Chapter 2 Studies on 1,3,4-oxadiazole derivatives

Transcription

1 Introduction and scope of present work Literature study reveals the importance of nitrogen containing heterocyclic compounds as essential antibacterial and antifungal drugs. 1 Among several different five membered heterocyclic rings, 1,3 4-oxadiazoles are the class of heterocyclic compounds with a wide range of pharmaceutical and biological action. 2-4 The different biological activity possessed by 1,3 4-oxadiazoles includes antimicrobial, 5,6 antifungal, 7,8 antiproliferative, 9 anticancer, 10,11 tyrosinase inhibitor 12 and Tiodazosin possessing oxadiazole nucleus is widely used as antihypertensive. 13 ther biological properties associated with 1,3,4-oxadiazole skeletal includes anti-inflammatory 14,15 as well as analgesic activity. 16,17 ome derivatives containing 1,3,4-oxadiazole as a core, are utilized as muscle relaxants, 18 tranquillizers 19 and cathepsin K inhibitors. 20 The molecule is also useful as angiogenesis inhibitor and IV integrase inhibitor. 21 In context to great medicinal significance; a number of routes have been developed for synthesizing 1,3,4- oxadiazoles everal synthetic methods under harsh conditions using different reagents viz. Cu(II) 27 and phosphorous oxy-chloride 28 have been established. Furthermore it is observed that the fluorine atom, if substituted with oxadiazole nucleus results in formation of potent bioactive molecules. 29,30 A series of 2-mercapto-5-phenyl-1,3,4-oxadiazole derivatives have been condensed with 4'-(bromomethyl)biphenyl-2-carbonitrilederivatives; resulting in the formation of 4'-(5-Aryl-[1,3,4]oxadiazol-2-ylsulfanylmethyl)-biphenyl-2- carbonitrilederivatives III a-h. The antimicrobial properties of the synthesized entities (III a-h ) measured as their MIC (Minimum Inhibitory Concentration) values were evaluated by using the broth dilution method against gram-positive bacteria (. aureus and E. faecalis), gram-negative bacteria (E. coli and P. aeruginosa) and fungi (C. albicans and A. niger). Ph.D. Thesis (2015) Abhishek R. Joshi, MC-GU, Patan

2 2 ylsulfanylmethyl)-biphenyl-2-carbonitrilederivatives III a-h C R 4'-(5-Aryl-[1,3,4]oxadiazol-2-ylsulfanylmethyl)-biphenyl-2-carbonitrile derivatives III a-h Table-1: List of 4'-(5-Aryl-[1,3,4]oxadiazol-2-ylsulfanylmethyl)- biphenyl-2-carbonitrile derivatives III a-h General structure for 4'-(5-Aryl-[1,3,4]oxadiazol-2- Compounds ubstituent omenclature III a m- 2 4'-((5-(3-nitrophenyl)-1,3,4-oxadiazol-2- ylthio)methyl)biphenyl-2-carbonitrile III b p- 2 4'-((5-(4-nitrophenyl)-1,3,4-oxadiazol-2- ylthio)methyl)biphenyl-2-carbonitrile III c p-cl 4'-((5-(4-chlorophenyl)-1,3,4-oxadiazol-2- ylthio)methyl)biphenyl-2-carbonitrile III d o-cl 4'-((5-(2-chlorophenyl)-1,3,4-oxadiazol-2- ylthio)methyl)biphenyl-2-carbonitrile III e m-cl 4'-((5-(3-chlorophenyl)-1,3,4-oxadiazol-2- ylthio)methyl)biphenyl-2-carbonitrile III f m-c 3 4'-((5-m-tolyl-1,3,4-oxadiazol-2-ylthio)methyl)biphenyl- 2-carbonitrile III g p-c 3 4'-((5-p-tolyl-1,3,4-oxadiazol-2-ylthio)methyl)biphenyl- 2-carbonitrile III h 4'-((5-phenyl-1,3,4-oxadiazol-2-ylthio)methyl)biphenyl- 2-carbonitrile Ph.D. Thesis (2015) Abhishek R. Joshi, MC-GU, Patan

