Antiradical Activity of Naphthazarin-Carbohydrate Nonglycoside Conjugates

Abstract

The ABTS^·+ scavenging activity of known (4, 6-8) and new (5, 9-12) naphthazarin-carbohydrate nonglycoside conjugates, and methoxynaphthazarins 1-3 was evaluated. The study of structure-activity relationships showed that the presence of carbohydrate fragments in the structure of naphthazarin-carbohydrate conjugates increased their antiradical activity compared with starting methoxynaphthazarins. Antiradical activity depends on the structure of carbohydrate fragments, their number, and position. Depending on the carbohydrate fragment the activity increased in the following order: methyl-α-d-glucopyranoside < d-glucofuranose < 1,2-O-isopropylidene-α-d-glucofuranose. The 2 sugar residues and their 3,7-position enhanced the activity of the conjugates.

A large group of naphthoquinone pigments from sea urchins includes the naphthazarin core in their structures. Due to their low solubility in water and product instability, limited bioavailability can be expected for these pigments. ¹ Naphthazarin-carbohydrate nonglycoside conjugates are water-soluble compounds where naphthazarin and sugar fragments are linked by an ether bond through the primary hydroxyl group of a carbohydrate. These conjugates are formed by the transesterification of methoxynaphthazarins with a carbohydrate. ^2-4 It was shown that naphthazarin-carbohydrate conjugates have cytotoxic and contraceptive activities to the sperm and eggs of the sea urchin Strongylocentrotus intermedius. ² Moreover, naphthazarin-carbohydrate conjugates induced apoptosis in human cancer HeLa and normal mouse JB6 P⁺ Cl41 cells with simultaneous inhibition of p53-dependent transcriptional activity. ⁴

It is well known that an antiradical activity is the most common property of naphthazarin pigments and their derivatives. ^1,5-7 In continuation of our study of structure-antiradical activity relationships of naphthazarin pigments and their derivatives, we also evaluated ABTS^·+ scavenging activity of naphthazarin-carbohydrate conjugates to clarify the effect of a sugar fragment on the activity. The antiradical activity of naphthazarin-carbohydrate nonglycoside conjugates is studied for the first time.

We analyzed antiradical activity and structure-antiradical activity relationships of methoxynaphthazarins 1-3 as well as naphthazarin-carbohydrate nonglycoside conjugates 4-12. Tested compounds 1-12 (Figure 1) were divided into 3 groups according to a structure of a naphthazarin core. The first group—the naphthazarin-carbohydrate conjugates 4 and 5 derived from the methyl ether of naphthopurpurin (1), the second group—the conjugates 6-9 derived from the dimethyl ether of ethylmompain (2), and the third group—the conjugates 10-12 derived from the dimethyl ether of ethylisomompain (3). Compounds 1-4, 6-8 we synthesized early, ^2,4–6 compounds 5, 9-12 were synthesized for the first time. The chemical structures of the synthesized compounds were confirmed by IR spectroscopy, ¹H, and ¹³C NMR spectroscopic data including 2D HMBC data and mass spectrometry.

Figure 1

Structure of compounds 1-12.

It was observed that the ABTS^·+ scavenging activity of compounds 1-12 was concentration-dependent and a gradual increase in concentration increased the activity. Concentrations that caused a 50% reduction of the absorbance of ABTS^·+ and Trolox equivalents are given in Table 1.

Table 1

Antiradical Activity of Compounds 1-12 in the ABTS Assay.

Compounds	EC50, µM a	TE b	Compounds	EC50, µM a	TE b
1	70.6 ± 5.7	0.17	7	30.8 ± 3.4	0.39
2	44.4 ± 4.5	0.27	8	36.4 ± 4.4	0.33
3	42.8 ± 3.1	0.28	9	18.1 ± 4.7	0.66
4	60.0 ± 4.8	0.20	10 +11	20.3 ± 4.7	0.59
5	41.4 ± 4.6	0.29	12	13.5 ± 3.2	0.89
6	38.7 ± 2.3	0.31	Trolox	12.0 ± 1.3	1

^aConcentration that caused a 50% reduction of the absorbance. Values are mean ± SD of 3 determinations.

