New jasmonate analogues as potential anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmc.2008.10.050

In an effort to develop new anti-inflammatory agents, methyl jasmonate analogues (2-20) were synthesized and evaluated for their inhibitory effects on the production of pro-inflammatory mediators (NO, IL-6, and TNF-α) in lipopolysaccharide (LPS)-activated RAW264.7 murine macrophage cells. The introduction of an enone functionality to the structure of a plant hormone (1) rendered the product (2) a significant anti-inflammatory activity. Analogues further derived from 2 (7, 9, 13, and 15) exhibited even more enhanced activity, and these compounds were much more potent than natural anti-inflammatory prostaglandins (PGA₁, PGA₂, and 15-deoxy-Δ^12,14-PGJ₂). Among them, compounds 9 and 15 showed the highest potency, while compounds 7 and 13 would be more desirable with respect to safety. This is the first study demonstrating the anti-inflammatory potential of jasmonate derivatives, and the present results suggest that α-haloenone jasmonates (7, 9, 13, and 15) may serve as potential anti-inflammatory leads.

Full text of DOI:10.1016/j.bmc.2008.10.050

Bioorganic & Medicinal Chemistry 16 (2008) 10228–10235
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New jasmonate analogues as potential anti-inflammatory agents
a
b
b
c
a
d
a,*
Hung The Dang , Hye Ja Lee , Eun Sook Yoo , Jongki Hong , Baoquan Bao , Jae Sue Choi , Jee H. Jung
a
College of Pharmacy, Pusan National University, Geumjeong-gu, Busan 609-735, Republic of Korea
College of Medicine, Cheju National University, Jeju, Republic of Korea
College of Pharmacy, Kyung Hee University, Seoul, Republic of Korea
Faculty of Food Science and Biotechnology, Pukyong National University, Busan, Republic of Korea
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
In an effort to develop new anti-inflammatory agents, methyl jasmonate analogues (2–20) were synthe-
Received 10 September 2008
Revised 21 October 2008
Accepted 22 October 2008
Available online 25 October 2008
sized and evaluated for their inhibitory effects on the production of pro-inflammatory mediators (NO,
IL-6, and TNF-a) in lipopolysaccharide (LPS)-activated RAW264.7 murine macrophage cells. The introduc-
tion of an enone functionality to the structure of a plant hormone (1) rendered the product (2) a
significant anti-inflammatory activity. Analogues further derived from 2 (7, 9, 13, and 15) exhibited even
more enhanced activity, and these compounds were much more potent than natural anti-inflammatory
Keywords:
12,14
prostaglandins (PGA
1
, PGA
2
, and 15-deoxy-
D
2
-PGJ ). Among them, compounds 9 and 15 showed the
Methyl jasmonate
Anti-inflammatory activity
Enone functionality
Cytokine
highest potency, while compounds 7 and 13 would be more desirable with respect to safety. This is the
first study demonstrating the anti-inflammatory potential of jasmonate derivatives, and the present
results suggest that
leads.
a
-haloenone jasmonates (7, 9, 13, and 15) may serve as potential anti-inflammatory
Ó 2008 Elsevier Ltd. All rights reserved.
1
. Introduction
In our previous study on the anti-inflammatory components of
lected as a starting material for the synthesis of cyclopentenone
prostaglandin-like compounds.
Jasmonic acid and methyl jasmonate (1) are fatty acid-derived
cyclopentanones occurring throughout the plant kingdom. Biosyn-
thesis of jasmonates in plants is distinct from that of prostaglan-
dins in mammals. In animals, prostaglandins are synthesized
from arachidonic acid (C20:4), while jasmonates are synthesized
the red alga Gracilaria verrucosa, several new prostaglandins and
oxygenated fatty acids were isolated. Among them, enone fatty
acids were confirmed as causative components for the inhibitory
effect on the production and gene expression of pro-inflammatory
1
7–19
mediators (NO, IL-6, and TNF-
a) in lipopolysaccharide (LPS)-acti-
from linolenic acid (C18:3) in plants.
Jasmonates in plants play
vated RAW264.7 murine macrophage cells. Thereafter, analogues
of these enone fatty acids were synthesized and evaluated for
anti-inflammatory activity and cytotoxicity. On the basis of this
study, we found that the a,b-unsaturated ketone (enone) function-
ality is essential for the activity. This finding has made us to engage
major role in a defense against insects and disease. Leaf tissues ex-
hibit a fast increase in jasmonate levels in response to various
stimuli and stresses, for example, wounding, herbivory, and infec-
tion. The widespread occurrence of jasmonates in plants and some
lower eukaryotes and their capacity to regulate processes in in-
sects support the notion that jasmonates are of general biological
1
in a study on anti-inflammatory and/or anti-tumor compounds
2
0
with enone functionality, as exemplified by sesquiterpene lactones
importance.
2
(
parthenolide), cyclopentenone prostaglandins (PGA
1
, PGA
and oleanane tritepenoids (CDDO).
In a quest of new leads with the enone functionality and higher
potency than natural anti-inflammatory prostaglandins (PGA
2
, and
Recently, it was reported that methyl jasmonate (1) exhibits
anticancer activity in vitro and in vivo. The initial report indicated
that methyl jasmonate induces suppression of cellular proliferation
and death in various human and mouse cancer cell lines, including
breast, prostate, melanoma, lymphoblastic leukemia, and lym-
1
2,14
3–12
13–
1
5-deoxy-
D
2
-PGJ ),
1
6
1
,
1
2,14
PGA
2
, and 15-deoxy-
D
2
-PGJ ), we intended to perform struc-
2
1
tural modification on prostaglandin analogues isolated from G.
verrucosa. However, due to limited amounts of the natural samples,
methyl jasmonate (1), a plant hormone, which shares the same
partial structure with anti-inflammatory prostaglandins, was se-
phoma cells. Furthermore, methyl jasmonate increased the life
span of EL-4 lymphoma-bearing mice, and exhibited selective cyto-
toxicity towards cancer cells while sparing normal blood lympho-
cytes, even when the latter were part of a mixed population of
leukemic and normal cells drawn from the blood of chronic lym-
phocytic leukemia (CLL) patients. Methyl jasmonate was also
found to suppress the proliferation or kill various other cancer cells
*
Corresponding author. Tel.: +82 51 510 2803; fax: +82 51 513 6754.
E-mail address: jhjung@pusan.ac.kr (J.H. Jung).
0
968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.050

H. T. Dang et al. / Bioorg. Med. Chem. 16 (2008) 10228–10235
10229
including lung and myeloid leukemia cells.22–24 Thus, methyl jasm-
onate is now considered as a promising lead for cancer treatment
moderate yield by addition of a catalytic amount of TFA (trifluro-
acetic acid) at 80 °C for 48 h (Scheme 1).
2
5–31
in humans.
