Dicaffeoyl- or digalloyl pyrrolidine and furan derivatives as HIV integrase inhibitors

Article abstract of DOI:10.1016/S0968-0896(01)00013-X

Human immunodeficiency virus (HIV) integrase (IN) catalyzes the integration of HIV DNA copy into the host cell DNA. Such integration is essential for the production of progeny viruses, and therefore therapeutic agents that can inhibit this process should be effective anti-HIV agents. We have previously reported the inhibitory activity of dicaffeoylglucoside against HIV IN. In the present study, we have synthesized and tested dicaffeoyl or digalloyl compounds joined through a five-membered heterocyclic ring as HIV IN inhibitors to explore the SARs of this family of compounds. The starting heterocyclic diols were prepared from L-tartaric acid, diethyl L-tartarate or D-(+)-ribonic γ-lactone. We found that the HIV IN inhibitory activities of dicaffeoyl derivatives were comparable to that of L-chicoric acid (IC₅₀ = 24.9μM). On the other hand, digalloyl derivatives were more potent than L-chicoric acid with IC₅₀ values of 4.7-15.6μM. Copyright

Full text of DOI:10.1016/S0968-0896(01)00013-X

Bioorganic & Medicinal Chemistry 9 (2001) 1429–1437
Dicaffeoyl- or Digalloyl Pyrrolidine and Furan Derivatives as
HIV Integrase Inhibitors
Dong Jin Hwang,a,b Sun Nam Kim,a Jung Hoon Choib and Yong Sup Leea,*
aDivision of Life Sciences, Korea Institute of Science & Technology, PO Box 131, Cheongryang, Seoul 130-650, South Korea
bDepartment of Chemistry, Hanyang University, Seoul 133-791, South Korea
Received 13 October2000; accepted 11 January 2001
Abstract—Human immunodeficiency virus (HIV) integrase (IN) catalyzes the integration of HIV DNA copy into the host cell
DNA. Such integration is essential for the production of progeny viruses, and therefore therapeutic agents that can inhibit this
process should be effective anti-HIV agents. We have previously reported the inhibitory activity of dicaffeoylglucosides against HIV
IN. In the present study, we have synthesized and tested dicaffeoyl or digalloyl compounds joined through a five-membered het-
erocyclic ring as HIV IN inhibitors to explore the SARs of this family of compounds. The starting heterocyclic diols were prepared
from l-tartaric acid, diethyl l-tartarate or d-(+)-ribonic g-lactone. We found that the HIV IN inhibitory activities of dicaffeoyl
derivatives were comparable to that of l-chicoric acid (IC50=24.9 mM). On the other hand, digalloyl derivatives were more potent
than l-chicoric acid with IC50 values of 4.7–15.6 mM. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
tested dicaffeoyl ordigalloyl compounds ( III) joined
through a functionalized pyrrolidine or furan ring linker
to explore the structure and HIV IN inhibitory activity
relationships (Scheme 1). Since the use of con-
formationally restricted molecules as a means to better
understand or improve the activity of the parent mole-
cule is a common theme in medicinal chemistry, we
selected five-membered ring, more rigid linker as a cen-
tral linker. Additionally, the linkers were designed to
functionalized five-membered rings which have hydro-
gen bond accepting ordonating gorups in place of
carboxylic acid moiety in l-tartaric acid.
Human immunodeficiency virus (HIV-1) is the prob-
able causative agent of acquired immune deficiency
syndrome (AIDS), which is one of the world’s most
serious health problems. HIV integrase (IN) catalyzes
the integration of HIV DNA copy into the host cell
DNA. Such integration is essential for the production of
progeny viruses, and therefore therapeutic agents that
can inhibit this process should be effective anti-HIV
agents.1ꢀ3
Recent studies showed that l-chicoric acid (1) and
dicaffeoylquinic acids (DCQAs, forexample, compound
(2) display potent activity against HIV IN and can
inhibit HIV replication with moderate anti-HIV activity
(Fig. 1).4ꢀ9 Extensive efforts have therefore been made
to develop caffeoyl-based HIV IN inhibitors. The com-
mon structural features of reported synthetic analogues
are caffeic acid esters separated by aliphatic, alicyclic, or
aromatic linker.10,11 We also recently reported a new
type of caffeoyl-based HIV IN inhibitors (4), which
have a glucose ring as a basic skeleton.12 However, there
was no report on the synthesis of caffeic acid esters
separated by a five-membered ring linker for HIV IN
inhibitors. In the present study, we have synthesized and
*Corresponding author. Tel.: +82-2-958-5167; fax: +82-2-958-5189;
e-mail: yslee@kist.re.kr
Figure 1.
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00013-X

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D. J. Hwang et al. / Bioorg. Med. Chem. 9 (2001) 1429–1437
Results and Discussion
aqueous HCl in 1,4-dioxane.15 The TBS protecting
groups were removed concomitantly during the lactone
ring formation stage.
The starting five-membered ring diols (I-a–e) were pre-
pared from l-tartaric acid as depicted in Scheme 2. N-
Methyl-3,4-dihydroxysuccinimide (I-a) was synthesized
by treatment of diacetylated compound 5, which was
derived from l-tartaric acid,13 with acetyl chloride in
ethanol (equation 1). 3,4-Dihydroxysuccinimide (I-b)
was also synthesized by the similar procedure from
The furan diol I-f was synthesized from diethyl l-tarta-
rate as previously reported.16 The trihydroxyfuranone I-g
was prepared in 43% yield from commercially available
d-(+)-ribonic g-lactone by selective mono-silylation of
primary alcohol.
compound
8
(eq (2)). N-Methyl-3,4-dihydrox-
ypyrrolidone (I-c) was prepared from acetoxylactam 6
by reduction with triethylsilane followed by deprotec-
tion of acetyl groups.14 The compound 6 was derived
from diacetoxysuccinimide 5 through NaBH4 reduction
and acetylation of the resulting hydroxyl group. 3,4-
Dihydroxypyrrolidone (I-d) was again synthesized simi-
larly from the compound I-b except forusing TBS pro-
tecting groups instead of acetyl groups. The TBS
protecting groups could also be removed in the final
step by using acetyl chloride in ethanol. 3,4-Dihydrox-
yfuranone (I-e) was synthesized by the reduction of
intermediary ring-opened aldehydo-amide, which equi-
librate with hydroxylactam 7, and ring closure of the
resulting hydroxyamide to form a lactone ring by using
The target compounds as typified by general structure
III were prepared by acylation of diols I with diace-
tylcaffeoyl chloride (Ac2-caffeoyl-Cl) followed by
removal of acetyl protecting groups in refluxing acetone
containing aqueous HCl (Scheme 1).11 The yields and
structures of compounds at each step, and HIV IN
inhibitory activities of dicaffeoyl compounds were
shown in Table 1.17
The inhibitory activity of l-chicoric acid was included
for comparison. In general, all compounds III-a–f
showed moderate HIV IN inhibitory activities with IC50
values in the range of 22.4–47.4 mM. Theirinhibitoyr
activities were comparable or somewhat inferior to
Scheme 1.
Scheme 2.

D. J. Hwang et al. / Bioorg. Med. Chem. 9 (2001) 1429–1437
1431
Table 1. Structures, yields, and HIV IN inhibitory activities of dicaffeoyl compounds (III-a–g)
Entry
1
Starting diols, I
Structure of II (yields from I)
Structure of III (yields from II)
IC50(mM)a of III
39.2
2
3
4
5
6
7
8
25.2
27.9
33.8
32.3
47.4
22.4
24.9
—
—
l-Chicoric acid (3)b
aAll values are averages of at least two runs.
bl-Chicoric acid was prepared by known method.10
l-chicoric acid. The nature of five-membered hetero-
cyclic ring linker did not markedly influence the inhibi-
tory activity. Among synthesized, compound III-g,
which contains a ribonolactone ring as a linker showed
the best inhibitory activity.
exhibited good inhibition against isolated HIV IN, none
of the dicaffeoyl and digalloyl compounds synthesized
in this study could inhibit the replication of HIV in cell
bases assay at nontoxic concentration (data not
shown)18 implicating the important role of carboxylic
acid in l-chicoric acid.
