Synthesis of Azole Nucleoside 5′-Monophosphate Mimics (P1Ms) and Their Inhibitory Properties of IMP Dehydrogenases

Article abstract of DOI:10.1081/NCN-120027838

IMPDH inhibitors have potential antimicrobial, anticancer and immunomodulatory effects. Nucleoside inhibitors of IMPDH exert their inhibitory effects via nucleoside 5′-MPs. Conversion of nucleoside analogs to NMPs by cellular nucleoside kinases is not ass

Full text of DOI:10.1081/NCN-120027838

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Synthesis of Azole Nucleoside 5′‐Monophosphate
Mimics (P1Ms) and Their Inhibitory Properties of IMP
Dehydrogenases
Guangyi Wang a , Kandasamy Sakthivel a , Vasanthakumar Rajappan a , Thomas W. Bruice
a , Kathleen Tucker a , Patrick Fagan a , Jennifer L. Brooks a , Tiffany Hurd a , Janet M.
Leeds a & P. Dan Cook a
a Biota, Inc., Carlsbad, California, 92008, USA
Version of record first published: 01 Jun 2007.
To cite this article: Guangyi Wang , Kandasamy Sakthivel , Vasanthakumar Rajappan , Thomas W. Bruice , Kathleen
Tucker , Patrick Fagan , Jennifer L. Brooks , Tiffany Hurd , Janet M. Leeds & P. Dan Cook (2004): Synthesis of Azole
Nucleoside 5′‐Monophosphate Mimics (P1Ms) and Their Inhibitory Properties of IMP Dehydrogenases , Nucleosides,
Nucleotides and Nucleic Acids, 23:1-2, 317-337
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 23, Nos. 1 & 2, pp. 317–337, 2004
Synthesis of Azole Nucleoside 5’-Monophosphate
Mimics (P1Ms) and Their Inhibitory Properties
of IMP Dehydrogenasesy
*
Guangyi Wang, Kandasamy Sakthivel, Vasanthakumar Rajappan,
Thomas W. Bruice, Kathleen Tucker, Patrick Fagan, Jennifer L. Brooks,
Tiffany Hurd, Janet M. Leeds, and P. Dan Cook
Biota, Inc., Carlsbad, California, USA
ABSTRACT
IMPDH inhibitors have potential antimicrobial, anticancer and immunomodulatory
effects. Nucleoside inhibitors of IMPDH exert their inhibitory effects via nucleoside
5’-MPs. Conversion of nucleoside analogs to NMPs by cellular nucleoside kinases is
not assured, and usually is inefficient. In order to bypass cellular phosphorylation, a
series of azole nucleoside 5’-MP mimics (P1Ms) based on ribavirin, EICAR and
bredinin were synthesized and screened against human and C. albicans IMP
dehydrogenises. P1Ms 8, 16, 25, 28 and 29 demonstrated substantial IMPDH
inhibition with Ki values in low micromolar range.
Key Words: Azole nucleoside; Monophosphate mimic; P1M; IMPDH.
yIn honor and celebration of the 70th birthday of Professor Leroy B. Townsend.
*
Correspondence: Guangyi Wang, Biota, Inc., Carlsbad, CA 92008, USA; E-mail: g.wang@biota-
inc.com.
317
DOI: 10.1081/NCN-120027838
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com
Copyright D 2004 by Marcel Dekker, Inc.

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INTRODUCTION
Inosine 5’-monophosphate dehydrogenases (IMPDH) catalyze the NAD-dependent
oxidation of IMP to XMP, which is the rate-limiting step of the de novo guanine
nucleotide biosynthesis. Human IMPDH exists in two isoforms. Type I isoform is
constitutively expressed and is the predominant isoform in normal cells. Type 2
isoform is selectively up-regulated in neoplastic and replicating cells and becomes the
predominant isoform.[1,2] There is great interest in selective inhibition of the Type 2
isoform for more potent and less toxic cancer therapeutics. Inhibition of IMPDH
effectively depletes guanine nucleotide pools in T cells and B cells that primarily
depend on the de novo biosynthesis for guanine nucleotides, and thereby results in
immunosuppression, which is useful in organ transplantation and for treatment of
autoimmune diseases.[3]
Two IMPDH binding sites are known to be of importance for drug targeting, the
substrate site (IMP site) and the cofactor site (NAD site). Among the potent
inhibitors[4–6] of the NAD site are mycophenolic acid, VX-497 and TAD analogs.
Mycophenolate mofetil, an ester prodrug of mycophenolic acid, has been used
clinically as an immunosuppressive drug.[7] Among the potent inhibitors[4,10,11] of the
IMP site are the 5’-monophosphates of ribavirin, bredinin and EICAR (Figure 1).
Ribavirin is a prescription drug for treatment of RSV and HCV infections. Ribavirin 5’-
MP (1) inhibits IMPDH at sub-micromolar concentrations, which is believed to
contribute, at least partially, to the antiviral activities.[8,9] Bredinin (Mizoribine) is used
clinically as an immunosuppressant in organ transplantation in Japan. Bredinin 5’-MP
(2) is the most potent, known IMPDH inhibitor of the IMP site,[10] although bredinin
does not show superior inhibitory effects over ribavirin in cell-based assays. EICAR
5’-MP (3) is a mechanism-based, irreversible inhibitor of IMPDH, and EICAR has
Figure 1. Known IMPDH inhibitors.

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319
broad-spectrum antiviral and anticancer activities in cell-based assays.[11,12] A few 2-
substituted inosine 5’-MPs 4–7 have demonstrated potent inhibition of IMPDH with
IC50 values around one micromolar.[13]
Although potent NMP inhibitors can be identified through enzymatic IMPDH
inhibition assays, the NMPs and their parent nucleosides may not be useful drugs. First,
efficient cellular phosphorylation of the parent nucleosides to NMP inhibitors is not
assured. Thus, the nucleosides are inactive if they fail to be phosphorylated. Second,
synthetic NMPs, as negatively charged chemical entities, have difficulty entering cells
by passive diffusion. A prodrug or a suitable carrier may help deliver NMPs into cells,
but their inherent instability to nucleotidases and phosphatases may regenerate the
parent nucleosides. Furthermore, NMPs may be anabolized to NDPs and NTPs, which
potentially can interact with unintended cellular targets, causing unpredictable and
often deleterious effects. Obviously, it is ideal to keep nucleoside inhibitors at the 5’-
MP level for selective and effective IMPDH inhibition.
To enhance the stability of NMPs, a useful approach is to employ phosphate
mimics (P1Ms) such as phosphonates, phosphoramidates and phosphorothioates. A
desirable nucleoside P1M drug candidate should bind with high affinity and specificity
to IMPDH and should have an adequate cellular half-life. So far, very few P1Ms of
nucleoside IMPDH inhibitors have been studied. One example is 1-(5,6-dideoxy-b-D-
ribo-hexofuranosyl-6-phosphonic acid)-1,2,4-triazole-3-carboxamide.[14] This compound
partially retains the inhibitory effect (Ki = 20 mM) of ribavirin 5’-MP. Another P1M is
ribavirin 5’-phosphoramidate,[15] which has showed a weak antiviral activity and the
Figure 2. Azole nucleoside 5’-monophosphate mimics (PIMs) synthesized.

