A carbolin-derived pad4 inhibitor and preparation method and application thereof

Abstract

The application provides a kind of karyolin derived PAD4 inhibitor and its preparation method and application, belong to biological medicine technical field.The application is based on the strategy of changing N-terminal electron cloud density and expanding conjugated system, PAD4 inhibitor is developed to a series of small molecules with significant in vitro enzyme activity inhibition and TNBC cell killing ability by modifying chloroacetamidine skeleton, relevant anti-enzyme and anticancer cell activity are shown, and moderate preference for PAD4 enzyme is shown.The in vitro anti-TNBC cell proliferation activity research shows that the karyolin derived PAD4 inhibitor provided by the application is superior to the lead compound 7 in in vitro anti-TNBC cell proliferation activity and safety, and has improved pharmacokinetic characteristics.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a carbaline-derived PAD4 inhibitor, its preparation method, and its application. Background Technology

[0002] Breast cancer (BC) is one of the most common types of cancer in women worldwide, and its incidence continues to rise in recent years. Triple-negative breast cancer (TNBC) is a specific subtype characterized by the negative expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), accounting for approximately 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. Furthermore, the aggressive nature, poor prognosis, and high recurrence rate of TNBC limit the efficacy of standard chemotherapy regimens such as anthracyclines, cisplatin, and paclitaxel. Therefore, there is an urgent need to identify new potential targets and develop effective targeted inhibitors for the treatment of TNBC.

[0003] Peptidylarginine deiminase 4 (PAD4) is a member of the calcium-dependent hydrolase PADs family and is overexpressed in various malignant tumors. PAD4 catalyzes the citrullination of nucleoproteins, particularly histones, converting positively charged arginine residues into neutrally charged citrulline residues. This weakens the interaction between histones and negatively charged DNA, leading to chromatin depolymerization and nuclear membrane rupture, ultimately resulting in the release of NETs by neutrophils. Recent studies have shown that TNBC cells with high PAD4 expression can also release the cancer extracellular chromatin network (CECN), thereby promoting cancer cell metastasis. Since NET degradation by DNase I alone does not affect circulating tumor cells, nor does it cause associated tissue damage and microthrombus formation, inhibiting endogenous PAD4 may be a potential targeted strategy for the treatment of highly metastatic TNBC. Summary of the Invention

[0004] In view of this, the present invention aims to provide a carbaline-derived PAD4 inhibitor, its preparation method, and its application. The carbaline-derived PAD4 inhibitor provided by the present invention exhibits 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 by immune cells and polarization of M1 macrophages, thereby reducing the proportion of suppressor cells. Furthermore, it can regulate the tumor microenvironment to an anti-tumor state by inhibiting the PAD4-H3cit-NETs pathway and reshaping the phenotype and function of neutrophils, thus effectively inhibiting the progression of triple-negative breast cancer.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a carbaline-derived PAD4 inhibitor having the structure shown in Formula I:

[0007]

[0008] In Equation I, R1 is One of them;

[0009] R2 is One of them;

[0010] X is One of them.

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

[0012] Z-Orn(Boc)-OH having the structure shown in Formula a, benzylamine, a condensing agent and an organic solvent were mixed and subjected to 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 give the compound having the structure shown in formula c.

[0014]

[0015] A compound having the structure shown in formula c, a compound having the structure shown in formula d, a condensing agent, and an organic solvent are mixed to 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 give the compound having the structure shown in formula f.

[0018]

[0019] A compound having the structure shown in Formula f, 2-chloroacetyliminoethyl ester, and an organic solvent were mixed and coupled to obtain a carbline-derived PAD4 inhibitor having the structure shown in Formula I.

[0020] In equations a and b, R3 is either Boc or Cbz, and R4 is either Cbz or Boc, and R3 and R4 are different.

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

[0022] Preferably, when R1 is In one of these cases, R3 is Cbz and R4 is Boc;

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

[0024] Preferably, when R1 is hour,

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

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

[0027] Preferably, when R1 is The method for preparing the compound having the structure shown in formula d includes the following steps:

[0028] A compound having the structure shown in formula g undergoes an esterification reaction with methanol to yield a compound having the structure shown in formula h.

[0029]

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

[0031] A compound having the structure shown in formula j is mixed with DDQ and an organic solvent and subjected to a dehydrogenation reaction to obtain a compound having the structure shown in formula k.

[0032]

[0033] A compound having the structure shown in formula k is subjected to hydrolysis and acidification to obtain a compound having the structure shown in formula d.

[0034] Preferably, the condensing agents are DCC and HOBt, and the temperature of the first condensation reaction and the second condensation reaction are room temperature, and the time is independently 6 to 10 hours.

[0035] Preferably, 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.

[0036] 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.

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

[0038] This invention provides the application of the above-mentioned carboline-derived PAD4 inhibitor in the preparation of antitumor drugs.

[0039] Preferably, the antitumor drug includes anti-breast cancer drugs and / or anti-lung cancer drugs.

[0040] This invention provides a carbaline-derived PAD4 inhibitor. Based on a strategy of altering the N-terminal electron cloud density and expanding the conjugated system, this invention modifies the chloroacetamidine backbone, developing a series of small-molecule PAD4 inhibitors with significant in vitro enzyme activity inhibition and TNBC cell killing capabilities. These inhibitors exhibit relevant anti-enzyme and anti-cancer cell activities, and a moderate preference for PAD4 enzymes. Furthermore, in vitro anti-TNBC cell proliferation activity studies show that the carbaline-derived PAD4 inhibitor provided by this invention has superior anti-TNBC cell proliferation activity and safety compared to lead compound 7 (structural formula below), while also possessing improved pharmacokinetic characteristics.

[0041]

[0042] The results of the examples show that the carbaline-derived PAD4 inhibitor provided by the present invention can upregulate DCs, especially cDCs, by inhibiting the PAD4-H3cit-NETs pathway and reducing the proportion of immunosuppressive cells M-MDSCs and G-MDSCs in the tumor microenvironment, thereby promoting the polarization of anti-tumor M1 macrophages and inducing 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 MHC-II antigen presentation of mature tumor-associated neutrophils. + The proportion of TANs, and the inhibition of the pro-tumor phenotype PD-L1 + / MHC-II + The ratio of TANs to MHC-II-TANs is used 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] This invention provides a method for preparing the above-mentioned carboline-derived PAD4 inhibitor. This method is simple to operate, low in cost, and easy to achieve industrial-scale mass production. Attached Figure Description

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

[0045] Figure 2 To demonstrate the in vitro antiproliferative and antimigration activities of compound 28;

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

[0047] Figure 4 To assess the in vivo antitumor activity of compound 28 in an in situ 4T1-luc xenograft model;

[0048] Figure 5 To assess the in vivo anti-metastasis activity of compound 28 against an in situ 4T1-luc xenograft model;

[0049] Figure 6 To evaluate the biocompatibility of compound 28 in an in situ 4T1-luc xenograft model;

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

[0051] Figure 8 The effect of compound 28 on the tumor immune microenvironment. Detailed Implementation

[0052] This invention provides a carbaline-derived PAD4 inhibitor having the structure shown in Formula I:

[0053]

[0054] In Equation I, R1 is One of them;

[0055] R2 is One of them;

[0056] X is One of them.

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

[0058] This invention provides a method for preparing the above-mentioned carbline-derived PAD4 inhibitor, comprising the following steps:

[0059] Z-Orn(Boc)-OH having the structure shown in Formula a, benzylamine, a condensing agent and an organic solvent were mixed and subjected to a first condensation reaction to obtain a compound having the structure shown in Formula b.

[0060] The compound having the structure shown in formula b undergoes a first deprotection reaction to give the compound having the structure shown in formula c.

[0061]

[0062] A compound having the structure shown in formula c, a compound having the structure shown in formula d, a condensing agent, and an organic solvent are mixed to carry out a second condensation reaction to obtain a compound having the structure shown in formula e.

[0063]

[0064] The compound having the structure shown in formula e undergoes a second deprotection reaction to give the compound having the structure shown in formula f.

[0065]

[0066] A compound having the structure shown in Formula f, 2-chloroacetyliminoethyl ester, and an organic solvent were mixed and coupled to obtain a carbline-derived PAD4 inhibitor having the structure shown in Formula I.

[0067] In equations a and b, R3 is either Boc or Cbz, and R4 is either Cbz or Boc, and R3 and R4 are different.

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

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

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

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

[0072] (2) When R2 is In one of the cases, R3 is Cbz and R4 is Boc.

[0073] In this invention, Z-Orn(Boc)-OH having the structure shown in Formula a, benzylamine, a condensing agent, and an organic solvent are mixed to perform a first condensation reaction to obtain a compound having the structure shown in Formula b. In this invention, the preferred molar ratio of Z-Orn(Boc)-OH having the structure shown in Formula a to the volume ratio of benzylamine is 10 mmol: 1.6 mL.

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

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

[0076] In this invention, the first condensation reaction is preferably carried out under alkaline conditions, preferably 8 hours. In this invention, the temperature of the first condensation reaction is preferably room temperature, and the time is preferably 6-10 hours, more preferably 8 hours. After the first condensation reaction, the resulting first condensation reaction solution is preferably post-treated, and the post-treatment preferably includes the following steps:

[0077] The first condensation reaction solution was subjected to vacuum filtration and concentration to obtain the residue.

[0078] The residue was dissolved using an organic solvent, and the resulting solution was washed, dried, and purified by column chromatography to obtain a pure compound having the structure shown in formula b.

[0079] In this invention, the organic solvent is preferably ethyl acetate (EA). In this invention, the washing agent is preferably a saturated NaHCO3 solution followed by a saturated NaCl solution, and the drying is preferably done with anhydrous Na2SO4. In this invention, the eluent for column purification is preferably 50% ethyl acetate.

[0080] In this invention, a compound having the structure shown in formula b undergoes a first deprotection reaction to obtain a compound having the structure shown in formula c. In this invention, when R3 is Cbz, the first deprotection reaction is preferably carried out in a Pd / C catalyst under a hydrogen atmosphere. In this invention, the mass of the Pd / C catalyst is preferably 10% of the mass of the compound having the structure shown in formula b. In this invention, when R3 is Cbz, the temperature of the first deprotection reaction is preferably room temperature, and the time is preferably 4 hours. After the first deprotection reaction, the resulting first deprotection reaction solution is preferably filtered under reduced pressure and evaporated to dryness.

[0081] In this invention, when R3 is Boc, the first deprotection reaction is carried out in an HCl / EA system. In this 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 this invention, when R3 is Boc, the temperature of the first deprotection reaction is preferably room temperature, and the time is preferably 4 hours. After the first deprotection reaction, the resulting first deprotection reaction solution is preferably evaporated to dryness under reduced pressure, and the residue is reconstituted using an organic solvent and evaporated to dryness again. In this invention, the organic solvent used for reconstitution is preferably dry ethyl acetate and anhydrous diethyl ether.

[0082] This invention involves mixing a compound having the structure shown in formula c, a compound having the structure shown in formula d, a condensing agent, and an organic solvent to perform a second condensation reaction, yielding a compound having the structure shown in formula e. In this invention, the condensing agent is preferably a DCC-HOBt system or an EDC-HOBt system; as a specific embodiment of this 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 this invention, the second condensation reaction is preferably carried out under alkaline conditions, preferably 8. In this invention, the temperature of the second condensation reaction is preferably room temperature, and the time is preferably 6–10 hours, more preferably 8 hours. In this invention, the post-treatment method of the second condensation reaction is similar to that of the first condensation reaction, and will not be described again here.

[0084] In this invention, a 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 this invention, 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. In this invention, the reaction conditions and post-treatment of the second deprotection reaction are similar to those of the first deprotection reaction, and will not be repeated here.

[0085] This invention involves mixing a compound having the structure shown in Formula f, 2-chloroacetyliminoethyl ester, and an organic solvent, and performing a coupling reaction to obtain a carbline-derived PAD4 inhibitor having the structure shown in Formula I. In this invention, the preferred structural formula of the 2-chloroacetyliminoethyl ester is as follows:

[0086]

[0087] In this invention, the molar ratio of the compound having the structure shown in formula f to 2-chloroacetyliminoethyl ester is preferably 1:5. In this invention, the coupling reaction is carried out under alkaline conditions, with a pH of 9-11, more preferably 10. In this invention, the coupling reaction temperature is preferably room temperature, and the reaction time is preferably 12 hours. After the coupling reaction, the resulting coupling reaction solution is preferably subjected to column purification, removal of organic solvent, and lyophilization. In this invention, the eluent for column purification is preferably 30% CH3OH. In this invention, the removal of organic solvent is preferably by rotary evaporation, and the drying method is preferably lyophilization.

[0088] In this invention, when R1 is The method for preparing the compound having the structure shown in formula d includes the following steps:

[0089] A compound having the structure shown in formula g undergoes an esterification reaction with methanol to yield a compound having the structure shown in formula h.

[0090]

[0091] A compound having the structure shown in formula h undergoes a cyclization reaction with a compound having the R2-CHO structure to obtain a compound having the structure shown in formula j.

[0092] A compound having the structure shown in formula j is mixed with DDQ and an organic solvent and subjected to a dehydrogenation reaction to obtain a compound having the structure shown in formula k.

[0093]

[0094] A compound having the structure shown in formula k is subjected to hydrolysis and acidification to obtain a compound having the structure shown in formula d.

[0095] In this invention, a 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. In this 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 concentrates the obtained esterification reaction solution under reduced pressure to obtain a residue; the obtained residue is then washed and dried. In the present invention, the washing reagents are preferably saturated NaHCO3 solution and saturated NaCl solution, and the drying is preferably drying with anhydrous Na2SO4 followed by rotary evaporation.

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

[0098] Following the cyclization reaction, the present invention preferably concentrates the resulting cyclization reaction solution under reduced pressure to obtain a residue; the residue is then reconstituted using an organic solvent, and the resulting solution is washed, dried, and purified by column chromatography. In this invention, the organic solvent used for reconstitution is preferably CH2Cl2; the washing reagents are preferably saturated NaHCO3 solution and saturated NaCl solution; and the drying is preferably drying with anhydrous Na2SO4. In this invention, the eluent for column purification is preferably 45% EA.

[0099] In this 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 this invention, DDQ is dichlorodicyanobenzoquinone. In this invention, the molar ratio of the compound having the structure shown in formula j to DDQ is preferably 1:2. In this invention, the organic solvent is preferably dichloroethane.

[0100] In this invention, the dehydrogenation reaction is preferably carried out at room temperature and for a duration of 4 hours.

[0101] Following the dehydrogenation reaction, the present invention preferably terminates the reaction using a saturated NaHCO3 aqueous solution, separates the organic phase, and then washes, dries, and purifies the resulting organic phase using a column. In this invention, the washing solution is preferably a saturated NaCl solution, and the drying is preferably anhydrous Na2SO4. In this invention, the eluent for column purification is preferably 4% CH3OH.

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

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

[0104] This invention provides the application of the above-mentioned carboline-derived PAD4 inhibitor in the preparation of antitumor drugs.

[0105] In this invention, the antitumor drug preferably includes an anti-breast cancer drug and / or an anti-lung cancer drug. In this invention, the anti-breast cancer drug is preferably an anti-triple-negative breast cancer drug.

[0106] The following detailed description, in conjunction with embodiments, illustrates the carbaline-derived PAD4 inhibitors, their preparation methods, and applications provided by this invention. However, these descriptions should not be construed as limiting the scope of protection of this invention.

[0107] The synthetic routes and methods for the intermediates used in the following embodiments are as follows:

[0108]

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

[0110] 4H-KLSS(M2a)

[0111] Prepare dilute sulfuric acid (pH=2) by adding 0.2 mL of concentrated sulfuric acid dropwise to 400 mL of distilled water in an ice-water bath. Then, add L-Trp (5.00 g, 24.5 mmol) and 10 mL of 37% formaldehyde solution, and react at room temperature for 5 h. After the reaction is complete as determined by TLC, the mixture is placed in an ice-water bath, and the pH is adjusted to 6 with concentrated ammonia. The mixture is then allowed to stand until a white solid precipitates. The mixture is filtered under reduced pressure, the filter cake is washed with H2O and dried to obtain M2a (4.980 g, 94.1%), 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, with acid gas removed under reduced pressure every 1 h. After the reaction was confirmed to be complete by TLC, the mixture was placed in an ice-water bath, the pH was adjusted to 7 with saturated KHSO4 solution, and the mixture was dried. 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 a medium-pressure preparative column (8% CH3OH) to give M3a (2.85 g, 90.2%) as a white solid.

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

[0115] SOCl2 (2.6 mL) was slowly added dropwise to 20 mL of CH3OH in an ice-water bath, and the mixture was activated for 30 min. Then M2a (2.16 g, 10 mmol) was added, and the reaction was carried out at room temperature for 12 h. After the reaction was confirmed to be complete by TLC, the mixture was concentrated under reduced pressure. The residue was dissolved in 40 mL of EA, and then washed successively with saturated NaHCO3 solution (20 mL × 3) and saturated NaCl solution (20 mL × 3). After drying with anhydrous Na2SO4, the residue was evaporated to dryness to obtain M4 (2.16 g, 93.9%), a light brown solid.

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

[0117] M4 (2.16 g, 9.4 mmol) was dissolved in a 100 mL eggplant flask with 40 mL CH3OH and 10 mL acetone until a light brown, clear solution was obtained. 0.8 g Pd / C was added, a single-pass filter was connected, and the mixture was stirred at room temperature for 7 days. The reaction was monitored by TLC (CH2Cl2:CH3OH = 10:1), and the disappearance of the starting material spot indicated complete reaction. Post-treatment: The mixture was filtered under reduced pressure (the filter residue was rinsed with CH2Cl2 and CH3OH), and purified by a medium-pressure preparative column (8–9% CH3OH was used to collect the product), yielding 1.690 g (79.6%) of M5 as a light brown solid. Simultaneously, a byproduct, identified as M5a, was collected with 7% CH3OH, totaling 0.200 g (9.2%), also a light brown solid.

[0118] KLSS(M6)

[0119] M5 (1.69 g, 7.5 mmol) was dissolved in 20 mL of CH3OH and 20 mL of CH2Cl2. The pH of the reaction system was adjusted to 10 with 2 M NaOH and the reaction was carried out in an ice bath for 4 h. After the reaction was confirmed to be complete by TLC, the pH was adjusted to 2 with saturated KHSO4 solution, the solution was concentrated under reduced pressure, and filtered. The filter cake was washed with H2O and a small amount of ice-cold CH3OH and dried to obtain M6 (1.45 g, 91.5%), a light brown solid. ESI-MS (m / z): 213.0 [M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 12.27 (s, 1H), 9.04 (s, 1H), 9.03 (s, 1H), 8.47 (d, J = 7.9H z,1H),7.72(t,J=8.2Hz,1H),7.67(dt,J=6.9Hz,0.9Hz,1H),7.37(dt,J=7.8Hz,0.8Hz,1H). 13C-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] The preparation method was the same as for M6, yielding M6a (0.18 g, 95.7%), a pale yellow solid. ESI-MS (m / z): 435.7 [MH] - ; 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 9.10 (s, 2H), 9.06 (s, 2H), 8.56 (d, J = 7.8Hz, 2H) ,7.92(d,J=8.4Hz,2H),7.73(t,J=7.6Hz,1H),7.56(s,2H),7.45(t,J=7.5Hz,2H); 13 C-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] L-Phe (3.30 g, 20 mmol) and 37% formaldehyde solution (10 mL) were suspended in 40 mL of concentrated hydrochloric acid and heated at 80 °C for 6 h. After the reaction was complete as detected by TLC, the mixture was cooled to room temperature and filtered under reduced pressure. The filter cake was rinsed with distilled water and dried to obtain M2b (3.33 g, 94.1%), a white solid.

