Synthesis and biological evaluation of bergenin derivatives as new immunosuppressants

Abstract

Bergenin, which is isolated from Bergenia species, exhibits various pharmacological properties. In the search for new types of immunosuppressants, a series of bergenin derivatives were designed and synthesized, and their immunosuppressive effects were evaluated by the CCK-8 assay. The experimental data demonstrated that compounds 7 and 13 showed the strongest inhibition effects on mouse splenocyte proliferation (IC₅₀ = 3.52 and 5.39 μM, respectively). Further studies revealed that the inhibitory effect may come from the suppression of both IFN-γ and IL-4 cytokines. Alkylated derivatives of bergenin with n-hexyl and n-heptyl on the two phenolic hydroxyl groups showed better inhibitory activities. The hydrophobicity of bergenin derivatives, the configuration of the 4-OH in bergenin, and the ability to form hydrogen bonds of the substituents on the C-4 position are important to the immunosuppressive activity. This work proved that the modifications of bergenin may represent a new route to the discovery of a new class of immunosuppressive agents.

A series of bergenin derivatives that may serve as new immunosuppressive agents have been synthesized. Among them, compounds 7 and 13 showed the strongest inhibition on mouse splenocyte proliferation (IC₅₀ = 3.52 and 5.39 μM, respectively).

Introduction

Immunosuppressive agents^1,2 are drugs that suppress the immune system. They are used in immunosuppressive therapy to prevent the rejection of transplanted organs and tissues, to treat autoimmune diseases like rheumatoid arthritis, multiple sclerosis, myasthenia gravis, vitiligo, systemic lupus erythematosus, or to treat some other non-autoimmune inflammatory diseases such as long term allergic asthma. The most often used immunosuppressive agents can be divided into three types: chemically synthesized small molecular drugs such as glucocorticoid hormone, cyclophosphamide and azathioprine, microbial products such as cyclosporin A and FK-506, and Chinese herbal medicines such as tripterygium glycosides. Although some new immunosuppressive drugs such as leflunomide, mycophenolate mofetil, sirolimus and tacrolimus have been discovered and developed for clinical use in organ-transplantation,³ more effective and safer immunosuppressive agents with novel structure skeletons are needed due to the chronic rejection after transplantation.

Bergenin (1) is a kind of isocoumarin compound. It is a fused β-C-glucoside of 4-O-methylgallic acid, which naturally occurs in some species such as B. purpurascens (Saxifragaceae) and A. japonica (Myrsinaceae) as well as several other plant species.⁴ It possesses various pharmacological effects such as anti-inflammatory,^5–7 neuroprotective,^8–10 hepatoprotective,^11–13 anti-platelet aggregation,¹⁴ antinociceptive,¹⁵ antiglycation,¹⁶ antiarrhythmic,¹⁷ anti-α-glucosidase¹⁸ and anticancer^19–21 activities. However, bergenin and its derivatives as immunosuppressive agents have been less explored. In fact, bergenin has been shown to have an effect on the generation of some kinds of cytokines in response to the immune system, inflammation, trauma, cancer, and reproduction.^22–24 In particular, bergenin exhibits a potent anti-arthritic activity by modulating Th1/Th2 cytokine production.²⁵ Since bergenin can modulate the cytokine production of Th1/Th2 cells, it could serve as a new scaffold for new types of immunosuppressive agents. Herein we report structural modifications of bergenin (Fig. 1), and the biological activities of synthetic bergenin derivatives as new immunosuppressants are also investigated.

Fig. 1. Bergenin and its structural modifications.

Results and discussion

Chemistry

There are multiple functional groups accessible for modification in commercially available bergenin (1). Previous studies by our group and others on iminosugars as a new type of immunosuppressant suggested that the immunosuppressive activities can be improved by increasing their hydrophobicity; in particular, iminosugars with an alkyl chain of seven carbons exhibit the best immunosuppressive activity.^26,27 In order to increase the lipophilicity of 1, the two phenolic hydroxyl groups in 1 were alkylated with various alkyl bromides under basic conditions, affording 8,10-disubstituted compounds 2a–2g. To further reduce the polarity of bergenin, compounds 1, 2d, and 2g were treated with benzaldehyde dimethyl acetal in the presence of camphorsulfonic acid, giving the 3,11-O-benzylidene bergenin derivatives 3, 4, and 7, respectively. The regioselective reductive cleavage of 3,11-O-benzylidene in compound 4 produced compound 5 or 6 (Fig. 2).

Fig. 2. Modification of bergenin 1 (synthesis of bergenin derivatives 2a–2g and 3–7). Reagents and conditions: a) for 2a, 2d, and 2f: RBr (1.5 equiv.), KI (10% mmol), K₂CO₃ (1.5 equiv.), DMF, 80 °C, overnight; for 2a, yield: 25%; for 2d, yield: 26%; for 2f, yield: 22%. b) For 2b, 2c, and 2e: RBr (2.5 equiv.), KI (10% mmol), K₂CO₃ (1.5 equiv.), DMF, 60 °C, overnight; for 2b, yield: 90%; for 2c, yield: 86%; for 2e, yield: 89%. c) For 2g: PhCH₂Br (2.5 equiv.), K₂CO₃ (8.0 equiv.), DMF, r.t., 12 h, yield: 90%. d) Benzaldehyde dimethyl acetal, camphorsulfonic acid, CH₃CN, r.t., 8 h; for 3, yield: 50%; for 4, yield: 83%; for 7, yield: 90%. e) 4 Å molecular sieves, TFA, Et₃SiH, CH₂Cl₂, 0 °C to r.t., 5 h, Ar, 63%. f) BH₃·THF, TMSOTf, CH₂Cl₂, 0 °C to r.t., 5 h, Ar, 61%.

