The synthesis of (Z)-4-oxo-4-(arylamino)but-2-enoic acids derivatives and determination of their inhibition properties against human carbonic anhydrase I and II isoenzymes

Abstract

The synthesis of (Z)-4-oxo-4-(arylamino)but-2-enoic acid (4) derivatives containing structural characteristics that can be used for the synthesis of several active molecules, is presented. Some of the butenoic acid derivatives (4a, 4c, 4e, 4i, 4j, 4k) are synthesized following literature procedures and at the end of the reaction. In addition, structures of all synthesized derivatives (4a–4m) were determined by ¹H-NMR, ¹³C-NMR and IR spectroscopy. Carbonic anhydrase is a metalloenzyme involved in many crucial physiologic processes as it catalyzes a simple but fundamental reaction, the reversible hydration of carbon dioxide to bicarbonate and protons. Significant results were obtained by evaluating the enzyme inhibitory activities of these derivatives against human carbonic anhydrase hCA I and II isoenzymes (hCA I and II). Butenoic acid derivatives (4a–4m) strongly inhibited hCA I and II with K_is in the low nanomolar range of 1.85 ± 0.58 to 5.04 ± 1.46 nM against hCA I and in the range of 2.01 ± 0.52 to 2.94 ± 1.31 nM against hCA II.

• Butenoic acid
• carbonic anhydrase
• enzyme inhibition
• enzyme purification
• isoenzymes

Introduction

The name maleimide (1) was derived from maleic acid (3) and imide functional group1. Maleimides are important structural blocks with unsaturated structures in organic synthesis. In polymer chemistry, such structure blocks are used as monomers. When double bonds in maleimide molecules are saturated with hydrogenation reaction, the succinimide structural blocks are obtained (2).

Literature offers several methods for synthesizing maleimide structures; among these methods, the main strategy is to obtain amino carboxylic acid (4) through the reaction of maleic anhydride with primary amines. But-2-enoic acid (4) derivatives are synthesized as the result of reaction of amine group with lactone carbonyl through acid catalysis2^,3. Opening of lactone ring is a critical step that can affect the reaction time and the cost of maleimides4–6.

Several derivatives involving the imide group have considerably high biological activities; however, the synthesis of imide groups in high yields and the development of low-cost synthesis methods for these groups are restricted to applications in synthetic and polymer chemistry. Cyclic structure involving the imide group occupy an important place in synthetic organic and drug chemistry. Especially, phthalimides are commonly used for the protecting amino acids in addition to their remarkable use in the field of medicine. Maleimides have easily usable characteristics in Michael Addition and Diels–Alder reactions in synthetic organic chemistry since 5-membered ring maleimides are more reactive compared to those with 6-membered and 7-memeberd rings2^,3. As maleimides can fit in the chemical structure of some proteins, they make up an important category of biological substrates3^,7. They can frequently be used as photon-initiator for the polymerization of free radicals in polymer chemistry3–8, as well as monomer in the synthesis of polymaleimides and their copolymers. They are among the most commonly mentioned chemical structures in the literature with their recently found functions, such as the use of their derivatives in the form of pharmaceutical mid-products because of their antibacterial property5^,9 and their characteristics of bonding with natural rubbers9^,10. In addition, they are used vastly in the space industry for resin encapsulation of IC-dyes and as structural adhesives for fiber-empowered composites9^,11. In recent times, some research groups12^,13 achieved a successful synthesis of 3-nitryl- and 3-acetoxy aryl maleimides. In the mentioned synthesis, antifungal activities of derivatives were investigated and were found to have biological activities also.

N-aryl maleimide molecules were obtained from aryl aromatic compounds and maleic anhydrate using the methods mentioned in literature. During the reaction, N-aryl maleimides associated with dehydration produced high yields (Scheme 1).

Scheme 1. General synthesis (Z)-4-oxo-4-(arylamino)but-2-enoic acids derivatives from maleic acid in catalytic acidic media.

