Design, synthesis, and biological evaluation of novel quinoline derivatives as HIV-1 Tat-TAR interaction inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.01.038

Thirty-two quinoline derivatives were designed and synthesized as HIV-1 Tat-TAR interaction inhibitors. All the compounds showed high antiviral activities in inhibiting the formation of SIV-induced syncytium in CEM174 cells. Nine of them with low cytotoxicities were evaluated by Tat dependent HIV-1 LTR-driven CAT gene expression colorimetric enzyme assay in human 293T cells, indicating effective inhibitory activities of blocking the Tat-TAR interaction. Molecular modeling experiments indicated that these compounds may inhibit Tat-TAR interaction by binding to Tat protein instead of TAR RNA.

Full text of DOI:10.1016/j.bmc.2009.01.038

Bioorganic & Medicinal Chemistry 17 (2009) 1948–1956
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of novel quinoline derivatives
as HIV-1 Tat–TAR interaction inhibitors
*
Shuguang Chen, Ran Chen, Meizi He, Ruifang Pang, Zhiwu Tan, Ming Yang
State Key Laboratory of Natural and Biomimetic Drugs, Peking University, PO Box 261, Beijing 100191, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Thirty-two quinoline derivatives were designed and synthesized as HIV-1 Tat–TAR interaction inhibitors.
All the compounds showed high antiviral activities in inhibiting the formation of SIV-induced syncytium
in CEM174 cells. Nine of them with low cytotoxicities were evaluated by Tat dependent HIV-1 LTR-driven
CAT gene expression colorimetric enzyme assay in human 293T cells, indicating effective inhibitory activ-
ities of blocking the Tat–TAR interaction. Molecular modeling experiments indicated that these com-
pounds may inhibit Tat–TAR interaction by binding to Tat protein instead of TAR RNA.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 9 December 2008
Accepted 19 January 2009
Available online 23 January 2009
Keywords:
HIV-1 Tat–TAR
Quinoline derivatives
Antiviral
Inhibitor
Molecular modeling
1. Introduction
diphenylfuran derivative DB60.10 Our laboratory also proposed a
molecular model of new Tat–TAR interaction inhibitors containing
Since the 1980s, Acquired Immure Deficiency Syndrome (AIDS)
has become a disastrous epidemic which greatly threatens the
health of humankind and brings us crisis and challenges in the
health care system, with more than 38 million people infected
worldwide.1 Today, there are more than 20 drugs available in clinic
for the treatment of HIV-1 infection. However, emergence of drug-
resistant viruses caused by mutations of the enzymes often results
in treatment failure with existing antiretrovirals.2 The cross-resis-
tance to the same class of compounds is also a problem for the
treatment process.3 Thus, efforts were made to develop new anti-
retroviral agents with different targets for inhibition of HIV-1 rep-
lication. One of the most promising targets is the process of Tat/
TAR RNA interaction. This Tat-mediated trans-activation process
requires the structural integrity of TAR RNA and the cooperative
interaction of human host cell proteins, such as positive transcrip-
tion elongation factor b (P-TEFb), a complex composed of cyclin T1
(CycT1) and cyclin-dependent kinase 9 (CDK9).4–7 CDK9 hyper-
phosphorylates the carboxy-terminal domain (CTD) of RNA poly-
merase II and induces efficient promoter clearance and
transcriptional elongation. Furthermore, CycT1 remodels the struc-
ture of Tat to enhance its affinity for the TAR RNA, and the TAR RNA
further enhances the interaction between Tat and CycT1.8 Due to
the crucial role that Tat/TAR complex plays, lots of compounds
which could efficiently inhibit the Tat/TAR RNA interaction have
been developed, such as acridine derivative CGP 40336A9 and
an ‘anchor’, a ‘linker’ and a positively charged ‘activator’.11 The pos-
itively charged ‘activator’, just like the role that the arginine-rich
region of Tat plays, reinforces the binding affinity between the
compound and the negatively charged TAR RNA, thus disrupting
the formation of Tat/TAR complex. According to this model we
have successfully designed series of inhibitors like
a,a-trehalose
derivatives,12 isoquinoline derivatives,13 b-carboline derivatives,14
and purine derivatives.11,15
When screening the compound library in our lab for inhibitory
activity of Tat–TAR interaction, we found that one compound
named CS3 revealed mild inhibitory activity and antiviral potency.
However, it was very interesting that CS3 bore no positively
charged group as in the molecular model mentioned above, sug-
gesting that it may inhibit Tat–TAR interaction in a different way.
We scrutinized the structure of CS3, and its features were found
as follows (as shown in Fig. 1): (a) it contained a planar aromatic
side chains
H3CO
aromatic ring
O
Cl
O
H
OCH3
N
H
O
N
* Corresponding author. Tel.: +86 10 82805264; fax: +86 10 82802724.
E-mail address: mingy@mail.bjmu.edu.cn (M. Yang).
