Caffeoylglycolic and caffeoylamino acid derivatives, halfmers of l-chicoric acid, as new HIV-1 integrase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2007.02.016

Human immunodeficiency virus (HIV) integrase (IN) catalyzes the integration of HIV DNA copy into the host cell DNA. l-Chicoric acid (1) has been found to be one of the most potent HIV-1 integrase inhibitor. Caffeoylglycolic and caffeoylamino acid derivati

Full text of DOI:10.1016/j.ejmech.2007.02.016

European Journal of Medicinal Chemistry 42 (2007) 1309e1315
http://www.elsevier.com/locate/ejmech
Short communication
Caffeoylglycolic and caffeoylamino acid derivatives, halfmers
of L-chicoric acid, as new HIV-1 integrase inhibitors
Seung Uk Lee a, Cha-Gyun Shin b, Chong-Kyo Lee c, Yong Sup Lee d,
*
a Life Sciences Division, Korea Institute of Science & Technology, P.O. Box 131 Cheongryang, Seoul 130-650, Republic of Korea
b Department of Biotechnology, Chung-Ang University, An-Sung 456-756, Republic of Korea
c Pharmaceutical Screening Center, Korea Research Institute of Chemical Technology, P.O. Box 107, Yusong, Taejeon 305-600, Republic of Korea
d Kyung Hee East-West Pharmaceutical Research Institute, College of Pharmacy, Kyung Hee University, 1 Hoegi-dong,
Dongdaemoon-ku, Seoul 130-701, Republic of Korea
Received 18 October 2006; received in revised form 20 February 2007; accepted 26 February 2007
Available online 12 March 2007
Abstract
Human immunodeficiency virus (HIV) integrase (IN) catalyzes the integration of HIV DNA copy into the host cell DNA. L-Chicoric acid (1)
has been found to be one of the most potent HIV-1 integrase inhibitor. Caffeoylglycolic and caffeoylamino acid derivatives’ halfmeric structures
of L-chicoric acid 2 were synthesized for the purpose of simplifying the structure of L-chicoric acid. Among synthesized, compounds 2c and 3f
showed HIV-1 IN inhibitory activities with IC50 values of 10.5 and 12.0 mM, respectively, comparable to that of parent compound L-chicoric acid
(IC50 ¼ 15.7 mM).
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Human immunodeficiency virus; Integrase inhibitor; L-Chicoric acid; Caffeoylglycolic acid; Caffeoylamino acid
1. Introduction
activity (Fig. 1). Accordingly, the development of synthetic
routes to 1 has been of interest to organic and medicinal
chemists. Unfortunately, the procedures still suffer from low
yields, lack of reproducibility or difficulty in the control of
stereogenic centers [3,7e9]. On the other hand, several ana-
logues of 1 have been synthesized to improve the anti-HIV
effect of 1. In general, the reported analogues of 1 were
dimeric forms of caffeic acid, which are linked by aliphatic,
alicyclic, or aromatic spacers [10]. Very recently, galloyl-
substituted derivatives with g-aminoalanine as a linker have
been found to have improved anti-HIV activities [11]. We
also reported several types of caffeoyl-based HIV-1 IN inhib-
itors that have glucose or five-membered heterocyclic ring or
catechol-substituted L-chicoric acid analogues as alternative
scaffolds [12e14].
The alarming spread of the acquired immune deficiency
syndrome (AIDS) epidemic has stimulated the discovery of
therapeutic agents to inhibit the replication of the causative
virus, human immunodeficiency virus (HIV-1). Advanced un-
derstanding of the viral cell cycle has made it possible to de-
fine targets to interrupt the life cycle of the virus. One such
target is viral integrase (IN), which is responsible for integra-
tion of proviral DNA into host cell DNA. This integration is
essential for the production of progeny virus, therefore, ther-
apeutic agents that can inhibit this process should be effective
anti-HIV agents [1,2]. Over the past few years, extensive ef-
forts have resulted in a large number of HIV-1 IN inhibitors
[3e6]. Among them, L-chicoric acid (1) is one of the most
potent HIV-1 IN inhibitors and has moderate anti-HIV
Simplification of the complex structures of natural products
is a promising strategy in the development of new leads for
medicinal applications [15]. This strategy could also provide
easier synthetic procedures to the target compounds since
the number of stereogenic centers on the parent compounds
* Corresponding author. Tel.: þ82 2 961 0370; fax: þ82 2 966 3885.
E-mail address: kyslee@khu.ac.kr (Y.S. Lee).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.02.016

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S.U. Lee et al. / European Journal of Medicinal Chemistry 42 (2007) 1309e1315
HO
HO
OH
OH
R1
O
R2O
R3O
X
CO2H
O
O
O
O
HO2C
CO2H
1, L-Chicoric acid
2, X = O
3, X = NH
Fig. 1. Design of new HIV-1 IN inhibitors from L-chicoric acid.
will be diminished. In this regard, we designed caffeoylgly-
colic acid derivatives 2, halfmeric structures of 1, by dividing
the structure of 1 into half to produce new HIV-1 IN inhibitors.
