Toward the discovery of dual HCMV-VZV inhibitors: Synthesis, structure activity relationship analysis, and cytotoxicity studies of long chained 2-uracil-3-yl-N-(4-phenoxyphenyl)acetamides

Article abstract of DOI:10.1016/j.bmc.2015.09.033

The need for novel therapeutic options to fight herpesvirus infections still persists. Herein we report the design, synthesis and antiviral evaluation of a new family of non-nucleoside antivirals, derived from 1-[ω-(4-bromophenoxy)alkyl]uracil derivatives - previously reported inhibitors of human cytomegalovirus (HCMV). Introduction of the N-(4-phenoxyphenyl)acetamide side chain at N³ increased their potency and widened activity spectrum. The most active compounds in the series exhibit submicromolar activity against different viral strains of HCMV and varicella zoster virus (VZV) replication in HEL cell cultures. Inactivity against other DNA and RNA viruses, including herpes simplex virus 1/2, points to a novel mechanism of antiviral action.

Full text of DOI:10.1016/j.bmc.2015.09.033

Accepted Manuscript
Toward the discovery of dual HCMV-VZV inhibitors: synthesis, structure ac-
tivity relationship analysis, and cytotoxicity studies of long chained 2-uracil-3-
yl-N-(4-phenoxyphenyl)acetamides
Denis A. Babkov, Anastasia L. Khandazhinskaya, Alexander O. Chizhov,
Graciela Andrei, Robert Snoeck, Katherine L. Seley-Radtke, Mikhail S.
Novikov
PII:
S0968-0896(15)30058-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.09.033
BMC 12580
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 August 2015
15 September 2015
19 September 2015
Please cite this article as: Babkov, D.A., Khandazhinskaya, A.L., Chizhov, A.O., Andrei, G., Snoeck, R., Seley-
Radtke, K.L., Novikov, M.S., Toward the discovery of dual HCMV-VZV inhibitors: synthesis, structure activity
relationship analysis, and cytotoxicity studies of long chained 2-uracil-3-yl-N-(4-phenoxyphenyl)acetamides,
Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.09.033
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Toward the discovery of dual HCMV-VZV
inhibitors: synthesis, structure-activity
relationship analysis, and cytotoxicity studies
of long chained 2-uracil-3-yl-N-(4-
Leave this area blank for abstract info.
phenoxyphenyl)acetamides
Denis A. Babkova, Anastasia L. Khandazhinskayab, Alexander O. Chizhovc, Graciela Andreid, Robert
Snoeckd, Katherine L. Seley-Radtkee*, and Mikhail S. Novikova
aDepartment of Pharmaceutical & Toxicological Chemistry, Volgograd State Medical University, Pavshikh
Bortsov Sq., 1, Volgograd 400131, Russia
bEngelhardt Institute of Molecular Biology, Russian Academy of Science, Vavilova 32, Moscow 119991, Russia
cZelinsky Institute of Organic Chemistry, Russian Academy of Science, Leninsky pr., 47, Moscow 119991,
Russia
dRega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, Leuven B-3000, Belgium
eDepartment of Chemistry & Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle,
Baltimore, MD 21250, USA

Bioorganic & Medicinal Chemistry
Toward the discovery of dual HCMV-VZV inhibitors: synthesis, structure activity
relationship analysis, and cytotoxicity studies of long chained 2-uracil-3-yl-N-(4-
phenoxyphenyl)acetamides
Denis A. Babkova, Anastasia L. Khandazhinskayab, Alexander O. Chizhovc, Graciela Andreid, Robert
Snoeckd, Katherine L. Seley-Radtkee*, and Mikhail S. Novikova
aDepartment of Pharmaceutical & Toxicological Chemistry, Volgograd State Medical University, Pavshikh Bortsov Sq., 1, Volgograd 400131, Russia
bEngelhardt Institute of Molecular Biology, Russian Academy of Science, Vavilova 32, Moscow 119991, Russia
cZelinsky Institute of Organic Chemistry, Russian Academy of Science, Leninsky pr., 47, Moscow 119991, Russia
dRega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, Leuven B-3000, Belgium
eDepartment of Chemistry & Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
The need for novel therapeutic options to fight herpesvirus infections still persists. Herein we
report the design, synthesis and antiviral evaluation of a new family of non-nucleoside
Revised
antivirals, derived from 1-[ω-(4-bromophenoxy)alkyl]uracil derivatives – previously
Accepted
Available online
reported inhibitors of human cytomegalovirus (HCMV). Introduction of the N-(4-
phenoxyphenyl)acetamide side chain at N3 increased their potency and widened activity
spectrum. The most active compounds in the series exhibit submicromolar activity against
different viral strains of HCMV and varicella zoster virus (VZV) replication in HEL cell
cultures. Inactivity against other DNA and RNA viruses, including herpes simplex virus 1/2,
points to a novel mechanism of antiviral action.2009 Elsevier Ltd. All rights reserved.
Keywords:
human cytomegalovirus
varicella zoster virus
antiviral
non-nucleoside inhibitor
uracil
* corresponding author: kseley@umbc.edu
1-410-455-8684 (O); 1-410-455-2608 (Fax)
1. Introduction
Human cytomegalovirus (HCMV, or human
herpesvirus-5) and varicella zoster virus (VZV, also known as
human herpesvirus-3) belong to the viral family known as
Herpesviridae. Despite modern prevention and treatment
strategies, they remain a common opportunistic pathogen
associated with serious morbidity and mortality, particularly
in immunocompromised individuals such as transplant
recipients1 and AIDS patients.2,3 All drugs currently licensed
for the treatment of HCMV and VZV infections (Figure 1)
target the viral DNA polymerase. Unfortunately they are
associated with severe toxicity issues, including marrow
toxicity for ganciclovir, valganciclovir, and cidofovir, and
renal toxicity for foscarnet and cidofovir.4,5 The emergence of
drug resistance is also a significant problem.6 Moreover, our
understanding of the full spectrum of risks of HCMV infection
and its interactions with the host immune system remains far
from complete.7 While the past two decades have seen
progress toward novel treatments for herpesviruses, the
need for better drugs that exhibit an improved toxicity profile
persists.8
Figure 1. Clinically approved anti-HCMV and anti-VZV drugs.
Previously, we have described a series of 1-[ω-
(phenoxy)alkyl]uracil derivatives that were found to exhibit
high specificity and promising inhibitory activity against
HCMV replication in HEL cell cultures with EC50 values within
5.5–12 µM range.9 These results provided strong impetus to
further explore structure-activity relationships and the
antiviral activity spectrum of similar scaffolds. The present
paper
describes
lead
development
of
N-(4-
phenoxyphenyl)acetamide derivatives (Figure 2).

Figure 2. Target compounds.
2. Results and Discussion
2.1. Chemistry
Scheme 1. Reagents and conditions: (a) K2CO3, acetone, reflux, 12 h; (b)
2,4-bis(trimethylsilyloxy)-5-R2-pyrimidine, neat, 160–170 °C, 1 h.
The synthesis of the compound library relied on
three key steps: synthesis of 1-[ω-(phenoxy)alkyl]uracil
derivatives and related compounds, synthesis of 2-chloro-N-
(4-phenoxyphenyl)acetamides, and their subsequent
conjugation to realize the target compounds. Based on the
successful synthesis of several previously reported
compounds,9
a series of analogues were synthesized
following the classical synthetic approach reported in Scheme
1. This first set of derivatives is characterized by focused
modifications on the aromatic ring. The synthesis of
compounds 4a-j started from commercially available phenols
1 (R1 = H, 3-Br, 4-Br, 4-Ph or 4-CN), which were treated with
4-fold excess of α,ω-dibromoalkanes 2 (n = 1–4, 6 or 8) to
produce bromides 3a-i according to known procedures.10-12
modified silyl Hilbert-Johnson reaction, i.e. condensation of
equimolar amounts of 2,4-
A
bis(trimethylsilyloxy)pyrimidines13 with bromides 3a-e, was
performed at 160–170 °C in the absence of solvent9 to afford
target compounds 4a-e in 76-88% yield. Compounds 4f-j
comprising 3, 4, 6, and 8 methylene units, respectively, were
obtained in an analogous manner as was used for compounds
4a-e (Scheme 1).
Scheme 2. Reagents and conditions: (a) chloroacetyl chloride, K2CO3,
DCE, 0 °C, 2 h; (b) 4a-i, K2CO3, DMF, 80 °C → RT, 24 h (for 7a-i and 7k-m)
or 4j, NaH, DMF, RT, 24 h (for 7j).
Intermediate 2-chloroacetamides 6a-c were readily
obtained via acylation of the commercially available 4-
(phenoxy)- (5a), 4-(benzyloxy)- (5b) and 4-benzyl- (5c)
anilines with chloroacetyl chloride promoted by anhydrous
K2CO3 in aprotic media.14 2-Chloroacetamide 6d was obtained
as we described previously.15 Treatment of potassium salts of
uracil derivatives 4a-j with 2-chloroacetamides 6a-d in
anhydrous DMF afforded target compounds 7a-i and 7k-m in
good yields (Scheme 2). To avoid possible complications due
to iodine elimination, compound 7j was obtained from 4j and
2-chloroacetamide 6a employing NaH as the base.16 Target
compound 7j was subsequently isolated in 86% yield.15
In order to obtain benzophenone derivative 9, the
appropriate building blocks were obtained in a somewhat
different order. First, acid 8 was synthesized quantitatively
via treatment of 4b with ethyl bromoacetate in the presence
of K2CO3, followed by hydrolysis using LiOH according to
published procedures.17 Subsequent conversion of acid 8 into
the corresponding acyl chloride, followed by condensation
with the N-trimethylsilyl derivative of 4-(benzoyl)aniline led
to 9 in 57% yield (Scheme 3).15 It should be noted that
utilization of base-free conditions for the synthesis of 9
avoids the by-products that typically arise from Knoevenagel
condensation between the benzophenone carbonyl and the
active methylene of the acetic acid residue.18

