Synthesis of proline derived benzenesulfonamides: A potent anti-Trypanosoma brucei gambiense agent

Article abstract of DOI:10.1016/j.ejmech.2018.05.017

Thousands of death in Africa and other developing nations are still attributed to trypanosomiasis. Excessive sleep has been associated with increased inflammation. We report herein, the synthesis, antitrypanosomal and anti-inflammatory activities of eight

Full text of DOI:10.1016/j.ejmech.2018.05.017

Accepted Manuscript
Synthesis of proline derived benzenesulfonamides: A potent anti-Trypanosoma brucei
gambiense agent
David I. Ugwu, Uchechukwu C. Okoro, Narendra K. Mishra
PII:
S0223-5234(18)30421-5
10.1016/j.ejmech.2018.05.017
EJMECH 10428
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 February 2018
Revised Date: 26 April 2018
Accepted Date: 12 May 2018
Please cite this article as: D.I. Ugwu, U.C. Okoro, N.K. Mishra, Synthesis of proline derived
benzenesulfonamides: A potent anti-Trypanosoma brucei gambiense agent, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.05.017.
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Graphical Abstract
ACCEPTED MANUSCRIPT
O
O
S
84% inhibition
1
N
R
R
O
OH
Novel compounds
IC50 2 nM
Acute inflammatory
African trypanosomiasis cells

ACCEPTED MANUSCRIPT
Synthesis of Proline Derived Benzenesulfonamides: a Potent anti-Trypanosoma
brucei gambiense agent
David I. Ugwu a,b*, Uchechukwu C. Okoro a and Narendra K. Mishra b
aDepartment of Pure and Industrial Chemistry, University of Nigeria, Nsukka.
bDepartment of Chemistry, Indian Institute of Technology, Kanpur, India
*email: izuchukwu.ugwu@unn.edu.ng

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Abstract
Thousands of death in Africa and other developing nations are still attributed to
trypanosomiasis. Excessive sleep has been associated with increased inflammation. We report
herein, the synthesis, antitrypanosomal and anti-inflammatory activities of eight new carboxamide
derivatives bearing substituted benzenesulfonamides. The base promoted reactions of L-proline and
L-4-hydroxyproline with substituted benzenesulfonyl chlorides gave the benzenesulfonamides (11a-
h) in excellent yields. Boric acid mediated amidation of the benzenesulfonamides (11a-h) and p-
aminobenzoic acid (12) gave the new carboxamides (13a-h) in excellent yields. The new
carboxamides were tested for their antitrypanosomal and anti-inflammatory activities against
Trypanosome brucei gambiense and inhibition of carrageenan-induced rat paw edema. Compound
13f was the most potent antitrypanosomal agent with an IC50 value of 2 nM as against 5 nM for
melarsoprol; whereas compound 13a was the most potent anti-inflammatory agent with percentage
inhibition of carrageenan-induced rat paw edema of 58, 60, 67 and 84% after 0.5 h, 1 h, 2 h and 3 h
administration respectively. The structure-activity relationship study revealed that substitution at the
para position in the benzenesulfonamide ring increased both the antitrypanosomal and anti-
inflammatory activities. The 4-hydroxyprolines (13a-d) showed higher anti-inflammatory activity
than the prolines (13e-h). In contrast, the prolines (13e-h) had higher antitrypanosomal activities
than the 4-hydroxyprolines. The link between excessive sleep and inflammation makes the report of
this class of compounds possessing both antitrypanosomal and anti-inflammatory activity
worthwhile. The pharmacokinetic studies showed that the compounds would not pose oral
bioavailability, transport and permeability problems.
Keywords: African trypanosomiasis; anti-inflammatory; carboxamides; catalysis; Trypanosome
brucei; sulfonamides.

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1. Introduction
African trypanosomiasis also known as sleeping sickness is an insect-borne parasitic disease
of humans caused by the protozoa specie Trypanosoma brucei. Of the two species of Trypanosoma
brucei, Trypanosoma brucei gambiense causes over 98% of reported cases [1]. Trypanosoma brucei
has over 800 genes that make proteins of the parasite mixes and matches to evade immune system
detection [2]. This has tampered with the development of vaccine for the diseases. There is
emerging increase in African trypanosoma due to growing poverty of the countries affected, the
need to take immediate action against human immunodeficiency virus/acquired immunodeficiency
syndrome (HIV/AIDS) and above all, the absence of non-toxic treatment that is easily administered,
reasonably priced and effective at every stage of the disease [3]. Suramin (1) and pentamidine (2)
are effective drugs used during the initial phase of the disease but are almost totally ineffective in
the neurological phase, since they cannot cross the blood-brain barrier [3]. Melarsoprol (3) was
found to be effective in all stages but it has associated drawback of being expensive (US$100 per
patient in 2000) and toxic. Eflornithine (4) has an advantage of being less toxic and as well active
against all stages of the disease but is overwhelmingly expensive (US$ 700 per patient in 2000) and
the recipient has to be hospitalized and given the drug by perfusion for at least two weeks. Robays
et al reported that the cost per life saved for melarsoprol was 600.4 Euros against 627.6 Euros for
eflornithine [4]. Nifurtimox (5) when taken orally for 1-2 months and alpha-difluoromethylornithine
(6, alpha-DFMO) with an administration scheme spread over 5 weeks including 14 days of
intravenous injections provides alternatives for all cases [5]. Although, eflornithine was better
tolerated than melarsoprol, it produces several side effects, including gastrointestinal problems,
fever, hypertension and convulsion [6].
The record of severe reactive encephalopathies in 5-10% of cases, death in 3-5% of the
patient against melarsoprol and side effects against eflornithine underscore the need for new anti-
trypanosomal agents [7]. The potential for resistance when used in monotherapy ushered in

