Fluorescent tricyclic analogues of acyclovir and ganciclovir. A structure-antiviral activity study

Article abstract of DOI:10.1021/jm010922s

Of a series of new guanine base modified tricyclic analogues of acyclovir (ACV, 1) and ganciclovir (GCV, 2), derivatives of the 3,9-dihydro-9-oxo-5H-imidazo[1,2-α]purine system, evaluated for activity against herpes simplex virus type 1 and 2, several fluorescent analogues, 6-(4-MeOPh)-TACV (8), 7-Me-6-Ph-TACV (17), 6-(4-MeOPh)-TGCV (27), and 7-Me-6-Ph-TGCV (28), were obtained that showed similar potency and selectivity as the parent compounds. The activity was found to be strongly dependent on the nature and steric demands of the substituents in the 6 and/or 7 position.

Full text of DOI:10.1021/jm010922s

4284
J . Med. Chem. 2001, 44, 4284-4287
Brief Articles
F lu or escen t Tr icyclic An a logu es of Acyclovir a n d Ga n ciclovir .
A Str u ctu r e-An tivir a l Activity Stu d y
Bozenna Golankiewicz,*,† Tomasz Ostrowski,† Tomasz Goslinski,† Piotr J anuszczyk,† J oanna Zeidler,†
Daniel Baranowski,† and Erik de Clercq#
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12-14, 61-704 Poznan˜, Poland, and
Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received May 9, 2001
Of a series of new guanine base modified tricyclic analogues of acyclovir (ACV, 1) and ganciclovir
(GCV, 2), derivatives of the 3,9-dihydro-9-oxo-5H-imidazo[1,2-a]purine system, evaluated for
activity against herpes simplex virus type 1 and 2, several fluorescent analogues, 6-(4-MeOPh)-
TACV (8), 7-Me-6-Ph-TACV (17), 6-(4-MeOPh)-TGCV (27), and 7-Me-6-Ph-TGCV (28), were
obtained that showed similar potency and selectivity as the parent compounds. The activity
was found to be strongly dependent on the nature and steric demands of the substituents in
the 6 and/or 7 position.
In tr od u ction
against cytomegalovirus (CMV) is lower by 1 order of
magnitude. Compound 5 is on the average approxi-
mately 15-fold less active than parent acyclovir.3
We have previously reported that linking the 2-NH2
and N-1 positions of guanine moiety of acyclovir (ACV,
1) and ganciclovir (GCV, 2) with an etheno bridge to
form derivatives of the tricyclic 3,9-dihydro-9-oxo-5H-
imidazo[1,2-a]purine system, TACV (3) and TGCV (4)
[For the systematic names 3,9-dihydro-3-[(2-hydroxy-
ethoxy)methyl]-9-oxo-5H-imidazo[1,2-a]purine and 3,9-
dihydro-3-[(1,3-dihydroxy-2-propoxy]-9-oxo-5H-imidazo-
[1,2-a]purine the abbreviations TACV and TGCV,
respectively, are used throughout this paper.], allows
for the modulation of physical and biological properties
of the parent antivirals.1-3 The appended ring by itself
diminishes the antiherpetic activity, depending on the
type and strain of the virus, from 100- to more than
2000-fold. Further substitutions in the 6 or 7 position
of this ring potentiate the activity and make the
compounds more selective toward particular herpes
viruses. The magnitude of the antiviral activity has been
found to depend on the position and type of the sub-
stituent, the virus type, and the nature of the acyclic
moiety in the 3 position of the heterocycle. Appropriate
substituents in the appended ring may simultaneously
endow 3 and 4 with advantageous physical properties:
e.g. a 6-methyl function improves solubility4 and 6-aryl
groups may confer fluorescence. Of a series of several
tricyclic analogues evaluated so far,1-3 the fluorescent
6-phenyl-TACV (5) and 6-phenyl-TGCV (6) have ap-
peared as the most promising. In particular, the activity
of compound 6 against herpes simplex virus type 1
(HSV-1) and type 2 (HSV-2), thymidine kinase deficient
(TK-) HSV-1, and varicella-zoster virus (VZV) is very
similar to that of parent ganciclovir; only its activity
The fact that some tricyclic analogues show selective
activity against herpes virus infections suggests that
they are preferentially metabolized by the virus-infected
cells and/or show a preferential affinity for virus-specific
enzymes. This assumption has recently been substanti-
ated by an NMR experiment in solution which demon-
strated the formation of 6-ethyl-TACV-HSV-1TK com-
plex.5 Fluorescent, antivirally active tricyclic analogues
related to TACV and TGCV may prove useful in the
noninvasive diagnosis of herpes virus infections. These
compounds and their metabolites could be monitored as
“tags” for the virus-infected cells and/or virus-specified
enzymes.
