Mathias P. Mertes

Potential ATPase mimics by polyammonium macrocycles: Criteria for catalytic activity

Abstract A series of polyammonium macrocycles, ranging in size from the 18-membered ring [18]aneN6 to the 36-membered [36]aneN12 were examined as potential ATPase mimics. The rates of hydrolysis of ATP were followed at pH 3.0 and 7.0 using 31P NMR and HPLC techniques. Stability constants as a function of degree of protonation, distribution curves for the ligands as a function of pH, and distribution curves for the mixed species of nucleotides, inorganic phosphate, and macrocycle were also determined. All of the macrocycles catalyzed the hydrolysis of ATP to some extent compared to noncatalyzed hydrolysis. A critical dependence on macrocyclic ring size was observed, with [21]aneN7 being the best catalyst at both pHs. Stability constants of the complexes formed between the phosphate species and macrocycle increase with increasing degree of protonation and decreasing ring size. The trend in stability constants for phosphate species was found to be PO43− > P2O74− > ATP4− > ADP3− > AMP2− for a given degree of protonation. The crystal structure of tetraprotonated [21]aneN7 was determined. The compound N7C14Cl4H41O crystallizes in the monoclinic space group P21 (#4) with unit cell dimensions a = 7.472(1), b = 19.480(2), c = 8.3638(9) A , β = 100.38(1)o, and V = 1197.4(3) A 3 . The structure was solved by direct methods and refined using full-matrix least-squares techniques to give a final R = 0.041 and Rw = 0.055.

Pharmacological specificity of synaptosomal and synaptic membrane γ-aminobutyric acid (GABA) transport processes☆

Abstract Amino cyclopentane and amino cyclohexane carboxylic acids were examined as potential inhibitors of γ-aminobutyric acid (GABA) uptake in brain synaptosomes and in synaptic membrane vesicles. The resealed synaptic plasma membrane vesicle preparation was used extensively in determining the potency inhibition of GABA uptake by these agents and in comparing their activities to the activities of various acyclic GABA analogs and to analogs of piperidyl-3-carboxylic acid (nipecotic acid). A number of the cyclic and acyclic GABA analogs also stimulated the carrier-mediated efflux of [ 3 b]GABA from preloaded synaptic membrane vesicles, whereas nipecotic acid did not increase the efflux of [ 3 H]GABA in a dose-dependent manner. These results suggest that there is a competitive type of interaction of the cyclic and acyclic GABA analogs with membrane uptake carriers and that a more complex type of action of nipecotic acid on these carriers occurs. The order of potency of uptake inhibition by some of these agents was compared with previously published orders of activation of physiologic GABA receptors by these compounds.

COMPARATIVE STUDY OF THE POTENCY AND SELECTIVITY OF ANTI-HERPES COMPOUNDS

Several anti-herpes compounds, including phosphonoacetic acid, 9-(2-hydroxyethoxymethyl) guanine, 9-β-D-arabinofuranosyladenine, 1-β-D-arabinofuranosylthymine, 5-iodo-5′-amino-2;5′-dideoxyuridine, 5-iodo-2′-deoxyuridine, 5-bromo-2′-deoxycytidine and various other 5-substituted 2′-deoxyuridines and 2′-deoxycytidines were compared in vitro (primary rabbit kidney cells) for both antiviral activity (based on the ID50 required to inhibit the cytopathogenicity of the KOS strain of type 1 herpes simplex virus) and antimetabolic activity (based on the ID50 required to inhibit the incorporation of 2′-deoxyuridine into host cell DNA). Of the whole set of compounds tested, E-5-(2-bromovinyl)- and E-5-(2-iodovinyl)-2′-deoxyuridine emerged as both the most potent and the most selective anti-herpes agents.

Antiviral, antitumor, and thymidylate synthetase inhibition studies of 5-substituted styryl derivatives of 2′-deoxyuridine and their 5′-phosphates

Abstract 2′-Deoxyuridine derivatives containing styryl, 3-nitrostyryl, 4-nitrostyryl, and phenylethyl groups substituted at the 5-position of the pyrimidine ring have been evaluated for their effects on vaccinia and herpes simplex virus replication (in primary rabbit kidney cell cultures) and mouse leukemia L-1210 cell culture growth. 5-Phenylethyl-2′-deoxyuridine inhibited herpes simplex (type 1 and 2) virus-induced cytopathogenicity by 50 per cent at a dose ( id 50 ) of 10–30 μg/ml. It was inactive against tumor cell growth. The corresponding styryl derivative showed an id 50 of 30–70 μg/ml for herpes simplex virus, 20 μg/ml for vaccinia virus, and 280 μg/ml for L-1210 cell growth. 5(E)-(3-Azidostyryl)-2′-deoxyuridine 5′-phosphate inhibited vaccinia replication with an IC 50 of 20 μg/ml and L-1210 cell culture growth with an id 50 of 80 μg/ml. The nucleotides of these compounds were all potent reversible inhibitors of thymidylate synthetase ( Lactobacillus casei ) with the following K i K m ratios: 3-nitrostyryl, 0.035; 4-nitrostyryl, 0.05; 3-azidostyryl, 0.06; styryl, 0.08; and phenylethyl, 0.31. The photodecomposition of the azidostyryl derivative, a photoaffinity labeling reagent for thymidylate synthetase, was examined at two wavelengths.

