Inhibition of cytokine production by hymenialdisine derivatives

Article abstract of DOI:10.1021/jm040013d

We describe herein the synthesis and biological activity of two indoloazepines that are structurally related to the marine sponge metabolite hymenialdisine. The natural product hymenialdisine was found to be a potent inhibitor of interleukin-2 (IC_50<

Full text of DOI:10.1021/jm040013d

3
700
J . Med. Chem. 2004, 47, 3700-3703
In h ibition of Cytok in e P r od u ction by Hym en ia ld isin e Der iva tives
Vasudha Sharma, Theresa A. Lansdell, Guangyi J in, and J etze J . Tepe*
Department of Chemistry, Michigan State University, East Lansing, Michigan 48824
Received J anuary 20, 2004
We describe herein the synthesis and biological activity of two indoloazepines that are
structurally related to the marine sponge metabolite hymenialdisine. The natural product
hymenialdisine was found to be a potent inhibitor of interleukin-2 (IC50 ) 2.4 µM) and tumor
necrosis factor R (IC50 ) 1.4 µM) production. One of the hymenialdisine derived indoloazepines
was found to also inhibit interleukin-2 (IC50 ) 3.5 µM) and tumor necrosis factor R (IC50 ) 8.2
µM) production.
Elevated levels of cytokines such as interleukin-1
(IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), inter-
leukin-8 (IL-8), and tumor necrosis factor R (TNF-R)
have been linked to many disorders including Crohn’s
disease and psoriasis. In addition, these proinflamma-
tory cytokines play an essential role in the pathogenesis
of rheumatoid arthritis and osteoarthritis.1 The inhi-
bition of cytokines, in particular TNF-R, has been
successful in several clinical trials for the treatment of
-3
4
F igu r e 1. Structures of hymenialdisine and its indoloazepine
derivatives 2 and 3.
rheumatoid arthritis. However, variability in responses
to these antiinflammatory drugs is found due to the
complex network of alternative cytokine mediated path-
potential pathway for this kinase inhibitor.13 In addi-
tion, Ireland and co-workers have recently also identi-
fied hymenialdisine as a very potent mitogen-activated
protein kinase kinase-1 inhibitor with low-nanomolar
5
ways. Inhibition of transcription factors that control
the expression of several proinflammatory mediators
such as the nuclear transcription factor NF-κB may
overcome these problems and provide an alternative
strategy in the treatment of these inflammatory disor-
1
4
IC50 values.
6
As part of our program to develop small-molecule
inhibitors of NF-κB mediated gene transcription, we
describe here the synthesis and biological evaluation of
two hymenialdisine-derived indoloazepines: 2 and 3.
Compound 2 was shown to inhibit IL-2 and TNF-R
production, whereas the methylated product 3 was
depleted of any significant activity. The natural product
ders. Because of its critical role in the regulation of
inflammatory responses, NF-κB has become an increas-
ingly significant therapeutic target in diseases such as
asthma, rheumatoid arthritis, multiple sclerosis, and
Alzheimer’s disease.7
The marine sponge metabolite hymenialdisine (1,
Figure 1) was originally isolated from the sponges
Axinella verrucosa and Acantella aurantiaca, and its
1
was found to inhibit IL-2 and TNF-R production as
well, with similar IC50 values.
8
structure was established using X-ray crystallography.
Hymenialdisine was found to inhibit various proinflam-
matory cytokines such as IL-1, IL-6, IL-8, and nitric
oxide in a variety of cell lines.9,10 Investigation of the
promising antiinflammatory properties of hymeniald-
isine revealed that the bromopyrrole alkaloid inhibits
cytokine production through inhibition of the NF-κB
signaling pathway. Gel shift analysis indicated that
hymenialdisine selectively reduced NF-κB nuclear bind-
ing and not the DNA binding of other transcription
factors such as C/EBP, AP-1, and SP1.11 Recently,
Meijer et al. reported that the potent NF-κB inhibitor
hymenialdisine acts as a competitive nanomolar inhibi-
Syn th esis a n d Biolgica l Eva lu a tion
The synthesis of the indol-aldisine or indoloazepine
sketeton was achieved via a slightly modified route as
reported in the synthesis of aldisine.
