Synthesis and anti-inflammatory activity of derivatives of coumarino-lignoid, cleomiscosin A and its methyl ether

Article abstract of DOI:10.1016/j.ejmech.2010.08.027

Six novel cleomiscosin A (a coumarino-lignoid), derivatives have been synthesized for the first time by using electrophilic substitution reaction to give nuclear nitrated and halogenated derivatives of cleomiscosin A in good yields. Structures of these compounds were established on the basis of IR, ¹H NMR, ¹³C NMR and Mass spectral data. Some of the synthesized derivatives were tested for in-vitro target based anti-inflammatory study using primary macrophages cell culture bioassay system. The results showed that the compounds 1a, 3a and 4a (1 and 10 μg/mL) exhibited potent anti-inflammatory activity.

Full text of DOI:10.1016/j.ejmech.2010.08.027

European Journal of Medicinal Chemistry 45 (2010) 5150e5156
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anti-inflammatory activity of derivatives of coumarino-lignoid,
cleomiscosin A and its methyl ether
,
b
b
Shelly Sharma a, S.K. Chattopadhyaya , Priyanka Trivedi , D.U. Bawankule
*
a Process Chemistry and Chemical Engineering Department, Central Institute of Medicinal and Aromatic Plants (CIMAP), (Council of Scientific and Industrial Research, CSIR),
P.O. CIMAP, Lucknow-226015, Uttar Pradesh, India
b Molecular Bioprospection Division, Central Institute of Medicinal and Aromatic Plants (CIMAP), (Council of Scientific and Industrial Research, CSIR), P.O. CIMAP,
Lucknow-226015, Uttar Pradesh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Six novel cleomiscosin A (a coumarino-lignoid), derivatives have been synthesized for the first time by
using electrophilic substitution reaction to give nuclear nitrated and halogenated derivatives of cleo-
miscosin A in good yields. Structures of these compounds were established on the basis of IR, 1H NMR,
13C NMR and Mass spectral data. Some of the synthesized derivatives were tested for in-vitro target based
anti-inflammatory study using primary macrophages cell culture bioassay system. The results showed
Received 7 May 2010
Received in revised form
9 August 2010
Accepted 10 August 2010
Available online 14 August 2010
that the compounds 1a, 3a and 4a (1 and 10
m
g/mL) exhibited potent anti-inflammatory activity.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Cleomiscosin A
Coumarino-lignoid
Electrophilic substitution reaction
Anti-inflammatory
ELISA
1. Introduction
established the antioxidant activity of cliv A and cliv C and there-
fore they could be beneficial in preventing oxidation of low density
lipoprotein in atherosclerotic lesions. Cliv C inhibited lipid perox-
idation comparable to vitamin E [4].
The plant Cleome viscosa (General name; Hurhur, Wild mustard)
is used in fever, inflammations, liver diseases, bronchitis, diarrhoea
and infantile convulsions. The seeds are small, dark brown or black
and glandular. The seeds are reported to have rubefacient, vesicant
and anthelminthic properties [1]. The seeds of the plant C. viscosa
have been chemically investigated and they were found to be the
first source of an interesting class of compounds known as cou-
marino-lignoids. Four coumarino-lignoids cleomiscosin A, B, C and
D were isolated from the seeds of the plant and cleomiscosin A (cliv
A) was the major compound followed by cleomiscosin B and C. We
have determined that a combination of three coumarino-lignoids
cleomiscosin A, B and C (cliv A, cliv B and cliv C) possessed signif-
icant liver protective activity [2]. It has also been reported by us that
the combination of cliv A, cliv B and cliv C which we have termed as
cliv 92 enhanced the body immune function by significantly
increasing the white blood cell counts, hem agglutination antibody
titer responses and reducing delayed type hypersensitivity
responses towards rabbit red blood cells [3]. Also, it has been
In a previous publication, Ray et al. have reported the prepara-
tion of seven derivatives of cleomiscosin A, which included, cleo-
miscosin A methyl ether, cleomiscosin A ethyl ether, cleomiscosin A
diacetate, cleomiscosin A ethyl ether monoacetate, o-methoxy-
trans cinnamic acid derivative from cleomiscosin A ethyl ether;
potassium permanganate oxidation product of cleomiscosin A
methyl ether of cleomiscosin A and substituted phenyl propionate
derivative from cleomiscosin A methyl ether [5]. On the basis of the
pharmacological results previously obtained by our group [6], it
was reported that the oral administration of coumarino-lignoid
mixture containing clemiscosin A, B and C isolated from the plant C.
viscosa inhibits the pro-inflammatory cytokines in dose dependent
manner in Swiss albino mice. The molecular manipulation of
promising lead compounds is still a major line of approach to
develop newer drugs. In order, to study further the chemistry and
pharmacological activity of coumarino-lignoids, we have tried
some electrophilic aromatic substitution reactions on the major
compound, cleomiscosin A. In this communication, we would like
to report the nitration and bromination reaction on cleomiscosin A
and its methyl ether derivative. The anti-inflammatory activity of
* Corresponding author. Tel.: þ91 9648029167; fax: þ91 522 2342666.