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combinatorial therapy in which nifurtimox-eflornithine combination was the most successful. The
work of Alirol et al however, observed that nifurtimox-eflornithine (NECT) cannot be considered as
an ideal treatment for African trypanosomiasis because it requires 7 days of intravenous
administration which is a huge challenge for primary healthcare systems in developing countries
[8]. Additionally, some adverse effects like frequent vomiting which may cause insufficient
bioavailability of nifurtimox, requiring frequent use of antiemetics and occasional readministration
of nifurtimox are worrisome. Although, psychotic behaviour and convulsions are less frequent, they
can be a source of distress for patients and relatives. NECT treatment regimen is usually
accompanied with painful lumbar punctures and difficult examination of the cerebrospinal fluid [9].
There is an urgent need for more effective treatments for Human African Trypanosomiasis
(HAT), as the current therapy has many shortcomings viz. high toxicity, prohibitive costs,
undesirable route of administration and poor efficacy. The above problems associated with the
current antitrypanosomal agents highlights the need for research aimed at developing new drugs for
the treatment of human African trypanosomiasis.
To direct the research, some proline derivatives have been reported to possess activity
against human African trypanosomiasis in micromolar concentration (IC50 30- 50 µM) [10,11].
Papadopoulou et al. reported a nitro based sulfonamide derivative possessing activity as low
as 0.218 µM against HAT [12]. Junqueira et al. reported novel galactosyl-
triazolobenzenesulfonamides possessing IC50 in micromolar concentration [13]. Brand et al.
reported the synthesis of pyrazole sulfonamides (7) that inhibited N-myristoyltransferase [14]. The
IC50 of the reported lead compound was 0.003 mM with blood brain barrier of 1.5. Sykes et al.
reported a lead agent against T. brucei gambiense having IC50 of 1.3 micromolar bearing
benzenesulfonamide moiety (8) [15]. Kulman et al. reported some series of sulfonamide derivatives
(9) having micromolar inhibition of tubulin of T. brucei with good selectivity [16]. This class of
compound presents a huge breakthrough in the chemotherapy of HAT because the toxicity of
current HAT drugs has been associated with poor selectivity between mammalian and parasitic

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tubulin [16]. Bhattacharya et al. reported N-phenyl-3,5-dinitro-N4,N4-di-n-butylsulfanilamide with
an IC50 value of 0.12 µM against T. brucei [17]
Some carboxamides have been reported to possess interesting antitrypanosomal activity
against T. brucei gambiense [18-20]. Report has shown that each additional hour of self-reported
sleep duration was associated with an eight percent increase in C-reactive protein (CRP) levels and
a seven percent increase in interleukin-6 (Il-6) which are two inflammatory mediators. Chronic
elevations in cytokines such as CRP and Il-6 have been associated with an increased risk of
problems such as diabetes and heart diseases [21]. It can therefore be adduced from these findings
that sleeping sickness can induce inflammatory diseases by raising the levels of CRP and Il-6.
In search for suitable coupling reagents for direct amidation reaction, Bala et al [22],
Mylavarapu et al [23], Tang et al [24] and Lanigan et al [25] have reported boric acid and its
derivatives as a good catalyst for the direct amidation of un-activated carboxylic acids. The work of
these groups was unable to check the feasibility of the catalyst system in the presence of other
functional groups.
HO S
3
SO H
3
O
O
O
NH
CH
NH
NH
NH
NH
NH
O
3
O
HO S
SO H
3
3
H C
HO S
3
SO H
3
3
1
NH
N
S
O
O
As
S
HN
NH
OH
H2N
NH2
2
HN
NH
NH
NH
3
F
F
F
O
F
NH2
O
NH2
O
S
O2N
H2N
O
H2N
N
N
O
4
HO
6
HO
H3C
5
Fig 1: examples of drugs in use for the treatment of HAT

