Development of α-glucosidase inhibitors by room temperature C-C cross couplings of quinazolinones

Article abstract of DOI:10.1039/c3ob40636a

Novel quinazolinone based α-glucosidase inhibitors have been developed. For this purpose a virtual screening model has been generated and validated utilizing acarbose as a α-glucosidase inhibitor. Homology modeling, docking, and virtual screening were successfully employed to discover a set of structurally diverse compounds active against α-glucosidase. A search of a 3D database containing 22 500 small molecules using the structure based virtual model yielded ten possible candidates. All ten candidates were N-3-pyridyl-2-cyclopropyl quinazolinone-4-one derivatives, varying at the 6 position. This position was modified by Suzuki-Miyaura cross coupling with aryl, heteroaryl, and alkyl boronic acids. A catalyst screen was performed, and using the best optimal conditions, a series of twenty five compounds was synthesized. Notably, the C-C cross coupling reactions of the 6-bromo-2-cyclopropyl-3- (pyridyl-3-ylmethyl)quinazolin-4(3H)-one precursor have been accomplished at room temperature. A comparison of the relative reactivities of 6-bromo and 6-chloro-2,3-disubstituted quinazolinones with phenyl boronic acid was conducted. An investigation of pre-catalyst loading for the reaction of the 6-bromo-2-cyclopropyl-3-(pyridyl-3-ylmethyl)quinazolin-4(3H)-one substrate was also carried out. Finally, we submitted our compounds to biological assays against α-glucosidase inhibitors. Of these, three hits (compounds 4a, 4t and 4r) were potentially active as α-glucosidase inhibitors and showed activity with IC₅₀ values <20 μM. Based on structural novelty and desirable drug-like properties, 4a was selected for structure-activity relationship study, and thirteen analogs were synthesized. Nine out of thirteen analogs acted as α-glucosidase inhibitors with IC₅₀ values <10 μM. These lead compounds have desirable physicochemical properties and are excellent candidates for further optimization.

Full text of DOI:10.1039/c3ob40636a

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Biomolecular Chemistry
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Development of α-glucosidase inhibitors by room
temperature C–C cross couplings of quinazolinones†
Cite this: Org. Biomol. Chem., 2013, 11,
4778
Ramesh Garlapati,a,b Narender Pottabathini,*a Venkateshwarlu Gurram,a
Kumara Swamy Kasani,a Rambabu Gundla,c Chiranjeevi Thulluri,d
Pavan Kumar Machiraju,c Avinash B. Chaudhary,a Uma Addepally,d
Raveendra Dayam,c Venkata Rao Chundurib and Balaram Patroa
Novel quinazolinone based α-glucosidase inhibitors have been developed. For this purpose a virtual
screening model has been generated and validated utilizing acarbose as a α-glucosidase inhibitor.
Homology modeling, docking, and virtual screening were successfully employed to discover a set of struc-
turally diverse compounds active against α-glucosidase. A search of a 3D database containing 22 500
small molecules using the structure based virtual model yielded ten possible candidates. All ten candi-
dates were N-3-pyridyl-2-cyclopropyl quinazolinone-4-one derivatives, varying at the 6 position. This posi-
tion was modified by Suzuki–Miyaura cross coupling with aryl, heteroaryl, and alkyl boronic acids. A
catalyst screen was performed, and using the best optimal conditions, a series of twenty five compounds
was synthesized. Notably, the C–C cross coupling reactions of the 6-bromo-2-cyclopropyl-3-(pyridyl-
3-ylmethyl)quinazolin-4(3H)-one precursor have been accomplished at room temperature. A comparison
of the relative reactivities of 6-bromo and 6-chloro-2,3-disubstituted quinazolinones with phenyl boronic
acid was conducted. An investigation of pre-catalyst loading for the reaction of the 6-bromo-2-cyclopropyl-
3-(pyridyl-3-ylmethyl)quinazolin-4(3H)-one substrate was also carried out. Finally, we submitted our
compounds to biological assays against α-glucosidase inhibitors. Of these, three hits (compounds 4a, 4t
and 4r) were potentially active as α-glucosidase inhibitors and showed activity with IC50 values <20 μM.
Based on structural novelty and desirable drug-like properties, 4a was selected for structure–activity
relationship study, and thirteen analogs were synthesized. Nine out of thirteen analogs acted as α-gluco-
sidase inhibitors with IC50 values <10 μM. These lead compounds have desirable physicochemical
properties and are excellent candidates for further optimization.
Received 30th March 2013,
Accepted 20th May 2013
DOI: 10.1039/c3ob40636a
www.rsc.org/obc
insulin, or both. This causes glucose to accumulate in the
blood, which often leads to various complications, such as
Introduction
Diabetes mellitus is one of the most common and serious blindness, kidney failure, or microvascular disease, which
metabolic disorders, characterized by high blood-glucose could lead to atherosclerosis, strokes, and other coronary heart
levels which result from defects in insulin secretion, or action, disease.2 α-Glucosidase is an enzyme that catalyzes the exo-
or both.1
hydrolysis of 1,4-α-glucosidic linkages with release of α-glucose.
Insulin enables cells to absorb glucose in order to turn it α-Glucosidase inhibitors decrease the absorption of carbo-
into energy. However, in diabetes, the body either does not hydrates from the digestive tract, thereby lowering the after-
respond properly to its own insulin or does not make enough meal glucose levels.3 For this reason, the α-glucosidase inhibi-
tors are clinically used as oral antihyperglycemic agents to
delay intestinal carbohydrate absorption and lessen postpran-
aMedicinal Chemistry Division, GVK Biosciences Pvt. Ltd, Plot 28A, IDA Nacharam,
dial increases in glucose levels.4 Some α-glucosidase inhibi-
Hyderabad, India. E-mail: narender.pottabathini@gvkbio.com;
tors, such as acarbose, miglitol, and voglibose, are currently
Fax: +91 40 6628 1505; Tel: +91 40 6628 1275
bDepartment of Chemistry, Sri Venkateshwara University, Tirupathi, India
cInformatics Division, GVK Biosciences Pvt. Ltd, Plot 79, IDA Mallapur, Hyderabad,
India
used in combination with either diet or other anti-diabetic
agents to control the blood glucose levels of patients (Fig. 1).
However, they often cause side effects such as flatulence and
diarrhea. Hence, natural and synthesized α-glucosidase inhibi-
tors have become an attractive therapeutic approach for
the treatment of postprandial hyperglycemia.5 α-Glucosidase
dCentre for Innovative Research, CBT, IST, JNTUH, Hyderabad, India
†Electronic supplementary information (ESI) available: Copies of the 1H NMR
and 13C NMR spectra are for all key intermediates and final products; additional
information as needed. See DOI: 10.1039/c3ob40636a
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C–C bond-forming reactions at the C-6 position of 2,3-disubsti-
tuted quinazolin-4(3H)-ones using both C-6 bromo and chloro
quinazolin-4(3H)-one derivatives under mild, room tempera-
ture conditions to install C-6 aryl/alkyl/heteroaryl groups. To
our knowledge, room temperature reactions at the C-6 position
of quinazolin-4(3H)-ones have not been reported to date. In
the broader context, accomplishing such reactions under
ambient temperatures generally represents a nontrivial under-
taking. In addition, we also compared the reactivities of C-6
bromo and chloro quinazolin-4(3H) ones, and evaluated the
pre-catalyst loading effect on reaction productivity. Further-
more, this report also demonstrates the stability of the cyclo-
propyl moiety to the Suzuki–Miyaura cross coupling reaction
conditions.13
Fig. 1 Known iminosugars as inhibitors of α-glucosidase.
inhibitors are also known for anti-HIV, anticancer and other
activities other than anti-diabetes.6
In this context we designed quinazolinone-based non-
carbohydrate mimetic inhibitors of the α-glucosidase enzyme.
C-6 substituted quinazolinone derivatives were screened as
α-glucosidase inhibitors by in silico high-throughput screen-
ing. As the three dimensional structure of α-glucosidase is not
available, it was derived through homology modeling. A virtual
library of C-6 substituted quinazolinone derivatives was
formed and docking simulation studies of C-6 substituted qui-
nazolinone derivatives were performed using the MODELER
program interfaced with Discovery Studio 3.5 (DS3.5). Among
the designed compounds, three were found to be more potent
than the standard drug acarbose.
Finally, we submitted our compounds to biological assays
against α-glucosidase inhibitors.
Results and discussion
Molecular modeling
Structure based virtual screening. Structure based virtual
screening was performed against the homology modeled
α-glucosidase protein using an in-house database (22 500 com-
pounds). The in-house database is filtered using a multiple
property filter, Lipinski’s rule, and ADMET, to prioritize com-
pounds with higher probabilities of possessing favourable
tissue absorption distribution profiles. The last step of screen-
ing involves a Pose filter using Schrödinger docking post-
processing, it is possible to simply filter the output file of
docked poses according to whether or not they fulfil certain
receptor ligand contacts or interactions. Each compound,
docked into the active site, was visually inspected, and those
displaying unfavourable interactions within the binding site
were discarded. Finally, ten compounds with high docking
scores and reasonable hydrogen bond interactions were syn-
thesized by either room temperature or elevated temperature
C–C cross-coupling reactions. These compounds were sub-
mitted for biological testing; from these ten compounds, three
compounds showed reasonable biological activity against
α-glucosidase. A flow diagram for structure based virtual
screening is shown in Fig. 2.
