Design and Development of Novel 4-(4-(1H-Tetrazol-5-yl)-1H-pyrazol-1-yl)-6-morpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine as Cardiotonic Agent via Inhibition of PDE3

Article abstract of DOI:10.1002/ardp.201500388

The classical phosphodiesterase 3A (PDE3A) inhibitors provide relaxation of the vasculature system via increasing the cellular level of cyclic adenosine monophosphate (cAMP) and proved to be useful in the management of congestive heart failure. Consequent

Full text of DOI:10.1002/ardp.201500388

RCHH HARM
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Arch. Pharm. Chem. Life Sci. 2016, 349, 268–276
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Full Paper
Design and Development of Novel 4-(4-(1H-Tetrazol-5-yl)-1H-
pyrazol-1-yl)-6-morpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-
amine as Cardiotonic Agent via Inhibition of PDE3
Zhi-Wei Mao1, Qiao-Ling Li2, Ping Wang1, Yang-Li Sun1, and Yu Liu1
1
Department of Cardiovasology, Zhengzhou Central Hospital Affiliated to Zhengzhou University,
Zhengzhou, Henan, P. R. China
Department of Kidney Rheumatology, The Fifth Affiliated Hospital of Zhengzhou University,
2
Zhengzhou, Henan, P. R. China
The classical phosphodiesterase 3A (PDE3A) inhibitors provide relaxation of the vasculature system via
increasing the cellular level of cyclic adenosine monophosphate (cAMP) and proved to be useful in the
management of congestive heart failure. Consequently, the present paper deals with the development of
novel pyrazole derivatives tethered with substituted 1,3,5-triazine derivatives in search for novel PDE3
inhibitors. The synthesis of designed inhibitors was realized in a multi-step reaction and the structures
were ascertained with the help of various spectroscopic techniques. Subsequently, these analogs were
tested for their inhibitory activities against PDE3 enzymes, where they exhibited considerable inhibition,
revealing 9g as the most promising inhibitor of the class. In a docking study, the morpholine fragment of
compound 9g was efficiently engulfed in the small pocket of the active site lined by Gly940 and Pro941.
The substituted aromatic ring of the core scaffold was found to be positioned deep in the cavity bordered
by Tyr829, Asn830, Leu850, Glu851, and Thr893. Moreover, it considerably improved the contractility of
cardiac muscles without altering the heart beat frequency in experimental subjects.
Keywords: 1,3,5-Triazine / Docking / PDE3 inhibitor / Synthesis
Received: October 26, 2015; Revised: January 20, 2016; Accepted: January 26, 2016
DOI 10.1002/ardp.201500388
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Among the CVDs, failure of the heart or congestive heart
failure (CHF) is regarded as a chronic manifestation which
Introduction
The morbidity and mortality associated with cardiovascular
diseases (CVDs) have put a selective pressure for their effective
treatment and prevention. It is a rapidly increasing public
health concern for both developed and developing nations.
The severity of the disease has been easily understood by its
widespread nature, which accounts for the loss of 17.5 million
people in 2012, representing 31% of all global deaths [1–3].
results in inadequate supply of the blood to meet the demand
of various organs due to diminished cardiac function. It is a
grave life-threatening situation, demonstrating the end-
stage of an innumerable other cardiac anomalies without a
remedial cure [4]. Concerning to this, novel and effective
medication are needed urgently.
Against CHF, presently two lines of therapeutic interven-
tions have been used, i.e., glycoside- and non-glycoside
agents. The glycoside drugs are mainly based on digitalis and
have been associated with serious side-effect, including
acute arrhythmias along with a low therapeutic index [5].
Correspondence: Dr. Ping Wang, Department of Cardiovasology,
Zhengzhou Central Hospital Affiliated to Zhengzhou University,
No. 195 Tonbai Middle Road, Zhengzhou, Henan 450000, P. R.
China.
E-mail: wangpingcr@hotmail.com
Fax: þ86-371-67690000
Zhi-Wei Mao and Qiao-Ling Li contributed equally to this work.
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However, the drugs belonging to the category of non-
glycoside agents do not have any significant extra-effects
and are able to act as a cardiotonic agent via selective
inhibition of cyclic nucleotide phosphodiesterase (PDE)
enzymes. It is deemed to be responsible for maintaining
the endogenous level of cyclic adenosine monophosphate
(cAMP) and cyclic guanosine monophosphate (cGMP), a
second messenger. According to the physiological functions
and substrate specificity, it was further sub-divided into 11
different families exerting numerous roles, viz., PDE1–11.
Among the enzyme class, particularly PDE3 is considered to
induce vascular contraction and its inhibition causes relaxa-
tion of the vasculature system via increasing the cellular level
of cAMP. It was further sub-divided into PDE3A and PDE3B
depending upon its occurrence, for instance, cardiac tissues
and hepatocytes, respectively. Thus, its inhibition offers
symptomatic relief in CHF [6, 7].
More recently, pyrazole-based compound has been reported
to exhibit potent PDE3 inhibitory activity (Fig. 1) [8, 9]. The
derivatives originating from 1,3,5-triazine are also behaving as
potent medicinal agents, such as, antibacterial [10], antimalar-
ial [11], anticancer [12], antifungal [13], antidiabetic [14], anti-
CFTR [15], carbonic anhydrase inhibitors [16], anti-HIV [17], and
others. Concerning to this, present paper deals with the
development of novel pyrazole derivatives tethered with
substituted 1,3,5-triazine derivatives in search of novel PDE3
inhibitor.
commercially available 2,4,6-trichloro-1,3,5-triazine (1) was
selected as starting template which later underwent
neucleophillic reaction with morpholine in the presence
of activating base to yield compound 2. Whereas, various
amines were selected depending upon the chemical nature
to be substituted on above-synthesized compound to
afford corresponding derivative 3. Moreover, the remain-
ing chloro of 1,3,5-triazine was easily replaced with
hydrazine under reflux to furnish compound 4 in excellent
yield. It was further allowed to react with malonaldehyde
bis-(dimethyl acetal) which results in the cyclization of
terminal hydrazine to pyrazole (6),
a core scaffold;
according to the method described by Finar and Godfrey.
