CN110407904B

CN110407904B - Dihydroartemisinin steroid conjugate and preparation method and application thereof

Info

Publication number: CN110407904B
Application number: CN201910705451.8A
Authority: CN
Inventors: 沈征武; 边弘竹
Original assignee: Yunbaiyao Zhengwu Technology Shanghai Co ltd
Current assignee: Yunbaiyao Zhengwu Technology Shanghai Co ltd
Priority date: 2019-07-31
Filing date: 2019-07-31
Publication date: 2023-06-16
Anticipated expiration: 2039-07-31
Also published as: CN110407904A

Abstract

The invention discloses a conjugate obtained by condensing dihydroartemisinin with steroid, or isomer, or medicinal salt, or prodrug molecule thereof, which has the following general formula (I); wherein, the 10-hydroxyl of the dihydroartemisinin and the 3-hydroxyl of the steroid compound are condensed and connected in an ether bond way. The invention discloses a preparation method of the compound and application of the compound in treating autoimmune diseases. The dihydroartemisinin steroid conjugate is a novel immunosuppressant, and the compound can be used for treating autoimmune diseases of human bodies singly or in combination with other medicines. The traditional Chinese medicine has the advantages of high curative effect, low toxicity and very broad application prospect.

Description

Dihydroartemisinin steroid conjugate and preparation method and application thereof

Technical Field

The invention relates to the technical field of pharmaceutical chemistry, in particular to a preparation method and application of conjugates formed by condensation of dihydroartemisinin 10-hydroxy and various steroid 3-hydroxy through ether bonds.

Background

Artemisinin is a rare sesquiterpene lactone containing a peroxy group isolated and extracted from the chinese drug artemia annua l. The arteannuin and its derivatives artemether, artesunate, dihydroarteannuin, etc. are used for treating malaria clinically, and have the characteristics of high efficiency, quick acting and low toxicity. In addition to their antimalarial effect, artemisinin and its derivatives have been found to have excellent immunosuppressive activity in clinical and laboratory studies for many years.

Immunosuppressant is a kind of medicine with immunosuppressant effect, and has the main function of inhibiting abnormal immune response, and is used in treating autoimmune diseases, allergic reaction and rejection after organ transplantation. Autoimmune diseases are the corresponding clinical symptoms caused by too strong or too long duration of autoimmune response to destroy the normal tissue structure of the autoimmune disease; allergic reaction is a pathological immune reaction which is generated after continuous stimulation of organism antigen or re-stimulation of the same antigen and mainly shows physiological dysfunction and tissue injury; following a clinical organ transplant procedure, the recipient immune system recognizes plant antigens and produces a response, and immune cells in the transplant can also recognize recipient tissue antigens and produce an immune response, which is a transplant rejection response. These symptoms are caused by an "inappropriate" immune response in the body, and immunosuppressants can play a therapeutic role to some extent, especially play a key role in the prevention and treatment of organ transplant rejection, and are widely used clinically.

Currently, all immunosuppressants can be divided into the following:

(1) Antimetabolites: azathioprine (Aza), methotrexate, mycophenolate Mofetil (MMF), and the like;

(2) Alkylating agent: cyclophosphamide and the like;

(3) Cortisol: prednisone, dexamethasone, and the like;

(4) Antibiotics: csA, FK506, rapamycin, etc.;

(5) Antibodies: anti-lymphoglobulin (ALG), monoclonal T lymphocyte antibody (OKT 3), etc.;

(6) Chinese herbal medicine: tripterygium glycosides, cordyceps sinensis preparations, etc.

The first three are clinically used immunosuppressants, and although the curative effects of the medicines are proved, only symptoms can be improved, the occurrence and the development of diseases cannot be effectively and fundamentally controlled, and the medicines have large toxic and side effects and cannot be continuously applied for a long time. Antibody immunosuppressants can act exclusively on lymphocytes, but are expensive and have serious adverse reactions, and further improvement is required.

With the recent increasing incidence of autoimmune diseases, there is an increasing need for treatment with immunosuppressive drugs. Autoimmune diseases are mostly chronic or progressive diseases, long-term administration is needed, and serious side effects are generally observed when the existing immunosuppressants are used for long-term administration, so that a new type of high-efficiency low-toxicity immunosuppressants are urgently needed for combined or alternate administration clinically. In recent years, in the research of immunosuppressive activity of artemisinin derivatives, the immunosuppressive activity of novel artemisinin derivatives synthesized by structural modification is greatly improved, and meanwhile, the artemisinin derivatives keep the characteristic of low toxicity in vivo, so that the artemisinin derivatives are expected to be developed into novel immunosuppressive agents.

To date, autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis still lack excellent therapeutic drugs, and this condition has become an internationally recognized medical problem today. Their pathogenesis and control mechanisms are not well understood, which presents great difficulties in the design of therapeutic and immunological intervention means. Current classical therapeutic approaches at home and abroad involve the use of hormones and immunosuppressants. Hormone therapy can temporarily relieve symptoms, but long-term application is easy to induce infection and produce a plurality of side effects, and relapse is easy to occur after drug withdrawal. Although the advent and use of immunosuppressants has increased clinical intervention and treatment of disease progression, long-term use can produce more toxic side effects in addition to being expensive. Therefore, aiming at autoimmune diseases such as systemic lupus erythematosus, research and development of novel immunosuppressants with independent intellectual property rights in China, high efficiency and low toxicity have become urgent subjects for current medical and pharmaceutical research.

Dry eye is an ocular surface inflammation characterized clinically by varying degrees of conjunctival congestion, and despite the varying etiology that initiates dry eye, immune-related inflammatory responses become the most important segments in the pathogenesis of dry eye when the tear film continues to be abnormal, ocular surface repair and defensive function is impaired. Studies have shown that long term abnormalities in the tear film cause decreased secretion of anti-inflammatory components (such as lactoferrin) in tears, while inflammatory cells in tissues such as ocular surfaces, lacrimal glands, etc. produce inflammatory factors and proteases, activate the MAPK signaling pathway, increase inflammatory factor content and protease activation in tears, initiate a series of inflammatory reactions, and cause T cell infiltration in the eyes [ J ]. Invest Ophthalmol Vis Sci,2004, 45: 4293-4301 ]. Therefore, there are attempts to treat dry eye with immunosuppressants both at home and abroad.

Artemisinin and its derivatives have been continuously demonstrated to have a certain immunosuppressive activity in clinical and laboratory studies. In order to research and obtain compounds with higher immunosuppressive activity, pharmaceutical workers have tried to introduce new groups on the artemisinin parent structure, and a large number of new artemisinin compounds are synthesized. Through research on the novel artemisinin derivatives, high-efficiency and low-toxicity immunosuppressants suitable for clinic can be found. The arteether maleate (SM 934) developed by Shanghai medicine of China academy of sciences has water solubility, bioavailability and immunosuppressive activity which are all obviously higher than those of dihydroartemisinin, is a better immunosuppressive candidate new medicine which appears in China in recent years, has a simple structure and a stable chemical structure, and is a candidate new medicine aiming at systemic lupus erythematosus. The dihydroartemisinin steroid conjugate represented by the compound 4 has far better immunosuppressive activity than SM934 through in vitro activity test. In two in vitro immunosuppressive activity studies experiments: experiments for inhibiting proliferation of spleen T lymphocytes of mice induced by concanavalin A (Con A) by the compound and experiments for inhibiting proliferation of spleen B lymphocytes of normal mice induced by endotoxin (LPS) by the compound. IC of Compound 4 ₅₀ The values were 0.015. Mu.M and 0.015. Mu.M, respectively. Whereas SM934 inhibits T lymphocyte proliferation ₅₀ 1.2. Mu.M, compound 4 was 80 times more active than it was; IC for inhibiting proliferation of B lymphocyte by SM934 ₅₀ At 2.6. Mu.M, compound 4 was 170 times more active. [ International Immunopharmacology (2009) 1509-1517 ]

Disclosure of Invention

The invention aims to solve the technical problem of providing a conjugate formed by condensation of dihydroartemisinin 10-hydroxy and steroid 3-hydroxy through ether bond and a derivative thereof, wherein the dihydroartemisinin steroid conjugate is used as a novel immunosuppressant, can effectively inhibit immune hyperfunction or over-expression, and can be used for treating various autoimmune diseases of human bodies by singly or jointly using the dihydroartemisinin steroid conjugate and other medicines.

In order to solve the technical problems, the invention is realized by the following technical scheme:

in one aspect of the present invention, there is provided a conjugate formed by condensing the 10-position hydroxyl group of dihydroartemisinin and 3-position hydroxyl groups of various steroids of the general formula (I) through ether linkage, and derivatives and isomers thereof, or pharmaceutically acceptable salts or prodrug molecules thereof,

wherein, the 10-hydroxyl of the dihydroartemisinin is condensed with 3-hydroxyl of various steroids to form new dihydroartemisinin derivatives;

The dihydroartemisinin has the structure of (II):

the 10-hydroxyl of dihydroartemisinin can be in alpha or beta configuration;

wherein, the 10-hydroxyl of the dihydroartemisinin and the 3-hydroxyl of the steroid are connected by ether bond. The 10-position of the dihydroartemisinin can be in alpha or beta configuration; the 3-position of the steroid may be in the alpha or beta configuration;

in the general formula (I), n=0 to 5;

in the general formula (I), R ₁ An H atom in the 6-or 7-position, or a hydroxyl, alkoxy or amino and amino group, or a ketocarbonyl group, or a double bond formed in the 6-and 7-positions; if the 6-or 7-position is hydroxyl or amino and amino, it may be in the alpha or beta configuration;

in the general formula (I), R ₂ Can be H atom or hydroxyl, alkoxy or amino and amino, or ketocarbonyl; r is R ₂ If hydroxyl or amino and amino, the alpha or beta configuration is possible;

in the general formula (I), R ₃ Examples of the compounds include hydrogen, carboxyl groups and derivatives thereof, hydroxyl groups and derivatives thereof, amino groups and derivatives thereof, halogen, alkyl groups of 1 to 10 carbon atoms and alkene or alkyne, alcohol or polyalcohol of 1 to 10 carbon atoms and derivatives thereof formed by hydroxyl groups of alcohol, carboxylic acid or polycarboxylic acid of 1 to 10 carbon atoms and derivatives thereof formed by carboxyl groups, alkylsulfonic acid of 1 to 10 carbon atoms and salts thereof, amine of 1 to 10 carbon atoms and derivatives thereof formed by amino groups, alkylimine and alkoxyimine of 1 to 10 carbon atoms, hydroxylamine sulfonate and salts thereof, and three-to eight-membered cyclic hydrocarbon or heterocyclic hydrocarbon containing or not containing hetero atoms, and derivatives formed by linking R3 and 16-carbon atoms into a ring.

In the general formula (I) of the invention, isomers of conjugates obtained by condensing the hydroxyl group at the 10-position of dihydroartemisinin and the hydroxyl group at the 3-position of steroid compounds include all isomers thereof, such as positional isomers, stereoisomers and optical isomers.

Preferably, the derivatives of formula (I) include compounds of the following specific structure:

in the present invention, the condensation of the hydroxyl group at the 10-position of dihydroartemisinin with the hydroxyl group at the 3-position of the steroid gives pharmaceutically acceptable salts of the conjugate, including lithium, potassium, sodium, calcium, magnesium salts, or organic salts with natural and unnatural organic nitrogen-containing compounds.

In the invention, conjugate prodrug molecules obtained by condensing 10-hydroxyl of dihydroartemisinin and 3-hydroxyl of steroid compounds refer to compounds which can be quickly converted into pharmacodynamic molecules in vivo after chemical structure modification of medicines, and the prodrug molecules are designed for improving bioavailability of the medicines, enhancing targeting, reducing toxic and side effects of the medicines and the like.

