IMP dehydrogenase : structural aspects of inhibitor binding.

Inosine monophosphate dehydrogenase (IMPDH, E.C. 1.1.1.205) is recognized as an important target for both antileukemic and immunosuppressive therapy. IMPDH catalyzes the NAD-dependent oxidation of inosine 5 monophosphate (IMP) to xanthosine 5 monophosphate. Several classes of IMPDH inhibitors are now in use or under development. These include agents that bind at either the substrate site (e.g. ribavirin and mizoribine) or at the NAD site (mycophenolic acid and thiazole-4-carboxamide adenine dinucleotide). All suffer from some degree of toxicity and/or susceptibility to metabolic inactivation. The finding that IMPDH exists as two isoforms, one of which (type II) is induced in tumor cells, has led to the search for potentially more effective isoform-specific agents. Recently, a number of crystal structures of IMPDH have become available. These include structures of the human type II, hamster, Tritrichomonas foetus, Streptococcus pyogenes and Borrelia burgdorferi enzymes. Each structure crystallizes as a tetramer of a/b barrels, with the active site located partly at the monomer-monomer interface. The substrate and cofactor bind in a continuous cleft on the C-terminal face of each barrel. The IMP base is well positioned to stack against the NAD nicotinamide ring to facilitate hydride transfer. The active site cleft is further bounded by a highly flexible flap and loop. These structures reveal enzyme-ligand interactions which suggest strategies for the design of improved inhibitors.

Carboxamide group conformation in the nicotinamide and thiazole-4-carboxamide rings: implications for enzyme binding.

Ab initio computations (RHF/6-31G* parallel 3-21G*) were performed on the thiazole-4-carboxamide group found in the antitumor drug tiazofurin and its dehydrogenase-binding anabolite thiazole-4-carboxamide adenine dinucleotide (TAD). Results indicate that the carboxamide group is constrained in the conformation in which the amino group is cis-planar to the ring nitrogen. This finding is consistent with carboxamide conformations observed in crystal structures of the thiazole nucleosides. In contrast, ab initio computations on the nicotinamide and dihydronicotinamide rings found in the cofactors NAD+ and NADH indicate two stable conformations for the carboxamide group. This finding confirms previous computational studies and is consistent with results from a survey of the Cambridge Structural Database. Natural bond orbital analysis indicates that the low-energy carboxamide conformers of all three heterocycles are stabilized by a combination of electrostatic and charge transfer interactions. A survey of the Protein Data Bank indicates that the carboxamide group conformation in TAD is constrained to that favored by dehydrogenase-bound NAD(P)(H).

Analogues of platelet activating factor. 5. Multiple oxygen substitution of the alkoxy chain.

Racemic analogues of platelet activating factor (PAF) in which the alkoxy chain (R = -O(CH2)15CH3) has been replaced with a chain containing multiple ether linkages near the end (17, R = -O(CH2)8O(CH2)2O(CH2)2OCH3) or at the beginning (18, R = -O(CH2)2O(CH2)2O(CH2)9CH3) of the chain have been prepared. Both compounds exhibit reduced hypotensive and platelet aggregation responses compared to racemic C16-PAF (1a). This reduction in the biological activities is more apparent when the new oxygen atoms are located near the end of the chain. This substitution of additional oxygen atoms into the alkoxy chain does not result in any dramatic gains in selectivity of the biological responses.

Analogues of platelet activating factor. 3. Replacement of the phosphate moiety with a sulfonylbismethylene group.

An analogue of platelet activating factor (PAF) in which the phosphate moiety has been replaced with a sulfonylbismethylene group has been prepared. A key step in the synthetic sequence is the preparation of 4-[[3-(dimethylamino)propyl]thio]-1-(hexadecyloxy)-2-butanol via a one-pot reaction involving a sequential Michael addition and reduction. In comparison to racemic C16-PAF, 8 showed no platelet aggregating activity and substantially reduced hypotensive activity.

Ara-tiazofurin: conservation of structural features in an unusual thiazole nucleoside.

Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide, NSC 286193) is a C-glycosyl thiazole nucleoside with antitumor activity. Crystal structures of tiazofurin and its alpha,2'-deoxy and xylo analogues all show close contacts between the thiazole sulfur (S) and the furanose ring oxygen (O1'). These contacts have been interpreted in terms of an attractive intramolecular S-O interaction in the thiazole nucleosides. Ara-tiazofurin (2-beta-D-arabinofuranosylthiazole-4-carboxamide, ara-T) is the inactive arabinose analogue of tiazofurin. The crystal structure of ara-T is reported. This structure provides evidence for an attractive S-O interaction not seen in the other thiazole nucleosides. The conformation about the C-glycosyl bond in ara-T is such that close contacts are formed between the thiazole sulfur and both O1' and the 2'-hydroxyl oxygen O2'. This conformation is interpreted in terms of an additional attractive interaction between S and O2'. This interpretation is supported by comparison of the conformation of ara-T with those of other ara-nucleosides. These findings provide further evidence for an attractive S-O interaction in the thiazole nucleosides. Ara-T also demonstrates a second conformational feature found in these compounds: the carboxamide nitrogen remains cis to the thiazole nitrogen. Implications of these potentially constrained conformational features are discussed in terms of the mechanism of activity of tiazofurin.

Dehydrogenase binding by tiazofurin anabolites.

Thiazole-4-carboxamide adenine dinucleotide (TAD) is the active anabolite of the new antitumor agent tiazofurin (NSC 286193). TAD is an analogue of NAD in which the nicotinamide ring has been replaced by a thiazole-4-carboxamide heterocycle. TAD putatively acts by inhibition of inosine monophosphate dehydrogenase (IMPd). In this study it is shown that TAD is a competitive inhibitor, with respect to NAD, of mammalian glutamate, alcohol, lactate, and malate dehydrogenases. TAD binds to these enzymes with 1-2 orders of magnitude less affinity than it binds to IMPd. Computer modeling studies suggest that dehydrogenase binding by TAD occurs at the regular cofactor site, the thiazole-4-carboxamide group mimicking the steric and hydrogen-bonding properties of the nicotinamide ring. Noncompetitive kinetics of TAD inhibition of the target enzyme IMPd are potentially due to a reverse order of addition of substrate and cofactor from that observed in the dehydrogenases studied here. The weaker binding of TAD to these dehydrogenases may be due to their inability to preserve a close sulfur-oxygen contact in the bound inhibitor.

Potent inhibitors of human inosine monophosphate dehydrogenase type II. Fluorine-substituted analogues of thiazole-4-carboxamide adenine dinucleotide.

Three analogues of thiazole-4-carboxamide adenine dinucleotide (TAD) (1-3) containing a fluorine atom at the C2' of the adenine nucleoside (in the ribo and arabino configuration) and at the C3' (in the ribo configuration) were synthesized in high yield from the corresponding 5'-monophosphates of 2'-deoxy-2'-fluoroadenosine (9), 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine (17), and 3'-deoxy-3'-fluoroadenosine (14), respectively. Pure 2',3'-O-isopropylidene-tiazofurin 5'-phosphorimidazolide (8) was obtained by phosphorylation of the protected tiazofurin followed by treatment with carbonyldiimidazole and HPLC purification. Reaction of 8 with 9 in DMF-d7 (monitored by 1H and 31P NMR) afforded the desired dinucleotide 12, which after deisopropylidenation gave 1 in 82% yield. Small amounts of symmetrical dinucleotides AppA (10, 7.2%) and TRppTR (11, 8.0%) were also isolated during HPLC purification of the major product 12. In a similar manner, compounds 2 and 3 were obtained by coupling of 8 with 14 and 17 in 80% and 76% yield, respectively. All newly prepared fluoro-substituted compounds as well as beta-CF2-TAD, earlier synthesized by us, showed good inhibitory activity against inosine monophosphate dehydrogenase type II, the isozyme which is predominant in neoplastic cells. Binding of 1 (Kis = 0.5 microM), 2 (Kis = 0.7 microM), and 3 (Kis = 2.9 microM) was comparable to that of TAD (Ki = 0.2 microM). The difluoromethylene bisphosphonate analogue, beta-CF2-TAD (Ki = 0.17 microM), was found to be equally effective as the best cofactor-type inhibitor, beta-CH2-TAD (Ki = 0.11 microM). Interestingly, the level of inhibition of horse liver alcohol dehydrogenase by these compounds was found to be much lower (0.1 mM for 1 and 2 and no inhibition up to 10 mM for 3). These findings show that inhibition of tumor-induced inosine monophosphate dehydrogenase type II is selective and may be of therapeutic interest.