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

[0125] The preparation method is the same as that for M3a. The product was separated and purified by a medium-pressure preparative column (8% CH3OH was used to collect the product) to obtain M3b (2.36 g, 85.0%), which was a white solid.

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

[0127] The preparation method was the same as that for M4, yielding M4b (1.80 g, 94.2%), a light brown solid.

[0128] IQ-OCH3(M5b)

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

[0130] IQ(M6b)

[0131] The preparation method was the same as for M6, yielding M6b (1.56 g, 88.5%), a white solid. ESI-MS (m / z): 174.1 [M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 9.66 (s, 1H), 8.87 (s, 1H), 8.45 (d, J = 8.1Hz, 1H), 8.36(d,J=8.1Hz,1H),8.10(dt,J=7.0Hz,0.9Hz,1H),8.00(dt,J=8.0Hz,0.9Hz,1H). 13 C-NMR (75MHz, 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, rt, 8h; (b) Pd / H2, CH3OH, rt, 4h; (c) EDC, HOBt, THF, NMM, rt, 8h; (d) 4M HCl / EA, 0℃, 4h; (e) Ethyl 2-chloroacetate hydrochloride, anhydrous methanol, DIPEA, rt, 12h.

[0136] Example 1

[0137] 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) were dissolved in 80 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 confirmed to be complete by TLC. Subsequently, the reaction solution was filtered under reduced pressure and concentrated. The residue was dissolved in 60 mL of EA, and successively extracted 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 chromatography (collecting the product with 50% EA) to give M8 (4.49 g, 98.7%) as a white solid.

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

[0139] M8 (4.49 g, 9.9 mmol) and 10% Pd / C (0.5 g) were dissolved in 60 mL of CH3OH. The reaction was stirred at room temperature for 4 h under a hydrogen atmosphere, and TLC was used to determine the completeness of the reaction. The mixture was then filtered under reduced pressure and evaporated to dryness to give M9 (3.99 g, 88.8%) as a white solid. Boc-4H-KLSS-Orn(Boc)-NBzl(M10a)

[0140] 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) 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 confirmed to be complete by TLC. The product was then purified by a medium-pressure preparative column (50% EA was used to collect the product) to give M10a (2.36 g, 70.9%) as a white solid. 4H-KLSS-Orn-NBzl·2HCl (M11a)

[0141] M10a (2.36 g, 3.8 mmol) was dissolved in 25 mL of 4 M HCl / EA in an ice-water bath and stirred for 4 h. After the reaction was complete as detected by TLC, the reaction solution was evaporated to dryness under reduced pressure. The residue was redissolved successively with dry EA (15 mL × 3) and anhydrous diethyl ether (15 mL × 1), and then evaporated to dryness again to obtain M11a (1.78 g, 94.6%), a pale yellow solid.

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

[0143] M11a (1.78 g, 3.6 mmol) and 2-chloroacetyliminoethyl ester (2.83 g, 18 mmol) were dissolved in 40 mL of anhydrous methanol in an ice-water bath, and the pH of the reaction system was adjusted to 10 with DIPEA. After reacting at room temperature for 12 h, the reaction was confirmed to be complete by TLC. After purification by a medium-pressure preparative column (product collected with 30% CH3OH), methanol was removed by rotary evaporation and lyophilized to give 8 (0.45 g, 25.3%) as a white solid. Purity: 95.54%. mp: 88.2–89.1 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):495.2270[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 10.75 (s, 1H), 9.98 (t, J = 4.7Hz, 1H), 9.12 (s, 1H), 8.47 (t, J = 5.7Hz, 1H), 8.44(d,J=8.5Hz,1H),7.43(d,J=7.6Hz,1H),7.32(d,J=5.5Hz,1H),7.26(m,5H),7.05(t,J=7.1Hz,1H),6.9 8(t,J=7.3Hz,1H),4.37(s,2H),4.34(m,1H),4.28(d,J=5.6Hz,2H),3.92(m,1H),3.89(m,2H),3.25(dt,J=5 .6Hz, 6.3Hz, 2H), 3.06 (dd, J=6.4Hz, 16.3Hz, 1H), 2.89 (dd, J=4.8Hz, 15.9Hz, 1H), 1.73 (m, 2H), 1.55 (m, 2H). 13 C-NMR (75MHz, 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 by separating and purifying M3b and M9 using the same preparation scheme as M10a via a medium-pressure preparative column (50-55% EA was used to collect the product), and was a white solid.

[0147] 4H-IQ-Orn-NBzl·2HCl(M11b)

[0148] The preparation method is the same as that for M11a, yielding M11b (1.14 g, 82.0%), which is a white solid.

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

[0150] M11b and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as 8, and purified by C18 silica gel column chromatography (product collected with 26% CH3OH) to give 9 (0.18 g, 15.7%), as a white solid. Purity: 95.23%. mp: 124.4–125.7 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):456.2161[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 10.45 (s, 1H), 9.34 (t, J = 8.2Hz, 1H), 9.07 (d, J = 6.9Hz, 1H), 8.76 (t, J = 5.8Hz, 1H), 7.30 (m, 9H), 4.49 (s, 2H), 4. 41(m,1H),4.34(m,1H),4.32(d,J=2.9Hz,2H),4.25(m,2H),3.40(m,1H), 3.32(m,2H),3.01(dd,J=16.2Hz,12.1Hz,1H),1.78(m,2H),1.68(m,2H). 13 C-NMR (75MHz, DMSO-d6): δ (ppm) = 171.1, 168.3, 162.6, 139.7, 131.5, 129.0, 128.7, 12 8.0,127.6,127.3,127.2,127.1,54.4,53.1,53.0,44.2,42.5,42.2,29.8,29.4,24.0.

[0151] Example 3

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

[0153] M10c (2.06 g, 86.4%) was obtained by separation and purification of 6-quinoxaloline carboxylic acid and M9 using the same preparation scheme as M10a via a medium-pressure preparative column (product collected with 6% CH3OH), and was a light brown solid.

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

[0155] The preparation method is the same as that for M11a, yielding M11c (1.75 g, 98.0%), which is a light brown solid.

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

[0157] M11c and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as 8, and purified by C18 silica gel column chromatography (product collected with 30% CH3OH) to give 10 (1.00 g, 52.3%), a light brown solid. Purity: 99.53%. mp: 106.5–107.4 °C. =-120.0(C=1mg / mL,CH3OH).HR-MS(m / z):453.1800[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 10.38 (s, 1H), 9.69 (m, 1H), 9.29 (m, 1H), 9. 10(d,J=7.7Hz,1H),9.04(d,J=3.0Hz,2H),8.77(s,1H),8.74(t,J=5.9Hz,1H) ,8.37(dd,J=8.7Hz,1.1Hz,1H),8.18(d,J=8.7Hz,1H),7.28(m,5H),4.59(m,1 H), 4.47 (s, 2H), 4.33 (d, J = 4.0Hz, 2H), 3.33 (m, 2H), 1.93 (m, 2H), 1.70 (m, 2H). 13 C-NMR (75MHz, DMSO-d6): δ (ppm) = 171.9, 166.2, 162.6, 147.4, 147.1, 143.9, 142.0, 1 39.9,135.6,129.6,129.4,129.3,128.7,127.5,127.1,54.0,42.5,42.4,29.1,24.5.

[0158] Example 4

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

[0160] M10d (0.88 g, 37.8%) was obtained by separation and purification of 2,1-benzisoxazole-3-carboxylic acid and M9 using the same preparation scheme as M10a via a medium-pressure preparative column (the product was collected with 5% CH3OH), and was a white solid.

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

[0162] The preparation method is the same as that for M11a, yielding M11d (0.75 g, 98.7%), which is a white solid.

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

[0164] M11d and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 22% CH3OH) according to the same preparation scheme as 8, yielding 10a (0.22 g, 26.8%) as a white solid. Purity: 98.08%. mp: 99.7–100.5 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):442.1640[M+H] + . 1 H-NMR (800MHz, DMSO-d6): δ (ppm) = 10.28 (t, J = 4.5Hz, 1H), 9.68 (s, 1H), 9.35 (d,J=7.8Hz,1H),9.24(s,1H),8.56(t,J=5.8Hz,1H),7.96(d,J=8.8Hz,1H),7 .79(d,J=9.1Hz,1H),7.52(dd,J=8.5Hz,6.4Hz,1H),7.27(m,6H),4.57(m,1H ),4.45(s,2H),4.32(d,J=5.6Hz,2H),3.32(m,2H),1.93(m,2H),1.66(m,2H). 13 C-NMR (200MHz, 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] M10e (1.24 g, 52.9%) was obtained by separation and purification of 2,1,3-benzoxadiazole-5-carboxylic acid and M9 using the same preparation scheme as M10a via medium-pressure preparative column (product collected with 6% CH3OH), as a pale yellow solid.

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

[0169] The preparation method is the same as that for M11a, yielding M11e (1.05 g, 98.4%), which is a yellow solid.

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

[0171] M11e and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 22% CH3OH) according to the same preparation scheme as 8, yielding 10b (0.65 g, 56.5%) as a pale yellow solid. Purity: 99.45%. mp: 93.7–94.6 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):443.1593[M+H] + . 1 H-NMR (800MHz, DMSO-d6): δ (ppm) = 10.36 (t, J = 3.9Hz, 1H), 9.65 (s, 1H), 9.28 (s, 1H), 9.19 (d, J = 7.6Hz, 1H), 8.78 (s, 1H), 8.74 (t, J = 5.9Hz, 1H), 8.15 ( d,J=9.4Hz,1H),8.02(dd,J=9.4Hz,0.8Hz,1H),7.27(m,5H),4.51(m,1H), 4.47(s,2H),4.32(d,J=5.7Hz,2H),3.33(m,2H),1.91(m,2H),1.71(m,2H). 13 C-NMR (200MHz, 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, rt, 8h; (b) 4M HCl / EA, 0℃, 4h; (c) EDC, HOBt, THF, NMM, rt, 8h; (d) Pd / H2, CH3OH, rt, 4h; (e) Ethyl 2-chloroacetate hydrochloride, anhydrous methanol, DIPEA, rt, 12h.

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

[0177] Boc-Orn(Z)-OH(M12) was prepared using the same method as M8, and purified by medium-pressure preparative column separation (50-60% EA was used to collect the product) to obtain M13 (4.40 g, 96.7%), which was a white solid.

[0178] HCl·Orn(Z)-NBzl(M14)

[0179] The preparation method was the same as that for M11a, yielding M14 (3.54 g, 93.4%), which was a white solid.

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

[0181] M6 and M14 were purified by medium-pressure preparative column separation (product collected with 6% CH3OH) according to the same preparation scheme as M10a to obtain M15a (1.35 g, 82.0%), which was a white solid.

[0182] KLSS-Orn-NBzl(M16a)

[0183] The preparation method is the same as that for M9, yielding M16a (0.93 g, 91.2%), which is a white solid.

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

[0185] M16a and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as in 8, and purified by C18 silica gel column chromatography (product collected with 48% CH3OH) to give 11 (0.44 g, 40.0%), a white solid. Purity: 99.46%. mp: 98.6–99.2 °C. =-60.0(C=1mg / 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), 8.9 5(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,7.9 Hz,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, 1 28.8,128.7,127.7,127.3,122.7,121.4,120.6,114.6,112.8,52.5,42.7,42.4,39.8,30.7,24.1.

[0186] Example 7

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

[0188] M15b (2.01 g, 78.7%) was obtained by separation and purification of 6-quinoxaloline carboxylic acid and M14 using the same preparation scheme as M10a via a medium-pressure preparative column (product collected with 6% CH3OH), and was a light gray solid.

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

[0190] The preparation method was the same as that for M9, yielding M16b (0.61 g, 81.8%), which was a light gray solid.

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

[0192] M16b and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 25% CH3OH) according to the same preparation scheme as 8, yielding 12 (0.15 g, 20.5%), a white solid. Purity: 98.55%. mp: 93.9–95.1 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):457.2113[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 9.99 (s, 1H), 9.56 (s, 1H), 9.12 (s, 1H), 8.47 (t, J = 5.6Hz, 1H), 8.12 (d, J = 7.7Hz, 1H), 7.44 (d, J = 8.7Hz, 1H), 7.4 0(s,1H),7.28(m,5H),6.61(d,J=8.3Hz,1H),4.47(m,1H),4.37(s,2H),4 .30(d,J=3.8Hz,2H),3.36(m,4H),3.27(m,2H),1.79(m,2H),1.60(m,2H). 13 C-NMR (75MHz, 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] M15c (1.65 g, 66.3%) was obtained by separation and purification of indole-5-carboxylic acid and M14 by C18 silica gel column chromatography (collecting the product with 40% CH3OH) according to the same preparation scheme as M10a, as a white solid.

[0196] ID5C-Orn-NBzl(M16c)

[0197] The preparation method was the same as that for M9, yielding M16c (1.10 g, 91.2%), which was a white solid.

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

[0199] M16c and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as 8, and purified by C18 silica gel column chromatography (product collected with 35% CH3OH) to give 13 (0.74 g, 55.8%), a white solid. Purity: 97.69%. mp: 109.0–109.5 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):440.1848[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 11.40 (s, 1H), 10.26 (s, 1H), 9.65 (s, 1H), 9.22 (t, J=5.1Hz,1H),8.59(t,J=5.7Hz,1H),8.39(d,J=7.9Hz,1H),8.25(s,1H),7.71(d,J=8 .5Hz,1H),7.43(d,J=7.1Hz,1H),7.42(d,J=3.0Hz,1H),7.26(m,5H),6.54(s,1H),4. 55(m,1H),4.44(s,2H),4.31(d,J=5.7Hz,2H),3.31(m,2H),1.85(m,2H),1.64(m,2H). 13 C-NMR (75MHz, DMSO-d6): δ (ppm) = 172.4, 168.1, 162.6, 139.9, 138.0, 128.7, 127.5, 1 27.4,127.1,125.4,121.3,120.8,111.3,102.5,53.5,42.5,42.4,39.6,29.3,24.5.

[0200] Example 9

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

[0202] M15d (1.64 g, 65.9%) was obtained by separation and purification of 3-indolecarboxylic acid and M14 using the same preparation scheme as M10a via a medium-pressure preparative column (product collected with 6% CH3OH), and was a white solid.

[0203] IA-Orn-NBzl(M16d)

[0204] The preparation method was the same as that for M9, yielding M16d (1.15 g, 95.9%), which was a white solid.

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

[0206] M16d and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 40% CH3OH) according to the same preparation scheme as 8, yielding 14 (1.00 g, 72.0%), a white solid. Purity: 99.71%. mp: 58.5–58.8 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):440.1848[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 11.64 (s, 1H), 9.96 (s, 1H), 9.53 (s, 1H), 9.10 (s ,1H),8.51(t,J=5.9Hz,1H),8.18(d,J=2.8Hz,1H),8.13(dd,J=7.1Hz,1.3Hz,1H),7 .95(d,J=8.1Hz,1H),7.44(dd,J=7.1Hz,1.5Hz,1H),7.26(m,5H),7.13(m,2H),4.57 (m,1H),4.36(s,2H),4.32(d,J=5.6Hz,2H),3.30(m,2H),1.83(m,2H),1.65(m,2H). 13 C-NMR (75MHz, DMSO-d6): δ (ppm) = 172.5, 165.2, 162.6, 139.9, 136.6, 128.7, 1 27.5,127.2,122.4,121.3,120.9,112.3,110.5,52.4,42.5,42.4,29.6,24.4.

[0207] Example 10

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

[0209] M15e (2.26 g, 87.6%) was obtained by separation and purification of 5-fluoroindole-3-carboxylic acid and M14 using the same preparation scheme as M10a via a medium-pressure preparative column (product collected with 6% CH3OH), as a white solid.

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

[0211] The preparation method is the same as that for M9, yielding M16e (0.80 g, 95.6%), which is a white solid.

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

[0213] M16e and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 40% CH3OH) according to the same preparation scheme as 8, yielding 15 (0.56 g, 58.4%) as a white solid. Purity: 99.87%. mp: 176.6–177.3 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):458.1754[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 11.83 (d, J = 1.9Hz, 1H), 10.02 (s, 1H), 9.58 (s, 1H), 9.15 (s,1H),8.53(t,J=5.9Hz,1H),8.28(d,J=2.3Hz,1H),8.07(d,J=8.1Hz,1H),7.83(dd,J=10 .2Hz,2.4Hz,1H),7.45(dd,J=8.8Hz,4.6Hz,1H),7.26(m,5H),7.01(dt,J=9.2Hz,2.4Hz,1H ),4.57(m,1H),4.38(s,2H),4.32(d,J=5.6Hz,2H),3.48(m,2H),1.81(m,2H),1.67(m,2H). 13 C-NMR (75MHz, 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 F-NMR (282MHz, DMSO-d6): δ (ppm) = -123.1.

[0214] Example 11

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

[0216] M15f (1.50 g, 60.1%) was obtained by separation and purification of 1H-benzimidazole-2-carboxylic acid and M14 using the same preparation scheme as M10a via a medium-pressure preparative column (product collected with 8% CH3OH), as a white solid.

[0217] BM2C-Orn-NBzl(M16f)

[0218] The preparation method was the same as that for M9, yielding M16f (1.06 g, 97.1%), a white solid.

[0219] BM2C-Orn(Cl)-NBzl(16)

[0220] M16f and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 37% CH3OH) according to the same preparation scheme as 8, yielding 16 (0.48 g, 37.4%) as a white solid. Purity: 99.71%. mp: 121.8–122.4 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):441.1800[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 13.36 (s, 1H), 10.21 (s, 1H), 9.66 (s, 1H), 9.22 (s, 1H), 8.79 (t, J = 5.4Hz, 1H), 8.73 (d, J = 8 .3Hz,1H),7.66(m,2H),7.28(m,7H),4.59(m,1H),4.43(s,2H),4.32(d,J=3.0Hz,2H),3.32(m,2H),1.90(m,2H),1.63(m,2H). 13 C-NMR (75MHz, 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] M15 g (1.49 g, 59.9%) was obtained as a white solid by separation and purification of 1H-benzimidazole-5-carboxylic acid and M14 using the same preparation scheme as M10a via a medium-pressure preparative column (product collected with 9% CH3OH).

[0224] BM5C-Orn-NBzl(M16g)

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

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

[0227] M16 g and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as 8, and purified by C18 silica gel column chromatography (product collected with 30% CH3OH) to give 17 (0.90 g, 73.2%), 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 confirmed to be complete by TLC. Subsequently, the product was purified by medium-pressure preparative column chromatography (5% CH3OH was used to collect the product) to give M15h (0.36 g, 77.4%) as a white solid.

