Inhibition of Carbonic Anhydrases (CAs) has been clinically exploited for many decades for a variety of therapeutic applications. Within a research project aimed at developing novel classes of CA inhibitors (CAIs) with a proper selectivity for certain isoforms, a series of derivatives featuring the 2-substituted-benzimidazole-6-sulfonamide scaffold, conceived as frozen analogs of Schiff bases and secondary amines previously reported in the literature as CAIs, were investigated. Enzyme inhibition assays on physiologically relevant human CA I, II, IX and XII isoforms revealed a number of potent CAIs, showing promising selectivity profiles towards the transmembrane tumor-associated CA IX and XII enzymes. Computational studies were attained to clarify the structural determinants behind the activities and selectivity profiles of the novel inhibitors.
Inhibition of Carbonic Anhydrases (CAs) has been clinically exploited for many decades for a variety of therapeutic applications. Within a research project aimed at developing novel classes of CA inhibitors (CAIs) with a proper selectivity for certain isoforms, a series of derivatives featuring the 2-substituted-benzimidazole-6-sulfonamide scaffold, conceived as frozen analogs of Schiff bases and secondary amines previously reported in the literature as CAIs, were investigated. Enzyme inhibition assays on physiologically relevant humanCA I, II, IX and XII isoforms revealed a number of potent CAIs, showing promising selectivity profiles towards the transmembrane tumor-associated CA IX and XII enzymes. Computational studies were attained to clarify the structural determinants behind the activities and selectivity profiles of the novel inhibitors.
Carbonic anhydrases (CA) are a family of ubiquitary zinc metalloenzymes that catalyze the
reversible reaction of hydration of CO2 to HCO3− . This simple transformation plays a
physiological regulatory role in a number of processes associated with pH control, ion
transport, fluid secretion and several biosynthetic pathways. Fifteen CA isoenzymes are encoded in humans and other
primates, that differ for their subcellular localization, catalytic activity, and
susceptibility to different classes of inhibitors. Specifically, cytosolic (CA I, CA II, CA III, CA VII, and CA
XIII), membrane-bound (CA IV, CA IX, CA XII and CA XIV), mitochondrial (CA VA and CA VB),
and secreted in saliva (CA VI) enzymes were characterized. Most of these CA isoforms represent interesting therapeutic
targets, and their inhibition has been exploited clinically for many decades for a variety
of applications in treating a multitude of diseases such as glaucoma, edema, epilepsy,
obesity, neuropathic pain and other neurological disorders. More recently, hCA IX and XII have
been implicated in tumor progression/metastasis, and their selective inhibition could
represent an additional opportunity for drug intervention against hypoxic cancers.Sulfonamides and their bioisosteres (sulfamates, sulfamides) are known, powerful CA
inhibitors (CAIs). Acetazolamide
1 (AAZ), methazolamide 2, ethoxzolamide 3,
brinzolamide 4 and dorzolamide 5 are among the CAIs used in
medicine mainly as diuretics and antiglaucoma agents (Chart
1).
Chart 1.
Structures of clinically used CAIs.
Structures of clinically used CAIs.Because of the ubiquity of CAs, the selectivity of the inhibitors for certain isoforms is a
crucial issue to be reached in a drug development campaign in order to target a disease
without relevant side effects. In
this respect, expanding the chemical space by the exploration of novel scaffolds may aid the
development of novel classes of CAIs featuring improved pharmacological properties in terms
of inhibition potency and isoform-selectivity.Among the several sulfonamide CAIs described, Schiff bases and secondary amines
incorporating aromatic/heterocyclic sulfonamide moieties in their structure (compounds of
type I in Figure 1) have been
extensively investigated in recent years. Comparing the activity of
imines and their secondary amine counterparts, it is evident that the molecular flexibility
markedly affects, positively or negatively, both activity and selectivity.
Figure 1.
Flowchart of our frozen analog approach.
Flowchart of our frozen analog approach.Prompted by these outcomes and exploiting our experience in the application of the frozen
analog approach,, we decided to further reduce the
flexibility of the Schiff bases constraining the N=C imine bond into a ring
(II, Figure 1). As a rigidifying
building block, we decided to introduce the benzimidazole (III, Figure 1), a privileged structure extensively used in
medicinal chemistry, which has been only scarcely explored for its potential in the
development of CAIs. As substituent
in the position 2 of benzimidazole core, we selected phenols and benzoic acid derivatives.
In fact, different studies showed that these scaffolds are effective CAIs, giving
interactions with the enzyme that may not involve a direct interaction with the active site
zing ion. Herein,
we report the synthesis of 2-substituted-benzimidazole-6-sulfonamides 6–18 and
the related carboxamide 19 and their inhibitory activity toward four
physiologically relevant enzymes, the cytosolic isoforms hCA I and II as well as the
transmembrane tumor-associated ones hCA IX and XII.
Materials and methods
Chemistry
All chemicals were purchased from Sigma Aldrich Srl (Milan, Italy) or from Fluorochem
Ltd. (Hadfield, UK) and were of the highest purity. All solvents were reagent grade and,
when necessary, were purified and dried by standard methods. All reactions requiring
anhydrous conditions were conducted under a positive atmosphere of nitrogen in oven-dried
glassware. Standard syringe techniques were used for anhydrous addition of liquids.
Reactions were routinely monitored by TLC performed on aluminum-backed silica gel plates
(Merck DC, Alufolien Kieselgel 60 F254) with spots visualized by UV light
(λ = 254, 365 nm) or using a KMnO4 alkaline solution. Solvents were removed
using a rotary evaporator operating at a reduced pressure of ∼10 Torr. Organic solutions
were dried over anhydrous Na2SO4. Chromatographic purification was
done on an automated flash-chromatography system (IsoleraTM Dalton 2000, Biotage) using
cartridges packed with KP-SIL, 60 Å (40–63 µm particle size). All microwave assisted
reactions were conducted in a CEM Discover® SP microwave synthesizer equipped
with a vertically focused IR temperature sensor. Analytical high performance liquid
chromatography (HPLC) was performed on a Shimadzu SPD 20 A UV/VIS detector (λ = 220 and
254 nm) using C-18 column Phenomenex Synergi Fusion - RP 80 A (75 × 4.60 mm; 4 µm) at
25 °C using a mobile phase A (water + 0.1% TFA) and B (ACN + 0.1% TFA) at a flow rate of
1 ml/min. 1H spectra were recorded at 400 MHz on a Bruker Ascend 400
spectrometer while 13C NMR spectra were obtained by distortionless enhancement
by polarization transfer quaternary (DEPTQ) spectroscopy on the same spectrometer.
Chemical shifts are reported in δ (ppm) relative to the internal
reference tetramethylsilane (TMS). Due to the existence of tautomers, some 1H
and 13C NMR signals could not be detected for some of the prepared
benzimidazoles so only the distinct signals are reported. Low resolution mass spectra were
recorded on a Finnigan LCQ DECA TermoQuest mass spectrometer in electrospray positive and
negative ionization modes (ESI-MS). High resolution mass spectra were recorded on a Bruker
solariX MRMS in electrospray positive ionization modes (ESI-FTMS). All tested compounds
possessed a purity of at least 95% established by HPLC unless otherwise noted. Acids
27 and 28a were commercially available, acid 28b
was obtained by previously reported procedure (see Supplementary
Data).
