Morroniside cinnamic acid conjugate as an anti-inflammatory agent

Article abstract of DOI:10.1016/j.bmcl.2010.06.095

A morroniside cinnamic acid conjugate was prepared and evaluated on E-selectin mediated cell-cell adhesion as an important role in inflammatory processes. 7-O-Cinnamoylmorroniside exhibited excellent anti-inflammatory activity (IC₅₀ = 49.3 μM) by inhibiting the expression of E-selectin; further, it was more active than another cinnamic-acid-conjugated iridoid glycoside (harpagoside; IC₅₀ = 88.2 μM), 7-O-methylmorroniside, and morroniside itself. As a result, 7-O- cinnamoylmorroniside was observed to be a potent inhibitor of TNF-α-induced E-selectin expression.

Full text of DOI:10.1016/j.bmcl.2010.06.095

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4855–4857
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Morroniside cinnamic acid conjugate as an anti-inflammatory agent
Yoshinori Takeda a, Naomi Tanigawa b, Fortunatus Sunghwa c, Masayuki Ninomiya d, Makoto Hagiwara b,
Kenji Matsushita b, Mamoru Koketsu d,
*
a Department of Chemistry, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
b Department of Oral Disease Research, National Institute, National Center for Geriatrics & Gerontology, Obu, Aichi 474-8511, Japan
c Department of Chemistry, College of Natural and Applied Sciences, University of Dar es salaam, Tanzania
d Department of Materials Science and Technology, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A morroniside cinnamic acid conjugate was prepared and evaluated on E-selectin mediated cell–cell
Received 24 April 2010
Revised 15 June 2010
Accepted 17 June 2010
Available online 20 June 2010
adhesion as an important role in inflammatory processes. 7-O-Cinnamoylmorroniside exhibited excellent
anti-inflammatory activity (IC50 = 49.3
more active than another cinnamic-acid-conjugated iridoid glycoside (harpagoside; IC50 = 88.2
O-methylmorroniside, and morroniside itself. As a result, 7-O-cinnamoylmorroniside was observed to
l
M) by inhibiting the expression of E-selectin; further, it was
l
M), 7-
be a potent inhibitor of TNF-
a
-induced E-selectin expression.
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Morroniside
7-O-Cinnamoylmorroniside
Anti-inflammatory activity
Inflammations are associated with the extravasation of leuko-
cytes from blood into tissue. Selectins are carbohydrate-binding
type-1 membrane glycoproteins that act as adhesion molecules
involved in the development of different inflammatory reactions.
E-selectin is particularly noteworthy in the case of inflammatory
diseases owing to its expression in activated endothelium and
bone–skin microvascular linings, and to its role in cell rolling, cell
signaling, and chemotaxis.1 E-selectin mediates cell tethering and
rolling interactions through the recognition of sialofucosylated
Lewis carbohydrates expressed on structurally diverse protein–lipid
ligands on circulating leukocytes; this phenomenon serves as an
important trigger in inflammatory response.2 Pro-inflammatory
COOMe
HO
HO
HO
O
O
O
O
O
OH
O
OH
O
HO
HO
HO
O
O
HO
OH
OH
Harpagoside
Morroniside 1
Figure 1. Chemical structures of harpagoside and morroniside.
cytokines, such as tumor necrosis factor-a(TNF-a), secreted by mac-
structure is considered to be not only chemically interesting, but
also medicinally important.
