Hydrazinyl arylthiazole based pyridine scaffolds: Synthesis, structural characterization, in?vitro α-glucosidase inhibitory activity, and in silico studies

Article abstract of DOI:10.1016/j.ejmech.2017.06.041

Acarbose, miglitol, and voglibose are the inhibitors of α-glucosidase enzyme and being clinically used for the management of type-II diabetes mellitus. However, many adverse effects are also associated with them. So, the development of new therapeutic agents is an utmost interest in medicinal chemistry research. Current study is based on the identification of new α-glucosidase inhibitors. For that purpose, hydrazinyl arylthiazole based pyridine derivatives 1–39 were synthesized via two step reaction and fully characterized by spectroscopic techniques EI-MS, HREI-MS, ¹H-, and ¹³C NMR. However, stereochemistry of the iminic bond was confirmed by NOESY. All compounds were subjected to in vitro α-glucosidase inhibitory activity and found many folds active (IC₅₀ = 1.40 ± 0.01–236.10 ± 2.20 μM) as compared to the standard acarbose having IC₅₀ value of 856.45 ± 5.60 μM. A limited structure-activity relationship was carried out in order to make a presumption about the substituent's effect on inhibitory activity which predicted that substituents of more negative inductive effect played important role in the activity as compared to the substituents of less negative inductive effect. However, in order to have a good understanding of ligand enzyme interactions, molecular docking study was also conducted. In silico study was confirmed that substituents like halogens (Cl) and nitro (NO₂) which have negative inductive effect were found to make important interactions with active site residues.

Full text of DOI:10.1016/j.ejmech.2017.06.041

Accepted Manuscript
Hydrazinyl arylthiazole based pyridine scaffolds: Synthesis, structural
characterization, in vitro α-glucosidase inhibitory activity, and in silico studies
Farman Ali, Khalid Mohammed Khan, Uzma Salar, Muhammad Taha, Nor Hadiani
Ismail, Abdul Wadood, Muhammad Riaz, Shahnaz Perveen
PII:
S0223-5234(17)30487-7
10.1016/j.ejmech.2017.06.041
EJMECH 9536
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 April 2017
Revised Date: 3 June 2017
Accepted Date: 23 June 2017
Please cite this article as: F. Ali, K.M. Khan, U. Salar, M. Taha, N.H. Ismail, A. Wadood, M. Riaz, S.
Perveen, Hydrazinyl arylthiazole based pyridine scaffolds: Synthesis, structural characterization, in vitro
α-glucosidase inhibitory activity, and in silico studies, European Journal of Medicinal Chemistry (2017),
doi: 10.1016/j.ejmech.2017.06.041.
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ACCEPTED MANUSCRIPT
Hydrazinyl arylthiazole based pyridine scaffolds: Synthesis, structural characterization,
in vitro -glucosidase inhibitory activity, and in silico studies
Farman Ali,a Khalid Mohammed Khan,a Uzma Salar,a Muhammad Taha,b,c Nor Hadiani
Ismail,b Abdul Wadood,d Muhammad Riazd and Shahnaz Perveen,e
aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University
of Karachi, Karachi-75270, Pakistan
bAtta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam
Campus, 42300 Bandar Puncak Alam, Selangor D. E. Malaysia
cFaculty of Applied Science Universiti Teknologi MARA, Shah Alam 40450, Selangor D. E., Malaysia
dDepartment of Biochemistry, Computational Medicinal Chemistry Laboratory, UCSS, Abdul Wali Khan
University Mardan, Pakistan
ePCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-75280, Pakistan
In vitro -glucosidase
inhibitory activity
Synthesis
In silico study
Amidine moiety
Lead Molecules
R
H
H
NH NH
NH2
N
N
N
N
N
N
R
N
N
N
S
S
H
H
N
Me
2-12
14-39
Metformin
Synthetic Derivatives 2-39
IC50 = 1.40 ± 0.01-236.10 ± 2.20 µM

ACCEPTED MANUSCRIPT
Hydrazinyl arylthiazole based pyridine scaffolds: Synthesis, structural characterization, in
vitro α-glucosidase inhibitory activity, and in silico studies
Farman Ali,a Khalid Mohammed Khan,a Uzma Salar,a Muhammad Taha,b,c Nor Hadiani Ismail,b
Abdul Wadood,d Muhammad Riaz,d and Shahnaz Perveen,e
aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
bAtta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM),
Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor D. E. Malaysia
cFaculty of Applied Science Universiti Teknologi MARA, Shah Alam 40450, Selangor D. E.,
Malaysia
dDepartment of Biochemistry, Computational Medicinal Chemistry Laboratory, UCSS, Abdul
Wali Khan University Mardan, Pakistan
ePCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-75280,
Pakistan
Abstract: Acarbose, miglitol, and voglibose are the inhibitors of α-glucosidase enzyme and
being clinically used for the management of type-II diabetes mellitus. However, many adverse
effects are also associated with them. So, the development of new therapeutic agents is an utmost
interest in medicinal chemistry research. Current study is based on the identification of new α-
glucosidase inhibitors. For that purpose, hydrazinyl arylthiazole based pyridine derivatives 1-39
were synthesized via two step reaction and fully characterized by spectroscopic techniques EI-
MS, HREI-MS, 1H-, and 13C-NMR. However, stereochemistry of the iminic bond was confirmed
by NOESY. All compounds were subjected to in vitro α-glucosidase inhibitory activity and
found many folds active (IC50 = 1.40 ± 0.01-236.10 ± 2.20 µM) as compared to the standard
acarbose having IC50 value of 856.45 ± 5.60 µM. A limited structure-activity relationship was
carried out in order to make a presumption about the substituent’s effect on inhibitory activity
which predicted that substituents of more negative inductive effect played important role in the
activity as compared to the substituents of less negative inductive effect. However, in order to
have a good understanding of ligand enzyme interactions, molecular docking study was also
conducted. In silico study was confirmed that substituents like halogens (Cl) and nitro (NO2)
which have negative inductive effect were found to make important interactions with active site
residues.
*Corresponding Author: khalid.khan@iccs.edu; drkhalidhej@gmail.com; Tel. 00922134824910; Fax.
00922134819018
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Keywords: Synthesis; pyridine; hydrazinyl arylthiazole; α-glucosidase; in vitro; structure-
activity relationship; molecular docking.
Introduction
Type-II diabetes mellitus is responsible for around 5% death of global population. α-Glucosidase
enzyme catalyzes the hydrolysis of α-glucosidal bond of complex carbohydrates and releases the
monosaccharide (α-D-glucose) which is absorbable in small intestine [1,2]. Hyperactivity of α-
glucosidase enzyme resulted in elevated level of blood glucose in diabetic patients and termed as
hyperglycemia [3]. Inhibition of α-glucosidase enzyme is one way to treat type-II diabetes
mellitus by suspending the absorption of glucose in intestine [4]. Acarbose, miglitol, and
voglibose are the drugs which are being clinically used for the treatment of type-II diabetes
mellitus (Figure-1). These all are the inhibitors of α-glucosidase enzyme [5,6] but 50% less
effective than the other anti-diabetic drugs such as metformin and sulfonylurea. However,
adverse effects are also linked to them such as abdominal discomfort, diarrhea, and flatulence
[7]. Therefore, these medications are often use in combination with other anti-diabetic agents to
improve the effectiveness. So, it is still an interest in medicinal chemistry to develop a safer
medication to cure diabetes mellitus.
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Figure-1: Clinical drugs for type II diabetes mellitus
Heterocyclic rings are the basic motifs of various biologically active compounds and natural
products [8-11] and have received much attention in drug discovery and lead optimization [12].
Specially, nitrogen-containing heterocycles have lots of applications in pharmaceuticals,
agrochemicals, and functional materials [13-14]. As far as pyridine is concerned, it is the basic
part of many natural products such as vitamin B3, coenzyme vitamin B6 family, and various
alkaloids. These natural products exhibit many interesting biological activities [15-17]
.
