Carboline-derived PAD4 inhibitor as well as preparation method and application thereof

Abstract

The invention provides a carboline-derived PAD4 inhibitor as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. On the basis of strategies of changing N-terminal electron cloud density and expanding a conjugated system, a chloroacetamidine skeleton is modified, a series of small molecule PAD4 inhibitors with remarkable in-vitro enzyme activity inhibition and TNBC cell killing ability are developed, and related anti-enzyme and anti-cancer cell activity and moderate preference for PAD4 enzyme are shown. The in-vitro anti-TNBC cell proliferation activity research shows that the in-vitro anti-TNBC cell proliferation activity and safety of the carboline-derived PAD4 inhibitor provided by the invention are superior to those of a lead compound 7, and meanwhile, the carboline-derived PAD4 inhibitor has improved pharmacokinetic characteristics.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a carboline-derived PAD4 inhibitor, a preparation method thereof, and an application thereof. Background Art

[0002] Breast cancer (BC) is one of the most common types of female cancers worldwide, and its incidence has still been increasing in recent years. Triple-negative breast cancer (TNBC) is a specific subtype characterized by negative expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), accounting for about 15-20% of all breast cancers. Due to the lack of relevant receptor markers, TNBC patients cannot benefit from endocrine therapy and HER2-targeted therapy. At the same time, due to the characteristics of TNBC such as strong invasiveness, poor prognosis, and high recurrence rate, the efficacy of standardized chemotherapy regimens such as anthracyclines, cisplatin, and paclitaxel in treating TNBC is limited. Therefore, there is an urgent need to find new potential targets and develop effective targeted inhibitors for treating TNBC.

[0003] Peptidylarginine deiminase 4 (PAD4) is a member of the calcium-dependent hydrolase PADs family and is overexpressed in a variety of malignant tumors. PAD4 can catalyze the citrullination of nuclear proteins, especially histones, converting positively charged arginine residues into neutrally charged citrulline residues, thereby weakening the interaction between histones and negatively charged DNA, resulting in chromatin depolymerization and nuclear membrane rupture, and ultimately the release of NETs by neutrophils. Recent studies have shown that TNBC cells with high expression of PAD4 can also release cancer extracellular chromatin network (CECN), thereby promoting cancer cell metastasis. Since only the degradation of NETs by DNase I does not affect circulating tumor cells, as well as the accompanying tissue damage and microthrombus formation, inhibiting endogenous PAD4 may be a potential targeted strategy for treating highly metastatic TNBC. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a carboline-derived PAD4 inhibitor, a preparation method thereof, and an application thereof. The carboline-derived PAD4 inhibitor provided by the present invention has good anti-TNBC cell proliferation activity and improved pharmacokinetics. It can induce a broad-spectrum anti-tumor phenotype in the tumor immune microenvironment by enhancing antigen presentation of immune cells and M1 macrophage polarization and reducing the proportion of inhibitory cells; it can also reshape the phenotype and function of neutrophils by inhibiting the PAD4-H3cit-NETs pathway to regulate the tumor microenvironment into an anti-tumor state, thereby effectively inhibiting the progression of triple-negative breast cancer.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a carbazole-derived PAD4 inhibitor having the structure shown in Formula I:

[0007]

[0008] In Formula I, R1 is one of;

[0009] R2 is one of;

[0010] X is one of.

[0011] The present invention provides a method for preparing the above-mentioned carbazole-derived PAD4 inhibitor, comprising the following steps:

[0012] Mix Z-Orn(Boc)-OH having the structure shown in Formula a, benzylamine, a condensing agent and an organic solvent, and carry out a first condensation reaction to obtain a compound having the structure shown in Formula b;

[0013] The compound having the structure shown in Formula b undergoes a first deprotection reaction to obtain a compound having the structure shown in c;

[0014]

[0015] Mix the compound having the structure shown in Formula c, the compound having the structure shown in Formula d, a condensing agent and an organic solvent, and carry out a second condensation reaction to obtain a compound having the structure shown in Formula e;

[0016]

[0017] The compound having the structure shown in Formula e undergoes a second deprotection reaction to obtain a compound having the structure shown in Formula f;

[0018]

[0019] Mix the compound having the structure shown in Formula f, 2-chloroacetyl iminoethyl ester and an organic solvent, and carry out a coupling reaction to obtain a carbazole-derived PAD4 inhibitor having the structure shown in Formula I;

[0020] In Formulas a and b, R3 is Boc or Cbz, R4 is Cbz or Boc, and R3 and R4 are different;

[0021] In Formulas c and e, R4 is Cbz or Boc.

[0022] Preferably, when the R1 is When it is one of the following, R3 is Cbz and R4 is Boc;

[0023] When the said R1 is one of the following, R3 is Boc and R4 is Cbz.

[0024] Preferably, when the said R1 is then

[0025] (1) When R2 is one of the following, R3 is Boc and R4 is Cbz;

[0026] (2) When R2 is one of the following, R3 is Cbz and R4 is Boc.

[0027] Preferably, when R1 is then, the preparation method of the compound with the structure shown in formula d includes the following steps:

[0028] The compound with the structure shown in formula g undergoes an esterification reaction with methanol to obtain a compound with the structure shown in formula h;

[0029]

[0030] The compound with the structure shown in formula h undergoes a cyclization reaction with the compound with the R2-CHO structure to obtain a compound with the structure shown in formula j;

[0031] The compound with the structure shown in formula j is mixed with DDQ and an organic solvent and undergoes a dehydrogenation reaction to obtain a compound with the structure shown in formula k;

[0032]

[0033] The compound with the structure shown in formula k undergoes hydrolysis and acidification reactions to obtain a compound with the structure shown in formula d.

[0034] Preferably, the condensing agent is DCC and HOBt, the temperature of the first condensation reaction and the second condensation reaction is room temperature, and the time is independently 6 - 10 h.

[0035] Preferably, when R3 is Cbz, the first deprotection reaction is carried out in the presence of a Pd / C catalyst and a hydrogen atmosphere; when R3 is Boc, the first deprotection reaction is carried out in the HCl / EA system;

[0036] When R4 is Boc, the second deprotection reaction is carried out in the HCl / EA system; when R4 is Cbz, the second deprotection reaction is carried out in the presence of a Pd / C catalyst under a hydrogen atmosphere.

[0037] Preferably, the coupling reaction is carried out under alkaline conditions, and the pH value of the alkaline conditions is 9-11.

[0038] The present invention provides the use of the above-mentioned carbazole-derived PAD4 inhibitor in the preparation of anti-tumor drugs.

[0039] Preferably, the anti-tumor drugs include anti-breast cancer drugs and / or anti-lung cancer drugs.

[0040] The present invention provides a carbazole-derived PAD4 inhibitor. Based on the strategy of changing the electron cloud density at the N-terminus and expanding the conjugated system, the chloroacetamidine skeleton is modified, and a series of small molecule PAD4 inhibitors with significant in vitro enzyme activity inhibition and TNBC cell killing ability are developed, showing relevant anti-enzyme and anti-cancer cell activities, and a moderate preference for the PAD4 enzyme. And the in vitro anti-TNBC cell proliferation activity study shows that the carbazole-derived PAD4 inhibitor provided by the present invention has better in vitro anti-TNBC cell proliferation activity and safety than the lead compound 7 (the structural formula is as follows), and at the same time has improved pharmacokinetic characteristics.

[0041]

[0042] The results of the examples show that the carbazole-derived PAD4 inhibitor provided by the present invention can inhibit the PAD4-H3cit-NETs pathway, reduce the proportion of immunosuppressive cells M-MDSCs and G-MDSCs in the tumor microenvironment, and up-regulate the proportion of DCs, especially cDCs, promote the polarization of anti-tumor M1 macrophages, so as to induce a broad-spectrum anti-tumor phenotype in the tumor immune microenvironment; it can also enhance the MHC-II antigen presentation of immune cells, by increasing the proportion of mature tumor-associated neutrophils MHC-II + TANs, while inhibiting the pro-tumor phenotypes PD-L1 + / MHC-II + TANs and MHC-II-TANs ratios to reshape the phenotype and function of neutrophils and transform the tumor immune microenvironment from a pro-tumor state to an anti-tumor state, thereby effectively inhibiting the progression of triple-negative breast cancer.

[0043] The present invention provides a preparation method of the above-mentioned carbazole-derived PAD4 inhibitor. This method is simple in operation, low in cost, and easy to realize industrial mass production. Description of the Drawings

[0044] Figure 1Structural design and molecular docking models of lead compounds 7, 11, and 28;

[0045] Figure 2 In vitro anti - proliferative and anti - migratory activities of compound 28;

[0046] Figure 3 Effects of compound 28 on histone citrullination and NET formation;

[0047] Figure 4 In vivo anti - tumor activity of compound 28 against the orthotopic 4T1 - luc xenograft model;

[0048] Figure 5 In vivo anti - metastatic activity of compound 28 against the orthotopic 4T1 - luc xenograft model;

[0049] Figure 6 Biocompatibility evaluation of compound 28 on the orthotopic 4T1 - luc xenograft model;

[0050] Figure 7 H&E staining of heart, liver, spleen, and kidney tissue sections of normal mice and 4T1 - luc tumor - bearing mice after different treatments;

[0051] Figure 8 Effects of compound 28 on the tumor immune microenvironment. Detailed implementation mode

[0052] The present invention provides a carboline - derived PAD4 inhibitor having the structure shown in Formula I:

[0053]

[0054] In Formula I, R1 is one of;

[0055] R2 is one of;

[0056] X is one of.

[0057] In the present invention, the dotted lines in R1, R2, and X represent the connection sites.

[0058] The present invention provides a preparation method of the above - mentioned carboline - derived PAD4 inhibitor, comprising the following steps:

[0059] Mix Z-Orn(Boc)-OH having the structure shown by formula a, benzylamine, a condensing agent and an organic solvent, and carry out a first condensation reaction to obtain a compound having the structure shown by formula b;

[0060] Carry out a first deprotection reaction on the compound having the structure shown by formula b to obtain a compound having the structure shown by c;

[0061]

[0062] Mix the compound having the structure shown by formula c, the compound having the structure shown by formula d, a condensing agent and an organic solvent, and carry out a second condensation reaction to obtain a compound having the structure shown by formula e;

[0063]

[0064] Carry out a second deprotection reaction on the compound having the structure shown by formula e to obtain a compound having the structure shown by formula f;

[0065]

[0066] Mix the compound having the structure shown by formula f, 2-chloroacetyl iminoethyl ester and an organic solvent, and carry out a coupling reaction to obtain a carbazole-derived PAD4 inhibitor having the structure shown by formula I;

[0067] In formula a and formula b, R3 is Boc or Cbz, R4 is Cbz or Boc, and R3 is different from R4;

[0068] In formula c and formula e, R4 is Cbz or Boc.

[0069] In the present invention, Z-Orn(Boc)-OH having the structure shown by formula a, benzylamine, a condensing agent and an organic solvent are mixed, and a first condensation reaction is carried out to obtain a compound having the structure shown by formula b. In the present invention, when the R1 is one of, R3 is preferably Cbz and R4 is preferably Boc;

[0070] When the R1 is one of, R3 is preferably Boc and R4 is preferably Cbz.

[0071] When the R1 is (1) when R2 is one of, R3 is Boc and R4 is Cbz;

[0072] (2) when R2 is When it is one of them, R3 is Cbz and R4 is Boc.

[0073] In the present invention, Z-Orn(Boc)-OH having the structure shown by formula a, benzylamine, a condensing agent and an organic solvent are mixed to carry out a first condensation reaction to obtain a compound having the structure shown by formula b. In the present invention, the molar amount of Z-Orn(Boc)-OH having the structure shown by formula a and the volume ratio of benzylamine are preferably 10 mmol:1.6 mL.

[0074] In the present invention, the condensing agent is preferably a DCC-HOBt system or an EDC-HOBt system. As a specific embodiment of the present invention, the molar ratio of Z-Orn(Boc)-OH having the structure shown by formula a to DCC and HOBt is preferably 10:12:12.

[0075] In the present invention, the organic solvent is preferably anhydrous THF.

[0076] In the present invention, the first condensation reaction is preferably carried out under alkaline conditions, and the alkaline conditions are preferably 8. In the present invention, the temperature of the first condensation reaction is preferably room temperature, and the time is preferably 6-10 h, more preferably 8 h. After the first condensation reaction, the present invention preferably performs post-treatment on the obtained first condensation reaction solution, and the post-treatment preferably includes the following steps:

[0077] Perform vacuum filtration and concentration on the first condensation reaction solution to obtain a residue;

[0078] Dissolve the residue with an organic solvent, wash, dry and column purify the obtained dissolved product to obtain a pure product of the compound having the structure shown by formula b.

[0079] In the present invention, the organic solvent is preferably ethyl acetate (EA). In the present invention, the detergents used for washing are preferably saturated NaHCO3 solution and saturated NaCl solution in sequence, and the drying is preferably anhydrous Na2SO4 drying. In the present invention, the eluent phase for column purification is preferably 50% ethyl acetate.

[0080] In the present invention, the compound having the structure shown by formula b undergoes a first deprotection reaction to obtain a compound having the structure shown by c. In the present invention, when R3 is Cbz, the first deprotection reaction is preferably carried out in the presence of a Pd / C catalyst and a hydrogen atmosphere. In the present invention, the mass of the Pd / C catalyst is preferably 10% of the mass of the compound having the structure shown by formula b. In the present invention, when R3 is Cbz, the temperature of the first deprotection reaction is preferably room temperature, and the time is preferably 4 h. After the first deprotection reaction, the present invention preferably performs vacuum filtration on the obtained first deprotection reaction solution and rotary evaporation to dryness.

[0081] In the present invention, when R3 is Boc, the first deprotection reaction is carried out in the HCl / EA system. In the present invention, the HCl / EA system is an ethyl acetate solution of HCl, and the concentration of HCl in the HCl / EA system is preferably 4 mol / L. In the present invention, when R3 is Boc, the temperature of the first deprotection reaction is preferably room temperature, and the time is preferably 4 h. After the first deprotection reaction, the present invention preferably evaporates the obtained first deprotection reaction solution to dryness under reduced pressure, and uses an organic solvent to redissolve and evaporate the obtained residue to dryness again. In the present invention, the organic solvent used for redissolution is preferably dry ethyl acetate and anhydrous diethyl ether.

[0082] The present invention mixes the compound having the structure shown in formula c, the compound having the structure shown in formula d, a condensing agent and an organic solvent, and carries out a second condensation reaction to obtain a compound having the structure shown in formula e. In the present invention, the condensing agent is preferably the DCC-HOBt system or the EDC-HOBt system; as a specific embodiment of the present invention, the molar ratio of the compound having the structure shown in formula c to EDC and HOBt is preferably 1:1:1.

[0083] In the present invention, the second condensation reaction is preferably carried out under basic conditions, and the basic conditions are preferably 8. In the present invention, the temperature of the second condensation reaction is preferably room temperature, the time is preferably 6 to 10 h, and more preferably 8 h. In the present invention, the post-treatment method of the second condensation reaction is similar to that of the first condensation reaction, and will not be elaborated here.

[0084] In the present invention, the compound having the structure shown in formula e undergoes a second deprotection reaction to obtain a compound having the structure shown in formula f. In the present invention, when R4 is Boc, the second deprotection reaction is carried out in the HCl / EA system; when R4 is Cbz, the second deprotection reaction is carried out in the presence of a Pd / C catalyst and a hydrogen atmosphere. In the present invention, the reaction conditions and post-treatment method of the second deprotection reaction are similar to those of the first deprotection reaction, and will not be elaborated here.

[0085] The present invention mixes the compound having the structure shown in formula f, 2-chloroacetyl iminoethyl ester and an organic solvent, and carries out a coupling reaction to obtain a carbazole-derived PAD4 inhibitor having the structure shown in formula I. In the present invention, the structural formula of the 2-chloroacetyl iminoethyl ester is preferably as follows:

[0086]

[0087] In the present invention, the molar ratio of the compound having the structure shown by formula f to ethyl 2-chloroacetimidate is preferably 1:5. In the present invention, the coupling reaction is carried out under alkaline conditions, and the pH value of the alkaline conditions is 9-11, more preferably 10. In the present invention, the temperature of the coupling reaction is preferably room temperature, and the time is preferably 12 h. After the coupling reaction, the present invention preferably performs column purification, removal of organic solvents and lyophilization on the obtained coupling reaction solution. In the present invention, the elution phase for column purification is preferably 30% CH3OH. In the present invention, the method for removing organic solvents is preferably rotary evaporation, and the drying method is preferably lyophilization.

[0088] In the present invention, when R1 is The preparation method of the compound having the structure shown by formula d includes the following steps:

[0089] The compound having the structure shown by formula g reacts with methanol in an esterification reaction to obtain a compound having the structure shown by formula h;

[0090]

[0091] The compound having the structure shown by formula h reacts with the compound having the structure of R2-CHO in a ring-forming reaction to obtain a compound having the structure shown by formula j;

[0092] The compound having the structure shown by formula j is mixed with DDQ and an organic solvent and undergoes a dehydrogenation reaction to obtain a compound having the structure shown by formula k;

[0093]

[0094] The compound having the structure shown by formula k undergoes hydrolysis and acidification reactions to obtain a compound having the structure shown by formula d.

[0095] In the present invention, the compound having the structure shown by formula g reacts with methanol in an esterification reaction to obtain a compound having the structure shown by formula h. In the present invention, the esterification reaction is preferably carried out in the presence of SOCl2; the temperature of the esterification reaction is preferably 0 °C, and the time is preferably 12 h.

[0096] After the esterification reaction, the present invention preferably performs reduced pressure concentration on the obtained esterification reaction solution to obtain a residue; the obtained residue is washed and dried. In the present invention, the reagents used for washing are preferably saturated NaHCO3 solution and saturated NaCl solution, and the drying is preferably rotary evaporation after drying with anhydrous Na2SO4.

[0097] In the present invention, a compound having the structure shown in formula h reacts with a compound having the structure of R2-CHO to undergo a cyclization reaction to obtain a compound having the structure shown in formula j. In the present invention, the cyclization reaction is preferably carried out in the presence of TFA. In the present invention, the temperature of the cyclization reaction is preferably room temperature, and the time is preferably 15 h.

[0098] After the cyclization reaction, the present invention preferably concentrates the obtained cyclization reaction solution under reduced pressure to obtain a residue; the obtained residue is redissolved with an organic solvent, and the obtained dissolved matter is washed, dried, and column-purified. In the present invention, the organic solvent used for redissolution is preferably CH2Cl2; the reagent used for washing is preferably saturated NaHCO3 solution and saturated NaCl solution, and the drying is preferably drying with anhydrous Na2SO4. In the present invention, the eluent phase for column purification is preferably 45% EA.

[0099] In the present invention, a compound having the structure shown in formula j is mixed with DDQ and an organic solvent to undergo a dehydrogenation reaction to obtain a compound having the structure shown in formula k. In the present invention, the DDQ is dichlorodicyanobenzoquinone. In the present invention, the molar ratio of the compound having the structure shown in formula j to DDQ is preferably 1:2. In the present invention, the organic solvent is preferably dichloroethane.

[0100] In the present invention, the temperature of the dehydrogenation reaction is preferably room temperature, and the time is preferably 4 h.

[0101] After the dehydrogenation reaction, the present invention preferably terminates the reaction with saturated aqueous NaHCO3 solution, separates the organic phase, and washes, dries, and column-purifies the obtained organic phase. In the present invention, the reagent used for washing is preferably saturated NaCl solution, and the drying is preferably drying with anhydrous Na2SO4. In the present invention, the eluent phase for column purification is preferably 4% CH3OH.

[0102] In the present invention, a compound having the structure shown in formula k undergoes hydrolysis and acidification reactions to obtain a compound having the structure shown in formula d. In the present invention, the hydrolysis is preferably carried out in an organic solvent, and the organic solvent is preferably CH3OH and / or CH2Cl2; in the present invention, the hydrolysis is preferably carried out in a basic environment, and the pH value of the basic environment is preferably 10. In the present invention, the temperature of the hydrolysis is preferably an ice bath, and the time is preferably 4 h.

[0103] In the present invention, the acidic reagent used for acidification is preferably KHSO4; in the present invention, the pH value of the acidification is preferably 2.

[0104] The present invention provides the use of the above-mentioned carbazole-derived PAD4 inhibitor in the preparation of anti-tumor drugs.

[0105] In the present invention, the anti-tumor drug preferably comprises an anti-breast cancer drug and / or an anti-lung cancer drug. In the present invention, the anti-breast cancer drug is preferably an anti-triple negative breast cancer drug.

[0106] The following examples are used to illustrate in detail the carbazole-derived PAD4 inhibitors provided by the present invention, their preparation methods and applications, but they should not be construed as limiting the protection scope of the present invention.

[0107] In the following examples, the synthetic routes and synthesis methods of the intermediates used are as follows:

[0108]

[0109] Reagents and conditions: (a) formaldehyde solution (37%), dilute sulfuric acid, r.t., 6 h; (b) (Boc)2O, Et3N, DMF, r.t., 12 h; (c) SOCl2, CH3OH, r.t., 12 h; (d) Pd / C, CH3OH / acetone, r.t., 4 h; (e) 2M NaOH, CH3OH / CH2Cl2, r.t., 4 h; (f) formaldehyde solution (37%), concentrated hydrochloric acid, 80 °C, 6 h; (g) DDQ, CH2Cl2, r.t., 4 h.

[0110] 4H-KLSS (M2a)

[0111] In an ice-water bath, 0.2 mL of concentrated sulfuric acid was added dropwise to 400 mL of distilled water to prepare dilute sulfuric acid with a pH of 2. Subsequently, L-Trp (5.00 g, 24.5 mmol) and 37% formaldehyde solution (10 mL) were added, and the reaction was carried out at room temperature for 5 h. After the reaction was detected to be complete by TLC, in an ice-water bath, the pH of the reaction system was adjusted to 6 with concentrated ammonia water and allowed to stand until a white solid precipitated. The product was filtered under reduced pressure, and the filter cake was rinsed with H2O and dried to obtain M2a (4.980 g, 94.1%), which was a white solid.

[0112] Boc-4H-KLSS (M3a)

[0113] In an ice-water bath, M2a (2.16 g, 10 mmol) and (Boc)2O (2.62 g, 12 mmol) were dissolved in 40 mL of DMF, and the pH of the reaction system was adjusted to 9 with TEA. The reaction was carried out at room temperature for 12 h, and the acidic gas was removed by reduced pressure pumping every 1 h. After the reaction was detected to be complete by TLC, in an ice-water bath, the pH was adjusted to 7 with saturated KHSO4 solution and dried by blowing. The residue was adjusted to pH 2 with saturated KHSO4 solution and extracted with EA (20 mL × 3). The organic layer was washed with saturated NaCl solution (20 mL × 3), dried over anhydrous Na2SO4, and purified by medium-pressure preparative column (8% CH3OH) to obtain M3a (2.85 g, 90.2%), which was a white solid.

[0114] 4H-KLSS-OCH3 (M4)

[0115] In an ice bath, slowly add SOCl2 (2.6 mL) dropwise to 20 mL of CH3OH and activate for 30 min. Then add M2a (2.16 g, 10 mmol) and react at room temperature for 12 h. After the reaction is complete as detected by TLC, concentrate under reduced pressure. Dissolve the residue in 40 mL of EA, wash successively with saturated NaHCO3 solution (20 mL × 3) and saturated NaCl solution (20 mL × 3), dry over anhydrous Na2SO4 and rotary evaporate to obtain M4 (2.16 g, 93.9%), which is a light brown solid.

[0116] KLSS-OCH3 (M5) and CH2-2KLSS-OCH3 (M5a)

[0117] Dissolve M4 (2.16 g, 9.4 mmol) in a 100 mL eggplant-shaped flask with 40 mL of CH3OH and 10 mL of acetone until it becomes light brown and clear; add 0.8 g of Pd / C, connect a single-pass, stir and react at room temperature for 7 days, monitor the reaction progress by TLC (CH2Cl2:CH3OH = 10:1), and observe that the starting material spot disappears, indicating that the reaction is complete. Post-treatment: Filter under reduced pressure (rinse the filter residue with CH2Cl2 and CH3OH), separate and purify by medium-pressure preparative column (collect the product with 8 - 9% CH3OH) to obtain 1.690 g (79.6%) of M5, which is a light brown solid. At the same time, collect the by-product with 7% CH3OH, which is identified as M5a, a total of 0.200 g (9.2%), which is a light brown solid.

[0118] KLSS (M6)

[0119] Dissolve M5 (1.69 g, 7.5 mmol) in 20 mL of CH3OH and 20 mL of CH2Cl2, adjust the pH of the reaction system to 10 with 2M NaOH and react in an ice bath for 4 h. After the reaction is complete as detected by TLC, adjust the pH to 2 with saturated KHSO4 solution, concentrate under reduced pressure and filter. Wash the filter cake with H2O and a small amount of ice-cold CH3OH and dry to obtain M6 (1.45 g, 91.5%), which is a light brown solid. ESI-MS (m / z): 213.0 [M + H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.27 (s, 1H), 9.04 (s, 1H), 9.03 (s, 1H), 8.47 (d, J = 7.9 Hz, 1H), 7.72 (t, J = 8.2 Hz, 1H), 7.67 (dt, J = 6.9 Hz, 0.9 Hz, 1H), 7.37 (dt, J = 7.8 Hz, 0.8 Hz). 1313C-NMR(75MHz, DMSO-d6): δ(ppm) = 166.2, 142.1, 137.5, 135.8, 132.6, 129.9, 129.3, 123.1, 121.2, 121.1, 118.2, 113.1.

[0120] CH2-2KLSS-OH (M6a)

[0121] This preparation protocol is the same as that of M6, obtaining M6a (0.18 g, 95.7%), which is a pale yellow solid. ESI-MS (m / z): 435.7 [M-H] - ; 1 1H-NMR(300MHz, DMSO-d6): δ(ppm) = 9.10 (s, 2H), 9.06 (s, 2H), 8.56 (d, J = 7.8 Hz, 2H), 7.92 (d, J = 8.4 Hz, 2H), 7.73 (t, J = 7.6 Hz, 1H), 7.56 (s, 2H), 7.45 (t, J = 7.5 Hz, 2H); 13 13C-NMR(75MHz, DMSO-d6): δ(ppm) = 166.3, 141.7, 138.4, 137.4, 131.9, 130.5, 130.0, 123.7, 122.3, 121.6, 118.1, 111.3, 53.0.