1
5,16
From the
commercially available 2-indolecarboxylic acid, conden-
sation with the ethyl ester of â-alanine in the presence
of EDCI and DMAP provided the indole 4. Methylation
of the indole nitrogen with MeI and K CO proceeded
2
3
in near-quantitative yields to give the N-methylindole,
which was used for the synthesis of 3. Hydrolysis of
esters 4 and 5 followed by the P2O5/MeSO3H mediated
cyclization provided the key intermediate aldisine de-
rivatives 8 and 9. TiCl4 mediated aldol condensation
with the phenyloxazolone 12 provided the oxazolone
successfully in 55% and 56% yield for 10 and 11,
respectively. Treatment of the oxazolone derivatives 10
and 11 with the S-benzylthiourea under basic conditions
provided the final products 2 and 3 in modest yields
(Scheme 1). Compound 3 was successfully crystallized
1
2
tor of the cyclin-dependent kinases GSK-3â and CK1.
Crystallographic data portrayed the binding of the
kinase inhibitor in the ATP binding pocket of the
kinases. Considering the potential relationship between
NF-κB activation and GSK-3, this might suggest a
*
To whom correspondence should be addressed. Phone: 517-355-
9
715, extension 147. Fax: 517-353-1793. E-mail: tepe@cem.msu.edu.
1
0.1021/jm040013d CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/21/2004

Brief Articles
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3701
Sch em e 1. Synthesis of 2 and 3
Ta ble 1. Inhibition of Cytokine (IL-2 and TNF-R) Production
Measured by EIA for PDTC and 1-3
from methanol, and its crystal structure is illustrated
in Figure 2. Clearly indicated in this crystal structure
IL-2 production
TNF-R production
a
compd
IC50 (µM)
IC50 (µM)
PDTC (control)
hymenialdisine 1
2
3
5.12((0.41)
2.41((0.71)
3.55((0.09)
>10
6.11((0.53)
1.36((0.25)
8.16((0.31)
>10
a
Values are mean of two experiments. Standard deviation is
given in parentheses.
F igu r e 2. X-ray crystal structure of 3‚MeOH.
is the Z double bond at the C(8)-C(9) position of 3 as
found in the natural product 1.
Hymenialdisine and indoloazepines 2 and 3 were
evaluated for their potential antiinflammatory activity
by examining the transcriptional activity of NF-κB in
human J urkat leukemia T-cells and THP-1 cells (human
monocytic leukemia cell line). Exposure of human
J urkat leukemia T-cells to phorbol myristate acetate
(PMA/PHA) and THP-1 cells to lipopolysaccharide (LPS)
activates the NF-κB mediated gene transcription of
several proinflammatory cytokines, including IL-2, IL-
6
, IL-8, and TNF-R. The antioxidant and nonselective
F igu r e 3. IL-2 production measured by EIA with PDTC and
1-3.
NF-κB inhibitor pyrrolidinedithiocarbamate (PDTC) has
been reported to repress activation of NF-κB and was
used as a control in all experiments. The effect of
hymenialdisine and the indoloazepines 2 and 3 on NF-
κB’s transcriptional activity was evaluated by measur-
ing the level of IL-2 and TNF-R production using a
competitive enzyme immunoassay (EIA). The inhibition
of IL-2 was evaluated in J urkat cells after PMA/PHA
activation.17 The inhibition of TNF-R was evaluated in
THP-1 cells after LPS activation. NF-κB mediated
transcription of IL-2 production was examined by
exposing the cells to PDTC or 1-3 30 min prior to PMA
activation. After 24 h, the cell-free supernatants were
collected and subjected to EIA for the quantification of
total IL-2 production. Hymenialdisine exhibited signifi-
cant inhibition of IL-2 production with an IC50 of 2.4
µM (Table 1). Treatment of the J urkat cells with 2 at
concentrations of 0.1-10 µM exhibited also a significant
dose-response inhibition of IL-2 production (Figure 3).
Compound 2 exhibited an inhibition of IL-2 production
with an IC50 of 3.5 µM. Interestingly, blockage of the
N-indole position with a methyl group (3) indicated no
significant inhibition of IL-2 production (Figure 3,
measured to 10 µM). This further supports the signifi-
cance of the pyrrolic N-H moiety in potential hydrogen-
bonding interactions.
We also examined 1-3 for their ability to inhibit
TNF-R production in LPS stimulated THP-1 cells.
Treatment of THP-1 cells with PDTC and 1-3 30 min
prior to LPS activation resulted in a significant inhibi-
tion of TNF-R production after a 3 h incubation period
(Table 1). Hymenialdisine was found to be a potent
inhibitor of TNF-R production with an IC50 of 1.4 µM.
