Synthesis of α-amyrin derivatives and their in vivo antihyperglycemic activity

Article abstract of DOI:10.1016/j.ejmech.2008.09.011

Ficus racemosa belongs to the family of Moraceae and is commonly known as 'Gular' in north India. Bio-activity guided isolation work on the fruits of F. racemosa resulted in the identification of antidiabetic active principle, α-amyrin acetate 7. Compound 7 lowered the blood glucose levels by 18.4 and 17.0% at 5 and 24 h, respectively, in sucrose challenged streptozotocin induced diabetic rat (STZ-S) model at the dose of 100 mg/kg body weight. Fifteen novel derivatives viz, 9-21, 24, 25 of α-amyrin 8 were prepared and their antihyperglycemic activity profile was assessed. The p-chlorobenzoic acid derivative 9 and nicotinic acid derivative 14 showed potent antihyperglycemic activity at 100 mg/kg body weight.

Full text of DOI:10.1016/j.ejmech.2008.09.011

European Journal of Medicinal Chemistry 44 (2009) 1215–1222
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of
a
-amyrin derivatives and their in vivo antihyperglycemic activityq
,
a
b
b
a
a
b,
**
T. Narender a , T. Khaliq , A.B. Singh , M.D. Joshi , P. Mishra , J.P. Chaturvedi , A.K. Srivastava
,
*
R. Maurya a, S.C. Agarwal c
a Division of Medicinal and Process Chemistry, Central Drug Research Institute, Chattar Manzil, Lucknow 226 001, Uttar Pradesh, India
b Division of Biochemistry, Central Drug Research Institute, Chattar Manzil, Lucknow 226 001, Uttar Pradesh, India
c Division of Botany, Central Drug Research Institute, Chattar Manzil, Lucknow 226 001, Uttar Pradesh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 April 2008
Received in revised form 22 August 2008
Accepted 4 September 2008
Available online 19 September 2008
Ficus racemosa belongs to the family of Moraceae and is commonly known as ‘Gular’ in north India. Bio-
activity guided isolation work on the fruits of F. racemosa resulted in the identification of antidiabetic
active principle,
and 24 h, respectively, in sucrose challenged streptozotocin induced diabetic rat (STZ-S) model at the
dose of 100 mg/kg body weight. Fifteen novel derivatives viz, 9–21, 24, 25 of -amyrin 8 were prepared
a-amyrin acetate 7. Compound 7 lowered the blood glucose levels by 18.4 and 17.0% at 5
a
and their antihyperglycemic activity profile was assessed. The p-chlorobenzoic acid derivative 9 and
nicotinic acid derivative 14 showed potent antihyperglycemic activity at 100 mg/kg body weight.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords:
a
-Amyrin acetate
Amyrin ester derivatives
Antihyperglycemic activity
STZ-S model
1. Introduction
diabetes [7,8]. However, effects with crude extracts remain
obscure if the active principles in the extracts are not character-
Diabetes mellitus is characterized by elevated plasma glucose
concentrations resulting from insufficient insulin production,
insulin resistance, or both [1]. The number of cases of non-insulin
dependent diabetes mellitus has increased dramatically due to the
changes in lifestyle, increasing prevalence of obesity and ageing of
populations [2]. In the year 2000, the number of diabetic patients
was 151 million and is estimated to rise to 300 million by 2025
[3,4]. The high prevalence and long-term complications of diabetes
mellitus have led to an intense search for new oral anti-
hyperglycemic agents from plants used as traditional medicine for
diabetes treatment [5]. The use of natural drugs, such as plants and
herbal remedies to treat diseases is very common in Asia and
developing countries, where the population is linked with the use
of traditional medicines, due to their efficiency or due to the costs of
the synthetic drugs and/or pharmaceuticals. Moreover, WHO study
groups emphasize strongly the optimal, rational uses of traditional
and natural indigenous medicines [6].
ized. Various groups have reported phytochemicals (Fig. 1) with
potential antihyperglycemic attributes. Glycyrin 1, one of the main
PPAR-
g ligands of licorice, significantly decreased the blood
glucose levels of genetically diabetic KK-Ay mice [9]. Charantin 2,
a steroidal saponin, obtained from Momordica charantia is known
to have an insulin-like activity, responsible for its hypoglycemic
effect [10]. Charantin stimulates the release of insulin and blocks
the formation of glucose in the bloodstream. A friedelane-type
C8H17
RO
O
O
O
OAc
OH
Glu
OMe
O
O
OH
HO
Glycyrin: 1
Charantin: 2
Kotalagenin 16-acetate: 3
A large number of crude plant extracts and purified substances
from plants have been tested in clinical trials for the treatment of
NH NH
H3CO
NH2
O
N
NH
N
N
H
N
H2N
H
OH
q
H
CDRI Comm. No.7334.
* Corresponding author. Tel.: þ91 522 2612411; fax: þ91 522 2623405/2623938.
Galegine: 4
Metformin: 5
Aegeline:6
** Corresponding author.
E-mail addresses: tnarender@rediffmail.com (T. Narender), drakscdri@rediffmail.
com (A.K. Srivastava).
Fig. 1. Naturally occurring antidiabetic agents 1–4 and 6; synthetic antidiabetic
agents 5.
