Synthesis and Biological Evaluation of 3-Styrylchromone Derivatives as Free Radical Scavengers and α-Glucosidase Inhibitors

Abstract

A series of 3-styrylchromone derivatives (4–20) were synthesized and the structure–activity relationships for α-glucosidase inhibition and antioxidant activities were analyzed. Among the synthesized compounds, compounds 15 and 20, which contain a catechol moiety, showed both potent 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity (15: EC₅₀=17 µM; 20: EC₅₀=23 µM) and α-glucosidase inhibitory activity (15: IC₅₀=16 µM; 20: IC₅₀=10 µM). Our data suggest that 3-styrylchromone derivatives are novel α-glucosidase inhibitors that have the potential to counteract diet-induced hyperglycemia in diabetes.

Diabetes mellitus is a metabolic disorder characterized by hyperglycemia resulting from insufficiency of secretion or action of endogenous insulin. α-Glucosidase has been recognized as a therapeutic target for modulation of postprandial hyperglycemia. α-Glycosidase inhibitors act to delay glucose absorption, making them potent drugs to control blood glucose levels.¹⁾ On the other hand, antioxidants function as free radical scavengers, chelating agents for pro-oxidant metals, quenchers of singlet oxygen formation, and reducing agents.^2,3) Because of their antioxidant characteristics, antioxidants are important in the prevention of human diseases. The accumulated evidence suggests that diabetic patients are under oxidative stress and that oxidative stress plays a major role in the pathogenesis of diabetes mellitus.⁴⁾ Recently, several researchers have evaluated α-glucosidase inhibitors possessing free-radical scavenging activity.^5–11)

4H-1-Benzopyran-4-ones (chromones) are an important class of oxygenated heterocyclic compounds, and have attracted the attention of organic chemists and biochemists due to their biological activities and occurrence in natural products. This chromone core structure is found in flavones, isoflavones, and 2-styrylchromones. Flavones and isoflavones are distributed in several species of plants. In contrast, 2-styrylchromones constitute a small group of naturally occurring chromones. Synthetic 2-styrylchromones possess a number of biological activities, including antioxidant,^12,13) anti-allergic,¹⁴⁾ anti-inflammatory,¹⁵⁾ antitumor,^16–18) and antiviral^19–21) effects. The synthesis and evaluation of biological activities of 2-styrylchromones has been extensively investigated, while studies dealing with 3-styrylchromones are few. Moreover, although there are some examples of the synthesis of 3-styrylchromones,^22–27) only a few studies have evaluated the biological activity of 3-styrylchromones.^22,28) In order to further explore novel biological activities, a series of 3-styrylchromone derivatives were synthesized, containing newly prepared compounds, and structure–activity relationships in regards to antioxidant activity and α-glycosidase inhibition were investigated.

Results and Discussion

Chemistry

3-Styrylchromone derivatives presented in this study were synthesized by Knoevenagel condensation of the appropriate 3-formylchromone with selected phenylacetic acid derivatives, by means of a modified previous procedure²²⁾ (Chart 1). This required the preparation of 3-formylchromone derivatives (2a, 2b and 2c) necessary for the synthesis of 3-styrylchromone derivatives (R¹=H, OMe and OH). Synthesis of 2a and 2b was prepared by Vilsmeier–Haack treatment of 2′-hydroxyacetophenones 1a and 1b, respectively, according to published procedures,^29,30) and 2c was also synthesized from 1c according to the same procedure. Having 2a, 2b and 2c in hand, the final step was condensation with phenylacetic acid derivatives (3a–g) in the presence of tert-BuOK in dry pyridine to provide the 3-styrylchromone derivatives (4–13) and allyl-protected products of 3-styrylchromones (14–20), respectively. 3-Styrylchromones having the allyloxy group were submitted to a deprotection step, using Pd(PPh₃)₄ in the presence of morpholine in degassed anhydrated THF, for removing the allyl group and affording 3-styrylchromones bearing the hydroxyl group (14–20).

Chart 1. Protocol for the Synthesis 3-Styrylchromone Derivatives 4–20

Reagents and conditions: (a) allylbromide, K₂CO₃, acetone, reflux; (b) DMF/POCl₃; (c) tert-BuOK, dry pyridine, reflux; (d) allylbromide, K₂CO₃, EtOH and then KOH; (e) Pd(PPh₃)₄, morpholine, THF, 60°C.

Biological Activity

All synthesized compounds were evaluated for their 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging and α-glucosidase inhibitory activities.

As shown in Table 1, modifications of 3-styrylchromones on the chromone ring (A-ring) and the phenyl group (B-ring) of the styryl moiety revealed some interesting structure–activity relationships. As a result, the introduction of the methoxy group or halogen atom (F or Cl) substituent (4–13) on the B-ring did not show DPPH radical scavenging activity, while the introduction of the hydroxyl group did. It was evident that the 3′,4′-dihydroxy derivatives (15 and 17) were more potent than the 4′-hydroxy derivatives (14 and 16). These compounds (15 and 17) have in common a catechol group in the B-ring, which is known to be an important structural feature of the antioxidant activity.^31,32) In contrast, it was noted that the introduction of a hydroxyl group at the 6-position on the chromone ring did not increase the DPPH radical scavenging activity of 3-styrylchromones (4, 14 and 15 versus 18, 19 and 20, respectively). These results indicate that hydroxyl groups on the B-ring play a more vital role than those on the A-ring.

