Synthesis and antiviral evaluation of certain novel pyrazinoic acid C- nucleosides

Article abstract of DOI:10.1021/jm970532z

Pyrazine (1,4-diazine) C-nucleosides constitute a rare class of nucleic acid analogues that has only recently been reported in the literature. As part of our ongoing investigation into the synthesis and reactivity of these compounds, we have developed an electrophilic esterification of a lithiated pyrazine C-nucleoside (1) to give, following deprotection, the versatile intermediate ethyl 3,5-dichloro-6-(β-D-ribofuranosyl)pyrazine-2-carboxylate (4). This intermediate was subjected to a variety of reaction conditions to generate a series of pyrazinoic acid C-nucleosides. These compounds, along with 3,5-dichloro-2-(β-D-ribofuranosyl)pyrazine (2) and 4, were evaluated for antiviral activity and cytotoxicity. No significant activity was observed for compounds 2 and 5-9, but 4 was active against two herpes viruses and cytotoxic in the micromolar range.

Full text of DOI:10.1021/jm970532z

1236
J . Med. Chem. 1998, 41, 1236-1241
Syn th esis a n d An tivir a l Eva lu a tion of Cer ta in Novel P yr a zin oic Acid
C-Nu cleosid es
J ohn A. Walker, II,† Weimin Liu,§ Dean S. Wise,‡ J ohn C. Drach, and Leroy B. Townsend*
Department of Medicinal Chemistry, College of Pharmacy, Department of Chemistry, College of Literature, Science, and Arts,
and Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan,
Ann Arbor, Michigan 48109-1065
Received August 11, 1997X
Pyrazine (1,4-diazine) C-nucleosides constitute a rare class of nucleic acid analogues that has
only recently been reported in the literature. As part of our ongoing investigation into the
synthesis and reactivity of these compounds, we have developed an electrophilic esterification
of a lithiated pyrazine C-nucleoside (1) to give, following deprotection, the versatile intermediate
ethyl 3,5-dichloro-6-(â-D-ribofuranosyl)pyrazine-2-carboxylate (4). This intermediate was
subjected to a variety of reaction conditions to generate a series of pyrazinoic acid C-nucleosides.
These compounds, along with 3,5-dichloro-2-(â-D-ribofuranosyl)pyrazine (2) and 4, were
evaluated for antiviral activity and cytotoxicity. No significant activity was observed for
compounds 2 and 5-9, but 4 was active against two herpes viruses and cytotoxic in the
micromolar range.
In tr od u ction
C-nucleosides nor on their potential chemotherapeutic
properties. We have published preliminary accounts of
two convergent synthetic routes that lead to 2′-deoxy-
pyrazine C-nucleosides14a and pyrazine C-ribosides14b
efficiently and in good yields. On the basis of this work,
we were interested in synthesizing some structurally
related compounds for biological evaluation. One of the
published ribosides, 3,5-dichloro-2-(2,3,5-tri-O-benzyl-
â-D-ribofuranosyl)pyrazine (1),14b was envisaged as a
very versatile compound if a carboxylic acid derivative
(i.e., acid, ester, amide, or nitrile) could be introduced
at the unoccupied position on the pyrazine ring. This
modification would provide a nucleoside that could lead
to a variety of functionalized pyrazinoic acid C-nucleo-
sides. We now report the synthesis of such pyrazinoic
acid C-nucleosides and provide the preliminary evalu-
ation for antiviral activity and cytotoxicity of these new
target compounds.
Nucleosides possess a broad spectrum of biological
functions, ranging from their primary roles as building
blocks in the genetic code to other functions such as
biosynthetic intermediates, energy donors, metabolic
regulators, and cofactors in enzymatic processes. Be-
cause of this, nucleosides and their synthetic analogues
have generated considerable scientific interest in their
chemistry and biology.1 Although N-nucleosides (in
which the glycosidic bond is a C1′-N linkage) are the
most abundant and therefore the most studied group
of analogues, there is also a group of naturally occurring
nucleosides in which the glycosidic bond is formed via
a C1′-C linkage.2 Within both classes of naturally
occurring nucleosides are compounds which possess a
carboxylic acid derivative (i.e., carboxylic acid, ester,
amide, or nitrile) residing meta to the site of glycosy-
lation. Many of these compounds, which have been
isolated from various organisms, possess very interest-
ing biological properties; e.g., compounds such as bredi-
nin,3 clitidine,4 and the C-nucleoside pyrazofurin5-7 are
some examples of biologically active nucleoside carboxy-
lic acid derivatives.
Resu lts a n d Discu ssion
Ch em istr y. Previous experience in our laboratories15
and others16 had established that trisubstituted pyra-
zines could be directly lithiated using nonnucleophilic
lithium dialkylamides at low temperature. These lithi-
ated pyrazines were reacted with various electrophilic
reagents to introduce groups at the unoccupied position
on the pyrazine ring. For our current investigation,
these groups would ideally be a carboxylic acid deriva-
tive that could readily be converted to other functional
groups at a subsequent stage of the synthetic route. We
also planned to effect a nucleophilic aromatic substitu-
tion of the chloro groups at a later stage in the synthetic
sequence.
A number of synthetic analogues which are structur-
ally related to these naturally occurring compounds
have been synthesized and reported to possess signifi-
cant biological properties, e.g., ribavirin,8 tiazofurin,9
and selenazofurin.10,11 The C-nucleoside 3-(â-D-ribo-
furanosyl)benzamide has been prepared and shown to
exhibit cytotoxicity in the nanomolar range to S49.1
lymphoma cells and leukemia K562 cells.12
Until very recently,13,14 there had been no reports in
the literature on the synthesis of pyrazine (1,4-diazine)
Therefore, we needed a reagent that would efficiently
provide a pyrazinoic acid derivative from a lithiopyra-
zine with a minimal amount of undesired side reactions.
A search of the literature revealed only one instance
where a direct formation of pyrazinoic acid derivatives
had occurred via a substituted lithiopyrazine. Que´-
† Present address: ProtoGene Laboratories, Inc., 1454 Page Mill Rd.,
Palo Alto, CA 94304.
‡ Present address: Berry & Associates, 2434 Bishop Circle East,
Dexter, MI 48130.
§ Present address: Shionogi BioResearch Corp., 45 Hartwell Ave.,
Lexington, MA 02173.
X Abstract published in Advance ACS Abstracts, December 1, 1997.
