Synthesis and antiviral activities of novel acylhydrazone derivatives targeting HIV-1 capsid protein

Article abstract of DOI:10.1016/j.bmcl.2009.02.116

HIV-1 capsid protein (CA) plays important roles in the viral replication cycle. A number of acylhydrazone derivatives that act as inhibitors of HIV-1 CA assembly, were designed and synthesized. The synthesized compounds were tested for their antiviral activities and cytotoxicities using CEM cells. Some derivatives also were assayed for their ability to inhibit HIV-1 CA assembly in vitro. Among them, compounds 14f and 14i display the most promising potency with EC₅₀ values of 0.21 and 0.17 μΜ, respectively.

Full text of DOI:10.1016/j.bmcl.2009.02.116

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2162–2167
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antiviral activities of novel acylhydrazone derivatives targeting
HIV-1 capsid protein
Baohe Tian a, Meizi He a, Shixing Tang b, Indira Hewlett b, Zhiwu Tan a, Jiebo Li a, Yinxue Jin 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 capsid protein (CA) plays important roles in the viral replication cycle. A number of acylhydrazone
derivatives that act as inhibitors of HIV-1 CA assembly, were designed and synthesized. The synthesized
compounds were tested for their antiviral activities and cytotoxicities using CEM cells. Some derivatives
also were assayed for their ability to inhibit HIV-1 CA assembly in vitro. Among them, compounds 14f
Received 11 November 2008
Revised 14 January 2009
Accepted 27 February 2009
Available online 4 March 2009
and 14i display the most promising potency with EC50 values of 0.21 and 0.17 lV, respectively.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Synthesis
Antiviral activity
Acylhydrazones
HIV-1 capsid
Human immunodeficiency virus type-1 (HIV-1) is the causative
agent of acquired immunodeficiency syndrome (AIDS) which has
become a major worldwide epidemic since 1981. Despite the ther-
apeutic success of highly active anti-retroviral therapy (HAART),
which includes the combined use of drugs targeting the viral en-
zymes reverse transcriptase (RT) and protease (PR), drug resistance
and poor compliance caused by side effects hinder the achieve-
ment of sustainable antiviral effects.1 With the advances in the
understanding of HIV-1 life cycle, in addition to RT and PR many
novel targets that may have potential antiviral intervention have
been identified, including the other viral enzymes and viral pro-
teins (e.g., integrase, envelope protein gp41 and regulatory protein
tat). Drug discovery and development for anti-HIV has progressed
significantly in the past decade, especially recently several novel
antiviral drugs, such as enfuvirtide (fusion inhibitor), maraviroc
(chemokine co-receptor antagonist) and raltegravir (integrase
inhibitor), have been approved for the treatment of AIDS.2 The suc-
cessful design of such inhibitors could provide hints for the devel-
opment of other new antiviral agents against HIV-1.
HIV CA assembly inhibitors, has been identified to bind to the CA
N-terminal domain (NTD) and inhibit capsid assembly during viral
maturation (Fig. 1).6
More recently, Prevelige and co-workers have reported a series
of new small molecule inhibitors that target HIV-1 CA through the
use of the high throughput assay (Fig. 2).7 Among these com-
pounds, we found that most of them have a core structure of acy-
lhydrazone moiety, and two substituted phenyl ring in each end
which seems to be the most important pharmacophores for their
antiviral activities. In addition there are a few peptides which have
been reported to bind to HIV-1 CA and interfere with the protein–
protein interactions in HIV-1 CA.8 Therefore, we designed novel
acylhydrazone derivatives bearing hydrophobic or lipophobic
groups in side chains by the introduction of different naturally
occurring amino acids (such as glycine, L-phenylalanine and L-ser-
ine) into the backbone of these molecules so as to improve their
binding affinities to the protein via hydrogen bonds, steric interac-
tions, hydrophobic contacts, etc. To further explore the antiviral
activities of these compounds, other molecular modifications were
required in which various steric, electron-donating, electron-with-
Recent studies reveal that HIV-1 capsid protein (CA) plays
important roles in the life cycle of HIV-1 and has been an attractive
target of high priority for anti-HIV.3 During the viral replication cy-
cle, HIV-1 CA can assemble into a conical core shell surrounding
the viral nucleocapsid/RNA genome complex which is critical for
viral replication.4 Recently, several inhibitors that bind to CA and
inhibit virus assembly have been reported.5 CAP-1, one of the
CH3
CH3
O
N
S
CH3
N
Cl
N
O
H
H
* Corresponding author. Tel.: +86 10 82805264; fax: +86 10 82802724.
