Design, synthesis, and biological evaluation of 1-[(2-benzyloxyl/alkoxyl) methyl]-5-halo-6-aryluracils as potent HIV-1 non-nucleoside reverse transcriptase inhibitors with an improved drug resistance profile

Article abstract of DOI:10.1021/jm201506e

Because the emergence of drug-resistant mutants has limited the efficacy of non-nucleoside reverse transcriptase inhibitors (NNRTIs), it is essential to develop new antivirals with better drug resistance and pharmacokinetic profiles. Here we designed and synthesized a series of 1-[(2-benzyloxyl/alkoxyl)methyl]- 5-halo-6-aryluracils, the HEPT analogues, and evaluated their biological activity using nevirapine and 18 (TNK-651) as reference compounds. Most of these compounds, especially 6b, 7b, 9b, 11b, and 7c, exhibited highly potent anti-HIV-1 activity against both wild-type and NNRTI-resistant HIV-1 strains. Compound 7b, which had the highest selectivity index (SI = 38 215), is more potent than nevirapine and 18. These results suggest that the introduction of a halogen at the C-5 position may contribute to the effectiveness of these compounds against RTI-resistant variants. In addition, meta substituents on the C-6 aromatic moiety could significantly enhance activity against NNRTI-resistant HIV-1 strains. These compounds can be further developed as next-generation NNRTIs with an improved antiviral efficacy and drug-resistance profile.

Full text of DOI:10.1021/jm201506e

Article
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of 1-[(2-Benzyloxyl/
alkoxyl)methyl]-5-halo-6-aryluracils as Potent HIV-1 Non-nucleoside
Reverse Transcriptase Inhibitors with an Improved Drug Resistance
Profile
Xiaowei Wang,†,# Jianfang Zhang,⊥,# Yang Huang,§,# Ruiping Wang,⊥ Liang Zhang,† Kang Qiao,† Li Li,†
Chang Liu,⊥ Yabo Ouyang,§ Weisi Xu,§ Zhili Zhang,† Liangren Zhang,‡ Yiming Shao,§ Shibo Jiang,¶
Liying Ma,*,§ and Junyi Liu*,†,‡
‡
†Department of Chemical Biology, School of Pharmaceutical Sciences; State Key Laboratory of Natural and Biomimetic Drugs,
Peking University, Beijing 100191;
§State Key Laboratory for Infection Disease Prevention and Control, National Center for AIDS/STD Control and Prevention
(NCAIDS), Chinese Center for Disease Control and Prevention, Beijing 102206
⊥College of Pharmaceutical Sciences, Capital Medical University, Beijing 100069
¶MOE/MOH Key Laboratory of Medical Molecular Virology, Shanghai Medical College and Institute of Medical Microbiology,
Fudan University, Shanghai 200032, China
S
* Supporting Information
ABSTRACT: Because the emergence of drug-resistant mutants has limited the
efficacy of non-nucleoside reverse transcriptase inhibitors (NNRTIs), it is essential to
develop new antivirals with better drug resistance and pharmacokinetic profiles. Here
we designed and synthesized a series of 1-[(2-benzyloxyl/alkoxyl)methyl]-5-halo-6-
aryluracils, the HEPT analogues, and evaluated their biological activity using
nevirapine and 18 (TNK-651) as reference compounds. Most of these compounds,
especially 6b, 7b, 9b, 11b, and 7c, exhibited highly potent anti-HIV-1 activity against
both wild-type and NNRTI-resistant HIV-1 strains. Compound 7b, which had the
highest selectivity index (SI = 38 215), is more potent than nevirapine and 18. These
results suggest that the introduction of a halogen at the C-5 position may contribute to
the effectiveness of these compounds against RTI-resistant variants. In addition, meta
substituents on the C-6 aromatic moiety could significantly enhance activity against
NNRTI-resistant HIV-1 strains. These compounds can be further developed as next-generation NNRTIs with an improved
antiviral efficacy and drug-resistance profile.
Like other types of anti-HIV drugs, the therapeutic efficacy of
NNRTIs has been limited by the emergence of mutants that
show cross-resistance to other structurally unrelated drugs.13−17
Therefore, it is essential to develop new NNRTIs with
improved pharmacokinetic and drug-resistance profiles.
Among more than 50 different series of NNRTIs that have
been reported so far, 1-[(2-hydroxyethoxy) methyl]-6-
(phenylthio)thymine (HEPT) was the first compound shown
to target specifically HIV-1 RT.18 Here we designed and
synthesized 22 HEPT (16) derivatives and detected their
inhibitory activity on HIV-1 RT activity. Then, we selected 11
active compounds for testing of antiviral activities against HIV-
INTRODUCTION
■
Reverse transcriptase (RT) of human immunodeficiency virus
type 1 (HIV-1) is an attractive target for development of drugs
used in the treatment of HIV-1 infection and AIDS. It is a
multifunctional enzyme critical in viral replication. Currently,
two functionally distinct classes of HIV-1 RT inhibitors
(nucleoside and non-nucleoside) have been discovered and
are being used clinically. Especially, non-nucleoside RT
inhibitors (NNRTIs), with a wide range of chemically diverse
structures, have gained a definitive and important place in
clinical use based on their unique antiviral potency with
generally low toxicity and favorable pharmacokinetic proper-
ties.1−3 To date, four NNRTIs have been approved for clinical
use: nevirapine, delavirdine, efavirenz, and etravirine.4−9
1
SF33, a laboratory-adapted HIV-1 X4/R5 dual tropic strain, and
HIV-1A17, an NNRTI-resistant variant. On the basis of the
results, we will delineate their structural features and gain
insight into how the major chemical substitutions on the
Crystal structures of several RT/NNRTI complexes clearly
show that NNRTIs bind to a hydrophobic pocket (the non-
nucleoside inhibitor binding pocket, NNIBP) in the p66 palm
subdomain near the polymerase active site. Upon binding,
NNRTIs cause a distortion of the catalytic aspartate site.10−12
Received: November 7, 2011
Published: January 27, 2012
© 2012 American Chemical Society
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pyrimidine backbone affect the activity of these inhibitors
against NNRTI-resistant HIV-1 strains.
ring may result in closer interactions with the non-nucleoside
binding pocket (NNBP) than can be achieved with 18.
To investigate the role of hydrogen bonds, we designed and
synthesized a series of compounds by exchanging C-4-OH for
-NH2, expecting that two hydrogen bonds could be formed
between the N-3/C-4 positions with the carbonyl group of
Lys101. Computational studies on the possible binding mode
of these 16 derivatives in the NNBP of HIV-1 RT suggest that
the introduction of a further anchor, which acts as both donor
and acceptor of hydrogen bonds, would potentially lead to
more activity against HIV-1 viruses.25
MOLECULAR DESIGN
■
Structure−activity relationship studies have developed promis-
ing 16 analogues such as 17 (MKC-442) and 18 (TNK-
651)19−21 (Figure 1), and the substituent on C-5 and C-6 of
To verify the structure−activity relationship of our design,
target compounds 1-[(2-benzyloxyl)methyl]-5-iodo-6-arylura-
cils (7a, 7b, and 7c) were flexibly docked into the binding site
of HIV-1 RT (PDB entry 1RT2, complexes with 18), using the
GOLD3.0.1 docking program. It was revealed that the binding
mode of 7a, 7b, and 7c has approximate conformations
comparable to that of the RT/18 complex (Figure 2).
