Synthesis, anti-HIV and anti-oxidant activities of caffeoyl 5,6-anhydroquinic acid derivatives

Article abstract of DOI:10.1016/j.bmc.2009.11.043

In our continued research on chlorogenic acid analogues and derivatives with improved bioactivity, we have synthesized some caffeoyl 5,6-anhydroquinic acid derivatives. The 1,7 acetonides of chlorogenic acid (15), and of the mono-caffeoyl 5,6-anhydroquini

Full text of DOI:10.1016/j.bmc.2009.11.043

Bioorganic & Medicinal Chemistry 18 (2010) 863–869
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, anti-HIV and anti-oxidant activities of caffeoyl 5,6-anhydroquinic
acid derivatives
b
c
c
Chao-Mei Ma a, Takuya Kawahata b, Masao Hattori a, , Toru Otake , Lili Wang , Mohsen Daneshtalab
*
a Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
b Osaka Prefectural Institute of Public Health, Osaka 537-0025, Japan
c School of Pharmacy, Memorial University of Newfoundland, St. John’s, Newfoundland and Labrador, Canada A1B 3V6
a r t i c l e i n f o
a b s t r a c t
Article history:
In our continued research on chlorogenic acid analogues and derivatives with improved bioactivity, we
have synthesized some caffeoyl 5,6-anhydroquinic acid derivatives. The 1,7 acetonides of chlorogenic
acid (15), and of the mono-caffeoyl 5,6-anhydroquinic acids (7–8) showed appreciable anti-HIV activity.
The 3,4-dicaffeoyl 5,6-anhydroquinic acid (12) exhibited an anti-HIV activity twice as that of 3,5-dica-
ffeoylquinic acid (22). The caffeoyl 5,6-anhydroquinic acid derivatives displayed potent anti-oxidant
activities. The mono-caffeoyl 5,6-anhydroquinic acids (10–11) were more than twice stronger than chlor-
ogenic acid (21) on SOD-like activity.
Received 16 September 2009
Revised 19 November 2009
Accepted 20 November 2009
Available online 3 December 2009
Keywords:
Caffeoyl 5,6-anhydroquinic acid
5,6-Anhydroquinic acid
Chlorogenic acid
Anti-oxidant
Ó 2009 Elsevier Ltd. All rights reserved.
Anti-HIV
1. Introduction
anti-diabetic agents.7 The current paper reports the synthesis,
anti-oxidant, and anti-HIV activities of chlorogenic acid analogues
Caffeoylquinic acids with the most well known compound
being chlorogenic acid (5-caffeoylquinic acid), are a group of natu-
ral products found in many medicinal and dietary plants. They are
potent antioxidants and might contribute to the prevention of Type
2 diabetes mellitus, cardiovascular disease and certain aging re-
lated diseases.1,2 Dicaffeoylquinic acids have been reported to
demonstrate a strong hepatoprotective activity in experimental li-
ver injury models.3 More interest in these compounds has come
from the report that dicaffeoylquinic acids showed anti-HIV activ-
ity by inhibiting the HIV integrase.4
Because of its wide availability and multifunctional groups in
the structure of chlorogenic acid, this compound is an ideal starting
material for the synthesis of derivatives with more potent or new
bioactivities. Chlorogenic acid derivatives with a lipophilic chain
at position 1 were reported to have anti-diabetic activity by inhib-
iting hepatic flucose-6-phosphate translocase.5 In our effort of syn-
thesis and bioactivity evaluation of chlorogenic acid derivatives
and analogues, we have reported that addition of a lipophilic chain
to position 7 of chlorogenic acid through amide bonds led to com-
pounds of potent anti-fungal activity;6 Addition of lipophilic chains
through acetal/ketal bonds to chlorogenic acid produced potent
with modification at the quinic acid moiety, that is, new caffeoyl
5,6-anhydroquinic acids.
2. Results and discussion
2.1. Chemistry
Quinic acid bisacetonide (1) was synthesized using the proce-
dure reported by Sefkow.8 Compound 2 was prepared by treating
1 with sulfuryl chloride in pyridine in the presence of DMAP.
Deprotection of 2 in 0.4 N HCl afforded 3 (Scheme 1). Condensation
of 3 with acetylcaffeoic acid chloride followed by deprotection re-
sulted in a mixture of 4, 5 and 6, which upon separation by column
chromatography afforded pure individual compounds. When large
excess amount of acetylcaffeoic acid chloride is used to react with
3, compound 6 was obtained almost quantitatively. Treatment of
4–6 with 0.8–1 N HCl at rt followed by column chromatography
O
OH
O
5
6
O
O
O
1
4
7
3
a
b
2
O
O
O
1'
O
O
1"
O
OH
OH
a-glucosidase inhibitors which may be useful for developing
O
O
1
2
3
* Corresponding author. Tel.: +81 76 434 7633; fax: +81 76 434 5060.
E-mail address: saibo421@inm.u-toyama.ac.jp (M. Hattori).
Scheme 1. Chemical structures and synthesis of 5,6-anhydroquinic acid deriva-
tives. (a) SO2Cl2, DMAP, pyridine; (b) 0.4 N HCl, rt, 1h.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.043

864
C.-M. Ma et al. / Bioorg. Med. Chem. 18 (2010) 863–869
Table 1
Cl
O
Anti-oxidant activities of caffeoyl 5,6-anhydroquinic and chlorogenic acid derivatives
+
OAc
OAc
O
O
#
IC50
EC50
#
IC50
EC50
OH
OH
O
3
1
3
5
7
9
11
13
15
17
19
21
>100.0
>100.0
2.9
1.5
2.0
0.7
1.1
1.6
2.0
>50
>50
>50
20
20
15
20
8
2
4
6
90.0
1.8
6.3
1.3
0.5
1.9
1.0
1.4
2.0
2.9
2.3
>50
>50
>50
18
16
11
19
20
30
28
a
OAc
OAc
O
R1=
4
R2=H
OAc
OAc
8
O
10
12
14
16
18
20
22
O
5
6
R1=H
R2=
OR2
OR1
O
O
OAc
OAc
R1=R2=
O
29
22
17
b
2.0
1.5
OH
OH
16
O
R =
7
8
9
R2=H
1 O
IC50: concentration (lg/ml) of SOD-like compound that inhibited the formation of
WST-1 formazan by 50%.
