Biomimetic Total Synthesis of ( + )-Ranuncoside,a Unique Tricyclic Spiroacetal Glycoside of Christmas Rose ( Helleborus niger L.)

Abstract

Christmas Rose (Helleborus niger L.) is an alpine plant belonging to the Ranunculaceae family. Typical for this popular ornamental plant are white flowers that appear during winter. Root extracts contain numerous medicinally active ingredients and potent cardiac glycosides. Recent findings indicate that Helleborus root extracts possess the potential for cancer treatment and inhibit the proliferation of tumor cells. In 2022, we published a practical extraction method of ( + )-ranucoside 1 from the dried leaves of H niger L. Here, we describe for the first time the total synthesis of 1. Starting from 5-hydroxylevulinic acid methyl ester 8 and acetobromo-α-D-glucose 13, the β-glycoside 14 is formed stereoselectively on the application of Koenigs-Knorr conditions. Subsequent cleavage of the acetyl-protecting groups and saponification of the ester function yields the pyrano dioxane derivative 17. Its lactonization gives ( + )-ranuncoside 1. The presented synthesis is practical and very efficient, consisting of 6 steps from D-glucose and giving 22% overall yield. Furthermore, it is biomimetic due to its concordance with the biosynthesis of ( + )-ranuncoside 1 in H foetidus.

Keywords

ranuncoside total synthesis spiroacetal altaicadispirolactone bioactivity antibiotic anticancer agent

Introduction

The Christmas Rose (Helleborus niger L.)¹ is a perennial flowering plant belonging to the subspecies Helleborus of the Ranunculaceae family. Its main distribution area includes the Eastern European Alps, where it can be found up to an altitude of 1800 m. Christmas Rose is also a popular ornamental plant in horticulture and various cultivars have been generated. It has a very early flowering period, ranging from December to April and its white flowers and green leaves often appear among snow and ice. Therefore, this alpine flower has been a symbol of life and death since middle age, and still today.²

A typical feature of the species H niger L. is its black rhizome from which it got its scientific name. Rhizome and roots (Radix Hellebori nigri) are used as medicine in allopathy and homeopathy.³ Furthermore, pharmacological studies revealed that the root extracts contain numerous medicinally active ingredients^4–7 as well as the powerful cardiac glycoside hellebrin^8–11 which possesses similar pharmacological properties to those of the Digitalis glycosides.^12–14 Recent findings indicate that Helleborus root extracts are safe and possess the potential for cancer treatment.¹⁵ Furthermore, extracts of roots and the whole plant have been shown to inhibit the proliferation of tumor cells.^16,17

In the 16th century, the Swiss physician and alchemist Paracelsus described Christmas Rose leaves as a dementia preventative par excellence.¹⁸ But despite these early accounts of their pharmacological potential, the aboveground parts of the plant have been little studied since then. In 1974, (+)-ranucoside 1 (structure Figure 1) was isolated by Martinek¹⁹ from dried stems, leaves, and flowers of H niger L., which had been collected in the Austrian Alps (South Carinthia). Recently, we²⁰ published a practical extraction method of (+)-ranucoside 1 from the dried leaves of cultivated H niger L. plants. These are cheap and easily accessible in large quantities from nurseries. The procedure is easy to carry out, rapid, efficient and gives 1 in good yield and in highly crystalline form. We also described the structure elucidation of 1 with the aid of 500 MHz NMR spectroscopy and confirmed the (R)-configuration of the spiro carbon by X-ray structure analysis. The latter finding is particularly significant because the spiroacetal ring system is responsible for the biological activity of numerous natural products.^21–23

Figure 1.

The toxic flower Helleborus niger L. (Christmas Rose) blooms in winter and belongs to the Ranunculaceae family. Its aboveground parts contain the spiroacetal glycoside ( + )-ranuncoside 1 (chemical structure on the right side). The photo was taken by the author (EC) in his garden in Seeheim, Germany (December 2019).

Based on our previously developed efficient and stereo-controlled synthetic routes to novel bicyclic motifs of spiroacetal natural products²⁴ as well as of tricyclic spiroacetal domain derivatives of the plant glycoside ( + )-ranuncoside 1,²⁵ we here describe the first time its total synthesis.

The synthesis is biomimetic, as it is an analogue to the biosynthesis of ( + )-ranuncoside 1 in H foetidus studied by Tscheche et al.²⁶ They also published the name ranuncoside for the first time after its isolation from H foetidus and structure determination.²⁷ Confusingly, the name ranuncoside was later also used for 2 completely different structures: (a) a pentacyclic triterpenoid glycosides from Hydrocotyle ranuncoloides (Apiaceae)²⁸ and (b) an olefinic glycoside from Ranunculus muricatus (Ranunculaceae) with unsolved stereochemistry in the whole hexose moiety.²⁹

Results and Discussion

Retrosynthesis

The retrosynthesis of ( + )-ranuncoside 1 is depicted in Scheme 1. The molecular structure of 1 is characterized by a central dioxane ring, fused on the right side 2-fold in trans-manner with a pyranose moiety and on the left side in spiroacetal manner with a saturated γ-lactone ring. The tricycle possesses 6 stereogenic centers, each with at least an oxygen hetero atom and (R)-configuration at the spiro center.

Scheme 1.

Retrosynthesis of ( + )-ranuncoside 1 by 2-fold hydrolytic cleavage at (A) the spiro carbon C-2 to the open chain glycoside 2 and (B) at the anomeric carbon C-4′a to the 5-hydroxylevulinic acid 3 and D-glucose 5. The cleavage sites are indicated by red lines and the water addition is marked in blue color.

