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

Figures & data

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.

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

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.

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.