N-(tert-butyl)-2-(4-hydroxyphenyl)-1H-benzo[d]imidazole-6-sulfonamide
26a (174 mg, 0.504 mmol) was dissolved in 2 ml of a solution DCM/TFA
(1:1) and the mixture was stirred for 18 h. The solvent was evaporated, and the
resulting solid was crystallized with ethanol to give the title compound as a brown
solid (110 mg, 75%). 1H NMR (400 MHz, DMSO-d6) δ
10.12 (s, 1H, exchangeable with D2O), 8.03 (d, J = 8.3 Hz,
2H), 7.99 (s, 1H), 7.72–7.64 (m, 2H), 7.28 (s, 2H, exchangeable with D2O),
6.95 (d, J = 8.3 Hz, 2H). 13C NMR (100 MHz, DMSO)
δ 159.92, 154.19, 137.88, 128.67, 119.73, 115.85. HRMS (ESI): m/z
[M + H]+ calcd for
C13H11N3O3S + H+, 290.05939;
found, 290.05938.
Compound 30a (245 mg, 0.626 mmol) was dissolved in 80 ml of toluene,
p-toluensulfonic acid (59 mg, 0.313 mmol) was added and the resulting
mixture was heated at reflux for 6 h. Solvent was evaporated and the crude residue was
taken up with a saturated solution of NaHCO3 (30 ml). The aqueous phase was
extracted with EtOAc (3 × 20 ml) and the collected organic phases were washed with
saturated solution of NaHCO3 (3 × 20 ml), brine (20 ml), anhydrified over
Na2SO4, filtered and concentrated under reduced pressure. The
title compound was obtained after crystallization with ethanol as a light brown solid
(130 mg, 65%). 1H NMR (400 MHz, DMSO-d6) δ 9.15
(s, 1H, exchangeable with D2O), 7.93 (s, 1H), 7.63–7.58 (m, 2H), 7.21 (s, 2H,
exchangeable with D2O), 7.01 (d, J = 8.1 Hz, 2H), 6.64 (d,
J = 8.1 Hz, 2H), 3.09 (t, J = 7.5 Hz, 2H), 2.99 (t,
J = 7.5 Hz, 2H). 13C NMR (100 MHz, DMSO)
δ 155.56, 137.19, 130.80, 129.05, 119.01, 115.09, 32.44, 30.88. HRMS
(ESI): m/z [M + H]+ calcd for
C15H15N3O3S + H+, 318.09069;
found, 318.09066
2-Phenyl-1H-benzo[d]imidazole-6-sulfonamide (9)
Compound 9 was obtained as a light brown solid (49 mg, 75%) by reaction of
26b (80 mg, 0.24 mmol) following the procedure described for
6. 1H NMR (400 MHz, DMSO-d6) δ
8.25–8.16 (m, 2H), 8.09 (d, J = 1.8 Hz, 1H), 7.82 (d,
J = 8.7 Hz, 1H), 7.77 (dd, J = 8.5, 1.8 Hz, 1H),
7.68–7.59 (m, 3H), 7.37 (s, 2H, exchangeable with D2O). 13C NMR
(100 MHz, DMSO) δ 153.36, 138.92, 131.25, 129.22, 128.00, 127.07,
120.64. HRMS (ESI): m/z [M + H]+ calcd for
C13H11N3O2S + H+, 274.06447;
found, 274.06445.
To a stirred solution of compound 10 (250 mg, 0.645 mmol) in 1.5 ml of THF
was added a water solution (1.5 ml) of LiOH (62 mg, 2.58 mmol). The reaction mixture was
stirred at room temperature for 3 h and then concentrated under vacuum. The aqueous
phase was washed with CHCl3 then acidified with 3N HCl until a white
precipitate formed. After filtration, the title compound was obtained as white solid
(200 mg, 83%).1H NMR (400 MHz, DMSO-d6) δ 8.32
(d, J = 8.2 Hz, 2H), 8.16–8.11 (m, 3H), 7.84–7.68 (m, 2H), 7.32 (s, 2H,
exchangeable with D2O). 13C NMR (100 MHz, DMSO) δ
166.76, 133.24, 132.14, 129.99, 126.83. HRMS (ESI): m/z
[M + H]+ calcd for
C14H11N3O4S + H+, 318.05430;
found, 318.05429.
Compound 35 (150 mg, 0.408 mmol) was dissolved in 2 ml of a solution
DCM/TFA (1:1) and the mixture was stirred for 24 h. The solvent was evaporated, and the
resulting solid was taken up with 2 ml of THF. To the resulting mixture, a water
solution (2 ml) of LiOH (62 mg, 2.58 mmol) was added, and the reaction mixture was
stirred at room temperature for 4 h. The reaction was concentrated under vacuum, the
aqueous phase was washed with CHCl3 then acidified with 3N HCl until a white
precipitate formed that was recovered by filtration. Compound 19 was
obtained as white solid (110 mg, 91%) after recrystallization from ethanol1H
NMR (400 MHz, DMSO-d6) δ 8.72 (d,
J = 2.4 Hz, 1H), 8.36–8.26 (m, 1H), 8.17 (s, 1H), 8.09 (s, 1H,
exchangeable with D2O), 7.88 (d, J = 8.4 Hz, 1H), 7.71–7.65
(m, 1H), 7.38 (s, 1H, exchangeable with D2O), 7.19 (d,
J = 8.7 Hz, 1H). 13C NMR (100 MHz, DMSO) δ
171.54, 168.31, 164.41, 152.09, 134.20, 130.25, 130.08, 123.63, 118.84, 115.01. HRMS
(ESI): m/z [M + H]+ calcd for
C15H11N3O4 + H+, 298.08223;
found, 298.08230.
To a stirred suspension of 24a (1.65 g, 6.04 mmol) in 250 ml of MeOH,
ammonium formate (7.61 g, 120.74 mmol) and palladium on carbon 10% wt. (160 mg) were
added. The resulting mixture was heated at reflux for 4 h. After cooling, the mixture
was filtered, and the solvent evaporated under reduced pressure. The crude material was
taken up with 100 ml of water and extracted with EtOAc (3 × 60 ml). The combined organic
phases were washed with brine, dried over Na2SO4, filtered and
evaporated. The product, obtained as a light brown solid (1.30 g, 88%), was used for the
next step without further purification. 1H NMR (400 MHz, DMSO-d6)
δ 6.96 (d, J = 2.1 Hz, 1H), 6.90–6.84 (m, 2H, 1H
exchangeable with D2O), 6.53 (d, J = 8.2 Hz, 1H), 5.13 (brs,
2H, exchangeable with D2O), 4.79 (brs, 2H, exchangeable with D2O),
1.08 (s, 9H). ESI m/z: 244 [M + H]+.
3,4-Diamino-N-ethylbenzenesulfonamide (20b)
Compound 20b was obtained as a light brown solid (910 mg, 94%) by reaction
of 24b (1.10 g, 4.48 mmol) following the procedure described for
20a. 1H NMR (400 MHz, DMSO-d6) δ
6.94–6.88 (m, 2H, 1H exchangeable with D2O), 6.81 (dd,
J = 8.0, 2.1 Hz, 1H), 6.53 (d, J = 8.0 Hz, 1H), 5.18
(brs, 2H, exchangeable with D2O), 4.82 (brs, 2H, exchangeable with
D2O), 2.75–2.62 (m, 2H), 0.94 (t, J = 7.2 Hz, 3H). ESI
m/z: 216 [M + H]+.
