Synthesis and anti-human immunodeficiency virus type 1 activity of (e)-n-phenylstyryl-n-alkylacetamide derivatives

Article abstract of DOI:10.3390/molecules14093176

series of (E)-N-phenylstyry l-N-alkylacetamides, 5, were synthesized by direct reduction-acetylation of β-arylnitroolefins, followed by N-alkylation. The title compounds were characterized by ¹H-NMR, EIMS and IR analysis. All the synthesized co

Full text of DOI:10.3390/molecules14093176

Molecules 2009, 14, 3176-3186; doi:10.3390/molecules14093176
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Anti-Human Immunodeficiency Virus Type 1
Activity of (E)-N-Phenylstyryl-N-alkylacetamide Derivatives
Pi Cheng 1,*, Ji-Jun Chen 2, Ning Huang 3, Rui-Rui Wang 3, Yong-Tang Zheng 3 and
Yi-Zeng Liang 1
1
School of Chemistry and Chemical Engineering, Central South University, Changsha 410083,
China
2
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of
Botany, the Chinese Academy of Sciences, Kunming 650204, China
3
Laboratory of Molecular Immunopharmacology, Key Laboratory of Animal Models and Human
Diseases Mechanisms, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming
650223, China
* Author to whom correspondence should be addressed; E-mail: picheng55@126.com;
Tel.: +86-731-8836376.
Received: 1 July 2009; in revised form: 30 July 2009 / Accepted: 26 August 2009 /
Published: 26 August 2009
Abstract: A series of (E)-N-phenylstyryl-N-alkylacetamides, 5, were synthesized by direct
reduction-acetylation of β-arylnitroolefins, followed by N-alkylation. The title compounds
1
were characterized by H-NMR, EIMS and IR analysis. All the synthesized compounds
were assayed as HIV-1 non-nucleoside reverse transcriptase inhibitors. A SAR study
revealed that when group R1 in 5 was ortho-substituted, the resulting compounds showed
better inhibitory activities against HIV-1 RT. Among the tested compounds, 5i (R1 = 2-Br,
R2 = 3,5-difluorobenzyl) exhibited the highest enzyme activity, with a 88.89% inhibitory
ratio against HIV-1 reverse transcriptase at the tested concentration. Further cell-based
anti-HIV-1 assays showed that compound 5i exhibited a SI value of 29 with an EC50 value
of 4 μM in C8166 cells.
Keywords: (E)-N-phenylstyryl-N-alkylacetamides; synthesis; reverse transcriptase; anti-
HIV-1 activity

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1. Introduction
Acquired immunodeficiency syndrome (AIDS), a disease resulting from infection with Human
Immunodeficiency Virus type 1 (HIV-1), is one of the world’s most serious health problems. Three
essential enzymes are encoded by the HIV-1 pol gene: (1) HIV-1 reverse transcriptase (RT), (2)
integrase (IN), and (3) protease (PR). These three enzymes compose the most important and validated
targets for developing novel antiretroviral agents [1–4]. Among RT inhibitors, non-nucleoside reverse
transcriptase inhibitors (NNRTIs) remain a high priority for medical research because of their
structural diversity [5–9].
Structure simplification of natural product has provided us an efficient way to find new and less
toxic anti-HIV-1 lead compounds [10]. In a previous paper [11], we reported that (E)-N-phenylstyryl
acetamides (Scheme 1), which contain the basic structural skeleton of hamigeroxalamic acid, could
exhibit HIV-1 RT and showed for the first time moderate anti-HIV-1 activities in cell culture.
Fundamental structure-activity relationships (SAR) were revealed and it was proposed that when
group R1 was an ortho-substituent, the resulting compounds showed better inhibitory activities against
HIV-1 RT. The ortho-substituted analogues thus represent a new template for further structure
modification with a view to finding more active anti-HIV-1 analogues.
Scheme 1. Structure of hamigeroxalamic acid, (E)-N-phenylstyryl acetamide and (E)-N-
phenylstyryl-N-alkylacetamide.
