WO2012079279A1 - Pregnane glycoside compounds containing orthoester groups and use thereof - Google Patents

Pregnane glycoside compounds containing orthoester groups and use thereof Download PDF

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WO2012079279A1
WO2012079279A1 PCT/CN2011/002023 CN2011002023W WO2012079279A1 WO 2012079279 A1 WO2012079279 A1 WO 2012079279A1 CN 2011002023 W CN2011002023 W CN 2011002023W WO 2012079279 A1 WO2012079279 A1 WO 2012079279A1
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sugar
extract
canadian
ethanol
hemp
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French (fr)
Chinese (zh)
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赵维民
左建平
杨以阜
王罗医
杨扬
陈振华
唐炜
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中国科学院上海药物研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/27—Asclepiadaceae (Milkweed family), e.g. hoya
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J17/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, having an oxygen-containing hetero ring not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J17/005—Glycosides

Definitions

  • the present invention relates to a class of pregnane glycosides containing orthoester groups, pharmaceutical compositions comprising such compounds, and their use in the preparation of immunosuppressive agents, and more particularly, a class of (Periploca) pregnane glycosides containing orthoester groups extracted from plants, pharmaceutical compositions comprising the same, and immunological rejection or autoimmunity thereof after preparation for prevention and/or treatment of cell/organ transplantation
  • autoimmune diseases include: rheumatoid arthritis, multiple sclerosis, and autoimmune hepatitis.
  • Immunosuppressors are a class of drugs that have an inhibitory effect on immune response and immune regulation. Different immunosuppressive agents act on different aspects of the immune response or immune regulation process. Clinically, immunosuppressive agents are mainly used to treat and/or prevent autoimmune diseases and rejection after cell/organ transplantation. Immunosuppressive agents are mainly classified into the following types: (1) chemical agents, such as nitrogen mustards of alkylating agents; (2) hormones, such as glucocorticoids; (3) fungal metabolites, such as cyclosporin A (cyclosporin A, CsA); (4) Chinese medicine and its active ingredients.
  • chemical agents such as nitrogen mustards of alkylating agents
  • hormones such as glucocorticoids
  • fungal metabolites such as cyclosporin A (cyclosporin A, CsA)
  • Chinese medicine and its active ingredients Chinese medicine and its active ingredients.
  • immunosuppressive drugs have obvious toxic side effects, mainly myelosuppression and liver and kidney toxicity. Since the action of immunosuppressive drugs is non-specific, it can lead to a decline in the immune function of the entire body, an increase in pathogenic microbial infections, and long-term application may increase the incidence of tumors. Therefore, it is important to find new high-efficiency and low-toxic immunosuppressive agents.
  • the bar willow also known as the snail, the goat's milk bar, etc., its root bark is the traditional Chinese medicine fragrant skin (Northern Five Plus Skin:), can be used for rheumatism, strong bones, strong waist and knee, can be used to treat rheumatoid joints Inflammation, muscle and bone pain and other diseases;
  • Southwest Bars also known as ⁇ ⁇ ⁇ , black bone vine (black bone, black keel:) is the root or whole plant of the plant southwestern bar, can be used to relax, ⁇ Rheumatism, treatment of rheumatoid arthritis, bruises, stomach pain, indigestion, amenorrhea, dysentery, etc. Summary of the invention
  • Another object of the present invention is to provide a pharmaceutical composition comprising a therapeutically effective amount of a pregnapide compound containing a orthoester group.
  • a further object of the present invention is to provide a method for extracting a pregnane glycoside compound containing an orthoester group from a genus Perilla.
  • the immunosuppressive agent is used for the treatment and/or prevention of rejection after cell/organ transplantation or autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis and autoimmune hepatitis.
  • the present invention separates a series of pregnane glycoside compounds containing orthoester groups from the bark and the soiled bark by biological activity tracking separation, and the compounds are represented by the following formula I:
  • Formula I Can be H or 4,6-dideoxy-3-0-methyl- ⁇ 3 -hexanone
  • R 2 is a sugar chain fragment composed of a sugar unit linked by an ether bond, and the sugar unit may be selected from, but not limited to, cymarose, canarose, Preferably, in the acylation product of the oleandrose, or the above monosaccharide, in the formula I: 11 or R 2 is
  • the specific combination of the orthoester group-containing pregnane glycoside compound of the present invention is as follows:
  • Ri is R 2 is Canadian cannabis kana sugar cannabis oleander nectar
  • the method for extracting a pregnane glucoside compound containing an orthoester group from the genus Perilla of the present invention includes the following steps:
  • the fragrant medicinal herbs are extracted three times with 95% ethanol, and the extract is concentrated under reduced pressure to obtain an ethanol extract.
  • the mixture is suspended in water and extracted with chloroform and n-butanol to obtain a chloroform extract and n-butanol extract, chloroform.
  • the extract was separated by silica gel (200 mesh:) column chromatography and eluted with a petroleum ether-acetone gradient (volume ratio 5:1, 2:1, 1 :1, 0:1) to obtain fractions Frl, Fr2, Fr3 And Fr4;
  • fraction Fr3 was separated by reversed-phase C 18 column chromatography and eluted with a methanol-water gradient (volume ratio of 1:1 ⁇ 1:0) to obtain secondary fractions Fr3.1, Fr3.2, Fr3.3, Fr3.4;
  • reverse phase C 18 column chromatography was performed using a Varian SD-1 model with a Merck NW25 C 18 column (10 ⁇ m, 20 mm 250 mm) and a ProStar 320 UV/Vis detector.
  • Black root vine root is extracted with 95% ethanol at room temperature, concentrated under vacuum distillation to form an extract, and dispersed in water, and then extracted with petroleum ether, ethyl acetate and n-butanol, respectively, to obtain a petroleum ether extract.
  • Fraction Fr5 was separated by Sephadex LH-20 (ie, background interference), and then silica gel column chromatography was carried out using chloroform-acetone (volume ratio 8: 1, 7:1, 6:1) as eluent. P-8, P-9, P-10 and Pl l. Wherein, the same ratio of solvent elution fractions is detected by thin layer chromatography and may contain multiple purified compounds.
  • the progesterate-containing progesterone sugar of the present invention is determined by various in vitro and in vivo experiments.
  • Glycosides have significant in vitro immunosuppressive activity and exhibit significant therapeutic effects in animal models of various autoimmune diseases, for example, they can significantly inhibit T cell proliferation; significantly inhibit dinitrofluorobenzene (DNFB) induction.
  • DTH Delayed type hypersensitivity
  • EAE experimental autoimmune encephalomyelitis
  • the severity and pathological changes of various aspects of the diseased joint tissue effectively weaken the liver damage caused by concanavalin (ConA) and reduce the level of cytokines in serum.
  • the present invention provides a crude extract of fragrant skin, which is obtained by the following method:
  • the fragrant medicinal material is extracted three times with 95% ethanol, and the extract is concentrated under reduced pressure to obtain an ethanol extract, which is added to the ethanol extract.
  • the water was centrifuged, the precipitate was washed with petroleum ether, dissolved in ethanol, allowed to stand, filtered, and the supernatant was taken to prepare a 70% ethanol solution, which was separated by macroporous resin column chromatography, and the eluted fraction of 90% ethanol was collected and concentrated. , that is.
  • Figure 1 shows the effect of compound P-1 on experimental autoimmune meningococcal disease (EAE) in mice (A: incidence of EAE in each group; B: disease score; C: body weight change, * ⁇ 0.05, * * ⁇ 0.01)
  • Figure 2 shows the effect of compound P-2 on experimental autoimmune meningococcal (EAE) symptoms in mice (A: incidence of EAE in each group; B: disease score; C: weight change) , *;? ⁇ 0.05).
  • Figure 3 shows the effect of compound P-1 on the severity of arthritis in mice (*/? ⁇ 0.05, **;? ⁇ 0 ⁇ 01).
  • Figure 4 shows the effect of compound P-1 on the pathological changes of arthritis in mice (A: ankle tissue section of normal group mice (400x); B: ankle joint tissue section of solvent control group (400x); C: P -1 treatment group mice ankle joint tissue section (400x); D: normal group mice ankle joint CT scan; E: solvent control group mice ankle CT tomography; F: P-1 treatment group mice Ankle CT tomography)).
  • Figure 5 shows the effect of Compound P-1 on the pathological changes of arthritis in mice (A: solvent control group mice ankle joint micro-CT; B: P-1 treatment group mice ankle joint ankle micro-CT).
  • Figure 6 shows the protective effect of Compound P-1 on ConA-induced hepatitis (A: serum ALT level; B: mouse survival rate; C: liver histopathology; D: pathology score, *p ⁇ 0.05, * * ⁇ 0.01, *** ⁇ 0 ⁇ 001).
  • Figure 7 shows the effect of compound P-1 on serum cytokine levels in hepatitis mice (A: serum IL-4 levels; B: serum IFN- ⁇ levels, *p ⁇ 0.05, **p ⁇ 0.01) .
  • Figure 8 shows the effect of crude extract of Xiangjiapi WJ-C on the severity of arthritis in mice.
  • the pulverized and dried fragrant medicinal herbs (15 kg) were refluxed with 95% ethanol three times (30 Lx3) for 2 hours each time.
  • the extract was concentrated under reduced pressure to obtain an ethanol extract, and about 2 L of water was suspended, followed by chloroform. Extracted with n-butanol to obtain a chloroform extract (762.5 and n-butanol extract (75.2 g).
  • the chloroform fraction was separated by silica gel (200 mesh:) column chromatography and eluted with a petroleum ether-acetone gradient (volume ratio in order) 5 ⁇ 2: 1, 1 :1, 0:1) The fractions Frl (601.3g), Fr2 (15.8g), Fr3 (48.2g) and Fr4 (3.6g) were obtained.
  • Fr3 was reversed by C 18 column chromatography. , eluted with a methanol-water gradient (volume ratios of 1:1, 2:1, 3:1, 4:1, and 1:0, sub-fractions Fr3.1, Fr3.2, Fr3.3, Fr3.4, wherein Fr3.4 (7.8 g) was prepared by reverse phase C18 preparative liquid chromatography eluting with a methanol-water gradient (volume ratio 2:3, 1:1, 2:1, 3:1, 4:1 and 1:0) give P-1 (2.0g), P-2 (1.5g), P-5 ( 18.7mg), P-6 (14.6mg).
  • Fr4 is prepared by reversed phase C 18 Chromatography, elution with methanol-water gradient (3:7, 2:3, 1 : 1, 2:1, 3:1 and 4:1 by volume) to give P-7 (33.0 mg), P-3 (40.1mg), P-4 ( 13.0mg).
  • methanol-water gradient 3:7, 2:3, 1 : 1, 2:1, 3:1 and 4:1 by volume
  • P-7 33.0 mg
  • P-3 40.1mg
  • P-4 13.0mg
  • the ethyl acetate fraction extract (32.0 g) was subjected to reverse phase C18 column chromatography eluting with a methanol-water gradient (volume ratio 1:9, 2:8, 3:7, 4:6, 5: 5, 6:4, 7:3, 8:2, 9:1 and 1:0, each gradient 1L), get six fractions (Frl-Fr6).
  • Fr5 was used to remove background interferences with Sephadex LH-20, and then subjected to silica gel column chromatography using chloroform-acetone (8:1, 7:1, 6:1 by volume) as the eluent to obtain compound P-8 (14.1 mg). ), P-9 (29.4 mg), P-10 (54.0 mg) and Pl l (35.1 mg).
  • the same ratio of solvent elution fractions is detected by thin layer chromatography and may contain multiple purified compounds.
  • mice were sacrificed by the spine method, the spleen was aseptically taken, ground to make a single cell suspension, and the red blood cells were removed with MTT lysate (10% SDS, 50% DMF), and RPMI containing 10% FBS was used.
  • MTT lysate (10% SDS, 50% DMF)
  • RPMI containing 10% FBS was used.
  • the -1640 medium was adjusted to a cell concentration of 5 x 10 5 /ml.
  • 5 ⁇ 10 5 cell suspension, 200 ⁇ RPMI-1640 medium and appropriate concentration of the sample to be tested were added to the 96-well plate, and cultured in a 37° C., 5 % C0 2 incubator for 48 hours, 5 hours before the end of the culture.
  • 90 ⁇ M of MTT lysate was added to each well, and after standing for 6-7 hours in the incubator, the OD 57 () value was measured at 570 nm using a micro
  • Lymphocyte proliferation assay 5xl0 5 fresh spleen cells according to 37 ° C, 5% C0 2 conditions for 48 hours, the culture was washed with 5 ⁇ ⁇ ⁇ of ConA or LPS induced proliferation, adding the appropriate concentration of sample to be tested To test its inhibitory activity against lymphocyte proliferation.
  • Cell proliferation was quantified by 3 H-TdR infiltration. 25 ⁇ of 3 H-TdR was added to each well 8 hours before the end of the culture, and the culture was completed. The cells were collected on a glass fiber membrane by a cell harvester, scintillation fluid was added, and 3 H-TdR infiltration in the DNA was detected by a liquid scintillation counter. The amount reflects the cell proliferation.
  • CC 5 was calculated based on the toxicity data OD 57Q of the compound against normal mouse spleen lymphocytes. (50% cytotoxic concentration), IC 50 (50% inhibitory concentration) was calculated based on the inhibitory effect of the compound on spleen lymphocyte proliferation in normal mice.
  • the safety index (SI) CC 5 o / IC 5 o is the main parameter for evaluating the safety of a compound. The larger the SI value, the safer the compound.
  • Example 3 Therapeutic effects of compounds P-1 and ⁇ -2 on animal models of autoimmune meningitis
  • Example 4 Therapeutic effect of Compound P-1 on an experimental rheumatoid arthritis animal model
  • the DBA/1 mouse arthritis model was induced using bovine type II collagen (CII).
  • CII bovine type II collagen
  • CII was dissolved in 0.1 M acetic acid solution at 4 ° C overnight.
  • an equal volume of CFA and CII collagen containing Mycobacterium tuberculosis strain H37Rv was fully emulsified and mixed, and 250 ⁇ 8 emulsifier was used for sensitization at the base of DBA/1 mice. After 3 weeks, the same The dose of emulsifier is attacked at the tail.
  • the model mice were confirmed to have an onset one week after the challenge, and oral perfusion administration was started once a day for 2 weeks, and the dose was 20 mg/kg.
  • the P1 treatment group was terminated on the 7th day after the second immunization with CII mice, and the end of the experiment was observed on the 21st day.
  • the severity of arthritis in each of the solvent control group and the P-1 treatment group was observed every day.
