Synthesis, α-glucosidase inhibition and antioxidant activity of the 7-carbo–substituted 5-bromo-3-methylindazoles

Article abstract of DOI:10.1016/j.bioorg.2020.103702

Series of 7-aryl- (3a–f), 7-arylvinyl- (3g–k) and 7-(arylethynyl)-5-bromo-3-methylindazoles (4a–f) have been evaluated through enzymatic assay in vitro for inhibitory effect against α-glucosidase activity and for antioxidant potential through the 2,2-diph

Full text of DOI:10.1016/j.bioorg.2020.103702

Journal Pre-proofs
Synthesis, α-glucosidase inhibition and antioxidant activity of the 7-carbo–
substituted 5-bromo-3-methylindazoles
Malose J. Mphahlele, Nontokozo M. Magwaza, Samantha Gildenhuys,
Itumeleng B. Setshedi
PII:
S0045-2068(19)32217-5
DOI:
https://doi.org/10.1016/j.bioorg.2020.103702
YBIOO 103702
Reference:
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Bioorganic Chemistry
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Revised Date:
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25 December 2019
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Please cite this article as: M.J. Mphahlele, N.M. Magwaza, S. Gildenhuys, I.B. Setshedi, Synthesis, α-glucosidase
inhibition and antioxidant activity of the 7-carbo–substituted 5-bromo-3-methylindazoles, Bioorganic Chemistry
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Synthesis, α-glucosidase inhibition and antioxidant activity of the 7-carbo–substituted 5-
bromo-3-methylindazoles
1
1
2
Malose J. Mphahlele, * Nontokozo M. Magwaza, Samantha Gildenhuys and Itumeleng B.
Setshedi2
1
Department of Chemistry, College of Science, Engineering and Technology, University of
South Africa, Private Bag X06, Florida 1710, South Africa
2
Department of Life & Consumer Sciences, College of Agriculture and Environmental
Sciences, University of South Africa, Private Bag X06, Florida 1710, South Africa
*
Author for correspondence; mphahmj@unisa.ac.za
Abstract: Series of 7-aryl- (3a–f), 7-arylvinyl- (3g–k) and 7-(arylethynyl)-5-bromo-3-
methylindazoles (4a–f) have been evaluated through enzymatic assay in vitro for inhibitory
effect against α-glucosidase activity and for antioxidant potential through the 2,2-diphenyl-1-
picrylhydrazyl (DPPH) radical scavenging assay. Compounds 3a–k and 4a–f showed
significant to moderate α-glucosidase inhibition with IC values in the range of 0.50–51.51 μM
5
0
and 0.42–23.71 μM compared with acarbose drug (IC = 0.82 μM), respectively. 5-Bromo-3-
5
0
methyl-7-phenyl-1H-indazole (3a), 5-bromo-3-methyl-7-styryl-1H-indazole (3h) and 5-
bromo-3-methyl-7-styryl-1H-indazole (4a) exhibited moderate to significant antigrowth effect
against the breast MCF-7 cancer cell line and reduced cytotoxicity against the human
embryonic kidney derived Hek293-T cells when compared to doxorubicin as reference
standard. Non-covalent (alkyl, π-alkyl and π-π T shaped), electrostatic (π-sulfur and/or π-anion)
and hydrogen bonding interactions are predicted to increase interactions with protein residues,
thereby enhancing the inhibitory effect of these compounds against α-glucosidase.
Keywords: polysubstituted indazoles; α-glucosidase; antioxidant activity; cytotoxicity;
molecular docking
1

1
. Introduction
Type 2 diabetes (T2D) mellitus is a chronic non-communicable metabolic disease, and its
prevalence has been rising steadily over the year to become a serious public health problem [1].
This disease is characterized by a defect in the secretion of insulin and resistance in its target
organs. The deficiency is attributed to increased glucose level in blood, also known as post-
prandial hyperglycemia (PPHG). PPHG has emerged as a prominent and early defect in type 2
diabetes [2]. Diabetic patients are more vulnerable to various forms of both short- and long-
term complications, such an increased risk of microbial infections [3] and cancer [4-6].
Hyperglycemia, has also been linked to increased cancer cell proliferation, inhibited cancer cell
apoptosis and increased cancer cell metastasis [7]. α-Glucosidase is a membrane bound enzyme
in small intestine responsible for inhibiting the hydrolysis of carbohydrates [8]. This enzyme
catalyzes the hydrolysis of 1,4-α bonds of the unabsorbed oligo- and disaccharides, and converts
them into glucose, which is absorbed in the upper jejunum resulting in hyperglycaemia [9].
Inhibitors of α-glucosidase can retard the decomposition and absorption of dietary
carbohydrates by restricting the breakdown of linear or branched oligosaccharide units like α-
limit dextrins, maltose and maltotriose to produce glucose, thereby preventing glucose
absorption into blood stream and suppressing PPHG [10,11]. Oxidative stress is implicated in
the early stages and progression of T2D due to the ability of free radicals to damage lipids,
proteins and DNA [12]. Preclinical and clinical studies, on the other hand, have revealed
complex associations between diabetes and breast cancer [7]. Inhibition of α-glucosidase has
also been found to have positive effects in the treatment of cancer [13]. Managing diabetes and
treating cancer using a combination of anti-diabetic, antioxidant and classical anti-cancer drugs
has proven to be more efficient in the treatment of diabetes-associated cancers. Metformin (1,1-
dimethylbiguanidehydrochloride), is the most widely used oral anti-diabetic drug for the
treatment of diabetes and has also shown promise in reducing cancer risk with little or no
2

negative effects [14]. Thus, drugs that mitigate PPHG and possess cytotoxic activity against
breast cancer may offer an opportunity for the development of novel therapeutics for diabetic
patients. Such a multi-target-directed ligand design strategy (MTDL) would probably be more
effective for the treatment of diabetes than one-target one-drug concepts and drug-combination
therapies.
The indazole moiety is a 2-azaindole bioisoster, and it continues to attract considerable
attention especially in the context of anti-diabetic [15-17] and anticancer [18,19] drug
development. Hydrophobic interactions, including alkyl, π-alkyl and π-π interactions between
inhibitors and α-glucosidase play a significant role in inhibitory activities of this enzyme [20].
Literature precedents revealed that a methyl group at the 3-position of the indazole ring is well
tolerated with respect to anti-diabetic potency in vitro [15,17]. Song et al. prepared series of
N-1 or N-2 substituted indazoles bearing an aryl group at either position 5 or 6, and with or
without a carbon-based substituent at the C-3 position and evaluated them in vitro and in vivo
as glucagon receptor antagonists [17]. The C-3 unsubstituted derivatives were found to be
strongly active than the 3-methyl substituted derivatives, which in turn, were more active than
the 3-aryl substituted analogues. The presence of bromine on the fused benzo ring of the 1H-
indazole framework, on the other hand, has been found to increase the inhibitory activity against
indoleamine 2,3-dioxygenase 1 (IDO1) activity [21]. Moreover, a bromine atom is known to
contain a region with positive charge, which is responsible for this atom’s directional and
stabilizing characteristics on the drug molecules [22]. It was envisaged that a bromine atom and
carbon-based substituents (methyl and aryl groups) on the 1H-indazole scaffold would increase
hydrophobic interactions and result in increased inhibitory effect against α-glucosidase. With
these assumptions in mind, series of 3-methyl-1H-indazoles substituted with a lipophilic
bromine atom at the 5-position and an aryl ring linked at postion-7 directly or via a π-conjugated
spacer (C=C or C≡C) were synthesized for evaluation of biological activity as potential anti-
diabetic agents. The 7-carbo–substituted 5-bromo-3-methyl-1H-indazoles have been prepared
3

via Suzuki-Miyaura cross-coupling of arylboronic and vinylboronic acids as well as
Sonogashira cross-coupling with phenylacetylene derivatives. The compounds were evaluated
via enzymatic assay in vitro for inhibitory effect against α-glucosidase activity and for
antioxidant potential using the DPPH radical scavenging assay. The most active compounds
against α-glucosidase were also evaluated for antigrowth effect against the breast MCF-7 cancer
cell line and also for cytotoxicity against the Hek293-T cells. Representative compounds from
each series were selected for a kinetic study and for molecular docking into the binding pocket
of the aforementioned enzyme to determine plausible protein-ligand interactions on a molecular
level.
2
. Results and Discussion
2
.1. Chemistry
The synthesis of the aforementioned compounds and their substitution patterns are
represented in Scheme 1 and Table 1, respectively. First 2-amino-5-bromo-3-iodoacetophenone
oxime (1) was subjected to methanesulfonyl chloride in the presence of trimethylamine in
dichloromethane at room temperature (RT) for 4 h to afford upon aqueous work-up and column
chromatography on silica gel 5-bromo-7-iodo-3-methylindazole (2). The analogous mixed
dihalogenated indazoles substituted with iodine at the C-3 position and bromine at the 5 or 7
positions have previously been transformed into polycarbo-substituted indazoles via palladium
catalyzed cross-coupling reactions [23]. Compound 2 was subjected to Suzuki-Miyaura cross-
coupling with various arylboronic and arylvinylboronic acids in the presence of
dichlorobis(triphenylphosphine)palladium(II) (PdCl (PPh ) ) as a source of active Pd(0)
2
3 2
species and potassium carbonate as a base in 4:1 toluene-ethanol (v/v) at 80 °C under reflux for
1
8 h. Aqueous work-up and purification by silica gel column chromatography afforded the
1
corresponding 7-aryl- 3a–g and arylvinyl substituted indazoles 3h–k (Scheme 1). The H NMR
4