Scheme 5. Reagents and conditions: (a) HO(CH2)4OH, KOH, 24 h; (b)
TsCl, Py, DCE, 0–5 °C; (c) uracil, K2CO3, DMF, 80 °C, 24 h; (d) 6a, K2CO3,
DMF, 80 °C → RT, 24 h.
Scheme 3. Reagents and conditions: (a) (i) ethyl bromoacetate, K2CO3,
DMF, 80 °C → RT, 24 h; (ii) LiOH, EtOH/H2O, RT, 2 h; (b) (i) SOCl2, DCE,
reflux, 1 h; (ii) DCE, −15 °C, overnight.
In addition, an analogue featuring two oxygen atoms
in the spacer chain was pursued as shown in Scheme 6.
Chloromethyl ether 18 was obtained via the classical Henry
method.21 Condensation with equimolar amount of 2,4-
To further explore the SAR of these compounds we
then designed a second set of analogues featuring a modified
spacer linking the N1 of the uracil with the aromatic moiety.
Synthesis of the various target structures demanded
different, but related approaches. Copper–catalyzed coupling
of phenylmagnesium bromide (10) with excess of 1,6-
dibromohexane19 in THF media produced bromide 11, which
was condensated with 2,4-bis(trimethylsilyloxy)pyrimidine
to afford 1-(6-phenylhexyl)uracil (12), however in an
unexpectedly mediocre 30% yield (Scheme 4). This was
somewhat surprising since our previous observations for this
reaction involving similar ω-(phenoxy)alkyl bromides 3a-i as
alkylating agents were, in contrast, quite efficient.9
bis(trimethylsilyloxy)pyrimidine
led
to
1-([2-(4-
bromobenzyl)ethoxy]methyl)uracil (19) in 67% yield. Being
more reactive than bromides 3a-i and 11, the reaction
proceeds under rather mild conditions in 1,2-dichloroethane
at ambient temperature.22
Scheme 6. Reagents and conditions: (a) HO(CH2)2OH, KOH, 12 h; (b)
(CH2O)n, HCl, DCE, 0 C, 2 h; (c) 2,4-bis(trimethylsilyloxy)pyrimidine,
DCE, RT, 16 h; (d) 6a, K2CO3, DMF, 80 C → RT, 24 h.
Next, the flexible spacer was replaced with a rigid 4-
(4-bromophenoxy)benzyl moiety, which was introduced at
the N1 of uracil employing previously reported procedures.23
As outlined in Scheme 7, 2,4-bis(trimethylsilyloxy)pyrimidine
and 4-(4-bromophenoxy)benzyl bromide (21) were reacted
in DCE at reflux for 20 h to give 23. Finally, to investigate the
potential role of an amide nitrogen, methylation of 7b with
methyl iodide/NaH in DMF successfully produced compound
24 (92% yield, Scheme 8).
Scheme 4. Reagents and conditions: (a) 1,6-dibromohexane, 5 mol%
Li2[CuICl2], THF, RT, 2 h; (b) 2,4-bis(trimethylsilyloxy)pyrimidine, neat,
160–170 °C, 1 h; (c) 6a, K2CO3, DMF, 80 °C → RT, 24 h.
Silyl Hilbert-Johnson reactions between 2,4-
bis(trimethylsilyloxy)pyrimidine and alkyl bromides are
known to be associated with the release of trimethylsilyl
bromide. This subsequently, and efficiently, cleaves dialkyl
ethers at elevated temperatures.20 As a result, in order to
obtain 1-[4-(4-bromobenzyloxy)butyl]uracil (16), it was
necessary to alkylate uracil with tosylate 15, which was
obtained via a Williamson ether synthesis between 1,4-
butanediol and 4’-bromobenzyl bromide18 followed by
condensation with 4-toluenesulfonyl chloride in the presence
of pyridine (Scheme 5).
Scheme 7. Reagents and conditions: (a) 2,4-bis(trimethylsilyl-
oxy)pyrimidine, DCE, reflux, 24 h; (b) 6a, K2CO3, DMF, 80 C → RT, 24 h.

strains), and herpes simplex virus 1 and 2 (HSV-1 and HSV-
2)] in HEL cell cultures. The results for HCMV and VZV are
summarized, respectively, in Tables 1 and 2. The results
reveal that the majority of the target compounds share
marked inhibitory properties. In examining the results for
HCMV, the SAR studies revealed that the nature of the R1
substituent significantly influences the antiviral activity: 7a
(R1 = H) ≈ 7c (R1 = 3-Br) < 7b (R1 = 4-Br) < 7e (R1 = 4-Ph) ≈
7d (R1 = 4-CN). Since compound 7b showed a selectivity
(ratio CC50/EC50) of about 9 for both HCMV strains and lower
toxicity (alteration of cell morphology) than compound 7d, it
was considered as the basis for further modifications.
Scheme 8. Reagents and conditions: (a) MeI, NaH, DMF, 0 C, 4 h.
2.2. Biological activities
2.2.1.
Anti-HCMV activity
The antiviral activity of the target compounds was
evaluated in vitro against different human herpesviruses [i.e.
HCMV (AD-169 and Davis strains), VZV (OKA and 07-1
Table 1. Anti-HCMV activity in human embryonic lung (HEL) cells
Antiviral activity, EC50 (μM)a
Cytotoxicity (μM)
Comp R1
Spacer
O(CH2)5
R2
X
Cell morphology (MCC)b Cell growth (CC50)c
AD-169 strain Davis strain
7a
7b
7c
H
H
H
H
H
H
H
H
H
H
I
O
O
3.6 ± 0.6
0.93 ± 0.75
2.0 ± 0.3
0.53 ± 0.12
0.51 ± 0
3.8 ± 2.0
1.79 ± 0
0.29 ± 0.06
<0.032
4.0 ± 2.8
0.97 ± 0.95
3.1 ± 1.3
0.44 ± 0.11
1.04 ± 0.33
3.9 ± 3.4
1.64 ± 0
0.20 ± 0.18
<0.032
≥20
20
5.4 ± 2.0
8.7 ± 5.4
6.3 ± 1.3
4.9 ± 1.3
2.8 ± 0.5
15.2 ± 6.8
7.7 ± 7.2
11.7 ± 11.7
-
4-Br
3-Br
4-CN
4-Ph
4-Br
4-Br
4-Br
4-Br
4-Br
4-Br
4-Br
-
O(CH2)5
O(CH2)5
O(CH2)5
O(CH2)5
O(CH2)3
O(CH2)4
O(CH2)6
O(CH2)8
O(CH2)5
O(CH2)5
O(CH2)5
-
O
≥100
≥4
7d
7e
7f
O
O
20
O
≥100
≥20
4
7g
7h
7i
O
O
O
≥0.16
100
4
7j
O
>20
>20
-
7k
7l
H
H
-
CH2
OCH2
-
0.44 ± 0.11
>20
0.57 ± 0.33
4
8.7 ± 1.7
-
≥20
20
7m
9
>4
>4
-
4-Br
H
O(CH2)5
(CH2)6
H
H
H
H
H
-
CO
O
1.79 ± 0
>20
1.64 ± 0
>20
≥20
100
≥4
5.8 ± 0.6
-
13
17
20
23
24
GCV
CDV
4-Br
CH2O(CH2)4
O
1. 8 ± 0.3
>20
≥1.2 ± 0.6
>20
4.8 ± 1.1
-
4-Br CH2O(CH2)2OCH2
O
100
4
4-Br
-
p-OC6H4CH2
O
>0.8
>0.8
-
-
-
0.98 ± 0.93
13.9 ± 10.6
0.83 ± 0.40
1.29 ± 1.27
7.8 ± 3.4
0.84 ± 0.29
≥4
6.1 ± 2.3
≥319 ± 102
≥208 ± 116
>350
>300
a Effective concentration required to reduce virus plaque formation by 50%. Virus input was 100 plaque forming units (PFU).
b Minimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology.
c Cytotoxic concentration required to reduce cell growth by 50%.

