Synthesis and an evaluation of the bioactivity of the C-glycoside of pseudopterosin a methyl ether

Article abstract of DOI:10.1021/jo801432t

(Chemical Equation Presented) The Suzuki-Miyaura cross-coupling protocol was applied to the synthesis of 1a, the C-glycoside analogue of PsA methyl ether. This marks the first construction of a C-glycoside for this class of marine natural products, thereby offering an opportunity to compare its bioactivity to the natural substances. Its activity profile resembled that of PsA (1) and PsA O-methyl ether (1b) when assayed for its anti-inflammatory activity and its ability to inhibit phagocytosis. We conclude that the intact structure is present when a pseudopterosin expresses its anti-inflammatory and phagocytosis inhibitory properties and that they are, therefore, not likely to be prodrugs. Results show that 1a is an effective binding agent toward the A_2A and A₃ adenosine receptors, displaying IC₅₀ values of 20 and 10 μM, respectively.

Full text of DOI:10.1021/jo801432t

Synthesis and an Evaluation of the Bioactivity of the C-Glycoside of
Pseudopterosin A Methyl Ether
Wei Zhong,† Claudia Moya,‡ R. S. Jacobs,‡ and R. Daniel Little*,†
Department of Chemistry and Biochemistry and Department of Ecology, EVolution, and Marine Biology,
UniVersity of California, Santa Barbara, California 93106
little@chem.ucsb.edu
ReceiVed June 30, 2008
The Suzuki-Miyaura cross-coupling protocol was applied to the synthesis of 1a, the C-glycoside analogue
of PsA methyl ether. This marks the first construction of a C-glycoside for this class of marine natural
products, thereby offering an opportunity to compare its bioactivity to the natural substances. Its activity
profile resembled that of PsA (1) and PsA O-methyl ether (1b) when assayed for its anti-inflammatory
activity and its ability to inhibit phagocytosis. We conclude that the intact structure is present when a
pseudopterosin expresses its anti-inflammatory and phagocytosis inhibitory properties and that they are,
therefore, not likely to be prodrugs. Results show that 1a is an effective binding agent toward the A2A
and A3 adenosine receptors, displaying IC50 values of 20 and 10 µM, respectively.
Introduction
The pseudopterosins, a class of diterpene glycosides isolated
from the marine octocoral Pseudopterogorgia elisabethae,1-3
show potent anti-inflammatory and wound healing properties.4,5
Pseudopterosin A (PsA, 1; Figure 1), a potent inhibitor of
phorbol myristate acetate (PMA)-induced topical inflammation
in mice,6 stabilizes cell membranes,4 prevents the release of
prostaglandins and leukotrienes from zymosan-stimulated mu-
rine macrophages, and inhibits degranulation of human poly-
morphonuclear leukocytes7 and also inhibits phagosome for-
FIGURE 1. Structures of PsA (1), the target structure 1a, and iso-PsE
(2).
mation in Tetrahymena cells.8 Its C-10 O-methyl ether, 1b,
offers a promising treatment for contact dermatitis.9
† Department of Chemistry and Biochemistry.
To explore the role of the O-glycoside linkage in the
expression of PsA biological activity, a synthetic route that
would provide access to C-glycoside analogues of the natural
products was required. C-Glycosides often display biochemical
profiles similar to those of their oxygenated relatives. We
reasoned that the properties of this glycoside might be altered
if hydrolytic stability is important to the maintenance of
bioactivity since cleavage of the sugar by a glycosidase enzyme
is not possible. On the other hand, should cleavage be required
to deliver the active form of the drug then the presence of the
carbon linkage ought to have deleterious consequences.
‡ Department of Ecology, Evolution, and Marine Biology.
(1) Look, S. A.; Fenical, W.; Matsumoto, G. K.; Clardy, J. J. Org. Chem.
1986, 51, 5140–5145.
(2) Roussis, V.; Wu, Z.; Fenical, W.; Strobel, S. A.; Van Duyne, G. D.;
Clardy, J. J. Org. Chem. 1990, 55, 4916–4922.
(3) Ata, A.; Kerr, R. G.; Moya, C. E.; Jacobs, R. S. Tetrahedron 2003, 59,
4215–4222.
(4) Ettouati, W.; Jacobs, R. S. Mol. Pharmacol. 1987, 31, 500–505.
(5) Montesinos, M. C.; Gadangi, P.; Longaker, M.; Sung, J.; Levine, J.;
Nilsen, D.; Reibman, J.; Li, M.; Jiang, C.-K.; Hirschhorn, R.; Recht, P. A.; Ostad,
E.; Levin, R. I.; Cronstein, B. N. J. Exp. Med. 1997, 186, 1615–1620.