E-mail address: chattsk@yahoo.com (S.K. Chattopadhyay).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.027

S. Sharma et al. / European Journal of Medicinal Chemistry 45 (2010) 5150e5156
5151
all the new molecules was evaluated and included in this publi-
cation (Tables 1 and 2).
into acetate. Compound 1b, C24H24O9, m.p. 160 ꢀC, was found to be
the monoacetate of cleomiscosin A and was confirmed from the
reported NMR data (Scheme 1) [5].
Treatment of cleomiscosin A in acetic anhydride with conc.
HNO3 and a catalytic amount for H2SO4 converted it into two
derivatives, 2a and 2b, (Scheme 1). The ESI-MS (positive mode) of
2a, C24H21O12N, m.p. 180e182 ꢀC; showed its [M þ H]þ at m/z 516.
In the 1H NMR spectrum of compound 2a, the C-5H was absent and
2. Chemistry
The new coumarino-lignoid derivatives 1ae5b were prepared
through the synthetic route reported in Schemes 1 and 2. Thus, the
reaction of bromination of cleomiscosin A (cliv A) with acetic acid at
100 ꢀC converted it into a monobromo derivative 1a, (C22H19O9Br,
m.p. 70e72 ꢀC), in which the primary alcoholic group was also
converted into an ester (Scheme 1). IR spectrum of the derivative
1a, showed the characteristic absorptions at 1735 cmꢁ1 of the
carbonyl group of the primary alcohol, in addition to the lactone
ring carbonyl at 1722 cmꢁ1. The 1H NMR spectrum showed the two
the C-4H underwent downfield shift at
confirming that the nitration took place at C-5H position. The 1H
NMR of 2a also showed two acetate groups at 2.01 and 2.27
d
7.64 (d, 1H, J ¼ 9.9 Hz)
d
(alcohol acetate and phenol acetate). Thus nitration of cliv A yielded
a nitro derivative in which both the phenolic and alcoholic group
were esterified. Compound 2b, C24H22O10, m.p. 175 ꢀC, was found to
be the diacetate of cleomiscosin A and was confirmed from the
reported NMR data (Scheme 1) [5].
We have also studied the reactivity of methyl ether of cleo-
miscosin A towards electrophilic aromatic substitution reactions
(Scheme 2). Reaction of the methyl ether of cleomiscosin A with
N-chlorosuccinimide in refluxing CHCl3 yielded a monochloro
derivative 3a, C21H19O8Cl, m.p. 172e174 ꢀC. The ESI-MS (positive
mode) spectrum of 3a showed [M þ H]þ and [Mþ2 þ H]þ peaks at
m/z 435 and 437. In the 1H NMR spectrum of 3a, the C-3 and C-4
coumarin protons at
d
6.37 and 8.02 (1H, d each, J ¼ 9.9 Hz) for C-3H
and C-4H; the downfield shift of C-4H in the 1H NMR spectrum of
the molecule indicated that a bromine group was at C-5 position.
This was also confirmed from the 1H NMR of the compound in
which C-5 H was absent. The 1H NMR spectrum also showed the
presence of an alcoholic acetate at
d 2.10 (s). The ESI-MS (positive
mode) spectrum showed [M þ H]þ and [Mþ2 þ H]þ at m/z 507 and
509 in the ratio of almost 1:1. Moreover, the MS/MS spectrum of the
molecule showed two diagnostic peaks at m/z 283 and 285 and at
m/z 222, the appearance of which can be explained due to retro
DielseAlder cleavage of the dioxane bridge of the molecule. The
DEPT 135ꢀ spectrum of 1a showed the presence of two coumarin
and three aromatic protons. From the 1H NMR and Mass spectrum
of compound it was confirmed that the Br group was introduced at
C-5 position and also the primary alcoholic group was converted
protons appeared at
d
6.27 and 7.90 (d each, 1H, J ¼ 9.6 Hz). The
downfield shift of the C-4H and the absence of the C-5H in the 1H
NMR spectrum of 3a indicated that chlorination took place in the
aromatic ring of coumarin at C-5. The same trend was also found in
the bromination reaction of the methyl ether of cleomiscosin A
with N-bromosuccinimide when refluxed with CHCl3 to yield the
Table 1
% Inhibition of pro-inflammatory cytokines of derivatives 1a, 1b, 2a, 3a and 4ae5b.
Compd.
R
R0
R00
R1
R2
Dose (
mg/mL)
Pro-inflammatory cytokines
TNF-
a
% inhibition
IL-6% inhibition
IL-1b % inhibition
Vehicle (DMSO)
1a
e
e
e
e
e
e
e
e
100.00
54.49
54.23
73.30
76.25
100.00**
60.17**
52.48
81.88
78.30
100.00*
55.27*
58.81
109.32
74.84
eBr
eOAc
eH
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
1b
eH
eNO2
e
eOAc
eOAc
eH
eH
e
e
2a
eOAc
eMe
eMe
eMe
eMe
e
e
e
70.47
87.16
70.38
67.81*
50.20*
49.58**
44.77**
41.99
64.84
60.61
76.60
77.98
82.03**
57.45**
56.21**
59.80**
60.03
81.36
79.04
86.44
80.18
65.26*
59.19**
40.12**
50.08**
44.69
59.08
53.28
72.02
70.20
3a
eCl
eBr
eNO2
eNO2
e
eH
eH
eNO2
eNO2
e
4a
e
eH
5a
e
eNO2
eH
5b
e
Diclofenac Sodium
e
e
46.46**
51.15**
38.51**
n ¼ 04, *P < 0.05, **P < 0.01 significance as compared to vehicle control (Student t-test).

