Acyclic nucleoside/nucleotide analogues with an imidazole ring skeleton

Article abstract of DOI:10.1081/NCN-100105250

Syntheses of a few acyclic nucleoside and acyclic nucleoside phosphonate analogues containing an imidazole ring have been reported. These analogues include methyl 1-(2-hydroxyethoxymethyl)imidazole-4, 5-dicarbo-xylate (1), 4,5-dicarbamoyl-1-(2-hydroxyetho

Full text of DOI:10.1081/NCN-100105250

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ACYCLIC NUCLEOSIDE/NUCLEOTIDE ANALOGUES WITH
AN IMIDAZOLE RING SKELETON
Huan-Ming Chen a & Ramachandra S. Hosmane b
a Laboratory for Drug Design and Synthesis, Department of Chemistry & Biochemistry ,
University of Maryland , Baltimore County, 1000 Hilltop Circle, Baltimore, MD, 21250, U.S.A.
b Laboratory for Drug Design and Synthesis, Department of Chemistry & Biochemistry ,
University of Maryland , Baltimore County, 1000 Hilltop Circle, Baltimore, MD, 21250, U.S.A.
Published online: 17 Aug 2006.
To cite this article: Huan-Ming Chen & Ramachandra S. Hosmane (2001) ACYCLIC NUCLEOSIDE/NUCLEOTIDE ANALOGUES WITH
AN IMIDAZOLE RING SKELETON, Nucleosides, Nucleotides and Nucleic Acids, 20:8, 1599-1614, DOI: 10.1081/NCN-100105250
To link to this article: http://dx.doi.org/10.1081/NCN-100105250
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(8), 159971614 (2001)
ACYCLIC NUCLEOSIDE=NUCLEOTIDE
ANALOGUES WITH AN IMIDAZOLE
RING SKELETON
Huan-Ming Chen and Ramachandra S.Hosmane *
Laboratory for Drug Design and Synthesis,
Department of Chemistry & Biochemistry, University of Maryland,
Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250
ABSTRACT
Syntheses of a few acyclic nucleoside and acyclic nucleoside phosphonate
analogues containing an imidazole ring have been reported. These
analogues include methyl 1-(2-hydroxyethoxymethyl)imidazole-4,
5-dicarbo-xylate (1), 4,5-dicarbamoyl-1-(2-hydroxyethoxymethyl)imid-
azole (2), 4,5-dicyano-1-(2-hydroxyethoxymethyl)imidazole (4), Methyl
1-(2-bromoethoxymethyl)imidazole-4,5-dicarboxylate (7), 4,5-dicyano-
(2-bromoethoxymethyl)imidazole (8), and Methyl 1-(2-phosphono-
methoxyethyl)imidazole (10). Also reported are a few potential prodrugs
of the above compounds, including the acetyl derivatives 5 and 6 (of 1
and 4, respectively), and the diethyl phosphonate ester 9 (of 10).
In addition, the corresponding benzyl-protected precursors 11 and 12
(of 1 and 4, respectively), along with their common hydrolysis product,
1-(2-benzyloxy-ethoxymethyl)-4,5-imidazoledicarboxylic acid (3), are
reported. Another potential prodrug included in the list is 1-(2-acetox-
yethyl)-4,5-dicyanoimidazole (15). The compounds were screened for
in vitro antiviral activity against a wide variety of herpes and respiratory
viruses. The most active compound was the phosphonate analogue 9
which exhibited an anti-measles virus activity with an EC50 of < 2.5
mg=mL and an SI value of > 176.
*Corresponding author.
1599
Copyright # 2001 by Marcel Dekker, Inc.
www.dekker.com

1600
CHEN AND HOSMANE
INTRODUCTION
Acyclic nucleoside179 and acyclic nucleotide10725 analogues have played
important roles as powerful antiviral and anticancer agents in modern
medicine1726. These are the analogues of natural nucleosides or nucleotides
whose pentafuranosyl sugar ring has been replaced with an acyclic moiety
that mimics part or whole of the natural sugar or sugar phosphate. Included
in this category are 9-(2-hydroxyethoxymethyl)guanine (acyclovir, ACV)1
and 9-[2-(phosphono-methoxy)ethyl]adenine (PMEA)10711, among host of
other nucleoside/nucleotide analogues discovered in recent years1726
.
Acyclovir is highly e€ective for treatment against herpes simplex viruses
(HSV-1 and HSV-2)7, varicella-zoster virus (VZV)678, and also shows good
activity against Epstein-Barr virus (EBV)26. PMEA10711 and its guanine
counterpart PMEG17718 are two of the most potent, broad spectrum acyclic
nucleotide analogues reported to date11,19720. The presence of a phospho-
nate group, instead of a phosphate moiety as present in natural nucleotides,
renders PMEA less prone to in vivo chemical or enzymatic hydrolysis, thus
increasing its therapeutic life time and ecacy. It has also been discovered
that the antiviral ecacy further increases if the compound is administered
as a prodrug, for example, as a phosphonate ester (e.g. Bis-POM-PMEA
where Rˆ(CH3)3CCO2CH2-) that has a better chance of penetration
through the cell membrane, but undergoes facile hydrolysis in the cell
interior to form the free acid10. Most, if not all, acyclic nucleosi-
de=nucleotide analogues require in vivo conversion into their triphosphate
derivatives by kinases so as to be incorporated into viral genome, and
subsequently cause nucleic acid chain termination17. In this regard, phos-
phonate derivatives o€er a distinct advantage over other conventional
nucleoside analogues in that they are able to bypass the crucial ®rst step of
phosphorylation, the lack of which has often been found to be the under-
lying cause of therapeutic failure of many otherwise promising nucleoside
analogues.
In spite of extensive research performed on acyclic purine and pyr-
imidine nucleoside=nucleotide analogues in recent years, relatively less is
known on the same analogues containing 5-membered heterocyclic ring