  • the severity of joint lesions was graded (0 points: normal; 1 point: mild redness or redness of the toe joints; 2 points: moderate redness and extension to the entire foot; 3 points: heavier redness and extension to the ankle joint; 4 points: toe, foot, and sputum are severely red and swollen, and the joint is straightened after the assessment.
  • each limb arthritis score is between 0 and 4, and the result is limbs.
  • the average form of the sum of the scores is expressed.
  • Figure 3 shows the effect of compound P-1 on the severity of arthritis in mice (*;? ⁇ 0.05, ** ⁇ 0.01).
  • the positive control drug is methotrexate (MTX).
  • FIG 4 and Figure 5 show the effect of P-1 on the pathological changes of arthritis in mice.
  • 4A is the ankle joint tissue section of the normal group (400x);
  • 4B is the ankle joint tissue section of the solvent control group (400x);
  • 4C For the compound P-1 treatment group, the ankle joint tissue section (400x);
  • 4D is the CT scan of the ankle joint of the normal group;
  • 4E is the CT scan of the ankle joint of the solvent control group;
  • 4F is the P-1 treatment Group of mouse ankle CT tomography.
  • Figure 5A shows the ankle joint micro-CT of the solvent control group;
  • Figure 5B shows the ankle joint micro-CT of the P-1 treatment group.
  • ConA was dissolved in physiological saline, ConA was formulated to a concentration of 15 mg/kg in a mouse liver injury test; ConA was formulated to a concentration of 20 mg/kg in a mouse survival rate experiment.
  • the prepared ConA physiological saline solution was passed through a 0.45 ⁇ m filter.
  • Mouse liver injury test Compound P-1 was administered four times in a dose of 10 mg/kg by intraperitoneal injection (ie, countdown 3, 2, 1 and 1 hour before ConA injection). .
  • Mouse sera were collected at various time points after injection of 0.2 ml of 15 mg/kg of ConA into the tail vein of mice, and alanine aminotransferase (ALT) levels were measured.
  • ALT alanine aminotransferase
  • the serum ALT level of the model group began to rise 3 hours after the injection of ConA (15 mg/kg), reached a peak at 12 hours, and then decreased again; the P-1 treatment group was at each time point. Can significantly reduce ALT levels, especially at 6, 12 and 24 hours.
  • mice in the model group After injection of a lethal dose of ConA (20 mg/kg), the mice in the model group all died within 9 hours, while the survival rate of the mice in the P-1 treatment group was 60%; After an hour, the survival rate of the mice in the P-1 treatment group was 40%.
  • NKT cells release a large number of cytokines, including IL-4, IL-5, IFN- ⁇ , and TNF- ⁇ , and the time at which each cytokine appears and peaks is different.
  • IL-4 and IFN- ⁇ have been shown to be key cytokines in the development of ConA-induced liver injury in mice, so we focused on whether P-1 can affect the levels of these two cytokines.
  • the P-1 pre-administered treatment group reduced 70% of IL-4 levels in serum at 2 hours and 6 hours; and reduced serum levels at 6 and 12 hours, respectively. % and 80% IFN- ⁇ levels.
  • Example 6 The crude extract of fragrant skin extract WJ-C for the treatment of experimental rheumatoid arthritis animal model
  • Source of experimental animals purchased from Shanghai Experimental Animal Center of Chinese Academy of Sciences, animal production license number: SCXK (Shanghai) 2007-0005
  • RPMI-1640 medium was purchased from Gibco BRL (Life Technologies, Grand Island, NY, USA); fetal bovine serum (FBS) was purchased from Hyclone (Logan, Utah, USA); bovine type II collagen Purchased from Collagen Research Center (Tokyo, Japan); Freund's complete adjuvant containing Mycobacterium tuberculosis strain H37Rv, available from Wako Pure Chemical Industries Ltd (Osaka, Japan).
  • Concanavalin A (Con A) was purchased from Sigma.
  • [Methyl-3H]thymidine (TdR) was purchased from the Shanghai Institute of Applied Physics (formerly Shanghai Institute of Nuclear Research).
  • mice in the model were confirmed one week after the challenge, and oral administration was continued for 2 weeks [WJ-C: 12.5 mg/kg, 25 mg/kg, 52 mg/kg; ammonia Hyperthyroidism (MTX): lmg/kg].
  • WJ-C 12.5 mg/kg, 25 mg/kg, 52 mg/kg
  • MTX ammonia Hyperthyroidism
  • the WJ-C treatment group ended on the 7th day after the second immunization with CII mice, and ended on the 21st day.
  • the solvent control group and WJ-C treatment were observed every day.
  • the severity of arthritic lesions in each mouse in the group were confirmed one week after the challenge, and oral administration was continued for 2 weeks [WJ-C: 12.5 mg/kg, 25 mg/kg, 52 mg/kg; ammonia Hyperthyroidism (MTX): lmg/kg].
  • the WJ-C treatment group ended on the 7th day after the second immunization with CII mice, and

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Abstract

The present invention relates to pregnane glycoside compounds containing orthoester groups, pharmaceutical compositions containing the compounds, and use thereof in the preparation of an immunosuppressant. More particularly, disclosed are pregnane glycoside compounds containing orthoester groups extracted from Periploca plants, pharmaceutical compositions containing the compounds, and use thereof in the preparation of an immunosuppressant for prevention and/or treatment of immunological rejection reactions following cell/organ transplantation or autoimmune diseases, wherein the autoimmune diseases comprise rheumatoid arthritis, multiple sclerosis and autoimmune hepatitis and the like.

Description

一类含有原酸酯基团的孕甾烷糖苷类化合物及其用途  A class of pregnane glycoside compounds containing orthoester groups and uses thereof
技术领域  Technical field
本发明涉及一类含有原酸酯基团的孕 烷糖苷类化合物、 包含该类化合 物的药物组合物、 及其在制备免疫抑制剂中的用途， 更具体而言， 涉及一类 从杠柳属 (Periploca)植物中提取的含有原酸酯基团的孕 烷糖苷类化合物、包 含该类化合物的药物组合物、 及其在制备预防和 /或治疗细胞 /器官移植后免 疫排斥反应或自身免疫性疾病的免疫抑制剂中的用途，自身免疫性疾病包括： 类风湿关节炎、 多发性硬化症和自身免疫性肝炎等。  The present invention relates to a class of pregnane glycosides containing orthoester groups, pharmaceutical compositions comprising such compounds, and their use in the preparation of immunosuppressive agents, and more particularly, a class of (Periploca) pregnane glycosides containing orthoester groups extracted from plants, pharmaceutical compositions comprising the same, and immunological rejection or autoimmunity thereof after preparation for prevention and/or treatment of cell/organ transplantation For use in immunosuppressive agents for diseases, autoimmune diseases include: rheumatoid arthritis, multiple sclerosis, and autoimmune hepatitis.
背景技术  Background technique
免疫抑制剂 （immunosuppressor) 是一类对免疫反应和免疫调节状态具 有抑制作用的药物， 不同的免疫抑制剂作用于免疫反应或免疫调节过程的不 同环节。在临床上， 免疫抑制剂主要用于治疗和 /或预防自身免疫性疾病和细 胞 /器官移植后的排斥反应。免疫抑制剂主要分为以下几种：（1 )化学制剂类， 如烷化剂类的氮芥； （2 ) 激素类， 如糖皮质激素； （3 ) 真菌代谢产物， 如环 孢菌素 A (cyclosporin A, CsA); (4) 中药及其有效成分。  Immunosuppressors are a class of drugs that have an inhibitory effect on immune response and immune regulation. Different immunosuppressive agents act on different aspects of the immune response or immune regulation process. Clinically, immunosuppressive agents are mainly used to treat and/or prevent autoimmune diseases and rejection after cell/organ transplantation. Immunosuppressive agents are mainly classified into the following types: (1) chemical agents, such as nitrogen mustards of alkylating agents; (2) hormones, such as glucocorticoids; (3) fungal metabolites, such as cyclosporin A (cyclosporin A, CsA); (4) Chinese medicine and its active ingredients.
免疫抑制药物大多具有明显的毒副作用， 主要是骨髓抑制和肝、 肾毒性 等。 由于免疫抑制药物的作用是非特异的， 所以可导致整个机体免疫功能的 下降， 病原微生物感染增加， 长期应用可能提高肿瘤发病率。 因此， 寻找新 的高效低毒的免疫抑制剂具有重要意义。  Most immunosuppressive drugs have obvious toxic side effects, mainly myelosuppression and liver and kidney toxicity. Since the action of immunosuppressive drugs is non-specific, it can lead to a decline in the immune function of the entire body, an increase in pathogenic microbial infections, and long-term application may increase the incidence of tumors. Therefore, it is important to find new high-efficiency and low-toxic immunosuppressive agents.
杠柳属是萝摩科 (Asclepiadaceae)的一个小属，全世界约有 12种， 我国产 4种： 杠柳 (Periploca sepium Bunge)、 西南杠柳 (R forrestii Schltr)、 青蛇藤 [R calophylla (Wight) Falc.]和多花青蛇藤 (R floribunda Tsiang)。 杠柳， 又名狭叶 萝摩、 羊奶条等， 其根皮为传统中药香加皮 (北五加皮：)， 能祛风湿、 壮筋骨、 强腰膝， 可用于治疗类风湿性关节炎、 筋骨痛等病症； 西南杠柳， 又名滇杠 柳， 黑骨藤 (黑骨头、 黑龙骨:)是植物西南杠柳的根或全株， 可舒筋活络、 祛 风湿， 治疗类风湿性关节炎、 跌打损伤、 胃痛、 消化不良、 闭经、 痢疾等。 发明内容 It is a small genus of Asclepiadaceae. There are about 12 species in the world. There are 4 species in China: Periploca sepium Bunge, R forrestii Schltr, and R calophylla. (Wight) Falc.] and R floribunda Tsiang. The bar willow, also known as the snail, the goat's milk bar, etc., its root bark is the traditional Chinese medicine fragrant skin (Northern Five Plus Skin:), can be used for rheumatism, strong bones, strong waist and knee, can be used to treat rheumatoid joints Inflammation, muscle and bone pain and other diseases; Southwest Bars, also known as 滇 柳 柳, black bone vine (black bone, black keel:) is the root or whole plant of the plant southwestern bar, can be used to relax, 祛 Rheumatism, treatment of rheumatoid arthritis, bruises, stomach pain, indigestion, amenorrhea, dysentery, etc. Summary of the invention
因此， 本发明的一个目的为提供由通式 I表示的含有原酸酯基团的孕甾 烷糖苷类化合物。  Accordingly, it is an object of the present invention to provide a pregnane aglycone compound containing an orthoester group represented by the general formula I.
本发明的另一目的为提供包含治疗有效量的含有原酸酯基团的孕 烷糖 苷类化合物的药物组合物。  Another object of the present invention is to provide a pharmaceutical composition comprising a therapeutically effective amount of a pregnapide compound containing a orthoester group.
本发明的又一目的为提供从杠柳属植物中提取含有原酸酯基团的孕甾烷 糖苷类化合物的方法。  A further object of the present invention is to provide a method for extracting a pregnane glycoside compound containing an orthoester group from a genus Perilla.
本发明的又一目的为提供含有原酸酯基团的孕 烷糖苷类化合物在制备 免疫抑制剂中的用途，  It is still another object of the present invention to provide a use of a pregnane glycoside compound containing an orthoester group for the preparation of an immunosuppressive agent,
其中， 所述免疫抑制剂用于治疗和 /或预防细胞 /器官移植后排斥反应或 自身免疫性疾病， 所述自身免疫性疾病包括多发性硬化症、 类风湿关节炎和 自身免疫性肝炎等。  Wherein the immunosuppressive agent is used for the treatment and/or prevention of rejection after cell/organ transplantation or autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis and autoimmune hepatitis.
本发明通过生物活性跟踪分离， 从杠柳、 西南杠柳中分离出一系列含有 原酸酯基团的孕 烷糖苷类化合物， 该类化合物由如下通式 I表示：  The present invention separates a series of pregnane glycoside compounds containing orthoester groups from the bark and the southwestern bark by biological activity tracking separation, and the compounds are represented by the following formula I:
Figure imgf000003_0001
Figure imgf000003_0001
通式 I 可为 H 或
Figure imgf000004_0001
4,6-二脱氧 -3-0-甲基 -Δ3-己酮糖基Formula I Can be H or
Figure imgf000004_0001
4,6-dideoxy-3-0-methyl-Δ 3 -hexanone
(4,6-dideoxy-3-0-methyl-A3-hexosulosyl); (4,6-dideoxy-3-0-methyl-A 3 -hexosulosyl);
R2为由糖单元通过醚键连接构成的糖链片段， 所述糖单元可选自但不限 大麻糖 (cymarose)、 卡那糖 ( canarose )、
Figure imgf000004_0002
竹桃糖 (oleandrose), 或上述单糖的酰化产物中 优选地， 通式 I中： 为11或
Figure imgf000004_0003
R2为 R 2 is a sugar chain fragment composed of a sugar unit linked by an ether bond, and the sugar unit may be selected from, but not limited to, cymarose, canarose,
Figure imgf000004_0002
Preferably, in the acylation product of the oleandrose, or the above monosaccharide, in the formula I: 11 or
Figure imgf000004_0003
R 2 is
Figure imgf000004_0004
Figure imgf000004_0004
Figure imgf000005_0001
Figure imgf000005_0001
..、、、 ,、'、、" ,、、、。 .., , , , , ',,", ,,,.
、 、八。  , ,Eight.
