1-[2-(2-Benzoyl- and 2-benzylphenoxy)ethyluracils as potent anti-HIV-1 agents

Article abstract of DOI:10.1016/j.bmc.2011.08.025

Non-nucleoside reverse transcriptase inhibitors (NNRTI) are key components in highly active antiretroviral therapy for treating HIV-1. Herein we present the synthesis for a series of N1-alkylated uracil derivatives bearing ω-(2-benzyl- and 2-benzoylphenoxy)alkyl substituents as novel NNRTIs. These compounds displayed anti-HIV activity similar to that of nevirapine and several of them exhibited activity against the K103N/Y181C RT mutant HIV-1 strain. Further evaluation revealed that the inhibitors were active against most nevirapine-resistant mono- and di-substituted RTs with the exception of the V106A RT. Thus, the candidate compounds can be regarded as potential lead compounds against the wild-type virus and drug-resistant forms.

Full text of DOI:10.1016/j.bmc.2011.08.025

Bioorganic & Medicinal Chemistry 19 (2011) 5794–5802
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
1-[2-(2-Benzoyl- and 2-benzylphenoxy)ethyl]uracils as potent anti-HIV-1 agents
Mikhail S. Novikov a, Olga N. Ivanova b, Alexander V. Ivanov b, Alexander A. Ozerov a,
Vladimir T. Valuev-Elliston b, Kartik Temburnikar d, Galina V. Gurskaya b, Sergey N. Kochetkov b,
Christophe Pannecouque c, Jan Balzarini c, Katherine L. Seley-Radtke d,
⇑
a Department of Pharmaceutical & Toxicological Chemistry, Volgograd State Medical University, Paversushikh Bortsov Sq., 1, Volgograd 400131, Russia
b Engelhardt Institute of Molecular Biology, Russian Academy of Science, Vavilov Str., 32, Moscow 119991, Russia
c Rega Institute for Medical Research, K.U. Leuven, Minderbroedersstraat 10, Leuven B-3000, Belgium
d Department of Chemistry & Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Non-nucleoside reverse transcriptase inhibitors (NNRTI) are key components in highly active antiretroviral
therapy for treating HIV-1. Herein we present the synthesis for a series of N1-alkylated uracil derivatives
Received 22 June 2011
Revised 9 August 2011
Accepted 11 August 2011
Available online 17 August 2011
bearing
x-(2-benzyl- and 2-benzoylphenoxy)alkyl substituents as novel NNRTIs. These compounds
displayed anti-HIV activity similar to that of nevirapine and several of them exhibited activity against
the K103N/Y181C RT mutant HIV-1 strain. Further evaluation revealed that the inhibitors were active
against most nevirapine-resistant mono- and di-substituted RTs with the exception of the V106A RT. Thus,
the candidate compounds can be regarded as potential lead compounds against the wild-type virus and
drug-resistant forms.
Keywords:
HIV-1
Non-nucleoside reverse transcriptase
inhibitors
Ó 2011 Elsevier Ltd. All rights reserved.
Reverse transcriptase
Uracil
Benzophenone
1. Introduction
protease and HIV integrase, respectively. NRTI and NNRTI are pri-
mary components of HAART.
A third decade of pandemic AIDS is underway, despite extensive
efforts from scientists across the globe. In the USA alone, almost
1.6 million patients require antiretroviral therapy, while in many
developing countries this number is an order of magnitude great-
er.1 To date 25 antiretroviral drugs have been approved for the
treatment of HIV infection using ‘highly active antiretroviral ther-
apy’ or HAART.2 These compounds inhibit HIV replication, however
their use is often hampered by toxic side-effects over long periods
of therapy, as well as a loss of activity when faced with emerging
drug-resistant virus strains. As a result, the search for new thera-
peutics with minimal side-effects and the ability to retain activity
against drug-resistant virus strains is regarded as a critical goal for
medicinal chemists.
These anti-HIV drugs comprise six different classes of com-
pounds: eight nucleoside (or nucleotide) reverse transcriptase
(RT) inhibitors (NRTIs), four non-nucleoside RT inhibitors (NNRTIs),
ten protease inhibitors (PIs), one integrase inhibitor (INI), one
fusion inhibitor (FI) and one CCR5 inhibitor.2 The first four classes
of inhibitors target three different viral enzymes: HIV–RT, HIV
One area of ongoing research in our laboratories has focused on
the design and synthesis of NNRTIs. These compounds are highly
specific, non-competitive inhibitors of HIV-1 RT, the key enzyme
involved in HIV replication. Since 1996, five NNRTIs (shown in
Fig. 1) have been approved by the FDA for clinical use: nevirapine
(ViramuneÒ, Boehringer Ingelheim), delavirdine (RescriptorÒ,
Pharmacia & UpJohn), and efavirenz (SustivaÒ, DuPont) are classi-
fied as first generation NNRTIs, whereas etravirine (IntelenceÒ,
Tibotec) and rilpivirine (TMC278, Tibotec) are considered as sec-
ond-generation NNRTIs, since they retain activity against a variety
of drug-resistant virus strains.3 NNRTIs have been reported to bind
to a non-nucleoside inhibitor binding pocket in HIV-1 RT (NNIBP),
which is located in the ‘palm’ domain of the p66 subunit at a dis-
tance of approximately 10 Å from the enzyme catalytic site.4,5 The
pocket is primarily hydrophobic and key amino acid residues in-
volved in inhibitor binding include aromatic (Y181, Y188, F227,
W229 and Y232), hydrophobic (P59, L100, V106, V179, F227,
L234 and P236) and hydrophilic (K101, K103, S105, D132 and
E224) residues in the p66 RT subunit, as well as three additional
amino acids (I135, E138 and T139) in the p51 subunit.3,6 One major
limitation of many NNRTIs however, is the rapid emergence of
HIV-1 variants resistant to these drugs due to one or more point
mutations in the RT binding site.
⇑
Corresponding author. Tel.: +1 410 455 8684; fax: +1 410 455 2608.
E-mail address: kseley@umbc.edu (K.L. Seley-Radtke).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.025

M. S. Novikov et al. / Bioorg. Med. Chem. 19 (2011) 5794–5802
5795
active against both wild-type HIV-1 and clinically significant drug-
resistant forms.21–23 This class of compounds is based on the ben-
zophenone fragment bridged by a short linker to an ortho-methyl-
aniline residue. SAR studies have further revealed that the substit-
uents in the two meta-positions of the benzoyl moiety as well as
the benzophenone fragment play crucial roles in retaining antiviral
activity against mutant strains.
Thus, by combining these leads, ‘chimeric’ molecules containing
the uracil residue such as that found in scaffold 1, as well as the
benzophenone fragment found in scaffold 2, may not only inhibit
HIV-1 RT, but also exhibit strong antiviral activity towards both
wild-type HIV-1 and drug-resistant virus strains (Fig. 2). Herein
we report the synthesis of N1-alkylated uracil derivatives and the
initial biological studies describing their antiviral activity.
2. Results and discussion
2.1. Chemistry
Synthesis of the target compounds was carried out by condensa-
tion of equimolar quantities 2,4-bis(trimethylsilyloxy)pyrimidine
or -5-methylpyrimidine 3 with 1-bromo-x-(2-benzylphenoxy)- 4
or 1-bromo- -(2-benzoylphenoxy)alkanes 5. Starting bromides 4
x
and 5 were prepared according to previously described methods.24
This synthetic approach has a key advantage: the reaction results in
the N1-substituted uracils as the only product. Formation of the N3-
substituted products was not observed, likely due to the steric
hindrance of the adjacent trimethylsilyl groups blocking the ap-
proach to the N3-nitrogen atom.
Figure 1. FDA-approved NNRTIs.
