Design, synthesis and pharmacological screening of novel renoprotective methionine-based peptidomimetics: Amelioration of cisplatin-induced nephrotoxicity

Article abstract of DOI:10.1016/j.bioorg.2021.105100

Cisplatin (CP) is an effective chemotherapeutic agent for treatment of various types of cancer, however efforts are needed to reduce its toxic side effect. Previous studies revealed promising effect of peptides in decreasing CP induced nephrotoxicity. Herein, novel Met-based peptidomimetics were synthesized using N-acylbenzotriazole as acylating agent in high yield. Evaluation of renoprotective effect of the synthesized targets on CP treated kidney cell line (LLC-PK1) revealed that pretreatment with 1/3 IC₅₀ of targets II, IIIa-g attenuated CP induced cell death where the IC₅₀ of CP was raised from 3.28 μM to 9.25-41.1 μM. The most potent compounds IIIg, II and IIIb exhibited antioxidative stress in CP-treated LLC-PK1 cells as confirmed by raising GSH/GSSG ratio and SOD concentration as well as decreasing ROS and MDA. Additionally, in vivo experiments using Sprague Dawley rats showed renoprotective effect of IIIg against CP-induced nephrotoxicity as evidenced by improved results of renal function tests and attenuated CP-induced renal structural injury. Moreover, antioxidant activity of IIIg was demonstrated via its ability to reduce renal MDA level and up-regulate renal antioxidant element GSH level. Further, immunohistochemistry of renal specimens showed the ability of IIIg to restore CP-induced suppression of Nrf2. Interestingly, in vivo and in vitro studies demonstrated that IIIg had no effect on CP antiproliferative activity. An assessment of the ADMET properties revealed that targets IIIg, II and IIIb showed good drug-likeness in terms of their physicochemical, pharmacokinetic properties. The findings presented here showcase that IIIg is a promising renoprotective candidate with antioxidative stress potential.

Full text of DOI:10.1016/j.bioorg.2021.105100

Bioorganic Chemistry 114 (2021) 105100
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and pharmacological screening of novel renoprotective
methionine-based peptidomimetics: Amelioration of
cisplatin-induced nephrotoxicity
,
b
,
,
,
,g
Khalid A. Aghaa *, Tarek S. Ibrahimb c, Nehal M. Elsherbinyd e, Mohamed El-Sherbinyf
,
Eatedal H. Abdel-Aalb, Zakaria K. Abdel-Samiib, Nader E. Abo-Dyab
,
d
a Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Fayoum University, Fayoum 63514, Egypt
b Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Zagazig University, Zagazig 44519, Egypt
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia
d Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Tabuk University, Tabuk 71491, Saudi Arabia
e Department of Biochemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
f Department of Anatomy, Faculty of Medicine, Mansoura University, Mansoura 35516, Egypt
g College of Medicine, Almaarefa University, Riyadh 11597, Saudi Arabia
A R T I C L E I N F O
A B S T R A C T
Keywords:
Cisplatin (CP) is an effective chemotherapeutic agent for treatment of various types of cancer, however efforts are
needed to reduce its toxic side effect. Previous studies revealed promising effect of peptides in decreasing CP
induced nephrotoxicity. Herein, novel Met-based peptidomimetics were synthesized using N-acylbenzotriazole as
acylating agent in high yield. Evaluation of renoprotective effect of the synthesized targets on CP treated kidney
cell line (LLC-PK1) revealed that pretreatment with 1/3 IC50 of targets II, IIIa-g attenuated CP induced cell death
where the IC50 of CP was raised from 3.28 µM to 9.25-41.1 µM. The most potent compounds IIIg, II and IIIb
exhibited antioxidative stress in CP-treated LLC-PK1 cells as confirmed by raising GSH/GSSG ratio and SOD
concentration as well as decreasing ROS and MDA. Additionally, in vivo experiments using Sprague Dawley rats
showed renoprotective effect of IIIg against CP-induced nephrotoxicity as evidenced by improved results of renal
function tests and attenuated CP-induced renal structural injury. Moreover, antioxidant activity of IIIg was
demonstrated via its ability to reduce renal MDA level and up-regulate renal antioxidant element GSH level.
Further, immunohistochemistry of renal specimens showed the ability of IIIg to restore CP-induced suppression
of Nrf2. Interestingly, in vivo and in vitro studies demonstrated that IIIg had no effect on CP antiproliferative
activity. An assessment of the ADMET properties revealed that targets IIIg, II and IIIb showed good drug-likeness
in terms of their physicochemical, pharmacokinetic properties. The findings presented here showcase that IIIg is
a promising renoprotective candidate with antioxidative stress potential.
Nephrotoxicity
Methionine
Peptidomimetics
Benzotriazole
Antioxidative stress potential
Cisplatin
1. Introduction
products. This induces nephrotoxicity by multitude of mechanisms
including DNA damage, mitochondrial apoptosis pathway and inflam-
Cis-diamminedichloroplatinum (Cisplatin; CP) is an effective
chemotherapeutic agent; it is effectively used in the treatment of
bladder, cervix, lung and head cancer [1]. However, the clinical
importance of CP is limited by dose dependent adverse effects. Kidney
toxicity is the main side effect, which may lead to renal failure and even
death on exposure to high dose or prolonged use of CP [2,3].
mation [4,5]. Oxidative stress has been also implicated in CP-induced
nephrotoxicity and lead to other associated damage mechanisms such
as inflammation and mitochondrial dysfunction [4,6]. Production of
reactive oxygen species (ROS) in the cell is balanced with antioxidant
defense system, however overproduction of ROS results in cellular
damage [7]. Therefore, anti-oxidant and anti-inflammatory agents pre-
sented a promising chemopreventive approach against CP-induced
nephrotoxicity [8].
The uptake of CP by renal tubular epithelial cells involves special
membrane transporters where it undergoes activation into more toxic
* Corresponding author at: Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Fayoum University, Fayoum 63514, Egypt.
E-mail address: Aghanet2010@yahoo.com (K.A. Agha).
https://doi.org/10.1016/j.bioorg.2021.105100
Received 20 April 2021; Received in revised form 13 June 2021; Accepted 14 June 2021
Available online 17 June 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

K.A. Agha et al.
