Discovery of dual inhibitors targeting both HIV-1 capsid and human cyclophilin A to inhibit the assembly and uncoating of the viral capsid

Article abstract of DOI:10.1016/j.bmc.2009.02.051

HIV-1 assembly and disassembly (uncoating) processes are critical for the HIV-1 replication. HIV-1 capsid (CA) and human cyclophilin A (CypA) play essential roles in these processes. We designed and synthesized a series of thiourea compounds as HIV-1 assembly and disassembly dual inhibitors targeting both HIV-1 CA protein and human CypA. The SIV-induced syncytium antiviral evaluation indicated that all of the inhibitors displayed antiviral activities in SIV-infected CEM cells at the concentration of 0.6-15.8 μM for 50% of maximum effective rate. Their abilities to bind CA and CypA were determined by ultraviolet spectroscopic analysis, fluorescence binding affinity and PPIase inhibition assay. Assembly studies in vitro demonstrated that the compounds could potently disrupt CA assembly with a dose-dependent manner. All of these molecules could bind CypA with binding affinities (Kd values) of 51.0-512.8 μM. Fifteen of the CypA binding compounds showed potent PPIase inhibitory activities (IC₅₀ values < 1 μM) while they could not bind either to HIV-1 Protease or to HIV-1 Integrase in the enzyme assays. These results suggested that 15 compounds could block HIV-1 replication by inhibiting the PPIase activity of CypA to interfere with capsid disassembly and disrupting CA assembly.

Full text of DOI:10.1016/j.bmc.2009.02.051

Bioorganic & Medicinal Chemistry 17 (2009) 3177–3188
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of dual inhibitors targeting both HIV-1 capsid and human
cyclophilin A to inhibit the assembly and uncoating of the viral capsid
Jiebo Li a, Zhiwu Tan a, Shixing Tang b, Indira Hewlett b, Ruifang Pang a, Meizi He a, Shanshan He a,
Baohe Tian a, Kan Chen a, Ming Yang a,
*
a State Key Laboratory of Natural and Biomimetic Drugs, Peking University, PO Box 261, Beijing 100191, China
b Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
HIV-1 assembly and disassembly (uncoating) processes are critical for the HIV-1 replication. HIV-1 capsid
(CA) and human cyclophilin A (CypA) play essential roles in these processes. We designed and synthe-
sized a series of thiourea compounds as HIV-1 assembly and disassembly dual inhibitors targeting both
HIV-1 CA protein and human CypA. The SIV-induced syncytium antiviral evaluation indicated that all of
Received 21 January 2009
Revised 19 February 2009
Accepted 21 February 2009
Available online 3 March 2009
the inhibitors displayed antiviral activities in SIV-infected CEM cells at the concentration of 0.6–15.8 lM
for 50% of maximum effective rate. Their abilities to bind CA and CypA were determined by ultraviolet
spectroscopic analysis, fluorescence binding affinity and PPIase inhibition assay. Assembly studies
in vitro demonstrated that the compounds could potently disrupt CA assembly with a dose-dependent
Keywords:
HIV-1
Capsid
Cyclophilin A
Assembly
Disassembly
Dual inhibitor
manner. All of these molecules could bind CypA with binding affinities (Kd values) of 51.0–512.8
lM. Fif-
teen of the CypA binding compounds showed potent PPIase inhibitory activities (IC50 values < 1
l
M)
while they could not bind either to HIV-1 Protease or to HIV-1 Integrase in the enzyme assays. These
results suggested that 15 compounds could block HIV-1 replication by inhibiting the PPIase activity of
CypA to interfere with capsid disassembly and disrupting CA assembly.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
inhibiting CA assembly.7 Kelly et al. elucidated the binding model
of CAP-1 and CA NTD, and found that the interaction between
Antiretroviral therapy has led to a significant improvement in
the prognosis for HIV-1-infected individuals. The currently used
anti-HIV-1 drugs target the envelope protein, reverse transcriptase
(RT), integrase (IN) and protease (PR), to block HIV-1 infection at
the stage of entry, reverse transcription, integration and matura-
tion, respectively.1 However, new anti-AIDS drugs are urgently
needed because of the growing problems of drug resistance and
the serious side-effects of the current drugs. Some new compounds
that target different viral proteins or various steps of viral life cy-
cles are widely investigated.2 However, the assembly and disas-
sembly processes of HIV-1 capsid (CA) protein have received less
attention as antiviral drug targets compared to IN, PR and RT.3–5
Recently, several inhibitors that bind to HIV-1 CA had been re-
ported,6,7 such as a helical peptide inhibitor8 (CAI) that binds be-
tween CA CTD(C-terminal domain) helices 8 and 11, and two
small molecule inhibitors9 (Fig. 1) that insert into a pocket formed
at the junction between helices 1, 2, 4 and 7 in the NTD (N-term-
inaldomain) of HIV-1 CA. These inhibitors bind within or adjacent
to the third interface and probably disrupt this interaction thereby
CAP-1 and CA NTD can displace Phe32 from the NTD core and open
a deep hydrophobic cavity to the small molecule inhibitor.10 The
aromatic ring of CAP-1 inserts into the cavity, and its urea NH
groups form hydrogen bonds with the backbone oxygen of Val59.
Recent studies11,12 have also shown that several small molecules
can bind to the ‘saddle’ pockets of CypA (pocket A and pocket B)
H
H
Cl
N
N
O
S
O
N
CAP-1
H
N
H
N
NO2
O
O
N
CAP-2
* Corresponding author. Tel.: +86 10 82805264; fax: +86 10 82802724.
E-mail address: yangm@bjmu.edu.cn (M. Yang).
Figure 1. Structures of CAP-1 and CAP-2.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.051

3178
J. Li et al. / Bioorg. Med. Chem. 17 (2009) 3177–3188
Table 1
Chemical structure, inhibition effect and cytotoxicity of the title compounds on SIV induced syncytium
H
N
H
N
Ar1
Ar2
S
Entry
Ar1
Ar2
EC50
(lM)
TC50
44.1
(lM)
TI (TC50/EC50)
NO2
H
N
T1
6.3
7
N
N
Cl
N
NO2
H
N
T2
T3
7.4
5.5
26.2
4
2
N
N
N
Br
Br
N
H
N
8.43
N
N
D1
D2
2.3
3.1
16.1
38.8
7
O
NO2
12
Cl
O
D3
D4
4.6
6.2
30.8
83.9
7
O
Br
Cl
O
14
O
O
D5
D6
4.2
4.7
86
20
20
O
Cl
O
91.9
O
O
Cl
O
D7
D8
3.0
6.1
24.4
69.1
8
Cl
F
O
Cl
O
11
O
F

J. Li et al. / Bioorg. Med. Chem. 17 (2009) 3177–3188
3179
Table 1 (continued)
Entry
Ar1
Ar2
EC50
11.6
(
lM)
TC50
16.0
(l
M)
TI (TC50/EC50)
O
D9
1
O
O
D10
6.3
60.2
83.7
10
O
F
F
O
D11
15.8
5
O
O
D12
D13
D14
D15
D16
D17
D18
2.8
4.6
46.9
18.7
75.4
88.0
91.9
37.8
41.9
17
O
O
4
O
Cl
O
6.9
11
S
NH
NH
NH
NH
NH
F
O
O
O
O
O
N
N
N
N
N
O
O
S
11.6
1.0
8
O
F
O
S
92
O
Cl
Cl
O
S
4.1
9
O
O
S
1.5
27
O
O
O
S
D19
6.2
48.4
8
NH
O
N
O
O
S
D20
D21
9.6
7.5
41.4
49.7
4
7
NH
NH
O
O
N
N
O
O
S
(continued on next page)
O

