Fluorescent molecule targeting folate receptor, preparation method therefor and use thereof

Abstract

Disclosed in the present invention are a fluorescent molecule targeting a folate receptor, a preparation method therefor and the use thereof. The present invention provides a compound represented by formula (I), a pharmaceutically acceptable salt thereof, or a tautomer thereof. The compound of the present invention can be used as a fluorescent contrast agent in near-infrared surgical navigation.

Description

A fluorescent molecule targeting folic acid receptors, its preparation method and applications

[0001] This application claims priority to Chinese patent application 2024119532600, filed on 2024 / 12 / 27. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the fields of targeted fluorescent probe technology and tumor diagnostic technology. Specifically, it relates to a fluorescent molecular conjugate that selectively targets the folate receptor, its preparation method, and its application in targeted tumor diagnosis, particularly in near-infrared surgical navigation. Background Technology

[0003] Surgical resection without positive surgical margins remains the cornerstone of solid tumor treatment. Surgeons primarily rely on intraoperative subjective assessment (such as tissue structure, texture, color, and feel) to distinguish tumor tissue from surrounding normal tissue, which easily leads to residual tumor or over-resection of normal tissue. Incomplete resection resulting in residual tumor is closely related to poor prognosis and cancer recurrence, while over-resection may damage surrounding normal tissue. Therefore, determining tumor boundaries is crucial. Targeted fluorescent probes can illuminate cancer cells in real time during surgery, helping doctors determine tumor boundaries and detect metastases; this concept is also known as fluorescence-guided surgery (FGS). Currently, non-targeted fluorescent molecules such as indocyanine green (ICG) and methylene blue (MB) are used in clinical practice as fluorescent contrast agents to mediate surgical procedures, but due to a lack of specific targeting to tumor tissue, their detection rate and false negative rate still have room for improvement.

[0004] Folate receptors (FRs) are common tumor-targeting receptors belonging to the glycoprotein family. They have a molecular weight of 35–40 kDa and several subtypes, including α, β, and γ. The α subtype is overexpressed in various human cancers, such as ovarian cancer, lung cancer, breast cancer, and brain cancer. Fluorescent dyes that bind to groups targeting FR-α can specifically accumulate in FR-overexpressing tumor tissues, while healthy tissues show low background fluorescence levels. Therefore, FR-α is an ideal target for intraoperative imaging. Thus, although folate receptor-targeting conjugates such as vintafolide and EC150, developed as therapeutic drugs, have repeatedly faced market setbacks, folic acid and its analogues are still frequently used in the design of tumor-targeting diagnostic probes, such as OTL038 (pafolacianine), which has already been approved by the US Food and Drug Administration.

[0005] Folic acid receptor-targeting near-infrared fluorescent small molecule conjugates have shown promising application prospects in clinical applications, especially in near-infrared surgical navigation. However, due to the presence of sites in their structural fragments that are susceptible to attack by metabolic enzymes, these conjugate molecules may undergo degradation in vivo due to reactions such as de-targeting of the ligand and removal of the pteridine ring, thereby reducing their targeting efficacy and diminishing their advantages over classic fluorescent contrast agents.

[0006] Therefore, it is of great significance to develop molecular fluorescent probes that are structurally stable, achieve specific targeting of tumor tissues with high affinity, and are metabolically stable in vivo. Summary of the Invention

[0007] The technical problem this invention aims to solve is to address the shortcomings of the prior art by providing a fluorescent probe molecule for targeting folate receptor-positive tumor tissues. This invention optimizes the in vivo metabolic stability of the targeted fluorescent molecule by selecting the targeting ligand of the small molecule conjugate, potentially improving its imaging window and enhancing imaging stability; alternatively, the fluorescent probe molecule of this invention exhibits improved chemical structural stability and affinity for FR-α as a drug; or, the fluorescent probe molecule of this invention can serve as a product replacement for OTL038, with different chemical structures but at least comparable to OTL038 in terms of affinity, structural stability, and imaging capabilities.

[0008] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0009] The first aspect of the present invention provides a compound as shown in formula (I), a pharmaceutically acceptable salt thereof, or a tautomer thereof;

[0010] in,

[0011] X is CH or N, Y is S; or Y is CH or N, X is S;

[0012] n is 2, 3, 4, 5 or 6;

[0013] It is a C / C single or double bond, and a ring. It has aromatic properties;

[0014] AR stands for "5-10 membered heteroaryl group having 1-4 heteroatoms independently selected from N and O", C6-C 10 Aryl, "a 5- to 10-membered heterocyclic alkenyl group having 1 to 3 heteroatoms independently selected from N, O, and S" or C5-C 10 Cycloalkyl, and AR is formed by the ring carbon atom with a C=O group and (CH2). n Group linkage; AR optionally linked by 1 to 3 R groups 0 Replace; the R mentioned above 0It is a halogen;

[0015] AA is The a-terminus is connected to the carbonyl group;

[0016] L 1 It is a C1-C6 alkylene or a C1-C4 alkylene-phenyl;

[0017] L 2 For O, S, or NH;

[0018] * indicates that the chirality of the carbon atom is S, R, or "a mixture of S and R";

[0019] FL is a fluorescent dye.

[0020] In certain embodiments of the present invention, the pharmaceutically acceptable salt and the compound represented by formula (I) contain AA The carboxyl group on it forms a salt.

[0021] In some embodiments of the present invention, the 5-10 heteroaryl group having 1-4 heteroatoms independently selected from N and O is a 5-6 heteroaryl group having 1 heteroatom independently selected from N and O, such as pyridyl, indolyl, indazole, furanyl, benzofuranyl, oxazolyl, tetrazolyl, and pyridine, for example.

[0022] In some embodiments of the present invention, the C6-C 10 The aryl group is phenyl or naphthyl, preferably phenyl.

[0023] In some embodiments of the present invention, the 5-10 membered heterocyclic alkenyl group having 1-3 heteroatoms independently selected from N, O and S is a 5-6 membered heterocyclic alkenyl group having 1 heteroatom independently selected from N, O and S.

[0024] In some embodiments of the present invention, the 5- to 10-membered heterocyclic alkenyl group contains one or two unsaturated double bonds, and the 5- to 10-membered heterocyclic alkenyl group is not aromatic.

[0025] In some embodiments of the present invention, the C5-C 10 The cycloalkyl group can be a monocyclic, fused, spirocyclic, or bridged ring, preferably a bridged ring.

[0026] In some embodiments of the present invention, the C5-C 10 The cycloalkyl group is a C6-C8 bridged cycloalkyl group, for example...

[0027] In some embodiments of the present invention, the halogen is Cl, F, Br, or I, preferably F.

[0028] In some embodiments of the present invention, the C1-C6 alkylene oxide is preferably a C1-C4 alkylene oxide, for example... Preferred More preferably

[0029] In certain embodiments of the present invention, the C1-C4 alkylene-phenyl group is preferably...

[0030] In some embodiments of the present invention, the structure of the compound represented by formula (I) is as shown in formula (II) or formula (III):

[0031] Where X is CH or N; Y is CH or N.

[0032] In some embodiments of this invention, n is 3 or 4.

[0033] In some embodiments of the present invention, R 0 It can be Cl, F, Br or I independently, with F being preferred.

[0034] In some embodiments of this invention, AR is either not replaced or is represented by one R. 0 Replacement; the preferred AR is the one that has not been replaced.

[0035] In some embodiments of the present invention, the present invention provides compounds of formula (II), wherein X is N.

[0036] In some embodiments of the present invention, the present invention provides compounds of formula (III), wherein Y is N.

[0037] In some embodiments of the present invention, AR is a 5- to 6-membered heteroaryl group having one heteroatom independently selected from N and O, surrounded by 1 to 3 R atoms. 0 The substituted phenyl, phenyl, or C6-C8 bridged cycloalkyl group is preferably a 5-6 membered heteroaryl group containing one nitrogen atom and surrounded by 1-3 R atoms. 0 Substituted phenyl, phenyl or C6-C8 bridged cycloalkyl.

[0038] In some embodiments of the present invention, AR is phenyl, F-substituted phenyl, pyridyl, or bicyclooctane (e.g., ).

[0039] In some embodiments of this invention, AR is...

[0040] In some embodiments of the present invention, AR is preferably...

[0041] In certain embodiments of this invention, the invention provides compounds of formula (II) in which X is N and AR is...

[0042] In some embodiments of the present invention, when L 1 When it is a C1-C4 alkylene-phenyl, L 1 phenyl and L 2 Connected.

[0043] In some embodiments of the present invention, L 1 It is a C1-C4 alkylene or C1-C3 alkylene-phenyl, preferably methylene, n-butylene or methylene-phenyl, more preferably methylene-phenyl.

[0044] In some embodiments of the present invention, L 2 It is O.

[0045] In some embodiments of the present invention, the chirality of the carbon atom is marked as S or R, preferably S.

[0046] In some embodiments of this invention, AA is... Preferred

[0047] In some embodiments of this invention, AA is... Preferred

[0048] In some embodiments of the present invention, FL is a dye having fluorescence excitation and emission spectra in the near-infrared range.

[0049] In some embodiments of this invention, FL is:

[0050] In the formula, R 1a and R 1b It can be independently O, S, -NH- or -CH2-, preferably -CH2-;

[0051] R 2 It is -CH2- or -CH2CH2-, preferably -CH2-;

[0052] R 3 and R 4 It is -(CH2)3SO3H;

[0053] R 5 and R 6Independently, it is a halogen, hydroxyl, amino, -NH (C1-C6 alkyl), -N (C1-C6 alkyl)2, C1-C6 perfluoroalkyl, C1-C6 alkyl, C1-C6 alkoxy or SO3H, preferably SO3H;

[0054] X 1 and X 2 Independent for CR 7 2. NR 8 O or S, R 7 and R 8 Independently selected from C1-C4 alkyl groups;

[0055] Preferred, X 1 and X 2 For CR 7 2, R 7 It is a methyl group.

[0056] Compared to existing technologies, the main objective of this invention is to optimize the chemical structure of the targeting ligand of the small molecule conjugate targeting folic acid receptor α, thereby improving the in vivo metabolic stability of the targeting fluorescent molecule, especially the target portion. Regarding the fluorescent dye portion of the folic acid targeting fluorescent molecule, although this invention only provides a specific embodiment with fluorescent dye S0456, those skilled in the art should be able to anticipate that other dyes with fluorescence excitation and emission spectra in the near-infrared range, especially fluorescent molecules similar to S0456 (e.g., the fluorescent molecules disclosed in US9624424B2 and CN105228628B), can all be used as FL in this invention to achieve the function of targeting diseased tissues and for tissue imaging.

