In vitro stability and in vivo anti-inflammatory efficacy of synthetic jasmonates

Article abstract of DOI:10.1016/j.bmc.2012.04.052

A chlorinated methyl jasmonate analog (J7) was elaborated as an in vitro anti-inflammatory lead. However, its in vitro efficacy profile was not reproduced in a subsequent in vivo evaluation, presumably due to its rapid enzymatic hydrolysis in a biological system. In an attempt to improve the metabolic stability of the lead J7 by replacement of its labile methyl ester with reasonable ester groups, several analogs resistant to enzymatic hydrolysis were synthesized. In vivo evaluation of the stability-improved analogs showed that these compounds displayed higher efficacy than the lead J7, suggesting that these new jasmonate analogs may serve as potential anti-inflammatory leads.

Full text of DOI:10.1016/j.bmc.2012.04.052

Bioorganic & Medicinal Chemistry 20 (2012) 4109–4116
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
In vitro stability and in vivo anti-inflammatory efficacy of synthetic jasmonates
Hung The Dang a, Yoon Mi Lee a, Gyeoung Jin Kang b, Eun Sook Yoo b, Jongki Hong c, Sun Mee Lee d,
Sang Kook Lee e, Yuna Pyee e, Hwa-Jin Chung e, Hyung Ryong Moon a, Hyung Sik Kim a, Jee H. Jung a,
⇑
a College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea
b School of Medicine, Jeju National University, Jeju 690-756, Republic of Korea
c College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea
d College of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea
e College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A chlorinated methyl jasmonate analog (J7) was elaborated as an in vitro anti-inflammatory lead. How-
ever, its in vitro efficacy profile was not reproduced in a subsequent in vivo evaluation, presumably due to
its rapid enzymatic hydrolysis in a biological system. In an attempt to improve the metabolic stability of
the lead J7 by replacement of its labile methyl ester with reasonable ester groups, several analogs resis-
tant to enzymatic hydrolysis were synthesized. In vivo evaluation of the stability-improved analogs
showed that these compounds displayed higher efficacy than the lead J7, suggesting that these new
jasmonate analogs may serve as potential anti-inflammatory leads.
Received 9 April 2012
Revised 27 April 2012
Accepted 27 April 2012
Available online 5 May 2012
Keywords:
Methyl jasmonate analogs
Anti-inflammatory activity
Metabolic stability
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Vehicle
J7
80
70
60
50
40
30
20
10
0
Indomethacin
Diclofenac
In our continuing study on the potential anti-inflammatory
leads derived from the red alga Gracilaria verrucosa, methyl jasmo-
nate (1) was employed as a prostaglandin-like template for struc-
tural modification.1,2 Introduction of an unsaturation to the
cyclopentanone ring of 1 yielded a highly electrophilic enone
functionality (2), and led to a significant enhancement of in vitro
activity.2 Methyl 4,5-didehydrojasmonate (2) exhibited in vitro
anti-inflammatory potency comparable to that of the most potent
**
**
**
*
**
**
natural anti-inflammatory prostaglandin 15d-D
12,14-PGJ2.2 This
**
type of jasmonate analog inhibited the production of pro-inflam-
matory mediators [nitric oxide (NO), interleukin (IL)-1b, IL-6, and
**
**
tumor necrosis factor (TNF)-
activity in LPS-activated murine macrophage (RAW264.7) cells.3
-Halogenation of the enone function of 2 enhanced its in vitro po-
tency even further.2 Subsequently, one of the
-haloenone analogs,
a] through down-regulation of NF-jB
*
**
**
a
0.5
1
2
4
6
a
Time (h)
methyl 5-chloro-4,5-didehydrojasmonate (J7) was subjected to in
vivo trial, employing the carrageenan-induced paw edema assay.
However, the in vitro activity profile of J7 was not reproduced in
the in vivo study. Though J7 displayed still significant activity, it
was not as effective as diclofenac or indomethacin (Fig. 1; in vivo
dosage regimen of J7 was determined as 50 mg/kg, based on the
in vitro potency and cytotoxicity to RAW264.7 cells, and standard
dosage regimen was used for diclofenac and indomethacin).2
Figure 1. Effect of single dose administration of J7, indomethacin, or diclofenac (50,
5 and 25 mg/kg, respectively, intraperitoneal injection) on carrageenan-induced
swelling of the rat hind paw. The dosage regimen of J7 was determined on the basis
of in vitro potency and cytotoxicity to RAW264.7 cells, and standard dosage
regimen was used for diclofenac and indomethacin. Each value represents the
mean S.D. from 8 to 10 rats per group. Asterisks indicate significant differences
from the control group (⁄p <0.05, ⁄⁄p <0.01).
However, in an in vitro assessment using the same dosage regimen
as that used for in vivo evaluation, J7 was more effective than
⇑
Corresponding author. Tel.: +82 51 510 2803; fax: +82 51 513 6754.
E-mail address: jhjung@pusan.ac.kr (J.H. Jung).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.052

4110
H. T. Dang et al. / Bioorg. Med. Chem. 20 (2012) 4109–4116
COX-2
β-actin
140
100
(A)
(B)
IL-6
NO
120
100
80
60
40
20
0
80
60
40
20
0
BL
LPS
J7
J7a
Dic Indo PGA1
BL LPS J7
J7a Dic Indo PGA1
Figure 2. Inhibitory effects of test samples on the mRNA expression of COX-2 and the production of NO and IL-6. (A) RAW264.7 cells (1.5 ꢀ 105 cells/mL) were stimulated
with LPS (1 lg/mL) alone or with test samples (the same dosage regimen as that of in vivo: J7 10 lM; J7a 10 lM; indomethacin 1 lM; diclofenac 5 lM; PGA1 10 lM) for 20 h
(BL, blank). Total RNA was isolated in an RNase-free environment and the COX-2 mRNA expression was determined by RT-PCR. (B) RAW264.7 cells (1.5 ꢀ 105 cells/mL) were
stimulated with LPS (1 lg/mL) alone or with test samples at a concentration of 10 lM. Nitric oxide production was determined using the Griess reagent method. The
production of IL-6 was determined by ELISA. The data represent the mean SD of triplicate experiments.
NO
IL-6
TNF-α
MTT
O
O
O
O
120
100
80
60
40
20
0
120
100
80
O
O
OCH3
OCH3
O
O
OH
OH
2
2a
60
40
20
0
Cl
Cl
J7
J7a
BL
LPS
2
2a
J7
J7a
Figure 3. Inhibitory effects of jasmonate analogs (2 and J7) and their corresponding acids (2a and J7a) on the production of NO, IL-6, and TNF-
lg/mL) alone or with test samples at concentrations of
12.5 lM for 24 h (BL, blank). Nitric oxide production was determined using the Griess reagent method, and the production of pro-inflammatory cytokines (IL-6 and TNF-a)
a
in LPS-activated RAW264.7
cells (data edited from our previous publication2). RAW264.7 cells (1.5 ꢀ 105 cells/mL) were stimulated with LPS (1
was determined by ELISA. Cell viability was determined using the MTT method. The data represent the mean SD of triplicate experiments.
diclofenac, indomethacin, or prostaglandin A1 (PGA1) in inhibiting
COX-2 (diclofenac and indomethacin are typical COX-2 inhibitors,
Fig. 2A). The lead J7 also showed more strong suppression of NO
and IL-6 than diclofenac, indomethacin, and PGA1 at the equal
replacement of the ester bond with an amide bond is not suitable
for this type of compound.2 Therefore, modifications of the meta-
bolically labile methyl ester moiety of compound 2 to linear alkyl
or aryl (3–10), hydroxyalkyl (11–13), methoxyalkyl (14–16), and
branched alkyl (17–22) esters were attempted to modulate their
lipophilicity/hydrophilicity and steric hindrance. The stability-im-
concentration of 10 lM (Fig. 2B).
