Natural killer T cell agonist containing sulfonamide group or part of sulfonamide group and having immunomodulatory activity and application

Abstract

The invention belongs to the technical field of chemistry and medicine, and relates to a natural killer T cell agonist which contains a sulfonamide group or a part of the sulfonamide group and has immunoregulatory activity and application of the natural killer T cell agonist. The agonist is a compound with a structure as shown in a formula (1) or a pharmaceutically acceptable salt thereof. According to the invention, a sulfonamide group is introduced to an acyl chain of alpha GalCer, and the binding force between glycolipid molecules and CD1d is enhanced by forming an additional hydrogen bond. By adopting the design concept, the NKT cell agonist with better immunomodulatory activity is obtained, the synthesis method of the series of compounds is simple and efficient, and the screened novel alpha GalCer analogue has obviously improved immunostimulatory activity compared with the original alpha GalCer, can induce immunoreactions with different biases, and has good immunomodulatory activity. Potential candidate drugs are provided for immunotherapy and immune modulation. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of chemistry and pharmaceutical technology, and in particular relates to a natural killer T cell agonist with immunomodulatory activity containing a sulfonamide group or a portion thereof, and its application, as well as a drug complex containing the agonist. Background Art

[0002] Natural killer T (NKT) cells are a unique subset of non-traditional T cells that serve as a bridge between innate and adaptive immunity. A growing number of studies have shown that NKT cells play an important role in the immunotherapy of various diseases, such as cancer, viral infections, and autoimmune diseases. α-Galactosylceramide (αGalCer) is the most representative agonist molecule for NKT cells. After binding to the CD1d protein, it is presented on the surface of immune cells to activate NKT cells, which then triggers the release of various cytokines, including pro-inflammatory Th1 cytokines (represented by IFN-γ) and anti-inflammatory Th2 cytokines (represented by IL-4). To meet different immune regulation requirements, researchers hope to achieve preferential activation of Th1 or Th2 responses by structurally modifying αGalCer, thereby exerting better immune effects in different types of diseases, while also exploring the structure-activity relationship of αGalCer analogs.

[0003] Over the past three decades, numerous αGalCer analogs have been synthesized and studied to obtain NKT cell agonists with stronger immunostimulatory activity or biased immune responses. Structural modifications of αGalCer primarily include modifications to the sugar ring, glycosidic bond, phytosphingosine chain, and acyl fatty acid chain. Several representative αGalCer analogs have been shown to exhibit potent biological effects in inducing Th1- or Th2-biased immune responses. Despite this, the availability of biased αGalCer analogs with high immunoactivity remains limited, as the mechanisms underlying their selective activation of Th1 or Th2 responses remain unclear. Furthermore, a universally applicable structural design strategy for NKT cell agonists is lacking. Summary of the Invention

[0004] The purpose of the present invention is to obtain a new NKT cell agonist with enhanced immunostimulatory activity and the ability to induce selective immune responses by structurally modifying αGalCer, thereby improving its immunoregulatory effects in anti-tumor, anti-infection and autoimmune diseases.

[0005] In order to achieve the above-mentioned object, the first aspect of the present invention provides a class of natural killer T cell agonists with immunomodulatory activity containing a sulfonamide group or a portion thereof, which is a compound having a structure represented by formula (1) or a pharmaceutically acceptable salt thereof:

[0006]

[0007] Said Y is any group represented by formula (A) to (E):

[0008]

[0009] R1 is -H or C1-C 30 of hydrocarbon groups;

[0010] X1, X2 and X3 are each independently C1-C 30 The hydrocarbon group contains 0-5 heteroatoms selected from N, O, F, Br and Cl;

[0011] Z1 and Z2 are each independently -OR2, -SR3, -NHR4, -SO2R5, -[-OCH2CH2-]n1-OCH3, -F, -Br, -Cl, C1-C 30 wherein R2 and R3 are each independently -H, C1-C 30 alkyl, aryl, heteroaryl or -CH2OSO3Na, R4 is R2, -COR2 or -CONHR2, R5 is -NHR2, C1-C 30 A hydrocarbon group, an aryl group, a heteroaryl group or -CH2OSO3Na, n1 is an integer from 0 to 20, and n2 is an integer from 0 to 10;

[0012] W1 and W2 are each independently -CH2-, -NH-, -O- or -S-;

[0013] W3 is -NHCO-, -NCH2CO-, -NHCONH-, -NHSO2-, -NHSO2NH-, -NHCS-, -O-, -S- or -OCO-;

[0014] Z3 and Z4 are each independently -OH, -H, -F, -Br or -Cl.

[0015] According to a preferred embodiment of the present invention, R1 is -H or C1-C 12 The alkyl group is further preferably -H or a C1-C3 alkyl group, and the C1-C6 alkyl group includes but is not limited to methyl, ethyl, n-propyl, and isopropyl.

[0016] According to a preferred embodiment of the present invention, X1 is C1-C 18 A straight chain or branched alkyl group, an alkenyl group or an alkynyl group containing an unsaturated double bond or triple bond, when it is an alkyl group, X1 is more preferably C 10 -C 12 of a straight chain alkyl group.

[0017] According to a preferred embodiment of the present invention, X2 is C1-C 18 Straight or branched alkyl, C2-C 18 Alkenyl or alkynyl containing unsaturated double or triple bonds, C6-C 18 Aryl, C7-C 18 Alkaryl or C1-C 18 and optionally substituted by C1-C4 alkyl, F, Br or Cl; preferably, X2 is C1-C 13 Straight or branched alkyl, C2-C 12 Alkenyl or alkynyl containing unsaturated double or triple bonds, C6-C 12 Aryl, C7-C 12 Alkaryl or C7-C 12 and optionally substituted by C1-C4 alkyl, F, Br or Cl.

[0018] Specifically, the C1-C 18 The alkyl group includes, but is not limited to, a linear or branched alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbon atoms. The alkenyl or alkynyl group containing an unsaturated double bond or triple bond is a group containing one or more carbon-carbon double bonds or triple bonds and having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbon atoms. 12 The aryl group includes but is not limited to a group with phenyl having 6, 7, 8, 9, 10, or 12 carbon atoms; 12 Alkaryl or C7-C 12 Aralkyl groups include, but are not limited to, groups having 7, 8, 9, 10, 12 carbon atoms and having an alkyl substituent and a phenyl group.

[0019] According to a preferred embodiment of the present invention, X3 is C6-C 18 A straight chain or branched alkyl group, an alkenyl group or an alkynyl group containing an unsaturated double bond or triple bond, preferably, when it is an alkyl group, X3 is C 10 -C 16 The alkyl group includes, but is not limited to, a linear or branched alkyl group having 10, 11, 12, 13, 14, 15, or 16 carbon atoms.

[0020] According to one embodiment of the present invention, the agonist is at least one of the following compounds:

[0021]

[0022] Among them, m can be any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18.

[0023] According to a specific embodiment of the present invention, the agonist is at least one of the following compounds:

[0024]

[0025] According to another specific embodiment of the present invention, the agonist is at least one of the following compounds:

[0026]

[0027] According to another specific embodiment of the present invention, the agonist is at least one of the following compounds:

[0028]

[0029]

[0030] In a specific embodiment, a method for synthesizing an αGalCer analog molecule represented by structural formula 1 is listed below. However, the present invention does not particularly limit the method for preparing the aforementioned αGalCer analog molecule. Those skilled in the art can determine a suitable method to prepare the αGalCer analog molecule in combination with the structural characteristics of the NKT cell agonist molecules in the art and conventional synthesis methods in the art. In addition, the examples of the present invention exemplarily list specific synthesis methods for some αGalCer analog molecules. Those skilled in the art can also determine the specific preparation methods for all αGalCer analog molecules of the present invention in combination with the exemplified synthesis methods of the present invention. However, those skilled in the art should not understand this as a limitation of the present invention.

[0031] The aforementioned αGalCer analog molecules can be prepared by the following method:

[0032]

[0033] X1 and X2 are as defined above. A1 is a protective group for hydroxyl, such as benzyl or the like.

[0034] The compound represented by Structural Formula 1 can be obtained by reacting the compounds represented by Structural Formulas 2 and 3 through steps (a) to (e). However, the present invention is not particularly limited to the method for synthesizing the compound represented by Structural Formula 1, and the reaction conditions of steps (a) to (e) can be reasonably replaced according to the target compound.

[0035] The second aspect of the present invention provides a drug complex comprising the above-mentioned agonist as an active ingredient and may also contain other active ingredients, or drug carriers and / or pharmaceutical excipients.

[0036] The drug carrier includes, but is not limited to, microcapsules, microspheres, nanoparticles, or liposomes. The pharmaceutical excipient may include an excipient. The excipient includes, but is not limited to, at least one of a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, an absorbent, a diluent, a flocculant, a deflocculant, a filter aid, and a release retardant. The drug may be in the form of a tablet, powder, granule, capsule, suspension, emulsion, syrup, or extract.

[0037] According to the test results of the present invention, the above compounds all have better activity than αGalCer and can therefore be used as immunotherapy drugs. Among them, GCS-12 and can significantly enhance the Th1 / 2 balanced cytokine response, and GCS2N-12-11 can also induce a Th1 / 2 balanced response, so they can be used as drugs to enhance the Th1 / 2 balanced cytokine response. GCS-11 and GCS-13 behave as potent Th1-biased immune agonists, and GCS2N-11-12 can also induce a Th1-biased cytokine response, so they can be used as drugs to induce a Th1-biased cytokine response. Compared to αGalCer, GCS-12b, GCS-12c, GCS-12d, GCS-12e, GCS-12f, GCS-12g, GCS-12-5, GCS-12-6, and GCS-12-7 significantly increased IL-4 secretion and are Th2-biased glycolipids. GCS2N-11-5, GCS2N-11-6, GCS2N-11-7, GCS2N-12-4, GCS2N-12-5, and GC2N-12-6 are also Th2-biased glycolipids, suggesting their potential use as agents for inducing Th2-biased cytokine responses. GCS-10m, GCS-11m, GCS-12m, GCS-10n, GCS-11n, and GCS-12n generally exhibit Th2-biased glycolipid responses.

[0038] The present invention has the following technical advantages:

[0039] (1) The present invention introduces a sulfonamide group into the middle position of the acyl chain of αGalCer (corresponding to the polar environment in the CD1d protein pocket), which can form additional hydrogen bond interactions between the sulfonamide and the CD1d protein, thereby enhancing the interaction between the glycolipid molecule / CD1d binary complex.

