Sulfanilamide Derivatives Effectiveness on Growth

Inhibition in E. coli and S. aureus Tiffani Ream, Department of Biological Sciences, York College of PA

Acetanilide

Methods

Ammonia N-butylamine Sec-butylamine Piperidine Morpholine

Derivatives

Antibiotic Disks (0-1000 µM)

E. coliS. aureus

Measure zone of inhibition

Determine EC50

Compound E. coli S. aureus

Sulfanilamide 10.78a No fit

Ammonia 9.47 145.8

N-butylamine 257.50 47.36

Sec-butylamine 178.70 5.16x106

Morpholine 19.99 40.82

Piperidine 5.91x106 1.23x108

Results

Sulfa drugs have been used in medicine since 1932 when Farbenindustrie patented Protonsil (Williamson, Mindard, & Masters 2007). The original sulfanilamide was synthesized in 1908, along with thousands of derivatives. In 1940, Domagk won the Nobel Prize for his research in sulfanilamide derivative development. However, due to the technology of the time, most of the papers and research he and other performed is not accessible or readily available.

Sulfa drugs are competitive inhibitors of p-aminobenzoic acid (PABA) (Woods 1941). Bacteria require PABA in the synthesis of folic acid, which is crucial for cell replication. Normally, PABA binds to dihydropteroate synthetase; however, when sulfanilamide is present, competition occurs between PABA and the drug (Yoneeyma & Katsumata 2006). The resulting compound of sulfanilamide/dihydropteroate synthetase complex is not functional in the folic acid synthesis pathway, thus causing cell cycle arrest.

A standard practice in pharmaceutics is to take an effective drug and modify it to uncover a more efficient product or one with more suitable qualities such as solubility or polarity. To do this, chain lengths are modified and functional groups are added.

The objective of this study is to examine derivatives of sulfanilamide for a more effective compound. Both chain lengths and functional groups were varied in this study in the attempt to find a more effective derivative of sulfanilamide.

Introduction

Questions Asked

1. Is the standard sulfanilamide the most effective sulfa drug?

2. Are trends evident within the different sulfanilamide derivatives?

3. Do differences between gram positive and gram negative bacteria exist?

Figure 1. Comparison of area of inhibition between E. coli (left) and S. aureus(right) in ammonia, n-butylamine, sec-butylamine derivatives of sulfanilamide and the standard. Means (n=6) and standard of error were calculated for each concentration. An EC50 for each derivative and the standard sulfanilamide was determined from these graphs.

http://www.chm.bris.ac.uk/motm/sulfanilamide/sulfa1.gif

Space filling model of sulfanilamide.

Figure 2. Comparison of area of inhibition between E. coli (left) and S. aureus(right) in morpholine and piperidine derivatives of sulfanilamide and the standard. Means (n=6) and standard of error were calculated for each concentration. An EC50 for each derivative and the standard sulfanilamide was determined from these graphs.

Area of Inhibition Measured

http://www.ljmilitaria.com/1c77fe2c0.jpg

Results

Literature Cited

•Williamson, K.L., R.D. Minard, and K.M. Masters. 2007. Macroscale and Microscale Organic Experiments. Houghton Mifflin: Boston, MA.

•Woods, W.B. 1941. Studies on the Antibacterial Action of Sulfonamide Drugs. JEM. 369-381.

•Yoneyama, H. and R. Katsumata. 2006. Antibiotic Resistance in Bacteria and Its Future for Novel Antibiotic Development. Bioscience, Biotechnology, Biochemistry. 70.5, 1060-1075.

Sulfanilamide is the most effective compound in E. coli (EC50=10.78 µM), while the morpholine derivative is most effective in S. aureus (EC50=40.82 µM).

Morpholine is 3x105 times more effective than Piperidine in both gram positive and gram negative.

Sec-butylamine (EC50=178.70) was more effective than N-butylamine (EC50=257.50) in E. coli only. N-butylamine (EC50=47.36) was 1x105 times more effective in S. aureus.

Table 1. EC50 Table of Sulfanilamide and Sulfanilamide derivatives.

a= µM

Acknowledgments

I would like to thank Dr. Kaltreider and Dr. Halligan for all of their help and guidance in this project. I would also like to thank Brian Kanaskie and Bethany Remeniuk for their help in the lab.

Conclusion Sulfanilamide is the most effective

compound in E. coli, while Morpholine is more effective in S. aureus.

Polar substitutents are more effective than non-polar functional groups.

Branched carbon chains are more effective in E. coli, while a straight chain has a lower EC50 in S. aureus. More of a contrast in effectiveness of chain length is evident in S. aureus.

Area of Inhibition in Sulfanilamide Derivatives in E. coli

-9.5 -9.0 -8.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.50

2000

4000

6000

8000

10000

12000

14000

16000

18000

MorpholinePiperidineStandard Sulfanilamide

Log Concentration (ug/mL)A

rea o

f In

hib

itio

n

(pix

els

)

Area of Inhibition in Sulfanilamide Derivatives in S. aureus

-9.5 -9.0 -8.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.50

4000

8000

12000

16000

20000

24000

28000

AmmoniaN-butylamineSec-butylamine

Standard Sulfanilamide

Log Concentration (ug/mL)

Are

a o

f In

hib

itio

n (

pix

els

)

Area of Inhibition in Sulfanilamide Derivatives in E. Coli

-9.5 -9.0 -8.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.50

4000

8000

12000

16000

20000

24000

28000AmmoniaN-butylamineSec-butylamineStandard Sulfanilamide

Log Concentration (ug/mL)

Are

a o

f In

hib

itio

n (

pix

els

)

Area of Inhibition in Sulfanilamide Derivatives in S. aureus

-9.5 -9.0 -8.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.50

2000

4000

6000

8000

10000

12000

14000

16000

18000MorpholinePiperidineStandard Sulfanilamide

Log Concentration (ug/mL)

Are

a o

f In

hib

itio

n (

pix

els)

The parent compound, sulfanilamide, is the most effective in E. coli, while the morpholine derivative is most effective in S. aureus.

Due to the disparity between gram positive and gram negative, further research is necessary to determine the cause. Structural differences in the dihydropteroate synthetase or the structural differences of the cell wall may be the cause of these different results.

Summary