3
38
M. Bozdag et al. / Bioorg. Med. Chem. 22 (2014) 334–340
the same range (hCA IX and XII, which are probably the most inter-
septet; t, triplet; q, quadruplet; m, multiplet; br s, broad singlet;
dd, double of doubles, appt, apparent triplet, appq, apparent
quartet. The assignment of exchangeable protons (OH and NH)
30
esting drug targets in the human CA family of enzymes). Thus,
the ring was important in this case for orientating the inhibitor
scaffold in a diverse manner compared to the classical benzenesul-
fonamide derivatives. The tail on the other hand led to an enhance-
ment of the enzyme inhibitory activity compared to the parent
compound 4, and through its interactions at the rim of the active
site, ensured a selectivity profile for compound 5. This is thus a
highly interesting example that combining the ring and the tail ap-
proach, interesting families of compounds can be obtained as
inhibitors of various types of CAs. Work is in progress in our labo-
ratories for obtaining a large series of such derivatives and to
investigate in detail their properties.
2
was confirmed by the addition of D O. Analytical thin-layer chro-
matography (TLC) was carried out on Merck silica gel F-254 plates.
Flash chromatography purifications were performed on Merck
Silica gel 60 (230–400 mesh ASTM) as the stationary phase and
ethylacetate/n-hexane or MeOH/DCM were used as eluents.
Melting points (mp) were carried out in open capillary tubes and
are uncorrected.
4.1.1. 2-Thiol-5-acetaminopyridine (1)
2-Mercapto-5-nitropyridine (Bionet Research) (10 g, 64 mmol)
was suspended in 100 mL of water and sodium hydrosulfite
(
(
2
39 g, 224 mmol) was added with stirring at 0 °C. Acetic anhydride
8.4 mL, 89.6 mmol) was then added. The mixture was stirred for
h, always kept in ice, and the yellow precipitate was filtered,
3
. Conclusion
-(3-Tosylureido)pyridine-2-sulfonamide (ts-PySA) and 4-tosy-
5
washed with water, and dried to give 1 as a bright yellow solid
(
(
lureido-benzenesulfonamide (ts-SA) only differ by the substitution
of a CH group from the benzene ring of the second compound by a
nitrogen in the first one, but they have very different inhibitory
properties against the many isoforms of CA found in humans,
hCA I–XIV. By means of X-ray crystallography on the hCA II adducts
of the two compounds these differences have been thoroughly
understood and rationalized. As all sulfonamides, the two com-
pounds bind in deprotonated form to the Zn(II) ion from the en-
zyme active site and their organic scaffolds extend throughout
the cavity, participating in many interactions with amino acid
residues and water molecules. However the pyridine derivative
ts-PySA undergoes a tilt of the heterocyclic ring compared to the
benzene analog, which leads to a very different orientation of the
two compounds when bound within the enzyme active site. This
tilt also leads to a clash between a carbon atom from the pyridine
ring of ts-PySA and the OH moiety of Thr200, leading to less
effective inhibitory properties of the pyridine versus the benzene
sulfonamide derivative. Indeed, ts-SA is a promiscuous, low nano-
molar inhibitor of 7 (hCA I, II, VII–XIV) out of 10 hCA isoforms,
whereas the pyridine sulfonamide ts-PySA is a low nanomolar
inhibitor only of the tumor-associated hCA IX and XII, being less
effective against other 9 isoforms. Thus, a difference of one atom
1
6
5.4 g, 50% yield). H NMR (DMSO-d ) d: 2.01 (s, 3H); 7.22–7.36
m, 2H); 8.22 (s, 1H); 10.07 (s, 1H).
4
.1.2. 5-Acetamino-2-pyridinesulfonyl chloride (2)
The 2-thiol-5-acetaminopyridine 1 (5.16 g, 30.6 mmol) was
added to 86 mL of H SO concd and cooled to 0 °C. Sodium
2
4
hypochlorite (13%, 146 mL, 306 mmol) was added dropwise. The
reaction was stirred for 1 h at 0 °C. The resulting solution was di-
luted with water and ice and the precipitate was filtered under
vacuum to obtain 2 as a white solid (4.97 g, 69% yield). 1H NMR
(
DMSO-d
6
) d: 2.15 (s, 3H); 8.12 (d, J = 8.6 Hz, 1H); 8.46 (d,
J = 8.6 Hz, 1H); 9.04 (s, 1H); 11.17 (s, 1H).