The fact that aryl substitution may confer on the
tricyclic analogues simultaneously fluorescence and
antiherpetic activity together with the continuous need
for the development of new antiviral drugs resulting
from the emergence of drug-resistant herpesviruses6
encouraged us to study a further series of 22 derivatives
of 3,9-dihydro-9-oxo-5H-imidazo[1,2-a]purine.
We first attempted to develop fluorescent tricyclic
analogues of acyclovir, with antiviral activity similar to
that of the parent compound, and then to synthesize
the ganciclovir counterparts of the most active ones. The
antivirally active TACV and TGCV derivatives de-
scribed so far3 consist mostly of 6-substituted com-
pounds with 7-Me-TACV as the only one example of
different substitution site. The present series (Chart 1)
included 6-substituted (7-16, 25-27), 6,7-disubstituted
(17-22, 28), and 7-substituted (23, 24) derivatives. 6-Et-
TACV (25) was included in our studies as an active
reference compound; it corresponds to the only HSV-1
TK substrate for which complex formation with the
enzyme was shown in solution.5
* To whom correspondence should be addressed. Tel: 4861 8528 503.
Fax: 4861 8520 532. E-mail: bogolan@ibch.poznan.pl.
† Polish Academy of Sciences.
#
Katholieke Universiteit Leuven.
10.1021/jm010922s CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/16/2001

Brief Articles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4285
Ch a r t 1
TACV 17 and 6-Ph-7-Me-TGCV 28, the intermediate
N-1 alkylation products: 1-(1-benzoylethyl)-ACV 17′
and 1-(1-benzoylethyl)-GCV 28′. The structures of 17′
and 28′ were identified on the basis of their HR MS and
1H and 13C NMR spectra. The mechanistic sequence of
the reaction of bromoacetone7 and other R-bromoke-
tones3 with substituted guanines involving first alky-
lation at N-1 of the guanine moiety followed by ring
closure and elimination has been established on the
basis of the structures of the tricyclic products. However,
the initial intermediate had never been isolated. Com-
pound 15 with the blocked OH function in the acyclic
moiety was obtained by routine acetylation of 14 with
acetic anhydride in pyridine. Compound 16 has been
described previously.8 The 7-hydroxymethyl derivative
23 was synthesized from acyclovir and glycidaldehyde
following the procedure described for guanosine.9 As in
the case of guanosine, compound 23 was accompanied
by the 2-NH2, N-1-etheno derivative of the parent
compound.
The remaining 6,7-disubstituted (19-22) and 7-sub-
stituted (24) TACV derivatives were obtained via un-
usual tritylation reactions, as previously described.10,11
Str u ctu r e-Activity Rela tion sh ip
The substituent introduced in the 6-phenyl group of
5 had a pronounced influence on its antiviral activity
and cytotoxicity (Table 1). The effect depended upon the
nature of the substituent and the site of its attachment.
Electron donating groups [methyl (compound 7); meth-
oxy (compound 8)] in the 4 position of 6-phenyl-TACV
increased its potency to that of acyclovir. Compound 9
with a 6-(2-methoxyphenyl) substituent and compound
10 with a 6-(3-methoxyphenyl) substituent had some
antiviral activity but 10-fold weaker than for the un-
substituted 6-phenyl-TACV. Introduction of an ad-
ditional ortho-methoxy group in the 6-phenyl substitu-
ent of compounds 8 and 10, to form compounds 11 and
12, respectively, decreased antiherpetic activity and
increased cytotoxicity. The effect was especially pro-
nounced for the highly effective 8, for which the selec-
tivity indices against HSV-1 and HSV-2 decreased from
1500 to 40 and from 1000 to 8, respectively. Introduction
of an electron withdrawing nitro group in the 4 position
of the 6-phenyl moiety was unfavorable and resulted in
the weakly active compound 13. This may not, neces-
sarily, be attributed to the electron withdrawing char-
acter of the substituent but rather to steric reasons,
since the previously examined 6-(4-bromophenyl)-TACV
showed antiviral activity pattern very similar to that
of unsubstituted 6-Ph-TACV.3 In contrast to the re-
placement of the phenyl group in the 6 position with
the linear 4-biphenylyl substituent, which on average
resulted only in about 5-fold decrease of activity against
HSV-1 and HSV-2,3 introduction of angular, condensed
2-naphthyl group (compd 14) resulted in approximately
15-fold decrease of activity.