[0057] In some embodiments of this invention, FL is

[0058] In some embodiments of the present invention, the structure of the compound represented by formula (I) is as shown in formula (IV):

[0059] In some embodiments of the present invention, the structure of the compound represented by formula (I) is as shown in formula (II);

[0060] in,

[0061] X is CH or N, preferably N;

[0062] n is 3 or 4;

[0063] AR is a 5- to 6-membered heteroaryl, phenyl, or C6-C8 bridged cycloalkyl group that contains one nitrogen atom;

[0064] AA is The a-terminus is connected to the carbonyl group;

[0065] L 1 It is a C1-C6 alkylene or a C1-C4 alkylene-phenyl;

[0066] L 2 For O, S, or NH;

[0067] FL is

[0068] In some embodiments of the present invention, the structure of the compound represented by formula (I) is as shown in formula (III);

[0069] in,

[0070] Y can be CH or N, preferably N;

[0071] n is 3 or 4;

[0072] AR is a 5- to 6-membered heteroaryl, phenyl, or C6-C8 bridged cycloalkyl group that contains one nitrogen atom;

[0073] AA is The a-terminus is connected to the carbonyl group;

[0074] L 1 It is a C1-C6 alkylene or a C1-C4 alkylene-phenyl;

[0075] L 2 For O, S, or NH;

[0076] FL is

[0077] In some embodiments of the present invention, the compound represented by formula (I) above is any of the following compounds:

[0078] Preferably, the compound represented by formula (I) above is any of the following compounds:

[0079] A second aspect of the present invention provides a method for preparing the compound represented by formula (IV), comprising the following steps:

[0080] (1) The compound shown in formula (VI) undergoes a deprotection reaction in the presence of acid, and the pH is adjusted to alkaline after the reaction is complete.

[0081] (2) In an alkaline aqueous solution, the product obtained in step (1) reacts with SO456 to obtain the compound shown in formula (IV);

[0082] Among them, R 1 It is a C1-C6 alkyl protecting group, preferably tert-butyl;

[0083] X, Y, n, AR, *, L 1 and L 2 The definition is as described above.

[0084] In some embodiments of the present invention, the C1-C6 alkyl protecting group is a C1-C6 alkyl group.

[0085] In some embodiments of the present invention, in step (1), the deprotection reaction may be carried out in the presence of a haloalkane solvent (e.g., DCM) or water, or directly in an acid.

[0086] In some embodiments of the present invention, in step (1), when water is present in the deprotection reaction, the volume ratio of water to acid is 1:(20-200), preferably 1:(30-100).

[0087] In some embodiments of the present invention, in step (1), the acid is a strong protic acid, such as trifluoroacetic acid or hydrochloric acid, preferably trifluoroacetic acid.

[0088] In some embodiments of the present invention, in step (1), the alkaline pH value is 9-12, for example 11.

[0089] In some embodiments of the present invention, step (1) includes the following steps: in a haloalkane solvent (e.g., DCM), the compound shown in (VI) undergoes a deprotection reaction in the presence of trifluoroacetic acid, and after the deprotection reaction is completed, the acid is removed under reduced pressure and then adjusted to alkalinity with sodium hydroxide solution.

[0090] In some embodiments of the present invention, step (1) includes the following steps: in a haloalkane solvent (e.g., DCM), the compound shown in (VI) undergoes a deprotection reaction in the presence of hydrochloric acid, and after the deprotection reaction is completed, it is directly adjusted to alkalinity with sodium hydroxide solution.

[0091] In some embodiments of the present invention, step (1) includes the following steps: the compound shown in (VI) undergoes a deprotection reaction in the presence of water and trifluoroacetic acid, and after the deprotection reaction is completed, the reaction solution is added to methyl tert-butyl ether and then adjusted to alkalinity with sodium hydroxide solution.

[0092] In some embodiments of the present invention, in step (2), the pH value of the alkaline aqueous solution is 9-12, for example 11.

[0093] In some embodiments of the present invention, in step (2), the temperature of the reaction can be 70-100°C, for example 90°C.

[0094] A third aspect of the present invention provides a compound of formula (VI),

[0095] Among them, R 1 It is a C1-C6 alkyl protecting group or hydrogen;

[0096] X, Y, n, AR, *, L 1 and L 2 The definition is as described above.

[0097] In one particular scheme, R 1 It is a C1-C6 alkyl protecting group, preferably tert-butyl.

[0098] In some embodiments of the present invention, the C1-C6 alkyl protecting group is a C1-C6 alkyl group.

[0099] In some embodiments of the present invention, R 1 The protecting group is a C1-C6 alkyl group, preferably a tert-butyl group.

[0100] In some embodiments of the present invention, R 1 It is hydrogen.

[0101] In certain embodiments of the present invention, the compound represented by formula (VI) is any of the following compounds:

[0102] A fourth aspect of the present invention provides a compound represented by formula (VII),

[0103] Among them, X, Y, The definitions of n and AR are as described above.

[0104] In some embodiments of the present invention, the compound represented by formula (VII) is any of the following compounds:

[0105] A fifth aspect of the present invention provides a pharmaceutical composition comprising the above-described compounds, further comprising at least one pharmaceutically acceptable carrier or excipient.

[0106] The sixth aspect of the present invention provides the use of the above-described compounds in the preparation of tumor diagnostic reagents for tumor-targeted imaging.

[0107] The seventh aspect of the invention provides the use of the above-described compound in the preparation of an image-guided agent for surgical procedures on a subject suffering from a disease, wherein the agent is visualized by irradiating a designated surgical site with infrared light, the disease being a tumor.

[0108] In some embodiments of the present invention, the tumor is breast cancer, lung cancer, ovarian cancer, endometrial cancer, bladder cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, head and neck cancer, or mesothelioma; preferably, the tumor is ovarian cancer or lung cancer.

[0109] Terminology Definition

[0110] The term "pharmaceutically acceptable salt" as used in this invention refers to a salt of the compounds of this invention, prepared by reacting the compounds of this invention with a relatively non-toxic acid or base. When the compounds of this invention contain relatively acidic functional groups, a base addition salt can be obtained by contacting the neutral form of the compounds of this invention with a sufficient amount of base in a pure solution or a suitable inert solvent. When the compounds of this invention contain relatively basic functional groups, an acid addition salt can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent.

[0111] Certain chemical groups defined herein are preceded by simplified symbols to indicate the total number of carbon atoms present in the group. For example, C1-C6 alkyl refers to alkyl groups having a total of 1, 2, 3, 4, 5, or 6 carbon atoms as defined below.

[0112] In this paper, the numerical ranges defined in the substituents, such as 5-10, 6-8, 5-6, 1-6, 1-4, etc., indicate the integers within that range. For example, 1-6 means 1, 2, 3, 4, 5 or 6, and 1 to 3 means 1, 2 or 3.

[0113] Those skilled in the art will understand that, according to conventions used in the art, the structural formulas of the groups described in this invention are... This refers to the fact that the corresponding group R is linked to other fragments or groups in the compound through this site.

[0114] When a listed group does not explicitly indicate that it has a substituent, the group refers only to the unsubstituted group. For example, when "C1-C6 alkyl" is not limited to "substituted or unsubstituted", it refers only to "C1-C6 alkyl" itself or "unsubstituted C1-C6 alkyl".

[0115] The "halogen" mentioned in this invention refers to F, Cl, Br, and I.

[0116] In this invention, the term "alkylene" refers to a straight-chain or branched, saturated divalent hydrocarbon group having a specified number of carbon atoms (e.g., C1-C6). The alkylene group in this invention is preferably a C1-C6 alkylene group, more preferably a C1-C4 alkylene group, and the alkylene group includes, but is not limited to:

[0117] The term "aryl" as used in this invention refers to a monocyclic or bicyclic group with 6-10 carbon atoms, wherein at least one ring in the system is aromatic (e.g., a 6-carbon monocyclic ring, a 10-carbon bicyclic ring, etc.); and wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted with substituents. Examples of aryl groups also include phenyl, naphthyl, etc.

[0118] The term "heteroaryl" as used in this invention refers to a cyclic, unsaturated monovalent group having a specified number of ring atoms (e.g., 5-10, 5-6), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified type of heteroatom (one or more of N and O). It can be monocyclic or polycyclic, and the polycyclic ring can be fused, spirocyclic, or bridged. Each ring of the heteroaryl is aromatic. The heteroaryl in this invention can be a 5-10 member heteroaryl having 1-4 heteroatoms independently selected from N, O, and S, preferably a 5-6 member heteroaryl having 1-2 heteroatoms independently selected from N and O. Examples of heteroaryl include pyridyl, indolyl, indazole, furanyl, benzofuranyl, oxazolyl, tetrazolyl, etc.

[0119] The term "cycloalkyl" as used in this invention refers to a saturated monocyclic, spirocyclic, bridged, or fused cyclic group having a specified number of carbon atoms in the ring (e.g., 5–10, 6–8), with the ring atoms consisting solely of carbon atoms, preferably a bridged ring. The cycloalkyl group in this invention is preferably a C6-C8 bridged cycloalkyl group. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Non-limiting examples of the bridged or fused cycloalkyl group include: bicyclo[1.1.0]butyl, bicyclo[2.1.0]pentyl, bicyclo[1.1.1]pentyl, bicyclo[3.1.0]hexyl, bicyclo[2.1.1]hexyl, bicyclo[3.2.0]heptyl, bicyclo[4.1.0]heptyl, bicyclo[2.2.1]heptyl, bicyclo[3.1.1]heptyl, bicyclo[4.2.0]octyl, bicyclo[3.2.1]octyl, bicyclo[2.2.2]octyl, etc.

[0120] The term "heterocyclic alkenyl" refers to an unsaturated monocyclic, spirocyclic, bridged, or fused-ring cyclic group having a specified number of ring atoms (e.g., 5–10, 5–6), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified type of heteroatom (e.g., one or more of N, P, O, and S). The heterocyclic alkenyl contains one or more unsaturated double bonds and is not aromatic.

[0121] The "aromaticity" described in this invention refers to a specific chemical property, namely, the tendency to readily undergo electrophilic substitution rather than electrophilic addition. From a molecular structure perspective, aromatic compounds tend to have more evenly distributed carbon-carbon single and double bond lengths, exhibit coplanarity, possess larger conjugation energies (delocalization energies), and have more stable molecular structures. In this invention, the aromaticity of compounds is determined using Hückel's rule.

[0122] The term "alkyl protecting group" refers to an alkyl group that can be selectively introduced under specific chemical conditions and removed under different specific reaction conditions, thereby exposing the protected functional group (e.g., hydroxyl, carboxyl, etc.). In this invention, C1-C6 alkyl protecting groups refer, for example, to straight-chain, branched, or cyclic saturated alkyl groups consisting of 1 to 6 carbon atoms, covalently linked to the protected functional group (e.g., hydroxyl, carboxyl, etc.) via carbon atoms. The C1-C6 alkyl protecting group is preferably a C1-C6 alkyl group, such as tert-butyl.

