Design, synthesis, and biological evaluation of novel nucleoside and nucleotide analogues as agents against DNA viruses and/or retroviruses

Article abstract of DOI:10.1021/jm010216r

A novel strategy was developed for the synthesis of N⁷-purine acyclic nucleosides 9 and 14. The key step involved the reaction between [2-(p- methoxyphenyloxy)ethoxy]methyl chloride and N⁹-tritylated nucleobases 6 or 11 followed by concomitant self-detritylation. N⁷-Guanine acyclic nucleoside 9 exhibited antiviral activity, but was phosphorylated by both HSV and Vero cell thymidine kinases. Thus, it showed more potent cellular toxicity than acyclovir (2). N⁷-Adenine acyclic nucleoside 14 was found to be an excellent antiviral agent as well as a good inhibitor of calf mucosal adenosine deaminase. This inhibitory property allows for a greater expression of antiviral activity of antiviral agents, such as N⁹-adenine acyclic nucleoside 1 and ara-A (3). Compound 14 was phosphorylated neither by herpes simplex virus (HSV) thymidine kinase nor by Vero cell thymidine kinase, yet it enhanced the rate constant for the monophosphorylation of acyclovir (2) by HSV thymidine kinase. Consequently, the combination of acyclovir (2) and 14 exhibited greater antiviral activity than acyclovir alone. 7-[2(Phosphonomethoxy)ethyl] adenine (20) was also synthesized. The key step involved the reaction of 9-(2-cyanoethyl)adenine (15) with methyl iodoacetate in the presence of lithium 2,2,6,6-tetramethylpiperidine in THF. Unlike 9-[2- (phosphonomethoxy)ethyl] adenine (PMEA, 4), the N⁷-isomer 20 was not phosphorylated effectively by 5-phosphoribosyl 1-pyrophosphate synthetase (PRPP synthetase). Thus, it did not exhibit pronounced antiviral activity. Dinucleotide 5′-monophosphate 24 and its butenolide ester 25 were also synthesized. Compound 24 showed substrate activity toward PRPP synthetase and exhibited notable activity against DNA viruses. The antiviral activity of the ester derivative 25 was found to be higher than that of the parent molecule 24. Dinucleotide 5′-monophosphate 24 is suseptible to degradation by snake venom and spleen phosphodiesterases. However, its respective butenolide ester derivative 25 was completely resistant to snake venom and spleen enzymes. Butenolide ester derivatives 28 and 29 were also synthesized and exhibited notable anti-DNA virus and anti-retrovirus activity in vitro. Compounds 2, 4, 9, 14, 20, 24, 25, and 28 were also evaluated for their inhibitory effect on HSV-1-induced mortality in NMRI mice. N⁷-adenine acyclic nucleoside 14 [LD₅₀ (intraperitoneal, ip) 950 mg/kg], nucleotide-containing butenolide 25 [LD₅₀ (ip) 675 mg/kg], and butenolide 28 [LD₅₀ (ip) 710 mg/kg] were found to be potent anti-HSV-1 agents in vivo. In addition, butenolide 28 efficiently decreased tumor formation induced by Moloney murine sarcoma virus (MSV) in NMRI mice while significantly increasing the survival time of MSV-infected mice.

Full text of DOI:10.1021/jm010216r

3710
J . Med. Chem. 2001, 44, 3710-3720
Design , Syn th esis, a n d Biologica l Eva lu a tion of Novel Nu cleosid e a n d
Nu cleotid e An a logu es a s Agen ts a ga in st DNA Vir u ses a n d /or Retr ovir u ses
Gholam Hossein Hakimelahi,*,† Tai Wei Ly,† Ali A. Moosavi-Movahedi,‡ Moti L. J ain,† Maryam Zakerinia,§
Hady Davari,§ Hui-Ching Mei,| Thota Sambaiah, Ali A. Moshfegh,§ and Shahram Hakimelahi†,∇
Institute of Chemistry, Academia Sinica, Taipei, Taiwan 115, Republic of China, Institute of Biochemistry-Biophysics,
Tehran University, Tehran, Iran, Department of Microbiology, Shiraz University of Medical Sciences, Shiraz, Iran,
Department of Medical Technology, China Medical College, Taichung 404, Taiwan, and Department of Medicinal Chemistry,
Purdue University, Lafayette, Indiana 47907
Received May 17, 2001
A novel strategy was developed for the synthesis of N7-purine acyclic nucleosides 9 and 14.
The key step involved the reaction between [2-(p-methoxyphenyloxy)ethoxy]methyl chloride
and N9-tritylated nucleobases 6 or 11 followed by concomitant self-detritylation. N7-Guanine
acyclic nucleoside 9 exhibited antiviral activity, but was phosphorylated by both HSV and Vero
cell thymidine kinases. Thus, it showed more potent cellular toxicity than acyclovir (2). N7-
Adenine acyclic nucleoside 14 was found to be an excellent antiviral agent as well as a good
inhibitor of calf mucosal adenosine deaminase. This inhibitory property allows for a greater
expression of antiviral activity of antiviral agents, such as N9-adenine acyclic nucleoside 1
and ara-A (3). Compound 14 was phosphorylated neither by herpes simplex virus (HSV)
thymidine kinase nor by Vero cell thymidine kinase, yet it enhanced the rate constant for the
monophosphorylation of acyclovir (2) by HSV thymidine kinase. Consequently, the combination
of acyclovir (2) and 14 exhibited greater antiviral activity than acyclovir alone. 7-[2-
(Phosphonomethoxy)ethyl]adenine (20) was also synthesized. The key step involved the reaction
of 9-(2-cyanoethyl)adenine (15) with methyl iodoacetate in the presence of lithium 2,2,6,6-
tetramethylpiperidine in THF. Unlike 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA, 4), the
N7-isomer 20 was not phosphorylated effectively by 5-phosphoribosyl 1-pyrophosphate syn-
thetase (PRPP synthetase). Thus, it did not exhibit pronounced antiviral activity. Dinucleotide
5′-monophosphate 24 and its butenolide ester 25 were also synthesized. Compound 24 showed
substrate activity toward PRPP synthetase and exhibited notable activity against DNA viruses.
The antiviral activity of the ester derivative 25 was found to be higher than that of the parent
molecule 24. Dinucleotide 5′-monophosphate 24 is suseptible to degradation by snake venom
and spleen phosphodiesterases. However, its respective butenolide ester derivative 25 was
completely resistant to snake venom and spleen enzymes. Butenolide ester derivatives 28 and
29 were also synthesized and exhibited notable anti-DNA virus and anti-retrovirus activity in
vitro. Compounds 2, 4, 9, 14, 20, 24, 25, and 28 were also evaluated for their inhibitory effect
on HSV-1-induced mortality in NMRI mice. N7-adenine acyclic nucleoside 14 [LD50 (intrap-
eritoneal, ip) 950 mg/kg], nucleotide-containing butenolide 25 [LD50 (ip) 675 mg/kg], and
butenolide 28 [LD50 (ip) 710 mg/kg] were found to be potent anti-HSV-1 agents in vivo. In
addition, butenolide 28 efficiently decreased tumor formation induced by Moloney murine
sarcoma virus (MSV) in NMRI mice while significantly increasing the survival time of MSV-
infected mice.
Ch a r t 1
In tr od u ction
Antiviral agents (Chart 1) including 9-[(2-hydroxy-
ethoxy)methyl]guanine (acyclovir, 2),1 9-[(1,3-dihydroxy-
2-propoxy)methyl]guanine (ganciclovir),2 9-[(2,3-dihy-
droxy-1-propoxy)methyl]guanine,3 (S)-9-[3-hydroxy-2-
(phosphonylmethoxy)propyl]adenine (HPMPA), 9-[2-
(phosphonomethoxy)ethyl]adenine (PMEA, 4), and 9-[2-
(phosphonomethoxy)ethyl]guanine (PMEG, 27) are N9-
isomers of acyclic nucleosides.4 The N7-isomers of purine
nucleosides constitute another class of antiviral agents.5
* To whom correspondence should be addressed: phone 886-2-
27898682; fax 886-2-27898554; e-mail hosein@chem.sinica.edu.tw.
† Institute of Chemistry, Academia Sinica, Taipei, Taiwan 115,
Republic of China.
‡ Tehran University.
§ Shiraz University of Medical Sciences.
| China Medical College.
Purdue University.
Under kinetic control,6 alkylation of purines gives
both the N7-isomers and the N9-isomers, with the latter
∇ Present address: Department of Cell Biology, Faculty of Medicine,
University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
10.1021/jm010216r CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/15/2001

Nucleoside/ Nucleotide Analogues as Antiviral Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3711
Sch em e 1
Sch em e 2a
a
Reagents and conditions: (a) (1) Me3SiNHSiMe3, (NH4)2SO4
(cat.), ∆, 24 h; (2) Ph3CCl, MeCN, 25 °C, 7.0 h. (b) Cl(CH2)2OCH2Cl,
DMF, 25 °C, 8.0 h. (c) p-MeO(C6H4)O(CH2)2OCH2Cl, DMF, 25 °C,
8.0 h. (d) CAN, MeCN/H2O (3:1), 0-25 °C, 1.0 h.
being the predominant component.7 Regioselective syn-
thesis of N2-acetyl-7-[(2-acetoxyethoxy)methyl]guanine
(65% yield) through alkylation of N2,N9-diacetylguanine
with 2-oxa-1,4-butanediol diacetate in the presence of
TiCl4 in AcOH has been reported.5b Unfortunately, the
synthesis of N6-acetyl-7-[(2-acetoxyethoxy)methyl]ad-
enine via N,6N9-diacetyladenine by the above strategy
failed. Herein we report a highly regioselective route
toward purine nucleosides with acyclic substituents at
the N-7 position. Our strategy involves the alkylation
of N9-tritylated purines, which is followed by concomi-
tant self-detritylation to yield the desired N7-alkylated
purine nucleosides (Scheme 1). Compounds 9 and 14
synthesized from this strategy were found to possess
significant activity against herpes simplex viruses
(HSV).
(Phosphonomethoxy)alkylpurines (i.e., PMEA, 4) ef-
fectively inhibit a wide array of DNA viruses (herpes-,
adeno-, irido-, and poxviruses) and retroviruses [Molo-
ney murine sarcoma virus (MSV), murine leukemia
virus (MuLV), and human immunodeficiency virus
(HIV)].4 We were interested in the preparation of the
N7-isomer of PMEA, compound 20, using the above
strategy. Unfortunately, the desired precursor 16 was
not produced effectively. Thus, an alternative, and more
efficient, method was devised as shown in Scheme 4.
Unlike PMEA (4), N7-acyclic adenine phosphonate 20
was found to not be a good substrate for PRPP syn-
thetase and did not exhibit potent antiviral activity. On
the other hand, newly synthesized dinucleotide 5′-
monophosphate 24, which is an aggregate of phospho-
nate 20 and adenosine monophosphate (AMP, 21), was
found to be an alternate substrate for PRPP synthetase
and exhibited notable antiviral activity.
hydrolysis of prodrugs is not required due to the
intrinsic activation provided by the anchimeric as-
sistance of C2-OMe. As such, compounds 25, 28, and 29
exhibited superior lipophilicity and notable antiviral
activity.