It is known that condensation of trimethylsilyl pyrimidines with
highly reactive alkylating agents occurs in a facile manner in aprotic
solvents under mild conditions and with high yields.25,26 In contrast,
treatment of 2,4-bis(trimethyl-silyloxy)pyrimidines with alkylating
agents of lower reactivity requires elevated temperatures. Conden-
sation of trimethylsilyl uracil derivatives with allylbromides27,28 or
benzylhalides28 successfully proceeds in 1,2-dichloroethane in the
presence of a catalytic amount of iodine. Heating 2,4-bis(trimethyl-
silyloxy)pyrimidines with an excess of alkyl halide leads to 1-
substituted uracils with good yields.29,30 Previously we have
reported a method of synthesis of 1-[2-(phenoxy)ethyl]-uracils
which was based on condensation of trimethylsilyl uracil deriva-
tives with 1-bromo-2-(phenoxy)ethanes.31
To date more than 50 different structural types of NNRTIs have
been designed and described.7–9 Several are currently regarded as
potential anti-HIV drugs, either directly or following additional
structural activity relationship (SAR) optimization of a lead com-
pound. One such scaffold that has produced strong antiviral lead
activity encompasses the uracil nucleobase ( Fig. 2). Uracil ana-
logues with various substituents in the C-6 position such as exem-
plified in scaffold 1 (Fig. 2) have proven to be fruitful leads for
further development. Many of these analogues, for example, the
6-phenylthio-10,11, 6-phenyl–selenyl-12–14, 6-phenoxy-15,16, 6-ben-
zyl-17–19, and 6-benzoyluracil-15,16,20 derivatives such as those
shown in Figure 2 have been shown to inhibit HIV replication in cell
culture in nanomolar concentrations. However, the emergence of
resistant virus strains19 has hampered their clinical use.
Analogous to this route, we have now synthesized a number of
1-[2-(2-benzylphenoxy)ethyl]-
and
1-[x-(2-benzoylphen-
oxy)ethyl]uracil derivatives 6–24. The synthesis (Scheme 1) was
accomplished by heating trimethylsilyl derivative 3 and bromide 4
or 5 at 160–170 °C for 2 h without a solvent. The target compounds
were obtained in 62–83% yields, and formation of the N3- and N1,N3-
disubstituted byproducts was not observed.
Recently another promising NNRTI scaffold (2, Fig. 2) has been
reported. Inhibitors of this structural type were found to be highly
The presence of a substituent at position 6 of the pyrimidine can
affect regioselectivity of 6-R-uracil alkylation.32 For example, con-
densation of 2,4-bis(trimethylsilyloxy)-6-methylpyrimidine with
4–5 molar excess of 1,4-dibromobutane leads to a complex mix-
ture of N1- and N3-mono- as well as to N1,N3-disubstituted uracils,
the ratio of which depends upon the reaction temperature.33 As a
result, an alternative approach for synthesis of 1-[2-[2-(3,5-dim-
ethylbenzoyl)-4-methylphenoxy]ethyl]-6-methyluracil 25 was
needed. Thus treatment of 6-methyluracil (5 molar excess) with
bromide 5 in the presence of K2CO3 in DMF (Scheme 1) afforded
25 in a 64% yield following separation from the N1,N3-disubsti-
tuted product.
To study the influence of the bridging carbonyl group on the
antiviral properties of the compounds, the reduced analogue of
compound 17 was pursued. As shown in Scheme 2, treatment of
17 with NaBH4 in the presence of NaOH resulted in the hydroxyl
derivative 26 (93%).
Figure 2. Uracil (1) and benzophenone (2) containing NNRTI scaffolds.

5796
M. S. Novikov et al. / Bioorg. Med. Chem. 19 (2011) 5794–5802
Scheme 1. Reagents and conditions: (i) 160–170 °C, 2 h; (ii) K2CO3, DMF, 70 °C, 8 h.
concentrations. Most compounds were found to be nontoxic in
MT-4/CEM cell cultures. Benzophenone 16, which bears a methyl
group at the R3-position, showed the highest toxicity albeit at a
minor level (CC50:179 lM).
The studied compounds were also investigated as potential
inhibitors of replication against a large panel of other DNA and
RNA viruses. However, none of the compounds displayed notable
activity against HIV-2 or any DNA or RNA viruses other than
HIV-1 (HSV-1, HSV-2, Vaccinia virus, Para-influenza-3 virus, Reovi-
rus-1, Sindbis virus, Vesicular stomatitis virus, Respiratory syncy-
tial virus, Coxsackie virus B4, Feline Corona Virus (FIPV), Yellow
Fever virus, Dengue virus, Rift Valley Fever virus, Punta Toro virus,
West Nile virus).
Scheme 2. Reagents and conditions: (i) NaBH4, NaOH, EtOH (aq.), 50 °C, 8 h.
2.2. Antiviral activity
Compounds 11–22 and 24–25 were also evaluated against a
drug-resistant (double mutant) HIV-1 strain bearing the K103N
and Y181C mutations in the RT. These mutations resulted in pro-
nounced resistance to nevirapine and, to a much lesser extent,
efavirenz. Most of the studied benzophenones were found to be
inactive against the mutant virus (Table 1). However, 20, 24 and
25 did inhibit HIV-1 replication, albeit at higher compound con-
centrations (50- to 150-fold). Compounds 12, 15 and 22 also re-
tained weak levels of activity against this mutant virus strain. At
this time we cannot explain the differences in binding affinity for
the closely related benzophenones towards the double mutant
HIV strain, however investigations are currently underway that
may provide additional information that may shed light on the dif-
fering levels of activity.
The anti-HIV properties of the uracil derivatives were studied in
human T-lymphocyte CEM (compounds 6–10) or MT-4 (com-
pounds 11–26) cell cultures infected with HIV-1(IIIB) or HIV-2
(ROD), or the double mutant (K103N + Y181C RT) virus strain (Ta-
ble 1). All compounds were found to be inactive against HIV-2,
however most compounds exhibited notable antiviral activity
against HIV-1. In examining the structure–activity relationship
(SAR) of the compounds, it became evident that 2-(2-benzylphen-
oxy)ethyl uracils 6–10 displayed moderate levels of activity
whereas their 2-(2-benzoylphenoxy)ethyl counterparts 11–22,
24, 25, which contain the bridging carbonyl linker, inhibit HIV rep-
lication at much lower compound concentrations (except for 11
and 12). Reduction of the carbonyl group of the benzophenone
fragment to the respective alcohol (26) or addition of a methylene
group to the linker (23) resulted in a dramatic decrease of antiviral
properties. Substituent R3 appears to play a crucial role in modulat-
ing antiviral activity of the compounds; benzophenones bearing a
methyl group or fluorine atom were found to be less active than
their counterparts bearing chlorine or bromine atoms (11 and 12
vs 13 and 14, and 16 vs 17 and 18).
An increase in antiviral activity was obtained by inserting one
or two substituents at the R4 position(s). As such, compound 17
bearing two methyl groups exhibited more pronounced activity
than the analogous 15 possessing only one methyl group or 13,
which has no methyl groups. Moreover, substitution of these
methyl groups by various halogens had no significant impact on
the antiviral activity as exemplified in 17 versus 19-21 and 18 ver-
sus 22. A similar level of anti-HIV activity was exhibited by the
compound 25 bearing a methyl group in the C-6-position of the
pyrimidine ring whereas introduction of the same substituent at
C-5 (24) slightly decreased the antiviral properties of the com-
pound. The most potent anti-HIV agents were found to be uracils
17, 20, 22 and 25 which inhibited viral replication at nanomolar
2.3. Enzymatic study
Next, the inhibitory properties of the uracil derivatives against
HIV-1 RT enzyme were studied in standard DNA- and RNA-depen-
dent DNA-polymerase assays using activated DNA and wild-type
p66/p66 homodimer RT or poly(rC)-oligo(dG) template–primer
and p66/p51 heterodimer recombinant enzyme. Noteworthy that
these enzyme preparations had similar activity: when activated
DNA was used as template–primer in assay conditions as described
in Section 4, p66/p66 and p66/p51 catalyzed incorporation of 0.29,
and 0.30 pmol dATP per second per 1 lg of the enzyme, respec-
tively. Nevirapine and efavirenz were used as reference standards
(Table 2). A preliminary investigation of their mechanism of action
was performed with compound 8 in p66/p66/activated DNA assay.
