Synthesis and characterisation of thiobarbituric acid enamine derivatives, and evaluation of their α-glucosidase inhibitory and anti-glycation activity

Abstract

A new series of thiobarbituric (thiopyrimidine trione) enamine derivatives and its analogues barbituric acid derivatives was synthesised, characterised, and screen for in vitro evaluation of α-glucosidase enzyme inhibition and anti-glycation activity. This series of compounds were found to inhibit α-glucosidase activity in a reversible mixed-type manner with IC₅₀ between 264.07 ± 1.87 and 448.63 ± 2.46 µM. Molecular docking studies indicated that compounds of 3g, 3i, 3j, and 5 are located close to the active site of α-glucosidase, which may cover the active pocket, thereby inhibiting the binding of the substrate to the enzyme. Thiopyrimidine trione derivatives also inhibited the generation of advanced glycation end-products (AGEs), which cause long-term complications in diabetes. While, compounds 3a–k, 5, and 6 showed significant to moderate anti-glycation activity (IC₅₀ = 31.5 ± 0.81 to 554.76 ± 9.1 µM).

Keywords:

• Thiopyrimidine trione
• α-glucosidase inhibitor
• antiglycation
• molecular docking

1. Introduction

Diabetes mellitus (DM) is a disease which caused by a breakdown of carbohydrate metabolism, which plays a significant role in the development of long-term diabetic complications. According to the International Diabetes Federation, 693 million people will suffer from this condition by 2045^1,2. DM can be categorised into three types: Type I (T1DM); Type II (T2DM); and gestational (GDM). About 80 − 90% of all DM patients are Type II (T2DM). Drug treatments of T2DM aim to decrease hepatic glucose production, enhance insulin action, and boost insulin secretion from β-pancreatic cells, or block α-glycosidase enzyme (carbohydrate digestive enzymes)^3–6. Therapeutic in individuals with this disease may lead to various complications, including kidney disease, disorders of the nervous system, leg amputation, heart disease and severe retinopathy up to blindness⁷.

Carbohydrate digestive enzymes are found in the brush border of the intestine. They catalyse the breaking down long-chain polysaccharides into absorbable monosaccharide units. Of these enzymes, α-glucosidases, which play a key role in the digestion and absorption of complex carbohydrates, and has emerged as target to maintain postprandial blood glucose control. α-Glucosidase inhibitors currently used to treat T2DM include acarbose (Precose), voglibose, and miglitol⁸. However, these drugs are associated with several side effects, such as flatulence, stomach-ache, diarrhoea, and liver damage⁹. Therefore, an increasing interest in exploring new drug candidates for glycosidase inhibition is needed^10–12.

Barbituric acid (BA) derivatives have been reported to have potential anti-hypertensive¹³, anti-cancer¹⁴, anti-convulsant¹⁵, anti-inflammatory¹⁶, anti-psychotic¹⁷, and antitumor properties^18–21. Recently, these derivatives have also been reported as anti-diabetic agents²². On the other hand, thiobarbituric acid (TBA) analogues has been described to exert anti-inflammatory^16,23, immunotropic²⁴, anticonvulsant²⁵, and anti-hypnotic^25,26, anti-neoplastic²⁷, and antitumor activities²⁸. De Belin et al.²⁹ reported a number of TBA derivatives as inhibitors of hypoxia-inducible factor 1 (HIF-1). Recently, Barakat et al.³⁰ described the synthesis of a new series of diethylammonium salts of aryl substituted TBA derivatives as α-glycosidase inhibitors. Therefore, given the relevance of TBA derivatives in medicinal chemistry, the design of new molecules containing the thiobarbituric moiety is an inspiring goal.

In continuation of our studies on the synthesis of biologically active compounds^22,30,31, herein, we synthesised 1,3-diethylthiobarbiturate enamine derivatives and evaluated their in vitro α-glucosidase inhibitory and anti-glycation activities. In addition, molecular docking studies were performed to study the interactions of the compounds with the catalytic site of the enzyme using acarbose and evaluated their α-glucosidase inhibition capacity and the anti-glycation properties.

2. Results and discussion

2.1. Synthesis of the target compounds

Enamine derivatives 2a³² and 2b³³ were prepared by reacting the commercially available compounds, 1,3-diethylthiobarbituric acid 1a or 1,3-dimethylbarbituric acid 1b with DMF in the presence of acetic anhydride as solvent for 2 h at 90 °C to afford 2a and 2b, respectively as a yellow crystalline solid in good yields. Compounds 2a³² and 2b³³ were reacted with different amines in ethanol at RT to afford the target products 3a–k and 4a–d, respectively (Scheme 1) in excellent yields and purities, as observed from their spectral data. The reaction of 2a (2 equiv.) or its analogues 2b (2 equiv.) with the commercially available material 4-(aminomethyl)aniline (1 equiv.) under the same conditions described above gave the dimeric products 6 and 5, respectively as shown in Scheme 1. The structures of the products obtained were deduced by ¹H- and 13C-NMR spectra (Supplementary material).

Scheme 1. Synthetic route for the synthesis of 3a–k, 4a–d, 5, and 6.

2.2. Biological activity

All the synthesised derivatives of TBA (3a–k), BA (4a–d), and the dimeric analogues 5 and 6 were evaluated for their capacity to inhibit α-glucosidase and protein glycation in vitro in comparison to acarbose (IC₅₀ = 875.75 ± 2.08 µM) and rutin (IC₅₀ = 54.59 ± 2.20 µM), as standard tested compounds (Table 1).