洋地黄毒糖 加拿 Τ大麻糖 I 加拿大麻糖 I 2-乙酰基洋地黄糖  Digitalis syrup, canada, cannabis sugar, cannabis sugar, cannabis I 2-acetyldigone
Figure imgf000005_0002
Figure imgf000005_0002
加拿大麻糖 II 卡那糖 I 加拿大麻糖 I 夹竹桃糖 更优选地， 本发明的含有原酸酯基团的孕 烷糖苷类化合物的具体化合 如下：Canadian Hemp Sugar Cana Sugar I Canadian Hemp Sugar I Oleander Sugar More preferably, the specific combination of the orthoester group-containing pregnane glycoside compound of the present invention is as follows:
Figure imgf000006_0001
P-6: 为
Figure imgf000006_0001
P-6: Yes
P-7: Ri为 P-7: Ri is
P-8: 为 P-8: Yes
P-9: Ri为
Figure imgf000007_0001
R2为 加拿大麻糖 卡那糖 加拿大麻糖 夹竹桃糖P-9: Ri is
Figure imgf000007_0001
R 2 is Canadian cannabis kana sugar cannabis oleander nectar
Figure imgf000007_0002
Figure imgf000007_0002
P-10: Ri ¾ H, R2为 加拿大麻糖 卡那糖 加拿大麻糖 夹竹桃糖 ； P-10: Ri ¾ H, R 2 is a sugar kanamycin Canada Canada Matang Matang oleander sugar;
P-11 : Ri为 H， R2为
Figure imgf000007_0003
P-11 : Ri is H, R 2 is
Figure imgf000007_0003
本发明的杠柳属植物中提取含有原酸酯基团的孕 烷糖苷类化合物的方 法包括下列歩骤： The method for extracting a pregnane glucoside compound containing an orthoester group from the genus Perilla of the present invention The law includes the following steps:
(一） 提取化合物 P-l、 P-2、 P-5、 P-6、 P-3、 P-4和 P-7  (1) Extraction of compounds P-l, P-2, P-5, P-6, P-3, P-4 and P-7
1、 香加皮药材用 95%乙醇回流提取三次， 减压浓缩提取液得到乙醇浸 膏， 加入水悬浮， 依次用氯仿和正丁醇萃取， 得到氯仿部位浸膏和正丁醇部 位浸膏，氯仿部位浸膏经硅胶 (200目:)柱层析分离，以石油醚-丙酮梯度洗脱 (体 积比为 5:1, 2:1, 1 :1, 0:1)得流分 Frl、 Fr2、 Fr3和 Fr4;  1. The fragrant medicinal herbs are extracted three times with 95% ethanol, and the extract is concentrated under reduced pressure to obtain an ethanol extract. The mixture is suspended in water and extracted with chloroform and n-butanol to obtain a chloroform extract and n-butanol extract, chloroform. The extract was separated by silica gel (200 mesh:) column chromatography and eluted with a petroleum ether-acetone gradient (volume ratio 5:1, 2:1, 1 :1, 0:1) to obtain fractions Frl, Fr2, Fr3 And Fr4;
2、流分 Fr3经反相 C18柱层析分离， 以甲醇 -水梯度洗脱 (体积比为 1 : 1→ 1 :0)得次流分 Fr3.1、 Fr3.2、 Fr3.3、 Fr3.4; 2. The fraction Fr3 was separated by reversed-phase C 18 column chromatography and eluted with a methanol-water gradient (volume ratio of 1:1→1:0) to obtain secondary fractions Fr3.1, Fr3.2, Fr3.3, Fr3.4;
3、次流分 Fr3.4经反相 C18制备液相， 以甲醇 -水梯度洗脱（体积比为 2:3 → 1 :0) 得 P-l、 P-2、 P-5、 P-6; 流分 Fr4经反相 C18制备液相色谱分离， 以 甲醇 -水梯度洗脱 (体积比为 3:7→4:1 ) 得 P-3、 P-4、 P-7 o 3, views Fr3.4 fractions by reverse phase C 18 preparative liquid, methanol - water gradient (volume ratio of 2: 3 → 1: 0) to give Pl, P-2, P- 5, P-6 The fraction Fr4 was separated by reverse phase C 18 preparative liquid chromatography, eluted with a methanol-water gradient (volume ratio of 3:7→4:1) to obtain P-3, P-4, P-7 o
其中，反相 C18柱层析分离采用 Varian SD-1型号，配有 Merck NW25 C18 色谱柱 （ 10 μιη, 20 mm 250 mm), 以及 ProStar 320 UV/Vis检测器。 Among them, reverse phase C 18 column chromatography was performed using a Varian SD-1 model with a Merck NW25 C 18 column (10 μm, 20 mm 250 mm) and a ProStar 320 UV/Vis detector.
(二） 提取化合物 P-8、 P-9、 P-10和 P-l l  (ii) Extraction of compounds P-8, P-9, P-10 and P-l l
1、 将黑骨藤根用 95 %的乙醇室温浸提， 减压蒸馏浓缩成浸膏， 并用水 分散悬浮， 之后依次用石油醚、 乙酸乙酯和正丁醇萃取， 分别得到石油醚部 位浸膏、 乙酸乙酯部位浸膏和正丁醇部位浸膏；  1. Black root vine root is extracted with 95% ethanol at room temperature, concentrated under vacuum distillation to form an extract, and dispersed in water, and then extracted with petroleum ether, ethyl acetate and n-butanol, respectively, to obtain a petroleum ether extract. Ethyl acetate extract and n-butanol extract;
2、 将乙酸乙酯部位浸膏进行反相 C18柱层析， 用甲醇-水梯度洗脱， 体 积比依次为 1 :9、 2:8、 3:7、 4:6、 5:5、 6:4、 7:3、 8:2、 9:1 和 1 :0， 每个梯度 1L， 得到 Frl-Fr6六个流分; 2. The ethyl acetate fraction extract was subjected to reverse phase C 18 column chromatography and eluted with a methanol-water gradient. The volume ratios were 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1 and 1:0, each gradient 1L, get six fractions of Frl-Fr6;
3、 流分 Fr5用 Sephadex LH-20除杂 （即背景干扰物）， 然后以氯仿-丙 酮 (体积比为 8: 1, 7:1, 6:1)为洗脱剂进行硅胶柱层析得到 P-8、 P-9、 P-10和 P-l l。 其中， 同一比例溶剂洗脱流分经过薄层色谱检测， 可以含有多个纯化 的化合物。  3. Fraction Fr5 was separated by Sephadex LH-20 (ie, background interference), and then silica gel column chromatography was carried out using chloroform-acetone (volume ratio 8: 1, 7:1, 6:1) as eluent. P-8, P-9, P-10 and Pl l. Wherein, the same ratio of solvent elution fractions is detected by thin layer chromatography and may contain multiple purified compounds.
经多种体外、 动物体内试验确定， 本发明的含有原酸酯基团的孕甾烷糖 苷类化合物具有显著的体外免疫抑制活性， 并在多种自身免疫性疾病的动物 模型上表现出显著疗效， 例如， 其能够明显抑制 T细胞增殖； 显著抑制二硝 基氟苯 (DNFB)诱导的小鼠迟发型超敏反应 (DTH); 明显降低小鼠实验性自身 免疫性脑脊髓膜炎 （EAE) 的发病率、 减轻疾病的严重程度； 显著减轻牛 II 型胶原诱导的小鼠关节炎病变的严重程度及病变关节组织各方面的病理改 变； 有效削弱伴刀豆蛋白 （ConA)引起的肝脏损伤， 降低血清中细胞因子水 平。 The progesterate-containing progesterone sugar of the present invention is determined by various in vitro and in vivo experiments. Glycosides have significant in vitro immunosuppressive activity and exhibit significant therapeutic effects in animal models of various autoimmune diseases, for example, they can significantly inhibit T cell proliferation; significantly inhibit dinitrofluorobenzene (DNFB) induction. Delayed type hypersensitivity (DTH) in mice; significantly reduced the incidence of experimental autoimmune encephalomyelitis (EAE) in mice and reduced the severity of the disease; significantly reduced the type II collagen-induced arthritis in mice The severity and pathological changes of various aspects of the diseased joint tissue; effectively weaken the liver damage caused by concanavalin (ConA) and reduce the level of cytokines in serum.
此外， 本发明提供了一种香加皮粗提物， 其通过下述方法得到： 将香加皮药材用 95%乙醇回流提取三次，减压浓缩提取液得到乙醇浸膏， 向乙醇浸膏加入水离心， 沉淀以石油醚洗涤， 再用乙醇溶解、 静置， 过滤， 取上清液， 配成 70%乙醇溶液， 采用大孔树脂柱色谱分离， 收集 90%乙醇的 洗脱流分， 浓缩， 即得。  In addition, the present invention provides a crude extract of fragrant skin, which is obtained by the following method: The fragrant medicinal material is extracted three times with 95% ethanol, and the extract is concentrated under reduced pressure to obtain an ethanol extract, which is added to the ethanol extract. The water was centrifuged, the precipitate was washed with petroleum ether, dissolved in ethanol, allowed to stand, filtered, and the supernatant was taken to prepare a 70% ethanol solution, which was separated by macroporous resin column chromatography, and the eluted fraction of 90% ethanol was collected and concentrated. , that is.
附图说明  DRAWINGS
图 1显示了化合物 P-1对小鼠实验性自身免疫性脑脊髓膜炎 （EAE) 的 影响（A:各组 EAE发病率； B:疾病评分； C:体重变化， *^<0.05 , **^<0.01 ) 0 图 2显示了化合物 P-2对小鼠实验性自身免疫性脑脊髓膜炎 （EAE) 症 状的影响 （A: 各组 EAE发病率； B: 疾病评分； C: 体重变化， *;?<0.05)。 Figure 1 shows the effect of compound P-1 on experimental autoimmune meningococcal disease (EAE) in mice (A: incidence of EAE in each group; B: disease score; C: body weight change, *^<0.05, * *^<0.01) 0 Figure 2 shows the effect of compound P-2 on experimental autoimmune meningococcal (EAE) symptoms in mice (A: incidence of EAE in each group; B: disease score; C: weight change) , *;?<0.05).
图 3 显示了化合物 P-1 对小鼠关节炎病变严重程度的影响 （*/?<0.05， **;?<0·01 )。  Figure 3 shows the effect of compound P-1 on the severity of arthritis in mice (*/?<0.05, **;?<0·01).
图 4显示了化合物 P-1对小鼠关节炎病理改变的影响 （A: 正常组小鼠 踝关节组织切片 （400x ); B: 溶剂对照组小鼠踝关节组织切片 （400x ); C: P-1治疗组小鼠踝关节组织切片 （400x ) ; D: 正常组小鼠踝关节 CT断层扫 描图； E: 溶剂对照组小鼠踝关节 CT断层扫描图； F: P-1 治疗组小鼠踝关 节 CT断层扫描图）。 图 5显示了化合物 P-1对小鼠关节炎病理改变的影响 （A: 溶剂对照组 小鼠踝关节显微 CT; B: P-1治疗组小鼠踝关节踝关节显微 CT)。 Figure 4 shows the effect of compound P-1 on the pathological changes of arthritis in mice (A: ankle tissue section of normal group mice (400x); B: ankle joint tissue section of solvent control group (400x); C: P -1 treatment group mice ankle joint tissue section (400x); D: normal group mice ankle joint CT scan; E: solvent control group mice ankle CT tomography; F: P-1 treatment group mice Ankle CT tomography)). Figure 5 shows the effect of Compound P-1 on the pathological changes of arthritis in mice (A: solvent control group mice ankle joint micro-CT; B: P-1 treatment group mice ankle joint ankle micro-CT).
图 6显示了化合物 P-1对 ConA引起的肝炎的保护作用 （A: 血清 ALT 水平； B: 小鼠生存率； C: 肝脏组织病理学检查； D: 病理学评分， *p<0.05， **ρ<0.01 , ***ρ<0·001 )。  Figure 6 shows the protective effect of Compound P-1 on ConA-induced hepatitis (A: serum ALT level; B: mouse survival rate; C: liver histopathology; D: pathology score, *p<0.05, * *ρ<0.01, ***ρ<0·001).
图 7显示了化合物 P-1对肝炎小鼠血清细胞因子水平的影响 （A: 血清 中 IL-4的水平； B: 血清中 IFN-γ的水平， *p<0.05， **p<0.01 )。  Figure 7 shows the effect of compound P-1 on serum cytokine levels in hepatitis mice (A: serum IL-4 levels; B: serum IFN-γ levels, *p<0.05, **p<0.01) .
图 8显示了香加皮粗提物 WJ-C对小鼠关节炎病变严重程度的影响。 具体实施方式  Figure 8 shows the effect of crude extract of Xiangjiapi WJ-C on the severity of arthritis in mice. detailed description
以下通过具体实施例来进一歩说明本发明。 应理解， 以下实施例仅用于 说明本发明而不用于限定本发明的范围。  The invention will now be further illustrated by the following examples. It is to be understood that the following examples are merely illustrative of the invention and are not intended to limit the scope of the invention.
实施例  Example
实施例 1 含有原酸酯基团的孕 ^烷糖苷类化合物的制备  Example 1 Preparation of a pregnane glycoside compound containing an orthoester group
(一) 化合物 P-l、 P-2、 P-5、 P-6、 P-7、 P-3和 P-4的制备  (1) Preparation of compounds P-l, P-2, P-5, P-6, P-7, P-3 and P-4
粉碎、 干燥的香加皮药材 (15 kg)用 95%乙醇回流提取三次 (30 Lx3), 每 次 2小时， 减压浓缩提取液得到乙醇浸膏， 加入约 2 L的水悬浮， 依次用氯 仿和正丁醇萃取， 得到氯仿部位浸膏 (762.5 和正丁醇部位浸膏 (75.2 g)。 氯 仿部位经硅胶 (200目:)柱层析分离，以石油醚-丙酮梯度洗脱 (体积比依次为 5丄 2: 1, 1 :1, 0:1)得流分 Frl ( 601.3g)、 Fr2 ( 15.8g)、 Fr3 (48.2g) 和 Fr4 (3.6g)。 Fr3经反相 C18柱层析， 以甲醇 -水梯度洗脱 (体积比为依次为 1 :1， 2:1， 3:1， 4:1和 1 :0， 得次流分 Fr3.1、 Fr3.2、 Fr3.3、 Fr3.4， 其中 Fr3.4 (7.8g) 经反相 C18制备液相色谱， 以甲醇 -水梯度洗脱 （体积比依次为 2:3， 1 :1 , 2:1， 3:1， 4:1和 1 :0) 得 P-1 (2.0g) 、 P-2 ( 1.5g) 、 P-5 ( 18.7mg) 、 P-6 ( 14.6mg) 。 Fr4经反相 C18制备液相色谱， 以甲醇 -水梯度洗脱 （体积比依次为 3:7， 2:3 , 1 : 1， 2:1， 3:1和 4:1 ) 得 P-7 (33.0mg) 、 P-3 (40.1mg) 、 P-4 ( 13.0mg) 。 (其中， 取上述乙醇浸膏， 加入适量水搅匀放置， 离心分离沉淀， 沉淀以 石油醚洗涤， 再用乙醇溶解、 静置， 过滤， 取上清液， 配成 70%乙醇溶液， 采用大孔树脂柱色谱分离， 收集 90%乙醇的洗脱流分， 浓缩， 即为香加皮粗 提物 WJ-C, 用于实施例 6)。 The pulverized and dried fragrant medicinal herbs (15 kg) were refluxed with 95% ethanol three times (30 Lx3) for 2 hours each time. The extract was concentrated under reduced pressure to obtain an ethanol extract, and about 2 L of water was suspended, followed by chloroform. Extracted with n-butanol to obtain a chloroform extract (762.5 and n-butanol extract (75.2 g). The chloroform fraction was separated by silica gel (200 mesh:) column chromatography and eluted with a petroleum ether-acetone gradient (volume ratio in order) 5丄2: 1, 1 :1, 0:1) The fractions Frl (601.3g), Fr2 (15.8g), Fr3 (48.2g) and Fr4 (3.6g) were obtained. Fr3 was reversed by C 18 column chromatography. , eluted with a methanol-water gradient (volume ratios of 1:1, 2:1, 3:1, 4:1, and 1:0, sub-fractions Fr3.1, Fr3.2, Fr3.3, Fr3.4, wherein Fr3.4 (7.8 g) was prepared by reverse phase C18 preparative liquid chromatography eluting with a methanol-water gradient (volume ratio 2:3, 1:1, 2:1, 3:1, 4:1 and 1:0) give P-1 (2.0g), P-2 (1.5g), P-5 ( 18.7mg), P-6 (14.6mg). Fr4 is prepared by reversed phase C 18 Chromatography, elution with methanol-water gradient (3:7, 2:3, 1 : 1, 2:1, 3:1 and 4:1 by volume) to give P-7 (33.0 mg), P-3 (40.1mg), P-4 ( 13.0mg). (In which, take the above ethanol extract, add appropriate amount of water and mix well, centrifuge to separate the precipitate, precipitate with petroleum ether, dissolve with ethanol, let stand, filter, take the supernatant, prepare 70% ethanol solution, use large The column was chromatographed on a column of resin, and the eluted fraction of 90% ethanol was collected and concentrated to obtain a crude extract of fragrant skin, WJ-C, for use in Example 6).
