Phosphoralaninate pronucleotides of pyrimidine methylenecyclopropane analogues of nucleosides: Synthesis and antiviral activity

Article abstract of DOI:10.1080/15257770500266867

□ The Z- and E-thymine and cytosine pronucleotides 3d, 4d, 3e, and 4e of methylenecyclopropane nucleosides analogues were synthesized, evaluated for their antiviral activity against human cytomegalovirus (HCMV), herpes simplex virus 1 and 2 (HSV-1 and HSV

Full text of DOI:10.1080/15257770500266867

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Phosphoralaninate Pronucleotides of Pyrimidine
Methylenecyclopropane Analogues of Nucleosides:
Synthesis and Antiviral Activity
Amalraj Ambrose a , Jiri Zemlicka a , Earl R. Kern b , John C. Drach c , Elizabeth Gullen d
Yung-Chi Cheng d
&
a Department of Chemistry , Developmental Therapeutics Program, Barbara Ann Karmanos
Cancer Institute, Wayne State University School of Medicine , Detroit, Michigan, USA
b Department of Pediatrics , The University of Alabama School of Medicine , Birmingham,
Alabama, USA
c Department of Biologic and Materials Sciences , School of Dentistry, University of
Michigan , Ann Arbor, Michigan, USA
d Department of Pharmacology , Yale University School of Medicine , New Haven,
Connecticut, USA
Published online: 16 Aug 2006.
To cite this article: Amalraj Ambrose , Jiri Zemlicka , Earl R. Kern , John C. Drach , Elizabeth Gullen & Yung-Chi Cheng (2005)
Phosphoralaninate Pronucleotides of Pyrimidine Methylenecyclopropane Analogues of Nucleosides: Synthesis and Antiviral
Activity, Nucleosides, Nucleotides and Nucleic Acids, 24:10-12, 1763-1774, DOI: 10.1080/15257770500266867
To link to this article: http://dx.doi.org/10.1080/15257770500266867
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Nucleosides, Nucleotides, and Nucleic Acids, 24:1763–1774, 2005
c
Copyright ꢀ Taylor & Francis Group, LLC
ISSN: 1525-7770 print/1532-2335 online
DOI: 10.1080/15257770500266867
PHOSPHORALANINATE PRONUCLEOTIDES OF PYRIMIDINE
METHYLENECYCLOPROPANE ANALOGUES OF NUCLEOSIDES:
Synthesis and Antiviral Activity
ᮀ
Amalraj Ambrose and Jiri Zemlicka
Department of Chemistry, Developmental
Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Wayne State University
School of Medicine, Detroit, Michigan, USA
ᮀ
Earl R. Kern
Department of Pediatrics, The University of Alabama School of Medicine,
Birmingham, Alabama, USA
ᮀ
John C. Drach
Department of Biologic and Materials Sciences, School of Dentistry,
University of Michigan, Ann Arbor, Michigan, USA
ᮀ
Elizabeth Gullen and Yung-Chi Cheng
Department of Pharmacology, Yale
University School of Medicine, New Haven, Connecticut, USA
ᮀ
The Z- and E-thymine and cytosine pronucleotides 3d, 4d, 3e, and 4e of methylenecyclo-
propane nucleosides analogues were synthesized, evaluated for their antiviral activity against
human cytomegalovirus (HCMV), herpes simplex virus 1 and 2 (HSV-1 and HSV-2), varicella
zoster virus (VZV), Epstein-Barr virus (EBV), human immunodeficiency virus type 1 (HSV-1),
and hepatitis B virus (HBV) and their potency was compared with the parent compounds 1d,
2d, 1e, and 2e. Prodrugs 3d and 4d were obtained by phosphorylation of parent analogues 1d
or 2d with reagent 8. A similar phosphorylation of N4-benzoylcytosine methylenecyclopropanes 9a
and 9b gave intermediates 11a and 11b. Deprotection with hydrazine in pyridine–acetic acid
gave pronucleotides 3e and 4e. The Z-cytosine analogue 3e was active against HCMV and EBV.
The cytosine E-isomer 4e was moderately effective against EBV.
Keywords Antivirals; HCMV; EBV; VZV; Methylenecyclopropane analogues; Pronu-
cleotides; Phenyl phosphoralaninates; Prodrugs
This paper is dedicated to the memory of John A. Montgomery.
Received 28 December 2004; accepted 28 April 2005.
We thank L.M. Hrihorczuk from the Central Instrumentation Facility, Department of Chemistry,
Wayne State University (D. M. Coleman, Director) for mass spectra and Katherine Z. Borysko for the
HCMV (Towne) and cytotoxicity assays. The work described herein was supported by program project
PO1-AI46390 (J.C.D.) and contracts NO1-AI85347 and NO1-30049 (E.R.K.) from the National Institute
of Allergy and Infectious Diseases, and U.S. Public Health Service grant RO1-CA44358 (Y.-C.C.) from
the National Cancer Institute, National Institutes of Health, Bethesda, Maryland.