From the sequence alignment of templates and target
protein, the key active site residues Asp199, Asp321, Arg517
and His591 are conserved in α-glucosidase and maltase-
glucoamylase (Asp203, Asp327, Arg526 and His600 from 2QLY,
3L4T). The Ramachandran plot for the predicted model reveals
that 90% residues were found in most favourable region, while
8.2% were found in the allowed region (see the Experimental
section for figure). The other violations for bond lengths,
amide torsions, side chain torsions and backbone torsions
showed any major deviations from the allowed values. Only
0.8% of residues were in the disallowed region, yet these resi-
dues were not located in the active site. The overall G factor
calculated on basis of the main chain parameter implied that
the modeled structure was acceptable for virtual screening.
Among the multitudinous biologically active heterocycles,
nitrogen containing heterocycles play a vital role.7 In fact,
recent surveys have reported that a large number of molecules
currently under investigation by researchers contain nitrogen
heterocycles, and of these, quinazoline and quinazolinone
derivatives constitute the most important family of com-
pounds.8 These quinazolines and quinazolinones have a
common unique structural framework, which we were able to
use as a scaffold for diversification, which in turn is useful for
the preparation of pharmacological drugs.9 So far, research on
quinazolin-4(3H)-ones has been carried out on the C-2, N-3
and C-4 positions of the core structure. A very few literature evi-
dences have been studied on the extension of C-6 position of
the core structure.10 Therefore, there has been a substantial
amount of interest in developing efficient methods for the
synthesis of C-6 substituted quinazolin-4(3H)-ones. Recently
we communicated a facile palladium catalyzed synthetic
method for the introduction of substituted amino groups at
the C-6 position of 2-cyclopropyl-3-(pyridyl-3ylmethyl)quinazo-
lin-4(3H)-one with excellent yields.11 In continuation of this
work, we conducted an extensive study on C–C cross coupling
reactions of 6-halo-2,3-disubstituted 3H-quinazolin-4-ones.
There are a few reports describing the C–C coupling of
6-halo substituted quinazolinones with moderate yields. Most
of these approaches are limited and there are no reports of
thorough study of Suzuki–Miyaura cross coupling reactions on
this scaffold.12 Herein we report an extensive study of Pd-
catalyzed Suzuki–Miyaura cross coupling reactions at C-6
of 2-cyclopropyl-3-(pyridyl-3-ylmethyl)quinazolin-4(3H)-one in
excellent yields. We explored the influence of catalytic systems
on the efficiency of the cross coupling reactions and evaluated
a much wider substrate scope than before. We also describe
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Fig. 2 Schematic representation of in silico screening protocol implemented in
the discovery of α-glucosidase inhibitors.
Fig. 4 First set of ten compounds after virtual screening with α-glucosidase
inhibitory activity, including biological assay results; IC50 values are in μM.
and the side chain OH group of the Ser593 residue (–OH⋯N,
2.16 Å). Compound 4t showed ∼7.31 μM inhibitory activity
against α-glucosidase and the predicted binding mode has a
hydrogen bond between nitrogen in the pyridine moiety of 4t
and the guanidine hydrogen of the Arg517 residue (–N⋯H–N,
2.00 Å). Compound 4a showed ∼10.21 μM inhibitory activity
against α-glucosidase and the predicted binding mode has a
hydrogen bond between the carbonyl group of 4a and the gua-
nidine hydrogen of the Arg517 residue (–CO⋯H–N, 2.89 Å).
The predicted binding mode of these three compounds with
the homology modeled protein is shown in Fig. 5.
Fig. 3 (a) Predicted binding mode of acarbose in modeled protein (3D rep-
resentation). (b) Predicted binding mode of acarbose.
The predicted binding mode of acarbose against the α-glu-
cosidase model also retained hydrogen bond interactions with
conserved amino acids i.e. three hydrogen bond interactions
with the Asp321, His591, and Asp199 residues of the homology
modeled protein. The first hydrogen bond is observed between
the hydroxyl group of acarbose and the COO group of the
Asp321 residue (–OH⋯O–CO, 2.44 Å). The second hydrogen
bond is observed between the hydroxyl group of the compound
and the N–H of His591 (–HO⋯HN, 1.73 Å). The third hydrogen
bond is observed between the hydroxyl group of the compound
and the COO group of the Asp199 residue (–OH⋯O–CO,
1.77 Å). The predicted binding mode and 2D interaction
diagram of acarbose in the homology modeled α-glucosidase
protein are shown in Fig. 3a and 3b.
Structure based virtual screening yielded ten compounds
from in-house virtual library compounds. These ten com-
pounds were synthesized and submitted for biological testing
(Fig. 4). Out of ten compounds, three compounds showed
<20 μM inhibitory activity against α-glucosidase and the
remaining are not active (NA) against α-glucosidase.
From the biological testing, 2-cyclopropyl-6-phenyl-3-
((pyridin-3-yl)methyl)quinazolin-4(3H)-one (4a) was considered
further to explore the relevance of various structural elements.
We designed fourteen analogs of 4a by substituting different
groups on the phenyl moiety present at the C-6 position of qui-
nazolin-4(3H)-one (Table 1).
3,4-Dimethylphenyl substituted analog 4c (7.44 μM),
3-fluoro-4-methylphenyl 4e (7.57 μM) and 3,4-dihydroxyphenyl
substituted quinazolinone 4n (7.68 μM) showed slight
increases in inhibitory activity against α-glucosidase. 2,4-Dihy-
droxyphenyl substituted derivative 4m (6.527 μM) showed
higher activity compared to 3,4-dihydroxy substituted phenyl
derivative 4n. The trihydroxyphenyl substituted derivative, 4o
(5.70 μM) showed the second highest inhibitory activity
against α-glucosidase among all the synthesized compounds.
The 2,6-dichloro substituted quinazolinone derivative 4f
showed the highest inhibition against α-glucosidase. The
derivatives of compound 4a along with their docking scores
and biological activities are given in Table 1. From the pre-
dicted binding mode of compound 4o, it is clear that it forms
two hydrogen bond interactions with the protein. The first
hydrogen bond is observed between the hydroxyl group at the
fourth position of the phenyl moiety present in the compound
The analog 4r showed ∼5.75 μM inhibitory activity against
α-glucosidase and the predicted binding mode has a hydrogen
bond between nitrogen in the indole group of compound 4r
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Fig. 5 Predicted binding mode of 4a, 4r, and 4t in modeled protein in a 3D
representation along with 2D representations.
Fig. 6 Predicted binding mode of 4f, 4n, and 4o in modeled protein 3D rep-
resentation along with 2D representation.
Table 1 IC50 values (μM), dock scores for 6-phenylquinazolin-4(3H)-one deriva-
tives against α-glucosidase
position of the phenyl moiety present in compound 4o and the
carbonyl group in the COO group of Asp321 (–OH⋯OC–O,
2.02 Å). The predicted binding mode of compound 4o with the
homology modeled protein is shown in Fig. 6.
Compound
code
Docking
score
IC50 (μM) of
α-glucosidase
Entry
R
Chemistry
1
2
3
4
5
6
7
8
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4l
4m
4n
4o
H
−8.15
−6.23
−9.57
−8.15
−9.13
−9.79
−7.87
−8.26
−7.68
−8.93
−7.93
−9.89
−8.86
−9.18
Standard
10.21 0.6
NA
7.44 0.73
NA
7.57 0.41
4.04 0.049
NA
NA
NA
NA
NA
The development of easily applicable synthetic strategies for
the construction of carbon–carbon bonds with palladium cata-
lyzed Suzuki–Miyaura cross-coupling reactions remains an
important challenge in organic synthesis.14 6-Bromo- and
6-chloro-2-cyclopropyl-3-(pyridyl-3-ylmethyl)quinazolin-4(3H)-ones
were chosen for the present study. The overall aim was not
only to determine the best conditions for Suzuki–Miyaura
cross coupling reactions of both precursors, but also to find
the optimal conditions that can be applied for the synthesis of
a broad range of 6-aryl, heteroaryl, and alkyl 2,3-disubstituted
quinazolinones. Both halo quinazoline-4(3H)-one precursors
3a and 3b (Fig. 7) are conveniently synthesized in two steps by
known procedures (see the ESI† for details).
2-OCH3
3,4-Dimethyl
2-COCH3
3-F and 4-CH3
2,6-Dichloro
4-CO2CH3
4-CN
3-NO2
4-NMe2
4-SO2Me
2,4-Di-OH
3,4-Di-OH
2,3,4-Tri-OH
9
10
11
12
13
14
15
6.52 0.41
7.68 0.06
5.70 0.09
6.04 0.17
Acarbose
All the test compounds were dissolved in DMSO to required
concentration; the test concentrations used were 2–10 μM.
We have previously utilized precursors 3a, 3b for amination
reactions. We followed similar screening reactions as
described in our recent communication on C–C cross-coupling
reactions of O6-alkyl-2-haloinosine derivatives.15 Optimization
4o and the electron rich oxygen in the COO group of the
Asp321 residue (–OH⋯O–CO, 2.20 Å). The second hydrogen
bond is observed between the hydroxyl group at the third experiments were carried out using Pd(OAc)2 and Pd2(dba)3 as
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Table 2 Initial optimization of C–C reaction experiments on 3a at room
temperaturea,b
Fig. 7 6-Halo-2-cyclopropyl-3-(pyridyl-3-ylmethyl)quinazolin-4(3H)-ones selected
for analysis.
Entry
Catalytic system, conditions, T = rt, t = 36 h
Yieldc (%)
1
2
3
4
5
6
7
8
9
5 mol% [PdCl2(dcpf)]/K3PO4, 1,4-dioxane
5 mol% [PdCl2(dcpf)]/K3PO4, THP
20
84
79
83
15
19
55
41
50
21
5 mol% [PdCl2(dtbpf)]/K3PO4, 1,4-dioxane
5 mol% [PdCl2(dtbpf)]/K3PO4, THP
5 mol% [PdCl2(dppf)]/K3PO4, THP
5 mol% Pd(OAc)2/7.5 mol% L1/K3PO4, THP
5 mol% Pd(OAc)2/7.5 mol% L2/K3PO4, THP
5 mol% Pd(OAc)2/7.5 mol% L3/K3PO4, THP
5 mol% Pd2(dba)3/7.5 mol% L4/K3PO4, THP
5 mol% Pd(OAc)2/7.5 mol% L5/K3PO4, THP
10
a Reaction conditions (entries 1–10) 3a precursor 0.0711 mM in
anhydrous solvent, 2.0 molar equiv. Ph-B(OH)2, 2.0 molar equiv. of
base. b Reactions were conducted in closed vial sparged with argon.
c % yields refer to isolated and purified products.