ACYCLIC NUCLEOSIDE/NUCLEOTIDE ANALOGUES
1601
counterparts. This is surprising in view of the fact that a number of imida-
zole, pyrazole, and triazole nucleoside analogues have long been documented
as potent antiviral, antibacterial, and antitumor agents27731. These include,
but are not limited to, bredinin32, a potent nucleoside antibiotic containing
an imidazole ring, and ribavirin29,33736, a broad spectrum antiviral nucleoside
possessing a triazole nucleus. We report here the synthesis of a few acyclic
nucleoside and nucleoside phosphonate analogues of imidazole, which con-
tain the acyclic sugar moiety of ACV or PMEA.
RESULTS AND DISCUSSION
Chemistry
The analogues reported herein include methyl 1-(2-hydroxyethoxy-
methyl)imidazole-4,5-dicarboxylate
(1),
4,5-dicarbamoyl-1-(2-hydroxy
ethoxymethyl)imidazole (2), 4,5-dicyano-1-(2-hydroxyethoxymethyl)imida
zole (4), Methyl 1-(2-bromoethoxymethyl)imidazole-4,5-dicarboxylate (7),
4,5-dicyano-(2-bromoethoxymethyl)imidazole (8), and Methyl 1-(2-phos-
phonomethoxyethyl)imidazole (10). Also reported are a few potential pro-
drugs of the above compounds, including the acetyl derivatives 5 and 6 (of 1
and 4, respectively), and the diethyl phosphonate ester 9 (of 10). In addition,
the corresponding benzyl-protected precursors 11 and 12 (of 1 and 4,
respectively), along with their common hydrolysis product, the dicarboxylic
acid 3, are reported. Another potential prodrug included in the list is 4,5-
dicyano-1-(2-acetoxyethyl)imidazole (15). Most of the chemistry employed in
here is either simple or well documented in the literature37.
Syntheses of 1, 2, 5, 7, 9, 10, and 11 are outlined in Scheme I, while
those of 4, 6, 12 and 15 are given in Scheme II. Synthesis of compound 3,
obtained from both 4,5-dicarbomethoxy- and 4,5-dicyanoimidazole pre-
cursors, is outlined in both schemes I and II. The acyclic nucleoside 1 was
prepared by sequential reactions involving alkylation of methyl 4,5-imida-
zoledicarboxylate (13)38 with 2-benzyloxyethoxymethyl chloride39 in the
presence of triethylamine or potassium carbonate in dimethylformamide (to
form 11), followed by catalytic hydrogenation over palladiun-charcoal in
methanol. The acyclic nucleoside 2 was synthesized by treatment of 5 with
ammonium hydroxide or methanolic ammonia. Compound 5, in turn, was
prepared by sequential silylation and alkylation of 13, using bis(trime-
thylsilyl)tri¯uoroacetamide (BTMSTFA)40 in pyridine and 2-acetoxyethox-
ymethyl bromide41, respectively. The acyclic nucleoside 4 was prepared by
‡
hydrolysis of 6, using an ion-exchange resin (H form) as shown in Scheme II.
Compound 6 was prepared from 4,5-dicyanoimidazole (14)40, using an
analogous procedure employed for 5. The acyclic nucleoside phosphonate
analogue 10 was synthesized by sequential reactions involving base-catalyzed

