Dr Gopal Natesan Department o f Medicinal Chemistry Department o f Medicinal Chemistry Faculty of Pharmacy MAHSA University, Kuala Lumpur

Bacteri a Bacterial Infection Antibiotic/ Antibacterial agents TREATMENT Resistant strain Adaptation & evolution INTRODUCTION

Methicillin-Resistant Staphylococcus aureus (MRSA ) Multidrug-resistance organism (MDRO) Bacteria develop resistance towards more than one class of antibiotics Bacteria develop resistance towards more than one class of antibiotics  Treatment effectiveness  Prolonged treatment duration

HeterocycliccompoundsHeterocycliccompounds (Source: Gaba, 2013 ) BenzimidazoleBenzimidazole Selected Pharmacophore

Usage of chemotherapeutic agents for infectious diseases treatment are LIMITED: Resistance strain, Toxicities, Adverse effects NEED : Newer chemical entities with better efficacy. BENZIMIDAZOLE pharmacophore: alternate for the problems associated with existing treatment. Problems Associated with existing drugs

To design, synthesise & study the antibacterial property of novel 1-[(1H-benzo[d]imidazol-1-yl) (4-substituted phenyl) methylene]-2- (mono/di/tri-substituted methoxy benzylidene) hydrazine derivatives. Aim & Objectives

Literature Review

 Azetidine-2-one derivative of 1H-benzimidazole (Source: Ansari &Lal 2009) Benzimidazole possess potential antibacterial activity.  Novel 2-(6-fluorochroman- 2-yl)-1-alkyl/acyl/aroyl-1H- benzimidazoles. (Source: Kumar et al 2006)  2-Piperidin-4-yl-benzimidazoles (Source: He et al 2003)

Novel 2-substituted benzimidazoles (Source: Sharma, Gangal and Rauf 2008) 1,2-substitution enhance the inhibitory activity  Benzimidazole derivatives with aliphatic carboxamidine (Source: Karatas et al 2012) Activity: Aromatic amidine substituent > Aliphatic amidine or amide substituent

 Hydrazone moiety bearing benzimidazole derivatives (Source: Ozkay et al. 2010) Schiff base and inhibitory activity electron withdrawing group in the aromatic ring did not exhibit antibacterial activity chloro, bromo or methyl substituent on the hydrazone aromatic ring increases the activity

Scheme for synthesis of title compound

Tested compounds (100 µg/ml) DMSO (Negative control) Norfloxacin (Positive control) Diffusion at room temperature for 2 hours Incubation of agar plates at 37ºC±10ºC for 24 hours Results: Inhibition zone Preparation of agar with bacterial suspension (Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli ) Three wells Antibacterial activity Evaluation (Agar well diffusion method)

RESULT & DISCUSSION (Mechanims involved in Step 1 & 2) STEP 1: Philips condensation STEP 1: ( STEP 1: Nucleophilic addition mechanism (Schotten baumann reaction) Nucleophilic attack - reformation of the carbon-oxygen double bond and a chloride ion are pushed off - removal of a hydrogen ion from the nitrogen - react with chloride ion, and released as HCl

Mechanism involved in Step 3 STEP 3: STEP 3: Primary undergo nucleophilic addition with ketones moiety to give carbinolamines intermediate which then dehydrate to give substituted imines

(Mechanism involved in Step 4) STEP 4: STEP 4: Nucleophilic addition followed by dehydration forms benzylidene cpd

RESULT & DISCUSSION ( Synthetic method- Step 1)  -Value signalsProtons 8.05 s H br NH m 4-H (Phenyl)

7.65 : N-H (Step 1) RESULT & DISCUSSION ( Synthetic method- Step 2)

RESULT & DISCUSSION ( Synthetic method- Step 3)  -Value signalsProtons 3.87 s OCH s -CH- (H-2) 6.97br -NH mH-2’, 6’ H-3’,5’ mdmd H-5,6 H-7,4

RESULT & DISCUSSION ( Synthetic method- Step 4)  -Value signalsProtons ssss -OCH 3 -CH ssss -CH- (H-2) -CH=N mmdmmd H-3’,5’,6” H-2’,6’ H-2”,5” mdmd H-5,6 H-7,4