3 List of derivatives synthesized III a-h Derivatives III a-d C 2 4'-((5-(3-nitrophenyl)-1,3,4-oxadiazol-2-ylthio)methyl)biphenyl-2-carbonitrile IIIa C 2 4'-((5-(4-nitrophenyl)-1,3,4-oxadiazol-2-ylthio)methyl)biphenyl-2-carbonitrile III b C Cl 4'-((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-ylthio)methyl)biphenyl-2-carbonitrile III c Cl C 4'-((5-(2-chlorophenyl)-1,3,4-oxadiazol-2-ylthio)methyl)biphenyl-2-carbonitrile III d Ph.D. Thesis (2015) Abhishek R. Joshi, MC-GU, Patan

4 Derivatives III e-h C Cl 4'-((5-(3-chlorophenyl)-1,3,4-oxadiazol-2-ylthio)methyl)biphenyl-2-carbonitrile III e C C 3 4'-((5-m-tolyl-1,3,4-oxadiazol-2-ylthio)methyl)biphenyl-2-carbonitrile III f C 4'-((5-p-tolyl-1,3,4-oxadiazol-2-ylthio)methyl)biphenyl-2-carbonitrile III g C 3 C 4'-((5-phenyl-1,3,4-oxadiazol-2-ylthio)methyl)biphenyl-2-carbonitrile III h Ph.D. Thesis (2015) Abhishek R. Joshi, MC-GU, Patan

5 5 2.2 Designing the derivatives Lipinski s rule of five Table-2: creening for drug-likeness of derivatives III a-h (tage 1) Entry Descriptors for Lipinski s rule of five o. of M.W. Log P o. of -bond donor o. of -bond acceptor violation III a III b III c III d III e III f III g III h C R 4'-(5-Aryl-[1,3,4]oxadiazol-2-ylsulfanylmethyl)-biphenyl-2-carbonitrile derivatives III a-h The atoms indicated with blue color stands for -bond acceptor sites and the red color for donor site in the above figure. In general, there is no donor site viz. the oxadiazole ring. -bond acceptor sites include the two nitrogen atoms and an oxygen atom of oxadiazole ring along with the sulfur. When -Cl and - 2 are substituted 6 and 6 -bond acceptor sites are found to be present respectively. All of the derivatives were found to violate one of the (descriptors) parameters of Lipinski s rule of five. enceforth the titled derivatives are feasible for further ynthesis and biological studies. Ph.D. Thesis (2015) Abhishek R. Joshi, MC-GU, Patan

6 6 2.3 Experimental - schematic representation and procedure for the synthesis of compounds III a-h ynthesis of 5-aryl-1,3,4-oxadiazole-2-thiol 2 a-h (step 1) Ethanol (40 ml) and potassium hydroxide (0.01 mol, gm/mol, 0.56 gm in 2 ml 2 ) were taken in a dry round bottom flask. Various aryl substituted benzoic acid hydrazide (0.01 mol) were added to it and stirred well to get a clear solution. Carbon disulfide (0.02 mol, gm/mol, 1.2 ml) was added to the clear solution obtained above and refluxed for h. The ethanol was distilled off and then cooled to room temperature. The content was poured into water and acidified with diluted Cl till the precipitates were separated. The separated solid was washed with cold water and dried to get the desired product. The completion of the reaction was monitored on TLC using ethyl acetate: benzene (6:4) as mobile phase. All the derivatives (2 a-h ) were obtained using the same procedure. (ee appendix-1 for physical constant data) tep 1 C 2 C 2 / K R Ethanol R 1 a-h 2 a-h ynthesis of 4'-(5-Aryl-[1,3,4]oxadiazol-2-ylsulfanylmethyl)-biphenyl-2- carbonitrile III a-h (step 2) In a round bottom flask (moisture free) containing 4'-(bromomethyl)biphenyl-2- carbonitrileii (0.01 mol, 272 gm/mol, 2.72 gm) in acetone, various aryl substituted 1,3,4-oxadiazole-2-thiol (0.01 mol) were added. K 2 C 3 (0.02 mol, 138 gm/mol, 2.76 gm) was added to neutralize the liberated hydrochloric acid during the reaction. This mixture was allowed to stir for 4 h at room temperature. The resulting material was poured onto crushed ice and stirred well for 30 min. The solid separated out was filtered and washed with cold water. The product obtained was dried and recrystallized from alcohol. All the targeted derivatives were obtained by the above stated procedure. The completion of the reaction was confirmed by using TLC with mobile phase n- hexane: ethyl acetate (8:2) and (7:3) (as per the resolution). Ph.D. Thesis (2015) Abhishek R. Joshi, MC-GU, Patan