On the basis of the above results, we then synthesized a
series of novel uracil derivatives (Figure 1), which are
characterized by methyl and fluorine at the meta positions of
the phenyl ring, and all possess a halo or dimethylamino moiety
at position 5 of the pyrimidine. Title compounds were tested
for their activities against HIV-1 in both cell-based assay and
reverse transcriptase (RT) assay in comparison with 18 and
nevirapine, which were used as reference drugs. The results are
described below.
Figure 1. Structure of 16 analogues and target compounds.
the pyrimidine ring has been revealed as very important for
antiviral activity.22,23 Interestingly, 17 and 18, with their bulky
isopropyl groups at the C-5 position, have been shown to
counteract the Tyr181 residue,24 thus forcing it into a position
where strong interaction with the 6-benzyl group of the
inhibitor can take place. In contrast, the C-5 methyl group in 16
does not produce any “trigger effect” but only brings a slight
perturbation of the Tyr181 side-chain conformation.
Although considerable research has been performed to
understand the structure−activity relationship of 16 analogues,
little information can be found to explain the effect of varying
both electronic effect and steric hindrance of the 16 series at
the C-5 position with a halogen. Compared to 18, for example,
the C-5 halogen atom has a large electron cloud density, which
could form an interaction with the binding site of RT and play
an important role in improving anti-HIV-1 activity. Moreover,
the large atomic radius of the C-5 halogen may also provide a
steric hindrance role, when compared with the C-5 isopropyl of
18, and could trigger the conformation of a C-6 aromatic ring.
In addition, modification to the C-6 by introduction of a m-
dimethyl- or m-difluorophenyl moiety onto the phenylmethyl
CHEMISTRY
■
A conventional strategy of synthesizing the newly designed
compounds is described in Scheme 1. We prepared the 3-oxo
esters 1a−c according to the method of Hannick and Kishi26 by
reaction of 2-bromoacetate with phenylacetonitrile or 3,5-
dimethylphenylacetonitrile or 3,5-difluorophenyl acetonitrile,
respectively. The β-ketoesters (1) were than converted by
reaction with thiourea and sodium in ethanol into 2-thiouracils
(2),27 which were refluxed with 10% chloroacetic acid to yield
6-(arylmethyl)uracils (3).28 Halogenations at the C-5 position
of 3 by reaction of PbO2 with halogen in glacial acetic acid at
room temperature led to 4a−5b in high yield. Compounds 4a−
5b were silylated in situ with N,O-bis(trimethylsilyl)acetamide
(BSA) and then N-1 alkylated with the appropriate alkyl
chloromethyl ether, in the presence of LiI, to give 6a−9b29,30 in
good yield, followed by reaction with aqueous dimethylamino
solution to get 10a−11b.31,32 Finally, the uracil moiety was
successfully converted to the 4-(1,2,4-triazolyl)pyrimidinone
derivatives. Without purification, subsequent ammonia treat-
ment yielded the target compounds 12a−15b.33−35 Structure
Figure 2. Superimposed stereoview of the docked 7a, 7b, and 7c, (blue) with 18 (green).
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a
Scheme 1
a
Reagents and conditions: (a) aryl acetonitrile, Zn/THF, reflux; (b) thiourea, EtONa, EtOH, reflux; (c) 10% ClCH2COOH, reflux; (d) halogen,
PbO2, glacial acetic acid; (e) alkyl halide, BSA, LiI, rt; (f) aq NH(CH3)2 solution, 60−80 °C; (g) POCl3, 1,2,4-1H-triazole, TEA, NH3·H2O, dioxane.
Figure 3. Docking results of 7a into the 1RT2 NNBP, and 7b into the NNBP of 1RT2 (a) and 1JLA Tyr181Cys (b). Hydrogen bonds are shown as
a green dashed line, and halogen bonds are shown as a green solid line.
assignments of these compounds were identified by NMR and
mass spectral data.
pyrimidine ring, has anisotropic surface charge distributions
with significant electropositive patches which could serve as a
Lewis acid. Thus, the introduction of electron-withdrawing
substituents on the pyrimidine ring might increase the
electrostatic interaction of the compound with the carbonyl
oxygen of Tyr188 residue. In addition, the iodine atom of 7a is
only 3.35 Å away from the carbonyl oxygen of Tyr188, which is
less than the van der Waals contact distance of 3.55 Å, and the
angle C−X···O (X is a halogen) of 7a is 157.93°, which is close
to the predicted ideal angle of 165°. (iii) Favorable π-stacking
interactions of the 7a phenyl ring with a hydrophobic pocket, as
defined by the side chains of Tyr188, Trp229, Tyr181, Leu100,
and Phe227, especially the Tyr188 and Tyr181 residues, were
observed.
Interestingly, the antiviral results show that the chain at the
C-6 position with different substituents on the aromatic moiety,
which is located in a hydrophobic region delimited by Tyr181,
Tyr188, Phe227, and Trp229, significantly enhances the activity
against HIV-1 and demonstrates improved resistance profiles.
We have investigated 7b, 9b, and 11b by introduction of m-
dimethyl substitution onto the phenyl ring intended to
optimize interactions with Trp229, which is less mutable in
immunodeficiency virus reverse transcriptase, by reducing the
RESULTS AND DISCUSSION
■
Docking studies of the title compounds into the HIV-1 NNBP
revealed that they could bind to the NNBP and displayed a
slightly different binding mode compared to HIV-1 RT/18
complex. In particular, the C-5-isopropyl group of 18, which is
responsible for the “trigger action”, does not coincide with the
halogen on the C-5 position of compounds 6a−9b. Moreover,
the disubstitution on the phenyl rings of 7b and 7c is at
different positions comparing to that of 18 (Figure 2).
Within the NNBP, target compounds establish several
ligand−receptor interactions. Inspection of the 7a/RT complex
(Figure 3) may be characterized as follows: (i) A hydrogen
bond donated by the 3-NH function of the pyrimidine to the
carbonyl oxygen of Lys101 is a crucial factor for anti-HIV-1
activity. (ii) Unusual interactions between the iodine atom and
an electronegative atom, such as the Tyr188 carbonyl oxygen,
were observed in the 7a /RT structure. Here, demonstrating
electrostatic dynamics, the iodine atom serves as an electron
acceptor for the Tyr188 carbonyl oxygen (Figure 3). The
results suggested that halogen, especially iodine on the
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dependence on binding to Tyr181. Molecular modeling
revealed that methyl groups attached to the 3′ and 5′ positions
of the 6-benzyl group would be buried in a deep hydrophobic
region near Trp229 and Phe227 (Figure 3).
structure-based modifications at this location might enhance
the activity.
