Synthesis of substituted 2H-benzo[e]indazole-9-carboxylate as a potent antihyperglycemic agent that may act through IRS-1, Akt and GSK-3β pathways

Article abstract of DOI:10.1039/c6md00467a

Based on high throughput screening of our chemical library, we identified two 4,5-dihydro-2H-benzo[e]indazole derivatives (5d and 5g), which displayed a significant effect on glucose uptake in L6 skeletal muscle cells. Based on these lead molecules, a ser

Full text of DOI:10.1039/c6md00467a

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Tamrakar, A. Srivastava and A. Goel, Med. Chem. Commun., 2016, DOI: 10.1039/C6MD00467A.
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ARTICLE TYPE
Synthesis of substituted 2Hꢀbenzo[e]indazoleꢀ9ꢀcarboxylate as potent
antihyperglycemic agent that may act through IRSꢀ1, Akt and GSKꢀ3β
pathways†
5
Gaurav Tanejaa, Chandra Prakash Guptaa, Shachi Mishraa, Rohit Srivastavab, Neha Rahujab, Arun
Kumar Rawatb, Jyotsana Pandeyb, Anand P. Guptac, Natasha Jaiswalb, Jiaur R. Gayenc, Akhilesh K.
Tamrakarb, Arvind Kumar Srivastavab and Atul Goela*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
10 DOI: 10.1039/b000000x
Based on high throughput screening of our chemical library, we identified two 4,5ꢀdihydroꢀ2Hꢀ
benzo[e]indazole derivatives (5d and 5g), which displayed significant effect on glucose uptake in L6
skeletal muscle cells. Based on these lead molecules, a series of benzo[e]indazole derivatives were
prepared. Among all the synthesized dihydroꢀ2Hꢀbenzo[e]indazoles, 8ꢀ(methylthio)ꢀ2ꢀphenylꢀ6ꢀpꢀtolylꢀ
15 4,5ꢀdihydroꢀ2Hꢀbenzo[e]indazoleꢀ9ꢀcarboxylate (5e) showed significant glucose uptake stimulation in L6
skeletal muscle cells even better than lead compounds. Additionally, 5e decreased glucagonꢀinduced
glucose release in HepG2 hepatoma cells. The 2Hꢀbenzo[e]indazole 5e exerted antihyperglycemic effect
in normal, sucrose challenged streptozotocinꢀinduced diabetic rats and type 2 diabetic db/db mice.
Treatment with 5e at the dose of 30 mg/kg in db/db mice caused a significant decrease in triglyceride and
20 total cholesterol levels, and increased the HDLꢀC level in a significant manner. The mechanistic studies
revealed that 2Hꢀbenzo[e]indazole 5e significantly stimulated insulinꢀinduced signaling at the level of
IRSꢀ1, Akt and GSKꢀ3β in L6 skeletal muscle cells and possibly by inhibiting protein tyrosine
phosphataseꢀ1B. This new 2Hꢀbenzo[e]indazole derivative has potential for the treatment of diabetes with
improved lipid profile.
HPLCꢀchromatogram. See DOI: 10.1039/b000000x/
25 Introduction
Diabetes mellitus is a debilitating metabolic disorder which
represents a huge social and economic burden owing to its long
term complications and morbidity. International Diabetes
Federation’s (IDF) most recent estimates have indicated that 415
30 million people have diabetes, and the number is set to rise beyond
642 million by 2040.1 Type 2 diabetes is characterized by
impaired insulin secretion from βꢀcells with associated increasing
insulin resistance in hepatic and peripheral tissues2ꢀ4 and by
chronic hyperglycemia.5 It possess a large risk of secondary
35 complications such as diabetic retinopathy,6 diabetic
neuropathy,7,8 diabetic nephropathy,9,10 cerebrovascular11,12 and
cardiovascular complications.13 Initially glycaemic control is
achieved by lifestyle changes such as increased physical activity
and dietary modifications but, when this is no longer sufficient,
40 pharmacological intervention is required.14
Current regimen of drugs for the treatment of type 2 diabetes
include use of sulfonylureas,15 biguanides,16,17 alphaꢀglucosidase
55 inhibitors,18 glitazones,19 glucagonꢀlike peptide
1
(GLPꢀ1)
analogues,20 gliptins21 and sodiumꢀglucose coꢀtransporter
(SGLT2) inhibitors.22 However,
major challenge in the
2
a
treatment of type 2 diabetes is that, glycaemic control may still
deteriorate, despite of the aggressive therapy followed.2
60 Furthermore, weight gain and hypoglycaemia are common
associated events.23 In addition, other adverse events such as
gastrointestinal discomfort with the use of biguanides, and
possible oedema, cardiac failure or fractures with the use of
thiazolidinediones, occur frequently during treatment.24ꢀ27
65 Therefore, new therapies that can correct glycaemia on a long
term basis without causing adverse events are highly desirable.
We initiated a program to identify new antiꢀdiabetic molecules
for the management of T2DM treatment. To discern new smallꢀ
molecule insulin sensitizers, a highꢀthroughput screening of our
70 chemical library of diverse scaffolds was conducted and it was
observed that two partially hydrogenated indazole derivatives
showed significant stimulation of glucose uptake in the L6
aMedicinal and Process Chemistry Division, CSIR-Central Drug
Research Institute, Lucknow 226031, India. Fax: +91-522-2771941;
Email: atul_goel@cdri.res.in
45 b Biochemistry Division, CSIR-Central Drug Research Institute, Lucknow
226031, India.
cPharmacokinetics and Metabolism Division, CSIR-Central Drug
Research Institute, Lucknow 226031, India.
skeletal muscle cells (Supplementary Figure S1†). On the basis of
literature survey, we found that benzene derivatives fused with
†The authors declare no competing interests.
