Synthesis and antiherpetic activity of (Z)- and (E)-9-(3- Phosphonomethoxyprop-1-en-yl)adenines

Article abstract of DOI:10.1007/s11171-005-0007-7

The isomeric (Z)- and (E)-9-(3-phosphonomethoxyprop-1-en-yl)adenines were synthesized. The stereoselectivity of double bond formation was studied by variation of sulfonyl groups. The resulting phosphonates exhibited a moderate antiherpetic activity in a c

Full text of DOI:10.1007/s11171-005-0007-7

Russian Journal of Bioorganic Chemistry, Vol. 31, No. 1, 2005, pp. 58–65. Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 65–72.
Original Russian Text Copyright © 2005 by Ivanov, Andronova, Galegov, Jasko.
Synthesis and Antiherpetic Activity
of (Z)- and (E)-9-(3-Phosphonomethoxyprop-1-en-yl)adenines
1
A. V. Ivanov*, V. L. Andronova**, G. A. Galegov**, and M. V. Jasko*
* Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia
** Ivanovskii Institute of Virology, Russian Academy of Medical Sciences, ul. Gamalei 16, Moscow, 123098 Russia
Received December 8, 2003; in final form, August 5, 2004
Abstract—The isomeric (Z)- and (E)-9-(3-phosphonomethoxyprop-1-en-yl)adenines were synthesized. The
stereoselectivity of double bond formation was studied by variation of sulfonyl groups. The resulting phospho-
nates exhibited a moderate antiherpetic activity in a culture of Vero cells infected with herpes simplex type 1
virus. The Z-isomer was shown to be a more effective inhibitor of virus reproduction in the case of both wild
and acyclovir-resistant strains.
Key words: herpes simplex virus, nucleotides, phosphonates
1
INTRODUCTION
The synthesis of (X) and (XII) consisted in obtain-
ing a glycerol-based alkylating agent (IVa) and its fur-
ther coupling with adenine sodium salt. The resulting
mixture of alkenes (Va) and (VIIa) was separated, and
the individual isomers were treated with acetic acid to
remove trityl groups. Phosphonomethylation of alco-
hols (VI) and (VIII) and subsequent deprotection
resulted in the target Z- and E-phosphonates (X) and
(XII).
Acyclic nucleoside and nucleotide analogues con-
stitute a relatively new group of antiviral agents. Both a
variety of structures and a wide range of antiviral activ-
2
ities are characteristic of them. For example, synade-
nol (I) and relative compounds inhibit the reproduction
of HSV, HIV, and hepatitis B viruses [1–3]. Com-
pounds containing the phosphonomethoxy fragment,
e.g., PMEA (II) and (S)-9-(3-hydroxy)-2-phospho-
nomethoxypropyl)adenine (III), which inhibit both the
HIV and HSV replication, belong to another group of
analogues [4]. Moreover, a number of such acyclic ana-
logues are already used in clinical practice as drugs,
e.g., acyclovir (Zovirax®), famcyclovir (Famvir®), pen-
ciclovir (Denavir®), etc. [5].
Several approaches to the synthesis of alkenes bear-
ing the double bond conjugated with heterocyclic aro-
matic system have been reported in literature. One of
the them involves the obtaining of substance with a
nonconjugated double bond and its further isomeriza-
tion by heating with a base. For example, (Z)- and (E)-
isomers of 9-(4-hydroxybut-1-en-1-yl)adenine were
obtained by heating of the corresponding but-2-en-1-yl
derivatives in the presence of potassium tert-butylate
[6]. Note that this method is inappropriate for obtaining
propene derivatives. Another strategy includes the alky-
lation of a heterocyclic base with synthons containing
vicinal bromine atoms and subsequent HBr elimination
[1]. We chose this approach for the synthesis of target
alkoxypropenyladenines (V) and (VII) (Scheme 2).
Alkylating agents (IVa)–(IVc) were synthesized from
glycerol through the protection of one of the primary
hydroxyl groups with trityl or TBDMS group and sub-
sequent introduction of two sulfonyl groups. The result-
ing synthons (IVa)–(IVc) were used for the alkylation
of adenine in the presence of a base, sodium hydroxide
or potassium bis(trimethylsilyl)amide.
RESULTS AND DISCUSSION
We herein report the synthesis of two new acyclic
nucleotide analogues containing the heterocyclic base-
conjugated double bond like that in synadenol (I) and
the phosphonomethoxy fragment like that in PMEA
(II). The Z- and E-isomers of 9-(3-phosphonomethox-
yprop-1-en-yl)adenine (X) and (XII) were actually
synthesized (Scheme 1) and their antiherpetic activities
were studied.
1
Corresponding author; phone: +7 (095) 135-1405; fax: +7 (095)
135-6065; e-mail: aivanov@yandex.ru
2
Abbreviations: HSV-1/L , herpes simplex virus type 1 strain L ;
2
2
HSV-1/L /R, acyclovir-resistant herpes simplex virus type 1
2
strain L ; ID and ID , doses inhibiting virus cytopathogenic
2
50
95
activity by 50 and 95%, respectively; IMP, ethyl iodometylphos-
phonate; Ms, mesyl (methanesulfonyl); PMEA, 9-(2-phospho-
The reaction of (IVa) with adenine in the presence
of sodium hydride yielded the mixture of Z- and E-iso-
mers of 9-(3-trityloxyprop-1-en-1-yl)adenine (Va) and
(VIIa). Note that the reaction in the presence of insuf-
ficient quantity of sodium hydride resulted in 9-(2-
nomethoxyethyl)adenine; SI, selectivity index (the TCD : ID
50
50
ratio); TBDMS, tert-butyldimethylsilyl; TCD , tissue cytotoxic-
50
ity dose responsible for the change in 50% of cell monolayer; Tr,
trityl (triphenylmethyl); and Ts, tosyl (p-toluenesulfonyl).