In addition, we also designed caffeoylamino acid derivatives 3
to examine the effect of amide on HIV-1 IN inhibitory activity.
In this series of compounds 3, more detailed SAR studies
would be possible since a variety of chiral a-amino acids
are readily available.
2.2. Biological activity
The resulting caffeoylglycolic and caffeoylamino acid de-
rivatives 2e3 were assayed for HIV IN inhibition (Table 1)
[13,18]. To compare inhibitory activities, L-chicoric acid (1)
was prepared by a known procedure [9] and its activity data
are included as a reference in the table. Although the catechol
moiety has previously been found to be important for HIV-1
IN inhibitory activity in the di-caffeoyl series of compounds,
3,4-dimethoxy-, 4-hydroxy-3-methoxy- and 3,4-dihydroxy-cin-
namic acid derivatives (4aec) were prepared and assayed to ex-
amine the substituent effect on HIV-1 IN inhibitory activity. As
expected, O-methylated compounds (2aeb) completely lacked
inhibitory activity (IC50 [ 300 mM), again indicating the im-
portance of the catechol group for activity. Interestingly, the in-
hibitory activity of caffeoylglycolic acid (2c) (IC50 ¼ 10.5 mM),
the simplest compound, was comparable to that of L-chicoric
acid (IC50 ¼ 15.7 mM), even though it contained one less
caffeoyl group than L-chicoric acid. On the other hand, in
C-1 methyl-substituted caffeoylglycolic acid (2d) and caffeoy-
lamino acid derivatives, the inhibitory activities were decreased
about 10-fold from L-chicoric acid. However, 3,4-dihydroxy-
benzyl-substituted caffeoylamino acid (3f) also exhibited
equipotent HIV-1 IN inhibitory activity to L-chicoric acid
(IC50 ¼ 12.0 mM), implicating the importance of the catechol
group for activity. These data indicate that the inhibitory activ-
ity can be retained or even increased upon simplification of
L-chicoric acid to halfmeric structures. However, more SAR
studies are needed on this series of compounds, since com-
pounds 2d and 3f did not show anti-HIVactivity in cell culture
assays with HIV-1IIIB infected MT-4 cells at non-toxic concen-
trations (200 mM) in spite of their potent HIV-1 IN inhibitory
activities [19]. On the other hand, L-chicoric acid exhibited
2. Results and discussion
2.1. Chemistry
Caffeoylglycolic and caffeoylamino acid derivatives were
synthesized from caffeic acid derivatives by alkylation with
methyl esters of a-bromo carboxylic acids or EDC coupling
with a-amino acids followed by demethylation of methyl ester
as shown in Schemes 1 and 2. Cinnamic acid derivatives 4aec
were treated with methyl a-bromo esters 5a and b using cesium
carbonate as a base to afford cinnamoylglycolic acid methyl
esters 6aed [16]. Demethylation of 6aed was accomplished
using the lithium iodide-promoted ester dealkylation method
since demethylation in alkaline hydrolysis conditions can afford
caffeic acid. Treatment of 6aed with lithium iodide in refluxing
pyridine gave caffeoylglycolic acid derivatives 2aed [17].
Caffeoylamino acids 3aef were synthesized as shown in
Scheme 2. The carbodiimide-based coupling reaction of caffeic
acid with amino acid methyl ester 7 using dicyclohexylcarbo-
diimide (DCC) and 4-(dimethylamino)pyridine (DMAP) in
CH2Cl2/THF afforded caffeoylamino acid methyl esters
8aef. The methyl ester group was hydrolyzed cleanly by
lithium hydroxide/water to give caffeoylamino acids 3aef.
R3
O
O
R3
R1O
R2O
OR4
R1O
R2O
Cs2CO3
DMF, rt
OR4
O
OH
+
Br
O
O
4
5
6, R = CH3
2, R = H
LiI, pyridine
reflux
Scheme 1.

S.U. Lee et al. / European Journal of Medicinal Chemistry 42 (2007) 1309e1315
1311
R3
O
O
R3
OR4
R1
R1O
R1O
DCC, HOBt, TEA
N
H
OR4
OH
+
H2N
O
THF,CHCl3
R2
O
4
7
8, R = CH3
3, R = H
LiOH
THF-H2O
Scheme 2.
anti-HIV activity with an EC50 value of 7.5 mM
(CC50 > 42.2 mM, TI > 5.6).
3.2. General procedure A for the synthesis of cinnamic
acid methoxycarbonylmethyl esters 6aed
Cs2CO3 (1.56 g, 4.8 mmol) was added to solutions of cin-
namic acid derivatives 4aec (4.8 mmol) in DMF (10 ml),
and the reaction mixture was then stirred for 5 min at rt.
Methyl bromoacetate 5 (4.8 mmol) was added dropwise to
the mixture at 0 ꢀC, which was then stirred at rt for 5 h. The
reaction mixture was diluted with ethyl acetate and washed
with water and saturated aqueous NaHCO3 solution. The or-
ganic layer was dried over anhydrous MgSO4, filtered, and
concentrated. The residue was purified by recrystallization or
flash column chromatography to provide cinnamic acid me-
thoxycarbonylmethyl esters 6aed.