In another aspect of the invention, the conjugate obtained by condensing the hydroxyl group at the 10-position of dihydroartemisinin and the hydroxyl group at the 3-position of the steroid further comprises a pharmaceutical composition formed with an auxiliary material/carrier, i.e. comprising a therapeutically effective amount of the above-mentioned dihydroartemisinin-steroid conjugate or an isomer thereof, or a pharmaceutically acceptable salt thereof, or a prodrug molecule thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

The acceptable carrier is non-toxic, can be administered adjunctively and has no adverse effect on the therapeutic efficacy of the conjugate. Such carriers may be any solid excipient, liquid excipient, semi-solid excipient, or gaseous excipient in aerosol compositions commonly available to those skilled in the art. Solid pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, stearyl glycerate, sodium chloride, anhydrous skim milk, and the like. Liquid and semi-solid excipients may be selected from glycerol, propylene glycol, water, ethanol and various oils, including those of petroleum, animal, vegetable or synthetic origin, for example peanut oil, soybean oil, mineral oil, sesame oil and the like, preferred liquid carriers, particularly for injectable solutions, including water, saline, aqueous dextrose and glycols. Other adjuvants such as flavoring agent, sweetener, etc. can also be added into the composition.

The medicament or pharmaceutical composition of the present invention may be administered orally or by other modes of administration, such as injection, transdermal administration, spray administration, rectal administration, vaginal administration, etc. The preferred mode of administration is injection or oral, which may be adjusted according to the extent of the disease.

The pharmaceutical compositions of the present invention may be used in combination with other immunosuppressants including antimetabolites: azathioprine (Aza), methotrexate, mycophenolate Mofetil (MMF), and the like; alkylating agent: cyclophosphamide and the like; cortisol: prednisone, dexamethasone, and the like; antibiotics: csA, FK506, rapamycin, etc.; antibodies: anti-lymphoglobulin (ALG), monoclonal T lymphocyte antibody (OKT 3), etc.; chinese herbal medicine: tripterygium glycosides, cordyceps sinensis preparations, etc.

The various dosage forms of the pharmaceutical composition of the present invention may be prepared according to conventional methods in the pharmaceutical arts. For example, the compound is admixed with one or more carriers and then formulated into a desired dosage form, such as a tablet, pill, capsule, semi-solid, powder, sustained release dosage form, solution, suspension, formulation, aerosol, or the like.

In another aspect of the invention, a preparation method of the conjugate is provided, wherein the condensation reaction of the 10-hydroxyl of dihydroartemisinin and the 3-hydroxyl of the steroid compound is carried out under the catalysis of acidic reagents including boron trifluoride diethyl ether, acidic resin, phosphotungstic acid, camphorsulfonic acid and the like.

In another aspect of the invention, the invention also provides application of the conjugate obtained by condensing the 10-hydroxyl of dihydroartemisinin and the 3-hydroxyl of steroid compound or isomer thereof, or pharmaceutically acceptable salt thereof, or prodrug molecule thereof in preparing medicines for treating autoimmune diseases.

The autoimmune diseases include lupus erythematosus, rheumatoid arthritis, multiple sclerosis, psoriasis, xerophthalmia, and other autoimmune diseases.

The autoimmune diseases of the human body also comprise rejection reaction after human organ transplantation.

1. The derivative of the invention has simple synthesis, good chemical stability and low toxicity, and shows very strong inhibition activity on T cell proliferation and interferon IFN-gamma in vitro experiments.

2. Because of the structural characteristics of artemisinin compounds, the half-life period of artemisinin compounds in vivo is only about 40 minutes, and the artemisinin compounds need to enter cells as soon as possible to play a role. In the derivative, the permeability and the permeability speed of artemisinin to cells are increased by conjugated cholic acid, so that the compound can quickly enter cells through ion channels on the surface of cell membranes: therefore, the biological activity of the artemisinin derivative is far stronger than that of other artemisinin derivatives, the dosage can be reduced, and the toxicity can be further reduced.

3. The compound obtained by the invention has higher immunosuppressive activity than traditional artemisinin, dihydroartemisinin, artemether, artesunate, beta-amino arteether maleate (SM 934) and the like, and has wide application prospect in the aspect of treating autoimmune diseases of human bodies.

Drawings

Fig. 1 is a graph of body weight change.

Fig. 2 is a graph of the right ear added thickness variation.

* Representing the statistical difference between G1 and G2-5: * <0.0001, <0.01.

Fig. 3 is a graph of body weight change.

Fig. 4 is a graph of foot pad thickness variation.

* Representing the statistical difference between G1 and G2-5: * <0.001, <0.01, <0.05.

Detailed Description

Example 1 preparation of compound 1:

boron trifluoride diethyl etherate (1 mL) was slowly added dropwise to a solution of dihydroartemisinin (569 mg,2.0 mmol) and cholic acid (817 mg,2.0 mmol) in diethyl ether (50 mL) at-78deg.C, and the reaction was allowed to warm to room temperature and stirred overnight. After the reaction is monitored by thin-layer silica gel chromatography, the reaction is slowly quenched by saturated sodium bicarbonate aqueous solution (30 mL), extracted by ethyl acetate (30 mL multiplied by 3), the organic phases are combined and then washed 1 time by water (50 mL), and then washed 1 time by saturated saline (50 mL), dried by anhydrous magnesium sulfate, filtered, and the filtrate is dried under reduced pressure to obtain a crude product. The crude product was purified by column on silica gel (petroleum ether: ethyl acetate=1:1) to give 726mg of compound as a white solid, yield: 53.8%.

¹ H NMR(400MHz,CDCl ₃ )：δ5.47(s,1H),4.93(d,J＝3.3Hz,1H),4.00(s,1H),3.88(s,1H),3.51(t,J＝11.0Hz,1H),2.64–2.56(m,1H),2.47–2.21(m,4H),2.10–2.02(m,2H),1.99–1.85(m,5H),1.72(m,8H),1.53(m,5H),1.45(m,6H),1.40–1.30(m,5H),1.31–1.23(m,4H),1.16(m,2H),0.99(m,7H),0.89(m,8H),0.71(s,3H)。

¹³ C NMR(100MHz,CDCl ₃ )：δ104.03(s),100.14(s),88.08(s),81.29(s),77.24(s),68.67(s),52.65(s),46.86(s),46.51(s),44.56(s),42.21(s),41.33(s),39.32(s),37.47(s),36.51(s),35.58(s),35.29(s),35.22(s),34.80(d),30.94–30.35(m),30.25(d),29.72(s),28.71(d),27.58(s),26.70(s),26.30(s),24.70(s),23.22(s),22.60(s),20.41(s),17.24(s),13.08(s),12.46(s)。

Example 2 preparation of Compound 2

A white solid was prepared in a similar manner as in example 1. Yield: 54.8%.

¹ H NMR(400MHz,CDCl ₃ )：δ5.47(s,1H),4.91(d,J＝3.4Hz,1H),4.02(s,1H),3.67–3.55(m,1H),2.65–2.56(m,1H),2.48–2.25(m,3H),2.07–2.01(m,1H),1.92–1.81(m,5H),1.77–1.70(m,3H),1.68–1.58(m,4H),1.54(m,2H),1.50–1.34(m,12H),1.27(m,8H),1.18–1.06(m,2H),1.01(d,J＝6.2Hz,3H),0.96(d,J＝6.3Hz,3H),0.93–0.85(m,7H),0.71(s,3H)。

¹³ C NMR(100MHz,CDCl ₃ )：δ179.27(s),104.03(s),100.49(s),88.10(s),81.30(s),77.24(s),73.32(s),52.62(s),48.36(s),47.33(s),46.46(s),44.55(s),42.00(s),37.43(s),36.51(s),36.06(s),35.26(s),35.04(s),34.66(s),34.30(s),33.79(s),32.64(s),31.05–30.54(m),29.86–29.32(m),28.98(s),27.35(d),26.29(s),26.06(s),24.64(d),23.62(s),23.34(s),20.38(s),17.33(s),13.20(s),12.77(s)。

EXAMPLE 3 preparation of Compound 3

A white solid was prepared in analogy to example 1, yield: 54.8%.

¹ H NMR(400MHz,CDCl ₃ )；δ5.46(s,1H),4.93(d,J＝3.1Hz,1H),3.88(s,1H),3.48(t,J＝10.8Hz,1H),2.65–2.55(m,1H),2.46–2.33(m,2H),2.27(m,1H),1.87(m,16H),1.52–1.13(m,21H),0.95(m,14H),0.66(s,3H)。

¹³ C NMR(100MHz,CDCl ₃ )；δ179.74(s),104.02(s),100.40(s),88.09(s),81.30(s),77.49(s),77.25(s),68.70(s),55.79(s),52.63(s),50.53(s),44.59(s),42.73(s),41.33(s),39.56(s),39.33(s),37.47(s),36.51(s),35.86(s),35.30(d),34.80(d),32.88(s),30.86(d),29.12(s),28.18(s),26.29(s),24.63(d),23.70(s),22.90(s),20.64(s),20.41(s),18.27(s),13.20(s),11.78(s)。

EXAMPLE 4 preparation of Compound 4

A white solid was prepared in analogy to example 1, yield: 56.8%.

¹ H NMR(400MHz,CDCl ₃ )：δ5.46(s,1H),4.92(d,J＝3.3Hz,1H),3.61(m,2H),2.67–2.55(m,1H),2.47–2.34(m,2H),2.28(ddd,J＝15.8,9.6,6.5Hz,1H),2.04(m,2H),1.94–1.71(m,9H),1.71–1.55(m,4H),1.54–1.41(m,11H),1.40–1.21(m,9H),1.19–1.01(m,3H),0.98–0.89(m,12H),0.70(s,3H)。

¹³ C NMR(100MHz,CDCl ₃ )：δ178.71(s),104.07(s),100.21(s),88.08(s),81.22(s),77.24(s),71.41(s),55.61(s),54.92(s),52.61(s),44.51(s),43.79(s),42.37(s),40.07(s),39.19(s),37.49(s),36.99(s),36.48(s),35.21(s),35.01(s),34.71(s),34.25(s),33.60(s),30.78(d),28.74(d),26.90(s),26.27(s),24.63(d),23.53(s),21.24(s),20.41(s),18.42(s),13.16(s),12.15(s)。

EXAMPLE 5 preparation of Compound 5

A white solid was prepared in analogy to example 1, yield: 65.5%.

¹ H NMR(400MHz,CDCl ₃ )：δ5.47(s,1H),4.92(d,J＝3.3Hz,1H),3.73–3.53(m,1H),2.66–2.56(m,1H),2.39(m,2H),2.27(ddd,J＝15.8,9.7,6.4Hz,1H),2.05(dt,J＝14.5,3.7Hz,1H),1.99–1.94(m,1H),1.93–1.70(m,8H),1.68–1.55(m,3H),1.51–1.21(m,21H),1.08(m,4H),1.00–0.89(m,13H),0.66(s,3H)。

¹³ C NMR(100MHz,CDCl ₃ )δ180.33(s),104.04(s),100.16(s),88.07(s),81.26(s),76.26(s),56.42(s),55.98(s),52.63(s),44.57(s),42.77(s),41.97(s),40.42(s),40.12(s),37.48(s),36.50(s),35.85(s),35.32(s),35.42(s),34.74(s),32.59(s),31.00(s),30.85(s),30.78(s),29.72(s),29.01(s),28.20(s),27.33(s),26.40(s),26.27(s),24.63(d),24.21(s),23.51(s),20.89(s),20.42(s),18.30(s),13.18(s),12.07(s)。

EXAMPLE 6 preparation of Compound 6

A white solid was prepared in analogy to example 1, yield: 49.5%.

