Synthesis and antiviral activities of new acyclic and "double-headed" nucleoside analogues

Article abstract of DOI:10.1016/j.bioorg.2006.11.003

To develop an understanding of the structure-activity relationships for the inhibition of orthopoxviruses by nucleoside analogues, a variety of novel chemical entities were synthesized. These included a series of pyrimidine 5-hypermodified acyclic nucleoside analogues based upon recently discovered new leads, and some previously unknown "double-headed" or "abbreviated" nucleosides. None of the synthetic products possessed significant activity against two representative orthopoxviruses; namely, vaccinia virus and cowpox virus. They were also devoid of significant activity against a battery of other DNA and RNA viruses. So far as the results with the orthopoxviruses and herpes viruses, the results may point to the necessity for nucleoside analogues 5′-phosphorylation for antiviral efficacy.

Full text of DOI:10.1016/j.bioorg.2006.11.003

Bioorganic Chemistry 35 (2007) 221–232
www.elsevier.com/locate/bioorg
Synthesis and antiviral activities of new acyclic
and ‘‘double-headed’’ nucleoside analogues
a,1
a,2
a
b
Xinying Zhang , Adel Amer , Xuesen Fan , Jan Balzarini ,
b
b
c
c
Johan Neyts , Erik De Clercq , Mark Prichard , Earl Kern ,
a,
*
Paul F. Torrence
a
Department of Chemistry and Biochemistry, Northern Arizona University, Flagstaff, AZ 86011-5698, USA
b
Rega Institute for Medical Research, Katholieke Universiteit Leuven, 3000 Leuven, Belgium
Department of Pediatrics, University of Alabama School of Medicine, Birmingham, AL, USA
c
Received 29 September 2006
Available online 2 December 2006
Abstract
To develop an understanding of the structure–activity relationships for the inhibition of ortho-
poxviruses by nucleoside analogues, a variety of novel chemical entities were synthesized. These
included a series of pyrimidine 5-hypermodified acyclic nucleoside analogues based upon recently
discovered new leads, and some previously unknown ‘‘double-headed’’ or ‘‘abbreviated’’ nucleo-
sides. None of the synthetic products possessed significant activity against two representative ortho-
poxviruses; namely, vaccinia virus and cowpox virus. They were also devoid of significant activity
against a battery of other DNA and RNA viruses. So far as the results with the orthopoxviruses
0
and herpes viruses, the results may point to the necessity for nucleoside analogues 5 -phosphoryla-
tion for antiviral efficacy.
ꢀ
2007 Elsevier Inc. All rights reserved.
Keywords: Smallpox; Vaccinia virus; Cowpox virus; Thymidine kinase
*
Corresponding author. Fax: +1 928 523 8111.
E-mail address: Paul.Torrence@nau.edu (P.F. Torrence).
On leave from the School of Chemistry and Environmental Sciences, Henan Normal University, Xinxiang,
1
Henan 453007, China.
2
On leave from the Department of Chemistry, Faculty of Science, Alexandria University, Egypt.
0
045-2068/$ - see front matter ꢀ 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2006.11.003

222
X. Zhang et al. / Bioorganic Chemistry 35 (2007) 221–232
1
. Introduction
The threat of bioterrorism has mandated that drug countermeasures be developed
against the ancient scourge of smallpox caused by the variola orthopoxvirus [1–5]. A pri-
ority of the US Government is to have two food and drug administration (FDA) approved
agents available with two more in the drug pipeline [6].
ꢁ
One drug, Cidofovir (Vistide ), licensed to treat cytomegalovirus (CMV) retinitis in
HIV-infected patients, is available through a special protocol (Investigational New Drug,
IND) for emergency treatment of smallpox or vaccine reactions (http://www.bt.cdc.gov/
agent/smallpox/vaccination/cidofovir.asp) if vaccine immune globulin (VIG) is not effec-
tive [7–10]. Some entities for the treatment of orthopoxvirus infections in pre-clinical or
clinical development include inhibitors of viral morphogenesis (TTP-6171) [11] and viral
release (ST-246) [12] as well as compounds interacting with cellular targets [i.e. Erb-1
kinase inhibitors (CI-1033) [13,14] and tyrosine kinase inhibitors (Gleevec, STI-571)] [15].
Recently, we have described several novel 5-substituted pyrimidine nucleosides (Fig. 1,
0
2
a–2c) all derived from the 5-formyl-2 -deoxyuridine (Fig. 1, 1) building block [16–19]. In
an attempt to improve upon these leads, we have prepared a series of analogues based
upon a structural motif that has been successful in other domains of antiviral chemother-
apy. We chose the acyclic nucleoside analogue structural motif; specifically, the 2-hydroxy-
ethoxymethyl substituent, exemplified by acyclovir [20,21]. Although the lead compounds
of Fig. 1 have been found active [16–19] against only orthopoxviruses, vaccinia virus and
cowpox virus, and some herpes viruses (Prichard et al., unpublished observations), to be
judicious, the synthetic products of this study were evaluated against a wide range of RNA
and DNA viruses.
