Nucleotides and pronucleotides of 2,2-bis(hydroxymethyl) methylenecyclopropane analogues of purine nucleosides: Synthesis and antiviral activity

Article abstract of DOI:10.1021/jm040149b

Phenylmethylphosphor-L-alaninate pronucleotides 7a, 7b, 8a, and 8b, cyclic phosphates 10a and 10b, and phosphates 11a and 11b derived from 2,2-bis(hydroxymethyl)methylenecyclopropane analogues 1a, 1b, 2a, and 2b were synthesized and evaluated for their an

Full text of DOI:10.1021/jm040149b

J. Med. Chem. 2005, 48, 91-99
91
Nucleotides and Pronucleotides of
2,2-Bis(hydroxymethyl)methylenecyclopropane Analogues of Purine
Nucleosides: Synthesis and Antiviral Activity
Zhaohua Yan,† Earl R. Kern,‡ Elizabeth Gullen,§ Yung-Chi Cheng,§ John C. Drach,| and Jiri Zemlicka*,†
Department of Chemistry, Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute,
Wayne State University School of Medicine, Detroit, Michigan 48201-1379, Department of Pediatrics,
The University of Alabama School of Medicine, Birmingham, Alabama 35233, Department of Pharmacology,
Yale University School of Medicine, New Haven, Connecticut 06510-8066, and Department of Biologic and Materials Science,
School of Dentistry, University of Michigan, Ann Arbor, Michigan 48019-1078
Received July 26, 2004
Phenylmethylphosphor-L-alaninate pronucleotides 7a, 7b, 8a, and 8b, cyclic phosphates 10a
and 10b, and phosphates 11a and 11b derived from 2,2-bis(hydroxymethyl)methylenecy-
clopropane analogues 1a, 1b, 2a, and 2b were synthesized and evaluated for their antiviral
activity. An improved protocol for the synthesis of analogues 1a, 1b, 2a, and 2b is also described.
Phosphate 11a was the most effective agent against human and murine cytomegalovirus (EC50
0.25-1.1 µM). The Z-pronucleotides 7a and 7b had EC50 3.6-25.2 and 3-18.4 µM, respectively.
The EC50 of cyclic phosphate 10a was 6.0-20 µM. The activity against Epstein-Barr (EBV)
was assay-dependent. Pronucleotides 7a and 7b and phosphate 11a had EC50 2.3-3.4 µM
against EBV/H-1, but 7b was cytotoxic (CC50 3.8 µM). Cyclic phosphate 10a was the only
compound effective against EBV/Daudi (EC50 0.96 µM), but it was inactive in H-1 cells.
Pronucleotide 7a was active against varicella zoster virus with EC50 6.3 and 7.3 µM, respectively,
and hepatitis B virus (HBV, EC50 4.1 µM). Cyclic phosphate 10a was the most effective analogue
against HBV (EC50 0.8 µM).
Scheme 1a
Recently, we have described (Z)- and (E)-2,2-bis-
(hydroxymethyl)methylenecyclopropane analogues of
nucleosides 1 and 2 as antiviral agents.1 Several of these
analogues are active in vitro at micromolar or submi-
cromolar concentration range1a,b against herpesviruses
such as human and murine cytomegalovirus (HCMV
and MCMV), Epstein-Barr virus (EBV), varicella zoster
virus (VZV), and human herpes virus 6 and 8 (HHV-6
and HHV-8). The guanine analogue 1a (ZSM-I-62,
cyclopropavir), the most potent agent against HCMV
and MCMV among the methylenecyclopropane ana-
logues known to date, is highly effective in reducing
MCMV replication in visceral organs of mice and HCMV
replication in implanted human tissues in SCID mice.
In four experimental CMV infections, cyclopropavir (1a)
was more active than ganciclovir.1c
Transformation of the first-generation purine meth-
ylenecyclopropane analogues 3 and 4 into phosphoro-
alaninate triester pronucleotides 5 and 6 increased
antiviral potency in several instances.2-6 It was there-
fore of interest to investigate the antiviral activity of
prodrugs 7 and 8 of the second-generation (Z)- and (E)-
2,2-bis(hydroxymethyl)methylenecyclopropane analogues
1 and 2. Our attention focused on guanine and adenine
pronucleotides 7a and 8a and 7b and 8b. In addition,
ganciclovir cyclic phosphate 9 is an effective antiherpetic
agent with a mechanism of action different from
ganciclovir.7-10 More recently, similar cyclic phosphates
a Reagents: (a) 1. Ti(i-PrO)4 (cat.), pyridine, I2, CH2Cl2. 2.
KMnO4, H2O. (b) (PhSe)2, NaOH, EtOH, NaBH4. (c) KMnO4, H2O.
(d) (i-Pr)2NEt, toluene, 80 °C. (e) 1. LiAlH4, Et2O. 2. Ac2O, pyridine.
derived from 2′,3′-dideoxy-4′-hydroxymethyl-3′-thiori-
bonucleosides were reported to exhibit in vitro anti-
HCMV effects.11 Because cyclopropavir (1a) can be re-
garded as a rigid analogue1a of ganciclovir, we extended
our investigation to cyclic phosphates 10a and 10b. The
synthesis and antiviral activity of analogues 7a, 7b, 8a,
8b, 10a, and 10b are the subject of this contribution.
Phosphates 11a and 11b, the starting materials for
synthesis of 10a and 10b, were also included in the
study. The structures of 1 and 2 as well as other
nucleotides discussed below are shown in Chart 1.
* Corresponding author. Telephone: (313) 833-0715. Fax: (313)
832-7294. E-mail: zemlicka@kci.wayne.edu.
† Wayne State University School of Medicine.
‡ The University of Alabama School of Medicine.
§ Yale University School of Medicine.
| University of Michigan.
10.1021/jm040149b CCC: $30.25 © 2005 American Chemical Society
Published on Web 12/14/2004

92 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Yan et al.
Chart 1a
a B ) purine, in formulas 1-8. Series a: B ) Gua. Series b: B ) Ade.
Scheme 2a
Scheme 3a
a Reagents: (a) 1-Methylimidazole, pyridine.
Synthesis. The previously described procedure start-
ing from diethyl isopropylidene malonate1a was ad-
equate for the preparation of limited amounts of 2,2-
bis(hydroxymethyl)cyclopropane analogues including
cyclopropavir (1a) for initial antiviral screening. The
major drawback of this protocol is a difficult chromato-
graphic separation of a key intermediate at an early
stage of synthesis. It was clear that in order to obtain
the larger amounts of 1a required for in vivo testing
and further preclinical studies a more efficient proce-
dure would have to be devised. Such a protocol is
described in Scheme 1. Commercially available diethyl
allylmalonate (12) was transformed into iodomethylcy-
clopropane dicarboxylate 13 in 72% yield using a
modification of the reported procedure.12,13 Intermediate
13 was then converted to phenylselenylmethylcyclopro-
pane 14 (83%). Oxidation with KMnO4 afforded phe-
nylselenoxide 15 (70%), which, in turn, was transformed
to methylenecyclopropane dicarboxylate 16 by a base-
catalyzed â-elimination (63% yield). Reduction and
acetylation1a were combined in a single step to give
diacetate 17 in 92% yield. The latter was used for
synthesis of analogues 1a and 2a and 1b and 2b as
described earlier.1a
a Series a: B ) Gua, Z-isomer. Series b: B ) Gua, E-isomer.
Series c: B ) Ade, Z-isomer. Series d: B ) Ade, E-isomer.
Reagents: (a) 1. MeC(OMe)3, MeSO3H, DMF. 2. NEt3. (b) 80%
AcOH. (c) 3,4-Dihydro-2H-pyran, MeSO3H, DMF. (d) NH4OH,
MeOH, ∆. (e) 1. 18, 1-methylimidazole, pyridine/THF. 2. 80%
AcOH. 3. 0.1 M Na2HPO4, pH 7.5.
reaction with trimethyl orthoacetate in dimethylforma-
mide (DMF) to the cyclic orthoacetate 19a, which was
smoothly hydrolyzed to monoacetate 20a in 84% overall
yield (Scheme 3). A similar method was used for
synthesis of monoacyl derivatives of ganciclovir.14 Ana-
logues 2a, 1b, and 2b gave the respective acetates 20b,
20c, and 20d (via cyclic ortho esters 19b, 19c, and 19d)
in 82, 87, and 63% yield, respectively. Direct phospho-
rylation of 20a, 20b, 20c, and 20d with reagent 18 was
not investigated because the subsequent removal of the
O-acetyl group is not compatible with an alkali-labile
phosphotriester alaninate moiety.15 Therefore, acetates
20a, 20b, 20c, and 20d were converted to tetrahydro-
pyranyl (THP) derivatives 21a, 21b, 21c, and 21d by
an acid-catalyzed reaction with dihydropyran in DMF
followed by deacetylation with NH4OH (yields 77-87%).
The phosphorylation of compounds 21a, 21b, 21c, and
21d with reagent 18 was uneventful, giving the pro-
nucleotides 7a and 8a and 7b and 8b in 29-45% yield
The first-generation pronucleotides 5 and 6 were
obtained by a direct phosphorylation2,6 of methylenecy-
clopropanes 3 and 4 with reagent 18 in pyridine using
1-methylimidazole as a catalyst (Scheme 2). To avoid
formation of mixtures of mono- and diphosphorylated
products, one of the hydroxy groups of analogues 1 and
2 was selectively protected as shown in Scheme 3.
Analogue 1a was transformed by an acid-catalyzed

Antiviral Nucleotides and Pronucleotides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 93
Scheme 4a
Table 1. Inhibition of HCMV and MCMV Replication by
Phosphate Derivatives of gem-Bis(hydroxymethyl)methylene-
cyclopropane Analogues
a Series a: Z-isomers. Series b: E-isomers. Reagents: (a) 1.