It was shown that this compound acted in a non-competitive man-
ner with regards to the dNTP substrate (Fig. S1). In addition, the
uracil derivative 8 retained its efficacy when the enzymatic reac-
tions were carried out in the presence of heparin, which was added

M. S. Novikov et al. / Bioorg. Med. Chem. 19 (2011) 5794–5802
5797
Table 1
Antiviral activity of the studied compounds against HIV-1
Compound
R1
R2
R3
R4
CC50
(l
M) CEM or MT-4
HIV-1 (IIIB) wild type
HIV-1(IIIB) K103N + Y181C
EC50
(
lM)
SIa
EC50
(
lM)
SIa
RIb
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
—
—
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
—
—
4-Me
4-Me
4-Me
4-Cl
4-Br
Me
F
Cl
Br
Cl
Me
Cl
Br
Cl
Cl
Cl
Br
Cl
Cl
Cl
H
48c
53c
66c
3.3c
14
241
507
>909
—
—
—
>200
—
—
—
—
1
—
1
>2
1
1
>6
<1
1
1
1
>251
1
>10
—
>74
>74
—
—
—
—
>909
—
3-Me
3,5-Me2
3,5-Me2
3,5-Me2
H
H
H
H
3-Me
3,5-Me2
3,5-Me2
3,5-Me2
3,5-F2
3,5-Cl2
3,5-Br2
3,5-Cl2
3,5-Me2
3,5-Me2
3,5-Me2
3,5-Me2
—
0.22c
0.13c
0.22c
0.22c
0.31
0.51
>200c
>220c
>357
>353
>337
>301
>325
179
>1000
>1136
>694
>3787
>4630
>6246
3081
>19588
>12260
>11381
>15794
>7390
>15188
>3.45
>6882
>15140
>1
>357
171
>337
>301
52
>179
>313
>282
>307
1.13
>237
24.8
—
>1136
330
0.089
0.065
0.052
0.058
0.016
0.023
0.027
0.018
0.032
0.017
5.8c
0.044
0.020
P20c
0.075
0.003
>3787
>4630
1000
>3081
>19588
>12260
>11381
63
>7390
1459
—
93
205
>313
>282
>307
>284
>236
>258
>20c
>303
>303
>20c
>15
22
23
24
25
4.09
4.09
—
>15
0.56
26
Cl
—
—
—
>200
186
Nevirapine39
Efavirenz39
>200
>2112
1
>113
—
>63
a
b
c
SI = selectivity index; CC50/EC50
RI = resistance index; EC50 (resistant virus)/EC50 (wild-type virus).
Data obtained in CEM cell cultures.
.
heparin prevents reinitiation of the polymerization reaction, this
indicates that the inhibitor only impacts the elongation process
of the RT reaction and has no effect on binding of the enzyme to
nucleic acids.34
Table 2
Inhibitory activity of the compounds against wild-type HIV-1 RT in DNA-dependent
and RNA-dependent polymerase activity assays
Compound
KI (lM)
The inhibition constants (KI) reflecting the affinity of each com-
pound for RT were assessed using a Dixon plot.35 Comparison of
the results revealed that the compounds displayed similar inhibi-
tory activity in activated DNA (p66/p66) and poly(rC)-oligo(dG)
(p66/p51) assays. The correlation (r) between the KI values of the
test compounds against HIV-1 RT in these assays was 0.979
(Fig. S3). Moreover, the correlation (r) between the EC50’s for
HIV-1 replication in cell culture and the KI’s for HIV-1 RT inhibition
using p66/p66 and p66/p51 were 0.911 and 0.952, respectively
(Fig. S4).
In examining the SAR of the compounds, the results show that,
in general, as also noticed in the HIV-1-infected cell culture system,
2-(2-benzoylphenoxy)ethyl uracils 11–22 exhibit greater inhibi-
tion of HIV–RT than their corresponding 2-(2-benzylphenoxy)ethyl
counterparts (compounds 6–10). The differences between the
benzophenones and the diphenylmethane series were especially
pronounced when comparing the efficacy of compounds 11 and
6. In contrast, the carbonyl group had almost no influence on the
activity of uracil derivatives bearing methyl groups at the R3 and
R4 positions (16 vs 8).
Activated DNA (p66/p66)
Poly(rC)-oligo(dG) (p66/p51)
6
7
8
9
76
36
5.9
5.0
11
5.6
2.9
0.98
2.0
0.49
2.4
18
6.3
2.7
2.1
3.1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
1.7
2.6
0.65
0.45
0.21
2.0
0.52
1.7
2.4
0.77
0.77
0.68
2.3
0.69
>50
1.2
0.36
0.82
0.47
0.16
>242
1.1
0.73
>50
1.7
>249
Nonetheless, considering that the reduced benzohydroxy ana-
logue 26 failed to inhibit HIV–RT, the benzophenone carbonyl frag-
ment appears to be crucial for retention of activity and must be
interacting with the enzyme in some fashion.
simultaneously with the inhibitor and dNTPs after a short pre-
incubation period of the RT with activated DNA (Fig. S2). Since

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M. S. Novikov et al. / Bioorg. Med. Chem. 19 (2011) 5794–5802
Table 3
Comparison of the antiviral activity data for the compounds
bearing various substituents at R3 led to surprising results. On
the one hand, substitution of the methyl group of 2-(2-(3,5-
dimethyl)benzylphenoxy)ethyl uracil with a chlorine or bromine
had almost no effect on the potency of the compounds (8 vs 9
and 10). However the same substitution on the benzophenone-
containing compounds led to a marked enhancement of activity.
The inhibitors bearing chlorine or bromine in R3 position inhibited
RT in lower concentrations in comparison to their counterparts
with methyl or fluorine substituents (13 and 14 vs 11 and 12, or
17 and 18 vs 16). It is notable that these results are in agreement
with the anti-HIV activity observed for the uracil derivatives.
Interestingly, several of the compounds (14, 17, and 18) showed
decreased inhibitory activity in one of the enzymatic assays. Since
there were no similarities between structure and the type of assay,
this discrepancy could be attributed to the poor solubility observed
for some of the halogenated compounds in aqueous media, in par-
ticular, in the enzymatic reaction conditions required for the
assays.
Inhibitory activity of inhibitors 17 and 20 against mutant HIV–RT’s in the activated
DNA assay
HIV-1 RT mutation(s)
KI (lM)
17
20
Nevirapine
Efavirenz
Wt
L100I
2.4
3.3
33
>1000
8.0
10
1.0
57
0.7
1.8
5.6
>1200
1.4
8.4
1.6
10
4.2
61
0.03
0.33
0.73
0.08
0.08
0.36
0.08
0.58
K103N
V106A
Y181C
Y188L
G190A
K103N/Y181C
1685
482
1655
>1000
1362
>1000
efavirenz. For the Y181C RT mutant, compounds 17 and 20 inhib-
ited the polymerase 3.3- and 2-fold less effectively, as compared
to a decrease of 395- and 2.6-fold for nevirapine and efavirenz,
respectively. In the case of the Y188L mutant RT, the activity of
benzophenone 17 was 4.3-fold lower than for the wild-type en-
zyme, while compound 20 exhibited 12-fold lower activity. Nevira-
pine and efavirenz were >250 and 12-fold less active, respectively.
Interestingly, benzophenone 17 displayed even higher inhibitory
activity toward the mutant G190A RT form, while 20 exhibited
somewhat lower activity (2.3-fold), more closely in line with efavi-
renz (2.7-fold decrease) but in sharp contrast to nevirapine (423-
fold decrease). Lastly, benzophenones 17 and 20, as well as efavi-
renz, exhibited a 23.8-, 14.9- and 19.3-fold lower efficacy towards
the K103N/Y181C double mutant RT, as compared to a >250-fold
lower inhibitory activity for nevirapine.
Thus, the data obtained are highly encouraging and appear to
point to a resistance susceptibility profile for the synthesized benz-
ophenones that is closer to efavirenz than to nevirapine. Moreover,
the resistance index for the compounds against the Y181C, Y188L
and K103N/Y181C mutant RTs was comparable to that of efavirenz,
and even lower for the L100I, K103N and G190A mutants, although
for the V106A mutant, efavirenz was more potent than 17 and 20.
While these data do not directly support the results of the antiviral
activity studies, which revealed different resistance susceptibility
profiles for the structurally-related benzophenones 17 and 20, it
is possible that this difference is due to differences in solubility
for some of the compounds in aqueous media, rather than to a dif-
ferent mode of interaction with mutant HIV–RTs.
