Synthesis and biological evaluation of 6-substituted 5-Alkyl-2-(phenylaminocarbonylmethylthio)pyrimidin-4(3H)-ones as potent HIV-1 NNRTIs

Article abstract of DOI:10.1002/cmdc.201000555

A series of new 5-alkyl-2-phenylaminocarbonylmethylthiopyrimidin-4(3H)-ones bearing variously substituted arylmethyl moieties at the C6 position of the pyrimidine ring were synthesized and evaluated for anti-HIV activity in MT-4 cells. Most of these new c

Full text of DOI:10.1002/cmdc.201000555

MED
DOI: 10.1002/cmdc.201000555
Synthesis and Biological Evaluation of 6-Substituted 5-
Alkyl-2-(phenylaminocarbonylmethylthio)pyrimidin-4(3H)-
ones as Potent HIV-1 NNRTIs
Mingyan Yu,[a] Zhenyu Li,[a] Shuai Liu,[a] Erkang Fan,[b] Christophe Pannecouque,[c]
Erik De Clercq,[c] and Xinyong Liu*[a]
A series of new 5-alkyl-2-phenylaminocarbonylmethylthiopyri-
midin-4(3H)-ones bearing variously substituted arylmethyl moi-
eties at the C6 position of the pyrimidine ring were synthe-
sized and evaluated for anti-HIV activity in MT-4 cells. Most of
these new congeners exhibited moderate to good activities
against the wild-type virus, with EC50 values in the range of
1.40–0.19 mm. Among them, 2-[(4-cyanophenylamino)carbonyl-
methylthio]-6-(2-chloro-6-fluorobenzyl)-5-ethylpyrimidin-4(3H)-
one 4b6 is one of the compounds endowed with the highest
broad-spectrum HIV-1 inhibitory activity, with EC50 values of
0.19Æ0.005 mm against the wild-type virus, 1.05Æ0.24 mm
(twofold resistance) against the E138K strain, and 2.38Æ
0.13 mm (4.5-fold resistance) against the Y181C strain. Further-
more, reverse transcriptase (RT) inhibition assays against wild-
type HIV-1 RT were performed with selected derivatives, con-
firming that the main target of these compounds is HIV-1 RT
and that these new S-DABO analogues act as non-nucleoside
RT inhibitors (NNRTIs). Structure–activity relationship and mo-
lecular modeling analyses of these new congeners are also dis-
cussed.
Introduction
Human immunodeficiency virus (HIV) infection affects nearly
60 million individuals worldwide. Since the first cases of indi-
viduals dying from a rare opportunistic infection was reported,
25 million people have died from this epidemic.[1] Although
the introduction of highly active antiretroviral therapy (HAART)
has dramatically decreased the morbidity and mortality result-
ing from the infection with HIV, AIDS has remained one of the
world’s most serious health problems.[2] In the research of anti-
HIV agents, HIV-1 non-nucleoside reverse transcriptase inhibi-
tors (NNRTIs) bind to an allosteric site on reverse transcriptase
(RT) and represent an important therapeutic class of inhibitors
used in the treatment of HIV-1 infection.[3–5] Among the
NNRTIs, dihydroalkyloxybenzyloxopyrimidines (DABOs) were re-
ported as potent HIV NNRTIs, with robust anti-HIV-1 activity
against both the wild-type (wt) and a panel of clinically rele-
vant HIV-1 mutants.[6] Three generations of DABO analogues
have been found to date: dihydroalkyloxybenzyloxopyrimi-
dines (O-DABOs), dihydroalkylthiobenzyloxopyrimidines (S-
stituent at C5. Herein we describe the synthesis, anti-HIV-1 ac-
tivity, and preliminary SAR studies of these new congeners.
The different degrees of potency displayed by the new mole-
cules against wild-type and mutant HIV-1 virus were also stud-
ied by molecular modeling approaches.
DABOs), and dihydroalkylaminodifluorobenzyloxopyrimidines
[7–11]
(N-DABOs),
from which many promising DABOs have been
developed.
Our recent work on S-DABOs disclosed that assembly of a
phenylaminocarbonylmethylthio moiety at the C2 position of
the pyrimidine ring has resulted in new lead compounds with
potent antiviral activities against HIV-1.[12] Continuing our re-
search to obtain new SAR information and to explore the
effect of the insertion of a C2-phenylaminocarbonylmethylthio
moiety in S-DABOs, we designed a novel series of S-DABOs
with structural characterization of a 2-phenylaminocarbonyl-
methylthio chain at C2, a 2-chloro-6-fluoro-, 1-naphthylmethyl,
or 2,6-difluorobenzyl group at C6, and a methyl or ethyl sub-
[a] Dr. M. Yu, Z. Li, S. Liu, Prof. Dr. X. Liu
Institute of Medicinal Chemistry, School of Pharmaceutical Sciences
Shandong University, No.44 Wenhuaxi Road, Jinan 250012 (P.R. China)
Fax: (+86)53188382731
E-mail: xinyongl@sdu.edu.cn
[b] E. Fan
Biomolecular Structure Center
University of Washington, 98195, Seattle, WA (USA)
[c] Prof. Dr. C. Pannecouque, Prof. Dr. E. De Clercq
Rega Institute for Medical Research
Katholieke Universiteit Leuven, 3000 Leuven (Belgium)
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DABO Analogues as HIV-1 NNRTIs
The results of the anti-HIV assay demonstrated that the ma-
jority of these series of compounds exhibited moderate to
good activities against HIV-1 strain IIIB with EC50 values in the
range of 1.40–0.19 mm, whereas compounds 4b3 and 4b4
were inactive. The most active S-DABO derivative was com-
pound 4b6 with an EC50 value of 0.19Æ0.005 mm and a SI
value of >169, which are much better than those of NVP, DDC,
and DDI. It is worth noting that the SI values of these S-DABO
derivatives are very variable, ranging from 10 to 785. Com-
pound 4a2 was identified as the most selective compound in
this series (SI=785), which is also much more selective than
NVP, DDC, and DDI.
Results and Discussion
Chemistry
The target compounds 4a1–4a6, 4b1–4b6, 4c1–4c6, 4d1–
4d6, and 4e1 were prepared as depicted in Scheme 1. The b-
keto esters 2a–e were prepared by a simple and high-yielding
The SAR analysis showed that the combined action of the
substituents at the C5 position and C6 position of the pyrimi-
dine ring influenced the anti-HIV-1 strain IIIB activity of the new
S-DABO analogues. When C5 was substituted by a methyl
group, the 2-chloro-6-fluorobenzyl analogues (4a1–4a6) were
more potent than the corresponding 2,6-difluorobenzyl ana-
logues (4c1–4c6) in inhibiting wt HIV-1, with the exception of
4a1. However, when C5 was substituted by an ethyl moiety,
the 2,6-difluorobenzyl analogues 4d1–4d6 were more potent
than the corresponding 2-chloro-6-fluoro analogues 4b1–4b6
with the exception of 4d5 and 4d6. Moreover, it was further
confirmed that the substituent at the C5 position of the pyri-
midine ring also influenced the anti-HIV-1 activity of these new
congeners. When the C5 substituents were changed from
methyl to ethyl, a marked increase in anti-HIV-1 activity was
observed for the 2,6-difluorobenzyl analogues (4c1–4c6 versus
4d1–4d6). The above observations contrasted sharply with
the 2-chloro-6-fluorobenzyl derivatives (4a1–4a6 versus 4b1–
4b6), in which the steric bulk of the C5 substituent is detri-
mental to HIV-1 inhibitory activity. In addition, when the C6
group was substituted by 1-naphthylmethyl (4e1), the com-
pound lost its anti-HIV-1 activity completely.
