Synthesis of novel triterpene and N-allylated/N-alkylated niacin hybrids as α-glucosidase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.01.053

Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia. α-Glucosidase (EC 3.2.1.20) inhibitors interfere with enzymatic action to slow down the liberation of d-glucose from oligosaccharides and disaccharides, resulting in delayed glucose absorption and decreased postprandial plasma glucose levels. In continuation of our drug discovery program on antidiabetic agents, we synthesized novel N-allylated/N-alkylated niacin and α-amyrin (4-9) and lupeol (12-16) hybrids and tested for their α-glucosidase inhibiting activity. Compounds 4-9 showed better activity profile than the marketed α-glucosidase inhibitor i.e. acarbose. Compound 4 possess the highest inhibitory action with IC₅₀ of 5 μM. Kinetic and CD studies revealed that 4 inhibited the α-glucosidase in a noncompetitive manner and caused conformational changes in secondary structure of the enzyme protein.

Full text of DOI:10.1016/j.ejmech.2013.01.053

European Journal of Medicinal Chemistry 63 (2013) 162e169
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel triterpene and N-allylated/N-alkylated niacin hybrids as
a-glucosidase inhibitors
,
a
b
a
b
Tadigoppula Narender a , Gaurav Madhur , Natasha Jaiswal , Manali Agrawal , Chandan K. Maurya ,
*
Neha Rahuja b, Arvind K. Srivastava b, Akhilesh K. Tamrakar b
,
*
a Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226 001, UP, India
b Biochemistry Division, CSIR-Central Drug Research Institute, Lucknow 226 001, UP, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia.
3.2.1.20) inhibitors interfere with enzymatic action to slow down the liberation of
gosaccharides and disaccharides, resulting in delayed glucose absorption and decreased postprandial
plasma glucose levels. In continuation of our drug discovery program on antidiabetic agents, we syn-
a
-Glucosidase (EC
Received 9 July 2012
Received in revised form
10 January 2013
Accepted 26 January 2013
Available online 8 February 2013
D
-glucose from oli-
thesized novel N-allylated/N-alkylated niacin and
a
-amyrin (4e9) and lupeol (12e16) hybrids and tested
-glucosidase inhibiting activity. Compounds 4e9 showed better activity profile than the
-glucosidase inhibitor i.e. acarbose. Compound 4 possess the highest inhibitory action with
M. Kinetic and CD studies revealed that 4 inhibited the -glucosidase in a noncompetitive
for their
marketed
IC50 of 5
a
a
m
Keywords:
a
a
-Amyrin acetate
manner and caused conformational changes in secondary structure of the enzyme protein.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Lupeol
Niacin
Triterpeneeniacin hybrids
a
-Glucosidase inhibitor
1. Introduction
derived active chemical compounds like terpenoids, flavonoids,
alkaloids, guanidines, steroids, carbohydrates and amino acids
exhibit activity against T2DM. Metformin is currently used as
antidiabetic agent in the treatment of type-2 diabetes. Metformin
and its analogs [4,5] were synthesized on the basis of a natural
product lead, galegine, which was isolated from the seeds of Galega
officinali [6]. The unusual amino acid hypoglycin B isolated from
Blighia sapid possesses antihyperglycemic activity [7]. Trigonelline
(N-methylniacin or N-methyl nicotinic acid) isolated from seeds of
Trigonella foenumgraecum was reported as hypoglycemic agents in
alloxan induced diabetic mice [8].
Ficus racemosa Linn. (Moraceae) commonly known as Gular [9]
has traditionally been used for the treatment of diabetes [10]. The
hypoglycemic potential of its leaf extract was demonstrated in
streptozotocin-induced diabetic rats [11]. Glucose lowering efficacy
of the stem bark in normal as well as alloxan-induced diabetic rats
was also reported [12]. As a part of our drug discovery program, we
Diabetes mellitus is a metabolic disorder of multiple etiologies
characterized by chronic hyperglycemia with disturbances of car-
bohydrate, fat and protein metabolism resulting from defects in
insulin secretion (Type 1 diabetes), insulin action (Type 2 diabetes)
or both. As a consequence, raised blood sugar leads to common
effect of uncontrolled diabetes with serious damage to many of the
body’s systems, especially the nerves and blood vessels. Type 2
diabetes mellitus (T2DM) accounts for approximately 80e90% of all
cases of diabetes and it is the fastest growing global threat to public
health [1]. The prevalence of diabetes for all age-groups worldwide
was estimated to be 2.8% in 2000 and 4.4% in 2030. The total
number of people with diabetes is projected to rise from 171 mil-
lion in 2000 to 366 million in 2030 [2]. It is usually associated with
older age, obesity, family history, impaired glucose metabolism,
physical inactivity, and race or ethnicity.
Plants have always been an ideal source of drugs and many of
the currently available drugs have been derived directly or devel-
oped from plants. The ethnobotanical information reports that
about 800 plants may possess anti-diabetic potential [3]. Plant-
identified a-amyrin acetate 1 as antihyperglycemic principle from
the fruits of F. racemosa [13] for the first time.
Lupeol and its derivatives hold the promising antiprotozoal, anti-
inflammatory, antitumor, cardioprotective, hepatoprotective, anti-
microbial activities [14]. Recently we reported antihyperglycemic
activity in hybrids prepared from these triterpenoids (a-amyrin and
lupeol) and niacin (nicotinic acid) [13,15]. N-methylated niacin also
known as trigonelline isolated from the seeds of T. foenumgraecum
* Corresponding authors. Tel.: þ91 522 2612411; fax: þ91 522 2623405.