(PhO)2P(O)H, pyridine, ∆. 2. NEt3, H2O. (b) 1. TMSCl, imidazole,
pyridine. 2. I2, pyridine. 3. H2O. 4. NH4OH. 5. Dowex 50 (H(+)). (c)
N,N′-Dicyclohexyl-4-morpholinecarboxamidine, DCC, pyridine, ∆.
upon prolonged treatment with 80% acetic acid to
remove THP groups and cleave any byproducts phos-
phorylated at the heterocyclic moiety.2
EC50/CC50 (µM)
HCMV/HFFa
MCMV/MEFa
Acetates 20a and 20b were used as convenient
starting materials for the synthesis of cyclic phosphates
10a and 10b (Scheme 4). Phosphitylation using diphen-
yl phosphite in pyridine according to the method de-
scribed previously16,17 for synadenol (3b) and its E-iso-
mer (4b) gave phosphites 22a and 22b as triethyl-
ammonium salts in 80 and 73% yield, respectively.
Silylation of 22a and 22b followed by oxidation with
iodine in pyridine and deacetylation afforded the phos-
phates 11a (86%) and 11b (75%). Cyclization by a
method previously described for ribonucleoside 3′,5′
cyclic phosphates18 using a N,N′-dicyclohexylcarboxa-
midinium salt of 11a and N,N′-dicyclohexylcarbodiimide
(DCC) in pyridine furnished cyclic phosphate 10a in
93% yield. In a similar fashion, the E-isomer 10b was
obtained from phosphate 11b (89%).
Antiviral Activity. Pronucleotides 7a, 8a, 7b, and
8b, cyclic phosphates 10a and 10b, and phosphates 11a
and 11b were assayed in vitro against the following
viruses: human and murine cytomegalovirus (HCMV
and MCMV), Epstein-Barr virus (EBV), varicella zoster
virus (VZV), hepatitis B virus (HBV), and herpes
simplex virus 1 and 2 (HSV-1 and HSV-2). All tested
analogues were inactive against human immunodefi-
ciency virus (HIV-1). The results are summarized in
Tables 1-3.
The Z-isomeric pronucleotide 7a was an inhibitor of
replication of HCMV/HFF (Towne and AD169 strains),
but its potency was about one log lower than that of the
parent compound, cyclopropavir (1a, Table 1). It was less
effective against MCMV/MEF. A similar decrease of ac-
tivity relative to synguanol (3a) was observed2 with
pronucleotide 5a. The lower activity may be the result
of less effective intracellular conversion to phosphate
11a according to the mechanism proposed for similar
analogues19 (Scheme 5), possibly in the phosphoamidase
step. Regardless of the precise mechanistic details of the
bioactivation of 5a and 7a, our results support the
hypothesis that the active metabolite of cyclopropavir
(1a) is phosphate 11a. Indeed, other findings have in-
dicated1d that viral kinase UL97 is required for the
phosphorylation of 1a. The same enzyme was implicated
in the phosphorylation of synguanol (3a).20 Pronucleo-
tides 7a, 7b, 8a, and 8b are substrates for porcine liver
esterase (PLE), a widely accepted19 model of intracellu-
lar esterases. The PLE-catalyzed hydrolysis of 7a led
to formation of diastereoisomeric phosphoramidate 23
compound
Towneb
AD169c
1ad
0.33/100-200e
2.4/>100f
3.6/>100
3/32
>100/>100
>100/>100
20/>100
>100/>100
0.25/100
0.49/>380
11.7/>404
8.1/>198
0.27/>380
9.7/>404
25.2/>198
NT
1bd
7a
7b
18.4/200
8a
>198/>198g
>205/>205g
6.0/>301
NT
8b
NT
7.2/>301
NT
0.26/175
NT
2.1/>392
10a
10b
11a
11b
ganciclovir
>299/>299g
1.1/>291
>100/>100
2.1/>100
>291/>291g
1.6/>392
a Plaque reduction assay. NT ) not tested. b Visual cytotoxicity.
c Cytotoxicity by neutral red uptake. d Data from ref 1a. e Average
of four experiments. f Average of three experiments. g Cytopathic
effect (CPE) inhibition assay.
(via intermediates 24 and 25) as shown by 31P NMR
spectra (Scheme 5). No evidence for a formation of cyclic
phosphate 26 or 10a was found. The activity of adenine
Z-pronucleotide 7b against HCMV was comparable with
that of parent analogue 1b in both AD169 and Towne
strains of the virus. With the latter strain, however, in-
creased cytotoxicity to the HFF host cells was observed.
Phosphate 11a is almost equally effective as cyclo-
propavir (1a) against HCMV and MCMV (Table 1), but
similar to other nucleotides,21 it is most likely dephos-
phorylated at the cell membrane to cyclopropavir (1a),
which is therefore rephosphorylated inside the cells. The
only advantage of phosphate 11a is then an increased
solubility at pH 7 over that of the parent analogue 1a.
Of interest is the potency of cyclic phosphate 10a
against HCMV (AD169) and MCMV in the micromolar
range even though it was somewhat less effective
against the Towne strain of HCMV. The efficacy of 10a
in the AD169 assay (EC50 6.0 µM) does not differ much
from that of ganciclovir cyclic phosphate 9 (EC50 2 and
5.9 µM, respectively).7,8
As observed previously with methylenecyclopropane
analogues 3 and pronucleotides 5, the activity against
EBV is quite often assay-dependent.1a,6,22 Thus, pro-
nucleotides 7a and 7b showed greater antiviral activity
in H-1 cells than 1a and 1b, although cytoxicity also
increased (Table 2). In contrast, 7a and 7b had no
significant activity in either viral or cellular prolifera-
tion when Daudi cells were used. The parent analogue
1a and phosphate 11a had a similar level of potency in
H-1 cells, but they lacked a significant effect in Daudi

94 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Yan et al.
Scheme 5
Table 2. Inhibition of EBV, VZV, and HBV Replication by
Phosphate Derivatives of gem-Bis(hydroxymethyl)methylene-
cyclopropane Analogues
0.96/>150, Daudi), was ineffective in H-1 culture. The
result in Daudi culture is not compatible with a partial
or total enzymatic hydrolysis to 11a or 1a because, as
already mentioned, the latter analogues lacked signifi-
cant activity. Instead, the mechanism of action of cyclic
phosphate 10a may resemble that of ganciclovir cyclic
phosphate 9. Compound 9 is an antiviral analogue per
se; it is converted to neither ganciclovir nor ganciclovir
phosphate.7-10 The intramolecular distance between the
heterocyclic moiety (N-9) and phosphorus atom deter-
mined from MM2 minimized model of 10a is very close
to that observed in a crystal structure of ganciclovir
cyclic phosphate23,24 (9, 5.944 versus 5.893 Å). This
further stresses the similarity between 10a and 9.
Against VZV/HFF, pronucleotide 7a was the only
potent analogue as shown in both the cytopathic and
plaque reduction assay (EC50 6.3 and 7.3 µM, respec-
tively). Cyclopropavir (1a) was inactive. A similar
potentiating effect of the adenine pronucleotide 7b
relative to 1b was also seen, albeit to a lesser extent.
The other analogues lacked significant potency. Inter-
estingly, cyclic phosphate 10a was a potent anti-HBV
agent (EC50 0.8 µM), surpassing both phosphate 11a
(EC50 15 µM) and cyclopropavir (1a, EC50 >10 µM). This
result is also indicative that the mechanism of action
of cyclic phosphate 10a is different from cyclopropavir
(1a). The potency of both pronucleotides 7a and 7b was
also lower than that of 10a (EC50 4.1 and 12.8 µM,
respectively) but higher than that of parent analogues
1a and 1b.
EC50/CC50 (µM)
EBV
VZV
HBV
2.2.15b,f
compound
Daudia
45/74
H-1b,c
HFFd,e
1a
7/>50
>380
>10
>10
4.1
12.8
>10
>20
0.8
>20
15
>20
1b
166/>202
>99/>198
>20/>50
>404
7a
3.4/35
>102/>102 3/3.8
6.3(7.3)g
>41
7b
8a
>99/>198
>20/>100 >198
8b
>102/>102 >20/>25
>205
10a
10b
11a
11b
control
0.96/>150
>20/>100 >301
>150/>150 >20/>100 >299
>146/>146 2.3/62 >291
>146/>146 >20/>100 >291
1.1/>222h 5i
1.6/>444h 0.05/>100j
a Viral capsid immunofluorescence (VCA) ELISA. b DNA hybrid-
ization assay. c Cytotoxicity was determined in CEM cells. d Cyto-
pathic effect (CPE) inhibition assay. e For CC50 values, see Table
1, footnote c. f For CC50 values, see EBV/H-1 assays. g Plaque
reduction assay. h Acyclovir. i Ganciclovir. j Lamivudine.
Although the efficacy of the tested analogues in
HSV-1 and HSV-2 assays was only moderate and assay-
dependent, some aspects of their antiviral properties
deserve discussion. Thus, the EC50 values of 10a were
16-22 µM in BSC-1 and Vero cells, whereas no activity
cells. By contrast, cyclic phosphate 10a, the most potent
and noncytotoxic anti-EBV agent listed in Table 2 (EC50

Antiviral Nucleotides and Pronucleotides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 95
Table 3. Inhibition of HSV-1 and HSV-2 Replication by
Phosphate Derivatives of gem-Bis(hydroxymethyl)methylene-
cyclopropane Analogues
saturated aqueous Na2S2O3 (2 × 1 L), brine (1 L), 4 M HCl (1
L), and brine (1 L). The organic phase was dried (MgSO4) and
evaporated to give crude product 13 (220 g) of which 15% was
unchanged 12 according to the 1H NMR and the rest was 13.