Compound 2 was also found to inhibit TNF-R produc-

3
702 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Brief Articles
Ta ble 2. Inhibition of Cell Growth with 1-3 in CEM
Exp er im en ta l Section
Leukemia T Cells
All commercial reagents were used without further purifica-
tion. All solvents were reagent grade. THF was freshly distilled
from sodium/benzophenone under nitrogen. CH
inhibition of cell growth
a
compd
GI50 (µM)
2 2
Cl was freshly
PDTC (control)
N/Tb
distilled from CaH under nitrogen. Column chromatography
2
hymenialdisine 1
2
3
1.61((0.62)
1.73((0.088)
14.3((2.41)
was carried out on silica gel 60 (230-400 mesh) supplied by
EM Science. Yields refer to chromatographically and spectro-
scopically pure compounds unless otherwise stated. Infrared
spectra were recorded on a Nicolet IR/42 spectrometer. Proton
and carbon NMR spectra were recorded on a Varian Gemini-
a
Values shown are average of two experiments. Standard
deviation is given in parentheses. b N/T: not tested.
3
00 spectrometer or a Varian VXR-500 spectrometer. High-
resolution mass spectra and FAB mass spectra were obtained
at the Mass Spectrometry Laboratory of the Michigan State
University with a J EOL AX-505H and a J EOL HX-110 double-
focusing mass spectrometer (J EOL USA, Peabody, MA),
respectively.
tion, albeit it was less potent than 1 (IC50 ) 8.2 µM).
The methylated indoloazepine 3 was again depleted of
any significant activity (tested to 10 µM, Table 1).
Inhibition of the DNA binding of NF-κB by 1-3 was
evaluated by examining the nuclear extracts of PMA/
PHA activated J urkat cells using gel electrophoresis
3
-[(1H-In d ole-2-ca r bon yl)a m in o]p r op ion ic Acid Eth yl
Ester , 4. To a mixture of 2-indolecarboxylic acid (5.01 g, 31.11
mmol) and 4-(dimethylamino)pyridine (6.44 g, 52.72 mmol) in
(EMSA). Nuclear extracts were prepared from J urkat
1
2 2
50 mL of anhydrous CH Cl was added N-(3-dimethylami-
cells that were treated with various concentrations of
PDTC and 1-3 for 30 min before PMA/PHA stimulation
for an additional 30 min. Compound 2 (5.0 µM) indicated
approximately 49% inhibition of NF-κB-DNA binding,
compared to approximately 46% for hymenialdisine (5.0
µM) (data not shown). Compound 3, however, failed to
show any significant inhibition. Similar to the studies
reported on hymenialdisine, no significant inhibition of
DNA binding was observed when compounds 1-3 were
nopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI‚HCl) (7.13
g, 37.21 mmol) and â-alanine ethyl ester hydrochloride (5.71
g, 37.21 mmol) at 0 °C. The mixture then was stirred at 0 °C
for 4 h and allowed to warm to room temperature for 20 h.
The solution was washed with water (50 mL) and 10% HCl
(50 mL). The reaction mixture was then dried over anhydrous
Na SO , filtered, and concentrated to yield 4 as a white solid
2
4
(7.95 g, 98%). The solid was recrystallized in methanol.
3-[(1-Met h yl-1H -in d ole-2-ca r b on yl)a m in o]p r op ion ic
Acid E t h yl E st er , 5. A solution of 4 (2.5 g, 9.57 mmol),
CO (5.94 g, 43.06 mmol), and iodomethane (1.49 mL, 23.92
K
2
3
tested for inhibition of DNA binding with the transcrip-
tion factor AP-1 (data not shown).11
mmol) in acetonitrile (75 mL) was stirred at reflux for 24 h.
The mixture was cooled and filtered, and the filtrate was
evaporated in vacuo. The solid was then washed with 1 N HCl
Hymenialdisine, 2, and 3 were tested for their cyto-
toxicity and were found to exhibit significant inhibition
of cell growth (Table 2). Cells were treated at different
concentrations from 10 nM to 5 µM over a 48 h time
period. Hymenialdisine and 2 indicated similar inhibi-
tion of cell growth with a GI50 of 1.61 and 1.73 µM,
respectively, whereas 3 indicated weaker inhibition of
cell growth (GI50 ) 14.3 µM). The inhibition of cell
growth with 1-3 could therefore account for some of
the inhibition of IL-2 production seen in the J urkat cells,
where IL-2 production was measured 24 h after PMA
stimulation. However, inhibition of TNF-R production
was measured within 3 h after LPS stimulation and no
significant inhibition of cell growth was apparent at this
time interval.
