Structure-activity relationships (SAR) research of thiourea derivatives as dual inhibitors targeting both HIV-1 capsid and human cyclophilin A

Article abstract of DOI:10.1111/j.1747-0285.2010.00981.x

HIV-1 capsid (CA) and human cyclophilin A (CypA) play important roles in HIV-1 assembly and disassembly processes, which are critical in HIV-1 replication. Based on the discovery of thiourea derivatives targeting both of the two proteins and indicating effective inhibitory activities in our group, we designed and synthesized a new class of thiourea derivatives. Their abilities to bind to capsid and cyclophilin A were determined by ultraviolet spectroscopic analysis, fluorescence binding affinity, and PPIase inhibition assay. Furthermore, the newly synthesized compounds were tested for their antiviral activities and cytotoxicities using CEM cells. According to the biological evaluation and subsequent molecular docking analyses, we studied the structure-activity relationships of thiourea derivatives. Three optimal compounds (K17, K24, K25) based on the achieved structure-activity relationships would be the basis for future optimization.

Full text of DOI:10.1111/j.1747-0285.2010.00981.x

ª 2010 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2010.00981.x
Chem Biol Drug Des 2010; 76: 25–33
Research Article
Structure–Activity Relationships (SAR)
Research of Thiourea Derivatives as Dual
Inhibitors Targeting both HIV-1 Capsid and
Human Cyclophilin A
Kan Chen1, Zhiwu Tan1, Meizi He1, Jiebo
Li1, Shixing Tang2, Indira Hewlett2, Fei
Yu1, Yinxue Jin1 and Ming Yang1*
viral agents with different targets for inhibition of HIV-1
replications. One of the most promising targets is HIV-1 capsid (CA)
protein, which is critical for the assembly and disassembly stage of
HIV-1 replication (4).
1The State Key Laboratory of Natural and Biomimetic Drugs, Peking
University, Beijing 100191, China
Recently, several inhibitors that bind to HIV-1 CA and inhibit assem-
bly have been reported (5–8). One of the assembly inhibitors, CAP-1
(Figure 1A), has been identified to bind to the CA N-terminal
domain (NTD) (9). Kelly and co-workers reported that a deep hydro-
phobic cavity was opened to CAP-1 because of the interaction
between the small molecule inhibitors and CA NTD. However, joint
NMR ⁄ X-ray structure showed that the buried portion of CAP-1
occupied only 73% of the cavity. Although CAP-1 exhibits antiviral
activity, its affinity for CA NTD is significantly below the levels
needed for therapeutic use (10).
2Center for Biologics Evaluation and Research, Food and Drug
Administration, Bethesda, MD 20892, USA
*Corresponding author: Ming Yang, yangm@bjmu.edu.cn
HIV-1 capsid (CA) and human cyclophilin A (CypA)
play important roles in HIV-1 assembly and disas-
sembly processes, which are critical in HIV-1 repli-
cation. Based on the discovery of thiourea
derivatives targeting both of the two proteins and
indicating effective inhibitory activities in our
group, we designed and synthesized a new class
of thiourea derivatives. Their abilities to bind to
capsid and cyclophilin A were determined by ultra-
violet spectroscopic analysis, fluorescence binding
affinity, and PPIase inhibition assay. Furthermore,
the newly synthesized compounds were tested for
their antiviral activities and cytotoxicities using
CEM cells. According to the biological evaluation
and subsequent molecular docking analyses, we
studied the structure–activity relationships of
thiourea derivatives. Three optimal compounds
(K17, K24, K25) based on the achieved structure–
activity relationships would be the basis for future
optimization.
Recent studies have also found that human cyclophilin A (CypA)
could facilitate the disassembly of HIV-1 CA complex. Compounds
bearing sulfanilamide (Figure 1B) bind to the 'saddle' pockets of
CypA (pocket A and B) can inhibit its activity catalyzing the cis ⁄
trans isomerization of the Gly89–Pro90 peptide bond, which plays
an important role in disassembly stage (11–13).
Based on the researches mentioned above, Jiebo Li et al. (14) in
our group designed a series of thiourea derivatives as dual inhibi-
tors targeting both HIV-1 CA and CypA. Its features were summa-
rized as follows (Figure 2): (i) It contained a substituted phenyl that
inserted into the deep hydrophobic cavity of CA NTD, as well as
Key words: capsid, HIV-1, human cyclophilin A, SAR, thiourea
A
derivatives
Received 28 December 2009, revised 25 March 2010 and accepted for
publication 28 March 2010
B
Since the 1980s, acquired immune deficiency syndrome (AIDS) has
become a major worldwide epidemic, which greatly threatens the
health of humankind (1). The currently used anti-HIV-1 drugs mainly
target the envelope protein, reverse transcriptase (RT), integrase
(IN), and protease (PR) to block the replication of HIV-1 (2). How-
ever, emergence of drug-resistant viruses caused by mutation of the
above proteins often results in treatment failure with existing anti-
retrovirals (3). Thus, efforts were made to develop new antiretro-
Figure 1: (A): Structure of CAP-1 (above). (B) Structure of one
cyclophilin A inhibitor bearing sulfanilamide (below).
