Irreversible enzyme inhibitors. LXXXIX. candidate active-site-directed irreversible inhibitors of dihydrofolic reductase. X. derivatives of 2-amino-5-benzamidopropyl-4-pyrimidinol

Since 2-amino-5-benzamidopropyl-6-methyl-4-pyrimidinol (VII) was a reasonably good reversible inhibitor of dihydrofolic reductase, the benzamido group was substituted with p-bromomethyl (XVIIa), m-bromomethyl (XVIIIa), and p-bromoacetyl (XIXa) groups; these compounds, and the corresponding 6-phenylpyrimidines, were synthesized from the proper 2-amino-5-aminopropyl-4-pyrimidinol (V) by devising methods that were compatible with the high reactivity of the halogen. Compounds XVII-XIX showed in-activation of dihydrofolic reductase; the fact that p-nitrobenzyl bromide inactivated the enzyme as rapidly as XVII and XVIII and phenacyl bromide inactivated the enzyme as rapidly as XIX indicated that these inactivations proceeded by a random bimolecular mechanism and not the desired active-site-directed mechanism.     

Irreversible enzyme inhibitors. LXXXIII. Candidate active-site-directed irreversible inhibitors of dihydrofolic reductase. VIII. Derivatives of 2,4-diaminopyrimidine. II

2,4-Diamino-6-(p-aminophenethyl)pyrimidines with a 5-phenylbutyl (XIX) and 5-(p-chlorophenyl) (VIII) substituent were synthesized by condensation of the corresponding pyrimidine-6-carboxaldehydes (XVI, X) with the Wittig reagent derived from p-nitro-benzyl bromide, followed by catalytic hydrogenation. Selective bromoacetylation of VIII and XIX afforded the candidate active-site-directed irreversible inhibitors of dihydrofolic reductase, namely, 6-(p-bromoacetamidophenethyl)-2,4-diaminopyrimidine with a 5-(p-chlorophenyl)- (IV) and 5-phenylbutyl- (III) substituents. Although III and IV were excellent reversible inhibitors of dihydrofolic reductase, neither showed any inactivation of the enzyme; in contrast, the corresponding 2-amino-6-(p-bromoacetamidophenethyl)-5-phenylbutyl-4-pyrimidinol (II) - which differs from III only in the 4-substituent (NH₂ vs. OH) - was an excellent active-site-directed irreversible inhibitor of dihydrofolic reductase, but II was a poor reversible inhibitor. Thus the conformations of II and III are most probably different when complexed to dihydrofolic reductase.     

Irreversible enzyme inhibitors. CXI. Candidate active-site-directed irreversible inhibitors of dihydrofolic reductase derived from 4,6-diamino-1,2-dihydro-l-phenyl-s-triazines. V.

Condensation of 5-(p-nitrophenyl)-2-pentanone with phenylbiguanide hydrochloride (V) gave a 2-methyl-2-(p-nitrophenylpropyl)-1,2-dihydro-s-triazine (IX); hydrogenation of the nitro group to amino followed by bromoacetylation afforded the candidate irreversible inhibitor of dihydrofolic reductase, namely, 2-(p-bromoacetamidophenylpropyl)-4,6-diamino-1,2-dihydro-2-methyl-s-triazine hydrochloride (VIII). Similarly, the o, m, and p-isomers of 5-nitrophenoxy-2-pentanone were converted to the corresponding 2-(bromoacetamidophenoxypropyl)-1,2-dihydro-s-triazines (XI). The four candidate irreversible inhibitors were evaluated on the dihydrofolic reductases from pigeon liver, Walker-256 rat tumor, and L-1210/FR8 mouse leukemia. Only VIII was an irreversible inhibitor; VIII slowly inactivated the L-121-/FR8 mouse leukemia enzyme with a half-life of 2–3 hours at 37°, but VIII showed no inactivation of the other two dihydrofolic reductases—a species specific inactivation.     