Scheme 1. Reagents and conditions: a) 1. R1CHBrCOOEt, Zn, THF, reflux,
2. K2CO3 (50%), 3. HCl (13%); b) thiourea, NaOEt, reflux, 6–12 h; c) appro-
priate N-phenylacetamide halides, K2CO3, DMF, room temperature, 12 h.
method reported by Hannick and Kishi through the reaction of
arylacetonitriles 1a–c with activated zinc dust and 2-bromoal-
kanoates.[13,14] Subsequent condensation of b-keto esters 2a–e
with thiourea in the presence of sodium ethoxide in ethanol at
reflux gave the key intermediates 5-alkyl-6-substituted thioura-
cil 3a–e.[15] Next, selective S-alkylation of 3a–e with the appro-
priate N-phenylacetamide halides (1:1.1) in the presence of po-
tassium carbonate in anhydrous N,N-dimethylformamide (DMF)
afforded the desired target compounds 4a1–4a6, 4b1–4b6,
4c1–4c6, 4d1–4d6, and 4e1. Both analytical and spectral
data for all compounds are in full agreement with the pro-
posed structures.
In fact, the variable activities of these novel series com-
pounds are attributed to the changed substituent at C2, in
which a phenylmethylthio group of the S-DABOs prototypes
was replaced by the phenylaminocarbonylmethylthio group.
The presence of a C2 phenylaminocarbonylmethylthio moiety
changes the original orientation of the S-DABOs in the non-nu-
cleoside binding site (NNBS) of HIV-1 RT. This issue is addressed
in the section concerning molecular modeling analysis.
In addition, the 15 selected compounds were also tested
against a panel of HIV-1 mutant RT strains E138K, F227L+
V106A, K103N, L100I, RES056 (K103N+Y181C), Y181C, and
Y188L (Table 2). To define the resistance profile of these com-
pounds, both the absolute activity against the HIV-1 mutants
(EC50 values) and the relative activity (fold resistance) need to
be considered. In this assay, the selected compounds retained,
in part, their activities against the E138K and Y181C mutant
strains, with EC50 values at the low micromolar level, whereas
they were inactive against the HIV-1 F227L+V106A, K103N,
L100I, RES056 (K103N+Y181C), and Y188L mutant strains.
Their fold resistance values ranged from 2 to 23.8 (E138K), and
from 4.5 to 97 (Y181C). Among the tested compounds, 4d6
was identified as the most active compound, having moderate
activity against mutant strains E138K (EC50 1.05Æ0.24 mm with
Anti-HIV activity in MT-4 cells
According to the MTT method,[16,17] the newly designed and
synthesized S-DABO analogues (compounds 4a–e) were tested
in MT-4 cells to evaluate their biological activity against wt
HIV-1 strain IIIB and HIV-2 strain ROD. The results, expressed as
EC50, CC50, and SI (selectivity index) values, are listed in Table 1.
Nevirapine (NVP), zidovudine (azidothymidine, AZT), dideoxycy-
tidine (DDC), and dideoxyinosine (DDI) were used as reference
drugs. Selected compounds were also tested against HIV-1
mutant RT strains E138K, F227L+V106A, K103N, L100I, RES056
(K103N+Y181C), Y181C, and Y188L. The results are listed in
Table 2 together with those of NVP, AZT, efavirenz (EFV), dela-
virdine (DLV), and etravirine (TMC125) as reference standards.
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Table 1. Anti-HIV activities of compounds 4a–e against wt HIV-1 IIIB and HIV-2 ROD strains in MT-4 cells.
EC50 [mm][a]
Compd
Ar
R1
R2
HIV-1 IIIB
HIV-2 ROD
>152.80
CC50 [mm][b]
SI (IIIB)[c]
4a1
4a2
4a3
4a4
4a5
4a6
4b1
4b2
4b3
4b4
4b5
4b6
4c1
4c2
4c3
4c4
4c5
4c6
4d1
4d2
4d3
4d4
4d5
4d6
4e1
NVP
AZT
DDC
DDI
2-Cl-6-F-Ph
2-Cl-6-F-Ph
2-Cl-6-F-Ph
2-Cl-6-F-Ph
2-Cl-6-F-Ph
2-Cl-6-F-Ph
2-Cl-6-F-Ph
2-Cl-6-F-Ph
2-Cl-6-F-Ph
2-Cl-6-F-Ph
2-Cl-6-F-Ph
2-Cl-6-F-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH2CH3
-CH2CH3
-CH2CH3
-CH2CH3
-CH2CH3
-CH2CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-F
-Cl
-Br
-OCH3
-NO2
-CN
-F
-Cl
-Br
-OCH3
-NO2
-CN
-F
-Cl
-Br
-OCH3
-NO2
-CN
-F
-Cl
-Br
0.77Æ0.49
0.27Æ0.03
0.30Æ0.05
0.44Æ0.32
1.14Æ0.01
0.45Æ0.24
1.26Æ0.17
0.50Æ0.32
152.80
ꢀ199
785
479
23
>210.80
>143.26
>10.25
>20.33
>73.15
>277.84
>5.10
210.80Æ78.13
143.26Æ38.48
10.24Æ4.43
20.33Æ4.84
73.15Æ62.29
277.84
18
162
>220
10
5.09Æ0.74
2.35Æ0.97
3.22Æ0.85
23.01Æ1.81
31.84Æ1.11
144.72Æ34.48
82.94Æ16.02
96.24Æ9.20
163.40Æ50.89
167.33Æ19.80
129.80Æ13.50
178.97Æ36.83
53.96Æ31.45
4.85Æ0.59
3.59Æ0.28
152.03Æ34.46
164.54Æ82.34
44.77Æ10.61
>15
ꢀ0.65
>3.23
0.39Æ0.15
>2.35
>3.23
ꢁ4
<1
>23.02
>31.84
>144.72
>82.94
>96.24
>163.40
>167.33
>129.80
>178.98
>53.97
>4.85
60
0.19Æ0.005
0.61Æ0.08
0.41Æ0.14
0.60Æ0.18
0.79Æ0.29
1.40Æ0.40
0.56Æ0.11
0.31Æ0.04
0.21Æ0.02
0.29Æ0.07
0.27Æ0.08
1.03Æ0.27
0.48Æ0.39
169
237
200
160
208
120
231
575
251
17
-CH3
-CH2CH3
-CH2CH3
-CH2CH3
-CH2CH3
-CH2CH3
-CH2CH3
-CH2CH3
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
1-Naph
-OCH3
-NO2
-CN
-F
>3.59
13
>152.03
>164.55
>44.76
>15
148
344
<1
>44.76
0.19Æ0.04
>80
>11587
>127
>13
0.0076Æ0.0014
0.75Æ0.55
0.0033Æ0.0006
>88.26
>94
>211
0.88Æ0.53
16Æ5.16
8.86Æ2.89
[a] Compound concentration required to effect 50% protection of MT-4 cells against HIV-induced cytotoxicity, as determined by the MTT method. [b] Com-
pound concentration required to decrease the viability of mock-infected cells by 50%, as determined by the MTT method. [c] Selectivity index: CC50/EC50
ratio (wt HIV-1 IIIB).
a twofold resistance ratio with respect to wt) and Y181C (EC50
Table 2. Anti-HIV-1 activity of selected compounds against HIV-1 mutant
2.38Æ0.13 mm with a 4.5-fold resistance ratio). Compared with
strains.