[0122] 4H-IQ (M2b)

[0123] Suspend L-Phe (3.30 g, 20 mmol) and 37% formaldehyde solution (10 mL) in 40 mL of concentrated hydrochloric acid and heat the reaction at 80 °C for 6 h. After detecting the completion of the reaction by TLC, cool it to room temperature and filter under reduced pressure. Wash the filter cake with distilled water and dry it to obtain M2b (3.33 g, 94.1%), which is a white solid.

[0124] Boc-4H-IQ (M3b)

[0125] This preparation protocol is the same as that of M3a. After separation and purification by medium-pressure preparative column (collect the product with 8% CH3OH), M3b (2.36 g, 85.0%) is obtained, which is a white solid.

[0126] 4H-IQ-OCH3 (M4b)

[0127] This preparation protocol is the same as that of M4, obtaining M4b (1.80 g, 94.2%), which is a light brown solid.

[0128] IQ-OCH3 (M5b)

[0129] The obtained M4b (1.80 g, 9.4 mmol) was placed in a 200 mL eggplant-shaped flask and dissolved in 80 mL of dry CH2Cl2 to form a light brown clear solution. In the fume hood, 4.279 g (18.8 mmol) of DDQ was added, and a drying tube was connected. The reaction was carried out at room temperature for 4 h. During this period, a solid precipitated. The reaction progress was monitored by TLC (CH2Cl2:CH3OH = 20:1, Rf = 0.30). When the starting material spot disappeared, the reaction was judged to be complete. Post-treatment: The reaction was terminated with excess saturated NaHCO3 until no bubbles were generated. CH2Cl2 was removed by rotary evaporation, and the mixture was filtered under reduced pressure. The filter cake was rinsed successively with H2O and a small amount of ice-cold CH3OH, and then dried in air to obtain M5b (1.56 g, 88.5%), which was a white solid.

[0130] IQ (M6b)

[0131] This preparation protocol was the same as that for M6, and M6b (1.56 g, 88.5%) was obtained as a white solid. ESI-MS (m / z): 174.1 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 9.66 (s, 1H), 8.87 (s, 1H), 8.45 (d, J = 8.1 Hz, 1H), 8.36 (d, J = 8.1 Hz, 1H), 8.10 (dt, J = 7.0 Hz, 0.9 Hz, 1H), 8.00 (dt, J = 8.0 Hz, 0.9 Hz, 1H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 164.9, 151.5, 138.1, 136.6, 134.3, 131.4, 129.6, 129.3, 128.9, 125.3.

[0132] Examples 1 - 5

[0133] The synthetic routes and methods for Compounds 8 - 10b are as follows:

[0134]

[0135] Reagents and conditions: (a) benzylamine, DCC, HOBt, THF, NMM, r.t., 8 h; (b) Pd / H2, CH3OH, r.t., 4 h; (c) EDC, HOBt, THF, NMM, r.t., 8 h; (d) 4M HCl / EA, 0 °C, 4 h; (e) ethyl 2-chloroacetate hydrochloride, anhydrous methanol, DIPEA, r.t., 12 h.

[0136] Example 1

[0137] Ice bath. Dissolve Z-Orn(Boc)-OH (3.66 g, 10 mmol), benzylamine (1.6 mL), DCC (2.47 g, 12 mmol) and HOBt (1.62 g, 12 mmol) in 80 mL of anhydrous THF, and adjust the pH of the reaction system to 8 with NMM. After stirring the reaction at room temperature for 8 h, TLC detects that the reaction is complete. Subsequently, the reaction solution is filtered under reduced pressure and concentrated. The residue is dissolved in 60 mL of EA, and washed successively with saturated NaHCO3 solution (30 mL×3) and saturated NaCl solution (30 mL×3), dried over anhydrous Na2SO4, and purified by medium-pressure preparative column (collect the product with 50% EA) to obtain M8 (4.49 g, 98.7%), which is a white solid.

[0138] Orn(Boc)-NBzl (M9)

[0139] Dissolve M8 (4.49 g, 9.9 mmol) and 10% Pd / C (0.5 g) in 60 mL of CH3OH. Under a hydrogen atmosphere, after stirring the reaction at room temperature for 4 h, TLC detects that the reaction is complete. Subsequently, filter under reduced pressure and rotary evaporate to dryness to obtain M9 (3.99 g, 88.8%), which is a white solid. Boc-4H-KLSS-Orn(Boc)-NBzl (M10a)

[0140] Ice bath. Dissolve M3a (1.70 g, 5.4 mmol), M9 (2.07 g, 6.5 mmol), EDC (1.33 g, 6.5 mmol) and HOBt (0.87 g, 6.5 mmol) in 40 mL of anhydrous THF, and adjust the pH of the reaction system to 8 with NMM. After stirring the reaction at room temperature for 8 h, TLC detects that the reaction is complete. Subsequently, purify by medium-pressure preparative column (collect the product with 50% EA) to obtain M10a (2.36 g, 70.9%), which is a white solid. 4H-KLSS-Orn-NBzl·2HCl (M11a)

[0141] Ice bath. Dissolve M10a (2.36 g, 3.8 mmol) in 25 mL of 4M HCl / EA and stir the reaction for 4 h. After TLC detects that the reaction is complete, evaporate the reaction solution to dryness under reduced pressure. The residue is redissolved successively with dry EA (15 mL×3) and anhydrous ether (15 mL×1), and then evaporated to dryness again to obtain M11a (1.78 g, 94.6%), which is a pale yellow solid.

[0142] 4H-KLSS-Orn(Cl)-NBzl (8)

[0143] Ice bath: Dissolve M11a (1.78 g, 3.6 mmol) and 2-chloroethyl chloroacetimidate (2.83 g, 18 mmol) in 40 mL of anhydrous methanol, and adjust the pH of the reaction system to 10 with DIPEA. After reacting at room temperature for 12 h, the reaction was monitored by TLC and found to be complete. After purification by medium-pressure preparative column (collect the product with 30% CH3OH), the methanol was removed by rotary evaporation and the residue was lyophilized to obtain 8 (0.45 g, 25.3%), which is a white solid. Purity: 95.54%. m.p.: 88.2 - 89.1 °C. =-60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 495.2270 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 10.75 (s, 1H), 9.98 (t, J = 4.7 Hz, 1H), 9.12 (s, 1H), 8.47 (t, J = 5.7 Hz, 1H), 8.44 (d, J = 8.5 Hz, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.32 (d, J = 5.5 Hz, 1H), 7.26 (m, 5H), 7.05 (t, J = 7.1 Hz, 1H), 6.98 (t, J = 7.3 Hz, 1H), 4.37 (s, 2H), 4.34 (m, 1H), 4.28 (d, J = 5.6 Hz, 2H), 3.92 (m, 1H), 3.89 (m, 2H), 3.25 (dt, J = 5.6 Hz, 6.3 Hz, 2H), 3.06 (dd, J = 6.4 Hz, 16.3 Hz, 1H), 2.89 (dd, J = 4.8 Hz, 15.9 Hz, 1H), 1.73 (m, 2H), 1.55 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.7, 171.2, 169.2, 162.8, 139.7, 136.5, 130.5, 128.7, 127.5, 127.2, 121.3, 119.0, 117.9, 111.5, 105.6, 61.1, 52.9, 51.9, 47.9, 42.6, 42.3, 29.3, 24.4, 20.1.

[0144] Example 2

[0145] Boc-4H-IQ-Orn(Boc)-NBzl (M10b)

[0146] M10b (1.78 g, 76.7%) was obtained as a white solid by separating and purifying M3b and M9 according to the same preparation protocol as M10a by medium-pressure preparative column (collect the product with 50 - 55% EA).

[0147] 4H-IQ-Orn-NBzl·2HCl (M11b)

[0148] This preparation protocol is the same as that of M11a, and M11b (1.14 g, 82.0%) is obtained as a white solid.

[0149] 4H-IQ-Orn(Cl)-NBzl(9)

[0150] From M11b and 2-chloroacetyl iminoethyl ester according to the same preparation protocol as 8, after separation and purification by C18 silica gel column chromatography (collect the product with 26% CH3OH), 9 (0.18 g, 15.7%) is obtained as a white solid. Purity: 95.23%. m.p.: 124.4 - 125.7 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 456.2161 [M + H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 10.45 (s, 1H), 9.34 (t, J = 8.2 Hz, 1H), 9.07 (d, J = 6.9 Hz, 1H), 8.76 (t, J = 5.8 Hz, 1H), 7.30 (m, 9H), 4.49 (s, 2H), 4.41 (m, 1H), 4.34 (m, 1H), 4.32 (d, J = 2.9 Hz, 2H), 4.25 (m, 2H), 3.40 (m, 1H), 3.32 (m, 2H), 3.01 (dd, J = 16.2 Hz, 12.1 Hz, 1H), 1.78 (m, 2H), 1.68 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.1, 168.3, 162.6, 139.7, 131.5, 129.0, 128.7, 128.0, 127.6, 127.3, 127.2, 127.1, 54.4, 53.1, 53.0, 44.2, 42.5, 42.2, 29.8, 29.4, 24.0.

[0151] Example 3

[0152] QN-Orn(Boc)-NBzl (M10c)

[0153] From 6-quinoxaline carboxylic acid and M9 according to the same preparation protocol as M10a, after separation and purification by medium-pressure preparative column (collect the product with 6% CH3OH), M10c (2.06 g, 86.4%) is obtained as a light brown solid.

[0154] QN-Orn(HCl)-NBzl(M11c)

[0155] This preparation protocol is the same as that of M11a, obtaining M11c (1.75 g, 98.0%), which is a light brown solid.

[0156] QN-Orn(Cl)-NBzl(10)

[0157] From M11c and 2-chloroacetimidoyl ethyl ester, according to the same preparation protocol as 8, separated and purified by C18 silica gel column chromatography (collecting the product with 30% CH3OH), 10 (1.00 g, 52.3%) was obtained, which is a light brown solid. Purity: 99.53%. m.p.: 106.5 - 107.4 °C. =-120.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 453.1800 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 10.38 (s, 1H), 9.69 (m, 1H), 9.29 (m, 1H), 9.10 (d, J = 7.7 Hz, 1H), 9.04 (d, J = 3.0 Hz, 2H), 8.77 (s, 1H), 8.74 (t, J = 5.9 Hz, 1H), 8.37 (dd, J = 8.7 Hz, 1.1 Hz, 1H), 8.18 (d, J = 8.7 Hz, 1H), 7.28 (m, 5H), 4.59 (m, 1H), 4.47 (s, 2H), 4.33 (d, J = 4.0 Hz, 2H), 3.33 (m, 2H), 1.93 (m, 2H), 1.70 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.9, 166.2, 162.6, 147.4, 147.1, 143.9, 142.0, 139.9, 135.6, 129.6, 129.4, 129.3, 128.7, 127.5, 127.1, 54.0, 42.5, 42.4, 29.1, 24.5.

[0158] Example 4

[0159] BIX3C-Orn(Boc)-NBzl(M10d)

[0160] From 2,1-benzisoxazole-3-carboxylic acid and M9, according to the same preparation protocol as M10a, separated and purified by medium-pressure preparative column (collecting the product with 5% CH3OH), M10d (0.88 g, 37.8%) was obtained, which is a white solid.

[0161] BIX3C-Orn(HCl)-NBzl(M11d)

[0162] This preparation scheme is the same as that of M11a, and M11d (0.75 g, 98.7%) is obtained as a white solid.

[0163] BIX3C-Orn(Cl)-NBzl(10a)

[0164] From M11d and 2-chloroacetimidoyl ethyl ester, according to the same preparation scheme as 8, separated and purified by C18 silica gel column chromatography (collect the product with 22% CH3OH), 10a (0.22 g, 26.8%) is obtained as a white solid. Purity: 98.08%. m.p.: 99.7 - 100.5 °C. = - 60.0 (C = 1 mg / mL, CH3OH). HR - MS (m / z): 442.1640 [M + H] + . 1 1H - NMR (800 MHz, DMSO - d6): δ (ppm) = 10.28 (t, J = 4.5 Hz, 1H), 9.68 (s, 1H), 9.35 (d, J = 7.8 Hz, 1H), 9.24 (s, 1H), 8.56 (t, J = 5.8 Hz, 1H), 7.96 (d, J = 8.8 Hz, 1H), 7.79 (d, J = 9.1 Hz, 1H), 7.52 (dd, J = 8.5 Hz, 6.4 Hz, 1H), 7.27 (m, 6H), 4.57 (m, 1H), 4.45 (s, 2H), 4.32 (d, J = 5.6 Hz, 2H), 3.32 (m, 2H), 1.93 (m, 2H), 1.66 (m, 2H). 13 13C - NMR (200 MHz, DMSO - d6): δ (ppm) = 171.2, 162.7, 157.4, 157.2, 156.8, 139.8, 132.4, 128.7, 127.6, 127.5, 127.2, 121.3, 118.8, 115.6, 53.2, 42.6, 42.3, 39.6, 28.8, 24.4.

[0165] Example 5

[0166] BFZ5C - Orn(Boc) - NBzl(M10e)

[0167] From 2,1,3 - benzoxadiazole - 5 - carboxylic acid and M9, according to the same preparation scheme as M10a, separated and purified by medium - pressure preparative column (collect the product with 6% CH3OH), M10e (1.24 g, 52.9%) is obtained as a pale yellow solid.

[0168] BFZ5C-Orn(HCl)-NBzl(M11e)

[0169] This preparation protocol is the same as that of M11a, obtaining M11e (1.05 g, 98.4%), which is a yellow solid.

[0170] BFZ5C-Orn(Cl)-NBzl(10b)

[0171] From M11e and 2-chloroethyl chloroacetimidate according to the same preparation protocol as 8, after purification by C18 silica gel column chromatography (collecting the product with 22% CH3OH), 10b (0.65 g, 56.5%) was obtained, which is a pale yellow solid. Purity: 99.45%. m.p.: 93.7 - 94.6 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 443.1593 [M + H] + . 1 1H-NMR (800 MHz, DMSO-d6): δ (ppm) = 10.36 (t, J = 3.9 Hz, 1H), 9.65 (s, 1H), 9.28 (s, 1H), 9.19 (d, J = 7.6 Hz, 1H), 8.78 (s, 1H), 8.74 (t, J = 5.9 Hz, 1H), 8.15 (d, J = 9.4 Hz, 1H), 8.02 (dd, J = 9.4 Hz, 0.8 Hz, 1H), 7.27 (m, 5H), 4.51 (m, 1H), 4.47 (s, 2H), 4.32 (d, J = 5.7 Hz, 2H), 3.33 (m, 2H), 1.91 (m, 2H), 1.71 (m, 2H). 13 13C-NMR (200 MHz, DMSO-d6): δ (ppm) = 171.7, 165.4, 162.7, 149.6, 149.3, 139.9, 137.9, 132.2, 128.7, 127.5, 127.1, 116.8, 116.6, 54.2, 42.5, 42.3, 39.6, 29.0, 24.5.

[0172] Examples 6 - 17

[0173] The synthetic routes and methods for Compounds 11 - 17e are as follows:

[0174]

[0175] Reagents and conditions: (a) benzylamine, DCC, HOBt, THF, NMM, r.t., 8 h; (b) 4 M HCl / EA, 0 °C, 4 h; (c) EDC, HOBt, THF, NMM, r.t., 8 h; (d) Pd / H2, CH3OH, r.t., 4 h; (e) ethyl 2-chloroacetate hydrochloride, anhydrous methanol, DIPEA, r.t., 12 h.

[0176] Example 6 Boc-Orn(Z)-NBzl (M13)

[0177] M13 (4.40 g, 96.7%) was obtained as a white solid by separating and purifying Boc-Orn(Z)-OH (M12) according to the same preparation protocol as M8 on a medium-pressure preparative column (collecting the product with 50 - 60% EA).

[0178] HCl·Orn(Z)-NBzl (M14)

[0179] M14 (3.54 g, 93.4%) was obtained as a white solid by the same preparation protocol as M11a.

[0180] KLSS-Orn(Z)-NBzl (M15a)

[0181] M15a (1.35 g, 82.0%) was obtained as a white solid by separating and purifying M6 and M14 according to the same preparation protocol as M10a on a medium-pressure preparative column (collecting the product with 6% CH3OH).

[0182] KLSS-Orn-NBzl (M16a)

[0183] M16a (0.93 g, 91.2%) was obtained as a white solid by the same preparation protocol as M9.

[0184] KLSS-Orn(Cl)-NBzl (11)

[0185] 11 (0.44 g, 40.0%) was obtained as a white solid by separating and purifying M16a and ethyl 2-chloroacetimidate according to the same preparation protocol as 8 on a C18 silica gel column chromatography (collecting the product with 48% CH3OH). Purity: 99.46%. m.p.: 98.6 - 99.2 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 491.1957 [M + H] + . 1H-NMR(300MHz, DMSO-d6): δ(ppm) = 12.08(s, 1H), 9.96(t, J = 4.7Hz, 1H), 9.51(s, 1H), 9.10(s, 1H), 8.95(d, J = 0.6Hz, 1H), 8.88(s, 1H), 8.78(t, J = 5.0Hz, 1H), 8.77(d, J = 7.7Hz, 1H), 8.42(d, J = 7.9Hz, 1H), 7.67(t, J = 8.2Hz, 1H), 7.62(dt, J = 0.8Hz, 6.9Hz, 1H), 7.31(m, 5H), 7.25(m, 1H), 4.70(dt, J = 5.3Hz, 7.9Hz, 1H), 4.36(s, 2H), 4.35(d, J = 5.2Hz, 2H), 3.30(dt, J = 5.8Hz, 5.9Hz, 2H), 1.86(m, 2H), 1.63(m, 2H). 13 C-NMR(75MHz, DMSO-d6): δ(ppm) = 171.6, 164.8, 162.8, 141.6, 139.6, 139.4, 137.8, 132.9, 129.2, 128.8, 128.7, 127.7, 127.3, 122.7, 121.4, 120.6, 114.6, 112.8, 52.5, 42.7, 42.4, 39.8, 30.7, 24.1.

[0186] Example 7

[0187] QN-Orn(Z)-NBzl (M15b)

[0188] M15b (2.01 g, 78.7%) was obtained as a light gray solid by separating and purifying 6 - quinoxalinecarboxylic acid and M14 according to the same preparation protocol as M10a on a medium - pressure preparative column (collecting the product with 6% CH3OH).

[0189] 4H - QN - Orn - NBzl (M16b)

[0190] M16b (0.61 g, 81.8%) was obtained as a light gray solid according to the same preparation protocol as M9.

[0191] 4H - QN - Orn(Cl) - NBzl (12)

[0192] 12 (0.15 g, 20.5%) was obtained as a white solid by separating and purifying M16b and 2 - chloroethyl iminoacetate according to the same preparation protocol as 8 on a C18 silica gel column chromatography (collecting the product with 25% CH3OH). Purity: 98.55%. m.p.: 93.9 - 95.1 °C. =-60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 457.2113 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 9.99 (s, 1H), 9.56 (s, 1H), 9.12 (s, 1H), 8.47 (t, J = 5.6 Hz, 1H), 8.12 (d, J = 7.7 Hz, 1H), 7.44 (d, J = 8.7 Hz, 1H), 7.40 (s, 1H), 7.28 (m, 5H), 6.61 (d, J = 8.3 Hz, 1H), 4.47 (m, 1H), 4.37 (s, 2H), 4.30 (d, J = 3.8 Hz, 2H), 3.36 (m, 4H), 3.27 (m, 2H), 1.79 (m, 2H), 1.60 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 172.3, 166.4, 162.7, 159.0, 158.5, 139.8, 128.7, 127.5, 127.2, 122.4, 118.5, 114.7, 114.1, 53.3, 42.5, 42.4, 38.8, 29.3, 24.4.

[0193] Example 8

[0194] ID5C-Orn(Z)-NBzl (M15c)

[0195] From indole-5-carboxylic acid and M14 according to the same preparation protocol as M10a, separated and purified by C18 silica gel column chromatography (collecting the product with 40% CH3OH), M15c (1.65 g, 66.3%) was obtained as a white solid.

[0196] ID5C-Orn-NBzl (M16c)

[0197] The preparation protocol was the same as M9, and M16c (1.10 g, 91.2%) was obtained as a white solid.

[0198] ID5C-Orn(Cl)-NBzl (13)

[0199] From M16c and 2-chloroethyl imidoester according to the same preparation protocol as 8, separated and purified by C18 silica gel column chromatography (collecting the product with 35% CH3OH), 13 (0.74 g, 55.8%) was obtained as a white solid. Purity: 97.69%. m.p.: 109.0 - 109.5 °C. =-60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 440.1848 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 11.40 (s, 1H), 10.26 (s, 1H), 9.65 (s, 1H), 9.22 (t, J = 5.1 Hz, 1H), 8.59 (t, J = 5.7 Hz, 1H), 8.39 (d, J = 7.9 Hz, 1H), 8.25 (s, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.43 (d, J = 7.1 Hz, 1H), 7.42 (d, J = 3.0 Hz, 1H), 7.26 (m, 5H), 6.54 (s, 1H), 4.55 (m, 1H), 4.44 (s, 2H), 4.31 (d, J = 5.7 Hz, 2H), 3.31 (m, 2H), 1.85 (m, 2H), 1.64 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 172.4, 168.1, 162.6, 139.9, 138.0, 128.7, 127.5, 127.4, 127.1, 125.4, 121.3, 120.8, 111.3, 102.5, 53.5, 42.5, 42.4, 39.6, 29.3, 24.5.

[0200] Example 9

[0201] IA-Orn(Z)-NBzl (M15d)

[0202] From 3-indolecarboxylic acid and M14 according to the same preparation protocol as M10a, purified by medium-pressure preparative column chromatography (collecting the product with 6% CH3OH), M15d (1.64 g, 65.9%) was obtained as a white solid.

[0203] IA-Orn-NBzl (M16d)

[0204] The preparation protocol was the same as that of M9, and M16d (1.15 g, 95.9%) was obtained as a white solid.

[0205] IA-Orn(Cl)-NBzl (14)

[0206] From M16d and 2-chloroethyl iminoacetate according to the same preparation protocol as 8, purified by C18 silica gel column chromatography (collecting the product with 40% CH3OH), 14 (1.00 g, 72.0%) was obtained as a white solid. Purity: 99.71%. m.p.: 58.5 - 58.8 °C. =-60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 440.1848 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 11.64 (s, 1H), 9.96 (s, 1H), 9.53 (s, 1H), 9.10 (s, 1H), 8.51 (t, J = 5.9 Hz, 1H), 8.18 (d, J = 2.8 Hz, 1H), 8.13 (dd, J = 7.1 Hz, 1.3 Hz, 1H), 7.95 (d, J = 8.1 Hz, 1H), 7.44 (dd, J = 7.1 Hz, 1.5 Hz, 1H), 7.26 (m, 5H), 7.13 (m, 2H), 4.57 (m, 1H), 4.36 (s, 2H), 4.32 (d, J = 5.6 Hz, 2H), 3.30 (m, 2H), 1.83 (m, 2H), 1.65 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 172.5, 165.2, 162.6, 139.9, 136.6, 128.7, 127.5, 127.2, 122.4, 121.3, 120.9, 112.3, 110.5, 52.4, 42.5, 42.4, 29.6, 24.4.

[0207] Example 10

[0208] 5FIA-Orn(Z)-NBzl (M15e)

[0209] From 5-fluoroindole-3-carboxylic acid and M14 according to the same preparation protocol as M10a, purified by medium-pressure preparative column chromatography (collecting the product with 6% CH3OH), M15e (2.26 g, 87.6%) was obtained as a white solid.

[0210] 5FIA-Orn-NBzl (M16e)

[0211] The preparation protocol was the same as that of M9, and M16e (0.80 g, 95.6%) was obtained as a white solid.

[0212] 5FIA-Orn(Cl)-NBzl (15)

[0213] From M16e and 2-chloroethyl imidoester according to the same preparation protocol as 8, purified by C18 silica gel column chromatography (collecting the product with 40% CH3OH), 15 (0.56 g, 58.4%) was obtained as a white solid. Purity: 99.87%. m.p.: 176.6 - 177.3 °C. =-60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 458.1754 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 11.83 (d, J = 1.9 Hz, 1H), 10.02 (s, 1H), 9.58 (s, 1H), 9.15 (s, 1H), 8.53 (t, J = 5.9 Hz, 1H), 8.28 (d, J = 2.3 Hz, 1H), 8.07 (d, J = 8.1 Hz, 1H), 7.83 (dd, J = 10.2 Hz, 2.4 Hz, 1H), 7.45 (dd, J = 8.8 Hz, 4.6 Hz, 1H), 7.26 (m, 5H), 7.01 (dt, J = 9.2 Hz, 2.4 Hz, 1H), 4.57 (m, 1H), 4.38 (s, 2H), 4.32 (d, J = 5.6 Hz, 2H), 3.48 (m, 2H), 1.81 (m, 2H), 1.67 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 172.5, 164.9, 162.7, 156.7, 139.9, 133.2, 130.7, 128.6, 127.5, 127.1, 113.5, 110.8, 110.4, 106.2, 52.6, 42.5, 42.4, 29.5, 24.4. 19 19F-NMR (282 MHz, DMSO-d6): δ (ppm) = -123.1.

[0214] Example 11

[0215] BM2C-Orn(Z)-NBzl (M15f)

[0216] From 1H-benzimidazole-2-carboxylic acid and M14 according to the same preparation protocol as M10a, purified by medium-pressure preparative column chromatography (collecting the product with 8% CH3OH), M15f (1.50 g, 60.1%) was obtained as a white solid.

[0217] BM2C-Orn-NBzl (M16f)

[0218] The preparation protocol was the same as that of M9, and M16f (1.06 g, 97.1%) was obtained as a white solid.

[0219] BM2C-Orn(Cl)-NBzl (16)[[ID=,27]]

[0220] Compound 16 was prepared from M16f and 2-chloroacetimidoyl ethyl ester according to the same preparation protocol as Compound 8, and purified by silica gel column chromatography (eluting with 37% CH3OH to collect the product), yielding 16 (0.48 g, 37.4%) as a white solid. Purity: 99.71%. m.p.: 121.8 - 122.4 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 441.1800 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 13.36 (s, 1H), 10.21 (s, 1H), 9.66 (s, 1H), 9.22 (s, 1H), 8.79 (t, J = 5.4 Hz, 1H), 8.73 (d, J = 8.3 Hz, 1H), 7.66 (m, 2H), 7.28 (m, 7H), 4.59 (m, 1H), 4.43 (s, 2H), 4.32 (d, J = 3.0 Hz, 2H), 3.32 (m, 2H), 1.90 (m, 2H), 1.63 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 172.2, 162.6, 159.1, 145.5, 139.6, 128.7, 127.6, 127.3, 124.0, 52.9, 42.6, 42.2, 29.6, 24.2.