3. Experimental section
3.1. Chemistry
All reactions were carried out under nitrogen atmosphere.
Flash column chromatography for purification was performed
with Merck Kiesegel 60 Art 9385 (230e400 mesh). All sol-
vents were purified according to standard procedures. H and
13C NMR spectra were recorded on a Gemini Varian-300
(300 and 75 MHz, respectively).
1
Table 1
HIV-1 integrase inhibitory activities of catechol-substituted compounds (2e3)
Inhibitors
IC50 (mM)
Inhibitors
IC50 (mM)
O
O
H3CO
OH
OH
HO
OH
O
>300
N
H
148.1
2a
2b
2c
3b
O
O
H3CO
HO
O
O
O
H3CO
HO
O
>300
129.6
95.0
3c
HO
OH
OH
N
H
O
O
O
HO
O
O
HO
HO
OH
OH
O
O
10.5
3d
HO
N
H
O
O
HO
OH
HO
HO
O
O
128.1
129.0
2d
3e
HO
OH
OH
N
H
O
O
HO
OH
OH
O
HO
OH
N
171.6
15.7
12.0
3a
3f
HO
H
O
N
H
HO
HO
L-Chicoric acid

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S.U. Lee et al. / European Journal of Medicinal Chemistry 42 (2007) 1309e1315
3.2.1. 3-(3,4-Dimethoxy-phenyl)-acrylic acid
methoxycarbonylmethyl ester (6a)
anhydrous MgSO4, filtered, and concentrated. The residue was
purified by recrystallization or flash column chromatography
to provide cinnamic acid carboxymethyl esters (2aed).
Treatment of 3,4-dimethoxycinnamic acid (1.0 g, 4.8 mmol)
with methyl bromoacetate (0.73 g, 4.8 mmol) according to gen-
eral procedure A provided the desired product 6a (0.85 g, 63%).
1H NMR (CDCl3) d 7.72 (1H, d, J ¼ 15.9 Hz), 7.13 (1H, dd,
J ¼ 8.3, 1.7 Hz), 7.07 (1H, d, J ¼ 1.7 Hz), 6.88 (1H, d,
J ¼ 8.3 Hz), 6.41 (1H, d, J ¼ 15.9 Hz), 4.76 (2H, s), 3.92 (6H,
s), 3.80 (3H, s); 13C NMR (CDCl3) d 168.4, 166.2, 151.3,
149.1, 146.2, 127.0, 122.9, 114.2, 111.0, 109.5, 60.5, 55.8,
55.7, 52.1.
3.3.1. 3-(3,4-Dimethoxy-phenyl)-acrylic acid carboxymethyl
ester (2a)
General procedure B using 6a (0.34 g, 1.2 mmol) afforded
2a (0.2 g, 63%). 1H NMR (acetone-d6) d 7.72 (1H, d,
J ¼ 15.9 Hz), 7.41 (1H, d, J ¼ 1.7 Hz), 7.13 (1H, dd, J ¼ 8.3,
1.7 Hz), 7.04 (1H, d, J ¼ 8.3 Hz), 6.55 (1H, d, J ¼ 15.9 Hz),
4.78 (2H, s), 3.94 (3H, s), 3.90 (3H, s); 13C NMR (acetone-
d6) d 173.4, 166.2, 145.9, 142.9, 133.6, 127.4, 123.3, 114.8,
111.7, 110.4, 60.3, 55.5.
3.2.2. 3-(4-Hydroxy-3-methoxy-phenyl)-acrylic acid
methoxycarbonylmethyl ester (6b)
Treatment of 4-hydroxy-3-methoxycinnamic acid (1.0 g,
5.2 mmol) with methyl bromoacetate (0.80 g, 5.2 mmol) ac-
cording to general procedure A provided the desired product
6b (1.1 g, 79%). 1H NMR (acetone-d6) d 7.70 (1H, d,
J ¼ 15.9 Hz), 7.42 (1H, s), 7.21 (1H, d, J ¼ 8.1 Hz), 6.92
(1H, d, J ¼ 8.1 Hz), 6.51 (1H, d, J ¼ 15.9 Hz), 4.78 (2H, s),
3.96 (3H, s), 3.80 (3H, s); 13C NMR (acetone-d6) d 169.1,
166.9, 150.3, 148.7, 147.0, 127.2, 124.3, 116.0, 114.5,
111.3, 61.0, 56.3, 52.2.
3.3.2. 3-(4-Hydroxy-3-methoxy-phenyl)-acrylic acid
carboxymethyl ester (2b)
General procedure B using 6b (0.94 g, 3.7 mmol) afforded
2b (0.4 g, 45%). 1H NMR (acetone-d6) d 8.06 (1H, br s), 7.47
(1H, d, J ¼ 15.9 Hz), 7.20 (1H, d, J ¼ 1.9 Hz), 7.00 (1H, dd,
J ¼ 8.1, 1.9 Hz), 6.69 (1H, d, J ¼ 8.1 Hz), 6.29 (1H, d,
J ¼ 15.9 Hz), 4.55 (2H, s), 3.74 (H, s); 13C NMR (CD3OD-
d6) d 171.7, 168.4, 150.8, 149.4, 147.7, 127.6, 124.3, 116.5,
114.6, 111.7, 61.6, 56.4.
