Sulfonamides

PHRM 304: Antibiotics and chemotherapeutic agents

 No relation with any natural product Not properly termed as antibiotic For example: Sulfonamides

Synthetic Antimicrobial Agents

Antibacterial agents which act against cell metabolism (antimetabolites)

Sulfonamides:
History The sulfonamide story began in 1935 when it was discovered that a red dye called prontosil had antibacterial properties in vivo (in animals). Strangely enough, no antibacterial effect was observed in vitro (in petri dish). This remained a mystery until it was discovered that prontosil was not in fact the antibacterial agent.

It was found that the dye was metabolized by bacteria present in the small intestine, and broken down to give a product called sulfanilamide. It was this compound which was the true antibacterial agent. Thus, prontosil was the first example of a prodrug. Sulfanilamide was synthesized in the laboratory and became the first synthetic antibacterial agent active against a wide range of infections.

Fig. In vivo metabolism of prontosil

Further developments led to a range of sulfonamides which proved effective against Grampositive organisms, especially pneumococci and meningococci. Despite their undoubted benefits, sulfa drugs have proved ineffective against infections such as Salmonella (Gram-negative)- the organism responsible for typhoid. Other problems have resulted from the way these drugs are metabolized, since toxic products are frequently obtained. This led to the sulfonamides mainly being superseded by penicillin.

Sulfonamides: Introduction
Sulfanilamide, a colorless cleavage product formed by reductive liver metabolism of the administered dye (Prontosil rubrum).

The Nobel Prize in Physiology or Medicine 1939 was awarded to Gerhard Domagk "for the discovery of the antibacterial effects of prontosil".

Sulfonamides: Introduction
Once known as wonder drug Once mainstays of antimicrobial chemotherapy The relative cheapness of the sulfonamides is one of their most attractive features and accounts for much of their persistence in the market.

Structure-activity relationships (SARs)

Fig. Sulfonamide analogues

The para-amino group is essential for activity and must be unsubstituted (i.e. R1=H). The only exception is when R1=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. Incorporation of other groups (halogen, alkyl, etc.) destroy the activity.

Enzyme Fig. Metabolism of acyl group to regenerate active compound

The aromatic ring and the sulfonamide functional group are both required. Total loss of antibacterial activity occurs if sulfonamide group is replaced by other acidic groups (sulfonic, phosphoric etc.). The aromatic ring must be para-substituted only. Extra substitution eliminates activity for steric reasons.

 The sulfonamide nitrogen must be primary (:sulfanilamide) or secondary (acidic proton is essential for antibacterial activity). R2 is the only possible site that can be varied in sulfonamides.

Sulfonamide analogues
R2 can be varied by incorporating a large range of heterocyclic or aromatic structures, which affects the extent to which the drug binds to plasma protein. This in turn controls the blood levels of the drug such that it can be short acting or long acting. Thus, a drug which binds strongly to plasma protein will be slowly released into the blood circulation and will be longer lasting. R2 affects pharmacokinetic properties but not the pharmacodynamic properties.

Sulfonamide analogues with reduced toxicity

For example, the metabolite formed from sulfathiazole is poorly soluble and can prove fatal if it blocks the kidney tubules.

Sulfathiazole
(insoluble)

Fig. Metabolism of sulfathiazole

It was discovered that the solubility problem could be overcome by replacing the thiazole ring in sulfathiazole with a pyrimidine ring to give sulfadiazine.

Sulfadiazine Its metabolites will also be water soluble

+ H+

The reason for the improved solubility lies in the acidity of the sulfonamide NH proton. In sulfathiazole, this proton is not very acidic. 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 resulting anion. Therefore, sulfadiazine and its metabolite are significantly ionized at blood pH. As a consequence, they are more soluble and less toxic.

Treatment of intestinal infections

The succinyl moiety contains an acidic group which means that the prodrug is ionized in the slightly alkaline conditions of the intestine. As a result, it is not absorbed into the bloodstream and is retained in the intestine. Slow enzymatic hydrolysis of the succinyl group then releases the active sulfathiazole where it is needed.

Substitution on the aniline nitrogen with benzoyl groups

Benzoyl substitution on the aniline nitrogen has also given useful prodrugs, which are poorly absorbed through the gut wall since they are too hydrophobic.

Mechanism of action
Folic acid metabolism:
Para-aminobenzoic acid (PABA)
[GTP]

+ Pteridine
Sulfonamides Competitive inhibition

Dihydropteroate synthetase

Dihydropteroic acid
L-Glutamate Dihydrofolate synthetase

2 NADPH 2 NADP+

Dihydrofolic acid (folic acid)

Tetrahydrofolic acid Purines DNA, RNA

Cofactors

Thymidine DNA

Methionine t RNA, Proteins

Folic acid (dihydrofolate) is the precursor for tetrahydrofolate- a compound which is crucial to cell biochemistry since it acts as the carrier for one-carbon units, necessary for many biosynthetic pathways. If tetrahydrofolate is no longer synthesized, then any biosynthetic pathway requiring one-carbon fragments is disrupted. The biosynthesis of nucleic acids (DNA, RNA) is particularly disrupted and this leads to the cessation of cell growth and division.