Analogues of platelet activating factor. 4. Some modifications of the phosphocholine moiety.

Racemic analogues of platelet activating factor (PAF) in which the methylene bridge separating the phosphate and trimethylammonium moieties is altered in length (7a-f) have been prepared. Increasing the length of this bridge results in a progressive decrease in the hypotensive and platelet aggregation responses. Analogues in which the phosphocholine group is substituted with a methyl group (7h and 7i) or a phenyl group (5j) or in which the methylene bridge is replaced with a meta-substituted benzyl group (5k) have been prepared. With respect to both the blood pressure and platelet aggregation responses, 7i and 5k showed little if any changes in potency compared to racemic C16-PAF (1a). While 7h is more potent than 1a with respect to both the hypotensive and platelet aggregation properties, 5j is less potent. Replacement of the phosphate moiety of C18-PAF (1b) with a phosphonate group (7g) leads to decreased activity in both assays. Analogue 11, in which the phosphocholine group has been replaced with a 4-(trimethylammonio)butoxy group, exhibited no detectable hypotensive or platelet aggregating activity. None of the analogues exhibited a separation of the blood pressure and platelet aggregation activities.

Analogues of platelet activating factor (PAF). 1. Some modifications of the alkoxy chain.

Analogues of platelet activating factor (PAF) in which the ether oxygen has been removed (6) and in which the alkoxy chain at C1 has been replaced with a o-, p-, or m-alkylphenoxy group (30, 31, and 35, respectively) have been prepared. Compound 6 shows reduced platelet aggregation and hypotensive activity in comparison with C16 and C18 PAF. The results obtained for compounds 30, 31, and 35 indicate that the hypotensive and platelet aggregating responses are sensitive to structural modification of the ether chain. The ortho analogue 30 shows no platelet aggregating activity and only a weak hypotensive response. The para analogue 31 exhibits a moderate decrease in activity in both assays. The meta analogue 35 is the most active of the three.

Chemical synthesis of benzamide adenine dinucleotide: inhibition of inosine monophosphate dehydrogenase (types I and II).

Treatment of 3-(2,3-O-isopropylidene-beta-D-ribofuranosyl)benzamide (6) with POCl3 in (EtO)3-PO afforded only little phosphorylation product (8, 5%), but the major product was 5'-chlorobenzamide riboside (7, 85%). Reaction of 6 with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite followed by 2-cyanoethanol/tetrazole treatment and oxidation with tert-butyl peroxide gave a 1:1 mixture of the desired 5'-O-bis(2-cyanoethyl) phosphate 9 and the chloro derivative 7. This mixture was treated with methanolic ammonia and partitioned between CHCl3 and water. The 2',3'-O-isopropylidenebenzamide mononucleotide (8) was obtained in 21.2% overall yield from the aqueous layer. Compound 8 was then converted into the corresponding imidazolide 11b which, upon coupling with 2',3'-O-acetonide of AMP, afforded the acetonide of benzamide adenine dinucleotide (15) in 94% yield together with small amounts of symmetrical pyrophosphates P1,P2-bis(2',3'-O-isopropylideneadenosin-5'-yl)pyrophosphate (13, 3%) and P1,P2-bis(2',3'-O-isopropylidene-3-(carbamoylphenyl)-5'-ribosyl)py rophosphate (14, 2%). Deprotection of 15 with Dowex 50/H+ in water afforded the desired benzamide adenine dinucleotide (BAD) in 93% yield. BAD inhibits inosine monophosphate dehydrogenase type I (IC50 = 0.78 microM) and type II (IC50 = 0.88 microM) with same degree of potency.