E-mail addresses: t_narendra@cdri.res.in, tnarender@rediffmail.com (T. Narender),
akhileshcdri@gmail.com (A.K. Tamrakar).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.053

T. Narender et al. / European Journal of Medicinal Chemistry 63 (2013) 162e169
163
seeds has been reported for its antihyperglycemic activity [8]. We
therefore planned and prepared N-allylated/N-alkylated derivatives
of -amyrin/lupeol and niacin hybrids [14,15] and screened for their
PTPase, glucose-6-phosphatase, glycogen phosphorylase and
glucosidase activity. Interestingly N-allylated/N-alkylated niacin and
amyrin hybrids showed strong -glucosidase inhibitory activity in
our studies. -Glucosidase inhibitors are one of the alternative
therapeutic approaches for T2DM. The digestion and absorption of
carbohydrate would be delayed by the inhibition of intestinal
glucosidase (Fig. 1) resulting in decreased postprandial hyperglyce-
mia [16]. In addition, -glucosidase inhibitors have also been
a
a-
a
a
a-
a
reported for other biological activities such as reducing triglycerides
[17] and anti-HIV activity [18]. Thus, during the past few decades,
considerable attention has been paid on designing potent
a-gluco-
sidase inhibitors in the chemical and medical research. Few
a-glu-
cosidase inhibiting drugs from natural sources, such as plants,
foodstuffs and microbes have also been identified [19e21] (Fig. 1).
We here report on the synthesis of hybrids of triterpenes viz.
amyrin/lupeol and N-allylated/N-alkylated niacin [15,22] and their
-glucosidase inhibiting activity.
a-
a
2. Results and discussion
2.1. Synthesis
Scheme 1. Synthesis of
a
-amyrin and N-allylated/N-alkylated niacin hybrids. Reagents
and conditions: (i) Sodium methoxide, methanol, r.t., 2 h, (ii) Nicotinic acid, DCC-
DMAP, DCM, r.t., 6 h, (iii) 3,3-Dimethylallyl bromide, Diethyl ether, r.t., 6-7 h, (iv)
Appropriate alkyl bromide, toluene, reflux, 36 h.
The synthesis of
N-allylated chain was accomplished by subjecting the
acetate 1 to deacetylation by freshly prepared sodium methoxide in
dry methanol. -Amyrin 2 thus, obtained was esterified using
niacin employing DCCeDMAP (Steglich esterification) protocol in
dry DCM to afford non-natural -amyrineniacin hybrid 3. This was
a
-amyrineniacin hybrids having N-alkylated or
a
-amyrin
a
hybrids showed significant inhibitory activity against
In the current series, compound 4 was found to be the most active,
showing concentration-dependent inhibition of -glucosidase ac-
tivity with 95% inhibition at 10 M concentration (p < 0.001). From
the doseeresponse curve, IC50 value of 4 was calculated as 5
(Fig. 2). Acarbose (marketed -glucosidase inhibitor), showed 70.9%
inhibitory activity at 100 M concentration under similar assay
a-glucosidase.
a
a
then subjected to N-allylation using prenyl bromide (3,3-
dimethylallyl bromide) in dry diethyl ether at room temperature
m
mM
to afford N-prenylated
using appropriate alkyl bromides in dry toluene at reflux condition
to afford -amyrineN-alkylated niacin hybrids 5e9 (Scheme 1).
a-amyrineniacin hybrid 4 and N-alkylation
a
m
a
conditions (p < 0.001). It is noteworthy to mention here that the
The similar protocol was used to prepare derivatives 12e16 from
lupeol (10) (Scheme 2).
2.2. In vitro
relationship
a-glucosidase inhibition and structure activity
The parent compounds 3 and 11 were not soluble; therefore their
-glucosidase inhibiting activity has not been done. Compounds 4e
a
9 and 12e16 prepared from 3 and 11 respectively have been tested
for their -glucosidase inhibiting activity. The results are summa-
rized in Table 1. As evident from the results, most of the amyrin
a
Scheme 2. Synthesis of lupeol and N-allylated/N-alkylated niacin hybrids. Reagents
and conditions: (i) Nicotinic acid, DCC-DMAP, DCM, r.t., 6 h, (ii) Appropriate allyl
bromide, Diethyl ether, r.t., 6-7 h. (iii) Appropriate alkyl bromide, reflux, 36 h.
Fig. 1.
a-Glucosidase inhibiting drugs available in the market.

164
T. Narender et al. / European Journal of Medicinal Chemistry 63 (2013) 162e169
Table 1
a
-Glucosidase inhibitory activity of compounds 4e9 and 12e16..
Compound no.
R
Concentration (
10
mM)
25
50
100
4
L94.9 ꢀ 3.8
L95.0 ꢀ 2.4
L98.0 ꢀ 1.5
L99.0 ꢀ 0.5
5
ꢁ41.4 ꢀ 5.5
ꢁ13.6 ꢀ 5.8
ꢁ64.2 ꢀ 4.2
ꢁ47.5 ꢀ 12.0
ꢁ71.2 ꢀ 6.8
ND
ꢁ75.3 ꢀ 4.1
ꢁ40.1 ꢀ 7.1
ꢁ84.6 ꢀ 7.2
ꢁ65.1 ꢀ 8.2
ꢁ81.5 ꢀ 8.7
ND
ꢁ77.4 ꢀ 3.1
ꢁ69.0 ꢀ 7.5
ꢁ99.6 ꢀ 13.8
ꢁ91.7 ꢀ 4.5
ꢁ83.5 ꢀ 10.1
ND
ꢁ84.0 ꢀ 4.5
ꢁ80.3 ꢀ 4.9
ꢁ99.0 ꢀ 0.5
ꢁ95.9 ꢀ 3.2
ꢁ80.9 ꢀ 2.0
ꢁ4.4
6
7
8
9
12
13
14
15
16
ND
ND
ND
ND
ND
ND
ND
ND
ND
þ3.8
þ8.8
þ4.5
ND
ND
ND
ND
ND
ND
ꢁ13.3
L70.9
Acarbose (std)
Values are mean ꢀ SD; N ¼ 3each performed in triplicate; ND ¼ not done.