The synthesis of compound 7 was achieved under Duff’s
condition via selective formylation of compound 6, while it
was further allowed to react with hydroxylamine to afford
compound 8. The synthesis of target analogs was achieved
via refluxing above synthesized compound with NaN3 using
AlCl3 as a catalyst in DMF. The compounds were obtained in
excellent yield and considerable purity as determined via
various analytical techniques.
The FT-IR absorption bands of final compounds are shown in
the experimental section. The N–H stretching frequency was
observed at 3431–3438 cmꢀ1. The strong band at 1621–
1649 cmꢀ1 is attributed to the C N stretching frequency. The
–
–
Ar C–H group stretching frequency was observed at
3052–3065 cmꢀ1. Moreover, the presence of Cl on the phenyl
ring was observed at 784–786 cmꢀ1. The (–N N–) group of
–
–
tetrazole was observed at 1571–1589 cmꢀ1, whereas OCH3
group on the phenyl ring was observed at 2924–2928 cmꢀ1
.
Results and discussion
The final compound showed another band at
1156–1162 cmꢀ1 which is attributed to primary C–F stretching
Chemistry
–
The synthesis of target analogs was achieved in numerous
consecutive steps as outlined in Scheme 1. Initially, the
frequency. Moreover, the presence of C N group of
–
tetrazole was confirmed at 1621–1649 cmꢀ1. The 1H NMR
Figure 1. Structures of the various pyrazole-
based analogs (A and B) and of the designed
inhibitor (C) developed in the present study.
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Scheme 1. Synthesis of the target compounds. Reagents and conditions: (a) aq. NaOH soln., 0–5°C, stirring; (b) aq. NaOH soln.,
40–45°C; (c) KHCO3, reflux; (d) HCl, EtOH, reflux; (e) HMTA, CF3COOH, reflux; (f) NaI, NH2OH, DMF, reflux; (g) NaN3, DMF, AlCl3.
spectra showed at 8.02–6.18 ppm which is attributed to the
resonance of Ar–H. The resonance of methyl proton was
found as a singlet in the region 2.18–2.35 ppm, whereas
another singlet corresponds to 3.82–3.84 ppm for proton of
methoxy group. The resonance of pyrazole protons was
found as a doublet in the region 8.84–8.84 ppm. Finally, all
the structures of final compounds were recognized by mass
and elemental analysis.
was disclosed by the compounds containing nitro as the
substituent with prominent inhibition in the case of compound
9g against PDE3A and PDE3B with IC50 0.33 ꢁ 0.01 and
5.20 ꢁ 0.09 mM, respectively. Whereas, its isomeric replacement
leads to the marginal loss of the activity as evidenced by the
inhibitory potency of compounds 9h and 9i. The activity was
further diminished in the case of the rest of the compound
having electron donating substituents, viz., –methyl (9j–l) and
–methoxy (9m–o). It was surprising to note that compounds
containing methyl as a substituent showed prominent reduc-
tion in activity against PDE3B than PDE3A in comparison to
compounds having electron-withdrawing groups.
The structure–activity relationship study corroborated that
the entire set of synthesized compounds showed a similar
pattern of inhibition against PDE3A than PDE3B, with more
prominent action against PDE3A. While in comparison to
standard, only few compounds exhibit much prominent
action than vesnarinone. Particulary, compounds containing
nitro (9g–i) and fluoro (9d–f) along with 9a were found to
inhibit PDE3 enzymes more significantly than their congeners.
Among the synthesized compounds, analogs containing
electron-withdrawing group showed considerable activity
compared to electron-donating compounds. Moreover, the
position of substituent was also found critical for the
generation of the bioactivity, for instance, a minor isomeric
variation in core scaffold renders compound non-active or less
Pharmacological activity
As presented in Table 1, various target derivatives were
assessed for possible inhibition of PDE3 (types A and B)
enzyme (see also the Supporting Information for the InChI
codes). It has been found that entire set of target compounds
exhibit considerable range of inhibition against both the
variants of PDE3, e.g., PDE3A and PDE3B. Among the
synthesized compounds, 9a showed moderate inhibition of
PDE3A (IC50 ¼ 9.10 ꢁ 0.04 mM) and PDE3B (IC50 ¼ 13.43 ꢁ 0.23
mM). While on varying the position of substituent, the
inhibitory potency was reasonably reduced as found in the
case of compounds 9b and 9c against both the variants of
PDE3. The presence of fluoro on the flanked aromatic ring of
the core scaffold showed considerable improvement in the
inhibitory potency which suggests that para-substituted
derivative (9d) was found more active than its isomeric
counterparts, viz., 9e and 9f. The highest inhibitory potency
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Table 1. Inhibitory activity of target compounds against
PDE3.
of PDE3 was selected to perform the docking of compound 9g
to elaborate the key intermolecular interactions necessary for
the inhibitory activity. As shown in Fig. 3, 9g was efficiently
accommodated inside the catalytic site of the PDE3 enzyme
via making prolific interactions with the neighboring
residues. It was found that the morpholine fragment was
engulfed in the small-pocket lined by Gly940 and Pro941.