Notably, the nature of the spacer between the uracil
and the left aromatic "wing" significantly influences the anti-
HCMV activity of the compounds. For example, elongation of
the spacer from 3 to 8 methylene groups progressively
lowers EC50 values, as demonstrated by 7f (n = 3) < 7g (n = 4)
< 7b (n = 5) < 7h (n = 6) < 7i (n = 8). Specifically, introduction
of five methylene groups to 7f to give 7i increases the activity
43-73 fold, however a concomitant increase in cytotoxicity
was also observed.
the cytotoxic methylene derivative 7k and the less potent 7l
(X = OCH2) and 9 (X = CO). In addition, N-methyl derivative
24 and compound 7m, both lacking the phenoxy core,
indicated that the N-(4-phenoxyphenyl)acetamide side chain
is likely a key element required for antiviral properties of the
scaffold.
Table 2. Anti-VZV activity in human embryonic lung (HEL)
cells
Antiviral activity, EC50 (μM)a
Cytotoxicity (μM)
Replacement of the oxygen atom for a methylene
(compound 13) resulted in a loss of activity, which points to
the potential role of an ether functionality in the spacer
region. A shift in position of the oxygen on the aromatic
moiety did not markedly affect activity (1.2 to 2-fold change
of 17 as compared to 7b), while introduction of an additional
oxygen (compound 20) renders the compound completely
inactive. Use of a rigid spacer (compound 23) also proved
unfavorable. Overall, incorporation of an alkoxyphenyl side
chain proved to be optimal.
Cell
morphology
(MCC)b
Cell
growth
(CC50
Comp
TK+ VZV
(OKA strain) 1 strain)
TK- VZV (07-
c
)
7a
7b
7c
>100
>100
>100
>100
>100
>100
100
-
1.6 ± 0.7
>100
2.34 ± 0.18
>100
8.7 ± 5.4
-
7d
7e
>100
>100
-
>20
>20
-
7f
5.4 ± 0.5
3.1 ± 0.1
0.8 ± 0
≥0.12 ± 0.05
>100
4.6 ± 0.9
2.8 ± 0.6
0.51 ± 0.01
≥0.16 ± 0
>100
≥100
>100
4
15.2 ± 6.8
Interestingly, the one modification made to the
uracil moiety proved deleterious. Substitution of the H5 of
the uracil with iodine rendered 7j completely inactive.
7g
7.7 ± 7.2
7h
7i
11.7 ± 11.7
0.8
11.7 ± 11.7
Next, the SAR studies involving modifications to the
right "wing" revealed that the presence of a second benzene
core in the acetamide side chain is mandatory for inhibitory
properties (compound 7k). Investigation of the role of the
linker between the aromatic residues shows that oxygen
provides the optimal activity profile. The corresponding
methylene analogue retains a similar level of activity, but is
more cytotoxic (7k), while use of a carbonyl (9) or OCH2 (7l)
group renders the compound significantly less active. N-
Methylation of the parent 7b has no significant influence on
the potency as compound 24 shows.
7j
>100
≥4
-
7k
7l
0.8
4
-
>4
11.7
≥20
-
7m
9
>4
15
≥20
-
>100
>100
>100
>100
>100
>100
20
-
13
17
20
23
24
Acyclovir
>100
>100
-
>100
>100
-
>100
>100
-
>4
>4
-
2.2.2.
Anti-VZV activity
8.0 ± 0.3
1.82 ± 0.67
7.7 ± 1.5
54.5 ± 50.6
≥100
>440
6.0 ± 2.4
>440
Similar activity trends were observed for the anti-
VZV properties of the target compounds. The 4-bromo-
substituted compound 7b blocks VZV replication at 1.14 µМ
(OKA strain), which is comparable with acyclovir. At the same
time, thymidine kinase deficient strain 07-1 was also
susceptible to 7b. Since thymidine kinase is required for the
activation of nucleoside analogues (e.g. acyclovir and
brivudin), it is likely these compounds are acting as
nonnucleoside inhibitors.24 Other substituents at R1 were
found inactive (7a, 7c-e). As was noted for the HCMV
inhibition, elongation of the spacer has a pronounced positive
impact on the anti-VZV properties. Compound 7i featuring 8
methylene units proved the most active, with EC50's of ≥0.12
µМ and ≥0.16 µМ for the OKA and 07-1 strains, respectively.
Again however, an increase in activity was accompanied by
higher cytotoxicity for both 7h and 7i. Other spacer
modifications, including oxygen (13), a position shift (17),
and introduction of additional oxygen (20), or a benzene
moiety (23) all resulted in a loss of activity. Thus, the nature
of the spacer once again plays a crucial role in the antiviral
properties.
0.029 ±
0.017
Brivudin
38.6 ± 45.5
>300
≥79.3
a Effective concentration required to reduce virus plaque
formation by 50%. Virus input was 100 plaque forming units
(PFU).
b Minimum cytotoxic concentration that causes a
microscopically detectable alteration of cell morphology.
c Cytotoxic concentration required to reduce cell growth by
50%.
Interestingly, the compounds were inactive against HSV-1
and HSV-2. This is somewhat surprising since most anti-
herpes drugs targeting viral DNA polymerase share a broad
spectrum activity against the various herpesviruses.25-29 The
mechanism of action for compounds 7a-m, 9, 13, 17, 20, 23,
and 24 however, remains unclear, thus further investigation
is warranted.
In addition, the target compounds were screened against a
large panel of additional DNA and RNA viruses. No activity
was observed for Vaccinia virus, Vesicular stomatitis virus,
In terms of substituents, introduction of
a
Coxsackie virus B4, Influenza
A
virus H1N1 subtype,
virus,
substituent at C5 of uracil renders compound 7j completely
inactive. Substitution of the oxygen linker between aromatic
residues on the acetamide side chain (right wing) led to more
Influenza virus H3N2 subtype, Influenza
A
B
Parainfluenza-3 virus, Reovirus-1, Sindbis virus, Punta Toro
virus, Feline Corona Virus, HIV-1 and HIV-2.

3. Conclusions
(1H, dd, J = 7.8 and 2.2 Hz, Ura-H-5), 6.93 (1H, dd, J = 8.3 and
2.2 Hz, H-6’), 7.06-7.14 (2H, m, H-2’, H-4’), 7.22 (1H, t, J = 8.2
Hz, H-5’), 7.65 (1H, d, J = 7.8 Hz, Ura-H-6), 11.22 (1H, s, NH);
13C NMR (100 MHz, DMSO-d6): δ 22.6, 28.4, 47.6, 67.9, 101.1,
114.3, 117.5, 122.4, 123.6, 131.5, 146.0, 151.3, 160.0, 164.1.
Herein we have described the synthesis, preliminary
biological evaluation and SAR studies for a series of novel
uracil derivatives as potential dual HCMV-VZV agents. The
majority of the synthesized compounds exhibited potent
antiviral activity in cell cultures. Experimental data