50 Electronic Supplementary Information (ESI) available: Biological details,
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nitrogen containing five membered heterocyclic moieties in a
the reaction of 1ꢀacetylꢀ2ꢀtetralone or related materials with
30 hydrazine,41ꢀ43 dilithiated 2ꢀtetralone phenylhydrazone and
aromatic esters44 or by condensation of dilithiated 2ꢀtetralone
carboalkoxyhydrazones with aromatic esters followed by acid
cyclization of Cꢀacylated intermediates.45 Sivaprasad et al.46
synthesized 2Hꢀbenzo[e]indazoles by VilsmeierꢀHaack reaction
35 of 2ꢀtetralone phenylhydrazones. The quest for new, simple,
efficient and cost effective synthesis is essentially required due to
limitations of the earlier procedures. Therefore, the development
of mild and novel approaches to 2Hꢀbenzo[e]indazole derivatives
is rewarding because of their extreme significance. We describe a
40 novel and direct synthesis of dihydroꢀ2Hꢀbenzo[e]indazoles 5aꢀh
in oneꢀstep through ring transformation of 6ꢀarylꢀ4ꢀ(methylthio)ꢀ
2ꢀoxoꢀ2Hꢀpyranꢀ3ꢀcarboxylates48 3aꢀh from 2ꢀphenylꢀ6,7ꢀ
dihydroꢀ2Hꢀindazolꢀ4(5H)ꢀone47 (4) at room temperature in
excellent yields (Scheme 1). The ring transformation proceeded
45 via Michael addition of the conjugate base of 4 to the lactones
3aꢀh at position 6 followed by intramolecular cyclization and
subsequent loss of CO2. Oxidation of methylthio group to
methylsulfonyl group was achieved in 58ꢀ75 % yield by stirring
an equimolar mixture of 5e,g with metaꢀchloro perbenzoic acid
50 (mꢀCPBA)49 in dry dichloromethane for 30ꢀ60 minute at ambient
temperature to afford the methyl 8ꢀ(methylsulfonyl)ꢀ2ꢀphenylꢀ6ꢀ
Figure 1: Biologically active benzo[e]indazole derivatives
5
rigid or flexible conformation constitute an interesting class of
compounds
such
as
benzimidazole,28
pyrazole,29
pyrazolopyrimidine,30 that possess potential antiꢀhyperglycemic
activity. Indazoles belong to privileged class of molecules with
diverse biological activity (Figure 1).31ꢀ34 Motivated by these
10 results, we thus, synthesized functionalized dihydroꢀ2Hꢀ
benzo[e]indazole derivatives with donor (D) and acceptor (A)
functionalities in anticipation to evaluate their antiꢀhyperglycemic
activity and gain mechanistic insight. Herein, we report
discovery35 of functionalized dihydroꢀ2Hꢀbenzo[e]indazole
15 derivative 5e, which exhibited good antihyperglycemic activity in
in vivo animal models.
arylꢀ4,5ꢀdihydroꢀ2Hꢀbenzo[e]indazoleꢀ9ꢀcarboxylates
6a,b
(Scheme 1). All the new synthesized derivatives of 2Hꢀ
benzo[e]indazoleꢀ9ꢀcarboxylates (5aꢀc,e,f,h and 6a,b) were
55 characterized by spectroscopic analyses. Furthermore, both the
lead compounds 5d and 5g were also prepared and evaluated for
biological activity together with newly synthesized 2Hꢀ
benzo[e]indazole derivatives.
Results and discussion
Chemistry
2HꢀBenzoindazoles have attracted significant interest in the
Biological evaluation
20 pharmaceutical industry owing to their potential biological
activity.
These
molecules
have
been
used
for
60 In vitro effect of compound on insulin response in target
tissues
anticryptosporidial,36 selective cyclooxygenaseꢀ2 inhibitor,37
human dopamine D4 receptor,38 antiproliferative,39 and antifungal
activity.40 The reported strategies in the literature for the
25 synthesis of 2Hꢀbenzo[e]indazoles, generally require several
steps which suffer with one or more drawbacks including reactive
starting materials, long reaction time, low yields and harsh
reaction conditions. These systems have been prepared earlier by
To evaluate the antiꢀhyperglycemic potential of above synthesized
compounds of the 4,5ꢀdihydroꢀ2Hꢀbenzo[e]indazole series, effect
on glucose uptake was monitored in L6 skeletal muscle cells
65
O
SMe
8
SMe
SMe
COOMe
9
N
4
7
COOMe
O
3
COOMe
O
CH3
O
N
MeS
1
KOH
5
4
6
Ar
2
N
2
+
Ar
6
KOH/DMF
3-6 h
DMSO
O1
3a-h
MeO
Ar
Br
5
N
4
2a-h
3
5a-h
1
m-CPBA
Dry DCM
rt, 30-60 min
Ar=
Cl
OMe
CH3
5c
5d
5b
5a
SO2Me
5e
COOMe
S
CH3
F
F
Ar
N
N
6b
5g
5h
6a
5f
6a-b
2
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Scheme 1. Synthesis of 4,5ꢀdihydroꢀ2Hꢀbenzo[e]indazoleꢀ9ꢀcarboxylates 5aꢀh and 6a,b.
