Synthesis of novel imbricatolic acid analogues via insertion of N-substituted piperazine at C-15/C-19 positions, displaying glucose uptake stimulation in L6 skeletal muscle cells

Article abstract of DOI:10.1016/j.bmcl.2012.05.097

A new class of N-substituted piperazine analogues of imbricatolic acid have been designed and synthesized by using the appropriate synthetic routes in excellent yield. All synthesised compounds were screened for their in vitro glucose uptake stimulatory activity. Among them compounds 4b, 4e, 8b, and 8e triggered L6 skeletal muscle cells for glucose uptake at 54.73%, 40.79%, 40.90%, and 39.55% stimulation, respectively. Compound 4b has emerged as important lead compound showing potential antidiabetic activity. Illustration about their synthesis and in vitro glucose uptake activity is described.

Full text of DOI:10.1016/j.bmcl.2012.05.097

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4636–4639
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of novel imbricatolic acid analogues via insertion of N-substituted
piperazine at C-15/C-19 positions, displaying glucose uptake stimulation
in L6 skeletal muscle cells
Mohammad Faheem Khan a, Padam Kumar a, Jyotsana Pandey b, Arvind Kumar Srivastava b,
Akhilesh Kumar Tamrakar b, Rakesh Maurya a,
⇑
a Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, CSIR, Lucknow 226001, India
b Biochemistry Division, CSIR-Central Drug Research Institute, CSIR, Lucknow 226001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new class of N-substituted piperazine analogues of imbricatolic acid have been designed and synthe-
sized by using the appropriate synthetic routes in excellent yield. All synthesised compounds were
screened for their in vitro glucose uptake stimulatory activity. Among them compounds 4b, 4e, 8b,
and 8e triggered L6 skeletal muscle cells for glucose uptake at 54.73%, 40.79%, 40.90%, and 39.55% stim-
ulation, respectively. Compound 4b has emerged as important lead compound showing potential antidi-
abetic activity. Illustration about their synthesis and in vitro glucose uptake activity is described.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 4 April 2012
Accepted 25 May 2012
Available online 5 June 2012
Keywords:
Diterpene
Imbricatolic acid
N-Substituted piperazine
Glucose uptake
Type 2 diabetes mellitus is a currently rising problem, affecting
approximately 194 million people all over world according to
World Health Organisation, and this number is projected to be
366 million by 2030.1 Over the last 30 years, an enormous number
of studies have been dedicated to unraveling the pathophysiology
of type 2 diabetes mellitus.2 A major pathogenic defect in the dis-
ease is insulin resistance, which is characterized by the impeded
capacity of peripheral tissues, most notably in skeletal muscle to
utilize glucose effectively in face of hyperinsulinemia.3 This leads
to a concomitant rise in blood glucose level and ends up with dia-
betes. To date, some antidiabetic drugs like pioglitazone and rosig-
litazone (Fig. 1) are in markets which were approved by the US FDA
for the treatment of type 2 diabetes in 1999.4 However, therefore,
development of newer and safer drugs is urgently needed to over-
come some factors like weight gain, congestive heart failure etc.,
that limit the use of these drugs.5
antiplasmodial,8 antiproliferative,9 antiviral,10 neuroprotective,11
trypanocidal,12 and cytotoxic.13 Some easily isolated labdane diter-
penes such as labdanolic acid, ozic acid and isocupressic acids are
common starting materials for synthesis of other natural products
and numerous diterpene derivatives with privileged motifs for fur-
ther modification and structure–activity relationship analyses.14
Among labdane-type diterpenes, the most relevant molecule
having bicyclic framework is the imbricatolic acid as
a
O
NH
O
N
S
NH
O
O
S
O
N
O
N
Rosiglitazone
Pioglitazone
OAc
Labdane-type terpenes are excellent example of natural prod-
ucts with important pharmaceutical potentials. They make a large
family of bicyclic diterpenes, mainly isolated from plant sources
and characterized by the presence of 4,10-dimethyl substituted
decalin system with exomethylene group at C-8 position.
O
OH
O
N
N
They have been reported to exhibit a wide range of potent
N
COOCH3
biological activities including antitubercular,6 antibacterial,7
COOH
1
O
O
N
8a
4b
⇑
Corresponding author. Tel.: +91 (522) 2612411 18x4235; fax: +91 (522)
2623405/2623938/2629504.
F
E-mail address: mauryarakesh@rediffmail.com (R. Maurya).
CDRI Communication No. 8259.
Figure 1. Structures of representative glucose uptake stimulators.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.097

