Novel thiazole–pyrazolone hybrids as potent ACE inhibitors and their cardioprotective effect on isoproterenol-induced myocardial infarction

Article abstract of DOI:10.1002/ardp.202000140

A facile synthesis of a group of novel thiazole–pyrazolone hybrids and their investigation for angiotensin-converting enzyme (ACE) inhibition are reported in this study. These compounds were synthesized using a well-known approach, based on the condensation of ethyl acetoacetate with thiazolylhydrazines, and characterized by various spectroscopic and analytical techniques. The entire set of compounds displayed a moderate-to-excellent inhibitory activity against ACE. In particular, compound 4i was found to be the most potent ACE inhibitor and was further studied for cardioprotective effects against isoproterenol (ISO)-induced myocardial infarction (MI) in rats. Compound 4i improved the cardiac function and prevented cardiac injury induced by ISO in Sprague Dawley rats. The levels of oxidative stress and proinflammatory cytokines were also restored to near normal by 4i as compared with the ISO group. In the Western blot analysis, compound 4i prevented mitochondrial apoptosis after MI by downregulating the expression of cleaved caspase-3 and Bax, with the upregulation of Bcl-2, as compared with the ISO group.

Full text of DOI:10.1002/ardp.202000140

Received: 4 May 2020
Revised: 23 June 2020
Accepted: 11 July 2020
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DOI: 10.1002/ardp.202000140
F U L L P A P E R
Novel thiazole–pyrazolone hybrids as potent ACE inhibitors
and their cardioprotective effect on isoproterenol‐induced
myocardial infarction
Hongwen You1 | Xinyou Su2 | Guoying Su3
1Department of Cardiology, Shandong
Provincial Hospital Affiliated to Shandong First
Abstract
Medical University, Jinan, Shandong, China
A facile synthesis of a group of novel thiazole–pyrazolone hybrids and their
investigation for angiotensin‐converting enzyme (ACE) inhibition are reported in this
study. These compounds were synthesized using a well‐known approach, based on
the condensation of ethyl acetoacetate with thiazolylhydrazines, and characterized
by various spectroscopic and analytical techniques. The entire set of compounds
displayed a moderate‐to‐excellent inhibitory activity against ACE. In particular,
compound 4i was found to be the most potent ACE inhibitor and was further studied
for cardioprotective effects against isoproterenol (ISO)‐induced myocardial infarc-
tion (MI) in rats. Compound 4i improved the cardiac function and prevented cardiac
injury induced by ISO in Sprague Dawley rats. The levels of oxidative stress and
proinflammatory cytokines were also restored to near normal by 4i as compared
with the ISO group. In the Western blot analysis, compound 4i prevented
mitochondrial apoptosis after MI by downregulating the expression of cleaved
caspase‐3 and Bax, with the upregulation of Bcl‐2, as compared with the ISO group.
2Department of Oncology, Jinan Central
Hospital, Cheeloo College of Medicine,
Shandong University, Jinan, Shandong, China
3Department of Cardiology, Jinan Central
Hospital, Cheeloo College of Medicine,
Shandong University, Jinan, Shandong, China
Correspondence
Guoying Su, Department of Cardiology, Jinan
Central Hospital, Cheeloo College of Medicine,
Shandong University, Jinan, 250013 Shandong,
China.
Email: suguoyingxn@sina.com
K E Y W O R D S
ACE, inflammation, myocardial infarction, oxidative stress, thiazole
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INTRODUCTION
meta‐analysis was found to improve the survival rate and disease
progression if administered within 3–16 days of infarction.[7,8] The
Cardiovascular diseases (CVDs) are ranked after cancer as a major
cause of morbidity and mortality across both developed and devel-
oping nations.[1] Myocardial infarction (MI) is the most common cause
of death among patients with CVDs.[2] According to an estimate, in the
past 30 years, the CVD risk has risen significantly in China due to
adapting of western lifestyle, aging population, and increased
urbanization.[3] In 2014, approximately 290 million patients with CVD
were reported in China, among which 2.5 million were MI patients.[4]
However, numerous clinical agents are currently in practice to curb
the menace of MI, but no single agent has been proven effective in its
management. Therefore, patients with acute MI could be managed
alone or in a combination of modalities including direct angioplasty or
fibrinolysis, aspirin, angiotensin‐converting enzyme (ACE) inhibitors,
beta blockers, and nitrates.[5,6] Particularly, the ACE inhibitor in a
ACE is an enzyme causing the endogenous conversion of angiotensin
(AT) I to AT II. This conversion triggers vasoconstriction, which results
in increased blood pressure. Therefore, to meet the normal blood
demand, the heart has to work more, which further weakens the
cardiac muscles. Thus, drugs that prevent the action of ACE (ACE
inhibitors) reduce vasoconstriction by preventing conversion to AT II
and exert beneficial effect against MI.[9–12]
Heterocyclic molecules are well known for their importance as
medicinal agents providing benefit against many diseases and pa-
thological conditions.[13] Pyrazole is a five‐membered heterocyclic
ring responsible for a variety of pharmacological effects such as
antibacterial,[14–16] antifungal,[17–19] antiviral,[20–22] and anticancer
effects.[23,24] However, thiazole is another five‐membered ring sys-
tem that possesses numerous medicinal properties.[25] In
a
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Arch Pharm. 2020;e2000140.
wileyonlinelibrary.com/journal/ardp
© 2020 Deutsche Pharmazeutische Gesellschaft
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pioneering study by Jiang et al.,[26] numerous thiazole‐bearing pyr-
azoles were synthesized as a potent inhibitor of ACE inhibitors.
