Sage Journals: Discover world-class research

Abstract

Indole-3-acetonitrile-6-O-β-d-glucopyranoside 1 is a simple alkaloid with anti-influenza A virus activity extracted from Radix isatidis. Herein, a concise and practical total synthetic route of 1 was presented, starting from 6-benzyloxyindole on a multigram scale. The pivotal reaction sequence involved the Mannich reaction, the fluoride ion-induced elimination-addition reaction, and phase-transfer-catalyzed glycosylation reaction as key steps. In addition, compounds 1 and two modified derivatives 8 and 16 were tested for anti-influenza efficacy and cytotoxicity in vitro performed on Madin-Darby canine kidney cells. The results revealed that the compounds exerted antiviral activity against the influenza A virus to a certain extent and displayed no cytotoxicity. These findings could contribute to the development of traditional Chinese medicine R. isatidis for treating the influenza virus.

Keywords

3-indoleacetonitrile synthesis anti-influenza activity cytotoxicity

“Ban langen” (Radix isatidis) is an official crude drug being recorded in Chinese Pharmacopeia for many times.¹ It has been also used in combination with other botanicals in traditional Chinese medicine and Ayurveda for the treatment of various diseases since ancient times, especially for preventing and treating influenza.² The chemical and biological diversity has been widely investigated and assessed with ethanol extracts or water decoctions of the roots and leaves of Isatis indigotica.^3,4 It contains various constituents with different structural features, such as alkaloids, lignans, indirubin, clemastanin B, ceramides, flavonoids, epigoitrin, and polysaccharides, etc.^5
-7 Meanwhile, the extracts of R. isatidis display extensive biological activity, for instance, antiviral,^8

-11 anti-inflammatory,¹² antipyretic,¹³ neuroprotective,¹⁴ antibiotic,¹⁵ and antidiabetic activity.¹⁶ However, it is difficult to fully elucidate which chemical component causes the corresponding pharmacological efficacy in the use of traditional Chinese medicine. The diversity of the chemical constituents from R. isatidis and the ambiguity of real effective ingredients provide a good opportunity for further exploration.

The indole scaffold, as a privileged structure, is considered as one of the most promising heterocycles in drug discovery and embedded within the backbones of numerous pharmaceuticals and biologically important natural alkaloids.¹⁷ Indole derivatives possess versatile biological activities, including antiviral, anti-inflammatory, antibacterial, antimalarial, anticonvulsant. and anticancer, etc.^18

-21 Due to their impressive natural structures and significant biological profiles, indole derivatives constantly call for the development of methods for their innovative synthetic strategies. Since the first synthesis of indole was completed by Adolf von Baeyer in 1866,²² many efficient synthetic approaches have been successfully discovered and applied for the construction of indole and its complex derivatives, such as classical name reactions represented by Fischer indole synthesis,²³ organic electrosynthesis,²⁴ catalytic asymmetric dearomatization reactions,²⁵ organocatalytic asymmetric synthesis,²⁶ transition-metal-catalyzed C-H functionalization,²⁷ aerobic oxidative functionalization,²⁸ and photoredox catalysis.²⁹ Nevertheless, novel strategies for the synthesis of indole derivatives are still urgently needed.

In order to clarify the antiviral active constituents of R. isatidis, numerous researchers had isolated single compound and conducted its bioactivity assay by the extracts of R. isatidis (organic solvents or water).^{7

-10,30
-32} Various indole alkaloids were isolated from R. isatidis and considered as the main active constituents in free and glycosidic forms.^5,33 Among them, indole-3-acetonitrile-6-O-β-d-glucopyranoside 1 was an alkaloid with a relatively simple skeleton.^34,35 Recent studies demonstrated that the single compound could inhibit influenza A virus (H3N2) activity in vitro and significantly cure lung tissue lesions and pneumonia induced by influenza virus in vivo.³⁶ Meanwhile, this compound also exerted remarkable inhibitory activity on lipopolysaccharide-induced nitric oxide production in macrophages with half-maximal inhibitory concentration (IC₅₀) of 5.87 µM to reduce the development of inflammation.³⁵

At present, it is time-consuming, laborious, and inefficient to extract enough amount of this indole alkaloid 1 from crude indigowoad root.³⁷ The scarcity of material has limited further pharmacological activity evaluation and, more generally, has hampered efforts to obtain potentially superior derivatives. Therefore, in this study, we designed and accomplished a novel synthetic route to meet the supply of this alkaloid for further bioactivity assay and mechanism exploration.

Results and Discussion

Our retrosynthetic analysis is illustrated in Figure 1. We envisioned the key glycosylation formation via nucleophilic attack of 6-hydroxyindole-3-acetonitrile 3 to a glycosylating reagent 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide 2 to acquire the target molecule 1 after hydrolysis of the glycosylated product. Compound 3 could be achieved from the deprotection of the benzyl group via Pd-catalyzed hydrogenation. The required precursor 4 should be ingeniously obtained from the nucleophilic displacement of the N,N-dimethylamino group of Mannich base 5 via the fluoride ion-induced elimination-addition reaction. Compound 5 might be easily accessible from commercially available 6-benzyloxyindole 6, formaldehyde, and dimethylamine via Mannich reaction.

Figure 1

Retrosynthetic strategy for synthesizing 3-indoleacetonitrile glucoside 1.

Preparation of the target compound 1 commenced with the synthesis of the crucial intermediate 3 from commercially available 6-benzyloxyindole 5 as shown in Figure 2. Initial step to introduce the C-3 side chain of 6-benzyloxyindole involved a sequence of a Mannich reaction, quaternization of the Mannich base 5, substitution of the trimethylamine moiety with a cyanide source to yield the cyanomethyl group. Several conditions (Table 1) were tried to optimize this transformation.

Figure 2

Synthetic route of 3-indoleacetonitrile glucoside 1.

Table 1

Optimization of Transforming the Trimethylamine Moiety to the Cyano Group.