2,6-difluorobenzyl analogues, the 2-chloro-6-fluorobenzyl ana-
Compd
EC50 [mm][a] (fold resistance)[b]
logues showed a lower fold resistance of antiviral activity from
wt to E138K and Y181C HIV-1 strains (with the exception of
4b6 versus 4d6). Furthermore, the newly synthesized com-
pounds 4a–e were also evaluated for their capacity to inhibit
HIV-2 (strain ROD) replication in MT-4 cells, but none were
found to be effective (Table 1).
E138K
Y181C
4a1
4a2
4a3
4a6
4b1
4b5
4b6
4c2
4c3
4c4
4c6
4d1
4d2
4d3
4d6
NVP
AZT
EFV
3.87Æ0.86 (6.1)
2.68Æ0.25 (7.7)
2.51Æ0.27 (7.5)
2.07Æ0.97 (4.9)
4.19Æ1.90 (2.6)
2.62Æ0.24 (6.4)
2.03Æ0.3 (7.2)
>152.80 (>240.4)
>210.80 (>610.2)
32.41Æ9.11 (97)
15.83Æ3.10 (37.3)
9.85Æ2.54 (6.2)
2.54Æ0.62 (6.2)
2.13Æ0.58 (7.6)
>82.94 (>192.0)
>96.24 (>175.0)
152.97 (>212.9)
>129.80 (>246.5)
>178.98 (>443.9)
>53.97 (>311.9)
>4.85 (>17.2)
10.26Æ8.84 (23.8)
12.13Æ12.07 (22)
3.78Æ0.62 (5.2)
3.76Æ0.75 (7.1)
2.13Æ0.02 (5.3)
1.64Æ0.59 (8.2)
ꢀ1.03 (ꢀ3.7)
Inhibition of HIV-1 RTwt
To directly prove that the newly synthesized compounds
target HIV-1 RT, we chose two compounds (4b6 and 4d2) to
evaluate HIV-1 RT inhibitory activity, using a poly(rA)/oligo(dT)15
homopolymer template with the HIV antigen detection ELISA
for quantifying expression of HIV-1 RTwt in culture medium,
and nevirapine as a reference compound (Table 3). The results
show that these new compounds do inhibit the activity of HIV-
1 RTwt with IC50 values of 3.89 and 16.8 mm for 4b6 and 4d2,
respectively. In addition, none of the newly synthesized com-
pounds was active against HIV-2 (ROD) in the cell assay. Based
on the chemical structure and the general characteristic that
NNRTIs inhibit HIV-1 but not HIV-2 replication, it could be con-
1.05Æ0.24 (2)
2.38Æ0.13 (4.5)
NA
0.0079Æ0.0012(0.5)
0.0055Æ0.000479 (1.4)
1.47Æ0.33 (45.4)
0.009Æ0.0048 (3.2)
0.18Æ0.057 (1.2)
0.02Æ0.009 (1.4)
0.0055Æ0.00078 (1.4)
0.06Æ0.023 (1.9)
0.008Æ0.002 (2.6)
DLV
TMC125
[a] Compound concentration required to effect 50% protection of MT-4
cells against cytopathicity induced by HIV-1 mutant strains, as determined
by the MTT method. [b] Ratio of EC50 value against drug-resistant strain
and EC50 of wt HIV-1 IIIB (EC50mut/EC50wt).
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DABO Analogues as HIV-1 NNRTIs
The analyses of the 4b6 docking results revealed that the
overall binding mode is not influenced by the Y181C mutation.
The two binding modes (Figure 1a, Figure 1c) show the fol-
lowing: 1) The 2-phenylaminocarbonylmethylthio substituent
is well accommodated in the large pocket mainly defined by
Val106, Pro225, Pro236, and Phe227. A hydrogen bond is
formed between the C=O group of the C2 side chain and sur-
rounding amino acids only in RTY181C. Lengthening the C2 side
chain leads the C2 side chain to go beyond the opening de-
fined by Pro225 and Pro236. Thus, the end of C2 side chain is
exposed to the solvent, causing the lack of its hydrophobic in-
teractions with the NNIBP. 2) The pyrimidine NH moiety at po-
sition 3 is engaged in a hydrogen bond with the C=O moiety
of Lys101. 3) The alkyl group at the C5 position is positioned in
the hydrophobic cavity formed by the Val179 side chains.
4) The benzyl substituent at C6 of the pyrimidine ring is ac-
commodated in a hydrophobic pocket mainly defined by the
aromatic side chains of Tyr181 (Cys181), Tyr188, Phe227, and
Trp229 as well as by Leu234. In particular, the phenyl ring in-
teracts favorably with the Tyr188 side chain, giving rise to a
positive p-stacking interaction.
Table 3. Selected compounds assayed against wt HIV-1 RT.
Compd
IC50 [mm][a]
4b6
4d2
NVP
3.89
16.8
0.30
[a] Compound concentration required to inhibit HIV-1 RTwt activity by
50%.
cluded that the newly synthesized S-DABO compounds target-
ed the HIV-1 RT, therefore acting as NNRTIs.
Molecular modeling analysis
To investigate the binding modes of the newly synthesized
compounds, molecular modeling study was performed by
means of AutoDock Vina for docking. Default parameters were
used as described in the AutoDock Vina manual unless other-
wise specified.
Derivative 4b6 was identified as the most potent compound
against wt and mutant HIV-1 strains. Thus, 4b6 was taken as a
representative compound to study the 2-phenylaminocarbo-
nylmethyl-S-DABO binding models by docking into the NNBS
of either RTwt or RTY181C. To check the structural role of substitu-
tion at the C2 side chain of S-DABO, the same docking proce-
Inspection of the binding modes of 4b6 reveals a fair super-
imposition with those of JMC17 and TNK-651 both in RTwt and
RTY181C, showing an overall common binding conformation
(Figure 1b, Figure 1c). Notably, all compounds share the same
binding modes in RTwt (Figure 1b). Although the three com-
pounds are still superimposable in RTY181C, the binding confor-
mations of the compounds are slightly less overlapped. In par-
ticular, the benzyl substituent at C6 of 4b6 does not overlap
the C6 position of compounds JMC17 and TNK-651 (Figure 1d).
These results appear to be due to the introduction, in 4b6, of
a new anchor point (C=O at C2 side chain) by forming a new
hydrogen bond with NNBP. The new hydrogen bond pattern
changes the original orientation of the S-ADBOs in the RTY181C
(Figure 1d). Meanwhile, the new hydrogen bond with NNBP at
the C2 side chain could compensate for the incoming lack of
positive hydrophobic ligand–NNBP interactions due to the
Tyr181 to Cys181 mutation. As a result, 4b6 could be expected
to be less susceptible than JMC17 to decreasing activity when
tested against the Y181C mutated strain of HIV-1. This resist-
ance profile of 4b6 (4.5-fold resistance) is slightly better than
that of JMC17 (17.3-fold resistance).[6]
dure was carried out on
a structurally related S-DABO
(JMC17)[6] and also on TNK-651, a potent 1-[(2-hydroxyethoxy)-
methyl]-6-(phenylthio)thymine (HEPT) analogue. The coordi-
nates of TNK-651 co-crystallized in either RTwt (PDB code:
1RT2)[18] or RTY181C (PDB code: 1JLA)[19] were used to define the
NNBSwt and NNBSY181C. The docking modes are shown in
Figure 1.