5152
S. Sharma et al. / European Journal of Medicinal Chemistry 45 (2010) 5150e5156
Table 2
In-vitro target based anti-inflammatory activity of 1a, 1b, 2a, 3ae5b in murine macrophage cell culture system, Quantification of Pro-inflammatory cytokines using sandwich
ELISA.
Compd.
R
R0
R00
R1
R2
Dose (
m
g/mL)
Pro-inflammatory cytokines
TNF-
a
Pg/mL
IL-6 Pg/mL
IL-1b Pg/mL
Vehicle (DMSO)
1a
e
e
e
e
e
e
e
e
334.33 ꢂ 23.40
543.00 ꢂ 23.11
103.44 ꢂ 11.79
57.17 ꢂ 8.75*
60.83 ꢂ 9.51*
113.08 ꢂ 33.93NS
77.42 ꢂ 5.34NS
72.81 ꢂ 5.36NS
67.50 ꢂ 4.79NS
61.22 ꢂ 7.17*
41.50 ꢂ 11.20**
51.81 ꢂ 7.05**
46.22 ꢂ 4.10**
61.11 ꢂ 15.52NS
55.11 ꢂ 9.39NS
74.50 ꢂ 2.58NS
72.61 ꢂ 8.94NS
39.83 ꢂ 5.00**
eBr
eOAc
eH
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
182.19 ꢂ 45.36NS
181.29 ꢂ 44.91NS
245.08 ꢂ 28.11NS
254.91 ꢂ 33.35NS
235.59 ꢂ 40.25NS
226.72 ꢂ 40.28NS
180.67 ꢂ 26.52*
165.77 ꢂ 19.71*
149.68 ꢂ 16.81**
140.38 ꢂ 17.12**
216.77 ꢂ 39.67NS
202.64 ꢂ 39.23NS
256.10 ꢂ 22.93NS
260.72 ꢂ 8.54NS
155.33 ꢂ 8.39**
326.75 ꢂ 21.13**
284.98 ꢂ 16.34**
444.60 ꢂ 47.97NS
414.77 ꢂ 31.21NS
473.27 ꢂ 16.69NS
445.44 ꢂ 11.28NS
311.98 ꢂ 23.80**
305.21 ꢂ 17.74**
324.69 ꢂ 14.18**
325.95 ꢂ 34.46**
441.81 ꢂ 27.20NS
429.17 ꢂ 30.71NS
469.36 ꢂ 16.01NS
435.38 ꢂ 25.99NS
277.73 ꢂ 20.61**
1b
eH
eNO2
e
eOAc
eOAc
eH
eH
e
e
2a
eOAc
eMe
eMe
eMe
eMe
e
e
e
3a
eCl
eBr
eNO2
eNO2
e
eH
eH
eNO2
eNO2
e
4a
e
eH
5a
e
eNO2
eH
5b
e
Diclofenac Sodium
e
e
n ¼ 04.
NS: Not Significance.
*P < 0.05, **P < 0.01 significance as compared to vehicle control (Student t-test).
C-5 bromo derivative 4a, C21H19O8Br, m.p. 105e107 ꢀC, [M þ H]þ
and [Mþ2 þ H]þ (positive mode) at m/z 479 and 481.
The target based anti-inflammatory activities of compounds
1ae5b (Tables 1 and 2) have been evaluated in in-vitro condition at
the initial dose of 1 g/mL and 10 g/mL. The compounds 1a, 3a and
4a showed the significant inhibition of pro-inflammatory targets in
lipo-polysaccharide (LPS)-induced inflammation in primary
macrophages cell culture model. The pro-inflammatory cytokines
Treatment of methyl ether of cleomiscosin A with conc. HNO3
at ꢁ5 ꢀC yielded two products; 5a and 5b (Scheme 2). In the 1H
m
m
NMR spectrum of trinitro derivative 5a, C21H17N3O14
, m.p.
145e147 ꢀC, C-4H, underwent a downfield shift and appeared at
7.70 (d, J ¼ 10.0 Hz) indicating the introduction of one nitro group
d
(TNF-a, IL-6, IL-1b) were quantified from cell culture supernatant
at C-5 position. The C-70 H showed a downfield shift and appeared
using enzyme-linked immunosorbent assay (ELISA). The
compounds 1a, 3a and 4a showed the significant inhibition of the
pro-inflammatory targets which are the mediators of various
chronic inflammatory disorders.
at
d
5.96 (d, J ¼ 6.4 Hz). Also, the 1H NMR spectrum showed two
singlets at
d
7.67 (C-50) and 7.14 (C-20) indicating the placement of
the second nitro group at C-60 position. Moreover, the primary
alcoholic group was found to be nitrated and its two protons
appeared as a double doublet at
14.8 Hz) and as a broad doublet merges with the C-80 H signal at
4.87. The C-90 H carbon showed a downfield shift in its 13C NMR
d
4.76 and 4.72 (J ¼ 5.6 Hz and
4. Conclusion
d
In summary, we report that nuclear bromination of cleomiscosin
A in acetic acid produced the monobromo derivative in which the
primary alcoholic group of cleomiscosin A was also esterified with
acetic acid. Furthermore, we have also studied the chlorination and
bromination reactions using N-chlorosuccinimide (NCS) and
N-bromosuccinimide (NBS) on the methyl ether of cleomiscosin A.