M. F. Khan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4636–4639
4637
representative compound 1 because of its abundance and wide-
spread occurrence.15 An initial literature survey revealed that it
possesses cytotoxic16 and gastroprotective effect.17 Korey Sakar
et. al. isolated 1 from the acidic fraction of the petroleum ether ex-
tract of Juniperus nana Willd unripe berries which showed strong
cytotoxic activity according to Brine Shrimp bioassay (LD50
yield. Corresponding amide derivatives 4a–h resulted from amida-
tion of carboxylic acid by the reaction of N-substituted piperazine
in the presence of dry CH2Cl2 and (C2H5)3N at room temperature
within two hours, on the basis of the published procedure.22
Similar optimized synthetic route was also used to obtain sec-
ond series of N-substituted piperazine with methylene linker as
outlined in Scheme 2. Reaction of 2 with CH3I and anhydrous
K2CO3 in appropriate solvent at 0 °C upto room temperature to
yielded compound 5 as described previously.23 Compound 6 was
synthesized in two different synthetic routes, by the direct methyl-
ation of carboxylic acid of C-19 used methyl iodide in basic med-
ium of anhydrous K2CO3 or by the estrification of hydroxyl group
of C-15 in alkyl chain followed by deprotection of hydroxyl group
in 10% methanolic KOH at room temperature. It was found that
protection of the hydroxyl groups as esters was very convenient
and necessary, since direct methylation of acid was low yielding
with the formation of undesired by products due to the interaction
of OH group. According to literature24 procedure slow addition of
bromo acetyl bromide at 0 °C in solution of 6 with dry CH2Cl2-
Et3N and stirred at room temperature, after 30 min compound 7
was obtained in good yield. Correspondingly, following the reac-
tion sequence as shown in Scheme 1 treatment of 7 with N-substi-
tuted piperazine in dry CH2Cl2 and (C2H5)3N at room temperature
for 1 hour yielded compounds 8a–h in almost quantitative yield.
All the synthesised compounds were analyzed by using IR, 1H
NMR, 13C NMR, and ESIMS analytical methods.
0.16 l
g/mL).16 Despite the many studies on the chemical modifica-
tion of this class of molecules, only few studies have been reported
for imbricatolic acid. In a recent progress, a study has shown that
imbricatolic acid and its amide derivative showed gastroprotective
effect in ethanol/HCl induced gastric lesions model in mice and
found to be cytotoxic against MRC-5, AGS and Hep G2 cell lines.18
Due to rarity and paucity of information in the literature concern-
ing chemical modification, this frameworks draw attention as syn-
thetic targets to realize
a thoroughly examination of their
pharmacological properties. Hence, we are keenly interested to ex-
plore it chemically with convenient synthetic pathways.
In continuation of our research into the chemical modification
of natural products in the treatment of type 2 diabetes mellitus,
as well as keeping the structural features of imbricatolic acid in
mind, we planned to synthesized analogues of 1 with insertion of
N-substituted piperazine moieties which were available commer-
cially. All the modified compounds were evaluated against type 2
diabetes mellitus via stimulating glucose uptake in L6 skeletal
muscle cells. In order to understand chemical as well as biological
properties of imbricatolic acid, we now report herein the results of
this effort.
We have isolated imbricatolic acid 1 as starting compound from
chloroform fraction of Cupressus sempervirens fruits by using exten-
sive column chromatography.19 The purity of starting compound
was proven on the basis of NMR spectral data which were also
accordance with published data in literature.20 To explore the glu-
cose uptake activity, the carboxylic acid at C-19 and hydroxyl
group at C-15 position of 1 was transformed into their amides
and esters analogues respectively. The reactions sequence to intro-
duce the N-substituted piperazine is shown in the Scheme 1 and 2.
Compound 1 was acetylated at C-15 in anhydrous pyridine with
addition of 1.1 equivalent of acetic anhydride and yielded com-
pound 2 (Scheme 1). Acid chloride 3 was obtained from 2 via reac-
tion of oxalyl chloride in dry CH2Cl2 at 0 °C according to
literature.21 Good yield of 2 was achieved by addition of dry DMF
in catalytic amount. This step was preferred in situ in terms of
The biological scope of 1 and its synthetic analogues were
examined and summarised in Table 1and 2. Each compound was
screened at 10 lM concentration for in vitro glucose uptake stimu-
latory effect in L6 skeletal muscle cells by using previously de-
scribed method.25 All tested samples were dissolved in DMSO
(0.1%). Twelve out of tested synthetic analogues along with
OH
a
OCOCH3
b
OCOCH3
COOH
COOH
2
COOCH3
1
5
c
OH
OH
a
OAc
b
OAc
d
X
COOCH3
e
6
COOH
1
COOH
2
COCl
3
OCOCH2R
OCOCH2Br
c
f
R
4a. 1-(4-fluorophenyl) piperazine
4b.
4c. 1-(4-methylphenyl) piperazine
OAc
1-(3-methoxyphenyl) piperazine
COOCH3
8a-h
COOCH3
4d.
4e.
1-phenyl piperazine
1-methyl piperazine
R
7
1-(4-fluorophenyl) piperazine
8b.1-(3-methoxyphenyl) piperazine
1-(4-methylphenyl) piperazine
1-phenyl piperazine
8a.
8e. 1-methyl piperazine
4f. 1-acetoxy piperazine
8f.
1-acetoxy piperazine
4g.
trans-1-cinnamyl piperazine
4h.1-(4-chlorophenyl) piperazine
8c.
8d.
8g.
8h.
trans-1-cinnamyl piperazine
1-(4-chlorophenyl) piperazine
COR
4a-h
Scheme 2. Reagents and conditions: (a) C6H5N, Ac2O, rt, 1 h (b) CH2Cl2, K2CO3, CH3I
(5.0 equiv), 0 °C, 6 h (c) 10% KOH, MeOH, rt, 1 h (d) CH2Cl2, K2CO3, CH3I (5.0 equiv),
0 °C, 6 h (e) CH2Cl2, Et3N, BrCOCH2Br (1.1 equiv), 0 °C, 1.5 h (f) CH2Cl2, Et3N,
substituted piperazine (1.0 equiv), rt, 2 h.
Scheme 1. Reagents and conditions: (a) C6H5N (ca. 1.1 equiv), Ac2O (ca. 1.1 equiv),
rt, 1 h.(b) CH2Cl2, (COCl)2 (ca. 1.1 equiv), 0 °C, 4 h (c) CH2Cl2, Et3N, substituted
piperazine (1.0 equiv), 0 °C to rt, 1.5 h.