O
R1
H
R2
N
R1
H
N
N
OH
O
O
O
HO
(E)-N-phenylstyryl acetamide
hamigeroxalamic acid
(E)-N-phenylstyryl-N-alkylacetamide
In this paper, a series of (E)-N-phenylstyryl-N-alkylacetamides (Scheme 1) were synthesized and
evaluated in vitro as NNRTIs of HIV-1. Besides, syncytium reduction and cytotoxicity activities of the
title compounds in cell based assay are provided. Detailed synthetic procedures and the anti-HIV-1
activities of these N-phenylstyryl-N-alkylacetamides are reported.
2. Results and Discussion
2.1. Chemistry
The synthetic route to the (E)-N-phenylstyryl-N-alkylacetamides 5 are shown in Scheme 2. First,
(E)-β-nitroolefins 2 were produced by refluxing various benzaldehydes with ammonium acetate and
nitromethane. Compounds 3 were synthesized through a one-pot reduction of compounds 2 using
Fe/AcOH/Ac2O. In this reaction, diacetyl compounds 4 were isolated as byproducts. These could also
be converted into compounds 3 by treatment of potassium hydroxide. Treatment of compounds 3 with
sodium hydride and various alkyl halides provided the title compounds 5 in moderate yields.
Structures of groups R1 and R2 were given in Scheme 2, and R1 were mainly substituted in the ortho-
position on the aromatic ring.

Molecules 2009, 14
Scheme 2. Synthetic route for (E)-N-phenylstyryl-N-alkylacetamides (5).
3178
R1
R1
(a)
NO2
CHO
R1 = 2-F
2-Cl
2
1
2-Br
(b)
2-methyl
2,4-dimethoxyl
O
R1
R1
H
N
N
(c)
R2 = methyl
O
O
O
3
ethyl
allyl
benzyl
2,6-difluorobenzyl
3,5-difluorobenzyl
2,6-dichlorobenzyl
4
(d)
R2
N
R1
5
Reagents and conditions: (a) NH4OAc, MeNO2, reflux; (b)Fe/AcOH/Ac2O, reflux, N2; (c) KOH,
MeOH; (d) NaH, R2X, THF, 0 °C.
A possible mechanism for the one-pot synthesis of (E)-N-phenylstyryl acetamides 3 by direct
reduction-acetylation of (E)-β-phenyl-nitroolefins 2 is illustrated in Figure 2.
Figure 2. One-pot synthesis of N-phenylstyryl acetamides 3.
R1
NH2
R1
R1
NH4OAc
CH3NO2
Fe
NO2
CHO
AcOH
Ac2O
6
7
R1
NHOH
2
1
OH-
O
R1
R1
Ac2O
NHCOCH3
N
O
3
Fe
AcOH
Ac2O
Ac2O
R1
4
NHOCOCH3
8
Compounds 2 would first be reduced to compounds 6 and/or 7. Compounds 7 would be trapped in
the presence of acetic anhydride to form oxime acetates 8, as would compounds 6, to yield the desired
(E)-N-phenylstyryl acetamides 3. Oxime acetates 8 should be in turn reduced to 3 under the reaction
conditions. Compounds 3 might be further acetylated to the diacetyl compound 4, a reaction that could
be reversed by treatment with methanolic potassium hydroxide. It was worth noting that the trans-

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conformation of the carbon-carbon double bonds was maintained in this reduction–acetylation
reaction.