Whereas, the substituted aromatic ring of core scaffold was
found to be positioned deep in the cavity bordered by Tyr829,
Asn830, Leu850, Glu851, Thr893, and Asp894 via formation of
one H-bond with Asn830. Apart from it, 9g revealed the
formation of two non-covalent p–p stacking interaction with
Phe991 via involvement of 1,3,5-triazine and pyrazole. The
results of the docking study indicated that designed inhibitor
was appreciably accommodated deep inside the catalytic site
of protein and was thereby able to inhibit its catalytic function
for inhibitory activity. To further find out the key structural
fragment necessary for the activity, based on the docked
orientation, a pharamcophore-based model was generated
and is presented in Fig. 4. These computational results are
found in accordance with the earlier studies and provide valid
evidence for the efficient inhibitory activity of 9g against
PDE3A [9].
IC50 (mM)
Compound
PDE3A
PDE3B
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9.10 ꢁ 0.04
12.32 ꢁ 0.11
14.23 ꢁ 0.12
4.43 ꢁ 0.13
8.34 ꢁ 0.15
7.45 ꢁ 0.11
0.33 ꢁ 0.01
1.23. ꢁ 0.32
2.23 ꢁ 0.05
18.22 ꢁ 0.12
23.32 ꢁ 0.23
28.45 ꢁ 0.33
33.18 ꢁ 0.17
42.64 ꢁ 0.15
52.56 ꢁ 0.22
10.22 ꢁ 0.21
13.43 ꢁ 0.23
18.16 ꢁ 0.12
19.03 ꢁ 0.18
4.10 ꢁ 0.04
6.22 ꢁ 0.02
14.03 ꢁ 0.16
5.20 ꢁ 0.09
6.45 ꢁ 0.14
12.32 ꢁ 0.13
36.36 ꢁ 0.11
45.19 ꢁ 0.17
67.86 ꢁ 0.20
ND
ND
ND
Vesnarinone
15.45 ꢁ 0.21
active. It has been further found that para-substituted
derivative showed significant inhibitory activity than either
of ortho and meta counterparts. The structure–activity
relationship (SAR) of the target analogs has been clearly
outlined in Fig. 2.
Effect of compound 9g as ionotropic and
chronotropic activities
Encouraged by the excellent PDE3A inhibitory activity of
compound 9g, it is worthwhile to perform its possible effect
on the force of contraction and the frequency rate of heart of
experimental rat. This study is more imperative to perform
because the PDE3A enzymes are highly expressed in cardiac
tissues. Thus, the effect of 9g on the above-discussed
parameters was determined by the impulsively beating atria
of rat. However, the endogenous catecholamine (CAs) release
can interfere with the study, so the animal was pre-treated
with reserpine to block the release of any CAs [19]. The study
was performed in comparison to vesnarinone as a standard
and the results are presented in Table 2. From the results, it
was easily concluded that 9g does not affect the frequency
rate and is not able to induce any arrhythmias. It is
noteworthy to mention that the effect of compound 9g
was considerably found lower than vesnarinone as standard.
On the other hand, as assumed, it showed drastic improve-
ment in contractility of cardiac muscles (79 ꢁ 3) and even
much better than standard (51 ꢁ 1) at 100 mM.
In silico study of compound 9g
Docking study is considered as a powerful computational tool
which can easily predict the preferred orientation of ligand
into the protein of interest via many algorithmic
approaches [18]. Thus, in the current study, protein model
Conclusion
We have developed a novel series of hybrid pyrazole-1,3,5-
triazine conjugates as potent inhibitors of PDE3 with more
selective inhibition against the PDE3A over PDE3B. Moreover,
these molecules proved to enhance the force of contraction
rather than frequency of heart beat, which makes them as a
prospective lead for future drug discovery. However, more
Figure 2. Structure–activity relationship of the target analogs
for PDE3 inhibition.
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Figure 3. Docked orientation of compound 9g
in PDE3.
studies are needed to be performed for exact elucidation of its
molecular mechanism.
melting points of the synthesized derivatives were quantified
in digital melting point apparatus and are uncorrected. TLC
was used to analyze the completion of the reaction using silica
gel-G coated Al-plates and were detected under UV (254 nm)
and in chamber containing iodine vapor. Flash-1112 series
elemental analyzer was used for determination of carbon,
hydrogen, and nitrogen. IR spectra (KBr) were recorded on a
PerkinElmer FT-IR. The 1H NMR and 13C NMR spectra were
recorded on a Bruker Avance-500 NMR spectrometer and
Experimental
Chemistry
All chemicals were obtained from commercial suppliers and
used directly without further purification unless specified. The
Figure
4. Structure-based
pharmacophores
model of 9g for inhibition of PDE3.
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Table 2. Pharmacological evaluation of compound 9g on the heart of rat.a)
Parameter
Compound
Base
1 ꢂ 10ꢀ6
1 ꢂ 10ꢀ5
1 ꢂ 10ꢀ4
Contractile force
9g
100 ꢁ 2
100 ꢁ 5
100 ꢁ 1
100 ꢁ 5
115 ꢁ 2
104 ꢁ 1
100 ꢁ 4
105 ꢁ 4
132 ꢁ 6
126 ꢁ 2
110 ꢁ 1
112 ꢁ 4
179 ꢁ 3
151 ꢁ 1
118 ꢁ 5
121 ꢁ 5
Vesnarinone
Frequency rate
6d
Vesnarinone
a)
The pharmacological effect of each concentration (mol/L) of compound along with standard was articulated by the disparity
between the force of retrenchment and frequency earlier than and after its inclusion to the bathing fluid, and were shown as a
percentage difference in contraction and frequency in comparison with the controls. Results are presented as means ꢁ SEM
from six atria.
Bruker Avance-100 NMR spectrometer in DMSO-d6 solution
using TMS as an internal reference, respectively. MS spectra
were recorded on an Agilent 1100 LC/MS.
426.83 [Mþ1]; Elemental analysis for C17H16ClN11O: Calculated:
C, 47.95; H, 3.79; N, 36.18; Found: C, 47.94; H, 3.80; N, 36.18.