1
3
and C NMR spectra of compounds 3a–k showed increased number of peaks in the aromatic
1
region. The H NMR spectra of 3h–k revealed additional set of doublets in the aromatic region
with coupling constant values J = 16.0 Hz typical for the trans vinyl moiety. Sonogashira cross-
coupling of compound 1 with terminal acetylenes in the presence of PdCl (PPh ) -copper iodide
2
3 2
catalyst mixture and cesium carbonate in aqueous tetrahydrofuran (THF) at room temperature
(RT) afforded after 3 h by aqueous work-up and purification by column chromatography on
1
silica gel, the homocoupled dimer and a single cross-coupled product 4a–f. The H NMR and
13C NMR spectra of the 7-arylethynyl substituted 5-bromo-3-methylindazoles 4a–f showed
increased number of peaks in the aromatic region confirming the formation of the cross-coupled
product. The alkynyl nature of compounds 4a–f was confirmed by the presence of additional
set of singlets in the region δ 83.0–86.0 ppm and δ 93.0–96.0 ppm corresponding to the carbon
2
atoms of the triple bond. Exclusive substitution at the Csp –I bond was confirmed in the
molecular ion region of the mass spectra of compounds 3 and 4 of M+ and M+2 in the ratio 1:1
7
9
81
due to the presence of the Br and Br isotopes in the molecular ions. The 7-phenyl- and 7-
styryl- substituted derivatives 3a and 3h were formed in comparable yield, 65% and 67%,
respectively. A similar trend was observed for the Suzuki-Miyaura cross-coupling of 2 to afford
7
-(4-methoxyphenyl)- 3e and the 7-(4-methoxystyryl)- derivative 3k in 52% and 50% yield,
respectively. Moderate yields were observed for the 7-(4-fluorostyryl)- and 7-(4-chlorostyryl)-
substituted derivatives 3i (56%) and 3j (54%) when compared to the corresponding 7-(4-
fluorophenyl)- and 7-(4-chlorophenyl)- substituted analogues 3b (81%) and 3c (66%),
respectively. Compound 3e substituted with a strongly electron withdrawing trifluoromethoxy
group at the para position of the phenyl ring and 3g substituted with a combination of strongly
electronegative 3-chloro and 4-fluoro atoms on the phenyl ring were isolated in 50% and 52%
yield, respectively. However, relatively high yield (64%) was observed for 3f substituted with
a strongly electron-withdrawing aminocarbonyl group on the meta position of the phenyl ring.
The presence of 3-(fluorophenyl)ethynyl- or 3-(chlorophenyl)ethynyl- group at the 7-position
5

of the indazole scaffold of 3b or 3d resulted in relatively higher yield (76% and 72%,
respectively) than the isomeric 4-(halogenophenyl)ethynyl derivatives 4c (64%) and 4e (69%).
Increased yield was observed for 4f (78%) bearing a strongly electron delocalizing methoxy
group at the para position of the phenyethynyl moiety.
Scheme 1: Suzuki-Miyaura and Sonogashira cross-coupling of 2 to afford 3a–k and 4a–f,
respectively.
NOH
Br
CH3
NH2
I
1
CH SO Cl, Et N, CH Cl
2
3
2
3
2
RT, 4 h
CH3
N
CH3
N
Br
CH3
Br
Br
R
H, PdCl (PPh ) , CuI
2 3 2
RB(OH) , PdCl (PPh ) , PCy
+
R
R
2
2
3 2
3
N
N
N
H
N
o
Cs CO , toluene-EtOH, 80 C, 5 h
Cs CO , THF (aq), RT, 3 h
H
2
3
2
3
H
R
I
R
3
a–k
2
4a–f
dimers
Table 1: Substitution patterns of compounds 3a–k and 4a–f.
3
4
: R =
: R =
C H - (3a); 4-FC H - (3b); 4-ClC H - (3c); 4-MeOC H - (3d); 4-CF OC H - (3e);
6 5 6 4 6 4 6 4 3 6 4
3
-NH C(O)C H - (3f); 3-Cl,4-FC H - (3g); C H CH=CH- (3h); 4-FC H CH=CH-
2 6 4 6 3 6 5 6 4
(
3i); 4-ClC H CH=CH- (3j); 4-CH OC H CH=CH- (3k)
6 4 3 6 4
C H - (4a); 3-FC H - (4b); 4-FC H - (4c); 3-ClC H - (4d); 4-ClC H - (4e); 4-
6
5
6
4
6
4
6
4
6
4
MeOC H - (4f)
6
4
2
.2. Biological evaluation of compounds 2, 3a–k and 4a–f.
Compounds 2, 3a–k and 4a–f were evaluated for inhibitory effect in vitro through
enzymatic assays against α-glucosidase activity to correlate between both structural variations
and biological activity. Since free radical-mediated oxidative stress is also implicated in the
early stages of T2D and cancer [24], these compounds were evaluated for antioxidant activity
in the DPPH radical scavenging assay. Moreover, the most active compounds against α-
6

glucosidase were also evaluated for potential cytotoxicity against the breast MCF-7 cancer cell
line and the normal Hek293-T cells. Structure activity relationship has been rationalized in
terms of substitution pattern on the phenyl group and its connectivity to the 7-position of the
indazole framework.
2
.2.1. α-Glucosidase inhibitory activity and antioxidant potential of 2, 3a–k and 4a–f
The inhibitory activities of compounds 2, 3a–k and 4a–f against α-glucosidase were
evaluated using the Saccharomyces cerevisiae form of enzyme against acarbose, a well-known
inhibitor of α-glucosidase [8]. The results are expressed as IC values, i.e., the concentration
5
0
of each sample required inhibit 50% of the α-glucosidase activity (Table 2). 5-Bromo-7-iodo-
-methylindazole (2) was found to be less active against this enzyme when compared to the
reference standard acarbose (IC = 0.82 μM) with an IC value of 11.69 μM. The three series
3
5
0
50
of 7-carbo substituted derivatives exhibited variable degree of activity with IC values ranging
5
0
from 0.42 to 51.50 μM when compared with the standard acarbose having IC value 0.82 μM.
5
0
Replacement of iodine with a phenyl group resulted in increased inhibitory effect for 3a against
α-glucosidase with an IC value of 0.53 μM. Except for the 7-(4-trifluoromethoxyphenyl)
5
0
substituted derivative 3e with an IC value of 0.75 μM, derivatives 3b–d and 3f substituted at
5
0
the para position of the 7-phenyl ring with an electron donating (-OCH ) or withdrawing (F, Cl
3
or NH C(O)-) group were found to be less active against this enzyme. The observed increased
2
activity for compound 3e is probably due to the presence of the trifluoromethoxy (–OCF3)
group, which is both more electron withdrawing and lipophilic than its methoxy analogue and
also far more lipophilic than the halogen atoms [25]. This group has previously been found to
increase biological activity of the molecule due to its enhanced lipid solubility and metabolic
stability [25]. Moreover, the strong electron-withdrawing effect of the halogen atoms helps in
forming halogen and/or hydrogen bonds that help to increase the number of interactions of drug
molecules with their protein targets, thereby enhancing biological activity [26]. The presence
of 3-chloro-4-fluorophenyl group on the indazole scaffold of 3g resulted in moderate inhibitory
7