4.2.1.2. 1-(6-Phenylhexyl)uracil (12)
undoubtedly
shows
that
ω-(4-bromophenoxy)alkyl
An equimolar mixture of the 1-bromo-6-phenylhexane
substituent at N1 and N-(4-phenoxyphenyl)acetamide side
chain at N3 of uracil are the key elements responsible for both
anti-HCMV and anti-VZV activity. The mode of action for the
reported series is not yet fully elucidated and will be
published elsewhere once it has been studied further. In the
meantime, the most active compounds in the series 7b, 7h
and 7i represent an excellent starting point for further
optimization.
(11)
(2.15
g,
8.91
mmol)
and
2,4-
bis(trimethylsilyloxy)pyrimidine (2.26 g, 8.81 mmol) was
heated at 160-170 C for 1 h. The resulting melt was
dissolved in EtOAc (50 mL) and treated with i-PrOH (10 mL).
Precipitate was filtered off, and the filtrate evaporated, taken
up into acetone (30 mL) and EtOAc (5 mL) and purified by
short-column flash chromatography using EtOAc to give 12
as white crystals (0.72 g, 30%); mp 78-79 °C, Rf 0.61 (ethyl
acetate); 1H NMR (400 MHz, DMSO-d6) δ 1.26-1.34 (4Н, m,
CH2), 1.55-1.59 (4Н, m, CH2), 2.55 (2H, t, J = 7.4 Hz, PhCH2),
3.64 (2H, t, J = 7.2 Hz, NCH2), 5.53 (1H, dd, J = 7.8 and 1.6 Hz,
Ura-H-5), 7.13-7.17 (3H, m, H-2’, H-4’, H-6’), 7.26 (2H, t, J =
7.4 Hz, H-3’, H-5’), 7.59 (2H, d, J = 7.8 Hz, Ura-H-6), 11.11 (1H,
s, NH); 13C NMR (100 MHz, DMSO-d6): δ 25.6, 28.2, 28.4, 30.7,
35.1, 47.5, 100.8, 125.6, 128.2, 142.2, 145.6, 151.0, 163.8.
4. Experimental
4.1. General
All reagents were obtained at the highest grade available
from Sigma and Acros Organics, and were used without
further purification unless otherwise noted. Anhydrous DMF
and isopropyl alcohol were purchased from Sigma-Aldrich
Co. Anhydrous acetone, DCE, and EtOAc were obtained by
distillation over P2O5. TLC was performed on Merck TLC Silica
gel 60 F254 plates eluting with the specified solvents and
samples were made visual with a UV lamp, VL-6.LC (France).
Acros Organics (Belgium) silica gel (Kieselgur 60–200 μm,
60A) was used for column chromatography. Yields refer to
spectroscopically (1H and 13C NMR) homogeneous materials.
Melting points were determined in glass capillaries on a Mel-
Temp 3.0 (Laboratory Devices Inc., USA). NMR spectra were
4.2.1.3. 1-[4-[(4-Bromobenzyl)oxy]butyl]uracil
(16)
A mixture of uracil (3.1 g, 27.66 mmol) and K2CO3 (1.40 g,
10.13 mmol) in anhydrous DMF (10 mL) was stirred at 80C
for 40 min. After cooling to room temperature, 4-(4-
bromobenzyloxy)butyl p-toluenesulfonate (15) (3.80 g, 9.19
mmol) was added and stirring continued for 24 h. The
reaction mixture was filtered, concentrated under reduced
pressure and purified by short-column flash chromatography
using a mixture of CHCl3/EtOH (10:1). Analytical sample was
recrystallized from EtOAc to give compound 16 as white
crystals (1.20 g, 37%), mp 95.5-97.5 °C, Rf 0.44 (ethyl
1
obtained using Bruker Avance 400 (400 MHz for H and 100
MHz for 13C) and Bruker Avance 600 (600 MHz for 1H and
150 MHz for 13C) spectrometers in DMSO-d6 or CDCl3 with
tetramethylsilane as an internal standard. High-resolution
mass spectra were measured on Bruker micrOTOF II
instruments using electrospray ionization (HRESIMS). The
measurements were run in positive ion mode (interface
capillary voltage –4500 V) in a mass range from m/z 50 to
m/z 3000 Da; external or internal calibration was performed
with ESI Tuning MixTM (Agilent Technologies). A syringe
injection was used for solutions in MeCN (flow rate = 3
1
acetate); H NMR (400 MHz, DMSO-d6) δ 1.51 (2H, quin, J =
7.4 Hz, CH2), 1.63 (2H, quin, J = 7.4 Hz, CH2), 3.42 (2H, t, J = 6.1
Hz, NCH2), 3.66 (2H, t, J = 7.1 Hz, OCH2), 4.41 (2H, s, CH2O),
5.53 (1H, d, J = 7.8 Hz, Ura-H-5), 7.27 (2H, d, J = 8.1 Hz, H-2, H-
6), 7.53 (2H, d, J = 8.1 Hz, H-3, H-5), 7.63 (1H, d, J = 7.8 Hz,
Ura-H-6), 11.21 (1H, s, NH); 13C NMR (100 MHz, DMSO-d6): δ
25.8, 26.4, 47.6, 69.6, 71.3, 101.1, 120.7, 129.8, 131.5, 138.4,
146.0, 151.3, 164.1.
μL/min). N2 was applied as
a dry gas; the interface
temperature was set at 180 °C.
4.2.1.4. 1-[[2-(4-Bromobenzyloxy)ethoxy]methyl] -
uracil (19)
4.2. Synthesis
To a solution of 2,4-bis(trimethylsilyloxy)pyrimidine (4.58
g, 17.84 mmol) in anhydrous methylene chloride (30 mL) was
4.2.1. Compounds 4a, 4b, 4d-k
The synthesis and characterization data for were reported
added
a
solution
of
[[2-(4-
previously.9
bromobenzyloxy)ethoxy]methoxy]methyl chloride (18) (5.0
g, 17.88 mmol) in methylene chloride (20 mL) and stirred for
16 h at room temperature. Ethanol (95%, 10 ml) was added
and the resulting mixture was stirred for 30 min, filtered,
evaporated to dryness under reduced pressure and purified
by short-column flash chromatography using a mixture of
CHCl3/EtOH (10:1). Analytical sample was recrystallized from
ethyl acetate to give compound 19 as white crystals (4.25 g,
67%), mp 103.5-104.5 °C, Rf 0.39 (ethyl acetate); 1H NMR
(400 MHz, DMSO-d6): δ 3.54 (2H, dd, J = 5.9 and 3.2 Hz, CH2),
3.66 (2H, dd, J = 5.5 and 3.5 Hz, CH2), 4.44 (2H, s, NCH2), 5.10
(2H, s, OCH2), 5.60 (1H, d, J = 7.8 Hz, Ura-H-5), 7.26 (2H, d, J =
8.3 Hz, H-2’, H-6’), 7.52 (2H, d, J = 8.3 Hz, H-3’, H-5’), 7.70 (1H,
d, J = 8.1 Hz, Ura-H-6), 11.33 (1H, s, NH); 13C NMR (100 MHz,
4.2.1.1. 1-[5-(3-Bromophenoxy)pentyl]uracil ( 4c)
An
equimolar
mixture
of
the
1-bromo-5-(3-
bromophenoxy)pentane (3c) (4.35 g, 13.51 mmol) and 2,4-
bis(trimethylsilyloxy)pyrimidine (3.43 g, 13.38 mmol) was
heated at 160-170 C for 1 h. The resulting melt was
dissolved in EtOAc (50 mL) and treated with i-PrOH (10 mL).
The precipitated product was collected and purified by short-
column flash chromatography using EtOAc/DCE (1:5).
Subsequent recrystallization from a mixture of EtOAc/hexane
(2:1) provided the desired product as white crystals (3.87 g,
82%); mp 124.5-126 °C; Rf 0.42 (ethyl acetate); 1H NMR (400
MHz, DMSO-d6): δ 1.38 (2H, quin, J = 7.6 Hz, CH2), 1.62 (2H,
quin, J = 7.5 Hz, CH2), 1.71 (2H, quin, J = 7.5 Hz, CH2), 3.66
(2H, t, J = 7.2 Hz, NCH2), 3.97 (2H, t, J = 6.5 Hz, OCH2), 5.53