S0022-2623(97)00532-3 CCC: $15.00 © 1998 American Chemical Society
Published on Web 03/18/1998

Novel Pyrazinoic Acid C-Nucleosides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 8 1237
Sch em e 1a
a
Conditions: (A) 2 equiv of NH3/1,4-dioxane, 40 °C, 42 h; (B) 2 equiv of MeNH2/THF, rt, 30 min; (C) satd NH3/MeOH, 50 °C, 20 h; (D)
satd NH3/MeOH, MeOH, rt, 48 h; (E) satd NH3/EtOH, 4 °C, 48 h; (F) satd NH3/EtOH, 20 °C, 70 h.
Ta ble 1. Reactivity of Nucleoside 4 toward Nucleophiles
guiner and co-workers had been successful in reacting
conditionsa
yieldb
3-chloro-2-lithiopyrazine with carbon dioxide to obtain
3-chloropyrazinoic acid (30% yield).16 Numerous re-
agents are known to react with organolithium species
to give carboxylic acid derivatives, and we initially felt
that this would be a straightforward process to obtain
our target compounds. However, lithiation of 1 followed
by treatment with carbon dioxide17 (solid or gaseous)
unfortunately resulted in a rapid decomposition of the
starting material. We then investigated the synthesis
of pyrazine esters using alkyl chloroformates or alkyl
carbonates. In all instances, the organolithium inter-
mediate was destroyed rapidly upon the addition of an
electrophile and only 1 was isolated from the reaction
mixture. Model studies in our laboratories with alkyl
isocyanates had indicated that these reagents would
react with the lithiopyrazines, but all attempts to cleave
the amide/alkyl bond via acidic hydrolysis ultimately
resulted in complete destruction of the compound.18
Parker reported that the reaction of trimethylsilyl
isocyanate with organomagnesium compounds can lead
to the isolation of a free carboxamide upon aqueous
workup and hydrolysis of the silylated amide product.19
Our use of this reagent at -94 °C resulted only in the
isolation of a product that was silylated on the hetero-
cycle. This finding was not contrary to literature reports
since it has been noted that mixtures of amides and silyl
products are produced unless the temperature of the
reaction is maintained between 85 and 95 °C.19 Due to
the instability of the lithiopyrazine intermediate, we
could not raise the temperature of our reaction this high.
Even at -78 °C, a large amount of highly colored,
uncharacterizable material was formed and yields of the
product were low. The use of chloroacetyl isocyanate
to give free amides has been reported,19 but in our hands
only decomposition products were observed when this
reagent was used. Other electrophiles, e.g., chlorosul-
fonyl isocyanate (CSI)20 and urethane (ethyl carbam-
ate),21 also did not provide the desired products under
these conditions.
product
5
A
E
F
B
C
E
F
59
38
30
57
23
35
17
30
12
6
7
8
9
D
F
a
b
See Scheme 1 for reaction conditions. Percent (%) isolated
product.
bofuranosyl)pyrazine-2-carboxylate 3 in a 78% yield
(Scheme 1). Debenzylation of 3 using boron trichloride
yielded ethyl 3,5-dichloro-6-(â-D-ribofuranosyl)pyrazine-
2-carboxylate (4) in an 89% yield. The preparation of 4
was achieved in a 50% overall yield from the com-
mercially available 2,6-dichloropyrazine.14b This method
compares very well to any nucleoside preparation in the
literature, emphasizes the utility of a convergent ap-
proach to C-nucleosides, and provides a versatile inter-
mediate from which a variety of analogues could be
prepared.
In addition to the reactive carbonyl center of the ethyl
ester, 4 also possesses two chloro groups that could be
activated toward nucleophilic aromatic displacement
due to the ester functionality ortho and para to these
halogens. In order to determine the order and reactivity
of these sites to nucleophilic addition, we chose to treat
4 with 2 equiv of ammonia in the aprotic solvent 1,4-
dioxane (condition A). After 42 h at 40 °C, TLC analysis
indicated a complete consumption of the starting mate-
rial and the formation of only one product. This
compound was isolated, purified by silica gel chroma-
tography, and identified as ethyl 5-amino-3-chloro-6-(â-
D-ribofuranosyl)pyrazine-2-carboxylate (5) (Table 1). The
1H NMR spectrum of 5 showed the presence of an
aromatic amino functionality (as a D2O exchangeable
broad singlet centered at δ 7.65) as well as showed that
the ethyl ester was still intact. The site of nucleophilic
aromatic displacement was unequivocally proven to
occur at the 5-position (para to the carboxylate) by a
single-crystal X-ray structural determination of com-
pound 5.24 It is interesting to note that under these
conditions (where the amount of nucleophile may be
controlled), the order of reactivity is very selective. For
example, treatment of 4 with 2 equiv of the more
nucleophilic reagent methylamine in tetrahydrofuran
Mander and co-workers have demonstrated that alkyl
cyanoformates are useful reagents to effect C-alkylation
of lithium enolates preferential to O-alkylation.22 Oth-
ers have successfully used these reagents to form
heterocyclic alkyl esters.23 We found that the treatment
of 1 with lithium 2,2,6,6-tetramethylpiperidide (LTMP)
at low temperature followed by quenching with 5 equiv
of ethyl cyanoformate gave, after aqueous workup and
chromatographic purification, the protected 6-(â-D-ri-

1238 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 8
Walker et al.
Ta ble 2. Antiviral Activity and Cytotoxicity of Pyrazine
at room temperature (condition B) led exclusively to the
formation of ethyl 3-chloro-5-(methylamino)-6-(â-D-ri-
bofuranosyl)pyrazine-2-carboxylate (6) in which the
5-chloro group had also been preferentially displaced by
the methylamino group. In an attempt to synthesize
substituted pyrazinamide ribosides from 4, we treated
4 with a saturated solution of ammonia in methanol
(condition C). At room temperature, TLC analysis of
the reaction after 1 h revealed the formation of several
products as well as a sizable amount of unreacted
starting material. Upon heating the reaction for 20 h
at 50 °C, TLC analysis showed only one product along
with some highly colored, unidentifiable baseline mate-
rial. Upon isolation of the product, it was determined
that ammonolysis of the ethyl ester had been achieved
as well as a nucleophilic substitution of the 5-chloro
substituent by ammonia to give 5-amino-3-chloro-6-(â-
D-ribofuranosyl)pyrazine-2-carboxamide (7). Compound
1 was isolated in only a 23% yield via this route.