Replacement of the dimethyl substitution in 7b by difluoro
substitution (7c), at the C-6-substituted aromatic ring, could
also result in high potency against wild HIV-1 and Tyr181Cys
mutant virus. This finding could be explained by a putative π-
stacking interaction which would be improved between the
electron-deficient benzene ring of the ligand and the electron-
rich benzene ring of Tyr188, through introduction of the 3,5-
difluorophenylmethyl moiety onto the C-6 position of the
aromatic ring. Moreover, the contact distance between the
iodine atom of 7c and the carbonyl oxygen of Tyr188 is 3.27 Å,
within the van der Waals distance, and the halogen bonding
angle ((C−X···O), 155.44°) is similar to the ideal angle 165°.
Upon replacement of the C-4-OH by -NH2, the binding
mode changed such that the distance between the iodine with
the carbonyl oxygen of Try188 (4.85 Å) became longer than
the van der Waals distance (3.55 Å). Furthermore, the π-
stacking interaction, which is usually present in our target
compounds, was not observed in compound 13a.
The significant difference in behavior of 7b against the drug-
resistant mutants is fully explained by the presence of the
additional m-methyl groups, sitting comfortly in a deep
hydrophobic region of the NNRTI binding site and, hence,
increasing the van der Waals contacts and σ−π interactions to
the conserved Trp229 and Phe227, as well as the other residues
in this region. As a result, the compound derives a fraction of its
binding energy from the interaction with the Tyr181 side chain.
Furthermore, the phenyl moiety at the terminus of the N-1
side chain extends into a tunnel that leads out of the NNIBP
toward the catalytic site composed of Tyr318 and Pro236
(Figure 4). Particularly, Tyr318 could provide the π−π
The title compounds were tested for their inhibitory activity
against HIV-1 RT using a poly(ra)/oligo(dT)15 homopolymer
template and nevirapine as a reference compound.36 As shown
in Table 1, nine compounds showed promising anti-RT
activities. Particularly, compound 7b is 1373- and 6.7-fold
more potent than nevirapine and 18, respectively. However, the
anti-RT activities of compounds 12a−15b significantly
decreased.
Subsequently, the anti-HIV-1 activity and cytotoxicity of 6a−
11b were determined, using nevirapine and 18 as reference
compounds. As shown in Table 2, all these compounds were
capable of inhibiting infection by HIV-1SF33 in MT4 cells with
EC50 values ranging from 2 nM to 2.071 μM. Among these
compounds, 7b, 11b, 9b, 6b, and 7c were about 325- to 46-fold
more potent than nevirapine. They had a higher antiviral
activity and selectivity index (SI) than that of 18. In addition,
the above-mentioned compounds displayed much higher
potency against HIV-1A17, the NNRTI-resistant mutant strain
Figure 4. Docking results of 7b into the 1RT2 NNBP.
interaction with the phenyl ring of the N-1 group. We presume
that the steric hindrance aroused by the dimethyl substitution
in 7b would change the conformation of the ligand binding
model in NNIBP with the rotation of the terminal phenyl
moiety at the N-1 side chain and result in significantly
improving the inhibition of HIV-1 RT, suggesting that
Table 1. Inhibitory Activity of the Target Compounds against HIV-1 RT (6a−15b)
a
a
compd
-C-4
R
R1
R2
IC50
compd
C-4
R
R1
R2
IC50
6b
O
3,5-dimethyl
3,5-dimethyl
3,5-dimethyl
3,5-dimethyl
3,5-dimethyl
3,5-dimethyl
3,5-dimethyl
3,5-dimethyl
3,5-dimethyl
3,5-dimethyl
Me
Ph
I
0.110
0.003
0.210
0.043
0.039
0.021
>100
7.217
>100
16.338
4.120
0.150
0.020
6a
O
H
H
H
H
H
H
H
H
H
H
F
Me
Ph
I
0.114
0.010
1.825
1.521
0.162
0.012
>100
>100
>100
>100
11.85
0.015
7b
O
I
7a
O
I
8b
O
Me
Ph
Br
Br
8a
O
Me
Ph
Br
Br
9b
O
9a
O
10b
11b
12b
13b
14b
15b
O
Me
Ph
N(CH3)2
10a
11a
12a
13a
14a
15a
6c
O
Me
Ph
N(CH3)2
O
N(CH3)2
O
N(CH3)2
NH2
NH2
NH2
NH2
Me
Ph
I
NH2
NH2
NH2
NH2
O
Me
Ph
I
I
I
Me
Ph
Br
Br
Me
Ph
Br
Br
I
b
NVP
Me
Ph
17
18
7c
O
F
I
a
b
Effective dose (μM) of the compounds required to inhibit HIV-1 RT activity by 50%. Nevirapine (NVP), 17, and 18 were used as the reference
compounds.
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a
Table 2. Inhibitory Activity of the Compounds on HIV-1SF33 and HIV-1A17 Infection in MT4 Cells
b
inhibition: EC50 (μM)
SI
compd
MW
CC50 (μM)
HIV-1SF33
HIV-1A17
>10
HIV-1SF33
HIV-1A17
7a
11a
10a
9a
448
365
303
401
339
476
393
429
414
422
484
111
302
74.84 1.67
91.82 4.01
166.34 8.75
67.36 8.01
155.27 6.05
98.31 2.66
55.81 2.19
152.18 9.60
141.52 1.86
300.00 9.21
121.25 4.27
325.13 0.02
84.60 3.25
0.045 0.014
0.345 0.134
0.614 0.423
1.720 0.962
2.071 1.321
0.002 0.001
0.014 0.009
0.009 0.006
0.006 0.005
0.537 0.243
0.006 0.002
0.068 0.230
0.022 0.015
1663
264
<7
<9
>10
>10
270
<16.6
<6.7
<15.5
1080
25
>10
39
8a
>10
75
7b
0.091 0.039
2.198 0.445
0.601 0.270
0.637 0.145
>10
38215
5201
16889
23667
559
11b
9b
253
222
<30
48
6b
6c
7c
2.543 0.404
>10
20208
4781
3845
b
NVP
<35
<6.6
18
>10
a
b
Each compound was tested in triplicate; the data were presented as the mean SD. SI was calculated based on the CC50 for MT4 cells and EC50
for inhibiting infection by HIV-1SF33 and HIV-1A17, respectively. Nevirapine (NVP) and 18 were used as the reference compounds.
The purity of tested compounds was determined by HPLC (Dionex
Ultimate 3000 HPLC system). All the assayed compounds displayed a
purity of ≥95% (Table S1, Supporting Information). Melting points
were determined on a WBS-1B type digital melting-point apparatus
and are uncorrected. NMR spectra were recorded on a Bruker Avance
300 or Avance 500 with tetramethylsilane (TMS) as an internal
standard, and chemical shifts are reported in δ (ppm). All the reactions
were routinely monitored by TLC, which was performed on
aluminum-backed silica gel plates (60 F254) with spots visualized by
UV light. Silica gel (0.040−0.064 mm) was used for column
chromatography and analytical silica gel for TLC plates. Unless
otherwise stated, all reagents were purchased from commercial
sources. When necessary, they were purified and dried by standard
methods. Organic solutions were dried over anhydrous sodium sulfate.