The discrepancy between the relative in vitro and in vivo effica-
cies of J7 might be due to a quick degradation of J7 to an inactive
free acid form (J7a) catalyzed by esterases in biological systems.
This was supported by the observation that leads 2 and J7 lost their
in vitro potency after hydrolysis to their corresponding acids 2a
and J7a, respectively (Fig. 3). The loss of activity via hydrolysis to
the corresponding acid has been encountered in many bioactive es-
ters,4–7 leading to low efficacy in in vivo trials.8,9 Therefore, bioac-
tive molecules of ester forms are usually subjected to structural
modification to improve their metabolic stability.4–7 One of the
strategies is to replace the metabolically labile ester moiety with
appropriately designed bioisosteres (such as amides, isoxazolones,
isoxazoles, or oxazolones) that would defy enzymatic degradation
while retaining the desired potency of the parent esters.10,11 An
alternative approach is to modify the polarity (lipophilicity/hydro-
philicity) and/or the steric properties (e.g., steric hindrance) of the
ester moiety.12,13 In our previous study, several amide derivatives
of 2 were prepared as bioisoteres. However, this modification led
to complete loss of in vitro activity, suggesting that bioisosteric
proved analogs were then subjected to subsequent a-chlorination
of the enone function to enhance their potency.14 In this article, the
synthesis, structure–activity/stability relationship, and anti-
inflammatory activity of new jasmonate analogs will be presented
and discussed.
2. Results and discussion
The synthesis of jasmonate derivatives is outlined in Scheme 1.
Esters 3–16 were prepared from methyl jasmonate (1) in three
steps. Transformation of compound 1 into methyl 4,5-didehydroj-
asmonate (2) was performed in 1 step, utilizing the palladium(II)
acetate/allyl diethyl phosphate dehydrogenation system.15 Hydro-
lysis of compound 2 by LiOH in aqueous THF yielded the corre-
sponding acid (2a), which was further coupled with various
alcohols in EtOAc using a very efficient coupling agent 2-(1H-ben-
zotriazole-1-yl)-1,2,3,3-tetramethyluronium
tetrafluoroborate
(TBTU)16 to generate esters 3–16. On the other hand, esters 17–22

H. T. Dang et al. / Bioorg. Med. Chem. 20 (2012) 4109–4116
4111
O
O
O
Cl
Cl
(iii)
OH
O
OH
J7a
O
O
11-Cl
(vi)
O
O
O
O
O
(iv)
(ii)
(i)
(v)
O
OR
O
OCH3
O
Cl
O
OCH3
O
OH
2
1
1a
1b
17a-22a
(ii)
(i)
O
O
O
(iii)
(vii)
Cl
O
OH
O
OR
O
OR
O
OR
2a
17-22
3-16
19-Cl: R = tert-butyl
22-Cl: R = cyclohexyl
3: R =
4: R =
5: R =
6: R =
-C2H5
-C3H7
-C4H9
-C5H11
-C2H4OH
11: R =
12: R =
17: R =
R =
-C2H4OC2H4OH
18:
R =
R =
7:
8:
-C2H4OC2H4OC2H4OH
13:
14: R = -C2H4OCH3
-C2H4OC2H4OCH3
R =
19: R =
20: R =
21: R =
22: R =
OCH3
9: R =
10:
OH
R =
15:
R =
-C2H4OC2H4OC2H4OCH3
16: R =
H3CO
Scheme 1. Synthesis of jasmonate analogs. Reagents and conditions: (i) Pd(OAc)2, diethyl allyl phosphate, sodium carbonate, DMF, 80 °C, 48 h; (ii) LiOH, aqueous THF, rt,
12 h; (iii) ROH, 2-(1H-benzotriazole-1-yl)-1,2,3,3-tetramethyluronium tetrafluoroborate (TBTU), TEA, EtOAc, RT, 12 h; (iv) oxalyl chloride, dichloromethane, 40 °C, 1 h; (v)
ROH, TEA, DMAP, dichloromethane, RT, 12 h; (vi) H2O2, 10% KOH, MeOH, ꢁ10 °C, 12 h, and then HCl/acetic acid, CHCl3, RT, 12 h (vii) H2O2, 10% KOH, MeOH, ꢁ10 °C, 12 h, and
then CeCl3, H2O/MeOH (1:3), reflux, 3 h.
were synthesized from methyl jasmonate (1) via four steps. The
reactive acyl chloride (1b) was prepared by treatment of acid (1a)
with oxalyl chloride in dichloromethane. The intermediate esters
17a–22a were consequently obtained by coupling the acyl chloride
(1b) with various alcohols in the presence of triethylamine and 4-
dimethylaminopyridine in dichloromethane. Finally, transforma-
tion of the intermediate esters 17a–22a into their corresponding
target esters 17–22 was performed using the palladium(II) ace-
tate/allyl diethyl phosphate dehydrogenation system as described
in vitro safety (TC50/EC50). The in vitro metabolic stability (ratio
of esters/acids), biological activity (EC50 and TC50), and calculated
logP value were employed as criteria for further lead selection (Ta-
ble 1).
