Indole derivatives kalyani bokde lncp bhopal

Indole derivatives kalyani bokde lncp bhopal

1. SYNTHESIS CHARACTERIZATION AND ANTIMICROBIAL EVALUATION OF INDOLE BASED HETEROCYCLIC COMPOUNDS A DISSERTATION SUBMITTED IN THE PARTIAL FULFILLMENT OF REQUIREMENT FOR THE AWARD OF MASTER OF PHARMACY IN PHARMACEUTICAL CHEMISTRY SUBMITTED TO RAJIV GANDHI PROUDYOGIKI VISHWAVIDYALAYA BHOPAL (M.P.) SUBMITTED BY KALYANI BOKDE 0137PY21MP05 UNDER THE GUIDANCE OF Dr. Mehta Parulben D. DIRECTOR PHARMACEUTICAL CHEMISTRY LAKSHMI NARAIN COLLEGE OF TECHNOLOGY BHOPAL (M.P.)

2. INTRODUCTION LITERATURE REVIEW AIM AND PLAN OF WORK EXPERIMENTAL BIOLOGICAL SCREENING RESULT CONCLUSION REFERENCES CONTENTS

3. INTRODUCTION Heterocyclic compounds   Design of novel and newer potent molecules has been the thirst of researchers worldwide. An important approach towards this incorporates the modification of the existing molecules with established biological actions utilizing the various approaches for drug designing. Heterocyclic compounds either of synthetic or natural origins have found applicability in several physiological neurotransmitters as well as in the modulators of certain neurotransmission processes. A cyclic organic compound containing all carbon atoms in ring formation is referred to as a carbocyclic compound. If at least one atom other than carbon forms a part of the ring system then it is designated as a heterocyclic compound [1]. Heterocyclic compounds containing nitrogen atoms are gifted with wide spectrum of biological actions. A plethora of sulphur and nitrogen possessing compounds are found in the biological and non-biological systems. Amongst the heterocyclic compounds containing sulphur and nitrogen, the six and five membered heterocycles have gathered the maximum interest, owing to their biological and industrial applications (Figure 1.1).

4. Generally nitrogen, oxygen and sulphur are known to be the part of heterocyclic ring. Wide variety of drugs, biologically active compounds having antitumor, antibiotic, anti-inflammatory, antidepressant, antiviral, antimalarial, anti-HIV, antimicrobial, antibacterial, antifungal, antidiabetic , herbicidal, fungicidal, and insecticidal agents are known to have heterocyclic ring in their structures.   Among these, heterocycles with nitrogen as heteroatom arrest the attention of research community due to their outstanding applications [2]. Amongst nitrogen containing heterocycles , Indole and its derivatives are well studied and documented heterocyclic compounds.

5. Figure 1.1 Major heterocycles (five and six membered ) Indole Indole is a benzopyrrole in which the benzene and pyrrole rings are fused at the 2-and 3-positions of the pyrrole nucleus. The Indole nucleus [Figure 1.1] has ten π-electrons which are circulating over nine atoms. Hence, Indole is an electron rich ring system Figure 1.1 Indole

6. LITERATURE REVIEW Zhao et al (2021 ) reported a novel method for synthesizing 10 H -indolo[1,2- a ] indole derivatives. by applying the intramolecular cyclization strategy at room temperature [22]. The reaction was realized through I 2 /ZnI 2 catalyzed intramolecular nucleophilic addition, hydrogen migration, and rearrangement procedures. The electronic effect and steric hindrance have a significant influence on the reactivity. Moreover, the synthetic value of 10 H -indolo[1,2- a ] indole was also exemplified by the selective decarboxylation and aromatization.