Morroniside (1) (Fig. 1), which is one of the iridoid glycoside, has
been isolated from several plant families (for e.g., Cornaceae,9,10
Caprifoliaceae,11,12 Sarraceniaceae,13 and Hydrangeaceae14); this
rophages, induce expression of E-selectin in human endothelial cells
and enhance vascular inflammation.3 Therefore, compounds that
exhibit the ability to regulate expression of E-selectin in endothelial
cells may be useful as therapeutic agents for the treatment of inflam-
matory diseases.
compound is reported to exhibit antioxidative, a-glucosidase-inhib-
Harpagoside, which is a phenylpropanoid-conjugated iridoid
glycoside, is known to be a natural anti-inflammatory agent.4 This
compound is a major constituent of Harpagophytum procumbens
(Devil’s Claw), and was found to be one of the active agents in the
extract, inhibiting the production of COX-2,5–7 IL-1b, IL-6, and
itory, and anti-inflammatory activities.15–17 The use of such natural
products as starting materials for the synthesis of biologically active
compounds has received considerable attention. Recently, we
reported a highly chemoselective etherification of unprotected 1,
catalyzed by molecular iodine. A series of alkyl ether derivatives
were obtained and tested for their cytotoxic activity in a colon 26-
L5 cell line. Among the tested compounds, 7-O-dodecylmorroniside
TNF-a
by RAW 264.7 cells.8 The structural characteristic of this com-
pound is that cinnamic acid attached to iridoid moiety (Fig. 1). This
showed moderated cytotoxic activity, having IC50 values of 20.9 lM;
this value was higher than that of morroniside 1.18 These results
motivated us to investigate synthetic approaches for deriving
* Corresponding author. Tel./fax: +81 58 293 2619.
E-mail address: koketsu@gifu-u.ac.jp (M. Koketsu).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.095

4856
Y. Takeda et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4855–4857
COOMe
O
COOMe
O
COOMe
O
COOMe
O
HO
MeO
HO
O
O
O
O
O
O
e
a
d
OH
O
OR
O
OBn
O
OR
O
BnO
BnO
HO
HO
RO
RO
O
O
O
O
RO
RO
OH
OR
OBn
OR
5
R = Bn
2
4
Morroniside 1
R = H
3 R = Bn
b, c
f
6 R = H
Scheme 1. Synthesis of morroniside cinnamic acid conjugate 6. Reagents and conditions: (a) iodine, MeOH, acetone, rt, 15 min, 84%; (b) BnBr, NaH, DMF, 0 °C ? rt, 1 h; (c)
Mel, K2CO3, DMF, 1 h, 99% in two steps; (d) concd HCl, AcOH, H2O, ꢀ10 °C ? rt, 6 h, 44%; (e) cinnamoyl chloride, Et3N, DMAP, THF, 60 °C, 5 h, 79%; (f) DDQ, 1,2-dichloroethane,
H2O, rt, 4 days, 64%.
bio-active compounds from 1. We now report both the preparation
and biological activity of such derivatives.
O-cinnamoylmorroniside (6) as a morroniside cinnamic acid conju-
gate (Scheme 1).29
Morroniside (1) was isolated from the methanolic root extract
of Strychnos cocculoides (Loganiaceae) by repeated silica gel vac-
uum liquid chromatography eluting with dichloromethane/metha-
nol (80:20).19 The 1H NMR spectrum of this compound revealed it
to be a mixture of anomeric forms.12,20
In order to examine the anti-inflammatory effects of the tested
compounds (harpagoside, 1, 2, and 6), human umbilical vein endo-
thelial cells (HUVECs) were stimulated with TNF-a in the presence
and absence of these compounds, and the expression level of E-
selectin was measured by an enzyme-linked immunosorbent assay
We developed a five-step synthesis route for obtaining a mor-
roniside cinnamic acid conjugate 6. As a key step, we adapted
chemoselective etherification of unprotected 1 using molecular
iodine.18 In other words, the treatment of 1 in acetone with meth-
anol in the presence of molecular iodine at room temperature
yielded 7-O-methylmorroniside (2)18; this compound was used
as a key intermediate for the subsequent reactions. The hydroxy
group of the glucoside of 2 was benzyl-protected. On the process
of benzylation, methyl ester of 7-O-methylmorroniside (2) was
deprotected. Therefore, methyl esterification by iodomethane
was carried out to obtain 20,30,40,60-tetra-O-benzyl-7-O-methyl-
morroniside (3) in two steps.21 The hydroxy group at C-7 in
20,30,40,60-tetra-O-benzylmorroniside (4) was obtained by the reac-
tion of 3 with concentrated hydrochloric acid in acetic acid/
water.22 Conjugation of cinnamic acid to 4 was achieved by reac-
tion of 4 in THF with cinnamoyl chloride in the presence of
DMAP.23 For deprotection, we first attempted to perform debenzy-
lation by employing Hanessian’s method,24 Lewis acid,25 and cata-
lytic hydrogenation (using Pt–C26 and Lindlar’s catalyst27).