1602
CHEN AND HOSMANE
condensation of 13 with diethyl (2-p-toluenesulfonyloxyethoxymethanephos
phonate42 (to form 9), followed by dealkylation of the phosphonate ester
groups using bromotrimethylsilane in acetonitrile. The acyclic nucleoside 3
containing a 4,5-dicarboxyimidazole nucleus was prepared by saponi®cation
of either the corresponding diester precursor 11 or the dinitrile precursor 12.
The acyclic nucleosides 7 and 8 were prepared by condensation of the sily-
lated derivatives of the respective imidazole precursors 13 and 14 with 2-
bromoethoxymethyl chloride43. Compound 15 was prepared by alkylation of
14 with 2-acetoxyethyl bromide40 in the presence of potassium carbonate in
dimethylformamide. The percent yields shown for each reaction in Schemes I
and II are for the pure products obtained either by recrystallization or by
chromatography. All products were characterized by spectroscopic and by
either microanaytical or high resolution mass spectral analyses.
Biological Screening
A majority of compounds of Schemes I and II were screened for invitro
antiviral activity against a wide variety of herpes and respiratory viruses
under the auspices of the antiviral drug screening program of the National
Institute of Allergy and Infectious Diseases (NIAID)44. While the herpes
family of viruses included HSV-1, HSV-2, HCMV, MCMV, VZV, and EBV,
the respiratory viruses screened against were adenovirus type I, measles
virus, parain¯uenza virus type 3, rhinovirus type 2, in¯uenza A (H1N1),
in¯uenza A (H3N2), and in¯uenza B. A number of the tested compounds
failed to show either activity or adequate selectivity over toxicity against
most of the viruses, with the exception of compound 9 which exhibited an
EC50 of < 2.5 mg=mL, an IC50 of 440 mg=mL, and a selectivity index (SI)
of > 176 against the measles virus in a Cytopathic E€ect Inhibition (CPE)
assay45. The compound tested positive in a Neutral Red (NR) uptake assay45
as well with an EC50 of 2.5 mg=mL, an IC50 of 59 mg=mL, and a selectivity
index (SI) of 24. Compound 10, which is anticipated to possess a similar
activity pro®le as its likely prodrug precursor 9, remains yet to be screened.
Compounds 9 and 10 are the target imidazole analogues of PMEA described
under Introduction.
EXPERIMENTAL
1
The H and 13C nmr spectra were recorded on a General Electric QE-
300 nmr spectrometer operating at 300 MHz for 1H and 75 MHz for 13C. The
data are reported in the following format: Chemical shift (all relative to
Me4Si), multiplicity (s ˆ singlet, d ˆ doublet, dt ˆ double triplet, dd ˆ double
doublet, t ˆ triplet, q ˆ quartet, m ˆ multiplet, b ˆ broad, coupling constants,
integration and assignment). Evaporations were done under reduced pressure

ACYCLIC NUCLEOSIDE/NUCLEOTIDE ANALOGUES
1603
Scheme I. Synthesis of acyclic nucleoside and nucleoside phosphonate analogues.

1604
CHEN AND HOSMANE
Scheme II. Synthesis of acyclic nucleoside analogues 3, 4, 6, 8, 12, and 15.
on a rotary evaporator. Thin layer chromatography was performed on
Merck Kieselgel 60F (0.2 mm thickness). Elemental microanalyses were
254
performed by Atlantic Microlab, Inc., Norcross, Georgia. Melting points
were determined on a Thomas Hoover capillary melting point apparatus and
are uncorrected. Dry solvents were prepared as follows: methanol was dis-
tilled from calcium hydride and was stored over molecular sieves (type 3A);
dimethylformamide was dried over calcium oxide and then distilled under
reduced pressure from calcium hydride, and was subsequently stored over
molecular sieves (type 3A). All starting materials were purchased from
Aldrich Chemical Co. All solvents were reagent grade and were purchased

ACYCLIC NUCLEOSIDE/NUCLEOTIDE ANALOGUES
1605
from VWR Scienti®c. All yields reported are for pure, dry compounds that
require no further puri®cation for use in other reactions.
Methyl 1-(2-Hydroxyethoxymethyl)-4,5-imidazoledicarboxylate(1)
Methyl
1-(2-benzyloxyethoxymethyl)-4,5-imidazoledicarboxylate(11)
(1.74 g, 5 mmol) was dissolved in a mixture of 20mL of methanol and 5 mL
of glacial acetic acid, and the solution was transferred to a hydrogenation
bottle. Palladium on activated carbon (10% (1.74 g) was added, and the
bottle shaken in a Parr Hydrogenation apparatus at 50psi for 48 h. The
reaction mixture was ®ltered through a pad of Celite.TM A TLC analysis
showed that the reaction was complete. The ®ltrate was evaporated to dry-
ness. The residue was puri®ed by silica gel ¯ash chromatography, eluting ®rst
with chloroform and then with chloroform-methanol (20: 1) to give 1 as a
pure waxy product (semi-solid) (1.1 g, 85%). Rf 0.37 (CHCl3-MeOH, 10: 1).
1H-NMR(DMSO-d6) d 8.12 (s, 1H, imidazole), 5.57 (s, 2H, NCH2O), 4.70
(brs, 1H, OH, exchangeable with D2O), 3.83 (s, 3H, OCH3), 3.78 (s, 3H,
OCH3), 3.40(m, 4H, OCH CH2OH); 13C-NMR (DMSO-d6) d 52.05, 52.69,
2
59.75, 70.44, 75.84, 136.27, 140.85, 140.97, 160.05, 162.73.
Anal: Calcd. for C10H14N2O6: C, 46.49; H, 5.47; N, 10.85. Found: C,
46.52; H, 5.52; N, 10.69.
4,5-Dicarbamoyl-1-(2-Hydroxyethoxymethyl)imidazole (2)
A solution of methyl 2-acetoxyethoxymethyl-4,5-imidazoledicarbox-
ylate (50mg) in 5 mL of 30% aqueous ammonia was stirred overnight. The
solvent was removed in vacuo, the residue was puri®ed by silica gel column
chromatography to give a waxy solid (23 mg, 60%), Rf 0.20, CHCl3:
Methanol (20: 1). 1H NMR (CDCl3) d 10.59 (brs, 1H, NH, exchangeable
with D2O), 8.09 (s, 1H, imidazole), 8.03 (brs, 1H, NH, exchangeable with
D2O), 7.85 (brs, 1H, NH, exchangeable with D2O), 7.65 (brs, 1H, NH,
exchangeable with D2O), 5.85 (s, 2H, NCH2O), 3.273.5 (m, 4H, OCH2-
‡
CH2OH); FABMS m=z: 229 [MH ].
Anal: HRMS (FAB): Calcd for C8H14N4O4 229.0937, Found; 229.0948.
1-(2-Benzyloxyethoxymethyl)-4,5-imidazoledicarboxylic Acid (3)
Method A: A mixture of 1-(2-benzyloxyethoxymethyl)-4,5-dicyanoimi-
dazole (13) (50mg, 0.18 mmol) and 6N aq. sodium hydroxide (5 mL) was
re¯uxed for 24 h. The reaction mixture was neutralized with 1 N hydrochloric
acid, ®ltered and the solid was washed with water to give a crude product,
which was recrystallized from water to obtain pure 3 (43 mg, 74%), mp >
250 ꢀC. Rf 0.55 (CHCl3-MeOH, 2 : 1).