Digalloyl compounds (III-h–k) were also synthesized
from selected diols by using triacetylgalloyl chloride
(Ac3-galloyl-Cl) in a similar procedure as above since
digalloyl-substituted tartaric acid derivatives were
found to be potent inhibitors of HIV IN.11,12 The yields
and structures of compounds at each step, and HIV IN
inhibitory activities of digalloyl compounds were shown
in Table 2.
In conclusion, we have synthesized and tested the inhi-
bitory activities of a new type of HIV IN inhibitors,
which have a pyrrolidine or furan ring as a basic skele-
ton. This work is the first example on the synthesis of
caffeic or gallic acid esters separated by a functionalized
five-membered heterocyclic ring as a central linker for
the development of HIV integrase inhibitors. We are
undergoing synthesis of furan ring-based digalloyl
compounds containing carboxylic acid functionality for
HIV IN inhibitors.
As was previously observed, upon substitution of digal-
loyl groups at five-membered heterocyclic ring instead
of caffeoyl groups, the inhibitory activities were
remarkably increased. Digalloyl compounds (III-h–k)
were 2 to 5-times more potent than l-chicoric acid
against HIV IN. In case of galloyl series, the inhibitory
activities of compounds III-j and III-k were more potent
than those of III-h and III-i indicating that furan ring is
better than pyrrolidine ring in enhancing HIV IN inhi-
bitory activity. Although the digalloyl compounds
Experimental
Melting points were determined with a Thomas-Hoover
capillary apparatus and uncorrected. IR spectra were

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D. J. Hwang et al. / Bioorg. Med. Chem. 9 (2001) 1429–1437
Table 2. Structures, yields, and HIV IN inhibitory activities of digalloyl compounds (III-h–k)
Entry
1
Starting diols, I
Structure of II (yields from I)
Structure of III (yields from II)
IC50 (mM)a of III
12.4
2
3
4
5
15.6
7.4
4.7
l-Chicoric acid (3)b
24.9
aAll values are averages of at least two runs.
bl-Chicoric acid was prepared by known method.10
obtained on a Perkin Elmer 16F PC FT-IR spectro-
meter. The NMR spectra were recorded on Varian
Gemini 300 FT or Brucker Avance 300 spectrometers at
cmꢀ1) 3448, 3372, 3234, 2926, 1728, 1400, 1338, 1160,
1094; 1H NMR (CD3OD) d 5.05 (s, 2H); 13C NMR
(CD3OD) d 175.36, 75.56.
1
300 MHz (for H NMR) and 75 MHz (for 13C NMR).
The chemical shifts are reported in ppm downfield rela-
tive to tetramethylsilane. High-resolution FABMS
(positive ion mode) were obtained on a Jeol JMS-AX
505WA mass spectrometer, and glycerol was used as a
matrix. Optical rotations were measured on an
AUTOPOL III automatic polarimeter and all con-
centrations are given in g/mL. All starting materials
were obtained from commercial supplies, and used
without further purification. Analytical thin layer chro-
matography was carried out on pre-coated silica gel
(Merck Kiesegel 60 F254 layerthickness 0.25 mm). Flash
column chromatography was carried out using Kiesegel
60 (230–400 mesh, Merck).
(3R,4R)-3,4,5-Triacetoxy-1-methyl-pyrrolidin-2-one (6).
To a solution of 5 (1.06 g, 46.3 mmol) in CH2Cl2
(20 mL) and CH3OH (20 mL) was added NaBH4
(227 mg, 60.1 mmol) at ꢀ78 ꢁC. The reaction mixture
was stirred at ꢀ10 ꢁC for3 h and then quenched by slow
addition of ice-water(ca. 0.5 mL). Afterevaporation of
solvents, insoluble solid was filtered through Celite and
washed with EtOAc (40 mL). The filtrate was dried
(MgSO4) and purified by flash column chromatography
(EtOAc/hexane=1:1) to afford (3R,4R)-3,4-diacetoxy-
5-hydroxy-1-methyl-pyrrolidin-2-one (716 mg, 67%) as
a white solid. Mp 85–87 ꢁC; IR (KBr, cmꢀ1) 3288, 2938,
1748, 1716, 1440, 1372, 1242, 1048; 1H NMR (CDCl3) d
5.05 (m, 1H), 4.92 (m, 2H), 2.96 & 2.97 (two s, 3H),
2.14–2.20 (m, 6H); 13C NMR (CDCl3) d 171.23, 170.95,
167.53, 86.17, 79.49, 75.02, 27.30, 21.13, 21.04.
(3R,4R)-3,4-Dihydroxy-1-methylpyrrolidine-2,5-dione
(I-a). To a solution of (3R,4R)-3,4-diacetoxy-1-methyl-
pyrrolidine-2,5-dione13 (5, 3 g, 13.1 mmol) in EtOH
(50 mL) was added dropwise acetyl chloride (2.8 mL,
38.3 mmol) at 0 ꢁC and further stirred at rt for 3 h. After
removing solvent under reduced pressure the resulting
solid was recrystallized from EtOAc–hexane to give I-a
(1.79 g, 94%) as a white solid. Mp 175–179 ꢁC; IR (KBr,
cmꢀ1) 3434, 2926, 2894, 1718, 1454, 1278, 1146, 1078;
1H NMR (CD3OD) d 4.39 (s, 2H), 2.95 (s, 3H).
To a solution of above compound (500 mg, 2.2 mmol) in
CH2Cl2 (20 mL) was added triethylamine (0.6 mL,
4.3 mmol) and acetic anhydride (0.41 mL, 4.3 mmol) at
rt. After stirring for 1 h, the reaction mixture was con-
centrated, and purified by flash column chromato-
graphy (EtOAc/hexane=1:3) to give acetoxylactam 6 as
a yellow oil (590 mg, quantitative). IR (KBr, cmꢀ1
)
3622, 3505, 2944, 1738, 1440, 1372, 1216, 1060, 1018; 1H
NMR (CDCl3) d 6.07 (m, 1H), 5.31 (m, 1H), 5.17 (m,
1H), 2.87 & 2.88 (two s, 3H), 2.12–2.16 (m, 9H); 13C
NMR (CDCl3) d 170.37, 170.03, 169.96, 168.15, 85.34,
76.29, 73.42, 28.08, 21.14, 20.99, 20.94.
(3R,4R)-3,4-Dihydroxy-pyrrolidine-2,5-dione (I-b). To a
solution of (3R,4R)-3,4-diacetoxy-pyrrolidine-2,5-dione
(8, 770 mg, 3.58 mmol) in EtOH (20 mL) was added
dropwise acetyl chloride (1 mL, 13.7 mmol) at 0 ꢁC and
further stirred at rt for 2 h. The reaction mixture was
concentrated and purified by flash column chromato-
graphy (EtOAc/MeOH=20:1) to give I-b (390 mg,
83%) as a white solid. Mp 208 ꢁC (dec.); IR (KBr,
(3R,4S)-3,4-Dihydroxy-1-methyl-pyrrolidin-2-one (I-c).
To a solution of 6 (400 mg, 1.5 mmol) in CH2Cl2
.
(10 mL) was added BF3 Et2O (0.36 mL, 2.9 mmol) at

D. J. Hwang et al. / Bioorg. Med. Chem. 9 (2001) 1429–1437
1433
ꢀ78 ꢁC and stirred for 10 min under N2 atmosphere.