Retrosynthetic cleavage of 2 bonds at the spiro center at carbon C-2 (red lines in Scheme 1) followed by the addition of 1 molecule of water (blue color in Scheme 1) forms the glycoside 2. A similar cleavage procedure at the anomeric center at carbon C-4′a and water addition results in 5-hydroxylevulinic acid 3 and D-glucose 4 (open chain form), which cyclizes to the more stable 5 (pyranose form).

Synthesis of the Levulinic Acid–Derived Starting Material 8

Starting from low-cost platform chemical levulinic acid 6,³⁰ the 5-hydroxylevulinic acid methyl ester 8 was synthesized in 2 steps (see Scheme 2). According to a procedure of Neier et al³¹ with bromine in methanol, the yield of bromide 7 could be increased from 45% to 62% by adding the bromine slowly and slightly increasing its amount. Literature precedents^32–34 had lower yields.

Scheme 2.

Synthesis of the glycosylation acceptor, the 5-hydroxylevulinic acid methyl ester 8, from levulinic acid 6 via bromide 7. Saponification of ester 8 yields 5-hydroxylevulinic acid 3. Both compounds, 3 and 8, could be dimerized, via the intermediates 9 and 10, to meso-altaicadispirolactone 11.

The further reaction of bromide 7 with sodium formate in aqueous ethanol to the methyl ester 8 reaches 95% yield according to Ma et al.³⁵ In contrast, Lindsey et al³⁶ reported that yields for this simple transformation were typically only around 20%. After several optimization attempts, we achieved a yield of 74%. Thus, the total yield of glycosyl acceptor 8 from levulinic acid 6 was 44%. Two other known syntheses of 8 require 2 or more steps with lower yield or without any yield specification.^37,38 In view of a biomimetic synthesis of ( + )-ranuncoside 1, it should be noted that the glycosyl acceptor 5-hydroxylevulinic acid methyl ester 8 was also isolated from the genus Clematis (Ranunculaceae)^39,40 as well as from the genus Drymaria (Caryophyllaceae).⁴¹

Altaicadispirolactone (1,6,9,13-tetraoxadispiro[4.2.4.2]tetradecane-2,10-dione) 11

The bicyclic spiro part of altaicadispirolactone 11 (red color in Scheme 2) has exactly the same structure and stereochemistry as the left half of ( + )-ranuncoside 1. Interestingly, in 2016, Lindsey et al³⁶ reported that the 5-hydroxylevulinic acid methyl ester 8 is relatively unstable, and the initially clear liquid forms crystals when left on the benchtop. Using X-ray diffraction, they determined the crystals to be altaicadispirolactone 11. The tricyclic compound is formed by dimerization of 2 molecules of ester 8 via 2-fold acetalization (9 → 10) and 2-fold lactonization under the elimination of 2 molecules of methanol (10 → 11). Compound 11 has R,S- or meso-configuration at the 2 spiro centers with axial orientation of the 2 butanolide ring oxygen atoms. Responsible for this structural feature is the action of 2 stabilizing anomeric effects, respectively (blue color in Scheme 2). Surprisingly, in our hands, the methyl ester 8 didn't form altaicadispirolactone 11. In order to compare the structural data with ( + )-ranuncoside 1, we prepared 11 from ester 8 after its saponification to 5-hydroxylevulinic acid 3 based on the method of Zhang et al³² (see Experimental Part).

From a historical point of view, it is interesting that in 1962 Rappe⁴² published the correct structure (without stereochemistry) of 11 by heating 5-hydroxylevulinic acid 3 to about 100 °C, according to a procedure described by Wolff⁴³ in 1891. In this procedure, the acid melts and a precipitate of 11 consisting of well-defined crystals forms sometime later.

The name altaicadispirolactone has been reported much later by Li and Li⁴⁴ according to the isolation of (5R,8R)-1,6,9,13-tetraoxadispiro[4.2.4.2]tetradecane-2,10-dione (altaicadispirolactone) from Anemone altaica, a flower native to the Altai Mountains (Central Asia). They described the synthesis of (±)-altaicadispirolactone by refluxing 5-hydroxylevulinic acid 3 for 17 h in the presence of p-toluene sulfonic acid and in acetonitrile as solvent. Ten years later, Zhang et al³² described the same reaction, but under Lewis acid catalysis (boron trifluoride etherate) by refluxing 3 for 4 h in dichloromethane. In their paper, an X-ray structure revealed that the obtained 11 was identical with those of Lindsey et al³⁶ (see above).

Synthesis of the D-Glucose-Derived Starting Material 13

In 1901, the first synthesis of acetobromo glucose 13 was described by Koenigs and Knorr⁴⁵ (see Scheme 3). They obtained dextrorotatory and analytical pure 13 in 58% yield and with a melting point of 88 °C to 89 °C (diethyl ether). Shortly after, Emil Fischer^46,47 described the synthesis of 13 by reacting pentaacetyl-β-D-glucose 12 with hydrogen bromide in glacial acetic acid in 76% to 83% yield. Interestingly, he noted about the procedure of Koenigs and Knorr: The method with acetyl bromide also gives quite good results if the conditions are right, and at first glance seems simpler, because no prior preparation of pentaacetate 12 is required. However, considering the lower price of the materials and the safety of the operation, especially when larger quantities are involved, our 2-step method will probably be preferred for the preparation of acetobromo glucose 13. Until today, numerous modified synthetic methods for 13, starting from D-glucose 5 or its pentaacetate 12, have been described.^48–51 Synthetic methods for α-D-glucose pentaacetate have been reviewed.⁵²

Scheme 3.

