Novel lung adenocarcinoma and nuclear factor-kappa B (NF-κB) inhibitors: Synthesis and evaluation of lantadene congeners

Article abstract of DOI:10.1016/j.ejmech.2013.12.052

The C-3, C-17 and C-22 congeners of pentacyclic triterpenoids reduced lantadene A (3), B (4) and 22β-hydroxyoleanolic acid (5) were synthesized and were tested in vitro for their NF-κB and IKKβ inhibitory potencies and cytotoxicity against A549 lung cancer cells. The lead congeners 12 and 13 showed IC₅₀ of 0.56 and 0.42 μmol, respectively against TNF-α induced activation of NF-κB. The congeners 12 and 13 exhibited inhibition of IKKβ in a single-digit micromolar dose and at the same time, 12 and 13 showed marked cytotoxicity against A549 lung cancer cells with IC₅₀ of 0.12 and 0.08 μmol, respectively. The lead ester congeners were stable in the acidic pH, while hydrolyzed readily in the human blood plasma to release the active parent moieties.

Full text of DOI:10.1016/j.ejmech.2013.12.052

European Journal of Medicinal Chemistry 74 (2014) 135e144
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel lung adenocarcinoma and nuclear factor-kappa B (NF-kB)
inhibitors: Synthesis and evaluation of lantadene congeners
,
Sharad Kumar Suthar a, Hong L. Boon b, Manu Sharma a
*
a Department of Pharmacy, Jaypee University of Information Technology, Waknaghat 173234, India
b Cancer Research Initiative Foundation, Drug Discovery Laboratory, 12A Jalan TP5, Taman Perindustrian UEP, 47600 Subang Jaya, Selangor Darul Ehsan,
Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
The C-3, C-17 and C-22 congeners of pentacyclic triterpenoids reduced lantadene A (3), B (4) and 22b-
Received 26 August 2013
Received in revised form
24 December 2013
Accepted 26 December 2013
Available online 8 January 2014
hydroxyoleanolic acid (5) were synthesized and were tested in vitro for their NF-
potencies and cytotoxicity against A549 lung cancer cells. The lead congeners 12 and 13 showed IC50 of
0.56 and 0.42 mol, respectively against TNF- induced activation of NF- B. The congeners 12 and 13
exhibited inhibition of IKK in a single-digit micromolar dose and at the same time, 12 and 13 showed
marked cytotoxicity against A549 lung cancer cells with IC50 of 0.12 and 0.08 mol, respectively. The lead
kB and IKKb inhibitory
m
a
k
b
m
ester congeners were stable in the acidic pH, while hydrolyzed readily in the human blood plasma to
release the active parent moieties.
Keywords:
Tumor necrosis factor-alpha
Nuclear factor-kappa B
Inhibitor of nuclear factor-kappa B kinase
A549 lung cancer cells
Lantadene congeners
Ó 2014 Elsevier Masson SAS. All rights reserved.
b
1. Introduction
In last two decades, number of compounds has been reported to
inhibit NF-kB by interacting with key molecules in the signaling
Transcription factor nuclear factor-kappa B (NF-
k
B) influences
pathway [7]. Recently, pentacyclic triterpenoids lantadene A (1)
and B (2) isolated from the leaves of weed Lantana camara L.
(Verbenaceae) have attracted a lot of interest because of their
anticancer properties [8e11]. These compounds along with
the gene expression events that impact a large number of biological
processes including immune response, cell survival, differentiation,
and proliferation [1]. Recent studies have revealed key roles of NF-
k
B signaling in pathological conditions such as oncogenesis and
inflammation. NF- B is commonly over-expressed and constitu-
tively activated in different types of hematologic cancers and solid
tumors [2]. Constitutive activation of NF- B promotes tumor pro-
liferation, invasion, and metastasis. Moreover, NF- B activation al-
reduced lantadene A (3), reduced lantadene B (4), and 22b-
hydroxyoleanonic acid (5) showed potent cytotoxic effects in
antitumor screening launched by the National Cancer Institute, USA
[12] and were found to be potent inhibitors of NF-kB [8,9,13]. The
promising results obtained with a diverse but limited series of
target compounds have prompted us to further explore the new
analogs of lantadenes. Based on the SAR information obtained in
our previous work, we have synthesized new lantadene analogs
with different substituents at C-3 position of ring A, C-17 and C-22
positions of the ring D and E, respectively of lantadenes. These
congeners were evaluated for their in vitro anticancer activity and
k
k
k
lows malignant cells to escape apoptosis and therefore, contributes
to the development of radiation and chemotherapy resistance in
cancer cells [3,4]. Many chemotherapeutic agent has been found to
activate NF-
and subsequent failure of chemotherapy [5,6]. There are number of
evidences which suggest that the inhibition of NF- B activation can
kB via various approaches, leading to chemo-resistance
k
prevent tumor resistance to chemotherapeutic agents, shift the
death-survival balance toward apoptosis, and improve the efficacy
of current chemotherapeutic regimens [5]. Therefore, designing of
TNF-
line A549. The most active compounds were evaluated in vitro for
their inhibitory potency against the recombinant IKK in a non-
a-induced NF-kB activation against lung adenocarcinoma cell
b
NF-
k
B inhibitors is an interesting approach to develop the new
radioisotope Kinase-Glo Luminescent Kinase Assays. The most
active ester congeners were also studied for their stability in the
acidic pH and hydrolysis in the human blood plasma. The lead
cancer therapeutics.
congener was further docked with the crystal structure of IKK
predict the possible binding mode of the congener.
b to
* Corresponding author. Tel.: þ91 1792 239219; fax: þ91 1792 245362.
E-mail address: lantadene@hotmail.com (M. Sharma).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.12.052

136
S.K. Suthar et al. / European Journal of Medicinal Chemistry 74 (2014) 135e144
2. Results and discussion
lantadene congeners on tumor necrosis factor-alpha (TNF-
NF- B activation. The compounds (1e22) were evaluated in a dose-
dependent manner to determine the concentration needed to
inhibit 50% of TNF- -induced NF- B activation (IC50). The parent
compounds 1e5 showed inhibition of TNF- -induced NF- B activa-
tion in the range of 6.42e0.98 mol. The reduction of C-3 keto group
of compounds 1 and 2 into C-3 hydroxyl group of compounds 4 and 5
led to increase in the activity, whereas hydrolysis of C-22 ester side
chain of compound 1 and 2 into compound 5 led to decrease in the
a)-induced
k
2.1. Chemistry
a
k
The sequence of steps involved in synthesis of lantadene conge-
ners (3e22) is summarized in Schemes 1 and 2. Pentacyclic tri-
terpenoid lantadene A (1) and B (2) were isolated from the leaves of
weed L. camara Linn. The isolated compounds 1 and 2 differ only in
arrangement of atoms in side chain with E conformation present in
the side of 1, while side chain of compound 2 possessed Z confor-
mation. The isolated compounds 1 and 2 were selectively reduced
into corresponding compounds 3 and 4 in 87.60e89.88% yield, using
sodium borohydride as reducing agent and methanol (MeOH)e
tetrahydrofuran (THF) mixture as solvent. The compound 5 was
synthesized in 76.47% yield by alkali hydrolysis of 1 and 2 using 10%
ethanolic potassium hydroxide. The compounds 6e9 and 14 were
synthesized in a single step process by reaction of carbonyl chlorides
with hydroxyl group of compounds 3, 4, and 5 in the presence of
pyridine and 4-dimethylaminopyridine (4-DMAP) at 92e95 ꢀC for
10e14 h. At the same time, compounds 10e13 and 15e16 were
synthesized in two-step process. In the step-1, carboxyl group of
compounds was converted into anhydride function by reacting with
acetyl chloride in the presence of pyridine. In the step-II, anhydride
function of respective compounds was reacted with hydroxyl group
of compounds 3, 4 and 5 in presence of 4-DMAP to yield compounds
10e13 and 15e16. The 3-oxo group of compound 5 was reduced into
hydroxyl group by using sodium borohydride to yield compound 17.
a
k
m
activity. Thecongeners of3 and 4 showed inhibitionofTNF-
NF- B activation in the range of >10e0.42 mol, whereas the con-
geners of compound 5 showed IC50 > 10 mol. It further confirmed
thatC-22ester sidechainplayeda criticalrole inthe inhibition ofTNF-
-induced NF- B activation in A549 lung adenocarcinoma cell line.
a-induced
k
m
m
a
k
The hydrolysis of C-22 ester functionality in lantadenes led to sig-
nificant reduction in activity, supporting the notion that the mecha-
nism of inhibition is likely through a covalent Michael addition of
nucleophiles (such as SH from cysteine) from protein candidate(s) to
lantadenes (Fig. 1). The introduction of C-3 cinnamoyloxy function-
ality in parent compound 3 and 4 led to increase in the activity. This
further confirmed the importance of strongly electrophilic a,b-un-
saturated C]O group at C-22 and C-3 positions; played an important
role in binding of compounds to the receptor site. The results of in-
hibition of TNF-a-induced NF-kB activation in A549 lung adenocar-
cinoma cell line by compounds 1e22 are shown in Table 1.
2.2.2. In vitro phosphorylation assay
The 3b,22b-diacetoyloxy substituted compound 18 was synthesized
The NF-
mostly through upstream kinase-based phosphorylation of I
The phosphorylation of I BR leads to degradation of proteasome
and nucleus enrichment with p65 [14]. As compounds 12 and 13
showed potent NF- B inhibition, therefore, we decided to evaluate
the effect of parent compounds 3, 4, and its congeners 12, and 13
against upstream kinase IKK that have been reported to activate
the NF- B pathway (Table 2). The compounds were evaluated
in vitro for their inhibitory potency against the recombinant IKK in
a non-radioisotope Kinase-Glo Luminescent Kinase Assays (Prom-
ega, U.S.A.). It was found that 12 and 13 remarkably inhibited IKK
with IC50 of 1.20 and 0.94 mol, respectively (Table 2).
k
B can be activated by various signaling pathways and
from compound 17 by reaction of acetyl chloride with hydroxyl
functions of 17 in the presence of 4-DMAP. Similarly, compound 19
was synthesized from compound 5 by means of esterification of
carboxylic group using dimethyl sulfate. Whereas, the compound 19
was reduced by using sodium borohydride to yield compound 20.
