Synthesis and antimicrobial activity of some new 1,2,4-triazine ... - NOPR

Indian Journal of Chemistry Vol. 53B, March 2014, pp. 325-331

Synthesis and antimicrobial activity of some new 1,2,4-triazine and benzimidazole derivatives Abha Bishnoi*a, Suruchi Singha, Anil K Tiwaria, Archna Ranib, Sapna Jainb & C K M Tripathic a

Department of Chemistry, Lucknow University, Lucknow 226 007, India

b

Department of Applied Chemistry, Delhi College of Engineering, New Delhi 110 042, India c

Fermentation Division, CSIR-Central Drug Research Institute, Lucknow 226 001, India E-mail: [email protected] Received 14 August 2012; accepted (revised) 7 November 2013

1-(4-Substituted phenyl)-3-(substituted)propan-1-(1H-benzo[d]imidazol-2-yl)hydrazines and 1-(4-substituted phenyl)3-(substituted)propan-1-(5H-[1,2,4]triazino[5,6-b]indol-3-yl) hydrazines have been synthesized by the fusion of triazine and benzimidazole derivatives with Mannich bases. The synthesized compounds have been characterized and screened against ITCC 5226 Sclerotium rolfsii and ITCC 0482 Macrophomina phaseolina and MTCC739 Escherichia coli, ATCC6533 Bacillus subtilis, ATCC9144 Staphylococcus aureus, ATCC25619 Pseudomonas aeruginosa and ATCC24433 Candida albicans. Keywords: Antimicrobial, animal pathogens, plant pathogens, benzimidazole, Mannich bases, triazine

Recent years have seen increased problem of microbial infection on human beings as well as on plants. In order to combat these infections, a wide range of antimicrobial agents are available but their continuous use has lead to development of resistance and resurgence of dangerous microbes. This has necessitated synthetic diversification through a new approach. In this regard development of broad spectrum antimicrobial agents can overcome the ever growing burden of newer and newer chemicals entering in the environment, designed against very specific microbes. Approaches to address this problem comprises the design of compounds, including more than one kind of active chemical moiety in a single molecule, to enhance antimicrobial activity and minimize the prevalence of resistance in microbes. Mannich bases have been reported as potent biological agents finding application in pharmaceutical chemistry as antibacterial, antimalarial, vasorelaxant, anticancer and analgesics as well as in agricultural chemistry as pesticides against various plant pathogens1-4. Similarly, literature revealed that benzimidazole or triazine exhibit broad spectrum of biological activity4-6 viz., fungicidal like benomyl (Benlate), carbendazim, propiconazole which have well known commercial value in protecting desirable plants from the development of fungal diseases. These are benzimidazole or triazine derivatives, containing nitrogen bearing side chain

(Figure 1). Imazalil, an imidazole fungicide (Figure 1d) used to control powdery mildews in cucumbers, marrows and ornamentals and Fusarium in sweet potatoes, also inhibits the growth of several other fungi7,8. Similarly, a series of 1,2,4-triazines has been discovered with potent antifungal activity9-11. These observations have prompted the design and synthesis of new series of Mannich bases containing 1,2,4-triazine or benzimidazole moieties in single molecular structure to explore their potency as agrochemicals and chemotherapeutics. Based on these findings and in continuation of the work on heterocycles12-15, 1-(4-substituted phenyl)-3(substituted) propan-1-(1H-benzo[d]imidazol-2-yl)hydrazines 7a-d and 1-(4-substituted phenyl)-3substituted propan-1-(5H-[1,2,4]triazino[5,6-b]indol3-yl)hydrazines 8a-c were synthesized and screened against multi host plant pathogenic fungi ITCC 5226 Sclerotium rolfsii, ITCC 0482 Macrophomina phaseolina, and animal fungi ATCC24433 Cadida albicans, along with some gram positive as well as gram negative bacterial strains, viz. MTCC739 Escherichia coli, ATCC6533 Bacillus subtilis, ATCC9144 Staphylococcus aureus and ATCC25619 Pseudomonas aeruginosa. Results and Discussion The preparation of new 1-(4-substituted phenyl)-3substituted propane-1-(1H benzo[d]imidazol-2yl)hydrazines 7a-d and 1-(4substituted phenyl)-3-