7. Heravi et al (2021) reviewed the synthesis of indole moiety present in selected alkaloids namely makaluvamine D, f tryprostatin A, physovenine , (-) debromoflustramine B, phalarine , lycogarubin C, lycogalic acid, (±)- minfiensine , (±)- aspidophylline A, (±)- scholarisine A, perophoramidine , dictyodendrins E, (-)- mersicarpine , racemosin B, asteropusazole , scholarisine K, alstolactine A, (-)- quebrachamine , (-)- pyrifolidine , (-)- aspidospermine , (-)- aspidospermidine , (-)- goniomitine , clauszoline K, glycozolicine , clausine C, clausine N, clausine M, clauraila A, clausenal , siamenol , (+)- strictamine , (-)-2(S)- cathafoline , (-)-Ψ- akuammigine , hyellazole , 4-deoxycarbazomycin B, fontanesine B, (-)- minovincine , (-)- aspidofractinine , (+)- uleine , (-)- tubifolidine and preparaherquamide among others. Most of these alkaloids involved modification of the Fisher Indole synthesis method [23].

8. Nguyen et al (2021) used ligand- and structure-based cheminformatics and experimental approaches for identifying the potential GPR17 ligand for GBM treatment [24]. They e identified a novel indoline -derived phenolic Mannich base as an activator of GPR17 using molecular docking of over 6000 indoline derivatives and synthesized one molecule through a multicomponent Petasis borono−Mannich reaction

9. Maeda et al (2021) studied he use of a Pd /C-ethylene system in the synthesis of anilines and indoles from cyclohexanones in the presence of NH 4 OAc [25]. Hydrogen transfer between cyclohexanone and ethylene generates the desired products. The reaction tolerates a variety of substitutions on the starting cyclohexanones .

10. PLAN OF THE WORK RESEARCH ENVISAGED AND PLAN OF WORK Research Envisaged In the past years, health-care associated infections have become an important cause of morbidity and mortality, whilst the incidence of antibiotic-resistant bacteria has increased dramatically and become a serious threat. In fact, the management of bacterial infections is getting increasingly tough due to augmented prevalence of MDR pathogens, which represent a major challenge to antimicrobial therapy. Microbial resistance is now frequently confronted to common antibiotics being used in clinical settings, and there is an imperative demand for newer anti-infective agents to overcome emerging multi-drug resistance [68]. Today, the need for novel antimicrobials has been greater than ever in the face of increasing resistance to the older ones and increasingly tough management of bacterial infections. In spite of urge for such agents, the scientific progression in terms of antimicrobial research and discovery of new antibacterial molecules has declined dramatically in the past few years.

11. Despite the fact that there is no existing antibiotic to which resistance does not develop the majority of research on antibiotics advances are in terms of upgrading previously existing antibiotic classes, and upgrades for novel classes are poor.   Hence it was decided upon to synthesize some indole derivatives linked with thiadiazole and assess their antimicrobial potential in culture medium. Objective The objective of the investigation was to   1. Synthesize indole derivatives linked with thiadiazole moiety 2. Assess the antimicrobial potential of the synthesized compounds

12. Plan of Work 1. Review of literature 2. Designing of a scheme for synthesis of indole derivatives 3. Procurement of reagents and chemical 4. Synthesis of indole derivatives 5. Characterization of synthesized molecules FTIR NMR Antimicrobial evaluation of the synthesized molecules ( in vitro ) Zone of inhibition by cup and plate method Minimum inhibitory concentration by serial dilution method

13. EXPERIMENTAL The primary objective of the present investigation was to synthesize a few indole derivatives and evaluate the antimicrobial activity of them. The synthetic scheme (Scheme 9) for the synthesis of indoles was designed using the schemes reported earlier.  