C-Nucleosides
50% inhibitory concentration (µM)
cytotoxicityc
antiviral activity
HFF
KB
cells,
substituent
R2 R5
Cl
CO2Et Cl
CO2Et NH2
CO2Et NHMe >100
CONH2 NH2
CONH2 OMe
CONH2 OEt
HCMV,a HSV-1,b cells,
compd
plaque ELISA visual growth
70 >100
3.2 3.2
>100
2
4
5
6
7
8
9
H
20
>100
>100
>100
>100
>100
20
>100
>100
>100
>100
>100
>100e
>100
>100
>100
>100
>100
>100
>100
>100
As stated above, TLC analysis of the reaction after 1
h had shown the presence of more than one product (or
intermediate) being formed. To isolate and characterize
these other products, 4 was dissolved in methanol and
treated with saturated methanolic ammonia (condition
D) at room temperature. After 24 h, TLC analysis
showed that all of 4 had been consumed and that only
one major product had been formed. Isolation and
preliminary identification of this compound showed that
transesterification had occurred to give the methyl
carboxylate and that a nucleophilic substitution of the
5-chloro substituent by a methoxy group had occurred.
The yield of 3-chloro-5-methoxy-6-(â-D-ribofuranosyl)-
pyrazine-2-carboxamide (8) was 30% overall. Under
these conditions, the formation of 7 was not observed.
The reaction of 4 in saturated methanolic ammonia for
2 days at room temperature resulted in the formation
and isolation of both 7 and 8. No evidence of the amino
ester compound 5 was observed. It is assumed that an
amino ester compound must be an intermediate in the
formation of 7 via a nucleophilic displacement of the
5-chloro group by ammonia followed by ammonolysis of
the ester. The reaction of 4 in ethanolic ammonia at 4
°C for 2 days (condition E) resulted in the formation of
5 and 7. There was no evidence that a displacement of
the 5-chloro group by ethanol to give 9 had occurred.
Raising the temperature of this reaction to 20 °C
(condition F) resulted in the isolation of not only 5 and
7 but also 3-chloro-5-ethoxy-6-(â-D-ribofuranosyl)pyra-
zine-2-carboxamide (9) in a 12% yield. These results
suggest that under more kinetic conditions ammonia is
the only viable nucleophile, while raising the temper-
ature results in a competition between ammonia and
the alcohol (or its conjugate base). Thus, from the
versatile intermediate 4, a series of novel substituted
pyrazinoic acid C-ribosides was prepared quite readily.
ganciclovir
(DHPG)
7.7d
1.8e >100d
a
Plaque reduction assays were performed in duplicate wells as
b
described in the text. Assayed by ELISA in quadruplicate wells.
c Visual cytotoxicity scored on HFF cells at time of HCMV plaque
enumeration. Inhibition of KB cell growth was determined as
d
described in the text in quadruplicate wells. Average of 88
experiments in which DHPG was used as a positive control.
e Average from 2-4 separate experiments.
active against HCMV, but the activity of compound 4
against both viruses occurred at cytotoxic concentra-
tions. Although the activity of 2 was weak, it did occur
at noncytotoxic concentrations suggesting some pos-
sibilities for additional synthesis. None of the com-
pounds with other substituents in the 5-position (5-9)
were active against either virus.
Exp er im en ta l Section
Unless otherwise noted, materials were obtained from
commercial suppliers and used as provided. Dichloromethane
(phosphorous pentoxide) and tetrahydrofuran (sodium/ben-
zophenone) were distilled from the indicated drying agent and
stored over activated 4-Å molecular sieves under a positive
pressure of argon prior to use (if not used immediately). The
phrase “evaporated in vacuo” is meant to imply the use of a
rotary evaporator with a bath temperature not exceeding 40
°C using a water aspirator. Thin-layer chromatography (TLC)
was carried out on Analtech 60F-254 silica gel plates, and
detection of components on TLC was made by UV light
absorption at 254 or 365 nm, staining with iodine vapor, or
heating to a char following treatment with 10% sulfuric acid
in methanol. Solvent systems are expressed as a percentage
of the more polar component with respect to total volume (v/v
%). Mallinckrodt SilicAR 230-400 mesh (40-63 µm) was used
for chromatography, which was carried out utilizing Ace Glass
Michel-Miller columns. A Rainin Rabbit HPX pump was used
for solvent delivery. UV light-active product-containing frac-
tions were detected on an Isco V4 UV detector and collected
by an Isco Foxy fraction collector. Flow rates, sample loading,
and fraction size were determined using the guidelines out-
lined by Still and co-workers.25 Melting points were deter-
mined on a Thomas-Hoover apparatus and are uncorrected.
The 1H (300 or 360 MHz) and 13C (90 or 125 MHz) NMR
spectra were recorded on Bruker instruments. The chemical
shift values are expressed in δ values (parts per million)
relative to tetramethylsilane as an internal standard for 1H
NMR and relative to the standard chemical shift of the solvent
for 13C NMR. Mass spectroscopy was performed by The
Biologica l Eva lu a tion . The capacity of the com-
pounds to inhibit the replication of human cytomega-
lovirus (HCMV) and herpes simplex virus type 1 (HSV-
1) was examined in an initial study of their antiviral
effects. To determine if the compounds had specific
antiviral activity, the cytotoxicities of compounds were
investigated in uninfected stationary human foreskin
fibroblasts (HFF cells) and growing KB cells (Table 2).
The two dichloro-substituted compounds (2, 4) were

Novel Pyrazinoic Acid C-Nucleosides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 8 1239
University of Michigan Chemistry Department. Elemental
analyses were performed by MHW Laboratories, Phoenix, AZ,
or by The University of Michigan Chemistry Department. The
presence of solvent as indicated by analysis was always
was evaporated in vacuo. The compound (absorbed on silica
gel) was subjected to column chromatography (SiO2, 40 × 220
mm, 7% methanol/chloroform, Rf ) 0.38) to give, following
solvent evaporation, 4 (139 mg, 89%) as a white solid. An
analytical sample was crystallized from acetone/petroleum
ether to afford a flocculent white solid: mp 122.5-123 °C; 1H
NMR (360 MHz, DMSO-d6) δ 5.32 (d, 1, J ) 5.3 Hz, D2O
exchangeable), 5.14 (d, 1, J ) 3.9 Hz), 5.03 (d, 1, J ) 6.0 Hz,
D2O exchangeable), 4.49 (dd, 1, J ) 4.5, 7.0 Hz, D2O exchange-
able), 4.41 (q, 2, J ) 7.1 Hz), 4.23 (dd, 1, J ) 4.8, 9.1 Hz), 3.98
(dd, 1, J ) 5.9, 10.9 Hz), 3.91 (dd, 1, J ) 4.5, 9.4 Hz), 3.61 (m,
1), 3.51 (m, 1), 1.34 (t, 3, J ) 7.1 Hz); 13C NMR (90 MHz,
DMSO-d6) δ 162.3, 151.1, 147.2, 143.3, 140.8, 84.8, 80.7, 74.6,
70.9, 62.5, 61.8, 13.8. Anal. (C12H14Cl2N2O6) C,H,N.