Enzyme activities of the synthesized derivatives were investigated, as well as significant results obtained by these syntheses were introduced in the literature. Carbonic anhydrases (CA, E.C.4.2.1.1) are ubiquitous metalloenzymes that catalyze a simple reaction, the interconversion between carbon dioxide (CO₂) and bicarbonate (HCO₃) generate H⁺ ions in the hydration reaction; thus, being one of the main players of pH regulation in many tissues, organs and organisms14–18. In humans, CAs are present in a large variety of tissues, such as the gastrointestinal tract, the nervous system, the reproductive tract, lungs, kidneys, skin and eyes19–24. This regulatory reaction supports many biochemical and physiological processes associated with pH control, fluid secretion and ion transport.

Six distinct genetic CA families, the α-, β-, γ-, δ-, ζ- and η-CAs, are known to date, constituting an interesting example of convergent evolution at the molecular level25–31. Also, up to now, 16 different x-CA isoenzymes have been described in various organisms32–35. These enzymes differ in their subcellular localization, catalytic activity and susceptibility to different classes of inhibitors. Some of them are cytosolic (CA I, CA II, CA III, CA VII and CA XIII), others are membrane bound (CA IV, CA IX, CA XII and CA XIV), two are mitochondrial (CA VA and CA VB) and one is secreted in saliva (CA VI)36–41. The disregulated activity of some of these isoenzymes leads to a large spectrum of diseases, including retinal or cerebral edema (in which hCA I is involved); epilepsy, glaucoma, edema, high altitude sickness (hCA II seems to be the main, but not the only isoenzyme involved in these conditions); oxidative stress (hCA III); retinitis pigmentosa (hCA IV); obesity (hCA VA/VB); carcinogenesis (hCA VI); epilepsy (hCA VII); tumorigenesis (hCA IX and XII; but hCA XII is also implicated in glaucoma); sterility (hCA XIII) and various retinopathies (in which hCA XIV is the main isoform involved)33^,42–47.

Inhibitors of carbonic anhydrase enzymes (CAIs) have a large number of applications in therapy, including anticancer, antiglaucoma and anti-osteoporosis agents. They are used as diuretics, anti-obesity and anti-infective drugs. These CAIs have also been used for managing Alzheimer's disease and a variety of neurological disorders. Many types of CAI derivatives have been reported recently, together with their potential applications39^,48. These chemical groups are used for the clinical treatment of some conditions for decades47^,49–52.

In this contest, many efforts have been made for the development of specific CAIs, and some remarkable results have been achieved in the past 15 years53–59. In this study, we investigated the inhibitory effects of butenoic acid derivatives (4a–4m) on hCA I and II.

Experimental

The known and unknown derivatives, having maleimide skeleton structure, were synthesized using the methods commonly known in the literature, and biological activities of such derivatives were investigated. Derivatives with amino carboxylic acid (4) skeleton structure were synthesized through the reaction of maleic anhydride with the related aromatic amine molecule. Due to their structural features, activities of the synthesized derivatives were investigated using enzymatic reactions. This work is the first to report the syntheses of 4a, 4c, 4e, 4i, 4j, 4k derivatives, and their structures were determinate by NMR spectroscopy (Supplementary material). Melting points for all these new derivatives are listed in Supplementary material, whereas that of other derivatives are provided in the literature (Table 1)60–68.

Table 1. The synthesis of (Z)-4-oxo-4-(aryl) but-2-enoic acid (4) and 3-chloro-aryl maleimide (5) skeleton structures.

First, for the synthesis of (Z)-4-oxo-4-(aryl)but-2-enoic acid skeleton structure, the derivatives 5–17 were treated with maleic anhydrate at room temperature. High-yield derivatives, having (Z)-4-oxo-4-(aryl)but-2-enoic acid (4) skeleton structures, were obtained via ring-opening reaction at room temperature. Finally, these derivatives were reacted with aromatic amine compounds for the synthesis of (Z)-4-oxo-4-(aryl)but-2-enoic acid (4). The yields of all the derivatives are showed in Table 1.