Polysubstituted pyridines have been used as antiprion, antibacterial, and anticancer agents. These
were also used as potassium channel openers for the cure of urinary incontinence [18-20].
Furthermore, some derivatives were aslo found to be highly selective ligands for adenosine
receptors [21], a potential targets for the identification of new drugs to treat Parkinson’s disease,
asthma, hypoxia/ischaemia, kidney diseases, and epilepsy [22]. Thiazole ring is the core scaffold
of medicinally important compounds [23] and possess significant biological potential such as
anticonvulsant, antiinflammatory, analgesic, antituberculosis, antiviral, pesticidal, antimicrobial,
anticancer, antitumor, and enzyme inhibition activities [24-32].
There are many reports available on the α-glucosidase inhibitory activity of isolated natural
products such as monoterpenoids, triterpenes, sesquiterpenoids, isoindolin-1-ones, p-terphenyl
scaffolds [33-37]. Our group has identified many heterocyclic compounds having promising α-
glucosidase inhibitory activity (Figure-2) [38-43]. Figure-3 displayed that the new synthetic
compounds have structural resemblance such as pyridine ring, thiazole ring and hydrazine
moiety, with the already identified lead candidates (Figure-2) which prompted us to screen these
hybrid scaffolds for α-glucosidase inhibitory potential. It is worth-mentioning that synthetic
compounds also have the same amidine moiety as in the antidiabetic agent “metformin” (Figure-
3).
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Figure-2: Identified leads for α-glucosidase inhibitory activity
Figure-3: Rationale of the Current Study
This manuscript described the synthesis, structural characterization, in vitro inhibition, and in
silico studies of arylated hydrazinyl thiazole based pyridine derivatives 1-39 as well as
identification of lead compounds against the α-glucosidase enzyme for the treatment of type-II
diabetes mellitus.
Results and Discussion
Chemistry
Arylated hydrazinyl thiazole based pyridine derivatives 1-39 were synthesized via two steps
reaction scheme. In the first step, 3-pyridinecarboxaldehyde or 2/3/4-acetylpyridine was treated
with thiosemicarbazide to afford thiosemicarbazone intermediates (1, 13, 22, and 31). Catalytic
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amount of glacial acetic acid was used in the reaction. In second step thiosemicarbazone
intermediates (1, 13, 22, and 31) undergo the cyclization reaction with substituted phenacyl
bromides to form thiazole ring via Hantzsch reaction (Scheme-1). Reaction progress was
checked by thin layer chromatography (TLC). After completion of the reaction, flask was kept
overnight at room temperature to afford precipitates. Resulting precipitates were washed with
distilled water and crystallized from ethanol to afford the pure products. Purity of compounds
were checked via TLC analysis. Spectroscopic techniques EI-MS, HREI-MS, 1H-, and 13C-NMR
were used to characterize the structures of synthetic compounds 1-39 (Table-1). However, 2D-
NMR (COSY, HSQC, HMBC, and NOESY) of representative and structurally new compound
18 was also carried out.
Scheme-1: Synthesis of hydrazinyl thiazole based pyridine derivatives 1-39
Characteristic spectral feature of representative compound 18
13
1
1H- and C-NMR spectra of compound 18 were recorded in DMSO-d6. In H-NMR spectrum,
the most downfield broad singlet of the NH which is directly attached to thiazole skeleton,
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appeared at δH 11.47. Another downfield signal of H-6 of pyridine skeleton was appeared at δH
8.58 (d, J6,5 = 4.4 Hz), showed ortho coupling with H-5. Downfield chemical shift is due to the
presence of more electronegative nitrogen atom at ortho position. H-3 of pyridine skeleton was
appeared in downfield region at δH 8.03 (d, J3,4 = 8.4 Hz), showed ortho coupling with H-4. A
singlet of H-2ʹʹ was appeared at δH 7.93. Signals of two protons H-4 and H-6ʹʹ were overlapped
and appeared at
δH 7.85. The characteristic singlet of H-5' of thiazole moiety was appeared at δH
7.60. H-5 was appeared as a triplet at 7.46 (t, J5(4,6) = 8.0 Hz). Signals of two H-4ʹʹ, H-5ʹʹ
overlapped and appeared at
2.40 (Figure-4).
δH 7.37. The protons of iminic methyl was appeared as a singlet at δH
Figure-4: 1H-NMR and 13C-NMR chemical shifts of compound 18
13C-NMR broad-band decoupled spectrum displayed total sixteen carbon signals, including six
quarternary, nine methine, and one methyl carbons. The quaternary carbon of thiazole ring,
attached to the more electronegative nitogen and sulfur atoms, was the most downfield signal
and appeared at
ring which is lying adjacent to nitrogen and imine bond and appeared at
C-4ʹ of thiazole and CH-6 of pyridine ring which are directly attached to electronagative nitrogen
atom, were resonated at C 149.1 and C 148.5, respectively. The quaternary carbon of imine was
resonated at C 138.3. Two quaternary carbons of phenyl C-3ʹʹ and C-1ʹʹ were resonated at δC
δ
C 169.4. The second most downfield signal was the quaternary C-2 of pyridine
δ
C 154.8. The quaternary
δ
δ
δ
133.4 and δC 124.2, respectively. Four aromatic methine carbons (CH-2ʺ, CH-4ʺ, CH-5ʺ, and
CH-6ʺ) and three methine carbons of pyridine ring (CH-3, CH-4, and CH-5) were appeared in
the usual aromatic region
δ
C 136-119. While characteristic C-5ʹ of thiazole ring was resonated at
C 12.2 (Figure-4).
δ
C 106.1. The most upfield iminic methyl was appeared at
δ
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The 13C/1H-NMR chemical shifts were assigned on the basis of COSY, HSQC, and HMBC
correlations. Stereochemistry of the imine group (C=N) was found as E-configuration after
analyzing the NOESY spectrum in which iminic methyl protons (CH3-C=N) showed NOESY
interaction with the NH (Figure-4).
EI-MS of compound 18 showed the molecular ion peak [M]+ at m/z 328 and isotopic peak
[M+2]+ at m/z 330 in a ratio of 3:1 which confirmed the presence of chlorine atom. HREI-MS of
compound 18 displayed M+ at m/z 328.0544 with a composition of C16H13ClN4S (Calcd.
328.0549) which confirmed the formation of the desired molecule. However, the key
fragmentation pattern obeserved in the spectrum showed that molecular ion undergo cleavage by
three ways (a-c).
(a)
Moleuclar ion M+ at m/z 328 undergoes homolytic cleavage by the loss of methyl radical
to yield a cation at m/z 313 which will further fragmented to yield another cation at m/z
223.
(b)
(c)
In second way, moleuclar ion undergoes homolytic cleavage by the loss of pyridiyl
radical to afford a cation at m/z 250 which is also the base peak in the spectrum.
In third way, moleuclar ion undergoes complex series of homolytic cleavage to afford a
radical cation at m/z 79 (Figure-5).
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Figure-5: Key EI-MS fragmentation of compound 18
In vitro α-glucosidase inhibitory activities
All arylated hydrazinyl thiazole based pyridine derivatives 1-39 were screened to evaluate in
vitro α-glucosidase inhibitory activity. All compounds demonstrated excellent and potent
inhibitory activity in the range of IC50 = 1.40 ± 0.01-236.10 ± 2.20 µM as compared to the
standard acarbose (IC50 = 856.45 ± 5.60 µM) (Table-1).
Table-1: In vitro α-glucosidase inhibitory activities of synthetic compounds 1-39
Comp.
No.
Comp.
No.