The starting material 12 was removed by slowly adding
KMnO4 (100 g, 0.63 mol) with external ice cooling to a stirred
suspension of crude product 13 in water (300 mL). After 50
min of being stirred at 0 °C, the solution was filtered, and the
filter cake was washed with hexane/ethyl acetate 2:1 mixture
(4 × 150 mL). The layers were separated, and the organic
phase was washed with water (300 mL), saturated Na2SO3
solution (300 mL), and brine (2 × 500 mL). After being dried
over MgSO4 and evaporation of the solvent, 170 g (72%) of pure
1
13 was obtained. The H and 13C NMR spectra corresponded
to those reported in the literature.25
Diethyl 1-(Phenylselenyl)methylcyclopropane-2,2-di-
carboxylate (14). Caution! Because selenium compounds
are toxic, contact with skin should be avoided. All
operations should be carried out in a well-ventilated
hood. Diphenyl diselenide26 (78 g, 0.25 mol) was refluxed in
ethanol (300 mL) until a clear solution was obtained. After
being cooled to room temperature, 4 M NaOH (125 mL, 0.5
mol) was added followed by solid NaBH4 (19 g, 0.5 mol) in
several portions. The resultant mixture was refluxed for 30
min, brought back to room temperature, and stirred while
adding dropwise to it a solution of compound 13 (163 g, 0.5
mol) in ethanol (200 mL). After 15 min, water (500 mL) was
added, and the mixture was extracted with hexane/ethyl
acetate 2:1 (1 L). The aqueous layer was saturated with NaCl
and reextracted with the same solvent mixture (2 × 500 mL).
The combined extracts were washed with brine (500 mL) and
dried over Na2SO4. Evaporation of the solvents provided a
residue that was purified by column chromatography (950 g
of silica gel) in hexane/ethyl acetate (1:0 to 10:1) to give 147 g
(83%) of product 14 as an oil containing 5% of malonate 12
EC50/CC50 (µM)
HSV-1
HFFb
HSV-2
HFFb,d Veroc
compound
BSC-1a
Veroc,d
1a
>100/>100 >380
50/>100 >404
>50
>50
>50
>40
>50
>30
>380
>404
>198
22.1c
>198
>205
>50
>50
>50
>40
>50
>30
22
>50
>50
>50
32.3f
1b
7a
>100/>100 >198
7b
55/3.5
70/>100
21.1c
>198
8a
8b
>100/>100 >205
10a
10b
11a
11b
acyclovir
20/>100
>301
16/>100 242
>100/>100 >299
>100/>100 9.9c
>100/>100 >291
>50
50
>299
(23.3)c
>291
6.7e
>50
1.5/>100
0.9e
13.5f
a ELISA. Cytotoxicity was determined in KB cells. b Cytopathic
effect (CPE) assay. For cytotoxicity see Table 1, footnote c. c Plaque
reduction assay. d For cytotoxicity, see Table 2, footnote c. e The
CC50 in HFF cells was >444 µM. f The CC50 in CEM cells was >200
µM.
1
according to the H NMR spectra. This product was directly
used in the next step. An analytical sample was obtained by
column chromatography of a 100-mg sample of 14 on silica
gel using hexane/ethyl acetate (25:1) to give 90 mg of com-
pound 14. 1H NMR (CDCl3) δ: 7.53 (m, 2H) and 7.24 (m, 3H,
phenyl), 4.09-4.28 (m, 4H, CH2O), 3.04 (dd, J ) 12.0 and 6.4
Hz, 1H) and 2.71 (dd, J ) 12.4 and 9.2 Hz, 1H, CH2Se), 2.22
(m, 1H, CH of cyclopropane), 1.44 (dd, J ) 8.8 and 6.0 Hz,1H)
and 1.38 (dd, J ) 8.8 and 6.8 Hz, 1H, CH2 of cyclopropane),
1.28 and 1.25 (2t, 6H, J ) 7.2 Hz, CH3). 13C NMR (75 MHz,
CDCl3): 169.8, 168.0 (CO), 134.0, 129.5, 129.3, 127.6 (phenyl),
61.9, 61.8 (CH2O), 36.0 (quarternary C of cyclopropane), 28.7,
26.8 (CH2Se, CH of cyclopropane), 22.3 (CH2 of cyclopropane),
14.4, 14.3 (CH3). EI-MS: 354, 356 (M, 7.4, 15.1), 153 (100.0).
Anal. (C16H20O4Se) C, H.
was observed in HFF cells (Table 3). Even more inter-
esting is the activity of phosphate 11a against HSV-1
and HSV-2 in HFF (EC50 9.9 and 23.3 µM, respectively)
although the parent analogue 1a was inactive. This may
indicate that phosphate 11a is capable under certain
circumstances of penetrating the cellular membrane.
The only moderately effective pronucleotide was adenine
derivative 7b with EC50 21.1 and 22.1 µM against
HSV-1 and HSV-2 in HFF culture. Compound 7b was
cytotoxic in KB cells with CC50 3.5 µM. The E-isomers
8a, 8b, 10b, and 11b were inactive.
Diethyl 1-(Phenylselenyl)methylcyclopropane 2,2-di-
carboxylate Selenium Oxide (15). KMnO4 (169 g, 1.07 mol)
was added slowly with stirring and ice cooling to a suspension
of compound 14 (147 g, 0.42 mol) in water (400 mL). After
being stirred for 2 h, the reaction mixture was filtered, and
the filter cake was washed with ethyl acetate (6 × 300 mL).
The layers were separated, and the organic phase was washed
with water (500 mL) and brine (2 × 500 mL). It was dried
over MgSO4. Evaporation of the solvent provided crude product
15 (134 g) containing traces27 of KMnO4. This material was
dissolved in THF (300 mL), and H2O2 (30%, 40 mL) was added
dropwise within 1 h with stirring. After an additional 10 min
of being stirred, ethyl acetate (300 mL) was added, and the
organic phase was washed with water (500 mL), saturated
NaHCO3 solution (300 mL), and brine (300 mL). It was dried
(MgSO4) and evaporated to give pure oxide 15 (109 g, 70%,
Experimental Section
General Methods. See ref 1a. The UV spectra were
measured in ethanol, and NMR spectra were determined at
400 MHz (1H), 100 MHz (13C), and 162 MHz (31P) NMR in CD3-
SOCD3 unless stated otherwise. Mass spectra were obtained
in an electrospray ionization mode (ESI-MS) in methanol-
NaCl. A stock solution of reagent 18 in THF instead of CH2-
Cl2 was prepared as described.2 Porcine liver esterase (lyoph-
ilized powder) was a product of Sigma, St. Louis, MO. The
O-substituted compounds 11a, 11b, and 20a-d are racemic
whereas 7a, 7b, 8a, 8b, and 21a-d are mixtures of four
diastereoisomers each. The alanine moiety in 7a, 7b, 8a, and
8b is of L-configuration.
1
two diastereoisomers) as an oil. H NMR (CDCl3) δ: 7.70 (m,
Diethyl 1-(Iodomethyl)cyclopropane-2,2-dicarboxy-
late (13). The procedure described for the corresponding
dimethyl ester12 was modified as follows. To a solution of a
diethyl allylmalonate (12, 144.6 g, 0.72 mol) in CH2Cl2 (500
mL) were added Ti(i-PrO)4 (43 mL, 0.15 mol), pyridine (117
mL, 1.44 mol), and iodine (550 g, 2.16 mol) under N2 at room
temperature with stirring. The stirring was continued for 45
h. The resultant solution was washed with 4 M HCl (1 L),
2H) and 7.50 (m, 3H, phenyl), 4.16 (m, 4H, CH2O), 3.08 (dd, J
) 12.4 Hz, J ) 7.6 Hz) and 2.75 (m, 2H, CH2SeO), 2.20 (m)
and 1.97 (m, 1H, CH of cyclopropane), 1.49 (m), 1.39 (dd, J )
8.0 Hz, J ) 7.0 Hz) and 1.23 (m, 8H, CH2 of cyclopropane and
CH3). 13C NMR (75 MHz, CDCl3): 169.1, 169.0, 168.1, 167.8
(CO), 140.0, 139.7, 131.9, 131.7, 129.91, 129.89, 126.4, 126.1
(phenyl), 62.3, 62.13, 62.09 (CH2O), 52.8, 52.7 (CH2SeO), 34.4,
33.8 (quarternary C of cyclopropane), 21.8, 21.6, 21.31, 21.27

96 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Yan et al.
(CH and CH2 of cyclopropane), 14.3, 14.2 (CH3). EI-MS: 371,
ester 19b. Mp: 261-262 °C. UV (EtOH) λmax
:
272 nm (ꢀ
373 (M + H, 1.2, 2.3), 57 (100.0). Anal. (C16H20O5Se) C, H.
10 500), 229 (ꢀ 29 600). 1H NMR δ: 10.89 (s, 1H, NH), 8.02 (s,
1H, H8), 7.24 (d, 1H, H1′), 6.74 (s, 2H, NH2), 4.95 (t, J ) 5.6
Hz, 1H, OH), 4.12 and 4.04 (AB, JAB ) 11.2 Hz, 2H, H5′), 3.44-
3.34 (m, overlapped with H2O, H5′′), 2.02 (s, 3H, CH3), 1.65
(m, 2H, H3′). 13C NMR: 171.1 (CO), 157.4 (C6), 154.7 (C2), 150.6
(C4), 134.3 (C8), 117.3, 116.9 (C2′, C5), 111.7 (C1′), 65.6 (C5′),
63.1 (C5′′), 26.3 (C4′), 21.4 (CH3), 15.0 (C3′). ESI-MS: 306 (100.0,
M + H), 328 (72.5, M + Na) 633 (16.2, 2M + Na). Anal.
(C13H15N5O4) C, H, N.
Diethyl Methylenecyclopropane-2,2-dicarboxylate (16).
A solution of compound 15 (217 g, 0.58 mol) and i-Pr2NEt (201
mL, 1.15 mol) in toluene (800 mL) was heated at 85-89 °C
for 24 h. After being cooled, the solution was washed with
water (400 mL). The aqueous layer was extracted with ethyl
acetate (3 × 400 mL). The combined organic phase was washed
with saturated NaHCO3 solution (500 mL) and brine (500 mL).
