Carbonic anhydrase inhibitory properties of some uracil derivatives

Abstract Inhibitors of carbonic anhydrase (CA) have been carried out in many therapeutic applications, especially antiglaucoma activity. In this study, we investigated some uracil derivatives (4–12) to inhibit human CA I (hCA I) and II (hCA II) isoenzymes. The KI values of the compounds 4–12 are in the range of 0.085–428 µM for hCA I and of 0.1715–645 µM against hCA II, respectively. It is concluded from the kinetic investigations, all compounds used in the study act as competitive inhibitors with substrate, 4-NPA. Uracil derivatives are emerging agents for the inhibiton of carbonic anhydrase which could be used in biomedicine.


Introduction
Uracil, one of the pyrimidine bases, is commonly present in ribonucleic acid (RNA) 1 . It is chemically weak acid and exposed to two tautomeric forms at pH 7.0. This amide-imidic acid tautomeric shifts from lactam which is the amide tautomer to the imidic acid tautomer is referred to as the lactim structure ( Figure 1) 2 . Uracils are used in the area of drug discovery due to their bioactivities, drug similarity in the perspectives of synthetic accessibility and ability. And it is reported that uracils show anti-viral and antitumour characteristics in addition to bactericidal, herbicidal and insecticidal features 3 . 5-Fluorouracil (5-FU) (1) is an anti-tumour agent used for the treatment of cancerous tumours in colon or/and breast 4 . Though 5-FU as a single agent has gained medical achievement, the molecule has been chemically modified with different processes to create new derivatives. Therefore, modified syntheses can improve their therapeutic indexes due to their well-known side effects 2 . 6-Amino-5-chlorouracil (2) and 6-amino-5-bromouracil (3) were the first thymidine phosphorylase inhibitors to be generated. However, their relatively less favourable IC 50 values did not allow them to be developed into drug candidates 2 .
Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes involving in numerous vital biochemical/physiological processes 5,6 based on reversible hydration/dehydration process of CO 2 /HCO À 3 7 . The enzyme is found in all living beings of the three domains of life 8 with six evolutionary gene families 9 . 15 CA isoenzymes encoded by a-CA gene family described in humans 10 . Some of these human CA isoenzymes such as CA I, II, III, VII and XIII are cytosolic, some forms, CA IV, IX, XII and XIV, are membrane bound, two ones as CA VA and VB are mitochondrial and CA VI is found in saliva. The last three forms of human CAs (CA VIII, X and XI) are determined as noncatalytic 5 .
The testing of CA inhibitors is carried out for the treatment of clinically important cases 11 . Many chemical ligands have been used to inhibit CAs such as anions 12 , bischalcones 13 , coumarins 14 , benzenesulfonamides 15 and phenoles 16 . And uracil derivatives have also been carried out to inhibit the catalytic activity of CAs 17 .

Purification of human carbonic anhydrase isozymes by affinity chromatography
Erythrocytes were purified from fresh human blood obtained from the Blood Centre of the Research Hospital at Atat€ urk University. The blood samples were centrifuged at 1500 rpm for 15 min and the plasma and buffy coat were removed. The red cells were isolated and washed twice with 0.9% NaCl, and hemolysed with 1.5 volumes of ice-cold water. The ghost and intact cells were removed by centrifugation at 20 000 rpm for 30 min at 4 C. The pH of the hemolysate was adjusted to 8.7 with solid Tris 18 . Firstly, benzoyl chloride and stirred for four hours at room temperature in CH 2 Cl 2 cellulose. After the spacer arm cellulose added as a benzyl group and finally diazotized sulfanilamide clamped to the para position of benzyl group as ligand. The hemolysate was applied to the prepared cellulose-benzyl-sulfanylamide affinity column equilibrated with 25 mM Tris-HCl/0.1 M Na 2 SO 4 (pH 8.7). The affinity gel was washed with 25 mM Tris-HCl/22 mM Na 2 SO 4 (pH 8.7). The human carbonic anhydrase (hCA I and hCA II) isozymes were eluted with 1 M NaCl/25 mM Na 2 HPO 4 (pH 6.3) and 0.1 M CH 3 COONa/0.5 M NaClO 4 (pH 5.6), respectively. All procedures were performed at 4 C 16 .