[0123] The term "pharmaceutically acceptable carrier" as used in this invention refers to a component in a pharmaceutical preparation or composition that is non-toxic to the subject, excluding the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0124] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0125] The reagents and raw materials used in this invention are all commercially available.

[0126] The positive and progressive effects of this invention are as follows: the folic acid receptor-targeted near-infrared fluorescent molecule of this invention has one or more of the following effects:

[0127] (1) The compound of the present invention has significantly improved the metabolic stability of hepatocytes, which is expected to improve its imaging window and enhance imaging stability;

[0128] (2) The compounds of the present invention are not metabolized by common CYP enzymes. They are mainly metabolized in their original form in the livers of five species: mice, rats, dogs, cynomolgus monkeys and humans, which also suggests improved metabolic stability.

[0129] (3) The compounds of the present invention also have one or more advantages such as high fluorescence quantum yield, stable chemical structure, active tumor targeting, high TBR value at the lesion site and high imaging capability.

[0130] (4) The present invention provides at least one product alternative to OTL038. The target structure of the present invention is different from the pteridine target structure of OTL038, and it can be comparable to or better than OTL038 in terms of affinity, chemical structure stability and imaging capability. Attached Figure Description

[0131] Figure 1 shows the stability of compounds 1-3, OTL038, and fluorescent dye SO456 under xenon lamp irradiation conditions.

[0132] Figure 2 shows the fluorescence distribution of ex vivo tissues after Compound 1 and Compound 2 of the present invention were injected into KB cells and A549 cells xenograft mouse models (the upper arrow represents KB tumors and the lower arrow represents A549 tumors).

[0133] Figure 3 shows the in vitro pseudo-color images of tumor tissue and tissue near the tumor at different time points after compounds 1-3 and compound 5 of the present invention were injected into a KB cell xenograft mouse model.

[0134] Figure 4 shows the in vitro grayscale images of tumor tissue and surrounding tissue at different time points after compounds 1-3 and compound 5 of the present invention were injected into a KB cell xenograft mouse model.

[0135] Figure 5 shows the tumor-to-background ratio (TBR) at different time points after compounds 1-3 and compound 5 of the present invention were injected into a KB cell xenograft mouse model.

[0136] Figure 6 shows pseudo-color and grayscale images of tumor tissue and surrounding tissues after 6 hours of administration of compounds 1-3 and compound 5 of the present invention injected into KB cells, A549 cells, and LNCaP xenograft mouse models.

[0137] Figure 7 shows the tumor-to-background ratio (TBR) 6 hours after injecting compounds 1-3 and compound 5 of the present invention into KB cells, A549 cells, and LNCaP xenograft mouse models. Detailed Implementation

[0138] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods not specifying specific conditions should be performed according to conventional methods and conditions, or as selected in the product instructions.

[0139] In the compound preparation method of this invention, the functional groups of the intermediate compound may need to be protected by suitable protecting groups. Functional groups requiring protection include hydroxyl, phenol, and carboxyl groups. Suitable protecting groups for hydroxyl or phenol groups include silane ethers, alkyl ethers, alkoxy ethers (acetals), and esters, such as tert-butyldimethylsilyl, tert-butyldiphenylsilyl, trimethylsilyl, tetrahydropyranyl, benzyl, substituted benzyl, tert-butyl, etc. Suitable protecting groups for carboxyl groups include alkyl esters, aryl esters, or arylalkyl esters. Protecting groups can be added or removed according to standard techniques well known to those skilled in the art.

[0140] The materials, analytical methods, and instruments involved in this invention, unless otherwise specified, use reagents and solvents obtained from commercial suppliers. Unless otherwise specified, known starting materials of this invention are assumed to have been purchased from the respective reagent companies (such as TCI, Sigma-Aldrich, MedChemExpress, etc.) through reagent procurement platforms or their official websites.

[0141] The analytical methods and conditions involved in this invention are as follows:

[0142] The structure of the compound was determined by nuclear magnetic resonance (NMR) hydrogen spectrum (1H NMR). 1 H-NMR (Bruker instrument, 400MHz) and / or mass spectrometry (MS) were used; the mass spectrometry was performed using a Waters Acquity Xevo G2-XS QT of UPLC / MS ultra-high performance liquid chromatography-high resolution mass spectrometry system. 1 H-NMR was performed using a Bruker AVANCE III 400MHz nuclear magnetic resonance spectrometer.

[0143] The intermediates and products involved in this invention are analyzed using rapid chromatography (column chromatography) and high-performance liquid chromatography (HPLC).

[0144] Suitable methods and starting materials for synthesizing the compounds of the present invention are provided in the following schemes and examples. Unless otherwise specified, all substituents are as defined above. Furthermore, unless clearly described otherwise, all reactions, reaction conditions, abbreviations, and symbols have meanings well-known to those skilled in the art of organic chemistry.

[0145] Table 1 shows the English abbreviations and Chinese names of the compounds involved in this invention.

[0146] Table 1

[0147] Example 1 Preparation of 4-{2-[(E)-2-((E)-2-[4-((S)-2-{4-[3-(2-amino-4-oxo-3,4-dihydrothiopheno[2,3-d]pyrimidin-6-yl)propyl]benzamido}-2-carboxyethyl)phenoxy]-3-{2-[(E)-3,3-dimethyl-5-sulfono-1-(4-sulfonobutyl)indol-2-ylidene]ethenyl}cyclohex-1-en-1-yl)vinyl]-3,3-dimethyl-5-sulfono-3H-indol-1-on-1-yl}butyl-1-sulfonic acid inner salt (compound 1)

[0148] Step 1: Preparation of ethyl 4-(5-oxo-n-pentyl)benzoate (intermediate 1)

[0149] Add the following to the reaction flask sequentially: Bu4NCl (6.45 g, 2.0 eq), LiCl (491 mg, 1.0 eq), LiOAc (1.91 g, 2.5 eq), DMF (25 mL), ethyl 4-iodobenzoate [CAS No.: 51934-41-9] (3.2 g, 1.0 eq), 5-hexen-1-ol [CAS No.: 821-41-0] (1 g, 1.0 eq), and Pd(OAc)2 (156 mg, 0.06 eq). Purge the mixture with nitrogen three times, heat to 70 °C, and react for 2 h. Monitor the reaction with LC-MS until completion. Cool the reaction system to 25 °C, and add H2O (50 mL) and EA (100 mL) sequentially. Collect the organic phase by separation. The organic phase was washed three times with saturated NaCl (20 mL * 3), dried over anhydrous sodium sulfate, concentrated under vacuum to remove the solvent, and purified by column chromatography (eluent PE:EA = 25:1 to 15:1, V / V) to obtain intermediate 1 (1.25 g, purity: 98.81%, yield: 45.9%).

[0150] 1 H NMR: (400MHz, CDCl3) δppm 9.76(t,J=2.0Hz,1H),7.96(d,J=8.0Hz,2H),7.24(d,J=8.0Hz,2H),4.36(q,J=6.8Hz,2H),2.69(t,J=7. 6Hz,2H),2.46(td,J=7.2,6.0Hz,2H),1.62-1.75(m,4H),1.39(t,J=6.8Hz,3H); MS(ESI)m / z=235.2[M+H] + .

[0151] Step 2: Preparation of ethyl 2-amino-5-{3-[4-(ethoxycarbonyl)phenyl]propyl}thiophene-3-carboxylate (intermediate 2)

[0152] Intermediate 1 (10.9 g, 1.0 eq), EtOH (60 mL), elemental sulfur S8 [CAS No.: 10544-50-0] (2.2 g, 1.5 eq), and TEA (4.7 g, 1.0 eq) were added sequentially to the reaction flask. The mixture was purged with nitrogen three times, and the temperature was raised to 80 °C for 2 h. The reaction was monitored by LCMS until the reaction was complete. The reaction system was cooled to 25 °C and filtered. The filtrate was concentrated under vacuum to remove the solvent, and purified by column chromatography (eluent PE:EA = 30:1 to 10:1, V / V) to obtain intermediate 2 (15.1 g, purity: 97.64%, yield: 90%).

[0153] 1 H NMR: (400MHz, CDCl3) δppm 7.96(d,J=8.0Hz,2H),7.24(d,J=8.0Hz,2H),6.66(s,1H),4.36(q,J=6.8Hz,2H),4.25(q,J=7.2Hz,2H),2.71(t,J=7.6Hz,2H ),2.61(td,J=7.2,6.0Hz,2H),1.93(t,J=7.2Hz,2H),1.39(t,J=6.8Hz,3H),1.33(t,J=7.2Hz,3H); MS(ESI)m / z=362.2[M+H] + .

[0154] Step 3: Preparation of ethyl 4-[3-(2-amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-yl)propyl]benzoate (intermediate 3)

[0155] Intermediate 2 (3 g, 1.0 eq), chloroformamidine hydrochloride [CAS No.: 29671-92-9] (4.36 g, 4.0 eq), and DMSO2 (17.8 g, 20.0 eq) were added sequentially to the reaction flask. The mixture was purged with nitrogen three times, and the temperature was raised to 140 °C for 4 h. LC-MS showed that some substrate was not completely reacted. The temperature was then raised to 160 °C and the reaction continued for another 4 h. LC-MS showed that the reaction was essentially complete. The reaction system was cooled to 25 °C, and H2O (150 mL) was added and dissolved by sonication. The pH was adjusted to 7-8 using ammonia. The mixture was then cooled to 0 °C and stirred for 1 h. After filtration, the filter cake was vacuum dried to obtain crude intermediate 3 (3.7 g, crude yield: 138%), which was used directly in the next reaction without purification. MS (ESI) m / z = 358.1 [M+H] + .

[0156] Step 4: Preparation of 4-[3-(2-amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-yl)propyl]benzoic acid (intermediate 4)

[0157] To the reaction flask, crude intermediate 3 (3.7 g), EtOH (80 mL), and sodium hydroxide (1 mol / L, 80 mL) were added sequentially. The mixture was stirred at room temperature for 14 h, and LC-MS showed that the reaction was complete. The reaction solution was concentrated under vacuum to remove ethanol, and the pH was adjusted to 3 with hydrochloric acid (1 mol / L). After filtration, the filter cake was dried under vacuum to obtain crude intermediate 4 (3.1 g, purity: 88.58%, crude product yield: 113%), which was directly used in the next step of the reaction.