prenylated hybrid (4) turned out to be ten times more potent than
the marketed a-glucosidase inhibiting drug i.e., acarbose. Among
activity. The above data indicated that allylated hybrid (4) and all the
alkylated hybrids (5e9) showed better activity profile than the
the straight chain alkylated hybrids (5e9), compound 7 with C-16
marketed a-glucosidase inhibiting drug i.e acarbose (Table 1). In our
alkyl chain exhibited strong and dose dependent activity. It inhibi-
previous studies niacin (nicotinic acid) lupeol hybrid 11 showed in-
vivo activity in STZ-S model [15], however surprisingly none of the
N-allylated or N-alkylated lupeoleniacin hybrids (12e16) showed
ted the
a
-glucosidase by 64.2% at 10
mM (p < 0.001), 84.6% at 25
mM
M
(p < 0.001), 99.6% at 50
mM (p < 0.001) and 99.0% at 100
m
(p < 0.001). The compound 8 with C-18 alkyl chain inhibited the
glucosidase by 47.5%, 65.1%, 91.7% and 95.9% at 10, 25, 50 and 100
concentrations respectively (p < 0.001) and compound 9 with C-20
alkyl chain inhibited the -glucosidase by 71.2%, 81.5%, 83.5% and
80.9% at 10, 25, 50 and 100 M (p < 0.001) concentrations respec-
tively. The compound 5 with C-12 alkyl chain and compound 6 with
C-13 also exhibited dose-dependent -glucosidase inhibiting
a
-
significant
2.3. Kinetics of
To further determine the kinetic parameters of
a
-glucosidase inhibiting activity.
mM
a
-glucosidase inhibition
a
m
a
-glucosidase
inhibition by 4, the enzyme activity was measured with increasing
concentrations of substrate (0.5e20 mM) in the absence (control)
a

T. Narender et al. / European Journal of Medicinal Chemistry 63 (2013) 162e169
165
Fig. 4. The influence of compound 4 on the secondary structure of
a
-glucosidase
analyzed by CD spectroscopy. The enzyme was incubated with increasing concentra-
tions of 4 followed by absorbance measurement at 250 and 190 nm. Values obtained
were processed using software, as described in experimental part (4.2.2).
2.4. CD analysis
Fig. 2. Dose response curve of
with increasing concentration of 4 and enzyme activity was measured as described in
experimental part (4.2.1.). Results shown are mean
experiments.
a-glucosidase inhibition by 4. Enzyme was incubated
We further examined the influence of 4 on the secondary
structure of
The secondary structure of
buffer solution without (control) or with different concentrations
(50, 100, 150, 200, 250 M) of 4. Data were collected for 1e2 min at
a
-glucosidase by circular dichroism (CD) spectroscopy.
ꢀ
SE of three independent
a
-glucosidase was characterized in the
m
1 s interval at 250 nm and at 190 nm. A loss in the secondary
structure of the enzyme was observed with the increasing
or presence of 4 at increasing concentrations (2e6 mM). The initial
rates of reaction were determined using calibration curve
constructed using varying concentrations of p-nitrophenol
produced. The type of inhibition, [Km] and [Vmax] values were
determined from LineweavereBurk plots and values were calcu-
lated according to MichaeliseMenten kinetics. It is evident from
the plot that the presence of 4 caused increase in [Km] value from
1.67 (control) to 3.33, along with the increase in [Vmax] value from
0.0286 to 0.049 indicating the pattern of noncompetitive type of
inhibition (Fig. 3). These results suggested that compound 4 binds
concentrations of 4 from 50
control (Fig. 4). These results suggested that the inhibition of the
enzyme -glucosidase by 4 might be due to changes in the con-
mM to 250 mM as compared to the
a
formation of the secondary structure of the enzyme protein.
2.5. In vivo antihyperglycemic activity
The compound
4 was further evaluated for its anti-
with the enzyme at a site other than the active site on the
cosidase and retard the conversion of substrate into product.
a-glu-
hyperglycemic activity in sucrose-loaded STZ-induced diabetic rats.
Fig. 5 shows the blood glucose profile of sucrose-loaded diabetic
rats at different time periods upon treatment with 4. Compound 4
caused 19.9% (p < 0.001) reduction in blood glucose after 5 h of
treatment at 100 mg/kg dose. From the above results, 4 seems to be
Fig. 3. LineweavereBurk plot of
Vmax. The enzyme activity was measured with increasing concentrations of substrate in
the absence (control) or presence of 4 at increasing concentrations (2e6 M).
a
-glucosidase inhibition by 4 used to calculate Km and
Fig. 5. Blood glucose lowering effect of 4 in sucrose-loaded STZ-induced diabetic rats.
Diabetic rats were administered the suspension of 4, followed by blood glucose
measurement at various time intervals. Values are mean ꢀ S.E. of six rats in group.
m

166
T. Narender et al. / European Journal of Medicinal Chemistry 63 (2013) 162e169
suppressing the postprandial hyperglycemia by inhibiting
cosidase enzyme.
a-glu-
8.71 (dd, J ¼ 4.82 & 1.08 Hz, 1H), 8.24 (m, 1H), 7.33 (m, 1H), 5.08 (t,
J ¼ 3.16 Hz, 1H), 4.73 (t, J ¼ 8.18 Hz, 1H), 2.02e1.86 (m, 4H), 1.77e
1.49 (m, 9H), 1.41e1.20 (m, 10H), 1.04 (s, 3H), 0.98 (s, 6H), 0.91 (s,
3. Conclusion
3H), 0.87 (m, 6H), 0.76 (s, 6H); 13C NMR (CDCl3, 50 MHz)
d 164.8,
153.1, 150.8, 139.6, 136.9, 126.7, 124.2, 123.2, 82.2, 58.9, 55.3, 47.6,
42.0, 41.5, 39.9, 39.6, 39.5, 38.4, 38.0, 36.8, 33.7, 32.8, 31.2, 28.7, 28.2,
28.1, 26.6, 23.6, 23.4, 23.2, 21.4, 18.2, 17.5, 16.9, 16.8, 15.7.
