Synthesis and Antiviral Evaluation of New N-acylhydrazones Containing Glycine Residue

Article abstract of DOI:10.1111/j.1747-0285.2010.01050.x

N-acylhydrazones containing glycine residue 3a-j and 8a-h were synthesized as HIV-1 capsid protein assembly inhibitors. The structures of the novel N-acylhydrazone derivatives were characterized using different spectroscopic methods. Antiviral activity de

Full text of DOI:10.1111/j.1747-0285.2010.01050.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2010.01050.x
Chem Biol Drug Des 2011; 77: 189–198
Research Article
Synthesis and Antiviral Evaluation of New
N-acylhydrazones Containing Glycine Residue
Baohe Tian1, Meizi He1, Zhiwu Tan1,
and have been studied extensively as potential therapeutic agents
in a number of pharmaceutical contexts. Furthermore, recent studies
have revealed that certain NAHs, such as N-(4-tert-butylbenzoyl)-2-
hydroxy-1-naphthaldehyde hydrazone (BBNH) and (E)-3,4-dihydroxy-
Shixing Tang2, Indira Hewlett2, Shuguang
Chen1, Yinxue Jin1 and Ming Yang1,*
1State Key Laboratory of Natural and Biomimetic Drugs, Peking
University Health Science Center, PO Box 261, Beijing 100191,
China
N¢-((2-methoxynaphthalen-1-yl)methylene)benzohydrazide (DHBNH),
also displayed potential anti-HIV activities through inhibiting the
RNA-dependent DNA polymerase and ⁄ or the RNase H (RNH) of
HIV-1 reverse transcriptase (RT) (Figure 1) (13–15). These com-
pounds represent a novel lead structure for the development of
antiviral agents.
2Center for Biologics Evaluation and Research, Food and Drug
Administration, Bethesda, MD 20892, USA
*Corresponding author: Prof Ming Yang, mingy@bjmu.edu.cn;
yangm@bjmu.edu.cn
More recently, Peter, Jr has reported a series of small-molecule HIV
inhibitors (16), and most of them contain a core structure of acylhyd-
razone moiety (Figure 2). These compounds were identified to bind to
HIV-1 capsid protein (CA) and inhibit capsid assembly during viral
maturation. HIV-1 CA plays important roles in the life cycle of HIV-1,
and its proper assembly is critical to the viral infectivity (17–21). Thus,
HIV-1 CA has become an attractive novel target of high priority for
anti-HIV (22), and NAH derivatives deserve further studies as poten-
tial HIV-1 CA assembly inhibitors. In addition, CAP-1, one of phenyl-
urea derivatives, has also been identified to bind to the N-terminal
domain (NTD) of HIV-1 CA and inhibit capsid assembly during viral
maturation (Figure 3) (23). Moreover, Kelly et al. have further deter-
mined the structure of the complex between CAP-1 and CA NTD (24),
which may be used as a tool for drug screening.
N-acylhydrazones containing glycine residue 3a–j
and 8a–h were synthesized as HIV-1 capsid protein
assembly inhibitors. The structures of the novel N-
acylhydrazone derivatives were characterized
using different spectroscopic methods. Antiviral
activity demonstrated that compound 8c bearing
4-methylphenyl moiety was the most active with
low cytotoxicity.
Key words: antiviral activity, HIV-1 capsid assembly, N-acylhydraz-
one derivatives, synthesis
Received 14 April 2010, revised 14 September 2010 and accepted for
publication 19 September 2010
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 (1). Over the past decade,
extensive efforts have been made to discover a large number of
molecules that can inhibit replication of HIV (2). Recently, major
progress has been made in the treatment of HIV infection by the
introduction of highly active antiretroviral therapy (HAART, a combi-
nation of nucleoside and non-nucleoside reverse transcriptase inhib-
itors, and ⁄ or protease inhibitors). Although HAART decreased the
number of AIDS cases dramatically, it is not able to eradicate HIV-1
from patients. Because of drug-related side-effects, inconvenient
dosing requirements, and ⁄ or the emergence of drug-resistant virus,
there is an urgent need for the development of novel classes of
anti-HIV agents targeting different steps of the viral life cycle (3–5).
In connection with our recent attempts to search for new HIV-1 CA
assembly inhibitors (25–27), a series of acylhydrazone derivatives
with new molecular structures were virtually screened for their abil-
ity to bind to HIV-1 CA using the docking technique. Molecular
docking studies were performed using AutoDock 4 software (28)
based on the X-ray crystal structure of HIV-1 CA NTD taken from
the Protein Data Bank (PDB ID: 2JPR) (24). Among them, two mole-
cules 3a and 8a containing glycine residue were found to be
docked into the same binding site of the protein in the similar man-
ner as CAP-1, especially in their binding models an aromatic ring
inserted into the same hydrophobic pocket as that of CAP-1 in the
complex between CAP-1 and HIV-1 CA (Figure 4). In comparison
with the binding free energy ()10.58 kcal ⁄ mol) and the inhibition
constant Ki (9.37 lM) of CAP-1, the low binding free energy
()11.05 and )12.41 kcal ⁄ mol, respectively) and inhibition constant
Ki (7.31 and 6.80 lM, respectively) of compounds 3a and 8a sug-
gested that they could tightly bind to HIV-1 CA and had good bind-
ing affinity with it in theory, because of hydrophobic forces,
electrostatic interactions, and hydrogen-bond interactions. Moreover,
the initial biological assay also showed that compounds 3a and 8a
both had potent antiviral activities. Based on this preliminary result,
N-Acylhydrazones (NAHs), which include the fragment (–CO–NH–
N=CH–), have attracted much attention in the last 20 years because
of their biological properties as well as their chelating properties
toward metal ions. Many of them have been reported to possess a
broad spectrum of biological activities, such as antifungal (6–8),
antimalarial (9), antitubercular (10), antiplatelet (11,12) activities,
189

Tian et al.