Ch em istr y
Syn th esis of N7-Gu a n in e Acyclic Nu cleosid es 7
a n d 9 (Sch em e 2). N9-tritylated guanine 6 was syn-
thesized in two steps. Silylation of guanine (5) with
hexamethyldisilazane (HMDS) in the presence of a
catalytic amount of (NH4)2SO4 at refluxing temperature
followed by condensation of the resultant silylated
guanine with trityl chloride in MeCN at 25 °C afforded
the desired N9-tritylated guanine 6 in 90% yield. Treat-
ment of 6 with (chloroethoxy)methyl chloride9 in DMF
at room temperature gave the corresponding N7-alkyl-
ated guanine 7 in 88% yield. Likewise, treatment of 6
with [2-(p-methoxyphenyloxy)ethoxy]methyl chloride10
led to the N7-isomer 8 in 82% yield. Removal of the
p-methoxyphenyl moiety was then achieved by treat-
ment with ceric ammonium nitrate (CAN)11 in a mixture
of MeCN and H2O (3:1) at 0-25 °C to afford compound
9 in 70% yield. Incidentally, acyclovir (2) is the N9-
isomer of the N7-alkylated guanine 9. These two regioi-
1
somers exhibited characteristic differences in their H
and 13C NMR spectra7,12 The signals of the H2C(1′) (5.81
ppm) and HC(8) (8.67 ppm) for the N7-isomer 9 were
found to be shifted downfield relative to the correspond-
ing signals of the N9-isomer 2, in which the H2C(1′) and
HC(8) resonated at 5.35 and 7.81 ppm, respectively. On
the other hand, the NH2 signal was observed to be
shifted upfield for the N7-isomer, 5.96 ppm for com-
pound 9, relative to the corresponding signal for com-
pound 2, which was observed at 6.52 ppm. The 13C NMR
signals for C(1′) (75.25 ppm) and C(8) (143.92 ppm) of
the N7-isomer 9 were found to be shifted downfield
relative to the corresponding signals of acyclovir (2),
which were observed, respectively, at 71.64 and 137.89
ppm. In contrast, the signal of C(5) of compound 9
resonated at 107.16 ppm, which was upfield from that
of the N9-isomer 2 at 116.52 ppm. The UV λmax of the
N7-isomer 9 appeared at 289 nm, whereas the corre-
sponding λmax of the N9-isomer 2 appeared at 253 and
273 (sh) nm.
One major problem often encountered in the use of
nucleotide analogues as potential therapeutics for viral
infections is one of low lipophilicity.8a We have therefore
synthesized novel butenolide ester derivatives 25, 28,
and 29 as membrane-soluble prodrugs.
Generally, phosphoesters are very stable against
nonenzymatic or even enzymatic hydrolysis.8a The
rationale behind the design of our new prodrugs is based
on the known fast hydrolysis of donor-substituted benzyl
phosphoesters.8a Similarly, the oxygen of the C-2 meth-
oxy group of the butenolide moiety can act as a donor8b
group to facilitate the hydrolysis of prodrugs to their
respective active species (see Scheme 6). This induces
the spontaneous cleavage of esters 25, 28, and 29 inside
the infected cells to yield the corresponding bioactive
nucleotides 24, 4, and 27 as well as (Z)-4-(2-hydroxy-
ethylidenyl)-2,3-dimethoxy-∆R,â-butenolide [LD50 (ip) >
1.0 g/kg]. Consequently, enzymatic activation for the
Syn th esis of N7-Ad en in e Acyclic Nu cleosid es 12
a n d 14 (Sch em e 3). By the same synthetic strategy
shown in Scheme 2, novel N7-alkylated adenines 12 and
14 were obtained from adenine (10) via the N9-tritylated
adenine 11. Reaction of 11 with (2-chloroethoxy)methyl

3712 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Hakimelahi et al.
Sch em e 3a
Sch em e 4a
a
Reagents and conditions: (a) Ph3CCl, pyridine/DMF (3:1), 25
°C, 7.0 h. (b)Cl(CH2)2OCH2Cl, CH2Cl2, 25 °C, 8.0 h. (c)p-MeO(C6H4)-
O(CH2)2OCH2Cl, CH2Cl2, 25 °C, 10 h. (d) CAN, MeCN/H2O (3:1),
0-25 °C, 1.0 h.
chloride in CH2Cl2 at 25 °C gave N7-adenine derivative
12 in 90% yield. Likewise, reaction of 11 with [2-(p-
methoxyphenyloxy)ethoxy]methyl chloride in CH2Cl2 at
25 °C gave N7-isomer 13 in 86% yield. Treatment of 13
with CAN then produced the deprotected compound 14
in 80% yield.
a
Reagents and conditions: (a) BrCH2CH2CN, NaH, DMF, 65
°C, 17.0 h. (b) ICH2COOME, LiTMP, THF, -20 to -25 °C, 16.0 h.
(c) NaBH4, wet THF, 25 °C, 4.0 h. (d) Diethyl (p-toluenesulfony-
loxymethane)phosphonate, tert-butoxide, DMF, 25 °C, 4.0 h. (e)
Me3SiBr, CH2Cl2/DMF, 25 °C, 7.0 h.
The structures of the N7-substituted adenines were
Sch em e 5a
1
assigned by H and 13C NMR spectra. In methanol-d4,
pronounced downfield shifts were observed for the
signals resulting from the H2C(1′) (5.79 ppm) and HC-
(8) (8.60 ppm) of the N7-isomer 14 when compared with
those of the N9-isomer 1, respectively observed at 5.67
and 8.27 ppm. In DMSO-d6, the NH2 signals of the N7-
alkylated product 14 was shifted downfield to ∼9.2 and
10.0 ppm, whereas the corresponding signals of the N9-
isomer 1 appeared as a broad peak at ∼7.3 ppm. The
13C NMR signals of the C(1′) (82.01 ppm) and C(8)
(147.68 ppm) for the N7-isomer 14 were found to be
shifted downfield relative to those of the N9-isomer 1,
which were observed respectively at 74.31 and 143.14
ppm. The signals of the C(2) (144.29 ppm) and C(6)
(152.12 ppm) for isomer 14, however, were found to be
shifted upfield relative to those of the N9-isomer 1,
observed respectively at 153.74 and 157.19 ppm. Fur-
thermore, the H-C(2) coupling constants for the N7- and
N9-isomers were respectively 220 and 204 Hz, whereas
the corresponding H-C(8) coupling constants were
respectively 224 and 217 Hz. The attachment of the side
chain at the N-7 position of adenine was confirmed by
heteronuclear multiple quantum coherence (HMQC)
spectroscopy, in which the H2C(1′) and C(6) exhibited a
strong interaction in 14 whereas the corresponding N9-
isomer 1 showed long-range coupling between H2C(1′)
and C(4).
a
Reagents and conditions: (a) ButMe2SiCl, AgNO3, pyridine/
CH3CN, 25 °C, 7.0 h. (b) CCl3SO2Cl, collidine, THF, 25 °C, 10.0 h.
(c) n-Bu4NF, THF, 25 °C, 30 min. (d) NaHCO3, DMF, 25 °C, 1.0 h.
Syn t h esis of N7-Ad en in e Acyclic Nu cleosid e
P h osp h on a te 20 (Sch em e 4). Alkylation of adenine
(10) with 3-bromopropionitrile in the presence of NaH
in DMF gave N9-(cyanoethyl)adenine 15 in 75% yield.
Reaction of 15 with methyl iodoacetate and lithium
2,2,6,6-tetramethylpiperidine (LiTMP) in THF afforded
a mixture of N7-alkylated product 16 (60% yield) and
N9-isomer 17 (20% yield). Reduction of the ester group
in 16 with NaBH4 in wet THF13 gave N7-(hydroxyethyl)-
adenine 18 in 55% yield. Conversion of 18 to phospho-
nate 19 (60% yield) was accomplished by use of diethyl
(p-toluenesulfonyloxymethane)phosphonate and sodium
tert-butoxide in DMF.14 Treatment of compound 19 with
Me3SiBr15 then afforded phosphonic acid 20 in 45%
yield.
lid e 28, a n d P ME G-Con t a in in g Bu t en olid e 29
(Sch em es 5 a n d 6). The nucleotide analogue 24 was
readily obtained in three steps from adenosine 5′-
monophosphate (21), starting with silylation of phos-
phate 21 in CH3CN with t-BuMe2SiCl in the presence
of AgNO3 and pyridine.16 The resulting trisilylated
compound 22 was condensed with phosphonic acid 20
by use of trichloromethanesulfonyl chloride in collidine
and THF to afford dinucleotide 5′-monophosphate 23 in
42% overall yield.17 Desilylation of 23 with n-Bu4NF in
THF at 25 °C gave dinucleotide 24 in 90% yield, which
was then reacted with (Z)-4-(2-chloroethylidenyl)-2,3-
dimethoxy-∆R,â-butenolide (26)8b in the presence of
NaHCO3 in DMF to afford the target molecule 25 in 85%
yield. Similarly, treatment of compound 26 with either
Syn th esis of Din u cleotid e 5′-Mon op h osp h a te 24,
Its Bu ten olide Ester 25, P MEA-Con tain in g Bu ten o-

Nucleoside/ Nucleotide Analogues as Antiviral Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3713
Ta ble 1. Substrate Activity and Inhibitory Property against
Sch em e 6
ADAa
substrate
Km (10-5 M)
rel Vmax
Ki (10-5 M)
1
14
4.3
43
20
>80
16
1.5 × 10-2
14
3 (araA)
4 (PMEA)
1.0
>80
0.83
>80
10
>80
>80
14
20
24
25
28
1.5 × 10-6
9.8 × 10-2
64
>80
a
The reaction velocity, V, in micromoles per minute per
milligram of the enzyme was determined,19 and a plot of 1/[S] ([S]
) substrate concentration) vs 1/V was made. Thus, by the method
of Lineweaver and Burk, the Michaelis (Km) and maximum velocity
constants (Vmax) were determined.19 For inhibition studies, in
addition to araA (3), substrate solutions contained 1, 4, 14, 20,
24, 25, or 28 and then Ki was measured for each substrate.20
Ta ble 2. Phosphorylation of Various Nucleosides and Thymidine
with HSV or Vero Cell Thymidine Kinasesa
HSV thymidine
kinase
Vero cell
thymidine kinase
substrate
Km (µM)
rel Vmax
Km (µM)
rel Vmax
<3.0
PMEA (4) or PMEG (27) in the presence of NaHCO3 in
DMF respectively gave an 80% or 88% yield of the
desired compound 28 or 29.
1
2.0 × 104
<3.0
39.2
<3.0
28.0
<3.0
80.9
100.0
>3.0 × 104
2.2 × 104
1.3 × 104
18.3
2 (acyclovir)
3 (araA)
9
1.5
<3.0
<3.0
12.0
<3.0
<3.0
1.5 × 104
12.8
Lip op h ilicity a n d Solu bility Tests. Lipophilicity
and water solubility were determined by the distribution
between 1-octanol and water according to the methods
reported by Baker et al.18 Adenine acyclic nucleosides
1 and 14 were observed to exhibit higher lipophilicity
as well as water solubility than those exhibited by N7-
guanine acyclic nucleoside 9, acyclovir (2), and ara-A
(3) (see Supporting Information, Table 7). Phosphonate
20 and butenolide ester derivatives 25, 28, and 29 also
exhibited higher lipophilicity and water solubility as
compared to PMEA (4) or PMEG (27). On the other
hand, even though the solubility of nucleotide analogue
24 in water was found to be higher than that of PMEA
(4), its lipophilicity was lower than that of 4. Further-
more, butenolide ester derivative 25 showed higher
lipophilicity as well as water solubility with respect to
the parent nucleotide 5′-monophosphate 24.
14
30.0
>3.0 × 104
2 + 14 (1:1 w/w)
thymidine
1.0
1.0
1.0 × 102
a
Apparent Km values were determined for HSV and Vero cell
thymidine kinases from reactions containing 50 mM Tris-HCl (pH
7.5), 2.0 mM ATP, 2.0 mM MgCl2, 1.0 mg/mL BSA, 1.5 µM
[
14C]thymidine, and 198 units of enzymes/mL.21 The results were
compared with those of thymidine. All reactions were performed
at 37 °C. The radiochemical nucleoside kinase coupled assay was
used in the determination of the relative substrate velocities.21
of the enzyme. Nucleotide analogue 24, however, was a
substrate for ADA, but its Vmax was about 95% less than
that of ara-A (3). The slow rate of deamination of
compound 24 by ADA may reflect the lack of substrate
activity of the acyclic nucleoside phosphonate moiety
therein in the active site of the enzyme.