3.2.3. 3-(3,4-Dihydroxy-phenyl)-acrylic acid
methoxycarbonylmethyl ester (6c)
3.3.3. 3-(3,4-Dihydroxy-phenyl)-acrylic acid carboxymethyl
ester (2c)
Treatment of 3,4-dihydroxycinnamic acid (1.0 g, 5.6 mmol)
with methyl bromoacetate (0.86 g, 5.6 mmol) according to gen-
eral procedure A provided the desired product 6c (0.85 g, 60%).
1H NMR (DMSO-d6) d 9.69 (1H, br s), 9.20 (1H, br s), 7.56 (1H,
d, J ¼ 15.9 Hz), 7.10 (1H, s), 7.55 (1H, d, J ¼ 8.1 Hz), 6.79 (1H,
d, J ¼ 8.1 Hz), 6.37 (1H, d, J ¼ 15.9 Hz), 4.77 (2H, s), 3.70 (3H,
s); 13C NMR (acetone-d6) d 169.1, 166.8, 148.9, 146.9, 146.2,
127.6, 127.3, 122.8, 116.3, 115.3, 114.3, 61.0, 52.2.
General procedure B using 6c (0.50 g, 2.0 mmol) afforded
2c (0.17 g, 35%). 1H NMR (DMSO-d6) d 7.48 (1H, d,
J ¼ 15.9 Hz), 7.08 (1H, s), 6.97 (1H, d, J ¼ 8.1 Hz), 6.77
(1H, d, J ¼ 8.1 Hz), 6.28 (1H, d, J ¼ 15.9 Hz), 4.63 (2H, s);
13C NMR (DMSO-d6 þ CD3OD) d 173.7, 167.5, 149.4,
146.5, 145.7, 126.5, 122.2, 116.4, 114.8, 100.6, 63.5.
3.3.4. 3-(3,4-Dihydroxy-phenyl)-acrylic acid
1-carboxy-ethyl ester (2d)
3.2.4. 3-(3,4-Dihydroxy-phenyl)-acrylic acid
1-methoxycarbonyl-ethyl ester (6d)
General procedure B using 6d (0.28 g, 1.1 mmol) afforded
2d (0.13 g, 50%). 1H NMR (DMSO-d6) d 7.50 (1H, d,
J ¼ 15.9 Hz), 7.01e7.06 (2H, m), 6.76 (1H, d, J ¼ 8.1,
1.9 Hz), 6.31 (1H, d, J ¼ 15.9 Hz), 4.98 (1H, q, J ¼ 7.1 Hz),
1.43e1.45 (3H, d, J ¼ 7.1 Hz); 13C NMR (DMSO-d6)
d 172.6, 166.4, 149.1, 146.4, 146.0, 125.8, 122.0, 116.2,
115.4, 113.7, 68.7, 17.3.
Treatment of 3,4-dihydroxycinnamic acid (1.0 g, 5.6 mmol)
with 2-bromopropionic acid methyl ester (0.94 g, 5.6 mmol)
according to general procedure A provided the desired product
6d (0.5 g, 30%). 1H NMR (acetone-d6) d 8.56 (2H, br s), 7.53
(1H, d, J ¼ 15.9 Hz), 7.13 (1H, d, J ¼ 1.9 Hz), 7.01 (1H, dd,
J ¼ 8.1, 1.9 Hz), 6.82 (1H, d, J ¼ 8.1 Hz), 6.27 (1H, d,
J ¼ 15.9 Hz), 5.06 (1H, q, J ¼ 7.2 Hz), 3.64 (3H, s), 1.43
(3H, d, J ¼ 7.2 Hz); 13C NMR (acetone-d6) d 171.9, 166.8,
149.1, 146.8, 146.4, 117.2, 122.6, 116.3, 115.2, 114.4, 69.1,
52.3, 17.2.