To investigate the influence of the C-4 substituents on the immunosuppressive activity, compounds 8–13 were designed and synthesized. Dess–Martin oxidation of bergenin derivative 7, which was followed by reduction with sodium borohydride, provided compound 8 with C-4 configuration inversion.²⁸ Compounds 7 and 8 were treated with trifluoromethanesulfonic anhydride in the presence of pyridine, affording the corresponding 4-triflate derivatives 9 and 10. Treatment of 10 with sodium azide in DMF gave azido-containing compound 11. Reduction of the azido group in 11 with H₂S gas (generated by the reaction of FeS and 10% H₂SO₄) provided compound 12, which was further treated with methanesulfonyl chloride, yielding sulfamide derivative 13 (Fig. 3).

Fig. 3. Modification at the C-4 position of compound 7. Reagents and conditions: a) Dess–Martin reagent, CH₂Cl₂, 0 °C to r.t., 6–8 h. b) NaBH₄, CH₂Cl₂/EtOH (1 : 2), 0 °C to r.t., 24–48 h; yield: 42% over two steps. c) Tf₂O, pyridine, CH₂Cl₂, 0 °C to r.t., 6–12 h, Ar; for 9, yield: 80%; for 10, yield: 76%; d) NaN₃, DMF, 80 °C, overnight, 93%. e) FeS, 10% H₂SO₄, pyridine, r.t., 12 h, 88%. f) MsCl, Et₃N, CH₂Cl₂, 0 °C to r.t., 4 h, 91%.

Biological evaluation

Mouse splenocyte proliferation inhibition assay

With the bergenin derivatives in hand, the effects of bergenin 1 and synthetic compounds 2a–2g and 3–13 on Con A induced mouse splenocyte proliferation were assessed by the cell-counting kit-8 (CCK-8) assay.²⁷ This assay is efficient and serves as a good indicator of anti-T-cell-proliferation activities. T cells play a major role in graft rejection.²⁹ The mouse splenocytes were induced with 5 μg mL⁻¹ of Con A, and were treated with 10 μM concentration of the tested compounds at 37 °C, 5% CO₂ for 48 h. The assay was conducted using the Con A-treated splenocytes as the experimental control and cyclosporine A (CSA, 1 μM, 93.5% inhibitory rate) and betamethasone (10 μM, 89.6% inhibitory rate) treated splenocytes as the positive control. The results are summarized in Table 1. As shown in Table 1, compared with the control, the inhibitory rate seems to be related to the length of linear carbon chain R¹, and the activity of the 6- or 7-carbon chain is the best, which can be seen by the fact that compound 2c and compound 2d showed an inhibitory rate of 98.10% and 99.03%, respectively. In contrast, there was no inhibitory activity when R¹ was the benzyl group (compound 2g). Neither compound 3 with the 3,11-O-benzylidene modification of bergenin nor compound 4 with the combined modification pattern in 2d and 3, showed immunosuppression activity. The ring opening analogues 5 and 6 showed the significantly increased inhibitory activity compared with compound 4. Compound 7, with the combined modification in 2g and 3, had an inhibitory rate of 91.44%, but the activity of compound 8 decreased sharply due to the inversion of configuration on the C-4 position. Compared with compound 7, the inhibitory rates of compounds 9, 11, 12, and 13 were −1.62%, −2.26%, 72.64%, and 84.86%, respectively. The inhibitory rate difference may arise from the different substitution at the C-4 position. Because 4β-OH (7), 4β-NH₂ (12), and 4β-NHSO₂CH₃ (13) can serve as both hydrogen bond donors and acceptors, in contrast, 4β-OSO₂CF₃ (9) and 4β-N₃ (11) can only act as hydrogen bond acceptors; this difference in the hydrogen binding pattern may lead to the difference of drug-receptor binding. Compound 9 showed the opposite activity to its 4α-triflate isomer 10.

Inhibitory rates of the bergenin derivatives on splenocyte proliferation^a.

					Inhibitory rate (±SEM) %
	R1	R2	R3	R4
1	–H	–H	–H		11.7 (±11.91)
2a	–(CH2)3CH3	–H	–H		−9.27 (±4.01)
2b	–(CH2)4CH3	–H	–H		5.40 (±5.10)
2c	–(CH2)5CH3	–H	–H		98.10 (±0.27)
2d	–(CH2)6CH3	–H	–H		99.03 (±0.15)
2e	–(CH2)7CH3	–H	–H		−21.50 (±4.73)
2f	–(CH2)9CH3	–H	–H		15.37 (±3.53)
2g	–CH2Ph	–H	–H		−5.95 (±4.84)
3	–H				−3.78 (±1.98)
4	–(CH2)6CH3				−6.83 (±4.59)
5	–(CH2)6CH3	–CH2Ph	–H		99.4 (±0.16)
6	–(CH2)6CH3	–H	–CH2Ph		69.8 (±10.71)
7	–CH2Ph				91.44 (±0.72)
8	–CH2Ph				11.24 (±4.86)
9	–CH2Ph				−1.62 (±13.18)
10	–CH2Ph				75.92 (±3.39)
11	–CH2Ph				−2.26 (±8.84)
12	–CH2Ph				72.64 (±3.46)
13	–CH2Ph				84.86 (±1.79)

^aEffects of compounds on Con A-induced mouse splenocyte proliferation were assessed by the CCK-8 assay. Cyclosporin A (CSA, 1 μM, 93.5% inhibitory rate) and betamethasone (10 μM, 89.6% inhibitory rate) were used as the positive control. The concentration of compounds was 10 μM. Data are means ± SEM of at least three independent experiments.

The 50% inhibitory concentrations (IC₅₀) of the synthetic compounds exhibiting an inhibitory rate over 50% at the concentration of 10 μM were tested and the results are summarized in Table 2. Among them, compounds 7 and 13 displayed promising IC₅₀ values of 3.52 and 5.39 μM, respectively.