Fig. 8 Five ligands and three Pd(II) pre-catalysts selected for the initial analysis.
metal sources, ligands L1–L5 were selected for combination
with the metal, and three ferrocenyl Pd(II) pre-catalysts (Fig. 8)
were selected for analysis.
Table 3 Initial optimization of C–C reaction experiments on 3a at room
temperaturea,b
The optimization experiments were performed with phenyl
boronic acid at room temperature for 36 h. We screened con-
ditions with K3PO4 as base and also compared with several sol-
vents (PhMe, 1,4-dioxane and THP). Precursor 3a was only
partially soluble in toluene; THP was superior to 1,4-dioxane.16
Results from our initial investigations are shown in Tables 2
and 3. From these data, Pd(OAc)2/L2 (CyJohnPhos) was
superior to the other biaryl-ligand-based catalysts, which gave
product yields in the range of 40–68% with a catalytic loading
of metal of 5 or 10 mol%.
Entry
Catalytic system, conditions, T = rt, t = 36 h
Yieldc (%)
1
2
3
4
5
6
7
8
9
10 mol% [PdCl2(dcpf)]/K3PO4, THP
89
95
92
95
28
28
68
55
58
24
10 mol% [PdCl2(dtbpf)]/K3PO4, THP
20 mol% [PdCl2(dcpf)]/K3PO4, THP
20 mol% [PdCl2(dtbpf)]/K3PO4, THP
Among the ferrocene-based pre-catalysts that were tested,
[PdCl2(dcpf)] and [PdCl2(dtbpf)] were found to be the best, and
were superior to Pd(OAc)2/L2.
10 mol% [PdCl2(dppf)]/K3PO4, THP
10 mol% Pd(OAc)2/15 mol% L1/K3PO4, THP
10 mol% Pd(OAc)2/15 mol% L2/K3PO4, THP
10 mol% Pd(OAc)2/15 mol% L3/K3PO4, THP
10 mol% Pd2(dba)3/15 mol% L4/K3PO4, THP
10 mol% Pd(OAc)2/15 mol% L5/K3PO4, THP
THP was a generally better solvent as compared to 1,4-
dioxane. The reaction of 5 mol% [PdCl2(dcpf)] in the presence
of THP was superior compared to 1,4-dioxane at room temp-
erature. Whereas, in the case of 5 mol% [PdCl2(dtbpf)], THP
and 1,4-dioxane were both good solvents. Room temperature
reactions were carried out with 10 or 20 mol% of catalyst using
THP as solvent and the results are tabulated in Table 3.
Finally, [PdCl2(dtbpf)] was determined to be a slightly better
catalytic system compared to [PdCl2(dcpf)]. The cost of
10
a Reaction conditions (entries 1–10) 3a precursor 0.0711 mM in
anhydrous solvent, 2.0 molar equiv. Ph-B(OH)2, 2.0 molar equiv. of
base. b Reactions were conducted in closed vial sparged with argon.
c % yields refer to isolated and purified products.
The final C–C coupling products of boronic acids with elec-
[PdCl2(dtbpf)] is higher than [PdCl2(dcpf)], so we have con- tron donating groups like 3,4-dimethyl phenyl boronic acid
sidered [PdCl2(dcpf)] for the evaluation of C–C cross coupling (entry 3, 81%), o-methoxy phenylboronic acid (entry 2, 88%)
with different boronic acids. The C–C cross coupling reactions
yields didn’t change appreciably when the reactions were con- 6, 89%) were produced in excellent yields. The naphthalene
and sterically hindered 2,6-dichlorophenyl boronic acid (entry
ducted over 36 h.
system also resulted in a good yield (entry 16, 79%). Even
when strongly electron depleting substituents were present
After finalizing the optimal conditions, several boronic
acids underwent cross coupling reactions at room temperature (m-nitro, o-acetyl), good yields were obtained. Reactions of
to synthesize the analogs shown in Table 1. From Table 4, it is boronic acids with electron withdrawing groups like 4-cyano-
clear that a wide assortment of aryl, heteroaryl and alkyl
boronic acids undergo Pd-catalyzed Suzuki–Miyaura cross like 3-fluoro-4-methyl (entry 5, 90%) and bis(3,5-trifluoro-
phenyl boronic acid (entry 8, 59%), fluorinated substituents
coupling reactions.
methyl) phenyl boronic acids (entry 11, 66%) also resulted in
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Table 4 Evaluation of the scope of C–C coupling reactions of various boronic
acids with 6-bromo- and 6-chloro-3-(pyridyl-3-ylmethyl)quinazolin-4(3H)-ones,
3a and 3ba
Table 4 (Contd.)
rt, 36 h,
THP, yieldb
80 °C, 8 h, 1,4-
Entry Amine
18
Product (%)
dioxane, yieldc (%)
rt, 36 h,
THP, yieldb
80 °C, 8 h, 1,4-
4r
NP
90 (86)d
Entry Amine
1
Product (%)
dioxane, yieldc (%)
4a
92
88
97 (82)d
98d
19
20
4s
4t
82
60
88
85 (69)d
2
4b
3
4
5
4c
4d
4e
81
80
90
85 (56)d
92
21
22
23
4u
4v
NR
NP
NP
96
55
97 (61)d
4w
65 (82)d
24
25
4x
4y
NP
25
87
91 (83)d
6
4f
89
98
a Reaction conditions (entries 1–25) bromo and chloro compound (3a,
3b) 0.0711 mM in anhydrous. b 2 molar equiv. boronic acid, 2.0 equiv.
K3PO4, 20 mol% of PdCl2(dcpf), reactions were conducted in closed
vial sparged with argon at rt. c 1.5 molar equiv. boronic acid, 2.0 equiv.
K3PO4, 20 mol% of PdCl2(dcpf), 80 °C, reactions were conducted in
closed vial sparged with argon. % yields refer to isolated and purified
products. d Yield obtained with chloro substrate 3b. e Final compound
synthesized in two steps, C–C coupling followed by deprotection of
methyl group. NR: no reaction; NP: not performed.
7
8
9
4g
4h
4i
56
59
62
89 (56)d
98 (67)d
97
10
11
4j
60
66
75
71
4k
good yields. Reactions with heteroaryl boronic acids resulted
in moderate to good yields from 60% to 82% (entries 17 to 22).
In a few cases unreacted Br-precursor 3a remained in the reac-
tion. The reaction with methyl boronic acid resulted a low
yield (entry 25, 25%). Thus, we decided to examine the cross
coupling reactions of aliphatic boronic acids and heteroaryl
boronic acids at elevated temperatures for the completion of
reaction, to generate material for biological assays.
12
13
4l
NR
NR
58
4m
88e
14
15
4n
4o
NR
NR
86e
75e
At this stage, we again explored the generality of the ele-
vated temperature cross coupling reaction conditions, and
[PdCl2(dcpf)] proved the best catalytic system.
Comparison of the product formation dependent on the
pre-catalyst loading
16
17
4p
4q
79
85
We investigated the effect of pre-catalyst loading on the reac-
tion of 6-bromo precursor (3a) with phenyl boronic acid,
drawing an interesting observation. Fig. 9 shows the reaction
rate comparison for the formation of 4a. The rate of reaction is
dependent on the catalyst loading. The reactions were
NP
81 (35)d
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Fig. 10 LC analysis of the reaction mixture from a competitive reaction of 3a
and 3b with PhB(OH)2 (showing the integrated percentages).
Fig. 9 Comparison of the formation of 4a with (
) 1 mol%, (
) 2 mol%,
(
) 5 mol%, ( ) 10 mol%, (
) 20 mol%, (
) 30 mol%; mol% of pre-
catalyst loading on 3a with PhB(OH)2 at 80 °C. Formation of 4a in percentages,
determined by LC/MS. K3PO4 as base and 1,4-dioxane as solvent.
conducted with 2.0 molar eq. K3PO4 in 1,4-dioxane at 80 °C.
All the six reactions were performed with different pre-catalyst
loadings of 1 mol%, 2 mol%, 5 mol%, 10 mol%, 20 mol% and
30 mol% in a parallel reactor. LC/MS analyses of all reactions
were taken at different time intervals i.e. 1 h, 2 h, 3 h, 4 h, 8 h,
16 h and 24 h. Reactions yields were improved significantly
from 1 mol% to 10 mol%. It was also observed that increasing
the catalytic loading from 10 mol% to 30 mol% didn’t result a
change in yield in the case of PhB(OH)2.
Relative reactivities of bromo quinazolinone (3a) and
chloro quinazolinone (3b)
It is evident that 3a is superior to 3b in terms of product yield
under the C–C bond forming reaction conditions (Table 4). We
decided to determine their relative reactivities in a competitive
experiment.
We conducted a reaction of an equimolar amount of 3a and
3b (1.5 molar equiv. each) with one molar equivalent of phenyl
boronic acid, under the optimized conditions. After the com-
plete consumption of phenyl boronic acid the experiment was
stopped. Product 4a as well as reactants 3a, 3b were collected
together by column chromatography and subjected to LC/MS
analysis.
In the mixture, 12.3% of the bromo quinazolinone 3a and
47% of the chloro quinazolinone 3b was observed. 3b was
totally non-reactive and the total quantity was recovered after
the reaction. This experiment shows two important factors.