(Supplementary Figure S2
†
). The activity profile of the screened
compounds revealed that 4ꢀmethylphenyl group (5e) at position
C6 showed significant glucose uptake stimulation, while other
derivatives in which this group was replaced by other aryl groups
showed less (5d, g) or no (5aꢀc, 5f, h) significant effect on
glucose uptake in L6 skeletal muscle cells. Oxidation of
methylthio group to methyl sulfonyl group (6a,b) also showed no
5
10 significant effect on glucose uptake. Hence, methyl 8ꢀmethylthioꢀ
2ꢀphenylꢀ6ꢀpꢀtolylꢀ4,5ꢀdihydroꢀ2Hꢀbenzo[e]indazoleꢀ9ꢀ
carboxylate 5e exhibited higher glucose uptake stimulation than
lead compounds 5d, g and was selected for further study. In the
L6 myotubes, 5e increased the rate of glucose uptake in a
15 concentrationꢀdependent fashion with significant effect at the
minimal concentration of 2.5 ꢁM (P < 0.05) and caused around
2.0ꢀfold stimulation at 10 ꢁM concentration (P < 0.001). Since
insulin is the physiological stimulator of glucose uptake in
skeletal muscle cell,50 we used it as positive control, which
20 caused around 2.3ꢀfold stimulation (P < 0.001) upon acute
exposure at 100 nM for 20 min (Figure 2).
45
Figure 3: Effect of 5e on glucose uptake in HepG2 liver cells.
Significance: *p < 0.05, ***p < 0.001 relative to control condition.
Figure 4: Effect of 5e on glucose release from HepG2 liver cells.
50 Significance: ###p < 0.001, ***p < 0.001 relative to control condition.
First, the effect of compound 5e on the improvement of oral
glucose tolerance post sucrose load (SLM) on normal rats was
evaluated. Figure 5 showed the acute effect of 5e on postprandial
hyperglycemia post sucrose load in normal albino rats. Treatment
55 with 5e significantly prevented the postprandial rise in blood
glucose. The percent inhibition on the rise of postprandial
hyperglycemia by 5e was calculated to be ~ 35.8% at the dose of
100 mg/kg. Further the compound was evaluated for oral glucose
tolerance in sucrose challenged streptozotocinꢀinduced diabetic
60 rats. Figure 6 shows the blood glucose profile of treated and
control animals at various time interval post sucrose load. It is
evident from the results that compound 5e inhibited the rise in
postprandial hyperglycemia. The percent improvement in glucose
tolerance by 5e was calculated to be ~ 23.1% after 5h post
65 sucrose load in STZꢀinduced diabetic rat.
3
Figure 2: Doseꢀdependent effect of 5e on Hꢀ2ꢀDeoxyglucose uptake by
L6 myotubes. Significance: *p < 0.05, ***p < 0.001 relative to control
25 condition.
The glucose uptake stimulatory effect of 5e was further
validated in hepatic cells, which is another major target tissue of
insulin action. In human hepatic cell lines (HepG2), 5e
significantly increased the uptake of glucose, the effect
30 comparable to standard antidiabetic drug metformin (Figure 3).
Metformin has been established to exert its antidiabetic potential
through regulating hepatic glucose metabolism.51 In hepatic
tissues, activation of insulin signaling inhibits the release of
glucose via gluconeogenesis.52 Here, treatment with 5e
35 significantly decreased the glucagonꢀmediated release of glucose
in HepG2 cells (P < 0.001) (Figure 4). Moreover, 5e exerted these
cellular effects without any significant change in cell viability, as
As an interesting chemical entity, the compound 5e was further
evaluated for antihyperglycemic effect in the db/db mice, a
genetic model of type 2 diabetes. The animals were dosed orally
established by MTT assay (Supplementary Figure S3†). Results
validate the potential of 5e to enhance insulin response in
40 sensitive tissues.
In vivo antiꢀdiabetic effect of compound in animal models
Before screening in in vivo models, compound 5e was checked
for its purity by HPLC method and was found >98% pure
70 Figure 5: Effect of 5e at oral dose of 100 mg/kg b.wt. on (A) blood
glucose profile, (B) glucose AUC (0–2h) of sucrose loaded (oral) normal
(Supplementary Figure S4, S5†).
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rats. Values are mean % change in 5 animals of a group; significance: **
p < 0.01 compared with control.
enhanced glucose utilization in peripheral tissues and decreased
50 hepatic glucose production.54 Given the sensitizary potential of
the 5e, we assessed its cellular activity in the insulin signal
Figure 6: Effect of 5e at oral dose of 100 mg/kg b.wt. on (A) blood
glucose profile, (B) glucose AUC (0–24h) of streptozotocinꢀinduced
diabetic rats with sucrose challenge. Values are mean % change in 5
animals of a group; significance: **p < 0.01 when compared with control.
5
Figure 7: Antihyperglycemic effect of 5e in db/db mice. Blood glucose
profile of db/db mice treated with vehicle, compound 5e at oral dose of 3
mg/kg, 10mg/kg, 30 mg/kg b.wt. and standard drug Pioglitazone at oral
dose of 10 mg/kg b.wt. Values are mean % change in 5 animals of a
group; significance: *p < 0.05, **p < 0.01 when compared with control.
with different doses (3, 10, 30 mg/kg) of compound or standard
10 pioglitazone (10 mg/kg) once a day for 15 consecutive days with
daily measurement of the blood glucose levels, and oral glucose
tolerance test (OGTT) at day 10 and day 15. The pioglitazone has
been established to stimulate insulin signaling,53 thereby used as
positive control to compare the antidiabetic efficacy of 5e. The
15 effect on blood glucose during the course of treatment was
illustrated in Figure 7. Treatment with compound 5e led to a
continuous fall in blood glucose level over time, in a doseꢀ
dependent manner. Significant decrease in blood glucose level
was observed from day 7 upon treatment with compound (30
20 mg/kg), whereas upon pioglitazone (10 mg/kg) significant
lowering in blood glucose level was observed from day 4 (Figure
7). There was no effect of compound on body weight and food
intake during the course of treatment (data not shown).
