Cardioprotective effect of novel sulphonamides-1,3,5-triazine conjugates against ischaemic–reperfusion injury via selective inhibition of MMP-9

Article abstract of DOI:10.1111/cbdd.12807

Diseases affecting cardiovascular system are ranked as a top most cause of morbidity and mortality. Herein, a novel class sulphonamides-1,3,5-triazine conjugates have been synthesized and tested for inhibitory activity against MMP-2 and MMP-9. The results of the study showed that these molecules efficiently inhibit MMP-9 than MMP-2, revealing compound 8e as the most potent inhibitor (IC₅₀?=?2.34?±?0.56?nm). Due to involvement of MMP-9 in many cardiovascular diseases, particularly in myocardial ischaemia (MI), compound 8e was further subjected for the determination of the protective effect on isoproterenol (ISO)-induced myocardial injury in rats.

Full text of DOI:10.1111/cbdd.12807

Received Date : 19-Apr-2016
Revised Date : 02-Jun-2016
Accepted Date : 12-Jun-2016
Article type : Research Article
Cardio protective effect of novel sulphonamides-1,3,5-triazine conjugates
against Ischemic-reperfusion injury via selective inhibition of MMP-9
1
,2
2
3
3
1,3
Xiao-zhu Zheng ,Jia-li Zhou , Jing Ye , Pei-pei Guo , Chun-shui Lin *
1
Southern Medical University, Guangzhou, 510515, Guangdong, China
2
Department of Anesthesiology, Zhejiang Hospital, Hangzhou, 310030, Zhejiang, China
3
Department of Anesthesiology, Southern Medical University, Nanfang Hospital, Guangzhou
510515, Guangdong, China
*
Corresponding author:
Chun-shui Lin
Tel&Fax: 0086-020-61641114
Email: linchunshui@hotmail.com
Running head title
Lin et al.: Cardio protective effect of novel sulphonamides-1,3,5-triazine conjugates
Abstract
Diseases affecting cardiovascular system are ranked as a top most cause of morbidity and
mortality. Herein, a novel class sulphonamides-1,3,5-triazine conjugates have been
synthesized and tested for inhibitory activity against MMP-2 and MMP-9. The results of the
study showed that, these molecules efficiently inhibit MMP-9 than MMP-2, revealing
compound 8e as the most potent inhibitor (IC = 2.34±0.56 nM). Due to involvement of
5
0
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
an 'Accepted Article', doi: 10.1111/cbdd.12807
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MMP-9 in many cardiovascular diseases, particularly in myocardial ischemia (MI),
compound 8e was further subjected for the determination of the protective effect on
isoproterenol (ISO)-induced myocardial injury in rats.
Keywords: Sulphonamide, 1,3,5-triazine, MMP inhibition, myocardial ischemia,
biochemical assay.
1
. Introduction
Diseases affecting cardiovascular system are ranked as a top most cause of morbidity and
mortality [1]. According to an estimate, by 2020, the heart disease and stroke will turn out to
be the principal causes of death and disability worldwide, with the number of losses proposed
to augment to over 24 million by 2030 [2]. This fatal killer does not confine to only
developed nation; yet, it also engulfed developing countries or resource poor economies with
major impact [3]. The severity of the condition could be easily understood by the fact that,
nearly 80% of deaths attributed to cardiovascular diseases (CVDs) are now occurring in low
and middle income countries [4]. Alone in China, nearly 230 million people are living with
CVDs with one in 5 adults has a disease of cardiovascular system [5]. According to Report
on Cardiovascular Disease in China released in 2011, approximately 2 million patients are
diagnosed with myocardial infarction (MI) [6]. It is often categorized as imbalance in
demand and supply of coronary blood, which results in myocardial ischemic injury [7]. It
later damages the cardiomyocytes. Numerous Preclinical and clinical studies showed that at
some stage in ischemic damage, oxidative stress produced by the production of reactive
oxygen species (ROS) plays a pivotal role in the advancement of MI [8,9].
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Despite of advances in medical science to provide better options to treat MI, antioxidant still
serve as a major agent to circumvent the problem of oxidative stress in MI [10]. However, it
frequently failed to provide adequate protection against the oxidative reaction [11].
Therefore, additional agents are needed urgently which could offer novel ways of protection.
Matrix metalloproteinase (MMPs) are the class of the enzymes belong to the zinc-dependent
endopeptidases [12] that participate in the breakdown of numerous extracellular matrix
(ECM) proteins [13]. It results in modulation of various physiological and pathological
processes including MI, atherosclerosis and congestive heart failure [14]. According to the
specificity of substrate, the major classes of MMPs are found in the heart include
collagenases (MMP-1, MMP-8 and MMP-13), gelatinases (MMP-2 and MMP-9),
stromelysins (MMP-3, MMP-7 and MMP-10) and the membrane- type MMPs (e.g., MMP
14, also known as MT1-MMP) [15]. Various studies reported that, the expression of MMP-2
and MMP-9 has been found highly elevated in clinical and experimental forms of heart
failures [16], with major significant concentration of MMP-9 has been observed within 24 h
of MI [17]. This was observed with the loss of inhibitory activity of natural MMP inhibitor
TIMP-1 [18]. In such circumstances, inhibitors of Matrix metalloproteinase-9 offer selective
advantages over other options for effective management of MI [17].