DMSO-d6): δ 68.4, 69.2, 71.5, 76.9, 101.9, 120.7, 129.8, 131.5,
138.2, 145.3, 151.4, 163.9.
A mixture of the appropriate 1-substituted uracil 4a-i, 12,
16, 19 or 22 (1.42 mmol) and K2CO3 (0.29 g, 2.10 mmol) in
anhydrous DMF (10 mL) was stirred at 80C for 40 min. After
cooling to room temperature, the corresponding 2-
chloroacetamide 6a-c (1.56 mmol) was added and stirring
continued for 24 h. The reaction mixture was filtered,
concentrated under reduced pressure and purified by short-
column flash chromatography using DCE. Analytical samples
were recrystallized from a mixture of hexane/ethyl acetate
(1:1).
4.2.1.5. 1-[4-(4-Bromophenoxy)benzyl]uracil ( 22)
A solution of 2,4-bis(trimethylsilyloxy)-pyrimidine (3.44 g,
13.40 mmol) and 4-(bromophenoxy)benzylbromide (21) (4.6
g, 13.45 mmol) in anhydrous DCE (50 mL) was heated at
reflux for 24 h, cooled to room temperature and treated with
i-PrOH (15 mL). The resulting precipitate was collected and
purified by short-column flash chromatography using a
mixture of CHCl3/EtOH (10:1). An analytical sample was
recrystallized from a mixture of DMF/i-PrOH/H2O (2:2:1) to
give compound 22 as white crystals (2.60 g, 52%), mp 164-
165.5 °C, Rf 0.46 (ethyl acetate); 1H NMR (400 MHz, DMSO-d6)
δ 4.85 (2H, s, CH2), 5.60 (Hz, dd, J = 7.8 and 2.2 1H, Ura-H-5),
6.95 (2H, d, J = 9.0 Hz, H-2”, H-6”), 7.03 (d, J = 8.6 Hz, 2H, H-3”,
H-6”), 7.35 (d, J = 8.8 Hz, 2H, H-2’, H-6’), 7.54 (d, J = 8.8 Hz,
2H, H-3’, H-5’), 7.78 (d, J = 8.1 Hz, 1H, Ura-H-6), 11.34 (d, J =
2.0 Hz, 1H, NH); 13C NMR (100 MHz, DMSO-d6): δ 50.0, 101.7,
115.4, 119.4, 120.9, 129.9, 132.7, 133.1, 145.9, 151.3, 156.0,
156.4, 164.0.
4.2.3.1. 2-[2,6-Dioxo-3-(5-phenoxypentyl)-3,6-
dihydropyrimidin-1(2H)-yl]-N-(4-phenoxyphenyl)
acetamide (7a)
Yield 92%, mp 114-115.5 °C, Rf 0.39 (DCE/ethyl acetate,
1
1:1); H NMR (400 MHz, DMSO-d6): δ 1.41 (2H, quin, J = 7.1
Hz, CH2), 1.67 (2H, quin, J = 7.1 Hz, CH2), 1.74 (2H, quin, J =
7.8 Hz, CH2), 3.76 (2H, t, J = 7.0 Hz, NCH2), 3.94 (2H, t, J = 6.3
Hz, OCH2), 4.60 (2H, s, COCH2), 5.75 (1H, d, J = 7.9 Hz, H-5),
6.87-6.91 (3H, m, H-4’, H-2”, H-6”), 6.95-6.99 (4H, m, H-2”’, H-
6”’, H-2’, H-6’), 7.10 (1H, dt, J = 7.3 and 1.0 Hz, H-4”’), 7.36
(2H, dt, J = 7.3 and 1.8 Hz, H-3”’, H-5”’), 7.36 (2H, dt, J = 8.6
and 1.3 Hz, H-3’, H-5’), 7.57 (2H, d, J = 9.0 Hz, H-3”, H-5”), 7.78
(1H, d, J = 7.8 Hz, H-6), 10.26 (1H, s, NH); 13C NMR (100 MHz,
DMSO-d6): δ 22.7, 28.5, 28.6, 43.4, 48.8, 67.4, 100.3, 114.7,
118.2, 119.8, 120.7, 121.0, 123.3, 130.0, 130.3, 135.0, 144.9,
151.4, 152.1, 157.6, 158.9, 162.6, 165.4; HRMS: found m/z
500.2181; calcd for C29H29N3O5 [M+H]+ 500.2180.
4.2.2. General procedure for the synthesis of 2 -
chloro-N-(4-substituted phenyl)acetamide 6a-c
Chloroacetyl chloride (0.47 mL, 5.90 mmol) was added
dropwise to a stirred mixture of the appropriate aniline 5a-c
(5.56 mmol) and K2CO3 (0.90 g, 6.51 mmol) in anhydrous DCE
(20 mL) at 0 °C. The reaction mixture was stirred for 2 hours
and allowed to warm to room temperature overnight. The
inorganic materials were filtered through a pad of silica gel
and washed with DCE (25 mL). The filtrate was evaporated
under reduced pressure and the residue was purified by
recrystallization from a mixture of hexane/ethyl acetate (3 :
2).
4.2.3.2. 2-[3-[5-(4-Bromophenoxy)pentyl] -2,6-
dioxo-3,6-dihydropyrimidin-1(2H)-yl]-N-(4-phen-
oxyphenyl)acetamide (7b)
Yield 87%, mp 128-130 °C, Rf 0.47 (DCE/ethyl acetate,
1
1:1); H NMR (400 MHz, DMSO-d6): δ 1.40 (2H, quin, J = 7.8
Hz, CH2), 1.66 (2H,quin, J = 7.2 Hz, CH2), 1.72 (2H, quin, J = 7.3
Hz, CH2), 3.76 (2H, t, J = 7.0 Hz, NCH2), 3.93 (2H, t, J = 6.4 Hz,
OCH2), 4.61 (2H, s, COCH2), 5.75 (1H, d, J = 7.8 Hz, H-5), 6.88
(2H, d, J = 8.9 Hz, H-2”, H-6”), 6.96 (2H, d, J = 8.2 Hz, H-2”’, H-
6”’), 6.98 (2H, d, J = 8.4 Hz, H-2’, H-6’), 7.09 (1H, dt, J = 7.3 and
0.9 Hz, H-4”’), 7.36 (2H, t, J = 7.6 Hz, H-3”’, H-5”’), 7.40 (2H, d, J
= 8.8 Hz, H-3’, H-5’), 7.57 (2H, d, J = 8.9 Hz, H-3”, H-5”), 7.77
(1H, d, J = 8.0 Hz, H-6), 10.27 (1H, s, NH); 13C NMR (100 MHz,
DMSO-d6): δ 22.3, 25.5, 28.1, 43.2, 48.5, 67.6, 100.0, 111.8,
116.7, 118.0, 119.5, 120.7, 123.0, 130.0, 132.1, 134.7, 144.6,
151.1, 151.8, 157.3, 157.9, 162.3, 165.1; HRMS: found m/z
578.1287; calcd for C29H28BrN3O5 [M+H]+ 578.1285.
4.2.2.1. 2-Chloro-N-(4-phenoxyphenyl)acetamide
(6a)
Yield 80%, mp 105-106 °C, Rf 0.62 (hexane/ethyl acetate,
1:1); 1H NMR (600 MHz, DMSO-d6): δ 4.24 (2H, s, COCH2),
6.97 (2H, d, J = 8.7 Hz, H-3’, H-5’), 7.00 (2H, d, J = 9.0 Hz, H-2”,
H-6”), 7.10 (1H, t, J = 7.4 Hz, H-4”), 7.36 (2H, t, J = 8.5 Hz, H-3”,
H-5”), 7.61 (2H, d, J = 9.0 Hz, H-2’, H-6’), 10.30 (1H, s, NH); 13
C
NMR (150 MHz, DMSO-d6): δ 47.7, 122.2, 123.6, 125.4, 134.2,
138.5, 156.6, 161.4, 168.7.
4.2.2.2. N-(4-Benzylphenyl)-2-chloroacetamide
(6b)
4.2.3.3. 2-[3-[5-(3-Bromophenoxy)pentyl] -2,6-
dioxo-3,6-dihydropyrimidin-1(2H)-yl]-N-(4-phen-
oxyphenyl)acetamide (7c)
Yield 78%, mp 142.5-144 °C, Rf 0.52 (hexane/ethyl acetate,
1:1); 1H NMR (400 MHz, DMSO-d6): δ 3.89 (2H, s, PhCH2), 4.23
(2H, s, COCH2), 7.15-7.21 (m, 5H, C6H5), 7.25-7.29 (2H, m,
aromatic H), 7.51 (2H, d, J = 8.6 Hz, H-2’, H-6’), 10.24 (1H, s,
NH); 13C NMR (100 MHz, DMSO-d6): δ 40.9, 43.9, 119.9, 126.3,
128.7, 128.9, 129.4, 136.8, 137.1, 141.7, 164.8.
Yield 91%, mp 125-126.5 °C, Rf 0.53 (DCE/ethyl acetate,
1
1:1); H NMR (400 MHz, DMSO-d6): δ ppm 1.40 (quin, J = 6.4
Hz, 2H, CH2), 1.66 (quin, J = 7.3 Hz, 2H, CH2), 1.73 (quin, J =
7.8 Hz, 2H, CH2), 3.76 (t, J = 7.3 Hz, 2H, CH2N), 3.96 (t, J = 6.4
Hz, 2H, CH2O), 4.61 (s, 2H, CH2CO), 5.76 (d, J = 7.8 Hz, 1H, H-
5), 6.93 (dd, J = 8.3, 2.3 Hz, 1H, H-4’), 6.95-7.00 (m, 4H, H-2”,
H-6”, H-2”’, H-6”’), 7.07-7.12 (m, 3H, H-2’, H-6’, H-4”’), 7.20 (t,
J = 8.0 Hz, 1H, H-5’), 7.36 (dt, J = 7.6, 2.4 Hz, 2H, H-3”’, H-5”’),
7.57 (d, J = 9.0 Hz, 2H, H-3”, H-5”), 7.77 (d, J = 8.0 Hz, 1H, H-6),
10.27 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): δ ppm 22.6,
28.4, 40.7, 43.4, 48.8, 67.9, 100.3, 114.3, 117.5, 118.2, 119.8,
121.0, 122.4, 123.3, 123.6, 130.3, 131.5, 135.0, 144.8, 151.4,
152.1, 157.6, 160.0, 162.6, 165.4; HRMS: found m/z
578.1280; calcd for C29H28BrN3O5 [M+H]+ 578.1285.
4.2.2.3. N-[4-(Benzyloxy)phenyl]-2-
chloroacetamide (6c)
Yield 88%, mp 142-144 °C, Rf 0.63 (hexane/ethyl acetate,
1:1); 1H NMR (600 MHz, DMSO-d6) δ 4.19 (2H, s, COCH2), 5.05
(2H, 2H, s, PhCH2), 6.97 (2H, d, J = 9.1 Hz, H-3’, H-5’), 7.31 (1H,
t, J = 7.4 Hz, H-4”), 7.37 (2H, t, J = 7.6 Hz, H-3”, H-5”), 7.42 (2H,
d, J = 7.0 Hz, H-2”, H-6”), 7.49 (2H, d, J = 9.0 Hz, H-2’, H-6’),
10.16 (1H, s, NH); 13C NMR (150 MHz, DMSO-d6): δ 47.7, 73.6,
119.2, 125.3, 131.9, 132.0, 132.6, 135.9, 141.3, 159.0, 168.5.
4.2.3. General procedure for the synthesis of 7a-l,
9, 13, 17, 20, and 23