[0040] (2) Enhanced glycolipid / CD1d binary complex interaction helps to trigger stronger immune effects and induce biased immune responses.

[0041] (3) Based on the concept of enhancing the interaction of binary complexes, αGalCer analogs with sulfonamide groups were designed. Compared with αGalCer, they not only significantly improved the immunostimulatory activity but also obtained NKT cell agonists with specific bias.

[0042] The novel αGalCer analogs and their synthesis methods provided by this invention provide important information for exploring the structure-activity relationship of αGalCer analogs and clarifying the mechanism of inducing biased immune responses. The sulfonamide-containing αGalCer analogs provided by this invention have demonstrated potent immune activation in animals and induced immune responses of varying biases. They are expected to be developed into effective immunotherapeutic drugs for the treatment of various diseases, including anti-tumor and autoimmune diseases.

[0043] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Exemplary embodiments of the present invention will be described in more detail with reference to the accompanying drawings.

[0045] Figure 1 Figure 3 shows the levels of (A) IL-4 (2 h) and (B) IFN-γ (24 h) secreted in mouse serum after administration of GCS-10, GCS-11, GCS-12, GCS-13, GCS-14, and αGalCer, as well as the fold increase in IL-4 and IFN-γ induced by αGalCer compared with different αGalCer analogs (C).

[0046] Figure 2 Figure 3 shows the levels of (A) IL-4 (2 h) and (B) IFN-γ (24 h) secreted in mouse serum after administration of GCS-12-2, GCS-12-4, GCS-12-5, GCS-12-6, GCS-12-7, GCS-12-8, GCS-12-10, GCS-12a, and αGalCer, as well as the fold increase in IL-4 and IFN-γ induced by αGalCer compared with different αGalCer analogs (C).

[0047] Figure 3 Figure 3 shows the levels of (A) IL-4 (2 h) and (B) IFN-γ (24 h) secreted in mouse serum after administration of GCS-12b, GCS-12c, GCS-12d, GCS-12e, GCS-12f, GCS-12g, and αGalCer, as well as the fold increase in IL-4 and IFN-γ induced by αGalCer compared with different αGalCer analogs (C).

[0048] Figure 4 The graph shows the (A) IL-4 (2 h) and (B) IFN-γ (24 h) secreted levels in mouse serum after administration of GCS2N-11-12, GCS2N-12-11, GCS2N-11-5, GCS2N-11-6, GCS2N-11-7, GCS2N-12-4, GCS2N-12-5, GC2N-12-6, and αGalCer, as well as the fold increase in IL-4 and IFN-γ induced by αGalCer compared with different αGalCer analogs (C).

[0049] Figure 5 Figure 3 shows the levels of (A) IL-4 (2 h) and (B) IFN-γ (24 h) secreted in mouse serum after administration of GCS-10m, GCS-11m, GCS-12m, GCS-10n, GCS-11n, GCS-12n, and αGalCer, as well as the fold increase in IL-4 and IFN-γ induced by αGalCer compared with different αGalCer analogs (C). DETAILED DESCRIPTION

[0050] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0051] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0052] Example 1: Synthesis of compound GCS-10

[0053]

[0054] Synthesis of compound 4a: Compound 2a (1.0 equiv) was dissolved in CH2Cl2, and compound 3a (1.5 equiv) and Et3N (1.0 equiv) were added at room temperature under argon protection, and stirred at room temperature for 6 hours. The organic solvent was removed, and the reaction mixture was purified by column chromatography to obtain compound 4a (yield 90%). 1H NMR (400MHz, CDCl3) δ4.03(s,1H),3.67(s,3H),3.10(q,J=6.8Hz,2H),3.06–2.93(m,2H),2.30(t, J=7.5Hz,2H),1.78(d,J=8.0Hz,2H),1.41(m,2H),1.28(d,J=14.1Hz,32H),0.88(t,J=6.6Hz,3H). 13 C NMR (150MHz, CDCl3) δ174.39,52.64,51.55,43.36,34.15,32.01,30.42,29.75,29 .63,29.44,29.36,29.23,29.16,28.42,26.64,24.99,23.77,22.78,14.22.ESI-MS calcd.for C 25 H 52 NO4S[M+H] + ,462.36;found,462.43.

[0055] The synthesis of compound GCS-10 comprises the following steps 1)-4):

[0056] First, compound 5a was synthesized as follows: 1) Compound 4a (1.0 equiv) was dissolved in THF, and an equal volume of aqueous NaOH (1 M) was added at room temperature with stirring for 8 hours. The reaction system was then neutralized with 1 M HCl. The precipitated solid intermediate was collected, washed with water, and dried. 2) The resulting intermediate was dissolved in dry toluene, and SO₂Cl₂ (1 mL) was added under argon at room temperature. The mixture was stirred at 80°C for 2 hours. The solution was then cooled to room temperature, and the organic solvent and unreacted SO₂Cl₂ were removed under argon. The resulting crude acid chloride 5a (quantitative reaction) was used immediately in the subsequent reaction step.

[0057] 3) The crude acid chloride 5a was dissolved in dry CHCl and added to a solution of compound 6a (1.0 equiv) in dry CHCl, and stirred at room temperature for 2 hours. The organic phase was then removed, and the reaction mixture was purified by column chromatography to obtain a solid intermediate. 4) The obtained intermediate was dissolved in a CHCl / MeOH mixture, and Pd(OH) / C (C, 20% wt) was added. The mixture was stirred under a hydrogen atmosphere at room temperature for 12 hours. The resulting mixture was filtered through celite and concentrated to obtain the crude product, which was then purified by column chromatography to obtain compound GCS-10 (yield 41%). 1H NMR(400MHz,CD3OD / CDCl3)δ4.91(d,J=3.7Hz,1H),4.21(d,J=9.5Hz,1H),3.96–3.8 5(m,2H),3.75(tdd,J=23.8,11.2,5.2Hz,7H),3.56(t,J=4.7Hz,2H),3.02(dt,J=18 .9,7.6Hz,4H),2.22(t,J=7.6Hz,2H),1.79(t,J=7.8Hz,2H),1.60(ddd,J=32.5,14. 7,8.1Hz,7H),1.44(q,J=5.8,5.1Hz,4H),1.37–1.22(m,61H),0.89(t,J=6.6Hz,6H). 13 C NMR(150MHz,CD3OD / CDCl3)δ175.13,100.36,70.84,69.51,62.28,52.61,43.53,36.88,32.46 ,30.81,30.24,30.17,29.88,29.79,29.68,28.86,27.12,26.40,24.08,23.18,14.33.ESI-MS calcd.for C 48 H 96 N2O 11 SNa[M+Na] + ,931.6627;found,931.6867.

[0058] Example 2: Synthesis of compound GCS-11

[0059]

[0060] Synthesis of compound 4b: The procedure was the same as that for synthesizing compound 4a (yield 98%). 1 H NMR (600MHz, CDCl3) δ4.03(s,1H),3.67(d,J=3.3Hz,3H),3.09(s,2H),2.99(s,2H),2.30(d,J= 7.9Hz,2H),1.79(s,2H),1.61(s,4H),1.41(s,2H),1.36–1.12(m,33H),0.87(d,J=6.9Hz,3H). 13C NMR (150MHz, CDCl3) δ174.41,52.66,51.55,43.39,34.18,32.02,30.45,29.74,29 .64,29.49,29.43,29.29,29.22,28.43,26.67,25.02,23.79,22.79,14.22.ESI-MS calcd.forC 25 H 52 NO4S[M+H] + ,462.36;found,462.90.

[0061] Synthesis of compound GCS-11: The procedure was the same as that for synthesizing compound GCS-10 (yield 35%). 1 H NMR(600MHz,CD3OD / CDCl3)δ4.91(s,1H),4.21(s,1H),3.93(s,2H),3.84–3.67(m,6H),3.60–3.47(m,4H),3.03(dd ,J=19.4,12.7Hz,4H),2.22(s,2H),1.79(s,2H),1.70–1.50(m,6H),1.29(d,J=21.0Hz,74H),0.89(d,J=6.4Hz,6H). 13 C NMR(150MHz,CD3OD / CDCl3)δ175.02,100.21,78.22,78.01,77.80,75.02,72.36,7 1.36,70.73,70.21,69.40,67.64,62.21,52.55,50.92,49.43,49.29,49.15,49.01 ,48.87,48.72,48.58,43.48,36.82,32.83,32.38,30.75,30.24,30.20,30.17,30 .09,29.98,29.84,29.80,29.63,28.77,27.07,26.34,24.00,23.11,14.33.ESI-MS calcd.for C 48 H 96 N2O 11 SNa[M+Na] + ,931.6627;found,931.6644.

[0062] Example 3: Synthesis of compound GCS-12

[0063]

[0064] Synthesis of compound 4c: The procedure was the same as that for synthesizing compound 4a (yield 98%). 1 H NMR (400MHz, CDCl3) δ4.36(t,J=6.1Hz,1H),3.65(s,3H),3.07(q,J=6.8Hz,2H),3.01–2.94(m,2H),2.29(t,J=7.5 Hz,2H),1.82–1.70(m,3H),1.56(dp,J=27.6,7.2Hz,5H),1.42(s,3H),1.35–1.18(m,33H),0.86(t,J=6.7Hz,3H). 13 C NMR (150MHz, CDCl3) δ174.28,52.40,51.38,43.20,34.02,31.84,30.26,29.55,29.48 ,29.40,29.32,29.28,29.17,29.08,28.27,26.53,24.87,23.60,22.62,14.06.ESI-MS calcd.for C 25 H 52 NO4S[M+H] + ,462.36;found,462.17.

[0065] Synthesis of compound GCS-12: The procedure was the same as that for synthesizing compound GCS-10 (yield 45%). 1 H NMR (400MHz, CD3OD / CDCl3) δ4.91(d,J=3.7Hz,1H),4.20(d,J=4.6Hz,1H),3.98–3.83(m,2H),3.83–3.65(m,6H),3.54(d,J=5.0Hz,2H),3 .02(dt,J=22.2,7.8Hz,4H),2.21(t,J=7.6Hz,2H),1.84–1.73(m,2H),1.72–1.48(m,6H),1.29(d,J=15.7Hz,61H),0.88(t,J=6.6Hz,6H). 13CNMR(150MHz,CD3OD / CDCl3)δ175.21,100.30,74.95,72.45,71.44,70.79,70.40,69.47,67.75,62.28,52.63,51.00,49.4 3,49.28,43.58,36.91,32.75,32.48,30.87,30.28,30.16,30.09,29.90,28.87,27.21,26.47,24.10,23.21,14.37.ESI-MS calcd.forC 48 H 96 N2O 11 SNa[M+Na] + ,931.6627;found,931.6648.