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N N N

O N

O

H N O

NH

O

N Cl

CH2

O O

Cl

N N

NH O

O

NH

Cl

c N

Cl a

d

b Figure 1 — a: Propiconazole, b: Benomyl, c: Carbendazim, d: Imazalil

substitutedpropane-1-(5H-[1,2,4]-triazino[5,6-b]indol3-yl)hydrazines 8a-c involves three steps: (i) Mannich reaction of different acetophenones and secondary amines, (ii) reduction of the keto compounds by sodium borohydride to the corresponding 3-N-substituted amino-1-phenyl-propanols 4a-e and (iii) the reaction of alcohol derivatives with thionyl chloride followed by addition of 2-hydrazinyl-1-Hbenzo[d]imidazoles 5/3-hydrazinyl-1-5H-[1,2,4]triazino[5,6-b]indole 6 in alcohol to produce compounds 7a-d and 8a-c (Scheme I). The structures of all products were in good agreement with the spectral data and elemental analysis.

of the organism. On the other hand, all the seven compounds were found to show random activity towards Sclerotium rolfsii. Most of the synthesized compounds have shown promising activity against S. aureus while very mild activity against P. aeruginosa was observed (Table I). Compounds 7c and 8a exhibited moderate activity against C. albicans, E. coli and B. subtilis. It may therefore be inferred that in order to elucidate a structure activity relationship, synthesis and antimicrobial evaluation of more such compounds with larger number of substituents is required and this work is in progress. Materials and Methods

Biological Activity Seven compounds were examined for their antifungal activity at a concentration of 500 ppm (Table I). Biological data against Macrophomina phaseolina indicate that benzimidazole derivatives of Mannich bases 7a-c exihibit slightly better inhibition than triazine derivatives of these bases 8a-c. It is very interesting to observe that two compounds 7b and 8c with substituents R (=OH) and R1R2 (=morpholine group) inhibited the growth of fungus to a considerable extent. It appears that these two groups, present together in a single molecular structure, play a greater role in conferring the antifungal activity to the compound than other groups studied in the current piece of work. Maximum inhibition of compound 7b against Macrophomina phaseolina suggests benzimidazole derivatives of Mannich base derived from p-hydroxy acetophenone and morpholine finds comparatively a better fit at the receptor site than the compounds bearing other group at these positions. Antifungal efficacy of 7b is also correlated with its energetically favoured conformation. It seems that the caged geometry of the compound 7b allows it to interact in a more favorable way with the receptor site

In vitro antifungal assay: Determination of percentage of inhibition Potato dextrose agar of HI MEDIA, MUO96 was used as culture medium. The compounds were dissolved in DMSO. Food poisoning method was followed to determine antifungal activity in terms of percentage of inhibition as described below16. Mother cultures of fungal species, grown and maintained on potato dextrose agar media at 28±1°C were used for the experiments. The compounds were mixed aseptically with PDA media and were sterilized. The medium with acetone was served as control. The Petri plates having 9 cm inner diameter were taken, and 25 mL of the sterilized PDA containing test samples were plated in the centre. After solidification, 0.5 cm diameter agar discs taken from a 48 hr old fungal mother culture was placed with the fungal side downward in the centre of each plate with the help of sterile cork borer and glass rod. The plates were incubated in dark at 28±1°C. Radial growths were determined by measuring the colony size along two diameters at right angles after complete growth in the control. Three replicates for

