, 1H, ArH), 6.91 (s, 1H, ArH), 7.40‐7.74 (m, 5H, ArH), 8.2 (s, 1H, Ar‐OH), 10.5 (s, 1H, Ar‐OH), 11.15 (b,1H, NH),12.61 (b, 1H, NH). Anal. calcd. for C37H44O4N12: C, 61.60; H, 6.10; N, 23.33. Found: C, 61.51; H, 5.98; N, 23.28%. 4‐[3’‐Methyl‐4’‐(p‐methoxyphenylazo)‐1’H‐pyrazole‐5’‐ ylazo]‐3,5‐[4,4’‐dihexyl‐1,1’,3,3’‐tetrahydroxy‐diphenylazo]‐1H‐ pyrazole (5b): Red. Yield: 81%. M.p.: 167‐168 oC. FT‐IR (KBr, cm‐1): 3462, 3453 (2 OH), 3284, 3197 (2 NH), 3094 (Ar‐H), 2986 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 0.90 (t, 3H, CH3 ),1.31‐1.59 (m, 6H, ‐CH2 ), 2.45 (s, 3H, CH3), 2.62 (s, 2H, ‐ CH2), 6.45 (s, 1H, ArH), 6.91 (s, 1H, ArH), 7.40‐7.74 (m, 5H, ArH), 8.2 (s, 1H, Ar‐OH), 10.5 (s, 1H, Ar‐OH), 11.15 (b,1H, NH),12.61 (b, 1H, NH). Anal. calcd. for C38H46O5N12: C, 60.80; H, 6.10; N, 22.41. Found: C, 60.20; H, 6.88; N, 22.37%. 4‐[3’‐Methyl‐4’‐(p‐chlorophenylazo)‐1’H‐pyrazole‐5’‐ylazo]‐ 3,5‐[4,4’‐dihexyl‐1,1’,3,3’‐tetrahydroxy‐diphenylazo]‐1H‐pyrazole (5c): Reddish Brown. Yield: 84%. M.p.: 249‐250 oC. 46 Patel / European Journal of Chemistry 3 (1) (2012) 44‐50 Scheme 3 FT‐IR (KBr, cm‐1): 3480, 3460 (2 OH), 3276, 3169 (2 NH), 3090 (Ar‐ H), 2994 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 0.90 (t, 3H, CH3 ), 1.31‐1.59 (m, 6H, ‐CH2 ), 2.45 (s, 3H, CH3), 2.62 (s, 2H, ‐CH2), 6.45 (s, 1H, ArH), 6.91 (s, 1H, ArH), 7.40‐7.74 (m, 5H, ArH), 8.2 (s, 1H, Ar‐OH), 10.5 (s, 1H, Ar‐OH), 11.15 (b, 1H, NH),12.61 (b, 1H, NH). Anal. calcd. for C37H43O4N12Cl: C, 58.88; H, 5.71; N, 22.28. Found: C, 58.82; H, 5.69; N, 22.23%. 4‐[3’‐Methyl‐4’‐(p‐methylphenylazo)‐1’H‐pyrazole‐5’‐ylazo]‐ 3,5‐[4,4’‐dihexyl‐1,1’,3,3’‐tetrahydroxy‐diphenylazo]‐1H‐pyrazole (5d): Yellow Brown. Yield: 62%. M.p.: 275 oC. FT‐IR (KBr, cm‐1): 3491, 3480 (2 OH), 3289, 3200 (2 NH), 3022 (Ar‐H), 2985 (Ar‐ H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 0.88 (t, 3H, CH3 ),1.26‐1.79 (m, 2H, CH2 ),2.40 (s, 3H, CH3), 2.76 (s, 3H, p‐CH3), 7.35‐7.73 (dd, 4H, ArH), 8.9 (s, 1H, Ar‐OH), 10.9 (s, 1H, Ar‐OH), 9.30 (b, 1H, NH), 10.23 (b, 1H, NH). Anal. calcd. for C38H46O4N12: C, 62.10; H, 6.23; N, 22.28. Found: C, 61.92; H, 6.18; N, 22.82%. 4‐[3’‐Methyl‐4’‐(m‐methoxyphenylazo)‐1’H‐pyrazole‐5’‐ ylazo]‐3,5‐[4,4’‐dihexyl‐1,1’,3,3’‐tetrahydroxy‐diphenylazo]‐1H‐ pyrazole (5e): Yellowish. Yield: 78%. M.p.: 168‐169 oC. FT‐IR (KBr, cm‐1): 3464, 3450 (2 OH), 3294, 3203 (2 NH), 3101 (Ar‐ H), 2978 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 0.85 (t, 3H, CH3 ), 1.31‐1.68 (m, 2H, CH2 ),2.46 (s, 3H, CH3), 3.82 (s, 3H, m‐OCH3), 6.98‐7.44 (m, 4H, ArH), 8.4 (s, 1H, Ar‐OH), 11.0 (s, 1H, Ar‐OH), 11.16 (b, 1H, NH), 12.63 (b, 1H, NH). Anal. calcd. For C38H46O5N12: C, 60.80; H, 6.10; N, 22.40. Found: C, 60.60; H, 6.01; N, 22.10%. 