We have found that the reactions of the methyl ether of cliv A with
NBS and NCS produced a monobromo and monochloro derivative
only. Nitration of cleomiscosin A with conc. HNO3 and a catalytic
amount of conc. H2SO4 in presence of acetic anhydride produced
the C-5 nitro derivative in which both the alcoholic and the
phenolic groups were esterified and also the diesterified cleo-
miscosin A were obtained. It is very interesting to see that the
nitration of cleomiscosin A methyl ether with conc. HNO3, two
spectrum and appeared at d 69.7. The structure of the second minor
dinitroderivative, C21H18N2O12, m.p. 200e202 ꢀC; was confirmed as
5b, as it showed the downfield shift of the C-4H which appeared at
d
7.73 (d, J ¼ 9.6 Hz) and also showed the absence of C-5H in its 1H
NMR spectrum. The other nitro group was put at C-60 as C-50 H and
C-20 H appeared as singlets at
d 7.57 and 7.12, respectively.
3. Result and discussion
Six new coumarino-lignoid derivatives of both cleomiscosin A
and cleomiscosin A methyl ether 1ae5b were reported for the first
time. Structures of the synthesized compounds were established on
the basis of IR, 1H NMR, 13C NMR and Mass spectral data.

S. Sharma et al. / European Journal of Medicinal Chemistry 45 (2010) 5150e5156
5153
Scheme 1. Synthesis of cleomiscosin A derivatives (1ae2b). Reagents and reaction conditions: (i) Cleomiscosin A, Acetic acid, Br2 in acetic acid, Reflux, 5 h. (ii) Cleomiscosin A,
Acetic anhydride, Conc. H2SO4, Conc. HNO3, 5 h, room temperature.
nitrated products were obtained. In both the products, nitration
took place at C-5 and C-60 positions and in one product the primary
alcoholic group was also nitrated. All the above derivatives 1ae5b
were made for the first time from cleomiscosin A and its methyl
ether derivative.
The preliminary in-vitro anti-inflammatory activity of these
novel series of derivatives has evidenced that coumarino-lignoid
derivative 1a, 3a and 4a showed significant inhibition of the pro-
inflammatory targets which are the mediators of various chronic
inflammatory disorders.
5.1.1. General procedure for the synthesis of (5-bromo-90-acetate)
cleomiscosin A (1a) and 90 acetate of cleomiscosin A (1b)
A mixture of cleomiscosin A (0.25 mmol) and Br2 in acetic acid
(0.5 mL, 19.74 mmol Br2 in 2.5 mL, 43.67 mmol acetic acid) in
a 100 mL round bottom flask was refluxed at 100 ꢀC for 5 h. After
completion of the reaction, reaction mixture was diluted with
water and 5% Na2SO3 solution and stirred for 30 min at room
temperature. It was then extracted with CHCl3. The residue
obtained after drying and concentrating the CHCl3 extract was
purified by column chromatography (silica gel, 60e120 mesh;
petroleum ether/ethyl acetate, 70:30) to afford the desired bro-
moacetate and a monoacetate derivative of cleomiscosin A.
5. Experimental protocols
5.1. Chemistry
5.1.1.1. (5-Bromo-90-acetate) cleomiscosin
A
(1a). White amoþr-
phous solid, m.p. 70e72 ꢀC, % yield: 38; Mass (ESIþ) (M þ H) /
(Mþ2 þ H)þ: m/z ¼ 507/509. IR (KBr): nmax ¼ 3448, 2940, 1735,
1722, 1602, 1562, 1435, 1125, 1056, 825 cmꢁ1. 1H NMR (300 MHz,
All the chemicals and solvents were obtained from Lobachemie,
Qualigens, Merck and used as such. Melting points were determined
in an open capillary tube on a JSGW melting point apparatus and are
uncorrected. The NMR spectra of synthesized derivatives 1ae4a
were recorded by using Bruker AVANCE 300 MHz whereas the NMR
spectra of derivatives 5a and 5b were recorded by using Bruker
CDCl3, 25 ꢀC):
d
¼ 6.37 (d, J ¼ 9.9 Hz, 1H, 3H), 8.02 (d, J ¼ 9.9 Hz, 1H,
4H), 7.16 (d, J ¼ 1.8 Hz, 1H, 20H), 6.97 (d, J ¼ 8.1 Hz, 1H, 50H), 6.90
(dd, J ¼ 8.1, 1.8 Hz, 1H, 60H), 5.01 (d, J ¼ 7.5 Hz, 1H, 70H), 4.09 (m, 1H,
80H), 4.37 (m, 2H, 90H), 3.87, 3.94 (2s, 2 ꢃ 3H, 2ꢃ eOCH3), 2.10
AVANCE 400 MHz FT NMR. IR spectra, KBr pellets, 600e4000 cmꢁ1
,
(s, 3H, 90-OCOCH3) ppm. 13C NMR (CDCl3, 25 ꢀC):
d
¼ 159.9 (C-2),
were recorded on PerkineElmer FTIR, BX Spectrophotometer. Mass
spectra (Electrospray ionization in positive mode, ESIþ) were
recorded on an API 3000 (Applied Biosystem) mass spectrometer;
chromatographic purifications were performed by silica gel 60e120
mesh (MERCK) column chromatography.
