Journal of Medicinal Chemistry
Article
NMR (400 MHz, DMSO-d6) δ(ppm): 8.63 (1H, br s), 7.80 (2H, d, J
= 7.82 Hz), 7.56 (2H, d, J = 8.35 Hz), 7.46 (2H, d, J = 7.85 Hz), 7.33
(2H, br s), 7.28 (2H, d, J = 8.34 Hz), 6.22 (1H, br s), 3.41−3.40 (2H,
m), 2.87 (2H, t, J = 6.61 Hz); 13C NMR (100 MHz, DMSO-d6)
δ(ppm): 155.8, 144.6, 143.0, 141.3, 138.1, 130.0, 126.6, 120.8, 84.4,
41.1, 36.4; MS (ESI positive) m/z: 446 [M + H]+.
this work should, in the longer term, help in the identification
of novel, lead molecules that selectively inhibit SmCA and
could become new, clinically useful, anti-schistosome ther-
apeutics to impede vital parasite biochemistry and debilitate
these important global pathogens.
Carbonic Anhydrase Inhibition. An applied photophysics
stopped-flow instrument was used to assay the CA-catalyzed CO2-
hydration activity.30 Phenol red (at a concentration of 0.2 mM) was
used as an indicator, working at an absorbance maximum of 557 nm,
with 20 mM HEPES (pH 7.4) as a buffer and 20 mM Na2SO4 (to
maintain constant ionic strength), following the initial rates of the
CA-catalyzed CO2-hydration reaction for a period of 10−100 s. The
CO2 concentrations ranged from 1.7 to 17 mM for the determination
of the kinetic parameters and inhibition constants.18 The enzyme
concentrations ranged between 5 and 12 nM. For each inhibitor, at
least six traces of the initial 5−10% of the reaction were used to
determine the initial velocity. The uncatalyzed rates were determined
in the same manner and subtracted from the total observed rates.
Stock solutions of the inhibitor (0.1 mM) were prepared with
distilled−deionized water and dilutions up to 0.01 nM were carried
out thereafter with the assay buffer. The inhibitor and enzyme
solutions were pre-incubated together for 15 min at room
temperature prior to the assay, to allow for the formation of the
E−I complex. The inhibition constants were obtained by non-linear
least squares methods using PRISM 3 and the Cheng−Prusoff
equation as reported earlier and represent the mean from at least
three different determinations. All CA isoforms were recombinant
proteins obtained in house, as reported earlier.16,34−36
Parasites and Mice. The Puerto Rican strain of S. mansoni was
used. Adult male and female parasites were recovered by perfusion
from female 6−8 week old Swiss Webster mice that were infected
with ∼90 cercariae, 9 weeks previously.37 All protocols involving
animals were approved by the Institutional Animal Care and Use
Committees (IACUC) of Tufts University. Pilot experiments, as
described,38 determined that the maximum tolerable dose for the
mice of compounds 7 and 16 was 100 mg/kg and for 22a was 50 mg/
kg. At day 42, 44, and 46 post infection, groups of mice were
administered each compound intraperitoneally at this dosage. Control
mice were treated with carrier (20% DMSO) alone. Two weeks after
the final treatment, all mice were perfused and worm numbers
recovered per animal were calculated.
Crystallization and X-ray Data Collection. The SmCA enzyme,
purified in the recombinant form, as described previously,16 was
crystallized at 296 K using the sitting-drop vapor-diffusion method in
96-well plates (CrystalQuick, Greiner Bio-One, Germany). Drops
were prepared using 1 μL protein solution mixed with 1 μL reservoir
solution and were equilibrated against 100 μL precipitant solution.
The concentration of the protein was 10 mg mL−1 in 50 mM Tris pH
8.3. Initial crystallization conditions were found using the JCSG-plus
screen (Molecular Dimensions) and were optimized. Crystals of the
native protein were prepared using a solution of 0.2 M zinc acetate
dihydrate, 0.1 M imidazole pH 8.0, and 20% w/v PEG 3000, and they
belonged to the primitive tetragonal space group P41212. Crystals of
the complex with compounds 18 and 8 were prepared using a solution
of 20% PEG 3350, 0.2 M di-ammonium hydrogen citrate. Crystals of
the complex with 22a−b, 23, and 27 were obtained using 20% PEG
6000 and 0.1 M citrate (pH 5.0). The crystals belonged to the
primitive trigonal space group P3221. The complexes were prepared
by soaking the SmCA native crystals in the mother liquor solution
containing the inhibitors at a concentration of 10 mM for two days.