¹ H NMR(400MHz,CDCl ₃ )；δ5.41(s,1H),4.96(d,J＝3.5Hz,1H),4.14–4.02(m,1H),3.64(dt,J＝15.5,5.5Hz,1H),2.68–2.58(m,1H),2.35(m,3H),2.12–1.95(m,2H),1.95–1.56(m,12H),1.56–1.21(m,16H),1.12(m,6H),1.02–0.89(m,10H),0.86(d,J＝7.3Hz,3H),0.65(s,3H)。

¹³ C NMR(100MHz,CDCl ₃ )；δ179.13(s),104.09(s),98.78(s),88.20(s),81.19(s),77.24(s),72.49(s),71.73(s),56.21(s),55.93(s),52.64(s),47.88(s),44.45(s),42.88(s),39.99(d),37.55(s),36.51(s),36.00(s),35.39(d),34.60(d),31.64(s),30.24(s),30.36(s),30.44(s),30.75(s),30.77(s),30.87(s),29.67(s),28.11(s),26.26(s),24.64(d),24.22(s),23.77(s),20.75(s),20.41(s),18.25(s),13.16(s),12.02(s)。

Example 7 preparation of compound 7:

preparation of intermediate 7-1:

under nitrogen protection, 8.0 g deoxycholic acid (20.38 mmol) and 100 ml absolute methanol were added to the reaction flask, followed by dropwise addition of 2 ml concentrated sulfuric acid. The reaction was refluxed for 2 hours until the reaction was complete by thin layer silica gel chromatography. The reaction mixture was cooled to room temperature, concentrated under reduced pressure, and washed with 5% aqueous sodium hydroxide and saturated brine after adding 200 ml of methylene chloride. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure and dried in vacuo to give 8.20 g of a white solid in 99% yield.

MS:[M+H] ⁺ ＝407.3

¹ H NMR(400MHz,CDCl ₃ )δ:3.99(s,1H),3.67(s,3H),3.58-3.67(m,1H),2.33-2.43(m,1H),2.20-2.30(m,1H),1.50-1.92(m,13H),1.20-1.50(m,10H),1.03-1.18(m,1H),0.98(d,J＝6.0Hz,3H),0.92(s,3H),0.69(s,3H).

Preparation of intermediate 7-2:

282 mg of deoxycholate methyl 7-1 (0.69 mmol) and 266 mg of dihydroartemisinin (0.94 mmol) were dissolved in 20 ml of anhydrous tetrahydrofuran in a three-necked round bottom flask under nitrogen. After the solution was cooled to-78 ℃, 392 mg of boron trifluoride diethyl etherate (2.76 mmol) was added dropwise. The reaction mixture was slowly warmed to room temperature and stirred for 16 hours. After the reaction was completed, the reaction mixture was quenched with 20 ml of saturated aqueous sodium bicarbonate and extracted three times with 20 ml of ethyl acetate. The organic phases were combined, washed with 50 ml of water and 50 ml of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate=10/1 to 3/1) to give 150 mg of a white solid in 32.1% yield.

¹ H NMR(400MHz,CD ₃ OD)δ:5.45(s,1H),3.97(s,1H),3.65(s,3H),3.50-3.60(m,1H),2.47-2.57(m,1H),2.20-2.43(m,3H),2.00-2.09(m,1H),1.70-1.98(m,11H),1.04-1.70(m,26H),0.85-1.02(m,12H),0.70(s,3H).

Preparation of compound 7:

30 mg of lithium borohydride (1.115 mmol) were suspended in 20 ml of anhydrous tetrahydrofuran in a three-necked round bottom flask under nitrogen. At 0deg.C, 150 mg of intermediate 7-2 (0.223 mmol) in 5 ml of anhydrous tetrahydrofuran was added dropwise. After the completion of the dropwise addition, the reaction solution was stirred at room temperature for 24 hours. After the reaction was completed, the reaction mixture was quenched with 30 ml of cold water and extracted three times with 30 ml of ethyl acetate. The organic phases were combined, washed with brine and dried over anhydrous sodium sulfate, evaporated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=10/1 to 2/1) to give 100 mg of a white solid in 69.5% yield.

mp：128.0-130.7℃

[α] ²² _D ：+94.8(C＝0.1,CH ₃ OH)

MS:[M-H+HCOOH] ^- ＝689.3

¹ H NMR(400MHz,CDCl ₃ )δ:5.46(s,1H),4.90(d,J＝3.2Hz,1H),4.02(s,1H),3.56-3.69(m,3H),2.55-2.65(m,1H),2.32-2.42(m,1H),2.00-2.08(m,1H),1.60-1.93(m,13H),1.30-1.60(m,19H),1.05-1.30(m,6H),1.02(d,J＝6.4Hz,3H),0.96(d,J＝6.4Hz,3H),0.85-0.95(m,6H),0.61(s,3H).

IR(KBr),cm ^-1 :2936,2922,2863,1740,1726,1447,1374,1099,1009,981.

EXAMPLE 8 preparation of Compound 8

Preparation of intermediate 8-1:

1.0 g glycine (13.32 mmol) was dissolved in a mixed solvent of 20 ml 1, 4-dioxane and 5 ml water at room temperature, followed by addition of 5.2 g 9-fluorenylmethyl chloroformate (20.1 mmol) and 3.3 g potassium carbonate (23.88 mmol). The reaction solution was stirred at room temperature for 12 hours. 1 mol/L of diluted hydrochloric acid was added dropwise to the reaction solution until the pH was about 1, followed by extraction with ethyl acetate three times. The organic phases were combined, washed successively with water and saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure and dried in vacuo to give 4.0 g of a colorless oil. The crude product was used directly in the next reaction without purification.

MS:[M+H] ⁺ ＝298.10

Preparation of intermediate 8-2:

to the reaction flask was added, under nitrogen blanket and at room temperature, 100 mg of compound 7 (0.155 mmol), 55 mg of intermediate 8-1 (0.185 mmol), 29 mg of N, N' -diisopropylcarbodiimide (0.229 mmol), 25 mg of 1-hydroxybenzotriazole (0.185 mmol), 9 mg of 4-dimethylaminopyridine (0.074 mmol) and 5 ml of anhydrous N, N-dimethylformamide. The reaction solution was stirred at room temperature for 12 hours. After the completion of the reaction, the reaction mixture was diluted with 50 ml of ethyl acetate, washed with water and then with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluent: ethyl acetate/petroleum ether=1/4) to give 110 mg of a white solid in 76.8% yield.

¹ H NMR(400MHz,CDCl ₃ )δ:7.78(d,J＝7.6Hz,2H),7.62(d,J＝7.6Hz,2H),7.42(t,J＝7.6Hz,2H),7.32(d,J＝7.6Hz,2H),5.46(s,1H),5.31(m,1H),4.91(d,J＝3.2Hz,1H),4.42(d,J＝7.2Hz,2H),4.25(d,J＝7.2Hz,1H),4.10-4.20(m,2H),4.00(m,2H),3.56-3.66(m,1H),2.55-2.65(m,1H),2.32-2.42(m,1H),2.00-2.08(m,1H),1.00-1.95(m,38H),0.99(d,J＝6.4Hz,3H),0.96(d,J＝6.4Hz,3H),0.84-0.93(m,6H),0.68(s,3H).

Preparation of Compound 8:

50 mg of intermediate 8-2 (0.054 mmol) was dissolved in 5 ml of acetonitrile, and 14 mg of piperidine (0.16 mmol) was added at room temperature and stirred for 12 hours. When thin layer silica gel chromatography showed the reaction was completed, the reaction solution was evaporated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: isopropanol/dichloromethane=1/5) to give 25 mg of a white solid in 65.9% yield.

mp：102.2-104.5℃

[α] ¹⁸ _D ：+97.8(C＝0.1,CH ₃ OH)

MS:[M+H] ⁺ ＝702.3

¹ H NMR(400MHz,CDCl ₃ )δ:5.46(s,1H),4.90(d,J＝3.2Hz,1H),4.05-4.16(m,2H),4.02(s,1H),3.56-3.66(m,1H),3.46(s,2H),2.55-2.65(m,1H),2.32-2.42(m,1H),2.00-2.08(m,1H),1.30-1.94(m,32H),1.02-1.30(m,6H),0.99(d,J＝6.4Hz,3H),0.96(d,J＝6.4Hz,3H),0.85-0.94(m,6H),0.69(s,3H).

IR(KBr),cm ^-1 :2936,2926,2863,1739,1447,1375,1194,1100,1010,965.

EXAMPLE 9 preparation of Compound 9

To the reaction flask was added, under nitrogen, 500 mg of compound 7 (0.775 mmol), 103 mg of nicotinic acid (0.837 mmol), 133 mg of N, N' -diisopropylcarbodiimide (1.054 mmol), 112 mg of 1-hydroxybenzotriazole (0.829 mmol), 42 mg of 4-dimethylaminopyridine (0.344 mmol) and 10 ml of anhydrous N, N-dimethylformamide in this order. The reaction solution was stirred at room temperature for 12 hours. After the completion of the reaction, the reaction mixture was diluted with 50 ml of ethyl acetate, washed with water and brine in this order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Purification of the residue by column chromatography on silica gel (eluent: ethyl acetate/petroleum ether=1/3) afforded 450 mg of a white solid in 77.4% yield.

mp：98.0-101.7℃

[α] ²⁰ _D ：+81.2(C＝0.1,CH ₃ OH)

MS:[M+H] ⁺ ＝750.5

¹ H NMR(400MHz,CDCl ₃ )δ:9.24(s,1H),8.79(d,J＝3.2Hz,1H),8.30(m,1H),7.41(dd,J＝3.2,7.6Hz,1H),5.46(s,1H),4.90(d,J＝3.6Hz,1H),4.30-4.40(m,2H),4.03(d,J＝2.8Hz,1H),3.58-3.68(m,1H),2.56-2.65(m,1H),2.32-2.42(m,1H),2.00-2.08(m,1H),1.20-1.95(m,36H),1.06-1.30(m,6H),1.04(d,J＝6.4Hz,3H),0.96(d,J＝6.4Hz,3H),0.83-0.95(m,6H),0.70(s,3H).

IR(KBr),cm ^-1 :2935,2923,2863,1725,1591,1447,1376,1281,1100,1021,1010,982.

Example 10 preparation of Compound 10

Preparation of intermediate 10-1:

to the reaction flask, 400 mg of compound 2 (0.607 mmol), 258 mg of benzyl glycinate p-toluenesulfonate (0.765 mmol), 277 mg of O- (7-azabenzotriazol-1-yl) -N, N, N' -tetramethylurea hexafluorophosphate (0.730 mmol) and 10 ml of anhydrous N, N-dimethylformamide were added sequentially at room temperature under nitrogen. 226 mg of N-ethyldiisopropylamine (1.75 mmol) are then added dropwise. The reaction solution was stirred at room temperature for 16 hours. After the completion of the reaction, 50 ml of water was added to the reaction mixture, followed by extraction with ethyl acetate three times. The organic phases were combined, washed three times with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Purification of the residue by column chromatography on silica gel (eluent: ethyl acetate/petroleum ether=20/1) gives 401 mg of a white solid in 82.0% yield

¹ H NMR(400MHz,CDCl ₃ )δ:7.32-7.42(m,5H),5.94(m,1H),5.46(s,1H),5.20(s,3H),4.90(d,J＝3.2Hz,1H),4.10(dd,J＝2.0,5.2Hz,2H),4.00(m,1H),3.76(t,J＝6.0Hz,1H),3.55-3.65(m,1H),2.56-2.65(m,1H),2.28-2.42(m,2H),2.10-2.21(m,1H),2.00-2.10(m,1H),1.70-1.92(m,13H),1.00-1.70(m,20H),1.00(d,J＝6.0Hz,3H),0.96(d,J＝6.0Hz,3H),0.82-0.93(m,6H),0.69(s,3H).