EC50: compound concentration (lg/ml) to produce 50% reduction of the DPPH.
OH
OH
R1=H
R2=
O
O
OR2
OR1
O
OH
OH
R1=R2=
O
+
O
OH
OH
of 3 carbons linked with C-1 and C-7 of quinic acid through oxygen
atoms (acetal/ketal) with the only difference in the stereochemis-
try of C-100. Compounds 13 and 14 with asymmetric alkyl groups
were found to be inactive against HIV. Though all possessing sym-
metric alkyl-groups, compounds 17–20 with alkyl-chains longer
than that of compound 15 did not show anti-HIV activity.
R1=
R2=H
OH
OH
10
O
HO
R2=
11 R
1=H
OR2
OR1
HO
O
OH
OH
12
R1=R2=
O
Scheme 2. Chemical structures and synthesis of caffeoyl 5,6-anhydroquinic acids.
(a) DMAP, pyridine; (b) 0.8 N HCl, rt, 24 h.
In the case of the dicaffeoyl compounds (9 and 12), the one with
free hydroxyl and carboxyl groups (3,4-dicaffeoyl 5,6-anhydroqui-
nic acid, 12) showed anti-HIV activity with IC100 of 6.25
that is twice as potent as 3,5-dicaffeoylquinic acid4 (22, IC100 of
12.5 g/ml, in the same experiment), a known anti-HIV agent.
These results suggest that the hydroxyl group(s) (at least the one
at position 5) of the quinic acid part is (are) not essential for the
anti-HIV activity of dicaffeoylquinic acids. Contrary to the anti-
HIV activity of the acetonides of mono-caffeoyl compounds (7, 8
and 4), the acetonide of the dicaffeoyl derivative (9) showed no
anti-HIV activity (see Fig. 1).
Since caffeoylquinic acid derivatives are well known to demon-
strate strong anti-oxidant activities, the newly synthesized caffeoyl
5,6-anhydroquinic acids were tested for their anti-oxidant activi-
ties, SOD-like activity using a SOD Assay Kit-WST and radical scav-
enging activity against DPPH (2,2-dipheny1-1-picrylhydrazy1). In
the SOD-like activity assay, WST-1 produces a water-soluble for-
mazan upon reduction with superoxide anion. The reduction rate
is linearly related to the xanthine oxidase (XO) activity, and is
inhibited by SOD or other antioxidants. The inhibitory activity of
the compounds was expressed as IC50 representing the concentra-
tion that inhibited the formation of WST-1 formazan by 50%. As
shown in Table 1, all the caffeoyl quinic/5,6-anhydroquinic acid
derivatives showed SOD-like activity. Compound 2, a 5,6-anhy-
droquinic acid derivative whose 3,4-hydroxyls are masked by an
acetal group showed appreciable SOD-like activity even though
there is no aromatic moiety in its structure. Despite the lack of free
phenolic hydroxyl in their structures, compounds 4–6 showed
lg/ml
afforded 7–12 (Scheme 2). The ketal derivatives (15–20) were pre-
pared as described in previous paper.7 Using the same method
compounds 13 and 14 were prepared (Scheme 3).
l
2.2. Biological activity
The anti-HIV activity of the compounds synthesized was
screened using MT-4 cells infected with HIV-1 (LAV-1) according
to the procedure described in a previous paper.9 The results are
shown in Figure 1. The 1,7 acetonides of chlorogenic acid (15),
3-caffeoyl 5,6-anhydroquinic acids (7) and 4-caffeoyl 5,6-anhydro-
quinic acid (8) showed appreciable anti-HIV activity, with IC100
being 25, 50 and 50
tained even when the phenolic groups were acetylated as demon-
strated by the activity of 4 (IC100 = 50 g/ml). However, the anti-
lg/ml, respectively. The anti-HIV activity re-
l
HIV activity was found to have diminished after the acetonide
groups were removed or only the carboxyl group was masked by
a methyl group since compounds 10, 11 and 16 did not exhibit
anti-HIV activity. The findings that the introduction of an acetonide
group in chlorogenic acid or its analogues led to anti-HIV activity
warranted further investigation on derivatives with longer or dif-
ferent orientations of ketal/acetal chains. For this purpose, com-
pounds 13 and 14 were synthesized and tested together with the
previously synthesized compounds 17–207 for their anti-HIV activ-
ity. Like compound 15, compounds 13 and 14 have an alkyl group
OH
HO
OH
OH
3'
OH
2'
OH
4'
5'
1'
7'
6'
O
b
a
O
8'
O
9'
O
O
O
O
4
O
O
6
O
5
H
OH
OH
1
3
2
O
HO
O
O
O
OH
OH
1"
OH
O
H
R1=CH2CH3
R2=H
R1=H R2=CH2CH3
chlorogenic acid
13
14
R1
R2
Scheme 3. Chemical structures and synthesis of the 1,7-acetal compounds. (a) propionaldehyde, TMSOTf; (b) 0.4 N HCl, rt, 1h.

C.-M. Ma et al. / Bioorg. Med. Chem. 18 (2010) 863–869
865
Structure
Structure
Code
and
Code
and
activity
1
CC >100
IC:NE
activity
2
CC >100
IC:NE
OH
O
O
O
O
O
O
O
O
O
O
O
O
3
4
CC >100
IC:NE
CC >100
IC>50
O
O
O
OH
OH
OH
O
O
O
OAc
OAc
O
O
5
6
O
O
Not tested
CC >6.25
IC>NE
O
OAc
OAc
O
O
O
O
O
O
OH
O
OAc
OAc
OAc
OAc
O
O
7
8
CC >100
IC>50
O
CC >100
IC>50
O
O
O
O
O
O
OH
O OH
O
OH
OH
OH
OH
O
9
10
CC >25
IC:NE
O
O
CC >25
IC>NE
HO
OH
OH
O
O
O
HO
OH
O
O
O
OH
OH
OH
OH
O
O
11
CC >25
IC:NE
12
CC >25
IC>6.25
O
HO
HO
O
OH
OH
O
HO
O
HO
O
OH
O
OH
OH
OH
OH
Figure 1. Structures and anti-HIV activities of caffeoyl 5,6-anhydroquinic and chlorogenic acid derivatives.