4‐[3’‐Methyl‐4’‐(m‐chlorophenylazo)‐1’H‐pyrazole‐5’‐ylazo]‐ 3,5‐[4,4’‐dihexyl‐1,1’,3,3’‐tetrahydroxy‐diphenylazo]‐1H‐pyrazole (5f): Red. Yield: 81%. M.p.: 224‐225 oC. FT‐IR (KBr, cm‐1): 3482, 3459 (2 OH), 3275, 3144 (2 NH), 3069 (Ar‐H), 2963 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 0.89 (t, 3H, CH3 ), 1.33‐1.89 (m, 2H, CH2 ),2.44 (s, 3H, CH3), 7.45‐7.70 (m, 4H, ArH), 8.7 (s, 1H, Ar‐OH), 11.2 (s, 1H, Ar‐OH), 11.13 (b, 1H, NH), 12.68 (b,1H, NH). Anal. calcd. for C37H43O4N12: C, 58.88; H, 5.70; N, 22.28. Found: C, 58.81; H, 5.60; N, 22.22%. 4‐[3’‐Methyl‐4’‐(m‐methylphenylazo)‐1’H‐pyrazole‐5’‐ylazo]‐ 3,5‐[4,4’‐dihexyl‐1,1’,3,3’‐tetrahydroxy‐diphenylazo]‐1H‐ pyrazole (5g): Red. Yield: 73%. M.p.: 190‐191 oC. FT‐IR (KBr, cm‐1): 3494, 3483 (2 OH), 3276, 3206 (2 NH), 3078 (Ar‐H), 2966 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 0.85 (t, 3H, CH3 ), 1.37‐1.79 (m, 2H, CH2 ), 2.41 (s, 3H,CH3), 2.45 (s, 3H, m‐ CH3), 7.22‐7.53 (m, 4H, ArH), 8.3 (s, 1H, Ar‐OH), 10.6 (s, 1H, Ar‐ OH), 11.15 (b, 1H, NH), 12.61 (b, 1H, NH). Anal. calcd. for C38H46O4N12: C, 62.10; H, 6.23; N, 22.88. Found: C, 61.89; H, 6.18; N, 22.85%. 4‐[3’‐Methyl‐4’‐(o‐methoxyphenylazo)‐1’H‐pyrazole‐5’‐ ylazo]‐3,5‐[4,4’‐dihexyl‐1,1’,3,3’‐tetrahydroxy‐diphenylazo]‐1H‐ pyrazole (5h): Red. Yield: 84%. M.p.: 183‐184 oC. FT‐IR (KBr, cm‐1): 3467, 3456 (2 OH), 3284, 3201 (2 NH), 3097 (Ar‐H), 2989 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 0.86 (t, 3H, CH3 ), 1.41‐1.67 (m, 2H, CH2 ), 2.46 (s, 3H,CH3), 3.91 (s, 3H, o‐ OCH3), 7.18‐7.52 (m, 4H, ArH), 7.9 (s, 1H, Ar‐OH), 11.0 (s, 1H, Ar‐OH), 10.99 (b, 1H, NH), 12.56 (b, 1H, NH). Anal. Calcd. for C38H46O5N12: C, 60.8; H, 6.10; N, 22.40. Found: C, 60.50; H, 5.89; N, 22.31%. 4‐[3’‐Methyl‐4’‐(o‐chlorophenylazo)‐1’H‐pyrazole‐5’‐ylazo]‐ 3,5[4,4’‐dihexyl‐1,1’,3,3’‐tetrahydroxy‐diphenylazo]‐1H‐pyrazole (5i): Yellowish Orang. Yield: 85%. M.p.: 235‐236 oC. FT‐IR (KBr, cm‐1): 3479, 3465 (2 OH), 3283, 3201 (2 NH), 3081 (Ar‐H), 2977 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 0.88 (t, 3H, CH3 ), 1.49‐1.89 (m, 2H, CH2 ), 2.48 (s, 3H, CH3), 7.40‐7.66 (m, 4H, ArH), 8.4 (s, 1H, Ar‐OH), 10.7 (s, 1H, Ar‐OH), 11.12 (b, 1H, NH), 12.65 (b, 1H, NH). Anal. calcd. for C37H43O4N12Cl: C, 58.88; H, 5.70; N, 22.28. Found: C, 58.80; H, 5.63; N, 22.23%. 4‐[3’‐Methyl‐4’‐(o‐methylphenylazo)‐1’H‐pyrazole‐5’‐ylazo]‐ 3,5‐[4,4’‐dihexyl‐1,1’,3,3’‐tetrahydroxy‐diphenylazo]‐1H‐pyrazole (5j): Redish Brown. Yield: 72 %. M.p.: 221‐222 oC. FT‐IR (KBr, cm‐1): 3497, 3486 (2 OH), 3288, 3198 (2 NH), 3093 (Ar‐H), 2991 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 0.90 (t, 3H, CH3 ), 1.35‐1.76 (m, 2H, CH2 ), 2.44 (s, 3H, CH3), 2.59 (s, 3H, ‐ CH3), 7.26‐7.53 (m, 4H, ArH), 8.8 (s, 1H, Ar‐OH), 11.2 (s, 1H, Ar‐ OH), 11.09 (b, 1H, NH), 12.58 (b, 1H, NH). Anal. calcd. for C38H46O4N12: C,62.10; H, 6.23; N, 22.88. Found: C, 61.81; H, 6.19; N, 22.84%. 