It was dried over MgSO4, and the solvents were evaporated
to give crude product 16 containing a small amount of selenide
14 that could not be removed by chromatography. To obtain
pure 16, the crude product 16 was dissolved in THF (1 L), and
H2O2 (30%, 400 mL) was added dropwise with stirring and
ice cooling. The stirring was then continued for 14 h at room
temperature. Ethyl acetate (500 mL) was added, the layers
were separated, and the organic phase was washed with water
(500 mL). The aqueous layer was saturated with NaCl and
extracted with ethyl acetate (2 × 400 mL). The combined
organic phase was washed with saturated NaHCO3 solution
(700 mL) and brine (700 mL). It was dried over MgSO4, the
solvents were evaporated, and the residue was chromato-
graphed on a silica gel column using hexane/ethyl acetate (10:
1) to afford compound 16 (73 g, 63%) identical with the product
obtained previously1a by a different procedure.
2,2-Bis(acetoxymethyl)methylenecyclopropane (17). A
solution of diester 16 (75 g, 0.37 mol) in ethyl ether (100 mL)
was added dropwise with stirring and external ice cooling to
a suspension of LiAlH4 (18.2 g, 0.47 mol) in ethyl ether (500
mL). The mixture was refluxed for 17 h. After being cooled to
0 °C, water (36 mL) and 20% NaOH (73 mL) were added
dropwise. Stirring was continued for 10 min, the solids were
filtered off, and the filter cake was washed with ethyl acetate
(10 × 150 mL). Combined washings were dried over Na2SO4,
and the solvent was evaporated to give 2,2-bis(hydroxymethyl)-
methylenecyclopropane (50 g) that was dissolved in pyridine.
Acetic anhydride (225 mL, 2.04 mol) was added slowly with
stirring and ice cooling to a solution of the above obtained
crude product (50 g) in pyridine (200 mL, 2.47 mol). Stirring
was then continued at room temperature for 14 h. The mixture
was cooled again to 0 °C, and water (500 mL) was added
slowly. The resulting mixture was extracted with ethyl ether
(2 × 500 mL). The combined extracts were washed with 10%
CuSO4 (500 mL), 2 M HCl (500 mL), saturated NaHCO3
solution (500 mL), and brine (500 mL). They were dried over
MgSO4, and the solvent was evaporated to give diacetate 17
(69 g, 92% in two steps) identical with the product described
previously.1a
(Z)-9-{[2-(Acetoxymethyl)-2-(hydroxymethyl)cyclopro-
pylidene]methyl}guanine (20a). A suspension of diol 1a
(1.82 g, 6.92 mmol) in DMF (100 mL) was sonicated for 10
min. Trimethyl orthoacetate (1.23 mL, 9.68 mmol) and meth-
anesulfonic acid (67 µL, 1.04 mmol) were then added, and the
mixture was stirred at room temperature for 3 h. After
addition of triethylamine (3 mL), the solvent was evaporated,
and the crude cyclic ortho ester 19a was dissolved in 80%
acetic acid (100 mL). The solution was stirred at room
temperature for 10 h, and the solvent was evaporated in vacuo.
The crude product was purified by column chromatography
on silica gel CH2Cl2/MeOH (10:1 to 6:1) to give O-acetate 20a
(1.78 g, 84%). Mp: 258-260 °C. UV λmax: 273 nm (ꢀ 11 000),
230 (ꢀ 27 500). 1H NMR δ: 10.69 (s, 1H, NH), 8.21 (s, 1H, H8),
7.14 (d, 1H J ) 1.6 Hz, H1′), 6.55 (s, 2H, NH2), 5.25 (t, J ) 5.2
Hz, 1H, OH), 4.16, 4.11 (AB, 2H, JAB ) 11.4 Hz, H5′), 3.71 and
3.44 (dAB, J ) 11.4 and 5.1 Hz, 2H, H5′′), 1.94 (s, 3H, CH3),
1.45 (m, 2H, H3′). 13C NMR: 170.8 (CO), 157.4 (C6), 154.8 (C2),
150.4 (C4), 134.8 (C8), 116.9, 116.7 (C2′, C5), 111.9 (C1′), 65.6
(C5′), 63.2 (C5′′), 28.1 (C4′), 21.2 (CH3), 12.3 (C3′). ESI-MS: 306
(100.0, M + H), 328 (39.8, M + Na). Anal. (C13H15N5O4) C, H,
N.
(Z)-9-{[2-(Acetoxymethyl)-2-(hydroxymethyl)cyclopro-
pylidene]methyl}adenine (20c). Trimethyl orthoacetate (3.0
mL, 23.5 mmol) and methanesulfonic acid (187 µL, 3.0 mmol)
were slowly added to an ice-cooled suspension of analogue (1b,
2.89 g, 11.7 mmol) in DMF (120 mL). The workup including
hydrolysis of intermediary ortho ester 19c followed the protocol
described for compound 20a. After chromatography on silica
gel CH2Cl2/MeOH (20:1), acetate 20c (2.95 g, 87%) was
obtained. Mp: 196-199 °C. UV λmax: 277 nm (ꢀ 8 700), 256 (ꢀ
1
12 400), 227 (ꢀ 25 500). H NMR δ: 8.63 (s, 1H, H8), 8.16 (s,
1H, H2), 7.42 (poorly resolved d, 1H, H1′), 7.35 (bs, 2H, NH2),
5.29 (t, J ) 5.2 Hz, 1H, OH), 4.24, 4.13 (AB, 2H, J ) 11.4 Hz,
H5′), 3.75, 3.48 (2AB, 2H, J ) 11.2 and 5.0 Hz, H5′′), 1.92 (s,
3H, CH3 of Ac), 1.50 (m, 2H, H3′). 13C NMR: 170.8 (CO), 156.7
(C6), 153.7 (C2), 148.7 (C4), 138.3 (C8), 119.0 (C5), 117.1 (C2′),
112.0 (C1′), 65.7 (C5′), 63.4 (C5′′), 28.2 (C4′), 21.2 (CH3), 12.3 (C3′).
ESI-MS: 290 (M + H, 100.0), 312 (M + Na, 54.7). Anal.
(C13H15N5O3) C, H, N.
(E)-9-{[2-(Acetoxymethyl)-2-(hydroxymethyl)cyclopro-
pylidene]methyl}adenine (20d). The procedure described
for compound 20c was followed with E-isomer 2b (1.90 g, 7.69
mmol) to give compound 20d (1.40 g, 63%) via ortho ester 19d.
Mp: 191-193 °C. UV λmax: 277 nm (ꢀ 9 000), 257 (ꢀ 12 200),
1
226 (ꢀ 28 500). H NMR δ: 8.49 (s, 1H, H8), 8.18 (s, 1H, H2),
7.53 (d, 1H, J ) 2.4 Hz, H1′), 7.39 (s, 2H, NH2), 4.95 (t, J ) 5.6
Hz, 1H, OH), 4.15, 4.10 (AB, 2H, J ) 11.5 Hz, 2H, H5′), 3.49,
3.44 (partly overlapped 2AB, J ) 11.2 and 6.0 Hz, 2H, H5′′),
2.04 (s, 3H, CH3), 1.72 (m, 2H, H3′). 13C NMR: 171.1 (CO),
156.7 (C6), 153.8 (C2), 148.9 (C4), 137.9 (C8), 119.1 (C5), 117.8
(C2′), 111.8 (C1′), 65.6 (C5′), 63.1 (C5′′), 26.5 (C4′), 21.4 (CH3),
15.2 (C3′). ESI-MS (MeOH): 290 (M + H, 100.0). Anal.
(C13H15N5O3) C, H, N.
(Z)-9-{[2-(Hydroxymethyl)-2-(2-tetrahydropyranyloxy-
methyl)cyclopropylidene]methyl}guanine (21a). A solu-
tion of methanesulfonic acid (0.14 mL, 2.23 mmol) in DMF (5
mL) was added to a stirred mixture of compound 20a (680 mg,
2.23 mmol) and 3,4-dihydro-2H-pyran (3.25 mL, 35.68 mmol)
in DMF (35 mL) at room temperature. The stirring was
continued for 5.5 h whereupon the reaction was quenched by
addition of triethylamine (0.3 mL). The solvent was evaporated
in vacuo (oil pump) at room temperature. The residue was
dissolved in MeOH (100 mL) and NH4OH (30%, 20 mL), and
the mixture was heated at 40-50 °C for 10 h. The volatile
components were evaporated, and the crude product was
chromatographed on a silica gel column using CH2Cl2 and CH2-
Cl2/MeOH (15:1 to 12:1 to 6:1) to give compound 21a (672 mg,
87%). Mp: 295-298 °C. UV λmax: 273 nm (ꢀ 10 500), 231 (ꢀ
27 300). 1H NMR δ: 10.64 (s, 1H, NH), 8.36 and 8.31 (2s, 1H,
H8), 7.11 (s, 1H, H1′), 6.53 (s, 2H, NH2), 5.06 (m, 1H, OH), 4.60
and 4.51 (2bs, 1:1, CHO of THP), 3.88 (d) and 3.70-3.28
(cluster of m, partially overlapped with H2O, 6H, CH2O of THP,
H5′′, H5′′), 1.70-1.31 (cluster of m, 8H, CH2 of THP, H3′). 13C
NMR: 157.3 (C6), 154.7 (C2), 150.3 (C4), 134.9 (C8), 117.6,
117.4, 116.9 (C5, C2′), 111.4 (C1′), 98.4 (CHO of THP), 68.9, 68.7
(CH2O of THP), 62.8, 62.6, 61.7, 61.3 (C5′, C5′′), 30.64, 30.58,
29.2, 29.0, 25.67, 25.6, 19.6, 19.3 (3 × CH2 of THP, C4′), 12.2,
12.1 (C3′). ESI-MS: 348 (100.0, M + H), 370 (M + Na). Anal.
(C16H21N5O4) C, H, N.