[0158] 1 H NMR: (400MHz, DMSO-d6) δppm 11.03(brs,1H),7.86(d,J=8.0Hz,2H),7.31(d,J=8.0Hz,2H),6.83(s,1H),6.5 6(brs,2H),2.58-2.72(m,4H),1.92(t,J=7.6Hz,2H); MS(ESI)m / z=330.1[M+H] + .

[0159] Step 5: Preparation of (S)-2-{4-[3-(2-amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-yl)propyl]benzamido}-3-[4-(tert-butoxy)phenyl]propionate tert-butyl ester (intermediate 5)

[0160] Intermediate 4 (3.4 g, 1.0 eq), DMSO (75 mL), HATU (5.9 g), DIPEA (4.1 g), and O-tert-butyl-L-tyrosine tert-butyl hydrochloride [CAS:17083-23-7] (4.1 g, 1.2 eq) were added sequentially to the reaction flask. The mixture was purged with nitrogen three times and reacted at 25 °C for 2 h. LC-MS showed that the reaction was complete. H2O (100 mL) and EA (150 mL) were added to the reaction flask. The mixture was separated, and the organic phase was collected. The aqueous phase was extracted once with EA (150 mL). The organic phases were combined, washed twice with saturated sodium chloride (100 mL * 2), dried over anhydrous sodium sulfate, and the organic phase was concentrated under vacuum to remove the solvent. The mixture was then slurried with ethyl acetate to obtain intermediate 5 (2.1 g, purity: 94.9%, yield: 33.6%).

[0161] 1H NMR: (400MHz, CDCl3) δppm 11.68(brs,1H),7.65(d,J=8.0Hz,2H),7.21(d,J=8.0Hz,2H),7.10(d,J=8. 4Hz,2H),6.91(d,J=8.4Hz,2H),6.87(s,1H),6.74(d,J=8.0Hz,1H),5.48(br s,2H),4.91(dd,J=8.0,4.0Hz,1H),3.19(d,J=4.0Hz,2H),2.66-2.82(m,4H ),2.00(t,J=7.6Hz,2H),1.42(s,9H),1.31(s,9H); MS(ESI)m / z=605.3[M+H] + .

[0162] Step 6: Preparation of 4-{2-[(E)-2-((E)-2-[4-((S)-2-{4-[3-(2-amino-4-oxo-3,4-dihydrothiopheno[2,3-d]pyrimidin-6-yl)propyl]benzamido}-2-carboxyethyl)phenoxy]-3-{2-[(E)-3,3-dimethyl-5-sulfono-1-(4-sulfonobutyl)indol-2-ylidene]ethenyl}cyclohex-1-en-1-yl)vinyl]-3,3-dimethyl-5-sulfono-3H-indol-1-on-1-yl}butyl-1-sulfonic acid inner salt (compound 1)

[0163] Intermediate 5 (50 mg, 1.0 eq), DCM (2 mL), and TFA (1 mL) were added sequentially to the reaction flask. The reaction was carried out at 25 °C for 2 h, and LC-MS showed that the reaction was complete. The reaction solution was concentrated under vacuum to remove the solvent, and H2O (1.5 mL) was added. The pH was adjusted to 11 with sodium hydroxide solution and set aside for later use.

[0164] SO456 (CAS: 12252007-83-2, 50 mg, 0.68 eq) was added to another reaction flask and dissolved in 1.5 mL of H2O. The solution adjusted to pH 11 was then added dropwise, and the mixture was heated to 90 °C and reacted for 22 h. LCMS showed that the target product was formed. The reaction solution was directly purified by preparative HPLC (eluent (0.1% NH4OH)H2O / CH3CN = 0-60%, V / V), and lyophilized to obtain compound 1 (15 mg, HPLC purity: 96.79%, yield: 19.8%).

[0165] 1H NMR: (400MHz, DMSO-d6) δppm 11.77(brs,1H),8.46(d,J=8.0Hz,1H),7.66-7.76(m,4H),7.59(d,J=8.0Hz,2H),7.55(s,2H),7.35(d, J=8.4Hz,2H),7.20-7.29(m,4H),7.02(d,J=8.4Hz,2H),6.84(s,1H),6.15(d,J=13.6Hz,2H),4.58-4.67 (m,1H),4.07(s,4H),3.11-3.20(m,1H),2.95-3.05(m,1H),2.64-2.72(m,4H),2.56-2.62(m,4H),2.53 (t,J=7.6Hz,2H),1.90(s,2H),1.60-1.79(m,10H),1.13(s,6H),1.02(s,6H); MS(ESI)m / z=1344.7[M+H] + 672.8 [M+2H] 2+ .

[0166] Example 2: Preparation of 4-{2-[(E)-2-((E)-2-[4-((S)-2-{5-[4-(2-amino-4-oxo-3,4-dihydrothiopheno[2,3-d]pyrimidin-6-yl)butyl]pyridine-2-carboxyethyl)phenoxy]-3-{2-[(E)-3,3-dimethyl-5-sulfonyl-1-(4-sulfonylbutyl)indol-2-ylidene]ethenyl}cyclohex-1-en-1-yl)vinyl]-3,3-dimethyl-5-sulfonyl-3H-indol-1-on-1-yl}butyl-1-sulfonic acid inner salt (compound 2)

[0167] Step 1: Preparation of methyl 5-(6-oxohexyl)pyridine-2-carboxylate (intermediate 6)

[0168] 5-Hexen-1-ol (10.7 g, 1 eq) was added to DMF (150 mL, 15 V); then Pd(OAc)₂ (1.2 g, 0.1 eq), LiCl (2.2 g, 1.1 eq), LiOAc (2.29 g, 2.5 eq), Bu₄NCl (8.3 g, 2 eq), and methyl 5-bromopyridine-2-carboxylic acid (CAS: 29682-15-3, 1.6 g, 1.2 eq) were added. After three purgings with N₂, the reaction mixture was heated to 80 °C and stirred for 12 h. The reaction was monitored by LC-MS until complete. The reaction mixture was then added to water (300 mL, 30 V), extracted three times with EA (300 mL, 30 V), washed with saturated brine (300 mL, 30 V), and dried over anhydrous Na₂SO₄. The organic phase was concentrated and purified by column chromatography (eluent PE / EA = 0-39%, V / V) to give colorless oily intermediate 6 (3.5 g, purity 99%, yield 32%).

[0169] 1 H NMR: (400MHz, CDCl3) δppm 9.77(t,J=2.0Hz,1H),8.57(d,J=2.0Hz,1H),8.06(d,J=8.0Hz,1H),7.65(dd,J=8.0,2.0Hz,1H),4.00(s,3H),2. 71(t,J=7.6Hz,2H),2.44(td,J=7.2,6.0Hz,2H),1.60-1.72(m,4H),1.32-1.44(m,2H); MS(ESI)m / z=236.0[M+H] + .

[0170] Step 2: Preparation of methyl 5-{4-[5-amino-4-(ethoxycarbonyl)thiophen-2-yl]butyl}pyridine-2-carboxylate (intermediate 7)

[0171] Intermediate 6 (6.5 g, 1 eq) was added to EtOH (65 mL, 10 V), followed by S8 (768 mg, 1.2 eq), TEA (2.02 g, 1 eq), and ethyl 2-cyanoacetate [CAS: 105-56-6] (2.26 g, 1 eq). The mixture was purged with nitrogen three times. The reaction mixture was heated to 80 °C and stirred for 2 h, with LCMS monitoring until the reaction was complete. The reaction mixture was then cooled and concentrated for purification by column chromatography (eluent PE / EA = 0.35%, V / V) to obtain a yellow oily intermediate 7 (9 g, purity 96%, yield 90%).

[0172] 1H NMR: (400MHz, CDCl3) δppm 8.49(d,J=2.0Hz,1H),7.99(d,J=8.0Hz,1H),7.56(dd,J=8.0,2.0Hz,1H),6.54(s,1H),5.73(brs,2H),4.18(q,J=7.6Hz,2H), 3.93(s,3H),2.63(t,J=7.6Hz,2H),2.54(t,J=7.2Hz,2H),1.53-1.65(m,4H),1.26(t,J=7.6Hz,3H); MS(ESI)m / z=363.0[M+H] + .

[0173] Step 3: Preparation of methyl 5-{4-[4-(ethoxycarbonyl)-5-guanidinothiophen-2-yl]butyl}pyridine-2-carboxylate (intermediate 8)

[0174] Intermediate 7 (5.3 g, 1 eq) was added to DMSO2 (13.7 g, 10 eq), followed by chloroformamidine hydrochloride (5.05 g, 3 eq). The mixture was substituted with N2 three times. The reaction solution was heated to 110 °C and stirred for 1 h. The reaction was monitored by LC-MS until complete. The reaction solution was cooled and dissolved in MeOH (100 mL). The solution was purified by column chromatography (eluent DCM / MeOH = 0-10%, V / V) to obtain intermediate 8 (8.5 g, yield 140%, crude product). MS (ESI) m / z = 405.1 [M+H] + .

[0175] Step 4: Preparation of methyl 5-[4-(2-amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-yl)butyl]pyridine-2-carboxylate (intermediate 9)

[0176] Intermediate 8 (8.5 g) was added to DMF (150 mL, 30 V) and stirred at 140 °C for 6 h. The reaction was monitored by LCMS until complete. The solvent was removed from the reaction solution using an oil pump, and the solution was dissolved in MeOH (100 mL). The solution was purified by column chromatography (eluent DCM / MeOH = 0-10%, V / V) to give a white solid intermediate 9 (3.4 g, purity: 96.7%, yield 60%).

[0177] 1H NMR: (400MHz, DMSO-d6) δppm 8.56(d,J=2.0Hz,1H),7.98(d,J=8.0Hz,1H),7.81(dd,J=8.0,2.0Hz,1H),6.80(s,1H),6.4 6(brs,2H),3.86(s,3H),2.73(t,J=7.6Hz,4H),1.53-1.70(m,4H); MS(ESI)m / z=359.1[M+H] + .

[0178] Step 5: Preparation of 5-[4-(2-amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-yl)butyl]pyridine-2-carboxylic acid (intermediate 10)

[0179] Intermediate 9 (3.4 g, 1 eq) was added to EtOH (10 mL, 3 V), followed by NaOH / H2O (10 mL, 2 M, 3 V). The mixture was stirred at room temperature for 12 h. The EtOH in the reaction solution was concentrated, and the pH of the reaction solution was adjusted to 5. A yellow solid precipitated out. The precipitate was filtered and dried to obtain a pale yellow solid intermediate 10 (2.1 g, purity: 73%, yield: 75%).

[0180] 1 H NMR: (400MHz, DMSO-d6) δppm 10.91(brs,1H),8.57(d,J=2.0Hz,1H),7.99(d,J=8.0Hz,1H),7.84(dd,J=8.0,2.0Hz,1H), 6.81(s,1H),6.55(brs,2H),2.65-2.82(m,4H),1.55-1.72(m,4H); MS(ESI)m / z=345.2[M+H] + .

