Synthesis and In Vitro Inhibition Effect of New Pyrido[2,3-d]pyrimidine Derivatives on Erythrocyte Carbonic Anhydrase I and II

In vitro inhibition effects of indolylchalcones and new pyrido[2,3-d]pyrimidine derivatives on purified human carbonic anhydrase I and II (hCA I and II) were investigated by using CO2 as a substrate. The results showed that all compounds inhibited the hCA I and hCA II enzyme activities. Among all the synthesized compounds, 7e (IC50 = 6.79 µM) was found to be the most active compound for hCA I inhibitory activity and 5g (IC50 = 7.22 µM) showed the highest hCA II inhibitory activity. Structure-activity relationships study showed that indolylchalcone derivatives have higher inhibitory activities than pyrido[2,3-d]pyrimidine derivatives on hCA I and hCA II. Additionally, methyl group bonded to uracil ring increases inhibitory activities on both hCA I and hCA II.


Introduction
Carbonic anhydrase (CA, EC 4.2.1.1) is a ubiquitous zinc enzyme. Basically, there are several mammalian cytosolic forms (CA-I, CA-II, CA-III, CA-VII, and CA-XIII), four membrane-bound forms (CA-IV, CA-IX, CA-XII, and CA-XIV), one mitochondrial form (CA-V), and a secreted CA form (CA-VI) [1,2]. They all catalyze a very simple physiological reaction, the interconversion between carbon dioxide and the bicarbonate ion, and are thus involved in crucial physiological processes connected with respiration and transport of CO 2 /bicarbonate between metabolizing tissues and the lungs, pH and CO 2 homeostasis, electrolyte secretion in a variety of tissues/organs, biosynthetic reactions (such as the gluconeogenesis, lipogenesis, and ureagenesis), bone resorption, calcification, tumorigenicity, and many other physiologic or pathologic processes [1][2][3]. CA inhibitors have now been a mainstay of human clinical intervention for several decades, with at least 25 clinically used drugs that are CA inhibitors [4]. Although there are many studies on this enzyme, the CA enzyme family continues to capture the attention of drug discovery scientists and clinicians as the knowledge regarding the therapeutic implications associated with this enzyme class continues to grow [4,5].
Indoles are one of the most important nitrogen containing heterocyclic molecules, found extensively in biological system which play vital role in biochemical processes. Indole ring constitutes an important template for drug design such as the classical nonsteroidal anti-inflammatory drugs (NSAIDs) indomethacin and indoxole [6]. Further indole derivatives have been reported to possess promising biological activities including analgesic [7], antipyretic [8], antifungal [9], antiinflammatory [10,11], antitumor [12], anticonvulsant [13], and selective COX-2 inhibitory activities [14]. Thus the efficient synthesis of novel substituted indole derivative compounds still represents highly pursued target.
In this study, a series of 7 indolylchalcone and 11 new pyrido [2,3-d]pyrimidine derivatives containing indole ring were synthesized and their effects on human carbonic anhydrase (hCA) I and II were evaluated. Additionally, structureactivity relationship was examined.

General Chemistry.
Melting points were taken on a Barnstead Electrothermal 9200. IR spectra were measured on a Shimadzu Prestige-21 (200 VCE) spectrometer. 1 H and 13 C NMR spectra were measured on a Varian Infinity Plus spectrometer at 300 and 75 Hz, respectively. 1 H and 13 C chemical shifts are referenced to the internal deuterated solvent. Mass spectra were obtained using MICROMASS Quattro LC-MS-MS spectrometer. Solvents were dried following standard methods. Sepharose 4B, L-tyrosine, sulfonamide, synthetic starting material, reagents, and solvents were purchased from Merck, Alfa Easer, Sigma-Aldrich, and Fluka.

Synthetic Procedures and Spectral Data
1H-Indole-3-carbaldehyde (2). Phosphorous oxychloride (1 mL) was added dropwise to cold anhydrous DMF (3 mL) and the mixture was stirred at 0 ∘ C for 1 h. The indole (1.17 g), dissolved in anhydrous DMF, was added dropwise to above formylation complex solution at below 10 ∘ C. The mixture was warmed to 35-40 ∘ C and stirred for 1 hour. Then NaOH (aq) (5.5 g NaOH, 14.6 mL water) was added. The mixture was warmed to 100 ∘ C and stirred for 1 h; then it was cooled, filtrated, and dried in vacuum oven for overnight. Pink powder was obtained in 94% yield. 1  Synthesis of Indolylchalcone Derivatives (5a-g). A solution of NaOH (aq) (40%, 5 mL) was added to mixture of 1-methyl-1Hindole-3-carbaldehyde (1 mmol) 3 and acetophenone derivatives (1 mmol) 4a-g in absolute ethanol. The mixture was stirred at room temperature for 2 hours. Then it was poured into ice-cold water, neutralized with acid, filtrated, and washed with water. The filtrate was dried in vacuum oven.