Table 1. Structures and anti-HIV-1 activities of (E)-N-phenylstyryl-N-alkylacetamides 5.a
RT
c
d
e
IC50
EC50
CC50
(μM)
SIf
Entry R1
R2
inhibitio
n (%)b
45.42
(μM)
(μM)
(CC50/EC50)
5a
5b
5c
3-Br
methyl
methyl
methyl
ND
ND
128 ± 20
ND
409 ± 106 3.2
4-Br
2-Br
16.59
ND
ND
85.27
339 ± 107 60 ±10
>789
>13.2
2-Br
2-Br
2-Br
2-Br
2-Br
2-Br
2-Br
2-Cl
ethyl
46.37
47.76
60.55
47.56
ND
48 ± 8
75 ± 13
8 ± 1
358 ± 88
7.5
5d
5e
allyl
ND
337 ± 154 4.5
acetyl
benzyl
320 ± 22
ND
85 ± 17
10.6
5f
9 ± 2
51 ± 13
5.7
5.9
29
5g
5h
5i
2,6-difluoro-benzyl 57.33
3,5-difluoro-benzyl 88.89
2,6-dichloro-benzyl 30.34
295 ± 9
97 ± 13
ND
29 ± 4
4 ± 0.5
49 ± 10
96 ± 20
91 ± 15
118 ± 28
172 ± 73
116 ± 13
190 ± 36
441 ± 18
424 ± 22
536 ± 41
664 ± 79
3.9
4.6
4.7
4.5
5.5
5j
methyl
ethyl
75.22
59.10
29.95
61.69
38.51
79.80
40.30
69.45
34.73
36.62
NDi
434 ± 44
521 ± 11
ND
5k
5l
2-Cl
2-Cl
allyl
5m
5n
5o
5p
5q
5r
2-F
methyl
allyl
477 ± 107 130 ± 33
2-F
ND
261 ± 40
116 ± 11
273 ± 43
32 ± 7
538 ± 138 2.0
609 ± 177 5.2
2-methyl
2-methyl
methyl
allyl
487 ± 27
ND
>930
>3.4
2,4-dimethoxy methyl
2,4-dimethoxy ethyl
2,4-dimethoxy allyl
373 ± 40
ND
256 ± 35
112 ± 10
290 ± 79
8.0
19 ± 3
5.9
5s
ND
55 ± 4
1.08×10-2 >509
5.3
5t
AZTg
PFAh
>47130
ND
ND
96.46
5
ND ND
b
a All data represent mean values for at least two separate experiments. All compounds were tested at 200
c
e
μg/mL. concentration that inhibit 50% HIV-1 RT. EC50 = Effective concentration required to protect
e
C8166 cells against the cytopathicity of HIV-1 by 50%. CC50 = Cytostatic concentration required to
Ⅲ
B
f
g
reduce C8166 cell proliferation by 50%. Selectivity index: CC50/EC50 ratio. AZT was used as positive
control. hPFA: Foscarnet was tested at 20 μg/mL as positive control. i ND: not determined.
2.2. Biological activity
(E)-N-phenylstyryl-N-alkylacetamides 5 were assayed for their inhibitory ratio against HIV-1 RT at
200 μg/mL. As outlined in Table 1, 5a and 5b showed significantly less enzymatic activity, compared
with 5c, which suggests that the presence of an ortho-substituent on the aromatic ring in the N-methyl
derivatives appears to increase enzyme inhibition. This positive influence of a group in ortho position
as to compounds 3 was already reported in our previous paper [11].
In some new developed NNRTIs such as the oxindole [12] and benzimidazole-2-one [13]
derivatives, NH unit in the amide is a proton-donating group in the hydrogen bond formation process

Molecules 2009, 14
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between the inhibitor and HIV-1 RT based on detailed docking studies, and N-alkyl analogues would
suffer dramatic loss of activities. In our experiment, the SAR of compounds 5 against HIV-1 RT was
similar to that of compounds 3 — the ortho substituted compounds possessed higher enzymatic
activity. Furthermore, compounds 5 possessed higher inhibitory ratio against HIV-1 RT compared with
their synthetic precursors 3. These suggested that compounds 5 and 3 probably have similar
mechanism of action towards HIV-1 RT, and the NH unit in compounds 3 was not crucial to potent
anti-HIV-1 RT activity.