CH3O
N
HO
N
O
O
N
H
N
H
(CH )
3 3C
HO
OH
(E)-3,4-dihydroxy-N'-((2-methoxynaphthalen-
1-yl)methylene)benzohydrazide (DHBNH)
N-(4-tert-butylbenzoyl)-2-hydroxy-1-
naphthaldehyde hydrazone (BBNH)
Figure 1: Structure of N-(4-tert-butylbenzoyl)-2-hydroxy-1-naphthaldehyde hydrazone and (E)-3,4-dihydroxy-N¢-((2-methoxynaphthalen-1-
yl)methylene)benzohydrazide.
Br
CH3
Br
O
OH
OH
O
N
S
O
N
N
N
H
O
O
H
Br
OH
OH
Br
Br
Br
Br
O
H
N
N
N
N
OH
N
H
H
Br
CH3
NO2
Br
Figure 2: Some small-molecule HIV-1 capsid protein inhibitors.
trophotometer (Thermo Nicolet Corporation, Madison, WI, USA)
using KBr pallets. 1H NMR spectroscopic analysis was performed
on a Varian 300 MHz spectrometer using tetramethylsilane (TMS)
as internal standard, and NMR chemical shifts were reported in d
ppm (parts per million) relative to internal standard. Splitting pat-
terns were assigned as follows: s = singlet, d = doublet, t = triplet,
and m = multiplet. Elemental analyses (C, H, N) were determined
on a Vario EL III instrument (Elementar company, Hanau, Germany).
Mass spectra were performed on a VG ZAB-HS mass spectrometer,
and the values are reported in m ⁄ z. The reported chemical yields
were not optimized.
CH
3
CH
CH
3
3
O
N
S
N
H
N
H
O
Cl
Figure 3: Structure of CAP-1.
to further improve their inhibitory activity other molecular modifica-
tions were made in which different kinds of substituents were
introduced into the phenyl ring at the right end of the two mole-
cules. Starting from the structures of 3a and 8a, we have synthe-
sized their derivatives, and all the derivatives were tested for their
antiviral activities. In addition, some representative derivatives were
also assayed for their abilities to inhibit HIV-1 CA assembly in vitro
using an ultraviolet spectrophotometry assay. Moreover, the struc-
ture–activity relationships (SAR) of this series of NAH compounds
were investigated.
Methyl N-(4-toluenesulfonyl)-glycinate (1)
Triethylamine (8.3 mL, 60 mmol), 4-toluenesulfonyl chloride (3.81 g,
20 mmol) and a catalytic amount of N,N-dimethylaminopyridine
(DMAP) were added to a stirred suspension of methyl glycinate
hydrochloride (2.51 g, 20 mmol) in CH2Cl2 (50 mL) in an ice bath.
The reaction mixture was stirred at room temperature for 20 h
while 2 N hydrochloric acid (80 mL) was added slowly. The organic
phase was separated and washed with water (10 mL), dried with
anhydrous Na2SO4 and evaporated in vacuo giving a residue, which
was washed with a mixture of methanol and water (v ⁄ v: 1 ⁄ 10) and
dried to afford the corresponding methyl ester 1 as a white solid.
Yield: 84.4%; m.p. 91–93 ꢀC.
Experimental section
Chemistry
All solvents and reagents were purchased from commercial sources
and used directly without purification. Progress of all the reactions
was monitored by thin-layer chromatography (TLC) with silica gel
GF254 plates, which were visualized by UV light. All melting points
were determined with X-4 melting point apparatus and are uncor-
rected. Infrared spectra were recorded on a NEXUS-470 FTIR spec-
N-(4-toluenesulfonyl)-glycinyl hydrazide (2)
Compound 1 (2.43 g, 10 mmol) was added in small portions to a
stirred solution of 85% hydrazine hydrate (3 mL) in 5 mL ethanol.
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Synthesis and Antiviral Evaluation of New N-acylhydrazones
A
B
C
Figure 4: (A) Model of 3a docked
into HIV-1 capsid protein (CA) N-termi-
nal domain (NTD). (B) Model of 8a
docked into HIV-1 CA NTD. (C) Model
of CAP-1 docked into HIV-1 CA NTD.
The mixture was heated under reflux for 6 h. While cooling to room
temperature, the resulting precipitate was filtered in vacuo, washed
with cold water, and dried to give the corresponding hydrazide 2 as
a white solid. Yield: 86.4%; m.p. 155–156 ꢀC.
Acetophenone N-(4-toluenesulfonyl)-glycinyl
hydrazone (3b)
White crystalline solid; yield: 83.4%; m.p. 154–155 ꢀC; 1H NMR
(DMSO-d6): d = 2.20 (s, 3H), 2.37 (s, 3H), 3.70, 4.06 (2d, 2H,
J = 5.7 Hz), 7.36–7.46 (m, 5H), 7.68–7.77 (m, 4H), 7.82, 8.00 (2t,
1H, J = 5.7 Hz), and 10.33, 10.73 (2s, 1H); IR (KBr) m: 3245, 3194,
3086, 2924, 1686, 1596, 1376, 1332, 1167 cm)1; EI-MS (m ⁄ z): 345
(M+); Anal. calcd for C17H19N3O3S: C 59.11, H 5.54, N 12.17; found
C 58.87, H 5.48, N 12.06.
General procedures for the preparation of 3a,
3b, 3e, 3f, 3h, 3i (Method A)
To a magnetically stirred suspension of compound 2 (121.5 mg,
0.5 mmol) in anhydrous methanol (3 mL) was added the appropriate
aldehyde ⁄ ketone (0.5 mmol). Shortly, the solution became homoge-
neous and within minutes the resulting hydrazone began to precipi-
tate. After the mixture was stirred for 1–2 h more 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 corresponding hydrazone.