Analogues of platelet activating factor (PAF). 2. Some modifications of the glycerine backbone.

Racemic analogues of platelet activating factor (PAF) that contain a methylene group between the C2 and C3 carbon atoms (39) or between the C1 and C2 carbon atoms (40) have been synthesized. These compounds show reduced platelet aggregation and hypotensive activity as measured against racemic C16 PAF. Compounds in which the C1 carbon atom of PAF is substituted with one or two methyl groups (41 and 42, respectively) or the C3 carbon is substituted with a single methyl group (43) have been synthesized. Platelet aggregation and hypotensive responses produced by these compounds are significantly less than those obtained with racemic C16 PAF. None of the above compounds exhibit a separation of the platelet aggregation and hypotensive activities.

C-glycosyl bond conformation in oxazofurin: crystallographic and computational studies of the oxazole analogue of tiazofurin.

Oxazofurin is the inactive oxazole analogue of the C-glycosyl thiazole antitumor agent tiazofurin. Replacement of the thiazole sulfur in tiazofurin with the oxazole oxygen in oxazofurin produces conformational effects that are examined using crystallographic and computational methods. The crystal structure of oxazofurin contains six molecules in the asymmetric unit and has been refined to a standard R value of 6.8% for all data. The six oxazofurin conformers show an average C-glycosidic torsion angle of 70(9) degrees. This value is significantly higher than the average absolute C-glycosidic torsion angle of 24(10) degrees obtained from previous thiazole nucleoside structures. Previous studies suggest that, in tiazofurin, an electrostatic interaction between a positively charged thiazole sulfur and negatively charged furanose oxygen constrains the C-glycosidic torsion angle to a relatively small value. Ab initio molecular orbital studies presented here suggest that the higher C-glycosidic angles observed in the oxazofurin structures result from a repulsive interaction between negatively charged oxazole and furanose oxygens. Thus, it is likely that differences in activity between oxazo- and tiazofurin are either (1) due directly to differences in electronic properties between the thiazole and oxazole rings or (2) due to the variation in C-glycosidic bond conformation resulting from the alteration in the charge distribution of the heterocycle.

Furanfurin and thiophenfurin: two novel tiazofurin analogues. Synthesis, structure, antitumor activity, and interactions with inosine monophosphate dehydrogenase.

The syntheses of furan and thiophene analogues of tiazofurin (furanfurin and thiophenfurin, respectively) are described. Direct stannic chloride-catalyzed C-glycosylation of ethyl 3-furan-carboxylate (6) or ethyl 3-thiophencarboxylate (18) with 1,2,3,5-tetra-O-acetyl-D-ribofuranose gave 2- and 5-glycosylated regioisomers, as a mixture of alpha- and beta-anomers, and the beta-2,5-diglycosylated derivatives. Deprotection of ethyl 5-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)furan-3-carboxylate (9 beta) and ethyl 5-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)thiophene-3-carboxylate (20 beta) with sodium ethoxide afforded ethyl 5-beta-D-ribofuranosylfuran-3-carboxylate (12 beta) and ethyl 5-beta-D-ribofuranosylthiophene-3-carboxylate (23 beta) which were converted into 5-beta-D-ribofuranosylfuran-3-carboxamide (furanfurin, 4) and 5-beta-D-ribofuranosylthiophene-3-carboxamide (thiophenfurin, 5) by reaction with ammonium hydroxide. The anomeric configuration and the site of glycosylation were established by 1H-NMR and proton-proton nuclear Overhauser effect difference spectroscopy. The structure of compound 23 beta was confirmed by X-ray crystallography. Thiophenfurin was found to be cytotoxic in vitro toward murine lymphocytic leukemia P388 and L1210, human myelogenous leukemia K562, human promyelocytic leukemia HL-60, human colon adenocarcinoma LoVo, and B16 melanoma at concentrations similar to that of tiazofurin. In the same test furanfurin proved to be inactive. Thiophenfurin was found active in vivo in BD2F1 mice inoculated with L1210 cells with a % T/C of 168 at 25 mg/kg. K562 cells incubation with thiophenfurin resulted in inhibition of inosine monophosphate (IMP) dehydrogenase (63%) and an increase in IMP pools (6-fold) with a concurrent decrease in GTP levels (42%). Incubation of adenosine-labeled K562 cells with tiazofurin, thiophenfurin, and furanfurin resulted in a 2-fold higher NAD analogue formulation by thiophenfurin than by tiazofurin. Furanfurin was converted to the NAD analogue with only 10% efficiency. The results obtained support the hypothesis that the presence of S in the heterocycle in position 2 with respect to the glycosidic bond is essential for the cytotoxicity and IMP dehydrogenase activity of tiazofurin, while the N atom is not.