The basic SAR prompted us to synthesize a series of N-alkyl analogues 5d –5t with ortho-
substituents R1 (Table 1). Compound 5i (R1 = 2-Br, R2 = 3,5-difluorobenzyl) exhibited the highest
enzyme activity. with a 88.89% inhibitory ratio against HIV-1 RT at the tested concentration, which
increased nearly one fold from the precursor compound 3 (R1 = 2-Br, R2 = H). Compounds 5 were then
tested for their syncytium reduction activities (EC50) and cytotoxities (CC50) in cell-based assays, in
addition the selectivity index (SI) was calculated (Table 1). The most active compound was (E)-N-(2-
bromostyryl)-N-3’,5’-difluorobenzylacetamide (5i, EC50 = 4 μm, SI = 29). Compound 5f (IC50 = 320 μm,
EC50 = 8 μm) is 40 times more potent in cell culture than in enzyme assays. One possibility for this
discrepancy was supposed to be that the structure of compound 5f was different from other N-alkylated
compounds. In compound 5f, two acetyl groups were substituted on the nitrogen atom, and this change
in structure might cause compound 5f active to other viral targets. Further structure modification of
this kind of compounds is currently underway in our lab.
3. Experimental
3.1. General
Unless otherwise stated, all reagents were commercial analytical or chemical pure grades and were
not additionally purified. IR spectra were measured on a Bio-Rad FTS-135 spectrometer (Bio-Rad,
Richmond, CA) with KBr pellets, ν in cm-1; EI-MS data were recorded on an AutoSped 300
instrument; 1H-NMR spectra were recorded in CDCl3 on Bruker AM 400 or DRX-500 spectrometers.
Chemical shifts are given in δ (ppm) with TMS as internal reference; column chromatography (CC):
silica gel (200–300 mesh; Qingdao Marine Chemical Inc.; Qingdao; China).
3.2. Preparation of (E)-β-phenylnitroolefins 2
An ortho-substituted benzaldehyde (2 mmol), ammonium acetate (0.1 g, 2.6 mmol) and
nitromethane (3 mL) were placed in a 10 mL round bottomed flask equipped with a condenser. The
mixture was refluxed and stirred for 2 hours and then evaporated to give a residue that was dissolved
in CH2Cl2 (5 mL), washed successively with saturated brine and water, and the organic layer was
evaporated to give the target compounds that were used without further purification (yields: 90–95%).
3.3. Preparation of (E)-N-phenylstyrylacetamides 3
Under a N2 atmosphere, the appropriate compound 2 (1 mmol), iron powder (1.12 g, 20 mmol),
acetic acid (0.2 mL) and acetic anhydride (6 mL) were added to a 20 mL round flask with a condenser.
The mixture was refluxed for 2 hours and then cooled to room temperature. Methanol (20 mL) was

Molecules 2009, 14
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added to dilute the mixture, which was filtered through Celite®. The filtrate was poured onto ice
cooled water (50 mL) and then extracted with ethyl acetate. The organic layer was evaporated to a
residue and dissolved in methanol (20 mL). The methanolic solution was adjusted to pH 12–14 with
methanolic potassium hydroxide to hydrolyze diacetyl compounds 4 to target compounds 3 and this
basic solution was poured onto water (50 mL) and extracted again with ethyl acetate. The organic layer
was washed successively with saturated brine and water, dried over anhydrous Na2SO4, and then
evaporated to give a crude product which was purified on a silica gel column eluted with hexane/ethyl
acetate (4:1, V/V) to give compounds 3 (yields: 45–50%).
3.4. General procedure for the preparation of (E)-N-phenylstyryl-N-alkylacetamides 5: Preparation of
(E)-N-(3-bromostyryl)-N-methylacetamide (5a)
A solution of compound 3 (R1 = 3-Br, 98 mg, 0.5 mmol) in dry tetrahydrofuran (5 mL) was cooled
to 0 °C, sodium hydride (0.6 mmol) was added, the mixture was kept stirring at 0°C for 30 min, then a
solution of iodomethane (0.6 mmol) in tetrahydrofuran (1 mL) was added. The mixture was stirred at
0°C for another 30 min, then warmed up to ambient temperature and stirred until the completion of the
reaction as monitored by TLC. The mixture was poured onto ice cooled water and extracted with ethyl
acetate, the organic layer was then washed with brine and dried with anhydrous Na2SO4. After
evaporation, the residue was purified on silica gel chromatography giving the target compound 5a as a
white powder; yield: 53%; 1H-NMR (CDCl3) δ: 8.11 (d, J = 14.9 Hz, 1H), 7.53–7.15 (m, 4H), 5.90 (d,
J = 14.9 Hz, 1H), 3.25 (s, 3H), 2.36 (s, 3H); IR νmax (KBr) cm-1: 1670, 1634; EI-MS m/z (%): 255
[M+2]+ (75), 253 [M]+ (79), 213 (95), 211 (100), 185 (13), 183(13), 131 (15). Compounds 5b–5t
(structures provided in Table 1) were synthesized using the above method.