2-Fluorobenzaldehyde N-(4-toluenesulfonyl)-
glycinyl hydrazone (3e)
White crystalline solid; yield: 74.2%; m.p. 207–208 ꢀC; 1H NMR
(CDCl3): d = 2.38 (s, 3H), 3.54, 4.03 (2s, 2H), 7.26–7.31 (t, 2H,
J = 7.8 Hz), 7.37–7.42 (t, 2H, J = 7.8 Hz), 7.45–7.49 (m, 1H), 7.70–
7.73 (m, 2H), 7.77–7.88 (m, 1H), and 8.14, 8.40 (2s, 1H); IR (KBr) m:
3202, 3108, 2997, 2881, 1673, 1614, 1414, 1330, 1292, 1156 cm)1
;
Benzaldehyde N-(4-toluenesulfonyl)-glycinyl
hydrazone (3a)
EI-MS (m ⁄ z): 349 (M+); Anal. calcd for C16H16FN3O3S: C 55.00, H
4.62, N 12.03; found C 54.89, H 4.56, N 11.95.
White crystalline solid; yield: 87.5%; m.p. 158–159 ꢀC; 1H NMR
(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); IR (KBr) m: 3241,
3077, 2944, 2863, 1688, 1603, 1367, 1346, 1166 cm)1; EI-MS (m ⁄ z):
331 (M+); Anal. calcd for C16H17N3O3S: C 57.99, H 5.17, N 12.68;
found C 57.70, H 5.13, N 12.43.
4-Bromobenzaldehyde N-(4-toluenesulfonyl)-
glycinyl hydrazone (3f)
White crystalline solid; yield: 89.3%; m.p. 213–214 ꢀC; 1H NMR
(DMSO-d6): d = 2.37 (s, 3H), 3.53, 4.03 (2d, 2H, J = 3.0 Hz), 7.36–
7.38 (m, 2H), 7.56–7.72 (m, 6H), 7.85, 8.04 (2t, 1H, J = 3.0 Hz),
Chem Biol Drug Des 2011; 77: 189–198
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Tian et al.
7.90, 8.12 (2s, 1H), and 11.47, 11.53 (2s, 1H); IR (KBr) m: 3228,
3071, 2982, 2931, 1680, 1599, 1319, 1272, 1158 cm)1; EI-MS (m ⁄ z):
409 (M ) 1+), 411 (M + 1+); Anal. calcd for C16H16BrN3O3S: C
46.84, H 3.93, N 10.24; found C 46.90, H 3.94, N 10.33.
4H), 7.80, 8.00 (2t, 1H, J = 5.7 Hz), 7.87, 8.08 (2s, 1H), and 11.25,
11.34 (2s, 1H); IR (KBr) m: 3226, 3070, 2921, 2838, 1684, 1612,
1600, 1364, 1341, 1165 cm)1; EI-MS (m ⁄ z): 361 (M+); Anal. calcd
for C17H19N3O4S: C 56.50, H 5.30, N 11.63; found C 56.62, H 5.30,
N 11.66.
4-Nitrobenzaldehyde N-(4-toluenesulfonyl)-
glycinyl hydrazone (3h)
3-Methoxy-4-hydroxybenzaldehyde N-(4-
toluenesulfonyl)-glycinyl hydrazone (3g)
White crystalline solid; yield: 74.8%; m.p. 168–170 ꢀC; 1H NMR
(DMSO-d6): d = 2.31 (s, 3H), 3.79 (s, 3H), 3.52, 3.98 (2d, 2H,
J = 5.1 Hz), 6.81 (d, 1H), 7.03 (t, 1H), 7.19 (d, 1H), 7.37 (t, 2H), 7.71
(d, 2H), 7.81, 8.02 (2t, 1H, J = 5.1 Hz), 9.48 (brs, 1H), and 11.14,
11.26 (2s, 1H); IR (KBr) m: 3228, 3071, 2982, 2931, 2835, 1680,
1599, 1319, 1272, 1158 cm)1; EI-MS (m ⁄ z): 377 (M+); Anal. calcd
for C17H19N3O5S: C 54.10, H 5.07, N 11.13; found C 53.87, H 4.99,
N 11.03.
Light yellow crystalline solid; yield: 91.5%; m.p. 249–250 ꢀC; 1H
NMR (DMSO-d6): d = 2.38 (s, 3H), 3.58, 4.08 (2d, 2H, J = 4.8 Hz),
7.37–7.41 (m, 2H), 7.70–7.73 (m, 2H), 7.88–7.93 (m, 2H), 7.96, 8.03
(2s, 1H), 8.26–8.28 (m, 2H), 8.08, 8.29 (2t, 1H, J = 4.8 Hz), and
11.72, 11.77 (2s, 1H); IR (KBr) m: 3238, 3077, 2949, 1687, 1587,
1520, 1341, 1165 cm)1; EI-MS (m ⁄ z): 376 (M+); Anal. calcd for
C16H16N4O5S: C 51.06, H 4.28, N 14.89; found C 50.90, H 4.23, N
14.62.
2-Furaldehyde N-(4-toluenesulfonyl)-glycinyl
hydrazone (3i)
3,4-Methylenedioxybenzaldehyde N-(4-
White crystalline solid; yield: 90.7%; m.p. 165–167 ꢀC; 1H NMR
(DMSO-d6): d = 2.37 (s, 3H), 3.52, 3.94 (2d, 2H, J = 6.0 Hz), 6.60–
6.63 (m, 1H), 6.86–6.90 (m, 1H), 7.37–7.41 (m, 2H), 7.69–7.73 (m,
2H), 7.81 (s, 1H), 7.83, 7.87 (2s, 1H), 8.04, 8.33 (2t, 1H, J = 6.0 Hz),
and 11.36, 11.43 (2s, 1H); IR (KBr) m: 3275, 3135, 3085, 2944, 1681,
1599, 1439, 1376, 1308, 1149 cm)1; EI-MS (m ⁄ z): 321 (M+); Anal.
calcd for C14H15N3O4S: C 52.33, H 4.70, N 13.08; found C 52.11, H
4.63, N 12.92.