(6) Look, S. A.; Fenical, W.; Jacobs, R. S.; Clardy, J. Proc. Natl. Acad. Sci.
U.S.A. 1986, 83, 6238–6240.
(7) Mayer, M. S. A.; Jacobson, P. B.; Fenical, W.; Jacobs, R. S.; Glaser,
K. B. Pharm. Lett. 1998, 62, 401–407.
(8) Moya, C. E.; Jacobs, R. S. Comp. Biochem. Physiol. C: Pharmacol.
Toxicol. 2006, 143, 436–443.
(9) Haimes, H.; Glasson, S.; Harlan, P.; Jacobs, R.; Fenical, W.; Jimenez, J.
Inflamm. Res. 1995, 44, 13–17. [Suppl 3W].
10.1021/jo801432t CCC: $40.75
Published on Web 08/19/2008
2008 American Chemical Society
J. Org. Chem. 2008, 73, 7011–7016 7011

Zhong et al.
SCHEME 1. Assembly of Enol Ether Coupling Partner 5 and the Suzuki-Miyaura Coupling Protocol Applied to a Model
System
TABLE 1. Optimization of Suzuki-Miyaura Cross-Coupling
Reaction
Iso-PsE (2), a new pseudopterosin with anti-inflammatory and
potential wound healing properties, was discovered recently.10
We envisioned that it could serve as a lead structure for the
synthesis of PsA-OMe C-glycoside (1a) since its aglycon is
identical to that of PsA (1). The plan, therefore, was to remove
the sugar from 2 and to replace it by the C-glycoside analog of
D-xylose. Herein we report the first synthesis of a pseudopterosin
C-glycoside, PsA-OMe C-glycoside (1a), via a generalizable
sequence of transformations and provide an assessment of its
pharmacology.
9-BBN hydroboration quenching triflatec
a
entry (equiv)
conditions
agentb
(equiv)
comment
1
2
3
4
2.6
2.5
1.2
1.5
70 °C, 5 h
60 °C, 2 h
0 °C, 5 h
0 °C to rt, 2 h
with sonication
0 °C to rt, 2 h
K3PO4
2
1
0.5
0.5
byproduct 8 only
byproduct 8 only
no reaction
NaHCO3
NaHCO3
NaHCO3
7, 64%
5
1.5
NaHCO3
0.5
7, 74%
a 0.5 M 9-BBN in THF. b 1 M aqueous solution. c 0.1 equiv of
PdCl2(dppf) and 6 in DMF.
Results and Discussion
performed at room temperature using 1.0 equiv of the enol ether
and 0.1 equiv of the catalyst PdCl2(dppf). As illustrated by
entries 1 and 2, attempts to form the alkylborane by using >2
equiv of 9-BBN at an elevated temperature resulted in the
formation of the acyclic byproduct 8, while no reaction occurred
at 0 °C (entry 3). In contrast, when the olefin was stirred or
sonicated in the presence of 1.5 equiv of 9-BBN at room
temperature, the desired C-glycoside 7 was formed in acceptable
yields (74% when stirred and 64% when sonicated); no acyclic
byproduct was produced.
Synthesis of the Target C-Glycoside 1a. We applied the
knowledge and experience gained from the model studies to
the synthesis of C-glycoside 1a using the cross coupling of aryl
triflate 11 and enol ether 5. A short, efficient preparation of
triflate 11 was achieved from iso-PsE (2), as shown in Scheme
2. Thus, methylation of iso-PsE (2), followed by acidic
Model Study. To achieve our objectives we examined the
Suzuki-Miyaura cross-coupling protocol as the key transforma-
tion in our synthetic scheme.11-15 A model coupling reaction
between the enol ether derived from 2,3,4-tri-O-methoxmethyl-
D-xylopyranoside, 5, and 2-methoxyphenyl triflate 6 was
explored (Scheme 1). Coupling partner 5 was prepared via the
oxidation of MOM-protected D-xylose structure 3 using TPAP
and NMO,16 followed by treatment of the resulting lactone 4
with freshly prepared Petasis’ reagent.17 With olefin 5 and triflate
6 in hand,18 we examined the Suzuki-Miyaura coupling. Our
initial attempts resulted in the formation of the acyclic product
8 rather than the desired adduct 7. This outcome is consistent
with observations made by Link and co-workers who noted the
formation of an acyclic byproduct in the coupling of aryl
bromides and triflates with the glucose-derived enol ether
analogue of 5.11 Since xylose is a more reactive sugar than
glucose, the formation of acyclic byproduct was, therefore, not
entirely unexpected.
SCHEME 2. Formation of Triflate Coupling Partner 11
Our attempts to optimize the coupling reaction are sum-
marized in Table 1. In each case, the cross-coupling was
(10) Hoarau, C.; Day, D.; Moya, C.; Wu, G.; Hackim, A.; Jacobs, R. S.;
Little, R. D. Tetrahedron Lett. 2008, 49, 4604–4606.