3180
J. Li et al. / Bioorg. Med. Chem. 17 (2009) 3177–3188
Table 1 (continued)
Entry
Ar1
Ar2
EC50
(lM)
TC50
58.7
(l
M)
TI (TC50/EC50)
O
D22
D23
0.9
0.6
66
S
NH
NH
O
O
N
N
O
Br
O
S
74.4
77.5
37.0
19.1
127
19
3
O
Cl
Cl
O
S
D24
D25
D26
D27
4.1
10.8
5.7
NH
O
O
O
S
S
NH
N
O
Cl
Cl
O
S
S
3
NH
NH
N
O
O
S
S
2.2
20.9
—
9
N
O
CsA
—
—
NAa
—
*
EC50, concentration required to protect cells against the cytopathogenicity of SIV by 50%.
NA, no activity.
a
and inhibit its activity catalyzing the cis/trans-isomerization of the
Gly89-Pro90 peptide bond, which facilitates the disassembly of the
HIV-1 CA complex. These researches open a new field to find new
inhibitor that target HIV-1 CA to disrupt the assembly and/or dis-
assembly process of HIV-1 CA.
kinds of substitute phenyls (o-CH3, m-F, m-Cl, m-Br, p-F, p-CH3,
p-OCH3 and p-Cl) were introduced to evaluate the influence of anti
assembly activity. Then, according to the binding model of CAP-1
and CA had been clearly interpreted7, we extrapolated that larger
phenyl groups and some hydrogen receptors outside the cavity
would form stronger hydrophobic interaction and hydrogen with
the protein. As an attempt, we introduced the pharmacophore-sul-
famethoxazole segment (D14–D23). Finally, the introductions of
the 4-metoxybenzene (D24) and thiazole (D25–D27) instead of
isoxazolyl were to investigate the groups’ assembly inhibitory.
Based on the references mentioned above, we initially selected
CAP-1 as leading compound to develop new anti-HIV agents. As
shown in Table 1, we maintained thiourea scaffold as linker to con-
nect two main groups, Ar1 and Ar2. Ar1 represented a series of
substituted phenyl groups; Ar2 stood for different types of aryl
groups. We designed a novel compound with thiourea as the core
structure, and a 3-chloro-4-methoxyl-phenyl (the activity group of
CAP-1) as Ar1 and a N4-(2-aminoethyl)-N2,N2-diethyl-6-methyl-5-
nitropyrimidine-2,4-diamine as Ar2 (T1). Secondly, we changed
the 3-bromide phenyl instead of 3-chloro-4-methoxyl-phenyl to
test whether the different kind of phenyl substituent would po-
tently influence the activity (T2). Thirdly, we kept the 3-bromide
phenyl and eliminated the nitro-group in pyrimidine to identify
the activity of nitro-group (T3). Therefore, the biological experi-
ments revealed that the modification of substituted phenyl group
could change the inhibiting capability of compounds. However,
the introduction of N4-(2-aminoethyl)-N2,N2-diethyl-6-methyl-5-
2. Results and discussion
The routes used for preparation of the title compounds were
illustrated in Scheme 1 and the synthetic procedures were ex-
plained in Section 4. Thirty compounds were obtained and their
MS, 1H NMR spectroscopy data were provided in Section 4.
As shown in Table 1, we evaluated these thiourea compounds
using SIV-induced syncytium assay in CEM cells to test their the
concentration for 50% of maximum effect (EC50), concentration
for 50% of maximum toxic (TC50) and therapeutic index (TI) values.
The thiourea compounds inhibited viral replication with a range of
nitropyrimidine-2,4-diamine could not improve the activity com-
pared with CAP-1, which inspired us to design new types of assem-
bly inhibitors.
Then, we linked the 3-chorile-4-methyl phenyl of CAP-1 and 3-
nitro-4-methyl phenyl of CAP-2 which had been reported as the
agents to inhibit CA assembly in vitro with thiourea scaffold. Con-
sequently, the 6-isobenzofuran-1(3H)-one was selected to substi-
tute the aromatic nitro group to avoid the cell toxicity. Different
EC50 values from 0.6 lM to 15.8 lM. Compound D23 showed best
EC50 value. Its TI value was also greater than 100. The compounds
(D16, D22 and D23) which consisted of meta-chlorine or bromine
mono-substituted phenyl at Ar1 and sulfamethoxazole group at
Ar2 showed very good antiviral activities (EC50 < 1 lM). Neverthe-
less, the compounds consisted of meta-fluorine substituted phenyl
at Ar1 and sulfamethoxazole group at Ar2 displayed the weakest
antiviral capability (EC50 > 10 lM). As the TRIM5a could interact

J. Li et al. / Bioorg. Med. Chem. 17 (2009) 3177–3188
3181
Scheme 1. Reagents and conditions: (a) (1) 1,4-diazabicyclo[2,2,2]-octane, CS2, rt, 12 h, (2) BTC, reflux, 2 h (for two steps); (b) HNO3, H2SO3, rt, 24 h; (c) POCl3, Me2NC6H4,
reflux, 5 h; (d) amine, ethanol, rt, 24 h; (e) POCl3, reflux, 3 h; (f) 1,2-diaminoethane, ethanol, rt, 26 h; (g) N(C6H5)3, reflux, 5h; (B) Reagents and conditions: (a) (1) 1,4-
diazabicyclo[2,2,2]-octane, CS2, rt, 12 h, (2) BTC, reflux, 2 h (for two steps); (b) C2H5OH, room temperature, 12 h; (C) Reagents and conditions: (a) NaBH4, CH3OH, THF; (b)
KNO3,H2SO4; (c) SnCl2, 37%HCl solution, 50 °C; (d) (1) 1,4-diazabicyclo[2,2,2]-octane, CS2, rt, 12 h, (2) BTC, reflux, 2 h (for two steps); (e) C2H5OH, room temperature, 12 h; (D)
Reagents and conditions: (a) (1) 1,4-diazabicyclo[2,2,2]-octane, CS2, rt, 12 h, (2) BTC, reflux, 2 h (for two steps); (b) C2H5OH, room temperature, 12–24 h.
with CypA and protect the monkey cells against virus infections,13
CsA (Cyclosporin A) at the concentration of 10 M, as the negative
control, showed less than 10% SIV inhibiting proportion. Therefore,
the antiviral results led us to further understand the exact inhibi-
tion mechanism of the compounds.
To clear whether the compound really target to HIV-1 CA, we
used a spectrophotometrically method that measured the sample
turbidity variation which was caused by the capsid assemble pro-
cession.9,14 This assay was used to probe for potential inhibitory ef-
fects of the thiourea compounds in this study. As shown in Figure
2, dissolution of native HIV-1 CA into assembly buffer (50 mM
phosphate buffer, 2.5 M NaCl, pH 8.0, 0.2%v/v DMSO) led to an in-
crease in absorbance at an initial rate of 102.60 mOD/min. Tang
et al had demonstrated that CAP-1 and its analogues could bind
with CA and affect the rate of assembly. We found that the initial
assembly rate in the presence of CAP-1 decreased to 52.46 mOD/
min. For the identification of new assembly inhibitors, we added
our test compounds and expected to delay the initial rate of CA
assembly.
The presence of compound T1 could moderate decrease the
assembly rate of CA to 91.03 mOD/min. The modification of 3-bro-
mine phenyl could slightly influence the assembly rate which de-
crease to 88.09 mOD/min in the existence of T2. Interestingly,
compared with T2, the elimination of the nitro-group in pyrimi-
dine (T3) thoroughly loss the inhibition capability. Like CAP-1,
the title compounds D1–D27 could more or less decrease the
l