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

[0232] The preparation method is the same as that for M9, yielding M16h (0.26 g, 96.5%), which is a white solid.

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

[0234] M16h (0.26 g, 0.3 mmol) and 2-chloroacetyliminoethyl ester (0.48 g, 3.1 mmol) were dissolved in 40 mL of anhydrous methanol in an ice-water bath, and the pH of the reaction system was adjusted to 10 with DIPEA. After reacting at room temperature for 12 h, the reaction was confirmed to be complete by TLC. After purification by a medium-pressure preparative column (product collected with 55% CH3OH), methanol was removed by rotary evaporation and lyophilized to give 17a (0.14 g, 45.7%) as a white solid. Purity: 97.72%; mp: 193.3–194.7 °C; =-60.0(C=1mg / mL,CH3OH); HR-MS(m / z):993.3841[M+H] + ; 1 H-NMR (800MHz, DMSO-d6): δ (ppm) = 10.16 (s, 1H), 9.62 (s, 1H), 9.17 (s, 1H), 8.94 (s, 2H), 8.9 2(s,2H),8.75(t,J=6.5Hz,2H),8.73(d,J=8.4Hz,2H),8.53(d,J=7.9Hz,2H),7.98(d,J=8.3H z,2H),7.73(t,J=7.7Hz,2H),7.55(s,2H),7.43(t,J=7.5Hz,2H),7.26(m,10H),4.63(dt,J=4 .2Hz, 8.2Hz, 2H), 4.40 (s, 4H), 4.29 (d, J = 5.5Hz, 4H), 3.29 (m, 4H), 1.85 (m, 4H), 1.57 (m, 4H); 13 C-NMR (200MHz, 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 by separating and purifying M6b and M14 using the same preparation scheme as M10a via a medium-pressure preparative column (product collected with 6% CH3OH), and was a white solid.

[0238] IQ-Orn-NBzl(M16i)

[0239] The preparation method was the same as that for M9, yielding M16i (1.52 g, 95.0%), which was a white solid.

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

[0241] M16i and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 45% CH3OH) according to the same preparation scheme as 8, yielding 17b (0.90 g, 49.3%) as a white solid. Purity: 96.71%. mp: 67.8–68.6 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):452.1848[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 9.95 (s, 1H), 9.53 (s, 1H), 9.42 (s, 1H), 9.10 (s, 1H), 8.90 (d, J=8.4Hz,1H),8.76(t,J=5.8Hz,1H),8.59(s,1H),8.27(d,J=8.0Hz,1H),8.22(d,J=8.1Hz,1H), 7.91(dt,J=1.3Hz,6.8Hz,1H),7.84(dt,J=1.1Hz,7.0Hz,1H),7.28(m,5H),4.69(dt,J=5.2Hz,7 .8Hz,1H),4.36(s,2H),4.34(d,J=5.2Hz,2H),3.30(t,J=6.5Hz,2H),1.89(m,2H),1.63(m,2H). 13 C-NMR (75MHz, DMSO-d6): δ (ppm) = 171.4, 164.2, 162.7, 152.2, 143.6, 139.5, 135.8, 1 32.0,129.8,128.8,128.5,128.3,127.7,127.3,120.4,52.6,42.6,42.3,30.4,24.1.

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

[0243] M15j (2.40 g, 72.0%) was obtained by separation and purification of 3-indoleacetic acid and M14 using the same preparation scheme as M10a via a medium-pressure preparative column (product collected with 6% CH3OH), and was a white solid.

[0244] IAA-Orn-NBzl(M16j)

[0245] The preparation method was the same as that for M9, yielding M16j (1.72 g, 97.1%), which was a white solid.

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

[0247] M16j and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 40% CH3OH) according to the same preparation scheme as in 8, yielding 17c (0.90 g, 77.3%) as a white solid. Purity: 99.86%. mp: 77.3–78.2 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):454.2004[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 10.89 (s, 1H), 9.98 (s, 1H), 9.56 (s, 1H), 9.11 (s ,1H),8.49(t,J=5.8Hz,1H),8.15(d,J=8.1Hz,1H),7.55(d,J=7.7Hz,1H),7.34(d, J=8.1Hz,1H),7.26(m,6H),7.06(t,J=7.3Hz,1H),6.96(t,J=7.3Hz,1H),4.37(s,2 H),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 C-NMR (75MHz, 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 by separation and purification of 5-azaindole-3-carboxylic acid and M14 using the same preparation scheme as M10a via a medium-pressure preparative column (product collected with 9% CH3OH), as a white solid.

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

[0252] The preparation method is the same as that for M9, yielding M16k (1.02 g, 99.6%), which is a white solid.

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

[0254] M16k and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as in step 8, and purified by C18 silica gel column chromatography (product collected with 30% CH3OH) to give 17d (0.20 g, 16.3%) as a white solid. Purity: 60.26%; mp: 65.3–66.8 °C; =-60.0(C=1mg / mL,CH3OH); ESI-MS(m / z):441.3[M+H] + ; 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 9.49 (s, 1H), 8.91 (s, 1H), 8.52 (d, J = 6.7Hz, 1H), 8.09 (d, J = 6.6H) z,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 by separation and purification of 1,4-benzodioxane-6-carboxylic acid and M14 using the same preparation scheme as M10a via medium-pressure preparative column (product collected with 6% CH3OH), as a white solid.

[0258] BO6C-Orn-NBzl(M16l)

[0259] The preparation method was the same as that for M9, yielding M16l (0.60 g, 89.0%), a white solid.

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

[0261] M16l and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 30% CH3OH) according to the same preparation scheme as in 8, yielding 17e (0.36 g, 49.8%) as a white solid. Purity: 99.13%. mp: 101.7–102.3 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):459.1794[M+H] + . 1 H-NMR (800MHz, DMSO-d6): δ (ppm) = 10.33 (m, 1H), 9.68 (t, J = 15.7Hz, 1H), 9.26 (t, J = 17.8Hz, 1H), 8.60(t,J=6.0Hz,1H),8.44(d,J=7.9Hz,1H),7.53(d,J=1.6Hz,1H),7.49(dd,J=8.3Hz,1.5Hz,1H ),7.30(t,J=7.6Hz,2H),7.26(d,J=7.4Hz,2H),7.22(t,J=7.2Hz,1H),6.92(d,J=8.4Hz,1H),4.4 8(m,1H),4.46(s,2H),4.29(d,J=5.2Hz,4H),4.27(m,2H),3.30(m,2H),1.86(m,2H),1.62(m,2H). 13 C-NMR (200MHz, 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, rt, 12h; (b) Aldehydes, TFA, CH2Cl2, rt, 15h; (c) DDQ, CH2Cl2, rt, 4h; (d) 2M NaOH, CH3OH / CH2Cl2, rt, 4h; (e) EDC, HOBt, THF, NMM, rt, 8h; (f) Pd / H2, CH3OH, rt, 4h; (g) 4M HCl / EA, 0℃, 4h; (h) 2-Chloroethyl acetate hydrochloride, anhydrous methanol, DIPEA, rt, 12h.

[0265] Example 18

[0266] Trp-OCH3(M18)

[0267] The preparation method was the same as that for M4, yielding M18 (4.32 g, 99.2%), a light brown solid.

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

[0269] M18 (2.18 g, 10 mmol) was dissolved in 40 mL of CH2Cl2 in an ice-water bath, and TFA (5 mL) was slowly added, followed by activation for 10 min. Then, 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 confirmed to be complete by TLC, the solution was concentrated under reduced pressure. The residue was dissolved in 40 mL of CH2Cl2, and then washed successively with saturated NaHCO3 solution (20 mL × 3) and saturated NaCl solution (20 mL × 3). After drying with anhydrous Na2SO4, the product was purified by a medium-pressure preparative column (45% EA was used to collect the product) 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 reacted with stirring at room temperature for 4 h. After the reaction was confirmed to be complete by TLC, the reaction was terminated with saturated NaHCO3 aqueous solution. The organic layer was separated, and the aqueous layer was extracted with CH2Cl2 (20 mL × 2) and the two layers were combined. The organic layer was washed with saturated NaCl solution (20 mL × 3), dried over anhydrous Na2SO4, and purified by medium-pressure preparative column chromatography (4% CH3OH was used to collect the product) to obtain M20a (0.59 g, 49.2%) as a white solid.

[0272] CH3-KLSS(M21a)

[0273] The preparation method was the same as that for M6, yielding M21a (0.52 g, 93.5%), which was a white solid.

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

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

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

[0277] The preparation method is the same as that for M9, yielding M23a (0.51 g, 99.8%), which is a white solid.

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

[0279] M23a and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as 8, and purified by C18 silica gel column chromatography (product collected with 55% CH3OH) to give 18 (0.38 g, 62.5%), a white solid. Purity: 98.63%. mp: 161.9–162.5 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):505.2113[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 12.16 (s, 1H), 10.31 (t, J = 5.4Hz, 1H), 9.67 (s, 1H), 9.2 3(s,1H),8.97(t,J=5.7Hz,1H),8.72(d,J=7.6Hz,1H),8.71(s,1H),8.36(d,J=7.9Hz,1H) ,7.67(d,J=8.2Hz,1H),7.59(t,J=7.5Hz,1H),7.29(m,6H),4.74(dt,J=5.2Hz,7.9Hz,1H) ,4.45(s,2H),4.35(d,J=5.6Hz,2H),3.35(m,2H),2.87(s,3H),1.87(m,2H),1.63(m,2H). 13C-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 by separating and purifying M18 and isobutyraldehyde using the same preparation scheme as M19a via a medium-pressure preparative column (25-30% EA was used to collect the product), and was a white solid.

[0283] KIbu-OCH3(M20b)

[0284] The preparation method is the same as that for M20a. The product was separated and purified by a medium-pressure preparative column (36% EA was used to collect the product) to obtain M20b (1.62 g, 89.1%), which was a light brown solid.

[0285] KIbu(M21b)

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

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

[0288] M22b (1.72 g, 49.6%) was obtained by separating and purifying M21b and M14 using the same preparation scheme as M10a via a medium-pressure preparative column (65-70% EA was used to collect the product), and was a pale yellow solid.

[0289] KIbu-Orn-NBzl(M23b)

[0290] The preparation method was the same as that for M9, yielding M23b (1.28 g, 96.6%), a pale yellow solid.

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

[0292] M23b and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 55% CH3OH) according to the same preparation scheme as 8, yielding 19 (0.80 g, 53.4%), a white solid. Purity: 99.83%. mp: 159.0–160.2 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):533.2426[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 12.08 (s, 1H), 10.22 (s, 1H), 9.63 (s, 1H), 9.19 (s, 1H), 8.94 (t, J=5.7Hz,1H),8.82(d,J=8.2Hz,1H),8.69(s,1H),8.36(d,J=7.8Hz,1H),7.77(d,J=8.2Hz,1H),7.5 9(t,J=7.5Hz,1H),7.29(m,6H),4.73(dt,J=5.3Hz,7.4Hz,1H),4.42(s,2H),4.36(m,2H),3.73(m, J=6.6Hz,1H),3.33(m,2H),1.88(m,2H),1.63(m,2H),1.44(d,J=6.4Hz,3H),1.43(d,J=6.4Hz,3H). 13 C-NMR (75MHz, DMSO-d6): δ (ppm) = 171.6, 165.0, 162.7, 162.7, 149.5, 141.4, 139.6, 138.6, 135.1, 128.8, 12 8.4,127.7,127.3,122.4,121.9,120.4,112.8,112.5,52.1,42.7,42.2,39.6,31.2,31.1,24.0,21.8,21.7.

[0293] Example 20

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

[0295] M19c (2.34 g, 81.8%) was obtained by separating and purifying M18 and trimethylacetaldehyde using the same preparation scheme as M19a via a medium-pressure preparative column (25-30% EA was used to collect the product), and was a white solid.

[0296] KPiv-OCH3(M20c)

[0297] The preparation method is the same as that for M20a. The product was separated and purified by a medium-pressure preparative column (36% EA was used to collect the product) to obtain M20c (1.80 g, 78.0%), which was a light brown solid.

[0298] KPiv(M21c)

[0299] The preparation method was the same as that for M6, yielding M21c (1.68 g, 98.2%), a pale yellow solid.

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

[0301] M21c and M14 were separated and purified by medium-pressure preparative column (product collected with 75% EA) according to the same preparation scheme as M10a to obtain M22c (3.04 g, 80.0%), which was a white solid.

[0302] KPiv-Orn-NBzl(M23c)

[0303] The preparation method was the same as that for M9, yielding M23c (2.25 g, 95.2%), which was a white solid.

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

[0305] M23c and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as in 8, and purified by C18 silica gel column chromatography (product collected with 45% CH3OH) to give 20 (0.66 g, 60.3%), a white solid. Purity: 99.75%. mp: 149.5–151.4 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):547.2583[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 11.67 (s, 1H), 10.26 (s, 1H), 9.66 (s, 1H), 9.21 (s, 1H),8.97(t,J=5.4Hz,1H),8.81(d,J=8.3Hz,1H),8.73(s,1H),8.36(d,J=7.8Hz,1H) ,7.76(d,J=8.2Hz,1H),7.59(t,J=7.6Hz,1H),7.29(m,6H),4.73(dt,J=4.7Hz,7.3Hz ,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). 13C-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 by separating and purifying M18 and isovaleraldehyde using the same preparation scheme as M19a via a medium-pressure preparative column (25-30% EA was used to collect the product), and was a white solid.

[0309] KIvr-OCH3(M20d)

[0310] The preparation method is the same as that for M20a. The product was separated and purified by a medium-pressure preparative column (30% EA was used to collect the product) to obtain M20d (2.10 g, 87.5%), which was a light brown solid.

[0311] KIvr(M21d)

[0312] The preparation method was the same as that for M6, yielding M21d (1.72 g, 86.4%), which was a yellow solid.

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

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

[0315] KIvr-Orn-NBzl(M23d)

[0316] The preparation method was the same as that for M9, yielding M23d (2.30 g, 93.8%), which was a white solid.

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

[0318] M23d and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 60% CH3OH) according to the same preparation scheme as 8, yielding 21 (0.68 g, 62.2%), a white solid. Purity: 99.67%. mp: 143.7–144.3 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):547.2583[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 12.06 (s, 1H), 10.22 (s, 1H), 9.62 (s, 1H), 9.20 (s, 1H), 8.94 (t, J = 5.4Hz ,1H),8.74(d,J=8.5Hz,1H),8.70(s,1H),8.36(d,J=7.7Hz,1H),7.66(d,J=8.1Hz,1H),7.59(t,J=7.3Hz,1 H),7.29(m,6H),4.74(dt,J=5.6Hz,7.3Hz,1H),4.42(s,2H),4.35(d,J=4.5Hz,2H),3.34(m,2H),3.07(d,J =7.1Hz,2H),2.35(m,J=6.6Hz,1H),1.87(m,2H),1.61(m,2H),1.00(d,J=6.0Hz,3H),0.99(d,J=6.0Hz,3H). 13 C-NMR (75MHz, 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] M19e (2.45 g, 85.1%) was obtained by separation and purification of M18 and 2-ethylbutanal using the same preparation scheme as M19a via a medium-pressure preparative column (product collected with 25% EA), and was a white solid.

[0322] KEb-OCH3(M20e)

[0323] The preparation method is the same as that for M20a. The product was separated and purified by medium-pressure preparative column (34% EA was used to collect the product) to obtain M20e (2.20 g, 91.0%), which was a light brown solid.

[0324] KEb(M21e)

[0325] The preparation method is the same as that for M6, yielding M21e (1.88 g, 89.7%), which is a yellow solid.

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

[0327] M21e and M14 were separated and purified by medium-pressure preparative column (product collected with 55% EA) according to the same preparation scheme as M10a to obtain M22e (3.20 g, 77.5%), which was a yellow solid.

[0328] KEb-Orn-NBzl(M23e)

[0329] The preparation method was the same as that for M9, yielding M23e (2.48 g, 99.1%), a pale yellow solid.

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

[0331] M23e and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 62% CH3OH) according to the same preparation scheme as 8, yielding 22 (0.95 g, 56.4%), a white solid. Purity: 98.79%. mp: 97.8–98.7 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):561.2739[M+H] + . 1H-NMR (300MHz, 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. 6Hz,1H),8.80(d,J=8.3Hz,1H),8.68(s,1H),8.37(d,J=7.8Hz,1H),7.67(d,J=8.1Hz,1H),7.59(t,J=7 .5Hz,1H),7.30(m,6H),4.73(dt,J=5.6Hz,7.0Hz,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=6.9Hz,3H),0.78(t,J=7.0Hz,3H). 13 C-NMR (75MHz, 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 by separating and purifying M18 and n-hexanal using the same preparation scheme as M19a via a medium-pressure preparative column (product collected with 25% EA), and was a yellow oily substance.

[0335] KH-OCH3(M20f)

[0336] The preparation method is the same as that for M20a. The product was separated and purified by a medium-pressure preparative column (35-40% EA was used to collect the product) to obtain M20f (1.00 g, 53.4%), which was a brown solid.

[0337] KH(M21f)

[0338] The preparation method was the same as that for M6, yielding M21f (0.81 g, 85.4%), which was a yellow solid.

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

[0340] M21f and M14 were separated and purified by medium-pressure preparative column (70% EA was used to collect the product) according to the same preparation scheme as M10a to obtain M22f (0.94 g, 52.9%), which was a yellow solid.

[0341] KH-Orn-NBzl(M23f)

[0342] The preparation method is the same as that for M9, yielding M23f (0.72 g, 97.7%), which is a yellow solid.

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

[0344] M23f and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 58% CH3OH) according to the same preparation scheme as 8, yielding 23 (0.45 g, 54.0%) as a white solid. Purity: 99.78%. mp: 144.4–145.3 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):561.2739[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 12.11 (s, 1H), 10.29 (s, 1H), 9.67 (s, 1H), 9.22 (s, 1H), 8.97 (t, J=5.6Hz,1H),8.74(d,J=8.4Hz,1H),8.70(s,1H),8.36(d,J=7.8Hz,1H),7.67(d,J=8.1Hz,1H),7.5 9(t,J=7.5Hz,1H),7.29(m,6H),4.75(dt,J=5.6Hz,7.2Hz,1H),4.45(s,2H),4.36(d,J=5.3Hz,2H) ,3.36(m,2H),3.19(t,J=7.4Hz,2H),1.87(m,4H),1.63(m,2H),1.41(m,4H),0.90(t,J=6.4Hz,3H). 13 C-NMR (75MHz, 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, 1 27.7,127.3,122.5,121.8,120.4,112.8,112.7,52.1,42.6,42.2,39.7,33.6,31.6,31.1,27.9,24.0,22.5,14.4.

[0345] Example 24

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

[0347] M18 and decanal were separated and purified by medium-pressure preparative column (product collected with 25% EA) according to the same preparation scheme as M19a to obtain M19g (1.96g, 55.1%), which is a yellow oily substance.

[0348] KDa-OCH3 (M20g)

[0349] The preparation method is the same as that for M20a. The product was separated and purified by a medium-pressure preparative column (35% EA was used to collect the product) to obtain M20g (1.24g, 64.2%), which was a yellow solid.