Sch em e 1a
a
Reagents: (i) 1. NaH, DMF, R2 COCH2Br or PhCOCH(Br)Ph;
2. NH4OH. (ii) 1. NaH, DMF, PhCOCH(Br)CH3; 2. NH4OH.
Ch em istr y
The tricyclic analogues 7-14, 17, 18, and 25-28 were
prepared by reacting the 1-sodium derivative of acyclo-
vir or ganciclovir in dimethylformamide with an ap-
propriate bromo ketone (Scheme 1) according to a
previously described method for an alkylation-conden-
sation reaction using other bromo ketones.2,3 In the case
of the reaction of 1 and 2 with 2-bromopropiophenone,
we isolated, in addition to the expected 6-Ph-7-Me-
The complete loss of selectivity of compound 14 after
blocking its side-chain hydroxy group (compound 15)
suggests that, as in acyclovir, antiviral activity requires
phosphorylation of the acyclic moiety. As observed for
6-Me-TACV,1 an unsubstituted N-5 position was crucial
for the activity of 6-aryl derivatives. 5-Benzyl-6-phenyl-
TACV (16) was totally inactive.

4286 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
Brief Articles
Ta ble 1. Activity Against Human Herpes Simplex Virus Type 1 and 2 and Cytotoxicity of Novel Tricyclic Analogues of Acyclovir and
Ganciclovir
minimal inhibitory concn (µg/mL)a
HSV-1
TK-
HSV-1
minimalb
cytotoxic
HSV-1
F
HSV-2
196
TK-/TK+
compd
KOS
0.13
McIntyre
G
Lyons
VV
VSV
ACVr
VMW 1837 concn (µg/mL)
ACV (1)
0.08
0.004
0.02
4
0.7
0.7
0.005
0.07
0.07
1.92
1.92
1.92
16
16
9.6
>16
ND
0.04
9.6
>16
ND
0.13
0.006
0.01
20
0.7
0.2
0.005
0.07
0.03
1.92
0.64
1.92
16
16
9.6
>16
ND
0.06
9.6
>16
ND
0.19
0.01
>100
0.07
0.02
>100
0.28
0.04
>100
10
0.7
>80
>80
19
0.48
90
400
400
ND
ND
9.2
5.9
>80
>80
>16
>16
>16
16
>16
>100
3.2
>16
>16
>10
>3.2
240
>16
>16
3.2
7.1
0.07
>100
ND
ND
g400
>100
g400
400
GCV (2)
BVDU
3
4
5
6
7
8
9
10
11
12
13
14
15
16
0.006
0.02
20
0.7
0.4
0.02
0.43
0.1
1.92
1.92
1.92
9.6
>100
>100
9.2 >200
400
20
2
1.3
70
2
0.2
0.02
0.2
0.2
9.6
3.2
1.92
NDc
ND
400
>100
>90
>40
>40
>80
>400
16
>16
>16
>16
>16
>100
>200
>16
>16
2
400
0.7
0.005
0.4
0.2
9.6
>100
>175
>40
>40
>80
240
>70
>100
>175
250
150
400
0.3
0.5
0.93
0.15
1.92
1.92
3.2
9.6
9.6
6.4
82
21
9.6
9.6
1.92
>16
>16
12.8
3.2
1.92
>400
16
80
80
80
>16
>16
>16
9.6
>16
ND
>16
>16
>16
>16
>100
>200
>16
>16
>10
>3.2
>400
>16
>80
>240
9.6
5.8
>16
16
g80
g80
g100
300
g16
80
>16
>100
0.11
1.92
>16
>10
>3.2
1.92
>16
>100
0.16
1.92
>16
>10
>3.2
9.6
0.13
0.03
0.03
>16
ND
0.1
9.6
>16
ND
>3.2
9.6
>16
>100
17
18
0.07
3.2
16
>16
ND
>3.2
9.6
>16
>16
10
>3.2
48
0.1
19-21, 24
22
23
25
26
27
28
40
>3.2
9.6
0.01
0.005
0.005
>3.2
9.6
0.08
0.005
0.005
>3.2
240
>16
>80
>400
g16
>400
80
g80
>400
0.02
0.005
0.02
0.13
0.08
0.005
0.08
0.02
0.02
0.04
0.02
a
b
Concentration required to reduce virus-induced cytopathicity by 50%. Concentration required to cause a microscopically detectable
change in normal cell morphology. The results listed are mean values of two or more independent determinations. c Not determined.