toluenesulfonyl)-glycinyl hydrazone (3j)
White crystalline solid; yield: 67.9%; m.p. 202–204 ꢀC; 1H NMR
(DMSO-d6): d = 2.38 (s, 3H), 3.51, 3.99 (2d, 2H, J = 6.0 Hz), 6.08 (s,
2H), 6.95–7.22 (m, 3H), 7.36–7.41 (m, 2H), 7.70–7.73 (m, 2H), 7.80,
8.00 (2t, 1H, J = 6.0 Hz), 7.82, 8.05 (2s, 1H), and 11.28, 11.36 (2s,
1H); IR (KBr) m: 3257, 3084, 2950, 2902, 1683, 1597, 1503, 1436,
1360, 1334, 1251, 1167, 1098 cm)1; EI-MS (m ⁄ z): 375 (M+); Anal.
calcd for C17H17N3O5S: C 54.39, H 4.56, N 11.19; found C 54.49, H
4.60, N 11.32.
General procedures for the preparation of 3c,
3d, 3g, 3j (Method B)
Ethyl 2-(2-naphthoxy) acetate (4)
To a stirred mixture of compound 2 (121.5 mg, 0.5 mmol) in anhy-
drous methanol (3 mL) was added the appropriate aldehyde ⁄ ketone
(0.5 mmol) and two drops of glacial acetic acid. The reaction mix-
ture was heated at reflux for 3–8 h. After cooling, the precipitate
was collected by vacuum filtration, washed with cold methanol and
dried. Recrystallization of the reaction product from methanol gave
the corresponding hydrazone.
A mixture of 2-naphthol (4.33 g, 30 mmol), ethyl 2-bromoacetate
(3.3 mL, 30 mmol) and anhydrous K2CO3 (4.15 g, 30 mmol) in ace-
tone (20 mL) was heated at reflux for 12 h. After cooling, inorganic
salts were separated by filtration, washed with a small quantity of
acetone, then the combined filtrate was evaporated in vacuo to dry-
ness. The residue was washed with water, subsequently with a
solution of petroleum ether ⁄ ethyl acetate (4:1) and dried to produce
the corresponding ethyl 2-(2-naphthoxy) acetate 4 as a pink solid.
Yield: 80%; m.p. 49 ꢀC.
4-Methylbenzaldehyde N-(4-toluenesulfonyl)-
glycinyl hydrazone (3c)
White crystalline solid; yield: 72.6%; m.p. 185–186 ꢀC; 1H NMR
(DMSO-d6): d = 2.32 (s, 3H), 2.36 (s, 3H), 3.51, 4.00 (2d, 2H,
J = 2.7 Hz), 7.22–7.37 (m, 4H), 7.48–7.56 (m, 2H), 7.69–7.71 (m,
2H), 7.81, 8.00 (2t, 1H, J = 2.7 Hz), 7.88, 8.09 (2s, 1H), and 11.31,
11.39 (2s, 1H); IR (KBr) m: 3285, 3238, 3085, 2972, 2920, 2856,
1675, 1609, 1558, 1395, 1325, 1160 cm)1; EI-MS (m ⁄ z): 345 (M+);
Anal. calcd for C17H19N3O3S: C 59.11, H 5.54, N 12.17; found C
58.96, H 5.51, N 12.13.
2-(2-Naphthoxy) acetyl hydrazide (5)
Compound 4 (4.6 g, 20 mmol) was added in small portions to a stir-
red solution of 85% hydrazine hydrate (6 mL) in 10 mL ethanol. The
mixture was heated under reflux for 6 h. While cooling to room
temperature, the resulting precipitate was filtered in vacuo, washed
with cold water and dried to give the corresponding hydrazide 5 as
a white solid. Yield: 92.6%; m.p. 174–175 ꢀC.
Methyl 2-naphthyloxyacetamidoacetate (6)
4-Methoxybenzaldehyde N-(4-toluenesulfonyl)-
glycinyl hydrazone (3d)
White crystalline solid; yield: 78.4%; m.p. 184–185 ꢀC; 1H NMR
(DMSO-d6): d = 2.37 (s, 3H), 3.79 (s, 3H), 3.51, 3.99 (2d, 2H,
J = 5.7 Hz), 6.97–7.01 (m, 2H), 7.36–7.41 (m, 2H), 7.54–7.73 (m,
Compound 5 (2.16 g, 10 mmol) was added to a stirred solution of
HOAc (60 mL), 1 N HCl (30 mL) and water (250 mL), then to the
resulted mixture in ice-salt bath a solution of NaNO2 (0.69 g,
10 mmol) in cold water (30 mL) was added. After stirring for
15 min in ice-salt bath, the yellow solution was formed. The result-
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Synthesis and Antiviral Evaluation of New N-acylhydrazones
ing azide was taken in cold ethyl acetate (300 mL), washed with 8.37, 8.56 (2t, 1H, J = 4.8 Hz), and 11.56, 11.64 (2s, 1H); IR (KBr) m:
3% solution of NaHCO3, washed with water, and finally dried
(Na2SO4). A solution of methyl glycinate hydrochloride (1.26 g,
10 mmol) in ethyl acetate (200 mL) containing 2 mL of Et3N was
stirred at 0 ꢀC for 30 min, filtered, and the filtrate was added to
the azide solution. The mixture was kept at )5 ꢀC for 12 h, then at
room temperature for additional 12 h, followed by washing with
0.5 N HCl, water, 3% solution of NaHCO3 and finally dried
(Na2SO4). The solution was evaporated to dryness to give product 6
as a light yellow oil. Yield: 53.7%.
3398, 3060, 2910, 2848, 1695, 1649, 1542, 1287, 1217, 1067 cm)1
;
EI-MS (m ⁄ z): 439 (M ) 1+), 441 (M + 1+); Anal. calcd for
C21H18BrN3O3: C 57.29, H 4.12, N 9.54; found C 57.37, H 4.19, N
9.61.