2 2
and extracted in CH Cl . The crude material was further
purified by column chromatography on silica gel (CH Cl -
2
2
EtOAc 3:1) to yield 5 (2.64 g) as white solid (100%). The
product was recrystallized with dichloromethane/ether (1:1)
3
-[(1H-In d ole-2-ca r bon yl)a m in o]p r op ion ic Acid , 6. A
mixture of 4 (6.1 g, 23.4 mmol) and LiOH‚H O (1.96 g, 46.7
2
mmol) was stirred at room temperature in ethanol (300 mL)
for 20 h, and the ethanol was evaporated to dryness in vacuo.
The residue was dissolved in water (50 mL), and the solution
was acidified to pH 3 with concentrated HCl to form a white
solid. The mixture was allowed to stand at 0 °C for 30 min,
and the product was isolated by filtration. The solid was
washed with water and recrystallized with methanol to afford
6
(5.03 g, 93%).
-[(1-Met h yl-1H -in d ole-2-ca r b on yl)a m in o]p r op ion ic
Acid , 7. The procedure is similar as for 6 to provide 7 from 5
2.16 g, 93%). The product was recrystallized in methanol.
,4-Dih yd r o-2H,10H-a zep in o[3,4-b]in d ole-1,5-d ion e, 8.
Compound 6 (2 g, 8.6 mmol) was added to a clear solution of
(1.33 g, 9.44 mmol) in MeSO H (70 mL) at 60 °C. The
3
(
Con clu sion
3
We have described herein the synthesis of a new
indoloazepine inhibitor of the proinflammatory protein
NF-κB. Inhibition of the transcriptional activity of
NF-κB by indoloazepines 2 and 3 was evaluated by
measuring IL-2 and TNF-R production in J urkat and
THP-1 cells. The free indoloazepine 2 and hymeniald-
isine exhibited potent inhibition of IL-2 and TNF-R
production, whereas the N-methylindoloazepine 3 was
depleted of any significant activity. Hymenialdisine and
indoloazepine 2 were found to inhibit DNA binding by
NF-κB. Gel electrophoresis assays indicated that none
of the compounds had any notable effect on the DNA
binding of the transcription factor AP-1. Therefore, the
indoloazepine 2 provides a new synthetic, readily avail-
able scaffold for further optimization for cytokine inhibi-
tion. The further clinical potential and details on the
mode of action of this new class of indoloazepines is
currently under investigation in our laboratory and will
be reported shortly.
P
2
O
5
3
mixture was heated to 110 °C for 1.5 h and then cooled to room
temperature. The mixture was poured into ice-water and
stirred for 30 min. The buff-colored solid was then filtered and
dissolved in acetone (250 mL) and filtered again, and the
filtrate was concentrated to give a light-brown solid that was
further purified by column chromatography (20% acetone in
CH
1
1
2
Cl
0-Met h yl-3,4-d ih yd r o-2H ,10H -a zep in o[3,4-b]in d ole-
,5-d ion e, 9. The procedure is similar as for 8 to provide 9
2
) on silica gel to yield 8 (1.56 g, 85%).
from 7 (1.3 g, 75%). The product was recrystallized in
methanol.
5-(5-Oxo-2-ph en yloxazol-4-yliden e)-3,4,5,10-tetr ah ydr o-
2H-a zep in o[3,4-b]in d ol-1-on e, 10. A solution of TiCl
mL, 18.68 mmol) in CH Cl (21 mL) was added into THF (65
4
(2.04
2
2
mL) at -10 °C. Compound 8 (1 g, 4.67 mmol) and 2-phenyla-
zlactone (1.50 g, 9.34 mmol) were subsequently added and
allowed to stir at 0 °C for 20 min. Pyridine (2.09 mL, 37.36
mmol) was then added over a 30 min period. The reaction
mixture was stirred at 0 °C for an additional 2 h and then
4
allowed to stir overnight at room temperature. NH Cl (80 mL,

Brief Articles
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3703
saturated solution in water) was added, and the mixture was
stirred for 10 min and subsequently extracted with ethyl
acetate (3×). The organic extracts were combined dried,
filtered, and concentrated, and the residue was purified using
column chromatography on silica gel (EtOAc/hexane 7:3) to
yield 10 (0.91 g, 55%) as a reddish-yellow solid.