Moving forward with the SAR evaluation, the role of the R4
substituent was less clear. On the one hand, for compounds lacking
the carbonyl group (6–10), introduction of one or two methyl sub-
stituents significantly enhanced the inhibitory activity against RT
(8 vs 7 vs 6). On the other hand, in the benzophenone series, the
corresponding 3,5-dimethyl or 3,5-dichloro-containing inhibitors
displayed activity similar to that of their unsubstituted counter-
parts (16 vs 11, 17 and 20 vs 13). These observations were in dis-
agreement with the activity of the compounds in infected cell
culture. A correlation was found only in the pairs of the benzophe-
nones 13 and 15 (all assays), 18 and 14 (activated DNA assay), and
22 and 14 (poly(rC)-oligo(dG) assay). In all other cases, the discrep-
ancies again could be attributed to two effects: the greater affinity
of the substituted benzophenones for the enzyme, and/or the low-
er solubility of the compounds bearing the halogen substituents.
Next, in order to more fully understand the activity of the benz-
ophenones against drug-resistant HIV strains as well as to verify
the reasons for the poor levels of activity of the compounds against
the K103N/Y181C RT double mutant virus, a panel of RT’s bearing
one or two of the most clinically significant mutations (L100I,
K103N, V106I, Y181C, Y188L, G190A, and K103N/Y181C) confer-
ring resistance of the virus to NNRTIs was constructed. Based on
the results of the previously highlighted antiviral studies, two
structurally-related benzophenones were selected for further
study. In that regard, compounds 17 and 20 both exhibited the
highest activity against the wild-type virus but exhibited quite dif-
ferent levels of activity towards the K103N/Y181C double mutant
HIV-1 strain (Table 3).
Both benzophenones 17 and 20 retained activity against most
HIV-RT forms with the exception of V106A (Table 3). Although
the absolute KI values were closer to that of nevirapine, the resis-
tance susceptibility profile of compounds 17 and 20 towards most
HIV–RT mutant forms more closely resembled the profile of efavi-
renz. For example, nevirapine and efavirenz exhibited a 15-fold
and 10-fold lower activity, respectively, towards the L100I RT mu-
tant compared to the wild-type enzyme, whereas benzophenones
17 and 20 retained almost full activity, exhibiting only a very min-
or degree of resistance (1.4-fold and 2.5-fold, respectively). Simi-
larly, against K103N, a common drug-resistant HIV–RT form, the
inhibitory activity of nevirapine was 400-fold lower than that to-
wards wtRT, whereas activity of efavirenz or the studied benz-
ophenones 17 and 20 was only 24-, 13.8 and 8-fold lower,
respectively.
In that regard, it should be noted that 10% DMSO was used in
the HIV-1 RT poly(rC)-oligo(dG) experiment, whereas cell cultures
only contained 0.1% DMSO at most. To test this possibility, we
determined solubility of several compounds in 10% DMSO solution
(as was used in the RT activity assay) or in 0.1% (as was used in the
cell culture experiments) by UV spectroscopy. It was found that the
maximum concentration of the compounds bearing halogens
(including 17 and 20) could not exceed 2–3 lM in 10% DMSO solu-
tion (Supplementary data, Table S2). Assuming that the resistance
index of the compounds 17–19 against K103N + Y181C HIV-1
strain is 50–200 as was shown for benzophenones 20, 24, and
25, they should display antiviral activity against this strain at
micromolar concentrations, that is, in the same range as they tend
to precipitate. If this is indeed the case, the next generation of
benzophenones should be designed with enhanced solubility char-
acteristics, which may lead to obtaining more effective antiretrovi-
ral agents active against a variety of drug-resistant viral strains.
3. Conclusions
Both compounds proved to be inactive against the V106A RT,
while efavirenz retained activity. In contrast to nevirapine, the
benzophenones retained partial activity against all other forms of
RT, and in some cases the decrease was lower than that of
Herein we have described the synthesis, SAR, and biological eval-
uation of a series of potential NNRTI anti-HIV agents. Several of the
uracil derivatives exhibited potent antiviral activity in infected cell

M. S. Novikov et al. / Bioorg. Med. Chem. 19 (2011) 5794–5802
5799
culture and effectively inhibited recombinant HIV–RT. Notably, the
compounds did not exhibit toxicity in cell culture, and thus have
high selectivity indices. Two of the uracil derivatives effectively
inhibited several mutant HIV-1 RTs and also partially retained anti-
viral activity against several drug-resistant HIV strains. Despite
these encouraging results, the poor solubility of some of the com-
pounds hindered true assessment of their potential biological activ-
ity. Althoughtheactivity levels ofthe compoundstowardswild-type
virus and HIV-1 RT were similar to that of nevirapine, they more clo-
sely resemble the activity profile of efavirenz by retaining their
inhibitory activity towards mutant RT forms resistant to nevirapine.
Given the promising and potent antiviral activity exhibited by these
compounds, it is clear that the benzophenone-linked uracil scaffold
is an excellent lead for the development of additional compounds. As
a result, current efforts are underway to improve the solubility of
some of the more insoluble compounds in order to design more
effective anti-HIV drugs. Those results will be reported elsewhere
as they become available.
(2) Ion-pairing reversed phase regime: solution [A]: 0.1% hepta-
fluorobutyric acid [CF3(CF2)2COOH] (pH 3); solution [B]: 80%
CH3CN. Gradient: 0% [B] for 5 min; 0% ?20% [B] for 10 min;
20% ?100% [B] for 15 min; 100% [B] for 5 min. UV spectra
were recorded on a Shimadzu UV-2401PC spectrophotome-
ter (Japan). The rate of elution was 0.5 ml/min.
4.2. Synthesis
4.2.1. General procedure for compounds 6–24
2,4-bis(trimethylsilyloxy)pyrimidine 3, prepared from uracil or
thymine (17.84 mmol) by refluxing in excess HMDS, was incubated
with bromides 4 or 5 (17.85 mmol) at 160–170 °C for 2 h and then
kept overnight at room temperature. The resulting product was
treated with EtOAc (40 mL) and isopropanol (10 mL), the mixture
kept at room temperature for 30 min then evaporated. The result-
ing residue was recrystallized twice from isopropanol or from a
mixture isopropanol–DMF (1:1).
4. Materials and methods
4.1. General
4.2.1.1. 1-[2-(2-Benzyl-4-methylphenoxy)ethyl]uracil (6).
Yield: 66%, mp: 146–147 °C, Rf 0.55 (elution with ethyl acetate);
1H NMR (DMSO-d6): d 2.18 (s, 3H, CH3), 3.81 (s, 2H, PhCH2), 4.07
(t, 2H, J = 6 Hz, N–CH2), 4.14 (t, 2H, J = 6 Hz, O–CH2), 5.48 (d, 1H,
J = 8 Hz, H-5), 6.84–6.98 (m, 3H, aromatic H), 7.12–7.25 (m, 5H,
aromatic H), 7.60 (s, 1H, J = 8 Hz, H-6), 11.31 (s, 1H, NH); 13C
NMR (DMSO-d6): d 18.1, 32.9, 45.3, 63.5, 98.8, 110.1, 124.2,
126.7, 127.2, 127.8, 129.1, 139.2, 144.5, 149.4, 151.9, 162.2; HRMS
calcd for C20H20N2O3, [M+H]+ 337.1547; found (FAB) m/z ([M+H]+):
337.1549.
Activated DNA was purchased from GE Healthcare (Little Chal-
font, UK) Oligonucleotides were obtained from Lytech (Moscow,
Russia). [a-
32P]dATP (5000 Ci/mmol) and [8-3H]dGTP were from
Izotop (Moscow, Russia), and Moravek Biochemicals, Brea, CA),
respectively. Ni-NTA-agarose resin and Rosetta(DE3) Escherichia
coli strain were from Novagen (Madison, WI). All other reagents
of highest grade were obtained from Sigma (St. Louis, MI), and
the enzymes were purchased from Sybenzyme (Novosibirsk, Rus-
sia) or Fermentas (Vilnus, Lithuania). All chemicals were obtained
from commercial sources and used without further purification
unless otherwise noted. Anhydrous DMF, isopropanol, and ethyl-
ene glycol were purchased from Sigma-Aldrich Co. Anhydrous ace-
tone, CH2Cl2, 1,2-dichloroethane, and ethyl acetate were obtained
by distillation over P2O5.