E-mail addresses: mingy@bjmu.edu.cn, yangm@bjmu.edu.cn (M. Yang).
Figure 1. Structure of CAP-1.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.116

B. Tian et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2162–2167
2163
CH
CH
3
OH
3
Br
H
O
N
N
S
N
N
H
O
O
Br
HO
O
Br
Br
O
O
OH
N
N
CH
O
3
N
H
OH
N
H
Br
CH
3
NO
2
CH
3
Figure 2. Small molecule inhibitors targeting HIV-1 CA.
drawing or hydrophobic groups were introduced into the aromatic
ring in the end of the molecules.
The synthetic route we applied for the synthesis of a variety of
acylhydrazone analogues is summarized in Schemes 1–4. In
Scheme 1, N-acylhydrazones 2(a–b) were synthesized according
to a general procedure described in the literature.9 Starting from
substituted benzoic acids, they were first converted to their corre-
sponding acid chlorides by reaction with thionyl chloride, followed
R1
O
C
O
C
H
a, b
c
N
R1
OH
R1
NH NH2
a. R1=CH3O
b. R1=NO2
C
O
N
1a-b
2a-b
d,e
R2
R2
R2
O
O
O
H
N
N
H
c
N
O
b
N
N
H
NH2
N
H
H
O
O
O
R1
R1
R1
3a-f
5a-f
4a-f
a. R1=CH3O, R2=H
d. R1=NO2, R2=H
e. R1=NO2, R2=CH2OH
f. R1=NO2, R2=CH2Ph
b. R1=CH3O, R2=CH2OH
c. R1=CH3O, R2=CH2Ph
Scheme 1. Synthesis of acylhydrazones and their derivatives. Reagents and conditions: (a) concentrated H2SO4, anhydrous CH3CH2OH, reflux, 20 h; (b) 85% NH2NH2ÁH2O,
CH3CH2OH, reflux, 6–8 h; (c) PhCHO, anhydrous CH3OH, rt, 1–2 h; (d) SOCl2, CHCl3, reflux, 6 h; (e) glycine/
L-phenylalanine/
L
-serine methyl ester hydrochloride, Et3N, CH2Cl2,
0 °C to rt, 5 h.
R1
O
S
O
S
a
b
H
N
R1
NHNH2
R1
Cl
a. R1=CH3
S
N
O
O
b. R1=Br
O
O
c. R1=CH3CONH
6a-c
7a-c
c
R2
R2
R2
O
O
O
O
O
O
H
H
S
N
S
N
S
O
d
b
N
N
N
N
NH2
H
H
H
O
O
O
R1
R1
R1
10a-i
8a-i
9a-i
g. R1=CH3CONH, R2=H
h. R1=CH3CONH, R2=CH2OH
i. R1=CH3CONH, R2=CH2Ph
d. R1=Br, R2=H
e. R1=Br, R2=CH2OH
f. R1=Br, R2=CH2Ph
a. R1=CH3, R2=H
b. R1=CH3, R2=CH2OH
c. R1=CH3, R2=CH2Ph
Scheme 2. Synthesis of sulfonylhydrazones and their derivatives. Reagents and conditions: (a) 85% NH2NH2ÁH2O, CH3CH2OH, H2O, rt, 5 h; (b) PhCHO, anhydrous CH3OH, rt, 1–
2 h; (c) glycine/ -phenylalanine/
-serine methyl ester hydrochloride, Et3N, CH2Cl2, 0 °C to rt, 20 h; (d) 85% NH2NH2ÁH2O, CH3CH2OH, reflux, 6–8 h.