However, the desired compound 6 was not obtained as there was
no reaction or decomposition. Finally, 20,30,40,60-tetra-O-benzyl-7-
O-cinnamoylmorroniside (5) was debenzylated using DDQ28 in
1,2-dichloroethane/water mixture to yield the corresponding 7-
(ELISA).30 E-selectin was induced by stimulation with TNF-
a in HU-
VECs. The induction was inhibited by addition of tested com-
pounds (harpagoside and 6). However, 1 and 2 did not inhibit
the expression of E-selectin by the stimulation with TNF-
goside selected as a reference anti-inflammatory agent showed
IC50 values of 88.2 M; in contrast, 6, found to be a potent inhibitor
of TNF- -induced E-selectin expression, showed IC50 values of
49.3 M. To determine if the treatment of HUVECs with these com-
pounds induced cell death and influenced TNF- -induced E-selec-
a. Harpa-
l
a
l
a
tin expression, we investigated the effects of harpagoside, 1, 2, and
6 on the viability of HUVECs using a lactose dehydrogenase (LDH)
assay. The viability of the cells was not affected by treatment with
these compounds (data not shown).
These results indicated that harpagoside and 6 exhibited anti-
inflammatory activity by inhibiting the expression of E-selectin.
In addition, 6 is the best anti-inflammatory agent in comparison
with the others (Fig. 2).
It is well known that iridoid glycosides exhibit anti-inflamma-
tory activity, but the mechanism by which they exhibit this activity
remains unclear.31 In this study, our data shows that unconjugated
iridoid glycosides (morroniside (1) and 7-O-methylmorroniside
(2)) do not inhibit the expression of E-selectin in endothelial cells.
However, cinnamic-acid-conjugated iridoid glycosides (harpago-
side and 7-O-cinnamoylmorroniside (6)) exhibit anti-inflammatory
activity by inhibiting the expression of E-selectin by stimulation
with TNF-a. This fact suggests that the cinnamoyl moiety in iridoid
glycoside derivatives contributes to the anti-inflammatory activity
in such compounds. As 6 is the most potent among the tested com-
pounds, it may be useful as a therapeutic agent for the regulation
of vascular inflammation.
In conclusion, we have shown that 6 indicates its impressive
activity. It is anticipated that further structural modifications
would result in products that will enhance anti-inflammatory
activity. These investigations on chemical modification using mor-
roniside 1 are ongoing. The results of these investigations will be
reported in the near future.
References and notes
1. Lasky, L. A. Annu. Rev. Biochem. 1995, 64, 113.
Figure 2. Inhibitory effects of tested compounds on TNF-
expression in HUVECs. HUVECs were incubated with 10 ng/ml of TNF-
presence and in the absence of the test specimens for 2 h. The expression level of E-
selectin in HUVECs was measured by an ELISA. (Mean SEM, n = 3.)
a
-induced E-selectin
in the
2. Bevilacqua, M. P.; Nelson, R. M. J. Clin. Invest. 1993, 91, 379.
3. Bevilacqua, M. P.; Pober, J. S.; Mendrick, D. L.; Cotran, R. S.; Gimbrone, M. A.
Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 9238.
a
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Kim, K. A.; Kim, C. J. J. Pharmacol. Sci. 2003, 93, 367.
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T. Biol. Pharm. Bull. 2008, 31, 1422.