2-Furaldehyde 2-naphthyloxyacetamidoaceto-
hydrazone (8g)
Pink crystalline solid; yield: 72.1%; m.p. 205–206 ꢀC; 1H NMR
(DMSO-d6): d = 3.90, 4.27 (2d, 2H, J = 5.7 Hz), 4.70 (s, 2H), 6.62 (d,
1H), 6.90 (d, 1H), 7.26–7.50 (m, 4H), 7.80–7.88 (m, 4H), 7.89, 8.10
(2s, 1H), 8.32, 8.54 (2t, 1H, J = 5.7 Hz), and 11.42, 11.51 (2s, 1H);
IR (KBr) m: 3372, 3200, 3062, 2938, 1698, 1669, 1631, 1540, 1232,
2-Naphthyloxyacetamidoacetohydrazide (7)
Compound 6 (1.37 g, 5 mmol) was added in small portions to a stir-
red solution of 85% hydrazine hydrate (2 mL) in 3 mL ethanol. The
mixture was heated under reflux for 6 h. While cooling to room
temperature, the resulting precipitate was filtered in vacuo, washed
with cold water and dried to give the corresponding hydrazide 7 as
;
1184, 1068 cm)1 EI-MS (m ⁄ z): 351 (M+); Anal. calcd for
C19H17N3O4: C 64.95, H 4.88, N 11.96; found C 64.68, H 4.87, N
11.84.
1
a white solid. Yield: 81.7%; m.p. 179–180 ꢀC; H NMR (DMSO-d6):
d = 3.76 (d, 2H, J = 5.7 Hz), 4.23 (s, 2H), 4.65 (s, 2H), 7.26–7.50 (m,
4H), 7.79–7.88 (m, 3H), 8.39 (t, 3H, J = 5.7 Hz), and 9.11 (s, 1H);
EI-MS (m ⁄ z): 273 (M+).
General procedures for the preparation of 8c,
8d, 8e, 8h
2-Naphthyloxyacetamidoacetohydrazone of 4-methylbenzaldehyde ⁄ 4-
methoxybenzaldehyde ⁄ 4-hydroxybenzaldehyde ⁄ 3,4-methylenedioxy-
benzaldehyde (8c, 8d, 8e, 8h) were prepared according to the
procedure in method B described earlier, starting from compound 7.
General procedures for the preparation of 8a,
8b, 8f, 8g
2-Naphthyloxyacetamidoacetohydrazones of benzaldehyde ⁄ acetophe-
none ⁄ 4-bromobenzaldehyde ⁄ 2-furaldehyde (8a, 8b, 8f, 8g) were
prepared according to the procedure in method A described earlier,
starting from compound 7.
4-Methylbenzaldehyde 2-naphthyloxyacetamido-
acetohydrazone (8c)
White crystalline solid; yield: 63.9%; m.p. 208–209 ꢀC; 1H NMR
(DMSO-d6): d = 2.33 (s, 3H), 3.90, 4.33 (2d, 2H, J = 4.8 Hz), 4.69 (s,
2H), 7.23–7.59 (m, 8H), 7.80–7.89 (m, 3H), 7.96, 8.16 (2s, 1H), 8.36,
8.55 (2t, 1H, J = 4.8 Hz), and 11.42, 11.51 (2s, 1H); IR (KBr) m: 3398,
3086, 2973, 2913, 1691, 1654, 1542, 1218, 1185, 1062 cm)1; EI-MS
(m ⁄ z): 375 (M+); Anal. calcd for C22H21N3O3: C 70.38, H 5.64, N
11.19; found C 70.17, H 5.53, N 11.02.
Benzaldehyde 2-naphthyloxyacetamidoaceto-
hydrazone (8a)
White crystalline solid; yield: 74.8%; m.p. 201–202 ꢀC; 1H NMR
(DMSO-d6): d = 3.92, 4.35 (2d, 2H, J = 5.7 Hz), 4.70 (s, 2H), 7.23–
7.50 (m, 7H), 7.68–8.00 (m, 5H), 8.05, 8.21 (2s, 1H), 8.36, 8.55 (2t,
1H, J = 5.7 Hz), and 11.48, 11.57 (2s, 1H); IR (KBr) m: 3347, 3204,
3065, 2916, 1672, 1631, 1540, 1217, 1183, 1070 cm)1; EI-MS (m ⁄ z):
361 (M+).
4-Methoxybenzaldehyde 2-naphthyloxyacetamido-
acetohydrazone (8d)
White crystalline solid; yield: 67.2%; m.p. 187–188 ꢀC; 1H NMR
(DMSO-d6): d = 3.79 (s, 3H), 3.90, 4.32 (2d, 2H, J = 5.4 Hz), 4.69 (s,
2H), 7.00 (t, 2H), 7.26–7.50 (m, 4H), 7.56 (d, 2H), 7.80–7.89 (m, 3H),
7.93, 8.14 (2s, 1H), 8.34, 8.53 (2t, 1H, J = 5.4 Hz), and 11.31, 11.45
(2s, 1H); IR (KBr) m: 3386, 3201, 3089, 2962, 2925, 1690, 1655,
1608, 1253, 1184, 1033 cm)1; EI-MS (m ⁄ z): 391 (M+); Anal. calcd
for C22H21N3O4: C 67.51, H 5.41, N 10.74; found C 67.29, H 5.37, N
10.73.
Acetophenone 2-naphthyloxyacetamidoaceto-
hydrazone (8b)
White crystalline solid; yield: 70.5%; m.p. 221–222 ꢀC; 1H NMR
(DMSO-d6): d = 2.26 (s, 3H), 4.04, 4.38 (2d, 2H, J = 5.7 Hz), 4.71 (s,
2H), 7.26–7.50 (m, 7H), 7.77–7.89 (m, 5H), 8.35, 8.51 (2t, 1H,
J = 5.7 Hz), and 10.50, 10.81 (2s, 1H); IR (KBr) m: 3413, 3197, 3060,
2928, 1678, 1631, 1522, 1219, 1187, 1052 cm)1; EI-MS (m ⁄ z): 375
(M+); Anal. calcd for C22H21N3O3: C 70.38, H 5.64, N 11.19; found C
70.13, H 5.53, N 11.07.