Irreversible enzyme inhibitors. LXXXV. On the mode of pyrimidine binding of 5-alkyl and 5-arylpyrimidines to dihydrofolic reductase

A series of 5-isoamyl- and 5-(p-chlorophenyl)pyrimidines substituted with amino, alkylamino, mercapto, benzyloxy, hydroxy, or hydrogen at the 2- and 4-positions and with amino or methyl at the 6-position have been synthesized for evaluation of the mode of pyrimidine binding to dihydrofolic reductase. The studies were performed in order to determine where a bulky group could be placed on the pyrimidine ring that would still allow good binding; such studies are essential to find a suitable position for placement of a covalent forming group for design of active-site-directed irreversible inhibitors. Two classes of candidate compounds have emerged for further study as irreversible inhibitors, namely, 2-amino-4-mercapto-6-(p-bromoacetamidophenylalkyl)-pyrimidines and 2,4-diamino-6-(p-bromoacetamidophenylalkyl)aminopyrimidines having a group such as phenyl, phenylbutyl or isoamyl at the 5-position that can give strong hydrophobic bonding to the enzyme.     

Irreversible enzyme inhibitors. LXXXI. Further observations on the mode of binding of the anilino moiety of 2-amino-5-(anilinopropyl)-6-methyl-4-pyrimidinol to dihydrofolic reductase and thymidylate synthetase

Replacement of phenyl group of 2-amino-5-(anilinopropyl)-6-methyl-4-pyrimidinol (III) by benzyl (XI) led to a large loss in binding to both dihydrofolic reductase and thymidylate synthetase; the binding by XI returned when the protonated benzylamino group was N-acetylated to XII, which removes the charge at pH 7.4 and changes the ground-state conformation of the benzene ring. Replacement of the benzyl group of the acetamide, XII, with the polar 2-, 3-, or 4-, picolyl groups also led to a loss in binding. Substitution of p-fluoro or m-trifluoromethyl on the anilino group of III, or replacement of the anilino of III by 3-pyridylamino, gave little - if any - enhancement in binding to the enzymes.     

Enzyme ribonucleotide reductase: unraveling an enigmatic paradigm of enzyme inhibition by furanone derivatives

Several 2'-substituted-2'-deoxyribonucleotides are potent inactivators of the enzyme ribonucleotide reductase (RNR), by destroying the essential tyrosyl radical located in subunit R2 or/and covalently alkylating the subunit R1. In the absence of external reductants, the inactivation is achieved by alkylation of subunit R1 by a methylene-3(2H)-furanone. The furanone is generated in solution through degradation of a keto-deoxyribonucleotide intermediate, produced during the inhibitory mechanism of a wide group of 2'-substituted inhibitors, and is easily detected experimentally by UV spectroscopy. Interestingly, the same keto-deoxyribonucleotide is also a proposed intermediate of the normal substrate pathway, but by some unknown reason, it does not dissociate from the active site and does not inactivate the enzyme. Therefore, if the currently accepted mechanism for substrate reduction is correct, there must be some specific reason that makes such a reactive intermediate behave differently, not dissociating from the active site during substrate reduction. In this article, we propose to validate the current substrate mechanism by showing that the keto-deoxyribonucleotide dissociates from the active site only in the case of the inhibitors, and therefore, it corresponds to a viable intermediate in the substrate mechanism. Furthermore, we answer unexplained experimental observations that concern the predomination of the normal reduction mechanism over the abnormal ketone formation in the FdNDP and the release of F(-), either in the normal or in the abnormal turnover. For that purpose, we have investigated the interaction between the enzyme and this keto-deoxyribonucleotide generated from the normal substrate and from two widely studied representative inhibitors. A model containing 140 atoms was used to represent the desired structures. The results allowed us to conclude that the solvation free energy of the 2'-substituents, its influence inside the active site, and the charge transfer mechanism from a protein side chain to solution are the thermodynamic driving forces for the intermediate dissociation and subsequent RNR inhibition. Such charge transfer cannot be accomplished by the natural substrate, preventing its dissociation. These results elucidate a paradox which has been unexplained for more than 20 years and further validates both the proposed substrate and inhibition chemical mechanisms.     

Synthesis of 3-aminorhodanine derivatives as aldose reductase inhibitors

A number of 5-condensated 3-acylaminorhodanines 3 was prepared as potential inhibitors of the aldose reductase by acylation of the amino group of 3-aminorhodanine 1 and subsequent condensation of the 5-methylene function with appropriate aldehydes. Some of these compounds displayed interesting activity.     