Preparation of compound 10:

250 mg of intermediate 10-1 (0.31 mmol) was dissolved in a mixed solution of 16 ml of tetrahydrofuran and 3 ml of water, 60 mg of lithium hydroxide (2.50 mmol) was added at 0℃and the reaction temperature was gradually raised to room temperature and stirred for 4 hours. After the reaction was completed by thin-layer silica gel chromatography, 0.1 mol/l hydrochloric acid was added dropwise to the reaction mixture to a pH of about 6. Extraction was performed three times with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Purification of the residue by column chromatography on silica gel (eluent: methanol/dichloromethane=1/100 to 5/100) afforded 147 mg of white solid in 66.2% yield.

mp：142.5-145.2℃

[α] ²² _D ：+93.6(C＝0.1,CH ₃ OH)

MS:[M-H] ^- ＝714.5

¹ H NMR(400MHz,DMSO-d ₆ )δ:12.51(br,1H),8.01(t,J＝5.2Hz,1H),5.33(s,1H),4.79(d,J＝3.2Hz,1H),4.12-4.30(brs,1H),3.79(s,1H),3.67(d,J＝5.2Hz,2H),3.40-3.52(m,1H),2.30-2.41(m,1H),2.10-2.22(m,2H),1.95-2.09(m,2H),1.45-1.85(m,15H),1.05-1.42(m,17H),0.85-1.06(m,16H),0.61(s,3H).

IR(KBr),cm ^-1 :3361,2936,2924,2864,1732,1656,1536,1448,1377,1194,1100,1021,1010,982.

EXAMPLE 11 preparation of Compound 11

Preparation of intermediate 11-1:

to the reaction flask were successively added 690 mg deoxycholic acid (1.76 mmol), 107 mg ammonium chloride (2.00 mmol), 1135 mg 2- (7-azobenzotriazole) -N, N' -tetramethylurea hexafluorophosphate (2.98 mmol), 386 mg diisopropylethylamine (2.98 mmol) and 12 ml N, N-dimethylformamide under nitrogen. The reaction solution was stirred at room temperature for 16 hours. Thin layer silica gel chromatography showed the end of the reaction, the reaction solution was diluted with ethyl acetate, washed three times with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate/petroleum ether=1/5 to 2/1) to give 679 mg of a white solid in 98.5% yield.

¹ H NMR(400MHz,CD ₃ OD)δ:3.97(m,1H),3.48-3.58(m,2H),2.22-2.32(m,1H),2.07-2.17(m,1H),1.73-1.96(m,7H),1.56-1.68(m,3H),1.06-1.56(m,13H),1.03(d,J＝6.4Hz,3H),0.94(s,3H),0.72(s,3H).

Preparation of intermediate 11-2:

a suspension of 188 mg lithium aluminum hydride (4.95 mmol) in 15 ml anhydrous tetrahydrofuran was cooled to 0deg.C under nitrogen and 500 mg of intermediate 11-1 (1.28 mmol) was added in portions. The temperature of the reaction solution was gradually raised to 80℃and refluxed at this temperature for 2 half an hour until thin layer silica gel chromatography showed complete consumption of the starting material. The reaction solution was cooled to 0℃and 50 ml of water was carefully added dropwise thereto, followed by extraction three times with 100 ml of ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purification of the residue by column chromatography on silica gel (eluent: dichloromethane/methanol=50/1 to 10/1) afforded 312 mg of a white solid in 64.6% yield.

¹ H NMR(400MHz,DMSO-d ₆ )δ:6.7-7.8(brs,2H),4.30-4.70(brs,1H),4.15-4.27(m,1H),3.78(s,1H),3.30-3.40(m,1H),3.16(s,1H),2.60-2.73(m,2H),1.70-1.86(m,4H),1.40-1.65(m,6H),1.20-1.40(m,10H),0.95-1.20(m,4H),0.96(d,J＝6.4Hz,3H),0.84(s,3H),0.61(s,3H).

Preparation of intermediate 11-3:

to the reaction flask was successively added 100 mg of intermediate 11-2 (0.265 mmol), 82 mg of 9-fluorenylmethylchloroformate (0.317 mmol), 5 ml of dichloromethane and 51 mg of triethylamine (0.504 mmol) at room temperature under nitrogen. The reaction solution was stirred at room temperature for 16 hours. After the reaction was completed by thin layer silica gel chromatography, the reaction solution was concentrated under reduced pressure, and the residue was purified by C18 silica gel column chromatography (eluent: water/acetonitrile=30% -100%) to obtain 95 mg of a white solid in 59.8% yield.

¹ H NMR(400MHz,CDCl ₃ )δ:7.78(d,J＝7.6Hz,2H),7.61(d,J＝7.6Hz,2H),7.38-7.44(m,2H),7.30-7.37(m,2H),4.70-4.80(m,1H),4.41(d,J＝6.8Hz,2H),4.20-4.30(m,1H),4.00(m,1H),3.58-3.67(m,1H),3.06-3.74(m,2H),1.65-1.91(m,8H),1.50-1.60(m,5H),1.33-1.50(m,9H),1.00-1.25(m,4H),0.99(d,J＝6.8Hz,3H),0.95(s,3H),0.69(s,3H).

Preparation of intermediate 11-4:

683 mg of intermediate 11-3 (1.14 mmol) was dissolved in 60 ml of anhydrous tetrahydrofuran under nitrogen and cooled to-78 ℃.1.00 g of boron trifluoride diethyl etherate (7.04 mmol) was added dropwise to the reaction solution, and the temperature of the reaction solution was then gradually raised to room temperature. The reaction solution was stirred at room temperature for 16 hours. When the thin layer silica gel chromatography showed the reaction was completed, 30 ml of saturated sodium bicarbonate was added dropwise to the reaction solution, and extraction was performed three times with ethyl acetate. The organic phases were combined, washed with water and saturated brine in this order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Purification of the residue by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate=20/1-3/1) afforded 613 mg of a white solid in 62.0% yield.

¹ H NMR(400MHz,CDCl ₃ )δ:7.78(d,J＝7.6Hz,2H),7.61(d,J＝7.6Hz,2H),7.38-7.44(m,2H),7.30-7.37(m,2H),5.46(s,1H),4.91(d,J＝3.6Hz,1H),4.70-4.80(m,1H),4.41(d,J＝6.8Hz,2H),4.20-4.30(m,1H),4.00(m,1H),3.56-3.67(m,1H),3.10-3.25(m,2H),2.55-2.65(m,1H),2.32-2.42(m,1H),2.00-2.05(m,1H),1.79-1.94(m,5H),1.68-1.79(m,4H),1.22-1.68(m,22H),1.02-1.22(m,3H),1.11(d,J＝6.4Hz,3H),0.98(d,J＝6.4Hz,3H),0.85-0.95(m,6H),0.70(s,3H).

Preparation of Compound 11:

605 mg of intermediate 11-4 (0.698 mmol) was dissolved in 3 ml of acetonitrile, 178 mg of piperidine (2.09 mmol) was added at room temperature and stirred at room temperature for 16 hours. Thin layer silica gel chromatography showed complete consumption of the starting material. The reaction solution was evaporated under reduced pressure, and the residue was dissolved in 20 ml of ethyl acetate, washed three times with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Purification of the residue by C18 silica gel column chromatography (eluent: water/acetonitrile=30% -100%) afforded 340 mg of a white solid in 75.6% yield.

mp：151.7-154.1℃

[α] ²⁵ _D ：+95.6(C＝0.1,CH ₃ OH)

MS:[M+H] ⁺ ＝644.4

¹ H NMR(400MHz,CDCl ₃ )δ:5.45(s,1H),4.91(d,J＝3.2Hz,1H),4.01(s,1H),3.55-3.67(m,1H),2.74-2.90(m,2H),2.55-2.64(m,1H),2.00-2.09(m,1H),1.68-1.94(m,10H),1.48-1.68(m,9H),1.30-1.48(m,11H),1.03-1.30(m,7H),1.12(d,J＝6.4Hz,3H),0.99(d,J＝6.4Hz,3H),0.82-0.93(m,6H),0.69(s,3H).

IR(KBr),cm ^-1 :2922,2863,1583,1447,1375,1343,1100,1010,982.

EXAMPLE 12 preparation of Compound 12

Preparation of intermediate 12-1:

to the reaction flask were successively added 500 mg deoxycholic acid (1.27 mmol), 734 mg 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (1.93 mmol), 157 mg dimethylamine hydrochloride (1.93 mmol), 20 ml anhydrous N, N-dimethylformamide and 499 mg diisopropylethylamine (3.86 mmol) under nitrogen atmosphere. The reaction was stirred at room temperature overnight. When thin layer silica gel chromatography showed complete consumption of starting material, the reaction was diluted with 150 ml of ethyl acetate and washed 4 times with 150 ml of water. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give 451 mg of a white crude product, which was directly used in the next reaction in 84.6% yield.

MS:[M+H] ⁺ ＝420.3

¹ H NMR(400MHz,DMSO-d ₆ )δ:4.45(d,J＝4.0Hz,1H),4.18(d,J＝4.0Hz,1H),3.78(m,1H),3.3-4.3(m,1H),2.94(s,3H),2.78(s,3H),2.23-2.43(m,1H),2.10-2.20(m,1H),1.40-1.85(m,10H),1.10-1.40(m,12H),0.8-1.08(m,8H),0.59(s,3H).

Preparation of intermediate 12-2:

a30 ml anhydrous tetrahydrofuran suspension of 156 mg lithium aluminum hydride (4.11 mmol) was cooled to 0deg.C under nitrogen, and 445 mg of intermediate 12-1 (1.06 mmol) was added in portions. After the addition, the reaction solution was gradually warmed to 80℃and reacted at that temperature for 2.5 hours. After the reaction was completed by thin layer silica gel chromatography, the reaction solution was cooled to 0 ℃, 50 ml of cold water was added dropwise to the reaction solution, and then extracted three times with 50 ml of ethyl acetate. The organic phases were combined, washed with brine and dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography on silica gel (eluent: dichloromethane/methanol=50/1 to 10/1) to give 392 mg of a white solid in 91.2% yield.

MS:[M+H] ⁺ ＝406.3

¹ H NMR(400MHz,CDCl ₃ )δ:3.99(m,1H),3.57-3.47(m,1H),2.38-2.47(m,2H),2.37(s,6H),1.03-1.90(m,26H),0.99(d,J＝6.4Hz,3H),0.92(s,3H),0.69(s,3H).

Preparation of Compound 12:

380 mg of intermediate 12-2 (0.937 mmol), 865 mg of dihydroartemisinin (3.04 mmol) and 60 ml of anhydrous tetrahydrofuran are added in sequence to the reaction flask under nitrogen. After the reaction mixture was cooled to-78 ℃, 1.27 g of boron trifluoride etherate (8.95 mmol) was added dropwise. After the addition, the reaction solution temperature was gradually raised to room temperature and stirred for 16 hours. After the reaction was completed by thin layer silica gel chromatography, the reaction mixture was cooled to 5℃with an ice-water bath, and 50 ml of saturated aqueous sodium hydrogencarbonate solution was added. The reaction solution was extracted 3 times with 50 ml of ethyl acetate. The organic phases were combined, washed successively with water, saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane/methanol=100/1 to 40/1) to give 182 mg of an off-white solid in a yield of 28.9%.

mp：122.2-123.8℃

[α] ¹⁷ _D ：+83.8(C＝0.1,CH ₃ OH)

MS:[M+H] ⁺ ＝672.4

¹ H NMR(400MHz,CDCl ₃ )δ:5.45(s,1H),4.90(d,J＝3.2Hz,1H),4.01(m,1H),3.55-3.66(m,1H),2.54-2.64(m,1H),2.30-2.45(m,6H),2.00-2.09(m,1H),1.57-1.92(m,13H),1.33-1.57(m,16H),1.02-1.32(m,12H),1.01(d,J＝6.4Hz,3H),0.95(d,J＝6.4Hz,3H),0.82-0.94(m,6H),0.69(s,3H).

IR(KBr),cm ^-1 :2922,2861,2777,1462,1448,1375,1100,1020,1010,982.