4638
M. F. Khan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4636–4639
Table 1
List of analogues at C-19 (4a–h) and C-15 positions (8a–h) of 1 and their in vitro glucose uptake activity at 10
l
M concentration
OH
OH
OH
OR
COOH
COOH
COOCH3
8a-h
COR
1
1
4a-h
(34.41% stimulation)
(34.41% stimulation)
Entry
Compd
R
% Glucose uptake stimulation
22.45
Entry
Compd
R
% Glucose uptake stimulation
28.86
N
N
N
N
F
N
N
N
N
F
1
4a
4b
9
8a
O
O
O
2
3
54.73
16.30
10
11
8b
8c
40.90
18.58
O
4c
N
N
N
N
N
N
N
N
N
N
O
O
O
O
O
O
N
N
N
N
N
4
5
6
7
4d
4e
4f
02.21
40.79
12
13
14
15
16
8d
8e
8f
15.27
39.55
Cl
Cl
N
N
N
N
N
24.14
29.51
N
N
4g
4h
À10.16
À01.96
8g
8h
À03.75
O
N
N
8
À05.90
O
Rosiglitazone
54.50
starting compound 1 exhibited from weak to significant glucose
uptake stimulatory effect, in comparison to standard drug rosiglit-
azone (as a positive control which causes 54.50% stimulation).
These observations as well as chemical structures of screened com-
pounds provided preliminary support of the study that suitably-
functionalized imbricatolic acid derivatives possess anti-diabetic
activity. The introduction of N-substituted piperazine moiety with
phenyl ring essentially preserved the activity with a trend towards
improved potency in the 4-F, 4-Cl and 3-methoxy substituted
congeners. In both series 4-fluro and 4-chloro substituted pair of
compounds 4a (22.45%), 4e (40.79%) and 8a (28.86%), 8e
(39.55%) respectively were found to be better tolerated and more
potent than the reference compound 1.
However, phenyl rings with no substituent’s were associated
with an almost linear reduction in potency of glucose uptake as
in case of compounds 4c (16.30%), 4f (24.14%), 8c (18.51%) and
8f (29.51%). The effect of methyl group at 4 position of phenyl ring
was examined in the compounds 4d and 8d and was found to be
50-fold less active at 2.21% and 15.27% stimulation than the refer-
ence compound 1. The incorporation of N-substituted piperazine
moiety with 3-methoxy phenyl group resulted in significant inten-
sification of activity. The presence of acetyl and methyl groups in-
stead of phenyl group within the piperazine moiety led to
compounds 4g, 4h, 8g and 8h which were failed to show any sig-
nificant effect on glucose uptake. However, the stimulation of glu-
cose uptake by compounds 4b and 8b was 54.73% and 40.90%
where compound 4b displayed a remarkable ability to stimulate
glucose uptake in L6 skeletal muscle cells after administration of
10 lM concentrations, compared to standard drug rosiglitazone.
It is evident from the screening results of compounds 4a–h and
8a–h that the presence of substituted phenyl ring within pipera-
zine moiety display glucose uptake stimulatory activity. Com-
pounds with no phenyl rings at piperazine moiety did not show
any remarkable activity.
In conclusion, two series of compounds characterized by amide
and ester linkage at C-19 and C-15 positions of imbricatolic acid
scaffold respectively were achieved by concise, efficient and eco-
nomical synthetic routes. Synthesis of such new analogues aims
(i) to explore the chemical modification of imbricatolic acid and
(ii) to investigate the role of N-substituted piperazine with diter-
penes pharmacophore in treatment of type 2 diabetes. Compound
with 1-(3-methoxy phenyl piperazine moiety was more potent
than its ester linkage counterpart. On the other hand 1-(4-flur-
ophenyl piperazine moiety displayed good activity rather than its
amide linkage counterpart. To the best of our knowledge, this is
the first report of antidiabetic activity for the synthesized imbrica-
tolic acid analogues toward the glucose uptake stimulation in skel-
etal muscle cell.
This is anticipated that modification of imbricatolic acid will not
only inspire researchers but also drives others in future with better

M. F. Khan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4636–4639
4639
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