2.2. Antimicrobial activity of heterocyclic tetraazo dyes 5a‐ 5j The antimicrobial activities of the newly synthesized compounds were evaluated using the micro broth dilution method [27] against a panel of eight microorganism species. The origin of microbial strains are Bacillus subtilis (NRRL B‐ 3711), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATTC 25922), Proteus vulgaris (NRRL B‐123), Candida albicans (NRRL Y‐2983), Candida glabrata as yeasts. Stock solutions of synthesized compounds were diluted in DMSO to give serial decreasing dilutions ranging from 4 to 0.0009 mg/mL. The dilutions were sterilized by filtration through 0.45 μm millipore filters and were transferred to 96‐well microtitre plates. Patel / European Journal of Chemistry 3 (1) (2012) 44‐50 47 Figure 1. Tautomeric equilibriums of dyes 5a‐5j. Overnight grown microbial suspensions adjusted to McFarland 0.5 standard solutions were used as inoculants. A 100 μl from each microorganism suspension was transferred into the wells. The well containing media, sterile distilled water and inoculums were used for positive growth control. The minimal inhibitory concentration (MIC) values were determined after incubation at 310.15 K for 18‐24 h. The MIC values were defined as the lowest compound concentration where absence of growth was recorded. Each test was repeated at least twice for all microorganisms. Streptomycin and fluconazole were used as reference antibiotics for bacteria and yeasts, respectively. All of the antimicrobial activity studies were performed in triplicate. 3. Results and discussion 3.1. Spectral characteristics and tautomerism Tetraazo dyes 5a‐5j can exist in six possible tautomeric forms, namely the Tetraazo form T1, the triazo‐hydrazo form T2, the hydrazo‐triazo form T3, the dihydrazo‐diazo form T4, T5 and the dihydrazo‐diazo form T6 as shown in (Figure 1). The FT‐IR spectra of dyes 5a‐5j showed intense intense two imino (NH) bands at 3294‐3144 cm‐1. The other λmax values of 3101‐3022 cm‐1 (aromatic C‐H) and 2994‐2963 cm‐1 (aliphatic C‐H) were recorded. 1H NMR spectra of dyes 5a‐5j showed four broad peaks at 12.75‐10.23 ppm (NH), 11.20‐9.30 ppm (NH) and two broad peaks at 7.9‐8.9 ppm (OH), 10.5‐11.2 ppm (OH), 0.86‐0.96 ppm (CH3), 1.24‐1.89 ppm (CH2), respectively. The other δ values of 2.48‐2.40 ppm (CH3) and 8.35‐6.98 ppm (aromatic H) were recorded. These results suggest that dyes 5a‐5j is present as one of the tautomeric forms in DMSO and the solid state. Previously, we established that the tautomeric structure of pyrazole dyes in the solid state and solution medium using FT‐IR and 1H NMR. The spectral data generally lead to the conclusion that the tautomeric equilibrium of these dyes was in favour of the hydrazo form [28‐30]. These suggest that these dyes are predominantly in triazo‐hydrazo form (T2), (T3) or dihydrazo‐ diazo form (T4), (T5) and (T6) in the solid state and DMSO. 3.2. Solvent effects on UV‐Vis spectra The UV‐Vis absorption spectra of dyes 5a‐5j were recorded over the range of λ between 300 and 700 nm, using a variety of solvents in concentrations (10‐6‐10‐8 M) and the results are summarized in (Table 1). The visible absorption spectra of the dyes did not correlate with the polarity of solvent. Dyes 5a‐5j gave a maximum absorption peak in all used solvents. This result suggests that dyes 5a‐5j is present in a single tautomeric form in all used solvents. 