In a second structural modification, we have synthesized several unusual ‘‘double-head-
ed’’ nucleosides. There are relatively few publications on the synthesis of these so called
Fig. 1. Structures of key intermediate (1) and nucleosides (2a, 2b, and 2c) with established anti-orthopoxvirus
activities.

X. Zhang et al. / Bioorganic Chemistry 35 (2007) 221–232
223
‘
‘double-headed nucleosides’’ or ‘‘abbreviated nucleosides’’. Nelson Leonard and associ-
ates [22,23], and in a separate simultaneous effort, Shen and colleagues [24] at Merck pro-
vided the first syntheses and characterizations of such molecules. Since then a number of
publications, a few of which are referenced here [25–29], have followed Leonard’s and
Shen’s leads.
While these ‘‘double-headed’’ nucleosides, in the embodiment presented in this study,
likely would not be phosphorylated by cellular or viral kinases because of the missing
0
5
-hydroxy moiety, they might be expected to exhibit promising biological activities for
other reasons. 1,2,4-Triazoles have demonstrated antiviral activity against HIV-1
30,31]. In addition, the closely related 1,2,3-triazole and benzotriazoles have shown activ-
[
ity as xanthine oxidase inhibitors, antimicrobial, anti-influenza, and anti-orthopoxvirus
agents [32–35]. These modified nucleosides recruit the potential of simultaneous recogni-
tion of two heterocyclic bases in a bisubstrate type of interaction. Alternatively,
‘
‘double-headed’’ nucleosides may interact with nucleic acids using one base for
Watson–Crick pairing and the second one as the intercalator stabilizing the base pair.
Indeed, the latter possibility has been realized in a recent study [36,37] (Fig. 2).
1
.1. Chemistry
The synthetic approach to acyclic pyrimidine nucleoside analogues 4a–7b involved first
the preparation of the known 1-[(2-acetoxyethoxy)methyl]-thymine (3a), followed by its
oxidation to 1-[(2-acetoxyethoxy)methyl]-5-formyluracil (4a) followed by acid-catalyzed
reaction with CH OH and deprotection to give the previously unknown 1-(2-hydroxyeth-
3
oxymethyl)-5-(dimethoxymethyl)uracil (5b). Alternatively, 1-[(2-acetoxyethoxy)methyl]-5-
formyluracil (4a) itself could be deacetylated to provide 1-(2-hydroxyethoxymethyl)-5-
formyluracil (4b), also previously unreported. This latter compound was the basis for
the preparation of 1-(2-hydroxyethoxymethyl)-5-(2,2-dicyanovinyl)uracil (6b) directly
from malononitrile. Likewise reaction of malononitrile with 1-[(2-acetoxyethoxy)meth-
yl]-5-formyluracil (4a) yielded 1-[(2-acetoxyethoxy)methyl]-5–(2,2-dicyanovinyl)uracil
Fig. 2. Acyclic nucleoside analogues prepared to be evaluated as potential antiviral agents.

224
X. Zhang et al. / Bioorganic Chemistry 35 (2007) 221–232
Fig. 3. Synthetic approach to ‘‘double-headed’’ nucleoside analogues.
(6a). Similarly, reaction 1-[(2-acetoxyethoxy)methyl]-5-formyluracil (4a) with malononitri-
le and 1,3-cyclohexanedione gave rise to 1-[(2-acetoxyethoxy)methyl]-5-(2-amino-3-cyano-
5
1
-oxo-5,6,7,8-tetrahydro-4H-chromen-4-yl)uracil (7a) and reaction of malononitrile and
,3-cyclohexanedione with 1-[(2-hydroxyethoxy)methyl]-5-formyluracil (4b) gave 1-[(2-
hydroxyethoxy)methyl]-5-(2-amino-3-cyano-5-oxo-5,6,7,8-tetrahydro-4H-chromen-4-yl)-
uracil (7b).
Synthesis of the ‘‘double-headed’’ nucleosides (9, 10, and 11) was based on the utiliza-
0
tion of carboxylic acid nucleosides 8a–8c at carbon 5 as the synthon for the second het-
erocyclic base. These uronic acid congeners were generated by application of reported
procedures [38]. Reaction of 8a, 8b, or 8c with phthalazin-1-yl-hydrazine hydrochloride
in dimethylformamide in the presence of diisopropylethylamine (DIEA) as a base and
2
-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate methana-
minium (HATU) as coupling reagent at 0–3ꢂ followed by treatment of the residual after
the work up gave the triazolophthalazine derivatives 9, 10, and 11, respectively, in accept-
able yields (Fig. 3).
1
.2. Antiviral activities
Synthetic products were evaluated for antiviral activity against HIV-1, HIV-2, vaccinia
virus, cowpox virus, herpes simplex viruses types 1 and 2, parainfluenza virus type 3, Punta
Toro virus, vescicular stomatitis virus, respiratory syncytial virus, Coxsackie B4 virus, reo-
virus type 1, and Sindbis virus (Tables 1 and 2).