Eth yl 5-Am in o-3-ch lor o-6-(â-D-r ibofu r a n osyl)p yr a zin e-
2-ca r boxyla te (5). Con d ition A: A 10-mL pressure tube was
charged with 4 (176 mg, 0.5 mmol). To this tube was added a
0.5 M solution of ammonia in 1,4-dioxane (Aldrich; 2.2 mL,
1.1 mmol), the tube was sealed, and the reaction continued at
40 °C for 42 h. At this point, TLC indicated that all starting
material had been consumed. A minimal amount of silica gel
was added, and the solvent was evaporated in vacuo. The
compound (absorbed on silica gel) was subjected to column
chromatography (SiO2, 20 × 180 mm, 10% methanol/chloro-
form, Rf ) 0.38) to give, following solvent evaporation, 5 (98
mg, 59%) as a white solid: mp 164-166 °C; 1H NMR (360
MHz, DMSO-d6) δ 7.65 (bs, 2, D2O exchangeable), 5.36 (pt, 1,
J ) 4.6 Hz, D2O exchangeable), 5.12 (d, 1, J ) 6.5 Hz, D2O
exchangeable), 4.99 (d, 1, J ) 4.8 Hz, D2O exchangeable), 4.72
(d, 1, J ) 7.6 Hz), 4.26 (m, 3), 4.01 (dd, 1, J ) 4.7, 8.4 Hz),
3.92 (m, 1), 3.61 (m, 2), 1.30 (t, 3, J ) 7.1 Hz); 13C NMR (90
MHz, DMSO-d6) δ 163.2, 153.6, 145.6, 136.3, 126.4, 86.0, 83.7,
71.8, 71.4, 61.0, 60.8, 14.1; HRMS calcd for C12H16ClN3O6
333.0728, found 333.0733. Anal. (C12H16ClN3O6) C,H,N.
1
confirmed by H NMR spectroscopy.
3,5-Dich lor o-2-(â-D-r ibofu r an osyl)pyr azin e (2). A flame-
dried, evacuated, 100-mL round-bottom flask was charged,
under argon, with 114b (550 mg, 1.0 mmol). This was dissolved
in dry dichloromethane (30 mL) and cooled to -70 °C. To this
solution was added boron trichloride (1.0 M solution in
dichloromethane, 10.0 mL, 10.0 mmol) dropwise, and stirring
was continued at -70 °C for 120 min. The reaction was then
warmed to room temperature and stirred for an additional 60
min. The excess boron trichloride was then quenched with
methanol/dichloromethane (1:1, 20 mL), and the solution was
adjusted to pH 7 with 2 N aqueous ammonium hydroxide. A
minimal amount of silica gel was added and the solvent
evaporated. The compound (absorbed on silica gel) was
subjected to column chromatography (SiO2, 40 × 150 mm, 5%
methanol/chloroform, Rf ) 0.25) to give, following solvent
evaporation, 2 (240 mg, 85%) as a white solid: mp 132-132.5
°C; 1H NMR (360 MHz, DMSO-d6) δ 8.87 (s, 1), 5.19 (d, 1, J )
5.7 Hz), 5.10 (d, 1, J ) 5.1 Hz, D2O exchangeable), 5.03 (d, 1,
J ) 5.6 Hz, D2O exchangeable), 4.65 (pt, 1, J ) 5.3, 6.1 Hz,
D2O exchangeable), 4.30 (dd, 1, J ) 5.3, 10.6 Hz), 3.99 (dd, 1,
J ) 5.3, 10.5 Hz), 3.88 (dd, 1, J ) 4.9, 9.6 Hz), 3.53 (m, 1),
3.43 (m, 1); 13C NMR (90 MHz, DMSO-d6) δ 150.8, 146.2, 145.5,
142.8, 85.3, 80.2, 74.3, 71.4, 61.6. Anal. (C9H10Cl2N2O4)
C,H,N.
Eth yl 3,5-Dich lor o-6-(2,3,5-tr i-O-ben zyl-â-D-r ibofu r a n -
osyl)p yr a zin e-2-ca r boxyla te (3). A flame-dried, evacuated,
3-neck, 250-mL round-bottom flask equipped with an addition
funnel was charged, under argon, with dry tetrahydrofuran
(60 mL) and cooled to -70 °C. To this flask was added
n-butyllithium (2.5 M solution in hexane, 3.2 mL, 8.0 mmol)
followed by 2,2,6,6-tetramethylpiperidine (1.52 mL, 9.0 mmol),
and the solution was warmed and allowed to stir at 0 °C for
60 min. After cooling to -94 °C, a solution of 114b (4.03 g, 7.3
mmol in 40 mL of dry tetrahydrofuran) was added dropwise
(ca. 20 min). The lithiation was allowed to take place at -94
°C for 120 min, at which time ethyl cyanoformate (3.61 mL,
37.0 mmol) was added neat in one portion. The condensation
was complete in 60 min (as determined by TLC), at which time
the reaction was quenched with 25 mL of water, warmed to
room temperature, and diluted with ethyl acetate to a total
volume of 200 mL. The organic layer was isolated, dried over
magnesium sulfate, and filtered, and the solvent was evapo-
rated in vacuo. The resultant orange oil was subjected to
column chromatography (SiO2, 50 × 220 mm, 15% ethyl
acetate/hexanes, Rf ) 0.38) to give, following solvent evapora-
tion, 3 (3.56 g, 78%) as a colorless oil: 1H NMR (360 MHz,
CDCl3) δ 7.35-7.23 (m, 15), 5.47 (d, 1, J ) 5.7 Hz), 4.67-4.37
(m, 10), 4.12 (pt, 1, J ) 4.8, 4.9 Hz), 3.59 (dd, 2, J ) 3.5, 4.6
Hz), 1.34 (t, 3, J ) 7.1 Hz); 13C NMR (90 MHz, CDCl3) δ 162.7,
149.6, 148.7, 145.0, 141.7, 138.3, 137.9, 137.7, 128.6, 128.5,
128.4, 128.3, 128.1, 128.0, 127.7, 82.8, 80.5, 79.1, 78.1, 73.5,
72.9, 72.5, 70.5, 62.7, 14.2; HRMS calcd for C33H36Cl2N2O6
622.1637, found 622.1617. Anal. (C33H36Cl2N2O6) H,N; C:
calcd, 63.57; found, 62.90.