SOD-like activity of the potency similar to that of compounds 7–9
in which the hydroxyl groups are not protected. The most potent
SOD-like activity was observed with the two caffeoyl anhydroqui-
nic acid derivatives (10–11), which showed more than twice stron-
ger SOD-like activity than chlorogenic acid did. On the other hand,
radical scavenging activities against DPPH were strictly limited to
those with free phenolic groups in their structures. Apparently,
the phenolic groups are the essential in serving as hydrogen-do-
nors to the hydrogen abstracting DPPH radical, which caused a de-
crease in absorption around 517 nm.10
5,6-anhydroquinic acids possessed equal or stronger anti-oxidant
activity compared to those of the known caffeoylquinic acid deriv-
atives. Due to the multi-hydroxyl groups in the quinic acid moiety,
the acyl quinic acids are very hydrophilic with mi Log P for chloro-
genic acid being ꢀ0.453 calculated using Molinspiration.11 The
5,6-anhydroquinic acid derivatives with one less hydroxyl group
in their structures are less hydrophilic. The mi Log P for caffeoyl
5,6-anhydroquinic acid was calculated to be 0.223. The favorable
mi Log P values for the acyl 5,6-anhydroquinic acids may suggest
that these compounds would have better pharmacokinetic profiles
than the acyl quinic acid counterparts.
3. Conclusions
4. Experimental
4.1. Chemistry
The current study described for the first time the synthesis of
caffeoyl 5,6-anhydroquinic acids. This method could be applied
to the synthesis of other acyl 5,6-anhydroquinic acids, such as p-
coumaroyl, feruloyl or galloyl 5,6-anhydroquinic acids. Generally,
acyl quinic acids are important antioxidants ingredients of coffee,
many fruits, and vegetables. Our results revealed that caffeoyl
HPLC grade solvents were purchased from Wako Pure Chemical
Industries, Ltd. Other chemical reagents were purchased from Sig-
ma–Aldrich, Inc. Column chromatography was carried out on

866
C.-M. Ma et al. / Bioorg. Med. Chem. 18 (2010) 863–869
OH
OH
13
CC >12.5
14
CC >12.5
OH
OH
IC: NE
IC: NE
O
O
O
O
O
O
OH
OH
O
OH
O
OH
O
O
H
H
O
O
16
CC >25
IC:NE
15
CC >50
IC>25
OH
OH
OH
OH
O
O
O
O
OH
OH
OH
OH
O
H3CO
O
OH
O
O
17
CC>25
IC: NE
18
CC>25
IC: NE
OH
OH
OH
OH
O
O
O
O
OH
OH
OH
OH
O
O
O
O
O
O
19
CC>25
IC: NE
20
CC>12.5
IC: NE
OH
OH
OH
OH
O
O
O
O
OH
OH
OH
O
O
OH
O
O
O
O
21*
Not
tested
22**
CC >50
IC>12.5
OH
OH
O
OH
OH
O
O
OH
OH
O
HO
OH
OH
HO
O
OH
OH
O
OH
CC: Cytotoxic concentration (µg/ml) at which reduced viability of MT-4 cells was
visualized.
IC: The inhibitory concentration (µg/ml) of tested compound required to completely
prevent the HIV-1-induced cytopathic effect.
*Chlorogenic acid was reported to have no anti-HIV acitivty.4
**Compound 22 is a known anti-HIV compound4 and was used as a positive control
in this experiment.
Fig. 1 (continued)
Wakogel 50C18 (38–63
lm, Wako Pure Chemical Industries, Ltd).
(3H, s) and 1.63 (6H, s) (4 ꢁ CH3), 2.00 (1H, dd, J = 9.5, 13.0 Hz,
Preparative HPLC was performed on a Tosoh CCPM-II system (Tos-
oh Co., Tokyo) with a UV 8020 detector. NMR spectra were mea-
sured with a Varian Unity 500 (1H, 500 MHz; 13C, 125 MHz) or a
Varian Gemini 300 (13C, 75 MHz) NMR spectrometer. TMS was
used as an internal standard and J values were reported in hertz.
Electrospray ionization mass (ESI-MS) spectra were obtained on
an Esquire 3000plus spectrometer (Bruker Daltonik GmbH, Bremen,
Germany). High-resolution-FAB-MS was measured on a Jeol JMS-
700 with a resolution of 5000 using m-nitrobenzyl alcohol as the
matrix. The purities of synthesized compounds were verified with
1H NMR, 13C NMR and HPLC.
H-2a), 2.30 (1H, dd, J = 13.5, 5.0 Hz, H-2b), 4.61 (1H, m, H-4),
4.70 (1H, dt, J = 14.0, 4.5 Hz, H-3), 5.82 (1H, d, J = 10.0 Hz, H-6),
6.07 (1H, dd, J = 3.5, 10.0 Hz, H-5). 13C NMR (CDCl3, 125 MHz) d
25.8 and 28.0 and 28.5 and 28.7 (4 ꢁ CH3), 36.1 (C-2), 69.9 (C-4),
70.5 (C-3), 78.3 (C-1), 110.4 (C-10), 110.9 (C-100), 129.5 (C-6),
130.5 (C-5), 172.8 (C-7). HR-EI-MS m/z 254.1176 (calcd For
C13H18O5, requires 254.1154).
Compound 2 (3.4 g) was treated with 4 ml 1 N HCl in 6 ml ace-
tone at rt for 1 h. To the mixture was added 1 N Na2CO3 till pH 6.
The acetone was evaporated and the water solution was passed
through ODS column (3 ꢁ 8 cm) to obtain 3 (1.3 g) from 30% to
40% MeOH eluted part and recovered some 2 (0.61 g).
4.2. Synthesis of the 5,6-anhydroquinic acid derivatives
Compound 3: (yield: 45%) amorphous powder; ½a D25
ꢂ
+220.3 (c
0.2, MeOH); 1H NMR (CDCl3, 500 MHz) d 1.63 (3H, s) and 1.64
(3H, s) (2 ꢁ CH3), 2.26 (2H, m, H-2a and H-2b), 4.19 (1H, m, H-4),
4.24 (1H, m, H-3), 5.70 (1H, d, J = 10.0 Hz, H-6), 6.06 (1H, dd,
J = 3.5, 10.0 Hz, H-5). 13C NMR (CDCl3, 125 MHz) d 28.5 and 28.7
(2 ꢁ CH3), 36.6 (C-2), 65.5 (C-3), 66.0 (C-4), 77.0 (C-1), 110.8 (C-
10), 127.1 (C-6), 133.7 (C-5), 172.9 (C-7). HR-EI-MS m/z 214.0828
(calcd For C10H14O5, requires 214.0841).