Table 1. Result of in vitro α-glucosidase enzyme inhibitor and anti-glycation activities.

Compound	Structure	Anti-glycation assay IC50 ± SEM (µM)	α-Glucosidase IC50 ± SEM (µM)
3a		88.57 ± 0.37^b	NA
3b		80.36 ± 0.74^b	NA
3c		82.22 ± 4.36^b	NA
3d		130.53 ± 3.15^b	NA
3e		77.28 ± 0.72^b	NA
3f		81.74 ± 1.39^b	NA
3g		82.36 ± 5.09^b	397.45 ± 0.98^a
3h		70.92 ± 1.84^b	NA
3i		75.13 ± 0.65^b	264.07 ± 1.87^a
3j		101.92 ± 1.7^b	433.33 ± 2.34^a
3k		ND	ND
4a		NA	NA
4b		NA	NA
4c		NA	NA
4d		NA	NA
5		554.76 ± 9.1^b	448.63 ± 2.46^a
6		31.5 ± 0.81^a	NA
Rutin		54.59 ± 2.20	–
Acarbose		–	875.75 ± 2.08

^a Significant activity.

^b Moderate activity.

ND: not determined; NA: not active.

The results summarised in Table 1 indicated that all the N,N′-dimethylbarbituric-based enamine acid derivatives 4a–d were completely inactive, while compounds 3a–k, 5, and 6 showed moderate to significant activity against protein glycation (IC₅₀ = 554.76 ± 9.1 to 31.5 ± 0.81 µM). The dimeric moiety of TBA 6 via diaminobenezene linkage (IC₅₀ = 31.5 ± 0.81 µM, Table 1) was the most protein glycation inhibitor in this series of compounds, and showed more activity than the standard rutin (IC₅₀ = 54.59 ± 2.20 µM). While, the dimeric analogues of BA 5 (IC₅₀ = 554.76 ± 9.1 µM) was the least active.

On the other hand, substituted phenyl with an electron-withdrawing group such as a chlorine atom (a weak deactivating group) at the ortho position, showed a better anti-glycation activity than the same atom at the para position. Therefore, the change in the position had a remarkable effect on the anti-glycation activity³⁴ (3 h vs 3f, Table 1). Halogen with a higher atomic weight and weaker electron-withdrawing effect, such as iodine at the ortho position, decreased the activity as compared to chlorine at the same position (3i vs 3 h). This observation could be attributed to the negative inductive effect^35,36. In contrast, a strong electron-withdrawing group, such as sulphonic acid at ortho position, decreased the activity compared to chlorine and iodine in the ortho position (3j vs 3 h). Electron-donating group such as methyl (a weak donating group) at the para position yielded slightly better and a moderate activity as compared to the chlorine at the same position (3e vs 3f). On the other hand, replacing the 4-methylphenyl 3e by 2-pyridylmethylene 3b or 3-methylpyridyl 3c decreased the anti-glycation activity, and showed a comparable activity to compounds 3g and 3f as shown in Table 1. Compound with pyrimidine benzenesulfonamide 3d moiety decreased the activity, which is consistent with the result obtained for 3j with a strong withdrawing group. While, compounds with 2-morpholinoethyl 3a and cyclohexyl 3g moieties showed moderate activity against protein glycation.

The results summarised in Table 1 indicated, once again, that none of the BA enamine derivatives showed any activity, while 3g, 3i, 3j, and 5 exerted a significant activity against α-glucosidase (IC₅₀ = 264.07 ± 1.87 to 448.63 ± 2.46 µM). Of the series of compounds, thiopyrimidine trione derivative with higher atomic weight halogen, such as iodine at the ortho position, was the most active, exhibiting 3.3-fold higher activity than the standard acarbose. Compounds with a cyclohexyl ring 3g, sulphonic acid 3j, and the dimeric analogue of BA 5 showed twice the activity of the standard drug. The rest of the compounds did not show any activity.

Finally, the most two active compounds from the series are shown in Figure 1. In conclusion, this work has demonstrated that the core of TBA-based enamine derivatives is a privileged structure for anti-glycation and α-glucosidase inhibition and thus deserves further investigation.

Figure 1. Lead compounds 3i and 6 with promising activities.

2.3. Molecular docking studies

Molecular docking provides significant insight into ligand-protein binding modes and mechanisms. Here, molecular docking studies were carried out to explore the binding modes of TBA derivatives with a notorious α-glucosidase, such as that of Baker’s yeast (Saccharomyces cerevisiae). We used our previously built homology model of α-glucosidase from the template (PDB ID: 3A4A)³⁰. Initially, the 3 D structures of all the ligands were built, protonated, and minimised by means of the MMFF94x force field³⁷, and using the molecular operating environment (MOE)³⁸ 2018.04. All recently synthesised TBA derivatives and a reference inhibitor (acarbose) were docked into the active site of the receptor using the default parameters in MOE. Each complex was visually analysed for ligand–protein interactions, and their images were prepared using UCSF chimaera software³⁹.