Ethyl 2-((2-nitrophenyl)amino)-2-oxoacetate (22)
To a solution of 2-nitroaniline (2.00 g, 14.48 mmol) in Et2O (100 ml), ethyl
chlorooxoacetate (2.17 g, 1.78 ml, 15.93 mmol) was added portionwise with continuous
stirring. Once the addition was complete, the resulting yellow suspension was stirred
for 18 h at room temperature and then concentrated under vacuo. The crude residue was
dissolved in EtOAc (100 ml), washed with saturated NaHCO3 (3 × 30 ml) and
with brine (30 ml). The organic phase was dried over anhydrous
Na2SO4, and evaporated to dryness, giving the desired product as
a yellow solid (3.38 g, 98%). 1H NMR (400 MHz, DMSO-d6)
δ 11.38 (s, 1H), 8.17–8.05 (m, 2H), 7.85–7.76 (m, 1H), 7.52–7.41 (m,
1H), 4.34 (q, J = 7.0 Hz, 2H), 1.33 (t, J = 7.0 Hz,
3H). ESI m/z: 239 [M + H]+.
4-Amino-3-nitrobenzenesulfonyl chloride (23)
A solution of ethyl 2-(2-nitrophenylamino)-2-oxoacetate 22 (2.00 g,
8.40 mmol) in 4.5 ml of chlorosulfonic acid was heated at 80 °C for 3 h. The red mixture
was poured slowly into ice − water (150 ml) and stirred for 30 min. The product was
extracted from the aqueous solution using Et2O (3 × 30 ml). The combined
organic phases were washed with brine (10 ml), dried (Na2SO4),
filtered, and concentrated in vacuo to give the title compound as a
brown solid which was immediately used for the next reaction without purification.
1H NMR (400 MHz, DMSO-d6) δ 8.16 (d,
J = 2.0 Hz, 1H), 7.56 (dd, J = 8.8, 2.0 Hz, 1H),
6.96 (d, J = 8.8 Hz, 1H).
To a stirred solution at 0 °C of crude 23 (1.04 g, 4.39 mmol) in dry THF
(25 ml) was added dropwise, under nitrogen atmosphere, tert-butylamine
(1.85 ml, 17.56 mmol). The reaction was allowed to reach room temperature and was
stirred for 18 h. The solvent was removed at reduced pressure and the residue was taken
up with 50 ml of water and extracted with EtOAc (3 × 20 ml). The combined organic phases
were washed with brine, dried over Na2SO4, filtered and evaporated
under reduced pressure. Purification by silica gel chromatography (DCM/MeOH) yield pure
24a (0.96 g, 80%) as a light yellow solid. 1H NMR (400 MHz,
DMSO-d6) δ 8.38 (d, J = 2.2 Hz, 1H), 7.95
(s, 2H, exchangeable with D2O), 7.70 (dd, J = 9.0, 2.2 Hz,
1H), 7.44 (s, 1H, exchangeable with D2O), 7.11 (d,
J = 9.0 Hz, 1H), 1.10 (s, 9H). ESI m/z: 274
[M + H]+.
4-Amino-N-ethyl-3-nitrobenzenesulfonamide (24b)
Compound 24b was obtained as a yellow solid (840 mg, 78%) by reaction of
23 (1.04 g, 4.39 mmol) with a 2 M THF solution of ethylamine (8.78 ml,
17.56 mmol) following the procedure described for 24a. 1H NMR
(400 MHz, DMSO-d6) δ 8.36 (d, J = 1.6 Hz, 1H), 8.01 (s, 2H,
exchangeable with D2O), 7.69 (dd, J = 9.0, 1.6 Hz, 1H), 7.54–7.44 (m, 1H,
exchangeable with D2O), 7.15 (d, J = 9.0 Hz, 1H), 2.82 – 2.72 (m, 2H), 0.99
(t, J = 7.2 Hz, 3H). ESI m/z: 246 [M + H]+.
To a stirred solution of 20a (150 mg, 0.62 mmol) in dry DMF (7 ml),
4-hydroxybenzaldehyde (75 mg, 0.61 mmol) and Na2S2O5
(0.165 g, 0.793 mmol) were added. The resulting mixture was stirred at 80 °C for 18 h.
After cooling at room temperature, water was added. The brown precipitate formed was
recovered by filtration and was washed several times with water and 1N HCl. After
recrystallization from EtOH, compound 26a was obtained as light brown solid
(160 mg, 72%). 1H NMR (400 MHz, DMSO-d6) δ 10.07
(s, 1H, exchangeable with D2O), 8.03 (d, J = 8.5 Hz, 2H),
8.01–7.96 (m, 1H), 7.69–7.61 (m, 2H), 7.43 (s, 1H, exchangeable with D2O),
6.94 (d, J = 8.5 Hz, 2H), 1.09 (s, 9H).ESI m/z: 346
[M + H]+.
Compound 26e was obtained as a light brown solid (222 mg, 71%) by reaction
of 20a (188 mg, 0.773 mmol) and methyl 5-formylsalicylate (139 mg,
0.773 mmol) following the procedure described for 26a. 1H NMR
(400 MHz, DMSO-d6) δ 8.65 (d, J = 2.3 Hz,
1H), 8.32 (dd, J = 8.7, 2.3 Hz, 1H), 8.01 (s, 1H), 7.76–7.64 (m, 2H),
7.46 (s, 1H, exchangeable with D2O), 7.19 (d, J = 8.7 Hz,
1H), 3.97 (s, 3H), 1.09 (s, 9H). ESI m/z: 404 [M + H]+.
N-(2-amino-4-(N-(tert-butyl)sulfamoyl)phenyl)-2-(4-hydroxyphenyl)acetamide and
N-(2-amino-5-(N-(tert-butyl)sulfamoyl)phenyl)-2-(4-hydroxyphenyl)acetamide (29)
To a stirred solution of 20a (354 mg, 1.45 mmol) and 4-hydroxyphenylacetic
acid 27 (200 mg, 1.31 mmol) in dry DMF (26 ml) were added, EDC
hydrochloride (427 mg, 2.23 mmol), HOBt (341 mg, 2.23 mmol) and 4-methylmorpholine
(4.84 ml, 4.45 mmol). The reaction was stirred at room temperature for 18 h. Water
(80 ml) was added the resulting mixture was extracted with EtOAc (3 × 40 ml). The
combined organic phases were washed with brine (3 × 50 ml), dried over
Na2SO4, filtered and evaporated under reduced pressure.
Purification by silica gel chromatography (DCM/MeOH) yield an isomeric mixture as a
white solid (0.360 g, 73%) that was used for the next step without separation. ESI
m/z: 378 [M + H]+.