115.6 (C-3), 143.1 (C-4), 107.9 (C-5), 143.7 (C-6), 131.7 (C-7), 129.7
(C-8), 141.2 (C-9), 112.6 (C-10), 127.5 (C-10), 109.4 (C-20), 148.1
(C-30), 144.9 (C-40), 124.5 (C-50), 123.9 (C-60), 75.8 (C-70), 76.9 (C-
80), 62.8 (C-90), 20.9 (CH3), 170.6 (CO, eOCOCH3), 61.6, 56.9
(2ꢃ eOCH3) ppm.

5154
S. Sharma et al. / European Journal of Medicinal Chemistry 45 (2010) 5150e5156
Scheme 2. Synthesis of cleomiscosin A methyl ether derivatives (3ae5b). Reagents and reaction conditions: (iii) Cleomiscosin A methyl ether, N-Chlorosuccinimide (NCS), CHCl3,
Reflux, 2 h. (iv) Cleomiscosin A methyl ether, N-Bromosuccinimide (NBS), CHCl3, Reflux, 2 h. (v) Cleomiscosin A methyl ether, Conc. HNO3, 2 h, ꢁ5ꢀ C.
5.1.1.2. 90 Acetate of cleomiscosin A (1b). White aþmorphous solid,
m.p. 160 ꢀC, % yield: 30; Mass (ESIþ) (M þ H) : m/z ¼ 428.9,
[M þ Na]þ ¼ 450.9. IR (KBr): nmax ¼ 3427, 2925, 1727, 1615, 1574,
acetate, 80:20) to afford the desired nitro diacetate and diacetate
product of cleomiscosin A. Yelloþw amorphous solid, m.p.
180e182 ꢀC, % yield: 45. Mass (ESI ) (M þ H)þ: m/z ¼ 516.3,
[M þ Na]þ: 538.2, [M þ K]þ: m/z ¼ 554.2. IR (KBR) nmax ¼ 3447,
1773.17, 1749.6, 1718.84, 1653, 1637, 1625, 1572.81, 1541.89, 1524.95,
1364.41, 1288.05, 1133.12 cmꢁ1 1H NMR (300 MHz, CDCl3, 25 ꢀC):
1448, 1138, 1058, 841 cmꢁ1 1H NMR (300 MHz, CDCl3, 25 ꢀC):
.
d
¼ 6.31 (d, J ¼ 9.6 Hz, 1H, 3H), 7.60 (d, J ¼ 9.6 Hz, 1H, 4H), 6.86 (d,
J ¼ 1.8 Hz, 1H, 20H), 6.92 (d, J ¼ 7.8 Hz, 1H, 50H), 6.90 (dd, J ¼ 8.5 Hz,
1H, 60H), 4.99 (d, J ¼ 8.1 Hz,1H, 70H), 4.37 (m,1H, 80H), 4.32, 4.05 (m,
2H, 90H), 3.89, 3.88 (2s, 2 ꢃ 3H, 2ꢃ eOCH3), 2.07 (s, 3H, 90-
d
¼ 6.41 (d, J ¼ 9.9 Hz, 1H, 3H), 7.64 (d, J ¼ 9.9 Hz, 1H, 4H), 6.90 (d,
J ¼ 2.1 Hz, 1H, 20H), 7.07 (d, J ¼ 8.1 Hz, 1H, 50H), 6.92 (dd, J ¼ 8.1 Hz,
2.1 Hz; 1H, 60H), 5.05 (d, J ¼ 7.8 Hz, 1H, 70H), 4.09 (m, 1H, 80H), 4.36
(m, 2H, 90H), 3.79, 3.92 (2s, 2 ꢃ 3H, 2ꢃ eOCH3), 2.01 (s, 3H, 90-OAc),
OCOCH3) ppm. 13C NMR (CDCl3, 25 ꢀC):
d
¼ 161.1 (C-2), 114.7 (C-3),
144.1 (C-4), 100.8 (C-5), 146.3 (C-6), 137.7 (C-7), 132.3 (C-8), 139.2
(C-9),112.2 (C-10),126.9 (C-10),110.1 (C-20),147.4 (C-30),145.3 (C-40),
115.3 (C-50), 121.5 (C-60), 75.8 (C-70), 77.3 (C-80), 63.2 (C-90), 21.1
(CH3), 170.9 (CO, eOCOCH3), 56.7, 56.4 (2ꢃ eOCH3) ppm.