1
(E)-N-(4-bromostyryl)-N-methylacetamide (5b): White powder, yield: 69%; H-NMR δ: 8.13 (d,
J = 14.9 Hz, 1H), 7.43 (d, J = 8.2 Hz, 2H), 7.20 (d, J = 8.2 Hz, 2H), 5.92 (d, J = 14.9 Hz, 1H), 3.26 (s,
3H), 2.36 (s, 3H); IR νmax cm-1: 1674, 1638; EI-MS m/z (%): 255 [M+2]+ (40), 253 [M]+ (43), 213
(90), 211 (100), 185 (10), 183(10), 168 (33), 149 (37), 131 (30), 117 (20).
1
(E)-N-(2-bromostyryl)-N-methylacetamide (5c): White powder, yield 50%; H-NMR δ: 8.00 (d,
J = 15.0 Hz, 1H,), 7.55–7.00 (m, 4H), 6.22 (d, J = 15.0 Hz, 1H), 3.24 (s, 3H), 2.28 (s, 3H); IR νmax cm-1:
1680, 1632; EI-MS m/z (%): 255 [M+2]+ (25), 253 [M]+, 213 (50), 211 (50), 185 (55), 183(55), 132
(100), 117 (67).
1
(E)-N-(2-bromostyryl)-N-ethylacetamide (5d): White powder, yield 65%; H-NMR δ : 7.85 (d,
J = 14.6 Hz, 1H), 7.54–7.02 (m, 4H), 6.27 (d, J = 14.6 Hz, 1H), 3.82 (q, J = 7.0 Hz, 2H), 2.28 (s, 3H),
1.31 (t, J = 7.0 Hz, 3H); IR νmax cm-1: 1677, 1632, 1399, 1246; EI-MS m/z (%): 268 [M+2]+ (45), 267
[M] (45), 227 (75), 225 (76), 210 (38), 188 (32), 130 (100).
(E)-N-(2-bromostyryl)-N-allylacetamide (5e): Viscous oil, yield 65%; 1H-NMR δ: 7.95 (d, J = 14.2 Hz,
1H), 7.55-7.04 (m, 4H), 6.33 (d, J = 14.2 Hz, 1H), 5.86-5.83 (m, 1H), 5.31-5.02 (m, 2H), 4.43-4.29 (m,
2H), 2.24 (s, 3H); IR νmax cm-1: 1678, 1632, 1395, 1225; EI-MS m/z (%): 281 [M+2]+ (90), 279 [M]
(100), 239 (87), 237 (94), 224 (40), 222 (48), 158 (80), 156 (37), 143 (27), 130 (51), 117 (83).

Molecules 2009, 14
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1
(E)-N-(2-bromostyryl)-N-acetylacetamide (5f): White powder, yield 55%; H-NMR δ: 7.56 (d,
J = 7.9 Hz, 2H), 7.30 (t, J = 7.4 Hz, 1H), 7.16 (t, J = 7.7 Hz, ), 6.85 (d, J = 14.4 Hz, 1H), 6.76 (d,
J = 14.4 Hz, 1H), 2.44 (s, 6H); IR νmax cm-1: 1715, 1644, 1468, 1426, 1368, 1274, 1228; FAB-MS m/z
(%): 282 [M+1]+ (100).