Affinity chromatography is the science of separation of biomolecules based on their specific interactions, such as that between enzyme and substrate to solid phase-coupled ligands. Due to its ability to enrich selective targets, affinity chromatography has remained a mainstay technique in separation chemistry69–72. In this study, both hCA I and II isoenzymes were purified by Sepharose-4B-l-tyrosine-sulfanilamide affinity chromatography73–76. The affinity chromatography consists Sepharose-4B-l-tyrosine-sulfanilamide that acts as an affinity matrix for selective retention of CA isoenzymes77–79. The column material was prepared according to a previous method80^,81. Thus, homogenate solution acidity was adjusted and supernatant was transferred to the previously prepared column82. The protein flow in the column eluates was spectrophotometrically determined at 280 nm. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was applied for the detection of both the isoenzymes' purity83–86. This technique is widely used in biochemistry, genetics, forensics, molecular biology and biotechnology for the separation of biological macromolecules including proteins, according to their electrophoretic mobility87–89. After visualizing the SDS-PAGE process, a single band was observed for hCA I and II isoenzymes. This protein-imaging method was previously described90–93. In this application, the imaging method was performed using 10 and 3% acrylamide for the running and the stacking gel, respectively, with 0.1% SDS94.

hCA I and II isoenzyme activities were determined according to the method of Verpoorte et al.95 The protein quantity was spectrophotometrically measured at 595 nm according to the Bradford method96. Bovine serum albumin was used as the standard protein90. For determining the inhibition effect of each butenoic acid derivative, an activity (%)-[butenoic acid derivatives] graph was drawn. To determine K_i values, three different butenoic acid derivative concentrations were tested. Also, different substrate concentrations were used and Lineweaver–Burk curves were drawn97, as previously described52.

Results and discussion

Carbonic anhydrase enzyme inhibitors (CAIs) have a large spectrum of applications in therapy, including blood pressure-lowering, anticancer, vasodilator effects, antiglaucoma and anti-osteoporosis agents. They are used as diuretics, anti-obesity and anti-infective drugs. It has been shown that the CA inhibition in smooth muscle cells results in a rise in pH, leading to KCa channel activation and vasorelaxation98^,99. Also, these CAIs have been used for managing Alzheimer's disease and a variety of neurological disorders. Many types of CAIs and new derivatives have been reported recently, together with their potential applications. In our study, physiologically relevant hCA I and II isomers are studied. A dozen of butenoic acid derivatives (4a–4m) were examined for their hCA I and II isoenzymes inhibition properties. All butenoic acid derivatives (4a–4m) have shown efficient inhibition against both isoforms. The chemical structures of butenoic acid derivatives (4a–4m) are given in Table 1. Also, CA I and II inhibiting effects of a dozen of butenoic acid derivatives (4a–4m) are summarized in Table 2. It is well known that developing isoenzyme-specific CAIs is highly beneficial in obtaining novel classes of drugs devoid of various undesired side effects.

Table 2. Human carbonic anhydrase isoenzymes I and II inhibition profile of some butenoic acid derivatives (4a–4m).

	IC50 (nM)				Ki (nM)
Bromophenols	hCA I	r^2	hCA II	r^2	hCA I	hCA II
4a	42.77	0.9933	34.49	0.959	41.18 ± 12.17	29.05 ± 5.21
4b	35.54	0.9902	26.04	0.9956	32.47 ± 7.72	21.35 ± 3.04
4c	30.43	0.9975	23.45	0.9931	33.00 ± 4.69	24.03 ± 7.52
4d	38.82	0.9924	29.64	0.9857	37.96 ± 5.51	21.74 ± 1.64
4e	28.72	0.9979	23.04	0.9973	29.05 ± 9.23	21.34 ± 6.26
4f	32.25	0.9948	32.71	0.9945	26.32 ± 6.49	23.52 ± 3.67
4g	30.43	0.9989	27.66	0.9921	22.15 ± 7.76	22.34 ± 4.65
4h	32.88	0.9986	32.63	0.9863	33.86 ± 10.30	28.31 ± 4.93
4i	31.87	0.9958	30.19	0.9823	29.19 ± 6.94	24.92 ± 4.24
4j	32.59	0.9990	27.48	0.9932	32.23 ± 9.73	20.91 ± 3.14
4k	37.42	0.9958	29.61	0.9889	35.18 ± 11.50	23.21 ± 5.22
4l	40.91	0.9933	27.79	0.9934	41.79 ± 13.19	23.03 ± 4.57
4m	41.07	0.9975	28.95	0.9855	31.95 ± 3.45	28.38 ± 5.18
AZA*	6.07	0.9154	5.50	0.9636	6.76 ± 2.55	5.85 ± 2.56

*AZA (Acetazolamide) was used as a standard inhibitor for both hCA enzymes.