4-Hydroxybenzaldehyde 2-naphthyloxyacetamido-
acetohydrazone (8e)
White crystalline solid; yield: 61.0%; m.p. 152–154 ꢀC; 1H NMR
(DMSO-d6): d = 3.89, 4.30 (2d, 2H, J = 6.0 Hz), 4.69 (s, 2H), 6.80–
6.83 (m, 2H), 7.26–7.53 (m, 6H), 7.80–7.87 (m, 3H), 7.89, 8.09 (2s,
1H), 8.32, 8.51 (2t, 1H, J = 6.0 Hz), 9.91 (s, 1H), and 11.26, 11.36
(2s, 1H); IR (KBr) m: 3389, 3163, 3060, 2924, 1659, 1603, 1276,
4-Bromobenzaldehyde 2-naphthyloxyacetamido-
acetohydrazone (8f)
White crystalline solid; yield: 78.3%; m.p. 219–220 ꢀC; 1H NMR
(DMSO-d6): d = 3.92, 4.34 (2d, 2H, J = 4.8 Hz), 4.69 (s, 2H), 7.26–
7.50 (m, 3H), 7.64 (s, 4H), 7.80–7.89 (m, 4H), 7.97, 8.18 (2s, 1H),
1216, 1053 cm)1
;
EI-MS (m ⁄ z): 377 (M+); Anal. calcd for
Chem Biol Drug Des 2011; 77: 189–198
193

Tian et al.
C21H19N3O4: C 66.83, H 5.07, N 11.13; found C 67.14, H 5.14, N
11.20.
the incubation was changed to 30 ꢀC for an additional 12 h. The
cells were collected by centrifugation and resuspended in 80 mL
lysis buffer (50 mM Mes, 10 mM MgCl2, 100 mM NaCl, 1 mM EDTA,
1 mM 1,4-Dithiothreitol). Cell lysis was carried on an Ultrasonic Dis-
rupter, and the cell debris was separated by centrifugation. Then
the CA was precipitated from the supernatant with 25% saturated
ammonium sulfate; the precipitate was collected by centrifugation,
resuspended in 12 mL dialysis buffer (50 mM Tris ⁄ HCl, 30 mM NaCl,
1 mM EDTA, 1 mM DTT) and then dialyzed against 300 mL of the
same buffer overnight. Cellulose DE-52 was equilibrated with the
dialysis buffer and the crude CA was loaded, eluted with the same
buffer. Flow-through fractions were analyzed by denaturing
SDS ⁄ polyacrylamide gel and the purified CA fraction was precipi-
tated again with 50% saturated ammonium sulfate, collected by
centrifugation, dialyzed against 300 mL H2O for two times. Protein
concentrations were determined by the Bradford method.
3,4-Methylenedioxybenzaldehyde 2-
naphthyloxyacetamidoacetohydrazone (8h)
White crystalline solid; yield: 71.4%; m.p. 210–212 ꢀC; 1H NMR
(DMSO-d6): d = 3.89, 4.31 (2d, 2H, J = 5.1 Hz), 4.69 (s, 2H), 6.08 (s,
2H), 6.95–6.99 (m, 1H), 7.10–7.13 (m, 1H), 7.26–7.50 (m, 5H), 7.80–
7.86 (m, 3H), 7.89, 8.11 (2s, 1H), 8.36, 8.54 (2t, 1H, J = 5.1 Hz), and
11.38, 11.40 (2s, 1H); IR (KBr) m: 3392, 3213, 3084, 2907, 1700,
1648, 1599, 1257, 1218, 1041 cm)1; EI-MS (m ⁄ z): 405 (M+); Anal.
calcd for C22H19N3O5: C 65.18, H 4.72, N 10.37; found C 64.98, H
4.74, N 10.32.
Biological activity
Antiviral activity and cytotoxicity
Ultraviolet spectrophotometry analysis
Inhibition of Simian Immunodeficiency Virus-induced syncytium in
CEM174 cell cultures was measured in a 96-well microplate con-
taining 1 · 105 CEM cells ⁄ mL infected with 100 TCID50 of Simian
Immunodeficiency Virus (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 methyl thiazolyl tetrazolium
reduction assay.
Ultraviolet spectrophotometry assay was performed at 350 nm on
an Agilent 8453 spectrophotometer. A 1.0 lL of concentrated ligand
in DMSO (1 mM) was added to a 75 lL aqueous solution (2 mL of
5 M NaCl mixed with 1 mL of 200 mM NaH2PO4, pH 8.0), and then
added 25 lL CA (40 lM) to initiate the reaction. Spectral measure-
ments were made every 10 seconds, following a short initial delay
to allow sample equilibration. Relative assembly rates were esti-
mated from initial slopes of the plots of absorbance versus time.
Results and discussion
Chemistry
The synthetic routes for the title compounds 3a–j are described in
Scheme 1. The 4-toluenesulfonyl chloride was first reacted with
methyl glycinate hydrochloride in the presence of Et3N, to give the
key intermediate methyl N-(4-toluenesulfonyl)-glycinate 1 in good
yield (29). This intermediate 1 was then converted to the corre-
sponding hydrazide 2 by refluxing with hydrazine hydrate in ethanol.