Preparation and Purification of Hemolysate from Blood
Red Cells. Blood samples (25 mL) were taken from healthy human volunteers. They were anticoagulated with acidcitrate-dextrose and centrifuged at 2000 g for 20 min at 4 ∘ C and the supernatant was removed. The packed erythrocytes were washed three times with 0.9% NaCl and then haemolysed in cold water. The ghosts and any intact cells were removed by centrifugation at 2000 g for 25 min at 4 ∘ C, and the pH of the haemolysate was adjusted to pH 8.5 with solid Tris-base. The 25 mL haemolysate was applied to an affinity column containing L-tyrosine-sulfonamide-sepharose-4B [21] equilibrated with 25 mM Tris-HCl/0.1 M Na 2 SO 4 (pH 8.5). The affinity gel was washed with 50 mL of 25 mM Tris-HCl/22 mM Na 2 SO 4 (pH 8.5). The hCA isozymes were then eluted with 0.1 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), which recovered hCA I and hCA II, respectively. Fractions of 3 mL were collected and their absorbance was measured at 280 nm.

CA Enzyme
Assay. CA activity was measured by the Maren method which is based on determination of the time required for the pH to decrease from 10.0 to 7.4 due to CO 2 hydration [22]. The assay solution was 0.5 M Na 2 CO 3 /0.1 M NaHCO 3 (pH 10.0) and Phenol Red was added as the pH indicator. CO 2 -hydratase activity (enzyme units (EU)) was calculated by using the equation 0 − / , where 0 and are the times for pH change of the nonenzymatic and the enzymatic reactions, respectively.

In Vitro Inhibition Studies.
For the inhibition studies of indolylchalcone and pyrido[2,3-d]pyrimidine derivatives, different concentrations of these compounds were added to the enzyme. Activity percentage values of CA for different concentrations of each pyrimidine derivatives were determined by regression analysis using Microsoft Office 2000 Excel. CA enzyme activity without these compounds was accepted as 100% activity.

Results and Discussion
3.1. Chemistry. The synthetic procedures are depicted in Scheme 1. The indolylchalcone derivatives 5a-g, prepared by the condensing various acetophenones and indolylaldehyde 3 with NaOH as a base, were reacted with 3-methyl-6aminouracil 6a and 6-aminouracil 6b to get pyrido[2,3d]pyrimidine derivatives (7a-k) at high yields. The large J value (15.5 Hz) clearly reveals the E-geometry for the chalcones. [2,3d]pyrimidine Derivatives for hCA I and hCA II Inhibitory Activities. For evaluating the hCA I and II inhibitory effect, all compounds were subjected to hCA I and II inhibition assay with CO 2 as a substrate. The result showed that all synthesized compounds (5a-g and 7a-k) inhibited the hCA I and hCA II enzyme activities.

Biological Evaluation of Indolylchalcone and Pyrido
The IC 50 values and inhibition constants of 5a-g and 7ak analogues against hCA I and hCA II were summarized in Table 1 and the IC 50 graphs were given in Figure 1.
We  5c  5d  5e  5f  5g  7a  7b  7c  7d  7e  7f  7g  7h  7i  7j 7k EtOH, rt,2h NaOH (aq) 40%, 6a: R 3 =CH 3 /6b: R 3 =H Scheme 1: Synthesis of pyrido [2,3-d]pyrimidine derivatives (7a-k).  Sulfonamides are coordinated to the zinc (II) ion within the hCA active site, whereas their organic scaffold fills the entire active site cavity, making an extensive series of van der Waals and polar interactions with amino acid residues delimiting this cavity [24,25]. As the synthesized compounds are very bulky and do not contain a classical zinc-binding group [4], it can be hypothesized that they are not able to bind near the zinc ion showing a different mechanism of action. Structural studies of the complexes that these compounds form with the human isoform II could clarify this important issue.

Conclusions
In conclusion, series of 7 indolylchalcone and 11 new pyrido[2,3-d]pyrimidine derivatives containing indole ring were synthesized. Their activities as hCA I and hCA II inhibitors and structure-activity relationships were examined. All compounds inhibited both hCA I and hCA II enzyme activities. Most of compounds containing electrondonating groups at phenyl ring were generally stronger inhibitors of hCA I and hCA II. Additionally, methyl group bonded to 3-position of uracil ring generally increased inhibitory activities on both hCA I and hCA II. Thus, the present study revealed that the type and position of substituent of the phenyl and uracil rings could be exploited to modulate the CA inhibitors efficacy.
In summary, enzyme inhibition is an important issue for drug design [26][27][28]. Our results showed that new pyrido [2,3d]pyrimidine derivatives inhibited the hCA I and II enzyme activity. Therefore, the compounds here investigated are likely to be adopted as good candidates as drugs and may be taken for further evaluation in in vivo studies.