To a pyridine solution (270 ml) of 1 (8 g, 29 mmol) was added
DMAP (672 mg, 5.5 mmol) and sulfuryl chloride (13.44 ml,
166 mmol) at 0 °C. After being stirred at rt for 21 h, the mixture
was evaporated to dryness. The residue was partitioned in chloro-
form and water. The chloroform layer was evaporated to dryness
and chromatographed on silica gel column (6 ꢁ 40 cm) eluted with
hexane–ethylacetate. The hexane–ethylacetate (8:2) eluted part
was re-chromatographed on ODS column (4.5 ꢁ 16 cm) to obtain
2 (4 g) from 60% methanol eluted part.
4.3. Synthesis of the caffeoyl 5,6-anhydroquinic acid derivatives
Compound 2: (yield: 54%) amorphous powder; ½a D25
ꢂ
+112.1 (c
To a solution of 3 (1 g, 4.7 mmol) and DMAP (180 mg, 1.5 mmol)
in CH2Cl2 (50 ml) were added pyridine (15 ml) and di-O-acety-
0.56, CHCl3); 1H NMR (CDCl3, 500 MHz) d 1.39 (3H, s) and 1.49

C.-M. Ma et al. / Bioorg. Med. Chem. 18 (2010) 863–869
867
lcaffeoyl chloride (5 g, 17. 7 mmol, 3.8 equiv.) at room tempera-
ture. The reaction mixture was stirred at rt for 5 h and acidified
with 1 N HCl to pH 3. The aqueous phase was re-extracted with
CH2Cl2. The combined organic extracts were concentrated and
purified by SiO2 column chromatography (6 ꢁ 40 cm) eluted with
hexane–ethylacetate. Compound 6 (750 mg) was obtained from
hexane–ethylacetate 9:1–8:2 eluted part. The hexane–ethylacetate
(2:3) eluted part was applied to ODS column (3 ꢁ 8 cm) eluted
with large amount of 40% MeOH to remove di-O-acetylcaffeic acid.
Compound 4 (450 mg) and a mixture of 4 and 5 were eluted out by
40% MeOH after di-O-acetylcaffeic acid. The mixture of 4 and 5 was
further chromatographed on SiO2 column (2.5 ꢁ 30 cm) to obtain
compound 4 (265 mg) and 5 (440 mg) from hexane–ethylacetate
(2:3) eluted part.
through ODS to get 10 (14 mg), 7 (12 mg) and recovered 4
(43 mg) from 10% to 30% and 40% MeOH eluted part, respectively.
Compound 5 (100 mg) was treated in the same way as for 4 to
obtain 11 (16 mg), 8 (13 mg) and recovered 5 (40 mg) from 10% to
30% and 40% MeOH eluted part of an ODS column, respectively.
Compound 7: (yield: 26% calculated from the reacted part of 4)
amorphous powder; ½a D24
ꢂ
+317.0 (c 0.2, MeOH); d 1.57 (3H, s)
and 1.61 (3H, s) (2 ꢁ CH3), 2.22 (1H, dd, J = 3.0, 13.5 Hz, H-2a),
2.46 (1H, dd, J = 14.0, 8.0 Hz, H-2b), 4.38 (1H, t, J = 3.5 Hz, H-4),
5.35 (1H, m, H-3), 5.78 (1H, d, J = 10.0 Hz, H-6), 6.09 (1H,
dd, J = 3.5, 10.0 Hz, H-5), 6.30 (1H, d, J = 15.5 Hz, H-80), 6.78 (1H,
d, J = 8.0 Hz, H-50), 6.94 (1H, dd, J = 2.0, 8.0 Hz, H-60), 7.06 (1H, d,
J = 2.0 Hz, H-20), 7.60 (1H, d, J = 15.5 Hz, H-70). 13C NMR (CD3OD,
125 MHz), 28.6 and 28.8 (2 ꢁ CH3), 34.7 (C-2), 64.8 (C-4), 69.6
(C-3), 79.2 (C-1), 111.9 (C-100), 115.1 (C- 20), 115.2 (C-50), 116.5
(C-80), 122.9 (C-10), 127.8 (C-6), 129.4 (C-60), 134.0 (C-5), 146.8
(C-70), 147.2 (C-40), 149.6 (C-30),168.7 (C-90),174.6 (C-7). ESI-MS
(negative): m/z 375.0 [MꢀH]ꢀ. HR-FAB-MS [MꢀH]ꢀ m/z 375.1106
(calcd For C19H19O8, requires 375.1080).
Repeated the above procedure with 3 (100 mg, 0.47 mmol),
DMAP (36 mg, 0.30 mmol), pyridine (3 ml), di-O-acetylcaffeoyl
chloride (1 g, 3.5 mmol, 7.6 equiv) and CH2Cl2 (10 ml) to obtain
300 mg of 6.
Compound 4: (yield: 33%) amorphous powder; ½a D24
ꢂ
+103.5 (c
1.6, CHCl3); 1H NMR (CDCl3, 500 MHz) d 1.56 (3H, s) and 1.60
(3H, s) (2 ꢁ CH3), 2.27 (1H, dd, J = 3.0, 13.5 Hz, H-2a), 2.31 (6H, s,
2 ꢁ COCH3), 2.56 (1H, dd, J = 14.0, 8.0 Hz, H-2b), 4.44 (1H, dd,
J = 4.5, 2.0 Hz, H-4), 5.41 (1H, m, H-3), 5.73 (1H, d, J = 10.0 Hz,
H-6), 6.09 (1H, dd, J = 3.5, 10.0 Hz, H-5), 6.42 (1H, d, J = 15.5 Hz,
H-80), 7.22 (1H, d, J = 8.0 Hz, H-50), 7.37 (1H, d, J = 2.0 Hz, H-20),
7.40 (1H, dd, J = 2.0, 8.0 Hz, H-60), 7.65 (1H, d, J = 15.5 Hz, H-70).