Entry	Cyanide source	Solvent	Temperature (°C)	Time (h)	Yield (%)
1	NaCN	MeOH/H₂O = 5:1	100	16	65
2	KCN	MeOH/H₂O = 5:1	100	16	70
3	TMSCN/TBAF	Toluene/CH₂Cl₂ = 2:1	25	1	65
4	TMSCN/TBAF	Toluene	25	1	60
5	TMSCN/TBAF	CH₂Cl₂	25	1	68
6	TMSCN/TBAF	THF	25	1	76
7	TMSCN/TBAF	THF	25	2	92
8	TMSCN/KF	THF	25	2	85
9	TMSCN/NaF	THF	25	2	80

Abbreviations: CH₂Cl₂, dichloromethane; H₂O, water; KCN, potassium cyanide; KF, potassium fluoride; MeOH, methanol; NaCN, sodium cyanide; NaF, sodium fluoride; TBAF, tetrabutylammonium fluoride; THF, tetrahydrofuran; TMSCN, trimethylsilyl cyanide.

Initial scouting of cyanomethylation was carried out by using sodium cyanide or potassium cyanide as a cyanide source by examining an array of conditions reported in the literature (Table 1, entry 1 and 2).³⁸ Unfortunately, only 65%-70% yield of the product 4 was obtained, and the common cyanides were highly toxic to exhaustedly manipulate. Therefore, trimethylsilyl cyanide (TMSCN), which was of lower toxicity and easy to handle,³⁹ turned out to be a better cyanide source affording 4 in 65% yield; the yield was not significantly improved with the prolongation of time (Table 1, entry 3). After screening several solvents (Table 1, entry 3-6), tetrahydrofuran (THF) was found to be the most efficient, and other solvents afforded low yields with the incomplete conversion. To our delight, we found that the yield increased to 92% with elongated reaction time (Table 1, entry 7). Replacing tetrabutylammonium fluoride (TBAF) with other fluorides such as potassium fluoride (KF) or sodium fluoride (NaF) did not improve the reaction (Table 1, entry 8 and 9). Consequently, the optimum reaction conditions were determined to be stirring quaternary ammonium salt in the presence of TMSCN/(n-Bu)₄NF in THF at room temperature for 2 hours. Most significantly, the reaction could be reliably scaled up to multigram scales. Namely, aminomethylation derivative 6 was obtained by treatment 7 with dimethylamine/aqueous formaldehyde in 1,4-dioxane at 25 °C in high yield without further purification. The resulting Mannich base 6 was reacted with methyl iodide in dichloromethane (CH₂Cl₂)/toluene and subsequently subjected to nucleophilic substitution using TMSCN/(n-Bu)₄NF to afford the nitrile 4. Compound 4 was selectively reductive removal of O-benzyl to provide the 6-hydroxyindole-3-acetonitrile 3 using palladium on carbon (Pd/C) as a catalyst.

It was another difficulty for us to perform the crucial glycosylation reaction, although various methods were developed for the construction of O-linked glucosides.⁴⁰ At first, Koenigs-Knorr glycosidation reaction⁴¹ was conducted by treating the commercially available glycosylating reagent tetra-O-acetyl-α-d-glucopyranosyl bromide 2 with phenol 3 in the presence of silver (I) oxide, but it did not work well (Table 2, entry 1). Under the reaction conditions using lithium hydroxide (LiOH)/dimethylformamide (DMF) system reported by Somei and co-workers, S_N2 reaction took place in 71% yield (Table 2, entry 2).⁴² Finally, the phase-transfer catalyzed glycosylation was practical and efficient. Namely, glucoside 1 was prepared by the phase-transfer-catalyzed (benzyltributylammonium chloride) glycosylation of 3 with 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide 2 in a biphasic system (chloroform [CHCl₃]/potassium carbonate [K₂CO₃] solution),⁴³ and subsequently subjected to hydrolysis using LiOH in THF/water (H₂O) (Table 2, entry 4 and 5).

Table 2

Optimizing the Condition of Preparation of O-Linked Glucoside 8.

Entry	Condition	Time(h)	Temperature(°C)	Yield (%)
1	Ag₂O, CH₃CN	6	35	58
3	LiOH, DMF	4	25	71
4	K₂CO₃/TBAB/CHCl₃/H₂O	48	25	78
5	K₂CO₃/TBAB/CHCl₃/H₂O	48	25	89

Abbreviations: Ag₂O, silver (I) oxide; CHCl₃, chloroform; CH₃CN, acetonitrile; DMF, dimethylformamide; H₂O, water; K₂CO₃, potassium carbonate; LiOH, lithium hydroxide; TBAB, tetrabutylammonium bromide.

Having established an efficient method to synthesize the 3-indoleacetonitrile glucoside as described above, we also had access to the 3-indoleacetonitrile 4-position derivative 15 (Figure 3, Cappariloside A), with an overall yield of 49.2% versus 41% previously reported by Masanori Somei.⁴² The pharmacological activity of cappariloside A lay in broad-spectrum antiviral efficacy and impairing the upregulations of proinflammatory factors in host cells induced by the influenza virus.⁴⁴ In addition, the arylation of compound 8 afforded the phenyl-substituted analog 16 through Chan-Lam-Evans coupling reaction⁴⁵ and hydrolysis reaction (Figure 4).

Figure 3

Synthetic route of cappariloside A.

Figure 4

N-arylation of 3-indoleacetonitrile glucoside 1.

The synthesized compounds 1, 8, and 16 were selectively evaluated for the anti-influenza virus effects against 2 influenza virus subtypes, namely, A/HK/68 (H3N2) and A/PR/8/34 (H1N1), on Madin-Darby canine kidney (MDCK) cells utilizing the cytopathic effect (CPE) assay. And cytotoxic effects were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In addition, oseltamivir carboxylate was used as a positive control in parallel. The inhibitory results were expressed as a half-maximal effective concentration (EC₅₀) and half-maximal toxic concentration (TC₅₀) summarized in Table 3.

Table 3

Anti-Influenza Virus Efficacy of the Compounds 1, 8, and 16 In Vitro.^a

Compound	Antiviral EC₅₀ ^b (μg/mL)		Cytotoxicity(TC₀ ^c, μg/mL)
Compound	A/HK/68	A/PR/8/34(H1N1)	Cytotoxicity(TC₀ ^c, μg/mL)
1	106.9 ± 6.1	123.8 ± 12.2	1097.3 ± 40.9
8	223.0 ± 15.1	243.0 ± 10.3	1892.1 ± 70.2
16	142.7 ± 20.5	155.0 ± 15.7	2234.9 ± 82.3
Cappariloside A⁴⁴	362.2 ± 9.5	288.0 ± 10.5	1256.2 ± 49.1
OSC	0.054 ± 0.01	0.051 ± 0.01	1759.2 ± 70.6

Abbreviations: CPE, cytopathic effect; EC₅₀, half-maximal effective concentration; OSC, oseltamivir carboxylate; TC₀, 50% cytotoxic concentration.

