untitled European Journal of Chemistry 2 (3) (2011) 388‐393 European Journal of Chemistry ISSN 2153‐2249 (Print) / ISSN 2153‐2257 (Online)  2011 EURJCHEM DOI:10.5155/eurjchem.2.3.388‐393.184 European Journal of Chemistry Journal homepage: www.eurjchem.com Synthesis and antimicrobial activity of novel oxime derivatives of phenothiazine Ashutosh Barvea, *, Malleshappa Noolvib, Niharika Subhedara, Vishnu Dev Guptaa and Gaurav Bhatiac a Department of Pharmaceutical Chemistry, Bhanwar Nahata College of Pharmacy, Mandsaur, 458001, India b Department of Pharmaceutical Chemistry, Amar Shaheed Baba Ajit Singh Jujhar Singh Memorial College of Pharmacy, Bela, 140111, India c Department of Pharmacognosy, Smriti College of Pharmaceutical Education, Indore, 452001, India *Corresponding author at: Department of Pharmaceutical Chemistry, Bhanwar Nahata College of Pharmacy, Mandsaur, 458001, India. Tel.: +91.9893107417; fax: +91.1881263655. E‐mail address: ashutoshbarve@ymail.com (A. Barve). ARTICLE INFORMATION ABSTRACT Received: XX November 2010 Received in revised form: XX January 2011 Accepted: XX January 2011 Online: 30 September 2011 KEYWORDS A series of 4‐methyl‐2‐oxo‐pyranyl‐phenothiazines (IIIa‐j) followed by 4‐methylpyrano‐(2, 3‐ β)‐phenothiazine‐2(11H)‐one oxime (IVa‐j) were synthesized by using 7‐hydroxy‐4‐ methylcoumarin (I). Further reaction of (I) was carried out with substituted aromatic amines (a‐j) to convert into 7‐arylamino‐4‐methyl‐coumarin (IIa‐j). Additionally (IIa‐j) was treated with sulphur in presence of iodine to obtain a series of novel 4‐methyl‐2‐oxo‐pyranyl‐ phenothiazine (IIIa‐j) derivatives, which on treatment with hydroxylamine hydrochloride afforded the title compounds i.e. 4‐methylpyrano‐(2,3‐β)‐phenothiazine‐2(11H)‐one oxime (IVa‐j). The structures of these compounds were confirmed by IR, NMR and Mass spectral analysis. The newly synthesized compounds were evaluated for antibacterial and antifungal activity. The results show that compound IIIa, IIIe, IIIh, IIIj, IVa, IVi and IVj exhibited moderate to good antibacterial and antifungal activity at 5‐100 mcg/mL. Coumarin Oxime Phenothiazine Antimicrobial activity Anti‐bacterial activity Anti‐fungal activity 1. Introduction In recent times microbial resistance against antimicrobial agents has increased remarkably. New prototype compounds are required to deal with this problem, so discovery of novel synthetic and semi‐synthetic product as antimicrobials is the prerequisite of present health scenario, and continuous effort in development of same is very much required. Pharmacological properties of coumarin aroused our interest to explore new analogs of coumarin for antimicrobial activity. Coumarin and its analogues are widely distributed in plants and they are responsible for wide variety of the activities especially antibacterial and antifungal [1‐10]. The in vitro antimicrobial activity of phenothiazine was first described by Paul Ehlich early in the twentieth century [11]. Various studies show and support those phenothiazines may be used for the management of bacterial or fungal infections [12‐21]. The study also shows that phenothiazine not only itself having antimicrobial effect but also it show synergistic interaction with other antimicrobial [22]. Moreover various heterocyclic compounds containing oxime, and the complexes of oximes with different transition metals are reported in the literature and. Found: to be active as antibacterial, antitubercular, antilepral, antiviral and antimalarial [23‐34] so we consider these reports and incorporate this functional moiety into our scheme. Therefore the development of facile synthetic routes to achieve access to these molecules is of prime interest. In view of the above mentioned pharmacological applications of phenothiazine coumarin and oxime and in continuation of our research on the synthesis of biologically active molecules, we considered undertaking the design and synthesis of hitherto unknown phenothiazine derivatives. Further, the increasing number of multidrug resistant pathogens has led us to screen the newly synthesized derivatives against the representative panel of Gram‐positive Gr (+) and Gram‐negative Gr (‐) bacteria and fungi. 