The 3-hydroxylimino substituted compounds 21 and 22 were syn-
thesized from 1 and 2, by the reaction of hydroxylamine hydro-
chloride with 3-oxo group of 1 and 2.
k
BR.
k
k
b
k
b
b
2.2. Biological screening
m
2.2.1. In vitro inhibition of TNF-
lung adenocarcinoma cell line
a-induced NF-kB activation in A549
2.2.3. Inhibitory activity against A549 lung cancer cells
Chronic inflammation of lungs results in lung carcinoma via
activation of NF- B [15]. Since, the lantadene congeners showed
The human lung adenocarcinoma A549 cell line transiently co-
transfected with NF- B-luc was used to monitor the effects of
k
k
Scheme 1. Reagents and conditions: (a) NaBH4, MeOHeTHF, stir 7 h; (b) 10% ethanolic KOH, reflux 6 h; (c) R0eCOeCl/R0COOCOCH3, 4-DMAP, pyridine, reflux 92e95 ꢀC, 10e14 h.

S.K. Suthar et al. / European Journal of Medicinal Chemistry 74 (2014) 135e144
137
Scheme 2. Reagents and conditions: (a) NaBH4, MeOHeTHF, stir 7 h; (b) CH3COCl, 4-DMAP, pyridine, reflux 92e95 ꢀC, 10 h; (c) K2CO3, (CH3O)2SO2, acetone, reflux, 12 h; (d)
NH4OH.HCl, pyridine, reflux 92e95 ꢀC, 8 h.
ꢀ
marked inhibition of NF-
k
B, they were further evaluated for their
1.9 A). Apart from hydrogen bonding, pentacyclic triterpenoid
scaffold of the ester congener moiety-A also showed hydrophobic
and van der Waal interactions with Leu-104, Val-152, Leu-153, Leu-
in vitro cytotoxicity against lung cancer cells A549. The cytotoxicity
profile of parent compounds (1e5) and ester congeners (6e22) are
reported in Table 3. The parent pentacyclic triterpenoids 1, 2, 3, and
4 showed cytotoxicity against A549 lung cancer cells with IC50
160, and Ile-161 residues of the target protein, while 22
ecioyloxy side chain demonstrated hydrophobic and van der Waal
interactions with Leu-21 and Val-41 residues of IKK (Fig. 2b).
Estimated free energy of binding of ester congener moiety-B to
IKK
b-sen-
values of 2.84, 1.19, 0.79, and 0.43
mmol, respectively, whereas the
b
compound 5 showed IC50 > 10
mmol. The C-3 cinnamoyloxy
congener of compounds 3 and 4 showed marked cytotoxicity with
IC50 of 0.12 and 0.08 mol respectively (Table 3). From the cyto-
b
was found to be ꢁ4.65 kcal/mol. Analyses of docked complex
m
(Fig. 2c,d) showed that hydroxyl oxygen of carboxylic function was
hydrogen bonded to Tyr-98 (O]CeO/HeO, 2.0 A), whereas
ꢀ
toxicity profiles of compounds 1e22, it is evident that removal of
the ester side chain at C-22 position led to decrease in the activity.
carbonyl oxygen of carboxylic function formed a hydrogen bond
ꢀ
The strongly electrophilic
a
,
b-unsaturated carbonyl group of the
with Gly-102 residue of IKK
b
(OeC]O/HeN, 2.1 A). In addition,
ester side chain seems to play an important role in binding of
compounds to the receptor site.
carbonyl oxygen of ester congener moiety-B exhibited hydrogen
bonding with Asp-103 residue of the target protein with a distance
of 2.2 A (OeC]O/HeN). Furthermore, both the oxygens of car-
ꢀ
2.3. Predicting binding of lead ester congener 13 with IKK
b
boxylic acid function also interacted with Lys-106 residue of the
target protein (O]CeO/HeN, 2.3 A and OeC]O/HeN, 1.9 A).
ꢀ
ꢀ
Virtual screening of most potent ester congener 13 to IKK
carried out to rationalize the obtained biological results and to find
out the possible binding mode of 13 with IKK . The lead ester
congener 13 is a hybrid compound of two potent anticancer moi-
eties i.e. reduced lantadene B and cinnamic acid, and at the site of
action it is supposed to hydrolyzed back into parent moieties.
Therefore, we docked both of the parent moieties, reduced lanta-
dene B (moiety-A) and cinnamic acid (moiety-B) into the active site
b
was
Aromatic scaffold of moiety-B exhibited hydrophobic and van der
Waal interactions with Met-96 and Ile-161 residues of the IKK
protein (Fig. 2d). Besides hydrogen, hydrophobic and van der Waal
interactions, the SH group of Cys-99 residue of IKK that acts as
nucleophile was projected towards -carbon of the 22 -sen-
ecioyloxy side chain of moiety-A. This indicates possible covalent
Michael addition reaction between Cys-99 and -carbon that might
inhibitory potency of lead
b
b
b
b
b
b
have played a central role in the IKK
b
of IKK
b
(PDB ID: 3QA8) [16] using AutoDock tools 1.5.4 (Fig. 2aed).
compound 13 (Figs. 1 and 2b).
The estimated free energy of binding of docked ester congener
moiety-A was found to be ꢁ5.87 kcal/mol. Analyses of docked
2.4. Stability studies of ester congeners (12 and 13)
complex (Fig. 2a,b) indicated that 22b-senecioyloxy side chain and
C-28 carboxylic group were critical for IKK
the oxygens of ester function of 22 -senecioyloxy side chain
interacted with amine hydrogens of Arg-31 by means of hydrogen
b
inhibitory activity. Both
2.4.1. Chemical hydrolysis of lead ester congeners in simulated
gastric fluid
b
A successful ester congener should be stable in the acidic pH of
stomach. The compounds 12 and 13 that emerged as lead ester
congeners from the synthesized series were further screened for
their chemical stability studies. Ester congeners 12 and 13 were
exposed to simulated gastric fluid of pH 2 for 0, 2, 5, 8, and 12 h.
Results of HPLC analysis of ester congeners exposed to the simu-
lated gastric fluid of pH 2 showed that only 0.0, 3.57, 9.33,15.26, and
23.42% of 12 and 0.0, 2.80, 7.52, 12.39, and 18.95% of 13 were
ꢀ
ꢀ
bonding (O]CeO/HeN, 2.3
A and OeC]O/HeN, 2.7 A).
Furthermore, carbonyl oxygen of ester group formed a hydrogen
ꢀ
bond with Gln-40 residue of target protein (OeC]O/HeN, 2.2 A).
The hydroxyl oxygen of C-28 carboxylic group showed a hydrogen
bond with hydroxyl hydrogen of Tyr-98 residue (O]CeO/HeO,
ꢀ
1.9 A), whereas carbonyl oxygen of acid function exhibited
hydrogen bonding with Gly-101 residue of IKKb (OeC]O/HeN,
Fig. 1. Proposed covalent binding of IKKb with ester congeners of lantadene, which leads to inhibition of NF-kB.

138
S.K. Suthar et al. / European Journal of Medicinal Chemistry 74 (2014) 135e144
Table 1
eventually undergoes hydrolysis. The ester congener 13 showed
slower rate of hydrolysis than 12, because it possessed geminal
methyl groups in the proximity of ester function. The geminal
methyl groups will have slightly higher ability to decrease the
electrophilicity of ester carbonyl carbon. On the contrary, methyl
groups of the side chain of ester congener 12 were present in the
vicinal position and therefore,12 exhibited slightly higher degree of
hydrolysis than 13.
TNF-
a
-induced NF-
k
B activation inhibitory activities (IC50 in
m
mol) of parent com-
IC50
pounds (1e5), congeners (6e22).
Compd.
IC50
Compd.
1
2
3
4
5
6
7
8
1.06 ꢂ 0.46
1.56 ꢂ 0.04
0.98 ꢂ 0.02
1.02 ꢂ 0.62
6.42 ꢂ 1.24
2.80 ꢂ 0.24
2.32 ꢂ 0.01
5.04 ꢂ 1.02
4.64 ꢂ 1.76
4.18 ꢂ 1.68
3.70 ꢂ 0.32
0.56 ꢂ 0.06
0.42 ꢂ 0.01
14
15
16
17
18
19
20
21
22
>10
>10
2.42 ꢂ 0.24
>10
>10
>10
>10
>10
3. Conclusion
9
>10
The C-3, C-22 and C-17 congeners of pentacyclic triterpenoids 3
and 4 showed marked cytotoxicity and inhibitory potential against
TNF-a-induced activation of NF-kB in lung cancer cell line A549.
The congeners of 5 showed feeble activity, supporting the percep-
tion that the mechanism of inhibition is likely through a covalent
Michael addition of nucleophiles (such as SH from cysteine) from
protein candidate(s) to reduced lantadenes. The ester congener 13
10
11
12
13
Results are given as the mean of at least three independent experiments with
triplicates in each experiment.
inhibited kinase activity of IKK
centration. The experimental and docking studies revealed that C-
22 ester side chain with -unsaturated carbonyl group was critical
for the activity. Moreover, ester congener 13 showed stability in the
acidic pH and was hydrolyzed readily in the human blood plasma to
release the two active moieties. The lead congener 12 and 13 are
promising anticancer candidates against lung cancer, warranting
further investigations.
b in a single-digit micromolar con-
hydrolyzed after the exposure time of 0, 2, 5, 8, and 12 h, respec-
tively (Table 4). The chemical structures of ester congeners 12 and
13 differ only at the ester side chain present in C-22 position. The
a,b
22
b
-angeloyloxy side chain is present in ester congener 12, while
-senecioyloxy side chain is present in the ester congener 13. It
22b
can be inferred from the results of chemical hydrolysis study that
ester congener 13 showed slightly higher resistances towards hy-
drolysis in comparison to the 12 and both of them survived the
stomach pH conditions.