6-(Arylmethyl)-1-alkyl-5-halouracils 6a−9b. General Proce-
dure. Activated zinc dust (18 g, 275 mmol) was suspended in
dry THF (125 mL) at reflux, and a few drops of ethyl 2-
bromoacetate were added to initiate the reaction. After the
appearance of a green color (ca. 45 min), arylacetonitrile (4.50
mmol) was added in one portion followed by dropwise addition
of ethyl 2-bromoacetate (10 mmol) over a period of 1 h. The
reaction mixture was refluxed for an additional 10 min, diluted
with THF (3 × 125 mL), and quenched with aqueous K2CO3
(50%, 54 mL). Rapid stirring for 45 min gave two distinct
layers. The upper layer was decanted, the residue was washed
with THF (2 × 100 mL), and the combined THF fractions
were treated with aqueous HCl (10%, 50 mL) at room
temperature for 45 min. The mixture was concentrated in
vacuo, diluted with CH2Cl2 (300 mL), and washed with
saturated aqueous NaHCO3 (200 mL) to pH 7. The organic
phase was dried (Na2SO4) and evaporated in vacuo to give an
oil, which was used without further purification for the
synthesis of compound 2a−c.
with K103N and Y181C mutations, than that of nevirapine and
18.
CONCLUSION
■
In summary, we have designed a series of 1-[(benzyloxyl/
alkoxyl)methyl]-5-halo-6-aryl uracil and 1-[(benzyloxyl/
alkyloxyl)methyl]-4-amino-5-halo-6-aryl uracil (6a−15b) as
inhibitors of HIV-1 RT and described an efficient method of
synthesis. Interestingly, compounds 7a−c, 9a,b, and 11a,b
showed antiviral activities much more potent than those of
nevirapine, both in enzyme and cell-based assays. An excellent
correlation was found between EC50 values for inhibiting HIV-1
infection and IC50 values for inhibiting HIV-1 RT activity,
confirming that these compounds act as inhibitors of the HIV-1
RT.
Some of the dimethyl-substituted compounds (6b, 7b, 9b,
and 11b) had antiviral activity against wild-type HIV-1
comparable to that of 18. However, they were more effective
than 18 against the NNRTI-resistant HIV-1 strain A17. This
may have resulted from the introduction of two methyl groups
to the C-6-substituted aromatic ring, which could increase
contacts to the conserved Trp229 and modulate the terminal
phenyl moiety at the N-1 side chain.
Meanwhile, the difluoro-substituted derivative (compound
7c) showed a significant anti-HIV-1 potency for HIV-1SF33 and
HIV-1A17 infection in MT4 cells, compared to that of
nevirapine and 18. These results appear to confirm our
hypothesis that a favorable effect of 3,5-difluorogenation,
related to an attractive π-stacking interaction, takes place with
the benzene rings of Tyr188. Also, it is consistent with the
hypothesis that the presence of two electron-withdrawing
groups at the meta positions of the phenyl ring would enhance
the beneficial electronic effect on the antiviral activity produced
by 3,5-difluorogenation.
Sodium (2.2 g, 96 mmol) was dissolved in anhydrous EtOH (80
mL), and thiourea (5.2 g, 68 mmol) and compounds 1a−c (4.5 mmol)
were added to the clear solution. The reaction mixture was refluxed for
4 h and evaporated under reduced pressure until nearly dry, and the
residue was redissolved in H2O (50 mL). The 2-thiouracil was
precipitated by acidification to pH 4 with 2 N aq HCl.
The precipitated 2-thiouracil was desulfurized by suspension in 10%
aqueous chloroacetic acid (100 mL) and subsequent reflux for 24 h.
After cooling to room temperature, the precipitate was filtered off,
washed with cold EtOH and Et2O, and finally dried in vacuo to give
compounds 3a−c as white solids.
The SI values of the major 16 derivatives for inhibiting HIV-
1SF33 infection are in the range of 39 to 38 000. Strikingly,
compound 7b, which has the highest SI value, is highly effective
against the NNRTI-resistant HIV-1 strain A17, suggesting great
potential for further development as a new anti-HIV-1 drug.
6-Arylmethyluracil 3a−c (0.5 mmol) was suspended in glacial
HOAc (3 mL). Pb(OAc)2 (0.22 mmol) was added in portions while
stirring, followed by the addition of halogen (0.22 mmol). The
suspension was stirred for 12 h at 50 °C and then diluted with H2O
EXPERIMENTAL SECTION
Chemistry. The structural characterization was performed with an
NMR spectrometer and a high resolution mass spectrometer (HRMS).
■
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(120 mL). The precipitate was isolated, washed with H2O, and dried
in vacuo to give 4a−c, 5a,b as a solid product.
1H, NH); 13C NMR (75 MHz, DMSO-d6) δ: 15.03 (CH2CH3), 42.08
(CH2Ph), 65.37 (OCH2CH3), 74.02 (NCH2O), 101.9 (C-5), 127.47,
127.64, 129.39, 133.50 (aryl), 151.43(C-6), 156.10 (C-2), 160.05 (C-
4); HRMS (m/z): calcd for C14H15BrN2O3 [M + H]+ 339.03388,
found 339.03409.
6-(3,5-Dimethylbenzyl)-5-bromo-1-ethoxymethyluracil (8b).
white solid, yield 82%; mp 192−193 °C; 1H NMR (500 MHz,
DMSO-d6) δ 1.24 (t, 3H, J = 7.0 Hz, CH2CH3), 2.32 (s, 6H, 2CH3),
3.66 (q, 2H, J = 7.0 Hz, OCH2CH3), 4.41 (s, 2H, CH2Ar), 5.23 (s, 2H,
NCH2O), 6.79−6.95 (m, 3H, aryl), 9.30 (s, 1H, NH); 13C NMR (125
MHz, DMSO-d6) δ: 15.05 (CH2CH3), 21.33 (CH3Ar-C6), 37.33
(CH2Ar-C6), 65.40 (OCH2CH3), 73.74 (NCH2O), 101.83 (C-5),
125.21, 129.33, 133.35, 139.08 (aryl), 151.07(C-6), 153.44 (C-2),
158.91 (C-4); HRMS (m/z): calcd for C16H19BrN2O3 [M + H]+
367.06518, found 367.06542
6-(Arylmethyl)-5-halouracil 4a−c and 5a,b (0.42 mmol) were
dissolved in dry CHCl3 (10 mL), and N,O-bis(trimethylsilyl)acetamide
(BSA, 1.05 mmol) was added. After 30 min, the appropriate alkyl
chloromethyl ether (0.51 mmol) and LiI (0.2 mmol) were added. The
solution was stirred at room temperature for 4 h and then quenched
with ice-cold sat. NaHCO3 (25 mL). The aqueous phase was extracted
with AcOEt (3 × 20 mL). The organic layer was dried with Na2SO4
and evaporated under reduced pressure. The residue was purified by
silica column chromatography, giving the product.