[0221] Example 12

[0222] BM5C-Orn(Z)-NBzl (M15g)

[0223] Compound M15g was prepared from 1H-benzoimidazole-5-carboxylic acid and M14 according to the same preparation protocol as M10a, and purified by medium-pressure preparative column chromatography (eluting with 9% CH3OH to collect the product), yielding M15g (1.49 g, 59.9%) as a white solid.

[0224] BM5C-Orn-NBzl (M16g)

[0225] The preparation protocol was the same as that for M9, yielding M16g (1.02 g, 93.2%) as a white solid.

[0226] BM5C-Orn(Cl)-NBzl (17)

[0227] Compound 17 (0.90 g, 73.2%) was obtained from M16g and 2-chloroacetimidoethyl ester according to the same preparation protocol as for 8. Purification by C18 silica gel column chromatography (using 30% CH3OH to collect the product) yielded 17 as a white solid. Purity: 98.28%. MP: 252.3-252.8°C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):441.1800[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 10.25 (s, 1H), 9.64 (s, 1H), 9.22 (s, 1H), 8.63 (m, 1H), 8.61 (d, J = 3.9Hz, 1H), 8.53 (s, 1H), 8.31 (s, 1H), 7.88 (d, J =8.5Hz,1H),7.67(d,J=8.5Hz,1H),7.26(m,5H),4.55(m,1H),4.44(s,2H ), 4.31 (d, J = 3.6Hz, 2H), 3.32 (t, J = 6.7Hz, 2H), 1.88 (m, 2H), 1.66 (m, 2H). 13 C-NMR (75MHz, DMSO-d6): δ (ppm) = 172.2, 167.1, 162.7, 143.7, 139.9, 128.7, 127.5, 127.1, 123.5, 115.8, 114.7, 53.8, 42.5, 42.4, 29.2, 24.5.

[0228] Example 13

[0229] CH2-2KLSS-2Orn(Z)-NBzl(M15h)

[0230] M6a (0.18 g, 0.4 mmol), M14 (0.39 g, 1.0 mmol), EDC (0.19 g, 1.0 mmol), and HOBt (0.14 g, 1.0 mmol) were dissolved in 40 mL of anhydrous THF in an ice-water bath, and the pH of the reaction system was adjusted to 8 with NMM. After stirring at room temperature for 8 h, the reaction was complete as determined by TLC. The product was then purified by medium-pressure preparative column chromatography (using 5% CH3OH to collect the product) to afford M15h (0.36 g, 77.4%) as a white solid.

[0231] CH2-2KLSS-2Orn-NBzl(M16h)

[0232] The preparation protocol was the same as for M9 to afford M16h (0.26 g, 96.5%) as a white solid.

[0233] CH2-2KLSS-2Orn(Cl)-NBzl(17a)

[0234] In an ice-water bath, M16h (0.26 g, 0.3 mmol) and 2-chloroethyl chloroacetimidate (0.48 g, 3.1 mmol) were dissolved in 40 mL of anhydrous methanol, and the pH of the reaction system was adjusted to 10 with DIPEA. After reacting at room temperature for 12 h, the reaction was detected to be complete by TLC. After purification by medium-pressure preparative column (collecting the product with 55% CH3OH), the methanol was removed by rotary evaporation and freeze-dried to obtain 17a (0.14 g, 45.7%), which was a white solid. Purity: 97.72%; m.p.: 193.3 - 194.7 °C; = -60.0 (C = 1 mg / mL, CH3OH); HR-MS (m / z): 993.3841 [M + H] + ; 1 1H-NMR (800 MHz, DMSO-d6): δ (ppm) = 10.16 (s, 1H), 9.62 (s, 1H), 9.17 (s, 1H), 8.94 (s, 2H), 8.92 (s, 2H), 8.75 (t, J = 6.5 Hz, 2H), 8.73 (d, J = 8.4 Hz, 2H), 8.53 (d, J = 7.9 Hz, 2H), 7.98 (d, J = 8.3 Hz, 2H), 7.73 (t, J = 7.7 Hz, 2H), 7.55 (s, 2H), 7.43 (t, J = 7.5 Hz, 2H), 7.26 (m, 10H), 4.63 (dt, J = 4.2 Hz, 8.2 Hz, 2H), 4.40 (s, 4H), 4.29 (d, J = 5.5 Hz, 4H), 3.29 (m, 4H), 1.85 (m, 4H), 1.57 (m, 4H); 13 13C-NMR (200 MHz, DMSO-d6): δ (ppm) = 171.6, 164.5, 162.7, 162.6, 141.5, 141.1, 139.6, 137.4, 131.7, 130.2, 129.8, 128.7, 127.6, 127.2, 123.5, 122.0, 121.7, 114.8, 111.2, 52.6, 42.6, 42.2, 39.6, 30.2, 24.1.

[0235] Example 14

[0236] IQ-Orn(Z)-NBzl (M15i)

[0237] M15i (2.17 g, 69.8%) was obtained as a white solid by separating and purifying M6b and M14 according to the same preparation protocol as M10a on a medium-pressure preparative column (collecting the product with 6% CH3OH).

[0238] IQ-Orn-NBzl (M16i)

[0239] M16i (1.52 g, 95.0%) was obtained as a white solid according to the same preparation protocol as M9.

[0240] IQ-Orn(Cl)-NBzl(17b)

[0241] 17b (0.90 g, 49.3%) was obtained as a white solid by separating and purifying M16i and 2-chloroacetyl iminoethyl ester according to the same preparation protocol as 8 on a C18 silica gel column chromatography (collecting the product with 45% CH3OH). Purity: 96.71%. m.p.: 67.8 - 68.6 °C. =-60.0 (C=1 mg / mL, CH3OH). HR-MS (m / z): 452.1848 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 9.95 (s, 1H), 9.53 (s, 1H), 9.42 (s, 1H), 9.10 (s, 1H), 8.90 (d, J = 8.4 Hz, 1H), 8.76 (t, J = 5.8 Hz, 1H), 8.59 (s, 1H), 8.27 (d, J = 8.0 Hz, 1H), 8.22 (d, J = 8.1 Hz, 1H), 7.91 (dt, J = 1.3 Hz, 6.8 Hz, 1H), 7.84 (dt, J = 1.1 Hz, 7.0 Hz, 1H), 7.28 (m, 5H), 4.69 (dt, J = 5.2 Hz, 7.8 Hz, 1H), 4.36 (s, 2H), 4.34 (d, J = 5.2 Hz, 2H), 3.30 (t, J = 6.5 Hz, 2H), 1.89 (m, 2H), 1.63 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.4, 164.2, 162.7, 152.2, 143.6, 139.5, 135.8, 132.0, 129.8, 128.8, 128.5, 128.3, 127.7, 127.3, 120.4, 52.6, 42.6, 42.3, 30.4, 24.1.

[0242] Example 15 IAA-Orn(Z)-NBzl (M15j)

[0243] M15j (2.40 g, 72.0%) was obtained as a white solid by separating and purifying 3-indoleacetic acid and M14 according to the same preparation protocol as M10a on a medium-pressure preparative column (collecting the product with 6% CH3OH).

[0244] IAA-Orn-NBzl (M16j)

[0245] M16j (1.72 g, 97.1%) was obtained as a white solid according to the same preparation protocol as M9.

[0246] IAA-Orn(Cl)-NBzl(17c)

[0247] 17c (0.90 g, 77.3%) was obtained as a white solid by separating and purifying M16j and 2-chloroethyl iminoacetate according to the same preparation protocol as 8 on a C18 silica gel column (collecting the product with 40% CH3OH). Purity: 99.86%. m.p.: 77.3 - 78.2 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 454.2004 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 10.89 (s, 1H), 9.98 (s, 1H), 9.56 (s, 1H), 9.11 (s, 1H), 8.49 (t, J = 5.8 Hz, 1H), 8.15 (d, J = 8.1 Hz, 1H), 7.55 (d, J = 7.7 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.26 (m, 6H), 7.06 (t, J = 7.3 Hz, 1H), 6.96 (t, J = 7.3 Hz, 1H), 4.37 (s, 2H), 4.32 (m, 1H), 4.27 (m, 2H), 3.60 (s, 2H), 3.23 (m, 2H), 1.69 (m, 2H), 1.54 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.9, 171.3, 162.7, 139.7, 136.6, 128.7, 127.7, 127.5, 127.2, 124.3, 121.4, 119.1, 118.7, 111.8, 109.2, 52.6, 42.5, 42.3, 42.2, 32.9, 29.8, 24.1.

[0248] Example 16

[0249] 5NIA-Orn(Z)-NBzl (M15k)

[0250] M15k (1.40 g, 56.1%) was obtained as a white solid by separating and purifying 5-azaindole-3-carboxylic acid and M14 according to the same preparation protocol as M10a on a medium-pressure preparative column (collecting the product with 9% CH3OH).

[0251] 5NIA-Orn-NBzl (M16k)

[0252] M16k (1.02 g, 99.6%) was obtained as a white solid according to the same preparation protocol as M9.

[0253] 5NIA-Orn(Cl)-NBzl(17d)

[0254] 17d (0.20 g, 16.3%) was obtained as a white solid by separating and purifying M16k and 2-chloroethyl iminoacetate according to the same preparation protocol as 8 on a C18 silica gel column (collecting the product with 30% CH3OH). Purity: 60.26%; m.p.: 65.3 - 66.8 °C; = -60.0 (C = 1 mg / mL, CH3OH); ESI-MS (m / z): 441.3 [M+H] + ; 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 9.49 (s, 1H), 8.91 (s, 1H), 8.52 (d, J = 6.7 Hz, 1H), 8.09 (d, J = 6.6 Hz, 1H), 7.27 (m, 5H), 4.54 (m, 1H), 4.46 (m, 2H), 4.32 (s, 2H), 3.31 (m, 2H), 1.85 (m, 2H), 1.70 (m, 2H).

[0255] Example 17

[0256] BO6C-Orn(Z)-NBzl (M15l)

[0257] M15l (0.91 g, 35.2%) was obtained as a white solid by separating and purifying 1,4-benzodioxane-6-carboxylic acid and M14 according to the same preparation protocol as M10a on a medium-pressure preparative column (collecting the product with 6% CH3OH).

[0258] BO6C-Orn-NBzl (M16l)

[0259] M16l (0.60 g, 89.0%) was obtained as a white solid according to the same preparation protocol as M9.

[0260] BO6C-Orn(Cl)-NBzl(17e)

[0261] Compound 17e (0.36 g, 49.8%) was obtained as a white solid by separating and purifying M16l and ethyl 2-chloroacetimidate according to the same preparation protocol as 8 through C18 silica gel column chromatography (collecting the product with 30% CH3OH). Purity: 99.13%. m.p.: 101.7 - 102.3 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 459.1794 [M+H] + . 1 1H-NMR (800 MHz, DMSO-d6): δ (ppm) = 10.33 (m, 1H), 9.68 (t, J = 15.7 Hz, 1H), 9.26 (t, J = 17.8 Hz, 1H), 8.60 (t, J = 6.0 Hz, 1H), 8.44 (d, J = 7.9 Hz, 1H), 7.53 (d, J = 1.6 Hz, 1H), 7.49 (dd, J = 8.3 Hz, 1.5 Hz, 1H), 7.30 (t, J = 7.6 Hz, 2H), 7.26 (d, J = 7.4 Hz, 2H), 7.22 (t, J = 7.2 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 4.48 (m, 1H), 4.46 (s, 2H), 4.29 (d, J = 5.2 Hz, 4H), 4.27 (m, 2H), 3.30 (m, 2H), 1.86 (m, 2H), 1.62 (m, 2H). 13 13C-NMR (200 MHz, DMSO-d6): δ (ppm) = 172.2, 166.2, 162.6, 146.6, 143.3, 139.9, 128.7, 127.5, 127.5, 127.1, 121.6, 117.2, 117.1, 64.8, 64.5, 53.6, 42.5, 42.3, 29.1, 24.4.

[0262] Examples 18 - 57

[0263] The synthetic routes and methods for Compounds 18 - 48i are as follows:

[0264] Reagents and conditions: (a) SOCl2, CH3OH, r.t., 12 h; (b) aldehydes, TFA, CH2Cl2, r.t., 15 h; (c) DDQ, CH2Cl2, r.t., 4 h; (d) 2 M NaOH, CH3OH / CH2Cl2, r.t., 4 h; (e) EDC, HOBt, THF, NMM, r.t., 8 h; (f) Pd / H2, CH3OH, r.t., 4 h; (g) 4 M HCl / EA, 0 °C, 4 h; (h) ethyl 2-chloroacetate hydrochloride, anhydrous methanol, DIPEA, r.t., 12 h.

[0265] Example 18

[0266] Trp-OCH3 (M18)

[0267] The preparation protocol was the same as that of M4, and M18 (4.32 g, 99.2%) was obtained as a light brown solid.

[0268] 4H-CH3-KLSS-OCH3 (M19a)

[0269] In an ice-water bath, M18 (2.18 g, 10 mmol) was dissolved in 40 mL of CH2Cl2, and TFA (5 mL) was slowly added, followed by activation for 10 min. Subsequently, 37% acetaldehyde solution (2 mL, 12 mmol) was added, and the reaction was carried out at room temperature for 15 h. After the reaction was completed as detected by TLC, it was concentrated under reduced pressure. The residue was dissolved in 40 mL of CH2Cl2, washed successively with saturated NaHCO3 solution (20 mL × 3) and saturated NaCl solution (20 mL × 3), dried over anhydrous Na2SO4, and purified by medium-pressure preparative column (collecting the product with 45% EA), to obtain M19a (2.22 g, 91.0%) as a white solid.

[0270] CH3-KLSS-OCH3 (M20a)

[0271] M19a (2.22 g, 9.1 mmol) and DDQ (4.13 g, 18.2 mmol) were dissolved in 60 mL of dry CH2Cl2, and the reaction was stirred at room temperature for 4 h. After the reaction was completed as detected by TLC, the reaction was terminated with saturated aqueous NaHCO3 solution. The organic layer was separated, and the aqueous layer was extracted with CH2Cl2 (20 mL × 2) and then combined. The organic layer was washed with saturated NaCl solution (20 mL × 3), dried over anhydrous Na2SO4, and purified by medium-pressure preparative column (collecting the product with 4% CH3OH), to obtain M20a (0.59 g, 49.2%) as a white solid.

[0272] CH3-KLSS (M21a)

[0273] This preparation scheme is the same as that of M6, obtaining M21a (0.52 g, 93.5%), which is a white solid.

[0274] CH3-KLSS-Orn(Z)-NBzl (M22a)

[0275] From M21a and M14 according to the same preparation scheme as M10a, separated and purified by medium-pressure preparative column (collecting the product with 6% CH3OH), M22a (0.67 g, 51.7%) was obtained, which is a white solid.

[0276] CH3-KLSS-Orn-NBzl (M23a)

[0277] This preparation scheme is the same as that of M9, obtaining M23a (0.51 g, 99.8%), which is a white solid.

[0278] CH3-KLSS-Orn(Cl)-NBzl (18)

[0279] From M23a and 2-chloroethyl imidoacetate according to the same preparation scheme as 8, separated and purified by C18 silica gel column chromatography (collecting the product with 55% CH3OH), 18 (0.38 g, 62.5%) was obtained, which is a white solid. Purity: 98.63%. m.p.: 161.9 - 162.5 °C. =-60.0 (C=1 mg / mL, CH3OH). HR-MS (m / z): 505.2113 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm)=12.16 (s, 1H), 10.31 (t, J=5.4 Hz, 1H), 9.67 (s, 1H), 9.23 (s, 1H), 8.97 (t, J=5.7 Hz, 1H), 8.72 (d, J=7.6 Hz, 1H), 8.71 (s, 1H), 8.36 (d, J=7.9 Hz, 1H), 7.67 (d, J=8.2 Hz, 1H), 7.59 (t, J=7.5 Hz, 1H), 7.29 (m, 6H), 4.74 (dt, J=5.2 Hz, 7.9 Hz, 1H), 4.45 (s, 2H), 4.35 (d, J=5.6 Hz, 2H), 3.35 (m, 2H), 2.87 (s, 3H), 1.87 (m, 2H), 1.63 (m, 2H). 1313C-NMR(75MHz, DMSO-d6): δ(ppm) = 171.7, 164.8, 162.7, 141.6, 141.4, 139.6, 138.8, 136.5, 128.8, 127.9, 127.7, 127.3, 122.6, 121.8, 120.4, 112.8, 52.2, 42.6, 42.2, 39.6, 31.0, 24.1, 21.0.

[0280] Example 19

[0281] 4H-KIbu-OCH3 (M19b)

[0282] M19b (1.85 g, 68.0%) was obtained as a white solid by separating and purifying M18 and isobutyraldehyde according to the same preparation scheme as M19a on a medium-pressure preparative column (collecting the product with 25 - 30% EA).

[0283] KIbu-OCH3 (M20b)

[0284] The preparation scheme was the same as that of M20a. After separating and purifying on a medium-pressure preparative column (collecting the product with 36% EA), M20b (1.62 g, 89.1%) was obtained as a light brown solid.

[0285] KIbu (M21b)

[0286] The preparation scheme was the same as that of M6. M21b (1.49 g, 96.8%) was obtained as a light brown solid.

[0287] KIbu-Orn(Z)-NBzl (M22b)

[0288] M22b (1.72 g, 49.6%) was obtained as a light yellow solid by separating and purifying M21b and M14 according to the same preparation scheme as M10a on a medium-pressure preparative column (collecting the product with 65 - 70% EA).

[0289] KIbu-Orn-NBzl (M23b)

[0290] The preparation scheme was the same as that of M9. M23b (1.28 g, 96.6%) was obtained as a light yellow solid.

[0291] KIbu-Orn(Cl)-NBzl (19)

[0292] Compound 19 (0.80 g, 53.4%) was obtained as a white solid by separating and purifying M23b and 2-chloroacetimidoyl ethyl ester according to the same preparation protocol as Compound 8 through C18 silica gel column chromatography (collecting the product with 55% CH3OH). Purity: 99.83%. m.p.: 159.0 - 160.2 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 533.2426 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.08 (s, 1H), 10.22 (s, 1H), 9.63 (s, 1H), 9.19 (s, 1H), 8.94 (t, J = 5.7 Hz, 1H), 8.82 (d, J = 8.2 Hz, 1H), 8.69 (s, 1H), 8.36 (d, J = 7.8 Hz, 1H), 7.77 (d, J = 8.2 Hz, 1H), 7.59 (t, J = 7.5 Hz, 1H), 7.29 (m, 6H), 4.73 (dt, J = 5.3 Hz, 7.4 Hz, 1H), 4.42 (s, 2H), 4.36 (m, 2H), 3.73 (m, J = 6.6 Hz, 1H), 3.33 (m, 2H), 1.88 (m, 2H), 1.63 (m, 2H), 1.44 (d, J = 6.4 Hz, 3H), 1.43 (d, J = 6.4 Hz, 3H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.6, 165.0, 162.7, 162.7, 149.5, 141.4, 139.6, 138.6, 135.1, 128.8, 128.4, 127.7, 127.3, 122.4, 121.9, 120.4, 112.8, 112.5, 52.1, 42.7, 42.2, 39.6, 31.2, 31.1, 24.0, 21.8, 21.7.

[0293] Example 20

[0294] 4H-KPiv-OCH3 (M19c)

[0295] Compound M19c (2.34 g, 81.8%) was obtained as a white solid by separating and purifying M18 and pivalaldehyde according to the same preparation protocol as M19a through medium-pressure preparative column chromatography (collecting the product with 25 - 30% EA).

[0296] KPiv-OCH3 (M20c)

[0297] This preparation protocol is the same as that of M20a. After separation and purification by a medium-pressure preparative column (collecting the product with 36% EA), M20c (1.80 g, 78.0%) was obtained as a light brown solid.

[0298] KPiv(M21c)

[0299] This preparation protocol is the same as that of M6. M21c (1.68 g, 98.2%) was obtained as a pale yellow solid.

[0300] KPiv-Orn(Z)-NBzl(M22c)

[0301] From M21c and M14 according to the same preparation protocol as M10a, after separation and purification by a medium-pressure preparative column (collecting the product with 75% EA), M22c (3.04 g, 80.0%) was obtained as a white solid.

[0302] KPiv-Orn-NBzl(M23c)

[0303] This preparation protocol is the same as that of M9. M23c (2.25 g, 95.2%) was obtained as a white solid.

[0304] KPiv-Orn(Cl)-NBzl(20)

[0305] From M23c and 2-chloroethyl chloroacetimidate according to the same preparation protocol as 8, after separation and purification by C18 silica gel column chromatography (collecting the product with 45% CH3OH), 20 (0.66 g, 60.3%) was obtained as a white solid. Purity: 99.75%. m.p.: 149.5 - 151.4 °C. =-60.0 (C=1 mg / mL, CH3OH). HR-MS (m / z): 547.2583 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 11.67 (s, 1H), 10.26 (s, 1H), 9.66 (s, 1H), 9.21 (s, 1H), 8.97 (t, J=5.4 Hz, 1H), 8.81 (d, J=8.3 Hz, 1H), 8.73 (s, 1H), 8.36 (d, J=7.8 Hz, 1H), 7.76 (d, J=8.2 Hz, 1H), 7.59 (t, J=7.6 Hz, 1H), 7.29 (m, 6H), 4.73 (dt, J=4.7 Hz, 7.3 Hz, 1H), 4.43 (s, 2H), 4.36 (m, 2H), 3.39 (m, 2H), 1.87 (m, 2H), 1.65 (m, 2H), 1.62 (s, 9H). 1313C-NMR(75MHz, DMSO-d6): δ(ppm) = 171.6, 164.9, 162.7, 162.6, 150.9, 141.2, 139.6, 137.5, 134.0, 129.6, 128.8, 127.6, 127.3, 122.1, 121.3, 120.5, 113.0, 112.8, 51.9, 42.6, 42.2, 39.6, 37.9, 31.3, 29.0, 23.9.

[0306] Example 21

[0307] 4H-KIvr-OCH3 (M19d)

[0308] M19d (2.44 g, 85.1%) was obtained as a white solid by separating and purifying M18 and isovaleraldehyde according to the same preparation scheme as M19a on a medium-pressure preparative column (collecting the product with 25 - 30% EA).

[0309] KIvr-OCH3 (M20d)

[0310] The preparation scheme was the same as that of M20a. M20d (2.10 g, 87.5%) was obtained as a light brown solid by separating and purifying on a medium-pressure preparative column (collecting the product with 30% EA).

[0311] KIvr (M21d)

[0312] The preparation scheme was the same as that of M6. M21d (1.72 g, 86.4%) was obtained as a yellow solid.

[0313] KIvr-Orn(Z)-NBzl (M22d)

[0314] M22d (3.15 g, 80.9%) was obtained as a white solid by separating and purifying M21d and M14 according to the same preparation scheme as M10a on a medium-pressure preparative column (collecting the product with 60% EA).

[0315] KIvr-Orn-NBzl (M23d)

[0316] The preparation scheme was the same as that of M9. M23d (2.30 g, 93.8%) was obtained as a white solid.

[0317] KIvr-Orn(Cl)-NBzl (21)

[0318] Compound 21 (0.68 g, 62.2%) was obtained as a white solid by separating and purifying M23d and 2-chloroacetimidoylethyl ester according to the same preparation protocol as 8 through C18 silica gel column chromatography (collecting the product with 60% CH3OH). Purity: 99.67%. m.p.: 143.7 - 144.3 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 547.2583 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.06 (s, 1H), 10.22 (s, 1H), 9.62 (s, 1H), 9.20 (s, 1H), 8.94 (t, J = 5.4 Hz, 1H), 8.74 (d, J = 8.5 Hz, 1H), 8.70 (s, 1H), 8.36 (d, J = 7.7 Hz, 1H), 7.66 (d, J = 8.1 Hz, 1H), 7.59 (t, J = 7.3 Hz, 1H), 7.29 (m, 6H), 4.74 (dt, J = 5.6 Hz, 7.3 Hz, 1H), 4.42 (s, 2H), 4.35 (d, J = 4.5 Hz, 2H), 3.34 (m, 2H), 3.07 (d, J = 7.1 Hz, 2H), 2.35 (m, J = 6.6 Hz, 1H), 1.87 (m, 2H), 1.61 (m, 2H), 1.00 (d, J = 6.0 Hz, 3H), 0.99 (d, J = 6.0 Hz, 3H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.6, 164.9, 162.7, 144.5, 141.4, 139.6, 138.8, 136.6, 128.8, 128.2, 127.7, 127.3, 122.5, 121.8, 120.4, 112.7, 112.5, 52.1, 42.6, 42.4, 42.2, 31.1, 28.3, 24.0, 22.9, 22.8.

[0319] Example 22

[0320] 4H-KEb-OCH3 (M19e)

[0321] Compound M19e (2.45 g, 85.1%) was obtained as a white solid by separating and purifying M18 and 2-ethylbutyraldehyde according to the same preparation protocol as M19a through medium-pressure preparative column chromatography (collecting the product with 25% EA).

[0322] KEb-OCH3 (M20e)

[0323] This preparation protocol is the same as that of M20a. After separation and purification by medium-pressure preparative column (collecting the product with 34% EA), M20e (2.20 g, 91.0%) was obtained as a light brown solid.

[0324] KEb (M21e)

[0325] This preparation protocol is the same as that of M6. M21e (1.88 g, 89.7%) was obtained as a yellow solid.

[0326] KEb-Orn(Z)-NBzl (M22e)

[0327] From M21e and M14 according to the same preparation protocol as M10a, after separation and purification by medium-pressure preparative column (collecting the product with 55% EA), M22e (3.20 g, 77.5%) was obtained as a yellow solid.

[0328] KEb-Orn-NBzl (M23e)

[0329] This preparation protocol is the same as that of M9. M23e (2.48 g, 99.1%) was obtained as a light yellow solid.

