10.1002/cmdc.202000500
ChemMedChem
FULL PAPER
173.02. ESI-MS: Calculated for C35H54N2O3S m/z: 582.39 (100.0%), found
583.5 [M+1]. Elemental Analysis: calculated C, 72.12; H, 9.34; N, 4.81; S,
5.50; found C, 72.10; H, 9.37; N, 4.80; S, 5.52.
(20% hexane in ethyl acetate to 100% ethyl acetate), affording 8d as
yellow semisolid (yield 0.021g, 31%) with Rf = 0.72 (ethyl acetate 100%).
1H-NMR (CDCl3, 400 MHz): 1.59 (15H, s, CH3), 1.67 (3H, s, CH3), 1.90-
2.10 (16H, m, CH2CH2), 2.31 (4H, s, CH2CH2CO), 3.65-3.69 (2H, m,
CH2NH), 4.08 (2H, t, J=5.0 Hz, CH2O), 5.01-5.26 (5H, m, CH), 5.98 (1H,
s, NH), 6.26 (1H, d, J = 9.5 Hz, ArH) ,6.80 (1H, d, J = 2.1 Hz, ArH), 6.83
(1H, dd, J = 8.6, 2.3 Hz, ArH), 7.38 (1H, d, J = 8.5 Hz, ArH), 7.63 (1H, d, J
= 9.5 Hz, ArH); 13C-NMR (100 MHz, CDCl3): 16.51, 16.62, 18.28, 26.29,
27.28, 28.87, 35.83, 39.29, 40.19, 40.34, 68.13, 102.54, 112.83, 113.57,
114.14, 125.02, 129.53, 134.08, 143.81, 156.44, 161.59, 162.27, 173.68.
ESI-MS: Calculated for C38H53NO4 m/z: 587.40 (100.0%), found 588.48
[M+1]. Elemental Analysis: calculated C, 77.64; H, 9.09; N, 2.38; found C,
77.62; H, 9.12; N, 2.40.
Synthesis
of
(4E,8E,12E,16E)-4,8,13,17,21-pentamethyl-N-(4-
sulfamoylbenzyl)docosa-4,8,12,16,20-pentaenamide (8b):
To derivate 7 solution in dry DMF (120 mg, 0.25 mmol, 1 equiv), Et3N (108
µL, 0.75 mmol,
3 equiv) and 4-(aminomethyl)benzenesulfonamide
hydrochloride (83.5 mg, 0.375 mmol, 1.5 equiv) were added and reaction
mixture was stirred overnight at room temperature under N2 flow. Next,
water and HCl solution were added (pH ~3-4) and extracted with ethyl
acetate. Organic layers were dried over Na2SO4, filtered and concentrated
to dryness using vacuum. Crude product was purified on silica gel column
chromatography using ethyl acetate as eluent. Pure product 8b was
obtained as a yellow semisolid (yield: 61.7 mg, 43%) Rf = 0.75 (EtOAc).
1H-NMR (CDCl3, 400 MHz): 1.59 (15H, s, CH3), 1.67 (3H, s, CH3), 1.92-
2.08 (16H, m, CH2CH2), 2.31-2.31 (4H, m, CH2CH2CO), 4.42 (2H, d, J=6
Hz, CH2NH), 5.07-5.18 (5H, m, CH), 5.31 (2H, s, NH2), 6.37 (1H, t, J=6.0
Hz, NH), 7.29 (2H, d, J= 8.3 Hz, ArH), 7.75 (2H, d, J= 8.3 Hz, ArH); 13C-
NMR (100 MHz, CDCl3): 15.93, 16.02, 16.08, 17.69, 25.70, 26.69, 26.74,
26.79, 28.28, 32.75, 34.40, 35.16, 35.29, 39.60, 39.74, 39.76, 42.84,
124.22, 124.25, 124.28, 124.41, 124.49, 125.30, 125.62, 126.63, 127.95,
131.25, 133.42, 134.89, 134.94, 135.21, 141.07, 143.72, 173.41, 177.20.
ESI-MS: Calculated for C34H52N2O3S m/z: 568.37 (100.0%), found 569.45
[M+1]. Elemental Analysis: calculated C, 71.79; H, 9.21; N, 4.92; S, 5.64;
found C, 71.77; H, 9.23; N, 4.91; S, 5.65.
