Inhibitor of dihydrofolate

In the context of preparing potential inhibitors of dihydrofolate reductase (DHFR), the group of Organ has developed a rapid microwave-assisted method for the preparation of biguanide libraries (Scheme 6.174) [330]. Initial optimization work was centered around the acid-catalyzed addition of amines to dicyandiamide. It was discovered that 150 °C was the optimum temperature for reaction rate and product recovery, as heating beyond this point led to decomposition. While the use of hydrochloric acid as catalyst led to varying yields of product, evaluation of trimethylsilyl chloride in acetonitrile as solvent led to improved results. As compared to the protic... 

Screening for inhibitors of dihydrofolate reductase using pulsed ultrafiltration mass spectrometry. Comb Chem High Throughput Screen 1998, 1, 47-55. 

Dihydrofolate reductase acts as an auxiliary enzyme for thymidylate synthase. It is involved in the regeneration of the coenzyme N, N -methylene-THF, initially reducing DHF to THF with NADPH as the reductant (see p. 418). The folic acid analogue methotrexate, a frequently used cytostatic agent, is an extremely effective competitive inhibitor of dihydrofolate reductase. It leads to the depletion of N, N -methylene-THF in the cells and thus to cessation of DNA synthesis. 

The diaminopyrimidines trimethoprim and pyrimethamine are synthetic, antibacterial drags and inhibitors of dihydrofolate reductase that are used both independently as well as in combination with sulfanilamides, in particular, with sulfamethoxazole (cotrimoxazole, bactrim, biseptol, sulfatrim, and many others). 

All of these compounds are inhibitors of dihydrofolate reductase in bacteria, plasmodia, and humans. Fortunately, they have a significantly higher affinity to bacterial and protozoal dihydrofolate reductase. Pyrimethamine, for example, inhibits dihydrofolate reductase in parasites in concentrations that are a several hundred times lower than that required to inhibit dihydrofolate reductase in humans. This is the basis of their selective toxicity. Selective toxicity can be elevated upon the host organism s production of folic acid, which parasites are not able to use. 

This powerful inhibitor of dihydrofolate reductase is used for preventing and treating malaria caused by plasmodia P. vivax, P. malariae, P. ovale, including P. falciparum. 

Combination of a sulfonamide with an inhibitor of dihydrofolate reductase (trimethoprim or pyrimethamine) provides synergistic activity because of sequential inhibition of folate synthesis (Figure 46-2). 

Sulfadiazine in combination with pyrimethamine is first-line therapy for treatment of acute toxoplasmosis. The combination of sulfadiazine with pyrimethamine, a potent inhibitor of dihydrofolate reductase, is synergistic because these drugs block sequential steps in the folate synthetic pathway blockade (Figure 46-2). The dosage of sulfadiazine is 1 g four times daily, with pyrimethamine given as a 75-mg loading dose followed by a 25-mg once-daily dose. Folinic acid, 10 mg orally each day, should also be administered to minimize bone marrow suppression. 

Other useful targets for pharmaceutical agents are thymidylate synthase and dihydrofolate reductase, enzymes that provide the only cellular pathway for thymine synthesis (Fig. 22-49). One inhibitor that acts on thymidylate synthase, fluorouracil, is an important chemotherapeutic agent. Fluorouracil itself is not the enzyme inhibitor. In the cell, salvage pathways convert it to the deoxynucleoside monophosphate FdUMP, which binds to and inactivates the enzyme. Inhibition by FdUMP (Fig. 22-50) is a classic example of mechanism-based enzyme inactivation. Another prominent chemotherapeutic agent, methotrexate, is an inhibitor of dihydrofolate reductase. This folate analog acts as a competitive inhibitor the enzyme binds methotrexate with about 100 times higher affinity than dihydrofolate. Aminopterin is a related compound that acts similarly. 

Methotrexate acts by inhibition of dihydrofolate reductase, the enzyme requisite for the reduction of dihydrofolic acid (3) to 5,6,7,8-tetrahydrofolic acid (4). In turn, (4) is a precursor to a series of enzyme cofactors (5-7) essential for the transfer of one carbon unit necessary for the biosynthesis of purines and pyrimidines and hence, ultimately, DNA. As an inhibitor of dihydrofolate reductase, methotrexate kills cells during the S phase of the cell cycle, when the cells are in the log phase of growth. Unfortunately, this cytotoxicity is non-selective, and rapidly proliferating normal cells, e.g., gastrointestinal epithelium cells and bone marrow, are dramatically affected as well. In addition, recent use of high dose methotrexate therapy with leucovorin rescue has led to additional clinical problems arising from a dose-related nephrotoxic metabolite, 7-hydroxy methotrexate (8). Finally, the very polar nature of methotrexate renders it virtually impenetrable to the blood-brain barrier, which can necessitate direct intrathecal injection in order to achieve therapeutic doses for the treatment of CNS tumours. 