^aThe effect of compounds was determined by measuring the survival of Madin-Darby canine kidney cells infected with the influenza A virus using the CPE assay.

^bEC₅₀ represents 50% effective concentration.

^cTC₀ represents the maximal nontoxic concentration, determined by CPE assay.

Compound 1 possessed inhibitory activity against H3N2 and H1N1 with an EC₅₀ value of 106.9 ± 6.1 µg/mL and 123.8 ± 12.2 µg/mL, respectively. Compared with acetylated product 8, it seemed that the free sugar ring was of importance to maintain anti-influenza activity. Meanwhile, N-arylindole derivative 16 exhibited weaker antiviral efficiency. It should be noted that the 3 compounds also possessed no cytotoxicity toward the MDCK cells.

Conclusions

In conclusion, we accomplished a facile and efficient synthesis of indole-3-acetonitrile-6-O-β-d-glucopyranoside 1 in 5 steps with 56.5% overall yield from the readily available 6-benzyloxyindole. And the phenyl-substituted analog 16 was obtained via an one-step Chan-Lam-Evans coupling reaction. Moreover, compounds 1, 8, and 16 were evaluated for anti-influenza activity and their cytotoxicity in vitro employing the CPE and MTT assay, respectively. The results indicated that simple structural modification of compound 1 could not increase antiviral effectiveness, for example, through acetylation of the glucose unit or N-arylation of the indole-3-acetonitrile moiety. At last, the tested compounds showed no cytotoxicity. This study had laid a solid foundation for further access to various 3-indoleacetonitrile glycosides and structure-activity relationship research.

Experimental

General

The chemicals and all solvents used were of analytical grade without further purification unless otherwise noted. Reactions were monitored by thin-layer chromatography using Qing Dao Hai Yang GF254 silica gel plates (5 × 10 cm); zones were detected visually under ultraviolet (UV) irradiation (254 nm). ¹H NMR spectra and ¹³C NMR were recorded on a Bruker NMR AVANCE 400 (400 MHz) or a Bruker NMR AVANCE 500 (500 MHz), using deuterated chloroform (CDCl₃) or dimethylsulfoxide (DMSO)-d ₆ as a solvent and tetramethylsilane as an internal standard. Low-resolution mass spectra were recorded on Agilent 6460 triple-quadrupole mass spectrometer with electrospray ionization (ESI). High-resolution mass spectra (HR-MS) were performed on an AB SCIEX TripleTOF 4600 instrument. Melting points (MPs) were determined using a WRS-1C melting point apparatus (Shanghai INESA Physico optical instrument Co. Ltd.) and were uncorrected.

Synthesis of Indole-3-acetonitrile-6-O-β-d-glucopyranoside (Compound 1)

Synthesis of 1-(6-(benzyloxy)-1H-indol-3-yl)-N,N-dimethylmethanamine (Compound 6 )

A 500 mL round-bottomed flask was charged with 37% formaldehyde solution (5.32 g, 4.92 mL, 65.59 mmol), dimethylamine 40% w/w in H₂O (9.5 g, 84.42 mmol), acetic acid (100 mL), and 1,4-dioxane (100 mL). A solution of 6-benzyloxyindole (14.5 g, 64.94 mmol) in 1,4-dioxane (100 mL) was added dropwise while keeping the internal temperature between 0 °C and 5 °C. The reaction mixture was then stirred for 2 hours and kept at room temperature for 36 hours. The mixture was diluted with H₂O (500 mL) and adjusted to pH 8-9 with aqueous sodium hydroxide (NaOH) solution (10%). The resulting precipitate was collected by filtration, and the filter cake was washed with ethyl acetate several times, dried in vacuum to afford the compound 6 (15.6 g, 85.7%) as a white solid. ¹H NMR (400 MHz, DMSO-d ₆): δ 10.68 (s, 1 H), 7.46-7.40 (m, 5 H), 7.32 (d, J = 5.2 Hz, 1 H), 7.04 (s, 1 H), 6.91 (s, 1 H), 6.71 (d, J = 6.8 Hz, 1 H), 5.10 (s, 2 H), 3.46 (s, 2 H), 2.12 (s, 6 H) (supplemental Figure S1).

Synthesis of 2-(6-(benzyloxy)-1H-indol-3-yl) acetonitrile (Compound 4 )

Methyl iodide (6.68 g, 2.93 ml, 47.08 mmol) was added to a suspension of compound 6 (6 g, 21.4 mmol) in a mixture of CH₂Cl₂/toluene (80/160 mL) under argon atmosphere. The mixture was vigorously stirred at room temperature for 16 hours. The reaction mixture was concentrated under reduced pressure, and THF (120 mL) was added to the residue. TMSCN (3.18 g, 32.1 mmol) and TBAF (1 M in THF, 85 mL, 85 mmol) were added dropwise to the resulted suspension successively. The reaction mixture was stirred at 25 °C for 2 hours and concentrated under reduced pressure. The residue was dissolved in ethyl acetate (180 mL). The mixture was washed with saturated sodium bicarbonate (NaHCO) solution (100 mL), brine (100 mL), dried over anhydrous magnesium sulfate (MgSO₄), and evaporated. The residue was purified by flash chromatography (petroleum ether/ethyl acetate, 5:1-1:1) to give the expected product 4 (5.16 g, 92%) as a white solid. ¹H NMR (400 MHz, DMSO-d ₆): δ 10.91 (s, 1 H), 7.52-7.36 (m, 5 H), 7.35-7.28 (m, 1 H), 7.19 (d, J = 2.2 Hz, 1 H), 6.96 (d, J = 2.2 Hz, 1 H), 6.80 (dd, J = 8.5, 2.2 Hz, 1 H), 5.12 (s, 2 H), 3.99 (s, 2 H) (supplemental Figure S2). Liquid chromatography (LC)/MS (ESI) m/z: [M + H]⁺ calcd for C₁₇H₁₄N₂O, 263.31; found, 263.3.

