Introduction

The basic mechanism of antibiotic action against bacterial cells are: (i) Inhibition of cell wall synthesis (e.g. penicillin) (ii) Inhibition of protein synthesis (e.g. tetracyclines) (iii) Alternation of cell membrane (e.g. polymixins) (iv) Inhibition of nucleic acid synthesis (e.g. quinolones) and (v) Antimetabolite action (e.g. sulphonamides)¹. Inhibition of cell wall synthesis is most common mechanism of antibiotic action. Synthetic compounds may effect the integrity of cell membrane leading to cell death ²^,³.

Sysnthetic antimicrobial agents form ingredients in commercial products⁴^,⁶ like soaps, paints etc. but require multiphase clinical trial to accept as commercial drug⁷. Although penicillin, methicillin and vancomycin have been used successfully to cure these infections, the resistance strains to these antibiotics have also evolved ⁸⁻⁹. It is seen when thiourea linkage present in certain compounds, produces antimicrobial activity¹⁰. Synthesized compounds possess thiourea & guanidine moieties. Biological activity of thiouriedo compounds have been studied by Pandeya & Co- workers¹¹⁻¹². Compounds with thiourea moiety have been reported to show antithyroidal¹³, hypoglycemic¹³, anticonvulsant¹⁴, anaesthetic¹⁵ and antibacterial¹²^,¹⁶ activities. Similarly compounds incorporating a guanidine moiety are reported to have various bioactivities¹⁷⁻¹⁹. Keeping these facts in mind it was planned to synthesize novel compounds possessing thiourea & guanidine moieties for better bioactivities.

Materials and Methods

All chemicals used were of analar grade. Substituted isothiocyanates were prepared according to literature method²⁰. A mixture of guanidine carbonate (0.05m) with respective isothiocyanates (0.1m) in acetone (25 ml) & ethanol (25ml) was refluxed for 12 hrs on a water bath to get crystals of respective thioamido derivatives. (scheme-1)

NH₂–C– NH₂    + 2RNCS   →  R–NH–C–NH–C–NH–C–NH–R

II                                                                II          II           II

NH.H₂CO₃                                                 S          NH       S

R=Phenyl                             (compd.a)

R=p-chlorophenyl            (compd.b)

R= p-Tolyl                            (compd.c)

scheme-1

Synthesized compounds were characterized by PMR , FT–IR , UV SPECTRA & MP and elemental analysis . Mueller Hinton Agar  (MHA ) were from Merck , amplicillin & nystatin from Himedia (Mumbai) While other chemicals were purchased from Merk of highest purity available .

Antomicrobial Activity

Four representative strains S.aureus (ATCC 23564), E.coli (ATCC 25922), C. albicans (ATCC 2091) and A.niger were used . Apure culture of A. niger was procured from Botany  Department BHU. Stock solutions of the testing compounds were prepared in dimethylformamide. The antibacterial study was carried by disc  diffusion techniques ^21,22.  Filter paper discs of uniform size impregnated with  concentration of test compounds (100µg/disc) are placed over inoculated agar surface containing microbial  strain. After keeping  for ~ 30minutes at room temperature , they were incubated at 37°c for 24 hrs.The zone of inhibition were measured in terms of diameter in mm and the values of antibacterial and antifungal activity were compared against standard references ampicillin (10µg) and nystatin (100 µg) , (Table -1). The presented values of inhibition zones are average of three separate experiments.

Minimum inhibitory concentrations (MICs) were determined by agar double ditution method. Graded amount of synthesized compounds were incorporated Into measured amount agar media. The media were subsequently inoculated and incorporated . Stock solutions were serially double diluted to get 200,100,50,25 12.5,6.25,3.12 etc. µg/ml concentration of test compounds  and used for study , 1ml of solution was added into each sterilized petri-dish containing graded amount of MH agar media (previously cooled at 60°c ). Under aseptic conditions dilute bacterial suspension was inoculated on agar surface using sterilized swabs . After inoculation , the dishes were incubated  at 37°C for 24 hrs. The MICs ,the lowest  concentration of drug that inhibited the growth of bacteria was noted (table -2).

Table 1: Antimicrobial activity data of compounds showing zones of  Inhibition (mm).

NZ: No Zone observed

Table 2: Antibacterial activity data of synthesized compounds showing MICs  (µg/ml).

