Pyrimidine-fused heterocycle derivatives as a novel class of inhibitors for α-glucosidase

Abstract

The needs for diverse inhibitors of α-glucosidase (α-Gls) encouraged us to synthesize five different poly-hydroxy functionalized pyrimidine-fused heterocyclic (PHPFH) molecules, having either aliphatic or aromatic side chains (C₁–C₅) and their inhibitory activities were examined spectroscopically against yeast and mouse intestinal α-Gls. The results revealed that aromatic substitution of the synthetic compounds has significant impact on their inhibitory properties. Moreover C₃ with the substituted moiety as 4-(4-aminophenylsulfonyl) phenyl (4-APSP) revealed strong inhibitory activity with non-competitive and competitive inhibition modes against yeast and mouse α-Gls, respectively. Furthermore, in the presence of increasing concentration of C₃, both Trp and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence intensities of yeast α-Gls were gradually decreased, suggesting that C₃ binding induced significant structural alteration which was accompanied with the reduction of hydrophobic surfaces. Also, the interaction between yeast α-Gls and C₃ was proved to be spontaneous and driven mainly by hydrophobic forces. Overall, this study suggests that aromatic substitution on pyrimidine-fused heterocyclic (PFH) scaffold may represent a novel class of promising inhibitors of α-Gls.

Keywords::

• Alpha-glucosidase (α-Gls)
• pyrimidine-fused heterocycles (PFH)
• inhibition
• structural alteration

Introduction

Diabetes mellitus is a serious chronic ailment whose occurrence rate is increasing worldwide with incidences of obesity and aging of the general population. Currently, it is estimated that 150 million people have diabetes over the world, increasing to 220 million by 2010 and 300 million by 2025.¹ A key goal of diabetes treatment is to prevent the long-term complications because ultimately it can harm the heart, blood vessels, eyes, kidneys, and nervous system. Postprandial hyperglycemia plays a significant role in development of type-II diabetes and its complications, accounting for >90% of the cases.² During prolonged uncontrolled high-blood glucose level, an extensive non-enzymatic glycation of long-lived proteins, results in generation of cytotoxic advanced glycation end-products in the different tissues which are the main cause of various complications of diabetes.³ One of the therapeutic approaches to reduce postprandial hyperglycemia is retarding absorption of glucose by inhibition of α-glucosidase (α-Gls). α-Glucosidase is an exo-acting carbohydrase which catalyzes the release of α-d-glucopyranose from non-reducing ends of various carbohydrate substrates. The enzyme is widely distributed in microorganisms, plants, and animal tissues, and their substrate specificity is known to differ greatly depending on their source.⁴ The clinically used α-Gls inhibitors delay the hydrolysis and absorption of carbohydrates.⁵ The α-Gls is also an important target for inhibition by antiviral agents that interfere with the formation of essential glycoproteins required in viral assembly, secretion and infectivity.⁶ Many efforts have been made so far to develop α-Gls inhibitors for treatment of carbohydrate-mediated diseases such as diabetes, certain forms of hyper-lipoproteinemia and obesity, as well as for treatment of viral infections and cancer.^7,8 However, most of developed glucosidase inhibitors are sugar mimics (aza sugars, isoxazoles, and aminosugars), needing tedious multi-steps from carbohydrate and non-carbohydrate sources.⁹ Furthermore, these compounds show various adverse effects ranging from abdominal discomforts to hepatotoxicity.¹⁰ It is therefore necessary to develop novel α-Gls inhibitors with no or less unfavorable effects. The α-Gls inhibitors can be either synthesized chemically or isolated naturally from plants and food products. Since the natural enzyme inhibitors are not easily produced in large scale, many attempts have been done to produce synthetic inhibitors of this enzyme. The needs for diverse inhibitors of α-Gls promote the search for novel classes of these molecules. As reported recently, pyrimidine-fused heterocyclic (PFH) compounds display various biological activities including anticancer activity,¹¹ antiviral activity,¹² antimicrobial activity,¹³ anti-inflammatory properties, antioxidant effects, and tyrosine kinase inhibitory activity.¹⁴ Because of their wide range of biological activities, PFH scaffold has been extensively used in drug design as a common source for developing new therapeutic agents. To the best of our knowledge there is to date, no report on PFH derivatives acting as inhibitor for α-Gls. Consequently in this study five different poly-hydroxy functionalized PFH (PHPFH) molecules having either aliphatic- or aromatic side-chains (C₁–C₅) were synthesized and their inhibitory activity against yeast and mouse α-Gls examined. The results revealed that aromatic substitution of the synthetic compounds having significant impact on their inhibitory properties against the α-Gls. Consequently, this study may suggest the aromatic derivatives of above mentioned molecular scaffold as a novel template for antidiabetic compounds with promising inhibitory activity against α-Gls.