N-(2-amino-4-(N-(tert-butyl)sulfamoyl)phenyl)-3-(4-hydroxyphenyl)propanamide and
N-(2-amino-5-(N-(tert-butyl)sulfamoyl)phenyl)-3-(4-hydroxyphenyl)propanamide
(30a)
Compounds 30a was obtained as a light yellow solid (300 mg, 64%) by
reaction of 20a (323 mg, 1.33 mmol) with 3-(4-hydroxyphenyl)propanoic acid
28a (200 mg, 1.2 mmol) following the procedure described for
29. ESI m/z: 392 [M + H]+.
Methyl
5-(3-((2-amino-4-(N-(tert-butyl)sulfamoyl)phenyl)amino)-3-oxopropyl)-2-hydroxybenzoate
and methyl
5-(3-((2-amino-5-(N-(tert-butyl)sulfamoyl)phenyl)amino)-3-oxopropyl)-2-hydroxybenzoate
(30b)
Compounds 30b was obtained as a light yellow solid (90 mg, 90%) by
reaction of 20a (60 mg, 0.245 mmol) with
3-(4-hydroxy-3-(methoxycarbonyl)phenyl)propanoic acid 28b (50 mg,
0.223 mmol) following the procedure described for 29. ESI
m/z: 450 [M + H]+.
In a 10 ml CEM pressure vessel equipped with a stirrer bar, compounds 29
(50 mg, 0.132 mmol) were dissolved in 1.3 ml of acetic acid. The microwave vial was
sealed and heated in a CEM Discover microwave synthesizer to 80 °C for 30 min. After
cooling to room temperature, the reaction mixture was taken up with solution of
NaHCO3 (60 ml) and the aqueous phase was extracted with EtOAc (3 × 20 ml).
The collected organic phases were washed with water (3 × 20 ml), NaHCO3
solution (3 × 20 ml), brine (20 ml), dried over Na2SO4 and
filtered. The solvent was removed under reduced pressure and the resulting crude
material was purified by silica gel chromatography (DCM/EtOAc) to give compound
31 a light brown solid (35 mg, 74%). 1H NMR (400 MHz,
DMSO-d6) δ 9.31 (s, 1H, exchangeable with D2O),
7.93 (s, 1H), 7.64–7.54 (m, 2H), 7.39 (s, 1H, exchangeable with D2O), 7.14
(d, J = 8.0 Hz, 2H), 6.71 (d, J = 8.0 Hz, 2H), 4.09
(s, 2H), 1.06 (s, 9H). ESI m/z: 360 [M + H]+.
N-(tert-butyl)-3,4-dinitrobenzamide (33)
A solution of 3,4-dinitrobenzoic acid 32 (1.00 g, 4.71 mmol) in thionyl
chloride (15 ml) was refluxed for 2 h under a nitrogen atmosphere. After the solution
was cooled at room temperature, the excess thionyl chloride was removed at reduced
pressure and the crude material dried under vacuum. To the residue, dissolved in dry THF
(20 ml) and cooled to 0 °C, was added dropwise a mixture of
tert-butylamine (525 µL, 5.00 mmol) and triethylamine (695 µL,
5.00 mmol) in dry THF (5 ml). The mixture was stirred at room temperature for 18 h,
filtered, and evaporated. The crude residue was dissolved in DCM (20 ml), washed with 1N
HCl, saturated NaHCO3, and water, dried, and concentrated in vacuum.
Recrystallization from EtOH yielded compound 33 (1.05 g, 84%) as a light
yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.57
(s, 1H), 8.35–8.29 (m, 2H), 1.40 (s, 9H). ESI m/z: 268
[M + H]+.
3,4-Diamino-N-(tert-butyl)benzamide (34)
Compound 34 was obtained as a light brown solid (729 mg, 94%) by reaction
of 33 (1.00 g, 3.74 mmol) following the procedure described for
20a. 1H NMR (400 MHz, DMSO-d6) δ
7.05 (s, 1H, exchangeable with D2O), 6.98 (d, J = 2.0 Hz,
1H), 6.90 (dd, J = 8.1, 2.1 Hz, 1H), 6.45 (d,
J = 8.0 Hz, 1H), 4.86 (s, 2H, exchangeable with D2O), 4.48
(s, 2H, exchangeable with D2O), 1.33 (s, 9H). ESI m/z: 208
[M + H]+.
Compound 35 was obtained as a light brown solid (304 mg, 72%) by reaction
of 34 (240 mg, 1.15 mmol) and 25e (208 mg, 1.15 mmol)
following the procedure described for 6. 1H NMR (400 MHz,
DMSO-d6) δ 11.04 (s, 1H, exchangeable with
D2O), 8.66 (d, J = 2.2 Hz, 1H), 8.30 (dd,
J = 8.8, 2.4 Hz, 1H), 8.09 (s, 1H), 7.90–7.78 (m, 2H, 1H exchangeable
with D2O), 7.68 (d, J = 8.4 Hz, 1H), 7.26 (d,
J = 8.7 Hz, 1H), 3.97 (s, 3H), 1.42 (s, 9H). ESI
m/z: 368 [M + H]+.
Enzyme activity assays
An Applied Photophysics stopped-flow instrument has been used for assaying the
CA-catalysed CO2 hydration activity. Phenol red (at a concentration of 0.2 mM) has been used as
indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as
buffer, and 20 mM Na2SO4 (for maintaining constant the ionic
strength), following the initial rates of the CA-catalysed CO2 hydration
reaction for a period of 10–100 s. CO2 concentrations ranged from 1.7 to 17 mM
for the determining inhibition constants. For each inhibitor, at least six traces of the
initial 5–10% of the reaction have been used for measuring the initial velocity.
Uncatalyzed rates were determined in the same manner and subtracted from the total
observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in buffer with a
maximum 3% DMSO, and dilutions up to 0.01 nM were done with the assay buffer. Inhibitor
and enzyme solutions were preincubated for 15 min at room temperature prior to assay, in
order to allow for the formation of the E–I complex. The inhibition constants were
obtained by nonlinear 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 ones obtained in-house as
reported earlier.
Molecular modeling methods
The latest version of the AD4 docking software (version 4.2) together with its GUI AutoDockTools (ADT) and the
AutoDock4(Zn) force field, were
employed. The hCA IX X-ray structure used for the experiment had the PDB code 5FL4. The protein structure was prepared
for the docking using the Protein Preparation Wizard of the Maestro suite that adds bond orders, adds hydrogen
atoms, deletes water molecules and produces the appropriate protonation states. The
co-crystal ligand of 5FL4 was separated from the cognate protein. The 2 D Sketcher tool of
Maestro was used to build compounds 13, 14 and 17.