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.09.011

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T. Narender et al. / European Journal of Medicinal Chemistry 44 (2009) 1215–1222
Table 1
In vivo antihyperglycemic activity of
a
-amyrin acetate 7 and its synthetic derivatives in STZ-S model
R
Compound number
R
Dose (mg/kg)
% Antihyperglycemic activity
5 h
24 h
O
O
7
8
100
100
18.4*
13.9*
17.0*
8.12*
OH
O
O
Cl
9
100
22.1*
25.6**
O
O2N
10
11
100
100
13.8*
17.5*
27.6**
15.6*
O
MeO
O
O
O
o
12
13
100
100
24.0**
12.7*
23.5**
22.6*
O
O
O
N
14
15
100
100
18.2*
19.6*
27.6**
17.8*
O
O
N
O
O
Br
O
16
17
100
100
ND
ND
O
OH
3.15*
12.1*
HN
O O

T. Narender et al. / European Journal of Medicinal Chemistry 44 (2009) 1215–1222
1217
Table 1 (continued )
Compound number
R
Dose (mg/kg)
% Antihyperglycemic activity
5 h
24 h
18
19
100
24.9**
11.0*
20.2*
16.5*
O O
HOOC
O
100
O O
OH
20
21
ND
ND
O O
O
100
24.3**
15.6*
O
O
Ph
NH
C=S
24a
100
1.20*,a
7.35*,a
NO2
N
N
O2N
NO2
NH
C=S
25
ND
ND
NO
2 N
N
O2N
NH NH
5
100
23.5**
26.5**
N
N
H
NH2
ND ¼ Not determined.
*P < 0.05 and **P < 0.01.
a
Evaluated in SLM model.
triterpene, kotalagenin 16-acetate 3, isolated from the roots of
Salacia oblonga exerted its action as potent -glucosidase inhib-
itor [11]. -Carbolines like harmane, norharmane and pinoline act
[19] activities whilst the bark, root and fruit have been found to
possess antidiabetic activity [20,21]. The hypoglycemic potential of
leaf extract [22] of the plant was demonstrated in streptozotocin
induced diabetic rats whilst glucose lowering efficacy of the stem
bark in normal as well as alloxan-induced diabetic rats was also
reported [23]. Though the antidiabetic activity of the extract of
different parts of the plant is reported in the literature, so far the
antihyperglycemic principle has not been identified. Based on the
importance of this medicinally valuable herb in the Indigenous
system of medicine and its therapeutic importance in ameliorating
plasma glucose, bio-assay directed fractionation was carried out
in order to localize the antihyperglycemic principle. Since the
a
b
as potent insulin secretagogues [12]. Metformin 5 is currently
used as antidiabetic agent in the treatment of type-2 diabetes.
Metformin 5 and its analogues [13,14] were synthesized on
the basis of a natural product lead viz, galegine 4, isolated from
the seeds of Galega officinalis [5]. Our group recently reported the
isolation of the dual acting antihyperglycemic and anti-
dyslipidemic agent, aegeline 6, from the leaves of Aegle marmelos
[15].
Ficus racemosa Linn. (Moraceae), commonly known as ‘Gular’,
‘Umbar’ or ‘Jagya-dumbar’ [16] is found throughout India. Tradi-
tionally, the juice of the leaves is used to treat dysentery, bilious
disorders, diarrhoea, diabetes and as a mouth wash [17]. The leaves
of the plant showed anti-inflammatory [18] and hepatoprotective
active principle,
non-natural ester and metformin related derivatives of deacety-
lated -amyrin to develop SAR and improve the potency of the lead
(Table 1).
a-amyrin acetate was an ester, we prepared several
a

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T. Narender et al. / European Journal of Medicinal Chemistry 44 (2009) 1215–1222
2. Results and discussion
2.1. Chemistry
a
b
O
O
The syntheses of non-natural ester derivatives of
acetate were accomplished by subjecting the naturally isolated
amyrin actetate 7 to deacetylation by freshly prepared sodium
methoxide in dry methanol. -Amyrin 8 thus, obtained was ester-
a-amyrin
O
HO
O O
OH
19
7
8
a
-
b
a
ified using various aromatic and aliphatic acids employing DCC–
DMAP esterification protocol in dry DCM to afford non-natural
esters of a-amyrin 9–18 (Scheme 1).
c
O
Excess of non-esterified fatty acids (NEFA) have been implicated
in the pathology of insulin resistance [24]. High levels of NEFA have
been linked in humans with insulin insensitivity [25]. Treatment
with nicotinic acid decreases NEFA and increases peripheral
glucose uptake, but this effect is not long lived [26]. Perhaps a more
specific analogue of nicotinic acid, such as acipimox gave better
results for controlling dyslipidemia and hyperglycemia [27].
Keeping this in view, we prepared few non-esterified aliphatic and
O
O
HO
O
O
O
O
21
20
Scheme 2. Reagents and conditions: (a) Sodium methoxide, methanol, r.t., 2 h. (b)
Appropriate anhydride, DMAP, DCM, r.t., 4 h. (c) -Amyrin, DCC–DMAP, DCM, r.t., 6 h.
a
aromatic acid derivatives of a-amyrin viz, 19, 20 from the respective
anhydrides in DMAP–DCM (Scheme 2).
acid catalyst, indium chloride (InCl3), hitherto unreported for
hydrazine formation was employed for the same in order to facil-
itate the formation of the hydrazine 23 using more deactivating
2,4-dinitrophenylhydrazine.
The plausible mechanism was delineated to be of adduct
formation of the catalyst with the sterically hindered ketone 22.
The corresponding hydrazine 23 was made to react with the
appropriate phenylthioisocyanates in dry DMF in the presence of
NaH resulting in the formation of corresponding thiourea deriva-
tives 24 and 25 (Scheme 3).