Synthesis of 2-(6-hydroxy-1H-indol-3-yl) acetonitrile (Compound 3 )

A 250 mL single-necked round-bottomed flask was charged with compound 4 (10 g, 38.1 mmol), 10% Pd/C (4.2 g), and methanol (100 mL), evacuated and refilled with hydrogen 3 times. The solution was vigorously stirred at room temperature for 10 hours. After evaporation of the solvent under reduced pressure, the residue was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (4:1) to give compound 3 as a light yellow solid (5.97 g, 91%). ¹H NMR (400 MHz, DMSO-d ₆): δ 10.69 (s, 1 H), 8.99 (s, 1 H), 7.33 (d, J = 8.5 Hz, 1 H), 7.09 (d, J = 2.2 Hz, 1 H), 6.74 (d, J = 2.2 Hz, 1 H), 6.58 (dd, J = 8.5, 2.2 Hz, 1 H), 3.94 (s, 2 H) (supplemental Figure S3). LC/MS (ESI) m/z: [M + H]⁺ calcd for C₁₀H₈N₂O, 173.18; found, 173.1.

Synthesis of (2R,3R,4S,5R,6S)-2-(acetoxymethyl)-6-((3-(cyanomethyl)-1H-indol-6-yl)oxy) tetrahydro-2H-pyran-3,4,5-triyl triacetate (Compound 8 )

A 500 mL round-bottomed flask was charged with compound 3 (5.68g, 33 mmol), 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide 2 (27.1, 66 mmol), CHCl₃ (120 mL), anhydrous K₂CO₃ (22.8 g, 165 mmol), and distilled water (12 mL). Benzyltributylammonium chloride (2.06 g, 6.6 mmol) was added in portions, and the mixture was stirred at room temperature for 48 hours. After removal of solvent by evaporation, the residue was dissolved in ethyl acetate and washed with H₂O and brine, dried over anhydrous MgSO₄, and evaporated. The crude product was purified by column chromatography on silica gel (petroleum ether/ethyl acetate, 1:1) to yield the title compound 8 (14.75g, 89 %) as a light yellow solid.

MP: 289.8-291.2 °C.

¹H NMR (500 MHz, DMSO-d ₆): δ 11.04 (s, 1 H), 7.50 (d, J = 8.6 Hz, 1 H), 7.28 (s, 1 H), 7.02 (s, 1 H), 6.77 (dd, J = 8.6, 2.2 Hz, 1 H), 5.49 (d, J = 8.0 Hz, 1 H), 5.44 (t, J = 9.6 Hz, 1 H), 5.06 (dd, J = 9.6, 8.0 Hz, 1 H), 5.00 (t, J = 9.6 Hz, 1 H), 4.25-4.19 (m, 2 H), 4.09-4.03 (m, 1 H), 4.01 (s, 2 H), 2.04 (s, 3 H), 2.03 (s, 3 H), 2.01 (s, 3 H), 1.97 (s, 3 H) (supplemental Figure S4).

¹³C NMR (125 MHz, DMSO-d ₆): δ 170.4, 170.0, 169.7, 169.5, 153.3, 136.9, 124.3, 122.7, 119.8, 119.1, 111.0, 104.2, 100.0, 99.1, 72.4, 71.4, 71.1, 68.6, 62.1, 20.9, 20.8, 20.7, 13.7 (supplemental Figure S5).

HRMS (ESI): [M + Na]⁺ calcd for C₂₄H₂₇N₂O ₁₀ Na, 525.1480.1660; found, 525.1485 (supplemental Figure S16).

Deprotection of Compound 8 (Compound 1 )

LiOH·H₂O (5.41 g, 129 mmol) was slowly added to a solution of ester (10.8 g, 215 mmol) in THF-MeOH-H₂O (180 mL, V _THF/V _MeOH/V _water = 3:2:1) at 0 °C in portions. The reaction mixture was stirred at room temperature for 2 hours. The solvent was removed in vacuo and neutralized by 1 M hydrochloric acid (HCl) after cooling to 0 °C. The mixture was diluted with H₂O and extracted with ethyl acetate (150 mL × 3). The combined organic extract was washed with brine, dried over anhydrous MgSO₄, and concentrated. Purification by silica gel column chromatography (CH₂Cl₂/MeOH, 20:1-10:1) afforded the target compound 1 (6.36 g, 88.5%) as white foam powder.

MP: 240.6 °C-242.1 °C.

¹³C NMR (125 MHz, DMSO-d ₆): δ 154.0, 136.7, 123.3, 121.5, 119.5, 118.3, 110.8, 103.6, 101.9, 99.0, 77.0, 76.6, 73.4, 69.8, 60.8, 13.3 (supplemental Figure S7).

HRMS (ESI): m/z [M + Na]⁺ calcd for C₁₆H₁₈N₂O₆Na: 357.1057; found: 357.1058 (supplemental Figure S17).

Synthesis of 2-(4-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl)oxy)-1H-indol-3-yl) acetonitrile 15 (cappariloside A)

A 500 mL round-bottomed flask was charged with 37% formaldehyde solution (3.83 mL, 50.5 mmol), dimethylamine 40% w/w in H₂O (7.32 g, 65 mmol), acetic acid (100 mL), and 1,4-dioxane (100 mL) at 0 °C. A solution of 6-benzyloxyindole (11.16 g, 50 mmol) in 1,4-dioxane (100 mL) was added dropwise while keeping the internal temperature between 0 °C and 5 °C. The reaction mixture was then stirred for 2 hours and kept at room temperature for 36 hours. The mixture was diluted with H₂O (500 mL) and adjusted to pH 8-9 with aqueous NaOH solution (10%). The resulting precipitate was collected by filtration, and the filter cake was washed with diethyl ether, dried in vacuum to afford the product 10 (13.2 g, 94%) as a white solid. ¹H NMR (400 MHz, DMSO-d ₆) δ 10.91 (s, 1 H), 10.91 (s, 1 H), 7.56 (d, J = 7.2 Hz, 2 H), 7.41 (t, J = 7.2 Hz, 2 H), 7.34 (t, J = 7.2 Hz, 1 H), 7.04 (s, 1 H), 6.95 (d, J = 3.2 Hz, 2 H), 6.55 (s, 1 H), 5.15 (s, 2 H), 3.63 (s, 2 H), 2.07 (s, 6 H) (supplemental Figure S8).

