Patent Application
20080221214
Enzyme inhibition is an important area of pharmaceutical research, since studies in this field have already led to the discovery of wide variety of drugs useful in a number of diseases. Specific inhibitors interact with enzymes and block their activity towards their corresponding natural substrates. Urease inhibitors have recently attracted much attention as potential new anti-ulcer drugs and comprehensive developments in the field were recently reviewed in reference.
Ureases (E.C.3.5.1.5) are enzymes decomposing urea to ammonia and carbamates, and the latter spontaneously decompose to ammonia and carbonic acid. Due to the formation of two molecules of ammonia and carbonic acid, the net effect is an increase in pH. Bacterial ureases are directly involved in the formation of infection stones and contribute to the pathogenesis of pyelonephritis, ammonia, encephalopathy, hepatic coma or urinary catheter encrustation and peptic ulceration. Therefore, strategies based on urease inhibition are now considered as the first line of treatment for infections caused by urease-producing bacteria. In agriculture, high urease activities cause significant environmental and economic problems by releasing abnormally large amounts of ammonia into the atmosphere during urea fertilization. This further induces plant damage primarily by depriving them from their essential nutrient and secondly ammonia toxicity increasing the pH of the soil. To reduce the problems encountered using urea fertilizers, several approaches have been suggested, and the most promising one is to apply urease inhibitors.
Trypsin and chymotrypsin are digestive enzymes and members of a family of enzymes known as serine proteases. They are synthesized as inactive zymogen precursors (trypsinogen and chymotrypsinogen) to prevent unwanted destruction of cellular proteins. The inactive zymogens are secreted into the duodenum, and are converted to the mature, active enzymes by proteolysis to split off a pro-peptide, either in a subcellular compartment or in an extracellelar space where they are required for digestion. Any disturbance of the balance between proteolytic enzymes and their inhibitors, or of the activation process, can result in pancreatits, where premature, intercellular activation of zymogens cause the auto-digestion of the pancreas. Pancreatits carries a 40% risk of pancreatic cancer, a very invasive cancer with high mortality rates. Pancreatic inflammation promotes intensive cell proliferation to regenerate the damaged pancreas, during which the amplification of pathological changes in DNA can occur. The serine proteases contain a uniquely reactive residue at their active site and are inhibited by diisopropylfluorophosphates and serine proteinase inhibitors. Serine proteases, such as chymotrypsin and trypsin, are involved in the destruction of certain fibrous proteins. Chronic infection by hepatitis C virus can lead to the progressive liver injury, cirrhosis, and liver cancer. Viral proteases are an absolute requirement in the life cycle of many viruses and HIV-specific protease inhibitors are designed to target these proteases of HIV-infected patients. Therefore, the search for new effective serine protease inhibitors is still an urgent need for drug development.
The “Urchym” was prepared by the reaction of 4-nitro phenyl isocyanate in the presences of tertiary arnines e.g., triethyl amine, pyridine or 2,6-lutidine in quantitative yield. The invented compound can also be prepared by the reaction of phenyl isocyanate with tertiary arnines e.g., triethyl amine, pyridine or 2,6-lutidine to give N, N′-diphenylurea, it is then treated with nitrating agent (concentrated nitric and sulfuric acid) to yield N-4-nitrophenyl-N′-4′-nitrophenylurea.
Urchym was tested against ureas, using thiourea as a standard inhibitor (IC50 value=21 μM). N-4-nitrophenyl-N′-4′-nitrophenylurea was found to be the most potent urease inhibitor having an IC50 value of 1.25 μM, and is thus superior in activity compared to the standard inhibitor thiourea.
Compound Urchym was also tested for their α-chymotrypsin inhibitory activity and found that it was an excellent α-chymotrypsin inhibitory property with an IC50 value of 3.15±0.14 μM which is far above that of the standard inhibitor chymostatin (IC50=7.00
It is a one pot reaction for the synthesis of N-4-nitrophenyl-N′-4′-nitrophenylurea.
Reaction mixtures comprising a 25 μL solution of enzyme (Jack bean urease, Sigma-Aldrich, specific activity 15 EU/mg) and 55 μL of buffer (0.01 M K2HPO43H2O, 1 MM EDTA 0.01 M LiCl; pH 8.2) containing 100 mM urea were incubated with 5 μL of the test compounds (0.01 μM-1 mM, dissolved in DMSO) at 30° C. for 15 min in 96-well plates. Urease activity was determined by measuring ammonia production using the indophenol method, 45 μL, of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 70 μL of alkali reagent (0.5% w/v NaOH and 0.1% w/v NaOCI) were added to each well (the final reaction volume was 200 μL). The increasing absorbance at 630 nm was measured after 50 min using a microplate reader (Molecular Device). All reactions were performed in triplicate. The results (change in absorbance per min) were processed using SoftMax Pro software (Molecular Device). Percent inhibitions were calculated using the formula 100−(ODtest well/ODcontrol×100). Thiourea was used as the standard inhibitor for urease.
For the determination of α-chymotrypsin inhibitory activity the literature protocol was followed. In brief α-chymotrypsin (9 units/mL in 50 mM Tris-HCl buffer, pH 7.6; Sigma-Aldrich) was pre-incubated with the compound (3.02×10−5 g/100 μL) for 30 min at 37° C. and then 100 μL of substrate solution (N-succinyl-phenylalanine-p-nitroanilide; 1 mg/mL) in 50 mM Tris-HCl buffer, pH 7.6) was added to start the enzyme reaction. The absorbance of released p-nitroaniline was continuously monitored at 410 nm until a significant color change had achieved. All reactions were performed in triplicate. Percent inhibitions were calculated using the formula 100-(ODtest well/ODcontrol×100). Chymostatin was used as standard inhibitor for α-chymotrypsin.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1510/2006 | Nov 2006 | PK | national |
| 0700816.2 | Apr 2007 | GB | national |
