New peptide derived antimalaria and antimicrobial agents bearing sulphonamide moiety

Article abstract of DOI:10.1080/14756366.2019.1651313

Fourteen novel dipeptide carboxamide derivatives bearing benzensulphonamoyl propanamide were synthesized and characterized using ¹H NMR, ¹³C NMR, FTIR and MS spectroscopic techniques. In vivo antimalarial and in vitro antimicrobial studies were carried out on these synthesized compounds. Molecular docking, haematological analysis, liver and kidney function tests were also evaluated to assess the effect of the compounds on the organs. At 200 mg/kg body weight, 7i inhibited the multiplication of the parasite by 81.38% on day 12 of post-treatment exposure. This was comparable to the 82.34% reduction with artemisinin. The minimum inhibitory concentration (MIC) in μM ranged from 0.03 to 2.34 with 7h having MIC of 0.03 μM against Plasmodium falciparium. The in vitro antibacterial activity of the compounds against some clinically isolated bacteria strains showed varied activities with some of the new compounds showing better activities against the bacteria and the fungi more than the reference drug ciprofloxacin and fluconazole.

Full text of DOI:10.1080/14756366.2019.1651313

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
New peptide derived antimalaria and
antimicrobial agents bearing sulphonamide
moiety
D. I. Ugwuja, U. C. Okoro, S. S. Soman, R. Soni, S. N. Okafor & D. I. Ugwu
To cite this article: D. I. Ugwuja, U. C. Okoro, S. S. Soman, R. Soni, S. N. Okafor & D. I.
Ugwu (2019) New peptide derived antimalaria and antimicrobial agents bearing sulphonamide
moiety, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 1388-1399, DOI:
10.1080/14756366.2019.1651313
To link to this article: https://doi.org/10.1080/14756366.2019.1651313
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 1388–1399
https://doi.org/10.1080/14756366.2019.1651313
RESEARCH PAPER
New peptide derived antimalaria and antimicrobial agents bearing
sulphonamide moiety
D. I. Ugwujaa, U. C. Okorob, S. S. Somanc, R. Sonid, S. N. Okaforc and D. I. Ugwub
b
aDepartment of Chemical Sciences, Federal University, Wukari, Nigeria; Department of Pure and Industrial Chemistry, University of Nigeria,
c
d
Nsukka, Nigeria; Department of Chemistry, The Maharaja Sayajirao University of Baroda, Vadodara, India; Department of Pharmaceutical and
Medicinal Chemistry, University of Nigeria, Nsukka, Nigeria
ABSTRACT
ARTICLE HISTORY
Received 14 May 2019
Revised 5 June 2019
Accepted 26 July 2019
Fourteen novel dipeptide carboxamide derivatives bearing benzensulphonamoyl propanamide were syn-
thesized and characterized using 1H NMR, 13C NMR, FTIR and MS spectroscopic techniques. In vivo anti-
malarial and in vitro antimicrobial studies were carried out on these synthesized compounds. Molecular
docking, haematological analysis, liver and kidney function tests were also evaluated to assess the effect
of the compounds on the organs. At 200mg/kg body weight, 7i inhibited the multiplication of the para-
site by 81.38% on day 12 of post-treatment exposure. This was comparable to the 82.34% reduction with
artemisinin. The minimum inhibitory concentration (MIC) in mM ranged from 0.03 to 2.34 with 7h having
MIC of 0.03 mM against Plasmodium falciparium. The in vitro antibacterial activity of the compounds against
some clinically isolated bacteria strains showed varied activities with some of the new compounds show-
ing better activities against the bacteria and the fungi more than the reference drug ciprofloxacin and
fluconazole.
KEYWORDS
Dipeptide carboxamide;
benzensulphonamoyl propa-
namide; synthesis;
antimalarial; antimicrobial;
molecular docking
1. Introduction
example, sulfadoxine, sulfadiazine and sulfalene are effective
malaria drugs that possess sulfonamide groups attached to a
heterocyclic ring.
Malaria stands among one of the terrible diseases that have been
known for more than 4000 years1. Malaria is an infectious disease
caused by protozoa of the genus Plasmodium and is transmitted
to humans by the Anopheles mosquito. Since this malady is
mainly present in tropical regions and primarily affects poor peo-
ple in developing countries, its occurrence is often associated with
socioeconomic problems2. At present, artemisinin and its combin-
ational therapy is the most appropriate drug therapy for malaria
in the malaria endemic region, but due to spread of artemisinin
resistance in five countries, Cambodia, the Lao People’s
Democratic Republic, Myanmar, Thailand and Viet Nam3, urges the
need for the development of new antimalaria. To this concern,
lots of antimalaria and other therapeutic approaches are in the
pipeline to impede the growth of this disease, including antima-
laria peptides4. Peptides are among the most versatile bioactive
molecules and play crucial roles in the human body and other
organism5. Because of their good solubility, permeability and bio-
availability, many peptide hormones and analogues of short pepti-
des exert their action by binding to membrane receptors6,7 or
possess signalling and regulatory functions. There are peptides
(cell-penetrating as well as non-penetrating) that can be conju-
gated to a cargo to deliver it to a specific organ,8 However, des-
pite the advantages of the antimalaria peptide of having high
target selectivity that reduces the usual side effects of small and
low molecular weight drug, peptides mostly offer various chal-
lenges before it can be used as a therapeutic drug.9
Many drugs possess the functionality of primary sulfonamides10
defined by (RSO2NH2) compounds. Secondary or tertiary sulfona-
mides (R-SO2NR1R2) are defined by the single or double N-alkyl or
N-aryl/heteroaryl substitution of the primary sulfonamide, respect-
ively. As there is a continuous need of molecule to be introduced
in the treatment of diseases, the use of substituted sulfonamide is
a valid approach to expand the number of new lead compound
to be further developed. Such results can be achieved as there is
easy accessibility of derivatives, low toxicity profile, and possibility
to manipulate chemical structures under mild conditions11.
Krungkrai and coworkers reported a library of aromatic/hetero-
aromatic sulfonamides with diverse scaffolds and assayed these
compounds for the inhibition of carbonic anhydrase from
Plasmodium falciparum, (pfCA)12,13. The literature describes several
other sulfonamides with antimalarial activity14–20. Some of these
studies involve the inhibition of folate metabolic enzymes that are
crucial for the growth of the malaria parasite. Some of these
enzymes are absent in humans and are therefore potential targets
for malarial chemotherapy. In general, the sulfonamide functional
group has an impressive effectiveness and assuredness history in
medicine. It usually presents interesting pharmacokinetic proper-
ties being well absorbed by several routes of administration, pre-
senting good penetration into tissues and fluids, and being easily
metabolized21,22. In particular, in the last decade, it has been
widely employed in the treatment of malaria23–25
The reported emergence of insecticide-resistant mosquitoes
.
Sulfonamides are widely used in medicinal chemistry because
of their low cost, low toxicity and excellent biological activity. For and artemisinin-resistant malaria parasite calls for concerted effort
CONTACT D. I. Ugwuja
danielizuchukwu77@gmail.com
Department of Chemical Sciences, Federal University, P.M.P. 1020 Wukari, Nigeria
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1389
in the development of new antimalarial drugs. Malaria is often 2.3. General procedure of preparing 7a–7n
times accompanied with microbial infections leading to complica-
A
mixture of 2-[phenylsulfonyl)amido]propanioc acid (3a–b,
tions; this necessitates the development of new antimalarial drugs
with additional antimicrobial activity. To respond to the problems
associated with current antimalarial drugs, a design of compounds
containing sulfonamide, carboxamide and peptide functionality is
ideal given their reported individual antimalarial properties. The
synergistic activity arising from the successful combinations of
these functionalities is exploited in this research. We report herein
the synthesis of short peptides containing sulfonamide and car-
boxamide functionalities with interesting antimalarial and anti-
microbial activity.