2.27 (s, 3H, 40-OAc) ppm. 13C NMR (CDCl3, 25 ꢀC):
d
¼ 158.9 (C-2),
111.9 (C-3), 137.9 (C-4), 106.6 (C-5), 141.3 (C-6), 134.4 (C-7), 129.8 (C-
8), 140.0 (C-9), 111.6 (C-10), 134.3 (C-10), 110.4 (C-20), 156.4 (C-30),
139.8 (C-40), 121.7 (C-50),117.6 (C-60), 72.7 (C-70), 76.5 (C-80), 62.3 (C-
90), 20.8 (2 ꢃ CH3), 170.9 (eCOe, 40-OAc), 168.5 (eCOe, 90-OAc),
57.2, 63.4 (2ꢃ eOCH3) ppm.
5.1.2. General procedure for the synthesis of (5-nitro-40,90-
diacetate) cleomiscosin A (2a)
A mixture of cleomiscosin A (0.25 mmol) in acetic anhydride
(2 mL, 22.39 mmol), conc. HNO3 (1 mL) and conc. H2SO4 (0.1 mL) in
a 100 mL round bottom flask was kept for 5 h at room temperature.
After completion of the reaction, the reaction mixture was diluted
with water and extracted with CHCl3 .The residue obtained after
drying and concentrating the CHCl3 extract was purified by column
chromatography (silica gel, 60e120 mesh; petroleum ether/ethyl
5.1.3. General procedure for the synthesis of 5-chloro-cleomiscosin
A methyl ether (3a)
To a 50 mL round bottom flask was added the cleomiscosin A
methyl ether (50 mg, 0.125 mmol), N-chlorosuccinimide (NCS)
(100 mg, 0.374 mmol) and CHCl3 (5 mL). The reaction mixture was
refluxed at 65 ꢀC for 2 h. After completion of the reaction, the

S. Sharma et al. / European Journal of Medicinal Chemistry 45 (2010) 5150e5156
5155
reaction mixture was diluted with water and 5% Na2SO3 was added
and stirred at room temperature for 30 min. The reaction mixture
was then extracted with CHCl3 .The residue obtained after drying
and concentrating the CHCl3 extract was purified by column
chromatography (silica gel, 60e120 mesh; chloroform/acetone,
98:2) to afford the desired chloro product of cleomiscosin A methyl
ether. White amorphous solid, m.p. 172e174 ꢀC, % yield: 55 (30 mg).
Mass (ESIþ) (M þ H)þ/(Mþ2 þ H)þ: m/z ¼ 435/437, [M þ Na]þ: m/
z ¼ 457/459, [M þ K]þ: m/z ¼ 473/475. IR (KBr): nmax ¼ 3450, 1716,
1707, 1605, 1352, 819, 790 cmꢁ1 1H NMR (300 MHz, CDCl3, 25 ꢀC):
NMR (400 MHz, CDCl3 þ DMSO, a few drops, 25 ꢀC):
d
¼ 6.49 (d,
J ¼ 10.0 Hz, 1H, 3H), 7.70 (d, J ¼ 10.0 Hz, 1H, 4H), 7.14 (s, 1H, 20H),
7.67 (br s, 1H, 50H), 5.96 (d, J ¼ 6.4 Hz, 1H, 70H), 4.87 (br d, 1H, 80H),
4.76, 4.72 (dd, J ¼ 5.6, 14.8 Hz, 2H, 90H), 4.02(s, 3H, eOCH3), 3.98,
3.96 (2s, 2 ꢃ 3H, 2ꢃ eOCH3) ppm. 13C NMR (CDCl3 þ DMSO, a few
drops, 25 ꢀC):
d
¼ 157.7 (C-2), 116.8 (C-3), 136.9 (C-4), 105.5 (C-5),
140.9 (C-6), 138.7 (C-7), 133.0 (C-8), 139.3 (C-9), 122.2 (C-10), 134.6
(C-10), 109.8 (C-20), 153.4 (C-30), 149.4 (C-40), 107.8 (C-50), 138.3 (C-
60), 71.7 (C-70), 74.8 (C-80), 69.8 (C-90), 62.8, 56.0, 56.3 (3ꢃ
eOCH3) ppm.
d
¼ 6.27 (d, J ¼ 9.6 Hz, 1H, 3H), 7.90 (d, J ¼ 9.6 Hz, 1H, 4H), 6.92 (br s,
1H, 20H), 6.97 (d, J ¼ 8.1 Hz,1H, 50H), 6.85 (d, J ¼ 8.1 Hz,1H, 60H), 5.11
(d, J ¼ 8.1 Hz,1H, 70H), 3.92 (m,1H, 80H), 3.52, 4.01 (m, 2H, 90H), 3.89
(s,1H, 90OH), 3.84 (s, 3H, eOCH3), 3.82 (2s, 2 ꢃ 3H, 2ꢃ eOCH3) ppm.