Bioorganic Chemistry 114 (2021) 105100
During the last three decades considerable interest has been focused
N-p-chlorobenzoylbenzotriazole Ia that was prepared by reported
Katritzky benzotriazole methodology [25] with L-methionine in the
presecence of TEA. AAs like arginine, glycine, cysteine, histidine,
phenylalanine and anthranilc acid that have antioxidant or metal
chelating activities were coupled to N-p-chloro-L-methionine II. To do
this, N-p-chlorobenzoyl-L-methionine was activated for coupling reac-
tion by its reaction with four equivalent of 1H- benzotriazole and one
equivalent thionylchloride in CH2Cl2 at 25 ◦C for 3 h to give the corre-
sponding N-acylbenzotriazole IIa in 85% yield (Scheme 1, Fig. 3).
on development of new therapeutics based on native and modified
peptides. Among these effort Stefanucci group investigated the anti-
nociceptive and antioxidant activities of some peptides derived from the
enzymatic digestion of soy proteins namely rubiscolin-6, soymorphin-6
and their C-terminal amides.[9] Also attempt to develop endomorphin
analogues was studied based on peptidomimetic approach.[10]
It was shown that amino acids (AAs) like methionine, cysteine,
arginine, glycine, glutamine and amino acid derivatives like N-benzoyl
or N-acetyl derivatives have the ability to attenuate CP induced neph-
rotoxicity (Fig. 1) [11,12]. A part of this effect may arise from acting as
scavenger of oxygen free radicals or it is related to enhancing GSH
production. The oxidation of surface-exposed methionine (Met) residues
of proteins by reactive oxygen species (ROS) generates mixtures of the R-
and S-isomers of methionine sulfoxide (MetO) [13]. Loss of biological
activity of the oxidized protein occurs, due to changes in hydrophobicity
and protein conformation [14,15]. However, unlike the ROS-dependent
oxidation of other AAs residues of proteins (except cysteine residues),
the oxidation of Met residues is readily reversed by the action of
methionine sulfoxide reductase (Msr) that catalyzes reduction of MetO
residues of proteins back to Met, thereby ROS loses its activity [16,17].
Synergistic antioxidant activity was observed upon screening of di-
peptides containing methionine like Met-Met and Met-Trp. Also,
administration of CP in the form of CP-Met substitution complex, can
reduce CP-induced nephrotoxicity without diminishing the antitumor
activity [18,19]. Met significantly improved polymyxin-induced neph-
rotoxicity and reduced mitochondrial superoxide production in renal
tubular cells [20]. Moreover, several studies had confirmed the anti-
inflammatory activities of Met and their derivatives in animal models
[21–23].
2.2. Pharmacokinetic and physicochemical screening
Upon administration of a drug, its pharmacokinetics and physico-
chemical properties influence its rate of absorption, distribution, meta-
bolism and excretion in human system [26–28]. The Lipinski’s rule of
five is generally used to predict the drug-likeness of a chemical com-
pound by measuring the biological activity, good oral bioavailability
together with the compounds tendency to cross various aqueous and
lipophilic barriers by adhering to certain conditions [29]. SwissADME
was used to predict the pharmacokinetic and physicochemical proper-
ties of the compounds [30]. As shown in Table 1, all the compounds
obey Lipinski rule of five. Compounds II, IIIa, IIIe and IIIg showed high
gastro-intestinal absorption. These favourable properties suggest that
these compounds could be potential drugs. On the other hand absence of
p-chlorobenzoyl moiety resulted in low value of LogPo/w indicating
highly hydrophilic compounds that will cleared rapidly from the body
resulting in poor pharmacokinetic properties.
2.3. Cell-based renoprotective screening
The present study was conducted to alleviate the nephrotoxicity of
CP by incorporating a renoprotective and antioxidant AA Met in a
dipeptide of additional antioxidant amino acids. p-Chlorobenzoyl group
was included to improve pharmacokinetics properties of peptides
(Fig. 2).
2.3.1. Cell based assay of renoprotection against CP-induced nephrotoxicity
using kidney epithelial cells (LLC-PK1)
To verify the renoprotective effect of the synthesized compounds,
cell based kidney protection screening was performed using kidney
epithelial cells (LLC-PK1). Cell Line cells were obtained from American
Type Culture Collection, cells were cultured using Dulbecco’s Modified
Eagle’s Medium (DMEM) (Invitrogen/Life Technologies) supplemented
with 10% fetal bovine serum (FBS) (Hyclone), 10 µg/ml of insulin
(Sigma), 1% penicillin and streptomycin. All of the other chemicals and
reagents were from Sigma, or Invitrogen. The LLC-PK1 cells were treated
with various concentrations (100, 25, 6.25, 1.56, 0.39 µM) of synthe-
sized compounds or CP then subjected to 3-(4,5-dimethyldiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) assay to determine their IC50
(Table 2). Subsequently LLC-PK1 cells were co-treated with safe doses 1/
3 IC50 of the synthesized compounds and different concentration of CP
(100, 25, 6.25, 1.56, 0.39 µM) then cell viability and IC50 of CP were
measured (Table 2, Fig. 4).
N-acylbenzotriazoles were used as acylating agents to prepare the
targets. N-acylbenzotriazoles showed numerous merits in peptide or
peptidomimetic synthesis such as: (i) chirality is preserved during the
course of their preparation and reaction (ii) they form crystals easily;
(iii) they are stable in air; and (iv) they are usually isolated in high yields
[24].
2. Results and discussion
2.1. Chemistry
N-p-Chlorobenzoyl-L-methionine II was prepared by the reaction of
Fig. 1. Example of renoprotective compounds.
2

K.A. Agha et al.
Bioorganic Chemistry 114 (2021) 105100
Fig. 2. Proposed mechanisms for renoprotective efficacy of peptidomimetics.
Scheme 1. Chemical synthesis of target compounds, Reagent condition: a) SOCl2 (1 eq) in CH2Cl2, rt, 3 h, b) L-methionine, TEA (1:1), in CH3CN / H2O (3:1), rt, 5 h.
c) various amino acids in Dioxane / H2O (3:1), TEA (1 eq), 60 ◦C, 5 h.
The IC50 of CP group was 3.28 ± 0.09 µM. Upon mixing CP with 1/3
IC50 of the target peptidomimetics cell viability was enhanced and the
IC50 (Fig. 4) was increased from three fold (IC50 = 9.2 µM,
CP + compound IIIa) to about 13 fold (IC50 = 41.1 µM, Cp + compound
IIIg). Such enhancement in cell viability by rise in CP IC50 confirms that
the Met-containing peptidomimetics protect LLC-PK1 cells against the
cytotoxic effects of CP. Compound IIIg provides the best renoprotective
effect amongst the tested compounds and its calculated physicochemical
and pharmacokinetic properties were optimum for a drug candidate.
MDA were measured at the beginning of the experiment, after 8 h and at
the end of the experiment (Table 3, Fig. 5). Effect of synthesized pep-
tidomimetics on oxidative stress is unblemished as following:
2.3.2.1. GSH/glutathione disulfide (GSSG) ratio. Glutathione is a tri-
peptide observed in all cells except erythrocytes and it plays a vital rules
in various processes as free radical scavenger, inhibitor of lipid peroxi-
dation and in detoxification [31]. The ratio of reduced GSH to oxidized
GSH (GSSG) is an indicator of cellular health; this ratio is reduced in
neurodegenerative diseases and in tissue or cells under oxidative stress
such as our model [32,33]. Treatment of LLC-PK1 cells with CP for 8 hrs,
significantly lowered the initial GSH/ GSSG (3.73 ± 0.153) to
0.53 ± 0.12. Upon increasing the time of exposure of LLC-PK1 to CP for
additional 24 hrs the ratio of GSH/ GSSG was further decreased to
0.37 ± 0.06. When cells were treated with compounds IIIg, II and IIIb
for 8 hrs, followed by subsequent treatment with CP for 24 hrs, targets
IIIg, II and IIIb maintained the level of GSH/ GSSG to fivefold, fourfold
2.3.2. Effect of the synthesized peptidomimetics on oxidative stress in CP-
treated LLC-PK1 cell
In an attempt to gain insight about the mechanism by which the
targets exert renoprotective effect, LLC-PK1 cell line was treated with 1/
3 IC50 of the most potent targets II, IIIb and IIIg respectively for 8 hr,
followed by addition of 4 µM of CP and the mixture was left for 24 hr.
