Synthesis and biological evaluation of Val–Val dipeptide–sulfonamide conjugates

Article abstract of DOI:10.1002/ardp.202000074

Novel Val–Val dipeptide–benzenesulfonamide conjugates were reported in this study. These were achieved by a condensation reaction of p-substituted benzenesulfonamoyl alkanamides with 2-amino-4-methyl-N-substituted phenyl butanamide using classical peptide

Full text of DOI:10.1002/ardp.202000074

Received: 10 March 2020
Revised: 19 April 2020
Accepted: 22 April 2020
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DOI: 10.1002/ardp.202000074
F U L L P A P E R
Synthesis and biological evaluation of Val–Val
dipeptide–sulfonamide conjugates
James A. Ezugwu1,2
Chinaraju Bhimapaka2
| Uchechukwu C. Okoro1
| Mercy A. Ezeokonkwo1
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| Sunday N. Okafor3 | David I. Ugwu1
| Daniel I. Ugwuja4
1Department of Pure and Industrial Chemistry,
University of Nigeria, Nsukka, Enugu State,
Nigeria
Abstract
Novel Val–Val dipeptide–benzenesulfonamide conjugates were reported in this study.
These were achieved by a condensation reaction of p‐substituted benzenesulfonamoyl
alkanamides with 2‐amino‐4‐methyl‐N‐substituted phenyl butanamide using classical
peptide‐coupling reagents. The compounds were characterized using Fourier transform
2Department of Organic Synthesis and Process
Chemistry Division, CSIR‐India Institute of
Chemical Technology, Hyderabad, Telangana,
India
3Department of Pharmaceutical and Medicinal
Chemistry, University of Nigeria, Nsukka,
Enugu State, Nigeria
1
infrared, H‐nuclear magnetic resonance (NMR), 13C‐NMR, and electrospray ionization–
high‐resolution mass spectrometry spectroscopic techniques. As predicted from in silico
studies, the Val–Val dipeptide–benzenesulfonamide conjugates exhibited antimalarial and
antioxidant properties that were analogous to the standard drug. The synthesized com-
pounds were evaluated for in vivo antimalarial activity against Plasmodium berghei. The
hematological analysis was also conducted on the synthesized compounds. At 50 mg/kg
body weight, compounds 8a, 8d, and 8g–i inhibited the multiplication of the parasite by
48–54% on Day 7 of posttreatment exposure, compared with the 67% reduction with
artemisinin. All the synthesized dipeptides had a good antioxidant property, but it was
less when compared with vitamin C. The dipeptides reported herein showed the ability to
reduce oxidative stress arising from the malaria parasite.
4Department of Chemical Sciences, Federal
University, Wukari, Taraba State, Nigeria
Correspondence
James A. Ezugwu, Department of Pure and
Industrial Chemistry, University of Nigeria,
Nsukka 410001, Enugu State, Nigeria.
Email: james.ezugwu@unn.edu.ng
Funding information
CSIR‐TWAS, Grant/Award Number:
22/FF/CSIR‐TWAS/2017
K E Y W O R D S
antimalarial, antioxidant, benzenesulfonamide, drug resistance, in silico studies, Val–Val
dipeptide
bacterial DNA replication.[18,19] However, sulfonamide containing amino
acid moiety has been reported as an antioxidant and antimicrobial
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1
INTRODUCTION
Sulfonamides comprise a substantial class of pharmacological agents
such as antitumor, antibacterial, anticarbonic anhydrase, diuretic, hy-
poglycemic, antioxidant agents,[1–13] and many others. Recently, a host
of structurally novel Ala–Gly dipeptide–sulfonamide conjugates have
been reported to show a potent antimalarial property in the in vitro and
in vivo studies.[14] Recently, dipeptide–sulfonamide conjugates have
been reported as strong human carbonic anhydrase enzyme in-
hibitors.[15,16] Sharma and Soman[17] reported the synthesis of novel
diamide derivatives of glycine containing sulfonamide as a potent di-
peptidyl peptidase‐4 inhibitor. Sulfonamides exhibit an antibacterial
activity by competitively inhibiting the dihydropteroate synthase en-
zyme, which is very important for folate synthesis. Thus, they inhibit
agent.[19] Sulfonamide carboxamides have been reported as anti-
helmintic,[20] antitubercular,[21] antitrypanosomal,[22] antimalarial
agent.[23] The implications of free radicals through oxidative stress in
the physiopathogenesis of malaria have been reported by many au-
thors.[24–30] Potter et al.[31] recommended that this involvement may be
linked to the pathogenic mechanisms triggered by the malaria parasite.
Keller et al.[32] reported the high nitric oxide production in children
affected by malaria. Current studies have suggested that the production
of reactive oxygen and nitrogen species related to oxidative stress plays
a vital role in the development of systemic complications caused by
malaria. Several substances such as chloroquine, primaquine, and arte-
misinin, among others, used as antimalarial agents are prooxidants,
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Arch Pharm. 2020;e2000074.
wileyonlinelibrary.com/journal/ardp
© 2020 Deutsche Pharmazeutische Gesellschaft
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which is the reason behind their pharmacological power. This effect may
be due to the drug's ability to promote the direct production of free
radicals.[33] The reported emergence of resistant mosquitoes and
artemisinin‐resistant malaria parasite calls for a concerted effort in the
development of new antimalarial drugs. This present work was designed
to synthesize new antimalarial agents possessing sulfonamides, car-
boxamides, and peptide moieties that have been reported to in-
dependently possess antimalarial and antioxidant potentials. The
hematological analysis was also carried out to asses the effect of the
compounds on the blood of the experimental mice. The interesting thing
about the design is that the compounds sought would possess the an-
timalarial property and antioxidant activity to capture reactive oxygen
species produced during malaria infection. The choice of the Val–Val
central scaffold was informed by the moderate antimalarial properties
of Ala–Gly dipeptides reported by Ugwuja et al.[14] The antimalarial
properties of some amino acids, dipeptides, or tripeptides containing
quinine derivatives were also reported.[34]
reacted with L‐valine in an aqueous basic medium to obtain the sub-
stituted benzenesulfonamoyl alkanamides (3a–c). The reaction of com-
mercially available Boc‐protected valine with amines using 1‐ethyl‐3‐
(3‐dimethylaminopropyl)carbodiimide
hydrochloride
(EDC·HCl),
1‐hydroxybenzotriazole (HOBt) with trimethylamine (TEA) in di-
chloromethane (DCM) afforded the carbamate derivatives of valine
(6a–e). The unprotected dipeptides 7a–e were obtained from the re-
action of compounds 6a–e with DCM/trifluoroacetic acid (TFA; 1:1%)
for 1 hr, respectively. The condensation reaction of compounds 3a–c
with the unprotected dipeptides 7a–e in the presence of peptide‐
coupling reagents, EDC·HCl, HOBt, and TEA gave the targeted pro-
ducts, 8a–i, in Scheme 1. The Fourier transform infrared (FTIR) spectra
of the dipeptides showed N–H bands between 3,357 and 3,200 cm−1
.