14. The reaction proceeds in four steps as mentioned below – Step 1 involves the synthesis of phenyl hydrazone Step 2 involves the cyclization of phenyl hydrazone to phenyl indole   Step 3 involves substitution of ethylacetate group to the nitrogen of phenyl indole   Step 4 involves cyclization of the ethyl ester to thiadiazole by reaction with thiosemicarbazide Reagents, Chemicals and Instruments Acetophenone , 3-hydroxy acetophenone , 4-hydroxy acetophenone , 4-methyl acetophenone , 4-nitro acetophenone , phenyl hydrazine, and polyphosphoric acid were obtained from Avra Chemicals. Ethyl chloroacetate was purchased from Sigma and thiosemicarbazide was purchased from Loba . Ethanol, methanol, glacial acetic acid, hydrochloric acid, acetone, chloroform and dimethyl sulfoxide were obtained from Rankem . Any other reagent used was of synthetic grade and used as procured.  

15. Vacuum desiccator ( polylab ), electrical heating mantle ( Biotechnics India), magnetic stirrer with hot plate (( Biotechnics India), water bath ( Biotechnics India), melting point apparatus ( Biotechnics India) and vacuum pump (Value) were used in the present study. All glassware used were of Borosilicate grade, washed using chromic acid cleaning mixture, rinsed with distilled water and dried in hot air oven ( Biotechnics India) before using. Synthetic method General method for synthesis of substituted phenyl hydrazone A mixture of 0.167 mol of appropriate acetophenone and 0.167 mol of phenyl hydrazine was prepared in 60 mL ethanol and a few drops of glacial acetic acid were added to it. The mixture as cooled to 0°C using an ice bath to obtain a solid. The solid was filtered and washed with dilute HCl and then by rectified spirit. The product was recrystallized using ethanol and white product obtained was filtered and stored in air tight container.

16. Synthesis of phenyl hydrazone , 1a   20 g of acetophenone was mixed with 18 g of phenyl hydrazine and the above process as in 4.2.1 was performed to obtain a colorless solid.   4.2.1.1 Synthesis of 3- hydroxyphenyl hydrazone , 1b   22.2 g of 3-hydroxy acetophenone was mixed with 18 g of phenyl hydrazine and the above process as in 4.2.1 was performed to obtain a colorless solid.   4.2.1.1 Synthesis of 4- hydroxyphenyl hydrazoneb , 1c   22.2 g of 4-hydroxy acetophenone was mixed with 18 g of phenyl hydrazine and the above process as in 4.2.1 was performed to obtain a pale yellow solid.   4.2.1.1 Synthesis of 4-methylphenyl hydrazone , 1d   22.4 g 4-methyl acetophenone was mixed with 18 g of phenyl hydrazine and the above process as in 4.2.1 was performed to obtain a pale yellow solid.   4.2.1.1 Synthesis of 4-nitrophenyl hydrazone , 1e   27.6 g 4-nitro acetophenone was mixed with 18 g of phenyl hydrazine and the above process as in 4.2.1 was performed to obtain a yellow solid.

17. General method for synthesis of substituted phenyl indole 0.15 mol of the substituted phenyl hydrazone was placed in a beaker containing excess of polyphosphoric acid (180 g). The mixture was heated on a boiling water bath, stirring the mixture and maintaining the temperature at 100-120°C for 10 min. To the reaction mixture was added 450 mL of cold water and it was well stirred in order to dissolve the polyphosphoric acid completely. The solid was filtered at pump and washed with cold water several times to remove any trace of acid. The solid was refluxed with 300 mL of rectified spirit and a little amount of decolorizing charcoal was added to it and filtered. The filtrate was cooled to room temperature to obtain the white crystals of phenyl indole which were dried in desiccator over anhydrous calcium chloride and stored in air tight container.