Address correspondence to Jiri Zemlicka, Department of Chemistry, Developmental Therapeutics
Program, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit,
Michigan 48201-1379. Fax: (313) 832-7294; E-mail: zemlicka@kci.wayne.edu
1763

1764
INTRODUCTION
A. Ambrose et al.
In recent years, much of our attention has been focused on methylenecy-
clopropane analogues of nucleosides as antiviral agents.[1,2] In this group
of compounds, the biological effects are mostly displayed by the purine
Z-isomers 1, whereas E-isomers 2 or pyrimidine derivatives 1 and 2 are gener-
ally less effective or inactive. In several cases, the antiviral efficacy of purine
analogues was increased by transformation to phenyl phosphoralaninate
pronucleotides.[3,4] This effect was most striking in adenine and 2,6-diami-
nopurine analogues 3a and 3b where a 300–500 times increase of anti-HIV
potency relative to parent analogues 1a and 1b was noted. A significant
potentiation of antiviral effects of the E-isomer 2a following a transforma-
tion to phenyl phosphoralaninate 4a was also observed.[5,6] In addition,
pronucleotides 3a and 3b have favorable cross-resistance patterns[7] with
anti-HIV nucleoside analogues and in case of 2-amino-6-methoxypurine ana-
logue 3c in vivo effect against murine cytomegalovirus (MCMV) was noted.[8]
As indicated above, the pyrimidine methylenecyclopropanes 1 and 2
were largely devoid of antiviral effects but some exceptions were observed.[9]
Thus, thymine analogue 1d was effective against herpes simplex virus type
1 (HSV-1/BSC-1 ELISA) with EC50/CC50 2.0/>100 µM, Epstein-Barr virus
(EBV/H-1 and varicella zoster virus [VZV/HFF, Table 1]). Cytosine ana-
logues 1e and 2e were equally potent against EBV in Daudi culture. The Z-
isomer 1e was also active against EBV/H-1, varicella zoster virus (VZV/HFF),
and it displayed some potency against human cytomegalovirus (HCMV).
Also, transformation of pyrimidine anti-HIV agents, thymidine analogues zi-
dovudine (AZT) and stavudine (d4T), into the corresponding phenyl phos-
phoralaninates provided effective and non-cytotoxic antivirals independent
on the first phosphorylation step (“kinase bypass”).[10] Investigation of the
pyrimidine phosphoralaninate analogues 3 and 4 is then of interest. In
this communication, synthesis and antiviral activity of thymine and cytosine
pronucleotide analogues 3d, 3e, 4d, and 4e are described.
RESULTS AND DISCUSSION
Synthesis
Synthymol (1d) and the E-isomer 2d served as convenient starting ma-
terials for pronucleotides 3d and 4d. Previously,[9,11] both isomers were
obtained by an alkylation of 2,4-bis-O-trimethylsilyl-5-methylpyrimidine (5)
with ethyl 2-bromo-2-bromomethylcyclopropropane carboxylate (6a) fol-
lowed by β-elimination and reduction (Scheme 1). We have now simplified
this protocol using acetate 6b as an alkylating agent.[12,13] Intermediate 7
was obtained in 66% yield. The reduction step was eliminated and base-

Nucleoside Analogues and Antiviral Activity
1765
TABLE 1 Inhibition of HCMV, EBV, and VZV Replication by Phosphoralaninate Pronucleotides of
Pyrimidine Methylenecyclopropane Analogues 3d, 3e, 4d, 4e and Parent Analogues 1d, 1e, 2d, and 2e
EC50/CC50 (µM)
HCMV/HFF
EBV
EC50
Compounda
Towneb,c
AD169d,e
Daudif
H-1g
VZV/HFFb,h
3d
1d
4d
2d
3e
1e
4e
2e
Control
>100/>100
>100/>100
>100/>100
>100/>100
1.5/100
28.5/>100
>100/>100
>100/>100
1.7/>100k
>300/>300
>480/437
243/>300
>96/>480
29.8/>300
3.4/>518
>60/>300
>518/>518
0.22/40k
>100/>100
>20/45
>10/>50j
>20/>100
>10/>50j
16/74
69.3
3.6
1.3/>240i
>100/>100
>240/>240i
0.65/>100
<0.41/>259i
14.4/>100
<0.41/>259i
0.73l
>300d
>100
56.2
2.5/>50j
12/>100
>50/>50j
5k
3.6
92.4
>518
0.22l
aMost of the results obtained with parent analogues 1d, 1e, 2d, and 2e were taken from Qiu et al.;[9]
the rest are new data.
