Synthesis of new heterocyclic lupeol derivatives as nitric oxide and pro-inflammatory cytokine inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.05.032

A series of heterocyclic derivatives including indoles, pyrazines along with oximes and esters were synthesized from lupeol and evaluated for anti-inflammatory activity through inhibition of lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW 264.7 and J774A.1 cells. All the synthesized molecules of lupeol were found to be more active in inhibiting NO production with an IC₅₀ of 18.4-48.7 μM in both the cell lines when compared to the specific nitric oxide synthase (NOS) inhibitor, L-NAME (IC₅₀ = 69.21 and 73.18 μM on RAW 264.7 and J774A.1 cells, respectively). The halogen substitution at phenyl ring of indole moiety leads to potent inhibition of NO production with half maximal concentration ranging from 18.4 to 41.7 μM. Furthermore, alkyl (11, 12) and p-bromo/iodo (15, 16) substituted compounds at a concentration of 20 μg/mL exhibited mild inhibition (29-42%) of LPS-induced tumor necrosis factor alpha (TNF-α) and weak inhibition (10-22%) towards interleukin 1-beta (IL-1β) production in both the cell lines. All the derivatives were found to be non-cytotoxic when tested at their IC₅₀ (μM). These findings suggest that the derivatives of lupeol could be a lead to potent inhibitors of NO.

Full text of DOI:10.1016/j.bmcl.2014.05.032

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of new heterocyclic lupeol derivatives as nitric oxide
and pro-inflammatory cytokine inhibitors
⇑
Pamita Bhandari, Neeraj Kumar Patel, Kamlesh Kumar Bhutani
Department of Natural Products, National Institute of Pharmaceutical and Education Research (NIPER), Sector 67, S.A.S. Nagar, Mohali 160 062, Punjab, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of heterocyclic derivatives including indoles, pyrazines along with oximes and esters were syn-
thesized from lupeol and evaluated for anti-inflammatory activity through inhibition of lipopolysaccha-
ride (LPS) induced nitric oxide (NO) production in RAW 264.7 and J774A.1 cells. All the synthesized
molecules of lupeol were found to be more active in inhibiting NO production with an IC50 of
Received 5 March 2014
Revised 9 May 2014
Accepted 12 May 2014
Available online xxxx
18.4–48.7
L-NAME (IC50 = 69.21 and 73.18
substitution at phenyl ring of indole moiety leads to potent inhibition of NO production with half
maximal concentration ranging from 18.4 to 41.7 M. Furthermore, alkyl (11, 12) and p-bromo/iodo
(15, 16) substituted compounds at a concentration of 20 g/mL exhibited mild inhibition (29–42%) of
LPS-induced tumor necrosis factor alpha (TNF- ) and weak inhibition (10–22%) towards interleukin
1-beta (IL-1b) production in both the cell lines. All the derivatives were found to be non-cytotoxic when
l
M in both the cell lines when compared to the specific nitric oxide synthase (NOS) inhibitor,
l
M on RAW 264.7 and J774A.1 cells, respectively). The halogen
Keywords:
Lupeol
Heterocyclic derivatives
Anti-inflammatory activity
Nitric oxide
l
l
a
Tumor necrosis factor-alpha
tested at their IC50
inhibitors of NO.
(l
M). These findings suggest that the derivatives of lupeol could be a lead to potent
Ó 2014 Published by Elsevier Ltd.
Injury leads to inflammation which involves the assemblage of
cells and exudates in irritated tissues.1 Chronic inflammatory
diseases such as psoriasis, ulcerative colitis and rheumatoid arthri-
tis usually occurred due to ungoverned production of pro-inflam-
matory cytokines.2 Among the cytokines, nitric oxide (NO) is a
potent pro-inflammatory mediator and at low concentration regu-
lates physiological homeostasis in the cardiovascular system.3
Overproduction of NO by inducible nitric oxide synthase (iNOS)
generally destroys functional normal tissues during acute and
and other plants, these compounds can be an integral part of the
human diet.6 Consequently, much research interest has been
focused on the pharmacological evaluation and efficient isolation
of triterpenoids from plants.7,8 Lupeol had been studied for more
than a century and is reported to exhibit a broad spectrum of phar-
macological activities against some prominent disease such as
inflammation, diabetes, arthritis, cardiovascular ailments, renal
disorders, hepatic toxicity, microbial infections and cancer.9–20 Ear-
lier studies on the synthetic modifications of lupeol resulted in the
preparation of antimalarial, antidiabetic and antihypoglycemaic
analogues.21,22 Accordingly, chemical modifications, of the struc-
ture of lupeol, were required to improve the biological activities
while showing less toxicity. Therefore, in order to find potent
anti-inflammatory derivatives, in present work, considerable struc-
tural modifications have been performed mainly on the A ring. The
Fischer indolization of lupeol, modifications of ring A at C-3 posi-
tion and the isoprenyl unit at C-30 resulted in the preparation of
different derivatives which were evaluated for anti-inflammatory
activity on RAW 264.7 and J774A.1 cells along with their
cytotoxicity.