Oxidative stress biomarkers namely, GSH/GSSG ratio, ROS, SOD and
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K.A. Agha et al.
Bioorganic Chemistry 114 (2021) 105100
Fig. 3. Chemical structure of the designed methionine based peptidomimetics.
Table 1
Predicted physicochemical properties of synthesized peptidomimetics.
Physiochemical properties
Compounds
II
IIIa
IIIb
IIIc
IIId
IIIe
IIIf
IIIg
Molecular weight (g/mol)
287.76
18
344.81
22
418.96
26
390.91
24
424.90
28
434.94
29
443.95
29
406.88
27
Num. heavy atoms
Num. arom. heavy atoms
6
6
6
6
11
12
6
12
Num. rotatable bonds
7
10
13
11
12
12
15
10
Num. H-bond acceptors
3
4
4
4
5
4
5
4
Num. H-bond donors
2
3
3
3
4
3
6
3
Molar Refractivity
73.04
91.70
2.24
ꢀ 0.59
High
No
85.65
120.80
1.76
ꢀ 0.84
High
No
107.67
146.10
2.56
0.08
Low
No
98.39
159.60
1.96
ꢀ 0.69
Low
No
107.09
149.48
1.72
ꢀ 0.78
Low
No
114.95
120.80
3.12
0.71
High
No
113.98
182.70
1.38
ꢀ 1.06
Low
No
107.25
120.80
3.18
1.03
High
No
TPSA (Å2)
Consensus LogPo/w
Consensus LogPo/w if p-chlorobenzoyl group removed from the desined peptidomimetic
GI absorption
BBB permeant
Lipinski
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
radicals decreases the level of SOD also CP treatment negatively affect
SOD level negatively [34,35]. Data in table 3 showed that CP lowered
SOD level in LLC-PK1cells, this effect was reversed by pretreatment with
compounds IIIg, II and IIIb. Compounds IIIg and II were highly active in
raising the level of SOD to more than four and threefold (21.43 ± 0.15,
24.87 ± 0.06) respectively with respect to CP only treated cells
(6.27 ± 0.15) (Fig. 5).
Table 2
Renoprotective effect of synthesized peptidomimetic on CP treated LLC-PK1
cells.
Compound
IC50 before mix (µM)
Safe conc, (µM)
Cisplatin IC50 after mix
II
93 ± 2.66
30
15
30
12
7
18.7 ± 0.54
9.27 ± 0.27
16.9 ± 0.48
11.0 ± 0.31
12.8 ± 0.37
12.6 ± 0.36
14.7 ± 0.42
41.1 ± 1.18
–
IIIa
55.8 ± 1.59
90.6 ± 2.59
38.7 ± 1.11
21.5 ± 0.61
28.8 ± 0.82
76 ± 2.17
IIIb
IIIc
2.3.2.3. Malondialdehyde (MDA). Free radicals initiate lipid peroxida-
tion that can affect ion exchange in the cell membrane and can lead to
adverse effects[36]. This process can be monitored by measuring the
level of their end product (MDA) [37]. CP increased the level of MDA
[38] to fourfold after 8 hr and up to six fold at the end of experiment.
Pretreatment with the synthesized peptidomimetics II, IIIb and IIIg 8
hrs prior to addition of CP reduced the level of produced MDA to
20.43 ± 0.15, 25.6 ± 0.2 and 18.67 ± 0.21 respectively (about half the
MDA level in CP group 38.5 ± 2.92) (Fig. 5).
IIId
IIIe
10
25
50
–
IIIf
IIIg
157 ± 4.5
Cisplatin
3.28 ± 0.09
and three fold that of CP group respectively (Fig. 5). Compound IIIg is
the most potent in inhibition of glutathione depletion and this may
contribute to explanation of its potent renoprotective effect against CP
toxicity.
2.3.2.4. Reactive oxygen species (ROS). Oxidative stress is characterized
by elevated intracellular levels of reactive oxygen species (ROS) that
produce damaging effect to lipids, proteins and DNA [39,40]. CP in the
2.3.2.2. Superoxide dismutase (SOD). SOD plays a key role in guarding
against oxygen radicals. It was proved that, increasing the level of free
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K.A. Agha et al.
Bioorganic Chemistry 114 (2021) 105100
Fig. 4. The protective effect of peptidomimetic on CP-induced nephrotoxiciy.
Table 3
Effect of synthesized peptidomimetics II, IIIb, IIIg on oxidative stress biomarkers GSH/GSSG ratio, SOD, MDA, ROS.
Oxidative stress
Group
biomarkers*
Control
Cisplatin (8
hr)
II (8 hr)
IIIb (8 hr)
IIIg (8 hr)
CP (24 hr)
CP þ II (24
CP þ IIIb (24
CP þ IIIg (24
hr)
hr)
hr)
GSH/GSSG RATIO*
SOD (U / ml)*
MDA (ng /ml)*
ROS (U/L)*
3.73 ± 0.153
41.37 + 0.31
6.9 + 0.2
0.53 ± 0.12
11.43 ± 0.15
26.83 ± 0.15
34.43 ± 0.21
2.9 ± 0.1
1.93 ± 0.06
19.97 ± 0.15
18.43 ± 0.15
22.97 ± 0.12
3.33 ± 0.12
29.87 ± 0.21
12.17 ± 0.15
15.37 ± 0.06
0.37 ± 0.06
6.27 ± 0.15
38.5 ± 2.92
45.8 ± 0.2
1.67 ± 0.12
21.43 ± 0.15
20.43 ± 0.15
26.17 ± 0.12
1.13 ± 0.06
14.67 ± 0.12
25.6 ± 0.2
1.97 ± 0.06
24.87 ± 0.06
18.67 ± 0.21
24.67 ± 0.12
27.33 ± 0.15
15.27 ± 0.15
17.77 ± 0.15
8.93 ± 0.153
29.97 ± 0.21
*GSH/GSSG glutathione/glutathione disulfide ratio, SOD Superoxide dismutase, MDA Malondialdehyde, ROS reactive oxygen species
current study generated ROS more than fivefold the untreated LLC-
PK1cells. Prior treatment of LLC-PK1cells with peptidomimetics II,
IIIb and IIIg before CP addition reduced ROS production to about half
with compounds IIIg, II and IIIb (24.67 ± 0.12, 26.17 ± 0.12 and
29.97 ± 0.21) respectively (Fig. 5). The results obtained during this
stage of experiment prove the presence of antioxidative stress properties
of the synthesized peptidomimetics that help as a guard against oxida-
tive stress produced by CP.
At the end of the experimental protocol, animals were sacrificed
using high dose thiopental (40 mg/kg), blood samples were withdrawn
and centrifuged for 10 min at 3000 rpm to separate serum for further
analysis of liver and kidney functions. Liver and kidney specimens were
excised and fixed in 10% buffered formlain for histological analysis.