The sulfonamides act as competitive enzyme inhibitors and block the biosynthesis of the vitamin folic acid in bacterial cells. They do this by inhibiting the enzyme (dihydropteroate synthetase) responsible for linking together the component parts of folic acid. The consequences of this are disastrous for the cell.

Sulfonamides act as inhibitors by mimicking paminobenzoic acid (PABA)- one of the normal constituents of folic acid. 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.

H N
o

H N

6.69 A

6.88 A

C O O
2.22 A PABA
o

S O
2.49 A Sulfanilamide
o

Fig: Interatomic distance in PABA and in sulfanilamide

on enzyme

Usually happen

on enzyme
When sulfonamide on action Fig. Sulfonamide prevents PABA from binding by mimicking PABA

Sulfonamide resistance
Sulfonamides are competitive enzyme inhibitors and as such the effect can be reversible. This is demonstrated by certain organisms such as staphylococci, pneumococci, and gonococci which can acquire resistance by synthesizing more PABA. The more PABA there is in the cell, the more effectively it can compete with the sulfonamide to reach the enzyme's active site. In such cases, the dose levels of sulfonamide have to be increased to bring back the same level of inhibition.

Note that sulfonamides do not actively kill bacterial cells. They do, however, prevent the cells dividing and spreading. This gives the body's own defense systems enough time to gather their resources and wipe out the invader.

Folic acid is clearly necessary for the survival of bacterial cells. However, folic acid is also vital for the survival of human cells, so why do the sulfa drugs not affect human cells as well ?

Human cells cannot make folic acid. They lack the necessary enzymes and so there is no enzyme for the sulfonamides to attack. Human cells acquire folic acid as a vitamin from the diet. Folic acid is brought through the cell membrane by a transport protein and this process is totally unaffected by sulfonamides. If human cells can acquire folic acid from the diet, why can't bacterial cells infecting the human body do the same? In fact, it is found that bacterial cells are unable to acquire folic acid since they lack the necessary transport protein required to carry it across the cell membrane. Therefore, they are forced to make it by themselves.

Trimethoprim

Trimethoprim (synthetic folate antagonist) is a diaminopyrimidine structure which has proved to be a highly selective, orally active, antibacterial, and antimalarial agent. Unlike the sulfonamides, it acts against dihydrofolate reductase- the enzyme which carries out the conversion of folic acid to tetrahydrofolate. The overall effect, however, is the same as with sulfonamides- the inhibition of DNA synthesis and cell growth.

 Dihydrofolate reductase is present in mammalian cells as well as bacterial cells, so we might wonder why trimethoprim does not affect our own cells. Trimethoprim is able to distinguish between the enzymes in either cell. Although this enzyme is present in both types of cell and carries out the same reaction, mutations over millions of years have resulted in a significant difference in structure between the two enzymes such that trimethoprim recognizes and inhibits the bacterial enzyme, but does not recognize the mammalian enzyme.

Trimethoprim, owing to its partial structural analogy with the dihydrofolic acid molecule, inhibits the reduction of dihydrofolic acid to the metabolically active tetrahydrofolic acid. Trimethoprim has a tremendous affinity for bacterial dihydrofolate reductase (100,000 times higher than for the mammalian enzyme); when bound to this enzyme, it inhibits the synthesis of tetrahydrofolate.

Sequential blocking

Sulfamethoxazole

Trimethoprim is often given in conjunction with the sulfonamide- sulfamethoxazole (in 1:5 ratio, cotrimoxazole ). 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'.

Fig. Use of sulfamethoxazole and trimethoprim in 'sequential blocking'

The combinations use has been declining due to reports of sulfamethoxazole bone marrow toxicity, resistance and lack of greater efficacy in treating common urine and chest infections, and side effects of antibacterial sulfonamides. As a consequence, the use of co-trimoxazole was restricted in 1995 following the availability of trimethoprim (not in combination) in 1980. With its greater efficacy against a limited number of bacteria, Co-trimoxazole remains indicated for some infections; for example, it is used as prophylaxis in patients at risk for Pneumocystis jirovecii pneumonia (e.g. AIDS patients and those with some hematological malignancies) and as therapy in Whipple's disease. Gram positive bacteria are generally or moderately susceptible.

Synergistic action
Trimethoprim and sulfamethoxazole have a greater effect when given together than when given separately; the reasons is because they inhibit successive steps in the folate synthesis pathway.