E t h yl 3,5-Dich lor o-6-(â-D-r ib ofu r a n osyl)p yr a zin e-2-
ca r boxyla te (4). A flame-dried, evacuated, 100-mL round-
bottom flask was charged with 3 (680 mg, 1.1 mmol) under
argon. This was dissolved in dry dichloromethane (25 mL)
and cooled to -70 °C. To this solution was added boron
trichloride (1.0 M solution in heptane, 6.5 mL, 6.5 mmol)
dropwise, and the bright yellow mixture was allowed to stir
at -70 °C for 150 min. The excess boron trichloride was then
quenched at -70 °C with ethanol/dichloromethane (1:1, 10 mL)
and warmed to room temperature, and the solution was
neutralized with 3 N aqueous ammonium hydroxide. The
solvent was evaporated to give a yellowish solid. This solid
was then taken up in hot ethyl acetate, and the insoluble
ammonium chloride was filtered off through Celite. A minimal
amount of silica gel was added to the filtrate, and the solvent
Con d ition E: Alternately, a 25-mL pressure tube was
charged with 4 (200 mg, 0.57 mmol). To this tube was added
ethanolic ammonia (saturated at 0 °C, 7.5 mL), the tube was
sealed, and the reaction continued at 4 °C for 48 h. At this
point, TLC indicated that all starting material had been
consumed. A minimal amount of silica gel was added, and
the solvent was evaporated in vacuo. The compound (absorbed
on silica gel) was subjected to column chromatography (SiO2,
40 × 150 mm, 10% methanol/chloroform, Rf ) 0.38) to give,
following solvent evaporation, 5 (73 mg, 38%) as a white
solid: mp, 1H NMR, and 13C NMR all correspond to the
compound prepared above.
Also isolated was 61 mg (35%) of 7 as a white solid: Rf, mp,
1H NMR, and 13C NMR all correspond to the compound
prepared below.
Eth yl 3-Ch lor o-5-(m eth yla m in o)-6-(â-D-r ibofu r a n osyl)-
p yr a zin e-2-ca r boxyla te (6). Con d ition B: A flame-dried,
evacuated 50-mL pressure tube was charged with 4 (353 mg,
1.0 mmol) under argon, and this was dissolved in dry tetrahy-
drofuran (10 mL). To this solution was added a solution of
methylamine (2.0 M in tetrahydrofuran, 1.1 mL, 2.2 mmol) in
one portion, and the tube was sealed. A precipitate formed
immediately and, after 60 min at room temperature, TLC
indicated that all starting material had been consumed.
A
minimal amount of silica gel was added, and the solvent was
evaporated in vacuo. The compound (absorbed on silica gel)
was subjected to column chromatography (SiO2, 20 × 180 mm,
10% methanol/chloroform, Rf ) 0.37) to give, following solvent
evaporation, 6 (198 mg, 57%) as white needles: mp 174-175
1
°C; H NMR (300 MHz, DMSO-d6) δ 8.01 (bd, 1, J ) 4.6 Hz,
D2O exchangeable), 5.54 (pt, 1, J ) 4.2 Hz, D2O exchangeable),
5.12 (d, 1, J ) 6.5 Hz, D2O exchangeable), 5.01 (d, 1, J ) 4.8
Hz, D2O exchangeable), 4.74 (d, 1, J ) 7.7 Hz), 4.26 (m, 3),
4.01 (dd, 1, J ) 5.0, 8.2 Hz), 3.95 (m, 1), 3.63 (m, 2), 2.85 (d,
3, J ) 4.6 Hz), 1.30 (t, 1, J ) 7.1 Hz); 13C NMR (125 MHz,
DMSO-d6) δ 164.3, 153.5, 146.8, 138.2, 126.1, 86.9, 84.8, 72.8,
72.4, 61.7, 61.6, 28.7, 15.0. Anal. (C13H18ClN3O6) C,H,N.
5-Am in o-3-ch lor o-6-(â-D-r ibofu r a n osyl)p yr a zin e-2-ca r -
boxa m id e (7). Con d ition C: A 25-mL pressure tube was
charged with 4 (177 mg, 0.5 mmol). To this tube was added

1240 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 8
Walker et al.
methanolic ammonia (saturated at 0 °C, 5 mL), the tube was
sealed, and the reaction continued at room temperature for 1
h. TLC indicated that the reaction was sluggish and producing
multiple products. An additional quantity of methanolic
ammonia (5 mL) was added, the tube sealed, and stirring
continued at 50 °C for 20 h. TLC indicated that there was
only one major mobile product. A minimal amount of silica
gel was added, and the solvent was evaporated in vacuo. The
compound (absorbed on silica gel) was subjected to column
chromatography (SiO2, 40 × 200 mm, 15% methanol/chloro-
form, Rf ) 0.27) to give, following solvent evaporation, 7 (35
mg, 23%) as a white, hygroscopic solid: mp 84.5-85 °C; 1H
NMR (360 MHz, DMSO-d6) δ 7.66 (bs, 1, D2O exchangeable),
7.40 (bs, 2, D2O exchangeable), 7.36 (bs, 1, D2O exchangeable),
5.30 (pt, 1, J ) 4.6 Hz, D2O exchangeable), 5.09 (d, 1, J ) 6.7
Hz, D2O exchangeable), 4.96 (d, 1, J ) 4.2 Hz, D2O exchange-
able), 4.72 (d, 1, J ) 7.8 Hz), 4.27 (dd, 1, J ) 6.5, 12.9 Hz),
4.01 (m, 1), 3.90 (m, 1), 3.58 (m, 2); 13C NMR (90 MHz, DMSO-
d6) δ 165.0, 153.6, 143.9, 135.1, 128.7, 86.0, 83.2, 71.8, 71.4,
61.1; HRMS calcd for C10H13ClN4O5 304.0574, found 304.0563.
Anal. (C10H13ClN4O5‚1/2H2O) C,H,N.
3-Ch lor o-5-m et h oxy-6-(â-D-r ib ofu r a n osyl)p yr a zin e-2-
ca r boxa m id e (8). Con d ition D: A 25-mL pressure tube was
charged with 4 (150 mg, 0.42 mmol), and this was dissolved
in 10 mL of methanol. To this solution was added methanolic
ammonia (saturated at 0 °C, 250 µL), the tube was sealed, and
the solution stirred at room temperature for 24 h. TLC
indicated that all starting material had been consumed. The
solvent was evaporated in vacuo, and the residue was redis-
solved in 5 mL of methanolic ammonia (saturated at 0 °C) and
allowed to react at room temperature for 24 h. A minimal
amount of silica gel was added to the solution, and the solvent
was evaporated in vacuo. The compound (absorbed on silica
gel) was subjected to column chromatography (SiO2, 20 × 150
mm, 10% methanol/chloroform, Rf ) 0.15) to give, following
solvent evaporation, 8 (40 mg, 30%) as a white solid: mp 169-
170.5 °C; 1H NMR (360 MHz, DMSO-d6) δ 7.95 (bs, 1, D2O
exchangeable), 7.74 (bs, 1, D2O exchangeable), 5.10 (d, 1, J )
5.6 Hz, D2O exchangeable), 4.96 (d, 1, J ) 4.9 Hz), 4.91 (d, 1,
J ) 5.7 Hz, D2O exchangeable), 4.67 (pt, 1, J ) 5.5, 5.8 Hz,
D2O exchangeable), 4.32 (dd, 1, J ) 5.1, 10.0 Hz), 4.02 (dd, 1,
J ) 5.5, 10.6 Hz), 3.98 (s, 3), 3.82 (dd, 1, J ) 4.9, 9.6 Hz), 3.56
(m, 1), 3.45 (m, 1); 13C NMR (90 MHz, DMSO-d6) δ 164.8,
157.3, 141.5, 141.0, 136.4, 84.7, 78.9, 73.4, 71.1, 61.6, 55.0;
HRMS calcd for C11H14ClN3O6 319.0571, found 319.0566.