As shown in Table 1, the in vitro metabolic stability was
strongly dependent on both the lipophilicity/hydrophilicity and
the steric hindrance of the ester moiety. Linear alkyl and aryl esters
(3–10) with the most lipophilic properties were highly susceptible
to enzymatic hydrolysis compared to other esters. At 30 min of
incubation with PLE, the peaks of these esters (3–7, 9, and 10) were
mostly replaced by the peaks of their corresponding acids. Among
them only benzyl ester (8) showed better stability than the methyl
ester (2) with a twice higher stability at 30 min (Table 1). These re-
sults suggested that extension of the linear alkyl chain (increment
of logP value) or replacement of the methyl moiety by other
aryl groups is detrimental to their metabolic stability. In contrast,
the metabolic stability of the hydroxy alkyl esters (11–13)
and the methoxy alkyl esters (14–16) was improved. In particular,
the improvement was notable for hydroxy alkyl esters (11–13),
suggesting that the more hydrophilic they are, the more resistant
to enzymatic hydrolysis. Elongation of the ethylene glycol or ethyl-
ene glycol methyl ether moieties led to increased stability
(stability order: 13 > 12 > 11 > 16, 15 > 14) (Table 1). Branched or
cyclic alkyl esters (17–22) were much more stable than linear
alkyl/aryl esters (3–10), even though they exhibited similar
lipophilicities, suggesting that bulkiness around the ester bond
suppresses enzymatic hydrolysis. Among them, the tert-butyl ester
(19) was the most stable. The strong resistance to enzymatic
for the synthesis of compound 2. An
a-chloroenone derivative
(11-Cl) was synthesized by treatment of the acid (J7a), which was
formed by treatment of the epoxide of 2 with HCl in acetic acid,2
with ethylene glycol in EtOAc using TBTU as described for the syn-
thesis of compounds 3–16 from 2a. On the other hand, compounds
19-Cl and 22-Cl were directly synthesized from the esters 19 and
22, respectively, by treating their corresponding epoxides with cer-
ium(III) chloride.17
All synthesized esters (2–22) were evaluated for their in vitro
metabolic stability using porcine liver esterase (PLE). The esters
were subjected to hydrolysis by incubating with PLE under physi-
ological conditions (phosphate buffer, pH 7.4, and 37 °C).4–7 At reg-
ular intervals, the reaction mixture was withdrawn and analyzed
by RP-HPLC to quantify the amount of the intact ester and its cor-
responding acid. The in vitro anti-inflammatory activity of the ana-
logs were evaluated by monitoring their inhibitory effects on the
production of NO in LPS-stimulated RAW264.7 cells. In addition
to the EC50 values of NO inhibition, the toxicity (TC50) of these es-
ters to the RAW264.7 cells was also measured to estimate their

4112
H. T. Dang et al. / Bioorg. Med. Chem. 20 (2012) 4109–4116
Table 1
In vitro metabolic stability, biological activity, and the structure–activity/stability relationships of jasmonate derivativesa
O
Stability
60 min
30 min
R=
LogP
Bulkiness
EC50
TC50
TC50/EC50
O
OR
2
–CH3
2.0 0.33
None
ꢂ
0.1 0.06
0
19
20
23
23
25
140
146
35
31
37
7
7
1
1
1
3
4
5
6
–C2H5
–C3H7
–C4H9
–C5H10
2.53 0.33
3.06 0.33
3.59 0.33
4.12 0.33
"
0
0
0
0
ND
ND
ND
ND
;
;
;
;
ꢂ
ꢂ
ꢂ
ꢂ
""
""
"""
ꢂ
ꢂ
ꢂ
7
8
3.37 0.32
3.75 0.34
""
""
ꢂ
ꢂ
0
ND
0
;
33
20
;
158
31
5
1
0.2 0.04
ꢂ
ꢂ
9
2.55 0.34
3.20 0.34
"
ꢂ
"
0
0
ND
ND
;
;
23
32
ꢂ
49
2
5
OH
10
""
;
161
H3CO
11
12
1.17 0.45
1.02 0.48
;
ꢂ
ꢂ
1.7 0.15
5.9 0.78
0.7
3.1
""
24
36
ꢂ
161
166
7
5
OH
OH
OH
;;
"""
;
O
13
14
15
16
0.66 0.54
1.74 0.45
1.38 0.52
1.02 0.58
;;;
ꢃ
;
ꢂ
ꢂ
ꢂ
ꢂ
7.6 0.65
0.1 0.04
0.3 0.08
0.3 0.07
3.5
0
"""
ꢂ
"
42
27
33
35
;;
;
162
160
161
163
4
6
5
5
(
(
)
2
O
OCH3
OCH3
0
;
O
OCH3
;
0
"
;
)
O
2
17
18
19
2.87 0.33
3.17 0.34
3.22 0.34
"
"
0.5 0.1
0.1
0.1
9.8
"
20
22
20
ꢂ
ꢂ
ꢂ
58
53
33
3
2
1
""
""
"
0.4 0.09
11.2 1.2
"
""
""""
20
21
22
2.08 0.33
4.1 0.34
4.06 0.33
ꢃ
"
"
"
0.5 0.15
1.4 0.25
4.1 0.46
0.2
0.4
1.1
"
23
18
22
ꢂ
ꢂ
ꢂ
161
31
7
2
2
O
"""
"""
""
"""
31
a
In vitro metabolic stability was examined by the esterase-catalyzed hydrolysis assay. Data are the ratio of intact esters and their corresponding acids at 30 and 60 min
expressed as the mean SD of two separate experiments; ND: not determined. In vitro anti-inflammatory activity was gauged by inhibitory activity on NO production in LPS-
activated RAW264.7 cells. EC50 and TC50 are expressed as M. LogP values were calculated using ACD/ChemSketch 12.0 software. Definition of ‘bulkiness’ is found in Ref. 21.
l
Comparison of logP, bulkiness, stability, and activity between 2 and other esters is shown by symbols: " (increased), ; (decreased), ꢂ (comparable).
hydrolysis of the tert-butyl ester 19 might be due to its inability to
enter the hydrolytic enzyme pocket caused by steric hindrance.12,13
The cyclohexyl ester (22) also displayed highly improved stability
compared to the branched alkyl esters 17 and 18.
Table 2
In vitro anti-inflammatory activity of
a
-chlorinated jasmonate derivativesa
Esters
logP
EC50
TC50
TC50/EC50
In terms of biological activity, all the lipophilic esters including
linear and branched alkyl or aryl analogs (3–10 and 17–22) with
J7
2.40 0.33
1.57 0.45
3.62 0.34
4.45 0.33
0.77
0.58
0.83
0.65
11.32
6.25
11.52
9.69
14.7
10.8
13.9
14.9
11-Cl
19-Cl
22-Cl
logP >2.0, were almost equipotent to the methyl ester 2 (EC50
ꢂ
20 M), suggesting that the methyl ester could be replaced by
l
any ester group without losing biological potency provided that
the logP value stays higher than that of 2 (logP 2.0). However, their
a
In vitro anti-inflammatory activity was gauged by inhibitory activity on NO
production in LPS-activated RAW264.7 cells. Data are expressed in EC50 and TC50
M) as the mean SD of triplicate experiments. The logP values were calculated
using ACD/ChemSketch 12.0 software.
(
l
cytotoxicity was rather variable with the safety index (TC50/EC50
)
ranging from 1 to 7. On the other hand, alkoxy esters, including

H. T. Dang et al. / Bioorg. Med. Chem. 20 (2012) 4109–4116
4113
iNOS, COX-2, and prostaglandins) are involved in two different
inflammatory phases, which include an early (before 6 h) and a
sustained phase of paw edema.19 During the accelerating phase
(1–6 h), COX-2 and the production of inflammatory prostaglandins
play a predominant role, whereas NO production by iNOS is in-
volved in the maintenance of the inflammatory response at the la-
70
60
50
40
30
20
10
0
Control
Indomethacin
J7
11-Cl
**
**
**
**
**
**
19-Cl
*
ter phase.21 Indomethacin is
a typical non-selective COX-2
inhibitor; whereas, jasmonate analogs suppress the production
and gene expression of several pro-inflammatory mediators (NO,
**
**
**
**
**
COX-2, IL-1b, IL-6, and TNF-a
).3 Therefore, it is possible that the
*
**
strong inhibition of edema before 6 h by indomethacin was corre-
lated with its primary inhibition of COX-2. On the other hand, the
higher inhibition rate of the jasmonate analogs at the later phase
might be due to their principal suppression of NO, iNOS, and
cytokines.