(2) Harris, E. D., J r. Rheumatoid arthritis. Pathophysiology and
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(
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(
4) Moreland, L. W. Inhibitors of tumor necrosis factor for rheuma-
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1
0-Meth yl-5-(5-oxo-2-p h en yloxa zol-4-ylid en e)-3,4,5,10-
(
tetr a h yd r o-2H-a zep in o[3,4-b]in d ol-1-on e, 11. The proce-
dure is similar as for 10 to provide 11 from 9 (0.73 g, 56%) as
a yellow solid.
5
-(2-Am in o -5-o x o -1,5-d ih y d r o im id a zo l-4-y lid e n e )-
,4,5,10-tetr a h yd r o-2H-a zep in o[3,4-b]in d ol-1-on e, 2. LiH
16 mg, 2 mmol) was dissolved in ethanol (60 mL), and to this
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3
(
solution S-benzylisothiouronium chloride (405 mg, 2 mmol)
was added. The mixture was stirred until the solution became
clear. Compound 10 (150 mg, 0.4 mmol) was added, and the
mixture was heated at reflux for 48 h. After the mixture cooled,
the solvent was evaporated in vacuo. Ethanol (20 mL) was
added and subsequently evaporated three times. Ethanol (5
mL) was added again, and the mixture was refluxed for 3 h.
The solvent was evaporated in a vacuum, 1 N HClaq (15 mL)
was added, the mixture was extracted with n-butanol (3 × 15
mL), the extracts were washed with brine (3 × 10 mL), dried,
and concentrated, and the product was purified twice by
(
8) Cimino, G.; De Rosa, S.; De Stefano, D.; Mazzarella, L.; Puliti,
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1
982, 23, 767-768.
(
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M.; Tsai, L. H.; Walter, J .; Cleverley, K. E.; Salinas, P. C.; Wu,
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Inhibition of cyclin-dependent kinases, GSK-3beta and CK1 by
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column chromatography on silica gel (CH
yield 2 (35 mg, 28%) as a light-yellow solid. 2‚HCl: Anal.
Cl) H, N. C: calcd, 54.30; found, 54.56.
-(2-Am in o-5-oxo-1,5-d ih yd r oim id a zol-4-ylid en e)-10-
2 2
Cl /MeOH, 4:1) to
15 14 5 2
(C H N O
5
m e t h yl-3,4,5,10-t e t r a h yd r o-2H -a ze p in o[3,4-b]in d ol-1-
on e, 3. The procedure is similar as for 2 to provide 3 from 11.
Product 3 was purified by column chromatography on silica
(
gel, eluting with 85:15 CH
Cl /MeOH to yield 3 as a light-yellow solid (96 mg, 31%). Anal.
) C, H, N. 3‚HCl: Anal. (C16 Cl) C, H, N.
2 2 2
Cl /MeOH followed by 88:12 CH -
2
(C
16
H
15
N
5
O
2
16 5 2
H N O
Ack n ow led gm en t. We thank Professors G. R. Pettit
7
, 51-63.
and L. Meijer for the generous gift of hymenialdisine.
We also thank Dr. Rui Huang for his excellent technical
assistance in X-ray crystallography. We thank the Mass
Spectrometry facility at Michigan State University for
assistance in acquiring FAB mass spectra. The authors
gratefully acknowledge the financial support provided
by the Petroleum Research Fund, administered by the
American chemical Society, and Michigan State Uni-
versity.
(13) Ali, A.; Hoeflich, K. P.; Woodgett, J . R. Glycogen synthase kinase-
3
2
: properties, functions, and regulation. Chem. Rev. 2001, 101,
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(
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1
). J . Med. Chem. 2002, 45, 529-532.
(
15) Annoura, H.; Tatsuoka, T. Total synthesis of hymenialdisine and
debromohymenialdisine: stereospecific construction of the
2
-amino-4-oxo2-imidazolin-5(Z)-disubstituted ylidene ring sys-
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures for ELISA IL-2, ELISA TNF-R, inhibition of cell
growth, H NMR, C NMR, IR, MS, and EA data for 2-11.
This material is available free of charge via the Internet at
http://pubs.acs.org.
tem. Tetrahedron Lett. 1995, 36, 413-416.
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13
4
56-464.
(
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