Methyl iodide (8.02 g, 3.52 mL, 56.5 mmol) was added to a suspension of compound 10 (6.61 g, 28.25 mmol) in a mixture of CH₂Cl₂/toluene (80/160 mL) under argon atmosphere. The reaction mixture was vigorously stirred at room temperature overnight. The reaction mixture was concentrated in vacuo, and THF (120 mL) was added to the residue. TMSCN (4.2 g, 4.91 mL, 42.37 mmol) and TBAF (1 M in THF, 85 mL, 85 mmol) was added dropwise to the reaction suspension successively. The reaction mixture was stirred at 25 °C for 2 hours and concentrated under reduced pressure. The residue was dissolved in ethyl acetate (160 mL). The mixture was washed with saturated NaHCO₃ solution (100 mL), brine (100 mL), dried over anhydrous MgSO₄, and evaporated. The residue was purified by flash chromatography (petroleum ether/ethyl acetate, 2:1) to give the desired product 12 (6.8 g, 91.8%) as a colorless viscous liquid. ¹H NMR (400 MHz, DMSO-d ₆) δ 11.11 (s, 1 H), 7.57 (d, J = 7.6 Hz, 2 H), 7.41 (t, J = 7.2 Hz, 2 H), 7.32 (t, J = 7.2 Hz, 1 H), 7.23 (s, 1 H), 7.05-6.93 (m, 2 H), 6.65-6.52 (m, 1 H), 5.21 (s, 2 H), 4.05 (s, 2 H) (supplemental Figure S9).

A 100 mL single-necked round-bottomed flask was charged with compound 12 (2.62 g, 10 mmol), 10% Pd/C (1 g), and methanol (50 mL), and evacuated and refilled with hydrogen 3 times. The solution was vigorously stirred at room temperature for 4 hours. After evaporation of the solvent under reduced pressure, the residue was purified by silica gel chromatography (petroleum ether/ethyl acetate, 1:1) to give the product 13 (1.36 g, 80%) as a gray solid. ¹H NMR (400 MHz, DMSO-d ₆) δ 10.91 (s, 1 H), 9.52 (s, 1 H), 7.13 (s, 1 H), 6.87(t, J = 7.6 Hz, 1 H), 6.81 (d, J = 7.0 Hz, 1 H), 6.35 (d, J = 7.4 Hz, 1 H), 4.04 (s, 2 H) (supplemental Figure S10).

A mixture of compound 13 (1.72 g, 10 mol), 2,3,4,6-tetra-O-acetyl-α-d-gluco-pyranosyl bromide 2 (8.2 g, 20 mmol), benzyltributylammonium chloride (0.6 g, 2 mmol), anhydrous K₂CO₃ (6.9 g, 50 mmol), H₂O (2 mL), and CHCl₃ (20 mL) was stirred at room temperature for 48 hours. After removal of the solvent by evaporation, the residue was dissolved in ethyl acetate and washed with H₂O and brine, dried over anhydrous sodium sulfate (Na₂SO₄), and evaporated. The crude product was purified by column chromatography on silica gel eluted with petroleum ether/ethyl acetate (1:1) to yield the title compound 14 as a white solid (4.3 g, 83%). ¹H NMR (400 MHz, DMSO-d ₆) δ 11.20 (s, 1 H), 7.26 (d, J = 2.3 Hz, 1 H), 7.09-7.01 (m, 7.6 Hz, 2 H), 6.64 (d, J = 6.8 Hz, 1 H), 5.76 (d, J = 8.0 Hz, 1 H), 5.47 (t, J = 9.6 Hz, 1 H), 5.22 (dd, J = 9.6, 8.0 Hz, 1 H), 5.02 (t, J = 9.6 Hz, 1 H), 4.38–4.25 (m, 1 H), 4.21 (dd, J = 12.4, 5.7 Hz, 1 H), 4.06 (dd, J = 12.4, 2.3 Hz, 1 H), 3.92 (s, 2 H), 2.04 (s, 3 H), 2.02 (s, 3 H), 1.99 (s, 3 H), 1.98 (s, 3 H) (supplemental Figure S11).

LiOH·H₂O (503.5 mg, 12 mmol) was slowly added to a solution of compound 14 (1 g, 2 mmol) in THF-MeOH-H₂O (12 mL, V _THF/V _MeOH/V _water = 3:2:1) at 0 °C in portions. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was evaporated in vacuo and neutralized by 1 M HCl after cooling to 0 °C. The mixture was diluted with H₂O and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and evaporated. The residue was purified by flash chromatography (CH₂Cl₂/MeOH 20:1-10:1) to afford cappariloside A (574 mg, 85.9%) as a white solid. The NMR spectrum coincided with that the previously reported in the literature.⁴²

MP: 234.8 °C-236.7 °C.

¹H NMR (500 MHz, DMSO-d ₆) δ 11.08 (s, 1 H), 7.20 (d, J = 2.4 Hz, 1 H), 7.04-6.97 (m, 2 H), 6.69 (dd, J = 7.1, 1.2 Hz, 1 H), 5.30 (d, J = 5.6 Hz, 1 H), 5.10 (d, J = 4.9 Hz, 1 H), 5.02 (d, J = 5.4 Hz, 1 H), 4.89 (d, J = 7.6 Hz, 1 H), 4.58 (t, J = 5.7 Hz, 1 H), 4.23-4.12 (m, 2 H), 3.73 (ddd, J = 11.7, 5.3, 2.0 Hz, 1 H), 3.50 (quin, J = 6.0 Hz, 1 H), 3.41–3.27 (m, 3 H), 3.21 (td, J = 9.2, 5.4 Hz, 1 H) (supplemental Figure S12).

¹³C NMR (125 MHz, DMSO-d ₆) δ 151.8, 138.0, 122.8, 122.5, 120.1, 116.7, 106.0, 103.8, 103.4, 101.3, 77.1, 76.7, 73.5, 69.8, 60.8, 14.9 (supplemental Figure S13).