IC50 ± SEMa [
µM]
IC50 ± SEMa [
µM]
R
R
Category “A”
-
120.20 ± 1.10
28.50 ± 0.25
46.20 ± 0.40
1
2
7
8
177.10 ± 1.10
236.10 ± 2.20
6.10 ± 0.01
3
9
61.60 ± 0.50
113.10 ± 1.10
96.20 ± 0.80
70.10 ± 0.68
23.50 ± 0.20
13.40 ± 0.10
4
5
6
10
11
12
Category “B”
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-
120.60 ± 1.10
24.10 ± 0.24
5.50 ± 0.10
1.40 ± 0.01
13
14
18
19
15
86.20 ± 0.80
20
21
13.10 ± 0.15
6.40 ± 0.10
54.40 ± 0.60
41.60 ± 0.45
16
17
Category “C”
-
86.40 ± 0.70
21.10 ± 0.20
2.50 ± 0.01
22
23
27
36.10 ± 0.30
28
163.20 ± 1.40
40.10 ± 0.40
35.60 ± 0.35
8.10 ± 0.10
24
25
29
30
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53.60 ± 0.50
26
Category “D”
-
115.50 ± 1.10
26.20 ± 0.24
11.60 ± 0.15
31
32
36
124.40 ± 1.20
37
168.50 ± 1.40
31.10 ± 0.30
33
38
63.10 ± 0.55
85.10 ± 0.80
10.10 ± 0.10
856.45 ± 5.60
34
35
39
Standardc = Acarbose
aIC50 (mean ± standard error of mean); Standardc (Inhibitor for α-Glucosidase).
Structure-activity relationship (SAR)
Limited structure-activity relationship (SAR) was rationalized by observing the different
substitution pattern at the aryl part (R). Although all constant structural features such as pyridine
ring, thiazole ring, and Schiff base moiety are cordially playing their part in demonstrating the
inhibitory potential, however, the varying features such as aryl ring “R” are responsible for
variation in the inhibitory activity (Figure-6).
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Figure-6: General structural features of synthetic compounds
For better understanding of structure-activity relationship, compounds were categorized into four
categories “A-D”, on the basis of four different thiosemicarbazone intermediates which are the
precursors of rest of the cyclized products. Thiosemicarbazone intermediates such as 1 (IC50 =
120.20 ± 1.10 µM), 13 (IC50 = 120.60 ± 1.10 µM), and 31 (IC50 = 115.50 ± 1.10 µM) showed
potent and comparable α-glucosidase inhibitory activity as compared to standard acarbose (IC50 =
856.45 ± 5.60 µM). Although intermediate 22 (IC50 = 86.40 ± 0.70 µM) showed more enhanced
activity than intermediate 1 and positional isomers 22 and 31. Might be the compound 22
attained the conformation which will better interact with the active site (Figure-7).
Figure-7: Structure-activity relationship of thiosemicarbazone intermediates 1, 13, 22, 31
Results depicted in Table-1 showed that most of the cyclized products displayed far better
activity than thiosemicarbazone intermediates. All compounds 2-12 belongs to category “A”
showed many folds enhanced activity in the range IC50 = 6.10-236.10 µM as compared to the
standard acarbose (IC50 = 856.45 ± 5.60 µM). Compound 9 (IC50 = 6.10 ± 0.01 µM) with m,p-
dichloro substitutions at aryl ring showed more than hundred folds better activity than standard
acarbose. Its structurally similar compound 10 (IC50 = 70.10 ± 0.68 µM) with o,p-dichloro
substitutions showed eleven times less activity than 9. It shows that dichloro groups are playing a
crucial part in the activity when present at meta and para positions. However, mono-chloro
compounds 7 (IC50 = 28.50 ± 0.25 µM) and 8 (IC50 = 46.20 ± 0.40 µM) showed many folds
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decreased activity as compared to dichloro compound 9 (Figure-8). It showed that number and
positions of the substituent like chloro played an important role in exhibiting the inhibitory
potential. Activity of compounds 7 (IC50 = 28.50 ± 0.25 µM) and 8 (IC50 = 46.20 ± 0.40 µM) can
compared with compounds 6 (IC50 = 96.20 ± 0.80 µM) and 5 (IC50 = 113.10 ± 1.10 µM),
respectively, having the bromo instead of chloro, about two fold decreased activity was
observed. In case of compounds 11 (IC50 = 23.50 ± 0.20 µM) and 12 (IC50 = 13.40 ± 0.10 µM)
having nitro and hydroxy groups, respectively, a better activity was observed. It was experienced
that compounds with substituents having more negative inductive effect such as Cl, NO2 and OH
showed more potent inhibition than the compounds with less negative inductive effect (Figure-
8).
Figure-8: Structure-activity relationship of compounds 5-12
Similar activity trend was observed in the category “B” like category “A”. m,p-Dichloro
substituted analog 19 (IC50 = 1.40 ± 0.01 µM) was found to be the most active derivative of the
whole series and six hundred times more active than the standard acarbose (IC50 = 856.45 ± 5.60
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µM). Mono-chloro derivative 18 (IC50 = 5.50 ± 0.10 µM) was found to be less active than
dichloro compound 19 (IC50 = 1.40 ± 0.01 µM). Similarly, bromo substituted compounds such as
16 (IC50 = 54.40 ± 0.60 µM) and 17 (IC50 = 41.60 ± 0.45 µM) showed less inhibitory activity.
Compounds having substituents like OH and NO2 such as compounds 20 (IC50 = 13.10 ± 0.15
µM) and 21 (IC50 = 6.40 ± 0.10 µM) demonstrated good inhibitory potential than the compound
14 (IC50 = 24.10 ± 0.24 µM) with no substitution and compound 15 (IC50 = 86.20 ± 0.80 µM)
with biphenyl as R (Figure-9).
Cl
Me
H
N
N
N
Br
NO2
N
S
Me
Me
H
H
N
N
N
N
N
18
N
IC50 = 5.50 ± 0.10 µM
N
S
N
S
16
20
IC50 = 54.40 ± 0.60 µM
IC50 = 13.10 ± 0.15 µM
Cl
Me
H
Me
H
OH
N
N
Me
Cl
N
H
N
N
N
Br
N
N
N
S
N
N
S
N
S
19
17
21
IC50 = 1.40 ± 0.01 µM
IC50 = 41.60 ± 0.45 µM
IC50 = 6.40 ± 0.10 µM
Me
Me
H
N
H
N
N
N
N
N
N
S
N
S
14
15
IC50 = 24.10 ± 0.24 µM
IC50 = 86.20 ± 0.80 µM
Figure-9: Structure-activity relationship of compounds 14-21
It is worth-noting that the most active derivative of category “C” was again found to be a
dichloro analog 28 (IC50 = 2.50 ± 0.01 µM). Absence of chloro group at para position as in
compound 27 (IC50 = 21.10 ± 0.20 µM) showed a decreased activity. Compound 30 (IC50 = 8.10
± 0.10 µM) with the m-hydroxy substitution also showed potent inhibition. Switching of hydroxy
to nitro group as in 29 (IC50 = 35.60 ± 0.35 µM), a decreased activity was observed. Similarly,
bromo substituted compounds 25 (IC50 = 40.10 ± 0.40 µM) and 26 (IC50 = 53.60 ± 0.50 µM) were
demonstrated less inhibition than hydroxy and nitro substituted derivatives. Compound 24 (IC50
= 163.20 ± 1.40 µM) with the biphenyl ring as “R” was found to be the least active among
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category “C”. Least activity of biphenyl derivative might be either due to the increased carbon
load or it is not fulfilling the conformational requirement to fit well in the active site (Figure-10).