55
Glucose tolerance pattern of each animal was tested on day 10
25 and day 15. The postprandial rise in blood glucose levels of
compound treated animals were found to be significantly lowered
compared to that of the control group, in a doseꢀdependent
manner. It showed significant improvement in their glucose
tolerance pattern in comparison to control animals. The overall
30 improvement in the glucose tolerance was calculated to be around
7.8 %, 13.9 %, and 25.7 % at the doses of 3, 10, and 30 mg/kg, at
day 10 (Figure 8A). The improvement was further enhanced to
13.2 %, 20.1 % and 31.6 % at the doses of 3, 10, and 30 mg/kg,
respectively at day 15 (Figure 8B). These effects of 5e in db/db
35 mice are associated with decrease in fasting blood glucose and
60
Figure 8: Dose dependent effect of compound 5e on oral glucose
tolerance (OGTT) of db/db mice. Values are mean % change in 5
animals of a group; significance: *p < 0.05, **p < 0.01 when compared
with control.
serum insulin levels, in
a doseꢀdependent manner and
improvement in lipid profile of the animals. As shown in Figure
9, treatment with 5e at the dose of 30 mg/kg caused a significant
decrease in triglyceride (18.2 %, p < 0.01) and total cholesterol
40 (22.7 %, p < 0.05) levels, and increased the HDLꢀC level (20.5%,
p < 0.05) in a significant manner.
Mechanism Studies in L6 Skeletal Muscle
To investigate the signaling pathways involved in the glucose
45 uptake by 5e in skeletal muscle, western blot analysis was
carried out. From literature, it is known that insulin stimulation
in target tissues results in phosphorylation and activation of the
signaling cascade IR/IRS1/PI3K/Akt/GSK3β leading to
65
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Figure 9: Dose dependent effect of compound 5e on serum lipid profile,
insulin and fasting blood glucose level in db/db mice. Values are mean
% change in 5 animals of a group; significance: *p < 0.05, **p < 0.01
when compared with control.
db/db animal models. Further treatment with 5e at the dose of 30
mg/kg caused a significant decrease in triglyceride and total
45 cholesterol levels, and increased the HDLꢀC level in a significant
manner. Mechanism of action of 5e was found to be mediated via
activation of IRSꢀ1, Akt and GSKꢀ3β pathways and possibly by
the inhibition of PTPꢀ1B, which is a major mediator of insulin
signalling and insulinꢀresistance. For drug development
50 perspectives, detailed pharmacokinetic and toxicity studies are in
progress.
Experimental
Analysis and instruments
55 All the reactions were carried out under anhydrous conditions and
were monitored by thinꢀlayer chromatography (TLC),
visualization was done with UVꢀlight (254 nm). 1H NMR and 13
C
NMR spectra were recorded using Bruker Supercon Magnet
DRXꢀ300 spectrometer (operating at 400MHz for 1H and 50,
60 MHz for 13C) using DMSOꢀd6 or CDCl3 as solvent and
tetramethylsilane (TMS) as internal standard. Chemical shifts are
reported in parts per million. Signal pattern are indicated as s,
singlet; d, doublet; dd, double doublet; t, triplet; m, multiplet.
Coupling constant (J) are given in hertz. Infrared (IR) spectra
65 were recorded in KBr disc and reported in wave number (cmꢀ1).
ESIMS, EI spectrometer was used for mass spectra analysis.
Infrared spectra were recorded on a PerkinꢀElmer FTꢀIR RXI
spectrophotometer. Electrospray ionization mass spectra (ESIꢀ
MS) were recorded on Thermo Lcq Advantage MaxꢀIT. High
70 resolution mass spectra (HRMS) were recorded on 6520 Agilent
Q T of LC MS/MS (Accurate mass). Additionally, purity of
compounds was measured by a RPꢀ HPLC with the following
conditions, and purity of the compounds was >98%. The Waters
HPLC system, Milford USA consisted of a binary pump (model
75 Waters 515 HPLC pump), auto sampler (model 2707 Auto
sampler) and PDA detector (Waters 2998). Data collection and
analysis were performed using Empower pro 2 software. The
resolution and better peak shape were achieved on a XꢀBridge
C18 (5µm, 4.6 X 250 mm) column, protected with a C18 guard
80 column. The system was analyzed in isocratic mode with a
mobile phase consisting of acetonitrileꢀtriple distilled water (60:
40, v/v) at a flow rate of 1.0 mL/min.
5
Figure 10: Effect of 5e on activation of the components of insulin
signaling cascade in L6 myotubes. Cells were treated with indicated
concentration of 5e for 16h and stimulated with insulin (100 nM) for 10
min, followed by cell lysis and western analysis. Shown are
10 representative immunoblots (A) and densitometric quantification of pꢀ
IRSꢀ1 (B), pꢀAKT (C) and pꢀGSK (D) relative to their respective total
forms. Significance: *P < 0.05, **P < 0.01 realtive to control; #P < 0.05,
##P < 0.01 relative to control insulin treated condition.
transduction pathway in skeletal muscle, the major depot for
15 postprandial glucose utilization. Treatment of L6 myotubes with
5e increased the insulinꢀstimulated tyrosine phosphorylation of
IRSꢀ1 in a concentrationꢀdependent manner, which is further
validated by the enhanced insulinꢀstimulated phosphorylation of
the downstream targets Akt (Serꢀ473) and GKSꢀ3β (Serꢀ9) (Figure
20 10). Results indicated the ability of 5e to activate insulin signaling
pathway in skeletal muscle cells.