2. Experimental 2.1. Instrumentation Melting points were determined in Thermonik melting point apparatus and are uncorrected. IR spectrum was recorded on Thermonicolet FT‐IR 200 spectrophotometer by using KBr pellet values are expressed in cm‐1. NMR spectra were recorded in DMSO‐d6 using varian 400 MHz mercury plus and chemical shift are reported in δ (ppm). Mass spectra were recorded on GCMS‐QP 2010 Shimaduzu and mass values are reported in m/z. All the chemicals used are of reagent grade and substituted aromatic amines and other chemicals used during synthesis are of synthesis fine grade. 2.2. Synthesis The synthesis of 7‐hydroxy‐4‐methylcoumarin (I) was carried out by the reaction of resorcinol and ethylacetoacetate; it is well established [35]. The various substituted 7‐arylamino‐ 4‐methyl‐coumarin (IIa‐j) were prepared by reacting with substituted aromatic amines (a‐j). The 7‐arylamino‐4‐methyl‐ coumarins (IIa‐j) were further converted into 4‐methyl‐2‐oxo‐ pyranyl‐phenothiazines (IIIa‐j) by the action of sulphur in presence of iodine. Further these novel 4‐methyl‐2‐oxo‐ pyranyl‐phenothiazines (IIIa‐j) derivatives, on treatment with hydroxylamine hydrochloride were converted to 4‐ Barve et al. / European Journal of Chemistry 2 (3) (2011) 388‐393 389 methylpyrano‐(2,3‐β)‐phenothiazine‐2(11h)‐one oxime (IVa‐j) derivatives as shown in Scheme 1. The reaction and purity of compounds were monitored by TLC using precoated silica gel. structures of the compounds were confirmed by spectral studies. 2.2.1. Preparation of compounds (IIIa‐j) [35,36] In 50 mL beaker, mixture of resorcinol (3.7 g) and ethylacetoacetate (4.4 mL) was added to concentrated sulphuric acid (15 mL) at 5 oC with constant stirring for 30 minutes. Mixture was poured on to the crushed ice (about 100 g), with vigorous stirring, 7‐hydroxy‐4‐methyl coumarin (I) was precipitated. Suspension was filtered; crude was dissolved in cold aq. sodium hydroxide (10%) solution and re‐ precipitated it by addition of dilute hydrochloric acid. Crude was decolorized and re‐crystallized from charcoal and ethanol respectively [35]. A mixture of 7‐hydroxy‐4‐methyl coumarin (I) (1 mol) and primary aromatic amine (1 mol) in absolute ethanol (20 mL) was heated under reflux in presence of anhydrous ZnCl2 (0.2 g) for 5 h. It was cooled and the separated crude mass was filtered, washed repeatedly with cold water, dried and recrystallized from methanol, the synthesized 7‐ arylamino‐4‐methyl‐coumarin (IIa‐j) and sulphur powder (1.0 g) were heated together at 150‐160 °C for 3 h in the presence of iodine (150 mg). The mixture was cooled to room temperature and treated with dilute hydrochloric acid (100 mL) to remove unreacted amine and washed repeatedly with warm water. Residue was dried in vacuum to yield phenothiazine analogs (IIIa‐j) [36]. 4‐methylpyrano [2,3‐β]phenothiazin‐2(11H)‐one (IIIa): Yield: 54.5%. M.p.: 180‐182 oC. FT‐IR (KBr, cm‐1): 3446 (N‐H), 3101 (C‐H), 2950 (C‐H), 1738 (C=O) (pyraneone), 1571 (C‐ N) (hetero), 1450 (C=C), 665 (C‐S) (hetero). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 6.92 (s, 1H, Ar‐H), 6.30‐6.88 (m, 4H, Ar‐H), 6.26 (s, 1H, Ar‐H), 5.67 (s, 1H, CO‐CH), 4.17 (s, 1H, C‐NH‐ C), 1.70 (s, 3H, C‐CH3). 