Cl
Me
H
N
N
N
Br
S
NO2
Me
Me
N
H
H
N
N
N
N
N
27
N
IC50 = 21.10 ± 0.20 µM
S
S
N
N
25
29
IC50 = 40.10 ± 0.40 µM
IC50 = 35.60 ± 0.35 µM
Cl
Me
H
Me
H
OH
N
N
Me
Cl
N
H
N
N
N
Br
N
N
S
N
N
S
S
N
N
28
26
30
IC50 = 2.50 ± 0.01 µM
IC50 = 53.60 ± 0.50 µM
IC50 = 8.10 ± 0.10 µM
Me
Me
H
H
N
N
N
N
N
N
S
S
N
N
23
24
IC50 = 36.10 ± 0.30 µM
IC50 = 163.20 ± 1.40 µM
Figure-10: Structure-activity relationship of compounds 23-30
In case of category “D”, compound 39 (IC50 = 10.10 ± 0.10 µM) with m-hydroxy substitution
was found to be the most active derivative and compound 37 (IC50 = 11.60 ± 0.15 µM) with m,p-
dichloro substitutions was the second most active member of this category. Compound 26 (IC50 =
26.20 ± 0.24 µM) with m-chloro group showed less activity than dichloro analog. Switching
from chloro to bromo group as in case of compounds 34 (IC50 = 63.10 ± 0.55 µM) and 35 (IC50 =
85.10 ± 0.80 µM), a less inhibition was experienced. Similarly, compound 38 (IC50 = 31.10 ±
0.30 µM) with m-nitro group also showed comparable activity with the most active compounds
of this category. However, compound 32 (IC50 = 124.40 ± 1.20 µM) with no substitution and
compound 33 (IC50 = 168.50 ± 1.40 µM) with biphenyl ring as “R”, were found to be least active
analogs of this category (Figure-11).
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Figure-11: Structure-activity relationship of compounds 32-39
In a nut shell, the whole series was found to be the more potent than standard acarbose.
However, the limited structure-activity relationship revealed that the compounds with
substituents having more negative inductive effect were found to be more potent than the
compounds with the substitutions with less negative inductive effect. To rationalize the
interaction of these compounds with the active site of enzyme, in silico studies were also carried
out.
Molecular docking studies
All synthetic derivatives 1-39 were docked into the binding pocket of α-glucosidase enzyme by
using the Molecular Operating Environment (MOE) software and Triangular Matching docking
method (default). Ten different conformations for each compound were generated. Ligands were
allowed to be flexible during the docking in order to acquire the minimum energy structures.
Ligands were ranked by the scores from the GBVI/WSA binding free energy calculation in the S
field by using the software which is the score of the last stage. For all the scoring functions,
lower scores show the more favorable pose. The top ranked conformation of each derivative was
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selected for further analysis on the basis of docking score (S). At the end of docking, the
predicted ligand-protein complexes were analyzed for molecular interactions and their 3D
images were taken by using LigPlot implemented in MOE.
Interactions details of synthetic compounds
The catalytic site of the modeled α-glucosidase on the basis of the crystallographic structure of
Saccharomyces cerevisiae isomaltase (PDB code 3AJ7) is composed of Asp214, Glu276, and
Asp349 residues. The molecular docking studies also disclosed some other important residues
like Pro309, Phe157, Asn347, Arg312, Glu304, His279, and His348 in the α-glucosidase
inhibition which showed a slightly different docking profile in the α-glucosidase inhibition. To
discuss the interactions details of ligands and protein, all the thirty nine synthesized compounds
were classified into four different groups on the basis of their structural similarities. The
comparative analysis of ligand-protein interactions for each group was carried out in detail.
Interactions of the hydrazonomethyl pyridine 4-arylthiazole group of compounds
Compounds 1 and 2-12 are classified as hydrazonomethyl pyridine 4-phenylthiazole compounds
on the basis of their structural similarities. All these compounds have common hydrazonomethyl
pyridine 4-phenylthiazole group in their structures. According to the IC50 value, compound 9
(IC50 = 6.10 ± 0.01 µM) was the most active compound of this group. Figure-12a showed that
compound 9 was well fit into the binding cavity of α-glucosidase showing four interactions with
the residues Pro309, Phe157, and Asn347. Pro309 residue is participated in side chain hydrogen
donor interaction with the para-chlorine of dichlorobenzene. Phe157 showed two π-hydrogen
interactions with the π-electrons of dichlorobenzene and thiazole moiety of the compound.
Asn347 was found in another π-hydrogen interaction with the π-electrons of terminal pyridine
group.
Compound 11 is the second most active compound (IC50 = 23.50 ± 0.20 µM) in this group
formed three noticeable interactions with the binding site residues Arg312, Glu304, and His348
as displayed in Figure-12b. Arg312 showed π-hydrogen interaction with the pyridine ring of
compound.
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A side chain hydrogen donor interaction was also percieved between Glu304 and the sulfur of
thiazole. The third interaction was backbone hydrogen acceptor interaction between His348 and
oxygen of the nitro group of nitrobenzene moiety.
Figure-12c showed that compound 7 (IC50 = 28.50 ± 0.25 µM) is forming three interactions with
active site residues Asn347, Phe577, and His279. Asn347 was involved in side chain hydrogen
acceptor interaction with the nitrogen of hydrazone group. Phe177 showed π-hydrogen
interaction with the thiazole group and His279 formed another π-hydrogen interaction with the
chlorobenzene ring of the compound.
Compound 8 with IC50 value 46.20 ± 0.40 µM is the fourth most active compound in this group
and showed two different interactions with the residues Glu276 and His279 as shown in Figure-
12d. Glu276 showed a hydrogen donor interaction with the sulfur of thiazole group. His279 was
observed in π-hydrogen interaction with the chlorobenzene group.
Structural features such as presence of a group with more negative inductive effect like halogen
and nitro (NO2) groups were found to show good interaction mode and active nature of
compound. Among halogens, Cl containing compounds were found superior than Br. Similarly,
NO2 group containing compound (11) showed good result than compounds having single Cl/Br.
However, compounds containing two Cl groups at meta-para position (8) showed best
interaction mode than nitro containing compound. The brief interaction detail of this group and
group 2 i.e. compound 1 to 11, 13 and 22 is given in Table-1S (see the supporting information).
In addition to the catalytic residues, molecular docking studies of hydrazonomethyl pyridine 4-
phenylthiazole compounds predicted that residues like Pro309, Phe157, Asn347, Arg312,
Glu304, His279, and His348 have also played an important role in the α-glucosidase inhibition.
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Figure-12: 3D pictures of the docked conformations, (a) compound 9, (b) compound 11, (c)
compound 7, and (d) compound 8
Interactions of the hydrazonomethyl pyridine methanethioamide group of compounds
In order to discuss the interaction detail, compounds 1, 13 and 22 were placed in the second
group on the basis of their structural similarity i.e. having a common hydrazonomethyl pyridine
methanethioamide group. Compound 1 showed two important interactions with the residues
Asp349 and His111 (Table-1S). Acidic Asp349 showed a side chain hydrogen donor interaction
with nitrogen of thiourea group and His111 formed a hydrogen acceptor interaction with the
sulfur of the same group of compound (Table-1S). Compound 13 showed single H-donor
interaction with Asp214 using nitrogen of thiourea group. Compound 22, the most active
compound of this group formed three interactions with the residues Asp349, His111, and Phe177
(Table-1S). Asp349 showed H-donor interaction with the nitrogen of thiourea linker and an H-
acceptor interaction was observed between the sulfur of thiourea and His111. Whereas, Phe177
formed a π-hydrogen bond with the π system of pyridine moiety of compound (Table-1S).
Thiourea group and pyridine with para attachment were observed as active moieties in this group
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of compounds. Interactions of the hydrazonomethyl pyridine methanethioamide group of
compounds predicted the possible inhibitory role of His111 and Phe177 in addition to the
catalytic site residues Asp214 and Asp349.