activity for this compound with an IC value of 5.49 μM. The presence of the carbon–carbon
5
0
double bond linker in the series 3h–k resulted in slightly increased inhibitory effect for the 7-
styryl substituted derivative 3h (IC = 0.50 μM) than 3a (IC = 0.53 μM) in which the phenyl
5
0
50
substituent is linked directly to the fused benzo ring of the indazole scaffold. The 4-
fluorophenylstyryl substituted derivative 3i behaved like the 4-fluorophenyl substituted
derivative 3b and both compounds were found to be less active against this enzyme with IC50
values of 51.51 μM and IC = 51.07 μ, respectively. The presence of the carbon–carbon double
5
0
bond linker between the indazole scaffold and 4-chlorophenyl group resulted in moderate
inhibitory effect of 3j (IC = 7.73 μM) when compared to the 4-chlorophenyl substituted
5
0
analogue 3c (IC = 22,68 μM). The presence of the more polar lipophilic methoxystyryl (R =
5
0
4
-(CH OC H )CH=CH-) group in compound 3k resulted in increased inhibitory effect for this
3 6 4
compound with an IC value of 0.80 μM, which is comparable to that of acarbose (IC = 0.82
5
0
50
μM). The presence of a carbon-carbon triple bond between the indazole scaffold and aryl group
resulted in variable degree of activity within the series 4a–f. The parent compound 4a was
found to be the most active within this series and among all the compounds tested with an IC50
value of 0.43 μM. The presence of a -C≡C- bridge also resulted in significant inhibitory activity
for the 3- and 4-halogenophenyl substituted derivatives 4b, 4c, 4d and 4e against α-glucosidase
with IC values 7.14, 5.27, 3.42 and 4.87 μM, respectively. However, loss of activity was
5
0
observed for compound 4f with a carbon–carbon triple bond linker between the indazole
scaffold and the 7-(4-methoxyphenyl) group. Within the three series of compounds, derivatives
in which the phenyl ring is linked to the 7-position of the indazole scaffold directly (3a: R =
C H -) or via a π-conjugated spacer, viz., C=C (3h: R = C H CH=CH-) or ) or C≡C (4a: R =
6
5
6
5
C H C≡C-) bond have been found to exhibit increased inhibitory effect against α-glucosidase
6
5
and the trend is as follows: 4a (IC = 0.42 μM) > 3h > (IC = 0.50 μM) > 3a (IC = 0.53 μM).
5
0
50
50
Famuyiwa et al. have recently demonstrated the link between anti-diabetic and antioxidant
activities of drugs through in vivo experiments and theoretical calculations using protocatechuic
8

acid and glibenclamide as models [27]. Increased reactive oxygen species (ROS) production in
the liver and adipose tissue of high fat diet-fed mice has been found to be linked with insulin
resistance [28], and this effect was reversed by the use of antioxidants [29]. Indazole and its
derivatives were previously found to significantly inhibit 2,2-diphenyl-1-picrylhydrazyl
(DPPH) free radical generation in a concentration dependent manner [30]. This literature
precedent encouraged us to evaluate compounds 2, 3a–k and 4a–f for free radical scavenging
activity in vitro using the DPPH free-radical scavenging method (Table 2). The natural
antioxidants ascorbic acid was used as a positive control [31]. Compound 2 was found to
exhibit significant antioxidant effect when compared to ascorbic acid (IC = 5.20 µM) with an
5
0
IC value of 6.79 µM. Compound 3a which is one the highest active derivatives against α-
5
0
glucosidase (IC = 0.53 μM) within the series 3a–k also exhibited increased free radical
5
0
scavenging activity with an IC50 value of 4.99 μM. The 7-(4-fluorophenyl) substituted
derivative 3b (IC = 50.52 μM) was found to exhibit reduced antioxidant properties when
5
0
compared to the 7-(4-chlorophenyl) analogue 3c (IC = 0.91 μM). No antioxidant activity was
5
0
observed for the 4-methoxyphenyl substituted derivative 3d (IC = 49.40 μM) whereas the
5
0
analogous 7-(4-trifluoromethylphenyl) analogue 3e exhibited increased antioxidant activity
when (IC = 4.84 μM) when compared to the reference standard. Compound 3f also
exhibited reduced antioxidant activity with an IC value of 26.33 μM. A combination of 3-
5
0
5
0
chloro and 4-fluoro atoms on the phenyl substituent of 3g resulted in increased antioxidant
activity for this compound (IC = 4.62 μM). The presence of a carbon-carbon double bond
5
0
bridge between the phenyl ring and the indazole scaffold resulted in significant antioxidant
effect for 3h (IC = 7.10 μM) though less when compared to 3a (IC = 4.99 μM) in which the
5
0
50
phenyl ring is linked directly to the heterocyclic framework. Compounds 3i and 3k substituted
with moderately and strongly electron delocalizing 4-fluorophenyl or 4-methoxyphenyl groups
at position-7 were found to exhibit moderate antioxidant activity with IC values 15.30 μM
5
0
and 14.46 μM, respectively. The 2-chlorostyryl derivative 3j was found to be the most active
9

within the styryl substituted series 3j–k, more so than ascorbic acid with an IC value of 1.06
5
0
μM. The presence of a carbon-carbon triple bond linker between the phenyl ring and the
indazole scaffold resulted in significantly increased antioxidant activity for 4a (IC = 1.00 μM).
5
0
The presence of a halogen atom on the meta position of the 7-phenyl ring resulted in reduced
antioxidant activities for compounds 4b and 4d with IC values of 88.66 μM and 66.20 μM,
5
0
respectively. However, significant antioxidant activity was observed for the isomeric 7-(4-
fluorophenyl) substituted derivative 4c with an IC value of 7.96 μM. The presence of the
5
0
strongly electron delocalizing 4-methoxyphenyl group at the 7-position of the indazole
framework, on the other hand, resulted in increased antioxidant activity for 4f with an IC value
5
0
of 4.92 μM. Compounds 3a, 3e, 3h and 4a exhibit dual anti-α-glucosidase and antioxidant
properties. Though strongly inhibiting against α-glucosidase, compound 3k was found to
exhibit moderate antioxidant effect when compared to ascorbic acid with an IC value of 14.46
5
0
μM.
Literature survey revealed several studies highlighting the fact that T2D is a risk factor for
cancer, with some oral hypoglycemic drugs such as metformin therapy associated with a
significant reduction in cancer incidence, especially gastrointestinal and breast cancers. Since
breast cancer is related to T2D [7], the most active compounds against α-glucosidase, namely,
3
a, 3e, 3h, 3k and 4a were also screened for antigrowth effects in vitro against the breast MCF-7
cancer cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
tetrazolium reduction assay. Doxorubicin (Doxo) was used as a positive control for this assay
because this drug was previously found to inhibit MCF-7 cell proliferation [32]. The
preliminary results of this assay revealed compound 3a, 3h and 4a to exhibit moderate to
significant cytotoxicity against this cell line when compared to Doxo (IC = 36.03 μM) and
5
0
the trend is as follows: 4a (IC = 35.13 μM) > 3h (IC = 39.33 μM) > 3a (IC = 49.90 μM).
5
0
50
50
These compounds were also evaluated for toxicity against the human embryonic kidney
Hek293-T) cell line using the clinical drug Doxo as reference standard. The IC values of 3a,
(
5
0
1
0

3
e, 3h, 3k and 4a against the normal Hek293-T cells were found to be 19.01 ± 0.14 μg/mL,
3.01 ± 0.22 μg/mL, 24.00 ± 0.18 μg/mL, 25.01 ± 0.15 μg/mL and 18.03 ± 0.09 μg/mL,
2
respectively, as compared to the clinical drug, doxorubicin (IC = 5.02 ± 0.07 μg/mL). The
5
0
non-cancer–derived Hek293-T cell line has been found to be highly sensitivity to free Doxo
33]. Compounds 3a, 3e, 3h, 3k and 4a exhibited reduced cytotoxicity against Hek293-T cells,
[
which suggests good cytocompatability for the test compounds.
Table 2: α-Glucosidase inhibitory and antioxidant activities of 2–4, and cytotoxicity against
3
a, 3e, 3h, 3k and 4a.
IC values (µM)
5
0
2
–4
2
α-Glucosidase
11.69 ± 0.010
0.53 ± 0.013
51.07 ± 0.001
22.68 ± 0.001
13.52 ± 0.006
0.75 ± 0.006
22.7 ± 0.012
5.49 ± 0.003
0.50 ± 0.010
51.51 ± 0.013
7.73 ± 0.004
0.80 ± 0.009
0.42 ± 0.019
7.14 ± 0.009
5.27 ± 0.004
DPPH
MCF-7 (*SI)
6.79 ± 0.003
4.99 ± 0.006
50.52 ± 0.001
0.91 ± 0.004
49.40 ± 0.003
4.84 ± 0.009
26.33 ± 0.005
4.62 ± 0.005
7.10 ± 0.004
15.30 ± 0.003
1.06 ± 0.013
14.46 ± 0.009
1.00 ± 0.007
88.66 ± 0.001
7.96 ± 0.001
-
3
a
49.90 ± 0.02 (2.62)
3
b
-
3
c
-
3
d
-
3
e
71.94 ± 0.05 (3.12)
3
f
-
3
g
-
3
h
39.33 ± 0.02 (1.64)
3
i
-
3
j
-
3
k
53.25 ± 0.05 (2.13)
4
a
35.13 ± 0.02 (1.95)
4
b
-
-
4
c
1
1