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Wang et al.
inhibition of IMPDH has not been reported. P1Ms of bredinin and EICAR have not
been reported in the literature. Thus, the feasibility of P1Ms as IMPDH inhibitors has
been explored very little, especially considering the various kinds of known and
possible phosphate mimics. It is probable that P1Ms also may have difficulty entering
cells. However, a prodrug approach should be well suited for P1Ms. In fact, the
combination of phosphonate mimics of acyclic NMPs and a prodrug approach has been
used successfully in the newly approved antiviral drugs, adefovir dipivoxil (bis-POM
prodrug of PMEA) and tenofovir disoproxil fumarate (bis-POC prodrug of
PMPA).[16,17]
As part of our drug discovery program, we have designed and synthesized a series
of nucleoside P1Ms as IMPDH inhibitors. For identifying suitable P1M moieties, our
program started with known nucleoside inhibitors of IMPDH. When certain promising
P1M moieties are identified, they will be extended to other nucleosides, which will be
selected based on their inhibitory properties in the catalytic site, but not on
phosphorylation by cellular enzymes. In this article we will describe the synthesis
and biochemical evaluation of certain azole nucleoside 5’-MP mimics (P1Ms) as shown
in Figure 2.
CHEMISTRY
A number of azole nucleoside P1Ms were prepared through direct condensations
with phosphorylating reagents, as shown in Scheme 1. Ribavirin and EICAR were
reacted with thiophosphoryl chloride, followed by hydrolysis, to give the 5’-
phosphorothioate 8 and 16, respectively, in good yields. Condensations of 2’,3’-O-
isopropylidene ribavirin[18] with various phosphonic acids (RP(O)(OH)2, R = methyl,
fluoromethyl, difluoromethyl, vinyl and phenyl) in the presence of DCC, followed by
deprotection using Dowex-H+, afforded the 5’-O-phosphonates 9–13. By a similar
procedure, the EICAR 5’-O-phosphonates 17–19 were synthesized. Condensation of
Scheme 1.

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321
2’,3’-di-O-benzoylribavirin with fluorophosphonic acid in the presence of DCC,
followed by treatment with aqueous ammonia, afforded the 5’-O-phosphofluoridate
14 in good yield. Diphenyl (H)-phosphate was condensed with 2’,3’-di-O-benzoylriba-
virin and the resulting intermediate was treated with TEA/water and then with aqueous
methylamine to give the 5’-O-H-phosphonate 15. Reaction of 2’,3’-di-O-benzoylriba-
virin with 9-fluorenemethyl H-phopshonothioate in the presence of trimethylacetyl
chloride afforded, after removal of benzoyl groups, the 5’-H-phosphonothioate 26.
Commercially available 30 was subjected to deacetylation with NaOMe in
methanol (Scheme 2). Subsequent treatment with dimethoxypropane in the presence of
perchloric acid gave the 2’,3’-O-isopropylidene derivative 31, which was condensed
with (diethoxyphosphinyl)methyl triflate[19] to yield 32. Treatment of 32 with
methanolic ammonia, followed by deprotection with TMSBr and Dowex-H+, afforded
5’-O-phosphonomethyl ribavirin (20).
Ribavirin (34) was converted to 2’,3’-isopropylideneribavirin,[18] which was reacted
with thiolacetic acid under Mitsunobu conditions to give 35 as shown in Scheme 3.
Deacetylation of 35 under oxygen-free conditions yielded the thiol compound 36.
Reaction of 36 with methylphosphonic acid in the presence of DCC, followed by
deprotection of isopropylidene, gave 23 (Figure 2). Reaction of 36 with (diethoxyphos-
phinyl)methyl triflate in the presence of sodium hydride, followed by deprotection
using TMSBr and Dowex-H+ resin, afforded 21. Compound 37 was prepared by
treatment of 34 with iodine in the presence of triphenylphosphine. Treatment of 37 with
sodium azide, followed by catalytic hydrogenolysis over palladium, gave 38. Reaction
of 38 with (diethoxyphosphinyl)methyl triflate in the presence of sodium hydride,
followed by deprotection with TMSBr, afforded 22.
The 5’-deoxy-5’-difluoromethylene sugar 39, prepared according to a published
procedure,[20] was condensed with the silylated derivative of 40 in the presence of tin
Scheme 2.

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Wang et al.
Scheme 3.
Scheme 4.

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323
Scheme 5.
(IV) chloride to give 41 (Scheme 4). Treatment of 41 with methanolic ammonia,
followed by deprotection using boron trichloride and then bromotrimethylsilane,
afforded the 5’-difluoromethylenephosphonate 24. Similarly, the sugar 39 was
condensed with the silylated derivative of 42[21] in the presence of TiCl4 in
nitromethane to give compound 43. Subsequent deprotection afforded the bredinin
5’-difluoromethylenephosphonate 25. In a similar manner, compound 29 (Figure 2) was
prepared from condensation of the silylated derivative of 42 and 1-O-acetyl-5-deoxy-
2,3-di-O-benzoyl-5-(diethoxyphosphinyl)-D-ribofuranose[22] in the presence of SnCl4
and TMSOTf and subsequent debenzoylation. The condensations of the silylated
derivative of 42 with sugars required vigorous reaction conditions (e.g. reflux in
acetonitrile) for completion.[23] However, the existence of the phosphonate ester
groups did not allow such vigorous conditions owing to their sensitivity to Lewis
acid at elevated temperature, thus compounds 25 and 29 were obtained in low to
moderate yields.
Scheme 5 shows the synthesis of the 5’-deoxynucleotides 27 and 28. Compound
44, prepared by benzoylation of 37, was subjected to Arbuzov reaction using
triethylphosphite at 100°C to give 45. Subsequent deprotection with bromotrimethylsil-
ane and then with saturated methanolic ammonia yielded 28. Similarly, compound 44
was reacted with bis(trimethylsilyl)-H-phosphinite, followed by deprotection, to
afford 27.
BIOLOGICAL EVALUATION
The assays employed to measure the inhibition of IMPDH activity were a
modification of a reported method.[12] Effectiveness of the P1Ms as inhibitors of

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Wang et al.
IMPDHs was determined by measuring the reduction of NAD, which is coupled to
the oxidation of IMP to XMP in the presence or absence of a P1M. This assay
measures the activity of IMPDH from several organisms, including human and fungal
isoforms. This reaction is monitored at 340 nm using a UV/VIS spectrophotometer or
at 474 nm using a fluorometer (excitation wavelength = 344 nm). All of the azole
nucleoside P1Ms in Figure 2 were screened first against human IMPDH Type II
isozyme and a fungal (C. albicans) IMPDH, and initial ‘‘hits’’ were then counter-
screened against human IMPDH Type I isozyme. Table 1 shows the Ki values of the
active azole nucleoside P1Ms. Other P1Ms having Ki values more than 100 mM
against Human Type II isozyme were not tested further against Human Type I and C.
albicans IMPDHs.
As can be seen from Table 1, P1M inhibitors were found for all three IMPDH
enzymes tested: human Type I, human Type II and Candida albicans. For instance, the
phosphonates 25, 28 and 29 and the phosphorothioate 8 are active against all three
IMPDHs, having Ki values in the low micromolar range. The majority of the P1Ms in
Figure 2 were significantly less potent than the parent NMP inhibitors, and the inactive
P1Ms are not included in Table 1. Evidence was obtained for experimentally significant
differences in specificity of inhibition of human and C. albicans IMPDHs by P1Ms.
For example, compound 16 is about 10 times more active against C. albicans IMPDH
than human IMPDH. With this small, initial exploratory library of P1Ms, no significant
specificity of inhibition of human Type II IMPDH in preference over Type I was
observed. Given the small number of the P1Ms and the promising preliminary results,
we believe that nucleoside P1M approach has the potential to allow for the discovery of
potent and specific IMPDH inhibitors.
Stability of selected azole nucleotide P1Ms was assessed in fetal calf serum at
37°C following a published procedure.[24] The P1Ms in the serum at different time
points were analyzed on a reverse phase HPLC (C18). Compared to ribavirin 5’-MP
(t1/ = 6 h), ribavirin P1Ms were significantly more stable, with t1/
phosphorothioate 8, and t1/ > 24 h for the phosphonates 9, 24 and 28.
= 18 h for the
2
2
2
Table 1. Inhibition of IMPDHs by azole nucleoside P1Ms.
KI (mM) human
type II
KI (mM) human
type I
KI (mM)
C. albicans
Compound
8
16
0.94
34.2
85.0
27.1
2.04
34.7
1.82
7.92
0.059
0.02
1.15
2.92
31.8
NT
1.34
3.41
NT
20
24
13.9
3.33
44.8
2.44
9.40
0.098
0.057
NT
20.4
15.5
10.7
1.48
1.98
0.054
0.023
1.34
25
26
27
29
Ribavirin 5’-MP
Bredinin 5’-MP
EICAR 5’-MP
NT = not tested.