bPlaque reduction assay.
cVisual cytotoxicity.
dCytopathic effect (CPE) inhibition assay.
eStationary HFF cells. Cytotoxicity was determined by neutral red uptake.
fViral capsid antigen (VCA) ELISA.
gDNA hybridization assay. Cytotoxicity was determined in CEM cells unless stated otherwise.
hFor CC50 values see HCMV(AD169)/HFF.
iViral capsid antigen immunofluorescence (VCA-IF) assay.
jCytotoxicity was determined in H-1 cells.
kGanciclovir. EC50 only.
lAcyclovir. EC50 only.
catalyzed elimination of elements of HBr from 7 combined with deacetyla-
tion afforded after chromatographic separation[9] Z- and E-isomers 1d and
2d in 33 and 19% yield, respectively. Phosphorylation with reagent 8 then
afforded pronucleotides 3d and 4d in 54 and 42% yield, respectively.
Although cytidine analogue lamivudine (3TC) was directly phosphory-
lated using tert-butylmagnesium chloride and reagent 8 in THF[14] it was
more convenient to start with N4-benzoylcytosine analogues 9a and 9b
(Scheme 2). Both compounds were intermediates in synthesis[9] of the Z-
and E-isomers 1e and 2e and, unlike a mixture of 1e + 2e, they were sepa-
rated by chromatography. Thus, alkylation-elimination of N4-acetylcytosine
(10) with acetate 6b afforded, after deacetylation, a mixture of the (Z,E)-
isomers 1e + 2e in the ratio of 1:2 and 53% yield. The N-benzoylation
and following chromatographic separation provided isomers 9a and 9b in
21 and 41% yield, respectively. Phosphorylation with reagent 8 gave N4-
benzoyl pronucleotides 11a and 11b in 65 and 52% yield, respectively. The
N-debenzoylation was effected with hydrazine in pyridine–acetic acid[15] to
afford pronucleotides 3e and 4e in 29 and 27% yield.

1766
A. Ambrose et al.
SCHEME 1
The 31P NMR spectra indicated the expected presence of four diastereo-
isomers in pronucleotides 3d, 4d, 3e, and 11a whereas in the E-isomers 4e
and 11b only three signals were observed due to overlapping peaks. This
stereoisomerism was also reflected in numerous signals of 1H and 13C NMR
spectra.
SCHEME 2

Nucleoside Analogues and Antiviral Activity
1767
SCHEME 3
Antiviral Activity
The antiviral activity of pyrimidine pronucleotides was restricted to
cytosine analogues 3e and 4e (Table 1). Thus, the Z-cytosine analogue 3e
was effective against HCMV. It was more effective than analogue 1e and as
potent as ganciclovir in Towne virus assay. The activity pattern was reversed
in AD169 strain where 1e was superior to pronucleotide 3e. The E-isomer
4e was devoid of anti-HCMV potency. Pronucleotide 3e was as potent against
EBV in Daudi cells as the parent compound 1e but less so in H-1 culture.
All tested analogues were ineffective against HSV-1, HSV-2, HIV-1 and HBV.
Pronucleotides 3d and 3e were significantly less active against VZV than
the parent compounds 1d and 1e. The E-isomer 4e was moderately active
against EBV in both cell cultures. It was more effective in H-1 culture but
less active in Daudi cells than the parent E-isomer 2e.
Taken together, strong potentiating effects of phenyl phosphoralaninate
group observed in the purine series[5,6] are absent in pronucleotides 3e
and 4e. It has been established[3,4] that two key enzymes, esterase and phos-
phoamidase, are important for intracellular activation of phenyl phospho-
ralaninate pronucleotides (Scheme 3). The esterase action leads to phos-
phoralaninate 12 which is then converted to the respective phosphate 13
by phosphoamidase. All pronucleotides reported herein including the N4-
benzoyl derivatives 11a and 11b were substrates for porcine liver esterase
(PLE) that is considered as a good model of intracellular esterases.[3,4] The
substrate activity followed the lipophilicity pattern: 11a, 11b > 2d, 3d > 2e,
3e. It is then interesting that in several cases the pronucleotides were less
potent than the parent analogues (Table 1). Possibly, a limited affinity of
intermediates 12 toward phosphoamidase enzyme may be responsible for a
decrease of activation effect of pyrimidine methylenecyclopropane pronu-
cleotides.