Since the active moiety was the amyrin scaffold itself, dimer-
ization of the active factor was envisaged so as to enhance its
antihyperglycemic activity. The synthesis was accomplished first by
treating amyrin 8 with succinic anhydride and then reaction
between the resultant succinyl derivative 20 and amyrin 8 to afford
the target molecule 21 (Scheme 2).
Metformin 5 remains a primary therapeutic option for Type II
diabetics [28]. New studies have described the effectiveness of
combination therapy using metformin and other standard anti-
hyperglycemics for the maintenance of blood glucose levels [29].
Keeping in view the efficacy of metformin in antidiabetic therapy,
we envisaged Scheme 3 wherein thiourea unit resembling the
2.2. Biology
All the compounds viz, the natural lead,
a-amyrin acetate as
metformin pharmacophore was incorporated in a-amyrin acetate
well as its synthetic derivatives were evaluated for their anti-
hyperglycemic activity in STZ-S model at the dose of 100 mg/kg
selecting male albino rats of SD strain (body weight 140 ꢀ 20 g).
Fresh solution of streptozotocin solution (60 mg/kg) in 100 mM
citrate buffer pH 4.5 and calculated amount of the fresh solution
was injected to overnight fasted rats (45 mg/kg) intraperitoneally.
Animals showing blood glucose values between 8 and 15 mM were
selected and divided into groups of six animals in each. Rats of
experimental groups were administered suspension of the desired
test samples orally (made in 1.0% gum acacia) at 100 mg/kg dose. A
sucrose load of 2.5 g/kg body weight was given after 30 min of drug
administration. After 30 min of post-sucrose load, blood glucose
in order to optimize the activity of the natural product lead.
The synthesis of the title compounds was accomplished by
deacetylation of
by oxidation of the resultant
DCM protocol. The usual methods available for hydrzone formation
of saturated ketones failed in case of -amyrinone. Hydrazine
hydrate and its corresponding salt and phenyl hydrazine and its salt
failed to convert the -amyrinone 22 into the corresponding
a
-amyrin acetate 7 by sodium methoxide followed
a
-amyrin 8 to -amyrinone 20 by PCC–
a
a
a
hydrazine derivative. So a strategy was devised in which a Lewis
a
b
a
O
O
HO
8
7
HO
O O
O
7
8
22
c
b
d
R1
H
NO
NO2
2 N
N
N
N
23
24, 25
O2N
O2N
R
9-18
Scheme 3. Reagents and conditions: (a) Sodium methoxide, methanol, r.t., 2 h. (b)
Pyridinium chlorochromate, DCM, r.t., 12 h. (c) 2,4-Dinitrophenylhydrazine, absolute
ethanol, indium chloride, reflux, 12 h. (d) Appropriate phenyl thioisocyanate, sodium
hydride, DMF, 0 ꢁC, r.t.
Scheme 1. Reagents and conditions: (a) Sodium methoxide, methanol, r.t., 2 h. (b)
Appropriate aromatic/aliphatic acid, DCC–DMAP, DCM, r.t., 4 h.

T. Narender et al. / European Journal of Medicinal Chemistry 44 (2009) 1215–1222
1219
level was again checked with the aid of glucometer after 30, 60, 90,
120, 180, 240, 300 min and at 24 h, respectively. Comparison of the
AUC of experimental and control groups determined the percent
antihyperglycemic activity.
The activities were found to be very promising in few deriva-
tives. a-Amyrin acetate 7 exhibited significant activity possessing
3-ylacetate 7 (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 reaction mixture was acidified with
amberlyst-120, filtered off under suction and the filtrate concen-
trated under reduced pressure to afford the desired compound.
Yield: 92%; mp 180–181 ꢁC; IR (KBr) 3349 (OH) and 1631 (C]C)
antihyperglycemic activity of 18.4% (P < 0.05) and 17.0% (P < 0.05)
at 5 and 24 h, respectively. Amongst the synthetic derivatives, the
high potentiators of plasma glucose alleviation were 9, 10, 12,
14 and 18 depicting respective lowering of 22.1% (5 h) and
25.6% (24 h; P < 0.01); 13.8% (5 h) and 27.6% (24 h; P < 0.01);
24.0% (5 h; P < 0.01) and 23.5% (24 h; P < 0.01); 18.2% (5 h) and
27.6% (24 h; P < 0.01); 24.9% (5 h; P < 0.01) and 20.2% (24 h;
P < 0.05), respectively. The higher efficacy of these derivatives may
be attributed to the presence of electron deactivating groups in 9,
14 and 19 and lipophilic units in 12 and 18. However, compound 15
bearing deactivating 2-quinonyl ring displayed diminished activity
viz, 19.6% (5 h) and 17.8% (24 h; P < 0.05) than 14, having 2-pyridyl
ring. Moderate to good activity was also exhibited by 13, the pro-
penyl derivative with the percent glucose lowering of 12.7% (5 h;
P < 0.05) and 22.6% (24 h; P < 0.05). The hydrophilic derivative 19
alleviated plasma glucose levels by 16.5% (P < 0.05) 24 h post-dose,
although no significant lowering (11.0%) was observed at 5 h post-
cmꢂ1. 1H NMR (200 MHz, CDCl3)
d 0.80 (s, 3H), 0.83 (s, 3H), 0.87 (s,
6H), 0.93 (s, 3H), 0.96 (s, 3H), 0.99 (s, 3H), 1.13 (s, 3H), 2.17 (m, 2H),
3.22 (m, 1H), 5.12 (br s, 1H). FAB MS m/z 427 (M þ 1).