Table 1
Antiviral activities of compounds 3a–11 against selected RNA viruses in Vero, HeLa, and HEL cells
a
Compound
EC50
Parainfluenza
virus type 3 (Vero) 1 (Vero)
Reovirus type Sindbis
Punta Toro
Vescicular stomatitis
virus (HEL/HeLa)
Coxsackie B4 virus Respiratory syncytial
virus (Vero) virus (Vero)
(HeLa/Vero)
virus (HeLa)
3
3
4
4
5
5
6
6
7
7
9
1
1
a
b
a
b
a
b
a
b
a
b
>200
>200
>40
>200
>200
>40
>200
>200
>200
>200
>200
>200
>100
>100
>100
>100
>100
>100
>100
>200
>200
>40
>200
>200
>200
>200
>200
>200
>100
>100
>100
>100
>100
>100
>100
>200
>200
>200
>200
>200
>200
>100
>100
>100
>100
>100
>100
>100
>200
>200
>200
>200
>200
>200
>100
>100
>100
>100
>100
>100
>100
>40
>40
>40
>200
>200
>100
>100
>100
>100
>100
>100
>100
>200
>200
>100
>100
>100
>100
>100
>100
>100
>200
>200
>100
>100
>100
>100
>100
>100
>100
0
1
a
Concentration (lM) required to effect a 50% reduction in virus-induced cytopathogenicity in either Vero cells, HEL or HeLa cells, as indicated.

226
X. Zhang et al. / Bioorganic Chemistry 35 (2007) 221–232
Table 2
Antiviral activities of compounds 3a–11 against HIV-1, HIV-2, vaccinia virus (VV), cowpox virus (CV), and
herpes viruses
Compound EC50
HIV-1
CEM)
HIV-2
(CEM)
VV
(HEL)
VV
(HSF)
CV
(HSF)
HSV-1 (KOS)
(HEL)
HSV-2 (G)
(HEL)
(
3
3
4
4
5
5
6
6
7
7
9
1
a
b
a
b
a
b
a
b
a
b
>250
>250
>250
>250
>250
>250
>50
>250
>250
>250
>250
>250
>250
>50
>200
>200
>200
>200
>200
>200
100
>100
>100
>100
>100
>100
>300
>300
>300
>300
>300
>300
>60
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>60
>300
>300
>300
>300
>300
>200
>200
>200
>200
>200
>200
>100
>100
>100
>100
>100
>100
>200
>200
>200
>200
>200
>200
100
>100
>100
>100
>100
>100
>50
>50
>250
>250
>250
>250
>250
>250
>250
>250
1
Concentration (lM) required to effect a 50% reduction in virus-induced cytopathogenicity in either CEM cells,
HEL or HSF cells, as indicated. Under the conditions employed above, both Acyclovir and Ganciclovir displayed
and a submicromolar EC50 against HSV-1 and HSV-2 and Brivudin showed an EC50 of 12 lM against vaccinia
virus assayed in HEL cells. Cowpox virus (CV) and vaccinia virus (VV) showed EC50s in the 10 lM range when
they were assayed in HSF cells.
2
. Discussion
Although the parent nucleosides 2a, 2b, and 2c all possess potent in vitro antiviral activ-
ity [16–19] against both of the orthopoxviruses vaccinia virus and cowpox virus, their acy-
clic analogues, 5b, 6b, and 7b, were devoid of activity. This may be a consequence of the
substrate specificity of the orthopoxviral thymidine kinase. Recently, we have presented
evidence that at least one of the prototype antivirals, 2c, depends upon the viral thymidine
kinase for antiviral activity [19]. Moreover, the active analogue 2c is a good substrate for
vaccinia thymidine kinase [19]. Even though these studies have indicated that orthopoxvi-
ral thymidine kinase may be much more promiscuous than previously believed, this pro-
miscuity may not extend to acceptance of such hypermodification of the sugar domain as
embodied in the structures of 5b, 6b, and 7b. In support of this hypothesis was the earlier
observation that acyclovir is not phosphorylated by vaccinia thymidine kinase [39].
This line of reasoning probably also explains the inactivity of 5b, 6b, and 7b against the
herpes simplex viruses evaluated. We have found that compounds such as 2c are antiviral-
ly active against HSV-1 and varicella zoster virus, again by virtue of their phosphorylation
by the respective viral thymidine kinases (Prichard et al., data not shown). Thus although
the relatively promiscuous herpes simplex virus thymidine kinase readily phosphorylates
the acyclic nucleoside analogue acyclovir, it may not accept the hypermodification repre-
sented by 7b and its congeners.
The inactivity of the ‘‘double-headed’’ nucleosides (9–11), at least against the ortho-
poxviruses and herpes viruses, is also most probably connected to the inability of these
0
compounds to be phosphorylated because of the missing 5 -hydroxyl moiety.

X. Zhang et al. / Bioorganic Chemistry 35 (2007) 221–232
227
While overall, this study revealed no intriguing antiviral activities, it does define more
clearly the boundaries in a future search for efficacious orthopoxvirus countermeasures.