Prompted by the above aspects, in the present manuscript, we intend
to synthesize novel thiazole‐bearing pyrazole derivatives as potent
inhibitors of ACE, with a cardioprotective effect on isoproterenol
(ISO)‐induced MI in rats.
aromatic chloro group appeared at 787 cm−1. All synthesized com-
pounds showed characteristic peaks for each proton in 1H NMR spectra.
For instance, compounds of series 4a–j showed a doublet peak corre-
sponding to the protons of aromatic ring, which appeared at
7.62–7.87 ppm. The C–H of the thiazole group appeared as a singlet at
the chemical shift range of 7.18–7.41 ppm. Furthermore, C–H protons
of pyrazole moiety appeared at 5.64–5.67 ppm as a singlet. The N–H
group of protons of pyrazole moiety appeared as a singlet in the che-
mical shift range of 3.96–3.99 ppm. The proton in the CH3 group linked
with pyrazole moiety appeared as a singlet at the chemical shift range of
2.24–2.36 ppm. In 13C NMR spectra, the entire set of compounds
showed a chemical shift in the range of 173.2–105.1 ppm due to the
thiazole carbon atom. The pyrazole carbon was found at
165.8–94.1 ppm. The chemical shift range of 13.9–13.4 ppm was at-
tributed to the methyl group at the pyrazole ring. Moreover, the aro-
matic carbon of target compounds appeared at 133.4–127.6 ppm.
Finally, the mass and elemental analysis of all the synthesized
compounds 4a–j was also performed to ascertain their structure.
The compounds were obtained in excellent yield and purity, as
determined by various spectroscopic and analytical procedures. The
inhibitory activity of these compounds at 1.0‐µM concentration of test
and standard compound (lisinopril) was determined against ACE, and
the results are presented in Table 1. It has been found that the entire
set of the synthesized compounds showed a moderate‐to‐considerable
ACE inhibition, ranging from 43% to 95%. It was observed that
compound 4a with no substitution on the phenyl ring showed the least
activity (43%) among the tested derivatives. The activity was sig-
nificantly improved in the case of compound 4b upon insertion of the
substituent (p‐hydroxy, 67%). However, the activity dropped markedly
after replacing hydroxy with methyl (4c). A mild increase in the
activity was reported by compound 4d upon the introduction of
para‐methoxy. Moreover, the percentage of inhibition was found to be
increased upon the insertion of an electron‐withdrawing group,
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RESULTS AND DISCUSSION
The compounds bearing various substituents were synthesized via a
facile procedure, as shown in Scheme 1. Initially, the synthesis was
started with the development of compounds 2a–j by brominating the
corresponding acetophenone derivatives 1a–j. Compounds 2a–j further
underwent the cyclocondensation reaction with thiosemicarbazide to
yield thiazolylhydrazines 3a–j. The synthesis of target compounds 4a–j
was achieved via refluxing compounds 3a–j with ethyl acetoacetate in
the presence of dimethylformamide (DMF).[27] All synthesized com-
pounds were characterized by different spectroscopic methods such as
Fourier‐transform infrared (FTIR) spectroscopy, 1H nuclear magnetic
resonance (NMR), 13C NMR, and mass and elemental analysis. The title
compounds showed characteristic peaks for each functional group in
FTIR spectra, such as one absorption band at 3,385–3,391 cm−1 due to
the presence of the N–H group in the pyrazole ring. Absorption bands
at 3,081–3,087 cm−1 were attributed to the stretching vibration of the
C–H group of the phenyl ring. Another FTIR peak at 2,952–2,959 cm−1
showed the presence of the CH3 group in the pyrazole ring. The ab-
sorption band appearing at 1,682–1,689 cm−1 suggested the presence
of C═N group of the thiazole ring. However, the stretching vibration at
1,709–1,719 cm−1 was found due to C═O group in the pyrazole ring.
The thiazole C–S group appeared at 634–639 cm−1. Another aromatic
OH group appeared at 3,427 cm−1. The stretching vibration of the
fluoro group linked to the phenyl ring appeared at 1,538 cm−1. The
SCHEME 1 Reagents and conditions: (a) Br2, reflux; (b) thiosemicarbazide, propane‐2‐ol, reflux; (c) dimethylformamide, reflux

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TABLE 1 In vitro angiotensin‐converting enzyme inhibition of
compounds 4a–j
halogen disubstituted molecule (4j). This suggests that the same
substitution on the ring does not favor activity. Encouraged by
the excellent ACE inhibitory activity of compound 4i and the im-
portance of ACE inhibitors in MI, it is worthwhile to determine
the cardioprotective activity of compound 4i in the isoproterenol
(ISO)‐induced MI in rats. The induction of MI in rats by ISO is a very
popular model in determining the cardioprotective effect of any new
medicinal agent. ISO chemically belongs to the class of catecholamine
and possesses β‐adrenergic agonistic activity. It causes severe stress in
the myocardial tissue, resulting in infarct‐like necrosis of the heart
muscle, which renders it suitable for the induction of MI‐like char-
acteristics in rats.