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325
In summary, we have designed and synthesized a series of azole nucleoside 5’-
monophosphate mimics (P1Ms), which were evaluated for IMPDH inhibition. The
procedures for synthesis of these P1Ms are expected to be applicable to other
nucleosides. A few P1Ms showed, although not as potent as the parent NMPs,
substantial inhibition of IMPDHs. This, along with the superior stability of P1Ms,
especially phosphonate P1Ms, should warrant further exploration of nucleoside P1Ms
as inhibitors of IMPDH.
EXPERIMENTAL SECTION
NMR spectra were recorded on a Varian Mercury 300 NMR spectrometer.
Tetramethysilane was used as internal reference for 1H NMR, and 85% phosphoric
acid was used as external reference for 31P NMR. Mass spectra and purity of compounds
were determined on a ThermoFinnigan Deca XP on-line HPLC mass spectrometer. A
Phenomenex Luna C18 75 ꢀ 2 mm column packed with 3-um particles was used for LC-
MS analysis. A 0 to 50% linear gradient (15 min) of acetonitrile in 10 mM
dimethylhexylammonium acetate buffer (pH = 7) was used in series with mass spectra
detection in the negative ionization mode. The azole nucleoside P1Ms were purified by
reverse-phase HPLC with a Luna C18 250 ꢀ 21 mm column (Phenomenex) using
acetonitrile in triethylammonium acetate (50 mM, pH = 7) as eluent. All the purified
azole nucleoside P1Ms of this work were subjected to the LCMS analysis. Amounts of
the nucleoside P1M products in this section are based on UV absorptions and calculated
based on their H-forms. The P1Ms, as triethylammonium salts, were stored at ꢁ 80°C.
1-(5-O-Thiophosphoryl- -D-ribofuranosyl)-1,2,4-triazole-3-carboxamide
(8). To a suspension of 1-b-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin,
122 mg. 0.5 mmol) in 2.5 mL of anhydrous pyridine was added proton sponge1 [1,8-
bis(dimethylamino)naphthalene] (107 mg. 0.5 mmol) at 0–5°C under argon
atmosphere, followed by addition of thiophosphoryl chloride (0.1 mL, 1mmol). The
mixture was stirred at this temperature for 30 minutes, quenched with 3 mL of 1 M
triethylammonium bicarbonate buffer (TEAB) and extracted with chloroform (2 mL).
The aqueous layer was subjected to reverse-phase HPLC purification. Collected
1
fractions were lyophilized to give 60 mg of 8 with 100% Purity. H NMR (D2O) d 8.71
(s, 1H), 5.87 (d, J = 3.5 Hz, 1H), 4.64 (m, 1H), 4.54 (dd, J = 3.75, 4 Hz, 1H), 4.22 (m,
1H), 3.96 (m, 2H); 31P NMR d 48.89 (s); MS m/z (Mꢁ + 1) 340.25.
1-(5-O-Methylphosphonyl- -D-ribofuranosyl-1,2,4-triazole-3-carboxamide
(9). 1-(2,3-O-Isopropylidene-b-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide[18] (142
mg, 0.5 mmol) was co-evaporated with anhydrous pyridine (3 ꢀ 5 mL) under reduced
pressure and taken into 5 mL of anhydrous pyridine. To the above solution under argon
atmosphere were added dicyclohexylcarbodiimide (DCC, 206 mg, 1.0 mmol) and methyl
phosphonic acid (58 mg, 0.6 mmol). The mixture was stirred at 38°C for 36 h, cooled
and quenched with water (5 mL). The resulting dicyclohexylurea (DCU) was filtered off
and the filtrate was concentrated under reduced pressure and filtered again. After
evaporation, the concentrate was co-evaporated with toluene to remove a trace of
pyridine. The crude product (105 mg) was dissolved in methanol (5 mL) and Dowex

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Wang et al.
50Wx8–100 resin (1 g, pre-washed with water and methanol) was added. The mixture
was stirred at 50°C for 2 h, filtered through a short pad of cotton and the resin was
thoroughly washed with water. The filtrate was concentrated to yield a viscous residue
which was purified on reverse-phase HPLC. The fractions collected were pooled and
lyophilized to give 34 mg of 9 with 100% purity. 1H NMR (D2O) d 8.62 (s, IH), 5.91 (d,
J = 3.7 Hz, 1H), 4.56 (m, 1H), 4.39(m, 1H), 4.20 (m, 1H), 3.92 (m, 2H), 1.11 (d,
J = 17.0 Hz, 3H); 31P NMR (D2O) d 28.38 (s); MS m/z (Mꢁ + 1) 322.30.
1-[5-O-(Fluoromethyl)phosphonyl- -D-ribofuranosyl]-1,2,4-triazole-3-carboxa-
mide (10). According to the same procedure as described for 9, the reaction of 1-(2,3-
O-isopropylidene-b-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (142 mg, 0.5 mmol)
with (fluoromethyl)phosphonic acid[21] (60 mg, 0.6 mmol) in the presence of DCC (202
mg, 1.0 mmol) at 38°C for 24 h gave, after reverse-phase HPLC purification, 25 mg of
1
10 with 100% purity. H NMR (D2O) d 8.59 (s, 1H), 5.87 (d, J = 3.25 Hz, 1H), 4.55
(m, 1H), 4.50 (d, J = 3.75, 1H), 4.38 (dd, J = 4.5, 4 Hz 1H), 4.38 (d, J = 3.5 Hz, 2H),
4.19 (m, 1H), 4.04(m, 1H); 31P NMR (D2O) d 15.45-14.97 (d, J = 121.75 Hz); MS m/z
(Mꢁ + 1) 340.00.
1-[5-O-(Difluoromethyl)phosphonyl- -D-ribofuranosyl]-1,2,4-triazole-3-carbox-
amide (11). Diethyl(difluoromethyl)phosphonate (500 mg, 2.66 mmol) and 0.88 mL
(96.66 mmol) bromotrimethylsilane were refluxed in 10 mL of anhydrous methylene
chloride for 15 h. Solvent was evaporated and the residue was repeatedly co-evaporated
with methanol. The residue (300 mg) was dissolved in 2 mL of anhydrous pyridine
to make a (difluoromethyl)phosphonic acid stock solution and stored under argon at
–20°C. This was used in the next step.
According to the same procedure as described for 9, the reaction of 1-(2,3-O-
isopropylidene-b-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (142 mg, 0.5 mmol)
and (difluoromethyl)phosphonic acid (70 mg, 0.6 mmol) in the presence of DCC (210
mg, 1.0 mmol) at 38°C for 24 h gave, after reverse-phase HPLC purification, 100 mg
1
of 11 with 97.8% purity. H NMR (D2O) d 8.58 (s, 1H), 5.82 (m, 2H), 4.55 (m, 1H),
4.4(m,1H), 4.38 (dd, J = 4.5, 4 Hz 1H), 4.19 (m, 1H), 4.0 (m, 2H); 31P NMR (D2O) d
4.89 (t); MS m/z (Mꢁ + 1) 358.20.
1-(5-O-Vinylphosphonyl- -D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (12).
According to the same procedure as described for 9, the reaction of 1-(2,3-O-
isopropylidene-b-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (142 mg, 0.5 mmol)
with vinylphosphonic acid (58 mg, 0.6 mmol) in the presence of DCC (202 mg, 1.0
mmol) at 38°C for 24 h gave, after reverse-phase HPLC purification, 55 mg of 12 with
1
100% purity. H NMR (D2O) d 8.59 (s, 1H), 5.82(m, 4H), 4.54 (m, 1H), 4.38 (m, 1H),
4.19 (m, 1H), 3.75(m, 2H); 31P NMR (D2O) d 16.23 (s); MS m/z (Mꢁ + 1) 334.30.
1-(5-O-(Phenylphosphonyl- -D-ribofuranosyl)-1,2,4-triazole-3-carboxamide
(13). According to the same procedure as described for 9, the reaction of 1-(2,3-O-
isopropylidene-b-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (142 mg, 0.5 mmol)
and phenylphosphonic acid (80 mg, 0.6 mmol) in the presence of DCC (202 mg, 1.0
mmol) at 38°C for 24 h gave, after reverse-phase HPLC purification, 109 mg 13 with
1
100% purity. H NMR (D2O) d 8.45 (s, 1H), 7.40 (m, H), 7.28 (m, 1H), 7.19 (m, 2H),