6-Benzyl-1-ethoxymethyl-5-iodouracil (6a). white solid, yield 90%;
1
mp 175−177 °C; H NMR (300 MHz, DMSO-d6) δ 1.22 (t, 3H, J =
6.9 Hz, CH2CH3), 3.64 (q, 2H, J = 6.9 Hz, OCH2CH3), 4.58 (s, 2H,
CH2Ph), 5.24 (s, 2H, NCH2O), 7.14−7.28 (m, 5H, aryl), 9.26 (s, 1H,
NH); 13C NMR (75 MHz, DMSO-d6) δ: 15.01 (CH2CH3), 42.05
(CH2Ph), 65.39 (OCH2CH3), 74.05 (NCH2O), 79.17 (C-5), 127.48,
127.66, 129.38, 133.55 (aryl), 151.45 (C-6), 156.07 (C-2), 160.00 (C-
4); m/z (ESI): HRMS (m/z) calcd for C14H15IN2O3 [M + H]+
387.02001, found 387.02021.
6-Benzyl-1-benzyloxymethyl-5-bromouracil (9a). white solid,
yield 80%; mp 168−170 °C; 1H NMR (300 MHz, DMSO-d6) δ
4.47 (s, 2H, CH2Ph), 4.68 (s, 2H, OCH2Ph), 5.56 (s, 2H, NCH2O),
7.27−7.40 (m, 10H, aryl), 9.07 (s, 1H, NH); 13C NMR (75 MHz,
DMSO-d6) δ: 37.51 (CH2Ph-C6), 72.11 (OCH2Ph), 73.71 (NCH2O),
101.97 (C-5), 127.54, 127.67, 127.85, 128.22, 128.56, 129.35, 133.54,
136.88 (aryl), 150.86 (C-6), 152.92 (C-2), 158.50 (C-4); HRMS (m/
z): calcd for C19H17BrN2O3 [M + H]+ 401.04953, found 401.05027.
6-(3,5-Dimethylbenzyl)-1-benzyloxymethyl-5-bromouracil (9b).
white solid, yield 78%; mp 157−159 °C; 1H NMR (500 MHz,
DMSO-d6) δ 2.30(s, 6H, 2CH3), 4.39 (s, 2H, CH2Ar), 4.69 (s, 2H,
OCH2Ph), 5.31 (s, 2H, NCH2O), 6.72−7.41 (m, 8H, aryl), 9.39 (s,
1H, NH); 13C NMR (125 MHz, DMSO-d6) δ: 21.32 (CH3Ar-C6),
37.37 (CH2Ar-C6), 72.07 (OCH2Ph), 73.68 (NCH2O), 101.92 (C-5),
125.19, 127.77, 127.82, 127.90, 128.46, 128.58, 129.36, 133.28, 136.94,
139.07 (aryl), 151.04 (C-6), 153.20 (C-2), 158.83 (C-4); HRMS (m/
z): calcd for C21H21BrN2O3 [M + H]+ 429.08083, found 429.08150.
6-(Arylmethyl)-1-alkyl-5-dimethylaminouracils 10a,b,
11a,b. General Procedure. A mixture of 1-alkyl-6-(arylmeth-
yl)-5-iodouracil (0.2 mmol), 33% aqueous NH(CH3)2 solution
(2.4 mL), and 1,4-dioxane (2.4 mL) was reacted in a high-
pressure reactor at 80 °C for 1 h. After cooling to room
temperature, the reaction mixture was extracted with CH2Cl2 (2
× 25 mL). The combined organic layer was dried with Na2SO4
and evaporated under reduced pressure. The residue was
purified by silica column chromatography, giving the products.
6-Benzyl-5-dimethylamino-1-ethoxymethyluracil (10a). white
6-(3,5-Dimethylbenzyl)-1-ethoxymethyl-5-iodouracil (6b). white
solid, yield 89%; mp 177−178 °C; 1H NMR (300 MHz, DMSO-d6) δ
1.23 (t, 3H, J = 7.0 Hz, CH2CH3), 2.32 (s, 6H, 2CH3), 3.65 (q, 2H, J =
7.0 Hz, OCH2CH3), 4.51 (s, 2H, CH2Ar), 5.25 (s, 2H, NCH2O),
6.79−6.96 (m, 3H, aryl), 9.29 (s, 1H, NH); 13C NMR (75 MHz,
DMSO-d6) δ: 15.06 (CH2CH3), 21.35 (CH3Ar-C6), 41.92 (CH2Ph),
65.39 (OCH2CH3), 74.05 (NCH2O), 79.07 (C-5), 127.13, 127.66,
133.30, 139.08 (aryl), 151.52 (C-6), 156.33 (C-2), 160.13 (C-4); m/z
(ESI): HRMS (m/z) calcd for C16H19IN2O3 [M + H]+ 415.05131,
found 415.05181.
6-(3,5-Difluorobenzyl)-1-ethoxymethyl-5-iodouracil (6c). white
1
solid, yield 70%; mp 198−200 °C; H NMR (400 MHz, DMSO-d6)
δ 1.05 (t, 3H, J = 7.0 Hz, CH2CH3), 3.50 (q, 2H, J = 7.0 Hz,
OCH2CH3), 3.97 (s, 2H, CH2Ar), 5.19 (s, 2H, NCH2O), 6.97−7.16
(m, 3H, aryl), 9.80 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ:
15.48 (CH2CH3), 41.82 (CH2Ar), 65.17 (OCH2CH3), 71.26
(NCH2O), 73.98 (C-5), 103.09, 112.32, 140.02, 164.15 (aryl),
151.39 (C-6), 153.39 (C-2), 161.29 (C-4); m/z (ESI): HRMS (m/
z) calcd for C14H13F2IN2O3 [M + H]+ 422.9939, found 423.00165.
6-Benzyl-1-benzyloxymethyl-5-iodouracil (7a). white solid, yield
89%; mp 165−166 °C; 1H NMR (300 MHz, DMSO-d6) δ 4.57 (s, 2H,
CH2Ph), 4.67 (s, 2H, OCH2Ph), 5.32 (s, 2H, NCH2O), 7.12−7.41
(m, 10H, aryl), 8.90 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6) δ:
42.10 (CH2Ph-C6), 72.11 (OCH2Ph), 74.01 (NCH2O), 79.14 (C-5),
127.47 127.68, 127.84, 128.22, 128.56, 129.38, 133.46, 136.89 (aryl),
151.20 (C-6), 155.85 (C-2), 159.64 (C-4); m/z (ESI): HRMS (m/z)
calcd for C19H17IN2O3 [M + H]+ 449.03566, found 449.03562.
1-Benzyloxymethyl-6-(3,5-dimethylbenzyl)-5-iodouracil (7b).
white solid, yield 85%; mp 166−167 °C; 1H NMR (300 MHz,
DMSO-d6) δ 2.31(s, 6H, 2CH3), 4.49 (s, 2H, CH2Ar), 4.68 (s, 2H,
OCH2Ph), 5.33 (s, 2H, NCH2O), 6.72−7.41 (m, 8H, aryl), 9.11 (s,
1H, NH); 13C NMR (75 MHz, DMSO-d6) δ: 21.35(CH3Ar-C6),
41.99 (CH2Ar-C6), 72.07 (OCH2Ph), 74.00 (NCH2O), 79.14 (C-5),
125.11, 127.03, 127.68, 127.88, 128.23, 128.58, 129.36, 133.21, 136.97,
139.08 (aryl), 151.41(C-6), 156.11 (C-2), 159.93 (C-4); m/z (ESI):
HRMS (m/z) calcd for C21H21IN2O3 [M + H]+ 477.06696, found
477.06701.