4
d
3.42 ± 0.002
4.87 ± 0.004
23.71 ± 0.004
0.82 ± 0.006
-
66.20 ± 0.01
27.83 ± 0.01
4.92 ± 0.01
-
-
4
e
-
4
f
-
Acarbose
Ascorbic acid
Doxorubicin
-
5.20 ± 0.006
-
-
-
36.03 ± 0.02 (7.18)
*
Selectivity index (SI) = IC MCF-7/IC Hek293-T
50 50
Kinetic studies were undertaken on compounds 3a, 3h and 4a in order to elucidate the
plausible mechanism of inhibition of α-glucosidase by these compounds.
2
.2.4 Kinetic studies of 3a, 3h and 4a against α-glucosidase
The mechanism of action of compounds 3a, 3h and 4a against α-glucosidase was evaluated
by building the Lineweaver–Burk double reciprocal plots and the Dixon plots at increasing
inhibitor and substrate concentrations (0.1, 0.5, 2.5 and 5 µM). Results of the Lineweaver-Burke
plots represented in Figures 1a, 2a and 3a show relatively unchanged Michaelis constant (Km)
values of 0.020 ± 0.008, 0.017 ± 0.007, 0.019 ± 0.009 for 3a, 3h and 4a, respectively. The
maximum velocity of reaction (Vmax) values for the three compounds decrease with increasing
inhibitor concentration (0.02 µM to 1µM) and these changes are as follows: 0.11 ± 0.013 to
0
.022 ± 0.011 for 3a, 0.10 ± 0.021 to 0.053 ± 0.021 for 3h, and 0.10 ± 0.034 to 0.01 ± 0.004
for 4a. Unchanged K and decreasing V values are indicative of non-competitive mode of
m
max
inhibition. The Dixon plots for 3a (Figure 1b), 3h (Figure 2b) and 4a (Figure 3b) all display
intercepting lines above the x-axis, which is indicative of competitive mode of inhibition. This
observation then supports mixed inhibition mode for 3a, 3h and 4a suggesting that they bind to
the active site and elsewhere on the enzyme. The calculated K values for 3a, 3h and 4a are 0.13
i
±
0.05, 0.13 ± 0.01 and 0.17 ± 0.007, respectively.
1
2

a
1
1
35
15
9
7
5
3
1
-
5
5
5
5
5
5
-
55
-5
45
/[S]
95
145
1
.
b
1
1
35
15
95
75
55
35
15
-5
-
0. 35
0. 15
0. 65
INHIBITION]
1. 15
1. 65
[
Figure 1: Lineweaver Burke (1a) and Dixon (1b) plots for compound 3a. In the Lineweaver
Burke plot inhibitor concentrations 0 µM, 0.02 µM, 0.2 µM and 0.5 µM are represented by
blue, orange, grey and yellow symbols and lines, respectively. For the Dixon plot varying
substrate concentrations of 0.01 µM, 0.1 µM, 0.5 µM and 1 µM are represented by yellow,
grey, orange and blue symbols and lines respectively.
1
3

55
50
45
40
35
30
25
20
15
10
5
a
-
60
-10
40
90
140
1
/[S]
b
5
4
3
2
1
5
5
5
5
5
5
-
0.7
-0.2 -5
0.3
0.8
1.3
[INHIBITOR]
Figure 2: Lineweaver Burke (2a) and Dixon (2b) plots for compound 3h. In the Lineweaver
Burke plot inhibitor concentrations 0 µM, 0.01 µM, 0.1 µM, 0.5 µM and 1 µM are represented
by red, blue orange, grey and yellow symbols and lines, respectively. For the Dixon plot varying
substrate concentrations of 0.01 µM, 0.1 µM, 0.5 µM and 1 µM are represented by blue, orange,
grey and yellow symbols and lines respectively.
1
4

3
2
2
1
1
20
70
20
70
20
a
7
2
0
0
-
50
-30 0
50
/[S]
100
150
1
b
2
2
1
1
80
30
80
30
8
3
0
0
-20
-
0. 42
0. 08
0. 58
INHIBITION]
1. 08
1. 58
[
Figure 3: Lineweaver Burke (3a) and Dixon (3b) plots for compound 4a. In the Lineweaver
Burke plot inhibitor concentrations 0 µM, 0.02 µM, 0.2 µM, 0.5 µM and 1 µM are represented
by yellow, red, blue, grey, orange and red symbols and lines, respectively. For the Dixon plot
varying substrate concentrations of 0.01 µM, 0.1 µM, 0.5 µM and 1 µM are represented by
blue, orange, yellow and grey symbols and lines, respectively.
To elucidate the protein-ligand interactions at molecular level, we performed a docking
study on the most active compounds 3a, 3e, 3h, 3k and 4a against the human lysosomal acid
α-glucosidase.
2
.2.5 Molecular docking studies of 3a, 3e, 3h, 3k and 4a
1
5

The X-ray crystal structure of human lysosomal acid α-glucosidase complexed with
acarbose was downloaded from Protein Data Bank (PDB code: 5NN8). Acarbose was docked
into the active site of this crystal and the top scoring docked pose with the calculated binding
free energy (BE) of -88.6514 kcal/mol was applied as starting point for molecular dockings
into α-glucosidase binding pocket. Interaction mode of acarbose with a human lysosomal acid-
α-glucosidase revealed several hydrogen bonding interactions of this drug with the protein
residues (Asp282, Asp518, Asp616, His674 and Trp481) in the active site of α-glucosidase
(Figure 4a). Compounds 3a, 3e, 3h, 3k and 4a were docked individually into the same active
pocket site of α-glucosidase as acarbose, and their 2-dimensional docking poses showing
interactions with the α-glucosidase protein residues and their distances are represented in
Figure 4. The calculated BEs for 3a, 3e, 3h, 3k and 4a are estimated to be -36.2257, -48.1500,
-36.0903, -32.8335 and -41.0423 kcal/mol, respectively. These values are higher than that of
acarbose, presumably because this reference drug has higher number of hydrogen bonding
interactions with protein residues in the active site of α-glucosidase. The docking poses of 3a
(Figure 4b) and 3e (Figure 4c) show the involvement of their heterocyclic ring in π-alkyl
interaction with Ala284. The latter protein residue and Leu283 (3a) or Leu650 (3e) are involved
in π-alkyl and alkyl interaction with the 3-methyl group, respectively. The 5-bromo atom of 3a
is involved in alkyl interaction with the protein residue Phe525. Hydrogen bonding interactions
are predicted between the active site residue Asp616 and NH of both compounds. The phenyl
ring in both cases is involved in π-π T-shaped interaction with Trp481, and also π-sulfur
interaction with the protein residues Met519 and Asp616. Additional hydrogen bonding and
halogen bonding interactions predicted between the trifluoromethoxy group with Asp404 and
His674 help to increase the number of interactions with their protein targets, thereby enhancing
biological activity of 3e more so than the analogous 4-methoxyphenyl substituted derivative
3
d. These additional hydrophilic interactions probably account for the lower calculated BE
value of 3e among the test compounds. This compound has lower calculated BE value than 3a,
1
6

which exhibited higher inhibitory effect against α-glucosidase activity among the 7-aryl
substituted derivatives. The calculated binding energies obtained through molecular docking
have been reported not to correlate well with the biological response observed in the in vitro
tests [34].
The indazole scaffold of 3h (Figure 4d) is involved in π-π T-shaped interaction with
Trp481 and π-anion interactions with Asp616. There is π-sulphur and sulphur-x interactions
between the fused benzene ring and N-1 with Met519, respectively. π-Alkyl interactions are
predicted between the heterocyclic ring and Met519, and also between the styryl ring and
Ala555. The styryl ring is also involved in π-π T-shaped interaction with Phe525. A hydrogen
bond is predicted between the protein residue Asp282 and NH. The docking pose of 3k (Figure
4
e), on the other hand, shows the involvement of the heterocyclic ring in π-donor hydrogen
bond interaction with the protein residue Ala284, which is also involved in π-alkyl interaction
with the fused benzo ring. The 5-bromo atom of 3k is involved in an alkyl interaction with
Ala555, while its 3-methyl group is involved in a π-alkyl interaction with Leu283 and Ala284.
The 4-methoxystyryl ring is involved in π-π T-shaped interaction with Trp481, π-sulphur
interaction with Met519 and π-anion interactions with Asp616. Carbon hydrogen bonding
interactions are predicted between the methoxy group of 3k and protein residues Asp404 and
Asp518. No hydrogen bonding is predicted between 3k and the protein residues in the active
site, which probably accounts for the high calculated BE value (-32.8335 kcal/mol) when
compared to 3h (-36.0903 kcal/mol). The enzyme-ligand complex for 3k is stabilized by
hydrophobic interactions (alkyl, π-alkyl and π-π T-shaped) and carbon hydrogen bonding
interaction. The docking pose of 4a, which is represented in Figure 5f shows both rings of the
planar indazole scaffold involved in π-anion interaction with Asp616, and also π-π T-shaped
interaction with Trp481. Met519 is involved in π-sulfur and sulphur-x interactions with the
fused benzo ring and N-1, respectively. The heterocyclic ring of this compound is involved in
π-alkyl interaction with Met519, while the 3-methyl group is involved in π-alkyl interaction
1
7

with Phe649. π-π T-shaped interaction and π-alkyl interaction exists between the phenyl ring
with Phe525 and Ala555, respectively. Hydrogen bonding is predicted between NH and
Asp282 as well as between N-2 and Arg600. Increased number of hydrophobic and hydrophilic
interactions correlate well with enhanced inhibitory activity of this compound as well as its
lowest calculated BE among the test compounds. Hydrophobic (alkyl, π-alkyl and π-π T
shaped) and electrostatic (π-sulfur and/or π-anion) interactions, which are retained by these
indazoles probably help anchor these compounds in the binding site of α-glucosidase, thereby
enhancing their biological activity. These hydrophobic and electrostatic interactions, which are
not established in acarbose–α-glucosidase complex presumably account for the higher activity
of these indazole derivatives.
Acarbose
3a
3
e
3h
1
8