For the three ligands, the protonation and tautomeric state, as well as their geometry,
were optimized through LigPrep, part of the same suite. Through Maestro, the X-ray
structures of hCA I (PDB 6F3B),
hCA II (PDB 3K34), and hCA XII
(PDB 5MSA), were downloaded and
superimposed on the structure of hCA IX. The ligands were translated in the AD4 specific
file format (PDBQT) using the python scripts prepare_ligand4.py and prepare_receptor4.py,
part of ADT, applying the standard settings. Following the AutoDock4(Zn) force field
protocol, to add the tetrahedral
zinc pseudo atoms to the receptor PDBQT the script zinc_pseudo.py, part of the material
provided with the force field, was employed. The docking area was centered on the active
site. The zinc-specific non bonded pairwise potentials were included in the creation of
the grid parameter file. A set of grids of 60 Å × 40 Å × 50 Å with 0.375 Å spacing was
calculated considering the docking area for all the ligands atom types employing
AutoGrid4. For every ligand, 200 independent docking simulations were achieved. Each
docking calculation comprised 20 million energy evaluations employing the Lamarckian
genetic algorithm local search (GALS) method. This latter assesses a population of viable
docking solutions and propagates the best individuals from each generation into the
following generation of feasible solutions. A low-frequency local search according to the
method of Solis and Wets was applied to every docking attempt to guarantee that the final
solution represented a local minimum. All dockings were performed with a population size
of 250, and 300 iterations of Solis and Wets local search were applied with a probability
of 0.06. A rate of mutation of 0.02 and a crossover rate of 0.8 were used to produce new
docking attempts for following generations, and the best individual from each generation
was propagated over the following generation. The docking results from every (200)
independent docking calculation were clustered based on the of root-mean-square deviation
(rmsd) (solutions differing by less than 2.0 Å) between the Cartesian coordinates of the
atoms and were ranked on the basis of free energy of binding (ΔGAD4).
Results and discussion
The primary (6–14 and 17–18) and the
N-ethyl-2-substituted-1H-benzo[d]imidazole-6-sulfonamides
(15 and 16) were synthesized starting from
3,4-diaminobenzenesulfonamides 20a and 20b, respectively,
obtained following the procedure outlined in Scheme
1.
Scheme 1.
(a) Ethyl chlorooxoacetate, Et2O, r.t. 18 h (98%); (b) ClSO3H,
80 °C, 3 h (77%); c) tert-butylamine or 2M THF solution of
ethylamine, THF, 0 °C to r.t., 18 h (80%); (d) ammonium formate, Pd/C 10%, MeOH,
reflux, 4 h (88–94%).
(a) Ethyl chlorooxoacetate, Et2O, r.t. 18 h (98%); (b) ClSO3H,
80 °C, 3 h (77%); c) tert-butylamine or 2M THF solution of
ethylamine, THF, 0 °C to r.t., 18 h (80%); (d) ammonium formate, Pd/C 10%, MeOH,
reflux, 4 h (88–94%).The amino group of 2-nitroaniline 21 was protected by acylation with ethyl
chlorooxoacetate in diethyl ether. Reaction of the resulting ethyl
2-(2-nitrophenylamino)-2-oxoacetate 22 with chlorosulfonic acid at 80 °C,
followed by aqueous workup, yielded the unprotected sulfonyl chloride derivative
23, which reacted with tert-butyl- or ethyl-amine to
afford the N-substituted-4-amino-3-nitrobenzenesulfonamides
24a and 24b, respectively. Catalytic hydrogenation with
ammonium formate and palladium catalyst converted the nitro derivatives into the
corresponding amino derivatives 20a and 20b.The synthetic route to
2-aryl-1H-benzo[d]imidazole-6-sulfonamides
6–14 is outlined in Scheme 2.
Condensation of
3,4-diamino-N-(tert-butyl)benzenesulfonamide
20a with aldehydes 25a-e in the presence of NaHSO3
in dry DMF at 80 °C gave the
2-aryl-N-(tert-butyl)-1H-benzo[d]imidazole-6-sulfonamides
26a-e in good yields (65–83%). Deprotection with trifluoracetic acid at room temperature
furnished the primary sulfonamides 6, 9, 10,
12 and 13. Finally, the carboxylic acid derivatives
11 and 14 were obtained by deprotecting the methyl esters
10 and 13, respectively, with lithium hydroxide.
Scheme 2.
(a) NaHSO3, dry DMF, 80 °C, 18 h (65%–83%); (b) DCM/TFA (1:1), r.t.,
18–24 h (70–75%); (c) LiOH, THF/H2O (1:1) r.t, 3 h (83–93%); (d) EDC
hydrochloride, HOBt, NMM, dry DMF, r.t., 18 h (64–90%); (e)
p-toluenesulfonic acid, toluene, reflux, 6 h (60–65%); (f) MW, AcOH,
80 °C, 30 min (74%).
(a) NaHSO3, dry DMF, 80 °C, 18 h (65%–83%); (b) DCM/TFA (1:1), r.t.,
18–24 h (70–75%); (c) LiOH, THF/H2O (1:1) r.t, 3 h (83–93%); (d) EDC
hydrochloride, HOBt, NMM, dry DMF, r.t., 18 h (64–90%); (e)
p-toluenesulfonic acid, toluene, reflux, 6 h (60–65%); (f) MW, AcOH,
80 °C, 30 min (74%).Coupling reaction of benzenesulfonamide 20a with 2-(4-hydroxyphenyl)acetic
acid 27 or 3-arylpropanoic acids 28a and 28b, in
the presence of the peptide coupling reagents hydroxybenzotriazole (HOBt) and
1-ethyl-3–(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and
N-methylmorpholine in dry DMF, furnished the amides 29,
30a and 30b as regioisomeric mixtures, which were purified
without separation of regioisomers. Benzimidazoles derivatives 8 and
17 were straightforwardly obtained by p-toluenesulfonic
acid-mediated cyclization and deprotection of the corresponding 2-amido anilines
30a and 30b in refluxing toluene. The carboxylic acid
18 was obtained by deprotecting the methyl ester 17 with
lithium hydroxide. Our attempts to cyclize compound 29 using the same
reaction conditions were not successful. On the other hand, benzimidazole 31
was obtained in good yield (74%) by using acetic acid under microwave irradiation.
Finally, deprotection with trifluoracetic acid at room temperature furnished the primary
sulfonamide 7.N-ethyl-2-aryl-1H-benzo[d]imidazole-6-sulfonamides
15 and 16 were obtained starting from
3,4-diamino-N-ethylbenzenesulfonamide 20b and aldehyde
25e following the same synthetic strategy used for the preparation of the
primary sulfonamide analogs 13 and 14 (Scheme 3).
Scheme 3.
(a) 25e, NaHSO3, dry DMF, 80 °C, 18 h (71%); (b) LiOH,
THF/H2O (1:1) r.t, 3 h (89%).
(a) 25e, NaHSO3, dry DMF, 80 °C, 18 h (71%); (b) LiOH,
THF/H2O (1:1) r.t, 3 h (89%).Finally,
2–(4-hydroxy-3-carboxy)-phenyl-1H-benzo[d]imidazole-6-carboxamide
19 was straightforwardly synthesized as described in Scheme 4. After activation of the commercially
available 3,4-dinitrobenzoic acid 32 with thionyl chloride, coupling with
tert-butyl amine yielded 3,4-dinitrobenzamide 33 which was
reduced by catalytic hydrogenation with ammonium formate and palladium catalyst to the key
intermediate 34. Condensation with aldehyde 25e in the presence
of NaHSO3 gave the benzimidazole derivative 35. Amide deprotection
with trifluoracetic acid and hydrolysis of the methyl ester with LiOH gave the desired
compound 19.
Scheme 4.
(a) SOCl2, reflux, 2 h, then tert-butylamine,
NEt3, THF, 0 °C to r.t., 18 h (84%); (b) ammonium formate, Pd/C 10%,
MeOH, reflux, 2 h (94%); (c) 25e, NaHSO3, dry DMF, 80 °C, 18 h
(72%); (d) DCM/TFA (1:1), r.t. 24 h, then LiOH, THF/H2O (1:1) r.t.,4 h
(91%).