2.4.2. Assessment of biochemical markers of kidney function
Serum levels of creatinine (Cr) and blood urea nitrogen (BUN)(Bio-
med, Egypt) were assessed based on manufacturers‘ insructions.
2.4. In vivo screening
2.4.3. Assessment of biochemical markers of liver function
Serum levels of alanine aminotransferase (ALT) and aspartate ami-
notransferease (AST) (Biodiagnostics, Giza, Egypt) were evaluated by
colorimetric method according to manufacturers‘ insructions.
2.4.1. Assessment of potential toxicity of IIIg
2.4.1.1. Experimental design. All experimental procedures were aligned
with the guidelines of the ethics committee at Faculty of Medicine,
Mansoura University, Egypt. Sprague-Dawley rats were maintained at
standard conditions of humidity and temperature with free access to
food and water. After one week of acclimaization, a toxicity study to
assess the possible in vivo toxic effect of IIIg was performed. For this
purpose, 24 animals were randomly assigned into three groups receiving
differnt concentration of IIIg as following:
2.4.4. Histopathological studies
Fixed liver and kidney specimens were embedded in paraffin sections
and cut into 5-µm thick sections. Sections were then stained with he-
matoxylin and eosin (H&E) and viewed under light microscope. Images
were captured using a digital camera-aided computer system (Nikon
digital camera, Tokyo, Japan).
Group I: received IIIg at a dose (0.068 gm/kg/day) for 10 days
Group II: received IIIg at a dose (0.136 gm/kg/day) for 10 days
Group III: received IIIg at a dose (0.273 gm/kg/day) for 10 days
Additional untreated group that received vehicle only for 10 days
was used as normal control.
2.4.5. Results
As shown in Table 4 and Fig. 6, the highest dose of IIIg produced
moderate liver and kidney toxicity, where rats in group III showed sig-
nificant increase in liver and kidney function indices when compared to
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K.A. Agha et al.
Bioorganic Chemistry 114 (2021) 105100
Fig. 5. Effect of synthesized Met-based peptidomimetic II, IIIb and IIIg on oxidative stress biomarkers (GSH/ GSSG ratio, SOD, MDA and ROS) in CP-treated
LLC-PK1cells.
control group (p < 0.05). Additionlly, histopathological study demon-
Table 4
strated glomerular, tubular and hepatic changes. Of note, one rat died
Effect of different doses of IIIg on liver and kidney function tests.
from group III during the experimental course.
GPT (U/L)
GOT (U/L)
Cr (mg/dl)
BUN (mg/dl)
Based on the aforementioned results, the middle dose (0.136 gm/kg/
day) was used to assess the potenial in vivo renoprotective effect of the
synthesized peptidomimetic IIIg in CP-induced renal toxicity according
to the following protocol:
Control
Group I
Group II
Group III
23.66 ± 5.8
24 ± 5.2
89.67 ± 4.05
99.33 ± 9.3
105.3 ± 9.3
126.7 ± 8.09*
o.52 ± 0.03
0.57 ± 0.04
0.61 ± 0.06
0.92 ± 0.1*
16.47 ± 1.4
16.67 ± 3.9
19.33 ± 4.9
34.67 ± 0.3*
28 ± 5.5
45.3 ± 1.7*
*Significant compared to control group at p ≤ 0.05
2.5. Assessment of potential renoprotective efficacy of IIIg using CP-
induced renal toxicity model
2.5.1. Experimental design
All experimental procedures adhered to the guidelines of the ethics
committee at Faculty of Medicine, Mansoura University, Egypt. Twenty
four male Sprague-Dawley rats were randomly assigned after acclima-
tization for one week into the following groups.
Normal control group: received vehicle only for 10 constitutive
days.
Cisplatin group: Rats received CP (10 mg/kg, once, ip) on day 6.
Cisplatin þ Methionine: received Met (100 mg/kg/day, ip) for
10 days and cisplatin on day 6 of drug administration.
Cisplatin þ IIIg: Rats received IIIg (0.136 gm/kg/day, ip) for 10
constitutive days and CP on day 6 of IIIg administration.
At the end of the experimental protocol, animals were sacrificed
using high dose thiopental (40 mg/kg), blood samples were withdrawn
and centrifuged for 10 min at 3000 rpm to separate serum for further
analysis of kidney functions. kidneys were excised. One part was ho-
mogenized in phosphate buffered saline for further analysis of MDA and
GSH, and the other part was fixed in 10% buffered formlain for histo-
logical analysis and immunohistochemical studies.
Fig. 6. Upper panel: Microscopic pictures of H&E stained kidney sections
showing normal glomeruli and tubules in group I, normal tubules and minute
dilation of Bowman’s capsule in group II and dilation of Bowman’s capsule with
eosinophilic material (black arrow) accompanied with mild tubular degenera-
tion (blue arrows) in group III. Lower panel: Microscopic pictures of H&E
stained liver sections showing normal radiating hepatic cords around central
veins (CV) in group I, normal hepatocytes and mildly congested CV (red arrows)
in group II and mildly congested CV (red arrows) and mild hepatocytes
degeneration in group III. X:400 bar 500. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of
this article.)
2.5.2. Assessment of oxidative stress in renal tissue
Level of MDA and GSH in renal tissue homogenate was assessed
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K.A. Agha et al.
Bioorganic Chemistry 114 (2021) 105100
using commercially available colorimetric kits purchased from Bio-
diagnostics (Giza, Egypt) following manufacturers‘ protocol.
Statistical significance was considered at p ≤ 0.05.
2.5.8. Results and discussion
2.5.3. Immunohistochemical study
As demonstrated in Table 5, cisplatin administartion produced
marked renal injury as shown by significant increase in serum levels of
creatinine and BUN compared to normal group (p < 0.05). On the other
hand, treatment with Met and Synthesized peptidomimetic IIIg
improved renal function compared to CP-treated group. However, better
results were observed in IIIg-treated group. Oxidative stress is a hall-
mark of CP-induced organ toxicity. Increased lipid peroxidation along
with compromised antioxidant defence mechanisms is well established
[42]. Similarly, our current study showed marked increase in renal MDA
level and significant decrease in renal GSH (p < 0.05) compared to
normal group. In contrast, treated groups showed significant suppres-
sion of MDA level and restoration of GSH level in renal tissue when
compared with CP-treated group. Of note, the antioxidant effect
observed in synthesized peptidomimetics IIIg-treated group was supe-
rior compared to methionine group (p < 0.05).
Five µm renal sections were deparafinized and blocked with 5%
bovine serum albumin for 2 hrs. Thereafter, sections were washed and
incubated with primary antibody for Nrf-2 (Abcam, USA, ab89443)
◦
overnight at 4 C. Following incubation, slides were washed and then
incubated with labeled secondary antibody for 1 hr and the immuno-
reaction was visualized using 3,3′-diaminobenzidine (DAB, 2%) as
chromogen. Slides were then counterstained by Mayer’s hematoxylin
and sections were inspected and images were captured using Olympus®
digital camera installed on Olympus® microscope.