1.84 mmol), 1-ethyl-3–(3-dimethyl aminopropyl carbodiimide
hydrochloride (0.53 g, 2.76 Mmol) EDC, 1-hydroxybenzotriazole
(0.248, 1.84 Mmol) HOBT, Triethylamine (TEA), deprotected boc-gly-
cine amine (6a–g, 1.84 Mmol) in dichloromethane (50 ml) DCM
was stirred at room temperature for 16 h. The reaction was moni-
tored using TLC. On completion of the reaction, it was washed
with water (2 ꢂ 20ml), brine (1 ꢂ 10ml), dried over anhydrous
sodium sulfate and the solvent was evapourated under reduced
pressure to give the crude product (7a–n) which was then puri-
fied by column chromatography using silica gel.
2. Material and methods
2.3.1. Compounds 7a–7n
Reagent-grade chemicals and solvents were purchased from com- 2.3.1.1. N-(2-((3-chlorophenyl)amino)-2-oxoethyl)-2-(phenylsulfona-
mido)propanamide(7a). Yield 71% mp 172–174 ꢁC FTIR(KBrcmꢀ1),
mercial supplier and used after purification. Thin-layer chromatog-
raphy (TLC) was performed on silica-gel F254 plates (Merck).
Merck silica gel (60–120 mesh) was used for column chromato-
graphic purification. All reactions were carried out in a nitrogen
atmosphere. Melting points are uncorrected and were measured
3348.6(NH), 3003.9(C-H aro), 2985.4, 2934.6 (C-H), 1665.1,
1593.1(C ¼ O), 1526.7, 1478.7, 1446.4(C ¼ C), 1377.5 (SO2), 1168.4
(SO2NH), 758.0 (C-Cl) 1HNMR (DMSO-d6 400 MHz): 8.2(s, 1H),
8.1–8.1(d, 1H), 7.8–7.7(d, 2H), 7.6–7.5(m, 1H), 7.5–7.5(m, 3H),
in open capillary tubes, using a Rolex melting-point apparatus. IR 7.4–7.4(d, J ¼ 8 Hz), 7.3–7.3(m, 4H), 7.1–7.0(d, J ¼ 7.6 Hz, 1H),
3.8–3.7(m, 1H), 1.0–1.0(D, J ¼ 6.8 Hz, 3H) 13CNMR (DMSO-d6
spectra were recorded as KBr pellets on a Perkin-Elmer RX 1 spec-
trometer and the wave numbers are reported in cmꢀ1 1H NMR
.
100 MHz); 19.0, 42.9, 52.2 (3 aliphatic carbon), 117.8, 118.8, 123.4,
and 13C NMR spectral data were recorded on Advance Bruker 400
spectrometer (1H 400 MHz/13C 100 MHz) with DMSO-d6 as solvent
and tetramethylsilane (TMS) as internal standard and reported in d
(ppm). J values are in hertz (Hz). Mass spectra were determined
by High Resolution Mass Specter (HRMS).
127.0, 129.4, 131.0, 132.8, 133.5, 140.6, 141.3 (aromatic carbon),
168.2, 172.1 (carbonyl carbon). HRMS-ESI found value is (m/z):
395.02, Mþ, calculated C17H19ClN3O4S value is 395.07.
2.3.1.2.
N-(2-((4-chlorophenyl)amido)-2-oxoethyl)-2-oxoethyl)-2-
(phenylsulfonamido)propanamide(7b). Yield 80%, mp 140–142 ꢁC.
FTIR(KBrcmꢀ1), 3325.3, 3270.8(2NH), 3066.7(C-H Ar), 2986.8,
2933.2(C-H), 1646.5, 1646.5(C ¼ O), 1598.0, 1530.8, 1492.5, 1448.0
(C ¼ C), 1400.0, 1328 (2 SO2), 1166.9 (SO2NH), 785.8(C-Cl)
1HNMR(DMSO-d6 400 MHz): 1.0–1.0(d, J ¼ 7.2 Hz, 3H), 3.7–3.8(m,
1H), 7.3–7.3(m, 3H), 7.5–7.5(m, 6H), 7.6–7.6(d, J ¼ 2H), 7.6–7.6(d,
J ¼ 1H), 7.7–7.8(m, 1H), 8.0(s, 1H), 8.2–8.2(m, 1H), 10.0(s, 1H)
13CNMR(DMSO-d6 100 MHz): 19.0, 43.0, 52.3(aliphatic carbon),
121.0, 124.6, 125.9, 126.8, 127.0, 128.6, 129.4, 129.5, 132.2, 132.8,
138.0, 141.3, 166.4 (aromatic carbon), 167.9, 172.1 (carbonyl car-
bon). HRMS-ESI found value is (m/z): 395.02, Mþ, calculated
C17H19ClN3O4S value is 395.07.
2.1. General procedure for the synthesis of substituted benzene
sulphonamoyl Alkanamides (3a and 3b)26
Sodium carbonate (Na2CO3, 1.590 g, 15 mmol) was added to asolu-
tion of amino acids (2,12.5 mmol) in water (15 mL) with continu-
ous stirring until all the solutes had dissolved. The solution was
cooled to5 ꢁC and the appropriate benzenesulphonyl chloride
(1a–b, 15 mmol) was added in four portions over a period of 1 h.
The slurry was further stirred at room temperature for about 4 h.
The progress of the reaction was monitored using TLC (MeOH/
DCM,1:9). Upon completion, the mixture was acidified using 20%
aqueous hydrochloric acid to pH 2. The crystals were filtered via
suction and washed with pH 2.2 buffer. The pure products (3a–b)
were dried over self-indicating fused silica gel in a desiccator.
2.3.1.3.
N-(2-((3-fluorophenyl)amido)-2-oxoethyl)-2-oxoethyl)-2-
(phenylsulfonamido)propanamide(7c). Yield 66%, mp 125–127 ꢁC.
FTIR(KBrcmꢀ1),3332.7(NH), 3088.7, 2980.0(C-H Ar), 2939.0,
2857.0(C-H), 1669.7, 1607.9(C ¼ 0), 1529.0, 1491.5, 1445,3(C ¼ C),
1374.1, 1320.4(2SO2), 1166.9(SO2NH), 686.9(C-Fl) 1HNMR(DMSO-d6
400 MHz): 1.0–1.0(d, J ¼ 6.8 Hz. 3H), 3.7–3.8(m,1H), 6.6–6.8(m, 1H),
7.2–7.3(m, 2H), 7.5–7.6(m, 4H), 7.8–7.8(d, J ¼ 7.2 Hz, 2H), 8.0–8.0(d,
J ¼ 8.4 Hz, 3H), 8.5–8.5(d, J ¼ 7.6 Hz, 1H), 8.2–8.2(m, 1H), 10.0(s, 1H)
13CNMR(DMSO-d6 100 MHz): 19.0, 45.0, 52.3(aliphatic carbon),
106.4, 110.3, 115.2, 127.0, 129.4, 130.9, 132.8, 140.8, 141.3, 161.4,
163.7 (aromatic carbon), 168.1, 172.1 (carbonyl carbon). HRMS-ESI
found value is (m/z): 378.09, M-H, calculated C17H19FN3O4S value
is 379.10
2.2. General procedure of preparing compound 6a–6e27
A mixture of substituted analytical grade of Boc-glycine (4, 0.45 g,
1.84 mmol), 1-ethyl-3–(3-dimethyl aminopropyl carbodiimide
hydrochloride (0.53 g, 2.76Mmol) EDC, 1-hydroxybenzotriazole
(0.248, 1.84Mmol) HOBT, Triethylamine (TEA), amines (0.515 g
1.84Mmol) in dichloromethane (50 ml) DCM was stirred at room
temperature for sixteen (16) h. The reaction was monitored using
TLC. On completion of the reaction, it was washed with water
(2 ꢂ 20 ml), brine (1 ꢂ 10 ml), dried over anhydrous sodium sulfate
and the solvent was evaporated under reduced pressure to give
the crude product(5a–g) which was then purified by column chro-
matography using silica gel. The products (5a–g) were then
deprotected by addition of 10% TFA in DCM and allow to stir for
more than 1 h and been monitored with TLC. When the reaction
is completed, the solvent is evaporated, and the products (6a–g)
were used directly for the next stage of reaction.
