New quinoline-2-one/pyrazole derivatives; design, synthesis, molecular docking, anti-apoptotic evaluation, and caspase-3 inhibition assay

Article abstract of DOI:10.1016/j.bioorg.2019.103348

We report the synthesis of new quinoline-2-one/pyrazole hybrids and their antiapoptotic activity. This effect was studied in sight of decreasing tissue damage induced by I/R in colon of rats using N-acetylcysteine (NAC) as anti-apoptotic reference. Compounds 6a, 6c and 6f showed significant improvement for oxidative stress parameters MDA, SOD, GSH and NOx in comparison with model group and greater than the reference NAC (N-acetylcysteine), whereas compounds 6d and 6e exhibited weaker antioxidant activity when compared with the reference NAC. Moreover, compounds 6a, 6c and 6f showed significant decrease in inflammatory mediators TNFα and CRB greater than NAC when compared to the model group especially compound 6c whose found CRB conc 1.90 (mg/dL) in comparison to NAC of conc 2.13 mg/dL. Additionally, colonic histopathological investigation was performed to all targeted compounds that indicates H&E sections of compounds 6a and 6f revealed apparent normal colonic cells while compound 6e showed dilated blood vessels with more apoptotic cells if compared with NAC. Caspase-3 inhibition assay revealed that compounds 6a, 6b and 6d weaken caspase-3 expression to an extent higher than NAC (1.063, 0.430, 0.731 and 1.115, respectively). Docking studies with caspase-3 revealed that most of the tested compounds showed good binding with the enzyme especially for compound 6d make several interactions better than that of the reference NAC.

Full text of DOI:10.1016/j.bioorg.2019.103348

Journal Pre-proofs
New quinoline-2-one/pyrazole Derivatives; Design, Synthesis, Molecular
Docking, Anti-apoptotic Evaluation, and Caspase-3 Inhibition Assay
Ashraf A. Aly, Samia M. Sayed, El-Shimaa M. N. Abdelhafez, Sara
Mohamed Naguib Abdelhafez, Walaa Yehia Abdelzaher, Mohamed A.
Raslan, Amira E. Ahmed, Khaled Thabet, Ahmed A. M. El-Reedy, Alan B.
Brown, Stefan Bräse
PII:
S0045-2068(19)30834-X
DOI:
https://doi.org/10.1016/j.bioorg.2019.103348
YBIOO 103348
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
22 May 2019
13 September 2019
5 October 2019
Please cite this article as: A.A. Aly, S.M. Sayed, E-S. M. N. Abdelhafez, S. Mohamed Naguib Abdelhafez, W.
Yehia Abdelzaher, M.A. Raslan, A.E. Ahmed, K. Thabet, A. A. M. El-Reedy, A.B. Brown, S. Bräse, New
quinoline-2-one/pyrazole Derivatives; Design, Synthesis, Molecular Docking, Anti-apoptotic Evaluation, and
Caspase-3 Inhibition Assay, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103348
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New quinoline-2-one/pyrazole Derivatives; Design, Synthesis, Molecular
Docking, Anti-apoptotic Evaluation, and Caspase-3 Inhibition Assay
Ashraf A. Aly,a* Samia M. Sayed,b El-Shimaa M. N. Abdelhafez,c Sara Mohamed Naguib
Abdelhafez,d Walaa Yehia Abdelzaher,e Mohamed A. Raslan,b Amira E. Ahmed,b
Khaled Thabet,f Ahmed A. M. El-Reedy,g Alan B. Brown,h and Stefan Bräsei,j
a Department of Organic Chemistry, Faculty of Science, Minia University, 61519-ElMinia, Egypt, b Aswan
University, Faculty of Science, Chemistry Department, 81529 Aswan, Egypt, c Department of Medicinal Chemistry,
Faculty of Pharmacy, 61519-El-Minia University, Egypt, d Department of Histology, Faculty of Medicine, Minia
University, 61519-ElMinia, Egypt, e Department of Pharmacology, Faculty of Medicine, Minia University, 61519-
El-Minia Egypt, f Department of Biochemistry, Faculty of Pharmacy, Minia University, 61519 El-Minia, Egypt,
g Basic and Applied Science Department, Faculty of Oral and Dental Medicine, Nahda University, Beni Suef,
Egypt, h Chemistry Program, Florida Institute of Technology, Melbourne, FL 32901, USA,
i Institute of Organic Chemistry, Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany, .j Institute of
Toxicology and Genetics, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany.
Corresponding authors: Prof Ashraf A Aly; e-mail: ashrafaly63@yahoo.com
and ashraf.shehata@mu.edu.eg
Prof Ashraf A. Aly, Prof of Organic Chemistry, Department of Organic Chemistry, Faculty of Science, Minia
University, 61519-El-Minia, Egypt, e-mail: e-mail: ashrafaly63@yahoo.com and ashraf.shehata@mu.edu.eg
Prof Samia M. Sayed: Aswan University, Faculty of Science, Chemistry Department, 81529 Aswan, Egypt; e-mail:
aomabdo@yahoo.com
Dr El-Shimaa M. N. Abdelhafez: Department of Medicinal Chemistry, Faculty of Pharmacy, 61519-El-Minia
University, Egypt; e-mail: shimaanaguib_80@mu.edu.eg
Dr Sara Mohamed Naguib Abdelhafez: Department of Histology, Faculty of Medicine, Minia University, 61519-El-
Minia, Egypt; e-mail: sara_histology@yahoo.com
Dr Walaa Yehia Abdelzaher: Department of Pharmacology, Faculty of Medicine, Minia University, 61519-El-Minia
Egypt; e-mail: walaayehia22@yahoo.com
Prof Mohamed A. Raslan: Aswan University, Faculty of Science, Chemistry Department, 81529 Aswan, Egypt;
e-mail: raslanma47@yahoo.com
Mrs. Amira E. Ahmed, M.Sc. Student, Aswan University, Faculty of Science, Chemistry Department, 81529 Aswan,
Egypt; e-mail: amiraessam394@yahoo.com
Dr Khaled Thabet: Department of Biochemistry, Faculty of Pharmacy, Minia University, 61519 El-Minia, Egypt; e-
mail: khaled_thabet@minia.edu.eg
Dr Ahmed A. M. El-Reedy: Basic and Applied Science Department, Faculty of Oral and Dental Medicine, Nahda
University, Beni Suef, Egypt; e-mail: ahmed.reedy78@gmail.com
Prof Alan B. Brown, Chemistry Program, Florida Institute of Technology, Melbourne, FL 32901, USA, e-mail:
abrown@fit.edu.
Prof Stefan Bräse, Prof of Organic Chemistry, Institute of Organic Chemistry, Karlsruhe Institute of Technology,
76131 Karlsruhe, German, e-mail: stefan.braese@kit.edu
1