Synthesis of 2-(1-phenyl-6-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl)oxy)-1H-indol-3-yl)acetonitrile (Compound 16 )

A suspension of phenylboronic acid (73.1 mg, 0.6 mmol, 2 equiv.), copper (II) acetate (109 mg, 0.6 mmol, 2 equiv.), pyridine (95 mg, 1.2 mmol, 4 equiv.), and 4 Å molecular sieves (200 mg) in CH₂Cl₂ (5 mL) was stirred for 5 minutes at room temperature. To this stirring suspension was added compound 8 (150.7 mg, 0.3 mmol, 1 equiv.). The mixture was stirred overnight at room temperature. The reaction mixture was diluted with ethyl acetate (3 mL) and filtered through a plug of silica gel eluting with additional ethyl acetate (20 mL). The filtrate was concentrated, and the resulting residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate, 5:1-1:1) to provide the ester (120.6 mg, 69.5%) as a white solid.

LiOH·H₂O (50.4 mg, 1.2 mmol, 6 equiv.) was slowly added to a solution of the resulting ester (120.6 mg, 0.21 mmol, 1 equiv.) in THF/MeOH/H₂O (3 mL, V _THF/V _MeOH/V _water = 3:2:1) at 0 °C. The reaction mixture was stirred at room temperature for 2 hours. The solvent was removed in vacuo and neutralized by 1 M HCl after cooling to 0 °C. The mixture was diluted with water and extracted with ethyl acetate (5 mL × 3). The combined organic layers were washed with brine, dried over anhydrous MgSO₄, and concentrated. The residue was purified by silica gel column chromatography (CH₂Cl₂/MeOH, 40:1-20:1) afforded the target compound 9 (76.9 mg, 89.3%) as a white powder.

MP: 256.4 °C-258.2 °C.

¹H NMR (400 MHz, DMSO-d ₆): δ 7.67-7.52 (m, 6 H), 7.39 (t, J = 6 Hz, 1 H), 7.28 (s, 1 H), 6.99 (d, J = 8.6 Hz, 1 H), 5.29 (d, J = 2.4 Hz, 1 H), 5.06 (s, 1 H), 4.99 (d, J = 4.4 Hz, 1 H), 4.79 (d, J = 6.4 Hz, 1 H), 4.62 (t, J = 5.6 Hz, 1 H), 4.11 (s, 2 H), 3.70 (dd, J = 10.8, 4.4 Hz, 1 H), 3.50-3.45 (m, 1 H), 3.30–3.21 (m, 3 H), 3.17–3.12 (m, 1 H) (supplemental Figure S14).

¹³C NMR (125 MHz, CDCl₃): δ 154.9, 138.6, 135. 8, 129.9, 126.4, 126.3, 123.5, 122.6, 119.3, 119.0, 111.9, 106.4, 101.7, 98.2, 77.1, 76.6, 73.3, 69.8, 60.8, 13.2 (supplemental Figure S15).

HRMS (ESI): m/z [M + K]⁺ calcd for C₂₂H₂₂N₂KO₆: 449.1109; found: 449.1112 (supplemental Figure S18).

CPE Assay Protocol

In CPE assay, MDCK cells were grown to a confluent monolayer in a 96-well culture plate at a concentration of 5 × 10⁴ cells/well for 24 hours. The medium was removed, and the cells were rinsed twice. For the anti-influenza activity assay, an infectious virus at 100 TCID₅₀ was inoculated into the MDCK cells, which were then incubated for 2 hours at 37 °C in 5% carbon dioxide (CO₂). The virus supernatant was then removed, followed by the addition of serial 2-fold dilutions of antiviral compounds in Dulbecco’s modified Eagle medium containing 1.5 µg/mL trypsin. After being incubated at 34 °C in 5% CO₂ for 48 hours, the infected cells displayed 100% CPE under the microscope, and the CPE percentages in the antiviral compound-treated groups were recorded. The EC₅₀ of virus-induced CPE was detected by the Reed-Muench method. All data are obtained for at least 3 independent experiments.

Cytotoxicity Assay

Cytotoxicity of compounds was assessed by MTT assay. MDCK cells on 96-well plates were washed with sterile phosphate-buffered saline. Then 100 µL of compounds in serum-free MEM at 2-fold dilution was added to the cells, and the cells were incubated in CO₂ at 37 °C. Cell controls were also performed. After 48 hours, 5 mg/mL of fresh MTT was added to each well, and the plates were incubated at 37 °C for 4 hours. Afterward, the medium was removed and formazan crystal was dissolved in DMSO (100 µL per well). Absorbance of each well at 490 nm was read by a CLARIOstar multi-mode microplate reader (BMG Labtech, Germany). The cell viability (%) = OD of compound well/average OD of control wells. The TC₅₀ of each compound was obtained using a nonlinear regression model in GraphPad Prism 6.

Supplemental Material

Supplementary file 1 - Supplemental material for Multigram Scale Synthesis and Anti-Influenza Activity of 3-Indoleacetonitrile Glucosides

Supplemental material, Supplementary file 1, for Multigram Scale Synthesis and Anti-Influenza Activity of 3-Indoleacetonitrile Glucosides by Jianghong Dong, Fengmei Yan, Shuang Li, Zewen Li and Yuanhang Qin in Natural Product Communications

Footnotes

Acknowledgments

We thank Professor Yang Zifeng’s Group in State Key Laboratory of Respiratory Disease of China for the help in bioactivity assay.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this work was supported by the Key Science and Technology Program of Henan Province, China (No.182102310258).

ORCID iD

Jianghong Dong

Supplemental Material

Supplemental material for this article is available online.

References

Commission CP . Pharmacopoeia of People’s Republic of China. part 1. China Medical Science Press; 2015.

Zhou

Zhang

X-Y

. Research progress of Chinese herbal medicine Radix isatidis (banlangen). Am J Chin Med. 2013;41(4):743-764.doi:10.1142/S0192415X1350050X

23895149

Jian

BO.

Hui

Jihua

et al. Screening of antiviral active fractions of Radix isatidis . Traditional Chinese Drug Research and Clinical Plarmacology. 2007;18:12-14.doi:10.19378/j.issn.1003-9783.2007.01.004

L-W.

Chen

J-W.