Figure 1. The structure of CS3.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.038

S. Chen et al. / Bioorg. Med. Chem. 17 (2009) 1948–1956
1949
Table 1
Inhibition effect and cytotoxicity of the title compounds on SIV induced syncytium
R1
R5
O
H
N
R2
H
R3
N
R4
Compounda
R1
H
R2
R3
R4
R5
Cl
EC50
6.6
(
lM)
TC50
(l
M)
SId (TC50/EC50
)
b
c
5a
H
H
>100
>100
>15
OH
5b
5c
5d
Cl
Cl
Cl
4.0
7.0
7.9
>25
>14
>12
OH
OH
OH
CH3
H
H
>100
>100
5e
5f
H
H
H
H
Cl
Cl
2.7
5.4
>100
>100
>37
>18
OH
O
O
O
O
5g
5h
CH3
H
H
Cl
Cl
5.7
1.3
81.3
23.3
.14
17
O
O
CH3
H
O
O
O
5i
H
H
H
Cl
Cl
Cl
9.4
5.6
7.9
49.1
17.6
>100
5.2
3.1
>13
O
O
O
5j
5k
O
O
5l
H
H
Cl
Cl
10.0
2.9
>100
68.4
>19
22
NH
O
O
S
5m
(continued on next page)

1950
S. Chen et al. / Bioorg. Med. Chem. 17 (2009) 1948–1956
Table 1 (continued)
Compounda
R1
R2
R3
H
R4
R5
Cl
EC50
7.0
(l
M)
TC50
(
lM)
SId (TC50/EC50
)
b
c
O
O
OH
5n
5p
>100
>100
>14
CH3
H
O
OH
H
Cl
1.1
>90
O
O
O
O
OH
OH
5q
5r
H
Cl
Cl
4.7
2.8
>100
>100
>21
>36
O
O
8-CH3
OH
5s
7-CH3
Cl
4.7
59.6
13
O
O
O
OH
OH
5t
6-CH3
8-Cl
Cl
Cl
11.6
6.7
>100
>100
>8.6
>15
O
O
5u
O
OH
5v
7-Cl
Cl
8.2
>100
>12
O
O
O
O
OH
OH
5w
5x
5y
6-Cl
6-F
Cl
Cl
Cl
12.0
5.8
>100
>100
>100
>8.3
>17
>11
O
O
O
OH
OH
6,8-F
8.8
O
5z
6a
6b
7-CH3
6-Phenoxy
H
Cl
13.0
4.9
>100
>100
>100
>14
>20
>27
CH3
O
H
OH
OH
H
H
H
H
Carbonyl
Carbonyl
3.7

S. Chen et al. / Bioorg. Med. Chem. 17 (2009) 1948–1956
1951
Table 1 (continued)
Compounda
R1
H
R2
R3
R4
H
R5
EC50
5.1
(
lM)
TC50
(lM)
SId (TC50/EC50
>19
)
b
c
OH
OH
6c
8a
6-Cl
Carbonyl
>100
>100
H
H
H
H
CH3
Carbonyl
Carbonyl
8.7
6.0
>11
>16
OH
8b
8c
>100
OH
OH
H
H
H
H
Carbonyl
Carbonyl
6.9
7.3
>100
>100
>14
>13
F
OH
8d
a
Indinavir was used as the positive control at a concentration of 10 lM here. Its EC50 was 0.0108 lM and TC50 was above 100 lM in this system.
b
c
EC50, concentration was required to protect cells against the cytopathogenicity of SIV by 50%.
TC50, concentration was required to inhibit uninfected cells proliferation by 50%.
SI, selective index.
d
ring, namely 4-chloroquinoline; (b) it contained two side chains
which were connected with the aromatic ring by an amide linker.
So we proposed these two features as our structure template for
further modification to improve the antiviral activity. In order to
perform the modification more efficiently and reasonably, the re-
search was divided into three steps: (1) With the aromatic ring
remaining the same as 4-chloroquinoline, the two side chains moi-
ety was modified. (2) Then substitution of the 4-chloroquinoline
ring was explored. (3) Finally, the nitrogen atom of the quinoline
ring was substituted, while the chloride atom of the quinoline ring
was replaced by hydroxyl or carbonyl at the same time. Ultimately,
we designed and synthesized thirty-two compounds possessing
the features above, as shown in Table 1.
2. Result and discussion
The route used for preparation of the title compounds (5a–z,
6a–c and 8a–e) is illustrated in Scheme 1 and the synthetic proce-
dure is explained in Section 4. Thirty-two compounds were ob-
tained and their MS, 1H NMR spectroscopy data are provided in
Section 4. The biological activities of all the title compounds were
evaluated by SIV-induced syncytium in CEM cells. Their EC50, TC50
and SI values are listed in Table 1.
,
As shown in Table 1, most of the title compounds possessed a
TC50 value more than 100 M except 5g, 5h, 6i, 5j, 5m, and 5s.
With regard to their antiviral activities, each of the title com-
pounds exhibited an EC50 value within the range from 1.1 M to
12.0 M, suggesting that all compounds possessed effective anti-
SIV activities while most of them possessed low cytotoxicity.
l
l
Thirty of all thirty-two title compounds (except 6b and 6c) were
synthesized for the first time and all of them were evaluated for
their antiviral activity, while nine compounds were evaluated for
their inhibitory ability to Tat–TAR interaction.
l
Compounds 5a–m were synthesized to investigate the struc-
ture-activity relationship (SAR) of the side chains moiety men-
O
OH
N
O
ii
OCH2CH3
i
R3
N
H
OCH2CH3
OCH2CH3
iv
R3
R3
NH2
O
3
2
1
v
iii
OH
O
O
O
O
OH
OH
N
OCH2CH3
7a-d
OH
H
R3
R3
R3
N
N
N
4a-z
6a-c
vi
R4
iv
Cl
O
R1
O
O
OH
N
H
R2
N
H
R3
N
R3
N
5a-z
8a-d
R4
Scheme 1. Synthesis of quinoline derivatives. Reagents and conditions: (i) EMME, 3 h, rt, 130 °C; (ii) diphenyl ether, 2 h, rt, 250 °C; (iii) 1 M NaOH, 2 h, reflux; acetic acid was
added until pH 2–3; (iv) ethanolamine, 2 h, rt, 170 °C; (v) R4I, DMF, 18 h, 60 °C; (vi) SOCl2, CHCl3, 4 h, rt, reflux; evaporate; NHR1R2 was added.