Abstract
We report the synthesis of new quinoline-2-one/pyrazole hybrids and their antiapoptotic activity.
This effect was studied in sight of decreasing tissue damage induced by I/R in colon of rats using
N-acetylcysteine (NAC) as anti-apoptotic reference. Compounds 6a, 6c and 6f showed
significant improvement for oxidative stress parameters MDA, SOD, GSH and NOx in
comparison with model group and greater than the reference NAC (N-acetylcysteine), whereas
compounds 6d and 6e exhibited weaker antioxidant activity when compared with the reference
NAC. Moreover, compounds 6a, 6c and 6f showed significant decrease in inflammatory
mediators TNFα and CRB greater than NAC when compared to model group exhibited
significant decrease in comparable to NAC especially compound 6c whose found CRB conc 1.90
(mg/dL) in comparison to NAC of conc 2.13 mg/dL. Additionally, colonic histopathological
investigation was performed to all targeted compounds that indicates H&E sections of
compounds 6a and 6f revealed apparent normal colonic cells while compound 6e showed dilated
blood vessels with more apoptotic cells if compared with NAC. Caspase-3 inhibition assay
revealed that compounds 6a, 6b and 6d weaken caspase-3 expression to an extent higher than
NAC (1.063, 0.430, 0.731 and 1.115, respectively). Docking studies with caspase-3 revealed that
most of the tested compounds show good binding with the enzyme especially for compound 6d
make several interactions better than that of the reference NAC.
Key words: Anti-apoptotic Quinoline; Pyrazole; Caspase-3, NAC, Histopathology, Antioxidant,
Colon, Docking.
1. Introduction
Apoptosis is an essential biological process in cells during animal development tissue [1]
homeostasis [2] and immune responses [3]. However, it is essential in normal physiological
2

events in healthy organism, imbalance probably occurs in some cases between apoptotic and anti
apoptotic mediators leading to some diseases which is attributed to overexpression or inhibition
of apoptotic mediators. it can be abrogated in pathological states as in cancer [4] and
autoimmunity [5], or exacerbated as in stroke [6], neurodegeneration [7], retinal cell death [8],
myocardial and liver ischemia [9,10], inflammatory diseases such as sepsis [11], osteoarthritis
(OA) [12], rheumatoid arthritis [13], and asthma [14].
One of these mediators, is caspases family which consists of 14 mammalian caspases. Among
them, apoptotic caspase -2, -3, -6, -7, -8, 9-, and -10. They use a nucleophilic cysteine in its
active site to cleave aspartic acid peptide bonds within proteins. They have been considered hot
targets of compound design for many years ago, because dysregulation of caspase activity leads
to many severe diseases. We focused our studying on the executioner caspase-3, as it was found
to be generally more promiscuous than caspase-7 and appears to be the major executioner
caspase during the demolition phase of apoptosis. So caspase-3 plays a key executioner role and
its inhibition can drastically prevent apoptosis in vitro and in vivo as well [15].
Currently, acute mesenteric ischemia is a life-threatening clinical complication results during
extravascular events like strangulated hernia, volvulus, intussusceptions, and adhesive
obstruction. Ischemia/reperfusion (I/R) injury is restoration of blood supply to organs after an
ischemic period leading to parenchymal damage [16]. I/R injury elevates mortality rates to 60–
80% incidence. Gut barrier disruption along with distribution of endotoxin into the circulation
are revealed while (I/R), leading to severe systemic inflammation and eventually and exacerbate
apoptosis leading to organ failure [17,18].
Several publications have appeared in recent years documenting quinoline and pyrazole scaffold
are widely found in diverse pharmacologically active substances working as antiapoptotic agents.
3

Rebamipide I is an antioxidative agent which attenuates the increase in apoptosis in the intestinal
mucosa after ischaemia-reperfusion but was not completely abolished [19].
O
HOOC
O
CH3
N
N
O
N
N
H
O
N
O
N
N
O
N
Cl
N
NH
S
O
N
O
N
H
O
H
N
Alluprinol
III
Edaravone
II
Rebamipide
I
IV
Figure 1. Structures of some antioxidant agents used against ischemia-reperfusion injury in the intestine
O
H3C
SCH3
O
Bioactive scaffold
Bioactive scaffold
N
H2N
N
Caspase-3
inhibition
R1
N
R2
O
6a-f
Figure 2. Design of the target compounds 6a-f
It was demonstrated that Edaravone II [20] as well as alluprinol III [21] a pyrazole containing
compounds, have potential free radical scavenging and antioxidant activities in a model of
intestinal I/R injury that can prevent the aggravation of the small intestinal mucosal damage
occurring during reperfusion. It’s interestingly when gathering both moieties as in compound IV,
it exhibited anti-apoptosis activity through inhibition of caspase-3 with an IC50 = 4 nM [22].
Because of the emergence of acute mesenteric ischemia and its on-going impact on global health
system, there has been a growing interest for development of novel anti-apoptotic agents in order
to combat this disorder. As a result, hybrids of quinoline and pyrazole moieties had been
considered as interesting targets for designing new anti-apoptotic agents such have the structure
of compounds 6a-f (Figure 2).
4

Recently, we prepared ethyl pyranoquinoline-4-carboxylates and dialkyl 2(4-oxo-1,4-
dihydroquinolin-3-yl)fumarates [23]. We also reacted 4-hydroxy-2-quinolones with 2-(2-oxo-
1,2-dihydroindol-3-ylidene)malononitrile in pyridine, and spiro(indoline-3,4’-pyranoquinoline)-
3’-carbonitriles were obtained in good to excellent yields [24]. Moreover, we synthesized bis(4-
hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)succinic acid derivatives by one-pot reaction of one
equivalent of aromatic amines with two equivalents of diethyl malonate in diphenyl ether and
catalyzed with triethylamine [25]. Reaction of four equivalents of 4-hydroxyquinolin-2(1H)-ones
with one equivalent of acenaphthoquinone gave 1,2-dihydroacenaphthylene)-1,1,2,2-tetrayl-
tetrakis(4-hydroxyquinolin-2(1H)-ones) in a good to excellent yields [26]. In the view of
biological and pharmaceutical activities, we reported on the design and synthesis of novel series
of fused naphthofuro[3,2-c]quinoline-6,7,12-triones and pyrano[3,2-c]quinoline-6,7,8,13-
tetraones as potential ERK inhibitors. The synthesized inhibitors were evaluated for their ability
to inhibit ERK1/2 in an in vitro radioactive kinase assay [27]. Also, two series of N-2,3-bis(6-
substituted-4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)naphthalene-1,4-diones and substituted
N-(methyl-/ethyl)bisquinolinone triethyl-ammonium salts were successfully synthesized. The
synthesized compounds were targeted as candidates to extracellular signal-regulated kinases
ERK1/2 with considerable antineoplastic activity. To further elucidate the mechanism of action
of these synthesized compounds selected to investigate for their MAP Kinases pathway
inhibition together with molecular docking using ATP-binding site of ERK2 [28]. Encouraged
by the biological and pharmaceutical activities of 2-quinolones, we have recently reported a
review article which discusses the recent synthetic approaches and the applications of this class
of compounds in the synthesis of related four-membered to seven-membered heterocycles,
most of them showing unique biological activities [29]. Two series of diethyl 2-[2-(substituted-
5