Immunosuppressive activity and cytotoxicity of selected compounds (IC₅₀ in μM)^a.

Compound	Inhibition of mouse splenocyte proliferation	Jurkat cytotoxicity
2c	8.16 (±0.99)	5.21 (±1.33)
2d	7.21 (±0.07)	4.18 (±0.89)
5	7.66 (±0.17)	10.1 (±0.65)
6	10.6 (±0.82)	16.08 (±1.91)
7	3.52 (±1.07)	21.0 (±3.79)
10	8.05 (±0.64)	44.16 (±7.4)
12	8.19 (±0.31)	39.33 (±7.86)
13	5.39 (±0.38)	33.06 (±3.92)

^aIC₅₀ values were determined from the results of at least three independent tests.

Cytotoxicity assay

The cytotoxicity of compounds 2c, 2d, 5, 6, 7, 10, 12, and 13 was also assayed by the CCK-8 method using Jurkat cells.³⁰ Jurkat cells are an immortalized line of human T-lymphocyte cells. Their replication does not depend on the activation of T-cell receptors,³¹ thus allowing us to determine toxicity in the relevant cells whilst excluding the immunosuppressive effects associated with T-cell activation. The cytotoxicity was determined as the concentration of a compound that decreased cell viability to 50% (IC₅₀), and the results are summarized in Table 2. Compound 7, which showed the best IC₅₀ towards spleen-proliferation, exhibited a selective index of 5.96, while compounds 2c and 2d displayed similar potencies and toxicities. Compounds 5 and 6 possessed the narrowest therapeutic window. Compound 13 showed more than 6-fold selectivity and the widest therapeutic window among all of the eight tested compounds, probably owing to its high lipophilicity. Although its selectivity was moderate, compound 13 might be used as a probe to elucidate the possible new mechanism of bergenin derivatives as immunosuppressive agents.

Cytokine-secretion assay

To further confirm the immunosuppressive activities of these compounds, we next tested their effects on the secretion of cytokines from mouse splenocytes. Mouse spleen cells that were induced by 5 μg mL⁻¹ Con A were incubated with each compound at two different concentrations at 37 °C and 5% CO₂ for 48 h.³² The amount of cytokines was measured from the supernatant of spleen cells with enzyme-linked immunosorbent assay (ELISA). The data are shown in Fig. 4. All of these eight compounds showed inhibitory activity to IFN-γ secretion. Compared with the control, the levels of IFN-γ secretion were reduced by 67.4%, 101.5%, 99.6%, 51.3%, 71.0%, 68.6%, 48.4%, and 71.1% when including 10 μM of compound 2c, 2d, 5, 6, 7, 10, 12, and 13, respectively (Fig. 4a, 99.3% for betamethasone at 10 μM, −6.2% for bergenin at 10 μM). The assay on the secretion of IL-4 from splenocytes was similar to the assay of IFN-γ. The supernatant of the spleen cells was detected by using the mouse IL-4 ELISA kit. All of these eight compounds showed inhibition to the IL-4 secretion. The levels of IL-4 secretion were reduced by 61.9%, 98.6%, 95.9%, 51.4%, 76.9%, 63.1%, 8.7%, and 67.5%, when using 10 μM of compound 2c, 2d, 5, 6, 7, 10, 12, and 13, respectively (Fig. 4b, 96.9% for betamethasone at 10 μM, 18.3% for bergenin at 10 μM). It was found that among the eight tested compounds, compounds 2d, 5, 7 and 13 displayed the strongest inhibitory effect on the release of both IFN-γ and IL-4 at the concentration of 10 μM. These results are generally consistent with the inhibitory rate of the splenocyte proliferation assay.

Fig. 4. a) Inhibitory rates on IFN-γ secretion of the tested compounds. b) Inhibitory rates on IL-4 secretion of the tested compounds. Each compound was tested at two different concentrations: 5 and 10 μM. Data are means ± SEM of at least three independent experiments.

Con A is a specific activator of the T cells that play a central role in the cell-mediated immunity. T cells contain two classes of T lymphocytes, T helper cells (Th) and T cytotoxicity (Tc) cells. Th cells are further classified into Th1 and Th2. Th1 cells produce pro-inflammatory cytokines like IFN-γ, TNF-β, and IL-2, while Th2 cells produce cytokines such as IL-4, IL-5, IL-6, and IL-13. IL-4, an anti-inflammatory cytokine secreted by the Th2 cells, is a key regulator in humoral and adaptive immunity.³³ It is the hallmark cytokine produced by the Th2 cells and has many biological roles, including stimulation of activated B-cells as well as proliferation and differentiation of T cells. In contrast, the cytokine IFN-γ, the hallmark cytokine of the Th1 cells, shows antiviral, immunoregulatory, and antitumor properties and is used to treat infectious diseases, although it may precipitate autoimmunity. It increases expression of the chemokine receptors, which can mediate migration of immune cells to the site of inflammation.³⁴ Th1 responses predominate in organ-specific autoimmune disorders, acute allograft rejection, and in some chronic inflammatory disorders. In contrast, Th2 responses predominate in transplantation tolerance, chronic graft versus host disease, Omenn syndrome, systemic sclerosis, and allergic diseases. Based on the assay of cytokine secretion, it seems that compounds 2d, 5, 7 and 13 suppress both Th1 and Th2 cells. Therefore, these compounds might show inhibition activities toward both humoral responses and cell-mediated immune responses, showing potential to treat different kinds of immune diseases.³⁵ Taking into account their inhibitory effects on splenocyte proliferation and cytokine secretion, as well as their selectivity toward Jurkat cells, this new class of bergenin derivatives is promising for further studies.

