SAR and molecular mechanism study of novel acylhydrazone compounds targeting HIV-1 CA

Article abstract of DOI:10.1016/j.bmc.2010.02.003

We synthesized a series of acylhydrazone compounds bearing naturally occurring amino acids' side chains as HIV assembly inhibitors. Biological evaluation indicated that the compounds had anti-SIV and capsid assembly inhibitory activities. The structure-activity relationship (SAR) study showed that compounds bearing proper aromatic side chains had potential antiviral activities. The molecular modeling experiments revealed the molecular mechanism that they could bind to CA in the same manner as CAP-1 and occupy two more grooves.

Full text of DOI:10.1016/j.bmc.2010.02.003

Bioorganic & Medicinal Chemistry 18 (2010) 2135–2140
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
SAR and molecular mechanism study of novel acylhydrazone compounds
targeting HIV-1 CA
Yinxue Jin a, Zhiwu Tan a, Meizi He a, Baohe Tian a, Shixing Tang b, Indira Hewlett b, 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:
We synthesized a series of acylhydrazone compounds bearing naturally occurring amino acids’ side
chains as HIV assembly inhibitors. Biological evaluation indicated that the compounds had anti-SIV
and capsid assembly inhibitory activities. The structure–activity relationship (SAR) study showed that
compounds bearing proper aromatic side chains had potential antiviral activities. The molecular model-
ing experiments revealed the molecular mechanism that they could bind to CA in the same manner as
CAP-1 and occupy two more grooves.
Received 12 December 2009
Revised 30 January 2010
Accepted 2 February 2010
Available online 6 February 2010
Keywords:
HIV-1
Ó 2010 Published by Elsevier Ltd.
Capsid assembly
Antiviral
Acylhydrazone derivatives
1. Introduction
the p-position of the right phenyl ring with electron-donating,
electron-withdrawing and hydrophobic groups, or replaced the
In the past few years, scientists have made dramatic efforts to
develop new targets of human immunodeficiency virus (HIV) and
their inhibitors. Recent studies have revealed that HIV-1 capsid
proteins can assemble into a cone-shaped structure encapsidating
the viral nucleocapsid/RNA genome complex which performs
essential function for efficient replication.1,2 Mutations in CA that
inhibit assembly are lethal, and mutations that alter capsid stabil-
ity severely attenuate replication, making the capsid protein an
attractive target.3
right phenyl ring with heterocyclic ring. Based on the above strat-
egy, we obtained a series of novel acylhydrazone derivatives and
evaluated their antiviral activities and assembly inhibitory activi-
ties. We also performed molecular modeling experiments with
our compounds to gain more insight into the molecular
mechanism.
2. Results and discussion
In our previous work we had found that acylhydrazone deriva-
tives containing naturally occurring amino acid moiety had good
anti-SIV activities and focused on compounds containing a L-phen-
The route used for preparation of the title compounds is illus-
trated in Scheme 1 and the detailed synthetic procedure is ex-
plained in Section 4. Twenty-three compounds were obtained
and their MS, 1H NMR spectroscopy data are provided in Section
4. According to the literature, this array of acylhydrazone com-
pounds are in form of E geometrical isomer for C@N double bond
in DMSO-d6, and the E geometrical isomer undergoes a rapid cis/
trans amide equilibrium at room temperature.5,6 Analytic results
of the 1H NMR spectra of our synthesized compounds answered
for the literature.
We tested the concentration for 50% of maximum effect (EC50),
concentration for 50% of maximum toxic (TC50) and therapeutic in-
dex (TI = TC50/EC50) values of our title compounds using SIV-in-
duced syncytium assay in CEM cells. The results are summarized
in Table 1.
ylalanine side chain which showed great potential (see Fig. 1).4
This paper is aimed to find more potent compounds by introducing
different naturally occurring amino acid side chains to the acylhyd-
razone compounds so as to further investigate the structure–activ-
ity relationship (SAR) and molecular mechanism of this class of
acylhydrazone compounds. In our previous work, we had found
that compounds bearing electro-donating group at p-position of
the left phenyl ring had good antiviral activities.4 Hence to study
the SAR more efficiently and reasonably, we kept the left ring
substituted with p-methoxyl group and performed the following
modifications: (1) We introduced different naturally occurring
amino acids to the acylhydrazone compounds. (2) We substituted
As shown in Table 1, each of the title compounds exhibited an
EC50 value within the range from 0.41
of them possessed a TC50 value more than 100, indicating that all
lM to 14.86 lM while most
* Corresponding author. Tel.: +86 10 82805264; fax: +86 10 82802724.
E-mail address: yangm@bjmu.edu.cn (M. Yang).
0968-0896/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2010.02.003

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Y. Jin et al. / Bioorg. Med. Chem. 18 (2010) 2135–2140
Figure 1. The structure of acylhydrazone compounds containing amino acid moiety.
compounds had effective anti-SIV activities while most of them
had low cytotoxicities.
assay was used to probe for potential inhibitory effects of the
acylhydrazone derivatives on in vitro capsid assembly. CAP-1, a
well-known capsid assembly inhibitor, was used as a reference
compound in this assay. The results are summarized in Table 2.