The two C═O bands appeared between 1,688 and 1,641 cm−1. In the
1H‐nuclear magnetic resonance (NMR) of 8a, the characteristic NH
resonance of the sulfonamide part of the dipeptide conjugates was
observed at the δ8.07‐ppm region as a doublet peak. Other amide NH
resonances of the dipeptide–sulfonamide were observed at
δ7.69–7.87 ppm with aromatic protons and at δ10.19 ppm region as
multiplet and singlet peaks, respectively. The carbon‐13 NMR showed
all the peaks that were expected from successful coupled products. For
compound 8a, the peaks for carbonyl carbon of amide appeared at
170.73 and 170.46 ppm. All the aromatic and aliphatic peaks were ac-
counted for in the carbon‐13 NMR. The high‐resolution mass spectro-
metry (HRMS) peak of the derivatives appeared as molecular ions
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2
RESULTS AND DISCUSSION
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2.1
Chemistry
To synthesize Val–Val dipeptide–sulfonamide conjugates, the commer-
cially available substituted benzenesulfonyl chlorides (1a–c) were first
SCHEME 1 The synthesis of dipeptides bearing sulfonamide. Reagents, and conditions: (i) Na2CO3, H2O, HCl, −5 to 0°C, r.t., 4 hr. (ii)
EDC·HCl, HOBt, TEA, DCM, r.t., 19–24 hr. (iii) TFA/DCM (1:1%). (iv) EDC.HCI, HOBt, TEA, r.t., 19–24 hr

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TABLE 1 Percentage inhibition of parasite in mice
TABLE 3 In vitro antioxidant studies (IC50)
% Parasitemia
Compound
IC50 (mg/ml)
1.06
before
% Parasitemia
after treatment
8a
Compound treatment
% Inhibition
8b
0.78
8a
52.0 0.57735
48.0 15.50627
65.0 0.57735
58.0 1.15470
38.0 1.15470
73.0 0.57735
75.0 2.88675
43.0 3.46410
43.0 1.73205
43.0 4.04145
27.0 1.76383
83.0 1.73205
41.0 18.59537
21.7 0.69282
30.1 1.38564
54.1 1.38564
12.0 0.72188
9.6 3.49301
48.1 4.33026
48.1 6.99937
48.1 2.07953
67.1 2.11660
0.0 0.0000
8c
0.86
8b
8c
63.0 0.57735
67.0 0.57735
67.0 0.57735
65.0 0.57735
63.0 0.88192
68.0 0.57735
62.0 1.15470
66.0 0.57735
62.0 0.57735
68.0 1.73205
8d
0.70
8e
0.79
8d
8e
8f
0.85
8g
1.52
8f
8h
0.72
8g
8i
0.78
8h
8i
Vitamin C
0.30
Arte.
NTC
untreated group and B is the parasitemia of the tested group. The data
in Table 1 revealed that compounds 8a, 8d, 8g, 8h, and 8i with 48–54%
inhibition were active, compared with the reference drug (with 67%
inhibition). Among the synthesized compounds it was shown clearly
that compound 8d (54%) was more active against P. berghei, compared
with others. In the structure–activity relationship, among the phe-
nylsulfonamide hybrids (8a–c), the effect of 4‐chloro, 4‐bromo, and
3‐fluoro substituent on the N‐phenylacetamide was studied, and it was
revealed that 4‐bromo‐N‐phenylacetamide derivative (8a, 41%) was
the most potent inhibitor against the P. berghei, whereas compounds
8b and 8c were considered partially active and inactive, respectively.
The evaluation of the effects of 4‐bromo, 4‐chloro, and 3‐fluoro sub-
stituent on the N‐phenylacetamide among the p‐bromo and
p‐methylbenzenesulfonamide analogs (8d–f and 8g–i) revealed that
compounds 8d and 8g with 54 and 48% inhibition, respectively, were
more active.
Note: Values are means of three determinations SEM.
Abbreviations: Arte, artemisinin; NTC, non‐treated control; SEM,
standard error of the means.
[M+H]+/[M+Na]+. The results corresponded to three decimals with the
calculated values. The spectra used for the characterization of the new
compounds are available as supporting documents.
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2.2
Pharmacology/Biology
To determine the in vivo activity, the compounds were tested against
Plasmodium berghei (NK65 strain)‐infected mice; the animals were ob-
tained from TwinVet® Laboratory, Department of Veterinary Anatomy,
Faculty of Veterinary Medicine, University of Nigeria, Nsukka. The
permission and approval for the use of animals in this experiment were
granted by the Animal Ethics Committee, Veterinary Medicine De-
partment, University of Nigeria, Nsukka for PG/PhD/16/80697.
Artemether was used as standard drug in these experiments. The
percentage inhibition of parasite multiplication was obtained using
equation [(A − B)/A] × 100,[14] where A is the parasitemia of the
Data in Table 2 revealed that there is an increase in packed cell
volume (PCV), but it is not accompanied by corresponding increases
in RBC counts and Hb concentration, compared with pretreatment
and control, which is in accordance with the report of Wasser[35] who
adduced that the increase may be relative polycythemia due to
hemoconcentration.
TABLE 2 Hemotological analysis before and after treatment
RBC (mm3) × 106
Before
PCV (%)
HB (g/dl)
Compound
After
Before
After
Before
After
8a
7.4 0.11547
7.2 0.11547
8.0 0.11547
8.6 0.11547
7.6 0.11547
7.4 0.23094
7.0 0.11547
6.4 0.23094
7.2 0.11547
10.6 0.11547
5.3 0.17321
11.6 0.11547
36.0 1.15470
41.0 0.57735
38.0 0.57735
40.0 1.73205
42.0 0.57735
40.0 1.15470
44.0 2.30940
46.0 1.15470
28.0 1.15470
48.0 1.15470
52.0 1.15470
43.0 1.73205
8.0 0.00000
9.0 0.17321
8.4 0.23094
8.4 0.11547
13.6 0.05774
8.4 0.11547
7.7 0.05774
8.2 0.11547
8.0 0.11547
14.8 0.11547
10.3 0.17321
13.5 0.05774
8d
8g
8h
8i
Arte.
Note: Values are means of three determinations SEM.
Abbreviations: Arte., artemisinin; HB, hemoglobin; PCV, packed cell volume; RBC, red blood cells; SEM, standard error of the means.

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FIGURE 3 The binding pose of compound 8i in the binding cavity
FIGURE 1 The binding pose of compound 8e in the binding cavity
of 1HD2
of 3QS1
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Molecular docking
The results of the in vitro antioxidant studies presented in
Table 3 revealed that all the novel compounds exhibited antioxidant
properties, though less than vitamin C. 2,2‐Diphenyl‐1‐picrylhydrazyl
(DPPH) assay is used to assess antioxidant properties by a mechan-
ism in which the tested compounds inhibit lipid oxidation, so
scavenging of DPPH radical can determine the free‐radical scaven-
ging capacity. This method is adopted in this study due to relatively
short time required for the analysis. The DPPH‐free radical is very
stable and reacts with compounds that can donate hydrogen atoms.