Conclusions

Bergenin is responsible for multiple actions for the improvement of human health. Despite the fact that synthesis and biological activities of some bergenin derivatives were recently reported, only a few of them referred to their evaluation in immunological and inflammatory diseases. Herein, we designed and synthesized a series of bergenin derivatives starting from the naturally-occurring bergenin by modifications of the sugar moiety, substitution of the phenolic hydroxyls, substitution and/or inversion of the C-4 configuration. All synthetic compounds were evaluated in vitro for the immunosuppressive effects on the Con A induced proliferation of mouse splenocytes by the CCK-8 assay. The results showed that compounds 7 and 13 displayed the strongest inhibition effect (IC₅₀ = 3.52 μM and 5.39 μM, respectively). A mouse-cytokine-secretion assay and a Jurkat cytotoxicity assay were used to further evaluate their biological profiles. These studies revealed that the inhibitory effects on splenocyte proliferation may come from the suppression of both IFN-γ and IL-4 cytokines. To our knowledge, this is the first comprehensive investigation on the correlation between immunosuppressive activity and structures of bergenin derivatives. Considering both the potency and toxicity, compounds 7 and 13 can serve as lead compounds for the discovery of novel immunosuppressive agents. Our findings may open a new avenue in the development of new types of drugs possessing immunosuppressive activity.

Experimental section

General

All chemicals purchased were of reagent grade and used without further purification unless otherwise mentioned. Dichloromethane (CH₂Cl₂) was distilled over calcium hydride (CaH₂). Methanol was distilled from magnesium and iodine. Toluene and tetrahydrofuran (THF) were distilled over sodium/benzophenone. Reactions were monitored by analytical thin-layer chromatography (TLC) on silica gel 60 F₂₅₄ precoated on aluminum plates (E. Merck). Spots were visualized under UV (254 nm) light and/or by staining with acidic ceric ammonium molybdate. Solvents were evaporated under reduced pressure and below 45 °C (water bath). ¹H NMR, ¹³C NMR and 2D NMR spectra were recorded on an Avance III Bruker-600 or an Avance III Bruker-400 spectrometer. Chemical shifts were referenced to an internal SiMe₄ or to the residual solvent protons. Mass spectra were recorded using a Waters Xevo G2 Q-TOF spectrometer. All the final compounds have a purity of at least 95%, as determined by analytical HPLC which was performed on an Agilent 1260 Infinity system equipped with a VWD and the data were collected at 280 nm.

Preparation of compounds

General procedure A for the preparation of compounds 2a, 2d, and 2f

To a solution of bergenin (100.0 mg, 0.30 mmol) in N,N-dimethylformamide (5.0 mL) were added potassium carbonate (62.2 mg, 0.45 mmol), KI (5.1 mg, 10% mmol) and the respective alkyl bromide (1.5 equiv., 0.45 mmol). The reaction mixture was heated to 80 °C and stirred overnight, then the solvent was evaporated under reduced pressure and water was added. The mixture was extracted with ethyl acetate and the combined organic layers were washed with brine, dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated and the residue was purified by column chromatography on silica gel (CH₂Cl₂/CH₃OH = 25 : 1) to give the desired compounds 2a, 2d, and 2f (22–26% yield) as white solids. For the experimental details and compound characterization data, see the ESI.†

General procedure B for the preparation of compounds 2b, 2c, and 2e

To a solution of bergenin (100.0 mg, 0.30 mmol) in N,N-dimethylformamide (5.0 mL) were added potassium carbonate (62.2 mg, 0.45 mmol), KI (5.1 mg, 10% mmol) and 1-bromopentane (93 μL, 0.75 mmol)/1-bromohexane (105 μL, 0.75 mmol)/1-bromooctane (130 μL, 0.75 mmol). The reaction mixture was heated to 60 °C and stirred overnight, then the solvent was evaporated under reduced pressure and water was added. The mixture was extracted with ethyl acetate and the combined organic layers were washed with brine, dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated and then the residue was purified by column chromatography on silica gel (CH₂Cl₂/MeOH = 25 : 1) to give 2b (128.4 mg, 90% yield)/2c (130.1 mg, 86% yield)/2e (149.8 mg, 89% yield) as white solids. For the experimental details and compound characterization data, see the ESI.†

Compound 2g was prepared by the similar procedure as described in the preparation of 2b.

(6aR,7S,7aS,11aR,12aS)-1,3,7-Trihydroxy-2-methoxy-9-phenyl-6a,7,7a,11,11a,12a-hexahydro-5H-[1,3]dioxino[4′,5′:5,6]pyra-no[3,2-c]isochromen-5-one (3)

To a solution of bergenin (1.00 g, 3.00 mmol) in CH₃CN (20.0 mL) was added camphorsulfonic acid (0.139 g, 0.60 mmol) and benzaldehyde dimethyl acetal (0.68 mL, 4.53 mmol) at room temperature. The reaction mixture was stirred overnight and quenched with Et₃N (amounts equal to camphorsulfonic acid). The mixture was concentrated and the residue was purified by column chromatography (CH₂Cl₂/MeOH = 30 : 1) to give 3 (0.625 g, 50% yield) as a white solid. [α]²⁵_D −31.1 (c 0.4, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 7.68 (s, 1H), 7.52–7.41 (m, 2H), 7.35–7.26 (m, 3H), 6.45 (s, 1H), 5.55 (s, 1H), 4.80 (d, J = 10.2 Hz, 1H), 4.49–4.39 (m, 1H), 4.29–4.12 (m, 3H), 4.02 (s, 3H), 3.84–3.74 (m, 2H), 3.64 (t, J = 8.5 Hz, 1H), 2.05 (brs, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 163.46, 150.13, 147.01, 140.10, 136.56, 129.44, 128.45, 126.35, 118.13, 115.36, 110.27, 102.20, 80.49, 79.87, 74.82, 71.79, 70.81, 68.12, 61.04. HRMS (ESI) calcd for C₂₁H₂₁O₉ [M + H]⁺, 417.1180; found, 417.1185.

