Structural characterization of naphthalene sulfonamides and a sulfonate ester and theirin vitroefficacy againstLeishmania tarentolaepromastigotes

Article abstract of DOI:10.1039/d0nj06320g

Leishmaniasis, a parasitic infectious disease transmitted by sandfly bites, is an extremely complex human and veterinary disease. In humans, it takes different forms ranging from self-healing cutaneous ulcers to severe, life-threatening visceral infections. Current treatments primarily rely on chemotherapies. The current chemotherapeutic drugs, developed decades ago, are costly, toxic, and have become problematic due to drug resistance. To this end, two naphthalene sulfonamides and one naphthalene sulfonate ester were subjected to a structure-activity relationship (SAR) study for inhibition ofLeishmania. The new chlorosulfonamide,N-(2′-chlorophenyl)-1-naphthalene sulfonamide (B), and sulfonate ester, 3-methyl-4-((naphthalen-1-ylsulfonyl)oxy)benzoic acid achieved (C) IC₅₀values of 9.5 μM and 7.4 μM, respectively, againstLeishmania tarentolaepromastigotesin vitro, which are notably lower than the IC₅₀value of 49 μM for our benchmark compound,N-(4′-carboxy-2′-methylphenyl)-1-napthalenesulfonamide (A). Additionally, all three compounds were analyzed by X-ray crystallography and show strong intermolecular interactions in the crystalline state. The new findings from this SAR study open new directions for advanced leishmaniasis treatment.

Full text of DOI:10.1039/d0nj06320g

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Structural characterization of naphthalene
sulfonamides and a sulfonate ester and their
in vitro efficacy against Leishmania tarentolae
Cite this: New J. Chem., 2021,
45, 4791
promastigotes†
a
Edward W. Li,ab Jade Katinas,a Marjorie A. Jones
and
a
Christopher G. Hamaker
*
Leishmaniasis, a parasitic infectious disease transmitted by sandfly bites, is an extremely complex human
and veterinary disease. In humans, it takes different forms ranging from self-healing cutaneous ulcers to
severe, life-threatening visceral infections. Current treatments primarily rely on chemotherapies. The current
chemotherapeutic drugs, developed decades ago, are costly, toxic, and have become problematic due to
drug resistance. To this end, two naphthalene sulfonamides and one naphthalene sulfonate ester were
subjected to a structure–activity relationship (SAR) study for inhibition of Leishmania. The new chlorosulfo-
namide, N-(20-chlorophenyl)-1-naphthalene sulfonamide (B), and sulfonate ester, 3-methyl-4-((naphthalen-
1-ylsulfonyl)oxy)benzoic acid achieved (C) IC50 values of 9.5 mM and 7.4 mM, respectively, against Leishmania
tarentolae promastigotes in vitro, which are notably lower than the IC50 value of 49 mM for our benchmark
compound, N-(40-carboxy-20-methylphenyl)-1-napthalenesulfonamide (A). Additionally, all three compounds
were analyzed by X-ray crystallography and show strong intermolecular interactions in the crystalline state.
The new findings from this SAR study open new directions for advanced leishmaniasis treatment.
Received 30th December 2020,
Accepted 10th February 2021
DOI: 10.1039/d0nj06320g
rsc.li/njc
emerging global health threat, there is urgent need for new
anti-leishmanial drug development.
Introduction
Leishmaniasis is an extremely complex human and veterinary
Sulfonamides, known as sulfa drugs, have been used as treat-
disease, transmitted by sandfly bites.1 In humans, the disease ments against bacterial infections and are also of interest in
takes different forms ranging from self-healing cutaneous ulcers treating leishmaniasis.9,10 In previous work, Peixoto and Beverley
to a severe life-threatening infection.1 According to the World studied the effects of sulfonamides and sulfones against Leishmania
Health Organization, leishmaniasis is endemic in 95 countries, major promastigotes in vitro. Some of their sulfonamides and a
from rain forests in Central and South America to deserts in the sulfone were found to be inhibitory against the parasite, though
Middle East and West.2 Some cases of the disease have also with rather high IC50 values, over 150 mM.11 Since then, interest
appeared in Mexico and Texas.2 The disease not only affects in sulfonamides and sulfones has grown for treatment against
people who live in countries where the disease is endemic but leishmaniasis; however previous candidates failed as a treatment
also poses a risk to people who travel in those areas.2 For solution.8,12–16 Metal dithiocarbamates have also been recently
example, the cutaneous form of the disease is a growing health examined for their anti-leishmanial activity.17 Recently, we
problem for U.S. military service members in sandfly-rich reported a new class of naphthalene sulfonamides (Fig. 1, General
Afghanistan and Iraq.2 First line treatments for leishmaniasis structure Z = NH) which gave promising in vitro anti-leishmanial
rely on pentavalent antimonials and amphotericin B, both of properties.18 It was found that the ortho and para substituents,
which are toxic to humans, and many strains of Leishmania are X and Y, play an important role in the sulfonamides’ leishmani-
gaining resistance to these oft-used drugs.3–8 To combat this cidal activities, water solubilities, and a possible mechanism of
action.18 In compounds with X = ꢀCH3 and Y = H, the sulfona-
mides have superior activities relative to those with X = H,
a Department of Chemistry, Illinois State University Normal, IL 61790-4160, USA.
ꢀOCH3, or ꢀSCH3, in spite of poor solubility. When Y is a
E-mail: chamaker@IllinoisState.edu
b William Fremd High School, Palatine, IL, USA
carboxylic acid group, the methyl sulfonamide solubility issue
was greatly improved, but inhibitory activity was reduced. These
† Electronic supplementary information (ESI) available. CCDC 1917626–1917628.
interesting results led us to speculate that the size of X (larger
than X = H but smaller than X = OCH3) and the polarity of X will
For ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/d0nj06320g
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Hydrogen atoms attached to carbon were assigned positions
based on the geometries of their attached carbons. Hydrogen
atoms bonded to oxygen and nitrogen were assigned positions
based on the Fourier difference map. See Table S1 (ESI†) for final
refinement parameters.