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5.82(d, J = 3.23 Hz, 1H), 5.45(m, 1H), 4.34 (m, 1H), 4.16 (m, 1H), 3.88(m, 1H), 3.76
(m, 1H); 31P NMR (D2O) d 15.94 (s); MS m/z (Mꢁ + 1) 384.40.
1-(5-O-Fluorophosphoryl-1- -D-ribofuranosyl)-1,2,4-triazole-3-carboxamide
(14). A solution of 1-b-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (2.4 g, 10.0
mmol) and tert-butyldimethylchlorosilane (1.65 g, 11.0 mmol) in anhydrous pyridine
(30 mL) was stirred at room temperature overnight. After completion of the reaction,
the mixture was poured into saturated sodium bicarbonate solution, extracted with ethyl
acetate, dried over sodium sulfate, and concentrated to dryness. The residue was
dissolved in pyridine (25 mL) and benzoyl chloride (2.6 mL, 22.0 mmol) was added.
The resulting mixture was stirred for 30 min, quenched by adding aqueous sodium
bicarbonate and extracted with ethyl acetate. The organic layer was dried over
anhydrous sodium sulfate and concentrated to dryness. Chromatography on silica gel
using 2 % methanol in dichloromethane gave 1-(5-O-tert-butyldimethylsilyl-2,3-di-O-
benzoyl-1-b-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (3.5 g) which was used as
such in the next reaction.
The product from the above reaction (2.8 g, 5.0 mmol) was dissolved in 25 mL of
THF and TBAF (1 M in THF, 15 mL) was added. The reaction mixture was stirred at
room temperature overnight, quenched with aqueous sodium bicarbonate (100 mL) and
extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate
and concentrated to dryness. The residue was purified by chromatography on silica gel
using 15% methanol in dichloromethane to give 1-(2,3-Di-O-benzoyl-1-b-D-ribofu-
ranosyl)-1,2,4-triazole-3-carboxamide (1.5 g). 1H NMR (DMSO-d6) d 8.92 (s, 1H),
7.39–7.91 (m, 12H), 6.55 (d, J = 4.11 Hz, 1H), 6.02 (m, 1H), 5.85 (m, 1H), 5.22 (m,
1H), 4.53 (m, 1H), 3.72 (m, 2H).
According to the same procedure as described for 9, part of the above product
(226 mg, 0.5 mmol) was reacted with fluorophosphonic acid (55 mg, 0.6 mmol) at
38°C for 24 hours. The resulting crude product was treated with 28 % aqueous
ammonia (3 mL) for 2 h. After evaporation the crude product was purified on reverse-
1
phase HPLC to give 125 mg of 14 with 100% purity. H NMR (D2O) d 8.57 (s, 1H),
5.91(d, J = 3.8 Hz, 1H), 4.56(m, 1H), 4.40 (m, 1H), 4.21 (m, 1H), 3.98–4,13(m, 2H).
31P NMR (D2O) d ꢁ 8.42–0.77 (d, J = 930.23 Hz); MS m/z (Mꢁ + 1) 326.30.
1-(5-O-Hydrogenphosphonyl- -D-ribofuranosyl-1,2,4-triazole-3-carboxamide
(15). According to the same procedure as described for 9, 1-(2,3-di-O-benzoyl-1-b-D-
ribofuranosyl)-1,2,4-triazole-3-carboxamide (226 mg, 0.5 mmol) was reacted with
diphenyl hydrogen phosphonate (349 mg, 1.5 mmol) at room temperature for 15 min.
The reaction mixture was quenched by adding water-triethylamine (1:1, v/v, 5 mL),
stirred for 1 min and concentrated. The residue was treated with 50% aqueous
methylamine (10 mL) for 1h. After removal of the solvent, the oily residue was
purified on reverse-phase HPLC to give 70 mg of 15 with 100% purity. 1H NMR
(D2O) d 8.62 (s, 1H),7.59 (s, 0.5H), 5.90 (d, J = 3.8 Hz, 1H), 4.45(s, 0.5H), 4.56 (m,
1H), 4.39 (m, 1H), 4.19 (m, 1H), 3.89–4.19 (m, 2H); 31P NMR (D2O) d 6.56 (s); MS
m/z (Mꢁ + 1) 308.20.
5-Ethynyl-1-(5-O-thiophosphono- -D-ribofuranosyl)imidazole-4-carboxamide
(16). According to the same procedure as described for 8, the reaction of 5-ethynyl-1-

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Wang et al.
b-D-ribofuranosylimidazole-4-carboxamide (EICAR, 50 mg, 0.187 mmol) with thiopho-
sphoryl chloride (63 mg, 38 mL, 0.37 mmol) in 1.2 mL of anhydrous pyridine in the
presence of proton sponge1 (115.5 mg, 0.54 mmol) at 0–5°C under argon atmosphere
for 30 min gave, after reverse-phase HPLC purification, 16.7 mg of 16 with 85.4%
1
purity. H NMR (D2O) d 7.89 (s, 1H), 5.67 (d, J = 4 Hz, 1H), 4.34 (dd, J = 4.5, 4.5 Hz,
1H), 4.21–4.003 (m, 5H); 31P NMR (D2O) d 18.05 (S); MS m/z (Mꢁ + 1) 363.30.
5-Ethynyl-1-(5-methylphopsphonyl- -D-ribofuranosyl)imidazole-4-carboxamide
(17). To a stirred suspension of 5-ethynyl-1-b-D-ribofuranosylimidazole-4-carbox-
amide (200 mg, 0.75 mmol) in 80 mL of dry acetone at 0°C under argon was added
drop-wise 0.02 mL of 70% perchloric acid. The mixture was warmed to room
temperature, stirred for 50 min, cooled and carefully neutralized using an equimolar
amount of aqueous ammonia. Solvent was evaporated and the residue was purified on a
silica gel column with 10% methanol in chloroform to give 160 mg (70%) of 5-
ethynyl-1-(2,3-O-isopropylidene-b-D-ribofuranosyl)imidazole-4-carboxamide.[25] This
compound was used as such in the next reaction.
According to the same procedure as described for 9, part of the above product (100
mg, 0.33 mmol) was reacted with methylphosphonic acid (38 mg, 0.39 mmol) in the
presence of DCC (161 mg, 0.78 mmol) at 38°C for 36 h and then isopropylidene group
was removed using Dowex-H+ ..Purification on reverse-phase HPLC gave 14 mg of 17
1
with 100% purity. H NMR (D2O) d 7.95 (s, 1H), 5.81 (d, J = 4.25 Hz), 4.44 (dd,
J = 4.25, 4 Hz, 1H), 4.25 (dd, J = 3, 4.5 Hz, 1H), 4.17 (m, 1H), 3.91 (m, 2H), 1.09 (d,
J = 17.0 Hz, 3H). 31P NMR (D2O) d 28.26 (S); MS m/z (Mꢁ + 1) 345.30.
5-Ethynyl-1-(5-O-fluoromethylphosphonyl- -D-ribofuranosyl)imidazole-4-car-
boxamide (18). According to the same procedure as described for 9, 5-ethynyl-1-(2,3-
O-isopropylidene-b-D-ribofuranosyl)imidazole-4-carboxamide (60 mg, 0.195 mmol)
was reacted with fluoromethylphosphonic acid (26 mg, 0.205 mmol) in the presence
of DCC (97 mg, 0.47 mmol) at 38°C for 24 h, and then the isopropylidene group was
removed using Dowex-H+ .. Purification on reverse-phase HPLC gave 9.3 mg of 18
with 100% purity . 1H NMR (D2O) d 7.94 (s, 1H), 5.8 (d, J = 4.5 Hz), 4.54 (d,
J = 3.75 Hz, 1H), 4.45 (dd, J = 4.5, 4.25 Hz, 1H), 4.39 (d, J = 3.5 Hz, 1H), 4.27 (dd,
J = 3.75, 4 Hz, 1H), 4.16 (m, 1H), 4.02 (m, 1H). 31P NMR (D2O) d 15.52 (d,
J = 121.75 Hz); MS m/z (Mꢁ + 1) 363.20.
1-[5-O-(Difluoromethyl)phosphonyl- -D-ribofuranosyl]-5-ethynylimidazole-4-
carboxamide (19). According to the same procedure as described for 9, 5-ethynyl-1-
(2,3-O-isopropylidene-1-b-D-ribofuranosyl)imidazole-4-carboxamide (60 mg, 0.195
mmol) was reacted with difluoromethyl phosphonate (27 mg, 0.205 mmol) in the
presence of DCC (97 mg, 0.47 mmol) at 38°C for 24 h, and then the isopropylidene
group was removed using Dowex-H+. Purification on reverse-phase HPLC gave 14.1
1
mg of 19 with 100% purity. H NMR (D2O) d 7.93 (s, br, 1H), 6.00–5.59 (m, 2H),
4.42 (dd, J = 4, 4.25 Hz, 1H), 4.25 (dd, J = 4, 4.25 Hz, 1H), 4.15–4.08 (m, 3H); 31P
NMR (D2O) d 4.94 (dd, J = 166.25, 167 Hz); MS m/z (Mꢁ + 1) 381.20.
Methyl 1-(2,3-isopropylidene- -D-ribofuranosyl)-1,2,4-triazole-3-carboxylate
(31). Methyl 1-(2,3,5-tri-O-acetyl-b-D-ribofuranosyl)-1,2,4-triazole-3-carboxylate 30