[0350] KDa(M21g)

[0351] The preparation method was the same as that for M6, yielding M21g (1.08g, 90.4%), which was a pale yellow solid.

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

[0353] M21g and M14 were separated and purified by medium-pressure preparative column (50-55% EA was used to collect the product) according to the same preparation scheme as M10a to obtain M22g (1.16g, 53.8%), which was a pale yellow solid.

[0354] KDa-Orn-NBzl(M23g)

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

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

[0357] M23g and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as 8, and purified by C18 silica gel column chromatography (product collected with 75% CH3OH) to give 24 (0.62g, 61.1%), a white solid. Purity: 99.60%. mp: 192.5–193.5℃. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):617.3365[M+H] + . 1H-NMR (300MHz, 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 .4Hz,1H),8.74(d,J=8.4Hz,1H),8.70(s,1H),8.35(d,J=7.9Hz,1H),7.67(d,J=8.1Hz,1H),7.59(t,J =7.6Hz,1H),7.39(m,6H),4.74(dt,J=4.7Hz,7.5Hz,1H),4.43(s,2H),4.36(d,J=4.9Hz,2H),3.33(m, 2H),3.18(t,J=7.0Hz,2H),1.88(m,4H),1.63(m,2H),1.40(m,4H),1.23(m,8H),0.83(t,J=5.5Hz,3H). 13 C-NMR (75MHz, 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 by separation and purification of M18 and 1,1,3,3-tetramethoxypropane using the same preparation scheme as M19a via a medium-pressure preparative column (product collected with 33% EA), and was a pale yellow solid.

[0361] KMMO-OCH3(M20h)

[0362] The preparation method is the same as that for M20a. The product was separated and purified by medium-pressure preparative column (30-40% EA was used to collect the product) to obtain M20h (0.82g, 54.3%), which is a pale yellow solid.

[0363] KMMO(M21h)

[0364] The preparation method is the same as that for M6, yielding M21h (0.68 g, 86.8%), which is a yellow solid.

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

[0366] M21h and M14 were separated and purified by medium-pressure preparative column (product collected with 65% EA) according to the same preparation scheme as M10a to obtain M22h (1.05 g, 72.7%), which was a yellow solid.

[0367] KMMO-Orn-NBzl(M23h)

[0368] The preparation method is the same as that for M9, yielding M23h (0.80 g, 95.9%), a pale yellow solid.

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

[0370] M23h and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as in 8, and purified by C18 silica gel column chromatography (product collected with 60% CH3OH) to give 25 (0.55 g, 60.1%), a pale blue solid. Purity: 98.74%. mp: 101.9–102.4 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):579.2481[M+H] + . 1 H-NMR (300MHz, 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 .7Hz,1H),8.75(d,J=8.0Hz,1H),8.74(s,1H),8.37(d,J=7.9Hz,1H),7.68(d,J=8.2Hz,1H),7.60(t,J =7.5Hz,1H),7.29(m,6H),5.18(t,J=5.6Hz,1H),4.74(dt,J=5.3Hz,7.4Hz,1H),4.44(s,2H),4.36(d, J=5.5Hz,2H),3.51(d,J=5.7Hz,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 by separating and purifying M18 and cyclopropaneformaldehyde using the same preparation scheme as M19a via a medium-pressure preparative column (collecting the product with 38-43% EA), and was a white solid.

[0374] KC3-OCH3(M20i)

[0375] The preparation method is the same as that for M20a. The product was separated and purified by medium-pressure preparative column (38% EA was used to collect the product) to obtain M20i (1.48 g, 71.3%), which was a pale yellow solid.

[0376] KC3(M21i)

[0377] The preparation method was the same as that for M6, yielding M21i (1.24 g, 88.6%), a pale yellow solid.

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

[0379] M21i and M14 were separated and purified by medium-pressure preparative column (product collected with 65% EA) according to the same preparation scheme as M10a to obtain M22i (2.60 g, 89.7%), which was a white solid.

[0380] KC3-Orn-NBzl(M23i)

[0381] The preparation method was the same as that for M9, yielding M23i (1.96 g, 97.6%), which was a white solid.

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

[0383] M23i and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as 8, and purified by C18 silica gel column chromatography (product collected with 55% CH3OH) to give 26 (1.56 g, 68.2%), a white solid. Purity: 97.25%. mp: 142.5–143.3 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):531.2270[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 12.34 (s, 1H), 10.26 (t, J = 4.6Hz, 1H), 9.65 (s, 1H), 9.20 (s, 1H), 8. 94(t,J=5.9Hz,1H),8.66(d,J=8.4Hz,1H),8.62(s,1H),8.36(d,J=7.9Hz,1H),7.69(d,J=8.1Hz,1H), 7.59(t,J=7.6Hz,1H),7.32(m,5H),7.24(m,1H),4.70(dt,J=5.1Hz,8.1Hz,1H),4.44(s,2H),4.36(m, 2H),3.35(dt,J=6.3Hz,6.1Hz,2H),2.78(m,1H),1.86(m,2H),1.60(m,2H),1.25(m,2H),1.18(m,2H). 13 C-NMR (75MHz, 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] M19j (2.65 g, 85.9%) was obtained by separating and purifying M18 and cyclohexane formaldehyde using the same preparation scheme as M19a via a medium-pressure preparative column (product collected with 22% EA), and was a white solid.

[0387] KC6-OCH3(M20j)

[0388] The preparation method is the same as that for M20a. The product was separated and purified by a medium-pressure preparative column (18% EA was used to collect the product) to obtain M20j (1.98 g, 75.7%), which was a white solid.

[0389] KC6(M21j)

[0390] The preparation method was the same as that for M6, yielding M21j (1.13 g, 59.8%), a pale yellow solid.

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

[0392] M21j and M14 were separated and purified by medium-pressure preparative column (50-55% EA was used to collect the product) according to the same preparation scheme as M10a to obtain M22j (1.84 g, 75.9%), which was a white solid.

[0393] KC6-Orn-NBzl(M23j)

[0394] The preparation method was the same as that for M9, yielding M23j (1.13 g, 78.0%), which was a white solid.

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

[0396] M23j and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as 8, and purified by C18 silica gel column chromatography (product collected with 55% CH3OH) to give 27 (0.99 g, 76.0%), a white solid. Purity: 99.35%. mp: 168.9–169.7 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):573.2739[M+H] + . 1H-NMR (300MHz, DMSO-d6): δ (ppm) = 12.07 (s, 1H), 10.27 (t, J = 5.4Hz, 1H), 9.64 (s, 1H), 9.19 (s, 1H) ),8.96(t,J=5.4Hz,1H),8.77(d,J=8.3Hz,1H),8.68(s,1H),8.34(d,J=7.8Hz,1H),7.67(d,J=8. 2Hz,1H),7.58(t,J=7.5Hz,1H),7.31(m,5H),7.25(m,1H),4.75(dt,J=5.3Hz,7.4Hz,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 C-NMR (75MHz, DMSO-d6): δ (ppm) = 171.6, 165.0, 162.7, 149.0, 141.3, 139.6, 138.7, 135.2, 128.8, 128.3, 12 7.6,127.3,122.4,121.9,120.3,112.7,112.5,52.0,42.6,42.2,41.1,39.6,31.5,31.2,26.5,26.2,24.0.

[0397] Example 28

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

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

[0400] KB-OCH3(M20k)

[0401] The preparation method is the same as that for M5b, yielding M20k (0.60 g, 75.1%), which is a white solid.

[0402] KB(M21k)

[0403] The preparation method was the same as that for M6, yielding M21k (0.40 g, 69.9%), a pale yellow solid.

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

[0405] M21k and M14 were separated and purified by medium-pressure preparative column (product collected with 7% CH3OH) according to the same preparation scheme as M10a to obtain M22k (0.47 g, 54.1%), which was a white solid.

[0406] KB-Orn-NBzl(M23k)

[0407] The preparation method was the same as that for M9, yielding M23k (0.32 g, 86.7%), a white solid.

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

[0409] M23k and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as 8, and purified by C18 silica gel column chromatography (product collected with 60% CH3OH) to give 28 (0.11 g, 28.4%), as a white solid. Purity: 99.65%. mp: 156.7–157.8 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):567.2270[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 11.95 (s, 1H), 10.20 (s, 1H), 9.62 (s, 1H), 9.19(s,1H),8.92(t,J=5.4Hz,1H),8.88(s,1H),8.83(d,J=8.6Hz,1H),8.44( d,J=7.8Hz,1H),8.11(d,J=7.2Hz,2H),7.66(m,5H),7.30(m,6H),4.77(m,1H ),4.41(s,2H),4.35(d,J=1.6Hz,2H),3.32(m,2H),1.90(m,2H),1.63(m,2H). 13 C-NMR (75MHz, 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] M18 and 4-pyridinecarboxaldehyde were prepared using the same method as M19a, separated and purified by a medium-pressure preparative column (product collected with 7% CH3OH), and reacted at room temperature for 48 h to obtain M20l (1.02 g, 33.7%), which was a yellow solid.

[0413] K4PD(M21l)

[0414] The preparation method is the same as that for M6, yielding M21l (0.85 g, 87.4%), which is a yellow solid.

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

[0416] M21l and M14 were prepared using the same method as M10a, and purified by medium-pressure preparative column separation (product collected with 9% CH3OH) to obtain M22l (1.68 g, 91.0%), which was a pale yellow solid.

[0417] K4PD-Orn-NBzl(M23l)

[0418] The preparation method was the same as that for M9, yielding M23l (0.94 g, 71.8%), a pale yellow solid.

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

[0420] M23l and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 55% CH3OH) according to the same preparation scheme as 8, yielding 29 (0.30 g, 27.5%) as a yellow solid. Purity: 96.96%. mp: 257.8–258.4 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):568.2222[M+H] + . 1H-NMR (300MHz, 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=5.8Hz,2H),8.85(d,J=7.4Hz,1H),8.84(t,J=9.0Hz,1H),8.48(d,J= 7.8Hz,1H),8.14(d,J=5.7Hz,2H),7.74(d,J=8.3Hz,1H),7.65(t,J=7.5Hz,1H),7.31(m,6H ),4.74(m,1H),4.39(s,2H),4.35(d,J=4.0Hz,2H),3.31(m,2H),1.91(m,2H),1.64(m,2H). 13 C-NMR (75MHz, 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] M18 and imidazole-2-carboxaldehyde were reacted at room temperature for 48 h using the same preparation scheme as M19a. The mixture was then separated and purified by a medium-pressure preparative column (80% EA was used to collect the product) to obtain M20m (1.05 g, 36.0%), which was a milky white solid.

[0424] K2IM(M21m)

[0425] The preparation method is the same as that for M6, yielding M21m (0.92 g, 92.0%), which is a yellow solid.

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

[0427] M21m and M14 were prepared using the same method as M10a, and purified by medium-pressure preparative column separation (product collected with 7% CH3OH) to obtain M22m (1.52 g, 70.1%), which was a pale yellow solid.

[0428] K2IM-Orn-NBzl(M23m)

[0429] The preparation method was the same as that for M9, yielding M23m (0.98 g, 82.4%), a pale yellow solid.

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

[0431] M23m and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 40% CH3OH) according to the same preparation scheme as 8, yielding 30 (0.26 g, 23.4%), a pale yellow solid. Purity: 99.32%. mp: 107.4–108.2 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):557.2175[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 11.83 (s, 1H), 9.95 (s, 1H), 9.51 (s, 1H), 9.26 (d, J =8.9Hz,1H),9.10(s,1H),8.84(s,1H),8.65(t,J=5.7Hz,1H),8.41(d,J=7.8Hz,1H),7 .96(d,J=8.2Hz,1H),7.60(t,J=7.5Hz,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=1.4Hz,2H),3.33(m,2H),2.00(m,2H),1.68(m,2H). 13 C-NMR (75MHz, 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] M19n (2.92 g, 93.6%) was obtained by separating and purifying M18 and thiophene-2-carboxaldehyde using the same preparation scheme as M19a via a medium-pressure preparative column (25-30% EA was used to collect the product), and was a light brown solid.

[0435] K2SF-OCH3(M20n)

[0436] The preparation method is the same as that for M5b, yielding M20n (1.98 g, 68.7%), which is a light brown solid.

[0437] K2SF(M21n)

[0438] The preparation method was the same as that for M6, yielding M21n (1.74 g, 91.8%), a light brown solid.

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

[0440] M21n and M9 were separated and purified by medium-pressure preparative column (55-60% EA was used to collect the product) according to the same preparation scheme as M10a to obtain M22n (2.10 g, 59.6%), which was a white solid.

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

[0442] The preparation method is the same as that for M11a, yielding M23n (1.68 g, 89.5%), which is a yellow solid.

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

[0444] M23n and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as 8, and purified by C18 silica gel column chromatography (product collected with 55% CH3OH) to give 31 (0.48 g, 26.6%), as a yellow solid. Purity: 99.97%. mp: 163.9–164.2 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):573.1834[M+H] + . 1H-NMR (300MHz, DMSO-d6): δ (ppm) = 12.01 (s, 1H), 10.24 (t, J = 4.7Hz, 1H), 9.64 (s, 1H), 9.20 (s, 1H) ),8.98(t,J=5.6Hz,1H),8.81(s,1H),8.73(d,J=8.3Hz,1H),8.44(d,J=7.8Hz,1H),8.26(d,J=3.4 Hz,1H),7.85(d,J=5.0Hz,1H),7.82(d,J=8.4Hz,1H),7.64(t,J=7.5Hz,1H),7.32(m,7H),4.76(dt ,J=5.4Hz,7.3Hz,1H),4.43(s,2H),4.37(d,J=3.7Hz,2H),3.56(m,2H),1.91(m,2H),1.66(m,2H). 13 C-NMR (75MHz, DMSO-d6): δ (ppm) = 171.5, 164.2, 162.7, 142.6, 142.2, 139.6, 139.0, 135.5, 132.6, 131.0, 1 29.4,129.2,128.8,127.7,127.3,127.3,122.5,121.6,121.1,113.4,113.4,52.1,42.7,42.2,31.1,23.9.

[0445] Example 32

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

[0447] M18 and p-chlorobenzaldehyde were separated and purified by medium-pressure preparative column (35-45% EA to collect the product) according to the same preparation scheme as M19a to obtain M19o (3.15 g, 92.6%), which was a white solid.

[0448] KB4Cl-OCH3(M20o)

[0449] The preparation method was the same as that for M5b, yielding M20o (2.37 g, 76.2%), a light brown solid.

[0450] KB4Cl(M21o)

[0451] The preparation method is the same as that for M6, yielding M21o (1.97 g, 86.7%), which is a yellow solid.

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

[0453] M21o and M9 were separated and purified by medium-pressure preparative column (55-60% EA was used to collect the product) according to the same preparation scheme as M10a to obtain M22o (3.25 g, 84.9%), which was a white solid.

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

[0455] The preparation method is the same as that for M11a, yielding M23o (2.80 g, 95.9%), a pale yellow solid.

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

[0457] M23o and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 54% CH3OH) according to the same preparation scheme as 8, yielding 32 (2.45 g, 81.9%) as a white solid. Purity: 99.96%. mp: 206.5–207.5 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):601.1880[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 12.02 (s, 1H), 10.29 (s, 1H), 9.65 (s, 1H), 9.22 (s, 1H), 8.95 (t,J=5.4Hz,1H),8.89(s,1H),8.80(d,J=8.4Hz,1H),8.45(d,J=7.9Hz,1H),8.14(d,J=8.3Hz,2 H),7.74(d,J=8.2Hz,2H),7.72(d,J=7.3Hz,1H),7.62(t,J=7.5Hz,1H),7.30(m,6H),4.76(dt,J =4.7Hz,7.9Hz,1H),4.44(s,2H),4.35(d,J=3.0Hz,2H),3.34(m,2H),1.92(m,2H),1.64(m,2H). 13 C-NMR (75MHz, 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, 1 30.6,129.4,129.3,128.8,127.6,127.3,122.6,121.6,120.8,114.0,113.2,52.3,42.6,42.2,39.6,30.7,24.1.

[0458] Example 33

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

[0460] M19p (2.80 g, 86.4%) was obtained by separating and purifying M18 and p-fluorobenzaldehyde using the same preparation scheme as M19a via a medium-pressure preparative column (collecting the product with 32-40% EA), and was a white solid.

[0461] KB4F-OCH3(M20p)

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

[0463] The preparation method was the same as that for M6, yielding M21p (1.85 g, 95.8%), a white solid.

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

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

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

[0467] The preparation method is the same as that for M11a, yielding M23p (1.23 g, 75.0%), a pale yellow solid.

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

[0469] M23p and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as 8, and purified by C18 silica gel column chromatography (product collected with 58% CH3OH) to give 33 (0.66 g, 50.0%), 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 by separating and purifying M18 and 4-cyanobenzaldehyde using the same preparation scheme as M19a via a medium-pressure preparative column (40-45% EA for product collection), as a white solid.

[0473] K4CB-OCH3(M20q)

[0474] The preparation method is the same as that for M5b, yielding M20q (2.02 g, 92.1%), which is a white solid.

[0475] K4CB(M21q)

[0476] The preparation method was the same as that for M6, yielding M21q (1.86 g, 96.2%), a pale yellow solid.

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

[0478] M21q and M9 were separated and purified by medium-pressure preparative column (45-55% EA was used to collect the product) according to the same preparation scheme as M10a to obtain M22q (2.96 g, 95.9%), which was a white solid.

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

[0480] The preparation method is the same as that for M11a, yielding M23q (2.60 g, 98.1%), a pale yellow solid.

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

[0482] M23q and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 45% CH3OH) according to the same preparation scheme as 8, yielding 34 (0.96 g, 34.5%) as a white solid. Purity: 98.88%. mp: 265.3–266.4 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):592.2222[M+H] + . 1 H-NMR (300MHz, 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=8.4Hz,1H),8.47(d,J=7.8Hz,1H),8.33(d,J=8.2Hz,2H),8 .16(d,J=8.2Hz,2H),7.73(d,J=8.2Hz,1H),7.64(t,J=7.5Hz,1H),7.31(m,6H),4.76(dt,J=5 .0Hz,7.9Hz,1H),4.44(s,2H),4.35(d,J=2.7Hz,2H),3.34(m,2H),1.91(m,2H),1.64(m,2H). 13C-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, 1 29.5,128.8,127.6,127.3,122.7,121.5,121.0,119.3,114.6,113.2,111.9,52.4,42.6,42.2,39.6,30.7,24.1.

[0483] Example 35

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

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

[0486] KB3N-OCH3(M20r)

[0487] The preparation method is the same as that for M5b, yielding M20r (2.47 g, 88.9%), which is a white solid.

[0488] KB3N(M21r)

[0489] The preparation method was the same as that for M6, yielding M21r (2.25 g, 94.9%), which was a yellow solid.

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

[0491] M22r (1.86 g, 43.3%) was obtained by separating and purifying M21r and M9 using the same preparation scheme as M10a via a medium-pressure preparative column (55-60% EA was used to collect the product), and was a pale yellow solid.