7 7 tep 2; 2 a-h + II Br R 2 a-h + C K 2 C 3 / acetone II C III a-h R Ph.D. Thesis (2015) Abhishek R. Joshi, MC-GU, Patan

8 8 2.4 Physical constants for the final derivatives III a-h Table-3: Physical constants data for derivatives III a-h Derivatives ubstituent Molecular formula Molecular weight gm/mol m.p. C % Yield Elemental Analysis % Carbon % ydrogen %itrogen Calcd. Found Calcd. Found Calcd. Found III a m- 2 C C 68% III b p- 2 C C 71% III c p-cl C Cl C 57% III d o-cl C Cl C 62% III e m-cl C Cl C 58% III f m-c 3 C C 71% III g p-c 3 C C 65% III h C C 59% Ph.D. Thesis (2015) Abhishek R. Joshi, MC-GU, Patan

9 9 2.5 Characterization pectral data and interpretation pectral status for III a-h derivatives IR spectra for compound III c Cl C '-((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-ylthio)methyl)biphenyl-2-carbonitrile IIIc Table-4: IR spectral data for compound III c Functional group Frequency (Cm -1 ) -C--C- ring (str.) 1012,1270 -C- aromatic ring(str.) C 2 methylene group (str.) C=C- aromatic ring (str.) C (str.) 2221 Ph.D. Thesis (2015) Abhishek R. Joshi, MC-GU, Patan

10 MR spectra for compound III c Cl C C '-((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-ylthio)methyl)biphenyl-2-carbonitrile IIIc Table-5: 1 MR spectral data for compound III c Assignment o. of protons Multiplicity value (ppm) -C 2 2 singlet 4.68 Ar- 12 multiplet Ph.D. Thesis (2015) Abhishek R. Joshi, MC-GU, Patan

11 Mass spectra for compound III c Table-6: Mass spectral data for compound III c Peak assignment m/z value M+1 peak Ph.D. Thesis (2015) Abhishek R. Joshi, MC-GU, Patan

12 pectral interpretation (IR, 1 MR, Mass spectra) IR spectral data obtained here lead to the confirmation about the formation of targeted compounds. The IR spectral data obtained for the final derivatives III a-h were useful to a great extent in confirming their structure. Let us try to observe the spectral details of compound III c and correlate the frequencies with the functional group present in the structure. Absorption peaks at 1012 and 1270cm -1 helped to confirm the presence of - C--C- in the oxadiazole nucleus. The absorption band at 1489 cm -1 proved the presence of -C=C- in the aromatic ring. The presence of the C functional group (carbonitrile) was also confirmed by a sharp peak at 2221cm -1. A sharp absorption band at 2979cm -1 helped to assign the presence of C 2 (methylene group) in the compound III c. The stretching frequency for aromatic -C- was noticed at 3058cm -1 in the compound III c. The 1 uclear Magnetic Resonance study was conducted for each of the derivatives and the δ values were recorded. The δ values recorded corresponded to the methylene group and to the protons of the aromatic ring. For compound III c, δ value at 4.68 as a singlet clearly proved the presence of two hydrogen atoms of the methylene group. A multiplet obtained at 7.57 to 7.99 ppm confirmed the presence of aromatic protons. In mass spectra, the molecular ion peak observed at m/z in III c ( ) confirmed the formation of desired structure. Ph.D. Thesis (2015) Abhishek R. Joshi, MC-GU, Patan