Crystals of hCA II were obtained using the hanging-drop vapor-
diffusion method using a 24-well Linbro plate. 2 μL of 10 mg/mL
solution of hCA II in 20 mM Tris−HCl (pH 8.0) was mixed with 2
μL of a solution of 1.5 M sodium citrate and 0.1 M Tris pH (8.0) and
was equilibrated against the same solution at 296 K. The complexes
were prepared by soaking the hCA II native crystals in the mother
liquor solution containing the inhibitors at a concentration of 10 mM
for two days. All crystals were flash-frozen at 100 K using a solution
obtained by adding 15% (v/v) glycerol to the mother liquor solution
EXPERIMENTAL SECTION
■
General. Anhydrous solvents and all reagents were purchased from
Sigma-Aldrich, VWR, and TCI. All reactions involving air- or
moisture-sensitive compounds were performed under a nitrogen
atmosphere. Nuclear magnetic resonance (1H NMR and 13C NMR)
spectra were recorded using a Bruker Advance III 400 MHz
spectrometer in DMSO-d6 or CDCl3. Chemical shifts are reported
in parts per million (ppm) and the coupling constants (J) are
expressed in Hertz (Hz). Splitting patterns are designated as follows:
s, singlet; d, doublet; t, triplet; m, multiplet; brs, broad singlet; and dd,
double of doubles. The assignment of exchangeable protons (NH)
was confirmed by the addition of D2O. Analytical thin-layer
chromatography (TLC) was carried out on Merck silica gel F-254
plates. Flash chromatography purifications were performed with
Merck silica gel 60 (230−400 mesh ASTM) as the stationary phase
and ethyl acetate, n-hexane, acetonitrile, and methanol were used as
eluents. The solvents used in MS measurements were acetone,
acetonitrile (Chromasolv grade), purchased from Sigma-Aldrich
(Milan, Italy), and Milli-Q water 18 MΩ, obtained from the
Millipore’s Simplicity system (Milan, Italy). The mass spectra were
obtained using a Varian 1200L triple quadrupole system (Palo Alto,
CA, USA) equipped with a electrospray source (ESI) operating on
both positive and negative ions. Stock solutions of analytes were
prepared in acetone at 1.0 mg mL−1 and stored at 4 °C. Working
solutions of each analyte were freshly prepared by diluting stock
solutions with a mixture of Milli-Q H2O/ACN 1/1 (v/v) up to a
concentration of 1.0 μg mL−1. The mass spectra of each analyte were
acquired by introducing, via syringe pump at 10/L min−1, the working
solution. The raw data were collected and processed using Varian
workstation, version 6.8, software. All compounds reported here are of
>95% purity.
General Procedure for the Synthesis of Seleno, Thio, and
Ureido Derivatives (5−19, 22a−b, and 23). The appropriate
isoselenocyanate (1a−i), thiocyanate (20b, 21), or isocyanate (20a)
(1 equiv) was dissolved in acetonitrile and treated with the
corresponding benzenesulfonamide 2−4 (1 equiv). The mixture was
stirred overnight at r.t, quenched with H2O, and the readily formed
precipitate was collected by filtration and dried in air to afford the
titled selenourea 5−19, ureido 22a, and thioureido 22b and 23. The
experimental data were in agreement with the reported data.29,31
4-(2-(3-(4-Iodophenyl)thioureido)ethyl)benzenesulfonamide
(23). Following the general procedure, the product was a white solid
1
23 with 71% yield. H NMR (400 MHz, DMSO-d6) δ(ppm): 9.65
(1H, br s), 7.90 (1H, br s), 7.81 (2H, d, J = 8.08 Hz), 7.66 (2H, d, J =
8.54 Hz) 7.48 (2H, d, J = 8.09 Hz), 7.35 (2H, br s), 7.23 (2H, d, J =
8.53 Hz) 3.76 (2H, m), 2.99 (2H, t, J = 7.14 Hz); 13C NMR (100
MHz, DMSO-d6) δ(ppm): 181.2, 144.4, 143.1, 140.0, 138.1, 130.1,
126.7, 126.0, 88.9, 45.8, 35.0; MS (ESI positive) m/z: 462 [M + H]+.
Synthesis of Phenyl (4-iodophenyl)carbamate (26). 4-Iodoaniline
(1 equiv) and phenyl chloroformate (1 equiv) were added in acetone
at 0 °C. Subsequently, K2CO3 (1.3 equiv) was added and the mixture
was stirred for 30 min. The solvent was removed under a vacuum,
water was added, and the precipitate was filtered off. The product was
1
a light purple solid 26 with yield 90%. H NMR (400 MHz, CDCl3)
δ(ppm): 7.26 (2H, d, J = 8.44 Hz), 7.37 (2H, apt, J = 7.72 Hz), 7.26−
7.19 (3H, m), 7.15 (2H, d, J = 8.09 Hz), 6.89 (1H, br s); 13C NMR
(100 MHz, CDCl3) δ(ppm): 151.7, 150.8, 138.4, 137.6, 138.1, 129.9,
126.2, 121.9, 121.0; MS (ESI positive) m/z: 340 [M + H]+.
4-(2-(3-(4-Iodophenyl)ureido)ethyl)benzenesulfonamide (27). A
mixture of (4-iodophenyl)carbamate (26) (1 equiv) and 4-(2-
aminoethyl)benzenesulfonamide (4) (1 equiv) in acetonitrile was
stirred at reflux overnight. Then, water was added, and the precipitate
was filtered off. The product was a white solid 27 with yield 80%. 1H
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J. Med. Chem. 2021, 64, 10418−10428