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

[0493] The preparation method is the same as that for M11a, yielding M23r (1.45 g, 92.5%), which is a pale yellow solid.

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

[0495] M23r and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 60% CH3OH) according to the same preparation scheme as 8, yielding 35 (1.32 g, 79.6%) as a yellow solid. Purity: 99.92%. mp: 200.7–201.4 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):612.2121[M+H] + . 1 H-NMR (300MHz, 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=5.6Hz,1H),8.83(s,1H),8.80(d,J=8.9Hz,1H),8.53(d,J=7.9Hz,1H),8.48(d,J= 8.3Hz,1H),8.45(m,1H),7.98(t,J=8.0Hz,1H),7.71(d,J=8.2Hz,1H),7.65(t,J=7.6Hz,1H),7.3 1(m,6H),4.74(m,1H),4.38(s,2H),4.35(d,J=5.3Hz,2H),3.31(m,2H),1.91(m,2H),1.64(m,2H). 13 C-NMR (75MHz, 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] M18 and p-hydroxybenzaldehyde were reacted with reflux at 60°C for 8 hours according to the same preparation scheme as M19a. The product was then separated and purified by a medium-pressure preparative column (55-65% EA was used to collect the product) to obtain M19s (1.17 g, 36.3%), which was a white solid.

[0499] KB4O-OCH3(M20s)

[0500] The preparation method is the same as that for M5b, yielding M20s (1.02 g, 88.3%), which is a white solid.

[0501] KB4O(M21s)

[0502] The preparation method was the same as that for M6, yielding M21s (0.91 g, 93.3%), a pale yellow solid.

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

[0504] M22s (0.85 g, 44.3%) was obtained by separating and purifying M21s and M14 using the same preparation scheme as M10a via a medium-pressure preparative column (70% EA was used to collect the product), and was a pale yellow solid.

[0505] KB4O-Orn-NBzl(M23s)

[0506] The preparation method was the same as that for M9, yielding M23s (0.58 g, 86.3%), a pale yellow solid.

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

[0508] M23s and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 30% CH3OH) according to the same preparation scheme as 8, yielding 36 (0.32 g, 48.0%), a pale yellow solid. Purity: 99.09%. mp: 171.5–172.2 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):583.2219[M+H] + . 1H-NMR (300MHz, DMSO-d6): δ (ppm) = 11.84 (s, 1H), 10.22 (t, J = 4.3Hz, 1H), 10.00 (s, 1H), 9.62 (s, 1H), 9.18(s,1H),8.93(t,J=5.7Hz,1H),8.82(d,J=8.8Hz,1H),8.79(s,1H),8.40(d,J=7.8Hz,1H),7.96(d ,J=8.5Hz,2H),7.72(d,J=8.2Hz,1H),7.59(t,J=7.5Hz,1H),7.29(m,6H),7.08(d,J=8.5Hz,2H),4.76 (dt,J=5.2Hz,8.0Hz,1H),4.42(s,2H),4.36(d,J=3.0Hz,2H),3.34(m,2H),1.89(m,2H),1.63(m,2H). 13 C-NMR (75MHz, 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] M18 and p-methoxybenzaldehyde were reacted with reflux at 60°C for 8 hours according to the same preparation scheme as M19a. The product was then separated and purified by a medium-pressure preparative column (35-45% EA was used to collect the product) to obtain M19t (2.70 g, 80.4%), which was a white solid.

[0512] KB4MO-OCH3(M20t)

[0513] The preparation method is the same as that for M5b, yielding M20t (2.15 g, 80.6%), which is a white solid.

[0514] KB4MO(M21t)

[0515] The preparation method is the same as that for M6, yielding M21t (1.89 g, 91.8%), which is a yellow solid.

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

[0517] M21t and M14 were separated and purified by medium-pressure preparative column (60-65% EA was used to collect the product) according to the same preparation scheme as M10a to obtain M22t (2.40 g, 91.6%), which was a pale yellow solid.

[0518] KB4MO-Orn-NBzl(M23t)

[0519] The preparation method was the same as that for M9, yielding M23t (1.64 g, 86.2%), a pale yellow solid.

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

[0521] M23t and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as 8, and purified by C18 silica gel column chromatography (product collected with 55% CH3OH) to give 37 (0.58 g, 30.8%), a white solid. Purity: 99.96%. mp: 183.6–184.5 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):597.2375[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 11.93 (s, 1H), 10.30 (t, J = 5.1Hz, 1H), 9.67 (s, 1H), 9.22 (s, 1H), 8.98 (t ,J=5.7Hz,1H),8.83(d,J=7.7Hz,1H),8.82(s,1H),8.42(d,J=7.8Hz,1H),8.08(d,J=8.6Hz,2H),7.73(d,J =8.2Hz,1H),7.61(t,J=7.5Hz,1H),7.33(t,J=6.3Hz,1H),7.27(m,5H),7.24(d,J=8.7Hz,2H),4.77(dt,J= 4.9Hz,7.9Hz,1H),4.44(s,2H),4.36(d,J=2.9Hz,2H),3.91(s,3H),3.36(m,2H),1.91(m,2H),1.64(m,2H). 13C-NMR (75MHz, 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, 1 30.1,129.0,128.8,127.6,127.3,122.5,121.7,120.7,114.8,113.2,113.1,55.9,52.2,42.6,42.2,30.9,24.0.

[0522] Example 38

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

[0524] M19u (3.17 g, 86.6%) was obtained by separation and purification of M18 and 2,6-dimethoxybenzaldehyde using the same preparation scheme as M19a via a medium-pressure preparative column (50-60% EA for product collection), as a white solid.

[0525] KB26MO-OCH3(M20u)

[0526] The preparation method is the same as that for M5b, yielding M20u (1.95 g, 62.2%), which is a white solid.

[0527] KB26MO(M21u)

[0528] The preparation method was the same as that for M6, yielding M21u (1.67 g, 89.1%), a pale yellow solid.

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

[0530] M22u (2.52 g, 76.8%) was obtained by separating and purifying M21u and M14 using the same preparation scheme as M10a via a medium-pressure preparative column (75-80% EA was used to collect the product), and was a white solid.

[0531] KB26MO-Orn-NBzl(M23u)

[0532] The preparation method was the same as that for M9, yielding M23u (1.78 g, 87.6%), a white solid.

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

[0534] M23u and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as 8, and purified by C18 silica gel column chromatography (product collected with 50% CH3OH) to give 38 (0.96 g, 47.4%), a pale yellow solid. Purity: 99.92%. mp: 173.0–174.1 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):627.2481[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 11.45 (s, 1H), 10.21 (t, J = 4.8Hz, 1H), 9.64 (s, 1H), 9.18 (s, 1 H),8.93(t,J=5.6Hz,1H),8.82(s,1H),8.56(d,J=8.5Hz,1H),8.38(d,J=7.9Hz,1H),7.55(m,2H) ,7.52(t,J=8.4Hz,1H),7.27(m,6H),6.90(d,J=8.3Hz,2H),4.74(dt,J=5.4Hz,7.8Hz,1H),4.41 (s,2H),4.32(d,J=5.6Hz,2H),3.67(s,3H),3.65(s,3H),3.33(m,2H),1.82(m,2H),1.60(m,2H). 13 C-NMR (75MHz, 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] M19v (2.50 g, 68.3%) was obtained by separation and purification of M18 and 3,4-dimethoxybenzaldehyde using the same preparation scheme as M19a via a medium-pressure preparative column (50-60% EA for product collection), as a white solid.

[0538] KB34MO-OCH3(M20v)

[0539] The preparation method is the same as that for M5b, yielding M20v (1.51 g, 61.1%), which is a white solid.

[0540] KB34MO(M21v)

[0541] The preparation method was the same as that for M6, yielding M21v (1.02 g, 70.3%), a pale yellow solid.

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

[0543] M21v and M14 were separated and purified by medium-pressure preparative column (65-70% EA was used to collect the product) according to the same preparation scheme as M10a to obtain M22v (1.07 g, 53.3%), which was a white solid.

[0544] KB34MO-Orn-NBzl(M23v)

[0545] The preparation method was the same as that for M9, yielding M23v (0.71 g, 82.5%), a white solid.

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

[0547] M23v and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 53% CH3OH) according to the same preparation scheme as 8, yielding 39 (0.38 g, 47.0%), a pale yellow solid. Purity: 99.22%. mp: 151.9–152.8 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):627.2481[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 11.95 (s, 1H), 10.24 (t, J = 5.2Hz, 1H), 9.64 (s, 1H), 9.2 0(s,1H),8.96(t,J=5.7Hz,1H),8.93(d,J=8.0Hz,1H),8.82(s,1H),8.43(d,J=7.9Hz,1H), 7.70(m,3H),7.61(t,J=7.6Hz,1H),7.29(m,7H),4.75(dt,J=5.0Hz,7.9Hz,1H),4.42(s,2H ),4.35(d,J=5.6Hz,2H),3.94(s,3H),3.90(s,3H),3.34(m,2H),1.90(m,2H),1.65(m,2H). 13C-NMR (75MHz, 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 by separating and purifying M18 and 2,4,6-trimethoxybenzaldehyde using the same preparation scheme as M19a via a medium-pressure preparative column (50-60% EA for product collection), as a light brown solid.

[0551] KB246MO-OCH3(M20w)

[0552] The preparation method is the same as that for M5b, yielding M20w (0.74 g, 61.4%), a light brown solid.

[0553] KB246MO(M21w)

[0554] The preparation method was the same as that for M6, yielding M21w (0.62 g, 87.3%), which was a yellow solid.

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

[0556] M21w and M14 were separated and purified by medium-pressure preparative column (70% EA was used to collect the product) according to the same preparation scheme as M10a to obtain M22w (0.73 g, 62.2%), which was a white solid.

[0557] KB246MO-Orn-NBzl(M23w)

[0558] The preparation method was the same as that for M9, yielding M23w (0.52 g, 86.8%), a white solid.

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

[0560] M23W and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as in 8, and purified by C18 silica gel column chromatography (product collected with 64% CH3OH) to give 40 (0.26 g, 44.7%), a white solid. Purity: 99.87%. mp: 179.7–180.7 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):657.2587[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 11.41 (s, 1H), 10.22 (s, 1H), 9.66 (s, 1H), 9.20 (m, 1H) ,8.93(t,J=5.4Hz,1H),8.79(s,1H),8.57(d,J=8.3Hz,1H),8.36(d,J=7.8Hz,1H),7.55(m ,2H),7.29(m,6H),6.47(s,2H),4.74(dt,J=5.5Hz,7.7Hz,1H),4.42(s,2H),4.32(d,J=5 .3Hz,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 C-NMR (75MHz, 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 by separating and purifying M18 and p-methylbenzaldehyde using the same preparation scheme as M19a via a medium-pressure preparative column (25-35% EA was used to collect the product), and was a white solid.

[0564] KB4M-OCH3(M20x)

[0565] The preparation method was the same as that for M5b, yielding M20x (2.05 g, 73.2%), a light brown solid.

[0566] KB4M(M21x)

[0567] The preparation method was the same as that for M6, yielding M21x (1.74 g, 89.1%), which was a yellow solid.

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

[0569] M21x and M14 were separated and purified by medium-pressure preparative column (55-65% EA was used to collect the product) according to the same preparation scheme as M10a to obtain M22x (2.94 g, 79.6%), which was a white solid.

[0570] KB4M-Orn-NBzl(M23x)

[0571] The preparation method was the same as that for M9, yielding M23x (2.18 g, 93.8%), a white solid.

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

[0573] M23x and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as 8, and purified by C18 silica gel column chromatography (product collected with 80% CH3OH) to give 41 (0.18 g, 15.9%), a pale pink solid. Purity: 95.45%. mp: 72.4–73.1 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):581.2426[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 11.86 (s, 1H), 8.84 (s, 1H), 8.77 (d, J = 5.5Hz, 1H), 8.75 (t, J = 6. 7Hz,1H),8.43(d,J=7.8Hz,1H),8.25(t,J=4.7Hz,1H),7.99(d,J=7.8Hz,2H),7.70(d,J=8.3Hz,2H) ,7.60(t,J=7.6Hz,1H),7.49(d,J=7.8Hz,2H),7.28(m,6H),4.69(dt,J=5.5Hz,7.7Hz,1H),4.34(d ,J=3.7Hz,2H),4.01(s,2H),3.13(dt,J=6.1Hz,6.5Hz,2H),2.47(s,3H),1.82(m,2H),1.51(m,2H). 13C-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 by separating and purifying M18 and 4-isopropylbenzaldehyde using the same preparation scheme as M19a via a medium-pressure preparative column (25-30% EA was used to collect the product), and was a white solid.

[0577] K4CM-OCH3(M20y)

[0578] The preparation method is the same as that for M5b, yielding M20y (2.45 g, 82.9%), a light brown solid.

[0579] K4CM(M21y)

[0580] The preparation method was the same as that for M6, yielding M21y (2.30 g, 97.7%), a light brown solid.

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

[0582] M21y and M14 were separated and purified by medium-pressure preparative column (product collected with 55% EA) according to the same preparation scheme as M10a to obtain M22y (1.50 g, 45.0%), which was a white solid.

[0583] K4CM-Orn-NBzl(M23y)

[0584] The preparation method is the same as that for M9, yielding M23y (1.14 g, 95.5%), which is a white solid.

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

[0586] M23y and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 65% CH3OH) according to the same preparation scheme as 8, yielding 45 (0.46 g, 35.2%), a light gray solid. Purity: 99.41%. mp: 157.5–158.4 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):609.2739[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 11.93 (s, 1H), 10.25 (t, J = 4.7Hz, 1H), 9.64 (s, 1H), 9.20 (s, 1H), 8.95 (t, J =5.6Hz,1H),8.85(s,1H),8.83(d,J=7.7Hz,1H),8.43(d,J=7.8Hz,1H),8.02(d,J=8.0Hz,2H),7.71(d,J=8.2 Hz,1H),7.61(t,J=7.3Hz,1H),7.55(d,J=8.1Hz,2H),7.29(m,6H),4.77(dt,J=5.1Hz,7.5Hz,1H),4.43(s,2H ),4.35(d,J=3.6Hz,2H),3.35(m,2H),3.06(m,J=6.8Hz,1H),1.89(m,2H),1.63(m,2H),1.32(d,J=6.9Hz,6H). 13 C-NMR (75MHz, 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 by separating and purifying M18 and 2-naphthaldehyde using the same preparation scheme as M19a via a medium-pressure preparative column (product collected with 30% EA), and was a white solid.

[0590] KN-OCH3(M20z)

[0591] The preparation method was the same as that for M5b, yielding M20z (2.14 g, 76.4%), a light brown solid.

[0592] KN(M21z)

[0593] The preparation method was the same as that for M6, yielding M21z (1.86 g, 90.7%), which was a yellow solid.

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

[0595] M22z (3.15 g, 84.8%) was obtained by separating and purifying M21z and M14 using the same preparation scheme as M10a via a medium-pressure preparative column (product collected with 35% EA), and was a white solid.

[0596] KN-Orn-NBzl(M23z)

[0597] The preparation method was the same as that for M9, yielding M23z (2.45 g, 97.0%), a white solid.

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

[0599] M23z and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as 8, and purified by C18 silica gel column chromatography (product collected with 65% CH3OH) to give 43 (1.18 g, 42.2%), a light brown solid. Purity: 99.85%. mp: 171.6–172.3 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):617.2426[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 12.11 (s, 1H), 8.91 (s, 1H), 8.89 (d, J = 5.7Hz, 1H), 8.88 (t, J=8.4Hz,1H),8.66(s,1H),8.47(d,J=7.7Hz,1H),8.23(m,2H),8.17(dd,J=7.1Hz,3.7Hz,1H) ,8.07(dd,J=3.9Hz,3.0Hz,1H),7.74(d,J=8.2Hz,1H),7.64(m,3H),7.31(m,6H),4.77(dt,J= 4.5Hz,7.8Hz,1H),4.39(s,2H),4.36(d,J=2.9Hz,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-carboxaldehyde were reacted at room temperature for 48 h using the same preparation scheme as M19a. The mixture was then purified by separation using a medium-pressure preparative column (75% EA was used to collect the product) to obtain M20aa (1.46 g, 41.4%), which was a yellow solid.

[0603] K1IQ(M21aa)

[0604] The preparation method is the same as that for M6, yielding M21aa (1.02 g, 72.7%), which is a yellow solid.

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

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

[0607] K1IQ-Orn-NBzl(M23aa)

[0608] The preparation method is the same as that for M9, yielding M23aa (0.88 g, 87.5%), which is a yellow solid.

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

[0610] M23aa and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as 8, and purified by C18 silica gel column chromatography (product collected with 45% CH3OH) to give 44 (0.26 g, 25.4%), as a yellow solid. Purity: 96.94%. mp: 95.3–96.0 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):618.2379[M+H]+ . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 12.01 (s, 1H), 9.93 (t, J = 4.2Hz, 1H), 9.49 (s, 1H), 9.08 (s, 1H), 9.05 (s, 1H), 8.97 (d, J = 8.6Hz, 1H ),8.84(d,J=5.6Hz,1H),8.81(t,J=5.9Hz,1H),8.68(d,J=8.6Hz,1H),8.51(d,J=7.9Hz,1H),8.17(d,J=8.2Hz,1H),8.09(d,J=5.6Hz ,1H),7.88(t,J=7.3Hz,1H),7.77(d,J=8.2Hz,1H),7.69(dt,J=0.6Hz,8.1Hz,1H),7.64(dt,J=0.8Hz,7.2Hz,1H),7.32(m,5H),7.24 (m,1H),4.78(dt,J=5.0Hz,7.7Hz,1H),4.35(d,J=5.0Hz,2H),4.33(s,2H),3.30(dt,J=5.1Hz,6.4Hz,2H),1.85(m,2H),1.64(m,2H). 13 C-NMR (75MHz, 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-carboxaldehyde were reacted at room temperature for 48 h using the same preparation scheme as M19a. The mixture was then separated and purified by a medium-pressure preparative column (the product was collected with 25% EA) to obtain M20ab (1.85 g, 52.4%), which was a yellow solid.

[0614] K2Q(M21ab)

[0615] The preparation method was the same as that for M6, yielding M21ab (1.44 g, 80.8%), which was a yellow solid.

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

[0617] M21ab and M14 were separated and purified by medium-pressure preparative column (product collected with 50% EA) according to the same preparation scheme as M10a to obtain M22ab (1.98 g, 69.4%), which was a yellow solid.

[0618] K2Q-Orn-NBzl(M23ab)

[0619] The preparation method was the same as that for M9, yielding M23ab (1.48 g, 93.3%), which was a yellow solid.