Table 1. DPPH Scavenging and α-Glucosidase Inhibitory Activities of 3-Styrylchromone Derivatives

Compound	R1	R2	R3	R4	DPPH scavenging activity EC50 (μM)	α-Glucosidase inhibitory activity IC50 (μM)
4	H	H	OMe	H	>200	>100
5	H	OMe	OMe	H	>200	>100
6	H	OMe	OMe	OMe	>200	>100
7	OMe	H	OMe	H	>200	>100
8	OMe	OMe	OMe	H	>200	>100
9	OMe	OMe	OMe	OMe	>200	>100
10	H	H	F	H	>200	23
11	H	H	Cl	H	>200	16
12	OMe	H	F	H	>200	>100
13	OMe	H	Cl	H	>200	57
14	H	H	OH	H	60	39
15	H	OH	OH	H	17	16
16	OMe	H	OH	H	78	>100
17	OMe	OH	OH	H	22	68
18	OH	H	OMe	H	>200	33
19	OH	H	OH	H	125	9
20	OH	OH	OH	H	23	10
Ascorbic acid					23
Acarbose						>100

It was also found that the number of hydroxyl groups preferably attached to the B-ring might play an important role in the α-glucosidase inhibitory activity (14 and 16 versus 15 and 17, respectively). Interestingly, the introduction of a halogen at the 4′-position also increased the activity (10 and 11). It is known that halogens can impose molecular conformation or influence the potency of a product due to their steric and/or electronic effects. Conti and Desideri also reported on the inhibition of anti-picornavirus activity with halogen-substituted 3-styrylchromone.²²⁾ It was also noted that the introduction of a methoxy group at the 6-position on the chromone ring decreased the activity (10 and 11 versus 12 and 13, 14 and 15 versus 16 and 17, respectively), while the introduction of a hydroxyl group on the chromone ring increased the activity (4, 14 and 15 versus 18, 19 and 20, respectively). These results indicate that the hydroxylation at the 6-position of the chromone ring is important for the α-glucosidase inhibitory activity. It has been reported that some flavonoids and polyphenols, as well as sugar derivatives, were found to have an effect on α-glucosidase inhibitory activity.^6,7,33) It appears that this effect is associated with the polyphenols present in 3-styrylchromones. Of the synthesized compounds, compounds 15, 19 and 20 showed potent activity, and exhibited superior antihyperglycemic activity to the commercial antihyperglycemic drug, acarbose, which has the structural features of a tetrasaccharide.

The potency of α-glucosidase inhibition and the antioxidant activity of the synthesized 3-styrylchromones shared similar chemical features and functional groups, such as the presence of hydroxyl and methoxy groups, as shown in Table 1. Introduction of the hydroxyl group on the phenyl group of the styryl moiety increased both α-glucosidase inhibition and antioxidant activity, and 3′,4′-dihydroxy derivatives were more potent than the 4′-hydroxy derivatives (14 and 19 versus 15 and 20, respectively). In addition, the introduction of a hydroxyl group at the 6-position on the chromone ring resulted in even greater increase in α-glucosidase inhibition (14 and 15 versus 19 and 20, respectively), but a slight decrease in antioxidant activity. In contrast, a methoxy substitution at the 6-position on the chromone ring resulted in a reduction of both activities (14 and 15 versus 16 and 17, respectively).

Conclusion

A series of 3-styrylchromone derivatives (4–20) were synthesized and evaluated for DPPH free radical scavenging and α-glucosidase inhibitory activities. As a result, compounds 15 and 20, possessing a catechol moiety in the B-ring, showed potent activity in both assays. This indicates that the introduction of a hydroxyl group on the phenyl group of the styryl moiety is important for the α-glucosidase inhibition and antioxidant activities. This is the first report identifying the DPPH free radical scavenging and α-glucosidase inhibitory activities of 3-styrylchromone derivatives. These results suggest that 3-styrylchromone derivatives are novel α-glucosidase inhibitors that may counteract diet-induced hyperglycemia.

Experimental

Chemistry

All reagents and solvents were purchased from commercial sources. Analytical thin-layer chromatography was performed on silica-coated plates (silica gel 60 F-254, Merck) and visualized under UV light. Column chromatography was carried out using silica gel (Wakogel C-200, Wako). All melting points were determined using a Yanagimoto micro-hot stage and are uncorrected. ¹H-NMR and ¹³C-NMR spectra were recorded on a Varian 400-MR spectrometer using tetramethylsilane as the internal standard. MS spectra were measured using a JEOL JMS-700 spectrometer. Elemental analyses were carried out on a Yanaco CHN MT-6 elemental analyzer.

Synthesis of 3-Formylchromones

3-Formylchromones 2a–c were synthesized according to previous methods.^29,30) The products (2a, 2b) were identified by their melting points and ¹H-NMR spectra.