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O
S
N
CH3
F
F
S
O
NO2
H3C
Cl
O
O
S
N((CH 2)nCH3)2
NO2
N
O
CH3
H3C
N
N
N
NH
O
RHNO 2S
CH
3 Cl
F
9
8
7
Fig 2: examples of recently reported antitrypanosomal agent
The presence of sulfonic acid group, sulfone and carboxamide functionalities in
conventional drugs used for the treatment of HAT informs the desire to make new compounds
containing sulfonamide and carboxamide functionalities. Proline was considered in this study
because of the earlier report on some proline derivatives that showed activities against T. brucei
gambiense. We report herein the boric acid catalysed amidation of un-activated carboxylic acids
bearing sulfonamide, carboxylic acids and alcoholic group. Additionally, the antitrypanosomal and
anti-inflammatory activities of the derivatives were also tested.
2. Results and Discussion
2.1 Chemistry
O
H
N
O
H
N
OH
O
S
O
S
O
OH
O
10a
HO
N
10b
N
SO2Cl
OH
i. Na2CO3, 3 h
ii. HCl
R
i. Na2CO3, 3 h
R
R
O
O
ii. HCl
11e-h
OH
O
11a-d
O
OH
9a-d
OH
OH
H2N
H2N
12
12
OH
CO2H
O
CO2H
N
O
O
S
S
N
O
O
R
NH
O
R
NH
13e-h
R= p-CH3, p-NO2, H and o-CH2 in e-h
13a-d
R= p-CH3, p-NO2, H and o-CH3 in a-d
Scheme 1: Protocol for the synthesis of new carboxamide derivatives bearing sulfonamide
functionalities.
Table 1: Description of substituents

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S/N
R
a
b
c
d
e
f
g
h
p-CH3
p-NO2
H
o-CH3
p-CH3
p-NO2
H
o-CH3
The reactions of substituted benzenesulfonyl chloride (9a-d) and L-4-hydroxyproline (10a)
o
or L-proline (10b) in the presence of sodium carbonate at 0 C after 3 h gave the substituted
benzenesulfonamides (11a-h) in excellent yields. Further reactions of the benzenesulfonamides
(11a-h) with p-aminobenzoic acid (PABA, 12) in the presence of catalytic amount of boric acid
under reflux gave the new carboxamides (13a-h) in excellent yields (scheme 1, Table 1). The
1
compounds were characterised using FTIR, H NMR, 13C NMR and high resolution mass
spectroscopy (HRMS). In the FTIR spectra, the hydroxyl band of compounds 13a-d appeared
between 3473-3460 cm-1. These bands were not observed in compounds 13e-h. The NH band
appeared between 3462-3363 cm-1 indicating the successful coupling of PABA with the
benzenesulfonamide to form the carboxamide. In addition to the NH band, the appearance of two
carbonyl bands between 1740-1688 cm-1 for the carboxylic acid and 1704-1660 cm-1 for the
1
carboxamide is diagnostic of successful coupling. In the H NMR spectra, the proton count
accounted for all the expected protons and additionally, the appearance of the NH peak between
5.93-5.03 ppm is supportive of successful synthesis of target molecules. In the carbon-13 NMR
spectra, the peaks between 174.6-172.9 ppm were assigned to the carbonyl of carboxylic acid. In
addition, the C-CO2H peaks appeared between 153.7-152.6 ppm.
2.2 Biological assay
2.2.1 Anti-trypanosoma activity
Table 2: In vitro antitrypanosomal activity potential of the newly synthesized derivatives (µM)
Compd. No.
13a
IC50 (µM)
0.155±0.12
0.144±0.11
LD50 (µM/kg)
12.01±2.05
10.28±1.08
13b

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0.160±0.15
12.07±2.68
9.98±1.10
11.92±2.76
8.65±0.98
10.06±1.43
13.09±3.12
8.75±1.04
13c
0.157±0.14
0.101±0.11
0.002±0.01
0.159±0.32
0.134±0.21
0.005±0.02
13d
13e
13f
13g
13h
melarsoprol
The in vitro antitrypanosomal activity (Table 2) showed that except compound 13f that had
twofold better activity than melarsoprol, other derivatives had good anti-Trypanosoma brucei
activities in micromolar concentration but not comparable with melarsoprol. The drug-likeness
studies revealed that five of the new carboxamides are drug-like inclusive of compound 13f. This
findings, therefore, calls for more experimental work aimed at developing any of the drug-like
derivatives into a potential drug. The structure-activity relationship studies showed that the L-
proline derivatives (13e-h) had better anti-Trypanosome brucei activities than L-hydroxyproline
derivatives (13a-d). The nitro-substituted benzene derivative 13f with an IC50 of 0.002 µM was the
most active derivative among the proline derivatives. The order of activity is 13f˃13e˃13h˃13g.
Among the 4-hydroxyproline derivatives (13a-d), 13b was the most active with an IC50 of 0.144
µM. The order of activity is 13b˃13a˃13d˃13c. Generally, substitution on the benzene ring
increased the anti-Trypanosome brucei activities. The presence of electron withdrawing NO2
improved the antitrypanosomal activity. The LD50 suggests that the compounds would not pose
toxicity problems in clinical trials.
2.2.2 In vivo anti-inflammatory activity
Table 3: In vivo anti-inflammatory activities of the newly synthesized carboxamide derivatives (%)
Compd. No.
30 min (%)
58.01
1 h (%)
59.95
2 h (%)
67.23
3 h (%)
84.01
13a