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

[0621] M23ab and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 55% CH3OH) according to the same preparation scheme as in 8, yielding 45 (0.78 g, 46.4%) as a yellow solid. Purity: 99.04%. mp: 168.6–169.1 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):681.2379[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 12.35 (s, 1H), 10.29 (t, J = 4.6Hz, 1H), 9.64 (s, 1H), 9.22 (s, 1H), 9.04 (s, 1H), 9.03(d,J=8.6Hz,2H),8.94(t,J=5.8Hz,1H),8.82(d,J=8.3Hz,1H),8.68(d,J=8.7Hz,1H),8.50(d,J=7.8Hz,1H), 8.10(t,J=7.8Hz,2H),7.94(dt,J=1.2Hz,7.0Hz,1H),7.72(dq,J=0.7Hz,7.9Hz,2H),7.36(m,5H),7.25(m,1H),4 .81(m,1H),4.45(s,2H),4.40(dd,J=5.3Hz,3.2Hz,2H),3.40(dt,J=5.5Hz,6.0Hz,2H),2.03(m,2H),1.72(m,2H). 13C-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, 1 29.5,128.8,128.3,128.1,127.8,127.7,127.3,122.6,121.3,121.0,119.4,115.7,114.0,52.6,42.7,42.3,39.7,30.5,24.2.

[0622] Example 46K3IQ-OCH3(M20ac)

[0623] M18 and isoquinoline-3-carboxaldehyde were reacted at room temperature for 48 h using the same preparation scheme as M19a. The mixture was then purified by separation using a medium-pressure preparative column (75% EA was used to collect the product) to obtain M20ac (1.30 g, 36.7%), which was a yellow solid.

[0624] K3IQ(M21ac)

[0625] The preparation method was the same as that for M6, yielding M21ac (0.95 g, 76.4%), which was a yellow solid.

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

[0627] M21ac and M14 were separated and purified by medium-pressure preparative column (product collected with 55% EA) according to the same preparation scheme as M10a to obtain M22ac (0.98 g, 51.5%), which was a yellow solid.

[0628] K3IQ-Orn-NBzl(M23ac)

[0629] The preparation method was the same as that for M9, yielding M23ac (0.58 g, 74.8%), which was a yellow solid.

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

[0631] M23ac and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as 8, and purified by C18 silica gel column chromatography (product collected with 45% CH3OH) to give 46 (0.20 g, 30.0%), as a yellow solid. Purity: 97.92%. mp: 86.7–87.4 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):618.2379[M+H] + .1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 12.38 (s, 1H), 9.99 (t, J = 4.6Hz, 1H), 9.67 (s, 1H), 9.52 (s, 1H), 9.23 (s, 1H), 9.13 (s, 1H) ,9.06(d,J=8.3Hz,1H),8.97(s,1H),8.80(t,J=5.8Hz,1H),8.47(d,J=7.8Hz,1H),8.37(d,J=8.0Hz,1H),8.16(d,J=7.9Hz, 1H),7.97(d,J=8.3Hz,1H),7.93(t,J=7.3Hz,1H),7.82(t,J=7.4Hz,1H),7.66(t,J=7.6Hz,1H),7.32(m,5H),7.26(m,1H),4 .77(dt,J=5.4Hz,7.8Hz,1H),4.41(d,J=2.2Hz,2H),4.37(s,2H),3.37(dt,J=6.0Hz,5.7Hz,2H),2.03(m,2H),1.74(m,2H). 13 C-NMR (75MHz, 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, 1 29.3,128.8,128.6,128.5,128.0,127.7,127.3,122.5,121.2,120.7,118.4,114.8,113.7,52.8,42.7,42.5,39.9,30.3,24.3.

[0632] Example 47

[0633] K2BM-OCH3(M20ad)

[0634] M18 and benzimidazole-2-carboxaldehyde were reacted at room temperature for 48 h using the same preparation scheme as M19a. The mixture was then separated and purified by a medium-pressure preparative column (the product was collected with 30% EA) to obtain M20ad (1.89 g, 55.3%), which was a yellow solid.

[0635] K2BM(M21ad)

[0636] The preparation method was the same as that for M6, yielding M21ad (1.58 g, 87.4%), which was a yellow solid.

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

[0638] M21ad and M14 were separated and purified by medium-pressure preparative column (product collected with 55% EA) according to the same preparation scheme as M10a to obtain M22ad (0.99 g, 30.8%), which was a pale yellow solid.

[0639] K2BM-Orn-NBzl(M23ad)

[0640] The preparation method was the same as that for M9, yielding M23ad (0.69 g, 87.3%), a pale yellow solid.

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

[0642] M23ad and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as in 8, and purified by C18 silica gel column chromatography (product collected with 50% CH3OH) to give 47 (0.44 g, 55.8%), a pale yellow solid. Purity: 99.96%. mp: 174.7–175.6 °C. =-60.0(C=1mg / mL,CH3OH).ESI-MS(m / z):607.2331[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 12.03 (s, 1H), 10.24 (t, J = 4.8Hz, 1H), 9.57 (s, 1H), 9.49 (d, J=8.6Hz,1H),9.26(s,1H),9.00(s,1H),8.89(t,J=5.9Hz,1H),8.48(d,J=7.8Hz,1H),8.06(d,J =8.3Hz,1H),7.87(m,2H),7.66(dt,J=0.6Hz,7.2Hz,1H),7.34(m,7H),7.21(m,1H),4.72(m,1H ),4.44(s,2H),4.35(d,J=5.8Hz,2H),3.38(dt,J=5.5Hz,6.4Hz,2H),2.18(m,2H),1.75(m,2H). 13 C-NMR (75MHz, 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-carboxybenzimidazole were reacted at room temperature for 48 h using the same preparation scheme as M19a. The product was then separated and purified by a medium-pressure preparative column (45% EA was used to collect the product) to obtain M20ae (0.86 g, 24.3%), which was a milky white solid.

[0646] K2BM1M(M21ae)

[0647] The preparation method was the same as that for M6, yielding M21ae (0.82 g, 98.7%), which was a milky white solid.

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

[0649] M21ae and M14 were prepared using the same method as M10a. After being evaporated to dryness, they were ultrasonically washed with methanol and 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 method was the same as that for M9, yielding M23ae (0.52 g, 59.4%), which was a white solid.

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

[0653] M23ae and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 65% CH3OH) according to the same preparation scheme as 8, yielding 48 (85 mg, 14.3%) as a white solid. Purity: 97.71%. mp: 236.7–237.6 °C. =-60.0(C=1mg / 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] In an ice-water bath, M19h (0.94 g, 3.0 mmol) was dissolved in 10 mL of glacial acetic acid, followed by the slow addition of 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 precipitated. The mixture was filtered under reduced pressure, the filter cake was rinsed with H2O, and dried to obtain 0.78 g of yellow solid for later use. Subsequently, in an ice-water bath, indole (0.70 g, 6.0 mmol) was dissolved in 40 mL of CH2Cl2, followed by the slow addition of 0.3 mL of concentrated sulfuric acid and the aforementioned yellow solid (0.78 g, 2.9 mmol). After reacting at room temperature for 8 h, the reaction was confirmed to be complete by TLC. The pH was adjusted to 7 with saturated NaHCO3 solution; the mixture was transferred to a 100 mL separatory funnel and extracted with CH2Cl2 (20 mL × 2); the CH2Cl2 layers were combined and washed with saturated NaCl solution (20 mL × 3); the CH2Cl2 layers were dried with anhydrous Na2SO4 for 2 h, filtered under reduced pressure, and purified by separation and purification using a medium-pressure preparative column (50% EA was used to collect the product) to obtain M20af (0.58 g, 39.9%), which was a pale yellow solid.

[0657] KMID(M21af)

[0658] The preparation method was the same as that for M6, yielding M21af (0.48 g, 81.3%), which was an orange-red solid.

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

[0660] M21af and M14 were separated and purified by medium-pressure preparative column (65-70% EA was used to collect the product) according to the same preparation scheme as M10a to obtain M22af (0.65g, 78.0%), which was a yellow solid.

[0661] KMID-Orn-NBzl(M23af)

[0662] The preparation method was the same as that for M9, yielding M23af (0.52 g, 95.0%), a yellow solid.

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

[0664] M23af and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as in 8, and purified by C18 silica gel column chromatography (product collected with 50% CH3OH) to give 48a (85 mg, 14.3%) as a yellow solid. Purity: 98.53%; mp: 169.9–171.1 °C; =-60.0(C=1mg / mL,CH3OH); HR-MS(m / z):749.3114[M+H] + ; 1H-NMR (300MHz, DMSO-d6): δ (ppm) = 12.18 (s, 1H), 10.73 (d, J = 15.1Hz, 2H), 10.21 (s, 1H), 9.63 (s, 1H), 9 .21(s,1H),8.94(d,J=5.7Hz,1H),8.93(s,1H),8.56(s,1H),8.29(d,J=7.8Hz,1H),7.66(m,3H),7.57(t ,J=7.6Hz,1H),7.31(m,10H),6.95(t,J=5.6Hz,2H),6.84(m,2H),5.63(t,J=7.5Hz,1H),4.72(dt,J=5.3 Hz,7.3Hz,1H),4.43(d,J=5.5Hz,2H),4.42(s,2H),4.11(m,2H),3.34(m,2H),1.88(m,2H),1.62(m,2H); 13 C-NMR (75MHz, 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 by separating and purifying M18 and benzodiazole-4-carboxaldehyde using the same preparation scheme as M19a via a medium-pressure preparative column (40-50% EA for product collection), as a yellow solid.

[0668] K4BFZ-OCH3(M20ag)

[0669] The preparation method is the same as that for M5b, yielding M20ag (2.25g, 93.7%), a light gray solid.

[0670] K4BFZ(M21ag)

[0671] The preparation method was the same as that for M6, yielding M21ag (2.04g, 94.5%), a pale yellow solid.

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

[0673] M21ag and M9 were separated and purified by medium-pressure preparative column (product collected with 75% EA) according to the same preparation scheme as M10a to obtain M22ag (1.38 g, 43.8%), which was a yellow solid.

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

[0675] The preparation method is the same as that for M11a, yielding M23ag (1.18g, 94.3%), a pale yellow solid.

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

[0677] M23ag and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 55% CH3OH) according to the same preparation scheme as 8, yielding 48b (0.44 g, 35.0%) as a yellow solid. Purity: 99.61%. mp: 256.2–257.3 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):609.2124[M+H] + . 1 H-NMR (300MHz, 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=5.6Hz,1H),8.83(d,J=8.4Hz,1H),8.51(d,J=7.9Hz,1H),8.29(d,J=9.0Hz,1 H),8.24(d,J=6.6Hz,1H),7.92(dd,J=8.9Hz,6.8Hz,1H),7.65(m,2H),7.30(m,6H),4.75(dt,J =5.3Hz,7.5Hz,1H),4.42(s,2H),4.33(d,J=5.6Hz,2H),3.34(m,2H),1.89(m,2H),1.66(m,2H). 13C-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 by separating and purifying M18 and benzo[c][1,2,5]oxadiazole-5-carboxaldehyde using the same preparation scheme as M19a via a medium-pressure preparative column (45-50% EA for product collection), as a yellow solid.

[0681] K5BFZ-OCH3(M20ah)

[0682] The preparation method is the same as that for M5b, yielding M20ah (1.92 g, 90.3%), a pale yellow solid.

[0683] K5BFZ(M21ah)

[0684] The preparation method was the same as that for M6, yielding M21ah (1.76 g, 96.8%), a pale yellow solid.

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

[0686] M21ah and M9 were separated and purified by medium-pressure preparative column (50-55% EA was used to collect the product) according to the same preparation scheme as M10a to obtain M22ah (3.05 g, 96.4%), which was a yellow solid.

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

[0688] The preparation method was the same as that for M11a, yielding M23ah (2.68 g, 97.7%), a pale yellow solid.

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

[0690] M23ah and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 50% CH3OH) according to the same preparation scheme as in 8, yielding 48c (1.15 g, 40.1%) as a yellow solid. Purity: 98.47%. mp: 175.7–176.2 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):609.2124[M+H] + . 1 H-NMR (300MHz, 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=5.8Hz,1H),8.83(d,J=8.4Hz,1H),8.77(s,1H),8.50(d,J=7.8Hz,1H),8.42(d,J=9.4Hz,1H),8 .34(d,J=9.4Hz,1H),7.75(d,J=8.1Hz,1H),7.66(t,J=7.5Hz,1H),7.37(t,J=7.3Hz,1H),7.28(m,5H),4 .77(dt,J=4.9Hz,8.1Hz,1H),4.45(s,2H),4.36(d,J=2.7Hz,2H),3.35(m,2H),1.95(m,2H),1.67(m,2H). 13 C-NMR (75MHz, 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] M19ai (2.62 g, 73.3%) was obtained by separation and purification of M18 and quinoxaline-6-carboxaldehyde using the same preparation scheme as M19a via a medium-pressure preparative column (45-50% EA for product collection), as a white solid.

[0694] K6QN-OCH3(M20ai)

[0695] The preparation method is the same as that for M5b, yielding M20ai (2.35 g, 90.5%), which is a white solid.

[0696] K6QN(M21ai)

[0697] The preparation method was the same as that for M6, yielding M21ai (2.18 g, 96.8%), a yellow solid.

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

[0699] M21ai and M9 were separated and purified by medium-pressure preparative column (product collected with 50% EA) according to the same preparation scheme as M10a to obtain M22ai (0.58 g, 30.1%), which was a pale yellow solid.

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

[0701] The preparation method is the same as that for M11a, yielding M23ai (0.48 g, 92.8%), which is a yellow solid.

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

[0703] M23ai and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 35% CH3OH) according to the same preparation scheme as in 8, yielding 48d (0.18 g, 34.7%) as a pale yellow solid. Purity: 98.72%. mp: 180.4–181.4 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):619.2331[M+H] + . 1H-NMR (300MHz, DMSO-d6): δ (ppm) = 12.21 (s, 1H), 10.16 (s, 1H), 9.56 (s, 1H), 9.10 (d, J = 7.9Hz, 2H), 8.97 ( s,1H),8.91(t,J=5.8Hz,1H),8.87(d,J=8.5Hz,1H),8.82(d,J=1.4Hz,1H),8.62(dd,J=8.6Hz,1.4Hz,1H) ,8.49(d,J=7.8Hz,1H),8.39(d,J=8.7Hz,1H),7.75(d,J=8.2Hz,1H),7.65(t,J=7.5Hz,1H),7.31(m,6H), 4.78(dt,J=5.3Hz,7.8Hz,1H),4.40(s,2H),4.36(d,J=2.9Hz,2H),3.32(m,2H),1.93(m,2H),1.66(m,2H). 13 C-NMR (75MHz, 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, 1 31.1,130.9,130.3,129.4,129.2,128.8,127.7,127.3,122.7,121.7,121.0,114.4,113.3,52.4,42.7,42.3,30.7,24.1.

[0704] Example 53

[0705] 4H-K6BO-OCH3(M19aj)

[0706] M19aj (1.98 g, 54.3%) was obtained by separation and purification of M18 and 1,4-benzodioxane-6-carboxaldehyde using the same preparation scheme as M19a via a medium-pressure preparative column (50% EA was used to collect the product), and was a white solid.

[0707] K6BO-OCH3(M20aj)

[0708] The preparation method is the same as that for M5b, yielding M20aj (1.77 g, 90.6%), which is a white solid.

[0709] K6BO(M21aj)

[0710] The preparation method was the same as that for M6, yielding M21aj (1.22 g, 71.7%), a pale yellow solid.

[0711] K6BO-Orn(Z)-NBzl(M22aj)

[0712] M22aj (1.52 g, 62.9%) was obtained by separating and purifying M21aj and M14 using the same preparation scheme as M10a via a medium-pressure preparative column (70% EA was used to collect the product), and was a white solid.

[0713] K6BO-Orn-NBzl(M23aj)

[0714] The preparation method was the same as that for M9, yielding M23aj (0.75 g, 61.6%), a white solid.

[0715] K6BO-Orn(Cl)-NBzl(48e)

[0716] M23ag and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 55% CH3OH) according to the same preparation scheme as in 8, yielding 48e (0.30 g, 35.1%) as a pale yellow solid. Purity: 99.13%. mp: 169.2–169.9 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):625.2325[M+H] + . 1 H-NMR (800MHz, DMSO-d6): δ (ppm) = 11.87 (s, 1H), 10.17 (t, J = 4.7Hz, 1H), 9.59 (s, 1H), 9 .16(s,1H),8.91(t,J=5.8Hz,1H),8.82(s,1H),8.80(d,J=7.2Hz,1H),8.41(d,J=7.9Hz, 1H),7.71(d,J=8.3Hz,1H),7.61(m,3H),7.29(m,6H),7.15(m,1H),4.76(dt,J=5.0Hz,8. 2Hz,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). 13C-NMR (200MHz, 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.

[0717] Example 54

[0718] 4H-KBNC-OCH3(M19ak)

[0719] M19ak (2.50 g, 71.6%) was obtained by separation and purification of M18 and p-dimethylaminobenzaldehyde using the same preparation scheme as M19a via a medium-pressure preparative column (45% EA was used to collect the product), and was a white solid.

[0720] KBNC-OCH3(M20ak)

[0721] The preparation method is the same as that for M5b, yielding M20ak (1.55 g, 62.7%), which is a white solid.

[0722] KBNC(M21ak)

[0723] The preparation method was the same as that for M6, yielding M21ak (1.29 g, 86.7%), a pale yellow solid.

[0724] KBNC-Orn(Z)-NBzl(M22ak)

[0725] M21ak and M14 were separated and purified by medium-pressure preparative column (65-70% EA was used to collect the product) according to the same preparation scheme as M10a to obtain M22ak (2.06 g, 79.3%), which was a white solid.

[0726] KBNC-Orn-NBzl(M23ak)

[0727] The preparation method was the same as that for M9, yielding M23ak (1.45 g, 87.8%), a pale yellow solid.

[0728] KBNC-Orn(Cl)-NBzl(48f)

[0729] M23ak and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 50% CH3OH) according to the same preparation scheme as in 8, yielding 48f (0.45 g, 27.2%) as a pale yellow solid. Purity: 99.39%. mp: 267.9–268.6 °C. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):610.2692[M+H] + . 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 11.84 (s, 1H), 10.27 (t, J = 5.3Hz, 1H), 9.66 (s, 1H), 9.21 (s, 1H), 8 .97(t,J=5.6Hz,1H),8.86(d,J=8.4Hz,1H),8.75(s,1H),8.39(d,J=7.9Hz,1H),8.01(d,J=8.6Hz,2H) ,7.73(d,J=8.2Hz,1H),7.59(t,J=7.5Hz,1H),7.30(m,6H),6.99(d,J=8.6Hz,2H),4.77(dt,J=5.1Hz ,8.0Hz,1H),4.43(s,2H),4.36(d,J=2.3Hz,2H),3.35(m,2H),3.05(s,6H),1.90(m,2H),1.64(m,2H). 13 C-NMR (75MHz, DMSO-d6): δ (ppm) = 171.7, 164.9, 162.7, 151.3, 141.9, 141.9, 139.6, 139.3, 134.4, 129.8, 1 29.8,128.8,127.6,127.3,122.3,121.8,120.6,113.2,112.8,112.3,52.1,42.6,42.2,39.6,31.0,24.0.

[0730] Example 55

[0731] 4H-KNNC-OCH3(M19al)

[0732] M19al (2.98 g, 74.6%) was obtained by separation and purification of M18 and 6-(dimethylamino)-2-naphthaldehyde using the same preparation scheme as M19a via a medium-pressure preparative column (product collected with 45% EA), and was a white solid.