(6aR,7S,7aS,11aR,12aS)-1,3-Bis(heptyloxy)-7-hydroxy-2-methoxy-9-phenyl-6a,7,7a,11,11a,12a-hexahydro-5H-[1,3]dioxino[4′,5′:5,6]pyrano[3,2-c]isochromen-5-one (4)

This compound was prepared in the same manner as described in the preparation of 3. Compound 2d (130.8 mg, 0.25 mmol), camphorsulfonic acid (16.4 mg, 0.07 mmol) and benzaldehyde dimethyl acetal (56 μL, 0.375 mmol) were used to afford 4 (126.9 mg, 83% yield) as a white solid. [α]²⁵_D −20.5 (c 0.3, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 7.52 (dd, J = 7.2, 2.0 Hz, 2H), 7.44 (s, 1H), 7.41–7.32 (m, 3H), 5.59 (s, 1H), 4.89–4.79 (m, 1H), 4.53–4.41 (m, 1H), 4.26–4.16 (m, 2H), 4.09–3.96 (m, 3H), 3.93 (s, 3H), 3.91–3.81 (m, 2H), 3.76–3.66 (m, 2H), 3.34 (brs, 1H), 1.89–1.80 (m, 2H), 1.79–1.66 (m, 2H), 1.52–1.42 (m, 4H), 1.41–1.29 (m, 12H), 0.90 (t, J = 6.3 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 163.77, 153.49, 150.71, 149.15, 136.91, 129.40, 128.45, 126.41, 125.22, 118.85, 110.86, 102.22, 80.97, 80.69, 74.75, 73.49, 71.56, 69.25, 68.71, 61.18, 31.96, 31.84, 30.36, 29.28, 29.16, 29.07, 26.09, 26.06, 22.74, 22.68, 14.19, 14.17. HRMS (ESI) calcd for C₃₅H₄₉O₉ [M + H]⁺, 613.3371; found, 613.3373.

(2R,3S,4S,4aR,10bS)-2-((Benzyloxy)methyl)-8,10-bis(heptyloxy)-3,4-dihydroxy-9-methoxy-3,4,4a,10b-tetrahydropyrano[3,2-c]iso-chromen-6(2H)-one (5)

To a mixture of 4 (76.5 mg, 0.13 mmol) and 4 Å molecular sieves (4.0 mg) in dry CH₂Cl₂ (2.0 mL), TFA (0.05 mL, 0.62 mmol) and Et₃SiH (0.1 mL, 0.62 mmol) were added at 0 °C under an argon atmosphere. The reaction mixture was allowed to warm to room temperature and stirred for 5 h. The mixture was concentrated and purified by column chromatography on silica gel (petroleum ether/CH₂Cl₂/ethyl acetate = 3 : 2 : 1) to give 5 (48.0 mg, 63% yield) as a white solid. [α]²⁵_D −19.8 (c 0.2, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 7.42 (s, 1H), 7.39–7.27 (m, 5H), 4.72 (d, J = 10.3 Hz, 1H), 4.61 (q, J = 12.1 Hz, 2H), 4.13 (t, J = 9.8 Hz, 1H), 4.08–3.92 (m, 5H), 3.91 (s, 3H), 3.90–3.84 (m, 1H), 3.84–3.76 (m, 2H), 3.75–3.67 (m, 1H), 3.05 (brs, 1H), 1.89–1.25 (m, 20H), 0.89 (t, J = 6.7 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 164.07, 153.48, 151.06, 149.16, 137.68, 128.66, 128.08, 127.92, 125.75, 118.95, 110.70, 79.88, 78.52, 75.07, 74.81, 73.88, 72.39, 71.90, 70.10, 69.25, 61.19, 32.07, 31.88, 30.38, 29.34, 29.20, 29.12, 26.12, 26.09, 22.80, 22.73, 14.27, 14.22. HRMS (ESI) calcd for C₃₅H₅₁O₉ [M + H]⁺, 615.3522; found, 615.3537.

(2R,3S,4S,4aR,10bS)-3-(Benzyloxy)-8,10-bis(heptyloxy)-4-hydroxy-2-(hydroxymethyl)-9-methoxy-3,4,4a,10b-tetrahydropyrano[3,2-c]isochromen-6(2H)-one (6)

Compound 4 (82.0 mg, 0.13 mmol) and 4 Å molecular sieves (4.0 mg) were added in CH₂Cl₂ (2.0 mL) under an argon atmosphere, BH₃·THF (0.26 mL, 0.27 mmol) and TMSOTf (2 μL, 0.01 mmol) were added at 0 °C under vigorous stirring. After stirring for 5 h, TLC showed complete consumption of the starting material and Et₃N was added, followed by careful addition of MeOH until H₂ ceased to form. The solvent was removed under reduced pressure and the product was purified by column chromatography on silica gel (petroleum ether/CH₂Cl₂/ethyl acetate = 3 : 2 : 1) to give 6 (50.3 mg, 61% yield) as a white solid. [α]²⁵_D −6.5 (c 0.3, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 7.43 (s, 1H), 7.41–7.28 (m, 5H), 4.98 (d, J = 11.3 Hz, 1H), 4.75 (d, J = 11.3 Hz, 1H), 4.71 (d, J = 10.0 Hz, 1H), 4.19–4.07 (m, 2H), 4.07–3.99 (m, 2H), 3.99–3.93 (m, 3H), 3.92 (s, 3H), 3.76 (dd, J = 11.9, 3.9 Hz, 1H), 3.67–3.56 (m, 2H), 3.05 (brs, 1H), 1.98 (brs, 1H), 1.89–1.80 (m, 2H), 1.79–1.68 (m, 2H), 1.52–1.39 (m, 4H), 1.39–1.27 (m, 12H), 0.92–0.85 (m, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 164.03, 153.55, 150.88, 148.85, 138.04, 128.70, 128.35, 128.20, 125.38, 118.94, 110.67, 80.28, 79.86, 77.23, 75.80, 75.12, 74.72, 72.10, 69.29, 62.46, 61.20, 31.96, 31.87, 30.40, 29.23, 29.18, 29.10, 26.08, 26.04, 22.74, 22.72, 14.21. HRMS (ESI) calcd for C₃₅H₅₁O₉ [M + H]⁺, 615.3528; found, 615.3533.