4.2.3.4. 2-[3-[5-(4-Cyanophenoxy)pentyl] -2,6-
dioxo-3,6-dihydropyrimidin-1(2H)-yl]-N-(4-phen-
oxyphenyl)acetamide (7d)
NMR (100 MHz, DMSO-d6): δ 25.5, 25.8, 43.5, 48.7, 67.7,
100.4, 112.2, 117.1, 118.2, 119.8, 121.0, 123.3, 130.3, 132.4,
135.0, 144.8, 151.4, 152.1, 157.6, 158.2, 162.5, 165.4; HRMS:
found m/z 564.1127; calcd for C28H26BrN3O5 [M+H]+
564.1129.
Yield 83%, mp 127-128.5 °C, Rf 0.33 (DCE/ethyl acetate,
1
1:1); H NMR (400 MHz, DMSO-d6): δ 1.41 (2H, quin, J = 8.5
4.2.3.8. 2-[3-[6-(4-Bromophenoxy)hexyl] -2,6-dioxo-
3,6-dihydropyrimidin-1(2H)-yl]-N-(4-phen-
oxyphenyl)acetamide (7h)
Hz, CH2), 1.67 (2H, quin, J = 7.4 Hz, CH2), 1.76 (2H, quin, J =
7.6 Hz, CH2), 3.76 (2H, t, J = 7.1 Hz, NCH2), 4.05 (2H, t, J = 6.4
Hz, OCH2), 4.60 (2H, s, COCH2), 5.75 (1H, d, J = 8.0 Hz, H-5),
6.96 (2H, d, J = 8.8 Hz, H-2”, H-6”), 6.98 (2H, d, J = 8.8 Hz, H-
2”’, H-6”’), 7.08 (2H, d, J = 9.1 Hz, H-2’, H-6’), 7.09 (1H, t, J =
7.6 Hz, H-4”’), 7.36 (2H, dt, J = 7.4 and 1.2 Hz, H-3”’, H-5”’),
7.57 (2H, d, J = 8.8 Hz, H-3’, H-5’), 7.72 (2H, d, J = 8.8 Hz, H-3”,
H-5”), 7.77 (1H, d, J = 7.8 Hz, H-6), 10.27 (1H, s, NH); 13C NMR
(100 MHz, DMSO-d6): δ 22.5, 28.3, 28.4, 43.4, 48.7, 68.2,
100.3, 103.0, 115.8, 118.2, 119.5, 119.8, 121.0, 123.3, 130.3,
134.5, 135.0, 144.8, 151.4, 152.1, 157.6, 162.4, 162.5, 165.4;
HRMS: found m/z 525.2126; calcd for C30H28N4O5 [M+H]+
525.2132.
Yield 77%, mp 143-144 °C, Rf 0.48 (DCE/ethyl acetate,
1
1:1); H NMR (400 MHz, CDCl3): δ 1.42 (2H, quin, J = 7.6 Hz,
CH2), 1.52 (2H, quin, J = 7.1 Hz, CH2), 1.73-1.79 (4H, m,
CH2CH2), 3.76 (2H, t, J = 7.8 Hz, NCH2), 3.93 (2H, t, J = 6.3 Hz,
OCH2), 4.77 (2H, s, COCH2), 5.77 (1H, d, J = 7.8 Hz, H-5), 6.76
(2H, d, J = 8.8 Hz, H-2”, H-6”), 6.93 (2H, d, J = 8.8 Hz, H-2”’, H-
6”’), 6.97 (2H, d, J = 8.3 Hz, H-2’, H-6’), 7.07 (1H, dt, J = 7.1 and
0.9 Hz, H-4”’), 7.14 (1H, d, J = 7.9 Hz, H-6), 7.28-7.37 (4H, m,
H-3”’, H-5”’, H-3’, H-5’), 7.44 (2H, d, J = 8.8 Hz, H-3”, H-5”),
7.95 (1H, s, NH); 13C NMR (100 MHz, CDCl3): δ 25.4, 25.9,
28.7, 28.8, 49.6, 67.9, 101.2, 116.3, 118.4, 119.3, 122.9, 129.5,
132.1, 142.5, 151.3, 162.6; HRMS: found m/z 592.1437; calcd
for C30H30BrN3O5 [M+H]+ 592.1442.
4.2.3.5. 2-[3-[5-(4-Phenylphenoxy)pentyl]-2,6-
dioxo-3,6-dihydropyrimidin-1(2H)-yl]-N-(4-phen-
oxyphenyl)acetamide (7e)
4.2.3.9. 2-[3-[8-(4-Bromophenoxy)octyl] -2,6-dioxo-
3,6-dihydropyrimidin-1(2H)-yl]-N-(4-phen-
oxyphenyl)acetamide (7i)
Yield 78%, mp 138.5-139.5 °C, Rf 0.42 (DCE/ethyl acetate,
1:1); H NMR (400 MHz, DMSO-d6): δ 1.43 (2H, quin, J = 8.1
1
Hz, CH2), 1.68 (2H, quin, J = 7.1 Hz, CH2), 1.76 (2H, quin, J =
7.6 Hz, CH2), 3.78 (2H, t, J = 7.0 Hz, NCH2), 3.99 (2H, t, J = 6.3
Hz, OCH2), 4.61 (2H, s, COCH2), 5.76 (1H, d, J = 8.0 Hz, H-5),
6.94-7.01 (6H, m, H-2’, H-6’, H-2”, H-6”, H-2”’, H-6”’), 7.09 (1H,
dt, J = 7.3 and 1.0 Hz, H-4”’), 7.29 (1H, dt, J = 7.3 and 1.0 Hz,
Ph-H4), 7.35 (dt, J = 7.6, 1.0 Hz, 2H, H-3”’, H-5”’), 7.41 (t, J = 7.3
Hz, 2H, Ph-H3, Ph-H5), 7.54-7.59 (6H, m, H-3’, H-5’, H-3”, H-5”,
Ph-H2, Ph-H6), 7.79 (1H, d, J = 7.8 Hz, H-6), 10.27 (1H, s, NH);
13C NMR (100 MHz, DMSO-d6): δ 22.7, 28.5, 28.6, 43.4, 45.4,
48.8, 67.6, 100.3, 115.2, 118.2, 119.8, 121.0, 123.3, 126.5,
127.0, 128.0, 129.2, 130.3, 132.7, 135.1, 140.2, 144.9, 151.4,
152.1, 157.7, 158.6, 162.6, 165.4; HRMS: found m/z
576.2487; calcd for C35H33BrN3O5 [M+H]+ 576.2493.
Yield 71%, mp 128-129 °C, Rf 0.54 (DCE/ethyl acetate,
1:1); 1H NMR (400 MHz, CDCl3): δ 1.34 (6H, m, CH2 × 6), 1.42
(2H, quin, J = 7.6 Hz, CH2), 1.71 (2H, quin, J = 7.1 Hz, CH2),
1.75 (2H, quin, J = 8.1 Hz, CH2), 3.73 (2H, t, J = 7.4 Hz, NCH2),
3.90 (2H, t, J = 6.6 Hz, OCH2), 4.81 (2H, s, COCH2), 5.80 (1H, d,
J = 7.8 Hz, H-5), 6.77 (2H, d, J = 9.0 Hz, H-2”, H-6”), 6.88 (2H, d,
J = 8.8 Hz, H-2”’, H-6”’), 6.94 (d, J = 7.8 Hz, 2H, H-2’, H-6’), 7.06
(t, J = 7.3 Hz, 1H, H-4”’), 7.17 (d, J = 7.8 Hz, 1H, H-6), 7.30-7.37
(4H, m, H-3”’, H-5”’, H-3’, H-5’), 7.43 (2H, d, J = 8.8 Hz, H-3”, H-
5”), 8.52 (1H, s, NH); 13C NMR (100 MHz, CDCl3): δ 25.7, 26.2,
28.8, 28.91, 28.94, 29.0, 44.2, 49.9, 68.0, 76.6, 76.9, 77.3,
101.2, 112.4, 116.2, 118.2, 119.3, 121.2, 122.8, 129.6, 132.1,
133.3, 143.0, 151.3, 153.1, 156.9, 158.1, 162.9, 164.8; HRMS:
found m/z 620.1749; calcd for C32H34BrN3O5 [M+H]+
620.1755.
4.2.3.6. 2-[3-[3-(4-Bromophenoxy)propyl] -2,6-
dioxo-3,6-dihydropyrimidin-1(2H)-yl]-N-(4-phen-
oxyphenyl)acetamide (7f)
4.2.3.10. 2-[3-[5-(4-Bromophenoxy)pentyl] -5-iodo-
2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl]-N-(4-
phenoxyphenyl)acetamide ( 7j)
Yield 71%, mp 127.5-129 °C, Rf 0.38 (DCE/ethyl acetate,
1:1); 1H NMR (400 MHz, CDCl3) d 1.96-2.13 (2H, m, CH2), 3.92
(2H, t, J = 6.7 Hz, NCH2), 3.96-4.06 (2H, m, OCH2), 4.60 (2H, s,
CH2CO), 5.74 (1H, d, J=7.9 Hz, H-5), 6.84-6.91 (2H, m, H-2”, H-
6” ), 6.93-7.03 (4 H, m, H-2”’, H-6”’, H-2’, H-6’), 7.07-7.14 (1H,
m, H-4”’), 7.30-7.47 (4H, m, H-3’, H-5’, H-3”’, H-5”’), 7.53 - 7.64
(2H, m, H-3”, H-5”), 7.74 (1 H, d, J=7.9 Hz, H-6), 10.25 (1 H, s,
NH); 13C NMR (100 MHz, CDCl3): δ 27.8, 43.1, 46.3, 65.2,
100.0, 100.8, 112.0, 116.7, 117.9, 119.4, 120.7, 123.0, 129.9,
132.0, 134.6, 144.6, 145.7, 151.1, 157.6, 162.2, 165.0; HRMS:
found m/z 550.0968; calcd for C27H24BrN3O5 [M+H]+
550.0972.
A mixture of 1-[5-(4-bromophenoxy)pentyl]-5-iodouracil
(4j) (0.48 g, 1.00 mmol) and NaH (60% dispersion in mineral
oil, 0.05 g, 1.25 mmol) in anhydrous DMF (15 mL) was stirred
at room temperature for 1 h followed by addition of 2-chloro-
N-(4-phenoxyphenyl)acetamide (6a, 0.26 g, 0.99 mmol). After
4 h, the reaction mixture was filtered, concentrated under
reduced pressure and purified by short-column flash
chromatography using DCE. Analytical sample was
recrystallized from a mixture of hexane/EtOAc (1:1) to give
compound 7j as a white powder (0.60 g, 86%), mp 151-153
°C, Rf 0.74 (DCE/ethyl acetate, 1:1); 1H NMR (400 MHz,
DMSO-d6): δ 1.39 (quin, J = 8.1 Hz, 2H, CH2), 1.66 (quin, J = 7.1
Hz, 2H, CH2), 1.72 (quin, J = 7.9 Hz, 2H, CH2), 3.78 (t, J = 7.0 Hz,
2H, NCH2), 3.93 (t, J = 6.6 Hz, 2H, OCH2), 4.65 (s, 2H, COCH2),
6.88 (d, J = 9.0 Hz, 2H, H-2”, H-6”), 6.96 (d, J = 8.1 Hz, 2H, H-
2”’, H-6”’), 6.98 (d, J = 8.8 Hz, 2H, H-2’, H-6’), 7.10 (dt, J = 7.3,
0.9 Hz, 1H, H-4”’), 7.36 (dt, J = 7.5, 1.0 Hz, 2H, H-3”’, H-5”’),
7.40 (d, J = 9.0 Hz, 2H, H-3’, H-5’), 7.56 (d, J = 9.0 Hz, 2H, H-3”,
H-5”), 8.37 (s, 1H, H-6), 10.28 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6): δ 22.5, 28.4, 28.5, 45.0, 49.1, 67.2, 67.9, 112.1,
117.1, 118.3, 119.8, 121.0, 123.4, 130.3, 132.4, 134.9, 149.1,
4.2.3.7. 2-[3-[4-(4-Bromophenoxy)butyl] -2,6-
dioxo-3,6-dihydropyrimidin-1(2H)-yl]-N-(4-phen-
oxyphenyl)acetamide (7g)
Yield 69%, mp 151-152 °C, Rf 0.39 (DCE/ethyl acetate,
1
1:1); H NMR (400 MHz, DMSO-d6): δ 1.72 (4H, m, CH2CH2),
3.80 (2H, t, J = 6.6 Hz, CH2), 3.97 (2H, t, J = 5.9 Hz, CH2), 4.60
(2H, s, CH2CO), 5.76 (1H, d, J = 7.8 Hz, H-5), 6.90 (2H, d, J = 9.1
Hz, H-2”, H-6”), 6.94-7.00 (4H, m, H-2”’, H-6”’, H-2’, H-6’), 7.10
(1H, dt, J = 7.2 and 1.0 Hz, H-4”’), 7.36 (2H, t, 7.6 Hz, H-3”’, H-
5”’), 7.42 (2H, d, J = 9.0 Hz, H-3’, H-5’), 7.57 (2H, d, J = 9.1 Hz,
H-3”, H-5”), 7.79 (1H, d, J = 8.1 Hz, H-6), 10.27 (1H, s, NH); 13C