According to Table 2, dissolution of native HIV-CA into assem-
bly buffer led to an increase in absorbance at an initial rate of
60.56 mOD/min, while in the presence of CAP-1 the initial assem-
bly rate decreased to 31.37 mOD/min. In the presence of each title
compound, the capsid assembly rate performed a value ranging
from 3.26 mOD/min to 49.45 mOD/min, suggesting that all com-
pounds could inhibit capsid assembly effectively.
Especially as shown in Figure 2, we found that in the presence of
compounds 8a and 8b which showed high activities, the turbidity
value decreased significantly. Meanwhile, in the presence of com-
pounds 5e and 6c which showed low activities, the turbidity value
slightly decreased. The results demonstrated that 8a and 8b could
inhibit capsid assembly more efficiently than 5e and 6c, reflecting
that the antiviral activity was corresponding to the ability to inhi-
bit capsid assembly.
In our preliminary study, compounds 5a–d and 5e–k were
synthesized to investigate the SAR of the side chains moiety and
the right ring portion, respectively. By taking a detailed look in
Table 1, we found that: (1) Compounds bearing alkane side chain
(5a–d) all exhibited good anti-SIV activities, of which the compound
bearing a hydroxyl alkane side chain performed better activity (5d
versus 5a–c), giving us a hint that hydrogen bond at the side chains
might benefit inhibitory activities. (2) Substituent changes of the
right phenyl ring had little influence on anti-SIV activity (5e–g ver-
sus 5c, 5i–k vs 5d). (3) Replacement of the right phenyl ring with a
furan ring led to better activity when the side chains were alkane
chain (5h vs 5c). Our previous work had found that acylhydrazone
derivatives containing a L-phenylalanine moiety at the side chain
possessed good anti-SIV activities and the phenyl ring of the side
chain functioned by occupying a groove.4 Therefore, we designed
compounds bearing side chain of L-tyrosine which had both phenyl
ring and hydroxyl group, and synthesized compounds 6a–e. They
possessed fair anti-SIV activity not as good as acylhydrazone deriv-
As mentioned before, the EC50 values of our compounds 8a and
atives bearing an
L
-phenylalanine side chain, and displayed high
8b (0.56
l
M and 0.41
l
M) were basically at the same level as the
M and 0.17 M).
cytotoxicities. Maybe it was the hydrogen bond interaction between
the hydroxyl group and the capsid protein that hindered the phenyl
ring from fitting into the groove. Taking all the above factors into
EC50 value of compounds 1a and 1b (0.2
l
l
However, the capsid assembly rate values in presence of 8a and
8b (3.26 1.03 mOD/min and 3.58 0.25 mOD/min) were
significantly lower than those of 1a and 1b (22.6 0.6 mOD/min
and 7.9 0.6 mOD/min),4 suggesting that compounds 8a and 8b
could inhibit capsid assembly more effectively than compounds
1a and 1b.
account, we chose small
L-histidine to design the side chain. The
imidazole ring of -histidine was not only an aromatic ring, but also
L
a hydrogen bond donor and receptor. Finally we synthesized 8a–g.
As we expected, all of them exhibited significant anti-SIV activity,
especially 8a and 8b, with an EC50 value of 0.56
respectively. However, there was no significant difference between
the EC50 values of 8a and 8b and the EC50 values of 1a and 1b
(EC50 = 0.2 lM and 0.17 lM).
l
M and 0.41
l
M,
To further study the molecular mechanism of these novel acy-
lhydrazone compounds, we performed molecular modeling exper-
iments using the program AUTODOCK 4.0. The free binding energies of
our tested compounds and CAP-1 are listed in Table 3.
To ascertain that the compounds could target HIV-1 CA, we per-
formed ultraviolet spectrophotometry analysis to measure sample
turbidity caused by capsid assemble protein.3,7 In our study this
As shown in Table 3, all of our tested compounds exhibited free
energies below À9 kal/mol and most of them possessed free energy
lower than that of CAP-1, confirming that the compounds could
bind to CA. Seeing through Table 3, we found that: (1) Compounds
bearing alkane side chains had close free energies (5a–d). (2) Com-
pounds bearing aromatic side chains (8a–b) displayed noticeable
low free energies, except that compound 6a showed relatively high
free energy. (3) The free energies of our tested compounds were in
agreement with their assembly rate values, as 8a and 8b with assem-
bly rate values of 3.26 mOD/min and 3.58 mOD/min exhibited free
energies of À12.73 kal/mol and À12.78 kal/mol while 5e and 5g
with assembly rate values of 33.38 mOD/min and 30.08 mOD/min
exhibited free energies of À9.35 kal/mol and À9.57 kal/mol.