(11) Link, J. T.; Sorensen, B. K. Tetrahedron Lett. 2000, 41, 9213–9217.
(12) Sasaki, M.; Fuwa, H.; Ishikawa, M.; Tachibana, K. Org. Lett. 1999, 1,
1075–1077.
(13) (a) Johns, B. A.; Pan, Y. T.; Elbein, A. D.; Johnson, C. R. J. Am. Chem.
Soc. 1997, 119, 4856–4865. (b) Miyaura, N.; Suzuki, A. Chem. ReV. 1995, 95,
2457–2483.
(14) Sasaki, M. Bull. Chem. Soc. Jpn. 2007, 80, 856–871.
(15) Walker, J. R.; Alshafie, G.; Abou-Issa, H.; Curley, R. W., Jr. Bioorg.
Med. Chem. Lett. 2002, 12, 2447–2450.
(16) Kireev, A. S.; Nadein, O. N.; Agustin, V. J.; Bush, N. E.; Evidente, A.;
Manpadi, M.; Ogasawara, M. A.; Rastogi, S. K.; Rogelj, S.; Shors, S. T.;
Kornienko, A. J. Org. Chem. 2006, 71, 5694–5707.
(17) (a) Walker, J. R.; Alshafie, G.; Nieves, N.; Ahrens, J.; Clagett-Dame,
M.; Abou-Issa, H.; Curley, R. W., Jr. Bioorg. Med. Chem. 2006, 14, 3038–
3048. (b) Petasis, N. A.; Bzowej, E. I. J. Am. Chem. Soc. 1990, 112, 6392–
6394.
hydrolysis resulted in the formation of PsA methyl ether aglycon
10 in an 81% yield overall.19 Treatment of 10 with triflic
(18) Cabri, W.; Candiani, I.; Bedeschi, A.; Penco, S.; Santi, R. J. Org. Chem.
1992, 57, 1481–1486.
(19) Roussis, V.; Wu, Z.; Fenical, W. J. Org. Chem. 1990, 55, 4916–4922.
7012 J. Org. Chem. Vol. 73, No. 18, 2008

BioactiVity of the C-Glycoside of Pseudopterosin A Methyl Ether
anhydride and pyridine resulted in the isolation of 97% of the
required triflate 11.
Unlike the coupling of 5 and triflate 6, the room temperature
reaction of triflate 11 with the hydroboration adduct derived
from 5 did not lead to coupling, presumably because 11 is
significantly more hindered than 6. On the other hand, the
addition of triflate 11 and PdCl2(dppf) in DMF at room
temperature to the hydroboration adduct obtained from 5 and
9-BBN followed by stirring overnight at 80 °C resulted in the
formation of desired C-glycoside in a 51% yield. The MOM
protecting groups were efficiently removed by using 6 N
aqueous HCl in MeOH, thereby revealing the C-glycoside of
PsA methyl ether, structure 1a (Scheme 3).
SCHEME 3. Suzuki-Miyaura Coupling Leading to the
Formation of the C-Glycoside 1a
FIGURE 3. Inhibition of mouse ear edema.
Biological Activity of PsA O-Me C-Glycoside (1a). We
assessed the anti-inflammatory activity and the ability of 1a to
inhibit phagocytosis in Tetrahymena since these characteristics
are hallmarks of the pseudopterosins. Should the pseudopterosins
be prodrugs that require a glycosidase enzyme to deliver the
tricyclic aglycon core as the active species, then the C-glycoside
1a ought to inhibit the enzymic process and the activity should
be lost (Figure 2).
FIGURE 4. Inhibition of phagocytosis in Tetrahymena cells.
Since the pseudopterosins have demonstrable wound healing
properties and because the involvement of adenosine receptors
has been implicated in wound healing,4,5 we elected to assess
the ability of the C-glycoside 1a to bind to adenosine receptors
A2A and A3. Adenosine receptors are ubiquitously distributed
throughout the body and are of great interest as therapeutics
for a growing number of conditions.20 In addition to wound
healing, the therapeutic potential for compounds that modulate
adenosine receptors includes hepato-protective, cardiac antiar-
rhythmic, anxiety disorders, neuroprotective, antitumor, and anti-
inflammatory uses and as antihyperalgesic agents.21
The ability of 1a to bind to cultured human embryonic kidney
cells (HEK-293) in competition with specific adenosine receptor
binding agents 5′-(N-ethylcarboxamido)adenosine (NECA, 14)
and IB-MECA (15) was examined.22 Like the pseudopterosins,
these compounds consist of a flat aromatic structural feature
that is linked to a sugar residue.