L
L

2164
B. Tian et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2162–2167
O
undergoes a rapid cis/trans amide equilibrium at room tempera-
O
ture, which is in agreement with the analytic results of the 1H
NMR spectra of our synthesized derivatives. For example, two sets
of methylene, imine and amide protons signals of cis/trans amide
conformers are observed in the 1H NMR spectra of compounds
5a, 5d, 10a, 10d and 10g in DMSO-d6.
b
a
N
CH3
(CH3)2 HCl
NH
N
11
12
O
S
O
S
N
c
N
R
12
R
Cl
+
We also evaluated the biological activities of all our synthesized
compounds using SIV-induced syncytium in CEM cells.19 Their EC50
(antiviral activity), TC50 (cytotoxicity) and TI (therapy index) are
listed in Table 1. Most of our synthesized compounds exhibited sig-
nificant inhibitory activity against viral replication. Among the
compounds (2a, 2b, 5a–5f, 7a–7c, 10a–10i), compounds 5c, 10c,
10d and 10i were the most potent with EC50 values ranging from
O
O
13a-b
a. R=NO2
b. R=Br
Scheme 3. Synthesis of 1-(substituted benzenesulfonyl)-3-phenyl-2-pyrazolines.
Reagents and conditions: (a) (CH2O)n, HN(CH3)2ÁHCl, concentrated HCl, CH3CH2OH,
reflux, 3 h; (b) 85% NH2NH2ÁH2O, 50% aqueous NaOH, CH3OH, reflux, 45 min; (c) dry
pyridine, on a boiling water bath, 20 min.
0.2 to 0.47 lM and TI values above 125. It is worth noting that each
of these compounds except compound 10d contains a L-phenylala-
by addition of a variety of methyl ester hydrochloride of natural
amino acids in the presence of triethylamine.10 The yielded inter-
mediates 3(a–f) reacted with hydrazine hydrate in ethanol to ob-
tain acid hydrazides 4(a–f), which underwent condensation with
benzaldehyde affording the target compounds 5(a–f).11
The synthesis of sulfonylhydrazones and their derivatives is
shown in Scheme 2. N-Arylsulfonyl hydrazones 7(a–c) were pre-
pared from substituted aromatic sulfonyl chlorides by the typical
method.12 Their derivatives 10(a–i) were obtained by a route anal-
ogous to 5(a–f), using the substituted aromatic sulfonyl chlorides
as starting materials to react with natural amine acids methyl ester
hydrochlorides,13 subsequent hydrazinolysis with hydrazine hy-
drate and condensation with benzaldehyde.14
In Scheme 3, the hydrochloride of 3-dimethylaminopropiophe-
none 11 as Mannich Base was prepared by refluxing acetophenone,
dimethylamine hydrochloride and paraformaldehyde in ethanol in
the presence of concentrated hydrochloric acid according to the
standard method.15 The key intermediate 11 reacted with hydra-
zine hydrate providing 3-phenyl-2-pyrazoline 12 using a literature
procedure, which was immediately treated with substituted aro-
matic sulfonyl chlorides to give the target compounds 13(a–b),
respectively.16
To further investigate the antiviral activities of the acylhydraz-
one derivatives, further molecular modifications were carried out.
The analogues possessing different substitutions in the right phe-
nyl ring were synthesized through the route outlined in Scheme
4, using a variety of aromatic (or heteroaromatic) aldehydes and
ketones as starting materials. The condensation of the acid hydra-
zides 9c with appropriate aldehydes and ketones resulted in the
formation of the corresponding acylhydrazone derivatives 14(a–
l).17
Table 1
Antiviral activity and toxicity of acylhydrazone derivatives on SIV-induced syncytium
Compounda
EC50
(l
M)
TC50 (lM)
TId
b
c
2a
2b
5a
5b
5c
5d
5e
5f
7a
31.05
5.59
2
>100
29.6
63.8
45.6
>100
>100
>100
18.71
31.7
>100
40
>3.22
5.3
31.9
76
>500
>97.1
>50
0.6
0.2
1.03
2
3
0.36
0.95
0.65
1.5
1.63
0.4
6.2
88.1
>105.3
61.5
55.7
3.2
125
283.8
2.2
>6.5
>6.6
>75.8
>212.8
>192.3
382
70.7
>10.1
1.5
>22.5
15
>476.2
3.4
7b
7c
10a
10b
10c
10d
10e
10f
10g
10h
10i
13a
13b
14a
14b
14c
14d
14e
14f
14g
14h
14i
14j
14k
14l
83.5
5.19
50
0.33
35.98
15.3
15.1
1.32
0.47
0.52
0.1
92.8
80.2
>100
>100
>100
>100
>100
38.2
21.2
>100
9.8
>100
82.5
>100
9.4
>100
>100
49.1
99.8
78.3
0.3
9.93
6.64
4.45
5.49
0.21
2.8
0.27
0.17
0.4
>365
>588.2
122.8
40.6
87.6
2.46
0.89
The purity of all the products was determined by TLC and melt-
ing points. The physical and spectral properties of previously re-
ported compounds, such as melting points, were in accord with
literature values, and the structures of the novel compounds were
confirmed by 1H NMR and EI-MS. According to the literature,18 this
class of acylhydrazones are in the form of E geometrical isomer for
C@N double bond in DMSO-d6, and the E geometrical isomer
a
This assay used the antiviral drug Indinavir at 1 Â 10À6 mol/L as positive
control.