[0066] Example 4: Synthesis of compound GCS-13

[0067]

[0068] Synthesis of compound 4d: The procedure was the same as that for synthesizing compound 4a (yield 95%). 1 H NMR (400MHz, CDCl3) δ4.13(t,J=6.2Hz,1H),3.60(s,3H),3.03(d,J=6.8Hz,2H),2.96–2.89(m,2H),2.23(t,J=7 .5Hz,2H),1.71(d,J=7.7Hz,2H),1.58–1.43(m,5H),1.34(s,2H),1.20(d,J=8.7Hz,31H),0.81(t,J=6.7Hz,3H). 13 C NMR (100MHz, CDCl3) δ174.36,52.50,51.44,43.27,34.09,31.88,30.33,29.56,29.53,29.51 ,29.47,29.40,29.31,29.23,29.16,29.12,28.32,26.59,24.93,23.66,22.67,14.10.ESI-MS calcd.for C 25 H 52 NO4S[M+H] + ,462.36;found,462.48.

[0069] Synthesis of compound GCS-13: The procedure was the same as that for synthesizing compound GCS-10 (yield 38%). 1H NMR (600MHz, CD3OD / CDCl3) δ4.91(s,1H),4.20(s,1H),3.91(d,J=28.3Hz,2H),3.75(dd,J=43.7,24.5Hz,7H),3.55(s,2 H),3.09–2.96(m,4H),2.21(s,2H),1.79(s,2H),1.58(d,J=37.3Hz,8H),1.28(d,J=9.2Hz,71H),0.88(d,J=7.7Hz,6H). 13 C NMR(150MHz,CD3OD / CDCl3)δ174.99,100.19,75.07,72.35,71.31,70.71,70.18,69.37,67.68,62.21,52.59,50.88 ,43.47,36.84,32.88,32.32,30.72,30.12,29.95,29.75,29.53,28.72,27.06,26.30,23.96,23.06,14.28.ESI-MS calcd.for C 48 H 96 N2O 11 SNa[M+Na] + ,931.6627;found,931.6636.

[0070] Example 5: Synthesis of compound GCS-14

[0071]

[0072] Synthesis of compound 4e: The procedure was the same as that for synthesizing compound 4a (yield 98%). 1 H NMR (600MHz, CDCl3) δ4.15(t,J=6.1Hz,1H),3.66(s,3H),3.09(d,J=6.8Hz,2H),3.01–2.95(m,2H),2.30(t,J=7.5Hz,2H),1.78(d ,J=8.6Hz,2H),1.60(d,J=6.7Hz,3H),1.54(d,J=7.3Hz,2H),1.40(d,J=7.7Hz,3H),1.26(t,J=9.6Hz,32H),0.88(t,J=6.9Hz,3H). 13C NMR (150MHz, CDCl3) δ174.26,52.40,51.34,43.18,34.00,31.76,30.23,29.45,29.39 ,29.32,29.23,29.15,29.08,29.03,28.23,26.50,24.84,23.56,22.56,14.00.ESI-MS calcd.for C 25 H 52 NO4S[M+H] + ,462.36;found,462.51.

[0073] Synthesis of compound GCS-14: The procedure was the same as that for synthesizing compound GCS-10 (yield 38%). 1 H NMR (600MHz, CD3OD / CDCl3) δ4.90(s,1H),4.21(s,1H),3.90(d,J=25.4Hz,2H),3.78(d,J=50.8Hz,6H),3.58(s,2H),3.02(d, J=25.1Hz,4H),2.23(d,J=8.3Hz,2H),1.78(s,2H),1.58(d,J=40.1Hz,5H),1.47–1.21(m,63H),0.89(dd,J=7.5,3.6Hz,6H). 13 C NMR (150MHz, CD3OD / CDCl3) δ175.23,100.39,75.05,72.44,71.61,70.88,70.36,69.56,67.73,62.31,52.63,51.09,43.59,36.95,32 .82,32.52,32.45,30.87,30.30,30.23,30.15,30.07,29.94,29.83,29.71,28.88,27.24,26.53,26.46,24.13,23.21,14.32.ESI-MS calcd.forC 48 H 96 N2O 11 SNa[M+Na] + ,931.6627;found,931.6646.

[0074] Example 6: Synthesis of compound GCS-12-2

[0075]

[0076] Synthesis of compound 4f: The procedure was the same as that for synthesizing compound 4a (yield 65%).1 H NMR (400MHz, CDCl3) δ4.77–4.67(m,1H),3.60(d,J=5.0Hz,3H),2.99(dq,J=18.8,7 .1Hz,4H),2.24(q,J=7.0Hz,2H),1.51(dt,J=23.0,7.1Hz,4H),1.33–1.16(m,17H). 13 C NMR (150MHz, CDCl3) δ174.37,51.44,46.62,43.26,34.06,29.41,29.33,29.18,29.11,29.07,26.55,24.90,8.30.ESI-MS calcd.for C 15 H 31 NO4SNa[M+Na] + 344.19,found 344.26.

[0077] Synthesis of compound GCS-12-2: The procedure was the same as that for synthesizing compound GCS-10 (yield 35%). 1 H NMR (400MHz, CDCl3 / CD3OD) δ4.90(s,1H),4.21(d,J=5.1Hz,1H),3.90(dd,J=17.1,3.8Hz,2H),3.85–3.78(m,2H),3.72(m,J=16.7,9.5,3.8Hz,4 H),3.58(q,J=9.9,8.0Hz,2H),3.04(td,J=7.4,2.4Hz,4H),2.22(t,J=7 .6Hz,2H),1.68–1.52(m,6H),1.38–1.25(m,41H),0.89(t,J=5.6Hz,3H). 13 C NMR (150MHz, CDCl3 / CD3OD) δ174.10,99.28,74.18,71.47,70.40,69.80,69.30,68.45,66.78,61.30,49.99,45.88,42 .53,35.89,32.03,31.45,29.78,29.22,28.87,28.81,28.72,28.62,26.06,25.38,25.34,22.18,13.40,7.44.ESI-MS calcd.forC 38 H 76 N2O 11 SNa[M+Na]+791.5062; found 791.5094.

[0078] Example 7: Synthesis of compound GCS-12-4

[0079]

[0080] Synthesis of compound 4g: The procedure was the same as that for synthesizing compound 4a (yield 70%). 1 H NMR (600MHz, CDCl3) δ4.73(t,J=6.1Hz,1H),3.67(s,3H),3.16–3.05(m,2H),3.01(t,J=8.0Hz,2H),2.31(t,J=7.6Hz,2H),1.78( p,J=7.7Hz,2H),1.62(p,J=7.4Hz,2H),1.55(q,J=7.4Hz,2H),1.46(h,J=7.4Hz,2H),1.37–1.23(m,14H),0.96(t,J=7.4Hz,3H). 13 C NMR (150MHz, CDCl3) δ174.40,52.18,51.50,43.29,34.11,30.33,29.47,29.39,29.24,29.17,29.13,26.60,25.66,24.95,21.58,13.65.ESI-MS calcd.for C 17 H 35 NO4SNa[M+Na] + 372.22,found372.50.

[0081] Synthesis of compound GCS-12-4: The procedure was the same as that for synthesizing compound GCS-10 (yield 36%). 1 H NMR (400MHz, CDCl3 / CD3OD) δ4.91(s,1H),4.20(s,1H),3.91(d,J=19.5Hz,2H ),3.80(s,2H),3.78–3.65(m,4H),3.54(d,J=4.7Hz,2H),3.08–2.97(m,4H),2 .21(t,J=7.8Hz,2H),1.77(t,J=8.4Hz,2H),1.61(s,2H),1.55(s,2H),1.47( s,2H),1.28(d,J=10.5Hz,40H),0.97(t,J=7.4Hz,3H),0.89(t,J=6.8Hz,3H). 13C NMR (150MHz, CDCl3 / CD3OD) δ174.30,99.43,74.34,71.68,70.49,69.98,69.53,68.63,66.99,61.55,51.62,50.11,42.79,36.16,32 .21,31.67,30.02,29.45,29.40,29.25,29.22,29.20,29.10,29.06,28.92,26.35,25.61,25.28,22.41,21.27,13.69,13.18.ESI-MS calcd.for C 40 H 80 N2O 11 SNa[M+Na] + 819.5375, found 819.5351.

[0082] Example 8: Synthesis of compound GCS-12-5

[0083]

[0084] Synthesis of compound 4h: The procedure was the same as that for synthesizing compound 4a (yield 72%). 1 H NMR (400MHz, CDCl3) δ4.07(t,J=5.8Hz,1H),3.67(s,3H),3.10(q,J=6.8Hz,2H),3.04–2.95(m,2H),2.30(t,J=7.5Hz,2H),1.80 (p,J=7.7Hz,2H),1.64–1.59(m,2H),1.56(d,J=7.2Hz,2H),1.38(dd,J=14.1,8.7Hz,4H),1.27(s,14H),0.92(t,J=7.0Hz,3H). 13 C NMR (100MHz, CDCl3) δ52.39,51.44,43.25,34.07,30.42,30.30,29.45,29.37,29.21,29.14,29.10,24.92,23.33,22.21,13.77.ESI-MS calcd.for C 18 H 37 NO4SNa[M+Na] + 386.24, found 386.44.