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Azole Nucleoside 5’-Monophosphate Mimics
329
(3.8 g, 10 mmol) was dissolved in anhydrous methanol (50 mL) and sodium methoxide
(25 wt.% in methanol, 12 mL) was added. The solution was stirred at room temperature
for 6 h and neutralized with Dowex 50WX8-100 ion-exchange resin. The resin was
filtered through a short pad of cotton, washed thoroughly with methanol. The methanol
solution was concentrated to dryness to give 2.5 g of methyl 1-b-D-ribofuranosyl-1,2,4-
triazole-3-carboxylate. To a suspension of methyl 1-b-D-ribofuranosyl-1,2,4-triazole-3-
carboxylate (1.3 g, 0.5 mmol) in dry acetone (20 mL) and dimethoxypropane (18 mL)
at 0°C under argon was added dropwise 0.2 mL of 70% perchloric acid. The mixture
was warmed to room temperature, stirred for 50 min, cooled with ice and neutralized
using an equimolar amount of ammonia. Solvent was evaporated and the residue was
loaded on a silica gel column and eluted with 10% methanol in chloroform to give
1
980 mg of 31. H NMR (CDCl3) d 8.36 (s, 1H), 6.01 (d, J = 2.4 Hz, 1H), 5.05 (m,
2H), 4.57 (s, br, 1H), 3.47–4.01 (m, 6H), 1.65 (s, 3H), 1.59 (s, 3H)
Methyl 1-[5-O-(diethoxyphosphinyl)methyl-2,3-isopropylidene-b-D-ribofuranos-
yl)-1,2,4-triazole-3-carboxylate (32). A mixture of methyl 1-(2,3-O-isopropylidene-
b-D-ribofuranosyl)-1,2,4-triazole-3-carboxylate (600 mg, 2 mmol) and sodium hydride
(100 mg) in anhydrous THF at ꢁ 78°C under argon was stirred for 30 minutes,
followed by a slow addition of a solution of (diethoxyphosphinyl)methyl triflate
(300 mg, 1 mmol) in THF (10 mL). The mixture was brought to room temperature for
1 h, neutralized with acetic acid, and then evaporated. The residue was taken in 20 mL
of chloroform and washed with 10 mL water. The chloroform layer was dried over
anhydrous magnesium sulfate, filtered, and concentrated to dryness. Reaction product
was taken to next step without further purification.
1-[5-O-(Dihydroxyphosphinyl)methyl- -D-ribofuranosyl]-1,2,4-triazole-3-car-
boxamide (20). A solution of 32 (400 mg) in 25 mL of methanolic ammonia in a
steel vessel was kept at room temperature overnight. Ammonia and methanol were
evaporated and the residue was purified on silica gel with 13% methanol in
dichloromethane to give 380 mg of the 3-carboxamide derivative 33. Bromotrimethyl-
silane (3 mL, 23 mmol) was added to a stirred solution of 33 (350 mg, 0.8 mmol) in
acetonitrile (25 mL) and the resulting mixture was stirred at room temperature for 15 h
and evaporated to a yellowish syrup, which was dissolved in 5 mL of methanol and
concentrated to dryness. This evaporation was repeated three times. The residue was
dissolved in 20 mL of methanol and Dowex 50Wx8-100 ion-exchange resin (1 g) was
added. The mixture was heated at 50°C for 2 h, filtered, and washed with water
thoroughly. The filtrate was concentrated and the residue was purified on reverse-phase
1
HPLC to give 42 mg of 20 with 96.6% purity. H NMR (D2O) d 8.64 (s, 1H), 5.88 (d,
J = 3.8 Hz, 1H), 4.51(m, 1H), 4.36 (m, 1H), 4.17 (m, 1H), 3.45–3.75(m, 4H); 31P
NMR (D2O) d 15.44 (s); MS m/z (Mꢁ + 1) 338.20.
1-(5-Acetylthio-5-deoxy-2,3-O-isopropylidene- -D-ribofuranosyl)-1,2,4-triazole-
3-carboxamide (35). Diisopropyl azodicarboxylate (1.53 mL, 7.74 mmol) and
triphenylphosphine (2.03 g, 7.74 mmol) were dissolved in anhydrous THF (20 mL)
at 0°C. After a white precipitate appeared, 1 g (3.52 mmol) of 1-(2,3-O-isopropylidene-
b-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide[18] in 15 mL of anhydrous THF and
0.56 mL (7.74 mmol) of thiolacetic acid in 5 mL of anhydrous THF were added