The binding models of our tested compounds showed that: (1)
The right ring inserted into the pocket vacated by Phe 32 in the
Scheme 1. Reagents and conditions: (a) SOCl2, CHCl3, reflux, 6 h; (b) L-amino acid
methyl ester hydrochloride, Et3N, CH2Cl2, 0 °C to rt, 5 h; (c) 85% NH2NH2ÁH2O,
CH3CH2OH, reflux, 6–8 h; (d) R2–CHO, anhydrous CH3OH, rt, 1–2 h.

Y. Jin et al. / Bioorg. Med. Chem. 18 (2010) 2135–2140
2137
Table 1
Inhibition effects and cytotoxicities of the title compounds on SIV-induced syncytium
Table 2
Assembly inhibitory data of acylhydrazone derivatives
Entry
Assembly ratea (mOD/min)
O
R1
H
N
CA
CAP-1
5c
5d
5e
5f
5g
5h
5i
60.56 3.77
31.37 1.16
25.44 0.84
21.45 1.08
33.38 0.76
10.23 1.54
30.08 0.39
5.82 0.37
11.88 0.47
49.45 2.34
24.88 0.56
32.53 2.07
20.41 0.68
24.26 0.42
8.77 0.19
19.05 0.35
3.26 1.03
3.58 0.25
6.05 0.16
12.59 0.27
8.89 0.18
24.66 0.40
29.32 0.47
N
N
R2
H
O
H3CO
Entry
R1
R2
TC50
EC50
6.41
TI
5a
5b
5c
5d
5e
5f
–CH3
>100
>100
77.87
>100
>100
>100
>100
>15.60
>24.51
10.18
–CH(CH3)2
4.08
7.65
0.77
5j
–CH(CH3)CH2CH3
–CH(CH3)OH
5k
6a
6b
6c
6d
6e
8a
8b
8c
>129.87
>7.20
–CH(CH3)CH2CH3
–CH(CH3)CH2CH3
–CH(CH3)CH2CH3
13.89
8.10
CH3
Cl
>12.35
>24.10
5g
4.15
OH
O
5h
–CH(CH3)CH2CH3
>100
1.70
>58.82
8d
8e
8f
5i
–CH(CH3)OH
–CH(CH3)OH
>100
>100
>100
8.17
4.98
8.13
>12.24
>20.08
>12.30
CH3
Cl
8g
5j
a
Each value is reported as the mean SD (standard deviation) from two exper-
iments in duplicate.
5k
–CH(CH3)OH
OH
H2
6a
6b
6c
6d
6e
>100
41.15
39.96
>100
30.56
4.61
5.21
>21.69
7.90
C
OH
OH
OH
OH
OH
H2
C
CH3
Cl
H2
C
14.86
7.81
2.69
H2
C
OH
>12.80
3.38
H2
C
O
9.03
H2
C
NH
NH
NH
NH
NH
NH
NH
8a
8b
8c
8d
8e
8f
>100
>100
>100
>100
>100
>100
>100
0.56
0.41
1.55
4.40
3.27
3.18
8.44
>178.57
>243.90
64.52
N
N
N
N
N
N
N
H2
C
OCH3
CH3
NO2
Cl
H2
C
H2
C
>22.73
>30.58
>31.45
>11.85
H2
C
H2
C
OH
Figure 2. Turbidity assay results showing the effects of CA-binding compounds on
in vitro capsid assembly
H2
C
O
8g
Table 3
Free binding energies
Entry
Free energy (kal/mol)
same manner as CAP-1. (2) The left ring occupied a hydrophobic
groove surrounded by Val24, Val27, Thr58 and Val59. (3) The al-
kane side chains appeared generally disordered while the aromatic
side chains occupied a groove surrounded by Val26, Val29, Ala31
and Phe32, except that the aromatic side chains with hydrogen
bond donor could form hydrogen bond with the backbone oxygen
of Glu28. As typical compounds, the stereo views of compounds 5c,
5d, 6a and 8a are illustrated in Figure 3.
Looking at Figure 3, we found that: (1) Compounds 5c and 5d
shared the exact model we described above and no hydrogen bond
was formed. (2) The imidazole ring of 8a could fit well into the
groove we mentioned above while the phenyl ring of 6a was hin-
dered by hydrogen bond just as we assumed before. All the above
CAP-1
5a
5b
5c
5d
5e
5g
5j
6a
8a
8b
À10.58
À10.79
À11.23
À10.95
À10.64
À9.35
À9.57
À10.06
À10.88
À12.73
À12.78
results were in agreement with the results obtained from Table 3.
And the stereo view of 8a to capsid protein was the same as that of

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Y. Jin et al. / Bioorg. Med. Chem. 18 (2010) 2135–2140
Figure 3. (A) Stereo view of 5c to capsid protein; (B) stereo view of 5d to capsid protein; (C) stereo view of 6a to capsid protein; (D) stereo view of 8a to capsid protein. The
hydrogen bond is highlighted with green dots.
the binding model of compound 1b.4 Based on the facts that com-
pounds 8a–b and 1a–b had good antiviral activities, we could
reach the conclusion that the side chain occupying a groove was
an important mechanism of these acylhydrozone molecules, giving
clues for further study.
followed by addition of 4-methoxybenzoyl chloride in the presence
of triethylamine in chloroform.9 The appropriate N-(4-methoxy-
benzoyl) amino acid methyl ester (20 mmol) was added in small
portions to a stirred solution of 85% hydrazine hydrate (5 mL) in
ethanol (10 mL). The mixture was heated under reflux for 6–8 h.