The assay measures the reducing ability of antioxidants toward the
DPPH radical.
2.3
Table 6 shows the binding free energy of the synthesized compounds
docked into the binding sites of the receptors 3QSI and 1HD2. There
were significant binding affinities of the compounds with the receptors
when compared with the standard drugs. Compound 8e showed com-
parable in silico antimalarial activity (−14.61 kcal/mol) as to the stan-
dard drug (−10.74 kcal/mol) and compound 8i (‐10.10 kcal/mol) showed
comparable in silico antioxidant activity to vitamin C as reference drug
(‐13.04 kcal/mol). These findings have further necessitated in‐depth
studies of 8e–3QSI and 8i–1HD2 complexes, with
a view of
FIGURE 2 Two‐dimensional
representation of the binding interactions of
compound 8e with 3QS1

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FIGURE 4 Two‐dimensional
representation of the binding interactions of
compound 8i with 1HD2
understanding their binding interactions with the receptors. Figures 1–4
show the various molecular interactions of these compounds with drugs
receptors. Compound 8e makes multiple contacts of different nature
with the active site residues of 3QSI, as shown in Figures 1 and 2.
However, Figure 1 shows the binding pose of 8e in the binding cavity of
3QSI, and Figure 2 shows the details of the chemical interactions in-
volved. There was three H‐bond interactions between compound 8e
and amino acid residue of 3QS1. 2(O‐10) atoms of 8e interacted with
Ser77 (4.98 Å) and Val76 (3.82 Å), respectively, whereas O‐15 of com-
pound 8e interacted with Thr218 (3.80 Å). There was a significant
π–alkyl interaction between the π electrons of the chlorophenyl moiety
of compound 8e and Leu128 through an atomic interaction of 6.19 Å.
Other amino acid residues involved in the interaction are as follows:
Ile300, Tyr189, Asp32, and Leu291. The details are shown in Figure 2
TABLE 4 Chemical interactions of compound 8e with amino acid
residues of 3QS1
Ligand
O‐11
O‐15
O‐15
6 Ring
C‐22
C‐22
C‐23
C‐30
Receptor
Thr218
Val76
Interaction
H‐Bond
Distance (Å)
3.80
H‐Bond
3.82
Ser77
H‐Bond
4.98
TABLE 6 Binding free energy,
∆
G (kcal/mol)
Leu128
Leu291
Tyr189
Ile300
π–Alkyl
6.19
Antimalaria: 3QS1
Antioxidant: 1HD2
Compound
(ΔG, kcal/mol)
(ΔG, kcal/mol)
Alkyl bond
Alkyl bond
Alkyl bond
Alkyl Bond
6.04
8a
−13.79
−11.91
−12.00
−11.98
−14.61
−12.79
−11.46
−11.91
−12.00
−10.74
ND
−9.69
−8.87
−9.44
−8.83
−9.08
−9.65
−8.89
−9.03
−10.10
ND
6.62
8b
6.12
8c
Asp32
6.04
8d
8e
TABLE 5 Chemical interactions of compound 8i with amino acid
residues of 1HD2
8f
Ligand
N‐9
Receptor
Thr147
Pro45
Interaction
H‐Bond
Distance (Å)
3.68
8g
8h
6 Ring
C‐22
C‐22
C‐23
π–Alkyl
5.20
8i
Pro45
Alkyl bond
Alkyl bond
Alkyl bond
4.72
Chloroquine
Vitamin C
Native ligand
Cys47
Pro45
5.76
−13.04
−8.78
4.70
−11.24

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(Table 4). Compound 8i chemically interacted with IHD2, as shown in
Figure 4. One hydrogen bond was formed in this interaction; N‐9 and
Thr147 interacted through an atomic interaction of 3.68 Å. Other re-
levant amino acid residues involved are as follows: Pro45 and Cys47.
The details are shown in Figure 4 (Table 5).
Q‐TOFLC/MS ESI‐Technique. Melting points were determined in
open glass capillary tubes on a Stuart melting point apparatus and
were uncorrected. All experiments were carried out at Dr. B. Chi-
naraju Laboratory, Organic Synthesis and Processing Chemistry
Division, CSIR‐Indian Institute of Chemical Technology, Hyderabad,
India. All reactions were monitored by thin‐layer chromatography
(TLC) on precoated silica gel 60 F254 (mesh); spots were visualized
under UV light and in oven with ninhydrin. Merck neutral aluminum
oxide, activated (60–325 mesh), was used for chromatography.
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting Information.
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3
CONCLUSION
A novel series of Val–Val dipeptide–sulfonamide carboxamides has
been designed and synthesized, and their roles as antimalarial and
antioxidant agents were evaluated. Compound 8d (54% inhibition)
was observed to be the most potent compound in the series against
P. berghei 7 days postinfection, instead of compound 8e that had the
highest binding free energy. Most of the compounds were active
against P. berghei 7 days postinfection by reducing the parasitemia by
at least 40% with a dose of 50 mg/kg (Table 1). The control drug
exhibited activities in all the mice by inhibiting the parasitemia
growth by more than 40%. However, in consideration of the com-
pounds' ability to inhibit parasitemia, compared with the control
drug, compounds 8d, 8g, 8h, and 8i exhibited activities analogous to
artemisinin. Compounds' molecular docking showed significant che-
mical interactions of the compounds with different receptors, re-
sulting in a high binding affinity. From the hematological analysis,
although there is a decrease in the value of RBC, PCV, and HB, it is
observed that there are not many changes in the parameters that
were analyzed for the control and the test compounds. Among some
tested compounds, the increase in PCV is not accompanied by cor-
responding increases in RBC counts and HB concentration, compared
with pretreatment and control. These findings are in agreement with
the findings of Wasser[35] that the increase may be relative poly-
cytemia due to hemoconcentration and it is recommended that more
research should be carried out on these compounds, as they showed
great potential for antimalarial and antioxidant properties. In the
antioxidant activity study, compound 8d (IC50 = 0.7 mg/ml) was found
to be the most potent antioxidant agent.
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4.1.2
General procedure for the synthesis of
substituted benzenesulfonamoyl alkanamides[21]
Sodium carbonate (Na2CO3, 1.82 mmol) was added to a solution of
L‐valine (1.5 mmol) in water (15 ml) with continuous stirring until all
the solutes were dissolved. The solution was cooled to −5°C, and an
appropriate substituted benzenesulfonyl chloride (1a–c, 1.82 mmol)
was added in four portions in a period of 1 hr. The slurry was further
stirred at room temperature for 4 hr. The progress of the reaction
was monitored using TLC (MeOH/DCM, 1:9). Upon completion, the
mixture was acidified using 20% aqueous hydrochloric acid at pH 2.
The crystals were filtered via suction and washed with pH 2.2 buffer.
The pure products, 3a–c, were dried over self‐indicating fused cal-
cium chloride in desiccators.