1068-1620/05/3101-0058 © 2005 Pleiades Publishing, Inc.

SYNTHESIS AND ANTIHERPETIC ACTIVITY
59
O
HO
O
Ade
Ade
Ade
OH
HO P
HO P
O
O
OH
OH
(I)
(II)
(III)
mesyloxy-3-trityloxypropyl)adenine (XIIIa) as the (Va). After the removal of the protecting group, Z-iso-
mer (VI) was purified by chromatography on silica gel.
main product, whereas the use of excess sodium
hydride excludes the intermediate (XIIIa). It seems
likely that the alkylation of heterocyclic base rather
than elimination of 2'-mesyl group is a rate-limiting
stage under these conditions.
The configuration of the double bond was deter-
mined by 1H NMR spectroscopy. In the case of Z-alk-
enes (Va) and (VI), the spin–spin coupling constant
3J1',2' was ~8.5 Hz, whereas E-isomers (VIIa) and
(VIII) had the constant exceeded 14 Hz.
The separation of Z- and E-isomers of trityl deriva-
tives (Va) and (VIIa) was achieved by crystallization
from chloroform. E-Alkene (VIIa) is much worse sol-
uble in chloroform and can be obtained as pure sub-
stance even after a single crystallization. To achieve a
more complete separation of the Z-isomer, the mother
liquors were evaporated, and the residue was repeatedly
crystallized from chloroform. Thus, the final mother
The ratio of Z- and E-isomers (Va) and (VIIa) in the
reaction mixture was 1 : 3.5 when using dimesyltrilyl
glycerol (IVa). To alter this ratio, we have substituted
ditosyltrityl glycerol (IVb) or ditosylsilyl derivative
(IVc) for dimesyltrityl glycerol (IVa). However, the
transition from mesyl to the more hydrophobic and
bulky tosyl groups did not change the ratio of isomers
(Va) and (VIIa), although a similar yield was achieved
liquor contained approximately 90% of the Z-isomer at a decreased reaction time (from 8 to 6 h). The ratio of
OTr
OMs
OMs
(IVa)
i
Ade
Ade
TrO
HO
O
+
(VIIa)
(Va)
TrO
HO
ii
ii
Ade
Ade
(VI)
(VIII)
iii
iii
O
O
Ade
(XI)
Ade
EtO P
EtO P
O
(IX)
OH
OH
iv
iv
O
O
Ade
(XII)
Ade
(X)
O
HO P
HO P
O
OH
OH
(i) AdeH, NaH/DMF; (ii) CH3COOH; (iii) IMP, NaH/DMF; (iv) Me3SiBr/DMF.
Scheme 1.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 1 2005

60
IVANOV et al.
OR
OX
OX
XO Ade
Ade
RO
Ade
i
+
RO
RO
(IV)
(XIII)
(V)
(VII)
(IVa): R = Tr, X = Ms
(IVb): R = Tr, X = Ts
(XIIIa): R = Tr, X = Ms
(XIIIb): R = Tr, X = Ts
(Va): R = Tr
(VIIa): R = Tr
(IVc): R = TBDMS, X = Ts (XIIIc): R = TBDMS, X = Ts (Vb): R = TBDMS (VIIb): R = TBDMS
(i) AdeH, NaH or (Me3Si)2NK/DMF
Scheme 2.
NH
NH2
N
N
N
O
N
N
N
P
(EtO)(HO)P(O)CH I
2
OR
HO
O
O
N
N
NaH, DMF
RO P
EtO P
O
O
OH
OH
(XI)
(XIV): R = Et
(XV): R = H
Me3SiBr
Scheme 3.
The 1H NMR spectra of phosphonates (IX) and (XI)
exhibit the doublet at ~3.8 ppm corresponding to the
methylene group at the phosphorus atom. In addition,
in 31P NMR spectra, these compounds exhibited a sig-
nal with a chemical shift at approximately 15–18 ppm,
which is characteristic of alkyloxymethylphosphonic
acids [10]. In the case of the E-isomer, the by-product
(XIV) was isolated besides the target phosphonate (XI)
(Scheme 3). The signals of two ethyl groups were
observed in 1H NMR spectrum of (XIV), whereas only
one broadened singlet was observed in its 31P NMR
spectrum. After the removal of the ethyl groups by the
treatment of (XIV) with trimethylbromosilane, 31P
NMR spectrum of (XV) contained two signals at 16.2
Z- and E-isomers also remained unchanged when the
TBDMS protecting group was substituted for trityl.
The use of potassium hexamethyldisilylamide instead
of NaH also caused no change in the ratio of Z/E-iso-
mers.
The traditional method of the phosphonomethyl
fragment introduction into a molecule consists in the
reaction of the corresponding alcohol with mono- or
dialkyl esters of iodo-, tosyloxy-, or trifluoromethane-
sulfonyloxymethylphosphonic acids in the presence of
sodium hydride [7–12]. We herein used ethyl iodome-
thylphosphonate or ethyl tosyloxymethylphosphonate,
which are readily available and stable substances. Fur-
thermore, their use enables the isolation of the reaction
product by ion-exchange or reversed-phase chromatog-
raphy.