Wang

T-L.

Wei

Yang

J-Y

. Chemical constituents from water extract of Radix isatidis . Yao Xue Xue Bao. 2006;41(12):1193-1196.17290620

Chen

Gan

Lin

et al. Alkaloids from the root of Isatis indigotica . J Nat Prod. 2012;75(6):1167-1176.doi:10.1021/np3002833

http://www.ncbi.nlm.nih.gov/pubmed/22694318

Yang

Wang

Zheng

et al. Antiviral activity of Isatis indigotica root-derived clemastanin B against human and avian influenza A and B viruses in vitro. Int J Mol Med. 2013;31(4):867-873.doi:10.3892/ijmm.2013.1274

http://www.ncbi.nlm.nih.gov/pubmed/23403777

Luo

Liu

L-F.

Wang

X-H

et al. Epigoitrin, an alkaloid from Isatis indigotica, reduces H1N1 infection in stress-induced susceptible model in vivo and in vitro . Front Pharmacol. 2019;10:78. doi:10.3389/fphar.2019.00078

http://www.ncbi.nlm.nih.gov/pubmed/30792656

Meng

Guo

Liu

et al. Indole alkaloid sulfonic acids from an aqueous extract of Isatis indigotica roots and their antiviral activity. Acta Pharm Sin B. 2017;7(3):334-341.doi:10.1016/j.apsb.2017.04.003

http://www.ncbi.nlm.nih.gov/pubmed/28540170

Xiao

Chen

. Antiviral activities against influenza virus (FM1) of bioactive fractions and representative compounds extracted from Banlangen (Radix Isatidis). J Tradit Chin Med. 2016;36(3):369-376.doi:10.1016/s0254-6272(16)30051-6

http://www.ncbi.nlm.nih.gov/pubmed/27468553

10.

Liu

Y-F.

Chen

M-H.

Guo

Q-L

et al. Antiviral glycosidic bisindole alkaloids from the roots of Isatis indigotica . J Asian Nat Prod Res. 2015;17(7):689-704.doi:10.1080/10286020.2015.1055729

http://www.ncbi.nlm.nih.gov/pubmed/26123248

11.

Nie

L-X.

Y-L.

Dai

S-C

. Antiviral activity of Isatidis Radix derived glucosinolate isomers and their breakdown products against influenza a in vitro/ovo and mechanism of action. J Ethnopharmacol. 2020;251:112550 doi:10.1016/j.jep.2020.112550

http://www.ncbi.nlm.nih.gov/pubmed/31918015

12.

Zhou

et al. Lariciresinol-4-O-β-D-glucopyranoside from the root of Isatis indigotica inhibits influenza A virus-induced pro-inflammatory response. J Ethnopharmacol. 2015;174:379-386.doi:10.1016/j.jep.2015.08.037

http://www.ncbi.nlm.nih.gov/pubmed/26320688

13.

Zhou

et al. Radix isatidis polysaccharides inhibit influenza a virus and influenza a virus-induced inflammation via suppression of host TLR3 signaling in vitro . Molecules. 2017;22(1):116 doi:10.3390/molecules22010116

http://www.ncbi.nlm.nih.gov/pubmed/28085062

14.

Liu

S-F.

Zhang

Y-Y.

Zhou

et al. Alkaloids with neuroprotective effects from the leaves of Isatis indigotica collected in the Anhui Province, China. Phytochemistry. 2018;149:132-139.doi:10.1016/j.phytochem.2018.02.016

http://www.ncbi.nlm.nih.gov/pubmed/29499466

15.

Liu

Cheng

Guo

et al. Effects of indigowoad root (Radix Isatidis) on the immune responses and HSP70 gene expression of medicinal leeches (Poecilobdella manillensis) under proteus mirabilis infection. Aquaculture. 2016;454:44-55.doi:10.1016/j.aquaculture.2015.12.012 doi:10.1016/j.aquaculture.2015.12.012

16.

J-P.

Yuan

Zhang

W-Y

et al. Effect of Radix isatidis polysaccharide on alleviating insulin resistance in type 2 diabetes mellitus cells and rats. J Pharm Pharmacol. 2019;71(2):220-229.doi:10.1111/jphp.13023

http://www.ncbi.nlm.nih.gov/pubmed/30298631

17.

Fernando Rodrigues de Sa

. Eliezer JB, Carlos Alberto Manssour F. From nature to drug discovery: the indole scaffold as a ‘privileged Structure’. Mini-Rev Med Chem. 2009;9:782-793.

18.

Dadashpour

Emami

. Indole in the target-based design of anticancer agents: a versatile scaffold with diverse mechanisms. Eur J Med Chem. 2018;150:9-29.doi:10.1016/j.ejmech.2018.02.065

http://www.ncbi.nlm.nih.gov/pubmed/29505935

19.

Garg

Maurya

RK.

Thanikachalam

PV.

Bansal

Monga

. An insight into the medicinal perspective of synthetic analogs of indole: a review. Eur J Med Chem. 2019;180:562-612.doi:10.1016/j.ejmech.2019.07.019

http://www.ncbi.nlm.nih.gov/pubmed/31344615

20.

Zhang

M-Z.

Chen

Yang

G-F

. A review on recent developments of indole-containing antiviral agents. Eur J Med Chem. 2015;89:421-441.doi:10.1016/j.ejmech.2014.10.065

http://www.ncbi.nlm.nih.gov/pubmed/25462257

21.

Qin

H-L.

Liu

Fang

W-Y.

Ravindar

Rakesh

. Indole-Based derivatives as potential antibacterial activity against methicillin-resistance staphylococcus aureus (MRSA). Eur J Med Chem. 2020;194:112245 doi:10.1016/j.ejmech.2020.112245

http://www.ncbi.nlm.nih.gov/pubmed/32220687

22.

Baeyer

Emmerling

. Synthese des indols. Ber Dtsch Chem Ges . 1869;2(1):679-682.doi:10.1002/cber.186900201268

23.

Bugaenko

DI.

Karchava

AV.

Yurovskaya

. Synthesis of indoles: recent advances. Russ Chem Rev. 2019;88(2):99-159.doi:10.1070/RCR4844

24.

Zhong

Jun‐Song.