2-oxo-1,2-dihydroquinolin-4-yl)hydrazono]succinates and 1-(2-oxo-1,2-dihydroquinolin-4-yl)-
1H-pyrazoles have been designed and synthesized. Seven compounds were further examined
against the most sensitive cell lines, leukemia CCRF-CEM, and MOLT-4.One compound was
shown high activity with IC50ꢀ=ꢀ1.35 uM and 2.42 uM against MOLT-4 and CCRF-CEM,
respectively. Also, it showed a remarkable inhibitory activity compared to Erlotinib on the
EGFR TK with IC50ꢀ=ꢀ247.14ꢀnM and 208.42ꢀnM, respectively [30].
The present work involves the synthesis of new quinolin-2-one/pyrazole hybrids 6a-f substituted
with either electron donating or withdrawing groups to study their electronic effect. In addition,
screening of their anti-apoptotic activity, molecular modelling studies and caspase-3 inhibition
assay have been carried out to study the plausible mechanism of action of the newly synthesized
compounds.
2. Results and Discussion
2.1.
Chemistry
Cl
O
Cl
R1
R1
R1
POCl3
refiux,
CH3COOH
reflux,5-8 h
NH2NH2, EtOH
N
R2
Cl
stirring, reflux,
10-15 h
N
R2
O
stirring, 1 h
N
R2
3a-f
O
2a-f
1a-f
4a'
CN
O
H3C
O
4b'
3a'
O
H3CS
H3CS
H3C
SCH3
NHNH2
O
SCH3
3'
N
4c'
5a'
O
CH3
4'
R1
5
O
N
5'
H2N
4a
H2N
R1
N
N
4
2'
EtOH, reflux, 10-15 h
N
R2
O
1'
3
5
6
1
2
a
; R =H, R =H
N
R2
O
1
2
7
b;
c;
4a-f
R =Cl, R =H
N
O
2
1
8a
8
H
1
2
R =Br, R =H
6a
1
2
d
; R =OCH3, R =H
6a-f
1
2
e
; R =CH3, R =H
6a
6d
6b
6e
6c
(77%), (88%)
(73%), (72%)
(70%),
(75%),
1
2
6f
f
; R =H, R =CH3
3.
4.
Scheme 1. Synthesis of the target compounds 6a-f
6

The strategy involved the reaction of POCl3 with quinoline-2,4-diones 1a-f to give the
corresponding dichlorotetrahydroquinoline 2a-f [31-33]. Oxidation of 2a-f gave the monochloro
derivatives 3a-f, which on heating with hydrazine hydrate yielded the quinolones 4a-f [33]
(Scheme 1). Refluxing 4a-f with ethyl 2-cyano-3,3-bis(methylthio)propenoate (5) in absolute
ethanol afford the target compounds 6a-f in good yields [31]. The newly synthesized compounds
were identified by 1D-NMR (1H NMR, 13C NMR), 2D-NMR (COSY, HSQC, HMBC) and mass
spectra. As an example, NMR spectra of compound 6a (Table 1) showed the ethyl ester signals
are distinctive at δH 1.29 (t; H-4c’) and 4.23 (q; H-4b’), and δC 14.46 (C-4c’) and 59.07 (C-4b’).
H-4b’ give HMBC correlation with one of the two carbons at δC 162.95 and 161.65, which is
assigned as C-4a’. The methylthio protons H-3a’ are also distinctive, at δH 2.35; their attached
carbon appears at δC 12.31. H-3a’ also give HMBC correlation with a carbon at δC 91.69,
assigned as C-4’; its upfield chemical shift is attributed to its position in a push-pull system. The
broad 3H signal at δH 6.60 gives both H-C HSQC correlation, with a carbon at δC 120.25, and H-
N HSQC correlation, with a nitrogen at δN 59.8. This proton “signal” is therefore assigned as co-
resonant signals from H-3 and H-6a’. A carbon at δC 152.44, which gives no correlations, is
assigned on chemical-shift grounds as C-5’; N-2’, which is four bonds from the nearest protons,
is not observed at all. The signals of the quinolinone part structure are assigned straight
forwardly, and are similar to those of recently-studied quinolinones with the same substitution
pattern. The signal at δH gives HMBC correlation with a carbon at δC 144.20, assigned as C-4. C-
4 gives HMBC correlation with a doublet at δH 7.32, assigned as H-5; its attached carbon appears
at δC 124.53. C-5 gives HMBC correlation with a “triplet” at δH 7.58, assigned as H-7; this is a
three-bond correlation. H-7 gives HSQC correlation with its attached carbon at δC 131.16. The
other “triplet” of the four-spin aromatic pattern, at δH 7.20, is assigned as H-6; its attached carbon
7

appears at δC 122.01. C-6 in turn gives HMBC correlation with the other doublet of the four-spin
pattern, at δH 7.41; this doublet is assigned as H-6, and its attached carbon appears at δC 115.56.
The non-protonated carbons at δC 139.56 and 116.52 are assigned as C-8a and C-4a respectively;
their chemical shifts and correlations are consistent with these assignments. Finally, the other of
the carbons at δC 162.95 and 161.65 is assigned, on chemical-shift grounds, as C-2.
Table 1. NMR spectroscopic data of compound 6a
1H NMR (DMSO-d6)
1H-1H COSY
Assignment
H-1
12.07 (bs; 1H)
6.60
7.58 (“t”, J = 7.6; 1H)
7.41 (d, J = 8.1; 1H)
7.32 (d, J = 8.0; 1H)
7.20 (“t”, J = 7.6; 1H)
6.60 (s; 3H)
7.41, 7.32, 7.20
7.85, 7.32, 7.20
7.85, 7.41, 7.20
7.85, 7.40, 7.32
12.07
H-7
H-8
H-5
H-6
H-3, 6a'
H-4b'
H-3a'
4.23 (q, J = 7.1; 2H)
2.35 (s; 3H)
1.29
1.29 (t, J = 7.0; 3H)
13C NMR (DMSO-d6)
4.23
H4c'
Assignment
HSQC
HMBC
162.95, 161.65
152.44
4.23
C-2, 4a'
C-5'
149.94
144.20
139.56
131.16
124.53
122.01
2.35
7.32, 6.60
7.58, 7.32
7.32
C-3'
C4
C-8a
C-7
C-5
C-6
7.58
7.32
7.20
7.58
7.41
120.25
6.60
C-3
116.52
115.56
112.41
91.69
59.07
14.46
12.31
12.07, 7.41, 7.20
C-4a
C-8
C≡N
C-4'
C-4b'
C-4c'
7.41
6.60
1.29
4.23
1.29
2.35
C-3a'
15N NMR(DMSO-d6)
HSQC
HMBC
Assignment
181.4
151.1
59.8
6.60
N-1'
N-1
N-6a'
12.07
6.60
In case of 6d, NMR spectroscopic data (Table 3) showed the methylthio protons H-3a’ are also
distinctive, at H 2.37; their attached carbon appears at C 12.38. H-3a’ also give HMBC
correlation with a carbon at C 91.81, assigned as C-4’; its upfield chemical shift is attributed to
8