18. Synthesis of 2-phenyl indole , 2a   31.52 g of the 1a was placed in a beaker 180 g polyphosphoric acid and the above process in 4.2.2 was carried out to obtain 2a . Color –Yellow; melting point – 190-195°C   4.2.2.1 Synthesis of 3’ hydroxy-2-phenyl indole , 2b   33.94 g of the 1b was placed in a beaker 180 g polyphosphoric acid and the above process in 4.2.2 was carried out to obtain 2b . Color –Yellow; melting point – 213-215°C   4.2.2.1 Synthesis of 4’-hydroxy-2-phenyl indole , 2c   33.94 g of the 1c was placed in a beaker 180 g polyphosphoric acid and the above process in 4.2.2 was carried out to obtain 2c . Color –Yellow; melting point – 218-220°C   4.2.2.1 Synthesis of 4’-methyl-2-phenyl indole , 2d   33.64 g of the 1d was placed in a beaker 180 g polyphosphoric acid and the above process in 4.2.2 was carried out to obtain 2d . Color –Yellow; melting point – 178-181°C   4.2.2.1 Synthesis of 4’-nitro-2-phenyl indole , 2e   38.29 g of the 1e was placed in a beaker 180 g polyphosphoric acid and the above process in 4.2.2 was carried out to obtain 2e . Color –Yellow; melting point – 203-205°C

19. General method for synthesis of N-substituted phenyl indole Anhydrous K 2 CO 3 (0.006 mol ) was added to a solution of the appropriate phenyl indole (0.003 mol ) and ethylchloroacetate (0.003 mol ) dissolved in anhydrous DMF (10 mL) in a round bottom flask and refluxed for 1-2 h. The mixture was poured onto crushed ice to precipitate the solid product. The product was filtered at pump using Buchner funnel.   Synthesis of Ethyl 2-(2-phenyl-1H-indol-1-yl)acetate, 3a,   0.58 g of 2a , 0.83 g of K 2 CO 3 and 1.22 g of ethylchloroacetate were added in 10 mL DMF and the process mentioned in 4.2.3 was carried out to obtain 3a. Synthesis of Ethyl 2-(2-(3-hydroxyphenyl)-1H-indol-1-yl)acetate, 3b 0.63 g of 2b , 0.83 g of K 2 CO 3 and 1.22 g of ethylchloroacetate were added in 10 mL DMF and the process mentioned in 4.2.3 was carried out to obtain 3b.   Synthesis of Ethyl 2-(2-(4-hydroxyphenyl)-1H-indol-1-yl)acetate, 3c   0.63 g of 2c , 0.83 g of K 2 CO 3 and 1.22 g of ethylchloroacetate were added in 10 mL DMF and the process mentioned in 4.2.3 was carried out to obtain 3c.   Synthesis of Ethyl 2-(2-p-tolyl-1H-indol-1-yl)acetate, 3d   0.62 g of 2d , 0.83 g of K 2 CO 3 and 1.22 g of ethylchloroacetate were added in 10 mL DMF and the process mentioned in 4.2.3 was carried out to obtain 3d.   Synthesis of Ethyl 2-(2-(4-nitrophenyl)-1H-indol-1-yl)acetate, 3e   0.72 g of 2e , 0.83 g of K 2 CO 3 and 1.22 g of ethylchloroacetate were added in 10 mL DMF and the process mentioned in 4.2.3 was carried out to obtain 3e.  

20. General method for the synthesis of thiazole -linked compounds A mixture of compound 3a-e (0.01 mol ), thiosemicarbazide (0.91 g ; 0.01 mol ) and (5 mL) phosphorus oxychloride was refluxed for 8 hrs. The cold reaction mixture was poured on crushed ice and neutralized by adding sodium hydroxide solution. The resulting solid was filtered and recrystallized from chloroform to give a white crystals of amino thiadiazole derivatives, 4a-e . Physicochemical characterization of synthesized compounds All the synthesized compounds were characterized for their melting point, solubility and retention factor ( R f value) by TLC. Melting point The temperatures at which the compounds melt were by open capillary method and the values obtained are uncorrected. The dry compound was filled in capillary tube sealed at one end and place in the heating head of the melting point apparatus. The melting temperature was recorded using thermometer.