Melting points were measured on Mel-Temp 3.0 Pro apparatus
(Laboratory Devices Inc.) and uncorrected. All 1H and 13C NMR spec-
tra were registered on a Varian Mercury 300B and AMXIII-400 Bru-
ker, operated at 300 and 400 MHz for 1H; 75 and 100 MHz for 13C,
respectively, and tetramethylsilane (TMS) was used as an internal
standard (0.0 ppm). The spin multiplicities are indicated by the
symbols s (singlet), d (doublet), dd (doublet of doublets), t (triplet),
q (quartet), m (multiplet), and br (broad). Reactions were monitored
by thin-layer chromatography (TLC) using 0.25 mm silica gel pre-
coated plates. Mass spectra were recorded at the Johns Hopkins
Mass Spectrometry Facility (Baltimore, MD). X-ray structure analy-
sis was performed on a Bruker SMART 1000 difractometer, and the
data were analyzed with the SHELX 97 program. Complete
structural data will be reported separately.
4.2.1.2. 1-[2-[2-(3-Methylbenzyl)-4-methylphenoxy]ethyl]uracil
(7). Yield: 83%, mp: 127–128 °C, Rf 0.35 (elution with ethyl ace-
tate); 1H NMR (DMSO-d6): d 2.17 (s, 3H, CH3), 2.23 (s, 3H, CH3),
3.79 (s, 2H, ArCH2), 4.06 (t, 2H, J = 4.9 Hz, N–CH2), 4.13 (t, 2H,
J = 5 Hz, O–CH2), 5.46 (dd, 1H, J = 7.8 and 2.2 Hz, H-5), 6.85–6.86
(m, 2H, aromatic H), 6.90 (m, 1H, aromatic H), 6.93–6.96 (m, 3H,
aromatic H), 7.09–7.12 (m, 1H, aromatic H), 7.58 (d, 1H,
J = 7.8 Hz, H-6), 11.29 (s, 1H, NH); 13C NMR (DMSO-d6): d 20.2,
21.1, 47.3, 65.4, 100.7, 111.7, 125.7, 126.5, 127.7, 128.1, 129.1,
129.2, 129.5, 131.0, 137.3, 140.9, 146.1, 151.1, 153.5, 163.7; HRMS
calcd for C21H22N2O3, [M+H]+ 351.1630; found (FAB) m/z ([M+H]+):
351.1698.
4.2.1.3. 1-[2-[2-(3,5-Dimethylbenzyl)-4-methylphenoxy]ethyl]
uracil (8). Yield: 74%, mp: 156–158 °C, Rf 0.58 (elution with ethyl
acetate); 1H NMR (DMSO-d6): d 2.10 (s, 3H, CH3), 2.12 (s, 6H, CH3),
3.68 (s, 2H, PhCH2), 4.02 (t, 2H, J = 6 Hz, N–CH2), 4.07 (t, 2H,
J = 6 Hz, O–CH2), 5.38 (d, 1H, J = 8 Hz, H-5), 6.67 (s, 2H, H-200, H-
600), 6.70 (s, 1H, H-400), 6.78 (m, 3H, H-30, H-50, H-60), 7.52 (d, 1H,
J = 8 Hz, H-6), 11.28 (s, 1H, NH); 13C NMR (DMSO-d6): d 18.4,
19.1, 32.8, 45.5, 63.6, 98.8, 110.0, 124.4, 124.6, 125.3, 125.5,
126.0, 127.3, 127.6, 128.9, 129.2, 135.3, 138.9, 144.2, 149.2,
151.6, 161.9; HRMS calcd for C22H24N2O3, [M+H]+ 365.1859; found
(FAB) m/z ([M+H]+): 365.1854.
Yields refer to chromatographically (HPLC) and spectroscopi-
cally (1H and 13C NMR) homogeneous materials. The purity of the
compounds was verified using HPLC and all compounds were
verified to be >98% pure with the only minor impurity being uracil.
Briefly, the compounds were dissolved in DMSO, and purified using
a Nucleosil 100 C-8 (5 m, 4 ꢀ 150 mm) column on a Gilson chro-
matograph (France) supplied with a digital Gilson GSIOC 506 con-
troller and a Gilson-315 UV detector with varied wavelengths. The
compounds were purified using the following two protocols:
4.2.1.4. 1-[2-[4-Chloro-2-(3,5-dimethylbenzyl)phenoxy]ethyl]
uracil (9). Yield: 80%, mp: 191–192.5 °C, Rf 0.59 (elution with
ethyl acetate); 1H NMR (DMSO-d6): d 2.14 (s, 6H, CH3), 3.71 (s,
2H, ArCH2), 4.03 (t, 2H, J = 4 Hz, N–CH2), 4.19 (t, 2H, J = 5 Hz, O–
CH2), 5.46 (dd, 1H, J = 7.9 and 2.2 Hz, H-5), 6.75 (s, 2H, H-200, H-
600), 6.79 (s, 1H, H-400), 6.95–6.97 (m, 1H, H-60), 7.20 (d, 1H,
J = 2.4 Hz, H-30), 7.32 (dd, 1H, J = 8.8 and 2.5 Hz, H-50), 7.60 (d,
1H, J = 7.8 Hz, H-6), 11.30 (s, 1H, NH); 13C NMR (DMSO-d6): d
19.0, 32.5, 45.3, 63.8, 98.9, 111.6, 122.5, 124.4, 124.6, 125.0,
125.7, 127.7, 127.9, 129.9, 135.5, 138.0, 144.4, 149.2, 152.5,
(1) Reversed phase regime: solution [A]: 5 mM sodium-phos-
phate buffer [NaPi] (pH 5.2); solution [B]: 80% CH3CN. Gradi-
ent: 0% [B] for 5 min; 0% ?20% [B] for 10 min; 20% ?100%
[B] for 15 min; 100% [B] for 5 min.

5800
M. S. Novikov et al. / Bioorg. Med. Chem. 19 (2011) 5794–5802
161.9; HRMS calcd for C21H21ClN2O3, [M+H]+ 385.1313; found
1H, J = 8 and 2 Hz, H-6), 11.10 (s, 1H, NH); 13C NMR (DMSO-d6):
d 20.7, 46.9, 66.1, 100.3, 114.8, 125.0, 126.5, 128.1, 128.5, 129.4,
130.3, 131.3, 134.5, 136.3, 138.1, 145.2, 150.6, 154.0, 163.4,
193.9; HRMS calcd for C20H17ClN2O4, [M+H]+ 385.0949; found
(FAB) m/z ([M+H]+): 385.0959 (35Cl), 387.0932 (37Cl).
(FAB) m/z ([M+H]+): 385.1319 (35Cl), 387.1297 (37Cl).
4.2.1.5. 1-[2-[4-Bromo-2-(3,5-dimethylbenzyl)phenoxy]ethyl]
uracil (10). Yield: 76%, mp: 189–190.5 °C, Rf 0.71 (elution with
ethyl acetate); 1H NMR (DMSO-d6): d 2.20 (s, 6H, CH3), 3.78 (s,
2H, ArCH2), 4.09 (t, 2H, J = 5 Hz, N–CH2), 4.13 (t, 2H, J = 4.5 Hz,
O–CH2), 5.40 (d, 1H, J = 8 Hz, H-5), 6.69 (s, 2H, H-200, H-600), 6.73
(s, 1H, H-400), 6.88–6.91 (m 1H, H-60), 7.14 (m, 1H, H-30), 7.25–
7.28 (m, 1H, H-50), 7.55 (d, 1H, J = 8 Hz, H-6), 11.28 (s, 1H, NH);
13C NMR (DMSO-d6): d 21.0, 47.1, 65.7, 100.7, 112.3, 114.0, 126.4,
127.6, 130.0, 132.3, 132.5, 137.3, 139.9, 146.1, 151.0, 154.9,
163.7; HRMS calcd for C21H21BrN2O3, [M+H]+ 429.0808; found
(FAB) m/z ([M+H]+): 429.0804 (79Br), 431.0796 (81Br).
4.2.1.11. 1-[2-[2-(3,5-Dimethylbenzoyl)-4-methylphenoxy)eth
yl]uracil (16). Yield: 66%, mp: 215.5–217 °C, Rf 0.53 (elution with
ethyl acetate); 1H NMR (DMSO-d6): d 2.22 (s, 9H, CH3), 3.68 (t, 2H,
J = 4.5 Hz, N–CH2), 4.04 (t, 2H, J = 4.5 Hz, O–CH2), 5.02 (d, 1H,
J = 7.5 Hz, H-5), 6.70 (d, 1H, J = 7.5 Hz, H-50), 7.02–7.05 (m, 2H, aro-
matic H), 7.16–7.27 (m, 4H, aromatic H, H-6), 11.11 (s, 1H, NH); 13
C
NMR (DMSO-d6): d 19.8, 20.6, 47.2, 65.7, 100.1, 112.9, 126.7, 128.7,
129.0, 130.2, 132.2, 134.9, 137.1, 137.7, 145.3, 150.6, 153.2, 163.4,
195.7; HRMS calcd for C22H22N2O4, [M+H]+ 379.1652; found (FAB)
m/z ([M+H]+): 379.1661.
