Synthesis of indolyl-3-acetonitrile derivatives and their inhibitory effects on nitric oxide and PGE2 productions in LPS-induced RAW 264.7 cells

Article abstract of DOI:10.1016/j.bmcl.2013.02.114

Arvelexin is one of major constituents of Brassica rapa that exerts anti-inflammatory activities. Several indolyl-3-acetonitrile derivatives were synthesized as arvelexin analogs and evaluated for their abilities to inhibit NO and PGE₂ productions in LPS-induced RAW 264.7 cells. Of the indolyl-3-acetonitriles synthesized, compound 2k, which possesses a hydroxyl group at C-7 position of the indole ring and an N-methyl substituent, more potently inhibited NO and PGE₂ productions and was less cytotoxic than arvelexin on macrophage cells.

Full text of DOI:10.1016/j.bmcl.2013.02.114

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2571–2574
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of indolyl-3-acetonitrile derivatives and their inhibitory
effects on nitric oxide and PGE2 productions in LPS-induced RAW
264.7 cells
Tae Hoon Kwon a,b, , Ik Hwan Yoon a,b, , Ji-Sun Shin b, Young Hun Lee a,b, Bong Jin Kwon a,b
,
Kyung-Tae Lee a,b, Yong Sup Lee a,b,
⇑
a Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 130-701, Republic of Korea
b Department of Pharmacy, College of Pharmacy, Kyung Hee University, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Arvelexin is one of major constituents of Brassica rapa that exerts anti-inflammatory activities. Several
indolyl-3-acetonitrile derivatives were synthesized as arvelexin analogs and evaluated for their abilities
to inhibit NO and PGE2 productions in LPS-induced RAW 264.7 cells. Of the indolyl-3-acetonitriles
synthesized, compound 2k, which possesses a hydroxyl group at C-7 position of the indole ring and an
N-methyl substituent, more potently inhibited NO and PGE2 productions and was less cytotoxic than
arvelexin on macrophage cells.
Received 10 November 2012
Revised 18 February 2013
Accepted 28 February 2013
Available online 7 March 2013
Keywords:
Arvelexin
Ó 2013 Elsevier Ltd. All rights reserved.
Indole-3-acetonitrile
Nitric oxide
Prostaglandin E2
Anti-inflammatory
Inflammation is one of the protective immune responses to tis-
sue injury caused by harmful stimuli, such as invading pathogens,
damaged cells or irritants.1 Paradoxically, persistent and excessive
immune response can promote tissue damage, resulting in chronic
inflammation. This chronic inflammation is a part of many human
diseases, including arteriosclerosis,2 inflammatory bowel disease,3
arthritis,4 cancer5 and Alzheimer’s disease.6 In the inflammatory
state, activated immune cells, such as macrophages secrete large
amounts of proinflammatory cytokines, nitric oxide (NO), and
prostaglandin E2 (PGE2). However, high levels of NO and PGE2 in
a chronic inflammation state can result in various pathological
conditions.7,8 Accordingly, control of the production of NO and
PGE2 in macrophages are current research topics for the develop-
ment of new anti-inflammatory agents.
Arvelexin (2-(4-methoxy-1H-indol-3-yl)acetonitrile, 1) is a cru-
cifer phytoalexin, biosynthesized de novo as a defense metabolite
in response to various forms of stress including salt, drought, UV
irradiation, or pathogens.9 Arvelexin has been isolated from the
plants of Brassicaceae family including Thlaspi arvense (stink-
weed),10 seeds of Thellungiella halophila 11 and the roots of Brassica
campestris ssp rapa.12 Arvelexin was known to possess antifungal
activity,11 however, studies on other biological activities are scarce.
Furthermore, structure–activity relationship studies were not
investigated intensively by the synthesis of arvelexin analogs.
Recently, we first reported that arvelexin is an anti-inflammatory
constituent of Brassica rapa because it down-regulates inflamma-
tory i-NOS, COX-2, TNF-
a, IL-6, and IL-1b gene expression via
NF-j
B inactivation.13
In the present study, we prepared various indolyl-3-aceto-
nitriles 2a–2n as arvelexin analogs with the aim to study struc-
ture–activity relationships, and thereby provide new leads to
compounds possessing enhanced inflammatory activity and
reduced cytotoxicity. More specifically, we synthesized indolyl-3-
acetonitriles possessing methoxy, benzyloxy, or hydroxyl substitu-
ent (R1) at the benzene ring of indole structure and introduced
methyl or several acyl substituents (R2) at nitrogen. The anti-
inflammatory activities of arvelexin analogs were evaluated by
CN
OMe
CN
4
3
5
6
R1
2
N
N
H 1
R2
7
⇑
1, arvelexin
Corresponding author. Tel.: +82 2 961 0370; fax: +82 2 966 3885.
E-mail address: kyslee@khu.ac.kr (Y.S. Lee).
2a-2n, target compounds
These authors equally contributed to this Letter.
Figure 1. Structures of arvelexin and target compounds.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.114