25

Chen et al.
the pocket A of CypA. (ii) It contained a big sulfanilamide segment
such as sulfamethoxazole, 4-methoxybenzene, and thiazole, which
bound to the pocket B of CypA. (iii) Thiourea group linked these
two segments and stabilized the interaction between ligand and
targeting protein. These features ensured that the compounds tar-
geted both HIV-1 CA and CypA. However, the structure–activity
relationship (SAR) of thiourea derivatives on HIV-1 CA and CypA is
unclear in the existing research.
Scheme 1B outlined the synthesis of alkyl 4-aminobenzoate, para
aminobenzoic acid was treated with corresponding alcohols in the
presence of excessive SOCl2 to afford alkyl 4-aminobenzoates (15),
which were then converted to isothiocyanates, and followed by
reacting with sulfamethoxazole to get compounds K20–K23 (the
same preparation as Scheme 1A).
Scheme 1C described the preparation of 6-aminophthalide, the
route included three steps. First, the preparation of isobenzofuran-
1(3H)-one (16): in the presence of NaBH4, isobenzofuran-1,3-dione
was reduced in THF (tetrahydrofuran). Then, we used potassium
nitrate in sulfuric acid to obtain the 6-nitrophthalide smoothly as
described in the literature (17). Finally, 6-nitrophthalide was purified
by recrystallization and reacted with stannous chloride in the pres-
ence of hydrochloric acid to get 6-aminophthalide, which was used
to synthesize compound K20 with the same preparation method
mentioned in Scheme 1A. All title compounds were recrystallized in
methanol.
To study the SAR and perform the modification more reasonably
and efficiently, we followed the existing model and chose sulfa-
methoxazole, which showed good inhibitory activity in biological
evaluation, as sulfanilamide segment. Our consequent researches
were divided into three steps: (i) Introducing various p -, o -, m -
substituents to investigate the influence of substitute places and
electric property effects on anti-assembly activity. (ii) Evaluating the
influences of steric effect by introducing large groups. (iii) Inserting
some methylenes between thiourea and substituted phenyl to pro-
long and ascertain optimal linker length. Ultimately, we designed
27 compounds (Table 1). Eighteen of the 27 compounds [the other
nine compounds have been reported elsewhere (14)] were synthe-
sized for the first time.
4-(3-(2-Chlorophenyl)thioureido)-N-(5-methyliso-
xazol-3-yl)benzenesulfonamide (K2)
White solid, mp 175–176 ꢀC, yield = 66.3%. 1H NMR (300 MHz,
DMSO-d6) d2.30 (s, 3H), 6.16 (s, 1H), 7.32 (d, J = 6.0Hz,2H), 7.53 (d,
J = 6.0Hz, 2H), 7.88 (s, 4H), 9.80 (s,1H), 10.36 (s, 1H), 11.42 (s, 1H);
ESI-MS: m ⁄ z = 420.9 (M-H+).
Methods and Materials
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 spectrometer
using the solvents described. Chemical shifts were reported in d
ppm (parts per million) relative to tetramethyl silane except for deu-
teriorated water (D2O), and the signals were quoted as s (singlet),
d (doublet), t (triplet), q (quartet), and m (multiplet). The mass spec-
tra (EI or ESI) were recorded. Melting points were determined on
an XA-4 instrument that was uncorrected.
4-(3-(2-Fluorophenyl)thioureido)-N-(5-methyliso-
xazol-3-yl)benzenesulfonamide (K3)
White solid, mp 174–175 ꢀC, yield = 64.9%. 1H NMR (300 MHz,
DMSO-d6) d2.30 (s, 3H), 6.15 (s, 1H), 7.20 (d, J = 5.4 Hz, 1H), 7.28
(dd, J = 6.6 Hz, 2H), 7.53 (d, J = 7.8 Hz, 1H), 7.58 (d, J = 6.0 Hz,
4H), 9.81 (s, 1H), 10.32 (s, 1H), 11.40 (s, 1H); ESI-MS: m ⁄ z = 404.9
(M-H+).
Chemical synthetic procedure
As shown in Scheme 1, the synthesis of the target compounds
required access to different isothiocyanates, which subsequently
reacted with sulfamethoxazole to afford the thiourea derivatives of
K1–K27.
4-(3-(2-Methoxyphenyl)thioureido)-N-(5-methyli-
soxazol-3-yl)benzenesulfonamide (K4)
White solid, mp 156–157 ꢀC, yield = 70.2%. 1H NMR (300 MHz,
DMSO-d6) d2.30 (s, 3H), 3.83 (s, 3H), 6.15 (s, 1H), 6.94 (dd,
J = 7.5 Hz, 1H), 7.06 ((d, J = 8.4 Hz, 1H), 7.20 (dd, J = 7.8Hz, 1H),
7.77 (d, J = 8.7 Hz, 4H), 7.82 (d, J = 8.4 Hz, 1H), 9.50 (s, 1H), 10.26
(s, 1H), 11.39 (s, 1H); ESI-MS: m ⁄ z = 417.0 (M-H+).
Scheme 1A outlined the preparation of isothiocyanates: various
amines (aromatic amines, substituted benzylamines, and phenethyl-
amines were included) were treated with CS2 in the presence of
TEDA (Triethylene diamine), followed by subsequent conversion to
isothiocyanates by reacting with BTC (Bis(trichloromethyl) carbo-
nate), the thiourea derivatives were obtained by reacting sulfa-
methoxazole with corresponding isothiocyanates.