(E)-9-{[2-(Hydroxymethyl)-2-(2-tetrahydropyranyloxy-
methyl)cyclopropylidene]methyl}guanine (21b). The pro-
cedure described for the Z-isomer 21a was followed with
E-isomer 20b (280 mg, 0.91 mmol) to give compound 21b (270
(E)-9-{[2-(Acetoxymethyl)-2-(hydroxymethyl)cyclopro-
pylidene]methyl}guanine (20b). The procedure described
for the Z-isomer 20a was followed with E-isomer 2a (2.40 g,
9.12 mmol) to afford acetate 20b (2.18 g, 82%) via cyclic ortho
mg, 85%). Mp: 233-235 °C. UV (EtOH) λmax
: 272 nm (ꢀ
11 700), 229 (ꢀ 31 900). 1H NMR δ: 10.67 (s, 1H, NH), 8.04 (s,

Antiviral Nucleotides and Pronucleotides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 97
1H, H8), 7.22 and 7.20 (s, 1H, H1′), 6.52 (s, 2H, NH2), 4.78 and
4.59 (2m, 1H, 1:1, CHO of THP, OH), 3.72-3.60 (m, 2H), 3.48-
3.40 (2 clusters of m, 6H, CH2O of THP, H5′, H5′′), 1.71, 1.58,
and 1.45 (3m, 8H, CH2 of THP, H3′). 13C NMR: 157.4 (C6), 154.6
(C2), 150.5 (C4), 134.3 (C8), 118.5, 118.2, 116.9 (C5, C2′), 111.1
(C1′), 98.4, 98.3 (CHO of THP), 68.4 (CH2O of THP), 63.1, 62.0,
61.9 (C5′, C5′′), 31.0, 27.3, 27.2, 25.7, 19.8 (3 × CH2 of THP,
C4′), 14.8, 14.6 (C3′). ESI-MS: 348 (100.0, M + H), 370 (64.3,
M + Na). Anal. (C16H21N5O4) C, H, N.
8.24, 8.23, and 8.18 (4s, 1H), 7.31 and 7.14 (2m, 6H, Ph, H1′),
6.57 (s, 2H, NH2), 6.01 (m, 1H, NH of Ala), 5.25 (m, 1H, OH),
4.26, 4.05, 3.80, and 3.39 (4m, 5H, H5′, H5′′, CH of Ala), 3.57,
3.533, and 3.529 (3s, 3H, OCH3), 1.46 (m, 2H, H3′), 1.17 (m,
3H, CH3 of Ala). 13C NMR: 174.4 (CO), 157.4 (C6), 154.6 (C2),
151.3 (Cipso, PhO), 150.4 (C4), 134.7 (C8), 130.2 (Cpara, PhO),
125.2 (Cortho, PhO), 120.9 (Cmeta, PhO), 116.9, 116.1 (C5, C2′),
112.0 (C1′), 67.4 (C5′′), 62.6, 62.5 (C5′), 52.6, 52.5 (OCH3), 50.4,
50.3 (CH of Ala), 29.1, 29.0 (C4′), 20.3 (CH3 of Ala), 11.9 (C3′).
31P NMR: 4.73, 4.57, 4.52, 4.33 (1.8:1.2:1.9:1.0). ESI-MS: 246
(100.0, M - OP(O)(OPh)NHCHMeCO2Me), 505 (47.8, M + H),
527 (88.4, M + Na). Anal. (C21H25N6O7P) C, H, N.
(E)-9-{[(2,2-Hydroxymethyl)cyclopropylidene]methyl}-
guanine (Methylphenylphosphoryl) P f N-L-Alaninate
(8a). The procedure for the Z-isomer 7a was followed with
E-isomer 21b (230 mg, 0.66 mmol) to furnish compound 8a
(142 mg, 42%). UV λmax: 270 nm (ꢀ 14 000) 229 (ꢀ 34 100). 1H
NMR δ: 10.70 (s, 1H, NH), 8.05 (s, 1H, H8), 7.32 and 7.17 (2m,
6H, PhO, H1′), 6.52 (s, 2H, NH2), 5.95 (m, 1H, NH of Ala), 4.91
(m, 1H, OH), 4.07, 3.84, and 3.45 (4m, 5H, H5′, H5′′, CH of Ala),
3.58, 3.56, 3.55, and 3.52 (4s, 3H, OCH3), 1.68 (m, 2H, H3′),
1.21 (m, 3H, CH3). 13C NMR: 174.4 (CO), 157.4 (C6), 154.6
(C2), 151.4 (Cipso, PhO), 150.6 (C4), 134.3 (C8), 130.3 (Cpara, PhO),
125.2 (Cortho, PhO), 120.9 (Cmeta, PhO), 117.0 (C5, C2′), 111.8
(C1′), 67.9 (C5′′), 62.6 (C5′), 52.5 (OCH3), 50.5, 50.4 (CH of Ala),
27.4, 27.3 (C4′), 20.3 (CH3 of Ala), 14.8 (C3′). 31P NMR: 4.69,
4.52, 4.27, 4.10 (1.4:1.2:1.4:1.0). ESI-MS: 505 (100.0, M + H),
527 (36.6, M + Na). Anal. (C21H25N6O7P) C, H, N.
(Z)-9-{[2-(Hydroxymethyl)-2-(2-tetrahydropyranyloxy-
methyl)cyclopropylidene]methyl}adenine (21c). A solu-
tion of methanesulfonic acid (1.18 mL, 18 mmol) in DMF (5
mL) was added to a stirred mixture of compound 20c (2.60 g,
8.99 mmol) and 3,4-dihydro-2H-pyran (9.1 mL, 90 mmol) in
DMF (120 mL) at 0 °C. The experiment was performed as
described for guanine derivative 21a except that the reaction
was quenched with triethylamine (6 mL). The crude product
was chromatographed on a silica gel column using CH2Cl2/
MeOH (30:1), and the resultant solid was recrystallized from
ethyl acetate (20 mL) to give compound 21c (2.3 g, 77%). Mp:
212-214 °C. UV λmax: 278 nm (ꢀ 8800), 256 (ꢀ 12 000), 226 (ꢀ
1
25 500). H NMR δ: 8.79 and 8.76 (2s, 1H, H8), 8.17 (s, 1H,
H2), 7.41 and 7.39 (2 overlapped s, 3H, H1′ and NH2), 5.11 (m,
1H, OH), 4.59 and 4.50 (2bs, 1H, CHO of THP), 3.97 (d, J )
9.6 Hz), 3.77-3.57, 3.52-3.47, and 3.36-3.29 (3 clusters of
m, 6H, H5′, H5′′, CH2O of THP), 1.70-1.26 (cluster of m, 8H,
CH2 of THP, H3′). 13C NMR: 156.7 (C6), 153.7 (C2), 148.7 (C4),
138.2 (C8), 119.0, 119.1, 117.91, 117.85 (C5, C2′), 111.5 (C1′),
98.5, 98.3 (CHO of THP), 69.2, 68.8 (CH2O of THP), 62.6, 61.6,
61.4 (C5′, C5′′), 30.6, 29.2, 29.1, 25.6, 25.5, 19.5, 19.4 (3 × CH2
of THP, C4′), 12.3, 12.2 (C3′). ESI-MS: 248 (M + H -
dihydropyran, 100.0), 332 (M + H, 37.2), 354 (M + Na, 61.3).
Anal. (C16H21N5O3) C, H, N.
(Z)-9-{[(2,2-Hydroxymethyl)cyclopropylidene]methyl}-
adenine (Methylphenylphosphoryl) P f N-L-Alaninate
(7b). A solution of reagent 18 (4.2 g, 15 mmol) and 1-meth-
ylimidazole (2.4 mL, 30 mmol) in pyridine (20 mL) was added
slowly with stirring to compound 21c (0.99 g, 3 mmol) in
pyridine (90 mL) at room temperature. Stirring was continued
for 14 h. The solvent was evaporated, and the oily residue was
chromatographed using CH2Cl2/MeOH (30:1). After being dried
at 0.1 Torr and room temperature, the crude product was
dissolved in 80% acetic acid (100 mL), and the solution was
stirred at room temperature for 4 days. The solvent was
evaporated in vacuo at room temperature, and the residue was
purified by column chromatography in CH2Cl2/MeOH (30:1 to
20:1). Appropriate fractions were concentrated, and ether (50
mL) was added to give Z-phosphoralaninate 7b (550 mg, 35%)
as a solid. UV λmax: 278 nm (ꢀ 8500), 256 (ꢀ 12 100), 228 (ꢀ
24 300), 210 (ꢀ 25 800). 1H NMR δ: 8.63 and 8.61 (2s, 1H, H8),
8.17 and 8.16 (2s, 1H, H2), 7.44 (m, 1H, H1′), 7.35 (s), 7.30 (m),
and 7.12 (m, 7H, PhO, NH2), 5.98 (m, 1H, NH of Ala), 5.31
(m, 1H, OH), 4.36-4.29 (m), 4.08, 3.99 (2dd) and 3.47-3.38
(cluster of m partly overlapped with H2O, 5H, H5′, H5′′, CH of
Ala), 3.56, 3.52 (2bs, 3H, CH3O), 1.54-1.47 (m, 2H, H3′), 1.18,
1.15 (2d, 3H, CH3 of Ala). 13C NMR: 174.4 (CO), 156.7 (C6),
(E)-9-{[2-(Hydroxymethyl)-2-(2-tetrahydropyranyloxy-
methyl)cyclopropylidene]methyl}adenine (21d). The pro-
cedure for Z-isomer 21c was followed with E-isomer 20d (1.20
g, 4.15 mmol) to afford product 21d (1.10 g, 80%). Mp: 212-
216 °C. UV λmax: 278 nm (ꢀ 10 800), 261 (ꢀ 14 200), 226 (ꢀ
1
32 400). H NMR δ: 8.49 (s, 1H, H8), 8.17 (s, 1H, H2), 7.49 (2
overlapped d, 1H, J ) 1.6 Hz, H1′), 7.38 (s, 2H, NH2), 4.82 (m,
1H, OH), 4.60 (bs, 1H, CHO of THP), 3.75-3.64 (m, 2H) and
3.56-3.40 (cluster of m partly overlapped with H2O, 4H, CH2O
of THP, H5′′, H5′), 1.71-1.59 (m, 4H) and 1.44 (bd, 4H, CH2 of
THP, H3′). 13C NMR: 156.7 (C6), 153.8 (C2), 148.9 (C4), 137.8
(C8), 119.1, 118.9, 118.6 (C5, C2′), 111.2 (C1′), 98.4, 98.3 (CHO
of THP), 68.4 (CH2O of THP), 63.1, 61.93, 61.87 (C5′, C5′′), 30.9,
27.5, 27.4, 25.7, 19.82, 19.77 (3 x CH2 of THP, C4′), 15.0, 14.8
(C3′). ESI-MS: 248 (M + H - dihydropyran, 48.8), 332 (M +
H, 90.5), 354 (M + Na, 100.0). Anal. (C16H21N5O3) C, H, N.