3
. Experimental section
Melting points were recorded with a Barnstead 1201D electrothermal melting point
1
13
apparatus and are uncorrected. H NMR and C NMR spectra were recorded on a Var-
ian 400 MHz spectrometer. CDCl , CD OD or DMSO-d was used as the NMR solvent
3
3
6
for different compounds. The chemical shifts of the deuterated solvent served as internal
standard. The mass spectra were performed on a HP 1100 MSD spectrometer at the
HT Laboratories, San Diego. The HRMS (High Resolution Mass Spectra) were per-
formed on a JEOL HX 110A spectrometer at the Department of Chemistry, University
of Arizona. Silica gel column chromatography was conducted with Sigma–Aldrich silica
gel (70 ꢀ 230 mesh). The preparation of (2-acetoxyethoxy)methyl bromide from 1,3-
dioxolane and acetyl bromide was according to the procedure of Robins and Hatfield [40].
3
.1. 1-[(2-Acetoxyethoxy)methyl]-thymine (3a)
A suspension of 0.63 g (5 mmol) of thymine in 15 mL of anhydrous CH Cl was added
2
2
3
mL (12 mmol) of N,O-bis(trimethylsilyl)acetamide (BSA) under nitrogen. The mixture
was stirred at room temperature. After the solution became clear, (2-acetoxyethoxy)meth-
yl bromide (1.0 g, 5 mmol) was added dropwise. After 2 h, the reaction liquid was poured
slowly into a mixture of cold saturated aqueous NaHCO (25 mL) and CHCl (50 mL).
3
3
The resulting emulsion was separated by filtration. The aqueous layer was extracted with
EtOAc (3 · 25 mL). The combined organic layers were dried over anhydrous Na SO and
2
4
evaporated under reduced pressure. After purification through a silica gel column with
CHCl ACH COCH (3:1) as the eluant, 1-[(2-acetoxyethoxy)methyl]-thymine (3a) was
3
3
3
obtained (0.94 g, 3.88 mmol, colorless solid, yield 78%): mp 115–117 ꢂC (Lit. [34]
1
1
23–125 ꢂC); H NMR (CDCl ) d: 1.89 (s, 3H, CH ), 2.02 (s, 3H, CH CO), 3.75(A B -
3
3
3
2 2
t, J = 4.7 Hz, 2H, AcOCH CH O), 4.16 (A B -t, J = 4.7 Hz, 2H, AcOCH CH C), 5.14
s, 2H, NCH O), 7.13 (s, 1H, C H), 9.92 (s, 1H, NH); C NMR (CDCl ) d: 12.5, 21.0,
2 6 3
3.2, 67.7, 76.7, 112.1, 139.1, 151.5, 164.1, 171.0; MS (ES) m/e: 241 [MꢁH] , 265
2
2
2
2
2
2
1
3
(
6
ꢁ
+
[
M+Na] .
3
.2. 1-(2-Hydroxyethoxymethyl)-thymine (3b)
NaOMe in MeOH (3 mL, 0.5 N) was added to a solution of 3a (0.3 g, 1.24 mmol) in
1
0 mL of anhydrous MeOH. The mixture was stirred at room temperature. After the reac-
tion was complete (TLC), Amberlite IR-120 acid resin was added to adjust the pH value to
. The resin then was filtrated, and the solvent was evaporated under reduced pressure to
provide a colorless solid. After purification by a silica gel column with CHCl AMeOH
7
3
(
5:1) as the eluant, the product 1-(2-hydroxyethoxymethyl)-thymine (3b) was obtained
as colorless crystals in a yield of 93% (0.23 g, 1.15 mmol): mp 140–141 ꢂC (Lit. [40]
1
1
50.5–152 ꢂC); H NMR (DMSO-d ) d: 1.75 (d, J = 1.2 Hz, 3H, CH ), 3.45–3.46 (A B
6
3
2 2
m, 4H, OCH CH O), 4.61–4.64 (m, 1H, OH), 5.04 (s, 2H, NCH O), 7.55 (d,
2
2
2
1
3
J = 1.2 Hz, 1H, C H), 11.28 (s, 1H, NH); C NMR (DMSO-d ) d: 12.5, 60.7, 71.1,
7.0, 109.8, 141.2, 151.8, 164.9; MS (ES) m/e: 199 [MꢁH] , 223 [M+Na] .
6
6
ꢁ
+
7

228
X. Zhang et al. / Bioorganic Chemistry 35 (2007) 221–232
3
.3. 1-[(2-Acetoxyethoxy)methyl]-5-formyluracil (4a)
A CH CN solution (10 mL) containing 1-[(2-acetoxyethoxy)methyl]thymine (3a)
3
(
0.61 g, 2.5 mmol) and 2,6-lutidine (1 mL) was added to a solution of K S O (1.35 g,
2 2 8
5
6
mmol) and CuSO Æ5H O (0.25 g, 1 mmol) in 10 mL H O. The mixture was stirred at
4 2 2
5ꢂ for 2 h. After completion, the mixture was concentrated to half of the initial volume
and the remaining solution was extracted with EtOAc (3 · 25 mL). The organic layers
were combined and washed with H O. The aqueous layer was back-extracted with CHCl .