chronic inflammation.4 Tumor necrosis factor-alpha (TNF-
a),
another pro-inflammatory cytokine plays an important role in
inflammation cascades, and induces itself as well as other
inflammatory cytokines.5
Natural products represent promising scaffolds with high
chemical and structural diversity and wide array of biological
activities. Out of the structurally diverse compounds from tropical
plants, the triterpenoids are often found in significant quantities,
and a wide array isolated and characterized. Lup-20(29)-en-3b-
ol, commonly known as lupeol, a pentacyclic triterpenoid with
30 carbon atoms, biosynthetically derived from the cyclization of
squalene has a vast occurrence in diverse plant families. Since
the triterpenes are widely distributed in food, medicinal herbs
Lupeol was isolated from the bark of Embelica officinalis in
good yield (1.4%). For the isolation of lupeol, the air dried bark
of E. officinalis was ground to fine powder and extracted with
n-hexane and the combined percolations were concentrated to
dryness under reduced pressure. The lupeol was isolated by
⇑
Corresponding author. Tel./fax: +91 172 2232208.
E-mail addresses: kkbhutani@niper.ac.in, kkbhutani@gmail.com (K.K. Bhutani).
http://dx.doi.org/10.1016/j.bmcl.2014.05.032
0960-894X/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Bhandari, P.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.05.032

2
P. Bhandari et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
column chromatography on silica gel with 5% EtOAc/n-hexane
and re-crystallization.
by silica gel chromatography has been applied to provide pyrazine
derivative. To introduce aldehyde group on the allylic position of
lupeol, allylic oxidation with SeO2 under reflux in moist dioxane
followed by silica gel chromatography was carried out to afford
compound 18. In NMR spectrum, a new proton at dH 9.86 appeared
along with olefinic protons.
Nitric oxide (NO) is an important mediator in the inflammatory
process and generated normally at the inflammatory sites by an
inducible nitric oxide synthase (NOS) enzyme. Macrophages alone
itself released low levels of NO and upon stimulation with gram
negative bacteria derived components such as lipopolysaccharide
(LPS), the production of NO raised appreciably up to 24 h. The
NO inhibitory effects of lupeol (1), and its derivatives (3-keto 2,
oximes 3,4, esters 5–7 heterocyclic derivatives 9–17, and aldehyde
18) on RAW 264.7 and J774A.1 cells was judged by using Griess
reaction23 while cell viability was estimated using conventional
MTT assay24 (Table 1). Results revealed that all the derivatives of
lupeol were found to be more active in inhibiting NO production
The synthesized derivatives are divided into six categories: 3-
keto lupeol 2; 3-oxime 3 and 4; 3-ester derivatives 5–7; sulfur
derivative 8; indole derivatives 9–16; pyrazine derivatives 17;
and allylic oxidation (an aldehyde) 18. Scheme 1 describes the syn-
thesis of derivatives 2–18. 3-Keto lupeol (2), a key intermediate,
was prepared by Jone’s oxidation of 1 in 91% yields. Compounds
3 and 4 were prepared by refluxing 2 with hydroxylamines in
the presence of pyridine (anhydrous) under a nitrogen atmosphere.