4. Experimental
2.4.2. Metabolic stability of lead ester congeners (12 and 13) in
human plasma
4.1. Material and methods
To study plasma hydrolysis or susceptibility of ester congeners
12 and 13 towards human plasma esterases, they were exposed to
80% human plasma for 0, 15, 30, 60, and 120 min and extent of
hydrolysis was monitored by HPLC. On exposure to human plasma,
ester congeners 12 and 13 were hydrolyzed at a notably higher rate
than the rate of their hydrolysis observed in the simulated gastric
fluid. A level of ester congeners hydrolyzed was found to be 0.0,
25.84, 45.32, and 53.19, and 63.25% of 12 and 0.0, 25.78, 44.50,
51.87, and 60.76% of 13 after the exposure period of 0,15, 30, 60, and
120 min, respectively (Table 5). Ester congener 13 showed slightly
lesser degree of hydrolysis than ester congener 12. HPLC results
indicated that ester congeners 12 and 13 underwent rapid hydro-
lysis in human plasma to liberate the parent drug molecules to
reach the site of action, while in the simulated gastric fluid of pH 2,
they survived the stomach conditions. Stability of an ester bond
depends on the reactivity of carbonyl carbon, as it gets attacked by
the reactive functional groups. The oxygen atom (oxygen atom
neighboring to carbonyl carbon of ester bond) of ester function of
synthesized ester congeners is an electron withdrawing in nature,
and therefore, it decreases the electron density on the carbonyl
carbon. In this course, electron deficient carbonyl carbon will
become more prone to experience a nucleophilic attack and
Merck TLC plates silica gel 60 F254 (Merck, Germany) were used
to monitor the progress of reactions. Melting points reported are
uncorrected and were recorded on a digital melting point appa-
ratus (Indosati scientific lab equipments, India). Chromatography
columns of appropriate sizes were used with silica gel (100e
200 mesh) as adsorbent. FT-IR spectra were recorded on a Perki-
nElmer spectrum 400 FT-IR and FT-NIR spectrometer using potas-
sium bromide pellets. 1H NMR and 13C NMR spectra were recorded
at 400 and 100 MHz, respectively with Bruker AVANCE II 400 NMR
spectrometer using CDCl3, DMSO-d6, and a mixture of CDCl3 and
DMSO-d6 as solvents, and tetramethylsilane was used as internal
standard. ESI-MS spectra were recorded with Waters Micromass Q-
T of micro Mass spectrometer using electrospray ionization at
70 eV. For elemental analysis of compounds, 2400 CHN analyzer
(PerkinElmer, USA) was used. All the reagents and anhydrous
Table 3
In vitro cytotoxicity profile (IC50 in
(6e22) against A549 cell line.
mmol) of parent compounds (1e5) and congeners
Compd.
IC50
Compd.
IC50
1
2
3
4
2.84 ꢂ 0.72
1.19 ꢂ 0.28
0.79 ꢂ 0.01
0.43 ꢂ 0.03
>10
14
15
16
17
>10
>10
3.64 ꢂ 1.24
Table 2
>10
In vitro IKKb inhibition by parent compounds (3 and
4) and congener (12 and 13).
5
18
>10
6
7
8
9
10
11
12
13
5.42 ꢂ 1.24
4.90 ꢂ 1.20
7.36 ꢂ 1.06
7.20 ꢂ 1.20
6.80 ꢂ 2.40
6.52 ꢂ 2.32
0.12 ꢂ 0.02
0.08 ꢂ 0.02
19
20
21
22
>10
>10
>10
>10
Compd.
IC50 (mmol)
3
4
12
13
2.62 ꢂ 0.82
4.24 ꢂ 0.94
1.20 ꢂ 0.42
0.94 ꢂ 0.04
Cisplatin
21.3 ꢂ 3.62
Results are given as the mean of at least three in-
dependent experiments with triplicates in each
experiment.
Results are given as the mean of at least three independent experiments with
triplicates in each experiment.

S.K. Suthar et al. / European Journal of Medicinal Chemistry 74 (2014) 135e144
139
Fig. 2. (a) Binding of ester congener moiety-A of 13 into the binding site of IKK
with ester congener moiety-A, are highlighted; (b) stereoview of the docked structure of ester congener moiety-A of 13 into the active site of IKK
in hydrogen, hydrophobic, and van der Waal interactions with ester congener moiety-A, are highlighted; (c) binding of ester congener moiety-B of 13 within the active site of IKK
The amino acid residues Tyr-98, Gly-102, Asp-103, and Lys-106 involved in the hydrogen bond interactions with ester congener moiety-B, are highlighted; (d) docking of ester
b. The amino acids Arg-31, Gln-40, Tyr-98, and Gly-101 involved in the hydrogen bond interactions
b. The amino acid residues involved
b.
congener moiety-B of 13 into the active site of IKKb. The amino acid residues engaged in hydrogen, hydrophobic, and van der Waal interactions with ester congener moiety-B, are
highlighted.
solvents were purchased from local Indian suppliers and were used
without further purification or distillation, unless otherwise stated.
For RP-HPLC analysis, Waters HPLC system comprised of Waters
4.6. Synthesis of 3
28-oic acids (6e16)
b-substituted and 22b-substituted olean-12-en-
717plus autosampler, Waters 515 HPLC pumps, Waters Spherisorb
Compounds 6e9, 14 were synthesized through a single step
process, while compounds 10e13 and 15e16 were synthesized via
two step processes. In the first step of synthesis of compounds 10e
13 and 15e16, acid function was converted into anhydride function
(Supplementary data: Scheme S1). The acid and acetyl chloride in
the presence of pyridine were refluxed in dichloromethane (DCM)
for 4e5 h. Reaction mixture was concentrated and washed with
chloroform (100 ml ꢃ 3) under reduced pressure at 60e65 ꢀC to
afford solid to semisolid anhydride products of respective acids,
which were used in the next step without further purification.
ꢀ
ODS2 (80 A, 5
detector, and empower software system 2.1 was used.
m
m, 4.6 ꢃ 250 mm) C18 column, Waters 2996 PDA
4.2. Plant material
Leaves of L. camara L. were collected in September 2010 from
Palampur (HP), India. The leaves were shade-dried and powdered.
A voucher specimen (LC; 097 JUIT) was deposited in the Herbarium
at the Jaypee University of Information Technology Waknaghat,
India.
In the synthesis of 3b-substituted (6e13) and 22b-substituted
(14e16) olean-12-en-28-oic acids step, compounds 3, 4, and 5 with
appropriate carbonyl chlorides/anhydrides were refluxed in pyri-
dine in the presence of 4-DMAP for 10e14 h at 92e95 ꢀC (Scheme
1). Reaction mixture was poured into 10% HCl solution and
precipitated product was extracted with DCM and washed further
three times with 10% HCl solution (100 ml ꢃ 3). Organic layer was
evaporated to dryness and the reaction mixture obtained was
chromatographed over silica gel (100e200 mesh) and eluted with
varying ratio of hexaneeethyl acetate to give purified products (6e
16).
4.3. Extraction and isolation of lantadene A (1) and B (2)
Extraction and isolation of lantadene A (1) and B (2) were car-
ried out as per our previously reported procedure with slight
modifications (Supplementary data: page 6e7) [17].
4.4. Synthesis of 3
b
-hydroxy-22
b-angeloyloxy-olean-12-en-28-oic
acid (3) and 3
(4)
b
-hydroxy-22 -senecioyloxy-olean-12-en-28-oic acid
b
4.6.1. 3b-Acetoyloxy-22b-angeloyloxy-olean-12-en-28-oic acid (6)
Reduced lantadene A (3) and B (4) were synthesized as per
procedure reported by us previously (Scheme 1) (Supplementary
data: page 7e8) [17].
Yield: 84.10%, mp: 178e180 ꢀC. Anal. calcd. for C37H56O6
(596.41): %C, 74.46; H, 9.46. Found: %C, 74.50; H, 9.45. IR (KBr,
cmꢁ1): 2950.19, 2877.28 (CeH), 1736.33 (C]O ester), 1719.91 (C]O
acid), 1649.60 (C]C). 1H NMR (400 MHz, CDCl3,
d ppm): 5.8822e
4.5. Synthesis of 22
b
-hydroxy-3-oxo-olean-12-en-28-oic acid (5)
5.9399 (1H, m, C-33-H), 5.2740e5.2889 (1H, t, J ¼ 2.98 Hz, C-12-H),
4.9972e5.0118 (1H, t, J ¼ 2.92 Hz, C-22-H), 4.4116e4.4512 (1H, t,
J ¼ 7.92 Hz, C-3-H), 2.9424e2.9862 (1H, dd, J ¼ 13.92, 3.96 Hz, C-18-
H), 1.9830 (3H, s, C-20-H), 1.0885 (3H, s, CH3), 0.9247 (3H, s, CH3),
0.8730 (3H, s, CH3), 0.8227 (3H, s, CH3), 0.8015 (3H, s, CH3), 0.7957
Compound 5 was synthesized from compound 1 and 2 as per
procedure described by us previously with slight modifications
(Scheme 1) (Supplementary data: page 8e9) [12].