Compound
Substituent
H
% inhibition
4a
43
67
55
62
70
80
76
90
95
84
99
4b
4‐OH
4c
4‐CH3
4‐OCH3
4‐NO2
4‐Cl
4d
4e
4f
4g
4‐Br
4h
4‐F
Initially, the effect of compound 4i was investigated on the
serum biomarkers associated with cardiac injury (lactate dehy-
drogenase [LDH] and creatine kinase [CK]‐MB) after induction
with ISO.[28] As expected, the ISO‐treated rats showed an in-
creased level of LDH and CK‐MB, which was later found to be
reduced in 4i‐treated rats as compared with the ISO‐treated group
in dose‐dependent manner (Figure 1).
4i
2‐Cl, 4‐F
2,4‐di‐Cl
4j
Lisinopril
that is, para‐nitro and para‐chloro in the case of compounds 4e and 4f,
respectively. On the contrary, the activity was decreased mildly in the
case of compound 4g having para‐bromo. In the next instance, the
percentage of inhibition was further improved significantly in the case
of compounds 4h and 4i having ortho‐chloro and para‐fluoro, respec-
tively. The last compound of the tested series showed a drop in the
inhibitory activity after the introduction of chloro in the place of para‐
fluoro (84%). Among the tested series, compound 4i was revealed as
the most potent ACE inhibitor having 95% inhibitory activity, whereas
none of the synthesized derivatives showed an inhibitory activity
comparable to or more than lisinopril as the standard drug.
The structure–activity relationship of the designed analogs suggested
that the electron‐withdrawing groups have a significant influence on
the inhibitory activity as compared with their electron‐releasing
counterparts. More important, the disubstituted molecule with
two distinct halogen atoms (4i) was found more active than the same
The cardiac function is found to be altered after MI, and the ACE
inhibitor was found effective in such conditions.[12,29] Therefore, it is
worth to investigate the effect of compound 4i on the cardiac func-
tions of the rats. As shown in Figure 2, compound 4i causes a sig-
nificant improvement in left ventricular end‐systolic pressure (LVSP)
and dp/dtmax; however, the infarct percentage was also found to be
decreased significantly. Moreover, as presented in Table 2, com-
pound 4i causes significant progression in the posterior wall thick-
ness of the LV with simultaneous augmentation in the mass of LV.
The end‐systolic volume was also found to be increased in the
4i‐treated group as compared with the ISO‐treated group.
Oxidative stress and inflammation are the characteristic features
of the MI, and various studies have shown the role of oxidative stress
in the progression of inflammation in MI.[30–34] Concerning this, the
effect of compound 4i was investigated on the various biomarkers
denoting the expression of oxidative stress (malondialdehyde [MDA],
FIGURE 1 The effect of compound 4i on serum (a) LDH and (b) CK‐MB levels. Data represent the mean ( SEM) of three independent
experiments. CK‐MB, creatine kinase‐MB; ISO, isoproterenol; LDH, lactate dehydrogenase; SEM, standard error of the mean. *p < .05 versus
ISO‐treated group; **p < .01 versus ISO group

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FIGURE 2 The effect of compound 4i on (a) LVSP, (b, c) dp/dtmax, and (d) infarct volume after MI. Data represent the mean ( SEM) of three
independent experiments. ISO, isoproterenol; LVSP, left ventricular end‐systolic pressure; MI, myocardial infarction; SEM, standard error
of the mean. *p < .05 versus ISO‐treated group; **p < .01 versus ISO group
myeloperoxidase [MPO], glutathione peroxidase [GPx], and superoxide
dismutase [SOD]) and myocardial inflammation (Tn‐T, tumor necrosis
factor‐α [TNF‐α], and interleukin‐6 [IL‐6] level). It has been found that
compound 4i causes a significant reduction in oxidative stress by in-
creasing the level of GPx and SOD, whereas the level of MPO and
MDA was found to be reduced significantly as compared with the
ISO‐treated group in a dose‐dependent manner (Figure 3a–d). As
shown in Figure 3e–g, the level of various inflammatory markers (Tn‐T,
TNF‐α, and IL‐6) was reduced significantly in 4i‐treated group as
compared with the ISO‐treated group.
cardiomyocytes undergo necrosis and initiate a cascade of events
involving mitochondrial genes (Bcl‐2, Bax, and caspase‐3).[35–37]
These genes are classified as proapoptotic (Bax) and apoptotic gene
(Bcl‐2), where their intricate balance controls apoptosis. Moreover,
caspase‐3 is another vital gene involved in the repair of DNA and
membrane integrity. Therefore, the effect of compound 4i was in-
vestigated on cardiac Bcl‐2, Bax, and caspase‐3 via the Western blot
analysis. Results suggested that the expression of cleaved caspase‐3
and Bax was found to be downregulated in the 4i‐treated group, with
the upregulation of Bcl‐2, as compared with the ISO‐treated group
(Figure 4).
Numerous studies have shown the role of apoptosis in CVDs. It
has been found that during hypoxia and ischemia/reperfusion, the
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CONCLUSION
TABLE 2 The effect of compound 4i on cardiac function in rats
(echo readings)
Collectively, our study showed the development of novel
thiazole–pyrazolone hybrids as potent ACE inhibitors, possessing a
mild‐to‐significant inhibitory activity. Among the tested series, com-
pound 4i was found as the most potent inhibitor of ACE. The effect of
compound 4i was also investigated in isoproternol‐induced MI in rats.