2
3
All the organic liquids were combined, dried over anhydrous Na SO , and then concen-
2
4
trated. The residue was purified through a silica gel column chromatography with
EtOAc–hexane (2:1) as eluant. The fractions were collected, concentrated, and the residue
recrystallized from EtOAc to give 1-[(2-acetoxyethoxy)methyl]-5-formyluracil (4a) as
1
colorless crystals (0.20 g, 0.78 mmol, 31%): mp 115–116 ꢂC; H NMR (CDCl ) d: 2.07
3
(
s, 3H, CH CO), 3.81–3.84 (A B m, 2H, AcOCH CH O), 4.21–4.23 (A B m, 2H,
3 2 2 2 2 2 2
AcOCH CH O), 5.29 (s, 2H, NCH O), 8.23 (s, 1H, C H), 9.25 (s, 1H, NH), 10.02 (s,
2
2
2
6
1
3
1
1
H, CHO); C NMR (CDCl ) d: 21.0, 63.0, 68.6, 78.1, 112.1, 148.3, 150.1, 162.0,
3
ꢁ
+
71.0, 186.1; MS (ES) m/e: 255 [MꢁH] , 279 [M+Na] ; HRMS (FAB) Calcd for
+
C H N O : 257.0773 [M+H] . Found: 257.0785.
1
0
13
2
6
3
.4. 1-[(2-Acetoxyethoxy)methyl]-5-(dimethoxymethyl)uracil (5a)
1
-[(2-Acetoxyethoxy)methyl]-5-formyluracil (4a) (0.37 g, 1.45 mmol) was dissolved in
anhydrous methanol (15 mL), and this solution refluxed with stirring in the presence of
washed dry Amberlite IR-120 acid resin (0.15 g) for 40 min. The mixture was filtered to
remove the resin and the filtrate was concentrated to give a colorless solid. The solid
was washed with anhydrous methanol and anhydrous ethyl ether and the resulting color-
less solid, 1-[(2-acetoxyethoxy)methyl]-5-(dimethoxymethyl)uracil (5a), was obtained in a
1
yield of 98% (0.43 g, 1.42 mmol): mp 113–114 ꢂC; H NMR (CD OD) d: 2.02 (s, 3H,
3
CH CO), 3.36 (s, 6H, (OCH ) ), 3.77–3.79 (A B m, 2H, AcOCH CH O), 4.17–4.19
3
3 2
2
2
2
2
(
A B m, 2H, AcOCH CH O), 5.22 (s, 2H, NCH O), 5.28 (d, J = 0.8 Hz, 1H,
2 2 2 2 2
1
3
CH(OCH ) ), 7.70 (d, J = 0.8 Hz, 1H, C H); C NMR (CD OD) d: 19.5, 52.8, 63.3,
6
3
2
6
3
ꢁ
7.4, 77.1, 98.2, 111.5, 142.9, 151.5, 163.4, 171.5; MS (ES) m/e: 301 [MꢁH] , 325
+
+
[
M+Na] ; HRMS (ESI) Calcd for C H N O Na: 325.1012 [M+Na] . Found: 325.1002.
12 18 2 7
3
.5. 1-(2-Hydroxyethoxymethyl)-5-(dimethoxymethyl)uracil (5b)
A NH /MeOH solution (2 mL, 7 N) was added to a solution of 1-[(2-acetoxyeth-
3
oxy)methyl]-5-(dimethoxymethyl)-uracil (5a) (0.30 g, 1 mmol) in anhydrous methanol
8 mL). The mixture was stirred in an ice bath for 3 h, then at ambient temperature for
h. The solvent then was removed under high vacuum. The residue was purified through
(
5
a silica gel column with CHCl AMeOH (6:1) as the eluant to give the corresponding prod-
3
uct 1-(2-hydroxyethoxymethyl)-5-(dimethoxymethyl)uracil (5b) in a yield of 96% (0.25 g,
1
0
(
.96 mmol, light yellow solid): mp 92–94 ꢂC; H NMR (CD OD) d: 3.35 (s, 6H,
3
OCH ) ), 3.61–3.66 (A B m, 4H, AcOCH CH O), 5.22 (s, 2H, NCH O), 5.28 (d,
3 2 2 2 2 2 2
J = 0.8 Hz, 1H, CH(OCH ) ), 7.72 (d, J = 0.8 Hz, 1H, C H); C NMR (CD OD) d:
1
3
3
2
6
3
ꢁ
5
[
2.8, 60.8, 70.9, 77.3, 98.1, 111.3, 143.1, 151.5, 163.4; MS (ES) m/e: 259 [MꢁH] , 283
+
+
M+Na] ; HRMS (ESI) Calcd for C H N O Na: 283.0906 [M+Na] . Found: 283.0904.
