2.1. Ocular Diseases
Open-angle glaucoma is a multifaceted disease associated with elevated IOP, damage of the optic nerve and loss of vision. IOP is based on the balance of aqueous humor production by the ciliary body and its drainage. The important role of NO in all compartments of the eye in maintaining basal resting tone of circulation has been recognized through cGMP-dependent pathways. Hypertensive glaucoma patients have a reduced NO/cGMP content in the aqueous humor. Despite these drugs are an underexplored class in the field of ocular therapeutics, glyceryl trinitrate decreases IOP and enhances cGMP levels
in situ, likely for aqueous humor homeostasis in normal and pathological conditions [
18,
19].
Conversely, selective activation of hCA II, expressed in the ciliary bodies and elicited by β-adrenergic stimulation, improves aqueous formation and transport as well as its release into the posterior chamber, thus increasing IOP. For this reason, selective hCA II inhibitors are another class of drugs that has been used to reduce IOP [
20,
21]. Then, sulfonamide (compounds
1–
16) and atypical (compounds
17–
28) hCA inhibitors linked to NO-donors can be an interesting approach in cases of glaucoma, as reported in
Table 1 [
22,
23,
24]. hCA IV and overexpressed hCA XII isoforms are also found in the ciliary processes within the eyes of normal and glaucomatous patients [
25,
26] and showed to be innovative and less explored targets for this disease, whereas hCA I represents the main off-target isoform.
Accordingly, topical selective hCA II inhibitors such as dorzolamide (Trusopt
®) or brinzolamide (Azopt
®) are clinically used to regulate aqueous humor production and drainage and IOP in hypertensive glaucoma. On the contrary, hCA inhibitors such as acetazolamide (Diamox
®), methazolamide, ethoxzolamide or dichlorophenamide have been used as systemic antiglaucoma drugs for more than 50 years, but suffered from side effects due to inhibition of the enzyme present in tissues other than the eye [
27,
28]: the possibility of a topical administration was also investigated, but with negative results. As a part of an ongoing research, some authors studied a new class of NO donating dorzolamide analogues (amides or carbamates) for topical administration that also presented inhibitory activity against hCA, such as compounds
1–
5 reported in
Table 1 [
23]. These are examples of single molecules endowed with a dual mechanism of action (the enhancement of NO/cGMP signaling and the inhibition of specific CA isozymes). The NO-donating moieties comprehended an NO-donor linker (incorporating a nitrate ester) to the amine functionality of dorzolamide and were firstly evaluated for their stability to the hydrolysis by eye esterases in rabbit corneal homogenate. The dinitrate ester
5 was included to eventually measure the effect of increasing the theoretical deliverable amount of NO to eye tissues, whereas dorzolamide was the reference standard. Compounds
1–
5 inhibited hCA II with
Ki in the low nanomolar range. The alkyl nitrate linkers were more selective for hCA II than the benzyl nitrate spacer. Interestingly, compounds
1 and
3 were selective and equipotent to dorzolamide for hCA II. Conversely, hCA I inhibitory activity of these derivatives was lower (dorzolamide and isosorbide mononitrate were inactive). Compound
2 was also active against hCA IV in the low nanomolar range as dorzolamide. Crystallographic analysis of
5 in the CA active site showed some hydrogen bonds between secondary nitrate and carboxamide hydrogens of Asn62, between ester oxygen and carboxamide hydrogens and between primary nitrate oxygen with the imidazole moiety of His64.
Table 1.
Dual agents endowed with NO donating properties and selective hCA isoform inhibitory activity for the treatment of open-angle glaucoma.
Table 1.
Dual agents endowed with NO donating properties and selective hCA isoform inhibitory activity for the treatment of open-angle glaucoma.
Compound | Structure | Ki (nM) |
---|
hCA I | hCA II | hCA IV |
---|
Dorzolamide |  | 50,000 | 3.2 | 43 |
Isosorbide mononitrate |  | >100,000 | >100,000 | >100,000 |
1 |  | 2950 | 14 | 4360 |
2 |  | 470 | 71 | 46 |
3 |  | 410 | 13 | 181 |
4 |  | 705 | 76 | 339 |
5 |  | 1520 | 63 | 3905 |
6 |  | 189 | 18 | 44 |
7 |  | 242 | 198 | 345 |
8 |  | 15 | 10 | 47 |
9 |  | 40 | 28 | 154 |
10 |  | 137 | 33 | 225 |
11 |  | 32 | 10 | 46 |
12 |  | 43 | 14 | 38 |
13 |  | 395 | 101 | 139 |
14 |  | 587 | 88 | 148 |
15 |  | 41 | 12 | 76 |
16 |  | 950 | 71 | 89 |
17 |  | >100,000 | >100,000 | nd |
18 |  | >100,000 | >100,000 | nd |
19 |  | >100,000 | >100,000 | nd |
20 |  | >100,000 | >100,000 | nd |
21 |  | >100,000 | >100,000 | nd |
22 |  | >100,000 | >100,000 | nd |
23 |  | >100,000 | >100,000 | nd |
24 |  | >100,000 | >100,000 | nd |
25 |  | >100,000 | >100,000 | nd |
26 |  | >100,000 | >100,000 | nd |
27 |  | >100,000 | >100,000 | nd |
28 |  | >100,000 | >100,000 | nd |
Dichlorophenamide |  | 1200 | 38 | 15,000 |
Acetazolamide |  | 250 | 12 | 74 |
Another series of derivatives
6–
16 was recently explored attaching “tails” to give better physico-chemical properties (water solubility, enhanced penetrability through the cornea) to five different sulfonamide scaffolds (4-carboxybenzenesulfonamide, 3- or 4-carboxybenzolamides, 4-(2-carboxyethyl)benzenesulfonamide and 4-hydroxybenzenesulfonamide) [
22]. The hydroxyl or carboxyl moieties were functionalized by means of ester moieties (aliphatic C3–C5 moieties or the aromatic ones) releasing both NO and sulfonamides
in vivo. Compounds
8,
9,
11,
12 and
15 showed effective inhibition in the nanomolar range against the slow cytosolic isoform hCA I (an off-target enzyme for antiglaucoma agents), whereas the remaining compounds were less effective inhibitors. Other new sulfonamides acted as very good inhibitors of hCA II isozyme, with
Kis in the range of 10–33 nM, in the same range as those of the systemically used antiglaucoma sulfonamides acetazolamide and dichlorophenamide or the topically acting dorzolamide. hCA IV was also effectively inhibited by some of them with respect to the clinically used drugs.
The last series of compounds
17–
28 lack the sulfamoyl zinc-binding group and represent a class of mono- or polycyclic organic nitrate esters. Unfortunately, despite slight hCA II selectivity, their IC
50 values ranged between 7.13 mM and 124 mM against hCA I and between 65.1 μM and 0.79 mM against hCA II (
Table 1) [
24]. hCA IV inhibitory activity for these compounds was not evaluated so far.