The reaction mixture was treated with triethylsilane
(1.17 mL, 7.3 mmol) and the temperature was gradually
raised to rt over 3 h. After stirring for 18 h, the mixture
was poured into ice water containing excess Na2CO3.
The mixture was extracted with CH2Cl2 and the com-
bined organic layer was dried (MgSO4), concentrated,
and purified by flash column chromatography (EtOAc/
hexane=1:1) to give (3R,4R)-3,4-diacetoxy-1-methyl-
pyrrolidin-2-one as a colorless oil (308 mg, 97%). [a]D23
+118.0ꢁ (c 0.28, CHCl3); IR (KBr, cmꢀ1) 2936, 1756,
1712, 1500, 1440, 1370, 1224, 1058; 1H NMR (CDCl3) d
5.41 (d, 1H, J=5.7 Hz), 5.29 (ddd, 1H, J=7.8, 5.7,
5.3 Hz), 3.82 (dd, 1H, J=10.5, 7.8 Hz), 3.26 (dd, 1H,
J=10.5, 5.3 Hz), 2.90 (s, 3H), 2.15 (s, 3H), 2.10 (s, 3H);
13C NMR (CDCl3) d 170.77, 170.35, 167.59, 74.69,
71.65, 51.80, 30.31, 21.12, 21.08.
174.12, 83.93, 82.46, 77.13, 26.12, 18.57, 18.32, ꢀ4.03,
ꢀ4.12, ꢀ4.36, ꢀ4.62.
(3R,4S)-3,4-Dihydroxy-pyrrolidin-2-one (1-d). To
a
solution of hydroxylactam 9 (700 mg, 1.94 mmol) in
CH2Cl2 (20 mL) was added triethylamine (1.5 mL,
11.6 mmol) and acetic anhydride (1 mL, 11.6 mmol) at
rt. After stirring for 6 h, the reaction mixture was con-
centrated and purified by flash column chromatography
(EtOAc/hexane=1:4) to give (3R,4R)-5-acetoxy-3,4-bis-
(tert-butyl-dimethyl-silyloxy)-pyrrolidin-2-one (680 mg,
87%) as a white solid. Mp 89–91 ꢁC; IR (KBr, cmꢀ1
)
1
3386, 2932, 2858, 1754, 1472, 1386, 1258, 1138, 842; H
NMR (CDCl3) d 6.52 (bs, 1H, CONH), 5.81 (dd, 1H,
J=5.0, 2.4 Hz), 4.33 (d, 1H, J=7.9 Hz), 4.24 (dd, 1H,
J=7.9, 5.0 Hz), 2.06 (s, 3H), 0.88 (m, 18H), 0.21–0.09
(m, 12H); 13C NMR (CDCl3) d 174.44, 171.43, 77.61,
75.78, 74.22, 26.08, 25.97, 18.62, 18.23, ꢀ4.52, ꢀ4.57.
To a solution of above compound (250 mg, 1.16 mmol)
in ethanol (5 mL) was added dropwise acetyl chloride
(2 mL, 27.4 mmol) at 0 ꢁC. After stirring at rt for 4 h, the
reaction mixture was concentrated and purified by flash
column chromatography (EtOAc/hexane=3:1) to
afford I-c as a white solid (138 mg, 92%). Mp 105–
To a solution of (3R,4R)-5-acetoxy-3,4-bis(tert-butyl-
dimethyl-silyloxy)-pyrrolidin-2-one (618 mg, 1.53 mmol)
in CH2Cl2 (20 mL) was added dropwise BF3.Et2O
(0.4 mL, 3.1 mmol) at ꢀ78 ꢁC over10 min underN
2
atmosphere. The mixture was stirred for 20 min and
then treated with triethylsilane (0.9 mL, 7.7 mmol). The
reaction mixture was stirred for 30 min at ꢀ78 ꢁC and
further stirred at 0 ꢁC for 3 h. The reaction mixture was
poured into ice-water containing Na2CO3. The mixture
was washed with CH2Cl2 and the organic layer was
dried (MgSO4), concentrated, and purified by flash col-
umn chromatography (EtOAc/hexane=1:4) to give
(3R,4R)-3,4-bis(tert-butyl-dimethyl-silyloxy)-pyrrolidin-
2-one (418 mg, 79%) as a white solid. Mp 40–41 ꢁC;
[a]D23 +59.3ꢁ (c 0.22, CHCl3); IR (KBr, cmꢀ1) 3242,
109 ꢁC; [a]D23 +106.1ꢁ (c 0.23, CH3OH); IR (KBr, cmꢀ1
)
1
3324, 1680, 1470, 1408, 1268, 1082; H NMR (CD3OD)
d 4.13 (m, 1H), 4.02 (d, 1H, J=6.4 Hz), 3.54 (dd, 1H,
J=9.9, 7.3 Hz), 3.12 (dd, 1H, J=9.9, 6.3 Hz), 2.82 (s,
3H); 13C NMR (CD3OD) d 173.62, 76.65, 72.58, 52.97,
29.01.
(3R,4R)-3,4-Bis(tert-butyl-dimethyl-silyloxy)-5-hydroxy-
pyrrolidin-2-one (9). To a solution of 1-b (633 mg,
4.83 mmol) and imidazole (987 mg, 14.5 mmol) in DMF
(20 mL) was added TBSCl (1.82 g, 12.1 mmol). After
stirring at rt for 1 h, the reaction mixture was poured
1
2942, 1724, 1466, 1254, 1126, 842; H NMR (CDCl3) d
7.25 (bs, 1H, CONH), 4.24 (m, 1H), 4.11 (d, 1H,
J=7.1 Hz), 3.43 (m, 1H), 3.01 (dd, 1H, J=9.4, 7.1 Hz),
0.87 & 0.91 (two s, 18H), 0.16–0.06 (m, 12H); 13C NMR
(CDCl3) d 174.62, 77.24, 75.95, 46.29, 25.78, 25.75,
18.32, 17.90, ꢀ4.85, ꢀ4.92.
into water(200 mL) and exatcrted with CH
2Cl2
(50 mLꢂ3). The combined organic layer was washed
with water, dried (MgSO4), concentrated, and purified
by flash column chromatography (EtOAc/hex-
ane=1:15) to give (3R,4R)-3,4-bis(tert-butyl-dimethyl-
silyloxy)-pyrrolidine-2,5-dione (1.28 g, 74%) as
a
To a solution of above compound (201 mg, 0.58 mmol)
in ethanol (5 mL) was added dropwise acetyl chloride
(1 mL, 14.1 mmol) at 0 ꢁC for 15 min. After stirring at rt
for 4 h, the reaction mixture was concentrated and
recrystallized from EtOAc to afford I-d as a white solid
(62.3 mg, 91%). Mp 135 ꢁC (dec.); [a]2D3 +86.1ꢁ (c 0.25,
CH3OH); IR (KBr, cmꢀ1) 3298, 3240, 2888, 1688, 1480,
colorless oil. [a]D23 +132.0ꢁ (c 1.30, CHCl3); IR (KBr,
cmꢀ1) 3518, 3262, 2942, 2862, 1736, 1468, 1324, 1254,
1
1136, 842; H NMR (CDCl3) d 9.45 (bs, 1H, CONH),
4.46 (s, 2H), 0.88 (m, 18H), 0.16 (m, 12H); 13C NMR
(CDCl3) d 177.69, 81.49, 29.54, 22.17, ꢀ1.06, ꢀ1.11.