The top ranked conformer of TBA derivatives and standard (acarbose) were selected based on docking score. The docking score of the ligands 3g, 3i, 3j, and 5 and acarbose were −3.081, −4.909, −5.19, −5.642, and −4.382, respectively. The docking study revealed that the acarbose, and all the ligands accommodated into the binding pocket of the C-terminal domain of α-glucosidase. The clustering of standard and synthetic compounds at the allosteric site of the C-terminal domain is shown in Figure 2.

Figure 2. Binding mode of thiobarbituric acid derivatives into the α-glucosidase binding cavity. For clarity, acarbose is shown in cyan. Compounds 3g, 3i, and 3j are indicated in pink, and 5 in green. The part of the enzyme in the background is shown as surface model.

Acarbose occupied a large cavity in the binding sites of α-glucosidase due to its larger size, as compared to the synthetic compounds. The oxygen functionality of acarbose formed two hydrogen bonds with the active site residues, Arg212 and Arg439. Ring structures were involved in the π–π interactions with Phe177, His239, and Pro309. Moreover, residues Glu276, Glu304, and Asp349 interacted hydrophobically with the ligand (Figure 3).

Figure 3. Interactions of acarbose with crucial residues of α-glucosidase.

The carbonyl oxygen of the thiobarbituric ring of 3g, 3i, and 3j showed hydrogen bond interactions with crucial residue Arg212. Another hydrogen bond was observed between the nitrogen atoms of 3g with Thr215. These compounds were further stabilised through π–π interactions with the crucial active site residues Tyr71, Phe157, and Phe177. Additionally, π–π interactions were observed with the thiol ring of 3j through Phe177 and Tyr71. In the case of compound 3i, Phe157 was involved in forming halogen–π interactions. Moreover, hydrophobic interactions with the active site residues Phe157, Thr215, Leu218, and Arg349 stabilised these compounds. In the case of 5, the 2,4,6-trione ring-bearing oxygen atom formed hydrogen bonds with His111 and Arg212. Meanwhile, the amine functionality of the ligand also formed a hydrogen bond with residue Arg312. The benzene ring was involved in π–π interactions with Phe157 and Phe300. The hydrophobic interaction with crucial residue Arg349 also contributed to the binding of 5 with α-glucosidase. The interaction diagrams of all the ligands are shown in Figure 4. The docking results of 5 were in good agreement with experimental results, thereby indicating that it could be a good candidate as α-glucosidase inhibitor.

Figure 4. The predicted binding interactions of compounds 3g, 3i, 3j, and 5 in the active site.

3. Conclusions

Several derivatives of barbitutic and thiobarbituric enamine derivatives were synthesised, characterised, and screened for in vitro evaluation of α-glucosidase enzyme inhibition and anti-glycation activity. The results reveal that the four monomeric compounds 4a–d derived from N,N′-dimethylbarbituric enamine derivatives showed no anti-glycation activity, while compounds derived from N,N′-diethylthiobarbituric enamine derivatives 3a–k exhibited moderate activity against protein glycation with IC₅₀ in the range 70–550 µM. The most potent anti-glycation activity was showed by the dimeric product from N,N′-diethylthiobarbituric enamine 6 with an IC₅₀ of 31.5 µM, while the dimeric analogue of N,N′-dimethylbarbituric enamine 5 showed less activity with an IC₅₀ of 554.8 µM. The reported series of compounds were found to inhibit α-glucosidase activity in a reversible mixed-type manner with IC₅₀ between 264 and 448 µM. The type and position of substituent on phenyl ring (enamine moiety) has great impact on the biological activity. In this regard, the moderate electron-withdrawing group, such as a chlorine atom at the ortho position 3 h showed greater activity compared to the same atom at the para position 3f. On the other hand, the presence of iodine at the ortho position decreased activity compared to chlorine in the same position (3i vs 3 h). The strong electron-withdrawing group, such as sulphonic acid showed decrease in activity compared to weak electron-donating group like methyl (3e).

The dimeric thiobarbituric derivative 6 showed better anti-glycation activity compared with the standard rutin, while thiobarbituric ortho-iodo-enamine derivative 3i showed a positive effect as α-glucosidase inhibitor compared to the standard acarbose.

Molecular docking studies indicated that compounds of 3g, 3i, 3j, and 5 are located close to the active site of α-glucosidase, which may cover the active pocket, thereby inhibiting the binding of the substrate to the enzyme.

This work has confirmed that the core of (thio)barbituric-based enamine derivatives are a privileged structure, because in addition of the previous described biological activity, they have shown activity for anti-glycation and α-glucosidase inhibition.

4. Experimental

4.1. General methods

All melting points were determined using Mel-Temp apparatus and are uncorrected. Thin layer chromatography (TLC) was performed on silica gel (Kiesel gel G, Merck) and spots were detected under UV light at 254 nm. FTIR Spectra were recorded in a KBr matrix on a Bruker Tensor 37 FTIR spectrophotometer. ¹H-NMR spectra were recorded with a JEOL 400 MHz, 13C-NMR were recorded using the JEOL spectrophotometers, and the chemical shifts (δ) are given in ppm.

4.2. General procedure for the synthesis of 3a–k, 4a–d, 5, and 6

A solution of 2a³² or 2b³³ (1 equiv.) was mixed with different amines (1 equiv.) in MeOH (10 ml) and stirred at room temperature for 10–120 min (TLC 20% EtOAc/n-hexane). The solvent was evaporated slowly, providing the corresponding solid products in excellent yields and purities.