Strategy for the synthesis of unsymmetrical N-substituted polyazamacrocycles

Abstract A convergent route is described for the preparation of unsymmetrical N-substituted polyammonium macrocycles that is potentially applicable for the synthesis of a wide variety of macrocycles of differing ring size and heteroatom substitution.

5-Nitro-2'-deoxyuridine 5'-monophosphate is a potent irreversible inhibitor of Lactobacillus caesi thymidylate synthetase.

Abstract 5-Nitro-2′-deoxyuridine 5′-monophosphate was found to be an active sitedirected irreversible inhibitor of thymidylate synthetase from Lactobacillus caesi . Its K I was determined as 2.9 × 10 −8 M from a double-reciprocal plot of velocity vs substrate concentration.

Reactions of formyl phosphate with linear and macrocyclic polyamines

Abstract The aminolysis of formyl phosphate by both linear and macrocyclic polyamines at pH 7 and 5°C was examined and found to proceed primarily by C-O cleavege to give the N-formylated amines. In the presence of excess bromide ion (ionic strength 0.3) the reaction was second-order with rate constants ranging from 7.1 to 12 liter m −1 min−1 for the macrocyclic amines 1,4,7,13,16,19-hexaaza-10,22-dioxacyclotetraeicosane ([24]N6O2, 1) and 1,4,7,10,13,16-hexaazacyclooctadecane ([18]N6, 4) and the linear amines 1,4,7,10,13-pentaazatridecane ([L]N5, 5) and 1,4,7,10-tetraazadecane ([L]N4, 6). In the absence of excess bromide ion the reaction was first-order with a rate constant of 0.30 min−1 for 1 and ranged from 0.26 to 0.54 min−1 for the other amines. The addition of a stable substrate analog (phosphonoacetaldehyde) to the reaction significantly decreased the rate of aminolysis. The reaction is proposed to proceed through a supramolecular complex of polyprotonated polyamine and formyl phosphate with a dissociation constant less than 1 × 10−3 m . A similar complex has been proposed for the reaction of acetyl phosphate and 1 which gives P-O, not C-O, cleavage. The rate as a function of pH for the aminolysis of formyl phosphate by 1 is reasonably constant in the mid-pH region and the rate decreases at the extremes of pH 4 and 9. The results of this study more fully characterize the activation of formate in neutral aqueous media by adenosine 5′-triphosphate in the presence of 1, a model for the reaction catalyzed by the enzyme N10-formyltetrahydrofolate synthetase for which formyl phosphate is a proposed intermediate.

Palladium(O) catalyzed coupling reactions in the synthesis of 5-arylpyrimidine nucleosides

Abstract The palladium(O) catalyzed coupling reaction of aryliodides and 3′,5′-di-O-acetyl-5-chloromercuri-2′-deoxyuridine (1) gave 5-aryl substituted 2′-deoxyuridines. Optimum yields were obtained in diglyme at 120° for 3 hours.

A novel intramolecular 1,3-dipolar cycloaddition reaction of a C-6 substituted uridine analog

Abstract A novel 1,3-dipolar cycloaddition reaction resulting in two multicyclic compounds was observed when 6-(4-butanal)-2′,3′-O-isopropylideneuridine ( 1 ) was reacted with tetrahydroisoquinoline in refluxing pyridine. The stereochemistries of the products were established by X-ray crystallography and NMR spectra.

The mechanism of formyl phosphate hydrolysis

Abstract The hydrolysis of formyl phosphate was studied in unbuffered and buffered solutions. In the absence of buffer the rate constant (8.8 × 10 −3 min −1 at 25°C and pH 7) varies little from about pH 5 to 8 and increases dramatically at low and high pH values. At pH 7 the hydrolysis appears to proceed via two mechanisms, nucleophilic attack on the carbonyl carbon and a mechanism involving PO bond cleavage. This is based on the facts that the reaction proceeds 45% by CO bond cleavage and 55% by PO bond cleavage (shown using H 2 18 O) ; there is a solvent isotope effect of 1.6; the Δ S ‡ (−11.8 e.u.) is intermediate between that expected for a unimolecular and a bimolecular reaction; the reaction rate is affected by organic solvent and buffer. At pH 1 and 11 the mechanism is entirely nucleophilic substitution at the carbonyl since the reaction proceeds 100% by CO cleavage, and the Δ S ‡ (−21.3 and −25.5 e.u.) is that expected for a bimolecular reaction. In the presence of Tris and glycine the formyl phosphate disappearance is accompanied by formylation of the primary amines with a 100% yield in the case of glycine. Imidazole and pyridine also catalyze formyl phosphate breakdown. In the former case the reaction proceeds primarily by CO cleavage, but no formylated product was observed. Differences in the hydrolyses of formyl phosphate and acetyl phosphate are discussed.