1
To a solution of above compound (1.15 g, 3.2 mmol) in
CH2Cl2 (20 mL) and MeOH (10 mL) was added NaBH4
(145 mg, 3.8 mmol) at ꢀ78 ꢁC. The mixture was stirred
for 1 h and further stirred at 0 ꢁC for2 h. The reaction
mixture was quenched by slow addition of water (ca.
1 mL) and diluted with CH2Cl2 (60 mL). The organic
layer was washed with water, dried (MgSO4), con-
centrated, and purified by flash column chromato-
graphy (EtOAc/hexane=1:2) to afford hydroxylactam 9
(822 mg, 71%) as a white solid. Mp 87–89 ꢁC; IR (KBr,
cmꢀ1) 3274, 2930, 1716, 1474, 1254, 1134, 838; 1H
NMR (CDCl3) d 6.88 (bs, 1H, CONH), 4.82 (dd, 1H,
J=10.1, 2.7 Hz), 4.02 (d, 1H, J=4.5 Hz), 3.95 (dd, 1H,
J=4.5, 2.7 Hz), 3.80 (d, 1H, OH, J=10.1 Hz), 0.83–0.92
(m, 18H), 0.12–0.17 (m, 12H); 13C NMR (CDCl3) d
1356, 1266, 1112; H NMR (CD3OD) d 4.18 (m, 1H),
4.02 (d, 1H, J=7.2 Hz), 3.49 (dd, 1H, J=9.7, 7.5 Hz),
3.02 (dd, 1H, J=9.7, 7.1 Hz); 13C NMR (CD3OD) d
175.41, 75.25, 73.75, 44.86.
(3R,4R)-3,4-Bis-(tert-butyl-dimethyl-silyloxy)-5-hydroxy-
1-methyl-pyrrolidin-2-one (7). To a solution of 1-a
(1.59 g, 10.9 mmol) and imidazole (2.23 g, 32.8 mmol) in
DMF (8 mL) was added TBSCl (4.12 g, 27.3 mmol).
After stirring at rt for 1 h, the reaction mixture was
poured into ice water (100 mL) and extracted with
CH2Cl2 (50 mLꢂ3). The combined organic layer was
washed with water, dried (MgSO4), concentrated, and
purified by flash column chromatography (EtOAc/hex-
ane=1:20) to give (3R,4R)-3,4-bis(tert-butyl-dimethyl-

1434
D. J. Hwang et al. / Bioorg. Med. Chem. 9 (2001) 1429–1437
silyloxy)-1-methyl-pyrrolidine-2,5-dione (4.02 g, 99%)
as a colorless oil. [a]2D3 +141.6ꢁ (c 0.55, CHCl3); IR
(KBr, cmꢀ1) 2944, 2870, 1724, 1454, 1370, 1260, 1086,
838; 1H NMR (CDCl3) d 4.45 (s, 2H), 2.95 (s, 3H), 0.92
(s, 18H), 0.15 & 0.2 (two s, 12H); 13C NMR (CDCl3) d
173.93, 77.43, 26.02, 24.88, 18.59, ꢀ4.09, ꢀ4.66.
minopyridine (20.6 mg, 0.17 mmol), and triethylamine
(0.94 mL, 6.75 mmol) in DMF (5 mL) was added TBSCl
(0.56 g, 3.71 mmol) at 0 ꢁC and stirred at rt for 16 h. The
reaction mixture was poured into water (20 mL) and
extracted with CH2Cl2 (10 mLꢂ3). The combined
organic layer was washed with water, dried (MgSO4),
concentrated, and purified by flash column chromato-
graphy (EtOAc/hexane=1:2) to give I-g (0.23 g, 43%)
as a white solid. Mp 89–91 ꢁC; [a]D23 +35.9ꢁ (c 0.54,
To a solution of above compound (3.84 g, 10.28 mmol)
in CH2Cl2 (30 mL) and CH3OH (15 mL) was added
NaBH4 (388 mg, 12.3 mmol) at ꢀ78 ꢁC. The reaction
mixture was stirred at ꢀ10 ꢁC for3 h and then quenched
by the slow addition of water. After evaporation of sol-
vents, insoluble solid was filtered out through Celite 545
and washed with EtOAc (100 mL). The filtrate was
dried (MgSO4), concentrated, and purified by flash col-
umn chromatography (EtOAc/hexane=1:3) to give 7 as
a white solid (3.22 g, 83%). Mp 141–143 ꢁC; IR (KBr,
cmꢀ1) 3374, 2946, 1682, 1470, 1396, 1254, 1138, 1066,
844; 1H NMR (CDCl3) d 4.45 (dd, 1H, J=10.9, 2.2 Hz),
3.78 (d, 1H, J=3.3 Hz), 3.69 (dd, 1H, J=3.3, 2.2 Hz),
2.71 (s, 3H), 2.43 (d, 1H, OH, J=10.9 Hz), 0.69–0.76
(m, 18H), ꢀ0.30–0.04 (m, 12H); 13C NMR (CDCl3) d
171.81, 89.23, 80.25, 77.15, 27.05, 26.12, 18.54, 18.36,
ꢀ4.02, ꢀ4.20, ꢀ4.34, ꢀ4.65.
1
CHCl3); H NMR (CDCl3) d 4.60 (t, 1H, J=4.4 Hz),
4.46 (m, 1H), 4.37 (d, 1H, J=5.5 Hz), 3.85 (m, 2H), 3.12
(bs, 1H, OH), 2.96 (bs, 1H, OH), 0.80 (s, 9H), ꢀ0.01 (s,
6H); 13C NMR (CDCl3) d 177.67, 86.14, 70.65, 69.85,
63.06, 26.14, 26.02, 18.54, ꢀ5.32, ꢀ5.35.
General procedure for the preparation of acetyl protected
dicaffeoyl and digalloyl compounds (II-a–k). A solution
of diols (0.25 mmol, I-a–g) in pyridine (1 mL) was trea-
ted with a solution of 3,4-diacetylcaffeoyl chloride
(1.25 mmol) or 3,4,5-triacetylgalloyl chloride (1.5 mmol)
in toluene (3 mL) at rt and stirred overnight. After
removal of solvents, the residue was purified by column
chromatography (EtOAc/hexane=3:1 or CH2Cl2/
CH3OH=40:1) to give II–aꢃII–k.
(3R,4S)-3,4-Dihydroxy-dihydrofuran-2-one (I-e). To a
solution of 7 (1.32 g, 3.5 mmol) in EtOH (50 mL) was
added NaBH4 (1.32 g, 35.5 mmol) at 0 ꢁC. The tem-
perature was raised to 80 ꢁC and then stirred for 4 h at
that temperature. The reaction mixture was cooled to rt
and quenched by dropwise addition of water (ca. 1 mL)
until disappearing of gas evolution. The mixture was
filtered through Celite 545 and the filtrate was dried
(MgSO4), concentrated, and purified by flash column
chromatography (EtOAc/hexane=1:3) to afford
(2R,3R)-2,3-bis(tert-butyl-dimethyl-silyloxy)-4-hydroxy-
N-methyl-butyramide (1.01 g, 76%) as a white solid. Mp
General Procedure for Preparation of Dicaffeoyl and Di-
galloyl Compounds (III–aꢃIII–k). To a solution of
above compounds (0.18 mmol, II-aꢃII–k) in acetone
(6 mL) was added 3N HCl (2 mL). The reaction mixture
was refluxed for 3ꢃ5 h. Aftercooling to rt, the solution
was diluted with EtOAc (50 mL), washed with brine, dried
(MgSO4), concentrated, and purified by flash column
chromatography (EtOAc/hexane/AcOH=3:1:0.002) to
give III-a–k.