[0181] Step 6: Preparation of (S)-2-{5-[4-(2-amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-yl)butyl]pyridine-2-carboxamido}-3-[4-(tert-butoxy)phenyl]propionate tert-butyl ester (intermediate 11)

[0182] Intermediate 10 (2.1 g, 1 eq) was added to DMSO (10 mL, 5 V), followed by O-tert-butyl-L-tyrosine tert-butyl hydrochloride (1.96 g, 1.1 eq), HATU (3.48 g, 1.5 eq), and DIPEA (2.36 g, 3 eq). The reaction mixture was stirred at room temperature for 2 h, and the reaction was monitored by LCMS until complete. The reaction mixture was then directly purified by reverse preparative HPLC [eluent (0.1% NH4OH)H2O / CH3CN = 0-60%, V / V] to obtain a white solid intermediate 11 (3 g, purity: 85%, yield 81%).

[0183] 1 H NMR: (400MHz, DMSO-d6)δppm 10.82(brs,1H),8.63(d,J=8.0Hz,1H),8.49(d,J=2.0Hz,1H),7.92(d,J=8.0Hz ,1H),7.82(dd,J=8.0,2.0Hz,1H),7.13(d,J=8.4Hz,2H),6.86(d,J=8.4Hz,2H) ,6.81(s,1H),6.45(brs,2H),4.58-4.70(m,1H),3.06-3.33(m,2H),2.65-2.80 (m,4H),1.53-1.68(m,4H),1.34(s,9H),1.23(s,9H); MS(ESI)m / z=620.4[M+H] + .

[0184] Step 7: Preparation of 4-{2-[(E)-2-((E)-2-[4-((S)-2-{5-[4-(2-amino-4-oxo-3,4-dihydrothiopheno[2,3-d]pyrimidin-6-yl)butyl]pyridine-2-carboxyethyl)phenoxy]-3-{2-[(E)-3,3-dimethyl-5-sulfonyl-1-(4-sulfonyl)indol-2-ylidene]ethenyl}cyclohex-1-en-1-yl)vinyl]-3,3-dimethyl-5-sulfonyl-3H-indol-1-on-1-yl}butyl-1-sulfonic acid inner salt (compound 2)

[0185] Intermediate 11 (100 mg, 1 eq) was added to DCM (5 mL, 50 V), followed by HCl (2 mL, 20 V). The mixture was stirred at room temperature for 4 h. The tert-butyl protecting group was monitored by LCMS to ensure complete removal. The reaction solution was concentrated to dryness, and NaOH / H2O (2 M) was added dropwise to adjust the pH to 10 for later use.

[0186] The above reaction solution was added to a solution of SO456 (100 mg, 1 eq) in H2O (2 mL, 20 V). The reaction solution was stirred at 90 °C for 2 h. The reaction was monitored by LCMS to ensure complete reaction. The reaction solution was directly purified by reverse preparative HPLC [eluent (0.1% NH4OH)H2O / CH3CN = 0-60%, V / V] to obtain compound 2 (51 mg, purity: 97.4%, yield 23%).

[0187] 1 H NMR: (400MHz, DMSO-d6) δppm 11.82(brs,1H),8.62(d,J=8.0Hz,1H),8.44(s,1H),7.91(s,2H),7.68(d,J=14.0Hz,2H),7.63(d,J=8.4Hz,2H),7.54(s ,2H),7.32(d,J=8.4Hz,2H),7.22(d,J=8.4Hz,2H),6.96(d,J=8.4Hz,2H),6.90(s,1H),6.18(d,J=14.0Hz,2H),4.65(dd ,J=12.8,8.4Hz,1H),4.12(brs,4H),3.17-3.24(m,1H),3.07-3.16(m,1H),2.73-2.81(m,2H),2.67-2.72(m,4H),2.57- 2.66(m,6H),1.91(t,J=6.4Hz,2H),1.65-1.81(m,8H),1.53(s,4H),1.13(s,6H),1.06(s,6H); MS(ESI)m / z=1359.9[M+H] + .

[0188] Example 3: Preparation of 4-{2-[(E)-2-((E)-2-[4-((S)-2-{4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)propyl]benzamido}-2-carboxyethyl)phenoxy]-3-{2-[(E)-3,3-dimethyl-5-sulfonyl-1-(4-sulfonylbutyl)indol-2-ylidene]ethenyl}cyclohex-1-en-1-yl)vinyl]-3,3-dimethyl-5-sulfonyl-3H-indol-1-on-1-yl}butyl-1-sulfonic acid inner salt (compound 3)

[0189] Step 1: Preparation of 4-(4-ethoxycarbonylphenyl)butyric acid (intermediate 12)

[0190] At room temperature, intermediate 1 (10 g, 1 eq) was dissolved in a mixed solution of tert-butanol (100 mL) and water (50 mL), followed by the sequential addition of 2-methyl-2-butene (CAS: 513-35-9, 15.9 g, 5 eq), NaH₂PO₄ (8.2 g, 1.5 eq), and NaClO₂ (14.4 g, 3.5 eq), and the reaction was carried out at room temperature. After the reaction was complete, the pH was adjusted to 2–3 with HCl, the aqueous phase was extracted with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was pulped and filtered to give intermediate 12 (7.9 g, white solid, yield 73%).

[0191] 1 H NMR: (400MHz, CDCl3) δppm 7.97(d,J=8.0Hz,2H),7.25(d,J=8.0Hz,2H),4.36(q,J=7.2Hz,2H),2.73(t,J=7.6Hz,2H),2. 38(t,J=7.6Hz,2H),1.39(qui,J=7.6Hz,2H),1.39(t,J=7.2Hz,3H); MS(ESI)m / z=236.8[M+H] + .

[0192] Step 2: Preparation of ethyl 4-{4-[(1-cyano-2-ethoxy-2-oxoethyl)amino]-4-oxobutyl}benzoate (intermediate 13)

[0193] At room temperature, intermediate 12 (7.9 g, 1 eq) and ethyl aminocyanoacetate p-toluenesulfonate (CAS: 37842-58-3, 10 g, 1 eq) were dissolved in DMF (100 mL), and DIPEA (21.6 g, 5 eq) and HATU (25.4 g, 2 eq) were added. The reaction mixture was allowed to react completely at room temperature. The reaction solution was poured into ice water, extracted with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain the crude product. The crude product was purified by pulping with petroleum ether to give intermediate 13 (5 g yellow solid, yield 43%).

[0194] 1H NMR: (400MHz, CDCl3) δppm 7.97(d,J=8.0Hz,2H),7.24(d,J=8.0Hz,2H),6.24-6.38(br,1H),5.51(d,J=5.2Hz,1H),4.34-4.38(m,4H),2.7 3(t,J=7.2Hz,2H),2.31(t,J=7.2Hz,2H),2.05(qui,J=7.2Hz,2H),1.34-1.41(m,6H); MS(ESI)m / z=347.1[M+H] + .

[0195] Step 3: Preparation of ethyl 5-amino-2-{3-[4-(ethoxycarbonyl)phenyl]propyl}thiazole-4-carboxylate (intermediate 14)

[0196] Intermediate 13 (7.67 g, 1 eq) was dissolved in toluene (100 mL), and then Lawrence's reagent (CAS: 19172-47-5, 5.39 g, 0.6 eq) was added. The reaction mixture was reacted overnight at 70–90 °C. After the reaction was complete, the reaction solution was evaporated to dryness, and water and ethyl acetate were added. The layers were separated, and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, evaporated to dryness, and purified by silica gel column chromatography to obtain 7 g of crude product. The crude product was purified by silica gel column chromatography using DCM / MeOH (300 / 1–100 / 1) to obtain intermediate 14 (2.68 g, pale yellow solid, yield 33%).

[0197] 1 H NMR: (400MHz, CDCl3) δppm 7.97(d,J=8.0Hz,2H),7.24(d,J=8.0Hz,2H),6.24-5.93(brs,2H),4.34-4.42(m,4H),2.87(t,J=7.2 Hz,2H),2.74(t,J=7.2Hz,2H),2.04(qui,J=7.6Hz,2H),1.37-1.42(m,6H); MS(ESI)m / z=363.3[M+H] + .

[0198] Step 4: Preparation of ethyl 5-(3-benzoylthiourea)-2-{3-[4-(ethoxycarbonyl)phenyl]propyl}thiazole-4-carboxylate (intermediate 15)

[0199] Intermediate 14 (2.62 g, 1 eq) was dissolved in acetone (100 mL), and then benzoyl isothiocyanate (CAS: 532-55-8, 1.81 g, 1.5 eq) was added. The mixture was refluxed overnight. The reaction solution was evaporated to dryness, purified by column chromatography (eluent PE:EA = 4:1 to 2:1, V / V), and concentrated to give intermediate 15 (3.62 g pale yellow solid, yield 83%).

[0200] 1 H NMR: (400MHz, DMSO-d6)δppm 14.78(s,1H),12.17(s,1H),8.01(dd,J=6.8,1.6Hz,2H),7.88(dd,J=6.8,1.6 Hz,2H),7.67(t,J=7.6Hz,1H),7.56(t,J=7.6Hz,2H),7.39(d,J=7.6Hz,2H),4. 40(q,J=7.2Hz,2H), 4.30(q,J=7.2Hz,2H), 2.93(t,J=7.6Hz,2H), 2.77(t,J=7. 6Hz,2H),2.04(qui,J=7.6Hz,2H),1.29-1.37(m,6H);MS(ESI)m / z=526.0[M+H] + .

[0201] Step 5: Preparation of ethyl 4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)propyl]benzoate (intermediate 16)

[0202] At room temperature, intermediate 15 (4.1 g, 1 eq) was dissolved in DMF (50 mL), followed by the sequential addition of NaH (CAS: 7646-69-7, 0.47 g, 1.5 eq) and methyl iodoform (1.66 g, 1.5 eq). The reaction was allowed to proceed at room temperature, and the reaction was monitored for completeness by LC-MS. The reaction solution was poured into water, extracted with ethyl acetate, and the organic phases were combined and washed with saturated ammonium chloride aqueous solution and saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain 4.4 g of crude product.

[0203] 4.4 g of the crude product was dissolved in ammonia-methanol solution (CAS: 7664-41-7, 50 mL) and reacted overnight at 60 °C. The reaction was monitored by LCMS until complete. The reaction solution was filtered, the solid was collected and dried to obtain 2 g of crude product. The crude product was further recrystallized in methanol to obtain intermediate 16 (1.09 g, white solid, yield 39%).