[0085] Synthesis of compound GCS-12-5: The steps were the same as those for synthesizing compound GCS-10 (yield 43%). 1H NMR (400MHz, CDCl3 / CD3OD) δ4.91 (d, J=3.8Hz, 1H), 4.21 (s, 1H), 3.96–3.85 (m ,2H),3.85–3.78(m,2H),3.78–3.66(m,4H),3.56(d,J=2.8Hz,2H),3.08–2.96( m,4H),2.22(t,J=7.6Hz,2H),1.80(p,J=7.6Hz,2H),1.66(d,J=16.8Hz,2H),1 .57(dd,J=16.2,9.0Hz,4H),1.48–1.17(m,42H),0.91(dt,J=19.0,7.1Hz,6H). 13 C NMR (150MHz, CDCl3 / CD3OD) δ174.16,99.31,74.04,71.42,70.51,69.81,69.32,68.49,66.73,61.26,51.56,50.03,42.54,35.89,31.82,31 .45,29.94,29.82,29.30,29.27,29.17,29.07,29.03,29.01,28.91,2 8.88,28.85,28.73,26.16,25.41,22.76,21.71,13.31,12.98.ESI-MS calcd.forC 41 H 82 N2O 11 SNa[M+Na] + 833.5537, found 833.5527.

[0086] Example 9: Synthesis of compound GCS-12-6

[0087]

[0088] Synthesis of compound 4i: The procedure was the same as that for synthesizing compound 4a (yield 70%). 1 H NMR (600MHz, CDCl3) δ4.45(t,J=6.1Hz,1H),3.67(d,J=2.6Hz,3H),3.09(q,J=6.9Hz,2H),3.04–2.96(m,2H),2.30(t,J=7.5Hz,2H),1.79(d q,J=12.9,7.3,5.4Hz,2H),1.61(p,J=7.2Hz,2H),1.55(p,J=7.3Hz,2H),1.42(p,J=7.4Hz,2H),1.34–1.24(m,18H),0.89(d,J=6.9Hz,3H).13 C NMR (150MHz, CDCl3) δ174.47,52.63,51.57,43.39,34.19,31.38,30.43,29.54 ,29.46,29.31,29.23,29.21,28.08,26.67,25.02,23.74,22.45,14.06.ESI-MS calcd.for C 19 H 39 NO4SNa[M+Na] + 400.25,found 400.56.

[0089] Synthesis of compound GCS-12-6: The procedure was the same as that for synthesizing compound GCS-10 (yield 45%). 1 H NMR (400MHz, CDCl3 / CD3OD) δ4.81 (d, J=3.8Hz, 1H), 4.12 (q, J=5.1, 4.4Hz, 1H), 3.87–3.77 (m,2H),3.74–3.67(m,2H),3.62(td,J=10.0,9.2,4.4Hz,4H),3.47(d,J=8.7Hz,2H),2.92 (dt,J=15.5,7.5Hz,4H),2.13(t,J=7.6Hz,2H),1.70(q,J=7.8Hz,2H),1.60–1.49(m,4H), 1.45(q,J=7.1Hz,4H),1.35(t,J=7.5Hz,6H),1.20(d,J=11.9Hz,36H),0.83–0.78(m,6H). 13 C NMR (150MHz, CDCl3 / CD3OD) δ174.07,99.25,71.26,70.47,69.25,68.38,61.14,51.39,42.38,35.72,31.34,30.74 ,29.70,29.11,29.06,28.96,28.93,28.76,28.62,27.39,26.04,25.37,22.93,22.05,21.75,13.11,12.97.ESI-MS calcd.for C 42 H 84 N2O 11 SNa[M+Na] + 847.5688, found 847.5673.

[0090] Example 10: Synthesis of compound GCS-12-7

[0091]

[0092] Synthesis of compound 4j: The procedure was the same as that for synthesizing compound 4a (yield 74%). 1 H NMR (600MHz, CDCl3) δ4.30(t,J=6.2Hz,1H),3.67(d,J=4.9Hz,3H),3.09(q,J=6.9Hz,2H),3.03–2.97(m,2H),2.30(t,J=7.6Hz,2H),1 .80(q,J=7.8Hz,2H),1.62(p,J=7.4Hz,2H),1.55(p,J=7.2Hz,2H),1.41(t,J=7.5Hz,2H),1.35–1.25(m,20H),0.89(t,J=6.8Hz,3H). 13 C NMR (150MHz, CDCl3) δ174.41,52.49,51.50,43.30,34.12,31.53,30.35,29.48,29 .41,29.25,29.18,29.14,28.83,28.31,26.62,24.96,23.69,22.57,14.06.ESI-MS calcd.for C 20 H 41 NO4SNa[M+Na] + 414.26,found 414.49.

[0093] Synthesis of compound GCS-12-7: The procedure was the same as that for synthesizing compound GCS-10 (yield 38%). 1 H NMR (400MHz, CDCl3 / CD3OD) δ4.91 (d, J=2.7Hz, 1H), 4.24–4.17 (m, 1H), 3.96–3. 85(m,2H),3.85–3.78(m,2H),3.77–3.66(m,4H),3.56(s,2H),3.02(dt,J=17.6, 7.6Hz,4H),2.22(t,J=7.5Hz,2H),1.78(q,J=7.8Hz,2H),1.73–1.61(m,2H),1. 60–1.50(m,4H),1.49–1.41(m,2H),1.28(d,J=11.3Hz,44H),0.95–0.84(m,6H). 13C NMR (150MHz, CDCl3 / CD3OD) δ174.26,99.43,74.29,71.61,70.55,69.95,69.44,68.61,66.94,61.45,51.85,50.15,42.73,36 .11,32.12,31.60,31.20,29.97,29.39,29.17,29.04,28.88,28.48,27.95,26.31,25.56,23.23,22.33,22.19,13.50.ESI-MS calcd.for C 43 H 86 N2O 11 SNa[M+Na] + 861.5850, found 861.5841.

[0094] Example 11: Synthesis of compound GCS-12-8

[0095]

[0096] Synthesis of compound 4k: The procedure was the same as that for compound 4a (yield 68%). 1 H NMR (600MHz, CDCl3) δ4.60(t,J=6.1Hz,1H),3.63(s,3H),3.04(q,J=6.8Hz,2H),2.98–2.93(m,2H),2.27(t,J=7.6Hz,3H),1.75( p,J=7.9Hz,2H),1.58(p,J=7.3Hz,2H),1.51(p,J=7.2Hz,2H),1.37(p,J=7.2Hz,2H),1.29–1.20(m,22H),0.84(t,J=6.8Hz,3H). 13 C NMR (150MHz, CDCl3) δ174.42,52.53,51.41,43.32,34.14,31.78,30.37,29.97,29.73,29.51,29.43, 29.27,29.16,29.04,28.89,28.38,26.64,24.98,23.72,22.66,14.18.LC-MS(ESI)(m / z):calcd.for C 21 H 43 NO4SNa[M+Na] + 428.28, found 428.62.

[0097] Synthesis of compound GCS-12-8: The procedure was the same as that for synthesizing compound GCS-10 (yield 40%). 1 H NMR (600MHz, CDCl3 / CD3OD) δ4.91(s,1H),4.21(s,1H),3.93(s,1H),3.88(s,1H),3.81(s,2H),3.75(d,J=13.6Hz,3H),3.69(s,2H),3.56(s,2H ),3.02(d,J=27.1Hz,4H),2.22(s,2H),1.79(s,2H),1.68(s,2H),1.62( s,2H),1.55(s,2H),1.43(s,4H),1.29(d,J=19.9Hz,44H),0.89(s,6H). 13 C NMR (150MHz, CDCl3 / CD3OD) δ174.27,74.25,68.58,51.83,50.05,42.73,32.12,31.61,31.42,29.98,29.40,29.40, 29.21,29.06,28.89,28.78,28.68,28.00,26.31,25.56,23.23,22.35,22.27,13.56.LC-MS(ESI)(m / z):calcd.for C 44 H 88 N2O 11 SNa[M+Na] + 875.6001found875.5914.

[0098] Example 12: Synthesis of compound GCS-12-10

[0099]

[0100] Synthesis of compound 41: The procedure was the same as that for synthesizing compound 4a (yield 67%). 1 H NMR(600MHz, CDCl3) δ4.21(d,J=11.7Hz,1H),3.67(s,3H),3.10(q,J=6.8Hz,2H),3.03–2.97(m,2H),2.30(t,J=7.5Hz,2H),1.79(p, J=7.9Hz,2H),1.62(dd,J=13.1,6.2Hz,2H),1.55(t,J=7.3Hz,2H),1.41(t,J=7.5Hz,2H),1.32–1.24(m,26H),0.88(t,J=7.0Hz,3H). 13C NMR (150MHz, CDCl3) δ174.34,52.56,51.46,43.30,34.08,31.84,30.34,29.42,29.35,29.30, 29.25,29.20,29.11,28.30,26.56,24.92,23.68,22.66,14.10.LC-MS(ESI)(m / z):calcd.forC 23 H 47 NO4SNa[M+Na] + 456.31, found 456.68.

[0101] Synthesis of compound GCS-12-10: The procedure was the same as that for synthesizing compound GCS-10 (yield 36%). 1 H NMR (400MHz, CDCl3 / CD3OD) δ4.82(d,J=3.7Hz,1H),4.12(s,1H),3.88–3.77(m,2H),3.76–3.56(m,6H),3.46(s,2H),2.93(dt,J=21.1, 7.7Hz,4H),2.12(t,J=7.7Hz,2H),1.75–1.65(m,2H),1.59–1.43(m,4H),1.34(s,2H),1.19(d,J=10.0Hz,52H),0.80(t,J=6.6Hz,6H). 13 CNMR (150MHz, CDCl3 / CD3OD) δ174.23,99.39,74.23,71.54,70.50,69.44,68.57,66.84,61.43,51.76,50.07,42.68 ,36.05,32.08,29.94,29.35,29.13,28.99,28.78,26.27,25.53,23.18,22.29,13.49.LC-MS(ESI)(m / z):calcd.for C 46 H 92 N2O 11 SNa[M+Na] + 903.6314,found903.6208.

[0102] Example 13: Synthesis of Compound GCS-12a

[0103]

[0104] Synthesis of compound 4m: The procedure was the same as that for compound 4a (yield 70%). 1H NMR (400MHz, CDCl3) δ3.60(s,3H),3.07(t,J=7.5Hz,2H),2.83(t,J=8.0Hz,2H),2.78(s,3H),2.23(t,J=7.6Hz,2H),1 .71(q,J=8.1Hz,2H),1.53(dd,J=16.5,9.3Hz,4H),1.33(t,J=7.7Hz,2H),1.26–1.17(m,30H),0.81(t,J=6.6Hz,3H). 13 C NMR (150MHz, CDCl3) δ174.29,51.40,49.94,49.71,34.36,34.04,31.85,29.55,29.45,29.34,29. 27,29.18,29.07,28.45,28.02,26.42,24.88,23.16,22.63,14.08.LC-MS(ESI)(m / z):calcd.for C 26 H 53 NO4SNa[M+Na] + 498.36,found498.51.

