Antibacterial Agents which Act Against Cell Metabolism (Antimetabolites) 10:03 AM PHC 428 Dr. Alafeefy AM

Mechanisms of antibacterial agents There are Five main mechanisms by which antibacterial agents act. Inhibition of bacterial cell wall synthesis: e.g. Penicillins, Cephalosporins and Vancomycin Inhibition of cell metabolism: e. g. Sulfonamides Interactions with the plasma membrane: e.g. Polymyxins Disruption of protein synthesis: e.g. Rifamycins, Aminoglycosides, Tetracyclines, and Chloramphenicol Inhibition of nucleic acid transcription and replication: e.g. Nalidixic acid and Proflavin 10:03 AM PHC 428 Dr. Alafeefy AM

Sulfonamides (Antimetabolites) 4-Amino-N-substituted-benzene sulfonamide 10:03 AM PHC 428 Dr. Alafeefy AM

The history of sulfonamides The sulfonamide story began in 1935 when it was discovered that a red dye called prontosil had antibacterial properties in vivo (i.e. when given to laboratory animals). Strangely enough, no antibacterial effect was observed in vitro. In other words, prontosil could not kill bacteria grown in the test tube. This remained a mystery until it was discovered that prontosil was not in fact the antibacterial agent. Instead, it was found that the dye was metabolized by bacteria present in the small intestine of the test animal, and broken down to give a product called sulfanilamide 10:03 AM PHC 428 Dr. Alafeefy AM

Mechanism of action Act as competitive enzyme inhibitors and block the biosynthesis of the vitamin folic acid in bacterial cells Sulfonamides do not actively kill bacterial cells Dihydrofolate Reductase Tetrahydrofolate is an enzyme cofactor that provides one carbon units for the synthesis of the pyrimidine nucleic acid bases required for DNA synthesis . If pyrimidine and DNA synthesis is blocked, then the cell can no longer grow and divide. They prevent the cells dividing and spreading Antibacterial agents which inhibit cell growth are classed as bacteriostatic, whereas agents which can actively kill bacterial cells (e.g. penicillin) are classed as bactericidal Tetrahydrofolic acid 10:03 AM PHC 428 Dr. Alafeefy AM

Mechanism of action (cont.) The sulfonamide molecule is similar enough in structure to PABA that the enzyme is fooled into accepting it into its active site. Once it is bound, the sulfonamide prevents PABA from binding. As a result, folic acid is no longer synthesized. Since folic acid is essential to cell growth, the cell will stop dividing. 10:03 AM PHC 428 Dr. Alafeefy AM

Mechanism of action (cont.) Why the enzyme does not join the sulfonamide to the other two components of folic acid to give a folic acid analogue containing the sulfonamide skeleton? Answer: This can in fact occur, but it isn’t accepted by the next enzyme (dihydrofolate reductase) in the biosynthetic pathway 10:03 AM PHC 428 Dr. Alafeefy AM

Structure-Activity Relationships (SAR) The synthesis of a large number of sulfonamide analogues led to the following conclusions. The p-amino group (N4)is essential for activity and must be unsubstituted . The only exception is when N-substituted with acyl (i.e. amides). The amides themselves are inactive but can be metabolized in the body to regenerate the active compound. Thus amides can be used as sulfonamide prodrugs. 10:03 AM PHC 428 Dr. Alafeefy AM

Structure-Activity Relationships (SAR) The aromatic ring and the sulfonamide functional group are both required. The – SO2NH2 group must be directly linked to the benzene ring The aromatic ring must be para-substituted only. The sulfonamide nitrogen must be primary or secondary. R" is the only possible site that can be varied in sulfonamides. Substitution with heterocyclic rings as at-N1-have variable effects on the antibacterial activity 10:03 AM PHC 428 Dr. Alafeefy AM

Acidity of Sulfonamides The strongly electron withdrawing character of the aromatic SO2 group makes the neighboring nitrogen atom to which it is directly attached partially electropositive, in turn, increases the acidity of the hydrogen atoms attached to the nitrogen and facilitates its expulsion as a proton so that this functional group is slightly acidic (pKa 10.4). Replacement of one of the NH2 hydrogens by an electron withdrawing heteroaromatic ring was not only consistent with antimicrobial activity but also greatly acidified the remaining hydrogen (pKa 5-6) and dramatically increased potency. 10:03 AM PHC 428 Dr. Alafeefy AM