Synthesis of compounds
N-(40-Carboxy-20-methylphenyl)-1-napthalenesulfonamide.
A, was synthesized as previously reported.18 Single crystals of
A were grown by slow evaporation of an acetone solution of the
compound. 1H NMR (d, ppm, DMSO-d6): 12.76 (br, 1H, COOH),
10.17 (s, 1H, NH), 8.73 (d, 1H, JHH = 8.6 Hz), 8.22 (d, 1H, JHH
=
8.3 Hz), 8.08 (m, 2H,), 7.68 (m, 2H), 7.59 (m, 3H), 7.20 (d, 1H,
JHH = 8.2 Hz), 1.96 (s, 3H, CH3). 13C NMR (d, ppm, DMSO-d6):
166.71 (COOH), 139.17, 135.12, 134.44, 133.79, 132.04, 131.71,
129.38, 129.07, 127.98, 127.60, 127.52, 127.38, 127.01, 124.46,
124.42, 123.67, 17.55 (CH3). C18H15NO4S, ESI-HRMS: m/z calc.
(found), intensity: [M + H]+, 342.0800 (342.0784), 25%; [M + Na]+,
364.0619 (364.0620), 100%; [M + K]+, 380.0359 (380.0342), 20%.
N-(20-Chlorophenyl)-1-naphthalenesulfonamide (B). To 20 mL
of pyridine was added 2.266 g (10.00 mmol) 1-naphthalenesulfonyl
chloride and 1.276 g (10.01 mmol) 2-chloroaniline. The mixture
was refluxed for 30 minutes and then poured into 100 mL water to
form a precipitate. The solid was filtered, washed with water, and
dried to yield 2.052 g (64.6%) of compound B as a white solid.
Single crystals of B were grown by slow evaporation of a 1,2-
dichloroethane solution of B. 1H NMR (d, ppm, CDCl3): 8.65 (dd,
1H, JHH = 8.6, 0.8 Hz), 8.21 (dd, 1H, JHH = 7.4, 1.2 Hz), 8.02 (d, 1H,
JHH = 8.3 Hz), 7.89 (dd, 1H, JHH = 8.7, 0.6 Hz), 7.65 (m, 1H), 7.65
(m, 1H), 7.56 (m, 2H), 7.45 (t, 1H, JHH = 8.1 Hz), 7.21 (br s, 1H,
NH), 7.14 (m, 2H), 6.94 (m, 1H). 13C NMR (d, ppm, CDCl3):
135.21, 134.48, 134.13, 133.78, 130.56, 129.57, 129.33, 128.73,
128.37, 127.97, 127.25, 125.66, 124.70, 124.48, 124.17, 121.61.
C16H12ClNO2S, ESI-HRMS: m/z calc. (found), intensity: [M + H]+,
318.0356 (318.0334), 60%; [M + H]+, 320.0326 (320.0302), 23%;
[M + Na]+, 340.0175 (340.0152), 100%; [M + Na]+, 342.0146
(342.0120), 35%; [M + K]+, 355.9914 (355.9890), 20%; [M + K]+,
357.9872 (357.9860), 8%.
Fig. 1 General structure and structures of compounds A, B, and C.
affect inhibitory activity and may bring new insight into the
structure–activity relationship (SAR). Looking beyond traditional
sulfonamides and sulfones for new chemistry at the Z position
was another inspiration for this anti-leishmanial study.
Encouraged by our previous results, and the fact that L.
tarentolae has been shown to be a very useful model for
screening antileishmanial agents,19 we have designed a novel
naphthalene halogenated sulfonamide and a naphthalene sulfonate
ester for the preliminary screening against L. tarentolae for this SAR
study. To this end, the new compounds (Fig. 1), chlorosulfonamide
B and sulfonate ester C, were synthesized. Both compounds B
and C resulted in remarkable inhibition against L. tarentolae
promastigotes compared to the benchmark naphthalene sulfona-
mide A reported previously.18
Experimental
Reagents and materials were purchased from commercial
sources and used directly unless stated otherwise. NMR spectra
were obtained at 302 K in CDCl3 or DMSO-d6 at a frequency of
500.13 MHz for 1H and 125.76 MHz for 13C spectra. Mass spectro-
scopy data were acquired using positive ion mode electrospray
ionization on a high resolution time of flight mass spectrometer.