[0105] Synthesis of compound GCS-12a: The procedure was the same as that for synthesizing compound GCS-10 (yield 35%). 1 H NMR (400MHz, CDCl3 / CD3OD) δ4.91 (d, J = 3.6Hz, 1H), 4.20 (s, 1H), 3.95 (s, 1H), 3.88(d,J=12.5Hz,1H),3.84–3.64(m,6H),3.57(s,2H),3.16(t,J=6.7Hz,2H), 3.01–2.89(m,2H),2.86(s,3H),2.21(t,J=6.9Hz,2H),1.77(d,J=7.7Hz,2H), 1.66–1.53(m,6H),1.42(d,J=6.9Hz,2H),1.26(s,54H),0.88(d,J=6.2Hz,6H). 13C NMR (100MHz, CDCl3 / CD3OD) δ174.34,99.37,74.24,71.68,70.43,69.94,69.55,68.59,67.00,61.51,36.18,33.97,32.08,31.61,29.42,29. 36,29.29,29.29,29.21,29.12,29.07,29.02,28.96,28.81,28.14,27.76,26.17,25.59,22.88,22.36,13.63.LC-MS(ESI)(m / z):calcd.for C 49 H 98 N2O 11 SNa[M+Na] + 945.6789,found945.6761.

[0106] Example 14: Synthesis of compound GCS-12b

[0107]

[0108] Synthesis of compound 4a-1: The procedure was the same as that for synthesizing compound 4a (yield 88%). 1 H NMR(600MHz, CDCl3) δ7.29(d,J=53.9Hz,6H),3.92(d,J=8.0Hz,1H),3.67(d,J=5.6Hz,3H) ,3.30(d,J=7.0Hz,2H),3.13(s,2H),3.00(s,2H),2.31(s,2H),1.44(s,2H),1.26(s,15H). 13 C NMR (150MHz, CDCl3) δ174.59,138.24,129.07,128.58,127.13,53.59,51. 67,43.50,34.29,30.38,30.21,29.65,29.57,29.43,29.32,26.77,25.13.

[0109] Synthesis of compound GCS-12b: The procedure was the same as that for synthesizing compound GCS-10 (yield 40%). 1H NMR (400MHz, CDCl3 / CD3OD) δ7.34(t,J=7.3Hz,2H),7.29–7.22(m,3H),4.91(d,J=3.8 Hz,1H),4.20(d,J=4.4Hz,1H),3.96–3.85(m,2H),3.85–3.66(m,6H),3.55(d,J=5.5H z,2H),3.40(s,1H),3.32–3.24(m,2H),3.14–3.07(m,2H),3.03(t,J=7.2Hz,2H),2.2 1(t,J=7.7Hz,2H),1.74–1.40(m,8H),1.28(d,J=9.2Hz,50H),0.89(t,J=6.7Hz,6H). 13 C NMR (150MHz, CDCl3 / CD3OD) δ137.64,128.16,127.71,126.19,99.19,99.19,71.27,70.39,69.16,68.35,6 1.11,52.74,44.04,42.42,31.30,29.61,29.08,29.02,28.73,26.00,25.26,22.02,21.94,21.37,13.15.

[0110] Example 15: Synthesis of compound GCS-12c

[0111]

[0112] Synthesis of compound 4a-2: The procedure was the same as that for synthesizing compound 4a (yield 90%). 1 H NMR (600MHz, CDCl3) δ7.07(d,J=3.2Hz,4H),3.87(d,J=6.0Hz,1H),3.60(s,3H),3.20(t,J=8.0Hz,2H),3.02(t,J=7.9Hz,2H),2.92( q,J=6.9Hz,2H),2.26(d,J=7.2Hz,3H),2.23(d,J=7.5Hz,2H),1.55(q,J=7.5Hz,2H),1.37(t,J=7.0Hz,2H),1.20(d,J=20.1Hz,14H). 13 C NMR (150MHz, CDCl3) δ174.52,136.80,134.95,129.70,128.39,53.53,51. 63,43.48,34.23,30.26,29.78,29.55,29.35,29.25,26.66,25.06,21.17.

[0113] Synthesis of compound GCS-12c: The procedure was the same as that for synthesizing compound GCS-10 (yield 35%). 1 H NMR (400MHz, CDCl3 / CD3OD) δ7.13 (s, 4H), 4.91 (d, J = 3.7Hz, 1H), 3.93 (d, J = 3.2Hz, 1H),3.89(dd,J=11.0,4.4Hz,1H),3.84–3.78(m,2H),3.72(dt,J=21.6,7.1Hz,3H) ,3.54(p,J=7.0Hz,2H),3.25(dd,J=10.5,6.1Hz,2H),3.10–2.97(m,4H),2.33(s,3 H),2.21(s,2H),1.73–1.45(m,6H),1.28(d,J=15.0Hz,39H),0.89(t,J=6.6Hz,3H). 13 C NMR (150MHz, CDCl3 / CD3OD) δ174.19,136.02,134.65,128.99,127.77,99.32,71.47,70.46,69.84,69.38,68.51, 66.77,61.34,52.99,50.02,42.62,35.97,31.92,31.51,29.80,29.30,29.01,26.21,25.48,22.24,20.25,13.43.

[0114] Example 16: Synthesis of compound GCS-12d

[0115]

[0116] Synthesis of compound 4a-3: The steps were the same as those for synthesizing compound 4a (yield 90%). 1 H NMR (400MHz, CDCl3) δ7.12(dd,J=8.5,5.4Hz,2H),6.99–6.89(m,2H),4.01(t,J=6.2Hz,1H),3.60(s,3H),3.22–3.14(m,2H ),3.00(dq,J=26.6,6.8,5.9Hz,4H),2.23(t,J=7.6Hz,2H),1.55(d,J=7.5Hz,3H),1.47–1.34(m,2H),1.27–1.14(m,14H). 13C NMR (150MHz, CDCl3) δ174.52,161.11,133.75,130.04,129.99,115.94,115.80,53 .79,51.63,43.47,34.22,30.36,29.54,29.47,29.41,29.33,29.23,26.67,25.05.

[0117] Synthesis of compound GCS-12d: The procedure was the same as that for synthesizing compound GCS-10 (yield 32%). 1 H NMR (400MHz, CDCl3 / CD3OD) δ7.20 (dd, J=8.3, 5.3Hz, 2H), 6.98 (t, J=8.6Hz, 2H), 4.87 (d, J=3.7Hz ,1H),4.17(q,J=4.8Hz,1H),3.91–3.81(m,2H),3.77(dt,J=9.3,5.0Hz,2H),3.68(ddt,J=17.1,10 .3,4.8Hz,4H),3.52(td,J=10.0,8.5,6.4Hz,2H),3.26–3.18(m,2H),3.02(ddd,J=14.2,10.4,5.9 Hz,4H),2.18(t,J=7.6Hz,2H),1.69–1.45(m,6H),1.25(d,J=11.3Hz,36H),0.85(t,J=6.6Hz,3H). 13 C NMR (151MHz, CDCl3 / CD3OD) δ174.42,129.76,115.59,115.44,99.48,71.83,70.07,61.69,53.31,48.97,48.83,48.6 8,48.54,48.40,48.26,48.11,42.91,42.91,36.24,31.76,30.09,29.55,29.21,28.92,26.45,25.71,22.51,13.78.

[0118] Example 17: Synthesis of compound GCS-12e

[0119]

[0120] Synthesis of compound 4a-4: The steps were the same as those for synthesizing compound 4a (yield 48%). 1H NMR (400MHz, CDCl3) δ7.38(d,J=1.8Hz,5H),4.24(d,J=1.7Hz,2H),4.19–4.08(m,1H),3.66(d,J=1.7Hz,3H),2.97(q,J=6 .8, 6.2Hz, 2H), 2.30 (td, J = 7.5, 1.7Hz, 2H), 1.60 (dd, J = 14.6, 7.0Hz, 2H), 1.45 (q, J = 7.0Hz, 2H), 1.26 (d, J = 13.6Hz, 15H). 13 C NMR (100MHz, CDCl3) δ174.41,130.63,129.54,128.87,128.76,58.65,51. 52,43.81,34.15,30.37,29.46,29.40,29.26,29.16,29.13,26.52,24.98.

[0121] Synthesis of Compound GCS-12e: 1) First, compound 5a-4 was synthesized using the same steps as for compound 5a. 2) The resulting acid chloride was dissolved in dry CHCl and added to a solution of compound 7a (1.0 equiv) in dry CHCl, and stirred at room temperature for 2 hours. The organic phase was then removed, and the reaction mixture was purified by column chromatography to obtain a solid intermediate. 3) The resulting intermediate was dissolved in a CHCl / MeOH mixture, DDQ (5.5 equiv) was added, and stirred at room temperature for 3 hours. The organic phase was then removed, and the reaction mixture was purified by column chromatography to obtain the intermediate after removal of the PMB protecting group. 4) The resulting intermediate was dissolved in a CHCl / MeOH mixture, TFA (40%) was added, and stirred at room temperature for 30 minutes. The organic phase was then removed, and the reaction mixture was purified by column chromatography to obtain compound GCS-12-2-4 (yield 40%). 1 H NMR (400MHz, CDCl3 / CD3OD) δ7.82–7.71(m,1H),7.51–7.31(m,5H),4.91(d,J=3.7Hz,1H),4.38–4.18(m,3H),3.96–3.67(m,8H),3.59(dt,J= 11.2, 6.8Hz, 2H), 2.91 (t, J = 7.0Hz, 2H), 2.23 (t, J = 7.6Hz, 2H), 1.55 (dt, J = 62.6, 7.9Hz, 6H), 1.31 (d, J = 17.1Hz, 35H), 0.90 (t, J = 6.6Hz, 3H). 13C NMR (100MHz, CDCl3 / CD3OD) δ174.51,130.62,129.79,128.40,128.26,99.73,74.28,71.68,71.03,70.19,69.70,68.87,66.98,61.56,58.1 4,50.42,43.29,36.14,31.96,31.81,30.24,29.57,29.36,29.23,29.04,26.40,25.81,25.76,22.51,13.50.LC-MS(ESI)(m / z):calcd.for C 43 H 79 N2O 11 S[M+H] + 831.5399, found 831.5394.