3-Formyl-6-(2-propenyloxy)-4H-1-benzopyran-4-one (2c): Yield 53%. Yellow needles. mp 118–119°C. ¹H-NMR (CDCl₃, 400 MHz) δ: 10.40 (1H, s, CHO), 8.54 (1H, s, H-2), 7.66 (1H, d, J=3.1 Hz, H-5), 7.48 (1H, d, J=9.2 Hz, H-8), 7.36 (1H, dd, J=9.2, 3.1 Hz, H-7), 6.07 (1H, ddt, J=17.3, 10.5, 5.3 Hz, –CH=CH₂), 5.47 (1H, dq, J=17.3, 1.5 Hz, –CH=CH₂), 5.35 (1H, dq, J=10.5, 1.5 Hz, –CH=CH₂), 4.66 (2H, dt, J=5.3, 1.5 Hz, CH₂). ¹³C-NMR (CDCl₃, 100 MHz) δ: 188.8, 175.8, 160.2, 156.8, 150.9, 132.2, 126.1, 124.8, 120.0, 119.6, 118.5, 106.5, 69.5. High resolution mass spectrum (HR-MS) m/z: Calcd for C₁₃H₁₀O₄ (M⁺): 230.0579; Found: 230.0571.

Synthesis of 4-(2-Propenyloxy)benzeneacetic Acid (3f)

The compound (3f) was synthesized by a modified previous procedure.³⁴⁾ Solid K₂CO₃ (16.6 g, 120 mol) was added to a solution of 4-hydroxyphenylacetic acid (3h, 4.6 g, 30 mmol) and allylbromide (9.4 g, 75 mmol) in EtOH (150 mL) at room temperature, and the mixture was refluxed for 5 h. To the cooled slurry was added KOH (6.7 g, 120 mmol) and stirring was continued for 12 h. The solvent was removed, the residue taken up in H₂O (500 mL), washed with Et₂O (100 mL), acidified with conc. HCl and extracted with EtOAc. The organic layer was dried over Na₂SO₄ and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane : AcOEt=1 : 1) to give the title compound (3f) in 92% yield. Colorless solid. mp 76–77°C (lit.³⁴⁾ mp 69.5–71.9°C). ¹H-NMR (CDCl₃, 400 MHz) δ: 7.19 (2H, d, J=8.6 Hz, H-2′ and H-6′), 6.88 (2H, d, J=8.6 Hz, H-3′ and H-5′), 6.05 (1H, ddt, J=17.2, 10.5, 5.3 Hz, –CH=CH₂), 5.41 (1H, dq, J=17.2, 1.5 Hz, –CH=CH₂), 5.28 (1H, dq, J=10.5, 1.5 Hz, –CH=CH₂), 4.52 (2H, dt, J=5.3, 1.5 Hz, CH₂), 3.58 (2H, s, CH₂). MS (electron ionization (EI)) m/z: 192 [M]⁺. The ¹H-NMR spectrum was similar to that previously reported.³⁴⁾

3,4-Bis(2-propenyloxy)benzeneacetic Acid (3g): According to the procedure for the preparation of compound (3f), 3,4-dihydroxyphenylacetic acid (3i, 6.8 g, 50 mmol) and allylbromide (24.2 g, 200 mmol) were treated with K₂CO₃ (41 g, 300 mol) followed by KOH (11.2 g, 200 mmol) to give the title compound (3g) in 92% yield. Colorless semisolid. ¹H-NMR (CDCl₃, 400 MHz) δ: 6.86–6.75 (3H, m, aromatic), 6.06 (2H, m, –CH=CH₂), 5.40 (2H, m, –CH=CH₂), 5.27 (2H, m, –CH=CH₂), 4.59 (4H, m, CH₂), 3.56 (2H, s, CH₂). HR-MS m/z: Calcd for C₁₄H₁₆O₄ (M⁺): 248.1049; Found: 248.1028.

General Procedure for Preparation of (E)-3-Styryl-4H-1-benzopyran-4-one Derivative (4–13)

To a solution of the corresponding 3-formylchromone (2, 2 mmol) and phenylacetic acid (3, 10 mmol) in dry pyridine (20 mL), tert-BuOK (3 mmol) was added. The mixture was refluxed until complete disappearance of 3-formylchromone (6–22 h). After the reaction mixture was diluted with ice-water and acidified to pH 2 with 5 N HCl, the sample was extracted with AcOEt. The combined organic layer was washed with saturated NaHCO₃ solution and then with brine. The organic layer was dried over Na₂SO₄ and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane : AcOEt=5 : 1) to give the corresponding 3-styryl-4H-1-benzopyran-4-one derivative.