^bTrolox equivalents: EC₅₀ (Trolox)/EC₅₀ (compound).

Comparison of the antiradical activity of compounds 1, 4, and 5 in the first group of compounds (Table 1) showed that the activity of conjugates 4 (TE = 0.20) and 5 (TE = 0.29) is higher than the activity of the methyl ether of naphthopurpurin (1) (TE = 0.17) from which they were derived. The activity of the conjugate 4 having methyl-α-d-glucopyranoside fragment was lower than the activity of the conjugate 5 with 1,2-O-isopropylidene-α-D-glucofuranose fragment.

To estimate an influence of a structure of carbohydrate groups on the antiradical activity we compared data for compounds 6-9 from the second group of conjugates derived from the dimethyl ether of ethylmompain (2) (Table 1). And again, as can be seen from Table 1, the activity of the conjugate 6 (TE = 0.31) with methyl-α-d-glucopyranoside fragment was lower than activity of 7 (TE = 0.39) with 1,2-O-isopropylidene-α-D-glucofuranose fragment and 8 (TE = 0.33) having D-glucofuranose part. The 2 sugar residues in the molecule 9 significantly enhanced the activity of the conjugate (TE = 0.66). The activity of conjugates 6-9 was higher than the activity of the dimethyl ether of ethylmompain (2) (TE = 0.27) from which they were derived.

The third group of tested conjugates which were formed by the transesterification of the dimethyl ether of ethylisomompain (3) with 1,2-O-isopropylidene-α-D-glucofuranose included the conjugate 12 and the inseparable mixture (3:2) of compounds 10 and 11. The activity of the conjugate 12 with 2 carbohydrate fragments (TE = 0.89) was higher than the activity of the mixture 10 and 11 (TE = 0.59) with 1 carbohydrate group. The dimethyl ether of ethylisomompain (3) differs from the dimethyl ether of ethylmompain (2) only by the position of 1 methoxyl group, at C-2 in 2 and at C-3 in 3. To clarify an influence of the position of the carbohydrate group in the naphthazarin core we compared activities of compounds 12 and 9, and activities of the compound 7 and the mixture of compounds 10 and 11. As can be seen, activities of conjugates 10 +11 (TE = 0.59) and 12 (TE = 0.89) derived from the dimethyl ether of ethylisomompain (3) were higher than activities of conjugates 7 (TE = 0.39) and 9 (TE = 0.66) derived from the dimethyl ether of ethylmompain (2), respectively. So, the position of 2 substituents at C-3 and C-7 is preferable for the antiradical activity in this case.

The investigation showed that the presence of sugar fragments on the naphthazarin core enhanced the antiradical activity of naphthazarin-carbohydrate nonglycoside conjugates compared with starting methoxynaphthazarins from which they were derived. Structure-activity relationships study showed that the structure of sugar moiety affected the radical scavenging activity of naphthazarin-carbohydrate nonglycoside conjugates. This activity increased according the carbohydrate fragment in the following order: methyl-α-d-glucopyranoside fragment <D-glucofuranose fragment <1,2-O-isopropylidene-α-D-glucofuranose fragment. The 2 sugar residues in the molecule of naphthazarin-carbohydrate nonglycoside conjugates as well as their 3,7-position enhanced the activity of the conjugates.