The syntheses of compounds 1, 2, 3, 4, 6, 7, and 8 were
performed in accordance with earlier reported procedures.
4-(4-(1H-Tetrazol-5-yl)-1H-pyrazol-1-yl)-N-(2-chlorophenyl)-
6-morpholino-1,3,5-triazin-2-amine (9c)
Yield: 76%; mp: 261–262°C; MW: 425.84; FT-IR (nmax; cmꢀ1 KBr):
–
–
General procedure for the synthesis of 4-(4-(1H-tetrazol-5-
yl)-1H-pyrazol-1-yl)-6-morpholino-N-(substituted phenyl)-
1,3,5-triazin-2-amine (9)
3431 (N–H), 3065 (Ar C–H), 1628 (C C), 1632 (C N tetrazole),
– –
ꢀ1
–
–
–
1584 (C N triazine), 1578 (–N N–), 1089, 786 (C–Cl) cm
;
–
1H NMR (400 MHz, DMSO, TMS) d ppm: 9.85 (s, 1H, tetrazole),
8.94 (d, 1H, J ¼ 1.8 Hz, pyrazole), 8.16 (d, 1H, J ¼ 1.5 Hz,
pyrazole), 7.76 (d, 1H, J ¼ 7.5 Hz, Ar–H), 7.45 (t, 1H, J ¼
7.2 Hz, Ar–H), 7.31 (d, 1H, J ¼ 7.3 Hz, Ar–H), 7.18 (d, 1H,
J ¼ 7.1 Hz, Ar–H), 3.74–3.68 (m, 8H, morpholine), 3.98
(s, 1H, N–H); 13C NMR (100 MHz, CDCl3) d ppm: 179.2, 177.6,
167.3, 163.6, 137.8, 136.4, 130.8, 129.2, 127.6, 125.5, 122.9,
122.1, 102.3, 66.4, 48.7; Mass: 426.85 [Mþ1]; Elemental analysis
for C17H16ClN11O: Calculated: C, 47.95; H, 3.79; N, 36.18; Found:
C, 47.96; H, 3.79; N, 36.19.
A mixture of 1-(4-morpholino-6-((substituted phenyl)amino)-
1,3,5-triazin-2-yl)-1H-pyrazole-4-carbonitrile (8) (0.1 mmol),
sodium azide (0.2 mmol), and ammonium chloride (0.2 mmol)
in 35 mL of DMF was heated at reflux temperature for 72 h.
The reaction mixture was then poured into water and
acidified with acid to make pH 5. The product was filtered
and dried to afford the desired corresponding target
derivatives in excellent yield.
4-(4-(1H-Tetrazol-5-yl)-1H-pyrazol-1-yl)-N-(4-chlorophenyl)-
6-morpholino-1,3,5-triazin-2-amine (9a)
4-(4-(1H-Tetrazol-5-yl)-1H-pyrazol-1-yl)-N-(4-fluorophenyl)-
6-morpholino-1,3,5-triazin-2-amine (9d)
Yield: 76%; mp: 258–259°C; MW: 425.84; FT-IR (nmax; cmꢀ1 KBr):
3432(N–H), 3064(ArC–H), 1628(C C), 1628(C Ntetrazole), 1589
Yield: 79%; mp: 238–239°C; MW: 409.38; FT-IR (nmax; cmꢀ1
–
–
–
–
ꢀ1
1H NMR
KBr): 3436 (N–H), 3057 (Ar C–H), 1623 (C C), 1627 (C N
–
–
–
–
–
–
(C N triazine), 1578 (–N N–), 1082, 786 (C–Cl) cm
;
–
–
–
–
(400 MHz, DMSO, TMS) d ppm: 9.82 (s, 1H, tetrazole), 8.94 (d, 1H,
J¼ 1.6 Hz, pyrazole), 8.16 (d, 1H, J¼ 1.5 Hz, pyrazole), 7.73–7.41
(m, 4H, ArH), 3.74–3.65 (m, 8H, morpholine), 3.94 (s, 1H, N–H);
13C NMR (100 MHz, CDCl3) d ppm: 179.4, 177.5, 167.3, 163.2,
137.6, 137.1, 129.8, 129.2, 127.7, 122.1, 102.4, 66.3, 48.7; Mass:
426.85[Mþ1];ElementalanalysisforC17H16ClN11O:Calculated:C,
47.95; H, 3.79; N, 36.18; Found: C, 47.96; H, 3.81; N, 36.17.
tetrazole), 1586 (C N triazine), 1571 (–N N–), 1159 (C–F)
–
–
1092 cmꢀ1; 1H NMR (400 MHz, DMSO, TMS) d ppm: 9.87 (s, 1H,
tetrazole), 8.93 (d, 1H, J ¼ 1.8 Hz, pyrazole), 8.17 (d, 1H,
J ¼ 1.5 Hz, pyrazole), 7.42–7.31 (m, 4H, Ar–H), 3.74–3.67
(m, 8H, morpholine), 3.97 (s, 1H, N–H); 13C NMR (100 MHz,
CDCl3) d ppm: 179.2, 177.6, 167.3, 163.7, 157.4, 137.7, 134.5,
129.3, 120.8, 116.3, 102.3, 66.3, 48.7; Mass: 410.39 [Mþ1];
Elemental analysis for C17H16FN11O: Calculated: C, 49.88; H,
3.94; N, 37.64; Found: C, 49.89; H, 3.94; N, 37.65.