1952
S. Chen et al. / Bioorg. Med. Chem. 17 (2009) 1948–1956
tioned in Section 1. By taking a detailed look at the data in Table 1,
we found that: (1) compounds bearing a hydroxyl alkane side
chain (5a–e) all exhibited good anti-SIV activities and low cytotox-
icities. (2) Compounds bearing a methyl ester side chain (5f–m)
also possessed effective anti-SIV activities, however, it exhibited
fairly high cytotoxicity if the other chain was an alkane (5g–j) or
a structure containing sulfur atom (5m). So based on the conclu-
sions above, we designed and synthesized two compounds bearing
both a hydroxyl alkane side chain and a methyl ester side chain (5n
and 5p), which turned out to possess good anti-SIV activities and
tioned above. Actually 5z (EC50: 13.0
lM) did possess higher EC50
value than 5s (EC50: 4.7 M). Finally, we synthesized compounds
l
bearing different substituent groups on 1-position (the nitrogen
atom) of quinoline ring while 4-position turned into carbonyl
(6a–c and 8a–d). And again, this kind of modification did not show
significant changes in anti-SIV activity and cytotoxicity.
To ascertain that the compounds we designed could inhibit
HIV-1 Tat–TAR interaction, we evaluated nine title compounds
with low cytotoxicities (CS3, 5d, 5f, 5n, 5p, 5s, 5z, 8a, 8b) by Tat
dependent HIV-1 LTR-driven CAT gene expression colorimetric en-
zyme assay (see Section 4). As a report gene, the depressed CAT
expression indicated the high inhibitory activity of the compound.
As shown in Figure 2, the range of inhibited CAT expression in-
duced by the nine compounds was from 44.2% to 92.7%. The com-
pounds bearing hydroxymethyl chain (5p and 5s) showed lower
depressed CAT expression (59.6% versus 82.4% and 53.3% versus
92.7%) than their 2-OH-ethyl counterparts (5n and 5z). This result
complied with that of the SIV assay. The decreased CAT activities in
the presence of these nine title compounds suggested that most
of them could effectively block the interaction of Tat–TAR in
low cytotoxicities, especially 5p (EC50: 1.1 lM). Maybe it was the
Hydrogen bond interaction being hindered by methyl that made
5n have higher EC50 value than 5p. To investigate SAR of the aro-
matic ring moiety, we synthesized a series of compounds (5q–y)
bearing the two side chains of 5p with various substituents of
quinoline ring and listed their data in Table 1. It was found that
substituent changes of quinoline ring had little influence on anti-
SIV activity and cytotoxicity, whether it was electron-donating or
electron-withdrawing substituent at any position. In addition, we
synthesized 5z to testify the steric hindrance effect of methyl men-
120
100
100
92.7
82.4
79
5f
80
60
40
20
0
71.9
5d
63.9
59.6
58.7
CS3
53.3
44.2
33.7
TAR
29.2
Tat
Tat+TAR
5n
5p
5s
5z
8a
8b
Figure 2. Effects of the nine title compounds on Tat-mediated transactivation in 293T cells.
Figure 3. (a, b) Interaction of compound 5p to Tat protein; (c) interaction of compound 8a to Tat protein; (d) interaction of compound 8b to Tat protein.

S. Chen et al. / Bioorg. Med. Chem. 17 (2009) 1948–1956
1953
cell-based assay. Among all the nine compounds, compound 8a
showed the lowest depressed CAT expression (44.2%).
(compound 3). In detail, substituted aniline (compound 1) was
heated with diethyl ethoxymethylenemalonate (EMME) at 130 °C
to give malonates (compound 2), which generally without purifica-
tion, was cyclized to 4-hydroxyquinoline-3-carboxylic acid ethyl
esters by heating at 250 °C in diphenyl ether, as shown in Scheme
1.16,17 Compound 3 was hydrolyzed to give compound 4. Then
compound 4 was treated with an excess of SOCl2 in the presence
of several drops of chloroform to afford 4-chloroquinoline-3-car-
bonyl chloride, which was then reacted with the amine to afford
5a–z. Compounds 6a–c were prepared by heating compound 3
with ethanolamine at 130 °C.18 Alkylation of compound 3 by treat-
ment with alkyl halides and anhydrous potassium carbonate gave
l-alkyl-1, 4-dihydro-4-oxoquinoline esters (compounds 7a–d).19
Compounds 8a–d were obtained by heating compounds 7a–d with
ethanolamine.
To ascertain our assumption, we performed molecular modeling
experiments of three title compounds with different substituents
(5p, 8a, and 8b) using Autodock 4.0 to study the interaction be-
tween these compounds and HIV-1 TAR RNA. However, the free
energies which reflected the binding affinity of the compounds
and the TAR RNA element all turned out to be above-5 kcal/mol,
which means these compounds may not inhibit Tat–TAR interac-
tion in the way of binding to TAR RNA. Then we studied the inter-
action between these compounds and Tat protein. As shown in
Figure 3, the bound conformations of all these compounds occu-
pied a pocket surrounded by amino acid residues M36, H81, P82,
Q35, C34 with fairly low free energies below À10 kcal/mol
(Fig. 3a). H-bond interactions were observed between the side
chains of 5p, 8a, 8b and the amide backbone of M36, H81, and
P82, indicating that the H-bond interaction may play an important
role in enhancing the binding preferences of the compounds
(Fig. 3b–d), thus confirming our assumption that it was the H-bond
interaction being hindered by methyl that made 5n have higher
EC50 value than 5p. Besides, the aromatic ring moiety of all the
three compounds were out of the pocket, which could explain
why the modifications on the quinoline ring did not significantly
influence the biological activity. We supposed that these com-
pounds could change the conformation of Tat protein by binding
to it, which may cause a failure in forming Tat–TAR RNA complex.