It has been found that compound 4i improves hemodynamic para-
meters with a reduction of infarct size in rats. It also causes inhibition
of oxidative stress and inflammation in cardiac tissues. Compound 4i
also prevented necrosis of cardiomyocytes by inhibiting apoptosis.
Parameters
PWT (mm)
ESV (µl)
Pre
Post
1.32 0.04
1.48 0.05
205 16
406 21
49.5 4.2
152
8
EDV (µl)
EF (%)
339 19
55.1 3.3
Abbreviations: EDV, end‐diastolic volume; EF, ejection fraction; ESV, end‐
systolic volume; PWT, posterior wall thickness.

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FIGURE 3 The effect of compound 4i on the cardiac biomarkers depicting oxidative stress and inflammation: (a) MPO, (b) MDA, (c) SOD, (d)
GPx, (e) Tn‐T, (f) TNF‐α, and (g) IL‐6. Data represent the mean ( SEM) of three independent experiments. GPx, glutathione peroxidase; IL‐6,
interleukin‐6; ISO, isoproterenol; MDA, malondialdehyde; MPO, myeloperoxidase; SEM, standard error of the mean; TNF‐α, tumor necrosis
factor‐α. *p < .05 versus ISO‐treated group; **p < .01 versus ISO group
However, more studies are necessary to establish the present class of
the compound as a novel ACE inhibitor against MI.
spectrometer at 400 MHz (1H) or 100 MHz (13C), respectively, in di-
methyl sulfoxide (DMSO)‐d6, using tetramethylsilane (TMS) as an internal
standard. The multiplicity of a signal is indicated as follows: s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; br, broad; dd, doublet of
doublets, and so forth. Coupling constants (J) are quoted in hertz and
reported to the nearest 0.1 Hz. Infrared spectra were recorded as a neat
thin film on a PerkinElmer Spectrum One FTIR spectrometer using
Universal ATR sampling accessories. Letters in the parentheses refer to
the relative absorbency as compared with the most intense peak: w,
weak, <40%; m, medium, 40–75%; s, strong, >75%. Melting points (MPs)
were obtained using an OptiMelt automated melting point system. MS
spectra were recorded on an Agilent 1100 LC/MS.
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EXPERIMENTAL
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4.1
Chemistry
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4.1.1
General
The chemicals used in the present study were obtained from Sigma‐
Aldrich. NMR spectra were recorded using a Bruker Avance NMR

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FIGURE 4 The effect of compound 4i on (a) the Bcl‐2 family proteins and a representative bar graph of (b) Bcl‐2, (c) Bax, and
(d) cleaved caspase‐3 as determined via the Western blot analysis. Data represent the mean ( SEM) of three independent experiments.
ISO, isoproterenol; SEM, standard error of the mean. *p < .05 versus ISO group; **p < .01 versus ISO‐treated group
The InChI codes of the investigated compounds, together
with some biological activity data, are provided as Supporting
Information Data.
with 10% sodium bicarbonate and saturated brine solution; then, the
organic layer was dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo. The residue was purified by recrystallization
using ethanol solvent.
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4.1.2
Synthesis of compounds 2a–j and 3a–j
5‐Methyl‐2‐(4‐phenylthiazol‐2‐yl)‐1,2‐dihydro‐3H‐pyrazol‐3‐
one (4a)
Compounds 2a–j and 3a–j were synthesized as per the earlier re-
ported procedure.[38,39]
Yield: 79%; white solid; MP: 193–196°C; molecular weight (MW):
257.31; retention factor (Rf): 0.69; FTIR (νmax; cm−1 KBr): 3,387 (N–H
stretching, –NH), 3,087 (Ar C–H stretching), 2,958 (alkyl C–H
stretching), 1,709 (C═O stretching), 1,682 (C═N aromatic), 1,625
(C═C stretching), 1,132 (aromatic C–C stretching), 1,508 (N–H bend,
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4.1.3
General procedure for the synthesis of the
title compounds 4a–j
NH), 1,049 (C–N stretching), 634 (C–S stretching) cm−1 1H NMR
;
(400 MHz, DMSO‐d6, TMS) δ ppm: 7.87 (d, 2H, J = 7.6 Hz, Ar‐H), 7.38
(d, 2H, J = 7.2 Hz, Ar‐H), 7,34 (t, 1H, J = 1.4 Hz, Ar‐H), 7.24 (s, 1H,
thiazole‐H), 5.65 (s, 1H, pyrazole‐H), 3.98 (s, 1H, pyrazole‐NH), 2.35
(s, 3H, pyrazole‐CH3); 13C NMR (100 MHz, DMSO‐d6) δ ppm: 173.3,
165.9, 152.1, 150.2, 133.2, 129.4, 128.7, 127.6, 105.1, 94.2, 13.9;
mass: 258.34 (M+1); elemental analysis for C13H11N3OS: calculated:
C, 60.68; H, 4.31; N, 16.33; found: C, 60.69; H, 4.35; N, 16.32.