its position in a push-pull system. The broad 2H signal at H 6.65 gives H-N HSQC correlation
with a nitrogen at N 60.7; this proton signal is assigned as H-6a’. The sharp 1H singlet at H 6.58
is assigned as H-3; its attached carbon appears at C 119.85.
Table 2. NMR spectroscopic data of compound 6d
1H NMR (DMSO-d6)
1H-1H COSY
Assignment
11.97 (s; 1H)
7.37 (d, J = 9.0; 1H)
7.26 (dd, J = 9.0, 2.6; 1H)
6.91 (d, J = 2.6; 1H)
6.65 (bs; 2H)
6.65
7.26
7.37, 6.91
7.26
H-1
H-8
H-7
H-5
H-6a’
H-3
11.97
6.58 (s; 1H)
4.23 (q, J = 7.1; 2H)
3.70 (s; 3H)
2.37 (s; 3H)
1.29
H4B’
H-6A
H-3A’
H-4C’
1.29 (t, J = 7.1; 3H)
4.23
13C NMR (DMSO-d6)
HSQC
HMBC
4.23
Assignment
C-4a’
162.97
161.15
C-2
7.37, 7.26, 6.91,
3.70
154.07
C-6
152.46
150.07
143.54
134.19
120.23
119.85
C-5’
C-3’
C-4
2.37
7.37, 6.91, 6.58
7.37, 7.26, 6.91
6.91
C-8a
C-7
7.26
6.58
C-3
11.97, 7.37, 6.91,
6.58
7.37, 6.91, 6.58
116.99, 116.93
7.37
6.91
C-4a,8
106.37
91.81
C-5
C-4’
6.65
59.11
55.35
14.44
4.23
3.70
1.29
1.29
4.23
C-4b’
C-6a
C-4C’
12.38
15N NMR (DMSO-d6)
182
2.37
HSQC
C-3a’
Assignment
N-1'
HMBC
6.65,658
150.1
60.7
11.97
6.65
7.37
N-1
N-6a'
A carbon at C 152.46, which gives no correlations, is assigned on chemical-shift grounds as C-
5’; N-2’, which is four bonds from the nearest protons, is not observed at all. The multiplicities
9

and coupling constants of the three protons in the benzene ring allow their immediate assignment
as H-5 (H 6.91), H-7 (H 7.26), and H-8 (H 7.37); the attached carbons appear at C 106.37 (C-
5), 120.23 (C-7), and either 116.99 or 116.93 (C-8). The methoxy protons H-6a are distinctive at
H 3.70; their attached carbon appears at C 55.35. H-6a give HMBC correlation with a non-
protonated carbon at C 154.07, assigned as C-6; C-6 also correlates with H-5, H-7, and H-8. The
remaining carbons are all non-protonated. The line at C 134.19 gives HMBC correlation with H-
5 and H-7 and (weakly) with H-8; the line at C 143.54 correlates with H-8, H-5, and H-3. These
lines are assigned as C-8a and C-4 in the order stated. The other of the lines at C 116.99 and
116.93, and the line at C 161.15, are assigned on chemical-shift grounds as C-4a and C-2
respectively. The ethyl ester signals are distinctive at H 1.29 (t; H-4c’) and 4.23 (q; H-4b’), and
C 14.46 (C-4c’) and 59.06 (C-4b’). H-4b’ give HMBC correlation with a carbon at C 162.92,
which is assigned as C-4a’ (All carbon lines in this sample are well-separated, thus all
correlations are unambiguous). Interestingly, H-4c’ gives a weak HMBC correlation with a
carbon at C 91.63, assigned on chemical-shift grounds as C-4’; its upfield chemical shift is
attributed to its position in a push-pull system. In other samples of this series, C-4’ gives
correlation with the methylthio protons, but in this sample it does not. In case of 6f, the NMR
spectroscopic data (Table 3) revealed the methylthio protons H-3a’at H 2.34; their attached
carbon appears at C 12.32. H-3a’ also give HMBC correlation with a carbon at C 150.00,
assigned as C-3’. A carbon at C 152.57, which gives no correlations, is assigned on chemical-
shift grounds as C-5’; N-2’, which is four bonds from the nearest protons, is not observed at all.
The broad 2H signal at H 6.59 gives H-N HSQC correlation with nitrogen at N 60.2; therefore
the proton signal is assigned as H-6a’ and the nitrogen is assigned as N-6a’. H-6a’ also give
10

HMBC correlation with a nitrogen at N 179.8, assigned as N-1’. N-1’ gives HMBC correlation
with the 1H singlet at H 6.75, assigned as H-3; its attached carbon appears at C 119.89.
Table 3. NMR spectroscopic data of compound 6f
1H NMR (DMSO-d6)
1H-1H COSY
Assignment
7.72 (dt, Jd = 1.6, Jt = 7.6; 1H)
7.67 (d, J = 8.1; 1H)
7.35 (dd, J = 7.9, 1.3; 1H)
7.30 (dt, Jd = 0.8, Jt = 7.3; 1H)
6.75(s; 1H)
7.67, 7.30
7.72
7.30
7.72, 7.35
H-7
H-8
H-5
H-6
H-3
6.59 (bs; 2H)
H-6a'
4.23 (q, J = 7.1; 2H)
1.29
4.23
H-4b'
3.70 (s; 3H)
2.34 (s; 1H)
1.29 (t, J = 7.0; 3H)
13C NMR (DMSO-d6)
H-1a
H-3a'
H-4c'
HSQC
HMBC
Assignment
162.92
160.93
152.57
150.00
4.23
6.75, 3.70
C-4a'
C-2
C-5'
C3'
2.34
143.16
140.33
131.64
7.67, 7.35, 6.75
7.72, 7.35, 7.30, 3.70
7.35, 7.30
C-4
C-8a
C-7
7.72
7.35
124.99
7.72
C-5
122.25
119.89
117.55
115.14
7.30
6.75
7.67, 6.75
C-6
C-3
C-4a
C-8
C-4'
C-4b'
7.72, 7.67, 7.30, 6.75
7.67
7.30, 3.70
1.29
91.63
59.06
4.23
3.70
1.29
29.35
6.75
C-1a
14.46
1.29
4.23
C-4c'
12.32
2.34
C-3a'
15N NMR (DMSO-d6)
179.8
HSQC
HMBC
6.75, 6.59
6.75,3.70
Assignment
N-1'
145.9
N-1
60.2
6.59
N-6a'
H-3 also gives HMBC correlation with a nitrogen at N 145.9, assigned as N-1; with non-
protonated carbons at C 143.16 and 117.55, assigned as C-4 and C-4a respectively; and with the
N-methyl carbon at C 29.35, assigned as C-1a. N-1 also gives HMBC correlation with the N-
methyl protons H-1a, at H 3.70; the N-methyl carbon and protons give HSQC correlation with
11

each other. H-1a also give HMBC correlation with the lactam carbonyl at C 160.93, assigned as
C-2; with a non-protonated carbon at C 140.33, assigned as C-8a; and with a protonated carbon
at C 115.14, assigned as C-8. C-8 gives HSQC correlation with a 1H doublet at H 7.67,
assigned as H-8. C-4 gives HMBC correlation with the other end signal of the four-spin aromatic
system, at H 7.35; this signal appears as a double-doublet with one large J, and gives HSQC
correlation with its attached carbon at C 124.99. The proton and carbon just mentioned are
assigned as H-5 and C-5 respectively.
The mechanism describes the formation of compounds 6a-f can be explained due to nucleophilic
attack of the nitrogen of the hydrazine to the active electrophilic carbon in 5 to give Zwitter ion 7
(Scheme 2). The intermediate 7 would be neutralized to give 8, accompanied with elimination of
a molecule of methylthiol to give 9 (Scheme 2). Subsequently, another nucleophilic attack of the
nitrogen lone pair of the second nitrogen of hydrazine to the carbon of nitrile group would give
10 (Scheme 2). Aromatization of 9 would give compounds 6a-f (Scheme 2).
CN
H3CS
CO2Et
SCH3
SCH3
H3CS
HN
EtO2C
N
SCH
H
HN
N
3
5
EtO2C
N
..
SCH3
H
NH2
NH2
N
- HSCH3
HN
N
R1
R1
R1
N
R2
4
O
O
O
R2
R2
8
7
SCH3
EtO2C
N
EtO2C
HN
SCH3
..
HN
N
N
N
6a-f
R1
R1
N
R2
O
N
R2
O
9
10
Scheme 2. Mechanism describes the formation of compounds 6a-f
12