[0204] 1H NMR: (400MHz, DMSO-d6) δppm 11.06(s,1H),7.89(d,J=8.0Hz,2H),7.38(d,J=8.0Hz,2H),6.67(brs,2H),4.30(q,J=7.2Hz,2H),2.90(t,J =7.6Hz,2H),2.75(t,J=7.6Hz,2H),2.02(qui,J=7.6Hz,2H),1.32(t,J=7.2Hz,2H); MS(ESI)m / z=359.0[M+H] + .

[0205] Step 6: Preparation of 4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)propyl]benzoic acid (intermediate 17)

[0206] Intermediate 16 (1.09 g, 1 eq) was dissolved in a mixed solution of 1 M NaOH aqueous solution (10 mL) and ethanol (10 mL), and the mixture was refluxed. The disappearance of the starting material was monitored by LCMS. After the reaction was complete, the ethanol in the reaction solution was evaporated to dryness, and water and ethyl acetate were added. The layers were separated, and the aqueous phase was collected. The pH of the aqueous phase was adjusted to 5-6 with HCl, and a white solid precipitated. The solid was filtered, collected, and dried to give intermediate 17 (941.5 mg, white solid, yield 93%).

[0207] 1 H NMR: (400MHz, DMSO-d6) δppm 12.79(brs,1H),11.12(brs,1H),7.87(d,J=8.0Hz,2H),7.35(d,J=8.0Hz,2H),6.70(brs,2H), 2.90(t,J=7.6Hz,2H),2.74(t,J=7.6Hz,2H),2.02(qui,J=7.6Hz,2H); MS(ESI)m / z=330.7[M+H] + .

[0208] Step 7: Preparation of (S)-2-{4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)propyl]benzamido}-3-[4-(tert-butoxy)phenyl]propionate tert-butyl ester (intermediate 18)

[0209] Intermediate 17 (177 mg, 1.0 eq), DMSO (1 mL), HATU (256 mg, 1.3 eq), DIPEA (212 mg, 3.0 eq), and O-tert-butyl-L-tyrosine tert-butyl hydrochloride (177 mg, 1.0 eq) were added sequentially to the reaction flask. The mixture was purged with nitrogen three times and reacted at 25 °C under LC-MS monitoring until the starting material was completely converted. The reaction solution was poured into water (30 mL), stirred to precipitate a solid, and centrifuged and filtered to obtain intermediate 18 (320 mg white solid, yield 98.8%).

[0210] 1 H NMR: (400MHz, DMSO-d6) δppm 11.06(brs,1H),8.62(d,J=8.0Hz,1H),7.75(d,J=8.0Hz,2H),7.31(d,J=8.0Hz,2H),7.19(d,J=8.0,2H),6.88(d,J=8.0,2H),6.66(brs,2H), 4.52(q,J=8.0Hz,1H),3.04(d,J=8.0Hz,2H),2.90(t,J=7.2Hz,2H),2.72(t,J=7.2Hz,2H),2.02(qui,J=7.2Hz,2H); MS(ESI)m / z=606.0[M+H] + .

[0211] Step 8: Preparation of 4-{2-[(E)-2-((E)-2-[4-((S)-2-{4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)propyl]benzamido}-2-carboxyethyl)phenoxy]-3-{2-[(E)-3,3-dimethyl-5-sulfonyl-1-(4-sulfonylbutyl)indol-2-ylidene]ethenyl}cyclohex-1-en-1-yl)vinyl]-3,3-dimethyl-5-sulfonyl-3H-indol-1-on-1-yl}butyl-1-sulfonic acid inner salt (compound 3)

[0212] Intermediate 18 (300 mg, 1.0 eq), TFA (4 mL), and water (0.1 mL) were added sequentially to the reaction flask, and the reaction was carried out at 25 °C for 3 h. The reaction was monitored by LC-MS until completion. The reaction solution was then added dropwise to MTBE (40 mL), causing solid precipitation. The solid was centrifuged, filtered, and dried for later use (300 mg). MS (ESI) m / z = 494.0 [M+H] + .

[0213] Add water (4 mL) to the solid obtained in the previous step, adjust the pH to 11 with 2M sodium hydroxide, add SO456 (150 mg), and heat to 90 °C for reaction. Monitor the reaction using LCMS. If intermediate 19 has not reacted completely, add SO456, adjust the pH to 11 again, and continue heating to 90 °C until the starting material has basically reacted. After filtration, prepare HPLC for separation (column: Welch xbidge xb 5um 21.2*250 mm; mobile phase: [aqueous phase (TFA 0.1 v / v%) - acetonitrile]; ACN v / v%: 10%-50%, gradient time 18 min), and lyophilize to obtain compound 3 (110 mg green solid, yield 40.3%).

[0214] 1 H NMR: (400MHz, DMSO-d6) δppm 8.47(d,J=8.4Hz,1H),7.67-7.77(m,4H),7.59(d,J=8.4Hz,2H),7.56(s,2H),7.35(d,J=8.4Hz,2H),7. 28(d,J=8.4Hz,2H),7.24(d,J=8.4Hz,2H),7.04(d,J=8.4Hz,2H),6.17(d,J=14.0Hz,2H),4.58-4.66(m ,1H),4.07(brs,4H),3.11-3.19(m,1H),2.97-3.05(m,1H),2.81(t,J=6.4Hz,2H),2.69(brs,4H),2.53 -2.60(m,6H),1.91(brs,2H),1.66-1.85(m,10H),1.16(s,6H),1.05(s,6H); MS(ESI)m / z=673.3[M+2H] 2+ .

[0215] Example 3b: Preparation of 4-{2-[(E)-2-((E)-2-[4-((R)-2-{4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)propyl]benzamido}-2-carboxyethyl)phenoxy]-3-{2-[(E)-3,3-dimethyl-5-sulfonyl-1-(4-sulfonylbutyl)indol-2-ylidene]ethenyl}cyclohex-1-en-1-yl)vinyl]-3,3-dimethyl-5-sulfonyl-3H-indol-1-on-1-yl}butyl-1-sulfonic acid inner salt (compound 3b)

[0216] Compound 3b is a chiral isomer of compound 3 in Example 3. Its chirality is introduced by the starting material O-tert-butyl-L-tyrosine tert-butyl hydrochloride or O-tert-butyl-D-tyrosine tert-butyl hydrochloride used in reaction step 7. Therefore, this invention prepared compound 3b using a preparation process that is basically the same as that in Example 3, except that in step 7, the starting material O-tert-butyl-L-tyrosine tert-butyl hydrochloride was replaced with O-tert-butyl-D-tyrosine tert-butyl hydrochloride (CAS: 1998701-20-4), yielding crude intermediate 18b (750 mg white solid, yield 81.8%); in step 8, intermediate 18 was replaced with intermediate 18b, yielding compound 3b (20 mg dark green solid, yield 12.7%).

[0217] Example 4: Preparation of 4-{2-[(E)-2-((E)-2-[4-((S)-2-{5-[4-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)butyl]pyridine-2-carboxyethyl)phenoxy]-3-{2-[(E)-3,3-dimethyl-5-sulfonyl-1-(4-sulfonylbutyl)indol-2-ylidene]ethenyl}cyclohex-1-en-1-yl)vinyl]-3,3-dimethyl-5-sulfonyl-3H-indol-1-on-1-yl}butyl-1-sulfonic acid inner salt (compound 4)

[0218] Step 1: Preparation of ethyl 2-cyano-2-(pent-4-enamino)acetate (intermediate 19)

[0219] 4-Penten-1-acid (CAS: 591-80-0) (4.55 g, 1 eq) and DMF (80 mL) were added to a 250 mL single-necked reaction flask, followed by ethyl aminocyanoacetate p-toluenesulfonate (20.3 g, 1.5 eq), DIPEA (29 g, 5 eq), and HATU (26 g, 1.5 eq). The mixture was stirred, purged with nitrogen, and reacted at room temperature for 4 hours. After the reaction was complete, water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness. The filtrate was purified by silica gel column chromatography (eluent PE:EA = 5:1 to 3:1, V / V) to give intermediate 19 (6 g white solid, 63% yield).

[0220] 1H NMR: (400MHz, DMSO-d6) δppm 9.22(d,J=7.2Hz,1H),5.75-5.80(m,1H),5.73(d,J=7.2Hz,1H),4.96-5.07(m,2H),4. 19(q,J=7.2Hz,2H),2.23-2.30(m,4H),1.21(t,J=7.2Hz,3H); MS(ESI)m / z=211.0[M+H] + .

[0221] Step 2: Preparation of ethyl 5-amino-2-(but-3-en-1-yl)thiazolyl-4-carboxylate (intermediate 20)

[0222] At room temperature, intermediate 19 (10.31 g, 1 eq) and toluene (150 mL) were added to a reaction flask, followed by Lawson's reagent (15.9 g, 0.8 eq). The mixture was stirred, purged with nitrogen, and refluxed overnight. After the reaction was complete, water was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, evaporated to dryness, and purified by silica gel column chromatography (eluent PE:EA = 8:1–5:1, V / V) to give intermediate 20 (5 g of pale yellow solid, 45% yield).

[0223] 1 H NMR: (400MHz, DMSO-d6) δppm 7.24(brs,2H),5.80-5.88(m,1H),4.99-5.11(m,2H),4.19(q,J=7.2Hz,2H),2.81(t,J= 7.6Hz,2H),2.37(td,J=7.6,1.2Hz,2H),1.26(t,J=7.2Hz,3H); MS(ESI)m / z=227.2[M+H] + .

[0224] Step 3: Preparation of ethyl 5-amino-2-{4-[6-(methoxycarbonyl)pyridin-3-yl]but-3-en-1-yl}thiazolyl-4-carboxylate (intermediate 21)

[0225] Intermediate 20 (2.0 g, 1 eq) and DMF (40 mL) were added to a reaction flask. Then, methyl 5-bromopyridine-2-carboxylic acid (3.8 g, 2 eq), tris(o-methylphenyl)phosphine (CAS: 6163-58-2, 0.54 g, 0.2 eq), DIPEA (3.4 g, 3 eq) and Pd(OAc)2 (0.2 g, 0.1 eq) were added to this mixture. Stirring was started, nitrogen was purged, and the mixture was refluxed overnight. After the reaction was complete, water was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with water, dried over anhydrous sodium sulfate, filtered, evaporated to dryness, and purified by silica gel column chromatography (eluent PE:EA = 3:1 to DCM:EA = 1:1, V / V) to give intermediate 21 (1.2 g orange solid, 38% yield).

[0226] 1 H NMR: (400MHz, DMSO-d6) δppm 8.71(s,1H),7.98-8.01(m,2H),7.24(brs,2H),6.58-6.67(m,1H),4.18(q,J=7.2Hz,2H),3.88 (s,3H),2.88-8.95(m,2H),2.50-2.62(m,2H),1.25(t,J=7.2Hz,3H); MS(ESI)m / z=362.3[M+H] + .