After being cooled to room temperature, the resulting precipitate
was filtered in vacuo, washed with cold water and dried to give
the corresponding acylhydrazine. To a magnetically suspension of
the above acylhydrazine (0.5 mmol) in anhydrous methanol
(2 mL) was added substituted benzaldehyde (0.5 mmol). Shortly
the solution became homogeneous (heating could promote this
process) and within minutes the resulting hydrazone began to pre-
cipitate. After the mixture was stirred for further 1–2 h at room
temperature, the precipitate was collected by filtration, washed
with a small quantity of cold methanol and dried. Recrystallization
of the reaction product from methanol gave the pure hydrazone.
3. Conclusion
In this work, all experiments reported here showed that the
newly designed acylhydrazone compounds could inhibit capsid
assembly and had potent inhibitory activities of SIV. The SAR study
revealed that the introduction of proper aromatic side chains could
enhance inhibitory activities significantly. Molecular modeling
experiments confirmed the molecular mechanism that compounds
bearing proper aromatic side chains could bind to capsid protein in
the same manner as CAP-1 and occupy two other hydrophobic
grooves. Compounds bearing an
antiviral activities, of which compound 8a and 8b exhibited the
most promising activities with EC50 values of 0.56 and
0.41 M and TI values of above 179 and 244, respectively.
L-histidine side chain had good
4.2.1. (S,E)-N-(1-(2-Benzylidenehydrazinyl)-1-oxopropan-2-yl)-
4-methoxybenzamide (5a)
l
M
Compound 5a was white solid, mp 182–183 °C, yield 82.8%. 1H
NMR (DMSO-d6) d 1.39–1.43 (dd, 3H, J = 7.5 Hz), 3.81 (s, 3H), 4.50,
5.31 (2 m, 1H), 6.98–7.01 (d, 2H), 7.43 (m, 3H), 7.68 (m, 2H), 7.89–
7.92 (d, 2H), 8.01, 8.25 (2s, 1H), 8.43–8.53 (2d, 1H), 11.37, 11.54
(2s, 1H); MS (ESI+) m/z calcd: 325.14, found: 324.0 [(MÀ1)+].
l
4. Experimental
4.1. Chemistry
4.2.2. (S,E)-N-(1-(2-Benzylidenehydrazinyl)-3-methyl-1-oxobutan-
2-yl)-4-methoxybenzamide (5b)
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 an XA-4 instrument which was
uncorrected.
Compound 5b was white solid, mp 224–225 °C, yield 53.5%. 1H
NMR (DMSO-d6) d 0.93–1.01 (m, 6H), 2.18–2.27 (m, 1H), 3.81 (s,
3H), 4.27, 5.37 (2 m, 1H), 6.97–7.01 (2d, 2H), 7.43–7.46 (m, 3H),
7.68–7.71 (m, 2H), 7.88–7.92 (m, 2H), 8.00, 8.25 (2s, 1H), 8.12,
8.40 (2d, 1H), 11.47, 11.67 (2s, 1H); MS (ESI+) m/z calcd: 353.17,
found: 352.2 [(MÀ1)+].
4.2.3. N-((2S,3R)-1-((E)-2-Benzylidenehydrazinyl)-3-methyl-1-
oxopentan-2-yl)-4-methoxybenzamide (5c)
Compound 5c was white solid, mp 241–242 °C, yield 84.9%. 1H
NMR (DMSO-d6) d 0.84–0.95 (m, 6H), 1.24, 1.57 (2 m, 2H), 2.04 (m,
1H), 3.81 (s, 3H), 4.33, 5.45 (2t, 1H), 6.97–7.01 (2d, 2H, J = 3), 7.43–
7.46 (m, 3H), 7.68–7.71 (m, 2H), 7.87–7.92 (m, 2H), 8.00, 8.25 (2s,
1H), 8.16, 8.43 (2d, 1H), 11.46, 11.69 (2s, 1H); ESI-MS: m/z calcd:
367.19, found: 366.3 [(MÀ1)+].