4.2.1.6. 1-[2-(2-Benzoyl-4-methylphenoxy)ethyl]uracil (11).
Yield: 66%, mp: 191–193 °C, Rf 0.48 (elution with ethyl acetate),
1H NMR (DMSO-d6): d 2.23 (s, 3H, CH3), 3.67 (m, 2H, N–CH2),
4.03 (m, 2H, O–CH2), 5.09 (d, 1H, J = 7.5 Hz, H-5), 6.66 (d, 1H,
J = 7.5 Hz, H-50), 7.02–7.08 (m, 2H, H-30, H-60), 7.26–7.29 (m, 1H,
H-400), 7.41–7.46 (m, 2H, H-200, H-600), 7.54–7.63 (m, 2H, H-500, H-
6), 11.11 (s, 1H, NH); 13C NMR (DMSO-d6): d 19.8, 47.0, 65.8,
100.3, 112.8, 128.5, 129.1, 130.1, 132.3, 133.3, 137.0, 145.3,
150.6, 153.3, 163.4, 195.6; HRMS calcd for C20H18N2O4, [M+H]+
351.1339; found (FAB) m/z ([M+H]+): 351.1336.
4.2.1.12. 1-[2-[4-Chloro-2-(3,5-dimethylbenzoyl)phenoxy]ethyl]
uracil (17). Yield: 64%, mp: 236–238 °C, Rf 0.45 (elution with
ethyl acetate); 1H NMR (DMSO-d6): d 2.29 (s, 6H, CH3), 3.77 (t,
2H, J = 5 Hz, N–CH2), 4.16 (t, 2H, J = 5 Hz, O–CH2), 5.07 (d, 1H,
J = 7.8 Hz, H-5), 6.77 (d, 1H, J = 7.9 Hz, H-50), 7.23–7.28 (m, 4H, H-
30, H-200, H-400, H-600), 7.35 (m, 1H, H-60), 7.55 (dd, 1H, J = 8.8 and
2.8 Hz, H-6), 11.09 (s, 1H, NH); 13C NMR (DMSO-d6): d 20.6, 47.0,
66.1, 100.2, 114.8, 125.0, 126.8, 128.1, 130.5, 131.2, 135.3, 136.4,
137.9, 145.1, 150.6, 154.0, 163.3, 193.9; HRMS calcd for
4.2.1.7. 1-[2-(2-Benzoyl-4-fluorophenoxy)ethyl]uracil (12).
Yield: 71%, mp: 170.5–172 °C, Rf 0.47 (elution with ethyl acetate);
1H NMR (DMSO-d6): d 3.69 (m, 2H, N–CH2), 4.07 (m, 2H, O–CH2),
5.08 (d, 1H, J = 7.5 Hz, H-5), 6.68 (d, 1H, J = 8 Hz, H-50), 7.16–7.22
(m, 2H, aromatic H), 7.30–7.36 (m, 1H, aromatic H), 7.43–7.51
(m, 2H, aromatic H, H-6), 7.53–7.58 (m, 1H, aromatic H), 7.57–
7.66 (m, 2H, aromatic H), 11.12 (s, 1H, NH); 13C NMR (DMSO-d6):
d 46.9, 66.3, 100.3, 114.6, 114.6, 115.2, 115.4, 117.9, 118.1, 128.6,
129.1, 129.7, 129.7, 133.7, 136.3, 145.2, 150.6, 151.5, 155.1,
C
21H19ClN2O4, [M+H]+ 399.1106; found (FAB) m/z ([M+H]+):
399.1108 (35Cl), 401.1091 (37Cl).
4.2.1.13. 1-[2-[4-Bromo-(3,5-dimethylbenzoyl)phenoxy]ethyl]
uracil (18). Yield: 69%, mp: 236–237 °C, Rf 0.50 (elution with
ethyl acetate); 1H NMR(DMSO-d6): d 2.23 (s, 6H, CH3), 3.71 (t,
2H, J = 4.5 Hz, N–CH2), 4.10 (t, 2H, J = 4.5 Hz, O–CH2), 4.99 (d, 1H,
J = 8 Hz, H-5), 6.96–6.99 (m, 1H, H-50), 7.21 (d, 1H, J = 9 Hz, H-60),
7.19–7.21 (m, 3H, H-200, H-400, H-600), 7.41 (d, 1H, J = 2 Hz, H-
30),7.63 (dd, 1H, J = 8 and 2 Hz, H-6), 11.10 (s, 1H, NH); 13C NMR
(DMSO-d6): d 20.6, 47.0, 66.0, 100.2, 112.6, 115.3, 126.8, 130.8,
134.1, 135.3, 136.4, 137.9, 145.1, 150.6, 154.4, 163.3, 193.8; HRMS
157.4, 163.4, 194.0; HRMS calcd for
C
19H15FN2O4, [M+H]+
355.1088; found (FAB) m/z ([M+H]+): 355.1086.
4.2.1.8. 1-[2-(2-Benzoyl-4-chlorophenoxy)ethyl]uracil (13).
Yield: 79%, mp: 219–220 °C, Rf 0.39 (elution with ethyl acetate);
1H NMR (DMSO-d6): d 3.70 (t, 2H, J = 5 Hz, N–CH2), 4.10 (t, 2H,
J = 5 Hz, O–CH2), 5.08 (dd, 1H, J = 8 and 2 Hz, H-5), 6.67 (d, 1H,
J = 8 Hz, H-50), 7.19 (d, 1H, J = 8 Hz, H-60), 7.35–7.65 (m, 7H, H-30,
H-200, H-400, H-600, H-6), 11.10 (s, 1H, NH); 13C NMR (DMSO-d6): d
46.8, 66.1, 100.4, 114.8, 125.0, 128.2, 128.6, 129.2, 130.1, 131.4,
133.7, 136.3, 145.2, 150.6, 154.0, 163.4, 193.9. HRMS calcd for
calcd for
C
19H20BrN2O4, [M+H]+ 444.0601; found (FAB) m/z
([M+H]+): 443.0606 (79Br), 445.0589 (81Br).
4.2.1.14. 1-[2-[4-Chloro-2-(3,5-difluorobenzoyl)phenoxy]ethyl]
uracil (19). Yield: 77%, mp: 233.5–235 °C, Rf 0.43 (elution with
ethyl acetate); 1H NMR (DMSO-d6): d 3.77 (t, 2H, J = 4.5 Hz, N–
CH2), 4.14 (t, 2H, J = 4.5 Hz, O–CH2), 5.13 (d, 1H, J = 7.5 Hz, H-5),
6.96 (d, 1H, J = 7.5 Hz, H-50), 7.20–7.26 (m, 3H, aromatic H), 7.39–
7.50 (m, 2H, aromatic H), 7.55 (dd, 1H, J = 8 and 2.5 Hz, H-6),
11.14 (s, 1H, NH); 13C NMR (DMSO-d6): d 47.1, 66.2, 100.2, 114.9,
122.9, 125.1, 128.4, 128.7, 130.6, 132.3, 138.2, 139.7, 145.2,
C
19H15ClN2O4, [M+H]+ 371.0793; found (FAB) m/z ([M+H]+):
371.0792 (35Cl), 373.0776 (37Cl).
4.2.1.9. 1-[2-(2-Benzoyl-4-bromophenoxy)ethyl]uracil (14).
Yield: 76%, mp: 236–237 °C, Rf 0.42 (elution with ethyl acetate);
1H NMR (DMSO-d6): d 3.69 (t, 2H, J = 4.5 Hz, N–CH2), 4.09 (t, 2H,
J = 4.5 Hz, O–CH2), 5.07 (d, 1H, J = 8 Hz, H-5), 6.66 (d, 1H, J = 8 Hz,
H-50), 7.14 (d, 1H, J = 8 Hz, H-60), 7.42–7.47 (m, 2H, aromatic H),
7.57–7.65 (m, 3H, aromatic H), 11.10 (s, 1H, NH); 13C NMR
(DMSO-d6): d 46.8, 66.0, 100.4, 112.5, 115.2, 128.5, 129.2, 130.5,
130.9, 133.7, 134.3, 136.3, 145.2, 150.6, 154.5, 163.4, 193.8; HRMS
150.6, 154.2, 163.3, 191.3; HRMS calcd for
C19H13ClF2N2O4,
[M+H]+ 407.0605; found (FAB) m/z ([M+H]+): 407.0608 (35Cl),
409.0589 (37Cl).