By reacting D-glucose 5 with a large excess of acetyl bromide, the acetobromo glucose 13 was first described by Koenigs and Knorr⁴⁵ in 1901. The Emil Fischer's 2-step protocol,^46,47 acetylation of 5 to 12 and further bromination to 13 was significantly simplified with respect to effectiveness, feasibility, and industrial production.

Here, we present 2 laboratory reaction protocols for the synthesis of 1,2,3,4,6-penta-acetyl-β-D-glucopyranose 12 and its transformation to acetobromo-α-D-glucose 13. They differ substantially from Emil Fischer's protocol^46,47 and all other previously described methods due to their significantly simplified application on a laboratory scale. An industrial production on a multi kg-scale was also realized without any difficulties.

Biomimetic Synthesis of ( + )-ranuncoside 1

The crucial step in our total synthesis was the stereoselective β-glycosylation of 5-hydroxylevulinic acid methyl ester 8 with acetobromo glucose 13 (Scheme 4). This could be achieved by the application of Koenigs-Knorr conditions in the presence of mercury cyanide and in a mixture of benzene and nitromethane as solvents. The desired β-glycoside 14 was obtained in 60% yield and in crystalline form. It was identical in all respects to 14 synthesized by Cottier et al⁵³ in 3 steps from hydroxymethylfurfural (HMF) and 8 with an overall yield of 7%.

Scheme 4.

Synthesis of ( + )-ranuncoside 1: Stereoselective β-glycosylation of 5-hydroxylevulinic acid methyl ester 8 with acetobromo-α-D-glucose 13 yielded the glycoside tetraacetate 14. Further deacetylation (14→15) followed by methyl ester saponification lead via 16 and ketalization the dioxane derivative 17. Finally, lactonization with p-toluenesulfonic acid gave ( + )-ranuncoside 1.

Further selective deprotection of the acetyl-protecting groups in the sugar portion under Zémplen conditions with catalytic amounts of sodium methoxide in methanol succeeded as expected without any problems. The yield of the 5-(β-D-glucopyranosyloxy)levulinic acid methyl ester 15 was nearly quantitative (94%).

The observed 2 carbonyl shifts in the ¹³C NMR spectrum at 173.71 and 208.35 ppm for an ester C = O and a ketone C = O function confirmed the open-chain structure of glycoside 15, while complete saponification of the acetyl groups was evidenced by the absence of any ¹H-NMR-acetyl-shifts and the new appearance of 4 hydroxyl shifts (see Figure 2).

Figure 2.

500 MHz ¹H NMR spectrum of 5-(β-D-glucopyranosyloxy)levulinic acid methyl ester 15 measured in DMSO-d₆.

The successful saponification of the methyl ester to the carboxylic acid was unequivocally evidenced by the absence of an ester methyl group in the ¹H and ¹³C NMR spectra. By means of ¹³C NMR data, it could be excluded that the open-chain compound 16 was present, due to the loss of the ketone-shift around δ > 200 ppm. Thus, the pyrano dioxane 17 must have been formed via ketalization.

The structure of 17 was proven by (a) the presence of a COOH-group with shifts at δ = 11.82 (¹H NMR) and δ = 174.12 ppm (¹³C NMR) for its OH and CO-function and (b) by the 2 well-separated doublets of the C-3′-dioxane protons at δ = 4.20 and δ = 4.32 ppm together with the large coupling constant of J_3′gem = 17.13 Hz. The stereochemistry of the newly formed anomeric center (C-2′) was not determined, but because of the action of the anomeric effect, an axial oriented hydroxyl group should be very probable. The dioxane ring formation of intermediate 16 by hemiacetalization to 17 could be explained by an acidic reaction medium, caused by the aliphatic carboxylic acid function in the molecule.

The last reaction step, the generation of the 5-membered butanolide ring, succeeded in an acidic environment with p-toluenesulfonic acid and tetrahydrofuran as solvent under relatively drastic reaction conditions (reflux 4 days). The natural product ( + )-ranuncoside 1 was thus obtained in 81% yield. In all respects, the synthesized product was identical with a sample isolated from H niger L. by Cuny and Klingler.²⁰ The elaborated synthesis is highly efficient, due to shortness (6 steps starting from D-glucose) and a high overall yield of 22%.

Conclusions

The Christmas Rose (H niger L.)¹ belongs to the subspecies Helleborus of the Ranunculaceae family and its rhizome and roots contain numerous medicinally active ingredidents^4–7 including potent cardiac glycosides, which possess similar pharmacological properties to those of the Digitalis glycosides.^12–14

Recently, we published a practical extraction method of ( + )-ranucoside 1 from the leaves of cultivated H niger L. plants and confirmed the structure by 500 MHz NMR spectroscopy and X-ray structure analysis.²⁰ Based on the fact that the spiroacetal ring system is responsible for the biological activity of numerous natural products^21–23 and stimulated by our developed synthetic routes to motifs of spiroacetal natural products²⁴ and ranuncoside derivatives,²⁵ we here describe for the first time the total synthesis of ( + )-ranucoside 1 by biomimetic means.