Yuan

. Recent advances in the electrochemical synthesis and functionalization of indole derivatives. Adv Synth Catal. 2020;362(11):2102-2119.doi:10.1002/adsc.201901520

25.

Zheng

You

SL.

dearomatization

Casymmetric

. (Cada) reaction-enabled total synthesis of indole-based natural products. Nat Prod Rep. 2019;36:1589-1605.

26.

Zhang

Y-C.

Jiang

Shi

. Organocatalytic asymmetric synthesis of indole-based chiral heterocycles: strategies, reactions, and outreach. Acc Chem Res. 2020;53(2):425-446.doi:10.1021/acs.accounts.9b00549

http://www.ncbi.nlm.nih.gov/pubmed/31820922

27.

Mancuso

Dalpozzo

. Recent progress in the transition metal catalyzed synthesis of indoles. Catalysts. 2018;8(10):458.doi:10.3390/catal8100458 doi:10.3390/catal8100458

28.

Liu

Zhao

Chen

Deng

Guo‐Jun.

Huang

. Aerobic oxidative functionalization of indoles. Adv Synth Catal. 2020 doi:10.1002/adsc.202000285

29.

Liu

X-Y.

Qin

. Indole alkaloid synthesis facilitated by photoredox catalytic radical cascade reactions. Acc Chem Res. 2019;52(7):1877-1891.doi:10.1021/acs.accounts.9b00246

http://www.ncbi.nlm.nih.gov/pubmed/31264824

30.

Meng

L-J.

Guo

Q-L.

C-B

et al. Diglycosidic indole alkaloid derivatives from an aqueous extract of Isatis indigotica roots. J Asian Nat Prod Res. 2017;19(6):529-540.doi:10.1080/10286020.2017.1320547

http://www.ncbi.nlm.nih.gov/pubmed/28475367

31.

Liu

Chen

Guo

et al. Aromatic compounds from an aqueous extract of “ban lan gen” and their antiviral activities. Acta Pharm Sin B. 2017;7(2):179-184.doi:10.1016/j.apsb.2016.09.004

http://www.ncbi.nlm.nih.gov/pubmed/28303224

32.

Huang

Cheng

L-H

et al. Antivirus constituents of radix of Isatis indigotica . Chin J Nat Med. 2005;3:359-360.

33.

Meng

L-J.

Guo

Q-L.

Zhu

C-G.

C-B.

Shi

J-G

. Isatindigodiphindoside, an alkaloid glycoside with a new diphenylpropylindole skeleton from the root of Isatis indigotica . Chin Chem Lett. 2018;29(1):119-122.doi:10.1016/j.cclet.2017.05.019

34.

Dong

Yang

et al. Chemical constituents in antiviral fraction of Isatidis Radix and their activities. Chin Tradit Herb Drugs. 2013;44:2960-2964.

35.

Yang

Wang

et al. Indole alkaloids from the roots of Isatis indigotica and their inhibitory effects on nitric oxide production. Fitoterapia. 2014;95:175-181.doi:10.1016/j.fitote.2014.03.019

http://www.ncbi.nlm.nih.gov/pubmed/24685504

36.

Zhu

Yang

ZF.

. Application of indole-3-acetonitrile-6-O-β-D-glucopyrano- side in pharmaceuticals. CN102875474A. 2012-12-26, 2012.

37.

Liang

Ito

. Separation and purification of two minor compoundsfrom Radix isatidis by integrative MPLC and HSCCC with preparative HPLC. J Liq Chromatogr Relat Technol. 2015;38(5):647-653.doi:10.1080/10826076.2014.936606

http://www.ncbi.nlm.nih.gov/pubmed/25745338

38.

Zlotos

DP.

Attia

MI.

Julius

Sethi

Witt-Enderby

. 2-[(2,3-dihydro-1H-indol-1-yl)Methyl]melatonin analogues: a novel class of MT2-selective melatonin receptor antagonists. J Med Chem. 2009;52(3):826-833.doi:10.1021/jm800974d

http://www.ncbi.nlm.nih.gov/pubmed/19193160

39.

Iwao

Motoi

. Methodology for the efficient synthesis of 3,4-differentially substituted indoles. fluoride ion-induced elimination-addition reaction of 1-triisopropylsilylgramine methiodides. Tetrahedron Lett. 1995;36(33):5929-5932.doi:10.1016/00404-0399(50)1197P-

40.

Kulkarni

SS.

Wang

C-C.

Sabbavarapu

et al. “One-Pot” protection, glycosylation, and protection–glycosylation strategies of carbohydrates. Chem Rev. 2018;118(17):8025-8104.doi:10.1021/acs.chemrev.8b00036

http://www.ncbi.nlm.nih.gov/pubmed/29870239

41.

Gouliaras

Lee

Chan

Taylor

. Regioselective activation of glycosyl acceptors by a diarylborinic acid-derived catalyst. J Am Chem Soc. 2011;133(35):13926-13929.doi:10.1021/ja2062715

http://www.ncbi.nlm.nih.gov/pubmed/21838223

42.

Somei

Yamada

. The first and simple total synthesis of Cappariloside a. Heterocycles. 2000;53(7):1573-1578.doi:10.3987/COM-00-8908 doi:10.3987/COM-00-8908

43.

Loganathan

Trivedi

. Phase-transfer-catalyzed d-glucosylation: synthesis of benzoylated aryl β-d-glucopyranosides and β-d-glucopyranosyl-substituted cinnamates. Carbohydr Res. 1987;162(1):117-125.doi:10.1016/0008-6215(87)80206-9 doi:10.1016/0008-6215(87)80206-9

44.

Zhao

Zhou

et al. Cappariloside a shows antiviral and better anti-inflammatory effects against influenza virus via regulating host IFN signaling, in vitro and vivo. Life Sci. 2018;200:115-125.doi:10.1016/j.lfs.2018.03.033

http://www.ncbi.nlm.nih.gov/pubmed/29555588

45.

West

MJ.

Fyfe

JWB.

Vantourout

JC.

Watson

AJB

. Mechanistic development and recent applications of the Chan-Lam amination. Chem Rev. 2019;119(24):12491-12523.doi:10.1021/acs.chemrev.9b00491

http://www.ncbi.nlm.nih.gov/pubmed/31756093

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

1.21 MB

0.00 MB