21. Solubility Solubility of the compounds was determined qualitatively in test tubes. A small amount of compound was placed in test tube and 1 mL of solvent (water, methanol, chloroform, DMF) was added. The test tube was shaken for few seconds and was observed for visible particles if any. Determination of R f value Precoated silica gel G plates were used for determining the R f value of the compounds. A dilute solution of the compound was prepared in ethanol and as spot was applied on the TLC plate using a tapered capillary tube. The plate was kept in the developing solvent (n- butanol -acetic acid-water, 12-3-5) and the solvent was allowed to run. The plates were removed from the solvent when a small distance was remaining on the plate to be soaked in solvent. The plates were air dried and visualized using TLC cabinet under UV light. The R f value was calculated using the formula R f = Distance traveled by spot/Distance traveled by solvent

22. Spectral Studies   Infra-red (IR) Spectral study   The dried compounds were directly place on the sample screen of the spectrophotometer and the infra-red spectrum was obtained. 1 H-NMR spectral study   The samples were dissolved in deuteriated chloroform and filled in the NMR tubes. The spectrum was recorded at 300 MHz frequency. Antimicrobial Study Microorganisms used   The microorganisms used for the antimicrobial study were procured from Institute of Microbial Technology, Chandigarh (MTCC). Escherichia coli (MTCC 40), and Staphylococcus aureus (MTCC 3160) were used for the present investigation.

23. Nutrient Broth preparation Ready to use broth powder was used for preparing the nutrient broth ( Microgen ). Composition of the nutrient broth powder (pH 6.8 ±0.2) Ingredients Amount (g/L) Beef Extract 1.5 Peptone 5.0 Yeast dried 1.5 Sodium chloride 5.0 13 g of the nutrient broth powder was dissolved in 1000 mL of distilled water with the aid of a slight heat and sterilized using autoclave at 121°C and 15 lbs pressure for 15 min. Revival of lyophilized cultures The lyophilized cultures obtained from IMT, Chandigarh were revived by adding 0.3 mL of nutrient broth to the culture ampoules to obtain a suspension of the bacteria.

24. Nutrient agar preparation Ready to use nutrient agar powder was used for preparing the nutrient agar medium ( Microgen ). Composition of the nutrient agar powder (pH 6.8 ±0.2) Ingredients Amount (g/L) Beef Extract 3.0 Peptone 5.0 Agar 15.0 23 g of the nutrient agar powder was dissolved in 1000 mL of distilled water with the aid of a slight heat and sterilized using autoclave at 121°C and 15 lbs pressure for 15 min.   Agar plates were prepared by pouring the sterilized medium into sterilized petridishes suitably marked and labeled. The plates were allowed to solidify in the laminar flow bench and stored packed for culturing with microbes and antimicrobial screening.

25. Preparation of test compounds   The synthesized Schiff’s bases were dissolved in DMSO to obtain the solutions of 25, 50, 75 & 100 µg/ mL. These solutions were used as the test samples.   Screening Procedure (zone of inhibition)   About 3 mm thick pre-poured nutrient agar plates were inoculated with a few drops of the bacterial suspension by swabbing on the surface of agar. The antimicrobial action was screened using disc diffusion method [69]. Wells were bored into the agar plate at equal distances using cork borer (10mm) and 200µL of the Schiff’s bases (25, 50, 75 & 100 µg/mL) were placed in each hole. The plates were incubated for 24h at 37 ± 0.1°C to allow for microbial growth. The zone of inhibition in each plate was measured in millimeters. Screening Procedure (Minimum inhibitory concentration)   The broth dilution technique was used to determine the minimum inhibitory concentration of the synthesized compounds. The final inoculum size (of bacterial culture) was maintained to 10 5 CFU/ mL. A set of tubes containing only the inoculated broth was used as the growth control, and one containing only the broth was used to ensure the sterility of the medium.