(a) SOCl2, reflux, 2 h, then tert-butylamine,
NEt3, THF, 0 °C to r.t., 18 h (84%); (b) ammonium formate, Pd/C 10%,
MeOH, reflux, 2 h (94%); (c) 25e, NaHSO3, dry DMF, 80 °C, 18 h
(72%); (d) DCM/TFA (1:1), r.t. 24 h, then LiOH, THF/H2O (1:1) r.t.,4 h
(91%).
CA inhibition assays and structure-activity relationship (SAR) considerations
Table 1 lists the enzyme inhibitory activities
of the newly synthesized compounds 6–19 against the human (h) CA I, II, IX
and XII isoforms, assessed by a stopped-flow CO2 hydrase assay [19]. AAZ
1 was used as the standard drug in the assay. Selectivity ratios (SR) for inhibiting the
tumor-associated transmembrane isoforms (hCA IX and XII) over the physiologically dominant
cytosolic one (hCA II) are also reported for the most active compounds.
Table 1.
Inhibition data of human CA I, II, IX, and XII isoforms with compounds 6–19 and the
standard sulfonamide inhibitor AAZ (1) by a stopped-flow CO2 hydrase
assay.
Compound
Ki
(nM)a
hCA I
hCA II
hCA IX
hCA XII
SRbIX/II
SRbXII/II
1 (AAZ)
250
12
25
5.7
0.48
2.1
6
237.9
101.2
17.4
44.1
5.8
2.3
7
442.1
91.9
73.9
63.8
1.2
1.4
8
213.6
47.8
14.4
9.8
3.3
4.9
9
92.8
104.1
47.6
78.9
2.2
1.3
10
208.2
185.4
29.6
72.2
6.3
2.6
11
97.6
133.2
64.3
57.0
2.1
2.3
12
169.3
79.0
41.6
8.5
1.9
5.8
13
125.5
50.8
2.2
22.3
23.1
2.3
14
95.6
68.6
34.2
3.8
2.0
18.1
15
>10000
8921.1
390.5
813.1
–
–
16
>10000
7582.9
665.1
464.2
–
–
17
427.3
52.7
5.9
7.9
8.9
6.7
18
441.4
24.8
7.6
4.2
3.3
5.9
19
>10000
>10000
>10000
>10000
–
–
Mean from 3 different assays, by a stopped-flow technique (errors were in the range
of ± 5–10% of the reported values).
SR: Selectivity Ratio.
Inhibition data of humanCA I, II, IX, and XII isoforms with compounds 6–19 and the
standard sulfonamide inhibitor AAZ (1) by a stopped-flow CO2 hydrase
assay.Mean from 3 different assays, by a stopped-flow technique (errors were in the range
of ± 5–10% of the reported values).SR: Selectivity Ratio.First, as putative leads for the development of selective CAIs, we synthesized compound
6 and its homologs with a methyl or ethyl linker between the benzimidazole
and the phenol moieties (compounds 7 and 8 in Table 1). They all resulted in medium-potency
inhibition of the slow cytosolic isoform hCA I, with Ki values ranging from
213.6 nM to 442.1 nM. The 4′-hydroxybenzyl derivative 7 showed good
inhibition activity for hCA II, IX, and XII but was not selective (Ki 91.9 nM
for hCA II, 73.9 nM for hCA IX, 63.8 nM for hCA XII). On the other hand, the
4′-hydroxyphenyl (6) and 4′-hydroxyphenylethyl (8) analogs were
effective inhibitors of hCA IX and XII, respectively, with Kis in the low
nanomolar range (6, Ki 17.4 nM for hCA IX, 44.1 nM for hCA XII;
8, Ki 14.4 nM for hCA IX, 9.8 nM for hCA XII) basically
comparable to those of the reference 1
(Ki 25 nM for hCA IX, 5.7 nM for hCA XII), and interesting selectivity ratio
(SR) vs hCA II.Based on these data, compound 7 was not further investigated, and a
structure-activity relationship (SAR) study was undertaken on 6. In
particular, different substitution patterns on the pendant 2-phenyl ring at 5-position of
benzimidazole were investigated (compounds 9–17). Deletion of the 4′-hydroxy
substituent (9), as well as its replacement with methoxycarbonyl
(10) or carboxy (11) groups, produces good but unselective
inhibitors; in fact, a modest gain (9, 11) or a subsistence
(10) in activity toward the isoform I and a general decrease in inhibitory
potency for hCA II, IX, and XII with respect to the lead 6 were observed,
suggesting a precise role played by the hydroxy group in the interaction with the enzyme.
This is confirmed by the inhibitory activities showed by compounds 12–14,
where the introduction of an o-substituent to the
p-hydroxy group on the 2-phenyl ring resulted in a moderate increase in
activity toward the two cytosolic CA evaluated, and a more considerable improvement for
the isoform XII, with compound 14 being the most potent hCA XII inhibitor
(Ki for hCA XII 3.8 nM), showing also a good SR with respect to hCA II (SR
18.1).Specifically, the introduction of a second polar group (OH or COOH) at the
ortho position to the phenol ring positively affected the interaction
with isoform XII (compounds 12 and 14), while the presence of
the methoxycarbonyl group (compound 13) resulted in a minor increase in hCA
XII inhibition with respect to parent compound 6.Concerning the IX isoform, an exactly opposite trend can be observed for compounds
12–14. Decoration of the 2-phenyl moiety with a 4′-hydroxy and
3′-methoxycarbonyl groups gives a highly effective hCA IX inhibitor (13),
which shows low nanomolar Ki (2.2 nM), with a relevant gain in activity
compared to the reference 1 (Ki 25 nM), and good SR vs hCA II. Differently,
compounds 12 and 14, featuring 3′,4′-dihydroxy and
3′-carboxy-4′-hydroxy substituents, although being rather effective hCA IX inhibitors
(Ki for hCA IX 41.6 nM and 34.2 nM for 12 and 14,
respectively), show a slight decrease in activity with respect to reference compound
6 (Ki for hCA IX 17.4 nM).These data suggested the ortho-carboxy phenol ring of compound
14 and the ortho-carboxymethyl phenol ring of compound
13 as proper scaffolds for activity and selectivity for hCA XII and hCA IX,
respectively. For these derivatives, further SARs were investigated.First, we explored structural modifications of the 5-sulfonamide moiety, including the
insertion of a small alkyl group at the nitrogen atom to produce secondary sulfonamides
(compounds 15 and 16), and replacement of the sulfonamide with a
carboxamide (19). The obtained compounds proved to be scarcely active or
completely inactive inhibitors of all the CA isoforms tested (Kis varying from
390.5 nM to micromolar values), strongly supporting the crucial role played by the primary
sulfonamide group in the interaction with the enzyme.Finally, we explored the effect of the combination of the ethyl linker between the
benzimidazole and the 2-substituted phenol moieties (compounds 17 and
18 in Table 1). The potency for
the isoform hCA I decreased, while hCA II was slightly more (18) or equally
inhibited (17) with respect to related compounds 14 and
13, respectively. Both 17 and 18 derivatives
resulted to be potent hCA IX and hCA XII inhibitors, showing low nanomolar Ki
values (17, Ki for hCA IX 5.9 nM, Ki for hCA XII
7.9 nM; 18, Ki for hCA IX 7.6 nM, Ki for hCA XII
4.2 nM). However, the presence of the ethylene linker between the benzimidazole scaffold
and the side phenyl ring abolished the selectivity for hCA IX and XII isoforms, that was
observed for compounds 13 and 14, respectively.Noteworthy, compounds 17 and 18 are better inhibitors than the
phenol derivative 8, proving the role of the
ortho-carboxymethyl phenol and the ortho-carboxy phenol
rings as proper scaffolds in the development of potent and selective hCA IX and hCA XII
inhibitors.