4-(3-(3-Methoxyphenyl)thioureido)-N-(5-methyli-
soxazol-3-yl)benzenesulfonamide (K6)
White solid, mp 169–170 ꢀC, yield = 78.6%. 1H NMR (300 MHz,
DMSO-d6) d2.31 (s, 3H), 3.73 (s, 3H), 6.09(s, 1H), 6.72 (s, 1H), 7.00-
7.28 (m, 3H), 7.72(d, J = 8.4Hz, 2H), 7.77(d, J = 8.4Hz, 2H), 10.14
(s,1H), 10.16 (s, 1H), 11.40 (s, 1H); ESI-MS: m ⁄ z = 416.9(M-H+).
N-(5-Methylisoxazol-3-yl)-4-(3-m-tolylthioureido)
benzenesulfonamide (K7)
White solid, mp 168–170 ꢀC, yield = 81.5%. 1H NMR (300 MHz,
DMSO-d6) d2.29 (s, 3H), 2.31 (s, 3H), 6.17 (s, 1H), 6.97 (s, 1H), 7.25
Figure 2: Structure of thiourea derivatives.
26
Chem Biol Drug Des 2010; 76: 25–33

Structure–Activity Relationships (SAR) Research
Table 1: Chemical structure of
thiourea derivatives
O O
S
S
N
N
O
H
Ar n N
H
N
H
Compound
K1*
Ar
n
0
Compound
K15*
Ar
Cl
n
0
H3CO
H3C
K2
K3
0
0
K16*
K17
0
0
Cl
Cl
F
Cl
N
K4
K5*
K6
0
0
0
0
K18
K19
K20
K21
0
0
0
0
OCH3
CH3
O
O
H3CO
O
C
K7
H3CO
H3C
O
K8*
K9*
K10
0
0
0
K22
K23
K24
0
0
0
C 2H5O C
F
O
n
C 3H7O C
Cl
O
Br
n
C 4H9O C
K11
K12*
K13*
K14*
0
0
0
0
K25
K26
K27
1
1
2
NC
F
F
Cl
H3C
K1* Compounds with * has been reported in literature (14).
(dd, J = 5.7Hz,3H), 7.72 (d, J = 9.0Hz, 2H), 7.77 (d, J = 9.0Hz, 2H),
10.10 (s, 1H), 10.14 (s, 1H), 11.41 (s, 1H); ESI-MS: m ⁄ z = 401.0(M-H+).
J = 8.7 Hz, 2H), 7.71 (d, J = 8.7 Hz, 2H), 7.78 (d, J = 8.7Hz, 2H), 10.22
(s, 1H), 10.26 (s, 1H), 11.42 (s, 1H); ESI-MS: m ⁄ z = 466.8 (M+H+).
4-(3-(4-Bromophenyl)thioureido)-N-(5-methyliso-
xazol-3-yl)benzenesulfonamide (K10)
White solid, mp 190–192 ꢀC, yield = 86.9%. 1H NMR (300 MHz,
DMSO-d6) d2.31 (s, 3H), 6.10 (s, 1H), 7.44 (d, J = 8.7 Hz,2H), 7.52 (d,
4-(3-(4-Cyanophenyl)thioureido)-N-(5-methyliso-
xazol-3-yl)benzenesulfonamide (K11)
White solid, mp 190–191 ꢀC, yield = 64.6%. 1H NMR (300 MHz,
DMSO-d6) d2.31 (s, 3H), 6.17 (s, 1H), 7.73 (d, J = 7.8 Hz,2H), 7.75
Chem Biol Drug Des 2010; 76: 25–33
27

Chen et al.
A
a
Ar
NH2
Ar
NCS
n
n
H
N
H
N
Ar
b
n
H
N
NH2
S
S
H
O
N
O
O
N
O
S
O
N
K1–K19;K25–K27
O
n = 0;1;2
Ar = Substituted phenyl;
naphthyl;
pyridyl
B
a
b
HOOC
NH2
ROOC
NH2
ROOC
NCS
NH2
H
N
H
N
c
H
N
S
H
N
ROOC
S
O
O
N
O
S
K21–K24
O
O
N
O
R = 1;2;3;4
O
C
O
O
O
O
O2N
b
H2N
SCN
a
d
c
O
O
O
O
O
H
N
H
O
O
N
O
e
H
N
NH2
S
S
H
N
O
O
N
O
S
K20
O
O
N
O
Scheme 1: Synthesis of thiourea compounds. Reagents and condition: A(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, 24 h; B(a)SOCl2, reflux, 4 h; evaporate; saturated NaHCO3 was added until pH = 7–8; (b)(1)
1,4-diazabicyclo[2,2,2]-octane, CS2, rt, 12 h, (2) BTC, reflux, 2 h (for two steps); (c) C2H5OH, room temperature, 24 h; C(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, 24 h.
(d, J = 9.6 Hz,4H), 7.80 (d, J = 7.8Hz, 2H), 10.50 (s, 1H), 10.51 (s,
1H), 11.42 (s, 1H); ESI-MS: m ⁄ z = 411.9 (M-H+).
(m,1H), 7.79–7.93 (m, 4H), 7.83–7.98 (m, 4H), 10.27 (s, 1H), 10.38 (s,
1H), 11.42 (s, 1H); ESI-MS: m ⁄ z = 437.0 (M-H+).