[0733] KNNC-OCH3(M20al)

[0734] The preparation method is the same as that for M20a. The product was separated and purified by medium-pressure preparative column (38% EA was used to collect the product) to obtain M20al (0.65 g, 41.1%), which was a pale yellow solid.

[0735] KNNC(M21al)

[0736] The preparation method is the same as that for M6, yielding M21al (0.40 g, 63.0%), which is a yellow solid.

[0737] KNNC-Orn(Z)-NBzl(M22al)

[0738] M21al and M14 were separated and purified by medium-pressure preparative column (product collected with 65% EA) according to the same preparation scheme as M10a to obtain M22al (0.43 g, 57.8%), which was a yellow solid.

[0739] KNNC-Orn-NBzl(M23al)

[0740] The preparation method is the same as that for M9, yielding M23al (0.28 g, 80.0%), a yellow solid.

[0741] KNNC-Orn(Cl)-NBzl(48g)

[0742] M23al and 2-chloroacetyliminoethyl ester were purified by C18 silica gel column chromatography (product collected with 35-40% CH3OH) according to the same preparation scheme as in 8, yielding 48 g (85 mg, 26.9%) as a light brown solid. Purity: 99.63%. mp: 171.0-172.1℃. =-60.0(C=1mg / mL,CH3OH).HR-MS(m / z):660.2848[M+H] + . 1H-NMR (300MHz, DMSO-d6): δ (ppm) = 12.03 (s, 1H), 10.12 (s, 1H), 9.56 (s, 1H), 9.14 (s, 1H), 8.89 (t, J = 5.7Hz, 1H),8.85(m,1H),8.84(s,1H),8.48(s,1H),8.44(d,J=7.8Hz,1H),8.11(d,J=8.8Hz,1H),7.97(d,J=9.9Hz,1 H),7.94(d,J=9.2Hz,1H),7.74(d,J=8.2Hz,1H),7.62(t,J=7.6Hz,1H),7.30(m,7H),7.08(s,1H),4.77(dt,J =5.2Hz,7.9Hz,1H),4.39(s,2H),4.37(d,J=2.7Hz,2H),3.34(m,2H),3.09(s,6H),1.92(m,2H),1.65(m,2H). 13 C-NMR (75MHz, 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, 1 28.8,128.1,127.7,127.3,127.1,126.8,122.4,121.8,120.7,117.0,113.2,113.1,52.3,42.6,42.3,40.7,30.8,24.1.

[0743] Example 56

[0744] 4H-K5SA(OCH3)-OCH3(M19am)

[0745] M19am (3.24 g, 85.1%) was obtained by separating and purifying M18 and methyl 5-formyl salicylate using the same preparation scheme as M19a via a medium-pressure preparative column (30-40% EA was used to collect the product), and was a white solid.

[0746] K5SA(OCH3)-OCH3(M20am)

[0747] The preparation method is the same as that for M5b, yielding M20am (2.81 g, 87.8%), which is a white solid.

[0748] K5SA(M21am)

[0749] The preparation method was the same as that for M6, yielding M21am (2.58 g, 99.2%), a yellow solid.

[0750] K5SA-2Orn(Z)-NBzl(M22am)

[0751] M21am and M14 were separated and purified by medium-pressure preparative column (product collected with 3% CH3OH) according to the same preparation scheme as M15h to obtain M22am (3.84 g, 75.1%), which was a white solid.

[0752] K5SA-2Orn-NBzl(M23am)

[0753] The preparation method was the same as for M9, yielding M23am (2.68 g, 94.6%), a yellow solid. K5SA-2Orn(Cl)-NBzl(48h)

[0754] M23am and 2-chloroacetyliminoethyl ester were prepared according to the same protocol as 17a, and purified by C18 silica gel column chromatography (product collected with 70% CH3OH) to give 48h (0.45 g, 14.0%), a pale yellow solid. Purity: 63.17%; mp: 185.9–186.8℃; =-60.0(C=1mg / mL,CH3OH); HR-MS(m / z):905.3415[M+H] + ; 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 12.47 (s, 1H), 12.11 (s, 1H), 10.21 (t, J = 5.6Hz, 1H), 10.17 (t, J = 5.8Hz, 1H), 9.59 (s, 2H), 9.31 (d, J = 7. 7Hz,1H),9.19(s,1H),9.17(s,1H),8.91(t,J=5.8Hz,1H),8.85(t,J=5.8Hz,1H),8.84(s,1H),8.80(d,J=8.6Hz,1H),8.70(d,J=1.8Hz,1H ),8.43(d,J=7.9Hz,1H),8.11(dd,J=8.5Hz,1.8Hz,1H),7.72(d,J=8.3Hz,1H),7.60(t,J=7.8Hz,1H),7.35(d,J=2.3Hz,1H),7.27(m,11H) ,4.74(dt,J=5.3Hz,8.2Hz,1H),4.67(dt,J=5.1Hz,8.3Hz,1H),4.40(s,4H),4.35(d,J=5.4Hz,4H),3.32(m,4H),1.90(m,4H),1.65(m,4H); 13C-NMR (75MHz, 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.

[0755] Example 57

[0756] 4H-B-2Klss-OCH3(M19an)

[0757] M19an (4.14 g, 77.4%) was obtained by separating and purifying M18 and terephthalaldehyde using the same preparation scheme as M19a via a medium-pressure preparative column (56-65% EA for product collection), as a white solid.

[0758] B-2Klss-OCH3(M20an)

[0759] The preparation method was the same as that for M5b, yielding M20an (2.44 g, 59.9%), a light brown solid.

[0760] B-2Klss(M21an)

[0761] The preparation method was the same as that for M6, yielding M21an (1.53 g, 66.2%), which was a yellow solid.

[0762] B-2Klss-2Orn(Z)-NBzl(M22an)

[0763] M21an and M14 were prepared using the same method as M15h, and purified by medium-pressure preparative column separation (product collected with 7% CH3OH) to obtain M22an (0.65 g, 27.7%), which was a white solid.

[0764] B-2Klss-2Orn-NBzl(M23an)

[0765] The preparation method is the same as that for M9, yielding M23an (0.45 g, 89.9%), a pale yellow solid.

[0766] B-2Klss-Orn(Cl)-NBzl(48i)

[0767] M23an and 2-chloroacetyliminoethyl ester were prepared using the same protocol as 17a, and purified by C18 silica gel column chromatography (product collected with 58% CH3OH) to give 48i (0.19 g, 35.2%), a pale yellow solid. Purity: 97.49%; mp: 199.6–200.5 °C; =-60.0(C=1mg / mL,CH3OH); HR-MS(m / z):1057.4033[M+H] + ; 1 H-NMR (800MHz, DMSO-d6): δ (ppm) = 12.19 (s, 1H), 10.24 (s, 1H), 9.62 (s, 1H), 9.22 (s, 1H) ),8.99(t,J=5.4Hz,2H),8.95(s,2H),8.82(d,J=8.3Hz,2H),8.48(d,J=7.8Hz,2H),8.4 2(s,4H),7.78(d,J=8.1Hz,2H),7.65(t,J=8.2Hz,2H),7.31(m,12H),4.83(dt,J=5.0Hz ,7.1Hz,2H),4.42(s,4H),4.37(d,J=2.3Hz,4H),3.37(m,4H),1.92(m,4H),1.68(m,4H); 13 C-NMR (200MHz, DMSO-d6): δ (ppm) = 171.7, 164.8, 162.7, 142.3, 140.7, 139.6, 138.4, 135.1, 130.6, 1 29.5,129.3,128.8,127.6,127.3,122.6,121.6,120.9,114.0,113.4,52.2,42.7,42.3,31.1,24.1.

[0768] Test Example 1: CADD Molecular Docking and Structure Optimization

[0769] The 3D structure of the target compound was constructed using the 3D-sketcher module of Discovery Studio 2019, with energy minimization performed under the CHARMm force field. The crystal structure of PAD4 (PDB:2DW5) was obtained from the Protein Database (PDB). Water molecules were removed and the protein was cleaned before docking. Hydrogen atoms were added to the protein, defining it as the receptor. The original ligands in the protein were then... Amino acid residues within the specified range are defined as binding sites. In the DockLigands|CDOCKER parameter settings, PAD4 protein is used as the receptor input, all optimized molecules are used as ligand inputs, TopHits is set to 10, PoseClusterRadius is set to 0.5, and the calculation is started by clicking run. The scoring results are recorded and advantageous compounds are screened to analyze the interaction between ligand molecules and receptor proteins.

[0770] 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 designs and molecular docking models (PDB:2DW5) of lead compounds 7, 11, and 28 are shown below. Figure 1 As shown, the amino acid backbone of compound 7 occupies the PAD4 active pocket as a peptide matrix through a stable hydrogen bond formed between the carbonyl group and Arg374; while the dimethylaminonaphthalene moiety only forms a weak pi-alkyl interaction with Arg639, and the active pocket here still has a large physical space, providing possibilities for further modification. Since Arg374 is conserved in PAD1 and PAD4 isoenzymes, while Arg639 is unique to PAD4, enhancing the interaction between small molecule compounds and Arg639 becomes the main strategy of this invention to improve the activity and selectivity of PAD4 inhibitors.

[0771] Based on the above strategy, this invention designed compound 11 by introducing a β-carbamoline ring at the N-terminus. This compound enhances the pi-alkyl interaction with Arg639 and forms a stable hydrogen bond with Trp347, thereby enhancing its affinity for PAD4. Figure 1 (and Table 1). This result sparked our interest in further optimization. By introducing a rotatable benzene ring at the α-position of the β-carbamoline ring in compound 11, this invention further yielded compound 28, which can form a strong pi-cation interaction with the imine nitrogen atom of the Arg639 guanidino group, thereby enhancing selectivity for PAD4. Furthermore, the stable salt bridge formed with the key residues Asp350 and Asp473 in the active pocket allows the chloroacetamidine tip of compound 28 to correctly reach the active site and form a tight hydrogen bond and halogen interaction with His640, which may explain the enhanced PAD4 inhibitory activity (…). Figure 1 Furthermore, the binding free energy (ΔGbind) of compound 28 to the PAD4 protein is negative, indicating that the binding of the inhibitor to PAD4 is energy-favorable (Table 1).

[0772] Table 1. Molecular docking simulation and property determination of key compounds

[0773]

[0774] Test Example 2: Evaluation of in vitro PAD4 enzyme activity and anti-tumor cell proliferation activity, and discussion of structure-activity relationship.

[0775] (1) PAD2 / 4 enzyme activity inhibition experiment

[0776] The activity of the PAD2 / 4 inhibitor was determined by a colorimetric method based on the inhibition of PAD2 / 4-catalyzed citrullination of BAEE (N-α-benzoyl-L-arginine ethyl ester). Different concentrations (60000, 20000, 6750, 2250, 750, 250, 85, 30 nmol / L) of the test sample were prepared in ultrapure water containing 1% DMSO. In 96-well plates, according to the grouping, 10 μL of 10× buffer (preparation method: 625 μL 1M Tris-HCl pH=7.6, 25 μL 2M CaCl2, 50 μL 1M DTT, 125 μL 100mM PMSF, 425 μL H2O), the corresponding volume of ultrapure water, 20 μL of the test sample, and 10 μL of active PAD2 / 4 enzyme were added sequentially. The plates were incubated at 37℃ and 400 rpm for 60 min. Add 10 μL of 20 mM BAEE solution (prepared with 1× buffer) to each well and incubate at 37 °C and 400 rpm for 90 min. Then add 25 μL of 5 M HClO4 to quench the reaction. Transfer 125 μL of the reaction solution to the corresponding labeled EP tubes, and add 125 μL of reagent A (0.2 g diacetyl oxime and 0.6 g NaCl dissolved in 40 mL H2O) and 250 μL of reagent B (0.2 g antipyrine, 60 mg FeCl3, 10 mL H2SO4, and 10 mL H3PO4 dissolved in 20 mL H2O). Boil at 100 °C for 30 min, then cool in an ice bath for 5 min. Reabsorb 250 μL into each well of the 96-well plate according to the groupings. Measure the absorbance at 465 nm, fit the inhibition curve using GraphPad Prism 9.5.1, and calculate the IC50. 50 value.

[0777] (2) Cell viability assay - MTT assay

[0778] Mouse TNBC cells 4T1 and 4T1-luc, human TNBC cells MDA-MB-468, and normal human mammary epithelial cells MCF-10A were all purchased from the Type Culture Bank 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 were cultured in DMEM medium containing 10% bovine serum and 1% penicillin-streptomycin; and MCF-10A were cultured in MCF-10A-specific medium. All cells were cultured in a cell incubator at 37°C and 5% CO2.

[0779] Cells in the logarithmic growth phase and in good condition were washed with PBS to remove the cell surface culture medium. After trypsin digestion with 0.25% EDTA until the cells became rounded, the appropriate culture medium was added to stop the digestion. The cells were then gently pipetted to completely detach and disperse the cells (suspension cells do not require digestion). The cells were transferred to 15 mL centrifuge tubes, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in culture medium and counted. Cells were then centrifuged at (3–5) × 10⁻⁶ cells / mL. 4 Cells were evenly seeded at a density of 100 μL / mL into 96-well plates and incubated at 37°C with 5% CO2 for 12 h. Cell adhesion and growth were observed. 25 μL of serially diluted test samples were added to each well, with 6 replicates per concentration. Blank and negative control groups were included in each plate. After 48 h of incubation, 25 μL of MTT solution (5 mg / mL) was added to each well, and incubation continued for 4 h. The supernatant was discarded, and 150 μL of DMSO was added to each well. The cells were shaken on a cell shaker for 10 min to fully dissolve the formazan. The absorbance (OD value) at 490 / 570 nm was measured using a microplate reader, and cell viability curves were plotted and IC50 calculated using GraphPad Prism 9.5.1 software. 50 Values. The negative control group was given 25 μL of drug-free culture medium, and the blank group contained no cells (to subtract the UV absorption at the bottom of the plate). Each experiment was repeated at least three times.

[0780] 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 assay, and their in vitro antiproliferative activity against TNBC cell lines 4T1 and MDA-MB-468, as well as the normal human breast cell line MCF-10A, was evaluated by MTT assay. The results are shown in Tables 2 and 3.

[0781] Table 2. Inhibitory effect of compound 7-17e on PAD and its in vitro antiproliferative activity.

[0782]

[0783]

[0784]

[0785] a Three samples were tested and the results are expressed as mean ± standard deviation (SD).

[0786] Table 3. Inhibitory effect of compound 18-48i on PAD and its in vitro antiproliferative activity.

[0787]

[0788]

[0789]

[0790]

[0791]

[0792] a Three samples were tested and the results are expressed as mean ± standard deviation (SD).

[0793] Table 4. Inhibitory effect of compound 48h-48i on PAD and its in vitro antiproliferative activity.

[0794]

[0795]

[0796] a Three samples were tested and the results are expressed as mean ± standard deviation (SD).

[0797] As can be seen, lead compound 7, as a pan-PAD inhibitor, can significantly inhibit the activity of PAD2 and PAD4 in vitro, IC50... 50 The concentrations were 5.28 ± 0.22 μM and 3.54 ± 0.19 μM, respectively, consistent with previous results. Furthermore, compound 7 significantly inhibited the in vitro proliferation of 4T1 and MDA-MB-468 cells, with an IC50 concentration of 5.28 ± 0.22 μM and 3.54 ± 0.19 μM, respectively, consistent with previous findings. 50 The effective concentrations were 7.34±0.65 μM and 5.16±0.22 μM, respectively, and it also showed high cytotoxicity against normal human mammary cells MCF-10A, with an IC50 of 7.34±0.65 μM and 5.16±0.22 μM. 50The concentration was 4.30 ± 0.67 μM. The poor isoenzyme selectivity and indiscriminate killing of cells by compound 7 prompted us to further develop new, highly efficient, and selective PAD4 inhibitors. In this part of the work, this invention first demonstrated that changes in the electron cloud density of the N-terminal modification group 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 this did not significantly improve the compound activity. Furthermore, compounds 10 and 12–17, obtained by changing the electron cloud density based on the binary conjugated ring, can improve the inhibitory activity against PAD4 (IC50 ± 0.67 μM). 50 <3 μM). In contrast, compound 11, obtained based on a strategy of increasing the number of conjugated rings, enhanced the in vitro PAD4 enzyme inhibitory activity (IC50). 50 While exhibiting appropriate anti-tumor cell proliferation activity (1.76±0.08μM), it is worth further optimization.

[0798] Further structural modifications were mainly focused on the α-position of compound 11, and the in vitro bioactivity of the synthesized derivatives was evaluated (see Table 3). Among them, the long carbon chain modification of compound 24 showed better safety against normal mammary gland cells MCF-10A; compounds 18–27 (except 24), obtained by introducing saturated or cyclic hydrocarbons at the α-position, all effectively enhanced the inhibitory activity against PAD4 (IC50). 50 <1.8μM) and anti-proliferative capacity against TNBC cells (IC50). 50 <20 μM). These compounds showed no selectivity for normal breast cells MCF-10A, demonstrating their safety for normal cells. Unlike saturated cyclic hydrocarbons, most compounds with conjugated ring modifications at the α-position exhibited varying degrees of reduced cytotoxicity to MCF-10A. Among them, compound 28, obtained by introducing a freely rotating benzene ring, showed significant inhibition of PAD2 and PAD4 activity, with an IC50 value of <20 μM. 50 The concentrations were 2.97±0.29 μM and 0.79±0.09 μM, respectively, demonstrating good selectivity for PAD4. Furthermore, compound 28 significantly inhibited the in vitro proliferation of TNBC cells (4T1:IC). 50 =2.39±0.54μM,MDA-MB-468:IC 50 =2.34±0.23μM; In contrast, it showed lower toxicity to normal mammary cells, MCF-10A:IC 50=8.39±0.60 μM, exhibiting a larger therapeutic window. Notably, the introduction of polar groups onto the benzene ring of compound 28, yielding compounds 32–36, retained good PAD enzyme inhibitory activity. Compounds 37–40, obtained by alkyl blocking, reversed this trend to some extent. Compounds 41–42, obtained by introducing short carbon chains onto the benzene ring, also retained good PAD enzyme inhibitory activity. In contrast, most compounds 43–48, which incorporated biaromatic ring structures, showed satisfactory ability to inhibit TNBC cell proliferation in vitro.

[0799] Based on structure-activity relationship discussion, compound 28 was identified as exhibiting the best correlation between PAD4 enzyme inhibitory activity and anti-TNBC cell proliferation activity, demonstrating moderate selectivity for PAD4. Furthermore, its in vitro anti-tumor cell proliferation activity and safety were superior to those of lead compound 7. Therefore, in this part of the work, compound 28 is considered a representative PAD4 inhibitor for the treatment of triple-negative breast cancer, and a tool for further research into the key functions of PAD4 in TNBC progression.