Alcohols (VI) and (VII) were alkylated with IMP in
DMF in the presence of NaH (Scheme 1). The products
were separated by reversed-phase chromatography
under elution with ammonium bicarbonate buffer. This
allowed us to obtain the target phosphonates in the form
of ammonium salts. The structures of the resulting
compounds were confirmed by the methods of UV and
NMR spectroscopy. The absorption maxima in the UV
spectra of products (IX) and (XI) were the same as
those in the UV spectra of starting (VI) and (VII),
which supports the absence of the heterocyclic base
modifications.
1
and 16.4 ppm, whereas the H NMR spectrum con-
tained well-resolved signals of two methylene groups at
phosphorus atoms. It should be mentioned that, in the
case of phosphonomethyl group at the oxygen atom,
2
the value of JCH , P was 8.7 Hz, whereas the coupling
2
constant value for another signal reached 11 Hz, which
is characteristic of phosphonomethyl group at the nitro-
gen atom of heterocyclic base [13]. Furthermore, the
transition from N1-nonsubstituted phosphonates (IX)
and (XII) to N1-substituted phosphonates (XIV) and
(XV) resulted in the long-wave shift of absorption max-
imum in UV spectrum by 7 nm.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 1 2005

SYNTHESIS AND ANTIHERPETIC ACTIVITY
61
Antiherpetic activity of (Z)- and (E)-9-(3-phosphonomethoxyprop-1-en-1-yl)adenine
HSV-1/L2
HSV-1/ACVR
ID95, mM
Compound
(X)
(XII)
Acyclovir
(II)*
TCD50, mM
ID50, mM
ID95, mM
SI
ID50, mM
SI
>3.5
>3.5
>2.2
>0.36
0.095
0.39
1.53
>34
>8
0.11
0.44
0.53
0.08
0.86
1.57
0.89
>34
>8
1.57
0.0017
0.08
0.0036
>1300
>2.5
>4.5
>4.5
* The data are taken from [12]. The authors used BWS HSV-1 strain in the Vero cell culture. The infection multiplicity was the same as
described in the Experimental section.
The complex of these data allowed us to assign the not phosphorylate modified nucleosides [15]. However,
structure of (E)-1-(phosphonomethyl)-9-(phospho- (X) and (XII) do not require the first act of kination for
nomethoxyprop-1-en-1-yl)adenine (XV) to the result- their transformation into the corresponding dNMP ana-
ing compound. The aforementioned information corre- logues and, therefore, retain their activity against the
late well with that reported in literature for a similar acyclovir-resistant strain.
compound in the series of nucleoside carbocyclic ana-
logues [9].
EXPERIMENTAL
The by-product (XIV) became main reaction prod-
Adenine was from Reanal (Hungary) and methane-
sulfonyl chloride, p-toluenesulfonyl chloride, chlorotri-
phenylmethane, bromotrimethylsilane, glycerol, NaH
(80% suspension in oil), tert-butylchlorodimethyl-
silane, and potassium bis(trimethylsilyl)amide were
from Aldrich (United States). Ethyl iodomethylphos-
phonate and ethyl tosyloxymethylphosphonate were
synthesized as described in [11, 16].
Column chromatography was carried out on silica
gel 60 (63–100 µm), LiChroprep RP-8 (40–63 µm),
and LiChroprep RP-18 (25–40 µm) (Merck, Germany).
uct at 2–3-fold excess of IMP as alkylating agent,
whereas the yield of target monophosphonate (XI)
reached 38% when using 1.5 equiv of IMP per (VIII)
and the portion of diester (XIV) did not exceed 15%.
According to literature, the protection of exocyclic
amino group of nucleic base helps avoid its modifica-
tion including the N1-alkylation [14]. However, in our
case, the protection of the adenine residue in (VIII)
using dimethylformamide diethyl acetal with the aim to
treat the resulting (XVI) with IMP had no effect on the
ratio of the mono- and bisalkylation products. The sub-
stitution of tosyloxymethylphophonate for IMP also
failed to decrease the proportion of the by-product
(XIV).
Monoesters (IX) and (XI) were transformed into the
corresponding acids (X) and (XII) by the treatment
with trimethylbromosilane in DMF. The reaction prod-
ucts were isolated by reversed-phase chromatography.
Thus, we have obtained the target Z- and E-isomers
of 9-(3-phosphonomethoxyprop-1-en-1-yl)adenine.
The antiviral activity of phosphonates (X) and (XII)
was studied using Vero cell culture infected with HSV-1.
Both compounds displayed a moderate antiherpetic
activity for both the wild and the acyclovir-resistant
strain (see the table). The Z-isomer (X) was four times
more active than the E-isomer (XII) in respect of inhi-
bition of HSV replication. The activity was therewith
compatible with that of 9-(2-phosphonomethoxy-
ethyl)adenine (II). The synthesized phosphonates (X)
and (XII) appeared to be less toxic than (II) and, there-
fore, exceeded it in SI.