A mixture of ethyl acetoacetate (5 mmol) and dimethylformamide
dimethyl acetal (5.5 mmol) was refluxed to 100°C on a water bath
until it changed color from yellow to orange. Then the reaction
mixture was cooled to room temperature and diluted with ethanol
(20 ml). The corresponding substituted thiazole derivatives 3a–j
(5 mmol) were diluted in 20 ml ethanol separately and added drop-
wise to the reaction mixture over 5 min. Then, it was heated to reflux
for 2 hr and monitored by using thin‐layer chromatography. After
completion of the reaction, the reaction mixture was allowed to cool
to room temperature and concentrated under reduced pressure. The
resulting oil was extracted by using dichloromethane and washed
2‐[4‐(4‐Hydroxyphenyl)thiazol‐2‐yl]‐5‐methyl‐1,2‐dihydro‐3H‐
pyrazol‐3‐one (4b)
Yield: 82%; black solid; MP: 242–243°C; MW: 273.31; Rf: 0.74; FTIR
(νmax; cm−1 KBr): 3,427 (OH stretching), 3,391 (N–H stretching, –NH),

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3,082 (Ar C–H stretching), 2,959 (alkyl C–H stretching), 1,711 (C═O
stretching), 1,684 (C═N aromatic), 1,626 (C═C stretching), 1,131
(aromatic C–C stretching), 1,507 (N–H bend, NH), 1,051 (C–N
stretching), 639 (C–S stretching) cm−1; 1H NMR (400 MHz, DMSO‐d6,
TMS) δ ppm: 9.34 (s, 1H, Ar‐OH), 7.62 (d, 2H, J = 7.8 Hz, Ar‐H), 7.18
(s, 1H, thiazole‐H), 7.05 (d, 2H, J = 6.3 Hz, Ar‐H), 5.64 (s, 1H, pyrazole‐
H), 3.97 (s, 1H, pyrazole‐NH), 2.36 (s, 3H, pyrazole‐CH3); 13C NMR
(100 MHz, DMSO‐d6, TMS) δ ppm: 173.3, 165.7, 158.3, 152.2, 150.4,
128.9, 125.4, 116.5, 105.1, 94.1, 13.9; mass: 274.37 (M+1); elemental
analysis for C13H11N3O2S: calculated: C, 57.13; H, 4.06; N, 15.37;
found: C, 57.16; H, 4.05; N, 15.39.
DMSO‐d6, TMS) δ ppm: 7.58 (d, 2H, J = 7.2 Hz, Ar‐H), 7.48 (d, 2H,
J = 6.1 Hz, Ar‐H), 7.17 (s, 1H, thiazole‐H), 5.64 (s, 1H, pyrazole‐H),
3.97 (s, 1H, pyrazole‐NH), 2.37 (s, 3H, pyrazole‐CH3); 13C NMR
(100 MHz, DMSO‐d6, TMS) δ ppm: 173.4, 165.9, 152.3, 150.2, 147.8,
139.2, 126.3, 124.5, 105.2, 94.3, 13.9; mass: 303.34 (M+1); elemental
analysis for C13H10N4O3S: calculated: C, 51.65; H, 3.33; N, 18.53;
found: C, 51.67; H, 3.38; N, 18.51.
2‐[4‐(4‐Chlorophenyl)thiazol‐2‐yl]‐5‐methyl‐1,2‐dihydro‐3H‐
pyrazol‐3‐one (4f)
Yield: 71%; white solid; MP: 192–194°C; MW: 291.75; Rf: 0.67; FTIR
(νmax; cm−1 KBr): 3,386 (N–H stretching, –NH), 3,084 (Ar C–H
stretching), 2,958 (alkyl C–H stretching), 1,714 (C═O stretching),
1,682 (C═N aromatic), 1,629 (C═C stretching), 1,139 (aromatic C–C
stretching), 1,504 (N–H bend, NH), 1,059 (C–N stretching), 787 (C–Cl
stretching), 638 (C–S stretching) cm−1; 1H NMR (400 MHz, DMSO‐d6,
TMS) δ ppm: 7.75 (d, 2H, J = 7.5 Hz, Ar‐H), 7.59 (d, 2H, J = 6.4 Hz,
Ar‐H), 7.35 (s, 1H, thiazole‐H), 5.65 (s, 1H, pyrazole‐H), 3.96 (s, 1H,
pyrazole‐NH), 2.36 (s, 3H, pyrazole‐CH3); 13C NMR (400 MHz,
DMSO‐d6, TMS) δ ppm: 173.5, 165.9, 152.3, 150.3, 134.4, 131.2,
129.6, 128.9, 105.2, 94.4, 13.9; mass: 292.78 (M+1); elemental ana-
lysis for C13H10ClN3OS: calculated: C, 53.52; H, 3.45; N, 14.40;
found: C, 53.54; H, 3.48; N, 14.42.
5‐Methyl‐2‐[4‐(p‐tolyl)thiazol‐2‐yl]‐1,2‐dihydro‐3H‐pyrazol‐3‐
one (4c)
Yield: 76%; light yellow solid; MP: 214–216°C; MW: 271.34; Rf: 0.79;
FTIR (νmax; cm−1 KBr): 3,387 (N–H stretching, –NH), 3,084 (Ar C–H
stretching), 2,952 (alkyl C–H stretching), 1,713 (C═O stretching),
1,687 (C═N aromatic), 1,621 (C═C stretching), 1,134 (aromatic C–C
stretching), 1,509 (N–H bend, NH), 1,053 (C–N stretching), 639 (C–S
stretching) cm−1 1H NMR (400 MHz, DMSO‐d6, TMS) δ ppm: 7.58 (d,
;
2H, J = 7.9 Hz, Ar‐H), 7.29 (s, 1H, thiazole‐H), 7.19 (d, 2H, J = 6.7 Hz,
Ar‐H), 5.65 (s, 1H, pyrazole‐H), 3.99 (s, 1H, pyrazole‐NH), 2.34 (s, 3H,
pyrazole‐CH3), 2.24 (s, 3H, Ar‐CH3); 13C NMR (100 MHz, DMSO‐d6,
TMS) δ ppm: 173.4, 165.7, 152.3, 150.2, 131.8, 130.1, 129.5, 125.8,
105.2, 94.1, 21.4, 13.8; mass: 272.36 (M+1); elemental analysis for
C14H13N3OS: calculated: C, 61.97; H, 4.83; N, 15.49; found: C, 61.99;
H, 4.85; N, 15.52.