1606
CHEN AND HOSMANE
1H-NMR (DMSO-d6) d 9.02 (s, 1H, imidazole), 7.2477.35 (m, 5H, Ph),
5.91 (s, 2H, NCH2O), 4.44 (s, 2H, PhCH2), 3.75 (t, 2H, J ˆ 4.8 Hz, OCH2-
CH2OCH2Ph), 3.53 (t, 2H, J ˆ 4.8 Hz, OCH2CH2OCH2Ph).
Anal: HRMS (FAB): Calcd for C15H17N2O6 321.1086. Found:
321.1075.
MethodB: Compound 3 was also obtained in 92% yield by hydrolysis of
methyl 1-(2-benzyloxyethoxymethyl)-4,5-imidazoledicarboxylate (11), using
the procedure described under Method A above.
1-(2-Hydroxyethoxymethyl)-4,5-dicyanoimidazole (4)
A mixture of 1-(2-acetoxyethoxymethyl)-4,5-dicyanoimidazole (6)
(0.5 g, 2.14 mmol) and Dowex 50WX8-200 ion-exchange resin (H form, pre-
‡
washed with ethanol) (0.5 g) in 10 mL of ethanol was re¯uxed with stirring
for 24 h until the reaction was complete. The mixture was ®ltered, the resin
washed with ethanol and the combined ®ltrate was concentrated in vacuo.
The residue was puri®ed by silica gel ¯ash chromatography, eluting with
chloroform-methanol (20: 1) to give 4 as a colourless liquid 0.35 g (85%). Rf
1
0.37 (CHCl3-MeOH, 10: 1). H-NMR(DMSO-d6) d 8.09 (s, 1H, imidazole),
5.41 (s, 2H, NCH2O), 4.65 (brs, 1H, OH, exchangeable with D2O), 3.50(m,
‡
4H, OCH2CH2OH); FABMS m=z: 193 [MH ].
Anal: HRMS (FAB): Calcd for C8H9N4O2 193.0726. Found:
193.0730.
Methyl 1-(2-Acetoxyethoxymethyl)-4,5-imidazoledicarboxylate (5)
A mixture of methyl 4,5-imidazoledicarboxylate (1.84 g, 10mmol),
bis(trimethylsilyl)- tri¯uoroacetamide (BTMSTFA) (10.3 g, 40 mmol) and
pyridine (5 mL, 60mmol) in 30mL of dry acetonitrile was stirred for 12 h at
room temperature. The solvent and excess reagents were evaporated invacuo
under anhydrous conditions, and the residue coevaporated with dry acet-
onitrile and toluene (3610mL) was kept in vacuum until a syrup was formed,
and then dissolved in 30mL of dry acetonitrile. To the solution was added
2-acetoxyethoxymethyl bromide(2.94 g, 15 mmol). The mixture was stirred
for 24 h at room temperature, and the reaction was quenched with methanol
(20mL) and stirred for 2 h. The solvents were evaporated and the residue was
puri®ed by column chromatography eluting with chloroform to give 5 as a
1
pure colourless liquid (2.4 g, 80%). H-NMR (CDCl3) d 7.76 (s, 1H, imida-
zole), 5.64 (s, 2H, NCH2O), 3.95 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 4.19 (t,
2H, J ˆ 6.0Hz, CH 2OAc), 3.74 (t, 2H, J ˆ 6.0Hz, OCH 2), 2.10(s, 3H, Ac);
‡
FABMS m=z: 301 [MH ].
Anal: HRMS (FAB): Calcd for C12H17N2O7 301.1036. Found:
301.1019.