3182
J. Li et al. / Bioorg. Med. Chem. 17 (2009) 3177–3188
the influence of the Ar2 group in the inhibitory potency of HIV-1 CA
assembly. Compared D1 (assembly rate = 66.65 mOD/min) with D6
(assembly rate = 59.14 mOD/min), it was found that introduction
of naphthalene ring resulted in a slight loss of activity. The assem-
bly rate of CA in the presence of compound D24 (assembly
rate = 26.30 mOD/min) with 4-metoxybenzene group was approx-
imate to the presence of compound D17 (assembly rate = 29.00 -
mOD/min) with a methoxazole group. Substitutions with thiazole
group conducted to attenuated activity. For example, compared
with the influences of D17 and D20, the assembly rates in the pres-
ence of D26 and D27 increased to 52.50 mOD/min and 58.97 mOD/
min, respectively. The introduction of isobenzofuran-1(3H)-one
group (D13, assembly rate = 19.57 mOD/min) could significantly
improve the inhibition of CA assembly when contrasted with initial
3-nitro-4-methyl phenyl group (D2, assembly rate = 60.00 mOD/
min).
As seen in Table 3, the dose ratio of test compounds and CA in-
creased sequentially as 0.5:1, 1:1, and 2:1, and the results revealed
that the assembly effect also correspondingly amplified, which
demonstrated that the assembly rate was changed in a dose-
dependent manner. These results indicated that these CA-binding
compounds could inhibit capsid assembly in vitro in a dose-depen-
dence manner.
It has been demonstrated that CypA efficiently catalyzed the cis/
trans isomerization of Gly-Pro within the isolated NTD of HIV-1
CA.15 And, CypA could also function by weakening the association
between CA strips, thereby promoting disassembly of the viral
core. Thus, the compound that can bind to CypA and inhibit its PPI-
ases activity could be a potent inhibitor of HIV-1 replication. With
the quick developments11,12,16 of CypA inhibitors, new discoveries
made by Jean-Francois11 indicated that the substituted phenyl can
insert into the pocket A of CypA. Li12 et al. also reported that sulfa-
methoxazole group was an effective segment which could insert
into the saddle pocket B of CypA. Since our compounds had similar
active segments as those found by Jean-Francois and Li, we subse-
quently evaluated their binding activities with CypA and their PPI-
ase inhibitory activities.
Figure 2. The effects of CA-binding compounds on in vitro CA assembly by the
turbidity assay. Initial rate of the reaction: CA only (102.60 mOD/min); CAP-1
(52.46 mOD/min); D16 (15.77 mOD); D22 (31.20 mOD/min); D23 (36.43 mOD/
min); The reaction can be balance at the end of time.
assembly rate of CA in our study (Table 2 and Fig. 2). We then eval-
uated the influence of substituted phenyl group at Ar1. As shown in
Table 2, for meta-substituted derivatives, incorporation of an elec-
tron with drawing group, for example, a chloride (D16, assembly
rate = 15.77 mOD/min) resulted in a significant decrease of the
CA assembly rate compared with none substituted phenyl com-
pound (D8, assembly rate = 39.21 mOD/min). And, we investigated
Table 2
Assembly inhibitory data of thiourea derivatives
It had been reported that the trytophan residue (Trp121)
around the CypA binding site could be used as an intrinsic probe
for studying the interaction between CypA and its ligand.17 The in-
dole side chain of Trp121 can be validated as a major determinant
in immunosuppressant drug recognition and the separation of PPI-
ase catalytic efficiency from CsA affinity by the complementary
gain and loss of CsA sensitivity to mutation to or from tryptophan.
Therefore, we investigated the binding affinity of thiourea com-
pounds against CypA by using the published intrinsic fluorescence
titration technique.18 The inhibitory activity of the compounds on
PPIase could be determined by a standard spectrophotometric
method19, in which the rate constants for the cis–trans conversion
were evaluated by fitting the data to the integrated first-order rate
equation through nonlinear least-square analysis. As the positive
control, CsA showed a concentration for 50% of the maximum
inhibitory (IC50) value of 9nM against the PPIase activity of CypA.
Our results indicated that except T3, all the thiourea compounds
could bind to CypA with binding affinities (Kd values) ranging from
Entry
Assembly rate (mOD/mina)
T1
T2
T3
D1
D2
D3
D4
D5
D6
D7
D8
D9
D10
D11
D12
D13
D14
D15
D16
D17
D18
D19
D20
D21
D22
D23
D24
D25
D26
D27
91.03
88.09
NAb
66.65
60.00
69.60
49.63
49.20
59.14
67.80
63.60
64.64
78.60
55.71
65.69
19.57
73.80
76.20
15.77
29.00
40.01
64.80
45.89
39.21
31.20
36.43
26.30
27.38
52.50
58.97
51.0 lM to 512.8 lM (Table 4). Among them, 15 compounds exhib-
Table 3
The CA assembly effect of different ratio compound:CA
D16 ratio
Compound:CA
Assembly rate
(mODa/min)
D17 ratio
Compound:CA
Assembly rate
(mOD/min)
0.5:1
1:1
2:1
43.20
38.40
11.40
0.5:1
1:1
2:1
52.20
42.00
19.37
a
mOD, millioptical density units.
NA, no activity.
b
a
mOD, millioptical density units.

J. Li et al. / Bioorg. Med. Chem. 17 (2009) 3177–3188
3183
Table 4
Kd = 100.7 lM) in para-phenyl only displayed very weak binding
affinities but no inhibitory activities.
Equilibrium affinity constants and in vivo IC50 values for interaction of thiourea
derivatives with CypA
Regarding the influence of the substitution at Ar2, except D2
and D13, compounds D1–D13 showed no PPIase inhibiting activi-
ties, indicating that naphthalene and isobenzofuran-1(3H)-one
group substituted for Ar2 did not contribute to the inhibition of
PPIase activity. Compared with isobenzofuran-1(3H)-one of D5,
the larger sulfonamide group of D18 can contribute to the amelio-
ration of PPIase inhibition. Remarkably, the introduction of sulfat-
Entry
Kda
(
l
M)
PPIase activity
Entry
Kd (
l
M)
PPIase activity
IC50 (lM)
b
IC50
(l
M)
T1
T2
T3
73.3
79.3
200.5
124.4
88.9
125.1
222.2
133.3
171.8
147.1
512.8
141.0
140.4
227.3
NA
0.74
0.81
NAc
NA
0.46
NA
NA
NA
NA
NA
D13
D14
D15
D16
D17
D18
D19
D20
D21
D22
D23
D24
D25
D26
D27
96.6
109.5
128.5
79.6
107.3
100.7
116.5
118.8
88.1
88.6
55.8
60.8
51.6
0.59
0.35
0.38
0.33
0.69
NA
0.60
NA
0.58
0.45
0.65
NA
D1
D2
D3
D4
D5
D6
D7
D8
D9
D10
D11
D12
hiazole group (D26, Kd = 51.0 lM, IC50 = 0.13 lM) showed the best
enzymatic inhibitory activity among the Ar2 groups.
Fifteen compounds in our study were found to inhibit both the
CA assembly and CypA PPIase activities. We also performed the
HIV-1 protease20 and integrase21,22 inhibitory assays with the thio-
urea compounds to check if they can interact with another two
critical proteins: HIV-1 protease and integrase. The results showed
that these compounds did not inhibit the activity of HIV-1 protease
since none of their final UV absorptions decreased more than 5%,
and all the compounds tested showed lower than 20% of inhibition
NA
NA
NA
NA
0.16
0.13
0.45
51.0
76.9
NA
a
Kd values were determined from intrinsic tryptophan fluorescence titration
assays at 25 °C.
of HIV-1 integrase activities at 20 lM concentration. Our results
b
c
PPIase IC50, values were determined from enzyme inhibition assays at 6 °C.
NA, no activity.
indicated that the thiourea compounds did not inhibit the activi-
ties of HIV-1 protease and integrase. The antiviral activity of the
compounds in our study might be due to the specific inhibition
of HIV-1 CA and CypA.
ited potent PPIase inhibitory activities (IC50 < 1 lM). These results
To understand the molecular mechanism of the inhibitors, we
suggested that the thiourea compounds might disrupt the disas-
sembly process of the HIV-1 CA complexes by inhibiting CypA’s
activity to catalyze the cis/trans-isomerization of Gly89-Pro90
peptide bond of HIV-1 CA.
The further evaluation of the CypA PPIase inhibitory activities of
the thiourea compounds showed that the substitution at Ar1 group
could significantly affect the inhibitory effect. The introduction of
chlorine atom in m-position phenyl resulted in a very active com-
carried out molecular docking analysis using the program AUTO-
23
DOCK
.
The docking results revealed that the thiourea derivatives
inserted into the hydrophobic cavity of HIV-1 CA and the saddle
pockets of CypA. As shown in Figure 3A, the docking pose of D23
represented one of the binding models of the inhibitors and
HIV-1 CA. The 3-chloro-4-methoxyl-phenyl group of compound
D23 inserted into the hydrophobic cavity, which composed by
residues Val 27, Ala31, His62, Ala65, Leu138 and Leu141. The bind-
ing model indicated that the chlorine or bromine in meta-position
of Ar1 could contribute to the strong hydrophobic interaction be-
tween the small inhibitor and the hydrophobic cavity. Both the
docking analysis data and the above experimental results revealed
that the thiourea compounds with chlorine or bromine in meta-
position of Ar1 were potent assembly inhibitors of HIV-1 CA.
pound D16 (Kd = 79.6
chloride atom from meta (D16) to para position (D17,
Kd = 107.3 M, IC50 = 0.69 M), could obviously decrease the bind-
lM, IC50 = 0.33 lM). However, shifting the
l
l
ing affinities, and attenuate the inhibitory activities correspond-
ingly (IC50 value: D17 > D16). In addition, the compounds with
methyl group (D20, Kd = 118.8 lM) or methoxyl group (D18,
Figure 3. The molecular models of D23 bind with CA NTD and CypA. (A) The molecular model of D23 binds with CA NTD. The substituted phenyl of D23 inserted into the
hydrophilic cavity; the sulfamethoxazole group exposed to the surface of HIV-1 CA. (B) The molecular model of D23 binds with CypA. The small molecule inserted into the
saddle pockets (pocket A and pocket B); the figures were produced by Pmv.