(二） 化合物 P-8、 P-9、 P-10、 P-l l的制备  (II) Preparation of compounds P-8, P-9, P-10, P-l l
黑骨藤根 10 kg粉碎后， 用 95 %的工业乙醇室温浸提 (30 Lx3), 减压蒸 馏浓缩成浸膏， 用 2 L水分散悬浮后， 分别用石油醚、 乙酸乙酯和正丁醇依 次萃取， 得到石油醚部位浸膏 250 g， 乙酸乙酯部位浸膏 32.0 g， 正丁醇部位 浸膏约 200 g。 将乙酸乙酯部位浸膏 (32.0 g)进行反相 C18柱层析， 用甲醇-水 梯度洗脱 (体积比依次为 1 :9、 2:8、 3:7、 4:6、 5:5、 6:4、 7:3、 8:2、 9:1和 1 :0， 每个梯度 1L)，得到六个流分 (Frl-Fr6)。 Fr5用 Sephadex LH-20除去背景干扰 物， 然后以氯仿 -丙酮 (体积比依次为 8:1, 7:1, 6:1)为洗脱剂进行硅胶柱层析得 到化合物 P-8 (14.1 mg)， P-9 (29.4 mg)， P-10 (54.0 mg) 和 P-l l (35.1 mg)。 其 中， 同一比例溶剂洗脱流分经过薄层色谱检测，可以含有多个纯化的化合物。 After 10 kg of black vine root was pulverized, it was leached with 95% industrial ethanol at room temperature (30 Lx3), distilled under reduced pressure to form an extract, dispersed in 2 L of water, and then successively treated with petroleum ether, ethyl acetate and n-butanol. Extraction, 250 g of petroleum ether extract, 32.0 g of ethyl acetate extract, and about 200 g of n-butanol extract. The ethyl acetate fraction extract (32.0 g) was subjected to reverse phase C18 column chromatography eluting with a methanol-water gradient (volume ratio 1:9, 2:8, 3:7, 4:6, 5: 5, 6:4, 7:3, 8:2, 9:1 and 1:0, each gradient 1L), get six fractions (Frl-Fr6). Fr5 was used to remove background interferences with Sephadex LH-20, and then subjected to silica gel column chromatography using chloroform-acetone (8:1, 7:1, 6:1 by volume) as the eluent to obtain compound P-8 (14.1 mg). ), P-9 (29.4 mg), P-10 (54.0 mg) and Pl l (35.1 mg). Wherein, the same ratio of solvent elution fractions is detected by thin layer chromatography and may contain multiple purified compounds.
P-1 : 分子式： C72H114027， 分子量： 1411， 白色粉末， [a]D 24 -1.2 (c 1.4, CHC13); ¾ NMR (CDCI3) δ: 0.72 (3Η, s), 1.00 (3Η, s), 1.37 (3H, d, J= 6.5 Hz), 1.51 (3H, d, J = 6.8 Hz), 3.41, 3.42, 3.43, 3.44, 3.45 (均为 3H, s), 3.57 (3H, s), 3.67(1H, m), 3.70 (1H, q, J = 6.5 Hz ), 4.38 (1H, d, J = 8.0 Hz), 4.58 (1H, dd, J = 9.5, 1.5 Hz), 4.70 (1H, ddq, J = 6.8, 3.0, 0.5 Hz), 4.74 (1H, ά, J = 7.5 Hz), 4.76 (1H, dd, J = 10.0, 1.5 Hz), 4.92 (1H, άά, J = 10.0, 1.5 Hz), 4.94 (1H, ά, J = 0.5 Hz), 5.09 (1H, dd, J= 10.0, 8.0 Hz), 5.14 (1H, d, J= 7.5 Hz), 5.35 (1H, br s), 5.77 (1H, d, J= 3.0 Hz). 13C NMR数据见表 1。 P-1 : Molecular formula: C 72 H 114 0 27 , Molecular weight: 1411, white powder, [a] D 24 -1.2 (c 1.4, CHC1 3 ); 3⁄4 NMR (CDCI3) δ: 0.72 (3Η, s), 1.00 (3Η, s), 1.37 (3H, d, J= 6.5 Hz), 1.51 (3H, d, J = 6.8 Hz), 3.41, 3.42, 3.43, 3.44, 3.45 (all 3H, s), 3.57 (3H , s), 3.67(1H, m), 3.70 (1H, q, J = 6.5 Hz ), 4.38 (1H, d, J = 8.0 Hz), 4.58 (1H, dd, J = 9.5, 1.5 Hz), 4.70 (1H, ddq, J = 6.8, 3.0, 0.5 Hz), 4.74 (1H, ά, J = 7.5 Hz), 4.76 (1H, dd, J = 10.0, 1.5 Hz), 4.92 (1H, άά, J = 10.0 , 1.5 Hz), 4.94 (1H, ά, J = 0.5 Hz), 5.09 (1H, dd, J= 10.0, 8.0 Hz), 5.14 (1H, d, J= 7.5 Hz), 5.35 (1H, br s) , 5.77 (1H, d, J = 3.0 Hz). 13 C NMR data are shown in Table 1.
P-2: 分子式： C65H106O24， 分子量： 1271， 白色粉末， [a]D 24 -7.5 (c 0.08, CHCI3); ^ NMR (CDCI3) δ: 0.73 (3Η, s), 1.02 (3H, s), 1.31 (3H, d, J= 6.0 Hz), 1.37 (3H, d, J = 6.5 Hz), 2.07 (3H, s), 3.41, 3.42, 3.43, 3.44 (x 2) (均为 3H, s), 4.38 (IH, d, J = 6.5 Hz), 4.57 (IH, dd, J = 9.5, 1.5 Hz), 4.74 (IH, d, J = 7.5Hz), 4.76 (2H, dd, J= 10.0, 1.5 Hz), 4.92 (IH, dd, J= 10.0, 1.5 Hz), 5.08 (IH, dd, J = 8.0, 9.8 Hz), 5.13 (IH, d, J= 7.5 Hz), 5.40 (IH, br s). 13C NMR数据见表 1。 P-2: Molecular formula: C 65 H 106 O 24 , Molecular weight: 1271, white powder, [a] D 24 -7.5 (c 0.08, CHCI3); ^ NMR (CDCI3) δ: 0.73 (3Η, s), 1.02 ( 3H, s), 1.31 (3H, d, J = 6.0 Hz), 1.37 (3H, d, J = 6.5 Hz), 2.07 (3H, s), 3.41, 3.42, 3.43, 3.44 (x 2) (all 3H, s), 4.38 (IH, d, J = 6.5 Hz), 4.57 (IH, dd, J = 9.5, 1.5 Hz), 4.74 (IH, d, J = 7.5Hz), 4.76 (2H, dd, J= 10.0, 1.5 Hz) ), 4.92 (IH, dd, J = 10.0, 1.5 Hz), 5.08 (IH, dd, J = 8.0, 9.8 Hz), 5.13 (IH, d, J = 7.5 Hz), 5.40 (IH, br s). The 13 C NMR data is shown in Table 1.
P-3: 分子式： C70H112O26， 分子量： 1369， 白色粉末， [a]D 24 -3.08 (c 0.26, CHC13); lH NMR (CDCI3) δ: 0.72 (3H, s), 0.99 (3H, s), 1.36 (3H, d, J= 6.5 Hz), 1.50 (3H, d, J = 6.5 Hz), 3.43 (x 2), 3.44(x 2), 3.51 (均为 3H, s), 3.63 (3H, s), 4.27 (IH, d, J= 8.0 Hz), 4.57 (IH, dd, J= 9.5, 1.5 Hz), 4.70 (IH, ddq, J= 6.8, 3.0 Hz), 4.74 (IH, d, J = 7.6 Hz), 4.76 (2H, dd, J = 10.0, 1.5 Hz), 4.92 (IH, dd, J = 10.0, 1.5 Hz), 5.04 (IH, s), 5.13 (IH, d, J= 7.5 Hz), 5.36 (IH, br s), 5.77 (IH, d, J = 3.0 Hz). 13C NMR数据见表 1。 P-3: Molecular formula: C 70 H 112 O 26 , Molecular weight: 1369, white powder, [a] D 24 -3.08 (c 0.26, CHC1 3 ); l H NMR (CDCI3) δ: 0.72 (3H, s), 0.99 (3H, s), 1.36 (3H, d, J= 6.5 Hz), 1.50 (3H, d, J = 6.5 Hz), 3.43 (x 2), 3.44(x 2), 3.51 (both 3H, s ), 3.63 (3H, s), 4.27 (IH, d, J= 8.0 Hz), 4.57 (IH, dd, J= 9.5, 1.5 Hz), 4.70 (IH, ddq, J= 6.8, 3.0 Hz), 4.74 (IH, d, J = 7.6 Hz), 4.76 (2H, dd, J = 10.0, 1.5 Hz), 4.92 (IH, dd, J = 10.0, 1.5 Hz), 5.04 (IH, s), 5.13 (IH, d, J = 7.5 Hz), 5.36 (IH, br s), 5.77 (IH, d, J = 3.0 Hz). See Table 1 for 13 C NMR data.
P-4: 分子式： C63H104O23， 分子量： 1229， 白色粉末， [a]D 24 +8.1 (c 0.07, MeOH); lR NMR (CDC13) 3： 0.72 (3H, s), 1.00 (3H, s), 1.31 (3H, d, J= 6.0 Hz), 1.35 (3H, d, J= 6.5 Hz), 3.43 (x 2), 3.44 (x 2), 3.52 (均为 3H, s), 4.28 (IH, d, J = 7.7 Hz), 4.57 (IH, dd, J = 9.5, 1.4 Hz), 4.74 (IH, d, J = 7.5 Hz), 4.76 (x 2), 4.92 (均为 IH, dd, J = 9.6, 1.5 Hz), 5.10 (IH, άά, J = 10.0, 8.0 Hz), 5.13 (IH, ά, J = 7.5 Hz), 5.30 (IH, dd, J= 3.2, 1.2 Hz), 5.38 (IH, m). 13C NMR数据见表 1。 P-4: Molecular formula: C 63 H 104 O 23 , Molecular weight: 1229, white powder, [a] D 24 +8.1 (c 0.07, MeOH); l R NMR (CDC1 3 ) 3: 0.72 (3H, s), 1.00 (3H, s), 1.31 (3H, d, J= 6.0 Hz), 1.35 (3H, d, J= 6.5 Hz), 3.43 (x 2), 3.44 (x 2), 3.52 (both 3H, s ), 4.28 (IH, d, J = 7.7 Hz), 4.57 (IH, dd, J = 9.5, 1.4 Hz), 4.74 (IH, d, J = 7.5 Hz), 4.76 (x 2), 4.92 (both IH, dd, J = 9.6, 1.5 Hz), 5.10 (IH, άά, J = 10.0, 8.0 Hz), 5.13 (IH, ά, J = 7.5 Hz), 5.30 (IH, dd, J= 3.2, 1.2 Hz ), 5.38 (IH, m). 13 C NMR data are shown in Table 1.
P-5: 分子式： C71H112027， 分子量： 1397， 白色粉末， [a]D 18 -9.9 (c 0.41, CHCI3); IR (KBr) vmax 3454, 2935， 1749， 1716， 1637， 1454， 1375， 1313， 1238， 1157， 1095， 1066， 1003 cm"1; ^ NMR (CDC13) 3： 0.72 (3H, s), 1.00 (3H, s), 1.33 (3H, d, J= 6.5 Hz), 1.46 (3H, d, J= 6.9 Hz), 2.02 (3H, s), 2.91 (t, 9.5 Hz), 3.36, 3.38 (x 2), 3.40, 3.58 (均为 3H, s), 4.34 (IH, d, J= 8.0 Hz), 4.45 (IH, d, J = 9.0 Hz), 4.53 (IH, d, J = 9.0 Hz), 4.69 (IH, d, 9.5 Hz), 4.88 (IH, d, J = 9.5 Hz), 5.09 (IH, d, J= 8.0 Hz), 5.31 (IH, br s), 5.72 (IH, d, 3.0 Hz). 13C NMR数据见表 1。 P-5: Molecular formula: C 71 H 112 0 27 , Molecular weight: 1397, white powder, [a] D 18 -9.9 (c 0.41, CHCI3); IR (KBr) v max 3454, 2935, 1749, 1716, 1637, 1454, 1375, 1313, 1238, 1157, 1095, 1066, 1003 cm"1; ^ NMR (CDC1 3 ) 3: 0.72 (3H, s), 1.00 (3H, s), 1.33 (3H, d, J= 6.5 Hz), 1.46 (3H, d, J= 6.9 Hz), 2.02 (3H, s), 2.91 (t, 9.5 Hz), 3.36, 3.38 (x 2), 3.40, 3.58 (both 3H, s), 4.34 (IH, d, J = 8.0 Hz), 4.45 (IH, d, J = 9.0 Hz), 4.53 (IH, d, J = 9.0 Hz), 4.69 (IH, d, 9.5 Hz), 4.88 (IH, d , J = 9.5 Hz), 5.09 (IH, d, J = 8.0 Hz), 5.31 (IH, br s), 5.72 (IH, d, 3.0 Hz). See Table 1 for 13 C NMR data.