First, that C-6 bromo quinazolinone precursor 3a is more
rapidly consumed than chloro analog 3b, and second, reduc-
tive dehalogenation does not appear to be significant. Fig. 10
shows the LC trace of the reaction mixture from the compe-
tition experiment. The LC/MS analysis data can be found in
the ESI.† Suzuki reactions with Cl precursor (3b) have a higher
activation energy due to the greater Ar–Cl bond strength com-
pared to Ar–Br, so lower reactivity is expected.
Fig. 11 The effect of inhibitor dosage on the response of α-glucosidase inhi-
bition. The concentrations of test compounds employed from lower to higher
(2 μM to 10 μM) to study the inhibition of α-glucosidase.
In vitro functional activity of selected analogs
All the synthesized compounds were evaluated for their in vitro
α-glucosidase inhibition activity, and the inhibitory effects of
the positive test compounds were concentration dependent.
Typical dose–response plots of α-glucosidase inhibition for the
constructed molecules are shown in Fig. 11. The response of
enzyme inhibition increased with increasing dosage of the
active compounds. Among 25 in vitro screened molecules,
9 molecules showed IC50 concentrations below 10 μM. In the
graph (Fig. 11), each fragment of the column corresponds to a
specific concentration of the respective compound. The α-glu-
cosidase inhibitor assay was analyzed by Dixon plot at two
different substrate concentrations (1 mM and 2 mM) corres-
ponding to two different −Ki values (−2.32 and −1.96) for the
active inhibitors, 4f and 4o (Fig. 12 and 13).
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position of 2-cyclopropyl-3-(pyridyl-3-ylmethyl)quinazolin-
4(3H)-ones both at room temperature and elevated temp-
erature. The combination of 10 mol% (dcpf)PdCl2/K3PO4 in
1,4-dioxane gave good to excellent yields in the coupling of
6-bromo and 6-chloro-2-cyclopropyl-3-(pyridyl-3-ylmethyl)qui-
nazolin-4(3H)-one with a variety of aryl, heteroaryl and alkyl
boronic acids. The methodology for the C–C cross coupling
appears to be broad in scope for 2,3-disubstituted quinazoline-
4-ones. To our knowledge, this is the first report on successful
C–C bond forming reactions at the C-6 position of 2,3-disubsti-
tuted quinazoline-4(3H)-ones at room temperature. In particu-
lar, a cyclopropyl substituent was shown to be stable under the
Suzuki–Miyaura cross coupling conditions. Finally, we have
studied the relative reactivities of the bromo and chloro quina-
zolinone precursors towards C–C cross coupling and it was
clear that former is superior. We have also been investigated
the pre-catalyst loading towards the reactivity of C–C cross
coupling reactions and 10 mol% is suitable for phenyl boronic
acid. The combination of these docking experiments and the
development of facile chemical synthesis gives access to the
development of other analogs as α-glucosidase inhibitors for
further research which is being pursued in our laboratory.
Fig. 12 Dixon plot of reciprocal rates of metabolite (1/v) formation as a func-
tion of inhibitor [I] (compound 4f ) concentrations.
Experimental section
Homology modeling and validation
Human lysosomal α-glucosidase amino acid sequence (FASTA
format) was retrieved from the manually annotated and
reviewed Swiss-Prot database (Swiss-Prot: P10253). The tem-
plate search was performed using the BLAST-P program. The
human maltase-glucoamylase (PDBID: 2QLY and 3L4T) two
reference sequences were selected from the BLAST-P program
(Fig. 14). α-Glucosidase has 44% sequence identity with two
templates (PDBID: 2QLY and 3L4T) with query coverage of
90% for each. The three-dimensional (3D) model lysosomal
α-glucosidase is constructed from 89 to 952 amino acid resi-
dues using human maltase-glucoamylase (PDBID: 2QLY and
3L4T) as template by the MODELER program interfaced with
Discovery Studio 3.5 (DS3.5). MODELER implements compara-
tive protein structure modeling by agreement with spatial
restraints.18 The 3D model is obtained by the optimal
Fig. 13 Dixon plot of reciprocal rates of metabolite (1/v) formation as a func-
tion of inhibitor [I] (compound 4o) concentrations.
Enzyme inhibition data were expressed as IC50 values (the
concentration of the inhibitor required to produce 50% inhi-
bition of the test models). From the results, the apparent IC50
values of the test compounds with respect to α-glucosidase
activity were estimated. In the course of the screening study on
α-glucosidase inhibitors, we found that the analogs syn-
thesized showed different inhibitory activities against the
enzyme.
Among 25 screened 2,3-disubstituted quinazolino-4(3H)-
one molecules, 9 molecules showed fruitful inhibition. In com-
parison with acarbose, the test compounds 4f, 4o, and 4r
showed promising α-glucosidase inhibitory effect. Acarbose
was used as reference compound.17
Conclusions
Pd-catalyzed C–C bond formation is effective for the introduc-
tion of substituted aryl/alkyl/heteroaryl groups at the C-6 Fig. 14 Ligand Plot of 3L4T.
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Fig. 15 Ramachandran plot of ψ/Φ ligand.
Fig. 16 Modeled human α-glucosidase.
satisfaction of spatial restraints of homology-derived and
stereochemically derived restraints from the alignment, and
expressed in terms of probability density function of featured
restraints. The stereochemical quality of the α-glucosidase
homology model was validated by Ramachandran plot analysis
performed using the PROCHECK program (Fig. 15).19
General experimental considerations
Thin-layer chromatography was performed on 250 mm silica
plates and column chromatographic purifications were per-
formed on 100–200 mesh silica gel. All boronic acids,
Pd(OAc)2, Pd2(dba)3, ligands L1–L5, PdCl2(dppf), PdCl2(dcpf),
and PdCl2(dtbpf), and all other reagents were obtained from
commercial suppliers and were used without further purifi-
cation. 1,4-Dioxane was distilled over NaBH4 and then stored
over Na. Prior to each reaction 1,4-dioxane was freshly distilled.
For syntheses of compounds 3a, and 3b as well as their precur-
sors, please see the ESI.† 1H NMR spectra were collected either
at 400 MHz or at 300 MHz and spectra are referenced to
residual protio solvent. 13C NMR spectra, collected either at
100 MHz or at 75 MHz, are referenced to the carbon resonance
of the deuterated solvent. Spectra were obtained either in de-
acidified CDCl3 (deacidification was performed by percolating
the solvent through a bed of solid NaHCO3 and basic alumina)
or in DMSO-d6 (see specific compound descriptions below).
High resolution mass spectrometry was performed at the Mass
Spectrometry Laboratory at GVK Biosciences Pvt Ltd. LC/MS
analyses were performed with electrospray ionization (ESI),
and operated in the positive ion mode. LC analysis was per-
formed using a diode array detector.
Docking studies
The docking studies were performed using Glide (Schrödinger)
and the favourable interactions between one or more ligand
molecules with receptor were analysed. In Glide, the combi-
nation of position and orientation of a ligand relative to the
receptor, along with its conformation in flexible docking, is
referred to as a ligand pose. The ligand poses that Glide gener-
ates pass through a series of hierarchical filters that evaluate
the ligand’s interaction with the receptor.
The initial filters test the spatial fit of the ligand to the
defined active site, and examine the complementarity of
ligand–receptor interactions using a grid-based method pat-
terned after the empirical ChemScore function. Poses that
pass these initial screens enter the final stage of the algorithm,
which involves evaluation and minimization of a grid approxi-
mation to the OPLS-AA non bonded ligand–receptor inter-
action energy.20 Final scoring is then carried out on the
energy-minimized poses. By default, Schrödinger’s proprietary
GlideScore multi-ligand scoring function is used to score the
poses. If GlideScore was selected as the scoring function, a
composite Emodel score is then used to rank the poses of each
ligand and to select the poses to be reported to the user
(Fig. 16).
General procedure for the cross coupling of the 6-bromo-
2-cyclopropyl-3-(pyridin-3-ylmethyl)quinazolin-4(3H)-one
room temperature. In an oven dried, screw-cap vial equipped
with stirring bar were placed 6-bromo-2-cyclopropyl-3-
at
a
(pyridin-3-ylmethyl)quinazolin-4(3H)-one (1 eq.), boronic acid
(2.0 eq.), and K3PO4 (2 eq.), dissolved in anhydrous 1,4-
dioxane (10 V). The vial was flushed with argon and, after
addition of PdCl2(dcpf) (0.2 eq.), sealed with a teflon-lined cap
and placed in a sand bath that was maintained at room temp-
erature. The reaction was monitored by TLC. Upon completion
(36 h), the mixture was diluted with CH2Cl2. The mixture was
washed with water and the organic layer was separated and
Docking simulations were performed using Glide program
with OPLS-AA force field. The binding region was defined
using a grid of 14 Å × 14 Å × 14 Å box centred on the centroid
of the overlaid ligand on to the modeled 3D structure of
protein, in order to confine the centroid of the docked inhibi-
tor. Default settings were used for all the remaining
parameters.
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dried over Na2SO4. Evaporation under reduced pressure pro- d6): δ = 8.61 (d, J = 2.1 Hz, 1H), 8.51 (dd, J = 5.1, 1.5 Hz, 1H),
vided the crude product, which was loaded onto a silica 8.32 (d, J = 2.1 Hz, 1H), 8.10 (dd, J = 8.4, 2.1 Hz, 1H), 7.67–7.64
column packed in CH2Cl2. Sequential elution with pet-ether, (m, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.55 (s, 1H), 7.49 (d, J =
followed by 70% EtOAc in pet-ether, afforded the requisite 8.4 Hz, 1H), 7.39–7.35 (dd, J = 7.5, 4.8 Hz, 1H), 7.27 (d, J =
compound. Finally, compounds were dried under high 7.8 Hz, 1H), 5.62 (s, 2H), 2.31 (s, 3H), 2.27 (s, 3H), 2.22–2.14
vacuum to remove traces of solvent (see specific compound (m, 1H), 1.11–1.07 (m, 2H), 0.98–0.92 (m, 2H). 13C NMR
headings below for details).