13 Therapeutic studies antimicrobial studies Antimicrobial assay The newly synthesized entities (III a-h ) were screened for their antimicrobial assay against a broad panel of gram-positive bacteria. aureus (ATCC o ), E. faecalis (ATCC o ), gram-negative bacteria E. coli (ATCC o ), P. aeruginosa (ATCC o ) and fungi C. albicans (ATCC o ), A. niger (ATCC o. 1015). The samples for antifungal as well as antibacterial evaluation were tested by standard protocols like micro dilution/broth titer method. The stock solution (1000 μg/ml) for each compound was prepared and screening for antimicrobial activity was carried out by diluting the stock solution and preparing the sets consecutively from 1000, 500, 250, 125, 62.5, 31.25, up to 7.8μg/ml. The tubes along with the control were then kept for incubation at 37 C for 24 h. uspensions were further inoculated on an appropriate media and growth was noted after 48 h. The obtained results (MIC) in μg/ml by observing the highest dilutions (low turbidity) were recorded and compared with the MIC value of standard drugs ciprofloxacin and fluconazole Broad panel of gram-positive bacteria (. aureus and E. faecalis), gram-negative bacteria (E. coli and P. aeruginosa) and fungal strains (C. albicans and A. niger) were utilized for testing the antimicrobial properties of the targeted compounds (III a-h ). The results of theantibacterial and antifungal activities in MIC (μg/ml) have been reported in Table- 15 respectively. It was observed that most of the compounds exhibited excellent activity as compared to the standards used and a very few of them exhibited moderate or poor activity. The MIC value of ciprofloxacin was recorded to be 62.5μg/ml against grampositive bacterial strain. aureus, whereas the same standard responded at 125 μg/ml; when used against E. faecalis, E. coli and P. aeruginosa. Fluconazole, when used as a standard drug against fungal strains C. albicans and A. niger, MIC value was observed at 125 μg/ml and 62.5 μg/ml respectively. In general, it was observed that most of the compounds have resulted to be potent anti-bacterial, and a very few of them were good anti-fungal. Ph.D. Thesis (2015) Abhishek R. Joshi, MC-GU, Patan

14 Table-7: Antimicrobial screening of the compounds III a-h Compounds Minimum Inhibitory Concentration (MIC) in µg/ml Gram-positive bacteria Gram-negative bacteria Fungi. aureus ATCC E. faecalis ATCC E. coli ATCC P. aeruginosa ATCC C. albicans ATCC A. niger ATCC 1015 III a (R:m- 2 ) III b (R:p- 2 ) III c (R:p-Cl) III d (R:o-Cl) III e (R:m-Cl) III f (R:m-C 3 ) III g (R:p-C 3 ) III h (R:) Fluconazole Ciprofloxacin MIC: Minimum Inhibitory Concentration td.: tandard drug fluconazole for antifungal and ciprofloxacin for antibacterial tests Ph.D. Thesis (2015) Abhishek R. Joshi, MC-GU, Patan