Recent studies suggested that compounds belonging to the category of sulphonamide exhibit
wide array of matrix metalloproteinase (MMP) inhibitory action [19, 20, 21]. This excellent
activity has been corroborated due to its ability to serve as a peptide surrogates with exclusive
physical properties which make it as an ideal functional group for the advancement of novel
non-peptide matrix metalloproteinase inhibitors (MMPIs). As a result, the diverse library of
chemical compounds bearing sulphonamide scaffold has been developed as MMP inhibitor
and more are still in the process. To this class, CGS-27023A is the first breakthrough
displaying potent MMP inhibitory activity against stromelysin (MMP-3), the gelatinases
(MMP-2, MMP-9) and collagenase-3 (MMP-13). Moreover, the derivatives of 1,3,5-triazine
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also showed various bioactivities including, antimalarial [22], antibacterial [23], anticancer
24], carbonic anhydrase inhibitor [25], DPP-4 inhibitor [26] and CFTR inhibitor [27].
Moreover, in recent study, the derivatives of 1,3,5-triazine showed potent inhibition of MMP-
in gelatine zymography assay [28]. Prompted by the excellent pharmacological activity of
[
9
above scaffolds, we intended to club these moieties together in search of more potent
analogues of MMP-2 and MMP-9. Therefore, the present study was intended to develop
novel heterocyclic conjugates of sulphonamide-1,3,5-triazine as MMP-2 and MMP-9
inhibitor.
2. Methods and Materials
The chemical used in the present study was procured from Sigma Aldrich (USA). The spectra
1
13
of H NMR and C NMR were recorded on Bruker Avance 400 and Bruker Avance 500
Spectrophotometer, respectively. The chemical shifts are expressed in parts per million
(ppm), and coupling constants are expressed in Hertz (Hz). The Low-resolution mass
spectrum (MS) was recorded on a Waters ZQ LC/MS single quadrupole system equipped
with an electrospray ionization (ESI) source. The elemental analysis of the final derivatives
was performed via Vario Elemental analyser. The Thin-layer chromatography was
performed on 0.25 mm Merck silica gel plates (60F-254) and visualized under UV light.
2.1. General procedure for the synthesis of 2-chloro-4,6-disubstitute-1,3,5-triazine. 2(a-j)
The synthesis of di-substituted 1,3,5-triazine was performed in accordance with earlier
reported procedure. The solution of 2,4,6-tri chloro-1,3,5-triazine (1) (0.1 mol) in 25 mL
acetone maintain the temperature 0-5°C. Different aromatic and aliphatic amine (a-j) (0.1
mol) solution were added constantly to above solution and stirred at 40-45°C for 6 h followed
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by addition of NaHCO . The products were filtered and washed with cold water and
3
recrystallized with ethanol to afford pure products [19].
2
.2. General procedure for the synthesis of 2-hydrazinyl-4,6-disubstitute-1,3,5-triazine. 3
a-j)
-chloro-4,6-disubstitute-1,3,5-triazine. 2(a-j) (0.1 mol) was added into 50 mL of 1,4
(
2
dioxane maintaining temperature 100–110°C. Hydrazine hydrate was added constantly to
above solution and reflex for 6-7 h followed by addition of K CO (0.1 mol). The product
2
3
was filtered and washed with cold water and recrystallized with ethanol to afford the
corresponding pure product 3 (a-j) [20].
2
.3. General procedure for the synthesis of N-(4-acetyl-N-(P-substituted) phenyl
Benzenesulfonamide (6)
-(4-aminophenyl)ethanone (4) (0.01 mol), p-toluenesulfonyl chloride (5) (0.01 mol) and
1
pyridine (0.01 mole) were added to in an ionic liquid (2 g). The reaction mixture was stirred
at 120 °C for 4h. The reaction was monitored by TLC. After completion, the reaction mass
was poured in ice cold water and was extracted with EtOAc. The solvent was removed and
crude product was crystallized from aqueous ethanol [29].
2
.4. General procedure for the synthesis of intermediate compound (7)
To stirring solution of N-(4-acetyl-N-(P-substituted) phenyl Benzenesulfonamide (6) (0.01
mol) in ethanol (25 mL) containing KOH (1.2 g), aromatic 4-methoxy benzaldheydes (0.01
mol) were added, the reaction mixture was stirred at room temperature for 24 h. The
precipitated solid was obtained by filtration and the products were recrystallized from
dioxane to give intermediate 7 respectively.
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2.5. General procedure for the synthesis of title compounds 8 (a-j)
A mixture of intermediate compound (7) (0.01 mol), 2-hydrazinyl-4,6-disubstitute-1,3,5-
triazine 3 (a-j) (0.01 mol) and 1.2 g KOH in 1,4-Dioxane (25 mL) and 10 drops TEA was
refluxed at 100-105 °C for 17 h, then left to cool and poured onto ice/water and neutralized
with dilute hydrochloric acid. The precipitated was filtered and recrystallized from ethanol to
give title compounds 8 (a-j)
2.5.1. N-(4-(1-(4,6-bis(methylamino)-1,3,5-triazin-2-yl)-5-(4-methoxyphenyl)-1H-pyrazol-3-
yl)phenyl)-4-methylbenzenesulfonamide (8a)
o
-1
Yield: 76%; MP: 289-290 C; MW: 556.64; R : 0.67; FT-IR (ν ; cm KBr): 3385 (N-H),
f
max
3
382 (N-H, SO NH), 3078 (Ar C-H), 2953 (Ar-CH ), 2846 (Aliph.C-H str.), 2827 (Ar-
2
3
-1 1
OCH ), 1621 (C=C), 1608 (C=N str.), 1365 (C-N Str.), 1167 (S=O), 786, cm ; H-NMR
3
(400MHz, DMSO, TMS) δ ppm: 7.74 (d, 2H, J = 8.3 Hz, ArH), 7.55 (d, 2H, J = 7.2 Hz,Ar-
H), 7.54-6.69 (m, 4H, Ar-H), 7.42 (d, 2H, J = 5.2 Hz, ArH), 7.09 (s, 1H, Pyrazole), 7.04 (d,
2
H, J = 5.1 Hz, ArH,), 3.98 (s, 3H, -NH x3), 3.83 (S, 3H, Ar-OCH ), 2.91 (s, 6H, CH x2),
3 3
1
3
2
.32 (s, 3H, Ar-CH ); C-NMR (100MHz, CDCl ) δ,ppm:175.9, 164.2, 160.8, 151.6, 143.8,
3
3
1
37.8, 137.4, 129.4, 128.7, 128.2, 125.4, 123.2, 116.8, 114.8, 103.8, 55.8, 28.7, 21.3; Mass :
5
57.68 (M+1); Elemental analysis for C H N O S: Calculated: C, 60.42; H, 5.07; N, 20.13;
2
8
28
8
3
Found: C, 60.38; H, 5.04; N, 20.15.