(75 MHz CDCl3): δ = 162.6, 156.7, 148.8, 148.2, 146.3, 139.2,
General procedure for the cross coupling of the 6-bromo 137.1, 137.0, 136.2, 134.7, 133.1, 132.4, 130.1, 128.2, 127.3,
and chloro-2-cyclopropyl-3-(pyridin-3-ylmethyl)quinazolin-4- 124.3, 123.7, 120.2, 44.2, 19.8, 19.3, 14.3, 8.9. HRMS (ESI): m/z
(3H)-one. In an oven dried, screw-cap vial equipped with a stir- calcd for C25H24N3O [M + H]+ 382.1919 found 382.1941.
ring bar were placed 6-bromo or chloro -2-cyclopropyl-3- Melting point: 176–180 °C.
(pyridin-3-ylmethyl)quinazolin-4(3H)-one (1 eq.), boronic acid
6-(2-Acetylphenyl)-2-cyclopropyl-3-(pyridin-3-ylmethyl)quina-
(1.5 eq.), and K3PO4 (2 eq.), dissolved in anhydrous 1,4- zolin-4(3H)-one (4d). Chromatography was performed using
dioxane (10 V) The vial was flushed with argon and, after 70% EtOAc in n-hexane to yield a pale yellow solid. Rf (70%
addition of PdCl2(dcpf) (0.2 eq.), sealed with a teflon-lined cap EtOAc in n-hexane) = 0.55; 1H NMR (400 MHz, CDCl3): δ = 8.63
and placed in a sand bath that was maintained at 80 °C. The (bs, 1H), 8.55 (bs, 1H), 8.28 (bs, 1H), 7.64 (bs, 4H), 7.56 (d, J =
reaction was monitored by TLC. Upon completion (8 h), the 7.4 Hz, 1H), 7.47–7.43 (m, 2H), 7.30–7.26 (m, 1H), 5.62 (s, 2H),
mixture was cooled and diluted with CH2Cl2. The mixture was 2.16 (s, 3H), 1.91–1.88 (m, 1H), 1.28–1.25 (m, 2H), 1.02–1.01
washed with water and the organic layer was separated and (m, 2H). 13C NMR (100 MHz, CDCl3): δ = 203.5, 162.4, 157.6,
dried over Na2SO4. Evaporation under reduced pressure pro- 149.1, 148.3, 146.9, 140.2, 139.4, 139.1, 135.3, 134.6, 132.2,
vided the crude product, which was loaded onto a silica 131.0, 130.7, 128.2, 127.8, 127.2, 126.4, 123.8, 120.2, 44.3, 30.3,
column packed in CH2Cl2. Sequential elution with pet-ether, 14.4, 9.2. HRMS (ESI): m/z calcd for C25H22N3O2 [M + H]+
followed by 70% EtOAc in pet-ether, afforded the requisite 396.1712 found 396.1717. Melting point: 130–132 °C.
compound. Finally, compounds were dried under high 2-Cyclopropyl-6-(3-fluoro-4-methylphenyl)-3-(pyridin-3-ylmethyl)-
vacuum to remove traces of solvent (see specific compound quinazolin-4(3H)-one (4e). Chromatography was performed
headings below for details).
using 70% EtOAc in n-hexane to yield a pale yellow solid. Rf
2-Cyclopropyl-6-phenyl-3-(pyridin-3-ylmethyl)quinazolin-4- (70% EtOAc in n-hexane) = 0.55; 1H NMR (300 MHz, DMSO-
(3H)-one (4a). Chromatography was performed using 70% d6): δ = 8.60 (s, 1H), 8.51 (d, J = 3.3 Hz, 1H), 8.35 (d, J = 2.1 Hz,
EtOAc in n-hexane to yield a pale yellow solid. Rf (70% EtOAc 1H), 8.14 (dd, J = 8.4, 2.1 Hz, 1H), 7.67–7.63 (m, 2H), 7.61 (d,
1
in n-hexane) = 0.55; H NMR (300 MHz, DMSO-d6): δ = 8.61 (s, J = 6.3 Hz, 1H), 7.55–7.52 (m, 2H), 7.43–7.35 (m, 2H), 5.62 (s,
1H), 8.51 (d, J = 3.3 Hz, 1H), 8.35 (d, J = 2.1 Hz, 1H), 8.13 (dd, 2H), 2.28 (s, 3H), 2.20–2.16 (m, 1H), 1.11–1.10 (m, 2H),
J = 0, 2.1 Hz, 1H), 7.78 (d, J = 7.5 Hz, 2H), 7.68 (m, 1H), 7.65 (d, 0.98–0.95 (m, 2H). 13C NMR (75 MHz, CDCl3): δ = 163.2, 159.9,
J = 8.4 Hz, 1H), 7.53 (t, J = 7.5 Hz, 2H), 7.43–7.35 (m, 2H), 5.61 157.2, 149.0, 148.4, 146.7, 139.1, 137.8, 134.6, 134.6, 133.0,
(s, 2H), 2.21–2.16 (m, 1H), 1.12–1.11 (m, 2H), 0.99–0.94 (m, 132.2, 131.8, 127.5, 124.5, 123.7, 122.2, 120.2, 113.5, 113.2,
2H); 13C NMR (75 MHz, CDCl3): δ = 162.6, 157.1, 149.0, 148.4, 44.3, 14.3, 9.0.] HRMS (ESI): m/z calcd for C24H21FN3O
146.6, 139.6, 139.2, 134.6, 133.3, 132.3, 128.9, 127.7, 127.5, [M + H]+ 386.1669 found 386.1645. Melting point: 120–123 °C.
127.1, 124.8, 123.7, 120.3, 44.3, 14.4, 9.0. HRMS (ESI): m/z
2-Cyclopropyl-6-(2,6-dichlorophenyl)-3-(pyridin-3-ylmethyl)-
calcd for C23H20N3O [M + H]+ 354.1606; found 354.1599. quinazolin-4(3H)-one (4f). Chromatography was performed
Melting point: 124–128 °C.
using 70% EtOAc in n-hexane to yield a pale yellow solid. Rf
2-Cyclopropyl-6-(2-methoxyphenyl)-3-(pyridin-3-ylmethyl)- (70% EtOAc in n-hexane) = 0.55; 1H NMR (300 MHz, DMSO-
quinazolin-4(3H)-one (4b). Chromatography was performed d6): δ = 8.60, (s, 1H), 8.51 (d, J = 3.3 Hz, 1H), 8.39 (d, J = 2.1 Hz,
using 70% EtOAc in n-hexane to yield a pale yellow solid Rf 1H), 8.19 (dd, J = 8.4, 2.1 Hz, 1H), 7.85 (d, J = 1.2 Hz, 2H),
1
(70% EtOAc in n-hexane) = 0.5; H NMR (300 MHz, DMSO-d6): 7.68–7.62 (m, 3H), 7.39 (dd, J = 7.8, 5.1 Hz, 1H), 5.62 (s, 2H),
δ = 8.60 (s, 1H), 8.50 (s, 1H), 8.20 (d, J = 2.1 Hz, 1H), 7.92 (dd, 2.23–2.17 (m, 1H), 1.14–1.10 (m, 2H), 0.99–0.95 (m, 2H).
J = 8.4 Hz, 2.1 Hz, 1H), 7.67 (m, 1H), 7.59 (d, J = 8.4 Hz, 1H), 13C NMR (75 MHz, CDCl3): δ = 162.4, 157.9, 149.1, 148.4,
7.42–7.35 (m, 3H), 7.17 (d, J = 8.4 Hz, 1H), 7.09–7.04 (m, 1H), 147.4, 142.5, 136.3, 135.5, 134.7, 132.9, 132.1, 127.9, 127.6,
5.61 (s, 2H), 3.79 (s, 3H), 2.22–2.14 (m, 1H), 1.12–1.07 (m, 2H), 125.5, 125.2, 123.8, 120.4, 44.4, 14.4, 9.3. HRMS (ESI): m/z
0.98–0.92 (m, 2H). 13C NMR (75 MHz, CDCl3): δ = 162.6, 156.9, calcd for C23H18Cl2N3O [M + H]+ 422.0827; found 422.0847.
156.4, 148.9, 148.3, 146.3, 136.9, 136.1, 134.5, 132.1, 130.8, Melting point: 90–92 °C.
129.2, 129.1, 127.3, 126.4, 123.8, 120.9, 119.8, 111.2, 55.5, 44.2,
14.4, 8.9. HRMS (ESI): m/z calcd for C24H22N3O2 [M + H]+ dihydroquinazolin-6-yl)benzoate (4g). Chromatography was
384.1712 found 384.1742. Melting point: 85–88 °C. performed using 70% EtOAc in n-hexane to yield a pale yellow
Methyl-4-(2-cyclopropyl-4-oxo-3-(pyridin-3-ylmethyl)-3,4-
1
2-Cyclopropyl-6-(3,4-dimethylphenyl)-3-(pyridin-3-ylmethyl)- solid. Rf (70% EtOAc in n-hexane) = 0.55; H NMR (300 MHz,
quinazolin-4(3H)-one (4c). Chromatography was performed DMSO-d6): δ = 8.61 (s, 1H), 8.51 (d, J = 4.2 Hz, 1H), 8.44 (d, J =
using 70% EtOAc in n-hexane to yield a pale yellow solid. Rf 1.8 Hz, 1H), 8.21 (dd, J = 8.4, 2.1 Hz, 1H), 8.09 (d, J = 8.4 Hz,
(70% EtOAc in n-hexane) = 0.55; 1H NMR (300 MHz, DMSO- 2H), 7.97 (d, J = 8.4 Hz, 2H), 7.94 (d, J = 8.4 Hz, 1H), 7.68
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(m, 1H), 7.66 (d, J = 8.1 Hz, 1H), 7.39 (dd, J = 8.4, 5.2 Hz, 1H), 132.6, 132.1, 128.2, 127.1, 125.5, 123.8, 121.4, 120.6, 44.5, 14.4,
5.63 (s, 2H), 3.89 (s, 3H), 2.27–2.20 (m, 1H), 1.12 (m, 2H), 0.99 9.4. HRMS (ESI): m/z calcd for C25H18F6N3O [M + H]+ 490.1354
(m, 2H). 13C NMR (75 MHz, CDCl3): δ = 166.7, 162.5, 157.6, found 490.1349. Melting point: 126–130 °C.
