Synthesis and biological evaluation of α,β-unsaturated lactones as potent immunosuppressive agents

Article abstract of DOI:10.1016/j.bmcl.2011.08.020

Compounds having α,β-unsaturated lactones display a variety of biological activities. Many research groups have tested both natural and unnatural α,β-unsaturated lactones for as-yet undiscovered biological properties. We synthesized α,β-unsaturated lacton

Full text of DOI:10.1016/j.bmcl.2011.08.020

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5726–5729
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of
immunosuppressive agents
a,b-unsaturated lactones as potent
Sun-Mi Lee a, Won-Gil Lee b, Young-Chul Kim a,b, Hyojin Ko a,
⇑
a Graduate Program of Medical System Engineering, Gwangju Institute of Science and Technology, 261 Cheomdan-gwagiro, Buk-gu, Gwangju 500-712, Republic of Korea
b Department of Life Science, Gwangju Institute of Science and Technology, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Compounds having
groups have tested both natural and unnatural
ical properties. We synthesized
a
,b-unsaturated lactones display a variety of biological activities. Many research
,b-unsaturated lactones for as-yet undiscovered biolog-
,b-unsaturated lactones with various substituents at the d-position and
studied their immunosuppressive effects, that is, the inhibition of Interleukin-2 (IL-2) production. Among
the compounds synthesized, the benzofuran-substituted ,b-unsaturated lactone 4h showed the best
inhibitory activity toward IL-2 production in Jurkat e6-1 T lymphocytes (IC50 = 66.9 nM) without cytotox-
icity at 10 M. The results indicated that 4h may be useful as a potent immunosuppressive agent, as well
as in IL-2-related studies.
Received 13 June 2011
Revised 24 July 2011
Accepted 3 August 2011
Available online 8 August 2011
a
a
a
Keywords:
l
a,b-Unsaturated lactones
Immunosuppressive effect
Interleukin-2 (IL-2) inhibition
Ó 2011 Elsevier Ltd. All rights reserved.
a
,b-Unsaturated lactone moieties display a variety of biological
and adamantine (Table 1). Acrylic acid under basic conditions
introduced an acrylate group to 2 to yield 3. A second-generation
activities, including in the context of HIV1, as protein phosphatase
inhibitors2, as antioxidants3, as antiprotozoals4, as antibacterials5,
and as anticancer agents.6 Goniothalamin7,8, Kazusamycin A9, fost-
Grubb’s catalyst completed the formation of the
a,b-unsaturated
lactone 4 via a ring-closing metathesis17 (Scheme 1).18
riecin10, and kavalactone3 are naturally occurring
lactones that show antibacterial, antifungal, anticancer, and protec-
a,b-unsaturated
We measured the production of IL-2 by enzyme-linked immuno-
sorbent assay (ELISA) to examine the immunosuppressive activity of
tive activity against oxidative stress-induced neuronal cell death.
the
a,b-unsaturated lactones synthesized. Jurkat T lymphocytes
Some previously described
anti-inflammatory and immunosuppressive effects in immune-re-
lated cells.11–13 Because the pharmacological effects of
,b-unsatu-
a
,b-unsaturated lactones have displayed
(1 Â 106 cells/mL) were seeded into 24-well plates. Cells in each well
were treated with 10 lM or 1 lM final concentration of test com-
a
pounds, and 5 nM ionomycin and 500 nM Phorbal 12-myristate
13-acetate (PMA) were added to stimulate the cells. Cells were incu-
bated for 13 h at 37 °C. The cells were centrifuged at 4800 rpm for
2 min, and supernatants from each well were collected. IL-2 was
measured by ELISA using an anti-human IL-2 antibody as the cap-
ture antibody and a biotinylated anti-human IL-2 antibody for
detection (BD, NJ, USA).
rated lactones have not been fully established, many research
groups have explored these properties. Herein, we investigate the
relationship between
a,b-unsaturated lactones and immunosup-
pressive effect by measuring activity toward inhibition of IL-2 pro-
duction.14–16
IL-2 is produced by activated T cells during an immune re-
sponse. IL-2/IL-2 receptor interactions then stimulate the growth,
differentiation, and survival of T cells by inducting of the expres-
sion of specific genes. Thus, IL-2 is a cytokine targeted as a means
by which the T lymphocytes-mediated immune response may be
modulated, including the auto-reactive T cells.
An MTT assay was performed to measure the cytotoxicity of the
compounds (4a–4x) in Jurkat e6-1 cells. These results were used to
select potent immunosuppressive and non-cytotoxic compounds.
The Jurkat cells were seeded into 96-well plates at a density of
1 Â 105 cells/mL/well. Cells were incubated with or without
We began the synthesis of
a
,b-unsaturated lactones by treating
10 lM of each compound for 3 days at 37 °C. After 3 days, 10 lL
allyl magnesium bromide or allyl zinc bromide with various alde-
hydes 1 to obtain an allyl alcohol 2. The R groups included naph-
thalene, quinoline, benzene, substituted benzene (methyl and
methoxy groups at the ortho-, meta-, and para-positions), benzofu-
ran, benzothiophene, furan, thiophene, benzyloxy, styryl, diphenyl,
of an MTT solution were added to each well, and the absorbance
at 450 nm was read. The optical density was directly correlated
with cell concentration.
The structures and biological activities of the synthesized
a,b-
unsaturated lactones are listed in Table 1. All derivatives contained
aromatic or heteroaromatic moieties except for the adamantane-
substituted 4x. Among the compounds containing naphthalene
(4a–4e), compounds 4a, 4d, and 4e showed IL-2 inhibitory activity
⇑
Corresponding author. Tel.: +82 62 715 3238; fax: +82 62 970 2484.
E-mail address: kohyojin@gist.ac.kr (H. Ko).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.020