Materials and Methods

Materials

α-Gls of Saccharomyces cerevisiae (EC.3.2.1.20) and para-nitrophenyl-α-d-glucopyranoside (pNPG) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other chemicals were purchased from Fluka (Neu-Ulm, Germany) and Aldrich (Gillingham, Dorset , UK ) Chemical Companies and were used without further purification. All the solutions were made fresh using double-distilled water.

Synthesis of the ligands

This study introduced, an efficient multicomponent approach for the preparation of 10-substituted-5-(1,2,3,4,5-pentahydroxypentyl)dihydro[2,3-d:6,5-d]dipyrimidine-tetraones (10-R-5PH-HDPT) derivatives (C₁–C₅). According to this protocol, when (+)-d-glucose and amines, reacted in ethanol with two equivalent of barbituric acid in the presence of 4-methylbenzenesulfonic acid (PTSA) as a catalyst, the excellent yield of products were obtained (Figure 1). This strategy offers a powerful tool for preparing a new pyrimidine scaffold which has high potential for application in medicinal chemistry. The synthetic compounds were also characterized using different spectroscopic techniques such as ¹H-NMR, ¹³C NMR, infrared (IR) and mass spectroscopies. The data clearly revealed that these molecules were produced successfully. As the purity of synthetic compounds examined using elemental analysis, they were highly pure (>98%).

Figure 1.  Synthesis of poly-hydroxy functionalized pyrimidine-fused heterocyclics (PHPFHs) using the reaction of d-(+)-glucose, barbituric acid and amines. The detailed description of the synthetic procedure is seen in the Materials and Methods (General procedure for the synthesis of compounds C₁–C₅ subsection). As shown in this figure, further substitutions (C₁–C₅) were also made at the position 10 (R-group) of the general structure of pyrimidine-fused heterocyclic (PFH) with a poly-hydroxy carbon chain to obtain either the aliphatic or aromatic derivatives.

General procedure for the synthesis of compounds C₁–C₅

A mixture of (+)-d-glucose (0.18 g, 1 mmol), barbituric acid (0.26 g, 2 mmol), amine (1 mmol), and PTSA (0.05g, 30 mol%) in EtOH (5 mL) was stirred at 50°C for 12 h. After cooling to room temperature of the reaction mixture, the precipitated product was filtered and washed with ethanol (15 mL) to afford the pure product.

Spectral data of synthesized compounds

Compound C₁: Yield: 88%. mp = 228–230°C. ¹H-NMR (250 MHz, DMSO-d₆/TMS): 3.22–3.41 (m, 3H), 3.53–3.73 (m, 3H), 4.48 (d, J = 3.7 Hz, 1H), 6.34 (d, J = 8.8 Hz, 2H), 7.07 (d, J = 8.5 Hz, 2H), 10.59 (brs, 1H), 10.76 (brs, 1H), 11.09 (brs, 1H), 11.33 (brs, 1H). ¹³C NMR (62.5 MHz, DMSO-d₆/TMS): 22.6, 65.3, 68.2, 73.1, 73.5, 74.0, 89.1, 117.9, 119.0, 135.1, 149.8, 150.8, 151.5, 168.6. MS: 491.12 (15.5%, M⁺). Anal. Calcd. for C₂₀H₂₁N₅O₁₀ (491.41): C, 48.88; H, 4.31; N, 14.25. Found: C, 48.93; H, 4.38; N, 14.35.