EXPERIMENTAL SECTION
General Methods
1
The H, 13C, and 31P NMR spectra were determined at 400, 100 and
162 MHz, respectively, in CD3SOCD3 as a solvent unless stated otherwise.

1768
A. Ambrose et al.
The 13C NMR assignments were verified by DEPT spectra. The UV spec-
tra were measured in ethanol. Mass spectrometry (MS) was perfomed in
an electron-impact (EI) or electrospray ionization mode (ESI) on MICRO-
MASS QUATTRO LC-MS instrument in MeOH-KOAc or NaCl. Porcine liver
esterase (PLE) was a product of Sigma, St. Louis, Missouri. The (Z,E)-1-
acetoxymethyl-2-bromo-2-bromoethylcyclopropane (6b) was prepared as
described.[12,13]
(Z,E)-1-{[(Acetoxymethyl)-2-bromocyclopropyl]methyl}thymine (7). A
mixture of 2,4-bis-O-(trimethylsilyloxy)-5-methylpyrimidine[16] (5, 4.62 g, 17
mmol), acetate 6b (4.89 g, 17 mmol) was refluxed in acetonitrile (40 mL)
under N2 for 144 h. After cooling, ethanol (40 mL) was added and solvents
were evaporated. The residue was triturated with CH2Cl2 (250 mL), the
insoluble portion was filtered off using a Celite bed and it was washed with
the same solvent (3 × 20 mL). The combined filtrate and washings were
evaporated and the crude product was chromatographed on silica gel using
CH2Cl2-MeOH (100:0 to 98:2) to give compound 7 as a white solid (3.7 g,
1
66%), mp 95–99◦C. UV λmax 269 nm (ε 10,600), 210 (ε 9,700). H NMR
ꢀ
ꢀ
(CDCl3) δ 1.04 (s) and 1.41 (d, 2H, H3 ), 1.52 (bs, 1H, H4 ), 1.92 (s, 3H,
ꢀ
5-CH3), 2.02, 2.06 (2s, 3H, CH3 of Ac), 3.87–4.05 (m, 2H, H5 ), 4.27–4.47
(m, 2H, H1 ), 7.21, 7.27 (2s, 1H, H6), 10.08 (bs, 1H, NH); 13C NMR 12.6 (5-
ꢀ
ꢀ
ꢀ
ꢀ
CH3), 19.9, 21.0 (C3 ), 21.1 (CH3 of Ac), 22.7, 27.4 (C4 ), 35.0, 38.4 (C2 ),
ꢀ
ꢀ
53.6, 57.6 (C1 ), 63.48, 66.49 (C5 ), 110.4, 110.5 (C5), 141.0, 141.1 (C6),
151.8, 151.9 (C2), 164.8, 164.9 (C4), 170.9, 171.1 (CO of Ac). EI-MS 332 and
330 (M, 9.2 and 9.2), 272 (M-OAc, 66.3), 250 (M-Br, 43.9), 191 (M-Br-OAc,
100.0). EI-HRMS Calcd. for C12H1579BrN2O4: 330.0215; found 330.0220.
Anal. Calcd. for C12H15BrN2O4; C, 43.52; H, 4.57; N, 8.46. Found: C, 43.70;
H, 4.70; N, 8.49.
(Z)- and (E)-1-({[2-Hydroxymethyl)cyclopropylidene]methyl}thymine
(1d) and (2d). Compounds 1d and 2d were prepared by a modification of
the described procedure.[9] A mixture of compound 7 (1.30 g, 3.92 mmol)
and K2CO3 (1.62 g, 11.76 mmol) in DMF (100 mL) was stirred at 100–110◦C
under N2 for 7 h. After cooling, methanol-water (9:1, 25 mL) was added
with stirring continued at room temperature for 1 h. The insoluble portion
was filtered off and it was washed with DMF (2 × 20 mL). The filtrate was
evaporated and the residue was chromatographed on a silica gel column
with hexane-ethyl acetate mixture (2:3 to 3:2) to give the Z-isomer 1d (269
mg, 33%) and E-isomer 2d (155 mg, 19%) as white solids. The 1H NMR and
UV spectra corresponded to those reported previously.[9]
(Z)-1-{[(Hydroxymethyl)cyclopropylidene]methyl}thymine(methylphen-
ylphosphoryl)-P→N-L-alaninate (3d). A suspension of the Z-isomer 1d (288

Nucleoside Analogues and Antiviral Activity