3-Methyl-4-[(naphthalen-1-ylsulfonyl)oxy]benzoic acid (C). To a
solution of 4-hydroxy-3-methylbenzoic acid (1.521 g, 10.00 mmol)
in 10 mL 2 M aqueous sodium hydroxide was added dropwise a
solution of 2.266 g (10.00 mmol) 1-naphthalenesulfonyl chloride in
10 mL acetone. The mixture was allowed to stir at room tempera-
X-ray crystallography
Data for B were collected at 297(2) K on a Bruker–Nonius CAD4/ ture for 24 hours when approximately 2 mL of 6 M aqueous
Mach3 diffractometer using MoKa radiation (l = 0.71073 Å). hydrochloric acid was added to give a white precipitate. The
Data collection and cell refinement were performed using CAD4 precipitate was isolated by vacuum filtration, washed with dilute
express.20 Data reduction was carried out using XCAD4.21 Unit HCl and dried to yield 3.022 g (88.3%) of compound C as a white
cell parameters were obtained from a least-squares refinement of powder. Single crystals of C were grown by solvent diffusion of
25 centered reflections. The data were corrected for absorption hexanes into a solution of C in acetone. 1H NMR (d, ppm, DMSO-d6):
through use of empirical psi-scans.22 Data for A and C were 8.66 (d, 1H, JHH = 8.6 Hz), 8.44 (d, 1H, JHH = 8.3 Hz), 8.20 (m, 2H),
collected on a Bruker APEX II CCD diffractometer at 100(2) K 7.87 (td, 1H, JHH = 8.3, 1.0 Hz), 7.82 (br s, 1H, NH), 7.77 (t, 1H,
using MoKa radiation (l = 0.71073 Å). The data were processed JHH = 7.6 Hz), 7.70 (t, 1H, JHH = 7.8 Hz), 7.65 (dd, 1H, JHH = 8.5,
using the Bruker SAINT software package and were corrected for 1.7 Hz), 6.79 (d, 1H, JHH = 8.5 Hz), 2.07 (s, 3H, CH3). 13C NMR
absorption using SADABS. Structures were solved by direct (d, ppm, DMSO-d6): 166.49 (COOH), 150.65, 133.86, 132.77, 131.06,
methods using SHELXS-2017.23 The data were refined using 131.05, 130.60, 130.40, 129.46, 129.33, 128.48, 127.66, 127.54,
SHELXL-2017. All non-H atoms were refined anisotropically. 124.69, 123.99, 121.27, 15.87 (CH3). 8.82 (dd, 1H), 8.18 (m, 2H),
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8.01 (d, 1H), 7.91 (d, 1H), 7.77 (m, 1H), 7.74 (dd, 1H), 7.68 (m, 1H), was determined by the MTT assay. The data are plotted with a
7.54 (t, 1H), 6.81 (d, 1H) C18H14O5S, ESI-HRMS: m/z calc. (found), graphing software, GraphPad Prism 7.03 (GraphPad Software, San
intensity: [M + H]+, 343.0641 (343.0625), 50%; [M + Na]+, 365.0460 Diego, CA, USA). The software was used to calculate the IC50 values
(365.0444), 100%; [M + K]+, 381.0199 (381.0183), 15%.
by fitting the data with a nonlinear regression with variable
concentration, and the IC50 values are reported with 95% con-
fidence interval.
Cell culture
For this study, all cell culture work was conducted in a sterile tissue
culture hood under standard sterilized conditions. Following the
method reported,24 L. tarentolae promastigotes (ATCC strain 30143)
were grown at room temperature (22 1C) in Brain Heart Infu-
sion (BHI) medium supplemented with 100 units Penicillin and
0.1 mg mLꢀ1 streptomycin (Sigma) and 10 mM hemin. To insure
uniformity in the parasite quantity and age per test cultures,
L. tarentolae promastigotes were initially grown in 60 mL volumes
(Falcon sterile culture flasks), and subsequently dispensed as uni-
form 5 or 10 mL aliquots into smaller (Falcon) sterile flasks for
experimental testing following the method of Katinas et al.18
For each test, a control flask with cells was prepared using
1% (final volume) of dimethyl sulfoxide (DMSO) without any
test compound additions. With addition of 1% DMSO alone, we
found that cell viability is not affected significantly (p 4 0.05)
relative to cells not treated with DMSO. All inhibitors were
prepared as 10 mM as stock solutions in DMSO and used to
achieve the desired final concentration for each test. Care was
taken to ensure that DMSO was added as needed to keep the
final concentration consistently at 1%.
Multiple dose study
The effect of two smaller doses in comparison to a single dose
was evaluated by two sequential dosing of 50 mM inhibitor
during log phase of growth at culture days 3 and 4 compared to
one single dose of 100 mM at day 3. MTT assays were performed
every 24 hours on all cultures starting on the first day of
incubation and ending on the fifth day of incubation. The
growth curves were plotted for each of the treatment groups.
Time of addition
A time of addition test was carried out by treating L. tarentolae
promastigotes with 100 mM of each inhibitory compound at
separate days in their growth curves to assess differing suscepti-
bilities of cells at separate stages. New parasite cultures (10 mL per
flask) were prepared, and they were grown for 7 consecutive days
for the study. MTT analysis on each sample with or without
additions was carried out the day after addition and each day
thereafter. All the experiments were completed in parallel using
the same cell stock, and the viability of each treated sample was
normalized against the control cell culture (DMSO addition only).
MTT viability assay and cell growth curve
MTT assay is a colorimetric assay that relies on the oxidoreductase
enzymes in live cells to reduce the yellow MTT reagent (5 mg mLꢀ1
water) into the purple, insoluble formazan.25 All assays were
carried out in 96 well polypropylene microtiter plates (Thermo-
Fisher) with 100 mL of the cell culture of interest per well
followed by the addition of 10 mL of MTT reagent, then incubated
for 1 hour following the method of Mosmann.25 Absorbance
values were recorded at 595 nm using the Bio-Rad iMark Micro-
plate Reader. The corrected absorbance was calculated by sub-
tracting the absorbance of a blank (BHI medium with 1% DMSO)
from the absorbance of the sample of interest. For all studies, at
least four replicates were performed at each data point and used
to report the mean and standard deviation.
Cell rescue assay using folic acid
To assess the effect that the addition of folate has on the
efficacy of the three inhibitory compounds, cell cultures were
prepared as previously described. In some flasks, 100 mM of
inhibitor, or 100 mM folate, or 100 mM of inhibitor and 100 mM
folate were added on the third day of incubation corresponding
to the early logarithmic growth phase. In all cases, solutes were
dissolved in DMSO and final DMSO concentration was 1%.
Following 24 hours incubation, MTT assays were carried out on
the cell cultures. These treatment groups were compared to
cells that had only the inhibitors added on the third day.
Statistics
L. tarentolae promastigote growth curves with and without
additions of test compounds, were determined by MTT assays
at 24 hours intervals. Corrected A595nm absorbance values,
interpreted to reflect cell viability, are plotted as a function of
cell culture age (days). Values are reported as the mean ꢁ SD for
n = 4 replicates.
All statistical analyses were done with the software GraphPad
Prism 7.03. The unpaired t-tests and one-way ANOVAs were
employed to assess statistical significance, and a p-value of 0.05
was used as the threshold for statistical significance.