4-(4-(1H-Tetrazol-5-yl)-1H-pyrazol-1-yl)-N-(3-chlorophenyl)-
6-morpholino-1,3,5-triazin-2-amine (9b)
Yield: 72%; mp: 263–264°C; MW: 425.84; FT-IR (nmax; cmꢀ1 KBr):
4-(4-(1H-Tetrazol-5-yl)-1H-pyrazol-1-yl)-N-(3-fluorophenyl)-
6-morpholino-1,3,5-triazin-2-amine (9e)
–
–
3434 (N–H), 3062 (Ar C–H), 1625 (C C), 1629 (C N tetrazole),
–
–
ꢀ1
–
–
–
1586 (C N triazine), 1572 (–N N–), 1086, 784 (C–Cl) cm
;
Yield: 74%; mp: 243–244°C; MW: 409.38; FT-IR (nmax; cmꢀ1
–
1H NMR (400 MHz, DMSO, TMS) d ppm: 9.86 (s, 1H, tetrazole),
8.93 (d, 1H, J ¼ 1.6 Hz, pyrazole), 8.18 (d, 1H, J ¼ 1.5 Hz,
pyrazole), 7.78 (d, 1H, J ¼ 8.1 Hz, Ar–H), 7.56 (t, 1H, J ¼ 7.4 Hz,
Ar–H),7.34(d,1H,J ¼ 8.2 Hz,Ar–H),7.25(d,1H,J ¼ 7.8 Hz,Ar–H),
3.74–3.67 (m, 8H, morpholine), 3.96 (s, 1H, N–H); 13C NMR
(100 MHz, CDCl3) d ppm: 179.2, 177.6, 167.3, 163.7, 143.9, 137.6,
135.2, 130.9, 129.3, 122.3, 116.7, 115.9, 102.4, 66.4, 48.9; Mass:
KBr): 3431 (N–H), 3058 (Ar C–H), 1627 (C C), 1621 (C N
–
–
–
–
–
–
–
tetrazole), 1589 (C N triazine), 1578 (–N N–), 1162 (C–F)
–
1098 cmꢀ1; 1H NMR (400 MHz, DMSO, TMS) d ppm: 9.89 (s, 1H,
tetrazole), 8.91 (d, 1H, J ¼ 1.6 Hz, pyrazole), 8.17 (d, 1H,
J ¼ 1.5 Hz, pyrazole), 7.72 (d, 1H, J ¼ 8.4 Hz, Ar–H), 7.52
(t, 1H, J ¼ 7.3 Hz, Ar–H), 7.29 (d, 1H, J ¼ 7.1 Hz, Ar–H), 6.98
(d, 1H, J ¼ 5.4 Hz, Ar–H), 3.74–3.68 (m, 8H, morpholine), 3.97
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(s, 1H, N–H); 13C NMR (100 MHz, CDCl3) d ppm: 179.2, 177.6,
167.6, 163.8, 163.5, 144.2, 137.8, 131.2, 129.3, 113.4, 110.7,
104.9, 102.4, 66.4, 48.7; Mass: 410.41 [Mþ1]; Elemental
analysis for C17H16FN11O: Calculated: C, 49.88; H, 3.94; N,
37.64; Found: C, 49.89; H, 3.95; N, 37.65.
tetrazole), 1583 (C N triazine), 1589 (–N N–), 1525 (NO str),
–
–
–
–
2
1091, 715 cmꢀ1 1H NMR (400 MHz, DMSO, TMS) d ppm: 9.95
;
(s, 1H, tetrazole), 8.84 (d, 1H, J ¼ 1.6 Hz, pyrazole), 8.14 (d, 1H,
J ¼ 1.5 Hz, pyrazole), 8.08 (d, 1H, J ¼ 7.8 Hz, Ar–H), 7.65 (d, 1H,
J ¼ 7.5 Hz, Ar–H), 7.64 (t, 1H, J ¼ 7.3 Hz, Ar–H), 7.41
(t, 1H, J ¼ 7.4 Hz, Ar–H), 3.74–3.68 (m, 8H, morpholine), 3.95
(s, 1H, N–H); 13C NMR (100 MHz, CDCl3) d ppm: 179.2, 177.5,
167.3, 163.4, 144.7, 137.8, 137.2, 129.3, 129.1, 125.8, 119.7,
110.5, 102.3, 66.4, 48.8; Mass: 437.42 [Mþ1]; Elemental
analysis for C17H16N12O3: Calculated: C, 46.79; H, 3.70; N,
38.52; Found: C, 46.80; H, 3.71; N, 38.52.
4-(4-(1H-Tetrazol-5-yl)-1H-pyrazol-1-yl)-N-(2-fluorophenyl)-
6-morpholino-1,3,5-triazin-2-amine (9f)
Yield: 69%; mp: 247–248°C; MW: 409.38; FT-IR (nmax; cmꢀ1
–
–
KBr): 3435 (N–H), 3056 (Ar C–H), 1624 (C C), 1625 (C N
–
–
–
–
tetrazole), 1584 (C N triazine), 1579 (–N N–), 1156 (C–F)
–
–
1094 cmꢀ1; 1H NMR (400 MHz, DMSO, TMS) d ppm: 9.91 (s, 1H,
tetrazole), 8.87 (d, 1H, J ¼ 1.6 Hz, pyrazole), 8.21 (t, 1H,
J ¼ 7.6 Hz, Ar–H), 8.16 (d, 1H, J ¼ 1.5 Hz, pyrazole), 7.26
(t, 1H, J ¼ 7.2 Hz, Ar–H), 7.04 (d, 1H, J ¼ 6.9 Hz, Ar–H), 7.02
(d, 1H, J ¼ 6.8 Hz, Ar–H), 3.74–3.67 (m, 8H, morpholine), 3.96
(s, 1H, N–H); 13C NMR (100 MHz, CDCl3) d ppm: 179.2, 177.6,
167.2, 163.6, 154.8, 137.8, 129.3, 128.2, 125.1, 123.8, 123.2,
116.4, 102.4, 66.4, 48.7; Mass: 410.36 [Mþ1]; Elemental
analysis for C17H16FN11O: Calculated: C, 49.88; H, 3.94; N,
37.64; Found: C, 49.90; H, 3.94; N, 37.64.