1769
mg, 1.384 mmol) in pyridine (35 mL) was sonicated for 5 min. Phospho-
rochloridate 8 in THF (0.184 M, 38.38 mL, 6.92 mmol) was then added drop-
wise with stirring at room temperature. After addition of 1-methylimidazole
(1.10 mL, 13.84 mmol) the mixture was stirred for 2 h. The solvents were
evaporated at room temperature and the residue was dried in vacuo over-
night. Chromatography on silica gel using CH2Cl2-MeOH (98.5:1.5) gave a
colorless syrup, which slowly solidified. Hexane (10 mL) was added, white
solid 3d (338 mg, 54%) was filtered off, and it was dried in vacuo. UV λmax
287 nm (ε 13,100), 234 nm (ε 14,400), 205 nm (ε 17,800); 1H NMR δ 1.13–
ꢀ
ꢀ
1.27 (m, 4H, CH3 of Ala, H3 ) and 1.44–1.50 (m, 1H, H3 ), 1.76, 1.77, 1.78 (3
ꢀ
poorly resolved d, 3H, 5-CH3), 2.30–2.39 (m, 1H, H4 ), 3.54, 3.55, 3.57 (3s,
ꢀ
3H, OCH3), 3.70–3.91 (m, 2H, H5 ), 4.08–4.27 (m, 1H, CH, Ala), 5.96–6.05
ꢀ
(m, 1H, NH, Ala), 7.10–7.16, 7.20–7.22, and 7.30–7.35 (3m, 6H, Ph, H1 ),
7.78–7.82 (m, 1H, H6), 11.47, 11.48, 11.50 (3s, 1H, NH, Thy); 13C NMR 6.2,
ꢀ
ꢀ
6.3, 6.4 (C3 ), 12.70. 12.73 (CH3, Thy), 16.9, 17.0, 17.1 (C4 ), 20.28, 20.34
(CH3, Ala), 50.2, 50.35, 50.4, 50.5 (CH, Ala), 52.52, 52.53, 52.6 (OCH3),
ꢀ
68.3, 68.6 (C5 ), 110.93, 110.96, 111.01, 111.05 and 111.21, 111.24, 111.3
ꢀ
ꢀ
(C2 and C5), 114.87, 114.93, 115.01, 115.04 (C1 ), 120.55, 120.6, 120.7, 120.8
(Ph, Cmeta), 125.1, 125.2 (Ph, Cortho), 130.2, 130.3 (Ph, Cpara), 136.1, 136.3
(C6), 150.1 (C2), 151.27, 151.30 (Ph, Cipso), 164.3 (C4), 174.5 (CO, Ala);
31P NMR 4.18, 4.30, 4.41, 4.60; ESI-MS 488 (M + K, 100.0), 450 (M + H,
18.5). Anal. Calcd. for C20H24N3O7P: C, 53.45; H, 5.38; N, 9.35. Found: C,
53.41; H, 5.66; N, 9.42.
(E)-1-{[(2-Hydroxymethyl)cyclopropylidene]methyl}thymine(methyl-
phenylphosphoryl)-P→(N-L-alaninate (4d). The experiment was performed
as described for the Z-isomer 1d with E-isomer 2d (187 mg, 0.9 mmol),
pyridine (22 mL), phosphorochloridate 8 in THF (0.184 M, 24.9 mL, 4.5
mmol) and 1-methylimidazole (0.72 mL, 8.99 mmol). Chromatography
afforded a colorless syrup 4d which solidified during drying in vacuo (170
mg, 42%). UV λmax 286 nm (ε 13,100), 233 nm (ε 13,500), 206 nm (ε
1
16,500); H NMR δ 1.21 (t, 3H, CH3 of Ala), 1.48–1.53 (m, 1H) and
ꢀ
ꢀ
1.76–1.79 (m, 1H, H3 ), 1.82 (s, 3H, 5-CH3), 1.97–2.04 (m, 1H, H4 ),
3.57, 3.586, 3.590 (3s, 3H, OCH3), 3.80–3.87 (m, 1H, CH of Ala), 3.90–
ꢀ
4.02 (m, 2H, H5 ), 5.93–6.03 (m, 1H, NH of Ala), 7.13–7.20 (m), 7.29–
ꢀ
7.37 (m, 6H, Ph, H1 ), 7.82 (poorly resolved d, 1H, H6), 11.50 (s, 1H,
13
ꢀ
ꢀ
NH, Thy); C NMR 10.1, 10.2 (C3 ), 12.8 (5-CH3), 14.0 (C4 ), 20.3, 20.4
ꢀ
(CH3), 50.3, 50.5 (CH), 52.5 (OCH3), 68.9 (C5 ), 111.0, 111.6, 111.7
ꢀ
ꢀ
(C2 , C5), 115.1 (C1 ), 120.8, 120.85, 120.9 (Ph, Cmeta), 125.1 (Ph, Cortho),
130.3 (Ph, Cpara), 136.2 (C6), 150.2 (C2), 151.5 (Ph, Cipso), 164.4 (C4),
174.5 (CO, Ala); 31P NMR 4.31, 4.33, 4.66, 4.69; EI-MS 449 (M, 2.1),
191 (M-O(PO)(OPh)NHCH(CH3)CO2CH3, 100.0), 126 (Thy, 26.9); EI-
HRMS calcd for C20H24N3O7P 449.1352, found 449.1347. Anal. Calcd.