A characteristic feature of a retrosynthetic approach was the cleavage of the central dioxane ring at (a) the spiro center (C-2) and (b) at the anomeric center (C-4′a) and with respective water addition. This resulted in 5-hydroxylevulinic acid 3 and D-glucose 5. The derivatization of these 2 starting compounds leads to 5-hydroxylevulinic acid methyl ester 8 and acetobromo-α-D-glucose 13. The 5-hydroxy levulinic acid 3 and its methyl ester 8 dimerized to altaicadispirolactone 11 possesssing the structure and stereochemistry of the left half of of ( + )-ranuncoside 1.

Glycosylation of 5-hydroxylevulinic acid methyl ester 8 with acetobromo glucose 13 under Koenigs-Knorr conditions yielded the desired β-glycoside 14 (60% yield). Succeeding cleavage of the acetyl protecting groups under Zémplen conditions gave the free glucosyl ester 15 (94%). Its saponification lead via the intermediate 16 the more stable dioxane derivative 17 (quantitative yield). The generation of the 5-membered butanolide ring and formation of the natural product ( + )-ranuncoside 1 succeeded with p-toluenesulfonic acid (81% yield). The structure of all synthesized products was verified by ¹H and ¹³C NMR spectra. All physical and spectroscopic data of synthesized 1 were identical with a sample isolated from H niger L. by Cuny and Klingler.²⁰

This is to our knowledge, the first report of a total synthesis of 1, needing only 6 steps, starting from D-glucose and having a remarkable overall yield of 22% (62% per step). Moreover, this synthesis is also biomimetic, due to its matches with biosynthetic studies by Tscheche et al.²⁶

Experimental

TLC was performed on POLYGRAM® SILG/UV₂₅₄ (Macherey Nagel & Co.). Preparative chromatographic separations were carried out on columns with Merck silica gel 60 (40-63 μm). Melting points were determined on a Büchi Tottoli apparatus and are uncorrected. Specific optical rotations were determined on a Perkin-Elmer Polarimeter 241 in 1 dm cuvettes at a wavelength of 589 nm. NMR spectra were measured on a Bruker 300 MHz spectrometer with Avance-II console and BBO probe as well as 500 MHz spectrometer with DPX console and BBFO probe at 303 K using TMS as internal reference or by calibration with the shift of the solvent of the sample.⁵⁴ The abbreviation of the multiplicities of the shifts is indicated as s for singlet, d for doublet, t for triplet, q for quartet, sxt for sextet, oct for octet, m for multiplet, br for broad, and p for pseudo. Mass spectra were run on a Bruker Impact II spectrometer.

Methyl 5-bromo-4-oxopentanoate (5-Bromolevulinic Acid Methyl Ester) 7

Bromine (16.1 mL, 50.4 g, 0.32 mol) was added dropwise to a solution of levulinic acid 6 (34.8 g, 0.30 mol) in methanol (300 mL) under cooling with ice and good stirring. The reaction mixture was stirred overnight at room temperature and then heated another 1.5 h to reflux. After the methanol was removed by distillation, the residue was dissolved in dichloromethane (200 mL) and washed successively with water (70 mL), saturated NaHCO₃ solution (70 mL), and water (3 × 70 mL). After drying (MgSO₄), filtration, and removal of the solvent under vacuum, the crude product was distilled with a Vigreux column under vacuum. After byproduct separation by the first fraction of about 17 g (54-80 °C/0.4 torr), the main product was obtained between 81°C and 92 °C/0.4 torr: 39 g (62%) of chromatographic uniform 7 as a colorless oil with R_f 0.2 (n-hexane / ethyl acetate 9:1); ref.:³¹ 45%; ref.:^32,33 38%; ref.:³⁴ 30%.

Methyl 5-hydroxy-4-oxopentanoate (5-Hydroxylevulinic Acid Methyl Ester) 8

Sodium formate (8.95 g, 131.6 mmol) was added to a solution of 5-bromolevulinic acid methyl ester (25 g, 119.6 mmol) in ethanol (99%, 161 g) and water (28 mL), and the solution was heated to reflux. The starting material was no longer visible by TLC after a reaction time of 3 h, and 2 product spots appeared at lower positions. After 18 h of reflux, only the more polar product of the 2 remained visible (R_f in n-hexane/ethyl acetate 6:4: starting material ∼0.8; intermediate product ∼0.45; product ∼0.2). After removal of the ethanol by distillation under vacuum, the residue was mixed with water (100 mL), the pH set to ∼5, and the residue extracted with ethyl acetate (10 × 30 mL). After the solvent was removed by distillation, 16.0 g of crude product was obtained. The product was purified from the few less polar contaminants by column chromatography on silica gel using n-hexane/ethyl acetate 7:3 as eluent: yield 12.85 g (74%) of chromatographically pure 8 as a colorless oil with R_f 0.2 (n-hexane/ethyl acetate 6:4); ref.:³⁷ 42%.

¹H NMR (500 MHz, CDCl₃) δ 2.67 (br s, 4H, 2-H₂, 3-H₂), 3.66 (s, 3H, CH₃), 4.29 (s, 2H, 5-H₂).

¹³C NMR (125.8 MHz, CDCl₃) δ 27.59 (C-2), 32.94 (C-3), 52.11 (CH₃), 68.3 (C-5), 172.95 (C-1), 208.32 (C-4).