3
k
4a
Figure 4. The predicted binding conformations of acarbose (4a) compounds 3a (4b), 3e (4c),
h (4d), 3k (4e) and 4a (4f and g) in the active site of α-glucosidase. Interactions with key
3
amino acids are indicated with dashed lines (purple for hydrophobic contacts, green for H-
bonds, and orange for charge attraction).
3
. Conclusions
From a structure-activity-relationship perspective, among all the 7-carbo substituted 5-
bromo-3-methylindazoles tested for the inhibition of α-glucosidase, derivatives in which the
phenyl ring is linked directly (3a) or through a π-conjugated spacer such as C=C (3h) or C≡C
(4a) bond exhibited increased inhibitory effect against α-glucosidase than all the other
analogues and the trend is as follows: 4a > 3h > 3a. The enzyme-ligand complexes appear to
be stabilized by hydrophobic (alkyl, π-alkyl and π-π T-stacked), electrostatic and hydrophilic
(hydrogen and/or halogen) bonding interactions. The presence of 5-bromo, 3-methyl and
conjugated carbon-based substituent at the 7-position help to increase the number of
hydrophobic interactions with protein residues in the active site of the human lysosomal acid
α-glucosidase, thereby enhancing biological activity of these compounds. These compounds
were also found to exhibit increased antioxidant activity in the DPPH free radical scavenging
assay and the trend is as follows 4a (IC = 1.00 µM) > 3a (IC = 4.99 µM) > 3h (IC = 7.10
5
0
50
50
1
9

µM). Compounds 3a, 3h and 4a were also found to exhibit moderate to increased cytotoxicity
against the breast MCF-7 cancer cell line. Compound 4a exhibited strong inhibition of the α-
glucosidase enzyme than acarbose, the most robust antioxidant activity in comparison with
ascorbic acid, and significant cytotoxicity against the MCF-7 cell line than doxorubicin. This
compound exhibits multiple biological activities, which carries great potential for the therapy
and prevention of diabetes and oxidative stress as well as cancer.
4
.
Experimental
4
.1. General
The melting points of the prepared compounds were recorded on a Thermocouple digital
melting point apparatus (Mettler Toledo LLC, Columbus, Ohio, USA) and are uncorrected.
Their IR spectra were recorded as powders on a Bruker VERTEX 70 FT-IR Spectrometer
(Bruker Optics, Billerica, MA, USA) with a diamond ATR (attenuated total reflectance)
accessory by using the thin-film method. The Merck kieselgel 60 (0.063–0.200 mm) (Merck
1
KGaA, Frankfurt, Germany) was used as stationary phase for column chromatography. The H-
1
3
and C-NMR spectra of the prepared compounds were obtained as DMSO-d solutions using
6
the Varian Mercury 300 MHz NMR spectrometer (Agilent Technologies, Oxford, UK) and the
chemical shifts are quoted relative to the tetramethylsilane (TMS) peak. The high-resolution
mass spectra were recorded using a Waters Synapt G2 Quadrupole Time-of-flight mass
spectrometer (Waters Corp., Milford, MA, USA) at an ionization potential of 70 eV. The
synthesis and analytical data for 2-amino-5-bromo-3-iodoacetophenone, which has been used
as a precursor in this investigation were reported in our previous investigation [35].
4
.2.1. Synthesis of 1-(2-amino-5-bromo-3-iodophenyl)ethanone oxime (1)
A stirred mixture of 2-amino-5-bromo-3-iodoacetophenone (2.00 g, 5.90 mmol) in pyridine
(30 mL) was treated slowly with ammonium hydroxyl chloride (0.49 g, 7.05 mmol), and then
2
0

heated under reflux for 2 h. Upon completion of the reaction (thin layer chromatography, TLC,
monitoring), the mixture was allowed to cool and then quenched with an ice cold water. The
resultant precipitate was filtered to afford 1 as a white solid (1.80 g, 80%), mp. 174–176 °C;
-1 1
νmax (ATR) 680, 755, 860, 1002, 1232, 1364, 1425, 1526, 1738, 2833, 3207, 3364 cm ; H-
NMR (DMSO-d ) 3.94 (3H, s, CH ), 6.24 (2H, s, NH ), 7.09 (1H, s , H-4), 7.36 (1H, s, H-6),
6
3
2
1
3
1
1.06 (1H, s, NH); C-NMR (DMSO-d ) 12.9, 86.9, 106.9, 120.5, 131.7, 140.3, 145.8, 155.1;
6
+
+
79
HRMS (ES ): m/z [M + H] calcd for C H NO BrI: 353.8865; found 353.8863. Anal. Calcd
8
9
for C H N OBrI: C, 27.07; H, 2.27; N, 7.89. Found: C, 27.04; H, 2.30; N, 7.93.
8
8
2
4
.2.2. Synthesis of 5-bromo-7-iodo-3-methyl-1H-indazole (2)
A stirred mixture of 1-(2-amino-5-bromo-3-iodophenyl)ethanone oxime (2.00 g, 7.88
mmol) and triethylamine (1.60 g, 15.8 mmol) in dichloromethane (100 mL) at 0 °C was treated
dropwise with a solution of methanesulfonyl chloride (1.01 g, 8.82 mmol) in dichloromethane
(5 mL). The reaction was allowed to stir at room temperature (RT) for 4 h with thin layer
chromatography (TLC) monitoring. Upon completion, the reaction mixture was quenched with
an ice cold water. The aqueous phase was extracted with ethyl acetate (3 x 50 mL) and the
combined organic layers were dried over anhydrous Na SO . The salt was filtered off and the
2
4
organic phase was concentrated under reduced pressure on a rotatory evaporator. The residue
was purified by silica gel column chromatography eluting with 4:1 toluene-ethyl acetate (v/v)
to afford 2 as a white solid (1.50 g, 58%), mp. 171–174 °C; νmax (ATR) 517, 601, 666, 703,
-1 1
8
53, 907, 990, 1270, 1393, 2342, 2360, 2916, 3250 cm ; H-NMR (DMSO-d ) 3.278 (3H, s,
6
1
3
CH ), 8.66 (1H, d, J = 1.2 Hz, H-4), 8.82 (1H, d, J = 1.2 Hz, H-6), 13.84 (1H, s, NH); C-NMR
3
+
+
(
DMSO-d ) 12.30, 76.38, 112.2, 122.9, 123.9, 136.5, 142.6, 142.7; HRMS (ES ): m/z [M + H]
6
7
9
calcd for C H N BrI: 336.8759; found 336.8838. Anal. Calcd for C H N BrI: C, 28.52; H,
8
7
2
8
6
2
1
.79; N, 8.31. Found: C, 28.49; H, 1.75; N, 8.33.
.3. Typical procedure for the Suzuki-Miyaura cross-coupling of 2 to afford 3a–j.
4
2
1

A mixture of 2 (1 equiv.), arylboronic acid (1.2 equiv.), PdCl (PPh ) (5 mol %), K CO
3
2
3 2
2
(1.5 equiv.) in 4:1 toluene-ethanol (v/v, 15 mL) was placed in a 2 necked flask equipped with
a stirrer bar, rubber septum and a condenser. The mixture was flushed with argon gas for 5 min
and a balloon filled with argon gas was connected to the top of a condenser. The mixture was
o
then stirred at 80 C for 5 h, cooled to room temperature and then quenched with an ice cold
water. The product was extracted with chloroform and the combined organic layers were dried
with MgSO , filtered and purified by silica gel column chromatography to afford 3 as a solid.
4
The following compounds were prepared in this fashion:
4
.3.1. 5-Bromo-3-methyl-7-phenyl-1H-indazole (3a)
Brown solid (0.22 g, 65%), mp. 162–165 °C; νmax (ATR) 603, 754, 851, 920, 987, 1257,
-1 1
1
316, 1478, 2853, 2922, 3234 cm ; H-NMR (DMSO-d ) 2.48 (3H, s, CH ), 7.52–7.45 (2H,
6
3
m, Ph), 7.55–7.52 (2H, m, H-4ʹ and H-6), 7.66 (2H, d, J = 3.0 Hz, Ph), 7.69 (1H, d, J = 1.8 Hz,
1
3
H-4), 12.95 (1H, s, NH); C-NMR (DMSO-d ) 12.1, 112.7, 121.9, 125.3, 126.5, 127.9, 128.7,
6
+
+
79
1
2
5
4
29.3, 129.6, 136.6, 137.8, 141.9; HRMS (ES ): m/z [M + H] calcd for C H N Br:
14 12 2
87.0106; found 287.0184. Anal. Calcd for C H N Br: C, 58.56; H, 3.86; N, 9.76, Found: C,
1
4
11
2
8.55; H, 3.81; N, 9.73.
.3.2. 5-Bromo-7-(4-fluorophenyl)-3-methyl-1H-indazole (3b)
Brown solid (0.29 g, 81%) mp. 205–207 °C; νmax (ATR) 602, 806, 835, 925, 1161, 1226,
-1 1
1
314, 1478, 1605, 2853, 2922, 3235 cm ; H-NMR (DMSO-d ) 2.57 (3H, s, CH ), 7.54 (2H,
6 3
dt, JHH = 8.7 Hz and J = 9.6 Hz, H-3ʹ, 5ʹ), 7.63 (1H, d, J = 1.2 Hz, H-6), 7.87 (2H, dt, JHH
=
HF
1
3
8
.7 Hz and JHF = 5.1 Hz, H-2′, 6′), 7.93 (1H, d, J = 1.2 Hz, H-4), 13.0 (1H, s, NH); C-NMR
2
3
(
DMSO-d ) 12.1, 112.7, 116.4 (d, J = 20.6 Hz), 122.0, 125.2, 125.5, 128.0, 130.8 (d, J =
6 CF CF
4
1
+
8
.0 Hz), 132.9 (d, J = 2.7 Hz), 137.9, 141.9, 162.5 (d, J = 243.8 Hz); HRMS (ES ): m/z
CF CF
+
19 79
[
M + H] calcd for C H N F Br: 305.0011; found 305.0090. Anal. Calcd for C H N FBr:
14 11 2 14 10 2
C, 55.10; H, 3.30; N, 9.18, Found: C, 55.00; H, 3.17; N, 9.20.
.3.3. 5-Bromo-7-(4-chlorophenyl)-3-methyl-1H-indazole (3c)
4
2
2