4-(3-(3,4-Dichlorophenyl)thioureido)-N-(5-methy-
lisoxazol-3-yl)benzenesulfonamide (K17)
White solid, mp 181–182 ꢀC, yield = 78.4%. 1H NMR (300 MHz,
DMSO-d6) d2.31 (s, 3H), 6.16 (s, 1H), 7.43–7.47 (q, 1H), 7.59 (d,
J = 8.4 Hz, 1H), 7.70 (d, J = 9.0 Hz, 2H), 7.79 (d, J = 9.0 Hz, 2H),
7.86 (d, J = 8.4 Hz, 1H), 10.30 (s, 1H), 10.37 (s, 1H), 11.42 (s, 1H);
ESI-MS: m ⁄ z = 454.9 (M-H+).
N-(5-Methylisoxazol-3-yl)-4-(3-(3-oxo-1,3-dihydr-
oisobenzofuran-5-yl)thioureido)benzenesulfon-
amide (K20)
White solid, mp 175–176 ꢀC, yield = 80.3%. d2.29 (s, 3H), 5.40 (s,
2H), 6.15 (s, 1H), 7.39 (d, J = 9.0Hz,2H), 7.50 (d, J = 9.0 Hz, 2H),
7.68–7.83 (m, 3H), 10.41 (s,1H), 10.45 (s,1H), 11.42 (s, 1H); ESI-MS:
m ⁄ z = 443.0 (M-H+).
N-(5-Methylisoxazol-3-yl)-4-(3-pyridin-2-ylthiou-
reido)benzenesulfonamide (K18)
Methyl 3-(3-(4-(N-(5-methylisoxazol-3-yl)sulfam-
oyl)phenyl)thioureido)benzoate (K21)
Yellow solid, mp 208–210 ꢀC, yield = 56.3%. 1H NMR (300 MHz,
DMSO-d6) d2.31 (s, 3H), 6.17 (s, 1H), 7.13–7.17 (m, 1H), 7.26 (d,
J = 8.4Hz,1H), 7.84-7.87 (m, 3H), 8.03-8.06 (m, 2H), 8.33-8.35 (m,
1H), 11.12 (s,1H), 11.46 (s, 1H), 14.27 (s,1H); ESI-MS: m ⁄ z = 387.8
(M-H+).
White solid, mp 184–186 ꢀC, yield = 84.1%. 1H NMR (300 MHz,
DMSO-d6) d2.39 (s, 3H), 3.82 (s, 3H), 6.16 (s, 1H), 7.66 (d,
J = 8.4Hz,2H), 7.72 (d, J = 7.2Hz,2H), 7.79 (d, J = 7.2Hz,2H), 7.90 (d,
J = 8.4Hz, 2H), 10.40 (s, 1H), 10.44 (s, 1H), 11.40 (s, 1H); ESI-MS:
m ⁄ z = 445.0 (M-H+).
N-(5-Methylisoxazol-3-yl)-4-(3-naphthalen-2-ylth-
ioureido)benzenesulfonamide (K19)
White solid, mp 171–172 ꢀC, yield = 88.4%. 1H NMR (300 MHz,
DMSO-d6) d2.31 (s, 3H), 6.17 (s, 1H), 7.44-7.50 (m, 2H), 7.58-7.61
Ethyl 3-(3-(4-(N-(5-methylisoxazol-3-yl)sulfamoyl)
phenyl)thioureido)benzoate (K22)
White solid, mp 169–170 ꢀC, yield = 82.5%. 1H NMR (300 MHz,
DMSO-d6) d2.76 (t, 3H), 2.85 (s, 3H), 4.25 (q, 2H), 6.15 (s, 1H), 7.65
28
Chem Biol Drug Des 2010; 76: 25–33

Structure–Activity Relationships (SAR) Research
(d, J = 6.9Hz,2H), 7.71 (d, J = 8.7Hz,2H), 7.78 (d, J = 8.7Hz,2H), 7.91
(d, J = 6.9Hz, 2H), 10.42 (s, 1H), 10.46 (s, 1H), 11.43 (s, 1H); ESI-
MS: m ⁄ z = 458.9 (M-H+).
protein (80 lM) to equal the final concentration of CA and ligand.
Then, the reaction was initiated and spectral measurements were
made every 5 seconds, following a short initial delay to allow sam-
ple equilibration. Kinetic analysis of assembly reactions were esti-
mated from initial slopes of the plots of absorbance versus time.
n-Propyl 3-(3-(4-(N-(5-methylisoxazol-3-yl)sulfa-
moyl)phenyl)thioureido)benzoate (K23)
White solid, mp 178–179 ꢀC, yield = 79.9%. 1H NMR (300 MHz,
DMSO-d6) d0.94 (t, 3H), 1.68 (m, 2H), 2.31 (s, 3H), 4.19 (t, 2H), 6.17
(s, 1H), 7.66 (d, J = 8.7Hz,2H), 7.73 (d, J = 9.0Hz,2H), 7.79 (d,
J = 9.0Hz, 2H), 7.92 (d, J = 8.7Hz, 2H), 10.41 (s, 1H), 10.46 (s, 1H),
11.43 (s, 1H); ESI-MS: m ⁄ z = 472.9 (M-H+).