2.3.1.4. N-(2-((3-florophenyl)amino)-2-oxoethyl)-2-(phenylsulfona-
mido)propanamide(7d). Yield 77% mp 168–170 ꢁC FTIR(KBrcmꢀ1),
3333.01(NH), 3087(C-H Ar), 2979.2, 2939.5(C-H), 1695.5, 1670.5
(C ¼ O), 1609.9, 1529.5, 1492.8, 1445.2(C ¼ C), 1375.4(SO2),
687.6(C-F) 1HNMR (DMSO-d6): 10.1(s, 1H), 8.2–8.2(triplet. 1H),
7.8–7.7(m, 2H), 7.6–7.5 (m, 3H), 7.5–7.5(m, 4H), 7.4–7.2(m, 8 H),

1390
D. I. UGWUJA ET AL.
6.8–6.8(ddd, 1H), 3.8–3.7(m,1H), 1.0(d, J ¼ 7.2 Hz 3H) 13CNMR 1449.3, 1430.3(C ¼ C), 1332.8, (SO2), 1164.(SO2NH), 711.7(C-Cl).
1HNMR(DMSO-d6, 400 MHz) 1.0(s, 3H), 3.7–3.8(d, J ¼ 8H, 2H), 7.3(s,
(DMSO-d6 1OOMHz):19.0, 42.9, 52.3 (aliphatic carbon), 106.1, 106.4,
110.1, 110.3, 115.2, 126.9, 129.5, 130.9, 132.9, 140.8, 140.9, 141.1,
161.3, 163.7, aromatic carbon, 168.16, 172.25 carbonyl carbon.
1H), 7.5–7.5(d, J ¼ 4 Hz 2H), 7.7(s, 1H), 8.1–8.2(M 2H, 10.01(S, 1H)
13CNMR(DMSO-d6, 100 MHz); 19.3, 42.9, 52.2,(aliphatic carbon)
HRMS-ESI found value is (m/z): 378.09, M-H, calculated
C17H19FN3O4S value is 379.10.
121.0, 127.3, 128.9, 129.1, 129.5, 137.6, 138, 140.3,(aromatic car-
bon) 167.8, 171.9(carbonyl carbon). HRMS-ESI found value is (m/z):
432.1, M þ Na, calculated C18H21ClN3O4S value is 409.1.
2.3.1.5. N-(2-oxo-2)-(p-tolylamino)ethyl)-2-(phenylsulfonamido)pro-
panamide(7e). Yield 73% mp 160–162 ꢁC. FTIR(KBrcmꢀ1),3354.5,
3290.0(NH), 3059.2(C-H aro), 2968.5, 2928.9(C-H), 1653.1,
1602.3(C ¼ O), 1534.3, 1447.1(C ¼ C), 1378.7(SO2), 171.1(SO2NH)
1HNMR (DMSO-d6 400 MHz): 9.7(s, 1H), 8.2–8.2(m, 1H), 7.8–7.7(m,
2H), 7.6–7.5(m, 3H), 7.5–7.5(m, 4H), 7.4–7.4(d, J ¼ 8.2 Hz, 1H), 2.2(m,
1H), 1.0–1.04(d, J ¼ 7.2 Hz, 3H) 13NMR (DMSO-d6 100 MHz): 19.0,
20.0, 42.9, 52.2 (aliphatic carbon), 119.4, 127.0, 129.5, 129.6, 132.6,
132.9, 136.7, 141.2,(aromatic carbon), 167.4, 172.0 (carbonyl car-
bon). HRMS-ESI found value is (m/z): 376.13, M þ H, calculated
value is 375.13.
2.3.1.9. N-(2-((3-fluorophenyl)amino)-2-oxoethyl)-2–(4-methylphe-
nylsulfonamido)propanamide(7j). Yield 75% mp 122–124 ꢁC
FTIR(KBrcmꢀ1): 3333.5(NH), 3089.8(C-H Ar), 2978.7, 2938.1(C-H),
1696.7, 1665.0(C ¼ O), 1601.9, 1535.6, 1494.3, 1445.0(C ¼ C),
1322(SO2),
1169.5(SO2NH).
684.9(C-Fl)
1HNMR(DMSO-d6,
400 MHz):1.0–1.1(d, J ¼ 32 Hz, 3H), 2.3–2.9(m, 1H), 3.77(s, 1H), 6.8(s,
3H), 7.1–7.4(m, 1H), 7.5–7.9(m, 2H), 8.2(s, 1H), 10.1(s, 1H)
13CNMR(DMSO-d6, 100 MHz):19.0, 21.3, 43.0, 52.3(aliphatic carbon),
106.1, 106.3, 110.1, 110.3, 115.2, 127.0, 129.9, 130.8, 130.9, 138.5,
140.9, 141.0, 143.1, 161.4, 163.8(aromatic carbon), 168.1, 172.2(car-
bonyl carbon) HRMS-ESI found value is (m/z): 393.11, Mþ, calcu-
lated C18H21FN3O4S value is 393.12.
2.3.1.6. N-(2-oxo-2-(phenylamino)ethyl)-2-(phenylsulfonamido)pro-
panamide(7f). Yield 68%, mp 140–142 ꢁC FTIR(KBrcmꢀ1),
3340.6(NH), 3084.7(C-H Ar), 2979.7,(C-H), 1693.3, 1664.8(C ¼ O),
1596.1, 1528.3, 1499.7, 1480.2(C ¼ C), 1375.8(S02), 1167.1(SO2NH)
1HNMR (DMSO-d6, 400 MHz):1.0–1.1(d, J ¼ 32 Hz, 3H), 2.4(s, 1H),
2.3.1.10. N-(2-((4-fluorophenyl)amino)-2-oxoethyl)-2–(4-methylphe-
nylsulfonamido)propanamide(7k). Yield 59% mp 176–178 ꢁC
3.0(s, 3H), 3.7–4.0(m, 2H), 7.0(s,1H), 7.2(s, 2H), 7.5–7.8(m, 3H), 8.1(s, FTIR(KBrcmꢀ1), 3331.0, 3284.9(NH), 3078.5(C-H Ar), 2985.2,
1H), 8.2(s, 2H), 9.8–9.8(m, 2H) 13CNMR (DMSO-d6, 100 MHz): 19.0,
2929.4(C-H), 1673.4, 1648.7(C ¼ O), 1599.6, 1551.9, 1528.4,
43.0, 52.3(aliphatic carbon), 119.4, 119.5, 123.7, 123.9, 126.8, 127.0,
1511.2(C ¼ O),
1376.9(SO2),
1163.7(SO2NH),
695.6(C-Cl)
1HNMR(DMSO-d6, 400 MHz): 1.0–1.0(d J ¼ 8 Hz 3H),1.1–1.2(m,1H),
1.4–1.4(d, J ¼ 4 Hz 2H) 1.5–1.5(d, J ¼ 4 Hz, 3H), 2.3–2.3(d, J ¼ 7.6 Hz
1H), 3.2(s, 1H), 3.4–3.7(m, 1H), 7.0–7.1(m, 2H), 2.2–7.3(m, 2H),
7.4–7.4(m,2H), 7.5–7.6(m, 1H), 7.9–8.02(d, J ¼ 8.8 Hz 2H), 8.2(s,1H)
13CNMR(DMSO-d6, 100 MHz):19.0, 21.4,42.9, 96.5(aliphatic carbon),
115.7,115.9, 121.1, 121.2, 127.0, 121.9, 135.6, 138.4, 142.9, 143.0,
157.2, 159.6(aromatic carbon)167.6, 172.1(carbonyl carbon). HRMS-
ESI found value is (m/z): 393.11, Mþ, calculated C18H21FN3O4S
value is 393.12.
129.2, 129.4, 132.8, 139.2, 141.2(aromatic amine) 167.7, 172.1(car-
bonyl carbon). HRMS-ESI found value is (m/z): 361.1, Mþ, calcu-
lated C17H19N3O4S value is 361.10.
2.3.1.6. N-(2-(naphthalene-2-ylamino)-2-oxoethyl)-2-(phenylsulfo-
namido)propanamide(7g).