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12. Calis, I.; Sticher, O. Phytochemistry 1984, 23, 2539.
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2009, 92, 273.
14. Sakai, H.; Kakuda, R.; Yaoita, Y.; Kikuchi, M. J. Nat. Med. 2007, 61, 226.
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23. Procedure for synthesis of 20,30,40,60-tetra-O-benzyl-7-O-cinnamoylmorroniside
(5): solution of (50.0 mg, 0.0652 mmol) in anhydrous THF (2.0 ml)
containing cinnamoylchloride (26.2 mg, 0.157 mmol), triethylamine (27.3 l,
A
4
l
0.196 mmol), and N,N-dimethylaminopyridine (8.0 mg, 0.0655 mmol) stirred
at 60 °C for 5 h. The resultant solution was concentrated in vacuo to obtain a
residue; this residue was purified by silica gel column chromatography (EtOAc/
n-hexane = 1:4–1:3) to afford 5 (58.5 mg, 79%) as a white amorphous solid. 1
H
NMR (CDCl3, 600 MHz): d 1.37 (3H, d, J = 6.8 Hz, H-10b), 1.46 (3H, d, J = 6.9 Hz,
H-10 ), 1.78–1.81 (1H, m, H-9b), 1.85–1.88 (1H, m, H-9 ), 2.04–2.08 (2H, m,
H-6b), 2.17–2.20 (2H, m, H-6 ), 2.94 (1H, dt, J = 4.8, 12.8 Hz, H-5 ), 3.20 (1H,
dt, J = 4.8, 13.2 Hz, H-5b), 3.46–3.69 (4H, m, H-20, H-30, H-40, and H-50), 3.71–
3.72 (1H, m, H-8 ), 3.74 (3H, s, COOCH3), 3.77–3.81 (1H, m, H-8b), 4.05 (1H, dd,
J = 2.1, 9.0 Hz, H-60a), 4.41 (1H, dd, J = 2.7, 7.5 Hz, H-60b), 4.58–4.97 (8H, m, Ph-
a
a
a
a
a
CH2), 4.85 (1H, d, J = 8.1 Hz, H-10), 5.85 (1H, d, J = 8.9 Hz, H-1
a), 5.90 (1H, d,
J = 8.9 Hz, H-1b), 5.93 (1H, d, J = 7.6 Hz, H-7), 6.31 (1H, d, J = 15.5 Hz, H-800),
7.16–7.43 (25H, m, Ar), 7.52 (3H, s, H-3), 7.68 (1H, t, J = 15.5 Hz, H-700); 13C
16. Yin, M.; Lou, F.; Cao, Q.; Han, J.; Ji, H. China Patent CN 1839869, 2006.
17. Park, C. H.; Yamabe, N.; Noh, J. S.; Kang, K. S.; Tanaka, T.; Yokozawa, T. Biol.
Pharm. Bull. 2009, 32, 1734.
18. Sunghwa, F.; Sakurai, H.; Saiki, I.; Koketsu, M. Chem. Pharm. Bull. 2009, 57, 112.
19. Sunghwa, F.; Koketsu, M. Nat. Prod. Res. 2009, 23, 1408.
20. Jensen, S. R.; Nielsen, B. J. Phytochemistry 1974, 13, 517.
21. Procedure for synthesis of 20,30,40,60-tetra-O-benzyl-7-O-methylmorroniside (3): A
solution of 2 (195.6 mg, 0.465 mmol) in anhydrous DMF (5.0 ml) containing
NMR (CDCl3, 150 MHz): d 19.3 (C-10), 26.6 (C-5b), 29.8 (C-6b), 30.4 (C-5
a), 32.7
(C-6a
), 39.8 (C-9), 51.5 (COOCH3), 68.8 (C-60), 72.9 (C-8), 75.7 (C-20), 73.7, 75.1,
75.2, and 75.8, (4C, Ph-CH2), 77.8 (C-40), 82.1 (C-50), 84.7 (C-30), 95.1 (C-7), 96.1
(C-1), 99.3 (C-10), 111.1 (C-4), 112.5 (C-800), 127.6–128.5 (24C, Ar of Bn, C-200, C-
300, C-500, and C-600), 131.6 (C-400), 136.0 (C-100), 138.2–138.7 (4C, Ar of Bn),
140.4 (C-700), 153.2 (C-3), 165.3 (C-900), 167.0 (C@O); FABMS m/z 880
[M+HꢀH2O]+.