**
**
**
0.5
1
2
4
6
24
Time (h)
3. Conclusion
Figure 4. Effect of single dose administration by an intraperitoneal injection of J7
(50 mg/kg), 11-Cl (50 mg/kg), 19-Cl (50 mg/kg), or indomethacin (20 mg/kg) on
carrageenan-induced swelling of the rat hind paw. Each value represents the
mean S.D. (n = 5). Asterisks indicate significant differences from the control group
(⁄p <0.05, ⁄⁄p <0.01).
This study was focused on the optimization of metabolic stabil-
ity of the anti-inflammatory lead J7 to improve its in vivo efficacy.
Replacement of the methyl ester with a sterically bulky or
hydrophilic esters produced stability-improved analogs 11-Cl and
19-Cl. These analogs showed higher in vivo efficacy than J7 in a
carrageenan-induced paw edema model. The anti-inflammatory
effect of 11-Cl was comparable to that of indomethacin, and the
efficacy was sustained. This study offers a reasonable starting point
for the further optimization of jasmonate analogs as potential
anti-inflammatory agents.
hydroxy alkyl esters (11–13) and methoxy alkyl counterparts (14–
16), showed lower potency than 2, but their cytotoxicity was al-
most constant, with a higher safety index than other esters. A clear
structure–activity/stability relationship was found for this type of
alkoxy esters. Elongation of the ethylene glycol or ethylene glycol
methyl moieties improved stability, but with loss of potency.
Among them, the most hydrophilic and stable ester (13) (logP
0.66) was only half as potent as the methyl ester (2) and the
hydroxyethyl ester (11), suggesting that enhancing the metabolic
stability by increasing hydrophilicity is a matter of compromise.
The binding site of metabolizing enzymes is generally lipophilic
in nature. Hence, these enzymes more readily accept and metabo-
lize lipophilic molecules. Lipophilicity also modulate the binding of
a ligand to the biological target. In our study, by introducing rea-
sonably polar (11) or non-polar bulky (19 and 22) groups, the met-
abolic stability of the molecule was improved without losing
in vitro potency. The structure–activity/stability relationship of
the jasmonate analogs is illustrated in Table 1.
4. Experimental section
4.1. General
The 1H and 13C nuclear magnetic resonance (NMR) spectra were
recorded on a Varian Inova 400 spectrometer. Chemical shifts were
reported with reference to the respective residual solvent or deu-
terated solvent peaks (dH 7.24 and dC 77.0 for CDCl3). The FABMS
data were obtained on a JEOL JMS SX-102A spectrometer. HPLC
was performed on a YMC ODS-H80 column (250 ꢀ 10 mm, 4
l
l
m,
m,
80 Å) and a C18-5E Shodex packed column (250 ꢀ 10 mm, 5
Taken together, by replacing the methyl ester of compound 2
with other moieties possessing different lipophilic/hydrophilic
and steric properties, the hydroxy alkyl ester 11, branched alkyl es-
ter 19, and cycloalkyl ester 22 showed improved metabolic stabil-
ity without losing potency. Thereafter, these stable esters (11, 19,
100 Å) using a Shodex RI-71 detector. Quantitative analysis for
metabolic stability was performed using Gilson 321 and 322
pumps/UV-8000 systems with
a
YMC ODS-H80 column
m, 80 Å). All chemical reagents were purchased
(250 ꢀ 4.6 mm, 4
l
from Sigma–Aldrich (St. Louis, MO, USA).
and 22) were transformed to final
Cl, 19-Cl, and 22-Cl, respectively)14 and their inhibitory effects on
the production of NO were evaluated. These -chloroenone ana-
a-chlorinated derivatives (11-
4.2. Synthesis of jasmonate analogs
a
logs exhibited strong in vitro potency, which was comparable to
that of the lead J7 (Table 2). Compounds 11-Cl and 19-Cl, having
a lower and higher logP value than J7, respectively, were selected
for in vivo evaluation using the carrageenan-induced paw edema
model.18 The maximal effect of carrageenan was produced at 4 h
after injection (Fig. 4). The stability-improved analogs 11-Cl and
19-Cl displayed higher in vivo efficacy than the methyl jasmonate
J7, suggesting stability-dependent in vivo efficacy. However, the
most stable ester 19-Cl exhibited lower efficacy than 11-Cl, sug-
gesting that not only metabolic stability but also other pharmaco-
kinetic parameters are important factors for their in vivo efficacy.
When compared to indomethacin (20 mg/kg), these compounds
exhibited lower efficacy at early time points (before 6 h). However,
up to 24 h, the rate of resolution of edema by the test jasmonates
seemed to gradually increase and was comparable to indomethacin
(Fig. 4). Compound 11-Cl (50 mg/kg) displayed higher suppression
of the paw edema (62.4%) than indomethacin (57.1%) at 24 h. Dif-
ferent pro-inflammatory mediators (e.g., histamine, cytokines, NO,
4.2.1. Synthesis of methyl 4,5-didehydrojasmonate (2)
Allyl diethyl phosphate (2.0 equiv), sodium carbonate
(2.4 equiv), and palladium(II) acetate (0.12 equiv) were added to
a stirred solution of methyl jasmonate 1 (1 equiv) in DMF (dimeth-
ylformamide). The mixture was stirred at 80 °C for 48 h, and after
cooling, the reaction mixture was diluted with H2O and extracted
with EtOAc (three times). The combined organic phase was washed
with water and brine. After being dried over MgSO4, the organic
layer was concentrated. The resulting residue was purified by re-
versed-phase HPLC (YMC ODS-H80) eluting with 65% aqueous
CH3CN to yield compound 2; a colorless oil; 1H NMR (CDCl3,
400 MHz): d 7.60 (1H, dd, J = 6.0, 2.8 Hz, H-4), 6.17 (1H, dd,
J = 6.0, 2.4 Hz, H-5), 5.46 (1H, m, H-10), 5.24 (1H, m, H-9), 3.70
(3H, s, OCH3), 2.99 (1H, m, H-3), 2.57 (1H, dd, J = 15.6, 6.8 Hz, H-
2b), 2.51 (1H, m, H-8b), 2.45 (1H, dd, J = 15.6, 8.4 Hz, H-2a), 2.29
(1H, m, H-8a), 2.09–2.01 (3H, m, H-7 and H-11), 0.94 (3H, t,
J = 8.0 Hz, H-12); FABMS m/z 223 [M+H]+.

4114
H. T. Dang et al. / Bioorg. Med. Chem. 20 (2012) 4109–4116
4.2.2. Synthesis of compounds 3–16
4.2.2.6. Benzyl 4,5-didehydrojasmonate (8).
Colorless oil;
Lithium hydroxide (LiOH) (2 equiv) was added to a solution of
compound 2 (1 equiv) in aqueous THF, and the reaction mixture
was stirred at room temperature for 12 h. The reaction mixture
was then poured into water, and this was followed by extraction
with diethyl ether. After removal of diethyl ether, the water layer
was neutralized with 1 N HCl, and the suspension solution was ex-
tracted with EtOAc (three times). The combined organic layer was
washed with brine, dried over Na2SO4 and concentrated to yield
the acid 2a, which was used without purification for the next step.