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T. H. Kwon et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2571–2574
O
H
CN
POCl3, NaOH
DMF
NaBH4, NaCN
R1
R1
R1
N
H
NH2CHO-MeOH (1:1)
N
H
N
H
3a, R1 = 5-OCH3
3b, R1 = 6-OCH3
4a,R1 = 5-OCH3
, R1 = 5-OCH3 54%
2a
,
4b, R1 = 6-OCH3
2b, R1 = 6-OCH3, 31%
, R1 = 7-OCH3
, R1 = 7-OCH3
, R = 4-OBn
3c
3d
4c
4d
2c, R1 = 7-OCH3, 34%
1
1
, R = 4-OBn
, R1 = 4-OBn, 17%
, R1 = 5-OBn, 37%
, R1 = 6-OBn, 82%
2d
2e
3e, R1 = 5-OBn
3f, R1 = 6-OBn
3g, R1 = 7-OBn
4e, R1 = 5-OBn
4f, R1 = 6-OBn
4g, R1 = 7-OBn
2f
2g, R1 = 7-OBn, 62%
Scheme 1. Synthesis of arvelexin analogs 2a–2g.
Treatment of these carboxaldehydes with NaCN in the presence of
NaBH4 in NH2CHO–MeOH afforded indolyl-3-acetonitriles 2a–2g.15
Indolyl-3-acetonitriles 2h–2k, which has a hydroxy substituent
at the benzene ring and a methyl substituent at nitrogen, were pre-
pared from 2d to 2g as shown in Scheme 2. The methylation at
nitrogen with methyl iodide and KOH in DMF followed by removal
of benzyl group in 2d–2g by the catalytic hydrogenation over 10%
Pd–C in MeOH and THF produced 2h–2k.16
To examine further the influence of the substituents at nitro-
gen on the anti-inflammatory activity, N-acyl or ester contain-
ing indolyl-3-acetonitriles 2l–2n were also synthesized
(Scheme 3). Treatment of (2-(4-benzyloxy-1H-indol-3-yl)acetoni-
trile 2f) with acetic anhydride, methyl chloroformate, or methyl
bromoacetate, followed by debenzylation with 5% Pd–C afforded
2l–2n. In case of using 10% Pd–C at the debenzylation step, the
isolation yields of products were very low probably due to side
reactions such as hydrogenation at the double bond of the in-
dole ring.
CN
CN
1. MeI, KOH, DMF
BnO
HO
2. 10% Pd/C, H2(g)
N
H
N
CH3
MeOH-THF(1:1)
2h
2i
2d
, 4-OH, 24%
, 4-OBn
2e
, 5-OH, 28%
, 5-OBn
2j
, 6-OH, 64%
2f, 6-OBn
2g, 7-OBn
2k
, 7-OH, 61%
Scheme 2. Synthesis of arvelexin analogs 2h–2k.
measuring the inhibitions of the productions of NO and PGE2 in
LPS-induced RAW 264.7 cells (Fig. 1).
Indolyl-3-acetonitrile derivatives 2a–2g, which has a methoxy
or benzyloxy substituent at the benzene ring, were synthesized
as shown in Scheme 1 using a modification of previously reported
procedure. Commercially available indole derivatives 3a–3g, which
already possess a methoxy or benzyloxy substituent at the benzene
ring, were used as starting materials. Compounds 3a–3g were con-
verted into indole-3-carboxaldehydes 4a–4g by a Vilsmeier–Haack
reaction with phosphorus oxychloride and dimethyl formamide.14
Inflammation is a protective immune response against tissue
damage induced by external stimulants in the body.2 However,
the inflammatory response can also result in considerable damage
to the host, because microbial components like lipopolysaccharide
CN
CN
b
N
N
BnO
HO
20%
CH3
CH3
O
O
a
5
6
2l
80%
CN
CN
CN
c
b
N
N
BnO
32%
HO
67%
OCH3
OCH3
N
H
BnO
O
O
2f
2m
d
46%
CN
CN
b
N
N
BnO
HO
OCH3
OCH3
71%
O
O
7
2n
Scheme 3. Synthesis of arvelexin analogs 2i–2n. Reagents and conditions: (a) Ac2O, NEt3, reflux; (b) 5% Pd/C, H2, MeOH–THF; (c) methyl chloroformate, NaH, THF; (d) methyl
bromoacetate, NaH, DMF.