4.1.2. General procedure for the synthesis of non-natural esters of
amyrin (9–19)
To
a stirred solution of 4,4,6a,8a,11,12,14b-heptamethyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen
-3-ol 8 (0.85 g, 2 mmol) in dry DCM (8 ml) was added dicyclohex-
ylcarbodiimide, DCC (0.82 g, 4 mmol), 4,4-N,N-dimethyl amino-
pyridine, DMAP (0.48 g, 4 mmol) and the appropriate acid
(4 mmol). The stirring was continued for 4 h at room temperature.
The reaction mixture was filtered off under suction and the filtrate
concentrated under reduced pressure and the residue was
subjected to silica gel chromatography using different polarity
systems of hexane:ethylacetate.
dose (P < 0.05).
a
-Amyrin 8 had a minimal effect on efficacy, 13.9%
4.1.3. General procedure for the synthesis of 4,4,8a,11,12,14b-
hexamethyl-1,4,4a,5,6,6a,6b,7,8, 8a,9,10,11,12,12a,14,14a,14b-
octadecahydro-2H-picen-3-one (22)
(5 h) and 8.12% (24 h; P < 0.05), substantiating the vitality of the
acetate moiety in glucose amelioration effect. p-Methoxy phenyl
acetic ester 11 reduced the glycemia by 17.5% (5 h; P < 0.05) and
15.6% (24 h; P < 0.05) revealing some insignificant role of the
electron donating units in the hypoglycemia. The amino acid
derivative 17 did not impart any prolific activity, 3.15% (5 h;
P < 0.05) and 12.1% (24 h; P < 0.05) to the amyrin skeleton implying
a lesser role for hydrophobicity in correcting hyperglycemia. In
these experiments, the reference drug, metformin 5 lowered the
blood glucose level by 23.5% (P < 0.01) at 5 h and 26.5% (P < 0.01) at
24 h at the same dose, respectively. Surprisingly, no marked activity
was displayed by the thiourea derivative viz, 24 in the SLM model.
To
a 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-icosahydropicen-
3-ol 8 (1.04 g, 2.5 mmol) in dry DCM (15 ml) was added pyridinium
chlorochromate, PCC (0.86 g, 4 mmol). The stirred was continued
for 12 h at room temperature. The solvent was evaporated under
reduced pressure and the residue was subjected to silica gel chro-
matography using hexane:ethylacetate (96:04) as the eluting
solvent. Yield: 90%; mp 176–178 ꢁC; IR (KBr) 1715 (CO) and 1631
(C]C) cmꢂ1. 1H NMR (200 MHz, CDCl3)
d 0.80 (s, 3H), 0.83 (s, 3H),
0.87 (s, 6H), 0.93 (s, 3H), 0.96 (s, 3H), 0.99 (s, 3H), 1.13 (s, 3H), 1.95
(m, 2H), 2.43 (m, 2H), 5.15 (br s, 1H). FAB MS m/z 425 (M þ 1).
3. Conclusion
4.1.4. General procedure for the synthesis of (E)-1-(2,4-
dinitrophenyl)-2-(4,4,6a,6b,8a,11,12,14b-octamethyl-
1,2,4a,5,6,6a,7,8,8a,9,10,11,12,12a,14,14a-hexadecahydropicen-3-
(4H,6bH,14bH)-ylidene)-hydrazine (23)
From the foregoing studies, it could be inferred that some of the
non-natural ester analogues viz, 9, 10, 12, 14 and 18 of the naturally
occurring
naturally isolated
a
-amyrin 10 are better glucose ameliorating than the
-amyrin acetate 7 and almost as equipotent as
a
To a stirred solution of 4,4,6a,6b,8a,11,12,14b-octamethyl-
metformin. Moreover, it might be concluded from the above find-
ings that lipophilicity and electron deficient moieties in the ester
functionality are pivotal in the determination of plasma glucose
alleviation. Further work is in progress to find out the mechanism
of action in antihyperglycemic activity.
1,2,4a,5,6,6a,7,8,8a,9,10,11,12,12a,14,14a-hexadeca hydropicen-
3(4H,6bH,14bH)-one 22 (0.42 g,1 mmol) in absolute ethanol (7 ml)
was added 2,4-dinitrophenylhydrazine (0.198 g, 1 mmol) and
indium chloride (0.022 g, 10 mol%). The reaction mixture was
refluxed for 12 h. The solvent was evaporated under reduced pres-
sure and the desired compound 23 was crystallized in methanol.
Yield: 92%; mp 165–166 ꢁC; IR (KBr) 3433 (NH) and 1631 (C]C)
4. Experimental section
cmꢂ1. 1H NMR (200 MHz, CDCl3)
d 0.80 (s, 3H), 0.93 (s, 3H), 1.07 (s,
4.1. General
6H), 1.16 (s, 3H), 1.25 (s, 3H), 1.60 (s, 6H), 1.94 (m, 2H), 2.18 (m, 2H),
5.16 (t, J ¼ 6.7 Hz,1H), 7.95 (d, J ¼ 7.6 Hz,1H), 8.29 (dd, J ¼ 7.3, 2.2 Hz,
1H), 9.12 (d, J ¼ 2.5 Hz, 1H), 11.17 (br s, 1H). FAB MS m/z 605 (M þ 1).
M.P. are uncorrected. IR spectra were recorded on a Perkin–
Elmer RX-1 spectrophotometer using either KBr pellets or in neat.