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4.1.3
General procedure for the synthesis of
compounds 6a–c
In a solution of Boc‐valine (3.0 g, 13.82 mmol) in DCM (20 ml), TEA
(20.7 mmol), EDC·HCl (16.0 mmol), and HOBt (13.82 mmol) were
added at 0°C, and after stirring for 15 min, substituted aniline
(13.82 mmol) was added. The resulting mixture was allowed to warm
to room temperature and stirred for 19–24 hr, as monitored with
TLC. On the completion of the reaction, the mixture was diluted with
DCM and washed with water (2 × 50 ml). The organic layer was then
washed with 1 N HCl (50 ml), 5% NaHCO3 (50 ml), and brine solution
(50 ml), and dried over Na2SO4. The solvent was removed under
reduced pressure and the crude product was purified by column
chromatography (ethyl acetate/hexane = 5:95).
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4
EXPERIMENTAL
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4.1
Chemistry
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4.1.1
General
All chemicals and solvents used were of analytical grade, and they
were purchased from Aldrich (Sigma‐Aldrich) and AVRA Chemicals
Pvt. Ltd (Hyderabad, India) and used without purification. 1H‐NMR
and 13C‐NMR spectra (see the Supporting Information) were re-
corded on Advance 300‐, 400‐, and 500‐MHz spectrometers in di-
methyl sulfoxide (DMSO)‐d6 using tetramethylsilane as an internal
standard. FTIR spectra were recorded on a Thermo Nicolet Nexus
670 spectrometer. Mass spectra were obtained on an Agilent LCMS
instrument. HRMS were measured on Agilent Technologies 6510,
tert‐Butyl N‐{(1S)‐1‐[(4‐bromophenyl)carbamoyl]‐2‐methyl‐1‐
propyl}carbamate (6a)
Yield (3.0 g, 58.7%), white solid, m.p = 161–162°C. 1H‐NMR
(500 MHz, DMSO‐d6) δ 10.11 (s, 1H), 7.58 (d, J = 8.9 Hz, 2H), 7.49
(d, J = 8.9 Hz, 2H), 6.92 (d, J = 8.5 Hz, 1H), 3.90 (t, J = 8.0 Hz, 1H), 1.98
(dd, J = 13.7, 6.8 Hz, 1H), 1.39 (s, 9H), and 0.92–0.85 (m, 6H).
13C‐NMR (101 MHz, DMSO‐d6) δ 171.05, 155.67, 138.16, 131.60,
121.24, 114.96, 78.22, 60.70, 30.31, 28.22, 19.19, and 18.50; elec-
trospray ionization–mass spectrometry (ESI–MS): m/z 371 [M+H]+.

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tert‐Butyl N‐{(1S)‐1‐[(4‐chlorophenyl)carbamoyl]‐2‐methyl‐1‐
propyl}carbamate (6b)
4.1.5
General procedures for the synthesis of
Val–Val dipeptide–sulfonamide conjugates
Yield 70.0%, white solid, m.p = 151–152°C. 1H‐NMR (500 MHz,
DMSO‐d6) δ 10.12 (s, 1H), 7.66–7.60 (m, 2H), 7.41–7.30 (m, 2H), 6.91
(d, J = 8.5 Hz, 1H), 3.90 (t, J = 8.0 Hz, 1H), 1.98 (dq, J = 13.5, 6.7 Hz,
1H), 1.39 (s, 9H), and 0.93–0.85 (m, 6H). 13C‐NMR (126 MHz, DMSO‐
d6) δ 170.66, 155.35, 137.52, 128.38, 126.57, 120.49, 77.83, 60.39,
30.01, 27.94, 18.92, and 18.22; ESI–MS: m/z 327 [M+H]+.
In
a solution of substituted benzenesulfonamoyl alkanamides
(1.0 mmol) in DCM (10 ml), TEA (1.49 mmol), EDC·HCl (1.19 mmol),
and HOBt (1.0 mmol) were added at 0°C, and after stirring for
15 min, compounds 7a–c (1.0 mmol) were added. The resulting mix-
ture was allowed to warm to room temperature and stirred for
19–24 hr, as monitored with TLC. On the completion of the reaction,
the mixture was diluted with DCM and washed with water
(2 × 30 ml). The organic layer was then washed with 1 N HCl (30 ml),
5% NaHCO3 (30 ml), and brine solution (30 ml), and was dried over
Na2SO4. The solvent was removed under reduced pressure and the
crude product was purified by column chromatography (ethyl acet-
ate/hexane = 5:95).
tert‐Butyl N‐{(1S)‐1‐[(3‐fluorophenyl)carbamoyl]‐2‐methyl‐1‐
propyl}carbamate (6c)
Yield 67.76%, white solid, m.p = 168–169°C. 1H‐NMR (400 MHz,
DMSO‐d6) δ 10.20 (s, 1H), 7.72–7.53 (m, 1H), 7.41–7.28 (m, 2H),
6.98–6.83 (m, 2H), 3.92 (t, J = 8.0 Hz, 1H), 1.99 (dq, J = 13.5, 6.7 Hz,
1H), 1.39 (s, 9H), and 0.90 (d, J = 6.6 Hz, 6H). 13C‐NMR (101 MHz,
DMSO‐d6) δ 171.66, 163.80, 161.40, 156.11, 141.07, 130.81, 115.41,
110.31, 110.10, 106.55, 106.29, 78.58, 61.17, 30.73, 28.66, 19.64,
and 18.94, ESI–MS: m/z 311 [M+H]+.
(S)‐N‐(4‐Bromophenyl)‐3‐methyl‐2‐({(S)‐3‐methyl‐2‐[(phenyl)‐
sulfonamido]butanamido})butanamide (8a)
Yield (0.28 g, 71%), white solid, m.p = 190–192°C. FTIR (KBr, cm−1):
3,333, 3,276, 3,200 (3NH), 2,966, (C–H aliphatic), 1,685, 1,644,
(2C═O, amide), 1,540, 1,490, 1,452 (C═C‐aromatic), 1,392, 1,317,
1,241 (SO2), 1,161, and 1,089 (C–N). 1H‐NMR (400 MHz, DMSO‐d6) δ
10.19 (s, 1H, NH of amide), 8.07 (d, J = 7.0 Hz, 1H, SO2–NH),
7.87–7.69 (m, 3H, NH of amide + Ar‐H), 7.55 (dd, J = 12.2, 8.0 Hz, 3H,
Ar‐H), 7.48 (dd, J = 8.1, 5.8 Hz, 4H, Ar‐H), 4.03 (t, J = 7.7 Hz, 1H,
CH–C═O), 3.70 (dd, J = 9.2, 6.9 Hz, 1H, CH–C═O), 1.86 (ddd, J = 19.9,
13.4, 6.7 Hz, 2H, 2 × (CH–(CH3)2)), 0.81 (d, J = 6.7 Hz, 3H, CH3), and
0.75 (dd, J = 10.4, 4.0 Hz, 9H, (CH3)3). 13C‐NMR (101 MHz, DMSO‐d6)
δ 170.73, 170.46, (2C═O), 141.71, 138.67, 132.54, 132.00, 129.23,
126.96, 121.58, 115.33 (eight aromatic carbons), 61.74, 59.21, 31.63,
30.93, 19.53, 18.83, and 18.58 (seven aliphatic carbons). ESI–MS:
m/z, 510 [M+H]+ and 522 [M+Na]+. HRMS–ESI: calcd. for
C22H28N3BrO4S [M+H]+ 510.1062; Found 510.1068.
|
4.1.4
General procedure for the synthesis of
compounds 7a–c
Dichloromethane/trifluoroacetic acid (1:1%) was added to com-
pounds 6a–c and stirred at room temperature for 1 hr, as monitored
with TLC. On the completion of the reaction, the solvent was eva-
porated under reduced pressure. The solid TFA salts were pre-
cipitated by adding diethyl ether and dried.