That may be the reason why compounds like CS3 could inhibit Tat–
TAR interaction although they bore no positively charged groups.
4.2.1. 4-Chloro-N-(3-hydroxypropyl) quinoline-3-carboxamide
(5a)
A mixture of compound 4a (0.19 g; 1 mmol), SOCl2 (3 ml) and
several drops of chloroform was stirred under reflux for 3 h and then
concentrated under reduced pressure to give 4-chloroquinoline-3-
carbonyl chloride. The chloride was then mixed with anhydrous
THF (5 ml) and added dropwise to the solution of propanolamine
(1.2 mmol) and triethylamine (2 mmol) in anhydrous THF in ice
bath. After stirring overnight, the mixture was filtered. The filtrate
was concentrated under reduced pressure then purified by column
chromatography (silica gel, dichloromethane–methanol (100:1, v/
v)), Compound 5a was obtained as yellow solid, mp 143–144 °C,
yield 84.9%. 1H NMR (CDCl3) d 9.09 (s, 1H), 8.34–8.31 (d, 1H), 8.19–
8.16 (d, 1H), 7.87–7.82 (t, 1H), 7.77–7.70 (t, 1H), 6.72–6.70 (d, 1H),
3.96–3.92 (m, 2H), 3.79–3.74 (t, 2H), 3.70–3.66 (t, 2H); MS (ESI+)
m/z calcd: 264.07, found: 265.1 [(M+1)+].
3. Conclusion
In this work, all experiments reported here showed that the
newly designed quinoline derivatives could block the Tat–TAR
interaction and had potent inhibition activity of SIV. Molecular
modeling experiments indicated that these compounds may inhi-
bit Tat–TAR interaction by binding to Tat protein instead of TAR
RNA. Different side chains of the title compounds could affect the
binding affinity with Tat protein in varying potency, thus bringing
about different effects on the biological activities. However, modi-
fications on the quinoline ring did not significantly influence the
biological activities of the title compounds, which implied that
the substituent groups of the quinoline ring were not helpful for
improving the binding affinity between compounds and Tat pro-
tein. These studies provide an insight into the SAR of quinoline der-
ivate as HIV-1 Tat–TAR interaction inhibitors.
4.2.2. (S)-4-Chloro-N-(1-hydroxy-3-phenylpropan-2-yl)
quinoline-3-carboxamide (5b)
Compound 5b was prepared in the same way as (5a), pale-
white solid, mp 149–151 °C yield 73.6%. 1H NMR (DMSO-d6) d
8.66 (s, 1H), 8.63 (s, 1H), 8.28–8.25 (d, 1H), 8.14–8.11 (d, 1H),
7.95–7.89 (t, 1H), 7.85–7.80 (t, 1H), 7.36–7.29 (m, 5H), 4.97 (s,
1H), 4.24–4.20 (m, 1H), 3.58–3.54 (m, 1H), 3.50–3.45 (m, 1H),
3.05–2.99 (m, 1H), 2.77–2.69 (m, 1H); MS (ESI+) m/z calcd:
340.10, found: 341.0 [(M+1)+].
4.2.3. (S)-4-Chloro-N-(1-hydroxypropan-2-yl) quinoline-3-
carboxamide (5c)
Compound 5c was prepared in the same way as (5a), yellow
wax, yield 85.4%. 1H NMR (DMSO-d6) d 8.86 (s, 1H), 8.58–8.56 (d,
1H), 8.31–8.28 (d, 1H), 8.16–8.13 (d, 1H), 7.96–7.91 (t, 1H), 7.86–
7.81 (t, 1H), 4.85–4.81 (t, 1H), 4.06–4.00 (m, 1H), 3.56–3.48 (m,
1H), 3.40–3.32 (m, 1H), 1.18–1.16 (m, 1H); MS (ESI+) m/z calcd:
264.07, found: 264.9 [(M+1)+].
4. Experimental
4.1. Chemistry
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.
4.2.4. (S)-4-Chloro-N-(1-hydroxy-4-methylpentan-2-yl)
quinoline-3-carboxamide (5d)
Compound 5d was prepared in the same way as (5a), white nee-
dles, mp 124–126 °C yield 90.3%. 1H NMR (CDCl3) d 8.97 (s, 1H),
8.23–8.20 (d, 1H), 8.10–8.07 (d, 1H), 7.83–7.77 (t, 1H), 7.69–7.64
(t, 1H), 6.61–6.58 (d, 1H), 4.39–4.32 (m, 1H), 3.90–3.85 (m, 1H),
3.75–3.70 (m, 1H), 2.78 (br s, 1H), 1.84–1.73 (m, 1H) 1.65–1.44
(m, 2H), 1.09–0.94 (m, 6H); MS (ESI+) m/z calcd: 306.11, found:
307.2 [(M+1)+].
4.2. General synthetic procedure
4.2.5. 4-Chloro-N-(2-hydroxyethyl) quinoline-3-carboxamide
(5e)
The synthesis of the target compounds required access to differ-
ent substituted 4-hydroyl-3-quinolinecarboxylic ester derivatives
Compound 5e was prepared in the same way as (5a), pale-yel-
low solid, mp 151–152 °C, yield 88.4%. 1H NMR (CDCl3) d 9.09 (s,

1954
S. Chen et al. / Bioorg. Med. Chem. 17 (2009) 1948–1956
1H), 8.36–8.31 (d, 1H), 8.19–8.15 (d, 1H), 7.89–7.83 (t, 1H), 7.79–
7.69 (t, 1H), 3.96–3.92 (m, 2H), 3.79–3.74 (m, 2H); MS (ESI+) m/z
calcd: 250.05, found: 251.2 [(M+1)+].