Again, from the literature it is known that PTPꢀ1B inhibition
plays a key role in insulin signaling through dephosphorylation of
the insulin receptor.55ꢀ57 Hence, the above observations of the
25 activation of insulin signalling cascade in L6 myotubes prompted
us to check the protein tyrosine phosphataseꢀ1B inhibitory (PTPꢀ
1B) activity of compound 5e.58 The compound 5e was found to
inhibit the activity of PTPꢀ1B enzyme in a concentrationꢀ
dependent manner and the IC50 value was calculated to be 7.12
General procedure for the synthesis of functionalized methyl
8ꢀ(methylthio)ꢀ2ꢀphenylꢀ6ꢀarylꢀ4,5ꢀdihydroꢀ2Hꢀbenzo[e]indaꢀ
85 zoleꢀ9ꢀcarboxylates (5aꢀh): A mixture of 6ꢀarylꢀ2Hꢀpyranꢀ2ꢀone
3aꢀh (1 mmol), 2ꢀphenylꢀ6,7ꢀdihydroꢀ2Hꢀindazolꢀ4(5H)ꢀone (4)
(1.2 mmol) and powdered KOH (1.2 mmol) in dry DMF (5 mL)
was stirred at room temperature for 3ꢀ6 h. The progress of
reaction was monitored by TLC, and on completion the reaction
90 mixture was poured onto crushed ice with vigorous stirring and
finally neutralized with 10% HCl. The precipitate obtained was
filtered and purified on a silica gel column using 10% chloroform
in hexane as the eluent.
30 µM (Supplementary Table 1†). Thus, PTPꢀ1B may be involved for
the action of compound 5e.
Conclusion
In conclusion, we discovered two dihydroꢀ2Hꢀbenzo[e]indazoles
through screening of our chemical library as antiꢀhyperglycemic
35 agents. A series of 2Hꢀbenzo[e]indazoles were prepared by
reacting substituted 2Hꢀpyranꢀ3ꢀcarboxylates with 2ꢀphenylꢀ6,7ꢀ
dihydroꢀ2Hꢀindazolꢀ4(5H)ꢀone in good yields and their in vitro 2ꢀ
DG uptake activity was evaluated. The methyl 8ꢀ(methylthio)ꢀ2ꢀ
phenylꢀ6ꢀpꢀtolylꢀ4,5ꢀdihydroꢀ2Hꢀbenzo[e]indazoleꢀ9ꢀcarboxylate
40 5e showed antiꢀhyperglycemic activity with 2ꢀfold stimulation in
2ꢀDG uptake at 10 ꢁM and exhibited better glucose tolerance,
fasting as well as postprandial blood glucose in SLM, STZ and
Methyl
8ꢀ(methylthio)ꢀ2,6ꢀdiphenylꢀ4,5ꢀdihydroꢀ2Hꢀ
95 benzo[e]indazoleꢀ9ꢀcarboxylate (5a): A mixture of 3a (276 mg,
1 mmol, 1 equiv), compound 4 (212 mg, 1 mmol, 1 equiv) and
KOH (84 mg, 1.5 mmol, 1.5 equiv) in dry DMF (5 mL) was
stirred at room temperature for 4h. Using the general procedure
described above 234 mg (55%) of 5a was obtained as an off
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white solid; mp = 173ꢀ175 oC; MS (ESI) 427 [M + H+]; IR (KBr)
ν = 3020, 1726, 1574 cmꢀ1; 1H NMR (300 MHz, CDCl3)
= 7.99
(s, 1H, ArH), 7.67 (d, J = 9.0 Hz, 2H, ArH), 7.38ꢀ7.50 (m, 5H,
ArH), 7.24ꢀ7.35 (m, 3H, ArH), 7.19 (s, 1H, ArH), 4.01 (s, 3H,
OCH3), 2.81ꢀ2.93 (m, 4H, 2CH2), 2.48 (s, 3H, SCH3), ppm; 13C
Methyl
8ꢀ(methylthio)ꢀ2ꢀphenylꢀ6ꢀpꢀtolylꢀ4,5ꢀdihydroꢀ2Hꢀ
benzo[e]indazoleꢀ9ꢀcarboxylate (5e): A mixture of 3e (290 mg,
1 mmol, 1 equiv), compound 4 (212 mg, 1 mmol, 1 equiv) and
60 KOH (84 mg, 1.5 mmol, 1.5 equiv) in dry DMF (5 mL) was
stirred at room temperature for 3.5h. Using the general procedure
described above 237 mg (54%) of 5e was obtained as an off white
solid; mp = 178ꢀ180 C; MS (ESI) 441 [M + H+]; IR (KBr) ν =
2984, 2918, 1718, 1504 cmꢀ1; H NMR (300 MHz, CDCl3)
65 7.98 (s, 1H, ArH), 7.63ꢀ7.72 (m, 2H, ArH), 7.46 (t, J = 11.6 Hz,
2H, ArH), 7.21ꢀ7.33 (m, 5H, ArH), 7.18 (s, 1H, ArH), 4.01 (s,
3H, OCH3), 2.80ꢀ2.94 (m, 4H, 2CH2), 2.48 (s, 3H, CH3), 2.43 (s,
3H, CH3) ppm; 13C NMR (50.3 MHz, CDCl3)
153.32, 143.92, 140.43, 138.33, 137.74, 133.08, 132.56, 130.75,
δ
5
NMR (50.3 MHz, CDCl3)
δ = 170.66, 153.37, 143.93, 141.31,
o
140.38, 133.20, 132.49, 129.97, 129.48, 129.26, 128.77, 128.03,
127.97, 126.82, 123.45, 119.37, 117.24, 53.37, 27.75, 22.16,
18.79 ppm; HRMS (EI) calculated mass for C26H23N2O2S:
1
δ =
10 427.1480 found 427.1470.