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44.10
33.85
38.58
28.75
42.12
23.56
35.78
56.92
48.48
24.71
21.18
31.49
45.88
26.34
39.38
63.53
52.25
27.34
23.44
36.27
50.08
29.44
36.33
64.84
62.81
1.85
13b
13c
25.31
35.28
52.33
31.15
40.05
63.58
13d
13e
13f
13g
13h
Indomethacin
The in vivo anti-inflammatory activity of the compounds in percentages (%) are presented in
Table 3. All the reported derivatives had anti-inflammatory activities reported as the percentage
decrease in edema thickness induced by carrageenan. The structure-activity relationship studies
showed that the hydroxyl group in the proline ring (13a-d) increased the anti-inflammatory
activities. This could be as a result of utilization of the lone pair of the hydroxyl group in the
formation of hydrogen bonds with the amino acids of the possible target. The substitution at the
para position was shown to contribute significantly to the anti-inflammatory activities of the
derivatives as evident in the decreased activity in compounds 13c, 13d, 13g and 13h when
compared with compounds 13a, 13b, 13e and 13f. The effect of electron availability is made
evident from the results of compounds 13a and 13b and 13e and 13f, the methyl substituent had
better activity than the nitro-substituted derivatives. The substitution in ortho position (13d and
13h) favoured anti-inflammatory activity than the unsubstituted derivatives (13c and 13g).
Compound 13a and 13b had comparable anti-inflammatory activity with indomethacin. The drug-
likeness showed that compound 13b is non-drug-like thereby making compound 13a the only likely
anti-inflammatory drug candidate.
2.2.3 Physicochemical Properties Study
Table 4: Physicochemical Properties calculation of the new analogs

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Compd. No.
MW
clogP
HBD
HBA TPSA (Å2) NRB
ABS
(%)
NV
404
435
390
404
388
419
374
388
0.418
-1.132
-0.253
0.418
-0.190
1.305
-0.103
1.305
3
3
2
3
1
2
3
2
6
6
6
6
5
5
5
5
79.90
123.04
79.90
79.90
79.90
123.04
79.90
79.90
6
7
6
7
6
6
6
6
81.43
0
0
0
0
0
0
0
0
13a
13b
13c
13d
13e
13f
66.55
81.43
81.43
81.43
66.55
81.43
81.43
13g
13h
MW= molecular weight, HBA= hydrogen bond acceptor, HBD= hydrogen bond donor, NRB=
number of rotatable bond, ABS= solubility, TPSA= topological polar surface area, NV= ro5
number of violations.
Lipophilicity has long been recognized as an important factor for successful passage of drugs
through clinical development [26]. Generally, calculated logP (clogP) is being used for assessment
of lipophilicity and the key events of molecular desolvation, transfer from aqueous phases to cell
membranes and protein binding sites [27]. With the evidenced role as a predictor of eventual
compound success, computation of logP (clogP) for lipophilicity is essential for the development of
a successful therapeutic compound. All the tested compounds complied with ro5, where logP values
ranged between -1.132 to 1.305 (˂5); MW range 374-435 (˂500); HBA range 5-6 (˂10) and HBD
range 1-3 (˂5), suggesting that these compounds would not pose oral bioavailability problems. All
the compounds showed NRB values range 6-7 (˂10), indicating their acceptable molecular
flexibility with consequent expected good permeability and oral bioavailability. In addition, all the
compounds evaluated showed TPSA values range 79.90-123.04 Å2 (˂140 Å2), showing good
permeability and transport of the compounds in the cellular plasma membrane. Furthermore, the