1
(E)-N-(2-bromostyryl)-N-benzylacetamide (5g): White powder, yield 67%; mp 86-88 °C; H-NMR δ:
8.08 (d, J = 14.3 Hz, 1H), 7.48-7.02 (m, 9H), 6.30-6.19 (m, 1H), 5.03, (s, 2H), 2.43 (s, 3H); IR νmax
cm-1: IR νmax cm-1: 1663, 1578, 1398, 1322, 1256, 1229; EI-MS m/z (%): 331 [M+2]+ (20), 329 [M]
(22), 289 (23), 287 (25), 250 (8), 208 (10), 91 (100).
1
(E)-N-(2-bromostyryl)-N-2’,6’-difluorobenzylacetamide (5h): White powder, yield 70%; H-NMR δ:
7.50-6.80 (m, 8H), 6.27 (d, J = 14.2 Hz, 1H), 5.20 (s, 2H), 2.38 (s, 3H); IR νmax cm-1: 1669, 1627,
1469, 1406, 1316. EI-MS m/z (%): 367 [M+2]+ (15), 365 [M]+ (15), 325 (35), 323 (27), 286 (7), 244
(15), 127 (100).
1
(E)-N-(2-bromostyryl)-N-3’,5’-difluorobenzylacetamide (5i): White powder, yield 66%; H-NMR δ:
7.52-6.67 (m, 8H), 6.17 (d, J = 14.5 Hz, 1H), 5.00 (s, 2H), 2.43 (s, 3H); IR νmax cm-1: 160, 1635, 1598,
1461, 1437, 1396, 1319; EI-MS m/z (%): 367 [M+2]+ (45), 365 [M]+ (48), 325 (83), 323 (90), 286
(20), 244 (57), 127 (100). 117 (95).
1
(E)-N-(2-bromostyryl)-N-2’,6’-dichlorobenzylacetamide (5j): White powder, yield 67%; H-NMR δ:
7.53-6.94 (m, 8H), 6.21 (d, J = 12.9 Hz, 1H), 5.35 (s, 2H), 2.35 (s, 3H); IR νmax cm-1: 1674, 128, 1580,
1435, 1402, 1340; EI-MS m/z (%): 401 [M+4]+ (10), 399 [M+2]+ (20), 397 [M]+ (15), 357 (40), 322
(64), 320 (58), 161 (68), 159 (100) 117 (42).
1
(E)-N-(2-chlorostyryl)-N-methylacetamide (5k): White powder yield: 67%; H NMR δ: 8.07 (d,
J = 14.9 Hz, 1H), 7.43-7.13 (m, 4H), 6.27 (d, J = 14.9 Hz), 3.26 (s, 3H); 2.30 (s, 3H); IR νmax cm-1:
1680, 1632, 1387, 1332, 1256; EI-MS m/z (%): 211 [M+2]+ (25), 209[M]+ (75), 169 (32), 167 (100),
132 (100), 130(35).
1
(E)-N-(2-chlorostyryl)-N-ethylacetamide (5l): White powder, yield: 73%; H-NMR δ: 7.89 (d,
J = 14.6 Hz, 1H), 7.39-7.07 (m, 4H), 6.29 (d, J = 14.6 Hz, 1H), 3.81(q, J = 6.9 Hz, 2H), 2.27 (s, 2H),
1.29 (t, J = 6.9 Hz, 3H); IR νmax cm-1: 1677, 1633, 1440, 1399, 1246; EI-MS m/z (%): 225 [M+2]+ (28),
223 [M]+ (85), 183 (33), 181 (100), 168 (24), 166 (68), 146 (32), 130 (64).
1
(E)-N-(2-chlorostyryl)-N-allylacetamide (5m): White powder, yield: 63%; H-NMR δ: 8.00 (d,
J = 14.5 Hz, 1H), 7.51-7.07 (m, 4H), 6.34-6.26 (m, 1H), 5.91-5.60 (m, 1H), 5.28-5.19 (m, 2H), 4.26 (d,
J = 5.0 Hz, 2H), 2.32 (s, 3H); IR νmax cm-1: 1674, 1632, 1440, 1397; EI-MS m/z (%): 237 [M+2]+ (5),
235 [M]+ (14), 193 (10), 178 (12), 141 (100), 139 ( 87), 77 (100).