T. H. Kwon et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2571–2574
2573
Table 1
Effects of arvelexin and its analogs 2a–2n on cytotoxicity and productions of NO and PGE2 in LPS-induced RAW 267.4 cells
CN
R1
N
R2
2a~2n
R1
R2
IC50 (lM)
a
Entry
Cytotoxicity
NO production
PGE2 production
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
5-OCH3
6-OCH3
7-OCH3
4-OBn
5-OBn
6-OBn
7-OBn
4-OH
5-OH
6-OH
7-OH
6-OH
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
COCH3
CO2CH3
CH2CO2CH3
99.61 5.01
161.14 5.72
>400
105.04 16.97
82.68 5.79
53.25 8.10
>400
>100
>100
>100
>100
83.57 10.33
>100
32.44 10.8
>25
>25
73.09 16.4
16.01 6.55
57.72 7.76
11.72 3.56
6.22 2.75
41.27 2.73
15.18 3.05
>100
45.61 9.64
19.45 0.14
20.44 3.87
48.74 2.10
57.08 16.69
>100
80.10 17.94
39.76 14.13
82.07 2.79
>100
>400
325.15 5.50
371.68 10.96
>400
>400
339.08 23.76
>400
6-OH
6-OH
>100
1, arvelexin
206.6 2.32
83.60 9.05
13.92 8.52
a
Data are presented as the means SDs of three independent experiments.
(LPS) stimulate macrophages to produce pro-inflammatory cyto-
kines, i-NOS, and COX-2. In particular, the large amounts of NO
and PGE2 secreted by activated immune cells in the inflammatory
state can induce various pathological conditions. Thus, inhibition
of the production of inflammatory mediators is a potential strategy
for the treatment of many acute and chronic inflammatory dis-
eases. Recently, we found that arvelexin inhibited production of
NO and PGE2.13 In the present study, we synthesized several indo-
lyl-3-acetonitriles as arvelexin analogs to determine whether any
compounds had better anti-inflammatory activity and less cyto-
toxicity than arvelexin. The synthesized indolyl-3-acetonitriles
2a–2n were assessed in terms of their ability to inhibit the produc-
tion of inflammatory mediators, NO and PGE2, in LPS-induced RAW
264.7 cells.17–19 Arvelexin (1) was used as a reference for compar-
isons. The cytotoxic effects of the indolyl-3-acetonitriles on RAW
264.7 cells were also evaluated using the MTT assay20 to test
whether the inhibitory effects on the productions of NO and
PGE2 were due to cytotoxic effects.
Indolyl-3-acetonitriles synthesized showed varying cytotoxici-
ties and inhibitory activities on the LPS-induced productions of
NO and PGE2 (Table 1). When the 4-methoxy substituent of the in-
dole ring in arvelexin (1) was moved to 5-, 6-, or 7-position to lead
indolyl-3-acetonitriles 2a–2c, inhibitory activities on LPS-induced
NO and PGE2 productions were decreased. On the other hand, com-
pounds 2d–2g, which possess a benzyloxy substituent instead of
methoxy group, more potently inhibited NO production than arve-
lexin. However, except for compound 2g, these inhibitory effects
appeared to be due in part to their cytotoxic effects. When the hy-
droxy group and methyl group were introduced at indole structure
(2h–2k), both NO and PGE2 production were more inhibited with
reduced cytotoxicity compared to arvelexin. Interestingly, com-
pounds with C-7 substituent (2c, 2g, 2k) had no cytotoxic effect
13.92
arvelexin.
lM), and cytotoxicity (IC50 = >400 lM vs 206.6 lM) than
In conclusion, we synthesized indolyl-3-acetonitriles as arve-
lexin analogs in order to explore the relationship between struc-
tures of indolyl-3-acetonitriles and their anti-inflammatory
activities. Synthesized compounds showed varying cytotoxicities
and inhibitory activities on the LPS-induced productions of
NO and PGE2. Introduction of benzyloxy or hydroxyl group at 7-
position of the indole ring enhanced NO productions and reduced
cytotoxicity on RAW 264.7 cells. Compound 2k exhibited the most
potent and enhanced inhibitory activities on both NO and PGE2
productions in LPS-induced RAW 264.7 cells with reduced cytotox-
icity compared to arvelexin.
Acknowledgments
This work was supported by the Basic Science Research Pro-
gram through the National Research Foundation of Korea (NRF)
funded by MEST (#2012-006431).
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on RAW 264.7 cells at concentrations up to 400 lM, irrespective
of the pattern of the substituents. Substitution of acyl or other ester
groups at nitrogen of 6-hydroxy-indolyl-3-acetonitrile (2l–2n) did
not enhance the inhibitory activities on NO and PGE2 productions.
Of the compounds, 2k exhibited well-balanced and enhanced
activity profiles with respect to the inhibitions of the productions
of NO (IC50 = 39.76 lM vs 83.60 lM) and PGE2 (IC50 = 6.22 lM vs