[0330] KEb-Orn(Cl)-NBzl (22)

[0331] From M23e and 2-chloroacetimidoyl ethyl ester according to the same preparation protocol as 8, after separation and purification by C18 silica gel column chromatography (collecting the product with 62% CH3OH), 22 (0.95 g, 56.4%) was obtained as a white solid. Purity: 98.79%. m.p.: 97.8 - 98.7 °C. α = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 561.2739 [M+H] + . 11H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.05 (s, 1H), 10.24 (s, 1H), 9.64 (s, 1H), 9.20 (s, 1H), 8.96 (t, J = 5.6 Hz, 1H), 8.80 (d, J = 8.3 Hz, 1H), 8.68 (s, 1H), 8.37 (d, J = 7.8 Hz, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.59 (t, J = 7.5 Hz, 1H), 7.30 (m, 6H), 4.73 (dt, J = 5.6 Hz, 7.0 Hz, 1H), 4.43 (s, 2H), 4.36 (m, 2H), 3.34 (m, 2H), 2.49 (m, 1H), 1.94 (m, 2H), 1.88 (m, 2H), 1.82 (m, 2H), 1.62 (m, 2H), 0.80 (t, J = 6.9 Hz, 3H), 0.78 (t, J = 7.0 Hz, 3H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.6, 165.0, 162.7, 147.9, 141.4, 139.6, 138.9, 137.0, 128.8, 128.2, 127.7, 127.3, 122.5, 121.9, 120.4, 112.7, 112.2, 52.0, 45.1, 42.6, 42.2, 39.6, 31.1, 27.4, 23.9, 12.4.

[0332] Example 23

[0333] 4H-KH-OCH3 (M19f)

[0334] M19f (1.89 g, 63.0%) was obtained as a yellow oil by separating and purifying M18 and n-hexanal according to the same preparation scheme as M19a on a medium-pressure preparative column (collecting the product with 25% EA).

[0335] KH-OCH3 (M20f)

[0336] The preparation scheme was the same as that of M20a. M20f (1.00 g, 53.4%) was obtained as a brown solid by separating and purifying on a medium-pressure preparative column (collecting the product with 35 - 40% EA).

[0337] KH (M21f)

[0338] The preparation scheme was the same as that of M6. M21f (0.81 g, 85.4%) was obtained as a yellow solid.

[0339] KH-Orn(Z)-NBzl (M22f)

[0340] M22f (0.94 g, 52.9%) was obtained as a yellow solid by separating and purifying M21f and M14 with a medium-pressure preparation column according to the same preparation protocol as M10a (collecting the product with 70% EA).

[0341] KH-Orn-NBzl (M23f)

[0342] M23f (0.72 g, 97.7%) was obtained as a yellow solid according to the same preparation protocol as M9.

[0343] KH-Orn(Cl)-NBzl(23)

[0344] 23 (0.45 g, 54.0%) was obtained as a white solid by separating and purifying M23f and 2-chloroethyl iminoacetate with a C18 silica gel column chromatography according to the same preparation protocol as 8 (collecting the product with 58% CH3OH). Purity: 99.78%. m.p.: 144.4 - 145.3 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 561.2739 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.11 (s, 1H), 10.29 (s, 1H), 9.67 (s, 1H), 9.22 (s, 1H), 8.97 (t, J = 5.6 Hz, 1H), 8.74 (d, J = 8.4 Hz, 1H), 8.70 (s, 1H), 8.36 (d, J = 7.8 Hz, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.59 (t, J = 7.5 Hz, 1H), 7.29 (m, 6H), 4.75 (dt, J = 5.6 Hz, 7.2 Hz, 1H), 4.45 (s, 2H), 4.36 (d, J = 5.3 Hz, 2H), 3.36 (m, 2H), 3.19 (t, J = 7.4 Hz, 2H), 1.87 (m, 4H), 1.63 (m, 2H), 1.41 (m, 4H), 0.90 (t, J = 6.4 Hz, 3H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.6, 164.9, 162.7, 145.3, 141.4, 139.6, 138.7, 136.1, 128.8, 128.8, 128.2, 127.7, 127.3, 122.5, 121.8, 120.4, 112.8, 112.7, 52.1, 42.6, 42.2, 39.7, 33.6, 31.6, 31.1, 27.9, 24.0, 22.5, 14.4.

[0345] Example 24

[0346] 4H-KDa-OCH3 (M19g)

[0347] M19g (1.96 g, 55.1%) was obtained as a yellow oil by separating and purifying M18 and decanal according to the same preparation scheme as M19a on a medium-pressure preparative column (collecting the product with 25% EA).

[0348] KDa-OCH3 (M20g)

[0349] The preparation scheme was the same as that of M20a. M20g (1.24 g, 64.2%) was obtained as a yellow solid by separating and purifying on a medium-pressure preparative column (collecting the product with 35% EA).

[0350] KDa (M21g)

[0351] The preparation scheme was the same as that of M6. M21g (1.08 g, 90.4%) was obtained as a light yellow solid.

[0352] KDa-Orn(Z)-NBzl (M22g)

[0353] M22g (1.16 g, 53.8%) was obtained as a light yellow solid by separating and purifying M21g and M14 according to the same preparation scheme as M10a on a medium-pressure preparative column (collecting the product with 50 - 55% EA).

[0354] KDa-Orn-NBzl (M23g)

[0355] The preparation scheme was the same as that of M9. M23g (0.89 g, 95.7%) was obtained as a light brown solid.

[0356] KDa-Orn(Cl)-NBzl (24)

[0357] 24 (0.62 g, 61.1%) was obtained as a white solid by separating and purifying M23g and 2-chloroacetyl iminoethyl ester according to the same preparation scheme as 8 on a C18 silica gel column chromatography (collecting the product with 75% CH3OH). Purity: 99.60%. m.p.: 192.5 - 193.5 °C. =-60.0 (C=1 mg / mL, CH3OH). HR-MS (m / z): 617.3365 [M + H] + . 11H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.06 (s, 1H), 10.23 (s, 1H), 9.64 (s, 1H), 9.20 (s, 1H), 8.94 (t, J = 5.4 Hz, 1H), 8.74 (d, J = 8.4 Hz, 1H), 8.70 (s, 1H), 8.35 (d, J = 7.9 Hz, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.39 (m, 6H), 4.74 (dt, J = 4.7 Hz, 7.5 Hz, 1H), 4.43 (s, 2H), 4.36 (d, J = 4.9 Hz, 2H), 3.33 (m, 2H), 3.18 (t, J = 7.0 Hz, 2H), 1.88 (m, 4H), 1.63 (m, 2H), 1.40 (m, 4H), 1.23 (m, 8H), 0.83 (t, J = 5.5 Hz, 3H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.6, 164.9, 162.7, 145.3, 141.4, 139.6, 138.8, 136.1, 128.8, 128.1, 127.7, 127.3, 122.5, 121.9, 120.4, 112.7, 112.6, 52.1, 42.6, 42.2, 39.6, 33.7, 31.7, 31.1, 29.5, 29.2, 28.3, 24.0, 22.5, 14.4.

[0358] Example 25

[0359] 4H-KMMO-OCH3 (M19h)

[0360] M19h (1.53 g, 48.1%) was obtained as a pale yellow solid by separating and purifying M18 and 1,1,3,3-tetramethoxypropane according to the same preparation scheme as M19a by medium-pressure preparative column (collecting the product with 33% EA).

[0361] KMMO-OCH3 (M20h)

[0362] M20h (0.82 g, 54.3%) was obtained as a pale yellow solid by separating and purifying according to the same preparation scheme as M20a by medium-pressure preparative column (collecting the product with 30 - 40% EA).

[0363] KMMO (M21h)

[0364] M21h (0.68 g, 86.8%) was obtained as a yellow solid by the same preparation scheme as M6.

[0365] KMMO-Orn(Z)-NBzl(M22h)

[0366] M22h (1.05 g, 72.7%) was obtained as a yellow solid by separating and purifying M21h and M14 according to the same preparation protocol as M10a on a medium-pressure preparative column (collecting the product with 65% EA).

[0367] KMMO-Orn-NBzl(M23h)

[0368] The same preparation protocol as M9 was used to obtain M23h (0.80 g, 95.9%) as a pale yellow solid.

[0369] KMMO-Orn(Cl)-NBzl(25)

[0370] 25 (0.55 g, 60.1%) was obtained as a light blue solid by separating and purifying M23h and 2-chloroethyl imidoacetate according to the same preparation protocol as 8 on a C18 silica gel column (collecting the product with 60% CH3OH). Purity: 98.74%. m.p.: 101.9 - 102.4 °C. =-60.0 (C=1 mg / mL, CH3OH). HR-MS (m / z): 579.2481 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.15 (s, 1H), 10.30 (s, 1H), 9.67 (s, 1H), 9.23 (m, 1H), 8.96 (t, J=5.7 Hz, 1H), 8.75 (d, J=8.0 Hz, 1H), 8.74 (s, 1H), 8.37 (d, J=7.9 Hz, 1H), 7.68 (d, J=8.2 Hz, 1H), 7.60 (t, J=7.5 Hz, 1H), 7.29 (m, 6H), 5.18 (t, J=5.6 Hz, 1H), 4.74 (dt, J=5.3 Hz, 7.4 Hz, 1H), 4.44 (s, 2H), 4.36 (d, J=5.5 Hz, 2H), 3.51 (d, J=5.7 Hz, 2H), 3.38 (m, 2H), 3.33 (s, 3H), 3.32 (s, 3H), 1.89 (m, 2H), 1.63 (m, 2H). 13C-NMR (75MHz, DMSO-d6): δ (ppm) = 171.7, 164.8, 162.7, 141.5, 140.7, 139.6, 136.7, 129.0, 128.8, 128.5, 127.7,127.3,122.6,121.7,120.5,113.0,112.8,103.5,53.4,53.4,52.2,42.6,42.2,37.6,30.9,24.0.

[0371] Example 26

[0372] 4H-KC3-OCH3(M19i)

[0373] M19i (2.10 g, 78.0%) was obtained from M18 and cyclopropanecarboxaldehyde according to the same preparation scheme as M19a, and separated and purified by medium-pressure preparative column (product was collected in 38-43% EA) as a white solid.

[0374] KC3-OCH3(M20i)

[0375] The preparation scheme was the same as that of M20a. M20i (1.48 g, 71.3%) was obtained as a light yellow solid by separation and purification via medium-pressure preparative column (product was collected in 38% EA).

[0376] KC3(M21i)

[0377] The preparation protocol was the same as for M6 to afford M21i (1.24 g, 88.6%) as a light yellow solid.

[0378] KC3-Orn(Z)-NBzl(M22i)

[0379] M21i and M14 were prepared in the same manner as M10a and separated and purified by medium-pressure preparative column (product was collected in 65% EA) to give M22i (2.60 g, 89.7%) as a white solid.

[0380] KC3-Orn-NBzl(M23i)

[0381] The preparation protocol was the same as for M9 to afford M23i (1.96 g, 97.6%) as a white solid.

[0382] KC3-Orn(Cl)-NBzl(26)

[0383] Compound 26 (1.56 g, 68.2%) was obtained as a white solid by separating and purifying M23i and ethyl 2-chloroacetimidate according to the same preparation protocol as 8 through C18 silica gel column chromatography (collecting the product with 55% CH3OH). Purity: 97.25%. m.p.: 142.5 - 143.3 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 531.2270 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.34 (s, 1H), 10.26 (t, J = 4.6 Hz, 1H), 9.65 (s, 1H), 9.20 (s, 1H), 8.94 (t, J = 5.9 Hz, 1H), 8.66 (d, J = 8.4 Hz, 1H), 8.62 (s, 1H), 8.36 (d, J = 7.9 Hz, 1H), 7.69 (d, J = 8.1 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.32 (m, 5H), 7.24 (m, 1H), 4.70 (dt, J = 5.1 Hz, 8.1 Hz, 1H), 4.44 (s, 2H), 4.36 (m, 2H), 3.35 (dt, J = 6.3 Hz, 6.1 Hz, 2H), 2.78 (m, 1H), 1.86 (m, 2H), 1.60 (m, 2H), 1.25 (m, 2H), 1.18 (m, 2H). 13 C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.6, 164.9, 162.7, 145.8, 141.4, 139.6, 138.8, 136.4, 128.8, 127.6, 127.3, 122.6, 121.9, 120.4, 112.8, 111.8, 52.0, 42.6, 42.2, 39.6, 31.0, 23.9, 12.8, 10.1.

[0384] Example 27

[0385] 4H-KC6-OCH3 (M19j)

[0386] Compound M19j (2.65 g, 85.9%) was obtained as a white solid by separating and purifying M18 and cyclohexanecarbaldehyde according to the same preparation protocol as M19a through medium-pressure preparative column chromatography (collecting the product with 22% EA).

[0387] KC6-OCH3 (M20j)

[0388] This preparation protocol is the same as that of M20a. After separation and purification by a medium-pressure preparative column (collecting the product with 18% EA), M20j (1.98 g, 75.7%) was obtained as a white solid.

[0389] KC6(M21j)

[0390] This preparation protocol is the same as that of M6. M21j (1.13 g, 59.8%) was obtained as a pale yellow solid.

[0391] KC6-Orn(Z)-NBzl(M22j)

[0392] From M21j and M14 according to the same preparation protocol as M10a, after separation and purification by a medium-pressure preparative column (collecting the product with 50 - 55% EA), M22j (1.84 g, 75.9%) was obtained as a white solid.

[0393] KC6-Orn-NBzl(M23j)

[0394] This preparation protocol is the same as that of M9. M23j (1.13 g, 78.0%) was obtained as a white solid.

[0395] KC6-Orn(Cl)-NBzl(27)

[0396] From M23j and 2-chloroethyl iminoacetate according to the same preparation protocol as 8, after separation and purification by C18 silica gel column chromatography (collecting the product with 55% CH3OH), 27 (0.99 g, 76.0%) was obtained as a white solid. Purity: 99.35%. m.p.: 168.9 - 169.7 °C. =-60.0 (C=1 mg / mL, CH3OH). HR-MS (m / z): 573.2739 [M+H] + . 11H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.07 (s, 1H), 10.27 (t, J = 5.4 Hz, 1H), 9.64 (s, 1H), 9.19 (s, 1H), 8.96 (t, J = 5.4 Hz, 1H), 8.77 (d, J = 8.3 Hz, 1H), 8.68 (s, 1H), 8.34 (d, J = 7.8 Hz, 1H), 7.67 (d, J = 8.2 Hz, 1H), 7.58 (t, J = 7.5 Hz, 1H), 7.31 (m, 5H), 7.25 (m, 1H), 4.75 (dt, J = 5.3 Hz, 7.4 Hz, 1H), 4.44 (s, 2H), 4.37 (m, 2H), 3.39 (m, 1H), 3.37 (m, 2H), 2.00 (m, 2H), 1.92 (m, 2H), 1.80 (m, 5H), 1.49 (m, 5H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.6, 165.0, 162.7, 149.0, 141.3, 139.6, 138.7, 135.2, 128.8, 128.3, 127.6, 127.3, 122.4, 121.9, 120.3, 112.7, 112.5, 52.0, 42.6, 42.2, 41.1, 39.6, 31.5, 31.2, 26.5, 26.2, 24.0.

[0397] Example 28

[0398] 4H-KB-OCH3 (M19k)

[0399] M19k (0.81 g, 26.5%) was obtained as a white solid by separating and purifying M18 and benzaldehyde according to the same preparation scheme as M19a on a medium-pressure preparative column (collecting the product with 30 - 40% EA).

[0400] KB-OCH3 (M20k)

[0401] Following the same preparation scheme as M5b, M20k (0.60 g, 75.1%) was obtained as a white solid.

[0402] KB (M21k)

[0403] Following the same preparation scheme as M6, M21k (0.40 g, 69.9%) was obtained as a pale yellow solid.

[0404] KB-Orn(Z)-NBzl (M22k)

[0405] M22k (0.47 g, 54.1%) was obtained as a white solid by separating and purifying M21k and M14 according to the same preparation protocol as M10a using a medium-pressure preparation column (collecting the product with 7% CH3OH).

[0406] KB-Orn-NBzl (M23k)

[0407] The same preparation protocol as M9 was used to obtain M23k (0.32 g, 86.7%) as a white solid.

[0408] KB-Orn(Cl)-NBzl(28)

[0409] 28 (0.11 g, 28.4%) was obtained as a white solid by separating and purifying M23k and 2-chloroethyl imidoacetate according to the same preparation protocol as 8 using a C18 silica gel column chromatography (collecting the product with 60% CH3OH). Purity: 99.65%. m.p.: 156.7 - 157.8 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 567.2270 [M + H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 11.95 (s, 1H), 10.20 (s, 1H), 9.62 (s, 1H), 9.19 (s, 1H), 8.92 (t, J = 5.4 Hz, 1H), 8.88 (s, 1H), 8.83 (d, J = 8.6 Hz, 1H), 8.44 (d, J = 7.8 Hz, 1H), 8.11 (d, J = 7.2 Hz, 2H), 7.66 (m, 5H), 7.30 (m, 6H), 4.77 (m, 1H), 4.41 (s, 2H), 4.35 (d, J = 1.6 Hz, 2H), 3.32 (m, 2H), 1.90 (m, 2H), 1.63 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.6, 164.8, 162.6, 142.1, 141.2, 139.6, 137.9, 134.9, 130.4, 129.5, 129.4, 129.2, 129.0, 128.8, 127.6, 127.3, 122.5, 121.6, 120.8, 113.6, 113.3, 52.2, 42.6, 42.2, 39.6, 30.8, 24.1.

[0410] Example 29

[0411] K4PD-OCH3 (M20l)

[0412] M20l (1.02 g, 33.7%) was obtained as a yellow solid by reacting M18 and 4-pyridinecarboxaldehyde according to the same preparation protocol as M19a, followed by purification using medium-pressure preparative column chromatography (collecting the product with 7% CH3OH) and reacting at room temperature for 48 h.

[0413] K4PD(M21l)

[0414] This preparation protocol is the same as M6, and M21l (0.85 g, 87.4%) was obtained as a yellow solid.

[0415] K4PD-Orn(Z)-NBzl(M22l)

[0416] M22l (1.68 g, 91.0%) was obtained as a pale yellow solid by reacting M21l and M14 according to the same preparation protocol as M10a, followed by purification using medium-pressure preparative column chromatography (collecting the product with 9% CH3OH).

[0417] K4PD-Orn-NBzl(M23l)

[0418] This preparation protocol is the same as M9, and M23l (0.94 g, 71.8%) was obtained as a pale yellow solid.

[0419] K4PD-Orn(Cl)-NBzl(29)

[0420] 29 (0.30 g, 27.5%) was obtained as a yellow solid by reacting M23l and 2-chloroethyl chloroacetimidate according to the same preparation protocol as 8, followed by purification using C18 silica gel column chromatography (collecting the product with 55% CH3OH). Purity: 96.96%. m.p.: 257.8 - 258.4 °C. =-60.0 (C=1 mg / mL, CH3OH). HR-MS (m / z): 568.2222 [M+H] + . 11H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.13 (s, 1H), 10.13 (s, 1H), 9.77 (s, 1H), 9.57 (s, 1H), 8.97 (s, 1H), 8.89 (d, J = 5.8 Hz, 2H), 8.85 (d, J = 7.4 Hz, 1H), 8.84 (t, J = 9.0 Hz, 1H), 8.48 (d, J = 7.8 Hz, 1H), 8.14 (d, J = 5.7 Hz, 2H), 7.74 (d, J = 8.3 Hz, 1H), 7.65 (t, J = 7.5 Hz, 1H), 7.31 (m, 6H), 4.74 (m, 1H), 4.39 (s, 2H), 4.35 (d, J = 4.0 Hz, 2H), 3.31 (m, 2H), 1.91 (m, 2H), 1.64 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.6, 164.6, 162.6, 150.4, 142.3, 139.6, 134.9, 131.1, 129.6, 128.8, 127.6, 127.3, 123.6, 122.7, 121.4, 121.1, 115.0, 113.2, 52.4, 42.6, 42.2, 30.6, 24.1.

[0421] Example 30

[0422] K2IM-OCH3 (M20m)

[0423] M20m (1.05 g, 36.0%) was obtained as a milky white solid by reacting M18 and imidazole-2-carbaldehyde according to the same preparation protocol as M19a at room temperature for 48 h and then separating and purifying by medium-pressure preparative column (collecting the product with 80% EA).

[0424] K2IM (M21m)

[0425] The preparation protocol was the same as that of M6, and M21m (0.92 g, 92.0%) was obtained as a yellow solid.

[0426] K2IM-Orn(Z)-NBzl (M22m)

[0427] M22m (1.52 g, 70.1%) was obtained as a light yellow solid by reacting M21m and M14 according to the same preparation protocol as M10a and then separating and purifying by medium-pressure preparative column (collecting the product with 7% CH3OH).

[0428] K2IM-Orn-NBzl (M23m)

[0429] This preparation protocol is the same as that of M9, obtaining M23m (0.98 g, 82.4%), which is a pale yellow solid.

[0430] K2IM-Orn(Cl)-NBzl(30)

[0431] From M23m and 2-chloroethyl imidoacetate according to the same preparation protocol as 8, after purification by C18 silica gel column chromatography (collecting the product with 40% CH3OH), 30 (0.26 g, 23.4%) was obtained, which is a pale yellow solid. Purity: 99.32%. m.p.: 107.4 - 108.2 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 557.2175 [M + H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 11.83 (s, 1H), 9.95 (s, 1H), 9.51 (s, 1H), 9.26 (d, J = 8.9 Hz, 1H), 9.10 (s, 1H), 8.84 (s, 1H), 8.65 (t, J = 5.7 Hz, 1H), 8.41 (d, J = 7.8 Hz, 1H), 7.96 (d, J = 8.2 Hz, 1H), 7.60 (t, J = 7.5 Hz, 1H), 7.51 (s, 2H), 7.30 (m, 5H), 7.24 (m, 1H), 4.73 (m, 1H), 4.35 (s, 2H), 4.34 (d, J = 1.4 Hz, 2H), 3.33 (m, 2H), 2.00 (m, 2H), 1.68 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.9, 165.2, 162.7, 145.6, 142.1, 139.9, 138.8, 133.2, 131.0, 130.1, 129.1, 128.7, 127.6, 127.2, 122.4, 121.4, 120.8, 114.0, 52.8, 42.5, 42.3, 29.4, 24.6.

[0432] Example 31

[0433] 4H-K2SF-OCH3 (M19n)

[0434] From M18 and thiophene-2-carbaldehyde according to the same preparation protocol as M19a, after purification by medium-pressure preparative column (collecting the product with 25 - 30% EA), M19n (2.92 g, 93.6%) was obtained, which is a light brown solid.

[0435] K2SF-OCH3 (M20n)

[0436] This preparation protocol is the same as that of M5b, and M20n (1.98 g, 68.7%) is obtained, which is a light brown solid.

[0437] K2SF(M21n)

[0438] This preparation protocol is the same as that of M6, and M21n (1.74 g, 91.8%) is obtained, which is a light brown solid.

[0439] K2SF-Orn(Boc)-NBzl(M22n)

[0440] From M21n and M9 according to the same preparation protocol as M10a, separated and purified by medium-pressure preparative column (collect the product with 55 - 60% EA), M22n (2.10 g, 59.6%) is obtained, which is a white solid.

[0441] K2SF-Orn(HCl)-NBzl(M23n)

[0442] This preparation protocol is the same as that of M11a, and M23n (1.68 g, 89.5%) is obtained, which is a yellow solid.

[0443] K2SF-Orn(Cl)-NBzl(31)

[0444] From M23n and 2-chloroacetimidoyl ethyl ester according to the same preparation protocol as 8, separated and purified by C18 silica gel column chromatography (collect the product with 55% CH3OH), 31 (0.48 g, 26.6%) is obtained, which is a yellow solid. Purity: 99.97%. m.p.: 163.9 - 164.2 °C. =-60.0 (C=1 mg / mL, CH3OH). HR-MS (m / z): 573.1834 [M+H] + . 11H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.01 (s, 1H), 10.24 (t, J = 4.7 Hz, 1H), 9.64 (s, 1H), 9.20 (s, 1H), 8.98 (t, J = 5.6 Hz, 1H), 8.81 (s, 1H), 8.73 (d, J = 8.3 Hz, 1H), 8.44 (d, J = 7.8 Hz, 1H), 8.26 (d, J = 3.4 Hz, 1H), 7.85 (d, J = 5.0 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.64 (t, J = 7.5 Hz, 1H), 7.32 (m, 7H), 4.76 (dt, J = 5.4 Hz, 7.3 Hz, 1H), 4.43 (s, 2H), 4.37 (d, J = 3.7 Hz, 2H), 3.56 (m, 2H), 1.91 (m, 2H), 1.66 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.5, 164.2, 162.7, 142.6, 142.2, 139.6, 139.0, 135.5, 132.6, 131.0, 129.4, 129.2, 128.8, 127.7, 127.3, 127.3, 122.5, 121.6, 121.1, 113.4, 113.4, 52.1, 42.7, 42.2, 31.1, 23.9.

[0445] Example 32

[0446] 4H-KB4Cl-OCH3 (M19o)

[0447] M19o (3.15 g, 92.6%) was obtained as a white solid by separating and purifying M18 and p-chlorobenzaldehyde according to the same preparation protocol as M19a using a medium-pressure preparative column (collecting the product with 35 - 45% EA).

[0448] KB4Cl-OCH3 (M20o)

[0449] The preparation protocol was the same as that of M5b, and M20o (2.37 g, 76.2%) was obtained as a light brown solid.

[0450] KB4Cl (M21o)

[0451] The preparation protocol was the same as that of M6, and M21o (1.97 g, 86.7%) was obtained as a yellow solid.

[0452] KB4Cl-Orn(Boc)-NBzl (M22o)

[0453] M22o was obtained (3.25 g, 84.9%) as a white solid by separating and purifying M21o and M9 with a medium-pressure preparative column according to the same preparation protocol as M10a (collecting the product with 55 - 60% EA).

[0454] KB4Cl-Orn(HCl)-NBzl(M23o)

[0455] This preparation protocol is the same as that of M11a, and M23o (2.80 g, 95.9%) was obtained as a pale yellow solid.