(3R,4R)-Bis(3,4-diacetoxy-phenyl-acryloyloxy)-1-methyl-
pyrrolidine-2,5-dione (II-a). Yield 83%; Mp 83–85 ꢁC;
[a]D23 +207.7ꢁ (c 0.20, CHCl3); IR (KBr, cmꢀ1) 2927,
53–55 ꢁC; [a]D23 +43.6ꢁ (c 0.66, CHCl3); IR (KBr, cmꢀ1
)
3276, 2928, 1646, 1544, 1252, 1078, 836; 1H NMR
(CDCl3) d 6.86 (bs,1H, CONH), 4.26 (td, 1H, J=6.9,
5.8 Hz), 4.12 (d, 1H, J=6.9 Hz), 3.63 (d, 2H,
J=5.8 Hz), 3.43 (bs, 1H, OH), 2.81 & 2.82 (two s, 3H),
0.88–0.86 (m, 18H), 0.07–0.02 (m, 12H); 13C NMR
(CDCl3) d 177.72, 76.95, 76.86, 69.21, 30.45, 30.25,
22.85, 22.54, ꢀ0.67, ꢀ0.66.
1778, 1726, 1442, 1377, 1198, 913; H NMR (CDCl3) d
1
7.73 (d, 2H, J=16.0 Hz), 7.43–7.22 (m, 6H), 6.44 (d,
2H, J=16.0 Hz) 5.70 (s, 2H), 3.15 (s, 3H), 2.30 (m,
12H); 13C NMR (CDCl3) d 169.48, 167.83, 167.72,
166.30, 145.74, 144.13, 142.61, 132.63, 126.63, 124.05,
123.10, 116.88, 73.22, 25.4, 20.58.
(3R,4R)-Bis(3,4-dihydroxy-phenyl-acryloyloxy)-1-methyl-
pyrrolidine-2,5-dione (III-a). Yield 69%; Mp 127–
To a solution of above compound (2.01 g, 5.3 mmol) in
1,4-dioxane (10 mL) was added 3N HCl (3 mL) and the
temperature was raised to 80 ꢁC for2 h. Aftercooling to
rt, solvent was removed under reduced pressure and the
residue of solvent was eliminated with toluene and
CCl4. The residue was purified by flash column chro-
matography (EtOAc/hexane=3:1) to give I-e (236 mg,
38%) as a white solid. Mp 62–64 ꢁC; [a]D23 +44.2ꢁ (c
0.63, CH3OH); IR (KBr, cmꢀ1) 3384, 2916, 1778, 1636,
129 ꢁC; [a]D23 +381.4ꢁ (c 0.27, CH3OH); IR (KBr, cmꢀ1
3405, 2957, 1726, 1611, 1521, 1447, 1273, 1158, 1058,
)
1
809; H NMR (CD3OD) d 7.68 (d, 2H, J=15.9 Hz),
7.10–6.81 (m, 6H), 6.40 (d, 2H, J=15.9 Hz), 5.84 (s,
2H), 3.10 (s, 3H); 13C NMR (CD3OD) d 172.30, 168.23,
150.54, 149.61, 147.29, 127.84, 123.88, 116.94, 115.78,
113.47, 74.71, 25.76. HRMS(FAB) calcd for
C23H19NO10(M+H+) 470.1087, found 470.1090.
1
1344, 1186, 1144, 1089, 1010, 904; H NMR (CD3OD) d
4.41 (dd, 1H, J=8.6, 6.6 Hz), 4.30 (ddd, 1H, J=7.4, 7.1,
6.6 Hz), 4.22 (d, 1H, J=7.4 Hz), 3.75 (dd, 1H, J=8.6,
7.1 Hz); 13C NMR (CD3OD) d 177.90, 75.18, 74.48, 71.50.
(3R,4R)-Bis(3,4-diacetoxy-phenyl-acryloyloxy)-pyrrolidine-
2,5-dionꢁe (II-b). Yield 58%; Mp 98–100 ꢁC; [a]D23
+214.2 (c 0.16, CHCl3); IR (KBr, cmꢀ1) 3246, 3100,
2932, 1744, 1636, 1506, 1372, 1208, 1112, 1074, 902,
836; 1H NMR (CDCl3) d 7.74 (d, 2H, J=16.0 Hz),
7.41–7.25 (m, 6H), 6.45 (d, 2H, J=16.0 Hz), 5.74 (s,
2H), 2.31 (s, 12H); 13C NMR (CDCl3) d 169.04, 168.45,
(3R,4R,5S)-5-(tert-Butyl-dimethyl-silyloxymethyl)-3,4-di-
hydroxy-dihydrofuran-2-one (I-g). To a solution of d-
(+)-ribonic g-lactone (0.5 g, 3.38 mmol), 4-dimethyla-

D. J. Hwang et al. / Bioorg. Med. Chem. 9 (2001) 1429–1437
1435
168.34, 165.73, 146.31, 144.45, 142.88, 132.99, 127.18,
124.47, 123.54, 117.07, 74.04, 21.05.
J=15.9 Hz), 7.06–6.77 (m, 6H), 6.32 (d, 1H, J=15.9 Hz),
6.28 (d, 1H, J=15.9 Hz), 5.64 (d, 1H, J=6.3 Hz), 5.56
(m, 1H), 3.90 (dd, 1H, J=10.4, 7.8 Hz), 3.60 (m, 1H);
13C NMR (CD3OD) d 172.50, 168.24, 167.97, 149.87,
148.42, 148.22, 146.84, 127.68, 127.64, 123.30, 116.60,
115.44, 115.39, 114.04, 75.58, 74.68, 45.56.
HRMS(FAB) calcd forC 22H19NO9(M+H+) 442.1138,
found 442.1121.
(3R,4R)-Bis(3,4-dihydroxy-phenyl-acryloyloxy)-pyrroli-
dine-2,5-dione (III-b). Yield 76%; Mp 144 ꢁC (dec.);
[a]D23 +348.2ꢁ (c 0.27, CH3OH); IR (KBr, cmꢀ1) 3306,
2947, 1815, 1731, 1606, 1521, 1273, 1123, 978; 1H NMR
(CD3OD) d 7.57 (d, 2H, J=15.9 Hz), 6.98–6.69 (m, 6H),
6.25 (d, 2H, J=15.9 Hz), 5.82 (s, 2H); 13C NMR
(CD3OD) d 172.88, 168.29, 150.49, 149.57, 147.24,
127.87, 123.91, 116.95, 115.80, 113.54, 75.48.
(3R,4S)-Bis(3,4-diacetoxy-phenyl-acryloyloxy)-dihydro-
furan-2-one (II-e). Yield 30%; Mp 93 ꢁC (dec.); [a]D23
+202.6ꢁ (c 0.04, CHCl3); IR (KBr, cmꢀ1) 2928, 1774,
HRMS(FAB) calcd
456.0931, found 456.1301.
forC
22H17NO10(M+H+)
1
1722, 1638, 1506, 1372, 1208, 1100, 1012, 902, 836; H
NMR (CDCl3) d 7.74 (d, 1H, J=15.9 Hz), 7.68 (d, 1H,
J=15.9 Hz), 7.43–7.26 (m, 6H), 6.45 (d, 1H, J=15.9 Hz),
6.40 (d, 1H, J=15.9 Hz) 5.60–5.67 (m, 2H), 4.89 (dd,
1H, J=9.9, 6.2 Hz), 4.27 (dd, 1H, J=9.9, 4.9 Hz), 2.30
(m, 12H); 13C NMR (CDCl3) d 169.82, 168.10, 166.78,
166.38, 145.99, 145.54, 144.45, 142.94, 132.34, 126.96,
124.41, 123.41, 117.49, 117.23, 73.10, 72.18, 69.49,
20.91.