Compound 8: (yield: 27% calculated from the reacted part of 5)
amorphous powder; ½a D24
ꢂ
+223.7 (c 0.2, MeOH); 1H NMR (CD3OD,
500 MHz) d 1.32 (3H, s) and 1.33 (3H, s) (2 ꢁ CH3), 2.26 (2H, m,
H-2), 4.39 (1H, m, H-3), 5.40 (1H, m, H-4), 5.88 (1H, d,
J = 10.0 Hz, H-6), 6.16 (1H, dd, J = 3.5, 10.0 Hz, H-5), 6.33 (1H, d,
J = 15.5 Hz, H-80), 6.78 (1H, d, J = 8.0 Hz, H-50), 6.95 (1H, dd,
J = 2.0, 8.0 Hz, H-60), 7.07 (1H, d, J = 2.0 Hz, H-20), 7.60 (1H, d,
J = 15.5 Hz, H-70). 13C NMR (CD3OD, 125 MHz), 28.7(2 ꢁ CH3),
38.1 (C-2), 64.8 (C-3), 68.4 (C-4), 79.8 (C-1), 112.0 (C-100), 114.9
(C- 20), 115.1 (C-50), 116.5 (C-80), 123.1 (C-10), 127.8 (C-6), 130.4
(C-60), 132.4 (C-5), 146.8 (C-70), 147.4 (C-40), 149.7 (C-30),ꢀ168.8
(C-90), 174.4 (C-7). ESI-MS (negative): m/z 375.0 [MꢀH] . HR-
FAB-MS [MꢀH]ꢀ m/z 375.1124 (calcd For C19H19O8, requires
375.1080).
13C NMR (CDCl3, 125 MHz)
d
20.6 (2 ꢁ COCH3), 28.4 and
28.7(2 ꢁ CH3), 33.6 (C-2), 64.8 (C-4), 68.7 (C-3), 76.9 (C-1), 110.7
(C-100), 118.6 (C- 80), 122.7 (C-10), 122.8 (C-20), 124.0 (C-50), 126.5
(C-60), 127.9 (C-6), 132.9 (C-5), 143.7 (C-70), 143.0 (C-30), 147.9
(C-40), 165.9 (C-90), 168.1 (2 ꢁ COCH3), 172.7 (C-7). ESI-MS (posi-
tive): m/z 461.0 ([M+H]+, 478.1 ([M+NH4]+.
Compound 5: (yield: 20%) amorphous powder; ½a D24
ꢂ
+161.6 (c
1.3, CHCl3); 1H NMR (CDCl3, 500 MHz) d 1.64 (3H, s) and 1.65
(3H, s) (2 ꢁ CH3), 2.31 (6H, s, 2 ꢁ COCH3), 2.32 (2H, m, H-2), 4.48
(1H, dt, J = 4.0, 9.0 Hz, H-3), 5.48 (1H, ddd, J = 4.0, 4.0, 1.0 Hz,
H-4), 5.82 (1H, d, J = 10.0 Hz, H-6), 6.10 (1H, dd, J = 4.0, 10.0 Hz,
H-5), 6.42 (1H, d, J = 15.5 Hz, H-80), 7.23 (1H, d, J = 8.5 Hz, H-50),
7.36 (1H, d, J = 2.0 Hz, H-20), 7.40 (1H, dd, J = 2.0, 8.5 Hz, H-60),
7.66 (1H, d, J = 15.5 Hz, H-70). 13C NMR (CDCl3, 125 MHz) d 20.6
(2 ꢁ COCH3), 28.5 and 28.7(2 ꢁ CH3), 36.9 (C-2), 64.1 (C-3), 68.3
(C-4), 77.4 (C-1), 110.8 (C-100), 118.4 (C-80), 122.9 (C-20), 124.0
(C-50), 126.5 (C-60), 129.3 (C-5), 130.2 (C-6), 132.9 (C-10), 142.4
(C-30), 143.7 (C-70), 144.0 (C-40), 166.1 (C-90), 168.0 (2 ꢁ COCH3),
172.7 (C-7). ESI-MS (positive): m/z 483.0 ([M+Na]+.
Compound 10: (yield: 34% calculated from the reacted part of 4)
amorphous powder; ½a D24
ꢂ
+191.6 (1.1, MeOH); 1H NMR (CD3OD,
500 MHz) d 2.27 (2H, m, H-2), 4.34 (1H, t, J = 4.0 Hz, H-4), 5.29 (1H,
m, H-3), 5.83 (1H, d, J = 10.0 Hz, H-6), 5.98 (1H, dd, J = 4.5, 10.0 Hz,
H-5), 6.32 (1H, d, J = 15.5 Hz, H-80), 6.77 (1H, d, J = 8.0 Hz, H-50),
6.94 (1H, dd, J = 1.5, 8.0 Hz, H-60), 7.05 (1H, J = 2.0 Hz, H-20), 7.59
(1H, d, J = 15.5 Hz, H-70). 13C NMR (CD3OD, 125 MHz) d 34.8 (C-2),
64.6 (C-4), 71.0 (C-3), 74.4 (C-1), 115.0 (C-20), 115.2 (C- 80), 116.4
(C-50), 122.9 (C-60), 127.8 (C-10), 130.8 (C-5),133.0 (C-6), 147.0
(C-70), 146.8 (C-30), 149.6 (C-40), 168.7 (C-90), 176.9 (C-7). ESI-MS
(negative): m/z 335.0 [MꢀH]ꢀ. HR-FAB-MS [MꢀH]ꢀ m/z 335.0748
(calcd For C16H15O8, requires 335.0767)
Compound 6: (yield: 23% and 91% when 3.8 and 7.6 equiv of acid
Compound 11: (yield: 37% calculated from the reacted part of 5)
chloride used, respectively) amorphous powder; ½a D24
ꢂ
+125.5 (c 1.0,
amorphous powder; ½a D24
ꢂ
+249.2 (c 2.0, MeOH); 1H NMR (CD3OD,
CHCl3); 1H NMR (CDCl3, 500 MHz) d 1.61 (3H, s) and 1.64 (3H, s)