2.2. Biological Activity
2.2.1. Assay of Antioxidant biomarkers in colon (MDA, SOD, GSH, NOX)
This study showed the anti-apoptotic effect of novel compounds on decreasing tissue damage
induced by I/R in colon of rats in a dose equivalent to 30 mg N-acetylcysteine (NAC) [34] as
anti-apoptotic reference [35], when they are administrated via intra-peritoneal (i.p) an hour
before ischemia (0.5 h) and reperfusion (0.5 h), after that the organs were cut. This was
supported by biochemical, histological and morphometric studies. Different biomarker
concentrations were measured in serum to detect colon injury due to ischemia/reperfusion (I/R).
Some of these biomarkers are increased and others decreased indicating apoptotic effect as
shown in Table 4.
Table 4. The expression of the biomarkers during apoptosis for colon
*Biomarker
Effect during apoptosis in Colon
Serum TNFα[36], MDA[37], CRB[38]
Increase
SOD[39], GSH[40], Nox[41]
Decrease
TNF = Tumor necrosis factor, MDA = Malondialdehyde, SOD = Superoxide dismutase
GSH = Glutathione, NOx = NADPH oxidase CRB= C-reactive protein (CRP)
There was a significant improvement in oxidative stress parameters MDA, SOD, GSH and NOX
in comparison with model group. The results showed obviously that compounds 6a ,6c and 6f
had excellent MDA inhibition effect with conc 37.89, 40.27 and 35.08 nmol/g tissue;
respectively, and more than NAC (37.89 nmol/g tissue). In addition, compounds 6a, 6c and 6f
had SOD excellent activation effect with 149.35, 155.70 and 161.38 U/mg protein, respectively
in comparison with NAC (158.61 U/mg protein) as illustrated in Table 5. Also compounds 6a
and 6f had GSH excellent activation effect with conc 70.09 and 75.93 µmol/mg protein;
respectively relative to NAC (80.25 µmol/mg protein). In addition, compounds 6a, 6c and 6f had
NOx excellent activation effect with conc 110.58, 112.67 and 109.73 nmol/mg protein;
respectively relative to NAC (110.58 nmol/mg protein) but compound 6d and 6e had very weak
13

MDA, SOD, GSH, and NOx activation effect with significant increase in MDA with significant
decrease in intestinal SOD, GSH and NOx in model group (Table 5).
Table 5. MDA, SOD, GSH and NOx concentrations in colon of I/R rats treated with compounds
6a-f and NAC
Intestinal MDA
(nmol/g tissue)
30.43± 2.89
Intestinal SOD
(U/mg protein)
164.72±4.56
Intestinal GSH
(µmol/mg protein)
78.34±3.91
Groups
Sham operated
Model group
115.43±5.34
69.47±4.31ac
110.58±4.89b
108.46±5.07b
88.54±3.46abc
112.67±6.90b
70.36±3.40ac
73.81±4.53ac
109.73±5.30b
98.09±3.26ac
37.89±1.86b
39.56±2.50b
65.19±3.06abc
40.27±3.18b
88.09±3.71ac
94.26±2.98ac
35.08±3.26b
71.34±3.60ac
158.61±3.27b
149.35±4.50b
119.38±3.99abc
155.70±4.07b
75.34±2.51ac
79.61±3.49ac
161.38±4.56b
42.38±4.01ac
80.25±2.59b
NAC
6a
6b
6c
6d
6e
6f
70.09±4.32b
68.45±2.91b
66.58±3.80b
44.70±3.60ac
49.99±2.97ac
75.93±4.07b
2.1.Assay of inflammatory biomarkers (Serum TNFα and CRB)
The assay of inflammatory parameters (TNFα and CRP) was done in comparison with sham and
NAC treated group and the results were outlined in Table 6. Compounds 6a ,6c and 6f treated
groups showed significant decrease in TNFα of conc 8.4, 9.07and 8.90 pg/ml greater than NAC
of conc 9.50 pg/ml when compared to model group of conc 26.67 pg/ml (table 6). Moreover,
they also exhibited significant decrease in CRB comparable to NAC especially compound 6c
whose found conc 1.90 (mg/dL) in comparison to NAC of conc 2.13 mg/dL Meanwhile, these
biomarkers were significantly increased in model, 6d and 6e groups in comparison with sham
and NAC treated group (Table 6). It could be concluded from the previous results that
unsubstituted quinoline at position 6 (compounds 6a and 6b) showed the highest activity and
there is no great activity when substituted with either electron donating groups (CH3, OCH3) or
withdrawing group (Cl , Br)
14

Table 6. TNFα and CRP concentrations in colon of I/R rats treated with compounds 6a-f and
NAC.
Groups
Serum TNFα (pg/ml) Serum CRP (mg/dL)
Sham operated
Model group
8.34±0.75
26.67±2.60ac
9.50±0.57b
8.4±1.07b
2.00±0.18
17.45±0.93ac
2.13±0.20b
2.40±0.17b
12.69±0.16ac
1.90±0.14b
15.90±0.19ac
17.98±0.18ac
2.21±0.09b
NAC
6a
6b
6c
6d
6e
6f
15.12±1.20abc
9.07±0.79b
23.89±1.91ac
27.47±1.80ac
8.90±0.61b
Results represent the mean ± S.E (n= 5). a Significant (P < 0.05) difference from sham operated group, b Significant
(P < 0.05) difference from model group, c Significant (P < 0.05) difference from NAC treated group.
2.2.
2. Histopathological Investigation
Motivated by previously mentioned above results, colonic histopathological investigation was
performed. the Colon sections from sham group showed normal mucosa, submucosa, musculosa
and serosa. The mucosa was folded, intact and continuous, lined with simple columnar epithelial
cells and had close regularly arranged crypts that appeared tubular in structure (Figure 3).
Figure 3. Photomicrographs of a section in the colon from a control rat (sham) showing A)
normal mucosa (black line), submucosa (yellow line), musculosa (green arrow) and serosa
(arrow). The mucosa is folded, intact and continuous. B) Notice the simple columnar epithelial
cells (red arrow) lining the mucosa and has close regularly arranged tubular crypts (C). (H&E, A
x100; BX 400).
While ischemic reperfusion group showed sever congestive blood vessels, distorted mucosa with
absence of folding. Submucosal cellular Infiltration was clearly noticed. Detached intraluminal
15

debris with massive intraluminal apoptotic cells (small dense eccentric nuclei) were also
observed (Figure 4). H&E sections of compound 6c revealed apparent normal colonic cells with
vesicular nuclei. Additionally, NAC as well as 6f revealed few apoptotic cells with apparent
normal colonic architecture. Compound 6a showed dilated blood vessels with more apoptotic
cells if compared with compound NAC (Figure 5).
Figure 4. Photomicrographs of sections in the colon from a rat of ischemic reperfusion show
congestive blood vessels (arrows). B) Distorted mucosa (star) with absence of folding. Notice
the cellular Infiltration (stars) in the mucosa. C) Detached intraluminal debris (green arrow).
D)Massive intraluminal apoptotic cells (small dense eccentric nuclei (circle) (H&E, A x B, C,
100; DX 400).
16