ORDER
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Wang et al.
simultaneously. The mixture was allowed to warm to room temperature and stirred for
5 h, neutralized with triethylamine, concentrated to dryness, dissolved in ethyl acetate
and washed with water. The aqueous layer was extracted with ethyl acetate (2 ꢀ 20 mL).
The combined organic layer was washed with brine, dried over anhydrous
magnesium sulfate, filtered and evaporated. Chromatography on a silica gel column
using chloroform/THF (5:1) and then using chloroform/methanol (10:1) afforded 600
1
mg of 35. H NMR (CDCl3) d 8.28 (s, 1H), 7.27 (br, s, 1H), 5.94 (br, s, 1H), 5.35 (d,
J = 4.75 Hz, 1H), 4.82 (dd, J = 1.75, 5 Hz, 1H), 4.35 (m, 1H), 3.24 (m, 1H), 2.97 (m,
1H), 1.61 (s, 3H), 1.55 (s, 3H), 1.35 (s, 3H).
1-(5-Deoxy-2,3-O-isopropylidene-5-thio- -D-ribofuranosyl)-1,2,4-triazole-3-car-
boxamide (36). A mixture of methanol and triethylamine (9:1, 7.5 mL) was bubbled
with argon at room temperature for 15 minutes and then 200 mg (0.56 mmol) of 35 and
172.7 mg (1.12 mmol) of dithiothreitol were added. The mixture was stirred at room
temperature for 5 h. Solvent was evaporated under argon atmosphere and the residue was
loaded on a silica gel column. The impurities were eluted using 2% methanol in
methylene chloride and then the product using methylene chloride/methanol (30:1).
1
Evaporation of the solvent afforded 130 mg of the 5’-thio nucleoside 36. H NMR
(CDCl3) d 8.30 (s, 1H), 7.08 (br, s, 1H), 6.25 (br, s, 1H), 6.05 (d, J = 1 Hz, 1H), 5.40 (dd,
J = 1, 5.25 Hz, 1H), 4.95 (m, 1H), 4.35 (m, 1H), 2.69 (m, 3H), 1.58 (s, 3H), 1.37 (s, 3H).
1-(5-Deoxy-5-S-phosphonomethyl-5-thio-1- -D-ribofuranosyl)-1,2,4-triazole-3-
carboxamide (21). To a solution of 36 (150 mg, 0.50 mmol) in 5 mL of anhydrous
DMF at ꢁ 20°C was added 20 mg (0.50 mmol) of 60% sodium hydride in mineral oil,
followed by addition of 223 mg (0.74 mmol) of (diethoxyphosphinyl)methyl triflate.
The mixture was stirred at this temperature for 1.5 h, concentrated, diluted with ethyl
acetate and then washed with brine. The organic phase was dried over MgSO4, filtered,
and concentrated to dryness. The residue (156 mg) was dissolved in 15 mL of
anhydrous methylene chloride and to this solution was added 1 mL of bromotri-
methylsilane and the mixture was stirred at room temperature for 12 h. Solvent was
evaporated and the residue was dissolved in 20 mL of methanol/water (1:1). The
solution was stirred at 50°C for 3 h and concentrated. Chromatography on reverse
1
phase HPLC afforded 13.5 mg of 21 with 83.6% purity. H NMR (D2O) d 8.58 (s,
8.58, 1H), 5.89 (d, J = 3.25 Hz, 1H), 4.57 (m, 1H), 4.35 (dd, J = 4.25, 4 Hz, 1H), 4.20
(m, 1H), 2.85 (m, 2H), 2.56 (d, J = 11.5 Hz, 2H). 31P NMR (D2O) d 17.64 (d, J = 125
Hz); MS m/z (Mꢁ + 1) 354.28.
1-(5-Deoxy-5-iodo - -D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (37).
A
solution of 1.7 g (6.5 mmol) of triphenylphosphine and 1.52 g (6.0 mmol) of iodine
in 10 mL of pyridine was stirred at room temperature for 20 min and then 1-b-D-
ribofuranosyl-1,2,4-triazole-3-carboxamide 34 (976 mg, 4.0 mmol) was added. The
reaction mixture was stirred at room temperature for 2 h, concentrated to dryness, and co-
evaporated with toluene twice. Chromatography on silica gel with methylene chloride/
1
methanol (20:1 to 7.5:1) yielded 1.1 g of 37 which was used in the next step. H NMR
(DMSO-d6) d 8.84 (s, 1H), 7.86 (br, s, 1H), 7.62 (br, s, 1H), 5.87 (d, J = 2.75 Hz, 1H), 5.70
(d, J = 4.75 Hz, 1H), 5.47 (d, J = 4.75 Hz, 1H), 4.45 (dd, J = 3, 4.25 Hz, 1H), 4.17 (dd,
J = 4.25, 4.25 Hz, 1H), 4.03 (m, 1H), 3.53 (m, 1H), 3.35 (m, 1H).

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Azole Nucleoside 5’-Monophosphate Mimics
331
1-(5-Amino-5-deoxy-1- -D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (38). A
reaction mixture of 1.5 g (3.65 mmol) of 37 and 3.65 g (5.35 mmol) of sodium azide in
DMF (15 mL) was heated at 90°C for 12 h. After evaporation, the residue was
adsorbed on silica gel and loaded onto a silica gel column. The product was eluted
using 10% methanol in methylene chloride to afford 1.2 g of 1-(5-azido-5-deoxy-1-b-D-
1
ribofuranosyl)-1,2,4-triazole-3-carboxamide. H NMR (DMSO-d6) d 8.84 (s, 1H), 7.83
(br, s, 1H), 7.62 (br, s, 1H), 5.91 (d, J = 2.75 Hz, 1H), 5.69 (d, J = 4.75 Hz, 1H), 5.38
(d, J = 4.75 Hz, 1H), 4.37 (dd, J = 3, 4.25 Hz, 1H), 4.27 (dd, J = 4.25, 4.25 Hz, 1H),
4.06 (m, 1H), 3.53 (m, 2H).
A mixture of the above product and 200 mg of 10% Pd/C (1 g, 3.77 mmol) in
50 mL of methanol was shaken at 30 psi hydrogen for 18 h. The catalyst was filtered
1
and the filtrate was concentrated to dryness to give 600 mg (67%) of 38. H NMR
(DMSO-d6) d 8.87 (s, 1H), 7.82 (br, s, 1H), 7.61 (br, s, 1H), 5.80 (d, J = 3.5 Hz, 1H),
4.36 (dd, J = 3, 4.25 Hz, 1H), 4.12 (dd, J = 4.25, 4.25 Hz, 1H), 3.88 (m, 1H), 2.68
(m, 2H).
1-(5-Deoxy-5-N-phosphonomethylamino-1- -D-ribofuranosyl)-1,2,4-triazole-3-
carboxamide (22). To a solution of the crude 38 (150 mg, 0.62 mmol) in anhydrous
DMF and pyridine (1:1, 10 mL) at 0°C and was added 279 mg (0.93 mmol) of
(diethoxyphosphinyl)methyl triflate. The mixture was stirred at this temperature for
1.5 h and then concentrated. The residue was taken in ethyl acetate and then washed
with brine. The organic phase was dried over MgSO4, filtered and concentrated to give
142 mg of a crude product. The crude product was dissolved in 15 mL of anhydrous
methylene chloride and 1 mL (7.5 mmol) of bromotrimethylsilane was added. The
mixture was stirred under argon at 40°C for 15 h. Solvent was evaporated and the crude
1
was purified on reverse-phase HPLC to give 13.5 mg of 22 with 100% purity. H NMR
(D2O) d 8.61 (s, 1H), 8.59 (s, 1H), 8.36 (m, 1H), 7.89 (m, 2H), 5.91 (m, 1H), 4.59 (d,
J = 10.5 Hz, 2H); 31P NMR (D2O) d 7.87 (s); MS m/z (Mꢁ + 1) 337.23.
1-(5-Deoxy-5-methylphosphono-5-thio- -D-ribofuranosyl)-1,2,4-triazole-3-car-
boxamide (23). According to the same procedure as described for 9, the reaction of
36 (100 mg, 0.33 mmol) with methylphosphonic acid (36 mg, 0.37 mmol) in the
presence of DCC (137 mg, 0.66 mmol) at 35°C for 24 h and subsequent deprotection of
isopropylidene using Dowex-H+ gave, after purification on reverse-phase HPLC, 6.2 mg
1
of 23 with 100% purity. H NMR (D2O) d 8.58 (s, 1H), 5.89 (d, J = 3.25 Hz, 1H), 4.63
(m, 1H), 4.58 (m, 1H), 4.36 (dd, J = 4.25, 4.25 Hz, 1H), 4.19 (m, 1H), 2.95 (m, 2H),
1.43 (d, J = 12.5 Hz, 3H); 31P NMR (D2O) d 42.66 (s); MS m/z (Mꢁ + 1) 338.40.
Methyl 1-[2-O-acetyl-3-O-benzyl-5,6-dideoxy-6-(diethoxyphosphinyl)-6,6-
difluoro- -D-ribofuranosyl]-1,2,4-triazole-3-carboxylate (41). Methyl-1,2,4-tria-
zole-4-carboxylate (300 mg, 2.5 mmol) in HMDS (5 mL) was refluxed in the presence
of ammonium sulfate (5 mg). Excess HMDS was evaporated under high vacuum. The
resulting 40 was dissolved in anhydrous acetonitrile and 39[19] (1.25 g, 2.5 mmol) was
added. After addition of tin (IV) chloride (0.9 mL, 7.5 mmol), the reaction mixture was
heated under reflux for 2 h, then cooled to room temperature, diluted with chloroform,
filtered through celite, and washed with saturated sodium bicarbonate. The organic
layer was dried over anhydrous sodium sulfate and concentrated to dryness. The crude