NMR spectra were registered on a Bruker AMX III-
400 (United States) spectrometer at a working fre-
1
quency of 400 MHz for H NMR (using Me4Si for
CDCl3 solutions and sodium 3-(trimethylsilyl)-1-pro-
panesulfonate for D2O solutions as internal references),
31
162 MHz for P NMR (with phosphorus–proton spin
decoupling using 85% phosphoric acid as the external
reference), and 100 MHz for 13C NMR. Chemical shifts
are given in ppm (δ scale) and spin coupling constants
in Hz. UV spectra were registered on a Shimadzu UV-
2401 P (Japan) spectrophotometer.
3-Trityloxypropane-1,2-diol was synthesized as
described in [17]; yield 81%; mp 90–92°ë (lit. 92–
94°ë); 1H NMR (ëDCl3): 7.46 (6 H, d, 3JÓ, m 7.2, o-CH,
Tr), 7.32 (6 H, t, 3Jm, p 7.8, m-CH, Tr), 7.3 (3 H, t, p-CH,
Tr), 3.87 (1 H, m, H2), 3.67 (1 H, dd, 2J1‡, 1· 11.2, 3J1‡, 2
3.4, ç1‡), 3.58 (1 H, dd, 3J1·, 2 6.2, ç1b), and 3.23 (2 H,
m, H3).
1,2-Bis(mesyloxy)-3-trityloxypropane
(IVa).
The retention of antiherpetic activity of (X) and Methanesulfonyl chloride (1.70 ml, 22.4 mmol) was
(XII) for the acyclovir-resistant virus strain may be added to a solution of 3-trityloxypropan-1,2-diol
connected with the fact that, in 95% cases, the resis- (3.00 g, 8.97 mmol) in pyridine (50 ml) at 0°ë. The
tance to acyclovir is due to the mutation of the viral thy- reaction mixture was stirred for 1 h at +4°ë and left at
midine kinase, which resulted in the enzyme that does room temperature for 20 h. The mixture was evaporated
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 1 2005

62
IVANOV et al.
in a vacuum; the residue was dissolved in ëël4 (50 ml); (IVa) (1.50 g, 3.06 mmol) was added in a DMF (15 ml)
and acetonitrile (5 ml) mixture. The reaction mixture
was refluxed for 8 h, cooled, and evaporated in a vac-
uum. The residue was dissolved in chloroform (40 ml),
washed with water (2 × 50 ml), dried with Na2SO4, and
evaporated in a vacuum. The residue was dissolved in
chloroform (4 ml) and chromatographed on a silica gel
column (3.8 × 10 cm) eluted with chloroform (200 ml)
and then 2% EtOH in chloroform (400 ml). The frac-
tions containing the mixture of Z- and E-alkenes (Va)
and the resulting solution was washed with aqueous
NaHCO3 (50 ml) and water (2 × 50 ml), dried over
Na2SO4, and evaporated in a vacuum. The oily residue
was crystallized from ëël4 and air-dried to yield 3.24 g
1
(74%) of (IVa); mp 107–110°ë (decomp.); H NMR
(ëDCl3): 7.41 (6 H, d, 3JÓ, m 7.8, o-CH), 7.32 (6 H, dd,
3Jm, p 7.2, m-CH), 7.26 (3 H, t, p-CH), 4.85 (1 H, m,
3J2, 3‡ 4.7, 3J2, 3b 5.3 H2), 4.40 (2 H, m, H1), 3.47 (1 H,
dd, 2J3‡, 3b 10.6, ç3‡), 3.40 (1 H, dd, H3b), 3.04 and 3.01 and (VIIa) was evaporated in a vacuum to get 710 mg
(2 × 3 H, 2 s, 2 CH3).
(54%) of 1 : 3.5 Z-/E-isomer mixture (according to 1H
NMR). Crystallization from chloroform resulted in
homogeneous E-isomer (VIIa). An additional quantity
of (VIIa) was obtained by concentrating the filtrates
and repeated crystallization. The total yield of pure E-
isomer (VIIa) after air-drying was 451 mg (34%). The
mother liquor after two crystallizations was evaporated
in a vacuum to give 93 mg (7%) of oily Z-isomer (Va)
(purity >90%).
1,2-Bis(tosyloxy)-3-trityloxypropane (IVb). A
solution of 3-trityloxypropane-1,2-diol (3.00 g,
8.97 mmol) and p-toluenesulfonyl chloride (5.13 g,
26.9 mmol) in pyridine (50 ml) was kept for 20 h at
room temperature and evaporated in a vacuum. The res-
idue was dissolved in CCl4 (70 ml), successively
washed with aqueous citric acid (30 ml), water (2 ×
30 ml), aqueous NaHCO3 (20 ml), and water (2 ×
30 ml), and dried with Na2SO4. The oily residue
obtained after evaporation was crystallized from CCl4
(25 ml) and air-dried to give 4.16 g (72%) of (IVb); mp
(Z)-9-(3-Trityloxyprop-1-en-1-yl)adenine (Va): UV
(CH3OH): λmax 261 nm; 1H NMR (NDCl3): 8.33 (1 I, s,
I8), 7.89 (1 I, s, I2), 7.42 (6 H, d, 3Jo, m 8.4, o-CH), 7.32–
7.13 (9 H, m, m-CH and p-CH), 6.96 (1 H, d, 3J1', 2' 9.0,
H1'), 5.60 (2 H, br. s, 6-NH2), 5.88 (1 H, dt, 3J2', 3' 6.9,
H2'), 3.78 (2 H, dd, 4J1', 3' 0.9, H3').