26. A set of six tubes was labelled as 1 to 6 and to each tube was added 3 mL of nutrient broth. To the first tube was added 300µL of 1mg/mL of the test sample. The contents were mixed by swirling between hands and 300µL of content from the 1 st tube was transferred to 2 nd tube. The process was repeated from 2 nd to 3 rd tube up to the 6 th tube. From the 6 th tube, 300µL of content was withdrawn and discarded. To each of these tubes was added 200µL of the bacterial inoculum. All the tubes were incubated at 37°C for 24- 48 h to allow for growth of micro-organism. After incubation, the optical density of the content from each tube was observed at 600 nm using UV-Visible spectrophotometer. The concentration that led to half of the optical density (50%) of the growth control tube was observed for each sample and standard ( norfloxacin )

27. RESULT The primary objective of the present investigation was to synthesize a few indole derivatives linked with thiadiazole and evaluate the antimicrobial activity of them. Chemical characterization The synthesis was carried out by utilizing the reaction pathway scheme depicted in Scheme 9.   In the first two steps, aryl hydrazone is prepared by condensation of phenyl hydrazine and aromatic ketone followed by Fisher indole synthesis of indole via cyclization in the presence of acid catalyst. In the last step the nitrogen of the phenyl indole is substituted with various alkyl groups.   The mechanism involved in formation of phenyl indole is presented in scheme

28. Mechanism of Fisher Indole Synthesis Five derivatives of indole were synthesized using five aromatic acetophenones . These compounds were substituted on the nitrogen of indole and modified to obtain thiadiazole linked compouns . The compounds were characterized by using TLC and IR. NMR and

29. mass spectral study was carried out on five compounds in order to assure the formation of proper products. The result of the yield, melting point and R f value of the synthesized compound were depicted in the Table 5.1 while the solubility profile is presented Characters of Synthesized Compounds     Code     Color   M.P ( ° C)   %   Yield   R f value   4a   Brown   233-235   63   0.67 4b Yellow 206-208 69 0.48 4c Yellow 249-251 70 0.63 4d Brown 263-265 72 0.54 4e Brown 259-261 75 0.43

30. Solubility of the synthesized indoles   Comp. Name   Solubility Profile   Water   Methanol CHCl 3   DMF 3a - + ++ - 3b - + ++ - 3c - + ++ - 3d - + ++ - 3e - + ++ - Insoluble, + partially soluble, ++ soluble Structure Elucidation 5-((2-phenyl-1H-indol-1-yl)methyl)-1,3,4-thiadiazol-2-amine, 4a

31. 1 H NMR Spectra (d, 300 MHz, CDCl 3 ): 7.2-7.5 (H aromatic), 6.5 (H- pyrrole ring), 3.60 (H-methyl)

32. IR ( KBr ): 3420.33 (C-H), 3161.06 cm -1 (CH Ar ), 2603.54 (-CH 2 -), 1600-1700 cm -1 (C-C Ar ), 1485.76 cm -1 (C=C), 1197.04 cm -1 (C-N) m/z: 306.38   3-(1-((5-amino-1,3,4-thiadiazol-2-yl)methyl)-1H-indol-2-yl)phenol , 4b  

33. 1 H NMR Spectra (d, 300 MHz, CDCl 3 ): 7.4 (CH-Benzene), 7.3 (CH-Benzene), 7.2 (CH- Benzene), 2.5 (CH 2 ), 7.0 (CH-Benzene), 7.1 (CH-Benzene)

34. IR ( KBr ): 3410.90 (O-H), 3121.21 (C-H Ar ), 2602.10 (C-H), 1500-1650 cm -1 (C-C Ar ), 1481.52 (C=C), 1282.15 (C-N), 1069.29 (C-O ) m/z: 322.38 4-(1-((5-amino-1,3,4-thiadiazol-2-yl)methyl)-1H-indol-2-yl)phenol, 4c  

35. 1 H NMR Spectra (d, 300 MHz, CDCl 3 ): 7.4 (CH-Benzene), 7.3 (CH-Benzene), 7.2 (CH- Benzene), 2.5 (CH 2 ), 7.0 (CH-Benzene), 7.1 (CH-Benzene)  