BISHNOI et al.: NEW 1,2,4-TRIAZINE AND BENZIMIDAZOLE DERIVATIVES

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3 and 4; a: R=H, R1=Morpholine, b: R=H, R1=N-benzyl methylamine, c: R=OH, R1= Morpholine, d: R=OH, R1=Pyrrolidine, e: R=NH2, R1= Pyrrolidine, 7; a: R=H, R1=Morpholine, b: R=OH, R1= Morpholine, c: R=OH, R1=Pyrrolidine, d: R=NH2, R1= Pyrrolidine, 8; a: R=H, R1=Morpholine, b: R=H, R1=N-benzyl methylamine, c: R=OH, R1= Morpholine. Scheme I

each treatment were prepared. The experiments were repeated three times. The zone of inhibition was then calculated17. In vitro antibacterial assay A loopful of culture was taken from the slants of individual bacterial strain and inoculated in 25 mL of sterile nutrient broth. Inoculation was carried out by dipping a sterile cotton-wool swab into the suspension and spread evenly over the entire surface of the

petriplate by swabbing in three directions. The plates were allowed to dry for some time before applying discs. Cultures were incubated at 37°C and 120 rpm in an orbital shaker for 10 hr. A volume of 100 µL of actively growing culture was evenly spread onto the surface of the nutrient agar plate. Impregnated discs were placed on the agar surface with sterile forceps and gently pressed down to ensure contact. A concentration of 1000 µg/µL of the compounds in ethyl alcohol was added to each disc (20 µL/disc)


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Table I — Antimicrobial activity of the synthesized compounds Antifungal activity (Zone of Inhibition in mm) (conc. 500 ppm)

Antibacterial activity (Zone of Inhibition in mm) (conc. 2 mg/disc )

Compd

ITCC 0482 M. phaseolina

ITCC5226 S. rolfsii

ATCC24433 C. albicans

MTCC739 E. coli

ATCC6533 B. subtilis

ATCC9144 S. aureus

ATCC25619 P. aeruginosa

7a 7b 7c 7d 8a 8b 8c

11 22 15 6 6 10 15

14 6 6 14 6 13 11

6 6 6 8 7 9 10

6 6 9 6 10 6 8

6 6 8 6 8 6 6

17 20 11 6 14 12 10

6 7 6 6 7 6 6

placed on a sterile Petri plate. The impregnated discs were dried for 3-5 min and placed on pre-inoculated agar surface. A control was also established by using just the solvent in which each compound was dissolved. The plates were incubated at 37°C for 24 hr. The degree of inhibition of the test compounds was observed by the formation of zones around the impregnated discs. It was measured in mm18. Experimental Section Melting points were determined in open capillaries in an electrically heated block and are uncorrected. The progress of the reaction was monitored by TLC, using TLC grade silica gel (G) and was developed by exposure to an atmosphere of iodine vapours. IR spectra were recorded on Perkin-Elmer 1430 spectrophotometer using KBr pellets and 1H and 13 C NMR spectra were obtained on Bruker F 400 MHz NMR spectrometer in CDCl3, CD3OD and DMSO-d6 using TMS as an internal standard (chemical shifts in δ, ppm). Mass spectra were recorded on a Va 70-70H spectrometer at 70 eV. Elemental analyses were carried out on Carlo Erba EA-1108 elemental analyzer. Synthesis: Compounds 5 and 6 were prepared by known procedures19,20. Synthesis of 3-N-substituted aminopropiophenones, 3a-e: Compound 3b was prepared by known procedure21. 3-Morpholino-1-phenylpropan-1-one, 3a. m.p. 172°C. Yield 78%; Rf value: 0.411 (9:1, Hexane:EtOAc); IR: 2808(C-H), 1719(C=O), 1360(C-N), 1050 cm-1 (C-O). Anal. Calcd for C13H17NO2: C, 71.23; H, 7.76; N, 6.39. Found: C, 71.33; H, 7.52; N, 6.19%. 1-(4-Hydroxyphenyl)-3-morpholinopropan-1-one, 3c. m.p.196°C. Yield 72%; Rf value: 0.393 (9:1,