5.1.5.2. 5,60-Dinitro-cleomiscosin A methyl ether (5b). þDark yelloþw
solid, m.p. 200e202 ꢀC, % yield: 32 (30 mg). Mass (ESI ) (M þ H) :
m/z ¼ 491. IR (KBr): nmax ¼ 3448, 2942, 1733, 1622, 1572, 1571, 1524,
13C NMR (CDCl3, 25 ꢀC):
d
¼ 160.4 (C-2), 111.7 (C-3), 140.8 (C-4),
1460, 1440, 1280, 1222, 1072 cmꢁ1 1H NMR (400 MHz,
.
110.8 (C-5), 142.6 (C-6), 142.0 (C-7), 131.5 (C-8), 135.0 (C-9), 117.1 (C-
10), 127.8 (C-10), 110.7 (C-20), 150.3 (C-30), 149.8 (C-40), 114.9 (C-50),
120.6 (C-60), 77.5 (C-70), 79.0 (C-80), 61.4 (C-90), 61.7, 56.4, 56.37 (3ꢃ
eOCH3) ppm.
CDCl3 þ DMSO, a few drops, 25 ꢀC):
¼ 6.48 (d, J ¼ 9.6 Hz, 1H, 3H),
d
7.73 (d, J ¼ 9.6 Hz, 1H, 4H), 7.12 (s, 1H, 20H), 7.57 (s, 1H, 50H), 5.96 (d,
J ¼ 7.2 Hz, 1H, 70H), 4.55 (distorted triplet, 1H, 80H), 3.72 (dd,
J ¼ 12.8, 4.4 Hz, 1H, 90H), 3.91 (br d, J ¼ 13.2 Hz, 1H, 90H), 3.99 (s, 3H,
eOCH3), 3.92 (2s,
2
ꢃ
3H, 2ꢃ eOCH3) ppm. 13C NMR
5.1.4. General procedure for the synthesis of 5-bromo-cleomiscosin
A methyl ether (4a)
(CDCl3 þ DMSO, a few drops, 25 ꢀC):
d
¼ 157.6 (C-2), 116.0 (C-3),
136.9 (C-4), 104.8 (C-5), 141.0 (C-6), 138.5 (C-7), 133.7 (C-8), 139.2
(C-9), 122.3 (C-10), 134.1 (C-10), 110.1 (C-20), 152.2 (C-30), 148.5 (C-
40), 107.5 (C-50), 138.1 (C-60), 72.7 (C-70), 78.4 (C-80), 59.5 (C-90), 61.9,
55.8, 55.7 (3ꢃ eOCH3) ppm.
To a 50 mL round bottom flask was added the cleomiscosin A
methyl ether (25 mg, 0.063 mmol), N-bromosuccinimide (NBS)
(50 mg, 0.279 mmol) and CHCl3 (5 mL). The reaction mixture was
refluxed at 65 ꢀC for 2 h. After completion of the reaction, the
reaction mixture was diluted with water and 5% Na2SO3 was added
and stirred at room temperature for 30 min. It was then extracted
with CHCl3 .The residue obtained after drying and concentrating the
CHCl3 extract was purified by column chromatography (silica gel,
60e120 mesh; chloroform/acetone, 98:2) to afford the desired
bromo product of cleomiscosin A methyl ether. White amorphous
solid, m.p. 105e107 ꢀC, % yield: 67 (30 mg). Mass (ESIþ) (M þ H)þ/
(Mþ2 þ H)þ: m/z ¼ 479/481, [M þ Na]þ: m/z ¼ 501/503, [M þ K]þ: m/
z ¼ 517/519. IR (KBr): nmax ¼ 3448, 2930, 2854,1734,1719,1600,1458,
1402, 1264, 1124, 1057, 1025, 812 cmꢁ1 1H NMR (300 MHz, CDCl3,
5.2. Pharmacology
Inflammation is a multi-step process that is mediated by acti-
vated inflammatory cells, including macrophages/monocytes [7]. In
the presence of stimuli such as lipo-polysaccharide (LPS), activated
macrophages induce the overproduction of pro-inflammatory
cytokines such as TNF-a, IL-1, IL-6 [8]. In this study, we used
macrophage cells stimulated with LPS for target based in-vitro anti-
inflammatory evaluation of compound 1ae5b.
25 ꢀC):
d
¼ 6.27 (d, J ¼ 9.9 Hz,1H, 3H), 7.92 (d, J ¼ 9.9 Hz,1H, 4H), 6.91
5.2.1. Primary cell culture and treatment
(br s,1H, 20H), 6.97 (d, J ¼ 8.1 Hz,1H, 50H), 6.85 (d, J ¼ 8.1 Hz,1H, 60H),
5.10 (d, J ¼ 8.1 Hz, 1H, 70H), 4.06 (m, 1H, 80H), 3.55, 3.84 (m, 2H, 90H),
3.91 (s, 1H, 90OH), 3.84 (s, 3H, eOCH3), 3.82 (2s, 2 ꢃ 3H, 2ꢃ
Primary macrophage cells were collected from the peritoneal
cavities of mice (8-week-old female Swiss albino mice) after an
intra-peritoneal (i.p.) injection of 1 mL of 1% peptone (BD USA) 3
days before harvesting. Mice were euthanised by cervical disloca-
tion under ether anesthesia and peritoneal macrophages were
obtained by intra-peritoneal (i.p.) injection of Phosphate Buffer
Saline (PBS), pH-7.4. The peritoneal macrophages at the concen-
tration of 2 ꢃ 106 live cells/mL were used for the experimentation.
