Synthesis of N-alkyl (aril)-tetra pyrimidine thiones and investigation of their human carbonic anhydrase I and II inhibitory effects

Abstract

Tetrahydropyrimidine thiones, which are cyclic thiocarbamides derivatives, were synthesised from thiourea, β-diketones and substituted benzaldehydes. A tautomeric form of these derivatives incorporates the thiol functionality, which is known to interact with metal ions from metalloenzymes active sites, such as the carbonic anhydrases (CAs, EC 4.2.1.1) among others. This is a superfamily of widespread enzymes, which catalyses a crucial biochemical reaction, the reversible hydration of carbon dioxide to bicarbonate and protons (H⁺). The newly synthesised N-alkyl (aril)-tetrahydropyrimidine thiones were tested for inhibition of the cytosolic human isoforms I and II (hCA I and II). Both isoforms were effectively inhibited by the newly synthesised thiones. K_i values were in the range of 218.5 ± 23.9–261.0 ± 41.5 pM for hCA I, and of 181.8 ± 41.9–273.6 ± 41.4 pM for hCA II, respectively. This under-investigated class of derivatives may bring interesting insights in the field of non-sulphonamide CA inhibitors.

• Carbonic anhydrase
• enzyme inhibition
• pyrimidine
• synthesis

Introduction

The search of physiologically active compounds with various applications expanded exponentially in the last period in the search of drugs with better efficacy and less side effects1–4. The pyrimidine-thiones possess relevant pharmacological activity, and the development of optimal synthetic methods for some of their classes is of interest5. The tetrahydropyrimidine thiones belong to the cyclic thiocarbamide class, and their carboxylic acid derivatives were poorly investigated to date. This is the reason why we underwent the exploration of some synthetic strategies for a small panel of such compounds. We report here the syntheses of allyl 6-methyl-2-thioxo-4-(p-tolyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1), 2-(methacryloyloxy)ethyl 6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (2), allyl 4-(4-(dimethylamino)phenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (3), 1-(6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)ethanone (4) and ethyl 6-ethoxy-2-thioxo-4-(p-tolyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (5). Our interest in this type of heterocyclic compounds is motivated by the fact that a tautomeric form of these thiones incorporates a thiol functionality and heterocyclic thiols are known to act as metalloenzyme inhibitors, binding to the metal centre of such proteins.

Indeed, the carbonic anhydrase (CA, EC. 4.2.1.1) is a metalloenzyme that catalyses the hydration of carbon dioxide (CO₂) and the corresponding dehydration of bicarbonate ()6–10. This is a rather simple but crucial process, as the products of this reaction are involved in fundamental physiological processes, including pH and CO₂ homeostasis, respiration, transport of CO₂/ between metabolising tissues and the lungs, electrolyte secretion, gluconeogenesis, ureagenesis, lipogenesis, bone calcification, tumorigenicity and some other pathologic and physiologic processes11–16. There are six distinct and unrelated CA classes, i.e., the α-, β-, γ-, δ-, ζ- and η-CAs, which are involved in crucial physiologic and pathologic processes in organisms all over the phylogenetic tree17–21. All of them are metalloenzymes, whereas α-, β-, δ- and η-CAs use Zn²⁺ ions at the active site, the γ-CAs are probably Fe²⁺ enzymes, but they are active also with bound Zn²⁺ or Co²⁺ ions. On the other hand, the ζ-class uses Cd²⁺ or Zn²⁺ to perform the physiologic reaction catalysis22–26.

To date, 16 different α-CA isoforms were discovered in vertebrates. α-CAs are among the most effective catalysts known in nature. The α-CAs are present in vertebrates, algae, protozoa, and cytoplasm compartment of green plants, as well as in some bacteria27–32. In humans, α-CAs are diffused in different tissues including the blood, kidneys, gastrointestinal tract, reproductive tract, the nervous central system, eyes, skin, lungs, etc.33–37. Two cytosolic human CA isoforms (hCA I, and II) are widespread throughout the human body and are drug targets for clinically used diuretics, anticonvulsants and antiglaucoma drugs38–42. Five of the vertebrate CA isoforms are cytosolic (CA I, II, III, VII and XIII), some others (CA IV, IX, XII, XIV and XV) are membrane bound, two isoenzymes (CA VA and VB) are mitochondrial, and one isoform (CA VI) is secreted in saliva43–48. It was recently reported that CA XV isoform is not expressed in humans or in living primates. However, it is found in rodents and other higher vertebrates. Three acatalytic forms are known and called CA-related proteins (CARP)49–51.