However, so far there was no report on the anti-
Introduction of an electronegative group such as nitrile, car-
inflammatory activity of this type of compound. Therefore, for
our continuous search for new anti-inflammatory agents, the jasm-
onate skeleton was employed as a model structure for the synthe-
sis of new cyclopentenone-bearing derivatives. In the present
study, we report the synthesis and anti-inflammatory activity of
jasmonate analogues.
boxyl, or halogens at the
to increase the electrophilicity of the b-carbon, henceforth increas-
a-position of the enone group is expected
13–16
ing bioactivity, as in the case of oleanane tritepenoids (CDDO)
4
0
and punaglandins. To introduce an
a-cyano enone group to 1,
formylation of 1 with ethyl formate in toluene in the presence of
NaH and a catalytic amount of MeOH afforded the hydroxymethyl-
ene 1a. Conversion of 1a into a cyano group was performed accord-
ing to a literature procedure, using O,N-bis-(trifluoroacetyl)-
hydroxylamine in a mixture of toluene and pyridine in reflux. This
gave 1b in 30% yield, and subsequent oxidation of 1b with 2,3-di-
chloro-5,6-dicyano-1,4-benzoquinone (DDQ) in refluxing toluene
afforded the required a-cyano enone 3. Carboxylation of 1 with di-
methyl carbonate in toluene in the presence of NaH and a catalytic
amount of MeOH afforded the intermediate 1c in 50% yield. Subse-
4
1
2
. Chemistry
The enone functionality of the aforementioned compounds, en-
1
2
one fatty acids, sesquiterpene lactones (parthenolide), cyclo-
1
2,14
pentenone prostaglandins (PGA
1
,
2
PGA ,
and 15-deoxy-
D
-
3–12
13–16
PGJ
2
),
and oleanane tritepenoids (CDDO),
is considered to
be the key structure for their bioactivity. From a biochemical point
of view, the enone group is an electrophilic center susceptible to
nucleophilic attack (Michael addition) by a sulfhydryl group of re-
duced glutathione or cysteine residues in proteins. Alkylation of
quent oxidation of 1c with DDQ in refluxing toluene gave the
methoxycarbonyl enone ester 4. Attempts to obtain an -carboxyl
enone analogue by hydrolysis of 1c were unfavorable and resulted
in the reversion to 1. The -haloenone derivatives (7–10) were
synthesized from 2 via two steps. The enone 2 was treated with
alkaline H
at À10 °C to generate the epoxide 2a. The vinyl chlo-
rides 7 and 8 were obtained concomitantly by treatment of the
a-
a
9
a
crucial cysteine residues can result in a loss of function or, on
the contrary, activation4 of the target proteins. Therefore, intro-
duction of an enone functionality is a critical step for design and
synthesis of new jasmonate derivatives. Although many methods
to introduce unsaturation adjacent to a carbonyl functionality have
,10
2 2
O
4
2
epoxide 2a with HCl in acetic acid and CHCl
mation of vinyl bromide 9 was not obtained under the same con-
dition (HBr/acetic acid/CHCl ). Therefore, other conditions were
tried, and the -haloenone derivatives 9 and 10 were successfully
prepared by treatment of 2a with halide salts LiBr and LiI, respec-
3
.
However, the for-
been developed over the years, the synthesis of
a,b-unsaturated
carbonyl compounds is often a tedious and sometimes challenging
3
3
2
a
transformation. Attempts to achieve one-step synthesis of 2 on
the basis of the reported methods using several reagents, such as
43
3
3
tively, under mildly acidic condition (silica gel support).
activated manganese dioxide (MnO
2
), benzeneselenic anhydride
1
3
4
In our previous study, transformation of esters of enone fatty
acids to corresponding acid or amide analogues was favorable to
enhance anti-inflammatory activity of enone fatty acid derivatives.
Therefore, this transformation of jasmonates is also expected to en-
hance potency. The acid analogue 5, which was obtained by hydro-
lysis of 2 with porcine liver esterase in a mixture of acetone and
phosphate buffer (pH 8), was the starting material for synthesis
of 17 and 18. Synthesis of amides from the acid 5 was tried using
several coupling reagents, such as N,N-dicyclohexylcarbodiimide
(
2
(PhSeO) O),
and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
DDQ) and p-toluenesulfonic acid (p-TsOH), were unsatisfactory.
35
(
In addition, synthesis of 2 via preparation of the silyl enol ether of 1
followed by palladium-catalyzed oxidation was also unsuccess-
3
6
ful. Recently, a new method for the synthesis of
a,b-unsaturated
carbonyl compounds in one simple step utilizing nontoxic IBX (o-
3
7,38
iodoxybenzoic acid) as an oxidizing reagent has been reported.
However, the reaction was totally incomplete when tried in stan-
dard conditions. Therefore, modification of the reaction condition
3
9
(
DCC), 1-hydroxybenzotriazole (HOBt), or 1-(3-dimethylaminopro-
was attempted, and the desired product 2 was obtained with a
4
4,45
pyl)-3-ethyl-carbodiimide HCl (DCCI).
However, the amides 17
HO
COOCH3
COOCH3
(ii)
COOCH3
(i)
COOCH3 (x)
COOH
O
O
1a
O
O
1
O
O
2
O
5
(iii)
(v)
COOCH3
(vii)
COOCH3
(xi)
COR
H CO
3
O
NC
NC
O
O
1
b
1c
(vi)
2a
(ix)
17 R = NHCH(CH )
(viii)
3 2
1
8 R = NHC H OH
2 4
(
iv)
H CO
3
COOCH3
COOCH3
COOCH3
COOR
R
Cl
O
O
O
O
O
3
4
9 R = Br
0 R = I
7 R = CH3
1
8 R = H
Scheme 1. Reagents and conditions: (i) IBX, TFA, DMSO, 80 °C, 48 h; (ii) NaH, MeOH (cat), ethyl formate, toluene, rt, 12 h; (iii) O,N-bis-(trifluoroacetyl)-hydroxylamine,
pyridine, toluene, reflux, 1 h; (iv) DDQ, toluene, reflux, 10 min; (v) NaH, MeOH (cat), dimethyl carbonate, toluene, reflux, 3 h, then rt, 12 h; (vi) DDQ, toluene, reflux, 5 h; (vii)
H
2
O
2 3 2 2
, 10% KOH, MeOH, 12 h; (viii) HCl/acetic acid, CHCl , 12 h; (ix) LiBr or LiI, silica gel, CH Cl , 24 h; (x) porcine liver esterase, phosphate buffer pH 8, acetone, rt, 12 h; (xi)
TBTU, TEA, EtOAc, 24 h.

1
0230
H. T. Dang et al. / Bioorg. Med. Chem. 16 (2008) 10228–10235
and 18 were only obtained by treatment of 5 with isopropylamine
and ethanolamine, respectively, in the presence of 2-(1H-benzotri-
azole-1yl)-1,2,3,3-tetramethyluronium tetrafluoroborate (TBTU)
NO: 100 uM
TNF: 50 uM
NO: 50 uM
IL-6: 50 uM
MTT: 50 uM
MTT: 100 uM
4
6
1
1
20
00
120
100
80
60
40
20
0
and triethylamine (TEA) in ethyl acetate at room temperature.
3
. Results and discussion
Methyl jamonate (1) and its derivatives (2–5 and 7–20) were
8
0
60
evaluated for their anti-inflammatory activity through measure-
ment of their inhibitory effects on the production of pro-inflamma-
4
2
0
0
tory mediators (NO, IL-6, and TNF-a) in murine macrophage cells
(Figs. 1–3). As shown inFigure 1, the potency of compound 2 was sig-
nificantly enhanced than the original compound (1), indicating the
0
importance of an enone group for bioactivity of jasmonate deriva-
BL
LPS
1
2
3
4
tives. Also, compound 2 was more active than the
3) and -methoxycarbonyl enone (4), which is in contrast to the
case of the -cyano derivatives of oleanane triterperpenoids
and this indicated that substitution of these groups at
-position is detrimental to bioactivity. However, in the case of
-position of the en-
one group, the activity was further enhanced than compound 2
Fig. 2). In addition, the -haloenones (7, 9, 13, and 15) were much
more potent than natural anti-inflammatory prostaglandins (PGA
a-cyano enone
Figure 1. Inhibitory effects of methyl jasmonate (1) and its analogues (2–4) on
productions of NO, IL-6, and TNF- in LPS-activated RAW264.7 cells. RAW264.7
cells (1.5 Â 10 cells/mL) were stimulated with LPS (1 g/mL) alone or with test
samples at concentrations of 50 and 100 M for 24 h (BL, blank). The nitric oxide
production was determined by Griess reagent method, and the production of pro-
inflammatory cytokines (IL-6 and TNF- ) was determined by ELISA method. Cell
(
a
a
a
5
l
1
3–16
(
CDDO),
l
the
a
halogen substitution (such as Cl, Br, and I) at the
a
a
viability was determined using MTT method. The data represent means ± SD of
duplicate experiments.