Carbonic anhydrase inhibition
An Applied Photophysics stopped-flow instrument has been used for
assaying the CA catalysed CO2 hydration activity.[38] 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-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. For each
inhibitor at least six traces of the initial 5–10% of the reaction have been
used for determining the initial velocity. The 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 distilled-
deionized water and dilutions up to 0.01 nM were done thereafter with the
assay buffer. Inhibitor and enzyme solutions were preincubated together
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
non-linear least-squares methods using PRISM 3 and the Cheng–Prusoff
equation, as reported earlier,[40-42] and represent the mean from at least
three different determinations. All CA isofoms were recombinant ones
obtained in-house as reported earlier. [40-42]
Synthesis of (4E,8E,12E,16E)-4,8,13,17,21-pentamethyl-N-(2-((2-oxo-
2H-chromen-6-yl)oxy)ethyl)docosa-4,8,12,16,20-pentaenamide (8c):
Derivative 7 (0.039 g, 0.078 mmol, 1 equiv, 0.47 mL) was diluted with 7
mL dry DMF under N2 flow. 4a (0.038 g, 0.118 mmol, 1.5 equiv) and Et3N
(0.024 g, 0.235 mmol, 3 equiv) were solubilized in 3 mL dry DMF under N2
flow and added dropwise over derivative 7 solution. The obtained mixture
was stirred for 24h at room temperature under N2 atmosphere until the
starting products consumed. Obtained yellow solution was worked up as
usually. The organic layers were washed with brine (2 x 30 mL), dried over
Na2SO4, filtered off and concentrated to dryness under vacuum to give a
yellow oil that was purified by silica gel column chromatography, eluting
with an appropriate mixture of solvents (20% hexane in ethyl acetate to
100% ethyl acetate), affording 8c as yellow semisolid (yield 0.0167g, 69%)
with Rf = 0.74 (ethyl acetate 100%). 1H-NMR (CDCl3, 400 MHz): 1.57-168
(18H, m, CH3), 1.91-2.10 (16H, m, CH2CH2), 2.31 (4H, s, CH2CH2CO),
3.65-3.69 (2H, m, CH2NH), 4.06 (2H t, J = 5.1 Hz, CH2O), 5.03– 5.24 (5H,
m, CH), 5.90-6.01 (1H, m, NH), 6.43 (1H d, J = 9.5 Hz, ArH), 6.92 (1H, d,
J = 2.6 Hz, ArH), 7.10 (1H, dd, J = 9.0, 2.8 Hz, ArH), 7.63 (1H, d, J = 9.6
Hz, ArH); 13C-NMR (100 MHz, CDCl3): 16.51, 16.61, 16.63, 16.66, 18.28,
26.29, 27.28, 27.39, 28.87, 35.85, 35.87, 39.49, 40.20, 40.34, 40.36, 68.16,
111.36, 113.15, 117.90, 118.66, 119.87, 120.43, 124.81, 124.85, 125.01,
125.09, 126.18, 131.86, 134.05, 135.43, 135.55, 135.82, 143.65, 149.33,
155.60, 161.46, 173.85. ESI-MS: Calculated for C38H53NO4 m/z: 587.40
(100.0%), found 588.51 [M+1]. Elemental Analysis: calculated C, 77.64; H,
9.09; N, 2.38; found C, 77.65; H, 9.10; N, 2.36.
Molecular modeling
The crystal structure of CA II (pdb 5LJT),[39] was prepared using the
Protein Preparation Wizard tool implemented in Maestro - Schrodinger
suite, assigning bond orders, adding hydrogens, deleting water molecules,
and optimizing H-bonding networks[43]. Energy minimization protocol with
a root mean square deviation (RMSD) value of 0.30 was applied using an
Optimized Potentials for Liquid Simulation (OPLS3e) force field. 3D ligand
structures were prepared by Maestro[43a] and evaluated for their
ionization states at pH 7.4 ± 0.5 with Epik.[43b] OPLS3e force field in
Macromodel[43c] was used for energy minimization for a maximum
number of 2500 conjugate gradient iteration and setting a convergence
criterion of 0.05 kcal mol-1Å-1. The docking grid was centered on the mass
center of the co-crystallized ligands and Glide used with default settings.
Ligands were docked with the standard precision mode (SP) of Glide[43c]
and the best 5 poses of each molecule retained as output. The best pose
for each compound, evaluated in terms of coordination, hydrogen bond
interactions and hydrophobic contacts, was refined with Prime[43d] with a
VSGB solvation model considering the target flexible within 3 Å around the
ligand[44-46].
Synthesis of (4E,8E,12E,16E)-4,8,13,17,21-pentamethyl-N-(2-((2-oxo-
2H-chromen-7-yl)oxy)ethyl)docosa-4,8,12,16,20-pentaenamide (8d):
Derivative 7 (0.058 g, 0.116 mmol, 1 equiv, 0.7 mL) was diluted with 7 mL
dry DMF under N2 flow. Compound 4b (0.056 g, 0.174 mmol, 1.5 equiv)
and Et3N (0.035 g, 0.35 mmol, 3 equiv) were solubilized in 3 mL dry DMF
under N2 flow and added dropwise over compound 7 solution. The
obtained mixture was stirred for 24h at room temperature under N2
atmosphere until the starting products consumed and the obtained yellow
solution was worked up as usually. The organic layers were washed with
brine (2 x 30 mL), dried over Na2SO4, filtered off and concentrated to
dryness under vacuum to give a yellow oil that was purified by silica gel
column chromatography, eluting with an appropriate mixture of solvents
Acknowledgements
This work was supported by a grant of the Romanian Ministry of
Research and Innovation, CNCS–UEFISCDI, project number PN-
III-P4-ID-PCCF-2016–0050, within PNCDI II and project number
PN-III-P1-1.1-TE-2016-1180, within PNCDI III..
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