Methyl 6ꢀ(4ꢀchlorophenyl)ꢀ8ꢀ(methylthio)ꢀ2ꢀphenylꢀ4,
5ꢀ
dihydroꢀ2Hꢀbenzo[e]indazoleꢀ9ꢀcarboxylate (5b): A mixture of
3b (310 mg, 1 mmol, 1 equiv), compound 4 (212 mg, 1 mmol, 1
δ = 170.61,
equiv) and KOH (84 mg, 1.5 mmol, 1.5 equiv) in dry DMF (5 70 129.88, 129.44, 129.36, 128.00, 126.76, 123.38, 119.38, 117.29,
15 mL) was stirred at room temperature for 3h. Using the general
procedure described above 266 mg (58%) of 5b was obtained as
an off white solid; mp = 160ꢀ162 oC; MS (ESI) 461 [M + H+]; IR
53.24, 27.75, 22.14, 21.62, 18.79 ppm; HRMS (EI) calculated
mass for C27H24N2O2S: 440.1559 found 440.1547.
Methyl
8ꢀ(methylthio)ꢀ2ꢀphenylꢀ6ꢀ(thiophenꢀ2ꢀyl)ꢀ4,5ꢀ
(KBr) ν = 1722, 1588 cmꢀ1; 1H NMR (300 MHz, CDCl3)
δ = 8.00
dihydroꢀ2H benzo[e]indazoleꢀ9ꢀcarboxylate (5f): A mixture of
75 3f (282 mg, 1 mmol, 1 equiv), compound 4 (212 mg, 1 mmol, 1
equiv) and KOH (84 mg, 1.5 mmol, 1.5 equiv) in dry DMF (5
mL) was stirred at room temperature for 5h. Using the general
procedure described above 211 mg (49%) of 5f was obtained as
an off white solid; mp = 174ꢀ176oC; MS (ESI) 433 [M + H+]; IR
(s, 1H, ArH), 7.69 (d, J = 7.7 Hz, 2H, ArH), 7.41ꢀ7.51 (m, 4H,
20 ArH), 7.14ꢀ7.35 (m, 4H, ArH), 4.05 (s, 3H, OCH3), 2.85ꢀ2.89 (m,
4H, 2CH2), 2.52 (s, 3H, SCH3) ppm; 13C NMR (50.3 MHz,
CDCl3)
δ = 170.44, 153.11, 142.69, 140.33, 139.60, 134.10,
133.47, 132.42, 130.85, 129.91, 129.02, 128.29, 126.93, 123.52,
119.49, 117.13, 53.35, 27.75, 22.09, 18.79 ppm; HRMS (EI)
25 calculated mass for C26H22ClN2O2S: 461.1091 found 461.1079.
1
80 (KBr) ν = 3065, 2917, 1716, 1570 cmꢀ1; H NMR (200 MHz,
CDCl3)
δ = 7.97 (s, 1H, ArH), 7.61ꢀ7.71 (m, 2H, ArH), 7.28ꢀ7.53
(m, 5H, ArH), 7.02ꢀ7.18 (m, 2H, ArH), 4.00 (s, 3H, OCH3), 3.02ꢀ
3.13 (m, 2H, CH2), 2.82ꢀ2.92 (s, 2H, CH2), 2.49 (s, 3H, SCH3),
Methyl
6ꢀ(4ꢀbromophenyl)ꢀ8ꢀ(methylthio)ꢀ2ꢀphenylꢀ4,5ꢀ
dihydroꢀ2Hꢀbenzo[e]indazoleꢀ9ꢀcarboxylate (5c): A mixture of
3c (355 mg, 1 mmol, 1 equiv), compound 4 (212 mg, 1 mmol, 1
ppm; 13C NMR (50.3 MHz, CDCl3)
δ = 170.36, 153.16, 141.90,
equiv) and KOH (84 mg, 1.5 mmol, 1.5 equiv) in dry DMF (5 85 140.39, 136.34, 133.55, 133.34, 131.48, 130.26, 129.90, 128.33,
30 mL) was stirred at room temperature for 3.5h. Using the general
procedure described above 297 mg (59%) of 5c was obtained as
an off white solid; mp = 164ꢀ166 oC; MS (ESI) 506 [M + H+]; IR
127.87, 127.64, 126.83, 126.37, 123.50, 119.43, 117.11, 53.28,
27.75, 22.08, 18.82 ppm; HRMS (EI) calculated mass for
C24H20N2O2S2: 432.0966 found 432.0970.