ACYCLIC NUCLEOSIDE/NUCLEOTIDE ANALOGUES
1-(2-Acetoxyethoxymethyl)-4,5-dicyanoimidazole (6)
1607
A mixture of 4,5-dicyanoimidazole (1.2 g, 10mmol), bis(trimethylsilyl)-
tri¯uoroacetamide (BTMSTFA) (10.3 g, 40 mmol) and pyridine (5 mL,
60mmol) in 30mL of dry acetonitrile was stirred for 12 h at room tem-
perature. The solvent and excess reagents were evaporated in vacuo under
anhydrous conditions, and the residue coevaporated with dry acetonitrile and
toluene (3610mL) was kept in vacuum until a syrup was formed, and then
dissolved in 30mL of dry acetonitrile. To the solution was added 2-acetox-
yethoxymethyl bromide(2.94 g, 15 mmol). The mixture was stirred for 24 h at
room temperature, and the reaction was quenched with methanol (20mL)
and stirred for 2 h. The solvents were evaporated and the residue was puri®ed
by column chromatography eluting with chloroform to give 6 as a pure
colourless liquid (2.0g, 85%). Rf 0.55 (CHCl -MeOH, 10:1). 1H-NMR
3
(CDCl3) d 7.85 (s, 1H, imidazole), 5.45 (s, 2H, OCH2N), 4.25 (t, 2H,
J ˆ 4.8 Hz, AcOCH2), 3.81 (t, 2H, J ˆ 4.8 Hz, OCH2), 2.10(s, 3H, Ac);
‡
FABMS m=z: 235 [MH ].
Anal: HRMS (FAB): Calcd for C10H11N4O3 235.0831. Found:
235.0836.
Methyl 1-(2-Bromoethoxymethyl)-4,5-imidazoledicarboxylate (7)
A mixture of methyl 4,5-imidazoledicarboxylate (1.84 g, 10mmol),
bis(trimethylsilyl)-tri¯uoroacetamide (BTMSTFA) (10.3 g, 40 mmol) and
pyridine (5 mL, 60mmol) in 30mL of dry acetonitrile was stirred for 12 h
at room temperature. The solvent and the excess reagents were evaporated
in vacuo under anhydrous conditions, and the residue coevaporated with
dry acetonitrile and toluene (3610mL) was kept in vacuum until a syrup
was formed, and then dissolved in 30mL of dry acetonitrile. To the
solution was added 2-bromoethoxymethyl chloride (2.58 g, 15 mmol). The
mixture was stirred for 24 h at room temperature, and the reaction was
quenched with methanol (20mL) and stirred for 2 h. The solvents were
evaporated and the residue was puri®ed by column chromatography
eluting with chloroform to give 7 as a pure colorless liquid (2.7 g, 84%). 1H-
NMR (CDCl3) d 7.78 (s, 1H, imidazole), 5.68 (s, 2H, NCH2O), 3.95 (s, 3H,
OCH3), 3.93 (s, 3H, OCH3), 3.81 (t, 2H, J ˆ 6.0Hz, OCH 2), 3.42 (t, 2H,
‡
J ˆ 6.0Hz, CH 2Br); FABMS m=z: 321, 323 [MH ].
Anal: HRMS(FAB): Calcd for C10H14N2O579Br 321.0086. Found:
321.0089; Calcd for C10H14N2O581Br 323.0066. Found: 323.0070.
1-(2-Bromoethoxymethyl)-4,5-dicyanoimidazole (8)
A mixture of 4,5-dicyanoimidazole (1.2 g, 10mmol), bis(trimethylsilyl)-
tri¯uoro-acetamide (BTMSTFA) (10.3 g, 40 mmol) and pyridine (5 mL,