(2 ꢁ CH3), 2.29 (6H, s, 2 ꢁ COCH3), 2.30 (6H, s, 2 ꢁ COCH3), 2.38
(1H, dd, J = 2.5, 14.0 Hz, H-2a), 2.54 (1H, dd, J = 10.0, 14.0 Hz, H-
2b), 5.65 (1H, dt, J = 4.0, 9.0 Hz, H-3), 5.71 (1H, ddd, J = 4.0, 4.0,
1.0 Hz, H-4), 5.86 (1H, d, J = 10.0 Hz, H-6), 6.13 (1H, dd, J = 4.0,
10.0 Hz, H-5), 6.36 (1H, d, J = 15.5 Hz) and 6.40 (1H, d,
J = 15.5 Hz) (H-80, 800),7.19 (1H, d, J = 8.5 Hz) and 7.21 (1H, d,
J = 8.5 Hz) (H-50,500), 7.38 (4H, m, J = 2.0 Hz, H-20,200,60,600), 7.60
(1H, d, J = 15.5 Hz) and 7.64 (1H, d, J = 15.5 Hz) (H-70,700). 13C
NMR (CDCl3, 75 MHz) d 20.7 (4 ꢁ COCH3), 28.6 and 28.8(2 ꢁ CH3),
34.2 (C-2), 65.6 (C-3), 66.0 (C-4), 77.3 (C-1), 110.7 (C-1000), 118.4
and 118.5 (C-80,800), 122.6 and 122.7 (C-20,200), 123.8 and 123.9 (C-
50,500), 126.5 (C-60,600), 129.2 (C-5), 130.2 (C-6), 132.9 and 133.0
(C-10,100), 142.4 (C-30,300), 143.4 and 143.5 and 143.7 (C-70,700,40,400),
165.2 and 165.4 (C-90,900), 167.8 and 167.9 (4 ꢁ COCH3), 172.2
(C-7). ESI-MS (positive): m/z 724.9 ([M+NH4]+.
500 MHz) d 2.14 (1H, t, J = 12.0 Hz, H-2a), 2.32 (1H, d, J = 12.0 Hz,
H-2b), 4.26 (1H, dt, J = 12.0, 4.0 Hz, H-3), 5.35 (1H, t, J = 4.0 Hz,
H-4), 5.91 (1H, d, J = 10.0 Hz, H-6), 6.00 (1H, dd, J = 4.0, 10.0 Hz,
H-5), 6.31 (1H, d, J = 15.5 Hz, H-80), 6.78 (1H, d, J = 8.0 Hz, H-50),
6.94 (1H, dd, J = 1.5, 8.0 Hz, H-60), 7.05 (1H, J = 2.0 Hz, H-20), 7.60
(1H, d, J = 15.5 Hz, H-70). 13C NMR (CD3OD, 125 MHz) d 38.2 (C-
2), 66.5 (C-3), 69.5 (C-4), 74.7 (C-1), 115.0 (C-20), 115.2 (C- 80),
116.5 (C-50), 123.0 (C-60), 127.8 (C-10), 127.0 (C-5),135.2 (C-6),
146.8 (C-30),147.1 (C-70), 149.6 (C-40), 168.9 (C-90), 176.9 (C-7).
ESI-MS (negative): m/z 335.0 [MꢀH]ꢀ. HR-FAB-MS [MꢀH]ꢀ m/z
335.0760 (calcd For C16H15O8, requires 335.0767).
Compound 6 (300 mg) was treated in 12 ml THF and 8 ml 2 N
HCl at rt for 48 h. To the solution was added 10 ml H2O and Na2CO3
powder till pH was around 5. The THF was evaporated and the
water suspension was passed through ODS column (2 ꢁ 8 cm)
eluted with 0.1% TFA H2O–MeOH. The 20–50% MeOH eluted part
was purified by preparative HPLC (TSKgel ODS 80Ts G001, 0.1%
TFA H2O–MeOH 20–100% 100 min at flow rate of 5 ml/min) to ob-
tain 12 (20 mg) from 65 to 67.5 min. Compound 12 was further
Compound 4 (100 mg) was treated with 2 ml THF and 8 ml 1 N
HCl at rt for 24 h then 1 N Na2CO3 was added to adjust the solution
to pH6. THF was evaporated and the suspension was passed

868
C.-M. Ma et al. / Bioorg. Med. Chem. 18 (2010) 863–869
purified on Sephadex LH 20 column (1.5 ꢁ 20 cm) to obtain pure
12 in 80% MeOH eluted part. The 50–100% MeOH eluted part from
the above ODS column was separated on preparative HPLC of the
same condition as above to afford 9 (90 mg) at 84–86 min, and re-
cover 6 (70 mg) after 90 min.
(1H, dd, J = 2.0, 8.5 Hz, H-60), 7.05 (1H, d, J = 2.0 Hz, H-20), 7.59 (1H,
13
d, J = 15.5 Hz, H-70). C NMR (CD3OD, 125 MHz), d 6.9 (C-300), 28.2
(C-200), 34.1 (C-2), 38.3 (C-6), 70.5 (C-3), 70.8 (C-4), 73.8 (C-5), 80.7
(C-1), 105.2 (C-100), 115.1 and 115.2 (C-20 and 80), 116.5 (C-50),
123.0 (C-60), 127.8 (C-10), 146.8 (C-40), 147.2 (C-70), 149.6 (C-30),
168.9 (C-90), 175.7 (C-7). Important NOE correlation: H-100 and H-2.
ESI-MS (negative): m/z 393.0 ([MꢀH]ꢀ, 100%).