Assessment of combined IIIg administration on CP antiproliferative
effect.
Next, we investigated if IIIg co-administration affected CP chemo-
therapeutic efficacy using in vivo and in vitro Ehrlich Ascites Carcinoma
(EAC).
The effect of Met and IIIg on renal morphological changes induced
by CP was shown in Fig. 7. While Fig. 8 showed results of Nrf2 immu-
nostaining in different experimental groups. Nrf2 is a transcription
factor that plays a crucial role in regulating redox homeostasis and
cellular oxidative defence [43]. A substantial body of literature has re-
ported the role of Nrf2 in safeguarding against CP-induced nephrotoxi-
city. Indeed, CP renal injury was exacerbated in Nrf2 Knocked out mice
[44]. On the other hand, Nrf2 activators demonstrated renoprotective
capability against CP-induced toxicity [45]. In this context, Met has been
reported to induce Nrf2 and enhance antioxidant defence in rats [46].
Similarly, our results showed decreased immunostaining of Nrf2 in renal
tissue of cisplatin treated rats. However, treatment with Met showed
mild restoration of Nrf2 immunostaining in renal tissue, while IIIg
produced more increase in Nrf2 immunostaining in renal tissue of
treated rats.
2.5.4. In vivo EAC model
Twnety four adult Swiss albino mice (20–30 gm) were used in this
study. The animals were housed under standard conditions of humidity
and temperature and allowed free access to regular rodent diet and
water. Animals were injected intraperitoneally with suspension EAC
cells (2 × 106 viable cells/mouse) under aseptic conditions. Ehrlich-
tumor cells were provided by National Cancer Institute (Cairo, Egypt)
and maintained by serial intra peritoneal passage in female Swiss albino
mice at 7–10 day intervals [41]. Following tumor cells transplantation,
the EAC mice were further subdivided into three groups as following:
Untreated EAC mice: received vehicle, ip, daily for 14 days from the
1st day of the experiment.
Cisplatin treated EAC mice: received CP (5 mg/kg, ip, single
treatment on the 1st day of the experiment), 1 h prior to the injection of
vehicle, ip, daily for 14 days from the 1st day of the experiment.
Cisplatin þ IIIg treated EAC mice: received CP (5 mg/kg, ip, single
treatment on the 1st day of the experiment), 1 h prior to the injection
0.136 gm/kg IIIg, ip, daily for 14 days from the 1st day of the
experiment.
To evaluate the effect of IIIg on CP anti-tumor activity, EAC cells
were used as in vivo and in vitro models. EAC is originally a murine
spontaneous breast cancer. It is undifferentiated hyperdiploid carci-
noma characterized by high transplantable capability, 100% malig-
nancy and rapid proliferation [47,48]. An ascites variant was obtained
from EAC spontaneous breast cancer. The ascites was commonly used
over the last four decades for assessment of anti-tumor efficacy of
various chemicals and natural products. Intraperitoneal inoculation of
the ascites produces an ascitic fluid rich in tumor cells. As shown in
Fig. 9A, CP antiproliferative efficacy was reflected as significant
decrease in tumor volume in cisplatin treated EAC mice compared with
untreated EAC mice (p < 0.05). However, no significant difference in
tumor volume was observed between cisplatin treated EAC mice and
cisplatin + IIIg treated EAC mice, indicating that IIIg had no effect on
antiproliferative efficacy of CP. Similar results were obtained in vitro,
where no significant difference was observed in percentage cell viability
between EAC cells treated with CP alone or in combination with IIIg at
various concentrations, Fig. 9B.
2.5.5. Assessment of tumor volume
At the end of the experimental procedure, animals were sacrificed
under anaesthesia and the ascites fluid was separated individually from
each animal by needle aspiration from the peritoneal cavity under
aseptic conditions and collected in graduated tubes for tumor volume
measurement.
2.5.6. In vitro EAC cells and trypan blue exclusion assay
EAC cells were cultured in 96-well plate (1x104 cells/well) and kept
at 37 ◦C for 24 h in 5% CO2 incubator. Cells were then shifted to serum
free medium and treated with CP (10, 50 ad 100 µg/ml) alone and in
combination with different concentration of IIIg (10, 50 and 100 µM).
Five wells were used for each treatment concentration in addition to five
wells treated with vehicle only (DMSO, 0.1%). The plate was incubated
for 48 h in 5% CO2 incubator. Effect of treatment on cell viability was
determined using trypan blue exclusion assay. Briefly, after appropriate
incubation with treatments, equal volumes of trypan blue was added to
each well and then Neubauer hemocytometer was used to count number
of the stained cells (dead cells) and unstained cells (alive cells). The % of
dead EAC cells was then calculated.
Table 5
Effect of methionine and prepared drug on markers of kidney function (creati-
nine, BUN) and oxidative stress (MDA, GSH).
Cr (mg/dl)
BUN (mg/dl)
MDA (nmol/g
tissue)
GSH (mmol/g
tissue)
2.5.7. Statistical analysis
Normal
Cisplatin
Methionine
IIIg
0.55 ± 0.03
1.5 ± 0.15*
0.9 ± 0.05*
0.8 ± 0.04
16.25 ± 3.7
82.5 ± 12.39*
46.8 ± 5.9*
21.8 ± 1.7#
6.7 ± 1.3
2.9 ± 0.15
1.2 ± 0.2*
1.7 ± 0.3*
2.8 ± 0.11#
Results for in vivo and in vitro experiments were represented as
mean ± SE. Statitical difference among different groups was analyzed by
one-way ANOVA test followed by Tukey‘s post hoc test once differences
exist among different experimental groups. GraphPad prism program
was used for performing statistcical analyses and graphical presentation.
33.66 ± 4.06*
25.9 ± 2.7*
14.2 ± 1.8#
*Significant compared to control group at p ≤ 0.05. # Significant compared to
methionine group at p ≤ 0.05.
7

K.A. Agha et al.
Bioorganic Chemistry 114 (2021) 105100
Fig. 7. Microscopic pictures of H&E stained kidney
sections showing normal glomeruli (G) and tubules
(T) in the control group. Meanwhile, kidney sections
from Cisplatin group showed tubular dilation, se-
vere tubular epithelial degeneration (black arrows)
in cortex, diffuse tubular epithelial degeneration
(black arrows), fibrosis (yellow arrow) with con-
gested blood vessels (red arrows) in medulla. Kidney
sections from methionine group showing mild
tubular dilation with mild tubular epithelial
degeneration (black arrows) in cortex, mild tubular
epithelial degeneration (black arrows) and mildly
congested blood vessels (red arrows) in medulla.
Kidney sections from IIIg group showed mild
tubular epithelial degeneration (black arrows) in
cortex, mild tubular epithelial degeneration (black
arrows) with very mildly congested blood vessels
(red arrows) in medulla. Low magnification X:
100 bar 100, high magnification X: 400 bar 50. (For
interpretation of the references to colour in this
figure legend, the reader is referred to the web
version of this article.)