1
104–106°C (decomp.); H NMR (CDCl3): 7.68 and
3
7.64 (2 × 2 H, 2 d, Jo, m 8.4, o-CH, Ts), 7.29–7.23
(19 H, m, Tr and m-CH of Ts), 4.55 (1 H, m, H2), 4.14
(2 H, m, H1), 3.26 (2 H, m, H3), 2.43 and 2.42 (2 × 3 H,
2 s, 2 CH3, Ts).
(E)-9-(3-Trityloxyprop-1-en-1-yl)adenine (VIIa):
UV (CH3OH): λmax 261 nm; mp 192–195°C; 1H NMR
(NDCl3): 8.39 (1 H, s, H8), 7.96 (1 H, s, H2), 7.49 (6 H,
d, 3Jo, m 7.5, o-CH), 7.33 (6 H, t, 3Jm, p 7.5, m-CH), 7.25
(4 H, m, p-CH and H1'), 6.53 (1 H, dt, 3J1', 2' 14.3, 3J2', 3'
5.6, H2'), 5.56 (2 H, br. s, 6-NH2), 3.86 (2 H, dd, 4J1', 3'
1.6, H3').
1,2-Bis(tosyloxy)-3-(tert-butyldimethylsilyloxy)-
propane (IVc). A solution of tert-butylchlorodimethyl-
silane (1.62 g, 10.8 mmol) and glycerol (12.5 ml,
200 mmol) in pyridine (80 ml) was kept for 18 h at
room temperature and then evaporated in a vacuum.
The residue was coevaporated with toluene (2 × 15 ml)
and dissolved in chloroform (50 ml). The solution was
washed with aqueous NaHCO3 (40 ml) and water (2 ×
40 ml) and dried with Na2SO4. The oily residue was
dissolved in pyridine (70 ml), p-toluenesulfonyl chlo-
ride (5.13 g, 26.9 mmol) was added, and the reaction
mixture was kept for 18 h at room temperature. Then
the reaction mixture was evaporated in a vacuum, the
residue was dissolved in chloroform (50 ml), and the
solution was washed with aqueous NaHCO3 (30 ml)
and water (2 × 30 ml) and dried with Na2SO4. The sol-
vents were removed under reduced pressure to give
2.82 g (51%) of oily product (IVc); 1H NMR (CDCl3):
Methods B and C. A suspension of adenine (1.5 g,
11 mmol) and NaH (467 mg, 15.6 mmol) in DMF
(50 ml) was stirred until the hydrogen liberation ceased
and heated for another 1 h at 60°C. Then a solution of
1,2-bis(tosyloxy)-3-trityloxypropane (IVb) (method
B) (5.7 g, 5.6 mmol) or (method C) 1,2-bis(tosyloxy)-
3-(tert-butylmethylsilyloxy)propane (IVc) (2.8 g,
5.5 mmol) in a DMF (25 ml) and acetonitrile (10 ml)
mixture was added. The mixture was refluxed for 6 h,
cooled, and treated as described in method A. The yield
of the mixture of target (Va) and (VIIa) by the method
B was 1.64 g (68%). The yield of the oily mixture of
(Vb) and (VIIb) was 0.98 g (58%) according to method
C. The Z/E isomer ratio was 1 : 3.5 in both cases
(according to 1H NMR spectroscopy).
7.71 (4 H, d, 3Jo, m 8.4, o-CH), 7.28 (4 H, d, m-CH), 4.57
(1 H, m, H2), 4.17 (2 H, m, H1), 3.30 (2 H, m, H3), 2.45
and 2.43 (2 × 3 H, 2 s, 2 CH3, Ts), 0.90 (9 H, s, Me3C),
0.11 (6 H, s, Me2Si).
Method D. A suspension of adenine (1.5 g,
11 mmol) and potassium bis(trimethylsilyl)amide
(Z)- and (E)-9-(3-Trityloxyprop-1-en-1-yl)ade- (3.11 g, 15.6 mmol) in DMF (50 ml) was stirred for 1 h
nine (Va) and (VIIa). Method A. A suspension of ade- and treated with a solution of 1,2-bis(mesyloxy)-3-tri-
nine (827 g, 6.12 mmol) and NaH (230 mg, 7.67 mmol) tyloxypropane (IVa) (5.7 g, 5.6 mmol) in a DMF
in DMF (30 ml) was stirred until the hydrogen libera- (25 ml) and acetonitrile (10 ml) mixture. The mixture
tion ceased and kept for another 1 h at 60°C. Then a was refluxed for 6 h, cooled and treated as described in
solution of 1,2-bis(mesyloxy)-3-trityloxypropane method A. Yield of the mixture of target (Va) and
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 1 2005

SYNTHESIS AND ANTIHERPETIC ACTIVITY
63
(VIIa) was 1.44 g (60%). The Z/E isomer ratio was 1 : solved in chloroform (1 ml), and chromatographed on a
3.5 (according to 1H NMR).
silica gel column (1 × 15.5 cm) eluted with CHCl3 and
then with 95 : 5 CHCl3–EtOH. Fractions containing the
target alcohol were evaporated in a vacuum, and the
residue was air-dried to give 30 mg (92%) of (XVI). 1H
NMR (NDCl3): 8.92 (1 H, s, N=CH–N), 8.53 (1 H, s,
(Z)-9-[3-(tert-Butyldimethylsilyloxy)prop-1-en-1-
yl]adenine (Vb): UV (CH3OH): λmax 261 nm; 1H NMR
(NDCl3): 8.38 and 8.10 (2 H, 2 s, H8 and H2), 6.97
(1 H, d, 3J1', 2' 9.0, H1'), 5.86 (1 H, dt, 3J2', 3' 6.9, H'), 5.86
(2 H, br. s, 6-NH2), 4.32 (2 H, dd, 4J1', 3' 1.2, H3'), 0.88
(9 H, s, Me3C), 0.06 (6 H, s, Me2Si).