S.-M. Lee et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5726–5729
5727
Table 1
Percent inhibition of IL-2 production and cell viability in the presence of
a
,b-unsaturated lactones
10
0.00
Compound number
R
l
Ma
1
l
Ma
Cell viabilityb
DMSO
0.00
100.00
100.00
81.06
Cyclosporin A
100.00
4a
4b
4c
4d
97.20
37.53
123.45
100.00
100.00
75.51
72.55
67.72
6.40
59.20
77.73
123.22
4e
4f
100.00
98.69
57.35
41.46
87.52
78.53
4g
4h
100.00
100.00
27.75
140.49
118.73
100.00
4i
100.00
21.66
N/I
77.08
N/I
41.66
114.13
103.97
113.68
116.69
4j
4k
4l
N/I
59.61
46.16
N/I
4m
N/I
4n
4o
4p
82.62
86.11
64.52
89.70
15.19
47.93
127.86
62.29
79.73
4q
4r
80.61
89.18
89.03
10.96
103.27
132.19
(continued on next page)

5728
S.-M. Lee et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5726–5729
Table 1 (continued)
Compound number
R
10
l
Ma
1
l
Ma
Cell viabilityb
c
4s
90.36
N/I
122.39
4t
87.22
98.90
90.13
39.84
27.99
12.72
144.69
118.95
145.15
4u
4v
4w
4x
89.18
18.74
57.39
91.79
100.00
111.91
Table 2
IC50 of IL-2 production inhibition
Compound number
10
l
Ma
1
l
Ma
Cell viabilityb
IC50 (nM)
4c
4h
4i
4n
4q
4x
100.00
100.00
100.00
82.62
80.61
100.00
67.72
77.73
118.73
41.66
127.86
103.27
111.91
395.9
66.9
907.8
165.9
141.5
1574
100.00
77.08
89.70
89.03
57.39
Scheme 1. Synthetic method of
a
,b-unsaturated lactones. Reagents and conditions:
(a) Allyl MgBr or allyl ZnBr, tetrahydrofuran (THF), 0 °C, 1 h then rt 2–12 h; (b)
triethylamine (TEA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),
acrylic acid, dichloromethane (DCM), rt 12 h; (c) Grubb’s catalyst 2nd generation,
DCM, rt 8 h.
a
Percent inhibition of IL-2 production at each concentration comparing with
DMSO, negative control.
b
Cell viability was measured by an MTT assay, higher value means less cyto-
toxicity. All biological evaluation were performed at least three times.
only at 10 lM, and 4b displayed activity both at 10 lM and 1 lM.
However, 4b, 4c, and 4e caused cell death due to cytotoxicity.
When we compared the methyl-substituted naphthalene with
the simple naphthalene, the unsubstituted naphthalene deriva-
tives (4a, 4b) were more potent than the methyl-substituted naph-
thalene derivatives (4d, 4e). Compound 4c, which includes a
The position of the substituent appeared to minimally affect the
activity in this class of compounds. Although the methoxy benzyl
derivatives (4r–4t) displayed weak activity at 1
ity and good activity were observed at 10
(benzyloxy moiety), 4v (styryl moiety), 4w (diphenyl moiety),
and 4x (adamantane moiety) were potent at 10 M but inactive
at 1 M without cytotoxicity.
l
M, low cytotoxic-
lM. Compounds 4u
methylene group between the
naphthalene, showed around 70% inhibitory effects over the range
1–10 M. This result may be derived from the cytotoxicity of 4c.