Figure 5. Photomicrographs of sections in the colon from a rat with compound 6c showed
apparent normal colonic cells with vesicular nuclei (rectangle). NAC and 6f showed few
apoptotic cells (circle) with apparent normal colonic architecture. Compound 6a showed dilated
blood vessels (arrows) with more apoptotic cells (circle); AX 100; BX400).
17

Figure 6. Photomicrographs of sections in the colon from rats together with compound 6b which
showed: inflammatory cell infiltrations (star) more numerous apoptotic cells (circle). Compound
6d showed distorted colonic architecture with inflammatory cell infiltration (star), extravation of
RBCs and congested blood vessels (V). Notice almost all colonic cells showed apoptotic cells
(circle). Compound 6e showed sever distorted colonic architecture with inflammatory cell
infiltration (star), extravation of RBCs and congested blood vessels (V). Notice almost all
colonic cells showing more numerous apoptotic and distorted cells (circle); AX 100; BX400).
H&E sections of compound 6b showed inflammatory cell infiltrations and more numerous
apoptotic cells than compound 6a. Compound 6d revealed distorted colonic architecture with
18

inflammatory cell infiltration. Extravation of RBCs and congested blood vessels were frequently
seen. In this group almost all colonic cells showed apoptotic cells. Furthermore, compound 6e
exhibited sever distorted colonic architecture with inflammatory cell infiltration, extravation of
RBCs and congested blood vessels. Almost all colonic cells showed more numerous apoptotic
and distorted cells (Figure 6).
2.2.3. Morphometric results
There was significant increase in apoptotic cells in compound 6e than compound 6d. Also it was
detected a significant increase in apoptotic cells in compound 6d than compound 6b.
Additionally there was a significant increase in apoptotic in compound 6b than 6a and NAC
(Figure 7).
Figure 7: showing the mean number of apoptotic cells arranged ascendingly for NAC and 6a-f
Results represent the mean ± S.E. (n= 8). a Significant (P < 0.05) difference from group I, b
Significant (P < 0.05) difference from group II.
2.3. Caspase-3 expression assay
The inhibitory activity of the newly designed derivatives was examined on caspase-3 mRNA
expression level by LPS stimulated Caco-2 cells using NAC as a reference compound. Figure 8
19

shows the effect of the available target compounds 6a-f on caspase-3 mRNA expression level by
LPS stimulated Caco-2 cells. The significant increase in Caspase-3 mRNA expression was found
in LPS stimulated cells without treatment (A, P=0.018), LPS +6c (E, P=0.006, caspase
expression = 2.534), and LPS +6e (G, P =0.018, Fold change =2.049) in comparison to non-
stimulated cells (Control). This means that bulky substituents are not favourable for caspase-3
inhibitory activity. Meanwhile treatment of LPS-stimulated cells with either N-acetyl cysteine
(B, P =0.4, Fold change =1.115) or 6a (C, P = 0.2, Fold change = 1.063) decreases the mRNA
expression level of caspase-3 and returns it to the control level without any significant
difference. While the significant decrease in caspase-3 mRNA expression was associated with
the treatment of LPS stimulated cells with 6b (D, P =0.02, Fold change =0.430) and 6d (F, P
=0.02, Fold change =0.731). Results were calculated by paired t-test using GraphPad Prism 7
Software, San Diego, CA) as shown in Table 7. The results indicate true inhibition of caspase-3.
Table 7. Fold change of caspase-3 expression 6a-f
Fold change in Caspase-3 expression
C+LPS+
NAC
control
control+LPS
6a
6b
6c
6d
6e
6f
1
1.988
1.115
1.063
0.430
2.534
0.731
2.049
2.376
Figure 8. Fold change of caspase-3 expression for targeted compounds 6a-e and NAC
3
* *
*
*
2
1
0
*
* *
D
A
B
C
E
F
C ontrol
G
2.4. Docking
Studies
Type of treatm ent
Enzyme-ligand docking was carried out for all derivatives 6a-f and NAC as a reference to
predict the binding interactions between the newly synthesized quinoline/pyrazole hybrids and
20

caspase-3 enzyme obtained from the protein data bank (PDB:6CKZ) using Molecular Operating
Environment (MOE®) version 2014.09 using NAC as reference for validation of the method.
The theoretical predictions from the molecular docking study were found to be in agreement for
some highly active derivatives such as 6b, 6d and 6a with the experimentally observed caspase-3
inhibition activity. All derivatives were successfully docked into the active site of caspase-3
enzyme. The binding free energies from the major docked poses are listed in Table 8 and the
most favourable poses of the tested compounds are shown in Figure 9. Most of the tested
compounds have high binding affinity to the enzyme as the binding free energy(ΔG) values of
them range from -0.6 to 3.2 Kcal/mol comparable to the reference NAC (ΔG = - 0.7 to – 2.9
Kcal/mole).
Table 8. Energy scores for the complexes formed by the tested compounds 6a-f and the reference
NAC in the active site of caspase-3 (PDB: 6CKZ)
ΔG
Ligand-receptor interaction
Type Length (Å)
Compound S score
(Kcal/mole)
-2.9
Residue
Arg 207
Gly122
Arg64
H-acceptor 3.64
H-acceptor 3.53
H-acceptor 4.28
H-acceptor 3.00
H-acceptor 3.19
H-acceptor 3.07
H-acceptor 3.20
NAC
6a
-4.03
-6.35
-0.7
-1.0
-2.6
Arg207
Gly122
Arg207
Gly122
Arg207
Arg64
-3.2
-2.6
6b
6c
-6.45
-4.52
-2.9
-2.6
Pi-cation
3.61
-1.8
H-acceptor 3.35
H-acceptor 3.51
H-acceptor 3.09
H-acceptor 3.05
-1.2
Arg64
6d
-6.65
-2.8
Arg207
Gly122
Trp206
Tyr204
Gly122
-3.0
-0.6
H-Pi
4.41
4.12
4.01
6e
6f
-4.56
-4.18
-0.6
H-Pi
-0.5
H-donor
ΔG (Kcal/mole)a; The binding free energies
The docking result of the reference compound NAC is completely consistent with the mode of
action of quinoline/pyrazole derivatives. The 2D diagram showed a crucial binding Arg207 and
21

Gly122 through quinoline C=O and pyrazole C=N functionality. Moreover, stabilization of the
reference NAC within the active site occurred through three strong hydrogen bond interactions
with amino acid residue Arg207, Gly122 and Arg64. Docking Results with caspase-3 revealed
that most of the tested compounds show good binding with the enzyme and make several
interactions comparable to that of the reference NAC. Compounds 6a and 6b (Figure 9)
exhibited the same interactions with the conserved amino acids as NAC lacked only one H-
bonding interaction with Arg64, however, compound 6d possess greater interaction than NAC to
interact with the same amino acids with additional H-Pi bond with Trp206. On the other hand,
compounds 6c and 6e (Figure 9) dismissed hydrogen binding interactions with the amino acid
residue Arg207, Gly122. Compound 6f kept only hydrogen bond interaction with Gly122 (Figure
9).
NAC
6a
22