The dimeric transmembrane glycoproteins hCA IX and XII are also isoforms having an extracellular active site and are markers for a broad spectrum of hypoxic tumors52–54. The overexpression of these isoforms contributes to the increased acidification of extracellular hypoxic environment in solid tumours (pH: 6.8) and normal tissues (pH: 7.4), thus promoting tumour cell survival in an acidic condition by decreasing uptake of weakly basic anticancer drugs55. They also help tumour growth by supplying to be used as substrate for cell growth as this anion is required in the synthesis of pyrimidine nucleotides such as cytosine, thymine, and uracil56. Thus, specifically targeting the tumour-related isozymes (hCA IX and XII) over the main off target isoenzymes hCA I, and II may be a new antitumor approach, with a sulphonamide CA inhibitor in Phase I clinical trials for the treatment of hypoxic tumors57–59. However, CA II is also a drug target for antiglaucoma agents, diuretics and high-altitude sickness among others60–62. The precise physiological role of CA I, a highly abundant protein in the blood and gastrointestinal tract, is not well understood62.

Experimental

Chemistry

Synthesis of allyl 6-methyl-2-thioxo-4-(p-tolyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1)

Thiourea (1.52 g, 0.02 mol) is dissolved in 3:1 ratio of acetylacetone (3 mL) and ethyl alcohol (1 mL), and allyl acetoacetate (2.74 mL, 0.02 mol) is added to it drop by drop. After being dissolved in magnetic stirrer for 5 minutes, tolualdehyde (2.36 mL, 0.02 mol) is added to it. Upon being dissolved in the stirring rod within 30 minutes, trifluoroacetic acid (TFAA) catalyst is inserted. The process of reaction is controlled through Sulifol UV-254 plate. After determining the reaction has been fully completed, the solvent is evaporated. It is distilled in ethyl alcohol solution. The yield is 2.15 g. White crystalline with melting temperature of 205 °C is formed. IR ν, sm⁻¹: 3237 (NH), 1223 (C=S), 786, 1545, 3107 (Ar-CH), 1651 (C=C), 1727 1707 (C(O)R).

Synthesis of 2-(methacryloyloxy)ethyl 6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate (2)

Thiourea (1.52 g, 0.02 mol) is dissolved in 3:1 ratio of acetylacetone (3 mL) and ethyl alcohol (1 mL), and (methacryloyloxy) ethyl acetoacetate (3.82 mL, 0.02 mol) is added to it drop by drop. After being dissolved in magnetic stirrer for 5 minutes, benzaldehyde (2.03 mL, 0.02 mol) is added. After determining that the reaction has been fully completed, the solvent is evaporated. Processing of the reaction mixture was carried out by washing the reaction mixture with ice water. Then the precipitate was filtered, washed with 500 mL of water, dried and recrystallized from ethanol (75 mL). The yield is 2.4 g, m.p. 211 °C. IR ν, sm⁻¹: 3175 (NH), 1709 (C=O), 1092 (C=S), 756, 852, 974, 1230 (CH), 1608 (C=C).

Synthesis of allyl 4-(4-(dimethylamino)phenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate (3)

Thiourea (0.76 g, 0.01 mol) is dissolved in 3:1 ratio of acetylacetone (3 mL) and ethyl alcohol (1 mL), and allyl acetoacetate (1.37 mL, 0.01 mol) is added to it drop by drop. After being dissolved in magnetic stirrer for 5 minutes, 4 -(dimethylamino) benzaldehyde (1.49 mL, 0.01 mol) is added. Upon being dissolved in the stirring rod within 30 minutes, TFAA catalyst is inserted. After determining that the reaction has been fully completed, the solvent is evaporated. It is distilled in an ethyl alcohol solution. The yield is 1.8 g, m.p. 208 °C. IR ν, sm⁻¹: 1618, 1560, 1526 (Ar), 3112 (CH₂ = C), 785 (Ar-CH), 1722, 1701 (C(O)R), 1377, 1370 (CH₃), 1651 (C=C), 3242 (NH), 1314 (C–N).