4.2.1.15. 1-[2-[4-Chloro-2-(3,5-dichlorobenzoyl)phenoxy]ethyl]
uracil (20). Yield: 70%, mp: 240–242 °C, Rf 0.35 (elution with
ethyl acetate); 1H NMR (DMSO-d6): d 3.81 (t, 2H, J = 5 Hz, N–
CH2), 4.20 (t, 2H, J = 5 Hz, O–CH2), 5.16 (d, 1H, J = 7.8 Hz, H-5),
6.98 (d, 1H, J = 7.8 Hz, H-50), 7.27 (d, 1H, J = 8.9 Hz, H-60), 7.46 (d,
1H, J = 2.7 Hz, H-30), 7.59–7.59 (m, 2H, H-200, H-400), 7.61 (dd, 1H,
J = 8.9 and 2.7 Hz, H-6), 7.84 (t, 1H, J = 1.9 Hz, H-600), 11.14 (br s,
1H, NH); 13C NMR (DMSO-d6): d 47.0, 66.2, 100.1, 114.9, 125.1,
127.4, 128.5, 128.6, 132.2, 132.9, 134.6, 139.3, 145.2, 150.6,
calcd for
C
19H15BrN2O4, [M+H]+ 416.0288; found (FAB) m/z
([M+H]+): 415.0293 (79Br), 417.0268 (81Br).
4.2.1.10.
1-[2-[4-Chloro-2-(3-methylbenzoyl)phenoxy]ethyl]
uracil (15). Yield: 79%, mp: 191–192 °C, Rf 0.37 (elution with
ethyl acetate); 1H NMR (DMSO-d6): d 2.28 (s, 3H, CH3), 3.71 (t,
2H, J = 4.5 Hz, N-CH2), 4.10 (t, 2H, J = 4.5 Hz, O–CH2), 5.04 (dd,
1H, J = 8 and 2 Hz, H-5), 6.69 (d, 1H, J = 7 Hz, H-50), 7.19 (d, 1H,
J = 9 Hz, H-60), 7.29–7.44 (m, 5H, H-30, H-200, H-400, H-600), 7.51 (dd,
154.2, 163.2, 191.4; HRMS calcd for
C
19H13Cl3N2O4, [M+H]+
439.00137; found (FAB) m/z ([M+H]+): 439.0023 (35Cl), 440.9998
(2 ꢀ 35Cl, 1 ꢀ 37Cl), 442.9972 (1 ꢀ 35Cl, 2 ꢀ 37Cl).

M. S. Novikov et al. / Bioorg. Med. Chem. 19 (2011) 5794–5802
5801
4.2.1.16. 1-[2-[4-Chloro-2-(3,5-dibromobenzoyl)phenoxy]ethyl]
(s, 6H, CH3), 3.85 (t, 2H, J = 5 Hz, N–CH2), 4.19 (t, 2H, J = 5 Hz, O–
CH2), 5.01 (s, 1H, H-5), 7.21 (m, 1H, H-50), 7.24–7.26 (m, 3H, H-30,
H-200, H-600), 7.55 (d, 1H, J = 2.6 Hz, H-400), 7.55 (dd, 1H, J = 8.9 and
2.8 Hz, H-60), 11.04 (s, 1H, NH); 13C NMR (DMSO-d6): d 19.4, 20.6,
43.0, 66.2, 100.9, 114.7, 125.0, 126.9, 127.7, 130.6, 131.2, 135.5,
135.9, 137.9, 151.3, 153.7, 153.9, 162.1, 194.0; HRMS calcd for
uracil (21). Yield: 75%, mp: 261–262 °C, Rf 0.47 (elution with
ethyl acetate); 1H NMP-spectrum (DMSO-d6):
d 3.75 (t, 2H,
J = 5 Hz, N–CH2), 4.13 (t, 2H, J = 5 Hz, O–CH2), 5.08 (d, 1H,
J = 8 Hz, H-5), 6.87 (d, 1H, J = 8 Hz, H-50), 7.21 (d, 1H, J = 8 Hz, H-
60), 7.41 (d, 1H, J = 1.2 Hz, H-400), 7.57 (d, 1H, J = 8 Hz, H-6), 6.69
(s, 2H, H-200, H-600), 8.03 (d, 1H, J = 1.5 Hz, H-400), 11.10 (s, 1H,
NH); 13C NMR (DMSO-d6): d 47.0, 66.2, 100.2, 114.9, 122.9, 125.1,
128.4, 128.7, 128.6, 130.6, 132.3, 138.2, 139.7, 145.2, 150.6,
154.2, 163.2, 191.2; HRMS calcd for C19H13Br2ClN2O4, [M+H]+
526.9003; found (FAB) m/z ([M+H]+): 526.9011 (2 ꢀ 79Br, 35Cl),
528.8991 (79Br, 81Br, 35Cl), 530.8974 (79Br, 81Br, 37Cl).
C
22H21ClN2O4, [M+H]+ 413.1263; found (FAB) m/z ([M+H]+):
413.1265 (35Cl), 415.1250 (37Cl).
4.3. Synthesis of 1-[2-[4-chloro-2-[(3,5-dimethylphenyl)hydrox
ymethyl]phenoxy]ethyl]uracil (26)
To a solution of 1-[2-[4-chloro-2-(3,5-dimethylbenzoyl)-phen-
oxy]ethyl]uracil (17) (0.98 g, 2.46 mmol) in a mixture of ethanol
(20 mL) and water (10 mL) containing NaOH (0.2 g, 5.0 mmol)
NaBH4 (0.1 g, 2.64 mmol) was added. The mixture was stirred dur-
ing 8 h at 50 °C and then kept at room temperature overnight. After
treatment with 2% aqueous HCl the flask was kept at 4 °C over-
night. The crystalline precipitate was filtered and recrystallized
twice from isopropanol:DMF (1:1) mixture. Yield: 93%, mp: 206–
207.5 °C, Rf 0.55 (elution with ethyl acetate); 1H NMR (DMSO-
d6): d 2.17 (s, 6H, CH3), 4.05 (t, 2H, J = 5.4 Hz, N–CH2), 4.14 (t, 2H,
J = 4.2 Hz, O–CH2), 5.44 (d, 1H, J = 7.8 Hz, H-5), 5.72 (d, 1H,
J = 3.9 Hz, CH), 5.83 (d, 1H, J = 3.9 Hz, OH), 6.77 (s, 1H, H-40), 6.85
(s, 2H, H-20, H-60), 6.96 (d, 1H, J = 8.7 Hz, H-600), 7.19 (d, 1H,
J = 8.7 Hz, H-500), 7.38 (s, 1H, H-300), 7.56 (d, 1H, J = 7.8 Hz, H-60),
11.31 (s, 1H, NH); 13C NMR (DMSO-d6): d 20.9, 47.0, 65.8, 67.4,
100.7, 113.5, 123.9, 124.7, 126.5, 127.3, 128.2, 136.1, 136.9,
144.3, 145.8, 151.0, 153.1, 163.6; HRMS calcd for C21H21ClN2O4,
4.2.1.17.
1-[2-[4-Bromo-(3,5-dichlorobenzoyl)phenoxy]ethyl]
uracil (22). Yield: 70%, mp: 258–259.5 °C, Rf 0.47 (elution with
ethyl acetate); 1H NMR (DMSO-d6): d 3.81 (t, 2H, J = 5.1 Hz, N–
CH2), 4.19 (t, 2H, J = 5.1 Hz, O–CH2), 5.16 (dd, 1H, J = 8 and
2.1 Hz, H-5), 6.91 (d, 1H, J = 8 Hz, H-50), 7.16 d (1H, J = 8 Hz, H-
60), 7.56–7.59 (m, 3H, H-30, H-200, H-400), 7.74 (dd, 1H, J = 8.9 and
2.7 Hz, H-6), 7.84–7.85 (m, 1H, H-400), 11.14 (s, 1H, NH); 13C NMR
(DMSO-d6): d 46.9, 66.1, 100.2, 112.6, 115.3, 127.4, 128.9, 131.4,
132.9, 134.6, 135.1, 139.3, 145.2, 150.5, 154.6, 163.1, 191.3; HRMS
calcd for C19H13BrCl2N2O4, [M+H]+ 482.9509; found (FAB) m/z
([M+H]+): 482.9511 (2 ꢀ 35Cl, 79Br), 484.9486 (alternative isotope),
486.9469 (alternative isotope).