1770
A. Ambrose et al.
for C20H24N3O7P: C, 53.45; H, 5.38; N, 9.35. Found: C, 52.63; H, 5.70;
N, 9.14.
(Z,E)-1-{[(2-Hydroxymethyl)cyclopropylidene]methyl}cytosine (1e +
2e). A mixture of N4-acetylcytosine (10, 1.72 g, 11.2 mmol), acetate 6b
(3.21 g, 11.2 mmol) and K2CO3 (9.27 g, 67.2 mmol) in DMF (170 mL)
was heated at 100–110◦C (bath temperature) with stirring under N2 for
13 h. The reaction mixture was cooled to 50◦C and methanol (12 mL) was
added with stirring continued for 7 h. After cooling, the insoluble portion
was filtered off and it was washed with DMF (3 × 20 mL). The filtrate was
evaporated in vacuo and the residue was chromatographed on a silica gel
column using CH2Cl2-MeOH (9:1) to give the title compound 1d + 2d
1
(1.13 g, 53%). The H NMR spectrum (except the isomeric ratio Z/E =
1:2) corresponded to that of the product obtained by another method.[9]
(Z)- and (E)-N4-Benzoyl-1-{[(2-hydroxymethyl)cyclopropylidene]meth-
yl}cytosine (9a) and (9b). Both isomers were prepared as described.[9]
(Z)-N4-Benzoyl-1-{[(2-hydroxymethyl)cyclopropylidene]methyl}cytosine
(Methylphenylphosphoryl)-P→N-L-alaninate (11a). A suspension of Z-iso-
mer 9a (280 mg, 0.94 mmol) in pyridine (30 mL) was sonicated for 5 min.
Phosphorochloridate 8 in THF (0.184 M, 26.1 mL, 4.71 mmol) was then
added dropwise with stirring at room temperature. After addition of 1-
methylimidazole (0.91 mL, 11.4 mmol) the stirring was continued for 2 h.
The solvents were evaporated at room temperature, the oily residue was
dried in vacuo overnight whereupon it was partitioned between ethyl acetate
(250 mL) and water (100 mL). The aqueous phase was extracted with
ethyl acetate (100 mL), the combined organic phase was washed with water
(4 × 120 mL) and brine (2 × 80 mL), It was dried over Na2SO4 and the
solvent was evaporated. Chromatography on silica gel with CH2Cl2-MeOH
(98.5:1.5) gave pronucleotide 11a as a colorless syrup, which solidified
during drying in vacuo. Hexane (10 mL) was added, the white solid was
filtered off and dried in vacuo (330 mg, 65%). UV λmax 330 nm (ε 14,000),
1
270 (ε 19,900), 205 (ε 29,100); H NMR δ 1.15–1.21 (m, 3H, CH3 of Ala),
ꢀ
ꢀ
1.33–1.37 (m, 1H) and 1.54–1.58 (m, 1H, H3 ), 2.38–2.46 (m, 1H, H4 ), 3.53,
3.54, 3.55, 3.57 (4s, 3H, OCH3), 3.70–3.90 (m, 1H, CH of Ala), 3.92–4.05 (m,
ꢀ
1H) and 4.06–4.19 (m, 1H, H5 ), 5.93–6.03 (m, 1H, NH of Ala), 7.14 (dd),
ꢀ
7.30 (dd), 7.41 (bs), 7.50 (t), 7.61 (t) and 7.99 (m, total 11H, Ph, Bz, H1
and H5), 8.36–8.40 (m, 1H, H6), 11.30 and 11.32 (2bs, 1H, BzNH); 13C NMR
ꢀ
ꢀ
6.6, 6.7, 6.8 (C3 ), 16.9, 17.0 (C4 ), 20.2, 20.3 (CH3, Ala), 50.2, 50.35, 50.41,
ꢀ
ꢀ
50.5 (CH, Ala), 52.5, 52.6 (OCH3), 68.2 (C5 ), 97.7 (C5), 116.0 (C2 ), 117.0
ꢀ
(C1 ), 120.69, 120.74, 120.8 (Ph, Cmeta), 125.1 (Ph, Cortho), 128.1, 128.9,
129.2, 130.2 (Ph, Cpara, Bz, Cmeta, Cortho), 133.5, 133.8 (Bz, Cpara, Cipso),

Nucleoside Analogues and Antiviral Activity
1771
145.3 (C6), 151.4 (Ph, Cipso), 154.1 (C4), 163.8 (C2), 168.1 (CO, Bz), 174.4
(CO, Ala); 31P NMR 4.20, 4.34, 4.46, 4.62; ESI-MS 577 (M + K, 100.0), 539
(M + H, 12.8). Anal. Calcd. for C26H27N4O7P: C, 57.99; H, 5.05; N, 10.40.