White solid (0.25 g, 66%), mp. 199–201 °C; νmax (ATR) 693, 827, 923, 984, 1092, 1254,
-1 1
1
315, 1476, 1595, 2852, 2919, 3247 cm ; H-NMR (DMSO-d ) 2.48 (3H, s, CH ), 7.47 (1H,
6 3
d, J = 1.2 Hz, H-6), 7.56 (2H, d, J = 8.7 Hz, H-3′, 5′), 7.68 (2H, d, J = 8.7 Hz, H-2′, 6′), 7.82
1
3
(
1H, d, J = 1.2 Hz, H-4), 13.01 (1H, s, NH); C-NMR (DMSO-d ) 12.1, 112.7, 117.7, 122.6,
6
+
+
1
24.2, 125.3, 129.5, 129.6, 130.5, 130.9, 137.8, 142.0; HRMS (ES ): m/z [M + H] calcd for
3
5
79
C H N Cl Br: 320.9716; found 320.9794. Anal. Calcd for C H N ClBr: C, 52.29; H,
1
4
11
2
14 10
2
3
4
.13; N, 8.71, Found: C, 52.29; H, 3.10; N, 8.74.
.3.4. 5-Bromo-7-(4-methoxyphenyl)-3-methyl-1H-indazole (3d)
White solid (0.20 g, 52%), mp. 179–182 °C; νmax (ATR) 700, 811, 836, 925, 990, 1033,
-
1 1
1
3
177, 1247, 1439, 1503, 1605, 2852, 2921, 3246 cm ; H-NMR (DMSO-d ) 2.48 (3H, s, CH ),
6 3
.81 (3H, s, OCH ), 7.06 (2H, d, J = 8.7 Hz, H-3′, 5′), 7.39 (1H, d, J = 1.2 Hz, H-6), 7.61 (2H,
3
1
3
d, J = 8.7 Hz, H-2′, 6′), 7.88 (1H, d, J = 1.2 Hz, H-4), 12.91 (1H, s, NH); C-NMR (DMSO-
d6) 12.1, 55.7, 112.7, 114.7, 114.9, 121.3, 125.2, 127.4, 128.9, 129.9, 137.9, 141.8, 159.8;
+
+
79
HRMS (ES ): m/z [M + H] calcd for C H N O Br: 317.0211; found 317.0290. Anal. Calcd
1
5
14
2
for C H N OBr: C, 56.80; H, 4.13; N, 8.83; Found: C, 56.69; H, 4.12; N, 8.82.
1
5
13
2
4
.3.5. 5-Bromo-7-(4-trifluoromethoxyphenyl)-3-methyl-1H-indazole (3e)
White solid (0.22 g, 50%), mp. 150–153 °C; νmax (ATR) 693, 836, 924, 1151, 1207, 1253,
-
1 1
1
8
479, 1503, 2852, 2922, 3248 cm ; H-NMR (DMSO-d ) 2.55 (3H, s, CH ), 7.39 (2H, d, J =
6 3
.7 Hz, H-3′, 5′), 7.48 (1H, d, J = 1.8 Hz, H-6), 7.66 (2H, d, J = 8.7 Hz, H-2′, 6′), 7.82 (1H, d,
1
3
J = 1.8 Hz, H-4), 10.50 (1H, s, NH); C-NMR (DMSO-d ) 12.0, 113.7, 121.9, 122.2 (q, J =
6
CF
+
2
56.5 Hz), 122.2, 124.6, 124.9, 128.9, 129.3, 135.4, 137.9, 143.5, 149.2; HRMS (ES ): m/z [M
+
79
+
H] calcd for C H N OF Br: 371.0029; found 371.0007. Anal. Calcd for C H N OF Br:
15 11 2 3 15 10 2 3
C, 48.54; H, 2.72; N, 7.55. Found: C, 48.52; H, 2.75; N, 7.52.
4
.3.6. 5-Bromo-7-(3-aminocarbonylphenyl)-3-methyl-1H-indazole (3f)
White solid (0.25 g, 64%), mp. 199–202 °C; νmax (ATR) 644, 691, 939, 1123, 1261, 1569,
-1 1
1
674, 2852, 2921, 3149 cm ; H-NMR (DMSO-d ) 2.50 (3H, s, CH ), 7.21 (1H, t, J = 7.8 Hz,
6
3
2
3

H-5′), 7.48 (1H, d, J = 1.2 Hz, H-6), 7.55 (1H, dd, J = 1.2 and 7.8 Hz, H-6′), 7.61 (1H, d, J =
.2 Hz, H-4), 7.82 (1H, d, 7.8 Hz, H-4′), 7.98 (1H, d, J = 1.2 Hz, H-2′), 8.10 (2H, s, NH ), 13.02
1
2
1
3
(
1H, s, NH); C-NMR (DMSO-d ) 12.2, 112.7, 122.3, 125.8, 127.3, 128.0, 128.3, 128.7, 129.3,
6
+
+
79
1
29.6, 131.4, 135.8, 136.5, 137.8, 168.3; HRMS (ES ): m/z [M + H] calcd for C H N O Br:
15 13 2
3
30.0164; found 330.0242. Anal calcd for C H N OBr: C, 54.56; H, 3.66; N, 12.73. Found:
15 12 2
C, 54.53; H, 3.63; N, 3.62.
4
.3.7. 5-Bromo-7-(3-chloro-4-fluorophenyl)-3-methyl-1H-indazole (3g)
White solid (0.18 g, 51%), mp. 209–212 °C; νmax (ATR) 692, 834, 925, 987, 1235, 1313,
-1 1
1
478, 1798, 1602, 2853, 2922, 3238 cm ; H-NMR (DMSO-d ) 2.58 (3H, s, CH ), 7.27 (1H,
6
3
d, J = 8.1 Hz, H-5′), 7.33 (1H, t, J = 2.1 and 8.1 Hz, H-6′), 7.45 (1H, d, J = 1.2 Hz, H-6), 7.65
1
3
(
1H, d, J = 2.1 Hz, H-2′), 7.83 (1H, d, J = 1.2 Hz, H-4); C-NMR (DMSO-d ) 12.5, 112.9,
6
1
1
18.6 (d, 2JCF = 20.6 Hz), 112.9, 123.3, 124.5, 125,6, 128.7 (d, 3JCF = 6.7 Hz), 129.8, 131.2,
4
1
+
+
34.5 (d, J = 2.9 Hz), 138.2, 142.1, 158.1 (d, J = 250.8 Hz); HRMS (ES ): m/z [M + H]
CF
CF
3
5
79
calcd for C H N F Cl Br: 338.9622; found 338.9700. Anal calcd for C, 49.52; H, 2.67; Br,
1
4
10
2
2
3.53; Cl, 10.44; F, 5.59, N, 8.25, Found: C, 10.39; H, 5.54; N, 8.25.
.3.8. 5-Bromo-3-methyl-7-styryl-1H-indazole (3h)
4
White solid (0.30 g, 67%), mp. 205–208 °C; νmax (ATR) 686, 734, 954, 1294, 1339, 1489,
-1 1
1
576, 1738, 2852, 2916, 3034, 3133 cm ; H-NMR (DMSO-d ) 2.48 (3H, s, CH ), 7.32 (1H,
6 3
d, J = 7.2 Hz, H-4′), 7.42 (2H, t, J = 7.5 Hz, H-3′, 5′), 7.50 (1H, d, Jtrans = 16.0 Hz, H ), 7.60
a
(
1H, d, Jtrans = 16.0 Hz, H ), 7.68 (2H, d, J = 7.2 Hz, H-2′, 6′), 7.79 (1H, d, J = 1.2 Hz, H-6),
b
1
3
7
1
.86 (1H, d, J = 1.2 Hz, H-4), 13.2 (1H, s, NH); C-NMR (DMSO-d ) 12.12, 112.9, 121.9,
6
+
22.7, 122.8, 124.9, 125.0, 127.2, 128.6, 129.2, 131.6, 137.4, 138.2, 141.9; HRMS (ES ): m/z
+
79
[
M + H] calcd for C H N Br: 313.0262; found 313.0340. Anal. Calcd for C H N Br: C,
16 14 2 16 13 2
6
4
1.36; H, 4.18; N, 8.94, Found: C, 61.35; H, 4.14; N, 8.92.
.4.9. 5-Bromo-7-(4-fluorostyryl)-3-methyl-1H-indazole (3i)
2
4