6b
6d
6e
Figure 9. 2D diagrams illustrate the binding modes of the reference NAC, 6a, 6b, 6d and 6e
interacted with the active site of caspase-3
3. Conclusions
New quinoline-2-one/ pyrazole derivatives 6a-6f were prepared and characterized by different
spectroscopic techniques. It is worth to mention that the tested compounds 6a, 6c and 6f showed
more potent activity toward antioxidant and anti-inflammatory biomarkers than quinoline-2-one-
/pyrazole derivatives 6d and 6e. Histopathological examination for the colonic specimens treated
with targeted compounds 6a-6f indicated that compounds 6a and 6f revealed apparent normal
23

colonic cells, however, compound 6e showed dilated blood vessels with more apoptotic cells if
compared with NAC. Moreover, caspase-3 expression was assayed to exhibit that compound 6a,
6b and 6d provide lower expression to an extent than NAC. Molecular docking studies with
caspase-3 enzyme showed comparable binding scores and similar binding interactions as that of
the reference NAC. that most of the tested compounds show good binding with the enzyme
especially for compound 6d make several interactions better than that of the reference NAC,
however, compound 6b and 6d showed more favourable interaction than of the other
compounds.
In summary, introduction of a substitution at position 6 of quinoline with either electron donating
or withdrawing keeps the same anti-apoptotic activity as unsubstituted derivatives or may be
decrease activity due to steric effect. Compounds 6a, 6c, and 6f are promising anti-apoptotic
agents that are recommended to be further studied.
4. Experimental
4.1.
Chemistry
Melting points were determined using open glass capillaries on a Gallenkamp melting point
apparatus (Weiss-Gallenkamp, Loughborough, UK), and are uncorrected. The IR spectra were
recorded from potassium bromide disks with a FT device, Minia University NMR spectra were
1
13
measured in DMSO-d6 on a Bruker AV-400 spectrometer (400 MHz for H, 100 MHz for C,
and 40.55 MHz for 15N); chemical shifts are expressed in  (ppm), versus internal
tetramethylsilane (TMS) = 0 for 1H and 13C, and external liquid ammonia = 0 for 15N. Coupling
constants are stated in Hz. Correlations were established using 1H-1H COSY, and 1H-13C and 1H-
15N HSQC and HMBC experiments. Mass spectra were recorded on a Finnigan Fab 70 eV,
Institute of Organic Chemistry, Karlsruhe University, Karlsruhe, Germany. TLC was performed
24

on analytical Merck 9385 silica aluminum sheets (Kieselgel 60) with Pf254 indicator; TLC's were
viewed at max = 254 nm. Elemental analyses were carried out at the Microanalytical Center,
Cairo University, Egypt.
4.1.1. General procedure describes the formation of compounds 6a-f
A mixture of aromatic amines 4a-d (1 mmol) and 5 (2 mmol) in absolute EtOH (100 mL) was
refluxed for 10-15 h. The reaction was followed up by TLC analysis. The formed precipitate was
kept standing up at room temperature for 24 h. The precipitate which was collected by filtration
was washed with 200 mL of EtOH and then dried well. The formed product was then
recrystallized from stated solvents.
Ethyl
5-amino-3-(methylthio)-1-(2-oxo-1,2-dihydroquinolin-4-yl)-1H-pyrazole-4-
o
carboxylate (6a). Colorless crystals (DMF/EtOH), yield: 210 mg (70 %), mp = 250-252 C. IR
(KBr, cm-1): 3460-3335 (Ar-NH & Ar-NH2), 2848 (CH3, str), 1710 (CO-ester). NMR (DMSO-
d6, δ ppm): Table 1. ESI-MS: m/z= 345.1023 (M + 1). Anal. Calcd. for C16H16N4O3S (344.39): C,
55.80; H, 4.68; N, 16.27. Found: C, 55.70; H, 4.60; N, 16.10.
Ethyl 5-amino-1-(6-chloro-2-oxo-1,2-dihydroquinolin-4-yl)-3-(methylthio)-1H-pyrazole-4-
carboxylate (6b). Colorless crystals (DMF/MeOH), yield: 225 mg (77 %), mp = 260-262 oC. IR
1
(KBr, cm-1): 3350-3320 (NH,NH2), 3000 (Ar-CH), 2980-2880 (Aliph-CH), 1710 (COOEt). H
NMR (400 MHz, DMSO-d6, δ ppm): 12.20 (s, 1H, NH), 7.64 (dd, 1H, J = 8.8, 2.0 Hz, H-7), 7.43
(d, 1H, J = 1.6 Hz, H-5), 7.35 (d, 1H, J = 8.8 Hz, H-8), 6.67 (bs, 3H, H-3,6a’), 4.23 (q, 2H, J =
13
7.0 Hz, H-4b’), 2.36 (s, 3H, SCH3), 1.29 (t, 3H, J = 7.0 Hz, CH3-ester). C NMR (100 MHz,
DMSO-d6, δ ppm): 162.9 (C-4a’), 161.43 (C-2), 152.53 (C-5’), 150.37 (C-3’), 143.04 (C-4),
138.35 (C-8a), 131.11 (C-7), 125.92 (C-6), 125.00 (C-5), 123.57 (C-3), 117.62 (C-8), 117.17 (C-
15
4a), 91.84 (C-4’), 59.13 (C-4b’), 14.44 (C-4c’), 12.26 (C-3a’). N (NMR, DMSO-d6, δ ppm):
25