149.1, 148.4, 147.2, 143.9, 137.7, 134.6, 133.1, 132.1, 130.2,
2-Cyclopropyl-6-(4-(methylsulfonyl)phenyl)-3-(pyridin-3-
129.2, 127.7, 126.9, 125.2, 123.7, 120.3, 52.1, 44.3, 14.4, 9.2. ylmethyl)quinazolin-4(3H)-one (4l). Chromatography was per-
HRMS (ESI): m/z calcd for C25H22N3O3 [M + H]+ 412.1661 formed using 70% EtOAc in n-hexane to yield a pale yellow
1
found 412.1688. Melting point: 170–174 °C.
solid. Rf (70% EtOAc in n-hexane) = 0.55; H NMR (400 MHz,
4-[2-Cyclopropyl-4-oxo-3-(pyridin-3-ylmethyl)-3,4-dihydroqui- CDCl3): δ = 8.63 (s, 1H), 8.57 (d, J = 4.0 Hz, 1H), 8.54 (d, J =
nazolin-6-yl]benzonitrile (4h). Chromatography was per- 2.4 Hz, 1H), 8.05 (d, J = 8.4 Hz, 2H), 7.99 (dd, J = 8.4, 2.4 Hz,
formed using 70% EtOAc in n-hexane to yield a pale yellow 1H), 7.89 (d, J = 8.8 Hz, 2H), 7.71 (d, J = 8.8 Hz, 1H), 7.63 (d, J =
1
solid. Rf (70% EtOAc in n-hexane) = 0.55; H NMR (300 MHz, 8.0 Hz, 1H), 7.31 (dd, J = 7.6, 3.2 Hz, 1H), 5.64 (s, 2H), 3.11 (s,
DMSO-d6): δ = 8.61 (s, 1H), 8.51 (d, J = 3.3 Hz, 1H), 8.43 (d, J = 3H), 1.95–1.91 (m, 1H), 1.31–1.25 (m, 2H), 1.07–1.02 (m, 2H).
2.1 Hz, 1H), 8.21 (dd, J = 8.4, 2.1 Hz, 1H), 8.02 (d, J = 8.1 Hz, 13C NMR (100 MHz, CDCl3): δ = 162.4, 158.1, 149.1, 148.3,
2H), 7.96 (d, J = 8.1 Hz, 2H), 7.68 (m, 2H), (d, J = 8.1 Hz, 1H), 147.5, 145.0, 139.3, 136.7, 134.6, 133.1, 132.0, 128.0, 127.9,
7.39 (dd, J = 8.4, 5.1 Hz, 1H), 5.62 (s, 2H), 2.22–2.17 (m, 1H), 125.5, 123.7, 120.4, 44.5, 29.6, 14.4, 9.3. HRMS (ESI): m/z calcd
1.13–1.12 (m, 2H), 0.99–0.95 (m, 2H). 13C NMR (75 MHz, for C24H22N3O3S [M + H]+ 432.1382 found 432.1385. Melting
CDCl3): δ = 162.4, 158.1, 149.2, 148.4, 147.6, 144.0, 136.8, point: 208–212 °C.
134.6, 133.0, 132.7, 128.0, 127.6, 125.4, 123.7, 120.5, 118.7,
111.3, 44.4, 14.4, 9.3. HRMS (ESI): m/z calcd for C24H19N4O quinazolin-4(3H)-one (4m). Chromatography was performed
[M + H]+ 379.1559 found 379.1561. Melting point: 110–113 °C.
using 70% EtOAc in n-hexane to yield a pale yellow solid. Rf
2-Cyclopropyl-6-(2,4-dihydroxyphenyl)-3-(pyridin-3-ylmethyl)-
2-Cyclopropyl-6-(3-nitrophenyl)-3-(pyridin-3-ylmethyl)quina- (70% EtOAc in n-hexane) = 0.55; 1H NMR (400 MHz, DMSO-
zolin-4(3H)-one (4i). Chromatography was performed using d6): δ = 9.55 (bs, 1H), 9.42 (bs, 1H), 8.58 (bs, 1H), 8.48 (bs, 1H),
70% EtOAc in n-hexane to yield a pale yellow solid. Rf (70% 8.24 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 7.6 Hz, 1H),
EtOAc in n-hexane) = 0.55; 1H NMR (400 MHz, CDCl3): δ = 8.64 7.51 (d, J = 8.4 Hz, 1H), 7.35 (bs, 1H), 7.15 (d, J = 8.4 Hz, 1H), 6.43
(s, 1H), 8.54 (s, 3H), 8.24 (d, J = 8.0 Hz, 1H), 8.03 (d, J = 8.0 Hz, (s, 1H), 6.34 (d, J = 7.6 Hz, 1H), 5.58 (s, 2H), 2.14 (bs, 1H), 1.06
1H), 7.99 (dd, J = 8.4, 2.0 Hz, 1H), 7.71–7.61 (m, 3H), 7.30–7.26 (bs, 2H), 0.92 (d, J = 3.6 Hz, 2H). 13C NMR (100 MHz, DMSO-d6):
(m, 1H), 5.64 (s, 2H), 1.99–1.90 (m, 1H), 1.31–1.29 (m, 2H), δ = 161.7, 158.2, 157.1, 155.3, 148.4, 148.2, 145.0, 136.7, 135.2,
1.05–1.03 (m, 2H). 13C NMR (100 MHz, CDCl3): δ = 162.4, 134.3, 132.7, 130.8, 126.1, 125.4, 123.7, 119.3, 117.5, 107.3, 103.0,
158.0, 149.2, 148.8, 148.4, 147.5, 141.3, 136.4, 134.6, 132.9, 43.7, 13.9, 9.0. HRMS (ESI): m/z calcd for C23H20N3O3 [M + H]+
132.0, 129.9, 128.0, 125.3, 123.7, 122.4, 121.8, 120.5, 44.4, 14.4, 386.1505 found 386.1505. Melting point: 258–261 °C.
9.3. HRMS (ESI): m/z calcd for C23H19N4O3 [M + H]+ 399.1457
found 399.1460. Melting point: 157–160 °C.
2-Cyclopropyl-6-(3,4-dihydroxyphenyl)-3-(pyridin-3-ylmethyl)-
quinazolin-4(3H)-one (4n). Chromatography was performed
2-Cyclopropyl-6-(4-(dimethylamino)phenyl)-3-(pyridin-3- using 70% EtOAc in n-hexane to yield a pale yellow solid. Rf
ylmethyl)quinazolin-4(3H)-one (4j). Chromatography was (70% EtOAc in n-hexane) = 0.55; 1H NMR (400 MHz, DMSO-
performed using 70% EtOAc in n-hexane to yield a pale yellow d6): δ = 8.74 (s, 1H), 8.63 (d, J = 4.8 Hz, 1H), 8.22 (d, J = 2.0 Hz,
1
solid. Rf (70% EtOAc in n-hexane) = 0.55; H NMR (400 MHz, 1H), 8.03 (dd, J = 8.4, 2.0 Hz, 2H), 7.61–7.53 (m, 2H), 7.14 (d,
CDCl3): δ = 8.63 (s, 1H), 8.56 (d, J = 4.0 Hz, 1H), 8.51 (d, J = J = 2.0 Hz, 1H), 7.05 (dd, J = 8.0, 2.0 Hz, 1H), 6.96 (m, 1H), 6.86
1.6 Hz, 1H), 8.0 (dd, J = 8.4, 1.6 Hz, 1H), 7.65 (d, J = 8.4 Hz, (d, J = 8.0 Hz, 1H), 6.64–6.62 (d, J = 7.6 Hz, 1H) 5.66 (s, 2H),
1H), 7.60 (m, 1H), 7.34 (t, J = 8.0 Hz, 1H), 7.29 (dd, J = 8.0, 2.18–2.15 (m, 1H), 1.09 (m, 2H), 0.91–0.89 (m, 2H). 13C NMR
4.8 Hz, 1H), 7.03 (d, J = 8.0 Hz, 1H), 7.0 (s, 1H), 6.78 (d, J = (100 MHz, DMSO-d6): δ = 161.7, 156.8, 152.3, 146.7, 145.8,
8.4 Hz, 1H), 5.63 (s, 2H), 3.02 (s, 6H), 1.91–1.89 (m, 1H), 1.2–1.28 145.6, 145.2, 139.6, 138.1, 134, 132.3, 129.9, 127.2, 124.8,
(m, 2H), 1.02–1.0 (m, 2H). 13C NMR (100 MHz, CDCl3): δ = 162.6, 122.3, 119.9, 117.7, 116.2, 113.7, 43.7, 13.9, 9.1. HRMS (ESI):
156.8, 150.9, 148.9, 148.3, 146.5, 141.0, 139.1, 134.6, 133.5, 132.3, m/z calcd for C23H20N3O3 [M + H]+ 386.1505 found 386.1524.