4.2.1.18. 1-[3-[4-Chloro-2-(3,5-dimethylbenzoyl)phenoxy]pro-
pyl]uracil (23). Yield: 62%, mp: 200–201 °C, Rf 0.70 (elution with
ethyl acetate); 1H NMR (DMSO-d6): d 1.70 (m, 2H, J = 6.7 Hz, CH2),
3.31 (t, 2H, J = 6.7 Hz, CH2), 3.95 (t, 2H, J = 6.1 Hz, CH2), 5.46 (dd,
1H, J = 7.8 and 1.9 Hz, H-5), 7.14 (dd, 1H, J = 7.8 and 1.2 Hz, H-
500), 7.19 (d, 1H, J = 8.9 Hz, H-600), 7.28 (s, 1H, H-40), 7.32 (s, 1H, H-
20, H-60), 7.19 (d, 1H, J = 8.7 Hz, H-500), 7.38 (d, 1H, J = 2.6 Hz, H-
300), 7.56 (dd, 1H, J = 8.9 and 2.8 Hz, H-6), 11.16 (s, 1H, NH); 13C
NMR (DMSO-d6): d 20.6, 27.7, 44.5, 65.5, 100.9, 114.9, 124.6,
126.7, 128.2, 130.4, 131.5, 134.8, 137.3, 137.9, 145.0, 150.6154.6,
163.6, 194.5; HRMS calcd for C22H21ClN2O4, [M+H]+ 413.1263;
found (FAB) m/z ([M+H]+): 413.1269 (35Cl), 415.1248 (37Cl).
[M]+ 400.1190; found (FAB) m/z ([M+H]+): 400.1190
(
35Cl),
402.1168 (37Cl).
5. Biological evaluation
5.1. Antiviral assays
The methodology of the anti-HIV assays was as follows: human
CEM (ꢁ3 ꢀ 105 cells/cm3) cells were infected with 100 CCID50 of
HIV(IIIB) or HIV-2(ROD)/ml and seeded in 200 lL wells of a micro-
4.2.1.19. 1-[2-[4-Chloro-2-(3,5-dimethylbenzoyl)phenoxy]ethyl]
thymine (24). Yield: 69%, mp: 244–245 °C, Rf 0.54 (elution with
ethyl acetate); 1H NMR (DMSO-d6): d 1.53 (d, 3H, J = 1.1 Hz, CH3),
2.29 (s, 6H, CH3), 3.75 (t, 2H, J = 5.1 Hz, N–CH2), 4.18 (t, 2H,
J = 5 Hz, O–CH2), 6.80–6.81 (m, 1H, H-50), 7.25–7.28 (m, 4H, H-30,
H-200, H-400, H-600), 7.32 (d, 1H, J = 2.7 Hz, H-60), 7.55 (dd, 1H,
J = 8.9 and 2.7 Hz, H-6), 11.08 (s, 1H, NH); 13C NMR (DMSO-d6): d
11.6, 20.6, 46.8, 66.2, 107.8, 115.0, 124.9, 126.8, 128.0, 130.5,
131.1, 135.2, 136.2, 137.8, 141.3, 150.6, 154.0, 163.9, 193.7; HRMS
titer plate containing appropriate dilutions of the test compounds.
After 4 days of incubation at 37 °C, HIV-induced CEM giant cell for-
mation was examined microscopically.
The methodology of the anti-HIV assays in MT-4 cell cultures
was as follows: virus stocks were titrated in MT-4 cells and ex-
pressed as the 50% cell culture infective dose (CCID50). MT-4 cells
were suspended in culture medium at 1 ꢀ 105 cells/ml and in-
fected with HIV at a multiplicity of infection of 0.02. Immediately
after viral infection, 100 ll of the cell suspension was placed in
calcd for
C
22H21ClN2O4, [M+H]+ 413.1263; found (FAB) m/z
each well of a flat-bottomed microtiter tray containing various
concentrations of the test compounds. The test compounds were
dissolved in 100% DMSO at 50 mM or higher. After 4 days of incu-
bation at 37 °C, the number of viable cells was determined using
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) method. The selection and characterization of mutant virus
strains have been performed previously.
([M+H]+): 413.1267 (35Cl), 415.1244 (37Cl).
4.2.2. Synthesis of 1-[2-[4-chloro-2-(3,5-dimethylbenzoyl)phen
oxy]ethyl]-6-methyluracil (25)
A suspension of 6-methyluracil (5.0 g, 39.65 mmol) and K2CO3
(1.7 g, 12.30 mmol) in 50 mL DMF was stirred at ꢁ70 °C for 1 h
and then treated with a solution of 1-bromo-2-[4-chloro-2-(3,5-
dimethylbenzoyl)phenoxy]ethane 5 (2.9 g, 7.89 mmol) in DMF
(10 mL). The mixture was stirred at ꢁ70 °C for 16 h, then was kept
at room temperature overnight and filtered. The inorganic precip-
itate was washed with DMF, and the filtrate was evaporated in vac-
uum. The residue was dissolved in water (80 mL), extracted with
chloroform (50 mL ꢀ 3), and the combined organic phases were
dried on CaCl2 and evaporated. The target compound 25 was ob-
tained by double recrystallization from a isopropanol:DMF (1:1)
mixture. Yield: 64%, mp: 192–194 °C, Rf 0.59 (elution with ethyl
acetate); 1H NMR (DMSO-d6): d 1.78 (d, 3H, J = 0.6 Hz, CH3), 2.27
5.2. Reverse transcriptase plasmids, RT expression and
purification
Plasmid encoding the HIV-1 reverse transcriptase (RT) was ob-
tained out as previously reported.36 Plasmids encoding RT in which
one or two amino acid residues were substituted (L100I, K103N,
V106A, Y181C, Y188L, G190A and K103N/Y181C) were constructed
by amplifying the gene in two fragments using the oligonucleo-
tides listed in Table S1. ‘Head’ and ‘Tail’ PCR-products were mixed

5802
M. S. Novikov et al. / Bioorg. Med. Chem. 19 (2011) 5794–5802
together in the absence of oligos and 30 additional amplification
cycles were performed in order to obtain the full-length genes.
The resulting products were digested with XhoI and XbaI restriction
endonucleases and ligated into pBRP-HR or pET-21d-2c vectors.37
RT genes containing mutations K103N and G190A were inserted
into a pBRP-HR vector, other mutant genes were subcloned into
pET-21d-2c. The wild-type and mutant RTs were expressed in
E. Coli Rosetta (DE3) strain and purified on Ni-NTA–agarose resin
as described earlier for HCV RNA polymerase.37
References and notes
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vated DNA, 0.05 g p66/p66 RT, 3
and dTTP, 1 Ci [
32P]dATP in a Tris-HCl buffer (50 mM, pH 8.1)
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lg of acti-
l
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a-
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poly(rC)-oligo(dG) as the template–primer complex were per-
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air the filters were washed twice with 10% trichloracetic acid, then
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air. The radioactivity was measured by the Cherenkov method.38
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type GF/C (GE Healthcare UK Limited, Buckinghamshire, UK) and
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This work was supported by Ministry of Education and Science
of Russian Federation (state contract 16.512.11.2192), by the
Russian Foundation for Basic Research (grant 10-04-0056), by the
Presidium of the Russian Academy of Sciences (Programme ‘Molec-
ular and Cellular Biology’) and by the K. U. Leuven (GOA no. 10/
014). A.I. was supported by grant of President of Russian Federa-
tion (MR-5035.2011.4). The authors like to thank Inna Karpenko
and Natalia Zakirova (EIMB), and Lizette van Berckelaer, Kristien
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Supplementary data
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Supplementary data (1H, 13C NMR and solubility data, as well as
Dixon plots for the compounds) associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2011.08.025.