Compound C₂: Yield: 89%. mp = 241–243°C. ¹H-NMR (250 MHz, DMSO-d₆/TMS): 3.16–3.42 (m, 3H), 3.53–3.78 (m, 3H), 4.53 (d, J = 3.8 Hz, 1H), 6.68 (d, J = 7.5 Hz, 2H), 7.09 (d, J = 7.8 Hz, 2H), 11.11 (brs, 2H), 11.21 (brs, 1H), 11.30 (brs, 1H). ¹³C NMR (62.5 MHz, DMSO-d₆/TMS): 21.8, 65.8, 68.1, 73.0, 73.9, 74.6, 88.5, 115.2, 120.0, 133.9, 141.7, 150.9, 151.3, 166.1. MS: 554.02 (21%, M⁺). Anal. Calcd for C₂₀H₂₀BrN₅O₉ (554.30): C, 43.34; H, 3.64; N, 12.63. Found: C, 43.34; H, 3.64; N, 12.63.

Compound C₃: Yield: 81%. mp = 245–247°C. ¹H-NMR (250 MHz, DMSO-d₆/TMS): 3.12–3.45 (m, 3H), 3.51–3.73 (m, 3H), 4.29 (d, J = 3.3 Hz, 1H), 6.69 (d, J = 7.6 Hz, 2H), 6.74 (d, J = 7.5 Hz, 2H), 7.88 (d, J = 8.1 Hz, 2H), 7.91 (d, J = 8.0 Hz, 2H), 11.04 (brs, 2H), 11.09 (s, 2H). ¹³C NMR (62.5 MHz, DMSO-d₆/TMS): 22.1, 65.8, 67.9, 73.8, 74.1, 74.8, 88.5, 119.2, 130.5, 132.9, 147.6, 150.4, 151.8, 154.8, 167.1. MS: 630.23 (17.8%, M⁺). Anal. Calcd for C₂₆H₂₆N₆O₁₁S (630.58): C, 49.52; H, 4.16; N, 13.33. Found: C, 49.73; H, 4.21; N, 13.46.

Compound C₄: Yield: 87%. mp = 222–224°C. ¹H-NMR (250 MHz, DMSO-d₆/TMS): 0.97 (s, 12H), 2.91-2.3.02 (m, 4H), 2.95–3.47 (m, 5H), 3.54–3.76 (m, 3H), 4.18 (d, J = 3.5 Hz, 1H), 10.05 (brs, 1H), 11.012 (brs, 2H), 11.25 (brs, 1H). ¹³C NMR (62.5 MHz, DMSO-d₆/TMS): 21.1, 64.3, 46.0, 48.9, 49.7, 64.8, 66.3, 71.8, 72.9, 73.2, 88.1, 146.3, 151.4, 165.7. MS: 526.54 (19%, M⁺). Anal. Calcd for C₂₂H₃₄N₆O₉ (526.54): C, 50.18; H, 6.51; N, 15.96. Found: C, 50.27; H, 6.59; N, 16.11.

Compound C₅: Yield: 85%. mp = 189–191°C. ¹H-NMR (250 MHz, DMSO-d₆/TMS): 2.01–2.19 (m, 4H), 2.35–2.71 (m, 5H), 3.09–3.44 (m, 6H), 3.50–3.76 (m, 4H), 4.24 (d, J = 3.6 Hz, 1H), 10.02 (brs, 1H), 11.04 (brs, 1H), 11.10 (brs, 1H), 11.19 (brs, 1H). ¹³C NMR (62.5 MHz, DMSO-d₆/TMS): 22.0, 47.5, 49.5, 52.3, 55.6, 65.3, 67.6, 72.9, 73.2, 73.9, 89.5, 145.9, 152.1, 165.4. MS: 511.28 (17.5%, M⁺). Anal. Calcd for C₂₀H₂₉N₇O₉ (511.49): C, 46.96; H, 5.71; N, 19.17. Found: C, 47.09; H, 5.80; N, 19.28.