(5R,8S)-1,6,9,13-Tetraoxadispiro[4.2.4.2]tetradecane-2,10-dione (meso-altaicadispirolactone) 11

A solution of potassium hydroxide (61 mg, 1.05 mmol) in methanol (5 mL) was added to a solution of 5-hydroxylevulinic acid methyl ester 8 (146 mg, 1.0 mmol) in methanol (5 mL). The reaction mixture was stirred at 35 °C for 4 h. The clear solution was adjusted to pH ∼4 with 1N HCl and evaporated in vacuo. The residue of 5-hydroxylevulinic acid 3 was codestillated 3 times with dichloromethane and finally dissolved in dichloromethane (10 mL). Afterwards, boron trifluoride etherate (40 µL) was added and the solution was stirred at room temperature for 3 days. The reaction mixture was washed with water (15 mL), and the organic phase dried with (MgSO₄) and evaporated in vacuo. The residue was chromatographed on a silica gel column with n-hexane/ethylacetate 70:30 → 50:50. The combined product fractions (R_f 0.4) were evaporated in vacuo and recrystallized from dichloromethane: 28 mg (25%) of pure colorless crystals of 11 with m.p. 262 °C; ref.:⁴² m.p. 265 °C to 268 °C, ref.:⁴³ m.p. 263 °C.

¹H NMR (500 MHz, DMSO-d₆) δ 2.05 (m, 2H, 4-H_A, 12-H_A), 2.13 (m, 2H, 4-H_B, 12-H_B), 2.56 (ddd, 2H, 3-H_A, 11-H_A), 2.71 (pt, 2H, 3-H_B, 11-H_B), 3.88 (d, 2H, 7-H_A, 14-H_A), 3.94 (d, 2H, 7-H_B, 14-H_B); J_3A,4A = J_11A,12A = 10.60, J_3A,4B = J_11A,12A = 10.30, J_3B,4A = J_11B,12A = 2.70, J_3B,4B = J_11B,12B = 10.30, J_3gem = J_11gem = 18.15, J_7gem = J_14gem = 12.42 Hz.

¹³C NMR (125.8 MHz, DMSO-d₆) δ 26.90 (C-3, C11), 29.09 (C-4, C12), 64.97 (C-7, C-14), 102.50 (C-5, C-8), 175.48 (C-2, C-10).

1,2,3,4,6-Penta-O-acetyl-β-D-glucopyranose 12

Water-free D-glucose (180.2 g, 1.0 mol) was stirred into acetic acid (180 mL). After addition of water-free sodium acetate (90.1 g, 1.1 mol), the suspension was heated to 90 °C under stirring. Then, acetic anhydride (630 mL, 6.7 mol) was added over 1 h under stirring and initial cooling. Stirring was continued for another 1 h at ca. 95 °C. The mixture was then cooled to ca. 70 °C and a solution of sulfuric acid (0.8 mL) in water (1.4 L), pre-warmed to ca. 70 °C, was slowly added under vigorous stirring. The solution was cooled whereby the product started to form crystals. Stirring was continued for another 1 h at ca. 10 °C. The crystallized product was then filtered by suction with a Büchner funnel and washed with water (3 × 200 mL). To obtain the pure β-compound, the wet product was recrystallized from ethanol (2 L). Upon drying in a vacuum oven at 60 °C, the pure pentaacetyl-β-D-glucose 12 was obtained: yield 239 g (61%), m.p. 128 °C to 129 °C, [α]_D²⁴ = + 3.9 (c = 5.0, CHCl₃); ref.:⁴⁷ 74%; ref.:⁵⁵ 50%.

¹H NMR (500 MHz, CDCl₃) δ 2.00 (s, 3H, 3-CH₃CO), 2.02 (s, 6H, 2-CH₃CO, 4-CH₃CO), 2.07 (s, 3H, 6-CH₃CO), 2.10 (s, 3H, 1-CH₃CO), 3.83 (ddd, 1H, 5-H), 4.10 (dd, 1H, 6-H_A), 4.28 (dd, 1H, 6-H_B), 5.12 (m, 2H, 2-H, 4-H), 5.24 (pt, 1H, 3-H), 5.70 (d, 1H, 1-H); J_1,2 = 8.32, J_2,3 = 9.38, J_3,4 = 9.38, J_4,5 = 9.95, J_5,6A = 2.05, J_5,6B = 4.62, J_6gem = 12.60 Hz.

¹³C NMR (125.8 MHz, CDCl₃) δ 20.72 (2-CH₃CO, 3-CH₃CO, 4-CH₃CO), 20.86 (6-CH₃CO), 20.96 (1-CH₃CO), 61.60 (C-6), 67.90 (C-4), 70.38 (C-2), 72.88 (C-5), 72.94 (C-3), 91.85 (C-1), 169.10 (1-CH₃CO), 169.40, 169.54 (2-CH₃CO, 4-CH₃CO), 170.25 (3-CH₃CO), 170.75 (6-CH₃CO).

2,3,4,6-Tetra-O-acetyl-α-D-glucopyranosyl bromide (Acetobromo-α-D-glucose) 13

Pentaacetyl-β-D-glucose 12 (39.0 g (0.1 mol) was stirred into water-free acetic acid (50 mL) and a solution of hydrogen bromide (74.0 g, 54.5 mL) in glacial acetic acid (33%) was added under stirring. After ca. 45 min, a suction evacuation was applied for ca. 30 min, while stirring was continued. Then, the reaction solution was added to a mixture of water (150 mL) and dichloromethane (150 mL) under stirring, and stirring was continued for another 30 min. After the phases had separated, the organic phase was mixed with a saturated NaHCO₃ solution (50 mL). Additional solid NaHCO₃ was added under constant stirring until the aqueous phase reached pH 7-8. After the phases had separated, the organic phase was washed with water (30 mL) and dried (MgSO₄). The solvent was removed under vacuum, and the product crystallized with tert-butyl methyl ether (50 mL). This yielded 26 g of the first crop of crystals and 6.4 g of secondary one in equal quality: A total yield 32.4 g (79%) of acetobromo-α-D-glucose 13 with m.p. 83 °C to 85 °C and [α]_D²⁰ = + 198.8 (c = 5.06, CHCl₃). The product was stored in a desiccator over potassium hydroxide and protected from light and is stable for months under these conditions; ref.:⁴⁷ 76%; ref.:⁵⁰ 80% to 85%.