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evaluated compounds showed good % ABS range 66.55-81.43% calculated from %ABS= 109-
0.345×TPSA [28], which indicates their good bioavailability upon oral administration.
3. Conclusions
The synthesis of eight new carboxamide derivatives has been reported. The compounds were
characterised using FTIR, NMR and HRMS. The anti-inflammatory and antitrypanosomal (T.
brucei gambiense) activities results showed that the compounds were active. Particularly,
compound 13f had twofold anti-Trypanosoma brucei gambiense activity when compared with
melarsoprol. Other derivatives had antitrypanosomal activities in micromolar concentration but
were not comparable to melarsoprol. Like the antitrypanosomal activities, the anti-inflammatory
activities showed that all the new derivatives were active and of particular attention were
compounds 13a and 13b which had comparable activity with indomethacin. The pharmacokinetic
study showed that the reported derivatives would not have oral bioavailability, transport or
permeability problems. The connectivity between excess sleep and inflammation-related diseases
makes it resounding to have a compound that could act as both antitrypanosomal agent and anti-
inflammatory agent.
4.0 Experimental
All reactions requiring inert atmosphere were carried out under nitrogen atmosphere. Drying
of solvents was achieved using molecular sieve for 48 h. All reagents were purchased from
commercial suppliers, Aldrich, Merck, Fluka, Avra, SD fine and Alfa Aesar. Thin layer
chromatography was carried out using silica plates purchased from Avra. The plates were visualized
under UV light (popular India). FT-IR spectroscopy of the compounds were run in PerkinElmer
Spectrum version 10.03.06 and the bands presented in wavenumber. Proton and carbon-13 NMR
spectroscopy were run in DMSOd6 and CD3OD, on either Jeol 500 MHz or 400 MHz. The chemical
shifts were reported in part per million with reference to tetramethylsilane. High Resolution Mass

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spectroscopy were carried out using micro TOF electrospray time of flight (ESI-TOF) mass
spectrometer, sodium formate was used as the calibrant. All experiments were carried out at Prof.
Sandeep Verma’s Laboratory, Department of Chemistry, Indian Institute of Technology, Kanpur.
Melting points were determined using digital melting point apparatus and were uncorrected.
4.1 Synthesis of substituted proline-based benzenesulfonamides
Sodium carbonate (Na2CO3, 1.590 g, 15 mmol) was added to a solution of amino acids (10a-
b, 12.5 mmol) in water (15 mL) with continuous stirring until all the solutes dissolved. The solution
o
was cooled to -5 C and the appropriate benzenesulfonyl chloride (9a-d, 15 mmol) was added in
four portions over a period of 1 h. The slurry was further stirred at room temperature for about 3 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 (11a-h) were dried over self-indicating
fused silica gel in a desiccator [29].
4.2 Boric Acid Catalysed Amidation of Un-Activated Carboxylic Acid and p-aminobenzoic
acid.
To a suspension of substituted benzenesulfonamides (11a-h) (1.0 mmol) in dry toluene (40
mL) equipped with Dean-Stark apparatus for azeotropic removal of water, was added 4-
aminobenzoic acid (1.0 mmol) and boric acid (0.1 mmol) at room temperature and then refluxed for
4 h. On completion (as monitored by TLC), reaction mixture was precipitated to amides by adding
about 40 mL n-hexane. The new proline derivatives were obtained via suction filtration, washed
with n-hexane and dried over fused silica gel or concentrated using rotary evaporator and dried over
vacuum in the case of oily products.
4.2.1 4-[4-Hydroxy-1-(4-methylbenzenesulfonyl)pyrrolidine-2-amido]benzoic acid (13a):
o
Yield (0.40 g, 100%), mp 155.30-155.60 C; FTIR (KBr) ѵ/cm-1 3460, 3364, 2550, 1740,
1
1666, 1601, 1574, 1442, 1327, 1313, 1174, 1157, 1072, 1000; H NMR (400 MHz, DMSOd6) δH
7.66-7.64 (d, 1H, J 8.24 Hz, ArH), 7.59-7.56 (d, 3H, J 8.68 Hz, ArH), 7.37-7.35 (d, 1H, J 8.24 Hz,