Anal. (C11H14ClN3O6) C,H,N.
3-Ch lor o-5-eth oxy-6-(â-D-r ibofu r a n osyl)p yr a zin e-2-ca r -
boxa m id e (9). Con d it ion F : A 25-mL pressure tube was
charged with 4 (353 mg, 1.0 mmol). To this tube was added
ethanolic ammonia (saturated at 0 °C, 10 mL), the tube was
sealed, and the reaction continued at 20 °C for 70 h. TLC
indicated that all starting material had been consumed, a
minimal amount of silica gel was added, and the solvent was
evaporated in vacuo. The compound (absorbed on silica gel)
was subjected to column chromatography (SiO2, 40 × 150 mm,
10% methanol/chloroform, Rf ) 0.24) to give, following solvent
evaporation, 9 (40 mg, 12%) as a clear glass which could be
triturated with methanol to give a white solid: mp 153.0-
154.5 °C; 1H NMR (300 MHz, DMSO-d6) δ 7.94 (bs, 1, D2O
exchangeable), 7.72 (bs, 1, D2O exchangeable), 5.09 (d, 1, J )
5.3 Hz, D2O exchangeable), 4.96 (d, 1, J ) 4.9 Hz), 4.92 (d, 1,
J ) 5.6 Hz, D2O exchangeable), 4.66 (pt, 1, J ) 5.5 Hz, D2O
exchangeable), 4.41 (q, 2, J ) 6.9 Hz), 4.33 (dd, 1, J ) 5.0,
10.0 Hz), 4.03 (dd, 1, J ) 5.4, 10.6 Hz), 3.82 (m, 1), 3.41 (m,
1), 1.37 (t, 3, J ) 6.9 Hz); 13C NMR (125 MHz, DMSO-d6) δ
164.8, 156.8, 141.4, 141.0, 136.2, 84.7, 79.2, 73.4, 71.9, 63.8,
61.7, 14.0. Anal. (C12H16ClN3O6‚MeOH) C,H,N.
sage of KB, BSC-1, and HFF cells was performed in monolayer
cultures using minimal essential medium (MEM) with either
Hanks salts [MEM(H)] or Earle salts [MEM(E)] supplemented
with 10% calf serum or 10% fetal bovine serum (HFF cells).
The sodium bicarbonate concentration was varied to meet the
buffering capacity required. Cells were passaged at 1:2 to 1:10
dilutions according to conventional procedures using 0.05%
trypsin plus 0.02% EDTA in a HEPES-buffered salt solution.
Vir ologica l P r oced u r es. The Towne strain, plaque-
purlfied isolate PO, of HCMV was kindly provided by Dr. Mark
Stinski, University of Iowa. The KOS strain of HSV- 1 was
used in most experiments and was provided by Dr. Sandra K.
Weller, University of Connecticut. Stock HCMV was prepared
by infecting HFF cells at a multiplicity of infection (moi) of
<0.01 plaque-forming units (pfu)/cell as detailed previously.26
High-titer HSV-1 stocks were prepared by infecting KB cells
at an moi of <0.1 also as detailed previously.26 Virus titers
were determined using monolayer cultures of HFF cells for
HCMV and monolayer cultures of BSC-1 cells for HSV-1 as
described earlier.27 Briefly, HFF or BSC-1 cells were planted
as described above in 96-well cluster dishes and incubated
overnight at 37 °C. The next day cultures were inoculated
with HCMV or HSV-1 and serially diluted 1:3 across the
remaining 11 columns of the 96-well plate. After virus
adsorption the inoculum was replaced with fresh medium, and
cultures were incubated for 7 days for HCMV and for 2 or 3
days for HSV-1. Plaques were enumerated under 20-fold
magnification in wells having the dilution which gave 5-20
plaques/well. Virus titers were calculated according to the
following formula: titer (pfu/mL) ) number of plaques × 5 ×
3n, where n represents the nth dilution of the virus used to
infect the well in which plaques were enumerated.
HCMV P la qu e Red u ction Assa y. HFF cells in 24-well
cluster dishes were infected with approximately 100 pfu of
HCMV/cm2 cell sheet using the procedures detailed above.
Following virus adsorption, compounds dissolved in growth
medium were added to duplicate wells in 4-8 selected
concentrations. After incubation at 37 °C for 10-12 days, cell
sheets were fixed and stained with crystal violet and micro-
scopic plaques enumerated as described above. Drug effects
were calculated as a percentage of reduction in number of
plaques in the presence of each drug concentration compared
to the number observed in the absence of drug.
HSV-1 ELISA. An ELISA was employed to detect HSV-
1;28 96-well cluster dishes were planted with 10 000 BSC-1
cells/well in 200 µL/well of MEM(E) plus 10% calf serum. After
overnight incubation at 37 °C, selected drug concentrations
in quadruplicate and HSV-1 at a concentration of 100 pfu/well
were added. Following a 3-day incubation at 37 °C, medium
was removed, plates were blocked and rinsed, and horseradish
peroxidase-conjugated rabbit anti-HSV-1 antibody was added.
Following removal of the antibody-containing solution, plates
were rinsed and then developed by adding 150 µL/well of a
solution of tetramethylbenzidine as substrate. The reaction
was stopped with H2SO4, and absorbance was read at 450 and
570 nm. Drug effects were calculated as a percentage of the
reduction in absorbance in the presence of each drug concen-
tration compared to absorbance obtained with virus in the
absence of drug.