Preparation of the acetone powder of mouse intestine

The acetone powder of mouse intestine was prepared according to a method reported previously with minor changes.¹⁵ Small intestines of six mice were rinsed with 30 mL of cold saline and all operations carried out at 4°C. The intestines were homogenized in a Potter-Elvehjem type of homogenizer in two volumes of 5 mM phosphate buffer (pH 7.0). After 1 h, 10 volumes of acetone that had been cooled to –20°C were added to the homogenates. The mixture was stirred and then allowed to stand for 10 min and subsequently centrifuged at 1500g for 30 min. The precipitates were washed with 15 volumes of cold acetone (kept at –20°C) and the mixture centrifuged at 1500g for 30 min. The precipitate was collected and spread on large filter paper. Then it was covered with an aluminum foil and allowed to dry overnight under the hood. Also, some holes were poked on the foil to allow easy evaporation of acetone.

The inhibition assay of yeast and mammalian α-Gls

To measure activity of α-Gls, the liberation of p-nitrophenol (pNP) from the enzyme substrate (p-nitrophenyl α-d-glycopyranoside:pNPG) was determined spectrometrically, using a T90⁺ UV/vis spectrophotometer instrument (PG Instrument, London, UK) equipped with Peltier Temperature Controller (Model PCT-2). The increment of absorbance at 410 nm as result of pNP release was considered directly proportional to α-Gls activity. The concentration of inhibitors (C₁-C₅) required for inhibiting 50% of the enzyme activity (IC₅₀ value) under the assay conditions was also determined graphically by a plot of α-Gls velocity vs. log of concentration of the tested compound. Also to reveal the inhibition type and to determine the inhibition constant (K_i), the obtained results were plotted according to the Lineweaver–Burk equations in double reciprocal form. In this study, the inhibition assay of yeast enzyme was performed in 100 mM phosphate buffer pH 7.0 at 25°C with minor changes, according to the methods reported previously.¹⁶ To obtain IC₅₀ value of the inhibitors, a reaction mixture of yeast α-Gls (0.025 U/mL), 0.1 mM pNPG and increasing concentration of each inhibitor was used. Also the inhibition mode was determined for those inhibitors exhibited promising inhibitory activity (C₁–C₃) by incubating the yeast enzyme solution with the increasing concentration of pNPG (0.1–2 mM), in the presence of different concentration of each inhibitor. Moreover, to explore role of the synthetic compounds on inhibition of mammalian enzyme, mouse α-Gls was extracted and prepared in 100 mM potassium phosphate buffer (pH 7.0), containing 5 mM EDTA.⁵ After centrifugation, the supernatant was dialyzed against 10 mM potassium phosphate buffer (pH 7.0) containing 0.4 mM EDTA for 48 h and the enzyme assay conducted at 37°C according to previous publication.¹⁷ In brief, 10 µL of the enzyme solution was added to a sample solution containing the synthetic compounds and pNPG, and the release of pNP from pNPG monitored at 410 nm for 30 min. The inhibition mode was determined by incubating mouse α-Gls (0.3 U/mL) in the presence of increasing concentration of pNPG (0.6–3mM) and different concentrations of synthetic inhibitor. Also in this study, acarbose which is an anti-diabetic drug, exhibiting the inhibitory action against α-Gls enzyme was used as a positive control.

Fluorescence studies of the interaction between C₃ and yeast α-Gls

In this study, the structural alteration of yeast α-Gls as results of interaction with the most promising synthetic compound (C₃) was assessed, using fluorescence technique. The experiments were carried out on a Cary-Eclipse spectrofluorimeter (Varian, Inc., Sydney, Australia), in 100 mM phosphate buffer, pH 7.0 at both 37 and 42°C. The α-Gls (5 µM) was pretreated with certain concentrations of C₃ (0–50 µM) for 15 min before each fluorescence measurement. During intrinsic fluorescence study, as the excitation wavelength was 295 nm, the emission spectra collected between 300 and 500 nm. Also the slit widths for both excitation and emission were set to 10 nm. When excited at 295 nm, C₃ had no obvious fluorescence emission between 300 and 500 nm; therefore the fluorescence interference contributed to C₃ compound was negligible. However, the background emission of the corresponding buffer sample was also subtracted. To gain more detailed structural insight into the interaction between C₃ and α-Gls, ANS fluorescence study was performed in a similar experimental setup as mentioned above. In order to perform ANS fluorescence measurements, α-Gls (2 µM) was incubated with certain concentrations of C₃ (0–50 µM) for 15 min at 37°C in the dark. The fluorescence of protein-bound dye was measured by excitation at 365 nm and measuring the emission between 400 and 600 nm.¹⁸ In this study, while ANS concentration was 30 µM, the bandwidths of 10/10 nm used in excitation/emission channels. Furthermore the background emission of ANS solution alone was subtracted from the obtained emission intensities. Also the concentration of ANS stock solution in water was determined, using an extinction coefficient of 4950 M^–1 at 350 nm.