129.5, 127.2, 124.7, 122.9, 120.1, 115.5, 111.9, 111.1, 44.2, 40.6, Melting point: 263–266 °C.
14.3, 8.9. HRMS (ESI): m/z calcd for C25H25N4O [M + H]+ 397.2028
found 397.2011. Melting point: 130–132 °C.
2-Cyclopropyl-3-(pyridin-3-ylmethyl)-6-(2,3,4-trihydroxyphe-
nyl)quinazolin-4(3H)-one (4o). Chromatography was per-
6-(3,5-Bis(trifluoromethyl)phenyl)-2-cyclopropyl-3-(pyridin-3- formed using 70% EtOAc in n-hexane to yield a pale yellow
1
ylmethyl)quinazolin-4(3H)-one (4k). Chromatography was per- solid. Rf (70% EtOAc in n-hexane) = 0.55; H NMR (400 MHz,
formed using 70% EtOAc in n-hexane to yield a pale yellow DMSO-d6): δ = 9.13 (s, 1H), 8.59 (s, 1H), 8.50 (m, 3H), 8.26 (d,
1
solid. Rf (70% EtOAc in n-hexane) = 0.55; H NMR (300 MHz, J = 2.0 Hz, 1H), 7.94 (dd, J = 8.4, 2.0 Hz, 1H), 7.64 (d, J =
CDCl3): δ = 8.64 (s, 1H), 8.58 (d, J = 3.3 Hz, 1H), 8.54 (d, J = 8.0 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.38 (dd, J = 8.0, 4.8 Hz,
2.1 Hz, 1H), 8.11 (s, 2H), 8.0 (dd, J = 8.4, 2.1 Hz, 1H), 7.89 (s, 1H), 6.69 (d, J = 8.4 Hz, 1H), 6.43 (d, J = 8.8 Hz, 1H), 5.60 (s,
1H), 7.73 (d, J = 8.4 Hz, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.31 (dd, 2H), 2.17–2.13 (m, 1H), 1.08–1.06 (m, 2H), 0.95–0.94 (m, 2H).
J = 7.8, 5.1 Hz, 1H), 5.64 (s, 2H), 1.98–1.90 (m, 1H), 1.33–1.28 13C NMR (100 MHz, DMSO-d6): δ = 161.7, 157.1, 148.4, 148.2,
(m, 2H), 1.08–1.02 (m, 2H). 13C NMR (75 MHz, CDCl3): δ = 146.0, 145.1, 144.0, 137.0, 135.3, 134.3, 133.1, 132.7, 126.0,
162.4, 158.3, 149.2, 148.4, 147.7, 141.7, 135.8, 134.6, 132.9, 125.5, 123.7, 119.7, 119.3, 118.6, 107.3, 43.7, 14.0, 9.1. HRMS
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(ESI): m/z calcd for C23H20N3O4 [M + H]+ 402.1454 found
402.1459. Melting point: 284–286 °C.
2-Cyclopropyl-3-(pyridin-3-ylmethyl)-6-(thiophen-3-yl)quinazo-
lin-4(3H)-one (4t). Chromatography was performed using 70%
2-Cyclopropyl-6-(naphthalen-1-yl)-3-(pyridin-3-ylmethyl)qui- EtOAc in n-hexane to yield a pale yellow solid. Rf (70% EtOAc
1
nazolin-4(3H)-one: (4p). Chromatography was performed in n-hexane) = 0.55; H NMR (300 MHz, DMSO-d6): δ = 8.61 (d,
using 70% EtOAc in n-hexane to yield a pale yellow solid. Rf J = 1.5 Hz, 1H), 8.51 (dd, J = 4.2, 1.5 Hz, 1H), 8.38 (d, J =
1
(70% EtOAc in n-hexane) = 0.55; H NMR (400 MHz, CDCl3): δ 2.1 Hz, 1H), 8.18 (dd, J = 8.4, 2.1 Hz, 1H), 8.05 (m, 1H), 7.70
= 8.64 (s, 1H), 8.56 (d, J = 4.0 Hz, 1H), 8.42 (d, J = 1.6 Hz, 1H), (m, 3H), 7.60 (d, J = 8.4, 1H), 7.40–7.36 (dd, J = 8.4, 5.1 Hz,
7.93–7.90 (m, 2H), 7.88 (dd, J = 8.8, 2.4 Hz, 2H), 7.71 (d, J = 1H), 5.61 (s, 2H), 2.22–2.13 (m, 1H), 1.12–1.07 (m, 2H),
8.4 Hz, 1H), 7.64 (d, J = 7.6 Hz, 1H), 7.57–7.42 (m, 4H), 0.98–0.92 (m, 2H). 13C NMR (75 MHz, CDCl3): δ = 162.5, 156.8,
7.30–7.26 (dd, J = 8.0, 4.8 Hz, 1H), 5.62 (s, 2H), 1.93–1.90 (m, 148.9, 148.2, 146.3, 140.7, 134.6, 133.8, 132.5, 132.2, 127.4,
1H), 1.33–1.28 (m, 2H), 1.06–1.01 (m, 2H). 13C NMR (100 MHz, 126.5, 126.0, 123.7, 120.9, 120.2, 44.2, 14.3, 8.9. HRMS (ESI):
CDCl3): δ = 162.6, 157.3, 149.1, 148.3, 146.6, 139.0, 138.7, m/z calcd for C21H18N3OS [M + H]+ 360.1171 found 360.1159.
136.5, 134.6, 133.7, 132.2, 131.3, 128.3, 128.1, 127.9, 127.3, Melting point: 146–150 °C.
126.9, 126.3, 125.9, 125.5, 125.3, 123.8, 120.1, 44.3, 14.4, 9.1.
HRMS (ESI): m/z calcd for C27H22N3O [M + H]+ 404.1763 found ylmethyl)quinazolin-4(3H)-one (4u). Chromatography was per-
404.1781. Melting point: 212–215 °C. formed using 70% EtOAc in n-hexane to yield a pale yellow
2-Cyclopropyl-6-(3,5-dimethyl-1H-pyrazol-4-yl)-3-(pyridin-3-
1
2-Cyclopropyl-3-(pyridin-3-ylmethyl)-6-(pyridin-4-yl)quinazo- solid. Rf (70% EtOAc in n-hexane) = 0.55; H NMR (300 MHz,
lin-4(3H)-one (4q). Chromatography was performed using DMSO-d6): δ = 12.39 (s, 1H), 8.60 (d, J = 1.5 Hz, 1H), 8.50 (d, J =
70% EtOAc in n-hexane to yield a pale yellow solid. Rf (70% 3.6 Hz, 1H), 7.97 (d, J = 2.1 Hz, 1H), 7.74 (dd, J = 8.4, 2.1 Hz,
1
EtOAc in n-hexane) = 0.55; H NMR (300 MHz, DMSO-d6): δ = 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.39 (dd, J
8.68 (d, J = 6.3 Hz, 2H), 8.61 (d, J = 1.5 Hz, 1H), 8.51 (m, 2H), = 8.4, 5.1 Hz, 1H), 5.60 (s, 2H), 2.23 (s, 6H), 2.19–2.12 (m, 1H),
8.25 (dd, J = 8.4, 2.1 Hz, 1H), 7.83–7.81 (m, 2H), 7.69 (m, 1H), 1.09–1.09 (m, 2H), 0.96–0.92 (m, 2H). 13C NMR (75 MHz,
7.61 (d, J = 8.4 Hz, 1H), 7.39 (dd, J = 7.8, 4.8 Hz, 1H), 5.63 (s, CDCl3): δ = 162.5, 156.8, 149.0, 148.3, 145.8, 142.1, 135.6,
2H), 2.27–2.18 (m, 1H), 1.15–1.10 (m, 2H), 1.0–0.96 (m, 2H). 134.6, 132.3, 132.2, 127.1, 126.7, 123.7, 120.2, 117.4, 44.3, 14.4,
13C NMR (75 MHz, CDCl3): δ = 162.4, 158.2, 150.0, 149.0, 11.6, 9.0. HRMS (ESI): m/z calcd for C22H22N5O [M + H]+
148.2, 147.9, 147.0, 135.7, 134.7, 132.7, 132.1, 128.0, 125.4, 372.1824 found 372.1837. Melting point: 138–141 °C.
123.8, 121.5, 120.4, 44.4, 14.4, 9.4. HRMS (ESI): m/z calcd for
C22H19N4O [M + H]+ 355.1559 found 356.1557. Melting point: ylmethyl)quinazolin-4(3H)-one (4v). Chromatography was per-
177–180 °C. formed using 70% EtOAc in n-hexane to yield a pale yellow
2-Cyclopropyl-6-(3,5-dimethylisoxazol-4-yl)-3-(pyridin-3-
1
2-Cyclopropyl-6-(1H-indol-6-yl)-3-(pyridin-3-ylmethyl)quinazo- solid. Rf (70% EtOAc in n-hexane) = 0.55; H NMR (400 MHz,
lin-4(3H)-one (4r). Chromatography was performed using 70% CDCl3): δ = 8.64 (bs, 1H), 8.57 (bs, 1H), 8.16 (d, J = 2.0 Hz, 1H),
EtOAc in n-hexane to yield a pale yellow solid. Rf (70% EtOAc 7.67 (d, J = 8.4 Hz, 1H), 7.64–7.62 (m, 1H), 7.62 (dd, J = 8.8,
1
in n-hexane) = 0.55; H NMR (300 MHz, DMSO-d6): δ = 11.2 (s, 2.0 Hz, 1H), 7.31 (bs, 1H), 5.62 (s, 2H), 2.44 (s, 3H), 2.30 (s,
1H), 8.61 (s, 1H), 8.51 (d, J = 4.2 Hz, 1H), 8.35 (d, J = 2.1 Hz, 3H), 1.96–1.90 (m, 1H), 1.30–1.25 (m, 2H), 1.07–1.04 (m, 2H).