White solid (0.28 g, 56%), mp. 208–211 °C; νmax (ATR) 803, 891, 955, 1158, 1227, 1484,
-1 1
1
509, 1580, 1738, 2919, 3037, 3148 cm ; H-NMR (DMSO-d ) 2.43 (3H, s, CH ), 7.63 (2H,
6 3
dd, JHH = 8.7 Hz and JHF = 9.6 Hz, H-3′, 5′), 7.86 (1H, Jtrans = 16.0 Hz, H ), 7.92 (1H, J
=
trans
a
1
8
6.0 Hz, H ), 8.08 (2H, J = 8.7 Hz and J = 5.4 Hz, H-2′, 6′), 8.12 (1H, d, J = 1.2 Hz, H-6),
b HH HF
1
3
.22 (1H, d, J = 1.2 Hz, H-4), 13.4 (1H, s, NH); C-NMR (DMSO-d ) 12.1, 112.9, 116.2 (d,
6
2JCF = 21.7 Hz), 121.9, 122.7, 122.8, 124.9, 125.1, 129.0 (d, 3JCF = 7.5 Hz), 130.4, 134.0 (d,
4
1
+
+
J = 2.9 Hz), 138.2, 141.9, 162.9 (d, J = 245.0 Hz); HRMS (ES ): m/z [M + H] calcd for
CF
CF
7
9
C H N F Br: 331.0168; found 331.0247. Anal. Calcd for C H N BrF: C, 58.03; H, 3.65;
1
6
13
2
16 12
2
N, 8.46. Found: C, 57.99; H, 3.67; N, 8.53.
4
.4.10. 5-Bromo-7-(4-chlorostyryl)-3-methyl-1H-indazole (3j)
White solid (0.28 g, 54%), mp. 243–246 °C; νmax (ATR) 806, 843, 947, 1097, 1301, 1489,
-
1 1
1
8
604, 2852, 2919, 3057, 3181 cm ; H-NMR (DMSO-d ) 2.48 (3H, s, CH ), 7.50 (2H, d, J =
6
3
.4 Hz, H-3′, 5′), 7.61 (1H, d, Jtrans = 16.0 Hz, H ), 7.68 (1H, d, J
= 16.0 Hz, H ), 7.69 (2H,
a
trans
b
d, J = 8.4 Hz, H-2′, 6′), 7.78 (1H, d, J = 1.8 Hz, H-6), 7.87 (1H, d, J = 1.8 Hz, H-4), 13.14 (1H,
1
3
s, NH); C-NMR (DMSO-d ) 12.10, 112.8, 122.2, 122.5, 123.6, 125.0, 125.3, 128.8, 129.3,
6
+
+
35 79
1
3
8
4
30.2, 132.9, 136.4, 138.2, 141.9; HRMS (ES ): m/z [M + H] calcd for C H N Cl Br:
16 13 2
46.9872; found 346.9951. Anal. Calcd for C H N ClBr: C, 55.28; H, 3.48; Br, 22.98; N,
1
6
11
2
.06, Found: C, 55.27; H, 22.94; N, 8.02.
.4.11. 5-Bromo-7-(4-methoxystyryl)-3-methyl-1H-indazole (3k)
White solid (0.10 g, 50%), mp. 219–222 °C; νmax (ATR) 837, 1110, 1174, 1247, 1300,
-1 1
1
510, 1574, 1605, 1737, 2918, 3033, 3146 cm ; H-NMR (DMSO-d ) 2.47 (3H, s, CH ), 3.77
6 3
(
3H, s, OCH ), 6.98 (2H, d, J = 8.7 Hz, H-3′, 5′), 7.44 (1H, d, J
= 16.0 Hz, H ), 7.55 (1H, d,
trans a
3
Jtrans = 16.0 Hz, H ), 7.61(2H, d, J = 9.0 Hz, H-2′, 6′), 7.72 (1H, d, J = 2.1 Hz, H-6), 7.80 (1H,
b
1
3
d, J = 2.1 Hz, H-4), 13.1 (1H, s, NH); C-NMR (DMSO-d ) 12.1, 55.7, 112.9, 114.7, 120.3,
6
+
1
21.3, 123.2, 124.5, 124.9, 128.6, 130.3, 131.3, 138.2, 141.8, 159.8; HRMS (ES ): m/z [M +
2
5

+
79
H] calcd for C H N O Br: 343.0368; found 343.0446. Anal. Calcd for C H N OBr: C,
1
7
16
2
17 15
2
5
4
9.49; H, 4.41; N, 8.16; Found: C, 59.47; H, 4.44; N, 8.20.
.4. Typical procedure for the Sonogashira cross-coupling of 2 to afford 4a–f
A mixture of 2 (1.0 equiv), PdCl (PPh ) (5 mol %), CuI (10 mol %), terminal alkyne (1.5
2
3 2
equiv.) and K CO (1.5 equiv.) in 4:1 THF-water mixture (v/v, 15 mL/mmol of 2) in a two-
2
3
necked round bottom flask equipped with a stirrer bar, rubber septum, and a condenser was
flushed with argon gas for 5 minutes. A balloon filled with argon gas was connected to the top
of the condenser and the mixture was stirred at room temperature for 3 h. The mixture was
quenched with an ice cold water and the product was extracted with chloroform. The combined
organic layers were dried over anhydrous Na SO . The salt was filtered off and then the solvent
2
4
was evaporated under reduced pressure on a rotatory evaporator. The residue was purified by
column chromatography on silica gel to afford 4. The following compounds were prepared in
this fashion:
4
.4.1. 5-Bromo-3-methyl-7-(phenylethynyl)-1H-indazole (4a)
Brown solid (0.28 g, 61%), mp 181–183.9 °C; νmax (ATR) 685, 754, 847, 888, 988, 1287,
-1 1
1
321, 1489, 1576, 1597, 2214, 2923, 3158 cm ; H-NMR (DMSO-d ) 2.50 (3H, s, CH ), 7.47
6
3
(3H, t, J = 3.9 Hz, Ph), 7,63 (1H, d, J = 1.2 Hz, H-6), 7.71 (2H, t, J = 3.9 Hz, Ph), 8.04 (1H, d,
1
3
J = 1.8 Hz, H-4); C-NMR (DMSO-d ) 12.2, 84.3, 95.5, 107.0, 111.6, 122,4, 124.2, 124.4,
6
+
+
79
1
3
6
4
29.3, 129.7, 129.2, 131.4, 140.1, 142.5; HRMS (ES ): m/z [M + H] calcd for C H N Br:
16 12 2
11.0106; found 311.0184. Anal. Calcd for C H N Br: C, 61.76; H, 3.56; N, 9.00, Found: C,
1
6
11
2
1.77; H, 3.56; N, 8.96.
.4.2. 5-Bromo-7-(3-fluorophenylethynyl)-3-methyl-1H-indazole (4b)
White solid (0.39 g, 76%), mp 215–217 °C; νmax (ATR) 688, 777, 856, 871, 922, 1292,
-1 1
1
467, 1578, 1604, 2918, 3160 cm ; H-NMR (DMSO-d ) 2.48 (3H, s, CH ), 7.31–7.35 (1H,
6 3
m, H-4ʹ), 7.49–7.51 (2H, m, H-2ʹ and H-6ʹ), 7.54–7.65 (2H, m, H-5ʹ and H-6), 8.06 (1H, d, J =
1
3
1
.8 Hz, H-4), 13.38 (1H, s, NH); C-NMR (DMSO-d ) 12.2, 85.2, 94.2, 106.5, 111.6, 116.9
6
2
6