Thus, the 2-phenylaminocarbonylmethyl-S-DABOs can be
considered as promising novel DABO candidates for further de-
velopment, and for further studies aimed at evaluating their ef-
fectiveness as anti-AIDS agents that are active against both
wild-type and mutated strains of HIV-1.
Conclusions
We designed and synthesized a series of novel 5-alkyl-6-aryl-
methyl-2-phenylaminocarbonylmethylthio substituted DABOs,
which were evaluated for their biological activity against wild-
type HIV-1 (IIIB strain) and HIV-2 (ROD strain) in MT-4 cells. Se-
lected compounds were also tested against HIV-1 mutant virus
strains E138K, F227L+V106A, K103N, L100I, RES056 (K103N+
Y181C), Y181C, and Y188L. Some of the new compounds dis-
Figure 1. a) Model of 4b6 docked into RTwt (PDB code: 1RT2). b) Superimpo-
sition of the docked conformations of 4b6 (white), JMC17 (red), and TNK-
651 (purple) in RTwt (PDB code: 1RT2). c) Model of 4b6 docked into RTY181C
(PDB code: 1JLA). d) Superimposition of the docked conformations of 4b6
(white), JMC17 (red), and TNK-651 (purple) in RTY181C (PDB code: 1JLA)
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X. Liu et al.
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under reduced pressure. The solid was washed sequentially with
H2O, EtOH, and Et2O, then dried to give 3a–e, which is directly
used in the next step without further purification.
played anti-wt-HIV-1 activity at low micromolar concentrations
along with low cytotoxicity. Among them, the most potent wt
HIV-1 inhibitor is 4b6 (EC50 =0.19Æ0.005 mm), which is more
effective than the reference drugs NVP, DDC, and DDI. In addi-
General procedure for the preparation of target compounds
4a–e: Compounds 3a–e (5 mmol) and appropriate N-phenylaceta-
mide halides (5.5 mmol) were suspended in anhydrous DMF
(25 mL) in the presence of anhydrous K2CO3 (0.759 g, 5.5 mmol) at
room temperature. The mixtures were irradiated at room tempera-
ture for 12 h. The reaction mixture was poured into cold H2O
(200 mL), the resulting precipitate was collected by filtration under
reduced pressure and washed sequentially with H2O, EtOH, and
Et2O, and then dried to give the corresponding crude product,
which was purified by crystallization to give the pure target com-
pounds 4a–e.
tion, 4b6 retains potency against the E138K and Y181C mu-
Y181C
tants (EC50E138K =1.05Æ0.24 mm, twofold resistance; EC50
=
2.38Æ0.13 mm, 4.5-fold resistance). Furthermore, enzyme inhib-
itory assays were performed with selected derivatives against
HIV-1 RTwt. The results showed that the main target of these
compounds is HIV-1 RT and these compounds act as NNRTIs.
The preliminary SAR among the newly synthesized congeners
and docking studies provided useful indications for guiding
the further rational design of new S-DABO analogues as more
active and selective HIV-1 inhibitors.
2-[(4-Fluorophenylamino)carbonylmethylthio]-6-(2-chloro-6-fluo-
robenzyl)-5-methylpyrimidin-4(3H)-one (4a1): Recrystallized from
EtOH/DMF as a white crystal, Yield: 23.6%; mp: 237–2398C (dec);
1H NMR ([D6]DMSO): d=12.74 (s, 1H, NH), 9.95 (s, 1H, NH), 7.52–
7.00 (m, 7H), 3.97 (s, 2H, S-CH2), 3.74 (s, 2H, CH2), 2.03 ppm (s, CH3,
3H); IR (KBr): n˜ =3251(uNH), 3050 (uNH), 1671(uC O), 1654(uC O),
Experimental Section
Chemistry
=
=
1228 (uCÀN), 1213 cmÀ1 (uCÀN); ESI-MS: m/z 436.1 [M+1]+, 458.1
All melting points were determined on a micromelting point appa-
ratus and are uncorrected. IR spectra were recorded with a Nexus
470 FTIR spectrometer. NMR spectra were obtained on a Bruker
Avance 600 NMR spectrometer in the indicated solvents. Chemical
shift values (d) are expressed in ppm, and (CH3)4Si was used as in-
ternal reference. MS data were collected on an LC Autosampler
device: Standard G1313A instrument. TLC was performed on silica
gel GF254 (Merck), and spots were visualized by I2 vapors or by irra-
diation with UV light (l 254 nm). Solvents were reagent grade and,
when necessary, were purified and dried by standard methods.
Concentration of the reaction solutions involved the use of rotary
evaporator at reduced pressure.
[M+Na]+; C20H16ClF2N3O2S (435.06).
2-[(4-chlorophenylamino)carbonylmethylthio]-6-(2-chloro-6-fluo-
robenzyl)-5-methylpyrimidin-4(3H)-one (4a2): Recrystallized from
EtOH/DMF as a white crystal, Yield: 22.9%; mp: 238–2408C (dec);
1H NMR ([D6]DMSO): d=12.74 (s, 1H, NH), 10.04 (s, 1H, NH), 7.50
(d, J=8.4 Hz, 2H), 7.37 (d, J=8.4 Hz, 2H), 7.11–7.00 (m, 3H), 3.96
(s, 2H, S-CH2), 3.75 (s, 2H, CH2), 2.02 ppm (s, CH3, 3H); IR (KBr): n˜ =
3245(uNH), 3048(uNH), 1670(uC O), 1656(uC O), 1262 (uCÀN), 1245 cmÀ1
=
=
(uCÀN); ESI-MS: m/z 452.1 [M+1]+, 474.1 [M+Na]+; C20H16Cl2FN3O2S
(451.03).
2-[(4-bromophenylamino)carbonylmethylthio]-6-(2-chloro-6-fluo-
robenzyl)-5-methylpyrimidin-4(3H)-one (4a3): Recrystallized from
EtOH/DMF as a white crystal, Yield: 28.6%; mp: 234–2368C (dec);
1H NMR ([D6]DMSO): d=12.75 (s, 1H, NH), 10.04 (s, 1H, NH), 7.50–
6.99(m, 7H), 3.95 (s, 2H, S-CH2), 3.75 (s, 2H, CH2), 2.02 ppm (s, CH3,
3H); IR (KBr): n˜ =3246(uNH), 3047(uNH), 1670(uC O), 1657(uC O),
General procedure for the preparation of b-keto esters 2a–e:
The activated Zn dust was prepared by washing Zn dust sequen-
tially with 3n HCl(aq), distilled H2O, EtOH, Et2O, and drying under
vacuum. Activated Zn dust (32.5 g, 500 mmol) was suspended in
dry THF (200 mL) and held at reflux under a N2 atmosphere. A few
drops of 2-bromoalkanoates were added to initiate the reaction.