(6aR,7S,7aS,11aR,12aS)-1,3-Bis(benzyloxy)-7-hydroxy-2-methoxy-9-phenyl-6a,7,7a,11,11a,12a-hexahydro-5H-[1,3]dioxino[4′,5′:5,6]pyrano[3,2-c]isochromen-5-one (7)

This compound was prepared in the same manner as described in the preparation of 3. To a solution of compound 2f (5.00 g, 9.84 mmol) in CH₃CN (40.0 mL) was added camphorsulfonic acid (0.457 g, 1.97 mmol) and benzaldehyde dimethyl acetal (2.2 mL, 14.66 mmol) at room temperature. The reaction mixture was stirred for 4 h and quenched with Et₃N (amounts equal to camphorsulfonic acid) and concentrated. The residue was purified by column chromatography on silica gel (CH₂Cl₂/MeOH = 30 : 1) to give 7 (5.28 g, 90% yield) as a white solid. [α]²⁵_D −25.0 (c 0.4, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 7.58 (s, 1H), 7.54–7.22 (m, 15H), 5.52 (s, 1H), 5.16 (q, J = 11.7 Hz, 2H), 5.08 (d, J = 10.8 Hz, 1H), 4.94 (d, J = 10.8 Hz, 1H), 4.62 (d, J = 9.6 Hz, 1H), 4.23–4.09 (m, 2H), 4.05 (dd, J = 10.2, 4.6 Hz, 1H), 3.97 (s, 3H), 3.71–3.59 (m, 2H), 3.58–3.47 (m, 1H), 3.26 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 163.53, 152.95, 150.16, 149.48, 137.54, 136.87, 135.99, 129.33, 128.77, 128.55, 128.51, 128.38, 128.19, 127.65, 127.61, 126.36, 126.18, 118.91, 111.87, 102.08, 80.91, 80.56, 75.93, 73.33, 71.44, 71.40, 71.08, 68.52, 61.39. HRMS (ESI) calcd for C₃₅H₃₃O₉ [M + H]⁺, 597.2119; found, 597.2134.

(6aR,7R,7aS,11aR,12aS)-1,3-Bis(benzyloxy)-7-hydroxy-2-methoxy-9-phenyl-6a,7,7a,11,11a,12a-hexahydro-5H-[1,3]dioxino[4′,5′:5,6]pyrano[3,2-c]isochromen-5-one (8)

To a solution of 7 (3.21 g, 5.38 mmol) in dry CH₂Cl₂ (40.0 mL) was added Dess–Martin periodinane (4.56 g, 10.76 mmol) at 0 °C under an argon atmosphere. The reaction was allowed to warm to room temperature and stirred for 6 h, then the mixture was extracted with CH₂Cl₂, washed with Na₂S₂O₃ (aq) and brine, dried over anhydrous sodium sulfate and concentrated. The residue was dissolved in dry CH₂Cl₂/dry EtOH (1 : 2), and NaBH₄ (305.3 mg, 8.07 mmol) was added at 0 °C under an argon atmosphere. The reaction was allowed to warm to room temperature and stirred until TLC indicated the starting material was completely consumed. The reaction was quenched by NH₄Cl (aq). The mixture was extracted with CH₂Cl₂, washed with brine, dried over anhydrous sodium sulfate and concentrated. Purification by column chromatography on silica gel (petroleum ether/CH₂Cl₂/ethyl acetate = 4 : 1 : 1) afforded compound 8 (1.351 g, 42% yield over two steps) as a white solid. [α]²⁵_D −67.7 (c 0.1, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 7.60 (s, 1H), 7.52–7.31 (m, 15H), 5.56 (s, 1H), 5.28–5.13 (m, 3H), 5.10 (d, J = 10.8 Hz, 1H), 4.97 (d, J = 10.8 Hz, 1H), 4.62 (t, J = 2.3 Hz, 1H), 4.33 (dd, J = 10.5, 2.3 Hz, 1H), 4.17–4.04 (m, 2H), 3.98 (s, 3H), 3.73–3.59 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 163.93, 152.92, 150.67, 149.63, 137.68, 136.91, 136.10, 129.50, 128.84, 128.63, 128.52, 128.43, 128.23, 127.67, 127.60, 126.67, 126.37, 118.82, 111.90, 102.29, 78.79, 78.38, 75.85, 71.16, 69.41, 69.04, 67.18, 66.82, 61.46. HRMS (ESI) calcd for C₃₅H₃₃O₉ [M + H]⁺, 597.2125; found, 597.2130.