Synthesis of isosteric analogues of nicotinamide adenine dinucleotide containing C-nucleotide of nicotinamide or picolinamide.

Two isosteric analogues of nicotinamide adenine dinucleotide, C-NAD (11) and C-PAD (12), in which the nicotinamide riboside portion is replaced by a C-nucleoside, were synthesized from 5-(beta-D-ribofuranosyl)nicotinamide (7) and 6-(beta-D-ribofuranosyl)picolinamide (8), respectively. Nucleoside 7 was prepared from the 2,3-O-isopropylidene-5-O-(tetrahydropyranyl)-D-ribonolactone (13) and 3-cyano-5-lithiopyridine as reported earlier. Nucleoside 8 was obtained by conversion of the bromo function of the 6-(2,3:4,5-di-O-isopropylidene-D-altro-pentitol-1-yl)-2-bromopyrid ine (14) into a carboxamido group followed by mesylation of the anomeric hydroxyl group to give derivative 18. Treatment of 18 with CF3COOH/CHCl3 caused deisopropylidenation with simultaneous cyclization into the desired 6-(beta-D-ribofuranosyl)picolinamide (8). NAD analogues, C-NAD (11) and C-PAD (12), were synthesized by imidazole-catalyzed coupling of the corresponding 5'-monophosphates of 7 and 8 with the adenosine-5'-monophosphate. Dinucleotide 11 was found to inhibit the proliferation of L1210 cells (IC50 = 7 microM) and to be a good competitive inhibitor of inosine monophosphate dehydrogenase (IMPDH, ID50 = 20 microM) as well as bovine glutamate dehydrogenase (GDH, Ki = 15 microM). Interestingly, C-NAD (11) caused extremely potent noncompetitive inhibition of horse liver alcohol dehydrogenase (ADH, Ki = 1.1 nM), whereas C-PAD (12) was found to be a much less potent competitive inhibitor (Ki = 20 microM) of ADH.

The practical synthesis of a methylenebisphosphonate analogue of benzamide adenine dinucleotide: inhibition of human inosine monophosphate dehydrogenase (type I and II).

beta-Methylene-BAD (8), a nonhydrolyzable analogue of benzamide adenine dinucleotide (BAD), was synthesized as potential inhibitor of human inosine monophosphate dehydrogenase (IMPDH). Treatment of 2',3'-O-isopropylideneadenosine 5'-methylenebisphosphonate (15) with DCC afforded P1,P4-bis(2',3'-O-isopropylideneadenosine) 5'-P1,P2:P3,P4-dimethylenetetrakisphosphonate (17). This compound was further converted with DCC to an active intermediate 18 which upon reaction with 3-(2',3'-O-isopropylidene-beta-D-ribofuranosyl)benzamide (19) gave, after hydrolysis and deisopropylidenation, the desired beta-methylene-BAD (8) in 95% yield. In a similar manner, treatment of 18 with 2',3'-O-isopropylidenetiazofurin (21) followed by hydrolysis and deprotection afforded beta-methylene-TAD (5) in 91% yield. Compound 8 (IC50 = 0.665 microM) was found to be a 6-8 times less potent inhibitor of IMPDH than 5 (IC50 = 0.107 microM) and was almost equally potent against IMPDH type I and type II. Although TAD and beta-methylene-TAD were bound by LADH with the same affinity, the binding affinity of 8 toward LADH (Ki = 333 microM) was found to be 50-fold lower than that of the parent pyrophosphate 7 (Ki = 6.3 microM).