(
a
1
,
group to other functional group such as acid (5, 8, 12, and 14)
or amide group (17–20) appeared to be unfavorable for anti-
inflammatory activity of these analogues.
1
2,14
PGA
2
, and 15-deoxy-
D
2
-PGJ ) (Fig. 4).
2
Regarding the influence of the unsaturation of the side chain on
the activity, it was observed that compounds with a saturated side
chain show higher potency, for example, 11 > 2; 12 > 5; 19 > 17;
4
5
COR1
R2
12
9
O
2
0 > 18. However, this trend was inverted for a-haloenone deriva-
9
4
tives, that is, derivatives with an unsaturated side chain showed
higher potency, for example, 7 > 13; 9 > 15; 10 > 16 (Figs. 2 and
3). Taken together, the results revealed that the presence of an en-
one moiety is essential for the activity, but other structural modi-
fications can dramatically modulate the activity of this type of
compounds (Fig. 5).
Methyl jasmonate (1) contains two asymmetric centers at C-3
and C-7. Thus, this compound exists in four stereoisomeric
forms, (3R,7S)- and (3S,7R)-cis-isomers, known as (+)- and (À)-
epiMJA, respectively, and (3R,7R)- and (3S,7S)-trans-isomers,
commonly called as (À)- and (+)-MJA, respectively (Fig. 6). The
cis-isomers (epiMJAs) are less stable than trans-isomer (MJAs)
due to higher steric hindrance between the cis side chains, and
easily epimerized to the trans-isomers. The naturally occurring
1
2
3
4
5
7
8
9
R = OCH
1
1
3
3
3
3
R = H
Δ
Δ
Δ
Δ
Δ
Δ
Δ
Δ
Δ
2
2
,9
R = OCH
R = H
4,9
4,9
4,9
R = OCH
R = CN
1
2
R = OCH
R = COOCH
1
2
3
R = OH
R = H
1
1
2
4
4
,9
,9
R = OCH
R = Cl
3
2
R = OH
R = Cl
1
2
4
4
4
4
,9
,9
R = OCH
R = Br
1
3
3
2
1
0
R = OCH
1
R = I
2
11
R = OCH
R
R
R
R
R
R
R
R
R
R
= H
= H
= Cl
= Cl
= Br
= I
Δ
Δ
Δ
Δ
Δ
Δ
Δ
Δ
Δ
Δ
1
3
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
2
3
4
5
6
7
8
9
R = OH
1
4
4
4
R = OCH
1
3
R = OH
1
R = OCH
1
3
3
4
4
R = OCH
1
,9
R = NHCH(CH )
= H
= H
= H
= H
1
3 2
4,9
4
4
R = NHC H OH
1
2 4
isomers in plants were reported to have the R stereochemistry
R = NHCH(CH )
1
3 2
47–49
at C-3 and either S or R at C-7.
The (3R,7S)-(+)-isomer,
2
0
R = NHC H OH
1
2 4
(
+)-epiMJA, is believed to be the initial product formed in the
biosynthesis of jasmonates in plants, and the (3R,7R)-(À)-isomer,
(
À)-MJA, is formed as a result of subsequent epimerization under
Regardless of other factors, the potency of
a-haloenone deriva-
physiological conditions in the cells or during isolation. Previous
studies, in which the activities of pure stereoisomers of MJA
were compared, revealed that (3R,7S)-(+)-epiMJA and (3R,7R)-
tives was in the order Cl > Br > I, corresponding to the order of
electronegativity. Their suppressive effects on the pro-inflamma-
tory mediators were concentration-dependent (Figs. 2 and 3). At
(
(
À)-MJA are the active isomers, while (3S,7R)-(À)-epiMJA and
concentrations of 3.12 and 6.25
the highest potency without any cytotoxicity (Fig. 3). However, at
higher concentrations (12.5 and 25 M), the brominated ana-
logues (9 and 15) began to exhibit cytotoxicity to the cells (Figs.
and 3). The potency of the chlorinated analogues (7 and 13)
lM, compounds 9 and 15 showed
3S,7S)-(+)-MJA are the inactive forms, and that the 3R configu-
ration is indispensable for bioactivities of jasmonates.5
Methyl jasmonate (1), a commercial product, was known to be
an equilibrium mixture of approximately 45% each of (3R,7R)-
0–54
l
2
(
À)-MJA and (3S,7S)-(+)-MJA and 5% each of (3R,7S)-(+)-epiMJA
52,54
was lower than the brominated derivatives (9 and 15); nonethe-
less, they would be safer because their cytotoxicity was negligible
and (3S,7R)-(À)-epiMJA.
Therefore, considering the consistent
-carbon of the cyclopentenone
absolute configuration at the
c
even at higher concentration (12.5
lM) (Fig. 3). Concerning the
1
moiety of natural anti-inflammatory prostaglandins (PGA ,
PGA
the production and gene expression of pro-inflammatory media-
tors (NO, COX-2, IL-6, IL-1b, and TNF-
in the racemic mixture 2 would contribute most to the anti-
inflammatory activity (Fig. 6).
1
2,14
role of the methyl ester group, the ester forms (2, 7, 11, and
3) were more active than the corresponding free acid (5, 8,
2, and 14) or amide forms (17–20) (Fig. 2). Hence, in contrast
2
, PGJ
2
, and 15-deoxy-
D
2
-PGJ ) with inhibitory effects on
1
1
3
–12
a),
the enantiomer 2b
to the activity of acid and amide derivatives observed for enone
fatty acids in our previous study,1 transformation of the ester

H. T. Dang et al. / Bioorg. Med. Chem. 16 (2008) 10228–10235
10231
NO: 25 uM
NO: 12.5 uM
MTT: 25 uM
IL-6: 12.5 uM
MTT: 12.5 uM
TNF: 12.5 uM
1
1
20
00
120
100
80
60
40
20
0
8
6
4
2
0
0
0
0
0
BL LPS
2
5
7
8
9
10
11
12
13
14
15
16
17
18
19
20
5
Figure 2. Inhibitory effects of jasmonate derivatives (2, 5, and 7–20) on productions of NO, IL-6, and TNF-
mL) were stimulated with LPS (1 g/mL) alone or with test samples at concentrations of 12.5 and 25.0 M for 24 h (BL, blank). The nitric oxide production was determined by
) was determined by ELISA method. Cell viability was determined using MTT
a in LPS-activated RAW264.7 cells. RAW264.7 cells (1.5 Â 10 cells/
l
l
Griess reagent method, and the production of pro-inflammatory cytokines (IL-6 and TNF-
a
method. The data represent means ± SD of triplicate experiments.
NO: 3.12 uM
MTT: 3.12 uM
NO: 6.25 uM
MTT: 6.25 uM
NO: 12.5 uM
MTT: 12.5 uM
COOCH3
COOCH3
COOCH3
Cl
Br
1
1
20
00
120
100
80
60
40
20
0
O
O
O
O
2
7
13
COOCH3
Cl
Br
8
6
4
2
0
0
0
0
0
15
COOH
15-deoxy-Δ12,14-PGJ2
COOCH3
O
O
9
O
O
COOH
BL
LPS
7
9
13
15
COOH
Figure 3. Inhibitory effects of compounds 7, 9, 13, and 15 on the production of NO
5
OH
PGA1
OH
PGA2
in LPS-activated RAW264.7 cells. RAW264.7 cells (1.5 Â 10 cells/mL) were stim-
ulated with LPS (1
and 12.5
lg/mL) alone or with test samples at concentrations of 3.12, 6.25,
M for 24 h (BL, blank). The nitric oxide production was determined by
l
Griess reagent method. Cell viability was determined using MTT method. The data
represent means ± SD of triplicate experiments.