¹H NMR (500 MHz, CDCl₃) δ 2.02, 2.03, 2.08, 2.09 (s, 12H, 4 CH₃CO), 4.11 (dd, 1H, 6-H_A), 4.28 (m, 1H, 5-H), 4.31 (m, 1H, 6-H_B), 4.82 (dd, 1H, 2-H), 5.14 (pt, 1H, 4-H), 5.54 (pt, 1H, 3-H), 6.59 (d, 1H, 1-H); J_1,2 = 4.01, J_2,3 = 9.80, J_3,4 = 9.66, J_4,5 = 9.66, J_5,6A = 1.11, J_6gem = 12.27 Hz.

¹³C NMR (125.8 MHz, CDCl₃) δ 20.69, 20.76, 20.79, 20.80 (4xCH₃CO), 61.09 (C-6), 67.31 (C-4), 72.28 (C-5), 70.31 (C-3), 70.74 (C-2), 86.72 (C-1), 169.59 (4-CH₃CO), 169.92 (2-CH₃CO), 169.98 (3-CH₃CO), 170.63 (6-CH₃CO).

Methyl 4-oxo-5-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]pentanoate, [2,3,4,6-Tetra-O-acetyl-5-(β-D-glucopyranosyloxy)levulinic acid methyl ester] 14

Mercury cyanide (2.2 g, 17.4 mmol) was added to a solution of 5-hydroxylevulinic acid methyl ester 8 (1.27 g, 8.7 mmol) in anhydrous benzene (150 mL) and nitromethane (150 mL) and heated to 60 °C. Using a dosing pump, a solution of acetobromoglucose 13 (3.58 g, 8.7 mmol) in benzene (50 mL) was then added with a flow rate of 10 mL/h. After 8 h, only about 50% had been converted (TLC). Again, mercury cyanide (1.1 g) and a solution of acetobromoglucose 13 (3.58 g, 8.7 mmol) in benzene (40 mL) were added (dosing pump, within 6 h). After 18 h of total reaction time, the reaction was complete (TLC).

The reaction mixture was then added to a saturated sodium bicarbonate (NaHCO₃) solution (200 mL) and stirred well for 15 min. The organic phase was separated and consecutively washed with water (100 mL) and potassium iodide solution (15%, 100 mL). Drying (Na₂SO₄) and removal of the solvent in vacuo left a crude product containing polar and less polar by-products. It was purified by column chromatography on silica gel and dichloromethane/methanol 99.5:0.5 as eluent. The mixed fractions were subjected to a second separation (methyl tert-butyl ether/n-hexane 60:40). The combined product fractions were recrystallized from ethanol: 2.5 g (60%) of 14 with R_f 0.2 (dichloromethane/methanol, 98:2), m.p. 93 °C to 96 °C and [α]_D²⁷ = −21.0 (c = 0.90, CH₂Cl₂); ref.:⁵³ m.p. 89 °C (EtOH), [α]_D²⁰ = −29 (c = 0.8, CHCl₃).

MS: HR-ESI 477.15990 [M + H]⁺ (calcd. for C₂₀H₂₈O₁₃ + H, 477.16027).

¹H NMR (500 MHz, CDCl₃) δ 2.00, 2.01, 2 × 2.08 (4 CH₃CO), 2.59 (m, 2H, 2-H₂), 2.78 (m, 2H, 3-H₂), 3.66 (s, 3H, CH₃CO), 3.69 (ddd, 1H, 5′-H), 4.11 (dd, 1H, 6′-H_A), 4.25 (m, 3H, 5-H₂, 6′-H_B), 4.56 (d, 1H, 1′-H), 5.07 (m, 2H, 2′-H, 4′-H), 5.21 (pt, 1H, 3′-H); J_1′,2′ = 7.86, J_2′,3′ = 9.41, J_{3′, 4′} = 9.41, J_4′,5′ = 9.98, J_5′,6′A = 2.22, J_5′,6′B = 4.74, J_6′gem = 12.37 Hz.

¹³C NMR (125.8 MHz, CDCl₃) δ 20.74, 20.75, 20.84(2x), (4 CH₃CO), 27.36 (C-2), 33.85 (C-3), 52.00 (CH₃CO), 61.86 (C-6′), 68.43 (C-4′), 71.16 (C-2′), 72.17 (C-5′), 72.70 (C-3′), 73.83 (C-5), 100,79 (C-1′), 169.55, 169.65 (2′-CH₃CO, 4′-CH₃CO), 170.33 (3′-CH₃CO), 170.77 (6′-CH₃CO), 173.15 (C-1), 206.37 (C-4).