CypA ⁄ inhibitor binding affinity assay
We used the fluorescence measurement to evaluate the
CypA ⁄ inhibitor binding affinity (19). This assay was performed on a
SHIMADZU RF-5301PC fluorescence spectrophotometer (Shimadzu
corporation, Tokyo, Japan). The experiments were carried out at
room temperature. The reaction mixture contained 20 mM Tris–HCl,
100 mM NaCl (pH 7.4), 6 lM of CypA, and 0–64 lM of the
compounds. The compounds were prepared in DMSO as a stock
solution of 10 mM. The excitation wavelength was set at 280 nm.
The emission range was from 300 to 380 nm; and the slit was set
at 5 nm (EX) and 3 nm (EM), respectively. Fluorescence readings
were taken at the wavelength of maximum emission (340 nm). The
fluorescence intensity versus the final compound concentration was
integrated to the following equation, where F0 stands for the initial
fluorescence intensity of the protein solution without compound and
F is the fluorescence intensity when added Q (M) compound.
(1 ⁄ F0)F = 1 ⁄ F0)KD ⁄ F0ÆQ). So the experimental data were analyzed
of binding constants (Ka = 1 ⁄ KD).
n-Butyl 3-(3-(4-(N-(5-methylisoxazol-3-yl)sulfam-
oyl)phenyl)thioureido)benzoate (K24)
White solid, mp 175–176 ꢀC, yield = 76.3%. 1H NMR (300 MHz,
DMSO-d6) d0.91(t, 3H), 1.33-1.48(m, 2H), 1.64-1.73(m, 2H), 2.38 (s,
3H), 4.24 (q, 2H), 6.17 (s, 1H), 7.55 (d, J = 8.4Hz,2H), 7.73 (d,
J = 11.7Hz,2H), 7.80 (d, J = 11.7Hz,2H), 7.99 (d, J = 8.4Hz, 2H),
10.41 (s, 1H), 10.46 (s, 1H), 11.43 (s, 1H); ESI-MS: m ⁄ z = 486.9 (M-
H+).
4-(3-Benzylthioureido)-N-(5-methylisoxazol-3-yl)
benzenesulfonamide (K25)
White solid, mp 185–186 ꢀC, yield = 82.0%. 1H NMR (300 MHz,
DMSO-d6) d2.98 (s, 3H), 4.73 (d, J = 5.4Hz,2H), 6.15 (s, 1H), 7.25-
7.35 (m, 5H), 7.76 (s, 4H), 8.55(s,1H), 10.02(s,1H), 11.38 (s, 1H); ESI-
MS: m ⁄ z = 401.0 (M-H+).
CypA PPIase inhibition activity assay
Inhibition of the PPIase activity of CypA by the compounds was
determined by a published method with modifications (20). Suc-
AAPF-pNA was dissolved in THF that contained 480 mM of LiCl. The
final concentration was 3 mM. Then, a-chymotrypsin was dissolved
in 1 mM HCl to a concentration of 45 mg ⁄ mL. The assay buffer
(93 lL of 50 mM HEPES, 100 mM NaCl; pH 8.0 at 0 ꢀC) and CypA
(2 lL of 6 lM stock solution) were pre-equilibrated for 3 h on ice.
Right before the assay, 2 lL of a-chymotrypsin solution and 2 lL
of peptide substrate were added to the assay mixture. After a delay
(usually 6 seconds), the absorbance at the wavelength of 390 nm
was recorded on an Agilent 8453 spectrophotometer (Agilent) for
20 seconds. The progress curves were analyzed by fitting the data
to the integrated first-order rate equation through non-linear least-
square analysis to evaluate the rate constants for the cis–trans
conversion. The respective IC50 was calculated with OriginLab
Corporation, Northampton, MA, USA.
4-(3-(4-Fluorobenzyl)thioureido)-N-(5-methyliso-
xazol-3-yl)benzenesulfonamide (K26)
White solid, mp 172–173 ꢀC, yield = 77.7%. 1H NMR (300 MHz,
DMSO-d6) d2.29 (s, 3H), 4.70 (d, J = 4.2Hz,2H), 6.13 (s, 1H), 7.23-
7.35 (m, 5H), 7.62 (d, J = 9.0Hz,2H), 7.72 (d, J = 9.0Hz,2H),
8.16(s,1H), 9.94(s,1H),11.35 (s, 1H); ESI-MS: m ⁄ z = 419.0 (M-H+).
N-(5-Methylisoxazol-3-yl)-4-(3-phenethylthioure-
ido)benzenesulfonamide (K27)
White solid, mp 168–169 ꢀC, yield = 70.4%. 1H NMR (300 MHz,
DMSO-d6) d2.34 (s, 3H), 2.88 (dd, J = 7.2Hz, 2H), 3.70 (s, 2H), 6.15
(s, 1H), 7.25-7.35 (m, 5H), 7.62 (d, J = 9.0Hz,2H), 7.72 (d,
J = 9.0Hz,2H), 8.55(s,1H), 10.02(s,1H), 11.38 (s, 1H); ESI-MS:
m ⁄ z = 416.9 (M+H+).