NO: 10 uM
NO: 30 uM
MTT: 10 uM
1
1
20
00
120
4
. Conclusion
100
80
60
40
20
0
8
6
4
0
0
0
In this study, we have synthesized a series of new jasmonate
derivatives and evaluated their anti-inflammatory activity. Of the
synthesized analogues, the -haloenones (7, 9, 13, and 15) exhib-
a
ited potent activity, and the activity of these compounds were
much more enhanced than natural anti-inflammatory prostaglan-
12,14
dins (PGA
pounds 9 and 15 showed the highest potency, while compounds
and 13 would be more desirable with respect to safety. The re-
sults showed that jasmonate derivatives, especially -haloenone
1
, PGA
2
, and 15-deoxy-
D
-PGJ
2
). Among them, com-
20
0
7
BL LPS 2
A
B
C
7
9
13 15
a
jasmonates (7, 9, 13, and 15), may serve as potential anti-inflam-
matory leads, and this is the first study demonstrating the anti-
inflammatory potential of jasmonate analogues.
Figure 4. Inhibitory effects of compounds 2, 7, 9, 13, 15, PGA
1
(A), PGA
2
(B), and 15-
12,14
deoxy-
RAW264.7 cells (1.5 Â 10 cells/mL) were stimulated with LPS (1
with test samples at concentrations of 10 and 30 M for 24 h (BL, blank). The nitric
D
-PGJ2 (C) on the production of NO in LPS-activated RAW264.7 cells.
5
lg/mL) alone or
l
oxide production was determined by Griess reagent method. Cell viability was
determined using MTT method. The data represent means ± SD of triplicate
experiments.
5
5
. Experimental
.1. General
ODS-H80 column (250 Â 10 mm, 4
packed column (250 Â 10 mm, 5
detector. Chemical reagents, including methyl jasmonate (1),
l
m, 80 Å) and a C18-5E Shodex
1H and 13C spectra were recorded on Varian Inova 400 spec-
lm, 100 Å) using a Shodex RI-71
trometer. Chemical shifts were reported with reference to the
respective residual solvent or deuterated solvent peaks (d 7.24
and d 77.0 for CDCl ). FABMS data were obtained on a JEOL JMS
1
2,14
H
PGA
1
, PGA
2
, and 15-deoxy-
D
2
-PGJ , were purchased from the
C
3
Sigma–Aldrich Chemical Company and used without further puri-
fication. Biological assays were performed at Cheju National
University.
SX-102A spectrometer. HRFABMS data were obtained on JEOL
JMS SX-101A spectrometer. HPLC was performed with a YMC

1
0232
H. T. Dang et al. / Bioorg. Med. Chem. 16 (2008) 10228–10235
dropwise methyl jasmonate 1 (50 mg). After stirring at 0 °C for
10 min, ethyl formate (7.0 equiv) was added dropwise and the
reaction mixture was stirred at room temperate overnight. Then,
the mixture was extracted with cold water and the aqueous solu-
tion was re-extracted once with ether to remove any unreacted
starting material. The aqueous solution was acidified with 10%
HCl, and the orange-colored oily layer was removed by extraction
Cl > Br > I
COOCH > COOH,
3
>
H > CN
CONHCH(CH ) ,
3
2
>
COOCH
CONHC H OH
2 4
3
R1
R2
O
with ether. This ethereal solution was washed with dilute NaHCO
to remove traces of excess acid and dried over MgSO . After evap-
3
4
when R2 = H, saturation enhanced activity
when R = Halogens, saturation reduced activity
oration of the ethereal solution, the residue (48 mg, 86%) obtained
2
1
was used for the next reaction without purification; yellow oil; H
Figure 5. Structure–activity relationships of jasmonate derivatives. Anti-inflam-
matory activity was gauged as inhibition on the production of pro-inflammatory
mediators (NO, IL-6, and TNF-a).
NMR (CDCl
.68 (3H, s), 2.75 (1H, m), 2.62 (1H, m), 2.44–2.14 (6H, m), 2.03
2H, m), 0.95 (3H, t, J = 7.6 Hz).
3
, 400 MHz): d 7.17 (1H, s), 5.47 (1H, m), 5.30 (1H, m),
3
(
3
7
R
R
COOCH3
3
7S
R
COOCH
3
5.2.3. O,N-Bis-(trifluoroacetyl)-hydroxylamine
O,N-Bis-(trifluoroacetyl)-hydroxylamine was prepared by
refluxing 3.2 moles of trifluoroacetic anhydride with 1 mole of
hydroxylamine hydrochloride for 1.5 h. After removal of trifluoro-
acetic acid and trifluoroacetyl chloride under vacuum, the residue
O
O
O
(
−)-MJA
(+)-epiMJA
COOCH3
3
S
COOCH3
3
S
2 2
was recrystallized from CH Cl . The product is an 80% yield of
7R
7S
hygroscopic and easily volatile needles of O,N-bis-(trifluoroace-
4
1
O
tyl)-hydroxylamine.
(
+)-MJA
(−)-epiMJA
3
7
S
R
COOCH3
5
.2.4. Methyl 5-cyano-jasmonate (1b)
O,N-Bis-(trifluoroacetyl)-hydroxylamine (3.0 equiv) was added
O
2b
to a solution of 1a (20 mg) and pyridine (6.0 equiv) in toluene
and the resulting mixture was heated under reflux for 1 h. After
cooling down, the organics were diluted with EtOAc, washed with
H O, then brine, dried (MgSO ), filtered, and concentrated under
2 4
reduced pressure. The crude product was purified by reversed-
8
2R
S
COOH
COOH
8
S
COOH
1
O
O
O
O
OH
PGJ2
15-deoxy-Δ12,14-PGJ
COOH
2
phase HPLC (YMC ODS-H80) eluting with 50% aqueous MeOH to
1
yield compound 1b (6.0 mg, 30%); colorless oil; H NMR (CDCl
3
,
8
R
8
R
400 MHz): d 5.51 (1H, m), 5.21 (1H, m), 3.70 (3H, s), 3.40 (1H,
dd, J = 8.4, 5.6 Hz, minor isomer), 3.10 (1H, dd, J = 12.8, 8.4 Hz, ma-
jor isomer), 2.73 (1H, m), 2.56–2.29 (6H, m), 2.13 (1H, m), 2.03 (2H,
m), 0.95 (3H, t, J = 7.2 Hz, minor isomer), 0.93 (3H, t, J = 7.2 Hz, ma-
jor isomer).
1
2
S
12S
OH
PGA1
OH
PGA
2
Figure 6. Stereochemistry of jasmonates and anti-inflammatory prostaglandins.
5
.2.5. Methyl 5-cyano-4,5-didehydrojasmonate (3)
5
.2. Synthesis of jasmonate analogues
A mixture of 1b (5 mg) and 2,3-dichloro-5,6-dicyanobenzoqui-
none (DDQ, 2.0 equiv) in anhydrous toluene was heated under re-
flux for 10 min. The insoluble matter was removed by filtration
through a pipette plugged with cotton. The filtrate was concen-
trated in vacuo to give a residue, which was purified by re-
versed-phase HPLC (YMC ODS-H80) eluting with 70% aqueous
MeOH to yield compound 3 (1.4 mg, 28%); colorless oil; 1H NMR
5
.2.1. Methyl 4,5-didehydrojasmonate (2)
To a stirred solution of methyl jasmonate 1 (50 mg) in DMSO
(
5 mL) were added IBX (o-iodoxybenzoic acid, 2.0 equiv) and triflu-
roacetic acid (0.3 equiv). The mixture was stirred at 80 °C for 48 h,
and after cooling down, the reaction mixture was diluted with sat-
urated NaHCO
The combined organic phase was washed with water and brine.