Determination of the thermodynamic parameters of C₃ binding to yeast α-Gls

To further study on the properties of the interaction between C₃ and yeast α-Gls, the relevant thermodynamic parameters were obtained. The values of K_sv were calculated by plotting F₀/F vs. [Q], according to Stern-Volmer equation:¹⁹

1

where F₀ and F are fluorescence intensities of α-Gls in the presence and absence of the inhibitor and [Q] stands for concentration of C₃ inhibitor. The binding constant (K_a) and number of binding sites (n) for C₃ inhibitor on the enzyme at both 37 and 42°C were obtained using the following equation:

2

In this study, the decrease in fluorescence intensity of yeast α-Gls was monitored at 345 nm. The plot of log (F₀ – F)/F vs. Log[Q] was used to determine the binding parameters; as the slope of this plot showing the number of binding sites (n), and the binding constant K_a was calculated by the intercept on the Y-axis.

Moreover the thermodynamic parameters of ▵H (changes in enthalpy) and ▵S (changes in entropy) were calculated by Van't Hoff equation as below:

3

where k is the binding constant at the corresponding temperature and R is the universal gas constant (8.3145 J/mol K). The following relationship was used to estimate the free energy change (▵G):

4

Results and Discussion

Chemistry of the synthetic ligands

From the view of biological significance, polycyclic compounds are often of much greater interest than monocyclic constituent compounds. Due to their wide variety of biological activities,²⁰ the PFH compounds attracted much attention. In this study, it was aimed to synthesis PFH compounds with a poly-hydroxy carbon chain to obtain new molecular scaffold of fused-heterocyclic system for further substitutions at the position 10 (Figure 1). The newly synthesized compounds (C₁–C₅) were screened for their inhibitory activity against both yeast and mouse α-Gls enzymes. According to the substitutions made at the position 10, the synthetic compounds are classified in two groups as aromatic (C₁–C₃) and aliphatic (C₄–C₅) substituted compounds (Figure 1). The substituted moieties in the aromatic compounds are 4-hydroxyphenyl (in C₁), 4-bromophenyl (in C₂) and 4-(4-aminophenylsulfonyl) phenyl (in C₃). Moreover, these moieties in the aliphatic compounds are 2-(diisopropylamino) ethyl (in C₄), and 2-piperazin-1-yl) ethyl (in C₅). While the aliphatic compounds displayed weak inhibitory activities against yeast α-Gls, the aromatic substitutions exhibited promising activities against this enzyme (Table 1). In this study C₃ compound with the substituted moiety as 4-APSP exhibited the significant inhibitory action against both yeast and mammalian α-Gls (Table 1). Since the information regarding the interaction between α-Gls and its inhibitors could provide more insight into the structure-activity relationship, consequently the structural alteration of yeast α-Gls was studied as result of the interaction with C₃ which is the most promising inhibitor used here.

Table 1.  The IC50, Ki and inhibition mode of the synthetic compounds.