In vitro cytotoxicity assay
The in vitro cytotoxicity of CEM (Combined eosinophil mast) cells
for each compound was determined in 96-well plates using 3-(4, 5)-
dimethylthiahiazo (-z-y1)-3, 5-di-phenyltetrazoliumbromide dye to
measure cell viability. Indinavir (IDV) was added to the wells corre-
sponding to positive controls (P ), and medium was added to wells
corresponding to negative controls (N ). The cytotoxicity was calcu-
lated using the following formula: % cytotoxicity = [(P ) N ) )
(E ) N )] ⁄ (P ) N ) · 100, where E represents experimental data in
the presence of a compound. The concentration corresponding
to 50% cytotoxicity (TC50) for CEM cells was calculated using
ORIGINPRO 7.5 software.
Assembly inhibitory assay
HIV-1 CA could assemble into tubes, which leads to increase in
sample turbidity that can be measured spectrophotometrically. This
assay was used to probe for potential inhibitory effects of these
thiourea compounds on in vitro CA assembly (9,18). The assay was
performed at 350 nm on an Agilent 8453 spectrophotometer (Santa
Clara, CA, USA). A 1.2 lL of concentrated ligand in DMSO (10 mM)
was added to a 450 lL aqueous solution (2 mL of 5 M NaCl mixed
with 1 mL of 200 mM NaH2PO4, pH8.0), and then added 150 lL CA
Chem Biol Drug Des 2010; 76: 25–33
29

Chen et al.
Antiviral evaluation
Table 2: Assembly inhibition data of CAP-1 and thiourea deriva-
tives
Inhibition of SIV (Simian Immunodeficiency Virus)-induced syncytium
in CEM174 cell cultures was measured in a 96-well microplate con-
taining 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 microscopi-
cally. The EC50 was defined as the compound concentration required
to protect cells against the cytopathogenicity of SIV by 50%. Experi-
mental values were determined from two assays in duplicate.
Compound
Assembly ratea (mOD ⁄ minb)
Nonec
CAP-1
K1
60.6
31.4
23.3
26.6
37.2
30.1
38.6
13.6
7.7
K2
K3
K4
K5
K6
K7
Molecular modeling
K8
K9
45.4
9.4
The initial structures of our compounds were subjected to minimiza-
tion using MOPAC in Chemoffice 2006, and the 3D structure of
HIV-1 CA 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 (21). The
advanced docking program (The Scripps Research Institute, La Jolla,
CA, USA) (22) was used to remove the small molecule and perform
the automatic molecular docking with our compounds. The number
of generations, energy evaluation, and docking runs were set to
370 000, 1 500 000, and 50, respectively, and the kinds of atomic
charges were taken as Kollman-all-atom for macromolecular and
Gasteiger-Hꢀcel for the compounds. Molecular graphics images were
produced using the UCSF Chimera package from the Resource for
Biocomputing, Visualization, and Informatics at the University of
California, San Francisco.
K10
K11
K12
K13
K14
K15
K16
K17
K18
K19
K20
K21
K22
K23
K24
K25
K26
K27
15.7
20.3
43.9
17.3
27.3
23.8
21.7
5.7
26.4
20.8
29.4
42.0
11.7
10.6
6.0
6.3
20.5
12.8
aEach value was determined from two experiments in duplicate.
bmOD, millioptical density units.
Results and Discussion
cNone, no compound.
Assembly inhibitory activities
We primarily focused on the assembly inhibitory activities of thio-
urea derivatives. Spectrophotometrical method could measure the
sample turbidity variation that was caused by the CA assemble pro-
cession. In our study, it was used to probe for potential inhibitory
effects of the thiourea compounds on in vitro CA assembly. As
shown in Table 2, dissolution of native HIV-1 into assembly buffer
led to an increase in absorbance at an initial rate of 60.6 milliopti-
cal density units ⁄ min (mOD ⁄ min), while in the presence of CAP-1,
the initial assembly rate decreased to 31.4 mOD ⁄ min.
According to the assay results above and interrelated SAR, we
designed and synthesized compound K17, which kept ortho posi-
tion unsubstituted and introduced chlorine in both meta and para
position. Compared with the optimal compound (K16) reported by
Li (14), its assembly inhibitory activity had a remarkable improve-
ment. The assembly rate of CA in the presence of K17 decreased
to a minimum value of 5.7 mOD ⁄ min. The result further demon-
strated the rationality of achieved SAR.
Among the title compounds with electron-withdrawing groups in
para-position, the ranking of activities was: bromide > chlo-
ride > fluoride. The assembly inhibitory activities increased posi-
tively with their increasing sizes. Based on the report that buried
portion of CAP-1 occupied only 73% of the cavity of CA (10), we
predicted that the introduction of large substituent in para position
could fill the active cavity better and result in remarkable improve-
ment in the inhibitory potency of CA assembly. Two groups of com-
pounds were designed to verify this assumption. In the first group,
we introduced some rigid groups such as naphthalene, pyridine, and
6-methylisobenzofuran-1(3H)-one to replace benzene (K18–K20).
However, no significant change was observed in CA assembly rate.