4-(4-(1H-Tetrazol-5-yl)-1H-pyrazol-1-yl)-6-morpholino-N-
(p-tolyl)-1,3,5-triazin-2-amine (9j)
Yield: 84%; mp: 243–244°C; MW: 405.42; FT-IR (nmax; cmꢀ1
–
KBr): 3434 (N–H), 3052 (Ar C–H), 2981 (CH3 str), 1625 (C C),
–
–
–
–
1629 (C N tetrazole), 1581 (C N triazine), 1586 (–N N–),
–
–
–
1093, 712 cmꢀ1; H NMR (400 MHz, DMSO, TMS) d ppm: 9.92
(s, 1H, tetrazole), 8.86 (d, 1H, J ¼ 1.6 Hz, pyrazole), 8.16 (d,
1H, J ¼ 1.5 Hz, pyrazole), 7.24–7.04 (m, 4H, Ar–H), 3.74–3.67
(m, 8H, morpholine), 3.97 (s, 1H, N–H), 2.34 (s, 3H, CH3);
13C NMR (100 MHz, CDCl3) d ppm: 179.2, 177.6, 167.3, 163.6,
137.6, 135.8, 131.2, 129.9, 129.3, 120.3, 102.4, 66.4, 48.8, 21.2;
Mass: 406.41 [Mþ1]; Elemental analysis for C18H19N11O:
Calculated: C, 53.33; H, 4.72; N, 38.00; Found: C, 53.35; H,
4.71; N, 38.02.
1
4-(4-(1H-Tetrazol-5-yl)-1H-pyrazol-1-yl)-6-morpholino-N-
(4-nitrophenyl)-1,3,5-triazin-2-amine (9g)
Yield: 82%; mp: 258–259°C; MW: 436.39; FT-IR (nmax; cmꢀ1
–
–
KBr): 3432 (N–H), 3059 (Ar C–H), 1627 (C C), 1628 (C N
–
–
–
–
tetrazole), 1587 (C N triazine), 1582 (–N N–), 1528 (NO str),
–
–
2
1098, 712 cmꢀ1
;
1H NMR (400 MHz, DMSO, TMS) d ppm: 9.92
4-(4-(1H-Tetrazol-5-yl)-1H-pyrazol-1-yl)-6-morpholino-N-
(m-tolyl)-1,3,5-triazin-2-amine (9k)
(s, 1H, tetrazole), 8.89 (d, 1H, J ¼ 1.6 Hz, pyrazole), 8.17 (d, 1H,
J ¼ 1.5 Hz, pyrazole), 8.12–7.36 (m, 4H, Ar–H), 3.74–3.66 (m,
8H, morpholine), 3.97 (s, 1H, N–H); 13C NMR (100 MHz, CDCl3) d
ppm: 179.2, 177.4, 167.3, 163.6, 145.2, 137.9, 137.7, 129.2,
124.7, 119.2, 102.3, 66.3, 48.7; Mass: 437.40 [Mþ1]; Elemental
analysis for C17H16N12O3: Calculated: C, 46.79; H, 3.70; N,
38.52; Found: C, 46.80; H, 3.69; N, 38.52.
Yield: 65%; mp: 250–251°C; MW: 405.42; FT-IR (nmax; cmꢀ1
–
KBr): 3438 (N–H), 3056 (Ar C–H), 2983 (CH3 str), 1627 (C C),
–
–
–
–
1636 (C N tetrazole), 1586 (C N triazine), 1578 (–N N–),
–
–
–
1091, 719 cmꢀ1
;
1H NMR (400 MHz, DMSO, TMS) d ppm: 9.95
(s, 1H, tetrazole), 8.87 (d, 1H, J ¼ 1.6 Hz, pyrazole), 8.18 (d, 1H,
J ¼ 1.5 Hz, pyrazole), 7.52 (d, 1H, J ¼ 8.2 Hz, Ar–H), 7.44
(d, 1H, J ¼ 7.2 Hz, Ar–H), 7.18 (d, 1H, J ¼ 6.4 Hz, Ar–H), 6.89
(d, 1H, J ¼ 5.2 Hz, Ar–H), 3.74–3.68 (m, 8H, morpholine), 3.98
(s, 1H, N–H), 2.35 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3) d
ppm: 179.2, 177.6, 167.3, 163.7, 142.3, 139.2, 137.8, 129.4,
129.1, 121.2, 119.4, 114.8, 102.4, 21.3, 66.4, 48.8, 21.3; Mass:
406.43 [Mþ1]; Elemental analysis for C18H19N11O: Calculated:
C, 53.33; H, 4.72; N, 38.00; Found: C, 53.32; H, 4.72; N, 38.01.
4-(4-(1H-Tetrazol-5-yl)-1H-pyrazol-1-yl)-6-morpholino-N-
(3-nitrophenyl)-1,3,5-triazin-2-amine (9h)
Yield: 69%; mp: 264–265°C; MW: 436.39; FT-IR (nmax; cmꢀ1
–
–
KBr): 3435 (N–H), 3061 (Ar C–H), 1624 (C C), 1622 (C N
–
–
–
–
tetrazole), 1589 (C N triazine), 1584 (–N N–), 1529 (NO str),
–
–
2
1094, 718 cmꢀ1; 1H NMR (400 MHz, DMSO, TMS) d ppm: 9.94 (s,
1H, tetrazole), 8.86 (d, 1H, J ¼ 1.6 Hz, pyrazole), 8.18 (d, 1H,
J ¼ 1.5 Hz, pyrazole), 8.02 (d, 1H, J ¼ 3.4 Hz, Ar–H), 7.62 (d, 1H,
J ¼ 8.3 Hz, Ar–H), 7.56 (d, 1H, J ¼ 7.4 Hz, Ar–H), 7.45
(t, 1H, J ¼ 7.1 Hz, Ar–H), 3.74–3.67 (m, 8H, morpholine), 3.96
(s, 1H, N–H); 13C NMR (100 MHz, CDCl3) d ppm: 179.2, 177.5,
167.3, 163.5, 148.8, 143.4, 137.6, 130.4, 129.2, 123.9, 113.8,
109.2, 102.3, 66.4, 48.7; Mass: 437.38 [Mþ1]; Elemental
analysis for C17H16N12O3: Calculated: C, 46.79; H, 3.70; N,
38.52; Found: C, 46.78; H, 3.70; N, 38.53.