(2S)‐2‐Amino‐N‐(4‐bromophenyl)‐3‐methyl‐butanamide (7a)
Yield 86.90%, white solid, m.p = 106–107°C. 1H‐NMR (400 MHz,
DMSO‐d6) δ 10.77 (s, 1H), 8.34 (br, 2H), 7.61 (d, J = 9.0 Hz, 2H), 7.55
(d, J = 9.0 Hz, 2H), 3.80 (d, J = 4.9 Hz, 1H), 2.17 (dt, J = 13.5, 6.8 Hz,
1H), and 0.99 (dd, J = 6.9, 3.8 Hz, 6H). 13C‐NMR (101 MHz, DMSO‐d6)
δ 167.58, 137.81, 131.92, 121.82, 116.34, 58.38, 30.08, 18.85, and
18.16; ESI m/z: [M+H]+ 271.
(S)‐N‐(4‐Chlorophenyl)‐3‐methyl‐2‐({(S)‐3‐methyl‐2‐[(phenyl)‐
sulfonamido]butanamido})butanamide (8b)
Yield (0.25 g, 66%), off‐white solid, m.p = 173–175°C. FTIR (KBr,
cm−1): 3,336, 3,275, 3,196 (3NH), 2,967 (C–H aliphatic), 1,684, 1,643,
(2C═O, amide), 1,543, 1,495, 1,455, (C═C‐aromatic), 1,393, 1,318,
1,239 (SO2), 1,161, and 1,092 (C–N). 1H‐NMR (500 MHz, DMSO‐d6) δ
10.30 (s, 1H, NH of amide), 8.16 (d, J = 7.6 Hz, 1H, SO2–NH), 7.83 (dd,
J = 13.1, 6.3 Hz, 1H, NH of amide), 7.79–7.75 (m, 2H, Ar‐H), 7.64 (d,
J = 8.5 Hz, 2H, Ar‐H), 7.58–7.52 (m, 1H, Ar‐H), 7.47 (t, J = 7.5 Hz, 2H,
Ar‐H), 7.36 (t, J = 10.7 Hz, 2H, Ar‐H), 4.04 (t, J = 7.8 Hz, 1H,
CH–C═O), 3.69 (dd, J = 9.2, 7.0 Hz, 1H, CH–C═O), 1.95–1.88 (m, 1H,
CH–(CH3)2), 1.84 (dt, J = 13.6, 6.8 Hz, 1H, CH–(CH3)2), 0.80 (d,
J = 6.7 Hz, 3H, CH3), and 0.75 (t, J = 7.6 Hz, 9H, (CH3)3). 13C‐NMR
(101 MHz, DMSO‐d6) δ 170.70, 170.45, (2C═O), 141.70, 138.34,
132.54, 129.23, 129.05, 126.95, 126.06, 121.19 (eight aromatic
carbons), 61.94, 59.29, 31.62, 30.94, 19.56, 19.52, 18.84, and 18.59
(eight aliphatic carbons). ESI–MS: m/z, 466.20 [M+H]+ and 488.20 [M
+Na]+. ESI–HRMS: calcd. for C22H28N3ClO4S [M+Na]+ 488.1387;
Found 488.1369.
(2S)‐2‐Amino‐N‐(4‐chlorophenyl)‐3‐methyl‐butanamide (7b)
Yield 92.0%, white solid, m.p = 149–150°C. 1H‐NMR (300 MHz,
DMSO‐d6) δ 10.81 (s, 1H), 8.36 (br 2H), 7.67 (d, J = 8.9 Hz, 2H), 7.43
(d, J = 8.8 Hz, 2H), 3.81 (br, 1H), 2.19 (dq, J = 13.4, 6.7 Hz, 1H,
CH–(CH3)2), and 0.99 (dd, J = 6.8, 2.7 Hz, 6H). 13C‐NMR (101 MHz,
DMSO‐d6) δ 167.01, 136.87, 128.81, 127.71, 121.07, 58.16, 39.42,
29.87, 18.30, and 17.61, ESI m/z: [M+H]+ 227.
(2S)‐2‐Amino‐N‐(3‐fluorophenyl)‐3‐methyl‐butanamide (7c)
Yield 87.90%, white solid, m.p = 162–163°C. 1H‐NMR (400 MHz,
DMSO‐d6) δ 10.86 (s, 1H), 8.34 (br, 2H), 7.61 (dt, J = 11.4 2.2 Hz, 1H),
7.39 (dt, J = 14.0, 8.2 Hz, 2H), 6.96 (t, J = 8.3 Hz, 1H), 3.81 (d,
J = 5.3 Hz, 1H), 2.19 (dq, J = 13.5, 6.8 Hz, 1H), and 1.04–0.95 (m, 6H).
13C‐NMR (101 MHz, DMSO‐d6) δ 167.79, 163.79, 161.38, 140.16,
140.06, 131.24, 131.15, 115.82, 111.28, 111.07, 106.99, 106.73,
58.76, 30.41, 18.86, and 18.11, ESI m/z: [M+H]+ 211.

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(S)‐N‐(3‐Fluorophenyl)‐3‐methyl‐2‐({(S)‐3‐methyl‐2‐[(phenyl)‐
sulfonamido]butanamido})butanamide (8c)
[M+Na]+. ESI–HRMS: calcd. for C22H27N3ClBrO4S [M+Na]+
566.0492; Found 566.0492.