1H), 8.14 (dd, J = 8.4, 2.1 Hz, 1H), 7.92 (s, 1H), 7.70 (d, J = 13C NMR (100 MHz, CDCl3): δ = 165.6, 162.2, 158.4, 157.6,
7.2 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.53–7.46 (m, 2H), 149.0, 148.2, 146.6, 134.9, 134.6, 132.1, 128.5, 127.6, 126.9,
7.41–7.35 (m, 2H), 6.55 (bs, 1H), 5.60 (s, 2H), 2.20–2.16 (m, 123.7, 120.3, 115.6, 44.3, 14.3, 11.5, 10.7, 9.2. HRMS (ESI): m/z
1H), 1.14–1.10 (m, 2H), 0.97–0.94 (m, 2H). 13C NMR (75 MHz, calcd for C22H21N4O2 [M + H]+ 373.1665 found 373.1685.
CDCl3): δ = 162.8, 156.4, 148.8, 148.2, 145.9, 140.7, 135.5, Melting point: 151–155 °C.
134.8, 133.7, 132.5, 132.0, 131.6, 128.4, 127.3, 125.1, 124.5,
2,6-Dicyclopropyl-3-(pyridin-3-ylmethyl)quinazolin-4(3H)-
123.8, 121.6, 120.2, 119.3, 111.5, 103.0, 44.3, 14.4, 8.8. HRMS one (4w). Chromatography was performed using 70% EtOAc
(ESI): m/z calcd for C25H21N4O [M + H]+ 393.1715 found in n-hexane to yield a pale yellow solid Rf (70% EtOAc in
1
393.1712. Melting point: 205–209 °C.
n-hexane) = 0.5; H NMR (400 MHz, CDCl3): δ = 8.62 (s, 1H),
6-(Benzofuran-2-yl)-2-cyclopropyl-3-(pyridin-3-ylmethyl)quina- 8.55 (d, J = 3.2 Hz, 1H), 7.93 (d, J = 2.0 Hz, 1H), 7.60 (d, J =
zolin-4(3H)-one (4s). Chromatography was performed using 8.0 Hz, 1H), 7.50 (d, J = 8.4 Hz, 1H), 7.46 (dd, J = 8.4, 2.0 Hz,
70% EtOAc in n-hexane to yield a pale yellow solid. Rf (70% 1H), 7.28–7.25 (m, 1H), 5.60 (s, 2H), 2.05–1.98 (m, 1H),
EtOAc in n-hexane) = 0.55; 1H NMR (400 MHz, CDCl3): δ = 8.74 1.89–1.83 (m, 1H), 1.25–1.19 (m, 2H), 1.06–0.98 (m, 2H),
(d, J = 1.6 Hz, 1H), 8.65 (s, 1H), 8.56 (s, 1H), 8.19 (dd, J = 8.4, 0.97–0.95 (m, 2H), 0.81–0.77 (m, 2H); 13C NMR (100 MHz,
1.6 Hz, 1H), 7.64–7.59 (m, 3H), 7.54 (d, J = 8.4 Hz, 1H), CDCl3): δ = 162.6, 155.9, 148.8, 148.2, 145.4, 142.8, 134.8,
7.30–7.22 (m, 3H), 7.13 (s, 1H), 5.62 (s, 2H), 1.92–1.84 (m, 1H), 132.5, 126.9, 123.8, 122.5, 119.3, 118.3, 44.2, 15.3, 14.4, 9.7,
1.28–1.22 (m, 2H), 1.04–1.0 (m, 2H). 13C NMR (100 MHz, 8.7; HRMS (ESI): m/z calcd for C20H20N3O [M + H]+ 318.1606;
CDCl3): δ = 162.4, 157.5, 154.9, 154.6, 149.0, 148.3, 147.3, found 318.1614. Melting point: 77–79 °C.
137.5, 134.7, 130.7, 129.3, 129.0, 128.8, 128.4, 127.6, 124.6,
2-Cyclopropyl-6-isobutyl-3-(pyridin-3-ylmethyl)quinazolin-4-
123.8, 123.0, 122.9, 121.4, 121.0, 120.4, 111.6, 101.2, 44.3, 14.4, (3H)-one: (4x). Chromatography was performed using 70%
9.2. HRMS (ESI): m/z calcd for C25H20N3O2 [M + H]+ 394.1556 EtOAc in n-hexane to yield a pale yellow solid. Rf (70% EtOAc
found 394.1537. Melting point: 175–177 °C.
in n-hexane) = 0.55; 1H NMR (400 MHz, CDCl3): δ = 8.62
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(bs, 1H), 8.55 (bs, 1H), 8.04 (s, 1H), 7.60 (d, J = 8.4 Hz, 1H), and estimate the Ki of the reaction. The plots were prepared of
7.53–7.49 (m, 2H), 7.28 (m, 1H), 5.60 (s, 2H), 2.61 (d, J = the reciprocal of rate of product formation (1/v) versus the
7.2 Hz, 2H), 1.97–1.90 (m, 1H), 1.89–1.84 (m, 1H), 1.23–1.20 inhibitor concentration at different substrate concentrations.22
(m, 2H,), 1.0–0.95 (m, 2H), 0.92 (d, J = 6.0 Hz, 6H). 13C The relationship is follows as,
NMR (100 MHz, CDCl3): δ = 162.7, 156.2, 149.0, 148.4,
145.7, 140.3, 135.8, 134.6, 132.5, 126.7, 126.4, 123.7, 119.7,
1=v ¼ ðKm ½Iꢂ=Vmax ½SꢂKiÞ þ 1=Vmax ð1 þ Km=½SꢂÞ
45.0, 44.2, 30.2, 22.2, 14.3, 8.7. HRMS (ESI): m/z calcd for
C21H24N3O [M + H]+ 334.1919 found 334.2566. Melting point:
The subsequent straight lines were analyzed by linear
regression using the ORIGIN (version 8) software. The Ki
values were obtained by simultaneously interpreting two sets
of equations successively; one equation for each of the straight
lines equated with the line that represented the highest sub-
strate concentration (this line has the smallest slope). The
point of intersection of these pairs of lines signifies the value
of Ki. The latter values are reported as means or as a range of
110–113 °C.
2-Cyclopropyl-6-methyl-3-(pyridin-3-ylmethyl)quinazolin-4-
(3H)-one (4y). Chromatography was performed using 70%
EtOAc in n-hexane to yield a pale yellow solid. Rf (70% EtOAc
in n-hexane) = 0.55; 1H NMR (400 MHz, CDCl3): δ = 8.61 (s,
1H), 8.54 (d, J = 4.4 Hz, 1H), 8.07 (s, 1H), 7.59 (d, J = 8.0 Hz,
1H), 7.55 (dd, J = 8.0, 2.0 Hz, 1H),7.49 (d, J = 8.0 Hz, 1H), 7.27
values.
(dd, J = 8.0, 4.4 Hz, 1H), 5.60 (s, 2H), 2.47 (s, 3H), 1.90–1.83
(m, 1H), 1.25–1.20 (m, 2H), 1.0–0.95 (m, 2H). 13C NMR
(100 MHz, CDCl3): δ = 162.6, 156.2, 149.0, 148.3, 145.4, 136.5,
135.9, 134.6, 132.4, 126.8, 126.3, 123.7, 119.8, 44.2, 21.2, 14.4,
Acknowledgements
8.7. HRMS (ESI): m/z calcd for C18H18N3O [M + H]+ 292.1450
Authors are grateful to GVK Biosciences Pvt Ltd, for the finan-
cial support and encouragement. We thank Prof. Mahesh
K. Lakshman, Department of Chemistry, The City College and
The City University of New York, New York, USA for his invalu-
able support and motivation.
found 292.1440. Melting point: 98–100 °C.
Assay of α-glucosidase inhibitory activity
The inhibitory effect of the synthesized compounds on α-glu-
cosidase was evaluated in 96-well plates employing the sub-
strate PNPG and 4-nitrophenyl α-D-glucopyranoside according
to the procedure previously reported.21 Prior to use, all test
compounds were solubilized in solvent, dimethylsulfoxide
(DMSO), and then further diluted in DMSO to acquire the
desired final maximum test concentration. In brief, each well
comprised substrate 4-nitrophenyl α-D-glucopyranoside in
2 mM phosphate buffer (pH 7.2) and different test concen-
trations (10–100 μg mL−1). The reaction was commenced by
the addition of enzyme (0.1 IU per well), α-glucosidase
(obtained from Sigma Aldrich, Bengaluru) and the plates were
incubated at 37 °C for 10 min. The absorbance was measured
spectrophotometrically at 410 nm (Spectra MAX Plus; Mole-
cular Devices; supported by SOFTmax PRO-3.0). The increase
in absorbance (ΔA) was compared with that of control (buffer
instead of test compound) to compute the inhibitory activity of
enzyme. The half maximal inhibitory concentrations (IC50)
were determined from two independent assays, performed in
duplicate. Acarbose, an illustrious inhibitor of α-glucosidase
was employed as positive control.
References
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Inhibition ð%Þ ¼ ΔAcontrol ꢀ ΔAsampleÞ=ΔAcontrol ꢁ 100%
The concentration of inhibition required for 50% of
α-glucosidase activity under the assay conditions was defined
as the IC50 value.
Inhibitions kinetics
There are a variety of methods available to compute the inhi-
bition constant (Ki) that characterizes substrate inhibition by a
competitive inhibitor. Among them, the Dixon plot is fre-
quently employed to investigate the mechanism of inhibition
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