EXAMPLE 13 preparation of Compound 13

Preparation of intermediate 13-1:

6.0 g of methyl cholate (14.20 mmol) and 5.48 g of dihydroartemisinin (19.27 mmol) were dissolved in 300 ml of anhydrous tetrahydrofuran in a three-necked round bottom flask under nitrogen. After the solution was cooled to-78 ℃, 8.05 g of boron trifluoride etherate (56.72 mmol) was added dropwise. The reaction mixture was slowly warmed to room temperature and stirred for 16 hours. After the reaction was completed, the reaction mixture was quenched with 300 ml of saturated aqueous sodium bicarbonate and extracted three times with 200 ml of ethyl acetate. The organic phases were combined, washed with 300 ml of water and 300 ml of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate=10/1 to 3/1) to give 4.48 g of a white solid in 45.79% yield.

¹ H NMR(400MHz,CDCl ₃ )δ:5.45(s,1H),4.92(d,J＝3.2Hz,1H),3.99(s,1H),3.86(s,1H),3.75(t,J＝6.4Hz,3H),3.68(s,3H),3.42-3.53(m,1H),2.54-2.68(m,1H),2.19-2.45(m,4H),1.0-2.18(m,31H),0.95(m,6H),0.90(m,6H),0.70(s,3H).

Preparation of Compound 13:

708 mg of lithium borohydride (31.51 mmol) were suspended in 120 ml of anhydrous tetrahydrofuran in a three-necked round bottom flask under nitrogen. At 0deg.C, 4.48 g of intermediate 13-1 (6.50 mmol) in 30 ml of anhydrous tetrahydrofuran was added dropwise. After the completion of the dropwise addition, the reaction solution was stirred at room temperature for 24 hours. After the reaction was completed, the reaction mixture was quenched with 120 ml of cold water and extracted three times with 100 ml of ethyl acetate. The organic phases were combined, washed with brine and dried over anhydrous sodium sulfate, evaporated to dryness under reduced pressure, and the residue was purified by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate=10/1 to 2/1) to give 3.11 g of a white solid in a yield of 72.3%.

mp：138.0-140.7℃

[α] ²² _D ：+91.2(C＝0.1,CH ₃ OH)

MS:[M-H+HCOOH] ^- ＝705.2

¹ H NMR(400MHz,CDCl ₃ )δ:5.45(s,1H),4.92(d,J＝3.2Hz,1H),4.01(s,1H),3.86(s,1H),3.63(t,J＝6.0Hz,2H),3.42-3.53(m,1H),2.54-2.64(m,1H),2.32-2.43(m,1H),2.18-2.28(m,1H),1.05-2.15(m,36H),1.02(d,J＝6.4Hz,3H),0.96(d,J＝6.4Hz,3H),0.89(m,6H),0.71(s,3H).

¹³ C NMR(400MHz,CDCl ₃ )δ:103.50,99.94,87.60,80.81,72.63,67.94,63.05,52.16,46.97,46.05,44.10,41.81,40.89,39.05,36.94,36.03,35.27,34.87,34.76,34.34,34.26,34.20,,31.29,30.42,28.88,28.60,27.97,27.06,26.43,25.80,24.20,24.13,22.65,22.23,19.88,17.23,12,66,12.08.

IR(KBr),cm ^-1 :3419,2935,2922,2868,1447,1375,1099,1010,985.

EXAMPLE 14 preparation of Compound 14

Preparation of intermediate 14-1:

to the reaction flask was added 500 mg of compound 13 (0.756 mmol), 120 mg of 1-hydroxybenzotriazole (0.888 mmol), 45 mg of 4-dimethylaminopyridine (0.368 mmol), 143 mg of N, N' -diisopropylcarbodiimide (1.133 mmol), 269 mg of Fmoc-glycine (0.905 mmol) and 10 ml of anhydrous N, N-dimethylformamide in this order under nitrogen protection. The reaction solution was stirred at room temperature for 12 hours. After the completion of the reaction, the reaction mixture was diluted with 100 ml of ethyl acetate and washed with a saturated aqueous sodium hydrogencarbonate solution and then with a saturated brine. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate/petroleum ether=1/2) to give 550 mg of a white solid in 77.4% yield.

¹ H NMR(400MHz,CDCl ₃ )δ:7.78(d,J＝7.6Hz,2H),7.61(d,J＝7.6Hz,2H),7.42(t,J＝7.6Hz,2H),7.33(t,J＝7.6Hz,2H),5.45(s,1H),5.31(m,1H),4,.91(d,J＝3.6Hz,1H),4.42(d,J＝7.2Hz,2H),4.26(t,J＝7.2Hz,1H),3.96-4.04(m,2H),3.78-3.90(m,3H),3.42-3.52(m,1H),2.55-2.64(m,1H),2.30-2.41(m,1H),2.13-2.24(m,1H),1.10-2.10(m,19H),1.00(d,J＝6.4Hz,3H),0.95(d,J＝6.4Hz,3H),0.85-0.95(m,6H),0.70(s,3H).

Preparation of compound 14:

500 mg of intermediate 14-1 (0.532 mmol) was dissolved in 45 ml of acetonitrile and 137 mg of piperidine (1.609 mmol) was added. The reaction was stirred at room temperature for 12 hours until thin layer silica gel chromatography showed the reaction to be complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: methanol/dichloromethane=1/50) to give 200 mg of a white solid in 52.4% yield.

mp：136.2-139.7℃

[α] ²² _D ：+77.4(C＝0.1,CH ₃ OH)

MS:[M+H] ⁺ ＝718.5

¹ H NMR(400MHz,DMSO-d ₆ )δ:5.334(s,1H),4.78(d,J＝3.6Hz,1H),4,15(d,J＝3.6Hz,1H),4.06(d,J＝3.2Hz,1H),3.94-4.04(m,2H),3.79(m,1H),3.61(m,1H),3.26(s,2H),2.30-2.40(s,1H),2.04-2.23(m,4H),1.90-2.03(m,2H),1.75-1.67(m,4H),1.50-1.76(m,8H),1.20-1.50(m,12H),0.95-1.20(m,4H),0.94(d,J＝6.4Hz,3H),0.89(d,J＝6.4Hz,3H),0.78-0.85(m,6H),0.59(s,3H).

IR(KBr),cm ^-1 :3402,2921,2866,1738,1447,1375,1194,1099,1010,986.

EXAMPLE 15 preparation of Compound 15

To the reaction flask was added, under nitrogen blanket and at room temperature, 500 mg of compound 13 (0.756 mmol), 100 mg of nicotinic acid (0.812 mmol), 41 mg of 4-dimethylaminopyridine (0.336 mmol), 130 mg of N, N' -diisopropylcarbodiimide (1.030 mmol), 137 mg of 1-hydroxybenzotriazole (1.014 mmol) and 10 ml of anhydrous N, N-dimethylformamide. The reaction solution was stirred at room temperature for 12 hours. When the thin layer silica gel chromatography showed the reaction was completed, the reaction solution was diluted with 150 ml of ethyl acetate, washed with 150 ml of water and 150 ml of saturated brine in this order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Purification of the residue by column chromatography on silica gel (eluent: ethyl acetate/petroleum ether=1/2) gave 400 mg of a white solid in 69.0% yield.

mp：124.9-126.8℃

[α] ²² _D ：+79.5(C＝0.1,CH ₃ OH)

MS:[M+H] ⁺ ＝766.5

¹ H NMR(400MHz,CDCl ₃ )δ:9.24(s,1H),8.78(m,1H),8.30(d,J＝8.0Hz,1H),7.40(m,1H),5.44(s,1H),4.90(d,J＝3.6Hz,1H),4.33(m,2H),4.00(s,1H),3.85(s,1H),3.40-3.50(m,1H),2.52-2.63(m,1H),2.32-2.42(m,1H),2.14-2.29(m,1H),1.07-2.13(m,36H),1.03(d,J＝6.8Hz,3H),0.95(d,J＝6.4Hz,3H),0.87(m,6H),0.70(s,3H).

IR(KBr),cm ^-1 :3419,2937,2925,2870,1724,1591,1375,1282,1099,1021,1011,986.

EXAMPLE 16 preparation of Compound 16

Preparation of intermediate 16-1:

to the reaction flask were successively added 400 mg of compound 1 (0.98 mmol), 151 mg of methyl glycine hydrochloride (1.2 mmol), 452 mg of diisopropylethylamine (3.5 mmol), 456 mg of 2- (7-azobenzotriazole) -N, N' -tetramethylurea hexafluorophosphate (1.2 mmol) and 10 ml of anhydrous N, N-diformyl formamide under nitrogen. The reaction was stirred at room temperature until thin layer silica gel chromatography showed the reaction was complete. The reaction solution was diluted with 100 ml of ethyl acetate, and then washed with 100 ml of water and 100 ml of saturated brine in this order. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purification of the residue by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate=10/1 to 1/1) afforded 380 mg of a white solid in 52.0% yield.

¹ H NMR(400MHz,CDCl ₃ )δ:6.18(t,J＝5.2Hz,1H),5.45(s,1H),4.92(d,J＝3.2Hz,1H),4.06(d,J＝5.2Hz,2H),3.98(m,1H),3,85(m,1H),3.77(s,3H),3.43-3.54(m,1H),2.55-2.64(m,1H),2.17-2.50(m,5H),1.10-2.17(m,H),0.93-1.00(m,6H),0.82-0.93(m,6H),0.70(s,3H).

Preparation of Compound 16:

120 mg of intermediate 16-1 (0.16 mmol) was dissolved in 5 ml of tetrahydrofuran and a solution of 20 mg of lithium hydroxide (0.84 mmol) in 1 ml of water was added. The reaction was stirred at room temperature for 4 hours until thin layer silica gel chromatography showed the reaction to be complete. The pH of the reaction solution was adjusted to about 6 with 1 mol/L sodium bisulfate aqueous solution. Three times with 80 ml ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purification of the residue by column chromatography on silica gel (eluent: dichloromethane/methanol=60/1 to 15/1) afforded 94 mg of a white solid in 80.3% yield.

mp：150.4-152.7℃

[α] ²² _D ：+75.2(C＝0.1,CH ₃ OH)

MS:[M-H] ^- ＝730.5

¹ H NMR(400MHz,DMSO-d ₆ )δ:7.81(brs,1H),5.32(s,1H),4.77(d,J＝2.8Hz,1H),4.10-4.20(br,1H),4.07(s,1H),3.78(s,1H),3.55-3.65(m,3H),2.30-2.40(m,1H),2.08-2.25(m,4H),1.90-2.08(m,3H),1.50-1.87(m,11H),1.10-1.50(m,16H),0.86-0.96(m,6H),0.80-0.86(m,6H),0.58(s,3H).

IR(KBr),cm ^-1 :3399,2938,2922,2870,1634,1447,1376,1099,1020,1010,986.

EXAMPLE 17 preparation of Compound 17

Preparation of intermediate 17-1:

to the reaction flask were successively added 4.0 g of cholic acid (9.79 mmol), 1.0 g of ammonium chloride (18.70 mmol), 3.8 g of O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethylurea (10 mmol), 40 ml of anhydrous N, N-dimethylformamide and 2.5 g of diisopropylethylamine (19.34 mmol) under nitrogen. The reaction solution was stirred at room temperature for 16 hours. After the reaction was completed, the reaction mixture was diluted with 200 ml of ethyl acetate and washed with 100 ml of water 4 times. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure and dried in vacuo to give 3.8 g of crude white solid which was used directly in the next reaction without purification in 95.2% yield.

MS:[M+H] ⁺ ＝408.2

¹ H NMR(400MHz,DMSO-d ₆ )δ:7.20(s,1H),6.62(s,1H),3.90-4.20(m,3H),3.78(m,1H),3.61(m,1H),3.35(m,1H),3.13-3.23(m,1H),1.10-2.27(m,23H),0.92(d,J＝6.4Hz,3H),0.83(s,3H),0.59(s,3H).