(3R,4S)-Bis(3,4-diacetoxy-phenyl-acryloyloxy)-1ꢁ-methyl-
pyrrolidin-2-one (II-c). Yield 68%; Mp 82–84 C; [a]D23
+231.8ꢁ (c 0.74, CHCl3); IR (KBr, cmꢀ1) 3535, 2937,
1791, 1711, 1646, 1497, 1222, 1183, 1010, 918; 1H NMR
(CDCl3) d 7.64 (d, 1H, J=16.0 Hz), 7.62 (d, 1H,
J=16.0 Hz), 7.31–7.17 (m, 6H), 6.39 (d, 1H,
J=16.0 Hz), 6.34 (d, 1H, J=16.0 Hz), 5.54 (d, 1H,
J=5.6 Hz), 5.41 (ddd, 1H, J=7.8, 5.6, 5.1 Hz), 3.86 (dd,
1H, J=10.6, 7.8 Hz), 3.30 (dd, 1H, J=10.6, 5.1 Hz),
2.88 (s, 3H), 2.24 (m, 12H); 13C NMR (CDCl3) d
168.28, 167.58, 166.19, 165.80, 145.04, 145.00, 144.29,
144.15, 142.90, 142.84, 133.35, 133.17, 126.99, 124.43,
123.37, 118.24, 118.13, 74.98, 72.08, 52.19, 30.41, 21.04
(2C), 21.00 (2C).
(3R,4S)-Bis(3,4-dihydroxy-phenyl-acryloyloxy)-dihydro-
furan-2-one (III-e). Yield 44%; Mp 98–100 ꢁC; [a]D23
+121.7ꢁ (c 0.06, CH3OH); IR (KBr, cmꢀ1) 3354, 2926,
1774, 1724, 1636, 1506, 1372, 1258, 1178, 1110, 1070,
902, 836; 1H NMR (acetone-d6) d 7.83 (d, 1H,
J=14.9 Hz), 7.79 (d, 1H, J=14.9 Hz), 7.71–7.38 (m,
6H), 6.69 (d, 1H, J=14.9 Hz), 6.57 (d, 1H, J=14.9 Hz),
5.87 (d, 1H, J=8.6 Hz), 5.46 (m, 1H), 5.20 (dd, 1H,
J=5.3, 2.4 Hz), 3.75 (dd, 1H, J=9.7, 2.4 Hz); 13C NMR
(acetone-d6) d 169.50, 168.47, 168.36, 145.19, 145.04,
133.81, 133.72, 133.69, 127.43, 124.93, 124.09, 124.04,
118.83, 83.00, 74.97, 71.71.
(3R,4S)-Bis(3,4-dihydroxy-phenyl-acryloyloxy)-1-methyl-
pyrrolidin-2-one (III-c). Yield 78%; Mp 131–134 ꢁC;
[a]D23 +407.3ꢁ (c 0.15, CH3OH); IR (KBr, cmꢀ1) 3176,
2927, 1701, 1606, 1517, 1282, 1158, 814; 1H NMR
(CD3OD) d 7.63 (d, 1H, J=15.9 Hz), 7.60 (d, 1H,
J=15.9 Hz), 7.05–6.77 (m, 6H), 6.32 (d, 1H,
J=15.9 Hz), 6.29 (d, 1H, J=15.9 Hz), 5.62 (d, 1H,
J=5.9 Hz), 5.48 (ddd, 1H, J=8.0, 5.9, 5.5 Hz), 3.93 (dd,
1H, J=10.4, 8.0 Hz), 3.43 (dd, 1H, J=10.4, 5.5 Hz),
2.91 (s, 3H); 13C NMR (CD3OD) d 170.35, 168.64,
168.28, 150.34, 148.62, 147.26, 127.93, 123.71, 116.92,
115.67, 114.31, 114.23, 76.57, 73.16, 47.84, 30.54.
HRMS(FAB) calcd forC 23H21NO9(M+H+) 456.1295,
found 456.1307.
(3R,4R)-Bis(3,4-diacetoxy-phenyl-acryloyloxy)-tetrahy-
drofuran (II-f). Yield 64%; Mp 158–160 ꢁC; [a]D23
+183.1ꢁ (c 0.20, CHCl3); IR (KBr, cmꢀ1) 1781, 1716,
1646, 1502, 1382, 1207, 1178, 1113, 1018, 903; 1H NMR
(CDCl3) d 7.65 (d, 2H, J=16.0 Hz), 7.37–7.21(m, 6H),
6.36 (d, 2H, J=16.0 Hz), 5.35 (m, 2H), 4.19 (dd, 2H,
J=10.6, 4.5 Hz), 3.87 (dd, 2H, J=10.6, 1.7 Hz), 2.27
(m, 12H); 13C NMR (CDCl3) d 167.81, 165.47, 144.13,
132.95, 124.00, 122.92, 118.22, 77.66, 72.10, 20.65,
20.60.
(3R,4S)-Bis(3,4-diacetoxy-phenyl-acryloyloxy)-pyrrolidinꢁ-
2-one (II-d). Yield 57%; Mp 108 ꢁC (dec.); [a]D23 +85.3
(c 0.19, CHCl3); IR (KBr, cmꢀ1) 3346, 2927, 1786, 1726,
1646, 1497, 1372, 1224, 1172, 1112; 1H NMR (CDCl3) d
7.73 (d, 1H, J=15.6 Hz), 7.68 (d, 1H, J=15.6 Hz), 7.39–
7.22(m, 6H), 6.63 (bs, 1H, NH), 6.47 (d, 1H,
J=15.6 Hz), 6.41 (d, 1H, J=15.6 Hz), 5.68 (d, 1H,
J=5.5 Hz), 5.58 (ddd, 1H, J=8.6, 6.0, 5.5 Hz), 4.00 (dd,
1H, J=10.4, 8.6 Hz), 3.35 (dd, 1H, J=10.4, 6.0 Hz),
2.31 (m, 12H); 13C NMR (CDCl3) d 169.86, 167.88,
166.67, 166.39, 144.78, 144.64, 143.95, 143.86, 142.58,
142.54, 132.98, 126.54, 124.04, 123.98, 123.02, 117.84,
117.80, 73.67, 73.44, 44.62, 20.65, 20.59, 20.52.
(3R,4R)-Bis(3,4-dihydroxy-phenyl-acryloyloxy)-tetrahy-
drofuran (III-f). Yield 72%; Mp 76–78 ꢁC; [a]D23
+306.5ꢁ (c 0.03, CH3OH); IR (KBr, cmꢀ1) 3284, 2926,
1696, 1602, 1518, 1278, 1156, 1112, 810; 1H NMR
(CD3OD) d 7.59 (d, 2H, J=15.8 Hz), 7.05–6.78 (m, 6H),
6.27 (d, 2H, J=15.8 Hz), 5.32 (m, 1H), 4.18 (dd, 2H,
J=10.5, 4.4 Hz), 3.85 (d, 2H, J=10.4 Hz); 13C NMR
(CD3OD) d 168.07, 149.83, 147.93, 146.86, 127.67,
123.17, 116.58, 115.33, 114.40, 78.80, 72.96.
HRMS(FAB) calcd forC 22H20O9(M+H+) 429.1186,
found 429.1181.
(3R,4S)-Bis(3,4-dihydroxy-phenyl-acryloyloxy)-pyrrolidin-
2-one (III-d). Yield 96%; Mp 132 ꢁC (dec.); [a]D23
+408.8ꢁ (c 0.16, CH3OH); IR (KBr, cmꢀ1) 3292, 2924,
1706, 1600, 1516, 1278, 1152, 1100, 974, 810; H NMR
(CD3OD) d 7.64 (d, 1H, J=15.9 Hz), 7.57 (d, 1H,
(3R,4R,5S)-Bis(3,4-diacetoxy-phenyl-acryloyloxy)-5-(tert-
butyl-dimethyl-silyloxymethyl)-dihydrofuran-2-one (II-g).