181.4 (N-1’), 151.3 (N-1), 60.2 (N-6a’). ESI-MS: m/z= 379 (M++1), 378 (M+, 34). Anal. Calcd.
for C16H15ClN4O3S (378.83): C, 50.73; H, 3.99; N, 14.79. Found: C, 50.60; H, 4.10; N, 14.60.
Ethyl 5-amino-1-(6-bromo-2-oxo-1,2-dihydroquinolin-4-yl)-3-(methylthio)-1H-pyrazole-4-
carboxylate (6c). Colorless crystals (DMF/CH3CN), yield: 255 mg (88 %), mp = 350-352 oC. IR
1
(KBr, cm-1): 3350-3310 (NH, NH2), 3010 (Ar-CH), 2970-2890 (Aliph-CH), 1712 (COOEt). H
(400 MHz, DMSO-d6, δ ppm): 12.20 (s, 1H, NH), 7.75 (dd, 1H, J = 8.7, 1.7 Hz, H-7), 7.50 (d,
1H, J = 1.5 Hz, H-5), 7.37 (d, 1H, J = 8.8 Hz, H-8), 6.67 (bs, 3H), 4.23 (q, 2H, J = 7.0 Hz, H-
4b’), 2.36 (s, 3H, SCH3), 1.29 (t, 3H, J = 7.0 Hz, CH3-ester). 13C NMR (100 MHz, DMSO-d6, δ
ppm): 162.88 (C-4a’), 161.41 (C-2), 152.53 (C-5’), 150.36 (C-3’), 142.93 (C-4), 138.66 (C-8a),
133.76 (C-7), 126.60 (C-5), 124.8 (C-6), 121.06 (C-3), 118.05, 117.85 (C-4a,8), 91.84 (C-4’),
59.14 (C-4b’), 14.44 (C-4c’), 12.37 (C-3a’). 15N (NMR, DMSO-d6, δ ppm): 180.6 (N-1’), 151.7
(N-1), 60.2 (N-6a’). ESI-MS: m/z = 423.0128 (M++1), 423 (M+, 32). Anal. Calcd. for
C16H15BrN4O3S (423.29): C, 45.40; H, 3.57; N, 13.24. Found: C, 45.60; H, 3.60; N, 13.30.
Ethyl 5-amino-1-(6-methoxy-2-oxo-1,2-dihydroquinolin-4-yl)-3-(methylthio)-1H-pyrazole-
o
4-carboxylate (6d). Colorless crystals (DMF/EtOH), yield: 230 mg (75 %), mp = 300-302 C
(decomp). IR (KBr, cm-1): 3340-3310 (NH, NH2), 3030-3010 (Ar-CH), 2980-2880 (Aliph-CH),
1710 (COOEt). NMR (DMSO-d6, δ ppm): Table 2. ESI-MS: m/z = 375.1126 (M + 1). Anal.
Calcd. for C17H18N4O4S (374.42): C, 54.53; H, 4.85; N, 14.96. Found: C, 54.70; H, 4.90; N,
14.80.
Ethyl 5-amino-1-(6-methyl-2-oxo-1,2-dihydroquinolin-4-yl)-3-(methylthio)-1H-pyrazole-4-
o
carboxylate (6e). Colorless crystals (DMF/H2O), yield: 222 mg (73 %), mp = 330-332 C. IR
1
(KBr, cm-1): 3330-3312 (NH, NH2), 3015 (Ar-CH), 2990-2890 (Aliph-CH), 1710 (COOEt). H
(400 MHz, DMSO-d6, δ ppm): 12.00 (s, 1H, NH), 7.41 (dd, 1H, J = 8.4 Hz, H-7), 7.32 (d, 1H, J
26

= 8.4 Hz, H-8), 7.13 (bs, 1H, H-5), 6.58 (bs, 2H), 6.55 (s, 1H, H-3), 4.23 (q, 2H, J = 7.1 Hz, H-
13
4b’), 2.35 (s, 3H, SCH3), 2.30 (s, 3H, CH3), 1.29 (t, 3H, J = 7.0 Hz, CH3-ester). C NMR (100
MHz, DMSO-d6, δ ppm): 163.10 (C-4a’), 161.50 (C-2), 152.34 (C-5’), 149.82 (C-3’), 144.02 (C-
4), 137.64 (C-8a), 132.45 (C-7), 131.00 (C-6), 123.82 (C-5), 120.20 (C-3), 116.43 (C-4a), 115.56
(C-8), 91.69 (C-4’), 59.07 (C-4b’), 20.59 (Ar-CH3), 14.46 (C-4c’), 12.36 (C-3a’). 15N (NMR,
DMSO-d6, δ ppm): 180.5 (N-1’), 150.8 (N-1), 59.8 (N-6a’). ESI-MS: m/z= 359.1176 (M + 1).
Anal. Calcd. for C17H18N4O3S (358.42): C, 56.97; H, 5.06; N, 15.63. Found: C, 57.10; H, 5.20;
N, 15.70.
Ethyl 5-amino-1-(1-methyl-2-oxo-1,2-dihydroquinolin-4-yl)-3-(methylthio)-1H-pyrazole-4-
o
carboxylate (6f). Pale yellow crystals (DMF/EtOH), yield: 218 mg (72%), mp = 310-312 C
(decomp). IR (KBr, cm-1): 3330-3310 (NH, NH2), 3040-3020 (Ar-CH), 2970-2860 (Aliph-CH),
1708 (COOEt). NMR (DMSO-d6, δ ppm): Table 3. ESI-MS: m/z= 359.1176 (M +1), 313.1 (M+-
SCH3). Anal. Calcd. for C17H18N4O3S (358.42): C, 56.97; H, 5.06; N, 15.63. Found: C, 57.08; H,
5.12; N, 15.72.
4.2.
Biological activity
4.2.1. Material and methods
1. The 200 mg rats were chosen and grouped for 7 groups (each contains 5 rats (n=5) as
following: G1 = Sham, G2 = Model (I/R), G3 = NAC, G4-9 of compounds 6a-f
2. The compounds were injected intraperitoneally for 1 h before anesthetization by 0.25 ml
urethane and surgery.
3. The rats were operated to close superior mesenteric vein by clamps for 0.5 h (ischemia)
and reopened (reperfusion) for 0.5 h.
27

4. After that, blood samples were collected from the ventricular of the heart centrifuged
(centrifuge Jantezki, T30, Germany), at 5000 rpm for 10 min for serum collection. Sera
were separated and kept in refrigerator at -80°C until assessment of various parameters.
5. At the end, the rats were killed to obtain colon for biological and histological studies.
6. Intestinal tissue of each rat was rapidly dissected, left to dry on filter paper, weighed and
prepared for histopathological and immunohistochemical examinations or stored at -80°C
for further measurements.
7. 200 Mg of intestine was homogenized in 2 ml of phosphate buffer solution (prepared by
dissolving 8.01 g NaCl, 0.20 g KCl, 1.78 g Na2HPO4, 2H2O and 0.27 g KH2PO4 in 1 liter
of distilled water and pH = 7.4) using homogenizer (Tri-R Stir-R homogenizer, Tri-R
Instruments, Inc., Rockville Centre, NY).
4.2.2. Determination of lipid peroxides in the form of Malondialdehyde (MDA) in the
intestine
MDA, a reactive aldehyde that is a measure of lipid peroxidation, bone marrow cardiac contents
of MDA were determined using the thiobarbituric acid method described by Buege and Aust
(1978) [41], which measures the thiobarbituric acid reactive substances concentration, sometimes
referred to as MDA concentration.
Principle
This method measures the intestinal MDA level which is the breakdown product of tissue lipid
peroxides.
Procedures
. To a 0.5 mL of 10 % homogenate of the tissue sample, 0.5 mL of distilled water was added.
. The reagent was freshly prepared (26 mmol/L thiobarbituric acid, 0.92 mol/L
trichloroacetic acid in 0.25 mol/L HCl) and 2 mL were added to each tube.
28

. The mixture was heated in a boiling water bath for 15 min.
. After cooling, the supernatant was removed by centrifugation at 5000 rpm for 10 min.
. The absorbance of the sample was determined at 535 nm against a blank using Beckmann
DU-64 spectrophotometer (Phoenix Equipment Inc, New York).
1,1,3,3Tetramethoxypropane was used as an external standard to prepare standard
concentrations of MDA (1,2,4,6,8 and 10 nmol/mL) and the procedure was repeated to prepare a
standard curve using 1,1,3,3tetramethoxypropane instead of colonic homogenates [41]. From
this curve, the MDA concentration in the unknown sample was extrapolated from the
corresponding absorbance using the regression line from the standard curve then multiplied in
tissue and expressed as nmol/g tissue.
Standard curve of MDA

0.800
0.600

0.400

0.200




0.000
0.00
10.00 20.00
Concentration (nmol/ml)
30.00
4.2.3. Determination of superoxide dismutase (SOD) in the intestine
SOD activity was determined according to the previously described method.
Principle
29