Results
Dose response and IC50
Synthesis and spectroscopic characterization
To determine the in vitro efficacy of the new inhibitors, a dose
response study was conducted to determine the IC50 values. On The compounds of interest were synthesized by the reaction of
day three of incubation, the stock culture was dispersed as uni- 1-naphthalenesulfonyl chloride with the appropriate substituted
form 5 mL aliquots into sterile culture flasks, and the flasks were aniline or phenol in the presence of base. Compound A was
treated with test inhibitors in DMSO at 16 different concentrations prepared as previously reported.18 Compound B was prepared by
from 1 to 100 mM and DMSO concentration was kept constant at refluxing the reactants in pyridine for 30 minutes followed by
1% in all flasks. Following a 24 hours incubation, parasite viability aqueous work up. Compound C was prepared by slow addition
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of an aqueous solution of the sodium phenoxide to the sulfonyl
chloride in acetone, followed by aqueous work up. Compounds B
and C were isolated in approximately 65% and 88% yields,
respectively, while compound A was prepared in 47% yield as
previously reported.
Both compounds B and C display 1H NMR spectra that are in
agreement with their structures. All three compounds were
characterized by ESI-HRMS in 50 : 50 methanol : 0.1% formic
acid solution. For all three compounds, the most intense peak
in the mass spectrum was the [M + Na]+ ion (m/z = 364.0620 for
A, m/z = 340.0152 for B, and m/z = 365.0444 for C) followed by
the [M + H]+ and [M + K]+ peaks. Interestingly, even for com-
pound B, which lacks a carboxylic acid group, the most intense
peak was the sodium cation adduct, showing that the sulfonyl
oxygens carry significant negative charges.
Fig. 3 Hydrogen bonding network in compound A, showing carboxylate
and sulfonamide dimer chains. Sulfonamide H-bonding interactions are
shown in purple and carboxylic acid H-bonding interactions are shown in
orange.
infinite 2-dimensional sheets via C–Hꢂ ꢂ ꢂO hydrogen bonds
(Fig. 4b).
Compound C, the only sulfonate ester in the present study,
Crystal structure analysis
%
crystallizes in the triclinic space group P1 with Z = 2 (Fig. 2C). As
Single crystals of all three compounds were grown and subjected
to single-crystal X-ray diffraction studies. The crystal refinement
parameters are given in Table S1 (ESI†) and bond distances and
angles for the compounds are given in Table S2 (ESI†).
with the previous molecules, all of the bond lengths and angles
(Table S2, ESI†) in C are within range for similar compounds.26
The angle between the naphthalene and phenyl rings in C is
63.84(5)1, significantly smaller than the angle in either A or B.
The C1–S11–O14–C15 torsion angle is 71.86(12)1, which is
larger than the analogous torsion angle in compounds A and
B. In the crystal, molecules of C form dimers via the common
R22(8) carboxylate hydrogen-bonding motif (Table S3, ESI† and
Fig. 5a). These dimers form into ribbons via C–Hꢂ ꢂ ꢂO hydrogen
bonds formed between aromatic C–H and the terminal sulfonyl
oxygen atom (Fig. 5b).
Compound A (Fig. 2A) crystallizes in the monoclinic space
group C2/c with Z = 4. All bond lengths and angles are in within
the normal range for other sulphonamides (Table S2, ESI†).26
In compound A, the angle between the naphthalene and phenyl
rings is nearly perpendicular at 88.58(4)1 and the C1–S11–N14–
C15 torsion angle is 65.53(12)1. In the crystal, compound A
forms infinite chains via R22(8) carboxylate dimers and R22(8)
sulfonamide dimers (Fig. 3, Table S3, ESI†). The hydrogen atom
of the carboxylic acid group is disordered over both possible
positions, but the structure was modelled without the disorder.
Crystal structure comparison
The chains are linked together into a 3-dimensional network Fig. 6 shows stick diagram overlays of the molecules to visualize
through C–Hꢂ ꢂ ꢂO hydrogen bonds.
their conformational differences.27 Fig. 6 shows overlay of the
Compound B crystallizes in the monoclinic space group two carboxylate compounds A and C which only differ at atom
P21/n with four molecules in the unit cell (Fig. 2B). All of the bond 14 (an NH group in A and an oxygen atom in C). Looking at the
length and angles are in the normal ranges for sulfonamide bond length data in Table S2 (ESI†), the S–N bond in A [1.6316(13)
molecules (Table S2, ESI†).26 The angle between the phenyl and Å] is about 0.03 Å shorter than the analogous S–O bond in C
naphthalene rings in compound B is 71.88(5)1 with a C1–S11–N14– [1.6021(12) Å]. Additionally, the S–N–C angle in A [125.97(10)1] is
C15 torsion angle of 56.54(14)1, both of which are smaller than approximately 4.51 larger than the corresponding S–O–C angle in C
in compound A. In the crystal lattice, the molecules form [121.47(10)1]. As a result, the phenyl ring is swung further away
infinite C(4) chains through N–HꢂꢂꢂO hydrogen bonds (Table S3, from the naphthalene ring in compound C as compared to
ESI† and Fig. 4a) running along the b-axis. These chains form compound A (Fig. 6). The longer S–Z (Z = O or NH) distance along
Fig. 2 ORTEP diagrams for compounds A, B, and C. Displacement ellipsoids are drawn at the 50% probability level. Hydrogen atoms are drawn as
spheres of arbitrary size.
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with the lack of steric interference between the ortho-methyl group
and the sulfonamide hydrogen, H14, allows the phenyl group in
compound C more freedom to rotate leading to a structure that is
less compact than in compound A.
The sulfonamide compounds A and B (Fig. 6) have similar
conformations. The S–N bond lengths are very similar [1.6316(13) Å
versus 1.6382(15) Å], but the S–N–C bond angle in B is significantly
smaller than the analogous angle in A [125.97(10)1 in A, 124.13(12)1
in B]. This is due to the increased pyramidalization of the nitrogen
in B as compared to A. In B, N14 sits 0.166 Å above the plane
defined by S11/H14/C15, while N14 sits only 0.122 Å above the
same plane in A; the increase in pyramidalization leads to smaller
bond angles. The difference in the degree of pyramidalization
between the two molecules is likely due to the electronic differ-
ences between the substituents on the phenyl rings in the two
compounds. Fig. 6 also shows an overlay of all three compounds.