Yield 82%; Mp 62–64 ꢁC; [a]D23 ꢀ65.7ꢁ (c 0.14, CHCl3);
IR (KBr, cmꢀ1) 2957, 1810, 1776, 1735, 1641, 1512,
1377, 1257, 1198, 1168, 1113, 1023, 854; 1H NMR
1

1436
D. J. Hwang et al. / Bioorg. Med. Chem. 9 (2001) 1429–1437
(CDCl3) d 7.66 (d, 1H, J=16.0 Hz), 7.59 (d, 1H,
J=16.0 Hz), 7.30–7.15 (m, 6H), 6.39 (d, 1H, J=16.0 Hz),
6.36 (d, 1H, J=16.0 Hz), 5.97 (d, 1H, J=6.1 Hz), 5.65
(d, 1H, J=6.1 Hz), 4.64 (m, 1H), 3.97 (m, 2H), 2.25 (m,
12H), 0.91 (s, 9H), 0.12–0.10 (m, 6H); 13C NMR
(CDCl3) d 168.37, 168.22, 155.21, 165.16, 145.48,
145.32, 143.48, 142.86, 132.97, 127.20, 124.48, 123.25,
117.80, 83.92, 77.61, 71.67, 67.88, 26.17, 21.03, 18.61,
ꢀ5.26.
(3R,4R)-Bis(3,4,5-trihydroxy-benzoyloxy)-tetrahydrofuran
(III-j). Yield 93%; Mp 131 ꢁC (dec.); [a]D23 +107.2ꢁ (c
0.07, CH3OH); IR (KBr, cmꢀ1) 3349, 2929, 1721, 1619,
1618, 1531, 1457, 1374, 1218; 1H NMR (CD3OD) d 6.97
(s, 2H), 5.33 (d, 2H, J=2.3 Hz), 4.14 (dd, 1H, J=10.5,
4.3 Hz), 3.84 (d, 1H, J=10.5 Hz). HRMS(FAB) calcd
forC 18H16O11 (M+H+) 409.0771, found 409.0766.
(3R,4R,5S)-Bis(3,4,5-triacetoxy-benzoyloxy)-5-(tert-butyl-
dimethyl-silyloxymethyl)-dihydrofuran-2-one (II-k). Yield
20%; Mp 94–96 ꢁC; [a]D23 ꢀ42.8ꢁ (c 0.84, CHCl3); IR
(KBr, cmꢀ1) 2958, 1780, 1741, 1438, 1384, 1330, 1193,
(3R,4R,5S)-Bis(3,4-dihydroxy-phenyl-acryloyloxy)-5-hy-
droxymethyl-dihydrofuran-2-one (III-g). Yield 58%; Mp
112–115 ꢁC; [a]D23 ꢀ124.3ꢁ (c 0.23, CH3OH); IR (KBr,
cmꢀ1) 3366, 1782, 1702, 1630, 1598, 1514, 1444, 1372,
1
1046; H NMR (CDCl3) d 7.74 (s, 2H), 7.72 (s, 2H),
6.13 (d, 1H, J=6.1 Hz), 5.81(d, 1H, J=6.1 Hz), 4.71 (m,
1H), 4.01 (m, 2H), 2.28–2.25 (m, 18H), 0.94 (s, 9H), 0.16
(s, 3H), 0.14 (s, 3H).
1
1260, 1156, 1112, 976, 810; H NMR (CD3OD) d 7.60
(d, 1H, J=15.9 Hz), 7.58 (d, 1H, J=15.9 Hz), 7.04–6.72
(m, 6H), 6.33 (d, 1H, J=15.9 Hz), 6.26 (d, 1H,
J=15.9 Hz), 6.02 (d, 1H, J=6.1 Hz), 5.69 (d, 1H,
J=6.1 Hz), 4.76 (m, 1H), 3.92 (m, 2H); 13C NMR
(CD3OD) d 168.09, 167.34, 150.42, 149.26, 149.05,
147.25, 127.79, 127.71, 124.20, 123.96, 116.91, 116.84,
115.56, 115.30, 114.02, 113.41, 86.05, 72.56, 69.05,
62.48. HRMS(FAB) calcd forC 23H20O11 (M+H+)
473.1084, found 473.1072.
(3R,4R,5S)-Bis(3,4,5-trihydroxy-benzoyloxy)-5-hydroxy-
methyl-dihydrofuran-2-one (III-k). Yield 50%; Mp
115 ꢁC (dec.); [a]2D3 ꢀ157.9ꢁ (c 0.02, CH3OH); IR (KBr,
cmꢀ1) 3330, 2968, 1795, 1746, 1614, 1335, 1198, 1056;
1H NMR (CD3OD) d 6.81 (s, 2H), 6.80 (s, 2H), 5.90 (d,
1H, J=6.1 Hz), 5.56 (d, 1H, J=6.1 Hz), 4.57 (d, 1H,
J=2.2 Hz), 3.73 (d, 2H, J=2.2 Hz). HRMS(FAB) calcd
forC 19H16O13 (M+H+) 453.0669, found 453.0654.
(3R,4R)-Bis(3,4,5-triacetoxy-benzoyloxy)-1-methyl-pyr-
rolidine-2,5-dione (II-h). Yield 93%; Mp 95 ꢁC (dec.);
[a]D23 +101.9ꢁ (c 0.10, CHCl3); IR (KBr, cmꢀ1) 2949,
1795, 1741, 1442, 1379, 1330, 1208, 1056; 1H NMR
(CDCl3) d 7.84 (s, 4H), 5.80 (s, 2H), 3.18 (s, 3H), 2.30
(m, 18H).
HIV integrase assay17
Recombinant human immunodeficiency virus type 1
(HIV-1) integrase was expressed in Escherichia coli and
purified using nickel-chelated column in a one-step
manner. Aliquots of HIV-1 integrase of 0.5 mg/mL as
stock solutions were stored at ꢀ70 ꢁC until used.
(3R,4R)-Bis(3,4,5-trihydroxy-benzoyloxy)-1-methyl-pyr-
rolidine-2,5-dione (III-h). Yield 48%; Mp 101 ꢁC (dec.);
[a]D23 +171.1ꢁ (c 0.27, CH3OH); IR (KBr, cmꢀ1) 3398,
Oligonucleotide substrates
1
1731, 1624, 1452, 1345, 1208, 1037; H NMR (CD3OD)
d 7.09 (s, 4H), 5.88 (s, 2H), 3.10 (s, 3H). HRMS(FAB)
calcd forC 19H15NO12 (M+H+) 450.0673, found
450.0663.
Two 20-meroligonucleotides whose sequences resemble
the end of U5-LTR were obtained from Korea Biotech.
Inc.: K16 (U5-LTR, +strand), 50-TGTGGAAAATC-
TCTAGCAGT-30; K17 (U5-LTR, -strand), 50-ACT-
GCTAGA-GATTTTCCACA-30. The oligonucleotides
were purified by 20% polyacrylamide gel before use. In
order to construct oligonucleotide substrate, oligonu-
cleotide K16 of 30 pmol was labeled at the 50 end using
[g-32P]-ATP of 250 mCi (3,000 Ci/mmol; 1 Ci=37 GBq;
Amersham) and T4 polynucleotide kinase (T4 PNK,
New England Biolabs) of 10 units in 40 mL of reaction
buffer(70 mM Tris–HCl [pH 7.6], 10 mM MgCl 2, 5 mM
dithiothreitol) at 37 ꢁC for15 min. The labeling reaction
was subjected to 10 mM EDTA, and heated to 85 ꢁC for
15 min to inactivate T4 PNK. Afteraddition of com-
plementary oligonucleotide K17 of 30 pmol, the reac-
tion mixture was boiled for 3 min and cooled down
slowly. Labeled substrate was separated from unin-
corporated nucleotide by passage through a Biospin 6
(Bio-Rad).