After being dried over MgSO , the organic layer was concentrated.
The resulting residue was purified by reversed-phase HPLC (YMC
ODS-H80) eluting with 65% aqueous CH CN to yield compound 2
15 mg, 30%); colorless oil; 1H NMR (CDCl
, 400 MHz): d 7.61
1H, dd, J = 6.0, 2.8 Hz, H-4), 6.17 (1H, dd, J = 6.0, 2.4 Hz, H-5),
.46 (1H, m, H-10), 5.24 (1H, m, H-9), 3.70 (3H, s, OCH ), 2.99
1H, m, H-3), 2.57 (1H, dd, J = 15.6, 6.8 Hz, H-2b), 2.53–2.49 (1H,
m, H-8b), 2.45 (1H, dd, J = 15.6, 8.4 Hz, H-2a), 2.29 (1H, m, H-8a),
3
solution and extracted with EtOAc (three times).
(CDCl
3
, 400 MHz): d 8.23 (1H, d, J = 2.4 Hz, H-4), 5.52 (1H, m, H-
), 3.13 (1H, m, H-3), 2.68
10), 5.19 (1H, m, H-9), 3.71 (3H, s, OCH
(1H, dd, J = 16.0, 6.0 Hz, H-2b), 2.54 (2H, m, H-2a and H-8b), 2.38
(1H, m, H-8a), 2.26 (1H, m, H-7), 2.02 (2H, m, H-11), 0.95 (3H, t,
J = 7.6 Hz, H-12); FABMS m/z 248 [M+H] ; HRFABMS m/z
3
4
+
3
+
(
(
5
3
248.1314 [M+H] .
3
5.2.6. Methyl 5-methoxycarbonyl-jasmonate (1c)
(
Methyl jasmonate 1 (20 mg) was added to a stirred suspension
of NaH (2.5 equiv) and a catalytic amount of MeOH in toluene at
room temperature. After stirring for 15 min, dimethyl carbonate
(0.5 mL) was added and the reaction mixture was heated under re-
flux for 3 h. Then the reaction was stirred overnight, acidified with
10% HCl, and extracted with EtOAc (three times). The combined ex-
1
3
2
.06 (3H, m, H-7 and H-11), 0.94 (3H, t, J = 8.0 Hz, H-12);
C
NMR (CDCl
3
, 100 MHz): d 210.2 (C-6), 171.8 (C-1), 165.4 (C-4),
), 50.9 (C-7),
3.2 (C-3), 38.1 (C-2), 27.6 (C-8), 20.5 (C-11), 14.1 (C-12); FABMS
1
34.5 (C-10), 133.7 (C-5), 124.4 (C-9), 51.8 (OCH
3
4
+
+
m/z 223 [M+H] ; HRFABMS m/z 223.1345 [M+H] .
4
tracts were washed with water and brine, dried over MgSO , and
filtered. The solvent was removed under reduced pressure and
the crude product purified by reversed-phase HPLC (YMC ODS-
5
.2.2. Methyl 5-hydroxymethylene-jasmonate (1a)
To a stirred suspension of NaH (3.0 equiv) and a catalytic
amount of MeOH in anhydrous toluene (50 mL) at 0 °C was added
H80) eluting with 70% aqueous CH
(12.5 mg, 50%); colorless oil; H NMR (CDCl , 400 MHz): d 5.45
3
CN to yield compound 1c
1
3

H. T. Dang et al. / Bioorg. Med. Chem. 16 (2008) 10228–10235
10233
(
4
2
1H, m), 5.21 (1H, m), 3.74 (3H, s), 3.70 (3H, s), 3.34 (1H, dd, J = 9.6,
.0 Hz, minor isomer), 3.13 (1H, dd, J = 12.4, 8.4 Hz, major isomer),
.70 (1H, m), 2.59–2.18 (6H, m), 2.13–2.00 (3H, m), 0.94 (3H, t,
5.2.10. 5-Chloro-4,5-didehydrojasmonic acid (8)
Compound 8 (0.8 mg, 16%) was formed concomitantly with
1
compound 7; colorless oil; H NMR (CDCl
d, J = 2.8 Hz, H-4), 5.50 (1H, m, H-10), 5.23 (1H, m, H-9), 2.98
1H, m, H-3), 2.68 (1H, dd, J = 16.0, 6.4 Hz, H-2b), 2.59–2.52 (1H,
3
, 400 MHz) d 7.54 (1H,
J = 7.6 Hz).
(
5
.2.7. Methyl 5-methoxycarbonyl-4,5-didehydrojasmonate (4)
A mixture of 1c (5 mg) and 2,3-dichloro-5,6-dicyanobenzoqui-
m, H-8b), 2.50 (1H, dd, J = 16.0, 7.6 Hz, H-2a), 2.37 (1H, m, H-8a),
2.25 (1H, m, H-7), 2.06 (2H, m, H-11), 0.94 (3H, t, J = 7.6 Hz, H-
12); C NMR (CDCl , 100 MHz): d 201.5 (C-6), 176.6 (C-1), 157.9
3
1
3
none (DDQ, 2.0 equiv) in anhydrous toluene was heated under re-
flux for 5 h. The insoluble matter was removed by filtration
through a pipette plugged with cotton. The filtrate was concen-
trated in vacuo to give a residue, which was purified by re-
(C-4), 135.9 (C-5), 135.3 (C-10), 123.2 (C-9), 50.2 (C-7), 40.1 (C-
3), 37.7 (C-2), 27.8 (C-8), 20.6 (C-11), 14.1 (C-12); FABMS m/z
+
+
243 [M+H] ; HRFABMS m/z 243.0765 [M+H] .
versed-phase HPLC (YMC ODS-H80) eluting with 50% aqueous
1
CH
3
CN to yield compound 4 (1.5 mg, 30%); colorless oil; H NMR
5.2.11. Methyl 5-bromo-4,5-didehydrojasmonate (9)
(
1
3
CDCl
3
, 400 MHz): d 8.31 (1H, d, J = 2.4 Hz, H-4), 5.48 (1H, m, H-
0), 5.22 (1H, m, H-9), 3.82 (3H, s, OCH ), 3.71 (3H, s, OCH ),
.13 (1H, m, H-3), 2.64(1H, dd, J = 16.0, 6.0 Hz, H-2b), 2.58–2.51
To a mixture of compound 2a (5 mg) and LiBr (3 equiv) in a few
mL of CH Cl was added silica gel (200 mg/mmol). The suspension
2 2
3
3
was shaken at room temperature for a while and evaporated to
dryness. After allowing to stand at this temperature for 24 h, silica
gel was removed by filtration and the crude product was purified
by reversed-phase HPLC (YMC ODS-H80) eluting with 65% aqueous
(
1H, m, H-8b), 2.49 (1H, dd, J = 16.0, 8.4 Hz, H-2a), 2.35 (1H, m,
H-8a), 2.25 (1H, m, H-7), 2.04 (2H, m, H-11), 0.94 (3H, t,
J = 7.6 Hz, H-12); FABMS m/z 281 [M+H] ; HRFABMS m/z
2
+
+
CN to yield compound 9 (1.0 mg, 16%); yellow oil; 1H NMR
81.1400 [M+H] .