Entry	IC50 (µM)		Kia (µM)		Kib (µM)		Type of inhibition
	Yeast	Mouse	Yeast	Mouse	Yeast	Mouse	Yeast	Mouse
C1	74.9	—	70	—	170	—	Mixed type	NI
C2	25.1	—	30	—	130	—	Mixed type	NI
C3	9.6	64.1	14	2.5	—	—	Noncompetitive	Competitive
C4	—	—	—	—	—	—	NI	NI
C5	—	—	—	—	—	—	NI	NI
Acarbose	275.3	13.64	ND	ND	ND	ND	ND	ND

The inhibitory effects of synthetic compounds against yeast α-Gls

Oral administration of specific glucosidase inhibitors could effectively improve hyperglycemia, as well as diabetic complications and may be useful to treat obese patients. In this study, the inhibitory activity against yeast α-Gls of two classes of the synthetic compounds as aliphatic (C₄–C₅) and aromatic (C₁–C₃) substituted agents were examined. The concentrations of synthetic compounds (C₁–C₅) resulting in 50% inhibition of the enzyme activity (IC₅₀ values) were determined graphically by plotting the activity vs. concentration of these inhibitors and the resulted values are summarized as Table 1. Moreover, the inhibition constant (K_i) of the synthetic compounds was calculated by analyzing Lineaweaver–Burk plot (Table 1). The result of this study indicates that the aromatic substitutions on the heterocyclic scaffold (C₁–C₃) possess significant impact on inhibitory properties of the synthetic compounds, while aliphatic substitutions (C₄–C₅) showed almost negligible inhibition against yeast α-Gls. Also as shown in Table 1, the synthetic compounds, C₁–C₃ exhibited significantly higher inhibitory action than Acarbose against the yeast enzyme. Furthermore, C₃ compound with 4-APSP as the substituted moiety was proven to be the most promising inhibitor of α-Gls (IC₅₀ value of 9.6 µM). Comparing the IC₅₀ values of C₁ (74.9 µM) and C₂ (25.1 µM) against yeast α-Gls, it can be suggested that hydrogen-bonding ability does not play a significant role to improve the inhibitory properties of the synthetic compounds. This suggestion may further support with the lack of inhibitory action of C₅, because this compound with its piperazin moiety has a good ability for the hydrogen bonding. The aromatic domination of 4-APSP moiety in C₃ may play important role in the inhibitory action of this compound against both yeast and the mammalian α-Gls. As the mode of inhibition was determined from the double-reciprocal plots of the inhibition kinetics, C₁ and C₂ compounds exhibited mixed inhibitory effect. Also C₃ compound behaved as a non-competitive inhibitor, because the V_max value was affected by increasing concentrations of the inhibitor and K_m value kept constant for this compound (Figure 2A). As mentioned before, the inhibition parameters K_ia and K_ib are respectively the dissociation constants of enzyme-inhibitor complex and enzyme–substrate-inhibitor complex. The values of K_ia and K_ib for C₁ and C₂ were determined from the slope and intercept on the vertical axis of a Lineweaver–Burk, and the obtained results suggest a mixed-type inhibition for these compounds. The Lineweaver–Burk plots revealed that the intersecting point for different concentrations of C₃ occurred on the 1/s axis (Figure 2A), suggesting that this compound is a non-competitive inhibitor of yeast α-Gls. Moreover, the inhibitory constant K_i for C₃ was also determined from the Lineweaver–Burk equation, and the obtained results were summarized as Table 1. Theoretically, the inhibitory effect of non-competitive inhibitors on enzyme is not affected at higher level of substrate concentrations. Consequently the inhibitory action of C₃ on yeast α-Gls could be preserved in the presence of high carbohydrate concentration.

Figure 2.  The Lineweaver–Burk plots derived from the inhibition of yeast and mouse α-glucosidases. (A) The yeast α-glucosidase (α-Gls) activity was measured as a function para-nitrophenyl-α-d-glucopyranoside (pNPG) concentration (0.1–2 mM) in the absence and presence of C₃ compound (0–20 µM). The experiments performed in 100 mM phosphate buffer pH 7.0, at 25°C for 10 min. The different symbols represent the absence (diamonds) and presence of 5 µM (squares), 10 µM (triangles), and 20 µM (circles) of C₃ inhibitor in the reaction mixtures. (B) The mouse α-Gls activity was measured as a function pNPG concentration (0.6–3 mM) in the absence and presence of C₃ compound (0–200 µM). The experiments performed in 10 mM phosphate buffer pH 7.0, at 37°C for 30 min. The different symbols represent the absence (diamonds) and presence of 50 µM (squares), 100 µM (triangles), and 200 µM (circles) of C₃ inhibitor in the reaction mixtures.