2-(1H-Benzotriazole-1-yl)-1,2,3,3-tetramethyluronium tetrafluo-
roborate (TBTU, 1 equiv) was added to a solution of compound
2a and triethylamine (TEA, 2 equiv) in EtOAc. After 10 min of stir-
ring at room temperature, alcohols (2 equiv) were separately
added and the reaction mixture was stirred for 12 h. The mixture
was washed with water, dried, and concentrated to give a residue
that was purified by reversed-phase HPLC (YMC ODS-H80) and
eluted with 80% aqueous CH3CN to yield the target esters 3–16
(yields of 65–75%).
1H NMR (CDCl3, 400 MHz): d 7.58 (1H, dd, J = 5.6, 2.4 Hz, H-4),
7.34 (5H, m, –OCH2C6H5), 6.14 (1H, dd, J = 5.6, 2.4 Hz, H-5), 5.43
(1H, m, H-10), 5.26 (1H, m, H-9), 5.13 (2H, s, –OCH2C6H5), 3.01
(1H, m, H-3), 2.60 (1H, dd, J = 16.0, 6.8 Hz, H-2b), 2.51 (2H, m, H-
8b and H-2a), 2.27 (1H, m, H-8a), 2.09–1.98 (3H, m, H-7 and H-
11), 0.93 (3H, t, J = 7.6 Hz, H-12); FABMS m/z 299 [M+H]+.
4.2.2.7. 4-Hydroxyphenyl 4,5-didehydrojasmonate (9).
Col-
orless oil; 1H NMR (CDCl3, 400 MHz): d 7.68 (1H, dd, J = 5.6,
2.4 Hz, H-4), 6.93–6.79 (4H, m, –OC6H4OH), 6.15 (1H, dd, J = 6.0,
2.4 Hz, H-5), 5.45 (1H, m, H-10), 5.25 (1H, m, H-9), 3.01 (1H, m,
H-3), 2.80 (1H, dd, J = 15.6, 6.4 Hz, H-2b), 2.67 (1H, dd, J = 15.6,
8.4 Hz, H-2a), 2.56 (1H, m, H-8b), 2.34 (1H, m, H-8a), 2.18 (1H,
m, H-7), 2.05 (2H, m, H-11), 0.94 (3H, t, J = 7.6 Hz, H-12); FABMS
m/z 301 [M+H]+.
4.2.2.8.
(10).
2-Methoxyphenyl
4,5-didehydrojasmonate
Colorless oil; 1H NMR (CDCl3, 400 MHz): d 7.74 (1H,
dd, J = 6.0, 2.8 Hz, H-4), 7.20–6.95 (4H, m, –OC6H5OCH3), 6.20
(1H, dd, J = 6.0, 2.4 Hz, H-5), 5.47 (1H, m, H-10), 5.29 (1H, m, H-
9), 3.80 (3H, m, –OC6H5OCH3), 3.11 (1H, m, H-3), 2.86 (1H, dd,
J = 15.6, 6.8 Hz, H-2b), 2.69 (1H, dd, J = 15.6, 8.4 Hz, H-2a), 2.58
(1H, m, H-8b), 2.36 (1H, m, H-8a), 2.21 (1H, m, H-7), 2.06 (2H, m,
H-11), 0.93 (3H, t, J = 7.2 Hz, H-12); FABMS m/z 315 [M+H]+.
4.2.2.1. Ethyl 4,5-didehydrojasmonate (3).
Colorless oil; 1H
NMR (CDCl3, 400 MHz): d 7.62 (1H, dd, J = 5.6, 2.4 Hz, H-4), 6.15
(1H, dd, J = 5.6, 2.4 Hz, H-5), 5.46 (1H, m, H-10), 5.24 (1H, m, H-
9), 4.15 (2H, m, –OCH2CH3), 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), 2.06 (3H, m, H-7 and
H-11), 1.25 (3H, t, J = 8.0 Hz, –OCH2CH3), 0.94 (3H, t, J = 7.2 Hz, H-
12); FABMS m/z 237 [M+H]+.
4.2.2.9. 2-Hydroxyethyl 4,5-didehydrojasmonate (11).
Col-
orless oil; 1H NMR (CDCl3, 400 MHz): d 7.62 (1H, dd, J = 5.6,
2.4 Hz, H-4), 6.16 (1H, dd, J = 5.6, 2.4 Hz, H-5), 5.46 (1H, m, H-
10), 5.22 (1H, m, H-9), 4.27 (2H, t, J = 4.4 Hz, –CH2CH2OH), 3.82
(2H, t, J = 4.4 Hz, –CH2CH2OH), 2.99 (1H, m, H-3), 2.62 (1H, dd,
J = 16.0, 6.8 Hz, H-2b), 2.54–2.46 (2H, m, H-2a and H-8b), 2.27
(1H, m, H-8a), 2.10–2.00 (3H, m, H-7 and H-11), 0.94 (3H, t,
J = 7.6 Hz, H-12); FABMS m/z 253 [M+H]+.
4.2.2.2. Propyl 4,5-didehydrojasmonate (4).
Colorless oil; 1H
NMR (CDCl3, 400 MHz): d 7.61 (1H, dd, J = 5.6, 2.4 Hz, H-4), 6.18
(1H, dd, J = 5.6, 2.4 Hz, H-5), 5.45 (1H, m, H-10), 5.22 (1H, m, H-
9), 4.07 (2H, m, –OCH2CH2CH3), 2.97 (1H, m, H-3), 2.57 (1H, dd,
J = 15.6, 6.8 Hz, H-2b), 2.51–2.49 (1H, m, H-8b), 2.47 (1H, dd,
J = 15.6, 8.4 Hz, H-2a), 2.30 (1H, m, H-8a), 2.05 (3H, m, H-7 and
H-11), 1.65 (2H, m, –OCH2CH2CH3), 0.93 (6H, m, –OCH2CH2CH3
and H-12); FABMS m/z 251 [M+H]+.
4.2.2.10. 2-(2-Hydroxyethoxy)ethyl 4,5-didehydrojasmonate
(12).
Colorless oil; 1H NMR (CDCl3, 400 MHz): d 7.62 (1H,
dd, J = 6.0, 2.8 Hz, H-4), 6.16 (1H, dd, J = 6.0, 2.4 Hz, H-5), 5.46
(1H, m, H-10), 5.26 (1H, m, H-9), 4.25 (2H, t, J = 4.4 Hz, –
CH2CH2OCH2CH2OH), 3.72 (2H, t, J = 4.4 Hz, –CH2CH2OCH2CH2OH),
3.68 (2H, t, J = 4.8 Hz, –CH2CH2OCH2CH2OH), 3.57 (2H, t, J = 4.8 Hz,
–CH2CH2OCH2CH2OH), 2.98 (1H, m, H-3), 2.61 (1H, dd, J = 16.0,
6.8 Hz, H-2b), 2.54–2.46 (2H, m, H-2a and H-8b), 2.30 (1H, m, H-
8a), 2.13 (1H, m, H-7), 2.06 (2H, m, H-11), 0.93 (3H, t, J = 7.6 Hz,
H-12); FABMS m/z 297 [M+H]+.