FIGURE 2. Are the pseudopterosins prodrugs?
As shown in Figure 3, PsA O-Me C-glycoside (1a) inhibits
phorbol myristate acetate (PMA) induced inflammation in mouse
ears in a dose-dependent manner (N ) 7). The ED50 is 17 µg/
ear with a Hill coefficient of 1.3 (correlation coefficient, R2 )
0.968) and is statistically significant (p < 0.01). This value is
of the same order of magnitude as that measured for iso-PsE
(2; 27 µg/ear) and PsA-OMe (1b; 22 µg/ear) and is not
significantly greater than it is for PsA (1; 8 µg/ear).
Figure 4 illustrates that 1a is also capable of inhibiting the
incidence of phagocytosis in Tetrahymena cells in a dose-
dependent manner (N ) 7). Its ED50 is 4 µM with a Hill
coefficient of 1 (correlation coefficient, R2 ) 0.931) and is
statistically significant (p < 0.01). In comparison, the ED50 value
for PsA (1)-inhibited phagocytosis is 6 µM, while that for the
methyl ether 1b is 10 µM (data not shown).
The experiment demonstrated that the C-glycoside binds to
the A2A receptor in a cooperative manner with a Hill coefficient,
J. Org. Chem. Vol. 73, No. 18, 2008 7013

Zhong et al.
TABLE 2. Binding Data for Compounds 1, 1a, and 2 toward the
Adenosine Receptors A2A and A3
CuSO4 (100 mL). The organic layer was dried over MgSO4, filtered,
and then concentrated to dryness. The residue was purified by
column chromatography over silica gel (hexane/ethyl acetate, 2:1,
v/v) to afford the desired product 4 (2.02 g, 7.21 mmol, 85%) as a
1
colorless oil: Rf 0.40 (hexane/ethyl acetate, 1:1, v/v); H NMR
(CDCl3, 200 MHz): δ 4.82 (d, J ) 6.6, 1H), 4.71-4.60 (m, 5H),
4.33-4.26 (m, 3H), 3.94 (dd, J ) 2.0, 1H), 3.85 (d, J ) 7.4, 1H),
3.35 (s, 3H), 3.33 (s, 3H), 3.30 (s, 3H); 13C NMR (CDCl3, 50 MHz)
δ 169.4, 95.9, 95.8, 94.7, 78.6, 73.4, 73.3, 66.4, 55.8, 55.6; ESI-
MS found 303.1053, C11H20O8Na+ calcd 303.1050.
2,3,4-Tri-O-methoxmethyl-D-xylopyranoside Olefin (5). To a
solution of 2,3,4-tri-O-methoxmethyl-D-xylopyranoside lactone (4,
0.45 g, 1.61 mmol) in dry toluene (8 mL) was added freshly made
Petasis’ reagent (Cp2Ti(CH3)2) in dry toluene (0.66 g in 1.5 mL,
3.12 mmol) dropwise to give a red solution. The resulting mixture
was stirred in the dark at 70 °C for 18 h. The black solution was
cooled and poured into hexanes (50 mL). The solution was filtered
through Celite to remove the resulting precipitate and then
concentrated to dryness in vacuo. The residue was purified by
column chromatography over silica gel (hexane/ethyl acetate, 6:1,
v/v) to afford desired product 5 (0.31 g, 1.11 mmol, 74%) as a
IC50
(µM)
Hill
coefficient
compd
receptor
ref compd
C-glycoside (1a)
PsA (1)
iso-PsE (2)
C-glycoside (1a)
PsA (1)
A2A
A2A
A2A
A3
A3
A3
20
13
13
10
4.1
6.6
1.2
1
1
2.3
2.2
2.5
NECA (14)
NECA (14)
NECA (14)
IB-MECA (15)
IB-MECA (15)
IB-MECA (15)
1
colorless oil: Rf 0.25 (hexane/ethyl acetate, 3:1, v/v); H NMR
(CDCl3, 200 MHz) δ 4.79-4.62 (m, 6H), 4.48 (t, J ) 0.8, 1.6,
1H), 4.10 (dd, J ) 4.0, 10.4, 1H), 4.03 (d, J ) 6.8, 1H), 3.79-3.52
(m, 4H), 3.40 (s, 3H), 3.39 (s, 3H), 3.34 (s, 3H); 13C NMR (CDCl3,
50 MHz): δ 155.9, 96.7, 96.4, 95.0, 93.6, 79.5, 75.8, 74.4, 67.5,
55.6, 55.5, 55.2; ESI-MS found 301.1251, C12H22O7Na+ calcd
301.1258.
iso-PsE (2)
n(H), of 1.2. It competitively inhibited the binding of the
reference compound, NECA (14), to the receptor with an IC50
equal to 20 µM and a binding constant, Ki, of 16 µM. The
C-glycoside 1a also binds the A3 receptor in a cooperative
manner with a Hill coefficient of 2.3. In this instance, it
competitively inhibited the binding of IB-MECA (15), the
reference compound, with an IC50 equal to 10 µM and a Ki
value of 6 µM. From this data, it is clear that 1a binds with a
2-fold greater affinity to the A3 receptor compared to the A2A
receptor. These results are summarized graphically; the Table
2 provides a comparison between 1a and the pseudopterosins
PsA (1) and iso-PsE (2).