(KBr) ν = 3021, 1725, 1511 cmꢀ1; 1H NMR (300 MHz, CDCl3)
δ
Methyl
6ꢀ(4ꢀfluorophenyl)ꢀ8ꢀ(methylthio)ꢀ2ꢀphenylꢀ4,5ꢀ
90 dihydroꢀ2Hꢀbenzo[e]indazoleꢀ9ꢀcarboxylate (5g): A mixture of
3g (294 mg, 1 mmol, 1 equiv), compound 4 (212 mg, 1 mmol, 1
equiv) and KOH (84 mg, 1.5 mmol, 1.5 equiv) in dry DMF (5
mL) was stirred at room temperature for 4h. Using the general
procedure described above 230 mg (52%) of 5g was obtained as
95 an off white solid; mp = 182ꢀ184oC; MS (ESI) 445 [M + H+]; IR
= 7.98 (s, 1H, ArH), 7.40ꢀ7.71 (m, 4H, ArH), 7.12ꢀ7.34 (m , 4H,
35 ArH), 4.01 (s, 3H, OCH3), 2.83ꢀ2.87 (m, 4H, 2CH2), 2.48 (s, 3H,
CH3), ppm; 13C NMR (50.3 MHz, CDCl3)
δ = 170.42, 153.14,
142.68, 140.41, 140.12, 133.47, 132.38, 132.00, 131.18, 129.90,
129.07, 128.29, 126.88, 123.51, 122.23, 119.40, 117.11, 53.29,
27.75, 22.11, 18.83 ppm; HRMS (EI) calculated mass for
40 C26H22BrN2O2S: 505.0585 found 505.0575.
(KBr) ν = 2950, 1726, 1507 cmꢀ1; 1H NMR (300 MHz, CDCl3)
δ
= 7.98 (s, 1H, ArH), 7.67 (d, J = 7.6 Hz, 2H, ArH), 7.46 (t, J =
7.8 Hz, 2H, ArH), 7.25ꢀ7.37 (m, 3H, ArH), 7.09ꢀ7.19 (m, 3H,
ArH), 4.01 (s, 3H, OCH3), 2.83ꢀ2.88 (m, 4H, 2CH2), 2.48 (s, 3H,
Methyl
6ꢀ(4ꢀmethoxyphenyl)ꢀ8ꢀ(methylthio)ꢀ2ꢀphenylꢀ4,5ꢀ
dihydroꢀ2Hꢀbenzo[e]indazoleꢀ9ꢀcarboxylate (5d): A mixture of
3d (306 mg, 1 mmol, 1 equiv), compound 4 (212 mg, 1 mmol, 1
equiv) and KOH (84 mg, 1.5 mmol, 1.5 equiv) in dry DMF (5 100 SCH3) ppm; 13C NMR (50.3 MHz, CDCl3)
δ = 170.10, 160.66,
45 mL) was stirred at room temperature for 3h. Using the general
procedure described above 269 mg (59%) of 5d was obtained as
an off white solid; mp = 172ꢀ174 oC; MS (ESI) 457 [M + H+]; IR
152.78, 142.48, 139.96, 136.75, 136.71, 132.88, 132.13, 130.75,
130.65, 129.51, 128.90, 127.74, 126.45, 123.03, 119.00, 116.73,
115.47, 115.18, 52.90, 27.30, 21.68, 18.38 ppm; HRMS (EI)
calculated mass for C26H22FN2O2S: 445.1386 found 445.1381.
105 Methyl 8ꢀ(methylthio)ꢀ2ꢀphenylꢀ6ꢀ(pyrenꢀ1ꢀyl)ꢀ4,5ꢀdihydroꢀ
2Hꢀbenzo[e]indazoleꢀ9ꢀcarboxylate (5h): A mixture of 3h (400
mg, 1 mmol, 1 equiv), compound 4 (212 mg, 1 mmol, 1 equiv)
and KOH (84 mg, 1.5 mmol, 1.5 equiv) in dry DMF (5 mL) was
stirred at room temperature for 5.5h. Using the general procedure
110 described above 319 mg (58%) of 5h was obtained as an off
(KBr) ν = 2923, 1722, 1503 cmꢀ1; 1H NMR (300 MHz, CDCl3)
δ
= 7.98 (s, 1H, ArH), 7.63ꢀ7.71 (m, 2H, ArH), 7.41ꢀ7.52 (m, 2H,
50 ArH), 7.16ꢀ7.34 (m, 4H, ArH), 6.95.ꢀ7.03 (m, 2H, ArH), 4.01 (s,
3H, OCH3), 3.88 (s, 3H, OCH3), 2.81ꢀ2.95 (m, 4H, 2CH2), 2.49
(s, 3H, CH3) ppm; 13C NMR (50.3 MHz, CDCl3)
δ = 183.07,
170.23, 159.08, 152.90, 143.19, 133.14, 132.61, 132.26, 130.19,
129.46, 129.14, 126.34, 122.95, 118.96, 116.87, 113.75, 55.36,
55 52.81, 27.33, 21.73, 18.44, ppm; HRMS (EI) calculated mass for
C27H25N2O3S: 457.1586 found 457.1557.
white solid; mp = 174ꢀ176oC; MS (ESI) 551 [M + H+]; IR (KBr)
1
ν = 2926, 1725, 1504 cmꢀ1; H NMR (300 MHz, CDCl3)
δ =
6
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8.16ꢀ8.29 (m, 3H, ArH), 8.14 (s, 2H, ArH), 7.99ꢀ8.08 (m, 3H,
ArH), 7.90 (d, J = 7.7 Hz, 1H, ArH), 7.77 (d, J = 9.1 Hz, 1H,
ArH), 7.68 (d, J = 7.5 Hz, 2H, ArH), 7.47 (t, J = 7.8 Hz, 2H,
calculated mass for C26H22FN2O4S: 477.1284 found 477.1277.