Methyl 5-(β-D-glucopyranosyloxy)-4-oxopentanoate, [5-(β-D-glucopyranosyloxy)levulinic acid methyl ester]15

The tetra-O-acetyl-5-(β-D-glucopyranosyloxy)levulinic acid methyl ester 14 (952 mg, 2 mmol) was suspended in methanol (20 mL), and a solution of sodium methoxide (0.02 eq., 0.04 mmol) in methanol (100 mL) was added while stirring. After stirring for 5 h at room temperature, the conversion was complete (TLC). Acidic ion exchanger (wet IR 120 H⁺, 85 mg) was added, and the mixture was stirred for 15 min. After filtration of the ion exchanger and washing with methanol, the combined filtrates were evaporated to dryness in vacuo. The residue was purified by column chromatography on silica gel (dichloromethane/methanol 95:5 → 80:20): 580 mg (94%) of 15 as a chromatographically pure foam with R_f 0.05 (chloroform/methanol 95:5), R_f 0.25 (chloroform/methanol 80:20 and [α]_D²⁷ = −20.8 (c = 0.77, MeOH).

MS: HR-ESI 331.09982 [M + Na]⁺ (calcd. for C₁₂H₂₀O₉ + Na, 331.09995).

¹H NMR (500 MHz, DMSO-d₆) δ 2.50 (t, 2H, 2-H₂) overlapped by DMSO-d₆, 2.80 (t, 2H, 3-H₂), 3.02 (m, 1H, 2′-H), 3.05 (m, 1H, 4′-H), 3.08 (m, 1H, 5′-H), 3.13 (m, 1H, 3′-H), 3.33 (s, H₂O), 3.42 (m, 1H, 6′-H_A), 3.58 (s, 3H, OCH₃). 3.65 (m, 1H, 6′-H_B), 4.20 (d, 1H, 1′-H), 4.23 (d, 1H, 5-H_A), 4.32 (d, 1H, 5-H_B), 4.46 (t, 1H, 6′-OH), 4.93 (d, 1H, 4′-OH), 4.98 (d, 1H, 3′-OH), 5.11 (d, 1H, 2′-OH); J_5gem = 17.08, J_1′,2′ = 7.80, J_5′,6′A = 6.31, J_5′,6′B = 1.72, J_6′gem = 11.94, J_2′,2′OH = 4.65, J_3′,3′OH = 4.81, J_4′,4′OH = 4.96, J_6′,6′OH = 6.01.

¹H NMR (500 MHz, DMSO-d₆, D₂O) δ 2.50 (br s, 2H, 2-H₂), 2.74 (br s, 2H, 3-H₂), 3.04 (m, 2H, 2′-H, 4′-H), 3.11 (m, 1H, 5′-H), 3.16 (pt, 1H, 3′-H), 3.41 (dd, 1H, 6′-H_A), 3.54 (s, 3H, OCH₃), 3.64 (d, 1H, 6′-H_B), 3.92 (s, H₂O), 4.18 (d, 1H, 1′-H), 4.23 (d. 1H, 5-H_A), 4.35 (d, 1H, 5-H_B); J_5gem = 17,25, J_1′,2′ = 7.79, J_2′,3′ = J_3′,4′ = 8.96, J_5′,6′A = 6.36, J_5′,6′B = 1.51, J_6′gem = 11.94 Hz. Characteristic differences to the spectrum without D₂O are the absence of the shifts for the protons of the hydroxyl groups and the deep field shift of H₂O (3.33 vs 3.92 ppm).

¹³C NMR (125.8 MHz, DMSO-d₆) δ 27.81 (C-2), 34.40 (C-3), 52.53 (CH₃O), 61.75 (C-6′), 70.90 (C-4′), 74.01 (C-5), 74.28 (C-2′), 77.47 (C-3′), 77.52 (C-5′), 103.62 (C-1′), 173.71 (C-1), 208.35 (C-4).

3-((2S,4aR,6R,7S,8S,8aR)-2,7,8-trihydroxy-6-(hydroxymethyl)hexahydro-6H-pyrano[2,3-b][1,4]dioxin-2-yl)propanoic acid 17

A solution of lithium hydroxide hydrate (1.1 eq., 0.174 mmol) in water (3 mL) was added to a solution of ester 15 (0.20 g, 0.65 mmol) in tetrahydrofuran (5 mL) under vigorous stirring. Vigorous stirring was continued for 1 h at room temperature, whereby complete conversion occurred (TLC). Acidic ion exchanger (wet IR 120 H⁺, about 2 eq., 300 mg) was then added, and the mixture was stirred for 15 min. After filtration of the ion exchanger and washing with tetrahydrofuran/water 1:1, the combined filtrates were evaporated to dryness in vacuum. The residue was co-distilled twice with tetrahydrofuran, and the crude product was purified by column chromatography on silica gel (dichloromethane/methanol/acetic acid 85:14:1): 196 mg (100%) of 17 as a chromatographically pure foam with R_f 0.1 (chloroform/methanol/acetic acid 80:19:1 and [α]_D²⁷ = −19.5 (c = 1.39, MeOH).

MS: HR-ESI 317.08408 [M + Na]⁺ (calcd. for C₁₁H₁₈O₉ + Na, 317.08430).

¹H NMR (500 MHz, DMSO-d₆) δ 2.41 (t, 2H, 3-H₂), 2.71 (br s, 2H, 2-H₂), 3.02 (m, 1H, 8′a-H), 3.04 (m, 1H, 7′-H), 3.09 (m, 1H, 6′-H), 3.13 (m, 1H, 8′-H), 3.41 (dd, 1H, 6′′-H_A), 3.64 (d, 1H, 6′′-H_B), 4.18 (d, 1H, 4′a-H), 4.20 (d, 1H, 3′-H_A), 4.32 (d, 1H, 3′-H_B), 3.30 and 5.50 (each br s, 2′-OH, 7′-OH, 8′-OH, 6′′-OH), 11.82 (br s, 1H, COOH); J_2,3 = 6.0, J_3′gem = 17.13, J_4′a,8′a = 7.64, J_{6′,6′′HA} = 6.32, J_{6′,6′′HB} = 0, J_6′′gem = 12.1 Hz.