Hexane:EtOAc); IR: 3225 (O-H), 2810 (C-H), 1720 (C=O), 1357 (C-N), 1078 (C-O), 760 cm-1 (Ar monosubs.); 1H NMR (400 MHz, CDCl3): δ 9.40 (4-OH), 8.31 (d, 2H, 2,6CH in ArH), 8.11 (d, 2H, 3,5CH in ArH), 4.33 (t, 2H, 2-CH2), 3.60 (t, 4H, CH2-N-CH2), 3.56 (t, 2H, 3-CH2N), 1.32 (t, 4H, CH2-N-CH2); 13C NMR (CDCl3): δ 198.0 (C-1), 67.5 (C-O-C), 53.3 (C-N-C), 49.3 (C-3), 29.6 (C-2); MS: m/z 235(M+). Anal. Calcd for C13H17NO3: C, 66.38; H, 7.23; N, 5.95. Found: C, 66.21; H, 7.11; N, 5.52%. 1-(4-Hydroxyphenyl)-3-(pyrrolidin-1-yl) propan-1one, 3d. m.p. 116°C. Yield 70%; Rf value: 0.224 (9:1, Hexane:EtOAc); IR: 3312 (O-H), 2886 (C-H), 1739 (C=O), 1357 (C-N), 776 cm-1 (Ar monosubs.). Anal. Calcd for C13H17NO2: C, 71.23; H, 7.76; N, 6.39. Found: C, 71.53; H, 7.63; N, 6.02%. 1-(4-Aminophenyl)-3-(pyrrolidin-1-yl) propan-1one, 3e. m.p. 51-62°C, Yield 83%; Rf value: 0.143 (8:2, Hexane:EtOAc); IR: 3584 (N-H), 2985 (C-H), 1747 (C=O), 1640 (N-H bend), 1351 (C-N), 765 cm-1 (Ar monosubs.). Anal. Calcd for C13H18N2O: C, 71.55; H, 8.25; N, 12.84. Found: C, 71.22; H, 8.14; N, 11.97%. Synthesis of 3-N-substituted amino-1-phenylpropanol, 4a-e: Free base of keto compound (3a-e; 0.026 mol) in methanol (50 mL) was cooled in icebath for 30 min and powdered NaBH4 (0.074 mol) was added in equal portions in 1 h. The reaction mixture was further stirred in an ice-bath for 30 min. It was then concentrated under reduced pressure and the residue was treated with water (10 mL) and ethyl acetate (60 mL). The ethyl acetate layer was separated, washed with water till neutral washings, dried (over anhyd. Na2SO4) and concentrated. The crude product 4a, 4c were purified by recrystallization