Fig. 8. Microscopic pictures of immunostained
renal sections against Nrf2 showing strong positive
brown tubular (black arrows) and glomerular (blue
arrows) reactions in the normal control group (A).
Nrf2 positive tubular and glomerular expression
dramatically decreased in Cisplatin group (B). Nrf2
positive tubular (black arrows) and glomerular
(blue arrows) expressions mildly increased in
Methionine group (C) and moderately increased in
IIIg group (D). IHC counterstained with Mayer’s
hematoxylin. Black arrows point to positive cells.
Low magnification X:100 bar 100 and high magni-
fication X:400 bar 50. (For interpretation of the
references to colour in this figure legend, the reader
is referred to the web version of this article.)
3. Experimental
Santa Clara, CA, USA) equipped with an autosampler (G7129A), a quat.
pump (G7104C) and a column comp (G7116A) at Faculty of pharmacy,
Fayoum University. The LC-HRMS analysis was carried out using Zorbax
RP-18 column from Agilent Technologies (dimensions: 150 mm × 3
mm, dp = 2.7 µm) and Zorbax Extend-C18 column from Agilent Tech-
nologies (dimensions: 50 mm × 2.1 mm, dp = 1.8 µm) in a flow rate of
0.1cm3/min. The mobile phase consisted of a combination of solvent A
(water + 0.1% formic acid) and solvent B (acetonitrile + 0.1% formic
acid). The elution was as follows: t = 0 min, 30% B; t = 1min, 30% B;
t = 10 min, 70% B. Elemental analysis was performed on the Thermo
Fisher Flash 2000 CHNS analyzer at the Regional Center for Mycology
and Biotechnology, Al-Azhar University. All reactions were monitored
3.1. General information
Starting materials and solvents were purchased from common com-
mercial sources and used without further purification. Melting points
were determined on Fisher melting apparatus and are uncorrected. 1H
NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded on
Bruker a 400 MHz NMR Spectrometer and using DMSO-d6 as solvent, at
Faculty of Pharmacy, Mansoura University. The chemical shift (δ) is
reported in ppm, and coupling constants (J) are given in Hz. The HRMS
and LC-HRMS were recorded on LC/Q-TOF, 6530 (Agilent Technologies,
8

K.A. Agha et al.
Bioorganic Chemistry 114 (2021) 105100
(400 MHz, DMSO‑d6) δ 8.77 (d, J = 7.6 Hz, 1H, NH), 7.93 (d, J = 8.4 Hz,
2H, OC-ortho-Ar-H), 7.57 (d, J = 8.4 Hz, 2H, CO-meta-Ar-H), 4.54 (q,
– – –
NH CH ), 2.65–2.56 (m, 2H,
– –
CH2 SCH3),
J = 8.0 Hz, 1H,
2.10–2.06 (m, 5H, CH2 CH2 SCH3). 13C NMR (100 MHz, DMSO‑d6)
– – –
δ 174.0 (COOH), 167.2 (CO), 134.4 (
–
–
–
–
–
C Cl), 131.9 ( C CO), 128.7
–
– –
–
(Ar-C), 127.9 (Ar-C), 52.1 ( CH COOH), 30.7 ( CH2CH2SCH3), 30.6
–
–
(
CH2CH2SCH3), 15.1 ( SCH3). HRMS (ESI): m/z cald for C12 H15 Cl N
O3 S ([M+H] +) 288.0456, found 288.0462.
3.3.3. (S)-N-(1-(1H-benzo[d][1,2,3]triazol-1-yl)-4-(methylthio)-1-
oxobutan-2-yl)-4-chlorobenzamide (IIa)
White microcrystals, yield 3.46 g (89%); mp 78–80 ◦C. 1H NMR
(400 MHz, DMSO‑d6) δ 9.34 (d, J = 6.4 Hz, 1H, NH), 8.31 (d, J = 8.0 Hz,
1H, Ar-H), 8.25 (d, J = 8.0 Hz, 1H, Ar-H), 7.97 (d, J = 8.4 Hz, 2H, OC-
ortho-Ar-H), 7.82 (t, J = 7.6 Hz, 1H, Ar-H), 7.66 (t, J = 7.6 Hz, 1H, Ar-H),
– –
7.60 (d, J = 8.4 Hz, 2H, OC-meta-Ar-H), 5.99–5.94 (m, 1H, CH ), 2.82
–
(dd, J = 7.6, 5.2 Hz, 1H, CH2SCH3), 2.73 (dd, J = 13.2, 7.6 Hz, 1H,
–
–
–
CH2SCH3), 2.41–2.30 (m, 2H, CH2CH2SCH3), 2.10 (s, 3H, SCH3).
13
– – – –
N ), 166.8 ( CONH),
C NMR (100 MHz, DMSO‑d6) δ 171.9 ( CO
–
–
–
–
–
–
– –
N N C), 137.2 ( C Cl), 132.3 ( C CO), 131.6 (Ar-C),
– –
145.8 (
131.2 (Ar-C), 130.1 (Ar-C), 129.0 (Ar-C), 127.2 (Ar-C), 120.7 (Ar-C),
– –
CH ), 30.4
–
CH2CH2SCH3), 29.8
114.5 (Ar-C), 53.3
(
(
–
(CH2CH2SCH3), 14.8 ( SCH3). Anal. Calcd. for C18H17ClN4O2S: C,
55.60; H, 4.41;N, 14.41, S, 8.24 found C, 55.70; H, 4.47; N, 14.43, S,
8.28
Fig. 9. Effect of co-administration of CP and IIIg on EAC in vivo and in vitro. A)
tumor volume (volume of ascites fluid aspirated from different experimental
groups). B) Percentage of cell death (values were means of five replicates). Data
were expressed as mean ± SE. *** significant compared to EAC at P < 0.001.
3.3.4. (4-Chlorobenzoyl)-L-methionylglycine IIIa
1
◦
White microcrystals, yield 1.57 g (91%); mp 161–163 C. H NMR
(400 MHz, DMSO‑d6) δ 12.64 (s, 1H, COOH), 8.68 (d, J = 8.0 Hz, 1H,
–
NHCH2), 8.35 (s, 1H, NHCH ), 7.95 (d, J = 8.4 Hz, 2H, Ar-H), 7.56 (d,
J = 8.4 Hz, 2H, Ar-H), 4.58 (dd, J = 13.4, 8.2 Hz, 1H, CH), 3.82 (dd,
J = 17.6, 5.8 Hz, 1H, NHCH2), 3.73 (dd, J = 17.6, 5.6 Hz, 1H, NHCH2),
by TLC with visualization by UV irradiation.