Experimental

General

The melting points were determined with a Boetius apparatus and are uncorrected. Analytical grade solvents were used. The IR absorption spectra were recorded on a Bruker Vector 22 spectrophotometer. ¹H and ¹³C NMR spectra were recorded on Bruker Avance-III-500 HD (500 and 125 MHz, respectively), and Avance-III-700 (700 and 176 MHz, respectively) spectrometers. The chemical shifts (δ) are in parts per million (ppm) relative to tetramethylsilane (δ = 0.0 ppm). The mass spectra were taken on an AMD 604S spectrometer (direct sample inlet, ionizing energy 70 eV and elevated temperature). High-resolution mass spectra (HRMS) were recorded on a Bruker maXis Impact II spectrometer using methanol solutions of 9, 12. The course of reactions was monitored and the purity of the compounds obtained was checked by thin-layer chromatography (TLC). Merck Kiesel gel 60 F-254 plates were preliminary treated with 0.05 M tartaric acid in MeOH and dried at 50°C for 2 to 3 hours; 3:1 n-hexane-acetone mixture was used as an eluent. Preparative TLC and CC were performed on silica gel (Chemapol, Czech Republic), 5/40 and 40/100 µM, respectively, using n-hexane-acetone. Yields were not optimized. Naphthopurpurin methyl ether (5,8-dihydroxy-2-methoxy-1,4-naphthoquinone) (1), ⁸ ethylmompain dimethyl ether (6-ethyl-5,8-dihydroxy-2,7-dimethoxy-1,4-naphthoquinone) (2), ⁹ ethylisomompain dimethyl ether (6-ethyl-5,8-dihydroxy-3,7-dimethoxy-1,4-naphthoquinone) (3), ⁹ 5,8-dihydroxy-2-(1′-methoxy-6′-α-D-glucopyranosyloxy)-1,4-naphthoquinone (4), ⁴ 6-ethyl-5,8-dihydroxy-7-methoxy-2-(1′-methoxy-6′-α-D-glucopyranosyloxy)-1,4-naphthoquinone (6), ⁴ 6-ethyl-5,8-dihydroxy-7-methoxy-2-(1′,2′-isopropylidene-6′-α-D-glucofuranosyloxy)-1,4-naphthoquinone (7), 6-ethyl-5,8-dihydroxy-7-methoxy-2-(6′-α-D-glucofuranosyloxy)-1,4-naphthoquinone (8), ² and 1,2-O-isopropylidene-α-D-glucofuranose ¹⁰ were synthesized according to described methods. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was purchased from Fluka; 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) from Sigma-Aldrich; UV/Vis spectra were recorded on an UV-mini 1240 spectrophotometer (Shimadzu).

Synthesis of Compounds 5, 9 to 12 (General Method)

A mixture of a corresponding quinone (1.5 mmol), 1,2-O-isopropylidene-α-D-glucofuranose (6 mmol), DMSO (4 mL), and 3 drops of 1 M solution of MeONa in MeOH was stirred at 85°C for 2 hours. After cooling the reaction mixture was diluted with water (50 mL) and acidified to pH∼5-6 (with 5% HCl). Then the mixture was extracted with EtOAc, and the organic layer was dried over anhydrous Na₂SO₄, concentrated, and the product isolated by preparative TLC (n-hexane-acetone, 1:1).

5,8-Dihydroxy-2-(1′,2′-O-isopropylidene-6′-α-D-glucofuranosyl-Oxy)-1,4-naphthoquinone (5)

Yield 10%.

MP: 128°C to 131°C.

IR (CHCl₃): 3488, 3058, 3004, 2931, 1607, 1456, 1455 cm⁻¹.

¹H NMR (500 MHz, acetone-d₆ ): 1.28 (3H, s, Me′), 1.43 (3H, s, Me′), 4.24 (1H, dd, J = 8.2, J = 2.7 Hz, H-4′), 4.27 (1H, dd, J = 10.8, J = 6.2 Hz, H-6_b′), 4.36 (2H, m, H-3′, H-5′), 4.39 (1H, dd, J = 10.8, J = 2.2 Hz, H-6_a′), 4.54 (1H, d, J = 3.7 Hz, H-2′), 5.89 (1H, d, J = 3.7 Hz, H-1′), 6.43 (1H, s, H-3), 7.30 (1H, d, J = 9.4 Hz, H-7), 7.35 (1H, d, J = 9.4 Hz, H-6), 12.16 (1H, s, α-OH), 12.71 (1H, s, α-OH).