1
solid, yield 58%; mp 80−81 °C; H NMR (500 MHz, DMSO-d6) δ
1.21 (t, 3H, J = 7.0 Hz, CH2CH3), 2.73 (s, 6H, N(CH3)2), 3.62 (q,
2H, J = 7.0 Hz, OCH2CH3), 4.43 (s, 2H, CH2Ph), 5.10 (s, 2H,
NCH2O), 7.14−7.36 (m, 5H, aryl), 9.44 (s, 1H, NH); 13C NMR (125
MHz, DMSO-d6) δ: 15.06 (CH2CH3), 32.28 (CH2Ph-C6), 43.64
(N(CH3)2), 65.02 (OCH2CH3), 73.17 (NCH2O), 126.75 (C-5),
126.88, 127.47, 136.18, 138.66 (aryl), 151.68 (C-6), 152.85 (C-2),
161.42 (C-4); HRMS (m/z): calcd for C16H21N3O3 [M + H]+
304.16557, found 304.16521.
6-(3,5-Dimethylbenzyl)-5-dimethylamino-1-ethoxymethyluracil
1
(10b). white solid, yield 50%; mp 171−173 °C; H NMR (500 MHz,
DMSO-d6) δ 1.22 (t, 3H, J = 7.1 Hz, CH2CH3), 2.31 (s, 6H, CH3Ph),
2.75 (s, 6H, N(CH3)2), 3.42 (q, 2H, J = 7.1 Hz, OCH2CH3), 4.36 (s,
2H, CH2Ar), 5.11 (s, 2H, NCH2O), 6.73−6.90 (m, 3H, aryl), 8.96 (s,
1H, NH); 13C NMR (125 MHz, DMSO-d6) δ: 15.06 (CH2CH3),
21.29 (CH3Ar-C6), 32.11 (CH2Ar-C6), 43.66 (N(CH3)2), 65.02
(OCH2CH3), 73.17 (NCH2O), 126.52 (C-5), 125.21, 128.52, 136.18,
138.66 (aryl), 151.47 (C-6), 153.16 (C-2), 161.11 (C-4); HRMS (m/
z): calcd for C18H25N3O3 [M + H]+ 332.19687, found 332.19622.
6-Benzyl-1-benzyloxymethyl-5-dimethylaminouracil (11a). white
6-(3,5-Difluorobenzyl)-1-benzyloxymethyl-5-iodouracil (7c).
white solid, yield 58%; mp 175−177 °C; 1H NMR (400 MHz,
DMSO-d6) δ 4.46 (s, 2H, CH2Ar), 4.61 (s, 2H, OCH2Ph), 5.24 (s, 2H,
NCH2O), 6.72−7.34 (m, 8H, aryl), 9.86 (s, 1H, NH); 13C NMR (100
MHz, DMSO-d6) δ: 41.66 (CH2Ar-C6), 72.09 (OCH2Ph), 74.01
(NCH2O), 79.99 (C-5), 103.31, 110.46, 127.97, 128.35, 128.60,
136.66, 137.35, 164.71 (aryl), 151.46 (C-6), 154.31 (C-2), 162.22 (C-
4); HRMS (m/z): calcd for C19H15F2IN2O3 [M + H]+ 485.0095,
found 485.01706.
6-Benzyl-5-bromo-1-ethoxymethyluracil (8a). white solid, yield
78%; mp 170−172 °C; 1H NMR (300 MHz, DMSO-d6) δ 1.21 (t, 3H,
J = 6.9 Hz, CH2CH3), 3.62 (q, 2H, J = 6.9 Hz, OCH2CH3), 4.54 (s,
2H, CH2Ph), 5.25 (s, 2H, NCH2O), 7.21−7.96 (m, 5H, aryl), 9.36 (s,
1
solid, yield 56%; mp 91−92 °C; H NMR (500 MHz, DMSO-d6) δ
2.72 (s, 6H, N(CH3)2), 4.43 (s, 2H, CH2Ph), 4.67 (s, 2H, OCH2Ph),
5.21 (s, 2H, NCH2O), 7.25−7.39 (m, 10H, aryl), 9.17 (s, 1H, NH);
13C NMR (125 MHz, DMSO-d6) δ: 32.27 (CH2Ph-C6), 43.64
(N(CH3)2), 71.94 (OCH2Ph), 73.29 (NCH2O), 126.76 (C-5),
126.92, 127.47, 127.77, 128.03, 128.35, 128.52, 129.10, 136.34,
137.39 (aryl), 151.68 (C-6), 152.64 (C-2), 161.12 (C-4); HRMS
(m/z): calcd for C21H23N3O3 [M + H]+ 366.18122, found 366.18047.
2247
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Journal of Medicinal Chemistry
Article
6-(3,5-Dimethylbenzyl)-1-benzyloxymethyl-5-dimethylaminoura-
(CH3Ar-C6), 37.22 (CH2Ar-C6), 64.32 (OCH2CH3), 74.08
(NCH2O), 91.68 (C-5), 125.48, 128.89, 134.99, 138.48 (aryl),
154.14 (C-6), 155.67 (C-2), 162.66 (C-4); m/z (ESI): 366.3 (M +
H)+.
4-Amino-6-benzyl-1-benzyloxymethyl-5-bromouracil (15a). yield
60%; mp 148−149 °C; 1H NMR (500 MHz, DMSO-d6) δ 4.30 (s, 2H,
CH2Ph), 4.55 (s, 2H, OCH2Ph), 5.24 (s, 2H, NCH2O), 7.11−7.34
(m, 10H, aryl), 8.00 (br, 2H, NH2-C4); 13C NMR (125 MHz, DMSO-
d6) δ: 37.39 (CH2Ph-C6), 70.77 (OCH2Ph), 74.17 (NCH2O), 91.86
(C-5), 127.41, 127.89, 128.09, 128.17, 128.70, 129.44, 135.05, 138.07
(aryl), 153.94 (C-6), 155.59 (C-2), 162.69 (C-4); m/z (ESI): 400.3,
402.2 (M + H)+.
1
cil (11b). white solid, yield 51%; mp 152−153 °C; H NMR (300
MHz, DMSO-d6) δ 2.28 (s, 6H, CH3Ph), 2.72 (s, 6H, N(CH3)2), 4.34
(s, 2H, CH2Ar), 4.67 (s, 2H, OCH2Ph), 5.20 (s, 2H, NCH2O), 6.70−
7.39 (m, 8H, aryl), 9.10 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6)
δ: 21.29 (CH3Ar), 32.07 (CH2Ar-C6), 43.62 (N(CH3)2), 71.90
(OCH2Ph), 73.31 (NCH2O), 126.61 (C-5), 125.19, 127.47, 128.00,
128.35, 128.49, 136.07, 137.44, 138.65 (aryl), 151.68 (C-6), 152.64
(C-2), 161.12 (C-4); HRMS (m/z): calcd for C23H27N3O3 [M + H]+
394.21252, found 394.21203.