24. Nicolaou, K. C.; Pavia, M. R.; Seitz, S. P. J. Am. Chem. Soc. 1982, 104, 2027.
25. Williams, D. R.; Brown, D. L.; Benbow, J. W. J. Am. Chem. Soc. 1989, 111,
1923.
benzylbromide (440.9 ll, 3.722 mmol) and sodium hydride (60% oil
suspension, 123.0 mg, 2.05 mmol) was stirred under N2 on ice for few
minutes, and then at room temperature for 1 h. Methanol (10 ml) was added
to this mixture and the reaction mixture was stirred for 10 min. The resultant
solution was partitioned with EtOAc, washed with H2O, dried over anhydrous
Na2SO4, and concentrated in vacuo to obtain a residue; this residue was then
26. Chong, J. M.; Sokoll, K. K. Org. Prep. Proced. Int. 1993, 25, 639.
27. Heathcock, C. H.; Ratcliffe, R. J. Am. Chem. Soc. 1971, 93, 1746.
28. Ikemoto, N.; Schreiber, S. L. J. Am. Chem. Soc. 1992, 114, 2524.
29. Procedure for synthesis of 7-O-cinnamoylmorroniside (6): A solution of 5 (5.0 mg,
0.00574 mmol) in 1.65 ml of 1,2-dichloroethane and H2O = (10:1) containing
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (25.2 mg, 0.111 mmol) was stirred
at room temperature for 4 days. The resultant solution was partitioned with
EtOAc, washed with saturated NaHCO3 aq, NaHSO3 aq, dried over anhydrous
Na2SO4, and concentrated in vacuo to obtain a residue; this residue was
purified by silica gel column chromatography (EtOAc/n-hexane = 1:3–
dissolved in anhydrous DMF (5.0 ml) containing methyl iodide (86.9 ll,
1.39 mmol) and potassium carbonate (191.9 mg, 1.39 mmol), and was stirred
for 1 h. This resultant reaction mixture was thereafter partitioned with EtOAc,
washed with H2O, dried over anhydrous Na2SO4, and concentrated in vacuo to
obtain another residue; this residue was purified by silica gel column
chromatography (EtOAc/n-hexane = 1:4–1:2) to afford 3 (360.0 mg, 99%) as a
white amorphous solid. 1H NMR (CDCl3, 500 MHz): d 1.36 (3H, d, J = 6.9 Hz, H-
1:1 ? CH2Cl2/MeOH = 10:1–10:2) to afford
amorphous solid. 1H NMR (CD3OD, 600 MHz): d 1.30 (3H, d, J = 6.9 Hz, H-
10b), 1.36 (3H, d, J = 6.9 Hz, H-10 ), 1.68–1.72 (1H, m, H-9b), 1.74–1.78 (1H, m,
H-9 ), 1.87–1.91 (2H, m, H-6b), 2.01–2.04 (2H, m, H-6 ), 2.82 (1H, dt, J = 4.1,
12.4 Hz, H-5
), 3.18 (1H, t, J = 8.6 Hz, H-5b), 3.26–3.49 (4H, m, H-20, H-30, H-40,