3-[(1E)-2-(4-Methoxyphenyl)ethenyl]-4H-1-benzopyran-4-one (4): Yield 40%. Pale yellow amorphous. mp 133–134°C (lit.²⁴⁾ 123–124°C). ¹H-NMR (CDCl₃, 400 MHz) δ: 8.30 (1H, dd, J=8.0, 1.7 Hz, H-5), 8.10 (1H, d, J=0.8 Hz, H-2), 7.67 (1H, ddd, J=8.5, 7.1, 1.7 Hz, H-7), 7.56 (1H, d, J=16.4 Hz, H-β), 7.47 (1H, m, H-8), 7.47 (2H, d, J=8.9 Hz, H-2′ and H-6′), 7.42 (1H, ddd, J=8.0, 7.1, 1.1 Hz, H-6), 6.90 (2H, d, J=8.9 Hz, H-3′ and H-5′), 6.87 (1H, dd, J=16.4, 0.8 Hz, H-α), 3.83 (3H, s, OMe). MS-EI m/z: 278 [M]⁺. The ¹H-NMR spectrum was similar to that previously reported.²⁴⁾

3-[(1E)-2-(3,4-Dimethoxyphenyl)ethenyl]-4H-1-benzopyran-4-one (5): Yield 63%. Pale yellow needles. mp 128–129°C. ¹H-NMR (CDCl₃, 400 MHz) δ: 8.31 (1H, dd, J=8.0, 1.7 Hz, H-5), 8.12 (1H, d, J=0.7 Hz, H-2), 7.68 (1H, ddd, J=8.5, 7.1, 1.7 Hz, H-7), 7.54 (1H, d, J=16.3 Hz, H-β), 7.48 (1H, dd, J=8.5, 1.1 Hz, H-8), 7.43 (1H, ddd, J=8.0, 7.1, 1.1 Hz, H-6), 7.10 (1H, d, J=2.0 Hz, H-2′), 7.07 (1H, dd, J=8.2, 2.0 Hz, H-6′), 6.88 (1H, dd, J=16.3, 0.7 Hz, H-α), 6.86 (1H, d, J=8.2 Hz, H-5′), 3.95 (3H, s, OMe), 3.91 (3H, s, OMe). ¹³C-NMR (CDCl₃, 100 MHz) δ: 176.7, 155.8, 152.5, 149.1, 133.5, 131.3, 130.4, 126.2, 125.2, 124.0, 122.0, 120.0, 118.0, 116.9, 111.1, 108.7, 55.9, 55.8. MS (EI) m/z: 308 [M]⁺. Anal. Calcd for C₁₉H₁₆O₄: C, 74.01; H, 5.23. Found: C, 74.26; H, 5.26.

3-[(1E)-2-(3,4,5-Trimethoxyphenyl)ethenyl]-4H-1-benzopyran-4-one (6): Yield 62%. Yellow solid. mp 156–157°C. ¹H-NMR (CDCl₃, 400 MHz) δ: 8.30 (1H, dd, J=8.0, 1.7 Hz, H-5), 8.13 (1H, d, J=0.8 Hz, H-2), 7.69 (1H, ddd, J=8.5, 7.1, 1.7 Hz, H-7), 7.58 (1H, d, J=16.2 Hz, H-β), 7.48 (1H, dd, J=8.5, 1.1 Hz, H-8), 7.43 (1H, ddd, J=8.0, 7.1, 1.1 Hz, H-6), 6.90 (1H, dd, J=16.2, 0.8 Hz, H-α), 6.76 (2H, s, H-2′ and H-6′), 3.92 (6H, s, OMe), 3.87 (3H, s, OMe). ¹³C-NMR (CDCl₃, 100 MHz) δ: 176.6, 155.8, 153.3, 153.0, 138.0, 133.6, 133.1, 131.6, 126.2, 125.3, 124.0, 121.6, 118.4, 118.0, 103.6, 61.0, 56.1. HR-MS m/z: Calcd for C₂₀H₁₈O₅ (M⁺): 338.1154; Found: 338.1127.

6-Methoxy-3-[(1E)-2-(4-methoxyphenyl)ethenyl]-4H-1-benzopyran-4-one (7): Yield 69%. Yellow needles. mp 137–138°C. ¹H-NMR (CDCl₃, 400 MHz) δ: 8.09 (1H, d, J=0.8 Hz, H-2), 7.65 (1H, d, J=3.1 Hz, H-5), 7.55 (1H, d, J=16.3 Hz, H-β), 7.47 (2H, d, J=8.7 Hz, H-2′ and H-6′), 7.41 (1H, d, J=9.1 Hz, H-8), 7.26 (1H, dd, J=9.1, 3.1 Hz, H-7), 6.90 (2H, d, J=8.7 Hz, H-3′ and H-5′), 6.88 (1H, dd, J=16.3, 0.8 Hz, H-α), 3.92 (3H, s, OMe), 3.83 (3H, s, OMe). ¹³C-NMR (CDCl₃, 100 MHz) δ: 176.4, 159.4, 156.9, 152.4, 150.7, 130.9, 130.2, 127.8, 124.6, 123.6, 121.2, 119.4, 116.9, 114.1, 105.1, 55.9, 55.3. MS-EI m/z: 308 [M]⁺. Anal. Calcd for C₁₉H₁₆O₄: C, 74.01; H, 5.23. Found: C, 74.12; H, 5.28.