P-6: 分子式： C71H112027， 分子量： 1397， 白色粉末， [a]D 18 -3.0 (c 0.28, CHCI3); IR (KBr) vmax 3448 , 2935 1751 1718 1637 1456 1375 1313 1238 1 157 1095 1059 1003 cm"1; ^ NMR (CDC13) 3： 0.72 (3H, s), 1.00 (3H, s), 1.33 (3H, d, J= 5.8 Hz), 1.48 (3H, d, J = 6.6 Hz), 2.02 (3H, s), 3.38, 3.40 (x 2): 3.42, 3.58 (均为 3H, s), 4.19 (IH, br s), 4.35 (IH, d, J = 8.0 Hz), 4.55 (IH, d, J = 9.0 Hz), 4.70 (IH, d, J = 9.5 Hz), 4.76 (IH, d, 9.5 Hz), 4.95 (IH, d, J = 9.2 Hz), 5.10 (IH, d, J= 7.6 Hz), 5.31 (IH, br s), 5.72 (IH, d, 3.0 Hz). 13C NMR数据见表 1 P-6: Molecular formula: C 71 H 112 0 27 , Molecular weight: 1397, white powder, [a] D 18 -3.0 (c 0.28, CHCI3); IR (KBr) v max 3448 , 2935 1751 1718 1637 1456 1375 1313 1238 1 157 1095 1059 1003 cm"1; ^ NMR (CDC1 3 ) 3: 0.72 (3H, s), 1.00 (3H, s), 1.33 (3H, d, J = 5.8 Hz), 1.48 (3H, d, J = 6.6 Hz), 2.02 (3H, s), 3.38, 3.40 (x 2) : 3.42, 3.58 (both 3H, s), 4.19 (IH, br s), 4.35 (IH, d, J = 8.0 Hz), 4.55 (IH, d, J = 9.0 Hz), 4.70 (IH, d, J = 9.5 Hz), 4.76 (IH, d, 9.5 Hz), 4.95 (IH, d, J = 9.2 Hz), 5.10 (IH, d, J = 7.6 Hz), 5.31 (IH, br s), 5.72 (IH, d, 3.0 Hz). 13 C NMR data See Table 1
P-7: 分子式： C70H110O27， 分子量： 1383, 白色粉末， [a]D 24 -14.0 (c 0.2, CHCI3); IR (KBr) vmax 3489 , 2935 1745 1716 1637 1454 1375 1317 1240 1 157 1093 1058 1004 cm"1; lR NMR (CDC13) 3： 0.70 (3H, s), 0.95 (3H, s), 1.33 (3H, d, J= 6.4 Hz), 1.49 (3H, d, J= 6.9 Hz), 2.05 (3H, s), 3.38, 3.42, 3.58 (均为 3H, s), 4.18 (IH, br s), 4.35 (IH, ά, J = 8.0 Hz), 4.50 (IH, d, J = 9.0 Hz), 4.55 (IH, d, J = 9.0 Hz), 4.95 (IH, d, J = 9.6 Hz), 5.10 (IH, d, J = 7.6 Hz), 5.31 (IH, br s), 5.72 (IH, ά, J = 3.0 Hz). 13C NMR数据见表 1 HRESIMS mlz 1405.71 12 [M + Na]+ (计算值： C7。H11()027Na, 1405.7132)。 P-7: Molecular formula: C 70 H 110 O 27 , Molecular weight: 1383, white powder, [a] D 24 -14.0 (c 0.2, CHCI3); IR (KBr) v max 3489 , 2935 1745 1716 1637 1454 1375 1317 1240 1 157 1093 1058 1004 cm"1; l R NMR (CDC1 3 ) 3: 0.70 (3H, s), 0.95 (3H, s), 1.33 (3H, d, J = 6.4 Hz), 1.49 (3H, d, J = 6.9 Hz), 2.05 (3H, s), 3.38, 3.42, 3.58 (all 3H, s), 4.18 (IH, br s), 4.35 (IH, ά, J = 8.0 Hz), 4.50 (IH, d, J = 9.0 Hz), 4.55 (IH, d, J = 9.0 Hz), 4.95 (IH, d, J = 9.6 Hz), 5.10 (IH, d, J = 7.6 Hz), 5.31 (IH, br s ), 5.72 (IH, ά, J = 3.0 Hz). For the 13 C NMR data, see Table 1 HRESIMS mlz 1405.71 12 [M + Na] + (calc. C 7 .H 11 () 0 27 Na, 1405.7132).
P-8 : 分子式： C71H112026， 分子量： 1380, 白色粉末， [a]D 24 -6 (c 0.19, CHCI3); IR (KBr) v 3448, 2935, 1735, 1639, 1454, 1375, 1240, 1 163, 1059, 1005 cm"1; ^ NMR (CDC13) 3： 0.70 (3H, s), 0.95 (3H, s), 1.29 (3H, d, J= 6.2 Hz): 1.48 (3H, d, J = 6.8 Hz), 2.05 (3H, s), 3.37, 3.40 (x 2), 3.42, 3.60 (均为 3H, s), 4.50 (IH, d, J = 9.2 Hz), 4.55 (IH, d, J = 9.1 Hz), 4.70 (IH, d, J = 9.6 Hz), 4.75 (IH, d, J = 9.6 Hz), 4.90 (IH, d, J = 9.5 Hz), 5.10 (IH, d, J = 7.7 Hz), 5.31 (IH, br s), 5.75 (IH, s). 13C NMR数据见表 1 HRESIMS mlz 1403.7330 [M + Na]+ (计算值： C71H112026Na, 1403.7340)。 P-8 : Molecular formula: C 71 H 112 0 26 , Molecular weight: 1380, white powder, [a] D 24 -6 (c 0.19, CHCI3); IR (KBr) v 3448, 2935, 1735, 1639, 1454, 1375 , 1240, 1 163, 1059, 1005 cm"1; ^ NMR (CDC1 3 ) 3: 0.70 (3H, s), 0.95 (3H, s), 1.29 (3H, d, J = 6.2 Hz) : 1.48 (3H , d, J = 6.8 Hz), 2.05 (3H, s), 3.37, 3.40 (x 2), 3.42, 3.60 (both 3H, s), 4.50 (IH, d, J = 9.2 Hz), 4.55 (IH , d, J = 9.1 Hz), 4.70 (IH, d, J = 9.6 Hz), 4.75 (IH, d, J = 9.6 Hz), 4.90 (IH, d, J = 9.5 Hz), 5.10 (IH, d , J = 7.7 Hz), 5.31 (IH, br s), 5.75 (IH, s). 13 C NMR data are shown in Table 1. HRESIMS mlz 1403.7330 [M + Na] + (calculated value: C 71 H 112 0 26 Na, 1403.7340).
P-9: 分子式： C69H110O25， 分子量： 1338, 白色粉末， [a]D 24 -17 (c 0.21, CHCI3); IR (KBr) v 3453, 2935, 1716, 1639, 1454, 1378, 1097, 1000, 1058, £1 P-9: Molecular formula: C 69 H 110 O 25 , Molecular weight: 1338, white powder, [a] D 24 -17 (c 0.21, CHCI3); IR (KBr) v 3453, 2935, 1716, 1639, 1454, 1378 , 1097, 1000, 1058, £1
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CZ0Z00/ll0ZN3/X3d 表 1 化合物 P-1~P-11的 13C NMR数据 （ 100MHz, CDC13) CZ0Z00/ll0ZN3/X3d Table 1 13 C NMR data of the compounds P-1 to P-11 (100 MHz, CDC1 3 )
Ρ^2 ? ? Ρ^5 Ρ^6 Ρ^7 Ρ^8 Ρ^9 Τ Ο Ρ Ρ^2 ? ? Ρ^5 Ρ^6 Ρ^7 Ρ^8 Ρ^9 Τ Ο Ρ
1 37.4 (t) 37.2 (t) 37.4 (t) 37.3 (t) 37.2 (t) 37.2 (t) 37.2 (t) 37.2 (t) 37.2 (t) 37.1 (t) 37.11 37.4 (t) 37.2 (t) 37.4 (t) 37.3 (t) 37.2 (t) 37.2 (t) 37.2 (t) 37.2 (t) 37.2 (t) 37.1 (t) 37.1
2 29.3 (t) 31.9 (t) 29.4 (t) 31.9 (t) 29.2 (t) 29.2 (t) 29.3 (t) 29.3 (t) 29.3 (t) 31.5 (t) 31.5 2 29.3 (t) 31.9 (t) 29.4 (t) 31.9 (t) 29.2 (t) 29.2 (t) 29.3 (t) 29.3 (t) 29.3 (t) 31.5 (t) 31.5
(d) 78.4 (d) 78.5 (d) 78.5 (d) 78.5 (d) 71.5 (d) 71.5 (  (d) 78.4 (d) 78.5 (d) 78.5 (d) 78.5 (d) 71.5 (d) 71.5 (
(t) 42.2 (s) 140.6 (d) 121.5 (t) 31.8 (d) 31.8 ( (d) 49.5 ( (s) 36.5 ( (t) 20.5 (t) 30.8 (s) 45.2 ( (d) 51.0 ( (t) 23.4 (t) 38.3 (s) 85.3 (
Figure imgf000015_0001
(t) 42.2 (s) 140.6 (d) 121.5 (t) 31.8 (d) 31.8 ( (d) 49.5 ( (s) 36.5 ( (t) 20.5 (t) 30.8 (s) 45.2 ( (d) 51.0 ( t) 23.4 (t) 38.3 (s) 85.3 (
Figure imgf000015_0001
8 47-· (e 8 47-· (e
Si Si
Figure imgf000016_0001
Figure imgf000016_0001
∞ ^ S ^ ^ ¾ *> o o o ∞ ^ S ^ ^ 3⁄4 *> ooo
e a a a a ¾ e a a a a 3⁄4
Figure imgf000016_0002
Figure imgf000016_0002
CZ0Z00/ll0ZN3/X3d 加拿大麻 加拿大麻 加拿大麻 加拿大麻 卡那糖 I 加拿大麻 卡那糖 I 卡那糖 I 糖 II 糖 π 糖 π 糖 π 糖 π CZ0Z00/ll0ZN3/X3d Canada hemp Canadian hemp Canadian hemp Canadian maca saccharide I Canada makana sugar I kana sugar I sugar II sugar π sugar π sugar π sugar π
1 99.8 (d) 99.8 (d) 99.7 (d) 99.7 (d) 101.3 (d) 98.5 (d) 100.3 (d) 101.5 (d) 1 99.8 (d) 99.8 (d) 99.7 (d) 99.7 (d) 101.3 (d) 98.5 (d) 100.3 (d) 101.5 (d)
2 35.6 (t) 35.6 (t) 35.6 (t) 35.6 (t) 38.3 (t) 35.6 (t) 38.3 (t) 38.5 (t)2 35.6 (t) 35.6 (t) 35.6 (t) 35.6 (t) 38.3 (t) 35.6 (t) 38.3 (t) 38.5 (t)
3 77.6 (d) 77.6 (d) 77.6 (d) 77.7 (d) 69.4 (d) 76.7 (d) 69.3 (d) 69.5 (d)3 77.6 (d) 77.6 (d) 77.6 (d) 77.7 (d) 69.4 (d) 76.7 (d) 69.3 (d) 69.5 (d)
4 82.5 (d) 82.5 (d) 82.5 (d) 82.5 (d) 87.7 (d) 83.4 (d) 87.6 (d) 88.0 (d)4 82.5 (d) 82.5 (d) 82.5 (d) 82.5 (d) 87.7 (d) 83.4 (d) 87.6 (d) 88.0 (d)
5 68.5 (d) 68.5 (d) 68.5 (d) 68.5 (d) 70.3 (d) 68.0 (d) 70.3 (d) 70.4 (d)5 68.5 (d) 68.5 (d) 68.5 (d) 68.5 (d) 70.3 (d) 68.0 (d) 70.3 (d) 70.4 (d)
6 18.2 (q) 18.3 (q) 18.2 (q) 18.2 (q) 17.7 (q) 18.0 (q) 17.7 (q) 17.8 (q)-OC 58.1 (q) 58.0 (q) 58.0 (q) 58.1 (q) 57.9 (q) 6 18.2 (q) 18.3 (q) 18.2 (q) 18.2 (q) 17.7 (q) 18.0 (q) 17.7 (q) 17.8 (q)-OC 58.1 (q) 58.0 (q) 58.0 (q) 58.1 (q) ) 57.9 (q)
H Π3、  H Π3,
加拿大麻 加拿大麻 加拿大麻 加拿大麻 加拿大麻 洋地黄毒 洋地黄毒 加拿大麻 糖 III 糖 in 糖 in 糖 III 糖 π 糖 糖 糖 in Canadian hemp Canadian hemp Canadian hemp Canadian hemp Canadian hemp digoxigenis digoxigenis cannabis sugar III sugar in sugar in sugar III sugar π sugar sugar sugar in
1 98.6 (d) 98.5 (d) 98.5 (d) 98.5 (d) 98.4 (d) 98.3 (d) 98.4 (d) 98.5 (d)1 98.6 (d) 98.5 (d) 98.5 (d) 98.5 (d) 98.4 (d) 98.3 (d) 98.4 (d) 98.5 (d)
2 36.7 (t) 36.7 (t) 36.7 (t) 36.8 (t) 35.6 (t) 36.5 (t) 37.0 (t) 35.8 (t)2 36.7 (t) 36.7 (t) 36.7 (t) 36.8 (t) 35.6 (t) 36.5 (t) 37.0 (t) 35.8 (t)
3 77.6 (d) 77.6 (d) 77.7 (d) 77.7 (d) 76.7 (d) 66.3 (d) 66.7 (d) 76.7 (d)3 77.6 (d) 77.6 (d) 77.7 (d) 77.7 (d) 76.7 (d) 66.3 (d) 66.7 (d) 76.7 (d)
4 82.5 (d) 82.5 (d) 82.5 (d) 82.5 (d) 82.4 (d) 82.1 (d) 82.5 (d) 82.6 (d)4 82.5 (d) 82.5 (d) 82.5 (d) 82.5 (d) 82.4 (d) 82.1 (d) 82.5 (d) 82.6 (d)
5 68.9 (d) 68.7 (d) 68.9 (d) 68.9 (d) 68.6 (d) 68.2 (d) 68.2 (d) 68.8 (d)5 68.9 (d) 68.7 (d) 68.9 (d) 68.9 (d) 68.6 (d) 68.2 (d) 68.2 (d) 68.8 (d)
6 18.2 (q) 18.3 (q) 18.2 (q) 18.2 (q) 18.1 (q) 18.1 (q) 18.1 (q) 18.1 (q)-OC 58.0 (q) 58.0 (q) 58.0 (q) 58.1 (q) 58.1 (q) 58.4 (q) ri3 6 18.2 (q) 18.3 (q) 18.2 (q) 18.2 (q) 18.1 (q) 18.1 (q) 18.1 (q) 18.1 (q)-OC 58.0 (q) 58.0 (q) 58.0 (q) 58.1 (q ) 58.1 (q) 58.4 (q) ri3
夹竹桃糖 夹竹桃糖 夹竹桃糖 夹竹桃糖 夹竹桃糖 夹竹桃糖 夹竹桃糖 夹竹桃糖 Oleander Sugar Oleander Sugar Oleander Sugar Oleander Sugar Oleander Sugar Oleander Sugar Oleander Sugar Oleander Sugar
1 113.7 (s) 113.7 (s) 113.7 (s) 113.7 (s) 113.5 (s) 113.5 (s) 113.6 (s) 113.7 (s)1 113.7 (s) 113.7 (s) 113.7 (s) 113.7 (s) 113.5 (s) 113.5 (s) 113.6 (s) 113.7 (s)