The sulfones and sulfonamides synergize with the inhibitors of dihydrofolate reductase, and the combinations have been effective in controlling malaria, toxoplasmosis, and coccidiosis. Fansidar, a combination of sulfadoxine and pyrimethamine, has been successful in controlling some strains of chloroquine-resistant Plasmodium falciparum malaria (see Chapter 53 Antiprotozoal Drugs). However, reports of Fansidar resistance have increased in recent years. New inhibitors effective against the sulfonamide-resistant 7,8-dihydropteroate synthase are needed. 

The role of drug-membrane interactions in overcoming, or at least in reducing, resistance will be discussed using two examples of antibacterials. A series of 5-(substituted) benzyl-2,4-diaminopyrimidines and 4,6-diamino-l,2-dihydro-2,2-dimethyl-l-(3-substituted)phenyl-s-triazines, inhibitors of dihydrofolate reductase (DHFR), were tested against sensitive and resistant E. coli cell cultures as well as against E. coli-derived DHFR [55] and compared with the effect on sensitive and resistant murine tu-... 

Another way to reduce the supply of deoxynucleotides for cell replication is to target the reduction of dihydrofolate to tetrahydrofo-late. Folate antagonists are used in antimicrobial and anticancer chemotherapy. These compounds are competitive inhibitors of dihydrofolate reductase because they resemble the natural substrate. For example, methotrexate is used in antitumor therapy. 

Methotrexate is a competitive inhibitor of dihydrofolate reductase because its structure is similar to those of folic acid and dihydrofolic acid. Competitive inhibitors are characterized by an increase in the Km of the enzyme (lowered affinity for the substrate) while Vmax remains unchanged. 

The chemical structures of four commonly used anticancer drugs are shown in Fig. 15-17. Methotrexate was the first "true anticancer drug, synthesized in 1949, and has been in clinical use for treatment of a variety of cancers since the early 1950s. Methotrexate is a potent inhibitor of dihydrofolate reductase with an inhibition constant (Kt) for interaction with the enzyme of 10 9Af. Inhibition of this enzyme in a cell leads to major accumulation of DHF to concentrations of 2.5 fiM, and minor decreases in THF. Marked decreases in THF may not be seen due to the release of bound THF in methotrexate-treated cells. The high levels of DHF are toxic to the cell, inhibiting the reaction catalyzed by thymidylate synthase,... 

Figure 13.13. Structures of dihydrofolate and of methotrexate, a competitive inhibitor of dihydrofolate reductase.

Methotrexate is a potent inhibitor of dihydrofolate reductase, with an affinity 1,000-fold greater than that of dUiydrofolate. Chemotherapy consists of alternating periods of administration of methotrexate and folate (normally as 5-formyl-tetrahydrofolate, leucovorin) to replete the normal tissues and avoid induction of folate deficiency- so-called leucovorin rescue. As well as depleting tissue pools of tetrahydrofolate, methotrexate leads to the accumulation of relatively large amounts of 10-formyl-dihydrofolate, which is apotentinhibitor of both thymidylate synthetase and glycinamide ribotide transformylase, an intermediate step in purine nucleotide synthesis. It is likely that this, rather than simple depletion of tetrahydrofolate, is the basis of the cytotoxic action of methotrexate (Barametal., 1988). 

The second equation is often applicable to equilibrium situations such as those found in vitro. Sometimes, different dependencies on tt are found for substituents in different parts of the molecule. The hydrolysis rates of substituted phenyl / -D-glucosides by emulsin, for instance, have been shown by Hansch (I) to depend on w for the para substituents but not on tt for the meta substituents. Some of Baker s results (2) on the 5,6-disubstituted-2,4-diaminopyrimidines as inhibitors of dihydrofolate reductase can be correlated with tt only by choosing tt for the most lipophilic of the two substituents (3). Only a part of the molecule need be desolvated on combining with the receptor. 

Methotrexate is a potent inhibitor of dihydrofolate reductase, with an affinity 1,000-fold greater than that of dihydrofolate. Chemotherapy consists of alternating periods of administration of methotrexate and folate (normally as... 

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