CA inhibition
Enzyme activity was determined spectrophotometrically by following the change in absorbance at 348 nm of 4-nitrophenylacetate  25,26 . The catalytic activity of these enzymes was calculated from Lineweaver-Burk plots, as reported earlier, and represent the mean from at least three different determinations. The CA I and II isoenzymes used here were purified from human blood as described earlier [19][20][21][22][23][24] .

Results and discussion
Some research groups have studied with phenol and several phenolic compound types on 12 mammalian CA isoenzymes to show inhibitory properties of phenols 27 . We have extended previous investigations with some phenolic compounds which are globally used in food and medical industries. These kinds of compounds in different phenolic structures with antioxidant characteristics are essential in quenching reactive oxygen species [28][29] . These type of compounds show several medical activities such as anticancer, anticarcinogenic, antimutagenic, antibacterial, antiviral or antiinflammatory 5 . Phenol, phenolic compounds and hydroxybenzoic acid derivatives are widely put into effect as prodrugs and/or drugs. Salicylic acid possesses the ability to reduce fevers and ease aches and pains. To date these medicinal features have been known and used as anti-inflammatory drug [27][28][29][30][31][32][33][34] .
In the present study we have purified CA I and II (hCA I and hCA II) from human erythrocytes and examined the in vitro inhibition effects of above mentioned uracil derivatives 4-12 on these enzymes (Table 1), using the esterase activity of hCA I and II, with 4-NPA as substrate. Uracil derivatives 4 and 5 were synthesized from commercially available uracil. For this purpose uracil was converted to the brom uracil by using 1.1 Eq bromine in DMF at 120 C 17 . Condensation of brom uracil 2 with methanesulfonyl chloride, p-toluenesulfonyl chloride and acetic anhydryde to afford uracil derivatives 3 which has antitomour activity, 4, and 5 in comparable yield 35,36 , respectively. The other tested compounds 6-15 were purchased from Sigma-Aldrich.
(i) Against the slow cytosolic isozyme hCA I, compounds 4-8, and 15 behave as weak inhibitors, with K i values in the range of 316.2-464 mM, similarly to the structurally related compounds 13 and 14 (K I s of 795 and 4003 mM). It is interesting that compounds 9-12 were much better hCA I inhibitors as compared to the corresponding compounds 7 and 9 from which it was derivated ( Figure 2). Kinetic investigations (Lineweaver-Burk plots, data not shown) indicate that similarly to sulfonamides and inorganic anions [12][13][14][15][16] , all the investigated natural compounds act as competitive inhibitors with 4-NPA as substrate.
(ii) A better inhibitory activity has been observed with compounds 9-12 investigated here for the inhibition of the rapid cytosolic isozyme hCA II (Table 1). Structure-activity relationship (SAR) is thus quite sharp for this small series of hydroxylic compounds: compounds 9-12 are effective leads, with two mono or di hidroxy moieties is already a submicromolar hCA II inhibitor. This effect is maintained when different groups are present in the meta position to the phenol OH moiety, such as in resorcinol. The best hCA II  inhibitor in this series of derivatives were compound 11 with a K I of 0.1715 mM.
In a recent study it was reported that catechol and resorcinol 27 act as a CAI inhibitor, and could represent the starting point for a new class of inhibitors that may have advantages for patients with sulfonamide allergies. The sulfonamide zinc-binding group is thus superior to the thiol one (from the thioxolone hydrolysis product) for generating CA inhibitors with a varied and sometimes isozymeselective inhibition profile against the mammalian enzymes. However, it is still important to explore further classes of potent CAIs in order to detect compounds with different inhibition profiles.
Compounds 3-12 used in this study affect the activity of CA isozymes due to the presence of the different functional groups (CH 3 , OH, Br, COOH, NH 2 , mesityl, and tosyl) present in their scaffold. Therefore, our findings indicate another class of possible CAIs of interest, in addition to the well-known inhibitors, the phenols/ biphenyl diphenols bearing bulky ortho moieties in their molecules. Some hyroxylic compounds investigated here exhibited effective hCA I and II inhibitory activity, in the low-micromolar range, by the esterase method which usually gives K I -s an order of magnitude higher as compared to the CO 2 hydrase assay. These findings point out that substituted hdroxylic compounds may be used as leads for generating potent CAIs eventually targeting other isoforms.

Disclosure statement
The authors report no declarations of interest