36. IR ( KBr ): 3240.81 (O-H), 3119.55 (C-H Ar ), 2981.44 (C-H), 1500-1650 cm -1 (C-C Ar ), 1484.01 (C=C), 1396.53 (O-H bending), 1294.08 (C-N), 1092.37 (C-O ) m/z: 322.38

37. 5-((2-(p- tolyl )-1H-indol-1-yl)methyl)-1,3,4-thiadiazol-2-amine, 4d 1 H NMR Spectra (d, 300 MHz, CDCl 3 ): 7.4 (CH-Benzene), 7.3 (CH-Benzene), 7.2 (CH- Benzene), 2.5 (CH 3 ), 7.0 (CH-Benzene), 7.1 (CH-Benzene)

38. IR ( KBr ): 3113.84 (C-H Ar ), 2943.23 (C-H), 1500-1650 cm -1 (C-C Ar ), 1461.21 (C=C), 1282.19 (C-N)

39. 5-((2-(4-nitrophenyl)-1H-indol-1-yl)methyl)-1,3,4-thiadiazol-2-amine, 4e 1 H NMR Spectra (d, 300 MHz, CDCl 3 ): 7.1-7.4 (H Aromatic), 8.25 (H adjacent to NO 2 ), 3.6 (H – methyl)

40. IR ( KBr ): 3116.02 (C-H Ar ), 2978.17, 2809.22 (C-H), 1500-1650 cm -1 (C-C Ar ), 1525.49 (N-O), 1454.47 (C=C), 1285.93 (C-N) The structure elucidation of the compounds was performed by IR, 1 HNMR and mass spectroscopy. The IR spectra of all the compounds exhibited the stretching vibration peaks due to C-N, C=C, C-H Ar , CH aliphatic at 1300-1100 cm -1 , 1500-1700 cm -1 , 3000- 3200 cm -1 and 2600-3000 cm -1 respectively. The other vibrations that appeared in the spectra included those from C-O (1000-1100 cm -1 ), O-H (3200-3500 cm -1 ) and N-O (1400-1500 cm -1 ).

41. The 1 HNMR spectra obtained displayed the peaks of aliphatic CH and aromatic CH as well as peak of O-H in the corresponding compounds ( 4b, 4c ). The mass spectra displayed the molecular ion peak and the isotopic peaks as calculated. Antimicrobial activity Zone of Inhibition The zone of inhibition was measured to assess the preliminary antibacterial activity of the imine-linked benzothiazine derivatives (Schiff’s bases). Four concentrations of the conjugates were tested for antibacterial action. Norfloxacin was used as the standard drug for antibacterial action (Table 5.1). Zone of inhibition exhibited by compounds

42.       Compound Code   Zone of Inhibition (mm)*   E. coli   S. aureus   25µg   50µg   75µg   100µg   25µg   50µg   750µg   100µg 4a - - 12 13 - - - 12 4b - - 13 14 - - 11 12 4c - - 16 17 - - 13 14 4d - - 14 16 - - 14 16 4e - - 19 25 - - 17 19 Norfloxacin 23 - - - 24 - - -

43. Culture plate of 4a exhibiting zone of inhibition against S. aure us Culture plate of 4a exhibiting zone of inhibition against E. coli Minimum Inhibitory Concentration (MIC) The MIC value of the test compounds was determined using broth dilution method by measuring the optical density of the broth solution incubated with diluted drug samples. The concentration that resulted in 50% optical density in comparison to the growth tube was taken as MIC of the test sample (Table 5.2 & 5.3).