CH
CDCl
10), 5.23 (1H, m, H-9), 3.70 (3H, s, OCH
3
3
(
3
, 400 MHz): d 7.72 (1H, d, J = 2.8 Hz, H-4), 5.45 (1H, m, H-
), 2.99 (1H, m, H-3), 2.60
5
.2.8. 4,5-Didehydrojasmonic acid (5)
To a solution of compound 2 (8 mg, 36
were added phosphate buffer (pH 8.0, 0.9 mL) and 12
l
M) in acetone (0.1 mL)
L (36 U)
porcine liver esterase (Sigma Chemical; Lot No. E2884-1KU;
.34 mL, 16.5 mg protein/mL, and 177 U/mg protein) at ambient
(1H, dd, J = 16.0, 6.0 Hz, H-2b), 2.56–2.51 (1H, m, H-8b), 2.45 (1H,
dd, J = 16.0, 8.4 Hz, H-2a), 2.36 (1H, m, H-8a), 2.22 (1H, m, H-7),
l
1
3
2.03 (2H, m, H-11), 0.94 (3H, t, J = 7.6 Hz, H-12); C NMR (CDCl
100 MHz): d 202.0 (C-6), 171.4 (C-1), 162.6 (C-4), 135.2 (C-10),
125.8 (C-5), 123.6 (C-9), 51.9 (OCH ), 49.8 (C-7), 42.4 (C-3), 37.8
3
,
0
temperature, and the mixture was stirred overnight. Then the solu-
tion was acidified with diluted HCl solution to pH 4, and the mix-
ture was extracted with EtOAc. The organic extracts were dried
3
(C-2), 27.9 (C-8), 20.6 (C-11), 14.1 (C-12); FABMS m/z 301/303
+
[M+H] .
over MgSO
was purified by reversed-phase HPLC (YMC ODS-H80) eluting with
5% aqueous CH CN to yield compound 5 (6 mg, 80%); colorless oil;
, 400 MHz): d 7.63 (1H, dd, J = 6.0, 2.4 Hz, H-4), 6.20
1H, dd, J = 6.0, 2.0 Hz, H-5), 5.45 (1H, m, H-10), 5.23 (1H, m, H-9),
4
, filtered, and evaporated to give a residue, which
5.2.12. Methyl 5-iodo-4,5-didehydrojasmonate (10)
6
3
Compound 2a (5 mg), LiI (3 equiv), and silica gel (200 mg/
1
H NMR (CDCl
3
2 2
mmol) in a few mL of CH Cl were treated as in the method for
(
preparation of compound 9 to afford 10 (1.5 mg, 21%); colorless
1
2
.99 (1H, m, H-3), 2.63 (1H, dd, J = 15.6, 6.8 Hz, H-2b), 2.55–2.50
oil; H NMR (CDCl
3
, 400 MHz): d 7.96 (1H, d, J = 2.4 Hz, H-4),
), 3.03
(
1H, m, H-8b), 2.49 (1H, dd, J = 15.6, 8.4 Hz, H-2a), 2.30 (1H, m,
5.48 (1H, m, H-10), 5.20 (1H, m, H-9), 3.70 (3H, s, OCH
3
H-8a), 2.09 (1H, m, H-7), 2.03 (2H, m, H-11), 0.93 (3H, t,
(1H, m, H-3), 2.58 (1H, dd, J = 16.0, 6.4 Hz, H-2b), 2.54–2.50 (1H,
m, H-8b), 2.45 (1H, dd, J = 16.0, 8.8 Hz, H-2a), 2.34 (1H, m, H-8a),
2.19 (1H, m, H-7), 2.04 (2H, m, H-11), 0.94 (3H, t, J = 7.6 Hz, H-
1
3
J = 7.2 Hz, H-12); C NMR (CDCl
3
, 100 MHz): d 210.3 (C-6), 177.0
(
C-1), 165. 2 (C-4), 134.6 (C-10), 133.9 (C-5), 124.2 (C-9), 50.9 (C-
1
3
7
), 42.9 (C-3), 37.9 (C-2), 27.6 (C-8), 20.5 (C-11), 14.1 (C-12); FAB-
12); C NMR (CDCl
(C-4), 135.1 (C-10), 123.7 (C-9), 102.6 (C-5), 51.9 (OCH
), 45.3 (C-3), 37.8 (C-2), 27.9 (C-8), 20.6 (C-11), 14.1 (C-12); FAB-
3
, 100 MHz): d 204.0 (C-6), 171.4 (C-1), 170.3
+
+
MS m/z 209 [M+H] ; HRFABMS m/z 209.1174 [M+H] .
3
), 48.5 (C-
7
+
+
5
.2.9. Methyl 5-chloro-4,5-didehydrojasmonate (7)
MS m/z 349 [M+H] ; HRFABMS m/z 349.0314 [M+H] .
To a stirred methanolic solution of 2 (20 mg) at À10 °C was added
a solution of 30% hydrogen peroxide (50
l
L) in one portion. To the
5.2.13. Methyl 4,5-didehydro-dihydrojasmonate (11)
above mixture was added dropwise 10% KOH (12
l
L) and kept stir-
Methyl dihydrojasmonate (50 mg), which was obtained by
hydrogenation of 1 under palladium–charcoal (10% Pd–C) catalysis,
IBX (2 equiv), and TFA (0.3 equiv) in DMSO were treated as in the
ring at this temperature for 12 h. The reaction mixture was neutral-
izedwith1 N HClandconcentratedby evaporatingoff methanol. The
residual oil was dissolved in EtOAc, washed with water, dried over
MgSO4, and concentrated to dryness in vacuo to give the epoxide
method for preparation of compound 2 to afford 11 (16 mg, 32%);
1
colorless oil; H NMR (CDCl
3
, 400 MHz): d 7.60 (1H, dd, J = 5.6,
),
2
a (20 mg), which was used for the next reaction without purifica-
tion. A solution of 2a (5 mg) in acetic acid including 1 M HCl and
CHCl was stirred at room temperature for 12 h. The mixture was di-
luted with CH Cl and washed with water three times. After working
2.4 Hz, H-4), 6.14 (1H, dd, J = 5.6, 2.4 Hz, H-5), 3.70 (3H, s, OCH
3
3.00 (1H, m, H-3), 2.56 (1H, dd, J = 16.0, 7.2 Hz, H-2b), 2.44 (1H,
dd, J = 16.0, 8.4 Hz, H-2a), 1.98 (1H, m, H-7), 1.70 (1H, m, H-8b),
1.45 (1H, m, H-8a), 1.29 (6H, m, H-9, H-10, and H-11), 0.85 (3H,
3
2
2
1
3
up according to the standard method, the residue was purified by
using reversed-phase HPLC (YMC ODS-H80) eluting with 65% aque-
t, J = 6.4 Hz, H-12); C NMR (CDCl
3
, 100 MHz): d 211.0 (C-6),
), 51.2 (C-7), 43.9
171.9 (C-1), 165.1 (C-4), 133.7 (C-5), 51.8 (OCH
3
1
ous CH
CDCl
.21 (1H, m, H-9), 3.70 (3H, s, OCH
J = 16.0, 6.4 Hz, H-2b), 2.56–2.51 (1H, m, H-8b), 2.45 (1H, dd, J = 16.0,
.6 Hz, H-2a), 2.35 (1H, m, H-8a), 2.22 (1H, m, H-7), 2.04 (2H, m,
3
CN to yield compound 7 (2.0 mg, 38%); colorless oil; H NMR
(C-3), 38.4 (C-2), 31.9 (C-10), 30.7 (C-8), 26.5 (C-9), 22.4 (C-11),
+
(
5
3
, 400 MHz): d 7.52 (1H, d, J = 2.4 Hz, H-4), 5.48 (1H, m, H-10),
), 2.96 (1H, m, H-3), 2.60 (1H, dd,
14.0 (C-12); FABMS m/z 225 [M+H] ; HRFABMS m/z 225.1500
+
3
[M+H] .