4.2.1. 1,3-Diethyl-5-(((2-morpholinoethyl)amino)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (3a)

Compound 3a was synthesised from 2a and 4–(2-aminoethyl)morpholine following the general procedure, affording the product as a yellow powder in 81% yield; mp 135 °C; IR (KBr, cm⁻¹): 3420, 2999, 2960, 2908, 2870, 1624, 1591, 1456; ¹H-NMR (CDCl₃, δ, ppm): 10.60 (brs, 1H, NH), 8.23 (d, 1H, J = 14.8 Hz, CH=), 4.50 (m, 4H, 2CH₂), 3.72 (q, 2H, CH₂), 3.54 (m, 4H, 2CH₂), 2.60 (m, 2H, CH₂), 2.50 (m, 4H, 2CH₂), 1.25 (m, 6H, 2CH₃); ¹³C-NMR (CDCl₃ δ, ppm): 179.1, 163.0, 161.2, 160.6, 93.0, 66.9, 57.6, 53.6, 47.1, 43.0, 42.3, 12.5, 12.4; LC/MS (ESI): 341.44 [M + 1]⁺; Anal. Calcd for C₁₅H₂₄N₄O₃S: C, 52.92; H, 7.11; N, 16.46; Found: C, 53.01; H, 7.25; N, 16.59.

4.2.2. 1,3-Diethyl-5-(((pyridin-2-ylmethyl)amino)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (3b)

Compound 3b was synthesised from 2a and 2-picolylamine following the general procedure, affording the product as a pink powder in 83% yield; mp 154 °C; IR (KBr, cm⁻¹): 3215, 3045, 2958, 2908, 2866, 1614, 1598, 1544,1463; ¹H-NMR (CDCl₃ δ, ppm): 11.01 (brs, 1H, NH), 8.63 (d, 1H, J = 5.2 Hz, Ar-H) , 8.39 (d, 1H, J = 14.0 Hz, CH=), 7.72 (t, 1H, J = 8.8 Hz, Ar-H), 7.27 (d, 1H, J = 8.8 Hz, Ar-H), 7.23 (m, 1H, Ar-H), 4.76 (d, 2H, J = 3.6 Hz, CH₂), 4.56 (m, 4H, 2CH₂), 1.28–124 (m, 6H, J = 16.4 Hz, 2CH₃); ¹³C-NMR (CDCl₃ δ, ppm): 179.1, 163.0, 161.2, 160.7, 154.1, 150.3, 137.3, 123.5, 121.7, 93.5, 55.1, 43.0, 42.3, 12.5, 12.4; LC/MS (ESI): 319.40 [M + 1]⁺; Anal. Calcd for C₁₅H₁₈N₄O₂S: C, 56.59; H, 5.70; N, 17.60; Found: C, 56.72; H, 5.81; N, 17.78.

4.2.3. 1,3-Diethyl-5-(((4-methylpyridin-2-yl)amino)methylene)-2-thioxodihydro pyrimidine-4,6(1H,5H)-dione (3c)

Compound 3c was synthesised from 2a and 2-amino-4-picoline following the general procedure, affording the product as a light yellow powder in 87% yield; mp 175 °C; IR (KBr, cm⁻¹) 3215, 3157, 3045, 2958, 2908, 2866, 1614, 1598, 1544, 1463; ¹H-NMR (CDCl₃ δ, ppm):12.25 (d, 1H, J = 13.2 Hz, NH), 9.40 (d, 1H, J = 13.2 Hz, CH=), 8.27 (d, 1H, J = 5.2 Hz, Ar-H), 6.98 (d, 1H, J = 5.2 Hz, Ar-H), 6.86(s, 1H, Ar-H), 4.55 (m, 4H, 2CH₂), 2.38 (s, 3H, CH₃), 1.29 (m, 6H, 2CH₃); ¹³C-NMR (CDCl₃ δ, ppm):179.1, 163.3, 160.7, 152.5, 150.7, 149.6, 149.0, 122.9, 113.6, 95.8, 43.2, 42.5, 21.2, 12.5, 12.4; LC/MS (ESI): 319.40 [M + 1]⁺; Anal. Calcd for C₁₅H₁₈N₄O₂S: C, 56.59; H, 5.70; N, 17.60; Found: C, 56.81; H, 5.78; N, 17.79.

4.2.4. 4-(((1,3-Diethyl-4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)amino)-N-(pyrimidin-2-yl)benzenesulfonamide (3d)

Compound 3d was synthesised from 2a and sulphadiazine following the general procedure, affording the product as a yellow powder in 85% yield; mp 204 °C; IR (KBr, cm⁻¹): 3421, 3116, 2958, 2860, 1618, 1591, 1508, 1440; ¹H-NMR (DMSO-d₆, δ, ppm): 12.20 (d, 1H, J = 14.0 Hz, NH), 8.72 (d, 1H, J = 14.0 Hz, NH), 8.52 (d, 1H, J = 4.4 Hz, CH=), 8.47 (d, 1H, J = 8.8 Hz, Ar-H), 8.0 (d, 1H, J = 8.8 Hz, Ar-H), 7.79 (d, 2H, J = 8.8 Hz, Ar-H), 7.10 (m, 1H, Ar-H), 6.57 (d, 2H, J = 8.8 Hz, Ar-H), 4.42 (m, 4H, 2CH₂), 1.21 (m, 6H, 2CH₃); ¹³C-NMR (DMSO-d₆, δ, ppm): 178.9, 162.2, 160.5, 158.8, 157.8, 157.3, 154.3, 153.6, 142.4, 130.4, 129.8, 125.4,119.8, 116.1, 112.7, 95.7, 42.9, 42.4, 12.8, 12.7; LC/MS (ESI): 461.53 [M + 1]⁺; Anal. Calcd for C₁₉H₂₀N₆O₄S₂: C, 49.55; H, 4.38; N, 18.25; Found: C, 49.66; H, 4.50; N, 18.41.