1H NMR and 13C NMR, DEPT 90, DEPT 135 spectra were run on
Bruker Advance DPX 200/300 MHz spectrometer in CDCl3 unless
stated otherwise. TMS was used as internal standard. FAB mass
spectra were recorded on JEOL SX 102/DA-6000. Silica gel 60–120
mesh was used as stationary phase to isolate the compounds.
4.1.5. General procedure for the synthesis of thiourea derivatives of
amyrin (24, 25)
To a stirred solution of (E)-1-(2,4-dinitrophenyl)-2-(4,4,6a,6b,
8a,11,12,14b-octamethyl-1,2,4a,5,6,6a,7,8,8a,9,10,11,12,12a,14,14a-
hexa decahydropicen-3-(4H,6bH,14bH)-ylidene)-hydrazine 23 (0.60 g,
1 mmol) in dry DMF (6 ml) was added sodium hydride (0.23 g,
10 mmol) at 0 ꢁC. The stirring was continued for 30 min at the same
temperature. Then, the appropriate phenyl thioisocyanate (4 mmol)
was added to the ice-cold mixture and stirring continued for over-
night at room temperature. The reaction mixture was plunged into
ice-cold water, filtered under suction and filtrate extracted with
4.1.1. General procedure for the 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 (8)
To
a 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-icosahydropicen-

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T. Narender et al. / European Journal of Medicinal Chemistry 44 (2009) 1215–1222
chloroform (4 ꢃ 25 ml). The organic layer was dried upon sodium
sulphate, concentrated under reduced pressure and the residue
was subjected to silica gel chromatography, eluting with hex-
ane:ethylacetate (90:10) solvent system.
4.2.5. Synthesis of (E)-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-2-methylbut-2-enoate (13)
By the analogous procedure as described for compound 9, was
obtained from 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 8 (0.43 g,
1 mmol) and 2-methyl butanoic acid (0.20 g, 2 mmol) by DCC–
DMAP in dry DCM. Yield: 46%; mp 160–162 ꢁC; IR (KBr) 1735 (CO)
4.2. Synthesis of derivatives
4.2.1. Synthesis of 4,4,6a,6b,8a,11,12,14b-octa methyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-
icosahydropicen-3-yl-4-chloro benzoate (9)
and 1631 (C]C) cmꢂ1. 1H NMR (200 MHz, CDCl3)
d 0.72 (s, 6H), 0.81
(s, 3H), 0.84 (s, 6H), 0.92 (s, 3H), 0.94 (s, 3H), 1.00 (s, 3H), 1.44 (m,
2H), 1.58 (s, 3H), 1.59 (s, 3H), 1.69–1.73 (m, 2H), 1.76–1.82 (m, 2H),
4.43–4.50 (m, 1H), 5.05 (t, J ¼ 6.7 Hz, 1H), 6.76 (q, J ¼ 6.9 Hz, 1H).
FAB MS m/z 509 (M þ 1).
To
a 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-icosahy-
dropicen-3-ol 8(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 4-chlorobenzoic acid
(0.56 g, 4 mmol). The stirring was continued for 4 h at room
temperature. The reaction was filtered off under suction, concen-
trated under reduced pressure and the concentrate was subjected
to silica gel chromatography using hexane:ethylacetate (94:06)
system as eluent. Yield: 80%; mp 161–163 ꢁC; IR (KBr) 1735 (CO)
4.2.6. 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 (14)
By the analogous procedure as described for compound 9,
was
obtained
from
4,4,6a,6b,8a,11,12,14b-octamethyl-1,2,3,
and 1631 (C]C) cmꢂ1. 1H NMR (200 MHz, CDCl3)
d
0.80 (s, 6H), 0.93
4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen-3-ol
10 (0.42 g, 1 mmol) and pyridine-2-carboxylic acid (0.24 g,
2 mmol) by DCC–DMAP in dry DCM. Yield: 53%; mp 220–221 ꢁC;
(s, 6H), 1.02 (s, 6H), 1.15 (s, 3H), 1.25 (s, 3H), 1.91 (m, 2H), 1.95 (m,
2H), 4.73 (t, J ¼ 5.7 Hz, 1H), 5.13 (br s, 1H), 7.41 (d, J ¼ 8.3 Hz, 2H),
7.97 (d, J ¼ 8.4 Hz, 2H). FAB MS m/z 565 (M þ 1).
IR (KBr) 1735 (CO) and 1631 (C]C) cmꢂ1 1H NMR (200 MHz,
.
CDCl3)
d 0.80 (s, 6H), 0.93 (s, 6H), 1.09 (s, 6H), 1.15 (s, 3H), 1.25
4.2.2. 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-yl-4-nitrobenzoate (10)
By the analogous procedure as described for compound 9,
was obtained from 4,4,6a,6b,8a,11,12,14b-octamethyl-1,2,3,4,4a,
(s, 3H), 1.93 (m, 2H), 1.96 (m, 2H), 4.78 (t, J ¼ 8.1 Hz, 1H), 5.14 (br
s, 1H), 7.36–7.42 (m, 1H), 8.29 (d, J ¼ 7.9 Hz, 1H), 8.76–8.78 (m,
1H), 9.23 (br s, 1H). FAB MS m/z 532 (M þ 1).