Com p a r ison of P h osp h or yla tion of Nu cleosid es
by HSV a n d Ver o Cell Th ym id in e Kin a ses. The
rates of phosphorylation of N9-adenine acyclic nucleoside
1, acyclovir (2), ara-A (3), N7-guanine acyclic nucleoside
9, and N7-adenine acyclic nucleoside 14, as well as a
mixture of 2 and 14 (w/w ) 1:1), a mixture of 1 and 2
(w/w ) 1:1), and a mixture of 2 and 3 (w/w ) 1:1), in
the presence of HSV or Vero cell thymidine kinase were
determined21 and the results were compared with those
of thymidine. It was observed that N7-guanine nucleo-
side 9, unlike acyclovir, can be phosphorylated by both
the HSV thymidine kinase and the host cell kinase. On
the other hand, these enzymes were found not to
phosphorylate adenine nucleosides 1, 14, and ara-A (3);
yet the N7-adenine nucleoside 14 induced a 2-fold
increase on the rate of phosphorylation of acyclovir (2)
(see Table 2). N9-adenine nucleosides 1 and 3, however,
did not induce any increase on the rate of phosphory-
lation of 2. It is believed that the N7-adenine nucleoside
14 binds (Km ) 30 µM) to a specific receptor at the active
site of the enzyme to exhibit the observed activatory
property toward the HSV thymidine kinase.
Biologica l Resu lts a n d Discu ssion
Kin etic Stu d ies of Com p etitive In h ibition of
Ad en osin e Dea m in a se by Acyclic Nu cleosid es a n d
Nu cleotid es. The rates of deamination of N9-alkylated
adenine 1, 9-(â-D-arabinofuranosyl)adenine (ara-A, 3),
PMEA (4), N7-alkylated adenine 14, phosphonate 20,
dinucleotide 5′-monophosphate 24, its butenolide ester
derivative 25, and PMEA-containing butenolide 28 in
the presence of calf mucosal adenosine deaminase (ADA,
EC 3.5.4.4) in buffer solutions were determined.19 Ad-
ditionally, the inhibition studies on these compounds
were carried out on the basis of the Kaplan method
(Table 1).20 The results showed that both the N9- and
N7-acyclic nucleosides 1 and 14 functioned as ADA
substrates. The Vmax of 14 was less than that of 1 by a
factor of 4. Compounds 1, 14, and 24 showed competitive
inhibition of ADA when ara-A was used as a substrate.
However, N7-isomer 14 was found to be more efficient
than the N9-isomer 1 and nucleotide analogue 24 as an
inhibitor of ADA. PMEA (4), acyclic nucleoside phos-
phonate 20, and nucleotide-containing butenolides 25
and 28 were neither a good substrate nor an inhibitor
En zym a tic Con ver sion of Acyclic Nu cleosid e
P h osp h on a tes a n d Nu cleotid e 5′-Mon op h osp h a te

3714 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Hakimelahi et al.
Ta ble 3. Kinetics of the PRPP Synthetase Reaction with Acyclic
Nucleoside Phosphonates 4 and 20, Nucleotide Analogue 24, and
AMP (21)a
as a normal substrate. In addition, we found that
nucleotide-containing butenolide 25 was completely
resistant to snake venom and spleen enzymes.
substrate
Km (mM)
Vmax (µmol unit-1 h-1
)
Kib (mM)
An ti-DNA Vir u s Activity in Vitr o. The newly
synthesized compounds were tested for inhibition of
cytopathogenicity of the herpes simplex type 1 virus
(HSV-1), herpes simplex type 2 virus (HSV-2), thymi-
dine kinase-positive (TK+) and thymidine kinase-
deficient (TK-) strains of varicella-zoster virus (VZV),
and human cytomegalovirus in Vero cell culture up to
a level as high as 128 µg/mL.22,23 Compounds tested
include N7-alkylated purines 7, 9, 12, 14, and 20, as well
as mixtures of 1 and 14 (w/w ) 1:1), 2 and 14 (w/w )
1:1), 3 and 14 (w/w ) 1:1), in addition to N9-substituted
adenine 1,24 acyclovir (2),25,26 ara-A (3),27 PMEA (4),4i
PMEG (27),4i nucleotide analogue 24, and nucleotide-
containing butenolides 25, 28, and 29. Toxicity of these
compounds was evaluated by their ability to cause
morphological changes in HeLa and Vero cells at dif-
ferent concentrations. The minimum inhibitory concen-
trations (IC50) were measured by use of the linear
regression method (see Supporting Information, Table
8).28,29
4 (PMEA)
20
21 (AMP)
24
1.5
4.8
0.24
0.57
0.096
0.013
14
3.0
17
12
0.79
a
The PRPP synthetase reaction mixture contained 10.0 mM
potassium phosphate buffer (pH 8.0), 5.0 mM MgCl2, 2.5 mM
PRPP, an appropriate amount of AMP (21) or test compounds, and
0.04 unit of PRPP synthetase. The formation of ATP, diphosphates
of PMEA (4), 20, or 24 was analyzed by HPLC according to the
method of Balzarini and De Clercq.4i Inhibition of AMP phos-
b
phorylation by the enzyme was measured in the presence of
different substrate (AMP) concentrations and appropriate concen-
trations of 4, 20, and 24. The reaction mixture was incubated at
37 °C for 15 min with 0.002 unit of PRPP synthetase. The
formation of ATP was followed by HPLC on a Partisphere anion-
exchange column as described.4i
An a logu e t o Th eir An t ivir a lly Act ive Dip h os-
p h a tes (Tr ip h osp h a te Equ iva len ts). N9-Acyclic nu-
cleoside phosphonate (PMEA, 4), N7-acyclic nucleoside
phosphonate 20, adenosine 5′-monophosphate (21), and
nucleotide 5′-monophosphate 24 were individually in-
cubated with PRPP and PRPP synthetase, during which
time the reaction proceeded linearly. The assays were
then terminated after 4 h by the addition of MeOH.
HPLC on an anion-exchange Partisphere column was
used to analyze the formation of ATP (21pp), PMEApp
(4pp), 20pp, and 24pp. Inhibitory effects of PMEA (4),
20, and 24 toward phosphorylation of AMP (21) was also
evaluated according to an established procedure.4i Re-
sults are summarized in Table 3.
The substrate affinity of N7-acyclic nucleoside phos-
phonate 20 (Km ) 4.8 mM) for the enzyme was found to
be 4 times less than that of PMEA (4) (Km ) 1.5 mM).
Similarly, the Vmax for conversion of 20 to 20pp is at
least 7 times lower than that for the conversion of 4 to
4pp. On the other hand, nucleotide analogue 24 was
found to be a better substrate than PMEA (4) for PRPP
synthetase (Km ) 0.57 mM). It can also be phosphory-
lated (Vmax ) 12) by the enzyme at a rate similar to that
of AMP (21) (Vmax ) 14).
The pronounced anti-DNA virus activity of 14 with
respect to the corresponding chloro derivative 12 showed
that the presence of a hydroxyl group is essential for
antiviral activity. Results from the biological tests also
indicated that adenine nucleoside 14 was not effectively
deaminated by ADA; yet it inhibited the deactivating
property of the enzyme and led to the observed increase
in the antiviral activity of 1 and ara-A (3). Furthermore,
use of adenine acyclic nucleoside 14 resulted in a 2-fold
increment in the rate of phosphorylation of acyclovir (2)
by HSV thymidine kinase. As such, a combination of
14 and 2 exhibited profound antiviral activity. N7-
Adenine nucleoside 14 was found to be less toxic than
the corresponding N9-isomer 1. On the other hand, both
HSV and cellular thymidine kinases can phosphorylate
N7-guanine nucleoside 9. As a result, this nucleoside
exhibited more toxicity than acyclovir (2).
The rate of phosphorylation of N7-acyclic nucleoside
phosphonate 20 to its antivirally active anabolite 20pp
by PRPP synthetase is 7 times less than that of the
PMEA (4). Thus in comparison to compound 4, com-
pound 20 exhibited less activity against DNA viruses.
On the other hand, nucleotide 5′-monophosphate ana-
logue 24, possessing a natural AMP moiety, was con-
verted to its diphosphate (triphosphate form) 24pp at a
rate comparable to that of AMP (21), which is about 120
times faster than the rate of conversion of PMEA (4) to
PMEApp (4pp). Consequently, nucleotide 5′-monophos-
phate 24 exhibited higher anti-DNA virus activity than
PMEA (4).
It has been hypothesized4i that the primary amino
group at the C-6 position of purines is essential for
hydrogen bonding with the enzyme. The NH2 group in
20 is sterically hindered by an adjacent side chain at
the N-7 position. As such, it cannot as effectively
interact with the active site of the enzyme when
compared to PMEA (4), natural substrate AMP (21), or
nucleotide analogue 24.
Activity of Sn a k e Ven om a n d Sp leen P h os-
ph odiester ases again st Nu cleotide An alogu es. Com-
pound 24, possessing the skeletons of both phosphonate
20 and 5′-adenosine monophosphate (21), was dephos-
phorylated at the 5′-position by snake venom in 80%
yield after 8 h.17 The resultant 9-(â-D-furanosyl)adenine
3′-[1-(adenin-7-ylethoxy)methyl]phosphonate was hy-
drolyzed further in the presence of spleen phosphodi-
esterase to afford adenosine and phosphonate 20 in
about 40-60% yield after 8 h.17 Spleen phosphodi-
esterase also degraded compound 24 to give 5′-adenosine
monophosphate (21) and phosphonate 20 in an overall
yield of 60% after 8 h. These results indicate that the
phosphodiesterases recognized dinucleotide analogue 24
The ability of a drug to penetrate a membrane and
exhibit biological activity can be correlated to its
lipophilicity.8a,17 Consequently, we prepared compounds
25, 28, and 29 possessing a butenolide ester functional-
ity as lipophilic prodrugs. These compounds displayed
superior antiviral activity relative to their respective
parent compounds nucleotide 5′-monophosphate 24,
PMEA (4), and PMEG (27). In addition, spleen phos-
phodiesterase can recognize and at least partly hydro-
lyze nucleotide analogue 24 to the biologically less active
phosphonate 20 inside the infected cells; whereas its

Nucleoside/ Nucleotide Analogues as Antiviral Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3715
Ta ble 4. Antiviral Effects of Compounds 2, 4, 9, 14, 20, 24, 25,
and 28 against HSV-1-Induced Mortality in NMRI Mice upon
Intraperitoneal Administrationa
Ta ble 5. Inhibitory Effects of Nucleotide Analogues on the
Cytopathogenicity of HIV-1 and HIV-2 in MT4 Cells, as Well as
on the Cytopathogenicity of MSV in CEM Cells and Cellular
Toxicity
mean day of
a
dose
no. of
symptom
mean day of
IC50 (µg/mL)
compound (mg kg-1 day-1) mice initiationb [%] animal deathc [%]
HIV-1
(IIIB)
HIV-2
(LAV-2)
MT4
cellb
CEM
cellb
2 (acyclovir)
250
150
100
250
150
100
250
150
100
250
150
100
250
150
100
250
150
100
250
150
100
250
150
100
0
20 >21 [100]
>21 [100]
compound
MSV
2.0
27
17
13
0.19
0.93
0.020
20 19.1 ( 1.3 [86] >21 [100]
15 15.6 ( 1.6 [65] 18.9 ( 2.1 [80]
4 (PMEA)
20
24
25
27 (PMEG)
28
29
4.1
7.8
5.9
4.9
16
3.8
9.1
6.4
4.2
18
274
>300
298
285
>300
>300
>300
12
4 (PMEA)
20 >21 [100]
>21 [100]
20 18.5 ( 1.9 [80] >21 [100]
15 14.7 ( 1.4 [56] 17.0 ( 1.1 [77]
20 15.1 ( 1.2 [67] 18.5 ( 1.0 [90]
20 13.0 ( 1.1 [57] 16.0 ( 1.5 [78]
15 10.9 ( 0.6 [48] 14.1 ( 0.8 [66]
>300
9
16
1.4
6.0
1.0
7.1
265
14
280
13
14
20
24
25
28
20 >21 [100]
20 19.9 ( 1.3 [88] >21 [100]
>21 [100]
a
Inhibitory concentrations (IC50) were determined by use of an
established procedure28,30 and represent the average of duplicate
15 17.5 ( 2.1 [80] 19.8 ( 1.7 [95]
20 16.8 ( 2.4 [75] 20.0 ( 1.7 [95]
20 15.0 ( 0.9 [62] 17.8 ( 1.5 [80]
15 12.8 ( 1.1 [51] 15.4 ( 1.3 [70]
b
determinations. Concentration of the compound required to
reduce the number of viable uninfected cells by 50%.