Synthesis of 1–(6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)ethanone (4)

Thiourea (1.52 g, 0.02 mol) is dissolved in 3:1 ratio of acetylacetone (3 mL) and ethyl alcohol (1 mL), and acetylacetone (2.05 mL, 0.02 mol) is added to it drop by drop. After being dissolved in magnetic stirrer for 5 minutes, benzaldehyde (2.03 mL, 0.02 mol) is added. Upon being dissolved in the stirring rod within 30 minutes, TFAA catalyst is inserted. After determining that the reaction has been fully completed, the solvent is evaporated. It is distilled in an ethyl alcohol solution. The yield is 2.5 g, m.p. 203 °C. IR ν, sm⁻¹: 1810–1951, 3000–3100 (Ar), 1493 (Ar C–H), 1614 (Ar C–C), 703 (CH), 1675, 1703 (C=O), 3257 (NH), 1236 (C=S).

Synthesis of ethyl 6-ethoxy-2-thioxo-4-(p-tolyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (5)

Thiourea (2.58 g, 0.0339 mol) is dissolved in 3:1 ratio of acetylacetone (3 mL) and ethyl alcohol (1 mL), and diethyl malonate (5.15 mL, 0.0339 mol) is added to it drop by drop. After being dissolved in magnetic stirrer for 5 minutes, p-tolualdehyde (4 mL, 0.0339 mol) is added to it. Upon being dissolved in the stirring rod within 30 minutes, TFAA catalyst is inserted. After determining that the reaction has been fully completed, the solvent is evaporated. It is distilled in an ethyl alcohol solution. The yield is 3.2 g. White crystalline with melting temperature of 196 °C is formed. IR ν, sm⁻¹: 1605 (Ar), 1743, 1710 (C(O)R), 3315–3463 (NH), 1236 (C=S).

CA inhibition

Both cytosolic hCA isoenzymes were purified by Sepharose-4B-L tyrosine-sulphanilamide affinity separation technique with a single purification step^7,8. Sepharose-4B-L tyrosine-sulphanilamide affinity gel was prepared and used according to the previous studies47^,63. At the outset, the pH of the homogenate was adjusted to 8.7 using solid Tris(hydroxymethyl)aminomethane. Then, an aliquot of the supernatant was transferred to a previously prepared affinity column. Then, the purification process was realised. The protein flow in the column effluents was determined spectrophotometrically at 280 nm as previously reported64–66. Both isoenzymes purities were controlled by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli’s method67. SDS-PAGE was widely used in biochemistry for separation of biological macromolecule including proteins and enzymes according to their electrophoretic mobility29. SDS-PAGE was applied after purification of hCA I, and II isoenzymes. Also, this method was previously described in the literature68–72. Briefly, it was performed in acrylamide for the running (10%) and the stacking gel (3%) containing SDS (0.1%)73–76. A single band was observed for each hCA isoenzyme.

Both CA isoenzymes activities were determined in accordance with the method of Verpoorte et al.77 described previously47^,49. The increasing absorbance at 348 nm of p-nitrophenylacetate (NPA) to p-nitrophenolate (NP) was recorded during a short period (3 minutes) at 25 °C using a spectrophotometer (Shimadzu, UV-VIS Spectrophotometer, UVmini-1240). The quantity of protein was spectrophotometrically determined at 595 nm during the purification steps according to the Bradford method78. Bovine serum albumin as a standard protein was used and has been described previously79–81. To determine the inhibition effect of each N-alkyl (aril)-tetra pyrimidine thiones, an activity (%)–[N-alkyl (aril)-tetra pyrimidine thiones] graph was drawn. For calculation of K_i values, three different N-alkyl (aril)-tetra pyrimidine thiones concentrations were used. In these experiments, NPA was used as a substrate at five different concentrations. Finally, the Lineweaver–Burk curves were drawn82. These drawings and calculations are described in detailed in other works83^,84.

Results and discussion

The synthesis of the new compounds is reported in Scheme 1. The reaction of substituted benzaldehydes, with methylene active compounds such as β-diketones and thiourea in the presence of NiCl₂.6H₂O led to the desired cyclic thioureas 1–5. The three-component condensation reactions come to an end within 2–4 h at 65–70 °C. The synthesised compounds were crystalline and their structure was confirmed by spectral and physico-chemical methods, among which were ¹H and ¹³C NMR spectroscopies.

Scheme 1. The synthesis route of new N-alkyl (aril)-tetra pyrimidine thiones (1–5).