The synthesis of 5–7 was accomplished by esterification of 1 with
anhydrides in presence of DMAP/dry pyridine (Scheme 1). Com-
pound 8 was prepared by thionation of 2 using Lawesson’s reagent
in toluene. The indole derivative (9) of lupeol was prepared by the
Fischer indolization of 2 with phenylhydrazine hydrochloride in
the presence of acetic acid. 1H and 13C NMR data of 9 suggested
the disappearance of H-2 proton and keto group at C-3 (d 217.2)
of 3-oxolupeol (2). The NMR spectrum data of 9 was closely similar
with lupeol with additional signals at dH 7.72 (NH), and aromatic
proton signals at dH 7.03–7.39. In order to understand the influence
on the activity of a substitution group at phenyl ring, compounds
10–16 were prepared by the same protocol as 9. It is noteworthy
that the electron donating groups substituted to the phenyl ring
enhances the anti-inflammatory activity. The reaction of ketone 2
with ethylenediamine and sulfur in refluxing morpholine followed
with an IC50 of 18.4 to 48.7
pared to the specific inhibitor, L-NAME (IC50 = 69.21 and
73.18 M on RAW 264.7 and J774A.1 cells, respectively). However,
in comparison to curcumin (IC50 = 11.02 and 18.52 M on RAW
264.7 and J774A.1 cells, respectively), only compound 15 showed
significant activity (IC50 = 23.9 and 18.4 M on RAW 264.7 and
lM in both the cell lines when com-
l
l
l
J774A.1 cells, respectively) towards NO inhibition. Compounds
i
ii
R1
HO
1
N
H
O
9
2
R1
R1
R1
R1
R1
R1
R1
=
=
=
=
=
=
=
p-CH3
2,5-CH3
2-CH2CH3
p-F
p-Cl
p-Br
10
11
12
13
14
15
16
iv
iii
v
R
p-I
RO
3
4
R
=
OH
N
5
6
R =
R =
OAc
CO
HO
R
=
N
COOH
CO
S
R =
COOH
8
7
N
N
vi
17
O
2
OHC
vii
HO
HO
1
18
Scheme 1. (i) Jone’s oxidation (ii) phenylhydrazine derivatives, AcOH, reflux, (iii) Hydroxylamine hydrochloride, pyridine (anhydrous), reflux, (iv) Anhydrides, DMAP,
pyridine (anhydrous), reflux, (v) Lawesson’s reagent, toluene, (vi) ethylenediamine, morpholine, sulfur, reflux, (vii) SeO2, dioxane (moist), reflux.
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P. Bhandari et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Table 1
NO inhibitory effects of compounds on RAW 264.7 and J774A.1 cells
Compounds
IC50
RAW 264.7
(l
M) for inhibition of NO production
Cell viabilitya (% of control)
J774A.1
RAW 264.7
J774A.1
bL-NAME
69.21 2.65
11.02 2.40
37.3 0.34
38.5 0.37
46.0 0.28
48.7 1.38
36.7 1.67
39.2 0.78
39.2 1.48
36.2 2.67
31.7 1.54
27.8 2.93
25.9 3.17
30.6 2.76
41.7 1.38
35.6 2.45
23.9 1.34
22.6 2.75
32.4 1.38
42.9 2.45
73.18 1.70
18.52 2.89
34.5 2.50
38.8 4.20
44.6 1.40
47.7 0.88
33.2 1.28
39.3 0.97
42.6 1.36
36.3 1.48
31.7 1.27
28.6 2.89
23.2 0.98
28.6 2.56
41.7 2.60
33.2 4.10
18.4 3.04
25.2 2.89
32.4 2.30
37.0 1.27
—
—
—
—
bCurcumin
1
2
3
4
5
6
7
8
87.30 1.89
89.60 3.29
95.72 2.50
79.30 1.43
80.35 3.82
91.29 0.67
87.56 2.06
94.29 0.04
91.83 4.10
89.30 3.18
93.24 1.29
91.47 2.84
95.20 1.76
93.27 2.85
91.38 0.94
95.23 1.05
95.23 1.05
95.20 1.76
88.27 1.63
87.72 1.90
93.64 3.78
82.47 2.42
83.51 1.98
90.13 2.82
83.64 1.75
91.93 2.72
90.34 1.19
87.02 2.39
91.46 3.20
91.74 1.95
92.08 2.07
92.74 1.19
90.85 3.76
93.34 2.03
93.34 2.03
92.08 2.07
9
10
11
12
13
14
15
16
17
18
Values are represented as mean SD of three different experiments in triplicates.
a
Cell viability was measured at IC50 (lM).