Interactions of the ortho-hydrazonoethyl pyridine 4-arylthiazole group of compounds
Compounds 14 to 20 were grouped on the basis of structural similarity in a common
hydrazonoethyl pyridine 4-phenylthiazole group. Compound 19 showed highest activity (IC50 =
1.40 ± 0.01 µM) in this group as well as in all of the synthesized compounds and showed four
important interactions with the residues Asp349, Asn241, His279, and Phe300 as shown in
Figure-13a. Asp349 formed H-donor interaction with sulfur of thiazole moiety and a similar
interaction was observed between Asn241 and para-Cl of dichlorobenzene group of the
compound. A π-hydrogen interaction was found between His279 and π system of
dichlorobenzene of compound. Another π-hydrogen bond was observed between Phe300 and
thiazole ring.
Compound 18 the second most active member (IC50 = 5.50 ± 0.10 µM) of this group was found
forming three interactions with the residues Asp408, Phe157, and Arg312 as shown in Figure-
13b. Acidic Asp408 showed H-donor interaction with sulfur of thiazole group and Phe157
formed π-hydrogen interaction with chlorobenzene ring of the compound. Another π-hydrogen
interaction was found between the same chlorobenzene ring and basic Arg312. Compound 20
with IC50 value of 13.10 ± 0.15 µM is the third most active compound in this group and showed
two different interactions with the residues Glu304 and Phe157 as shown in Figure-13c. Glu304
showed a hydrogen donor interaction with the sulfur of thiazole group. Phe157 was observed in
π-π interaction with the nitrobenzene group. Compound 14, according to IC50 value, the fourth
member (IC50 = 24.10 ± 0.24 µM) of this group showed single H-acceptor interactions with the
Asn347 using the lone pair of N of pyridine ring as shown in Figure-13d. The addition of a
methyl group adjacent to pyridine ring in the members of this group was observed to improve the
interaction mode particularly for the compounds having Cl/NO2 groups i.e. 19, 18 and 20. The
summary of interactions of compounds of this group and group 4 (14 to 39) is given in Table-2S
(see the supporting information). The docking profile of the ortho-hydrazonoethyl pyridine 4-
arylthiazole group of compounds discloses the inhibition role of some additional important
residues like Arg312, glu304, Phe157, Asn241, His279, and Phe300, making the target more
interesting for further investigation.
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Figure-13: 3D pictures of the docked conformations, (a) compound 19, (b) compound 18, (c)
compound 20, and (d) compound 14
Interactions of the para/meta-hydrazonoethyl pyridine 4-arylthiazole group of compounds
On the basis of structural similarity in para/meta-hydrazonoethyl pyridine 4-phenylthiazole
groups, compounds 12, 21, 23, to 39 were grouped together. According to the IC50 value,
compound 28 (IC50 = 2.50 ± 0.01 µM) is the most active compound of this group. It is clear from
Figure-14a that compound 28 is showing three interactions in the binding cavity of α-glucosidase
with the residues Glu304, Phe157, and Arg312. Glu304 is involved in side chain H-donor
interaction with the sulfur of thiazole moiety of compound. Phe157 showed a π-hydrogen
interactions with the π-electrons of dichlorobenzene ring of the compound. Arg312 was found in
another π-hydrogen interaction with the π-electrons of the same dichlorobenzene group.
Compound 21 with IC50 value of 6.40 ± 0.10 µM also showed considerable interactions with the
active site residues. The binding mode of compound 28 is shown in Figure-14b. Compound 21
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showed interactions with Asn241, Phe177, and His279. Asn241 formed H-donor interaction with
the OH group of phenolic moiety of compound while Phe177 and His279 formed π-hydrogen
interactions with the π system of thiazole and phenolic ring, respectively. Comparing the binding
mode of compound 21 and 28 it was observed that dichlorobenzene group in compound 28 and
phenolic group in 21 imparts almost similar interaction strength i.e. one polar and two π-
hydrogen interactions. Compound 30, a para analog of compound 21, showed three π-hydrogen
interactions with the active site residues His245, Phe158, and Arg312 as depicted in Figure-14c.
His245, Phe158, and Arg312 formed π-hydrogen interactions with the π system of pyridine,
thiazole, and phenolic ring, respectively. Para attachment of pyridine ring in compound 30
resulted in π-interactions only with no hydrogen bond as compared to compound 21. The meta
analog of compound 21 and 30, compound 39 also showed three interactions, a hydrogen bond
and two π-interactions as presented in Figure-14d. Asp408 performed H-donor bonding with the
lone pair of sulfur of thiazole. Whereas Phe157 and Arg312 formed π-hydrogen interactions with
thiazole and phenolic ring, respectively. In addition to OH and Cl groups, the para attachment of
pyridine ring in this group of compounds were observed accountable for their good interaction
modes. Regarding the binding site residues involved, the docking profile of the para/meta-
hydrazonoethyl pyridine 4-arylthiazole group of compounds was observed almost similar to the
docking profile of hydrazonomethyl pyridine 4-phenylthiazole compounds. The interaction detail
given in Table-1S and -2S clearly predict the inhibiting capability of all these synthetic
compounds for α-glucosidase enzyme. The docking study of the thirtynine synthetic compounds
and the valuable variations of their binding site residues in addition to the catalytic site of the
enzyme may lead to further investigate the importance of these very interesting interactions.
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Figure-14: 3D pictures of the docked conformations, (a) compound 28, (b) compound 21, (c)
compound 30, and (d) compound 39
Conclusion
Synthetic hydrazinyl arylthiazole based pyridine derivatives 1-39 were screened for α-
glucosidase inhibitory activity in vitro. All analogs exhibited potent inhibition in the range of
IC50 = 1.40-236.10 µM as compared to the standard acarbose (IC50 = 856.45 ± 5.60 µM).
Molecular docking study was conducted to understand the ligand-enzyme interactions and the
structure-activity relationship. This study identified a library of lead molecules which can be
used in further advance research in order to obtain a powerful inhibitor for α-glucosidase enzyme
for the development of insulin-independent antidiabetic agents.
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Experimental
Materials and Methods
All reagents and solvents were purchased from Sigma-Aldrich, USA. Thin layer chromatography
(TLC) was performed on pre-coated silica gel, GF-254 (Merck, Germany). Spots were visualized
under ultraviolet light at 254 and 366 nm. Mass spectra were recorded under on MAT 312 and
MAT 113D mass spectrometers. The 1H-, 13C-NMR were recorded on Bruker AM
spectrometers, operating at 300, 400 and 500 MHz. The chemical shift values are presented in
ppm (δ), relative to tetramethylsilane (TMS) as an internal standard and the coupling constant (J)
are in Hz.
General procedure for the synthesis of thiosemicarbazone derivatives of pyridine (1, 13, 22,
and 31)
3-Pyridinecarboxaldehyde or 2/3/4-acetylpyridine (10 mmol), thiosemicarbazide (10 mmol) and
2-4 drops of glacial acetic acid in ethanol (10 mL) into a 100 mL round-bottomed flask. Reaction
mixture was refluxed for 3 hours. Reaction progress was checked by periodic TLC. After
reaction completion, solvent was evaporated and crude product was washed with distilled water
and then hexane. Product was crystallized from ethanol.
General procedure for the synthesis of hydrazinyl arylthiazole based pyridine derivatives
1-39
Thiosemicarbazones 1/13/22/31 (1 mmol), phenacyl bromides (1 mmol) and triethyl amine (1
mmol) in ethanol (15 mL) were refluxed for 3 h into a 100 mL round-bottommed flask. Reaction
progress was checked by periodic TLC. Precipitates were formed after keeping the flask
overnight at room temperature. The solid product was filtered, washed with excess distilled
water and dried in vacuum. Product was crystallized from ethanol to afford pure title compounds.