2
2
3
4
(
d, J = 21.9 Hz), 118.8 (d, J = 22.9 Hz), 124.5 (d, J = 8.03 Hz), 124.6, 128.4 (d, J =
CF CF CF CF
3
1
2
.7 Hz), 131.3 (d, J = 8.03 Hz), 131.4, 131.6, 140.2, 142.6, 163.9 (d, J = 248.8 Hz); HRMS
CF
CF
+
+
79
(
ES ): m/z [M + H] calcd for C H N F Br: 329.0011; found 329.0090. Anal. Calcd for
1
6
11
2
C H N BrF: C, 58.38; H, 3.06; N, 8.51. Found: C, 58.41; H, 3.11; N, 8,46.
1
6
10
2
4
.4.3. 5-Bromo-7-(4-fluorophenylethynyl)-3-methyl-1H-indazole (4c)
White solid (0.33 g, 64%), mp. 204–206 °C; νmax (ATR) 831, 991, 1231, 1468, 1511, 1583,
-1 1
2
921, 3220 cm ; H-NMR (DMSO-d ) 1.35 (3H, s, CH ), 6.16 (2H, dt, J = 8.7 Hz and JHF
6 3 HH
=
9.7 Hz, H-3ʹ, 5ʹ), 6.48 (1H, d, J = 1.8 Hz, H-6), 6.65 (2H, dt, JHH = 8.7 Hz and J = 5.4 Hz,
HF
1
3
H-2ʹ, 6ʹ), 6.89 (1H, d, J = 1.8 Hz, H-4), 12.22 (1H, s, NH); C-NMR (DMSO-d ) 12.5, 84.4,
6
9
1
4.9, 107.3, 111.9, 116.9, (d, 2JCF = 21.8 Hz), 119.2 (d, 4JCF = 2.7 Hz), 124.5, 124.7, 131.7,
3
4
+
+
34.9 (d, J = 8.5 Hz), 140.4, 142.8, 162.4 (d, J = 245.2 Hz); HRMS (ES ): m/z [M + H]
CF
CF
7
9
calcd for C H N F Br: 329.0011; found 329.0090. Anal calcd for C H N FBr: C, 58.38;
16
11
2
16 10
2
H, 3.06; N, 8.51. Found: C, 58.29; H, 3.02; N, 8.47.
4
.4.4. 5-Bromo-7-(3-chlorophenylethynyl)-3-methyl-1H-indazole (4d)
White solid (0.37 g, 72%), mp. 226–229 °C; νmax (ATR) 715, 758, 859, 881, 899, 1090,
-
1 1
1
7
270, 1290, 1318, 1474, 1592, 2921, 3034 cm ; H-NMR (DMSO-d ) 2.50 (3H, s, CH ), 7.47–
6 3
.49 (2H, m, Ar), 7.65 (2H, overlapping signals, Ar′), 7.89 (1H, d, J = 1.2 Hz, H-6), 8.01 (1H,
1
3
d, J = 1.2 Hz, H-4), 13.4 (1H, s, NH); C-NMR (DMSO-d ) 12.2, 85.5, 93.9, 106.5, 111.6,
6
+
1
24.4, 124.4, 124.6, 129.7, 130.6, 131.1, 131.6, 131.7, 133.7, 140.2, 142.6; HRMS (ES ): m/z
+
35 79
[
M + H] calcd for C H N Cl Br: 344.9716; found 344.9794. Anal. Calcd for
16 11 2
C H N ClBr: C, 55.60; H, 2.92; N, 8.11. Found: C, 55.57; H, 2.95; N, 8.21.
1
6
10
2
4
.4.5. 5-Bromo-7-(4-chlorophenylethynyl)-3-methyl-1H-indazole (4e)
White solid (0.32 g, 69%), mp. 225–228 °C; νmax (ATR) 710, 716, 823, 885, 989, 1092,
-1 1
1
268, 1317, 1466, 1488, 1580, 2921, 3214 cm ; H-NMR (DMSO-d ) 2.48 (3H, s, CH ), 7.54
6
3
(2H, d, J = 8.1 Hz, H-3ʹ, 5ʹ), 7.63 (1H, d, J = 1.2 Hz, H-6), 7.74 (2H, d, J = 8.1 Hz, H-2ʹ, 6ʹ),
1
3
8
.04 ( 1H, d, J = 1.2 Hz, H-4), 13.39 (1H, s, NH); C-NMR (DMSO-d ) 12.2, 85.3, 94.4, 106.7,
6
2
7

+
1
06.7, 111.6, 121.3, 124.4, 129.4, 131.5, 133.8, 134.4, 140.1, 142.5; HRMS (ES ): m/z [M +
+
35 79
H] calcd for C H N Cl Br: 344.9776; found 344.9794. Anal. Calcd for C H N ClBr: C,
1
6
11
2
16 10
2
5
4
5.60; H, 2.92; N, 8.11. Found: C, 55.58; H, 2.93; N, 8.14.
.4.6. 5-Bromo-7-(4-methoxyphenylethynyl)-3-methyl-1H-indazole (4f)
White solid (0.36 g, 78%), mp. 172–175 °C; νmax (ATR) 828, 888, 1030, 1172, 1247, 1292,
-1 1
1
570, 1578, 1604, 2208, 2919, 3132 cm ; H-NMR (DMSO-d ) 2.49 (3H, s, CH ), 3.80 (3H,
6 3
s, OCH ), 7.01 (2H, d, J = 8.7 Hz, H-3ʹ, 5ʹ), 7.58 (1H, d, J = 1.2 Hz, H-6), 7.67 (2H, d, J = 8.7
3
1
3
Hz, H-2ʹ, 6ʹ), 7.99 (1H, d, J = 1.2 Hz, H-4), 13.34 (1H, s, NH); C-NMR (DMSO-d ) 12.2,
6
5
5.8, 83.0, 95.9, 107.6, 111.7, 114.3, 114.8, 123.6, 124.8, 130.9, 133.8, 140.0, 142.4, 160.3;
+
+
79
HRMS (ES ): m/z [M + H] calcd for C H N O Br: 341.0211; found 341.0290. Anal. Calcd
1
7
14
2
for C H N OBr: C, 59.84; H, 3.84; N, 8.21. Found: C, 59.80; H, 3.85; N, 8.23.
1
7
13
2
4
.5. Inhibition of α-glucosidase activity
The α-glucosidase inhibitory activity of all synthetic compounds was assayed according to
the modified procedure by Shi et al. [36] with slight modification. α-Glucosidase type1 from
baker’s yeast (G5003), p-nitrophenyl-α-D-glucopyranoside (N1377), acarbose (A8980) were
purchased from Sigma Aldrich (Pty) Ltd. (Modderfontein, Johannesburg, South Africa). The
stock solutions of the test compounds (500 µM) were prepared in DMSO followed by dilution
with 100 mM phosphate buffer to obtain the concentrations of 200 µM. The selected assay
concentrations for the test compounds (2, 3a–k & 4a–f) and acarbose as positive control were
1
, 5, 10, 25 and 50 µM. The enzyme solution (0.48 u/mL α-glucosidase, 17 µL); phosphate
buffer (100 mM, pH 6.8; 50 µL) and test sample in DMSO (17 µL) were incubated at 37 °C for
0 minutes. After pre-incubation, 17 µL of 2 mM PNP-G was added. All tests and analyses
1
were performed in triplicates and the absorbances were recorded in a 96 well microplate at 400
nm using Varioskan flash microplate spectrophotometer (Thermo Scientific, Waltham, MA,
USA). IC value was defined as the concentration of compound exhibiting 50% inhibition of
5
0
α-glucosidase activity under the assay conditions. The values were calculated by the nonlinear
2
8

regression analysis and expressed as the mean SD of three distinct experiments using graph pad
prism.
4
.6. Kinetic studies against α-glucosidase
Compounds 3a, 3h and 4a were selected for the kinetics studies with various substrate
concentrations. The experimental process of the enzyme inhibition kinetics was similar to the
above-mentioned conditions with inhibitors concentrations of 0.02, 0.2, 0.5, 1 µM, and the
ranges of final substrate concentration were 0.01–1 mM. The type of inhibition was determined
by using Lineweave-Burk plot (the inverse of velocity (1/v) against the inverse of the substrate
concentration (1/[S]). The inhibitor constant was obtained by Dixon plot (the inverse of velocity
(1/v) against concentration of inhibitor at each substrate concentration) [37].
1
1
1
=
+
vo
vo
vmax[S]
4
.7. DPPH Assays of Compounds 2, 3a–k and 4a–f.
DPPH radical scavenging activity of compounds 2, 3a–k and 4a–f was evaluated following
the literature method [38]. Ascorbic acid (Sigma Aldrich, Saint Louis, Missouri, USA) was
used as a positive control. The test compounds and control at various concentrations (0 µM to
5
0 µM) in DMSO were mixed with a solution of DPPH (0.20 mM) in methanol. The mixtures
were incubated in the dark for 45 minutes and the absorbances were recorded at 512 nm using
Varioskan flash microplate spectrophotometer reader. All tests and analyses were run in
triplicate and averaged. The inhibition was calculated in terms of percentage using the following
formula:
AbC ‒ AbS
DPPH radical scavenged (%) =
× 100
AbC
where AbC is absorbance of control and AbS the absorbance of the test sample. A graph of
percentage inhibition of free radical activity was plotted against concentration of the sample
and the IC (compound concentration required to reduce the absorbance of the DPPH control
5
0
solution by 50%) was obtained from the graph.
2
9