Results and Discussion

In disc diffusion method bis-(N-p-chlorophenyl thioamido) derivative (100µg) showed  strong  antibacterial  activity  against  S.  aureus  & E.  coli  producing  zones  of  inhibition  24  mm  & 16 mm  respectively  and  were  comparable  to ampicillin  (10 µg)  which  produced  inhibition  zones 25 mm  and 12 mm  respectively . In  the  same  experiment  bis –(N–p – tolyl)  derivative  produced inhibition  zone  14 mm  against  E.  coli.  MIC  of  p –  chlorophenyl derivative  was  found  25 µg  &  50µg  against  S.aureus  &  E. coli  for  complete inhibition  of  bacterial growth. In antifungal activity  bis–(N- phenyl) derivative  (100µg) & bis-(N-p-tolyl) derivative  (100µ g) produced inhibition zones 20 mm and 22 mm against A. niger and C. albicans respectively and were comparable to Nystatin (100 units) which  produced  zones  of  inhibition 17 mm & 20 mm in the same  experiment.

The  results   indicate  that  compound  b is potent  antibacterial  agent  against  S. aureus & E. coli  while compound  a  & compound  c  showed  good  antifungal  activity  against  A. niger  and  C. albicans.  Therefore  compounds  deserve  further  studies  for  utilizing  as antiseptic  agent.

Acknowledgement 

Authors are thankful to dept. of Pharmaceutics, Saroj Institute of Technology & Management, Lucknow & dept. of Chemistry, Al- Margeb University , Al-Khums for providing experimental facilities. One of us S.N. Pandeya has been Emeritus Fellow of AICTE, New Delhi (India).

References

1. Greenwood D & Whitley R J, (2003), in Antibiotic & Chemotheraphy ( Finch R G, Greenwood D , Norrby   S R & Whitley R , eds), 11-19 ,Churchil Livingstone.
2. Aguilera O, ostolaza H , Quiros L M & Fierro J F, (1999), FEBS  lett, 462, 273-277.
3. Dwivedi R, singh V, Fahmi N & singh R V , (2003), Int J chem. Sci , 1(3), 233.
4. Lakshar J H , Helary G and Sauvet G , Makromol  symp, (1991), 47,383.
5. Johnson S A , Goddard P A , Iliffe C, Timmins B , Rickard A H , Robson G & Handley P S, (2002), J Appl Microbiol , 93, 336-344.
6. Muller M & Schimz K L (1999) , Cell Mol Life Sci , 56, 280-285.
7. Smith C, Burley C, Ireson M , Johnson T , Jordan D , Knight S, Mason T , Massey D , Moss J & Williams K , (1998) , Antimicrob chemother , 41,467-480.
8. Michel M & Gutmann L , (1997) , Lancet ,349, 1901-1906.
9. Chen Y L , Fang K C Sheu J Y , Hsu S L & Tzeng C C ,(2001) , J Med Chem, 44, 2374-2377 .
10. Khan M H & Giri S , Indian J Pharm , Sci , (1992) , 54, 128.
11. Pandeya S N , Misra V, singh P N & Rupainwar D C, (1998), Pharmacol Res , 37 (1), 17-22.
12. Gupta M K , Sachan A K , Pandeya S N & Gangwar V S , (2006), Asian J Chem , 18 (4) 2559-2962.
13. Pandeya S N , Ram P & Shankar V , (1981) , J Sci Indust Res , 40, 458.
14. Gupta M K Pandeya S N , Gangwar R A & Kumar A , (2004), Orien J Chem , 20 (3) ,  605-608.
15. Ranise A et al , (2003) , Farmaco , 58 (9), 765-780.
16. Patel N B , Lilakar J D & Chaudhari R C , (2004) , 20(3) , 543-546.
17. Aguilar M , Paula D P , Isabel M , Ortiz C & Fernandez J M G , (2008) , JOC , 73 (5), 1995-1998.
18. Singh G R & Mehra S C , (2006) Asian J Chem , 18 (4) , 3132-3134.
19. Sun J Y , Zhu  M Z , Wang S W , Malio S , Xie Y H & Wang J B , (2007), Phytomedicine , 14, 353.
20. A I Vogel , Text Book of Practical Organic Chemistry including Qualitative Organic Analysis , ELBS –Longman Green & Co.Ltd. p. 615 (1954).
21. Cappuccino J G & Sherman N , Microbiology : A Laboratory Manual 5^th Edition , Benzamin / Gumming Science Publishing , California (1999) , 254.
22. Dey P M & Harbone J B , Methods in Plant Biochemistry , Academic Press , London (1991) , 1 , 47-58.
23. Barry A L , Corian (Ed), Antibiotics in laboratory Medicines , Williams & Wilkins , Baltimore (2001).