4.2.7. Synthesis of 4,4,6a,6b,8a,11,12,14b-octa-methyl-
5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropicen-3-ol
8
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-
(0.85 g, 2 mmol) and 4-nitrobenzoic acid (0.66 g, 4 mmol) by DCC–
icosahydropicen-3-yl-isoquinoline-1-carboxylate (15)
DMAP in dry DCM. Yield: 72%; mp 192–193 ꢁC; IR (KBr) 1735 (CO)
By the analogous procedure as described for compound 9, was
obtained from 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 8 (0.42 g,
1 mmol) and quinoline-2-carboxylic acid (0.37 g, 2 mmol) by
DCC–DMAP in dry DCM. Yield: 51%; mp 205–206 ꢁC; IR (KBr)
and 1631 (C]C) cmꢂ1. 1H NMR (200 MHz, CDCl3)
d 0.80 (s, 6H), 0.93
(s 6H), 1.03 (s, 6H), 1.15 (s, 3H), 1.25 (s, 3H), 1.91 (m, 2H), 1.93 (m,
2H), 4.73 (t, J ¼ 5.7 Hz, 1H), 5.14 (br s, 1H), 8.19 (d, J ¼ 8.8 Hz, 2H),
8.29 (d, J ¼ 8.7 Hz, 2H). FAB MS m/z 576 (M þ 1).
1735 (CO) and 1631 (C]C) cmꢂ1 1H NMR
. d 0.81 (s, 3H), 0.92 (s,
4.2.3. Synthesis of 4,4,6a,6b,8a,11,12,14b-octa-methyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-
icosahydropicen-3-yl-2-(4-methoxy ph enyl)acetate (11)
3H), 1.00 (s, 6H), 1.04 (s, 6H,), 1.09 (s, 6H), 1.81 (m, 2H), 1.92–
1.96 (m, 2H), 4.92 (t, J ¼ 8.2 Hz, 1H), 5.14 (br s, 1H), 7.65 (d,
J ¼ 7.6 Hz, 1H), 7.76 (d, J ¼ 8.1 Hz, 1H), 7.87 (d, J ¼ 8.0 Hz, 1H),
8.11 (d, J ¼ 8.4 Hz, 1H), 8.29 (1H, d, J ¼ 8.2 Hz, H-70), 8.33 (1H, d,
J ¼ 8.2 Hz, H-40). FAB MS m/z 582 (M þ 1).
By the analogous procedure as described for compound 9,
was obtained from 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 8
(0.42 g, 1 mmol) and 4-methoxyphenyl acetic acid (0.33 g,
2 mmol) by DCC–DMAP in dry DCM. Yield: 54%; mp 148–150 ꢁC;
4.2.8. Synthesis of 4,4,6a,6b,8a,11,12,14b-octa-methyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-
icosahydropicen-3-yl-2-bromopropanoate (16)
IR (KBr) 1735 (CO) and 1631 (C]C) cmꢂ1
.
1H NMR (200 MHz,
CDCl3) 0.76 (s, 6H), 0.79 (s, 3H), 0.80 (s, 3H), 0.90 (s, 3H), 0.95
d
By the analogous procedure as described for compound 9,
was obtained from 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 8
(0.43 g, 1 mmol) and 2-bromopropanioc acid (0.30 g, 2 mmol) by
DCC–DMAP in dry DCM. Yield: 35%; mp 196–199 ꢁC; 562
(s, 3H), 0.99 (s, 3H), 1.05 (s, 3H), 1.56–1.64 (m, 2H), 1.86–1.92 (m,
2H), 3.54 (s, 2H), 3.78 (s, 3H), 4.48 (t, J ¼ 5.8 Hz, 1H), 5.11 (t,
J ¼ 6.8 Hz, 1H), 6.84 (d, J ¼ 8.6 Hz, 2H), 7.19 (d, J ¼ 8.5 Hz, 2H).
FAB MS m/z 575 (M þ 1).
(M þ 2); IR (KBr) 1735 (CO) and 1631 (C]C) cmꢂ1
.
1H NMR
4.2.4. Synthesis of 4,4,6a,6b,8a,11,12,14b-octamethyl-
(200 MHz, CDCl3) d 0.79 (s, 6H), 0.90 (s, 9H), 0.99 (s, 3H), 1.01 (s,
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-
icosahydropicen-3-yl-5-oxo-5-phenyl pentanoate (12)
3H), 1.07 (s, 3H), 1.25 (s, 3H), 1.66–1.68 (m, 2H), 1.80–1.85 (m,
2H), 4.36 (q, J ¼ 7.5 Hz, 1H), 4.52–4.59 (m, 1H), 5.13 (t, J ¼ 3.5 Hz,
1H). FAB MS m/z 561 (M þ 1).
By the analogous procedure as described for compound 9,
was obtained from 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 8
(0.42 g, 1 mmol) and 4-benzoyl butyric acid (0.38 g, 2 mmol) by
DCC–DMAP in dry DCM. Yield: 72%; mp 187–190 ꢁC; IR (KBr)
4.2.9. Synthesis of 4,4,6a,6b,8a,11,12,14b-octa-methyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-
icosahydropicen-3-yl-2-benzamido acetate (17)
1735 (CO) and 1631 (C]C) cmꢂ1
.
1H NMR (200 MHz, CDCl3)
By the analogous procedure as described for compound 9,
was obtained from 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 8
(0.42 g, 1 mmol) and N-benzoylglycine (0.35 g, 2 mmol) by DCC–
DMAP in dry DCM. Yield: 43%; mp 94–96 ꢁC; IR (KBr) 1735 (CO),
d
0.79 (s, 6H), 0.86 (s, 6H), 0.91 (s, 3H), 0.97 (s, 3H), 1.00 (s 3H),
1.06 (s, 3H), 1.91 (m, 2H), 1.88–1.93 (m, 2H), 2.10 (m, 2H), 2.42 (t,
J ¼ 7.0 Hz, 2H), 3.05 (t, J ¼ 7.1 Hz, 2H), 4.53 (t, J ¼ 7.7 Hz, 1H), 5.12
(br s, 1H), 7.42–7.56 (m, 3H), 7.96 (d, J ¼ 7.1 Hz, 2H). FAB MS m/z
601 (M þ 1).