2‐[4‐(4‐Bromophenyl)thiazol‐2‐yl]‐5‐methyl‐1,2‐dihydro‐3H‐
pyrazol‐3‐one (4g)
Yield: 77%; brownish black solid; MP: 206–209°C; MW: 336.21; Rf:
0.73; FTIR (νmax; cm−1 KBr): 3,389 (N–H stretching, –NH), 3,081 (Ar
C–H stretching), 2,959 (alkyl C–H stretching), 1,718 (C═O stretch-
ing), 1,685 (C═N aromatic), 1,628 (C═C stretching), 1,132 (aromatic
C–C stretching), 1,507 (N–H bend, NH), 1,052 (C–N stretching), 758
2‐[4‐(4‐Methoxyphenyl)thiazol‐2‐yl]‐5‐methyl‐1,2‐dihydro‐3H‐
pyrazol‐3‐one (4d)
Yield: 69%; yellow solid; MP: 206–208°C; MW: 287.34; Rf: 0.83; FTIR
(νmax; cm−1 KBr): 3,386 (N–H stretching, –NH), 3,085 (Ar C–H
stretching), 2,954 (alkyl C–H stretching), 2,824 (OCH3 stretching),
1,716 (C═O stretching), 1,689 (C═N aromatic), 1,623 (C═C stretch-
ing), 1,131 (aromatic C–C stretching), 1,508 (N–H bend, NH), 1,055
(C–Br stretching), 635 (C–S stretching) cm−1 1H NMR (400 MHz,
;
DMSO‐d6, TMS) δ ppm: 7.64 (d, 2H, J = 7.8 Hz, Ar‐H), 7.56 (d, 2H,
J = 6.2 Hz, Ar‐H), 7.30 (s, 1H, thiazole‐H), 5.67 (s, 1H, pyrazole‐H),
3.99 (s, 1H, pyrazole‐NH), 2.34 (s, 3H, pyrazole‐CH3); 13C NMR
(100 MHz, DMSO‐d6, TMS) δ ppm: 173.5, 165.9, 152.3, 150.3, 132.3,
132.1, 128.4, 123.2, 105.1, 94.2, 13.8; mass: 337.24 (M+1); elemental
analysis for C13H10BrN3OS: calculated: C, 46.44; H, 3.00; N, 12.50;
found: C, 46.48; H, 3.02; N, 12.52.
(C–N stretching), 637 (C–S stretching) cm−1 1H NMR (400 MHz,
;
DMSO‐d6, TMS) δ ppm: 7.46 (d, 2H, J = 7.5 Hz, Ar‐H), 7.16 (s, 1H,
thiazole‐H), 7.01 (d, 2H, J = 6.3 Hz, Ar‐H), 5.67 (s, 1H, pyrazole‐H),
3.98 (s, 1H, pyrazole‐NH), 3.82 (s, 3H, Ar‐OCH3), 2.35 (s, 3H,
pyrazole‐CH3); 13C NMR (100 MHz, DMSO‐d6, TMS) δ ppm: 173.3,
165.9, 160.7, 152.3, 150.3, 128.7, 125.4, 114.9, 105.2, 94.3, 55.9,
13.8; mass: 288.38 (M+1); elemental analysis for C14H13N3O2S: cal-
culated: C, 58.52; H, 4.56; N, 14.62; found: C, 58.54; H, 4.58;
N, 14.61.
2‐[4‐(4‐Fluorophenyl)thiazol‐2‐yl]‐5‐methyl‐1,2‐dihydro‐3H‐pyrazol‐
3‐one (4h)
Yield: 73%; brown solid; MP: 251–252°C; MW: 275.30; Rf: 0.85; FTIR
(νmax; cm−1 KBr): 3,385 (N–H stretching, –NH), 3,083 (Ar C–H
stretching), 2,952 (alkyl C–H stretching), 1,714 (C═O stretching),
1,687 (C═N aromatic), 1,629 (C═C stretching), 1,156 (C–F stretch-
ing), 1,136 (aromatic C–C stretching), 1,509 (N–H bend, NH), 1,053
5‐Methyl‐2‐[4‐(4‐nitrophenyl)thiazol‐2‐yl]‐1,2‐dihydro‐3H‐pyrazol‐
3‐one (4e)
Yield: 78%; yellow solid; MP: 253–254°C; MW: 302.31; Rf: 0.72; FTIR
(νmax; cm−1 KBr): 3,389 (N–H stretching, –NH), 3,082 (Ar C–H
stretching), 2,953 (alkyl C–H stretching), 1,719 (C═O stretching),
1,685 (C═N aromatic), 1,628 (C═C stretching), 1,538 (NO2 stretch-
ing), 1,136 (aromatic C–C stretching), 1,509 (N–H bend, NH), 1,057
(C–N stretching), 637 (C–S stretching) cm−1 1H NMR (400 MHz,
;
DMSO‐d6, TMS) δ ppm: 7.59 (d, 2H, J = 7.4 Hz, Ar‐H), 7.24 (s, 1H,
thiazole‐H), 7.16 (d, 2H, J = 6.3 Hz, Ar‐H), 5.66 (s, 1H, pyrazole‐H),
3.96 (s, 1H, pyrazole‐NH), 2.36 (s, 3H, pyrazole‐CH3); 13C NMR
(100 MHz, DMSO‐d6, TMS) δ ppm: 173.5, 165.9, 162.7, 152.3, 150.3,
130.8, 128.7, 116.1, 105.2, 94.2, 13.8; mass: 276.32 (M+1); elemental
(C–N stretching), 639 (C–S stretching) cm−1 1H NMR (400 MHz,
;

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analysis for C13H10FN3OS: calculated: C, 56.72; H, 3.66; N, 15.26;
found: C, 56.71; H, 3.64; N, 15.28.
