Inhibition studies

Also for these calixarene-based compounds inhibition studies were carried out by Supuran’s coworkers at University of Florence. All the mentioned compounds were tested towards the six different isoforms (hCAI, hCAII, hCAIX, VchCAβ, Can2, MgCA) already used for the two

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monovalent compounds described in the previous Chapter2. Also, in this case, AAZ was used as reference compound.

As it can be seen in Table 3.3.5.1, almost all tested calixarenes appear mainly selective for Can2 CA for which they show their lowest Ki values although higher with respect to AAZ. Analogously, most of them show a Ki towards MgCA lower than AAZ.

Towards Can2, almost all calixarenes showed to be high-nanomolar inhibitors, except for compounds 63, 42bx, 56b and 56a, with which even low-nanomolar Ki similar to AAZ are reached. In this context, if we compare compound 42bx with compound 42b, we notice that introducing a further propyl-benzenesulfonamide arm, the inhibition constant increases of one order of magnitude, whose behaviour, by the way, is kept in MgCA. This suggests that the thinner propyl-ammonium arm makes no significant clashes with enzyme without excluding its possibility to bind negative-charged moieties or electron-donating atoms, which would justify this interesting gain. Moving to ammonium analogues, it can be seen that tetra-ammonium derivative V in cone conformation is a nanomolar-range inhibitor towards Can2 and MgCA, meanwhile the same molecule in 1,3-alternated conformation 62 is totally inactive. As already commented in XRD structure of intermediate 60, amidic nitrogen (and so sulfonamidic nitrogen as well) is just few far away from calixarene scaffold and so in order to reduce steric clashes, it would be better lengthen more the aliphatic chain. With this idea in mind, maybe, the molecule in this conformation would be able to simultaneously chelate multiple copies of enzyme boosting its affinity. Among these, it is significant underline which type of contribute make either propyl or ethoxyethyl chains to inhibition. Paying attention to monofunctionalized and difunctionalized calixarenes, it is clear how propyl chains are favourite to ethoxyethyl chains. In fact passing from dipropyl to diethoxyethyl derivatives, in particular from 42a to 42c, and from tripropyl to triethoxyethyl derivatives, in particular from 49a to 49c and from 48a to 48b, Ki

increase due to expected clashes between ethoxyethyl chains and enzyme walls. This effect is mainly enhanced in the ammonium derivative 48a-48b. Here, probably, the chain length is a very discriminating factor for inhibition. The reason underneath this evidence is not still clear.

Further studies will be carried out to understand, which factor, among steric hindrance and polarity, is predominant in the binding. Particularly interesting is compound 56b, which shows remarkable inhibition properties also towards two out of the three hCAs. In fact, towards hCAI (Ki= 84.9 nM) this upper rim monofunctionalized compound is more active than AAZ and towards hCAII (Ki= 48.1 nM) is very close to the efficiency of the drug. In this context it is significant to compare this ligand with the analogue lower rim monofunctionalized 49b, as 56b blocked in the cone geometry, and the analogue upper rim monofunctionalized 56a, conformationally mobile. All three are equipped with a sulfonamide moiety linked to the calixarene through a thiourea unit, but only 56b presents a defined cavity oriented to the same part of the space as the binding unit. Apart the case of hCAIX and Can2 for which there are no substantial differences, both 49b and 56a result less active than 56b with an increase of Ki of one or two orders of magnitude and being completely inactive towards VchCAβ. The possible absence in 56b of steric hindrance due to the ethoxyethylchains that on the contrary in 49b are directing towards the active site funnel together with the sulfonamide unit, wh and the presence of the macrocyclic cavity in 56b potentially oriented together with the binding unit towards the funnel and then available for additional interactions with lipophilic side chains of the enzyme seem to be important factors in the binding.

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Table 3.3.5.1. Inhibition data of calixarene derivatives towards the selected isoforms, obtained from stopped-flow technique. Each value is a mean from 3 different assays (errors were in the range of  5-10 % of the reported values). All data are separated by a straight line when the degree of functionalization changes (in green, thioureidic derivatives; in red, amide derivatives; in black, ammonium derivatives).