[0227] Step 4: Preparation of ethyl 5-amino-2-{4-[6-(methoxycarbonyl)pyridin-3-yl]butyl}thiazolyl-4-carboxylate (intermediate 22)

[0228] Intermediate 21 (2.1 g, 1 eq), Pd(OH)2 (2 g, 2.4 eq) and DMF were added to a reaction flask, stirred, and hydrogen gas was introduced. The mixture was stirred at room temperature for 4 hours. Once the reaction was complete, it was filtered and purified by silica gel column chromatography (eluent PE:EA = 2:1 to DCM:EA = 1:1, V / V) to obtain intermediate 22 (1.8 g of yellow solid, yield 86%).

[0229] 1 H NMR: (400MHz, DMSO-d6) δppm 8.57(d,J=1.6Hz,1H),7.98(d,J=8.0Hz,1H),7.83(dd,J=8.0,1.6Hz,1H),7.23(brs,2H),4.18(q,J=7.2H z,2H),3.86(s,3H),2.66-2.78(m,4H),1.53-1.70(m,4H),1.25(t,J=7.2Hz,3H); MS(ESI)m / z=364.7[M+H] + .

[0230] Step 5: Preparation of ethyl 5-(3-benzoylthiourea)-2-{4-[6-(methoxycarbonyl)pyridin-3-yl]butyl}thiazole-4-carboxylate (intermediate 23)

[0231] Intermediate 22 (2.5 g, 1 eq), benzoyl isothiocyanate (1.68 g, 1.5 eq), and acetone (50 mL) were added to a reaction flask, stirred, purged with nitrogen, and refluxed overnight. After the reaction was complete, the mixture was filtered to obtain intermediate 23 (2.6 g white solid, 72% yield).

[0232] 1 H NMR: (400MHz, DMSO-d6) δppm 14.77(s,1H),12.16(s,1H),8.57(d,J=1.6Hz,1H),7.97-8.01(m,3H),7.8 4(dd,J=8.0,1.6Hz,1H),7.68(t,J=7.6Hz,1H),7.56(dd,J=8.4,7.6Hz,2H) ,4.40(q,J=6.8Hz,2H),3.86(s,3H),2.95(t,J=6.8Hz,2H),2.75(t,J=6.8 Hz,2H),1.61-1.78(m,4H),1.36(t,J=6.8Hz,3H); MS(ESI)m / z=528.0[M+H] + .

[0233] Step 6: Preparation of 5-{[(benzoimino)(methylthio)methyl]amino}-2-{4-[6-(methoxycarbonyl)pyridin-3-yl]butyl}thiazolyl-4-carboxylic acid ethyl ester (intermediate 24)

[0234] Intermediate 23 (2.5 g, 1 eq), NaH (3.1 g, 1.5 eq), and DMF (45 mL) were added to a reaction flask, stirred, and reacted at room temperature for 30 minutes. Then, iodomethane (1.1 g, 1.5 eq) was added, and the reaction was continued at room temperature for 3 hours. After the reaction was complete, the reaction solution was poured into a saturated ammonium chloride solution, water was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered to obtain intermediate 24 (2.7 g of pale yellow solid, 96% yield). MS (ESI) m / z = 541.2 [M+H] + .

[0235] Step 7: Preparation of methyl 5-[4-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)butyl]pyridine-2-carboxylate (intermediate 25)

[0236] Intermediate 24 (1.2 g, 1 eq) and ammonia-methanol solution (30 mL) were added to a reaction flask and refluxed. After the reaction was completed, the crude product was obtained by filtration. The crude product was purified by reverse preparative HPLC (eluent: 5% acetonitrile aqueous solution to 100% acetonitrile, v%) to obtain intermediate 25 (500 mg white solid, 50% yield).

[0237] 1 H NMR: (400MHz, DMSO-d6) δppm 12.47-12.54(br,1H),12.03-12.11(br,1H),8.58(d,J=2.0Hz,1H),8.05(d,J=7.6Hz,2H),7.98(d,J=8.0Hz,1H),7.84(dd,J=8.0,2.0Hz,1H),7. 68(d,J=7.6Hz,1H),7.56(t,J=7.6Hz,2H),3.86(s,3H),3.07(t,J=7.2Hz ,2H),2.76(t,J=7.2Hz,2H),1.68-1.82(m,4H); MS(ESI)m / z=464.4[M+H] + .

[0238] Step 8: Preparation of 5-[4-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)butyl]pyridine-2-carboxylic acid (intermediate 26)

[0239] Intermediate 25 (895 mg, 1 eq), LiOH monohydrate (CAS: 1310-66-3, 406 mg, 5 eq), and MeOH (30 mL) were added to a reaction flask and reacted overnight at room temperature. After the reaction was complete, the mixture was evaporated to dryness, water was added, and the mixture was extracted with ethyl acetate. The pH of the aqueous phase was adjusted to 5–6 with HCl, resulting in the precipitation of a solid. The solid was filtered to obtain intermediate 26 (533 mg white solid, 80% yield).

[0240] 1 H NMR: (400MHz, DMSO-d6) δppm 11.67-11.78(br,1H),8.37(s,1H),7.91(d,J=8.0Hz,1H),7.73(dd,J=8.0,1.2Hz,1H),7.05(br s,2H),2.91(t,J=6.4Hz,2H),2.69(t,J=6.4Hz,2H),1.60-1.77(m,4H); MS(ESI)m / z=346.0[M+H] + .

[0241] Step 9: Preparation of (S)-2-{5-[4-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)butyl]pyridine-2-carboxamido}-3-[4-(tert-butoxy)phenyl]propionate tert-butyl ester (intermediate 27)

[0242] Intermediate 26 (90 mg, 1.0 eq), DMSO (5 mL), HATU (130 mg, 1.3 eq), DIPEA (100 mg, 3.0 eq), and O-tert-butyl-L-tyrosine tert-butyl hydrochloride (105 mg, 1.2 eq) were added sequentially to the reaction flask. The mixture was purged with nitrogen three times and reacted at 25 °C for 2 h. The reaction was monitored by LC-MS until completion. The reaction solution was poured into water (30 mL), and a solid precipitated. This solid was centrifuged, filtered, and lyophilized to obtain intermediate 27 (150 mg solid, 92.6% yield). MS (ESI) m / z = 621.0 [M+H] + .

[0243] Step 10: Preparation of 4-{2-[(E)-2-((E)-2-[4-((S)-2-{5-[4-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)butyl]pyridine-2-carboxyethyl)phenoxy]-3-{2-[(E)-3,3-dimethyl-5-sulfonyl-1-(4-sulfonylbutyl)indol-2-ylidene]ethenyl}cyclohex-1-en-1-yl)vinyl]-3,3-dimethyl-5-sulfonyl-3H-indol-1-on-1-yl}butyl-1-sulfonic acid inner salt (compound 4)

[0244] Intermediate 27 (150 mg, 1.0 eq), TFA (5 mL), and water (0.05 mL) were added sequentially to the reaction flask, and the reaction was carried out at 25 °C for 2 h. The reaction was monitored by LC-MS until the reaction was complete. The reaction solution was then added dropwise to MTBE (40 mL), and a solid precipitated. The solid was centrifuged, filtered, and the filter cake was concentrated and dried under vacuum for later use.

[0245] Add water (5 mL) directly to the solid obtained in the previous step, adjust the pH to 11 with 2M sodium hydroxide, add SO456 (215 mg), and heat to 90 °C for reaction. Monitor the reaction using LCMS. If intermediate 27 has not reacted completely, add SO456, adjust the pH to 11 again, and continue heating to 90 °C until the starting material has basically reacted. After filtration, prepare HPLC separation (column: Welch xbidge xb 5um 21.2*250 mm; mobile phase: [aqueous phase (TFA 0.1 v / v%) - acetonitrile]; ACN v / v%: 10%-35%, gradient time 18 min), and lyophilize to obtain compound 4 (100 mg, yield 30.4%).

[0246] 1 H NMR: (400MHz, DMSO-d6) δppm 8.68(d,J=8.0Hz,1H),8.43(s,1H),7.93(d,J=8.0Hz,1H),7.89(d,J=8.0Hz,1H),7.72(d,J=14.0Hz,2H),7.62(d,J=8. 4Hz,2H),7.55(s,2H),7.32(d,J=8.4Hz,2H),7.26(d,J=8.4Hz,2H),7.01(d,J=8.4Hz,2H),6.18(d,J=14.0Hz,2H),4.6 9(dd,J=13.2,8.4Hz,1H),4.10(brs,4H),3.17-3.25(m,1H),3.08-3.16(m,1H),2.91(t,J=7.2Hz,2H),2.57-2.74(m,1 0H),1.91(t,J=6.0Hz,2H),1.60-1.80(m,10H),1.49-1.58(m,2H),1.16(s,6H),1.08(s,6H); MS(ESI)m / z=680.0[M+2H] 2+ .

[0247] Example 5: Preparation of 4-{2-[(E)-2-((E)-2-[4-((S)-2-{4-[4-(2-amino-4-oxo-3,4-dihydrothiopheno[2,3-d]pyrimidin-6-yl)butyl]bicyclo[2.2.2]octane-1-carboxamido}-2-carboxyethyl)phenoxy]-3-{2-[(E)-3,3-dimethyl-5-sulfonyl-1-(4-sulfonyl)indol-2-ylidene]ethenyl}cyclohex-1-en-1-yl)vinyl]-3,3-dimethyl-5-sulfonyl-3H-indol-1-on-1-yl}butyl-1-sulfonic acid inner salt (compound 5)

[0248] Step 1: Preparation of methyl 4-(hydroxymethyl)bicyclo[2.2.2]octane-1-carboxylate (intermediate 28)

[0249] 4-(methoxycarbonyl)bicyclo[2.2.2]octane-1-carboxylic acid (CAS: 18720-35-9, 10.3 g, 47.1 mmol, 1.0 eq) was dissolved in tetrahydrofuran (100 mL). Borane dimethyl sulfide (CAS: 13292-87-0, 10.7 g, 141.4 mmol, 3.0 eq) was added at 0 °C. The reaction mixture was stirred at 0 °C for two hours. After the reaction was monitored, methanol (80 mL) was slowly added to quench the reaction, and the mixture was evaporated to dryness under vacuum to obtain crude intermediate 28 (9.30 g, yellow oily liquid, yield 99%), which was used directly in the next reaction without purification. MS (ESI) m / z = 199.1 [M+H] + .