In conclusion we have prepared few novel N-allylated/N-alky-
lated derivatives (4e9 and 12e16) of triterpenes (amyrin and
lupeol) and niacin (nicotinic acid) hybrids for the first time and
discovered
hybrid 4 turned out to be ten times more potent
inhibitor than the marketed -glucosidase inhibiting drug i.e.
acarbose. All other alkylated derivatives (5e9) of amyrin and niacin
hybrids also showed better activity profile than marketed -glu-
cosidase inhibiting drug. N-allylated/N-alkylated lupeol hybrids
a
-glucosidase inhibiting activity. One of the allylated
4.1.4. Synthesis of 1-(3-methylbut-2-enyl)-3-((4,4,6a,6b,8a,11,12,14b-
octamethyl-1,2,3,4,4a,5,6, 6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-
icosahydropicen-3-yloxy)carbonyl)pyridinium bromide (4)
To the stirred solution of 4,4,6a,6b,8a,11,12,14b-octamethyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9, 10,11,12,12a,14,14a,14b-icosahydropic-
en-3-yl-nicotinate 3 (0.53 g,1 mmol) in dry diethyl ether was added
3,3-dimethylallyl bromide (0.30 g, 3 mmol). The reaction mixture
was stirred for 6e7 h at room temperature. Solid mass as a pre-
cipitate was obtained which was filtered off, washed with ether and
dried to afford desired product 4. Yield: 92%; m.p.:151 ꢂC; 1H NMR
a-glucosidase
a
a
(12e16) failed to inhibit
4. Experimental part
4.1. Chemistry
a-glucosidase.
(CDCl3, 200 MHz)
d
9.86 (d, J ¼ 5.88 Hz, 1H), 9.09 (s, 1H), 8.85 (d,
J ¼ 8.08 Hz, 1H), 8.43 (m, 1H), 5.68e5.56 (m, 3H), 5.12 (m, 1H), 4.77
(m, 1H), 1.90 (s, 3H), 1.84 (s, 3H), 1.76e1.69 (m, 4H), 1.58e1.49 (m,
4H), 1.38e1.21 (m, 10H), 1.05 (s, 3H), 0.99e0.97 (m, 8H), 0.91e0.84
1H NMR spectra were run on Bruker Advance DPX 300 and
200 MHz in CDCl3, 13C NMR spectra were recorded at 75 MHz and
50 MHz in CDCl3. Chemical shifts are reported as values in ppm
relative to CHCl3 (7.26) in CDCl3. TMS was used as internal standard.
ESI mass spectra were recorded on JEOL SX 102/DA-6000. Chro-
matography was executed with silica gel (60e120 or 100e200
mesh) using mixtures of methanol and chloroform as eluants.
Methanol and chloroform were distilled and dried prior to use.
(m, 12H), 0.77 (m, 6H); 13C NMR (CDCl3, 50 MHz)
d 160.9, 149.3,
146.3, 144.8, 143.5, 139.6, 130.8, 129.2, 124.1, 115.4, 84.9, 60.1, 59.0,
55.3, 47.6, 42.1, 41.5, 39.9, 39.6, 39.5, 38.3, 38.0, 36.7, 33.7, 32.8, 31.2,
28.7, 28.3, 28.0, 26.6, 25.9, 23.5, 23.3, 23.2, 21.3, 19.0, 18.2, 17.4, 16.9,
16.8, 15.7; ESI MS m/z 600 (Mþ), 618 (Mþ þ H2O).
4.1.5. General procedure for synthesis of N-alkylated amyrine
niacin hybrids (5e10)
4.1.1.
a-Amyrin acetate (1)
1H NMR (CDCl3, 200 MHz)
d
5.13 (t, J ¼ 3.56 Hz, 1H), 4.56e4.47
The mixture of 4,4,6a,6b,8a,11,12,14b-octamethyl-1, 2, 3, 4, 4a, 5,
6, 6a, 6b, 7, 8, 8a, 9, 10, 11, 12, 12a, 14, 14a, 14b-icosahydropicen-3-yl-
nicotinate 3 and appropriate alkyl bromide (1:4) in toluene was
stirred for 36 h under reflux condition. The reaction mixture was
concentrated under vacuum to evaporate toluene. The crude
product was then subjected to silica gel chromatography using
methanol:chloroform as a mobile phase to afford compounds 5e10.
(m, 1H), 2.06 (s, 3H), 1.95e1.89 (m, 3H), 1.68e1.52 (m, 10H), 1.43e
1.26 (m, 10H), 1.07 (s, 3H), 1.02 (s, 3H), 0.99 (s, 3H), 0.92 (s, 3H),
0.88 (s, 3H), 0.87 (s, 3H), 0.81 (s, 6H); 13C NMR (CDCl3, 50 MHz)
d
171.4, 140.1, 124.8, 81.4, 59.5, 55.7, 48.1, 42.5, 41.9, 40.5, 40.1, 40.0,
38.9, 38.1, 37.2, 34.2, 33.3, 31.7, 29.2, 28.5, 28.5, 27.0, 24.0, 23.8, 23.6,
21.8, 21.7, 18.7, 17.9, 17.3, 17.1, 16.1.
4.1.2. Synthesis of 4,4,6a,6b,8a,11,12,14b-octamethyl-1,2,3,4,4a,5,6,6a,
6b,7,8,8a,9,10,11,12,12a, 14,14a,14b-icosahydropicen-3-ol (2)
4.1.5.1. 1-Dodecyl-3-((4,4,6a,6b,8a,11,12,14b-octamethyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14, 14a,14b-icosahydropicen-
3-yloxy)carbonyl)pyridinium bromide 5. Yield: 78%; m.p. ¼ 91 ꢂC;
To a stirred solution of a-amyrin acetate 1 (4.68 g, 1 mmol) in
dry methanol (7 ml) was added sodium methoxide (2.16 g, 4 mmol).