Larger rigid groups could not adapt to the cavity pattern and caused
certain steric hindrance, which might be an understandable reason
We firstly evaluated the influence of o-substituents (K2–K5): com-
pared with unsubstituted compound (K1, assembly rate = 23.3 -
mOD ⁄ min), the synthesized substituted compounds lost their
assembly inhibitory activities of various degree and the assembly
rate of CA distributed in a range of 26.6–38.6 mOD ⁄ min. We sus-
pected that the substituents in ortho-position could result in certain
steric hindrance, which prevented the phenyl group from inserting
into the binding cavity. For meta-substituted derivatives, chloride
(K9, assembly rate = 9.4 mOD ⁄ min) and methide (K7, assembly
rate = 7.7 mOD ⁄ min) led to a significant decrease of the CA assem-
bly rate compared with K1. For para-substituted derivatives, elec-
tron-withdrawing groups (bromide, chloride, prussiate) showed
better assembly inhibitory activities than electron-donating groups
(methide, methoxide).
30
Chem Biol Drug Des 2010; 76: 25–33

Structure–Activity Relationships (SAR) Research
for the result. In the second group, a series of flexible groups were
introduced (K21–K24). Along with the extension of carbon chain,
assembly inhibitory activities increased gradually. For example,
compared with the influences of K21, the assembly rates in the
presence of K23 and K24 decreased to 10.6 and 6.0 mOD ⁄ min,
respectively. Experimental results confirmed that incorporation of
large flexible group could improve assembly inhibitory activity
effectively, as we expected.
Table 3: Equilibrium affinity constants and in vitro IC50 values
for interaction of chosen thiourea derivatives with cyclophilin A
b
Compound
Kda (lM)
PPIase activity IC50 (lM)
K1
88.1
109.5
55.80
14.31
12.48
9.00
9.07
9.45
8.24
32.73
39.08
0.58
0.35
0.65
0.50
NAc
0.78
NA
K12
K16
K17
K19
K21
K22
K23
K24
K25
K26
Ultimately, we evaluated the influence of the length of linker on
the inhibitory activity of CA assembly by introducing 1 or 2 methyl-
enes between phenyl and thioureido. Compared with the influence
of K1, the assembly rates decreased to 6.3 and 12.8 mOD ⁄ min in
the presence of K25 (1 methylene) and K27 (2 methylenes),
respectively. In addition, compound K26 (assembly rate = 20.5 -
mOD ⁄ min) showed significant improvement when contrasted with
non-extended compound K12 (assembly rate = 43.9 mOD ⁄ min).
These results indicated that it was optimal to increase one methy-
lene between phenyl and thiourea.
NA
0.78
0.58
0.29
aKd values were determined from two intrinsic tryptophan fluorescence titra-
tion assays in duplicate.
bPPIase IC50 values were determined from two enzyme inhibition assays in
duplicate.
cNA, no activity.
By investigating the influence of substitute places, electric property
effects, steric effect, and linker length, we obtained the SAR of
thiourea derivatives on assembly inhibitory activities. Three optimal
compounds (K17, K24, and K25) based on the SAR showed most
favorable CA assembly inhibitory activities among all the designed
compounds.
Antiviral activities
The antiviral activities of the newly synthesized compounds were also
evaluated by SIV-induced syncytium in CEM cells. Their EC50, TC50,
and SI values were listed in Table 4. Experimental results showed
that the newly synthesized compounds exhibited antiviral activities
with a range of EC50 values from 1.2 to 13.6 lM and possessed TC50
values more than 100 lM, which indicated that all compounds pos-
sessed effective anti-SIV activities and low cytotoxicities. Further-
more, compound K24 (EC50 = 1.2 lM) that exhibited good assembly
inhibitory activity also showed the best antiviral activity among all
PPIase inhibitory activities
As mentioned in the introduction, the compound that bound to
CypA and inhibited its PPIase activity could be a potent inhibitor
of HIV-1 replication. We subsequently investigated the binding
affinity of the chosen thiourea compounds against CypA by using
the intrinsic fluorescence titration technique. Then, the IC50 val-
ues for inhibition of PPIase activities of the compounds were
determined by a standard spectrophotometric method reported in
literature (20). The experiment data (As shown in Table 3)
showed that all of the tested molecules could bind to CypA with
binding affinities (Kd values) from 8.24 to 109.5 lM, of which,
most compounds exhibited potent PPIase inhibitory activities
(IC50 < 1 lM).
Table 4: Antiviral activity and toxicity of thiourea derivatives on
SIV-induced syncytium
Compounda
EC50 (lM)
TC50 (lM)
TId
b
c
K2
K3
8.0
8.9
6.4
4.0
2.1
3.9
4.1
4.4
8.0
13.6
8.7
1.7
5.0
4.5
1.2
7.8
9.9
4.0
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>12
>1
K4
K6
>15
>25
>47
>25
>24
>22
>12
>7
>11
>58
>20
>22
>83
>12
>10
>25
K7
K10
K11
K17
K18
K19
K20
K21
K22
K23
K24
K25
K26
K27
Experimental results also showed that the incorporation of large
flexible group in para-position (K21–K24) could significantly affect
the inhibitory effect with Kd values of 8.24–9.45 lM, which repre-
sented the best binding affinities among all tested compounds. In
addition, the introduction of one methylene between phenyl and
thioureido could obviously increase the binding affinities, as well as
the inhibitory activities. For example, compounds K25
(Kd = 32.73 lM, IC50 = 0.58 lM) and K26 (Kd = 39.08 lM,
IC50 = 0.29 lM) displayed stronger binding affinities and better
inhibitory activities, compared with corresponding non-extended
compounds. Besides, optimal compound K17 (Kd = 14.31 lM,
IC50 = 0.50 lM) also showed good activity in this respect. Some
consistencies of activities between PPIase inhibition and assembly
inhibition were found, which suggested that these structural
changes could also influence the interaction between ligand and
active pocket of CypA.
aIndinavir was used as the positive control at a concentration of 1 · 10)6
here. Its EC50 was 0.0108 lM and TC50 was above 100 lM in this system.