2.5.2. N-(4-(1-(4,6-bis(ethylamino)-1,3,5-triazin-2-yl)-5-(4-methoxyphenyl)-1H-pyrazol-3-
yl)phenyl)-4-methylbenzenesulfonamide (8b)
o
-1
Yield: 68%; MP: 294-295 C; MW: 584.69; R : 0.73; FT-IR (ν ; cm KBr): 3389 (N-H),
f
max
3
379 (N-H, SO NH), 3073 (Ar C-H), 2958 (Ar-CH ), 2847 (aliph. C-H str.), 2824 (Ar-
2
3
-1
1
OCH ), 1627 (C=C), 1604 (C=N str.), 1362 (C-N Str.), 1165 (S=O), 789 cm ; H-NMR
3
(400MHz, DMSO, TMS) δ ppm: 7.72 (d, 2H, J = 8.2 Hz, ArH), 7.56 (d, 2H, J = 7.3 Hz,Ar-
H), 7.53-6.68 (m, 4H, Ar-H), 7.41 (d, 2H, J = 5.1 Hz, ArH), 7.11 (s, 1H, Pyrazole), 7.03 (d,
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2
H, J = 4.8 Hz, ArH,), 3.97 (s, 3H, -NH x3), 3.81 (S, 3H, Ar-OCH ), 3.48 (s, 4H,Methylene),
3
1
3
2
.32 (s, 3H,Ar-CH ), 1.16 (s, 6H, Methyl); C-NMR (100MHz, CDCl ) δ,ppm:175.8, 164.2,
3
3
1
60.7, 151.5, 143.8, 137.9, 137.5, 136.8, 129.4, 128.6, 128.2, 125.3, 123.1, 116.8, 114.9,
1
03.7, 55.9, 35.8, 21.4, 15.2; Mass : 585.71 (M+1); Elemental analysis for C H N O S:
3
0
32
8
3
Calculated: C, 61.63; H, 5.52; N, 19.16; Found: C, 61.59; H, 5.54; N, 19.18.
2.5.3. N-(4-(1-(4,6-bis((3-aminopropyl)amino)-1,3,5-triazin-2-yl)-5-(4-methoxyphenyl)-1H-
pyrazol-3-yl)phenyl)-4-methylbenzenesulfonamide (8c)
o
-1
Yield: 59%; MP: 305-306 C; MW: 642.77; R : 0.59; FT-IR (ν ; cm KBr): 3427 (NH
f
max
2
str.), 3384 (N-H), 3371 (N-H, SO NH), 3078 (Ar C-H), 2962 (Ar-CH ), 2852 (aliph. C-H
2
3
-
str.), 2829 (Ar-OCH ), 1624 (C=C), 1607 (C=N str.), 1364 (C-N Str.), 1163 (S=O), 784 cm
3
1
1
;
H-NMR (400MHz, DMSO, TMS) δ ppm: 7.73 (d, 2H, J = 8.2 Hz, ArH), 7.56 (d, 2H, J =
7
.2 Hz,Ar-H), 7.54-6.70 (m, 4H, Ar-H), 7.38 (d, 2H, J = 4.3 Hz, ArH), 7.13 (s, 1H, Pyrazole),
.04 (d, 2H, J = 4.3 Hz, ArH,), 5.08 (s, 4H, Amine), 3.96 (s, 3H, -NH x3), 3.84 (S, 3H, Ar-
7
13
OCH ), 3.34-1.82 (m, 12H,Methylene), 2.34 (s, 3H,Ar-CH ); C-NMR (100MHz, CDCl )
3
3
3
δ,ppm:175.8, 164.2, 160.8, 151.5, 143.8, 137.9, 137.4, 136.9, 129.3, 128.5, 128.3, 125.4,
23.1, 116.9, 114.8, 103.6, 55.8, 39.5, 31.4, 21.3; Mass : 643.79 (M+1); Elemental analysis
1
for C H N O S: Calculated: C, 59.79; H, 5.96; N, 21.79; Found: C, 59.75; H, 5.98; N,
3
2
38 10
3
2
1.82.