4-Amino-6-(arylmethyl)-1-alkyl-5-halouracils 12a,b, 13a,b,
14a,b, 15a,b. General Procedure. 1-Alkyl-6-arylmethyl-5-
halouracil (0.067 mmol) was added to the solution of 1,2,4-
triazole (1.34 mmol) and POCl3 (0.53 mmol) in the presence
of triethylamine. After being stirred for about 30 min, the
solution became dark-red. Concentrated ammonia (1 mL, 25−
28%) was added dropwise to the mixture and reacted for
another 1 h. The reaction mixture was extracted with
dichloromethane, and the combined organic layer was dried
with Na2SO4 and evaporated in vacuo. The residue was purified
by silica column chromatography to yield the products.
4-Amino-6-benzyl-1-ethoxymethyl-5-iodouracil (12a). white
4-Amino-1-benzyloxymethyl-6-(3,5-dimethylbenzyl)-5-bromour-
1
acil (15b). white solid, yield 84%; mp 137−138 °C; H NMR (500
MHz, DMSO-d6) δ 2.19 (s, 6H, 2CH3), 4.27 (s, 2H, CH2Ar), 4.60 (s,
2H, OCH2Ph), 5.32 (s, 2H, NCH2O), 6.69−7.43 (m, 8H, aryl), 7.89
(br, 2H, NH2-C4); 13C NMR (125 MHz, DMSO-d6) δ: 21.22
(CH3Ar-C6), 41.70 (CH2Ar-C6), 71.98 (OCH2Ph), 74.20 (NCH2O),
87.14 (C-5), 126.34, 127.5, 127.96, 128.18, 128.45, 128.87, 129.65,
133.43, 137.07, 140.09 (aryl), 153.47 (C-6), 156.14 (C-2), 163.23 (C-
4); m/z (ESI): 428.4 (M + H)+.
Biological Assays. Assay for Measuring the Inhibitory Activity
of Compounds against HIV-1 RT. Oligo(dT) (TaKaRa Co.,
Japan) was immobilized via its 5′-terminal phosphate to
Covalink-NH microtiter plates (NUNC Co., Denmark). The
biotin-dUTP was incorporated by reverse transcriptase
(Sigma). Briefly, a serial concentration of inhibitor was added
to the mixture, which contained 50 mmol/L Tris-HCl (pH
1
solid, yield 46%; mp 150−152 °C; H NMR (300 MHz, DMSO-d6)
δ 1.12 (t, 3H, J = 6.9 Hz, CH2CH3), 3.46 (q, 2H, J = 6.9 Hz,
OCH2CH3), 4.38 (s, 2H, CH2Ph), 5.17 (s, 2H, NCH2O), 7.12−7.37
(m, 5H, Ph), 8.23 (br, 2H, NH2-C4); 13C NMR (75 MHz, DMSO-d6)
δ: 15.33 (CH2CH3), 41.98 (CH2Ph), 64.26 (OCH2CH3), 67.60
(NCH2O), 74.21 (C-5), 127.32, 127.83, 129.41, 133.22 (aryl), 151.16
(C-6), 156.89 (C-2), 164.75 (C-4); m/z (ESI): 386.2 (M + H)+.
4-Amino-6-(3,5-dimethylbenzyl)-1-ethoxymethyl-5-iodouracil
8.3), 3 mmol/L MgCl2, 75 mmol/L KCl, 5 mmol/L DTT (D,L-
dithiothreitol), 0.13 mg/mL BSA (Albumin Bovine V), 10 μg/
mL poly (A), 0.75 μM biotin-11-dUTP, and 1.5 μM dTTP.
The reaction mixture was incubated at 37 °C for 1 h and
washed with a buffer containing 50 mmol/L Tris-HCl (pH7.5),
0.15 mol/L NaCl, 0.05 mmol/L MgCl2, and 0.02% Tween-20.
After 100 μL of 1% BSA was added to each well and incubated
at room temperature for another 30 min, the plate was washed
with the same buffer. Before further incubation at 37 °C for 1 h,
50 μL of SA-ALP (alkaline phosphatase streptavidin) solution
(100 ng/mL) was added to each well, and the plate was washed
again as above. Finally, 50 μL of PNPP (p-nitrophenyl
phosphate, disodium) (1 mg/mL, pH 9.5) was added and
incubated at 37 °C for 30 min. The reaction was stopped by
addition of 0.5 M NaOH. The inhibitory activity of the
compounds was detected and quantified using a colorimetric
streptavidin−alkaline phosphatase reporter system.
1
(12b). white solid, yield 78%; mp 113−115 °C; H NMR (300 MHz,
DMSO-d6) δ 1.04 (t, 3H, J = 7.0 Hz, CH2CH3), 2.24 (s, 6H, 2CH3),
3.49 (q, 2H, J = 7.0 Hz, OCH2CH3), 4.31 (s, 2H, CH2Ar), 5.17 (s, 2H,
NCH2O), 6.73−6.89 (m, 3H, aryl), 8.49 (br, 2H, NH2-C4); 13C NMR
(75 MHz, DMSO-d6) δ: 15.32 (OCH2CH3), 21.37 (CH3Ar-C6),
41.66 (CH2Ar-C6), 64.33 (OCH2CH3), 67.55 (NCH2O), 74.26 (C-
5), 125.44, 128.86, 135.01, 138.48 (aryl), 156.28 (C-6), 157.05 (C-2),
164.75 (C-4); m/z (ESI): 414.3 (M + H)+.
4-Amino-6-benzyl-1-benzyloxymethyl-5-iodouracil (13a). white
solid, yield 58%; mp 138−139 °C; 1H NMR (500 MHz, DMSO-d6) δ
4.39 (s, 2H, CH2Ph), 4.55 (s, 2H, OCH2Ph), 5.25 (s, 2H, NCH2O),
7.09−7.34 (m, 10H, aryl), 7.98 (br, 2H, NH2-C4); 13C NMR (125
MHz, DMSO-d6) δ: 41.85 (CH2Ph-C6), 67.61 (OCH2Ph), 70.80
(NCH2O), 74.41 (C-5), 127.35 127.87, 128.06, 128.13, 128.70,
129.43, 135.13, 138.12 (aryl), 156.14 (C-6), 156.85 (C-2), 164.80 (C-
4); m/z (ESI): 448.3 (M + H)+.