4.2.2.3. Butyl 4,5-didehydrojasmonate (5).
Colorless oil; 1H
NMR (CDCl3, 400 MHz): d 7.60 (1H, dd, J = 5.6, 2.4 Hz, H-4), 6.16
(1H, dd, J = 6.0, 2.4 Hz, H-5), 5.44 (1H, m, H-10), 5.23 (1H, m, H-
9), 4.08 (2H, m, –OCH2CH2CH2CH3), 2.97 (1H, m, H-3), 2.56 (1H,
dd, J = 15.6, 6.4 Hz, H-2b), 2.54–2.50 (1H, m, H-8b), 2.46 (1H, dd,
J = 15.6, 8.4 Hz, H-2a), 2.28 (1H, m, H-8a), 2.08 (3H, m, H-7 and
H-11), 1.60 (2H, m, –OCH2CH2CH2CH3), 1.35 (2H, m,
–
OCH2CH2CH2CH3), 0.93 (6H, m, –OCH2 CH2CH2CH3 and H-12); FAB-
MS m/z 265 [M+H]+.
4.2.2.11. 2-[2-(2-Hydroxyethoxy)ethoxy]ethyl 4,5-didehydroj-
asmonate (13).
Colorless oil; 1H NMR (CDCl3, 400 MHz): d
4.2.2.4. Pentyl 4,5-didehydrojasmonate (6).
Colorless oil; 1H
7.62 (1H, dd, J = 5.6, 2.4 Hz, H-4), 6.16 (1H, dd, J = 5.6, 2.4 Hz, H-
5), 5.45 (1H, m, H-10), 5.22 (1H, m, H-9), 4.25 (2H, t, J = 4.0 Hz, –
CH2CH2OCH2CH2OCH2CH2OH), 3.71–3.57 (10H, m, –CH2CH2OCH2-
CH2OCH2CH2OH), 2.98 (1H, m, H-3), 2.60 (1H, dd, J = 16.0, 6.8 Hz,
H-2b), 2.53–2.44 (2H, m, H-2a and H-8b), 2.30 (1H, m, H-8a),
2.08 (1H, m, H-7), 2.03 (2H, m, H-11), 0.92 (3H, t, J = 8.0 Hz, H-
12); FABMS m/z 341 [M+H]+.
NMR (CDCl3, 400 MHz): d 7.63 (1H, dd, J = 5.6, 2.4 Hz, H-4), 6.15
(1H, dd, J = 5.6, 2.4 Hz, H-5), 5.45 (1H, m, H-10), 5.21 (1H, m, H-
9), 4.08 (2H, m, –OCH2CH2CH2 CH2CH3), 3.01 (1H, m, H-3), 2.56
(1H, dd, J = 15.6, 6.4 Hz, H-2b), 2.55–2.50 (1H, m, H-8b), 2.43 (1H,
dd, J = 15.6, 8.4 Hz, H-2a), 2.31 (1H, m, H-8a), 2.04 (3H, m, H-7
and H-11), 1.60 (2H, m, –OCH2CH2CH2 CH2CH3), 1.30 (4H, m, –
OCH2CH2CH2CH3), 0.92 (6H, m, –OCH2CH2 CH2CH2CH3 and H-12);
FABMS m/z 279 [M+H]+.
4.2.2.12.
(14).
2-Methoxyethyl
4,5-didehydrojasmonate
Colorless oil; 1H NMR (CDCl3, 400 MHz): d 7.62 (1H,
4.2.2.5. Phenyl 4,5-didehydrojasmonate (7).
Colorless oil; 1H
dd, J = 6.0, 2.8 Hz, H-4), 6.16 (1H, dd, J = 6.0, 2.4 Hz, H-5), 5.46
(1H, m, H-10), 5.22 (1H, m, H-9), 4.27 (2H, t, J = 4.4 Hz, –
CH2CH2OCH3), 3.57 (2H, t, J = 4.4 Hz, –CH2CH2OCH3), 3.36 (3H, s,
–CH2CH2OCH3), 2.99 (1H, m, H-3), 2.61 (1H, dd, J = 15.6, 6.4 Hz,
H-2b), 2.54–2.44 (2H, m, H-2a and H-8b), 2.29 (1H, m, H-8a),
2.10–2.00 (3H, m, H-7 and H-11), 0.93 (3H, t, J = 7.2 Hz, H-12); FAB-
MS m/z 267 [M+H]+.
NMR (CDCl3, 400 MHz): d 7.70 (1H, dd, J = 5.6, 2.8 Hz, H-4), 7.37-7.07
(5H, m, –OC6H5), 6.22(1H, dd, J = 5.6, 2.4 Hz, H-5), 5.44(1H, m, H-10),
5.26 (1H, m, H-9), 3.11 (1H, m, H-3), 2.84 (1H, dd, J = 16.0, 6.4 Hz,
H-2b), 2.70 (1H, dd, J = 15.6, 8.0 Hz, H-2a), 2.60–2.53 (1H, m,
H-8b), 2.38–2.31 (1H, m, H-8a), 2.18 (1H, m, H-7), 2.06 (2H, m,
H-11), 0.91 (3H, t, J = 7.6 Hz, H-12); FABMS m/z 285 [M+H]+.

H. T. Dang et al. / Bioorg. Med. Chem. 20 (2012) 4109–4116
4115
4.2.2.13. 2-(2-Methoxyethoxy)ethyl 4,5-didehydrojasmonate
(15).
Colorless oil; 1H NMR (CDCl3, 400 MHz): d 7.62 (1H,
dd, J = 5.6, 2.4 Hz, H-4), 6.16 (1H, dd, J = 6.0, 2.4 Hz, H-5), 5.46
(1H, m, H-10), 5.26 (1H, m, H-9), 4.25 (2H, t, J = 4.4 Hz, –
CH2CH2OCH2CH2OCH3), 3.72 (2H, t, J = 4.4 Hz, –CH2CH2OCH2-
CH2OCH3), 3.68 (2H, t, J = 4.8 Hz, –CH2CH2OCH2CH2OCH3), 3.57
2.99 (1H, m, H-3), 2.63–2.39 (3H, m, H-2 and H-8b), 2.29 (1H, m, H-
8a), 2.10–2.01 (3H, m, H-7 and H-11), 1.22 (3H, d, J = 8.4 Hz, –
CH(CH3)CH2OCH3), 0.94 (3H, t, J = 7.6 Hz, H-12); FABMS m/z 281
[M+H]+.
4.2.3.5. 1-Methylbenzyl 4,5-didehydrojasmonate (21).
Col-
(2H, t, J = 4.8 Hz, –CH2CH2OCH2CH2OCH3), 3.36 (3H, s,
–
orless oil; 1H NMR (CDCl3, 400 MHz): d 7.61 (1H, dd, J = 6.0,
2.8 Hz, H-4), 7.30 (5H, m, –OCH(CH3)C6H5), 6.18 (1H, dd, J = 6.0,
2.4 Hz, H-5), 5.89 (1H, m, –OCH(CH3)C6H5), 5.45 (1H, m, H-10),
5.24 (1H, m, H-9), 2.98 (1H, m, H-3), 2.64–2.39 (3H, m, H-2 and
H-8b), 2.26 (1H, m, H-8a), 2.08–1.97 (3H, m, H-7 and H-11), 1.54
(3H, d, J = 7.4 Hz –OCH(CH3)C6H5), 0.92 (3H, t, J = 7.6 Hz, H-12);
FABMS m/z 313 [M+H]+.