2 36.7 (t) 36.7 (t) 36.7 (t) 36.8 (t) 36.7 (t) 36.7 (t) 36.5 (t) 36.7 (t)2 36.7 (t) 36.7 (t) 36.7 (t) 36.8 (t) 36.7 (t) 36.7 (t) 36.5 (t) 36.7 (t)
3 78.3 (d) 77.0 (d) 77.0 (d) 78.3 (d) 78.1 (d) 77.5 (d) 77.6 (d) 77.6 (d)3 78.3 (d) 77.0 (d) 77.0 (d) 78.3 (d) 78.1 (d) 77.5 (d) 77.6 (d) 77.6 (d)
4 82.6 (d) 82.6 (d) 82.6 (d) 82.6 (d) 82.4 (d) 82.2 (d) 82.7 (d) 82.6 (d)4 82.6 (d) 82.6 (d) 82.6 (d) 82.6 (d) 82.4 (d) 82.2 (d) 82.7 (d) 82.6 (d)
5 69.8 (d) 69.9 (d) 69.8 (d) 69.8 (d) 69.8 (d) 69.8 (d) 69.8 (d) 69.9 (d)5 69.8 (d) 69.9 (d) 69.8 (d) 69.8 (d) 69.8 (d) 69.8 (d) 69.8 (d) 69.9 (d)
6 18.0 (q) 18.0 (q) 18.0 (q) 18.0 (q) 18.0 (q) 18.1 (q) 18.1 (q) 18.2 (q)-OC 57.7 (q) 57.7 (q) 57.7 (q) 57.7 (q) 57.4 (q) 57.5 (q) 57.5 (q) 57.6 (q)6 18.0 (q) 18.0 (q) 18.0 (q) 18.0 (q) 18.0 (q) 18.1 (q) 18.1 (q) 18.2 (q)-OC 57.7 (q) 57.7 (q) 57.7 (q) 57.7 (q ) 57.4 (q) 57.5 (q) 57.5 (q) 57.6 (q)
H3 H 3
卡那糖 卡那糖 卡那糖 卡那糖 卡那糖 II 卡那糖 卡那糖 II 卡那糖 II 卡那糖 II 卡那糖 II 卡那糖 IIKana Sugar Kana Sugar Kana Sugar Kana Sugar Kana Sugar II Kana Sugar Kana Sugar II Kana Sugar II Kana Sugar II Kana Sugar II Kana Sugar II
1 100.8 (d) 100.8 (d) 100.8 (d) 100.8 (d) 100.7 (d) 100.7 (d) 100.8 (d) 100.8 (d) 100.8 (d) 100.8 (d) 100.8 (d)1 100.8 (d) 100.8 (d) 100.8 (d) 100.8 (d) 100.7 (d) 100.7 (d) 100.8 (d) 100.8 (d) 100.8 (d) 100.8 (d) 100.8 (d)
2 38.4 (t) 38.4 (t) 38.4 (t) 38.4 (t) 36.7 (t) 36.8 (t) 36.8 (t) 36.9 (t) 36.9 (t) 36.9 (t) 36.9 (t)2 38.4 (t) 38.4 (t) 38.4 (t) 38.4 (t) 36.7 (t) 36.8 (t) 36.8 (t) 36.9 (t) 36.9 (t) 36.9 (t) 36.9 (t)
3 77.0 (d) 78.3 (d) 78.3 (d) 77.0 (d) 77.0 (d) 78.1 (d) 78.2 (d) 78.3 (d) 78.2 (d) 78.2 (d) 78.3 (d)3 77.0 (d) 78.3 (d) 78.3 (d) 77.0 (d) 77.0 (d) 78.1 (d) 78.2 (d) 78.3 (d) 78.2 (d) 78.2 (d) 78.3 (d)
4 79.2 (d) 79.2 (d) 79.2 (d) 79.2 (d) 79.1 (d) 79.0 (d) 79.1 (d) 79.2 (d) 79.1 (d) 79.1 (d) 79.2 (d)4 79.2 (d) 79.2 (d) 79.2 (d) 79.2 (d) 79.1 (d) 79.0 (d) 79.1 (d) 79.2 (d) 79.1 (d) 79.1 (d) 79.2 (d)
5 70.0 (d) 70.0 (d) 70.0 (d) 70.0 (d) 69.8 (d) 69.6 (d) 69.7 (d) 69.8 (d) 69.7 (d) 69.7 (d) 69.8 (d)5 70.0 (d) 70.0 (d) 70.0 (d) 70.0 (d) 69.8 (d) 69.6 (d) 69.7 (d) 69.8 (d) 69.7 (d) 69.7 (d) 69.8 (d)
6 17.0 (q) 17.0 (q) 17.1 (q) 17.1 (q) 17.9 (q) 17.9 (q) 17.9 (q) 18.0 (q) 18.0 (q) 18.0 (q) 18.0 (q)6 17.0 (q) 17.0 (q) 17.1 (q) 17.1 (q) 17.9 (q) 17.9 (q) 17.9 (q) 18.0 (q) 18.0 (q) 18.0 (q) 18.0 (q)
3-OC 86.4 (t) 86.4 (t) 86.4 (t) 86.4 (t) 86.2 (t) 86.2 (t) 86.3 (t) 86.4 (t) 86.3 (t) 86.3 (t) 86.4 (t)3-OC 86.4 (t) 86.4 (t) 86.4 (t) 86.4 (t) 86.2 (t) 86.2 (t) 86.3 (t) 86.4 (t) 86.3 (t) 86.3 (t) 86.4 (t)
H20 H 2 0
实施例 2 化合物 P-l至 P-l l的体外免疫抑制活性测试 Example 2 In vitro immunosuppressive activity test of compounds P-l to P-l l
淋巴细胞毒性评价： 脊椎法处死小鼠， 无菌取其脾脏， 磨碎制成单细胞 悬液， 用 MTT溶解液 (10 %SDS， 50 %DMF)去除红细胞后， 用含 10 %FBS 的 RPMI-1640培养液将细胞浓度调成 5xl05个 /ml。96孔板中加入 5xl05细胞 悬液、 200 μΐ RPMI-1640培养液和适当浓度的待测试样品， 放入 37°C、 5 % C02培养箱中培养 48小时，在结束培养前 5小时，每孔加入 5 mg/ml MTT 18 μ1。 结束培养时， 每孔加入 90 μΐ MTT溶解液， 在培养箱中放置 6-7小时后， 用酶标仪于 570 nm处测定 OD57()值。 Evaluation of lymphocyte toxicity: The mice were sacrificed by the spine method, the spleen was aseptically taken, ground to make a single cell suspension, and the red blood cells were removed with MTT lysate (10% SDS, 50% DMF), and RPMI containing 10% FBS was used. The -1640 medium was adjusted to a cell concentration of 5 x 10 5 /ml. 5 × 10 5 cell suspension, 200 μΐ RPMI-1640 medium and appropriate concentration of the sample to be tested were added to the 96-well plate, and cultured in a 37° C., 5 % C0 2 incubator for 48 hours, 5 hours before the end of the culture. Add 5 mg/ml MTT 18 μl per well. At the end of the culture, 90 μM of MTT lysate was added to each well, and after standing for 6-7 hours in the incubator, the OD 57 () value was measured at 570 nm using a microplate reader.
淋巴细胞增殖实验： 5xl05个新鲜的脾脏细胞按照 37°C、 5 %C02条件培 养 48小时， 培养液用 5 μ^ιηΐ的 ConA或者 LPS来诱导细胞的增殖， 加入适 当浓度的待测试样品以测试其对淋巴细胞增殖的抑制活性。 以 3H-TdR渗入 法定量测定细胞的增殖。在培养结束前 8个小时每孔加入 25 μα的 3H-TdR， 培养结束， 用细胞收集仪收集细胞于玻璃纤维膜上， 加入闪烁液， 用液体闪 烁计数仪检测 DNA中 3H-TdR渗入量来反映细胞增殖情况。 Lymphocyte proliferation assay: 5xl0 5 fresh spleen cells according to 37 ° C, 5% C0 2 conditions for 48 hours, the culture was washed with 5 μ ^ ιηΐ of ConA or LPS induced proliferation, adding the appropriate concentration of sample to be tested To test its inhibitory activity against lymphocyte proliferation. Cell proliferation was quantified by 3 H-TdR infiltration. 25 μα of 3 H-TdR was added to each well 8 hours before the end of the culture, and the culture was completed. The cells were collected on a glass fiber membrane by a cell harvester, scintillation fluid was added, and 3 H-TdR infiltration in the DNA was detected by a liquid scintillation counter. The amount reflects the cell proliferation.
根据化合物对正常小鼠脾脏淋巴细胞的毒性数据 OD57Q值计算 CC5。 ( 50 %细胞毒性浓度），根据化合物对正常小鼠脾脏淋巴细胞增殖的抑制作用计算 IC50 ( 50%抑制浓度）。 安全指数 (safety index,SI)=CC5o/IC5o， 是评价化合物安 全性的主要参数， SI值越大， 化合物越安全。 CC 5 was calculated based on the toxicity data OD 57Q of the compound against normal mouse spleen lymphocytes. (50% cytotoxic concentration), IC 50 (50% inhibitory concentration) was calculated based on the inhibitory effect of the compound on spleen lymphocyte proliferation in normal mice. The safety index (SI) = CC 5 o / IC 5 o is the main parameter for evaluating the safety of a compound. The larger the SI value, the safer the compound.
测试结果如表 2所示。 The test results are shown in Table 2.
表 2本发明中化合物的体外免疫抑制活性测试结果 Table 2 Test results of in vitro immunosuppressive activity of the compounds of the present invention
Τ细胞增殖抑制  Cell proliferation inhibition
化合物编号 ( 50 (μΜ) -Compound number ( 50 (μΜ) -
Κ50 (μΜ) SI (选择指数） 雷帕霉素 0.19 环孢霉素 0.27 Κ 50 (μΜ) SI (selection index) rapamycin 0.19 cyclosporine 0.27
P-1 18.7 0.51 36.7  P-1 18.7 0.51 36.7
P-2 10.1 0.64 15.8  P-2 10.1 0.64 15.8
P-3 15.7 0.82 19.1  P-3 15.7 0.82 19.1
P-4 4.0 1.13 3.5  P-4 4.0 1.13 3.5
P-5 7.78 2.71 2.9  P-5 7.78 2.71 2.9
P-6 3.51 0.46 7.6  P-6 3.51 0.46 7.6
P-7 9.4 1.01 9.3  P-7 9.4 1.01 9.3
P-8 48.2 1.32 36.5  P-8 48.2 1.32 36.5
P-9 6.9 0.52 13.3  P-9 6.9 0.52 13.3
P-10 15.7 0.82 19.1  P-10 15.7 0.82 19.1
P-11 12.3 1.97 6.2  P-11 12.3 1.97 6.2
结果表明， 本发明的化合物淋巴细胞毒性低， 对淋巴细胞的增殖具有显 著抑 。 并且， 从表 2中可以看出， 在本发明的化合物中， 无论 为11 或为
Figure imgf000020_0001
对相应化合物的免疫抑制活性均无明显影响。 实施例 3 化合物 P-1和 Ρ-2对自身免疫性脑脊髓膜炎动物模型的治疗作 用 The results showed that the compound of the present invention had low lymphocyte toxicity and markedly inhibited the proliferation of lymphocytes. And, as can be seen from Table 2, in the compound of the present invention, whether it is 11 or
Figure imgf000020_0001
There was no significant effect on the immunosuppressive activity of the corresponding compounds. Example 3 Therapeutic effects of compounds P-1 and Ρ-2 on animal models of autoimmune meningitis
以 MOG35_5J*段免疫 C57BL/6雌鼠以建立急性诱导实验性自身免疫性 脑脊髓膜炎 （EAE) 模型， 从免疫当天起给 C57BL/6小鼠 （10只 /组） 口服 等体积的 P-l ( 50mg/kg/天、 25mg/kg/天） 或 P-2 ( 10mg/kg/天） 、 阳性对照 药 选 择 一 种 新 型 免 疫 抑 制 剂 FTY720 ( 化 学 名 2-amino-2 [2-(4-octylphenyl)ethyl] -1,3- propanediol hydrochloride, 2 -氨基- 2 [2C57BL/6 female mice were immunized with MOG 35 _ 5 J* to establish an acute induced experimental autoimmune encephalomyelitis (EAE) model, and C57BL/6 mice (10/group) were orally administered from the day of immunization. Equal volume of Pl (50mg/kg/day, 25mg/kg/day) or P-2 (10mg/kg/day), positive control drug selected a novel immunosuppressive agent FTY720 (chemical name 2-amino-2 [2] -(4-octylphenyl)ethyl] -1,3-propanediol hydrochloride, 2 -amino-2 [2
- (4 -辛基苯基)乙基 -1]-1,3-丙二醇盐酸盐） （0.5mg/kg/天） 或溶剂对照。 每 天每只小鼠按照所定标准评分（0分： 没有发病迹象； 1分： 尾巴无力或后肢 无力； 2分： 尾巴无力和后肢无力； 3分： 后肢部分瘫痪； 4分： 后肢完全瘫 痪； 5分： 处于濒死状态或死亡） 和称重。 - (4-octylphenyl)ethyl-l]-1,3-propanediol hydrochloride) (0.5 mg/kg/day) or solvent control. Each mouse was scored according to the established criteria (0 points: no signs of onset; 1 point: weakness of the tail or weakness of the hind limbs; 2 points: weakness of the tail and weakness of the hind limbs; 3 points: partial paralysis of the hind limbs; 4 points: complete paralysis of the hind limbs; 5 Points: in a state of sudden death or death) and weighing.