H2), 8.02 (1 H, s, H8), 7.21 (1 H, br. d, 3J1', 2' 14.3, H1'),
6.58 (1 H, dt, 3J2', 3' 5.6, H2'), 4.37 (2 H, dd, 4J1', 3' 1.6,
H3'), 3.24 and 3.19 (6 I, 2 s, Me2N).
(E)-9-[3-(tert-Butyldimethylsilyloxy)prop-1-en-1-
yl]adenine (VIIb): UV (CH3OH): λmax 261 nm; H
NMR (CDCl3): 8.39 and 7.94 (2 H, 2 s, H8 and H2),
7.17 (1 H, dt, 3J1', 2' 14.3, 4J1', 3' 1.9, H1'), 6.60 (1 H, dt,
3J2', 3' 5.0, H2'), 5.86 (2 H, br. s, 6-NH2), 4.40 (2 H, dd,
H3'), 0.92 (9 H, s, Me3C), 0.13 (6 H, s, Me2Si).
1
(E)-9-[3-(O-Ethylphosphonomethoxyprop-1-en-
1-yl]adenine (XI). MethodA. Sodium hydride (30 mg,
1.0 mmol) was added to a solution of (E)-9-(3-hydroxy-
prop-1-en-1-yl)adenine (VIII) (50 mg, 0.26 mmol) and
monoethyl
iodomethylphosphonate
(90
mg,
0.36 mmol) in DMF (40 ml). The mixture was stirred
for 18 h at room temperature, quenched with acetic acid
(60 µl), and evaporated in a vacuum. The residue was
dissolved in water (1 ml) and chromatographed on a
LiChroprep RP-8 column (1.8 × 22 cm) eluted with
0.05 M aqueous NH4HCO3. The fractions containing
the target monoester were evaporated in a vacuum; the
residue was coevaporated with water (4 × 15 ml) and
dried in a vacuum to give 32 mg (38%) of (XI); UV
(H2O, pH 7): λmax 261 nm; 1H NMR (D2O): 8.02 (1 H,
(E)-9-(3-Hydroxyprop-1-en-1-yl)adenine (VIII).
Method A. A solution of (E)-9-(3-trityloxyprop-1-en-
1-yl)adenine (VIIa) (267 mg, 0.62 mmol) in 80% aque-
ous acetic acid (25 ml) was refluxed for 2 h, cooled, and
evaporated. The residue was coevaporated with toluene
(2 × 25 ml) and dissolved in 50% aqueous methanol.
The solution was washed with chloroform (2 × 15 ml)
and evaporated in a vacuum; yield of (VIII) 98 mg
(83%); UV (CH3OH): λmax 261 nm; mp 224–227°C; 1H
NMR (D2O): 8.33 (1 H, s, H8), 8.26 (1 H, s, H2), 7.18
s, H8), 7.87 (1 H, s, H2), 6.90 (1 H, d, 3J1', 2' 14.3, H1'),
(1 H, dt, 3J1', 2' 14.3, 4J1', 3' 1.2, H1'), 6.54 (1 H, dt, 3J2', 3'
5.9, H2'), 4.40 (2 H, dd, H3').
3
6.24 (1 H, dt, J2', 3' 6.5, H2'), 4.22 (2 H, d, H3'), 3.92
3
3
(2 H, dq, JCH , CH ≈ JCH , P 7.2, CH3CH2), 3.67 (2 H,
2
3
2
Method B. A solution of (E)-9-[3-(tert-butyldime-
thylsilyloxy)prop-1-en-1-yl]adenine (VIIb) (150 mg,
0.49 mmol) in 80% aqueous acetic acid (25 ml) was
refluxed for 2 h, cooled, and evaporated in a vacuum.
The residue was coevaporated with toluene (2 × 25 ml),
crystallized from aqueous methanol, and dried in a vac-
uum to give 71 mg (76%) of (VIII).
2
d, JCH , P 8.7, PCH2), 1.20 (3 H, t, CH3CH2). 31ê NMR
2
(D2O): 18.17 s.
Method B. A mixture of (E)-9-(3-hydroxyprop-1-
en-1-yl)adenine (VIII) (50 mg, 0.26 mmol), monoethyl
tosyloxymethylphosphonate (90 mg, 0.5 mmol) and
NaH (50 mg, 1.7 mmol) in DMF (50 ml) was stirred for
18 h at room temperature, quenched with acetic acid
(100 µl), and evaporated in a vacuum. The residue was
dissolved in water (1 ml) and chromatographed on a
LiChroprep RP-8 column (1.8 × 22 cm) eluted with
0.05 M aqueous NH4HCO3. The fractions containing
monoester (XI) were evaporated in a vacuum; the resi-
due was coevaporated with water (4 × 15 ml) and dried
in a vacuum to give 35 mg (40%) of ester (XI).