Ethical statement
The work with all these animals was cleared by Institutional
ArH), 7.23ꢀ7.35 (m, 2H, ArH), 4.08 (s, 3H, OCH3), 2.61ꢀ2.78 (m, 60 Animal Ethics Committee (IAEC) and was conducted in
5
4H, 2CH2), 2.49 (s, 3H, SCH3) ppm; 13C NMR (50.3 MHz,
CDCl3) = 170.32, 152.92, 142.25, 140.00, 135.72, 133.45,
133.13, 131.42, 131.00, 130.92, 130.31, 129.53, 129.13, 128.99,
128.04, 127.75, 127.62, 127.36, 126.97, 126.46, 126.25, 125.47,
125.28, 124.92, 124.62, 123.11, 119.06, 116.82, 52.99, 27.32,
accordance with the guidelines of the Committee for the purpose
of Control and Supervision of Experiments on Animals
(CPCSEA) formed by the Government of India in 1964.
δ
65 Acknowledgments
10 21.60, 18.11 ppm; HRMS (EI) calculated mass for C36H27N2O2S:
551.1793 found 551.1797.
We gratefully acknowledge financial support by the Council of
Scientific and Industrial Research (CSIR), New Delhi under 12th
Five Year Plan network project 'THUNDER' (BSC0102). We are
thankful to CSIR, New Delhi and UGC, New Delhi for research
70 fellowships. The spectroscopic data provided by Sophisticated
and Analytical Instrumentation Facilities (SAIF) of the Institute is
gratefully acknowledged. The CDRI communication number for
the manuscript is 9344.
General procedure for the synthesis of functionalized methyl
8ꢀ(methylsulfonyl)ꢀ2ꢀphenylꢀ6ꢀarylꢀ4,5ꢀdihydroꢀ2Hꢀ
benzo[e]indazoleꢀ9ꢀcarboxylates (6aꢀb): A mixture of methyl 8ꢀ
15 (methylthio)ꢀ2ꢀphenylꢀ6ꢀarylꢀ4,5ꢀdihydroꢀ2Hꢀbenzo[e]indazoleꢀ
9ꢀcarboxylates 5 (1 mmol) and mꢀchloro perbenzoic acid (mꢀ
CPBA) (5 mmol) in dry DCM (5 mL) was stirred at room
temperature for 30ꢀ60 min. The progress of reaction was
monitored by TLC, and on completion the reaction mixture was
20 neutralized with NaHCO3 solution. The neutralized solution
obtained was extracted with DCM solution; the organic layer was
separated and purified on a silica gel column using 40%
Chloroform in hexane as the eluent to afford 6aꢀc.
Methyl 8ꢀ(methylsulfonyl)ꢀ2ꢀphenylꢀ6ꢀ(pꢀtolyl)ꢀ4,5ꢀdihydroꢀ
25 2Hꢀbenzo[e]indazoleꢀ9ꢀcarboxylate (6a): A mixture of methyl
8ꢀ(methylthio)ꢀ2ꢀphenylꢀ6ꢀpꢀtolylꢀ4,5ꢀdihydroꢀ2Hꢀbenzo[e]indꢀ
azoleꢀ9ꢀcarboxylate (5e) (440 mg, 1 mmol) and mꢀCPBA (855
mg, 5 mmol) in dry DCM (5 mL) was stirred at room temperature
for 60 min. Using the general procedure described above 354 mg
30 (75%) of 6a was obtained as an off white solid; mp = 228ꢀ230 oC;
MS (ESI) 473 [M + H+]; IR (KBr) ν = 2927, 1729, 1599 cmꢀ1; 1H
Notes and references
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Hz, 1H, ArH), 7.68 (d, J = 7.2 Hz, 2H, ArH), 7.48 (t, J = 6.7 Hz,
2H, ArH), 7.19ꢀ7.36 (m, 5H, ArH), 4.03 (s, 3H, OCH3), 3.19 (s,
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95
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169.27, 152.62, 143.76, 140.22, 139.79, 138.05, 136.44, 135.52,
129.59, 129.24, 128.91, 128.12, 127.19, 126.76, 123.43, 119.08,
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40 calculated mass for C27H25N2O4S: 473.1535 found 473.1530.
Methyl 6ꢀ(4ꢀfluorophenyl)ꢀ8ꢀ(methylsulfonyl)ꢀ2ꢀphenylꢀ4,5ꢀ
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2Hꢀbenzo[e]indazoleꢀ9ꢀcarboxylate (5g) (444 mg, 1 mmol) and
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white solid; mp = 245ꢀ247 oC; MS (ESI) 477 [M + H+]; IR (KBr)
100
105 16. Huttunen, K.M., Mannila, A., Laine, K., Kemppainen, E.,
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ν = 2927, 1729, 1599 cmꢀ1; 1H NMR (300 MHz, CDCl3)
δ = 8.02
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7.48 (m, J = 7.5 Hz, 2H, ArH), 7.12ꢀ7.24 (m, 5H, ArH), 4.03 (s,
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2.92 (m, 2H, CH2) ppm; 13C NMR (50.3 MHz, CDCl3)
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169.12, 152.47, 142.66, 140.23, 139.74, 135.67, 135.34, 130.84,
55 130.67, 129.61, 128.61, 128.02, 127.55, 126.83, 123.50, 119.10,
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MedChemComm
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DOI: 10.1039/C6MD00467A
TOC
Synthesis, in vitro and in vivo antihyperglycemic activity of substituted 2H-benzo[e]indazole-9-
carboxylate are described.