3.4. General procedure C for the syntheses of
N-methoxycarbonylmethyl cinnamamides 8aef
To solutions of cinnamic acids 4aec (4.8 mmol) in THF
(12 ml) cooled to 0 ꢀC were added successively a-amino acid
methyl ester hydrochlorides 7 (4.8 mmol) and solutions of trie-
thylamine (0.49 g, 4.8 mmol) in CHCl3 (5 ml), 1-hydroxyben-
zotriazole (HOBt, 0.65 g, 4.8 mmol) in THF (10 ml), and
dicyclohexylcarbodiimide (DCC, 1.09 g, 5.3 mmol) in CHCl3
(10 ml). After stirring for 1 h at 0 ꢀC, the mixture was warmed
to rt and stirred overnight. After filtration of insoluble material
(dicyclohexylurea) from the mixture, the filtrate was evapo-
rated and dissolved in ethyl acetate. The ethyl acetate solution
was washed with water, 10% citric acid aqueous solution, 10%
3.3. General procedure B for the syntheses of cinnamic
acid carboxymethyl esters 2aed
Lithium iodide (9.68 mmol) was added to solutions of cin-
namic acid methoxycarbonylmethyl esters 6aed (1.21 mmol)
in pyridine (10 ml) under N2 atmosphere, and the resulting solu-
tion was refluxed for 4.5 h. After cooling to rt, the reaction mix-
ture was diluted with ethyl acetate and washed three times with
water and 3 N HCl solution. The organic layer was dried over

S.U. Lee et al. / European Journal of Medicinal Chemistry 42 (2007) 1309e1315
1313
NaHCO3 aqueous solution, and finally, with brine. The organic
3.4.5. 2-[3-(3,4-Dihydroxy-phenyl)-acryloylamino]-3-
phase was dried over anhydrous MgSO4, filtered, and concen-
trated. The residue was purified by recrystallization or flash
column chromatography to provide N-methoxycarbonylmethyl
cinnamamides 8aef.
(4-hydroxy-phenyl)-propionic acid methyl ester (8e)
General procedure C using 3,4-dihydroxycinnamic acid (1.0 g,
5.6 mmol) and L-tyrosine methyl ester hydrochloride (1.30 g,
5.6 mmol) afforded 8e (1.8 g, 89%). 1H NMR (acetone-d6)
d 7.44 (1H, d, J ¼ 15.9 Hz), 7.10 (1H, d, J ¼ 2.2 Hz), 7.09 (2H,
d, J ¼ 8.1 Hz), 6.87 (1H, dd, J ¼ 8.4, 2.2 Hz), 6.87 (1H, d,
J ¼ 8.4 Hz), 6.79 (2H, d, J ¼ 8.1 Hz), 6.39 (1H, d,
J ¼ 15.7 Hz), 4.83 (1H, m, J ¼ 5.6 Hz), 3.70 (3H, s), 3.08
(1H, dd, J ¼ 13.8, 5.6 Hz), 2.84 (1H, dd, J ¼ 13.8, 9.0 Hz);
13C NMR (DMSO-d6) d 173.1, 166.2, 156.7, 148.2, 146.3,
140.7, 130.7, 127.9, 126.9, 121.3, 118.3, 116.5, 115.8, 114.6,
54.8, 52.5, 36.9.
3.4.1. [3-(3,4-Dihydroxy-phenyl)-acryloylamino]-acetic
acid methyl ester (8a)
General procedure C using 3,4-dihydroxycinnamic acid
(1.0 g, 5.6 mmol) and glycine methyl ester hydrochloride
(0.70 g, 5.6 mmol) afforded 8a (0.58 g, 42%). 1H NMR
(DMSO-d6) d 9.44 (1H, br s), 9.20 (1H, br s), 8.46 (1H,
t, J ¼ 5.4 Hz), 7.29 (1H, d, J ¼ 15.9 Hz), 7.17 (1H,
d, J ¼ 1.5 Hz), 7.04 (1H, dd, J ¼ 8.1, 1.5 Hz), 6.82 (1H, d,
J ¼ 8.1 Hz), 6.42 (1H, d, J ¼ 15.9 Hz), 3.97 (2H, d,
J ¼ 5.4 Hz), 3.42 (3H, s); 13C NMR (DMSO-d6) d 170.6,
166.0, 147.6, 145.6, 140.0, 126.2, 120.7, 117.6, 115.8,
113.9, 51.8, 40.8.
3.4.6. 3-(3,4-Dihydroxy-phenyl)-2-[3-(3,4-dihydroxy-
phenyl)-acryloylamino]-propionic acid methyl ester (8f)
General procedure C using 3,4-dihydroxycinnamic acid
(1.0 g, 5.6 mmol) and L-3,4-dihydroxyphenylalanine methyl
1
ester (1.10 g, 5.6 mmol) afforded 8f (1.2 g, 58%). H NMR
(DMSO-d6) d 8.37 (1H, d, J ¼ 7.5 Hz), 7.25 (1H, d,
J ¼ 15.7 Hz), 6.97 (1H, d, J ¼ 1.8 Hz), 6.87 (1H, d, J ¼ 8.2,
1.8 Hz), 6.77 (1H, d, J ¼ 8.2 Hz), 6.63e6.65 (2H, m,
J ¼ 7.8 Hz), 6.49 (1H, dd, J ¼ 8.0, 1.9 Hz), 6.60 (1H, s),
6.43 (1H, d, J ¼ 15.7 Hz), 4.47 (1H, m, J ¼ 5.2 Hz), 3.60
(3H, s, eOCH3), 2.88 (1H, dd, J ¼ 13.7, 5.6 Hz), 2.81 (1H,
dd, J ¼ 13.7, 9.0 Hz); 13C NMR (DMSO-d6) d 173.1, 166.2,
148.2, 146.3, 145.7, 144.7, 140.7, 128.6, 126.9, 126.9,
121.3, 120.5, 118.4, 117.1, 116.5, 116.1, 114.6, 54.9, 52.5,
37.1.