13C NMR (400 MHz, CDCl3, δ, ppm): 160.9 (1C, >C=O), 152.8‐110.1 (14C, Ar‐C), 21.3 (1C, C‐CH3). MS (DIPMS, m/z): 280.05 and HMS calculated for C16H11NO2S: 281.051. Found: 281.0519. 10‐hydroxy‐4‐methylpyrano [2,3‐β] phenothiazin‐2(11H)‐ one (IIIb): Yield: 59%. M.p.: 182‐184 oC. FT‐IR (KBr, cm‐1): 3453 ( N‐H) (hetero), 3350 (O‐H) (phenol), 3037 (C‐H), 2877 (C‐ H) (aliphatic), 1741 (C=O) (pyraneone), 1525 (C‐N) (hetero), 1456 (C=C) (aromatic), 1263 (C‐O) (aromatic), 702 (C‐S) (hetero). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 6.30‐7.02 (m, 5H, Ar‐H), 5.73 (s, 1H, CO‐CH), 5.00 (s, 1H, Ar‐OH), 4.13 (s, 1H, C‐NH‐C), 1.78 (s, 3H, C‐CH3). 13C NMR (400 MHz, CDCl3, δ, ppm): 161.1 (1C, >C=O), 152.5‐109.2 (14C, Ar‐C), 21.2 (1C, C‐CH3). MS (DIPMS, m/z): 296.04 (M+) and HMS calculated for C16H11NO3S: 297.046. Found: 297.0456. 9‐hydroxy‐4‐methylpyrano [2,3‐β] phenothiazin‐2(11H)‐one (IIIc): Yield: 45.5%. M.p.: 178‐180 oC. FT‐IR (KBr cm‐1): 3108 (C‐H) (aromatic), 2866 (C‐H) (aliphatic), 1446 (C=C) (aromatic), 1483 (C‐N) (hetero), 3300 (N‐H) (hetero), 1716 (C=O) (pyraneone), 1242 (C‐O) (aromatic), 3218 (O‐H) (phenol), 669 (C‐S) (hetero). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 6.35‐7.13 (m, 5H, Ar‐H), 5.87 (s, 1H, CO‐CH), 5.06 (s, 1H, Ar‐OH), 4.17 (s, 1H, C‐NH‐C), 1.07(s, 3H, C‐CH3). 13C NMR (400 MHz, CDCl3, δ, ppm): 160.4 (1C, >C=O), 151.3‐111.5 (14C, Ar‐C), 21.6 (1C, ‐CH3). MS (DIPMS, m/z): 296.04 (M+) and HMS calculated for C16H11NO3S: 297.046. Found: 297.0456. 8‐hydroxy‐4‐methylpyrano[2,3‐β]phenothiazin‐2(11H)‐one (IIId): Yield: 50%. M.p.: 176‐179 oC. FT‐IR (KBr, cm‐1): 3477 (O‐H) (phenol), 3356 (N‐H) (hetero), 3049 (C‐H) (aromatic), 2867 (C‐H) (aliphatic), 1720 (C=O) (pyraneone), 1510 (C‐N) (hetero), 1460 (C=C) (aromatic), 1230 (C‐O) (aromatic), 675 (C‐S) (hetero). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 6.31‐7.01 (m, 5H, Ar‐H), 5.64 (s, 1H, CO‐CH), 5.06 (s, 1H, Ar‐OH), 4.17 (s, 1H, C‐NH‐C), 1.68 (s, 3H, C‐CH3). 13C NMR (400 MHz, CDCl3, δ, ppm): 160.8 (1C, >C=O), 152.2‐110.3 (14C, Ar), 21.4 (‐CH3 of α‐pyranone). MS (DIPMS, m/z): 296.04 (M+) and HMS calculated for C16H11NO3S: 297.046. Found: 297.0456. 10‐chloro‐4‐methylpyrano[2,3‐β]phenothiazin‐2(11H)‐one (IIIe): Yield: 59.2%. M.p.: 183‐185 oC. FT‐IR (KBr, cm‐1): 3446 (N‐H) (hetero), 3059 (C‐H) (aromatic), 2850 (C‐H) (aliphatic), 1749 (C=O) (pyraneone), 1571 (C‐N) (hetero), 1450 (C=C) (aromatic), 1090 (C‐Cl) (aromatic), 666 (C‐S) (hetero). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 6.24‐7.10 (m, 5H, Ar‐H), 5.74 (s, 1H, CO‐CH), 4.15 (s, 1H, C‐NH‐C), 1.68 (s, 3H, C‐CH3). 13C NMR (400 MHz, CDCl3, δ, ppm): 160.0 (1C, >C=O), 152.7‐110.1 (14C, Ar‐C), 21.7 (1C, ‐CH3). MS (DIPMS, m/z): 314.01 (M+) and HMS calculated for C16H10ClNO2S: 314. 0121. Found: 314.0127. 9‐chloro‐4‐methylpyrano[2,3‐β]phenothiazin‐2(11H)‐one (IIIf): Yield: 51.8%. M.p.: 175‐177 oC. FT‐IR (KBr, cm‐1): 3402 (N‐H) (hetero), 3057 (C‐H) (aromatic), 2883 (C‐H) (aliphatic), 1745 (C=O) (pyraneone), 1595 (C‐N) (hetero), 1456 (C=C) (aromatic), 1088 (C‐Cl) (aromatic), 698 (C‐S) (hetero). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 6.56‐6.93 (m, 4H, Ar‐H), 6.06 (s, 1H, Ar‐H), 5.81 (s, 1H, CO‐CH), 4.16 (s, 1H, C‐NH‐C), 1.67 (s, 3H, C‐CH3). 13C NMR (400 MHz, CDCl3, δ, ppm): 160.5 (1C, >C=O), 152.2‐110.9 (14C, Ar‐C), 21.1 (1C, ‐CH3). MS (DIPMS, m/z): 314.01 (M+) and HMS calculated for C16H10ClNO2S: 314. 0121. Found: 314.0127. 8‐chloro‐4‐methylpyrano[2,3‐β]phenothiazin‐2(11H)‐one (IIIg): Yield: 44.4%. M.p.: 170‐172 oC. FT‐IR (KBr, cm‐1): 3363 (N‐H) (hetero), 3074 (C‐H) (aromatic), 2933 (C‐H) (aliphatic), 1710 (C=O) (pyraneone), 1606 (C‐N) (hetero), 1086 (C‐Cl) (aromatic), 1415 (C=C) (aromatic), 705 (C‐S) (hetero). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 7.02‐7.07 (m, 1H, Ar‐H), 6.82 (s, 1H, Ar‐H), 6.61‐6.63 (d, J=7.6 Hz, 1H, Ar‐H), 6.34 (s, 1H, Ar‐H), 5.98 (s, 1H, Ar‐H), 5.90 (s, 1H, CO‐CH), 4.20 (s, 1H, C‐NH‐C), 1.65 (s, 3H, C‐CH3). 13C NMR (400 MHz, CDCl3, δ, ppm): 161.9 (1C, >C=O), 152.6‐110.1 (14C, Ar‐C), 21.3 (1C, ‐ CH3). MS (DIPMS, m/z): 314.01 (M+) and HMS calculated for C16H10ClNO2S: 314. 0121. Found: 314.0127. 4‐methyl‐9‐nitropyrano[2,3‐β]phenothiazin‐2(11H)‐one (IIIh): Yield: 51%. M.p.: 180‐182 oC. FT‐IR (KBr, cm‐1): 3318 (N‐H) (hetero), 3105 (C‐H) (aromatic), 2864 (C‐H) (aliphatic), 1719 (C=O) (pyraneone), 1552 (C‐N) (Nitro aromatic), 1510 (C‐N) (hetero), 1458 (C=C) (aromatic), 675 (C‐S) (hetero). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 7.73‐7.80 (dd, J=8.8 Hz, 1H, Ar‐H), 7.42 (s, 1H, Ar‐H), 7.16‐7.18 (d, J=8 Hz, 1H, Ar‐H), 6.76 (s, 1H, Ar‐H), 6.07 (s, 1H, Ar‐H), 5.93 (s, 1H, CO‐ CH), 4.18 (s, 1H, C‐NH‐C), 1.73 (s, 3H, C‐CH3). 13C NMR (400 MHz, CDCl3, δ, ppm): 160.0 (1C, >C=O), 152.4‐110.4 (14C, Ar‐C), 21.7 (1C, ‐CH3). MS (DIPMS, m/z): 325.03 (M+) and HMS calculated for C16H10N2O4S: 326.0361. Found: 326.0350. 4‐methyl‐8‐nitropyrano[2,3‐β]phenothiazin‐2(11H)‐one (IIIi): Yield: 54.5%. M.p.: 178‐180 oC. FT‐IR (KBr, cm‐1): 3256 (N‐H) (hetero), 3072 (C‐H) (aromatic), 2866 (C‐H) (aliphatic), 1715 (C=O) (pyraneone), 1515 (C‐N) (hetero), 1319 (C‐N) (Nitro aromatic), 1461 (C=C) (aromatic), 675 (C‐S) (hetero). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 7.80‐7.87 (dd, J=8 Hz, 1H, Ar‐H), 6.91‐6.92 (d, J=5.6 Hz, 1H, Ar‐H), 7.64 (s, 1H, Ar‐H), 6.61 (s, 1H, Ar‐H), 6.05 (s, 1H, Ar‐H), 5.72 (s, 1H, CO‐ CH), 4.10 (s, 1H, C‐NH‐C), 1.68 (s, 3H, C‐CH3). 13C NMR (400 MHz, CDCl3, δ, ppm): 160.3 (1C, >C=O), 153.4‐110.9 (14C, Ar‐C), 21.1 (1C, ‐CH3). MS (DIPMS, m/z): 325.03 (M+) and HMS calculated for C16H10N2O4S: 326.0361. Found: 326.0350. 4‐methyl‐2‐oxo‐2, 11‐dihydropyrano[2,3‐β]phenothiazine‐8‐ carboxylic acid (IIIj): Yield: 60%. M.p.: 176‐179 oC. FT‐IR (KBr, cm‐1): 3413 (N‐H) (hetero), 3035 (C‐H) (aromatic), 2923 (C‐ H) (aliphatic), 1714 (C=O) (pyraneone), 1680 (C=O acid stretching), 1475 (C‐N) (hetero) , 1427 (C=C) (aromatic), 390 Barve et al. / European Journal of Chemistry 2 (3) (2011) 388‐393 H2N R (I) (IIIa-j) S N O CH3 NOH H (IVa-j) NH2OH a) b) c) f) g) a, R= H b, R= 2-OH c, R= 3-OH d, R= 4-OH e, R= 2-Cl f, R= 3-Cl g, R= 4-Cl h, R= 3-NO2 i, R= 4-NO2 j, R= 4-COOH a) Concentrated H2SO4 b) Absolute C2H5OH c) Anhydrous ZnCl2 d) Sulphur e) Iodine f) C2H5OH g) Pyridine (a-j) OO OOHHO R S N O CH3 O H R HO O CH3 O (IIa-j) N O CH3 O H R d) e) Scheme 1 1395 (C‐O) (acid), 1271 (O‐H) (acid), 690 (C‐S) (hetero). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 10.57 (s, 1H, O‐H‐COOH), 7.87‐7.92 (dd, J=8 Hz, 1H, Ar‐H, ), 7.84 (s, 1H, Ar‐H), 6.91 (s, 1H, Ar‐H), 6.89 (s, 1H, Ar‐H), 6.16 (s, 1H, Ar‐H), 5.82 (s, 1H, CO‐CH), 4.12 (s, 1H, C‐NH‐C), 1.71 (s, 3H, C‐CH3). 13C NMR (400 MHz, CDCl3, δ, ppm): 160.2 (1C, >C=O), 169.8 (1C, Ar=COOH), 152.8‐ 110.1 (14C, Ar‐C), 21.3 (1C, ‐CH3). MS (DIPMS, m/z): 324.04 (M+) and HMS calculated for C17H11NO4S: 325. 0409. Found: 325.0452. 2.2.2. Preparation of compounds (IVa‐j) [37,38] To a solution of the keto‐ester (1.44 g, 10 mmol) and benzyl‐hydroxylamine (1.60 g, 10 mmol) in ethanol (30 mL) was added pyridine (5.0 mL, 62 mmol) in 1 portion. The reaction mixture was heated at 55 oC for 24 h and then concentrated on a rotary evaporator. The residue was partitioned between ether (150 mL) and water (50 mL). The organic layer was sequentially washed with hydrochloric acid (0.5 N, 60 mL) and water (30 mL), and then dried over Magnesium sulphate. Concentration in vacuo provided the oxime (2.50 g, 100%) as a 3:1 mixture of E Z isomers as a solids or liquid. 