4.2.5. 1,3-Diethyl-2-thioxo-5-((p-tolylamino)methylene)dihydropyrimidine-4,6(1H,5H)-dione (3e)

Compound 3e was synthesised from 2a and 4-methylanline uracil following the general procedure, affording the product as a yellow powder in 89% yield; mp 139 °C; IR (KBr, cm⁻¹): 3448, 3215, 3169, 2953, 2866, 1595, 1570, 1554, 1476, 1435, 1440; ¹H-NMR (CDCl₃, δ, ppm): 12.32 (d, 1H, J = 13.8 Hz, NH), 8.70 (d, 1H, J = 14.0 Hz, CH=), 7.27 (d, 2H, J = 8.0 Hz, Ar-H), 7.22 (dd, 2H, J = 8.0 Hz, Ar-H), 4.60(m, 4H, 2CH₂), 2.36 (s, 3H, CH₃), 1.30 (m, 6H, 2CH₃); ¹³C-NMR (CDCl₃, δ, ppm): 178.8, 163.2, 160.9, 152.9, 137.3, 135.5, 130.9, 118.2, 94.6, 43.2, 42.5, 12.4, 12.2; LC/MS (ESI): 317.41 [M + 1]⁺; Anal. Calcd for C₁₆H₁₉N₃O₂S: C, 60.55; H, 6.03; N, 13.24; Found: C, 60.32; H, 6.00; N, 13.43.

4.2.6. 5-(((4-Chlorophenyl)amino)methylene)-1,3-diethyl-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (3f)

Compound 3f was synthesised from 2a and 4-chloroanline following the general procedure, affording the product as a yellow powder in 78% yield; mp 215 °C; IR (KBr, cm⁻¹): 3302, 2958, 2908, 2866, 1614, 1587, 1545, 1504, 1438, 1409; ¹H-NMR (CDCl₃, δ, ppm): 12.32 (d, 1H, J = 14.0 Hz, NH), 8.65 (d, 1H, J = 14.0 Hz, CH=), 7.38 (d, 2H, J = 8.0 Hz, Ar-H), 7.25 (d, 2H, J = 8.8 Hz, Ar-H), 4.55 (m, 4H, 2CH₂), 1.30 (t, 6H, J = 7.9 Hz, 2CH₃); ¹³C-NMR (CDCl_3, δ, ppm): 178.9, 163.2, 160.8, 152.8, 136.6, 132.6, 130.4, 119.4, 95.3, 43.2, 42.5, 12.5, 12.3; LC/MS (ESI): 338.82 [M + 1]⁺; Anal. Calcd for C₁₅H₁₆ClN₃O₂S: C, 53.33; H, 4.77; N, 12.44; Found: C, 53.54; H, 4.80; N, 12.63.

4.2.7. 5-((Cyclohexylamino)methylene)-1,3-diethyl-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (3g)

Compound 3g was synthesised from 2a and cyclohexylamine following the general procedure, affording the product as a white powder in 84% yield; mp 105 °C; IR (KBr, cm⁻¹): 3302, 2958, 2908, 2866, 1614, 1587, 1545, 1504, 1438, 1409; ¹H-NMR (DMSO-d₆, δ, ppm): 10.61 (brs, 1H, NH), 8.25 (d, 1H, J = 15.6 Hz, CH=), 4.52 (m, 4H, 2CH₂), 3.40 (m, 1H, CH), 1.98 (m, 2H, CH₂), 1.83 (m, 2H, CH₂), 1.78 (m, 2H, CH₂), 1.47 (m, 2H, 2CH₂), 1.28 (m, 2H, 2CH₃); ¹³C-NMR (DMSO-d₆, δ, ppm): 179.0, 163.2, 161.3, 158.4, 92.6, 59.3, 42.9, 42.3, 33.5, 24.9, 24.3, 12.5, 12.4; LC/MS (ESI): 310.43 [M + 1]⁺; Anal. Calcd for C₁₅H₂₃N₃O₂S: C, 58.23; H, 7.49; N, 13.58; Found: C, 58.39; H, 7.53; N, 13.78.