¹³C NMR (125 MHz, CDCl₃): 26.4 (Me′), 27.0 (Me′), 67.6 (C-5′), 73.45 (C-6′), 74.94 (C-3′), 80.68 (C-4′), 86.07 (C-2′), 105.86 (C-1′), 111.52 (C-4α), 111.54 (C-3) 111.86 (CMe₂′), 112.50 (C-8α), 128.55 (C-7), 130.88 (C-6), 157.20 (C-5), 158.48 (C-8), 161.54 (C-2), 183.66 (C-1), 189.67 (C-4).

MS (EI, 70 eV): m/z (%) =408 [M⁺] (20), 350 (7), 408 (15), 293(7),

HRMS-EI: m/z [M⁺] calcd for C₁₉H₂₀O₁₀: 408.1056; found: 408.1053.

6-Ethyl-5,8-dihydroxy-2,7-di(1 ′,2′-O-isopropylidene-6′-α-D-glucofuranosyloxy)-1,4-naphthoquinone (9)

This substance is the side product of 7 synthesis ² and has not been published earlier.

Yield 6%.

MP: 103°C to 108°C.

IR (CHCl₃): 3605, 3487, 2994, 2938, 1672, 1602, 1437 cm⁻¹.

¹H NMR (700 MHz, acetone-d ₆): 1.15 (3H, t, J = 7.5 Hz, Me), 1.267 (3H, s, Me′), 1.274 (3H, s, Me′), 1.42 (3H, s, Me′), 1.43 (3H, s, Me′), 2.79 (2H, q, J = 7.5 Hz, CH₂), 4.16 (1H, dd, J = 8.2, J = 2.7 Hz, H-4′′), 4.24 (1H, dd, J = 8.2, J = 2.7 Hz, H-4′), 4.26 (2H, m, H-5′′, H-6_b′), 4.30 (1H, d, J = 2.4 Hz, H-3′′), 4.34 (1H, d, J = 2.4 Hz, H-3′), 4.39 (2H, m, H-6_a′, H-6_b′′), 4.52 (1H, d, J = 3.75 Hz, H-2′′), 4.53 (1H, d, J = 3.75 Hz, H-2′), 4.61 (1H, dd, J = 10.6, J = 2.7 Hz, H-6_a′′), 5.88 (1H, d, J = 3.75 Hz, H-1′′), 5.89 (1H, d, J = 3.75 Hz, H-1′), 6.55 (1H, s, H-3), 12.81 (1H,s, α-OH), 13.44 (1H, s, α-OH).

¹³C NMR (176 MHz, acetone-d₆ ): 13.5 (Me), 17.6 (CH₂), 26.2 (Me′), 26.9 (Me′), 26.4 (Me′), 27.0 (Me′), 67.7 (C-5′), 68.9 (C-5′′),73.3 (C-6′), 74.9 (C-3′), 75.0 (C-3′′), 76.8 (C-6′′), 80.6 (C-4′′), 80.7 (C-4′), 86.03 (C-2′′), 86.05 (C-2′), 105.83 (C-1′′), 105.85 (C-1′),106.4 (C-4a), 110.4 (C-3), 111.6 (CMe₂′), 111.7 (C-8a), 111.8 (CMe₂′′), 138.6 (C-6), 155.1 (C-7), 160.0 (C-2), 165.4 (C-8), 168.7 (C-5), 171.8 (C-1), 178.3 (C-4).

MS (EI, 70 eV): m/z (%) =454 [M⁺] (4), 418 (5), 264 (5), 264 (62).

HRESIMS: m/z [M-H]⁻ calcd for C₃₀H₃₈O₁₆: 653.7087; found: 653.7087.