from ethanol, 4b from chloroform and 4d, 4e from absolute ethanol as solvents. The TLCs were obtained using hexane:ethyl acetate (8.5:1.5 and 8.2:1.8 v/v) as mobile phase. 3-Morpholino-1-phenylpropan-1-ol, 4a. The copmpound 4a was obtained as an oil. Yield 75%; Rf value: 0.47 (8.5:1.5, Hexane:EtOAc); IR: 3300 (O-H), 3010 (C-H str. Ar), 2889 (C-H), 1343 (C-N), 1047 cm-1 (C-O). Anal. Calcd for C13H19NO2: C, 70.58; H, 8.59; N, 6.33. Found: C, 70.42; H, 8.69; N, 6.1%. 3-(N-Benzyl-N-methyl amino)-1-phenylpropan1-ol, 4b. m.p.164°C. Yield 66%; Rf value: 0.332 (8.2:1.8, Hexane:EtOAc); IR: 3250 (O-H), 3078 (C-H str. Ar), 2927 (C-H), 1458 cm-1 (Ar stretch); 1H NMR (400 MHz, CDCl3): δ 7.38-7.21 (m, 10H, ArH), 4.944.90 (m, 1H, 1-CH), 3.68-3.46 (q, 2H, CH2-Benzyl JA=12.9Hz, JB=12.6Hz), 2.87-2.57 (m, 2H, 3-CH2), 2.27 (s, 3H, N-CH3), 2.05-1.86 (m. 2H, 2-CH2); MS: m/z 255(M+), 134, 120, 91. Anal. Calcd for C17H21NO: C, 77.92; H, 9.09; N, 6.06. Found: C, 77.36; H, 9.14; N, 5.72%. 4-(1-Hydroxy-3-morpholinopropyl) phenol, 4c. m.p. 169.5°C. Yield 76%; Rf value: 0.513(8.5:1.5, Hexane:EtOAc); IR: 3323 (O-H), 3046 (C-H str. Ar), 2911 (C-H), 1334 (C-N), 1053 cm-1 (C-O). Anal. Calcd for C13H19NO3: C, 65.82; H, 8.01; N, 5.90. Found: C, 65.21; H, 8.25; N, 4.33%. 4-(1-Hydroxy-3-(pyrrolidin-1-yl) propyl) phenol, 4d. The compound 4d was obtained as an oil. Yield 41.12%; Rf value: 0.4 (8.5:1.5, Hexane:EtOAc); IR: 3411 (O-H), 3033 (C-H str. Ar), 2885 (C-H), 1372 cm-1 (C-N). Anal. Calcd for C13H19NO2: C, 70; H, 8.59; N, 6.33. Found: C, 70.24; H, 8.63; N, 5.96%. 1-(4-Aminophenyl)-3-(pyrrolidin-1-yl) propan-1ol, 4e. The compound 4e was obtained as an oil. Yield 80%; Rf value: 0.28 (8.2:1.8, Hexane:EtOAc); IR: 3396 (O-H), 3028 (C-H str. Ar), 2812 (C-H), 1348 cm-1 (C-N). Anal. Calcd for C13H20N2O: C, 70.90; H, 9.09; N, 12.72. Found: C, 70.42; H, 9.04; N, 11.97%. Synthesis of 1-(4-substituted phenyl)-3(substituted)propan-1-(1H-benzo[d]imidazol-2-yl) hydrazines, 7a-d, and 1-(4-substituted phenyl)-3substituted propan-1-(5H-[1,2,4]triazino[5,6b]indol-3-yl)hydrazines, 8a-c: SOCl2 (0.04 mol) in benzene (10 mL) was added to 3-N-substituted amino-1-phenyl-propanol 4a-e (0.0015 mol) and the reaction mixture was heated on a water-bath for about 6 hr. Excess of SOCl2 was removed by azeotropic distillation with dry benzene. The chloride derivative of 4a, 4c, 4d, 4e without purification were mixed with 2-hydrazinyl-1-H-benzo[d]imidazole 5 (0.01 mol) and