The stirring was continued for 2 h at room temperature. The re-
action mixture was acidified with amberlyst-120, filtered off under
suction and the filtrate concentrated under reduced pressure to
afford the desired compound 2. Yield: 88%; 1H NMR (CDCl3,
1H NMR (CDCl3, 300 MHz)
d
9.98 (d, J ¼ 2.22 Hz, 1H), 9.23 (s,
1H), 8.86 (d, J ¼ 5.14 Hz, 1H), 8.41 (t, J ¼ 4 Hz, 1H), 5.10 (m, 1H),
5.04 (m, 2H), 4.84-4.79 (m, 1H), 2.03e1.98 (m, 2H), 1.92e1.69
(m, 6H), 1.57e1.52 (m, 3H), 1.20 (m, 32H), 1.05 (s, 3H), 0.99 (s,
6H), 0.92 (s, 3H), 0.88 (s, 3H), 0.86e0.81 (m, 6H), 0.77 (m, 6H);
200 MHz)
d
5.1 (t, J ¼ 3.56 Hz, 1H), 3.26e3.19 (m, 1H), 2.02e1.8 (m,
13C NMR (CDCl3, 50 MHz)
d 160.8, 149.3, 144.7 (2C), 139.6, 130.9,
4H), 1.68e1.47 (m, 10H), 1.41e1.25 (m, 9H), 1.07 (s, 3H), 1.01 (s, 3H),
129.3, 124.1, 85.1, 62.9, 58.9, 55.3, 47.6, 42.0, 41.5, 39.9, 39.5 (2C),
38.3, 38.1, 36.7, 33.7, 31.9 (3C), 29.6 (5C), 29.3 (3C), 29.0, 28.7,
28.3, 28.0, 26.6, 26.1, 23.6, 23.2, 21.4, 18.2, 17.4, 17.1, 16.8, 15.7,
14.1; ESI MS m/z 700.4.
0.99 (s, 3H), 0.97e0.91 (m, 6H), 0.79 (m, 9H); 13C NMR (CDCl3,
50 MHz)
d 140.0, 124.8, 79.5, 59.5, 55.6, 48.1, 42.5, 41.9, 40.4, 40.1,
40.0, 39.2, 37.3, 34.2, 33.4, 31.7, 30.1, 29.2, 28.5 (2C), 27.7, 27.1, 23.8,
23.7, 21.8, 18.8, 17.9, 17.3, 16.1, 16.0.
4 . 1. 5 . 2 . 3 - ( ( 4 , 4 , 6 a , 6 b , 8 a , 11, 1 2 , 1 4 b - O c t a m e t h y l -
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen-
3-yloxy)carbonyl)-1-tridecylpyridinium bromide 6. Yield: 79%;
4.1.3. Synthesis of 4,4,6a,6b,8a,11,12,14b-octamethyl-1,2,3,4,4a,5,6,
6a,6b,7,8,8a,9,10,11,12,12a, 14,14a,14b-icosahydropicen-3-yl
nicotinate (3)
amorphous solid; 1H NMR (CDCl3, 300 MHz)
d 9.84 (bs, 1H), 9.22 (s,
To
a stirred solution of 4,4,6a,6b,8a,11,12,14b-octamethyl-
1H), 8.90 (d, J ¼ 7.62Hz, 1H), 8.34 (bs, 1H), 5.14 (m, 1H), 4.94e4.85
(m, 3H), 2.05e1.69 (m, 11H), 1.62e1.53 (m, 4H), 1.42e1.34 (m, 10H),
1.25 (m, 20H), 1.09 (s, 3H), 1.04 (s, 6H), 0.96 (s, 3H), 0.93 (s, 3H),
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahy-
dropicen-3-ol 2 (0.85 g, 2 mmol) in dry DCM (10 ml) was added DCC
(0.82 g, 4 mmol), DMAP (0.48 g, 4 mmol) and nicotinic acid (0.56 g,
4 mmol). The stirring was continued for 4 h at room temperature.
The reaction mixture was filtered off under suction, concentrated
under reduced pressure and the concentrate was subjected to silica
gel column chromatography using ethyl acetateehexane to obtain
0.88e0.86 (m, 6H), 0.82 (m, 6H); 13C NMR (CDCl3, 50 MHz)
d 160.8,
149.4, 144.71 (2C), 139.7, 130.9, 129.2, 124.1, 85.1, 62.9, 59.0, 55.3,
47.6, 42.1, 41.5, 40.0, 39.6, 39.5, 38.4, 38.1, 36.8, 33.7, 31.9 (2C), 29.6
(6C), 29.5, 29.3 (4C), 29.0, 28.7, 28.3, 28.1, 26.6, 26.1, 23.2, 22.7, 19.1,
17.5, 16.9, 16.8, 15.7, 14.1; ESI MS m/z 714.6.
3. Yield: 85%; 1H NMR (CDCl3, 200 MHz)
d
9.18 (d, J ¼ 1.4 Hz, 1H),

T. Narender et al. / European Journal of Medicinal Chemistry 63 (2013) 162e169
167
4.1.5.3. 1-Hexadecyl-3-((4,4,6a,6b,8a,11,12,14b-octamethyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a, 14,14a,14b-icosahydropicen-
3-yloxy)carbonyl)pyridinium bromide 7. Yield: 75%; amorphous
35.6, 34.2, 29.8, 28.1, 27.4, 25.1, 23.7, 20.9, 19.3, 18.2, 17.9, 16.8, 16.2,
15.9, 14.5.
solid; 1H NMR (CDCl3, 300 MHz)
d
9.55 (bs, 1H), 9.20 (s, 1H), 8.86 (d,
4.1.8. General procedure for synthesis of N-allylated lupeoleniacin
hybrids (12,13)
J ¼ 8.0 Hz, 1H), 8.32 (t, J ¼ 6.8 Hz, 1H), 5.11 (m, 1H), 4.84 (m, 3H),
2.00e1.90 (m, 5H), 1.85e1.66 (m, 4H), 1.56e1.52 (m, 3H), 1.39e1.22
(m, 39H), 1.06 (s, 3H), 1.00 (s, 6H), 0.92 (s, 3H), 0.89 (s, 3H), 0.87e
The mixture of 3a,5a,5b,8,8,11a-hexamethyl-1-(prop-1-en-2-
yl)-2,3,3a,4,5,5a,5b,6,7,7a,8,9,10,11,11a,11b,12, 13b-octadecahydro-
1H-cyclopenta[a]chrysen-9-yl nicotinate 11 and appropriate allyl
bromide (1:4) in dry diethyl ether was stirred at room temperature
for 6e7 h. The product was precipitated which was filtered off and
washed with diethyl ether to afford the pure compounds 12, 13.