Fig. 4 Hydrogen bonding networks in compound B, showing (top, a)
sulfonamide C(4) chains and (bottom, b) the 3-dimensional network
formed via C–Hꢂ ꢂ ꢂO hydrogen bonds. Sulfonamide H-bonding interac-
tions are shown in purple and C–Hꢂ ꢂ ꢂO interactions are shown in orange.
Hirshfeld analysis
All three structures were analysed using CrystalMaker.28 Fig. 7
shows the fingerprint plot and Hirshfeld surface mapped over
dnorm for compound A. Looking more closely at the interactions in
A, the most common interactions are HꢂꢂꢂH interactions (35.6%),
followed by OꢂꢂꢂH/HꢂꢂꢂO (27.7%), and CꢂꢂꢂH/HꢂꢂꢂC (24.7%). The
strongest interactions, seen as spikes in the fingerprint plots, are
the OꢂꢂꢂH/HꢂꢂꢂO interactions. The carboxylate and sulfonamide
hydrogen-bonding interactions are seen as the red areas in the
Hirshfeld surface plot for compound A, with the stronger
carboxylate interactions showing up as larger, deeper red spots
than the sulfonamide interactions.
Fig. 8 shows the fingerprint plot and Hirshfeld surface for
compound B. The first different to note between compounds B
and A, are that the hydrogen-bond spikes in the fingerprint plot
for B are shorter and wider than the spikes in the plot for A.
This is due to the lack of a carboxylic acid group in compound
B, which only has N–Hꢂ ꢂ ꢂO hydrogen bonds from the sulfona-
mide group, which are generally weaker than the N–Hꢂ ꢂ ꢂO
hydrogen bonds of carboxylic acid groups. The major interac-
tions in B are Hꢂ ꢂ ꢂH (33.4%), Cꢂ ꢂ ꢂH/Hꢂ ꢂ ꢂC (26.2%), Hꢂ ꢂ ꢂO/
Oꢂ ꢂ ꢂH (18.1%), and Hꢂ ꢂ ꢂCl/Clꢂ ꢂ ꢂH (11.5%) interactions. The
decrease in the occurrence of Hꢂ ꢂ ꢂO/Oꢂ ꢂ ꢂH interactions in B
compared to compounds A and C is mainly due to the lack of
the carboxylic acid group. In the Hirshfeld surface plot for B,
the strongest interactions, indicated by larger red areas on the
surface, are located near the sulfonamide hydrogen and oxygen
atoms involved in the N–Hꢂ ꢂ ꢂO hydrogen bonding. Additionally,
there is a small area of red near H19, which is involved in
Fig. 5 Hydrogen bonding networks in compound C, showing (top, a) the
carboxylate dimer and (bottom, b) the 2-dimensional sheets formed via
C–Hꢂ ꢂ ꢂO hydrogen bonds. Carboxylic acid H-bonding interactions are
shown in orange, and C–Hꢂ ꢂ ꢂO interactions are shown in dark green.
Fig. 7 Fingerprint plot and two views of the Hirshfeld surface for com-
pound A.
Fig. 6 Structure overlays for compounds
C (blue).
A (green), B (red), and
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Fig. 8 Fingerprint plot and two views of the Hirshfeld surface for com-
pound B.
Fig. 10 L. tarentolae promastigote growth curves in vitro at differing initial
inoculation cell concentrations. (a) Low initial cell volume inoculation;
(b) Two-times higher initial cell volume inoculation.
number of cells used shifts the growth curve. Therefore, all
experiments using test compounds were compared to control
cells from the same original stocks so that cell number and
viability were controlled. It is clear that the cells on days 0 to 2,
days 2 to 4, day 5, and days 6 to 7 are in the lag phase, log phase,
stationary phase and senescence phase, respectively, for cells
grown from a low initial volume inoculation. Two days after a
100 mM addition of compound, compound A showed an 80%
inhibition relative to the control cells while compounds B and
C showed a 95% inhibition relative to control cells.
Fig. 9 Fingerprint plot and two views of the Hirshfeld surface for com-
pound C.
C–Hꢂ ꢂ ꢂO hydrogen bonding to link the chains into the two-
dimensional sheets in the crystal structure.
The fingerprint plot and Hirshfeld surface for compound C
are shown in Fig. 9. The hydrogen bond spikes in the fingerprint
plot of C are narrower at the base then those in the fingerprint
plot of A due to the lack of N–Hꢂ ꢂ ꢂO hydrogen bonds in the
sulfonate ester. Otherwise, the overall shape for the fingerprint
plot of C is very similar to that of A. The important interactions
in C are Hꢂ ꢂ ꢂH (38.8%), Hꢂ ꢂ ꢂO/Oꢂ ꢂ ꢂH (31.2%), and Hꢂ ꢂ ꢂC/Cꢂ ꢂ ꢂH
(16.6%) interactions. The Hirshfeld surface plot for compound C
shows large red areas corresponding to the carboxylate hydrogen
bonding interactions. Also, smaller red areas are seen near the
terminal sulfonate oxygen atoms and H17 and H21C, which
correspond to the C–Hꢂ ꢂ ꢂO hydrogen bonds the link sulfonate
ester dimers into ribbons in the crystal.