1608
CHEN AND HOSMANE
60mmol) in 30mL of dry acetonitrile was stirred for 12 h at room
temperature. The solvent and the excess reagents were evaporated in vacuo
under anhydrous conditions, and the residue coevaporated with dry acetoni-
trile and toluene (3610mL) was kept in vacuum until a syrup was formed,
and then dissolved in 30mL of dry acetonitrile. To the solution was
added 2-bromoethoxymethyl chloride (2.58 g, 15 mmol). The mixture was
stirred for 24 h at room temperature, and the reaction was quenched with
methanol (20mL) and stirred for 2 h. The solvents were evaporated and the
residue was puri®ed by column chromatography eluting with chloroform to
give 8 as a pure colorless liquid (2.2 g, 87%). Rf 0.59, Chloroform:Methanol
1
(10: 1). H-NMR (CDCl3) d 7.90(s, 1H, imidazole), 5.57 (s, 2H, NCH O),
2
3.93 (t, 2H, J ˆ 5.4 Hz, OCH2), 3.49 (t, 2H, J ˆ 5.4 Hz, CH2Br); FABMS m=z:
‡
254, 256 [MH ].
Anal: HRMS (FAB): Calcd for C8H8N4O79Br 254.9880. Found:
254.9883; Calcd for C8H8N4O81Br 256.9861. Found: 256.9858.
Methyl 1-(2-diethoxyphosphonylmethoxyethyl)-4,5-
imidazoledicarboxylate (9)
A mixture of methyl imidazole-4,5-dicarboxylate (1.0g, 5.4 mmol) and
potassium carbonate (0.75g, 5.4 mmol) in 40 mL of anhydrous DMF was
stirred at 100 ꢀC for 3 h, then a solution of diethyl 2-p-toluenesulfonylox-
yethoxymethanephosphonate (2.75 g, 7.5 mmol) in 5 mL of dry DMF was to
the above mixture and continued to be stirred for 48 h at 100 ꢀC. The
reaction mixture was evaporated in vacuo. The residue dissolved in chloro-
form and washed with 0.1 M hydrochloric acid and water. After drying over
MgSO4, the chloroform solution was evaporated to dryness in vacuo. The
resulting residue was puri®ed by column chromatography, eluted with
chloroform to give 9 as a colorless liquid (1.33 g, 65%). Rf 0.74, Chlor-
1
oform:Methanol (10: 1). H NMR (CDCl3) d 7.67 (s, 1H, imidazole), 4.44
(t, 2H, J ˆ 4.8 Hz, H-10), 4.10(dq, 4H, J P-OCH ˆ 8.1 Hz, J ˆ 6.9 Hz, CH2CH3),
3.92 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.88 (t, 2H, J ˆ 4.8 Hz, H-20), 3.75
(d, 2H, JP-CH ˆ 8.1 Hz, OCH2P), 1.31 (t, 6H, J ˆ 6.9 Hz, CH2CH3); 13C NMR
(CDCl3, 75.5 MHz) d 16.40(s, CH ), 16.48 (s, CH3), 46.79 (s, C-10), 52.17
3
(s, OCH3), 52.40(s, OCH 3), 62.33 (s, OCH2CH3), 62.41 (s, OCH2CH3), 65.17
0
0
(d, JP,C ˆ 166.4 Hz, OCH2P), 71.50(d, J
ˆ 9.9 Hz, C-2 ), 137.08 (C-4 or
P,C-2
5), 139.81 (C-5 or 4), 140.63 (C-2), 160.44 (CˆO), 162.74 (CˆO); FABMS
‡
m=z: 379 [MH ]; Anal: Calcd for C14H23N2O8PH2O: C, 42.64; H, 6.39; N,
7.10. Found: C, 42.29; H, 6.26; N, 6.84.; HRMS(FAB): Calcd for
C14H24N2O8P 379.1270, Found 379.1268.
Compound 9 was also prepared from pure diethyl 2-bromoethoxy-
methanephosphonate according to the above procedure.