4.2.13. (R)-Methyl 2-(4-chloroquinoline-3-carboxamido)-3-
(methylthio) propanoate (5m)
Compound 5m was prepared in the same way as (5a), yellow
sticky jelly, yield 83.2%. 1H NMR (CDCl3) d 9.09 (s, 1H), 8.37–8.34
(d, 1H), 8.19–8.16 (d, 1H), 7.90–7.85 (t, 1H), 7.77–7.72 (t, 2H),
7.16–7.14 (d, 1H), 5.05–5.01 (m, 1H), 3.86–3.85 (d, 3H), 2.70–
2.62 (m, 2H), 2.41–2.36 (m, 2H); MS (ESI+) m/z calcd: 352.06,
found: 353.0 [(M+1)+].
4.2.6. Methyl 2-(4-chloroquinoline-3-carboxamido) acetate (5f)
Compound 5f was prepared in the same way as (5a), pale-yel-
low solid, mp 128–130 °C, yield 79.0%. 1H NMR (CDCl3) d 9.11 (s,
1H), 8.36–8.32 (d, 1H), 8.19–8.14 (d, 1H), 7.90–7.82 (t, 1H), 7.80–
7.70 (t, 1H), 7.05 (s, 1H), 4.38–4.36 (d, 2H), 3.85 (s, 3H); MS (EI+)
m/z calcd: 278.05, found: 278 [(M)+].
4.2.14. (2S,3R)-Methyl 2-(4-chloroquinoline-3-carboxamido)-3-
hydroxybutanoate (5n)
Compound 5n was prepared in the same way as (5a), yellow so-
lid, mp 220–222 °C, yield 92.4%. 1H NMR (CDCl3) d 9.11 (s, 1H),
8.38–8.35 (d, 1H), 8.20–8.17 (d, 1H), 7.90–7.85 (t, 1H), 7.77–
7.73(d, 1H), 7.14–7.11 (d, 1H), 4.94–4.91 (dd, 1H), 4.56 (br s, 2H),
3.86 (s, 3H), 1.42–1.40 (d, 3H); MS (ESI+) m/z calcd: 322.07, found:
323.0 [(M+1)+].
4.2.7. (S)-Methyl 2-(4-chloroquinoline-3-carboxamido)
propanoate (5g)
Compound 5g was prepared in the same way as (5a), pale-yel-
low solid, mp 129–131 °C, yield 85.9%. 1H NMR (CDCl3) d 9.07 (s,
1H), 8.36–8.31 (d, 1H), 8.18–8.14 (d, 1H), 7.90–7.81 (t, 1H), 7.77–
7.68 (t, 1H), 7.08–7.06 (d, 1H), 4.94–4.87 (m, 1H), 3.87 (s, 3H),
1.66–1.60 (d, 3H); MS (ESI+) m/z calcd: 292.06, found: 293.1
[(M+1)+].
4.2.15. (S)-Methyl 2-(4-chloroquinoline-3-carboxamido)-3-
hydroxypropanoate (5p)
Compound 5p was prepared in the same way as (5a), pale-yel-
low solid, mp 191–192 °C, yield 79.5%. 1H NMR (CDCl3) d 9.12 (s,
1H), 8.37–8.35 (d, 1H), 8.20–8.17 (d, 1H), 7.91–7.85 (t, 1H), 7.78–
7.73 (t, 1H), 5.02–4.98 (m, 1H), 4.19 (s, 2H), 3.88 (s, 3H); MS
(ESI+) m/z calcd: 308.06, found: 309.0 [(M+1)+].
4.2.8. (2S,3S)-Methyl 2-(4-chloroquinoline-3-carboxamido)-3-
methylpentanoate (5h)
Compound 5h was prepared in the same way as (5a), pale-
yellow sticky jelly, yield 93.6%. 1H NMR (CDCl3) d 9.05 (s,
1H), 8.32–8.29 (dd, 1H), 8.16–8.13 (d, 1H), 7.87–7.82 (dt, 1H),
7.74–7.70 (dt, 1H), 7.05–7.04 (d, 1H), 4.95–4.91 (m, 1H), 3.82
(s, 3H), 2.15–2.10 (m, 1H), 1.60–1.53 (m, 1H), 1.37–1.26 (m,
3H), 1.08–0.95 (m, 6H); MS (EI+) m/z calcd: 334.11, found:
334 [(M)+].
4.2.16. (S)-Ethyl 2-(4-chloroquinoline-3-carboxamido)-3-
hydroxypropanoate (5q)
Compound 5q was prepared in the same way as (5a), yellow
wax, yield 77.7%. 1H NMR (CDCl3) d 9.15–9.12 (s, 1H), 8.86 (s,
1H), 8.33–8.30 (d, 1H), 8.17–8.14 (d, 1H), 7.98–7.92 (t, 1H), 7.87–
7.84 (t, 1H), 5.14–5.10 (t, 1H), 4.61–4.59 (q, 1H), 4.21–4.14 (q,
2H), 3.83–3.78 (m, 2H), 1,27–1.22(t, 3H); MS (ESI+) m/z calcd:
322.07, found: 322.9 [(M+1)+].