Comparing the three structures, Hꢂ ꢂ ꢂH interactions are the
predominant interactions, ranging from 33.4% to 38.7% of the
intermolecular interactions in the crystals (Fig. S1, ESI†). Inter-
estingly, the Hꢂ ꢂ ꢂO/Oꢂ ꢂ ꢂH interactions have the greatest amount
of variability between the three compounds. Compound B has
the lowest amount of Hꢂ ꢂ ꢂO/Oꢂ ꢂ ꢂH interactions at 18.1%, while
compound C has the largest, at 31.2%. It is somewhat interesting
that the Hꢂ ꢂ ꢂO/Oꢂ ꢂ ꢂH interactions of C make up a larger percen-
tage of the overall intermolecular interactions than in compound
A, given that A has an additional hydrogen-bond donor group
(N–H). However, the more open nature of the C–SO2–O–C moiety
in C compared to the C–SO2–NH–C moiety in A allows greater
hydrogen-bonding access to terminal sulfonyl oxygen atoms for
C–Hꢂ ꢂ ꢂO hydrogen bonds. None of the compounds display
significant p-stacking between naphthalene or benzene rings,
as evidenced by the low percentage of Cꢂ ꢂ ꢂC contacts in the
Hirshfeld surface analyses (A: 6.3%, B: 6.2%; C: 8.1%).
Dose response and IC50. Dose response of L. tarentolae in the
logarithmic phase for each of the test compounds was assayed
at 16 concentrations ranging from 1 mM to 100 mM (Fig. 11). The
corrected responses were plotted as a function of concentra-
tions, and IC50 values were determined within 95% confidence
interval. The IC50 for the benchmark compound A was deter-
mined as 49 mM (Fig. 11a). For the chloro-substituted sulfonamide
B, the IC50 was determined to be 9.5 mM, which is 5 times lower
than that of benchmark compound A (Fig. 11b). Treatment with
the sulfonate ester, C, provided an IC50 of 7.4 mM (Fig. 11c).
Interestingly, the sulfonate ester C appears to reach a local
maximum of inhibition between 20 and 40 mM where it inhibits
cell viability at 75%. However, as the concentration of this
compound was increased, inhibition decreased until 55–65 mM
of the compound only inhibited about 50% of parasite viability.
Then, as concentration was increased to 100 mM, inhibition
increased to 85% reduction in cell viability. This non-typical dose
response is highly unusual but has been reported by others for
endocrine disruptors.29,30 The range of concentrations where B
reaches 75% inhibition coincides with when it begins crystalizing;
we observed noticeable crystallization in vitro in concentrations
above 30 mM. The poor solubility may negatively affect its efficacy
at higher concentration. Nevertheless, B is much more inhibitory
than benchmark compound A, despite the poor solubility. Both
compounds B and C have IC50 values in the range reported by
Taylor et al.31 of the EC50 value for amphotericin B effectiveness
against axenic Leishmania amazonensis which was 9.2 ꢁ 2.1 mM.
Dose accumulation study. To determine the effect of dose
accumulation, the dose additive study was started during the cell
logarithmic phase with a relatively lower cell concentration culture.
Evaluation of inhibitory effects on L. tarentolae
Growth curve. Typical growth curves for L. tarentolae are One 50 mM dose of compound or DMSO on day 3 of the cell growth
shown in Fig. 10 and indicate that a difference in the initial (Fig. 12) was followed by an equal second dose (50 mM) on day 4;
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Fig. 11 Dose response curves from 0–100 mM (16 concentrations, n = 4) in 1% DMSO after 24 hours incubation. The points were fit in Graphpad Prism
7.03, and all IC50 values were reported with the 95% confidence interval: (a) dose response curve for compound A; (b) dose response curve for
compound B; (c) dose response curve for compound C.
this was compared to one single dose of 100 mM on day 3 (Fig. 12). 100 mM. For control tests, 1% DMSO was added on any of the
Results show that on day 5 the sulfonate ester C and benchmark A first five days of incubations and in all the tests, the DMSO did
are dose additive relative to the single 100 mM dose. Remarkably, not affect any statistically significant change in parasite viability
after the first 50 mM dose the novel sulfonate ester effectively (p o 0.05, data not shown). The data were plotted as percent of
inhibits the cell growth comparable to the effectiveness of 100 mM control viability as a function of incubation time in days (Fig. 13).
one dose of the same compound, and both reached the lowest cell Overall, the novel chlorosulfonamide B and sulfonate ester lead to
viability (Fig. 13, III, 98–99%, p o 0.05) after 5 days. Surprising, cell inhibition significantly better than the benchmark compound,
chlorosulfonamide B reveals a different pattern with less reduction sulfonamide A.
in cell viability 24 hours after the 100 mM treatment, as well as after
Cells in early log phase appeared to be more sensitive to
the first 50 mM treatment. This delayed inhibition, compared to inhibition by all three compounds than cells in senescence.
compounds A and C, one day after treatment with 100 mM of the However, cells treated in log phase with compound A appeared
compound could be related to the lower water solubility of to have a late stage recovery in cell viability.
compound B. As previously observed for the dose response study
Effects of folic acid addition. Leishmania are known to be
with chlorosulfonamide B, crystals appeared after the first dose auxotrophic for folate and pterin, relying on their mammalian
and persisted after the second dose, similar to the 100 mM dose hosts to supply these essential cofactors.11,32 Sulfonamides are
test. We suspect the poor solubility may negatively affect the known competitive inhibitors for enzymes that utilize PABA to
cellular uptake and effectiveness of sulfonamide B.
synthesize 7,8-dihydrofolate from pterin, which is further meta-
Time of addition. The time of addition study is designed to bolized to tetrahydrofolate.33 Tetrahydrofolate is an important
evaluate inhibitory effectiveness for compounds A, B, and C one-carbon (1C) donor and acceptor in the 1C-metabolic pathway
additions at different times or phases during the cell culture and is essential in the biosynthesis of thymidine and purines. We
cycle from day one (lag phase) to day five (stationary phase) at hypothesized that our novel sulfonamides may also be inhibiting
Fig. 12 Dose accumulation study with compounds added on days 3 and 4 totalling 100 mM of compound compared to a single dose of 100 mM on day 3:
(a) compound A; (b) compound B; (c) compound C. The black triangles are 1% DMSO control, the blue diamonds are a single 100 mM addition on day 3,
and the red triangles are 50 mM additions on each of day 3 and day 4.