[0122] Example 18: Synthesis of compound GCS-12f

[0123]

[0124] Synthesis of compound 4a-5: The procedure was the same as that for compound 4a (yield 45%). 1H NMR (400 MHz, CDCl3) δ 7.27 (d, J = 7.4 Hz, 2H), 7.19 (d, J = 7.8 Hz, 2H), 4.20 (s, 2H), 4.08 (d, J = 6.1 Hz, 1H), 3.66 (s, 3H), 2.98 (q, J = 6.8 Hz, 2H), 2.36 (s, 3H), 2.30 (t, J = 7.5 Hz, 2H), 1.62 (p, J = 6.9 Hz, 3H), 1.46 (t, J = 7.2 Hz, 2H), 1.26 (d, J = 12.8 Hz, 14H). 13C NMR (100MHz, CDCl3) δ174.42,138.70,130.47,129.58,126.46,58.26,51. 53,43.79,34.16,30.37,29.48,29.42,29.28,29.17,26.54,24.99,21.28.

[0125] Synthesis of compound GCS-12f: The procedure was the same as that for synthesizing compound GCS-12e (yield 46%). 1H NMR (400 MHz, CDCl3 / CD3OD) δ7.60 (s, 1H), 7.34–7.13 (m, 4H), 4.91 (d, J = 3.8 Hz, 1H), 4.22 (d, J = 9.3 Hz, 3H), 4.01–3.49 (m, 10H), 2.91 (t, J = 7.2 Hz, 2H), 2.36 (s, 3H), 2.22 (t, J = 7.7 Hz, 2H), 1.54 (d, J = 63.8 Hz, 6H), 1.29 (d, J = 12.8 Hz, 36H), 0.89 (t, J = 6.6 Hz, 3H). 13C NMR (100MHz, CDCl3 / CD3OD) δ174.54,138.33,130.49,129.20,126.50,99.6 9,74.45,71.81,70.84,70.21,69.76,68.87,67.10,61.68,58.02,50.39,4 9.01,43.41,36.28,32.25,31.84,30.27,29.62,29.56,29.44,29.39,29.2 7,29.08,26.44,25.82,22.56,20.68,13.70.LC-MS(ESI)(m / z):calcd.for C44H81N2O11S[M+H]+845.5556,found 845.5550.

[0126] Example 19: Synthesis of compound GCS-12g

[0127]

[0128] Synthesis of compound 4a-6: The steps were the same as those for synthesizing compound 4a (yield 38%). 1 H NMR (400MHz, CDCl3) δ7.47–7.35(m,2H),7.17–7.04(m,2H),4.23(s,2H),3.69(s,3H),3.00(q,J=6.8H z,2H),2.33(t,J=7.5Hz,2H),1.64(dd,J=10.3,4.9Hz,2H),1.50(t,J=7.1Hz,2H),1.37–1.24(m,15H). 13C NMR (100MHz, CDCl3) δ174.41,164.25,161.78,132.41,132.33,125.40,125.37,116.01,11 5.79,57.91,51.52,43.82,34.14,30.41,29.45,29.39,29.25,29.15,29.13,26.51,24.97.

[0129] Synthesis of compound GCS-12g: The procedure was the same as that for synthesizing compound GCS-12e (yield 42%). 1 H NMR (400MHz, CDCl3 / CD3OD) δ7.74(d,J=2.3Hz,1H),7.43(ddd,J=8.4,5.2,2.2Hz,2H),7.10(td,J=8.8,2.4Hz,2H),4.90(t,J=3.1Hz ,1H),4.27(d,J=2.3Hz,3H),3.98–3.66(m,8H),3.59(d,J=6.6Hz,2H),2.98–2.89(m,2H),2.23(t,J=7.4Hz,2H),0.95–0.87(m,3H). 13 CNMR (100MHz, CDCl3 / CD3OD) δ174.54,164.15,161.69,132.53,132.45,125.95,115. 30,115.09,99.74,74.25,71.68,71.09,70.18,69.71,68.88,67.01,61.53,57.15,50 .45,43.23,36.11,31.89,31.79,30.26,29.61,29.56,29.51,29.40,29.35,29.26,29 .21,29.17,29.04,26.39,25.81,25.75,22.49,13.45.LC-MS(ESI)(m / z):calcd.forC 43 H 78 FN2O 11 S[M+H] + 849.5305,found849.5298.

[0130] Example 20: Synthesis of compound GCS2N-11-12

[0131]

[0132] Synthesis of compound GCS2N-11-12: Compound 6a (1.0 equiv) was dissolved in CHCl. ​​Compound 6b-1 (1.5 equiv) and DIPEA (1.5 equiv) were added at room temperature under argon, and the mixture was refluxed at 45°C with stirring for 4 hours. The organic solvent was removed, and the reaction mixture was purified by column chromatography to obtain a solid intermediate. The obtained intermediate was dissolved in a CHCl / MeOH mixture, and Pd(OH) / C (C, 20% wt) was added. The mixture was stirred under a hydrogen atmosphere at room temperature for 12 hours. The resulting mixture was filtered through celite and concentrated to obtain the crude product, which was then purified by column chromatography to obtain compound GCS2N-11-12 (yield 40%). 1 H NMR (400MHz, CDCl3 / CD3OD) δ4.91(d,J=3.8Hz,1H),4.20(d,J=4.4Hz,1H),3.94(d,J=3.2Hz,1H),3.88(d,J=4.6Hz,1H),3.80(q,J=4.3Hz,2H),3.78– 3.66(m,4H),3.57–3.52(m,2H),2.98(t,J=7.2Hz,4H),2.21(t,J=7.6Hz,2 H), 1.55 (t, J = 7.1Hz, 6H), 1.29 (d, J = 14.0Hz, 62H), 0.89 (t, J = 6.6Hz, 7H). 13 C NMR (100MHz, CDCl3 / CD3OD) δ174.43,99.59,71.89,70.65,70.15,69.67,68.80,67.20,61.71,50.29,42.83,36.29,32 .46,31.79,29.67,29.58,29.52,29.50,29.47,29.43,29.29,29.22,29.15,29.04,26.70,26.62,25.69,22.54,13.81.

[0133] Example 21: Synthesis of compound GCS2N-12-11

[0134]

[0135] Synthesis of compound GCS2N-12-11: The steps were the same as those for synthesizing compound GCS2N-11-12 (yield 35%). 1HNMR (400MHz, CDCl3 / CD3OD) δ4.91(d,J=3.8Hz,1H),4.20(d,J=4.5Hz,1H),3.94(d,J=3. 2Hz,1H),3.89(dd,J=10.7,4.7Hz,1H),3.81(d,J=4.3Hz,1H),3.79(d,J=3.9Hz,1H),3.78 –3.73(m,2H),3.72–3.66(m,2H),3.55(d,J=5.4Hz,2H),2.97(t,J=7.2Hz,4H),2.21(t,J =7.7Hz, 2H), 1.66 (dd, J = 21.2, 11.8Hz, 2H), 1.55 (t, J = 7.1Hz, 5H), 0.89 (t, J = 6.6Hz, 6H). 13 C NMR (100MHz, CDCl3 / CD3OD) δ174.51,99.67,74.62,71.93,70.75,70.22,69.74,68.88,67.25,61.76,50.37,42.89 ,36.37,32.47,31.86,29.73,29.65,29.59,29.54,29.47,29.38,29.29,29.21,29.17,26.77,25.80,22.60,13.85.

[0136] Example 22: Synthesis of compound GCS2N-11-5

[0137]

[0138] Synthesis of compound GCS2N-11-5: The steps were the same as those for synthesizing compound GCS2N-11-12 (yield 35%). 1 H NMR (400MHz, CDCl3 / CD3OD) δ4.91(d,J=3.8Hz,1H),4.21(s,1H),3.94(d,J=3.2Hz,1H),3.89(dd,J=10.8,4.7Hz,1H),3.83–3.66(m,7H),3.58–3 .51(m,2H),2.97(t,J=7.2Hz,4H),2.21(t,J=7.6Hz,2H),1.57(dd,J=20.7,12.5Hz,9H),1.28(d,J=14.4Hz,53H),0.89(dd,J=12.9,6.8Hz,7H). 13C NMR (101MHz, CDCl3 / CD3OD) δ170.22,98.53,71.82,70.85,70.26,69.74,68.98,67.34,62.10,51.25,50.07,50.04,49.86 ,49.84,43.98,37.77,33.48,31.35,31.29,31.16,31.11,31.02,30.87,30.64,28.54,27.66,24.59,24.24,16.17,16.04.

[0139] Example 23: Synthesis of compound GCS2N-11-6

[0140]

[0141] Synthesis of compound GCS2N-11-6: The steps were the same as those for synthesizing compound GCS2N-11-12 (yield 41%). 1 H NMR (400MHz, CDCl3 / CD3OD) δ4.91(d,J=3.8Hz,1H),4.21(d,J=4.4Hz,1H),3.94(d,J=3.3Hz,1H),3.88(s,1H),3.83–3.76(m,3H),3.7 6–3.66(m,4H),3.57–3.51(m,2H),2.98(t,J=7.2Hz,4H),2.21(t,J=7.6Hz,2H),1.73–1.50(m,9H),1.32(s,47H),0.93–0.86(m,6H). 13 CNMR(100MHz,Methanol-d4)δ174.45,99.67,74.68,71.91,70.73,70.21,69.69,68.87,67.26,61.75,50.36,42.86,36.33,32.52 ,31.82,31.35,29.69,29.64,29.60,29.55,29.40,29.33,29.25,29.18,29.06,26.65,26.38,25.72,22.56,22.43,13.81,13.71.

[0142] Example 24: Synthesis of compound GCS2N-11-7

[0143]

[0144] Synthesis of compound GCS2N-11-7: The steps were the same as those for synthesizing compound GCS2N-11-12 (yield 36%).1 H NMR (400MHz, Methanol-d4) δ4.82(d,J=3.8Hz,1H),4.11(d,J=4.5Hz,1H),3.85(d,J=3.1Hz,1H),3.80(dd,J=10.8,4.5Hz,1H),3.75–3.57(m,6H),3.46( d,J=6.3Hz,2H),2.88(t,J=7.2Hz,4H),2.13(t,J=7.7Hz,2H),1.63–1.51(m, 3H), 1.46 (p, J=7.7, 7.2Hz, 6H), 1.19 (d, J=15.2Hz, 54H), 0.84–0.74 (m, 9H). 13 C NMR (100MHz, CDCl3 / CD3OD) δ174.53,99.69,74.56,71.87,70.79,70.20,69.73,68.87,67.17,61.72,42.84 ,36.33,32.42,31.87,31.71,29.65,29.60,29.46,29.31,29.14,28.89,26.73,25.81,22.60,22.52,13.82.