(Z)-9-{[(2,2-Hydroxymethyl)cyclopropylidene]methyl}-
guanine (Methylphenylphosphoryl) P f N-L-Alaninate
(7a). A suspension of the Z-isomer 21a (410 mg, 1.18 mmol)
in pyridine (80 mL) was sonicated for 10 min whereupon a
solution of methyl chlorophenylphosphoryl P f N-L-alaninate
(18) in THF (0.18 M, 40 mL, 7.2 mmol) and 1-methylimidazole
(1.1 mL, 14.2 mmol) were added at room temperature. The
resultant mixture was stirred at this temperature for 4 h. The
solvents were evaporated in vacuo, and the residue was
chromatographed on a silica gel column using CH2Cl2/MeOH
(20:1) to give a crude product which was dissolved in acetic
acid (80%, 20 mL). After the solution was stirred at room
temperature for 4 days, the solvent was evaporated, and the
residue was dissolved in MeOH (20 mL) and 0.1 M Na2HPO4
(pH 7.5, 10 mL) was added. The resultant suspension was
stirred at room temperature for 22 h. The solvent was
evaporated at room temperature, and the residue was dis-
solved in CH2Cl2/MeOH (8:1, 100 mL). The solids were filtered
off, and they were washed with the same solvent (2 × 20 mL).
The combined organic layer was concentrated, and the residue
was chromatographed on a silica gel column using CH2Cl2/
MeOH (20:1 to 15:1 to 10:1). Evaporation of solvents afforded
a gum that solidified after trituration with ethyl ether (10 mL)
to give Z-phosphoralaninate 7a (200 mg, 33%). UV λmax: 270
153.7 (C2), 151.3 (Cipso, PhO), 148.7 (C4), 138.2 (C8), 130.2 (Cpara
,
PhO), 125.2 (Cortho, PhO), 120.90, 120.85, 120.8 (Cmeta, PhO),
119.1 (C5), 116.62, 116.58 (C2′), 112.1 (C1′), 67.6 (C5′′), 62.9, 62.8
(C5′), 52.6, 52.5 (CH3O), 50.4, 50.3, 50.2 (CH of Ala), 29.3, 29.2
(C4′), 20.4, 20.3, 20.2 (CH3 of Ala), 11.9 (C3′). 31P NMR: 4.74,
4.58, 4.49, 4.31 (3.7:1.3:3.4:1.0). ESI-MS: 230 (M - OP(O)-
(OPh)NHCHMeCO2Me), 489 (M + H, 39.8), 511 (42.1, M +
Na). Anal. (C21H25N6O6P) C, H, N.
(E)-9-{[(2,2-Hydroxymethyl)cyclopropylidene]methyl}-
adenine (Methylphenylphosphoryl) P f N-L-Alaninate
(8b). A protocol for the preparation of Z-isomer 7b was
followed with compound 21d (900 mg, 2.72 mmol). The crude
product was first chromatographed using CH2Cl2/MeOH (30:1
to 20:1 to 10:1) as an eluent and, after treatment with 80%
acetic acid, in ethyl acetate/MeOH (10:1) to give E-phospho-
roalaninate 8b (380 mg, 29%) as a solid. UV λmax: 278 nm (ꢀ
1
9300), 262 (ꢀ 12 900), 227 (ꢀ 28 900), 210 (ꢀ 27 900). H NMR
δ: 8.48 (s, 1H, H8), 8.18 (s, 1H, H2), 7.52 (bs, 1H, H1′), 7.35
and 7.18 (2m, 7H, PhO, NH2), 5.99 (m, 1H, NH of Ala), 4.94
(m, 1H, OH), 4.13 and 4.04 (2m), 3.85 (m), 3.59-3.48 (m
overlapped with 4s, 8H, H5′, H5′′, CH of Ala, CH3O), 1.71 (m,
2H, H3′), 1.21 (m, 3H, CH3 of Ala). 13C NMR: 174.4 (CO), 156.7
(C6), 153.8 (C2), 151.5 (Cipso, PhO), 149.0 (C4), 137.8 (C8), 130.2
1
nm (ꢀ 11 800), 230 (ꢀ 28 400). H NMR δ: 10.71 (s, 1H, NH),

98 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Yan et al.
(Cpara, PhO), 125.2 (Cortho, PhO), 121.0, 120.9 (Cmeta, PhO), 119.1
(C5), 117.4 (C2′), 111.9 (C1′), 67.9 (C5′′), 62.6 (C5′), 52.5 (CH3O),
50.5, 50.4 (CH of Ala), 27.5, 27.4 (C4′), 20.4, 20.3 (CH3 of Ala),
15.0 (C3′). 31P NMR: 4.66, 4.61, 4.26, 4.22 (1.3:1.0:1.6:1.1). ESI-
MS: 230 (M - OP(O)(OPh)NHCHMeCO2Me, 57.7), 489 (M +
H, 34.9), 511 (M + Na, 100.0). Anal. (C21H25N6O6P) C, H, N.
(78.1, M + H), 348 (100.0, M + Na). Anal. (C11H12N5O5P‚
0.4H2O) C, H, N.
(E)-9-{[2-(Acetoxymethyl)-2-(phosphinyloxymethyl)-
cyclopropylidene]methyl}guanine (22b). The procedure
described for the Z-isomer 22a was followed with E-isomer 20b
(1.50 g, 4.91 mmol) to give phosphite 22b (1.59 g, 73%
containing 0.73 mol of triethylamine28). UV (EtOH) λmax: 271
nm (ꢀ 9200), 231 (ꢀ 25 600). 1H NMR (D2O) δ: 7.94 (s, 1H,
(Z)-9-{[2-(Acetoxymethyl)-2-(phosphinyloxymethyl)-
cyclopropylidene]methyl}guanine (22a). A suspension of
compound 20a (1.0 g, 3.27 mmol) in pyridine (90 mL) was
sonicated for 10 min. Diphenyl phosphite (85-90%, 4.5 mL,
20 mmol) was then added at room temperature. The resulting
mixture was stirred at 60°C for 2 h. After the mixture was
cooled, triethylamine (15 mL) and water (10 mL) were added,
and the stirring was continued for 30 min. The volatile
components were then evaporated in vacuo and the crude
product was chromatographed on a silica gel column CH2Cl2/
MeOH ) 3:1 to 1.5:1) to furnish 1.13 g of phosphite 22a (1.13
1
H8), 7.19 (d, 1H, J ) 2.4 Hz, H1′), 6.74 (d, 1H, JH,P ) 636.5
Hz, P-H), 4.22 and 4.18 (AB, 2H, J ) 12.0 Hz, CH2OAc), 3.94
(m, 2H, CH2OPO2H2), 3.12 (q, J ) 7.3 Hz, CH2 of NEt3), 2.10
(s, 3H, Ac), 1.76 (poorly resolved m, 2H, H3′), 1.19 (t, J ) 7.4
Hz, CH3 of NEt3). 13C NMR: 174.5 (CO), 158.2 (C6), 153.9 (C2),
149.8 (C4), 135.9 (C8), 117.3 (C5), 115.2 (C2′), 111.9 (C1′), 66.3,
65.7 (C5′, C5′′), 46.8 (CH2 of NEt3), 24.15 (3J4′,P ) 8.1 Hz, C4′),
20.6 (Ac), 15.1 (C3′), 8.4 (CH3 of NEt3). 31P NMR: 7.12. Negative
ESI-MS: 368 (100.0, M - H).
(E)-9-{[2,2-Bis(hydroxymethyl)cyclopropylidene]meth-
yl}guanine Phosphate (11b). The procedure described for
the Z-isomer 11a was followed with the E-isomer 22b (235
mg, 0.53 mmol) to afford phosphate 11b (137 mg, 75%). Mp:
220 °C (dec). UV (H2O) λmax: 267 nm (ꢀ 12 700), 228 (ꢀ 34 000).
1H NMR (D2O, sodium salt) δ: 7.97 (s, 1H, H8), 7.29 (poorly
resolved d, 1H, H1′), 4.00, 3.75, 359 (poorly resolved AB
systems, 4H, H5′, H5′′), 1.62 and 1.54 (AB, JAB ) 8.8 Hz, 2H,
H3′). 13C NMR: 158.2 (C6), 153.7 (C2), 149.2 (C4), 135.4 (C8),
118.1 (C5), 115.0 (C2′), 110.5 (C1′), 66.0 (poorly resolved d, C5′),
63.4 (C5′′), 26.8 (3J4′,P ) 9.7 Hz, C4′), 14.3 (C3′). 31P NMR: 5.25.
ESI-MS: 344 (100.0, M + H). Anal. (C11H14N5O6P) C, H, N.
(E)-9-{[2,2-Bis(hydroxymethyl)cyclopropylidene]meth-
yl}guanine Cyclic Phosphate (10b). The procedure de-
scribed for the Z-isomer 10a was followed with E-isomer 11b
(240 mg, 0.70 mmol) to give cyclic phosphate 10b (202 mg,
89%). For analysis, it was recrystallized from H2O. Mp: 265
°C (dec). UV (H2O) λmax: 268 nm (ꢀ 12 400), 228 (ꢀ 33 900). 1H
NMR (D2O, sodium salt, 400 MHz) δ: 8.03 (s, 1H, H8), 7.40 (s,
1H, H1′), 4.31 and 4.08 (2 apparent t, J ) 10.2 and 12.6 Hz,
4H, H5′, H5′′), 1.84 (s, 2H, H3′). 13C NMR: (D2O, sodium salt,
100 MHz) 168.1 (C6), 161.3 (C2), 150.0 (C4), 134.2 (C8), 117.2
(C5), 113.8 (C2′), 112.0 (C1′), 71.8 (d, 2J5′,5′′,P ) 4.8 Hz, C5′′, C5′′),
22.0 (d, 3J4′,P ) 5.9 Hz, C4′), 15.2 (C3′). 31P NMR (D2O, sodium
salt): -2.16. Negative ESI-MS: 325 (100.0, M - H). Anal.