3.4.2. 2-[3-(3,4-Dihydroxy-phenyl)-acryloylamino]-
propionic acid methyl ester (8b)
General procedure C using 3,4-dihydroxycinnamic acid
(1.0 g, 5.6 mmol) and L-alanine methyl ester hydrochloride
(0.78 g, 5.6 mmol) afforded 8b (0.50 g, 34%). 1H NMR
(DMSO-d6) d 9.32 (2H, br s), 8.44 (1H, d, J ¼ 6.6 Hz), 7.29
(1H, d, J ¼ 15.9 Hz), 7.00 (1H, d, J ¼ 1.5 Hz), 6.89 (1H, dd,
J ¼ 8.1, 1.5 Hz), 6.79 (1H, d, J ¼ 8.1 Hz), 6.42 (1H, d,
J ¼ 15.9 Hz), 4.41 (1H, m), 3.67 (3H, s), 1.36 (3H, d,
J ¼ 7.2 Hz); 13C NMR (DMSO-d6) d 173.4, 165.3, 147.6,
145.6, 140.0, 126.4, 120.6, 117.6, 115.8, 113.8, 51.9, 47.7,
17.1.
3.5. General procedure D for the syntheses of
compounds 3aef
3.4.3. 2-[3-(3,4-Dihydroxy-phenyl)-acryloylamino]-4-
methyl-pentanoic acid methyl ester (8c)
Lithium hydroxide (5.40 mmol) was added to solutions of
N-methoxycarbonylmethyl cinnamamides 8aef (1.08 mmol)
in THF and water (1:1, 14 ml) at 0 ꢀC. The mixture was stirred
at rt for 40 min. The solution was diluted with water and acid-
ified to pH 1 by addition of 3 N HCl. The mixture was ex-
tracted with ethyl acetate, and the organic layer was dried
over anhydrous MgSO4, filtered, and concentrated. The resi-
due was purified by recrystallization or flash column chroma-
tography to provide N-carboxymethyl cinnamamides 3aef.
General procedure C using 3,4-dihydroxycinnamic acid
(1.0 g, 5.6 mmol) and L-leucine methyl ester hydrochloride
(1.02 g, 5.6 mmol) afforded 8c (1.1 g, 66%). 1H NMR
(CDCl3) d 7.42 (1H, d, J ¼ 15.9 Hz), 7.00 (1H, s), 6.94 (1H,
d, J ¼ 7.5 Hz), 6.75 (1H, d, J ¼ 7.5 Hz), 6.26 (1H, d,
J ¼ 15.9 Hz), 4.77 (1H, q, J ¼ 5.1 Hz), 3.75 (3H, s), 1.63e
1.75 (3H, m), 0.96 (6H, d, J ¼ 5.1 Hz); 13C NMR (CDCl3)
d 174.4, 167.7, 146.9, 144.3, 142.8, 127.0, 122.1, 116.3,
115.4, 114.2, 52.6, 51.3, 41.1, 24.9, 22.8, 21.7.
3.5.1. [3-(3,4-Dihydroxy-phenyl)-acryloylamino]-acetic
acid (3a)
3.4.4. 2-[3-(3,4-Dihydroxy-phenyl)-acryloylamino]-3-
phenyl-propionic acid methyl ester (8d)
General procedure C using 3,4-dihydroxycinnamic acid
(1.0 g, 5.6 mmol) and L-phenylalanine methyl ester hydrochlo-
1
ride (1.20 g, 5.6 mmol) afforded 8d (1.5 g, 81%). H NMR
(acetone-d6) d 7.28 (1H, d, J ¼ 15.7 Hz), 7.31e7.18 (5H,
m), 6.94 (1H, d, J ¼ 1.8 Hz), 6.83 (1H, dd, J ¼ 8.1, 1.8 Hz),
6.74 (1H, d, J ¼ 8.1 Hz), 6.38 (1H, d, J ¼ 15.7 Hz), 4.59
(1H, q, J ¼ 5.6 Hz), 3.57 (3H, s), 3.08 (1H, dd, J ¼ 13.7,
5.5 Hz), 2.96 (1H, dd, J ¼ 13.7, 9.2 Hz); 13C NMR (acetone-
d6) d 172.9, 166.4, 148.0, 146.2, 141.5, 138.0, 130.1, 129.2,
128.2, 127.5, 121.7, 118.8, 116.3, 114.9, 54.6, 52.2, 38.4.
General procedure D using 8a (0.30 g, 1.20 mmol) afforded
3a (0.09 g, 31%). 1H NMR (DMSO-d6) d 8.33 (1H, t,
J ¼ 6.0 Hz), 7.28 (1H, d, J ¼ 15.9 Hz), 6.98 (1H, d,
J ¼ 1.5 Hz), 6.88 (1H, dd, J ¼ 8.1, 1.5 Hz), 6.77 (1H, d,
J ¼ 8.1 Hz), 6.43 (1H, d, J ¼ 15.9 Hz), 3.88 (2H, d,
J ¼ 6.0 Hz); 13C NMR (DMSO-d6) d 171.5, 165.8, 147.5,
145.6, 139.8, 126.3, 120.6, 117.9, 115.8, 113.8, 40.8.