CH2CH2OCH2CH2OCH3), 2.98 (1H, m, H-3), 2.61 (1H, dd, J = 16.0,
6.8 Hz, H-2b), 2.54–2.46 (2H, m, H-2a and H-8b), 2.30 (1H, m, H-
8a), 2.13 (1H, m, H-7), 2.06 (2H, m, H-11), 0.93 (3H, t, J = 7.2 Hz,
H-12); FABMS m/z 311 [M+H]+.
4.2.2.14. 2-[2-(2-Methoxyethoxy)ethoxy]ethyl 4,5-didehydroj-
asmonate (16).
Colorless oil; 1H NMR (CDCl3, 400 MHz): d
7.63 (1H, dd, J = 5.6, 2.8 Hz, H-4), 6.16 (1H, dd, J = 5.6, 2.4 Hz, H-5),
5.45 (1H, m, H-10), 5.24 (1H, m, H-9), 4.24 (2H, t, J = 4.0 Hz, –
CH2CH2OCH2CH2OCH2CH2OCH3), 3.69–3.51 (10H, m, –CH2CH2OCH2
CH2OCH2CH2OCH3), 3.36 (3H, s, –CH2CH2OCH2CH2OCH2CH2OCH3),
2.99 (1H, m, H-3), 2.62 (1H, dd, J = 16.0, 6.8 Hz, H-2b), 2.54–2.44
(2H, m, H-2a and H-8b), 2.30 (1H, m, H-8a), 2.08–2.00 (3H, m, H-7
and H-11), 0.94 (3H, t, J = 8.0 Hz, H-12); FABMS m/z 315 [M+H]+.
4.2.3.6. Cyclohexyl 4,5-didehydrojasmonate (22).
Colorless
oil; 1H NMR (CDCl3, 400 MHz): d 7.61 (1H, dd, J = 6.0, 2.8 Hz, H-
4), 6.16 (1H, dd, J = 6.0, 2.4 Hz, H-5), 5.46 (1H, m, H-10), 5.26
(1H, m, H-9), 4.75 (1H, m, –OCHC5H10), 2.98 (1H, m, H-3), 2.54
(1H, dd, J = 15.2, 6.4 Hz, H-2b), 2.48 (1H, m, H-8b), 2.41 (1H, dd,
J = 15.2, 8.2 Hz, H-2a), 2.30 (1H, m, H-8a), 2.10–2.02 (3H, m, H-7
and H-11), 1.84–1.33 (10H, m, –OCHC5H10), 0.94 (3H, t, J = 7.6 Hz,
H-12); FABMS m/z 291 [M+H]+.
4.2.3. Synthesis of compounds 17–22
Acyl chloride 1b, which was prepared by reaction of the acid 1a
with an excess amount of oxalyl chloride in dichloromethane at
40 °C for 1 h, was treated with different alcohols in the presence
of TEA (1 equiv) and dimethylaminopyridine (0.1 equiv) in dichlo-
romethane at RT for 12 h to yield the intermediate esters 17a–22a.
The esters 17a–22a were consequently transformed into their cor-
responding target esters 17–22, respectively by the same proce-
dure used for the preparation of ester 2 from methyl jasmonate
1. Purification of these target esters was performed using re-
versed-phase HPLC (YMC ODS-H80) and eluting with 80% aqueous
CH3CN (product yields of 50–65%).
4.2.4. Synthesis of
Cl, and 22-Cl
a-chloroenone jasmonate analogs 11-Cl, 19-
The acid, J7a, which was obtained by treatment of the epoxide
of 2 with HCl in acetic acid,2 was coupled with ethylene glycol
(2 equiv) in EtOAc using TBTU (1 equiv) and TEA (2 equiv), as de-
scribed for the synthesis of esters 3–16 from 2a in order to prepare
compound 11-Cl. On the other hand, compounds 19-Cl and 22-Cl
were directly synthesized from the esters 19 and 22, respectively,
by treatment of their corresponding epoxides with CeCl3
(1.2 equiv) in a mixture of H2O/MeOH (1:3) under reflux for 3 h.
After processing according to the standard method, the final prod-
ucts were purified by reversed-phase HPLC (YMC ODS-H80) and
eluting with 80% aqueous CH3CN.
4.2.3.1. Isopropyl 4,5-didehydrojasmonate (17).
Colorless
oil; 1H NMR (CDCl3, 400 MHz): d 7.61 (1H, dd, J = 6.0, 2.4 Hz, H-
4), 6.15 (1H, dd, J = 6.0, 2.4 Hz, H-5), 5.46 (1H, m, H-10), 5.25
(1H, m, H-9), 5.01 (1H, m, –OCH(CH3)2), 2.97 (1H, m, H-3), 2.56
(1H, dd, J = 15.6, 6.4 Hz, H-2b), 2.54–2.47 (1H, m, H-8b), 2.39 (1H,
dd, J = 15.6, 8.4 Hz, H-2a), 2.29 (1H, m, H-8a), 2.09–2.00 (3H, m,
H-7 and H-11), 1.22 (6H, d, J = 8.0 Hz, –OCH(CH3)2), 0.94 (3H, t,
J = 7.6 Hz, H-12); FABMS m/z 251 [M+H]+.
4.2.4.1. 2-Hydroxyethyl 5-chloro-4,5-didehydrojasmonate (11-
Cl).
Colorless oil; 1H NMR (CDCl3, 400 MHz): d 7.53 (1H, d,
J = 2.8 Hz, H-4), 5.48 (1H, m, H-10), 5.22 (1H, m, H-9), 4.23 (2H, t,
J = 4.4 Hz, –CH2CH2OH), 3.81 (2H, t, J = 4.4 Hz, –CH2CH2OH), 2.97
(1H, m, H-3), 2.65 (1H, dd, J = 16.0, 6.0 Hz, H-2b), 2.57–2.46 (2H,
m, H-2a and H-8b), 2.34 (1H, m, H-8a), 2.23 (1H, m, H-7), 2.03
(2H, m, H-11), 0.93 (3H, t, J = 7.2 Hz, H-12); FABMS m/z 287 [M+H]+.
4.2.3.2. sec-Butyl 4,5-didehydrojasmonate (18).
Colorless
oil; 1H NMR (CDCl3, 400 MHz): d 7.61 (1H, dd, J = 6.0, 2.8 Hz, H-
4), 6.15 (1H, dd, J = 6.0, 2.4 Hz, H-5), 5.45 (1H, m, H-10), 5.24
(1H, m, H-9), 4.86 (1H, m, –OCH(CH3)(C2H5)), 2.98 (1H, m, H-3),
2.59–2.26 (4H, m, H-2 and H-8), 2.09–2.01 (3H, m, H-7 and H-
11), 1.56 (2H, m, –OCH(CH3)(CH2CH3)), 1.22 (3H, d, J = 7.8 Hz, –
OCH(CH3)(C2H5)), 0.94 (3H, t, J = 8.0 Hz, H-12), 0.87 (3H, t,
J = 7.6 Hz, –OCH(CH3)(CH2CH3)); FABMS m/z 265 [M+H]+.
4.2.4.2. tert-Butyl 5-chloro-4,5-didehydrojasmonate (19-
Cl).