and H-50), 3.72 (3H, s, COOCH3), 3.86 (1H, dd, J = 2.0, 6.3 Hz, H-8
), 3.94 (1H,
dd, J = 2.0, 6.3 Hz, H-8b), 4.09 (1H, dd, J = 6.9, 14.3 Hz, H-60a), 4.52–4.56 (1H, m,
6 (1.9 mg, 64%) as a white
10b), 1.43 (3H, d, J = 6.9 Hz, H-10
H-9 ), 1.92–1.96 (2H, m, H-6b), 2.04–2.07 (2H, m, H-6
13.0 Hz, H-5
), 3.07–3.12 1Y, m, H-5b), 3.47–3.77 (4H, m, H-20, H-30, H-40, and
a
), 1.70–1.74 (1H, m, H-9b), 1.76–1.79 (1H, m,
a
a), 2.82 (1H, dt, J = 4.5,
a
a
a
a
H-50), 3.36 (3H, s, 7-OMe), 3.72 (3H, s, COOCH3), 3.74–3.75 (1H, m, H-8
a
), 3.78–
a
3.80 (1H, m, H-8b), 4.26 (1H, dd, J = 3.0, 7.5 Hz, H-60a), 4.42–4.45 (1H, m, H-
60b), 4.61–4.93 (8H, m, Ph-CH2), 4.85 (1H, d, J = 8.2 Hz, H-10), 4.95 (1H, d,
J = 4.0 Hz, H-7), 5.84 (1H, d, J = 8.6 Hz, H-1), 7.20–7.36 (20H, m, Ar of Bn), 7.48
(3H, s, H-3); 13C NMR (CDCl3, 125 MHz): d 19.3 (C-10), 26.6 (C-5b), 29.8 (C-6b),
a
H-60b), 4.82 (1H, d, J = 8.2 Hz, H-10), 5.82 (1H, d, J = 9.2 Hz, H-1
a), 5.87 (1H, d,
J = 8.9 Hz, H-1b), 5.96 (1H, d, J = 12.4 Hz, H-7), 6.36 (1H, d, J = 14.9 Hz, H-800),
7.24–7.31 (3H, m, H-300, H-400, H-500), 7.37 (d, J = 6.9 Hz, H-200, H-600), 7.51 (3H, s,
H-3), 7.60 (1H, t, J = 14.9 Hz, H-700); 13C NMR (CD3OD, 150 MHz): d 19.9 (C-10),
30.4 (C-5a), 32.7 (C-6a
), 39.8 (C-9), 51.4 (COOCH3), 54.7 (7-OMe), 68.9 (C-60),
72.6 (C-8), 75.7 (C-20), 73.6, 75.1, 75.2, and 75.8, (4C, Ph-CH2), 77.9 (C-40), 82.2
(C-50), 84.7 (C-30), 94.8 (C-7), 98.0 (C-1), 99.3 (C-10), 111.2 (C-4), 127.6–129.0
27.5 (C-5b), 32.0 (C-5
a
), 34.5 (C-6b), 37.2 (C-6
a
), 40.2 (C-9
a
), 41.0 (C-9b), 51.8
(20C, Ar of Bn), 138.2–138.7 (4C, Ar of Bn), 153.1 (C-3), 167.1 (C@O); ½a D26
ꢁ
(COOCH3), 62.8 (C-60), 66.0 (C-8b), 71.7 (C-20), 74.0 (C-40), 75.6 (C-8
a), 77.6 (C-
ꢀ40.1 (c 1.0, CHCl3); FABMS m/z 804 [M+H]+.
50), 78.4 (C-30), 96.0 (C-7), 97.1 (C-1), 100.1 (C-10), 111.4 (C-4), 112.3 (C-800),
128.5 (C-200, C-600), 129.2 (C-300, C-500), 131.4 (C-400), 135.7 (C-100), 140.1 (C-700),
22. Procedure for synthesis of 20,30,40,60-tetra-O-benzylmorroniside (4): A solution of 3
(116.5 mg, 0.149 mmol) in AcOH (2.5 ml) and H2O (0.15 ml) was treated with
154.4 (C-3), 165.4 (C-900), 168.6 (C@O); ½a 2D6
ꢀ11.2 (c 1.0, MeOH); FABMS m/z
ꢁ
concd HCl (20
l
l) at ꢀ10 °C, and stirred for 6 h at room temperature. The
519 [M+HꢀH2O]+.