Preparation of intermediate 17-2:

a60 ml anhydrous tetrahydrofuran suspension of 1.38 g lithium aluminum hydride (36.36 mmol) was cooled to 0deg.C under nitrogen and 3.8 g intermediate 17-1 (9.32 mmol) was added in portions. The temperature of the reaction solution was gradually raised to 80℃and reacted at that temperature for 2.5 hours. After the reaction was completed, the reaction mixture was cooled to 0℃and 20 ml of water was slowly added dropwise, followed by extraction with 100 ml of ethyl acetate three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure and dried in vacuo to give 3.67 g of a white solid which was used in the next reaction without purification in 100% yield.

MS:[M+H] ⁺ ＝394.5

Preparation of intermediate 17-3:

2.0 g of intermediate 17-2 (5.08 mmol) was dissolved in 90 ml of dichloromethane under nitrogen at room temperature, followed by the addition of 1.33 g of 9-fluorenylmethylchloroformate (5.14 mmol) and 823 mg of triethylamine (8.13 mmol). The reaction solution was stirred at room temperature for 16 hours. After the completion of the reaction, the reaction mixture was concentrated under reduced pressure by thin layer silica gel chromatography, and the residue was purified by reverse phase C-18 silica gel column chromatography (30% acetonitrile aqueous solution as eluent) to obtain 1.12 g of an off-white solid in 35.9% yield.

MS:[M+H] ⁺ ＝616.2

Preparation of intermediate 17-4:

890 mg of intermediate 17-3 (1.44 mmol) and 570 mg of dihydroartemisinin (2.0 mmol) are dissolved in 50 ml of anhydrous tetrahydrofuran under nitrogen. After the reaction solution was cooled to-78 ℃, 838 mg of boron trifluoride diethyl etherate (5.90 mmol) was added dropwise to the reaction solution. The reaction solution temperature was gradually raised to room temperature and stirred at room temperature for 16 hours. After the reaction was completed by thin-layer silica gel chromatography, the reaction solution was cooled to 0 ℃, 30 ml of saturated aqueous sodium bicarbonate solution was added dropwise to the reaction solution, and extraction was performed three times with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=20/1 to 3/1) to give 430 mg of a white solid in 33.8% yield.

MS:[M+NH ₄ ] ⁺ ＝899.4

Preparation of compound 17:

to a solution of 430 mg of intermediate 17-4 (0.487 mmol) in 20 ml of acetonitrile at room temperature was added 120 mg of piperidine (1.41 mmol) and stirred at this temperature for 16 hours. After the reaction was completed, 70 ml of saturated aqueous sodium carbonate solution was added to the reaction mixture, and the mixture was extracted three times with 70 ml of ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Purification of the residue by C18 silica gel column chromatography (eluent: water/acetonitrile=30% -100%) afforded 140 mg of a white solid in 43.6% yield.

mp：164.2-166.9℃

[α] ²⁴ _D ：+87.8(C＝0.1,CH3OH)

MS:[M+H]+＝660.4

¹ H NMR(400MHz,DMSO-d ₆ )δ:5.70-6.40(br,2H),5.32(s,1H),4.78(d,J＝3.2Hz,1H),4.13-4.20(m,1H),4.07(d,J＝3.2Hz,1H),3.75-3.84(m,1H),3.58-3.66(m,1H),2.48-2.60(m,1H),2.30-2.40(m,1H),2.06-2.23(m,3H),1.90-2.03(m,2H),1.20-1.90(m,28H)m,0.95-1.20(m,4H),0.94(d,J＝6.4Hz,3H),0.91(d,J＝6.4Hz,3H),0.77-0.87(m,6H),0.60(s,3H).

IR(KBr),cm ^-1 :3411,2935,2866,1448,1375,1100,1021,1011,986.

EXAMPLE 18 preparation of Compound 18

Preparation of intermediate 18-1:

to the reaction flask were successively added 1.2 g of cholic acid (2.94 mmol), 1.69 g of O- (7-azabenzotriazol-1-yl) -N, N, N, N' -tetramethylurea hexafluorophosphate (4.44 mmol), 752 mg of N-ethyldiisopropylamine (5.82 mmol), 46 ml of anhydrous N, N-dimethylformamide and 2.2 ml of a 2M anhydrous tetrahydrofuran solution (4.40 mmol) under nitrogen. The reaction was stirred at room temperature for 16 hours until thin layer silica gel chromatography showed the reaction to be complete. The reaction solution was concentrated and evaporated to dryness, and the residue was dissolved in 100 ml of ethyl acetate, washed with a saturated ammonium chloride solution, a saturated sodium bicarbonate aqueous solution and a saturated brine in this order, dried over anhydrous sodium sulfate, and concentrated by filtration to give 1.28 g of an off-white solid which was directly used for the next reaction in 100% yield.

¹ H NMR(400MHz,DMSO-d ₆ )δ:4.32(d,J＝4.4Hz,1H),4.10(d,J＝3.6Hz,1H),4.01(d,J＝3.2Hz,1H),3.78(d,J＝2.4Hz,1H),3.60(s,1H),3.10-3.23(m,1H),2.95(s,3H),2.78(s,3H),2.10-2.33(m,4H),1.90-2.03(m,1H),1.55-1.85(m,6H),1.10-1.47(m,10H),0.94(d,J＝6.4Hz,3H),0.80(s,3H),0.59(s,3H).

Preparation of intermediate 18-2:

to the reaction flask was added 405 mg of lithium aluminum hydride (10.67 mmol) and 60 ml of anhydrous tetrahydrofuran under nitrogen. 1.28 g of intermediate 18-1 (2.94 mmol) was added in portions. After no gas was generated in the reaction flask, the suspension was slowly warmed to 80 ℃ and refluxed at that temperature for 2.5 hours. After the reaction is finished by thin-layer silica gel chromatography, cooling the reaction liquid to 0-5 ℃, adding sodium sulfate decahydrate in batches, and stirring vigorously. The mixture was filtered and the filter cake was washed with 150 ml of ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and dried in vacuo to give 1.1 g of an off-white solid which was used directly in the next reaction in 89% yield.

¹ H NMR(400MHz,CDCl ₃ )δ:4.00(s,1H),3.86(s,1H),3.40-3.54(m,2H),1.05-2.35(m,31H),1.00(d,J＝6.0Hz,3H),0.90(s,3H),0.70(s,3H).

Preparation of compound 18:

to the reaction flask was added, in order, 1.1 g of intermediate 18-2 (2.61 mmol), 985 mg of dihydroartemisinin (3.46 mmol) and 50 ml of anhydrous tetrahydrofuran under nitrogen. After the reaction mixture was cooled to-78 ℃, 1.45 g of boron trifluoride diethyl etherate (10.22 mmol) was added dropwise to the reaction mixture. After the completion of the dropping, the reaction solution was slowly warmed to room temperature and stirred for 16 hours. After the reaction was completed, 20 ml of saturated aqueous sodium bicarbonate solution was slowly dropped into the reaction solution, followed by extraction with ethyl acetate three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluent: dichloromethane/methanol=100/1 to 40/1) to give 140 mg of an off-white solid in 7.8% yield.

mp：118.5-121.3℃

[α] ¹⁶ _D ：+75.7(C＝0.1,CH ₃ OH)

MS:[M+H] ⁺ ＝688.4

¹ H NMR(400MHz,CDCl ₃ )δ:5.46(s,1H),4.93(d,J＝3.2Hz,1H),3.96(s,1H),3.85(s,1H),3.40-3.57(m,1H),2.93-3.15(m,2H),2.85-2.95(m,6H),2.30-2.68(m,4H),1.10-2.10(m,35H),0.93-1.09(m,6H),0.80-0.93(m,6H),0.66(s,3H).

IR(KBr),cm ^-1 :3412,2924,2868,1464,1446,1376,1098,1009,985.

Example 19 inhibition of proliferation of T cells in CD3+CD28+ antibody stimulated PBMC by Compounds

Experimental principle: cd4+ and cd8+ are two important surface markers for T lymphocytes, cd4+ T cells are capable of assisting T lymphocytes to become effector T cells and promoting B lymphocytes to macrophages and plasma cells, while cd8+ T cells are a cytotoxic T cell that can secrete a variety of cytokines and have an important role in the cellular immune response of the body, so the number of cd4+ T and cd8+ T cells is critical to maintaining the balance of immune functions of the body. The pathogenesis of autoimmune diseases is mainly due to the massive production of immune cells, including cd4+ T and cd8+ T cells, resulting in immune dysfunction of the body. Thus, the immunosuppressive activity of the drugs can be primarily evaluated by detecting the proliferation inhibitory activity of the drugs on CD4+ T and CD8+ T cells.

Experimental reagents and materials:

ImmunoCult ^TM human CD3/CD28T Cell Activator: supplier Stemcell; goods number 10971

EasySep ^TM Human T Cell Isolation Kit kit: vendor Stemcell; cargo number 17951

PMBC (peripheral blood mononuclear cells): shang Aosai ers biotechnology is supplied; goods number PB004FC

CellTrace Violet, supplier Thermofisher, cat# C34557

Cyclosporin a (CsA): supplier Sigma-Aldrich; goods number 239835

The experimental method comprises the following steps: human total T cell isolation kit (EasySep) ^TM Human T Cell Isolation Kit, stemcell, negative selection) from Peripheral Blood Mononuclear Cells (PBMC), T cell expansion was stimulated using CellTrace-labeled T cells, and compound 1-18 and control sample cyclosporin a (CsA) were incubated with T cells for 120 hours, respectively, and then proliferation of cd4+ T cells and cd8+ T cells was analyzed using a flow cytometer, while CellTrace-labeled T cells + anti-CD 3/CD28 antibodies (medium+cd3/CD 28) and CellTrace-labeled T cells were set as two groups without anti-CD 3/CD28 antibodies as a system control, and proliferation inhibitory activity of compound 1-18 on cd4+ T cells and cd8+ T cells was observed.

The experimental steps are as follows:

1. preparation of test samples

An appropriate amount of compound 1-18 was weighed separately, dissolved in DMSO (0.1 ml) and kept for later use with PBS to a concentration of 40 mM.

2. Isolation of human total T cells from Peripheral Blood Mononuclear Cells (PBMC)

According to human total T cell isolation kit (EasySep) ^TM Human T Cell Isolation Kit, stemcell), human total T cells were isolated from PBMCs, experimental procedures were as follows:

1) And taking out frozen PBMC from the liquid nitrogen tank, and rapidly placing the frozen PBMC in a water bath at 37 ℃ to ensure that the frozen pipe cover is above the water surface so as to prevent pollution. The frozen tube was shaken continuously by hand to allow the frozen PBMCs to thaw as quickly as possible.

2) The surface of the frozen tube was sprayed with 75% alcohol and wiped clean and transferred to a fume hood.

3) Thawed PBMCs were transferred to 50ml centrifuge tubes.

4) 10mL 1640 medium (+10% heat-inactivated bovine serum+1% PBS) was slowly added to 50mL into the centrifuge tube and mixed with shaking.

5) Centrifuge at 400g for 5min.

6) The supernatant was aspirated, and cells were suspended in 10mL 1640 medium (+10% heat-inactivated bovine serum+1% PBS), centrifuged at 400g for 5min, and the supernatant was aspirated.

7) Referring to STEM CELL total human T cell isolation kit, an isolation solution containing 1% heat-inactivated bovine serum and 1mM EDTA was prepared.

8) PBMCs were counted using a cytometer and suspended to 5x 10 using a separation solution ⁷ cells/mL。

9) Enrichment Cocktail, 50. Mu.L/mL added to the kit.

10 Mixing well, and incubating for 5min at room temperature.

11 RapidSpheres in the extraction kit ^TM The beads, vortex for 30s.

12 RapidSpheres in the kit ^TM The beads were added to the sample at 40. Mu.L/mL.

13 Mixing well, and incubating for 2min at room temperature.

14 The mixture was transferred into a flow tube.

15 Flow tube is inserted into the magnetic pole and is stationary for 3min.

16 Pouring the supernatant into a new centrifuge tube for later use.

3. Labeling T cells

T cells were labeled with CelltraceTM Violet (Cell trace Violet Cell Proliferation Kit, thermo) according to the product instructions, the experimental procedure was as follows:

(1) 20ul of DMSO was mixed with Celltrace reagent to a concentration of 5mM.