–
2.61–2.54 (m, 2H, CH2SCH3), 2.06 (s, 3H, SCH3), 2.02–1.95 (m, 2H,
3.2. General procedure for synthesis of compound Ia and IIa
CHCH2 ). 13C NMR (100 MHz, DMSO‑d6) δ 172.2 (COOH), 171.6
–
–
–
–
–
C
–
–
–
–
C
(CHCO), 165.9 (
CO), 136.6 (Cl
), 133.2 (COC ), 130.1 (Ar-
–
To 4.76 g BtH (40 mmol) dissolved in 50 cm3 CH2Cl2, 0.73 cm3 SOCl2
C), 128.7 (Ar-C), 53.1 (CH), 41.2 (NHCH2), 31.8 (CHCH2), 30.4 (SCH2),
◦
(10 mmol) were added. The mixture was stirred at 25 C for 30 min,
15.1 (SCH3). HRMS (ESI): m/z cald for C14 H18 Cl N2 O4 S ([M+H] +
)
followed by the addition of the corresponding acid (10 mmol) and the
reaction was allowed to stir for an additional 3 hrs at 25 ◦C. The reaction
was diluted with 50 cm3 CH2Cl2 and the organic layer was washed with
saturated Na2CO3 (3 × 20 cm3), 20 cm3 H2O, and 10 cm3 brine. The
organic layer was dried over anhydrous sodium sulfate, 50 cm3 hexane
was added to the filtrate, and then the solid obtained was dried under
vacuum to give compound Ia and IIa.
345.067, found 345.06809.
3.3.5. (4-Chlorobenzoyl)-L-methionyl-L-methionine (IIIb)
Oily, yield 1.72 g (82%). 1H NMR (400 MHz, DMSO‑d6) δ 12.64 (s,
–
– – –
NH CO C ), 8.38 (d,
–
1H, COOH), 8.78 (d, J = 7.6 Hz, 1H,
– – –
J = 8.4 Hz, 1H, NH CH COOH), 7.93 (d, J = 8.0 Hz, 2H, –CO-ortho-
Ar-H), 7.57 (d, J = 8.0 Hz, 2H, –CO-meta-Ar-H), 4.56–4.49 (m, 1H,
– –
– –
CH ), 4.37–4.32 (m, 1H, CH ), 3.10–3.05 (m, 1H, CH2SCH3),
–
2.63–2.54 (m, 3H, CH2SCH3), 2.07–2.02 (m, 10H, 2( CH2CH2SCH3).
3.3. General procedure for Synthesis of target compounds II and IIIa-g
13C NMR (100 MHz, DMSO‑d6)
δ
173.9 (COOH), 172.0
–
–
C
–
– – – –
– –
–
(
(
NH CO CH ), 166.1
(
CO
C
), 136.7 (Cl
), 133.1
–
The corresponding amino acids (5 mmol) was dissolved in water (3
mL) with 1 equivalent triethylamine (5 mmol, 70 mL) and added to the
solution of the N-acylbenzotriazoles Ia or IIa (5 mmol) in CH3CN (9 mL)
or Dioxane (9 ml). The mixture was stirred for 5hrs at room temperature
but incase of anthranilc acid heating at 60 ◦C was needed (until complete
consumption of N-acylbenzotriazole as monitored by TLC). The pH was
adjusted to 5 using 2 N HCl, and the solvent was evaporated under
reduced pressure. The residue was then extracted with ethyl acetate (2 ×
20 mL) and the organic layer was washed with 4 N HCl (3×10 mL),
water (10 mL), and brine (10 mL) then dried over anhydrous sodium
sulfate. The organic layer was evaporated under reduced pressure to
give the desired compounds II and IIIa-g.
–
– –
CO
– – –
C ), 129.9 (Ar-C), 128.8 (Ar-C), 52.2 ( CH ), 45.9 (CH ),
–
31.0 (Aliph-C), 30.5 (Aliph-C), 30.3 (Aliph-C) , 30.1 (Aliph-C), 15.0
–
–
(
SCH3), 9.0 ( SCH3). HRMS (ESI): m/z cald for C17 H24 Cl N2 O4 S2
([M+H] +) 419.0861, found 419.08717.
3.3.6. (4-Chlorobenzoyl)-L-methionyl-L-cysteine (IIIc)
1
Oily, yield 1.56 g (80%). H NMR (400 MHz, DMSO‑d6) δ 8.78 (d,
–
– – –
J = 8 Hz, 1H, NH CO
C ), 7.97 (s,1H, NH), 7.93 (d, J = 8.8 Hz, 2H,
–
–CO-ortho-Ar-H), 7.57 (d, J = 8.8 Hz, 2H, –CO-meta-Ar-H), 4.65–4.50
– –
– –
(m, 2H, CH ), 3.31 (dd, J = 14.4, 5.2 Hz, 1H, HS CH2 ), 3.08 (dd,
– –
– –
J = 7.2, 5.2 Hz, 1H, HS CH2 ), 2.65–2.54 (m, 2H, CH2 SCH3),
2.10–2.07 (m, 5H,
CH2CH2SCH3), 1.20 (s, 1H, SH). 13C NMR
–
–
(100 MHz, DMSO‑d6) δ 173.9 (COOH), 166.9 (NHCOCH ), 166.1
3.3.1. (1H-Benzo[d][1,2,3]triazol-1-yl)(4-chlorophenyl)methanone (Ia)
White microcrystals, yield 2.4 g (93 %); mp 138–140 ◦C (138–139
◦C) [49].
–
–
–
–
–
–
C
(
C
CO), 136.7 (Cl
), 133.1 (Ar-C), 129.7 (Ar-C), 128.8 (Ar-C),
–
– –
–
52.2 (CH COOH), 45.9 (NH CH CONH ), 30.6 (Aliph-C), 30.5
–
( SCH3). Anal. Calcd. for
(Aliph-C), 28.9 (Aliph-C), 15.0
15H19ClN2O4S2: C, 46.09; H, 4.90; N, 7.17; S, 16.40 found C, 46.18; H,
4.97; N, 7.17; S, 16.49
C
3.3.2. (4-Chlorobenzoyl)-L-methionine II
White microcrystals, yield 1.28 g (89%); mp 101–103 ◦C. 1H NMR
9

K.A. Agha et al.
Bioorganic Chemistry 114 (2021) 105100
3.3.7. (4-Chlorobenzoyl)-L-methionyl-L-histidine (IIId)
3.5. Measurement of GSH/GSSG ratio
Oily, yield 1.76 g (83%). 1H NMR (400 MHz, DMSO‑d6) δ 10.51 (s,
–
–
–
), 8.78 (d, J = 7.6 Hz, 1H,
1H, NH), 9.05 (s, 1H,
CH
N
The GSH/GSSG ratio was determined following a method previously
described by Irfan Rahman and his coworkers [50].
–
–
– – –
– – –
C ), 8.66 (d, J = 7.2 Hz, 1H, NH CH COOH), 7.96 (d,
–
NH CO
J = 8.4 Hz, 2H, –CO-ortho(Ar-H)), 7.55 (d, J = 8.4 Hz, 2H, –CO-meta(Ar-
–
– –
= 15.6, 5.2 Hz, 1H,
– –
H)), 7.46 (s, 1H, NH CH ), 4.59–4.47 (m, 2H, CH ), 3.21 (dd,
–
3.6. Measurement of ROS production
– – –
J
CH2 CH COOH), 3.04 (d, br, 1H,
– – –
–
CH2 CH COOH), 2.56–2.42 (m, 2H, CH2SCH3), 2.04–1.92 (m,
Human reactive oxygen species (ROS) ELISA Kit (BIOASSAY TECH-
NOLOGY – CHINA) that uses a double-antibody sandwich enzyme-
linked immunosorbent assay (ELISA) to assay the level of reactive oxy-
gen species (ROS) was used following manufacturer’s instructions.