[0456] KB4Cl-Orn(Cl)-NBzl(32)

[0457] 32 (2.45 g, 81.9%) was obtained as a white solid by separating and purifying M23o and 2-chloroacetimidoylethyl ester with a C18 silica gel column chromatography according to the same preparation protocol as 8 (collecting the product with 54% CH3OH). Purity: 99.96%. m.p.: 206.5 - 207.5 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 601.1880 [M + H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.02 (s, 1H), 10.29 (s, 1H), 9.65 (s, 1H), 9.22 (s, 1H), 8.95 (t, J = 5.4 Hz, 1H), 8.89 (s, 1H), 8.80 (d, J = 8.4 Hz, 1H), 8.45 (d, J = 7.9 Hz, 1H), 8.14 (d, J = 8.3 Hz, 2H), 7.74 (d, J = 8.2 Hz, 2H), 7.72 (d, J = 7.3 Hz, 1H), 7.62 (t, J = 7.5 Hz, 1H), 7.30 (m, 6H), 4.76 (dt, J = 4.7 Hz, 7.9 Hz, 1H), 4.44 (s, 2H), 4.35 (d, J = 3.0 Hz, 2H), 3.34 (m, 2H), 1.92 (m, 2H), 1.64 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.7, 164.7, 162.7, 142.1, 139.9, 139.6, 139.6, 136.7, 134.8, 134.3, 130.9, 130.6, 129.4, 129.3, 128.8, 127.6, 127.3, 122.6, 121.6, 120.8, 114.0, 113.2, 52.3, 42.6, 42.2, 39.6, 30.7, 24.1.

[0458] Example 33

[0459] 4H-KB4F-OCH3(M19p)

[0460] M19p (2.80 g, 86.4%) was obtained as a white solid from M18 and p-fluorobenzaldehyde using a medium-pressure preparative column (32-40% EA to collect the product).

[0461] KB4F-OCH3(M20p)

[0462] The preparation protocol was the same as for M5b to give M20p (2.02 g, 73.1%) as a light brown solid. KB4F (M21p)

[0463] The preparation protocol was the same as for M6 to afford M21p (1.85 g, 95.8%) as a white solid.

[0464] KB4F-Orn(Boc)-NBzl(M22p)

[0465] M21p and M9 were prepared in the same manner as M10a and separated and purified by medium-pressure preparative column (55-60% EA was used to collect the product) to obtain M22p (1.83 g, 49.7%) as a white solid.

[0466] KB4F-Orn(HCl)-NBzl(M23p)

[0467] The preparation protocol was the same as for M11a to afford M23p (1.23 g, 75.0%) as a light yellow solid.

[0468] KB4F-Orn(Cl)-NBzl(33)

[0469] M23p and 2-chloroacetimidoethyl ester were prepared according to the same protocol as for 8. Purification by C18 silica gel column chromatography (58% CH3OH to collect the product) yielded 33 (0.66 g, 50.0%) as a white solid. Purity: 98.72%. MP: 131.7-132.2°C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):585.2176[M+H] + . 1H-NMR(300MHz, DMSO-d6): δ(ppm) = 11.96(s, 1H), 10.14(s, 1H), 9.58(s, 1H), 9.16(s, 1H), 8.87(s, 1H), 8.87(m, 1H), 8.80(d, J = 8.4Hz, 1H), 8.44(d, J = 7.9Hz, 1H), 8.15(dd, J = 8.4Hz, 5.6Hz, 2H), 7.71(d, J = 8.2Hz, 1H), 7.62(t, J = 7.4Hz, 1H), 7.52(dd, J = 8.7Hz, 8.7Hz, 2H), 7.29(m, 6H), 4.75(dt, J = 4.7Hz, 7.8Hz, 1H), 4.39(s, 2H), 4.35(d, J = 2.6Hz, 2H), 3.30(m, 2H), 1.90(m, 2H), 1.63(m, 2H). 13 C-NMR(75MHz, DMSO-d6): δ(ppm) = 171.6, 164.8, 162.7, 142.1, 140.2, 139.6, 134.8, 134.3, 131.3, 131.2, 130.4, 129.2, 128.8, 127.6, 127.3, 122.5, 121.6, 120.8, 116.5, 116.2, 113.7, 113.2, 52.3, 42.6, 42.3, 30.7, 24.1. 19 F-NMR(282MHz, DMSO-d6): δ(ppm) = -112.4.

[0470] Example 34

[0471] 4H-K4CB-OCH3 (M19q)

[0472] M19q (2.22 g, 67.1%) was obtained as a white solid by separating and purifying M18 and 4-cyanobenzaldehyde according to the same preparation scheme as M19a on a medium-pressure preparative column (collecting the product with 40 - 45% EA).

[0473] K4CB-OCH3 (M20q)

[0474] The preparation scheme was the same as that of M5b, and M20q (2.02 g, 92.1%) was obtained as a white solid.

[0475] K4CB (M21q)

[0476] The preparation scheme was the same as that of M6, and M21q (1.86 g, 96.2%) was obtained as a light yellow solid.

[0477] K4CB-Orn(Boc)-NBzl(M22q)

[0478] M22q (2.96 g, 95.9%) was obtained as a white solid by separating and purifying M21q and M9 according to the same preparation protocol as M10a on a medium-pressure preparative column (collecting the product with 45 - 55% EA).

[0479] K4CB-Orn(HCl)-NBzl(M23q)

[0480] The same preparation protocol as M11a was used to obtain M23q (2.60 g, 98.1%) as a light yellow solid.

[0481] K4CB-Orn(Cl)-NBzl(34)

[0482] 34 (0.96 g, 34.5%) was obtained as a white solid by separating and purifying M23q and 2-chloroethyl chloroacetimidate according to the same preparation protocol as 8 on a C18 silica gel column (collecting the product with 45% CH3OH). Purity: 98.88%. m.p.: 265.3 - 266.4 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 592.2222 [M + H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.14 (s, 1H), 10.29 (s, 1H), 9.65 (s, 1H), 9.23 (s, 1H), 8.95 (s, 1H), 8.94 (m, 1H), 8.81 (d, J = 8.4 Hz, 1H), 8.47 (d, J = 7.8 Hz, 1H), 8.33 (d, J = 8.2 Hz, 2H), 8.16 (d, J = 8.2 Hz, 2H), 7.73 (d, J = 8.2 Hz, 1H), 7.64 (t, J = 7.5 Hz, 1H), 7.31 (m, 6H), 4.76 (dt, J = 5.0 Hz, 7.9 Hz, 1H), 4.44 (s, 2H), 4.35 (d, J = 2.7 Hz, 2H), 3.34 (m, 2H), 1.91 (m, 2H), 1.64 (m, 2H). 1313C-NMR(75MHz, DMSO-d6): δ(ppm) = 171.7, 164.7, 162.7, 142.2, 139.8, 139.6, 139.0, 135.0, 133.3, 131.0, 130.0, 129.5, 128.8, 127.6, 127.3, 122.7, 121.5, 121.0, 119.3, 114.6, 113.2, 111.9, 52.4, 42.6, 42.2, 39.6, 30.7, 24.1.

[0483] Example 35

[0484] 4H-KB3N-OCH3 (M19r)

[0485] M19r (2.81 g, 92.6%) was obtained as a yellow solid by separating and purifying M18 and 3-nitrobenzaldehyde according to the same preparation protocol as M19a on a medium-pressure preparative column (collecting the product with 35 - 45% EA).

[0486] KB3N-OCH3 (M20r)

[0487] M20r (2.47 g, 88.9%) was obtained as a white solid according to the same preparation protocol as M5b.

[0488] KB3N (M21r)

[0489] M21r (2.25 g, 94.9%) was obtained as a yellow solid according to the same preparation protocol as M6.

[0490] KB3N-Orn(Boc)-NBzl (M22r)

[0491] M22r (1.86 g, 43.3%) was obtained as a pale yellow solid by separating and purifying M21r and M9 according to the same preparation protocol as M10a on a medium-pressure preparative column (collecting the product with 55 - 60% EA).

[0492] KB3N-Orn(HCl)-NBzl (M23r)

[0493] M23r (1.45 g, 92.5%) was obtained as a pale yellow solid according to the same preparation protocol as M11a.

[0494] KB3N-Orn(Cl)-NBzl (35)

[0495] Compound 35 (1.32 g, 79.6%) was obtained as a yellow solid by separating and purifying M23r and 2-chloroacetimidate ethyl ester according to the same preparation protocol as 8 through C18 silica gel column chromatography (collecting the product with 60% CH3OH). Purity: 99.92%. m.p.: 200.7 - 201.4 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 612.2121 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.13 (s, 1H), 10.10 (s, 1H), 9.57 (s, 1H), 9.16 (s, 1H), 8.96 (s, 1H), 8.85 (t, J = 5.6 Hz, 1H), 8.83 (s, 1H), 8.80 (d, J = 8.9 Hz, 1H), 8.53 (d, J = 7.9 Hz, 1H), 8.48 (d, J = 8.3 Hz, 1H), 8.45 (m, 1H), 7.98 (t, J = 8.0 Hz, 1H), 7.71 (d, J = 8.2 Hz, 1H), 7.65 (t, J = 7.6 Hz, 1H), 7.31 (m, 6H), 4.74 (m, 1H), 4.38 (s, 2H), 4.35 (d, J = 5.3 Hz, 2H), 3.31 (m, 2H), 1.91 (m, 2H), 1.64 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.5, 164.6, 162.6, 148.7, 142.0, 139.8, 139.6, 139.3, 138.8, 135.6, 134.9, 131.1, 130.9, 129.6, 128.8, 127.7, 127.3, 124.2, 123.8, 122.7, 121.6, 121.0, 114.6, 113.1, 52.3, 42.5, 42.2, 30.6, 24.1.

[0496] Example 36

[0497] 4H-KB4O-OCH3 (M19s)

[0498] Compound M19s (1.17 g, 36.3%) was obtained as a white solid by refluxing M18 and p-hydroxybenzaldehyde at 60 °C for 8 h according to the same preparation protocol as M19a and then separating and purifying through medium-pressure preparative column (collecting the product with 55 - 65% EA).

[0499] KB4O-OCH3 (M20s)

[0500] This preparation protocol is the same as M5b, obtaining M20s (1.02 g, 88.3%), which is a white solid.

[0501] KB4O (M21s)

[0502] This preparation protocol is the same as M6, obtaining M21s (0.91 g, 93.3%), which is a pale yellow solid.

[0503] KB4O-Orn(Z)-NBzl (M22s)

[0504] From M21s and M14 according to the same preparation protocol as M10a, separated and purified by medium-pressure preparative column (collecting the product with 70% EA), M22s (0.85 g, 44.3%) was obtained, which is a pale yellow solid.

[0505] KB4O-Orn-NBzl (M23s)

[0506] This preparation protocol is the same as M9, obtaining M23s (0.58 g, 86.3%), which is a pale yellow solid.

[0507] KB4O-Orn(Cl)-NBzl (36)

[0508] From M23s and 2-chloroethyl iminoacetate according to the same preparation protocol as 8, separated and purified by C18 silica gel column chromatography (collecting the product with 30% CH3OH), 36 (0.32 g, 48.0%) was obtained, which is a pale yellow solid. Purity: 99.09%. m.p.: 171.5 - 172.2 °C. =-60.0 (C=1 mg / mL, CH3OH). HR-MS (m / z): 583.2219 [M+H] + . 11H-NMR (300 MHz, DMSO-d6): δ (ppm) = 11.84 (s, 1H), 10.22 (t, J = 4.3 Hz, 1H), 10.00 (s, 1H), 9.62 (s, 1H), 9.18 (s, 1H), 8.93 (t, J = 5.7 Hz, 1H), 8.82 (d, J = 8.8 Hz, 1H), 8.79 (s, 1H), 8.40 (d, J = 7.8 Hz, 1H), 7.96 (d, J = 8.5 Hz, 2H), 7.72 (d, J = 8.2 Hz, 1H), 7.59 (t, J = 7.5 Hz, 1H), 7.29 (m, 6H), 7.08 (d, J = 8.5 Hz, 2H), 4.76 (dt, J = 5.2 Hz, 8.0 Hz, 1H), 4.42 (s, 2H), 4.36 (d, J = 3.0 Hz, 2H), 3.34 (m, 2H), 1.89 (m, 2H), 1.63 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.7, 164.9, 162.7, 159.0, 142.0, 141.6, 139.6, 139.3, 134.5, 130.3, 130.0, 128.9, 128.8, 128.7, 127.7, 127.3, 122.4, 121.7, 120.6, 116.2, 113.2, 112.8, 52.2, 42.6, 42.2, 30.9, 24.1.

[0509] Example 37

[0510] 4H-KB4MO-OCH3 (M19t)

[0511] M19t (2.70 g, 80.4%) was obtained as a white solid by refluxing M18 and p-methoxybenzaldehyde at 60 °C for 8 h according to the same preparation protocol as M19a and then separating and purifying by medium-pressure preparative column (collecting the product with 35 - 45% EA).

[0512] KB4MO-OCH3 (M20t)

[0513] M20t (2.15 g, 80.6%) was obtained as a white solid according to the same preparation protocol as M5b.

[0514] KB4MO (M21t)

[0515] M21t (1.89 g, 91.8%) was obtained as a yellow solid according to the same preparation protocol as M6.

[0516] KB4MO-Orn(Z)-NBzl (M22t)

[0517] M22t was obtained (2.40 g, 91.6%) as a pale yellow solid by separating and purifying M21t and M14 with a medium-pressure preparation column according to the same preparation protocol as M10a (collecting the product with 60 - 65% EA).

[0518] KB4MO-Orn-NBzl (M23t)

[0519] M23t was obtained (1.64 g, 86.2%) as a pale yellow solid according to the same preparation protocol as M9.

[0520] KB4MO-Orn(Cl)-NBzl(37)

[0521] 37 (0.58 g, 30.8%) was obtained as a white solid by separating and purifying M23t and 2-chloroethyl imidoacetate with a C18 silica gel column chromatography according to the same preparation protocol as 8 (collecting the product with 55% CH3OH). Purity: 99.96%. m.p.: 183.6 - 184.5 °C. = - 60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 597.2375 [M + H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 11.93 (s, 1H), 10.30 (t, J = 5.1 Hz, 1H), 9.67 (s, 1H), 9.22 (s, 1H), 8.98 (t, J = 5.7 Hz, 1H), 8.83 (d, J = 7.7 Hz, 1H), 8.82 (s, 1H), 8.42 (d, J = 7.8 Hz, 1H), 8.08 (d, J = 8.6 Hz, 2H), 7.73 (d, J = 8.2 Hz, 1H), 7.61 (t, J = 7.5 Hz, 1H), 7.33 (t, J = 6.3 Hz, 1H), 7.27 (m, 5H), 7.24 (d, J = 8.7 Hz, 2H), 4.77 (dt, J = 4.9 Hz, 7.9 Hz, 1H), 4.44 (s, 2H), 4.36 (d, J = 2.9 Hz, 2H), 3.91 (s, 3H), 3.36 (m, 2H), 1.91 (m, 2H), 1.64 (m, 2H). 1313C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.7, 164.8, 162.7, 160.5, 142.0, 141.1, 139.6, 139.4, 134.6, 130.4, 130.3, 130.1, 129.0, 128.8, 127.6, 127.3, 122.5, 121.7, 120.7, 114.8, 113.2, 113.1, 55.9, 52.2, 42.6, 42.2, 30.9, 24.0.

[0522] Example 38

[0523] 4H-KB26MO-OCH3 (M19u)

[0524] M19u (3.17 g, 86.6%) was obtained as a white solid by separating and purifying M18 and 2,6-dimethoxybenzaldehyde according to the same preparation protocol as M19a on a medium-pressure preparative column (collecting the product with 50 - 60% EA).

[0525] KB26MO-OCH3 (M20u)

[0526] M20u (1.95 g, 62.2%) was obtained as a white solid according to the same preparation protocol as M5b.

[0527] KB26MO (M21u)

[0528] M21u (1.67 g, 89.1%) was obtained as a pale yellow solid according to the same preparation protocol as M6.

[0529] KB26MO-Orn(Z)-NBzl (M22u)

[0530] M22u (2.52 g, 76.8%) was obtained as a white solid by separating and purifying M21u and M14 according to the same preparation protocol as M10a on a medium-pressure preparative column (collecting the product with 75 - 80% EA).

[0531] KB26MO-Orn-NBzl (M23u)

[0532] M23u (1.78 g, 87.6%) was obtained as a white solid according to the same preparation protocol as M9.

[0533] KB26MO-Orn(Cl)-NBzl (38)

[0534] Compound 38 (0.96 g, 47.4%) was obtained as a pale yellow solid by separating and purifying M23u and 2-chloroacetimidoyl ethyl ester according to the same preparation protocol as 8 through C18 silica gel column chromatography (collecting the product with 50% CH3OH). Purity: 99.92%. m.p.: 173.0 - 174.1 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 627.2481 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 11.45 (s, 1H), 10.21 (t, J = 4.8 Hz, 1H), 9.64 (s, 1H), 9.18 (s, 1H), 8.93 (t, J = 5.6 Hz, 1H), 8.82 (s, 1H), 8.56 (d, J = 8.5 Hz, 1H), 8.38 (d, J = 7.9 Hz, 1H), 7.55 (m, 2H), 7.52 (t, J = 8.4 Hz, 1H), 7.27 (m, 6H), 6.90 (d, J = 8.3 Hz, 2H), 4.74 (dt, J = 5.4 Hz, 7.8 Hz, 1H), 4.41 (s, 2H), 4.32 (d, J = 5.6 Hz, 2H), 3.67 (s, 3H), 3.65 (s, 3H), 3.33 (m, 2H), 1.82 (m, 2H), 1.60 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.6, 165.0, 162.7, 159.0, 141.6, 139.5, 139.0, 138.1, 137.2, 131.2, 128.8, 128.4, 127.7, 127.3, 122.4, 121.6, 120.3, 115.2, 113.5, 112.8, 105.4, 56.4, 52.2, 42.6, 42.2, 39.6, 31.0, 24.1.

[0535] Example 39

[0536] 4H-KB34MO-OCH3 (M19v)

[0537] Compound M19v (2.50 g, 68.3%) was obtained as a white solid by separating and purifying M18 and 3,4-dimethoxybenzaldehyde according to the same preparation protocol as M19a through medium-pressure preparative column chromatography (collecting the product with 50 - 60% EA).

[0538] KB34MO-OCH3 (M20v)

[0539] This preparation protocol is the same as that of M5b, and M20v (1.51 g, 61.1%) is obtained as a white solid.

[0540] KB34MO (M21v)

[0541] This preparation protocol is the same as that of M6, and M21v (1.02 g, 70.3%) is obtained as a pale yellow solid.

[0542] KB34MO-Orn(Z)-NBzl (M22v)

[0543] From M21v and M14, following the same preparation protocol as M10a, and separated and purified by medium-pressure preparative column (collecting the product with 65 - 70% EA), M22v (1.07 g, 53.3%) is obtained as a white solid.

[0544] KB34MO-Orn-NBzl (M23v)

[0545] This preparation protocol is the same as that of M9, and M23v (0.71 g, 82.5%) is obtained as a white solid.

[0546] KB34MO-Orn(Cl)-NBzl (39)

[0547] From M23v and 2-chloroacetyl iminoethyl ester, following the same preparation protocol as 8, and separated and purified by C18 silica gel column chromatography (collecting the product with 53% CH3OH), 39 (0.38 g, 47.0%) is obtained as a pale yellow solid. Purity: 99.22%. m.p.: 151.9 - 152.8 °C. = - 60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 627.2481 [M + H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 11.95 (s, 1H), 10.24 (t, J = 5.2 Hz, 1H), 9.64 (s, 1H), 9.20 (s, 1H), 8.96 (t, J = 5.7 Hz, 1H), 8.93 (d, J = 8.0 Hz, 1H), 8.82 (s, 1H), 8.43 (d, J = 7.9 Hz, 1H), 7.70 (m, 3H), 7.61 (t, J = 7.6 Hz, 1H), 7.29 (m, 7H), 4.75 (dt, J = 5.0 Hz, 7.9 Hz, 1H), 4.42 (s, 2H), 4.35 (d, J = 5.6 Hz, 2H), 3.94 (s, 3H), 3.90 (s, 3H), 3.34 (m, 2H), 1.90 (m, 2H), 1.65 (m, 2H). 1313C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.7, 164.8, 162.7, 150.2, 149.4, 142.0, 141.2, 139.6, 139.3, 134.6, 130.5, 130.2, 129.0, 128.8, 127.7, 127.3, 122.4, 121.7, 121.5, 120.7, 113.2, 113.0, 112.5, 56.3, 55.9, 52.2, 42.6, 42.2, 39.6, 31.0, 24.0.

[0548] Example 40

[0549] 4H-KB246MO-OCH3 (M19w)

[0550] M19w (1.21 g, 30.6%) was obtained as a light brown solid by separating and purifying M18 and 2,4,6-trimethoxybenzaldehyde according to the same preparation scheme as M19a on a medium-pressure preparative column (collecting the product with 50 - 60% EA).

[0551] KB246MO-OCH3 (M20w)

[0552] M20w (0.74 g, 61.4%) was obtained as a light brown solid according to the same preparation scheme as M5b.

[0553] KB246MO (M21w)

[0554] M21w (0.62 g, 87.3%) was obtained as a yellow solid according to the same preparation scheme as M6.

[0555] KB246MO-Orn(Z)-NBzl (M22w)

[0556] M22w (0.73 g, 62.2%) was obtained as a white solid by separating and purifying M21w and M14 according to the same preparation scheme as M10a on a medium-pressure preparative column (collecting the product with 70% EA).

[0557] KB246MO-Orn-NBzl (M23w)

[0558] M23w (0.52 g, 86.8%) was obtained as a white solid according to the same preparation scheme as M9.

[0559] KB246MO-Orn(Cl)-NBzl (40)

[0560] 40 was obtained as a white solid (0.26 g, 44.7%) by separating and purifying M23W and 2-chloroacetimidoyl ethyl ester through C18 silica gel column chromatography (collecting the product with 64% CH3OH) according to the same preparation protocol as 8. Purity: 99.87%. m.p.: 179.7 - 180.7 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 657.2587 [M + H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 11.41 (s, 1H), 10.22 (s, 1H), 9.66 (s, 1H), 9.20 (m, 1H), 8.93 (t, J = 5.4 Hz, 1H), 8.79 (s, 1H), 8.57 (d, J = 8.3 Hz, 1H), 8.36 (d, J = 7.8 Hz, 1H), 7.55 (m, 2H), 7.29 (m, 6H), 6.47 (s, 2H), 4.74 (dt, J = 5.5 Hz, 7.7 Hz, 1H), 4.42 (s, 2H), 4.32 (d, J = 5.3 Hz, 2H), 3.91 (s, 3H), 3.67 (s, 3H), 3.64 (s, 3H), 3.33 (m, 2H), 1.84 (m, 2H), 1.59 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.6, 165.0, 162.6, 162.4, 159.7, 141.5, 139.5, 138.2, 137.5, 128.8, 127.7, 127.3, 122.4, 121.7, 120.3, 112.8, 92.1, 56.4, 56.0, 52.2, 42.6, 42.2, 31.0, 24.1.

[0561] Example 41

[0562] 4H-KB4M-OCH3 (M19x)

[0563] M19x (2.84 g, 88.6%) was obtained as a white solid by separating and purifying M18 and p-methylbenzaldehyde through medium-pressure preparative column (collecting the product with 25 - 35% EA) according to the same preparation protocol as M19a.

[0564] KB4M-OCH3 (M20x)

[0565] Following the same preparation protocol as M5b, M20x (2.05 g, 73.2%) was obtained as a light brown solid.

[0566] KB4M (M21x)

[0567] This preparation protocol is the same as that of M6, obtaining M21x (1.74 g, 89.1%), which is a yellow solid.

[0568] KB4M-Orn(Z)-NBzl(M22x)

[0569] From M21x and M14 according to the same preparation protocol as M10a, separated and purified by a medium-pressure preparative column (collecting the product with 55 - 65% EA), M22x (2.94 g, 79.6%) was obtained, which is a white solid.

[0570] KB4M-Orn-NBzl(M23x)

[0571] This preparation protocol is the same as that of M9, obtaining M23x (2.18 g, 93.8%), which is a white solid.

[0572] KB4M-Orn(Cl)-NBzl(41)

[0573] From M23x and 2-chloroacetimidoylethyl ester according to the same preparation protocol as 8, separated and purified by C18 silica gel column chromatography (collecting the product with 80% CH3OH), 41 (0.18 g, 15.9%) was obtained, which is a light pink solid. Purity: 95.45%. m.p.: 72.4 - 73.1 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 581.2426 [M + H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 11.86 (s, 1H), 8.84 (s, 1H), 8.77 (d, J = 5.5 Hz, 1H), 8.75 (t, J = 6.7 Hz, 1H), 8.43 (d, J = 7.8 Hz, 1H), 8.25 (t, J = 4.7 Hz, 1H), 7.99 (d, J = 7.8 Hz, 2H), 7.70 (d, J = 8.3 Hz, 2H), 7.60 (t, J = 7.6 Hz, 1H), 7.49 (d, J = 7.8 Hz, 2H), 7.28 (m, 6H), 4.69 (dt, J = 5.5 Hz, 7.7 Hz, 1H), 4.34 (d, J = 3.7 Hz, 2H), 4.01 (s, 2H), 3.13 (dt, J = 6.1 Hz, 6.5 Hz, 2H), 2.47 (s, 3H), 1.82 (m, 2H), 1.51 (m, 2H). 1313C-NMR(75MHz, DMSO-d6): δ(ppm) = 171.8, 166.2, 164.6, 142.0, 141.3, 139.6, 139.1, 135.2, 134.8, 132.0, 130.2, 130.0, 129.1, 128.9, 128.8, 127.6, 127.3, 122.5, 121.7, 120.7, 113.4, 113.2, 65.5, 52.5, 43.1, 42.6, 30.5, 25.8, 21.4.

[0574] Example 42

[0575] 4H-K4CM-OCH3 (M19y)

[0576] M19y (2.99 g, 85.9%) was obtained as a white solid by separating and purifying M18 and 4-isopropylbenzaldehyde according to the same preparation scheme as M19a on a medium-pressure preparative column (collecting the product with 25 - 30% EA).

[0577] K4CM-OCH3 (M20y)

[0578] The preparation scheme was the same as that of M5b, and M20y (2.45 g, 82.9%) was obtained as a light brown solid.

[0579] K4CM (M21y)

[0580] The preparation scheme was the same as that of M6, and M21y (2.30 g, 97.7%) was obtained as a light brown solid.

[0581] K4CM-Orn(Z)-NBzl (M22y)

[0582] M22y (1.50 g, 45.0%) was obtained as a white solid by separating and purifying M21y and M14 according to the same preparation scheme as M10a on a medium-pressure preparative column (collecting the product with 55% EA).