4.2.8. 5-(((2-Chlorophenyl)amino)methylene)-1,3-diethyl-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (3 h)

Compound 3h was synthesised from 2a and 2-chloroanline following the general procedure, affording the product as a yellow powder in 89% yield; mp 160 °C; IR (KBr, cm⁻¹): 3302, 2958, 2908, 2866, 1614, 1587, 1545, 1504, 1438, 1409; ¹H-NMR (CDCl₃, δ, ppm): 12.62 (d, 1H, J = 13.2 Hz, NH), 8.73 (d, 1H, J = 14.0 Hz, CH=), 7.49 (m, 2H, Ar-H), 7.39 (t, 1H, J = 7.9 Hz, Ar-H), 7.21 (t, 1H, J = 8.0 Hz, Ar-H), 4.56 (m, 4H, 2CH₂), 1.30(m, 6H, 2CH₃); ¹³C-NMR (CDCl₃, δ, ppm): 178.9, 163.0, 160.9, 152.1, 135.3, 130.7, 128.5, 127.3, 125.0, 116.9, 96.1, 43.2, 42.6, 12.5, 12.4; LC/MS (ESI): 338.82 [M + 1]⁺; Anal. Calcd for C₁₅H₁₆ClN₃O₂S: C, 53.33; H, 4.77; N, 12.44; Found: C, 53.53; H, 4.92; N, 12.60.

4.2.9. 1,3-Diethyl-5-(((2-iodophenyl)amino)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (3i)

Compound 3i was synthesised from 2a and 2-iodoanline following the general procedure, affording the product as a yellow powder in 83% yield; mp 175 °C; IR (KBr, cm⁻¹): 3302, 2958, 2908, 2866, 1614, 1587, 1545, 1504, 1438, 1409; ¹H-NMR (CDCl₃, δ, ppm): 12.42 (d, 1H, J = 13.2 Hz, NH), 8.67 (d, 1H, J = 14.0 Hz, CH=), 791 (d, 1H, J = 7.2 Hz, Ar-H), 7.45 (t, 1H, J = 7.9 Hz, Ar-H), 7.36 (d, 1H, J = 8.0 Hz, Ar-H), 6.98 (t, 1H, J = 7.2 Hz, Ar-H), 4.53 (m, 4H, 2CH₂), 1.30 (m, 6H, 2CH₃); ¹³C-NMR (CDCl₃, δ, ppm): 179.0, 162.8, 160.9, 153.0, 139.9, 130.0, 128.2,125.1, 117.7, 95.9, 89.9, 43.2, 42.5, 12.6, 12.4; LC/MS (ESI): 430.28 [M + 1]⁺; Anal. Calcd for C₁₅H₁₆IN₃O₂S: C, 41.97; H, 3.76; N, 9.79; Found: C, 41.88; H, 3.81; N, 10.01.

4.2.10. 2-(((1,3-Diethyl-4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl) amino)benzenesulfonic acid (3j)

Compound 3j was synthesised from 2a and 2-aminobenzenesulfonic acid following the general procedure, affording the product as a yellow powder in 80% yield; mp 243 °C; IR (KBr, cm⁻¹): 3302, 2958, 2908, 2866, 1614, 1587, 1545, 1504, 1438, 1409; ¹H-NMR (DMSO-d₆, δ, ppm): 13.01 (d, 1H, J = 14.8 Hz, NH), 8.62 (d, 1H, J = 14.4 Hz, CH=), 7.78 (d, 1H, J = 7.9 Hz, Ar-H), 7.64 (d, 1H, J = 8.0 Hz, Ar-H), 7.49 (t, 1H, J = 8.6 Hz, Ar-H), 7.30 (t, 1H, J = 8.4 Hz, Ar-H), 4.43 (m, 4H, 2CH₂), 1.20 (m, 6H, 2CH₃); ¹³C-NMR (DMSO-d₆, δ, ppm): 178.9, 161.1, 160.9, 153.9, 138.6, 135.9, 130.7, 128.1, 124.6, 118.3, 95.3, 42.9, 42.2, 12.8. LC/MS (ESI): 384.44 [M + 1]⁺; Anal. Calcd for C₁₅H₁₇N₃O₅S₂: C, 46.99; H, 4.47; N, 10.96; Found: C, 47.09; H, 4.53; N, 11.13.

4.2.11. 1,3-diethyl-5-((pyridin-2-ylamino)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (3k)

Compound 3k was synthesised from 2a and pyridin-2-amine following the general procedure, affording the product as a yellow powder in 83% yield; mp 243 °C; IR (KBr, cm⁻¹): 3302, 2958, 2908, 2866, 1614, 1587, 1545, 1504, 1438, 1409; ¹H-NMR (DMSO-d₆, δ, ppm): 13.03 (d, 1H, J = 14.8 Hz, NH), 8.62 (d, 1H, J = 14.4 Hz, CH=), 7.76 (d, 1H, J = 7.6 Hz, Ar-H), 7.63 (d, 1H, J = 8.8 Hz, Ar-H), 7.49 (t, 1H, J = 9.6 Hz, Ar-H), 7.28 (t, 1H, J = 8.4 Hz, Ar-H), 4.46 (m, 4H, 2CH₂), 1.22 (m, 6H, 2CH₃); ¹³C-NMR (DMSO-d₆, δ, ppm): 178.4, 160.5, 159.2, 138.1, 135.6, 130.7, 118.3, 94.6, 42.4, 41.7, 12.2. LC/MS (ESI): 305.35 [M + 1]⁺; Anal. Calcd for C₁₄H₁₆N₄O₂S: C, 55.25; H, 5.30; N, 18.41; Found: C, 55.38; H, 5.41; N, 18.59.