Cytotoxicity Assa ys. Two different assays were used to
explore cytotoxicity of selected compounds using methods we
have detailed previously. (i) Cytotoxicity produced in station-
ary HFF cells was determined by microscopic inspection of cells
not affected by the virus used in plaque assays.26 (ii) The effect
of compounds during two population doublings of KB cells was
determined by crystal violet staining and spectrophotometric
quantitation of dye eluted from stained cells as described
earlier.29 Briefly, 96-well cluster dishes were planted with KB
cells at 3000-5000 cells/well. After overnight incubation at
37 °C, test compound was added in quadruplicate at 6-8
concentrations. Plates were incubated at 37 °C for 48 h in a
CO2 incubator, rinsed, fixed with 95% ethanol, and stained
with 0.1% crystal violet. After drying, 0.1 N HCl in ethanol
Also isolated was 101 mg (30%) of 5 as a white solid: Rf,
mp, 1H NMR, and 13C NMR all correspond to the compound
prepared above.
Also isolated was 53 mg (17%) of 7 as a white solid (Rf )
0.10 in 10% methanol/chloroform): mp, 1H NMR, and 13C NMR
all correspond to the compound prepared above.
Cell Cu ltu r e P r oced u r es. The routine growth and pas-

Novel Pyrazinoic Acid C-Nucleosides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 8 1241
(9) (a) Fuertes, M.; Garcia-Lopez, T.; Garcia-Munoz, G.; Stud, M.
Synthesis of C-Glycosyl Thiazoles. J . Org. Chem. 1976, 41,
4074-4077. (b) Srivastava, P. C.; Pickering, M. V.; Allen, L. B.;
Streeter, D. G.; Campbell, M. T.; Witowski, J . T.; Sidwell, R.
W.; Robins, R. K. Synthesis and Antiviral Activity of Certain
Thiazole C-Nucleosides. J . Med. Chem. 1977, 20, 256-262.
(10) Srivastava, P. C.; Robins, R. K. Synthesis and Antitumor Activity
of 2-â-D-Ribofuranosylselenazole-4-carboxamide and Related
Derivatives. J . Med. Chem. 1983, 26, 445-448.
was added, and the plates were read at 570 nm in a spectro-
photometer designed to read 96-well ELISA assay plates.
Da ta An a lysis. Dose-response relationships were con-
structed by linearly regressing the percent inhibition of
parameters derived in the preceding sections against log drug
concentrations; 50% inhibitory concentrations (IC50) or IC90’s
were calculated from the regression lines. Samples containing
positive controls (acyclovir for HSV-1, ganciclovir for HCMV,
and 2-acetylpyridine thiosemicarbazone for cytotoxicity) were
used in all assays.
(11) (a) Tricot, G.; J ayaram, H. N.; Weber, G.; Hoffman, R. Tiazofu-
rin: Biological and Clinical Uses. Int. J . Cell Cloning 1990, 8,
161-170. (b) Robins, R. K.; Finch, R. A.; Avery, T. L. Nucleoside
and Nucleotide Modulation of Oncogenic Expression: A New
Approach to Cancer Chemotherapy. In Anticancer Drug Discov-
ery and Developments: Natural Products and New Molecular
Models; Valeriote, F. A., Corbett, T. H., Baker, L. H., Eds.;
Kluwer Academic Publishers: Boston, 1994; pp 149-182.
(12) (a) Krohn, K.; Heins, H.; Wielckens, K. Synthesis and Cytotoxic
Activity of C-Glycosidic Nicotinamide Riboside Analogues. J .
Med. Chem. 1992, 35, 511-517. (b) J ayaram, H. N.; Ghareh-
baghi, K.; J ayaram, N. H.; Rieser, J .; Krohn, K.; Paull, K. D.
Cytotoxicity of a New IMP Dehydrogenase Inhibitor, Benzamide
Riboside, to Human Myelogenous Leukemia K562 Cells. Bio-
chem. Biophys. Res. Commun. 1992, 186, 1600-1606.
(13) (a) Voegel, J . J .; Benner, S. A. Synthesis and Characterization
of Non-Standard Nucleosides and Nucleotides Bearing the
Acceptor-Donor-Donor Pyrimidine Analog 6-Amino-3-meth-
ylpyrazin-2(1H)-one. Helv. Chim. Acta 1996, 79, 1863-1880 and
references therein. (b) von Krosigk, U.; Benner, S. A. pH-
Independent Triple Helix Formation by an Oligonucleotide
Containing a Pyrazine Donor-Donor-Acceptor Base. J . Am.
Chem. Soc. 1995, 117, 5361-5362.
Ack n ow led gm en t. The authors would like to thank
Dr. J eff Kampf of The University of Michigan Depart-
ment of Chemistry for determining the X-ray crystal
structure of compound 5. The authors also thank
members of Dr. Drach’s research group, especially J ulie
M. Breitenbach and Roger G. Ptak, for the antiviral
evaluations. This investigation was funded by Grant
U01-AI31718 and Grant ROI-CA56842 from the Na-
tional Institutes of Health. We would also like to thank
Marina Savic for the expert preparation of this manu-
script.
Su p p or tin g In for m a tion Ava ila ble: X-ray structures
and relevant data for 5 (13 pages). Ordering information is
given on any current masthead page.
(14) (a) Chen, J . J .; Walker, J . A., II; Liu, W.; Wise, D. S.; Townsend,
L. B. An Efficient and Stereospecific Synthesis of Novel Pyrazine
C-Nucleosides. Tetrahedron Lett. 1995, 36, 8363-8366. (b) Liu,
W.; Walker, J . A., II; Chen, J . J .; Wise, D. S.; Townsend, L. B.
Synthesis of Pyrazine C-Ribosides via Direct Metalation. Tet-
rahedron Lett. 1996, 37, 5325-5328.
Refer en ces
(15) Liu, W.; Walker, J . A., II; Chen, J . J .; Wise, D. S.; Townsend, L.
B. A Short and Efficient Synthesis of Some Novel Pyrazine
C-Nucleosides. Presented at the 211th National Meeting of the
American Chemical Society, New Orleans, LA, March 1996;
Paper ORGN 265.
(16) Turck, A.; Mojovic, L.; Que´guiner, G. A New Route to 2,3-
Disubstituted Pyrazines; Regioselective Metalation of Chloro-
pyrazine. Synthesis 1988, 11, 881-884.
(17) Wakefield, B. J . Organolithium Methods; Academic Press, Inc:
San Diego, CA, 1988.
(1) For a review on the chemistry and biology of nucleosides, see:
(a) Nucleosides as Biological Probes; Suhadolnik, R. J ., Ed.; J ohn
Wiley & Sons: New York, 1979. (b) Chemistry of Nucleosides
and Nucleotides; Townsend, L. B., Ed.; Plenum Press: New
York, 1988, Vol. 1; 1991, Vol. 2. (c) Nucleosides and Nucleotides
as Antitumor and Antiviral Agents; Chu, C. K., Baker, D. C.,
Eds.; Plenum Press: New York, 1993. (d) Chemistry of Nucleo-
sides and Nucleotides; Townsend, L. B., Ed.; Plenum Press: New
York, 1994; Vol. 3.