Molecular docking studies
To clarify the reasons for the activities displayed by the newly designed compounds,
molecular docking studies were attained. Docking calculations were performed using a
protocol already successfully applied in our previous work on CA inhibitors. Namely, AutoDock4.2 (AD4), was employed together with the AutoDock4(Zn)
forcefield, which was
specifically designed to accurately predict the binding interactions of ligands docking to
zinc metalloproteins.Ligands 13, 14 and 17 were selected for the in
silico experiments as representative of the whole set. These compounds were first docked
in the active site of hCA IX. For the latter, a high-resolution X-ray crystal structure
bound to a small molecule inhibitor (PDB code 5FL4) was chosen. According to our theoretical model (Figures 2(A), 3(A) and 4(A)), in the three
inspected ligands, the negative nitrogen of the sulfonamide group chelates the zinc ion of
the active site. The sulfonamide also engages an H-bond with the backbone of T200.
Furthermore, the benzimidazole nitrogen is in a potential H-bond accepting position with
Q92 side chain. The benzimidazole core also engages in contacts with the V121, L199, and
T201 sidechains (Figure 2(A), Figure 3(A) and Figure 4(A)).
Notably, it would appear that the pendant 2-phenyl ring, in the three ligands, points
towards what has been defined as a “selectivity hot spot” in CAs (Figures
2(A), 3(A) and 4(A)): a high-variability region in CAs binding site, that can be
exploited for the rational design of selective compounds among different CAs. Here, ligand
13 phenyl ring and its 3′-methoxycarbonyl moiety are able to establish
favorable contacts with the lipophilic sidechains of L91 and V130 (Figure 2(A)). On the other hand, the 4′-hydroxy group is pointing
outside of the binding site, probably establishing a network of stabilizing H-bonding
interactions with the solvent water molecules (Figure
2(A)). It can be argued that this accounts for the higher potency displayed by
compounds featuring the 4′-hydroxy group. The same holds true for compound
14. As for ligand 17, while the benzimidazole core binding mode
is conserved, the ethyl linker engenders greater flexibility which allows the side phenyl
ring to expand further into the hotspot gorge. This allows enhancing the positive contacts
with L91 and V130. Moreover, an additional H-bond between the 3′-methoxycarbonyl group and
Q71 is formed (Figure 4(A)).
Figure 2.
(a) 13/hCA IX (PDB 5FL4) theoretical complex as calculated by docking
simulations. The protein is shown as cyan ribbons and sticks while the ligand as
salmon sticks. Critical residues are labeled. H-bonds are depicted as red dashed lines
while coordination bonds as green dashed lines. (b) 13 hCA IX theoretical
binding pose within the hCA XII (PDB 5MSA) X-ray structure. The protein is shown as light blue ribbons and
sticks while the ligand as salmon sticks. Critical residues are labeled. H-bonds are
depicted as red dashed lines while coordination bonds as green dashed lines. (c)
13 hCA IX theoretical binding pose within the hCA I (PDB 6F3B)
structure. The protein is shown as green ribbons and its molecular surface in
transparent gray. The ligand is represented as salmon sticks. (d) 13 hCA
IX theoretical binding pose within the hCA II (PDB 3K34) structure. The protein is
shown as pink ribbons and its molecular surface in transparent gray. The ligand is
represented as salmon sticks. The images were rendered using the UCSF Chimera
software.
Figure 3.
(a) 14/hCA IX (PDB 5FL4) theoretical complex as calculated by docking
simulations. The protein is shown as cyan ribbons and sticks while the ligand as
orange sticks. Critical residues are labeled. H-bonds are depicted as red dashed lines
while coordination bonds as green dashed lines. (b) 14 hCA IX theoretical
binding pose within the hCA XII (PDB 5MSA) X-ray structure. The protein is shown as
light blue ribbons and sticks while the ligand as orange sticks. Critical residues are
labeled. H-bonds are depicted as red dashed lines while coordination bonds as green
dashed lines. (c) 14 hCA IX theoretical binding pose within the hCA I
(PDB 6F3B) X-ray structure. The protein is shown as green ribbons and its molecular
surface as transparent gray. The ligand is shown as orange sticks. (d) 14
hCA IX docked binding pose within the hCA II (PDB 3K34) structure. The protein is
shown as pink ribbons and its molecular surface in transparent gray. The ligand is
depicted as orange sticks. The images were rendered using the UCSF Chimera
software.
Figure 4.
(a) 17/hCA IX (PDB 5FL4) theoretical complex as calculated by docking
simulations. The protein is shown as cyan ribbons and sticks while the ligand as
yellow sticks. Critical residues are labeled. H-bonds are depicted as red dashed lines
while coordination bonds as green dashed lines. (b) 17 hCA IX theoretical
binding pose within the hCA XII (PDB 5MSA) X-ray structure. The protein is shown as
light blue ribbons and sticks while the ligand as yellow sticks. Critical residues are
labeled. H-bonds are depicted as red dashed lines while coordination bonds as green
dashed lines. (c) 17 hCA IX theoretical binding pose within the hCA I
(PDB 6F3B) X-ray structure. The protein is shown as green ribbons and its molecular
surface as transparent gray. The ligand is shown as yellow sticks. (d) 17
hCA IX docked binding pose within the hCA II (PDB 3K34) structure. The protein is
shown as pink ribbons and its molecular surface in transparent gray. The ligand is
depicted as yellow sticks. The images were rendered using the UCSF Chimera
software.