Yield
70%,
mp
170–172 ꢁC
FTIR(KBrcmꢀ1), 3346.7, 3253.6(NH), 3066.9(C-H Ar), 2977.6,
2935.9(C-H), 1649.1, 1598.6(C ¼ O), 1529.8, 1502.3, 1461.7,
1447.5(C ¼ C), 1375.3(SO2), 1170.9(SO2NH). 1HNMR (DMSO-d6,
400 MHz: 1.0(d, J ¼ 5.2 Hz, 3H), 4.0–3.9(M, 1H), 4.0(br
s 2H),
7.6–7.4(M, 8 H), 7.8–7.7(m, 3H), 7.9–7.9(m, 1 H) , 8.1–8.0(m, 2H),
8.3(S, 1H). 13NMR(DMSO-d6 100 MHz): 19.0, 43.1, 52.3 (aliphatic car-
bon), 121.9, 123.1, 125.8, 126.0, 126.3, 126.5, 127.0, 128.1, 128.5,
129.3, 129.4, 129.6, 132.8, 133.5, 134.1, 141.3(aromatic carbon),
168.5, 172.2 carbonyl carbon. HRMS-ESI found value is (m/z):
412.1, M þ H, calculated C21H21N3O4S value is 411.1.
2.3.1.11.
2–(4-methylphenylsulfonamido)-N-(2-oxo-2-(p-tolylami-
no)ethyl)propanamide(7l). Yield 82% mp 120–122 ꢁC FTIR
(KBrcmꢀ1), 3337.1, 3291.8(NH), 3045(C-H Ar), 2982.6, 2919(C-H),
1707.0, 1669.0(C ¼ O), 1599.4, 1532.6, 1448.9, 1430.2(C ¼ C),
1388.2(SO2), 1164.3(SO2NH) 1HNMR(DMSO-d6, 400 MHz) 1.0-1.0(d,
J ¼ 4 Hz, 3H), 2.3(S, 3H), 3.7(S, 3H), 7.3–7.3(d, J ¼ 8 Hz, 1H),
7.5–7.6(d, J ¼ 8 Hz 2H), 7.6–7.6(d, J ¼ 8 Hz 3H), 8.1(s, 1H), 9.9(S, 1H)
13CNMR(DMSO-d6, 100 MHz); 19.0, 21.4, 43.0, 52.3(aliphatic carbon),
121.0, 127.0, 127.3, 129.1, 129.9, 138.1, 138.4(aromatic carbon),
167.9, 172.2(carbonyl carbon). HRMS-ESI found value is (m/z):
388.053, M-H, calculated C19H23N3O4S value is 389.14.
2.3.1.7. N-(2-((3-chlorophenyl)amino)-2-oxoethyl)-2–(4-methylphe-
nylsulfonamido)propanamide(7h). Yield 66% mp 120–122 ꢁC
FTIR(KBrcmꢀ1), 3361.4, 3324.9, (2NH), 3078.2, 2979.0(C-H Ar),
2939.3, 2857.8(C-H), 1857.0, 1667.3(2 C ¼ O), 1593.3, 1526.6,
1478.87, 1446.5(C ¼ C), 1376.9, (SO2) 1169.2(SO2NH), 1780.2(C-Cl)
1HNMR (DMSO-d6 400 MHz): 1.0–1.0(d, J ¼ 6.8 Hz, 3H), 2.3(s, 4H),
3.8(s, 3H), 7.0–7.1(d, J ¼ 6.8 Hz, 1H), 7.3–7.3(d, 7.2 Hz, 4H), 7.4–7.4(d,
J ¼ 7.6 Hz, 2H), 7.6–7.6(m, 3H), 7.7(s, 1H), 7.9–7.9(m, 1H), 8.2(s, 1H),
10.1(s, 1H). 13CNMR(DMSO-d6 100 MHz); 19.0, 21.4, 43.0, 52.3 (ali-
phatic carbon), 117.8, 118.9, 123.4, 127.0, 129.9, 130.9, 133.5,
138.4, 140.6, 143.0(aromatic carbon), 168.1, 172.2(carbonyl carbon).
HRMS-ESI found value is (m/z): 432.14, M þ Na, calculated
C18H21ClN3O4S value is 409.09.
2.3.1.12. 2–(4-methylphenylsulfonamido)-N-(2-oxo-2-(phenylamino)
ethyl)propanamide(7m). Yield 74% mp 164–166 ꢁC FTIR(KBrcmꢀ1),
3340.7(NH), 3081.5(C-H Ar), 2978.65(C-H), 1693.1, 1664.7,(C-H Ar),
1597.7, 1530.3, 1498.9, 1445.3(C ¼ C), 1324.2(SO2), 1167.1(SO2NH)
1HNMR(DMSO-d6 400 MHz): 1.0–1.0(d, J ¼ 8 Hz, 3H), 2.3(s, 3H),
3.3–3.4(d, 3H), 3.7–3.8(m, 3H), 7.0–7.1(m, 1H), 7.2–7.3(m, 4H),
7.5–7.5(d, J ¼ 8 Hz, 2H), 7.6–7.7(d, J ¼ 8 Hz, 2H), 7.9–7.9(d, J ¼ 7.2 Hz
1H), 9.8(s, 1H) 13CNMR(DMSO-d6 100 MHz): 19.0, 21.4, 43.05,
52.3(aliphatic carbon), 119.4, 123.7, 127.0, 129.2, 129.2, 129.9,
138.4, 139.2, 143.1(aromatic carbon), 167.7, 172.2(carbonyl carbon)
HRMS-ESI found value is (m/z): 376.04, M þ H, calculated
2.3.1.8. N-(2-((3-chlorophenyl)amino)-2-oxoethyl)-2–(4-methylphe-
nylsulfonamido)propanamide(7i). Yield 82%, mp 160–162 ꢁC
FTIR(KBrcmꢀ1), 3344.0, 3250.3(2NH), 3115.4, 3067.5(C-H Ar), 2985.1,
2920.8(C-H), 1675.1, 1648.0(2 C ¼ O), 1596.1, 1526.5, 1493.12, C18H21N3O4S value is 375.1.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1391
2.3.1.13. 2–(4-methylphenylsulfonamido)-N-(2-(naphthalene-2-yla- were added from 1000 (DMSO) stocks. After 48 h, the culture
mino)-2-oxoethyl)propanamide(7n). Yield 84%, mp 174–176 ꢁC
medium was removed and replaced with 1% formaldehyde in PBS
pH 7.4 for an additional 48 h at room temperature to fix cells.
FTIR(KBrcmꢀ1), 3333.3, 3288.0(NH), 3056.6(C-H Ar), 2981.3,
2969.1(C-H), 1743.5 1669.5(C ¼ O), 1598.3, 1534.5, 1461.2, 1448.2 Fixed parasites were transferred into 0.1% Triton-X-100 in PBS
(C ¼ C), 1371 (SO2), 1169.9(SO2NH) 1HNMR (DMSO-d6 400 MHz):
containing 1 nM YOYO-1 dye (Molecular Probes). Parasitemia was
determined from dot plots (forward scatter vs. fluorescence) ac-
quired on a FACS sort flow cytometer using Cell Quest software
(Beckton Dickinson). The MIC of compounds was the minimum
1.07–1.09(s, J ¼ 6.8 Hz), 2.34(s, 3H), 3.81–4.0(m, 3H), 7.3–7.3
(J ¼ 8 Hz, 2H), 7.4–7.5(m, 6H), 7.6—7.7(dd, 4H), 7.9–7.9(d, J ¼ 5.2 Hz,
3H), 8.0–8.0(d, J ¼ 7.6 Hz), 8.0–8.0(d, J ¼ 5.2 Hz, 1H), 8.2(s, 1H), 9.3(s,
1H) 13CNMR (DMSO-d6 100 MHz): 19.0, 21.4, 43.1, 52.4 (aromatic
concentration at which more than 99% of the parasites, relative to
carbon), 121.9, 123.1, 125.8, 126.0, 126.3, 127.1, 128.1, 128.5, 129.9,
133.5, 134.1, 138.4, 143.1 (aromatic carbon) 168.5, 172.3 (carbonyl
carbon). HRMS-ESI found value is (m/z): 426.0, M þ H, calculated
C21H21N3O4S value is 425.1.
the control, were inhibited from developing to schizonts (parasites
with six or more chromatin dots)29.