6-Ethyl-5,8-dihydroxy-3-methoxy-7-(1′,2′-O-isopropylidene-6′-α-D-glucofuranosyloxy)-1,4-naphthoquinone (10) and 6-ethyl-5,8-dihydroxy-7-methoxy-3-(1′,2′-O-isopropylidene-6′-α-D-glucofuranosyloxy)-1,4-naphthoquinone (11)

Compounds 10 and 11 as inseparable mixture (3:2, according to NMR data).

Yield 45%.

MS (EI, 70 eV): m/z (%) =466 [M⁺] (60).

Compound (10)

¹H NMR (500 MHz, acetone-d₆ ): 1.14 (3H, t, J = 7.5 Hz, Me), 1.27 (3H, s, Me′), 1.42 (3H, s, Me′), 2.77 (2H, q, J = 7.5 Hz, CH₂), 3.99 (3H, s, OMe), 4.16 (1H, dd, J = 8.4, J = 2.7 Hz, H-4′), 4.25 (1H, m, H-5′), 4.30 (1H, d, J = 2.7 Hz, H-3′), 4.47 (1H, dd, J = 10.6, J = 6.0 Hz, H-6_b′), 4.52 (1H, d, J = 3.6 Hz, H-2′), 4.68 (1H, dd, J = 10.6, J = 2.6 Hz, H-6_a′), 5.88 (1H, d, J = 3.6 Hz, H-1′), 6.45 (1H, s, H-2), 13.14 (1H,s, α-OH), 13.27 (1H, s, α-OH).

¹³C NMR (125 MHz acetone-d₆ ): 13.4 (Me), 17.34 (CH₂), 26.2 (Me′), 26.9 (Me′), 57.1 (OMe), 68.9 (C-5′), 75.0 (C-3′), 76.9 (C-6′), 80.6 (C-4′), 86.02 (C-2′), 105.83 (C-1′), 108.5 (C-8a), 108.7 (C-2), 109.5 (C-4a), 111.6 (CMe₂′), 135.8 (C-6), 157.2 (C-7), 161.4 (C-3), 164.3 (C-8), 169.8 (C-5), 171.4 (C-4), 178.6 (C-1).

Compound (11)

¹H NMR (500 MHz, acetone-d₆ ): 1.12 (3H, t, J = 7.5 Hz, Me), 1.28 (3H, s, Me′), 1.43 (3H, s, Me′), 2.67 (2H, q, J = 7.5 Hz, CH₂), 4.12 (3H, s, OMe), 4.23 (1H, dd, J = 8.1, J = 2.7 Hz, H-4′), 4.26 (1H, m, H-6_b′), 4.35 (1H, d, J = 2.6 Hz, H-3′), 4.36 (1H, m, H-5′), 4.39 (1H, dd, J = 10.2, J = 2.1 Hz, H-6_a′), 4.54 (1H, d, J = 3.6 Hz, H-2′), 5.90 (1H, d, J = 3.6 Hz, H-1′), 6.51 (1H, s, H-2), 13.09 (1H,s, α-OH), 13.26 (1H, s, α-OH).

¹³C NMR (125 MHz, acetone-d₆ ): 13.5 (Me), 17.25 (CH₂), 26.4 (Me′), 27.0 (Me′), 61.6 (OMe), 67.7 (C-5′), 73.39 (C-6′), 74.94 (C-3′), 80.7 (C-4′), 86.04 (C-2′), 105.84 (C-1′), 108.9 (C-8a), 109.4 (C-4a), 109.7 (C-2), 111.8 (CMe₂′), 135.4 (C-6), 157.4 (C-7), 161.0 (C-3), 162.5 (C-8), 169.4 (C-5), 171.8 (C-4), 180.3 (C-1).

6-Ethyl-5,8-dihydroxy-3,7-di(1′,2′-O-isopropylidene-6-′-α-glucofuranosyloxy)-1,4-naphthoquinone (12)

Yield 15%.