Synthesis, conformational analysis, and biological activity of C-thioribonucleosides related to tiazofurin.

The syntheses of furanthiofurin [5beta-D-(4'-thioribofuranosyl)furan-3-carboxamide, 1] and thiophenthiofurin [5beta-D-(4'-thioribofuranosyl)thiophene-3-carboxamide, 2], two C-thioribonucleoside analogues of tiazofurin, are described. Direct trifluoroacetic acid-catalyzed C-glycosylation of ethyl furan-3-carboxylate with 1-O-acetyl-2,3,5-tri-O-benzyl-4-thio-D-ribofuranose gave 2- and 5-glycosylated regioisomers, as a mixture of alpha and beta anomers. Ethyl 5-(2,3,5-tri-O-benzyl)-beta-D-(4'-thioribofuranosyl)furan-3-carboxylate (6beta) was debenzylated and then converted into the corresponding amide (furanthiofurin) by reaction with ammonium hydroxide. A similar C-glycosylation of ethyl thiophene-3-carboxylate with 1,2,3,5-tetra-O-acetyl-4-thio-D-ribofuranose catalyzed by stannic chloride afforded an anomeric mixture of 2- and 5-glycosylated regioisomers. Deacetylation of ethyl 5-(2,3,5-tri-O-acetyl)-beta-D-(4'-thioribofuranosyl)thiophene-3-carboxylate (13beta) with methanolic ammonia and treatment of the ethyl ester with ammonium hydroxide gave thiophenthiofurin. The glycosylation site and anomeric configuration were established by (1)H NMR spectroscopy. Thiophenthiofurin was found to be cytotoxic in vitro toward human myelogenous leukemia K562, albeit 39-fold less than thiophenfurin, while furanthiofurin proved to be inactive. K562 cells incubated with thiophenthiofurin resulted in inhibition of inosine 5'-monophosphate dehydrogenase (IMPDH) and an increase in IMP pools with a concurrent decrease in GTP levels. From computational studies it was deduced that, among the C-nucleoside analogues of tiazofurin, activity requires an electrophilic sulfur adjacent to the C-glycosidic bond and an energetically favorable conformer around chi = 0 degrees. Among these, the more constrained (least flexible) compounds (tiazofurin and thiophenfurin) are more active than the less constrained thiophenthiofurin. Those compounds which contain a nucleophilic oxygen in place of the thiazole or thiophene (oxazofurin, furanfurin, and furanthiofurin) show the least activity.

Synthesis of nonhydrolyzable analogues of thiazole-4-carboxamide and benzamide adenine dinucleotide containing fluorine atom at the C2' of adenine nucleoside: induction of K562 differentiation and inosine monophosphate dehydrogenase inhibitory activity.

Thiazole-4-carboxamide adenine dinucleotide (TAD) analogue 7 containing a fluorine atom at the C2' arabino configuration of the adenine nucleoside moiety was found to be a potent inducer of differentiation of K562 erythroid leukemia cells. This finding prompted us to synthesize its hydrolysis-resistant methylenebis(phosphonate) and difluoromethylenebis(phosphonate) analogues 8 and 9, respectively. Since both TAD and benzamide adenine dinucleotide (BAD) are potent inhibitors of inosine monophosphate dehydrogenase (IMPDH), the corresponding fluorine-substituted methylenebis(phosphonate) analogue 12 of BAD was also synthesized. Thus, 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine (13) was converted in five steps into the corresponding methylenebis(phosphonate) analogue 18. Dehydration of 18 with DCC led to the formation of the bicyclic trisanhydride intermediate 19a, which upon reaction with 2',3'-O-isopropylidenetiazofurin (20) or -benzamide riboside (21) followed by hydrolysis and deprotection afforded the desired methylene-bridged dinucleotides 8 and 12, respectively. The similar displacement of the 5'-mesyl function of 2',3'-O-isopropylidene-5'-O-mesyltiazofurin (24) with the difluoromethylenebis(phosphonic acid) derivative gave the phosphonate 25 which was coupled with 13 to afford 26. The desired difluoromethylenebis(phosphonate) analogue 9 was obtained by deprotection with Dowex 50/H+. This compound as well as beta-CF2-TAD (4) showed improved differentiation-inducing activity over beta-CH2-TAD (3), whereas analogues containing the -CH2-linkage (8 and 12) were inactive.