4.2.12. 5-(((4-Chlorophenyl)amino)methylene)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione (4a)

Compound 4a was synthesised from 2b and 4-chloroanline following the general procedure, affording the product as a white powder in 87% yield; mp 197 °C; IR (KBr, cm⁻¹): 3302, 2958, 2908, 2866, 1614, 1587, 1545, 1504, 1438, 1409;¹H-NMR (CDCl₃, δ, ppm):12.00 (d, 1H, J = 13.6 Hz, NH), 8.60 (d, 1H, J = 14.0 Hz, CH=), 7.36 (d, 2H, J = 8.8 Hz, Ar-H), 7.16 (d, 2H, J = 8.8 Hz, Ar-H), 3.32 (s, 6H, 2CH₃); ¹³C-NMR (CDCl₃, δ, ppm): 165.1, 162.6, 151.8, 136.8, 132.1, 130.3,119.2, 93.4, 28.1, 27.4; LC/MS (ESI): 294.71 [M + 1]⁺; Anal. for C₁₃H₁₂ClN₃O₃; Calcd: C, 53.16; H, 4.12; N, 14.31; Found: C, 53.15; H, 4.12; N, 14.33.

4.2.13. 5-(((2-Chlorophenyl)amino)methylene)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione (4b)

Compound 4b was synthesised from 2b and 2-chloroanline following the general procedure, affording the product as a white powder in 87% yield; mp 202 °C; IR (KBr, cm⁻¹): 3637, 3423, 3197, 2960, 2935, 1583, 1570, 1510, 1462; ¹H-NMR (CDCl₃, δ, ppm): 12.44 (d, 1H, J = 12.8 Hz, NH), 8.73 (d, 1H, J = 13.2 Hz, CH=), 7.48 (m, 2H, Ar-H), 7.36 (t, 1H, J = 7.2 Hz, Ar-H), 7.18 (m, 1H, Ar-H), 3.38 (s, 3H, CH₃), 3.36 (s, 3H, CH₃); ¹³C-NMR (CDCl₃, δ, ppm): 164.9, 162.7, 151.9, 151.1, 135.4, 130.6, 128.5, 126.9, 124.7, 116.6, 94.4, 28.2, 27.5; LC/MS (ESI): 294.71 [M + 1]⁺; Anal. for C₁₃H₁₂ClN₃O₃; Calcd: C, 53.16; H, 4.12; N, 14.31; Found: C, 53.17; H, 4.11; N, 14.29.

4.2.14. 5-(((2-Iodophenyl)amino)methylene)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione (4c)

Compound 4c was synthesised from 2b and 2-iodoanline following the general procedure, affording the product as a white powder in 89% yield; mp 285 °C; IR (KBr, cm⁻¹): 3086, 3053, 2953, 2885,1598, 1560, 1516, 1463, 1371; ¹H-NMR (CDCl₃, δ, ppm): 12.77 (d, 1H, J = 14.8 Hz, NH), 8.55 (d, 1H, J = 14.0 Hz, CH=), 7.77 (d, 1H, J = 7.2 Hz, Ar-H), 7.57 (d, 1H, J = 8.0 Hz, Ar-H), 7.46 (t, 1H, J = 7.6 Hz, Ar-H), 7.26 (t, 1H, J = 7.2 Hz, Ar-H), 3.20 (s, 6H, 2CH₃); ¹³C-NMR (CDCl₃, δ, ppm):163.3, 162.9, 152.2, 151.9, 138.3, 136.4, 131.3, 128.1, 125.9, 118.5, 93.9, 28.2, 27.6; LC/MS (ESI): 386.16 [M + 1]⁺; Anal. for C₁₃H₁₂IN₃O₃; Calcd: C, 40.54; H, 3.14; N, 10.91; Found: C, 40.55; H, 3.15; N, 10.90.

4.2.15. 1,3-Dimethyl-5-((pyridin-2-ylamino)methylene)pyrimidine-2,4,6(1H,3H,5H)-trione (4d)

Compound 4d was synthesised from 2b and 2-aminopyridine following the general procedure, affording the product as a white powder in 85% yield; mp 287–290 °C; IR (KBr, cm⁻¹): 3420, 2999, 2960, 2908, 2870, 1624, 1591, 1456; ¹H-NMR (CDCl₃, δ, ppm): 12.11 (brs, 1H, NH), 9.41 (d, 1H, J = 13.2 Hz, CH=), 8.43 (d, 1H, J = 4.4 Hz, Ar-H), 7.75 (dd, 1H, J = 8.0, 2.4 Hz, Ar-H), 7.16 (t, 1H, J = 7.6, Hz, Ar-H), 6.99 (d, 2H, J = 8.0 Hz, Ar-H), 3.36 (s, 6H, 2CH₃); ¹³C-NMR (CDCl₃, δ, ppm): 165.3, 162.6, 152.0, 151.3, 149.7, 149.3, 138.9, 121.4, 112.7, 94.3, 28.2, 27.5; LC/MS (ESI): 261.25 [M + 1]⁺; Anal. for C₁₂H₁₂N₄O₃; Calcd: C, 55.38; H, 4.65; N, 21.53; Found: C, 55.38; H, 4.64; N, 21.51.