4.2.9. (S)-Methyl 2-(4-chloroquinoline-3-carboxamido)-4-
methylpentanoate (5i)
Compound 5i was prepared in the same way as (5a), pale-yel-
low solid, mp 81–83 °C, yield 77.9%. 1H NMR (CDCl3) d 10.54 (s,
1H), 8.32–8.34 (d, 1H), 7.85–7.80 (m, 1H), 7.69–7.63 (m, 1H),
7.56–7.53 (d, 1H), 7.38–7.33 (d, 1H), 4.74–4.71 (m, 1H), 3.77 (s,
3H), 3.44–3.38 (m, 2H); MS (ESI+) m/z calcd: 334.11, found: 335.1
[(M+1)+].
4.2.17. (S)-Methyl 2-(4-chloro-8-methylquinoline-3-
carboxamido)-3-hydroxypropanoate (5r)
Compound 5r was prepared in the same way as (5a), pale-yel-
low solid, mp 186–188 °C, yield 89.3%. 1H NMR (CDCl3) d 12.09
(br s, 1H), 9.07 (s, 1H), 8.22–8.18 (d, 1H), 7.93 (s, 1H), 7.57–7.53
(d, 2H), 4.98–4.95 (m, 1H), 4.17 (s, 2H), 3.87 (s, 3H), 2.61 (s, 3H);
MS (ESI+) m/z calcd: 322.07, found: 323.1 [(M+1)+].
4.2.10. (S)-Methyl 2-(4-chloroquinoline-3-carboxamido)-3-
methylbutanoate (5j)
Compound 5j was prepared in the same way as (5a), pale-yel-
low sticky jelly, yield 83.2%. 1H NMR (CDCl3) d 9.03 (s, 1H), 8.29–
8.25 (d, 1H), 8.15–8.11 (d, 1H), 7.87–7.80 (m, 2H), 7.55–7.54 (m,
1H), 4.80–4.73 (m, 1H), 3.75 (s, 3H), 2.12–2.00 (m, 1H), 1.92–1.77
(m, 2H), 1.05–0.97 (m, 6H); MS (ESI+) m/z calcd: 320.09, found:
321.1 [(M+1)+].
4.2.18. (S)-Methyl 2-(4-chloro-7-methylquinoline-3-
carboxamido)-3-hydroxypropanoate (5s)
Compound 5s was prepared in the same way as (5a), pale-yel-
low solid, mp 167–169 °C, yield 79.3%. 1H NMR (CDCl3) d 11.98
(br s, 1H), 9.06 (s, 1H), 8.19–8.17 (d, 1H), 7.93 (s, 1H), 7.67–7.64
(d, 1H), 7.55–7.53 (d, 1H), 4.97–4.94 (m, 1H), 4.19–4.17 (m, 2H),
3.91–3.89 (d, 3H), 2.66–2.61 (m, 3H); MS (ESI+) m/z calcd:
322.07, found: 323.1 [(M+1)+].
4.2.11. (S)-Methyl 2-(4-chloroquinoline-3-carboxamido)-3-
phenylpropanoate (5k)
Compound 5k was prepared in the same way as (5a),
pale-yellow solid, mp 116–118 °C, yield 81.0%. 1H NMR
4.2.19. (S)-Methyl 2-(4-chloro-6-methylquinoline-3-
carboxamido)-3-hydroxypropanoate (5t)
(CDCl3)
d 8.99 (s, 1H), 8.33–8.30 (d, 1H), 8.16–8.13 (d,
Compound 5t was prepared in the same way as (5a), pale-yel-
low solid, mp 193–195 °C, yield 88.6%. 1H NMR (CDCl3) d 9.11 (s,
1H), 8.20–8.18 (d, 1H), 7.71–7.69 (d, 1H), 7.63–7.58 (t, 1H), 7.39–
7.37 (d, 1H), 5.00–4.97 (t, 1H), 4.18 (s, 2H), 3.87 (s, 3H), 2.83 (s,
3H); MS (ESI+) m/z calcd: 322.07, found: 323.1 [(M+1)+].
1H), 7.88–7.82 (t, 1H), 7.75–7.70 (t, 1H), 7.35–7.25 (m,
3H), 7.22–7.19 (m, 2H), 6.86–6.83 (d, 1H), 5.22–5.15 (m
1H), 3.84 (s, 3H), 3.44–3.23 (dd,2H); MS (EI+) m/z calcd:
368.09, found: 368 [(M)+].
4.2.12. (S)-Methyl 2-(4-chloroquinoline-3-carboxamido)-3-(1H-
inden-3-yl) propanoate (5l)
4.2.20. (S)-Methyl 2-(4,8-dichloroquinoline-3-carboxamido)-3-
hydroxypropanoate (5u)
Compound 5l was prepared in the same way as (5a), yellow so-
lid, mp 84–86 °C, yield 91.0%. 1H NMR (CDCl3) d 8.98 (s, 1H), 8.31–
8.29 (d, 1H), 8.13 (s, 2H), 7.85–7.83 (t, 1H), 7.73–7.70 (t, 2H), 7.63–
7.60 (d, 1H), 7.40–7.36 (d, 1H), 7.22–7.10 (m, 3H), 6.93–6.90 (d,
1H), 5.24–5.21 (m, 1H), 3.79 (s, 3H), 3.68–3.50 (dd, 2H); MS (EI+)
m/z calcd: 407.10, found: 407 [(M)+].
Compound 5u was prepared in the same way as (5a), yellow so-
lid, mp 290–291 °C, yield 90.6%. 1H NMR (CDCl3) d 9.08 (s, 1H),
8.21–8.18 (d, 1H), 7.93 (s, 1H), 7.57 (s, 1H), 7.53 (s, 1H), 5.00–
4.94 (q, 1H), 4.17 (s, 2H), 3.87 (s, 3H); MS (ESI+) m/z calcd:
344.01, found: 345.1 [(M+1)+].