KI (nM)

Compound hCAI hCAII hCAIX VchCAβ Can2 MgCA

I (cone-calix[6]-hexa-amide) 3349.1 497.2 872.5 5950 138 >100000

II (cone-calix[6]-hexa-thiourea) 5953.8 2397.4 817.8 7370.1 >100000 >100000 III

(cone-calix[6]-hexa-ammonium) >100000 >100000 >100000 6812.5 107 7500

IV (cone-calix[4]-tetra-amide) 8565.7 3878.9 4026.3 757.1 125 7100

V

(cone-calix[4]-tetra-ammonium) >100000 >100000 >100000 3144.9 136 7600 62

(1,3-alternated-calix[4]-ammonium) >100000 >100000 >100000 >100000 >100000 >100000 63

(1,3-alternated-calix[4]-thiourea) 542.3 401.4 1795 >100000 63.4 >100000

42b

(cone-calix[4]-dipropyl-diamide) 5521 533.6 1107 8555.1 270 8800

42bx

(cone-calix[4]-dipropyl-monoamide-monoammonium) >100000 4466 >100000 >100000 92.1 2585 42a

(cone-calix[4]-dipropyl-dithiourea) 8286.6 1214.2 246.4 8163.3 >100000 3900

41a

(cone-calix[4]-dipropyl-diammonium) >100000 >100000 >100000 9411.6 267 7800 42c

(cone-calix[4]-diethoxyethyl-dithiourea) 2692.3 342.7 2180.3 7488.4 260 8900

49a

(cone-calix[4]-tripropyl-monoamide) >100000 7377.6 1979.8 8151.8 253 5600

48a

(cone-calix[4]-tripropyl-monoammonium) >100000 >100000 >100000 7322.3 290 937 49c

(cone-calix[4]-triethoxyethyl-monoamide) 5909.4 3090.2 1487.1 8819.2 247 8800

49b

(cone-calix[4]-triethoxyethyl-monothiourea) 5567.9 894.7 1772.2 >100000 170 6700

56b

(cone-calix[4]-GABA-thiourea) 84.9 48.1 1937 6745 50.4 931

56a

(mobile-calix[4]-GABA-thiourea) 3751 859.7 1657 >100000 45.1 5325

48b

(cone-calix[4]-triethoxyethyl-monoammonium) >100000 >100000 >100000 >100000 237 >100000 55

(cone-calix[4]-GABA-ammonium) >100000 >100000 >100000 >100000 >100000 >100000

acetazolamide (AAZ) 250 12 25 451 10 74000

As far as linker nature is concerned, thioureidic linker shows a little stabilizing effect with respect to the amide one. Keeping constant the nature of permanent protecting chains, if we pass from compound 42b to 42a and from compound 49c to 49b, inhibition constants get lower, which is probably imputable to additional interactions between S atom and enzyme aminoacid side-chains.

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Since compounds 4a, 4b and 4c represent the exact monomeric acyclic model of all synthesized calixarenes, we analysed the inhibition data also in view of verifying the possible occurrence of a multivalent effect. In the case of hCAs, not only this is not the case, but the calixarene derivatives are even worse inhibitors than the monovalent ligands. Moreover, also in the cases where the calixarene-based ligands are better than the monovalent, i.e. against MgCA, it is clear that no multivalent effect occurs in the binding. In fact, calixarenes with a different number of the same active unit show very close Ki values, like for instance compounds IV, 42b and 49a bearing 4, 2 and 1 sulfonamide units, respectively, or compounds 48a and 41a exposing 1 and 2 ammonium heads, respectively. However, towards this CA isoform, the data suggest that the insertion of the active unit, both ammonium and sulfonamide, in the macrocyclic structure improves its biological activity.

Table 3.3.5.2. Inhibition data of calixarene derivatives towards the selected isoforms, obtained from stopped-flow technique. Each value is a mean from 3 different assays (errors were in the range of  5-10 % of the reported values). All data are separated by a straight line when the linker or binding unit changes (in red, amide derivatives; in black, ammonium derivatives).

KI (nM)

Compound hCAI hCAII hCAIX VchCAβ Can2 MgCA

IV (cone-calix[4]-tetra-amide) 8565.7 3878.9 4026.3 757.1 125 7100

42b

(cone-calix[4]-dipropyl-diamide) 5521 533.6 1107 8555.1 270 8800

49a

(cone-calix[4]-tripropyl-monoamide) >100000 7377.6 1979.8 8151.8 253 5600

4c (amide monomer) 8.4 4.1 21.8 4143.8 >10000 >10000

41a

(cone-calix[4]-dipropyl-diammonium) >100000 >100000 >100000 9411.6 267 7800 48a

(cone-calix[4]-tripropyl-monoammonium) >100000 >100000 >100000 7322.3 290 937 4a (ammonium monomer) >10000 >10000 >10000 >10000 >10000 >10000

acetazolamide (AAZ) 250 12 25 451 10 74000

3.3.6 Solid state structure of hCAII/49b, hCAII/56b and