3184
J. Li et al. / Bioorg. Med. Chem. 17 (2009) 3177–3188
Subsequently, we analyzed the interaction of the Ar2 group of the
thiourea compounds with HIV-1 CA. The oxygen of isoxazole of
compound D23 formed a hydrogen bond with the backbone hydro-
gen of Gly61, while another hydrogen bond was formed between
the hydrogen of sulfamide and the oxygen of Thr58. The two
hydrogen bonds may facilitate the binding affinity between the
inhibitor and HIV-1 CA protein. Moreover, we studied the interac-
tion between other substitutions of Ar2 and HIV-1 CA. Interest-
ingly, for the compound D25, the backbone hydrogen of Gly61
could also form hydrogen bonds with the nitrogen of thiazole.
Remarkably, all the sulfamide groups could form a hydrogen bond
with HIV-1 CA. Therefore, in the presence of the same Ar1, the dif-
ferences of inhibitory activities relied on the number of hydrogen
bonds between the Ar2 group and HIV-1 CA. These data indicated
that the most effective assembly inhibitors of HIV-1 CA must be
the compounds that contained chloride substitution in meta-phe-
nyl of Ar1 group, and their Ar2 group should have strong interaction
with HIV-1 CA protein.
We also performed docking analysis for the compounds that
bound to CypA (Fig. 3B). As a typical example, the compound
D23 inserted into the active saddle grooves of CypA. The 3-
chloro-4-methoxyl-phenyl of D23 directed toward the pocket A
composed of residues Gly72, Ser110, Gln111 and Lys82. The 3-
chloro-4-methoxyl-phenyl could occupy more suitable space of
pocket A, and contribute to the strong binding affinity. In addition,
additional van der Waals was developed between the 3-chloride
atom of D23 and the side chain of Lys82. Meanwhile, the sulfa-
methoxazole group of D23 could insert into the pocket B which
consisted of Arg55, Phe60, Ala101, Ala103, Phe113, Leu122, and
His126. The sulfamethoxazole group could provide two aryl rings
for hydrophobic interaction with CypA. The oxygen of isoxazole
could form hydrogen bonds with the guanidyl hydrogen atoms of
Arg55. As an active pocket, the pocket B needs more hydrogen
bonds to enhance the binding affinity. Consequently, the Ar2 which
had more hydrogen receptors and donors would facilitate the bind-
ing. In this study, sulfathiazole and sulfamethoxazole substitutions
could give sulfamide group which contains hydrogen and oxygen
as the hydrogen donor and receptor. Furthermore, thiazole and
isoxazole also had some hetero-atoms as good hydrogen receptors.
Therefore, it is clear that the introduction of sulfathiazole and sul-
famethoxazole group could improve the binding affinity. Based on
these analyses, we could say that the compounds composed of the
chloride substitution in meta-position phenyl at Ar1 and sulfa-
methoxazole group at Ar2 might display distinguished binding
activity with CypA.
insert into both the cavity of HIV-1 CA and pocket A of CypA, and
another segment such as sulfamethoxazole or sulfthiazole to form
hydrogen bonds and hydrophobic interactions with HIV-1 CA and
CypA. The inhibitory data of our dual inhibitors were summarized
in Table 5.
3. Conclusion
In this study, we synthesized a series of thiourea derivatives tar-
geting both HIV-1 CA and CypA to disrupt the processes of the
assembly and uncoating of HIV-1 CA. The biological experiment re-
sults confirmed that 15 compounds could block HIV-1 replication
by binding to CypA to inhibit its PPIase activity and uncoating,
and by inserting into HIV-1 CA to disrupt its assembly. In addition,
molecular docking analyses were also used to elucidate the binding
models of the inhibitors to the two targets, HIV-1 CA and CypA. The
compounds consisted of Ar1 with chloride substitution in meta-po-
sition phenyl and Ar2 with sulfamethoxazole group displayed po-
tent assembly inhibitory activities, CypA binding affinities, the
PPIase inhibitory activities and antiviral activities. The further
characterizations of these compounds in vivo are on-going.
4. Experimental
4.1. Chemistry
All materials were commercially available and used without
further purification. All the titled compounds were characterized
by 1H NMR spectra on a Varian 300 MHz or Bruke AM-300 spec-
trometer using the solvents described. Chemical shifts were re-
ported in d ppm (parts per million) relative to tetramethyl silane
(TMS) except for deuteriorated water (D2O) and the signals were
quoted as s (singlet), d (doublet), t (triplet), q (quartet), and m
(multiplet). The mass spectra (EI or ESI) were recorded. Melting
points were determined on
uncorrected.
a XA-4 instrument which was
4.2. General synthetic procedures
As shown in Scheme 1, the compounds could be prepared by
different types of amines and corresponding isothiocyanates.
Scheme 1A outlined the preparation of the thiourea derivatives
of T1–T3. The route includes three steps: First, the preparation of
corresponding phenyl isothiocyanates. In the presence of 1,4-
diazabicyclo[2,2,2]-octane, salts of dithiocarbamic acid were pre-
pared by reacting arylamines with carbon disulfide, followed by
subsequent conversion to aromatic isothiocyanates by reacting
with bis(trichloromethyl)carbonate (BTC). And the phenyl isothio-
cyanates were isolated by column chromatography or vacuum
distillation. Second, the preparation of N4-(2-aminoethyl)-N2,N2-
diethyl-6-methyl-5-nitropyrimidine-2,4-diamine and N4-(2-amino-
ethyl)-N2,N2-diethyl-6-methylpyrimidine-2,4-diamine. 4-Chloro-
N,N-diethyl-6-methyl-5-nitropyrimidin-2-amine was synthesized
as Scheme 1A described, and compounds was prepared from it
by amination. 4-Chloro-N,N-diethyl-6-methylpyrimidin-2-amine
was synthesized by 2-(diethylamino)-6-methylpyrimidin-4-ol
reacting with POCl3.24 N4-(2-Aminoethyl)-N2,N2-diethyl-6-methyl-
pyrimidine-2,4-diamine was synthesized from 4-chloro-N,N-
diethyl-6-methylpyrimidin-2-amine and 1,2-diaminoethane in
the presence of Na2CO3. Finally, title compounds T1–T3 were syn-
thesized from and N4-(2-aminoethyl)-N2,N2-diethyl-6-methyl-5-
nitropyrimidine-2,4-diamine or N4-(2-aminoethyl)-N2,N2-diethyl-
6-methylpyrimidine-2,4-diamine corresponding phenyl isothiocy-
anates in the presence of triethylamine. Scheme 1B–D outlined
the preparation of the thiourea derivatives of D1–D27. As shown
According to molecular docking analyses, the binding models of
the dual inhibitors should contain one fragment as suitable substi-
tuted phenyl, such as chloride atom substitution in meta-phenyl to
Table 5
The list of dual inhibitors and their inhibitory data
Entry
Capsid assembly rate (mOD/min)
Cyclophilin A PPIase IC50 (lM)
T1
T2
D2
91.03
88.09
60.00
19.57
73.80
76.20
15.77
29.00
64.80
39.21
31.20
36.43
27.38
52.50
58.97
0.74
0.81
0.46
0.59
0.35
0.38
0.33
0.69
0.60
0.58
0.45
0.65
0.16
0.13
0.45
D13
D14
D15
D16
D17
D19
D21
D22
D23
D25
D26
D27