4‐methylpyrano[2,3‐β]phenothiazin‐2(11H)‐one oxime (IVa): Yield: 46.6%. M.p.: 186‐188 oC. FT‐IR (KBr, cm‐1): 3396 (N‐H) (hetero), 3252 (O‐H) (oxime), 3007 (C‐H) (aromatic), 2840 (C‐H) (aliphatic), 1622 (C=N) (oxime), 1487 (C‐N) (hetero), 1412 (C=C) (aromatic), 703 (C‐S) (hetero). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 6.92(s, 1H, Ar‐H), 6.26‐6.88 (m, 4H, Ar‐H), 6.07 (s, 1H, Ar‐H), 5.05 (s, 1H, N=C‐C‐H), 4.17 (s, 1H, C‐NH‐C), 2.07 (s, 1H, C= N‐OH), 1.68 (s, 3H, C‐CH313C NMR (400 MHz, CDCl3, δ, ppm): 168.3 (1C, >C=N‐OH), 153.3‐106.4 (14C, Ar‐C), 22.5 (1C, ‐CH3). MS (DIPMS, m/z): 295.06 (M+) and HMS calculated for C16H12N2O2S: 296.061. Found: 296.0609. 10‐hydroxy‐4‐methylpyrano[2,3‐β]phenothiazin‐2(11H)‐one oxime (IVb): Yield: 45.5%. M.p.: 184‐186 oC. FT‐IR (KBr, cm‐1): 3429 (O‐H) (phenol), 3419 (N‐H) (hetero), 3315 (O‐H) (oxime), 3053 (C‐H) (aromatic), 2885 (C‐H) (aliphatic), 1656 (C=N) (oxime), 1515 (C‐N) (hetero), 1450 (C=C) (aromatic), 1211 (C‐O) (aromatic), 696 (C‐S) (hetero). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 6.30‐6.72 (m, 5H, Ar‐H), 5.18 (s, 1H, N=C‐C‐H), 5.00 (s, 1H, Ar‐OH), 4.13 (s, 1H, C‐NH‐C), 2.03 (s, 1H, C= N‐OH), 1.78 (s, 3H, C‐CH3). 13C NMR (400 MHz, CDCl3, δ, ppm): 168.1 (1C, >C=N‐OH), 153.1‐105.8 (14C, Ar‐C), 22.3 (1C, ‐ CH3). MS (DIPMS, m/z): 311.05 (M+) and HMS calculated for C16H12N2O3S: 312.056. Found: 312.0550. 9‐hydroxy‐4‐methylpyrano[2,3‐β]phenothiazin‐2(11H)‐one oxime (IVc): Yield: 45.4%. M.p.: 194‐196 oC. FT‐IR (KBr, cm‐1): 3384 (N‐H) (hetero) , 3332 (O‐H) (phenol), 3211 (O‐H) (oxime), 3053 (C‐H) (aromatic), 2889 (C‐H) (aliphatic), 1637 (C=N) (oxime), 1533 (C‐N) (hetero), 1438 (C=C) (aromatic), 1220 (C‐O) (aromatic), 665 (C‐S) (hetero). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 6.15‐6.69 (m, 5H, Ar‐H), 5.19 (s, 1H, N=C‐C‐H), 5.06 (s, 1H, Ar‐OH), 4.17 (s, 1H, C‐NH‐C), 2.04 (s, 1H, C= N‐OH), 1.07 (s, 3H, C‐CH3). 13C NMR (400 MHz, CDCl3, δ, ppm): 168.5 (1C, >C=N‐OH), 154.0‐103.7 (14C, Ar‐C), 22.6 (1C, ‐ Barve et al. / European Journal of Chemistry 2 (3) (2011) 388‐393 391 CH3). MS (DIPMS, m/z): 311.05 (M+) and HMS calculated for C16H12N2O3S: 312.056. Found: 312.0550. 8‐hydroxy‐4‐methylpyrano[2,3‐β]phenothiazin‐2(11H)‐one oxime (IVd): Yield: 43.6%. M.p.: 178‐180 oC. FT‐IR (KBr, cm‐1): 3373 (O‐H) (phenol), 3222 (N‐H) (hetero), 3101 (O‐H) (oxime), 3029 (C‐H) (aromatic), 2862 (C‐H) (aliphatic), 1631 (C=N) (oxime), 1520 (C‐N) (hetero) , 1415 (C=C) (aromatic), 1253 (C‐O) (aromatic), 700 (C‐S) (hetero). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 6.13‐6.71 (m, 5H, Ar‐H), 5.18 (s, 1H, N=C‐C‐H), 5.06 (s, 1H, Ar‐OH), 4.17 (s, 1H, C‐NH‐C), 2.10 (s, 1H, C= N‐OH), 1.68 (s, 3H, C‐CH3). 13C NMR (400 MHz, CDCl3, δ, ppm): 167.4 (1C, >C=N‐OH), 152.5‐106.2 (14C, Ar‐C), 21.3 (1C, ‐CH3). MS (DIPMS, m/z): 311.05 (M+) and HMS calculated for C16H12N2O3S: 312.056. Found: 312.0550. 10‐chloro‐4‐methylpyrano[2,3‐β]phenothiazin‐2(11H)‐one oxime (IVe): Yield: 43%. M.p.: 192‐194 oC. FT‐IR (KBr, cm‐1): 3338 (N‐H) (hetero), 3255 (O‐H) (oxime), 3049 (C‐H) (aromatic), 2920 (C‐H) (aliphatic), 1653 (C=N) (oxime), 1591 (C‐N) (hetero), 1499 (C=C) (aromatic), 1088 (C‐Cl) (aromatic), 686 (C‐S) (hetero). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 7.00‐7.07 (m, 1H, Ar‐H), 6.82‐6.84 (d, J=8 Hz, 1H, Ar‐H), 6.61‐6.64 (t, J =6 Hz, 1H, Ar‐H), 6.74 (s, 1H, Ar‐H), 6.06 (s, 1H, Ar‐H), 5.10 (s, 1H, N=C‐C‐H), 4.15 (s, 1H, C‐NH‐C), 1.97 (s, 1H, C= N‐OH), 1.68 (s, 3H, C‐CH3). 13C NMR (400 MHz, CDCl3, δ, ppm): 166.2 (1C, >C=N‐OH), 153.6‐104.3 (14C, Ar‐C), 22.7 (‐ CH3). MS (DIPMS, m/z): 329.02 (M+) and HMS calculated for C16H11ClN2O2S: 330.023. Found: 330.0220. 9‐chloro‐4‐methylpyrano[2,3‐β]phenothiazin‐2(11H)‐one oxime (IVf): Yield: 45.8%. M.p.: 198‐200 oC. FT‐IR (KBr, cm‐1): 3427 (N‐H) (hetero) , 3152 (O‐H) (oxime), 3028 (C‐H) (aromatic), 2881 (C‐H) (aliphatic), 1618 (C=N) (oxime), 1500 (C‐N) (hetero), 1402 (C=C) (aromatic), 1098 (C‐Cl) (aromatic), 703 (C‐S) (hetero). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 6.56‐6.93 (m, 4H, Ar‐H), 6.06 (s, 1H, Ar‐H), 5.08 (s, 1H, N=C‐C‐H), 4.16 (s, 1H, C‐NH‐C), 1.93 (s, 1H, C= N‐OH) 1.67 (s, 3H, C‐CH3). 13C NMR (400 MHz, CDCl3, δ, ppm): 168.0 (1C, >C=N‐OH), 152.3‐105.4 (14C, Ar‐C), 22.2 (1C, ‐CH3). MS (DIPMS, m/z): 329.02 (M+) and HMS calculated for C16H11ClN2O2S: 330.023. Found: 330.0220. 8‐chloro‐4‐methylpyrano[2,3‐β]phenothiazin‐2(11H)‐one oxime (IVg): Yield: 48.6%. M.p.: 176‐178 oC. FT‐IR (KBr, cm‐1): 3220 (N‐H) (hetero) , 3154 (O‐H) (oxime), 3020 (C‐H) (aromatic), 2957 (C‐H) (aliphatic), 1625 (C=N) (oxime), 1566 (C‐N) (hetero) , 1400 (C=C) (aromatic), 1094 (C‐Cl) (aromatic), 692 (C‐S) (hetero). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 7.02‐7.07 (m, 1H, Ar‐H), 6.82 (s, 1H, Ar‐H), 6.61‐6.63 (d, J=7.6 Hz, 1H, Ar‐H, ), 6.34 (s, 1H, Ar‐H), 5.98 (s, 1H, Ar‐H), 5.17 (s, 1H, N=C‐C‐H), 4.20 (s, 1H, C‐NH‐C), 1.97 (s, 1H, C=N‐OH), 1.65 (s, 3H, C‐CH3). 13C NMR (400 MHz, CDCl3, δ, ppm): 168.9 (1C, >C=N‐OH), 151.6‐106.0 (14C, Ar‐C), 22.9 (1C, ‐CH3). MS (DIPMS, m/z): 329.02 (M+) and HMS calculated for C16H11ClN2O2S: 330.023. Found: 330.0220. 4‐methyl‐9‐nitropyrano[2,3‐β]phenothiazin‐2(11H)‐one oxime (IVh): Yield: 40%. M.p.: 196‐198 oC. FT‐IR (KBr, cm‐1): 3228 (N‐H) (hetero) , 3145 (O‐H) (oxime), 3045 (C‐H) (aromatic), 2891 (C‐H) (aliphatic), 1633 (C=N) (oxime), 1542 (C‐N) (nitro aromatic asym.), 1479 (C‐N) (hetero), 1427 (C=C) (aromatic), 1338 (C‐N) (nitro aromatic sym.), 898 (C‐ N), 671 (C‐S) (hetero). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 7.73‐7.80 (dd, J=8.8 Hz, 1H, Ar‐H), 7.42 (s, 1H, Ar‐H), 7.16‐7.18 (d, J=8 Hz, 1H, Ar‐H), 6.76 (s, 1H, Ar‐H), 6.07 (s, 1H, Ar‐H), 5.10 (s, 1H, N=C‐C‐H), 1.98 (s, 1H, C= N‐OH), 1.73 (s, 3H, C‐CH3), 4.18 (s, 1H, C‐NH‐C). 13C NMR (400 MHz, CDCl3, δ, ppm): 168.7 (1C, >C=N‐OH), 152.4‐105.0 (14C, Ar‐C), 22.7 (1C, ‐CH3). MS (DIPMS, m/z): 340.04 (M+) and HMS calculated for C16H11N3O4S: 341.047. Found: 341.0460. 4‐methyl‐8‐nitropyrano[2,3‐β]phenothiazin‐2(11H)‐one oxime (IVi): Yield: 45.2%. M.p.: 166‐168 oC. FT‐IR (KBr, cm‐1): 3218 (N‐H) (hetero), 3154 (O‐H) (oxime), 3031 (C‐H) (aromatic), 2842 (C‐H) (aliphatic), 1660 (C‐N) (nitro aromatic asym.), 1645 (C=N) (oxime), 1600 (C‐N) (hetero), 1406 (C=C) (aromatic), 1338 (C‐N) (nitro aromatic sym.), 860 (C‐N), 673 (C‐S) (hetero). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 7.80‐7.87 (dd, J=8 Hz, 1H, Ar‐H), 7.64 (s, 1H, Ar‐H), 6.91‐ 6.92 (d, J=5.6 Hz, 1H, Ar‐H), 6.61 (s, 1H, Ar‐H), 6.05 (s, 1H, Ar‐ H), 5.19 (s, 1H, N=C‐C‐H), 4.10 (s, 1H, C‐NH‐C), 2.08 (s, 1H, C= N‐OH), 1.68 (s, 3H, C‐CH3). 13C NMR (400 MHz, CDCl3, δ, ppm): 168.3 (1C, >C=N‐OH), 155.2‐103.8 (14C, Ar‐C), 22.1 (1C, ‐CH3). MS (DIPMS, m/z): 340.04 (M+) and HMS calculated for C16H11N3O4S: 341.047. Found: 341.0460. 2‐(hydroxyimino)‐4‐methyl‐2,11‐dihydropyrano[2, 3]phenothiazine‐8‐carboxylic acid (IVj): Yield: 48.3%. M.p.: 168‐ 170 oC. FT‐IR (KBr, cm‐1): 3263 (N‐H) (hetero) , 3252 (O‐H) (oxime), 3053 (C‐H) (aromatic), 2894 (C‐H) (aliphatic), 1685 (C=O) (acid), 1622 (C=N) (oxime), 1587 (C‐N) (hetero), 1450 (C‐O) (acid), 1402 (C=C) (aromatic), 1319 (O‐H) (acid), 689 (C‐S) (hetero). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 10.57 (s, 1H, Ar‐COOH), 7.87‐7.92 (dd, J=8 Hz, 1H, Ar‐H), 6.91 (s, 1H, Ar‐H), 7.84 (s, 1H, Ar‐H), 6.89 (s, IH, Ar‐H), 6.00 (s, 1H, Ar‐H), 5.06 (s, 1H, N=C‐C‐H), 4.12 (s, 1H, C‐NH‐C), 2.00 (s, 1H, C= N‐OH), 1.71 (s, 3H, C‐CH3). 13C NMR (400 MHz, CDCl3, δ, ppm): 168.2 (1C, >C=N‐OH), 169.8 (1C, =COOH), 155.8‐105.7 (14C, Ar‐C), 22.3 (1C, ‐CH3). MS (DIPMS, m/z): 339.05 (M+) and HMS calculated for C17H12N2O4S: 340.051. Found: 340.0528. 