Found: C, 58.12; H, 5.17; N, 10.42.
(E)-N4-Benzoyl-1-{[(2-hydroxymethyl)cyclopropylidene]methyl}cytosine
(Methylphenylphosphoryl)-P→N-L-alaninate (11b). The experiment was
performed as described for the Z-isomer 11a with E-isomer 9b (339 mg,
1.14 mmol), phosphorochloridate 8 (0.184 M, 32 mL, 5.7 mmol) and 1-
methylimidazole (0.91 mL, 11.4 mmol), reaction time 7 h. Chromatography
afforded product 11b as a colorless syrup, which was converted to a white
solid (332 mg, 52%) by trituration with ether (10 mL). UV λmax 330 nm (ε
1
14,700), 270 nm (ε 20,400), 205 nm (ε 28,200); H NMR δ 1.20–1.24 (m,
ꢀ
3H, CH3 of Ala), 1.55 (m, 1H) and 1.78–1.82 (m, 1H, H3 ), 2.04–2.12 (m,
ꢀ
1H, H4 ), 3.58, 3.59 (2s, 3H, OCH3), 3.82–3.88 (m, 1H, CH of Ala), 3.95–
ꢀ
4.02 (m, 2H, H5 ), 5.96–6.06 (m, 1H, NH of Ala), 7.14–7.15 (m), 7.20–7.36
ꢀ
(m), 7.49 (t), 7.61 (t) and 8.00 (d, total 11H, Ph, Bz, H1 and H5, 8.46
(poorly resolved d, 1H, H6), 11.33 (s, 1H, BzNH); 13C NMR 9.9, 10.0 (C3 ),
ꢀ
ꢀ
14.3 (C4 ), 20.3, 20.4 (CH3, Ala), 50.4, 50.5 (CH, Ala), 52.5 (CH3O), 68.7
ꢀ
ꢀ
ꢀ
(C5 ), 97.8 (C5), 116.0 (C2 ), 116.8 (C1 ), 120.87, 120.91 (Ph, Cmeta), 125.1
(Ph, Cortho), 129.1, 129.2, 130.3 (Ph, Cpara, Bz, Cmeta, Cortho), 133.4, 133.8
(Bz, Cpara, Cipso,), 145.1 (C6), 151.4, 151.5 (Ph, Cipso), 154.1 (C4), 163.8
(C2), 168.2 (CO); 31P NMR 4.34, 4.37, 4.73; ESI-MS 577 (M + K, 22.2), 539
(M + H, 100.0). Anal. Calcd. For C26H27N4O7P: C, 57.99; H, 5.05; N, 10.40.
Found: C, 57.78; H, 5.21; N, 10.28.
(Z)-1-{[(2-Hydroxymethyl)cyclopropylidene]methyl}cytosine (Methyl-
henylphosphoryl)-P→N-L-alaninate (3e). A mixture of 11a (330 mg, 0.61
mmol) and hydrazine hydrate (0.35 mL, 4.88 mmol) in pyridine–acetic acid
(10 mL, 4:1 v/v) was stirred at room temperature for 27 h. The solvents were
removed at room temperature in vacuo at room temperature and the oily
residue was partitioned between CH2Cl2 (250 mL) and water (200 mL).
The aqueous phase was extracted with the same solvent (2 × 25 mL). The
combined organic phase was washed with brine (2 × 100 mL) and water
(2 × 100 mL), it was dried over Na2SO4 and evaporated to a yellow syrup.
Chromatography on silica gel with CH2Cl2-MeOH (94:6) gave product 3e
as a pale yellow solid (78 mg, 29%) after washing with hexane-ethyl acetate
(99:1, 10 mL) and drying in vacuo. UV λmax 297 nm (ε 13,100), 228 nm (ε
1
ꢀ
14,300), 205 nm (ε 26,400); H NMR δ 1.15–1.21 (m, 4H, CH3 of Ala, H3 ),
ꢀ
ꢀ
1.40–1.47 (m, 1H, H3 ), 2.21–2.28 (m, 1H, H4 ), 3.55, 3.57, 3.58 (3s, 3H,
ꢀ
OCH3), 3.74–3.86 (m, 1H, CH of Ala), 3.88–4.10 (m, 2H, H5 ), 5.76–5.80
(m, 1H, H5), 5.95–6.05 (m, 1H, NH of Ala), 7.12–7.18 and 7.32–7.40 (2m,
13
ꢀ
ꢀ
8H, Ph, NH2, H1 ), 7.88–7.94 (m, 1H, H6); C NMR 6.2, 6.4 (C3 ), 16.75,