[0583] K4CM-Orn-NBzl (M23y)

[0584] The preparation scheme was the same as that of M9, and M23y (1.14 g, 95.5%) was obtained as a white solid.

[0585] K4CM-Orn(Cl)-NBzl (42)

[0586] 45 was obtained as a light gray solid (0.46 g, 35.2%) by separating and purifying M23y and 2-chloroacetimidoyl ethyl ester according to the same preparation protocol as 8 through C18 silica gel column chromatography (collecting the product with 65% CH3OH). Purity: 99.41%. m.p.: 157.5 - 158.4 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 609.2739 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 11.93 (s, 1H), 10.25 (t, J = 4.7 Hz, 1H), 9.64 (s, 1H), 9.20 (s, 1H), 8.95 (t, J = 5.6 Hz, 1H), 8.85 (s, 1H), 8.83 (d, J = 7.7 Hz, 1H), 8.43 (d, J = 7.8 Hz, 1H), 8.02 (d, J = 8.0 Hz, 2H), 7.71 (d, J = 8.2 Hz, 1H), 7.61 (t, J = 7.3 Hz, 1H), 7.55 (d, J = 8.1 Hz, 2H), 7.29 (m, 6H), 4.77 (dt, J = 5.1 Hz, 7.5 Hz, 1H), 4.43 (s, 2H), 4.35 (d, J = 3.6 Hz, 2H), 3.35 (m, 2H), 3.06 (m, J = 6.8 Hz, 1H), 1.89 (m, 2H), 1.63 (m, 2H), 1.32 (d, J = 6.9 Hz, 6H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.7, 164.8, 162.7, 149.9, 142.1, 141.4, 139.6, 139.5, 135.6, 134.8, 130.2, 129.1, 129.0, 128.8, 127.6, 127.4, 127.3, 122.5, 121.7, 120.7, 113.4, 113.2, 52.2, 42.6, 42.2, 39.6, 33.9, 30.9, 24.3, 24.0.

[0587] Example 43

[0588] 4H-KN-OCH3 (M19z)

[0589] M19z (2.82 g, 79.4%) was obtained as a white solid by separating and purifying M18 and 2-naphthaldehyde according to the same preparation protocol as M19a through medium-pressure preparative column chromatography (collecting the product with 30% EA).

[0590] KN-OCH3 (M20z)

[0591] This preparation protocol is the same as that of M5b, obtaining M20z (2.14 g, 76.4%), which is a light brown solid.

[0592] KN(M21z)

[0593] This preparation protocol is the same as that of M6, obtaining M21z (1.86 g, 90.7%), which is a yellow solid.

[0594] KN-Orn(Z)-NBzl(M22z)

[0595] From M21z and M14, following the same preparation protocol as M10a, and purified by medium-pressure preparative column chromatography (collecting the product with 35% EA), M22z (3.15 g, 84.8%) was obtained, which is a white solid.

[0596] KN-Orn-NBzl(M23z)

[0597] This preparation protocol is the same as that of M9, obtaining M23z (2.45 g, 97.0%), which is a white solid.

[0598] KN-Orn(Cl)-NBzl(43)

[0599] From M23z and 2-chloroacetimidoyl ethyl ester, following the same preparation protocol as 8, and purified by C18 silica gel column chromatography (collecting the product with 65% CH3OH), 43 (1.18 g, 42.2%) was obtained, which is a light brown solid. Purity: 99.85%. m.p.: 171.6 - 172.3 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 617.2426 [M + H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.11 (s, 1H), 8.91 (s, 1H), 8.89 (d, J = 5.7 Hz, 1H), 8.88 (t, J = 8.4 Hz, 1H), 8.66 (s, 1H), 8.47 (d, J = 7.7 Hz, 1H), 8.23 (m, 2H), 8.17 (dd, J = 7.1 Hz, 3.7 Hz, 1H), 8.07 (dd, J = 3.9 Hz, 3.0 Hz, 1H), 7.74 (d, J = 8.2 Hz, 1H), 7.64 (m, 3H), 7.31 (m, 6H), 4.77 (dt, J = 4.5 Hz, 7.8 Hz, 1H), 4.39 (s, 2H), 4.36 (d, J = 2.9 Hz, 2H), 3.32 (m, 2H), 1.92 (m, 2H), 1.66 (m, 2H). 13C-NMR(75MHz, DMSO-d6): δ(ppm) = 171.6, 164.8, 162.7, 142.2, 141.2, 139.6, 135.2, 133.6, 133.3, 130.4, 129.3, 129.2, 129.0, 128.8, 128.4, 128.1, 127.7, 127.5, 127.3, 127.0, 126.7, 122.6, 121.7, 120.8, 113.7, 113.2, 52.4, 42.6, 42.3, 30.8, 24.1.

[0600] Example 44

[0601] K1IQ-OCH3 (M20aa)

[0602] M18 and isoquinoline-1-carbaldehyde were reacted at room temperature for 48 h according to the same preparation protocol as M19a, and then separated and purified by medium-pressure preparative column chromatography (collecting the product with 75% EA), to obtain M20aa (1.46 g, 41.4%), which was a yellow solid.

[0603] K1IQ (M21aa)

[0604] The preparation protocol was the same as that of M6, to obtain M21aa (1.02 g, 72.7%), which was a yellow solid.

[0605] K1IQ-Orn(Z)-NBzl (M22aa)

[0606] M21aa and M14 were reacted according to the same preparation protocol as M10a, and then separated and purified by medium-pressure preparative column chromatography (collecting the product with 55% EA), to obtain M22aa (1.26 g, 61.7%), which was a yellow solid.

[0607] K1IQ-Orn-NBzl (M23aa)

[0608] The preparation protocol was the same as that of M9, to obtain M23aa (0.88 g, 87.5%), which was a yellow solid.

[0609] K1IQ-Orn(Cl)-NBzl (44)

[0610] M23aa and 2-chloroethyl chloroacetimidate were reacted according to the same preparation protocol as 8, and then separated and purified by C18 silica gel column chromatography (collecting the product with 45% CH3OH), to obtain 44 (0.26 g, 25.4%), which was a yellow solid. Purity: 96.94%. m.p.: 95.3 - 96.0 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 618.2379 [M + H]+ . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.01 (s, 1H), 9.93 (t, J = 4.2 Hz, 1H), 9.49 (s, 1H), 9.08 (s, 1H), 9.05 (s, 1H), 8.97 (d, J = 8.6 Hz, 1H), 8.84 (d, J = 5.6 Hz, 1H), 8.81 (t, J = 5.9 Hz, 1H), 8.68 (d, J = 8.6 Hz, 1H), 8.51 (d, J = 7.9 Hz, 1H), 8.17 (d, J = 8.2 Hz, 1H), 8.09 (d, J = 5.6 Hz, 1H), 7.88 (t, J = 7.3 Hz, 1H), 7.77 (d, J = 8.2 Hz, 1H), 7.69 (dt, J = 0.6 Hz, 8.1 Hz, 1H), 7.64 (dt, J = 0.8 Hz, 7.2 Hz, 1H), 7.32 (m, 5H), 7.24 (m, 1H), 4.78 (dt, J = 5.0 Hz, 7.7 Hz, 1H), 4.35 (d, J = 5.0 Hz, 2H), 4.33 (s, 2H), 3.30 (dt, J = 5.1 Hz, 6.4 Hz, 2H), 1.85 (m, 2H), 1.64 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.5, 164.7, 162.8, 155.1, 142.1, 142.0, 139.6, 138.4, 137.5, 136.5, 131.0, 130.8, 129.4, 128.8, 128.7, 127.8, 127.7, 127.7, 127.3, 126.9, 122.6, 122.0, 121.5, 120.8, 114.8, 113.3, 52.3, 42.7, 42.4, 30.9, 24.0.

[0611] Example 45

[0612] K2Q-OCH3 (M20ab)

[0613] M18 and quinoline-2-carbaldehyde were reacted at room temperature for 48 h according to the same preparation protocol as M19a, and then separated and purified by medium-pressure preparative column (collecting the product with 25% EA) to obtain M20ab (1.85 g, 52.4%), which was a yellow solid.

[0614] K2Q (M21ab)

[0615] The preparation protocol was the same as that of M6 to obtain M21ab (1.44 g, 80.8%), which was a yellow solid.

[0616] K2Q-Orn(Z)-NBzl(M22ab)

[0617] M22ab (1.98 g, 69.4%) was obtained as a yellow solid by separating and purifying M21ab and M14 according to the same preparation protocol as M10a on a medium-pressure preparative column (collecting the product with 50% EA).

[0618] K2Q-Orn-NBzl(M23ab)

[0619] The same preparation protocol as M9 was used to obtain M23ab (1.48 g, 93.3%) as a yellow solid.

[0620] K2Q-Orn(Cl)-NBzl(45)

[0621] 45 (0.78 g, 46.4%) was obtained as a yellow solid by separating and purifying M23ab and 2-chloroethyl chloroacetimidate according to the same preparation protocol as 8 on a C18 silica gel column (collecting the product with 55% CH3OH). Purity: 99.04%. m.p.: 168.6 - 169.1 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 681.2379 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.35 (s, 1H), 10.29 (t, J = 4.6 Hz, 1H), 9.64 (s, 1H), 9.22 (s, 1H), 9.04 (s, 1H), 9.03 (d, J = 8.6 Hz, 2H), 8.94 (t, J = 5.8 Hz, 1H), 8.82 (d, J = 8.3 Hz, 1H), 8.68 (d, J = 8.7 Hz, 1H), 8.50 (d, J = 7.8 Hz, 1H), 8.10 (t, J = 7.8 Hz, 2H), 7.94 (dt, J = 1.2 Hz, 7.0 Hz, 1H), 7.72 (dq, J = 0.7 Hz, 7.9 Hz, 2H), 7.36 (m, 5H), 7.25 (m, 1H), 4.81 (m, 1H), 4.45 (s, 2H), 4.40 (dd, J = 5.3 Hz, 3.2 Hz, 2H), 3.40 (dt, J = 5.5 Hz, 6.0 Hz, 2H), 2.03 (m, 2H), 1.72 (m, 2H). 1313C-NMR(75MHz, DMSO-d6): δ(ppm) = 171.8, 164.8, 162.7, 156.8, 147.6, 142.1, 139.7, 139.2, 137.7, 136.6, 135.7, 131.5, 130.3, 129.5, 128.8, 128.3, 128.1, 127.8, 127.7, 127.3, 122.6, 121.3, 121.0, 119.4, 115.7, 114.0, 52.6, 42.7, 42.3, 39.7, 30.5, 24.2.

[0622] Example 46 K3IQ - OCH3 (M20ac)

[0623] M20ac (1.30 g, 36.7%) was obtained as a yellow solid by reacting M18 and isoquinoline - 3 - carbaldehyde according to the same preparation protocol as M19a at room temperature for 48 h and then separating and purifying by medium - pressure preparative column (collecting the product with 75% EA).

[0624] K3IQ (M21ac)

[0625] M21ac (0.95 g, 76.4%) was obtained as a yellow solid according to the same preparation protocol as M6.

[0626] K3IQ - Orn(Z) - NBzl (M22ac)

[0627] M22ac (0.98 g, 51.5%) was obtained as a yellow solid by reacting M21ac and M14 according to the same preparation protocol as M10a and then separating and purifying by medium - pressure preparative column (collecting the product with 55% EA).

[0628] K3IQ - Orn - NBzl (M23ac)

[0629] M23ac (0.58 g, 74.8%) was obtained as a yellow solid according to the same preparation protocol as M9.

[0630] K3IQ - Orn(Cl) - NBzl (46)

[0631] 46 (0.20 g, 30.0%) was obtained as a yellow solid by reacting M23ac and 2 - chloroacetyl iminoethyl ester according to the same preparation protocol as 8 and then separating and purifying by C18 silica gel column chromatography (collecting the product with 45% CH3OH). Purity: 97.92%. m.p.: 86.7 - 87.4 °C. α = - 60.0 (C = 1 mg / mL, CH3OH). HR - MS (m / z): 618.2379 [M + H] + .1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.38 (s, 1H), 9.99 (t, J = 4.6 Hz, 1H), 9.67 (s, 1H), 9.52 (s, 1H), 9.23 (s, 1H), 9.13 (s, 1H), 9.06 (d, J = 8.3 Hz, 1H), 8.97 (s, 1H), 8.80 (t, J = 5.8 Hz, 1H), 8.47 (d, J = 7.8 Hz, 1H), 8.37 (d, J = 8.0 Hz, 1H), 8.16 (d, J = 7.9 Hz, 1H), 7.97 (d, J = 8.3 Hz, 1H), 7.93 (t, J = 7.3 Hz, 1H), 7.82 (t, J = 7.4 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.32 (m, 5H), 7.26 (m, 1H), 4.77 (dt, J = 5.4 Hz, 7.8 Hz, 1H), 4.41 (d, J = 2.2 Hz, 2H), 4.37 (s, 2H), 3.37 (dt, J = 6.0 Hz, 5.7 Hz, 2H), 2.03 (m, 2H), 1.74 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.8, 165.1, 162.8, 152.3, 150.4, 142.0, 139.7, 139.1, 137.6, 136.3, 135.4, 131.8, 131.2, 129.3, 128.8, 128.6, 128.5, 128.0, 127.7, 127.3, 122.5, 121.2, 120.7, 118.4, 114.8, 113.7, 52.8, 42.7, 42.5, 39.9, 30.3, 24.3.

[0632] Example 47

[0633] K2BM-OCH3 (M20ad)

[0634] M20ad (1.89 g, 55.3%) was obtained as a yellow solid by reacting M18 and benzimidazole-2-carboxaldehyde at room temperature for 48 h according to the same preparation protocol as M19a and then separating and purifying by medium-pressure preparative column (collecting the product with 30% EA).

[0635] K2BM (M21ad)

[0636] Following the same preparation protocol as M6, M21ad (1.58 g, 87.4%) was obtained as a yellow solid.

[0637] K2BM-Orn(Z)-NBzl (M22ad)

[0638] M22ad (0.99 g, 30.8%) was obtained as a pale yellow solid by separating and purifying M21ad and M14 on a medium-pressure preparative column (collecting the product with 55% EA) according to the same preparation protocol as M10a.

[0639] K2BM-Orn-NBzl (M23ad)

[0640] M23ad (0.69 g, 87.3%) was obtained as a pale yellow solid according to the same preparation protocol as M9.

[0641] K2BM-Orn(Cl)-NBzl(47)

[0642] 47 (0.44 g, 55.8%) was obtained as a pale yellow solid by separating and purifying M23ad and 2-chloroethyl imidoacetate on a C18 silica gel column (collecting the product with 50% CH3OH) according to the same preparation protocol as 8. Purity: 99.96%. m.p.: 174.7 - 175.6 °C. = -60.0 (C = 1 mg / mL, CH3OH). ESI-MS (m / z): 607.2331 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.03 (s, 1H), 10.24 (t, J = 4.8 Hz, 1H), 9.57 (s, 1H), 9.49 (d, J = 8.6 Hz, 1H), 9.26 (s, 1H), 9.00 (s, 1H), 8.89 (t, J = 5.9 Hz, 1H), 8.48 (d, J = 7.8 Hz, 1H), 8.06 (d, J = 8.3 Hz, 1H), 7.87 (m, 2H), 7.66 (dt, J = 0.6 Hz, 7.2 Hz, 1H), 7.34 (m, 7H), 7.21 (m, 1H), 4.7 (m, 1H), 4.44 (s, 2H), 4.35 (d, J = 5.8 Hz, 2H), 3.38 (dt, J = 5.5 Hz, 6.4 Hz, 2H), 2.18 (m, 2H), 1.75 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 172.2, 165.2, 162.7, 151.0, 142.3, 140.0, 139.3, 134.7, 130.8, 130.2, 129.5, 128.7, 127.6, 127.1, 123.3, 122.6, 121.4, 121.0, 115.6, 114.1, 53.9, 42.6, 42.4, 29.2, 24.8.

[0643] Example 48

[0644] K2BM1M-OCH3 (M20ae)

[0645] M18 and 1-methyl-2-formylbenzimidazole were reacted at room temperature for 48 h according to the same preparation protocol as M19a, and then separated and purified by medium-pressure preparative column (collecting the product with 45% EA) to obtain M20ae (0.86 g, 24.3%), which was a milky white solid.

[0646] K2BM1M (M21ae)

[0647] The preparation protocol was the same as M6 to obtain M21ae (0.82 g, 98.7%), which was a milky white solid.

[0648] K2BM1M-Orn(Z)-NBzl (M22ae)

[0649] M21ae and M14 were reacted according to the same preparation protocol as M10a. After evaporation to dryness, it was washed by ultrasonic grinding with methanol and then filtered under reduced pressure to obtain M22ae (1.09 g, 67.0%), which was a white solid.

[0650] K2BM1M-Orn-NBzl (M23ae)

[0651] The preparation protocol was the same as M9 to obtain M23ae (0.52 g, 59.4%), which was a white solid.

[0652] K2BM1M-Orn(Cl)-NBzl (48)

[0653] M23ae and 2-chloroethyl iminoacetate were reacted according to the same preparation protocol as 8, and then separated and purified by C18 silica gel column chromatography (collecting the product with 65% CH3OH) to obtain 48 (85 mg, 14.3%), which was a white solid. Purity: 97.71%. m.p.: 236.7~237.6℃. =-60.0 (C=1 mg / mL, CH3OH). HR-MS (m / z): 621.2488 [M+H] + . 11H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.14 (s, 1H), 10.32 (s, 1H), 9.66 (s, 1H), 9.23 (s, 1H), 9.03 (m, 1H), 9.02 (s, 1H), 8.80 (d, J = 8.2 Hz, 1H), 8.50 (d, J = 7.8 Hz, 1H), 8.02 (d, J = 8.2 Hz, 1H), 7.96 (d, J = 7.7 Hz, 1H), 7.85 (d, J = 7.6 Hz, 1H), 7.67 (t, J = 7.6 Hz, 1H), 7.45 (t, J = 7.0 Hz, 1H), 7.33 (m, 7H), 4.81 (dt, J = 4.4 Hz, 7.5 Hz, 1H), 4.50 (s, 3H), 4.44 (s, 2H), 4.40 (d, J = 4.0 Hz, 2H), 3.39 (m, 2H), 1.95 (m, 2H), 1.67 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.6, 164.5, 162.7, 148.8, 142.5, 142.0, 139.6, 138.7, 137.1, 136.0, 131.7, 130.7, 129.5, 128.8, 127.7, 127.3, 124.1, 123.1, 122.6, 121.5, 121.1, 120.1, 115.1, 114.0, 111.4, 52.2, 42.7, 42.2, 39.6, 34.0, 31.1, 23.9.

[0654] Example 49

[0655] KMID-OCH3 (M20af)

[0656] Ice - water bath: Dissolve M19h (0.94 g, 3.0 mmol) in 10 mL of glacial acetic acid, slowly add 1 mL of distilled water and 2 mL of concentrated hydrochloric acid. After stirring at room temperature for 5 h, a large amount of yellow solid precipitates. Filter under reduced pressure, wash the filter cake with H2O, air - dry the filter cake to obtain 0.78 g of yellow solid for later use. Subsequently, in an ice - water bath, dissolve indole (0.70 g, 6.0 mmol) in 40 mL of CH2Cl2, slowly add 0.3 mL of concentrated sulfuric acid and the above - mentioned yellow solid (0.78 g, 2.9 mmol). After reacting at room temperature for 8 h, TLC detects that the reaction is complete. Adjust the pH to 7 with saturated NaHCO3 solution; transfer to a 100 - mL separatory funnel, extract with CH2Cl2 (20 mL×2); combine the CH2Cl2 layers, wash with saturated NaCl solution (20 mL×3); dry the CH2Cl2 layer with anhydrous Na2SO4 for 2 h, then filter under reduced pressure, and separate and purify by medium - pressure preparative column (collect the product with 50% EA), to obtain M20af (0.58 g, 39.9%), which is a pale - yellow solid.

[0657] KMID(M21af)

[0658] This preparation scheme is the same as that of M6, to obtain M21af (0.48 g, 81.3%), which is an orange - red solid.

[0659] KMID - Orn(Z) - NBzl(M22af)

[0660] From M21af and M14, according to the same preparation scheme as M10a, separate and purify by medium - pressure preparative column (collect the product with 65 - 70% EA), to obtain M22af (0.65 g, 78.0%), which is a yellow solid.

[0661] KMID - Orn - NBzl(M23af)

[0662] This preparation scheme is the same as that of M9, to obtain M23af (0.52 g, 95.0%), which is a yellow solid.

[0663] KMID - Orn(Cl) - NBzl(48a)

[0664] From M23af and 2 - chloroacetyl iminoethyl ester, according to the same preparation scheme as 8, separate and purify by C18 silica gel column chromatography (collect the product with 50% CH3OH), to obtain 48a (85 mg, 14.3%), which is a yellow solid. Purity: 98.53%; m.p.: 169.9 - 171.1 °C; α=-60.0 (C = 1 mg / mL, CH3OH); HR - MS (m / z): 749.3114[M + H] + ; 11H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.18 (s, 1H), 10.73 (d, J = 15.1 Hz, 2H), 10.21 (s, 1H), 9.63 (s, 1H), 9.21 (s, 1H), 8.94 (d, J = 5.7 Hz, 1H), 8.93 (s, 1H), 8.56 (s, 1H), 8.29 (d, J = 7.8 Hz, 1H), 7.66 (m, 3H), 7.57 (t, J = 7.6 Hz, 1H), 7.31 (m, 10H), 6.95 (t, J = 5.6 Hz, 2H), 6.84 (m, 2H), 5.63 (t, J = 7.5 Hz, 1H), 4.72 (dt, J = 5.3 Hz, 7.3 Hz, 1H), 4.43 (d, J = 5.5 Hz, 2H), 4.42 (s, 2H), 4.11 (m, 2H), 3.34 (m, 2H), 1.88 (m, 2H), 1.62 (m, 2H); 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.7, 164.8, 162.7, 144.0, 141.3, 139.6, 136.7, 136.6, 128.8, 128.0, 127.8, 127.4, 127.0, 122.6, 122.5, 121.8, 121.1, 120.4, 119.5, 119.3, 119.1, 118.4, 112.7, 112.2, 111.7, 52.1, 42.7, 42.3, 38.8, 32.2, 31.1, 23.9.

[0665] Example 50

[0666] 4H-K4BFZ-OCH3 (M19ag)

[0667] M19ag (2.43 g, 69.8%) was obtained as a yellow solid by separating and purifying M18 and benzodiazole-4-carbaldehyde according to the same preparation scheme as M19a on a medium-pressure preparative column (collecting the product with 40 - 50% EA).

[0668] K4BFZ-OCH3 (M20ag)

[0669] Following the same preparation scheme as M5b, M20ag (2.25 g, 93.7%) was obtained as a light gray solid.

[0670] K4BFZ (M21ag)

[0671] Following the same preparation scheme as M6, M21ag (2.04 g, 94.5%) was obtained as a pale yellow solid.

[0672] K4BFZ-Orn(Boc)-NBzl(M22ag)

[0673] M22ag (1.38 g, 43.8%) was obtained as a yellow solid by separating and purifying M21ag and M9 according to the same preparation protocol as M10a using a medium-pressure preparative column (collecting the product with 75% EA).

[0674] K4BFZ-Orn(HCl)-NBzl(M23ag)

[0675] The preparation protocol was the same as that of M11a, and M23ag (1.18 g, 94.3%) was obtained as a pale yellow solid.

[0676] K4BFZ-Orn(Cl)-NBzl(48b)

[0677] 48b (0.44 g, 35.0%) was obtained as a yellow solid by separating and purifying M23ag and 2-chloroacetimidoylethyl ester according to the same preparation protocol as 8 using a C18 silica gel column chromatography (collecting the product with 55% CH3OH). Purity: 99.61%. m.p.: 256.2 - 257.3 °C. =-60.0 (C=1 mg / mL, CH3OH). HR-MS (m / z): 609.2124 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 11.99 (s, 1H), 10.21 (s, 1H), 9.62 (s, 1H), 9.20 (s, 1H), 9.01 (s, 1H), 8.91 (t, J=5.6 Hz, 1H), 8.83 (d, J=8.4 Hz, 1H), 8.51 (d, J=7.9 Hz, 1H), 8.29 (d, J=9.0 Hz, 1H), 8.24 (d, J=6.6 Hz, 1H), 7.92 (dd, J=8.9 Hz, 6.8 Hz, 1H), 7.65 (m, 2H), 7.30 (m, 6H), 4.75 (dt, J=5.3 Hz, 7.5 Hz, 1H), 4.42 (s, 2H), 4.33 (d, J=5.6 Hz, 2H), 3.34 (m, 2H), 1.89 (m, 2H), 1.66 (m, 2H). 1313C-NMR(75MHz, DMSO-d6): δ(ppm) = 171.5, 164.6, 162.7, 150.1, 148.4, 141.9, 139.6, 139.4, 136.3, 135.4, 133.4, 133.1, 130.7, 129.6, 128.8, 127.6, 127.3, 126.8, 122.8, 121.4, 121.0, 117.3, 114.8, 113.0, 52.4, 42.6, 42.2, 30.8, 24.1.

[0678] Example 51

[0679] 4H-K5BFZ-OCH3 (M19ah)

[0680] M19ah (2.14 g, 61.6%) was obtained as a yellow solid by separating and purifying M18 and benzo[c][1,2,5]oxadiazole-5-carbaldehyde according to the same preparation protocol as M19a on a medium-pressure preparative column (collecting the product with 45 - 50% EA).

[0681] K5BFZ-OCH3 (M20ah)

[0682] The preparation protocol was the same as M5b, and M20ah (1.92 g, 90.3%) was obtained as a light yellow solid.

[0683] K5BFZ (M21ah)

[0684] The preparation protocol was the same as M6, and M21ah (1.76 g, 96.8%) was obtained as a light yellow solid.

[0685] K5BFZ-Orn(Boc)-NBzl (M22ah)

[0686] M22ah (3.05 g, 96.4%) was obtained as a yellow solid by separating and purifying M21ah and M9 according to the same preparation protocol as M10a on a medium-pressure preparative column (collecting the product with 50 - 55% EA).

[0687] K5BFZ-Orn(HCl)-NBzl (M23ah)

[0688] The preparation protocol was the same as M11a, and M23ah (2.68 g, 97.7%) was obtained as a light yellow solid.