b
c
Antiviral activity, concentration which inhibits viral replication by 50%.
Cellular toxicity, concentration which is toxic to 50% of SIV-induced syncytium.
Therapeutic index, TC50 value divided by EC50 value (TC50/EC50).
d
b. R1=H, R2=4-CH3Ph
d. R1=H, R2=2-FPh
f. R1=H, R2=3-CH3O,4-OHPh
h. R1=Ph, R2=Ph
a. R1=CH3, R2=Ph
c. R1=H, R2=4-NO2Ph
e. R1=H, R2=4-BrPh
g. R1=H, R2=4-ClPh
i. R1=H, R2=
R1
O
O
H
O
O
H
a or b
S
N
S
N
O
O
j. R1=CH3, R2=
N
NH2
R2
N
N
H
H
O
O
k. R1=H, R2=
O
l. R1=
R2=
CH3
CH3
9c
14a-l
Scheme 4. Synthesis of acylhydrazone derivatives of various aldehydes and ketones. Reagents and conditions: (a) R1COR2, anhydrous CH3OH, rt, 1–2 h; (b) R1COR2,
anhydrous CH3OH, glacial acetic acid, reflux, 3–8 h.

B. Tian et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2162–2167
2165
nine moiety which bears a benzyl side chain, and an electron-
donating substituent (such as methyl, methoxy) in the left phenyl
ring. One possible explanation is that the presence of these moie-
ties enhance the binding affinity of the compounds to HIV-1 CA.
In addition, we also synthesized the D-enantiomer of the acylhyd-
razone derivative 10c as an example and evaluated for its antiviral
activity. We found that it was nearly inactive (data not shown),
which demonstrated that the configuration of these compounds
greatly influenced their biological activities, and the
showed good antiviral activities in comparison with their
tiomers, so we just focused on the studies of these acylhydrazone
derivatives with -configuration in this paper. From this prelimin-
L
-enantiomers
D
-enan-
L
ary study, on the basis of compound 10c, then we investigated the
effect of structural modification of the right phenyl ring on their
biological activities, which included incorporation of different elec-
tron-donating, electron-withdrawing, hydrophobic and steric sub-
stituents, such as methyl, halide, nitro groups, and replacement of
the phenyl ring by a heterocyclic aromatic ring. Among the com-
pounds 14a–14l, compound 14f and 14i displayed most potent
inhibitory activity compared with the parent 10c with EC50 values
from 0.4 lM to 0.21 lM and 0.17 lM, respectively, and the cyto-
Figure 3. Turbidity assay results showing the effects of CA-binding compounds on
in vitro capsid assembly.
toxicities were markedly reduced, resulting in increase of therapy
index (TI) value from 125 to the above 476 and 588, respectively.
Furthermore, the cyclization of NHN@CH moiety in sulfonylhyd-
razones produced 2-pyrazoline derivatives possessing a rigid con-
formation which led to a considerable increase in antiviral potency.
For example, the compound 13b showed significantly higher activ-
the presence of 14b and 14c, which showed low activities with
EC50 values of 9.93 lM and 6.64 lM, respectively, the assembly
rates slightly decreased to 45.99 mOD/min and 31.92 mOD/min.
These results indicated that these CA-binding compounds could in-
hibit capsid assembly in varying degrees and hence exhibit differ-
ent level of antiviral activities, and the stronger ability to inhibit
capsid assembly the compound had, the higher antiviral activity
it showed. We next investigated the ability of acylhydrazone com-
pounds 14f and 14i to inhibit HIV-1 capsid assembly under differ-
ent concentrations, the result revealed the assembly rates were
diminished in a dose-dependent manner, and increasing the doses
of compounds could enhance the inhibition of HIV-1 CA assembly.