3.5.2. 2-[3-(3,4-Dihydroxy-phenyl)-acryloylamino]-
propionic acid (3b)
General procedure D using 8b (0.35 g, 1.30 mmol) afforded
3b (0.10 g, 30%). 1H NMR (DMSO-d6) d 8.19 (1H, d,

1314
S.U. Lee et al. / European Journal of Medicinal Chemistry 42 (2007) 1309e1315
J ¼ 6.9 Hz), 7.44 (1H, d, J ¼ 15.9 Hz), 7.05 (1H, d,
J ¼ 1.8 Hz), 6.94 (1H, dd, J ¼ 8.3, 1.8 Hz), 6.80 (1H, d,
J ¼ 8.3 Hz), 6.47 (1H, d, J ¼ 15.9 Hz), 4.54 (1H, m,
J ¼ 6.9 Hz), 1.48 (3H, d, J ¼ 7.2 Hz); 13C NMR (DMSO-d6)
d 176.2, 168.9, 148.8, 146.7, 142.8, 128.2, 117.9, 116.4,
115.1, 49.5, 17.8.
3.6. HIV integrase assay [12,17]
Recombinant human immunodeficiency virus type 1 (HIV-
1) integrase was expressed in Escherichia coli and purified in
a one-step process using a nickel-chelated column. Aliquots of
0.5 mg/ml HIV-1 integrase stock solutions were stored at
ꢁ70 ꢀC until used.
3.5.3. 2-[3-(3,4-Dihydroxy-phenyl)-acryloylamino]-4-
methyl-pentanoic acid (3c)
3.6.1. Oligonucleotide substrates
Two complementary 20-mer oligonucleotides whose se-
quences resemble the end of U5-LTR were obtained from Korea
Biotech, Inc.: K16 (U5-LTR, þstrand), 50-TGTGGAAAATC
TCTAGCAGT-30; K17 (U5-LTR, ꢁstrand), 50-ACTGCTAGA
GATTTTCCACA-30. The oligonucleotides were purified in
20% polyacrylamide gels before use. To construct the duplex
oligonucleotide substrate, 30 pmol of oligonucleotide K16
were labeled at the 50-end using 250 mCi of [g-32P]-ATP
(3000 Ci/mmol; 1 Ci ¼ 37 GBq; Amersham) and 10 units of
T4 polynucleotide kinase (T4 PNK, New England Biolabs) in
40 ml of reaction buffer (70 mM triseHCl [pH 7.6], 10 mM
MgCl2, 5 mM dithiothreitol) at 37 ꢀC for 15 min. The labeling
reaction was supplemented with 10 mM EDTA and heated to
85 ꢀC for 15 min to inactivate T4 PNK. After addition of
30 pmol of oligonucleotide K17, the reaction mixture was
boiled for 3 min and cooled slowly. Labeled substrate was sep-
arated from unincorporated nucleotide on a Biospin 6 column
(Bio-Rad).
General procedure D using 8c (0.50 g, 1.60 mmol) afforded
3c (0.32 g, 67%). 1H NMR (DMSO-d6) d 8.18 (1H, d,
J ¼ 8.1 Hz, eCONHCHe), 7.26 (1H, d, J ¼ 15.9 Hz, e
CH]CHe), 6.97 (1H, d, J ¼ 1.7 Hz, phenyl-H2), 6.86 (1H,
dd, J ¼ 8.3, 1.7 Hz, phenyl-H6), 6.77 (1H, d, J ¼ 8.3 Hz, phe-
nyl-H5), 6.45 (1H, d, J ¼ 15.9 Hz), 4.35 (1H, q, J ¼ 5.8 Hz),
1.69e1.54 (3H, m), 0.90 (6H, d, J ¼ 4.7 Hz); 13C NMR
(DMSO-d6) d 174.6, 165.4, 147.4, 145.6, 139.6, 126.4,
120.5, 118.1, 115.8, 113.9, 50.6, 24.4, 22.9, 21.4, 14.1.
3.5.4. 2-[3-(3,4-Dihydroxy-phenyl)-acryloylamino]-3-
phenyl-propionic acid (3d)
General procedure D using 8d (0.50 g, 1.47 mmol) afforded
3d (0.32 g, 66%). 1H NMR (DMSO-d6) d 8.30 (1H, d,
J ¼ 8.1 Hz), 7.32e7.20 (5H, m), 7.21 (1H, d, J ¼ 15.9 Hz),
6.95 (1H, d, J ¼ 2.4 Hz), 6.85 (1H, dd, J ¼ 8.1, 2.4 Hz), 6.75
(1H, d, J ¼ 8.1 Hz), 6.41 (1H, d, J ¼ 15.9 Hz), 4.56 (1H, m),
3.13 (1H, dd, J ¼ 13.8, 4.9 Hz), 2.94 (1H, dd, J ¼ 13.8,
9.6 Hz). 13C NMR (DMSO-d6) d 173.7, 165.8, 157.1, 147.9,
146.0, 140.5, 140.2, 138.2, 129.5, 128.7, 126.9, 126.7,
121.0, 118.4, 118.0, 116.2, 114.3, 54.1, 37.4.