Molecules 2009, 14
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1
(E)-N-(2-fluorostyryl)-N-methylacetamide (5n): White powder; yield: 70%; H-NMR δ: 8.12 (d,
J = 15.0 Hz, 1H), 7.51–7.03 (m, 4H), 6.07 (d, J = 15.0 Hz, 1H), 3.23 (s, 3H); 2.30 (s, 3H); IR νmax cm-1:
1671, 1637, 1488, 1457, 1385, 1314; EI-MS m/z (%): 193 [M]+ (60), 151 (100), 109(50).
1
(E)-N-(2-fluorostyryl)-N-allylacetamide (5o): White powder, yield: 63%; H-NMR δ: 8.10 (d,
J = 14.7 Hz, 1H), 7.44-7.01 (m, 4H), 6.07-6.00 (m, 1H), 5.90-5.81 (m, 1H), 5.28-5.04 (m, 2H), 4.27 (d,
J = 5.0 Hz, 2H), 2.34 (s, 3H); IR νmax cm-1: 1679, 1636, 1490, 1395; EI-MS m/z (%): 219 [M]+ (78),
177 (67), 162 (57), 148 ( 28), 135 (35), 109 (100), 101 (26), 68 (72).
1
(E)-N-(2-methylstyryl)-N-methylacetamide (5p): White powder, yield: 65%; H-NMR δ: 7.97 (d,
J = 15.0 Hz, 1H), 7.45–7.12 (m, 4H), 6.09 (d, J = 15.0 Hz), 3.24 (s, 3H); 2.35 (s, 3H); 2.29 (s, 3H); IR
ν
max cm-1: 1668, 1633, 1468, 1440, 1382, 1310, ; EI-MS m/z (%): 189 [M]+ (67), 147 (100), 132 (20),
106 (30). 73 (57).
1
(E)-N-(2-methylstyryl)-N-allylacetamide (5q): White powder, yield: 61%; H-NMR δ: 7.90 (d,
J = 14.7 Hz, 1H), 7.45-7.07 (m, 4H), 6.18-6.11 (m, 1H), 5.87-5.84 (m, 1H), 5.28-5.18 (m, 2H), 4.44 (d,
J = 5.0 Hz, 2H), 2.33 (s, 3H), 2.30 (s, 3H); IR νmax cm-1: 1675, 1635, 1396; EI-MS m/z (%): 215 [M]+
(100), 173 (42), 158 (53), 130 (44), 117 (46), 105 (41) 68 (42).
1
(E)-N-(2,4-dimethoxystyryl)-N-methylacetamide (5r): White powder, yield: 73%; H-NMR δ: 7.90 (d,
J = 15.0 Hz, 1H), 7.39-7.18 (m, 2H), 6.47 (s, 1H), 6.11 (d, J = 15.0 Hz, 1H), 3.85, 3.81 (s, each 3H),
3.21 (s, 3H), 2.28 (s, 3H); IR νmax cm-1: 1672, 1634, 1580, 1508, 1461, 1416, 1390, 1332, 1288; EI-MS
m/z (%): 235 [M]+ (100), 193 (20), 192 (20), 178 (30), 161 (30), 137 (22).
1
(E)-N-(2,4-dimethoxystyryl)-N-ethylacetamide (5s): White powder, yield: 69%; H-NMR δ: 7.85 (d,
J = 14.7 Hz, 1H), 7.23-7.17 (m, 2H), 6.46 (s, 1H), 6.15 (d, J = 14.7 Hz, 1H), 3.83, 3.80 (s, each 3H),
3.68 (q, J = 6.7 Hz, 2H), 2.24 (s, 3H), 1.19 (t, J = 6.7 Hz, 3H); IR νmax cm-1: 1668, 1633, 1608, 1579,
1505, 1462, 1402; EI-MS m/z (%): 249 [M]+ (100), 207 (15), 206 (19), 175 (17), 160 (13), 151 (15),
70 (57).