Inhibitors of carbonic anhydrase enzymes (CAIs) have a large number of applications in therapy, such as anticancer, antiglaucoma and anti-osteoporosis agents85. The classical CAIs are the primary sulphonamides, which are in clinical use for more than 50 years as diuretics and systemically acting antiglaucoma drugs. In addition to the established role of these CAIs as diuretics and antiglaucoma agents, it has recently emerged that they have potential as anticonvulsant, antiobesity, anticancer, antipain, and anti-infective drugs86–88. Many types of CAIs have been reported recently, together with their potential applications27^,28. Some newly synthesised N-alkyl (aril)-tetra pyrimidine thiones (1–5) were screened for the inhibition of both human CA isoforms involved in important physiologic or pathologic processes, i.e. the cytosolic, hCA I, and II. Table 1 shows inhibition data of newly synthesised N-alkyl (aril)-tetra pyrimidine thiones (1–5) reported here and acetazolamide AZA, which is a standard CA inhibitor against hCA I and II. The following structure–activity relationship may be noted regarding the inhibition data of Table 1.

Table 1. Inhibition data of human isoforms hCA I, and II with N-alkyl (aril)-tetrathiopyrimidines 1–5.

	KI (pM)
Compounds	hCA I	hCA II
1	218.5 ± 23.9	155.4 ± 25.9
2	261.0 ± 41.5	190.2 ± 32.8
3	312.6 ± 61.1	273.6 ± 41.4
4	219.8 ± 30.8	181.8 ± 41.9
5	231.0 ± 44.3	197.3 ± 30.6
AZA*	369.4 ± 68.5	271.8 ± 54.5

*Acetazolamide (AZA) was used as a standard inhibitor for all both CA isoenzymes and demonstrated as nanomolar (nM) inhibition.

Cytosolic hCA I is expressed in the body and can be found in high concentrations in the blood and gastrointestinal tract89. The slow cytosolic isoform hCA I was effectively inhibited by newly synthesised N-alkyl (aril)-tetra pyrimidine thiones (1–5) with K_i values ranging of 218.5 ± 23.9–312.6 ± 61.1 p.m. (Table 1). On the other hand, acetazolamide (AZA) is a medium potency hCA I inhibitor demonstrated a K_i of 369.4 ± 68.5 p.m. However, the most powerful hCA I inhibition was observed by allyl 6-methyl-2-thioxo-4-(p-tolyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1) with K_i value of 218.5 ± 23.9 p.m. Also, AZA is an effective antiglaucoma agent, but has a limited use due to numerous side effects that arise by inhibition of CAs other than those present in the eye ciliary processes leading to kidney stones, fatigue, and paresthesias86.

hCA II, the dominant physiologic isoform90^,91, was potently inhibited by all newly synthesised N-alkyl (aril)-tetra pyrimidine thiones (1–5) with K_is in the range of 181.8 ± 41.9–273.6 ± 41.4 p.m. Also, similar to hCA I, the most powerful hCA II inhibition property was shown by allyl 6-methyl-2-thioxo-4-(p-tolyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1) with K_i value of 155.4 ± 25.9 p.m. Also, AZA, which was used as clinical CA inhibitor, showed a K_i value of 271.8 ± 54.5 p.m. This result clearly showed that all newly synthesised N-alkyl (aril)-tetra pyrimidine thiones (1–5) are rather effective inhibitors for the cytosolic isoform hCA II. It was demonstrated that the CA II inhibition is due to the ability of an inhibitor to mimic the tetrahedral transition state when binding to the catalytic Zn²⁺ located in the active site29.

Conclusion

This study clearly showed that the newly synthesised N-alkyl (aril)-tetrapyrimidinethiones (1–5) possess effective inhibition profiles against both CA I and II isoenzymes. Picomolar K_i values were observed for all compounds against these two isoforms (218.5 ± 23.9–312.6 ± 61.1 pM against hCA I, and 181.8 ± 41.9–273.6 ± 41.4 pM against hCA II, respectively). These findings signify that these derivatives may be used as leads for generating interesting CAIs. The allyl 6-methyl-2-thioxo-4-(p-tolyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1) demonstrated the most effective inhibition profile against hCA I and II isoenzymes.

Acknowledgements

IG and SHA would like to extend his sincere appreciation to the Research Chairs Program at King Saud University for funding this research.

Declaration of interest

The authors have declared no conflict of interest.