(2) Labeled buffer (DPBS+2% heat-inactivated bovine serum+2 mM EDTA) was prepared, and 10mL of the buffer was used to suspend T cells, and 400g was centrifuged for 5min.

(3) The supernatant was aspirated off, 1mL of the buffer was added, 1uL CelltraceTM Violet was added, and incubated at room temperature for 20min in the absence of light.

(4) 1640 medium (+10% heat-inactivated bovine serum+1% PBS) was added and incubated at room temperature for 5min.

(5) Centrifuge at 400g for 5min.

(6) The supernatant was aspirated, and the cells were suspended in 1640 medium (+10% heat-inactivated bovine serum+1% PBS) and centrifuged at 400g for 5min.

(7) CellTrace labeled T cells were suspended using 1640 medium (+10% heat-inactivated bovine serum+1% PBS), counted, and diluted to 5X 10 ⁶ cells/ml。

(8) Adding 1/200 volume of ImmunoCurt to CellTrace labeled T cell suspension ^TM Human CD3/CD28T Cell Activator, mixing.

(9) Cells were plated in 96-well flat bottom plates (Corning, 3599), 200uL per well.

(10) 1uL of the test compound was added to each well of the test compound (200X, 32. Mu.M, 8. Mu.M, 2. Mu.M, 0.5. Mu.M, 0.125. Mu.M, etc. final concentration) at a concentration of 6.4mM,1.6mM,0.4mM,0.1mM,0.025mM, respectively, and mixed well.

(11) Simultaneously setting T cells without CellTrace mark and T cells with CellTrace mark+ImmunoCurt ^TM Human CD3/CD28T Cell Activator, cellTrace-labeled T cells were not added with ImmunoCurt ^TM Human CD3/CD28T Cell Activator was used as a control for three groups of systems.

(12) The 96-well plates were placed in a cell incubator for incubation (37 degrees celsius, 5% carbon dioxide). Incubate for 5 days.

(13) After 5 days, the plates were removed, centrifuged and the supernatant was aspirated for detection of cytokine ifnγ secretion (Human IFN- γ ELISA Kit).

(14) Proliferation of T cells was detected using FACS.

FACS detection of T cell proliferation

(1) 200ul FACS buffer (DPBS with 2% heat-inactivated bovine serum) was added to each well to suspend T cells.

(2) Centrifuge at 400g for 5min.

(3) The supernatant was discarded, centrifuged for 5min with 200ul FACS buffer,400g added, and the supernatant was discarded.

(4) 50ul of staining antibody (1ul cell reactive far red reactive dye,50. Mu.L BV605mouse anti-human CD4, 50. Mu.L APC-Cy7mouse anti-human CD8 antibody) was added to each well, mixed well and stained at room temperature for 30min.

(5) mu.L of FACS buffer was added to each well, and the supernatant was discarded after centrifugation at 400g for 5 min.

(6) Repeating the step (5), and discarding the supernatant.

(7) 100uL FACS buffer was added to each well and the cells were suspended and checked on the machine.

The statistical method comprises the following steps: all data are expressed as mean ± standard deviation, and all index inspection results were processed by Excel 2000 and Graphpad Prism 5 software.

Experimental results:

compounds 1-18 against CD4 ⁺ T cell proliferation and CD8 pair ⁺ The proliferation of T cells has stronger inhibition activity and certain dose dependency, and the higher the concentration is, the stronger the inhibition effect is.

/>

EXAMPLE 20 inhibitory Activity of Compounds on T cell secretion of IFN-gamma

Experimental principle: IFN-gamma is secreted by Th1 cells, and its biological function is to induce CD4+ T cells to convert into Th1 cells, promote T, B lymphocyte differentiation, etc. Studies have shown that elevated levels of IFN- γ secretion can promote the onset of inflammation and injury to body tissues, thereby exacerbating the condition of autoimmune disease. Therefore, the inhibition activity of the drug on IFN-gamma can be detected to evaluate the immunosuppressive activity. The inhibitory activity of each sample on IFN-gamma was observed by detecting the concentration of IFN-gamma secreted by T cells in the supernatant after each of compounds 1-18 was applied to the cells by enzyme-linked immunosorbent assay (ELISA) using IFN-gamma ELISA Kit (Human IFN-gamma ELISA Kit).

Experimental reagents and materials: human IFN-gamma Precoated ELISA kit kit, available from Davidae under the accession number 1110002

The experimental method comprises the following steps: adding cell supernatant into 96-well plate provided by kit, adding gradient dilution of human IFN gamma standard substance and blank well as control, incubating at room temperature, adding antibody (HRP-labeled) against human IFN gamma after washing the plate, continuing incubating at room temperature, washing unbound antibody (HRP-labeled) against IFN gamma, adding substrate, developing at room temperature for 15-20 min, and reading OD after stopping developing ₄₅₀ Numerical values. A standard curve was made using the data of human ifnγ standards and the concentration of human ifnγ in each sample to be tested was calculated.

The experimental steps are as follows:

1. all reagents in the assay kit were equilibrated at room temperature for 20 minutes.

2. The 50x wash in the kit was configured as a 1x wash using deionized water.

3. Cell supernatants were 2.4-fold diluted using the dilutions in the kit.

4. Human IFNgamma standard substances with the concentration of 400pg/mL,200pg/mL,100pg/mL,50pg/mL,25pg/mL and 12.5pg/mL are respectively prepared.

5. mu.L of standard and 100. Mu.L of 2.4-fold diluted cell supernatant were added to a 96-well plate. A blank (100. Mu.L of diluent was added) was also set

6. The kit was diluted 50-fold with Biotinylated antibody in the diluent, 50 μl was added per well and incubated for 2 hours at room temperature.

7. Plates were washed three times with wash solution, 300 μl per well. The liquid remained in the holes needs to be buckled and dried each time.

8. The strepavidin-HRP in the kit was 100-fold diluted with the diluent, 100. Mu.L was added to each well, and incubated at room temperature for 20 minutes.

9. And 7, repeating the step 7, and washing the plate three times.

10. TMB (tetramethylbenzidine) chromogenic substrate added to the kit was incubated at 100. Mu.L per well for 15 minutes at room temperature in the absence of light.

11. 100. Mu.L of stop solution was added to the kit per well and the OD was read using a plate reader within 10 minutes ₄₅₀ Values.

Experimental results:

the concentration of IFN-gamma secreted by T cells in a culture solution is detected by using an enzyme-linked immunosorbent assay (ELISA), and the compound is found to have a strong inhibition effect on IFN-gamma secreted by the T cells and is dose-dependent, wherein the higher the concentration is, the stronger the inhibition effect is.

EXAMPLE 21 Compound experiments on Canavalia-induced proliferation of T lymphocytes in mice

By using ³ H-thymidine ³ H-thymodine) the effect of compounds 1-18 on Con A (Concanavalin A) -induced proliferation function of spleen lymphocytes in normal mice was examined by the H-thymodine incorporation method, and the in vitro immunosuppressive activity of the samples was evaluated.

Experimental materials and reagents: female BALB/c inbred mice, 18-20 g, source: BK (BK)

Canavalin a (concanavalin a, con a): supplier Sigma-Aldrich, cat# C7555

RPMI-1640 medium was purchased from Gibco;

³ h-thymidine: shanghai, inc. of Tech, wuhan Yitai, inc.;

the test steps are as follows:

1. preparation of mouse spleen lymphocytes: mice were despinified, their spleens were aseptically removed to prepare a single cell suspension, erythrocytes were removed from the erythrocyte lysate, washed 3 times with PBS containing 2% FBS, 1 time with RPMI-1640 medium containing 10% FBS and the cell concentration was adjusted to 4X 10 ⁶ /ml。

2. ³ H-TdR incorporation assay the effect of the compounds of examples 1-18 on mouse spleen lymphocyte proliferation function was examined: mouse spleen lymphocyte suspension 100. Mu.l (4X 10) ⁵ Per well) was inoculated in 96-well plates, 50 μl of Con A (final concentration 10 μg/ml), 50 μl of the compounds of examples 1-18 at different concentrations, 50, 12.5, 3.125, 0.781, 0.195, 0.0488 μM, three wells per concentration, total volume 200 μl, and corresponding Con A free control wells and drug free control wells were set. 37 ℃,5% CO ₂ Culturing in an incubator for 48 hours. 8 hours before the end of the incubation, 25. Mu.l of each well was added ³ H-thymidine (10. Mu. Ci/ml). Culturing is continued until the experiment is finished. The cells were collected on a glass fiber membrane using a cell collector, and the DNA incorporated into the cells was read by a Beta counter (Microbeta Trilux, perkinelmer) after adding a scintillation fluid ³ The amount of H-TdR, expressed as cpm, represents the proliferation of the cells.

The statistical method comprises the following steps: all data are expressed in mean ± standard deviation, and all index inspection results are processed by Excel 2000 and SPSS 11.0 statistical software packages.

Experimental results: the inhibitory activity of the compounds on mouse spleen T cell proliferation was tested using normal mouse spleen T lymphocytes, co-incubated with the series of compounds in the presence of the inducer Con a. The results show that the compound has strong activity of inhibiting the proliferation of the spleen T lymphocytes of the mice.

Example 22 inhibition experiments of bacterial lipopolysaccharide LPS induced proliferation of spleen B lymphocytes in Normal mice

³ H-thymidine ³ H-Thymidine) the effect of compounds 1-18 on LPS (lipopolysaccharid) -induced proliferation of spleen B lymphocytes in normal mice was examined and the in vitro immunosuppressive activity of the compounds was evaluated.

Experimental materials and reagents: female BALB/c inbred mice, 18-20 g, supplier: BK (BK)

Lipopolysaccharide LPS (LIPOPOLYSACCHARIDE): supplier J & K, goods number L2762

RPMI-1640 medium: supplier Gibco

³ H-thymidine, available as sea division of the technology Co.Ltd

The test steps are as follows:

1. preparation of mouse spleen lymphocytes: mice were despinified, their spleens were aseptically removed to prepare a single cell suspension, erythrocytes were removed from the erythrocyte lysate, washed 3 times with PBS containing 2% FBS, 1 time with RPMI-1640 medium containing 10% FBS and the cells were adjusted to 5X 10 ⁶ /ml。

2. ³ The effect of compounds 1-18 on mouse spleen lymphocyte proliferation function was examined by H-TdR incorporation: mouse spleen lymphocyte suspension 100. Mu.l (4X 10) ⁵ Well) was inoculated into 96-well plates, 50. Mu.l LPS (final concentration 10. Mu.g/ml) was added, 1-18. Mu.l of different concentrations of compound was added, the final concentrations of compound in the test wells were 50, 12.5, 3.125, 0.781, 0.195, 0.0488. Mu.M, three wells per concentration, the total volume was 200. Mu.l, and corresponding LPS-free control wells and drug-free control wells were set. 37 ℃,5% CO ₂ Culturing in an incubator for 48 hours. 8 hours before the end of the incubation, 25. Mu.l of each well was added ³ H-thymidine (10. Mu. Ci/ml). Culturing is continued until the experiment is finished. The cells were collected on a glass fiber membrane using a cell collector, and the DNA incorporated into the cells was read by a Beta counter (Microbeta Trilux, perkinelmer) after adding a scintillation fluid ³ The amount of H-TdR, expressed as cpm, represents the proliferation of the cells.