After the appearance of a green color, arylacetonitriles (100 mmol)
were added in one portion followed by the dropwise addition of
2-bromoalkanoates (260 mmol) over 1 h. The reaction mixture was
held at reflux for an additional 2 h, diluted with THF (600 mL), and
quenched with K2CO3(aq) (50%, 125 mL). Rapid stirring for 60 min
gave two distinct layers. The THF layer was decanted, the residue
washed with THF (150 mL), and the combined THF fractions were
treated with HCl(aq) (10%, 150 mL) at room temperature for 45 min.
The mixture was concentrated under reduced pressure, diluted
with CH2Cl2 (300 mL), and washed with saturated NaHCO3 (3ꢁ
350 mL) and brine (3ꢁ350 mL). The organic phase was dried over
anhydrous Na2SO4, filtered, and concentrated to give the crude
products 2a–e, which were directly used in the following step
without further purification.
=
=
1261 (uCÀN), 1245 cmÀ1 (uCÀN); ESI-MS: m/z 497.9 [M+1]+, 520.0
[M+Na]+; C20H16BrClFN3O2S (496.98).
2-[(4-methoxyphenylamino)carbonylmethylthio]-6-(2-chloro-6-
fluorobenzyl)-5-methylpyrimidin-4(3H)-one (4a4): Recrystallized
from EtOH/DMF as a white crystal, Yield: 26.3%;mp: 240–2428C
1
(dec); H NMR ([D6]DMSO): d=12.73(s, 1H, NH), 10.30 (s, 1H, NH),
7.41–6.88(m, 7H), 3.98 (s, 2H, S-CH2), 3.72 (s, 2H, CH2), 3.71(s, OCH3,
3H), 2.02 ppm (s, CH3, 3H); IR (KBr): n˜ =3255(uNH), 3045(uNH),
1656(uC O), 1247 cmÀ1 (uCÀN); ESI-MS: m/z 448.1 [M+1]+, 470.1
=
[M+Na]+; C21H19ClFN3O3S (447. 08).
2-[(4-nitrophenylamino)carbonylmethylthio]-6-(2-chloro-6-fluo-
robenzyl)-5-methylpyrimidin-4(3H)-one (4a5): Recrystallized from
EtOH/DMF as a white crystal, Yield: 25.2%; mp: 235–2378C (dec);
1H NMR ([D6]DMSO): d=12.71 (s, 1H, NH), 10.54 (s, 1H, NH), 8.23
(d, J=9.0 Hz, 2H), 7.74 (d, J=9.0 Hz, 2H), 7.03–6.94 (m, 3H), 3.90
(s, 2H, S-CH2), 3.83 (s, 2H, CH2), 2.02 ppm (s, CH3, 3H); IR (KBr): n˜ =
General procedure for the preparation of 5-alkyl-6-substituted
thiouracil 3a–e: Na0 (8.2 g, 356 mmol) was dissolved in absolute
EtOH (50 mL), and thiourea (19 g, 249 mmol) and b-keto esters
2a–e (178 mmol) were added to the clear solution at room tem-
perature. The reaction mixture was held at reflux for 6–12 h
(checked by TLC) under a N2 atmosphere. The reaction mixture
was cooled to room temperature. Then, solvent was evaporated
and the residues were dissolved in H2O and were precipitated by
addition of concentrated HCl(aq) and subsequent acidification to
pH 4 with glacial AcOH. The resulting precipitate was filtered
3324(uNH), 3080(uNH), 1644(uC O), 1249 cmÀ1 (uCÀN); ESI-MS: m/z
=
463.4 [M+1]+, 485.5 [M+Na]+; C20H16ClFN4O4S (462.06).
2-[(4-cyanophenylamino)carbonylmethylthio]-6-(2-chloro-6-fluo-
robenzyl)-5-methylpyrimidin-4(3H)-one (4a6): Recrystallized from
EtOH/DMF as a white crystal, Yield: 24.8%; mp: 243–2458C (dec);
1H NMR ([D6]DMSO): d=12.77 (s, 1H, NH), 10.37(s, 1H, NH), 7.79 (d,
J=9.0 Hz, 2H), 7.68 (d, J=9.0 Hz, 2H), 7.04–6.95 (m, 3H), 3.94 (s,
830
www.chemmedchem.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 826 – 833

DABO Analogues as HIV-1 NNRTIs
2H, S-CH2), 3.80 (s, 2H, CH2), 2.02 ppm (s, CH3, 3H); IR (KBr): n˜ =
2-[(4-fluorophenylamino)carbonylmethylthio]-6-(2,6-difluoroben-
zyl)-5-methylpyrimidin-4(3H)-one (4c1): Recrystallized from EtOH/
DMF as a white crystal, Yield: 24.9%; mp: 246–2488C (dec);
1H NMR ([D6]DMSO): d=12.75 (s, 1H, NH), 10.01 (s, 1H, NH), 7.79
(d, J=9 Hz, 2H), 7.68 (d, J=9 Hz, 2H), 7.08–6.79 (m, 3H), 3.85 (s,
2H, S-CH2), 3.83 (s, 2H, CH2), 2.00 ppm (s, CH3, 3H); IR (KBr): n˜ =
3276 (uNH), 3050 (uNH), 1672 (uC O), 1651 (uC O), 1265 cmÀ1 (uCÀN);
3253(uNH), 3049(uNH), 2228 (uCꢂN), 1639(uC O), 1245 cmÀ1 (uCÀN); ESI-
=
MS: m/z 443.6 [M+1]+, 465.3 [M+Na]+; C21H16ClFN4O2S (442.07).
2-[(4-fluorophenylamino)carbonylmethylthio]-6-(2-chloro-6-fluo-
robenzyl)-5-ethylpyrimidin-4(3H)-one (4b1): Recrystallized from
EtOH/DMF as a white crystal, Yield: 25.1%; mp: 212–2148C (dec);
1H NMR ([D6]DMSO): d=12.71 (s, 1H, NH), 9.94 (s, 1H, NH), 7.50 (d,
J=9.0 Hz, 2H), 7.16 (d, J=9.0 Hz, 2H), 7.13–7.01 (m, 3H), 3.99 (s,
2H, S-CH2), 3.72 (s, 2H, CH2), 2.51 (q, J=7.2 Hz, CH2CH3, 2H),
1.04 ppm (t, J=7.2 Hz, CH2CH3, 3H); IR (KBr): n˜ =3252 (uNH),
=
=
ESI-MS: m/z 420.3 [M+1]+; C20H16F3N3O2S (419.09).
2-[(4-chlorophenylamino)carbonylmethylthio]-6-(2,6-difluoro-
benzyl)-5-methylpyrimidin-4(3H)-one (4c2): Recrystallized from
EtOH/DMF as a white crystal, Yield: 27.1%; mp: 248–2508C (dec);
1H NMR ([D6]DMSO): d=12.86 (s, 1H, NH), 10.15 (s, 1H, NH), 7.54
(d, J=9 Hz, 2H), 7.37 (d, J=9 Hz, 2H), 7.15–6.85 (m, 3H), 3.85 (s,
2H, S-CH2), 3.80 (s, 2H, CH2), 2.00 ppm (s, CH3, 3H); IR (KBr): n˜ =
3043(uNH), 1658(uC O), 1245 cmÀ1 (uCÀN); ESI-MS: m/z 450.1 [M+1]+,
=
472.1 [M+Na]+; C21H18ClF2N3O2S (449.08).