2.5.4. N-(4-(1-(4,6-bis(phenylamino)-1,3,5-triazin-2-yl)-5-(4-methoxyphenyl)-1H-pyrazol-3-
yl)phenyl)-4-methylbenzenesulfonamide (8d)
o
-1
Yield: 82%; MP: 312-313 C; MW: 680.78; R : 0.62; FT-IR (ν ; cm KBr): 3387 (N-H),
f
max
3
375 (N-H, SO NH), 3072 (Ar C-H), 2965 (Ar-CH ), 2833 (Ar-OCH ), 1627 (C=C), 1603
2
3
3
-1
1
(
C=N str.), 1367 (C-N Str.), 1162 (S=O), 789 cm ; H-NMR (400MHz, DMSO, TMS) δ
ppm: 7.75 (d, 2H, J = 8.2 Hz, ArH), 7.65-6.81 (m, 10H, Ar-H), 7.57 (d, 2H, J = 7.3 Hz,Ar-
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H), 7.55-6.68 (m, 4H, Ar-H), 7.37 (d, 2H, J = 4.2 Hz, ArH), 7.11 (s, 1H, Pyrazole), 7.05 (d,
H, J = 4.3 Hz, ArH,), 3.97 (s, 3H, -NH x3), 3.85 (S, 3H, Ar-OCH ), 2.32 (s, 3H,Ar-CH );
2
3
3
1
3
C-NMR (100MHz, CDCl ) δ,ppm:175.8, 167.3, 160.7, 151.4, 143.8, 138.7, 137.8, 137.4,
3
1
36.7, 129.5, 129.1, 128.4, 128.2, 125.3, 123.1, 122.4, 117.8, 116.9, 114.7, 103.6, 55.8, 21.4;
Mass : 681.80 (M+1); Elemental analysis for C H N O S: Calculated: C, 67.04; H, 4.74; N,
3
8
32
8
3
1
6.46; Found: C, 67.07; H, 4.72; N, 16.48.
2.5.5. N-(4-(1-(4,6-bis((4-hydroxyphenyl)amino)-1,3,5-triazin-2-yl)-5-(4-methoxyphenyl)-1H-
pyrazol-3-yl)phenyl)-4-methylbenzenesulfonamide (8e)
o
-1
Yield: 75%; MP: 321-322 C; MW: 712.78; R : 0.69; FT-IR (ν ; cm KBr): 3526 (O-H),
f
max
3
386 (N-H), 3378 (N-H, SO NH), 3079 (Ar C-H), 2962 (Ar-CH ), 2831 (Ar-OCH ), 1629
2 3 3
-
1
1
(
C=C), 1607 (C=N str.), 1369 (C-N Str.), 1164 (S=O), 781 cm ; H-NMR (400MHz,
DMSO, TMS) δ ppm: 7.72 (d, 2H, J = 8.1 Hz, ArH), 7.56 (d, 2H, J = 7.3 Hz,Ar-H), 7.54-
.71 (m, 4H, Ar-H), 7.48-6.71 (m, 8H, Ar-H), 7.39 (d, 2H, J = 4.1 Hz, ArH), 7.12 (s, 1H,
Pyrazole), 7.04 (d, 2H, J = 4.2 Hz, ArH,), 5.41 (s, 2H, Ar-OH), 3.95 (s, 3H, -NH x3), 3.82
6
1
3
(
S, 3H, Ar-OCH ), 2.35 (s, 3H,Ar-CH ); C-NMR (100MHz, CDCl ) δ,ppm:175.8, 167.3,
3 3 3
1
60.7, 151.4, 148.6, 143.7, 137.7, 137.4, 136.8, 131.4, 129.4, 128.6, 128.2, 125.4, 123.1,
22.1, 116.8, 116.5, 114.8, 103.6, 55.8, 21.3; Mass : 713.76 (M+1); Elemental analysis for
1
C H N O S: Calculated: C, 64.03; H, 4.53; N, 15.72; Found: C, 64.01; H, 4.51; N, 15.69.
38
32
8
5
2.5.6. N-(4-(1-(4,6-bis((4-methoxyphenyl)amino)-1,3,5-triazin-2-yl)-5-(4-methoxyphenyl)-
1H-pyrazol-3-yl)phenyl)-4-methylbenzenesulfonamide (8f)
o
-1
Yield: 79%; MP: 334-335 C; MW: 740.83; R : 0.73; FT-IR (ν ; cm KBr): 3389 (N-H),
f
max
3
371 (N-H, SO NH), 3084 (Ar C-H), 2968 (Ar-CH ), 2835 (Ar-OCH ), 1622 (C=C), 1609
2
3
3
-1
1
(
C=N str.), 1372 (C-N Str.), 1167 (S=O), 787 cm ; H-NMR (400MHz, DMSO, TMS) δ
ppm: 7.74 (d, 2H, J = 8.2 Hz, ArH), 7.57 (d, 2H, J = 7.2 Hz,Ar-H), 7.54-6.69 (m, 4H, Ar-H),
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7.52-7.04 (m, 8H, Ar-H), 7.41 (d, 2H, J = 3.8 Hz, ArH), 7.14 (s, 1H, Pyrazole), 7.06 (d, 2H,
1
3
J = 4.1 Hz, ArH,), 3.98 (s, 3H, -NH x3), 3.84 (S, 9H, Ar-OCH x3), 2.32 (s, 3H,Ar-CH ); C-
3
3
NMR (100MHz, CDCl ) δ,ppm:175.8, 167.3, 160.7, 153.4, 151.5, 143.8, 137.9, 137.5, 136.8,
3
1
31.3, 129.4, 128.6, 128.4, 125.3, 123.1, 121.8, 116.8, 115.2, 114.9, 103.7, 55.9, 21.4; Mass :
7
41.85 (M+1); Elemental analysis for C H N O S: Calculated: C, 64.85; H, 4.90; N, 15.13;
4
0
36
8
5
Found: C, 64.82; H, 4.93; N, 15.11.