Cytosolic isoenzyme hCA I is found in many tissues; however, it was demonstrated that this isoenzyme is involved in retinal and cerebral edema, and its inhibition may be a valuable tool for fighting these conditions. Also, it was reported that the K_i value of a dozen of butenoic acid derivatives (4a–4m) was less than 50 nM (K_is < 50 nM). The results obtained from this study clearly indicate that a dozen of butenoic acid derivatives (4a–4m) had potent inhibition profile against slow hCA I, and cytosolic dominant rapid isozymes hCA II with low nanomolar range (K_is < 30 nM). These compounds derived from butenoic acid (4a–4m) bind to hCA I in the low nanomolar range and were found as stronger inhibitors of this isoform, with K_is ranging between 22.15 ± 7.76 and 41.79 ± 13.19 nM. However, the most powerful inhibition effect was found in butenoic acid derivative 4g derived from 3-nitroaniline (11), with K_i value of 22.15 ± 7.76 nM. Butenoic acid derivative 4g possess two carbonyl groups (–C=O), an amine group (–NH₂), a hydroxyl group (–OH) and a nitro group (–NO₂). It is well known that these groups are biologically very reactive and the molecules in these groups demonstrated effective CA isoenzyme inhibition properties. On the other hand, acetazolamide (AZA) is considered a broad-specificity CA inhibitor due to its widespread inhibition of CAs, showing K_i value of 6.76 ± 2.55 nM against hCA I isoenzyme. The inhibition effects of all butenoic acid derivatives (4a–4m) are close to acetazolamide. AZA is considered a good CA inhibitor and approved for the treatment of a range of conditions, including glaucoma, epilepsy and altitude sickness5.

CA II is involved in several diseases, including glaucoma, epilepsy, altitude sickness and edema. Compared to the physiologically dominant isoform hCA II, all butenoic acid derivatives (4a–4m) showed K_i values ranging from 20.91 ± 3.14 to 29.05 ± 5.21 nM (Table 2). The butenoic acid derivative 4j, derived from 3-aminobenzoic acid, had two carbonyl groups (–C=O), an amine group (–NH₂), a hydroxyl group (–OH) and an acetate group (–COO), being the best hCA II inhibitor (K_i: 20.91 ± 3.14 nM). The molecules with acetate, azide, cyanate, sulfide and cyanide groups bind the active site of CA100. In another study, the binding of acetate ions to bovine CA was investigated by NMR and inhibition measurements. It was found that two acetate ions were found to interact with the protein, but only one is linked with the inhibition of the esterase activity of the bovine CA101. However, all butenoic acid derivatives (4a–4m) demonstrated similar hCA II inhibition properties. Also, the results obtained from this study showed that butenoic acid derivatives (4a–4m) had generally higher affinity toward hCA II than that of hCA I isoform. Also, AZA, which may interact with the distinct hydrophobic and hydrophilic halves of the CA II active site, showed K_i value of 5.85 ± 2.56 nM.

Conclusion

Although (Z)-4-oxo-4-(aryl)but-2-enoic acid molecules have very simple skeletons, they are very active biological compounds, so their monomer property is used in polymer chemistry. Therefore, the synthesis of these molecules is an important issue for researches. (Z)-4-Oxo-4-(aryl)but-2-enoic acid (4) derivatives used in this study influence the activity of CA enzymes due to the presence of different functional groups (–Et, –Me, –COOH, –SO₂NH₂, –CN, –NO₂, and –NH₂) in their aromatic scaffold. These findings signify that substituted but-2-enoic acid derivatives may be used as leads for generating potent CAIs. In this study, a dozen of butenoic acid derivatives (4a–4m) were evaluated against cytosolic human carbonic anhydrase isoenzyme I and II. All the butenoic acid derivatives (4a–4m) have shown low nanomolar inhibition against cytosolic CA I and II. Both isoenzymes were potently inhibited by butenoic acid derivatives (4a–4m) with K_is in the range of 22.15 ± 7.76–41.79 ± 13.19 nM against hCA I, and 20.91 ± 3.14–29.05 ± 5.21 nM against hCA II.

Supplementary material available online

Declaration of interest

The authors report there is no conflict of interests.