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ArH), 6.51-6.49 (d, 3H, J 8.24 Hz, ArH), 5.81 (s, 1H, NH), 4.17 (s, 1H, OH), 4.02-3.98 (t, 1H, J
7.80 Hz, CH-C=O), 3.43-3.40 (m, 1H, CH-OH), 3.06-3.04 (d, 2H, J 10.56 Hz, CH2N), 2.34 (s, 3H,
CH3), 1.92-1.89 (m, 2H, CH2); 13C NMR (100 MHz, DMSOd6) δC 173.8, 168.0, 153.7, 143.7,
134.9, 131.8, 130.1, 127.9, 117.4, 113.1, 68.9, 60.2, 56.8, 21.5; HRMS (FTMS+ESI) m/z, observed:
405.1122; C19H20N2O6S (M+H), requires: 405.1121.
4.2.2 4-[4-Hydroxy-1-(4-nitrobenzenesulfonyl)pyrrolidine-2-amido]benzoic acid (13b):
o
Yield (0.44 g, 99.98%), mp 164.70-164.90 C; FTIR FTIR (KBr) ѵ/cm-1 3460, 3365, 2968,
1
2555, 1713, 1667, 1602, 1442, 1423, 1575, 1528, 1327, 1312, 1174, 1130, 1093, 1071, 1011; H
NMR (400 MHz, DMSO-d6) δH 12.07 (s, 1H, OH of COOH), 8.38-8.36 (d, 2H, J 7.36 Hz, ArH),
8.04-8.02 (d, 2H, J 6.88 Hz, ArH), 7.58-7.56 (d, 2H, J 6.88 Hz, ArH), 6.51-6.49 (d, 2H, J 6.88 Hz,
ArH), 5.83 (s, 1H, NH), 4.17 (s, 1H, OH of alcohol), 4.13-4.09 (t, 1H, J 6.42 Hz, CH-C=O), 3.47-
3.44 (m, 1H, CH-OH), 3.21-3.19 (d, 2H, J 8.68 Hz, CH2N), 2.03-2.00 (m, 1H, CH of CH2), 1.93-
13
1.87 (m, 1H, CH of CH2); C NMR (100 MHz, DMSOd6) δC 173.5, 168.0, 153.7, 150.3, 146.9,
131.7, 129.5, 124.8, 117.4, 113.1, 69.0, 60.4, 57.1; HRMS (FTMS+ESI) m/z, observed: 454.0912;
C18H20N3O9S (M+H3O), requires: 454.0913.
4.2.3 4-[1-(benzenesulfonyl)-4-hydroxypyrrolidine-2-amido]benzoic acid (13c):
o
Yield (0.39 g, 100%); mp 142.50-142.70 C; FTIR (KBr) ѵ/cm-1 3461, 3365, 2956, 2672,
1
1700, 1666, 1601, 1574, 1443, 1422, 1385, 1313, 1174, 1128, 1099, 1070, 1010; H NMR (400
MHz, DMSO-d6) δH 7.78-7.76 (d, 2H, J 6.88 Hz, ArH), 7.65-7.54 (m, 5H, ArH), 6.52-6.50 (d, 2H, J
8.68 Hz, ArH), 5.83 (s, 1H, NH), 4.18 (s, 1H, OH), 4.06-4.02 (t, 1H, J 8.00 Hz, CH-C=O), 3.45-
3.41 (m, 1H, CH-OH), 3.11-3.08 (d, 2H, J 5.26 Hz, CH2N), 1.95-1.89 (m, 2H, CH2); 13C NMR (100
MHz, DMSOd6) δC 173.8, 168.1, 153.7, 137.8, 133.5, 131.8, 129.6, 128.7, 127.9, 125.8, 117.4,
113.1, 68.9, 60.2, 56.8, 21.6; HRMS (FTMS) m/z, observed: 413.0788; C18H18N2O6SNa (M+Na),
requires: 413.0786.
4.2.4 4-[4-Hydroxy-1-(2-methylbenzenesulfonyl)pyrrolidine-2-amido]benzoic acid (13d):

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o
Yield (0.40 g, 100%); mp 134.50-134.60 C; FTIR (KBr) ѵ/cm-1 3473, 3415, 3029, 2988,
1
1692, 1669, 1601, 1442, 1422, 1313, 1290, 1174, 1154, 1093, 1072, 1015; H NMR (400 MHz,
CD3OD ) δH 7.77-7.69 (m, 2H, ArH), 7.50-7.43 (m, 1H, ArH), 7.33-7.28 (m, 2H, ArH), 7.19-7.16 (t,
1H, J 7.32 Hz, ArH), 7.12-7.07 (t, 1H, J 9.38 Hz, ArH), 6.62-6.60 (d, 2H, J 8.68 Hz, ArH), 4.45 (s,
1H, OH), 4.30-4.28 (t, 1H, J 2.32 Hz, CH-C=O), 3.56-3.51 (m, 1H, CH-OH), 3.38-3.25 (m, 2H,
13
CH2N), 2.60 (s, 3H, CH3-Ar), 2.16-2.10 (m, 2H, CH2); C NMR (100 MHz, CD3OD) δC 172.9,
169.4, 153.2, 138.2, 131.5, 129.3, 128.6, 127.6, 125.8, 124.9, 117.7, 113.0, 69.3, 59.4, 38.8, 20.2,
19.5; HRMS (FTMS+ESI) m/z, observed: 403.0968; C19H19N2O6S (M-H); requires: 403.0963.
4.2.5 4-[1-(4-Methylbenzenesulfonyl)pyrrolidine-2-amido]benzoic acid (13e):
o
Yield (0.36 g, 93.25%); mp 161.40-161.60 C; FTIR (KBr) ѵ/cm-1 3462, 3060, 2984, 2879,
1
2559, 1738, 1669, 1603, 1422, 1556, 1524, 1345, 1291, 1174, 1134, 1092, 1072, 1025; H NMR
(400 MHz, DMSOd6) δH 7.75-7.69 (m, 4H, ArH), 6.65-6.63 (m, 4H, ArH), 5.02 (s, 1H, NH), 4.16-
4.12 (t, 1H, J 6.40 Hz, CH-C=O), 3.45-3.39 (m, 1H, CHa of CH2N), 3.22-3.16 (m, 1H, CHb of
CH2N), 2.40-2.30 (m, 3H, CH3-Ar), 1.92-1.82 (m, 3H), 1.60-1.56 (m, 1H, CH); 13C NMR (100
MHz, DMSOd6) δC 174.6, 169.3, 152.6, 144.1, 134.6, 131.5, 128.6, 127.9, 125.6, 118.2, 60.7, 30.7,
24.3, 20.2, 20.0; HRMS (FTMS+ESI) m/z, observed: 389.1172; C19H20N2O5S (M+H), requires:
389.1178.
4.2.6 4-[1-(4-Nitrobenzenesulfonyl)pyrrolidine-2-amido]benzoic acid (13f):
o
Yield (0.42 g, 99.90%); mp 132.40-132.50 C; FTIR (KBr) ѵ/cm-1 3378, 3082, 2980, 2874,
1
2540, 1745, 1704, 1604, 1446, 1417, 1529, 1349, 1315, 1177, 1162, 1105, 1076, 1023, 1011; H
NMR (400 MHz, DMSOd6) δH 12.35 (OH of COOH), 8.38-8.36 (d, 2H, J 9.16 Hz, ArH), 8.07-8.05
(d, 2H, J 8.68 Hz, ArH), 7.58-7.56 (d, 2H, J 8.24 Hz, ArH), 6.51-6.48 (d, 2H, J 8.64 Hz, ArH), 5.93
(s, 1H, NH), 4.19-4.16 (dd, 1H, J 3.64, 3.04 Hz, CH-C=O), 3.40-3.34 (m, 1H, CHa of CH2N), 3.23-
3.17 (m, 1H, CHb of CH2), 2.00-1.91 (m, 1H, CH of CH2), 1.86-1.75 (m, 2H, CH2), 1.65-1.59 (m,
1H, CH); 13C NMR (100 MHz, DMSOd6) δC 173.4, 168.0, 153.7, 150.4, 143.7, 131.7, 129.2, 125.2,