2
.5.7. N-(4-(1-(4,6-bis(o-tolylamino)-1,3,5-triazin-2-yl)-5-(4-methoxyphenyl)-1H-pyrazol-3-
yl)phenyl)-4-methylbenzenesulfonamide (8g)
o
-1
Yield: 71%; MP: 296-297 C; MW: 708.83; R : 0.79; FT-IR (ν ; cm KBr): 3392 (N-H),
f
max
3
378 (N-H, SO NH), 3089 (Ar C-H), 2962 (Ar-CH ), 2838 (Ar-OCH ), 1628 (C=C), 1604
2
3
3
-1
1
(
C=N str.), 1374 (C-N Str.), 1169 (S=O), 781 cm ; H-NMR (400MHz, DMSO, TMS) δ
ppm: 7.76 (d, 2H, J = 8.1 Hz, ArH), 7.55 (d, 2H, J = 7.1 Hz,Ar-H), 7.53-6.67 (m, 4H, Ar-H),
.38 (d, 2H, J = 3.9 Hz, ArH), 7.15-6.48 (m, 8H, Ar-H), 7.13 (s, 1H, Pyrazole), 7.04 (d, 2H,
J = 4.3 Hz, ArH), 3.96 (s, 3H, -NH x3), 3.83 (S, 3H, Ar-OCH ), 2.35 (s, 3H,Ar-CH ) 2.14 (s,
7
3
3
1
3
6
1
1
H,Ar-CH x2); C-NMR (100MHz, CDCl ) δ,ppm:175.9, 167.8, 160.7, 151.5, 143.8, 142.2,
3
3
37.9, 137.4, 136.8, 131.4, 129.4, 129.1, 128.6, 128.4, 126.5, 125.4, 123.8, 123.4, 122.9,
16.9, 114.8, 103.7, 55.8, 21.3, 17.6; Mass: 709.82 (M+1); Elemental analysis for
C H N O S: Calculated: C, 67.78; H, 5.12; N, 15.81; Found: C, 67.75; H, 5.14; N, 15.79.
40
36
8
3
2.5.8. N-(4-(1-(4,6-bis((4-nitrophenyl)amino)-1,3,5-triazin-2-yl)-5-(4-methoxyphenyl)-1H-
pyrazol-3-yl)phenyl)-4-methylbenzenesulfonamide (8h)
o
-1
Yield: 82%; MP: 325-326 C; MW: 770.77; R : 0.65; FT-IR (ν ; cm KBr): 3386 (N-H),
f
max
3
374 (N-H, SO NH), 3093 (Ar C-H), 2965 (Ar-CH ), 2834 (Ar-OCH ), 1623 (C=C), 1607
2 3 3
-1
1
(
C=N str.), 1528 (NO str.), 1378 (C-N Str.), 1164 (S=O), 785 cm ; H-NMR (400MHz,
2
DMSO, TMS) δ ppm: 8.01-6.94 (m, 8H, Ar-H), 7.75 (d, 2H, J = 8.1 Hz, ArH), 7.57 (d, 2H, J
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=
7.2 Hz,Ar-H), 7.55-6.71 (m, 4H, Ar-H), 7.41 (d, 2H, J = 3.8 Hz, ArH), 7.12 (s, 1H,
Pyrazole), 7.05 (d, 2H, J = 4.2 Hz, ArH), 3.98 (s, 3H, -NH x3), 3.81 (S, 3H, Ar-OCH ), 2.32
3
1
3
(
s, 3H,Ar-CH ); C-NMR (100MHz, CDCl ) δ,ppm:175.8, 167.3, 160.8, 151.4, 145.2, 143.8,
3
3
1
1
5
37.9, 137.7, 137.2, 136.8, 129.4, 128.5, 128.2, 125.4, 124.8, 123.1, 119.2, 116.9, 114.8,
03.6, 55.8, 21.4; Mass: 771.79 (M+1); Elemental analysis for C H N O S: Calculated: C,
3
8
30 10
7
9.21; H, 3.92; N, 18.17; Found: C, 59.24; H, 3.89; N, 18.19.
2.5.9. N-(4-(1-(4,6-di(piperazin-1-yl)-1,3,5-triazin-2-yl)-5-(4-methoxyphenyl)-1H-pyrazol-3-
yl)phenyl)-4-methylbenzenesulfonamide (8i)
o
-1
Yield: 69%; MP: 282-283 C; MW: 660.80; R : 0.87; FT-IR (ν ; cm KBr): 3382 (N-H),
f
max
3
379 (N-H, SO NH), 3095 (Ar C-H), 2962 (Ar-CH ), 2838 (Ar-OCH ), 1625 (C=C), 1601
2
3
3
-1
1
(
C=N str.), 1373 (C-N Str.), 1169 (S=O), 783 cm ; H-NMR (400MHz, DMSO, TMS) δ
ppm: 7.73 (d, 2H, J = 8.2 Hz, ArH), 7.55 (d, 2H, J = 7.1 Hz,Ar-H), 7.54-6.73 (m, 4H, Ar-H),
7.39 (d, 2H, J = 3.9 Hz, ArH), 7.09 (s, 1H, Pyrazole), 7.03 (d, 2H, J = 4.1 Hz, ArH), 3.96 (s,
1
H, -NH), 3.84 (S, 3H, Ar-OCH ), 3.21-2.81 (m, 16H, Piperazine), 2.36 (s, 3H,Ar-CH ), 1.93
3
3
1
3
(
s, 2H, NHx2, Piperazine); C-NMR (100MHz, CDCl ) δ,ppm:180.9, 175.8, 160.7, 151.4,
3
1
43.8, 137.7, 137.4, 136.5, 129.3, 128.5, 128.4, 125.2, 123.2, 116.9, 114.8, 103.6, 55.9, 48.2,
4
5.9, 21.3; Mass: 667.78 (M+1); Elemental analysis for C H N O S: Calculated: C, 61.24;
3
4
38 10
3
H, 5.74; N, 21.01; Found: C, 61.21; H, 5.76; N, 21.04.