4-(4-(1H-Tetrazol-5-yl)-1H-pyrazol-1-yl)-6-morpholino-N-
(o-tolyl)-1,3,5-triazin-2-amine (9l)
Yield: 62%; mp: 254–255°C; MW: 405.42; FT-IR (nmax; cmꢀ1
–
KBr): 3432 (N–H), 3054 (Ar C–H), 2981 (CH3 str), 1629 (C C),
–
–
–
–
1639 (C N tetrazole), 1585 (C N triazine), 1572 (–N N–),
–
–
–
1095, 712 cmꢀ1
;
1H NMR (400 MHz, DMSO, TMS) d ppm: 9.91
(s, 1H, tetrazole), 8.85 (d, 1H, J ¼ 1.6 Hz, pyrazole), 8.17 (d, 1H,
J ¼ 1.5 Hz, pyrazole), 7.15 (d, 1H, J ¼ 2.4 Hz, Ar–H), 7.02 (t, 1H,
J ¼ 1.8 Hz, Ar–H), 6.76 (t, 1H, J ¼ 1.9 Hz, Ar–H), 6.48
(d, 1H, J ¼ 1.3 Hz, Ar–H), 3.74–3.68 (m, 8H, morpholine),
3.97 (s, 1H, N–H), 2.18 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3) d
ppm: 179.2, 177.6, 167.2, 163.7, 142.4, 137.7, 131.4, 129.2,
128.9, 126.4, 123.8, 123.7, 102.3, 66.4, 48.7, 17.6; Mass: 406.41
4-(4-(1H-Tetrazol-5-yl)-1H-pyrazol-1-yl)-6-morpholino-N-
(2-nitrophenyl)-1,3,5-triazin-2-amine (9i)
Yield: 65%; mp: 267–268°C; MW: 436.39; FT-IR (nmax; cmꢀ1
–
–
KBr): 3432 (N–H), 3064 (Ar C–H), 1621 (C C), 1627 (C N
–
–
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1,3,5-Triazine as Cardiotonic Agent
Archiv der Pharmazie
[Mþ1]; Elemental analysis for C18H19N11O: Calculated: C,
7 pm). In order to prevent any stress in the experimental animal,
they had the free access to a standard laboratory diet and water.
To obtain a trial fragments, the rats were pre-treated intraperi-
toneally with reserpine (5 mg/kg body weight) to prevent any
interference of endogenous CAs on the results [10]. In every
experimentation, the rats were made unconscious with intra-
peritoneal injection of thiopental (a barbiturate derivative) in
80 mg/kg. Under the influence of anesthesia, midline thoracot-
omy was performed and the heart was rapidly removed. The
isolated heart without delay was rapidly transferred into
dissection plate packed with oxygenated Krebs–Henseleit
solution. The required enzymes were obtained from BPS
BioScience, Inc.
53.33; H, 4.72; N, 38.00; Found: C, 53.33; H, 4.73; N, 38.02.
4-(4-(1H-Tetrazol-5-yl)-1H-pyrazol-1-yl)-N-(4-methoxyphenyl)-
6-morpholino-1,3,5-triazin-2-amine (9m)
Yield: 83%; mp: 271–272°C; MW: 421.42; FT-IR (nmax; cmꢀ1
–
KBr): 3437 (N–H), 3052 (Ar C–H), 2924 (OCH str), 1636 (C C),
–
3
–
–
–
1642 (C N tetrazole), 1589 (C N triazine), 1576 (–N N–),
–
–
–
1096, 713 cmꢀ1
;
1H NMR (400 MHz, DMSO, TMS) d ppm: 9.95
(s, 1H, tetrazole), 8.89 (d, 1H, J ¼ 1.6 Hz, pyrazole), 8.14 (d, 1H,
J ¼ 1.5 Hz, pyrazole), 7.26–6.62 (m, 4H, Ar–H), 3.72–3.67 (m,
8H, morpholine), 3.95 (s, 1H, N–H), 3.84 (s, 3H, OCH3); 13C NMR
(100 MHz, CDCl3) d ppm: 179.2, 177.5, 167.3, 163.6, 153.4,
137.7, 131.3, 129.3, 121.8, 115.2, 102.4, 66.5, 55.9, 48.8;
Mass: 422.44 [Mþ1]; Elemental analysis for C18H19N11O2:
Calculated: C, 51.30; H, 4.54; N, 36.56; Found: C, 51.31; H, 4.54;
N, 36.54.