MP: 106°C to 110°C.

IR (CHCl₃): 3604, 3452, 2992, 2938, 1599, 1435 cm⁻¹.

¹H NMR (500 MHz, acetone-d₆ ): 1.14 (3H, t, J = 7.5 Hz, Me), 1.266 (3H, s, Me′), 1.273 (3H, s, Me′), 1.274 (3H, s, Me′), 1.42 (3H, s, Me′), 1.43 (3H, s, Me′), 2.78 (2H, q, J = 7.5 Hz, CH₂), 4.16 (1H, dd, J = 8.2, J = 2.7 Hz, H-4′′), 4.23 (1H, dd, J = 8.2, J = 2.7 Hz, H-4′), 4.24 (1H, m, H-5′′), 4.27 (1H, m, H-6_b′), 4.29 (1H, d, J = 2.4 Hz, H-3′′), 4.34 (1H, d, J = 2.4 Hz, H-3′), 4.36 (1H, m, H-5′), 4.40 (H, m, H-6_a′), 4.47 (1H, m, H-6_b′), 4.51 (1H, d, J = 3.75 Hz, H-2′′), 4.53 (1H, d, J = 3.75 Hz, H-2′), 4.68 (1H, dd, J = 10.6, J = 2.6 Hz, H-6_a′′), 5.87 (1H, d, J = 3.75 Hz, H-1′′), 5.89 (1H, d, J = 3.75 Hz, H-1′), 6.56 (1H, s, H-2), 13.18 (1H, s, α-OH), 13.30 (1H, s, α-OH).

¹³C NMR (125 MHz, acetone-d₆ ): 13.4 (Me), 17.3 (CH₂), 26.2 (Me′), 26.9 (Me′), 26.4 (Me′), 27.0 (Me′), 67.7 (C-5′), 68.9 (C-5′′),73.4 (C-6′), 74.9 (C-3′), 75.0 (C-3′′), 76.9 (C-6′′), 80.6 (C-4′′), 80.7 (C-4′), 86.01 (C-2′′), 86.04 (C-2′), 105.82 (C-1′′), 105.84 (C-1′), 108.6 (C-8a), 109.5 (C-2), 109.6 (C-4a), 111.6 (CMe₂′), 111.8 (CMe₂′′), 135.8 (C-6), 157.3 (C-7), 160.8 (C-3), 164.4 (C-8), 169.9 (C-5), 171.5 (C-4), 178.6 (C-1).

HRESIMS: m/z [M−H]⁻ calcd for C₃₀H₃₈O₁₆: 653.7087; found: 653.7085.

ABTS^·+ Scavenging Assay

To measure the antioxidant activity of compounds the ABTS^·+ assay was adapted from a published method. ¹¹ Briefly, a stock solution of ABTS^·+ radical cation was prepared by dissolving ABTS (7 mM, 4.713 mL in deionized water) with potassium persulfate (60 mM, 0.2 mL; 2.45 mM final concentration). The reaction mixture was left to stand at room temperature overnight (16 h) in the dark. The intensely colored ABTS^·+ radical cation was diluted with EtOH to an absorbance of 0.70 (±0.02) at 734 nm. To 20 µL of EtOH solutions of the test compound at different concentrations (0-100 µM final concentrations) 1.98 mL of the diluted ABTS^·+ solution was added and the absorbance was measured at 734 nm after 6 min. Inhibition was calculated as follows: Inhibition (%) = 100 − (A_reaction × 100)/ A_control, where A_reaction is the absorbance of the reaction mixture, and A_control is the absorbance of the ABTS^·+ solution. EtOH was used as a blank. Trolox was used as a positive control standard. The inhibition was plotted as a function of concentrations of antioxidant compounds and a concentration that caused a 50% reduction of the absorbance (EC₅₀) was found. Trolox equivalents was calculated as follows: (TE) = EC₅₀ (Trolox)/EC₅₀ (compound).