Compound9 (yield:39.4%): amorphouspowder;½a D24
ꢂ
+248.1(c 1.9,
CH3OH); 1H NMR (CDCl3, 500 MHz) d 1.61 (3H, s) and 1.64 (3H, s)
(2 ꢁ CH3), 2.38 (1H, dd, J = 3.0, 14.0 Hz, H-2a), 2.47 (1H, dd,
J = 10.0, 13.5 Hz, H-2b), 5.62 (1H, dt, J = 4.0, 10.0 Hz, H-3), 5.69 (1H,
t, J = 4.0 Hz, H-4), 5.95 (1H, d, J = 10.0 Hz, H-6), 6.12 (1H, dd, J = 4.0,
10.0 Hz, H-5), 6.19 (1H, d, J = 16.0 Hz) and 6.30 (1H, d, J = 15.5 Hz)
(H-80,800), 6.72 (1H, d, J = 8.5 Hz) and 6.76 (1H, d, J = 8.5 Hz) (H-
50,500), 6.84 (1H, dd, J = 2.0, 8.0 Hz) and 6.92 (1H, dd, J = 2.0, 8.0 Hz)
(H-60,600), 7.00 (1H, d, J = 2.0 Hz) and 7.05 (1H, d, J = 2.0 Hz)
(H-20,200), 7.52 (1H, d, J = 16.0 Hz) and 7.56 (1H, d, J = 15.5 Hz)
(H-70,700). 13C NMR (CDCl3, 125 MHz) d 28.6 and 28.7(2 ꢁ CH3), 35.3
(C-2), 66.3 (C-4), 67.1 (C-3), 79.1 (C-1), 112.2 (C-10), 114.4, 114.5
(C-80,800), 114.9 and 115.1 (C-20,200), 116.4 and 116.5 (C-50,500), 123.3
(C-60,600), 127.6 (C-10,100), 130.1 (C-5), 132.2 (C-6), 146.8 (C-30,300),
147.6 and 147.8 (C-70,700), 149.8 (C-40,400), 168.0 and 168.2 (C-90,900),
174.1 (C-7). ESI-MS (negative): m/z 537.1 [MꢀH]ꢀ. HR-FAB-MS
[MꢀH]ꢀ m/z 537.1415 (calcd For C28H25O11, requires 537.1397).
Compound 14: white solid (yield: 12%). ½a D24
ꢀ36.8 (c 0.20,
ꢂ
CH3OH). 1H NMR (CD3OD, 500 MHz), d 1.02 (3H, t, J = 7.5 Hz,
H-300), 1.77 (1H, dd, J = 11.5, 13.5 Hz, H-6a), 1.86 (2H, m, H-200),
2.06 (1H, m, H-2a), 2.13 (1H, dd, J = 3.5, 14.5 Hz, H-2b), 2.41 (1H,
m, H-6b), 3.75 (1H, dd, J = 3.5, 9.0 Hz, H-4), 4.26 (1H, br s, H-3),
5.35 (1H, m, H-5), 5.69 (1H, t, J = 4.5 Hz, H-100), 6.28 (1H, d,
J = 15.5 Hz, H-80), 6.77 (1H, d, J = 8.5 Hz, H-50), 6.94 (1H, dd,
J = 2.0, 8.5 Hz, H-60), 7.04 (1H, d, J = 2.0 Hz, H-20), 7.59 (1H, d,
13
J = 15.5 Hz, H-70). C NMR (CD3OD, 125 MHz), d 6.9 (C-300), 28.1
(C-200), 33.6 (C-2), 38.0 (C-6), 69.8 (C-3), 70.9 (C-4), 73.3 (C-5),
80.5 (C-1), 105.1 (C-100), 115.1 (C-20 and 80), 116.5 (C-50), 123.0
(C-60), 127.8 (C-10), 146.8 (C-40), 147.3 (C-70), 149.6 (C-30), 168.9
(C-90), 175.7 (C-7). Important NOE correlation: H-100 and H-6.
ESI-MS (negative): m/z 393.0 [MꢀH]ꢀ.
Compound 12: (yield: 9.5%) amorphous powder; ½a D24
ꢂ
+406.4 (c
4.5. Screening for anti-HIV-1 activity9
0.39, CH3OH); 1H NMR (CDCl3, 500 MHz) d 2.29 (1H, t, J = 12.0 Hz,
H-2a), 2.39 (1H, dd, J = 3.0, 12.0 Hz, H-2b), 5.54 (1H, dt, J = 4.0,
11.4 Hz, H-3), 5.67 (1H, m, H-4), 5.95 (2H, br s, H-5, 6), 6.19 (1H,
d, J = 16.0 Hz) and 6.30 (1H, d, J = 16.0 Hz) (H-80 and H-800), 6.70
(1H, d, J = 8.5 Hz) and 6.77 (1H, d, J = 8.5 Hz) (H-50,500), 6.83 (1H,
dd, J = 2.0, 8.0 Hz) and 6.93 (1H, dd, J = 2.0, 8.0 Hz) (H-60,600), 7.00
(1H, d, J = 2.0 Hz) and 7.05 (1H, d, J = 2.0 Hz) (H-20,200), 7.49 (1H, d,
J = 16.0 Hz) and 7.56 (1H, d, J = 15.5 Hz) (H-70,700). 13C NMR (CDCl3,
125 MHz) d 35.3 (C-2), 66.3 (C-4), 68.7 (C-3), 74.4 (C-1), 114.7
and 114.8 and 114.9 and 115.1 (C-80,800,20,200), 116.4 and 116.5
(C-50,500), 123.3 and 123.3 (C-60,600), 126.8 (C-10,100), 127.6 (C-5),
136.0 (C-6), 146.8 (C-30,300), 147.6 and 147.8 (C-70,700), 149.7 and
149.8 (40,C-400), 168.0 and 168.5 (C-90,900), 176.5 (C-7). ESI-MS (neg-
ative): m/z 497.1 [MꢀH]ꢀ. HR-FAB-MS [MꢀH]ꢀ m/z 497.1080 (calcd
For C25H21O11, requires 497.1084).
MT-4 cells were infected for 1 h with HIV-1 (HTLV-IIIB) at a 50%
tissue culture infected dose (TCID50) of 0.001/cell. The cells were
then resuspended in RPMI-1640 medium at 1 ꢁ 105 cells/ml. The
cell suspension was (200 ll/well) cultured for 5 days in 96-well
plates containing various concentrations of the test compounds.
Control assays were performed in the absence of test compound in
HIV-1-infected and -uninfected cells. The inhibitory concentration
(IC) at which the test compound completely inhibited the HIV-1- in-
duced cytopathic effect was determined on day 5 through an optical
microscope and the cell growth was visualized to give a cytotoxic
concentration (CC) that reduced the viability of MT-4 cells.