2.6. In vivo antimalaria test
Experimental Design and Treatment of Mice. Methods of Peter
et al.30 and Kalra et al.31 for antiplasmodial assay against
Plasmodium berghei infection in mice with some modifications
were employed. About eighty infected mice were randomly div-
ided into five groups, each having fourteen mice. The inoculum
was prepared from a donor mouse with rising parasitemia of
60.42%. After 7 days of infection, animals begin to receive treat-
ment (100 and 200 mg/kg b wt.) of the synthesized compounds
(7a–7n) for 12 days with constant check of the percentage of
parasitemia after a 4-day interval. Artemisinin (100 and 200 mg/kg
body weight.) was given to the other mice in group three as posi-
tive control, group four was not treated and group five was not
infected. All the compounds and the drugs were given orally by
using a standard intragastric tube. For all parasitemia determin-
ation, blood samples were collected from tail snip of each mouse
and thin smears prepared and stained with 10% Giemsa solution.
The uniform fields of each stained slide (for each mouse) were
examined under microscope with an oil immersion objective of
100X magnification power and average percent of parasitemia
was determined.
2.4. Antimicrobial activity
2.4.1. Sensitivity test
In-vitro antimicrobial activities of the synthesized compounds
were carried out against six bacteria and two fungi of tropical
interest. Agar cup diffusion techniques with modification as
described by Ugwuja et al.28 was used to determine the anti-
microbial activity of the compounds. Sensitivity test Muller nutri-
ent agar and plates were seeded with 0.1 ml of overnight culture
of micro-organism. The seeded plates were allowed to set after
which cups were made in each sector previously drawn from the
backside of the bottom plate using marker. Using the sterile pip-
ette, each cup was filled with six drops of their corresponding syn-
thesized compound (2 mg/mL). The solubility solvent was DMF. All
the plates were incubated at 37 ꢁC for 24 h and 48 h, respectively,
for fungi and bacteria. Zones of clearance round each cup means
inhibition and the diameter of such zones were measured. The
graph of Inhibition Zone Diameter (IZD) against the log of concen-
tration was plotted for each compound. The anti-log of the inter-
cept on x-axis gives the (MIC). The procedure was repeated for
fluconazole and ciprofloxacin (standard antifungal and antibacter-
ial agent), and (DMF) (solvent)
2.7. Haematological analysis
2.4.2. Minimum inhibitory concentration (MICs) tests
Twenty-four hours before the injecting the compounds, the blood
sample of five mice was taken and it was also taken after the last
day of treatment, the animals were sacrificed by cervical disloca-
tion and the blood samples were collected by heart puncture. The
blood samples for haematological parameters (red blood cell
(RBC) count, white blood cell (WBC) count, packed cell volume
(PCV), and hemoglobin (HGB)) were collected into EDTA bottles
and analyzed using an automated machine (Automated CBC
Analyzer: Sysmex KX-21).
The (MIC) was determined by further dilution of test sample found
to be sensitive against a particular organism. Serial dilutions of
the sulfonamides was made to obtain 2.0–0.125 mg/mL. After dilu-
tion, the test solutions were added into their corresponding cups
previously made in the molten agar starting from the lowest con-
centration (0.125–2 mg/mL). This was followed by the incubation
at the appropriate incubation temperature and time. The resultant
inhibition zones diameter (IZD) were measured and values was
subtracted from the diameter of the borer (8 mm) to give the IZD.
The MIC was also determined by plotting the graph of IZD2
against logarithm of concentration for each plate containing a
specific compound and micro-organism. The antilogarithm of the
intercept on the x-axis gives the MIC.
2.8. Liver function tests (LFTs)
The liver function tests carried out with the blood of the rats fed
with the sulfonamide derivatives were aspartate aminotransperase
(AST), alanine transaminase (ALT) and alkaline phosphatase (ALP).
Standard laboratory procedure according to Reitman and Franke32
was used for the determination of parameters.
2.5. In vitro antimalarial assay
The antimalarial activities of the new carboxamides were deter-
mined by their inhibition of parasite growth using chloroquine-
resistant strain of P. falciparum. Effects of inhibitors on parasite
development were determined as follows. Sorbitol synchronized,
0.1% parasitemia, ring stage P. falciparum strain W2 parasites were
cultured under the atmosphere of 3% O2, 6% CO2 and 91% N2 in
RPMI-1640 medium supplemented with 10% human serum in the
2.9. Renal or kidney function test
Kidney function tests carried out with the blood of the rats fed
with the sulfonamide derivatives were, creatinine and albunim.
The method reported by Kaplan and Tengv33 was used in the
presence of inhibitors for 48 h without media change. Inhibitors determination of creatinine.

1392
D. I. UGWUJA ET AL.
2.10. In silico studies
acid (alanine) in the presence of Na2CO3 as a base, the acid
formed after 4 h reaction and on acidification of the reacting mix-
ture. The diamide derivatives of glycine were synthesized by react-
ing commercially available boc-protected glycine with amines in
the presence of 1-ethyl-3–(3-dimethylaminopropyl) carbodiimide
hydrochloride (EDCI), 1-hydroybenzotriazole (HOBT), and triethyl-
amine (TEA) to yield various C-substituted amide derivatives of
glycine. (EDCI) alone could not sufficiently activate the carboxylic
acid group of glycine and thus addition of (HOBt) was necessary
for the coupling reaction. Compound 5 was converted into its salt
by addition of 10% trifluoroacetic acid(TFA) in DCM. The salt on
further reaction with acid in the presence of peptide coupling
agents EDCI, HOBt, TEA or DMAP, gave the desired diamide
derivative of the glycine (Scheme 1). The structure of compound
7a–7n was confirmed by characterization using (FTIR), (1HNMR),
(13NMR). The IR showed strong band at 3361 and 3324 showing
the presence of two NH (amide) bond of the derivatives, it also
shows strong bonds at 1673 and 1647 for amide carbonyls and a
band at 1355 cmꢀ1 for sulfonamide group which indicate success-
2.10.1. Physicochemical evaluation
The physicochemical properties used for the evaluation of the
drug-likeness of the synthesized compounds were calculated. The
molecular descriptors calculated include: molecular weight (MW),
partition coefficient (log P), hydrogen bond acceptor (HBA), hydro-
gen bond donor (HBD), topological polar surface area (TPSA) and
number of rotatable bond (NoRB), number of acid (nAc), molar
refractivity (MR) and number of atoms (nA). Four receptors were
used for this study: two each for antimalarial and antibacterial
studies, respectively. The receptors for antimalarial study include
Plasmepsin I (PDB ID: 3QS1) and Plasmepsin IV (PDB ID: 1SME) all
from P. falciparum. The receptors for antibacterial study include
penicillin-binding protein 1B (PDB ID: 2Y2G) and E. coli DNA
gyrase (PDB ID: 5MMN). The co-crystallized inhibitors for each
receptor are as follows: 3QS1 – KNI-10006; 1SME – pepstatin A;
2Y2G – alkyl boronate, and 5MMN – 1-ethyl-3-[8-methyly-5–(2-
methylpyridin-4-yl)-isoquinolin-3-yl]-urea. The 3D crystal structures
of these receptors with their co-crystallized ligands were from the
online protein data bank repository (https://www.rcsb.org/). The
chemical structures of the synthesized compounds were drawn
using ChemSketch. Further preparations of the protein and the
ligands were done using the discovery studio. These preparations
included deleting of multiple chains, water of crystallization from
the protein and energy minimization of the structures. The pre-
pared ligands were docked into the binding cavity of the recep-
tors and their interactions visualized Discovery Studio
Visualizer, v16.1.0.15350.
1
ful formation of the compounds. H NMR of 7a shows the methy-
lene group of glycine exhibiting multiplet at dd3.5–4.00 due to the
interaction CH2 and NH of the glycine. There is a doublet at
1.0–1.2 due to the CH AND CH3 interaction of the alanine amide.