0.82 (m, 6H), 0.78 (m, 6H); 13C NMR (CDCl3, 50 MHz)
d 160.8,
149.1, 144.8, 144.6, 139.5, 130.9, 129.1, 124.0, 84.8, 62.7, 58.9, 55.2,
47.5, 41.9, 41.3, 39.9, 39.6, 39.5, 38.3, 37.9, 36.6, 33.5, 31.7 (2C), 31.5,
29.5 (6C), 29.4 (3C), 29.2 (2C), 28.9, 28.6, 28.1, 27.9, 26.4, 25.9, 23.4,
23.2, 23.1, 21.2, 18.1, 17.3, 16.8, 16.7, 15.5, 13.9; ESI MS m/z 756.
4.1. 8.1. 3-(1R, 3aR, 5aR, 5bR, 7aR,11aR,11bR,13aS,13bS)-
3a,5a,5b,8,8,11a-Hexamethyl-1-(prop-1-en-2-yl)icosahydro-1H-
cyclopenta[a]chrysen-9-yloxy)carbonyl)-1-(3-methylbut-2-enyl)pyr-
idinium bromide 12. Yield: 91%; m.p. ¼ 256 ꢂC; 1H NMR (CDCl3,
4.1.5.4. 1-Octadecyl-3-((4,4,6a,6b,8a,11,12,14b-octamethyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a, 14,14a,14b-icosahydropicen-
3-yloxy)carbonyl)pyridinium bromide 8. Yield: 78%; m.p. ¼ 84 ꢂC;
1H NMR (CDCl3, 300 MHz)
d
9.57 (bs, 1H), 9.21 (s, 1H), 8.88 (d,
300 MHz)
d
9.84 (d, J ¼ 5.55 Hz, 1H), 9.13 (s, 1H), 8.79 (d, J ¼ 7.89 Hz,
J ¼ 7.95 Hz, 1H), 8.34 (t, J ¼ 6.72 Hz, 1H), 5.13 (m, 1H), 4.87e4.81 (m,
3H), 2.01e1.92 (m, 5H), 1.82e1.73 (m, 4H), 1.57e1.54 (m, 4H), 1.40e
1.24 (m, 42H), 1.08 (s, 3H), 1.02 (s, 6H), 0.94 (s, 3H), 0.91 (s, 3H),
1H), 8.38 (t, J ¼ 6.63 Hz,1H), 5.65 (m, 2H), 5.55 (m,1H), 4.69 (m,1H),
4.61 (s, 1H), 4.49 (s, 1H), 2.26 (m, 2H), 1.86 (s, 3H), 1.79 (s, 3H), 1.69e
1.60 (m, 9H), 1.44e1.29 (m, 11H), 1.21e1.09 (m, 7H), 0.97 (s, 3H),
0.89e0.88 (m, 6H), 0.83e0.82 (m, 6H), 0.71 (s, 3H); 13C NMR (CDCl3,
0.88e0.84 (m, 6H), 0.79 (m, 6H); 13C NMR (CDCl3, 50 MHz)
d 160.9,
149.2, 144.8, 144.6, 139.6, 130.9, 129.2, 124.1, 84.8, 62.8, 58.9, 55.2,
47.6, 41.9, 41.4, 39.9, 39.6, 39.5, 38.4, 38.0, 36.7, 33.6, 31.8 (2C), 31.5,
29.6 (7C), 29.4 (3C), 29.3, 29.2 (2C), 28.9, 28.6, 28.2, 27.9, 26.5, 26.0,
23.5, 23.3, 23.2, 21.3, 18.1, 17.4, 16.9, 16.8, 15.6, 14.0; ESI MS m/z
784.6.
75 MHz) d 160.8, 150.7, 149.0, 146.0, 144.7, 143.7, 130.7, 129.1, 115.5,
109.3, 84.8, 59.9, 55.3, 50.2, 48.3, 47.9, 42.9, 42.8, 40.8, 39.9, 38.2,
38.1, 37.9, 37.0, 35.5, 34.1, 29.8, 28.2, 27.4, 25.9, 24.9, 23.6, 20.9, 19.3,
19.0, 18.1, 17.9, 16.7, 16.1, 15.9, 14.5; ESI MS m/z 600.5.
4.1.8.2. 1-((E)-3,7-Dimethylocta-2,6-dienyl)-3-(1R,3aR,5a-
R,5bR,7aR,11aR,11bR,13aS,13bS)-3a,5a,5b, 8,8,11a-hexamethyl-1-(prop-1-
en-2-yl)icosahydro-1H-cyclopenta[a]chrysen-9-yloxy)carbonyl) pyr-
idinium bromide 13. Yield: 89%; m.p. ¼ 253 ꢂC; 1H NMR (CDCl3,
4.1.5.5. 1-Icosyl-3-((4,4,6a,6b,8a,11,12,14b-octamethyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14, 14a,14b-icosahydropicen-
3-yloxy)carbonyl) pyridinium bromide 9. Yield: 80%; m.p. ¼ 135 ꢂC;
1H NMR (CDCl3, 300 MHz)
d
9.96 (bs, 1H), 9.25 (s, 1H), 8.84 (d,
300 MHz)
d
9.89 (bs, 1H), 9.10 (s, 1H), 8.8 (d, J ¼ 6.87 Hz, 1H), 8.39
J ¼ 7.38 Hz, 1H), 8.44 (t, J ¼ 6.51 Hz, 1H), 5.07e5.04 (m, 3H), 4.77 (m,
1H), 2.00e1.67 (m, 9H), 1.62e1.49 (m, 5H), 1.35e1.27 (m, 11H), 1.19
(m, 34H), 1.03 (s, 3H), 0.97 (m, 9H), 0.89 (s, 3H), 0.86 (s, 3H), 0.83e
(m, 1H), 5.68 (m, 2H), 5.53 (m, 1H), 4.99 (s, 1H), 4.74 (t, J ¼ 6.69 Hz,
1H), 4.64 (bs, 1H), 4.52 (s, 1H), 2.37e2.28 (m, 1H), 2.10 (s, 3H), 1.88
(s, 3H), 1.72e1.60 (m, 15H), 1.53 (s, 3H), 1.37e1.26 (m, 13H), 1.17e
1.13 (m, 3H), 1.00 (s, 3H), 0.92 (m, 6H), 0.86 (m, 6H), 0.74 (s, 3H);
0.79 (m, 6H), 0.74 (s, 3H); 13C NMR (CDCl3, 50 MHz)
d 160.8, 149.4,
144.74 (2C), 139.6, 130.9, 129.4, 124.1, 85.0, 62.9, 58.9, 55.3, 47.6,
42.0, 40.8, 39.9, 39.6, 38.4, 38.1, 36.7, 33.7, 31.9 (2C), 31.7, 29.7 (8H),
29.6 (4H), 29.5, 29.3 (2C), 29.0, 28.7, 28.3, 28.2, 28.0, 26.5, 26.0, 23.6,
23.3, 23.2, 21.3, 18.2, 17.4, 17.1, 16.8, 15.7, 14.1; ESI MS m/z 812.6.