J. Li et al. / Bioorg. Med. Chem. 17 (2009) 3177–3188
3185
in Scheme 1C, isobenzofuran-1(3H)-one was prepared by the reac-
tion between isobenzofuran-1,3-dione and NaBH4 in THF.25 We
used potassium nitrate in sulfuric acid to get the 6-nitrophthalide
smoothly as described in the literature.26 Then, 6-nitrophthalide
was reduced by stannous chloride in hydrochloric acid to 6-amin-
ophthalide. Naphthylamine and sulfonamide derivatives could be
gotten from commercial Company (Alfa). The thiourea derivates
were synthesized from aromatic isothiocyanate (the same prepara-
tion as Scheme 1A) and substituted aromatic amino. All the title
compounds D1–D27 were recrystallized in methanol. The struc-
tures of these compounds were confirmed by ESI-MS/EI-MS and
1H NMR. The purities of these compounds were identified by HPLC.
4.2.6. 1-(3-Bromophenyl)-3-(3-oxo-1,3-dihydroisobenzofuran-
5-yl)thiourea (D3)
Compound D3 was prepared in the same way as D1, white solid,
mp 166–168 °C, yield = 76.3%. 1H NMR (300 MHz, DMSO-d6) d 5.38
(s, 2H), 7.29–7.32 (t, 2H), 7.40–7.42 (m, 1H), 7.60–7.64 (d, 1H), 7.76
(d, J = 3 Hz, 1H), 7.81–7.79 (t, 1H), 7.98 (t, J = 3 Hz, 1H); EI-MS:
m/z = 362, 364. RP-HPLC: tR = 3.3 min (10% water in menthol),
purity > 98%. RP-HPLC: tR = 3.3 min (10% water in menthol),
purity > 98%.
4.2.7. 1-(4-Chlorophenyl)-3-(3-oxo-1,3-dihydroisobenzofuran-
5-yl)thiourea (D4)
Compound D4 was prepared in the same way as D1, white solid,
mp 201–202 °C, yield = 78.5%. 1H NMR (300 MHz, DMSO-d6) d 5.41
(s, 2H), 7.40–7.66 (dd, 4H), 7.80–7.83 (dd, 2H), 8.02 (s, 1H), 10.09–
10.10 (d, 2H); EI-MS: m/z = 318. RP-HPLC: tR = 3.3 min (10% water
in menthol), purity > 98%.
4.2.1. 1-(3-Chloro-4-methylphenyl)-3-(2-(2-(diethylamino)-6-
methyl-5-nitropyrimidin-4-ylamino)ethyl)thiourea (T1)
A mixture of compound 3-chloro-4-methylphenyl isothiocya-
nate (0.183 g; 1 mmol) and N4-(2-aminoethyl)-N2,N2-diethyl-6-
methyl-5-nitropyrimidine-2,4-diamine (0.27 g; 1 mmol) was stir-
red in acetone. Then, N(C6H5)3 (0.02 g, 0.1 mmol) was added into
the mixture. After reflux stirring 5 h, the mixture was concentrated
under reduced pressure then purified by column chromatography
(silica gel, dichloromethane/methanol = 4:1, v/v). Compound T1
was obtained as yellow solid, mp 178 °C, yield = 64.9%.
4.2.8. 1-(3-Chlorophenyl)-3-(3-oxo-1,3-dihydroisobenzofuran-
5-yl)thiourea (D5)
Compound D5 was prepared in the same way as D1, white solid,
mp 175–176 °C, yield = 73.2%. 1H NMR (300 MHz, DMSO-d6) d 5.40
(s, 2H), 7.18–7.32 (m, 1H), 7.36–7.39 (m, 2H), 7.61–7.68 (t, 2H),
7.78–7.83 (m, 1H), 8.06 (s, 1H), 10.12–10.19 (d, 2H); EI-MS:
m/z = 318. RP-HPLC: tR = 3.3 min (10% water in menthol),
purity > 99%.
1H NMR (300 MHz, CDCl3) d 1.13–1.20 (m, 6H), 2.33 (s, 3H), 2.72
(s, 3H), 3.45–3.53 (m, 2H), 3.64–3.68 (m, 2H), 3.85–3.90 (m, 4H),
6.52 (s, 1H), 6.91–6.95 (m, 1H), 7.11–7.15 (m, 2H), 7.58 (s, 1H),
8.83 (s, 1H); ESI-MS: m/z = 452.15 (M+H+); RP-HPLC: tR = 4.4 min
(10% water in menthol), purity > 94%.
4.2.9. 1-(4-Methoxyphenyl)-3-(3-oxo-1,3-
dihydroisobenzofuran-5-yl)thiourea (D6)
4.2.2. 1-(3-Bromophenyl)-3-(2-(2-(diethylamino)-6-methyl-5-
nitropyrimidin-4-ylamino)ethyl)thiourea (T2)
Compound D6 was prepared in the same way as D1, white solid,
mp 183–184 °C, yield = 74.5%. Compound D6: 1H NMR (300 MHz,
DMSO-d6) d 3.74 (s, 3H), 5.38 (s, 2H), 6.90–7.35 (dd, 4H), 7.58 (d,
J = 12 Hz, 1H), 7.77 (d, J = 12 Hz, 1H), 8.01 (s, 1H), 9.84–9.87 (d,
2H); EI-MS: m/z = 314. RP-HPLC: tR = 2.9 min (10% water in men-
thol), purity > 99%.
Compound T2 was prepared in the same way as T1, yellow nee-
dle solid, mp 144–146 °C, yield = 63.6%. 1H NMR (300 MHz,CDCl3) d
1.13–1.20 (m, 6H), 2.70 (s, 3H), 3.44–3.51 (m, 2H), 3.62–3.70 (m,
2H), 3.85–3.91 (m, 4H), 6.53 (s, 1H), 7.05–7.380 (m, 4H), 7.57 (s,
1H), 8.84 (s, 1H); ESI-MS: m/z = 482.10, 484.09 (M+H+). RP-HPLC:
tR = 6.3 min (10% water in menthol), purity > 96%.
4.2.10. 1-(2,4,5-Trichlorophenyl)-3-(3-oxo-1,3-
dihydroisobenzofuran-5-yl)thiourea (D7)
4.2.3. 1-(3-Bromophenyl)-3-(2-(2-(diethylamino)-6-
methylpyrimidin-4-ylamino)ethyl)thiourea (T3)
Compound D7 was prepared in the same way as D1, white solid,
mp 185–186 °C, yield = 81.2%. 1H NMR (300 MHz, DMSO-d6) d 5.41
(s, 2H), 7.64–7.80 (dd, 2H), 7.82–7.97 (s, 2H), 8.06 (s, 1H), 9.80 (s,
1H), 10.36 (s, 1H); ESI-MS: m/z = 386.2 (M+H+). RP-HPLC:
tR = 4.3 min (10% water in menthol), purity > 99%.
Compound T3 was prepared in the same way as T1, yellow nee-
dle solid, mp 117–118 °C yield = 63.6%. 1H NMR (300 MHz, CDCl3) d
1.21 (s, 6H), 2.38 (s, 3H), 3.56–3.94 (m, 8H), 5.83 (s, 1H), 7.15–
7.720 (m, 4H), 8.20 (s, 1H), 9.57 (s, 1H), 11.11 (s, 1H); ESI-MS:
m/z = 437.11 (M+H+); RP-HPLC: tR = 3.8 min (10% water in men-
thol), purity > 99%.
4.2.11. 1-(3,5-Difluorophenyl)-3-(3-oxo-1,3-
dihydroisobenzofuran-5-yl)thiourea (D8)
Compound D8 was prepared in the same way as D1, white solid,
mp 190–191 °C, yield = 69.1%. 1H NMR (300 MHz, DMSO-d6) d 5.40
(s, 2H), 7.07–7.14 (t, 1H), 7.31–7.50 (t, 1H), 7.52–7.77 (m, 1H),
7.79–8.03 (dd, 2H), 9.65 (s, 1H), 10.17 (s, 1H); ESI-MS:
m/z = 321.1 (M+H+). RP-HPLC: tR = 2.9 min (10% water in menthol),
purity > 99%.
4.2.4. 1-(4-Methoxyphenyl)-3-(naphthalen-2-yl)thiourea (D1)
A
mixture of compound 4-methoxylphenyl isothiocyanate
(0.167 g; 1 mmol) and naphthalen-2-amine (0.14 g; 1 mmol) was
stirred in ethanol. After stirring overnight, the mixture was filtered.
The precipitation was recrystallized in methanol. Compound D1
was obtained as white solid, mp 179–180 °C, yield = 64.9%. Com-
pound D1: 1H NMR (300 MHz, DMSO-d6) d 3.75 (s, 3H), 6.91–
6.93 (d, J = 6.0 Hz, 2H), 7.34–7.36 (d, J = 6.0 Hz, 2H), 7.46–7.51
(m, 2H), 7.59–7.62 (d, 2H), 7.83–7.87 (t, 3H), 7.98 (s, 1H), 9.73–
9.86 (d, 2H), EI-MS: m/z = 308. RP-HPLC: tR = 4.4 min (10% water
in menthol), purity > 97%.
4.2.12. 1-(3-Oxo-1,3-dihydroisobenzofuran-5-yl)-3-m-
tolylthiourea (D9)
Compound D9 was prepared in the same way as D1, white solid,
mp 183–185 °C, yield = 78.2%. 1H NMR (300 MHz, DMSO-d6) d 2.28
(s, 3H), 5.37 (s, 2H), 6.90–7.35 (dd, 4H), 7.58–7.82 (dd, 2H), 8.01 (s,
1H), 9.98 (s, 2H); EI-MS: m/z = 298. RP-HPLC: tR = 3.1 min (10%
water in menthol), purity > 99%.
4.2.5. 1-(3-Chloro-4-methylphenyl)-3-(4-methyl-3-
nitrophenyl)thiourea (D2)
Compound D2 was prepared in the same way as D1, yellow so-
lid, mp 186–187 °C, yield = 85.4%. 1H NMR (300 MHz, DMSO-d6) d
2.28 (s, 3H), 7.27–7.30 (dd, 2H), 7.42–7.45 (d, 1H), 7.58 (s, 1H),
7.66 (d, J = 2.4 Hz, 1H), 8.22 (d, J = 2.4 Hz, 1H); EI-MS: m/z = 335.
RP-HPLC: tR = 4.4 min (10% water in menthol), purity > 97%.
4.2.13. 1-(3-Fluorophenyl)-3-(3-oxo-1,3-
dihydroisobenzofuran-5-yl)thiourea (D10)
Compound D10 was prepared in the same way as D1, white so-
lid, mp 190–191 °C, yield = 72.3%. 1H NMR (300 MHz, DMSO-d6) d
5.39 (s, 2H), 7.13 (t, J = 2.4 Hz, 1H), 7.33–7.38 (t, 2H), 7.47–7.49