[0145] Example 25: Synthesis of compound GCS2N-12-4

[0146]

[0147] Synthesis of compound GCS2N-12-4: The steps were the same as those for synthesizing compound GCS2N-11-12 (yield 37%). 1 H NMR (400MHz, CDCl3 / CD3OD) δ4.91(d,J=3.2Hz,1H),4.20(s,1H),3.94(s,1H),3.88(t,J=6.9Hz,1H),3.76(dt,J=24.4,9.5Hz,6H),3.54(d,J= 4.9Hz,2H),2.98(dt,J=7.5,4.1Hz,4H),2.27–2.16(m,2H),1.73–1.48(m,9H),1.46–1.19(m,50H),0.95(dd,J=7.6,2.0Hz,3H),0.89(s,4H). 13C NMR (100MHz, CDCl3 / CD3OD) δ174.24,99.45,74.35,71.61,70.56,69.97,69.45,68.63,66.95,61.46,50.14,42 .55,42.24,36.05,31.55,31.23,29.37,29.33,29.27,29.14,28.98,26.43,25.50,22.28,19.56,13.46,13.05.

[0148] Example 26: Synthesis of compound GCS2N-12-5

[0149]

[0150] Synthesis of compound GCS2N-12-5: The steps were the same as those for synthesizing compound GCS2N-11-12 (yield 45%). 1 H NMR (400MHz, CDCl3 / CD3OD) δ4.91(t,J=2.7Hz,1H),4.20(d,J=4.2Hz,1H),3.94(s,1H),3.89(dd,J=11.3,4.5Hz,1H),3.74(ddt,J=24.9,13.9,7.9 Hz, 6H), 3.54 (d, J = 4.8Hz, 2H), 2.98 (t, J = 7.3Hz, 4H), 2.21 (t, J = 7.8Hz, 2 H),1.73–1.50(m,9H),1.39–1.21(m,46H),0.90(dt,J=13.0,6.7Hz,7H). 13 C NMR (100MHz, CDCl3 / CD3OD) δ174.50,99.69,74.64,71.90,70.77,70.22,69.70,68.88,67.23,61.75,50.38,42.84,36.36,31 .84,29.71,29.66,29.63,29.57,29.43,29.39,29.36,29.27,29.23,29.13,28.88,26.70,25.78,22.58,22.20,13.82,13.68.

[0151] Example 27: Synthesis of compound GCS2N-12-6

[0152]

[0153] Synthesis of compound GCS2N-12-6: The steps were the same as those for synthesizing compound GCS2N-11-12 (yield 35%). 1H NMR (400MHz, CDCl3 / CD3OD) δ4.91(d,J=3.8Hz,1H),4.20(d,J=4.5Hz,1H),3.94(d,J=3.2Hz,1H),3.89(dd,J=10.8,4.7Hz,1H),3.84–3.66(m,7H ),3.55(d,J=5.3Hz,2H),2.98(t,J=7.2Hz,4H),2.21(t,J=7.7Hz,2H),1.56(dd,J=19.3,12.2Hz,9H),1.42–1.21(m,50H),0.89(q,J=6.6Hz,7H). 13 C NMR (100MHz, CDCl3 / CD3OD) δ173.18,138.85,138.75,138.69,138.61,128.67,128.60,128.57, 128.55,128.34,128.15,128.07,127.95,127.83,126.54,101.24,99.78,79.95,79.70,75.90,7 4.54,74.07,73.54,72.09,71.91,69.63,63.13,50.53,43.46,32.16,31.59,30.04,29.96,29.93,29.91,29.81,29.77,29.61,29.38,26.92,26.62,26.06,25.91,22.93,22.76,14.38,14.24.

[0154] Example 28: Synthesis of compound GCS-10m

[0155]

[0156] Synthesis of compound GCS-10m: The procedure was the same as that for synthesizing compound GCS-12e (yield 28%). 1H NMR (400MHz, CDCl3 / CD3OD) δ6.57(dd,J=16.6,10.0Hz,1H),6.18(d,J=16.6Hz,1H),5.96(d,J=10.0Hz, 1H),4.91(d,J=3.7Hz,1H),4.22(d,J=5.2Hz,1H),3.94(d,J=3.2Hz,1H),3.89(dd,J=10.8,4.6Hz,1H), 3.85–3.78(m,2H),3.75(t,J=5.4Hz,3H),3.69(dd,J=11.0,4.7Hz,1H),3.57(d,J=3.9Hz,2H),2.94(t, J=7.1Hz,2H),2.22(t,J=7.6Hz,2H),1.74–1.47(m,7H),1.30(d,J=19.3Hz,40H),0.89(t,J=6.6Hz,4H). 13 C NMR (100MHz, CDCl3 / CD3OD) δ174.55,136.09,125.53,99.69,77.89,77.57,7 7.25,74.44,71.82,70.84,70.19,69.77,68.85,67.09,65.80,61.65,50.39 ,42.75,36.20,32.23,31.81,29.70,29.66,29.58,29.52,29.23,29.19,29. 12,28.98,26.46,25.75,22.52,14.56,13.62.LC-MS(ESI)(m / z):calcd.for C 35 H 70 ClN2O 11 S[M+H] + 761.4384, found 761.4379.

[0157] Example 29: Synthesis of compound GCS-11m

[0158]

[0159] Synthesis of compound GCS-11m: The procedure was the same as that for synthesizing compound GCS-12e (yield 30%). 1H NMR (400MHz, CDCl3 / CD3OD) δ6.55 (dd, J=16.4, 10.0Hz, 1H), 6.19 (d, J=16.6Hz, 1H), 5.96(d,J=9.7Hz,1H),4.91(d,J=3.8Hz,1H),4.21(d,J=4.8Hz,1H),3.99–3.86(m,2H ),3.86–3.66(m,6H),3.56(d,J=5.7Hz,2H),3.39(s,1H),2.95(t,J=7.1Hz,2H),2.22 (t,J=7.6Hz,2H),1.73–1.48(m,7H),1.29(d,J=11.9Hz,44H),0.89(t,J=6.6Hz,4H). 13 C NMR (100MHz, CDCl3 / CD3OD) δ174.53,135.98,125.81,99.67,77.73,77.41,77.09,74.59,71.90,70.74,70.22,69.76,68.87,67.18,50.35 ,42.83,36.33,32.45,31.86,29.75,29.64,29.59,29.36,29.29,29.24,29.05,26.53,25.79,22.59,13.82.LC-MS(ESI)(m / z):calcd.forC 36 H 72 ClN2O 11 S[M+H] + 775.4540, found 775.4588.

[0160] Example 30: Synthesis of compound GCS-12m

[0161]

[0162] Synthesis of compound GCS-12m: The procedure was the same as that for synthesizing compound GCS-12e (yield 32%). 1H NMR (400MHz, CDCl3 / CD3OD) δ6.55 (dd, J=16.6, 10.0Hz, 1H), 6.19 (d, J=16.6Hz, 1H), 5. 95(d,J=10.0Hz,1H),4.91(d,J=3.8Hz,1H),4.21(t,J=4.6Hz,1H),4.02–3.85(m,2H), 3.85–3.66(m,6H),3.55(dd,J=5.7,2.5Hz,2H),2.95(t,J=7.2Hz,2H),2.21(t,J=7.7H z,2H),1.59(ddt,J=34.8,14.3,8.3Hz,6H),1.41–1.19(m,40H),0.89(t,J=6.7Hz,3H). 13 C NMR (100MHz, CDCl3 / CD3OD) δ174.65,162.60,162.24,125.92,118.18,115.27,77.70,77.39,77.06,72.03,70.51,69.94,67.17,6 1.69,42.86,36.30,32.37,31.86,29.65,29.59,29.37,29.29,29.07,26.54,25.82,22.60,13.83.LC-MS(ESI)(m / z):calcd.forC 37 H 74 ClN2O 11 S[M+H] + 789.4697,found 789.4689.

[0163] Example 31: Synthesis of compound GCS-10n

[0164]

[0165] Synthesis of compound GCS-10n: The procedure was the same as that for synthesizing compound GCS-12e (yield 29%). 1H NMR (400MHz, CDCl3 / CD3OD) δ4.91(d,J=3.7Hz,1H),4.57(s,2H),4.21(q,J=4.8Hz,1H),3.94 (d,J=3.1Hz,1H),3.89(dd,J=10.8,4.7Hz,1H),3.84–3.78(m,2H),3.72(dt,J=20.5,6.1Hz,4 H),3.55(dd,J=6.0,2.8Hz,2H),3.35(p,J=1.6Hz,4H),3.14(t,J=7.1Hz,2H),2.22(t,J=7.6 Hz, 2H), 1.61 (ddt, J=21.7, 14.6, 8.6Hz, 6H), 1.30 (d, J=22.5Hz, 42H), 0.89 (t, J=6.6Hz, 4H). 13 C NMR (100MHz, CDCl3 / CD3OD) δ174.53,99.69,77.80,77.48,77.15,74.57,71.85,70.81,70.22,69.73,68.87,67.13,61.70,54.94,50.38, 43.52,36.27,32.40,31.85,30.13,29.62,29.57,29.27,29.20,29.13,28.97,26.33,25.75,22.57,13.75.LC-MS(ESI)(m / z):calcd.for C 36 H 71 N2O 11 S[M+H] + 738.4700, found 738.4723.

[0166] Example 32: Synthesis of compound GCS-11n

[0167]

[0168] Synthesis of compound GCS-11n: The procedure was the same as that for synthesizing compound GCS-12e (yield 40%). 1H NMR (400MHz, CDCl3 / CD3OD) δ4.91 (d, J=3.6Hz, 1H), 4.58 (s, 2H), 4.21 (q, J=4. 8Hz,1H),3.96–3.86(m,2H),3.81(q,J=5.1Hz,2H),3.77–3.66(m,3H),3.57(q ,J=6.1,5.5Hz,2H),3.35(p,J=1.6Hz,5H),3.14(t,J=7.1Hz,2H),2.22(t,J=7 .6Hz,2H),1.72–1.52(m,6H),1.29(d,J=16.6Hz,47H),0.89(t,J=6.6Hz,4H). 13 C NMR (100MHz, CDCl3 / CD3OD) δ174.54,99.71,77.84,77.52,77.20,70.88,70.21,54.91,50.40,43.51,36.27, 31.83,30.16,29.61,29.55,29.26,29.00,26.71,26.37,25.78,22.55,13.69.LC-MS(ESI)(m / z):calcd.for C 37 H 73 N2O 11 S[M+H] + 753.4930, found 753.4943.