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that of 4a, 4b, 4c with 3-hydrazinyl-1-5H[1,2,4]triazino[5,6-b]indole 6 (0.01 mol) in absolute ethanol and refluxed for 10-15 hr on water bath. The reaction mixture was concentrated under reduced pressure and the crude product was purified by recrystallization from absolute ethanol. Thus seven new compounds were synthesized and characterized. The TLCs were checked using Hexane:EtOAc (8:2 and 7.5:2.5 v/v) as mobile phase. 2-(1H-Benzo[d]imidazol-2-yl)-1-(3- morpholino1-phenylpropyl) hydrazine, 7a. m.p. 110-120°C. Yield 65%; Rf value: 0.66 (8:2, Hexane:EtOAc); IR: 3010 (C-H str. Ar), 2874 (C-H), 1671 (N-H bend), 1351 (C-N), 1154 (C-O), 810 cm-1 (Ar monosubs.); 1H NMR (400 MHz, DMSO-d6): δ 7.70-7.51 (m, 4H, ArH imidazole), 7.12-7.08 (m, 5H, ArH), 5.48 (s, 1H,-NH imidazole), 3.82-3.78 (s, 1H, 1-NH hydrazine), 3.60 (m, 2H, 2-CH2; JA= 8.32Hz, JB= 12.86 Hz), 3.37 (t, 4H,-CH2-O-CH2), 3.20-3.11 (s, 1H, 2-NH hydrazine), 2.73 (m, 2H, 3-CH2N), 2.732.66 (m, 2H, 3-CH2N), 2.57 (t, 4H,-CH2-N-CH2); 13C NMR (400 MHz, DMSO-d6): δ 141.0, 136.6 (C-3,4 imidazole), 65.6 (C-O-C), 60.4 (C-N-C), 57.1 (C-1), 36.1 (C-2), 48.9 (C-3), 36.1 (C-2); MS: 351(M+). Anal. Calcd for C20H25N5O: C, 68.37; H, 7.12; N, 19.94. Found: C, 68.42; H, 7.23; N, 18.26%. 2-(1H-Benzo[d]imidazol-2-yl)-1-(3-morpholino-1p-hydroxy-phenylpropyl)hydrazine, 7b. m.p. 148°C. Yield 74%; Rf value: 0.43 (8:2, Hexane:EtOAc); IR: 3330 (O-H), 3210 (C-H str. Ar), 2990 (C-H), 1690 (N-H bend), 1390 (C-N), 1084 (C-O), 792 cm-1 (Ar monosubs.); 1H NMR (400 MHz, DMSO-d6): δ 8.007.86 (m, 4H, ArH imidazole), 6.95-6.78 (m, 4H, ArH), 4.50-4.32 (s, 1H, OH), 4.12-3.95 (s, 1H,-NH imidazole), 3.89 (s, 1H, 1-NH hydrazine), 3.20-3.18 (m, 1H, 1-CH), 2.62 (s, 1H, 2-NH hydrazine), 2.57 (m, 4H,-CH2-O-CH2), 2.43 (m, 4H,-CH2-N-CH2), 2.40 (m, 2H, 3-CH2N), 1.49 (m, 2H, 2-CH2; JA= 8.68Hz, JB= 12.53Hz); 13C NMR (400 MHz, DMSOd6): δ 141.0, 133.8 (C-3,4 imidazole), 67.5 (C-O-C), 63.3 (C-N-C), 56.6 (C-1), 49.3 (C-3), 34.9 C-2); MS: 367(M+). Anal. Calcd for C20H25N5O2: C, 65.39; H, 6.81; N, 19.07. Found: C, 64.99; H, 7.00; N, 18.39%. 2-(1H-Benzo[d]imidazol-2-yl)-1-(1-p-hydroxyphenyl-3-(pyrrolidin-1-yl)propyl)hydrazine, 7c. m.p. 90-110°C. Yield 70%; Rf value: 0.55 (8:2, Hexane:EtOAc); IR: 3552 (N-H, str.), 3274 (O-H), 3027 (C-H str. Ar), 2789 (C-H), 2720 (C-H), 1550 (N-H bend), 729 cm-1 (Ar monosubs.); 1H NMR (400 MHz, DMSO-d6): δ 7.86-7.60 (m, 4H, ArH imidazole), 7.00-6.21 (m, 4H, ArH), 4.35 (s, 1H, OH;

