Carbonic anhydrase activation profile of indole-based derivatives

Figures & data

Figure 1. Structures of known and novel indole-based CAAs.

Scheme 1. Reagents and conditions: (i) anhydrous Et₂O, oxalyl chloride, room temperature, 2h; (ii) anhydrous toluene, N,N-dimethyl-1,3-propylenediamine (for 12) or N,N-diethylethylenediamine (for 13), NEt₃, room temperature, overnight; iii) a) anhydrous DCM, BBr₃, -10 °C, 30 min.; (b) room temperature, overnight; (iv) acetone, Cs₂CO₃, 2-bromoethylacetate, reflux, overnight; (v) 1,4-dioxane, 1M NaOH, room temperature, 5h.

Scheme 2. Reagents and conditions: (i) 1,4-dioxane, 3-amino-1-propanol (for 18) or ethanolamine (for 19), room temperature, 3 h; (ii) (a) anhydrous DCM, BBr₃, −10 °C, 30 min.; (b) room temperature, overnight; (iii) anhydrous DMF, Cs₂CO₃, 3-dimethylamino-1-propylchloride hydrochloride (for 3) or 2-chloro-N,N-diethylethylamine hydrochloride (for 4), 60 °C, 5h.

Scheme 3. Reagents and conditions: (i) MeOH/H₂O, LiOH H₂O, 80 °C, overnight; (ii) anhydrous DMF, TBTU, DIPEA, ethanolamine (for 5 and 6) or benzylamine (for 7), room temperature, overnight.

Scheme 4. Reagents and conditions: (i) abs. EtOH, conc. H₂SO₄, 80 °C, 20 h; (ii) (a) NaH, dry DMF, 0 °C, 30 min.; (b) BzBr, room temperature, 5 h; (iii) (a) anhydrous DCM, BBr₃, -10 °C, 30 min.; (b) room temperature, overnight; (iv) anhydrous DMF, Cs₂CO₃, 3-dimethylamino-1-propylchloride hydrochloride (for 8) or 2-chloro-N,N-dimethylethylamine hydrochloride (for 9) or 2-chloro-N,N-diethylethylamine hydrochloride (for 22) or 60 °C, 5h.

Table 1. CA activation of isoforms hCA I, II, VA, and VII with compounds 1–9, by a stopped-flow CO2 hydrase assay.

KA (µM)^a
Cpd	R1	R2	hCA I [hCA VA/hCA VII]^b	hCAII [hCA II/hCA VII]^b	hCA VA [hCA VA/hCA VII]^b	hCA VII
1	(CH2)3N(CH3)2	(CH2)2OH	>100 [>9.3]	>100 [>9.3]	25.0 [2.3]	10.8
2	(CH2)2N(CH2CH3)2	(CH2)2OH	88.9 [8.9]	>100 [>10.0]	28.7 [2.9]	10.0
3	(CH2)3OH	(CH2)3N(CH3)2	>100 [>9.4]	>100 [>9.4]	50.9 [4.8]	10.6
4	(CH2)2OH	(CH2)2N(CH2CH3)2	>100 [>11.0]	>100 [>11.0]	34.4 [3.8]	9.1
5	(CH2)2OH	(CH2)3N(CH3)2	>100 [>9.6]	>100 [>9.6]	28.0 [2.7]	10.4
6	(CH2)2OH	(CH2)2N(CH3)2	>100 [>9.3]	>100 [>9.3]	26.8 [2.5]	10.7
7	CH2C6H5	(CH2)2N(CH2CH3)2	>100 [>13.3]	>100 [>13.3]	24.4 [3.2]	7.5
8	–	(CH2)3N(CH3)2	69.1[9.6]	>100 [>13.9]	59.8 [8.3]	7.2
9	–	(CH2)2N(CH3)2	>100 [>12.2]	>100 [>12.2]	29.1 [3.5]	8.2
Histamine			2.1	125	0.010	37.5

^aMean from 3 different assays, by a stopped flow technique (errors were in the range of ±5–10% of the reported values). ^bK_A ratio for the indicated enzyme isoforms.

Figure 2. The best binding pose of 7 before (A) and after (B) the proton shuttling step in the hCA VII active site (PDB ID 3MDZ). The ligand is represented as orange (A) or yellow (B) sticks, while the protein structure is depicted in sea-green ribbons and sticks. Interacting residues are labelled. The Zn²⁺ ion is represented as a violet sphere. H-bonds are represented with the red dashed lines.

Figure 3. Binding poses calculated for compound 7 (orange and yellow) rigidly translated into the hCA I structure (PDB ID 6EVR, pink surface, A) and hCA II structure (PDB ID 3K34, green surface, B). All the pictures were rendered using UCSF Chimaera software⁶¹.

Table 2. Calculated physicochemical and pharmacokinetic properties of 7.

Parameter	7	Range or recommended values^a
#rotor^b	11.000	0.0/15.0
Mol MW^c	455.599	130.0/725.0
Dipole^d	6.972	1.0/12.5
DonorHB^e	1.000	0.0/6.0
AccptHB^f	5.250	2.0/20.0
QPlogPo/w^g	6.455	−2.0/6.5
QPlogS^h	−6.598	−6.5/0.5
QPPCaco^i	1209.191	<25 Poor, >500 great
QPlogBB^j	−0.076	−3.0/1.2
QPPMDCK^k	672.043	<25 Poor, >500 great
QPlogKhsa^l	1.261	−1.5/1.5
PercentHumanOralAbsorption^m	100.000	(<25% is Poor)

^aFor 95% of known drugs. ^bNumber of non-trivial (not CX3), non-hindered (not alkene, amide, small ring) rotatable bonds. ^cMolecular weight of the molecule. ^fComputed dipole moment of the molecule. ^dComputed dipole moment of the molecule. ^eEstimated number of hydrogen bonds that would be donated by the solute to water molecules in an sueous solution. ^fEstimated number of hydrogen bonds that would be accepted by the solute from water molecules in an aqueous solution. ^gPredicted octanol/water partition coefficient. ^hPredicted aqueous solubility, log S. S in mol dm⁻³ is the concentration of the solute in a saturated solution that is in equilibrium with the crystalline solid. ⁱPredicted apparent Caco-2 cell permeability in nm/sec. ^jPredicted brain/blood partition coefficient. ^kPredicted apparent MDCK cell permeability in nm/sec. MDCK cells are considered to be a good mimic for the blood-brain barrier. ^lPrediction of binding to human serum albumin. ^mPredicted qualitative human oral absorption.

Figure 4. Treatment with compound 7 increases the level of BDNF released from human microglial cells. (A) C20 cells were exposed for 24 h at different concentrations of compounds 7, 8 and 9. Then the medium was replaced, and an MTS assay was performed to test cell viability. Results are reported as the percentage of the control group, treated with vehicle (DMSO). Data are represented as means ± SEMs of three independent experiments. The significance of the differences was determined by one-way ANOVA, which was followed by Bonferroni’s post-test: ** p < 0.01 vs. CTRL. B) The supernatant derived from C20 cells treated with 10 μM of compound 7 was collected and used for BDNF quantification by ELISA assay. Levels of BDNF (pg/ml) were then normalised on crystal violet absorbance. Data are represented as means ± SEMs of three independent experiments and the significance of the differences was determined by Student’s t-test analysis.

Pettersen EF, Goddard TD, Huang CC, Couch GS, et al. UCSF Chimera-a visualization system for exploratory research and analysis. J Comput Chem 2004;25:1605–12.

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