J = 2.4 Hz, H-4), 5.46 (1H, m, H-10), 5.24 (1H, m, H-9), 2.92 (1H,
m, H-3), 2.55–2.49 (2H, m, H-2b and H-8b), 2.38–2.32 (2H, m, H-
2a and H-8a) 2.23 (1H, m, H-7), 2.04 (2H, m, H-11), 1.43 (9H, s, –
O(CH3)3), 0.93 (3H, t, J = 7.6 Hz, H-12); FABMS m/z 299 [M+H]+.
Colorless oil; 1H NMR (CDCl3, 400 MHz): d 7.51 (1H, d,
4.2.4.3. Cyclohexyl 5-chloro-4,5-didehydrojasmonate (22-
4.2.3.3. tert-Butyl 4,5-didehydrojasmonate (19).
Colorless
Cl).
Colorless oil; 1H NMR (CDCl3, 400 MHz): d 7.53 (1H, d,
oil; 1H NMR (CDCl3, 400 MHz): d 7.61 (1H, dd, J = 5.6, 2.4 Hz, H-
4), 6.15 (1H, dd, J = 6.0, 2.4 Hz, H-5), 5.45 (1H, m, H-10), 5.24
(1H, m, H-9), 2.95 (1H, m, H-3), 2.52–2.46 (2H, m, H-2b and H-
8b), 2.37–2.31 (2H, m, H-2a and H-8a) 2.09–2.01 (3H, m, H-7 and
H-11), 1.44 (9H, s, –O(CH3)3), 0.93 (3H, t, J = 7.2 Hz, H-12); FABMS
m/z 265 [M+H]+.
J = 2.8 Hz, H-4), 5.45 (1H, m, H-10), 5.27 (1H, m, H-9), 4.76 (1H,
m, –OCHC5H10), 2.98 (1H, m, H-3), 2.54 (1H, dd, J = 15.6, 6.8 Hz,
H-2b), 2.48 (1H, m, H-8b), 2.41 (1H, dd, J = 15.6, 8.6 Hz, H-2a),
2.30 (1H, m, H-8a), 2.24 (1H, m, H-7), 2.05 (2H, m, H-11), 1.85–
1.35 (10H, m, –OCHC5H10), 0.94 (3H, t, J = 7.2 Hz, H-12); FABMS
m/z 325 [M+H]+.
4.2.3.4. 1-Methyl-2-methoxyethyl 4,5-didehydrojasmonate
(20).
4.3. Calculation of logP
Colorless oil; 1H NMR (CDCl3, 400 MHz): d 7.63 (1H,
dd, J = 5.6, 2.8 Hz, H-4), 6.15 (1H, dd, J = 6.0, 2.4 Hz, H-5), 5.47
(1H, m, H-10), 5.24 (1H, m, H-9), 5.11 (1H, m, –CH(CH3)CH2OCH3),
3.40 (2H, m, –CH(CH3)CH2OCH3), 3.36 (3H, m, –CH(CH3)CH2OCH3),
The calculation of the octanol–water partition coefficient logP
was performed using ACD/ChemSketch 12.0 software (Advanced
Chemistry Development, Inc., Toronto, ON, Canada)

4116
H. T. Dang et al. / Bioorg. Med. Chem. 20 (2012) 4109–4116
4.4. In vitro stability assessment
The mixture of each ester (ꢃ0.5 mg) and 50
11-Cl, and 19-Cl (50 mg/kg) or indomethacin (25 mg/kg) dissolved
in 5% cremophor and 5% ethanol in PBS were administered by
intraperitoneal injection, and the solvent alone served as a vehicle
control. Thirty minutes after the administration of the test com-
pounds, vehicle, or indomethacin, paw edema was induced by sub-
plantar injection of 0.1 mL of 1% freshly prepared carrageenan
suspension in normal saline into the right hind paw of each rat.
The left hind paw was injected with 0.1 mL of normal saline. The
paw volume was measured before (0 h) and at intervals of 0.5, 1,
2, 4, 6, and 24 h after carrageenan injection using a plethysmome-
ter (Ugo Basile, Comerio, Italy).
l
L of porcine liver
esterase (Sigma Chemical; Lot no. E2884-1KU: 1000 U/0.34 mL;
1 unit (1 U) of the enzyme hydrolyzes 1
0.01 M phosphate buffer solution (pH 7.4, 1 mL) and acetone
(10 L) was incubated at 37 °C. At regular intervals (30 and
60 min), the samples (100 L) were withdrawn and directly in-
jected into the Gilson 321 and 322 pumps/UV-8000 system with
m, 80 Å) for analysis
lmol of an ester) in
l
l
a YMC ODS-H80 column (250 ꢀ 4.6 mm, 4
l
using 70% aq MeOH as the mobile phase, with a flow rate of
0.5 mL/min, and UV detection at 210 nm. The peak areas of intact
esters and their hydrolyzed acids were analyzed using the Gilson
Unipoint ProgramVersion 5.1.
4.7. Statistics
All experiments were repeated at least 3 times. Data are pre-
sented as means SD for the indicated number of independently
performed experiments. The statistical significances within a
parameter were evaluated by one-way and multiple analysis of
variation (ANOVA).
4.5. In vitro anti-inflammatory assay
4.5.1. Cell culture
RAW 264.7 murine macrophage cells were purchased from
ATCC (Rockville, MD) and cultured in Dulbecco’s modified Eagle’s
medium (DMEM), supplemented with 10% (v/v) heat-activated fe-
Acknowledgments
tal bovine serum, streptomycin (100 lg/mL), and penicillin (100 U/
mL) at 37 °C in a 5% CO2 incubator.
This study was financially supported by a Grant from the Na-
tional Research Foundation of Korea (No. 20090083538) and a
Grant from the Marine Biotechnology Program funded by Ministry
of Land, Transport, and Maritime Affairs, Korea.
4.5.2. Cytotoxicity assay
The cytotoxic effects were evaluated using the MTT (3-[4,5-
dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide) assay.
Cells were seeded in 96-well plates and were treated with different
conditions for 24 h. MTT solution was then added to each well, and
the plate was incubated for 4 h. The culture supernatants were re-
moved, and the formazan crystals in each well were dissolved in
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.04.052.
200 lL of dimethyl sulfoxide (DMSO) for 30 min. The absorbances
of the contents of each well were measured at 540 nm using Ver-
saMax ELISA microplate reader (Molecular Devices, Sunnyvale,
CA, USA).
References and notes
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The production of NO was measured, as described previously by
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4.6.1. Animal
Male Sprague–Dawley (SD) rats (140–160 g, 5-weeks old) were
purchased from Central Laboratory Animal Inc. (Seoul, Korea). Ani-
mals were housed under standard laboratory conditions with free
access to food and water. The temperature was thermostatically
regulated to 22 °C 2 °C, and a 12-h light/dark schedule was main-
tained. Prior to their use, they were allowed 1 week for acclimati-
zation within the work area environment. All animal experiments
were carried out in accordance with the Institutional Animal Care
and Use Committee Guidelines of Seoul National University (SNU-
201110-4).
4.6.2. Carrageenan-induced paw edema
The carrageenan-induced hind paw edema model in rats was
used to assess anti-inflammatory activity.21 Test compounds J7,
21. Yesßilada, E.; Küpeli, E. J. Ethnopharmacol. 2002, 79, 237.