2-[(4-chlorophenylamino)carbonylmethylthio]-6-(2-chloro-6-fluo-
robenzyl)-5-ethylpyrimidin-4(3H)-one (4b2): Recrystallized from
EtOH/DMF as a white crystal, Yield: 25.6%; mp: 215–2178C (dec);
1H NMR ([D6]DMSO): d=12.74 (s, 1H, NH), 10.61(s, 1H, NH), 7.53–
7.00 (m, 7H), 4.04 (s, 2H, S-CH2), 3.73 (s, 2H, CH2), 2.52 (q, CH2CH3,
2H), 1.04 ppm (t, CH2CH3, 3H); IR (KBr): n˜ =3289 (uNH), 3056 (uNH),
3262 (uNH), 3044 (uNH), 1658 (uC O), 1267 (uCÀN), 1247 cmÀ1 (uCÀN);
=
ESI-MS: m/z 436.4 [M+1]+; C20H16ClF2N3O2S (435.06).
2-[(4-bromophenylamino)carbonylmethylthio]-6-(2,6-difluoro-
benzyl)-5-methylpyrimidin-4(3H)-one (4c3): Recrystallized from
EtOH/DMF as a white crystal, Yield: 26.6%; mp: 251–2538C (dec);
1H NMR ([D6]DMSO): d=12.72 (s, 1H, NH), 10.10 (s, 1H, NH), 7.50–
6.87 (m, 7H), 3.85 (s, 2H, S-CH2), 3.82 (s, 2H, CH2), 2.00 ppm (s, CH3,
1648(uC O), 1241 cmÀ1 (uCÀN); ESI-MS: m/z 466.1 [M+1]+, 488.1
=
[M+Na]+; C21H18Cl2FN3O2S (465.05).
3H); IR (KBr): n˜ =3261 (uNH), 3041(uNH), 16594(uC O), 1267 (uCÀN),
=
2-[(4-bromophenylamino)carbonylmethylthio]-6-(2-chloro-6-fluo-
robenzyl)-5-ethylpyrimidin-4(3H)-one (4b3): Recrystallized from
EtOH/DMF as a white crystal, Yield: 24.1%; mp: 207–2098C (dec);
1H NMR ([D6]DMSO): d=12.73 (s, 1H, NH), 10.20 (s, 1H, NH), 7.57–
7.02 (m, 7H), 4.04 (s, 2H, S-CH2), 3.79 (s, 2H, CH2), 2.52(q, J=7.8 Hz,
CH2CH3, 2H), 1.04 ppm (t, J=7.8 Hz, CH2CH3, 3H); IR (KBr): n˜ =3253
1247 cmÀ1 (uCÀN); ESI-MS: m/z 480.2 [M+1]+, 502.2 [M+Na]+;
C20H16BrF2N3O2S (479.01).
2-[(4-methoxyphenylamino)carbonylmethylthio]-6-(2,6-difluoro-
benzyl)-5-methylpyrimidin-4(3H)-one (4c4): Recrystallized from
EtOH/DMF as a white crystal, Yield: 23.5%; mp: 233–2358C (dec);
1H NMR ([D6]DMSO): d=12.74 (s, 1H, NH), 9.83 (s, 1H, NH), 7.42–
6.87 (m, 7H), 3.87 (s, 2H, S-CH2), 3.77 (s, 2H, CH2), 3.60 (s, OCH3,
3H), 2.00 ppm (s, CH3, 3H); IR (KBr): n˜ =3257 (uNH), 3050 (uNH),
(uNH), 303n8(uNH), 1655 (uC O), 1245 cmÀ1 (uCÀN); ESI-MS: m/z 512.5
=
[M+1]+, 534.3 [M+Na]+; C21H18BrClFN3O2S (511.00).
2-[(4-methoxyphenylamino)carbonylmethylthio]-6-(2-chloro-6-
fluorobenzyl)-5-ethylpyrimidin-4(3H)-one (4b4): Recrystallized
from EtOH/DMF as a white crystal, Yield: 28.4%; mp: 241–2438C
(dec); 1H NMR ([D6]DMSO): d=12.72 (s, 1H, NH), 9.77(s, 1H, NH),
7.41–6.88 (m, 7H), 4.01 (s, 2H, S-CH2), 3.72 (s, 2H, CH2), 3.69 (s, 3H,
OCH3), 2.51(q, J=7.2 Hz, CH2CH3, 2H), 1.04 ppm (t, J=7.2 Hz,
1656 (uC O), 1248 cmÀ1 (uCÀN); ESI-MS: m/z 432.4 [M+1]+, 454.3
=
[M+Na]+; C21H19F2N3O3S (431.11).
2-[(4-nitrophenylamino)carbonylmethylthio]-6-(2,6-difluoroben-
zyl)-5-methylpyrimidin-4(3H)-one (4c5): Recrystallized from EtOH/
DMF as a white crystal, Yield: 25.6%; mp: 258–2608C (dec);
1H NMR ([D6]DMSO): d=12.77 (s, 1H, NH), 10.58 (s, 1H, NH), 8.24
(d, J=9 Hz, 2H), 7.74 (d, J=9 Hz, 2H), 7.05–6.79 (m, 3H), 3.88 (s,
2H, S-CH2), 3.83 (s, 2H, CH2), 2.00 ppm (s, CH3, 3H); IR (KBr): n˜ =
CH2CH3, 3H); IR (KBr): n˜ =3256 (uNH), 3043(uNH), 1652(uC O),
=
1242 cmÀ1 (uCÀN); ESI-MS: m/z 462.4 [M+1]+, 484.4 [M+Na]+;
C22H21ClFN3O3S (461.1).
3271 (uNH), 3055 (uNH), 1651 (uC O), 1285 (uCÀN), 1251 cmÀ1 (uCÀN);
=
ESI-MS: m/z 447.3 [M+1]+; C20H16F2N4O4S (446.09).
2-[(4-nitrophenylamino)carbonylmethylthio]-6-(2-chloro-6-fluo-
robenzyl)-5-ethylpyrimidin-4(3H)-one (4b5): Recrystallized from
EtOH/DMF as a white crystal, Yield: 26.4%; mp: 224–2268C (dec);
1H NMR ([D6]DMSO): d=12.75(s, 1H, NH), 10.50 (s, 1H, NH), 8.24 (d,
J=9.0 Hz, 2H), 7.74(d, J=9.0 Hz, 2H), 7.03–6.93 (m, 3H), 3.97 (s,
2H, S-CH2), 3.81 (s, 2H, CH2), 2.53 (q, CH2CH3, 2H), 1.04 ppm (t, J=
2-[(4-cyanophenylamino)carbonylmethylthio]-6-(2,6-difluoroben-
zyl)-5-methylpyrimidin-4(3H)-one (4c6): Recrystallized from EtOH/
DMF as a white crystal, Yield: 27.9%; mp: 252–2548C (dec);
1H NMR ([D6]DMSO): d=12.87 (s, 1H, NH), 10.44 (s, 1H, NH), 7.52–
6.89 (m, 7H), 3.85 (s, 2H, S-CH2), 3.80 (s, 2H, CH2), 2.00 ppm (s, CH3,
3H); IR (KBr): n˜ =3288 (uNH), 3041(uNH), 1675 (uC O), 1654 (uC O),
7.2 Hz, CH2CH3, 3H); IR (KBr): n˜ =3301 (uNH), 3073 (uNH), 1645 (uC O),
=
1249 cmÀ1 (uCÀN); ESI-MS: m/z 477.1 [M+1]+, 499.1 [M+Na]+;
=
=
2224 (uCꢂN), 1261 (uCÀN), 1251 cmÀ1 (uCÀN); ESI-MS: m/z 427.3
C21H18ClFN4O4S (476.07).