Acidity of Sulfonamides (Cont.) With suitable groups in place, the pKa came down to the same range as that of PABA itself. The pKa of the carboxyl group of PABA is approximately 6.5 Acidity Increases Electronegativity of heteroatoms: S (2.5), N (3), O (3.5) 10:03 AM PHC 428 Dr. Alafeefy AM

Metabolism of Sulfonamides The resulting amides have reduced solubility which can lead to toxic effects. The metabolite formed from sulfathiazole (an early sulfonamide) is poorly soluble and can prove fatal if it blocks the kidney tubules. 10:03 AM PHC 428 Dr. Alafeefy AM

The solubility problem could be overcome by replacing the Thiazole ring in sulfathiazole with a Pyrimidine ring to give Sulfadiazine. The reason for the improved solubility lies in the acidity of the sulfonamide NH proton In sulfathiazole, this proton is not very acidic (high pKa). Therefore, sulfathiazole and its metabolite are mostly un-ionized at blood pH Replacing the Thiazole ring with a more electron withdrawing Pyrimidine ring increases the acidity of the NH proton by stabilizing the anion which results. Therefore, sulfadiazine and its metabolite are significantly ionized at blood pH. As a consequence, they are more soluble and less toxic. Sulfadiazine was also found to be more active than sulfathiazole and soon replaced it in therapy. 10:03 AM PHC 428 Dr. Alafeefy AM

Crystalluria The poor water solubility of the earliest sulfonamides led to occasional crystallization in the urine (crystalluria) and resulted in kidney damage because the molecules were un-ionized at urine pH values. Water- insoluble acetylsulfathiazole ( metabolic product) causes crystalluria and renal toxicity It is still recommended to drink increased quantities of water to avoid crystalluria when taking certain sulfonamides but this form of toxicity is now comparatively uncommon with the more important agents used today because they are at least partly ionized and hence reasonably water soluble at urinary pH values. Increasing the pH of the urine by oral ingestion of sodium bicarbonate used occasionally and still used. 10:03 AM PHC 428 Dr. Alafeefy AM

Classes of Sulfonamides Systemic Sulfonamides Short-acting Sulfonamides Intermediate- acting sulfonamides Long-acting sulfonamides Intestinal sulfonamides Ophthalmic sulfonamides Sulfonamides for burn therapy 10:03 AM PHC 428 Dr. Alafeefy AM

I. Systemic Sulfonamides According to their duration of action they are divided to: Short-acting Sulfonamides Intermediate-acting sulfonamides Long-acting sulfonamides 10:03 AM PHC 428 Dr. Alafeefy AM

A) Short-acting Sulfonamides They are rapidly absorbed and rapidly excreted. Their half lives from 4-7 hours and they are administered every 4 to 8 hours. 10:03 AM PHC 428 Dr. Alafeefy AM

B) Intermediate-acting sulfonamides They are absorbed and excreted more slowly than short-acting. Their half lives range from 10 to 12 hours so they are given twice daily. 10:03 AM PHC 428 Dr. Alafeefy AM

C) Long-acting sulfonamides They are rapidly absorbed but slowly excreted their half lives are 35 to 40 hours. 10:03 AM PHC 428 Dr. Alafeefy AM

II. Intestinal sulfonamides Water soluble latent forms which are poorly absorbed from the GIT (5%) and thus reach a high concentration in the colon lumen, By means of either bacterial or enzymatic hydrolysis releases the parent sulfonamide. Examples: sulfasalazine, phthalylsulfathiazole and succinylsulfathiazole (Prodrugs). 10:03 AM PHC 428 Dr. Alafeefy AM

Intestinal sulfonamides (cont.) Sulfasalazine is mainly used for treatment of inflammatory bowel disease, including ulcerative colitis and Crohn's disease. It is also effective in several types of arthritis, particularly rheumatoid arthritis Reductive metabolism by means of azoreductase enzyme converts the drug to sulfapyridine and 5-aminosalicylic acid (anti-inflammatory) both components are active 10:03 AM PHC 428 Dr. Alafeefy AM

Ophthalmic sulfonamides They are used in treatment of conjunctivitis and other superficial ocular infections. 10:03 AM PHC 428 Dr. Alafeefy AM

IV. Sulfonamides for Burn Therapy Mafenide is not a true sulfonilamide, it is not effective systemically, but is particularly effective topically in the treatment of burns or for healing infected wounds. PHC 428 Dr. Alafeefy AM 10:03 AM