(6aS,7S,7aR,11aR,12aS)-1,3-Bis(benzyloxy)-2-methoxy-5-oxo-9-phenyl-6a,7,7a,11,11a,12a-hexahydro-5H-[1,3]dioxino[4′,5′:5,6]pyrano[3,2-c]isochromen-7-yl trifluoromethanesulfonate (9)

To a solution of compound 7 (308.1 mg, 0.52 mmol) in dry CH₂Cl₂ (5.0 mL), dry pyridine (166 μL, 2.06 mmol) and Tf₂O (174 μL, 1.03 mmol) in dry CH₂Cl₂ (2.0 mL) were added at 0 °C under an argon atmosphere. Stirring was continued until TLC indicated the starting material was consumed completely. The reaction mixture was partitioned between CH₂Cl₂ and H₂O. The organic layer was combined and washed with 2 N HCl (aq), NaHCO₃ (aq) and brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 6 : 1) to give 9 (301 mg, 80% yield) as a white solid. [α]²⁵_D −48.8 (c 0.1, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 7.59 (s, 1H), 7.51–7.32 (m, 15H), 5.57 (s, 1H), 5.27–5.13 (m, 3H), 5.09 (d, J = 10.9 Hz, 1H), 4.96 (d, J = 10.9 Hz, 1H), 4.62 (d, J = 10.3 Hz, 1H), 4.40 (t, J = 9.9 Hz, 1H), 4.11 (dd, J = 10.5, 4.8 Hz, 1H), 4.00 (s, 3H), 3.85 (t, J = 9.4 Hz, 1H), 3.71 (t, J = 10.3 Hz, 1H), 3.59–3.50 (m, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 161.78, 153.43, 150.26, 149.73, 137.43, 136.20, 135.93, 129.39, 128.91, 128.73, 128.56, 128.50, 128.45, 127.80, 127.73, 126.00, 124.94, 118.63, 112.25, 101.75, 83.57, 78.40, 76.15, 74.05, 71.75, 71.33, 68.29, 61.50. ¹⁹F NMR (376 MHz, CDCl₃) δ −73.68. HRMS (ESI) calcd for C₃₆H₃₂F₃O₁₁S [M + H]⁺, 729.1612; found, 729.1623.

(6aS,7R,7aR,11aR,12aS)-1,3-Bis(benzyloxy)-2-methoxy-5-oxo-9-phenyl-6a,7,7a,11,11a,12a-hexahydro-5H-[1,3]dioxino[4′,5′:5,6]pyrano[3,2-c]isochromen-7-yl trifluoromethanesulfonate (10)

This compound was prepared in the same manner as described in the preparation of 9. Compound 8 (200.0 mg, 0.34 mmol) was used instead of 7 to afford 10 (185.6 mg, 76% yield) as a white solid. [α]²⁵_D −36.3 (c 0.1, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 7.60 (s, 1H), 7.51–7.30 (m, 15H), 5.58 (s, 1H), 5.55 (s, 1H), 5.19 (dd, J = 19.0, 11.7 Hz, 2H), 5.11 (d, J = 11.0 Hz, 1H), 4.96 (d, J = 10.7 Hz, 2H), 4.46 (dd, J = 10.5, 1.8 Hz, 1H), 4.06–4.01 (m, 1H), 4.00 (s, 3H), 3.83–3.75 (m, 1H), 3.64 (t, J = 10.1 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 162.31, 153.34, 150.59, 149.81, 137.47, 136.26, 135.92, 129.61, 128.89, 128.70, 128.51, 128.39, 127.71, 127.58, 126.33, 125.44, 118.42, 112.06, 102.51, 80.44, 75.93, 75.90, 75.03, 71.25, 70.33, 68.71, 68.03, 61.53. ¹⁹F NMR (376 MHz, CDCl₃) δ −73.94. HRMS (ESI) calcd for C₃₆H₃₂F₃O₁₁S [M + H]⁺, 729.1612; found, 729.1624.

(6aR,7S,7aS,11aR,12aS)-7-Azido-1,3-bis(benzyloxy)-2-methoxy-9-phenyl-6a,7,7a,11,11a,12a-hexahydro-5H-[1,3]dioxino[4′,5′:5,6]pyrano[3,2-c]isochromen-5-one (11)

A stirred mixture of 10 (185.6 mg, 0.26 mmol) and NaN₃ (82.79 mg, 1.27 mmol) in N,N-dimethylformamide (6.0 mL) was heated to 80 °C and stirred overnight. The reaction mixture was partitioned between ethyl acetate and H₂O. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 6 : 1) to give 11 (46.3 mg, 93% yield) as a white solid. [α]²⁵_D −50.7 (c 0.1, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 7.59 (s, 1H), 7.52–7.30 (m, 15H), 5.56 (s, 1H), 5.18 (q, J = 11.6 Hz, 2H), 5.08 (d, J = 10.9 Hz, 1H), 4.94 (d, J = 10.9 Hz, 1H), 4.63 (d, J = 10.2 Hz, 1H), 4.14–4.05 (m, 2H), 4.03–3.94 (m, 1H), 3.99 (s, 3H), 3.71–3.63 (m, 1H), 3.62–3.53 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 162.95, 153.19, 150.20, 149.63, 137.51, 136.60, 135.97, 129.40, 128.87, 128.66, 128.51, 128.35, 127.77, 127.69, 126.10, 125.76, 118.79, 112.02, 101.87, 80.11, 78.72, 76.08, 74.23, 72.14, 71.21, 68.56, 62.98, 61.48. HRMS (ESI) calcd for C₃₅H₃₂N₃O₈ [M + H]⁺, 622.2184; found, 622.2188.

