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Novel mycophenolic adenine dinucleotide (MAD) analogues have been prepared as potential inhibitors of inosine monophosphate dehydrogenase (IMPDH). MAD analogues resemble nicotinamide adenine dinucleotide binding at the cofactor binding domain of IMPDH; however, they cannot participate in hydride transfer and therefore inhibit the enzyme. The methylenebis(phosphonate) analogues C2-MAD and C4-MAD were obtained by coupling 2‘,3‘-O-isopropylideneadenosine 5‘-methylenebis(phosphonate) (22) with mycophenolic alcohols 20 and 21 in the presence of diisopropylcarbodiimide followed by deprotection. C2-MAD was also prepared by coupling of mycophenolic methylenebis(phosphonate) derivative 30 with 2‘,3‘-O-isopropylideneadenosine. Compound 30 was conveniently synthesized by the treatment of benzyl-protected mycophenolic alcohol 27 with a commercially available methylenebis(phosphonic dichloride) under Yoshikawa's reaction conditions. C2-MAD and C4-MAD were found to inhibit the growth of K562 cells (IC50 = 0.7 μM and IC50 = 0.1 μM, respectively) as potently as mycophenolic acid (IC50 = 0.3 μM). In addition, C2-MAD and C4-MAD triggered vigorous differentiation of K562 cells an order of magnitude more potently than tiazofurin, and MAD analogues were resistant to glucuronidation in vitro. These results show that C2-MAD and C4-MAD may be of therapeutic interest in the treatment of human leukemias.

The syntheses of furanthiofurin [5β-d-(4‘-thioribofuranosyl)furan-3-carboxamide, 1] and thiophenthiofurin [5β-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 α and β anomers. Ethyl 5-(2,3,5-tri-O-benzyl)-β-d-(4‘-thioribofuranosyl)furan-3-carboxylate (6β) 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)-β-d-(4‘-thioribofuranosyl)thiophene-3-carboxylate (13β) with methanolic ammonia and treatment of the ethyl ester with ammonium hydroxide gave thiophenthiofurin. The glycosylation site and anomeric configuration were established by ¹H 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 χ = 0°. 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.

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. Deisopropylidenation of 8 with CF3COOH/H2O afforded the methylenebis(phosphonate) analogue 3 of mycophenolic adenine dinucleotide (MAD). Compound 3, β-methylene-MAD, was found to be a potent inhibitor of inosine monophosphate dehydrogenase (IMPDH) type II (Ki = 0.3 μM) as well as an inhibitor of growth of K562 cells (IC50 = 1.5 μM). In contrast to MPA and mycophenolic alcohol, β-methylene-MAD was not converted into the glucuronide when incubated with uridine 5‘-diphosphoglucuronyltransferase.

Treatment of 3-(2,3-O-isopropylidene-β-d-ribofuranosyl)benzamide (6) with POCl3 in (EtO)3PO 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)pyrophosphate (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 μM) and type II (IC50 = 0.88 μM) with same degree of potency.

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-β-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 β-CF2-TAD (4) showed improved differentiation-inducing activity over β-CH2-TAD (3), whereas analogues containing the −CH2− linkage (8 and 12) were inactive.

The synthesis and biological activity of selenophenfurin (5-β-d-ribofuranosylselenophene-3-carboxamide, 1), the selenophene analogue of selenazofurin, are described. Glycosylation of ethyl selenophene-3-carboxylate (6) under stannic chloride-catalyzed conditions gave 2- and 5-glycosylated regioisomers, as a mixture of α- and β-anomers, and the β-2,5-diglycosylated derivative. Deprotected ethyl 5-β-d-ribofuranosylselenophene-3-carboxylate (12β) was converted into selenophenfurin by ammonolysis. The structure of 12β was determined by ¹H- and ¹³C-NMR, crystallographic, and computational studies. Selenophenfurin proved to be antiproliferative against a number of leukemia, lymphoma, and solid tumor cell lines at concentrations similar to those of selenazofurin but was more potent than the thiophene and thiazole analogues thiophenfurin and tiazofurin. Incubation of K562 cells with selenophenfurin resulted in inhibition of IMP dehydrogenase (IMPDH) (76%) and an increase in IMP pools (14.5-fold) with a concurrent decrease in GTP levels (58%). The results obtained confirm the hypothesis that the presence of heteroatoms such as S or Se in the heterocycle in position 2 with respect to the glycosidic bond is essential for both cytotoxicity and IMP dehydrogenase inhibitory activity in this type of C-nucleosides.

β-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-β-d-ribofuranosyl)benzamide (19) gave, after hydrolysis and deisopropylidenation, the desired β-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 β-methylene-TAD (5) in 91% yield. Compound 8 (IC50 = 0.665 μM) was found to be a 6−8 times less potent inhibitor of IMPDH than 5 (IC50 = 0.107 μM) and was almost equally potent against IMPDH type I and type II. Although TAD and β-methylene-TAD were bound by LADH with the same affinity, the binding affinity of 8 toward LADH (Ki = 333 μM) was found to be 50-fold lower than that of the parent pyrophosphate 7 (Ki = 6.3 μM).