140
S.K. Suthar et al. / European Journal of Medicinal Chemistry 74 (2014) 135e144
Table 4
CH3), 1.0142 (3H, s, CH3), 1.0058 (3H, s, CH3), 0.9493 (3H, s, CH3),
0.9077 (3H, s, CH3), 0.8131 (3H, s, CH3). 13C NMR (100 MHz, CDCl3,
d
Chemical stability of ester congeners (12e13) in simulated gastric fluid of pH 2.
ppm): 181.10 (C-28), 166.52 (C-31), 166.31 (C-10), 142.30 (C-13),
Time
% Ester congener remaining in simulated gastric
fluid
139.84 (C-33), 132.74 (C-50), 130.44 (C-20), 129.54 (C-30 & C-70),
128.71 (C-32), 128.33 (C-40 & C-60), 123.35 (C-12), 81.58 (C-3), 75.97
(C-22), 55.45 (C-5), 50.61 (C-17), 47.66 (C-9), 45.90 (C-19), 42.13 (C-
14), 39.53 (C-8), 39.30 (C-18), 38.77 (C-4), 38.12 (C-1), 37.99 (C-21),
37.01 (C-10), 33.71 (C-29), 32.62 (C-7), 30.06 (C-20), 28.21 (C-15),
27.63 (C-23), 26.18 (C-27), 25.88 (C-30), 25.75 (C-16), 23.59 (C-11),
23.47 (C-2), 20.45 (C-6), 18.21 (C-35), 16.98 (C-26), 16.73 (C-24),
15.63 (C-34),15.52 (C-25). ESI-MS (negative-ion mode, m/z): 658.00
(Mꢁ), 657.20 (Mꢁ ꢁ 1).
Compd. 12
Compd. 13
0 h
2 h
5 h
8 h
12 h
100
100
96.43
90.67
84.74
76.58
97.20
92.48
87.61
81.05
(3H, s, CH3), 0.6959 (3H, s, CH3). 13C NMR (100 MHz, CDCl3,
d ppm):
179.56 (C-28), 171.12 (C-10), 166.34 (C-31), 143.08 (C-13), 138.79 (C-
33),127.69 (C-32),122.62 (C-12), 80.88 (C-3), 75.96 (C-22), 55.26 (C-
5), 50.54 (C-17), 47.55 (C-9), 45.97 (C-19), 41.83 (C-14) 39.22 (C-8),
38.35 (C-18), 38.28 (C-4), 38.07 (C-1), 37.70 (C-21), 36.95 (C-10),
33.70 (C-29), 32.55 (C-7), 30.04 (C-20), 28.04 (C-15), 27.55(C-23),
26.15 (C-27), 25.85 (C-30), 24.20 (C-16), 23.51 (C-11), 23.43 (C-2),
21.35 (C-20), 20.56 (C-6), 18.11 (C-35), 17.01 (C-26), 16.70 (C-24),
15.65 (C-34), 15.51 (C-25). ESI-MS (negative-ion mode, m/z): 596.30
(Mꢁ), 595.30 (Mꢁ ꢁ 1).
4.6.4. 3b-Benzoyloxy-22b-senecioyloxy-olean-12-en-28-oic acid
(9)
Yield: 88.17%, mp: 115e116 ꢀC. Anal. calcd. for C42H58O6
(658.42): %C, 76.56; H, 8.87. Found: %C, 76.60; H, 8.85. IR (KBr,
cmꢁ1): 2950.46, 2877.90, 2665.64 (CeH), 1716.35 (C]O), 1649.60
(C]C). 1H NMR (400 MHz, CDCl3,
d ppm): 8.0327e8.0921 (2H, m, C-
30 & C-70-Ar-H), 7.5285e7.6294 (1H, m, C-50-Ar-H), 7.4187e7.4861
(2H, m, C-40 & C-60-Ar-H), 5.5628e5.5689 (1H, t, J ¼ 1.22 Hz, C-32-
H), 5.3730e5.3896 (1H, t, J ¼ 3.32 Hz, C-12-H), 5.0464e5.0615 (1H,
t, J ¼ 3.02 Hz, C-22-H), 4.7309e4.7716 (1H, m, C-3-H), 3.0210e
3.0653 (1H, dd, J ¼ 13.72, 4.04 Hz, C-18-H), 1.1943 (3H, s, CH3),
1.0264 (3H, s, CH3), 1.0167 (3H, s, CH3), 1.0062 (3H, s, CH3), 0.9499
(3H, s, CH3), 0.8970 (3H, s, CH3), 0.8201 (3H, s, CH3). 13C NMR
4.6.2. 3b-Acetoyloxy-22b-senecioyloxy-olean-12-en-28-oic acid (7)
Yield: 82.09%, mp: 172e174 ꢀC. Anal. calcd. for C37H56O6
(596.41): %C, 74.46; H, 9.46. Found: %C, 74.49; H, 9.47. IR (KBr,
cmꢁ1): 2949.03 (CeH),1720.98 (C]O acid),1653.63 (C]C). 1H NMR
(100 MHz, CDCl3, d
ppm): 179.16 (C-28),166.31 (C-31),165.34 (C-10),
157.12 (C-33),143.06 (C-13),132.74 (C-50),130.95 (C-20),129.54 (C-30
& C-70), 128.33 (C-40 & C-60), 122.57 (C-12), 115.99 (C-32), 81.52 (C-
3), 75.28 (C-22), 55.38 (C-5), 50.63 (C-17), 47.59 (C-9), 46.01 (C-19),
41.96 (C-14), 39.31 (C-8), 38.47 (C-18), 38.19 (C-4), 38.11 (C-1), 37.68
(C-21), 37.01 (C-10), 33.79 (C-29), 32.64 (C-7), 30.09 (C-20), 28.22
(C-15), 27.63 (C-23), 27.42 (C-35), 26.31 (C-27), 25.88 (C-30), 24.14
(C-16), 23.59 (C-11), 23.51 (C-2), 20.22 (C-6), 18.20 (C-34), 16.98 (C-
26), 16.96 (C-24), 15.54 (C-25). ESI-MS (negative-ion mode, m/z):
658.30 (Mꢁ), 657.00 (Mꢁ ꢁ 1).
(400 MHz, CDCl3,
d
ppm): 5.4733e5.4789 (1H, t, J ¼ 1.12 Hz, C-32-
H), 5.2685e5.2841 (1H, t, J ¼ 3.12 Hz, C-12-H), 4.9927e5.0069 (1H,
t, J ¼ 2.84 Hz, C-22-H), 4.4113e4.4508 (1H, t, J ¼ 7.90 Hz, C-3-H),
2.9395e2.9834 (1H, dd, J ¼ 13.92, 3.92 Hz, C-3-H), 1.9825 (3H, s, C-
20-H), 1.0870 (3H, s, CH3), 0.9227 (3H, s, CH3), 0.8722 (3H, s, CH3),
0.8213 (3H, s, CH3), 0.8007 (3H, s, CH3), 0.7959 (3H, s, CH3), 0.6922
(3H, s, CH3). 13C NMR (100 MHz, CDCl3,
d ppm): 180.09 (C-28),171.14
(C-10), 166.33 (C-31), 157.16 (C-33), 143.09 (C-13), 122.59 (C-12),
115.95 (C-32), 80.88 (C-3), 75.96 (C-22), 55.25 (C-5), 50.55 (C-17),
47.55 (C-9), 45.97 (C-19), 41.81 (C-14) 39.21 (C-8), 38.29 (C-18),
38.24 (C-4), 38.06 (C-1), 37.69 (C-21), 36.95 (C-10), 33.70 (C-29),
32.53 (C-7), 30.04 (C-20), 28.04 (C-15), 27.53 (C-23), 27.49 (C-35),
26.15 (C-27), 25.85 (C-30), 24.20 (C-16), 23.50 (C-11), 23.43 (C-2),
21.34 (C-20), 20.56 (C-6), 18.39 (C-34), 17.02 (C-26), 16.69 (C-24),
15.50 (C-25). ESI-MS (negative-ion mode, m/z): 596.30 (Mꢁ),
595.30 (Mꢁ ꢁ 1).
4.6.5. 3
b-(2-Chlorobenzoyloxy)-22b-angeloyloxy-olean-12-en-28-
oic acid (10)
Yield: 85.53%, mp: 176e178 ꢀC. Anal. calcd. for C42H57ClO6
(692.38): %C, 72.76; H, 8.29. Found: %C, 72.82; H, 8.31. IR (KBr,
cmꢁ1): 2948.62, 2877.90 (CeH), 1720.58 (C]O), 1548.70 (C]C). 1H
NMR (400 MHz, CDCl3,
d
ppm): 7.7959e7.8191 (1H, m, C-70-Ar-H),
7.3828e7.4741 (2H, m, C-40 & C-50-Ar-H), 7.2916e7.3325 (1H, m, C-
60-Ar-H), 5.9508e6.0081 (1H, m, C-33-H), 5.3552e5.3709 (1H, t,
J ¼ 3.14 Hz, C-12-H), 5.0753e5.0892 (1H, t, J ¼ 2.78 Hz, C-22-H),
4.7682e4.8085 (1H, m, C-3-H), 3.0253e3.0687 (1H, dd, J ¼ 13.68,
3.72 Hz, C-18-H), 1.1884 (3H, s, CH3), 0.9928 (3H, s, CH3), 0.9828
(3H, s, CH3), 0.9761 (3H, s, CH3), 0.9450 (3H, s, CH3), 0.9006 (3H, s,
4.6.3. 3b-Benzoyloxy-22b-angeloyloxy-olean-12-en-28-oic acid (8)
Yield: 90.00%, mp: 117e119 ꢀC. Anal. calcd. for C42H58O6
(658.42): %C, 76.56; H, 8.87. Found: %C, 76.55; H, 8.88. IR (KBr,
cmꢁ1): 3064.72, 2950.36, 2876.88 (CeH), 1716.97 (C]O), 1650.27
(C]C). 1H NMR (400 MHz, CDCl3,
d ppm): 8.0325e8.0887 (2H, m,
C-30 & C-70-Ar-H), 7.5310e7.6216 (1H, m, C-50-Ar-H), 7.4215e7.4646
(2H, m, C-40 & C-60-Ar-H), 5.9550e6.0677 (1H, m, C-33-H), 5.3776e
5.3950 (1H, t, J ¼ 3.48 Hz, C-12-H), 5.0955e5.1105 (1H, t,
J ¼ 3.00 Hz, C-22-H), 4.7317e4.7724 (1H, m, C-3-H), 3.0441e3.0865
(1H, dd, J ¼ 13.64, 4.40 Hz, C-18-H),1.1947 (3H, s, CH3),1.0267 (3H, s,
CH3), 0.7889 (3H, s, CH3). 13C NMR (100 MHz, CDCl3,
d ppm): 181.40
(C-28), 166.35 (C-10), 165.71 (C-31), 143.11 (C-13), 138.78 (C-33),
133.48 (C-50), 132.30 (C-30), 131.26 (C-70), 131.05 (C-40), 130.93 (C-
20), 127.67 (C-32), 126.56 (C-60), 122.59 (C-12), 82.63 (C-3), 75.99 (C-
22), 55.38 (C-5), 50.59 (C-17), 47.57 (C-9), 45.98 (C-19), 41.87 (C-14),
39.22 (C-8), 38.25 (C-18), 38.17 (C-4), 37.96 (C-1), 37.69 (C-21),
36.99 (C-10), 33.70 (C-29), 32.55 (C-7), 30.07 (C-20), 28.21 (C-15),
27.55 (C-23), 26.15 (C-27), 25.91 (C-30), 24.22 (C-16), 23.50 (C-11),
23.46 (C-2), 20.56 (C-6), 18.13 (C-35), 17.02 (C-26), 16.70 (C-24),
15.65 (C-34), 15.53 (C-25). ESI-MS (negative-ion mode, m/z): 691.60
(Mꢁ).