(Z)-9-(3-Hydroxyprop-1-an-1-yl)adenine (VI). A
solution of (Z)-9-(3-trityloxyprop-1-en-1-yl)adenine
(Va) (100 mg, 0.23 mmol) in 80% aqueous acetic acid
(20 ml) was refluxed for 2 h, cooled, and evaporated in
a vacuum. The residue was coevaporated with toluene
(2 × 15 ml), dissolved in 50% aqueous methanol
(30 ml), washed with chloroform (2 × 15 ml), and evap-
orated in a vacuum. The residue was dissolved in 93 : 7
chloroform–methanol and chromatographed on a silica
gel column (1 × 15.5 cm). Fractions absorbing in UV
light were concentrated and dried in a vacuum to give
34 mg (76%) of (VI); UV (CH3OH): λmax 261 nm; mp
Method C. Sodium hydride (25 mg, 0.83 mmol)
was added to a solution of N6-dimethylaminometh-
ylidene-9-(3-hydroxyprop-1-en-1-yl)adenine (XVI)
(30 mg, 0.12 mmol) and ethyl iodomethylphosphonate
(60 mg, 0.24 mmol) in DMF (40 ml), and the mixture
was stirred for 18 h at room temperature, quenched
with acetic acid (60 µl), and evaporated in a vacuum.
The residue was dissolved in 5% ammonia, the result-
ing solution was kept for 18 h at room temperature, and
evaporated in a vacuum. The residue was dissolved in
water (1 ml) and chromatographed on a LiChroprep
RP-8 column (1.8 × 22 cm) eluted with 0.05 M aqueous
NH4HCO3. The fractions containing monoester (XI)
1
195–198°C; H NMR (D2O): 8.28 (1 H, s, H8), 8.16
3
4
(1 H, s, H2), 6.77 (1 H, dt, J1', 2' 8.4, J1', 3' 1.6, H1'),
6.02 (1 H, dt, 3J2', 3' 6.5, H2'), 4.06 (2 H, dd, H3').
N6-Dimethylaminomethylidene-9-(3-hydroxyprop-
1-en-1-yl)adenine (XVI) A solution of (E)-9-(3-
hydroxyprop-1-en-1-yl)adenine (VIII) (25 mg,
0.13 mmol) and dimethylformamide diethylacetal
(400 µl, 2.33 mmol) in DMF (10 ml) was kept for 18 h
at room temperature and evaporated in a vacuum. The
residue was coevaporated with water (2 × 10 ml), dis-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 1 2005

64
IVANOV et al.
3
were evaporated in a vacuum, coevaporated with water
(4 × 15 ml), and lyophilized. Yield 5 g (22%).
120.88 (s, C2'), 120.71 (s, C5), 72.73 (d, JC, P 12.1,
C3'), 68.57 (d, 1JC, P 156.6, CP).
(E)-1-(O-Ethylphosphonomethyl)-9-(3-O-ethyl-
phosphonomethoxyprop-1-en-1-yl)adenine (XIV).
Sodium hydride (30 mg, 1.0 mmol) was added to a
solution of (E)-9-(3-hydroxyprop-1-en-1-yl)adenine
(VIII) (50 mg, 0.26 mmol) and monoethyl iodometh-
ylphosphonate (150 mg, 0.60 mmol) in DMF (5 ml).
The mixture was stirred for 18 h at room temperature,
quenched with acetic acid (60 µl), and evaporated in a
vacuum. The residue was dissolved in water (1 ml) and
chromatographed on a LiChroprep RP-8 column (1.8 ×
22 cm) eluted with 0.05 M aqueous NH4HCO3. The
fractions containing diester (XIV) were evaporated in a
vacuum.Yield of (XIV) 40 mg (35%); UV (H2O, pH 7):
(Z)-9-(3-Phosphonomethoxyprop-1-en-1-yl)ade-
nine (X) was obtained from ester (IX) by the method
used for the synthesis of (XII) in 84% yield; UV (H2O,
1
pH 7): λmax 261 nm; H NMR (D2O): 8.00 and 7.99
(2 H, 2 s, H8 and H2), 6.79 (1 H, d, 3J1', 2' 8.4, H1'), 5.95
(1 H, dt, 3J2', 3' 6.5, H2'), 4.09 (2 H, d, H3'), 3.45 (2 H, d,
2JCH , P 8.7, PCH2); 31P NMR (D2O): 15.88 s; 13C NMR
2
(D2O): 156.64 (s, C6), 153.81 (s, C2), 150.00 (s, C4),
143.07 (s, C8), 126.34 (s, C1'), 123.10 (s, C2'), 119.19
(s, C5), 68.39 (d, 3JC, P 11.1, C3'), 66.96 (d, 1JC, P 156.6,
CP).