[M+1]+; C21H16F2N4O2S (426.1).
2-[(4-cyanophenylamino)carbonylmethylthio]-6-(2-chloro-6-fluo-
robenzyl)-5-ethylpyrimidin-4(3H)-one (4b6): Recrystallized from
EtOH/DMF as a white crystal, Yield: 26.7%; mp: 195–1978C (dec);
1H NMR ([D6]DMSO): d=12.77 (s, 1H, NH), 10.35(s, 1H, NH), 7.79–
6.94 (m, 7H), 4.04 (s, 2H, S-CH2), 3.85 (s, 2H, CH2), 2.57 (q, J=
7.2 Hz, CH2CH3, 2H), 1.07 ppm (t, J=7.2 Hz, CH2CH3, 3H); 13C NMR
(150 MHz, [D6]DMSO): d=12.9 (CH3), 18.2 (CH2), 34.9 (CH2-Ph), 37.5
(SCH2), 109.8 (1C, Ph),114.1 (1C, Ph), 116.7 (C-5),119.1(CꢂN), 120.4
(2C, Ph),123.6 (1C, Ph), 126.1(1C, Ph), 128.6 (1C, Ph),131.9 (2C, Ph),
136.6 (1C, Ph), 143.5 (1C, Ph),151.6 (C-6), 160.4 (C-2), 164.6 (C-4),
165.5 (1C, Ph), 169.3 ppm (C=O); IR (KBr): n˜ =3347 (uNH), 3045 (uNH),
2-[(4-fluorophenylamino)carbonylmethylthio]-6-(2,6-difluoroben-
zyl)-5-ethylpyrimidin-4(3H)-one (4d1): Recrystallized from EtOH/
DMF as a white crystal, Yield: 23.7%; mp: 225–2278C (dec);
1H NMR ([D6]DMSO): d=12.67(s, 1H, NH), 10.02(s, 1H, NH), 7.52–
6.87 (m, 7H), 3.88 (s, 2H, S-CH2), 3.79 (s, 2H, CH2), 2.52 (q, J=
7.2 Hz, CH2CH3, 2H), 1.01 ppm (t, J=7.2 Hz, CH2CH3, 3H); IR (KBr):
n˜ =3288 (uNH), 3053 (uNH), 1672 (uC O), 1648 (uC O), 1251 (uCÀN),
=
=
1214 cmÀ1 (uCÀN); ESI-MS: m/z 434.4 [M+1]+; C21H18F3N3O2S
(433.11).
2-[(4-chlorophenylamino)carbonylmethylthio]-6-(2,6-difluoro-
benzyl)-5-ethylpyrimidin-4(3H)-one (4d2): Recrystallized from
EtOH/DMF as a white crystal, Yield: 26.6%; mp: 222–2248C (dec);
2225 (uCꢂN), 1640(uC O), 1249 cmÀ1 (uCÀN); ESI-MS: m/z 457.5
=
[M+1]+, 479.4 [M+Na]+; C22H18ClFN4O2S (456.08).
ChemMedChem 2011, 6, 826 – 833
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
831

X. Liu et al.
MED
1H NMR ([D6]DMSO): d=12.73 (s, 1H, NH), 10.10 (s, 1H, NH), 7.54
(d, J=9 Hz, 2H), 7.37 (d, J=9 Hz, 2H), 7.10–6.85 (m, 3H), 3.86 (s,
2H, S-CH2), 3.80 (s, 2H, CH2), 2.51 (q, J=7.2 Hz, CH2CH3, 2H),
1.01 ppm (t, J=7.2 Hz, CH2CH3, 3H); 13C NMR (150 MHz,
[D6]DMSO): d=13.1 (CH3), 18.4 (CH2), 34.1 (CH2-Ph), 37.7 (SCH2),
112.1 (2C, Ph), 112.6 (1C, Ph), 116.3 (C-5), 120.7 (2C, Ph), 127.1(1C,
Ph),128.9 (2C, Ph), 132.8 (1C, Ph), 137.6 (1C, Ph), 152.1 (C-6), 160.2
(C-2), 164.1 (C-4), 165.1 (2C, Ph), 169.9 ppm (C=O); IR (KBr): n˜ =
3288 (uNH), 3054 (uNH), 1673 (uC O), 1650 (uC O), 1269 (uCÀN),
was performed using the MTT assay as previously described.[16,17]
Stock solutions (10ꢁ final concentration) of test compounds were
added in 25 mL volumes to two series of triplicate wells so as to
allow simultaneous evaluation of their effects on mock-and HIV-in-
fected cells at the beginning of each experiment. Serial fivefold di-
lutions of test compounds were made directly in flat-bottomed 96-
well microtiter trays using a Biomek 3000 robot (Beckman instru-
ments, Fullerton, CA, USA). Untreated control HIV-and mock-infect-
ed cell samples were included for each sample.
=
=
1244 cmÀ1 (uCÀN); ESI-MS: m/z 450.3 [M+1]+; C21H18ClF2N3O2S
HIV-1(IIIB),[20] HIV-2 (ROD),[21] or HIV-1 mutant strains stock (50 mL) at
100–300 CCID50 (cell culture infectious dose) or culture medium
was added to either the infected or mock-infected wells of the mi-
crotiter tray. Mock-infected cells were used to evaluate the effect
of the test compound on uninfected cells to assess the cytotoxicity
of the test compound itself. Exponentially growing MT-4 cells[22]
were centrifuged for 5 min at 1000 rpm (220 g), and the superna-
tant was discarded. The MT-4 cells were resuspended at 6ꢁ
105 cellsmLÀ1, and 50 mL volumes were transferred to the microtiter
tray wells. Five days after infection, the viability of mock- and HIV-
infected cells was examined spectrophotometrically by the MTT
assay.
(449.08).
2-[(4-bromophenylamino)carbonylmethylthio]-6-(2,6-difluoro-
benzyl)-5-ethylpyrimidin-4(3H)-one (4d3): Recrystallized from
EtOH/DMF as a white crystal, Yield: 23.1%; mp: 223–2258C (dec);
1H NMR ([D6]DMSO): d=12.70 (s, 1H, NH), 10.09 (s, 1H, NH), 7.50
(d, J=9 Hz, 2H), 7.47 (d, J=9 Hz, 2H), 7.10–6.85 (m, 3H), 3.86 (s,
2H, S-CH2), 3.80 (s, 2H, CH2), 2.53 (q, J=7.2 Hz, CH2CH3, 2H),
1.01 ppm (t, J=7.2 Hz, CH2CH3, 3H); IR (KBr): n˜ =3284 (uNH), 3056
(uNH), 1657 (uC O), 1266 (uCÀN), 1243 cmÀ1 (uCÀN); ESI-MS: m/z 494.2
=
[M+1]+; C21H18BrF2N3O2S (493.03).