44. Optical density of test samples against S. aureus     T1   T2   T3   T4   T5   T6   Control Concentration (µg/mL)     100     10     1     0.1     0.01     0.001     -   Optical Density at 600 nm 4 a 0.521 0.578 0.684 0.806 0.869 0.937 0.963 4 b 0.658 0.774 0.911 0.934 0.957 0.962   4 c 0.628 0.739 0.884 0.928 0.954 0.961   4 d 0.641 0.702 0.811 0.847 0.895 0.916   4 e 0.354 0.515 0.621 0.737 0.803 0.866   Norfloxacin 0.234 0.308 0.419 0.547 0.695 0.721  

45. Optical density of test samples against E.coli     T1   T2   T3   T4   T5   T6   Control Concentration (µg/mL)     100     10     1     0.1     0.01     0.001     Optical Density at 600 nm 4 a 0.553 0.616 0.683 0.76 0.822 0.903 1.178 4 b 0.732 0.788 0.928 0.974 1.013 1.159   4 c 0.712 0.769 0.885 0.958 0.989 1.147   4 d 0.721 0.837 0.917 0.989 1.019 1.165   4 e 0.531 0.584 0.656 0.733 0.794 0.865   Norfloxacin 0.144 0.197 0.304 0.438 0.579 0.625  

46. Calculated IC 50 values of the test samples     Test sample   IC50 (µg/mL)   E. coli   S. aureus 4 a 78.03 107.33 4 b 147.08 161.27 4 c 140.11 146.20 4 d 141.25 169.38 4 e 66.94 62.21 Norfloxacin 0.89 0.01 As visible from the results, the IC 50 value for 4e was the lowest against both the pathogen (66.94 µg/mL for E. coli and 62.21 µg/mL for S. aureus ). Also form the results it was very evident that attachment of electron donating groups in the molecule increased the IC50 (reduced antimicrobial potency) as seen in compounds 4b, 4c and 4d. In contrast non substituted compound, 4a was having a higher antibacterial activity in comparison to the electron donor substitution.

47. CONCLUSION AND SUMMARY In the present work a few 2-phenylindole derivatives were synthesized and conjugated with thiadiazole nucleus via a linker and the compounds were assessed for their antimicrobial potential. The synthesis was achieved in four steps where in the first two steps, aryl hydrazone is prepared by condensation of phenyl hydrazine and aromatic ketone followed by Fisher indole synthesis of indole via cyclization in the presence of acid catalyst. In the third step the nitrogen of the 2-phenyl indole is substituted with ethylacetate group and in the final step of the reaction, the acetate group was cyclized to thiadiazole by reaction with thiosemicarbazide . The structure of the compounds was elucidated using IR, Mass and 1H NMR spectral studies while TLC was carried out to monitor the completion of reactions. Antidepressant studies of these compounds indicated that the compounds were having significant antidepressant activity. The IR spectra of all the compounds exhibited the stretching vibration peaks due to C-N, C=C, C-H Ar , CH aliphatic at 1300-1100 cm -1 , 1500-1700 cm -1 , 3000-3200 cm -1 and 2600-3000 cm -1 respectively. The other vibrations that appeared in the spectra included those from C-O (1000-1100 cm -1 ), O-H (3200-3500 cm -1 ) and N-O (1400-1500 cm -1 ).

48. The 1 HNMR spectra obtained displayed the peaks of aliphatic CH and aromatic CH as well as peak of O-H in the corresponding compounds ( 4b, 4c ). The mass spectra displayed the molecular ion peak and the isotopic peaks as calculated.   The antimicrobial action of the synthesized compounds was testing using two bacteria, Escherichia coli and staphylococcus aureus . The standard drug norfloxacin and the test compounds 4e and 4a were found to significantly inhibit the growth of the microbes in culture medium presenting IC 50 value of 66.94 µg/mL for E. coli and 62.21 µg/mL for S. aureus (4e) and 78.03 µg/mL for E. coli and 107.33 µg/mL for S. aureus (4a)

49. Conclusions The present work focused on synthesizing 2-phenyl indole derivatives conjugated with thiadiazole possessing antimicrobial potential. The synthesized compounds with diverse substitution pattern were able to exhibit antimicrobial action. Further studies on new compounds of similar structure would be carried out in order to derive a relation between the structure and activity of the nucleus.

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