Experimental results: taking normal mouse spleen B lymphocytes to incubate with a compound to be tested in the presence of an inducer, and detecting the inhibitory activity of the compounds 1-18 on proliferation of the mouse spleen B lymphocytes at different concentrations. The results show that compounds 1-18 can strongly inhibit mitogen bacteria Lipopolysaccharide (LPS) -induced proliferation of spleen B lymphocytes in normal mice:

/>

EXAMPLE 23 assay of Compounds for PHA-stimulated in vitro proliferation inhibition Activity of human PBMC

The inhibitory activity of compounds 1-18 on PHA stimulated proliferation of human PBMC in vitro was examined.

Experimental reagents and materials:

seperate TM-50: a vendor STEMCELL Technologies; goods number 86460

Lymphoprep: a vendor STEMCELL Technologies; goods number 7861

DPBS: vendor Biosera; cargo number LM-S2041/500

RPMI-1640: vendor Gibco; cargo No. 11875-093

FBS (French Origin): vendor Biosera; goods number FB-1280/500

Penicillin-Streptomycin: vendor Gibco; goods number 15140122

2-Mercaptoethanol: supplier Sigma-Aldrich; goods number M3148

PHA: supplier Sigma-Aldrich; goods number L8902-5MG

96-well Flat Clear Bottom White Polystyrene TC-treated Microplates: supplier Corning; goods number 3903

Luminescent Cell Viability Assay: vendor Promega; goods number G7572

Experimental method of isolation of human peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMC) to adjust cell concentration to 1×10 ⁶ Per mL,100 μl per well was seeded into 96-well plates. The compounds of examples 1-18 were dissolved to the appropriate concentration in a mother liquor to give a solution of 1:4 gradient dilutions, giving 5 concentration gradients (2. Mu.M, 0.5. Mu.M, 0.125. Mu.M, 0.031. Mu.M, 0.008. Mu.M) were added to the corresponding wells, three wells were set for each concentration, the positive control well was 2. Mu.M cyclosporin A (CSA), the negative control well was RPMI-1640 complete medium (RPMI 1640+10% FBS+1% Penicilliin-Streptomycin+55. Mu.M. Beta. Mercaptoethanol), and 1. Mu.g/mL PHA was added to each well to stimulate PBMC cells. After 72h incubation in the incubator, CTG was added to each well and proliferation of the cells was detected.

The experimental steps are as follows:

isolation of human Peripheral Blood Mononuclear Cells (PBMC)

1) Fresh blood samples were diluted with the same volume of DPBS, 15mL of Lymphoprep was added to the Sepmate tube, and 30mL of diluted blood sample was slowly added over Lymphoprep, taking care not to disrupt the interface.

2) The post-addition Sepmate tube was centrifuged at 1000. 1000x g for 25min at room temperature.

3) The leukocyte layer containing PBMCs was collected into a new 50mL centrifuge tube, washed twice with 40mL DPBS, and centrifuged for 5min at 350x g.

4) The supernatant was discarded, and the cells were resuspended in RPMI-1640 complete medium to a concentration of 1X 10 ⁶ /mL。

5) 100 μl/well of PBMC cells were seeded into 96-well plates at 37deg.C with 5% CO ₂ Incubate for 15min.

6) The compound to be tested is dissolved to a mother liquor with proper concentration by using RPMI-1640 complete culture solution, and the ratio of the mother liquor to the mother liquor is 1:4, carrying out gradient dilution to obtain 5 concentration gradients, wherein three compound holes are arranged in each concentration.

7) The positive control group is CSA, and the negative control group is RPMI-1640 complete medium.

8) Formulated 4-fold concentration of CSA (50. Mu.L/well in volume), 4-fold concentration of test compound (50. Mu.L/well in volume) were added to the corresponding wells while PBMC cells were stimulated with 1. Mu.g/mL PHA (50. Mu.L/well in volume) in a total volume of 200. Mu.L per well.

9) 96-well plates were placed at 37℃in 5% CO ₂ Culturing in an incubator for 72 hours.

10 After 72h, CTG was added to each well to examine the proliferation of cells.

11 Data analysis was performed using GraphPad Prism 6.0 software.

Data analysis data were analyzed with Graphpad Prism 6.0 software. Data are presented as mean and standard error (standard error of the mean, SEM).

Experimental results:

in this experiment, in PHA stimulated human PBMC proliferation system, positive control 2. Mu.M CSA can inhibit PBMC proliferation. Compounds 1-18 all inhibit PBMC proliferation and exhibit a degree of dose-dependence, IC ₅₀ The method comprises the following steps:

EXAMPLE 24 pharmacodynamic study experiment to test Compound 4 on DNFB (2, 4-dinitrofluorobenzene) induced mouse ear swelling model

Experimental materials and reagents: 50 female ICR mice weighing 25-30 grams; the source is as follows: BK (BK)

DNFB (2, 4-dinitrofluorobenzene): sigma-Aldrich (St.Louis, MO, USA), cat: D1529.

Acetone: shanghai Taitan technologies Co., ltd., cat:67-64-1.

Olive oil: shanghai Lingfeng chemical Co., ltd., cat:8001-25-0.

Positive control compound-Dexamethasone (Dex): DAMAS-BETA, purity 98%, cat:50-02-2.

The experimental method comprises the following steps: mice were randomly divided into 5 groups of 10 mice each by body weight. The model group, dex (2 MPK, qd) group, compound 4 high dose (12 MPK, qd) group, compound 4 medium dose (6 MPK, qd) group, and compound 4 low dose (3 MPK, qd) group, respectively. The method for model induction is as follows: all groups were challenged by applying 50 μl of 1% dnfb solution to the abdomen on day 0 and day one of the experiment, and 10 μl of 0.5% dnfb was applied to the right ear surface on day 5, and the right ear thickness before and after challenge was measured, and the efficacy of the compound was evaluated using the difference.

The experimental steps are as follows:

1. 1% DNFB was prepared as acetone: the olive oil proportion is (4:1), namely, 1mL of olive oil is added into 4mL of acetone, and the mixture is uniformly mixed for standby. 50uLDNFB is added into the mixed solution, and after being mixed evenly, the mixed solution is split into 10 tubes, each tube is 0.5ml, the sealing is carried out, the light is prevented, and one tube is used for each cage of mice.

2. Sensitization: all animals were anesthetized and shaved on day-1, on day 0 and on day 1, and 50 μl of freshly prepared 1% DNFB (4:1 acetone: olive oil) was applied to the abdomen.

3. On day 5, all animals measured right ear thickness data, and then 20 μl of 0.5% DNFB (10 ul/side) was smeared.

4. After the administration of the medicine on the 6 th day for another 4 hours, the medicine meets the requirement that the DNFB coated on the right ear on the 5 th day is 0.5% and the medicine is coated on the right ear for 24 hours (after the DNFB coated on the right ear for 0.5% and 24 hours), the thickness of the right ear of each mouse is measured by a spiral micrometer, and the difference obtained by subtracting the thickness of the right ear before the 5 th day attack is taken as a swelling value.

5. Administration: the compounds, including the positive drug (Dex), were administered orally once a day for 7 consecutive days.

6. Weight of: weigh before daily dosing.

Statistical analysis: the data collected for each group are expressed as Mean and standard deviation (mean±sem). Various changes were analyzed using GraphPad Prism software. p <0.05 was considered significant differences and p <0.01 was considered extremely significant differences.

Experimental results:

1. weight change

As shown in fig. 1, after grouping, the body weights of all animals in the initial stage of the experiment tended to decrease slowly due to the influence of disease modeling, and by the day of the experiment 2, the body weights of all animals (except G2) began to rise slowly and remained steady until the end of the experiment. G2, the desx-dosed group, always had a tendency to slowly decrease in body weight, presumably due to side effects of desx. Experiments show that all three concentrations of compound 4 have better safety.

Thickness of right ear

On day 5 of the experiment, the thickness of the right ear was measured after all animals had been anesthetized, and the average was taken three times. On day 6 of the experiment, the thickness of the right ear was measured after all animals had been anesthetized, and the average was taken three times. And carrying out statistical analysis on the thickness difference data of the right ear before and after attack. As shown in fig. 2, the difference in thickness of the right ear before and after challenge is significantly reduced in the Dex treatment group (2 MPK) compared to the model group (P < 0.0001) and the three low, medium and high dose treatment groups of compound 4 also significantly reduced the difference in thickness of the right ear before and after challenge (P < 0.01) with a certain dose dependent effect. The results indicate that the compound 4 can significantly inhibit DNFB-induced ear swelling, and the drug effect has a certain correlation with the dosage.

Example 25 pharmacodynamic study experiment to detect delayed hypersensitivity to plantar swelling caused by Compound 4 in sheep Red blood cells

Experimental materials and reagents: 50 female ICR mice; weight of: 25-30 g; the source is as follows: BK (BK)

Sheep red blood cells (SRBC, concentration 50%): cargo number: HQ80073, lot number: 190617.

polyethylene glycol 400 (PEG 400): alfa, cat No.: 25322-68-3.

Positive control compound: prednisone acetate tablet (PNS) containing 5mg of effective component.

The experimental method comprises the following steps: mice were randomly divided into 5 groups of 10 mice each according to body weight; the model group, PNS (5 MPK, qd) group, compound 4 high dose (6 MPK, qd) group, compound medium dose (2 MPK, qd) group, and compound 4 low dose (0.5 MPK, qd) group, respectively. All groups were challenged by intraperitoneal injection of sheep erythrocytes on the first day of the experiment and subcutaneous injection of sheep erythrocytes again on day 5, the thickness of the left and right rear plantar pads after challenge was measured and the efficacy of the compounds was evaluated using the difference.

The experimental steps are as follows:

1. all animals in Day1, G1-G5 groups were intraperitoneally injected with 2% (v/v) SRBC, each with 0.2 ml (about 1 x 10 ⁸ SRBC), animal immunization.

2. Administration is performed immediately after immunization.

3. At Day 5, the thickness of the left hind paw of all animals in groups G1-G5 was measured three times and averaged; then, 20% (v/v) SRBC were subcutaneously injected into all animal measurement sites of the G1-G5 group, 20ul (about 1X 10) ⁸ SRBC) are under attack.

4. Dosing was performed 18-20 hours post challenge, 24 hours measurement (Day 6), and all animals were anesthetized and the thickness of the left post-injection plantar region and the thickness of the right post-injection plantar region were measured three times each, and averaged. The difference between the thickness of the left rear plantar foot pad and the thickness of the right plantar foot pad after the attack is taken as the foot swelling degree, and the data are analyzed respectively.

5. Administration: the compound is prepared and used at present every day; oral administration; once a day, and administered continuously for 6 days.

6. Weight of: weigh before daily dosing.

Experimental results:

1. weight change

As shown in fig. 3, after grouping, all groups of animals showed a tendency to slowly decrease in weight due to the influence of disease modeling, with the decrease in weight being most pronounced in PNS-administered groups. There was no statistically significant difference (P > 0.05) between the 4 dosing groups compared to the model group, respectively.

2. Thickness of foot pad

On day 6 of the experiment, the thickness of the left and right hind paw was measured after all animals had been anesthetized, three times, and an average was taken. Statistical analysis was performed on the data of the thickness difference between the left rear plantar pad and the right plantar pad. As shown in fig. 4, the thickness differences between the left and right foot soles were also significantly reduced in the middle-high dose treatment group of compound 4 compared to the model group (P < 0.01), with significant statistical differences. The results suggest that compound 4 can significantly inhibit delayed hypersensitivity of plantar swelling caused by sheep erythrocytes, and that the drug effect is dose-dependent.

The foregoing examples merely illustrate embodiments of the invention and are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. Use of a compound having a structure selected from the group consisting of the 18 structural formulas:

。

2. The use of claim 1, the medicament further comprising one or more pharmaceutically acceptable carriers, diluents or excipients.

3. The use according to claim 1 or 2, wherein the autoimmune disease of the human body comprises enteritis, psoriasis, lupus erythematosus, rheumatoid arthritis, allergic dermatitis, multiple sclerosis, sepsis.

4. The use according to claim 3, characterized by the use of the preparation of immunosuppressive drugs against rejection reactions after human organ transplantation.