2574
T. H. Kwon et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2571–2574
15. Yamada, F.; Tamura, M.; Hasegawa, A.; Somei, M. Chem. Pharm. Bull. 2002, 50,
or presence of various concentrations (3.15, 6.25, 12.5, 25, 50 and 100
tested samples for 24 h. Nitrite levels in culture media were determined using
the Griess reaction and presumed to reflect NO levels. Briefly, 100 L of cell
culture medium was mixed with 100 L of Griess reagent [equal volumes of 1%
lM) of
92.
16. Physical and spectral data of selected compounds. Compound 2j: mp 160 °C (dec);
l
IR (KBr, cmÀ1
)
m
3386, 2937, 2364, 1626, 1223; 1H NMR (400 MHz, CD3OD) d
l
7.27 (1H, d, J = 8.5 Hz, H-4), 6.88 (s, 1H, H-2), 6.63 (1H, d, J = 2.0 Hz, H-7), 6.66
(1H, dd, J = 8.5, 2.0 Hz, H-5), 3.76 (3H, s, –NCH3), 3.56 (2H, s, –CH2CN); 13C NMR
(100 MHz, CD3OD) d 155.02, 139.86, 127.16, 121.87, 120.03, 119.78, 113.23,
111.25, 110.74, 104.48, 103.35, 95.97, 32.70; HR-DART-MS m/z 187.0860 (calcd
for C11H11N2O [M+H]+, 187.0871). Compound 2k: mp 140 °C (dec); IR (KBr,
(w/v) sulfanilamide in 5% (v/v) phosphoric acid and 0.1% (w/v)
naphtylethylenediamine–HCl], and incubated at room temperature for
10 min. Absorbance was then measured at 540 nm using a microplate reader
(Perkin Elmer Cetus, Foster City, CA, USA). Fresh culture media were used as
blanks in all experiments. The amount of nitrite in the samples was measured
with the sodium nitrite serial dilution standard curve and nitrite production
was measured.
cmÀ1 3379, 2938, 2367, 1581, 1274; 1H NMR (400 MHz, CD3OD) d 6.89–6.91
) m
(2H, m, H-2,4), 6.75 (1H, t, J = 7.9 Hz, H-5), 6.42 (1H, d, J = 7.9 Hz, H-6), 3.94
(3H, s, –NCH3), 3.74 (2H, s, –CH2CN); 13C NMR (100 MHz, CD3OD) d 144.90,
129.30, 127.96, 126.59, 119.87, 118.57, 109.08, 106.57, 102.85, 34.86, 12.76;
HR-DART-MS m/z 187.0860 (calcd for C11H11N2O [M+H]+, 187.0871).
19. PGE2 assay: RAW 264.7 cells were pretreated with tested samples for 1 h and
then stimulated with LPS (1 lg/mL) for 24 h. Levels of PGE2 in the culture
media was quantified using EIA kits (R&D Systems, Minneapolis, MN, USA)
according to the manufacturer’s instructions.
17. Cell culture and sample treatment: the RAW 264.7 macrophage cell line was
obtained from the Korea Cell Line Bank (Seoul). Cells were grown at 37 °C in
Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal
bovine serum (FBS), penicillin (100 units/mL), and streptomycin sulfate
20. MTT assay for cell viability: RAW 264.7 cells were plated at a density of
105 cells/well in 96-well plates. To determine the appropriate concentration
not toxic to cells, cytotoxicity studies were performed 24 h after treating cells
with various concentrations of tested compounds. Cell viabilities were
determined using colorimetric MTT assays, as described previously.21
21. Kim, J. Y.; Park, S. J.; Yun, K. J.; Cho, Y. W.; Park, H. J.; Lee, K.-T. Eur. J. Pharmacol.
2008, 584, 175.
(100 lg/mL) in a humidified 5% CO2 atmosphere. Cells were incubated with
various concentrations of tested samples and then stimulated with LPS 1
mL for the indicated time.
lg/
18. Nitrite determination: RAW 264.7 cells were plated at 4 Â 105 cells/well in 24
well-plates and then incubated with or without LPS (1 lg/mL) in the absence