Standards used for the present study.
b
Table 2
TNF- and IL-1b inhibitory effects of compounds on RAW 264.7 and J774A.1 cells at
10–12, 15 and 16 were observed to be more potent than other syn-
thesized derivatives and parent compound. Compounds 15 and 16,
(substitution of halogens in the phenyl ring of indole moiety)
a
concentration of 20
lg/mL
Compounds
TNF-
a
(% inhibition)
IL-1b (% inhibition)
showed most potent inhibition (IC50 = 23. 9 and 22.6
264.7 cells and IC50 = 18. 4 and 25.2 M in J774A.1 cells, respec-
tively) of NO production followed by compounds 11 and 12
(IC50 = 25. 9 M and 30.6 M in RAW 264.7 cells and IC50 = 23.2
and 28.6 M in J774A.1 cells, respectively). Furthermore, in com-
parison to the parent compound lupeol (1), activity reduces with
2 and 5–7 with a half maximal concentration 38.5 to 39.2
lM in RAW
l
RAW 264.7
J774A.1
RAW 264.7 J774A.1
aCurcumin
1.25 2.15
0.016 1.67
32.8 1.32
39.1 4.20
39.2 2.94
42.3 4.38
2.08 3.56
0.165 1.29
31.1 2.34
29.4 0.39
31.5 3.49
37.0 1.28
6.44 1.55
1.84 2.35
22.8 1.29
11.4 3.17
19.7 1.27
22.9 3.29
24.82 2.93
aDexamethasone
9.09 3.51
17.2 3.29
10.1 1.36
16.2 1.25
17.3 3.25
l
l
11
12
15
16
l
lM
while no apparent change in NO inhibition (IC50 ꢀ41.7–48.7
lM)
Values are represented as mean SD of three different experiments in triplicates.
was observed with 3, 4, 13 and 18. Also, compounds 8, 9, 14 and
a
Standards used for the present study (IC50 in lg/mL).
17 depicted moderate to mild inhibition of NO with IC50 ranging
from 27.8 to 36.2 lM. The results revealed that indole moieties
containing only one nitrogen atom had a noticeable impact on
the activity. However, substitution of the halogen group (bromine
and iodine) on the phenyl ring of indole moiety resulted in the
significant inhibition. It is important to note that substitution of
electron donating group at the phenyl ring of indole moiety that
is ethyl, methyl and dimethyl also exhibit potent inhibition
effects.
Similar to NO, macrophages also released basal levels of inflam-
matory cytokines and upon stimulation with LPS, the production of
these mediators increased markedly starting from 4 h and lasts
mostly up to 18 h. In view of significant NO inhibitory effects, com-
group (methyl ester) at the C-3 of the lupeol could reduce the
activity.
Therefore taking the above results together, a conclusive study
showed that substitution of the alkyl and halogen groups on the
phenyl ring of indole moiety enhances the inhibition towards NO
production. Compounds 10–12, 15 and 16 exhibited potent anti-
inflammatory activity mainly through inhibition of NO release
from RAW 264.7 and J774A.1 cells. It seems that presence of six
membered heterocyclic ring with two nitrogen atoms at C-2 and
C-3 of lupeol did not impact on the activity. However, substituted
electron donating groups at the phenyl ring of indole moiety pro-
motes the activity. The alkyl (11, 12) and halogen substituted
pounds 11, 12, 15 and 16 at a concentration of 20
ther tested for the reduction of LPS-stimulated TNF-
l
g/mL were fur-
and IL-1b
a
indole (15, 16) also found to be moderately active towards TNF-
a
release of RAW 264.7 and J774A.1 cells using the protocol provided
in the enzyme linked immune-sorbent assay (ELISA) kit.24 The
results revealed that these compounds exhibited mild inhibition
(29–42%) of LPS-induced tumor necrosis factor alpha (TNF-a)
whereas found to be inactive (10–22%) towards IL-1b inhibition
release while none of the synthesized derivatives was active
towards IL-1b on RAW 264.7 and J774A.1 cells. The aforemen-
tioned results suggested that derivatives of lupeol could be a lead
to potent inhibitors of NO.
(Table 2).
Acknowledgements
Taking the above results together, a preliminary structure
activity relationship (SAR) for lupeol derivatives could be out-
lined as follows: (1) A carbonyl group at C-3 of lupeol could
enhance the activity. (2) Substituted groups on the phenyl ring
of indole moiety could promote the activity. (3) A hydrophobic
The authors gratefully acknowledge the financial support from
the Director, NIPER, SAS Nagar, India for the research work. N.K.P.
is a recipient of DST-INSPIRE Senior Research Fellowship.
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