10 16 2 6

X. Zhang et al. / Bioorganic Chemistry 35 (2007) 221–232
229
3
.6. 1-(2-Hydroxyethoxymethyl)-5-formyluracil (4b)
-(2-Hydroxyethoxymethyl)-5-(dimethoxymethyl)uracil (5b) (0.26 g, 1 mmol) was add-
1
ed to a solution of 15 mL acetic acid and 3 mL H O. The mixture was stirred at 50ꢂ over-
2
night. The solvent was evaporated under reduced pressure, and the residue was purified
through a silica gel column with CHCl AMeOH (6:1) as eluant. The fractions were collect-
3
ed, concentrated, and the residue was recrystallized from EtOH to give 1-(2-hydroxyeth-
oxymethyl)-5-formyluracil (4b) as light yellow solid (0.21 g, 0.98 mmol 98%): mp
1
1
3
8
6
38–139.5 ꢂC; H NMR (DMSO-d ) d: 3.44–3.48 (A B m, 2H, OCH CH OH), 3.52–
6
2
2
2
2
.54 (A B m, 2H, OCH CH OH), 4.66 (t, J = 5.2 Hz, 1H, OH), 5.22 (s, 2H, NCH O),
2
2
2
2
2
1
3
.49 (s, 1H, C H), 9.77 (s, 1H, CHO), 11.76 (s, 1H, NH); C NMR (DMSO-d ) d:
6
6
ꢁ
0.7, 71.7, 78.3, 111.4, 150.8, 151.8, 162.7, 187.1; MS (ES) m/e 213 [MꢁH] , 237
+
+
[
M+Na] ; HRMS (FAB) Calcd for C H N O : 215.0668 [M+H] . Found: 215.0659.
8 11 2 5
3
.7. 1-[(2-Acetoxyethoxy)methyl]-5-(2,2-dicyanovinyl)uracil (6a)
Malononitrile (92 mg, 1.4 mmol) and 1-[(2-acetoxyethoxy)methyl]-5-formyluracil (4a)
(
179 mg, 0.7 mmol) were dissolved in anhydrous EtOH (8 mL). The mixture was stirred
at 50ꢂ overnight. The resulting reaction mixture was concentrated, and the residue was
purified through silica gel column chromatography with EtOAc–hexane (2:1) as eluant.
The fractions were collected, concentrated, and the residue was recrystallized from EtOH
to give 1-[(2-acetoxyethoxy)methyl]-5-(2, 2-dicyanovinyl)uracil (6a) as pale yellow crystals
1
(
157 mg, 0.52 mmol, 74%): mp 137–139 ꢂC; H NMR (CD OD) d: 2.02 (s, 3H, CH CO),
3
3
3
2
6
.83–3.85 (A B m, 2H, AcOCH CH O), 4.19–4.22 (A B m, 2H, AcOCH CH O), 5.31 (s,
2 2 2 2 2 2 2 2
H, NCH O), 7.97 (s, 1H, CH@), 8.76 (s, 1H, C H); C NMR (CD OD) d: 19.5, 63.2,
8.1, 78.3, 79.5, 107.7, 113.0, 113.8, 148.5, 150.0, 151.4, 161.3, 171.5; MS (ES) m/e: 303
1
3
2
6
3
ꢁ
+
[
MꢁH] ; HRMS (FAB) Calcd for C H N O : 305.0886 [M+H] . Found: 305.0890.
1
3
13
4
5
3
.8. 1-(2-Hydroxyethoxymethyl)-5-(2, 2-dicyanovinyl)uracil (6b)
Malononitrile (79 mg, 1.2 mmol) and 1-(2-hydroxyethoxymethyl)-5-formyluracil (4b)
(
128.4 mg, 0.6 mmol) were dissolved in anhydrous EtOH (10 mL). The mixture was stirred
at room temperature overnight. The resulting reaction mixture was concentrated, and the
residue was purified through silica gel column chromatography with CHCl AMeOH (6:1)
3
as eluant. The fractions were collected, concentrated, and the residue was recrystallized
from EtOH to give 1-(2-hydroxyethoxymethyl)-5-(2,2-dicyanovinyl)uracil (6b) as a yellow
1
solid (102 mg, 0.39 mmol, 65%): mp 124–125 ꢂC; H NMR (CD OD) d: 3.65–3.71 (A B
3
2
2
1
3
m, 4H, OCH CH O), 5.32 (s, 2H, NCH O), 7.96 (s, 1H, CH@), 8.78 (s, 1H, C H);
C
2
2
2
6
NMR (CD OD) d: 60.7, 71.7, 78.5, 79.2, 107.5, 113.1, 113.9, 148.7, 150.0, 151.4, 161.4;
3
ꢁ
+
MS (ES) m/e: 261 [MꢁH] , 285 [M+Na] ; HRMS (FAB) Calcd for C H N O :
1
1
11
4
4
+
2
63.0780 [M+H] . Found: 263.0790.