3.6.2. HIV-1 integrase reaction
Standard endonucleolytic activity assays were performed
in the presence of potential inhibitor with 0.1 pmol of duplex
oligonucleotide substrate and 15 pmol of HIV-1 integrase in
15 mM triseHCl (pH 7.4), 100 mM NaCl, 1 mM MnCl2,
2 mM 2-mercaptoethanol, 2.5 mM CHAPS, 0.1 mM EDTA,
0.1 mM PMSF, 1% glycerol, and 10 mM imidazole in a total
volume of 10 ml. Inhibitors or drugs were dissolved in 100%
DMSO and added to the reaction for a final concentration of
5% DMSO. Reaction mixtures were incubated at 33 ꢀC for
90 min and stopped by addition of 4 ml of 95% formamide,
20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene
cyanol FF. The reaction mixtures were heated to 90 ꢀC for
3 min and electrophoresed on 20% denaturing polyacrylamide
gels. Reaction products were visualized by autoradiography of
wet gels. IC50 was calculated by scanning bands on Kodak-5
film (Image Master VDS, Pharmacia Biotech).
3.5.5. 2-[3-(3,4-Dihydroxy-phenyl)-acryloylamino]-3-
(4-hydroxy-phenyl)-propionic acid (3e)
General procedure D using 8d (0.50 g, 1.40 mmol) afforded
3d (0.16 g, 38%). 1H NMR (DMSO-d6) d 8.13 (1H, d,
J ¼ 8.0 Hz), 7.18 (1H, d, J ¼ 15.7 Hz), 7.02 (2H, d,
J ¼ 8.3 Hz), 6.94 (1H, s), 6.83 (1H, d, J ¼ 8.1 Hz), 6.74 (1H,
d, J ¼ 8.1 Hz), 6.64 (2H, d, J ¼ 8.3 Hz), 6.42 (1H, d,
J ¼ 15.7 Hz), 4.43 (1H, q, J ¼ 5.4 Hz), 3.00 (1H, dd,
J ¼ 13.8, 4.9 Hz), 2.81 (1H, dd, J ¼ 13.8, 9.2 Hz); 13C NMR
(DMSO-d6) d 176.6, 169.0, 157.2, 148.8, 146.7, 142.7,
131.3, 129.5, 128.3, 122.3, 118.1, 116.5, 116.2, 115.1, 56.5,
37.9.
3.5.6. 3-(3,4-Dihydroxy-phenyl)-2-[3-(3,4-dihydroxy-
phenyl)-acryloylamino]-propionic acid (3f)
4. Conclusion
General procedure D using 8f (0.50 g, 1.34 mmol) afforded
3f (0.12 g, 25%). 1H NMR (DMSO-d6) d 7.76 (1H, d,
J ¼ 7.2 Hz), 7.18 (1H, d, J ¼ 15.9 Hz), 6.97 (1H, s), 6.83
(1H, d, J ¼ 7.8 Hz), 6.74 (1H, d, J ¼ 7.8 Hz), 6.65 (1H, s),
6.57 (1H, d, J ¼ 7.8 Hz), 6.46 (1H, d, J ¼ 7.8 Hz), 6.47 (1H,
d, J ¼ 15.9 Hz), 4.33 (1H, q, J ¼ 5.1 Hz), 2.98 (1H, dd,
J ¼ 13.5, 4.2 Hz), 2.76 (1H, dd, J ¼ 13.5, 7.8 Hz); 13C NMR
(DMSO-d6) d 175.7, 165.5, 148.1, 146.4, 145.5, 144.2,
139.5, 130.4, 127.1, 121.0, 120.7, 119.7, 117.5, 116.6,
115.9, 114.8, 56.2, 37.8.
We synthesized and tested the HIV-1 IN inhibitory activi-
ties of caffeoylglycolic and caffeoylamino acid derivatives 2
and 3. Compounds 2c and 3f showed equal or slightly in-
creased HIV-1 IN inhibitory activity compared to parent com-
pound L-chicoric acid. It is notable that while dimerization of
a pharmacophore of bioactive compounds through a linker is
relatively well-known strategy for obtaining more potent activ-
ity, compounds in this work retained activity even upon sim-
plification into halfmeric structures.

S.U. Lee et al. / European Journal of Medicinal Chemistry 42 (2007) 1309e1315
[8] H. Zhao, T.R. Burke Jr., Synth. Commun. 28 (1998) 737.
1315
Acknowledgements
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C. Cornell, J. Kuan, T.R. Kim, W.E. Robinson Jr., J. Med. Chem. 42
(1999) 497e509.
We are grateful to the Ministry of Education and Human
Resources Development for the support through the Post-
BK21 program.
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