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117.4, 113.1, 61.1, 48.9, 30.9, 24.7; HRMS (FTMS+ESI) m/z, observed: 438.0953; C18H20N3O8S
(M+H3O), requires: 438.0957.
4.2.7 4-[1-(Benzenesulfonyl)pyrrolidine-2-amido]benzoic acid (13g):
o
Yield (0.37 g, 99.98%); mp 171.50-171.60 C; FTIR (KBr) ѵ/cm-1 3363, 2958, 2688, 1688,
1
1677, 1602, 1573, 1446, 1422, 1354, 1313, 1201, 1163, 1095, 1073, 1016; H NMR (400 MHz,
DMSOd6) δH 7.80-7.78 (d, 2H, J 7.32 Hz, ArH), 7.67-7.63 (t, 1H, J 7.32 Hz, ArH), 7.59-7.55 (t,
2H, J 7.24 Hz, ArH), 7.21-7.07 (m, 2H, ArH), 6.52-6.50 (d, 2H, J 8.68 Hz, ArH), 5.83 (s, 1H, NH),
4.08-4.05 (dd, 1H, J 4.60, 5.04 Hz, CH-C=O), 3.34-3.28 (m, 1H, CH of CH2N), 3.13-3.08 (m, 1H,
CH of CH2N), 1.85-1.71 (m, 3H), 1.51-1.47 (m, 1H, CH); 13C NMR (100 MHz, DMSOd6) δC 173.7,
168.1, 153.7, 133.6, 131.8, 129.9, 129.4, 128.7, 127.6, 125.8, 117.4, 113.1, 60.9, 48.9, 30.9,
24.7466; HRMS (FTMS+ESI) m/z, observed: 393.1121; C18H21N2O6S (M+H3O), requires:
393.1122.
4.2.8 4-[1-(2-Methylbenzenesulfonyl)pyrrolidine-2-amido]benzoic acid (13h):
o
Yield (0.39 g, 100%); mp 119.90-120.10 C; FTIR (KBr) ѵ/cm-1 3382, 3020, 2981, 2632,
1
1692, 1688, 1604, 1574, 1443, 1422, 1313, 1290, 1158, 1131, 1091, 1016; H NMR (400 MHz,
CD3OD) δH 7.75-7.72 (m, 2H, ArH), 7.44-7.40 (t, 1H, J 6.64 Hz, ArH), 7.34-7.28 (m, 1H, ArH),
7.19-7.13 (m, 2H, ArH), 7.11-7.05 (m, 2H, ArH), 4.15-4.13 (t, 1H, J 4.30 Hz, ArH), 3.45-3.43 (m,
2H, CH2N), 2.59 (m, 3H, CH3-Ar), 1.85-1.66 (m, 2H, CH2), 1.59-1.42 (m, 2H, CH2); 13C NMR
(100 MHz, CD3OD) δC 174.3, 169.4, 153.2 (C-COOH), 137.6, 132.9, 131.5, 129.6, 127.9, 126.0,
125.0, 117.8, 113.1, 60.1, 30.9, 24.3, 20.2, 19.5; HRMS (FTMS+ESI) m/z, observed: 389.1163;
C19H21N2O5S (M+H), requires: 389.1164.
4.3 Biological Assay
4.3.1 In vitro antitrypanosomal activities
In vitro antitrypanosomal activities against T. b. gambiense strain was described by Otoguro
et al [30]. In brief, T. b. gambiense strain was cultured in IMDM with various supplements and 10%
heat-inactivated FBS at 37 °C, under 5.0% CO2/95% air according to the method of Baltz et al [31].