¹³C NMR (125.8 MHz, DMSO-d₆) δ 27.42 (C-3), 33.52 (C-2), 61.05 (C-6′′), 69.96 (C-7′), 73.12 (C-3′), 73.36 (C-8′a), 76.61 (C-8′), 77.05 (C-6′), 102.70 (C-2′, C-4′a), 174.12 (C-1).

( + )-Ranuncoside Monohydrate, [(2R,4ʹaR,6ʹR,7ʹS,8ʹS,8ʹaR)-7ʹ,8ʹ-Dihydroxy-6ʹ-(Hydroxymethyl)hexahydro-3H-Spiro[furan-2,2ʹ-Pyrano[2,3-b][1,4]dioxin]-5(4H)-One - Hydrate (1)]

p-Toluenesulfonic acid monohydrate (20 mg) was added to a solution of pyranodioxane 17 (200 mg, 0.68 mmol) in anhydrous tetrahydrofuran (25 mL). The mixture was refluxed over 4 days, whereby most of 17 had been converted to 1 (TLC). The mixture was evaporated in vacuo and the residue was purified by column chromatography on silica gel (dichloromethane/methanol 95:5). The obtained product was recrystallized from ethanol/water 80:20: 152 mg (81%) of pure colorless crystals of ( + )-ranuncoside 1 with m.p. 216 °C to 218 °C and [α]_D²⁷ = + 51.7 (c = 0.40, MeOH). The synthesized natural product was identical in all respects to a sample isolated from H niger by Cuny and Klingler.²⁰

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221145919 - Supplemental material for Biomimetic Total Synthesis of ( + )-Ranuncoside, a Unique Tricyclic Spiroacetal Glycoside of Christmas Rose (Helleborus niger L.)

Supplemental material, sj-docx-1-npx-10.1177_1934578X221145919 for Biomimetic Total Synthesis of ( + )-Ranuncoside, a Unique Tricyclic Spiroacetal Glycoside of Christmas Rose (Helleborus niger L.) by Eckehard Cuny, Jörg Fohrer and Franz-Dietrich Klingler in Natural Product Communications

Footnotes

Acknowledgements

The author EC thanks Prof. Dr Michael Reggelin for the opportunity to work in his group. The author FDK thanks Dr Vibhuti Klingler-Dabral for providing the facilities for product synthesis and proofreading of the manuscript.

Author Contributions

FDK performed the synthesis of ( + )-ranuncoside. All 3 authors performed the structural elucidation. EC prepared the figures and schemes and wrote the manuscript.

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) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Eckehard Cuny

Supplemental Material

Supplemental material for this article is available online.

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Supplementary Material

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Biomimetic Total Synthesis of ( + )-Ranuncoside,a Unique Tricyclic Spiroacetal Glycoside of Christmas Rose ( Helleborus niger L.)

Abstract

Keywords

Introduction

Results and Discussion

Retrosynthesis

Synthesis of the Levulinic Acid–Derived Starting Material 8

Altaicadispirolactone (1,6,9,13-tetraoxadispiro[4.2.4.2]tetradecane-2,10-dione) 11

Synthesis of the D-Glucose-Derived Starting Material 13

Biomimetic Synthesis of ( + )-ranuncoside 1

Conclusions

Experimental

Methyl 5-bromo-4-oxopentanoate (5-Bromolevulinic Acid Methyl Ester) 7

Methyl 5-hydroxy-4-oxopentanoate (5-Hydroxylevulinic Acid Methyl Ester) 8

(5R*,8S*)-1,6,9,13-Tetraoxadispiro[4.2.4.2]tetradecane-2,10-dione (meso-altaicadispirolactone) 11

1,2,3,4,6-Penta-O-acetyl-β-D-glucopyranose 12

2,3,4,6-Tetra-O-acetyl-α-D-glucopyranosyl bromide (Acetobromo-α-D-glucose) 13

Methyl 4-oxo-5-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]pentanoate, [2,3,4,6-Tetra-O-acetyl-5-(β-D-glucopyranosyloxy)levulinic acid methyl ester] 14

Methyl 5-(β-D-glucopyranosyloxy)-4-oxopentanoate, [5-(β-D-glucopyranosyloxy)levulinic acid methyl ester]15

3-((2S,4aR,6R,7S,8S,8aR)-2,7,8-trihydroxy-6-(hydroxymethyl)hexahydro-6H-pyrano[2,3-b][1,4]dioxin-2-yl)propanoic acid 17

( + )-Ranuncoside Monohydrate, [(2R,4ʹaR,6ʹR,7ʹS,8ʹS,8ʹaR)-7ʹ,8ʹ-Dihydroxy-6ʹ-(Hydroxymethyl)hexahydro-3H-Spiro[furan-2,2ʹ-Pyrano[2,3-b][1,4]dioxin]-5(4H)-One - Hydrate (1)]

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221145919 - Supplemental material for Biomimetic Total Synthesis of ( + )-Ranuncoside, a Unique Tricyclic Spiroacetal Glycoside of Christmas Rose (Helleborus niger L.)

Footnotes

Acknowledgements

Author Contributions

Declaration of Conflicting Interests

Funding

ORCID iD

Supplemental Material

References

Supplementary Material

(5R,8S)-1,6,9,13-Tetraoxadispiro[4.2.4.2]tetradecane-2,10-dione (meso-altaicadispirolactone) 11