Vanillin enones as selective inhibitors of the cancer associated carbonic anhydrase isoforms IX and XII. The out of the active site pocket for the design of selective inhibitors?

Figures & data

Table 1. Synthesis of vanillin enones 1–7.

Ketone	Catalyst	Temperature	Reaction time (h)	Product	Yield (%)
C-glucosyl	Et3N/L-Pro	Reflux	48	1	45
C-galactosyl	Et3N/L-Pro	Reflux	96	2	35
Acetone	NaOH	40 °C	48	3	63
Butanone	Pyrrolidine	Rt	48	4	79
Methyl isobutyl ketone	Pyrrolidine	Rt	72	5	76
Acetophenone	Et3N/L-Pro	Reflux	44	6	70
p-Bromoacetophenone	Et3N/L-Pro	Reflux	72	7	40

Scheme 1. Preparation of vanillin enones 1–7 discussed in the paper

Figure 1. Structure of compounds 1–7.

Table 2. Inhibition of hCA I, II, IX, XII with vanillin enones 1–7.^a,b

	Ki (µM)^b				Selectivity ratio
Cmpd	hCA I	hCA II	hCA IX	hCA XII	I/IX	I/XII	II/IX	II/XII
1	71.6	>100	30.8	4.8	2.3	15.0	>3.2	>21
2	80.5	>100	20.8	2.5	3.9	32.2	>4.8	>40
3	86.3	>100	24.0	3.2	3.6	27.0	>4.2	>31
4	>100	>100	32.0	2.5	>3.1	>40	>3.1	>40
5	>100	>100	26.3	1.7	>3.8	>59	>3.8	>59
6	76.3	96.4	29.1	0.63	2.6	121.1	3.3	153.0
7	92.6	82.0	>100	6.4	<0.9	14.5	<0.8	12.8
AAZ	0.25	0.012	0.025	0.006	0.03	41.6	0.001	2

^aAll CAs are recombinant enzymes obtained in the authors’ laboratory as reported earlier^46–50.

^bErrors in the range of 5–10% of the reported value, from three different determinations.

Figure 2. Binding poses of 7 to four CA isozymes obtained through molecular docking using Smina program. The ligand poses can be seen inside the catalytic site. In addition, some poses show the inhibitor bound in the adjacent pocket (oriented to the left) to the entrance at the active site cavity in the case of isoforms CA I, II, and XII.

Table 3. Binding free energies (kcal mol⁻¹) of ligands with hCA I and hCA II calculated with MM-GBSA.

Ligand	Pose	hCA I	hCA II
1	Down	41.0	33.4
	Up	–	–
	Pocket	25.6	25.9
2	Down	33.3	7.0
	Up	–	–
	Pocket	32.3	28.0
3	Down	20.1	24.9
	Up	29.3	20.8
	Pocket	19.7	Unstable
4	Down	Unstable	Unstable
	Up	35.0	Unstable
	Pocket	22.5	31.3
5	Down	32.9	32.8
	Up	Unstable	–
	Pocket	27.7	31.3
6	Down	Unstable	30.3
	Up	Unstable	–
	Pocket	18.6	17.0
7	Down	37.7	23.8
	Up	Unstable	–
	Pocket	18.8	21.5

Figure 3. Per-residue decomposition of binding energy (only the enthalpy term considered) of ligand-bound hCA I and II, located in the pocket adjacent to the catalytic site. These calculations were performed with the MMPBSA.py module and plotted using Python's Matplotlib library. The residues shown were those that evidenced at least one interaction of < 0.3 kcal mol⁻¹ with a ligand.

Figure 4. Poses of compound 7 bound in the adjacent pocket to the active site entrance of hCA I (above) and hCA II (below). Hydrogen bonds with residues Lys170, Pro 240, and Gln242 are highlighted in yellow. Images were generated with Pymol. The selected structures of all ligands with CAs are presented in the Supplementary Material.