Assessment of PDE3A and PDE3B inhibitory activities
Briefly, at room temperature, the desired enzymatic reac-
tion was performed for 60 min in 50 mL mixture containing
IMAP reaction buffer, 100 nM FAM-cAMP, 1 ng PDE3A or
PDE3B, and the synthesized analogs. The reactions were
performed in the presence of binding reagent made in 1:600
dilution in a reagent binding buffer containing 85% Binding
Buffer A and 15% Binding Buffer B at room temperature
for 60 min. The assays were executed twice at each
concentration and the resultant fluorescence intensity,
determined by microplate reader, is converted into fluores-
cence polarization. The highest value of fluorescence
polarization (FPt) in each data set was assumed as 100%
activity, in the absence of PDE3, it was assumed to be 0%
activity. The percentage of activity in the presence of
the compound was calculated with the help of the following
equation: %binding ¼ (FP ꢀ FPb)/(FPt ꢀ FPb) ꢂ 100, where FP
is the fluorescence polarization (with compound), FPb is the
fluorescent polarization (absence of PDE3), and FPt corre-
sponds to the highest fluorescent polarization in each data
set. The IC50 values were determined accordingly from the
percent activity of the enzymes at different concentrations
of synthetic inhibitors.
4-(4-(1H-Tetrazol-5-yl)-1H-pyrazol-1-yl)-N-(3-methoxyphenyl)-
6-morpholino-1,3,5-triazin-2-amine (9n)
Yield: 74%; mp: 278–279°C; MW: 421.42; FT-IR (nmax; cmꢀ1
–
KBr): 3431 (N–H), 3059 (Ar C–H), 2928 (OCH str), 1631 (C C),
–
3
–
–
–
1649 (C N tetrazole), 1586 (C N triazine), 1572 (–N N–),
–
–
–
1098, 712 cmꢀ1
;
1H NMR (400 MHz, DMSO, TMS) d ppm: 9.93
(s, 1H, tetrazole), 8.87 (d, 1H, J ¼ 1.6 Hz, pyrazole), 8.18 (d, 1H,
J ¼ 1.5 Hz, pyrazole), 7.21 (d, 1H, J ¼ 5.8 Hz, Ar–H), 7.08 (t, 1H,
J ¼ 5.3 Hz, Ar–H), 6.38 (d, 1H, J ¼ 4.3 Hz, Ar–H), 6.18
(d, 1H, J ¼ 4.1 Hz, Ar–H), 3.73–3.69 (m, 8H, morpholine),
3.96 (s, 1H, N–H), 3.82 (s, 3H, OCH3); 13C NMR (100 MHz, CDCl3)
d ppm: 179.2, 177.5, 167.3, 163.6, 161.4, 143.4, 137.8, 130.5,
129.2, 110.8, 110.2, 102.4, 99.6, 66.4, 55.9, 48.9; Mass: 422.40
[Mþ1]; Elemental analysis for C18H19N11O2: Calculated: C,
51.30; H, 4.54; N, 36.56; Found: C, 51.33; H, 4.55; N, 36.55.
4-(4-(1H-Tetrazol-5-yl)-1H-pyrazol-1-yl)-N-(2-methoxyphenyl)-
6-morpholino-1,3,5-triazin-2-amine (9o)
Yield: 68%; mp: 275–276°C; MW: 421.42; FT-IR (nmax
;
cmꢀ1 KBr): 3437 (N–H), 3064 (Ar C–H), 2924 (OCH3 str),
–
–
–
1634 (C C), 1642 (C N tetrazole), 1586 (C N triazine), 1574
Evaluation of inotropic and chronotropic activities
The whole atrium was detached from the ventricle and
mounted vertically in a 50 mL organ-bath containing Krebs–
Henseleit solution which was regularly gassed by 95% O2 and
5% CO2 in 35–37°C with a pH of 7.35–7.45. Whereas, the
bathing solution was prepared (in mM) using NaCl 118, KCl
4.5, CaCl2 1.36, MgSO4 1.21, NaH2PO4 1.22, NaHCO3 25, and
glucose 11. The resting tension was attuned to about 0.5 g in
the whole atria and the initial equilibration period was
achieved for each preparation. To verify the blockage of
endogeneous CAs, the atria that did not show any positive
inotropic effect induced by tyramine (1.5 mM) were selected
for further experimentation. The test compounds were added
in the graded dose of 1–100 mM and the responses of each
concentration were documented up to the maximum limit.
The force transducer (type E. Zimmermann, Leipzig, Berlin)
connected to PowerLab 8/30 (model 870) was utilized to
record developed tension.
–
–
–
ꢀ1
1H NMR (400 MHz, DMSO, TMS) d
–
(–N N–), 1095, 713 cm
;
–
ppm: 9.97 (s, 1H, tetrazole), 8.89 (d, 1H, J ¼ 1.6 Hz,
pyrazole), 8.18 (d, 1H, J ¼ 1.5 Hz, pyrazole), 6.89 (d, 1H,
J ¼ 4.8 Hz, Ar–H), 6.78 (t, 1H, J ¼ 4.5 Hz, Ar–H), 6.68
(d, 1H, J ¼ 4.2 Hz, Ar–H), 6.48 (d, 1H, J ¼ 3.2 Hz, Ar–H),
3.74–3.67 (m, 8H, morpholine), 3.97 (s, 1H, N–H), 3.84
(s, 3H, OCH3); 13C NMR (100 MHz, CDCl3) d ppm: 179.2,
177.5, 167.2, 163.5, 147.4, 137.6, 132.7, 129.2, 122.7, 121.9,
113.4, 112.1, 102.4, 66.4, 55.9, 48.8; Mass: 422.43 [Mþ1];
Elemental analysis for C18H19N11O2: Calculated: C, 51.30; H,
4.54; N, 36.56; Found: C, 51.32; H, 4.54; N, 36.56.
Pharmacological activity
Animals, drugs, chemicals, and instruments
The adult male Wistar rats weighing 250–350 g were obtained
from the animal house of the institute and kept in optimum
conditions (temperature: 23 ꢁ 2°C; light–dark cycle: 7 am to
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2016, 349, 268–276
Z.-W. Mao et al.
Archiv der Pharmazie
Molecular docking studies
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~
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The authors have declared no conflicts of interest.
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www.archpharm.com
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