The dose-dependence data for 14f and 14i are shown in Table 3.
In order to gain more insight into the interaction between this
new series of acylhydrazone derivatives and HIV-1 capsid protein,
molecular docking studies for the target compounds 5c, 10c, 10d,
13b, 14f and 14i were performed using AUTODOCK 4 software based
on the X-ray crystal structure of HIV-1 CA taken from the Protein
Data Bank (PDB ID: 2JPR).6b We found that all the molecules in
our series were docked into the binding pocket of the protein in
the same manner as CAP-1. In addition they have also occupied
two more other binding sites of the protein. The binding conforma-
tion with the lowest binding free energy (À13.49 kcal/mol) for the
representative compound 14i, one of the most active compounds,
is shown in Figure 4, where the 3,4-methylenedioxyphenyl group
of 14i inserts into the deep hydrophobic pocket vacated by
Phe32 just like the aromatic ring of CAP-1 (Fig. 4A). In addition,
ity with EC50 value of 0.1
lM in comparison with the sulfonylhy-
drazone 7b with EC50 value of 0.95
l
M.
HIV-1 CA can assemble into tubes, which leads to increase in
sample turbidity that can be measured spectrophotometrically.6a
In our study this assay was used to probe for potential inhibitory
effects of the acylhydrazone derivatives on in vitro capsid assem-
bly.20 As shown in Table 2, dissolution of native HIV-1 CA into
assembly buffer led to an increase in absorbance at an initial rate
of 59.16 mOD/min, while in the presence of CAP-1 the initial
assembly rate decreased to 33.37 mOD/min. The assembly rates
in the presence of the more potent compounds 5c, 10c, 10d, 10i,
and 13b decreased to 22.58 mOD/min, 18.90 mOD/min,
22.50 mOD/min, 20.78 mOD/min and 17.33 mOD/min, respec-
tively, which are consistent with the antiviral activities of these
compounds. Especially as shown in Table 2 and Figure 3, we found
that in the presence of 14f and 14i, which showed high activities
with EC50 values of 0.21 lM and 0.17 lM, respectively, the assem-
bly rates decreased to 10.56 mOD/min and 7.89 mOD/min, but in
Table 2
Effects of the target compounds on HIV-1 CA assembly
Compound
Assembly ratea (mOD/min)
it seemed that the
p–p stacking interaction between the phenyl
None
5a
5b
59.2 0.3
43.5 1.4
21.7 1.3
22.6 0.6
23.1 0.6
24.4 0.5
18.9 0.8
22.5 1.9
5c
Table 3
10a
10b
10c
10d
Effects of different concentrations of the target compounds on HIV-1 CA assembly
a
b
Compound
Concentration (
lM)
Ratio
Assembly rate (mOD/min)
14f
10
20
40
0.5:1
1:1
2:1
14.0 0.1
10.9 0.5
7.7 0.1
CAP-1
10i
13b
14b
14c
14f
33.4 1.2
20.8 0.5
17.3 0.5
46.0 1.2
31.9 1.2
10.6 0.7
7.9 0.6
14i
10
20
40
0.5:1
1:1
2:1
13.2 1.2
7.4 0.6
2.5 0.1
14i
a
Ratio, compound/HIV-1 CA.
a
b
Each value is reported as the mean SD (standard deviation) from two exper-
iments in duplicate.
Each value is reported as the mean SD (standard deviation) from two exper-
iments in duplicate.

2166
B. Tian et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2162–2167
Figure 4. The docking result of compound 14i in the complex with HIV-1 CA. (A) Stereo view of the complex of 14i with HIV-1 CA. (B) The interactions between 14i and CA in
this model in which only a subset of residues closest to the ligand is displayed for sake of clarity. This figure highlights essential interactions:
light yellow column), close contacts (indicated by colored circular regions).