3186
J. Li et al. / Bioorg. Med. Chem. 17 (2009) 3177–3188
(d, 1H), 7.61–7.66 (t, 1H), 7.79 (m, J = 2.4 Hz, 1H), 8.03 (s, 1H), 9.
99–10.01 (d, 2H); ESI-HRMS: m/z = 303.05980 (M+H+, calcd for
C15H11N2O2S1F1, 303.05253). RP-HPLC: tR = 3.1 min (10% water in
menthol), purity > 99%.
J = 10.5 Hz, 2H), 7.65–7.82 (dd, 4H), 10.20–10.23 (d, 2H), 11.40 (s,
1H); ESI-MS: m/z = 423.03469 (M+H+, calcd for C17H15N4O3S2CL1,
423.02741). RP-HPLC: tR = 10.9 min (10% water in menthol),
purity > 99%.
4.2.14. 1-(4-Fluorophenyl)-3-(3-oxo-1,3-
4.2.21. 4-(3-(4-Methoxyphenyl)thioureido)-N-(5-
dihydroisobenzofuran-5-yl)thiourea (D11)
methylisoxazol-3-yl)benzenesulfonamide (D18)
Compound D11 was prepared in the same way as D1, white so-
lid, mp 174–175 °C, yield = 70.6%. 1H NMR (300 MHz, DMSO-d6) d
5.40 (s, 2H), 7.16–7.22 (t, 2H), 7.46–7.50 (t, 2H), 7.61 (d,
J = 8.1 Hz, 1H), 7.79–7.82 (d, J = 8.1 Hz, 1H), 8.012 (s, 1H), 9.95–
10.01 (d, 2H); ESI-MS: m/z = 303.1 (M+H+). RP-HPLC: tR = 3.0 min
(10% water in menthol), purity > 97%.
Compound D18 was prepared in the same way as D1, white so-
lid, mp 180–181 °C, yield = 80.4%. 1H NMR (300 MHz, DMSO-d6) d
2.30 (s, 3H), 3.78 (s, 3H), 6.15 (s, 1H), 6.90 (d, J = 8.7 Hz, 2H), 7.31
(d, J = 8.7 Hz, 2H), 7.72 (d, J = 8.7 Hz, 2H), 7.75 (d, J = 8.7 Hz, 2H),
9.97–10.01 (d, 2H), 11.37 (s, 1H); EI-MS: m/z = 418. RP-HPLC:
tR = 9.0 min (10% water in menthol), purity > 97%.
4.2.15. 1-(3-Oxo-1,3-dihydroisobenzofuran-5-yl)-3-o-
tolylthiourea (D12)
4.2.22. N-(5-Methylisoxazol-3-yl)-4-(3-o-
tolylthioureido)benzenesulfonamide (D19)
Compound D12 was prepared in the same way as D1, white so-
lid, mp 183–185 °C, yield = 78.2%. 1H NMR (300 MHz, DMSO-d6) d
2.26 (s, 3H), 5.35 (s, 2H), 6.95–7.00 (t, 1H), 7.14 (d, J = 7.8 Hz,
1H), 7.18 (d, J = 7.8 Hz, 1H), 7.57 (d, J = 10 Hz, 1H), 7.63 (d,
J = 10 Hz, 1H), 7.79–7.82 (d, 1H), 8.02 (s, 1H), 8.12 (s, 1H), 9. 36
(s, 1H); ESI-MS: m/z = 299.1 (M+H+). RP-HPLC: tR = 3.3 min (10%
water in menthol), purity > 99%.
Compound D19 was prepared in the same way as D1, white so-
lid, mp 187–188 °C, yield = 78.2%. 1H NMR (300 MHz, DMSO-d6) d
2.23 (s, 3H), 2.30 (s, 3H), 6.14 (s, 1H), 7.18 (d, J = 8.7 Hz, 2H), 7.22
(d, J = 8.7 Hz, 2H), 7.78 (m, 4H), 9.67 (s, 1H), 10.05 (s, 1H), 11.36
(s, 1H); ESI-MS: m/z = 403.1 (M+H+). RP-HPLC: tR = 9.4 min (10%
water in menthol), purity > 98%.
4.2.23. N-(5-Methylisoxazol-3-yl)-4-(3-p-
4.2.16. 1-(3-Chloro-4-methylphenyl)-3-(3-oxo-1,3-
dihydroisobenzofuran-5-yl)thiourea (D13)
tolylthioureido)benzenesulfonamide (D20)
Compound D20 was prepared in the same way as D1, white
solid, mp 195–196 °C, yield = 69.2%. 1H NMR (300 MHz, DMSO-
d6) d 2.28–2.30 (d, 6H), 6.15 (s, 1H), 7.14–7.34 (dd, 4H), 7.72–
7.79 (m, 4H), 10.04–10.06 (d, 2H), 11.36(s, 1H); ESI-MS:
m/z = 403.1(M+H+). RP-HPLC: tR = 9.7 min (10% water in menthol),
purity > 97%.
Compound D13 was prepared in the same way as D1, white
solid, mp 189–190 °C, yield = 75.1%. 1H NMR (300 MHz, DMSO-
d6) d 2.28 (s, 3H), 5.38 (s, 2H), 7.29–7.30 (t, J = 3 Hz, 2H), 7.60–
7.63 (d, 2H), 7.79–7.80 (d, J = 3 Hz, 1H), 7.79 (s, 1H); EI-MS:
m/z = 332. RP-HPLC: tR = 4.4 min (10% water in menthol),
purity > 95%.
4.2.24. N-(5-Methylisoxazol-3-yl)-4-(3-
4.2.17. 4-(3-(4-Fluorophenyl)thioureido)-N-(5-methylisoxazol-
3-yl)benzenesulfonamide (D14)
phenylthioureido)benzenesulfonamide (D21)
Compound D21 was prepared in the same way as D1, white so-
lid, mp 166–167 °C, yield = 68.1%. 1H NMR (300 MHz, DMSO-d6) d
2.30 (s, 3H), 6.15 (s, 1H), 7.15–7.18 (m, 1H), 7.35 (d, J = 11.8Hz,
2H), 7.46 (d, J = 11.8 Hz, 2H), 7.78–7.79 (dd, 4H), 10.14–10.16 (d,
2H), 11.39 (s, 1H); EI-MS: m/z = 388. RP-HPLC: tR = 10.1 min (10%
water in menthol), purity > 99%.
Compound D14 was prepared in the same way as D1, white so-
lid, mp 188–190 °C, yield = 77.2%. 1H NMR (300 MHz, DMSO-d6) d
2.29 (s, 3H), 6.16 (s, 1H), 7.16–7.22 (t, 2H), 7.44–7.49 (t, 2H),
7.71 (d, J = 9 Hz, 2H), 7.78 (d, J = 9 Hz, 2H), 10.11–10.18 (d, 2H),
11.41 (s, 1H); ESI-MS: m/z = 407.1 (M+H+). RP-HPLC: tR = 10.6 min
(10% water in menthol), purity > 97%.
4.2.25. 4-(3-(3-Bromophenyl)thioureido)-N-(5-methylisoxazol-
3-yl)benzenesulfonamide (D22)
4.2.18. 4-(3-(3-Fluorophenyl)thioureido)-N-(5-methylisoxazol-
3-yl)benzenesulfonamide (D15)
Compound D22 was prepared in the same way as D1, white so-
lid, mp 165–166 °C, yield = 69.8%. 1H NMR (300 MHz, DMSO-d6) d
2.29 (s, 3H), 6.16 (s, 1H), 7.27–7.36 (m, 2H), 7.42–7.45 (m, 1H),
7.45–7.82 (m, 5H), 10.23–10.31 (d, 2H), 11.41 (s, 1H); ESI-MS:
m/z = 467.0, 469.0 (M+H+). RP-HPLC: tR = 10.4 min (10% water in
menthol), purity > 95%.
Compound D15 was prepared in the same way as D1, white so-
lid, mp 169–170 °C, yield = 81.3%. 1H NMR (300 MHz, DMSO-d6) d
2.29 (s, 3H), 6.16 (s, 1H), 7.13–7.18 (t, 1H), 7.35 (d, J = 7.5 Hz,
2H), 7.45–7.48 (d, J = 7.5 Hz, 2H), 7.72 (d, J = 9 Hz, 2H), 7.77 (d,
J = 9 Hz, 2H), 10.15–10.17 (d, 2H), 11.39 (s, 1H); ESI-MS:
m/z = 407.1 (M+H+). RP-HPLC: tR = 6.9 min (10% water in menthol),
purity > 98%.
4.2.26. 4-(3-(3-Chloro-4-methylphenyl)thioureido)-N-(5-
methylisoxazol-3-yl)benzenesulfonamide (D23)
4.2.19. 4-(3-(3-Chlorophenyl)thioureido)-N-(5-methylisoxazol-
3-yl)benzenesulfonamide (D16)
Compound D23 was prepared in the same way as D1, white so-
lid, mp 172–173 °C, yield = 81.2%. 1H NMR (300 MHz, DMSO-d6) d
2.30–2.33 (d, 6H), 6.16 (s, 1H), 7.30–7.33 (t, 2H), 7.62 (s, 1H),
7.71 (d, J = 9 Hz, 2H), 7.78 (d, J = 9 Hz, 2H), 10.15–10.22 (d, 2H),
11.39 (s, 1H); EI-MS: m/z = 436; RP-HPLC: tR = 12.1 min (10% water
in menthol), purity > 98%.
Compound D16 was prepared in the same way as D1, white so-
lid, mp 179–180 °C, yield = 83.7%. 1H NMR (300 MHz, DMSO-d6) d
2.28 (s, 3H), 6.16 (s, 1H), 7.19–7.22 (m, 2H), 7.37–7.42 (m, 2H),
7.67–7.82 (m, 5H), 10.25–10.31 (d, 2H), 11.41 (s, 1H); ESI-MS:
m/z = 423.0(M+H+). RP-HPLC: tR = 10.4 min (10% water in men-
thol), purity > 99%.
4.2.27. 4-(3-(4-Chlorophenyl)thioureido)-N-(4-
methoxyphenyl)benzenesulfonamide (D24)
4.2.20. 4-(3-(4-Chlorophenyl)thioureido)-N-(5-methylisoxazol-
3-yl)benzenesulfonamide (D17)
Compound D17 was prepared in the same way as D1, white so-
lid, mp 177–178 °C, yield = 77.9%. 1H NMR (300 MHz, DMSO-d6) d
2.29 (s, 3H), 6.16 (s, 1H), 7.37 (d, J = 10.5 Hz, 2H), 7.48 (d,
Compound D24 was prepared in the same way as D1, white so-
lid, mp 190–192 °C, yield = 68.2%. 1H NMR (300 MHz, DMSO-d6) d
3.67 (s, 3H), 6.79 (d, J = 9 Hz, 2H), 6.98 (d, J = 9 Hz, 2H), 7.38 (d,
J = 10 Hz, 2H), 7.48 (d, J = 10 Hz, 2H), 7.60 (d, J = 10 Hz, 2H), 7.66
(d, J = 10 Hz, 2H), 9.87 (s, 1H), 10.13–10.16 (d, 2H); EI-MS:

J. Li et al. / Bioorg. Med. Chem. 17 (2009) 3177–3188
3187
m/z = 436. RP-HPLC: tR = 3.4 min (10% water in menthol),
purity > 99%.
were taken at the wavelength of maximum emission (340 nm).
The fluorescence intensity versus the final compound concentra-
tion was integrated to the following equation, where F0 stands
for the initial fluorescence intensity of the protein solution without
compound (F0/F = 1 + KaꢁC). So the experimental data was analyzed
of binding constants (Ka = 1/Kd).
4.2.28. 4-(3-(3-Chloro-4-methylphenyl)thioureido)-N-(thiazol-
2-yl)benzenesulfonamide (D25)
Compound D25 was prepared in the same way as D1, white so-
lid, mp 190–192 °C, yield = 76.4%. 1H NMR (300 MHz, DMSO-d6) d
2.30 (s, 3H), 6.82 (d, J = 4.8 Hz, 1H), 7.27 (d, J = 4.8 Hz, 1H), 7.38–
7.66 (dd, 4H), 7.73–7.76 (dd, 4H), 10.04–10.15 (d, 2H), 12.70 (s,
1H); ESI-MS: m/z = 439.0 (M+H+). RP-HPLC: tR = 7.2 min (10% water
in menthol), purity > 95%.
4.3.4. CypA PPIase inhibitory assay
Suc-AAPF-pNA was dissolved in THF that contained LiCl
(480 mM) to a concentration of 3 mM. Then,
dissolved in 1 mM HCl to a concentration of 45 mg/ml. The assay
buffer (93 l of 50 mM HEPES, 100 mM NaCl; pH 8.0 at 0 °C) and
CypA (2 l of 6 M stock solution) were pre-equilibrated for 3 h
on ice. Immediately before the assay was started, 2 l of -chymo-
trypsin solution and 2 l of peptide substrate were added to the as-
a-chymotrypsin was
l
4.2.29. 4-(3-(4-Chlorophenyl)thioureido)-N-(thiazol-2-
yl)benzenesulfonamide (D26)
l
l
l
a
Compound D26 was prepared in the same way as D1, white
solid, mp 199–200 °C, yield = 67.3%. 1H NMR (300 MHz, DMSO-
d6) d 6.82 (d, J = 4.8 Hz, 1H), 7.25 (d, J = 4.8 Hz, 1H), 7.38–7.53
(dd, 4H), 7.628–7.758 (d, 4H), 10.09–10.15 (d, 2H), 12.70 (s, 1H);
ESI-HRMS: m/z = 424.99620 (M+H+, calcd for C16H13N4O2S1Cl1,
424.98892). RP-HPLC: tR = 6.4 min (10% water in menthol),
purity > 95%.
l
say mixture. After a delay from the onset of mixing (usually 6 s),
absorbance value at the wavelength of 390 nm had been recorded
on an Agilent 8453 spectrophotometer for 20 s. The progress
curves were analyzed by fitting the data to the integrated first-or-
der rate equation through nonlinear least-square analysis to eval-
uate the rate constants for the cis–trans conversion and the
respective IC50 was calculated with ORIGINPRO 7.5 software.
4.2.30. N-(Thiazol-2-yl)-4-(3-p-
tolylthioureido)benzenesulfonamide (D27)
4.3.5. HIV-1 protease inhibitory assay
Compound D27 was prepared in the same way as D1, white so-
lid, mp 196–197 °C, yield = 81.2%. 1H NMR (300 MHz, DMSO-d6) d
2.28 (s, 3H), 6.82–6.83 (d, 1H), 7.13–7.16 (d, 2H), 7.24–7.26 (d,
2H), 7.31–7.34 (d, 2H), 7.63–7.74 (dd, 4H), 9.94–9.98 (d, 2H),
12.70 (s, 1H) ESI-MS: m/z = 405.1 (M+H+). RP-HPLC: tR = 6.2 min
(10% water in menthol), purity = 95%.
We tested the inhibition data for HIV-1 protease by ultraviolet
spectrophotometry assay at 280 nm on a WFZ800-D2 spectropho-
tometer. The inhibitors (10
lM) were dissolved in 4.0
ll DMSO and
incubated with HIV-1 protease (5
l
M) in 200 l assay buffer (0.1 M
l
MES, 0.2 M NaCl, 5 mM EDTA, pH 5.5) at room temperature for
20 min. Then protease substrate (Lys-Ala-Arg-Val-Leu-Phe(NO2)-
Glu-Ala-Met, 100 lM) was added to initiate the reaction. The
4.3. Biological evaluation
hydrolysis of the substrate was recorded as a linear decrease in
UV absorbance at 280 nm over a 40-min time period. Inhibition ra-
tio was determined from the Ai/A0, where Ai and A0 were the
absorption at 280 nM in the presence and absence of the inhibitor.
4.3.1. Antiviral evaluation
Inhibition of SIV-induced syncytium in CEM174 cell cultures
was measured in a 96-well microplate containing 2 ꢀ 105 CEM
cells/ml infected with 100 TCID50 of SIV per well and containing
appropriate dilutions of the tested compounds. After 5 days of
incubation at 37 °C in 5% CO2 containing humidified air, CEM giant
(syncytium) cell formation was examined microscopically. The
EC50 was defined as the compound concentration required to pro-
tect cells against the cytopathogenicity of SIV by 50%. AZT was
Indinavir (10 lM) was used as positive control with final absorp-
tion decreased more than 90%.20 Both D5 and D23 showed no
inhibition of protease for their final absorptions decreased less
than 5%.
4.3.6. HIV-1 integrase inhibitory assay
The recombinant HIV-1 integrase was purified from Escherichia
coli. The oligonucleotides (d1, 50-ACC CTT TTA GTC AGT GTG GAA
AAT CTC TAC CAG T-30, d2, 50-ACT GGT AGA GAT TTT CCA CAC
TGA CTA AAA G-30, t1, 50-TGA CCA AGG GCT AAT TCA CT-30 and
t2, 50-AGT GAA TTA GCC CTT GGT CA-30) were purchased from
SBS Genetech Co., Ltd. The oligonucleotide d1 was synthesized to
include a 50-phosphorylated three base overhangs on the 50 end
of the strand and the oligonucleotides t1 and t2 were biotinylated.
The oligonucleotides d1 and d2 as well as t1 and t2 were mixed
and boiled for 3 min. Then the mixture was cooled slowly and pro-
vided the donor substrate and the target substrate, respectively.
The enzyme-linked immunosorbent assay was performed to detect
the activity of integrase. The donor oligonucleotides were immobi-
lized onto Covalink polystyrene microtiter plate (NUNC, Naperville,
IL) under the catalysis of 0.05 M EDC (1-ethyl-3-(3-dimethyl-
used as the positive control at a concentration of 10
lM here. Its
EC50 is 0.0122 M and TC50 is above 100 M in this system.
l
l
4.3.2. Assembly inhibitory assay
Ultraviolet spectrophotometry assay was performed at 350 nm
on a WFZ800-D2 spectrophotometer. A 1.2 l of concentrated li-
gand in DMSO (10 mM) was added to a 450 l aqueous solution
(2 ml of 5 M NaCl mixed with 1 ml of 200 mM NaH2PO4, pH 8.0),
and 150 l capsid protein (80 M) was added to initiate the reac-
l
l
l
l
tion. Spectral measurements were made every 10 s, following a
short initial delay to allow sample equilibration. Relative assembly
rates were estimated from initial slopes of the plots of absorbance
versus time.
4.3.3. CypA/inhibitor binding affinity assay
amino-propyl)-carbodiimide)
in
100
ll
1-methlyimidazole
Fluorescence measurements were performed on a SHIMADZU
RF-5301PC fluorescence spectrophotometer. The experiments
were carried out at room temperature in 20 mM Tris–HCl,
100 mM NaCl (pH 7.4) with the protein concentration set at
(10 mM pH 7) incubated at 50 °C for 5 h. Then the plates were
washed with PBS buffer three times and stored with blocking buf-
fer (PBS with 1% BSA and 0.2% sodium azide) at 4 °C over night. The
coated plates were washed with PBS buffer three times and was
6
l
M and the compound concentrations varied from 0 to 64
l
M.
added with 100
NaCl 25 mM, MgCl2 7.5 mM, b-mercaptoethanol 5 mM and BSA
50 g/ml) containing the purified integrase with a final concentra-
tion of 0.05 mM. After incubation at 37 °C for 30 min, the target oli-
gonucleotides (t1 and t2 mixture) were added and incubated for
ll TM buffer per well (Tris–HCl 20 mM, pH 7.8,
The compounds were prepared in dimethylsulfoxide (DMSO) as a
stock solution of 10 mM. The excitation wavelength was set at
280 nm, the emission range was from 300 nm to 380 nm, and the
slit was set at 5 nm (EX) and 3 nm (EM). Fluorescence readings
l