48 Patel / European Journal of Chemistry 3 (1) (2012) 44‐50 Table 1. Influence of solvent on λmax (nm) of dyes 5a‐5j. Dye no DMSO DMF Acetonitrile Methanol Acetic acid Chloroform 5a 473 468 454 422 437 444 5b 467 464 455 420 432 440 5c 503 505 459 437 449 455 5d 457 449 436 408 406 432 5e 472 463 452 414 416 445 5f 483 482 463 423 422 456 5g 472 463 453 422 418 445 5h 475 466 455 429 427 458 5i 487 476 466 433 428 457 5j 467 465 447 403 414 440 Table 2. Absorption maxima (λmax, nm) of dyes 5a‐5j in acidic and basic solutions. Dye no Methanol Methanol + KCl Methanol + HCl Chloroform Chloroform + Piperidine Acetic acid 5a 473 468 454 422 437 444 5b 467 464 455 420 432 440 5c 503 505 459 437 449 455 5d 457 449 436 408 406 432 5e 472 463 452 414 416 445 5f 483 482 463 423 422 456 5g 472 463 453 422 418 445 5h 475 466 455 429 427 458 5i 487 476 466 433 428 457 5j 467 465 447 403 414 440 It was observed that in DMSO, DMF and acetonitrile, λmax of dyes 5a‐5j shifted bathochromically with respect to the λmax in chloroform (e.g. for dye 5d λmax is 432 nm in chloroform, 457 nm in DMSO, 449 nm in DMF and 436 nm in acetonitrile) (Figure 2). But, when we compare the bathochromic shifts of λmax of dyes 5a‐5j in acetonitrile are less than in DMSO and DMF solvent. On the other hand, it was observed that in acetic acid and methanol, λmax of dyes 5a‐5j shifted hypsochromically with respect to the λ max in chloroform (e.g. for dye 5b λ max is 440 nm in chloroform, 432 nm in acetic acid and 420 nm in methanol) (Figure 3). It was also observed that hypsochromic shifts of λmax of dyes 5a‐5c and 5j in acetic acid are less than hypsochromic shifts of λmax of dyes 5a‐5c and 5j in methanol. Hypsochromic shifts of λmax of dyes 5d‐5i in acetic acid and methanol are similar. Figure 2. Absorption spectra of dye 5d in various solvent. 3.3. Acid and base effects on UV‐Vis spectra The effects of acid and base on the absorption of dye solutions were investigated and the results are shown in (Table 2). The absorption spectra of the dyes in methanol were also quite sensitive to the addition of base (potassium hydroxide, 0.1 M), with λmax of dyes 5a‐5j showing bathochromic shifts and absorption curves of the dyes resembled those in DMSO and DMF (Figure 3). This result suggests that these dyes are present in a different tautomeric form in methanol+KOH than that in methanol and this tautomeric form resembled those in DMSO and DMF. When piperidine was added to dye solutions in chloroform, λmax of dyes 5a‐5j did not change significantly except for dye 5c (Figure 4). λmax of the dye 5c showed bathochromic shift when a small amount of piperidine was added to dye 5c solution in chloroform. When hydrochloric acid (0.1 M) was added to dye solutions in methanol, λmax of dyes 5a‐5c and 5j showed little bathochromic shifts with respect to the λmax in methanol and the absorption spectra of dyes resembled those in acetic acid (Figure 5). It was also observed that when hydrochloric acid (0.1 M) was added to dye solutions in methanol, λmax of dyes 5d‐5i did not change significantly. Figure 3. Absorption spectra of dye 5b in various solvent. 