2-(Pyridin-3-ylmethylene)hydrazinecarbothioamide (1)
Yield: 89%; m.p: 227-229 ᵒC; 1H-NMR (400 MHz, DMSO-d6) δ 11.55 (s, 1H, NH), 8.91 (d, J2,4
= J2,6 = 2.0 Hz, 1H, H-2), 8.55 (d, J6,4 = 1.6 Hz, J6,5 = 4.8 Hz, 1H, H-6), 8.26 (m, 2H, H-4, NHa),
8.10 (brd.s, 1H, NHb), 8.05 (s, 1H, H-7), 7.24 (m, 1H, H-5); 13C-NMR (125 MHz, DMSO-d6):
δ
178.2, 150.2, 148.7, 139.2, 133.8, 130.1, 123.7; EI-MS m/z (% rel. abund.): 179.9 (M+, 100),
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120.0 (57), 105.0 (74), 76.0 (60), 51.0 (17); HREI-MS Calcd for C7H8N4S: m/z = 180.0470,
Found 180.0477.
4-Phenyl-2-(2-(pyridin-3-ylmethylene)hydrazinyl)thiazole (2)
Yield: 89%; m.p: 204-205 ᵒC; 1H-NMR (400 MHz, DMSO-d6) δ 12.47 (s, 1H, NH), 8.89 (d, J2,4
= 1.6 Hz, 1H, H-2), 8.64 (dd, J6,4 = 1.6 Hz, J6,5 = 5.2 Hz 1H, H-6), 8.27 (d, J4,5 = 8.0 Hz, 1H, H-
4), 8.09 (s, 1H, H-7), 7.86 (d, J2'', 3'' = J6'', 5'' = 7.2 Hz, 2H, H-2'', 6''), 7.65 (m, 1H, H-5), 7.24 (t, J3′′
(2′′, 4′′) = J5′′ (4′′, 6′′) = 7.6 Hz, 2H, H-3′′, 5′′), 7.37 (s, 1H, H-5'), 7.31 (t, J4′′ (3′′, 5′′) = 7.6 Hz, 1H, H-4′′);
13C-NMR (100 MHz, DMSO-d6):
δ 167.8, 146.7, 145.0, 136.9, 135.7, 134.5, 131.7, 128.6, 128.6,
127.6, 125.5, 125.5, 125.1, 117.2, 104.3; EI-MS m/z (% rel. abund.): 280.0 (M+, 68), 176.0
(100), 134.0 (77); HREI-MS Calcd for C15H12N4S: m/z = 280.0783, Found 280.0797.
4-(Biphenyl-4-yl)-2-(2-(pyridin-3-ylmethylene)hydrazinyl)thiazole (3)
Yield: 89%; m.p: 203-205 ᵒC; 1H-NMR (300 MHz, DMSO-d6) δ 12.38 (s, 1H, NH), 8.81 (s, 1H,
H-2), 8.56 (d, J6,5 = 4.8 Hz, 1H, H-6), 8.07 (ovp, 2H, H-4, 7), 7.95 (d, J2'', 3'' = J6'', 5'' = 8.1 Hz, 2H,
H-2'', 6''), 7.73 (d, J3'', 2'' = J5'', 6'' = J2′′′, 3′′′ = J6′′′, 5′′′ = 8.4 Hz, 4H, H-3'', 5'', 2′′′, 6′′′), 7.49 (ovp, 4H,
H-5', 3′′′, 4′′′, 5′′′), 7.38 (t, J5 (4, 6) = 7.2 Hz, 1H, H-5); 13C-NMR (100 MHz, DMSO-d6):
δ 168.0,
150.2, 149.5, 147.6, 139.6, 139.1, 138.1, 133.7, 132.9, 130.5, 128.9, 128.9, 127.5, 126.8, 126.8,
126.5, 126.5, 126.1, 126.1, 124.1, 104.2; EI-MS m/z (% rel. abund.): 356.0 (M+, 77), 252.0
(100), 210 (44); HREI-MS Calcd for C21H16N4S: m/z = 356.1096, Found 356.1118.
4-(2-(2-(Pyridin-3-ylmethylene)hydrazinyl)thiazol-4-yl)benzonitrile (4)
Yield: 89%; m.p: 252-254 ᵒC; 1H-NMR (300 MHz, DMSO-d6) δ 12.42 (s, 1H, NH), 8.80 (s, 1H,
H-2), 8.55 (d, J6,5 = 4.5 Hz, 1H, H-6), 8.07 (s, 1H, H-7), 8.04 (d, J4,5 = J2'', 3'' = J6'', 5'' = 8.1 Hz, 3H,
H-4, 2′′, 6′′), 7.87 (d, J3'', 2'' = J5'', 6'' = 8.1 Hz, 2H, H-3'', 5''), 7.66 (s, 1H, H-5'), 7.45 (m, 1H, H-5),
7.24 (t, J3′′(2′′, 4′′) = J5′′(4′′,6′′) = 7.6 Hz, 2H, H-3′′, 5′′), 7.37 (s, 1H, H-5'), 7.47 (m, 1H, H-5); 13C-
NMR (100 MHz, DMSO-d6):
δ 168.3, 149.9, 148.8, 147.9, 138.6, 132.6, 132.6, 132.6, 130.1,
126.0, 126.0, 123.9, 118.9, 118.9, 109.6, 107.8; EI-MS m/z (% rel. abund.): 305.0 (M+, 78),
201.0 (100), 159.0 (81),79.0 (9), 51.0 (6); HREI-MS Calcd for C16H11N5S: m/z = 305.0735,
Found 305.0760.
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4-(3-Bromophenyl)-2-(2-(pyridin-3-ylmethylene)hydrazinyl)thiazole (5)
Yield: 89%; m.p: 206-208 ᵒC; 1H-NMR (300 MHz, DMSO-d6) δ 12.37 (s, 1H, NH), 8.80 (s, 1H,
H-2), 8.55 (d, J6,5 = 4.2 Hz, 1H, H-6), 8.06 (ovp, 3H, H-4, 7, 6''), 7.86 (d, J4'', 5'' = 7.5 Hz, 1H, H-
4''), 7.51 (s, 1H, H-5'), 7.47 (ovp, 2H, H-2'',5''), 7.39 (t, J5 (4, 6) = 7.8 Hz, 1H, H-5); 13C-NMR (125
MHz, DMSO-d6):
δ 168.1, 149.8, 148.8, 147.9, 138.4, 136.8, 132.6, 130.8, 130.2, 130.1, 128.1,
124.3, 123.9, 122.0, 105.6; EI-MS m/z (% rel. abund.): 358.1 (M+, 50), 360.1 (M+ + 2 , 50),
254.0 (100), 174.0 (100), 51.1 (6); HREI-MS Calcd for C15H11BrN4S: m/z = 357.9888, Found
357.9894.
4-(4-Bromophenyl)-2-(2-(pyridin-3-ylmethylene)hydrazinyl)thiazole (6)
Yield: 89%; m.p: 222-224 ᵒC; 1H-NMR (300 MHz, DMSO-d6) δ 12.36 (s, 1H, NH), 8.80 (s, 1H,
H-2), 8.55 (d, J6,5 = 4.5 Hz, 1H, H-6), 8.06 (s, 1H, H-7), 8.06 (d, J4,5 = 8.4 Hz, 1H, H-4), 7.81 (d,
J2'', 3'' = J6'', 5'' = 8.4 Hz, 2H, H-2'', 6''), 7.60 (d, J3'', 2'' = J5'', 6'' = 8.4 Hz, 2H, H-3'', 5''), 7.47 (t, J5 (4, 6)
= 5.1 Hz, 1H, H-5), 7.43 (s, 1H, H-5'); 13C-NMR (100 MHz, DMSO-d6):
δ 168.1, 149.9, 149.4,
147.9, 138.4, 133.8, 133.8, 132.6, 131.5, 131.5, 130.3, 127.5, 123.9, 120.5, 104.9; EI-MS m/z (%
rel. abund.): 358.1 (M+, 52), 360.1 (M+ + 2, 50), 256.1 (91), 174.1 (100), 79.0 (10); HREI-MS
Calcd for C15H11BrN4S: m/z = 357.9888, Found 357.9900.