7
5.2.14. 4,5-Didehydro-dihydrojasmonic acid (12)
Compound 11 (8 mg) and porcine liver esterase (12 lL) in a
mixture of acetone (0.1 mL) and phosphate buffer (pH 8.0,
13
H-11), 0.94 (3H, t, J = 7.6 Hz, H-12); C NMR (CDCl
d 201.4 (C-6), 171.4 (C-1), 158.1 (C-4), 135.7 (C-5), 135.2 (C-10),
23.6 (C-9), 52.0 (OCH ), 50.2 (C-7), 40.4 (C-3), 37.9 (C-2), 27.8
C-8), 20.6 (C-11), 14.1 (C-12); FABMS m/z 257 [M+H] ; HRFABMS
3
, 100 MHz):
1
3
0.9 mL) were treated as in the method for preparation of com-
+
1
(
pound 5 to afford 12 (6 mg, 80%); colorless oil; H NMR (CDCl
3
,
+
m/z 257.0937 [M+H] .
400 MHz): d 7.64 (1H, dd, J = 5.2, 2.4 Hz, H-4), 6.18 (1H, dd,

1
0234
H. T. Dang et al. / Bioorg. Med. Chem. 16 (2008) 10228–10235
J = 5.2, 2.4 Hz, H-5), 3.02 (1H, m, H-3), 2.63 (1H, dd, J = 16.0, 6.4 Hz,
H-2b), 2.49 (1H, dd, J = 16.0, 8.8 Hz, H-2a), 2.03 (1H, m, H-7), 1.73
1,2,3,3-tetramethyluronium tetrafluoroborate (TBTU, 1 equiv).
After stirring for 1 h at room temperature, isopropylamine (2
equiv) was added and the reaction mixture was stirred for
12 h. The mixture was washed with water, dried, and concen-
trated to give a residue which was purified by reversed-phase
(
1H, m, H-8b), 1.47 (1H, m, H-8a), 1.30 (6H, m, H-9, H-10, and H-
1
2
1), 0.85 (3H, t, J = 6.8 Hz, H-12); 13C NMR (CDCl
11.1 (C-6), 177.1 (C-1), 165.0 (C-4), 133.9 (C-5), 51.2 (C-7), 43.6
3
, 100 MHz): d
(
1
C-3), 38.2 (C-2), 31.9 (C-10), 30.7 (C-8), 26.5 (C-9), 22.4 (C-11),
HPLC (YMC ODS-H80) eluting with 65% aqueous CH
3
CN to yield
+
1
4.0 (C-12); FABMS m/z 211 [M+H] ; HRFABMS m/z 211.1349
compound 17 (1.5 mg, 63%); colorless oil;
H
NMR (CDCl
3
,
+
[
M+H] .
400 MHz): d 7.65 (1H, dd, J = 5.6, 2.8 Hz, H-4), 6.14 (1H, dd,
J = 5.6, 2.4 Hz, H-5), 5.45 (1H, m, H-10), 5.26 (1H, m, H-9), 4.07
5
.2.15. Methyl 5-chloro-4,5-didehydro-dihydrojasmonate (13)
3 2
(1H, septet, J = 6.4 Hz, –CH(CH ) ), 3.08 (1H, m, H-3), 2.50 (1H,
Compound 11a (5 mg), which was obtained by epoxidation of
1, in acetic acid including 1 M HCl and CHCl were treated as in
3
m, H-8b), 2.35 (1H, dd, J = 14.4, 6.8 Hz, H-2b), 2.29 (1H, m, H-
8a), 2.22 (1H, dd, J = 14.4, 8.4 Hz, H-2a), 2.05 (3H, m, H-7 and
1
the method for preparation of compound 7 to afford 13 (2.1 mg,
0%); colorless oil; 1H NMR (CDCl
, 400 MHz): d 7.53 (1H, d,
J = 2.4 Hz, H-4), 3.71 (3H, s, OCH ), 2.99 (1H, m, H-3), 2.60 (1H,
dd, J = 16.0, 6.0 Hz, H-2b), 2.46 (1H, dd, J = 16.0, 8.8 Hz, H-2a),
.14 (1H, m, H-7), 1.75 (1H, m, H-8b), 1.53 (1H, m, H-8a), 1.29
H-11), 1.14 (6H, d, J = 6.8 Hz, –(CH
H-12); C NMR (CDCl
3
3
)
2
), 0.94 (3H, t, J = 7.2 Hz,
, 100 MHz): d 210.6 (C-6), 169.3 (C-1),
166.4 (C-4), 134.4 (C-10), 133.4 (C-5), 124.5 (C-9), 51.1 (C-7),
43.9 (C-3), 41.5 (C-2), 40.9 (–CH(CH ), 27.8 (C-8), 22.8
(–(CH ), 20.6 (C-11), 14.2 (C-12); FABMS m/z 250 [M+H] ;
13
4
3
3
3 2
)
+
2
3 2
)
1
3
+
(
6H, m, H-9, H-10, and H-11), 0.86 (3H, t, J = 6.8 Hz, H-12);
C
HRFABMS m/z 250.1798 [M+H] .
NMR (CDCl
3
, 100 MHz): d 202.0 (C-6), 171.5 (C-1), 157.9 (C-4),
), 50.4 (C-7), 41.2 (C-3), 38.2 (C-2), 31.8
C-10), 30.7 (C-8), 26.3 (C-9), 22.4 (C-11), 14.0 (C-12); FABMS m/z
1
35.7 (C-5), 52.0 (OCH
3
5.2.20. N-Hydroxyethyl-4,5-didehydrojasmonylamide (18)
Compound 5 (2 mg), TBTU (1 equiv), TEA (2 equiv), and eth-
anolamine (2 equiv) in EtOAc were treated as in the method for
(
2
+
+
59 [M+H] ; HRFABMS m/z 259.1103 [M+H] .
preparation of compound 17 to afford 18 (1.3 mg, 54%); yellow
1
5
.2.16. 5-Chloro-4,5-didehydro-dihydrojasmonic acid (14)
oil; H NMR (CDCl
3
, 400 MHz): d 7.66 (1H, dd, J = 5.6, 2.4 Hz,
Compound 14 (0.7 mg, 14%) was formed concomitantly with
H-4), 6.15 (1H, dd, J = 5.6, 2.0 Hz, H-5), 5.89 (1H, br s, NH),
5.46 (1H, m, H-10), 5.26 (1H, m, H-9), 3.73 (2H, t, J = 5.6 Hz, –
CH CH OH), 3.44 (2H, quint, J = 6.0 Hz, –CH OH), 3.08 (1H,
2 2 2
compound 13; colorless oil; 1H NMR (CDCl
(
, 400 MHz): d 7.55
1H, d, J = 2.4 Hz, H-4), 2.99 (1H, m, H-3), 2.68 (1H, dd, J = 16.4,
3
6
.0 Hz, H-2b), 2.51 (1H, dd, J = 16.4, 9.6 Hz, H-2a), 2.17 (1H, m,
m, H-3), 2.50 (1H, m, H-8b), 2.46 (1H, dd, J = 14.8, 6.8 Hz,
H-7), 1.76 (1H, m, H-8b), 1.55 (1H, m, H-8a), 1.31 (6H, m, H-9, H-
H-2b), 2.30 (2H, m, H-2a and H-8a), 2.06 (3H, m, H-7 and
0, and H-11), 0.87 (3H, t, J = 6.8 Hz, H-12); 13C NMR (CDCl
,
H-11), 0.94 (3H, t, J = 7.6 Hz, H-12);
13
1
1
5
9
3
3
C NMR (CDCl , 100
00 MHz): d 202.1 (C-6), 176.7 (C-1), 157.7 (C-4), 135.5 (C-5),
0.4 (C-7), 40.9 (C-3), 38.0 (C-2), 31.8 (C-10), 30.7 (C-8), 26.3 (C-
MHz): d 210.6 (C-6), 171.3 (C-1), 166.2 (C-4), 134.5 (C-10),
133.5 (C-5), 124.5 (C-9), 62.2 (–CH OH), 51.1 (C-7), 43.8 (C-3),
42.2(C-2), 40.5 (–CH CH OH), 27.8 (C-8), 20.6 (C-11), 14.2
2
+
), 22.4 (C-11), 14.0 (C-12); FABMS m/z 245 [M+H] ; HRFABMS
2
2
+
+
+
m/z 245.0961 [M+H] .