3.9. 1-[(2-Acetoxyethoxy)methyl]-5-(2-amino-3-cyano-5-oxo-5,6,7,8-tetrahydro-4H-
chromen-4-yl)uracil (7a)
1
-[(2-acetoxyethoxy)methyl]-5-formyluracil (4a) (102 mg, 0.4 mmol), malononitrile
(
33 mg, 0.5 mmol), and 1,3-cyclohexanedione (57 mg, 0.5 mmol) were dissolved in

230
X. Zhang et al. / Bioorganic Chemistry 35 (2007) 221–232
anhydrous ethanol (8 mL). The mixture was stirred at 50 ꢂ overnight. The colorless precip-
itate that formed was filtered off and rinsed with ethanol and ethyl ether to give the product
1
4
-[(2-acetoxy-ethoxy)-methyl]-5-(2-amino-3-cyano-5-oxo-5,6,7,8-tetrahydro-4H-chromen-
-yl)uracil (7a) (100 mg). The filtrate was concentrated and the residue was purified through
silica gel column chromatography with CHCl AMeOH (5:1) as eluant to yield an additional
3
3
5 mg quantity of compound 7a. Thus, the product 7a was obtained in a total yield of 81%
1
(
135 mg, 0.32 mmol): mp 202–204 ꢂC; H NMR (DMSO-d ) d: 1.82–1.95 (m, 2H, CH ), 1.98
6
2
(
s, 3H, CH CO), 2.20–2.32 (m, 2H, CH ), 2.45–2.52 (m, 2H, CH ), 3.66–3.68 (A B m, 2H,
3
2
2
2 2
AcOCH CH O), 4.01(s, 1H, CH), 4.05–4.08 (A B m, 2H, AcOCH CH O), 5.08 (s, 2H,
2
2
2
2
2
2
1
3
NCH O), 6.89 (s, 2H, NH ), 7.58 (s, 1H, C H), 11.3 (s, 1H, NH); C NMR (CD OD) d:
2
2
6
3
2
1
0.5, 21.3, 27.3, 29.4, 36.9, 55.9, 63.6, 67.5, 77.2, 111.8, 115.7, 120.4, 142.5, 151.5, 160.1,
ꢁ
+
62.8, 166.1, 171.0, 196.6; MS (ES) m/e: 415 [MꢁH] , 439 [M+Na] ; HRMS (FAB) Calcd
+
for C H N O : 417.1410 [M+H] . Found: 417.1410.
1
9
21
4
7
3.10. 1-[(2-Hydroxyethoxy)methyl]-5-(2-amino-3-cyano-5-oxo-5,6,7,8-tetrahydro-4H-
chromen-4-yl)uracil (7b)
1
-[(2-hydroxyethoxy)methyl]-5-formyluracil (4b) (107 mg, 0.5 mmol), malononitrile
(
40 mg, 0.6 mmol) and 1,3-cyclohexanedione (67 mg, 0.6 mmol) were dissolved in anhy-
drous ethanol (8 mL). The mixture was stirred at 50ꢂ overnight. The colorless precipitate
that formed was filtered off and rinsed with ethanol and ethyl ether to give the product
1
4
-[(2-hydroxyethoxy)methyl]-5-(2-amino-3-cyano-5-oxo-5,6,7,8-tetrahydro- 4H-chromen-
-yl)uracil (7b) (110 mg). The filtrate was concentrated and the residue was purified
through silica gel column chromatography with CHCl AMeOH (5:1) as eluant to give
3
an additional 45 mg quantity of compound 7b. Thus, the product 7b was obtained in a
1
total yield of 83% (155 mg, 0.41 mmol): mp 208–209.5 ꢂC; H NMR (DMSO-d ) d:
6
1
.82–1.95 (m, 2H, CH ), 2.20–2.28 (m, 2H, CH ), 2.45–2.52 (m, 2H, CH ), 3.43–3.49
2 2 2
(
A B m, 4H, AcOCH CH O), 4.02(s, 1H, CH), 4.63–4.66 (m, 1H, OH), 5.07 (s, 2H,
2 2 2 2
1
3
NCH O), 6.88 (s, 2H, NH ), 7.56 (s, 1H, C H), 11.2 (s, 1H, NH); C NMR (DMSO-
2
2
6
d ) d: 20.5, 27.3, 29.3, 37.0, 56.0, 60.7, 71.2, 77.4, 111.8, 115.6, 120.4, 142.6, 151.4,
6
ꢁ
+
1
60.1, 162.9, 166.0, 196.6; MS (ES) m/e: 373 [MꢁH] , 397 [M+Na] ; HRMS (FAB) Calcd
+
for C H N O : 375.1304 [M+H] . Found: 375.1299.
1
7
19
4
6
3.11. General procedure for coupling of carboxylic acid nucleosides with hydralazine
hydrochloride
Step 1. A mixture of carboxylic acid nucleoside (1 mmol), HATU (2-(1H-7-aza-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate methan-aminium,
0
0
.38 g, 1 mmol) and DIPEA (diisopropylethylamine) (0.34 mL, 2 mmol) was stirred at
ꢂC for 3 min. [preactivation period] in 4 mL anhydrous DMF (dimethylformamide).