Purification of the recombinant CA
A 2-liter culture was grown to 37 ꢀC in Lysogeny broth (LB), a nutri-
tionally rich medium containing ampicillin (100 ug ⁄ mL) to an A600 of
0.8, isopropyl-b-D-thiogalactopyranoside was added to 0.2 mM and
O
O
O
S
O
S
OCH3
a
N
H
b
CH3
Cl
O
CH3
1
R
H
N
O
O
O
O
H
S
S
N
N
c or d
NH2
N
H
N
Ar
H
O
O
CH3
CH3
2
3
b. R = CH3, Ar = Ph
a. R = H, Ar = Ph
c. R = H, Ar = 4-CH3Ph
e. R = H, Ar = 2-FPh
g. R = H, Ar = 3-CH3O,4-OHPh
i. R = H, Ar =
d. R = H, Ar = 4-CH3OPh
f. R = H, Ar = 4-BrPh
h. R = H, Ar = 4-NO2Ph
O
j. R = H, Ar =
O
O
Scheme 1: Synthesis of 4-toluenesulfonylhydrazone derivatives containing glycine residue. Reagents and conditions: (a) glycine methyl
ester hydrochloride, Et3N, CH2Cl2, 0 ꢀC to rt, 20 h; (b) 85% NH2NH2ÆH2O, CH3CH2OH, reflux, 6 h; (c) RCOAr, anhydrous CH3OH, rt, 1–2 h; (d)
RCOAr, anhydrous CH3OH, glacial acetic acid, reflux, 3–8 h.
194
Chem Biol Drug Des 2011; 77: 189–198

Synthesis and Antiviral Evaluation of New N-acylhydrazones
The condensation of the hydrazide 2 with various aromatic alde-
hydes and ketones in methanol afforded the title compounds 3a–j.
ular ion recorded in the MS was also in agreement with the molec-
ular weight of the desired compounds.
The title compounds 8a–h were prepared according to the syn-
thetic strategy outlined in Scheme 2. 2-Naphthol was first O-alkyl-
ated with ethyl 2-bromoacetate in the presence of anhydrous
K2CO3 in acetone at refluxing temperature, to give ethyl 2-(2-
naphthoxy) acetate 4, which was reacted with hydrazine hydrate
in ethanol to obtain the corresponding hydrazide 5 (30). The
hydrazide 5 was then treated with aqueous NaNO2 in HOAc and
1 N HCl at )5 ꢀC to obtain the azide via the azide-coupling
method, which was reacted directly with methyl glycinate hydro-
chloride in ethyl acetate in the presence of Et3N at 0 ꢀC for
30 min to give, after neutralization, the desired intermediate 6
(31). Treatment of 6 with hydrazine hydrate in ethanol under
reflux afforded the corresponding hydrazide 7 as key intermediate,
which underwent condensation with a variety of aromatic alde-
hydes and ketones, resulting in the formation of the corresponding
acylhydrazone derivatives 8a–h by a route analogous to that for
3a–j.
Antiviral activity and cytotoxicity
The title compounds 3a–j and 8a–h were assayed for their antivi-
ral activities using SIV-induced syncytium in CEM174 cells, and their
cytotoxicities were also evaluated in vitro in uninfected CEM174
cells. Their EC50 (antiviral activity), TC50 (cytotoxicity), and therapeu-
tic index (TI) are listed in Table 1. As shown in Table 1, each of the
tested compounds possessed an EC50 value within the range from
0.47 lM to 20.2 lM, and a TC50 value ranging from 29.4 lM to
more than 100 lM. Analyzing the activities of the synthesized com-
pounds, the following SAR was observed.
In series 3a–j, compared with the leading compound 3a
(EC50 = 1.5 lM), all other derivatives showed equivalent or
decreased antiviral activities. Although the introduction of halogen
groups (F in 3e, Br in 3f) into the phenyl ring at the right end
resulted in approximately equal activities, their cytotoxicities were
both significantly decreased with TC50 values of more than 100 lM.
For example, 3e and 3f showed EC50 values of 1.7 and 2.1 lM,
resulting in TI values of more than 58.8 and 47.6, respectively. Sim-
ilarly, the substituents, such as methyl and methoxy groups on the
phenyl ring in compounds 3c and 3d, caused slight decrease in an-
tiviral activities, but both of them showed lower cytotoxicity with
TC50 value of more than 100 lM. Only compound 3i in which the
phenyl ring in 3a was replaced with the furyl ring displayed slightly
higher activity (EC50 = 1.37 lM), but was more cytotoxic with a
TC50 value of 31.6 lM when compared with 3a.
The purity of all the products was determined by TLC, melting
points and elemental analysis. The physical and spectral properties
of previously reported compounds, such as melting points, were in
accord with literature values, and the structures of the novel com-
1
pounds were confirmed by H NMR, EI-MS and IR. According to the
literature, this class of acyl hydrazones are in the form of E geo-
metrical isomer for C=N double bond in DMSO-d6, and the E geo-
metrical isomer undergoes a rapid cis ⁄ trans amide equilibrium at
room temperature (32,33), which is consistent with the analytical
results of the 1H NMR spectra of our synthesized derivatives, in
which two sets of methylene, imine and amide protons signals of
cis ⁄ trans amide conformers were observed. Furthermore, the molec-
The test results of the series 8a–h indicated that all the deriva-
tives with different substituted groups on the phenyl ring exhibited
O
O
OH
a
b
OC2H5
4
O
O
O
OCH3
O
NH2
d
c
N
H
N
H
O
6
5
R
O
O
H
N
H
N
O
O
e or f
N
NH2
N
Ar
N
H
H
O
O
8
7
a. R = H, Ar = Ph
b. R = CH3, Ar = Ph
c. R = H, Ar = 4-CH3Ph
e. R = H, Ar = 4-OHPh
g. R = H, Ar =
d. R = H, Ar = 4-CH3OPh
f. R = H, Ar = 4-BrPh
O
O
h. R = H, Ar =
O
Scheme 2: Synthesis of 2-naphthoxyacyl hydrazone derivatives containing glycine residue. Reagents and conditions: (a) ethyl 2-bromoace-
tate, K2CO3, acetone, reflux, 12 h; (b) 85%NH2NH2ÆH2O, CH3CH2OH; reflux, 6 h; (c) (1) NaNO2, HCl, AcOH, H2O; (2) glycine methyl ester hydro-
chloride, Et3N, ethyl acetate, )5 ꢀC to rt; (d) 85%NH2NH2ÆH2O, CH3CH2OH, reflux, 6 h; (e) RCOAr, anhydrous CH3OH, rt, 1–2 h; (f) RCOAr,
anhydrous CH3OH, glacial acetic acid, reflux, 3–8 h.