4-(4-Chlorophenyl)-2-(2-(pyridin-3-ylmethylene)hydrazinyl)thiazole (7)
Yield: 89%; m.p: 223-225 ᵒC; 1H-NMR (300 MHz, DMSO-d6) δ 12.36 (s, 1H, NH), 8.80 (s, 1H,
H-2), 8.55 (d, J6,5 = 4.5 Hz, 1H, H-6), 8.06 (s, 1H, H-7), 8.06 (d, J4,5 = 9.3 Hz, 1H, H-4), 7.87 (d,
J2'', 3'' = J6'', 5'' = 8.4 Hz, 2H, H-2'', 6''), 7.47 (ovp, 4H, H-5, 5', 3'', 5''); 13C-NMR (125 MHz,
DMSO-d6): δ, 168.1, 149.8, 149.3, 147.9, 138.3, 133.4, 132.6, 131.9, 130.2, 128.6, 128.6, 127.2,
127.2, 123.9, 104.8; EI-MS m/z (% rel. abund.): 314.0 (M+, 53), 316.0 (M+ + 2, 20), 210.0 (100),
168.0 (42), 51.0 (7); HREI-MS Calcd for C15H11ClN4S: m/z = 314.0393, Found 314.0415.
4-(3-Chlorophenyl)-2-(2-(pyridin-3-ylmethylene)hydrazinyl)thiazole (8)
Yield: 89%; m.p: 202-204 ᵒC; 1H-NMR (300 MHz, DMSO-d6) δ 12.37 (s, 1H, NH), 8.80 (s, 1H,
H-2), 8.55 (d, J6,5 = 3.9 Hz, 1H, H-6), 8.06 (ovp, 2H, H-4, 7), 7.89 (s, 1H, H-2''), 7.83 (d, J6'', 5''
=
7.5 Hz, 1H, H-6''), 7.51 (s, 1H, H-5'), 7.45 (m, 2H, H-4'', 5''), 7.40 (t, J5 (4, 6) = 7.8 Hz, 1H, H-5);
13C-NMR (75 MHz, DMSO-d6):
δ
168.1, 149.8, 149.0, 147.9, 138.4, 136.5, 133.4, 132.6, 130.5,
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130.2, 127.2, 125.1, 124.0, 123.9, 105.6; EI-MS m/z (% rel. abund.): 313.9 (M+, 47), 315.9 (M+ +
2 , 18), 209.9 (100), 167.9 (100), 79.0 (6), 51.0 (6); HREI-MS Calcd for C15H11ClN4S: m/z =
314.0393, Found 314.0406.
4-(3,4-Dichlorophenyl)-2-(2-(pyridin-3-ylmethylene)hydrazinyl)thiazole (9)
Yield: 89%; m.p: 237-239 ᵒC; 1H-NMR (300 MHz, DMSO-d6) δ 12.38 (s, 1H, NH), 8.80 (s, 1H,
H-2), 8.55 (d, J6,5 = 4.5 Hz, 1H, H-6), 8.06 (ovp, 3H, H-4, 7, 2''), 7.85 (dd, J6'', 2'' = 1.5 Hz, J6'', 5''
8.4 Hz, 1H, H-6''), 7.67 (d, J5'', 6'' = 8.4 Hz, 1H, H-5''), 7.57 (s, 1H, H-5'), 7.47 (m, 1H, H-5); 13C-
NMR (75 MHz, DMSO-d6): 168.2, 149.9, 148.0, 147.9, 138.6, 135.1, 132.6, 131.4, 130.8,
=
δ
130.2, 129.7, 127.1, 125.5, 123.9, 106.2; EI-MS m/z (% rel. abund.): 347.9 (M+, 83), 349.9 (M+ +
2 , 71), 243.9 (100), 207.9 (69), 79.0 (10), 51.0 (6); HREI-MS Calcd for C15H10Cl2N4S: m/z =
348.0003, Found 347.9995.
4-(2,4-Dichlorophenyl)-2-(2-(pyridin-3-ylmethylene)hydrazinyl)thiazole (10)
Yield: 89%; m.p: 166-168 ᵒC; 1H-NMR (300 MHz, DMSO-d6) δ 12.36 (s, 1H, NH), 8.80 (s, 1H,
H-2), 8.55 (d, J6,5 = 4.2 Hz, 1H, H-6), 8.06 (ovp, 2H, H-4, 7), 7.90 (d, J6'', 5'' = 5.4 Hz, 1H, H-6''),
7.69 (d, J3'', 5'' = 1.8 Hz, 1H, H-3''), 7.51 (ovp, 3H, H-5, 5', 5''); 13C-NMR (100 MHz, DMSO-d6):
δ
167.2, 149.9, 147.9, 138.4, 132.6, 132.5, 132.1, 132.0, 131.5, 130.2, 130.2, 129.7, 127.5, 123.9,
109.5; EI-MS m/z (% rel. abund.): 348.0 (M+, 36), 350.0 (M+ + 2 , 21), 243.9 (100), 208.0 (37),
79.0 (7), 51.0 (6); HREI-MS Calcd for C15H10Cl2N4S: m/z = 348.0003, Found 348.0003.
4-(3-Nitrophenyl)-2-(2-(pyridin-3-ylmethylene)hydrazinyl)thiazole (11)
Yield: 89%; m.p: 240-242 ᵒC; 1H-NMR (300 MHz, DMSO-d6) δ 12.47 (s, 1H, NH), 8.81 (s, 1H,
H-2), 8.67 (s, 1H, H-2''), 8.56 (d, J6,5 = 4.5 Hz, 1H, H-6), 8.31 (d, J4'', 5'' = 7.5 Hz, 1H, H-4''), 8.16
(d, J6'', 5'' = 8.4 Hz, 1H, H-6''), 8.07 (ovp, 2H, H-4, 7), 7.73 (ovp, 2H, H-5', 5''), 7.47 (m, 1H, H-5);
13C-NMR (100 MHz, DMSO-d6):
δ 168.4, 150.0, 148.3, 148.2, 147.9, 138.7, 136.1, 132.7, 131.6,
130.2, 130.2, 124.0, 122.1, 119.9, 106.8; EI-MS m/z (% rel. abund.): 325.2 (M+, 40), 221.1
(100), 175.1 (29), 78.0 (7), 51.0 (6); HREI-MS Calcd for C15H11N5O2S: m/z = 325.0633, Found
325.0647.
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3-(2-(2-(Pyridin-3-ylmethylene)hydrazinyl)thiazol-4-yl)phenol (12)
Yield: 89%; m.p: 231-233 ᵒC; 1H-NMR (400 MHz, DMSO-d6) δ 12.30 (s, 1H, NH), 9.41 (s, 1H,
OH), 8.79 (s, 1H, H-2), 8.54 (d, J6,5 = 3.6 Hz, 1H, H-6), 8.05 (ovp, 2H, H-4, 7), 7.46 (m, 1H, H-
5), 7.27 (ovp, 3H, H-5′, 2′′, 6′′), 7.19 (t, J5′′ (4′′,6′′) = 7.6 Hz, 1H, H-5′′), 6.70 (d, J4'', 5'' = 8.0 Hz, 1H,
H-4''); 13C-NMR (75 MHz, DMSO-d6):
δ
165.8, 152.1; EI-MS m/z (% rel. abund.): 296.1 (M+,
84), 192.0 (100), 164.0 (29), 150.0 (81), 79.1 (20), 51.0 (10); HREI-MS Calcd for C15H12N4OS:
m/z = 296.0732, Found 296.0720.