20 >21 [100]
>21 [100]
pounds 2, 4, 14, 24, 25, and 28 clearly demonstrated
that they are taken up effectively by cells to exert in
vivo activity. None of the compounds were toxic to the
mice at the highest dose tested.
20 19.3 ( 1.6 [85] >21 [100]
15 16.0 ( 1.7 [70] 19.5 ( 1.2 [90]
20 >21 [100]
20 >21 [100]
>21 [100]
>21 [100]
15 19.6 ( 1.5 [94] >21 [100]
The LD50 values of the most active compounds 14, 25,
and 28 in mice were also determined. As such, N7-acyclic
nucleoside 14 and butenolide ester derivatives 25 and
28 were administered at different doses intraperito-
neally. They did not show any toxicity up to a concen-
tration level as high as 400 mg/kg. All mice were
controlled in good conditions after 6 months of admin-
istration. Nevertheless, LD50 (ip) values of 950, 675, and
710 mg/kg were determined for 14, 25, and 28, respec-
tively. Moreover, no discernible abnormality was ob-
served in the histological appearance of the viscera of
either the control or tested groups of mice that received
the drugs ip (250 mg kg-1 day-1) for 10 days. Further-
more, there were no physiological changes in their
cardiovascular or central nerve systems.
20 >21 [100]
20 >21 [100]
15 >21 [100]
>21 [100]
>21 [100]
>21 [100]
saline
20 3.38 ( 0.7 [0] 9.4 ( 0.6 [0]
a
Mice were inoculated intraperitoneally with HSV-1 (KOS).
Treatment was initiated 4 h postinfection and continued for 6
b
consecutive days. Experiments were terminated at day 21. Val-
ues in brackets represent percentage of HSV-1-infected mice
without symptoms at day 21 postinfection. c Values in brackets
represent percentage of HSV-1-infected mice that were alive at
day 21 postinfection.
butenolide ester derivative 25 was found to be stable
toward phosphodiesterases. Thus, in comparison with
nucleotide analogue 24, its prodrug 25 possesses supe-
rior bioavailability and greater stability both in vitro
and in vivo.
An ti-Retr ovir u s Activity in Vitr o. Compounds 4,
20, 24, 25, 27, 28, and 29 were tested for inhibition of
cytopathogenicity against the human immunodeficiency
viruses HIV-1 (III-B) and HIV-2 (LAV-2) in MT4 cells.
These compounds were also screened for their antiviral
activity against Moloney murine sarcoma virus (MSV)
in CEM cells in a cell-protection assay.30 Toxicity of
these compounds was evaluated by their ability to cause
morphological changes in MT4 or CEM cells at different
concentrations. The minimum inhibitory concentrations
(IC50) were measured by the use of the linear regression
method (Table 5).28
In comparison to the rate of phosphorylation of PMEA
(4) to PMEApp (4pp) by PRPP synthetase, the conver-
sion of dinucleotide 24 to its anabolically active form
24pp is 120 times faster; yet PMEA (4) exhibited higher
activity than 24 as well as the butenolide ester deriva-
tive 25 against retroviruses. Thus, the HIV and MSV
reverse transcriptases may have higher affinity for
PMEA (4) than dinucleotide analogue 24. PMEA (4) was
also found to be more active than its N7-isomer 20
against retroviruses. On the other hand, butenolide
ester derivatives 28 and 29 displayed superior antiviral
activity relative to their respective parent molecules 4
and 27. Thus in comparison to PMEA (4) and PMEG
(27), their respective lipophilic prodrugs 28 and 29
possess superior bioavailability and greater anti-retro-
virus activity. As shown in Scheme 6, we believe that
An ti-HSV-1 Activity in Vivo a n d Deter m in a tion
of LD50 for N7-Ad en in e Acyclic Nu cleosid e 14,
Nu cleotid e-Con ta in in g Bu ten olid e 25, a n d P MEA-
Con ta in in g Bu ten olid e 28 in Mice. Acyclovir (2),
PMEA (4), N7-guanine acyclic nucleoside 9, N7-adenine
acyclic nucleoside 14, N7-acyclic nucleoside phosphonate
20, nucleotide 5′-monophosphate 24, and butenolide
ester derivatives 25 and 28 were evaluated for their
inhibitory effects on HSV-1-induced mortality in NMRI
mice (Table 4).4a The butenolide derivative of PMEA,
28, appeared to be the most potent anti-HSV-1 agent
in vivo, followed by nucleotide-containing butenolide 25,
nucleoside analogue 14, nucleotide analogue 24, acy-
clovir (2), PMEA (4), phosphonate 20, and nucleoside
analogue 9. Since compound 28 is less active in vitro
against HSV-1 when compared to compounds 25 and
14, the in vitro potency does not directly translate to in
vivo potency. These results confirms previous find-
ings.4c,4d,4h
All compounds were administered intraperitoneally
(ip, 100-250 mg kg-1 day-1) for 6 consecutive days.
Compounds 2, 4, 14, 24, and 25 gave full protection
against HSV-induced mortality at the 150 mg/kg dose
level. The same level of protection was provided by
compound 28 at a dose of 100 mg/kg. Survival times of
all treated groups were found to be significantly differ-
ent from the placebo-treated control group (see Table
4). The potent anti-HSV-1 activity exhibited by com-

3716 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Hakimelahi et al.
Ta ble 6. Inhibitory Effects of Acyclic Nucleoside Phosphonate
4 and Its Prodrug 28 on MSV-Induced Tumor Formation and
Associated Death in NMRI Mice upon Intraperitoneal
Administrationa
phosphate 24 were converted directly to their antivirally
active anabolites in the presence of PRPP synthetase,
whereby a pyrophosphate group is transferred from
PRPP to their phosphonate (i.e., 4 and 20) or phosphate
(i.e., 24) moieties. Indeed, the substrate affinity for
PRPP synthetase seemed to be dependent on the avail-
ability of the primary amine group at the C-6 position
of purines. With respect to this structural requirement,
the ability of compounds 4, 20, and 24 to inhibit
cytopathogenicity of the DNA viruses was found to
correlate well with their rate of phosphorylation by
PRPP synthetase. Finally, prodrugs 25, 28, and 29,
possessing a butenolide moiety, were designed. These
butenolide ester derivatives showed superior bioavail-
ability and profound antiviral activity relative to their
respective precursors. In comparison to nucleotide 24,
prodrug 25 also exhibited higher stability toward phos-
phodiesterases. Moreover, compounds 2, 4, 9, 14, 20, 24,
25, and 28 were evaluated for their inhibitory effect on
HSV-1-induced mortality in NMRI mice. Nucleotide-
containing butenolide 25 and PMEA-containing buteno-
lide 28 were found to be remarkable anti-HSV-1 agents
in vivo. Survival times of all treated groups were
determined to be significantly longer than that of the
control group. Compounds 4, 20, 24, 25, 28, and 29 were
also tested against HIV-1 (III-B), HIV-2 (LAV-2), and
MSV. Prodrugs 28 and 29 were found to possess notable
anti-retrovirus activity in vitro as well as in vivo.
dose
mean day of
tumor
mean day of
animal
(mg kg-1 no. of
compound
PMEA (4)
day-1
)
mice initiationb [%]
deathc [%]
50
20
10
50
20
10
0
15 12.5 ( 1.6 [84] 17.8 ( 2.0 [96]
15 12.0 ( 1.3 [58] 15.9 ( 1.7 [76]
10
9.6 ( 1.5 [19] 13.0 ( 1.1 [42]
prodrug (28)
15 18.4 ( 1.4 [95] >21 [100]
15 17.9 ( 1.7 [80] >21 [100]
10 14.0 ( 2.1 [60] 18.9 ( 1.8 [97]
30 3.82 ( 0.95 [0] 7.8 ( 1.3 [0]
control
untreated controld
0
40
>21 [100]
>21 [100]
a
b
All mice received two injections within 2 days. Values in
brackets represent percentage of MSV-infected mice without
tumors at day 21 postinfection c Values in brackets represent
percentage of MSV-infected mice that were alive at day 21
d
postinfection. Untreated control group was neither treated with
MSV nor with the drugs.
the oxygen of the methoxy group at the C-2 position of
the butenolide moiety is responsible for the ease of
conversion of these novel prodrugs 28 and 29 to their
corresponding potential drugs PMEA (4) and PMEG (27)
inside the infected cells.
In h ibitor y Effects of P MEA (4) a n d Its Bu ten o-
lid e Ester Der iva tive 28 on MSV-In d u ced Tu m or
F or m a tion in Vivo. Compounds 4 and 28 were evalu-
ated for their inhibitory effect on MSV-induced tumor
formation in NMRI mice (Table 6).31 The compounds
were administered intraperitoneally (50 mg kg-1 day-1
)
Exp er im en ta l Section
for 2 consecutive days. Prodrug 28 exhibited much
higher anti-MSV activity than PMEA (4) in vivo. At a
dose of 10 mg kg-1 day-1, compound 28 prevented tumor
formation in 60% of the MSV-infected mice, whereas
with compound 4 at the same dosage level, only a 19%
prevention was observed. In surviving animals treated
with 28, about 2 g of weight loss was observed. In the
case of PMEA-treated mice, the weight loss of the
surviving animals was at least 2 times more.
Gen er a l. For anhydrous reactions, glassware was dried
overnight in an oven at 120 °C and cooled in a desiccator over
anhydrous CaSO4 or silica gel. Reagents were purchased from
Fluka and enzymes from Sigma Chemical Co. Solvents,
including dry ether and tetrahydrofuran (THF), were obtained
by distillation from the sodium ketyl of benzophenone under
nitrogen. Other solvents, including chloroform, dichloromethane,
ethyl acetate, and hexanes, were distilled over CaH2 under
nitrogen. Absolute methanol and ethanol were purchased from
Merck and used as received.
Melting points were obtained with a Bu¨chi 510 melting point
apparatus. Infrared (IR) spectra were recorded on a Beckman
IR-8 spectrophotometer. The wavenumbers reported are ref-
erenced to the 1601 cm-1 absorption of polystyrene. Proton
NMR spectra were obtained on a Varian XL-300 (300 MHz)
spectrometer. Chloroform-d and dimethyl sulfoxide-d6 were
used as solvent; Me4Si (δ 0.00 ppm) was used as an internal
standard. All NMR chemical shifts are reported as δ values
in parts per million (ppm), and coupling constants (J ) are given
in hertz (Hz). The splitting pattern abbreviations are as
follows: s, singlet; d, doublet; t, triplet; q, quartet; br, broad;
m, unresolved multiplet due to the field strength of the
instrument; and dd, doublet of doublets. UV spectroscopy was
carried out on an HP8452A diode-array spectrophotometer.
Mass spectra were carried out on a VG 70-250 S mass
spectrometer. Microanalyses were performed on a Perkin-
Elmer 240-B microanalyzer.
Purification on silica gel refers to gravity column chroma-
tography on Merck silica gel 60 (particle size 230-400 mesh).
Analytical thin-layer chromatography (TLC) was performed
on precoated plates purchased from Merck (silica gel 60 F254).
Compounds were visualized by use of UV light, I2 vapor, or
2.5% phosphomolybdic acid in ethanol with heating.