Synthesis of a methylenebis(phosphonate) analogue of mycophenolic adenine dinucleotide: a glucuronidation-resistant MAD analogue of NAD.

Mycophenolic alcohol (MPAlc), obtained by reduction of the carboxylic group of mycophenolic acid (MPA), was coupled with 2',3'-O-isopropylideneadenosine 5'-methylenebis(phosphonate) (4) in the presence of diisopropylcarbodiimide (DIC) to give P1-(2',3'-O-isopropylideneadenosin-5'-yl)-P2-(mycophenolic alcohol-6'-yl)methylenebis(phosphonate) (8) in 32% yield. Deisopropy-lidenation of 8 with CF3COOH/H2O afforded the methylenebis(phosphonate) analogue 3 of mycophenolic adenine dinucleotide (MAD). Compound 3, beta-methylene-MAD, was found to be a potent inhibitor of inosine monophosphate dehydrogenase (IMPDH) type II (Ki = 0.3 microM) as well as an inhibitor of growth of K562 cells (IC50 = 1.5 microM). In contrast to MPA and mycophenolic alcohol, beta-methylene-MAD was not converted into the glucuronide when incubated with uridine 5'-diphosphoglucuronyltransferase.

CNAD: a potent and specific inhibitor of alcohol dehydrogenase.

CNAD (5-beta-D-ribofuranosylnicotinamide adenine dinucleotide) is an isosteric and isomeric analogue of NAD, in which the nicotinamide ring is linked to the sugar via a C-glycosyl (C5-C1') bond. CNAD acts as a general dehydrogenase inhibitor but shows unusual specificity and affinity for liver alcohol dehydrogenase (ADH, EC 1.1.1.1). The pattern of inhibition is congruent to 4 nM, with NAD as the variable substrate. These values are 3-5 orders of magnitude smaller than those obtained for CNAD in other dehydrogenases and are comparable to values observed for the tightest binding ADH inhibitors known. The specificity and affinity of CNAD for ADH are likely due to coordination of the zinc cation at the ADH catalytic site by the CNAD pyridine nitrogen. This is supported by kinetic and computational studies of ADH-CNAD complexes. These results are compared with those for a related analogue, CPAD. In this analogue, displacement of the pyridine nitrogen to the opposite side of the ring removes the specificity for ADH.

Synthesis of 3-deazaneplanocin A, a powerful inhibitor of S-adenosylhomocysteine hydrolase with potent and selective in vitro and in vivo antiviral activities.

The neplanocin A analogue 3-deazaneplanocin A (2b) has been synthesized. A direct SN2 displacement on the cyclopentenyl mesylate 3 by the sodium salt of 6-chloro-3-deazapurine afforded the desired regioisomer 4 as the major product. After deprotection, this material was converted to 3-deazaneplanocin A in two steps. X-ray crystallographic analysis confirmed the assigned structure. Consistent with its potent inhibition of S-adenosylhomocysteine hydrolase, 3-deazaneplanocin A displayed excellent antiviral activity in cell culture against vesicular stomatitis, parainfluenza type 3, yellow fever, and vaccinia viruses. Antiviral activity was also displayed in vivo against vaccinia virus by using a mouse tailpox assay. The significantly lower cytotoxicity of 3-deazaneplanocin A, relative to its parent compound neplanocin A, may be due to its lack of conversion to 5'-triphosphate and S-adenosylmethionine metabolites.