terminated by the addition of 0.1 ml of 1 M HCl, and HA thus produced
in the reaction was then allowed to react with 0.2 ml of pyridine and
0.1 ml of benzene sulfonyl chloride (Sigma‐Aldrich) to develop yellow
color. This specific color was measured at 410 nm. The ACE inhibition
was expressed as percentage inhibition and calculated from the following
equation: Inhibition% = 100 − [T/C] × 100, where T is the absorbance of
test reaction and C is the absorbance of control reaction. The therapeutic
drug lisinopril was used as a reference ACE inhibitor.
2‐[4‐(2‐Chloro‐4‐fluorophenyl)thiazol‐2‐yl]‐5‐methyl‐1,2‐dihydro‐
3H‐pyrazol‐3‐one (4i)
Yield: 79%; red brown solid, MP: 319–320°C; MW: 309.74; Rf: 0.81;
FTIR (νmax; cm−1 KBr): 3,388 (N–H stretching, –NH), 3,082 (Ar C–H
stretching), 2,956 (alkyl C–H stretching), 1,718 (C═O stretching), 1,689
(C═N aromatic), 1,623 (C═C stretching), 1,157 (C–F stretching), 1,138
(aromatic C–C stretching), 1,507 (N–H bend, NH), 1,052 (C–N stretch-
ing), 789 (C–Cl stretching), 639 (C–S stretching) cm−1
;
1H NMR
4.2.1
Animals
|
(400 MHz, DMSO‐d6, TMS) δ ppm: 7.86 (d, 1H, J = 7.9 Hz, Ar‐H), 7.43 (d,
1H, J = 6.1 Hz, Ar‐H), 7.37 (s, 1H, thiazole‐H), 7.07 (d, 1H, J = 1.5 Hz, Ar‐
H), 5.64 (s, 1H, pyrazole‐H), 3.99 (s, 1H, pyrazole‐NH), 2.35 (s, 3H,
pyrazole‐CH3); 13C NMR (100 MHz, DMSO‐d6, TMS) δ ppm: 173.4,
165.9, 164.5, 152.2, 147.9, 133.9, 130.5, 125.6, 118.4, 114.2, 105.1, 94.1,
13.7; mass: 310.76 (M+1); elemental analysis for C13H9ClFN3OS: cal-
culated: C, 50.41; H, 2.93; N, 13.57; found: C, 50.43; H, 2.91; N, 13.59.
After obtaining from the institutional animal house, the adult male
Sprague Dawley (240–270 g) rats were housed under controlled
temperature and humidity. The rats were acclimatized to the la-
boratory environment via exposing them to alternate, dark and light
cycle with ad libitum supply of food and water.
|
Ethics statement
2‐[4‐(2,4‐Dichlorophenyl)thiazol‐2‐yl]‐5‐methyl‐1,2‐dihydro‐3H‐
pyrazol‐3‐one (4j)
4.2.2
Yield: 73%; white solid, MP: 356–357°C; MW: 326.20; Rf: 0.77; FTIR
(νmax; cm−1 KBr): 3,389 (N–H stretching, –NH), 3,085 (Ar C–H
stretching), 2,959 (alkyl C–H stretching), 1,717 (C═O stretching),
1,684 (C═N aromatic), 1,626 (C═C stretching), 1,139 (aromatic C–C
stretching), 1,508 (N–H bend, NH), 1,056 (C–N stretching), 787 (C–Cl
stretching), 638 (C–S stretching) cm−1; 1H NMR (400 MHz, DMSO‐d6,
TMS) δ ppm: 7.87 (d, 1H, J = 7.8 Hz, Ar‐H), 7.67 (d, 1H, J = 6.5 Hz, Ar‐
H), 7.41 (s, 1H, thiazole‐H), 7.32 (d, 1H, J = 1.8 Hz, Ar‐H), 5.67 (s, 1H,
pyrazole‐H), 3.98 (s, 1H, pyrazole‐NH), 2.34 (s, 3H, pyrazole‐CH3);
13C NMR (100 MHz, DMSO‐d6, TMS) δ ppm: 173.4, 165.9, 152.3,
147.9, 135.9, 133.6, 130.9, 130.1, 128.2, 127.4, 105.2, 94.1, 13.8;
mass: 327.24 (M+1); elemental analysis for C13H9Cl2N3OS: calcu-
lated: C, 47.87; H, 2.78; N, 12.88; found: C, 47.89; H, 2.76; N, 12.89.