2
.5.10. N-(4-(1-(4,6-dimorpholino-1,3,5-triazin-2-yl)-5-(4-methoxyphenyl)-1H-pyrazol-3-
yl)phenyl)-4-methylbenzenesulfonamide (8j)
o
-1
Yield: 79%; MP: 289-291 C; MW: 668.77; R : 0.74; FT-IR (ν ; cm KBr): 3386 (N-H),
f
max
3
374 (N-H, SO NH), 3093 (Ar C-H), 2966 (Ar-CH ), 2838 (Ar-OCH ), 1627 (C=C), 1604
2
3
3
-1
1
(
C=N str.), 1375 (C-N Str.), 1161 (S=O), 789 cm ; H-NMR (400MHz, DMSO, TMS) δ
ppm: 7.75 (d, 2H, J = 8.3 Hz, ArH), 7.57 (d, 2H, J = 7.2 Hz,Ar-H), 7.56-6.71 (m, 4H, Ar-H),
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7.41 (d, 2H, J = 3.8 Hz, ArH), 7.11 (s, 1H, Pyrazole), 7.04 (d, 2H, J = 4.3 Hz, ArH), 3.98 (s,
1
H, -NH), 3.82 (S, 3H, Ar-OCH ), 3.74-3.64 (m, 16H, Morpholine), 2.34 (s, 3H,Ar-CH );
3
3
1
3
C-NMR (100MHz, CDCl ) δ,ppm:180.8, 175.9, 160.6, 151.5, 143.8, 137.7, 137.4, 136.8,
3
1
29.4, 128.5, 128.4, 125.4, 123.2, 116.9, 114.8, 103.7, 66.4, 55.9, 48.7, 21.4; Mass: 669.80
(
M+1); Elemental analysis for C H N O S: Calculated: C, 61.06; H, 5.43; N, 16.76; Found:
34 36 8 5
C, 61.03; H, 5.45; N, 16.72.
2
.6. Pharmacological Evaluation
.6.1. MMP Inhibition Study
2
The inhibitory activities of these constitutive compounds were determined by a fluorimetric
assay, where a pro-fluorescing peptide is used as substrate, and the fluorogenic activity of its
cleavage product is measured after co-incubation with the appropriate MMP with test
compounds. For this assay, the reagents (Calbiochem) used as follows. The Substrate
corresponds to Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg 25 mM in assay buffer (Calbiochem).
This low-fluorescent peptide is cleaved by MMPs to provide Mca-Pro-Leu-Gly, which is
intensely fluorescent at an excitation at 340 nm and emission at 405 nm. While the enzymes:
pro-MMP-2 250 pM and MMP-9 (recombinant catalytic sequence) 500 pM in assay buffer.
Whereas, the assay buffer consists of triseHCl 0.1 M, pH 7.5, NaCl 0.1 M, CaCl 10 mM,
2
0.05% v/v BRIJ35, APMA (p-aminophenylmercury acetate) 1 mM. This was carried out in
96-well plate, a solution of inhibitor (50 mL) at the appropriate concentration and enzyme (50
mL) was incubated together at 25 °C for 30 min; then 10 mL of substrate solution was added
and the plate was further incubated 3 h at 37 °C. The incubation solutions were quenched
with acetic acid (100 mL of 3% soln.) and their fluorescence were recorded with the help of a
plate reader spectrometer. The inhibitory activities were calculated against a control and IC50
curves, derived from 6 different inhibitor concentrations, extrapolated by means of statistical
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analysis program GraphPad Prism (v. 4.0). Each inhibitor concentration was used in triplicate
and the experiments run twice.
2
.6.2. Chemicals
Isoproterenol hydrochloride was purchased from Sigma Chemical Co., St. Louis, MO, USA
while the assay kits used for biochemical assays were obtained from Randox Laboratories
Limited, Ardmore Co., Antrim, United Kingdom. Moreover, all other chemicals and reagents
used were of analytical grade.
2
.6.3. Animals
The male albino Wistar rats with a mean weight of 155 ± 5.24 g were obtained from the
Central Experimental Animal House and entire experimental protocol has been duly
approved by Institutional Animal Ethical Committee (IAEC). The animals were housed in
polypropylene cages under a 20–24 °C temperature with recommended relative humidity of
55-70 % accompanied by light and a dark cycle of 12 h each. The animals were fed with
standard laboratory diet and water ad libitum.
2.6.4. Experimental procedure
Randomly, four groups of animals were formed with total twenty-four male rats, containing
six animals in each group. These groups were as follows, the rat belongs to Group 1 (normal
control) were administered with distilled water via p.o. route. Whereas, the Group 2
corresponds to ISO-treated (60 mg/ kg, s.c.) at an interval of 24 h for two consecutive days.
The rest of the groups, i.e., Groups 3 and 4 were administered with 5 and 10 mg/kg body
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weight/day p.o. of compound 8e, respectively for next 30 days followed by ISO at an interval
of 24 h for two days. After 12h of the second injection of ISO, the rats were anaesthetized
and later killed. The heart was sliced up from the body for further examination. The
surrounding area of the neck of the animal was shaved to expose the jugular vein which later
cut for blood collection (5ml). The collected blood was then centrifuged at 1282×g for 5min
and serum was taken for further analysis.
2
.6.5. Biochemical estimation
According to the procedure provided by Randox assay kits, the activities of LDH, AST, ALT
and ALP were determined in the serum.
2
.6.6. Estimation antioxidant parameter
Briefly, after washing the heart with ice cold normal saline, the homogenate was prepared in
.1 N Tris HCl buffer (pH 7.4) and cleared supernatant was collected for the further assay.