(C-12); FABMS m/z 252 [M+H] ; HRFABMS m/z 252.1588 [M+H] .
5
.2.17. Methyl 5-bromo-4,5-didehydro-dihydrojasmonate (15)
Compound 11a (5 mg), LiBr (3 equiv), and silica gel (200 mg/
5.2.21. N-Isopropyl-4,5-didehydro-dihydrojasmonylamide (19)
Compound 12 (2 mg), TBTU (1 equiv), TEA (2 equiv), and iso-
propylamine (2 equiv) in EtOAc were treated as in the method
mmol) in a few mL of CH
2 2
Cl were treated as in the method for
preparation of compound 9 to afford 15 (1.1 mg, 18%); yellow
for preparation of compound 17 to afford 19 (1.5 mg, 63%); col-
1
1
oil; H NMR (CDCl
3
, 400 MHz): d 7.73 (1H, d, J = 2.8 Hz, H-4),
), 2.99 (1H, m, H-3), 2.60 (1H, dd, J = 16.0,
.4 Hz, H-2b), 2.46 (1H, dd, J = 16.0, 8.8 Hz, H-2a), 2.14 (1H, m,
orless oil; H NMR (CDCl
3
, 400 MHz): d 7.63 (1H, dd, J = 5.2,
3
6
.72 (3H, s, OCH
3
2.4 Hz, H-4), 6.12 (1H, dd, J = 5.2, 1.6 Hz, H-5), 5.29 (1H, br s,
NH), 4.08 (1H, septet, J = 6.8 Hz, –CH(CH ), 3.11 (1H, m, H-
3 2
)
H-7), 1.76 (1H, m, H-8b), 1.50 (1H, m, H-8a), 1.29 (6H, m, H-9, H-
3), 2.33 (1H, dd, J = 14.0, 6.8 Hz, H-2b), 2.22 (1H, dd, J = 14.0,
8.4 Hz, H-2a), 1.93 (1H, m, H-7), 1.68 (1H, m, H-8b), 1.47 (1H,
m, H-8a), 1.30 (6H, m, H-9, H-10, and H-11), 1.15 (6H, d,
1
1
5
0, and H-11), 0.86 (3H, t, J = 6.8 Hz, H-12); 13C NMR (CDCl
00 MHz): d 202.0 (C-6), 171.4 (C-1), 162.3 (C-4), 125.6 (C-5),
2.0 (OCH ), 50.0 (C-7), 43.2 (C-3), 38.0 (C-2), 31.8 (C-10), 30.9
3
,
13
3
J = 6.8 Hz, –(CH
(CDCl 100 MHz):
133.3 (C-5), 51.4 (C-7), 44.5 (C-3), 41.5 (C-2), 41.2 (–CH(CH
1.9 (C-10), 30.8 (C-8), 26.5 (C-9), 22.8 (–(CH ), 22.4 (C-11),
3
)
2
), 0.85 (3H, t, J = 6.4 Hz, H-12);
211.5 (C-6), 169.3 (C-1), 166.1 (C-4),
),
C NMR
(
[
C-8), 26.3 (C-9), 22.4 (C-11), 14.0 (C-12); FABMS m/z 303/305
M+H] .
3
,
d
+
3 2
)
3
3 2
)
+
5
.2.18. Methyl 5-iodo-4,5-didehydro-dihydrojasmonate (16)
14.0 (C-12); FABMS m/z 252 [M+H] ; HRFABMS m/z 252.1945
[M+H] .
+
Compound 11a (5 mg), LiI (3 equiv), and silica gel (200 mg/
2 2
mmol) in a few mL of CH Cl were treated as in the method for
preparation of compound 9 to afford 16 (2.1 mg, 30%); colorless
5.2.22. N-Hydroxyethyl-4,5-didehydro-dihydrojasmonylamide
(20)
Compound 12 (2 mg), TBTU (1 equiv), TEA (2 equiv), and eth-
anolamine (2 equiv) in EtOAc were treated as in the method for
1
oil; H NMR (CDCl
3
, 400 MHz): d 7.97 (1H, d, J = 2.4 Hz, H-4),
), 3.05 (1H, m, H-3), 2.58 (1H, dd, J = 15.6,
.6 Hz, H-2b), 2.45 (1H, dd, J = 15.6, 8.8 Hz, H-2a), 2.14 (1H, m,
3
5
.71 (3H, s, OCH
3
H-7), 1.73 (1H, m, H-8b), 1.55 (1H, m, H-8a), 1.28 (6H, m, H-9, H-
preparation of compound 17 to afford 20 (1.2 mg, 50%); yellow
0, and H-11), 0.86 (3H, t, J = 6.8 Hz, H-12); 13C NMR (CDCl
00 MHz): d 204.0 (C-6), 171.4 (C-1), 170.0 (C-4), 102.7 (C-5),
2.0 (OCH ), 48.7 (C-7), 46.1 (C-3), 38.0 (C-2), 31.8 (C-10), 30.9
oil; H NMR (CDCl
1
1
1
5
3
,
3
, 400 MHz): d 7.65 (1H, dd, J = 6.0, 2.4 Hz,
H-4), 6.13 (1H, dd, J = 6.0, 2.0 Hz, H-5), 5.94 (1H, br s, NH),
3.73 (2H, t, J = 5.2 Hz, –CH CH OH), 3.58 (2H, quint, J = 6.0 Hz,
–CH OH), 3.11 (1H, m, H-3), 2.44 (1H, dd, J = 14.8, 6.8 Hz, H-
2b), 2.30 (1H, dd, J = 14.8, 8.4 Hz, H-2a), 1.96 (1H, m, H-7),
.70 (1H, m, H-8b), 1.48 (1H, m, H-8a), 1.30 (6H, m, H-9, H-
3
2
2
(
[
C-8), 26.3 (C-9), 22.4 (C-11), 14.0 (C-12); FABMS m/z 351
M+H] ; HRFABMS m/z 351.0450 [M+H] .
2
+
+
1
1
3
5
.2.19. N-Isopropyl-4,5-didehydrojasmonylamide (17)
10, and H-11), 0.85 (3H, t, J = 6.8 Hz, H-12); C NMR (CDCl
100 MHz): d 211.4 (C-6), 171.4 (C-1), 165.9 (C-4), 133.6 (C-5),
62.2 (–CH OH), 51.4 (C-7), 44.4 (C-3), 42.2 (C-2), 40.9
3
,
To a mixture of compound 5 (2 mg) in EtOAc and triethyl-
amine (TEA,
2
equiv), was added 2-(1H-benzotriazole-1yl)-
2

H. T. Dang et al. / Bioorg. Med. Chem. 16 (2008) 10228–10235
10235
(
(
–CH
2
CH
2
OH), 31.9 (C-10), 30.8 (C-8), 26.5 (C-9), 22.5 (C-11), 14.0
6. Mandal, A. K.; Zhang, Z.; Kim, S. J.; Tsai, P. C.; Mukherjee, A. B. J. Immunol. 2005,
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+
+
C-12); FABMS m/z 254 [M+H] ; HRFABMS m/z 254.1763 [M+H] .
7
8
.
.
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1
l
g/mL), and 100
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4
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6
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