p–p interaction (indicated by
6. (a) Tang, C.; Loeliger, E.; Kinde, I.; Kyere, S.; Mayo, K.; Barklis, E.; Sun, Y.; Huang,
M.; Summers, M. F. J. Mol. Biol. 2003, 327, 1013; (b) Kelly, B. N.; Kyere, S.; Kinde,
I.; Tang, C.; Howard, B. R.; Robinson, H.; Sundquist, W. I.; Summers, M. F.; Hill,
C. P. J. Mol. Biol. 2007, 373, 355.
of 3,4-methylenedioxyphenyl group and that of Phe32 further sta-
bilizes the binding. Moreover, the 4-methylphenyl group of 14i can
fully occupy the hydrophobic groove surrounded by Val24, Val27,
7. Prevelige, P. J. PCT Patent, WO2007048042, 2007.
Thr58 and Val59, and the benzyl side chain of 14i with
ration just fits into the other groove formed by Val26, Glu29,
Ala31 and Phe32. Figure 4B shows that besides the interaction
the binding between 14i and HIV-1 CA was dominated by Van der
Waals force, electrostatic and hydrophobic interactions. All of
these reinforce the binding affinity, and could explain the high bio-
logical activity of compound 14i theoretically.
In conclusion, a novel class of acylhydrazone derivatives was
synthesized and assessed for their antiviral activities and cytotox-
icities in vivo. The preliminary study shows that several com-
L-configu-
8. (a) Hoglund, S.; Su, J.; Reneby, S. S.; Vegvari, A.; Hjerten, S.; Sintorn, I. M.; Foster,
H.; Wu, Y. P.; Nystrom, I.; Vahlne, A. Antimicrob. Agents Chemother. 2002, 46,
3597; (b) Garzon, M. T.; Lidonmoya, M. C.; Barrera, F. N.; Prieto, A.; Gomez, J.;
Mateu, M. G.; Neira, J. L. Protein Sci. 2004, 13, 1512.
9. Dimmock, J. R.; Sidhu, K. K.; Tumber, S. D.; Basran, S. K.; Chen, M.; Quail, J. W.;
Yang, J.; Rozas, I.; Weaver, D. F. Eur. J. Med. Chem. 1995, 30, 287.
p–p
10. Prein, M.; Manley, P. J.; Padwa, A. Tetrahedron 1997, 3, 7777.
11. General procedure for the preparation of benzaldehyde N-(substituted benzoyl)
amino acid hydrazones 5(a–f). The appropriate N-(substituted benzoyl) amino
acid methyl ester 3(a–f) (20 mmol) was added in small portions to a stirred
solution of 85% hydrazine hydrate (5 ml) in 10 ml ethanol. The mixture was
heated under reflux for 6–8 h. while cooling to room temperature, the resulting
precipitate was filtered in vacuo, washed with cold water and dried to give the
corresponding hydrazide 4(a–f). To a magnetically suspension of the above
hydrazide (0.5 mmol) in anhydrous methanol (2 ml) was added benzaldehyde
(0.5 mmol) dropwise. Shortly the solution becomes homogeneous and within
minutes the resulting hydrazone began to precipitate. After the mixture was
stirred for further 1–2 h at room temperature, the precipitate was collected by
pounds which contain a L-phenylalanine moiety and an electron-
donating substituent in the left phenyl ring have good antiviral
activities. Structural modification of compound 10c produced a
series of new analogues, of which compound 14f and 14i exhibited
the most potent activities with IC50 values of 0.21 and 0.17 lM and
filtration, washed with
a small quantity of cold methanol and dried.
TI values of above 476 and 588, respectively. The structure–activity
relationships and molecular mechanisms of action of this novel
class of antiviral agents will be further studied in our laboratory.
Recrystallization of the reaction product from methanol gave the
corresponding hydrazone 5(a–f). The analytical data of representative
compounds 5a and 5d were as follows: Compound 5a: mp: 195–196 °C; 1H
NMR (300 MHz, DMSO-d6): d = 3.82 (s, 3H), 3.96, 4.40 (2d, 2H, J = 5.7 Hz), 7.03
(d, 2H), 7.43–7.48 (m, 3H), 7.69–7.72 (m, 2H), 7.86–7.90 (m, 2H), 8.01, 8.24 (2s,
1H), 8.57, 8.75 (2t, 1H, J = 5.7 Hz), and 11.52 (s, 1H); EI-MS (m/z): 311
(M+).Compound 5d: mp: 234–236 °C; 1H NMR (300 MHz, DMSO-d6): d = 4.03,
4.46 (2d, 2H, J = 6.0 Hz), 7.43–7.46 (m, 3H), 7.72 (t, 2H), 8.14 (d, 2H), 8.02, 8.24
(2s, 1H), 8.36 (d, 2H), 9.12, 9.28 (2t, 1H, J = 6.0 Hz), and 11.60 (s, 1H); EI-MS (m/
z): 326 (M+).