M
bEC50, concentration was required to protect cells against the cytopathoge-
nicity of SIV by 50%.
cTC50, concentration was required to inhibit uninfected cells proliferation by 50%.
dTherapeutic index, TC50 value divided by EC50 value (TC50 ⁄ EC50).
Chem Biol Drug Des 2010; 76: 25–33
31

Chen et al.
A
Figure 3: The docking model:
(A) The left figure showed stereo
view of the complex of K17 with
HIV-1 capsid, the right figure
highlighted its essential
B
interactions: hydrogen bonds
(indicated by gray line), close
contacts (indicated by wireframe
model). (B) Stereo view and
essential interactions of the
complex of K24 with
cyclophilin A.
tested compounds, suggesting that there were definite relations
between assembly inhibitory activities and antiviral activities.
stabilizing the binding. Furthermore, the oxygens of 5-methylisoxa-
zole and sulfanilamide could form two hydrogen bonds with two
guanidyl hydrogen atoms of Arg55 respectively, which further
enhanced the interaction between the sulfamethoxazole group and
the active pocket. Meanwhile, the 4-(butoxycarbonyl) phenyl could
fully occupy the hydrophobic pocket A of CypA, which was com-
posed of residues Gly72, Gly75, Lys82, Gly109, Ser110, and Gln111.
Two oxygens of ester group could form three hydrogen bonds with
hydrogen atoms of Gly109, Ser110, and Gln111, respectively. In
addition, Van der Waals force and hydrophobic interactions
between 4-(butoxycarbonyl) phenyl and the above residues were
observed, as shown in Figure 3B. In summary, the 4-(butoxycarbonyl)
phenyl could occupy more suitable place of pocket A and form extra
hydrogen bonds with nearby residues. All of these could explain its
high binding affinity against CypA theoretically.
Docking analysis
To gain more insight into the acquired SAR, we performed molecu-
lar modeling experiments using Autodock 4.0 (22). As shown in Fig-
ure 3A, the docking conformation of K17 represented one of the
most common binding modes between thiourea derivatives and HIV-
1 CA. The 3,4-dichlorophenyl group inserted into the deep hydro-
phobic pocket vacated by Phe32 while the thioureido linker
extended outside the pocket. The binding between 3,4-dichlorophe-
nyl group of K17 and CA active pocket was dominated by Van der
Waals force, electrostatic, and hydrophobic interactions. Compared
with compound K16, K17 showed lower binding free energy and
the chlorine in para-position could contribute to extra Van der Wa-
als force with residue Ile141, which might explain its better assem-
bly inhibitory activity. Furthermore, the binding model of K21–K24
indicated that the large flexible group in para-position could facili-
tate to occupy the whole pocket and lead to stronger hydrophobic
interactions. Both the docking analysis date and the assembly inhi-
bition experimental results ascertained our assumptions and corre-
sponding SAR. Simultaneously, we analyzed the influence of
sulfamethoxazole group on binding model, the sulfamethoxazole
group outside the pocket could form a series of hydrophobic inter-
actions with nearby residues such as Asn57, Thr58, Val59, and
Gly60. In addition, the oxygen of 5-methylisoxazole could form a
hydrogen bond with the backbone hydrogen of Gly61. All of these
reinforced the binding affinity and could explain the high assembly
inhibitory activities of compounds with sulfamethoxazole group.
Conclusions and Future Directions
In this work, a class of thiourea derivatives was synthesized and
assessed for their abilities to bind to CA and CypA as well as PPI-
ase inhibitory activities. The biological experiment results confirmed
that all compounds could target both HIV-1 CA and CypA while
most of them exhibited potent PPIase inhibitory activities
(IC50 < 1 lM). In addition, we studied the SAR of thiourea deriva-
tives by investigating the influence of substituted places, electronic
effects, steric effect, and linker length. Three optimal compounds
(K17, K24, K25) based on the achieved SAR showed most favor-
able CA assembly inhibitory activities among all the compounds.
Furthermore, molecular docking was used to analyze and explain
the achieved SAR. These studies on SAR of thiourea derivatives
would provide an insight into further research of dual inhibitors tar-
geting both HIV-1 CA and CypA.
We subsequently carried out docking analysis for the compounds
that bound to CypA. Compound K24 was selected as a typical
example because of its highest binding affinity against CypA among
all the tested compounds. As shown in Figure 3B, the sulfamethox-
azole group of K24 inserted into the pocket B of CypA that con-
sisted of Arg55, Phe60, Phe113, Leu122, and His126. Van der
Waals force and hydrophobic interactions between sulfametho-
xazole group and these residues played important roles in
Acknowledgments
This project was supported by the National Natural Science Founda-
tion of China (No. 30670415).
32
Chem Biol Drug Des 2010; 76: 25–33

Structure–Activity Relationships (SAR) Research
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