1689 (CONH) and 1631 (C]C) cmꢂ1 1H NMR (200 MHz, CDCl3)
;

T. Narender et al. / European Journal of Medicinal Chemistry 44 (2009) 1215–1222
1221
d
0.80 (s, 3H), 0.82 (s, 3H), 0.85 (s, 3H), 0.98 (s, 3H), 1.01 (s, 3H),
4.2.14. Synthesis of (E)-2-(2,4-dinitrophenyl)-1-
1.07 (s 3H), 1.09 (s, 3H), 1.25 (s, 3H), 1.90 (m, 2H), 1.95 (m, 2H),
4.24 (d, J ¼ 4.9 Hz, 2H), 4.62 (t, J ¼ 7.8 Hz, 1H), 5.12 (t, J ¼ 3.3 Hz,
1H), 6.72 (br s, 1H), 7.44–7.52 (m, 3H), 7.81 (dd, J ¼ 8.1, 1.2 Hz,
2H). FAB MS m/z 588 (M þ 1).
(4,4,6a,6b,8a,11,12,14b-octamethyl-
1,2,4a,5,6,6a,7,8,8a,9,10,11,12,12a,14,14a-hexadecahydropicen-
3(4H,6bH,14bH)-ylidene)-4-phenylthiosemicarbazide (24)
By the analogous procedure as described in the repre-
sentative method for thiourea derivatives, was obtained from
(E)-1-(2,4-dinitrophenyl)-2-(4,4,6a,6b,8a,11,12,14b-octa-
methyl-1,2,4a,5,6,6a,7,8,8a,9,10,11,12,12a,14,14a-hexadecahy-
dropicen-3(4H,6bH,14bH)-ylidene) hydrazine 23 (0.60 g, 1 mmol)
and phenyl thioisocyanate (0.54 g, 4 mmol) by NaH in dry DMF.
Yield: 18%; mp 152–153 ꢁC; IR (KBr) 3421 (NH), 1624 (C]C)
4.2.10. 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-2,5-dimethyl benzoate (18)
By the analogous procedure as described for compound 9,
was obtained from 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 8
(0.43 g, 1 mmol) and 2,5-dimethylcarboxylic acid (0.30 g,
2 mmol) by DCC–DMAP in dry DCM. The stirring was continued
for 4 h at room temperature. The reaction was filtered off under
suction, concentrated under reduced pressure and the desired
compound was isolated by silica gel chromatography. Yield: 43%;
cmꢂ1 1H NMR (200 MHz, CDCl3)
; d 0.77 (s, 6H), 0.79 (s, 6H), 1.03
(s, 9H), 1.10 (s, 3H), 1.90 (t, J ¼ 8.2 Hz, 2H), 5.10 (br s, 1H), 6.71
(d, J ¼ 8.9 Hz, 1H), 7.42–7.49 (m, 3H), 7.55 (dd, J ¼ 7.4, 1.3 Hz, 2H),
8.00 (dd, J ¼ 8.9, 2.2 Hz, 1H), 8.25 (d, J ¼ 2.1 Hz, 1H). FAB MS m/z
740 (M þ 1).
mp 202–203 ꢁC; IR (KBr) 1735 (CO) and 1631 (C]C) cmꢂ1
.
1H
4.2.15. Synthesis of (E)-2-(2,4-dinitrophenyl)-4-(2-nitrophenyl)-1-
(4,4,6a,6b,8a,11,12,14b-octamethyl-
NMR (200 MHz, CDCl3) 0.80 (s, 3H), 0.91 (s, 3H), 0.96 (s, 6H),
d
0.99 (s, 3H), 1.02 (s, 3H), 1.08 (s 3H), 1.25 (s, 3H), 1.81 (m, 2H),
1.92–1.96 (m, 2H), 2.34 (s, 3H), 2.55 (s, 3H), 4.75 (t, J ¼ 8.1 Hz,
1H), 5.12 (br s, 1H), 7.09–7.21 (m, 2H), 7.69 (s, 1H). FAB MS m/z
558 (M)þ.
1,2,4a,5,6,6a,7,8,8a,9,10,11,12,12a,14,14a-hexadecahydropicen-3-
(4H,6bH,14bH)-ylidene)-thiosemicarbazide (25)
By the analogous procedure as described for compound 24, was
obtained from (E)-1-(2,4-dinitrophenyl)-2-(4,4,6a,6b,8a,11,12,14b-
octamethyl-1,2,4a,5,6,6a,7,8,8a,9,10,11,12,12a,14,14a-hexadecahy-
4.2.11. Synthesis of 2-(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-benzoic acid (19)
dropicen-3-(4H,6bH,14bH)-ylidene)-hydrazine
23
(0.45 g,
0.75 mmol) and 2-nitrophenylthioisocyanate (0.72 g, 4 mmol) by
NaH in dry DMF. Yield: 12%; semisolid; IR (KBr) 3433 (NH), 1631
To
a
stirred solution of 4,4,6a,6b,8a,11,12,14b-octamethyl-
(C]C) cmꢂ1; 1H NMR (200 MHz, CDCl3)
d 0.79 (s, 6H), 0.82 (s, 3H),
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-icosahydropi-
cen-3-ol 8 (0.42 g, 1 mmol) in dry DCM (10 ml) was added DMAP
(0.48 g, 4 mmol) and phthalic anhydride (0.59 g, 4 mmol). Yield:
42%; mp 193–194 ꢁC; IR (KBr) 1735 (CO), 1740 (COOH) and 1631
0.85 (s, 6H), 1.25 (s, 9H), 2.14 (t, J ¼ 6.7 Hz, 2H), 5.10 (br s, 1H), 6.46
(d, J ¼ 8.9 Hz, 1H), 7.36–7.43 (m, 3H), 7.53 (d, J ¼ 8.8 Hz, 1H), 8.02
(dd, J ¼ 8.7, 2.2 Hz, 1H), 8.28 (d, J ¼ 2.1 Hz, 1H). FAB MS m/z 785
(M þ 1).