结果如图 1、 图 2所示 （A: 各组 EAE发病率； B: 疾病评分； C: 体重 变化， *;?<0.05， **^<0.01 ) ， 研究结果显示， 模型组小鼠 100%发病， 疾病 症状严重。 相反， P-l 50mg/kg和 25mg/kg给药组， P-2 10 mg/kg给药组小鼠 发病率明显降低， 而且发病时间延迟 (图 1A、 IB; 图 2A、 2B )。 此外， EAE 发病的另外一个客观指标是体重减轻， P-l、 P-2口服给药能明显阻止小鼠体 重下降 (图 1C; 2C), 实验重复 3次， 得到类似结果。 这些结果提示， P-1和 P-2能够明显降低 EAE的发病率， 减轻疾病的严重程度。 实施例 4 化合物 P-1对实验性类风湿性关节炎动物模型的治疗作用 采用牛 II型胶原 （CII) 诱导 DBA/1小鼠关节炎模型。 CII溶于 0.1M醋 酸溶液， 于 4°C过夜。 实验当天将等体积的含结核分枝杆菌菌株 (Mycobacterium tuberculosis strain) H37Rv的 CFA与 CII胶原充分乳化混匀， 以 250μ8乳化剂于 DBA/1小鼠尾基部进行致敏， 3周后以同样剂量的乳化剂 于尾部进行攻击。 在攻击后一周确认模型小鼠发病， 开始每天口服灌胃给药 1次， 连续 2周， 给药剂量 20mg/kg。 P-l治疗组在以 CII第二次免疫攻击小 鼠后的第 7天开始到第 21天实验结束，每天观察溶剂对照组和 P-1治疗组中 的每只小鼠关节炎病变严重程度。关节病变的严重程度通过评分标准（0分: 正常； 1分： 轻度红肿或足趾关节红肿； 2分： 中度红肿并延伸至整个足部； 3分： 较重红肿并延伸至踝关节； 4分： 趾、 足、 踝均严重红肿， 消退后关节 强直） 评定确定， 每一肢体关节炎评分指数在 0-4分之间， 结果以四肢评分 总和的平均值形式表示。 在第二次免疫攻击后 21天， P-1给药 2周后， 取各 组小鼠后肢， 固定于中性福尔马林溶液中， 石蜡包埋， 切片， 作苏木精 -伊红 Hematoxylin & eosin)染色， 光镜下观察病变关节组织的病理改变， 根据病变 组织的病变程度进行评分。 图 3为化合物 P-1对小鼠关节炎病变严重程度的 影响 （*;?<0.05， **^<0.01 ) 。 阳性对照药为氨甲喋吟 （MTX) 。 图 4、 图 5 为 P-1对小鼠关节炎病理改变的影响， 其中 4A为正常组小鼠踝关节组织切 片（400x ) ; 4B为溶剂对照组小鼠踝关节组织切片（400x ); 4C为化合物 P-1 治疗组小鼠踝关节组织切片 （400x ); 4D为正常组小鼠踝关节 CT断层扫描 图； 4E为溶剂对照组小鼠踝关节 CT断层扫描图； 4F为 P-1治疗组小鼠踝关 节 CT断层扫描图。 图 5A为溶剂对照组小鼠踝关节显微 CT; 图 5B为 P-1 治疗组小鼠踝关节显微 CT。 The results are shown in Figure 1 and Figure 2 (A: incidence of EAE in each group; B: disease score; C: weight change, *;? <0.05, **^<0.01), the results of the study showed that the model group of mice 100 % onset, serious symptoms of the disease. In contrast, in the P1 50 mg/kg and 25 mg/kg administration groups, the incidence of mice in the P-2 10 mg/kg administration group was significantly reduced, and the onset time was delayed (Fig. 1A, IB; Figs. 2A, 2B). In addition, another objective indicator of the onset of EAE is weight loss. Oral administration of Pl and P-2 can significantly prevent weight loss in mice (Fig. 1C ; 2C). The experiment was repeated 3 times and similar results were obtained. These results suggest that P-1 and P-2 can significantly reduce the incidence of EAE and reduce the severity of the disease. Example 4 Therapeutic effect of Compound P-1 on an experimental rheumatoid arthritis animal model The DBA/1 mouse arthritis model was induced using bovine type II collagen (CII). CII was dissolved in 0.1 M acetic acid solution at 4 ° C overnight. On the day of the experiment, an equal volume of CFA and CII collagen containing Mycobacterium tuberculosis strain H37Rv was fully emulsified and mixed, and 250 μ 8 emulsifier was used for sensitization at the base of DBA/1 mice. After 3 weeks, the same The dose of emulsifier is attacked at the tail. The model mice were confirmed to have an onset one week after the challenge, and oral perfusion administration was started once a day for 2 weeks, and the dose was 20 mg/kg. The P1 treatment group was terminated on the 7th day after the second immunization with CII mice, and the end of the experiment was observed on the 21st day. The severity of arthritis in each of the solvent control group and the P-1 treatment group was observed every day. The severity of joint lesions was graded (0 points: normal; 1 point: mild redness or redness of the toe joints; 2 points: moderate redness and extension to the entire foot; 3 points: heavier redness and extension to the ankle joint; 4 points: toe, foot, and sputum are severely red and swollen, and the joint is straightened after the assessment. The assessment determines that each limb arthritis score is between 0 and 4, and the result is limbs. The average form of the sum of the scores is expressed. Twenty-one days after the second immunization challenge, 2 weeks after P-1 administration, the hind limbs of each group of mice were fixed in neutral formalin solution, embedded in paraffin, and sectioned for hematoxylin-eosin. Hematoxylin & eosin) staining, pathological changes of the diseased joint tissue were observed under light microscope, and scored according to the lesion degree of the diseased tissue. Figure 3 shows the effect of compound P-1 on the severity of arthritis in mice (*;?<0.05, **^<0.01). The positive control drug is methotrexate (MTX). Figure 4 and Figure 5 show the effect of P-1 on the pathological changes of arthritis in mice. 4A is the ankle joint tissue section of the normal group (400x); 4B is the ankle joint tissue section of the solvent control group (400x); 4C For the compound P-1 treatment group, the ankle joint tissue section (400x); 4D is the CT scan of the ankle joint of the normal group; 4E is the CT scan of the ankle joint of the solvent control group; 4F is the P-1 treatment Group of mouse ankle CT tomography. Figure 5A shows the ankle joint micro-CT of the solvent control group; Figure 5B shows the ankle joint micro-CT of the P-1 treatment group.
实验结果表明， P-1 口服能够显著减轻关节炎病变的严重程度 （图 3 )， 显著减轻病变关节组织各方面的病理改变 （图 4、 图 5)， 溶剂对照组小鼠病 变部位骨关节破坏， 血管裔形成， 滑膜增生， 周围软组织中大量炎症细胞浸 润 （图 4B)， 出现明显骨溶解现象， 后肢各部位骨骼骨密度下降， 踝关节骨 组织严重变形（图 4E)。 P-1能够显著减轻骨溶解现象， 无明显骨密度下降现 象， 关节部位无明显病理改变 （图 4C、 4F)。 实施例 5 化合物 P-1对 ConA诱导的自身免疫性肝炎模型的预防和治 疗作用  The results of the experiment showed that P-1 oral administration can significantly reduce the severity of arthritic lesions (Fig. 3), significantly reducing the pathological changes in all aspects of the diseased joint tissues (Fig. 4, Fig. 5), and the bone and joint destruction of the lesions in the solvent control group. Vascularization, synovial hyperplasia, infiltration of a large number of inflammatory cells in surrounding soft tissues (Fig. 4B), marked osteolysis, decreased bone mineral density in various parts of the hind limbs, and severe deformation of the ankle bone tissue (Fig. 4E). P-1 can significantly reduce osteolysis, no significant decrease in bone density, and no obvious pathological changes in joints (Fig. 4C, 4F). Example 5 Compound P-1 prevents and treats ConA-induced autoimmune hepatitis model
采用 ConA诱导的肝炎模型。 将 ConA溶解于生理盐水， 在小鼠肝损伤 实验中， 将 ConA配制成 15 mg/kg浓度； 在小鼠生存率实验中， 将 ConA配 制成 20 mg/kg浓度。 配制的 ConA生理盐水溶液过 0.45μιη滤膜。 小鼠肝损伤实验： 以生理盐水为对照， 化合物 P-1按 10 mg/kg的剂量以 腹腔注射的方式提前给药四次（即在 ConA注射前倒数 3， 2， 1天和 1小时）。 在小鼠尾静脉注射 0.2 ml的 15 mg/kg的 ConA后的不同时间点收集小鼠血清， 检测丙氨酸转氨酶 (ALT)水平。 结果如图 6A所示， 模型组小鼠的血清 ALT 水平在注射 ConA ( 15 mg/kg) 后 3小时开始升高， 12小时到达高峰， 然后 再下降； P-1治疗组在各时间点均可以显著降低 ALT水平， 特别是在 6， 12 和 24小时。 A hepatitis model induced by ConA was used. ConA was dissolved in physiological saline, ConA was formulated to a concentration of 15 mg/kg in a mouse liver injury test; ConA was formulated to a concentration of 20 mg/kg in a mouse survival rate experiment. The prepared ConA physiological saline solution was passed through a 0.45 μm filter. Mouse liver injury test: Compound P-1 was administered four times in a dose of 10 mg/kg by intraperitoneal injection (ie, countdown 3, 2, 1 and 1 hour before ConA injection). . Mouse sera were collected at various time points after injection of 0.2 ml of 15 mg/kg of ConA into the tail vein of mice, and alanine aminotransferase (ALT) levels were measured. As a result, as shown in Fig. 6A, the serum ALT level of the model group began to rise 3 hours after the injection of ConA (15 mg/kg), reached a peak at 12 hours, and then decreased again; the P-1 treatment group was at each time point. Can significantly reduce ALT levels, especially at 6, 12 and 24 hours.
小鼠生存实验： 在小鼠尾静脉注射 0.2 ml 的致死剂量 （20 mg/kg) 的 ConA, 检测 P-1是否能够提高小鼠的生存率。 结果如图 6B所示， 在注射致 死剂量的 ConA (20 mg/kg) 后， 模型组小鼠在 9小时内全部死亡， 而此时 P-1治疗组小鼠生存率为 60%;在 48小时后， P-1治疗组小鼠生存率为 40%。  Mouse Survival Assay: A 0.2 ml lethal dose (20 mg/kg) of ConA was injected into the tail vein of mice to determine whether P-1 could improve survival in mice. The results are shown in Fig. 6B. After injection of a lethal dose of ConA (20 mg/kg), the mice in the model group all died within 9 hours, while the survival rate of the mice in the P-1 treatment group was 60%; After an hour, the survival rate of the mice in the P-1 treatment group was 40%.
肝脏组织学检查结果显示出 P-1可以显著减缓 ConA引起的肝脏损伤。 如图 6C所示， 在溶剂治疗的肝炎小鼠中， 肝脏出现大面积坏死和炎性细胞 浸润； 10 mg/kg P-1预防给药明显缓解肝细胞坏死的症状。 病理学评分结果 也可以看出 P-1对肝脏坏死和炎性细胞浸润具有显著保护作用 （图 6D)。  Liver histology revealed that P-1 significantly slowed the liver damage caused by ConA. As shown in Fig. 6C, in the solvent-treated hepatitis mice, extensive necrosis and inflammatory cell infiltration occurred in the liver; 10 mg/kg P-1 prophylactic administration significantly ameliorated the symptoms of hepatocyte necrosis. Pathological scoring results also showed that P-1 had significant protective effects on hepatic necrosis and inflammatory cell infiltration (Fig. 6D).
根据报道，小鼠在对 ConA的应答中， NKT细胞释放了大量的细胞因子， 包括 IL-4， IL-5 , IFN-γ以及 TNF-α, 各细胞因子出现和达到高峰的时间有所 不同。 IL-4和 IFN-γ被证明是在 ConA诱导的小鼠肝损伤的发生发展过程中 起着关键作用的细胞因子， 因此我们着重检测了 P-1是否能够影响这两种细 胞因子的水平。 如图 7所示， 在注射 ConA后， P-1预先给药治疗组在 2小 时和 6小时均能降低血清中 70%的 IL-4 水平； 在 6小时和 12小时能分别降 低血清中 60%和 80%的 IFN-γ水平。  According to reports, in response to ConA, NKT cells release a large number of cytokines, including IL-4, IL-5, IFN-γ, and TNF-α, and the time at which each cytokine appears and peaks is different. . IL-4 and IFN-γ have been shown to be key cytokines in the development of ConA-induced liver injury in mice, so we focused on whether P-1 can affect the levels of these two cytokines. As shown in Figure 7, after injection of ConA, the P-1 pre-administered treatment group reduced 70% of IL-4 levels in serum at 2 hours and 6 hours; and reduced serum levels at 6 and 12 hours, respectively. % and 80% IFN-γ levels.
实施例 6 香加皮粗提物 WJ-C对实验性类风湿性关节炎动物模型的治 疗作用  Example 6 The crude extract of fragrant skin extract WJ-C for the treatment of experimental rheumatoid arthritis animal model
实验动物来源：购自中国科学院上海实验动物中心，动物生产许可证号： SCXK (沪） 2007-0005 Source of experimental animals: purchased from Shanghai Experimental Animal Center of Chinese Academy of Sciences, animal production license number: SCXK (Shanghai) 2007-0005
主要试剂： RPMI-1640培养基购自 GibcoBRL公司 （ Life Technologies， Grand Island, NY, USA) ; 胎牛血清（fetal bovine serum, FBS )购自 Hyclone 公司（Logan, Utah, USA);牛 II型胶原购自 Collagen Research Center ( Tokyo， Japan) ;弗氏完全佐剂含 Mycobacterium tuberculosis strain H37Rv,购自 Wako Pure Chemical Industries Ltd ( Osaka, Japan)。 刀豆蛋白 A (concanavalin A, Con A) 购自 Sigma公司。 [甲基 -3H]胸腺嘧啶核苷 (TdR) 购自中国科学院上 海应用物理研究所 （原上海原子核研究所） 放药中心。  Primary Reagents: RPMI-1640 medium was purchased from Gibco BRL (Life Technologies, Grand Island, NY, USA); fetal bovine serum (FBS) was purchased from Hyclone (Logan, Utah, USA); bovine type II collagen Purchased from Collagen Research Center (Tokyo, Japan); Freund's complete adjuvant containing Mycobacterium tuberculosis strain H37Rv, available from Wako Pure Chemical Industries Ltd (Osaka, Japan). Concanavalin A (Con A) was purchased from Sigma. [Methyl-3H]thymidine (TdR) was purchased from the Shanghai Institute of Applied Physics (formerly Shanghai Institute of Nuclear Research).