151.0, 152.2, 157.6, 158.2, 160.2, 165.2; HRMS: found m/z
3”, H-5”), 7.75 (1H, d, J = 7.8 Hz, Ura-H-6), 10.27 (1H, s, NH);
13C NMR (100 MHz, DMSO-d6): δ 25.9, 28.5, 28.6, 31.1, 35.3,
43.4, 48.9, 100.2, 118.2, 119.8, 121.0, 123.3, 125.9, 128.5,
128.5, 130.3, 135.0, 142.5, 144.8, 151.4, 152.1, 157.6, 162.6,
165.4; HRMS: found m/z 498.2386; calcd for C30H31N3O4
[M+H]+ 498.2387.
704.0248; calcd for C29H27BrIN3O5 [M+H]+ 704.0252.
4.2.3.11. N-(4-Benzylphenyl)-2-[3-[5-(4-
bromophenoxy)pentyl] -2,6-dioxo-3,6-
dihydropyrimidin-1(2H)-yl]acetamide (7k)
Yield 80%, mp 117-118 °C, Rf 0.42 (DCE/ethyl acetate,
1:1); H NMR (400 MHz, DMSO-d6): δ ppm 1.39 (2H, quin, J =
4.2.3.15. 2-[3-[4-(4-Bromobenzyloxy)butyl]-2,6-
dioxo-3,6-dihydropyrimidin-1(2H)-yl]-N-(4-phen-
oxyphenyl)acetamide (17)
1
6.9 Hz, CH2), 1.65 (2H, quin, J = 7.1 Hz, CH2), 1.72 (2H, quin, J
= 7.1 Hz, CH2), 3.75 (2H, t, J = 6.9 Hz, NCH2), 3.87 (2H, s,
PhCH2), 3.92 (2H, t, J = 6.4 Hz, OCH2), 4.59 (2H, s, COCH2), 5.75
(1H, d, J = 7.8 Hz, H-5), 6.88 (2H, d, J = 9.1 Hz, H-2”, H-6”),
7.13-7.20 (5H, m, aromatic H), 7.25-7.28 (2H, m, aromatic H),
7.40 (2H, d, J = 9.0 Hz, H-3”, H-5”), 7.46 (2H, d, J = 8.5 Hz, H-3’,
H-5’), 7.77 (1H, d, J = 7.8 Hz, H-6), 10.20 (1H, s, NH); 13C NMR
(100 MHz, DMSO-d6): δ ppm 22.6, 28.4, 43.4, 48.7, 67.8,
100.28, 112.1, 117.0, 119.4, 126.2, 128.7, 128.9, 129.3, 132.4,
136.5, 137.1, 141.7, 144.8, 151.4, 158.2, 162.6, 165.4; HRMS:
found m/z 576.1487; calcd for C30H30BrN3O4 [M+H]+
576.1492.
Yield 63%, mp 114-116 °C, Rf 0.28 (DCE/ethyl acetate,
1
1:1); H NMR (400 MHz, DMSO-d6): δ 1.54 (2H, quin, J = 6.3
Hz, CH2), 1.67 (2H, quin, J = 7.4 Hz, CH2), 3.44 (2H, t, J = 6.1 Hz,
CH2), 3.75 (2H, t, J = 7.1 Hz, CH2), 4.42 (2H, s, ArCH2), 4.60
(2H, s, CH2CO), 5.75 (1H, d, J = 7.9 Hz, H-5), 6.96 (2H, d, J = 8.1
Hz, H-2”’, H-6”’), 6.98 (2H, d, J = 9.1 Hz, H-2”, H-6”), 7.09 (t, J =
7.3 Hz, 1H, H-4”’), 7.27 (d, J = 8.3 Hz, 2H, H-2’, H-6’), 7.36 (2H,
dt, J = 7.6 and 2.0 Hz, H-3”’, H-5”’), 7.52 (2H, d, J = 8.3 Hz, H-3’,
H-5’), 7.57 (2H, d, J = 7.1 Hz, H-3”, H-5”), 7.76 (1H, d, J = 7.8
Hz, H-6), 10.27 (1H, s, NH); 13C NMR (100 MHz, DMSO-d6): δ
25.7, 26.4, 43.4, 45.4, 48.8, 69.6, 71.3, 100.3, 118.2, 119.8,
120.7, 121.0, 123.3, 129.9, 130.3, 131.5, 135.0, 138.4, 144.8,
151.4, 152.1, 157.6, 162.6, 165.4; HRMS: found m/z
578.1287; calcd for C29H28BrN3O5 [M+H]+ 578.1285.
4.2.3.12. N-[4-(Benzyloxy)phenyl]-2-[3-[5-(4-
bromophenoxy)pentyl] -2,6-dioxo-3,6-dihydro-
pyrimidin-1(2H)-yl]acetamide (7l)
Yield 80%, mp 172.5-174.5 °C, Rf 0.46 (DCE/ethyl acetate,
1:1); H NMR (400 MHz, DMSO-d6): δ 1.38 (2H, quin, J = 7.8
4.2.3.16. 2-[3-[[2-(4-
1
Bromobenzyloxy)ethoxy]methyl]-2,6-dioxo-3,6-
dihydropyrimidin-1(2H)-yl]-N-(4-
phenoxyphenyl)acetamide ( 20)
Hz, CH2), 1.64 (2H, quin, J = 7.4 Hz, CH2), 1.71 (2H, quin, J =
7.5 Hz, CH2), 3.74 (2H, t, J = 7.1 Hz, NCH2), 3.91 (2H, t, J = 6.4
Hz, OCH2), 4.58 (2H, s, COCH2), 5.05 (2H, s, OCH2Ph), 5.74 (1H,
d, J = 8.1 Hz, H-5), 6.87 (2H, d, J = 9.1 Hz, H-3’, H-5’), 6.94 (2H,
d, J = 8.2 Hz, H-2”’, H-6”’), 7.31 (1H, t, J = 7.1 Hz, H-4”), 7.35-
7.46 (8H, m, aromatic H), 7.73 (1H, d, J = 8.0 Hz, H-6), 10.13
(1H, s, NH); 13C NMR (100 MHz, DMSO-d6): δ 22.6, 28.4, 43.4,
48.8, 67.9, 69.7, 100.3, 112.0, 115.2, 117.0, 120.8, 128.0,
128.1, 128.7, 132.4, 137.4, 144.8, 151.4, 154.6, 158.2, 162.6,
165.1; HRMS: found m/z 592.1437; calcd for C30H30BrN3O5
[M+H]+ 592.1442.
Yield 75%, mp 147.5-149.5 °C, Rf 0.53 (DCE/ethyl acetate,
1
1:1); H NMR (400 MHz, DMSO-d6): δ 3.55 (2H, t, J = 5.9 Hz,
CH2), 3.68 (2H, t, J = 4.9 Hz, CH2), 4.45 (2H, s, ArCH2), 4.61
(2H, s, CH2CO), 5.19 (2H, s, NCH2O), 5.82 (1H, d, J = 7.8 Hz, H-
5), 6.96 (2H, d, J = 9.0 Hz, H-2”’, H-6”’), 6.98 (d, J = 9.0 Hz, 2H,
H-2”, H-6”), 7.09 (t, J = 7.3 Hz, 1H, H-4”’), 7.27 (d, J = 8.3 Hz,
2H, H-2’, H-6’), 7.36 (2H, dt, J = 8.6 and 1.0 Hz, H-3”’, H-5”’),
7.52 (2H, d, J = 8.3 Hz, H-3’, H-5’), 7.56 (2H, d, J = 7.1 Hz, H-3”,
H-5”), 7.83 (1H, d, J = 7.9 Hz, H-6), 10.30 (1H, s, NH); 13C NMR
(100 MHz, DMSO-d6): δ 43.4, 68.6, 69.2, 71.5, 78.0, 101.1,
118.3, 119.8, 120.7, 121.0, 123.3, 129.9, 130.3, 131.5, 135.0,
138.2, 144.2, 151.6, 152.1, 157.6, 162.4, 165.3; HRMS: found
m/z 580.1073; calcd for C28H26BrN3O6 [M+H]+ 580.1078.
4.2.3.13. 2-[3-[5-(4-Bromophenoxy)pentyl] -2,6-
dioxo-3,6-dihydropyrimidin-1(2H)-yl]-N-phenyl-
acetamide (7m)
Yield 78%, mp 157-158.5 °C, Rf 0.37 (DCE/ethyl acetate,
1:1); 1H NMR (400 MHz, DMSO-d6) δ 1.39 (2H, quin, J = 7.6 Hz,
CH2), 1.72 (2H, quin, J = 7.2 Hz, CH2), 1.66 (2H, quin, J = 7.6
Hz, CH2), 3.75 (2H, t, J = 7.2 Hz, NCH2), 3.93 (2H, t, J = 6.4 Hz,
OCH2), 4.61 (2H, s, CH2CO), 5.76 (1H, d, J = 7.8 Hz, Ura-H-5),
6.88 (2H, d, J = 8.8 Hz, H-2, H-6), 7.04 (1H, t, J = 7.3 Hz, H-4’),
7.30 (2H, t, J = 8.1 Hz, H-3’, H-5’), 7.40 (2H, d, J = 9.0 Hz, H-3,
H-5), 7.55 (2H, dd, J = 8.4 and 1.1 Hz, H-2, H-6), 7.77 (1H, d, J
= 8.1 Hz, Ura-H-6), 10.25 (1H, s, NH); 13C NMR (100 MHz,
DMSO-d6): δ 22.6, 28.4, 43.5, 48.8, 67.8, 100.3, 112.1, 117.0,
119.3, 123.6, 129.1, 132.4, 139.1, 144.8, 151.4, 158.2, 162.6,
165.6; HRMS: found m/z 486.1020; calcd for C23H24BrN3O4
[M+H]+ 486.1023.
4.2.3.17. 2-[3-[4-(4-Bromophenoxy)benzyl] -2,6-
dioxo-3,6-dihydropyrimidin-1(2H)-yl]-N-(4-phen-
oxyphenyl)acetamide (23)
Yield 64%, mp 201.5-202.5 °C, Rf 0.43 (DCE/ethyl acetate,
1:1); 1H NMR (400 MHz, DMSO-d6): δ 4.62 (2H, s, COCH2),
4.95 (2H, s, ArCH2), 5.82 (2H, 1H, d, J = 7.8 Hz, H-5), 6.94-6.98
(6H, m, H-2”, H-6”, H-2”’, H-6”’, H-3””, H-5””), 7.04 (d, J = 8.5
Hz, H-2’, H-6’), 7.09 (1H, dt, J = 7.3 and 1.0 Hz, H-4”’), 7.36
(2H, dt, J = 8.6, 1.2 Hz, H-3”’, H-5”’), 7.38 (2H, d, J = 8.5 Hz, H-
3’, H-5’), 7.54 (2H, d, J = 8.8 Hz, H-2””, H-6””), 7.57 (2H, d, J =
9.1 Hz, H-3”, H-5”), 7.91 (1H, d, J = 7.8 Hz, H-6), 10.30 (1H, s,
NH); 13C NMR (100 MHz, DMSO-d6): δ 43.6, 51.2, 100.9, 115.5,
118.2, 119.4, 119.8, 121.0, 123.3, 130.0, 130.3, 132.4, 133.1,
135.0, 144.7, 151.5, 152.1, 156.1, 156.3, 157.6, 162.5, 165.4;
HRMS: found m/z 598.0969; calcd for C31H24BrN3O5 [M+H]+
598.0972.
4.2.3.14. 2-[2,6-Dioxo-3-(6-phenylhexyl)-3,6-
dihydropyrimidin-1(2H)-yl]-N-(4-phenoxyphenyl) -
acetamide (13)
Yield 90%, mp 135-136 °C, Rf 0.48 (DCE/ethyl acetate,
1
1:1); H NMR (400 MHz, DMSO-d6): δ 1.22-1.37 (4H, m, CH2),
4.2.4. 2-(3-(5-(4-Bromophenoxy)pentyl)-2,6-
dioxo-3,6-dihydropyrimidin-1(2H)-yl)acetic acid
(8)
A mixture of 1-[5-(4-bromophenoxy)pentyl]uracil (4b)
(1.42 mmol) and K2CO3 (0.29 g, 2.10 mmol) in anhydrous
DMF (10 mL) was stirred at 80 C for 40 min. After cooling to
1.49-1.65 (4H, m, CH2), 2.55 (2H, t, J = 7.4 Hz, CH2), 3.72 (2H,
t, J = 7.1 Hz, CH2), 4.61 (2H, s, CH2), 5.74 (1H, d, J = 7.8 Hz, Ura-
H-5), 6.93-7.00 (4H, m, H-3’, H-5’, H-2”, H-6”), 7.07-7.12 (1H,
m, H-4”), 7.13-7.19 (3H, m, H-4, H-2, H-6), 7.21-7.27 (2H, m,
H-3, H-5), 7.33-7.39 (2H, m, H-2’, H-6’), 7.54-7.60 (2H, m, H-