a,b-unsaturated lactones and the
l
l
l
The compounds containing quinoline (4f, 4g) yielded almost the
same inhibitory activity toward IL-2 production, but the position
of the substituents resulted in large differences in cytotoxicity.
Benzofuran 4h and benzophiophene 4i were very potent and
showed IC50s of 66.9 nM and 907.8 nM for IL-2 production, respec-
tively, as shown in Table 2. Compound 4h showed 100% inhibition
at both concentrations, and the cell viability was better than that
observed in the presence of cyclosporine A under the given condi-
tions. On the other hand, compound 4i showed a high cytotoxicity
with less than 50% cell viability. Furan (4j, 4k) and thiophene (4l,
4m) derivatives displayed neither inhibitory activity nor cytotoxic-
ity. It should be noted that 4m and 4i yielded very different inhib-
itory activities and cytotoxicities because they include a thiophene
moiety at the same position. Among the methyl- and methoxy-
substituted benzyl derivatives (4n–4t), the unsubstituted benzyl
and 4-methyl benzyl-substituted compounds (4n, 4q) were active,
non-cytotoxic, and showed good IC50s (165.9 nM and 141.5 nM).
The IC50s of selected compounds (4c, 4h, 4i, 4n, 4q, and 4x)
were measured, as shown in Table 2. The simple aromatic com-
pounds 4c, 4n, and 4q showed moderate or good IC50s. The IC50
of benzothiophene 4i was not good, even with 100% inhibition at
10 lM and more than 70% inhibition at 1 lM. We expected com-
pound 4x to provide good inhibitory effects because its structure
was relatively wide. Introduction of other nonaromatic substitu-
ents may be productive, but is outside the scope of this study.
As mentioned above, benzofuran-substituted
lactones 4h displayed the best inhibitory activity toward IL-2 pro-
duction (IC50 = 66.9 nM, Fig. 1) without cytotoxicity at 10 M. The
a,b-unsaturated
l
results indicated that 4h may be a potent immunosuppressive
agent, and it could be used in other IL-2 related studies. Although
the other compounds showed less potency toward inhibiting IL-2
production or less cell viability than that observed in the presence
of 4h, some compounds may potentially be developed as potent
immunosuppressive agents.

S.-M. Lee et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5726–5729
5729
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.08.020.
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This study was supported by a grant of the Korea Healthcare
Technology R&D Project, the Ministry for Health and Welfare,
Republic of Korea (A090352), by a National Research Foundation
of Korea (NRF) grant funded by the Korea government (MEST)
(No. 20100017290), and by a grant from the Institute of Medical
System Engineering (iMSE) in the GIST.