(2) For a review of the chemistry and biology of C-glycosides, see:
(a) Watanabe, K. A. The Chemistry of C-Nucleosides. In Chem-
istry of Nucleosides and Nucleotides; Townsend, L. B., Ed.;
Plenum Press: New York, 1994; Vol. 3, Chapter 5. (b) Hacksell,
U.; Daves, G. D., J r. The Chemistry and Biochemistry of
C-Nucleosides and C-Arylglycosides. Prog. Med. Chem. 1985, 22,
1-65. (c) Buchanan, J . G. The C-Nucleoside Antibiotics. Prog.
Chem. Org. Nat. Prod. 1983, 44, 243-299. (d) Goodchild, J . The
Biochemistry of Nucleoside Antibiotics. Top. Antibiot. Chem.
1982, 6, 99-227.
(3) (a) Mizuno, K.; Tsujino, M.; Takeda, M.; Hayashi, M.; Atsumi,
K.; Asano, K.; Matsuda, T. Studies on Bredinin. I. Isolation,
Characterization, and Biological Properties. J . Antibiot. 1974,
27, 775-782. (b) Amemiya, H.; Itoh, H. Mizoribine (Bredi-
nin®): Mode of Action and Effects on Graft Rejection. In
Immunosupressive Drugs. Developments in Anti-Rejection Therapy;
Thomson, A. W., Starzl, T. E., Eds.; Edward Arnold: London,
1994; pp 161-176.
(4) (a) Konno, K.; Hayano, K.; Shirahama, H.; Saito, H.; Matsumoto,
T. Structure and Synthesis of Clitidine, a New Pyridine Nucleo-
side from Clitocybe acromelalga. Tetrahedron Lett. 1977, 481-
482. (b) Konno, K.; Hayano, K.; Shirahama, H.; Saito, H.;
Matsumoto, T. Clitidine. A New Toxic Pyridine Nucleoside from
Clitocybe acromelalga. Tetrahedron 1982, 38, 3281-3284.
(5) (a) Gerzon, K.; Williams, R. H.; Hoehn, M.; Gorman, M.; DeLong,
D. C. Pyrazomycin, A C-Nucleoside with Antiviral Activity.
Presented at the Second International Congress on Heterocyclic
Chemistry, Montpellier, France, J uly 1969; Abstract C-30. (b)
Williams, R. H.; Hoehn, M. U.S. Patent 3,802,999, 1974.
(6) Sweeney, M. J .; Davis, F. A.; Gutowski, G. E.; Hamill, R. L.;
Hoffman, D. H.; Poore, G. A. Experimental Antitumor Activity
of Pyrazomycin. Cancer Res. 1973, 33, 2619-2623.
(18) Unpublished results.
(19) Parker, K. A.; Gibbons, E. G. A Direct Synthesis of Primary
Amides from Grignard Reagents. Tertrahedron Lett. 1975, 16,
981-984.
(20) For reviews of CSI, see: (a) Rasmussen, J . K.; Hassner, A. Recent
Developments in the Synthetic Uses of Chlorosulfonyl Isocyan-
ate. Chem. Rev. 1976, 76, 389-408. (b) Dhar, D. N.; Murthy, K.
S. K. Recent Advances in the Chemistry of Chlorosulfonyl
Isocyanate. Synthesis 1986, 437-449.
(21) Chakraborty, D. P.; Mandal, A. K.; Roy, S. K. Ethyl Carbamate
as an Aminocarbonylating Agent: Modification of Gattermann’s
Amidation Reaction. Synthesis 1981, 977-979.
(22) Mander, L. N.; Sethi, S. P. Regioselective Synthesis of â-Ke-
toesters from Lithium Enolates and Methyl Cyanoformate.
Tetrahedron Lett. 1983, 24, 5425-5428.
(23) Rho, T.; Abuh, Y. F. One-Pot Synthesis of Pyrimidine-5-carbox-
aldehyde and Ethyl Pyrimidine-5-carboxylate by Utilizing Py-
rimidin-5-yl-lithium. Synth. Commun. 1994, 24, 253-256.
(24) See the Supporting Information for details of the X-ray structural
determination of compound 5.
(25) Still, W. C.; Kahn, M.; Mitra, A. A Rapid Chromatographic
Technique for Preparative Separations with Moderate Resolu-
tion. J . Org. Chem. 1978, 43, 2923-2925.
(26) Turk, S. R.; Shipman, C., J r.; Nassiri, M. R.; Genzingler, G.;
Krawczyk, S. H.; Townsend, L. B.; Drach, J . C. Pyrrolo[2,3-d]-
pyrimidine Nucleosides as Inhibitors of Human Cytomegalovi-
rus. Antimicrob. Agents Chemother. 1987, 31, 544-550.
(27) Prichard, M. N.; Turk, S. R.; Coleman, L. A.; Engelhardt, S. L.;
Shipman, C., J r.; Drach, J . C. A Microtiter Virus Yield Reduction
Assay for The Evaluation of Antiviral Compounds Against
Human Cytomegalovirus and Herpes Simplex Virus. J . Virol.
Methods 1990, 28, 101-106.
(28) Prichard, M. N.; Shipman, C., J r. A Three-Dimensional Model
To Analyze Drug-Drug Interactions. Antiviral Res. 1990, 14,
181-206.
(29) Prichard, M. N.; Prichard, L. E.; Baguley, W. A.; Nassiri, M. R.;
Shipman, C., J r. Three-Dimensional Analysis of the Synergistic
Cytotoxicity of Ganciclovir and Zidovudine. Antimicrob. Agents
Chemother. 1991, 35, 1060-1065.
(7) Gutowski, G. E.; Sweeney, M. J .; DeLong, D. C.; Hamill, R. L.;
Gerzon, K.; Dyke, R. W. Biochemistry and Biological Effects of
the Pyrazofurins (Pyrazomycins): Initial Clinical Trial. Ann. N.
Y. Acad. Sci. 1975, 255, 544-551.
(8) Witkowski, J . T.; Robins, R. K.; Sidwell, R. W.; Simon, L. N.
Design, Synthesis, and Broad-Spectrum Antiviral Activity of 1-â-
D-Ribofuranosyl-1,2,4-triazole-3-carboxamide and Related Nu-
cleosides. J . Med. Chem. 1972, 15, 1150-1154.
J M970532Z