4.2. General synthetic procedure
Starting from natural occurring amino acids, they were first
converted to their corresponding N-(4-methoxybenzoyl) amino
acid methyl esters by reaction with thionyl chloride in methanol,8

Y. Jin et al. / Bioorg. Med. Chem. 18 (2010) 2135–2140
2139
4.2.4. N-((2S,3R)-1-((E)-2-Benzylidenehydrazinyl)-3-hydroxy-1-
oxobutan-2-yl)-4-methoxybenzamide (5d)
4.2.11. N-((2S,3R)-3-Hydroxy-1-((E)-2-(4-hydroxybenzylidene)-
hydrazinyl)-1-oxobutan-2-yl)-4-methoxybenzamide (5k)
Compound 5k was pale white solid, mp 133–134 °C, yield
93.4%. 1H NMR (DMSO-d6) d 1.12–1.20 (2d, 3H, J = 6.0 Hz), 3.83
(s, 3H), 4.12, 4.24 (2 m, 1H), 4.41, 5.30 (2q, 1H), 4.86, 5.03 (2d,
1H), 6.80–6.85 (dd, 2H), 7.03–7.06 (d, 2H), 7.50–7.53 (d, 2H),
7.87–7.92 (dd, 2H), 8.00–8.03 (d, 1H), 8.13 (s, 1H), 9.79, 9.96 (2s,
1H), 11.29–11.33 (d, 1H); ESI-MS: m/z calcd: 371.15, found:
370.1 [(MÀ1)+].
Compound 5d was white solid, mp 160–161 °C, yield 85.0%. 1H
NMR (DMSO-d6) d 1.13–1.21 (2d, 3H, J = 6.0 Hz), 3.83 (s, 3H), 4.12,
4.24 (2 m, 1H), 4.43, 5.33 (2q, 1H), 4.86, 5.03 (2d, 1H), 7.02–7.05 (d,
2H), 7.42–7.50 (m, 3H), 7.68–7.70 (m, 2H), 7.87–7.92 (m, 2H), 7.80,
7.98 (2d, 1H), 8.02, 8.26 (2s, 1H), 11.46–11.49 (d, 1H); ESI-MS: m/z
calcd: 355.15, found: 354.4 [(MÀ1)+].
4.2.5. 4-Methoxy-N-((2S,3R)-3-methyl-1-((E)-2-(4-methylben-
zylidene)hydrazinyl)-1-oxopentan-2-yl)benzamide (5e)
Compound 5e was white solid, mp 235 °C, yield 87.6%. 1H NMR
(DMSO-d6) d 0.84–0.94 (m, 6H), 1.24, 1.56 (2 m, 2H), 2.01 (m, 1H),
3.81 (s, 3H), 4.32, 5.44 (2t, 1H), 6.98–7.01 (d, 2H), 7.24–7.27 (d,
2H), 7.57–7.60 (d, 2H), 7.89–7.92 (d, 2H), 7.97, 8.20 (2s, 1H),
8.15, 8.42 (2d, 1H), 11.40, 11.63 (2s, 1H); ESI-MS: m/z calcd:
381.21, found: 380.2 [(MÀ1)+].
4.2.12. (S,E)-N-(1-(2-Benzylidenehydrazinyl)-3-(4-hydroxyphen-
yl)-1-oxopropan-2-yl)-4-methoxybenzamide (6a)
Compound 6a was white solid, mp 185–186 °C, yield 50.0%. 1H
NMR (DMSO-d6) d 2.97–2.99 (d, 2H), 3.81 (s, 3H), 4.64, 5.47 (2 m,
1H), 6.65–6.68 (d, 2H), 6.98–7.01 (d, 2H), 7.17–7.20 (d, 2H),
7.44–7.50 (m, 2H), 7.70–7.75 (m, 2H), 7.83–7.86 (d, 2H), 8.05,
8.24 (2s, 1H), 8.49–8.63 (2d, 1H), 9.21 (s, 1H), 11.47, 11.70 (2s,
1H); EI-MS: m/z calcd: 417.17, found: 417.
4.2.6. N-((2S,3SR)-1-((E)-2-(4-Chlorobenzylidene)hydrazinyl)-3-
methyl-1-oxopentan-2-yl)-4-methoxybenzamide (5f)
4.2.13. (S,E)-N-(3-(4-Hydroxyphenyl)-1-(2-(4-methylbenzylidene)
hydrazinyl)-1-oxopropan-2-yl)-4-methoxybenzamide (6b)
Compound 6b was white solid, mp 201–202 °C, yield 86.9%. 1H
NMR (DMSO-d6) d 2.43, 2.44 (2s, 3H), 2.96–2.98 (d, 2H), 3.81 (s,
3H), 4.63, 5.46 (2 m, 1H), 6.64–6.67 (m, 2H), 6.98–7.01 (d, 2H),
7.15–7.18 (d, 2H), 7.25–7.32 (2d, 2H), 7.58–7.64 (dd, 2H), 7.82–
7.85 (d, 2H), 8.00, 8.19 (2s, 1H), 8.47–8.61 (2d, 1H), 9.20 (s, 1H),
11.39, 11.62 (2s, 1H); ESI-MS: m/z calcd: 431.18, found: 430.2
[(MÀ1)+].