1
(E)-N-(2,4-dimethoxystyryl)-N-allylacetamide (5t): White powder, yield: 65%; H-NMR δ: 7.94 (d,
J = 15.1 Hz, 1H), 7.34-7.15 (m, 2H), 6.45 (s, 1H), 6.15 (d, J = 15.1 Hz, 1H), 5.87-5.80 (m, 1H), 5.25-
5.10 (m, 2H), 3.39 (d, J = 4Hz, 2H), 3.83, 3.73 (s, each 3H), 2.29 (s, 3H); IR νmax cm-1: 1671, 1634,
160, 1579, 1505, 1398; EI-MS m/z (%): 261 [M]+ (92), 221 (45), 219 (53), 177 (42), 162 (41), 147
(81), 109 (42), 73 (100).
3.5. Biological activity assays
Compounds 5 were assayed for their anti-HIV-1 activities as reported previously [14–16].
HIV-1 reverse transcriptase assay: The inhibition of compounds 5 on recombinant HIV-1 RT activity
was determined with a commercially available ELISA kit (Roche Molecular Biochemicals). The
compounds were incubated with DIG-labeled-reaction mixture at 37 °C for 15 h, and then anti-DIG-

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3184
POD solution was added, followed by substrate ABTS. Foscarnet was used as a positive compound.
The absorbance at 405 nm/490 nm (A405/490) was read in the ELISA reader mentioned above.
Inhibition ratio on recombinant RT activity was determined from the dose–response curve.
Cell based anti-HIV-1 bioassay: C8166 cells were maintained in RPMI-1640 supplemented with 10%
heat inactivated newborn calf serum (Gibco), The cells used in all experiments were in log-phase
growth. 3′-Azido-3′-deoxythymidine (AZT), the positive control, was purchased from Sigma (USA).
Syncytium reduction assay: In the presence of 100 µL of various concentrations of compounds, C8166
cells (4 × 105 mL-1) were infected with HIV-1IIIB at a multiplicity of infection (MOI) of 0.06. The final
volume per well was 200 µL. AZT was used as a positive control. After 3 days of culture, the
cytopathic effect (CPE) was measured by counting the number of syncytia (multinucleated giant cell)
in each well under an inverted microscope. Percentage inhibition of syncytial cell number in treated
culture to that in infected control culture and 50% effective concentration (EC50) was calculated.
Cytotoxicity assay: The cellular toxicity of compounds on C8166 cells was assessed by MTT methods.
Briefly, cells were seeded on a microplate in the absence or presence of various concentrations of
compounds in triplicate and incubated at 37 °C in a humid atmosphere of 5% CO2 for 72 h. The
supernatants were discarded and MTT reagent (5 mg/mL in PBS) was added to each wells, then
incubated for 4 h, 100 µL of 50% DMF-20% SDS was added. After the formazan was dissolved
completely, the plates were read on a Bio-Tek ELx 800 ELISA reader at 595/630 nm. The cytotoxic
concentration that caused the reduction of viable cells by 50% (CC50) was calculated from dose-
response curve.
4. Conclusions
We have synthesized a series of (E)-N-phenylstyryl-N-alkylacetamides. Evaluation of the title
compounds as NNRTIs of HIV-1 in vitro showed that compound 5c showed a higher TI value than its
isomer 5a, which suggested that higher enzymatic inhibitory ratio would lead to increased anti-HIV
activity in cell-based assay. Compound 5i exhibited the highest inhibition activity in both enzyme and
cell based assays. Although the TI values of the tested compounds were suboptimal, the title
compounds possessed low molecular weight and contained a naturally occurring structure unit
(hamigeroxalamic acid). The SAR exploration provided us a guide for further anti-HIV-1 study of the
derivatives in this family. Compound 5i represented a new lead for the design and synthesis of more
potent and selective analogues act as NNRTIs. Further structure modification and anti-HIV-1 activities
of compounds 5 are underway.
Acknowledgements
This work was partly supported by young teachers’ scientific researching foundation for Dr Pi
Cheng in Centre South University.

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Sample Availability: Samples of the compounds 5a–5t are available from the authors.
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