The inhibitory effects of synthetic compounds against mammalian α-Gls

Since the prevalence of diabetes has risen at an alarming rate, considerable research effort has been devoted recently to the discovery and development of novel active α-Gls inhibitors, being potential therapeutic agents for diabetes. The α-Gls activity of mouse intestinal acetone powder closely mimics the mammalian system and therefore may be a better model to identify, design and develop anti hyperglycemic agents particularly for the management of postprandial hyperglycemia in diabetes.⁵ Therefore, in this study, the inhibitory effect of synthetic compounds (C₁–C₅) was also examined against mammalian α-Gls. As seen in Table 1, among all the tested compounds, only C₃ with IC₅₀ value of 64.1 µM exerted inhibitory action against the mammalian enzyme which is higher than that of Acarbose. Mammalian α-Gls is located in the brush-border surface membrane of intestinal cells and plays as a key enzyme in catalysis of the final step of carbohydrate digestion.⁴ Hence, inhibitors of intestinal α-Gls can retard the liberation of D-glucose from complex dietary carbohydrates and delay glucose absorption, reducing plasma glucose levels and suppressing postprandial hyperglycemia.⁵ Therefore, C₃ compound with promising inhibitory properties on both yeast and mammalian α-Gls (Table 1), may present capable therapeutic potential controlling hyperglycemia. The Lineweaver–Burk plots revealed that the intersecting point of different C₃ concentrations occurred on the 1/v axis (Figure 2B), suggesting that C₃ is a competitive inhibitor of mouse α-Gls. Also the inhibition constant (K_i) on mouse α-Gls of C₃ was calculated to be 2.5 µM.

The thermodynamic study on the interaction between C₃ compound and α-Gls

The C₃ is a compound structurally different from other members of the aromatic inhibitors used in this study and thus inhibits both yeast- and mouse α-Gls in non-competitive and competitive modes respectively. The 4-APSP moiety in structure of C₃ compound possesses both polar and non-polar characteristics which is important to interact effectively with similar groups in the structure of target protein, enhancing the inhibitory properties of this compound. As shown in Figure 3A, yeast α-Gls possesses a fluorescence peak at near 345 nm, which belongs to Trp residues of this enzyme. However, while α-Gls incubated with increasing concentration of C₃ compound, the fluorescence intensity was quenched gradually. These results suggest that there is an interaction between C₃ and α-Gls, and that the interaction results in changes of the microenvironment around Trp residues. The changes in intrinsic fluorescence are similar with other studies, in which many enzymatic inhibitors could induce fluorescence quenching of their target enzymes. Moreover, the quenching constant (K_sv) and the rate constant in the process of double molecular quenching (K_q) were calculated, using equation 1 and the insets of Figure 3A. Moreover, binding constant (K_a) and number of binding sites (n) at 37°C and 42°C were obtained through the plots of lg[(F₀ – F)/F] vs. lg[Q]. The above-mentioned parameters were summarized as Table 2. Additionally, the calculated Stern–Volmer quenching constant and thermodynamic parameters were obtained at two different temperatures (37 and 42°C). The results show that the Stern–Volmer quenching constant (K_SV) increases with temperature, indicating that the likely quenching mechanism of C₃/α-Gls binding initiates with dynamic collision rather than by the complex formation with α-Gls. Moreover, in the interaction between C₃ and α-Gls, the molecular ratio of enzyme to inhibitor was lower than unit (one), showing no complex is formed and C₃ binding is accompanied with the conformational changes in the enzyme. Additionally the number of binding site on the enzyme for C₃ was decreased as a result of temperature elevation. The thermodynamic parameters of enthalpy (▵H), entropy (▵S) and free energy change (▵G) are the main evidences to estimate the binding type of a ligand to protein.¹⁹ In order to elucidate the binding mode between C₃ and α-Gls, these parameters were obtained in this study. The changes in enthalpy (▵H) and entropy (▵S) were calculated according to van’t Hoff equation by plotting the value of LnK vs. 1/T. The value of ▵G was further calculated from the values of ▵H and ▵S, using Equations (3) and (4). The negative ▵H value is observed whenever there is hydrogen bonding in the interactions, consequently the positive value obtained here for the enthalpy changes (Table 2), indicates that hydrogen bonding does not play important role in the interaction of C₃ with α-Gls. The negative value of the free energy (▵G), as seen in Table 2, supports the assertion that the binding process is spontaneous. Also the positive value of ▵S is frequently taken as evidence for the hydrophobic interaction because the water molecules that are arranged in an orderly fashion around the ligand and protein acquire a more random configuration.¹⁹ As shown in Table 2, the values of ▵G were negative, while those of ▵H and ▵S were positive. However these results indicated that the interaction between α-Gls and C₃ compound is a spontaneous process with increasing entropy and driven mainly by hydrophobic force. In this study, the ANS fluorescence of α-Gls in the presence of C₃ was also employed. ANS is a useful probe in measuring surface hydrophobicity of proteins due to its own hydrophobicity and environmental sensitivity. As shown in Figure 3B, the relative ANS fluorescence intensities of α-Gls were decreased as function of concentration of C₃ compound. These results suggest that C₃ binding induces significant structural alteration in α-Gls which is accompanied with the reduction of hydrophobic surface of this protein. The decrease in the hydrophobicity in the presence of C₃ as seen in Figure 3B supports the notion that poor hydrophobic surroundings may lead to the failure of the formation of active center. Also this study provides an insight into understanding of the interaction between yeast α-Gls and C₃ compound as the most promising inhibitor applied.