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Fig. 13 Effect of time of addition for inhibitory compounds at 100 mM: (a) compound A; (b) compound B; (c) compound C.
Table 1 Comparison of IC50 values (mM) of the studied compounds and
reference compounds
Compound
IC50 value (mM)
Ref.
Species studied
A
B
C
49
9.5
7.4
9.2 (EC50
31.4
This work
This work
This work
31
16
16
L. tarentolae
L. tarentolae
L. tarentolae
L. amazonesis
L. amazonesis
L. amazonesis
Amphotericin B
Miltefosine
NiQNBSa
)
27
a
N-Isoquinolin-1-yl-4-nitrobenzenesulfonamide.
the early log phase of in vitro cell growth. Based on IC50 values,
L. tarentolae promastigotes are much more sensitive to the newly
synthesized chlorosulfonamide B and sulfonate ester C than to our
benchmark sulfonamide A; the IC50 values of the novel molecules
are at least 5 times lower than the benchmark A compound.
Interestingly, the dose response curve of compound C does
not follow the classical shape that is usually observed in most
dose response curves, including compounds A and B (Fig. 11c).
Essentially, this means that slope of the dose response plot
changes sign at least once in the tested concentration range.29
In this instance, the curve changes slope in two places; increasing
at 20–25 mM and decreasing at 60–65 mM. Consequently, 20 mM of
C is more toxic to the parasites than double or even triple that
concentration. This unusual (non-monotonic) dose response rela-
tionship has been previously reported for other compounds but
there is no universal mechanism that explains these results.30 We
speculate that in the intermediate concentrations, the parasites
detect the toxic compounds and have some as yet unknown
compensatory response(s). At the lower end (1–20 mM) of the
Fig. 14 Effects of folate addition on cell viability 24 hours after addition of
100 mM folic acid (FA), or 100 mM test compound A, B, or C, or simulta-
neous addition of 100 mM folic acid and 100 mM test compound to cell
cultures. Data are the mean ꢁ SD for n = 4 replicates.
these enzymes, and we proposed that if the addition of folic acid
rescues cells from inhibition by these molecules we bypass the
requirement for pterin metabolism for dihydrofolate production.
Based on the relative cell viability assay, folate addition to the
control cells increased growth by 13% (Fig. 14). However, folate
addition to cells with test compounds only modestly protects the
cells from the inhibitory effects of compounds A, B, and C which
alone inhibit 80, 70 and 90%, respectively. This suggests that
these compounds are not strong folate pathway antagonists and
other mechanisms are involved.
Discussion
With all three compounds tested in this study, a single 100 mM scale, the concentration may not be high enough to trigger the
dose substantially reduced in vitro Leishmania tarentolae viability as compensatory response. At the higher end, the concentration may
shown in Table 1. The IC50 values reported for our novel sulfona- overwhelm the parasites’ compensation efforts. Although we have
mide compounds and sulfonate ester compound are substantially no indication that the sulfonate ester behaves as an electrophile,
lower than those reported by Peixoto and Beverley11 for different future work will involve testing whether this is important in the
sulfonamide and sulfone compounds which were in the range of mechanism(s) of cell viability inhibition. Further investigation is
150 mM or higher. The new compounds tested here thus have good necessary to better understand this mechanism and determine
anti-Leishmania properties, especially for compounds B and C ways to improve the molecule to resist this cellular response.
from which the parasites do not appear to easily recover (as shown
Since compounds B and C have comparable negative effects
in Fig. 14). This is especially true when the compounds are given in on L. tarentolae in vitro, but the chlorinated compound (B) is
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likely limited by its observed lower solubility at concentrations senescence phase. This, coupled with the observation that the
above 30 mM, we speculate that compound B could be even compounds were most effective in the early logarithmic phase,
more inhibitory than shown in these experiments. Indeed, we suggests interference with cell proliferation as a possible mecha-
saw that in the dose response, time of addition, and folate nism of inhibition. In the literature, parasite clumping, and shape
effects studies, the effect of compound B as concentration change are identifiers for late stage Leishmania promastigotes
increased from 50 mM to 100 mM did not increase as expected. (stationary and senescence phases) as nutrients are depleted and
Additionally, B appears unable to induce more than B95% waste increases.40 In the cells treated with the three compounds
inhibition at any concentration after 24 h unlike the other two early in their growth curves in the time-of-addition study, we did
compounds, which could also be attributed to a solubility issue. not observe clumping several days after treatment, but we did
After 48 hours, the inhibitory effect of B increases, apparently see the parasites change shape to a more circular morphology,
overcoming its solubility problem. We speculate that this is due reflecting cell death.18
to the continuous dissolution of the crystalized compound as
There is a five-fold improvement in IC50 values between
the aqueous phase molecule interacts with the parasites. When A and B as shown in Table 1. It appears substitution of the
two days or more incubation is permitted, compound B cumu- methyl group by chlorine plays an important role in increasing
latively inhibits parasite viability more than compound A and the compound’s inhibitory ability. Halogenation has been
comparably with compound C. Fortunately, at lower concentra- reported to improve the protein-binding ability of agonists
tions near its IC50, B is a very good inhibitor, and it appears to through the formation of strong non-bonding interactions,
be sufficiently soluble. To gain better access to the abilities of including halogen bonds.41,42 In the crystal structure of B,
chlorosulfonamides, future work should focus on increasing there are no significant Clꢂ ꢂ ꢂCl interactions, but Clꢂ ꢂ ꢂH inter-
their solubility which may include synthesizing derivatives that actions are significant, accounting for 11.5% of the total inter-
include an addition carboxyl or other hydrophilic group.34
molecular interactions. This may point to Clꢂ ꢂ ꢂH interactions
Folic acid pathways are increasingly attractive targets for being important in the inhibitory mechanism for compound B.