The experimental procedures involving animals in this study were
approved by the Animal Ethics Committee of the Institute and
Experimental Animal Regulation by the National Science and
Technology Commission, China, for the use of laboratory animals.
|
4.2.3
Initiation of experimental MI
Experimental MI was induced via subcutaneous injection of ISO in
rats after dissolving in normal saline (100 mg/kg) at a period of 24 hr
for 2 days.
|
4.2.4
Experimental design
|
4.2
In vitro ACE inhibition study
The animals were randomly divided into five groups (12 rats each).
Group 1: normal control; Group 2: ISO rats; Group 3: compound
4i (5 mg/kg) + ISO; Group 4: compound 4i (10 mg/kg) + ISO; Group 5:
compound 4i (15 mg/kg) + ISO. Compound 4i after suspending in
0.5% carboxymethyl cellulose was orally administered once a day to
rats for the duration of 10 days (once a day) using an intragastric
tube. The rats were killed to isolate serum and plasma by cervical
decapitation after 12 hr of the second dose of ISO. The heart tissues
were isolated and washed in chilled normal saline. The supernatant
obtained after homogenization of heart tissues in Tris‐HCl buffer (pH
7.4) was used for the estimation of various biochemical parameters.
In vitro ACE inhibition assay was performed using the colorimetric
method, where 10 ml of 0.05 M sodium borate buffer, pH 8.2, containing
0.3 M NaCl and 0.5% Triton X‐100, was used to suspend 1 g Rabbit Lung
Acetone Powder (Sigma‐Aldrich), a source of ACE enzyme, at 4°C for
24 hr, followed by centrifugation at 15,000 rpm for 60 min at 4°C. The
resultant supernatant was used as a source of ACE enzyme for this assay.
The release of hippuric acid (HA) resulting from the hydrolysis of the
substrate hippuryl‐histidyl‐leucine (HHL; Sigma‐Aldrich) directly corre-
lated with the inhibitory activity of the compound. The test and standard
drug lisinopril was made in a solution at 1‐mM concentration and was
preincubated with 7 ml of the ACE enzyme for 10 min at 37°C. The final
volume was adjusted using 0.05 M sodium borate buffer (pH 8.2) con-
taining 0.3 M NaCl to obtain identical concentration. Furthermore, the
enzyme reaction was then initiated by adding 50 ml of 5 mM substrate
(HHL), followed by incubation at 37°C for 30 min. The reaction was then
|
4.2.5
Evaluation of hemodynamic parameters
After the first ISO injection, the blood pressure and heart rate were
taken at 48 hr using a computerized, noninvasive tail‐cuff system,

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Visitech BP‐2000 Blood Pressure Analysis System™ (Visitech Sys-
tems, Apex, NC, USA). Thereafter, all the rats were anesthetized with
urethane to measure the left ventricular function. The LVSP, left
ventricular end‐diastolic pressure, and left ventricular maximum rate
of positive or negative pressure development ( LVdp/dtmax) were
recorded using a BL‐420E monitor system (Chengdu, China).
sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and
shifted to the polyvinylidene fluoride membrane, and probed with
primary antibodies (1:1,000 in 1% bovine serum albumin [BSA]/TBS‐
T) overnight at 4°C. The membranes were then washed twice for
15 min each in TBS‐T and incubated with horseradish peroxidase‐
conjugated goat anti‐mouse or rabbit antibodies (1:10,000 in 1%
BSA/TBS‐T).
|
4.2.6
Infarct size measurement
|
4.2.11
Statistical analysis
For infarct size measurement, rats from different groups were
carefully dissected and 2‐mm‐thick sections were sliced. The infarct
size of rats was determined with 5‐triphenyltetrazolium chloride
(Sigma‐Aldrich Co.) at 37°C for 30 min in the dark. The region of the
heart with no color was supposed as the ischemic heart muscles,
whereas the area with brick red color was deemed as regular myo-
cardium. The area of infarct size was recorded by the volume and
weight as a percentage of the left ventricle.
Data were analyzed by SPSS 17.0 software and expressed as the
mean standard error of the mean. Differences were analyzed by
one‐way analysis of variance, followed by Dunnett's test for in-
dividual comparisons between each group mean. A p‐value of .05 was
considered statistically significant.
CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interests.
|
4.2.7
Enzyme‐linked immunosorbent assay of LDH,
DATA AVAILABILITY STATEMENT
CK‐MB, cTn‐T, TNF‐α, and IL‐6
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
Immediately, blood samples were centrifuged at 12,000g for 10 min
at 4°C, and then the supernatant was collected for measurement at
−20°C. Activities of LDH, CK‐MB, Tn‐T, TNF‐α, and IL‐6 were per-
formed using a suite of commercial kits in accordance with the
manufacturer's instructions (Beyotime Institute of Biotechnology,
Nanjing, China).
ORCID
Guoying Su
http://orcid.org/0000-0002-4462-2587
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How to cite this article: You H, Su X, Su G. Novel
thiazole–pyrazolone hybrids as potent ACE inhibitors and
their cardioprotective effect on isoproterenol‐induced
myocardial infarction. Arch Pharm. 2020;e2000140.
https://doi.org/10.1002/ardp.202000140
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