[0689] K5BFZ-Orn(Cl)-NBzl (48c)

[0690] Compound 48c (1.15 g, 40.1%) was obtained as a yellow solid by separating and purifying M23ah and 2-chloroacetimidoyl ethyl ester according to the same preparation protocol as 8 through C18 silica gel column chromatography (collecting the product with 50% CH3OH). Purity: 98.47%. m.p.: 175.7 - 176.2 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 609.2124 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.31 (s, 1H), 10.33 (s, 1H), 9.66 (s, 1H), 9.24 (s, 1H), 8.99 (s, 1H), 8.96 (t, J = 5.8 Hz, 1H), 8.83 (d, J = 8.4 Hz, 1H), 8.77 (s, 1H), 8.50 (d, J = 7.8 Hz, 1H), 8.42 (d, J = 9.4 Hz, 1H), 8.34 (d, J = 9.4 Hz, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.66 (t, J = 7.5 Hz, 1H), 7.37 (t, J = 7.3 Hz, 1H), 7.28 (m, 5H), 4.77 (dt, J = 4.9 Hz, 8.1 Hz, 1H), 4.45 (s, 2H), 4.36 (d, J = 2.7 Hz, 2H), 3.35 (m, 2H), 1.95 (m, 2H), 1.67 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.7, 164.6, 162.7, 149.7, 149.3, 142.2, 140.8, 139.7, 138.3, 135.3, 134.6, 131.1, 129.6, 128.8, 127.6, 127.3, 122.7, 121.5, 121.1, 117.1, 116.2, 115.0, 113.2, 52.4, 42.6, 42.2, 39.6, 30.6, 24.1.

[0691] Example 52

[0692] 4H-K6QN-OCH3 (M19ai)

[0693] Compound M19ai (2.62 g, 73.3%) was obtained as a white solid by separating and purifying M18 and quinoxaline-6-carbaldehyde according to the same preparation protocol as M19a through medium-pressure preparative column chromatography (collecting the product with 45 - 50% EA).

[0694] K6QN-OCH3 (M20ai)

[0695] This preparation protocol is the same as that of M5b, and M20ai (2.35 g, 90.5%) is obtained as a white solid.

[0696] K6QN (M21ai)

[0697] This preparation protocol is the same as that of M6, and M21ai (2.18 g, 96.8%) is obtained as a yellow solid.

[0698] K6QN-Orn(Boc)-NBzl (M22ai)

[0699] From M21ai and M9 according to the same preparation protocol as M10a, purified by medium-pressure preparative column chromatography (collecting the product with 50% EA), M22ai (0.58 g, 30.1%) is obtained as a pale yellow solid.

[0700] K6QN-Orn(HCl)-NBzl (M23ai)

[0701] This preparation protocol is the same as that of M11a, and M23ai (0.48 g, 92.8%) is obtained as a yellow solid.

[0702] K6QN-Orn(Cl)-NBzl (48d)

[0703] From M23ai and 2-chloroethyl imidoacetate according to the same preparation protocol as 8, purified by C18 silica gel column chromatography (collecting the product with 35% CH3OH), 48d (0.18 g, 34.7%) is obtained as a pale yellow solid. Purity: 98.72%. m.p.: 180.4~181.4℃. =-60.0 (C=1mg / mL, CH3OH). HR-MS (m / z): 619.2331 [M+H] + . 11H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.21 (s, 1H), 10.16 (s, 1H), 9.56 (s, 1H), 9.10 (d, J = 7.9 Hz, 2H), 8.97 (s, 1H), 8.91 (t, J = 5.8 Hz, 1H), 8.87 (d, J = 8.5 Hz, 1H), 8.82 (d, J = 1.4 Hz, 1H), 8.62 (dd, J = 8.6 Hz, 1.4 Hz, 1H), 8.49 (d, J = 7.8 Hz, 1H), 8.39 (d, J = 8.7 Hz, 1H), 7.75 (d, J = 8.2 Hz, 1H), 7.65 (t, J = 7.5 Hz, 1H), 7.31 (m, 6H), 4.78 (dt, J = 5.3 Hz, 7.8 Hz, 1H), 4.40 (s, 2H), 4.36 (d, J = 2.9 Hz, 2H), 3.32 (m, 2H), 1.93 (m, 2H), 1.66 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.6, 164.7, 162.7, 146.9, 142.9, 142.7, 142.3, 139.9, 139.6, 139.6, 139.3, 135.3, 131.1, 130.9, 130.3, 129.4, 129.2, 128.8, 127.7, 127.3, 122.7, 121.7, 121.0, 114.4, 113.3, 52.4, 42.7, 42.3, 30.7, 24.1.

[0706] Example 53

[0707] 4H-K6BO-OCH3 (M19aj)

[0708] M19aj (1.98 g, 54.3%) was obtained as a white solid by separating and purifying M18 and 1,4-benzodioxane-6-carbaldehyde according to the same preparation scheme as M19a on a medium-pressure preparative column (collecting the product with 50% EA).

[0709] K6BO-OCH3 (M20aj)

[0710] The preparation scheme was the same as that of M5b, and M20aj (1.77 g, 90.6%) was obtained as a white solid.

[0711] K6BO (M21aj)

[0712] The preparation scheme was the same as that of M6, and M21aj (1.22 g, 71.7%) was obtained as a light yellow solid.

[0713] K6BO-Orn(Z)-NBzl(M22aj)

[0714] M22aj (1.52 g, 62.9%) was obtained as a white solid by separating and purifying M21aj and M14 according to the same preparation protocol as M10a on a medium-pressure preparative column (collecting the product with 70% EA).

[0715] K6BO-Orn-NBzl(M23aj)

[0716] The same preparation protocol as M9 was used to obtain M23aj (0.75 g, 61.6%) as a white solid.

[0717] K6BO-Orn(Cl)-NBzl(48e)

[0718] 48e (0.30 g, 35.1%) was obtained as a pale yellow solid by separating and purifying M23ag and 2-chloroacetimidoylethyl ester according to the same preparation protocol as 8 on a C18 silica gel column chromatography (collecting the product with 55% CH3OH). Purity: 99.13%. m.p.: 169.2 - 169.9 °C. =-60.0 (C=1 mg / mL, CH3OH). HR-MS (m / z): 625.2325 [M+H] + . 1 1H-NMR (800 MHz, DMSO-d6): δ (ppm) = 11.87 (s, 1H), 10.17 (t, J = 4.7 Hz, 1H), 9.59 (s, 1H), 9.16 (s, 1H), 8.91 (t, J = 5.8 Hz, 1H), 8.82 (s, 1H), 8.80 (d, J = 7.2 Hz, 1H), 8.41 (d, J = 7.9 Hz, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.61 (m, 3H), 7.29 (m, 6H), 7.15 (m, 1H), 4.76 (dt, J = 5.0 Hz, 8.2 Hz, 1H), 4.41 (s, 2H), 4.38 (s, 4H), 4.34 (m, 2H), 3.34 (m, 2H), 1.90 (m, 2H), 1.63 (m, 2H). 1313C-NMR (200 MHz, DMSO-d6): δ (ppm) = 171.6, 164.8, 162.7, 144.9, 144.1, 142.1, 140.8, 139.6, 139.3, 134.6, 131.1, 130.2, 129.1, 128.8, 127.7, 127.3, 122.5, 122.1, 121.7, 120.7, 118.0, 117.6, 113.3, 64.8, 64.6, 52.2, 42.6, 42.2, 30.9, 24.1.

[0719] Example 54

[0720] 4H-KBNC-OCH3 (M19ak)

[0721] M19ak (2.50 g, 71.6%) was obtained as a white solid by separating and purifying M18 and p-dimethylaminobenzaldehyde according to the same preparation protocol as M19a using a medium-pressure preparative column (collecting the product with 45% EA).

[0722] KBNC-OCH3 (M20ak)

[0723] The preparation protocol was the same as that of M5b, and M20ak (1.55 g, 62.7%) was obtained as a white solid.

[0724] KBNC (M21ak)

[0725] The preparation protocol was the same as that of M6, and M21ak (1.29 g, 86.7%) was obtained as a pale yellow solid.

[0726] KBNC-Orn(Z)-NBzl (M22ak)

[0727] M22ak (2.06 g, 79.3%) was obtained as a white solid by separating and purifying M21ak and M14 according to the same preparation protocol as M10a using a medium-pressure preparative column (collecting the product with 65 - 70% EA).

[0728] KBNC-Orn-NBzl (M23ak)

[0729] The preparation protocol was the same as that of M9, and M23ak (1.45 g, 87.8%) was obtained as a pale yellow solid.

[0730] KBNC-Orn(Cl)-NBzl (48f)

[0731] 48f (0.45 g, 27.2%) was obtained as a pale yellow solid by separating and purifying M23ak and 2-chloroacetimidoylethyl ester according to the same preparation protocol as 8 through C18 silica gel column chromatography (collecting the product with 50% CH3OH). Purity: 99.39%. m.p.: 267.9 - 268.6 °C. = -60.0 (C = 1 mg / mL, CH3OH). HR-MS (m / z): 610.2692 [M+H] + . 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 11.84 (s, 1H), 10.27 (t, J = 5.3 Hz, 1H), 9.66 (s, 1H), 9.21 (s, 1H), 8.97 (t, J = 5.6 Hz, 1H), 8.86 (d, J = 8.4 Hz, 1H), 8.75 (s, 1H), 8.39 (d, J = 7.9 Hz, 1H), 8.01 (d, J = 8.6 Hz, 2H), 7.73 (d, J = 8.2 Hz, 1H), 7.59 (t, J = 7.5 Hz, 1H), 7.30 (m, 6H), 6.99 (d, J = 8.6 Hz, 2H), 4.77 (dt, J = 5.1 Hz, 8.0 Hz, 1H), 4.43 (s, 2H), 4.36 (d, J = 2. / Hz, 2H), 3.35 (m, 2H), 3.05 (s, 6H), 1.90 (m, 2H), 1.64 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.7, 164.9, 162.7, 151.3, 141.9, 141.9, 139.6, 139.3, 134.4, 129.8, 129.8, 128.8, 127.6, 127., 122.3, 121.8, 120.6, 113.2, 112.8, 112.3, 52.1, 42.6, 42.2, 39.6, 31.0, 24.0.

[0732] Example 55

[0733] 4H-KNNC-OCH3 (M19al)

[0734] M19al (2.98 g, 74.6%) was obtained as a white solid by separating and purifying M18 and 6-(dimethylamino)-2-naphthaldehyde according to the same preparation protocol as M19a through medium-pressure preparative column chromatography (collecting the product with 45% EA).

[0735] KNNC-OCH3 (M20al)

[0736] This preparation protocol is the same as that of M20a. After separation and purification by a medium-pressure preparative column (collecting the product with 38% EA), M20al (0.65 g, 41.1%) was obtained as a pale yellow solid.

[0737] KNNC (M21al)

[0738] This preparation protocol is the same as that of M6. M21al (0.40 g, 63.0%) was obtained as a yellow solid.

[0739] KNNC-Orn(Z)-NBzl (M22al)

[0740] From M21al and M14 according to the same preparation protocol as M10a, after separation and purification by a medium-pressure preparative column (collecting the product with 65% EA), M22al (0.43 g, 57.8%) was obtained as a yellow solid.

[0741] KNNC-Orn-NBzl (M23al)

[0742] This preparation protocol is the same as that of M9. M23al (0.28 g, 80.0%) was obtained as a yellow solid.

[0743] KNNC-Orn(Cl)-NBzl (48g)

[0744] From M23al and 2-chloroacetyl iminoethyl ester according to the same preparation protocol as 8, after separation and purification by C18 silica gel column chromatography (collecting the product with 35 - 40% CH3OH), 48g (85 mg, 26.9%) was obtained as a light brown solid. Purity: 99.63%. m.p.: 171.0 - 172.1 °C. =-60.0 (C=1 mg / mL, CH3OH). HR-MS (m / z): 660.2848 [M+H] + . 11H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.03 (s, 1H), 10.12 (s, 1H), 9.56 (s, 1H), 9.14 (s, 1H), 8.89 (t, J = 5.7 Hz, 1H), 8.85 (m, 1H), 8.84 (s, 1H), 8.48 (s, 1H), 8.44 (d, J = 7.8 Hz, 1H), 8.11 (d, J = 8.8 Hz, 1H), 7.97 (d, J = 9.9 Hz, 1H), 7.94 (d, J = 9.2 Hz, 1H), 7.74 (d, J = 8.2 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.30 (m, 7H), 7.08 (s, 1H), 4.77 (dt, J = 5.2 Hz, 7.9 Hz, 1H), 4.39 (s, 2H), 4.37 (d, J = 2.7 Hz, 2H), 3.34 (m, 2H), 3.09 (s, 6H), 1.92 (m, 2H), 1.65 (m, 2H). 13 13C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.7, 164.9, 162.7, 142.1, 141.8, 139.6, 139.5, 135.5, 134.9, 130.9, 130.2, 129.0, 128.8, 128.1, 127.7, 127.3, 127.1, 126.8, 122.4, 121.8, 120.7, 117.0, 113.2, 113.1, 52.3, 42.6, 42.3, 40.7, 30.8, 24.1.

[0745] Example 56

[0746] 4H-K5SA(OCH3)-OCH3 (M19am)

[0747] M19am (3.24 g, 85.1%) was obtained as a white solid by separating and purifying M18 and methyl 5-formylsalicylate according to the same preparation protocol as M19a on a medium-pressure preparative column (collecting the product with 30 - 40% EA).

[0748] K5SA(OCH3)-OCH3 (M20am)

[0749] Following the same preparation protocol as M5b, M20am (2.81 g, 87.8%) was obtained as a white solid.

[0750] K5SA (M21am)

[0751] Following the same preparation protocol as M6, M21am (2.58 g, 99.2%) was obtained as a yellow solid.

[0752] K5SA-2Orn(Z)-NBzl(M22am)

[0753] M22am (3.84 g, 75.1%) was obtained as a white solid by separating and purifying M21am and M14 according to the same preparation protocol as M15h on a medium-pressure preparative column (collecting the product with 3% CH3OH).

[0754] K5SA-2Orn-NBzl(M23am)

[0755] The same preparation protocol as M9 was used to obtain M23am (2.68 g, 94.6%) as a yellow solid. K5SA-2Orn(Cl)-NBzl(48h)

[0756] 48h (0.45 g, 14.0%) was obtained as a pale yellow solid by separating and purifying M23am and 2-chloroethyl chloroacetimidate according to the same preparation protocol as 17a on a C18 silica gel column (collecting the product with 70% CH3OH). Purity: 63.17%; m.p.: 185.9 - 186.8 °C; = -60.0 (C = 1 mg / mL, CH3OH); HR-MS (m / z): 905.3415 [M + H] + ; 1 1H-NMR (300 MHz, DMSO-d6): δ (ppm) = 12.47 (s, 1H), 12.11 (s, 1H), 10.21 (t, J = 5.6 Hz, 1H), 10.17 (t, J = 5.8 Hz, 1H), 9.59 (s, 2H), 9.31 (d, J = 7.7 Hz, 1H), 9.19 (s, 1H), 9.17 (s, 1H), 8.91 (t, J = 5.8 Hz, 1H), 8.85 (t, J = 5.8 Hz, 1H), 8.84 (s, 1H), 8.80 (d, J = 8.6 Hz, 1H), 8.70 (d, J = 1.8 Hz, 1H), 8.43 (d, J = 7.9 Hz, 1H), 8.11 (dd, J = 8.5 Hz, 1.8 Hz, 1H), 7.72 (d, J = 8.3 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.35 (d, J = 2.3 Hz, 1H), 7.27 (m, 11H), 4.74 (dt, J = 5.3 Hz, 8.2 Hz, 1H), 4.67 (dt, J = 5.1 Hz, 8.3 Hz, 1H), 4.40 (s, 4H), 4.35 (d, J = 5.4 Hz, 4H), 3.32 (m, 4H), 1.90 (m, 4H), 1.65 (m, 4H); 1313C-NMR (75 MHz, DMSO-d6): δ (ppm) = 171.7, 171.5, 168.4, 164.9, 162.7, 160.3, 142.0, 141.0, 139.8, 139.6, 139.4, 134.7, 134.4, 132.1, 130.0, 129.1, 128.8, 128.7, 127.6, 127.6, 127.3, 127.2, 122.5, 121.7, 120.7, 118.4, 116.8, 113.4, 113.3, 53.3, 52.4, 42.6, 42.3, 30.7, 29.6, 24.3, 24.1.

[0757] Example 57

[0758] 4H-B-2Klss-OCH3 (M19an)

[0759] M19an (4.14 g, 77.4%) was obtained as a white solid by separating and purifying M18 and terephthalaldehyde according to the same preparation scheme as M19a on a medium-pressure preparative column (collecting the product with 56 - 65% EA).

[0760] B-2Klss-OCH3 (M20an)

[0761] M20an (2.44 g, 59.9%) was obtained as a light brown solid according to the same preparation scheme as M5b.

[0762] B-2Klss (M21an)

[0763] M21an (1.53 g, 66.2%) was obtained as a yellow solid according to the same preparation scheme as M6.

[0764] B-2Klss-2Orn(Z)-NBzl (M22an)

[0765] M22an (0.65 g, 27.7%) was obtained as a white solid by separating and purifying M21an and M14 according to the same preparation scheme as M15h on a medium-pressure preparative column (collecting the product with 7% CH3OH).

[0766] B-2Klss-2Orn-NBzl (M23an)

[0767] M23an (0.45 g, 89.9%) was obtained as a light yellow solid according to the same preparation scheme as M9.

[0768] B-2Klss-Orn(Cl)-NBzl (48i)

[0769] 48i (0.19 g, 35.2%) was obtained as a pale yellow solid by separating and purifying M23an and 2-chloroacetimidoylethyl ester according to the same preparation protocol as 17a through C18 silica gel column chromatography (collecting the product with 58% CH3OH). Purity: 97.49%; m.p.: 199.6 - 200.5 °C; = -60.0 (C = 1 mg / mL, CH3OH); HR-MS (m / z): 1057.4033 [M+H] + ; 1 1H-NMR (800 MHz, DMSO-d6): δ (ppm) = 12.19 (s, 1H), 10.24 (s, 1H), 9.62 (s, 1H), 9.22 (s, 1H), 8.99 (t, J = 5.4 Hz, 2H), 8.95 (s, 2H), 8.82 (d, J = 8.3 Hz, 2H), 8.48 (d, J = 7.8 Hz, 2H), 8.42 (s, 4H), 7.78 (d, J = 8.1 Hz, 2H), 7.65 (t, J = 8.2 Hz, 2H), 7.31 (m, 12H), 4.83 (dt, J = 5.0 Hz, 7.1 Hz, 2H), 4.42 (s, 4H), 4.37 (d, J = 2.3 Hz, 4H), 3.37 (m, 4H), 1.92 (m, 4H), 1.68 (m, 4H); 13 13C-NMR (200 MHz, DMSO-d6): δ (ppm) = 171.7, 164.8, 162.7, 142.3, 140.7, 139.6, 138.4, 135.1, 130.6, 129.5, 129.3, 128.8, 127.6, 127.3, 122.6, 121.6, 120.9, 114.0, 113.4, 52.2, 42.7, 42.3, 31.1, 24.1.

[0770] Test Example 1 CADD Molecular Docking and Structure Optimization

[0771] The three-dimensional structure of the target compound was constructed by the 3D-sketcher module of DiscoveryStudio 2019 and energy-minimized under the CHARMm force field. The crystal structure of PAD4 (PDB: Amino acid residues within the range are defined as the binding site. In the DockLigands|CDOCKER parameter settings, the PAD4 protein is input as the receptor, all optimized molecules are input as ligands, TopHits is set to 10, PoseClusterRadius is set to 0.5, and click run to start the calculation. Record the scoring results and screen for dominant compounds, and analyze the interaction between the ligand molecules and the receptor protein.

[0772] The lead compound YW3-56 (Compound 7) and the target product were subjected to CDOCKER molecular docking with the PAD4 protein (PDB: 2DW5). The structural design and molecular docking model (PDB: 2DW5) of lead compound 7, Compound 11, and 28 are as Figure 1 shown. The amino acid backbone of Compound 7 forms a stable hydrogen bond with Arg374 through a carbonyl group, occupying the PAD4 active pocket as a peptide substrate; while the dimethylaminonaphthalene moiety only forms a weak pi-alkyl interaction with Arg639, and there is still a large physical space left in the active pocket here, providing the possibility for further modification. Since Arg374 is conserved in PAD1 and PAD4 isozymes, while Arg639 is unique to PAD4. Therefore, enhancing the interaction between small molecule compounds and Arg639 has become the main strategy for improving the activity and selectivity of PAD4 inhibitors in the present invention.

[0773] Based on the above strategy, Compound 11 was designed in the present invention by introducing a β-carboline ring at the N-terminus, which enhanced the pi-alkyl interaction with Arg639 and formed a stable hydrogen bond with Trp347, thereby enhancing the affinity for PAD4 ( Figure 1 and Table 1). This result inspired our interest in further optimization. By introducing a rotatable benzene ring at the α-position of the β-carboline ring of Compound 11, Compound 28 was further obtained in the present invention. This compound can form a strong pi-cation interaction with the imine nitrogen atom of the guanidine group of Arg639, thereby enhancing the selectivity for PAD4. In addition, the stable salt bridges formed with the key residues Asp350 and Asp473 in the active pocket enable the chloroacetamidine warhead of Compound 28 to reach the active site correctly and form a tight hydrogen bond and halogen interaction with His640, which may explain the enhanced inhibitory activity against PAD4 ( Figure 1 ). In addition, the binding free energy (ΔGbind) of Compound 28 with the PAD4 protein is negative, indicating that the binding of the inhibitor to PAD4 is energetically favorable (Table 1). [[ID=!3]]

[0774] Table 1 Molecular Docking Simulations and Property Determinations of Key Compounds

[0775]

[0776] Evaluation of in vitro PAD4 enzyme activity and anti-tumor cell proliferation activity in Test Example 2 and discussion of structure-activity relationship

[0777] (1) PAD2 / 4 enzyme activity inhibition experiment

[0778] The activity of PAD2 / 4 inhibitor was determined by a colorimetric method that inhibits the citrullination of BAEE (N-α-benzoyl-L-arginine ethyl ester) by PAD2 / 4. Test samples with different concentrations (60000, 20000, 6750, 2250, 750, 250, 85, 30 nmol / L) were prepared in ultrapure water containing 1% DMSO. According to the grouping, 10 μL of 10× buffer (preparation method: 625 μL of 1M Tris-HCl pH = 7.6, 25 μL of 2M CaCl2, 50 μL of 1M DTT, 125 μL of 100 mM PMSF, 425 μL of H2O), the corresponding volume of ultrapure water, 20 μL of the test sample, and 10 μL of PAD2 / 4 active enzyme were added successively to a 96-well plate and incubated at 37 °C with rpm = 400 for 60 min. 10 μL of 20 mM BAEE solution (prepared with 1× buffer) was added to each well, and incubation continued at 37 °C with rpm = 400 for 90 min. Subsequently, 25 μL of 5M HClO4 was added to quench the reaction. 125 μL of the reaction solution was transferred to an EP tube with the corresponding label, and 125 μL of reagent A (0.2 g of diacetylmonoxime and 0.6 g of NaCl dissolved in 40 mL of H2O) and 250 μL of reagent B (0.2 g of antipyrine, 60 mg of FeCl3, 10 mL of H2SO4, and 10 mL of H3PO4 dissolved in 20 mL of H2O) were added. After boiling at 100 °C for 30 min, it was cooled in an ice bath for 5 min, and then aspirated back into the 96-well plate according to the grouping, 250 μL per well. The absorbance at 465 nm was measured, and the inhibition curve was fitted using GraphPad Prism 9.5.1, and the IC 50 value was calculated.

[0779] (2) Cell viability assay - MTT method

[0780] Mouse TNBC cells 4T1 and 4T1-luc, human TNBC cell MDA-MB-468, and human normal mammary epithelial cell MCF-10A were all purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). 4T1 and 4T1-luc were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin; MDA-MB-468 was cultured in DMEM medium containing 10% bovine serum and 1% penicillin-streptomycin; MCF-10A was cultured in a special medium for MCF-10A cells. All cells were cultured in a cell incubator at 37°C and 5% CO2.

[0781] Cells in the logarithmic growth phase with good status were washed with PBS to remove the medium on the cell surface. After digestion with trypsin containing 0.25% EDTA until the cell morphology became round, the corresponding medium was added to terminate the digestion, and the cells were gently pipetted to completely detach and disperse (suspension cells did not need to be digested). Transferred to a 15 mL centrifuge tube, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in the medium and counted. The cells were evenly seeded in a 96-well plate at a density of (3 - 5)×10 4 cells / mL, 100 μL per well, and cultured in an incubator at 37°C and 5% CO2 for 12 h. The cell attachment and growth were observed, and 25 μL of the test sample diluted with the medium in a gradient was added according to the preset, with 6 replicates for each concentration, and blank and negative control groups were set for each plate. After culturing for 48 h, 25 μL of MTT solution (5 mg / mL) was added to each well and cultured for another 4 h. The supernatant was discarded, 150 μL of DMSO was added to each well, and shaken on a cell shaker for 10 min to fully dissolve the formazan. The absorbance (OD value) at a wavelength of 490 / 570 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and a cell viability curve was plotted using GraphPad Prism 9.5.1 software and the IC 50 value was calculated. The negative control group was added with 25 μL of medium without the drug solution, and the blank group did not contain cells (used to deduct the ultraviolet absorption at the bottom of the plate). Each experiment was repeated and measured at least three times.

[0782] Considering the extensive histological distribution of PAD2 in vivo and its important role in breast cancer, all compounds were screened for inhibitory activity against PAD2 and PAD4 by in vitro chemiluminescence method, and the in vitro anti-proliferative activities against TNBC cell lines 4T1 and MDA-MB-468, and human normal mammary cell line MCF-10A were evaluated by the MTT method. The results are shown in Tables 2 and 3.

[0783] Table 2 Inhibitory effects of compound 7-17e on PAD and its in vitro anti-proliferative activities

[0784]

[0785]

[0786]

[0787] a The samples were tested in triplicate and expressed as mean ± standard deviation (SD).

[0788] Table 3 Inhibitory effects of compounds 18 - 48i on PAD and their in vitro anti - proliferative activities

[0789]

[0790]

[0791]

[0792]

[0793]

[0794] a The samples were tested in triplicate and expressed as mean ± standard deviation (SD).