4.2.16. 5-(((4-(((1,3-Dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl) amino)benzyl)amino)methylene)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione (5)

Compound 5 was synthesised from 2b (2 equiv.) and 4-aminobenzylamine (1 equiv.) following the general procedure, affording the product as a white powder in 90% yield; mp 195 °C; IR (KBr, cm⁻¹): 3302, 2958, 2908, 2866, 1614, 1587, 1545, 1504, 1438, 1409; ¹H-NMR (DMSO-d₆, δ, ppm): 10.48 (brs, 2H, NH), 8.25(d, 2H, J = 14.0 Hz, 2CH=), 7.01 (d, 2H, J = 8.0 Hz, Ar-H), 6.55 (d, 2H, J = 9.0 Hz, Ar-H), 4.50 (d, J = 6.4 Hz, 2H, CH₂), 3.12 (s, 12H, 4CH₃); ¹³C-NMR (DMSO-d₆, δ, ppm): 168.1, 165.5, 160.6, 151.9, 134.4, 118.7, 145.2, 95.3, 28.1, 27.4; LC/MS (ESI): 455.44 [M + 1]⁺; Anal. Calcd for C₂₁H₂₂N₆O₆: C, 55.50; H, 4.88; N, 18.49; Found: C, 55.65; H, 4.93; N, 18.70.

4.2.17. 5-(((4-(((1,3-Diethyl-4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)amino)benzyl)amino)methylene)-1,3-diethyl-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (6)

Compound 6 was synthesised from 2a (2 equiv.) and 4-aminobenzylamine (1 equiv.) following the general procedure, affording the product as a yellow powder in 86% yield; mp 247 °C; IR (KBr, cm⁻¹): 3302, 2958, 2908, 2866, 1614, 1587, 1545, 1504, 1438, 1409; ¹H-NMR (CDCl₃, δ, ppm): 12.34 (d, 1H, J = 13.6 Hz, NH), 10.77 (m, 1H, NH), 8.68 (d, 1H, J = 14.0 Hz, CH=), 8.29 (d, 1H, J = 13.6 Hz, CH=), 7.39 (m, 4H, Ar-H), 4.65 (d, 2H, J = 6.4 Hz, CH₂), 4.46 (m, 8H, 4CH₂), 1.35 (m, 12H, 4CH₃); 13C-NMR (CDCl₃, δ, ppm): 179.0, 178.8, 163.3, 163.2, 161.0, 160.7, 160.4, 152.7, 138.4, 133.6, 129.7, 129.6, 118.9, 118.8, 95.4, 93.6, 53.6, 43.2, 43.0, 42.5, 42.4, 12.5, 12.4, 12.3, 12.2; LC/MS (ESI): 543.67 [M + 1]⁺; Anal. Calcd for C₂₅H₃₀N₆O₄S₂: C, 55.33; H, 5.57; N, 15.49; Found: C, 55.54; H, 5.69; N, 15.66.

4.3. Protocol for in vitro α-glucosidase inhibition assay

The assay protocol for was performed spectrophotometrically following the reported method²², where α-glucosidase from S. cerevisiae (G0660-750UN, Sigma Aldrich) was dissolved in phosphate buffer (pH 6.8, 50 mM). Test compounds were dissolved in 70% DMSO. 20 μL of test sample, 20 μL of enzyme and 135 μL of buffer were added to 96-well plates and incubated for 15 min at 37 °C. After incubation, 25 μL of p-nitrophenyl-α-d-glucopyranoside (0.7 mM, Sigma Aldrich) was added and changes in absorbance were monitored for 30 min at 400 nm. The test compound was replaced by DMSO (7.5% final) as control. Acarbose (Acarbose, Sigma Aldrich) was used as a standard inhibitor.

4.4. Protocol for anti-glycation assay^40,41

The assay was performed following Gutierrez. R. M. P, with slight modifications. In brief, Bovine Serum Albumin solution (10 mg/mL) was prepared in 100 mM of phosphate buffer pH 7.4 containing 3 mM sodium azide as antimicrobial agent. A methylglyoxal solution of 14 mM was also prepared in the same buffer. 1-mM concentrations of the test compounds and standard inhibitor were prepared in dimethyl sulfoxide (DMSO). Each well of a 96-well plate contained 20 µL of inhibitor, 50 µL of BSA, 50 µL of methylglyoxal and 80 µL of phosphate buffer, while the control contained 20 µl of DMSO instead of test compound. The total reaction volume was 200 µL. The reaction mixture was then incubated for 9 days at 37 °C. After incubation, each sample was examined for the development of specific fluorescence (excitation 330 nm; emission 420 nm) against a blank on a microplate reader (Spectramax M2 Devices, CA, USA).

4.5. Calculation of inhibitory activity

The percentage inhibition of advanced glycation end (AGEs) products formation by the test sample versus control was calculated using the following formula: $$\text{The}\hspace{0.5em}\%\hspace{0.5em}\text{inhibition\hspace{0.17em}\hspace{0.17em}of\hspace{0.17em}\hspace{0.17em}AGE\hspace{0.17em}\hspace{0.17em}formation}=1-\left[\left(\frac{\text{fluorescence\hspace{0.17em}\hspace{0.17em}of\hspace{0.17em}\hspace{0.17em}the\hspace{0.17em}\hspace{0.17em}test\hspace{0.17em}\hspace{0.17em}group}}{\text{fluorescence \hspace{0.17em}\hspace{0.17em}of \hspace{0.17em}\hspace{0.17em}the \hspace{0.17em}\hspace{0.17em}control\hspace{0.17em}\hspace{0.17em}group}}\right)\right]\times 100\%$$

Acknowledgements

The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for providing funding to this research group (RGP-234, Saudi Arabia).

Disclosure statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.