S. Chen et al. / Bioorg. Med. Chem. 17 (2009) 1948–1956
1955
4.2.21. (S)-Methyl 2-(4,7-dichloroquinoline-3-carboxamido)-3-
hydroxypropanoate (5v)
12.79 (br s, 1H), 10.01–9.97 (t, 1H), 8.77 (s, 1H), 8.16–8.17 (m,
1H), 7.82–8.72 (m, 2H), 4.85 (br s, 1H), 3.53–3.57 (m, 2H),
3.39–3.45 (m, 2H); MS (ESI+) m/z calcd: 266.05, found: 266.9
[(M+1)+].
Compound 5v was prepared in the same way as (5a), yellow so-
lid, mp 277–279 °C, yield 84.5%. 1H NMR (CDCl3) d 9.11 (s, 1H),
8.21–8.19 (d, 1H), 7.92 (s, 1H), 7.66 (s, 1H), 7.55–7.53 (d, 1H),
4.99–4.93 (q, 1H), 4.18 (s, 2H), 3.89 (s, 3H); MS (ESI+) m/z calcd:
344.01, found: 345.1 [(M+1)+].
4.2.29. N-(2-Hydroxyethyl)-1-methyl-4-oxo-1,4-
dihydroquinoline-3-carboxamide (8a)
A mixture of compound 3 (2 mmol), anhydrous potassium car-
bonate (3 mmol) and an excess of iodomethane was stirred in DMF
(30 ml) at 60 °C for 24 h then extracted with 20 ml  3 dichloro-
methane, the extraction was concentrated under reduced pressure.
Then the residue was recrystallized from ethanol to give 7a as
white needles, which was heated with an excess of ethanolamine
under reflux for 2 h and then the ethanolamine was evaporated un-
der reduced pressure. The residue was recrystallized from ethanol
to give 6a as white needles, mp > 250 °C, yield 74.7%. 1H NMR
(DMSO-d6) d 10.13–10.09 (t, 1H), 8.88 (s, 1H), 8.37–8.33 (d, 1H),
7.89–7.85 (m, 2H), 7.61–7.56 (t, 1H), 4.89–4.84 (t, 1H), 4.02 (s,
3H), 3.58–3.50 (m, 2H), 3.45–3.37 (m, 2H); MS (ESI+) m/z calcd:
246.10, found: 246.9 [(M+1)+].
4.2.22. (S)-Methyl 2-(4,6-dichloroquinoline-3-carboxamido)-3-
hydroxypropanoate (5w)
Compound 5w was prepared in the same way as (5a), pale-yellow
solid, mp 303–305 °C, yield 92.3%. 1H NMR (CDCl3) d 9.09 (s, 1H),
8.21–8.18 (d, 1H), 7.73–7.70 (d, 1H), 7.63–7.59 (t, 1H), 7.38–7.34
(d, 1H), 5.00–4.96 (q, 1H), 4.18 (s, 2H), 3.89 (s, 3H); MS (ESI+) m/z
calcd: 344.01, found: 345.1 [(M+1)+].
4.2.23. (S)-Methyl 2-(4-chloro-6-fluoroquinoline-3-
carboxamido)-3-hydroxypropanoate (5x)
Compound 5x was prepared in the same way as (5a), pale-yel-
low solid, mp 303–305 °C, yield 74.1%. 1H NMR (CDCl3) d 9.09 (s,
1H), 8.21–8.18 (d, 1H), 7.72–7.70 (d, 1H), 7.63–7.60 (t, 1H), 7.38–
7.34 (d, 1H), 4.99–4.96 (q, 1H), 4.18 (s, 2H), 3.88 (s, 3H); MS
(ESI+) m/z calcd: 326.05, found: 327.1 [(M+1)+].
4.2.30. 1-Benzyl-N-(2-hydroxyethyl)-4-oxo-1,4-
dihydroquinoline-3-carboxamide (8b)
Compound 8c was prepared in the same way as (8a), white nee-
dles, mp > 250 °C, yield 43.2%.. 1H NMR (DMSO-d6) d 10.44 (s, 1H),
8.94 (s, 1H), 8.55–8.52 (d, 1H), 7.65–7.60 (t, 1H), 7.50–7.42 (m, 2H),
7.38–7.32 (m, 3H), 7.18–7.15 (d, 2H), 5.48 (s, 1H), 3.89–3.84 (m,
2H), 3.70–3.65 (m, 2H), 3.59–3.56 (t, 1H); MS (ESI+) m/z calcd:
322.13, found: 323.0 [(M+1)+].
4.2.24. (S)-Methyl 2-(4-chloro-6, 8-difluoroquinoline-3-
carboxamido)-3-hydroxypropanoate (5y)
Compound 5y was prepared in the same way as (5a), pale-yel-
low solid, mp 219–221 °C, yield 81.8%. 1H NMR (CDCl3) d 9.09 (s,
1H), 7.73–7.69 (d, 1H), 7.63–7.60 (d, 1H), 7.38–7.34 (d, 1H),
4.99–4.96 (q, 1H), 4.18 (s, 2H), 3.87 (s, 3H); MS (ESI+) m/z calcd:
344.04, found: 345.1 [(M+1)+].
4.2.31. 1-(4-Fluorobenzyl)-N-(2-hydroxyethyl)-4-oxo-1,4-
dihydroquinoline-3-carboxamide (8c)
4.2.25. (2S,3R)-Methyl 2-(4-chloro-7-methylquinoline-3-
carboxamido)-3-hydroxybutanoate (5z)
Compound 8d was prepared in the same way as (8a), white nee-
dles, mp > 250 °C, yield 39.8%. 1H NMR (DMSO-d6) d 10.40 (s, 1H),
8.92 (s, 1H), 8.54–8.52 (d, 1H), 7.64–7.61 (t, 1H), 7.50–7.45 (t, 1H),
7.41–7.38 (d, 1H), 7.18–7.13 (m, 2H), 7.08–7.02 (m, 2H), 5.45 (s,
2H), 3.88–3.84 (m, 2H), 3.74–3.66 (m, 2H), 1.63 (br s, 1H); MS
(ESI+) m/z calcd: 340.12, found: 341.0 [(M+1)+].