Organometallic derivatives of 2,6-di-tert-butylphenols as specific inhibitors of methyl oleate oxidation

2,6-Di-tert-butylphenols containing the Pt—SnCl₃ and Pt—GeCl₃ groups in the para position exert a dual effect on the oxidation of methyl oleate by molecular oxygen. Initially, these compounds act as antioxidants producing the corresponding phenoxyl radicals whose decomposition is accompanied by elimination of SnCl₂ and GeCl₂, which are oxidation promoters.     

Dihydrofolate reductase: structural aspects of mechanisms of enzyme catalysis and inhibition

The mechanism of catalytic reduction of folic and dihydrofolic acids to tetrahydrofolate, which proceeds under the action of dihydrofolate reductase and the coenzyme NADPH, is considered. The roles of the enzyme active site, the coenzyme, individual amino acid residues of the enzyme, and water molecules in the catalytic reaction are discussed. Interactions of the enzyme with competitive inhibitors many of which are widely used in medicine as antitumor and antibacterial drugs are examined. The factors controlling the selectivity of inhibitor binding to bacterial forms of the enzyme are analyzed. The results of X-ray diffraction and NMR spectroscopic studies of the structures of the enzyme and its complexes with the substrate and inhibitors are surveyed. The role of specific interactions and molecular motions of the protein and ligands in the mechanism of catalysis and in the binding of the ligands to the enzyme is discussed.     

Irreversible enzyme inhibitors. XC. candidate active-site-directed irreversible inhibitors of thymidylate synthetase. I. 2-amino-5-(benzenesulfonamidopropyl)-6-methyl-4-pyrimidinol with substituents on the benzene ring

2-Amino-5-[p- (bromoacetamidomethyl)benzenesulfonamidopropyl]-6-methyl-4-pyrimidinol (XV) was synthesized by acylation of 2-amino-5-aminopropyl-6-methyl-4-pyrimidinol (III) with p-cyanobenzenesulfonyl chloride followed by catalytic reduction and reaction of the resultant aminomethyl group with p-nitrophenyl bromoacetate. A second irreversible inhibitor of thymidylate synthetase, namely 2-amino-5-[p-(bromoacetyl)benzene-sulfonamidopropyl]-6-methyl-4-pyrimidinol (XVI), was synthesized by acylation of in with p-acetylbenzenesulfonyl chloride followed by bromination. Both XV and XVI were good reversible inhibitors of thymidylate synthetase and inactivated the enzyme when the candidate compound was incubated with the enzyme. Iodoacetamide, which does not form a complex with enzyme, could inactivate thymidylate synthetase almost as well as XV; therefore it appears that XV inactivated the enzyme by a random bimolecular mechanism rather than by the desired active-site-directed mechanism via an enzyme-inhibitor complex. Similar conclusions were reached with XVI since phenacyl bromide could inactivate the enzyme somewhat more rapidly than XVI.     

Irreversible enzyme inhibitors. XCI. candidate active-site directed irreversible inhibitors of thymidylate synthetase. II. 2-amino-6-methyl-5-(p-tolylsulfonamidopropyl)-4-pyrimidinols with N-substituents on the sulfonamide moiety

Three derivatives of 2-amino-6-methyl-5-(p-tolylsulfonamidopropyl)-4-pyrimidinol (I) with N - substituents on the sulfonamide group, namely bromoacetamidopropyl (XVI), m-bromoacetamidobenzyl (XXIVa), and p-bromoacetamidobenzyl (XXIVb), were synthesized as candidate active-site-directed irreversible inhibitors of thymidylate synthetase. The bromoacetamidopropyl derivative, (XVI), the p-bromoacetamidobenzyl derivative (XXIVb), and iodoacetamide showed irreversible inhibition of thymidylate synthetase, but XXIVa did not. Since iodoacetamide did inactivate the enzyme, but XXIVa did not, it cannot be ascertained whether XXIVb and XVI inactivate the enzyme by a random bimolecular mechanism or by the active-site-directed mechanism without evaluation of additional candidate inhibitors. Two synthetic routes were employed. The key intermediates for the bromoacetamidobenzyl sulfonamides (XXIV) were the corresponding nitrobenzyl sulfonamides (XXI); the latter were best prepared by reductive alkylation of 2-amino-5-aminopropyl-6-methyl-4-pyrimidinol (XXV) with a nitrobenzaldehyde followed by tosylation. The key intermediate for XVI was a toluenesulfonamide with a carbobenzoxyaminopropyl substituent on the nitrogen (XIV); the latter was synthesized via N-carbobenzoxy-N'-tosyl-1,3-diaminopropane (XI).     