ACYCLIC NUCLEOSIDE/NUCLEOTIDE ANALOGUES
1609
Methyl 1-(2-Phosphonylmethoxyethyl)-4,5-imidazoledicarboxylate (10)
To a solution of methyl 1-(2-diethoxyphosphonylmethoxyethyl)-4,5-
imidazoledicarboxylate (9) (0.48 g, 1.3 mmol) in 10 mL of dry acetonitrile was
added bromotrimethylsilane (0.40 g, 2.6 mmol), and the mixture was stirred
for 24 h at room temperature. The reaction mixture was evaporated to dry-
ness, the residue was codistilled with acetonitrile (3620mL), mixed with
water (30mL), and adjusted to pH 8 with triethylamine. The mixture was
allowed to stand for 1 h and then evaporated in vacuo and the residue
codistilled with methanol (2620mL), and then applied to a silica gel column.
The column was eluted ®rst with chloroform and then with chloroform-
methanol (10: 1). Evaporation of appropriate fractions a€orded 10 as a
colorless liquid (350 mg, 84%). Rf 0.13, Chloroform:Methanol:30%
Ammonium Hydroxide (2:1:0.3).
1H NMR (DMSO-d6) d 7.98 (s, 1H, imidazole), 5.64 (brs, 2H, OH,
exchangeable with D2O), 4.34 (t, 2H, J ˆ 4.8 Hz, H-10), 3.81 (s, 3H, OCH3),
3.77 (s, 3H, OCH3), 3.77 (t, 2H, J ˆ 4.8 Hz, H-20), 3.55 (d, 2H, JP-CH ˆ 8.4 Hz,
OCH2P); 13C NMR (DMSO-d6, 75.5 MHz) d 46.41 (s, C-10), 52.51 (s, OCH3),
0
53.07 (s, OCH3), 66.36 (d, JP,C ˆ 160.2 Hz, OCH2P), 71.09 (d, JP,C-2
ˆ
10.2 Hz, C-20), 135.32 (C-4 or 5), 141.43 (C-5 or 4), 141.43 (C-2), 160.45
‡
(CˆO), 162.95 (CˆO); FABMS m/z: 323 [MH ].
Anal: HRMS(FAB): Calcd for C10H16N2O8P 323.0644. Found:
323.0649.
Methyl 1-(2-Benzyloxyethoxymethyl)-4,5-Imidazoledicarboxylate (11)
A mixture of methyl 4,5-imidazoledicarboxylate (0.92 g, 5 mmol) and
K2CO3 (0.7 g, 5 mmol) was stirred for 1 h at 80 ꢀC. Then a solution of
1-benzyloxy-2-chloromethoxyethane (1.5 g, 7.5 mmol) in 20mL of DMF
was dropwise added, and the mixture was continued to stir for 24 h at
90 ꢀC. The reaction mixture was evaporated under reduced pressure, and
the residue was dissolved in 30mL of water and then neutralized with 2M
hydrochloric acid. The solution was extracted with chloroform. The
extract was evaporated in vacuo, and the residue was puri®ed by silica gel
column chromatography, eluting with chloroform to give 11 as a colourless
liquid, 1.22 g (70%); Rf 0.31 (chloroform-methanol (30:1)).
1H-NMR (CDCl3) d 7.72 (s, 1H, imidazole), 7.24±7.35 (m, 5H, Ph), 5.66
(s, 2H, NCH2O), 4.52 (s, 2H, PhCH2), 3.92 (s, 3H, OCH3), 3.91 (s, 2H,
OCH3), 3.63 (m, 4H, OCH2CH2O); 13C NMR (CDCl3) d (52.16, 53.10, 69.06,
69.71, 73.25, 76.44, 127.58, 127.58, 127.66, 127.66, 128.32, 128.32, 137.65,
139.38, 139.47, 160.21, 162.45.
Anal: Calcd for C17H20N2O6: C, 58.61; H, 5.79; N, 8.04. Found: C,
58.81; H, 5.88; N, 7.77.

1610
CHEN AND HOSMANE
1-(2-Benzyloxyethoxymethyl)-4,5-dicyanoimidazole (12)
A mixture of 4,5-dicyanoimidazole (0.6 g, 5 mmol) and K2CO3 (0.7 g,
5 mmol) was stirred for 1 h at 80 ꢀC. Then a solution of 1-benzyloxy-2-
chloromethoxyethane (1.5 g, 7.5 mmol) in 20mL of DMF was dropwise added
and continue to stirred for 24 h at 90 ꢀC. The mixture was evaporated under
reduced pressure, and the residue was dissolved in 30mL of water and neu-
tralized with 2M hydrochloric acid. The solution was extracted with chloro-
form. The extract was evaporated in vacuo, and the residue was puri®ed by
column chromatography, eluting with chloroform to give a colourless liquid,
1
0.85 g (60%); Rf 0.44 (chloroform-methanol (30 : 1)). H NMR (CDCl3) d
7.79 (s, 1H, imidazole), 7.26-7.39 (m, 5H, Ph), 5.53 (s, 2H, NCH2O), 4.51 (s,
‡
2H, PhCH2), 3.64 (m, 4H, OCH2CH2O); FABMS m/z: 283 [MH ].
Anal: HRMS(FAB): Calcd for C15H15N4O2 283.1195. Found:
283.1197.
1-(2-Acetoxyethyl)-4,5-dicyanoimidazole (15)
A mixture of 4,5-dicyanoimidazole (0.6 g, 5 mmol) and K2CO3 (0.7 g,
5 mmol) was stirred for 1 h at 80 ꢀC. Then a solution of 2-bromoethyl acetate
(1.25 g, 7.53 mmol) in 20mL of DMF was drop wise added, and the mixture
was continued to stir for 24 h at 90 ꢀC. The reaction mixture was evaporated
under reduced pressure, and the residue was dissolved in 30mL of water and
then neutralized with 2M hydrochloric acid. The solution was extracted with
chloroform. The extract was evaporated in vacuo, and the residue was pur-
i®ed by silica gel column chromatography, eluting with chloroform to give 15
as a colourless liquid, 0.71 g (70%), which crystallized upon standing, mp
1
92793 ꢀC. Rf 0.62 (CHCl3-MeOH, 10: 1). H NMR (CDCl3) d 8.25 (s, 1H,
imidazole), 4.67 (t, 2H, J ˆ 4.8 Hz, AcOCH2), 4.49 (t, 2H, J ˆ 4.8 Hz, CH2N),
2.05 (s, 3H, Ac); MS (EI) m/z: 204, 145, 144 (100), 132.
Anal: Calcd for C9H8N4O2: C, 52.94; H, 3.95; N, 27.44. Found: C,
52.78; H, 3.95; N, 27.28.
Biological Screening Protocols
The Cytopathic E€ect Inhibition (CPE) Assay. The CPE (virus e€ects on cell
morphology) inhibition assay used in this study was done as described by
Barnard et al45. Compounds, using a 1=2 log dilution scheme, were serially
diluted in a 96-well plate containing con¯uent cell monolayers (16104
cells=well) with MEM without serum as diluent. Virus in MEM
supplemented with 2% FBS (‡20mM MgCl 2 for rhinovirus; no FBS was
included in the in¯uenza virus assays, instead 0.2% trypsin was included to