Compound 5f was white solid, mp 215–216 °C, yield 92.3%. 1H
NMR (DMSO-d6) d 0.84–0.94 (m, 6H), 1.23, 1.57 (2 m, 2H), 2.02
(m, 1H), 3.81 (s, 3H), 4.32, 5.42 (2t, 1H), 6.97–7.01 (2d, 2H),
7.50–7.53 (d, 2H), 7.71–7.74 (d, 2H), 7.89–7.93 (2d, 2H), 7.99,
8.24 (2s, 1H), 8.21, 8.46 (2d, 1H), 11.54, 11.79 (2s, 1H); ESI-MS:
m/z calcd: 401.15, found: 400.2 [(MÀ1)+].
4.2.7. N-((2S,3R)-1-((E)-2-(4-Chlorobenzylidene)hydrazinyl)-3-
methyl-1-oxopentan-2-yl)-4-methoxybenzamide (5g)
Compound 5g was white solid, mp 144–146 °C, yield 93.5%. 1H
NMR (DMSO-d6) d 0.86–0.94 (m, 6H), 1.24, 1.56 (2 m, 2H), 2.03 (m,
1H), 3.81 (s, 3H), 4.30, 5.42 (2t, 1H), 6.80–6.85 (dd, 2H), 6.98–7.01
(d, 2H), 7.50–7.53 (d, 2H), 7.66–7.91 (dd, 2H), 8.13 (s, 1H), 8.40–
8.43 (d, 1H), 11.27, 11.49 (2s, 1H); ESI-MS: m/z calcd: 383.18,
found: 382.3 [(MÀ1)+].
4.2.14. (S,E)-N-(1-(2-(4-Chlorobenzylidene)hydrazinyl)-3-(4-
hydroxyphenyl)-1-oxopropan-2-yl)-4-methoxybenzamide (6c)
Compound 6c was white solid, mp 197–199 °C, yield 90.1%. 1H
NMR (DMSO-d6) d 2.98 (s, 2H), 3.81 (s, 3H), 4.62, 5.47 (2 m, 1H),
6.66 (m, 2H), 6.98–7.00 (m, 2H), 7.20 (m, 2H), 7.54–7.57 (m, 2H),
7.73–7.84 (m, 4H), 8.01, 8.23 (2s, 1H), 8.50–8.61 (2d, 1H), 9.19 (s,
1H), 11.50, 11.74 (2s, 1H); ESI-MS: m/z calcd: 451.13, found:
450.2 [(MÀ1)+].
4.2.8. N-((2S,3R)-1-((E)-2-(4-Chlorobenzylidene)hydrazinyl)-3-
methyl-1-oxopentan-2-yl)-4-methoxybenzamide (5h)
Compound 5h was pale brown solid, mp 220–221 °C, yield
81.2%. 1H NMR (DMSO-d6) d 0.83–0.93 (m, 6H), 1.24, 1.55 (2 m,
2H), 2.00 (m, 1H), 3.81 (s, 3H), 4.30, 5.34 (2t, 1H), 6.62–6.64 (m,
1H), 6.87–6.91 (2d, 1H), 6.98–7.01 (d, 2H), 7.83–7.92 (m, 3H),
8.13 (s, 1H), 8.43–8.46 (d, 1H), 11.41, 11.65 (2s, 1H); ESI-MS: m/z
calcd: 357.17, found: 356.2 [(MÀ1)+].
4.2.15. (S,E)-N-(1-(2-(4-Hydroxybenzylidene)hydrazinyl)-3-(4-
hydroxyphenyl)-1-oxopropan-2-yl)-4-methoxybenzamide (6d)
Compound 6d was white solid, mp 142–143 °C, yield 89.6%. 1H
NMR (DMSO-d6) d 2.94–2.96 (d, 2H), 3.80 (s, 3H), 4.61, 5.42 (2 m,
1H), 6.63–6.67 (2d, 2H), 6.80–6.88 (2d, 2H), 6.97–7.00 (d, 2H),
7.15–7.18 (2d, 2H), 7.51–7.58 (2d, 2H), 7.81–7.84 (d, 2H), 7.93,
8.11 (2s, 1H), 8.44–8.59 (2d, 1H), 9.20 (s, 1H), 9.94–9.96 (d, 1H),
11.26, 11.47 (2s, 1H); ESI-MS: m/z calcd: 433.16, found: 432.2
[(MÀ1)+].
4.2.9. N-((2S,3R)-1-((E)-2-Benzylidenehydrazinyl)-3-hydroxy-1-
oxobutan-2-yl)-4-methoxybenzamide (5i)
4.2.16. (S,E)-N-(1-(2-(Furan-2-ylmethylene)hydrazinyl)-3-(4-
hydroxyphenyl)-1-oxopropan-2-yl)-4-methoxybenzamide (6e)
Compound 6e was brown solid, mp 134–136 °C, yield 78.3%. 1H
NMR (DMSO-d6) d 2.95–2.97 (d, 2H), 3.81 (s, 3H), 4.61, 5.33 (2 m,
1H), 6.63–6.68 (m, 3H), 6.91 (m, 1H), 6.98–7.01 (d, 2H), 7.13–
7.25 (2d, 2H), 7.82–7.84 (d, 2H), 7.92–7.94 (d, 1H), 8.12 (s, 1H),
8.43–8.61 (2d, 1H), 9.19 (s, 1H), 11.41, 11.61 (2s, 1H); ESI-MS: m/
z calcd: 407.15, found: 406.2 [(MÀ1)+].