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Wang et al.
product was purified on silica gel using 5% methanol in methylene chloride to give
1
1.1 g of 41. H NMR (CDCl3) d 8.28 (s, 1H), 7.35 (m, 5H), 5.98 (d, J = 1.7 Hz, 1H),
5.60 (m, 1H), 4.52–4.63 (m, 3H), 4.19–4.37 (m, 5H), 4.00 (s, 3H), 2.22–2.58 (m, 2H),
2.14 (s, 3H), 1.36 (s, 3H); 31P NMR (D2O) d 7.48 (t).
1-[5,6-Dideoxy-6-(dihydroxyphosphinyl)-6,6-difluoro- -D-allofuranosyl]-1,2,4-
triazole-3-carboxamide (24). A solution of 41 (1.0 g) and methanolic ammonia
saturated at 0°C in a steel vessel was kept at room temperature overnight. Excess
ammonia and solvent were evaporated to give 1-[3-O-benzyl-5,6-dideoxy-6-(diethoxy-
phosphinyl)-6,6-difluoro-b-D-ribofuranosyl]-1,2,4-triazole-3-carboxamide (725 mg) as a
solid crude product. Part of the crude product (505 mg, 1.0 mmol) was dissolved in
anhydrous dichloromethane (25 mL) and cooled to ꢁ 78°C. Boron trichloride (2 M in
dichloromethane, 2.1 mL) was added. The reaction mixture was stirred at room
temperature for 1 h. Methanol (10 mL) was added and evaporated and this process was
repeated three times. The residue was taken in acetonitrile and DMF (1:1 v/v, 20 mL)
and bromotrimethylsilane (2.2 mL, 16.8 mmol) was added. The mixture was stirred
under argon atmosphere at room temperature for 40 h. After evaporation, the oily
residue was co-evaporated with methanol (3 ꢀ 10 mL) and toluene(3 ꢀ 10 mL).
1
Purification on reverse-phase HPLC gave 50 mg of 24 with 100% purity. H NMR
(D2O) d 8.55 (s, 1H), 5.88(d, J = 3.52 Hz, 1H), 4.52(m, 1H), 4.41 (m, 1H), 4.31 (m,
1H), 2.31–2.51(m, 2H); 31P NMR (D2O) d 5.58 (t); MS m/z (Mꢁ + 1) 358.20.
4-Carbamoyl-1-[2-O-acetyl-3-O-benzyl-5,6-dideoxy-6-(diethoxyphosphinyl)-6,6-
difluoro- -D-ribofuranosyl]-1,3-imidazolium-5-olate (43). 4-Carbamoylimidazo-
lium-5-olate (127 mg 1.0 mmol) in HMDS (5 mL) and xylene (5 mL) was refluxed
in presence of ammonium sulfate (2 mg). Excess HMDS was evaporated under high
vacuum. The resulting silylated imidazolium 42 was dissolved in anhydrous
nitromethane and 39 (500 mg, 1.0 mmol) was added. After addition of titanium(IV)
chloride (0.15 mL, 1.3 mmol), the reaction mixture was stirred at room temperature for
42 h, poured into a suspension of 4 g of sodium carbonate in methanol. The methanol
solution was filtered through celite and evaporated. The residue was purified on silica
1
gel using 20% methanol and 0.5% TEA in ethyl acetate to give 280 mg of 43. H NMR
(CDCl3) d 7.84 (s, 1H), 7.32 (m, 5H), 6.24 (bs, 1H), 4.83 (d, J = 11.7 Hz, 1H), 4.6 (m,
1H), 4.45 (d, J = 11.7 Hz, 1H), 4.22(m, 5H), 3.61 (m, 1H), 2.22–2.61 (m, 2H), 1.40
(m, 6H); 31P NMR (D2O) d 7.5 (t).
4-Carbamoyl-1-[5,6-dideoxy-6-(dihydroxyphosphinyl)-6,6-difluoro- -D-ribo-
furanosyl]-1,3-imidazolium-5-olate (25). Compound 43 (280 mg 0.5 mmol) was
subject to the same treatment as 41, with boron trichloride at room temperature for 2 h
and then with bromotrimethylsilane at room temperature for 48 h. Purification on
1
reverse-phase HPLC gave 30 mg of 25 with 90% purity. H NMR (D2O) d 8.20 (s,
1H), 5.63 (d, J = 3.8 Hz, 1H), 4.48 (m, 1H), 4.35 (m, 1H), 4.18 (m, 1H), 2.22–2.61 (m,
2H); 31P NMR (D2O) d 5.14(t); MS m/z (Mꢁ + 1) 373.05.
1-[5-O-(H-Thiophosphonyl)- -D-ribofuranosyl]-1,2,4-triazole-3-carboxamide
(26). 1-(2,3-Di-O-benozyl-1-b-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (226 mg.
0.5 mmol, for preparation, see Compound 14) and 9-fluorenemethyl (H)-phospho-
nothioate (400 mg, 1.5 mmol) were dissolved in 10% pyridine in dichloromethane

ORDER
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Azole Nucleoside 5’-Monophosphate Mimics
333
(10 mL). Trimethylacetylchloride (0.07 mL, 0.7 mmol) was added and the mixture was
stirred at room temperature for 5 min. Then, triethylamine (10 mL) was added and
stirred for further 20 min. Solvent was evaporated and the residue was treated with
aqueous methylamine (50%, 5 mL) for 1 h. The solution was concentrated and the
residue was purified on reverse-phase HPLC to give 25 mg of 26 with 100% purity
(diastereoisomeric mixture 1:1). 1H NMR (D2O) d 8.62 (m, 1.5H), 6.64 (s, 0.5H)
5.89(d, J = 3.5 Hz, 1H), 4.56(m, 1H), 4.42 (m, 1H), 4.21 (m, 1H), 3.91–4.13 (m, 2H);
31P NMR (D2O) d 56.76 and 56.85 (diastereomers); MS m/z (Mꢁ + 1) 324.03.
1-(2,3-Di-O-benzoyl-5-deoxy-5-iodo- -D-ribofuranosyl)-1,2,4-triazole-3-carbox-
amide (44). A solution of 37 (1.8 g, 5.0 mmol) was dissolved in anhydrous pyridine
(10 mL) was cooled to 0°C and benzoyl chloride (1.3 mL, 11.0 mmol) was added.
After 1 h at same temperature, the mixture was poured into saturated sodium
bicarbonate and extracted with ethyl acetate. The organic phase was dried over sodium
sulfate and evaporated. Purification on silica gel column using 3% methanol in
dichloromethane gave 2.1 g of 44. (CDCl3) 8.46 (s, 1H), 7.94 (m, 4H), 7,58 (m, 2H),
7.37 (m, 4H), 7.02 (s, br, 1H), 6.27 ((d, J = 3.2 Hz, 1H),6.13 (m, 1H),5.85 (m, 2H),
4.64 (m, 1H), 3.50–3.66 (m, 2H).
1-[5-Deoxy-5-(hydroxyl-H-phosphinyl)- -D-ribofuranosyl]-1,2,4-triazole-3-car-
boxamide (27). A mixture of ammonium phosphinate (0.4 g. 5.0 mmol) and HMDS
(1.07 mL, 5.0 mmol) was heated at 100°C for 2 h under argon. The resulting
bis(trimethylsilyl)phosphonite was cooled to 0°C and compound 44 (560 mg, 1.0 mmol)
in 20 mL of dichloromethane was added. The reaction mixture was stirred at room
temperature overnight, filtered and concentrated. The oily residue was dissolved in
5 mL of dichloromethane and 5 mL of methanol, stirred for 2 h at room temperature
and evaporated. Aqueous ammonium (28%, 10 mL) was added to the oily residue and
stirred at room temperature for 4 h. The mixture was concentrated to dryness and
purified on reverse-phase HPLC to give 20 mg of 27 with 100% purity. 1H NMR
(D2O) d 8.56 (s, 1H), 7.59 (s, 0.5H), 5.86(d, J = 3.8 Hz, 1H), 4.58(m, 1H), 4.24 (m,
2H), 1.85–2.0(m, 2H); 31P NMR (D2O) d ꢁ 20.06 (s); MS m/z (Mꢁ + 1) 292.00.
1-[5-Deoxy-5-(dihydroxyphosphinyl)- -D-ribofuranosyl]-1,2,4-triazole-3-car-
boxamide (28). Compound 44 (1.6 g) was dissolved in triethylphosphite (5 mL) and
heated to 100°C for 50 h. Excess reagent was evaporated to dryness under high vacuum
and the residue was adsorbed on a small amount of silica gel. The silica gel bearing the
product was loaded on a silica gel column and eluted with 3% methanol in
dichloromethane to give 500 mg of 45. (CDCl3) d 8.44 (s, 1H), 7.96 (m, 4H), 7.54 (m,
2H), 7.26 (m, 4H) 7.05 (s, br, 1H) 6.25 (d, J = 3.02 Hz 1H) 6.04 (m, 1H), 5.86 (m,
1H), 5.68 (s, br, 1H), 4.95 (m, 1H), 4.08 (m, 4H), 2.01–2.30 (m, 2H) 1.26 (m, 6H). 31P
NMR (D2O) d 26.35 (s).
Compound 45 (500 mg, 0.9 mmol) was dissolved in DMF and acetonitrile (1:1, 10
mL) and bromotrimethylsilane (0.60 mL, 4.5 mmol) was added. The reaction mixture
was stirred at room temperature for 6 h and then concentrated under high vacuum. The
residue was co-evaporated with methanol and toluene three times. Aqueous ammonia
(28%, 15 mL) was added to the residue and stirred at room temperature for 6 h. After
evaporation of aqueous solution, the residue was purified on reverse-phase HPLC to
1
give 50 mg of 28 with 100% purity. H NMR (D2O) d 8.56 (s, 1H), 5.83(d, J = 3.8 Hz,