Concluding Remarks. We have successfully synthesized the
C-glycoside analogue of PsA methyl ether, 1a, using a
Suzuki-Miyaura cross-coupling of an alkylborane generated
in situ from enol ether 5 with the aryl triflate 11 derived from
iso-PsE (2). Since the bioactivity of 1a is maintained despite
the presence of the C-linked sugar, we conclude that the intact
structure is present when a pseudopterosin expresses its anti-
inflammatory and phagocytosis inhibitory properties and,
therefore, that for these indications, the pseudopterosins are not
prodrugs. In addition, the C-glycoside is an effective binding
agent toward adenosine receptors A2A and A3.
C-Glycoside 7. To a dried flask containing olefin 5 (94 mg,
0.34 mmol) under argon was added 0.5 M 9-BBN in THF (1.02
mL, 0.51 mmol, 1.5 equiv) at 0 °C. The reaction mixture was
allowed to warm to room temperature and stirred for 2 h.
Aqueous NaHCO3 (1 M, 1.22 mL) was then added, and the
resulting solution was allowed to stir for 15 min. PdCl2(dppf)
(28 mg, 0.034 mmol, 0.1 equiv) and 6 (44 mg, 0.17 mmol, 0.5
equiv) in DMF (2 mL) were added dropwise. The resulting
mixture was stirred in the dark at room temperature for 18 h
and then poured into Et2O (10 mL). The organic layer was
washed with H2O (10 mL) and brine (10 mL). The aqueous layer
was extracted with ether (3 × 10 mL). The combined organic
layers were dried over MgSO4, filtered, and concentrated to
dryness in vacuo. The residue was purified by column chroma-
tography over silica gel (hexane/ethyl acetate, 5:1, v/v) to afford
the C-glycoside 7 (49 mg, 0.13 mmol, 74%) as a colorless oil:
1
Rf 0.60 (hexane/ethyl acetate, 1:1, v/v); H NMR (CDCl3, 200
MHz) δ 7.26-7.17 (m, 2 H), 6.94-6.84 (m, 2 H), 5.02-4.63
(m, 6 H), 4.05-3.97 (dd, 1 H, J ) 4.8, 11.4), 3.83 (s, 3 H),
3.62-3.56 (m, 2 H), 3.50 (s, 3 H), 3.45 (s, 3 H), 3.44 (m, 3 H),
3.31 (s, 3 H), 3.06 (m, 1 H), 2.52 (dd, 1 H, J ) 9.4, 10.0);
ESI-MS found 409.1841, C19H30O8Na+ calcd 409.1833.
Acyclic Byproduct 8. To a dried flask containing olefin 5 (100
mg, 0.36 mmol) under argon was added 0.5 M 9-BBN in THF
(1.80 mL, 0.9 mmol, 2.5 equiv). The reaction mixture was
allowed to reflux at 60 °C and stirred for 2 h. Aqueous NaHCO3
(1 M, 1.07 mL) was then added and the mixture allowed to stir
for 15 min. PdCl2(dppf) (30 mg, 0.036 mmol, 0.1 equiv) and 6
(92 mg, 0.36 mmol, 1.0 equiv) in DMF (4 mL) were added
dropwise. The resulting mixture was stirred in the dark at room
temperature for 18 h and then poured into Et2O (20 mL). The
organic layer was washed with H2O (20 mL) and brine (20 mL).
The aqueous layer was extracted with ether (3 × 20 mL). The
combined organic layers were dried over MgSO4, filtered, and
concentrated to dryness in vacuo. The residue was purified by
column chromatography over silica gel (hexane/ethyl acetate,
2:1, v/v) to afford adduct 8 (74 mg, 0.19 mmol, 53%) as a
Experimental Section
2,3,4-Tri-O-methoxmethyl-D-xylopyranoside Lactone (4). To
a solution of 2,3,4-tri-O-methoxmethyl-R/ꢀ-D-xylopyranosides (2.40
g, 8.5 mmol), NMO (1.49 g, 12.73 mmol), and freshly activated
powdered 4 Å molecular sieves (4.26 g) in CH2Cl2 (17 mL) was
added TPAP (152 mg, 0.43 mmol) at room temperature under argon.