A solution of hydralazine.HCl (0.186 g), and DIPEA (0.18 mL, mmol) in DMF (4 mL)
was added to this solution at 0ꢂ. The reaction mixture was stirred at 0 ꢂC for 3 h and left
overnight at room temperature. The reaction mixture was diluted with ethyl acetate
(
80 mL), and the mixture was washed with 5% aqueous citric acid solution (2 · 15 mL),
saturated sodium bicarbonate solution (2 · 15 mL) and saturated sodium chloride solu-
tion (2 · 15 mL). The organic layer was dried over anhydrous sodium sulphate, filtered
and the solvent was removed in vacuo.

X. Zhang et al. / Bioorganic Chemistry 35 (2007) 221–232
231
Step 2. The residual solid was treated with methylene chloride (5 mL), TFA (5 mL) and
water (0.2 mL). The mixture was stirred at room temperature for 2 h. The solvent was
evaporated in vacuo and the residue was subjected to column chromatography on silica
gel, eluting with CHCl AMeOH (4:1). The following compounds were obtained after sol-
3
vent evaporation in vacuo.
From compound 8a, compound 9, mp 175–177 ꢂC (dec) was obtained in a yield of 20%:
1
0
0
H NMR (DMSO-d ) d: 4.52 (‘‘dd’’, 1H, H-2 ), 4.56 (‘‘dd’’, 1H, H-3 ), 5.53 (d, J = 3.2 Hz,
6
0
1
H, H-4 ), 5.73 (d, 1H, H-5), 5.75 (br s, 2H, 2OH, D O exchangeable), 6.10 (d, J = 6 Hz, 1
H, H-1 ), 7.94, 8.09, 8.22, 8.50, 9.14 (t, m, d, d, s, 6H, ArH and H-6), 11.37 (s, 1H, NH,
2
0
1
3
D O exchangeable); C NMR (DMSO-d ) d: 73.32, 73.93, 76.10, 88.80, 103.06, 122.89,
2
6
1
23.22, 123.88, 129.92, 132.06, 135.23, 141.22, 143.52, 149.08, 149.56, 151.51, 163.71;
+
+
HRMS–FAB Calcd for C H N O : 383.1113 [M+H] . Found: 383.1104.
1
7
15
6
5
1
From compound 8b, compound 10, mp 231–233 ꢂC was obtained in a yield of 19%: H
0
0
NMR (DMSO-d ) d: 2.56–2.62 (m, 4H, 2H-2 , and 2H-3 ), 5.64 (‘‘d’’, 1H, H-5), 5.67 (‘‘dd’’,
1
H-6), 11.31 (s, 1H, NH, D O exchangeable); C NMR (DMSO-d ) d: 31.67, 31.95,
6
0
0
H, H-4 ), 6.24 (‘‘dd’’, 1H, H-1 ), 6.98, 8.08, 8.23, 8.52, 9.14 (m, t, d, d, s, 6H, ArH, and
1
3
2
6
7
1
2.27, 86.61, 102.32, 122.91, 123.27, 123.87, 129.89, 132.03, 135.19,, 141.16, 143.63,
49.40, 149.98, 151.18, 163.83; HRMS–FAB Calcd for C H N O : 351.1206 [M+H] .
17 15 6 3
+
+
Found: 351.1216 .
1
From compound 8c, compound 11, mp 198–200 ꢂC, was obtained in a yield of 25%; H
0
0
NMR (DMSO-d ) d: 4.90 (‘‘dd’’, 1H, H-3 ), 4.97 (‘‘dd’’, 1H, H-2 ), 5.60 (d, J = 4 Hz, 1H,
6
0
0
H-4 ), 5.85, 5.87 (2d overlapped ꢀt, 2H, 2OH, D O exchangeable), 6.19 (d, 1H, J = 5.6 Hz,
2
H-1 ), 7.27 (br s, 2H, NH , D O exchangeable), 7.93, 8.06, 8.11, 8.21, 8.49, 8.51, 9.10 (t, t, s,
2
2
1
3
d, s, d, s, 7H, ArH); C NMR (DMSO-d ) d: 69.37, 70.90, 72.29, 84.06, 115.52, 118.98,
6
1
1
19.28, 119.92, 125.96, 128.09, 131.27, 135.70, 139.66, 145.05, 145.46, 146.46, 149.55,
+
+
52.79; HRMS–FAB Calcd for C H N O : 406.1376 [M+H] . Found: 406.1373.
1
8
16
9
3
4
. Antiviral evaluation procedures
These were carried out according to previously published procedures [16–19,41] pursu-
ant to the footnotes of Tables 1 and 2.
Acknowledgments
The authors acknowledge contract USAMRIID DAMD 17-03-C-0081 from the US
Army Medical Research Materiel Command, contract 8019 from the Arizona Biomedical
Research Commission, a grant from the North Atlantic Treaty Organization, Public
Health Service contract NO1-AI-30049 from the NIAID, NIH, and the State of Arizona
Proposition 301 Funds for financial support, The authors also thank Kathy Keith and
Robert Smith for expert technical assistance.
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