(C]C) cmꢂ1; 1H NMR (200 MHz, CDCl3)
d 0.80 (s, 6H), 0.91 (s, 6H),
0.98 (s, 6H), 1.01 (s, 3H), 1.08 (s, 3H), 1.89 (t, J ¼ 11.3 Hz, 2H), 4.77 (m,
1H), 5.13 (br s, 1H), 7.58 (d, J ¼ 7.3 Hz, 2H), 7.73 (d, J ¼ 4.7 Hz, 1H),
7.90 (d, J ¼ 4.6 Hz, 1H). FAB MS m/z 575 (M þ 1).
4.3. Antihyperglycemic assay
4.3.1. Sucrose-loaded (SLM) model
Overnight fast male albino rats were used for sucrose-loaded
experiment. Blood was collected initially and thereafter test
compounds were given to the test group consisting of five rats by
oral gavage at a dose of 100 mg/kg body weight. After half an hour
post-test treatment, a sucrose load of 10 mg/kg body weight was
given to each rat. Blood was collected at 30, 60, 90 and 120 min
post-sucrose load. The % fall in blood glucose level was calculated
according to the AUC method.
4.2.12. Synthesis of 4-(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)-4-oxo-butanoic acid (20)
By the analogous procedure as described for compound 19, was
obtained from 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 8 (0.85 g,
2 mmol) and succinic anhydride (0.20 g, 2 mmol) by DMAP in dry
DCM. Yield: 48%; mp 170–175 ꢁC; IR (KBr) 1735 (CO), 1740 (COOH)
and 1631 (C]C) cmꢂ1
;
1H NMR (200 MHz, CDCl3,)
d
0.79 (s, 6H),
4.3.2. Streptozotocin (STZ-S) model
0.86 (s, 6H), 0.91 (s, 3H), 0.97 (s, 3H), 1.00 (s, 3H), 1.06 (s, 3H), 1.91 (t,
J ¼ 8.5 Hz, 2H), 2.66 (m, 4H), 4.54 (m, 1H), 5.12 (br s, 1H). FAB MS m/
z 527 (M þ 1).
Male albino rats of SD strain (body weight 140 ꢀ 20 g) were
selected for this study. Streptozotocin was dissolved in 100 mM
citrate buffer pH 4.5 and calculated amount of the fresh solution
was injected to overnight fasted rats (45 mg/kg) intraperitoneally.
Blood was checked 48 h later by glucostrips and animals showing
blood glucose values between 8 and 15 mM were selected and
divided into groups of six animals in each. Rats of experimental
groups were administered suspension of the desired test samples
orally (made in 1.0% gum acacia) at 100 mg/kg dose. Animals of
control group were given an equal amount of 1.0% gum acacia. A
sucrose load of 2.5 g/kg body weight was given after 30 min of drug
administration. After 30 min of post-sucrose load, blood glucose
level was again checked at 30, 60, 90, 120, 180, 240, 300 min and at
24 h, respectively. Comparison of the AUC of experimental and
control groups determined the percent antihyperglycemic activity.
4.2.13. Synthesis of 4,4,6a,8a,11,12,14b-hepta-methyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-
icosahydropicen-3-yl-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-succinate (21)
By the analogous procedure as described for compound 9, was
obtained from 4-(4,4,6a,6b,8a,11,12,14b-octamethyl-1,2,3,4,4a,5,
6,6a,6b,7,8,8a,9,10,11,12,12, 14,14a,14b-icosahydropicen-3-yloxy)-4-
oxobutanoic acid 20 (0.26 g, 0.5 mmol) and 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-ico-
sahydropicen-3-ol 8 (0.21 g, 0.5 mmol) by DCC–DMAP in dry
DCM. Yield: 52%; mp >210 ꢁC; IR (KBr) 1735 (CO) and 1631 (C]C)
cmꢂ1
;
1H NMR (200 MHz, CDCl3)
d
0.79 (s, 12H,), 0.86 (s, 12H),
Acknowledgements
0.91 (s, 6H), 0.97 (s, 6H), 1.00 (s, 6H), 1.06 (s, 6H), 1.91 (t,
J ¼ 8.2 Hz, 4H), 2.63 (m, 4H), 4.53 (m, 2H), 5.12 (br s, 2H). FAB MS
m/z 921 (M þ 1).
Authors are grateful to the Director, CDRI, Lucknow, India for
constant encouragement for the program on antihyperglycemic

1222
T. Narender et al. / European Journal of Medicinal Chemistry 44 (2009) 1215–1222
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financial support.
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Appendix. Supplementary material
Supplementary material associated with this article can be
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