(a) 13/hCA IX (PDB 5FL4) theoretical complex as calculated by docking
simulations. The protein is shown as cyan ribbons and sticks while the ligand as
salmon sticks. Critical residues are labeled. H-bonds are depicted as red dashed lines
while coordination bonds as green dashed lines. (b) 13 hCA IX theoretical
binding pose within the hCA XII (PDB 5MSA) X-ray structure. The protein is shown as light blue ribbons and
sticks while the ligand as salmon sticks. Critical residues are labeled. H-bonds are
depicted as red dashed lines while coordination bonds as green dashed lines. (c)
13 hCA IX theoretical binding pose within the hCA I (PDB 6F3B)
structure. The protein is shown as green ribbons and its molecular surface in
transparent gray. The ligand is represented as salmon sticks. (d) 13 hCA
IX theoretical binding pose within the hCA II (PDB 3K34) structure. The protein is
shown as pink ribbons and its molecular surface in transparent gray. The ligand is
represented as salmon sticks. The images were rendered using the UCSF Chimera
software.(a) 14/hCA IX (PDB 5FL4) theoretical complex as calculated by docking
simulations. The protein is shown as cyan ribbons and sticks while the ligand as
orange sticks. Critical residues are labeled. H-bonds are depicted as red dashed lines
while coordination bonds as green dashed lines. (b) 14 hCA IX theoretical
binding pose within the hCA XII (PDB 5MSA) X-ray structure. The protein is shown as
light blue ribbons and sticks while the ligand as orange sticks. Critical residues are
labeled. H-bonds are depicted as red dashed lines while coordination bonds as green
dashed lines. (c) 14 hCA IX theoretical binding pose within the hCA I
(PDB 6F3B) X-ray structure. The protein is shown as green ribbons and its molecular
surface as transparent gray. The ligand is shown as orange sticks. (d) 14
hCA IX docked binding pose within the hCA II (PDB 3K34) structure. The protein is
shown as pink ribbons and its molecular surface in transparent gray. The ligand is
depicted as orange sticks. The images were rendered using the UCSF Chimera
software.(a) 17/hCA IX (PDB 5FL4) theoretical complex as calculated by docking
simulations. The protein is shown as cyan ribbons and sticks while the ligand as
yellow sticks. Critical residues are labeled. H-bonds are depicted as red dashed lines
while coordination bonds as green dashed lines. (b) 17 hCA IX theoretical
binding pose within the hCA XII (PDB 5MSA) X-ray structure. The protein is shown as
light blue ribbons and sticks while the ligand as yellow sticks. Critical residues are
labeled. H-bonds are depicted as red dashed lines while coordination bonds as green
dashed lines. (c) 17 hCA IX theoretical binding pose within the hCA I
(PDB 6F3B) X-ray structure. The protein is shown as green ribbons and its molecular
surface as transparent gray. The ligand is shown as yellow sticks. (d) 17
hCA IX docked binding pose within the hCA II (PDB 3K34) structure. The protein is
shown as pink ribbons and its molecular surface in transparent gray. The ligand is
depicted as yellow sticks. The images were rendered using the UCSF Chimera
software.With the aim of rationalizing the selectivity of the compounds, the crystal structures of
hCA I (PDB 6F3B), hCA II (PDB
3K34), and hCA XII (PDB
5MSA) were downloaded and their
binding sites analyzed. To ascertain how the predicted docked poses of ligands
13, 14 and 17 in the hCA IX active site would fit
in the other CAs, the 3 D structures of the enzymes were superimposed. This analysis
revealed that the recognition pattern achieved for hCA IX, conducive of potent enzyme
inhibition, is unlikely to be confirmed for hCA I and hCA II due to major steric clashes
(Figures 2(C,D), 3(C,D), and 4(C,D)). Indeed,
their binding sites in the hot spot region feature bulky substituents that would
unfavorably affect the binding mode of the compounds, especially in the case of ligand
17 (Figure 4(C,D)).Conversely, hCA XII and hCA IX binding sites share a higher degree of homology. As such,
the binding poses found for 13, 14 and 17 in hCA IX
also fit in hCA XII (Figures 2(B) and 3(B)). Still, few key differences in the hot spot
region can be found. Specifically, some hydrophobic residues in hCA IX are replaced by
polar ones (L91, L123, V130 in CA IX become T88, Y121, S130 in hCA XII, respectively).
Importantly, in hCA XII the positive K69 takes the place of Q71 in hCA IX. It is possible
to infer that the more hydrophilic and positively charged binding site of hCA XII provides
a better fit for compounds with a 3′-carboxyl group on the pendant 2-phenyl ring (see
compound 14, Figure 3(B)). Instead,
compounds bearing the 3′-methoxycarbonyl moiety can interact more favorably with the
lipophilic and neutral hot spot region of hCA IX. Purportedly, the presence of the ethyl
linker grants the possibility for the ligand to maximize the favorable interactions in hCA
IX and hCA XII, with both substitution patterns on the 2-phenyl ring, the 3′-carboxyl
(18) or the 3′-methoxycarbonyl (17) moiety. On the other hand,
the same ethyl linker, by enhancing the ligand flexibility, should also allow for a better
fit into the hCA I and hCA II isoform structures, thereby negatively impacting on the
ligand selectivity profile (Figures 2, 3 and
4).
Conclusions
Several Schiff bases and secondary amines incorporating aromatic sulfonamide moieties in
their structure has been extensively studied as CAIs. Starting from these classes of
compounds and according to the frozen analog approach, we designed a series of derivatives
featuring the 2-substituted-benzimidazole-6-sulfonamide scaffold, a chemical template only
scarcely exploited in the CAIs’ medicinal chemistry field. A library of 14 derivatives was
synthesized and tested for their enzyme inhibitory activity against the physiologically
relevant humanCA I, II, IX, and XII isoforms. Computational studies were attained to
rationalize the SAR in terms of inhibitory activity and selectivity profile.Of note, the identification of a number of newly synthesized derivatives featuring high
potency against the hCA IX and or XII isoforms, combined with promising selectivity
profiles. These findings could result interesting for the development of novel anticancer
agents with limited side effects. Indeed, hCA IX and XII enzymes have recently emerged as
excellent targets for the design of novel therapeutic strategies for cancer, due to their
involvement in the tumor cells survival as well as in insurgence of resistance to classical
anticancer protocols. Extensive SAR analysis and cellular studies are ongoing to increase
the knowledge within this series of CAIs inhibitors.
Authors: J Borras; A Scozzafava; L Menabuoni; F Mincione; F Briganti; G Mincione; C T Supuran Journal: Bioorg Med Chem Date: 1999-11 Impact factor: 3.641
Authors: Serdar Durdagi; Murat Şentürk; Deniz Ekinci; Halis Türker Balaydın; Süleyman Göksu; Ö İrfan Küfrevioğlu; Alessio Innocenti; Andrea Scozzafava; Claudiu T Supuran Journal: Bioorg Med Chem Date: 2011-01-14 Impact factor: 3.641
Authors: Karen Köhler; Alexander Hillebrecht; Johannes Schulze Wischeler; Alessio Innocenti; Andreas Heine; Claudiu T Supuran; Gerhard Klebe Journal: Angew Chem Int Ed Engl Date: 2007 Impact factor: 15.336
Authors: Garrett M Morris; Ruth Huey; William Lindstrom; Michel F Sanner; Richard K Belew; David S Goodsell; Arthur J Olson Journal: J Comput Chem Date: 2009-12 Impact factor: 3.376
Authors: Mohammed I A Hamed; Khaled M Darwish; Raya Soltane; Amani Chrouda; Ahmed Mostafa; Noura M Abo Shama; Sameh S Elhady; Hamada S Abulkhair; Ahmed E Khodir; Ayman Abo Elmaaty; Ahmed A Al-Karmalawy Journal: RSC Adv Date: 2021-11-03 Impact factor: 4.036
Authors: Nadia Arif; Zahid Shafiq; Khalid Mahmood; Muhammad Rafiq; Sadia Naz; Sohail Anjum Shahzad; Umar Farooq; Ali H Bahkali; Abdallah M Elgorban; Muhammad Yaqub; Ahmed El-Gokha Journal: ACS Omega Date: 2022-08-04
Chart 1.
Figure 1.
Scheme 1.
Scheme 2.
Scheme 3.
Scheme 4.
Figure 2.
Figure 3.
Figure 4.