The resulting mixture was stirred for 1 h and then diluted with
CH2Cl2 (50 mL). The mixture was washed with aqueous saturated
Na2SO3 solution (100 mL), brine (100 mL), and aqueous saturated
1
(20) Perez Gonzalez, M.; Teran, C.; Teijeira, M. Med. Res. ReV. 2008, 28
No. 3, 239–371.
(21) Jacobson, K.; Gao, Z. Nat. ReV. 2006, 5, 247–264.
(22) These studies were performed by Cerep, Inc., Seattle, WA, and Paris,
France.
colorless oil: Rf 0.15 (hexane/ethyl acetate, 1:1, v/v); H NMR
(CDCl3, 200 MHz) δ 7.26-7.12 (m, 2 H), 6.89-6.80 (m, 2 H),
5.82-5.65 (m, 1 H), 5.36-5.28 (m, 2 H), 4.92-4.54 (m, 8 H),
4.31 (t, 1 H, J ) 7.0, 7.4), 3.80 (s, 3 H), 3.79-3.72 (m, 4 H),
7014 J. Org. Chem. Vol. 73, No. 18, 2008

BioactiVity of the C-Glycoside of Pseudopterosin A Methyl Ether
3.43 (s, 3 H), 3.38 (s, 3 H), 3.36 (s, 3 H), 2.77-2.65 (m, 1 H),
1.98 (br, 1 H); ESI-MS found 411.1978, C19H32O8Na+ calcd
411.1989.
desired product (25 mg, 0.043 mmol, 51%) as a colorless oil:
Rf 0.30 (hexane/ethyl acetate, 3:1, v/v); H NMR (CDCl3, 200
1
MHz) δ 5.22 (d, 1 H, J ) 9.2), 5.07 (d, 1 H, J ) 9.6), 4.91-4.62
(m, 5 H), 4.07-3.90 (m, 2 H), 3.70 (s, 3 H), 3.64-3.54 (m, 3
H), 3.49 (s, 3 H), 3.45 (s, 3 H), 3.44 (m, 3 H), 3.40-3.36 (m,
2 H), 3.31 (s, 3 H), 3.00 (m, 1 H), 2.81 (dd, 1 H, J ) 10.0,
14.0), 2.31-2.22 (m, 2 H), 2.10 (s, 3 H), 1.76 (s, 3 H), 1.69 (s,
3 H), 1.64-1.45 (m, 2 H), 1.32-1.22 (m, 2 H), 1.18 (d, 3 H, J
) 7.4), 1.06 (d, 3 H, J ) 5.0); 13C NMR (CDCl3, 50 MHz) δ
155.2, 142.1, 137.6, 132.4, 130.0, 129.4, 126.5, 126.3, 98.8, 98.1,
97.2, 83.0, 81.4, 80.1, 77.8, 68.9, 60.4, 56.6, 56.2, 55.4, 40.1,
39.2, 35.9, 31.4, 29.5, 28.9, 27.7, 26.6, 25.7, 22.6, 21.2, 17.6,
11.4; ESI-MS found 599.3574, C33H52O8Na+ calcd 599.3554.
Iso-PsE Methyl Ether C-Glycoside (1a). To a solution of
MOM-protected starting material (25 mg, 0.043 mmol) in MeOH
(1 mL) was added 6 N aqueous HCl (0.75 mL). The reaction
mixture was stirred at room temperature for 18 h, and then
concentrated to dryness in vacuo. The residue was purified by
column chromatography over silica gel (CH2Cl2/MeOH, 25:1, v/v)
to afford the desired target structure 1a (15 mg, 0.034 mmol, 79%)
Iso-PsE Methyl Ether Aglycon (10). To a solution of iso-PsE
(1b, 0.25 g, 0.56 mmol) in dry acetone (30 mL) were added
MeI (0.105 mL, 1.68 mmol) and K2CO3 (0.235 g, 1.68 mmol).
The resulting mixture was refluxed at 60 °C for 24 h and then
cooled to room temperature. The solution was concentrated to
dryness in vacuo, followed by the addition of H2O (20 mL).
The aqueous layer was extracted with CHCl3 (3 × 20 mL). The
combined organic layers were dried over MgSO4, filtered, and
concentrated to dryness in vacuo. The residue was purified by
column chromatography over silica gel (CH2Cl2/MeOH, 20:1,
v/v) to afford the desired product (0.25 g, 0.54 mmol, 97%) as
a white solid. To a solution of iso-PsE methyl ether (0.25 g,
0.54 mmol) in MeOH (30 mL) was added 1 N aqueous HCl (24
mL). The resulting mixture was stirred at 50 °C for 4 h and
then cooled to room temperature. The solution was concentrated
to dryness in vacuo, followed by the addition of H2O (20 mL).