Introduction of a methyl group in the 7 position of
the 6-phenyl derivative gave rise to compound 17, the
most potent fluorescent tricyclic analogue of acyclovir
found so far. It turned out to be even slightly more active
against various strains of TK+ HSV-1 and HSV-2 and
distinctly more active against TK- HSV-1 than acyclo-
vir. Unfortunately none of the other 6-aryl-7-substituted
TACV derivatives (18-20) showed any selective anti-
viral activity. For compounds 19 and 20, bearing bulky
benzhydrylphenyl group in the 7-position this may be
due to steric reasons, which, as discussed above, proved
important for mono 6-substituted TACV derivatives.
Another bulky group, trityl, in the 7 position of com-
pound 21 similarly inactivated 6-methyl-TACV. When
the OH group of the acyclic moiety of 21 was blocked
with TBDMS group, compound 22 thus formed became
quite toxic but displayed anti-VV activity (SI 20). The
substituent in the 7 position may have a significant role
in the biological activity of TACV. The activity is very
sensitive to structural changes of substituents in this
position. Replacement of 7-methyl of 17 with 7-phenyl
group resulted in compound 18, which was nonselective
and highly cytotoxic. The effect was not a steric one
because replacement of the 7-methyl group of 7-Me-
TACV (moderately active against HSV-1 and HSV-2, on
average ∼100 fold less than ACV)3 by hydroxymethyl
resulted in 23, which was totally nonselective and toxic.
6-Et TACV5 turned out to be about 10-100-fold less
active than ACV and nontoxic. The tricyclic ganciclovir
congeners 27 and 28, corresponding to 8 and 17, were
active against TK+HSV-1 and TK+HSV-2 similarly to
parent GCV.
Con clu sion s
From the 22 compounds synthesized, the fluorescent
tricyclic analogues of acyclovir 6-(4-MeOPh)-TACV 8
and 7-Me-6-Ph-TACV 17 as well as of ganciclovir 6-(4-
MeOPh)-TGCV 27 and 7-Me-6-Ph-TGCV 28 showed
similar antiherpetic potency as the parent compounds,
acyclovir and ganciclovir. The antiherpetic activity was
found to be strongly dependent on the nature and steric
demands of the substituents in the 6 and/or 7 position.
To obtain 6-Ar-TACV and 6-Ar-TGCV with improved
properties (stronger fluorescence, better solubility, bet-
ter transport), variations in the 4 position of phenyl ring
should be investigated. Present data and further studies
on the modulation of biological activity of TACV system
with nonsteric demanding substituents in the 7 position
may contribute to a better understanding of the struc-
ture-activity relationships of the tricyclic analogues of
ACV and GCV.
Exp er im en ta l Section
Gen er a l Meth od s. The methods are as described previ-
ously.11
Syn th etic P r oced u r es. General procedure for the prepa-
ration of 3,9-dihydro-3-[(2-hydroxyethoxy)methyl]-9-oxo-5H-
imidazo[1,2-a]purines 7-14, 17, 18, and 3,9-dihydro-3-[(1,3-
dih ydr oxy-2-pr opoxy)m et h yl]-9-oxo-5H -im ida zo[1,2-a ]-
purines 25-28 substituted in the 6 or 6,7 positions was
according to ref 3. 3,9-Dihydro-3-[(2-hydroxyethoxy)methyl]-
7-hydroxymethyl-9-oxo-5H-imidazo[1,2-a]purine 23 was pre-
pared from 1 following the procedure described for guanosine.9
In the case of the reaction of 1 and 2 with 2-bromopro-
piophenone additional reaction products (17′ and 28′) were
isolated chromatographically as fractions moving faster than
the main product. 1-(1-Benzoylethyl)-9-[(2-hydroxyethoxy)-

Brief Articles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4287
methyl]guanine (17′): yield 16%; mp 73-75 °C (propanol-2);
UV (MeOH) λmax 245 nm (20.9), 283 nm (10.6); 1H NMR
(DMSO-d6) δ 1.57 (d, 3H, CH3), 3.43-3.50 (m, 4H, CH2CH2),
4.67 (t, 1H, OH), 5.42 (s, 2H, NCH2O), 6.25 (brs, 2H, NH2),
6.47 (q, 1H, CH), 7.54-7.71, 8.02-8.05 (2 × m, 5H, Ph), 8.03
(s, 1H, 8-H); 13C NMR (DMSO-d6) δ 17.41 (CH3), 59.71 (CH2-
OH), 70.34 (CH2CH2OH), 72.01 (NCH2O), 72.73 (CH-CH3),
113.29 (C-5), 128.26, 128.90, 133.52, 134.33 (Ph), 140.37 (C-
8), 154.55 (C-4), 159.02, 159.32 (C-2, C-6), 197.67 (C)O); MS
(nba): calcd for (M + 1)+ 358.15151, found 358.15152. 1-(1-
Benzoylethyl)-9-[(1,3-dihydroxy-2-propoxy)methyl]guanine
(28′): yield 19%; mp 142-145 °C (propanol-2); UV (MeOH)
7-15, 17, 18, 23, 25-28. This material is available free of
charge via the Internet at http://pubs.acs.org.