2-[(4-methoxyphenylamino)carbonylmethylthio]-6-(2,6-difluoro-
benzyl)-5-ethylpyrimidin-4(3H)-one (4d4): Recrystallized from
EtOH/DMF as a white crystal, Yield: 27.1%; mp: 232–2348C (dec);
1H NMR ([D6]DMSO): d=12.66(s, 1H, NH), 9.85 (s, 1H, NH), 7.42–
6.87 (m, 7H), 3.89 (s, 2H, S-CH2), 3.76 (s, 2H, CH2), 3.72(s, OCH3,
3H), 2.51 (q, J=7.2 Hz, CH2CH3, 2H), 1.02 ppm (t, J=7.2 Hz,
The MTT assay is based on the reduction of yellow colored 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Acros
Organics, Geel, Belgium) by mitochondrial dehydrogenase of meta-
bolically active cells to a blue–purple formazan that can be mea-
sured spectrophotometrically. The absorbances were read in an
eight-channel computer-controlled photometer (Multiscan Ascent
Reader, Labsystems, Helsinki, Finland), at two wavelengths (540
and 690 nm). All data were calculated using the median OD (opti-
cal density) value of three wells. The 50% cytotoxic concentration
(CC50) was defined as the concentration of the test compound that
reduced the absorbance (OD540) of the mock-infected control
sample by 50%. The concentration achieving 50% protection from
the cytopathic effect of the virus-infected cells was defined as the
50% effective concentration (EC50).
CH2CH3, 3H); IR (KBr): n˜ =3250 (uNH), 3050 (uNH), 1656 (uC O),
=
1241 cmÀ1 (uCÀN); ESI-MS: m/z 446.3 [M+1]+, 468.3 [M+Na]+;
C22H21F2N3O3S (445.13).
2-[(4-nitrophenylamino)carbonylmethylthio]-6-(2,6-difluoroben-
zyl)-5-ethylpyrimidin-4(3H)-one (4d5): Recrystallized from EtOH/
DMF as a white crystal, Yield: 23.3%; mp: 253–2558C (dec);
1H NMR ([D6]DMSO): d=12.76 (s, 1H, NH), 10.57 (s, 1H, NH), 8.24–
6.80 (m, 7H), 3.87 (s, 2H, S-CH2), 3.85 (s, 2H, CH2), 2.52 (q, J=
7.2 Hz, CH2CH3, 2H), 1.02 ppm (t, J=7.2 Hz, CH2CH3, 3H); IR (KBr):
n˜ =3271 (uNH), 3056 (uNH), 1651 (uC O), 1265 (uCÀN), 1251 (uCÀN); ESI-
=
MS: m/z 461.4 [M+1]+, 483.3 [M+Na]+; C21H18F2N4O4S (460.10).
2-[(4-cyanophenylamino)carbonylmethylthio]-6-(2,6-difluoroben-
zyl)-5-ethylpyrimidin-4(3H)-one (4d6): Recrystallized from EtOH/
DMF as a white crystal, Yield: 25.3%; mp: 226–2288C (dec);
1H NMR ([D6]DMSO): d=12.93 (s, 1H, NH), 10.40 (s, 1H, NH), 7.78
(d, J=9 Hz, 2H), 7.68 (d, J=9 Hz, 2H), 7.03–6.81 (m, 3H), 3.88 (s,
2H, S-CH2), 3.84 (s, 2H, CH2), 2.51 (q, J=7.2 Hz, CH2CH3, 2H),
1.00 ppm (t, J=7.2 Hz, CH2CH3, 3H); IR (KBr): n˜ =3288 (uNH), 3043
RT inhibition assay
Compounds were tested for antiviral activity against HIV-1 RTwt,
using a poly(rA)/oligo(dT)15 homopolymer template with HIV anti-
gen detection ELISA for quantifying expression of HIV-1 RT in cul-
ture medium. Oligo(dT) was immobilized via its 50-terminal phos-
phate to Covalink-NH microtiter plates. The biotin-dUTP was incor-
porated by reverse transcriptase. The reaction mixture contained
50 mm Tris-HCl (pH 8.3), 3 mm MgCl2, 75 mm KCl, 5 mm DTT (d,l-
dithiothreitol), 0.13 mgmLÀ1 BSA, 10 mgmLÀ1 poly (A), 0.75 mm
biotin-11-dUTP, and 1.5 mm dTTP. After incubation at 378C for 1 h,
the plate was washed three times with a wash buffer containing
50 mm Tris-HCl (pH7.5), 0.15m NaCl, 0.05 mm MgCl2, and 0.02%
Tween-20. After 100 mL of 1% BSA was added to each well and in-
cubated for 30 min at room temperature, the plate was washed
with the same buffer. Subsequently 50 mL of SA-ALP (alkaline phos-
phatase streptavidin) solution (100 ngmLÀ1) was added per well
and then incubated for 1 h at 378C. The plate was washed as
above and then 50 mL of PNPP (p-nitrophenyl phosphate, disodi-
um) (1 mgmLÀ1, pH 9.5) was added, after 30 min at 378C the reac-
tion was stopped by addition of 0.5m NaOH. The products were
detected and quantified using a colorimetric streptavidin alkaline
phosphatase reporter system.
(uNH), 2227 (uCꢂN), 1646 (uC O), 1269 (uCÀN), 1249 cmÀ1 (uCÀN); ESI-
=
MS: m/z 441.4 [M+1]+, 463.4 [M+Na]+; C22H18F2N4O2S (440.11).
2-[(4-fluorophenylamino)carbonylmethylthio]-6-(1-naphthyl-
methyl)-5-ethylpyrimidin-4(3H)-one (4e1): Recrystallized from
EtOH/DMF as a white crystal, Yield: 23.6%; mp: 200–2028C (dec);
1H NMR ([D6]DMSO): d=12.77 (s, 1H, NH), 10.10 (s, 1H, NH), 8.15
(d, J=8.4 Hz, 1H), 7.89 (d, J=7.8 Hz, 1H), 7.73 (d, J=7.8 Hz, 1H),
7.54–7.03 (m, 8H), 4.31 (s, 2H, S-CH2), 3.93 (s, 2H, CH2), 2.52 (q, J=
7.2 Hz, CH2CH3, 2H), 0.85 ppm (t, J=7.2 Hz, CH2CH3, 3H); IR (KBr):
n˜ =3262 (uNH), 3044 (uNH), 1668 (uC O), 1649 (uC O), 1252(uCÀN),
=
=
1217 cmÀ1 (uCÀN); ESI-MS: m/z 448.2 [M+1]+, 470.2 [M+Na]+;
C25H22FN3O2S (447.14)
Anti-HIV activity in MT-4 cells
Evaluation of the antiviral activity of the compounds against HIV-1
strain IIIB, HIV-2 strain (ROD), and HIV-1 mutant strains in MT-4 cells
832
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 826 – 833

DABO Analogues as HIV-1 NNRTIs
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Foundation
of
Shandong
University
(GIIFSDU
No.
21310070613090), the Joint Research Fund for Overseas Chinese
Young Scholars of the Natural Science Foundation of China
(NSFC No. 30928032), the National Natural Science Foundation
of China (NSFC Nos. 30873133, 30772629, and 30371686), the
Key Project of NSFC for International Cooperation (No.
30910103908), and the Research Fund for the Doctoral Program
of Higher Education of China (No. 20070422083) are gratefully
acknowledged.
Keywords: antiviral agents
chemistry · NNRTIs
· DABOs · HIV-1 · medicinal
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Published online on February 14, 2011
ChemMedChem 2011, 6, 826 – 833
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
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