[0795] Table 4 Inhibitory effects of compounds 48h - 48i on PAD and their in vitro anti - proliferative activities

[0796]

[0797]

[0798] a The samples were tested in triplicate and expressed as mean ± standard deviation (SD).

[0799] It can be seen that as a pan - PAD inhibitor, lead compound 7 can significantly inhibit PAD2 and PAD4 activities in vitro, with IC 50 values of 5.28 ± 0.22 μM and 3.54 ± 0.19 μM respectively, which is consistent with the previous research results. In addition, compound 7 can significantly inhibit the in vitro proliferation of 4T1 and MDA - MB - 468 cells, with IC 50 values of 7.34 ± 0.65 μM and 5.16 ± 0.22 μM respectively, and also shows relatively high cytotoxicity to human normal breast cells MCF - 10A, with its IC 50was 4.30 ± 0.67 μM. The poor isoenzyme selectivity of compound 7 and its indiscriminate killing of cells prompted us to further develop new highly efficient and highly selective PAD4 inhibitors. In this part of the work, the present invention first demonstrated that the change in the electron cloud density of the N-terminal modification group would affect the affinity and selectivity of the inhibitor for PAD4 (see Table 2). Compounds 8 and 9 were obtained by reducing the electron cloud density of the N-terminal conjugated ring, but the activity of the compounds was not significantly improved. In addition, compounds 10 and 12-17 obtained by changing the electron cloud density on the basis of the binary conjugated ring could improve the inhibitory activity against PAD4 (IC 50 < 3 μM). In contrast, compound 11 obtained based on the strategy of increasing the number of conjugated rings showed appropriate anti-tumor cell proliferation activity while enhancing the in vitro PAD4 enzyme inhibitory activity (IC 50 = 1.76 ± 0.08 μM), which was worthy of further optimization.

[0800] Further structural modifications mainly focused on the α-position of compound 11, and the in vitro biological activities of the synthesized derivatives were evaluated (see Table 3). Among them, the long carbon chain modification of compound 24 had better safety for normal breast cells MCF-10A; compounds 18-27 (except 24) obtained by introducing saturated hydrocarbons or cycloalkanes at the α-position effectively enhanced the inhibitory activity against PAD4 (IC 50 < 1.8 μM) and the anti-proliferation ability against TNBC cells (IC 50 < 20 μM). These compounds had no selectivity for normal breast cells MCF-10A, indicating their safety for normal cells. Different from saturated cycloalkanes, most of the compounds with conjugated ring modifications introduced at the α-position weakened the cytotoxicity to MCF-10A to varying degrees. Among them, after introducing a freely rotatable benzene ring, compound 28 showed significant inhibition of the activities of PAD2 and PAD4, with IC 50 being 2.97 ± 0.29 μM and 0.79 ± 0.09 μM respectively, showing good selectivity for PAD4. In addition, compound 28 could significantly inhibit the in vitro proliferation of TNBC cells, 4T1: IC 50 = 2.39 ± 0.54 μM, MDA-MB-468: IC 50 = 2.34 ± 0.23 μM; in contrast, its toxicity to normal breast cells was relatively low, MCF-10A: IC 50= 8.39 ± 0.60 μM, with a larger therapeutic window. It is worth noting that polar group modifications were introduced on the benzene ring of compound 28, and compounds 32 - 36 obtained retained good PAD enzyme inhibitory activity. However, compounds 37 - 40 obtained by alkyl blocking reversed this trend to a certain extent. Compounds 41 - 42 obtained by introducing short carbon chains on the benzene ring also retained good PAD enzyme inhibitory activity. In contrast, most compounds 43 - 48 introduced with a bi - aromatic ring structure showed satisfactory ability to inhibit the in vitro proliferation of TNBC cells.

[0801] Based on the structure - activity relationship discussion, compound 28 was identified to have the best correlation between PAD4 enzyme inhibitory activity and anti - TNBC cell proliferation activity, showing moderate selectivity for PAD4, and its in vitro anti - tumor cell proliferation activity and safety were superior to those of the lead compound 7. Therefore, in this part of the work, compound 28 was used as a representative inhibitor of PAD4 for the treatment of triple - negative breast cancer and a tool drug for further studying the key functions of PAD4 in TNBC progression.

[0802] Test Example 2 In vitro anti - proliferation and anti - migration activities of compound 28

[0803] (1) Transwell chamber assay

[0804] The pre - starved 4T1 cells were dispersed in serum - free RPMI 1640 medium and evenly seeded in the upper chamber of the Transwell at a density of 5×10 4 cells / well, and treated with the test samples prepared with serum - free medium. 600 μL of normal medium was added to the lower chamber. It should be noted that there should be no air bubbles between the lower medium and the bottom membrane of the chamber. After incubation in an incubator at 37 °C and 5% CO2 for 12 h, the medium and cells in the upper chamber were gently wiped off with a cotton swab, and the cells that had migrated across the membrane were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 15 min. Using the Zeiss inverted microscope Zen Blue 3.1 imaging system, 9 different fields of view were selected for each well to take pictures, and the number of cells migrating across the membrane was counted using Image J.

[0805] (2) Cell scratch / wound - healing assay

[0806] The 4T1 cells in the logarithmic growth phase were seeded at 5×10 5Inoculate evenly at a density of cells / holes in a six-well plate and culture in an incubator at 37 °C and 5% CO2 for 12 h until the confluence is over 90%. Scratch with the tip of a sterilized 100 μL pipette, rinse the cell debris with PBS buffer, and treat with normal medium containing different concentrations of the sample to be tested. Set at least three replicates for each group. After continuing to culture for 24 h, use the Zeiss inverted microscope Zen Blue 3.1 imaging system to take pictures of 3 - 6 different fields of view for each well, and use Image J to statistically analyze the healing area.

[0807] The in vitro anti - proliferation and anti - migration activities of compound 28 are as Figure 2 shown. Figure 2 In it, (A) is the correlation analysis of the inhibitory activities against PAD4 and 4T1 cells; (B) is the effect of compound 28 on the survival of TNBC cells. Cell viability was determined by the 3 - (4,5 - dimethylthiazol - 2 - yl) - 2,5 - diphenyltetrazolium bromide (MTT) assay. (C) and (D) are the representative results of transwell assay and analysis. Scale bar: 1000 μm. (E) and (F) are the representative results of wound healing assay and analysis. Scale bar: 1000 μm. Data are expressed as mean ± standard deviation. Statistical analysis was performed by one - way ANOVA ***p < 0.001.

[0808] By performing a correlation analysis on the in vitro PAD4 enzyme inhibitory activity and the anti - 4T1 cell proliferation ability, compound 28 was screened out to have the most relevant anti - enzyme and anti - cancer cell activities ( Figure 2 A in it), and was used for subsequent research. Using lead compound 7 as a positive control, compound 28 showed enhanced ability to inhibit the in vitro proliferation of TNBC cells such as 4T1 and MDA - MB - 468 ( Figure 2 B in it), which may be because compound 28 has enhanced PAD4 inhibitory effect and higher cell membrane permeability. Transwell and scratch experiments showed that compound 28 inhibited the in vitro migration of 4T1 cells in a dose - dependent manner. And, at the same dose, the number of migrated cells and the scratch healing rate of compound 28 were significantly lower than those of compound 7, indicating that compound 28 has enhanced anti - migration activity ( Figure 2 C - F in it). These results indicate that the in vitro anti - tumor ability of compound 28 is superior to that of lead compound 7.

[0809] Test Example 3 Compound 28 inhibits H3cit and NET formation

[0810] (1) H3cit expression in tumor cells

[0811] Seed 4T1 cells at 2×10 5Cells were evenly inoculated in confocal dishes at a density of [[number]] cells per dish, cultured in an incubator at 37 °C and 5% CO2 for 12 h, and then the corresponding test samples were added and incubated for another 48 h. After washing twice with PBS, they were fixed with 4% paraformaldehyde (containing 0.2% Triton X-100) at 4 °C for 15 min and blocked with 5% BSA at room temperature for 1 h. Subsequently, they were incubated overnight at 4 °C with anti-histone H3 (citrulline R2+R8+R17) antibody (ab5103, 1:400), and then with Alexa 568 Goat anti-rabbit IgG H&L (ab175471, 1:800) at room temperature for 1 h. The nuclear DNA was stained with Hoechst 33342 for 3 min, washed twice with PBS, and then PBS containing an anti-fluorescence quencher was added. Images were taken using a STED super-resolution confocal microscope, and the average fluorescence intensity of each field was statistically analyzed using Image J.

[0812] (2) Neutrophil extracellular trap (NET) release assay

[0813] After euthanizing female BALB / c mice aged 6 - 8 weeks, bone marrow was aseptically isolated from the femurs and tibias, suspended in PBS, and filtered through a 70 μm nylon membrane to obtain a single-cell suspension. Neutrophils were isolated using a mouse neutrophil isolation kit (TBDSceicge, LZS1100) and evenly inoculated in confocal dishes at a density of 5×10 5 cells per dish. The corresponding test compounds were added respectively, and the cells were incubated in an incubator at 37 °C and 5% CO2 for 2 h. Subsequently, the calcium ionophore A23187 (5 μmol / L) was added and incubation continued for 2 h to induce NETosis. Centrifuge at 4 °C, 500 g for 5 min without braking deceleration, and carefully aspirate the culture medium. The cells were incubated with a fluorescently labeled anti-mouse Ly6G monoclonal antibody (ThermoFisher Scientific, 11-9668-82, 1:400) at room temperature in the dark for 30 min. Centrifuge at 4 °C, 500 g for 5 min without braking deceleration, and carefully aspirate the antibody. Fix with 4% paraformaldehyde (containing 0.2% Triton X-100) for 15 min and block with 5% BSA at room temperature for 1 h. Subsequently, incubate with anti-histone H3 (citrulline R2+R8+R17) antibody (ab5103, 1:400) overnight at 4 °C, and then with Alexa 568Goat anti-rabbit IgG H&L(ab175471, 1:800) was incubated at room temperature for 1 h. Nuclear DNA was stained with Hoechst 33342 for 3 min. After washing twice with PBS, PBS containing an anti-fluorescence quencher was added. Images were taken using a STED super-resolution confocal microscope, and the average fluorescence intensity of each field was statistically analyzed using Image J.

[0814] To further investigate the inhibitory effect of compound 28 on PAD4 in cells, immunoblotting and immunofluorescence analysis were used to evaluate the citrullination level in 4T1 cells. The effects of compound 28 on histone citrullination and NET formation are shown as Figure 3 follows. Figure 3 In (A) and (B), representative results of immunoblotting assays and analysis are shown, in which the expression of H3cit and PAD4 in 4T1 cells treated with 7, 27, and 28 are presented. Actin was used as a loading control; in (C) and (D), representative results of immunofluorescence assays and analysis are shown, in which the expression of H3cit (red) in 4T1 cells treated with 7 and 28 are presented. Scale bar: 50 μm; in (D) and (E), the expression of H3cit (red) and NET formation (enlarged) in mouse bone marrow neutrophils treated with 7 or 28 activated by the calcium ionophore A23187 were evaluated, in which DNA was stained with Hoechst 33342 (blue), and neutrophils were identified by high staining of Ly6G (green). Scale bar: 50 μm. Data are presented as mean ± standard deviation. Statistical analysis was performed by one-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001.

[0815] As expected, the immunoblotting results showed that compound 28 could inhibit H3cit in 4T1 cells in a dose-dependent manner; moreover, at the same dose, this inhibitory effect was significantly greater than that of the lead compound 7 and the compound 27 modified with a saturated hydrocarbon ring ( Figure 3 A–B in). This result was consistent with the in vitro enzyme inhibition experiment, indicating that compound 28 obtained by introducing a conjugated aromatic ring on the basis of compound 11 more effectively inhibited the activity of PAD4 than the compound 27 modified with a saturated hydrocarbon ring, which might benefit from the strong pi-cation interaction between the aromatic ring and Arg639. Interestingly, compared with the positive control 7, the expression of PAD4 was upregulated in 4T1 cells treated with compound 28 ( Figure 3 A–B in). This might be because compound 28 mainly focused on inhibiting the activity of PAD4, thus increasing the expression level of PAD4 through negative feedback. In addition, immunofluorescence analysis further confirmed that compound 28 significantly inhibited H3 citrullination mediated by PAD4 in 4T1 cells, and its inhibitory ability was superior to that of compound 7 at the same dose.Figure 3 in C–D).

[0816] Next, the inhibitory effect of compound 28 on mouse bone marrow neutrophil NETosis was detected. The calcium ionophore A23187 was used to induce an increase in intracellular calcium concentration. It was observed that the H3cit of neutrophils in the Control group was significantly enhanced, and a filamentous or reticular substance was released, which was co-localized by blue DNA and red H3cit, called NETs ( Figure 3 in E–F). In contrast, treatment with compound 7 or 28 before induction with the calcium ionophore could effectively inhibit histone H3 citrullination and NET formation. Notably, compared with the positive control 7, neutrophils treated with compound 28 showed lower H3cit levels while maintaining a high degree of cell membrane integrity (judged by green Ly6G), which may mean that the function and activity of neutrophils as immune cells were retained. Taken together, these results demonstrated that compound 28 is an excellent PAD4 inhibitor that can effectively block histone H3 citrullination and NET formation and ensure the integrity of neutrophils.

[0817] Test Example 4 Pharmacokinetics of Compound 28

[0818] Based on its good in vitro bioactivity, the pharmacokinetic properties of compound 28 in Sprague–Dawley (SD) rats were further evaluated (see Table 4). Compound 28 at 3.5 mg / kg was administered via the tail vein, and it was observed to have a moderately improved terminal elimination half-life (T1 / 2, 0.25 h), peak concentration (Cmax, 297.71 ng / mL), clearance (CL, 105.24 L / h / kg), and drug exposure (AUC 0- , 17346.55 h·ng / L); these properties reflected the pharmacokinetic optimization of compound 28 relative to compound 7. At the same time, considering the good in vitro activity of compound 28, it is of great significance to evaluate its potential for anti-tumor and anti-metastasis in vivo.

[0819] Table 5 Pharmacokinetic characteristics of compounds 7 and 28 in vivo

[0820]

[0821] a Samples in triplicate were taken at each time point.

[0822] Test Example 5 Anti-tumor and anti-metastasis activities in vivo--4T1-luc orthotopic tumor-bearing mouse model

[0823] Male ICR mice, male C57BL / 6 mice, and female BALB / c mice at 6 - 8 weeks of age with a body weight of 20 ± 2 g, and male Sprague Dawley (SD) rats with a body weight of 220 ± 10 g were all purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal experiments were approved by the Institutional Animal Care and Use Committee of Capital Medical University, with the ethics number: AEEI - 2018 - 174.

[0824] 4T1 - luc orthotopic breast cancer model in BALB / c mice. After the 4T1 - luc cells cultured in vitro were passaged to a certain number, they were digested with trypsin and dispersed into a cell suspension of 5×10 6 cells / mL with PBS, and 0.1 mL was injected into the fourth mammary pad of female BALB / c mice. When the tumor volume reached approximately 25 mm 3 (usually on the 5th day after tumor inoculation), the tumor - bearing mice with uniform tumor sizes were randomly grouped. All test samples were dissolved in physiological saline containing 5% DMSO and 20% HP - β - CD, and administered via tail vein injection every two days according to the groups. Since the administration, the body weight and tumor volume of the mice were measured every two days. After the last administration for 24 h, the mice were weighed, sacrificed by cervical dislocation after ether anesthesia, the tumor tissues and main organs were dissected, photographed, weighed, and immediately frozen in liquid nitrogen or fixed with formalin for standby. Serum ALT, AST, UREA, and CREA - S levels were detected using an automatic biochemical analyzer and corresponding kits. The grouping for the survival curve experiment was the same as the above - mentioned grouping, and the mice were raised under the same conditions and continuously administered until the mice died or exceeded the specified time.

[0825] Table 6 Administration schedule for the 4T-luc orthotopic tumor model

[0826]

[0827] i.v. Intravenous injection for administration

[0828] To evaluate the anti - tumor and anti - metastatic activities of compound 28 in vivo, a 4T1 - luc orthotopic tumor model in BALB / c mice was established. When the tumor volume reached about 25 mm 3 or so, the tumor - bearing mice were randomly divided into 6 groups (10 mice in each group): blank control Control, positive control DOX (1.16 mg / kg) and 7 (10 mg / kg), and the dosing groups 28 (10, 5, and 1 mg / kg). All samples were dissolved in physiological saline containing 5% DMSO and 20% HP - β - CD, and injected intravenously every two days for a total of 9 times.

[0829] Figure 4Antitumor activity in vivo of compound 28 against the orthotopic 4T1-luc xenograft model. (A) and (B) show the treatment regimens and survival curves of orthotopic 4T1-luc tumor-bearing mice. In the control group, DOX (1.16 mg / kg), 7 (10 mg / kg), and 28 (10, 5, and 1 mg / kg) were intravenously injected every two days. (C) shows in vivo images and analysis of orthotopic tumors in 4T1-luc tumor-bearing mice on day 1 and day 17 of treatment. (D) shows in vivo images and analysis of orthotopic tumors in 4T1-luc tumor-bearing mice on day 1 and day 17 of treatment. (E) and (F) show the weights and representative pictures of tumors harvested on day 18. Data are presented as mean ± standard deviation. Statistical analysis was performed by one-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001.

[0830] Figure 5 Antimetastatic activity in vivo of compound 28 against the orthotopic 4T1-luc xenograft model. (A) and (B) show in vivo images and analysis of lung metastases in 4T1-luc tumor-bearing mice on day 17 of treatment. (C) and (D) show representative images (indicated by arrows) and analysis of metastases in lung tissues. (E) shows H&E staining of lung sections. (F) and (G) show immunohistochemical staining and analysis of H3cit and Ly6G in tumor sections. Scale bar: 50 μm. Data are presented as mean ± standard deviation. Statistical analysis was performed by one-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001.

[0831] Tumor-bearing mice treated with high-dose (10 mg / kg) 28 had the best survival curve ( Figure 4 in A). More importantly, in vivo imaging, tumor volume monitoring, and tumor weight statistics of 4T1-luc tumor-bearing mice all confirmed that compound 28 could inhibit the growth of primary tumors of mouse TNBC in a dose-dependent manner ( Figure 4 in C–F). Statistical results showed that the tumor growth inhibition rate of the 10 mg / kg 28 treatment group was 61.8%, that of the positive control DOX group was 54.6%, and that of the 10 mg / kg 7 treatment group was 36.2%. In addition, compound 28 could inhibit the lung metastasis of mouse TNBC in a dose-dependent manner; and compared with the DOX and 7 treatment groups, the anti-lung metastasis ability of the 10 mg / kg 28 treatment group was significantly enhanced, and the H&E staining results of its lung tissue sections were similar to those of normal mice ( Figure 5 in A–E). Notably, immunohistochemical staining of H3cit and Ly6G in tumor sections showed that at the same dose, the inhibitory effect of the 28 treatment group on histone H3 citrullination and NET formation was comparable to that of the 7 treatment group, and the 28 treatment group significantly increased neutrophil infiltration in tumor tissues ( Figure 5In Figures F - G). Inspired by this discovery, the present invention further evaluated the effect of compound 28 intervention on the tumor immune microenvironment of triple-negative breast cancer.

[0832] Figure 6 It is the biocompatibility evaluation of compound 28 on the in-situ 4T1-luc xenograft model. (A) is the body weight curve of 4T1-luc tumor-bearing mice during different treatments. (B)-(F) are the visceral-somatic ratios of the main organs (heart, liver, spleen, kidney, brain) after different treatments. (G)-(J) are the serum biochemical indexes (ALT, AST, UREA, CREA-S) after different treatments.

[0833] Figure 7 It is the H&E staining of the heart, liver, spleen and kidney tissue sections of normal mice and 4T1-luc tumor-bearing mice after different treatments.

[0834] In the biosafety evaluation of compound 28 treatment, it was observed that the body weights of mice in each group remained stable during different treatments; however, the statistical results of the visceral-somatic ratios of mice showed that after the treatment ended, the weights of the livers and kidneys of mice in the positive control DOX group and the 7 treatment group decreased significantly ( Figure 6 in Figures A - F). Compared with the Control group, there were no significant differences in the visceral-somatic ratios and serum biochemical indexes (ALT, AST, UREA and CREA-S) of mice in the 10 mg / kg 28 treatment group ( Figure 6 in Figures B - J). In addition, no obvious morphological or structural damage was observed in the heart, liver, spleen and kidney tissues of mice in the compound 28 treatment group in the H&E staining results ( Figure 7 ). In summary, at the same dose (10 mg / kg), the anti-tumor and anti-metastatic effects of compound 28 in the 4T1-luc orthotopic tumor model of mice were significantly enhanced compared with the lead compound 7; compound 28 is a promising anti-TNBC candidate drug and has no obvious physiological toxicity.

[0835] Test Example 6 Effect of compound 28 on the tumor immune microenvironment

[0836] In the tumor immune microenvironment, PD-L1+TANs show a tumor-promoting phenotype by inhibiting T cell cytotoxicity, leading to poor prognosis and low survival rate of patients. Single-cell mass cytometry was used to detect the immune cell composition in the tumor tissues of 4T1-luc tumor-bearing mice, and the cells were grouped and counted according to the specific antibody expression of immune cells (Figure 4.30A).

[0837] Figure 8Effect of compound 28 on the tumor immune microenvironment. (A) tSNE plot of immune cells in tumor tissues detected by single-cell flow cytometry-mass spectrometry. (B)-(C) Proportions of immune cells (DC, cDC, M1 macrophages, G-MDSCs, and M-MDSCs) and tumor-associated neutrophils in tumor tissues. (D)-(E) Expression levels of PD-L1 and MHC-II in tumor-associated neutrophils.

[0838] The results showed that compound 28 treatment could induce a broad-spectrum anti-tumor phenotype in the tumor immune microenvironment by downregulating the proportions of myeloid-derived immunosuppressive cells M-MDSCs and G-MDSCs, upregulating the proportion of dendritic cells (DCs), especially conventional dendritic cells (cDCs), and promoting the polarization of anti-tumor M1 macrophages ( Figure 8 as shown in B). In addition, 28 treatment increased the proportion of mature tumor-associated neutrophils MHC-II + TANs and inhibited the proportion of pro-tumor phenotype PD-L1 + / MHC-II + TANs and MHC-II-TANs ( Figure 8 as shown in C). Interestingly, a significant decrease in PD-L1 expression was observed in MHC-II + TANs including PD-L1 + , indicating a weakened immunosuppressive function of these cells ( Figure 8 as shown in D). Moreover, the expression of major histocompatibility complex class II molecules in MHC-II + TANs and MHC-II-TANs was enhanced after 28 treatment, indicating an enhanced function as antigen-presenting cells (APCs) ( Figure 8 as shown in E). In summary, these results suggest that compound 28 mainly transforms the tumor immune microenvironment from a pro-tumor state to an anti-tumor environment by regulating the proportion of immune cells and reshaping the phenotype and function of neutrophils.

[0839] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A carboline-derived PAD4 inhibitor having the structure shown in Formula I: In formula I, R1 is one of; R2 is one of; X is one of 2. A method for preparing the carboline-derived PAD4 inhibitor according to claim 1, comprising the following steps: Mix Z-Orn(Boc)-OH having the structure shown in Formula a, benzylamine, a condensing agent and an organic solvent, and carry out a first condensation reaction to obtain a compound having the structure shown in Formula b; The compound having the structure shown in Formula b undergoes a first deprotection reaction to obtain a compound having the structure shown in c; Mix the compound having the structure shown in Formula c, the compound having the structure shown in Formula d, a condensing agent and an organic solvent, and carry out a second condensation reaction to obtain a compound having the structure shown in Formula e; The compound having the structure shown in Formula e undergoes a second deprotection reaction to obtain a compound having the structure shown in Formula f; Mix the compound having the structure shown in Formula f, 2-chloroethyl iminoacetate and an organic solvent, and carry out a coupling reaction to obtain the carboline-derived PAD4 inhibitor having the structure shown in Formula I; In Formula a and Formula b, R3 is Boc or Cbz, R4 is Cbz or Boc, and R3 is different from R4; In Formula c and Formula e, R4 is Cbz or Boc.

3. The preparation method according to claim 2, characterized in that, When the R1 is one of the following, R3 is Cbz and R4 is Boc; When R1 is one of , R3 is Boc and R4 is Cbz.

4. The preparation method according to claim 2, characterized in that, When the R1 is , (1) When R2 is one of , R3 is Boc and R4 is Cbz; (2) When R2 is one of , R3 is Cbz and R4 is Boc.

5. The preparation method according to claim 2, wherein When R1 is The preparation method of the compound having the structure shown in formula d includes the following steps: The compound having the structure shown in Formula g undergoes an esterification reaction with methanol to obtain a compound having the structure shown in Formula h; The compound having the structure shown in Formula h undergoes a cyclization reaction with a compound having the structure of R2-CHO to obtain a compound having the structure shown in Formula j; The compound having the structure shown in Formula j is mixed with DDQ and an organic solvent, and undergoes a dehydrogenation reaction to obtain a compound having the structure shown in Formula k; The compound having the structure shown in Formula k undergoes hydrolysis and acidification reactions to obtain a compound having the structure shown in Formula d.

6. The preparation method according to claim 2, wherein The condensing agent is a DCC-HOBt system or an EDC-HOBt system; The temperature of the first condensation reaction and the second condensation reaction is room temperature, and the time is independently 6 to 10 h.

7. The preparation method according to claim 2, wherein When R3 is Cbz, the first deprotection reaction is carried out in a Pd / C catalyst and a hydrogen atmosphere; when R3 is Boc, the first deprotection reaction is carried out in an HCl / EA system; When R4 is Boc, the second deprotection reaction is carried out in an HCl / EA system; when R4 is Cbz, the second deprotection reaction is carried out in a Pd / C catalyst and a hydrogen atmosphere.

8. The preparation method according to claim 2, characterized in that The coupling reaction is carried out under alkaline conditions, and the pH value of the alkaline conditions is 9 to 11.

9. Use of the carboline-derived PAD4 inhibitor according to claim 1 or the carboline-derived PAD4 inhibitor prepared by the preparation method according to any one of claims 2 to 8 in the preparation of an anti-tumor drug.

10. The application according to claim 9, characterized in that, The anti-tumor drug includes an anti-breast cancer drug and / or an anti-lung cancer drug.

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