Refer en ces
(1) Boryski, J .; Golankiewicz, B.; De Clercq, E. Synthesis and
antiviral activity of novel N- substituted derivatives of acyclovir.
J . Med. Chem. 1988, 31, 1351-1355.
(2) Boryski, J .; Golankiewicz, B.; De Clercq, E. Synthesis and
antiviral activity of 3-substituted derivatives of 3,9-dihydro-9-
oxo-5H-imidazo[1,2-a]purines, tricyclic analogues of acyclovir
and ganciclovir. J . Med. Chem. 1991, 34, 2380-2383.
(3) Golankiewicz, B.; Ostrowski, T.; Andrei, G.; Snoeck, R.; De
Clercq, E. Tricyclic analogues of acyclovir and ganciclovir.
Influence of substituents in the heterocyclic moiety on the
antiviral activity. J . Med. Chem. 1994, 37, 3187-3190, and
references therein.
1
λmax 245 nm (15.2), 283 nm (7.7); H NMR (DMSO-d6) δ 1.57
(d, 3H, CH3), 3.27-3.47 (m, 4H, 2 × CH2), 3.52-3.58 (m, 1H,
CH), 4.64 (t, 2H, 2 × OH), 5.52 (s, 2H, NCH2O), 6.23 (brs, 2H,
NH2), 6.48 (q, 1H, CH), 7.54-7.71, 8.03-8.06 (2 × m, 5H, Ph),
8.03 (s, 1H, 8-H); 13C NMR (DMSO-d6) δ 17.48 (CH3), 60.86
(CH2CHCH2), 71.52 (NCH2O), 72.59 (CH-CH3), 80.16 (CH2CH-
CH2), 113.38 (C-5), 128.38, 128.94, 133.48, 134.56 (Ph), 140.32
(C-8), 154.68 (C-4), 159.03, 159.50 (C-2, C-6), 197.60 (C)O);
MS (nba): calcd for (M + 1)+ 388.15950, found 388.16208.
Other 6,7-disubstituted (19-22) and 7-substituted (24)
TACV derivatives were obtained as described previously.10,11
(4) Zielenkiewicz, W.; Golankiewicz, B.; Perlovich, G. L.; Koz´biał,
M. Aqueous solubilities, infinite dilution activity coefficients and
octanol-water partition coefficients of tricyclic analogues of
acyclovir. J . Solution Chem. 1999, 28, 731-745.
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A. A transferred NOE study of a tricyclic analogue of acyclovir
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base) and wyosine (Yt) from Torulopsis utilis phenyl transfer
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Ack n ow led gm en t. This work was supported by the
Polish Committee for Scientific Research (KBN) (grants
no 4PO5F03008 and 4PO5F00516), the Flemish FWO
(Fonds voor Wetenschappelijk Onderzoek) (Krediet
G.0104.98) and the Geconcerteerde Onderzoeksacties
(Vlaamse Gemeenschap)(grant 00/12). We thank Anita
Van Lierde and Frieda De Meyer for excellent technical
assistance.
(8) Ostrowski, T.; Zeidler, J .; Goslinski, T.; Golankiewicz, B. Sub-
stituent-directed aralkylation and alkylation reactions of the
tricyclic analogues of acyclovir and guanosine. Nucleosides,
Nucleotides Nucleic Acids 2000, 19, 1911-1929.
(9) Nair, V.; Offerman, R. J . Ring-extended products from the
reaction of epoxy carbonyl compounds and nucleic acid bases.
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(10) Zeidler, J .; Golankiewicz, B. A case of unusual sterically driven
C-tritylation reaction of tricyclic analogues of acyclovir. Tetra-
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(11) Goslinski, T.; Zeidler, J .; Golankiewicz, B. Unusual tritylation
reactions of tricyclic analogues of acyclovir and an attempt to
elucidate their mechanism. Helv. Chim. Acta 2000, 83, 373-379.
Su p p or tin g In for m a tion Ava ila ble: Preparation condi-
tions, physical properties, spectral (1H NMR, UV, fluorescence)
characteristics and elemental analysis data of compounds
J M010922S