Deter m in a tion of Solu bility. Each compound (70 mg)
listed in Table 7 was agitated in a 25-mL volumetric flask with
phosphate buffer (0.10 M, pH 6.8, 5.0 mL) for 20 h. This
solution was filtered from undissolved solid through a sintered
glass funnel (4.0-5.5 mesh ASTM) and the concentration of
the solution was determined by UV absorbance (see Table 7,
Supporting Information).
Su m m a r y a n d Con clu sion s
A series of new compounds were synthesized and their
biological activities were determined. These compounds
include N7-guanine acyclic nucleosides 7 and 9, N7-
adenine acyclic nucleosides 12 and 14, N7-acyclic nu-
cleoside phosphonate 20, dinucleotide 5′-monophosphate
analogue 24, nucleotide-containing butenolide 25, PMEA-
containing butenolide 28, and PMEG-containing buteno-
lide 29. Unlike acyclovir (2), which was phosphorylated
by HSV-specified thymidine kinase, its N7-isomer 9 was
recognized by both HSV and cellular thymidine kinases.
As such, compound 9 was found to be less active and
more toxic than compound 2 in vitro. Compound 14 was
not phosphorylated in the presence of thymidine ki-
nases. Nevertheless, with respect to the lack of activity
of chloro derivatives 7 and 12, it is reasonable to
conclude that the hydroxy group in 14 is essential for
its antiviral activity. Consequently, either phosphory-
lation of this compound is not essential for biological
activity or the phosphorylation is performed by other
enzymes. Moreover a combination of N7-adenine acyclic
nucleoside 14 and N9-adenine acyclic nucleoside 1,
acyclovir (2), and ara-A (3) in a ratio of 1:1 (w/w) was
found to possess profound activity against DNA viruses.
PMEA (4), its N7-isomer 20, and nucleotide 5′-mono-

Nucleoside/ Nucleotide Analogues as Antiviral Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3717
Deter m in a tion of P a r tition Coefficien ts (Lip op h ilic-
ity). A solution of each compound (10 mL) in Table 7 (see
Supporting Information) in phosphate buffer (0.10 M) pos-
sessing an UV absorbance of 2.2-3.3 at 258-267 nm, for
adenine, or at 270-290 nm, for guanine, was shaken with
1-octanol (10 mL) in a separatory funnel for 1.5 h. The layers
were separated and their concentrations were determined by
an UV spectrophotometer. The partition coefficient was cal-
postinfection were observed (see Table 4). Deaths were re-
corded for 21 days after infection.
(B) An ti-MSV Activity in Vivo. The inhibitory effects of
the compounds 4 and 27 on the initiation of MSV-induced
tumor formation and survival of MSV-induced mice (10-15
animals/group) were evaluated as previously described.31
Results are summarized in Table 6.
9-[(2-Hyd r oxyeth oxy)m eth yl]a d en in e (1). Compound 1
was prepared by a standard procedure:23 mp 198-199 °C; Rf
(hexanes/EtOAc ) 1:2) 0.23; UV (EtOH) λmax 259 (ꢀ 14 000);
1H NMR (CD3OD) δ 3.62 (s, 4 H, O(CH2)2O), 5.67 (s, 2 H,
H2C1_), 8.22 (s, 1 H, HC2), 8.27 (s, 1 H, HC8); 13C NMR (CD3-
OD) δ 61.86 (CH2OH), 72.08 (OCH2) 74.31 (C1′), 119.98 (C5),
143.14 (C8), 150.90 (C4), 153.74 (C2), 157.19 (C6); MS m/z 209
(M+).
culated as P ) [S]1-octanol/[S]H
.
O
2
En zym e Assa ys: (A) Ad en osin e Dea m in a se. The re-
ported procedures19,20 were used for ADA, and the results are
summarized in Table 1.
(B) HSV a n d Ver o Cell Th ym id in e Kin a ses. Phospho-
rylation of nucleosides with HSV or Vero cell thymidine kinase
was studied as described previously.21 Results are summarized
in Table 2.
9-(Tr ip h en ylm eth yl)gu a n in e (6). Guanine 5 (1.51 g, 9.99
mmol) and (NH4)2SO4 (100 mg) were suspended in hexameth-
yldisilazane (HMDS) (150 mL) and refluxed for 24 h. The
solvent was evaporated under reduced pressure and the
residue was dissolved in CH3CN (150 mL). Triphenylmethyl
chloride (2.79 g, 10.0 mmol) was added and the reaction
mixture was stirred at 25 °C for 7.0 h. The solution was
concentrated under reduced pressure and the residue was
purified by use of column chromatography (hexanes/EtOAc )
1.5:8.5) to afford 6 (3.54 g, 8.99 mmol) in 90% yield: mp 268-
270 °C; Rf (hexanes/EtOAc ) 1:2) 0.34; UV (EtOH) λmax 254 (ꢀ
13 870), 278 (sh); 1H NMR (DMSO-d6) δ 5.97 (s, 2 H, NH2),
7.09-7.39 (m, 16 H, HC8 + C(C6H5)3), 10.45 (br s, 1H, NH);
MS m/z 393 (M+). Αnal. Calcd (C24H19N5O): C, H, N.
7-[(2-Ch lor oeth oxy)m eth y]gu a n in e (7). To a solution of
6 (1.77 g, 4.49 mmol) in DMF (30 mL) was added (2-
chloroethoxy)methyl chloride (0.65 g, 5.0 mmol). The reaction
mixture was stirred at 25 °C for 8.0 h. The solution was then
partitioned between EtOAc (100 mL) and water (100 mL). The
EtOAc solution was washed with water (4 × 100 mL); then it
was dried over MgSO4 (s) and filtered. Evaporation under
reduced pressure and purification of the residue by use of
column chromatography (EtOAc) afforded 7 (1.07 g, 4.39 mmol)
in 88% yield: mp >280 °C (decomp); Rf (hexanes/EtOAc ) 1:2)
0.20; UV (EtOH) λmax 288 (ꢀ 15 100); 1H NMR (DMSO-d6) δ
3.67 (t, J ) 6.10 Hz, 2 H, CH2Cl), 3.75 (t, J ) 6.10 Hz, 2 H,
OCH2), 5.62 (s, 2 H, H2C1_), 6.15 (s, 2 H, NH2), 7.58 (s, 1 H,
NH), 8.16 (s, 1 H, HC8); 13C NMR (DMSO-d6) δ 43.39 (CH2Cl),
68.50 (OCH2), 74.71 (C1′), 107.71 (C5), 143.87 (C8), 153.15 (C4),
154.37 (C2), 159.12 (C6); MS m/z 243 (M+, Cl cluster). Αnal.
Calcd (C8H10ClN5O2): C, H, N, Cl.
(C) 5-P h osp h or ibosyl 1-P yr op h osp h a te Syn th eta se.
Substrate affinities of acyclic nucleoside phosphonates 4, 20,
21, and dinucleotide analogue 24 for PRPP synthetase as well
as their inhibitory effect against the enzyme were evaluated
according to the established procedures.4i Results are il-
lustrated in Table 3.
(D) Sn a k e Ven om P h osp h od iester a se. Snake venom
phosphodiesterase (200 units) was dissolved in tris(hydroxym-
ethyl)aminomethane buffer (1.0 mL), which was adjusted to
pH 9.2 with 0.1 N HCl. The enzyme solution (0.10 mL) was
added to the nucleotide 24 or 25 (0.70 mg) and the mixture
was incubated at 37 °C for 8 h. The solution was then applied
to Whatman 3-MM paper as a band, which was developed with
a mixture of i-PrOH, concentrated NH4OH, and H2O (9:1:2).
Degradation products or unreacted starting materials were
separated. Dinucleotide 5′- monophosphate 24 gave 9-(â-D-
furanosyl)adenine 3′-[1-(adenin-7-ylethoxy)methyl]phospho-
nate in about 80% yield. On the other hand, dinucleotide-
containing butenolide 25 was completely resistant to the
enzyme.
(E) Sp leen P h osp h od iester a se. Spleen phosphodiesterase
(20 units) was dissolved in sodium pyrophosphate buffer (0.01
M, 1.0 mL), which was adjusted to pH 6.5 with phosphoric
acid. Nucleotide 24, 25, or 9-(â-D-furanosyl)adenine 3′-[1-
(adenin-7-yl-ethoxy)methyl]phosphonate (0.70 mg) was dis-
solved in ammonium acetate buffer (0.05 M, 0.20 mL), which
was adjusted to pH 6.5 with acetic acid. An aliquot of the
enzyme solution (0.1 mL) was added to the nucleotide solution
and the mixture was incubated at 37 °C for 8 h. The solution
was then applied to Whatman 3-MM paper as a band and
developed with a mixture of i-PrOH, concentrated NH4OH, and
H2O (9:1:2). Bands containing products were cut out, which
were eluted with H2O, and the resultant mixture was freeze-
dried. The isolated products were characterized by comparison
with authentic samples. Dinucleotide 24 afforded phosphonate
20 and adenosine 5′-monophosphate (21) in about 60% yield.
9-(â-D-Furanosyl)adenine 3′-[1-(adenin-7-ylethoxy)methyl]phos-
phonate gave adenosine (40% yield) and 20 (60% yield).
Dinucleotide analogue 25, having a butenolide ester unit, was
found to be stable to the enzyme.
An ti-DNA Vir u s Activity a n d An ticellu la r Eva lu a -
tion s. The methods for measuring viruses-induced cytopatho-
genicity in Vero cell culture, as well as the toxicity of the tested
compounds toward HeLa and Vero cells, have been described
previously.22,23,28 Results are summarized in Table 8 (see
Supporting Information).
An ti-Retr ovir u s Activity a n d An ticellu la r Eva lu a -
tion s. The methods for measuring viruses-induced cytopatho-
genicity in MT4 cells or CEM cells, as well as the toxicity of
the tested compounds toward MT4 and CEM cells, have been
described previously.30 Results are summarized in Table 5.
An im a l Stu d ies: (A) An ti-HSV-1 Activity. Two-week-old
NMRI mice (15-20 animals/group), weighing ca. 7 g each, were
infected ip with 4 × 104 units of HSV-1 (KOS).4a Compounds
in Table 4 were administered ip once a day for 6 consecutive
days, starting 4 h postinfection. Percentage of HSV-1-infected
mice without symptoms and those that were alive at day 21
7-[[2-(p-Meth oxyph en yloxy)eth oxy]m eth yl]gu an in e (8).
Compound 8 (2.72 g, 8.20 mmol) was prepared in 82% yield
from 6 (3.64 g, 9.25 mmol) and [2-(p-methoxyphenyloxy)ethoxy]-
methyl chloride (2.18 g, 10.1 mmol) in DMF (100 mL) by the
method used for the synthesis of 7: mp >250 °C (decomp); Rf
(hexanes/EtOAc ) 1:2) 0.18; UV (EtOH) λmax 290 (ꢀ 16,500);
1H NMR (DMSO-d6) δ 3.70 (s, 3 H, OCH3), 4.02-4.10 (m, 4 H,
O(CH2)2O), 5.74 (s, 2 H, H2C1_), 5.88 (s, 2 H, NH2), 6.67, 6.70
(AA′BB′, J ) 9.30 Hz, 4 H, C6H4), 7.50 (s, 1 H, NH), 8.85 (s,1
H, HC8); 13C NMR (DMSO-d6) δ 56.12 (CH3), 68.74 (OCH2),
70.20 (CH2OPh), 75.31 (C1′), 107.25 (C5), 144.02 (C8), 153.25
(C4), 154.48 (C2), 159.26 (C6), 115.55, 116.38, 153.51, 155.56
(C6H4); MS m/z 331 (M+). Αnal. Calcd (C15H17 N5O4): C, H, N.
7-[(2-Hyd r oxyeth oxy)m eth yl]gu a n in e (9). To a solution
of 8 (1.36 g, 4.10 mmol) in a mixture of CH3CN (30 mL) and
water (10 mL) was added CAN (2.25 g, 4.10 mmol) at 0 °C.