Yield (0.45 g, 86.5%), white solid, m.p = 186–188°C. FTIR (KBr, cm−1):
3,346, 3,298, 3,222 (3NH), 2,967, 2,882 (C–H aliphatic), 1,683, 1,644,
(2C═O, amide), 1,548, 1,485, 1,444 (C═C‐aromatic), 1,380, 1,322,
1,215 (SO2), 1,163, 1,093 (C–N). 1H‐NMR (300 MHz, DMSO‐d6) δ
10.25 (s, 1H, NH of amide), 8.06 (d, J = 7.6 Hz, 1H, SO2–NH), 7.78 (d,
J = 6.0 Hz, 3H, NH of amide + Ar‐H), 7.53 (dd, J = 19.5, 8.8 Hz, 4H, Ar‐
H), 7.32 (d, J = 13.5 Hz, 2H, Ar‐H), 6.89 (d, J = 7.5 Hz, 1H, Ar‐H), 4.03
(t, J = 7.0 Hz, 1H, CH–C═O), 3.81–3.59 (m, 1H, CH–C═O), 1.85 (dd,
J = 13.2, 6.5 Hz, 2H, 2 × (CH–(CH3)2)), and 0.79 (d, J = 17.1 Hz, 12H,
(CH3)4). 13C‐NMR (101 MHz, DMSO‐d6) δ 170.79, 170.67, (2C═O),
163.77, 161.37, 141.73, 140.95 (d, Jc,F = 11.1 Hz), 132.54, 130.89 (d,
Jc,F = 9.4 Hz), 129.23, 126.96, 115.37, 110.36, 110.15, 106.48, and
106.22 (aromatic carbons), 61.63, 59.13, 31.64, 30.91, 19.51, 18.81,
18.56 (seven aliphatic carbons). ESI–MS: m/z, 450 [M+H]+ and 472
[M+Na]+. HRMS–ESI: calcd. for C22H28N3FO4S [M+Na]+ 472.1682;
Found 472.1685.
(S)‐N‐(3‐Fluorophenyl)‐3‐methyl‐2‐({(S)‐3‐methyl‐2‐[(4‐
bromophenyl)sulfonamido]butanamido})butanamide (8f)
Yield (0.37 g, 78.8%), white solid, m.p = 196–197°C. FTIR (KBr, cm−1):
3,353, 3,294, 3,219 (3NH), 2,968, 2,932, 2,877 (C–H aliphatic), 1,682,
1,643, (2C═O, amide), 1,546, 1,485, 1,447 (C═C‐aromatic), 1,382,
1,330, 1,275, 1,213 (SO2), 1,164, 1,083, 1,015 (C–N). 1H‐NMR
(300 MHz, DMSO‐d6) δ 10.25 (s, 1H, NH of amide), 8.06 (d, J = 8.1 Hz,
1H, SO2–NH), 7.95 (d, J = 9.5 Hz, 1H, NH of amide), 7.71–7.69 (m, 4H,
Ar‐H), 7.56 (d, J = 11.6 Hz, 1H, Ar‐H), 7.31 (dd, J = 14.0, 7.8 Hz, 2H,
Ar‐H), 6.87 (t, J = 7.9 Hz, 1H, Ar‐H), 4.02 (t, J = 7.6 Hz, 1H, CH–C═O),
3.70 (dd, J = 8.9, 7.2 Hz, 1H, CH–C═O), 1.84 (dt, J = 13.4, 6.7 Hz, 2H,
2(CH–(CH3)2)), 0.83 (d, J = 6.6 Hz, 3H, CH3), and 0.75 (dd, J = 13.2,
7.4 Hz, 9H, (CH3)3). 13C‐NMR (126 MHz, DMSO‐d6) δ 170.62, 170.58,
(2C═O), 163.54, 161.62, 140.95, 140.91, 140.87, 132.49, 132.31,
130.93, 130.85, 129.12, 128.21, 126.43, 115.39, 110.35, 110.19,
106.46, 106.25 (aromatic carbons), 61.51, 59.04, 31.72, 30.90, 19.51,
19.42, 18.64, and 18.58 (eight aliphatic carbons). ESI–MS: m/z, 529
[M+H]+ and 551 [M+Na]+. HRMS–ESI: calcd. for C22H27N3O4FSBr [M
+Na]+ 550.0787; Found 550.0792.
(S)‐N‐(4‐Bromophenyl)‐3‐methyl‐2‐({(S)‐3‐methyl‐2‐[(4‐
bromophenyl)sulfonamido]butanamido})butanamide (8d)
Yield (0.145 g, 41%), white solid, m.p = 128–130°C. FTIR (KBr, cm−1):
3,351, 3,291, 3,212 (3NH), 2,966, (C–H aliphatic), 1,685, 1,642,
(2C═O, amide), 1,539, 1,489, 1,461 (C═C‐aromatic), 1,395, 1,329,
1,243 (SO2), 1,168, 1,071, and 1,010 (C–N). 1H‐NMR (400 MHz,
DMSO‐d6) δ 10.15 (s, 1H, NH of amide), 8.04 (d, J = 8.3 Hz, 1H,
SO2–NH), 7.94 (d, J = 9.5 Hz, 1H, NH of amide), 7.70 (s, 4H, Ar‐H),
7.58–7.51 (m, 2H, Ar‐H), 7.50–7.42 (m, 2H, Ar‐H), 4.01 (t, J = 7.7 Hz,
1H, CH–C═O), 3.69 (dd, J = 9.4, 6.9 Hz, 1H, CH–C═O), 1.83 (dt,
J = 14.0, 6.9 Hz, 2H, 2 × (CH–(CH3)2)), 0.82 (d, J = 6.7 Hz, 3H, CH3),
and 0.79–0.68 (m, 9H, (CH3)3). 13C‐NMR (101 MHz, DMSO‐d6) δ
170.55, 170.40, (2C═O), 140.91, 138.57, 132.31, 132.04, 129.12,
126.43, 121.56, 115.38 (eight aromatic carbons), 61.52, 59.03, 31.72,
30.90, 19.51, 19.42, 18.67, and 18.59 (eight aliphatic carbons).
ESI–MS: m/z, 588 [M+H]+ and 610 [M+Na]+. HRMS–ESI: calcd. for
C22H27N3Br2O4S [M+H]+ 588.0167; Found 588.0175.
(S)‐N‐(4‐Bromophenyl)‐3‐methyl‐2‐({(S)‐3‐methyl‐2‐[(4‐
methylphenyl)sulfonamido]butanamido})butanamide (8g)
Yield (0.226 g, 58%), off‐white solid, m.p = 168–169°C. FTIR (KBr,
cm−1): 3,309, 3,264, 3,106 (3NH), 2,964, (C–H aliphatic), 1,646 (C═O,
amide), 1,531, 1,450 (C═C‐aromatic), 1,387, 1,332 (SO2), 1,162, 1,087,
and 1,013 (C–N). 1H‐NMR (400 MHz, DMSO‐d6) δ 10.17 (s, 2H, NH of
amide), 8.02 (d, J = 8.0 Hz, 1H, SO2–NH), 7.72–7.61 (m, 3H, NH of
amide + Ar‐H), 7.56 (d, J = 8.8 Hz, 2H, Ar‐H), 7.48 (d, J = 8.8 Hz, 2H,
Ar‐H), 7.26 (d, J = 8.1 Hz, 2H, Ar‐H), 4.04 (t, J = 7.7 Hz, 1H, CH–C═O),
3.64 (dd, J = 9.2, 6.8 Hz, 1H, CH–C═O), 2.29 (s, 3H, CH3‐Ar), 1.83 (tt,
J = 13.1, 6.6 Hz, 2H, 2 × (CH‐(CH3)2)), 0.81 (d, J = 6.7 Hz, 3H, CH3), and
0.74 (t, J = 6.5 Hz, 9H, (CH3)3). 13C‐NMR (101 MHz, DMSO‐d6) δ
170.73, 170.41, (2C═O), 142.67, 138.76, 138.63, 132.02, 129.66,
127.08, 121.55, 115.36 (eight aromatic carbons), 61.71, 59.07, 31.67,
30.99, 21.38, 19.55, 19.45, 18.70, and 18.58 (nine aliphatic carbons).