Chem Biol Drug Des 2011; 77: 189–198
195

Tian et al.
Table 1: Antiviral activity and cytotoxicity of acylhydrazone
Table 2: Effects of title compounds on capsid assembly
Assembly ratea
derivatives on SIV-induced syncytiuma
Compound
EC50 (lM)b
TC50 (lM)c
TId
Compound
(mOD ⁄ min)
3a
3b
3c
3d
3e
3f
1.5
1.53
2.5
3.9
1.7
2.1
20.2
6.1
1.37
5.8
2.25
5.42
0.47
1.1
83.5
30.9
>100
>100
>100
>100
53.8
32.9
31.6
>100
77.0
45.0
>100
>100
29.4
60.8
>100
67.9
55.7
20.2
>40
>25.6
>58.8
>47.6
2.7
None
3a
60.6 € 12.1
22.2 € 0.5
8.7 € 0.2
28.0 € 0.7
26.1 € 0.5
31.4 € 1.2
8.1 € 0.2
4.5 € 0.1
6.6 € 0.1
28.7 € 1.2
3c
3j
3e
CAP-1
8a
8c
3g
3h
3i
5.4
23.1
>17.2
34.2
8.3
>212.8
>90.9
7.0
60.2
>18.8
34.0
8d
8e
3j
8a
8b
8c
8d
8e
8f
aEach value is reported as mean € SD (standard deviation) from two experi-
ments in duplicate.
4.2
1.01
5.33
2
capsid assembly. The assembly rate slowed when the molecule
bound strongly because the strongly binding compound prevented or
slowed the assembly. 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 60.6 € 12.1 mOD ⁄ min, while in the presence of
CAP-1 the initial assembly rate decreased to 31.4 € 1.2 mOD ⁄ min.
The assembly rates in the presence of our synthesized compounds
decreased to the range of 4.5 € 0.1 to 28.7 € 1.2 mOD ⁄ min. Espe-
cially, we found that in the presence of 8c and 8d, which showed
high activities with EC50 values of 0.47 and 1.1 lM, the assembly
rates decreased to 4.5 € 0.1 mOD ⁄ min and 6.6 € 0.1 mOD ⁄ min,
respectively, but in the presence of 3j and 8e, which showed low
activities with EC50 values of 5.8 and 4.2 lM, the assembly rates
slightly decreased to 28.0 € 0.7 and 28.7 € 1.2 mOD ⁄ min, respec-
tively. These results, in which the abilities to inhibit capsid assem-
bly were consistent with the antiviral activities of these
compounds, showed the CA-binding compounds could inhibit capsid
assembly in varying degrees and hence exhibit different levels of
antiviral activities.
8g
8h
aThis assay used the antiviral drug Indinavir at 1 · 10)6 M as positive con-
trol.
bAntiviral activity, concentration that inhibits viral replication by 50%.
cCytotoxicity, concentration that is toxic to 50% of SIV-induced syncytium.
dTherapeutic index, TC50 value divided by EC50 value (TC50 ⁄ EC50).
moderate to good antiviral activities with EC50 values in the range
of 0.47–5.42 lM, in which the compound 8c was found to be the
most active. Compounds 8c and 8d (EC50 = 0.47 and 1.1 lM) in
which methyl and methoxy groups were introduced into the phenyl
ring were greater than the leading compound 8a (EC50 = 2.25 lM).
Furthermore, they possessed lower cytotoxicities with TC50 values
of more than 100 lM, safer than compound 8a. Bromosubstitution
on the phenyl ring led to compound 8f (EC50 = 1.01 lM), which
showed over twofold higher inhibitory activity than 8a, but its cyto-
toxicity was similar to 8a. In addition, contrary to 3i, the replace-
ment with furyl ring in compound 8g, gave rise to a significant
decrease in both activity and toxicity (EC50 = 5.33 lM,
TC50 > 100 lM).
Conclusion
To summarize, we have designed and prepared a novel series of
NAH derivatives containing glycine residue, most of which dem-
onstrated potent antiviral activities. Among them, derivative 8c
showed the highest inhibitory activity with an EC50 value of
0.47 lM and TI value of more than 212.8. Ultraviolet spectropho-
tometry analysis confirmed that the abilities of the title com-
pounds to inhibit capsid assembly showed a strong correlation
with their antiviral activities. Therefore, this series should be
evaluated as a suitable hit for the design of new anti-HIV
agents, and further research in this area is in progress in our
laboratory.
These structure–activity relationship analyses indicated that the
lipophilic groups on the phenyl ring were critical for the high antivi-
ral activity, such as 3c, 3e, 3f, 8e, and 8f, which might be
explained by the increase in the molecular hydrophobicity that
enhanced the hydrophobic interaction between the molecule and
HIV-1 CA, thus resulting in better antiviral activity. Furthermore, the
proper molecular size caused by the presence of these substituents
could also contribute to their high affinity with the protein.
Inhibition of capsid assembly in vitro
Among all the title compounds, eight representative derivatives
were selected to be further evaluated for their inhibitory effects on
in vitro capsid assembly. In accordance with the literature (23), in
our study the turbidity in direct relation to the assembly rate was
monitored using ultraviolet spectrophotometry. The assembly rate
indicates how strongly a molecule binds to the protein and inhibits
Acknowledgment
This project was supported by the National Natural Science Founda-
tion of China (No. 30670415).
196
Chem Biol Drug Des 2011; 77: 189–198

Synthesis and Antiviral Evaluation of New N-acylhydrazones
ture with RNase H inhibitor dihydroxy benzoyl naphthyl hydraz-
one bound at a novel Site. ACS Chem Biol;1:702–712.
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