采用牛 II型胶原 （CII) 诱导 DBA/1小鼠关节炎模型。 CII溶于 0.1M醋酸溶 液， 于 4°C过夜。 实验当天将等体积的含结核分枝杆菌菌株 (Mycobacterium tuberculosis strain) H37Rv的 CFA与 CII胶原充分乳化混匀，以 25(^g乳化剂 于 DBA/1小鼠尾基部进行致敏，3周后以同样剂量的乳化剂于尾部进行攻击。 在攻击后一周确认模型小鼠发病， 开始进行连续 2周的口服给药治疗 [WJ-C: 12.5mg/kg、 25mg/kg、 52mg/kg; 氨甲喋吟 (MTX): lmg/kg]。 WJ-C治疗组在 以 CII第二次免疫攻击小鼠后的第 7天开始到第 21天实验结束，每天观察溶 剂对照组和 WJ-C治疗组中的每只小鼠关节炎病变严重程度。 关节病变的严 重程度通过评分标准 （0分： 正常； 1分： 轻度红肿或足趾关节红肿； 2分： 中度红肿并延伸至整个足部； 3分： 较重红肿并延伸至踝关节； 4分： 趾、足、 踝均严重红肿， 消退后关节强直）评定确定， 每一肢体关节炎评分指数在 0-4 分之间， 结果以四肢评分的总和表示。 图 8为 WJ-C对小鼠关节炎病变严重 程度的影响， 结果显示， WJ-C浓度依赖性抑制小鼠关节炎的发展。 An arthritis model of DBA/1 mice was induced using bovine type II collagen (CII). The CII was dissolved in 0.1 M acetic acid solution at 4 ° C overnight. On the day of the experiment, an equal volume of CFA and CII collagen containing Mycobacterium tuberculosis strain H37Rv was fully emulsified and mixed, and 25 (^g emulsifier was sensitized to the base of DBA/1 mouse, 3 weeks later. The same dose of emulsifier was used to attack the tail. The mice in the model were confirmed one week after the challenge, and oral administration was continued for 2 weeks [WJ-C: 12.5 mg/kg, 25 mg/kg, 52 mg/kg; ammonia Hyperthyroidism (MTX): lmg/kg]. The WJ-C treatment group ended on the 7th day after the second immunization with CII mice, and ended on the 21st day. The solvent control group and WJ-C treatment were observed every day. The severity of arthritic lesions in each mouse in the group. The severity of joint lesions was graded (0 points: normal; 1 point: mild redness or redness of the toe joints; 2 points: moderate redness and extended to the entire foot Department; 3 points: heavier redness and extension to the ankle joint; 4 points: toe, foot, and sputum are severely red and swollen, and the joint is straightened after the assessment). The score of each limb arthritis score is between 0 and 4 points. Expressed as the sum of the limbs scores. Figure 8 shows The effect of WJ-C on the severity of arthritis in mice showed that WJ-C concentration-dependently inhibited the development of arthritis in mice.

Claims

权利要求 类含有原酸酯基团的孕甾烷糖苷类化合物， 其由如下通式 I表  Claims Pregnancy glucoside compounds containing orthoester groups, which are represented by the following formula I
Figure imgf000025_0001
Figure imgf000025_0001
产物通过醚键连接构成的糖链片段。 The product is linked by a ether linkage to a sugar chain fragment.
2、如权利要求 1所述的含有原酸酯基团的孕 烷糖苷类化合物，其特征 在于， 通式 I中：  The pregnane group-containing pregnane glycoside compound according to claim 1, wherein in the formula I:
Figure imgf000025_0002
Figure imgf000026_0001
Figure imgf000025_0002
Figure imgf000026_0001
加拿大麻糖 加拿大麻糖 加拿大麻糖 乙酰基洋地黄糖
Figure imgf000026_0002
Canadian Hemp Sugar Canada Hemp Sugar Canada Hemp Acetyl Digitalis
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加拿大麻糖 加拿大麻糖 加拿大麻糖 洋地黄糖
Figure imgf000026_0003
Canadian Hemp Sugar Canada Hemp Sugar Canada Hemp Candy Digitalis
Figure imgf000026_0003
洋地黄毒糖 加拿大麻糖 加拿大麻糖 乙酰基洋地黄糖
Figure imgf000026_0004
Figure imgf000027_0001
Digitalis syrup canadian cannabis cannabis acetyl digoxigen
Figure imgf000026_0004
Figure imgf000027_0001
加拿大麻糖 I 卡那糖 I 加拿大麻糖 11 加拿大麻糖 I (4-乙酰基）
Figure imgf000027_0002
Canadian Hemp Sugar I Caramel I Canadian Hemp Sugar 11 Canadian Hemp Sugar I (4-Acetyl)
Figure imgf000027_0002
II 卡那糖 I 加 I 夹竹桃糖 。  II Kana sugar I plus I oleander sugar.
3、如权利要求 1所述的含有原酸酯基团的孕 烷糖苷类化合物，其选自 下列化合物中：  The pregnane group-containing pregnane glycoside compound according to claim 1, which is selected from the group consisting of the following compounds:
Figure imgf000027_0003
Figure imgf000027_0003
P-2: R, H, R2 加拿大麻糖 111 加拿大麻糖 11 加拿大麻糖 I 2-乙酰基洋地黄糖 P-2: R, H, R 2 Canadian Hemp Sugar 111 Canadian Hemp Sugar 11 Canadian Hemp Sugar I 2-Acetyldigone
P-3:R
Figure imgf000027_0004
， R2为 加拿大麻糖 111 加拿大麻糖 11 加拿大麻糖 I 洋地黄糖 P-3: R
Figure imgf000027_0004
, R 2 is sesame chips 111 Canada Canada Canada sesame chips 11 Matang I digitalis sugar
Figure imgf000028_0001
Figure imgf000028_0001
Figure imgf000028_0002
Figure imgf000028_0002
27 。 、 、 Y ."0H 27 . , , Y ." 0H
P-9: Ri为
Figure imgf000029_0001
R2为 加拿大麻糖 卡那糖 加拿大麻糖 夹竹桃糖 P-9: Ri is
Figure imgf000029_0001
R 2 is Canadian cannabis kana sugar cannabis oleander nectar
Figure imgf000029_0002
Figure imgf000029_0002
P-10: Ri H, R2为 加拿大麻糖 卡那糖 加拿大麻糖 夹竹桃:
Figure imgf000029_0003
P-10: Ri H, R 2 is Canadian cannabis kana sugar cannabis oleander:
Figure imgf000029_0003
P-11: Ri为 H， R2为
Figure imgf000029_0004
P-11: Ri is H, R 2 is
Figure imgf000029_0004
4、一种药物组合物，其包含治疗有效量的如权利要求 1至 3中任一项所 述的一种或多种含有原酸酯基团的孕 烷糖苷类化合物作为活性成分。 A pharmaceutical composition comprising as an active ingredient a therapeutically effective amount of one or more pregnane group-containing pregnane glycoside compounds according to any one of claims 1 to 3.
5、一种从杠柳属植物中提取如权利要求 3所述的含有原酸酯基团的孕甾 烷糖苷类化合物的方法， 其包括下列歩骤：  A method for extracting a pregnane group-containing progesterone glycoside compound according to claim 3, which comprises the following steps:
(一) 提取化合物 P-l、 P-2、 P-5、 P-6、 P-7、 P-3和 P-4  (1) Extraction of compounds P-l, P-2, P-5, P-6, P-7, P-3 and P-4
(1) 将香加皮药材用 95%乙醇回流提取三次， 减压浓缩提取液得到乙醇 浸膏， 加入水悬浮， 依次用氯仿和正丁醇萃取， 得到氯仿部位浸膏和正丁醇 部位浸膏， 氯仿部位浸膏经硅胶柱层析分离， 以石油醚 -丙酮体积比依次为 5:1、 2:1、 1:1、 0:1梯度洗脱得流分 Frl、 Fr2、 Fr3和 Fr4;  (1) The fragrant medicinal herbs are extracted three times with 95% ethanol, and the extract is concentrated under reduced pressure to obtain an ethanol extract, which is added to water suspension, and sequentially extracted with chloroform and n-butanol to obtain a chloroform extract and a n-butanol extract. The chloroform extract was separated by silica gel column chromatography and eluted with a gradient of 5:1, 2:1, 1:1, 0:1 in volume ratio of petroleum ether-acetone. The fractions Frl, Fr2, Fr3 and Fr4 were obtained.
(2) 流分 Fr3经反相 C18柱层析，以甲醇-水梯度洗脱，体积比依次为 1:1、 2:1、 3:1、 4:1、 1:0， 得次流分 Fr3.1、 Fr3.2、 Fr3.3、 Fr3.4; (2) The fraction Fr3 was subjected to reversed-phase C 18 column chromatography and eluted with a methanol-water gradient. The volume ratio was 1:1, 2:1, 3:1, 4:1, 1:0, and the secondary flow was obtained. Fr3.1, Fr3.2, Fr3.3, Fr3.4;
(3) 次流分 Fr3.4经反相 C18制备液相色谱， 以甲醇-水梯度洗脱， 体积比 依次为 2:3、 1:1、 2:1、 3:1、 4:1和 1:0， 得 P-l、 P-2、 P-5、 P-6; 流分 Fr4经 反相 C18制备液相色谱， 以甲醇-水梯度洗脱， 体积比依次为 3:7、 2:3、 1 : 1、 2: 1、 3:1和 4:1， 得 P-3、 P-4、 P-7; (3) The secondary fraction Fr3.4 was prepared by reverse phase C 18 preparative liquid chromatography, eluting with a methanol-water gradient, and the volume ratio was 2:3, 1:1, 2:1, 3:1, 4:1. And 1:0, get Pl, P-2, P-5, P-6; fraction Fr4 Prepared by reverse phase C 18 preparative liquid chromatography, eluting with a methanol-water gradient. The volume ratios were 3:7, 2:3, 1:1, 2:1, 3:1 and 4:1, resulting in P-3. P-4, P-7;
(二） 提取化合物 P-8、 P-9、 P-10和 P-l l  (ii) Extraction of compounds P-8, P-9, P-10 and P-l l
(1) 将黑骨藤根用 95 %的乙醇室温浸提， 减压蒸馏浓缩成浸膏， 并用水 分散悬浮， 之后依次用石油醚、 乙酸乙酯和正丁醇萃取， 分别得到石油醚部 位浸膏、 乙酸乙酯部位浸膏和正丁醇部位浸膏；  (1) The black-bone root was extracted with 95% ethanol at room temperature, distilled under reduced pressure to form an extract, and dispersed in water, followed by extraction with petroleum ether, ethyl acetate and n-butanol to obtain a petroleum ether extract. , ethyl acetate extract and n-butanol extract;
(2) 将乙酸乙酯部位浸膏进行反相 C18柱层析， 用甲醇-水梯度洗脱， 体 积比依次为 1 :9、 2:8、 3:7、 4:6、 5:5、 6:4、 7:3、 8:2、 9:1和 1 :0， 得到 Frl-Fr6 六个流分； (2) The ethyl acetate fraction extract was subjected to reverse phase C 18 column chromatography and eluted with a methanol-water gradient in a volume ratio of 1:9, 2:8, 3:7, 4:6, 5:5. , 6:4, 7:3, 8:2, 9:1 and 1:0, get six fractions of Frl-Fr6;
(3) 流分 Fr5用 Sephadex LH-20除杂， 然后以氯仿 -丙酮体积比依次为 8: 1、 7:1、 6:1的洗脱剂进行硅胶柱层析得到 P-8、 P-9、 P-10和 P-l l。  (3) The fraction Fr5 was depurated with Sephadex LH-20, and then subjected to silica gel column chromatography with an eluent of 8:1, 7:1, 6:1 in a volume ratio of chloroform-acetone to obtain P-8, P-. 9, P-10 and Pl l.
6、如权利要求 1至 3中任一项所述的含有原酸酯基团的孕^烷糖苷类化 合物在制备免疫抑制剂中的用途。  Use of the orthoester group-containing compound of the orthoester group according to any one of claims 1 to 3 for the preparation of an immunosuppressive agent.
7、 如权利要求 6所述的用途， 其中， 所述免疫抑制剂用于治疗和 /或预 防细胞 /器官移植后排斥反应或自身免疫性疾病。  7. The use according to claim 6, wherein the immunosuppressant is for treating and/or preventing rejection after a cell/organ transplant or an autoimmune disease.
8、如权利要求 7所述的用途， 其中， 所述自身免疫性疾病为多发性硬化 症、 类风湿关节炎和自身免疫性肝炎。  The use according to claim 7, wherein the autoimmune diseases are multiple sclerosis, rheumatoid arthritis, and autoimmune hepatitis.
9、一种富含含有原酸酯基团的孕 烷糖苷的香加皮粗提物，其通过下述 方法得到：  9. A crude extract of fragrant skin of a pregnane glycoside containing an orthoester group, which is obtained by the following method:
将香加皮药材用 95%乙醇回流提取三次，减压浓缩提取液得到乙醇浸膏， 向乙醇浸膏加入水， 放置沉淀， 离心分离， 沉淀以石油醚洗涤， 再用乙醇溶 解、静置， 过滤， 取上清液， 配成 70%乙醇溶液， 进行大孔树脂柱色谱分离， 收集 90%乙醇的洗脱流分， 浓缩， 即得。  The fragrant medicinal herbs were extracted three times with 95% ethanol, and the extract was concentrated under reduced pressure to obtain an ethanol extract. Water was added to the ethanol extract, and the precipitate was placed, centrifuged, and the precipitate was washed with petroleum ether, dissolved in ethanol, and allowed to stand. Filtration, take the supernatant, and prepare a 70% ethanol solution, perform macroporous resin column chromatography, collect 90% ethanol elution fraction, and concentrate.
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