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334
Wang et al.
1H), 4.55(m, 1H), 4.25 (m, 2H), 1.91–2.0(m, 2H); 31P NMR (D2O) d 20.46 (s); MS m/
z (Mꢁ + 1) 308.20.
4-Carbamoyl-1-[5-deoxy-5-(dihydroxyphosphinyl)- -D-ribofuranosyl]-1,3-imi-
dazolium-5-olate (29). A suspension of 4-carbamoyl-1,3-imidazolium-5-olate[21] (188
mg, 1.48 mmol) and sodium sulfate (20 mg) in HMDS (3 mL) and anhydrous xylene (3
mL) was heated under reflux for 3 h and converted to a clear solution. After
evaporation of the volatiles, the residue was dried under high vacuum for 30 min, then
dissolved in 4 mL of anhydrous 1,2-dichloroethane. Stannic tetrachloride (140 mL, 1.18
mmol) was added, followed by addition of 1-O-acetyl-5-deoxy-2,3-di-O-benzoyl-5’-
(diethoxyphosphinyl)-D-ribofuranose[22] (700 mg, 1.33 mmol) in 1,2-dichloroethane (2
mL) and then trimethylsilyl triflate (85 mL, 0.44 mmol). The resulting mixture was
stirred at room temperature under argon for 6 days. After cooling with ice, the stirred
mixture was diluted with chloroform and 5% aqueous sodium bicarbonate and then
filtered through celite. The organic layer was washed with aqueous sodium bicarbonate,
dried over sodium sulfate, and concentrated to dryness. Chromatography on silica gel
with 10% MeOH and 4% TEA in methylene chloride gave 310 mg of 4-carbamoyl-1-
[5-deoxy-2,3-di-O-benzoyl-5-(diethoxyphosphinyl)-b-D-ribofuranosyl]-1,3-imidazolium-
1
5-olate as a colorless foam; H NMR d: 1.32, 1.29 (2t, 6H, J = 7.2 Hz, OCH2CH3),
2.53 (m, 2H, H-5’), 4.11 (m, 4H, OCH2CH3), 4.70 (m, 1H, H-4’), 5.90 (t, 1H, J = 5.4
Hz, H-2’); 6.09 (t, 1H, J = 5.7 Hz, H-3’), 6.15 (d, 1H, J = 4.8 Hz, H-1’), 7.28–7.96 (m,
10H, Bz), 8.19 (s, 1H); 31P NMR (CDCl3) 27.11; MS (m/z) 586 (M+ ꢁ 1).
A solution of the above product (203 mg, 0.337 mmol) and bromotrimethylsilane
(144 mL, 1.1 mmol) in anhydrous acetonitrile (1 mL) was kept at room temperature
overnight and then concentrated to dryness. The residue was dissolved in saturated
methanolic ammonia and stirred at room temperature for 12 h. After evaporation of
volatiles, the residue was subjected to purification on reverse-phase HPLC to yield
21.2 mg of 29 (purity: 94.7%); 31P NMR (D2O) d 19.8 (s); MS m/z (Mꢁ + 1) 322., 645
(MM+ ꢁ 1).
Assay for Inhibition of IMPDH Activity
Human IMPDH Type I and Type II isozymes and C. albicans IMPDH were
acquired from the Department of Biochemistry, Brandeis University. The IMPDH assay
followed a modification of a reported procedure.[12] Assay buffer conditions: 200 uL-total/
reaction (50 mM Tris-HCl, pH 8.0, 100 mM KCl, 3 mM EDTA, 1 mM DTT, 50 uM
IMP, 150 uM NAD, 30 nM purified human type I or II IMPDH, or 7.5 nM purified
C. albicans IMPDH). The P1Ms were tested at various concentrations up to 500 uM
final concentration. The assay was performed in duplicate in 96-well plates (Corning).
An appropriate volume of assay buffer, containing the substrates IMP and NAD, was
pipetted into the plate wells. The P1Ms were added to the reactions at the desired
concentrations. The reactions were initiated by addition of the enzymes. The reactions
were allowed to proceed for 5 minutes at 25°C. Blank reactions were prepared in
parallel with the test reactions in which enzyme was omitted from the reactions,
substituted by an appropriate volume of the buffer. The oxidation of inosine 5’-
monophosphate (IMP) to xanthosine 5’-monophosphate (XMP) by IMPDH was coupled
to the reduction of nicotinamide adenine dinucleotide (NAD). Production of NADH

ORDER
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Azole Nucleoside 5’-Monophosphate Mimics
335
was monitored at 340 nm using a UV/VIS microplate spectrophotometer (SpectraMax
Plus 384, Molecular Devices Corp.) or at 474 nm (excitation wavelength = 344 nm)
using a microplate fluorometer (SpectraMax Gemini XS, Molecular Devices Corp.).
Initial velocity data (mA minꢁ 1) was collected for each experiment, performed at least
two times, and fit to standard equations for % inhibition and Ki for competitive
inhibition or irreversible inhibition, where appropriate. The same screening assay
format was used for determinations of % inhibition by P1Ms of IMPDH from several
organisms–including Human isoforms I and II, and fungal isoforms–except that
amounts of different enzymes added were adjusted so as to achieve similar activities.
Serum Stability Assessment
The stability of nucleotide mimics was assessed in fetal calf serum, generally
following a published procedure.[25] Fetal calf serum purchased from HyClone
Corporation was mixed 1:1 with each compound in Tris-HCl buffer containing MgCl2.
The final concentrations of the reaction components were as follows: 50 mM Tris-HCl,
pH 7.4, 0.1 mM MgCl2, 500 mM nucleotide mimic, 10% (v/v) fetal calf serum. The
reaction mixtures were incubated at 37°C. At appropriate times, aliquots of 25 ml were
removed and added to 65 ml ice-cold methanol. These solutions were incubated for
at least one hour at ꢁ 20°C. After incubation, samples were centrifuged for 20 minutes
at high speed in a microcentrifuge. The supernatant was transferred to a clean tube and
the extract was concentrated to dryness under vacuum in a LabConco Centrivap
Concentrator. The dried extracts were resuspended in water and filtered to remove
particulates and then analyzed on reverse phase HPLC. The reverse phase HPLC columns
used for the analysis were a Phenomenex C18 Aqua column (3 ꢀ 150 mm). The HPLC
flow rate was 0.5 ml/min with the following buffer system: 5 mM tetrabutylammonium
acetate, 50 mM ammonium phosphate, and an acetonitrile gradient running from 5% up
to as high as 60%. The amount of remaining parent compound at each time point was
used to determine the half-life of the compound. Time points were only taken through
24 hours so that if greater than 50% of a compound was still intact after 24 hours
incubation the half-life was expressed as > 24 hours. Unmodified nucleoside 5’-
monophosphates were used as positive controls. Under these conditions unmodified
nucleoside 5’-monophosphates had half-lives of approximately three to six hours.
ACKNOWLEDGMENT
We wish to thank Professor Lizbeth Hedstrom of Brandeis University for providing
IMPDH enzymes and useful discussion.
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Received August 15, 2003
Accepted October 17, 2003

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