Table 5
Metabolic stability of ester congeners (12e13) in human plasma.
Time
% Ester congener remaining in human plasma
Compd. 12
Compd. 13
0 min
100
100
4.6.6. 3
oic acid (11)
b-(2-Chlorobenzoyloxy)-22b-senecioyloxy-olean-12-en-28-
15 min
30 min
60 min
120 min
74.16
54.68
46.81
36.75
74.72
55.50
48.13
39.24
Yield: 84.52%, mp: 170e172 ꢀC. Anal. calcd. for C42H57ClO6
(692.38): %C, 72.76; H, 8.29. Found: %C, 72.79; H, 8.30. IR (KBr,

S.K. Suthar et al. / European Journal of Medicinal Chemistry 74 (2014) 135e144
141
cmꢁ1): 2949, 2877 (CeH), 1717 (C]O), 1651 (C]C). 1H NMR
(400 MHz, CDCl3,
ppm): 7.7950e7.8183 (1H, m, C-70-Ar-H),
27.99 (C-23), 27.49 (C-35), 26.10 (C-27), 25.83 (C-30), 24.17 (C-16),
23.57 (C-11), 23.40 (C-2), 20.52 (C-6), 18.06 (C-34), 16.99 (C-26),
16.82 (C-24),15.48 (C-25). ESI-MS (negative-ion mode, m/z): 683.70
(Mꢁ).
d
7.3818e7.4814 (2H, m, C-40 & C-50-Ar-H), 7.2909e7.3319 (1H, m, C-
60-Ar-H), 5.5659e5.5721 (1H, t, J ¼ 1.24 Hz, C-32-H), 5.3732e5.3897
(1H, t, J ¼ 3.30 Hz, C-12-H), 5.0436e5.0578 (1H, t, J ¼ 2.84 Hz, C-22-
H), 4.7666e4.8069 (1H, m, C-3-H), 3.0136e3.0580 (1H, dd,
J ¼ 13.56, 4.44 Hz, C-18-H), 1.1922 (3H, s, CH3), 1.0137 (3H, s, CH3),
0.9908 (6H, s, 2ꢃ CH3), 0.9799 (3H, s, CH3), 0.8939 (3H, s, CH3),
4.6.9. 22b-Benzoyloxy-3-oxo-olean-12-en-28-oic acid (14)
Yield: 55.15%, mp: 123e124 ꢀC. Anal. calcd. for C37H50O5
(574.37): %C, 77.31; H, 8.77. Found: %C, 77.25; H, 8.75. IR (KBr,
cmꢁ1): 2949.41 (CeH), 1720.54 (C]O), 1603.96 (C]C). 1H NMR
0.8124 (3H, s, CH3). 13C NMR (100 MHz, CDCl3,
d ppm): 178.49 (C-
28), 165.41 (C-10), 164.66 (C-31), 156.13 (C-33), 142.02 (C-13), 132.46
(C-50), 131.34 (C-30), 130.21 (C-70), 130.01 (C-40), 129.94 (C-20),
125.52 (C-60), 121.49 (C-12), 114.92 (C-32), 81.61 (C-3), 74.22 (C-22),
54.37 (C-5), 49.57 (C-17), 46.54 (C-9), 44.95 (C-19), 40.89 (C-14),
38.25 (C-8), 37.37 (C-18), 37.17 (C-4), 36.93 (C-1), 36.66 (C-21),
35.96 (C-10), 32.72 (C-29), 31.56 (C-7), 29.04 (C-20), 27.17 (C-15),
26.56 (C-23), 26.42 (C-35), 25.27 (C-27), 25.11 (C-30), 24.85 (C-16),
23.17 (C-11), 23.11 (C-2),19.20 (C-6),17.13 (C-34),15.98 (C-26),15.94
(C-24), 14.49 (C-25). ESI-MS (negative-ion mode, m/z): 691.60 (Mꢁ).
(400 MHz, CDCl3, d
ppm): 7.7746e7.7974 (2H, m, C-30 & C-70-Ar-H),
7.3507e7.3908 (1H, m, C-50-Ar-H), 7.2097e7.2294 (2H, m, C-40 & C-
60-Ar-H), 5.3667e5.3839 (1H, t, J ¼ 3.44 Hz, C-12-H), 5.1223e5.1373
(1H, t, J ¼ 3.00 Hz, C-22-H), 3.0982e3.1430 (1H, dd, J ¼ 13.80,
4.12 Hz, C-18-H), 2.4412e2.5270 (1H, m, C-2-Ha), 2.2664e2.3317
(1H, m, C-2-Hb), 1.1857 (3H, s, CH3), 1.1249 (3H, s, CH3), 1.0218 (3H,
s, CH3), 0.9854 (3H, s, CH3), 0.8841 (3H, s, CH3), 0.8210 (3H, s, CH3),
0.7370 (3H, s, CH3). 13C NMR (100 MHz, CDCl3,
d ppm): 217.68 (C-3),
178.11 (C-28), 165.12 (C-10), 143.09 (C-13), 132.90 (C-50), 130.06 (C-
20),129.47 (C-30 & C-70),128.28 (C-40 & C-60),122.63 (C-12), 76.82 (C-
22), 55.32 (C-5), 50.69 (C-17), 47.44 (C-9), 46.87 (C-4), 45.96 (C-19),
42.09 (C-14), 39.23 (C-8), 39.11 (C-18), 38.52 (C-1), 37.60 (C-21),
36.78 (C-10), 34.12 (C-2), 33.66 (C-29), 32.19 (C-7), 29.98 (C-20),
27.61 (C-15), 26.46 (C-23), 26.26 (C-27), 25.80 (C-30), 24.01 (C-16),
23.53 (C-11), 21.49 (C-6), 19.54 (C-26), 16.81 (C-24), 15.11 (C-25).
ESI-MS (negative-ion mode, m/z): 574.30 (Mꢁ), 573.30 (Mꢁ ꢁ 1).
4.6.7. 3b-Cinnamoyloxy-22b-angeloyloxy-olean-12-en-28-oic acid
(12)
Yield: 89.50%, mp: 174e176 ꢀC. Anal. calcd. for C44H60O6
(684.44): %C, 77.16; H, 8.83. Found: %C, 77.13; H, 8.85. IR (KBr,
cmꢁ1): 3266 (OeH of COOH), 2950, 2877 (CeH), 1719 (C]O), 1649
(C]C). 1H NMR (400 MHz, CDCl3,
d ppm): 7.6478e7.6878 (1H, d,
J ¼ 16 Hz, C-30-H), 7.5207e7.5446 (2H, m, C-50 & C-90-Ar-H),
7.3725e7.3890 (3H, m, C-60, C-80 & C-70-Ar-H), 6.4243e6.4643 (1H,
d, J ¼ 16 Hz, C-20-H), 5.9749e6.0328 (1H, m, C-33-H), 5.3658e
5.3833 (1H, t, J ¼ 3.50 Hz, C-12-H), 5.0882e5.1019 (1H, t, J ¼ 2.74 Hz,
C-22-H), 4.6313e4.6713 (1H, t, J ¼ 8.00 Hz, C-3-H), 3.0294e3.0740
(1H, dd, J ¼ 13.96, 3.92 Hz, C-18-H),1.1817 (3H, s, CH3),1.0093 (3H, s,
CH3), 0.9796 (3H, s, CH3), 0.9532 (3H, s, CH3), 0.9234 (3H, s, CH3),
0.9029 (3H, s, CH3), 0.7933 (3H, s, CH3). 13C NMR (100 MHz, CDCl3,
4.6.10. 22b-(2-Chlorobenzoyloxy)-3-oxo-olean-12-en-28-oic acid
(15)
Yield: 49.57%, mp: 193e195 ꢀC. Anal. calcd. for C37H49ClO5
(608.33): %C, 72.94; H, 8.11. Found: %C, 72.93; H, 8.13. IR (KBr,
cmꢁ1): 2955.55, 2927.40, 2874.30 (CeH), 1735.52 (C]O keto),
1703.58 (C]O), 1591.67 (C]C). 1H NMR (400 MHz, CDCl3,
d ppm):
7.8866e7.9101 (1H, m, C-70-Ar-H), 7.3686e7.4244 (2H, m, C-40 & C-
50-Ar-H), 7.2473e7.2884 (1H, m, C-60-Ar-H), 5.3250e5.3424 (1H, t,
J ¼ 3.48 Hz, C-12-H), 5.2060e5.2206 (1H, t, J ¼ 2.92 Hz, C-22-H),
3.0374e3.0820 (1H, dd, J ¼ 13.76, 4.24 Hz, C-18-H), 2.4468e2.5326
(1H, m, C-2-Ha), 2.2759e2.3412 (1H, m, C-2-Hb),1.1811 (3H, s, CH3),
1.1258 (3H, s, CH3), 1.0210 (3H, s, CH3), 0.9776 (3H, s, CH3), 0.8789
(3H, s, CH3), 0.8395 (3H, s, CH3), 0.7925 (3H, s, CH3). 13C NMR
d
ppm): 179.30 (C-28), 165.85 (C-31), 165.28 (C-10), 143.35 (C-30),
142.06 (C-13),137.75 (C-33),133.47 (C-40), 129.15 (C-70), 127.83 (C-60
& C-80), 127.02 (C-50 & C-90), 126.63 (C-32), 121.57 (C-12), 116.30 (C-
20), 79.89 (C-3), 74.94 (C-22), 54.27 (C-5), 49.54 (C-17), 46.53 (C-9),
44.95 (C-19), 40.80 (C-14), 38.21 (C-8), 37.20 (C-18), 37.07 (C-4),
36.92 (C-1), 36.66 (C-21), 35.95 (C-10), 32.66 (C-29), 31.51 (C-7),
29.01 (C-20), 27.08 (C-15), 26.51 (C-23), 25.11 (C-27), 24.85 (C-30),
23.19 (C-16), 22.58 (C-11), 22.41 (C-2), 19.52 (C-6), 17.09 (C-35),
16.02 (C-26), 15.84 (C-24), 14.61 (C-34), 14.47 (C-25). ESI-MS
(negative-ion mode, m/z): 683.70 (Mꢁ).