(C11H12N5O5P‚0.5H2O) C, H, N.
1
g, 80%) containing 0.63 mol of triethylamine according to H
NMR spectra.28 UV (EtOH) λmax: 272 nm (ꢀ 9400), 231 nm (ꢀ
1
23 200). H NMR (D2O) δ: 8.01 (s, 1H, H8), 6.94 (s, 1H, H1′),
6.68 (d, 1H, 1JH,P ) 635.7 Hz, P-H), 4.40 and 3.93 (AB, 2H, J
) 12.0 Hz, CH2OAc), 3.97 (d, 2H, 3JP-H ) 7.2 Hz, CH2OPO2H2),
3.12 (q, J ) 7.3 Hz, CH2 of NEt3), 1.93 (s, 3H, Ac), 1.62 and
1.56 (AB, 2H, JAB ) 8.6 Hz, H3′), 1.56 (d, J ) 7.8 Hz, CH3 of
NEt3). 13C NMR: 173.8 (CO), 158.4 (C6), 153.8 (C2), 150.0 (C4),
136.6 (C8), 118.6 (C5), 115.5 (C2′), 111.9 (C1′), 66.6, 65.7 (C5′,
C5′′), 46.8 (CH2 of NEt3), 26.0 (d, 3J4′,P ) 8.9 Hz, C4′), 20.4 (CH3),
12.7 (H3′), 8.5 (CH3 of NEt3). 31P NMR: 6.96. ESI-MS: 370
(100.0, M + H), 392 (28.0, M + Na).
(Z)-9-{[2,2-Bis(hydroxymethyl)cyclopropylidene]meth-
yl}guanine Phosphate (11a). A suspension of partial tri-
ethylammonium salt 22a (193 mg, 0.445 mmol) and imida-
zole29 (151 mg, 2.22 mmol) in pyridine (30 mL) was sonicated
for 10 min. Trimethylsilyl chloride (0.56 mL, 4.40 mmol) was
added, and stirring at room temperature was continued for
10 min. After addition of iodine (227 mg, 0.89 mmol) in
pyridine (2 mL), the stirring was continued for 10 min. Water
(10 mL) was then added, and after 40 min, the volatile
components were evaporated in vacuo and the residue was
dissolved in water (150 mL). The aqueous solution was
extracted with CH2Cl2 (5 × 40 mL), NH4OH (30%, 20 mL) was
added to the aqueous layer, and the mixture was kept for 24
h at room temperature. The volatile components were evapo-
rated, the residue was dissolved in water (5 mL), the pH was
adjusted to 9-9.5 with NH4OH, and the solution was loaded
onto the top of a Dowex 50 column (WX2, 200 mesh, H(+) form,
40 mL). Elution with water followed by concentration of the
appropriate fractions gave phosphate 11a (131 mg, 86%). Mp:
260 °C (dec). UV (H2O) λmax: 269 nm (ꢀ 10 600), 229 (ꢀ 26 700).
1H NMR (D2O, sodium salt) δ: 8.40 (s, 1H, H8), 7.06 (s, 1H,
H1′), 4.17, 3.79 and 4.16, 3.81 (2AB, J ) 10.6 Hz, 2H, H5′), 3.87
and 3.64 (AB, JAB ) 12.2 Hz, 2H, H5′′), 1.53 and 1.50 (AB, 2H,
JAB ) 8.7 Hz, H3′). 13C NMR: 158.5 (C6), 153.9 (C2), 149.4 (C4),
136.7 (C8), 118.7 (C5), 115.4 (C2′), 110.8 (C1′), 66.2 (C5′′), 63.2
(C5′), 29.0 (3J4′,P ) 8.2 Hz, C4′), 11.8 (C3′). 31P NMR: 4.75. ESI-
MS: 344 (100.0, M + H), 366 (32.1, M + Na). Anal.
(C11H14N5O6P) C, H, N.
Hydrolysis of Pronucleotides with Porcine Liver Es-
terase (PLE). 1. TLC Analysis. Pronucleotide 7a, 7b, 8a, or
8b (0.75 mg, 1.5 µmol) was stirred with PLE (200 units) in
0.02 M Na2HPO4 (pH 7.4, 1.0 mL) at room temperature. The
reaction was periodically checked by TLC in CH2Cl2/MeOH )
5:1 (7a, 8a) or 7:1 (7b, 8b). Quantitative hydrolysis was
observed after 24 h (7a, 8a), 40 h (7b), and 3 h (8b).
2. Analysis by 31P NMR Spectra.30 Pronucleotide 7a (7.5
mg, 15 µmol) was incubated with PLE (2,000 units) in 0.5 M
TRIZMA‚HCl in water (pH 7.6, 0.8 mL) containing acetone
(0.1 mL) at room temperature. After 72 h, three signals in the
31P NMR spectrum were observed: 3.53 (corresponding to a
peak observed in the enzyme solution alone), 7.92, and 8.04
(diastereoisomers of phosphoramidate 23).
Biological Assays. The antiviral assays were performed
as described previously.31 The HCMV assays were run in HFF
culture with Towne and AD169 strains of virus in a plaque
reduction or cytopathic effect (CPE) inhibition assay. The
MCMV was assayed in MEF cells by plaque reduction. The
HSV-1 was run in BSC-1 cells by ELISA. In addition, HSV-1
and HSV-2 assays were performed in HFF (CPE and plaque
reduction) and Vero cells (plaque reduction assay). The VZV
was assayed in HFF (CPE or plaque reduction) and hepatitis
B virus (HBV) in 2.2.15 cells by DNA hybridization. The EBV
assays were performed in Daudi cells by viral capsid antigen
(VCA) ELISA and in H-1 cells by DNA hybridization assay.
The cytotoxicity assays were performed in HFF, KB, and CEM
cells. For further details, see Tables 1-3. All assays are results
of single experiments unless specified otherwise.
(Z)-9-{[2,2-Bis(hydroxymethyl)cyclopropylidene]meth-
yl}guanine Cyclic Phosphate (10a). A mixture of phosphate
11a (140 mg, 0.40 mmol), DCC (560 mg, 2.7 mmol), and N,N′-
dicyclohexyl-4-morpholinecarboxamidine (140 mg, 0.47 mmol)
in pyridine (100 mL) was refluxed under N2 with stirring for
10 h. Pyridine was evaporated, and the crude product was
worked up as described for phosphate 11a to give cyclic
phosphate 10a (123 mg, 93%). For analysis, it was recrystal-
lized from H2O. Mp: 250 °C (dec). UV (H2O) λmax: 266 nm (ꢀ
1
13 500), 228 (ꢀ 29 000). H NMR (D2O, sodium salt) δ: 7.88
(s, 1H, H8), 6.90 (s, 1H, H1′), 4.22 and 4.13 (2 apparent t, J )
11.0 and 12.2 Hz, 4H, H5′, H5′′), 1.51 (s, 2H, H3′). 13C NMR:
165.0 (C6), 158.9 (C2), 150.2 (C4), 135.9 (C8), 116.8, 116.5 (C5,
2
Acknowledgment. We thank L. M. Hrihorczuk
from the Central Instrumentation Facility, Department
C2′), 111.7 (C1′), 71.94 (d, J5′,5′′,P ) 5.2 Hz, C5′, C5′′), 24.0 (d,
3J4′,P ) 5.9 Hz, C4′), 11.5 (C3′). 31P NMR: - 2.23. ESI-MS: 326

Antiviral Nucleotides and Pronucleotides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 99
(10) Germershausen, J.; Bostedor, R.; Liou, R.; Field, A. K.; Wagner,
A. F.; MacCoss M.; Tolman, R. L.; Karakas, J. D. Comparison
of the Modes of Action of 2′-Nor-deoxyguanosine and Its Cyclic
Phosphate, 2′-Nor-cyclic GMP. Antimicrob. Agents Chemother.
1986, 29, 1025-1031.
(11) Tse, H. L. A.; Knight, D. J.; Coates, J. A. V.; Mansour, T. S.
Constrained Analogues of 2′-Nor Cyclic Nucleoside Monophos-
phates. Bioorg. Med. Chem. Lett. 1997, 7, 1387-1392.
(12) Kitagawa, O.; Inoue, T.; Hirano, K.; Taguchi, T. Ionic Iodocar-
bocyclization Reactions of 4-Alkenyl- and 4-Alkylmalonate De-
rivatives. J. Org. Chem. 1993, 58, 3106-3112.
(13) The reaction was performed on a much larger (>1000 times)
scale using diethyl instead of dimethyl allylmalonate and
Ti(i-PrO)4 instead of Ti(t-BuO)4 as the catalyst. The reported12
yield of 13 (dimethyl ester) was 48%.
(14) Gao, H.; Mitra, A. K. Regioselective Synthesis of Various
Prodrugs of Ganciclovir. Tetrahedron Lett. 2000, 41, 1131-1136.
(15) Winter, H.; Maeda, Y.; Mitsuya, H.; Zemlicka, J. Phosphodiester
Amidates of Allenic Nucleoside Analogues: Anti-HIV Activity
and Possible Mechanism of Action. J. Med. Chem. 1996, 39,
3300-3306.
of Chemistry, Wayne State University (D. M. Coleman,
Director) for mass spectra. Our thanks are also due to
K. B. Birnbaum, National Research Council of Canada,
Ottawa, Canada and D. Shugar, Institute of Biochem-
istry and Biophysics, Polish Academy of Sciences,
Warszawa, Poland for discussion of the structure of
ganciclovir cyclic phosphate 9. The work described
herein was supported by U. S. Public Health Service
grants RO1-CA32779 (J.Z.) and RO1-CA44358 (Y.-C.C.)
from the National Cancer Institute, contracts NO1-
AI85347 and NO1-30049 (E.R.K.), and program project
PO1-AI46390 (J.C.D.) from the National Institute of
Allergy and Infectious Diseases, National Institutes of
Health, Bethesda, MD.