Reductivetrans-2,6-diallylation of pyridines with allylboranes. Synthesis oftrans- andcis-2,6-diallyl-1,2,5,6-tetrahydropyridines and their deuterated derivatives

The reductivetrans-2,6-diallylation of pyridines with triallyl- and allyl(dialkyl)boranes has been discovered. Heating (40–100 °C) of pyridine, deuteropyridine, or 3-bromopyridine complexes with triallylborane in the presence of alcohols (ROH or CH₃OD), water, or Et₂NH results in the respectivetrans-2,6-diaIlyl-1,2,5,6-tetrahydropyridines (2,3,22, or25) in 20–97 % yields. A preparative method for the isomerization oftrans-2,6-diallyl compounds2 and25 into the respectivecis-isomers4 and28 by heating them with triallyl- or allyl(dialkyl)boranes (125–150 °C) has been suggested. The hydrogenation oftrans- orcis-2,6-diallyl-1,2,5,6-tetrahydropyridines gavetrans- orcis-2,6-dipropylpiperidines, respectively. Thecis- andtrans-configurations of compounds2 and4 were established by analyzing the NMR spectra ofN-benzyl (7 and13) andN,N-dimethyl (6 and 14) derivatives of piperidine derivatives5 and8. A possible mechanism for the reductive diallylation of pyridines has been discussed.Translated fromIzyestiya Akademii Nauk. Seriya Khimicheskaya, No. 4, pp. 693–704, April, 1994.This study was financially supported by the Russian Foundation for Basic Research (Project 93-03-18193).     

1-Aryl-4,6-diamino-1,2-dihydro-s-triazines. Contrasting effects on intestinal helminths,bacteria, and dihydrofolic reductase

The preparation of a variety of novel 1-aryl-4,6-diamino-1,2-dihydro-s-triazines is described. These compounds exhibit an array of contrasting effects on intestinal helminths, bacteria, and dihydrofolic reductase. It is concluded that potent anthelmintic activity among the dihydro-triazines is not dependent solely on the bulky-substituent hypothesis advanced by other investigators, and indeed cannot reliably be predicted on this basis.     

Exploring the potential of xanthene derivatives as trypanothione reductase inhibitors and chloroquine potentiating agents

Xanthene derivatives were synthesized and evaluated for their potential as trypanothione reductase (TryR) inhibitors and chloroquine (CQ) potentiating agents. Some derivatives displayed inhibitory activity against TryR comparable to known tricyclic anti-depressants. On the other hand a number of derivatives increased CQ accumulation and potentiating effects in a resistant strain of Plasmodium falciparum with one compound also displaying strong intrinsic antimalarial activity.     

N-long-chain monoacylated derivatives of 2,6-diaminopyridine with antiviral activity

N-Monoacyl-2,6-diaminopyridines (2a-c) and N,N'-diacyl-2,6-diaminopyridines (3a-c) were synthesized from 2,6-diaminopyridine by acylation with the corresponding acyl halide or by dehydration with the corresponding carboxylic acid using 1,3-dicyclohexylcarbodiimide (DCC). The antiviral activities of N-monoacyl- and N,N'-diacyl-2,6-diaminopyridines (2a-c and 3a-c) were estimated using plaque reduction assay with HSV-1. All N-monoacyl derivatives (2a-c) showed significant anti-herpes simplex virus (HSV)-1 activity (EC(50) = 15.3-18.5 microg/ml). The CC(50) values of 2a-c measured using Vero cells ranged at 37.5-50.0 microg/ml. These compounds showed no significant antibacterial activities with Escherichia coli or Staphylococcus aureus even at a concentration of 1 mg/ml. The N,N'-diacyl derivatives (3a-c) showed no significant anti-HSV-1 activity.     

Novel synthesis of 2,6-benzothiazonine derivatives

The reaction of 1-alkylamino-1-alkylthio-3-phenyl-3-thioxopropenes with phthaloyl chloride in toluene at 60°C, followed by treatment with triethylamine, afforded 6-alkyl-3-phenyl-2,6-benzothiazonine-1,5,7-triones in good to excellent yields.