λ
max 268 nm. 1H NMR (D2O): 8.21 and 8.14 (2 H, 2 s,
(E)-1-(Phosphonomethyl)-9-(3-phosphonometho-
xyprop-1-en-1-yl)adenine (XV) was obtained from
diethyl ester (XIV) by the method described for (XII);
H8 and H2), 7.11 (1 H, d, 3J1', 2' 14.3, H1'), 6.33 (1 H,
3
dt, J2', 3' 6.5, H2'), 4.18 (2 H, d, H3'), 3.86–3.58 (6 H,
2
1
yield 76%; UV (H2O, pH 7): λmax 269 nm; H NMR
m, 2 CH3CH2 and PCH2N), 3.58 (2 H, d, J
8.4,
CH2, P
(D2O): 8.34 and 8.32 (2 H, 2 s, H8 and H2), 7.28 (1 H,
3
PCH2O), 1.12 (3 H, t, J
6.9, CH3CH2 at 3'-O]),
3
3
CH2, CH3
d, J1', 2' 14.0, H1'), 6.50 (1 H, dt, J2', 3' 6.5, H2'), 4.38
3
2
0.98 (3 H, t, JCH , CH 7.2, CH3CH2 at N1]); 31P NMR
(2 H, d, H3'), 3.83 (2 H, d, JCH , P 11.8, PCH2N), 3.73
2
3
2
(2 H, d, 2JCH , P 8.7, PCH2O); 31P NMR (D2O): 16.40 (s,
(D2O): 18.17 br. s.
2
(Z)-9-(3-I-Ethylphosphonomethoxyprop-1-en-1-
yl)adenine (IX) was synthesized by the method
described for phosphonate (XI) in 37% yield; UV
1P) + 16.20 (s, 1P).
Experiments in cell systems. The Vero cell culture
(kidney cell culture from African green monkeys)
obtained from the Laboratory of Cell Cultures
(Ivanovskii Institute of Virology, Russian Academy of
Medical Sciences) was used. Cells were maintained in
the Eagle MEM nutrition medium (PANEKO, Mos-
cow) containing 7% fetal calf serum.
1
(H2O, pH 7): λmax 261 nm; H NMR (D2O): 8.04 and
8.02 (2 H, 2 s, H8 and H2), 6.82 (1 H, d, 3J1', 2' 8.4, H1'),
3
5.96 (1 H, dt, J2', 3' 6.5, H2'), 4.11 (2 H, d, H3'), 3.96
3
3
(2 H, dq, JCH , CH ≈ JCH , P 7.0, CH3CH2), 3.46 (2 H,
2
3
2
2
d, JCH , P 8.7, PCH2), 1.22 (3 H, t, CH3CH2); 31P NMR
2
The HSV-1/L2 wild strain from the Virus Museum
(Ivanovskii Institute of Virology, Russian Academy of
Medical Sciences) was used. The mutant acyclovir-
resistant strain HSV-1/ACVR was obtained by serial
passages in the presence of increasing acyclovir con-
centrations. The resulting viral strain was used for
obtaining the acyclovir-resistant virus clone [18]. The
viral strains were passaged in an Eagle mixture and 199
media (1 : 1) containing 2% of fetal calf serum. The
96-well plates (Linbro, Flow Lab., UK) were used for
cell culturing and subsequent infection.
(D2O): 15.82 s.
(E)-9-(3-Phosphonomethoxyprop-1-en-1-yl)ade-
nine (XII). A solution of ethyl ester of (E)-9-(3-
phophonomethoxyprop-1-en-1-yl)adenine (XI) (30 mg,
0.095 mmol) in a mixture of water (20 ml), DMF
(10 ml), and tributylamine (200 µl) was evaporated in a
vacuum to a volume of ~3 ml and coevaporated with
DMF (4 × 10 ml). DMF (15 ml) and Me3SiBr (51 µl,
0.38 mmol) were added to the residue, and the mixture
was kept for 18 h at room temperature and evaporated
in a vacuum. The residue was dissolved in water (1 ml)
and chromatographed on a LiChroprep RP-18 column
(2 × 17 cm) eluted with 0.05 M aqueous NH4HCO3.
The fractions containing phosphonate (XII) were evap-
orated in a vacuum to give 22 mg (81%) of the product
(XII); UV (H2O, pH 7): λmax 261 nm; 1H NMR (D2O):
Antiviral activity was estimated according to the
ability of the compounds under study to inhibit the
development of virus-induced cytopathogenic response
by 50 and 95% as compared with total cell death in ref-
erence infected cultures [19]. The infection multiplicity
was 0.1 TCD50/cell. The cells were incubated for 48 h,
until 95–100% cytotoxic effect developed.
7.99 (1 H, s, H8), 7.85 (1 H, s, H2), 6.85 (1 H, d, 3J1', 2'
14.3, H1'), 6.18 (1 H, dt, 3J2', 3' 6.2, H2'), 4.14 (2 H, d,
H3'), 3.51 (2 H, d, JCH , P 8.7, PCH2). 31ê NMR (D2O):
2
The Trypane Blue test was used for the detection of
dead cells [20]. The concentration that resulted in 50%
2
15.66 s; 13C NMR (D2O): 157.25 (s, C6), 154.49 (s, cell survival after 72-h contact with the compound
under study, was taken as TCD50.
C2), 150.00 (s, C4), 142.37 (s, C8), 125.30 (s, C1'),
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 1 2005

SYNTHESIS AND ANTIHERPETIC ACTIVITY
65
10. Jasko, M.V. and Tarussova, N.B., Collect. Czech. Chem.
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ACKNOWLEDGMENTS
The work was supported by the Russian Foundation
for Basic Research, project no. 04-04-49354, and the
Program of Basic Research of the Presidium of RAS
Fundamental Sciences to Medicine.
11. Yas’ko, M.V., Novikov, N.A., and Tarusova, N.B.,
Bioorg. Khim., 1994, vol. 20, pp. 50–54.
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cock, M.J.M., Ghazzouli, I., and Martin, J.C., J. Med.
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