2-(1-(Pyridin-2-yl)ethylidene)hydrazinecarbothioamide (13)
Yield: 89%; m.p: 158-160 ᵒC; 1H-NMR (400 MHz, DMSO-d6) δ 10.28 (s, 1H, NH), 8.56 (d, J6,5
= 3.6 Hz, 1H, H-6), 8.41 (d, J3,4 = 6.3 Hz, 1H, H-3), 8.37 (s, 1H, NHa), 8.11 (brd.s, 1H, NHb),
7.78 (t, J4 (3, 5) = 6.0 Hz, 1H, H-4), 7.34 (m, 1H, H-5), 2.37 (s, 3H, CH3); 13C-NMR (75 MHz,
DMSO-d6):
δ 179.0, 154.6, 148.3, 148.1, 136.3, 123.8, 120.8, 12.0; EI-MS m/z (% rel. abund.):
194.1 (M+, 100), 179.1 (71), 134.1 (59), 106.0 (68), 78.0 (82), 51.0 (26); HREI-MS Calcd for
C8H10N4S: m/z = 194.0626, Found 194.0630.
4-Phenyl-2-(2-(1-(pyridin-2-yl)ethylidene)hydrazinyl)thiazole (14)
Yield: 89%; m.p: 125-126 ᵒC; 1H-NMR (400 MHz, DMSO-d6) δ 11.45 (s, 1H, NH), 8.57 (d, J6,5
= 4.0 Hz, 1H, H-6), 8.03 (d, J3,4 = 8.4 Hz, 1H, H-3), 7.88 (ovp, 3H, H-4, 2'', 6''), 7.42 (ovp, 4H,
13
H-5', 3'', 4'', 5''), 7.31 (t, J5 (4, 6) = 7.6 Hz, 2H, H-5), 2.40 (s, 3H, CH3); C-NMR (75 MHz,
DMSO-d6):
δ 169.3, 154.6, 149.2, 148.3, 138.3, 136.8, 133.7, 129.0, 128.5, 127.5, 125.5, 124.2,
123.4, 104.4, 21.7, 12.2; EI-MS m/z (% rel. abund.): 294.1 (M+, 100), 216.1 (53), 189.1 (85),
134.0 (39), 79.1 (47), 51.0 (10); HREI-MS Calcd for C16H14N4S: m/z = 294.0939, Found
294.0927.
4-(Biphenyl-4-yl)-2-(2-(1-(pyridin-2-yl)ethylidene)hydrazinyl)thiazole (15)
Yield: 89%; m.p: 173-176 ᵒC; 1H-NMR (400 MHz, DMSO-d6) δ 11.47 (s, 1H, NH), 8.58 (d, J6,5
= 4.4 Hz, 1H, H-6), 8.04 (d, J3,4 = 8.0 Hz, 1H, H-3), 7.98 (d, J2'', 3'' = J6'', 5'' = 8.0 Hz, 2H, H-2'', 6''),
7.86 (t, J4 (3,5) = 8.0 Hz, 1H, H-4), 7.73 (ovp, 4H, H-5, 5′, 3'', 5''), 7.49 (ovp, 3H, H-2′′′, 4′′′, 6′′′),
7.38 (t, J3′′′ (4′′′,5′′′) = J5′′′ (4′′′,6′′′) =7.2 Hz, 2H, H-3′′′, 5′′′), 2.41 (s, 3H, CH3); 13C-NMR (100 MHz,
DMSO-d6):
δ
169.4, 154.9, 148.6, 147.0, 139.0, 136.6, 133.9, 128.9, 128.9, 127.5, 126.8, 126.8,
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ACCEPTED MANUSCRIPT
126.4, 126.4, 126.2, 126.1, 126.1, 124.3, 123.4, 119.5, 104.8, 12.3; EI-MS m/z (% rel. abund.):
370.2 (M+, 100), 292.2 (41), 265.1 (69),79.1 (23), 51.0 (5); HREI-MS Calcd for C22H18N4S: m/z
= 370.1252, Found 370.1226.
4-(3-Bromophenyl)-2-(2-(1-(pyridin-2-yl)ethylidene)hydrazinyl)thiazole (16)
1
Yield: 89%; m.p: 90-92 ᵒC; H-NMR (400 MHz, DMSO-d6) δ 11.47 (s, 1H, NH), 8.58 (d, J6,5
=
4.4 Hz, 1H, H-6), 8.09 (s, 1H, H-2''), 8.03 (d, J3,4 = 8.0 Hz, 1H, H-3), 7.89 (d, J6'', 5'' = 7.6 Hz, 1H,
H-6''), 7.86 (t, J4 (3,5) = 8.4 Hz, 1H, H-4), 7.53 (s, 1H, H-5'), 7.49 (d, J4'', 5'' = 8.0 Hz, 1H, H-4''),
13
7.39 (ovp, 2H, H-5, 5''), 2.40 (s, 3H, CH3); C-NMR (100 MHz, DMSO-d6):
δ 169.5, 154.8,
148.5, 147.6, 136.5, 130.8, 130.7, 130.1, 128.2, 124.5, 124.3, 123.4, 119.5, 106.0, 105.8, 12.3;
EI-MS m/z (% rel. abund.): 372.1 (M+, 92), 374.0 (M+ + 2 , 100), 296.0 (70), 267.0 (82), 174.0
(M+ , 49), 79.0 (88), 51.0 (10); HREI-MS Calcd for C16H13BrN4S: m/z = 372.0044, Found
372.0076.
4-(4-Bromophenyl)-2-(2-(1-(pyridin-2-yl)ethylidene)hydrazinyl)thiazole (17)
Yield: 89%; m.p: 177-180 ᵒC; 1H-NMR (400 MHz, DMSO-d6) δ 11.47 (s, 1H, NH), 8.57 (d, J6,5
= 4.4 Hz, 1H, H-6), 8.03 (d, J3,4 = 8.0 Hz, 1H, H-3), 7.84 (d, J4, 3 = J2'', 3'' = J6'', 5'' = 8.4 Hz, 3H, H-
4, 2'', 6''), 7.61 (d, J3'', 2'' = J5'', 6'' = 8.4 Hz, 2H, H-3'', 5''), 7.45 (s, 1H, H-5'), 7.37 (t, J5 (4,6) = 5.6 Hz,
13
1H, H-5), 2.39 (s, 3H, CH3); C-NMR (100 MHz, DMSO-d6):
δ 169.4, 154.8, 149.6, 148.5,
138.3, 136.5, 131.5, 131.5, 127.6, 127.6, 124.3, 123.4, 120.5, 119.5, 105.4, 12.2; EI-MS m/z (%
rel. abund.): 372.1 (M+, 86), 374.0 (M+ + 2 , 100), 296.0 (50), 266.9 (84), 174.0 (M+ , 40), 79.1
(37), 51.0 (8); HREI-MS Calcd for C16H13BrN4S: m/z = 372.0044, Found 372.0041.
4-(3-Chlorophenyl)-2-(2-(1-(pyridin-2-yl)ethylidene)hydrazinyl)thiazole (18)
Yield: 89%; m.p: 128-130 ᵒC; 1H-NMR (400 MHz, DMSO-d6) δ 11.47 (s, 1H, NH), 8.58 (d, J6,5
= 4.4 Hz, 1H, H-6), 8.03 (d, J3,4 = 8.4 Hz, 1H, H-3), 7.93 (s, 1H, H-2''), 7.85 (ovp, 2H, H-4, 6''),
7.60 (s, 1H, H-5'), 7.46 (t, J5 (4, 6) = 8.0 Hz, 1H, H-5), 7.37 (ovp, 2H, H-4'', 5''), 2.40 (s, 3H, CH3);
13C-NMR (100 MHz, DMSO-d6):
δ 169.4, 154.8, 149.1, 148.5, 138.3, 136.5, 133.4, 130.5, 127.2,
125.2, 124.2, 124.0, 123.4, 119.5, 106.1, 12.2; EI-MS m/z (% rel. abund.): 328.0 (M+, 100),
330.0 (M+ + 2 , 38), 250.0 (58), 223.0 (95), 79.0 (76), 51.0 (15); HREI-MS Calcd for
C16H13ClN4S: m/z = 328.0549, Found 328.0544.
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