(100 MHz, CDCl3, d
ppm): 217.99 (C-3), 180.46 (C-28), 170.27 (C-10),
142.91 (C-13), 133.34 (C-50), 132.37 (C-30), 131.41 (C-70), 130.96 (C-
40), 129.28 (C-20), 126.62 (C-60), 122.61 (C-12), 77.37 (C-22), 55.22
(C-5), 50.67 (C-17), 47.41 (C-9), 46.83 (C-4), 45.80 (C-19), 41.95 (C-
14), 39.21 (C-8), 39.04 (C-18), 38.27 (C-1), 37.57 (C-21), 36.73 (C-10),
34.09 (C-2), 33.62 (C-29), 32.05 (C-7), 30.02 (C-20), 27.56 (C-15),
26.41 (C-23), 26.32 (C-27), 25.81 (C-30), 24.04 (C-16), 23.48 (C-11),
21.43 (C-6), 19.47 (C-26), 16.78 (C-24), 15.06 (C-25). ESI-MS (nega-
tive-ion mode, m/z): 609.50 (Mꢁ þ 1), 607.50 (Mꢁ ꢁ 1).
4.6.8. 3b-Cinnamoyloxy-22b-senecioyloxy-olean-12-en-28-oic acid
(13)
Yield: 85.99%, mp: 165e166 ꢀC. Anal. calcd. for C44H60O6
(684.44): %C, 77.16; H, 8.83. Found: %C, 77.23; H, 8.84. IR (KBr,
cmꢁ1): 3240 (OeH of COOH), 2947, 2875 (CeH), 1745 (C]O ester),
1715 (C]O acid), 1635 (C]C). 1H NMR (400 MHz, CDCl3,
d
ppm):
4.6.11. 22b-Cinnamoyloxy-3-oxo-olean-12-en-28-oic acid (16)
7.6488e7.6887 (1H, d, J ¼ 15.96 Hz, C-30-H), 7.5206e7.5445 (2H, m,
C-50 & C-90-Ar-H), 7.3722e7.3885 (3H, m, C-60, C-80 & C-70-Ar-H),
6.4254e6.4654 (1H, d, J ¼ 16 Hz, C-20-H), 5.5490e5.5552 (1H, t,
J ¼ 1.24 Hz, C-32-H), 5.3506e5.3683 (1H, t, J ¼ 3.54 Hz, C-12-H),
5.0753e5.0874 (1H, t, J ¼ 2.37 Hz, C-22-H), 4.6323e4.6722 (1H, t,
J ¼ 7.98 Hz, C-3-H), 3.0231e3.0646 (1H, dd, J ¼ 13.92, 3.52 Hz, C-18-
H), 1.1778 (3H, s, CH3), 1.0006 (3H, s, CH3), 0.9776 (3H, s, CH3),
0.9539 (3H, s, CH3), 0.9230 (3H, s, CH3), 0.8987 (3H, s, CH3), 0.7826
Yield: 56.59%, mp: 281e283 ꢀC. Anal. calcd. for C39H52O5
(600.38): %C, 77.96; H, 8.72. Found: %C, 77.99; H, 8.73. IR (KBr,
cmꢁ1): 3210 (OeH of COOH), 2950, 2872 (CeH), 1738 (C]O keto),
1699 (C]O), 1632 (C]C). 1H NMR (400 MHz, CDCl3,
d ppm):
7.4707e7.5108 (1H, d, J ¼ 16.04 Hz, C-30-H), 7.3828e7.4063 (2H, m,
C-50 & C-90-Ar-H), 7.3229e7.3530 (3H, m, C-60, C-80 & C-70-Ar-H),
6.1513e6.1914 (1H, d, J ¼ 16.04 Hz, C-20-Ar-H), 5.4020e5.4190 (1H,
t, J ¼ 3.40 Hz, C-12-H), 5.0958e5.1106 (1H, t, J ¼ 2.96 Hz, C-22-H),
3.1014e3.1460 (1H, dd, J ¼ 13.84, 4.20 Hz, C-18-H), 2.5140e2.5996
(1H, m, C-2-Ha), 2.3373e2.4024 (1H, m, C-2-Hb), 1.1825 (3H, s,
CH3), 1.0890 (3H, s, CH3), 1.0422 (3H, s, CH3), 1.0372 (3H, s, CH3),
1.0086 (3H, s, CH3), 0.8936 (3H, s, CH3), 0.8448 (3H, s, CH3). 13C NMR
(3H, s, CH3). 13C NMR (100 MHz, CDCl3,
d ppm): 180.12 (C-28),
166.83 (C-31), 166.27 (C-10), 157.13 (C-33), 144.33 (C-30), 143.04 (C-
13), 134.45 (C-40), 130.14 (C-70), 128.82 (C-60 & C-80), 128.00 (C-50 &
C-90), 122.57 (C-12), 118.71 (C-20), 115.89 (C-32), 80.87 (C-3), 75.91
(C-22), 55.25 (C-5), 50.51 (C-17), 47.51 (C-9), 45.92 (C-19), 41.78 (C-
14), 39.18 (C-8), 38.20 (C-18), 38.05 (C-4), 37.90 (C-1), 37.64 (C-21),
36.93 (C-10), 33.65 (C-29), 32.50 (C-7), 29.99 (C-20), 28.06 (C-15),
(100 MHz, CDCl3, d
ppm): 218.03 (C-3), 180.44 (C-28), 165.56 (C-10),
144.94 (C-30), 143.05 (C-13),134.33 (C-40),130.74 (C-70), 128.94 (C-60
& C-80), 128.36 (C-50 & C-90), 122.57 (C-12), 118.10 (C-20), 76.52 (C-

142
S.K. Suthar et al. / European Journal of Medicinal Chemistry 74 (2014) 135e144
22), 55.32 (C-5), 50.82 (C-17), 47.47 (C-9), 46.90 (C-4), 45.94 (C-19),
42.08 (C-14), 39.28 (C-8), 39.16 (C-18), 38.46 (C-1), 37.73 (C-21),
36.77 (C-10), 34.16 (C-2), 33.69 (C-29), 32.18 (C-7), 30.10 (C-20),
27.64 (C-15), 26.45 (C-23), 26.34 (C-27), 25.79 (C-30), 24.01 (C-16),
23.57 (C-11), 21.10 (C-6), 19.54 (C-26), 16.69 (C-24), 15.12 (C-25).
ESI-MS (negative-ion mode, m/z): 599.60 (Mꢁ).
38.14 (C-18), 38.10 (C-1), 37.68 (C-4), 37.68 (C-21), 36.94 (C-10),
33.69 (C-29), 32.56 (C-7), 30.01 (C-20), 28.02 (C-15), 27.56 (C-23),
26.29 (C-27), 25.86 (C-30), 23.91 (C-16), 23.50 (C-11), 23.44 (C-2),
21.31 (C-20), 21.10 (C-40), 18.09 (C-6), 17.05 (C-26), 16.67 (C-24),
15.49 (C-25). ESI-MS (negative-ion mode, m/z): 556.20 (Mꢁ),
555.20 (Mꢁ ꢁ 1).
4.7. Synthesis of 3
b
,22b
-dihydroxy-olean-12-en-28-oic acid (17)
4.9. Synthesis of methyl 22b-hydroxy-3-oxo-olean-12-en-28-ate
(19)
1 mmol (470.68 mg) of compound 5 was stirred with 1 mmol
(37.83 mg) of sodium borohydride in 50 ml solution of methanol
(25 ml) and tetrahydrofuran (25 ml) for 7 h (Scheme 2). After
completion of reaction, dilute HCl solution was added to the reac-
tion mixture to quench the NaBH4. The organic solvents were
removed under reduced pressure and precipitated product was
extracted with DCM. The solvent was removed under reduced
pressure to afford a compound 17, which was further purified by
using column chromatography (silica gel of 100e200 mesh and
gradient mobile phase of hexaneeethyl acetate).
Compound 5 (1 mmol, 470.68 mg) was refluxed with potassium
carbonate (3 mmol, 414.61 mg) in acetone for 1 h. After that
dimethyl sulfate (2 mmol, 189.66
ml) was added to the reaction
mixture and refluxing was continued for another 11 h (Scheme 2).
After completion of reaction, acetone was removed under reduced
pressure, reaction mixture was poured into water, and precipitated
product was extracted with DCM. Organic solvent was removed in
rotary evaporator and crude product was chromatographed over
silica gel (100e200 mesh) and eluted with gradient mobile phase of
hexaneeethyl acetate to yield a final purified product (19).