Supporting Information Available: Elemental analyses.
This information is available online free of charge via the
Internet at http://pubs.acs.org.
(16) Wang, R.; Corbett, T. H.; Cheng, Y.-C.; Drach J. C.; Kern, E.;
Mitsuya, H.; Zemlicka, J. Tryptophanyl Phosphoramidates as
Prodrugs of Synadenol and Its E-Isomer. Bioorg. Med. Chem.
Lett. 2002, 12, 2467-2470.
References
(1) (a) Zhou, S.; Breitenbach, J. M.; Borysko, K. Z.; Drach, J. C.;
Kern, E. R.; Gullen, E.; Cheng, Y.-C.; Zemlicka, J. Synthesis and
Antiviral Activity of (Z)- and (E)-2,2-[Bis(hydroxymethyl)cyclo-
propylidene]methylpurines and -pyrimidines: Second-Genera-
tion Methylenecyclopropane Analogues of Nucleosides. J. Med.
Chem. 2004, 47, 566-575. (b) Kushner, N. L.; Williams-Aziz, S.
L.; Hartline, C. B.; Harden, E. A.; Zhou, S.; Yan, Z.; Zemlicka,
J.; Kern, E. R. In Vitro Activity of Second Generation Methyl-
enecyclopropane Analogs of Nucleosides against Herpesvirus
Replication. Antiviral Res. 2004, 62, A68. (c) Quenelle, D. C.;
Bidanset, D. J.; Collins, B. P.; Herrod, B. P.; Hartline, C. B.;
Yan, Z.; Zemlicka, J.; Kern, E. R. Effect of Oral Treatment with
a Methylenecyclopropane Analog, ZSM-I-62, on Replication of
Human Cytomegalovirus (HCMV) or Murine Cytomegalovirus
(MCMV) Infection in Animal Models. Antiviral Res. 2004, 62,
A56. (d) Prichard, M. N.; Williams, A. D.; Hartline, C. B.; Yan,
Z.; Zemlicka, J.; Britt, W. A.; Kern, E. R. Identification of the
Mode of Action of Methylenecyclopropane Analogs Using Chemo-
typic Clustering Analysis. Antiviral Res. 2004, 62, A56.
(2) Qiu, Y.-L.; Ptak, R. G.; Breitenbach, J. M.; Lin, J.-S.; Cheng,
Y.-C.; Drach, J. C.; Kern, E. R.; Zemlicka, J. Synthesis and
Antiviral Activity of Phosphoralaninate Derivatives of Methyl-
enecyclopropane Analogues of Nucleosides. Antiviral Res. 1999,
43, 37-53.
(3) Uchida, H.; Kodama, E. N.; Yoshimura, K.; Maeda, Y.; Kosala-
raksa, P.; Maroun, V.; Qiu, Y.-L.; Zemlicka, J.; Mitsuya, H. In
Vitro Anti-Human Immunodeficiency Virus Activities of Z- and
E-Methylenecyclopropane Nucleoside Analogues and Their Phos-
phoro-L-Alaninate Diesters. Antimicrob. Agents Chemother.
1999, 43, 1487-1490.
(4) Yoshimura, K.; Feldman, R.; Kodama, E.; Kavlick, M. F.; Qiu,
Y.-L.; Zemlicka, J.; Mitsuya, H. In Vitro Induction of Human
Immunodeficiency Virus Type 1 Variants Resistant to Phospho-
ralaninate Prodrugs of Z-Methylenecyclopropane Nucleoside
Analogues. Antimicrob. Agents Chemother. 1999, 43, 2479-2483.
(5) Rybak, R. J.; Hartline, C. B.; Qiu, Y.-L.; Zemlicka, J.; Harden,
E.; Marshall, G.; Sommadossi, J.-P.; Kern, E. R. In Vitro
Activities of Methylenecyclopropane Analogues of Nucleosides
and Their Phosphoralaninate Prodrugs against Cytomegalovirus
and Other Herpesvirus Infections. Antimicrob. Agents Chemoth-
er. 2000, 44, 1506-1511.
(17) Wang, R.; Harada, S.; Mitsuya, H.; Zemlicka, J. Inhibition of
Human Immunodeficiency Virus Reverse Transcriptase by Syn-
adenol Triphosphate and Its E-Isomer. J. Med. Chem. 2003, 46,
4799-4802.
(18) Smith, M.; Drummond, G. I.; Khorana, H. G. Cyclic Phosphates.
IV. Ribonucleoside-3′,5′ Cyclic Phosphates. A General Method
of Synthesis and Some Properties. J. Am. Chem. Soc. 1961, 83,
698-706.
(19) Zemlicka, J. Lipophilic Phosphoramidates as Antiviral Pronucle-
otides. Biochim. Biophys. Acta 2002, 1587, 276-286.
(20) Baldanti, F.; Sarasini, A.; Drach, J. C.; Zemlicka, J.; Gerna, G.
Z-Isomers of Hydroxymethylcyclopropylidenemethyladenine (Syn-
adenol) and Guanine (Synguanol) Are Active Against Ganciclo-
vir- and Foscarnet-Resistant Human Cytomegalovirus UL97
Mutants. Antiviral Res. 2002, 56, 273-278.
(21) Wagner, C. R.; Iyer, V. V.; McIntee, E. J. Pronucleotides: Toward
the In Vivo Delivery of Antiviral and Anticancer Nucleotides.
Med. Res. Rev. 2000, 20, 417-451.
(22) Chen, X.; Kern, E. R.; Drach, J. C.; Gullen, E.; Cheng, Y.-C.;
Zemlicka, J. Structure-Activity Relationships of (S, Z)-2-Ami-
nopurine Methylenecyclopropane Analogues of Nucleosides.
Variation of Purine-6 Substituents and Activity against Herp-
esviruses and Hepatitis B Virus. J. Med. Chem. 2003, 46, 1531-
1537.
(23) Birnbaum, K. B.; Stolarski, R.; Shugar, D. Structure and
Conformation of the Cyclic Phosphate of Ganciclovir, a Broad-
Spectrum Antiviral Agent. Biochim. Biophys. Acta 1994, 1200,
55-63.
(24) Birnbaum, K. B. Crystal Structures of Some Acyclonucleosides
with Antiviral Activity and Related Compounds. Acta Biochim.
Polon. 1996, 43, 65-76.
(25) Beckwith, A. L. J.; Tozer, M. J. The Iodination and Iodocycliza-
tion of Some Alkenylmalonates. Tetrahedron Lett. 1992, 33,
4975-4978.
(26) This reagent was obtained from commercial sources or it was
prepared as described: Reich, H. J.; Cohen, M. L.; Clark, P. S.;
Vinogradoff, A.; Lee, A. W. M.; Stevens, R. V. Reagents for
Synthesis of Organoselenium Compounds: Diphenyl Diselenide
and Benzeneselenyl Chloride. Org. Synth., Coll. Vol. VI (Noland,
W. E., Ed.), Wiley: New York, 1988; p. 533-537.
(27) In subsequent experiments, the residual KMnO4 in 15 was
removed by washing the organic phase with H2O2 solution prior
to evaporation of the solvents.
(6) Qiu, Y.-L.; Geiser, F.; Kira, T.; Gullen, E.; Cheng, Y.-C.; Ptak,
R. G.; Breitenbach, J. M.; Drach, J. C.; Hartline, C. B.; Kern, E.
R.; Zemlicka, J. Synthesis and Enantioselectivity of the Antiviral
Effects of (R, S)-,(S, Z)-Methylenecyclopropane Analogues of
Purine Nucleosides and Phosphoralaninate Prodrugs: Influence
of Heterocyclic Base, Type of Virus and Host Cells. Antiviral.
Chem. Chemother. 2000, 11, 191-202.
(7) Tolman, R. L.; Field, A. K.; Karkas, J. D.; Wagner, A. F.;
Germershausen, J.; Crumpacker, C.; Scolnick, E. M. 2′-nor-
cGMP: A seco-Cyclic Nucleotide with Powerful Anti-DNA-Viral
Activity. Biochem. Biophys. Res. Commun. 1985, 128, 1329-
1335.
(8) Duke, A. E.; Smee, D. F.; Chernow, M.; Boehme, R.; Matthews,
T. R. In Vitro and In Vivo Activities of Phosphate Derivatives
of 9-(1,3-Dihydroxy-2-propoxymethyl)-guanine against Cytome-
galovirus. Antiviral Res. 1986, 6, 299-308.
(9) Oliver, S.; Bubley, G.; Crumpacker, C. Inhibition of HSV-
Transformed Murine Cells by Nucleoside Analogs, 2′-NDG and
2′-Nor-cGMP. Mechanism of Inhibition and Reversal by Exog-
enous Nucleosides. Virology 1985, 145, 84-93.
(28) Calculated from integration of CH2 and CH3 signals of triethy-
lamine.
(29) Absence of imidazole led to lower yields of phosphate 11a.
(30) McGuigan, C.; Bidois, L.; Hiouni, A.; Ballatore, C.; De Clercq,
E.; Balzarini, J. Antiviral Chem. Chemother. 2000, 11, 111-116.
(31) Qiu, Y.-L.; Ksebati, M. B.; Ptak, R. G.; Fan, B. Y.; Breitenbach,
J. M.; Lin, J.-S.; Cheng, Y.-C.; Kern, E. R.; Drach, J. C.; Zemlicka,
J. (Z)- and (E)-2-(Hydroxymethyl)cyclopropylidenemethyladenine
and -guanine. New Nucleoside Analogues with a Broad Spectrum
of Antiviral Activity. J. Med. Chem. 1998, 41, 10-23.
JM040149B