The activity of superoxide dismutase (SOD), reduced glutathione, glutathione reductase
GR), glutathione peroxidase (GPx) was determined by the previous reported procedures.
Whereas, the lipid peroxidation (LPO) was determined by the formation of malondialdehyde
MDA)-thiobarbituric acid reactive substances (TBARS) adduct.
0
(
(
2
.6.7. Histopathological analysis
The hearts tissues were fixed in buffered formalin (10%) and cut in to four segments and
embedded in paraffin wax. From the formalin fixed paraffin embedded sections, the serial
thin sections of 4 μm thickness were cut and stained. After hematoxylin and eosin (H & E)
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staining, these sections were examined using light microscope (Nikon, Tokyo, Japan, 100x)
for myocardial necrosis, inflammatory cell infiltration and edema
2
.6.8. Statistical analysis
The data were presented as meanꢀ±ꢀSEM. Statistical differences between more than two
groups were assessed by using a one-way ANOVA followed by a post-hoc test. Data of two
groups were analyzed by unpaired t-test. A value of pꢀ<ꢀ0.05 was considered statistically
significant. All statistical analysis was performed with the help of Graphpad Prism software
(Graphpad Software, Inc, San Diego, USA).
3
3
. Results and Discussion
.1.Chemistry
The synthesis of the target molecules are realized in excellent yields and presented in Scheme
. As shown in scheme 1, initially, the synthesis of di-substituted 1,3,5-triazines 2(a-j) was
1
achieved by the nucleophilic substitution (SNAr) of Cl atoms of the 2,4,6-trichloro-1,3,5-
triazine (1) with different aliphatic and aromatic amines in good yield. Whereas, the synthesis
of tri-substituted 1,3,5-triazines 3 (a-j) were accomplished by the nucleophilic substitution
(SNAr) of the mono chloro of di-substituted 1,3,5-triazines 2(a-j) with hydrazine, step
second. In the third step, synthesis of N-(4-acetyl-N-(p-substituted) phenyl
benzenesulfonamide (6) was accomplished by reaction of p-substituted phenyl benzene
sulfonil chloride (5) with 1-(4-aminophenyl)ethanone (4) in the presence of pyridine.
Moreover, in the fourth step, the synthesis of intermediate 7 was accomplished by the
reaction of N-(4-acetyl-N-(p-substituted) phenyl benzenesulfonamide (6) with 4-methoxy
benzaldehyde at a room temperature. Finally the target compounds 8(a-j) were synthesized
by reacting intermediate 7 with tri-substituted 1,3,5-triazines 3 (a-j).
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The intermediate compounds synthesized for the generation of title compounds were obtained
in excellent yield (59-91 %) according to the procedure reported elsewhere. Whereas, the
completion of the reaction has been monitored by TLC. The structure of the synthesized title
1
12
derivatives were ascertained by the H-NMR, C-NMR, mass, FT-IR and elemental analysis.
The FT-IR spectra of all title compounds 8(a-j) were characterized by the appearance of a
-1
strong bands at 3382-3389 cm attributed to N-H group. Many strong absorption bands
-1
appear at 3382 cm which confirm the existence of N-H group of SO NH. The band
2
-1
corresponds to C=N triazine ring appears at 1608-1617 cm . Furthermore, another band at
-1
2
953cm attributed to the stretching vibrations of the aromatic CH group. The OCH group of
3 3
-1
-1
aromatic ring appears at 2827 cm . Furthermore, another band at 1528 cm attributed to the
1
stretching vibrations of the aromatic NO group. The H NMR spectrums of title compounds
2
8
(a-j) reveal a doublet corresponding to phenyl ring at 7.55-6.71 ppm. The 1,3,5-triazine NH
proton appear at 3.98 ppm. The resonance due to piperazine proton found in the range of
.21-2.81ppm. The pyrazole ring proton appeared as singlet peaks at 7.11-7.13 ppm.
3
MMP Inhibitory Activity
The above synthesized target compounds were tested for their efficacy to inhibit MMP-2 and
MMP-9 via enzyme based fluorimetric assay and the results are presented in Table 1.
As shown in Table 1, the entire set of the synthesized compounds showed wide array of
inhibitory activity against tested gelatinases (MMP-2, MMP-9). For instance, compound 8a
and 8b, bearing allyl amine as a substituent showed least inhibitory activity against tested
enzymes, with marked reduction in the case of compound 8a. On changing the pattern of
substitution, as seen in the case of compound 8c, having propyl amine, showed noticeable
improvement in the activity, with marked inhibitory activity against MPP-9. Whereas, the
introduction of aromatic group as seen in the case of compound 8d showed mild
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improvement in inhibitory activity. The most potent activity was displayed by compound 8e
against both the tested geletinase with most prominent inhibition against MMP-9 with IC of
5
0
2.34±0.56 nM. The activity was slightly decreased in the case of compound 8f. The inhibitory
potency was further dropped in the case of 8g and 8h against both the tested enzyme, with
marked reduction in the case of compound 8h bearing p-nitro. Moreover, considerable
improvement in activity was reported by the rest of compounds having basic aromatic groups,
i.e., 8i and 8j.
From the comparative inhibitory activity, it was evident that, all of the synthesized
compounds showed better inhibition against MMP-9 than MMP-2. The structure-activity
relationship studies suggest that, allyl amine substituted compounds showed least inhibitory
activity. Whereas, the most prominent activity was revealed in the case of electron donating
aromatic compounds, viz, 8e, 8f and 8g. Moreover, the basic aromatic substituent showed
mild to moderate inhibitory activity. Thus, it was easy to understand that, minor structural
variation could bring significant difference in the inhibitory activity of the target compounds.