15 Results and discussion Antibacterial activity Antibacterial potential of the synthesized compounds III a-h were tested against grampositive bacteria. auerus (ATCC no ) and E. faecalis (ATCC no ) and gram-negative bacteria E. coli (ATCC no ) and P. aeruginosa (ATCC no ). Compounds III b (p- 2 ), III c (p-cl), III d (o-cl) and III e (m-cl) were found to exhibit excellent or equivalent activity as compared to the standard ciprofloxacin. ther compounds III a (m- 2 ), III h (), III f (m-c 3 ) and III g (p-c 3 ) were found to possess lower activity as compared to the standard drug and even poor than the former mentioned derivatives. When the same set of derivatives IIIa-h were tested against another gram-positive bacterial strain E. faecalis, all the derivatives other than compound III h () and III g (p-c 3 ), resulted in equivalent or excellent activity. Compounds III a- h were exposed to gram-negative bacteria E. coli and III e was found to show excellent activity with MIC value μg/ml. Compounds III c (p-cl) and III d (o-cl) were exhibiting μg/ml MIC values, indicating better antibacterial potential than the standard ciprofloxacin. Derivatives III a (m- 2 ), III b (p- 2 ), III h () and IIIf (m- C 3 ) were also found to show better activity than the standard drug. When the synthesized -derivatives III a-h were tested against gram-negative bacteria P. aeurginosa it was found that compound III d (o-cl) and III e (m-cl) exhibited excellent activity (31.25 μg/ml). Along with other derivatives III a (m- 2 ), III c (p-cl), III h () and III g (p- C 3 ) exhibiting equivalent activity (125 μg/ml) to that of the standard, derivative III b (p- 2 ) was found to show higher activity (62.5 μg/ml) than the standard drug. The only derivative possessing poor activity as compared to standard drug was compound III f (m-c 3 ) against the gram-negative bacterial strain P. aeruginosa Antifungal activity The antifungal tests were performed against two different fungal strains A. niger and C. albicans, where fluconazole was used as a standard drug. The antifungal property of the synthesized derivatives III a-h was found to be more effective on A. niger as compared to C. albicans. n testing the compounds against C. albicans, synthesized derivative III d (o- Cl) was found to show excellent activity as compared to the standard drug fluconazole. Ph.D. Thesis (2015) Abhishek R. Joshi, MC-GU, Patan

16 16 Few other derivatives III a (m- 2 ), III c (p-cl) and III e (m-cl) were found to possess activity equivalent to that of the standard drug, while others were found to be poor in activity. ame derivatives when tested for their antifungal property against a different fungal strain A. niger resulted in comparatively much better results than the former (C. albicans). Compounds III c and III d possessing -Cl as a substituent exhibited excellent activity (31.25 and 62.5 μg/ml), even better than the standard drug fluconazole. Also the derivatives possessing - 2 were found to possess equivalent activity (62.5 μg/ml) to that of the std. fluconazole. Derivatives possessing C 3 as a functional group were found to show poor activity (125 and 250 μg/ml) as compared to std. as well as other derivatives AR study - tructure Activity Relationship A larger difference was observed in the biological activity between the compounds bearing electron-withdrawing and electron-donating functional group as substituent (- R). From the MIC results in table 7, it was very clear that the derivatives bearing electron-withdrawing substituent (-Cl, 2 ) were exhibiting higher antimicrobial potential as compared to electron-donating substituent (-C 3 ). n comparing the MIC results between the derivatives possessing electron-withdrawing functional groups (III a-c and III d,e ) and the derivative III h () for most of the bacterial and fungal strains, it was clearly observed that compound III h with atom as substituent exhibited poor activity as compared to the derivatives with electron-withdrawing groups. Even the Minimum Inhibitory Concentration results of compounds III f and III g showed poor activity than derivatives having -Cl and - 2 substituent. AR study helps to understand that the presence of electron-withdrawing functional groups can help in enhancing the antibacterial and antifungal properties. Ph.D. Thesis (2015) Abhishek R. Joshi, MC-GU, Patan

17 Conclusion The compounds possessing electron-withdrawing substituent were found to exhibit excellent antibacterial and antifungal properties. everal derivatives were also showing even better results than the standard drug ciprofloxacin viz. compound III d (o-cl) showed MIC value even higher than standard drug against all bacterial and fungal strains ( μg/ml) and compound III e (m-cl) was found to exhibit excellent property against gram-negative bacteria E. coli and P. aeruginosa. Compounds with excellent biological properties can be further optimized by introducing electron with-drawing functional groups in order to develop new potent leads. Ph.D. Thesis (2015) Abhishek R. Joshi, MC-GU, Patan

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