eOCH3) ppm. 13C NMR (CDCl3, 25 ꢀC):
d
¼ 160.4 (C-2), 111.7 (C-3),
143.4 (C-4), 107.6 (C-5), 143.8 (C-6), 141.1 (C-7), 132.1 (C-8), 141.9 (C-
9), 112.1 (C-10), 127.8 (C-10), 110.7 (C-20), 150.3 (C-30), 149.8 (C-40),
115.1(C-50),120.6 (C-60), 77.5 (C-70), 79.1 (C-80), 61.4 (C-90), 61.5, 56.8,
56.4 (3ꢃ eOCH3) ppm.
5.1.5. General procedure for the nitration reaction of cleomiscosin A
methyl ether, (5a and 5b)
5.2.2. Quantification of pro-inflammatory mediators using enzyme-
linked immunosorbent assay (ELISA)
To a 50 mL round bottom flask was added the cleomiscosin A
methyl ether (70 mg, 0.175 mmol), and conc. HNO3 (3 mL). The
reaction mixture was kept at 0ꢀ to ꢁ5 ꢀC for 2 h. After completion of
the reaction, crushed ice was added to the reaction mixture and
yellow precipitate of nitro derivative was obtained. The reaction
mixture was then extracted with CHCl3 .The residue obtained after
drying and concentrating the CHCl3 extract was purified by column
chromatography (silica gel, 60e120 mesh; petroleum ether/ethyl
acetate, 80:20; and petroleum ether/ethyl acetate, 60:40) to afford
the desired trinitro and dinitro product of cleomiscosin A methyl
ether. The prepared compound was confirmed by 1H NMR, 13C
NMR, ESI-MS (positive mode) and FTIR analysis.
The cells were suspended in RPMI 1640 medium (sigma
chemicals co. USA) containing 10% heat-inactivated fetal calf
serum (Gibco, USA), 100 U/mL of penicillin and 100
mg/mL of
streptomycin and incubated in a culture plate (Nunc, Germany) at
37 ꢀC in 5% CO2 in an incubator. Non-adherent cells were removed
after 2 h and the adherent cells were resuspended in RPMI 1640
medium. Cells were pretreated with 1 and 10
mg/mL of test
compounds for 30 min and then stimulated with lipo-poly-
saccharide (LPS, 0.5 mg/mL). After incubation with LPS for 24 h,
supernatants were collected and immediately frozen at ꢁ80 ꢀC.
Harvested supernatants were tested for quantification of pro-
inflammatory mediators (IL-1
b
, IL-6 and TNF-
a
) by ELISA using
commercial kits for mouse IL-1
synthesized derivatives were dissolved in Dimethyl sulphoxide
(DMSO) and cells treated with 10 L of DMSO were considered as
a vehicle control. In-vitro anti-inflammatory activity of the
compounds was compared with vehicle control [6].
b
, IL-6 and TNF-
a (BD USA). All the
5.1.5.1. 5,60,90-Trinitro-cleomiscosin
A
methyl ether (5a). Light
yellow solid, m.p. 145e147 ꢀC, % yield: 42 (40 mg). Mass (ESIþ)
(M þ H)þ: m/z ¼ 536, [M þ Na]þ: m/z ¼ 559. IR (KBr): nmax ¼ 3448,
2945, 1737, 1638, 1571, 1525, 1460, 1440, 1281, 1220, 1071 cmꢁ1. 1H
m

5156
S. Sharma et al. / European Journal of Medicinal Chemistry 45 (2010) 5150e5156
[3] D.U. Bawankule, S.K. Chattopadhayay, A. Pal, K. Saxena, S. Yadav, N.P. Yadav,
Acknowledgments
D. Mani, A.K. Tripathi, S. Beg, A. Srivastava, A.K. Gupta, S.P.S. Khanuja, Nat. Prod.
Commun. 2 (2007) 923e926.
The authors are grateful to the director, CIMAP (CSIR), Lucknow, for
providing necessary facilities and encouragement. Financial assistance
from the CSIR networking project NWP-09 is gratefully acknowledged.
The communication number of this manuscript is 2010-67J.
[4] W.Y. Jin, P.T. Thuong, N.D. Su, B.S. Min, K.H. Son, H.W. Chang, H.P. Kim, S.S. Kang,
D.E. Sok, K. Bae, Arch. Pharm. Res. 30 (2007) 275e281.
[5] A.B. Ray, S.K. Chattopadhyay, S. Kumar, C. Konno, Y. Kisso, H. Hikino, Tetrahe-
dron 41 (1985) 209e214.
[6] D.U. Bawankule, S.K. Chattopadhyay, Anirban Pal, K. Saxena, S. Yadav, U. Faridi,
M.P. Darokar, A.K. Gupta, S.P.S. Khanuja, Inflammopharmacology 16 (2008)
272e277.
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