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Ninety five mL of T. brucei gambiense suspension (2.0-2.5 trypanosomes/mL for strain GUTat 3.1)
was seeded in a 96-well microplate, and 5.0 mL of a test compound solution (dissolved in 5.0%
dimethylsulfoxide) was added followed by incubation for 72 h. Ten mL of the fluorescent dye
Alamar Blue was added to each well. After incubation for 3-6 h, the resulting solution was read at
528/20 nm excitation wavelength and 590/35 nm emission wavelength by a FLx800 fluorescent
plate reader (Bio-Tek Instrument, Inc. Vermont, USA). Data were transferred into a spreadsheet
program (Excel). The IC50 values were determined using fluorescent plate reader software (KC-4,
Bio-Tek). Successive subcultures were done in 24-well tissue culture plates under the same
conditions.
4.3.2 In vivo anti-inflammatory activities determination
Edema was produced by using 1% carrageenan solution. Foot volumes were measured in
plethysmometer by mercury displacement. The instrument was calibrated before performing the
experiment using standard calibrated probe number and standard drug used was indomethacin.
Anti-inflammatory activity was determined by carrageenan induced rat hind paw edema method.
The tested compounds and reference drug (indomethacin) were administered orally at a dose level
of 100 mg/kg and 25 mg/kg respectively. After an hour of oral medication, all rats were injected
with 1% carrageenan suspension (0.05 mL/animal) into the sub-planter surface of the right hind
paw. The thickness of both paws were measured at different time intervals of zero, 1 h, 2 h and 3 h,
after carrageenan injection. The anti-inflammatory activity of the tested compounds and
indomethacin were calculated as the percentage decrease in carrageenan-induced edema thickness
and was determined with the following formula [32]. In vivo efficacy studies in rat were conducted
according to the rules and regulations for the protection of animal rights of Nigeria. They were
approved by the veterinary office of University of Nigeria, Nsukka, Nigeria.
Percentage inhibition= ꢀꢁꢂꢀꢃ ∗ 100 where Vc = volume of control, Vt = volume of test
ꢀꢁ
sample.
4.3.3 Determination of LD50 for the active anti-inflammatory compounds

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Male mice were divided into various groups and test compounds were administered in various doses
intraperitoneally. Following treatments, the animals were observed for up to 6 h continuously and
were then kept under observation for 72 h. All behavioral changes and deaths during the
observation periods were recorded. The percentage of death at each dose level was then calculated,
converted to probits and the LD50 (µM/kg) values were calculated [33]. To observe the health status
of the mice, they were monitored 4 times a day. Humane endpoints were used when the animal
shows sign of weight loss, weakness accompanied by the inability to get food, complete anorexia
and convulsion for 24 h. For the purpose of ameliorating the suffering of the dying mice, CO2
euthanasia was applied. All the dead mice were disposed in bio-safety containers in accordance
with local standard protocols. The mortality rate in each group was calculated according to the
formula:
Mortality rate (%)= (the number of dead mice/the number of mice in the group)×100.
4.3.4 Physicochemical Properties
The prediction was based on molecular weight (MW), calculated logP, topological polar
surface area (TPSA), hydrogen bond donors (HBD), hydrogen bond acceptor (HBA) and number of
rotatable bond (NRB) and number of violation (NV) which is a record of the number of violations.
The Physicochemical Properties Study was carried out using molinspiration, MedChem designer
3.0 and DrugLiTo software.
5.0 Supplementary Information
The 1H NMR, 13C NMR and HRMS spectra are available
6.0 Acknowledgements
Authors are thankful to Prof Sandeep Verma of the Department of Chemistry, Indian
Institute of Technology, Kanpur for providing the facilities for synthesis and spectral
characterization. We are also thankful to the Department of Biochemistry, University of Nigeria for

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the anti-inflammatory studies. We are thankful to the Department of Veterinary Parasitology and
Entomology, University of Nigeria for the anti-trypanosomal activities study.
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1
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Highlights
1. Structure-based design and synthesis of novel proline derived sulphonamides is reported
2. The link between excessive sleep and inflammation calls for the synthesis of antitrypanosomal
agents with additional anti-inflammatory properties.
3. In vitro and in vivo studies reveal that the reported compounds possessed good
antitrypanosomal and anti-inflammatory activities.
4. Physicochemical properties prediction was carried out to assess the oral bioavailability,
transport and permeability properties of the compounds.