Bio-inspired synthesis and bioactivities of phenylethanoid glycoside crenatosides

Abstract

Herein, we report an effective approach for the synthesis of phenylethanoid glycosides bearing a fused 1,4-dioxane motif, employing a bio-inspired oxidative cyclization of the glucose 2′-OH and benzylic carbon of phenylethanol aglycone as a key step. Thus, crenatoside (1), isocrenatoside (2), and 20 analogs with varied substitution on the peripheral phenyl rings are synthesized. Some of the synthetic glycosides show considerable anti-inflammatory and immunosuppressive activities, and compound 36 exhibited the most potent immunosuppressive activity, with an IC₅₀ value of 19.9 μM on B lymphocyte proliferation responses.

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Introduction

Phenylethanoid (or phenylpropanoid) glycosides (PhGs) are a distinct class of natural products widely distributed in folk medicinal plants, particularly those belonging to the dicotyledonous group.¹ Structurally, PhGs are characterized by cinnamate and hydroxyphenylethyl moieties attached to D-glucopyranose through ester and β-glycosidic linkages, respectively.¹ To date, more than 500 PhGs have been isolated and characterized, which exhibit a wide range of pharmacological activities, such as antibacterial, antitumor, antiviral, anti-inflammatory, antioxidant, neuroprotective, and cardioprotective effects.² Being a prototypical PhG, acteoside was first extracted from dicotyledonous plants in 1963 (Fig. 1a); five years later, its structure was determined to be β-(3,4-dihydroxyphenyl)ethyl α-L-rhamnopyranosyl (1′′→3′)-β-D-(4′-O-caffeoyl)glucopyranoside.³ Two decades later, β-hydroxyacteoside (namely campneoside II), an oxidized derivative of acteoside, was isolated from a traditional Chinese medicinal plant Forsythiae fructus.⁴ Having similar structural characteristics to campneoside II, crenatoside (1) was extracted from the aerial parts of Orobanche crenata in 1993.⁵ In fact, crenatoside (1) is the first phenylethanoid glycoside disclosed to possess a fused dioxane skeleton, characterized by an ether linkage connecting O′-2 of glucose to the benzylic carbon of 3,4-dihydroxyphenylethanol aglycone. It was suggested that 1,4-dioxanyl and caffeoyl moieties play pivotal roles in the selective inhibition of neuraminidase (NA) of influenza A virus (H1N1).⁶ In 1998, isocrenatoside (2) was identified as the 6′-O-caffeoyl isomer of crenatoside from the fresh whole plant of Orobanche coerulescens.⁷

Fig. 1 (a) Representative phenylethanoid glycosides. (b) Proposed biotransformation from acteoside to crenatosides. (c) Oxidative cyclization approach for constructing the 1,4-dioxane skeleton in this work.

All these four PhGs share common structural features, including the central D-glucose, 3-O-L-rhamnose, the 4-O-caffeoyl moiety, and 3,4-dihydroxyphenylethyl aglycone. The primary difference lies in the oxidation state of the benzylic carbon in 3,4-dihydroxyphenylethanol aglycone. Among them, acteoside is the most abundant disaccharide caffeoyl ester PhG.⁸ We hypothesize that the minor β-hydroxyacteoside and crenatoside (1) could be derived from acteoside through an oxidative transformation (Fig. 1b). Thus, acteoside underwent phenol oxidation to generate 2,5-cyclohexadienone intermediate A; subsequent addition of water led to the formation of β-hydroxyacteoside. Alternatively, an intramolecular nucleophilic addition of glucose 2′-OH resulted in the formation of 1,4-dioxane fused crenatoside (1). Moreover, the caffeic acid moiety could undergo acyl migration from glucose O-4′ to O-6′ to afford isocrenatoside (2). Over the past few decades, only acteoside and a few analogous PhGs have been synthesized.^9,10 It is noteworthy that Zeng et al. constructed a glucose fused 1,4-dioxane skeleton via a NaH-mediated intramolecular alkylation of glucose O′-2 with the bromide of the phenylethanol moiety, albeit without stereoselectivity.⁶ Herein, we present the first synthesis of crenatoside (1), isocrenatoside (2) and their analogs through an oxidative cyclization approach (Fig. 1c).¹¹

Results and discussion

We embarked on an examination of the bio-inspired oxidative cyclization step to construct the glucose fused 1,4-dioxane skeleton (Scheme 1). Thus, perbenzoyl glucopyranosyl trichloroacetimidate 3 was coupled with 3-bromo-4-acetoxy-phenylethanol 4 under the catalysis of TMSOTf to provide phenylethanoid glycoside 5 (93%), which was then transformed to the desired cyclization precursor 6 by the removal of benzoyl and acetyl groups followed by the formation of 4,6-O-benzylidene. After a thorough examination of various reaction parameters, including benzoquinone, solvent, and concentration, we successfully obtained the desired 1,4-dioxane glycoside 7, employing 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) as an oxidant and acetonitrile as a solvent at 60 °C, albeit with a moderate 26% yield (see the ESI† for details).¹¹ We attributed the low yield to the electron-withdrawing properties of the bromide substituent in the phenyl moiety, which was installed to facilitate later-stage derivatization. Thus, we turned our attention to phenylethanoid glycosides (i.e., 8–10), which bear electron rich phenyl moieties. Indeed, under similar conditions, the cyclization reaction of 3-methoxy-4-hydroxy-phenylethanoid glycoside 8 proceeded smoothly, leading to 11 in a good 72% yield. When the 4-hydroxy group was replaced with the 4-methoxy group (in 9), the yield of the oxidative cyclization was reduced to 35%. Considering that the realistic structures of crenatoside (1) and isocrenatoside (2) feature 3,4-dihydroxy substituents, we prepared 3,4-dihydroxyphenylethanoid glycoside 10. Nevertheless, compound 10 was found poorly soluble in acetonitrile due to the presence of four hydroxyl groups. Fortunately, the oxidative cyclization of 10 could be successfully carried out in tetrahydrofuran, affording the desired 1,4-dioxane fused glycoside 13 in a satisfactory 63% yield.