ESI–MS: m/z, 524 [M+H]+ and 546 [M+Na]+. ESI–HRMS: calcd. for
C23H30N3BrO4S [M+Na]+ 546.1038; Found 546.1042.
(S)‐N‐(4‐Chlorophenyl)‐3‐methyl‐2‐({(S)‐3‐methyl‐2‐[(4‐
bromophenyl)sulfonamido]butanamido})butanamide (8e)
Yield (0.035 g, 71%), off‐white solid, m.p = 157–158°C. FTIR (KBr,
cm−1): 3,356, 3,293, 3,210 (3NH), 2,966 (C–H aliphatic), 1,685,
1,641, (2C═O, amide), 1,540, 1,492, 1,457, (C═C‐aromatic),
1,394, 1,330, 1,245 (SO2), 1,168, and 1,091 (C–N). 1H‐NMR
(400 MHz, DMSO‐d6) δ 10.17 (s, 1H, NH of amide), 8.04 (t,
J = 11.0 Hz, 1H, SO2–NH), 7.96 (t, J = 11.8 Hz, 1H, NH of amide),
7.74–7.66 (m, 4H, Ar‐H), 7.60 (d, J = 8.9 Hz, 2H, Ar‐H), 7.41–7.29
(m, 2H, Ar‐H), 4.02 (t, J = 7.7 Hz, 1H, CH–C═O), 3.70 (t, J = 7.8 Hz,
1H, CH–C═O), 1.83 (dd, J = 14.2, 7.1 Hz, 2H, CH–(CH3)2), 0.83 (t,
J = 7.2 Hz, 3H, CH3), and 0.79–0.69 (m, 9H, (CH3)3). 13C‐NMR
(101 MHz, DMSO‐d6) δ 170.54, 170.37 (2C═O), 140.91, 138.17,
132.31, 129.12, 128.21, 127.36, 126.43, 121.17 (eight aromatic
carbons), 61.53, 59.02, 31.72, 30.92, 19.51, 19.43, 18.67, and
18.59 (eight aliphatic carbons). ESI–MS: m/z, 544 [M+H]+ and 566
(S)‐N‐(4‐Chlorophenyl)‐3‐methyl‐2‐({(S)‐3‐methyl‐2‐[(4‐
methylphenyl)sulfonamido]butanamido})butanamide (8h)
Yield (0.31 g, 58%), off‐white solid, m.p = 147–148°C. FTIR (KBr,
cm−1): 3,357, 3,294, 3,217 (3NH), 2,965 (C–H aliphatic), 1,687, 1,643,
(2C═O, amide), 1,540, 1,493, 1,454, 1,401 (C═C‐aromatic), 1,314,
1,245 (SO2), 1,164, and 1,092 (C–N). 1H‐NMR (400 MHz, DMSO‐d6) δ
10.17 (s, 1H, NH of amide), 8.02 (d, J = 8.3 Hz, 1H, SO2–NH),
7.71–7.57 (m, 5H, NH of amide + Ar‐H), 7.35 (d, J = 8.8 Hz, 2H, Ar‐H),
7.26 (d, J = 8.0 Hz, 2H, Ar‐H), 4.04 (t, J = 7.7 Hz, 1H, CH–C═O), 3.64
(dd, J = 9.2, 6.9 Hz, 1H, CH–C═O), 2.29 (s, 3H, CH3‐Ar), 1.91–1.75 (m,
2H, 2 × (CH–(CH3)2)), 0.81 (d, J = 6.7 Hz, 3H, CH3), and 0.74 (t,
J = 6.4 Hz, 9H, (CH3)3). 13C‐NMR (101 MHz, DMSO‐d6) δ 170.73,

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170.37 (2C═O, of amide), 142.67, 138.77, 138.20, 129.66, 129.13,
127.35, 127.08, 121.15 (eight aromatic carbons), 61.68, 59.01, 31.67,
31.00, 21.38, 19.55, 19.45, 18.70, and 18.58 (nine aliphatic carbons).
ESI–MS: m/z, 480 [M+H]+ and 502 [M+Na]+. HRMS–ESI: calcd. for
C23H30N3ClO4S [M+H]+ 480.1717; Found 480. 1724.
cervical dislocation and the blood samples were collected by heart
puncture. EDTA bottles were used to collect the blood samples for he-
matological parameters (RBC count, PCV, and HGB) on the compounds
that showed appreciable percentage inhibition. PCV was determined by
microhematocrit technique using a capillary tube, as described by Scha-
lam.[37] The RBC counts were determined as described by Brown.[38] The
HB concentrations were determined according to Hewitt.[39]
(S)‐N‐(3‐Fluorophenyl)‐3‐methyl‐2‐({(S)‐3‐methyl‐2‐[(4‐
methylphenyl)sulfonamido]butanamido})butanamide (8i)
Yield (0.405 g, 79.4%), white solid, m.p = 153–154°C. FTIR (KBr, cm−1):
3,349, 3,298, 3,229 (3NH), 2,966, (C–H aliphatic), 1,688, 1,644,
(2C═O, amide), 1,546, 1,490, 1,445 (C═C‐aromatic), 1,380, 1,318,
1,212 (SO2), 1,160, and 1,092 (C–N). 1H‐NMR (400 MHz, DMSO‐d6) δ
10.23 (s, 1H, NH of amide), 8.01 (d, J = 8.1 Hz, 1H, SO2–NH), 7.73–7.63
(m, 3H, NH of amide + Ar‐H), 7.57 (d, J = 11.6 Hz, 1H, Ar‐H), 7.37–7.25
(m, 4H, Ar‐H), 6.87 (t, J = 7.8 Hz, 1H, Ar‐H), 4.04 (t, J = 7.6 Hz, 1H,
CH–C═O), 3.65 (dd, J = 8.8, 7.1 Hz, 1H, CH–C═O), 2.29 (s, 3H, CH3‐
Ar), 1.91–1.75 (m, 2H, 2 × (CH–(CH3)2)), 0.81 (d, J = 6.7 Hz, 3H, CH3),
and 0.75 (t, J = 6.3 Hz, 9H, (CH3)3). 13C‐NMR (101 MHz, DMSO‐d6) δ
170.77, 170.62, (2C═O), 163.79, 161.39, 142.67, 141.00, 140.89,
138.78, 130.94, 130.84, 129.66, 127.09, 126.10, 115.37, 110.36,
110.15, 106.48, 106.21 (aromatic carbons), 61.68, 59.03, 31.68, 30.99,
21.36, 19.54, 19.43, 18.67, and 8.56 (nine aliphatic carbons). ESI–MS:
m/z, 464 [M+H]+ and 487 [M+Na]+. HRMS–ESI: calcd. for
C23H30N3FO4S [M+H]+ 464.2019; Found 464.2023.