resultant solution was partitioned with EtOAc, washed with saturated NaHCO3
aq, dried over anhydrous Na2SO4, and concentrated in vacuo to obtain a
residue; this residue was purified by silica gel column chromatography (EtOAc/
30. ELISA analysis: For quantification of E-selectin protein expression, an ELISA-
based method was used. 4 ꢂ 104 cells of HUVEC were seeded into a 96-well
microtiter plate-and grown with EBM-2 overnight. Medium was replaced to
n-hexane = 1:3–1:1) to afford 4 (50.0 mg, 44%) as a white amorphous solid. 1
H
DMEM before the cells were pretreated with 10 ng/ml of TNF-
a. Two hours
NMR (CDCl3, 500 MHz): d 1.34 (3H, d, J = 6.9 Hz, H-10b), 1.42 (3H, d, J = 6.3 Hz,
H-10 ), 1.69–1.72 (1H, m, H-9b), 1.77–1.81 (1H, m, H-9 ), 1.93–1.97 (2H, m,
H-6b), 2.11–2.14 (2H, m, H-6 ), 2.81 (1H, dt, J = 4.6, 12.6 Hz, H-5 ), 3.16 (1H,
dt, J = 4.9, 13.6 Hz, H-5b), 3.45–3.71 (4H, m, H-20, H-30, H-40, and H-50), 3.73
after the addition of TNF- , the medium was removed and the cell layer was
a
a
a
washed three times with ice-cold PBS/1% BSA and fixed with 4%
paraformaldehyde phosphate buffer. After being washed with PBS five times,
the cells were incubated with PBS/1% BSA for 60 min. After being washed with
PBS three times, the cells were incubated with an anti-E-selectin antibody at
4 °C overnight (1:1000 in PBS/1% BSA). After being washed with PBS three
times, the AP-conjugated secondary antibody (1:500 in PBS/1% BSA) was added
for 120 min. After the addition of p-nitrophenyl phosphate (pNPP) solution, the
enzymatic reaction was allowed to proceed for 60 min at room temperature;
the optical density (OD: 405 nm) was measured using the ELISA plate reader.
At least three independent experiments (each performed in triplicate) were
used for the statistical interpretation of the data (means SEM).
a
a
(3H, s, COOCH3), 3.76–3.78 (1H, m, H-8a), 3.79–3.80 (1H, m, H-8b), 3.87–3.89
(1H, m, H-60a), 4.52–4.54 (1H, m, H-60b), 4.56–4.94 (8H, m, Ph-CH2), 4.84 (1H,
d, J = 8.6 Hz, H-10), 4.95 (1H, d, J = 2.9 Hz, H-7), 5.82 (1H, d, J = 8.6 Hz, H-1),
7.21–7.35 (20H, m, Ar of Bn), 7.48 (3H, s, H-3); 13C NMR (CDCl3, 125 MHz): d
19.3 (C-10), 26.6 (C-5b), 29.8 (C-6b), 30.4 (C-5a), 32.7 (C-6a), 39.8 (C-9), 51.5
(COOCH3), 68.8 (C-60), 72.9 (C-8), 75.7 (C-20), 73.7, 75.1, 75.2, and 75.8, (4C, Ph-
CH2), 77.8 (C-40), 82.1 (C-50), 84.7 (C-30), 95.1 (C-7), 96.1 (C-1), 99.3 (C-10),
111.1 (C-4), 127.6–128.5 (20C, Ar of Bn), 138.2–138.7 (4C, Ar of Bn), 153.2 (C-
3), 167.0 (C@O); ½a D26
ꢁ
ꢀ36.0 (c 1.0, CHCl3); FABMS m/z 767 [M+H]+.
31. Recio, M. C.; Giner, R. M.; Manez, S.; Rios, J. L. Planta Med. 1994, 60, 232.