6-Methoxy-3-[(1E)-2-(3,4-dimethoxyphenyl)ethenyl]-4H-1-benzopyran-4-one (8): Yield 63%. Light brown solid. mp 135–136°C. ¹H-NMR (CDCl₃, 400 MHz) δ: 8.11 (1H, d, J=0.8 Hz, H-2), 7.66 (1H, d, J=3.1 Hz, H-5), 7.53 (1H, d, J=16.3 Hz, H-β), 7.41 (1H, d, J=9.1 Hz, H-8), 7.27 (1H, dd, J=9.1, 3.1 Hz, H-7), 7.10 (1H, d, J=1.9 Hz, H-2′), 7.07 (1H, dd, J=8.2, 1.9 Hz, H-6′), 6.89 (1H, dd, J=16.3, 0.8 Hz, H-α), 6.86 (1H, dd, J=8.2 Hz, H-5′), 3.95 (3H, s, OMe), 3.92 (3H, s, OMe), 3.91 (3H, s, OMe). ¹³C-NMR (CDCl₃, 100 MHz) δ: 176.4, 156.9, 152.4, 150.7, 149.04 149.01, 131.1, 130.5, 124.6, 123.6, 121.1, 120.0, 119.4, 117.1, 111.1, 108.7, 105.1, 55.9 (2C), 55.8. MS-EI m/z: 338 [M]⁺. Anal. Calcd for C₂₀H₁₈O₅: C, 71.00; H, 5.36. Found: C, 71.04; H, 6.39.

6-Methoxy-3-[(1E)-2-(3,4,5-trimethoxyphenyl)ethenyl]-4H-1-benzopyran-4-one (9): Yield 83%. Pale yellow amorphous. mp 128–129°C. ¹H-NMR (CDCl₃, 400 MHz) δ: 8.12 (1H, d, J=0.8 Hz, H-2), 7.66 (1H, d, J=3.1 Hz, H-5), 7.57 (1H, d, J=16.3 Hz, H-β), 7.42 (1H, d, J=9.0 Hz, H-8), 7.27 (1H, dd, J=9.0, 3.1 Hz, H-7), 6.91 (1H, dd, J=16.3, 0.8 Hz, H-α), 6.76 (2H, s, H-2′ and H-6′), 3.93 (3H, s, OMe), 3.92 (6H, s, OMe), 3.87 (3H, s, OMe). ¹³C-NMR (CDCl₃, 100 MHz) δ: 176.4, 157.0, 153.3, 152.8, 150.6, 138.0, 133.1, 131.4, 124.6, 123.7, 120.8, 119.5, 118.6, 105.1, 103.6, 61.0, 56.1, 55.9. MS-EI m/z: 368 [M]⁺. Anal. Calcd for C₂₁H₂₀O₆: C, 68.47; H, 5.47. Found: C, 68.67; H, 5.51.

3-[(1E)-2-(4-Fluorophenyl)ethenyl]-4H-1-benzopyran-4-one (10): Yield 80%. Colorless amorphous. mp 142–143°C. ¹H-NMR (CDCl₃, 400 MHz) δ: 8.31 (1H, dd, J=8.0, 1.7 Hz, H-5), 8.11 (1H, d, J=0.7 Hz, H-2), 7.68 (1H, ddd, J=8.5, 7.1, 1.7 Hz, H-7), 7.62 (1H, d, J=16.3 Hz, H-β), 7.48 (1H, dd, J=8.5, 1.1 Hz, H-8), 7.48 (2H, m, H-2′ and H-6′), 7.43 (1H, ddd, J=8.0, 7.1, 1.1 Hz, H-6), 7.05 (2H, m, H-3′ and H-5′), 6.88 (1H, dd, J=16.3, 0.7 Hz, H-α). ¹³C-NMR (CDCl₃, 100 MHz) δ: 176.6, 162.4 (d, ¹J_C-F=248 Hz), 155.8, 153.1, 133.6, 133.5, 130.5, 128.1 (d, ³J_C-F=8 Hz), 126.2, 125.3, 124.1, 121.6, 118.8, 118.0, 115.5 (d, ²J_C-F=22 Hz). MS-EI m/z: 266 [M]⁺. Anal. Calcd for C₁₇H₁₁FO₂: C, 76.68; H, 4.16. Found: C, 76.64; H, 4.21.

3-[(1E)-2-(4-Chlorophenyl)ethenyl]-4H-1-benzopyran-4-one (11): Yield 82%. Colorless amorphous. mp 157–158°C (lit.²⁵⁾ 159–160°C). ¹H-NMR (CDCl₃, 400 MHz) δ: 8.30 (1H, dd, J=8.0, 1.7 Hz, H-5), 8.11 (1H, d, J=0.8 Hz, H-2), 7.68 (1H, ddd, J=8.5, 7.1, 1.7 Hz, H-7), 7.64 (1H, d, J=16.3 Hz, H-β), 7.48 (1H, dd, J=8.5, 1.1 Hz, H-8), 7.46–7.41 (1H, m, H-6), 7.45 (2H, d, J=8.5 Hz, H-2′ and H-6′), 7.32 (2H, d, J=8.5 Hz, H-3′ and H-5′), 6.93 (1H, dd, J=16.3, 0.8 Hz, H-α). ¹³C-NMR (CDCl₃, 100 MHz) δ: 176.5, 155.7, 153.4, 135.9, 133.6, 133.4, 130.4, 128.8, 127.7, 126.2, 125.3, 124.0, 121.4, 119.7, 118.1. MS-EI m/z: 282 [M]⁺. The ¹H and ¹³C-NMR spectra were similar to that previously reported.²⁵⁾