13C NMR (CDCl3, 50 MHz)
d 160.8, 150.8, 149.7, 149.2, 144.8, 143.6,
132.4, 130.8, 129.1, 122.9, 115.1, 109.3, 84.9, 59.9, 55.3, 50.2, 48.2,
47.9, 42.9, 42.8, 40.8, 39.9, 39.5, 38.2, 38.1, 37.9, 37.0, 35.5, 34.1, 29.8,
28.2, 27.4, 26.1, 25.6, 24.9, 23.7, 20.9, 19.3, 18.1, 17.9, 17.7, 17.5, 16.8,
16.1, 15.9, 14.5; ESI MS m/z 668.6.
4.1.6. Lupeol 10
1H NMR (CDCl3, 300 MHz)
3.15 (m, 1H), 2.42e2.33 (m, 1H), 1.95 (m, 1H), 1.68 (m, 10H), 1.52e
1.32 (m, 10H), 1.26e1.12 (m, 6H), 1.03 (s, 3H), 0.96 (s, 3H), 0.94 (s,
3H), 0.83 (s, 3H), 0.79 (s, 3H), 0.76 (s, 3H); 13C NMR (CDCl3, 50 MHz)
150.8, 109.4, 78.9, 55.3, 50.4, 48.3, 47.9, 42.9, 42.8, 40.8, 40.0, 38.8,
38.7, 38.4, 37.1, 35.6, 34.3, 29.8, 28.0, 27.5, 27.4, 25.1, 20.9, 19.3, 18.3,
18.0, 16.1, 15.9, 15.4, 14.6.
d
4.68 (bs, 1H), 4.56 (bs, 1H), 3.20e
4.1.9. General procedure for synthesis of N-alkylated lupeoleniacin
hybrids (14e16)
The mixture of 3a,5a,5b,8,8,11a-hexamethyl-1-(prop-1-en-2-
yl)-2,3,3a,4,5,5a,5b,6,7,7a,8,9,10,11,11a,11b,12, 13b-octadecahydro-
1H-cyclopenta[a]chrysen-9-yl nicotinate (11) and appropriate alkyl
bromide (1:4) in toluene was stirred for 36 h under reflux condi-
tion. The reaction mixture was concentrated under vacuum to
evaporate toluene. The crude product was then subjected to silica
gel chromatography using methanol:chloroform mobile phase to
afford the pure compounds 14e16.
d
4.1.7. Synthesis of 3a,5a,5b,8,8,11a-hexamethyl-1-(prop-1-en-2-
yl)-2,3,3a,4,5,5a,5b,6,7,7a,8,9, 10,11,11a,11b,12,13b-octadecahydro-
1H-cyclopenta[a]chrysen-9-yl nicotinate (11)
To a stirred solution of lupeol 10 (0.43 g, 1 mmol) in dry DCM
(8 ml) was added DCC (0.41 g, 2 mmol), DMAP (0.24 g, 2 mmol) and
nicotinic acid (0.27 g, 2 mmol). The stirring was continued for 4 h at
room temperature. The reaction was filtered off under suction,
concentrated under reduced pressure and the concentrate was
subjected to silica gel chromatography using ethyl acetate:hexane
4.1.9.1. 1-Dodecyl-3-((3a,5a,5b,8,8,11a-hexamethyl-1-(prop-1-en-2-
yl)icosahydro-1H-cyclopenta[a]
chrysen-9-yloxy)carbonyl)pyr-
idinium bromide 14. Yield: 81%; m.p. ¼ 177 ꢂC; 1H NMR (CDCl3,
300 MHz)
d
10.02 (d, J ¼ 5.61 Hz, 1H), 9.37 (s, 1H), 8.81 (d,
J ¼ 7.95 Hz,1H), 8.41 (t, J ¼ 6.27 Hz,1H), 5.08 (t, J ¼ 6.41 Hz, 2H), 4.71
(m, 1H), 4.59 (s, 1H), 4.47 (s, 1H), 2.28 (m, 1H), 1.98 (m, 2H), 1.79e
1.59 (m, 9H), 1.33e1.23 (m, 16H), 1.16e1.14 (m, 20H), 0.96 (s, 3H),
0.92 (s, 3H), 0.87 (s, 3H), 0.82e0.78 (m, 9H), 0.70 (s, 3H); 13C NMR
to obtain 11. Yield: 89%; 1H NMR (CDCl3, 300 MHz)
d 9.22 (s, 1H), 8.8
(d, J ¼ 3.33 Hz, 1H), 8.29 (d, J ¼ 7.77 Hz, 1H), 7.41e7.36 (m, 1H), 4.8e
4.73 (m, 1H), 4.70 (s, 1H), 4.58 (s, 1H), 2.43e2.34 (m, 1H), 2.01e1.90
(m, 3H), 1.77e1.69 (m, 10H), 1.54e1.18 (m, 15H), 1.06 (s, 3H), 1.00 (s,
3H), 0.97 (s, 3H), 0.93 (s, 3H), 0.91 (s, 3H), 0.80 (s, 3H); 13C NMR
(CDCl3, 50 MHz) d 160.8,150.7,149.1,145.0,144.7,130.9,129.3,109.4,
84.9, 62.6, 55.3, 50.2, 48.1, 47.9, 42.9, 42.7, 40.7, 39.9, 38.3, 38.1, 37.9,
36.9, 35.5, 34.1, 31.8 (2C), 29.5 (3C), 29.4, 29.3, 29.2, 29.0, 28.2, 27.4,
25.9, 24.9, 23.7, 22.6, 20.9, 19.3, 18.1, 17.9, 16.9, 16.1, 15.9, 14.5, 14.1;
ESI MS m/z 700.5.