3188
J. Li et al. / Bioorg. Med. Chem. 17 (2009) 3177–3188
1 h at 37 °C. After three washes with PBS containing 0.05% Tween
20 and two washes with PBS, the plates were blocked with 1%
BSA at room temperature for 30 min. Then the integration products
were detected and quantified using a pNPP Phosphatase Assay Kit
(Biochain). The absorption at 405 nm was detected on Bio-Rad
Model 550. For the inhibition assay, the integrase was incubated
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The initial structures of our compounds were subjected to min-
imization using MOPAC in Chemoffice 2005 and the 3D structure of
HIV-1 Capsid in complex with its inhibitor CAP-1 was recovered
from the Protein Database (http://www.rcsb.org) with the code
as 2JPR.10 Another 3D structure of CypA in complex with CsA
was downloaded from Protein Database with the code 1CWA.28
The advanced docking program AUTO-DOCK 4.0 was used to remove
the small molecule and perform the automatic molecular docking
with our compounds. The number of generations, energy evalua-
tion, and docking runs were set to 370,000, 1,500,000, and 50,
respectively, and the kinds of atomic charges were taken as Koll-
man-all-atom for macromolecular and Gasteiger-Hücel for the
compounds. Molecular graphics images were produced using the
UCSF Chimera package from the Resource for Biocomputing, Visu-
alization, and Informatics at the University of California, San Fran-
cisco (supported by NIH P41 RR-01081).
Acknowledgments
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This project was supported by the National Natural Science
Foundation of China (No. 30670415). We thank Professor Fangqiu
Li for her help in this work.