3-[(1E)-2-(4-Fluorophenyl)ethenyl]-6-methoxy-4H-1-benzopyran-4-one (12): Yield 86%. Pale yellow amorphous. mp 154–155°C. ¹H-NMR (CDCl₃, 400 MHz) δ: 8.09 (1H, d, J=0.8 Hz, H-2), 7.65 (1H, d, J=3.1 Hz, H-5), 7.61 (1H, d, J=16.3 Hz, H-β), 7.49 (2H, m, H-2′ and H-6′), 7.41 (1H, d, J=9.1 Hz, H-8), 7.27 (1H, dd, J=9.1, 3.1 Hz, H-7), 7.05 (2H, m, H-3′ and H-5′), 6.90 (1H, dd, J=16.3, 0.8 Hz, H-α), 3.92 (3H, s, OMe). ¹³C-NMR (CDCl₃, 100 MHz) δ: 176.4, 162.4 (d, ¹J_C-F=247 Hz), 157.0, 152.9, 150.6, 133.6, 130.3, 128.1 (d, ³J_C-F=8 Hz), 124.6, 123.7, 120.8, 119.5, 119.0, 115.5 (d, ²J_C-F=22 Hz), 105.1, 55.9. MS-EI m/z: 296 [M]⁺. Anal. Calcd for C₁₈H₁₃FO₃: C, 72.97; H, 4.42. Found: C, 73.20; H, 4.47.

3-[(1E)-2-(4-Chlorophenyl)ethenyl]-6-methoxy-4H-1-benzopyran-4-one (13): Yield 80%. Pale yellow needles. mp 134–135°C. ¹H-NMR (CDCl₃, 400 MHz) δ: 8.10 (1H, d, J=0.8 Hz, H-2), 7.65 (1H, d, J=3.1 Hz, H-5), 7.63 (1H, d, J=16.3 Hz, H-β), 7.45 (2H, d, J=8.5 Hz, H-2′ and H-6′), 7.42 (1H, d, J=9.1 Hz, H-8), 7.33 (2H, d, J=8.5 Hz, H-3′ and H-5′), 7.27 (1H, dd, J=9.1, 3.1 Hz, H-7), 6.95 (1H, dd, J=16.3, 0.8 Hz, H-α), 3.92 (3H, s, OMe). ¹³C-NMR (CDCl₃, 100 MHz) δ: 176.3, 157.1, 153.2, 150.6, 135.9, 133.3, 130.2, 128.8, 127.7, 124.6, 123.7, 120.6, 119.9, 119.5, 105.1, 55.9. MS-EI m/z: 312 [M]⁺. Anal. Calcd for C₁₈H₁₃ClO₃: C, 69.13; H, 4.19. Found: C, 69.31; H, 4.19.

General Procedure for Preparation of 3-Styrylchromones Having the Hydroxyl Group (14–20)

According to the general procedure for preparation of (E)-3-styryl-4H-1-benzopyran-4-one derivative, the corresponding 3-formylchromone (2, 2 mmol) and phenylacetic acid (3a, 3f or 3g, 10 mmol) were treated with tert-BuOK (3 mmol) and then the residue was passed once through a short silica gel column (hexane : AcOEt=5 : 1) and the solvent was evaporated. The obtained allyl protected compound (1.0 mmol) was dissolved in degassed anhydrated tetrahydrofuran (THF) (50 mL) and morpholine (10 equiv. per allyl group to be cleaved) was added Pd(PPh₃)₄ (5 mol%). The green mixture was stirred at 60°C (monitored by TLC) and concentrated under reduced pressure. The residue was treated with saturated NH₄Cl solution and extracted with AcOEt. The organic layer was dried over Na₂SO₄ and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane : AcOEt=2 : 1) to give the title compound.

3-[(1E)-2-(4-Hydroxyphenyl)ethenyl]-4H-1-benzopyran-4-one (14): Yield 30% (2 steps). Ocher amorphous. mp 209–211°C. ¹H-NMR (DMSO-d₆, 400 MHz) δ: 9.63 (1H, br s, OH), 8.64 (1H, d, J=0.7 Hz, H-2), 8.15 (1H, dd, J=8.0, 1.7 Hz, H-5), 7.81 (1H, ddd, J=8.5, 7.1, 1.7 Hz, H-7), 7.68 (1H, dd, J=8.5, 1.0 Hz, H-8), 7.61 (1H, d, J=16.4 Hz, H-β), 7.52 (1H, ddd, J=8.0, 7.1, 1.1 Hz, H-6), 7.37 (2H, d, J=8.6 Hz, H-2′ and H-6′), 6.84 (1H, dd, J=16.4, 0.7 Hz, H-α), 6.78 (2H, d, J=8.6 Hz, H-3′ and H-5′). ¹³C-NMR (DMSO-d₆, 100 MHz) δ: 176.2, 157.9, 155.7, 154.9, 134.4, 131.2, 128.7, 128.1, 125.9, 125.8, 123.8, 121.4, 118.9, 116.5, 116.1. HR-MS m/z: Calcd for C₁₇H₁₂O₃ (M⁺): 264.0786; Found: 264.0774.