Compd code RR1R1 R2R2 R3R3 Molecular Formula Molecular Weight (g/mol) Melting Point (°C) Rf Value Percentage yield (%) IV a OCH 3 HH C 23 H 20 N 4 O IV b OCH 3 C 25 H 24 N 4 O IV c OCH 3 H C 24 H 21 N 4 O IV d CH 3 HHOCH 3 C 23 H 20 N 4 O IV e CH 3 OCH 3 C 25 H 24 N 4 O IV f CH 3 HOCH 3 C 24 H 21 N 4 O IVgCH 3 HH C 22 H 18 N IV h OCH 3 HHCH 3 C 22 H 18 N 4 O Physical data of synthesis of 1-[(1H-benzo[d]imidazol-1-yl) (4’- substituted phenyl) methylene]-2-(mono/di/tri-substituted methoxybenzylidene) hydrazine derivatives (IV a -IV h )

Antibacterial activity Antibacterial activity Note: - = no activity; + = poor activity (1-5 mm); ++ = moderate activity (6-10 mm); +++ = good activity (11-15 mm); ++++ = excellent activity (16-20mm) RESULT & DISCUSSION Serial No. Compound code Zone of Inhibition (mm) Gram positiveGram negative Staphylococcus aureus Bacillus cereus Pseudomonas aeruginosa Escherichia coli 1III a III b IV a IV b IV c IV d IV e IV f IV g IV f Norfloxacin++++

 10 Novel benzimidazole derivatives were synthesised successfully and in good yield.  All the newly synthesised compound’s structure was established on the basis of NMR and Mass Spectroscopy and the structure assigned on them are satisfactory.  All the test compounds exhibited mild to moderate antibacterial activities only.  Compounds (IV b ) and (IV e ) showed better antibacterial activity compared to other test compounds with additional poor activity against gram negative bacteria. CONCLUSION

To explore other pharmacological properties of the synthesised compound. Synthesise more number of different analogs to study the SAR of the prosposed compounds. Evaluation of antimicrobial properties in higher doses and also in different organisms. Study of the molecular mechanism of action of these synthesised compounds. FUTURE SCOPE OF STUDY

1.Ansari, K.F., and Lal, C. 2009, Synthesis, physicochemical properties and antimicrobial activity of some new benzimidazole derivatives, European Journal of Medicinal Chemistry. 44: Ansari, K.F., and Lal, C. 2009, Synthesis and evaluation of some new benzimidazole derivatives as potential antimicrobial agents, European Journal of Medicinal Chemistry. 44: Goker, H., Ozden, S., Yildiz, S., and Boykin, D.W. 2005, Synthesis and potent antibacterial activity against MRSA of some novel 1,2-disubstituted-1H-benzimidazole-N-alkylated-5-carboxamidines, European Journal of Medicinal Chemistry. 40: Karatas, H., Alp, M., Yildiz, S., and Goker, H. 2012, Synthesis and Potent In-vitro activity of novel 1H-Benzimidazoles as anti-MRSA agents, Chemical Biology &Drug Design. 80: Kumar, B. V. S., Vaidya, S.D., Kumar, R. V., Bhirud, S. B., and Mane, R. B. 2006, Synthesis and anti- bacterial activity of some novel 2-(6-flurochroman-2-yl)-1-alkyl/acyl/aroyl-1H-benzimidazole, European Journal of Medicinal Chemistry. 41: Ozkay, Y., Tunali, Y., Karaca, H., Isikdag, I. 2010, Antimicrobial activity and a SAR study of some novel benzimidazole derivatives bearing hydrazone moiety, European Journal of Medicinal Chemistry. 45: Sharma, S., Gangal, S. and Rauf, A. 2009, Convenient one-pot synthesis of novel 2-substituted benzimidazoles, tetrahydrobenzimidazoles and imidazoles and evaluation of their in vitro antibacterial and antifungal activities, European Journal of Medicinal Chemistry. 44: REFERENCES

NDD Team Mr Saravanan, Ms. Choo Shuet Yee, Ms. Lim Pei Cheng and Ms. Jecintha THANK YOU…