The aqueous layer was extracted with CHCl3 (3 × 20 mL). The
combined organic layers were dried over MgSO4, filtered, and
concentrated to dryness in vacuo. The residue was purified by
column chromatography over silica gel (hexane/ethyl acetate,
20:1, v/v) to afford desired product 10 (143 mg, 0.45 mmol,
84%) as a white solid: Rf 0.50 (hexane/ethyl acetate, 10:1, v/v);
1H NMR (CDCl3, 200 MHz) δ 5.68 (s, 1 H), 5.15 (td, 1 H, J )
1.6, 9.2), 3.76 (s, 3 H), 3.61 (td, 1 H, J ) 3.6, 9.6), 3.36 (dd, 1
H, J ) 6.8, 15.6), 2.22-2.13 (m, 3 H), 2.10 (s, 3 H), 2.02 (m,
1 H), 1.77 (d, 3 H, J ) 1.2), 1.70 (d, 3 H, J ) 1.0), 1.67-1.39
(m, 4 H), 1.29 (d, 3 H, J ) 7.0), 1.21-1.12 (dd, 1 H, J ) 6.2,
1
as a white solid: Rf 0.52 (CH2Cl2/MeOH, 20:1, v/v); H NMR
(CD3OD, 400 MHz) δ 5.09 (d, 1 H, J ) 9.2), 3.69 (dd, 1 H, J )
2.4, 8.8), 3.62 (m, 1 H), 3.60 (s, 3 H), 3.48-3.38 (m, 2 H),
3.27-3.17 (m, 6 H), 3.09 (t, 1 H, J ) 8.8), 2.88 (t, 1 H, J ) 10.8),
2.63 (dd, 1 H, J ) 9.6, 14.0), 2.27-2.11 (m, 2 H), 2.02 (s, 3 H),
1.81-1.76 (m, 1 H), 1.73 (s, 3 H), 1.65 (s, 3 H), 1.63-1.53 (m, 4
H), 1.12-1.08 (m, 1 H), 1.07 (d, 3 H, J ) 7.2), 1.00 (d, 3 H, J )
5.6); 13C NMR (CD3OD, 100 MHz) δ 156.4, 143.3, 138.7, 133.6,
131.5, 131.1, 127.9, 127.2, 83.2, 79.9, 77.0, 71.9, 71.3, 60.9, 42.1,
40.6, 37.2, 32.7, 30.7, 30.3, 29.3, 28.4, 26.1, 22.3, 21.7, 17.9, 11.8;
ESI-MS found 467.2776, C27H40O5Na+ calcd 467.2768.
1
11.0), 1.08 (d, 3 H, J ) 6.0). H NMR spectral data was the
same as that reported in the literature.17
Measurement of Phagocytic Activity in Tetrahymena thermo-
phila Cells. The effect of drugs on phagosome formation in
Tetrahymena cells was measured by visualizing newly formed
phagosomes containing India ink by light microscopy. For the
experiment, cells were washed twice with 10 mM HEPES buffer
by centrifuging at 450g for 5 min. The pellet was then
resuspended in 10 mM HEPES at pH 7.4. The final cell
concentration for each experimental treatment was 250000 cells/
mL in a total volume of 4 mL. Cell suspensions were placed in
13 × 100 mm test tubes in a 25 °C water bath and allowed to
acclimate for 45 min to 1 h. Drugs were prepared at the desired
concentrations in 0.4 mL volumes. For control samples, 0.4 mL
of buffer and vehicle were added to the incubation mixture. In
order to visualize the newly formed phagosomes, 0.45 mL of
diluted India ink (1:25, v/v) was added to each of the test drug
volumes. The experiment was started when the drug/ink mixture
was added to the Tetrahymena cells and was terminated after
10 min when 500 µL of cell suspension (approximately 125,000
cells/mL) was removed and fixed in 100 µL of formalin solution.
A minimum of 100 cells from each treatment was examined for
the incidence of phagosome formation under light microscopy
(40 x magnifications). Phagocytic activity was assessed by
calculating the ratio of cells with food vacuoles compared to
the cells with no food vacuoles.