Acknowledgment
This project was supported by the National Natural Science
Foundation of China (No. 30670415).
12. (a) Cusack, N. J.; Reese, C. B.; Risius, A. C.; Roozpeikar, B. Tetrahedron 1976, 32,
2157; (b) Aggarwal, V. K.; Alonso, E.; Bae, I.; Hynd, G.; Lydon, K. M.; Palmer, M.
J.; Patel, M.; Porcelloni, M.; Richardson, J.; Stenson, R. A.; Studley, J. R.; Vasse, J.
L.; Winn, C. L. J. Am. Chem. Soc. 2003, 125, 10926.
13. Casaschi, A.; Grigg, R.; Sansano, J. M. Tetrahedron 2001, 57, 607.
14. Benzaldehyde N-(substituted benzenesulfonyl) amino acid hydrazones 10(a–i)
were prepared as described for benzaldehyde N-(substituted benzoyl) amino
acid hydrazones 5(a–f), starting from N-(substituted benzenesulfonyl) amino
acid methyl esters 8(a–i). The analytical data of representative compounds
10a, 10d and 10g were as follows:Compound 10a: mp: 158–159 °C; 1H NMR
(300 MHz, DMSO-d6): d = 2.37 (s, 1H), 3.54, 4.03 (2d, 2H, J = 5.7 Hz), 7.36–7.44
(m, 5H), 7.60–7.73 (m, 4H), 7.82, 8.02 (2t, 1H, J = 5.7 Hz), 7.93, 8.15 (2s, 1H),
and 11.38, 11.46 (2s, 1H); EI-MS (m/z): 331 (M+).Compound 10d: mp: 195–
References and notes
1. Louie, M.; Markowitz, M. Antiviral Res. 2002, 55, 15.
2. Flexner, C. Nat. Rev. Drug Disc. 2007, 6, 959.
3. Li, J.; Tang, S.; Hewlett, I.; Yang, M. Infect. Disorders-Drug Targets 2007, 7(3), 238.
4. (a) Tang, S.; Murakami, T.; Agresta, B. E.; Campbell, S.; Freed, E. O.; Levin, J. G. J.
Virol. 2001, 75, 9357; (b) Forshey, B. M.; Schwedler, U. K.; Sundquist, W. I.;
Aiken, C. J. Virol. 2002, 70, 8645; (c) Scholz, I.; Arvidson, B.; Huseby, D.; Barklis,
E. J. Virol. 2005, 79, 1470.
5. Sticht, J.; Humbert, M.; Findlow, S.; Bodem, J.; Muller, B.; Dietrich, U.; Werner,
J.; Krausslich, H.-G. Nat. Struct. Mol. Biol. 2005, 12, 671.

B. Tian et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2162–2167
2167
197 °C; 1H NMR (300 MHz, DMSO-d6): d = 3.61, 4.10 (2d, 2H, J = 5.1 Hz), 7.42–
7.44 (m, 3H), 7.61–7.83 (m, 6H), 7.93, 8.14 (2s, 1H, J = 5.1 Hz), 8.10, 8.26 (2t,
1H), and 11.42, 11.47 (2s, 1H); EI-MS (m/z): 397 (M+1+), 395 (MÀ1+).Compound
10g: mp: 225–227 °C; 1H NMR (300 MHz, DMSO-d6): d = 2.08 (s, 3H), 3.54, 4.03
(2d, 2H, J = 5.1 Hz), 7.42–7.44 (m, 4H), 7.61–7.76 (m, 6H), 7.93, 8.14 (2s, 1H,
J = 5.1 Hz), 10.32 (s, 1H), and 11.39, 11.47 (2s, 1H); EI-MS (m/z): 374 (M+).