(6aR,7S,7aS,11aR,12aS)-7-Amino-1,3-bis(benzyloxy)-2-methoxy-9-phenyl-6a,7,7a,11,11a,12a-hexahydro-5H-[1,3]dioxino[4′,5′:5,6]py-rano[3,2-c]isochromen-5-one (12)

Into a solution of 11 (15.3 mg, 0.025 mmol) in dry pyridine, H₂S gas (generated in situ by the reaction of FeS and 10% H₂SO₄) was gently bubbled until the starting material was completely consumed. The mixture was concentrated and purified by column chromatography on silica gel (petroleum ether/acetone = 5 : 1) to give 12 (12.9 mg, 88% yield) as a light yellow solid. [α]²⁵_D −10.4 (c 0.1, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 7.59 (s, 1H), 7.47 (dd, J = 7.9, 6.1 Hz, 4H), 7.43–7.33 (m, 11H), 5.52 (s, 1H), 5.18 (q, J = 11.7 Hz, 2H), 5.09 (d, J = 10.8 Hz, 1H), 4.96 (d, J = 10.8 Hz, 1H), 4.67 (d, J = 10.4 Hz, 1H), 4.14–4.03 (m, 2H), 3.99 (s, 3H), 3.67 (t, J = 9.9 Hz, 1H), 3.62–3.54 (m, 1H), 3.53–3.48 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 163.76, 152.98, 150.26, 149.92, 149.56, 137.62, 137.02, 136.05, 129.40, 128.82, 128.64, 128.60, 128.48, 128.43, 128.22, 127.70, 127.66, 126.43, 126.27, 123.85, 118.97, 111.89, 102.08, 81.34, 75.97, 73.64, 71.79, 71.15, 68.70, 61.44, 54.63. HRMS (ESI) calcd for C₃₅H₃₄NO₈ [M + H]⁺, 596.2279; found, 596.2298.

N-((6aR,7S,7aS,11aR,12aS)-1,3-Bis(benzyloxy)-2-methoxy-5-oxo-9-phenyl-6a,7,7a,11,11a,12a-hexahydro-5H-[1,3]dioxino[4′,5′:5,6]py-rano[3,2-c]isochromen-7-yl)methanesulfonamide (13)

To a solution of 12 (21.8 mg, 0.04 mmol) in dry CH₂Cl₂ (2.0 mL), Et₃N (5.0 μL, 0.07 mmol) and MsCl (4.0 μL, 0.06 mmol) were added at 0 °C under an argon atmosphere. The mixture was stirred for 4 h, concentrated and purified by column chromatography on silica gel (petroleum ether/CH₂Cl₂/ethyl acetate = 2 : 2 : 1) to give 13 (22.6 mg, 91% yield) as a white solid. [α]²⁵_D −35.2 (c 0.1, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 7.58 (s, 1H), 7.53–7.29 (m, 15H), 5.50 (s, 1H), 5.19 (dd, J = 22.7, 11.5 Hz, 2H), 5.09 (d, J = 10.9 Hz, 1H), 4.94 (d, J = 10.9 Hz, 1H), 4.85 (d, J = 6.2 Hz, 1H), 4.66 (d, J = 10.1 Hz, 1H), 4.18 (t, J = 10.1 Hz, 1H), 4.08–4.00 (m, 2H), 3.99 (s, 3H), 3.69–3.55 (m, 3H), 3.13 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 163.03, 153.20, 150.30, 149.74, 137.50, 136.55, 136.01, 129.90, 129.52, 129.16, 128.89, 128.80, 128.70, 128.55, 128.41, 128.14, 127.76, 127.72, 126.27, 125.95, 118.77, 112.04, 102.19, 79.17, 78.88, 76.08, 74.67, 72.82, 71.26, 68.54, 61.50, 57.20, 43.16. HRMS (ESI) calcd for C₃₆H₃₆NO₁₀S [M + H]⁺, 674.2054; found, 674.2064.

Biological evaluation

Mouse splenocyte proliferation inhibition assay

The assay was performed according to the procedures previously reported by our laboratory with minor modification. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Peking University Health Science Center and approved by the Animal Ethics Committee of Peking University Health Science Center. Briefly, spleens isolated from healthy mice (BALB/c mouse, male, 6–8 weeks) were harvested for the preparation of splenocytes after lysis of the red blood cells with 0.84% ammonium chloride. These splenocytes were plated on 96-well microplates at a density of 5 × 10⁵ cells per well and cultured in RPMI-1640 medium containing 10% (v/v) fetal bovine serum and 1% (v/v) streptomycin–penicillin (Hyclone), 5 μg mL⁻¹ Con A (Sigma) alone or along with 10 μM synthetic bergenin derivatives at 37 °C, 5% CO₂ for 48 h. Splenocytes from BALB/c mice stimulated with CSA or betamethasone were used as the positive control. CCK-8 (Dojindo) was added to each well and the plates were further incubated for 3 h at 37 °C. The optical density was measured using a microplate reader (Tecan) at 450 nm. All data were presented as mean ± SEM of at least three independent experiments.

Cytotoxicity assay

The human peripheral blood leukemia cell line Jurkat (the cell line was obtained from the Cell Resource Center, Peking Union Medical College) was cultured in RPMI-1640 medium, supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) streptomycin–penicillin. Jurkat cells were seeded at a density of 2.5 × 10⁴ cells per well in 96-well plates and exposed to serially diluted test compounds in RPMI-1640 medium (control) at 37 °C for 48 h. The cell viability was determined by using a CCK-8 assay, as described above.

Mouse cytokine secretion inhibition assay

Male BALB/c mouse splenocytes were plated on 96-well microplates at a density of 5 × 10⁵ cells per well and pretreated with Con A (5 μg mL⁻¹) and each compound (5 μM or 10 μM). The 96-well microplates were incubated for 48 h in 5% CO₂ at 37 °C, then the plates were centrifuged at 4 °C, 1500 rpm for 5 min to precipitate the cells. The cell-free supernatant was carefully transferred to the 96-well plates which were precoated with the capture antibody of IFN-γ or IL-4. Finally, the concentration of IFN-γ or IL-4 was detected with instant ELISA kits (eBioscience) according to the manufacturer's protocol.

Conflicts of interest

The authors declare no competing financial interest.