Sulfonamides for burn therapy (cont.) Silver sulfadiazine is used as effective topical antimicrobial agents, especially against Pseudomonas s. in burn therapy, where treatment failure with other drugs may occur. PHC 428 Dr. Alafeefy AM 10:03 AM

Mixtures of sulfonamides Sulfonamides alone The main purpose is to reduce the risk of crystalluria Trisulfapyrimidine Each drug is administered in one third of the total dose, they behave independently concerning solubility but their therapeutic effects are additive. 10:03 AM PHC 428 Dr. Alafeefy AM

b. Multiple (or Triple) sulfas They are a 1:1:1 combination of sulfabenzamide, sulfacetamide, and sulfathiazole. The combination is primarily used as topical cream for Gardnerella vaginalis in vaginal infections 10:03 AM PHC 428 Dr. Alafeefy AM

2) Mixtures of sulfonamides with other drugs Sulfamethoxazole and Trimethoprim There is a synergistic effect obtained from such combination. It is usually given orally and I.V. administration. When taken orally the tablet has a standard ratio of 5 : 1 40 mg of the sulfa and 8 mg trimethoprim. Both drugs are excreted in urine their half lives are about 8-10 hours 10:03 AM PHC 428 Dr. Alafeefy AM

This approach has been described as Trimethoprim is often given in conjunction with the sulfonamide sulfamethoxazole. The latter inhibits the incorporation of PABA into folic acid, while the former inhibits dihydrofolate reductase. Therefore, two enzymes in the one biosynthetic route are inhibited. This is a very effective method of inhibiting a biosynthetic route and has the advantage that the doses of both drugs can be kept down to safe levels. To get the same level of inhibition using a single drug, the dose level of that drug would have to be much higher, leading to possible side-effects. This approach has been described as 'sequential blocking'. 10:03 AM PHC 428 Dr. Alafeefy AM

Pharmacokinetic factors of the combination Pairing these two particular antibacterial agents was based upon pharmacokinetic factors and convenient availability. For such a combination to be useful in vivo the two agents must arrive at the necessary infected tissues at the correct time and in the right ratio. It is used for oral treatment of urinary tract infections, shigellosis, otitis media, traveler's diarrhea and bronchitis. 10:03 AM PHC 428 Dr. Alafeefy AM

Selectivity of Trimethoprim There is a significant differences between the bacterial and the mammalian dihydrofolate reductases away from the active site. The bacterial enzyme is sensitive to inhibition by trimethoprim by up to 100,000 times lower concentrations than is the mouse enzyme. This difference explains the useful selective toxicity of trimethoprim. 10:03 AM PHC 428 Dr. Alafeefy AM

Advantages of this combination The combination of sulfamethoxazole-trimethoprim is not only synergistic in vitro but is less likely to induce bacterial resistance than either agent alone. Thus, these agents block sequentially at two different steps in the same essential pathway, and this combination is extremely difficult for a naive microorganism to survive. It is also comparatively uncommon that a microorganism will successfully mutate to resistance at both enzymes during the course of therapy. It is useful and comparatively nontoxic for AIDS patients who are infected with the pneumonia causing opportunistic pathogen Pneumotystis carinii. 10:03 AM PHC 428 Dr. Alafeefy AM

Homework & Probable Quiz, Midterm and/or Final Exam Questions Contrary to bacterial cells, sulfonamides has no lethal effect on human cell growth. Explain this fact verbally and chemically. Illustrate by chemical equations how you could prepare (sulfacetamide sodium and/or silver sulfacetamide) from 4-aminobenzene sulfonamide. Explain how an ‘azo-dye’ breaks down in vivo to yield sulphanilamide? N1-substitution in sulphanilamide is more effective and useful than N4-. Explain. Classify sulphonamide on the basis of their site of action. Give the structure, chemical name, uses and side effects of ONE drug each class. Comment on the probable mechanisms of bacterial resistance to sulphonamides. Write a short note on ‘chemotherapeutic consideration of sulphonamides’. Describe the synthesis of a sulphonamide drug that could be used mostly in: 1. 1. a. Gas-gangrene. b. Second-and third degree burns. 10:03 AM PHC 428 Dr. Alafeefy AM

The end Thanks for Attention 10:03 AM PHC 428 Dr. Alafeefy AM