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exchangeable with D2O), 3.41 (s, 1H, 2-NH hydrazine), 3.90 (s, 1H, NH imidazole), 2.69-2.62 (s, 1H, 1-NH hydrazine), 2.67 (m,1H, 1-CH), 2.36 (t, 2H, 3-CH2), 2.32-2.25 (m, 4H, 2,5-CH2 pyrollidine), 2.20 (m, 2H, 2-CH2; JA= 8.92Hz, JB= 12.64Hz), 1.59 (m, 4H, 3,4-CH2 pyrollidine); 13 C NMR (400 MHz, DMSO-d6): δ 140.6, 136.2 (C-3,4 imidazole), 59.1 (C-1), 54 (2,5 in pyrollidine), 48.2 (C-3), 28.5(3,4 in pyrollidine); MS: m/z 351(M+). Anal. Calcd for C20H25N5O: C, 68.37; H, 7.12; N, 19.94. Found: C, 68.42; H, 7.09; N, 19.06%. 2-(1H-Benzo[d]imidazol-2-yl)-1-(1-p-aminophenyl3-(pyrrolidin-1-yl)propyl)hydrazine, 7d. The compound 7d was obtained as an oil. Rf value: 0.50 (8:2, Hexane:EtOAc); IR: 3429 (N-H str.), 2981 (C-H str. Ar), 2981 (C-H str. Ar), 2749 (C-H), 1450 (N-H bend), 735 cm-1 (Ar monosubs); 1H NMR (400 MHz, DMSO-d6): δ 7.70-7.26 (m, 4H, ArH imidazole), 6.95-6.68 (m, 4H, ArH), 5.36 (s, 1H, OH, D2O exchangeable), 4.98 (s, 1H, NH imidazole), 4.20 (s, 1H, 2-NH hydrazine), 3.92 (m, 1H, 1-CH), 2.36 (t, 2H, 3-CH2), 2.11 (m, 4H, 2,5CH2 pyrollidine), 1.99 (s, 1H, 1-NH hydrazine), 1.94 (m, 2H, 2-CH2; JA= 8.15Hz, JB=11.93Hz), 1.42 (m, 4H, 3,4 CH2 pyrollidine); 13C NMR (400 MHz, DMSO-d6): δ 142.2, 136.2 (C-3,4 imidazole), 56.1 (C-1), 54.3 (2,5 in pyrollidine), 46.2 (C-3), 38.5 (3,4 in pyrollidine), 33.5 (C-2); MS: m/z 350(M+). Anal. Calcd for C20H26N6: C, 68.57; H, 7.28; N, 24.00. Found: C, 68.12; H, 7.38; N, 23.63%. 2-(5H-[1,2,4]Triazino[5,6-b]indol-3-yl)-1-(3-morpholino-1-phenylpropyl)hydrazine, 8a. m.p. 216220°C. Yield 67%; Rf value:0.34 (7.5:2.5, Hexane:EtOAc); IR: 3520 (N-H str.), 2760 (C-H), 1680 (N-H bend), 770 cm-1 (Ar monosubs.); 1H NMR (400 MHz, CD3OD): δ 8.92 (s, 1H,-NH indole), 7.55-7.30 (m, 4H, ArH indole), 7.21-7.08 (m, 5H, ArH), 3.90-3.84 (m, 1H, 1-CH), 3.89 (s, 1H, 1-NH hydrazine), 3.71-3.67 (t, 4H,-CH2-O-CH2), 2.59 (t, 4H,-CH2-N-CH2), 2.59-2.37 (t, 4H,-CH2-N-CH2), 2.31-2.26 (t, 2H, 3-CH2), 1.97-1.941 (m, 2H, 2-CH2; JA= 8.76Hz, JB= 11.73Hz), 1.92 (s, 1H, 2-NH hydrazine); 13C NMR (400 MHz, DMSO-d6): δ199.9 (C-4, 5 indole), 144.2, 142.6, 102.1 (C-2, 3 indole); MS: m/z 403(M+). Anal. Calcd for C22H25N7O: C, 65.50; H, 6.20; N, 24.31. Found: C, 65.30; H, 6.12; N, 23.63%. 3-(2-(5H-[1,2,4]Triazino[5,6-b]indol-3-yl)hydrazinyl)-N-benzyl-N-methyl-3-phenylpropan-1-amine, 8b. m.p. 93-100°C. Yield 70.5%; Rf value:0.45 (8:2, Hexane:EtOAc); IR: 2962 (C-H str. Ar), 2740 (C-H),