The stirred reaction mixture was allowed to warm up to 25
°C within 1.0 h. Water (30 mL) was added to afford a solid.
Filtration and crystallization of the solid from EtOH/water (4:
1) gave 9 (0.65 g, 2.88 mmol) in 70% yield: mp >280 °C
(decomp); Rf (hexanes/EtOAc ) 1:2) 0.08; UV (EtOH) λmax 289
(ꢀ 16 000); 1H NMR (DMSO-d6) δ 3.61-3.82 (m, 4 H, O(CH2)2O),
4.85 (br s, 1H, OH), 5.81 (s, 2 H, H2C1′), 5.96 (br s, 2 H, NH2),
6.61 (s, 1 H, NH), 8.67 (s, 1 H, HC8); 13C NMR (DMSO-d6) δ
60.91 (CH2OH), 70.01 (OCH2) 75.25 (C1′), 107.16 (C5), 143.92
(C8), 153.20 (C4), 154.40 (C2), 159.30 (C6); MS m/z 225 (M+).
Αnal. Calcd (C8H11N5O3): C, H, N.

3718 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Hakimelahi et al.
9-(Tr ip h en ylm eth yl)a d en in e (11). To a solution of 10
(1.35 g, 9.99 mmol) and DMF (10 mL) in pyridine (30 mL) was
added Ph3Cl (2.79 g, 10.0 mmol). The reaction mixture was
stirred at 25 °C for 7.0 h. It was then diluted with EtOAc (150
mL) and water (100 mL). The organic layer was separated and
then washed with water (4 × 100 mL). It was dried over
MgSO4 (s) and concentrated under reduced pressure to yield
a foam. Purification was carried out by use of column chro-
matography (EtOAc/hexanes ) 8:2) to afford compound 11
(3.58 g, 9.48 mmol) in 95% yield: mp 260-262 °C; Rf (hexanes/
and water (40 mL). The organic layer was dried over MgSO4
(s) and concentrated under reduced pressure. Purification of
the residue by use of column chromatography (EtOAc/hexanes
) 8:2) gave compound 17 (0.041 g, 0.20 mmol) in 20% yield.
Further elution of the column with EtOAc/hexanes (9:1)
afforded 16 (0.123 g, 0.594 mmol) in 60% yield. For 16: mp
112-114 °C; Rf (hexanes/EtOAc ) 1:2) 0.16; UV (EtOH) λmax
267 (ꢀ 14 000); 1H NMR (CD3OD) δ 3.83 (s, 3H, CH3), 4.37 (s,
2 H, H2C1_), 8.41 (s, 1 H, HC2), 8.75 (s, 1 H, HC8); MS m/z 207
(M+). Anal. Calcd (C8H9N5O2): C, H, N. For 17: mp 148-149
°C; Rf (hexanes/EtOAc ) 1:2) 0.29; UV (EtOH) λmax 260 (ꢀ
1
EtOAc ) 1:2) 0.38; UV (EtOH) λmax 260 (ꢀ 14 600); H NMR
1
(CDCl3) δ 5.60 (s, 2 H, NH2), 7.15-7.31 (m, 15 H, C(C6H5)3),
7.74 (s, 1 H, HC2), 8.05 (s, 1 H, HC8); MS m/z 377 (M+). Αnal.
Calcd (C24H19N5): C, H, N.
13 750); H NMR (CD3OD) δ 3.75 (s, 3H, CH3), 4.15 (s, 2 H,
H2C1′), 8.29 (s, 1 H, HC2), 8.38 (s, 1 H, HC8); MS m/z 207 (M+).
7-(2-Hyd r oxyeth yl)a d en in e (18). To a stirred solution
containing 16 (0.207 g, 0.999 mmol) and water (0.50 mL) in
THF (12 mL) was added NaBH4 (0.38 g, 10.0 mmol). After
being stirred for 4 h at 25 °C, the reaction mixture was
neutralized to pH ) 7.0 by use of 10% HCl aqueous solution.
Solvent was evaporated under reduced pressure and the
residue was purified by column chromatography (EtOAc/
hexanes ) 9:1) to give 18 (0.10 g, 0.55 mmol) in 55% yield:
mp 132-133 °C; Rf (hexanes/EtOAc ) 1:2) 0.09; UV (EtOH)
λmax 265 (ꢀ 14 110); 1H NMR (CD3OD) δ 3.68 (t, J ) 5.9 Hz, 2
H, CH2O), 3.66 (t, J ) 5.9 Hz, 2 H, CH2N), 8.34 (s, 1 H, HC2),
8.68 (s, 1 H, HC8); MS m/z 179 (M+). Anal. Calcd (C7H9N5O):
C, H, N.
7-[(2-Ch lor oeth oxy)m eth yl]a d en in e (12). To a solution
of 11 (1.79 g, 4.74 mmol) in CH2Cl2 (70 mL) was added (2-
chloroethoxy)methyl chloride (0.65 g, 5.0 mmol). The reaction
mixture was stirred at 25 °C for 8.0 h to afford a solid.
Filteration and crystallization from MeOH gave 12 (1.66 g,
4.27 mmol) in 90% yield: mp 184-186 °C; Rf (hexanes/EtOAc
) 1:2) 0.28; UV (EtOH) λmax 267 (ꢀ 14 600); 1H NMR (DMSO-
d6) δ 3.73 (t, J ) 4.5 Hz, 2 H, CH2Cl), 3.86 (t, J ) 4.5 Hz, 2 H,
OCH2), 5.76 (s, 2 H, H2C1′), 8.49 (s, 1 H, HC2), 8.74 (s, 1 H,
HC8), 9.32, 10.05 (2 br s, 2 H, NH2); 13C NMR (DMSO-d6) δ
43.13 (CH2Cl), 68.82 (OCH2), 78.10 (C1′), 118.20 (C5), 143.53
(C2), 146.50 (C8), 149.39 (C4), 151.60 (C6); MS m/z 227 (M+, Cl
cluster). Αnal. Calcd (C8H10ClN5O): C, H, N, Cl.
7-[2-(Dieth ylp h osp h on om eth oxy)eth yl]a d en in e (19).
To a solution of 18 (0.18 g, 0.10 mmol) in DMF (15 mL) was
added sodium tert-butoxide (0.150 g, 1.56 mmol). After 5 min,
diethyl (p-toluenesulfonyloxymethane)phosphonate (0.42 g, 1.3
mmol) was added and the reaction mixture was stirred at 35
°C for 4.0 h. The reaction was then quenched with acetic acid
(5.0 mL) and the mixture was partitioned between EtOAc (50
mL) and water (50 mL). The organic layer was separated and
then washed with water (5 × 60 mL), dried over MgSO4 (s),
filtered, and concentrated under reduced pressure. Purification
by use of column chromatography (EtOAc/MeOH ) 9:1) gave
19 (0.20 g, 0.60 mmol) in 60% yield as a white foam: Rf
(hexanes/EtOAc ) 1:2) 0.16; UV (EtOH) λmax 266 (ꢀ 15 321);
1H NMR (CD3OD) δ 1.39 (t, J ) 6.7 Hz, 6H, 2 CH3), 3.74 (t, J
) 6.3 Hz, 2 H, CH2N), 3.76 (d, J ) 9.0 Hz, 2 H, CH2P), 3.97-
4.29 (m, 6 H, CH2O + 2 CH2OP), 8.37 (s, 1 H, HC2), 8.77 (s, 1
H, HC8); MS m/z 329 (M+). Anal. Calcd (C12H20N5O4P): C, H,
N.
7-[[2-(p-Meth oxyph en yloxy)eth oxy]m eth yl]aden in e (13).
After 10 h, compound 13 (2.79 g, 8.86 mmol) was synthesized
in 86% yield from 11 (3.90 g, 10.3 mmol) and [2-(p-methox-
yphenyloxy)ethoxy]methyl chloride (2.25 g, 10.4 mmol) in CH2-
Cl2 (100 mL) by the method used for the synthesis of 12: mp
129-130 °C; Rf (hexanes/EtOAc ) 1:2) 0.21; UV (EtOH) λmax
1
270 (ꢀ 15 100); H NMR (CD3OD) δ 3.68 (s, 3 H, OCH3), 4.04
(br s, 4 H, O(CH2)2O), 5.83 (s, 2 H, H2C1′), 6.58, 6.68 (AA′BB′,
J ) 9.0 Hz, 4 H, C6H4), 8.29 (s, 1 H, HC2), 8.61 (s,1 H, HC8);
13C NMR (CD3OD) δ 56.03 (CH3), 68.70 (OCH2), 70.18 (CH2-
OPh), 82.27 (C1′), 119.59 (C5), 144.17 (C2), 147.57 (C8), 150.25
(C4), 151.97 (C6), 115.50, 116.35, 153.44, 155.53 (C6H4); MS
m/z 315 (M+). Αnal. Calcd (C15H17 N5O3): C, H, N.
7-[(2-Hyd r oxyeth oxy)m eth yl]a d en in e (14). Compound
14 (0.74 g, 3.54 mmol) was prepared in 80% yield from 13 (1.39
g, 4.43 mmol) and CAN (2.43 g, 4.43 mmol) in CH3CN (30 mL)
and water (10 mL) by the method used for the synthesis of 9:
mp 122-123 °C; Rf (hexanes/EtOAc ) 1:2) 0.12; UV (EtOH)
7-[2-(P h osp h on om eth oxy)eth yl]a d en in e (20). To a solu-
tion of 19 (3.29 g, 10.0 mmol) in CH2Cl2 (130 mL) and DMF
(10 mL) was added Me3SiBr (4.95 g, 30.0 mmol); then the
solution was stirred at 25 °C for 7.0 h. A mixture of MeOH
and water (5:1, 40 mL) was added, and solvents were evapo-
rated. Purification by use of column chromatography (EtOAc/
MeOH ) 6:4) afforded 20 (1.23 g, 4.50 mmol) in 45% yield:
mp 253 °C (decomp); Rf (hexanes/EtOAc ) 1:2) 0.05; UV
1
λmax 265 (ꢀ 14 800); H NMR (CD3OD) δ 3.69-3.79 (m, 4 H,
O(CH2)2O), 5.79 (s, 2 H, H2C1′), 8.32 (s, 1 H, HC2), 8.60 (s, 1 H,
HC8); 13C NMR (CD3OD) δ 61.80 (CH2OH), 72.27 (OCH2) 82.01
(C1′), 119.62 (C5), 144.29 (C2), 147.68 (C8), 150.32 (C4), 152.12
(C6); MS m/z 209 (M+). Αnal. Calcd (C8H11N5O2): C, H, N.
9-(2-Cya n oet h yl)a d en in e (15). To a suspension of 57%
NaH in mineral oil (0.962 g, 22.8 mmol) in dry DMF (100 mL)
was added 10 (3.40 g, 24.9 mmol) under nitrogen, and the
mixture was heated at 80 °C for 1.0 h. A solution of 3-bro-
mopropionitrile (2.81 g, 21.0 mmol) in DMF (5.0 mL) was
added at 25 °C, and the reaction was heated at 65 °C for 17 h.
It was then diluted with EtOAc (250 mL) and 5% aqueous HCl
solution (150 mL). The organic layer was separated and then
washed with water (4 × 100 mL). It was dried over MgSO4 (s)
and concentrated under reduced pressure. Purification of the
residue was carried out by use of column chromatography
(EtOAc/hexanes ) 7.5:2.5) to afford compound 15 (3.510 g,
18.67 mmol) in 75% yield: mp 148-150 °C; Rf (hexanes/EtOAc
) 1:2) 0.38; UV (EtOH) λmax 260 (ꢀ 13 900); 1H NMR (CD3OD)
δ 2.92 (t, J ) 5.8 Hz, 2 H, CH2CN), 3.43 (t, J ) 5.8 Hz, 2 H,
CH2N), 8.35 (s, 1 H, HC2), 8.69 (s, 1 H, HC8); MS m/z 188 (M+).