The SAR has been clearly depicted in Figure 1.
3
.2.Protective effect of compound 8e in Isoproternol Induced MI
Myocardial infarction (MI) is often recognized as heart attack, a sensitive situation of
necrosis of myocardium that developed in consequence of imbalance between demand and
supply of oxygen. Therefore, prompted by the excellent MMP inhibitory activity of
compound 8e, it is worthwhile to evaluate its activity in isoproterenol-induced MI in rats. It
has been well established that, the elevated concentration of serum enzymes, such as, lactate
dehydrogenease (LDH), Aspartate amino transferase (AST), Alanine amino transferase
(ALT) and Alkaline phosphatise (ALP) serve as vital biochemical marker to indicate the
progression of MI. As shown in Figure 2, after administration of ISO in rats, obvious
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significant increase in the serum concentration of heart marker enzymes was observed in the
case of LDH, AST, ALT and ATP. This increased level inveterate the inception of necrosis of
cardiomyocytes. However, as the treatment started with compound 8e, the level of these
enzymes was reached near to normal level. This observation signifies that, compound 8e was
able to exert protective role against the cardiac damage induced by isoproterenol via
maintaining the elevated expression of the enzymes (Figure 2 A-D).
Numerous studies have confirmed the role of free radical in the tissue damage in initiation of
injure via induction of peroxidation of unsaturated fatty acids. In iso-treated group, the level
of peroxidation was enhanced, as a result of myocardial injury due to generation of free
radicals. These radicals lead to the peroxidation of polyunsaturated fatty acids of membranes
ensuing in loss of its integrity. As confirmed in Figure 3, the level of MDA is significantly
elevated in the iso-treated group than control. Whereas, on incorporation of 8e, the level has
been reduced to nearly a normal level, suggesting that compound 8e exert protective action
via antioxidant behaviour and might have cardio-protective activity Figure 3 A. On the other
hand, Glutathione (GSH), a vital antioxidant which considered as a radical scavenger
necessary for preventing intracellular damage by free radicals or peroxides. Earlier studies
indicated that, in injury of heart, its level has been considerably reduced may due to increased
oxidative stress. Thus, as shown above, compound 8e found to exhibit antioxidant activity;
so, its effect was also assessed on glutathione levels. As shown in the Figure 3, the pre-
treatment of compound 8e causes significant improvement in the concentration of GSH,
which might improve the antioxidant status also Figure 3 B. It was also inferred that, it causes
the activation of glutathione-dependent enzymes such as glutathione peroxidase (GPx) and
glutathione reductase (GR), Figure 3 C and D. Moreover it also showed improvement in the
level of superoxide dismutase Figure 3 E. Overall, it could be suggested that, the effect of
compound 8e was more prominent at higher tested dose.
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In histopathological examination as shown in Figure 4, it was observed that, iso-treated rats
showed distinct features than control, such as, necrosis of myocardial cells, separation of
myocardial fibres accompanied with increased infiltration of mononuclear inflammatory
cells. Whereas, in compound 8e pre-treated group, these changes were reversed to normal in
dose-dependent manner.
4
. Conclusion
As a concluding remark, we have devised a series of sulphonamide-1,3,5-triazine conjugates
as potent inhibitor of MMPs. The inhibitory comparative chart confirmed that, these
derivatives are efficient and selective inhibitor of MMP-9 than MMP-2. It has been further
found that, highly potent MMP-9 inhibitor, i.e., compound 8e, was able to exert protective
effect in isoproterenol induced myocardial infarction in rats possibly via number of
mechanisms. However, more investigation are sought to clearly rationalize the molecular
mechanism for this effect, which may be useful for the discovery of novel agents of this class.
Conflicts of Interest
The authors declare no conflict of interest.
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Figures, Tables and Schemes captions
Figure 1: Structure-activity relationship activity of target compounds as MMP inhibitor 8 (a-
j).
Figure 2: Effect of compound 8e on the changes in cardiac serum marker enzymes.
Figure 3: Effect of compound 8e on the cardiac oxidative stress markers.
Figure 4: Histopathological analysis of cardiac tissue, where, A) Normal, B) Iso., C) Iso. +
8e (5mg/kg); D) Iso. + 8e (10mg/kg), magnification: 100x.
Table 1: Inhibitory activity of target derivatives on Iso-form of MMPs.
°
Scheme 1: Reagents and conditions: R-H (a-j) various amines. (i) Acetone, stir 3 h at 0-5 C
and 6h at 40-45 °C , NaHCO (ii) NH -NH , 1,4 dioxane, Reflux at 100-110 °C for 6-7 h,
3
2
2
K CO (iii) Pyridine, stirred at 120 °C for 4h (iv) 4-methoxy benzaldheydes, stir 24 h at room
2
3
°
temp. (v) 1,4-Dioxane, reflux for 17- h at 100-105 C.
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List of Figures:
Scheme 1
Figure 1
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Figure 2
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Figure 3
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Figure 4
List of Table
Table 1:
b
Code
IC50 (in nM)*
MMP-2
MMP-9
8
8
8
8
8
8
8
8
8
8
a
b
c
d
e
f
g
h
i
665.56±0.73 625.34±0.21
332.34±0.73 302.23±0.35
189.38±43
123.40±0.36 105.31±0.57
8.12±0.32 2.34±0.56
156.43±064
18.58±0.67 10.21±0.15
67.11±0.75 58.22±0.66
76.56±0.23 68.53±0.58
55.31±0.63 47.11±0.23
42.26±0.32 31.21±0.46
j
*
All experiments were performed thrice, and the results are presented as Mean±SD.
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