Leishmania therapies.6,35 Leishmania may be auxotrophic for Synthesis and testing of F, Br, I and CF3 substituted analogues
folate, however they can scavenge the molecule from its growth would grant us insight into the structure–activity relationships
medium.11,36 The data from our folate study indicate that the of size and polarity. We can confidently infer that chloro- and
addition of folate along with our three inhibitors does not perhaps other halogen sulfonamides or sulfonate esters are
protect the parasites, which suggests a mechanism that may promising compounds for future SAR studies and inhibitory
not involve substantial inhibition of one or more folate pathway testing.
enzymes. This is in agreement with Peixoto and Beverley,11
The IC50 value of the sulfonate ester C is also lower than that
who reported active sulfa drugs effects were not relieved by of sulfonamide A. We can attribute the significant improve-
folate addition. Folate is produced in the parasites by two ment in inhibitory activity to the substitution of the NH for an
enzymes: dihydrofolate reductase (DHFR) and pteridine reduc- oxygen. The substitution decreases the polarity and steric
tase 1 (PTR1).36 To effectively inhibit the folate pathway, both hindrance, potentially giving more chance for conformation
enzymes must be inhibited. Other likely sulfonamide targets in adaption to a putative active site. In addition to decreasing
Leishmania are reported to include inhibition of carbonic polarity, the substitution changes the linking atom from a
anhydrase37 and disruption of cell proliferation by disruption hydrogen-bond donor group to a potential hydrogen-bond
of microtubule activity.38
acceptor group. Altering the linkage between the naphthalene
Our additive study results indicated that for A and C, but not and the benzene with and without halogen substitution on the
for B, two sequential doses, 24 hours apart, of 50 mM was benzene are of great interest to further evaluate the potency of
equivalent to a single 100 mM dose, suggesting that the effects the molecules as inhibitors of Leishmania.
of the compounds are additive. This is a desirable trait for
Fig. 15 presents a summary of our observations on the
in vivo leishmanial treatments because multiple small doses inhibitory ability of naphthalene sulfonyl compounds studied
over time generally cause fewer side effects than a single large by us. First, substitution of in the para-position of the phenyl
dose.39
ring, Y, does not appear to significantly alter the potency of the
Our time-of-addition study allowed us to conclude that the molecules, but addition of a carboxylic acid group greatly
parasites were consistently most vulnerable to the compounds enhances the water solubility. Next, the substituent at the X
in their lag and logarithmic phases, especially in the early position (ortho on the phenyl ring), does appear to be important
logarithmic phase, which was day 2 in these experiments. to function. Presence of larger groups, such as methoxy and
Though we saw that day 2 additions were most effective for thiomethyl, or a smaller hydrogen group, at the X position
24 hours inhibition periods, day 1 additions were most effective lowers the effectiveness of the sulfonamide molecules overall.18
in the long term. However, those cells that were treated with a As shown in this and our previous study, compounds with
single dose of compound A early in their growth curve recovered chloro or methyl groups in the X position are good inhibitors of
considerably after several days. Neither compound B nor C Leishmania. Additionally, placement of either an oxygen atom
treated cells exhibited noticeable recovery back to control cell levels or an N–H group at position Z, linking the naphthene sulfonyl
indicating more complete viability inhibition of the parasites. Each group and the aryl ring, appears to lead to molecules that are
compound tested was less effective when added in the stationary or excellent inhibitors of Leishmania. Finally, unpublished results
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Fig. 15 Generic structure of naphthalene sulfonyl compounds studied by
our group, with important areas of substitution highlighted. Our studies
indicate that the position of substitution on the naphthalene ring (orange
oval) and the size of the ortho substituent, X, are important for anti-
leishmanial activity. The identity of the group in the para-position, Y, and
the identity of the Z group (O or NH) does not affect the potency.
13 C. Barea, A. Pabon, D. Castillo, M. Zimic, M. Quiliano,
S. Galiano, S. Perez-Silanes, A. Monge, E. Deharo and
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from our laboratories have shown that 2-naphthalene sulfonyl
compounds are poor inhibitors of Leishmania.
Conclusions
We have demonstrated that the two newly synthesized novel
molecules, naphthalene chlorosulfonamide B and sulfonate
ester C, are potent inhibitory compounds against Leishmania
tarentolae promastigotes in vitro. The high toxicity of the novel
compounds B and C against L. tarentolae brings new perspec-
tive to structure–activity relationships that are beneficial for the
treatment of leishmaniasis. We believe that continued SARs
studies on halogen substituted sulfonamides and sulfonate
esters is a fruitful direction to develop new therapies toward
treating this disease.
´
16 C. Galiana-Rosello, P. Bilbao-Ramos, M. A. Dea-Ayuela,
´
´
´
´
˜
M. Rolon, C. Vega, F. Bolas-Fernandez, E. Garcıa-Espana,
´
J. Alfonso, C. Coronel and M. E. Gonzalez-Rosende, J. Med.
Chem., 2013, 56, 8984–8998.
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18 J. Katinas, R. Epplin, C. Hamaker and M. A. Jones, Anti-Infect.
Agents, 2017, 15, 57–62.
Conflicts of interest
˜
˜
´
19 V. M. Taylor, D. L. Munoz, D. L. Cedeno, I. D. Velez,
M. A. Jones and S. M. Robledo, Exp. Parasitol., 2010, 126,
471–475.
There are no conflicts to declare.
20 Enraf-Nonius, CAD4 Express Software, Delft, The Netherlands,
1994.
21 K. Harms and S. Wocadlo, XCAD-4: Program for Processing
CAD-4 Diffractometer Data, University of Marburg, Germany,
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Acknowledgements
CGH wishes to thank Illinois State University (URG-FRA award)
and we all thank the Illinois State University Chemistry Depart-
ment for financial support. We also thank the NSF (Grant no.
CHE-1039689) for funding the X-ray diffractometer.
22 A. C. T. North, D. C. Phillips and F. S. Mathews, Acta
Crystallogr., 1968, A24, 351–359.
23 G. M. Sheldrick, Acta Crystallogr., 2015, A71, 3–8.
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