3.4. Substituent effects on UV‐Vis spectra As seen in (Table 1), generally, electron‐accepting chloro groups in all positions for dyes 5c, 5f and 5i cause bathochromical shifts in all used solvents when compared with dye 5a. λmax of dyes 5f and 5i in acetic acid hypsochromically shifted when compared with λmax of dye 5a in acetic acid. Electron‐donating methoxy and methyl groups in para position for dyes 5b and 5d and methyl group in ortho position for dye 5j because hypsochromical shifts in all used solvents when compared with dye 5a. Visible absorption spectra of dyes 5e, 5g and 5h did not regularly change with the substituent effect in all used solvents when compared with dye 5a. Patel / European Journal of Chemistry 3 (1) (2012) 44‐50 49 Table 3. Biological activities of dyes 5a‐5j (μg/mL). Dye no Bacteria Yeasts Bacillus subtilis Staphylococcus aureus Escherichia coli Proteus vulgaris Candida albicans Candida glabrata 5a 125 125 1000 1000 500 3.90 5b 2000 1000 2000 1000 2000 3.90 5c >4000 2000 1000 >4000 3.90 3.90 5d 1000 5000 1000 1000 1000 1000 5e 1000 1000 2000 2000 3.90 3.90 5f 1000 2000 1000 2000 3.90 3.90 5g 2000 1000 1000 1000 1000 1000 5h 1000 2000 2000 2000 2000 2000 5i 2000 2000 1000 2000 2000 1000 5j 2000 2000 1000 2000 2000 2000 Streptomycin 15.62 0.97 3.90 31.25 ‐ ‐ Fluconazole ‐ ‐ ‐ ‐ 3.90 7.81 Figure 4. Absorption spectra of dye 5c in different solutions. Figure 5. Absorption spectra of dye 5c in basic solutions. 3.5. Antimicrobial activity of the synthesized dyes Although, there are some reports about antimicrobial activity of monoazo dyes [31‐33], research on biological activity of disazo dyes has just started [34]. In the current study, in vitro antimicrobial activities of the newly synthesized disazo dyes were also reported. The results of antimicrobial screening of the synthesized compounds and standard antibiotics are given in (Table 3). The MIC values of the dyes are generally within the range 3.90‐2000 μg/mL against all tested microorganisms. Results showed that none of the synthesized dyes have important antibacterial activities when compared with control antibiotic, streptomycin. Not only gram negative but also Gram‐positive bacteria were resistant to all synthesized dyes with the exception of dye 5a. This compound showed activity against B. subtilis and S. aureus at the dose of 125 mg/mL. In contrast, different levels of antifungal activities were observed for some dyes against the yeasts. Dyes 5a‐5c, 5e and 5f exhibited stronger antifungal activity than not only other dyes but also fluconazole against C. glabrata. Among the all tested dyes, 5c, 5e and 5f showed the highest