15. Wellinga, K.; Grosscurt, A. C.; van Hes, R. J. Agric. Food Chem. 1977, 25, 987.
16. General procedure for the preparation of 1-(substituted benzenesulfonyl)-3-
11.26, 11.30 (2s, 1H); EI-MS (m/z): 435 (M+).Compound 14k: mp: 182–184 °C;
1H NMR (300 MHz, DMSO-d6): d = 2.25, 2.31 (2s, 3H), 2.68, 2.90 (2m, 2H), 3.93,
4.87 (2m, 1H), 6.62–6.65 (m, 1H), 6.87 (t, 1H, J = 3.9 Hz), 7.14–7.21 (m, 7H),
7.37 (m, 1H), 7.48 (m, 1H), 7.78, 7.82 (2s, 1H), 7.90 (s, 1H), 8.11, 8.29 (2d, 1H,
J = 9.0 Hz), and 11.28, 11.30 (2s, 1H); EI-MS (m/z): 411 (M+).
18. (a) Palla, G.; Predieri, G.; Domiano, P. Tetrahedron 1986, 42(13), 3649; (b) Li, Y.
J.; Wang, Y.; Jin, K.; Peng, Q. J.; Liu, S. N.; Zhang, Z. G. Chin. J. Magn. Reson. 2005,
22(1), 7.
phenyl-2-pyrazolines 13(a–b). To
a
mixture of 14 ml of 85% hydrazine
19. (a) Chuang, A. J.; Killarn, K. F., Jr.; Chuang, R. Y.; Rice, W. G.; Schaeffer, C. A.;
Mendeleyev, J.; Kun, E. FEBS Lett. 1993, 326, 140; (b) Antiviral inhibition assay:
Inhibition of SIV-induced syncytium in CEM174 cell cultures was measured in
hydrate, 7.2 ml of 50% aqueous sodium hydroxide and 18 ml of methanol, a
solution of of 3-dimethylaminopropiophenone hydrochloride (11) (20 mmol)
in 70 ml of methanol was added over 10 min. After refluxing for 45 min, the
methanol was distilled off at reduced pressure. The residue was dissolved in
CH2Cl2 and washed with water. Evaporation of the solvent gave the crude 3-
phenyl-2-pyrazoline (12), which could be used without further purification.
The crude 2-pyrazoline above obtained was dissolved in dry pyridine (20 ml).
To the resulting solution was added in small portions the appropriate
substituted benzenesulfonyl chloride (20 mmol). After the addition was
a
96-well microplate containing 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) cell formation was examined
microscopically. The EC50 was defined as the compound concentration
required to protect cells against the cytopathogenicity of SIV by 50%. The
TC50 assay was performed in uninfected CEM174 cells under the same
condition as above, and the value was determined as the concentration
required to inhibit CEM cells proliferation by 50% in MTT reduction assay.
20. Ultraviolet spectrophotometry analysis: Ultraviolet spectrophotometry assay
complete, the reaction mixture was heated on
a boiling water bath for
20 min, cooled and then poured onto crushed ice (30 g). The solid product
separated was filtered, washed with water, dried and crystallized from ethanol.
The analytical data of representative compound 13a was as follows:Compound
13a: mp: 178–180 °C; 1H NMR (300 MHz, CDCl3): d = 3.15 (t, 2H, J = 9.6 Hz),
3.74 (t, 2H, J = 9.6 Hz), 7.26–7.45 (m, 3H), 7.66–7.69 (m, 2H), 8.13–8.16(m, 2H),
and 8.35–8.38 (m, 2H); EI-MS (m/z): 331 (M+).
was performed at 350 nm on a Agilent 8453 spectrophotometer. A 1.0
concentrated ligand in DMSO (1 mM) was added to a 75 l aqueous solution
(2 ml of 5 M NaCl mixed with 1 ml of 200 mM NaH2PO4, pH 8.0), and then
added 25 capsid protein (40 M) to initiate the reaction. Spectral
ll of
l
l
l
l
17. The analytical data of representative compounds 14b and 14k were as
follows:Compound 14b: mp: 148–150 °C; 1H NMR (300 MHz, DMSO-d6):
d = 2,23–2.36 (m, 6H), 2.71, 2.90 (2m, 2H), 3.96, 4.99 (2m, 1H), 7.15–7.34 (m,
9H), 7.41–7.55 (m, 4H), 7.82, 7.99 (2s, 1H), 8.15, 8.27 (2d, 1H, J = 8.7 Hz), and
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.