Assay for Measuring the Inhibitory Activity of Compounds on
HIV-1SF33 and HIV-1A17 Replication in MT4. MT4 cells, HIV-1SF33
,
4-Amino-1-benzyloxymethyl-6-(3,5-dimethylbenzyl)-5-iodouracil
1
HIV-1IIIB, and HIV-1A17 were obtained from the NIH AIDS Research
and Reference Reagent Program (Germantown, MD). The inhibitory
activity of the compounds on infection by a laboratory-adapted HIV-1
strain SF33 and an NNRTI-resistant HIV-1 strain A17 was tested in
MT4 cells. Briefly, MT4 cells (4 × 104/well) were infected by addition
of 200 TCID50 of HIV-1, followed by incubation for 2 h at 37 °C
before addition of compounds at serial dilutions. After further
incubation at 37 °C for 7 days, p24 was measured using a commercial
enzyme-linked immunosorbent assay (ELISA) kit (Vironostika HIV-1
(13b). white solid, yield 58%; mp 129−130 °C; H NMR (500 MHz,
DMSO-d6) δ 2.21(s, 6H, 2CH3), 4.29 (s, 2H, CH2Ar), 4.55 (s, 2H,
OCH2Ph), 5.23 (s, 2H, NCH2O), 6.70−7.36 (m, 8H, aryl), 8.60 (br,
2H, NH2-C4); 13C NMR (125 MHz, DMSO-d6) δ: 21.37(CH3Ar-
C6), 41.89 (CH2Ar-C6), 68.06 (OCH2Ph), 72.30 (NCH2O), 78.04
(C-5), 126.11, 127.30, 127.86, 128.08, 128.32, 128.68, 129.40, 133.27,
136.97, 139.18 (aryl), 152.41(C-6), 155.01 (C-2), 160.03 (C-4); m/z
(ESI): 476.4 (M + H)+.
4-Amino-6-benzyl-5-bromo-1-ethoxymethyluracil (14a). white
1
́
Microelisa system; BioMerieux; Marcy l’Etoile, France).The concen-
solid, yield 48%; mp decomposed >200 °C; H NMR (500 MHz,
tration of a compound for inhibiting 50% viral replication (EC50) was
determined by nonlinear regression using GraphPad Prism 5.01.
Assessment of in Vitro Cytotoxicity in MT-4 Cell. An XTT assay, as
previously described,33was used to assess the cytotoxicity of target
compounds to MT4 cells. Briefly, a compound at graded
concentrations was added to MT4 cells (5 × 104/well), followed by
an incubation at 37 °C for 3 days. Ten microliters of CCK-8 reagent
was added to the cells. After incubation at 37 °C for 4 h to allow color
development of the XTT formazan product, the absorbance of each
well was then read at 450 nm in a Victor2 1420 Multilabel Counter
(Wallace-PerkinElmer Life and Analytical Sciences Inc., Boston, MA).
The percent of cytotoxicity and CC50 (concentration causing 50%
cytotocity) were calculated as previously described.37
DMSO-d6) δ 1.05 (t, 3H, J = 6.9 Hz, CH2CH3), 3.58 (q, 2H, J = 6.9
Hz, OCH2CH3), 4.46 (s, 2H, CH2Ph), 5.23 (s, 2H, NCH2O), 7.151−
7.87 (m, 5H, aryl), 8.30 (br, 2H, NH2-C4);; 13C NMR (125 MHz,
DMSO-d6) δ: 15.07 (CH2CH3), 41.96 (CH2Ph), 64.73 (OCH2CH3),
74.01 (NCH2O), 90.89 (C-5), 125.67, 128.14, 133.39, 139.50 (aryl),
152.43 (C-6), 157.01 (C-2), 159.05 (C-4); m/z (ESI): 338.3, 340.2
(M + H)+.
4-Amino-6-(3,5-dimethylbenzyl)-5-bromo-1-ethoxymethyluracil
1
(14b). white solid, yield 91%; mp 111−112 °C; H NMR (500 MHz,
DMSO-d6) δ 1.04 (t, 3H, J = 7.0 Hz, CH2CH3), 2.24 (s, 6H,
2CH3Ph), 3.50 (q, 2H, J = 7.0 Hz, OCH2CH3), 4.22 (s, 2H, CH2Ar),
5.15 (s, 2H, NCH2O), 6.74−6.89 (m, 3H, aryl), 8.40 (br, 2H, NH2-
C4); 13C NMR (125 MHz, DMSO-d6) δ: 15.31 (OCH2CH3), 21.35
2248
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Journal of Medicinal Chemistry
Article
Molecular Modeling. The docking studies were conducted by
using software GOLD3.0.1 and two protein crystal structures of wild-
type RT/1 complex (PDB ID: 1RT2) and Y181C mutant RT/2
complex (PDB: 1JLA). Following the default setting of Chemscore in
the software tool, top scored docked poses were visually inspected for
each ligand in the DS 2.5 based on relevant molecules. The radius of
the binding site sphere was defined by the original ligand as 8.9 Å, thus
ensuring that the docking method was reliable.
(6) Esnouf, R.; Ren, J.; Ross, C.; Jones, Y.; Stammers, D.; Stuart, D.
Mechanism of inhibition of HIV-1 reverse transcriptase by nonnucleo-
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ASSOCIATED CONTENT
■
S
* Supporting Information
Additional information as noted in the text. This material is
available free of charge via the Internet at http://pubs.acs.org.
(9) Das, K.; Clark, A. D. Jr.; Lewi, P. J.; Heeres, J.; de Jonge, M. R.;
Koymans, L. M. H.; Vinkers, H. M.; Daeyaert, F.; Ludovici, D. W.;
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reverse transcriptase inhibitors that are highly potent and effective
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AUTHOR INFORMATION
■
Corresponding Author
*Phone/fax: +86-10-82805203, e-mail: jyliu@bjmu.edu.cn
(J.L.); or phone: +86-10-58900976, fax: +86-10-58900980, e-
mail: mal@chinaaids.cn (L.M.).
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Lu, X.; Hsiou, Y.; Jacobo-Molina, A.; Andries, K.; Et, A. Structure of
HIV-1 reverse transcriptase in a complex with the non-nucleoside
inhibitor alpha-APA R 95845 at 2.8 Å resolution. Structure 1995, 3,
365−379.
Author Contributions
#These authors have contributed equally.
Notes
The authors declare no competing financial interest.
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Janssen, P. A.; Hughes, S. H.; Arnold, E. Structure of HIV-1 RT/TIBO
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ACKNOWLEDGMENTS
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We thank the NIH AIDS Research and Reference Reagent
Program in the USA for providing MT4 cells, HIV-1SF33, and
HIV-1A17. This study was supported by the National Science
Foundation of China (20972011, 21042009, 21172014,
30872232, and 81172733) and grants from the Ministry of
Science and Technology of China (2009ZX09301-010) and
SKLID development grant (2011SKLID102) for financial
support.
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ABBREVIATIONS USED
■
CC50, concentration for 50% cytotoxicity; EC50, effective
concentration for 50% inhibition; ELISA, enzyme-linked
immunosorbent assay; HAART, highly active antiretroviral
therapy; HIV-1, human immunodeficiency virus type 1; HEPT,
(1-[2-hydroxyethoxymethyl]-6-(phenylthio)thymine]; NNBP,
non-nucleoside binding pocket; NNIBP, the non-nucleoside
inhibitor binding pocket; NNRTI, non-nucleoside reverse
transcriptase inhibitor; PDB, protein database; RT, reverse
transcriptase; RTI, reverse transcriptase inhibitor; SI, selectivity
index (ratio of CC50/EC50)
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