4.7.1. 3b,22b-Dihydroxy-olean-12-en-28-oic acid (17)
Yield: 87.79%, mp: 282e284 ꢀC. Anal. calcd. for C30H48O4
(472.36): %C, 76.23; H, 10.24. Found: %C, 76.29; H, 10.23. IR (KBr,
cmꢁ1): 3435.07 (OeH), 2948.50, 2876.33 (CeH), 1705.76 (C]O),
1648.59 (C]C). 1H NMR (400 MHz, CDCl3 þ DMSO-d6 mixture,
4.9.1. Methyl 22b-hydroxy-3-oxo-olean-12-en-28-ate (19)
Yield: 72.00%, mp: 190e192 ꢀC. Anal. calcd. for C31H48O4
(484.36): %C, 76.82; H, 9.98. Found: %C, 76.83; H, 9.96. IR (KBr,
cmꢁ1): 3518 (OeH), 2945 (CeH), 1729 (C]O keto), 1708 (C]O
d
ppm): 11.4773 (1H, s (br), C-28-H (COOH)), 5.2267e5.2441 (1H, t,
ester). 1H NMR (400 MHz, CDCl3,
d ppm): 5.3678e5.3843 (1H, t,
J ¼ 2.48 Hz, C-12-H), 4.1543 (1H, s (br), C-22-OH), 3.7499e3.7654
(1H, t, J ¼ 3.10 Hz, C-22-H), 3.5768 (1H, s (br), C-3-OH), 3.0544e
3.0934 (1H, t, J ¼ 7.80 Hz, C-3-H), 2.9195e2.9626 (1H, dd, J ¼ 13.84,
3.56 Hz, C-18-H), 1.1270 (3H, s, CH3), 1.0925 (3H, s, CH3), 0.9397 (3H,
s, CH3), 0.8953 (3H, s, CH3), 0.8513 (3H, s, CH3), 0.7982 (3H, s, CH3),
0.7250 (3H, s, CH3). 13C NMR (100 MHz, CDCl3 þ DMSO-d6 mixture,
J ¼ 3.30 Hz, C-12-H), 3.8837e3.8989 (1H, t, J ¼ 3.04 Hz, C-22-H),
3.6702 (3H, s, C-31-H), 3.0396e3.0843 (1H, dd, J ¼ 13.84, 4.04 Hz, C-
18-H), 2.5138e2.5995 (1H, m, C-2-Ha), 2.3312e2.3962 (1H, m, C-2-
Hb), 1.1555 (3H, s, CH3), 1.1262 (3H, s, CH3), 1.0862 (3H, s, CH3),
1.0628 (3H, s, CH3), 1.0473 (3H, s, CH3), 0.8997 (3H, s, CH3), 0.8244
(3H, s, CH3). 13C NMR (100 MHz, CDCl3,
d ppm): 217.83 (C-3), 176.14
d
ppm): 176.23 (C-28), 143.82 (C-13), 120.96 (C-12), 77.16 (C-3),
(C-28), 143.43 (C-13), 122.19 (C-12), 74.62 (C-22), 55.33 (C-5), 52.43
(C-31), 51.58 (C-17), 47.46 (C-9), 46.87 (C-4), 45.97 (C-19), 42.11 (C-
14), 41.39 (C-8), 39.27 (C-1), 39.19 (C-18), 38.26 (C-21), 36.75 (C-10),
34.18 (C-2), 33.92 (C-29), 32.21 (C-7), 30.15 (C-20), 27.79 (C-15),
27.20 (C-23), 26.41 (C-27), 25.74 (C-30), 24.49 (C-16), 23.55 (C-11),
21.50 (C-6), 19.57 (C-26), 16.72 (C-24), 15.09 (C-25). ESI-MS (m/z):
507.50 (M þ Na)þ, 991.90 (2M þ Na)þ.
72.70 (C-22), 54.80 (C-5), 51.00 (C-17), 47.10 (C-9), 46.06 (C-19),
41.63 (C-14), 41.11 (C-8), 38.80 (C-4), 38.30 (C-18), 38.10 (C-1), 37.93
(C-21), 36.52 (C-10), 33.71 (C-29), 32.42 (C-7), 29.79 (C-20), 27.96
(C-2), 27.32 (C-15), 27.06 (C-23), 26.80 (C-27), 25.38 (C-30), 23.89
(C-16), 22.90 (C-11), 17.90 (C-6), 16.69 (C-26), 15.63 (C-24), 15.03 (C-
25). ESI-MS (negative-ion mode, m/z): 472.30 (Mꢁ) 471.20 (Mꢁ ꢁ 1).
4.8. Synthesis of 3
b
,22b
-diacetoyloxy-olean-12-en-28-oic acid (18)
4.10. Synthesis of methyl 3b,22b-dihydroxy-olean-12-en-28-ate
(20)
Compound 17 (1 mmol, 472.70 mg) and acetyl chloride (3 mmol,
213.30 l) were refluxed in pyridine in the presence of 4-DMAP for
m
Equimolar amount of compound 19 (1 mmol, 484.71 mg) and
sodium borohydride (1 mmol, 37.83 mg) was stirred in a 50 ml
solution of methanoletetrahydrofuran (25 ml MeOH þ 25 ml THF)
for 7 h (Scheme 2). At the end of reaction, sodium borohydride
remained was quenched with dilute HCl solution. Organic solvents
were removed in rotary evaporator and precipitated product was
extracted with DCM. The DCM was removed under reduced pres-
sure and crude product was further purified with the help of col-
umn chromatography (silica gel: 100e200 mesh) using gradient
mobile phase of hexaneeethyl acetate to give a final purified
product (20).
10 h at 92e95 ꢀC (Scheme 2). At the end of reaction 10% HCl so-
lution was added to the reaction mixture and precipitated product
was extracted with DCM. The product was washed further three
times with 10% HCl solution (100 ml ꢃ 3) and purified by using
column chromatography (silica gel: 100e200 mesh) in a gradient
mobile phase of hexaneeethyl acetate.
4.8.1. 3b,22b-Diacetoyloxy-olean-12-en-28-oic acid (18)
Yield: 59.25%, mp: 301e303 ꢀC. Anal. calcd. for C34H52O6
(556.38): %C, 73.34; H, 9.41. Found: %C, 73.36; H, 9.40. IR (KBr,
cmꢁ1): 3355.26 (OeH of COOH), 2990.34, 2950.25, 2923.26,
2876.39, 2847.49 (CeH), 1731.60 (C]O ester). 1H NMR (400 MHz,
4.10.1. Methyl 3b,22b-dihydroxy-olean-12-en-28-ate (20)
CDCl3,
d
ppm): 5.3430e5.3599 (1H, t, J ¼ 3.38 Hz, C-12-H), 5.0028e
Yield: 86.70%, mp: 179e180 ꢀC. Anal. calcd. for C31H50O4
(486.37): %C, 76.50; H, 10.35. Found: %C, 76.47; H, 10.33. IR (KBr,
cmꢁ1): 3566.56 (OeH), 3368.50 (OeH), 3270.80 (OeH of COOH),
2949.35, 2931.00, 2872.49 (CeH), 1711.48 (C]O), 1565.60 (C]C).
5.0172 (1H, t, J ¼ 3.88 Hz, C-22-H), 4.4800e4.5196 (1H, t,
J ¼ 7.92 Hz, C-3-H), 2.9854e3.0298 (1H, dd, J ¼ 13.76, 4.00 Hz, C-18-
H), 2.0498 (3H, s, C-20-H), 1.9411 (3H, s, C-200-H), 1.1530 (3H, s, CH3),
1.0225 (3H, s, CH3), 0.9472 (3H, s, CH3), 0.8944 (3H, s, CH3), 0.8693
(3H, s, CH3), 0.8606 (3H, s, CH3), 0.7650 (3H, s, CH3). 13C NMR
1H NMR (400 MHz, CDCl3,
d ppm): 5.3429e5.3608 (1H, t,
J ¼ 3.58 Hz, C-12-H), 3.8716e3.8883 (1H, t, J ¼ 3.84 Hz, C-22-H),
3.6632 (3H, s, C-31-H), 3.1955e3.2352 (1H, dd, J ¼ 11.36, 5.00 Hz, C-
3-H), 3.0220e3.0672 (1H, dd, J ¼ 13.88, 4.20 Hz, C-18-H), 1.1420
(3H, s, CH3), 1.1216 (3H, s, CH3), 0.9877 (3H, s, CH3), 0.9221 (3H, s,
(100 MHz, CDCl3, d
ppm): 180.23 (C-28),171.08 (C-10),169.70 (C-100),
142.86 (C-13), 122.71 (C-12), 80.85 (C-3), 76.14 (C-22), 55.25 (C-5),
50.56 (C-17), 47.52 (C-9), 45.76 (C-19), 41.80 (C-14), 39.24 (C-8),

S.K. Suthar et al. / European Journal of Medicinal Chemistry 74 (2014) 135e144
143
CH3), 0.8978 (3H, s, CH3), 0.7835 (3H, s, CH3), 0.7667 (3H, s, CH3). 13
NMR (100 MHz, CDCl3, ppm): 176.21 (C-28), 143.33 (C-13), 122.44
C
22.89 (C-11), 20.08 (C-6), 16.39 (C-26), 16.33 (C-34), 15.15 (C-24),
d
14.43 (C-25). ESI-MS (negative-ion mode, m/z): 568.30 (Mꢁ þ 1).
(C-12), 78.99 (C-3), 74.70 (C-22), 55.22 (C-5), 52.44 (C-31), 51.55 (C-
17), 47.62 (C-9), 46.02 (C-19), 42.00 (C-14), 41.38 (C-8), 39.29 (C-18),
38.75 (C-4), 38.48 (C-1), 38.18 (C-21), 37.02 (C-10), 33.91 (C-29),
32.69 (C-7), 30.14 (C-20), 28.09 (C-2), 27.79 (C-15), 27.21 (C-23),
27.18 (C-27), 25.85 (C-30), 24.53 (C-16), 23.46 (C-11), 18.32 (C-6),
16.80 (C-26), 15.58 (C-24), 15.38 (C-25). ESI-MS (m/z): 509.40
(M þ Na)þ.