The compound also exhibited multiplet from 7.1–7.9 representing
aromatic protons. 13C NMR shows strong peak at 168.16 and
172.25 showing the presence of two carbonyl carbons. The
appearance of peak at 19.02, 42.99, 53.30 shows the presence of
three aliphatic carbon. The peak at 117.82, 118.88, 123.48, 127.00,
129.49, 131.02, 132.89, 133.56, 140.67 and 141.30 shows aro-
matic carbon.
Over a century ago, Medicinal Chemists have used the physico-
chemical properties of compounds to predict or estimate pharma-
cokinetic properties34. Drug-likeness has also been used as a
3. Results and discussion
The first stage for the preparation of the desired diamide deriva-
tive of the glycine is the synthesis of phenylsulphonamido acids parameter to predict the balance among the molecular properties
by reacting substituted benzenesulphonyl chloride and an amino of compound that influences its pharmacodynamics and
a
Scheme 1. Synthesis of glycinediamide derivatives, reagents and conditions. (i) Na2CO3, DCM/H2O, HCl, 0 ꢁC, r.t, Ph 2, 4 h. (ii) EDCI, HOBt, TEA, DCM, amine,16h. (iii)
10% TFA in DCM. (V)EDCI, HOBt, TEA or DMAP, 16h.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1393
Table 1. Physicochemical properties of the synthesized compounds.
Mol HBA aAc aA HBD Nrb logP (o/w) MR TPSA MW LNV RNV
Table 2. Free binding energy (DG, kcal/mol) of compounds.
Malaria Receptors
Bacterial receptors
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
4
4
4
4
4
4
4
4
4
4
4
4
4
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
44
44
44
44
47
44
50
47
47
47
47
50
47
53
3
3
3
3
3
3
3
3
3
3
3
3
3
3
9
9
9
9
9
9
9
9
9
9
9
9
9
9
2.00
1.96
1.56
1.52
1.67
1.37
2.63
2.30
2.26
1.86
1.82
1.97
1.67
2.93
10.17 104.37 395.87
10.17 104.37 395.87
9.74 104.37 379.41
9.74 104.37 379.41
10.12 104.37 375.45
9.67 104.37 361.42
11.29 104.37 411.48
10.63 104.37 409.89
10.63 104.37 409.89
10.20 104.37 393.44
10.20 104.37 393.44
10.57 104.37 389.48
10.12 104.37 375.45
11.74 104.37 425.51
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
S/N
Compound
3QS1
1SME
2Y2G
5MMN
1
2
3
4
5
6
7
8
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
ꢀ11.96
ꢀ11.66
ꢀ12.09
ꢀ11.83
ꢀ12.16
ꢀ12.50
ꢀ12.91
ꢀ12.34
ꢀ11.60
ꢀ11.65
ꢀ12.01
ꢀ11.63
ꢀ12.22
ꢀ11.99
ꢀ13.45
ꢀ11.67
ꢀ12.61
ꢀ11.37
ꢀ11.75
ꢀ11.78
ꢀ12.51
ꢀ11.43
ꢀ12.55
ꢀ12.61
ꢀ11.24
ꢀ11.89
ꢀ11.72
ꢀ11.99
ꢀ12.24
ꢀ11.81
ꢀ8.82
ꢀ12.88
ꢀ13.43
ꢀ13.09
ꢀ13.34
ꢀ12.48
ꢀ12.98
ꢀ13.69
ꢀ12.59
ꢀ12.09
ꢀ12.41
ꢀ12.24
ꢀ11.91
ꢀ12.60
ꢀ13.30
ꢀ13.24
ꢀ12.32
ꢀ10.52
ꢀ10.58
ꢀ10.38
ꢀ10.98
ꢀ10.72
ꢀ10.62
ꢀ10.75
ꢀ9.95
9
ꢀ9.90
10
11
12
13
14
15
16
ꢀ10.98
ꢀ10.81
ꢀ10.62
ꢀ10.83
ꢀ11.11
ꢀ11.50
ꢀ9.92
MW: molecular weight; HBA: hydrogen bond acceptor; HBD: hydrogen bond
donor; TPSA: total polar surface area; nRB: number of rotatable bond; nAc: num-
ber of acid; MR: molar refractivity; nA: number of atoms; LNV: Lipinski’s number
of violations; RNV: Reckitt’s number of violation.
7n
Native ligand
Standard drug
ꢀ11.29
Standard drugs: Chloroquine for antimalarial and penicillin for antibacterial.
pharmacokinetic properties35. The absorption, distribution, metab-
olism, and excretion of drug can be optimized using the physico-
chemical parameters. The drug-likeness of our compounds were
evaluated using Lipinski’s rule of five (ro5) and Reckitt’s rule. For
bioavailability of orally administered drug, ro5 implies that the
drug must have molecular weight value of ꢃ500, hydrogen bond
donor ꢃ5, hydrogen bond acceptor ꢃ10, and partition coefficient
(Log P) value ꢃ5.” Reckitt, which is a modified Lipinski’s rule of 5
(ro5), stated that a likely drug molecule should have an octanol-
water partition coefficient (log P) between –0.4 and 5.6, molar
refractivity (AMR) between 40 and 130, number of atoms (nA)
between 20 and 70, hydrogen bond donor (HBD) ꢃ 5 taken as
equivalent to the number of –OH and –NH groups, hydrogen
bond acceptor (HBA) ꢃ10 taken as equivalent to the number of
oxygen and nitrogen atoms and molecular weight (MW) not more
than 500. A violation of more than one of these physicochemical
parameters disqualifies a compound from being a likely drug can-
didate. From Table 1, there was no violation of Lipinski’s ro5 and
not more than one violation of Reckitt rule. This, therefore all the
compounds reported have good balance between compound
solubility and its penetration of the lipid bilayers, and hence are
likely going to have good oral bioavailability. However, com-
pounds that will serve as substrate for biological transporters do
not obey this rule. They can have violations up to 436. This then
imply that violations of more than one rule do not totally rule out
a compound as a likely drug candidate. Log P shows the degree
of lipophilicity of drug molecules, which affects the solubility,
absorption, distribution, metabolism, and excretion properties. The
log P of compounds in Table 1 ranges from 1.37–2.93 which is
within the optimal limit. When a drug molecule is highly lipo-
philic, it will partition into the lipid interior of membranes and is
retained there. When log P is higher than the upper limit, the
drug molecule will have low solubility whereas in lower log P, the
drug has difficulty to penetrate the lipid membranes37. It has
been reported that the number of rotatable bond (NoRB) influen-
ces bioavailability in rats and a cut off of NoRB ꢃ10 is recom-
BBB likeness states that a CNS drug must have HBA value in the
range of 8–10, the MW must be in the range of 400–500 and the
number of acid must be zero. All the synthesized compounds
reported herein fulfilled the conditions and as such can cross the
blood brain barrier and might be useful as to treat brain
inflammations.
To gain further insight in the binding interactions of our com-
pounds and the drug receptors, we carried out molecular docking
studies. Table 2 shows the free binding energy, DG (kcal/mol) of
the compounds against each selected drug receptor. These DG
were compared to both the co-crystallized inhibitor and the
standard drug. All the compounds studied had a good binding
affinity with all the four drug receptors used in this study.
Compound 7g had a better binding affinity (ꢀ12.91 kcal/mol) with
plasmepsin I receptor from P. falciparum (3QS1) when compared
to the standard drug, chloroquine (ꢀ11.67 kcal/mol). Its binding
affinity was not significantly different from the co-crystallized
inhibitor. Similarly compounds 7a and 7h had a better binding
affinity with plasmepsin II receptor from P. falciparum (1SME)
when compared to both the co-crystallized inhibitor and the
standard drug. Compounds 7b and 7n showed good binding
affinities with penicillin-binding protein 1B (2Y2G) and E. coli DNA
gyrase (5MMN).
These findings have further necessitated in-depth studies of
7g-3QS1, 7a-1SME, 7g-2Y2G and 7n-5MMN complexes with a
view of understanding their binding interactions with the recep-
tors. Figures 1–6 show the various molecular interactions of these
compounds woth drug receptors.
Compound 7g makes multiple contacts of different natures
with the active site residues of 3QS1 as shown in Figures 1 and 2.