antifungal activity against both the yeasts. These dyes had similar activity level with fluconazole against C. albicans. On the other hand, as an impressive result, active concentration of these dyes against C. glabrata was lower than fluconazole. The rest of dyes had no important inhibitory activity against the yeasts C. albicans and C. glabrata. In previous studies about antimicrobial activity of monoazo or disazo dyes, synthesized compound had antibacterial activity [35,36]. It was reported weak or no antimicrobial activity for these compounds against fungi [37]. However, in the present study, synthesized dyes were determined more active against fungal organisms. Therefore the results obtained in the present study can be accepted as promising to develop new antifungal compound(s). 4. Conclusion A series of novel disazo dyes 4a‐4j based on heterocyclic rings were synthesized by heating pyrazolylhydrazonomalono nitriles with hydrazine monohydrate and disazo dyes 4a‐4j were further diazotized and coupled with 4‐hexylbenzene‐1,3‐ diol to produce the tetraazo dyes 5a‐5j. All newly synthesized tetraazo dyes were well characterized and acid‐base influence on the wavelength of maximum absorption have been studied. In the present paper, heterocyclic tetraazo dyes showed solvatochromic effects. When we compare the absorption maxima of such dyes in different solvent then it clearly indicate bathochromic shifts in DMSO and DMF rather than the other four solvents. It was also observed that the absorption spectra of these tetraazo dyes in methanol were quite sensitive to the addition of base. Our study demonstrated clearly that, novel 5c, 5e and 5f tetraazo dyes had significant antifungal activity when compared with control antibiotic, fluconazole. As a consequence, we can conclude that especially the newly synthesized 5c, 5e and 5f dyes could be lead for the development of new antifungal drugs. Acknowledgements The author is thankful to Mr. Ashok Chotalal Kapadia and Mr. Pradeep Natvarlal Mistry of Colortax (Private) Ltd., Surat, India for characterization of tetraazo dyes and Shashikant Babarbhai Patel of Sumit‐Industry, Pandesara, Surat, India for providing standard of analysis work. References [1]. Elnagdi, M. H.; Sallam, M. M. M; Fahmy, H. M.; Ibrahim, S. A. M.; Elias, M. A. M. Helv. Chim. Acta 1976, 59(2), 551‐557. [2]. Elnagdi, M. H.; Elgemeie, G. E. H.; Abdelaal, F. A. E. Heterocycles 1985, 23(12), 3121‐3153. [3]. Freeman, F. Synthesis‐Stuttgart 1981, 12, 925‐954. [4]. Tominaga, Y.; Honkawa, Y.; Hara, M.; Hosomi, A. J. Heterocycl. Chem. 1990, 27(3), 775‐783. [5]. Mohareb, R. M.; Sherif, S. M.; Gaber, H. M.; Ghabrial, S. S.; Aziz, S. I. Hetero. Chem. 2004, 15(1), 15‐20. [6]. Hanefeld, U.; Rees, C. W.; White, A. J. P.; Williams, D. J. J. Chem. Soc. Perkin Trans. 1996, 13, 1545‐1552. [7]. Ho, Y. W. Dyes Pigm. 2005, 64(3), 223‐230. [8]. Karcı, F.; Demirçalı, A. 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