4.12. In vitro cell culturing and cytotoxicity assay
The A549 lung cancer cell line was obtained from American
Type Culture Collection (ATCC, USA) and was grown in RPMI me-
dium supplemented with 5% fetal bovine serum and 1% penicillin-
streptomycin (Gibco, Invitrogen, USA). The A549 (4000 cells/well)
were seeded into 96-well plates and incubated overnight for cell
attachment. For treatment, compounds were added at concentra-
4.11. Synthesis of 3
b-hydroxylimino-substituted 22b-angeloyloxy/
22b-senecioyloxy-olean-12-en-28-oic acids (21e22)
tions ranging from 0.01 to 100
mmol and incubated for 48 h. At the
end of incubation, 20 l/well of 5 mg/ml 3-(4,5-dimethythiazol-2-
m
1 mmol (552.78 mg) of lantadene (1, 2) was refluxed with
yl)-2,5-diphenyltetrazolium bromide (MTT) (Amresco, USA) was
added and cells were further incubated for 4 h. Supernatant was
then removed and the purple formazan which has formed was
dissolved using 100 ml of DMSO (Fisher Scientific, UK). Absorbance
was read at 570 nm using Spectra Max M4 microplate reader
(Molecular Devices Inc., US).
10 equivalent of hydroxylamine hydrochloride (10 mmol,
694.90 mg) in pyridine at 92e95 ꢀC for 8 h (Scheme 2). The reaction
mixture was poured into 10% HCl solution and product was
extracted with DCM and washed further three times with 10% HCl
solution (100 ml ꢃ 3). The organic solvent was removed under
reduced pressure to dryness and crude product obtained was
subjected to column chromatography using silica gel (100e
200 mesh) and gradient mobile phase of hexaneeethyl acetate to
yield a final purified product (21e22).
4.13. In vitro inhibition of TNF-a-induced NF-kB activation in A549
lung cells
The A549 cells were cultured in 12-well plates and transiently
co-transfected with 0.2 g of a pNF- B-Luc vector (Stratagene, La
Jolla, CA) and 0.2 g of pSV- -galactosidase dissolved in 3 l lip-
ofectamineÔ or lipofectamineÔ 2000 (Invitrogen, Carlsbad, CA) as
the internal control. The plasmids were transfected according to the
manufacturer’s instructions. After 6 h, the medium was changed
4.11.1. 22
(21)
b
-Angeloyloxy-3-hydroxylimino-olean-12-en-28-oic acid
m
k
m
b
m
Yield: 59.88%, mp: 235e236 ꢀC. Anal. calcd. for C35H53NO5
(567.39): %C, 74.04; H, 9.41. Found: %C, 74.00; H, 9.40. IR (KBr,
cmꢁ1): 3271.11 (OeH), 2951.50 (CeH), 1718.30 (C]O), 1647.24 (C]
C). 1H NMR (400 MHz, CDCl3 þ DMSO-d6 mixture,
d
ppm): 11.7341
and cells were cultured for 6 h. Cells were then treated with TNF-
(15 ng/ml) and test compounds simultaneously for 7 h. The A549
cells treated with TNF- alone served as positive controls, while
cells without TNF- treatment served as negative controls. Lucif-
a
(1H, s (br), C-28-H (COOH)), 5.9792e6.0290 (1H, m, C-33-H),
5.3471e5.3645 (1H, t, J ¼ 3.48 Hz, C-12-H), 5.0170e5.0317 (1H, t,
J ¼ 2.94 Hz, C-22-H), 2.2019e2.2610 (1H, m, C-2-Ha), 2.0594e
2.1419 (1H, m, C-2-Hb), 1.1498 (3H, s, CH3), 1.0272 (3H, s, CH3),
1.0189 (6H, s, 2ꢃ CH3), 1.0096 (3H, s, CH3), 0.8932 (3H, s, CH3),
0.8598 (3H, s, CH3). 13C NMR (100 MHz, CDCl3 þ DMSO-d6 mixture,
a
a
erase activities from these cells were then measured by using the
Bright-Glo Luciferase Assay kit from Promega (Madison, WI),
following the manufacturer’s protocol. The relative NF-kB activities
of the cells treated by test compounds were obtained as the ratio of
its luciferase activity to that from the positive controls, both of
which have been corrected with background (signals from negative
controls) and cell viability. In these experimental conditions, none
of the test compounds induced significant toxicity to A549 cells
(<5% reduction of cell viability). The IC50 of each fraction was
d
ppm): 175.35 (C-28), 165.77 (C-31), 164.22 (C-3), 143.11 (C-13),
137.35 (C-33), 127.57 (C-32), 121.46 (C-12), 75.60 (C-22), 55.29 (C-
5), 49.65 (C-17), 46.60 (C-9), 45.53 (C-19), 41.51 (C-14), 39.50 (C-4),
38.81 (C-8), 38.12 (C-18), 37.79 (C-1), 37.39 (C-21), 36.50 (C-10),
33.31 (C-29), 31.99 (C-7), 29.55 (C-20), 27.18 (C-15), 27.06 (C-23),
25.76 (C-27), 25.27 (C-30), 23.64 (C-16), 23.10 (C-2), 22.89 (C-11),
20.07 (C-6), 18.51 (C-35), 16.42 (C-26), 16.34 (C-34), 15.14 (C-24),
14.40 (C-25). ESI-MS (negative-ion mode, m/z): 567.30 (Mꢁ), 566.30
(Mꢁ ꢁ 1).
determined by fitting the relative NF-kB activity to the drug con-
centration by using a sigmoidal doseeresponse model of varied
slope in GraphPad Prism 6. The IC50 reported herein is the average
of at least three replicates.
4.11.2. 22b-Senecioyloxy-3-hydroxylimino-olean-12-en-28-oic acid
(22)
4.14. In vitro phosphorylation assay
Yield: 59.53%, mp: 231e232 ꢀC. Anal. calcd. for C35H53NO5
(567.39): %C, 74.04; H, 9.41. Found: %C, 74.10; H, 9.42. IR (KBr,
cmꢁ1): 3256.38 (OeH), 2953.32, 2926.07, 2859.85 (CeH), 1738.33
(C]O), 1720.33 (C]O), 1647.17 (C]C). 1H NMR (400 MHz,
The cDNA encoding human IKK
b was isolated by PCR with
primers containing sequences encoding a FLAG tag in the carboxy-
terminal region and subcloned into an insect cell expression vector,
pFASTBAC1 (Invitrogen, U.S.A.). The Sf21 cells were infected with
CDCl3 þ DMSO-d6 mixture,
d ppm): 11.7373 (1H, s (br), C-28-H
(COOH)), 5.5564 (1H, s, C-32-H), 5.3506 (1H, s, C-12-H), 5.0073e
5.0238 (1H, t, J ¼ 3.30 Hz, C-22-H), 2.2105e2.2568 (1H, m, C-2-Ha),
2.0545e2.1524 (1H, m, C-2-Hb), 1.1478 (3H, s, CH3), 1.0251 (3H, s,
CH3), 1.0167 (6H, s, 2ꢃ CH3), 1.0086 (3H, s, CH3), 0.8872 (3H, s, CH3),
0.8578 (3H, s, CH3). 13C NMR (100 MHz, CDCl3 þ DMSO-d6 mixture,
IKK
cells were lysed and FLAG-tagged IKK
finity chromatography using anti-FLAG M2 affinity gel (Sigma,
U.S.A.). The kinase reaction of purified human IKK was performed
b
recombinant baculovirus and cultured at 28 ꢀC for 72 h. The
b
protein was purified by af-
b
at room temperature for 1 h in kinase reaction buffer (25 mmol/l
HEPES, pH 7.5, 10 mmol/l magnesium acetate, 1 mmol dithio-
threitol, 0.01% bovine serum albumin, 0.01% Tween20) containing
d
ppm): 181.24 (C-28), 165.73 (C-31), 164.08 (C-3), 156.41 (C-33),
143.12 (C-13),121.49 (C-12),116.77 (C-32), 75.59 (C-22), 55.28 (C-5),
49.52 (C-17), 46.59 (C-9), 45.55 (C-19), 41.52 (C-14), 39.48 (C-4),
38.81 (C-8), 38.00 (C-18), 37.80 (C-1), 37.40 (C-21), 36.51 (C-10),
33.33 (C-29), 32.00 (C-7), 29.56 (C-20), 27.21 (C-15), 27.06 (C-23),
26.00 (C-35), 25.77 (C-27), 25.27 (C-30), 23.57 (C-16), 23.12 (C-2),
500 nmol ATP, and bacterially expressed GST-IkBa, and then
terminated by adding the same volume of Kinase-GloÔ reagent
(Promega, U.S.A.). After incubating at room temperature for 10 min
the luminescent signal correlated with the amount of ATP

144
S.K. Suthar et al. / European Journal of Medicinal Chemistry 74 (2014) 135e144
remaining in solution following the reaction was measured by
Wallac Arvo HTS multilabel counter (PerkinElmer, U.S.A.).
Conflict of interest
The author(s) confirm that this article content has no conflict of
4.15. Predicting binding mode of lead ester congener 13 to IKK
b
interest.
Crystal structure of IKK was downloaded from Protein Data
b
Acknowledgments
Bank (PDB ID: 3QA8) [16]. Structures of ligands in PDB format were
prepared using CS ChemDraw Ultra 8.0. Molecular docking study
was performed using AutoDock tools 1.5.4. Protein was firstly
prepared by adding polar hydrogen atoms at pH 7.4 followed by
assigning of the Gasteiger charges. Nonpolar hydrogens were
merged and partial charges to their parent carbon atoms were
added. The search space was identified as a cubic box with di-
mensions 40 A ꢃ 40 A ꢃ 40 A and center x ¼ ꢁ20, y ¼ ꢁ15, z ¼ 30.
The Lamarckian genetic algorithm (LGA) was applied to explore
conformers with lowest binding energy. Results of molecular
docking analysis were obtained as estimated free energy of binding
in kcal/mol (docking score).
Authors are thankful to Department of Science and Technology,
Government of India for financial assistance for this research work.
We are also thankful to Sophisticated Analytical Instrumentation
Facility (SAIF), Panjab University, Chandigarh, India for spectral
analyses.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.12.052.
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