Table 2.  The binding parameters of interaction between C3 inhibitor and α-Gls.

T(K)	KSV	N	Lnk	▵G (kj/mol)	▵H (kj/mol)	▵S (j/k.mol)
310	0.083	1.729	18.6	–47.9	81.1	416.53
315	0.174	1.68	19.1	–50.02	81.1	416.53

Figure 3.  Fluorescence studies of the interaction between C₃ and yeast α-glucosidase (α-Gls). (A) The intrinsic fluorescence quenching of α-Gls induced by C₃ inhibitor. The enzyme was incubated with increasing concentration of C₃ (0–50 µM) for 15 min at 37°C and 42°C. As the experiments performed in 100 mM phosphate buffer pH 7.0, the excitation wavelength was 295 nm and emission spectra were acquired by scanning from 300 to 400 nm. Only the emission spectra obtained at 37°C are shown in this figure. Also the Stern–Volmer plots based on the fluorescence quenching at 37 and 42°C are shown as the inset figures. (B) The 8-anilinonaphthalene 1-sulphonate (ANS)-fluorescence of α-Gls as function of concentration of C₃ inhibitor. The ANS fluorescence measurements were performed in 100 mM phosphate buffer pH 7.0 while enzyme and ANS concentration were 2 and 30 µM, respectively. The enzyme was incubated with certain concentrations of C₃ inhibitor (0–50 µM) for 15 min, at 37°C in the dark, and fluorescence of protein-bound dye measured by excitation at 365 nm and measuring the emission between 400 and 600 nm.

Conclusions

The results of this study revealed that the aromatic substitutions on the heterocyclic scaffold (C₁–C₃) possess significant impact on the inhibitory properties of the synthetic compounds. Moreover, C₃ compound with 4-APSP as the substituted moiety was proven to be the most promising inhibitor of yeast and mammalian α-Gls. Also the interaction between yeast α-Gls and C₃ was verified to be spontaneous and driven by hydrophobic forces. Overall, these findings prove that the aromatic substitutions on the bipyrimidine heterocyclic scaffold may represent a new class of α-Gls inhibitors. Thus it is possible that C₃ compound and its analogous could be developed as another class of hypoglycemic agents.

Acknowledgments

We would like to thank financial support of Iran National Science Foundation (INSF)-Grant nos. 88001578 and 88001894. Also the financial supports of research councils of Shiraz University and Shiraz University of Technology are gratefully acknowledged.

Declaration of interest

The authors declared no conflicts of interest.

Notes

ND, not determined; NI, no inhibition.