3-[(1E)-2-(3,4-Dihydroxyphenyl)ethenyl]-4H-1-benzopyran-4-one (15): Yield 49% (2 steps). Ocher amorphous. mp 186–188°C. ¹H-NMR (DMSO-d₆, 400 MHz) δ: 9.11 (2H, br s, OH), 8.66 (1H, s, H-2), 8.14 (1H, dd, J=8.0, 1.7 Hz, H-5), 7.82 (1H, ddd, J=8.5, 7.1, 1.7 Hz, H-7), 7.68 (1H, dd, J=8.5, 1.0 Hz, H-8), 7.52 (1H, d, J=16.4 Hz, H-β), 7.52 (1H, m, H-6), 6.95 (1H, d, J=2.0 Hz, H-2′), 6.81 (1H, dd, J=8.1, 2.0 Hz, H-6′), 6.76 (1H, d, J=16.4 Hz, H-α), 6.73 (1H, d, J=8.1 Hz, H-5′). ¹³C-NMR (DMSO-d₆, 100 MHz) δ: 176.0, 155.7, 154.7, 146.2, 145.9, 134.4, 131.4, 129.2, 125.9, 125.8, 123.8, 121.4, 118.9, 118.8, 116.22, 116.20, 113.4. MS-EI m/z: 280 [M]⁺. Anal. Calcd for C₁₇H₁₂O₄: C, 72.85; H, 4.32. Found: C, 72.75; H, 4.39.

3-[(1E)-2-(4-Hydroxyphenyl)ethenyl]-6-methoxy-4H-1-benzopyran-4-one (16): Yield 64% (2 steps). Ocher needles. mp 209–211°C. ¹H-NMR (DMSO-d₆, 400 MHz) δ: 9.61 (1H, br s, OH), 8.62 (1H, d, J=0.7 Hz, H-2), 7.65 (1H, d, J=9.1 Hz, H-8), 7.58 (1H, d, J=16.4 Hz, H-β), 7.50 (1H, d, J=3.1 Hz, H-5), 7.41 (1H, dd, J=9.2, 3.1 Hz, H-7), 7.37 (2H, d, J=8.6 Hz, H-2′ and H-6′), 6.84 (1H, dd, J=16.4, 0.7 Hz, H-α), 6.78 (2H, d, J=8.6 Hz, H-3′ and H-5′), 3.88 (3H, s, OMe). ¹³C-NMR (DMSO-d₆, 100 MHz) δ: 175.7, 157.8, 157.0, 154.6, 150.5, 131.0, 128.7, 128.1, 124.5, 123.6, 120.6, 120.5, 116.6, 116.1, 105.4, 56.2. MS (EI) m/z: 294 [M]⁺. Anal. Calcd for C₁₈H₁₄O₄: C, 73.46; H, 4.80. Found: C, 73.27; H, 4.81.

3-[(1E)-2-(3,4-Dihydroxyphenyl)ethenyl]-6-methoxy-4H-1-benzopyran-4-one (17): Yield 55% (2 steps). Ocher amorphous. mp 219–221°C. ¹H-NMR (DMSO-d₆, 400 MHz) δ: 9.10 (2H, br s, OH), 8.64 (1H, s, H-2), 7.65 (1H, d, J=9.1 Hz, H-8), 7.51 (1H, d, J=3.1 Hz, H-5), 7.50 (1H, d, J=16.4 Hz, H-β), 7.41 (1H, dd, J=9.1, 3.1 Hz, H-7), 6.95 (1H, d, J=2.0 Hz, H-2′), 6.80 (1H, m, H-6′), 6.77 (1H, d, J=16.4 Hz, H-α), 6.74 (1H, m, H-5′), 3.88 (3H, s, OMe). ¹³C-NMR (DMSO-d₆, 100 MHz) δ: 175.7, 157.0, 154.5, 150.5, 146.2, 145.9, 131.2, 129.3, 124.4, 123.6, 120.6, 120.5, 118.9, 116.3, 116.2, 113.3, 105.4, 56.2. MS-EI m/z: 310 [M]⁺. Anal. Calcd for C₁₈H₁₄O₅: C, 69.67; H, 4.55. Found: C, 69.60; H, 4.65.