7-[(Meth oxycar bon yl)m eth yl]aden in e (16) an d 9-[(Meth -
oxyca r bon yl)m eth yl]a d en in e (17). To a stirred solution
containing 15 (0.134 g, 1.00 mmol) and methyl iodoacetate
(0.30 g, 1.5 mmol) in dry THF (20 mL) was added a THF
solution of LiTMP (2.8 mL, 1.2 mmol) dropwise under an argon
atmosphere at -20 °C. The reaction mixture was warmed to
25 °C within 1.0 h; then it was stirred at room temperature
for 15 h. The solution was partitioned between EtOAc (40 mL)
1
(EtOH) λmax 265 (ꢀ 14 700); H NMR (CD3OD) δ 3.66 (t, J )
6.4 Hz, 2 H, CH2N), 3.71 (d, J ) 8.7 Hz, 2 H, CH2P), 4.19 (t,
J ) 6.4 Hz, 2 H, CH2O), 8.36 (s, 1 H, HC2), 8.78 (s, 1 H, HC8);
MS m/z 273 (M+). Anal. Calcd (C8H12N5O4P): C, H, N.
9-[2′-O-(ter t-bu tyld im eth ylsilyl)-5′-O-(p h osp h on o)-â-D-
fu r an osyl]aden in e 3′-[1-(Aden in -7-yleth oxy)m eth yl]ph os-
p h on a te (23). To a solution of adenosine 5′-monophosphate
(21) monohydrate (3.65 g, 9.99 mmol) in a mixture of pyridine
(150 mL) and CH3CN (160 mL) was added AgNO3 (6.63 g, 39.0
mmol). After 10 min, tert-butyldimethylsilyl chloride (5.70 g,
37.8 mmol) was added. The mixture was stirred at 25 °C for
7.0 h and then filtered to remove AgCl. The filtrate was
evaporated and the resultant crude product 22 was dissolved
in dry THF (40 mL). In another flask, collidine (3.66 g, 30.0
mmol) was added to a solution of THF (45 mL) containing 20
(2.73 g, 10.0 mmol) at -10 °C. To this solution was added CCl3-
SO2Cl (2.20 g, 10.0 mmol) in THF (15 mL) dropwise. After
crude 22 in THF was added to the mixture, it was stirred at
25 °C for 10 h. The solvents were removed, and the residue
was dissolved in AcOEt (100 mL) and washed with water (3
× 100 mL). The organic layer was concentrated, and the
residue was purified by use of column chromatography (EtOAc/

Nucleoside/ Nucleotide Analogues as Antiviral Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3719
MeOH ) 6:4) to afford 23 (3.0 g, 4.2 mmol) in 42% overall
yield: mp 223-225 °C; UV (EtOH) λmax 264 (ꢀ 17 300); 1H NMR
(CD3OD) δ 0.16 (br s, 6 H, (CH3)2Si), 1.05 (s, 9 H, (CH3)3C),
3.67-4.27 (m, 8 H, CH2N + CH2O + CH2OP + CH2P), 4.32-
4.5 (m, 3H, HC2′ + HC3′ + HC4′), 6.58 (d, J ) 4.9 Hz, 1 H,
HC1′), 8.12, 8.42 (2s, 2 H, 2 HC2), 8.27, 8.89 (2s, 2 H, 2 HC8).
Anal. Calcd (C24H38N10O10P2Si): C, H, N.
9-[5′-O-(p h osp h on o)-â-D-fu r a n osyl]a d en in e 3′-[1-(Ad -
en in -7-yleth oxy)m eth yl]p h osp h on a te (24). To a solution
of 23 (0.36 g, 0.50 mmol) in THF (5.0 mL) was added n-Bu4-
NF (1.0 M solution in THF, 0.31 g, 1.2 mmol). Acetic acid (0.50
mL) was added to the mixture after it was stirred at 25 °C for
30 min. The solvents were removed, and the residue was
purified by use of Whatman 3-MM paper with a mixture of
i-PrOH, NH4OH, and H2O (9:1:2) as the eluent. The band at
ca. Rf 0.35 was eluted with H2O and collected by lyophilization
to give 24 (0.27 g, 0.45 mmol) in 90% yield: mp >250 °C
(decomp); UV (EtOH) λmax 264 (ꢀ 18 200); 1H NMR (CD3OD) δ
3.75-4.18 (m, 8 H, CH2N + CH2O + CH2OP + CH2P), 4.29-
4.70 (m, 3H, HC2′ + HC3′ + HC4′), 6.48 (d, J ) 4.5 Hz, 1 H,
HC1′), 7.99, 8.39 (2s, 2 H, 2 HC2), 8.26, 8.83 (2s, 2 H, 2 HC8).
Anal. Calcd (C18H24N10O10P2): C, H, N.
nucleotide analogues and anti-DNA virus and anticellular
activities of nucleoside and nucleotide analogues in tissue
culture. This material is available free of charge via the
Internet at http://pubs.acs.org.
Refer en ces
(1) Schaeffer, H. J .; Beauchamp, L.; de Miranda, P.; Elion, G. B.;
Bauer, D. J .; Collins, P. 9-(2-Hydroxyethoxymethyl)guanine
Activity Against Viruses of the Herpes Group. Nature (London)
1978, 272, 583-585.
(2) Ogilvie, K. K.; Cheriyan, U. O.; Radatus, B. K.; Smith, K. O.;
Galloway, K. S.; Kennell, W. L. Biologically Active Acyclonucleo-
side Analogues. II. The Synthesis of 9-[[2-Hydroxy-1-(hydroxym-
ethyl)ethoxy]methyl]guanine (BIOLF-62). Can. J . Chem. 1982,
60, 3005-3010.
(3) Ashton, W. T.; Canning, L. F.; Reynolds, G. F.; Tolman, R. L.;
Karkas, J . D.; Liou, R.; Davies, M.-E.; DeWitt, C. M.; Perry, H.
C.; Field, A. K. Synthesis and Antiherpetic Activity of (S)-, (R)-,
and (()-9-[(2,3-Dihydroxy-1-propoxy)methyl]guanine, Linear Iso-
mers of 2′-Nor-2′-deoxyguanosine. J . Med. Chem. 1985, 28, 926-
933.
(4) (a) Kim, C. U.; Misco, P. F.; Luh, B. Y.; Hitchcock, M. J . M.;
Ghazzouli, I.; Martin, J . C. A New Class of Acyclic Phosphonate
Nucleotide Analogues: Phosphonate Isosters of Acyclovir and
Ganciclovir Monophosphonates as Antiviral Agents. J . Med.
Chem. 1991, 34, 2286-2294. (b) Kim, C. U.; Luh, B. Y.; Misco,
P. F.; Bronson, J . J .; Hitchcock, M. J . M.; Ghazzouli, I.; Martin,
J . C. Acyclic Purine Phosphonate Analogues as Antiviral Agents.
Synthesis and Structure-Activity Relationships. J . Med. Chem.
1990, 33, 1207-1213. (c) De Clercq, E.; Holy, A.; Rosenburg, I.;
Sakuma, T.; Balzarini, J .; Maudgal, P. C. A Novel Selective
Broad-Spectrum Anti-DNA Virus Agents. Nature (London) 1986,
323, 464-467. (d) Pauwels, R.; Balzarini, J .; Schols, D.; Baba,
M.; Desmyter, J .; Rosenberg, I.; Holy, A.; De Clercq, E. Phos-
phonylmethoxyethyl Purine Derivatives, A New Class of Anti-
Human Immunodeficiency Virus Agents. Antimicrob. Agents
Chemother. 1988, 32, 1025-1030. (e) De Clercq, E.; Sakuma,
T.; Baba, M.; Pauwels, R.; Balzarini, J .; Rosenberg, I.; Holy, A.
Antiviral Activity of Phosphonylmethoxyalkyl Derivatives of
Purine and Pyrimidines. Antiviral Res. 1987, 8, 261-272. (f)
Baba, M.; Mori, S.; Shigeta, S.; De Clercq, E. Selective Inhibitory
Effect of (S)-9-(3-Hydroxy-2-Phosphonylmethoxypropyl)adenine
and 2′-Nor-Cyclic GMP on Adenovirus Replication In Vitro.
Antimicrob. Agents Chemother. 1987, 31, 337-339. (g) Snoeck,
R.; Sakuma, T.; De Clercq, E.; Rosenberg, I.; Holy, A. (S)-1-(3-
Hydroxy-2-Phosphonylmethoxypropyl)cytosine, A Potent and
Selective Inhibitor of Human Cytomegalovirus Replication.
Antimicrob. Agents Chemother. 1988, 32, 1839-1844. (h) Bron-
son, J . J .; Ghazzouli, I.; Hitchcock, M. J . M.; Webb, R. R.; Martin,
J . C. Synthesis and Antiviral Activity of the Nucleotide Analogue
(S)-1-[3-Hydroxy-2-(phosphonylmethoxy)propyl]cytosine. J . Med.
Chem. 1989, 32, 1457-1463. (i) Balzarini, J .; De Clercq, E.
5-Phosphoribosyl 1-Pyrophosphate Synthetase Converts the
Acyclic Nucleoside Phosphonates 9-(3-Hydroxy-2-phosphonyl-
methoxypropyl)adenine and 9-(2-Phosphonylmethoxyethyl)ad-
enine Directly to Their Antivirally Active Diphosphate Deriva-
tives. J . Biol. Chem. 1991, 266, 8686-8689 and references
therein.
9-[[(Z)-4-(Eth ylid en yl)-2,3-d im eth oxy-∆r,â-bu ten olid e]-
5′-O-p h osp h on o-â-D-fu r a n osyl]a d en in e 3′-[1-(Ad en in -7-
yleth oxy)m eth yl]p h osp h on a te (25). To a solution of 24
(0.300 g, 0.499 mmol) in DMF (20 mL) was added NaHCO3
(0.30 g, 3.6 mmol). The reaction mixture was stirred at 25 °C
under N2 for 10 min. Then, butenolide 26 (0.10 g, 0.50 mmol)
was added and the mixture was stirred under N2 for 1.0 h.
The solution was diluted with EtOAc (50 mL) and aqueous
HCl solution (1%, 40 mL). The organic layer was separated
and washed with H2O (50 mL). Then, it was dried over MgSO4
(s), filtered, and concentrated under reduced pressure. Puri-
fication by use of silica gel column chromatography with
EtOAc/MeOH (6:4) as eluant afforded 25 (0.32 g, 0.42 mmol)
in 85% yield: mp >237 °C (decomp); UV (EtOH) λmax 215 (ꢀ
16 000), λmax 264 (ꢀ 18 540); 1H NMR (CD3OD) δ 3.69-4.12
(m, 16 H, CH2N + CH2O + 2 CH2OP + CH2P + C2OCH3
+
C3OCH3), 4.31-4.78 (m, 3H, HC2′ + HC3′ + HC4′), 5.38 (t, J )
7.0 Hz, 1 H, dCH), 6.51 (d, J ) 4.8 Hz, 1 H, HC1′), 8.02, 8.40
(2s, 2 H, 2 HC2), 8.28, 8.86 (2s, 2 H, 2 HC8). Anal. Calcd
(C26H32N10O14P2): C, H, N.
[1-(Ad en in -9-ylet h oxy)m et h yl]p h osp h on o-(Z)-4-(et h -
ylid en yl)-2,3-d im eth oxy-∆r,â-bu ten olid e (28). Compound
28 (3.90 g, 8.80 mmol) was prepared in 88% yield from 4 (2.73
g, 9.99 mmol) and 26 (2.20 g, 10.0 mmol) in the same manner
that 25 was prepared from 24: mp >241 °C (decomp); Rf
(hexanes/EtOAc ) 1:2) 0.12; UV (EtOH) λmax 218 (ꢀ 13 097),
1
λmax 259 (ꢀ 14 700); H NMR (CD3OD) δ 3.57(t, J ) 6.0 Hz, 2
H, CH2N), 3.69 (d, J ) 9.0 Hz, 2 H, CH2P), 3.89 (m, 5 H, C2-
OCH3 + CH2OP), 4.06, (t, J ) 6.0 Hz, 2 H, CH2O), 4.13 (s, 3
H, C3OCH3), 5.41 (t, J ) 7.0 Hz, 1H, dCH), 8.12 (s, 1 H, HC2),
(5) (a) J a¨hne, G.; Kroha, H.; Mu¨ller, A.; Helsberg, M.; Winkler, I.;
Gross, G.; Scholl, T. Regioselective Synthesis and Antiviral
Activity of Purine Nucleoside Analogues with Acyclic Substit-
uents at N7. Angew. Chem., Int. Ed. Engl. 1994, 33, 562-563
and references therein. (b) Singh, D.; Wani, M. J .; Kumar, A. A.