Compound 5i was white solid, mp 155–156 °C, yield 89.6%. 1H
NMR (DMSO-d6) d 1.13–1.20 (2d, 3H, J = 6.0 Hz), 2.34 (s, 3H), 3.83
(s, 3H), 4.11, 4.22 (2 m, 1H), 4.42, 5.32 (2q, 1H), 4.88, 5.04 (2d,
1H), 7.03–7.05 (d, 2H), 7.24–7.29 (dd, 2H), 7.57–7.59 (d, 2H),
7.87–7.93 (dd, 2H), 7.83, 8.03 (2d, 1H), 7.98, 8.21 (2s, 1H),
11.43–11.47 (d, 1H); ESI-MS: m/z calcd: 369.17, found: 368.1
[(MÀ1)+].
4.2.10. N-((2S,3R)-1-((E)-2-(4-Chlorobenzylidene)hydrazinyl)-3-
hydroxy-1-oxobutan-2-yl)-4-methoxybenzamide (5j)
4.2.17. (S,E)-N-(1-(2-Benzylidenehydrazinyl)-3-(1H-imidazol-4-
yl)-1-oxopropan-2-yl)-4-methoxybenzamide (8a)
Compound 5j was white solid, mp 168–170 °C, yield 90.3%.
Compound 8a was white solid, mp 203–204 °C, yield 73.7%. 1H
NMR (DMSO-d6) d 3.04 (s, 2H), 3.81 (s, 3H), 4.69, 5.53 (2 m, 1H),
6.87 (s, 1H), 7.00–7.03 (d, 2H), 7.43–7.45 (m, 3H), 7.57 (s, 1H),
7.66–7.76 (m, 2H), 7.83–7.87 (2d, 2H), 8.00, 8.25 (2s, 1H), 8.59–
8.67 (m, 1H), 11.41, 11.60 (2s, 1H), 11.81 (s, 1H); ESI-MS: m/z
calcd: 391.16, found: 392.0 [(M+1)+].
1H NMR (DMSO-d6)
d 1.13–1.20 (2d, 3H, J = 6.3 Hz), 3.83
(s, 3H), 4.12, 4.20 (2 m, 1H), 4.43, 5.32 (2q, 1H), 4.88–5.08
(2d, 1H), 7.03–7.05 (d, 2H), 7.50–7.55 (dd, 2H), 7.71–7.73 (d,
2H), 7.88–7.93 (dd, 2H), 8.01, 8.25 (2s, 1H), 8.05–8.08 (d, 1H),
11.57–11.63 (d, 1H); ESI-MS: m/z calcd: 389.11, found: 388.0
[(MÀ1)+].

2140
Y. Jin et al. / Bioorg. Med. Chem. 18 (2010) 2135–2140
4.2.18. (S,E)-N-(3-(1H-Imidazol-4-yl)-1-(2-(4-methoxybenzylidene)
hydrazinyl)-1-oxopropan-2-yl)-4-methoxybenzamide (8b)
Compound 8b was white solid, mp 216–217 °C, yield 69.4%. 1H
NMR (DMSO-d6) d 3.03 (s, 2H), 3.80 (s, 6H), 4.67, 5.51 (2 m, 1H),
6.86 (s, 1H), 6.98–7.00 (d, 4H), 7.58–7.68 (m, 3H), 7.82–7.85 (m,
2H), 7.93, 8.16 (2s, 1H), 8.52–8.62 (2d, 1H), 11.25, 11.43 (2s, 1H);
ESI-MS: m/z calcd: 421.18, found: 422.0 [(M+1)+].
4.3. Biological evaluation
Antiviral evaluation: inhibition of SIV-induced syncytium in
CEM174 cell cultures was measured in a 96-well microplate contain-
ing 1 Â 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)cellformationwas examinedmicroscopically.
The EC50 wasdefinedasthe compoundconcentrationrequiredto pro-
tect cells against the cytopathogenicity of SIV by 50%. TC50 assay was
performed in uninfected CEM174 cells under the same condition
above, and the value was determined as the concentration required
to inhibit CEM cells proliferation by 50% in MTT reduction assay.
Assembly inhibitory assay: ultraviolet spectrophotometry assay
was performed at 350 nm on a Agilent 8453 spectrophotometer. A
4.2.19. (S,E)-N-(3-(1H-Imidazol-4-yl)-1-(2-(4-methylbenzylidene)
hydrazinyl)-1-oxopropan-2-yl)-4-methoxybenzamide (8c)
Compound 8c was white solid, mp 242–243 °C, yield 67.5%. 1H
NMR (DMSO-d6) d 2.34 (s, 3H), 3.04 (s, 2H), 3.81 (s, 3H), 4.68, 5.52
(2 m, 1H), 6.87 (s, 1H), 6.99–7.02 (d, 2H), 7.24–7.26 (d, 2H), 7.55–
7.64 (m, 3H), 7.83–7.87 (m, 2H), 7.96, 8.20 (2s, 1H), 8.58–8.65 (m,
1H), 11.34, 11.53 (2s, 1H), 11.81 (s, 1H); ESI-MS: m/z calcd: 405.18,
found: 406.0 [(M+1)+].