5H, CH2CH2SCH3). 13C NMR (100 MHz, DMSO‑d6) δ 172.4 (COOH),
–
–
–
–
– – – –
– –
–
), 136.7 (Cl C
172.0 ( NH CO CH ), 166.0 ( CO
C
), 133.8
–
–
– –
C N ), 133.1 (Ar-C), 130.13 (Ar-C), 130.1 (Ar-C), 128.7 (Ar-C),
–
(
–
–
–
117.4 (Ar-C), 53.4 ( CHCOOH), 45.8( NHCHCO ), 31.6 (Aliph-C),
–
30.3(Aliph-C), 15.1(Aliph-C), 8.9 ( SCH3). HRMS (ESI): m/z cald for
3.7. Measurement of SOD and MDA
C
18 H22 Cl N4 O4 S ([M+H] +) 425.1045, found 425.11029.
The SOD and MDA activity were determined according to the in-
structions of the assay kits (Lifespan Biosciences, Seattle, USA).
3.3.8. (4-Chlorobenzoyl)-L-methionyl-L-phenylalanine (IIIe)
Oily, yield 1.85 g (85%). 1H NMR (400 MHz, DMSO‑d6) δ 12.83 (s,
–
– – –
C CO NH ), 8.33 (d,
1H, COOH), 8.77 (d, J = 7.6 Hz, 1H,
–
4. Conclusions
J = 8.0 Hz, 1H, NHCHCOOH), 7.93 (d, J = 8.4 Hz, 2H, –CO-ortho(Ar-H)),
7.57 (d, J = 8.4 Hz, 2H, –CO-meta(Ar-H)), 7.34–7.21 (m, 5H, Ar-H),
We have designed and synthesized a novel nephroprotective pepti-
domimetic having the ability to attenuate CP-induced nephrotoxicity
probably by acting as guardians against oxidative stress. Among the
synthesized compounds, IIIg showed the most potent renoprotective
effect with antioxidant activity supported by invivo improvement in
results of renal function tests and attenuated CP-induced renal structural
injury in Sprague Dawley rats, proper physicochemical and pharmaco-
kinetic properties. Moreover, IIIg did not affect CP chemotherapeutic
effect both in vivo and in vitro. Thus, N-p-chlorobenzoyl-Methionyl an-
thranilic acid (IIIg), N-p-chlorobenzoyl-Met-OH (II) and N-p-chlor-
obenzoyl-Met-Met-OH (IIIb) are promising candidate compounds for
further investigation to develop better renoprotective agents.
–
–
– –
C CH2 ), 2.98–2.89
4.56–4.43 (m, 2H, 2CH), 3.11–3.05 (m, 1H,
–
–
– – –
(m, 1H,
C CH2 ), 2.65–2.56 (m, 2H, SCH2 ), 2.07 (s, 3H, SCH3),
2.01–1.83 (m, 2H, SCH2CH2). 13C NMR (100 MHz, DMSO‑d6) δ 173.9
–
–
–
–
–
C
–
C
(COOH), 173.3 (CHCONH), 166.1 (
CO), 137.9 (Cl
), 136.7
(Ar-C), 133.1 (Ar-C), 129.9 (Ar-C), 129.6 (Ar-C), 128.8 (Ar-C), 128.7
– – – –
(Ar-C), 126.9 (Ar-C), 52.2 (CH-COOH), 46.0 ( NH CO CH ), 31.9
–
–
– –
C CH2), 30.6 (SCH2CH2), 27.2 (SCH2 ), 15.0 (SCH3). HRMS (ESI):
(
m/z cald for C21 H24 Cl N2 O4 S ([M+H] +) 435.114, found 435.11496.
3.3.9. (4-Chlorobenzoyl)-L-methionyl-L-arginine (IIIf)
1
Oily, yield 1.93 g (87%). H NMR (400 MHz, DMSO‑d6) δ 8.83 (d,
–
– – –
C CO NH ), 8.48 (d, J = 7.2 Hz, 1H,
–
J
= 7.2 Hz, 1H,
– – –
NH CH COOH), 7.95 (d,
J = 7.6 Hz, 2H, –CO-ortho-Ar-H),
Declaration of Competing Interest
–
7.56–7.09 (m, 5H, –CO-meta-Ar-H, NH , NH), 4.62–4.51 (m, 1H,
–
2
–
– –
– – – –
CH COOH), 4.18 (s, br, 1H,
C
CO NH CH ), 3.75–3.62 (m,
–
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
– – –
2H,
NH CH2 ), 3.12 (s, br, 2H, CH2SCH3), 2.06 (s, br, 5H,
CH2CH2SCH3), 1.72 (d, br, 2H, CH2CHCOOH), 1.53 (d, br, 2H,
CH2CH2CHCOOH), 1.22 (s, 1H, NH). 13C NMR (100 MHz, DMSO‑d6) δ
–
–
–
– –
C
–
–
–
173.8 (COOH), 172.0 ( NHCOCH ), 166.1 (
CO ), 157.6
C ), 133.2 ( CO C ), 130.1 (Ar-C), 128.7(Ar-
–
Acknowledgements
–
–
–
– –
–
–
C
(C NH), 136.7 (Cl
–
–
–
–
–
–
( CHCOOH),
C),
53.5
(
CONHCH ),
52.2
40.7
We thank HRMS facility, Faculty of pharmacy, Fayoum University,
Egypt, for mass analysis.
–
–
(
CH NHC NH), 32.1 (Aliph-C), 30.6 (Aliph-C), 28.5 (Aliph-C), 25.7
–
(Aliph2-C), 15.2 (SCH3). HRMS (ESI): m/z cald for C18 H27 Cl N5 O4 S
([M+H] +) 444.1467, found 444.14801.
Appendix A. Supplementary material
Full experimental detail, 1H and 13C NMR spectra can be found via
the “Supplementary Content” section of this article’s webpage. Supple-
mentary data to this article can be found online at https://doi.org/10.10
16/j.bioorg.2021.105100.
3.3.10. (S)-2-(2-(4-Chlorobenzamido)-4-(methylthio)butanamido)benzoic
acid IIIg
Buff microcrystals, yield 1.67 g (82%); mp 205–207 ◦C. 1H NMR
–
–
C NH),
(400 MHz, DMSO‑d ) δ 11.80 (s, 1H, COOH), 9.15 (s, 1H,
–
6
– – –
7.64–7.54 (m, 3H, Ar-H), 7.17 (t, J = 7.4 Hz, 1H, –NH-para-Ar-H),
8.67 (d, J = 8.4 Hz, 1H, CH NH ), 8.03–7.98 (m, 3H, Ar-H),
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– – –
– – –
4.71–4.65 (m, 1H, NH CH ), 2.67–2.57 (m, 2H, CH2 SCH3 ),
2.31–2.12 (m, 2H, CH2CH2SCH3), 2.08 (s, 3H, SCH3). 13C NMR
–
–
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– – – –
–
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–
–
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