Simple Solution to the Age Old Problem of Regioselective
Functionalization of Guanine: First Practical Synthesis of
Acyclic N9- and/or N7-Guanine Nucleosides Starting from N2,N9-
Diacetylguanine. J . Org. Chem. 1999, 64, 4665-4668.
(6) Geen, G. R.; Grinter, T. J .; Kincey, P. M.; J arvest, R. L. The
Effect of the C-6 Substituent of the Regioselectivity of N-
Alkylation of 2-Aminopurines. Tetrahedron 1990, 46, 6903-
6914.
8.21 (s,
1
H, HC8); MS m/z 441 (M+). Anal. Calcd
(C16H20N5O8P): C, H, N.
[1-(Gu a n in -9-ylet h oxy)m et h yl]p h osp h on o-(Z)-4-(et h -
ylid en yl)-2,3-d im eth oxy-∆r,â-bu ten olid e (29). Compound
29 (3.7 g, 8.0 mmol) was prepared in 80% yield from 27 (2.89
g, 9.99 mmol) and 26 (2.20 g, 10.0 mmol) in the same manner
that 25 was prepared from 24: mp >260 °C (decomp); Rf
(hexanes/EtOAc ) 1:2) 0.05; UV (EtOH) λmax 252 (ꢀ 11 097),
λmax 273 (ꢀ 8100); 1H NMR (CD3OD) δ 3.71(t, J ) 7.2 Hz, 2 H,
CH2N), 3.79 (d, J ) 9.1 Hz, 2 H, CH2P), 3.92 (m, 5 H, C2OCH3
+ CH2OP), 4.10, (t, J ) 7.2 Hz, 2 H, CH2O), 4.18 (s, 3 H, C3-
OCH3), 5.50 (t, J ) 6.8 Hz, 1 H, dCH), 8.76 (s, 1 H, HC8); MS
m/z 457 (M+). Anal. Calcd (C16H20N5O9P): C, H, N.
(7) Kjellberg, J .; J ohansson, N. G. Studies of the Alkylation of
Derivatives of Guanine. Nucleosides Nucleotides 1989, 8, 225-
256.
(8) (a) Meier, C.; Lorey, M.; De Clercq, E.; Balzarini, J . cyclo-Sal-
2′,3′-dideoxy-2′, 3′-didehydrothymidine Monophosphate (cycloSal-
d4TMP): Synthesis and Antiviral Evaluation of a New d4TMP
Delivery System. J . Med. Chem. 1998, 41, 1417-1427 and
references therein. (b) Hakimelahi, G. H.; Mei, N.-W.; Moosavi-
Movahedi, A. A.; Davari, H.; Hakimelahi, S.; King, K.-Y.; Hwu,
J . R.; Wen, Y.-S. Synthesis and Biological Evaluation of Purine-
Containing Butenolides. J . Med. Chem. 2001, 44, 1749-1757.
(9) Hakimelahi, G. H.; Khalafi-Nezhad, A. Catalytic Effect of
Tetrabutylammonium Fluoride in the Preparation of Secoribo-
nucleosides. Helv. Chim. Acta 1989, 72, 1495-1550.
Ack n ow led gm en t. For financial support, we thank
Academia Sinica, Shiraz Saadi Hospital, Shiraz and
Tehran Universities, Daropakhsh Pharmaceutical Com-
pany, and National Science Council of Republic of
China.
Su p p or tin g In for m a tion Ava ila ble: Two tables showing
results of the solubility and lipophilicity of nucleoside and
(10) Khorshidi, A. Doctoral Thesis in Pharmacy, Faculty of Medicine,
Isfahan University, Isfahan, Iran, 1986.

3720 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Hakimelahi et al.
(11) Fukuyama, T.; Laird, A. A.; Hotchkiss, L. M. p-Anisyl Group:
A Versatile Protecting Group for Primary Alcohols. Tetrahedron
Lett. 1985, 26, 6291-6292.
(20) Moosavi-Movahedi, A. A.; Rahmani, Y.; Hakimelahi, G. H.
Thermodynamic and Kinetic Studies of Competitive Inhibition
of Adenosine Deaminase By Ring Opened Analogues of Adenine
Nucleoside. Int. J . Biol. Macromol. 1993, 15, 125-129 and
references therein.
(21) Keller, P. M.; Fyfe, J . A.; Beauchamp, L.; Lubbers, C. M.;
Furman, P. A.; Schaeffer, H. J .; Elion, G. B. Enzymatic Phos-
phorylation of Acyclic Nucleoside Analogues and Correlations
with Antiherpetic Activities. Biochem. Pharmacol. 1981, 30,
3071-3077 and references therein.
(22) De Clercq, E.; Descamps, J .; Verheist, G.; Walker, R. T.; J ones,
A. S.; Torrence, P. F.; Sugar, D. Comparative Efficacy of
Different Antiherpes Drugs Against Different Strains of Herpes
Simplex Virus. J . Infect. Dis. 1980, 141, 563-574.
(23) Davari, H. Doctoral Thesis, School of Veterinary Medicine,
Shiraz University, Shiraz, Iran, 1989.
(24) Schaeffer, H. J .; Gurwara, S.; Vince, R.; Bittner, S. Novel
Substrate of Adenosine Deaminase. J . Med. Chem. 1971, 14,
367-369.
(25) Elion, G. B.; Furman, P. A.; Fyfe, J . A.; de Miranda, P.;
Beauchamp, L.; Schaeffer, H. J . Selectivity of Action of an
Antiherpetic Agent 9-(2-Hydroxyethoxymethyl)guanine. Proc.
Natl. Acad. Sci. U.S.A. 1977, 74, 5716-5720.
(26) Scott, A. The Search for a Viral Penicillin. New Sci. 1983, 10,
642-645.
(27) Hakimelahi, G. H.; Zarrinehzad, M.; J arrahpour, A. A.; Sharghi,
H. Ring-Open Analogues of Adenine Nucleoside. Aminoacyl
Derivatives of Cyclo- and Acyclo-nucleosides. Helv. Chim. Acta
1987, 70, 219-231.
(12) (a) Kjellberg, J .; J ohansson, N. G. Characterization of N7- and
N9-Alkylated Purine Analogues by 1H and 13C NMR. Tetrahedron
1986, 42, 6541-6544. (b) For 1H and 13C NMR of acyclovir, see
Shiragami, H.; Koguchi, Y.; Tanaka, Y.; Takamatsu, S.; Uchida,
Y.; Ineyama, T.; Izawa, K. Synthesis of 9-(2-Hydroxyethoxym-
ethyl)guanine (Acyclovir) From Guanosine. Nucleosides Nucle-
otides 1995, 14, 337-340. (c) Bailey, S.; Harnden, M. R. Novel
Nucleosides. Part 2. 2,3-Dihydroxy-1-methoxypropyl- and 3-Hy-
droxy-1-methoxypropyl-Substituted Purines. Nucleosides Nucle-
otides 1987, 6, 555-574.
(13) Hakimelahi, G. H.; Moosavi-Movahedi, A. A.; Tsay, S.-C.; Tsai,
F.-Y.; Wright, J . D.; Dudev, T., Hakimelahi, S,; Lim, C. Design,
Synthesis, and SAR of Novel Carbapenem Antibiotics with High
Stability to Xanthomonas maltophilia Oxyiminocephalospori-
nase Type II. J . Med. Chem. 2000, 43, 3632-3640.
(14) Schultze, L. M.; Chapman, H. H.; Dubree, J . P.; J ones, R. J .;
Kent, K. M.; Lee, T. T.; Louie, M. S.; Postich, M. J .; Prisbe, E.
J .; Rohloff, J . C.; Yu, R. H. Practical Synthesis of the anti-HIV
Drug, PMPA. Tetrahedron Lett. 1998, 39, 1853-1856.
(15) (a) Tanaka, H.; Fukui, M.; Haraguchi, K.; Masaki, M.; Miyasaka,
T. Cleavage of A Nucleoside Oxetane With Carbanions: Syn-
thesis of A Highly Promising Candidate for Anti-HIV Agents. A
Phosphonate Isoster of AZT 5′-Phosphate. Tetrahedron Lett.
1989, 30, 2567-2570. (b) Barton, D. H. R.; Ge´ro, S. D.; Quiclet-
Sire, B.; Samadi, M. Stereoselectivity In Radical Reactions of
2′-Deoxynucleosides. A Synthesis of An Isoster of 3′-Azido-3′-
deoxythymidine 5′-monophosphate (AZT 5′-monophosphate).
Tetrahedron Lett. 1989, 30, 4969-4972.
(16) (a) Ogilvie, K. K.; McGee, D. P. C.; Boisvert, S. M.; Hakimelahi,
G. H.; Proba, Z. A. The Preparation of Protected Arabinonucleo-
sides. Can. J . Chem. 1983, 61, 1204-1212. (b) Ogilvie, K. K.;
Hakimelahi, G. H.; Proba, Z. A.; McGee, D. P. C. Silylated
Derivatives of Arabinonucleosides. Tetrahedron Lett. 1982, 23,
1997-2000.
(17) Hakimelahi, G. H.; Moosavi-Movahedi, A. A.; Sadeghi, M. M.;
Tsay, S.-C.; Hwu, J . R. Design, Synthesis, and Structure-
Activity Relationship of Novel Dinucleotide Analogues as Agents
Against Herpes and Human Immunodeficiency Viruses. J . Med.
Chem. 1995, 38, 4648-4659 and references therein.
(18) Baker, D. C.; Haskell, T. H.; Putt, S. R. Prodrugs of 9-â-D-
Arabinofuranosyladenine. 1. Synthesis and Evaluation of Some
5′-(O-Acyl) Derivatives. J . Med. Chem. 1978, 21, 1218-1221.
(19) Ogilvie, K. K.; Nguyen-Ba, N.; Gillen, M. F.; Radatus, B. K.;
Cheriyan, U. O.; Hanna, H. R.; Smith, K. O.; Galloway, K. S.
Synthesis of a Purine Acyclonucleoside Series Having Pro-
nounced Antiviral Activity. The Glyceropurines. Can. J . Chem.
1984, 62, 241-252 and references therein.
(28) Armitage, P. Statistical Methods in Medical Research; Blackwell
Scientific Publications: Oxford, U.K., 1983.
(29) Hakimelahi, G. H.; Mohanazadeh, F.; Davari, H.; Zakerinia, M.
Tetra-N-Butylammonium Fluoride as a Novel Reagent for the
Preparation of Acyclonucleosides. J . Sci. Iran 1990, 1, 186-191.
(30) Averett, D. R. Anti-HIV Compound Assessment by Two Novel
High Capacity Assays. J . Virol. Methods 1989, 23, 263-276.
(31) Balzarini, J .; Holy, A.; J indrich, J .; Naesens, L.; Snoeck, R.;
Schols, D,; De Clercq, E. Differential Antiherpes Virus and
Antiretrovirus Effects of the (S) and (R) Enantiomers of Acyclic
Nucleoside Phosphonates: Potent and Selective In Vitro and In
Vivo Antiretrovirus Activities of (R)-9-(2-Phosphonomethoxypro-
pyl)-2,6-Diaminopurine. Antimicrob. Agents Chemother. 1993,
37, 332-338 and references therein.
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