[0800] Test Example 2: In vitro antiproliferative and antimigration activities of compound 28

[0801] (1) Transwell chamber experiment

[0802] Pre-starved 4T1 cells were dispersed in serum-free RPMI 1640 medium at a concentration of 5 × 10⁶ cells / mL. 4 Cells were evenly seeded in the upper chamber of a Transwell microscope at a density of [number] cells / chamber, and treated with the test sample prepared in serum-free medium. 600 μL of normal culture medium was added to the lower chamber. Care must be taken to ensure there are no air bubbles between the lower culture medium layer and the bottom membrane of the chamber. After incubation at 37°C and 5% CO2 for 12 hours, the culture medium and cells in the upper chamber were gently wiped away with a cotton swab. Cells that had migrated across the membrane were fixed with 4% paraformaldehyde for 15 minutes and stained with 0.1% crystal violet for 15 minutes. Using a Zeiss Zen Blue 3.1 inverted microscope imaging system, nine different fields of view were selected from each chamber for imaging, and the number of transmembrane-migrating cells was counted using ImageJ.

[0803] (2) Cell scratch / healing test

[0804] 4T1 cells in logarithmic growth phase were fed with 5 × 10⁻⁶ cells. 5Cells were evenly seeded at a density of [number] cells / well in six-well plates and incubated at 37°C with 5% CO2 for 12 hours until confluence exceeded 90%. Cells were scratched using the tip of a sterile 100 μL pipette, washed with PBS buffer to remove cell debris, and treated with normal culture medium containing different concentrations of the test sample, with at least three replicates per group. After culturing for another 24 hours, images were taken from 3–6 different fields of view per well using a Zeiss Zen Blue 3.1 inverted microscope, and the healing area was statistically analyzed using ImageJ.

[0805] Compound 28 exhibits in vitro antiproliferative and antimigration activities such as Figure 2 As shown. Figure 2 In the table, (A) shows the correlation analysis of inhibitory activity on PAD4 and 4T1 cells; (B) shows the effect of compound 28 on TNBC cell survival. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (C) and (D) are representative results of transwell assays and analyses. Scale bar: 1000 μm. (E) and (F) are representative results of wound healing assays and analyses. Scale bar: 1000 μm. Data are expressed as mean ± standard deviation. Statistical analysis was performed by one-way ANOVA. ***p < 0.001.

[0806] Correlation analysis of in vitro PAD4 enzyme inhibitory activity and anti-4T1 cell proliferation ability revealed that compound 28 had the most relevant anti-enzyme and anti-cancer cell activities. Figure 2 Compound 28 (A) was used in subsequent studies. Using lead compound 7 as a positive control, compound 28 showed enhanced ability to proliferate TNBC cells against 4T1 and MDA-MB-468 in vitro. Figure 2 (B) This may be because compound 28 has enhanced PAD4 inhibitory activity and higher cell membrane permeability. Transwell and scratch assays showed that compound 28 inhibited the in vitro migration of 4T1 cells in a dose-dependent manner. Furthermore, at the same dose, the number of metastatic 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). These results indicate that compound 28 exhibits superior in vitro antitumor activity compared to lead compound 7.

[0807] Test Example 3: Compound 28 inhibits the formation of H3cit and NET.

[0808] (1) H3cit expression in tumor cells

[0809] 4T1 cells were fed at a rate of 2 × 10 5Inoculated at a uniform density of 1 sample per dish in confocal dishes and incubated at 37°C with 5% CO2 for 12 h, followed by incubation for another 48 h with the corresponding test sample. After washing twice with PBS, fixation was performed with 4% paraformaldehyde (containing 0.2% Triton X-100) at 4°C for 15 min, followed by blocking with 5% BSA at room temperature for 1 h. Subsequently, incubation was performed overnight at 4°C with anti-histone H3 (citrulline R2+R8+R17) antibody (ab5103, 1:400). The results were then analyzed using 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, washed twice with PBS, and then PBS containing an anti-fluorescence quencher was added. Images were captured using a STED super-resolution confocal microscope, and the average fluorescence intensity of each field was statistically analyzed using ImageJ.

[0810] (2) Neutrophil NETs release experiment

[0811] Female BALB / c mice aged 6–8 weeks were euthanized, and bone marrow was aseptically isolated from the femur and tibia. The resulting single-cell suspension was obtained by suspending the cells in PBS and filtering through a 70 μm nylon membrane. Neutrophils were isolated using a mouse neutrophil isolation kit (TBDSceicge, LZS1100) and cultured at 5 × 10⁻⁶ cells / mL. 5 Cells were evenly seeded at a density of cells / dish in confocal dishes, and the corresponding test compounds were added. The dishes were incubated at 37°C with 5% CO2 for 2 hours. Subsequently, calcium ion carrier A23187 (5 μmol / L) was added, and incubation continued for another 2 hours to induce NETosis. Cells were centrifuged at 500g for 5 minutes at 4°C without braking, and the culture medium was carefully aspirated. Cells were then incubated with fluorescently labeled anti-mouse Ly6G monoclonal antibody (ThermoFisher Scientific, 11-9668-82, 1:400) at room temperature in the dark for 30 minutes. Cells were centrifuged at 500g for 5 minutes at 4°C without braking, and the antibody was carefully aspirated. Cells were fixed with 4% paraformaldehyde (containing 0.2% Triton X-100) for 15 minutes and blocked with 5% BSA at room temperature for 1 hour. Subsequently, it was incubated overnight at 4°C with anti-histone H3 (citrulline R2+R8+R17) antibody (ab5103, 1:400), and then analyzed using 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, washed twice with PBS, and then PBS containing an anti-fluorescence quencher was added. Images were captured using a STED super-resolution confocal microscope, and the average fluorescence intensity of each field was statistically analyzed using ImageJ.

[0812] To further investigate the inhibitory effect of compound 28 on PAD4 in cells, Western blotting and immunofluorescence analysis were used to assess citrullination levels in 4T1 cells. The effects of compound 28 on histone citrullination and NET formation are as follows: Figure 3 As shown. Figure 3 In the table, (A) and (B) are representative results of immunoblotting and analysis, showing the expression of H3cit and PAD4 in 4T1 cells treated at 7, 27, and 28. Actin was used as a loading control; (C) and (D) are representative results of immunofluorescence and analysis, showing the expression of H3cit (red) in 4T1 cells treated at 7 and 28. Scale bar: 50 μm; (D) and (E) assess the expression of H3cit (red) and NET formation (expansion) in mouse bone marrow neutrophils treated at 7 or 28 by activation with calcium ionophore A23187, with DNA stained with Hoechst 33342 (blue) and neutrophils identified by Ly6G hyperstaining (green). Scale bar: 50 μm. Data are expressed as mean ± standard deviation. Statistical analysis was performed by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

[0813] As expected, the immunoblotting results showed that compound 28 inhibited H3cit in 4T1 cells in a dose-dependent manner; and, at the same dose, this inhibitory effect was significantly greater than that of lead compound 7 and compound 27 modified with a saturated hydrocarbon ring. Figure 3 (A-B). This result is consistent with in vitro enzyme inhibition experiments, indicating that compound 28, obtained by introducing a conjugated aromatic ring into compound 11, inhibited PAD4 activity more effectively than compound 27, obtained by modification with a saturated hydrocarbon ring. This may be attributed to the strong pi-cation interaction between the aromatic ring and Arg639. Interestingly, compared to positive control 7, PAD4 expression was upregulated in 4T1 cells treated with compound 28. Figure 3 (A-B). This may be because compound 28 primarily targets the inhibition of PAD4 activity, thereby increasing PAD4 expression through negative feedback. Furthermore, immunofluorescence analysis further confirmed that compound 28 significantly inhibited PAD4-mediated H3 citrullination in 4T1 cells, and its inhibitory effect was superior to that of compound 7 at the same dose. Figure 3 (C to D)

[0814] Next, the inhibitory effect of compound 28 on NETosis in mouse bone marrow neutrophils was examined. Induction of intracellular calcium concentration with the calcium ionophore A23187 resulted in a significant increase in H3cit in neutrophils in the Control group, and the release of a filamentous or network-like substance co-localized with blue DNA and red H3cit, termed NETs. Figure 3 (E-F). In contrast, treatment with compounds 7 or 28 prior to calcium ionophore induction effectively inhibited histone H3 citrullination and NET formation. Notably, neutrophils treated with compound 28 exhibited lower H3cit levels compared to the positive control 7, while maintaining high cell membrane integrity (as indicated by green Ly6G), which may suggest that the function and activity of neutrophils as immune cells were preserved. In summary, these results demonstrate that compound 28 is an excellent PAD4 inhibitor that effectively blocks histone H3 citrullination and NET formation while preserving neutrophil integrity.

[0815] Pharmacokinetics of Compound 28 in Test Example 4

[0816] Based on its favorable in vitro bioactivity, the pharmacokinetic characteristics of compound 28 in SD rats were further evaluated (see Table 4). After tail vein administration of 3.5 mg / kg of compound 28, 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) were observed. 0- (17346.55 h·ng / L); These properties reflect the pharmacokinetic optimization of compound 28 compared to compound 7. Furthermore, considering the good in vitro activity of compound 28, evaluating its in vivo antitumor and antimetastatic potential is of great significance.

[0817] Table 5. Pharmacokinetic characteristics of compounds 7 and 28 in vivo.

[0818]

[0819] a Three copies of the sample were prepared for each time point.

[0820] Test Example 5: In vivo antitumor and antimetastatic activity – 4T1-luc orthotopic tumor-bearing mouse model

[0821] Male ICR mice, male C57BL / 6 mice, and female BALB / c mice, aged 6–8 weeks and weighing 20±2g, as well as male Sprague Dawley (SD) rats weighing 220±10g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal experiments were approved by the Institutional Animal Care and Utilization Committee of Capital Medical University (ethics approval number: AEEI-2018-174).

[0822] Orthotopic 4T1-luc breast cancer model in BALB / c mice. After passage to a certain number of cultured 4T1-luc cells, they were trypsinized and dispersed in PBS to form 5 × 10⁶ cells / mL. 6 A cell suspension of 0.1 mL / mL was injected into the fourth mammary pad of each female BALB / c mouse. The injection continued until the tumor reached approximately 25 mm². 3 (Generally, 5 days after tumor implantation) Mice with uniformly distributed tumors were randomly grouped. All test samples were dissolved in physiological saline containing 5% DMSO and 20% HP-β-CD, and administered via tail vein every two days according to group. From the start of administration, mouse weight and tumor volume were measured every two days. 24 hours after the last administration, mice were weighed, euthanized by cervical dislocation under ether anesthesia, and tumor tissue and major organs were dissected, photographed, weighed, and immediately frozen in liquid nitrogen or fixed in formalin for later use. Serum ALT, AST, UREA, and CREA-S levels were detected using a fully automated biochemical analyzer and corresponding kits. Survival curve experiments were conducted with the same groupings as above, with mice kept under the same conditions and continuously administered the drugs until death or exceeding the prescribed time.

[0823] Table 6. Dosing regimen for the orthotopic 4T1-luc tumor model.

[0824]

[0825] IV tail vein injection

[0826] To evaluate the antitumor and antimetastatic activity of compound 28 in vivo, a BALB / c mouse 4T1-luc orthotopic tumor model was established. The tumor volume was increased to 25 mm². 3 At approximately 10:00 AM, tumor-bearing mice were randomly divided into 6 groups (n=10 per group): a blank control (Control), positive controls (DOX 1.16 mg / kg and 7 (10 mg / kg), and treatment groups (10, 5, and 1 mg / kg). All samples were dissolved in physiological saline containing 5% DMSO and 20% HP-β-CD and administered intravenously every two days for a total of 9 times.

[0827] Figure 4This study investigated the in vivo antitumor activity of compound 28 in an orthotopic 4T1-luc xenograft model. (A) and (B) show the treatment regimens and survival curves in orthotopic 4T1-luc tumor-bearing mice. Control groups received DOX (1.16 mg / kg), 7 (10 mg / kg), and 28 (10, 5, and 1 mg / kg) intravenously every two days. (C) shows in vivo images and analysis of orthotopic tumors in 4T1-luc tumor-bearing mice on days 1 and 17 of treatment. (D) shows in vivo images and analysis of orthotopic tumors in 4T1-luc tumor-bearing mice on days 1 and 17 of treatment. (E) and (F) show the weight and representative images of tumors harvested on day 18. Data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

[0828] Figure 5 This study examines the in vivo anti-metastatic activity of compound 28 against an 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 after treatment. (C) and (D) show representative images (indicated by arrows) and analysis of metastases in lung tissue. (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 expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

[0829] High-dose (10 mg / kg) 28 treatment resulted in the best survival curves in tumor-bearing mice. Figure 4 More importantly, in vivo imaging, tumor volume monitoring, and tumor weight statistics in 4T1-luc tumor-bearing mice all confirmed that compound 28 could inhibit the growth of primary TNBC tumors in mice in a dose-dependent manner. Figure 4 (C-F). Statistical results showed that the tumor growth inhibition rate in the 10 mg / kg 28 treatment group was 61.8%, compared to 54.6% in the positive control DOX group and 36.2% in the 10 mg / kg 7 treatment group. Furthermore, compound 28 dose-dependently inhibited lung metastasis of TNBC in mice; and compared to the DOX and 7 treatment groups, the 10 mg / kg 28 treatment group showed significantly enhanced anti-lung metastasis ability, with H&E staining results of lung tissue sections similar to those of normal mice. Figure 5 (A-E). Notably, immunohistochemical staining of H3cit and Ly6G in tumor sections showed that, at the same dose, the 28 treatment group had comparable inhibitory effects on histone H3 citrullination and NET formation as the 7 treatment group, and the 28 treatment group significantly increased neutrophil infiltration in tumor tissue. Figure 5(F~G). Inspired by this discovery, this invention further evaluated the effects of compound 28 intervention on the tumor immune microenvironment of triple-negative breast cancer.

[0830] Figure 6 This study evaluates the biocompatibility of compound 28 in an orthotopic 4T1-luc xenograft model. (A) shows the body weight curves of 4T1-luc tumor-bearing mice during different treatment periods. (B)–(F) show the visceral-to-somatic cell ratios of major organs (heart, liver, spleen, kidney, and brain) after different treatments. (G)–(J) show serum biochemical parameters (ALT, AST, UREA, and CREA-S) after different treatments.

[0831] Figure 7 H&E staining of heart, liver, spleen and kidney tissue sections from normal mice and 4T1-luc tumor-bearing mice after different treatments.

[0832] In the biosafety assessment of compound 28 treatment, the body weight of mice in each group remained stable during different treatment periods; however, statistical results of organ weight ratios showed that after treatment, the liver and kidney weights of mice in the positive control DOX group and the 7 treatment group decreased significantly. Figure 6 (A-F). Compared with the Control group, there were no statistically significant differences in the organ-to-body ratio and serum biochemical indicators (ALT, AST, UREA, and CREA-S) in the 10 mg / kg 28 treatment group. Figure 6 (Middle B-J). Furthermore, H&E staining results did not reveal any significant morphological or structural damage to the heart, liver, spleen, and kidney tissues of mice treated with compound 28. Figure 7 In summary, at the same dose (10 mg / kg), compound 28 showed significantly enhanced antitumor and antimetastatic effects in the mouse 4T1-luc orthotopic tumor model compared to lead compound 7; compound 28 is a promising candidate drug for anti-TNBC and has no obvious physiological toxicity.

[0833] Test Example 6: Effects of Compound 28 on the Tumor Immune Microenvironment

[0834] In the tumor immune microenvironment, PD-L1+TANs exhibit a tumor-promoting phenotype by inhibiting T-cell cytotoxicity, leading to poor prognosis and low survival rates in patients. Single-cell mass cytometry was used to detect the composition of immune cells in tumor tissues of 4T1-luc tumor-bearing mice, and the cells were clustered and statistically analyzed based on the expression of specific antibodies against the immune cells (Figure 4.30A).

[0835] Figure 8The effects of compound 28 on the tumor immune microenvironment are shown in Figure 28. (A) is a tSNE diagram of immune cells in tumor tissue detected by single-cell flow mass spectrometry. (B)–(C) show the proportions of immune cells (DCs, cDCs, M1 macrophages, G-MDSCs, and M-MDSCs) and tumor-associated neutrophils in tumor tissue. (D)–(E) show the expression levels of PD-L1 and MHC-II in tumor-associated neutrophils.

[0836] The results showed that compound 28 treatment could induce a broad-spectrum anti-tumor phenotype in the tumor immune microenvironment by downregulating the proportion of myeloid-derived immunosuppressive cells (M-MDSCs) and G-MDSCs, while upregulating the proportion of dendritic cells (DCs), especially conventional dendritic cells (cDCs), and promoting the polarization of anti-tumor M1 macrophages. Figure 8 (Middle B). Furthermore, treatment with 28 increased mature tumor-associated neutrophil MHC-II levels. + The proportion of TANs was increased, and the pro-tumor phenotype PD-L1 was suppressed. + / MHC-II + The ratio of TANs to MHC-II-TANs ( Figure 8 (C). Interestingly, in the case of PD-L1... + MHC-II + A significant decrease in PD-L1 expression was observed in TANs, indicating that these cells have weakened immunosuppressive function. Figure 8 (Middle D). Furthermore, MHC-II + The expression of major histocompatibility complex class II molecules of TANs and MHC-II-TANs was enhanced after 28 days of treatment, indicating that their function as antigen-presenting cells (APCs) was enhanced. Figure 8 (E). In summary, these results indicate that compound 28 primarily transforms the tumor immune microenvironment from a pro-tumor state to an anti-tumor state by regulating the proportion of immune cells and reshaping the phenotype and function of neutrophils.

[0837] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A carbaline-derived PAD4 inhibitor having the structure shown in Formula I: Equation I; In Equation I, R1 is , , , , , , and One of them; R2 is , , , , , and One of them; X is and One of them.

2. The method for preparing the carbline-derived PAD4 inhibitor according to claim 1, comprising the following steps: Z-Orn(Boc)-OH having the structure shown in Formula a, benzylamine, a condensing agent and an organic solvent were mixed and subjected to 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 give the compound having the structure shown in formula c. Formula a; Formula b; Formula c; A compound having the structure shown in formula c, a compound having the structure shown in formula d, a condensing agent, and an organic solvent are mixed to carry out a second condensation reaction to obtain a compound having the structure shown in formula e. Formula d; Formula e; The compound having the structure shown in formula e undergoes a second deprotection reaction to give the compound having the structure shown in formula f. Formula f; A compound having the structure shown in Formula f, 2-chloroacetyliminoethyl ester, and an organic solvent were mixed and coupled to obtain a carbline-derived PAD4 inhibitor having the structure shown in Formula I. In equations a and b, R3 is either Boc or Cbz, and R4 is either Cbz or Boc, and R3 and R4 are different. In equations c and e, R4 is either Cbz or Boc.

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

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

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

6. The preparation method according to claim 2, characterized in that, The condensing agent is a DCC-HOBt system or an EDC-HOBt system; The first condensation reaction and the second condensation reaction were carried out at room temperature for 6 to 10 hours each.

7. The preparation method according to claim 2, characterized in that, 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, wherein the pH value of the alkaline conditions is 9-11.

9. The use of the carboline-derived PAD4 inhibitor of claim 1 or the carboline-derived PAD4 inhibitor prepared by any one of claims 2 to 8 in the preparation of antitumor drugs.

10. The application according to claim 9, characterized in that, The antitumor drugs include anti-breast cancer drugs and / or anti-lung cancer drugs.

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