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Fig. 1. Chemical structures of ribavirin and derivatives

2',3'-Dideoxy-2',3'-didehydroribavirin (d4R), 2',3'-dideoxyribavirin (ddR), and 2',3'-anhydro-ribavirin (epoxyR) (Fig. 1) were synthesized from ribavirin according to the procedures described by Upadhya et al. (1990). 2'-Deoxyribavirin (2'dR) (Pochet and Dugué, 199 and 3'-deoxyribavirin (3'dR) (Upadhya et al., 1990) were obtained in four steps by radical deoxygenation of appropriately bis-silylated ribavirin at position 2' and 3', respectively, as described for the preparation of the 3'-deoxyadenosine (Meier and Huynh-Dinh, 1991). Conversion of ribavirin and analogs into their corresponding triphosphate was performed according to the method of Ludwig and Eckstein (1989). Spectra from electrospray ionization mass spectrometry were recorded in the negative-ion mode. HPLC analyses were performed on a PerkinElmer series 200 unit using a reverse-phase analytical column (Uptisphere UPS HDO, C18, 120 Å, 4.6 × 250 mm, 5 µm; Interchim, Montluçon, France) with detection at 230 nm. The eluants used were acetonitrile/10 mM triethylammonium acetate buffer, pH 6.6, 1/19 (A) and acetonitrile/10 mM triethylammonium acetate buffer, pH 6.6, 3/17 (B). A linear gradient from A to B over 20 min was used with a flow rate of 1 ml/min. Characteristic data for the target compounds are as follows: ribavirin 5'-triphosphate: HPLC, 4.91 min; C8H15N4O14P3 (483.9 m/z 483 (M-H); 2'-deoxyribavirin 5'-triphosphate: HPLC, 5.56 min; C8H15N4O13P3 (467.9 m/z 467 (M-H); 3'-deoxyribavirin 5'-triphosphate: HPLC, 4.87 min; C8H15N4O13P3 (467.9 m/z 467 (M-H); 2',3'-dideoxy-2',3'-didehydroribavirin 5'-triphosphate: HPLC, 5.99 min; C8H11N4O12P3 (449.97) m/z 449 (M-H); 2',3'-dideoxyribavirin 5'-triphosphate: HPLC, 6.01 min, C8H15N4O12P3 (451.99) m/z 451 (M-H); and 2',3'-anhydroribavirin 5'-triphosphate: HPLC, 5.79 min; C8H13N4O13P3 (465.97) m/z 465 (M-H).



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Chemistry

Physically ribavirin is similar to the sugar D-ribose from which it is derived. It is freely soluble in water, and is re-crystallized as fine silvery needles from boiling methanol. It is only sparingly soluble in anhydrous ethanol.

Classically ribavirin is prepared from natural D-ribose by blocking the 2', 3' and 5' OH groups with benzyl groups, then derivatizing the 1' OH with an acetyl group which acts as a suitable leaving group upon nucleophilic attack. The ribose 1' carbon attack is accomplished with 1,2,4 triazole-3-carboxymethyl ester, which directly attaches the 1' nitrogen of the triazole to the 1' carbon of the ribose, in the proper 1-β-D isomeric position. The bulky benzyl groups hinder attack at the other sugar carbons. Following purification of this intermediate, treatment with ammonia in methanolic conditions then simultaneously deblocks the ribose hydroxyls, and converts the triazole carboxymethyl ester to the carboxamide. Following this step, ribavirin may be recovered in good quantity by cooling and crystallization.

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Derivatives

Ribavirin is possibly best viewed as a ribosyl purine analogue with an incomplete purine 6-membered ring. This structural resemblance historically prompted replacement of the 2' nitrogen of the triazole with a carbon (which becomes the 5' carbon in an imidazole), in an attempt to partly "fill out" the second ring--- but to no great effect. Such 5' imidazole riboside derivatives show antiviral activity with 5' hydrogen or halide, but the larger the substituent, the smaller the activity, and all proved less active than ribavirin.[2] Note that two natural products were already known with this imidazole riboside structure: substitution at the 5' carbon with OH results in pyrazomycin/pyrazofurin, an antibiotic with antiviral properties but unacceptable toxicity, and replacement with an amino group results in the natural purine synthetic precursor 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR), which has only modest antiviral properties.

Derivatization of the triazole 5' carbon, or replacement of it with a nitrogen (i.e., the 1,2,4,5 tetrazole 3-carboxamide) also results in substantial loss of activity, as does alkyl derivatization of the 3' carboxamide nitrogen.

The 2' deoxyribose version of ribavirin (the DNA nucleoside analogue) is not active as an antiviral, suggesting strongly that ribavirin requires RNA-dependent enzymes for its antiviral activity.

Antiviral activity is retained for acetate and phosphate derivation of the ribose hydroxyls, including the triphosphate and 3', 5' cyclic phosphates, but these compounds are no more active than the parent molecule, reflecting the high efficiency of esterase and kinase activity in the body.