2.3. Biological activity The synthesized compounds were screened for the antibacterial and antifungal activity by using the agar‐cup technique in nutrients agar and potato dextrose agar media, respectively [39‐50]. Ciprofloxacin and gresiofluvin were used as standard drug for the antibacterial and antifungal activity respectively and zone of inhibition of all newly synthesized compounds (IIIa‐j) and (IVa‐j) was measured against these standard drugs (Table 1 and 2). The novel synthesized compounds have shown moderate activity against bacterial strain compared to standard drug. The title compounds have showed the better antibacterial activity and antifungal activity compared with the standard drug. We used microbial strains‐ Staphylococcus aureus (NCIM 2602); Bacillus subtilis (NCIM 2613) as Gr(+) bacterial strains, Escherichia coli (NCIM 2666); Pseudomonas aeruginosa (NCIM 5225) Gr (‐) bacterial strains as Saccharomyces cerevisiae (NCIM 3220); Candida albicans (NCIM 3471); Aspergillus niger (NCIM 813) as fungal strains for biological test. 3. Result and discussion We have synthesized a series of ten novel 4‐methyl‐2‐oxo‐ pyranyl‐phenothiazines (IIIa‐j) derivatives and their oxime derivatives i.e. 4‐methylpyrano‐(2,3‐β)‐phenothiazine‐2(11H)‐ one oxime (IVa‐j) using 7‐hydroxy‐4methyl‐coumarin. Structures of the synthesized compounds were established on the basis of IR, 1H NMR, Mass and HMS spectral data in order to substantiate the structures of the compounds. Compounds (IIIa‐j) have showed presence of absorption band at 670‐715 cm‐1 conformed the formation of phenothiazine ring (C‐S hetero stretching), sharp bands at 1715‐1775 cm‐1 gave conformation of >C=O in α‐pyrone ring in their respective spectra. Furthermore compounds (IVa‐j) have showed presence of absorption band ranging from 1620‐1690 cm‐1 for oxime (C=N) (oxime), and also showed presence of absorption band ranging from 3150‐3300 cm‐1 for oxime (O‐H) (oxime) gave conformation of formation of oxime (>C=N‐OH) oxime group in their respective spectra. 392 Barve et al. / European Journal of Chemistry 2 (3) (2011) 388‐393 Table 1. Antibacterial and antifungal activity of synthesized novel series of 4‐methyl‐2‐oxo‐pyranyl‐phenothiazines (IIIa‐j) derivatives by cup‐plate (agar cup) method*. Compound R Zone of inhibition in mm Bacterial strains Fungal strains Gr (+) Gr (‐) Bs Sa Ec Pa Sc Ca An IIIa ‐H 14 NA 17 20 18 19 10 IIIb ‐2OH NA 10 NA 9 8 12 15 IIIc ‐3OH 7 NA NA NA 8 10 8 IIId ‐4OH 12 NA NA 16 7 NA NA IIIe ‐2Cl 9 18 NA NA 22 NA 17 IIIf ‐3Cl 11 12 12 10 14 10 NA IIIg ‐4Cl 14 13 13 NA 15 14 10 IIIh ‐3NO2 18 17 NA 8 NA 11 NA IIIi ‐4NO2 14 8 9 15 15 13 10 IIIj ‐4COOH NA 9 22 17 12 16 16 Ciprofloxacin ‐ 18 18 25 30 ‐ ‐ ‐ Gresiofluvin ‐ ‐ ‐ ‐ ‐ 23 17 19 * Gr (+) bacterial strains: Bs: B. subtilis, Sa: S. aureus Gr (‐) bacterial strains: Ec: E. coli, Pa: P. aeruginosa, Fungal strains: Sc: S. cerevisiae, Ca: C. albicans, An: A. niger. The concentration of test compounds were 100 μg/mL. Solvent used DMF. NA = Not active. Table 2. Antibacterial and antifungal activity of synthesized novel series of 4‐methylpyrano (2,3‐β) phenothiazine‐2(11H)‐one oxime (IVa‐j) derivatives by cup‐ plate (agar cup) method. S O CH3 NOHN H R (IVa-j) Compound R Zone of inhibition in mm Bacterial strains Fungal strains Gr (+) Gr (‐) Bs Sa Ec Pa Sc Ca An IVa ‐H 14 18 23 14 27 15 NA IVb ‐2OH NA 10 NA NA 8 12 10 IVc ‐3OH 9 NA 8 15 NA 11 13 IVd ‐4OH NA 18 11 NA NA 12 14 IVe ‐2Cl 11 NA NA 10 16 14 NA IVf ‐3Cl 12 13 NA NA 12 10 8 IVg ‐4Cl 11 NA NA 13 11 14 NA IVh ‐3NO2 13 12 NA 18 NA 17 10 IVi ‐4 NO2 10 10 NA 12 7 10 16 IVj ‐4COOH 15 17 10 19 10 14 15 Ciprofloxacin ‐ 18 18 25 30 ‐ ‐ ‐ Gresiofluvin ‐ ‐ ‐ ‐ 23 17 18 * Gr (+) bacterial strains: Bs: B. subtilis, Sa: S. aureus Gr (‐) bacterial strains: Ec: E. coli, Pa: P. aeruginosa, Fungal strains: Sc: S. cerevisiae, Ca: C. albicans, An: A. niger. The concentration of test compounds were 100 μg/mL. Solvent used DMF. NA = Not active. In particular, it must be pointed out that in 1H NMR the characteristic peaks at δ 1.70 ppm (CH3 group of α‐pyrone ring), δ 4.17 ppm (N‐H group of phenothiazine), δ 5.67 ppm (C‐ H‐Pyran), 6.26 (C‐H‐Ar) and a multiplate at δ 6.30‐6.88 ppm (4H, C‐H‐Ar) indicate the presence of above groups in their respective structure. The compounds (IIIa‐j) showed prominent singlet at δ 6.90‐9.20 ppm for α‐pyrone, peak at δ 6.30‐7.20 ppm for aromatic protons. Compound (IVa‐j) have shown presence of a singlet between δ 9.10‐10.9 ppm indicate the formation of oxime (>C=N‐OH) by simple condensation process in all the spectra has confirmed the formation of oxime derivative and remaining peak for rest of the structure have been observed similar to that of compound (IIIa‐j). In 13C NMR the characteristic peaks of keto group at 160.90 ppm conforms the carbonyl group at pyranone ring, peaks appeared at 110‐152 ppm showed the peaks of aromatic carbon in molecule, peaks at 21.20 conforms the methyl group on pyranone ring. Compound (IVa‐j) have shown 13C NMR data at 168.20 conforms the formation of oxime, remaining have appeared similar to the compounds for (IIIa‐j). Final conformation of derivative have been done by DIPMS data, electron impact mass spectra showed an accurate molecular ion peak at m/z 281.3256, 297.3276, 297.3276, 297.3276, 314.0127, 314.0127, 314.0127, 326.0350, 326.0350, 325.0420 for title compounds IIIa‐j, respectively, and electron impact mass spectra showed an accurate molecular ion peak at m/z 296.060, 312.055, 312.055, 312.055, 330.022, 330.022, 330.022, 341.046, 341.046, and 340.052 for title compounds IVa‐j, respectively. The synthesized compounds were evaluated for in vitro antibacterial and antifungal activity against various strains Gr(+) bacterial strains: B. subtilis; S. aureus, Gr(‐) bacterial strains: E. coli; P. aeruginosa, fungal strains: S. cerevisiae; A. niger, C. albicans using nutrient agar cup plate method. The results are given in Table 1 and 2. We studied the effect of various phenothiazine and its oxime derivatives of coumarins containing various substituents like nitro (‐NO2), chloro (‐Cl), hydroxyl (‐OH), carboxylic (‐ Barve et al. / European Journal of Chemistry 2 (3) (2011) 388‐393 393 COOH) groups. The results showed that compounds (IIIa), (IIIe), (IIIh), (IIIj), (IVa), (IVi), and (IVj) exhibited comparable antibacterial and antifungal activity with the standard antibiotics ciprofloxin and greseofluvin. It has been observed that compound (IVa) show better antibacterial and antifungal activity compare to individual coumarin and phenothiazine and compounds (IIIa) and (IVa) (contains no substitution at ring 4) were shown better activity compare to rest of compounds with various substitution like ‐OH, ‐Cl, ‐NO2 and ‐COOH at different positions like ortho, meta, and para. 4. Conclusions In this paper, we report the synthesis and antimicrobial activity of novel series of 4‐methyl‐2‐oxo‐pyranyl‐ phenothiazines (IIIa‐j) and 4‐methylpyrano‐(2,3‐β)‐ phenothiazine‐2(11H)‐one oxime (IVa‐j). The preliminary in vitro antimicrobial activity of these novel series of derivative has evidenced that some of newly synthesized derivatives have shown very prominent potential as antimicrobial agents. The possible improvement of antimicrobial activity of these derivatives can be further modified based on modulation of ring substituent and/or additional fictionalization warrants further investigation. 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