(CDCl3, 50 MHz)
d 164.9, 153.1, 1508 (2C), 137.0, 126.8, 123.2, 109.4,
82.4, 55.4, 50.3, 48.3, 47.9, 42.9, 42.8, 40.8, 39.9, 38.3, 38.2, 38.0, 37.1,

168
T. Narender et al. / European Journal of Medicinal Chemistry 63 (2013) 162e169
4.1.9.2. 1-Hexadecyl-3-(((1R,3aR,5aR,5bR,7aR,11aR,11bR,13aS,13bS)-
3a,5a,5b,8,8,11a-hexamethyl-1-(prop-1-en-2-yl)icosahydro-1H-
was given to all groups 30 min post administration of the test
sample/vehicle. Blood glucose levels were checked at 30 min,
60 min, 90 min, 2 h, 3 h, 4 h, 5 h and 24 h time point post sucrose
administration. Food but not water was withheld from the cages
during the course of experimentation.
cyclopenta[a]chrysen-9-yloxy)carbonyl)pyridinium
bromide
15.
Yield: 77%; m.p. ¼ 179 ꢂC; 1H NMR (CDCl3, 300 MHz)
d
10.16 (d,
J ¼ 5.76 Hz, 1H), 9.21 (s, 1H), 8.88 (d, J ¼ 8.04 Hz, 1H), 8.45 (m, 1H),
5.11 (t, J ¼ 6.93 Hz, 2H), 4.84e4.79 (m, 1H), 4.68 (s, 1H), 4.56 (s, 1H),
2.22 (m, 1H), 2.05 (m, 2H), 1.77e1.68 (m, 9H), 1.51e1.31 (m, 14H),
1.30e1.24 (m, 30H), 1.04 (s, 3H), 0.99 (s, 3H), 0.95 (s, 3H), 0.91e0.84
4.2.4. Statistical analysis
Values are given as mean ꢀ SE. Analysis of statistical significance
of differences in measurements between samples was done by one-
way ANOVA with Dunnets post hoc test (GraphPad Prism version
3). P < 0.05 was considered statistically significant.
(m, 9H), 0.78 (s, 3H); 13C NMR (CDCl3, 75 MHz)
d 160.8, 150.8, 149.3,
144.7,144.6,130.9,129.4,109.3, 85.1, 62.9, 55.4, 50.3, 48.2, 47.9, 42.9,
42.8, 40.8, 39.9, 38.3, 38.2, 37.9, 37.0, 35.5, 34.1, 31.9 (2C), 31.7, 29.8,
29.6 (4C), 29.6 (2C), 29.5, 29.3, 29.0, 28.2, 27.4, 26.0, 25.0, 23.7, 22.6,
20.9, 19.3, 18.1, 17.9, 16.9, 16.1, 15.9, 14.5, 14.1; ESI MS m/z 756.6.
Acknowledgment
Authors are thankful to Dr. T. K. Chakraborty, Director, CDRI for
constant encouragement on the program on natural products’
synthesis, SAIF for spectral data, Miss. Preeti and J. P. Chaturvedi for
technical support and DST and CSIR, New Delhi for the financial
support. This is CDRI manuscript No.272/2012/TN and communi-
cation No. 8397.
4.1.9.3. 1-Docosyl-3-((3a,5a,5b,8,8,11a-hexamethyl-1-(prop-1-en-2-
yl)icosahydro-1H-cyclopenta[a] chrysen-9-yloxy)carbonyl)pyridini-
um bromide 16. Yield: 76%; m.p. ¼ 145 ꢂC; 1H NMR (CDCl3,
300 MHz)
d
9.91 (bs, 1H), 9.23 (s, 1H), 8.87 (d, J ¼ 7.26 Hz, 1H), 8.39
(s, 1H), 4.99 (m, 2H), 4.82e4.79 (m, 1H), 4.68 (s, 1H), 4.56 (s, 1H),
2.36 (m, 1H), 2.03 (m, 2H), 1.77e1.68 (m, 12H), 1.41e1.35 (m, 18 H),
1.24 (m, 35H), 1.04 (s, 3H), 0.99 (s, 3H), 0.95 (s, 3H), 0.91e0.89 (m,
6H), 0.86e0.84 (m, 3H), 0.79 (s, 3H); 13C NMR (CDCl3, 75 MHz)
Appendix A. Supplementary data
d
160.8, 150.8, 149.4, 144.7 (2C), 131.0, 129.3, 109.4, 85.1, 62.9, 55.4,
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.01.053.
50.3, 48.2, 47.9, 42.9, 42.8, 40.8, 39.9, 38.3, 38.2, 37.9, 37.1, 35.5, 34.1,
31.9 (2C), 31.7, 29.7 (12C), 29.3 (3C), 29.0, 28.2, 27.4, 26.1, 25.0, 23.7,
22.7, 20.9, 19.3, 18.2, 17.9, 16.8, 16.1, 15.9, 14.5, 14.1; ESI MS m/z 840.7.
References
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