room temperature, ethyl bromoacetate (0.17 mL, 1.56 mmol)
was added and stirring was continued for 24 h. The reaction
mixture was filtered, concentrated under reduced pressure
and purified by short-column flash chromatography using
DCE. Crude product was dissolved in EtOH (20 mL). Then
LiOH (0.20 g, 8.35 mmol) and water (10 mL) was added, and
the resulting mixture was stirred at room temperature for 2
h. After adjusting pH to 2 with addition of 1 M HCl resulting
precipitate was filtered and recrystallized from a mixture of
hexane/i-PrOH (1:2) to give the desired product as white
powder (2.68 g, 100%), Rf 0.54 (i-PrOH/EtOAc/NH4OH,
9:6:5), mp 142.5-145C; 1H NMR (400 MHz, DMSO-d6): δ 1.37
(2H, quin, J = 7.4 Hz, CH2), 1.55 (2H, quin, J = 7.4 Hz, CH2),
1.71 (2H, quin, J = 7.4 Hz, CH2), 3.79 (2H, t, J = 7.3 Hz, NCH2),
3.92 (2H, t, J = 6.5 Hz, OCH2), 4.45 (2H, s, CH2), 5.72 (1H, d, J =
7.8 Hz, Ura-H-5), 6.89 (2H, d, J = 8.8 Hz, H-2, H-6), 7.41 (2H, d,
J = 8.8 Hz, H-3, H-5), 7.65 (1H, d, J = 7.8 Hz, NH); 13C NMR
(100 MHz, DMSO-d6): δ 23.1, 27.1, 28.5, 50.0, 67.8, 100.4,
112.0, 117.0, 132.4, 144.9, 151.4, 158.2, 162.7, 169.8.
J = 7.8 Hz, H-5), 6.88 (2H, d, J = 8.5 Hz, H-2”, H-6”), 7.06-7.11
(4H, m, H-2’, H-6’, H-2”’, H-6”’), 7.18 (1H, t, J = 7.5 Hz, H-4”’),
7.39-7.43 (6H, m, H-3’, H-5’, H-3”, H-5”, H-3”’, H-5”’), 7.73 (1H,
d, J = 7.8 Hz, H-6); 13C NMR (100 MHz, DMSO-d6): δ 22.6, 28.4,
37.4, 42.3, 48.7, 60.1, 67.8, 100.2, 112.1, 117.0, 119.4, 119.8,
124.3, 129.5, 130.5, 132.4, 133.0, 137.8, 144.7, 151.2, 156.7,
158.2, 162.4, 166.2; HRMS: found m/z 592.1439; calcd for
C30H30BrN3O5 [M+H]+ 592.1442.
4.3. Biological assays
4.3.1.
Antiviral Activity Assays other than
HIV
The compounds were evaluated against the following
viruses: herpes simplex virus type 1 (HSV-1) strain KOS,
thymidine kinase-deficient (TK−) HSV-1 KOS strain resistant
to ACV (ACVr), herpes simplex virus type 2 (HSV-2) strains
Lyons and G, human cytomegalovirus (HCMV) (strains AD-
169 and Davis), varicella-zoster virus (strains OKA and YS),
vaccinia virus Lederle strain, respiratory syncytial virus
(RSV) strain Long, vesicular stomatitis virus (VSV), Coxsackie
B4, Parainfluenza 3, Influenza virus A (subtypes H1N1,
H3N2), influenza virus B, Reovirus-1, Sindbis and Punta Toro.
The antiviral assays were based on inhibition of virus-
induced cytopathicity or plaque formation in human
embryonic lung (HEL) fibroblasts, African green monkey cells
(Vero), human epithelial cells (HeLa) or Madin-Darby canine
kidney cells (MDCK). Confluent cell cultures in microtiter 96-
well plates were inoculated with 100 CCID50 of virus (1
CCID50 being the virus dose to infect 50% of the cell cultures)
or 10 or 100 plaque forming units (PFU) (for VZV and HCMV)
in the presence of varying concentrations of the test
compounds. Viral cytopathicity or plaque formation was
recorded as soon as it reached completion in the control
virus-infected cell cultures that were not treated with the test
compounds. Antiviral activity was expressed as the EC50 or
compound concentration required to reduce virus-induced
cytopathogenicity or viral plaque formation by 50%.
4.2.5. N-(4-Benzoylphenyl)-2-[3-[5-(4-bromo-
phenoxy)pentyl]-2,6-dioxo-3,6-dihydropyrimidin-
1(2H)-yl]acetamide (9)
A mixture of acid 8 (0.70 g, 1.70 mmol) and thionyl
chloride (0.15 mL, 2.06 mmol) in anhydrous DCE (10 mL)
was refluxed with the exclusion of moisture for 2 h. The
volatile materials were evaporated under reduced pressure
and the residue was dissolved in DCE (10 mL) and cooled to –
15 C. The resulting solution was added dropwise to a stirred
solution of N-(trimethylsilyl)-4-benzoylaniline, which was
prepared in situ by heating 4-benzoylaniline (0.34 g, 1.72
mmol) with excess of HMDS. The reaction mixture was
stirred overnight at room temperature and diluted with i-
PrOH (8 mL). Solvents were evaporated under reduced
pressure and crude product was purified with short-column
flash chromatography using hexane/EtOAc (1:2) to give
compound 9 as a white powder (0.57 g, 57%), mp 162.5-164
°C, Rf 0.39 (DCE/ethyl acetate, 1:1); 1H NMR (400 MHz,
DMSO-d6): δ 1.40 (2H, quin, J = 8.1 Hz, CH2), 1.66 (2H, quin, J =
7.1 Hz, CH2), 1.72 (2H, quin, J = 7.8 Hz, CH2), 3.76 (2H, t, J = 7.1
Hz, NCH2), 3.92 (2H, t, J = 6.3 Hz, OCH2), 4.67 (2H, s, COCH2),
5.77 (1H, d, J = 7.8 Hz, H-5), 6.87 (2H, d, J = 9.1 Hz, H-2’, H-6’),
7.39 (2H, d, J = 9.0 Hz, H-3’, H-5’), 7.54 (2H, t, J = 7.3 Hz, H-3”’,
H-5”’), 7.65 (1H, dt, J = 7.6 and 1.5 Hz, H-4”’), 7.69-7.75 (6H,
m, H-2”, H-6”, H-3”, H-5”, H-2”’, H-6”’), 7.79 (1H, d, J = 8.0 Hz,
H-6), 10.67 (1H, s, NH); 13C NMR (100 MHz, DMSO-d6): δ 22.6,
28.4, 43.7, 48.8, 67.8, 100.3, 112.1, 117.0, 118.6, 128.8, 129.7,
131.6, 131.8, 132.4, 132.6, 137.9, 143.2, 144.9, 151.4, 158.2,
162.5, 166.3, 194.9; HRMS: found m/z 590.1283; calcd for
C30H28BrN3O5 [M+H]+ 590.1285.
4.3.2.
Anti-HIV Activity Assays
Inhibition of HIV-1(IIIB)- and HIV-2(ROD)-induced
cytopathicity in CEM cell cultures was measured in microtiter
96-well plates containing ~3 × 105 CEM cells/mL infected
with 100 CCID50 of HIV per milliliter and containing
appropriate dilutions of the test compounds. After 4−5 days
of incubation at 37 °C in a CO2-controlled humidified
atmosphere, CEM giant (syncytium) cell formation was
examined microscopically. The EC50 (50% effective
concentration) was defined as the compound concentration
required to inhibit HIV-induced giant cell formation by 50%.
4.3.3.
Cytostatic Activity Assays
4.2.6. 2-[3-[5-(4-Bromophenoxy)pentyl] -2,6-
dioxo-3,6-dihydropyrimidin-1(2H)-yl]-N-methyl-N-
(4-phenoxyphenyl)acetamide ( 24)
NaH as a 60% dispersion in mineral oil (0.03 g, 0.75 mmol)
was added to a solution of compound 7b in DMF (10 mL) at
0C followed with MeI (0.08 mL, 1.29 mmol) after 20 min.
Stirring was continued for 4 h. DMF was evaporated under
reduced pressure, residue was dissloved in DCE (20 mL),
washed with a 5% solution of Na2SO3 (80 mL), water (50 mL),
Cytotoxicity measurements were based on the inhibition
of cell growth. HEL cells were seeded at a rate of 5 x 103
cells/well into 96-well microtiter plates and allowed to
adhere and proliferate for 24 h. Then, medium containing
different concentrations of the test compounds was added.
After 3 days of further incubation at 37 °C, the cell number
was determined with a Coulter counter. The cytostatic
concentration was calculated as the CC50, or the compound
concentration required reducing cell proliferation by 50%
relative to the number of cells in the untreated controls. CC50
values were estimated from graphic plots of the number of
and evaporated. Recrystallisation from
a mixture of
hexane/EtOAc (1:1) gave compound 23 as a white powder
(0.33 g, 92%), mp 135.5-137 °C, Rf 0.75 (DCE/ethyl acetate,
cells (percentage of control) as
a function of the
1
1:1); H NMR (400 MHz, DMSO-d6): δ 1.37 (2H, quin, J = 7.8
concentration of the test compounds. Alternatively,
cytotoxicity of the test compounds was expressed as the
minimum cytotoxic concentration (MCC) or the compound
Hz, CH2), 1.63 (2H, quin, J = 7.1 Hz, CH2), 1.71 (2H, quin, J =
7.1 Hz, CH2), 3.14 (3H, s, CH3), 3.72 (2H, t, J = 7.0 Hz, NCH2),
3.93 (2H, t, J = 6.3 Hz, OCH2), 4.26 (2H, s, COCH2), 5.70 (1H, d,

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Acknowledgments
The authors wish to express their gratitude to Ms. Therese
Ku, Mrs. Leentje Persoons, Mrs. Frieda De Meyer, Mrs. Lies
Van den Heurck, and Mrs. Lizette van Berckelaer for excellent
technical assistance. This work was supported by grant of
Russian Foundation for Basic Research (13-04-01391A). The
biological screening was supported by KU Leuven (GOA
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