[0169] Example 33: Synthesis of compound GCS-12n

[0170]

[0171] Synthesis of compound GCS-12n: The procedure was the same as that for synthesizing compound GCS-12e (yield 33%). 1 H NMR (400MHz, CDCl3 / CD3OD) δ4.91(d,J=3.8Hz,1H),4.57(s,3H),4.21(q,J=4.8Hz,1H ),3.94(d,J=3.2Hz,1H),3.89(dd,J=10.7,4.7Hz,1H),3.84–3.78(m,2H),3.77–3.67( m,4H),3.59–3.52(m,2H),3.35(p,J=1.6Hz,4H),3.14(t,J=7.2Hz,2H),2.22(t,J=7.6 Hz,2H),1.60(dp,J=22.2,8.4,7.5Hz,7H),1.41–1.24(m,43H),0.89(t,J=6.7Hz,4H).13 C NMR (100MHz, CDCl3 / CD3OD) δ174.54,99.71,77.77,77.45,77.13,74.57,71.85,70.85,70.23,69.70,68.88,67.16,61.71,54.95,50.41, 43.57,36.34,32.39,31.85,30.18,29.63,29.57,29.42,29.37,29.28,29.05,26.41,25.80,22.58,13.77.LC-MS(ESI)(m / z):calcd.for C 38 H 75 N2O 11 S[M+H] + 767.5086, found 767.5124.

[0172] Test Case

[0173] (1) Administration to mice

[0174] Different αGalCer analogs were prepared in liposome form: αGalCer and its analogs were mixed with DSPC and cholesterol in a molar ratio of 1:5:4 and dissolved in 2 mL of dichloromethane / methanol (volume ratio 1 / 1). The solvent was removed by rotary evaporation, leaving a thin layer of lipid film on the wall of the bottle; 2 mL of PBS was added and shaken at 55°C under argon protection for 1 hour; finally, the resulting emulsion was sonicated for 15 minutes to obtain the desired liposomes.

[0175] The in vivo bioactivity of the resulting αGalCer analogs was evaluated in a mouse model. Intraperitoneal injection was used, with a dose of 2.33 nmol per glycolipid per mouse. Blood was collected by tail puncture at 2 and 24 hours. Serum was separated by centrifugation.

[0176] (2) Determination of cytokine levels in serum of administered mice

[0177] Cytokine levels in mouse serum were measured using ELISA kits (IL-4 and IFN-γ, BD Biosciences) according to the manufacturer's instructions. Capture antibody (250-fold dilution) was diluted in coating buffer (7.13 g NaHCO₃, 1.59 g Na₂CO₃, qs to 1.0 L; pH 9.5) and added to a 96-well plate at a volume of 100 μL per well. The plates were sealed with plastic wrap and incubated overnight at 4°C. The plates were then washed with 250 μL of PBST (0.05% Tween-20 in PBS). After the final wash, the plates were inverted and blotted with absorbent paper to remove any residual buffer. 200 μL of 10% FBS in PBS was added to each well, sealed with plastic wrap, and incubated at room temperature for 1 hour. The wash step was repeated. The corresponding diluted standards and serum samples (10% FBS in PBS as the diluent) were then added to the 96-well plate in duplicate. After sealing with plastic wrap, incubate at room temperature with shaking for 2 hours; repeat the washing step; dilute the detection antibody and enzyme-labeled secondary antibody 250 times with 10% FBS PBS solution, add 100 μL to each well, seal with plastic wrap, and incubate at room temperature with shaking for 1 hour; repeat the washing step; add 100 μL TMB color development solution (solution A + solution B) to each well, and develop at room temperature in the dark for 30 minutes; add 50 μL 1M H2SO4 solution to each well to stop the color development, and use a microplate reader to measure the absorbance at 450 nm. The results are as follows Figure 1-Figure 5 shown.

[0178] from Figure 1 It can be seen that 2 hours and 24 hours after administration to mice, GCS-12 caused a significant increase in IL-4 secretion (4.7 times) compared with αGalCer, and also triggered a 7.0-fold increase in IFN-γ secretion level, indicating that GCS-12 can significantly enhance the Th1 / 2 balanced cytokine response, and its activity is significantly improved compared with αGalCer; for Th1-biased cytokine response, GCS-11 and GCS-13 triggered a 6.1- and 4.0-fold increase in IFN-γ secretion level, respectively, while the IL-4 secretion level was comparable to αGalCer, so GCS-11 and GCS-13 behaved as potent Th1-biased immune agonists. Figure 2 It can be seen that GCS-12-5, GCS-12-6 and GCS-12-7 significantly increased the secretion level of IL-4 (the secretion level of IFN-γ was lower) compared with αGalCer. Among them, GCS-12-6 is almost the most active Th2-biased glycolipid molecule (compared to the increase in the activity of αGalCer in vivo) among the reported αGalCer analogs. Figure 3It can be seen that GCS-12b, GCS-12c, GCS-12d, GCS-12e, GCS-12f and GCS-12g are all Th2-biased glycolipid molecules, and their activity is significantly improved compared to αGalCer. Figure 4 It can be seen that GCS2N-11-12 can induce a Th1-biased cytokine response, while GCS2N-12-11 can induce a Th1 / 2 balanced response; GCS2N-11-5, GCS2N-11-6, GCS2N-11-7, GCS2N-12-4, GCS2N-12-5 and GC2N-12-6 are all Th2-biased glycolipid molecules, and their activity is improved to varying degrees compared to αGalCer. Figure 5 It can be seen that although the activity of GCS-10m, GCS-11m, GCS-12m, GCS-10n, GCS-11n and GCS-12n is not improved compared with αGalCer, they generally behave as Th2-biased glycolipid molecules.

[0179] In summary, the αGalCer analogs provided by the present invention produce different cytokine elicitation patterns compared to the classic NKT cell agonist αGalCer, including Th1-biased, Th2-biased, or Th1 / 2-balanced immune responses, and have varying degrees of enhanced immunostimulatory activity. These results indicate that the αGalCer analogs provided by the present invention modified with a sulfonamide group (or a portion thereof) can achieve the design of NKT cell agonists with different functions and significantly enhanced activity.

[0180] While various embodiments of the present invention have been described above, the above description is intended to be illustrative, not exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A natural killer T cell agonist with immunomodulatory activity containing a sulfonamide group or a portion thereof, which is a compound having a structure represented by formula (1) or a pharmaceutically acceptable salt thereof: Said Y is any group represented by formula (A) to (E): R1 is -H or C1-C 30 of hydrocarbon groups; X1, X2 and X3 are each independently C1-C 30 The hydrocarbon group contains 0-5 heteroatoms selected from N, O, F, Br and Cl; Z1 and Z2 are each independently -OR2, -SR3, -NHR4, -SO2R5, -[-OCH2CH2-]n1-OCH3, -F, -Br, -Cl, C1-C 30 A hydrocarbon group, an aryl group or a heteroaryl group, wherein R2 and R3 are each independently -H, C1-C 30 alkyl, aryl, heteroaryl or -CH2OSO3Na, R4 is R2, -COR2 or -CONHR2, R5 is -NHR2, C1-C 30 A hydrocarbon group, an aryl group, a heteroaryl group or -CH2OSO3Na, n1 is an integer from 0 to 20, and n2 is an integer from 0 to 10; W1 and W2 are each independently -CH2-, -NH-, -O- or -S-; W3 is -NHCO-, -NCH2CO-, -NHCONH-, -NHSO2-, -NHSO2NH-, -NHCS-, -O-, -S- or -OCO-; Z3 and Z4 are each independently -OH, -H, -F, -Br or -Cl.

2. The natural killer T cell agonist according to claim 1, wherein R1 is -H or C1-C 12 The alkyl group is preferably -H or a C1-C3 alkyl group.

3. The natural killer T cell agonist according to claim 1, wherein X1 is C1-C 18 A straight chain or branched alkyl group, an alkenyl group or an alkynyl group containing an unsaturated double bond or triple bond, preferably, X1 is C 10 -C 12 of straight-chain alkyl; X2 is C1-C 18 Alkyl, C2-C 18 Alkenyl or alkynyl containing unsaturated double or triple bonds, C6-C 18 Aryl, C7-C 18 Alkaryl or C7-C 18 and optionally substituted by C1-C4 alkyl, F, Br or Cl; preferably, X2 is C1-C 13 Straight or branched alkyl, C2-C 12 Alkenyl or alkynyl containing unsaturated double or triple bonds, C6-C 12 Aryl, C7-C 12 Alkaryl or C7-C 12 and optionally substituted with C1-C4 alkyl, F, Br or Cl; X3 is C6-C 18 A straight chain or branched alkyl group, an alkenyl group or an alkynyl group containing an unsaturated double bond or triple bond, preferably, X3 is C 10 -C 16 of alkyl.

4. The natural killer T cell agonist according to claim 1, wherein The agonist is at least one of the following compounds:

5. The natural killer T cell agonist according to claim 1, wherein The agonist is at least one of the following compounds: The natural killer T cell agonist according to claim 1, wherein The agonist is at least one of the following compounds:

7. A drug complex, characterized in that The drug complex contains the agonist according to any one of claims 1 to 6 as an active ingredient.

8. Use of the natural killer T cell agonist according to claim 4 in the preparation of an immunotherapy drug, wherein the immunotherapy drug is preferably a drug that enhances Th1 / 2 balanced cytokine response.

9. Use of the natural killer T cell agonist according to claim 5 in the preparation of an immunotherapeutic drug, wherein the immunotherapeutic drug is preferably a drug that induces a Th1-biased cytokine response.

10. Use of the natural killer T cell agonist according to claim 6 in the preparation of an immunotherapeutic drug, wherein the immunotherapeutic drug is preferably a drug that induces a Th2-biased cytokine response.

Images