1625 (C-N, N-benzyl), 798 cm-1 (Ar monosubs.); 1H NMR (400 MHz, CD3OD): δ 9.42 (S, 1H,-NH indole), 8.00-7.92 (m, 4H, ArH indole), 7.82-7.76 (m, 10H, ArH), 4.20-4.10 (s, 1H, 1-NH hydrazine). 3.82 (m, 1H, 1-CH), 2.52(t, 2H, CH2-N), 2.41 (s, 3H, NCH3), 1.98 (s, 1H, 2-NH hydrazine), 1.30-1.22 (m, 2H, 2-CH2; JA= 8.71Hz, JB= 12.38Hz); 13C NMR (400 MHz, DMSO-d6): δ 210.3, 143 (C-4, 5 indole), 140.1, 100.6 (C-2, 3 indole), 62.7 (N-CH2), 43.3 (N-CH3); MS: m/z 437(M+). Anal. Calcd for C26H27N7: C, 71.39; H, 6.17; N, 22.42. Found: C, 71.52; H, 6.23; N, 20.86%. 2-(5H-[1,2,4]Triazino[5,6-b]indol-3-yl)-1-(3-morpholino-1-p-hydroxy-phenylpropyl) hydrazine, 8c. m.p. 204°C. Yield 65%; Rf value: 0.421 (7.5:2.5, Hexane:EtOAc); IR: 3300 (O-H), 2950 (C-H str. Ar), 2768 (C-H) 1640 (C-N, indole), 1392 (C-O), 786 cm-1 (Ar monosubs.); 1H NMR (400 MHz, CD3OD): δ 9.86 (s, 1H,-NH indole), 8.15-8.08 (m, 4H, ArH), 7.82-7.35 (m, 4H, ArH indole), 3.90 (m, 1H, 1-CH), 3.89-3.87 (s, 1H, OH), 3.49 (s, 1H, 1-NH hydrazine), 3.30-3.31 (t, 4H,-CH2-O-CH2), 3.20-3.23 (t, 4H,-CH2N-CH2), 2.82-2.80 (t, 2H, 3-CH2-N), 2.39 (s, 1H, 2NH hydrazine), 1.25-1.27 (m, 2H, 2-CH2; JA= 8.37Hz, JB= 12.63Hz); 13C NMR (400 MHz, DMSO-d6): δ 196.5, 143.2 (C-4, 5 indole), 98.8 (C-2, 3 indole); MS: 419(M+), 333, 169, 86. Anal. Calcd for C22H25N7O: C, 65.39; H, 6.81; N, 23.50. Found: C, 65.40; H, 6.91; N, 22.62%. Acknowledgement The authors express their sincere thanks to the Head, Department of Chemistry, Lucknow University, Lucknow, for providing laboratory facilities. They are also thankful to the Director, CDRI, Lucknow for elemental analysis and spectral data. Authors are also grateful to Head, Division of Agrochemicals, Indian Agricultural Research Institute, Pusa Road, New Delhi, for biological screening. References 1 Gul H I, Ojanen T & Hanninen O, Biol Pharm Bull, 25, 2002,1307. 2 Gul H I, Sahin F, Gul M, Ozturk S & Yerdelen K O, Arch Pharm (Weinheim), 338(7), 2005, 335. 3 Sharma B K, Srivastava J S, Prithviraj B, Singh U P & Pandeya S N, Folia Microbiol, 43(4), 1998, 393. 4 Sridhar S K, Pandeya S N, Stables J P & Ramesh A, Eur J Pharm Science, 26, 2002, 129. 5 Dimmock J R & Kumar P, Curr Med Chem, 4, 1997, 1. 6 Cremlyn R J, “Chiral imidazole fungicide composition and their use”, in Agrochemicals (John Wiley and Sons, New York), 1991, pp.157-216.


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