1772
A. Ambrose et al.
ꢀ
16.81 (C4 ), 20.26, 20.32, 20.4 (CH3, Ala), 50.2, 50.4, 50.46, 50.50 (CH, Ala),
ꢀ
ꢀ
52.6 (OCH3), 68.4, 68.6 (C5 ), 95.8, 95.9 (C5), 109.7, 109.89, 109.93 (C2 ),
ꢀ
117.2, 117.3 (C1 ), 120.8, 120.9 (Ph, Cmeta), 125.2 (Ph, Cortho, 130.3 (Ph,
C
para), 140.7, 140.8, 140.9 (C6), 151.4 (Ph, Cipso), 154.5 (C4), 166.1 (C2),
174.3, 174.5 (CO, Ala); 31P NMR 4.20, 4.34, 4.55, 4.68; ESI-MS 891 (2M +
Na, 41.1), 869 (2M + H, 100.0), 457 (M + Na, 74.4), 435 (M + H, 100.0).
Anal. Calcd. for C19H23N4O6P: C, 52.54; H, 5.34; N, 12.90. Found: C, 52.60;
H, 5.51; N, 12.97.
(E)-1-{[(2-Hydroxymethyl)cyclopropylidene]methyl}cytosine (Methyl-
phenylphosphoryl)-P→N-L-alaninate (4e). A mixture of 11b (337 mg, 0.625
mmol), hydrazine hydrate (0.4 mL, 6.4 mmol) in pyridine–acetic acid (18
mL, 4:1 v/v) was stirred at room temperature for 17 h. The reaction mixture
was worked up as described for the Z-isomer 3e. Chromatography on silica
gel with CH2Cl2-MeOH (92:8) gave product 4e as a pale yellow solid (74 mg,
27%). UV λmax 296 nm (ε 13,000), 229 nm (ε 13,700), 204 nm (ε 27,400);
1H NMR δ 1.19–1.23 (m, 3H, CH3 of Ala), 1.42–1.46 (m, 1H), 1.69–1.73
ꢀ
ꢀ
(m, 1H, H3 ), 1.93–1.99 (m, 1H, H4 ), 3.57, 3.58 (2s, 3H, OCH3), 3.80–3.88
3
ꢀ
(m, 1H, CH of Ala), 3.90–4.02 (m, 2H, H5 ), 5.83 (d, 1H, J5,6 7.6 Hz, H5),
5.95–6.05 (m, 1H, NH of Ala), 7.12–7.20 (m), 7.33–7.39 (m) and 7.45 (s,
13
ꢀ
ꢀ
total 8H, Ph, NH2, H1 ), 7.95–7.98 (m, 1H, H6); C NMR 9.8, 9.9 (C3 ), 13.7
ꢀ
ꢀ
(C4 ), 20.3, 20.4 (CH3), 50.4, 50.5 (CH, Ala), 52.5 (OCH3), 69.1 (C5 ), 95.9
ꢀ
ꢀ
(C5), 110.4 (C2 ), 117.1 (C1 ), 120.9 (Ph, Cmeta), 125.1 (Ph, Cortho), 130.3
(Ph, Cpara), 140.8 (C6), 151.5 (Ph, Cipso), 154.6 (C4), 166.1 (C2), 174.5 (CO,
Ala); 31P NMR 4.32, 4.37, 4.70; ESI-MS 869 (2M + H, 23.5), 457 (M + Na,
42.9), 435 (M + H, 100.0). Anal. Calcd. for C19H23N4O6P: C, 52.54; H, 5.34;
N, 12.90. Found: C, 52.39; H, 5.39; N, 12.72.
Hydrolysis of Phosphoralaninate Pronucleotides with
Porcine Liver Esterase (PLE)
Compounds 3d, 3e, 4d, 4e, 11a, and 11b (1.6 µmol each) were stirred
with PLE (200 U, 5 mg) in 0.01 M Na2HPO4 (pH 7.5, 1 mL) at room
temperature. Aliquots were periodically withdrawn and checked by TLC in
CH2Cl2-MeOH (19:1 or 9:1). The N4-benzoyl pronucleotides 11a, 11b were
hydrolyzed within 1 h, thymine analogues 3d, 4d within 16–20 h and cytosine
derivatives 3e, 4e within 36–40 h.
Antiviral Assays
The antiviral assays were described in details in the previous communica-
tions.[9,17,18] The HCMV (Towne and AD169 strains) and VZV assays were
performed in HFF culture using a plaque reduction or cytopathic effect

Nucleoside Analogues and Antiviral Activity
1773
(CPE) inhibition assay. 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 and CEM cells. For
further details see Table 1. For comparison, antiviral data for the parent
analogues 1d, 2d, 1e, and 2e are also given.
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