While Figure 1 specifically showed the binding pose of 7g in the
binding cavity of 3QS1, Figure 2 illustrated the details of the
chemical interactions involved. There were four different H-bond
interactions observed. The O-atom of 7g and interacted with SER
77 at an intermolecular distance of 4.56 Å. Another O-atom inter-
mended for good oral bioavailability38. All the compounds acted with VAL 76 (3.96 Å). N-13 interacted with O of GLY 34
reported here have NoRB ꢃ10. Total polar surface area (TPSA) has
(3.29 Å) while N-16 interacted with OD2 of ASP 215 (5.43 Å). There
was p-p T shaped interaction between the aromatic 6-membered
also been used as a surrogate property for cell permeability. A
molecule with TPSA ꢃ140 Å2 would be able to permeate the cell. ring of 7g and TYR 189. p-alkyl interaction occurred between the
Again, all the compounds had TPSA less than 140 and as such can same 6-membered ring and LEU 128 at an intermolecular distance
permeate the cell membranes. To examine whether or not a drug of 4.65. There were also four different p-donor H bonding involv-
molecule will be able to permeate through the blood brain barrier ing TYR 75, 2(ASP 32) and SER 219. There were other conserved
(BBB) for the treatment of central nervous system (CNS) related amino acid residues which were involved in hydrophobic interac-
diseases, certain parameters in the BBB likeness are considered. tions with 7g as shown in Figure 2. These numerous and

1394
D. I. UGWUJA ET AL.
significant interactions of compound 7g explain the observed two different H-acceptor interactions: 1. Between O-25 atom of 7a
high binding affinity with 3QS1.
and N SER 79 at an intermolecular distance of 3.20 Å; 2. Between
the same O-25 atom and OG SER 79 at a distance and energy of
2.96 Å and ꢀ2.0 kcal/mol, respectively. The chlorobenzene ring of
compound 7a was involved with two different p-H bonding inter-
actions with the amino acid residues of 1SME. Firstly, it interacted
with CA TYR 77 at an intermolecular distance of 4.69 Å, and sec-
ondly, it interacted with the OH TYR 192 at 3.42 Å.
Figure 4 shows the binding pose of compound 7g in the bind-
ing cavity of 2Y2G while Figure 5 represents the chemical interac-
tions involved in the 2Y2G-7g complex. The various chemical
interactions contribute to the observed strong binding affinity
between compound 7g and the penicillin-binding receptor. Four
different H-bonding interactions were observed. Firstly, the O-27
atom of the compound combined with THR 654 through an inter-
molecular distance of 4.32 Å. Secondly, O-28 atom combined with
THR 652 (4.67 Å). Thirdly, another O-atom interacted with THR 654
(4.33 Å), and lastly, the same O-atom interacted with SER 460
(4.07 Å). There was also found amide-p stacked interaction. MET
497 interacted with the p-electrons of the naphthalene ring. Other
forms of interactions between compound 7g and 2Y2G are shown
in Figures 4 and 5.
In Figure 3, different atoms of 7a interacted with the 1SME
receptor. N-10 and C-11 of 7a interacted, respectively, with the
OG1 THR 217 through H-donor interactions. The distance and
energy of interaction of N-10 and THR 217 are 2.94 Å and
ꢀ0.6 kcal/mol, respectively, while those of C-11 and THR 217 are
3.07 Å and ꢀ0.2 kcal/mol, respectively. Also, Cl-23 of 7a, through
H-donor interaction, had a contact with O LEU 131. There were
Figure 6 shows the 2D representation of the chemical interac-
tions of 7n with the amino acid residues of 5MMN. A lot of signifi-
cant interactions were involved. There were two different H-donor
interactions observed. C-14 atom of 7n interacted with OE1 GLU
50 (distance of interaction ¼ 3.50 Å). Also, C-19 of the compound
interacted with OD2 ASP 73 (distance of interaction ¼ 3.37 Å). O-9
atom of 7n interacted through H-acceptor interaction with NH1
ARG 76. Three other H-acceptor interactions occurred between
O-9, O-27 and O-28, and NH2 ARG 76, CD PRO 79 and NH1 ARG
136, respectively. Finally, there was an H-bonding interaction
Figure 1. A stereoview showing the active site of the 7g-2Y2G complex. 7g is
shown in ball-and-stick model (orange). The amino acids are shown as lines
(black). Hydrogen and p-H bonds between 7g and the protein are shown as
green and purple dotted lines, respectively.
Figure 2. Representation of the chemical interactions of 7g with the amino acid residues of 3QS1.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1395
Figure 3. 2D Representation of the chemical interactions of 7a with the amino acid residues of 1SME.
some of the test compound have better activity than ciprofloxa-
cin. In fungi, the test compounds showed great activities against
Aspergilus as well as against Candida. Some of the compounds
were more active than fluconazole(standard drug used) as shown
in Table 3.
3.2. In vitro antimalarial
To determine the in vitro antimalarial activity of the primary sulfo-
namides, minimum inhibition assay was employed using a chloro-
quine sensitive P. falciparum malaria parasite line (Table 4)
Chloroquine and artemesinin were used as standard antimalarial
drugs to compare those of the test compounds. Compounds 7d,
7e and 7h (MIC 0.03, 0.03 and 0.05 mM) displayed activity better
than chloroquine (MIC 0.08 mM), whereas about five of the total
newly synthesized sulfonamides inhibited the activity of P. falcip-
arum parasite at lower MIC range than artemisinin On the overall,
compound 7h emerged as having the best activity among the
newly synthesized compound and relative to both considered
standard antimalarial drugs.
Figure 4. A stereoview showing the active site of the 7g-2Y2G complex. 7g is
shown in ball-and-stick model (orange). The amino acids are shown as lines
(black). Hydrogen and p-H bonds between 7g and the protein are shown as
green and purple dotted lines, respectively.
3.3. In vivo antimalarial
To determine the in vivo activity, the compounds were tested
against P. berghei NK(65 Strain) infection mice., the animals were
obtained from Nigeria institute for Trypanosoma and onchocercar-
ioses Research (NTIR) Vom, Plateau State Nigeria. The permission
and approval for the use of animals in this experiment were
granted by the Animal Ethics Committee, Federal College of
Vetrenary Medical Laboratory, Vom, Plateau State. Method of
Peter et al.30, for antiplasmodial assay against P. berghei in mice
with some modification were employed. Artemisimin was used as
treated control of the experiments. The percentage inhibition of
between the 6-membered aromatic ring of 7n and the CG2 THR
165 (4.49 Å).
3.1. Antimicrobial activities
The antibacterial activities of the test compounds are shown in
Table 3. The compound showed great antibacterial activities
against all the micro-organism. Some of the test compound
showed better activities against the bacteria than the control
drug(ciprofloxacin). Compound 7b, 7f, 7g and 7m showed greater parasite multiplication was calculated comparing the treated
activity against Bacillus than ciprofloxacin. In all the bacteria used, group with untreated group by means of the following formula39

1396
D. I. UGWUJA ET AL.
Figure 5. Representation of the chemical interactions of 7g with the amino acid residues of 2Y2G.
Figure 6. 2D Representation of the chemical interactions of 7n with the amino acid residues of 5MMN.
[(A ꢀ B)/A] ꢂ 100; where A ¼ parasitaemia in the untreated group partially active and inactive, respectively40. Most of the com-
and B ¼ parasitemia in the test group. Compounds that reduced pounds were active against P. berghei after 7 days post-infection
parasitemia by 40% were considered active, whereas those that by reducing the parasiteamia by at least 40% when in a dose of
reduce parasitaemia by 30–40% or less than 30% were deemed 100 mg/kg (Table 5). The inhibition of parasitaemia increased on

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1397
Table 3. MIC (minimum inhibitory concentration) mg/ml.