1.0
75
l
L of concentrated ligand in DMSO (1 mM) was added to a
lL aqueous solution, then added 25 L capsid protein (40 M)
l
l
4.2.20. (S,E)-N-(3-(1H-Imidazol-4-yl)-1-(2-(4-nitrobenzylidene)
hydrazinyl)-1-oxopropan-2-yl)-4-methoxybenzamide (8d)
Compound 8d was yellow solid, mp 236–237 °C, yield 69.7%. 1H
NMR (DMSO-d6) d 3.05 (s, 2H), 3.81 (s, 3H), 4.70, 5.56 (2 m, 1H),
6.92 (s, 1H), 7.00–7.02 (d, 2H), 7.58 (s, 1H), 7.84–7.87 (m, 2H),
7.93–7.95 (d, 1H), 8.03–8.04 (d, 1H), 8.09, 8.36 (2s, 1H), 8.27–
8.30 (2d, 1H), 11.68, 11.80, 11.90 (3s, 2H); ESI-MS: m/z calcd:
436.15, found: 437.0 [(M+1)+].
to initiate the reaction. 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.
4.4. Molecular modeling
The initial structure 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 with the code as 2JPR.10 The advanced
docking program AUTODOCK 4.0 was used to remove the small mole-
cule and perform the automatic molecular docking with our com-
pounds. The number of generations, energy evaluation, and
docking runs were set to 370,000, 1,500,000, and 20, respectively,
and the kinds of atomic charges were taken as Kollman-all-atom
for macromolecular and Gasteiger–Hücel for the compounds.
4.2.21. (S,E)-N-(1-(2-(4-Chlorobenzylidene)hydrazinyl)-3-(1H-
imidazol-4-yl)-1-oxopropan-2-yl)-4-methoxybenzamide (8e)
Compound 8e was yellow solid, mp 248–249 °C, yield 82.8%. 1H
NMR (DMSO-d6) d 3.04 (s, 2H), 3.81 (s, 3H), 4.69, 5.53 (2 m, 1H),
6.88 (s, 1H), 7.00–7.02 (d, 2H), 7.49–7.51 (d, 2H), 7.56 (s, 1H),
7.69–7.71 (d, 1H), 7.76–7.78 (d, 1H), 7.83–7.86 (m, 2H), 7.98,
8.24 (2s, 1H), 8.63 (m, 1H), 11.43, 11.64, 11.79 (3s, 2H); ESI-MS:
m/z calcd: 425.13, found: 426.0 [(M+1)+].
4.2.22. (S,E)-N-(1-(2-(4-Hydroxybenzylidene)hydrazinyl)-3-(1H-
imidazol-4-yl)-1-oxopropan-2-yl)-4-methoxybenzamide (8f)
Compound 8f was white solid, mp 162–163 °C, yield 66.8%. 1H
NMR (DMSO-d6) d 3.04 (s, 2H), 3.81 (s, 3H), 4.67, 5.50 (2 m, 1H),
6.80–6.83 (2d, 2H), 6.86 (s, 1H), 7.48–7.59 (m, 3H), 7.83–7.86 (m,
2H), 7.90, 8.13 (2s, 1H), 8.51–8.60 (2d, 1H), 9.88 (s, 1H), 11.17,
11.34, 11.90 (3s, 2H); ESI-MS: m/z calcd: 407.16, found: 408.0
[(M+1)+].
Acknowledgment
The project was supported by the National Natural Science
Foundation of China (No. 30670415).
References and notes
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6. Pallaa, G.; Predierib, G.; Domiano, P. Tetrahedron 1986, 42, 3649.
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8. West, J. B.; Wong, C. H. J. Org. Chem. 1986, 51, 2728.
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4.2.23. (S,E)-N-(1-(2-(Furan-2-ylmethylene)hydrazinyl)-3-(1H-
imidazol-4-yl)-1-oxopropan-2-yl)-4-methoxybenzamide (8g)
Compound 8g was white solid, mp 210–211 °C, yield 59.2%.
1H NMR (DMSO-d6) d 3.04 (s, 2H), 3.81 (s, 3H), 4.67, 5.45
(2 m, 1H), 6.61–6.64 (m, 1H), 6.87–6.92 (m, 2H), 7.59 (s, 1H),
7.82–7.87 (m, 3H), 7.90, 8.14 (2s, 1H), 8.55–8.63 (2d, 1H),
11.33, 11.51, 11.95 (3s, 2H); ESI-MS: m/z calcd: 381.14, found:
382.0 [(M+1)+].