6-Hydroxy-3-[(1E)-2-(4-methoxyphenyl)ethenyl]-4H-1-benzopyran-4-one (18): Yield 53% (2 steps). Colorless amorphous. mp 236–237°C. ¹H-NMR (DMSO-d₆, 400 MHz) δ: 10.06 (1H, br s, OH), 8.59 (1H, d, J=0.7 Hz, H-2), 7.65 (1H, d, J=16.4 Hz, H-β), 7.55 (1H, d, J=9.0 Hz, H-8), 7.41 (1H, d, J=3.0 Hz, H-5), 7.48 (2H, d, J=8.7 Hz, H-2′ and H-6′), 7.24 (1H, dd, J=9.0, 3.0 Hz, H-7), 6.90 (1H, dd, J=16.4, 0.7 Hz, H-α), 6.96 (2H, d, J=8.7 Hz, H-3′ and H-5′), 3.78 (3H, s, OMe). ¹³C-NMR (DMSO-d₆, 100 MHz) δ: 175.8, 159.4, 155.3, 155.0, 149.5, 130.40, 130.37, 127.9, 124.7, 123.5, 120.2, 120.1, 118.0, 114.7, 108.2, 55.6. MS (EI) m/z: 294 [M]⁺. Anal. Calcd for C₁₈H₁₄O₄: C, 73.46; H, 4.80. Found: C, 73.22; H, 4.83.

6-Hydroxy-3-[(1E)-2-(4-hydroxyphenyl)ethenyl]-4H-1-benzopyran-4-one (19): Yield 41% (2 steps). Ocher amorphous. mp 293–295°C. ¹H-NMR (DMSO-d₆, 400 MHz) δ: 10.00 (1H, br s, OH), 9.63 (1H, br s, OH), 8.57 (1H, d, J=0.7 Hz, H-2), 7.58 (1H, d, J=16.4 Hz, H-β), 7.54 (1H, d, J=9.0 Hz, H-8), 7.40 (1H, d, J=3.0 Hz, H-5), 7.36 (2H, d, J=8.6 Hz, H-2′ and H-6′), 7.23 (1H, dd, J=9.0, 3.0 Hz, H-7), 6.82 (1H, dd, J=16.4, 0.7 Hz, H-α), 6.77 (2H, d, J=8.6 Hz, H-3′ and H-5′). ¹³C-NMR (DMSO-d₆, 100 MHz) δ: 175.8, 157.8, 155.3, 154.7, 149.5, 130.8, 128.8, 128.0, 124.7, 123.4, 120.3, 120.2, 116.8, 116.1, 108.2. MS (EI) m/z: 280 [M]⁺. Anal. Calcd for C₁₇H₁₂O₄: C, 72.85; H, 4.32. Found: C, 72.66; H, 4.45.

6-Hydroxy-3-[(1E)-2-(3,4-dihydroxyphenyl)ethenyl]-4H-1-benzopyran-4-one (20): Yield 36% (2 steps). Brown solid. mp 258–260°C. ¹H-NMR (DMSO-d₆, 400 MHz) δ: 9.90 (1H, br s, OH), 9.18 (2H, br s, OH), 8.59 (1H, d, J=0.8 Hz, H-2), 7.54 (1H, d, J=9.0 Hz, H-8), 7.49 (1H, d, J=16.4 Hz, H-β), 7.40 (1H, d, J=3.0 Hz, H-5), 7.23 (1H, dd, J=9.0, 3.0 Hz, H-7), 6.94 (1H, d, J=2.0 Hz, H-2′), 6.79 (1H, dd, J=8.1, 2.0 Hz, H-6′), 6.74 (1H, dd, J=16.4, 0.8 Hz, H-α), 6.73 (1H, d, J=8.1, H-5′), 3.88 (3H, s, OMe). ¹³C-NMR (DMSO-d₆, 100 MHz) δ: 175.8, 155.3, 154.6, 149.5, 146.1, 145.9, 131.0, 129.4, 124.7, 123.4, 120.3, 120.2, 118.8, 116.6, 116.2, 113.3, 108.2. HR-MS m/z: Calcd for C₁₇H₁₂O₅ (M⁺): 296.0685. Found: 296.0672.

Biological Assay

α-Glucosidase from Saccharomyces cerevisiae and 4-nitrophenyl α-D-glucopyranoside (PNP-G) were purchased from Sigma Chemical Co., St. Louis, MO, U.S.A. DPPH radical was purchased from Tokyo Chemical Industry Co., Tokyo, Japan.

DPPH Radical Scavenging Assay

DPPH radical scavenging activity was measured according to the method of Nile et al.³⁵⁾ with minor modifications. Briefly, 180 µL of 100 µM DPPH solution in MeOH was mixed with 20 µL of various concentrations of the sample solution in MeOH. The absorbance of the mixture was measured at 517 nm using a micro-plate reader (Molecular Devices SPECTRA MAX 190). The sample solution was replaced by MeOH as a control. Ascorbic acid was used as a positive control.

α-Glucosidase Inhibitory Assay

α-Glucosidase inhibitory activity was assayed using the method of Mabkhot et al.³⁶⁾ with minor modifications. Briefly, 210 µL of 50 mM phosphate buffer (pH 7.0) containing 100 mM NaCl, 30 µL of 0.25 U/mL α-glucosidase dissolved in the buffer, 30 µL of 7 mM PNP-G as a substrate dissolved in the buffer, and 30 µL of various concentrations of samples dissolved in dimethyl sulfoxide (DMSO) were mixed and the increment in absorption at 400 nm, due to the hydrolysis of PNP-G by α-glucosidase, was monitored continuously with a micro-plate reader (Molecular Devices SPECTRA MAX 190). The sample solution was replaced by DMSO as a control. Acarbose was used as a positive control.

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