ACYCLIC NUCLEOSIDE/NUCLEOTIDE ANALOGUES
1611
cleave hemagglutinin of virus to achieve a better attachment) was added
within ®ve minutes of drug addition in an equal volume to drug to the
appropriate wells at a multiplicity of infection (MOI) ˆ 0.001±0.01,
depending on the virus. The plates were incubated at 37 ꢀC (33 ꢀC for
rhinovirus) for 4±7 days until 100% CPE was detected in the virus
infected, untreated, control cells by light microscopy.
The positive control drugs that were employed are as follows: For
Measles Virus, Parain¯uenza Type 3 Virus and Respiratory syncytial virus
and In¯uenza viruses, the positive control drug is ribavirin, a 5-membered
heterocyclic nucleoside analog of guanine. For rhinovirus, the positive con-
trol drug was pirodavir, a compound that binds in the protein ``canyons'' of
the virus to prevent uncoating of the protein capsid surrounding the RNA of
the virus.
The compounds and the positive control drugs were assayed for virus
inhibition in quadruplicate and for cytotoxicity in duplicate. For each
compound, two wells were set aside as uninfected, untreated cell controls
per test and four wells per test received virus only and represented controls
for virus replication. Changes due to viral cytopathic e€ect were graded on
a scale of 174, grade 4 representing a scenario in which the entire (100%)
monolayer in a well showed viral cytopathic e€ect. For all CPE-based
assays, the 50% e€ective concentration (EC50) was calculated by regression
analysis using the means of the CPE ratings at each concentration of
compound.
Morphological changes due to compound cytotoxicity were graded on a
scale of 075; grade 5 was de®ned as 100% cytotoxicity. The 50% cytotoxic
dose (IC50) was calculated by regression analysis. After determining the EC50
and IC50 values, a selective index (SI) for each compound was then calculated
by using the formula, SI ˆ IC50=EC50.
Neutral Red (NR) Uptake Assay. This was done as described by Barnard
et al45. Medium was removed from each well of each plate used for the CPE
inhibition assay and 0.2 mL of NR (0.034% in physiological saline) was
added to the wells of the plates and incubated for 2 h at 37 ꢀC in the dark.
The NR solution was then removed from the wells and each plate was
rinsed two times with phosphate-bu€ered saline (pH 7.4). Equal volumes
(0.1 mL) of absolute ethanol and Sorenson citrate bu€er (0.1 M sodium
citrate, 0.1 M HCl, pH 4.2) were mixed together and added to the wells of
each plate. The plates were incubated in the dark for 30min at room
temperature to solubilize the dye. The plates were then gently mixed on a
96-well plate adapted vortexer for 1 min. Absorbance at 540nm and 450nm
was read with a microplate reader (Bio-Tek EL 1309; Bio-Tek Instruments,
Inc., Winooski, VT). Absorbance values were expressed as percent of
untreated controls and EC50 and IC50 values were calculated by
regression analysis.

1612
CHEN AND HOSMANE
ACKNOWLEDGMENTS
This research was supported by a grant from the National Institutes of
Health (# 1RO1CA71079). Mass spectral data were run at the Michigan
State University Mass Spectrometry Facility which was supported, in part,
by a grant (DRR-00480) from the Biotechnology Research Technology
Program, National Center for Research Resources, National Institutes of
Health. The authors gratefully acknowledge Dr. Chris Tseng of NIAID, Dr.
Earl Kern of the University of Alabama, Birmingham, and Drs. Robert
Sidwell, Dale Barnard, and D. Smee of the Utah State University, Logan.
For all the antiviral screening results reported in this paper, and described in
reference 44.
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the Program Ocer for Antiviral Research and Antimicrobial Chemistry of
the Virology Branch at the Division of Microbiology and Infectious Diseases,
the National Institute of Allergy and Infectious Diseases (NIAID), NIH,
Bethesda, Maryland. While screenings for the herpeses family of viruses were
performed by Dr. Earl Kern and his colleagues of the Department of Pediatrics
of the University of Alabama at Birmingham, screenings for the respiratory
viruses were carried out by Dr. Robert Sidwell, Dr. Dale Barnard, and Dr. D.
Smee of the Institute of Antiviral Research at the Utah State University, Logan.

1614
CHEN AND HOSMANE
45. Barnard, D.L.; Hill, C.L.; Gage, T.; Matheson, J.E.; Hu€man, J.H.;
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Received July 5, 2000
Accepted February 23, 2001