|
4.2.3
Antioxidant activity by DPPH method
The new dipeptides were screened for free‐radical scavenging activity
by DPPH method.[40] The antioxidant behavior of the synthesized
compounds was measured in vitro by the inhibition of generated
stable DPPH‐free radical. The DPPH solution was prepared by dis-
solving 1.9 mg of DPPH in 100 ml of ethanol. Three different con-
centrations (500, 1,000, and 2,000 µg/ml) of the compounds solution
were prepared using DMSO. The standard solution of vitamin C was
prepared in a similar manner. Also, 1 ml of DPPH solution was added
to a 2‐ml solution of the compounds and vitamin C. The reaction
mixture was vortexed thoroughly and left in the dark at room tem-
perature for 30 min. The absorbance of the mixture was measured
spectrophotometrically at 517 nm against the corresponding blank
solution. The percentage scavenging DPPH radical inhibitions were
calculated by using the following formula:
Abscontrol − Abssample
DPPH radical scavenging activity (%) =
Abscontrol
|
4.2
Pharmacological/biological assays
(1)
× 100,
|
4.2.1
Experimental design and treatment of mice
where Abscontrol was the absorbance of DPPH radical and Abssample
was the absorbance of DPPH radical and sample/standard. The ex-
periment was performed in triplicate. The IC50 value was obtained by
plotting the graph of % inhibition against the concentration (mg/ml)
using the GraphPad Prism 8.3.0 software.
In this study, methods of Okokon and Nwafor[36] for antiplasmodial as-
say against P. berghei infection in mice were adopted. About 40 infected
mice were randomly divided into 10 groups, each having four mice. A
stock of parasitized erythrocytes was obtained from infected mice, with
a minimum peripheral parasitemia of 20% by cardiac puncture in ethy-
lenediaminetetraacetic acid (EDTA)‐coated tube. The percentage para-
sitemia was determined by counting the number of parasitized RBCs
against the total number of RBCs. The cell concentration of the stock
was determined and diluted with physiological saline, such that 0.2 ml of
the final inoculum contained 1 × 107 parasitized RBCs, which are the
standard inoculums for the infection of a single mouse. After 7 days of
infection, animals began to receive treatment (50 mg/kg) of the syn-
thesized compounds (8a–i), with constant check of the percentage of
parasitemia after a 4‐day interval. Artemisinin (50 mg/kg body weight)
was given to the other mice in group 10 as a positive control, and group
11 was not treated. All the compounds and the drugs were given orally
by using a standard intragastric tube.
|
4.3
Molecular docking
The physicochemical properties used for the evaluation of the drug
likeness of the synthesized compounds were calculated. The calculated
molecular descriptors include the following: molecular weight, partition
coefficient, hydrogen bond acceptor, hydrogen bond donor, topological
polar surface area, the number of rotatable bond, and molar re-
fractivity (Table 7). Two receptors were used for this study: one each
for antimalarial and antioxidant studies, respectively. The receptor for
antimalarial study includes Plasmepsin I (PDB ID: 3QS1) from Plas-
modium falciparum. The receptor for antioxidant study is (PDB ID:
1HD2). The cocrystallized inhibitors for each receptor are as follows:
3QS1: KNI‐10006, and 1HD2: human peroxiredoxin 5. The three‐
dimensional crystal structures of these receptors with their cocrys-
tallized ligands were obtained from the online protein data bank re-
pository (https://www.rcsb.org/). The chemical structures of the
synthesized compounds were drawn using ChemSketch. Further
|
4.2.2
Hematological analysis
The blood sample was taken at two occasions: before injecting the
compounds and on the last day of treatment; the animals were killed by

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TABLE 7 Physicochemical properties of the compounds
mol HBA HBD NoRB log P (o/w) MR TPSA
[4] S. Nishitani, C. Ijichi, K. Takehana, S. Fujitani, I. Sonaka, Biochem.
Biophys. Res. Commun. 2004, 313, 387.
Weight
[5] E. A. Mohamed, M. F. Eman, M. S. Nermien, E. A. Abd El‐Galil,
Z. Naturforsch. 2016, 71, 583.
8a
8b
8c
8d
8e
8f
4
4
4
4
4
4
4
4
4
3
3
3
3
3
3
3
3
3
11
11
11
11
11
11
11
11
11
4.57
4.37
3.97
5.37
5.17
4.76
4.87
4.67
4.26
12.72 104.37 510.45
12.47 104.37 466.00
12.04 104.37 449.55
13.45 104.37 589.35
13.24 104.37 544.90
12.80 104.37 528.44
13.17 104.37 524.48
12.92 104.37 480.03
12.49 104.37 463.57
[6] T. Owa, T. Nagasu, Expert Opin. Ther. Pat. 2000, 10, 1725.
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Abbreviations: HBA, hydrogen bond acceptor; HBD, hydrogen bond
donor; MR, molar refractivity; NoRB, number of rotatable bond; TPSA,
topological polar surface area.
preparations of the protein and the ligands were done using Discovery
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water of crystallization from the protein, and energy minimization of
the structures. The prepared ligands were docked into the binding
cavity of the receptors and their interactions were visualized using
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ACKNOWLEDGMENTS
The authors expressed their sincere gratitude to The World Academy
of Science (TWAS) and Council for Science and Industrial Research
(CSIR), New Delhi, for the joint sponsorship of the experimental work
at CSIR‐India Institute of Chemical Technology, Hyderabad, Telangana,
India, under the 2017 CSIR‐TWAS Postgraduate Fellowship Award,
Department of Veterinary Anatomy, Faculty of Veterinary Medicine,
University of Nigeria, Nsukka, for the in vivo antimalarial analysis, and
Department of Pharmaceutical and Medicinal Chemistry, University of
Nigeria, Nsukka, for in vitro antioxidant studies.
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CONFLICT OF INTERESTS
[30] B. A. Q. Gomes, Master's Thesis. Federal University of Para (Belem,
PA, Brazil) 2011.
The authors declare that there are no conflicts of interests.
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doi.org/10.1111/cbdd.12134
James A. Ezugwu
http://orcid.org/0000-0001-6607-6954
http://orcid.org/0000-0002-4116-7144
http://orcid.org/0000-0003-2773-8470
http://orcid.org/0000-0001-6905-809X
Uchechukwu C. Okoro
Mercy A. Ezeokonkwo
Chinaraju Bhimapaka
David I. Ugwu
http://orcid.org/0000-0002-2370-6191
http://orcid.org/0000-0003-4115-1019
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Haifa, Mt. Camel, Haifa, Isreal 2005, p. 603.
Daniel I. Ugwuja
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How to cite this article: Ezugwu JA, Okoro UC, Ezeokonkwo
MA, et al. Synthesis and biological evaluation of Val–Val
dipeptide–sulfonamide conjugates. Arch Pharm.
SUPPORTING INFORMATION
2020;e2000074. https://doi.org/10.1002/ardp.202000074
Additional supporting information may be found online in the
Supporting Information section.