Comps
B. subtilis
E. coli
S. typhi
P. aeruginosa
K. pneumoniae
S. aureus
C. albicans
A. niger
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
CIP
FLU
0.219
0.129
3.467
0.194
–
0.155
0.141
0.214
0.214
0.245
0.371
0.245
0.129
–
0.489
0.467
0.372
0.521
6.327
0.131
–
0.239
0.239
0.275
0.316
0.169
0.123
0.123
0.131
–
0.199
0.602
2.691
0.476
0.194
4.677
0.079
1.288
1.288
0.129
3.161
0.057
0.057
0.617
0.128
–
0.398
0.257
0.457
0.295
3.802
0.354
–
0.616
0.616
0.380
3.162
0.169
0.144
0.129
0.232
–
0.251
1.69
0.129
0.177
–
5.011
0.166
5.248
5.248
0.083
0.079
0.107
0.138
–
4.677
0.288
–
0.489
0.389
0.190
–
0.234
0.234
–
0.387
0.115
0.229
–
0.0561
0.135
3.467
0.0343
0.0288
0.214
0.0297
0.034
0.034
0.371
0.0631
0.174
7.94
1.585
0.1000
–
1.995
2.089
0.1047
0.158
0.0316
0.0316
–
0.316
0.123
0.056
0.039
–
0.155
–
0.0217
0.184
–
0.113
–
0.252
–
0.0471
– : No activity; CIP means Ciprofloxacin; FLU means Fluconazole.
Table 4. In vitro antimalarial study.
Table 6. Liver function test.
Compounds
MIC (mM)
ALT (m/L)
AST (m/L)
T.P. (g/dl) Bilirubin (mg/dl) Glucose (g/l)
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
0.37
0.87
0.72
0.08
0.05
2.32
3.23
0.03
0.77
0.88
0.53
0.93
1.87
2.34
0.08
0.72
Comp Before After Before After Before After Before After Before After
7e
7d
7j
7h
24.0 22.0 64.0 60.5 5.8
24.2 22.5 64.0 68.5 5.7
26.3 25.5 65.0 60.0 6.0
25.4 24.5 65.0 69.5 6.0
20.5 23.5 62.0 61.5 5.8
4.5
4.0
5.8
5.5
5.5
0.3
0.3
0.4
0.4
0.3
0.2
0.2
0.2
0.3
0.5
5.8
5.8
5.9
5.9
5.6
5.0
5.5
5.7
5.5
5.0
7c
Control
24.2
65.5
5.9
0.3
6.3
Table 7. Kidney function test.
Creatinine (mg/dl)
Albumin (g/dl)
7m
7n
Chloroquine
Artemisimin
Compound
Before
After
Before
After
7e
7d
7j
7h
7c
Control
0.3
0.4
0.4
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
4.1
4.0
3.9
3.9
3.5
3.9
3.5
3.5
4.0
3.9
3.0
Table 5. Percentage inhibition of parasite in mice.
% inhibition
7 days
% inhibition
7 days
% inhibition
12 days
% inhibition
12 days
post-treatment
(100 mg/kg)
post-treatment
(200 mg/kg)
post-treatment
(100 mg/kg)
post-treatment
(200 mg/kg)
Comp
3.4. Liver function tests evaluation
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
Arte
NTC
NIC
36.23
51.33
48.32
53.62
62.34
38.23
27.54
61.36
51.73
77.93
40.35
38.23
33.46
47.8
52.11
57.62
40.22
48.73
40.22
70.97
54.69
49.85
66.57
77.36
54.92
60.72
71.36
63.8
49.97
55.23
51.33
81.72
66.71
41.87
36.83
57.77
46.37
82.68
44.28
41.73
29.33
49.11
86.22
–
61.37
53.4
Liver function tests are a group of blood tests that detect inflam-
mation and damage to the liver. They can check how well the
liver is working. The liver function tests studied in this research
are AST, ALT and ALP. It is observed from Table 6 that the admin-
istration of 10 mg/kg of the sulfonamide to the tested mice did
not show significant increment or decrease in the serum levels of
AST, ALT and T.P when compared with the control.
48.71
55.98
50.71
73.29
51.93
55.22
81.38
73.55
56.43
74.62
73.98
71.66
82.34
–
3.5. Kidney function test evaluation
Kidney function tests are common lab tests used to evaluate how
well the kidneys are working. In this research work, the kidney
function tests used are albumine, creatinine. In the mice fed with
10 mg/kg of sulfonamide derivatives, there were no significant
changes for the serum levels of creatinine and albumin when
compared with the control (Table 7).
75.67
–
–
78.87
–
–
–
–
Arte: Artemisinin; NIT: Non treated control; NIC: Non infected control.
12th day post infection for almost all the compounds. The control
drug showed activities on all the mice by inhibiting the parasit-
aemia growth more than 40%. However in consideration of the
compounds ability to inhibit parasitaemia to the control drug.
Compound 7d, 7e, 7j, 7h and 7e have almost the same or better
activity compared to the artemisinin. These compounds should be
considered for further studies because of its promising activities.
3.6. Haematological analysis
The blood samples for haematological parameters (red blood cell
(RBC) count, white blood cell (WBC) count, packed cell volume
(PCV), and haemoglobin (HGB)) were collected into EDTA bottles
and analyzed using an automated machine (Automated CBC

1398
D. I. UGWUJA ET AL.
Table 8. Heamotological analysis before and after treatment.
RBC (mm3) WBC (mm3)
PCV (%)
Comp Before After Before After Before After
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5. Day T, Greenfield SA. Bioactivity of a peptide derived from
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7e
7d
7j
6.85 ꢂ 106 6.0 ꢂ 106 36
6.82 ꢂ 106 6.8 ꢂ 106 37
7.1 ꢂ 106 6.5 ꢂ 106 39
7.3 ꢂ 106 5.9 ꢂ 106 39
7.5 ꢂ 106 6.0 ꢂ 106 38
35 12.2 ꢂ 103 10.5 ꢂ 103 14.1 11.0
34 13.5 ꢂ 103 9.5 ꢂ 103 13.5 10.5
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34
34
8.0 ꢂ 103 9.1 ꢂ 103 13.5 10.0
9.4 ꢂ 103 9.3 ꢂ 103 13.4 10.0
7h
7c
33 10.3 ꢂ 103 9.0 ꢂ 103 14.5
9.8
Analyzer: Sysmex KX-21) (Table 8). From the analysis, even though
there is a decrease in the value of RBC, PCV and HB, it is observed
that there is no significant changes in the parameters that were
analyzed for the control and the test compounds.
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4. Conclusion
Fourteen new ala-gly dipeptide sulfonamide carboxamides deriva-
tives were synthesis, characterization. The spectral data confirmed
the successful preparation of these derivatives. The in vitro anti-
bacterial activity of the compounds against some clinically isolated
bacteria strains showed varied activities with some of the new
compounds showing better activities against the bacteria and the
fungi more than the reference drug ciprofloxacin and fluconazole.
Compound 7b, 7f, 7g and 7m showed greater activity against
Bacillus than ciprofloxacin. In all the bacteria used, some of the
test compound has better activity than ciprofloxacin. In fungi, the
test compounds showed great activities against Aspergilus as well
as against Candida. The compounds exhibited antimalarial prop-
erty in vitro. Compounds 7d, 7e and 7h (MIC 0.03, 0.03 and
0.05 mM) displayed activity better than chloroquine (MIC
0.08 mM). 7h possessed the least MIC of 0.03 mM against
Plasmodium falciparium. The compounds also exhibited antima-
laria activity in vivo against P. berghei in mice. Most of the com-
pounds were active against P. berghei after 7 days post-infection
by reducing the parasiteamia by at least 40% when in a dose of
100 mg/kg (Table 5). The inhibition of parasitaemia increased on
12th day post infection for almost all the compounds. The control
drug showed activities on all the mice by inhibiting the parasit-
aemia growth more than 40%. However, in consideration of the
compounds ability to inhibit parasitaemia to the control drug,
compounds 7d, 7e, 7j, 7h and 7e have almost the same or better
activity compared to the artemisinin. Compound Molecular dock-
ing revealed significant chemical interactions of the compounds
with different receptors resulting in high binding affinity. From
the haematological analysis, even though there is a decrease in
the value of (RBC), (PCV) and (HB), it is observed that there are no
significant changes in the parameters that were analyzed for the
control and the test compounds. The studies on the liver and kid-
ney showed no significant damage to the organs by the new
compounds and it is recommended that more research should be
carried out on these compounds as it showed great potential for
antimicrobial and antimalarial properties.
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