(3R,4S)-Bis(3,4,5-triacetoxy-benzoyloxy)-1-methyl-pyr-
rolidin-2-one (II-i). Yield 25%; Mp 98 ꢁC (dec.); [a]D23
+90.1ꢁ (c 0.07, CHCl3); IR (KBr, cmꢀ1) 2949, 1785,
1
1731, 1506, 1335, 1198, 1601; H NMR (CDCl3) d 7.83
(s, 2H), 7.81 (s, 2H), 5.76 (d, 1H, J=5.4 Hz), 5.58 (td,
1H, J=7.8, 5.4 Hz), 4.02 (dd, 1H, J=10.8, 7.8Hz), 3.41
(dd, 1H, J=10.8, 5.4 Hz), 2.97 (s, 3H), 2.30–2.29 (m, 18H).
(3R,4S)-Bis(3,4,5-trihydroxy-benzoyloxy)-1-methyl-pyr-
rolidin-2-one (III–i). Yield 44%; Mp 98 ꢁC (dec.); [a]D23
+154.0ꢁ (c 0.05, CH3OH); IR (KBr, cmꢀ1) 3320, 2939,
1
1707, 1619, 1457, 1335, 1213, 1037; H NMR (CD3OD)
d 7.11 (s, 2H), 7.09 (s, 2H), 5.74 (d, 1H, J=5.6 Hz), 5.58
(m, 1H), 4.04 (dd, 1H, J=10.6, 8.0 Hz), 3.52 (dd, 1H,
J=10.6, 5.4 Hz), 2.96 (s, 3H). HRMS(FAB) calcd for
C19H17NO11(M+H+) 436.0880, found 436.0886.
(3R,4R)-Bis(3,4,5-triacetoxy-benzoyloxy)-tetrahydrofuran
(II-j). Yield 11%; Mp 57–60 ꢁC; [a]D23 +76.6ꢁ (c 0.14,
CHCl3); IR (KBr, cmꢀ1) 2968, 1790, 1741, 1613, 1506,
1443, 1384, 1340, 1198, 1056; H NMR (CDCl3) d 7.80
(s, 2H), 5.53 (d, 2H, J=2.0 Hz), 4.29 (dd, 2H, J=10.7,
4.5 Hz), 3.97 (dd, 2H, J=10.7, 2.0 Hz), 2.31 (s, 18H).
HIV-1 integrase reaction
A standard reaction assay of the endonucleolytic activ-
ity was carried out in the presence of potential inhibitor
containing 0.1 pmol of duplex oligonucleotide substrate
and 15 pmol of HIV-1 integrase in 15 mM Tris–HCl
1

D. J. Hwang et al. / Bioorg. Med. Chem. 9 (2001) 1429–1437
1437
(pH 7.4), 100 mM NaCl, 1 mM MnCl2, 2 mM 2-mer-
captoethanol, 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 to be 5%
DMSO in the final volume. Reaction mixtures were
incubated at 33 ꢁC for90 min and stopped by addition
of 4 mL of 95% formamide, 20 mM EDTA, 0.05% bro-
mophenol blue, 0.05% xylene cyanol FF. The reactions
were heated to 90 ꢁC for 3 min and electrophoresed on a
20% denaturing polyacrylamide gel. Reaction products
were visualized by autoradiography of the wet gel. IC50
was calculated by scanning bands on Kodak-5 film
(Image MasterVDS, Pharmacia Biotech.).
8. Neamati, N.; Hong, H.; Mazumder, A.; Wang, S.; Sunder,
S.; Nicklaus, M. C.; Milne, G. W. A.; Proska, B.; Pommier, Y.
J. Med. Chem. 1997, 40, 942.
9. Neamati, N.; Hong, H.; Sunder, S.; Milne, G. W. A.;
Pommier, Y. Mol. Pharmacol. 1997, 52, 1041.
10. King, P. J.; Ma, G.; Miao, W.; Jia, Q.; McDougall, B. R.;
Reinecke, M. G.; Cornell, C.; Kuan, J.; Kim, T. R.; Robinson,
W. E., Jr. J. Med. Chem. 1999, 42, 497.
11. Lin, Z.; Neamati, N.; Zhao, H.; Kiyru, Y.; Turpin, J. A.;
Abderham, C.; Strebel, K.; Kohn, K.; Witvrouw, M.; Panne-
couque, C.; Debyser, Z.; Clercq, E. D.; Rice, W. G.; Pmmier,
Y.; Burke, T. R., Jr. J. Med. Chem. 1999, 42, 1401.
12. Kim, S. N.; Lee, J. Y.; Kim, H. J.; Shin, C.-G.; Park, H.;
Lee, Y. S. Bioorg. Med. Chem. Lett. 2000, 10, 1879.
13. Yoda, H.; Shirakawa, K.; Takabe, K. Tetrahedron Lett.
1991, 32, 3401.
14. Wetter, H.; Oertle, K. Tetrahedron Lett. 1985, 26, 5515.
15. Yoda, H.; Katagiri, T.; Takabe, K. Tetrahedron Lett.
1991, 32, 6771.
Acknowledgements
16. Dulphy, H.; Gras, J.-L.; Lejon, T. Tetrahedron 1996, 52,
8517.
17. The enzyme inhibition assay of compounds against HIV-1
integrase was carried out as described previously Oh, J.-W.;
Shin, C.-G. Mol. Cells 1996, 6, 96.
The authors are grateful to Professor Cha-Gyun Shin at
Chung Ang University for measuring HIV integrase
inhibitory activities. We also thank Dr. Chong-Kyo Lee
at Korea Research Institute of Chemical Technology
for performing cell-based anti-HIV assay of com-
pounds. High-resolution mass spectral data were obtained
at Korea Basic Science Institute of Seoul National Uni-
versity. This work was supported by grants from the Min-
istry of Science and Technology, Korea (2N22130).
18. Anti-HIV and cytotoxicity assay procedures. Standard
virus-induced cytopathic effect (CPE) inhibition assay which
employed 3-(4,5-dimethyl thiazoly-2)-2,5-diphenyltetrazolium
bromide (MTT) to measure inhibition of virus-mediated cell
death (R4) was used with slight modification. MT-4 cells on
log phase were pelleted and infected with virus at an M.O.I.
(multiplicity of infection) of 100 CCID50 (50% cell culture
inhibitory dose) per well. The cells were immediately re-sus-
pended with RPMI 1640/10% FBS at the concentration of
1.2ꢂ105 cells/mL. 100 mL of the re-suspended cells were drop-
ped to the wells of 96 well plate containing 100 mL of 2ꢂ con-
centrated compounds in duplicate. After 5 days incubation at
37 ꢁC, the cells were observed microscopically and quantified
by using MTT assay. The liquid was aspirated until 50 mL of
liquid and all cells remained and 20 mL of 7.5 mg/mL MTT
solution was added. The plates were further incubated for 1 h,
and 100 mL of acidified isopropanol was added and shook on a
microplate shaker until the formazan form completely dis-
solved. The absorbance at 540 nm by using 690 nm as refer-
ence wavelength was measured with a microplate reader
(Vmax, Molecular Devices). The antiviral effective concentra-
tion was expressed as the EC50 orconcentration of the com-
pound required to inhibit virus-induced CPE by 50%. To
measure the effect of compounds on host cell growth, mock-
infected cells were applied to the compound containing wells
of the same plates in duplicate. The cytotoxic concentration
was expressed as the CC50 orconcentration of the compound
required to inhibit cell growth by 50%.
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