Scheme 1 Synthesis of 1,4-dioxane fused glycosides 7, 11–13.

To achieve the total synthesis of crenatosides, 2,3-dibenzoyl-4,6-O-benzylidene glucosyl trichloroacetimidate 14 was coupled with 3,4-OTBS-phenylethanol 15 under the conventional conditions to deliver phenylethanoid glycoside 16 on a decagram scale (Scheme 2). Subsequent saponification and desilylation generated ring-closing precursor 10 (7.4 g). Utilizing the previously optimized DDQ-mediated oxidative cyclization in THF, the desired 1,4-dioxane fused glycoside 13 was obtained in 63% yield (on a 1.4 g scale). To selectively protect the phenolic hydroxyls, levulinic anhydride and potassium bicarbonate were used to give 17 (80%), which bears free 3′-OH.¹² Under the conventional conditions (TMSOTf, 4 Å MS, CH₂Cl₂, 0 °C → RT), monosaccharide 17 was coupled with peracetylrhamnosyl trichloroacetimidate 18 to provide α-linked disaccharide 19 in 83% yield. Following the acidic removal of the 4,6-O-benzylidene group in 19, we selectively masked the 6-OH with acetic anhydride in the presence of diazabicyclononene (DBN), giving 4′-ol 20.¹³ Subsequent esterification of the remaining 4-OH with TBS-protected caffeic acid led to the fully protected crenatoside 22 in 64% yield. The final stage for the synthesis of crenatoside 1 called for selective deprotection of TBS and acyl groups. We achieved delicate removal of TBS groups in 22 using Olah's reagent in THF. Finally, selective deacylation with potassium t-butoxide at 0 °C furnished crenatoside 1 (31%), along with isocrenatoside 2 (35%).¹⁴ Evidently, the resultant crenatoside 1 underwent caffeinic migration from O-4 to O-6 under basic conditions to lead to isocrenatoside 2. The NMR spectroscopic data of the synthetic crenatosides were virtually identical to those recorded for the authentic phenylethanoid glycosides (see Tables S3–S6† for details).

Scheme 2 Synthesis of crenatoside (1), isocrenatoside (2), and their analogs (23–42).

At this juncture, we shifted our focus towards preparing analogs of crenatosides and conducting bioactivity tests (see the ESI† for details). Leveraging common intermediate 20, we conveniently obtained crenatoside analogs 23–25 and isocrenatoside analogs 26–30. Employing 1,4-dioxane fused glycoside 11 as the precursor, we successfully executed a collective synthesis of analogs 31–42 in a similar approach. These 22 phenylethanoid glycosides (1, 2, and 23–42) were subjected to bioactivity assays (see Tables S7–S9† for details). Upon bioactivity screening, most of the 22 phenylethanoid glycosides were tested for anti-inflammatory activity in the lipopolysaccharide stimulated mouse macrophage, RAW 264.7 cells, which could reduce the production of tumor necrosis factor (TNF)-α in the cells at 20 μM. Additionally, these phenylethanoid glycosides exhibited immunosuppressive activity, in which compound 36 exhibited the most potent immunosuppressive activity, with an IC₅₀ value of 19.9 μM on B lymphocyte proliferation responses. Some of the crenatoside derivatives (i.e., 27 and 36) possessed better biological activities compared to crenatoside (1) and isocrenatoside (2). These findings suggest that mild structural alterations, such as the substituents on the benzene rings, could significantly impact their bioactivities.

Conclusions

In summary, we have achieved the first chemical synthesis of phenylethanoid glycosides, crenatoside (1) and isocrenatoside (2), which bear a characteristic glucose fused 1,4-dioxane motif. By utilizing DDQ as an oxidant, the bio-inspired oxidative cyclization reactions could proceed effectively, resulting in the formation of 1,4-dioxane structures. The successful application of a range of substituted cinnamyl and hydroxyphenylethyl building blocks has enabled the synthesis of 20 crenatoside analogs, which demonstrated that mild structural alterations on the peripheral benzene rings could significantly impact their bioactivities. The availability of these 1,4-dioxane fused glycosides paves the way for further biological and pharmacological studies on PhGs.

Data availability

The data underlying this study are available in the published article and its ESI.†

ESI statement:

The ESI is available free of charge on the RSC Publications website.

Experimental details and NMR spectra are available in PDF.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

Financial support from the National Natural Science Foundation of China (22031011 & 22177125), the Youth Innovation Promotion Association of CAS (2020258), the Key Research Program of Frontier Sciences of CAS (ZDBS-LY-SLH030), the Science and Technology Commission of Shanghai Municipality (23ZR1476400), the Shanghai Municipal Science and Technology Major Project, and the Hangzhou Leading Innovation and Entrepreneurship Team Project (TD2022002) is acknowledged.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4qo01062k

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