4
.1.3. 5-Acetamino-2-pyridinesulfonamide (3)
To a cooled solution (0 °C) of 2 (3.5 g, 15 mmol) in acetonitrile
(
1.6 mL) aqueous ammonia (4.8 mL) was added dropwise. The
reaction was stirred for 30 min at rt. The resulting solution was di-
luted with water and extracted with EtOAc. The aqueous phase
was filtered to collect the precipitate, a white solid (1.06 g). The or-
ganic phase was evaporated, treated with acetone and filtered to
collect 3 as a white solid (476 mg). The two solid fractions were re-
united to give 1.53 g of the desired compound 3 (47% yield). 1
H
(
N vs CH) in two isostructural sulfonamides leads to drastic differ-
NMR (DMSO-d
1
1
6
) d: 2.11 (s, 3H); 7.32 (s, 2H); 7.88 (d, J = 8.6 Hz,
= 8.6 Hz, J = 2.3 Hz, 1H); 8.80 (d, J = 2.3 Hz, 1H);
ences of activity, phenomenon understood at the atomic level
through the high resolution crystallographic structure and kinetic
measurements reported in the paper. Combining the tail and the
ring approaches in the same chemotype leads to isoform-selective,
highly effective sulfonamide CA inhibitors.
H); 8.23 (dd, J
0.53 (s, 1H).
1
2
4
.1.4. 5-Aminopyridine-2-sulfonamide (4)
Into a round-bottom flask equipped with a stir bar and N were
2
2
placed 3 (1.06 g, 4.9 mmol), H O (0.5 mL), and EtOH (11 mL). Con-
4
4
. Experimental protocols
centrated HCl (4.4 mL) was added, and the mixture was refluxed
for 3 h. The reaction mixture was cooled to rt, quenched with sat-
urated NaHCO3 solution and extracted with EtOAc. The organic
.1. Chemistry
2 4
layer was washed with water and brine, dried over Na SO , filtered,
Anhydrous solvents and all reagents were purchased from
and concentrated in vacuo to afford 4 (583 mg, 68.5%) as a pale yel-
1
Sigma–Aldrich, Alfa Aesar and TCI. All reactions involving air- or
moisture-sensitive compounds were performed under a nitrogen
atmosphere using dried glassware and syringes techniques to
transfer solutions. NaH 60% in oil dispersion was washed with n-
hexane until a homogeneous white solid was obtained, dried and
low solid. H NMR (DMSO-d ) d: 6.05 (s, 2H); 6.96 (dd, J = 8.4 Hz,
6
1
J = 2.2 Hz, 1H); 7.02 (s, 2H); 7.56 (d, J = 8.4 Hz, 1H); 7.93 (d,
2
1
9
J = 2.2 Hz, 1H). Compound 4 was reported in the lit.
4.1.5. 5-(3-Tosylureido)pyridine-2-sulfonamide 5
stored under a nitrogen atmosphere prior to use. Infrared (IR) spec-
A suspension of 2-sulfamyl-5-aminopyridine 4 (0.2 g, 1.0 equiv)
in dry acetonitrile (4 mL) was treated with 4-methylbenzenesulfo-
nyl isocyanate (1.0 equiv) according to the general procedure
À1
tra were recorded as KBr plates and are expressed in
m
(cm ).
1
13
Nuclear magnetic resonance ( H NMR,
DEPT-90, HSQC, HMBC) spectra were recorded using a Bruker Ad-
vance III 400 MHz spectrometer in CDCl , MeOH-d or in DMSO-
. Chemical shifts are reported in parts per million (ppm) and
C NMR, DEPT-135,
7
previously reported. The reaction was quenched with H O
2
3
4
(1.0 mL) and the solvents were evaporated under vacuo to give a
residue that was purified by silica gel column chromatography
eluting with MeOH/DCM (10% v/v), to afford the titled compound
5 as a white solid.
d
6
the coupling constants (J) are expressed in Hertz (Hz). Splitting
patterns are designated as follows: s, singlet; d, doublet; sept,