Inhibition of Inflammation: In Vivo Studies. Compounds were
topically applied in acetone to the inside pinnae of the ears of
mice in a solution containing the edema-causing irritant, phorbol
12-myristate 13-acetate (PMA). Mice were briefly anesthetized
with halothane, PMA alone (2 µg/ear) or in combination with
various dilutions of test compoundwas applied to the left ears
(5 mice per treatment group), and acetone was applied to all
right ears. After 3 h and 20 min incubation, the mice were
euthanized, the ears removed, and a 6 mm biopsy was taken
from the center of the ear and immediately weighed. The ears
were then flash frozen and stored in liquid nitrogen for
myeloperoxidase activity studies. Edema was measured by
subtracting the weight of the right ear (acetone control) from
the weight of the left ear (treated). Results were recorded as %
Iso-PsE Methyl Ether Aglycon Triflate (11). To a solution of
iso-PsE methyl ether aglycon (10, 64 mg, 0.20 mmol) in dry
dichloromethane (2 mL) was added dry pyridine (92 µL, 1.17
mmol) under argon at room temperature. Tf2O (95 µL, 0.56
mmol) was added dropwise. The reaction mixture was stirred at
room temperature for 1 h. H2O (5 mL) was then added, and the
resulting mixture was extracted with dichloromethane (3 × 5
mL). The combined organic layers were washed with 1 N HCl
(10 mL), H2O (10 mL), and brine (10 mL). The organic layer
was dried over MgSO4, filtered, and concentrated to dryness in
vacuo. The residue was purified by column chromatography over
silica gel (hexane/ethyl acetate, 30:1, v/v) to afford the desired
product 11 (88 mg, 0.20 mmol, 97%) as a colorless oil: Rf 0.50
1
(hexane/ethyl acetate, 15:1, V/V). H NMR (CDCl3, 200 MHz):
δ 5.11 (td, 1 H, J ) 1.2, 9.2), 3.74 (s, 3 H), 3.65 (d, 1 H, J )
8.4), 3.38-3.29 (m, 1 H), 2.32-2.18 (m, 2 H), 2.12 (s, 3 H),
2.06-1.96 (m, 2 H), 1.77 (s, 3 H), 1.70 (s, 3 H), 1.66-1.52 (m,
4 H), 1.24 (d, 3 H, J ) 7.2), 1.08 (d, 3 H, J ) 5.6); 13C NMR
(CDCl3, 50 MHz) δ 147.4, 139.4, 139.1, 134.7, 133.4, 131.1,
129.1, 128.5, 121.9, 61.0, 41.9, 39.0, 35.8, 30.1, 29.0, 27.5, 26.9,
25.6, 22.3, 20.8, 17.7, 11.2; ESI-MS found 469.1647,
C22H29O4F3SNa+ calcd 469.1631.
MOM-Protected Iso-PsE Methyl Ether C-Glycoside. To a
dried flask containing olefin 5 (61 mg, 0.22 mmol) under argon
was added 0.5 M 9-BBN in THF (0.66 mL, 0.33 mmol, 1.5
equiv) at 0 °C. The reaction mixture was allowed to warm to
room temperature and stirred for 2 h. Aqueous NaHCO3 (1 M,
0.8 mL) was then added and the mixture allowed to stir for 15
min. PdCl2(dppf) (20 mg, 0.024 mmol, 0.1 equiv) and 11 (38
mg, 0.085 mmol) in DMF (1 mL) were added dropwise. The
resulting mixture was stirred in the dark at 80 °C for 18 h and
then poured into Et2O (10 mL). The organic layer was washed
with H2O (10 mL) and brine (10 mL). The aqueous layer was
extracted with ether (3 × 10 mL). The combined organic layers
were dried over MgSO4, filtered, and concentrated to dryness
in vacuo. The residue was purified by column chromatography
over silica gel (hexane/ethyl acetate, 6:1, v/v) to afford the
J. Org. Chem. Vol. 73, No. 18, 2008 7015

Zhong et al.
decrease (inhibition) or % increase (potentiation) in edema
relative to the PMA control group edema.
were also performed for A1 receptors (expressed in CHO cells)
and the A2B receptor.
In Vitro Binding Studies of C-Glycoside 1a to Adenosine
Receptors. These studies were performed by Cerep, Inc.22 A3
receptor/binding affinity of the C-glycoside to the A3 receptor
was measured in human recombinant (HEK-293 cells). The A3
specific radiolabeled agonist, [125I]AB-MECA, and the specific
A3 agonist, IB-MECA, were used to estimate the binding affinity
of the C-glycoside to the receptor. A2A receptor/binding affinity
of the C-glycoside to the A2A receptor was measured in human
recombinant (HEK-293 cells). The A2A specific radiolabeled
agonist [3H]CGS 21680 and the specific A2A agonist NECA were
used to estimate the binding affinity of the C-glycoside to the
receptor. Binding affinity studies of one dose of the C-glycoside
Acknowledgment. We gratefully acknowledge the U.S. Army
Medical Research Program (Grant No. W81XWH-06-1-0089)
for its support of this research.
Supporting Information Available: 1H and 13C NMR data
for compounds 4, 5, 11, the tris-MOM ether precursor of
1
1a, and 1a and the H NMR spectral data for 7 and 8. This
material is available free of charge via the Internet at
http://pubs.acs.org.
JO801432T
7016 J. Org. Chem. Vol. 73, No. 18, 2008