4.6. Test for superoxide dismutase (SOD)-like activity
Superoxide dismutase (SOD)-like activity of the synthesized
compounds was evaluated in 96-well plates using a SOD Assay Kit-
WST (Dojindo Chemical, Kumamoto, Japan). To each well was added
4.4. Synthesis of acetal chlorogenic acid derivatives 13 and 14
To the solution of chlorogenic acid (789 mg) and propionalde-
hyde (0.6 ml) in tetrahydrofuran (5 ml) in an ice bath, trimethyl-
silyl trifluoromethanesulfomate (TMSOTf, 0.5 ml) was slowly
added. The mixture was stirred at rt overnight. After adding
10 ml ice and 20 drops of 1 N NaOH, the mixture was evaporated
under vacuum to remove THF and TMSOTf and then the water sus-
pension was applied to an ODS column (2 ꢁ 8 cm) eluted with gra-
dient MeOH–H2O to obtain the diacetal compound (238 mg, yield
25%) from 60% to 70% MeOH eluted part. The diacetal compound
(188 mg) was deprotected with 0.4 N HCl in a MeOH–H2O (8:2)
solution at rt for 1 h. The mixture was neutralized with 1 N NaOH
to pH 6 and was subjected to evaporation to remove MeOH. The
residue was purified using an ODS column (2 ꢁ 7 cm) to obtain a
mixture of 13 and 14 (60 mg) from 50% to 60% MeOH eluted part.
A part of the mixture of 13 and 14 (30 mg) was applied to prepara-
tive HPLC (20 ꢁ 200 mm 5C18-AR-II Waters HPLC column) with a
flow rate of 5 ml/min and the mobile phase of 40–70% MeOH in
60 min, 70–100% MeOH in 20 min to obtain 14 (10 mg) at
30–37 min and 13 (10 mg) at 41–43 min.
10
l
l of test compound solution (DMSO) and 100
l
l of WST working
solution. The reaction was initiated by the addition of 10
ll of
xanthine oxidase solution. After incubating at 37 °C for 20 min, the
absorbance at 450 nm was measured with an InterMed
ImmunoReader (Nippon InterMed K.K. Tokyo, Japan). The SOD-like
activity was calculated as: inhibition rate % = {[(Ablank 1 ꢀ Ablank 3) ꢀ
(Acompound ꢀ Ablank 2)]/(Ablank 1 ꢀ Ablank 3)} ꢁ 100, where blank
1
contained DMSO in place of compound solution; blank 2 contained
buffer in place of enzyme; blank 3 contained DMSO and buffer only.
Each compound was tested at 4 concentrations and the IC50 values
were calculated by plotting the inhibition rate% against
concentrations.
4.7. Test for radical scavenging activity against DPPH
Radical scavenging activity of the newly synthesized com-
pounds was evaluated on 96-well plates. Each well contained
10 ll of compound solution (DMSO) and 190 ll of (0.1 mM) DPPH
ethanol solution. The mixture was incubated for 20 min at rt and
the absorbance at 540 nm was measured using a plate reader.
The radical scavenging activity was calculated as (effective rate
Compound 13: white solid (yield: 12%). ½a D24
ꢀ3.2 (c 0.4, CH3OH).
ꢂ
1H NMR (CD3OD, 500 MHz), d 1.02 (3H, t, J = 7.5 Hz, H-300), 1.86 (3H,
m, H-2a, 200), 1.96 (1H, dd, J = 6.5, 13.0 Hz, H-6a), 2.23 (1H, m, H-6b),
2.27 (1H, dd, J = 3.5, 14.0 Hz, H-2b), 3.73 (1H, dd, J = 3.5, 9.0 Hz, H-4),
4.21 (1H, br s, H-3), 5.35 (1H, m, H-5), 5.67 (1H, t, J = 4.5 Hz, H-100),
6.29 (1H, d, J = 15.5 Hz, H-80), 6.79 (1H, d, J = 8.5 Hz, H-50), 6.94
%) = 100 ꢁ (Acontrol ꢀ Acompound)/Acontrol
, where control contained
DMSO in place of compound solution. Compounds were tested at
4 concentrations and the EC50 values were calculated by plotting
the effective rate % against compound concentrations.

C.-M. Ma et al. / Bioorg. Med. Chem. 18 (2010) 863–869
869
2. Morton, L. W.; Abu-Amsha Caccettah, R.; Puddey, I. B.; Croft, K. D. Clin. Exp.
Pharmacol. Physiol. 2000, 27, 152.
Acknowledgment
3. Basnet, P.; Matsushige, K.; Hase, K.; Kadota, S.; Namba, T. Biol. Pharm. Bull. 1996,
19, 1479.
4. Robinson, W. E.; Reinecke, M. G.; Abdel-Malek, S.; Jia, Q.; Chow, S. A. Proc. Natl.
Acad. Aci. U.S.A. 1996, 93, 6326.
5. Hemmerle, H.; Burger, H.-J.; Below, P.; Schubert, G.; Rippel, R.; Schindler, P. W.;
Paulus, E.; Herling, A. W. J. Med. Chem. 1997, 40, 137.
6. Ma, C. M.; Kully, M.; Khan, J. K.; Hattori, M.; Daneshtalab, M. Bioorg. Med. Chem.
2007, 15, 6830.
We are grateful to the Japanese Science Society for their finan-
cial support to present part of this work on The 22nd International
Congress on Heterocyclic Chemistry.
Supplementary data
7. Ma, C. M.; Hattori, M.; Daneshtalab, M.; Wang, L. J. Med. Chem. 2008, 51, 6188.
8. Sefkow, M. Eur. J. Org. Chem. 2001, 1137.
9. Min, B. S.; Kim, Y. H.; Tomiyama, M.; Nakamura, N.; Miyashiro, H.; Otake, T.;
Hattori, M. Phytother. Res. 2001, 15, 481.
10. Shahidi, F.; Ho, C.-T. Antioxidant Measurement and Applications an Overview.
In Antioxidant Measurement and Application; Shahidi, F., Ho, C.-T., Eds.; ACS
Symposium Series 956; American Chemical Society: Washington, DC, 2007; p
5.
Supplementary data (NMR spectra of the synthesized new com-
pounds) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.bmc.2009.11.043.
References and notes
11. http://www.molinspiration.com/cgi-bin/properties.
1. Johnston, K. L.; Clifford, M. N.; Morgan, L. M. Am. J. Clin. Nutr. 2003, 78, 728.