Compound 5z was prepared in the same way as (5a), pale-yel-
low solid, mp 195–196 °C, yield 81.4%. 1H NMR (CDCl3) d 9.11 (s,
1H), 8.18–8.16 (d, 1H), 7.93 (s, 1H), 7.66 (s, 1H), 7.57–7.52 (d,
1H), 7.15–7.10 (d, 1H), 4.95–4.89 (q, 1H), 4.60–4.62 (m, 1H), 3.86
(s, 3H), 2.64 (s, 3H), 1.39–1.43 (d, 3H); MS (ESI+) m/z calcd:
336.09, found: 337.2 [(M+1)+].
4.2.32. N,1-Bis(2-hydroxyethyl)-4-oxo-1,4-dihydroquinoline-3-
carboxamide (8d)
4.2.26. N-(2-Hydroxyethyl)-4-oxo-6-phenoxy-1,4-
dihydroquinoline-3-carboxamide (6a)
Compound 8e was prepared in the same way as (8a), white nee-
dles, mp > 250 °C, yield 61.7%. 1H NMR (DMSO-d6) d 10.12–10.09 (t,
1H), 8.77 (s, 1H), 8.38–8.35 (d, 1H), 7.94–7.91 (d, 1H), 7.82–7.80 (t,
1H), 7.57–7.52 (t, 1H), 5.04–5.01 (t, 1H), 4.86–4.82 (t, 1H), 4.54–
4.51 (m, 2H), 3.75–3.74 (m, 2H), 3.55–3.50 (m, 2H), 3.43–3.35
(m, 2H); MS (ESI+) m/z calcd: 276.11, found: 277.0 [(M+1)+].
A mixture of compound 3 (2 mmol) and an excess of ethanol-
amine was stirred under reflux for 2 h and then the ethanolamine
was evaporated under reduced pressure, then the residue was
recrystallized from ethanol to give 6a as white needles,
mp > 250 °C, yield 57.9%. 1H NMR (DMSO-d6) d 12.75 (s, 1H),
10.07–10.03 (t, 1H), 8.72 (s, 1H), 7.79–7.76 (d, 1H), 7.61–7.53 (m,
2H), 7.48–7.43 (t, 2H), 7.26–7.21 (t, 1H), 7.13–7.10 (d, 2H), 4.80–
4.77 (t, 1H), 3.51–3.47 (m, 2H), 3.40–3.37 (m, 2H); MS (ESI+) m/z
calcd: 324.11, found: 325.0 [(M+1)+].
4.3. Biological evaluation
Transient transfection and CAT assays: 293T cells were grown
as monolayer in Dulbecco’s modified Eagle’s medium (DMEM)
(Gibco-BRL) supplemented with 10% (v/v) fetal calf serum, penicil-
lin (100 U mlÀ1), and streptomycin (100 U mlÀ1) at 37 °C in 5% CO2
containing humidified air. The cells were seeded at a six-well plate
24 h prior to transfection which was performed by standard cal-
cium phosphate coprecipitation techniques with optimum
amounts of the plasmids pLTRCAT and pSVCMVTAT. Twenty four
hours later, the culture medium was removed and the cells were
washed twice with phosphate-buffered saline (PBS). Then the
transfected cells were added to fresh medium together with di-
4.2.27. N-(2-Hydroxyethyl)-4-oxo-1,4-dihydroquinoline-3-
carboxamide (6b)
Compound 6b was prepared in the same way as (6a), dilute-
brow needles, mp > 250 °C, yield 66.6%. 1H NMR (DMSO-d6) d
12.63 (br s, 1H), 10.15–10.11 (t, 1H), 8.75 (s, 1H), 8.27–8.25 (m,
1H), 7.80–7.68 (m, 2H), 7.51–7.46 (m, 1H), 4.83 (br s, 1H), 3.55–
3.52 (m, 2H), 3.44–3.38 (m, 2H); MS (ESI+) m/z calcd: 232.08,
found: 232.9 [(M+1)+].
luted compounds at final concentration of 30 lM, respectively,
4.2.28. 6-Chloro-N-(2-hydroxyethyl)-4-oxo-1,4-
dihydroquinoline-3-carboxamide (6c)
and incubated for another 24 h. After 48-h post-transfection, the
cells were harvested and analyzed for CAT activity using a com-
mercial CAT ELISA kit (Roche Molecular Biochemicals) in accor-
dance with the manufacturer’s protocol. All data were reported
Compound 6c was prepared in the same way as (6a), dilute-
brow needles, mp > 250 °C, yield 75.1%. 1H NMR (DMSO-d6) d

1956
S. Chen et al. / Bioorg. Med. Chem. 17 (2009) 1948–1956
as a percentage of CAT activity ( SD). Results shown were repre-
sentative of three independent experiments
Medical Microbiology, University of Manitoba, Canada) for his help
in biological evaluation work.
Inhibition of SIV-induced syncytium in CEM174 cell cultures
was measured in a 96-well microplate containing 2 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 pro-
tect cells against the cytopathogenicity of SIV by 50%. Indinavir
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This project was supported by the National Natural Science
Foundation of China (No. 30670415). We thank Professor Xiaojian
Yao (Laboratory of Molecular Human Retrovirology, Department of