[0250] Step 2: Preparation of methyl 4-formylbicyclo[2.2.2]octane-1-carboxylate (intermediate 29)

[0251] Intermediate 28 (13.4 g, 67.6 mmol, 1.0 eq) was dissolved in dichloromethane (600 mL), and Dess-Martin reagent (CAS: 87413-09-0, 44.3 g, 101.4 mmol, 1.5 eq) was slowly added. The reaction mixture was stirred at room temperature for 12 hours. After the reaction was monitored, sodium bicarbonate solution was slowly added at 0 °C to quench the reaction. Then, dichloromethane and water were added for extraction, and the organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under vacuum, stirred with silica gel, and purified by column chromatography (eluting PE / EA = 14:1, V / V) to give intermediate 29 (8.30 g, white solid, yield 63%). MS (ESI) m / z = 197.3 [M+H] + .

[0252] Step 3: Preparation of 6-{4-(methoxycarbonyl)bicyclo[2.2.2]octane-1-yl}hex-5-enoic acid (intermediate 30)

[0253] 4-(carboxybutyl)triphenylphosphine bromide (CAS: 17814-85-6, 31.1 g, 68.8 mmol, 1.5 eq) was dissolved in tetrahydrofuran (120 mL). Potassium tert-butoxide (15.4 g, 137.6 mmol, 3.0 eq) was slowly added at 0 °C. The mixture was stirred at 0 °C for half an hour, and then a tetrahydrofuran solution of intermediate 29 (9.00 g, 45.9 mmol, 1.0 eq) was slowly added. The reaction mixture was stirred at room temperature for 12 hours. After the reaction was monitored, ethyl acetate and water were added for extraction. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under vacuum, stirred with silica gel, and purified by column chromatography (eluent PE / EA = 3:1, V / V) to give intermediate 30 (9.00 g, yellow oily liquid, yield 70%). MS(ESI)m / z = 281.2[M+H] + .

[0254] Step 4: Preparation of 6-{4-(methoxycarbonyl)bicyclo[2.2.2]octane-1-yl}hexanoic acid (intermediate 31)

[0255] Intermediate 30 (9.00 g, 32.1 mmol, 1.0 eq) was dissolved in methanol (40 mL), and palladium on carbon (6.83 g, 3.21 mmol, 0.1 eq) was added. The mixture was purged three times with hydrogen, and stirred at room temperature for 12 hours. After the reaction was monitored, the reaction solution was filtered through diatomaceous earth, and the filtrate was evaporated to dryness under vacuum to obtain crude intermediate 31 (9.00 g, yellow oily liquid, yield 99%), which was used directly in the next reaction without purification. MS (ESI) m / z = 283.2 [M+H] + .

[0256] Step 5: Preparation of methyl 4-(6-hydroxyhexyl)bicyclo[2.2.2]octane-1-carboxylate (intermediate 32)

[0257] Intermediate 31 (9.00 g, 31.9 mmol, 1.0 eq) was dissolved in tetrahydrofuran (150 mL), and borane dimethyl sulfide (9.68 g, 127.5 mmol, 4.0 eq) was added. The reaction mixture was stirred at room temperature for 12 hours. After the reaction was monitored, methanol was slowly added to quench the reaction. The mixture was then evaporated to dryness under vacuum to obtain crude intermediate 32 (8.50 g, yellow oily liquid, yield 99%), which was used directly in the next reaction without purification. MS (ESI) m / z = 269.2 [M+H] + .

[0258] Step 6: Preparation of methyl 4-(6-oxohexyl)bicyclo[2.2.2]octane-1-carboxylate (intermediate 33)

[0259] Intermediate 32 (9.00 g, 33.5 mmol, 1.0 eq) was dissolved in dichloromethane (300 mL), and Dysmart reagent (21.8 g, 50.3 mmol, 1.5 eq) was slowly added. The reaction mixture was stirred at room temperature for 12 hours. After the reaction was monitored, sodium bicarbonate solution was slowly added at 0 °C to quench the reaction. Then, dichloromethane and water were added for extraction, and the organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under vacuum, mixed with silica gel, and purified by column chromatography (eluting PE / EA = 30:1, V / V) to give intermediate 33 (5.00 g, yellow oily liquid, yield 56%). MS (ESI) m / z = 267.3 [M+H] + .

[0260] Step 7: Preparation of methyl 2-amino-5-(4-{4-(methoxycarbonyl)bicyclo[2.2.2]octane-1-yl}butyl)thiophene-3-carboxylate (intermediate 34)

[0261] Intermediate 33 (4.35 g, 16.3 mmol, 1.0 eq) and methyl 2-cyanoacetate [CAS: 105-34-0] (1.70 g, 16.3 mmol, 1.0 eq) were dissolved in methanol (150 mL). Triethylamine (1.67 g, 16.3 mmol, 1.0 eq) and sulfur (526 mg, 2.04 mmol, 0.125 eq) were added. The reaction mixture was stirred at 70 °C for 2 hours. After the reaction was completed, the reaction mixture was evaporated to dryness under vacuum, mixed with silica gel, and purified by column chromatography (eluent PE:EA = 30:1–10:1, V / V) to give intermediate 34 (4.4 g, yellow solid, yield 71%). MS (ESI) m / z = 380.2 [M+H] + .

[0262] Step 8: Preparation of methyl 4-[4-(2-amino-4-oxo-3,4-dihydrothiopheno[2,3-d]pyrimidin-6-yl)butyl]bicyclo[2.2.2]octane-1-carboxylate (intermediate 35)

[0263] Intermediate 34 (3.20 g, 8.43 mmol, 1.0 eq), chloroformamidine hydrochloride (2.50 g, 21.1 mmol, 2.5 eq), and dimethyl sulfone (4.01 g, 42.16 mmol, 5.0 eq) were added sequentially to a reaction flask, and the mixture was stirred at 130 °C for 2 hours. After cooling to room temperature, ammonia water (30 mL) was added, and the mixture was stirred for another hour. After the reaction was completed, the reaction solution was filtered, and the filter cake was dried under vacuum to obtain crude intermediate 35 (3.28 g, white solid, yield 99%).

[0264] 1 H NMR: (400MHz, DMSO-d6) δppm 8.54(brs,1H),6.77(s,1H),6.49(brs,2H),3.55(s,3H),2.68(t,J=7.2Hz,2H),1.61-1.71(m,6H),1.5 2(qui,J=7.2Hz,2H),1.27-1.37(m,6H),1.15-1.25(m,2H),1.05-1.11(m,2H); MS(ESI)m / z=390.2[M+H] + .

[0265] Step 9: Preparation of 4-[4-(2-amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-yl)butyl]bicyclo[2.2.2]octane-1-carboxylic acid (intermediate 36)

[0266] Intermediate 35 (2.00 g, 5.13 mmol, 1.0 eq) was dissolved in tetrahydrofuran (20 mL) and water (10 mL). Lithium hydroxide monohydrate (1.10 g, 25.67 mmol, 5.0 eq) was slowly added at 0 °C. The reaction mixture was stirred at 25 °C for 16 hours. After the reaction was monitored, water (100 mL) was added, and the pH of the mixture was adjusted to 4 using 6 M hydrochloric acid. Ethyl acetate was then added for extraction, and the organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under vacuum, and ethanol (50 mL) was added, followed by stirring at room temperature for half an hour. The precipitated solid was collected by filtration and dried under vacuum to give compound 10 (1.10 g, off-white solid, yield 57%, purity 96%).

[0267] 1H NMR: (400MHz, DMSO-d6) δppm 11.94(brs,1H),11.03(brs,1H),6.77(s,1H),6.55(brs,2H),2.68(t,J=7.2Hz,2H),1.58-1.69(m,6H),1 .51(qui,J=7.2Hz,2H),1.27-1.38(m,6H),1.13-1.26(m,2H),1.03-1.12(m,2H); MS(ESI)m / z=376.1[M+H] + .

[0268] Step 10: Preparation of (S)-2-{4-[4-(2-amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-yl)butyl]bicyclo[2.2.2]octane-1-carbamate}-3-[4-(tert-butoxy)phenyl]propionate tert-butyl ester (intermediate 37)

[0269] Intermediate 36 (95 mg, 1.0 eq), DMSO (3 mL), HATU (140 mg, 1.3 eq), DIPEA (150 mg, 3.0 eq), and O-tert-butyl-L-tyrosine tert-butyl hydrochloride (140 mg, 1.2 eq) were added sequentially to the reaction flask. The mixture was purged with nitrogen three times and reacted at 25°C for 2 h. The reaction was monitored by LC-MS until completion. The reaction solution was poured into water (30 mL), resulting in the precipitation of a viscous solid. This solid was centrifuged, filtered, and lyophilized to obtain intermediate 37 (240 mg crude product, 145% yield), which was used directly in the next reaction without purification. MS (ESI) m / z = 651.0 [M+H] + .

[0270] Step 11: Preparation of 4-{2-[(E)-2-((E)-2-[4-((S)-2-{4-[4-(2-amino-4-oxo-3,4-dihydrothiopheno[2,3-d]pyrimidin-6-yl)butyl]bicyclo[2.2.2]octane-1-carboxamido}-2-carboxyethyl)phenoxy]-3-{2-[(E)-3,3-dimethyl-5-sulfonyl-1-(4-sulfonyl)indol-2-ylidene]ethenyl}cyclohex-1-en-1-yl)vinyl]-3,3-dimethyl-5-sulfonyl-3H-indol-1-on-1-yl}butyl-1-sulfonic acid inner salt (compound 5)

[0271] Add the crude intermediate 37 (240 mg, 1.0 eq) obtained in the previous step, TFA (3 mL), and 2 drops of water to the reaction flask sequentially, and react at 25°C for 2 h. Monitor the reaction using LCMS until it is complete. Add the reaction solution dropwise to MTBE (40 mL), and a solid will precipitate. Centrifuge and filter, then concentrate and dry the filter cake under vacuum for later use.

[0272] Add water (5 mL) directly to the solid obtained in the previous step, adjust the pH to 11 with 2M sodium hydroxide, add SO456 (420 mg), and heat to 90 °C for reaction. Monitor the reaction using LCMS. If intermediate 37 has not reacted completely, add more SO456, adjust the pH to 11 again, and continue heating to 90 °C until the starting material has basically reacted. After filtration, the reaction solution is separated by HPLC (column: Welch xbidge xb 5um 21.2*250mm; mobile phase: [aqueous phase (TFA 0.1 v / v%) - acetonitrile]; ACN v / v%: 10%-35%, gradient time 18 min), and lyophilize to obtain compound 5 (100 mg, yield 30.4%).

[0273] 1 H NMR: (400MHz, DMSO-d6) δppm 7.79(d,J=14.0Hz,2H),7.58-7.64(m,4H),7.34(d,J=8.0Hz,2H),7.21(d,J=8.4Hz,2H),7.11(d,J=8.0Hz,1H ),7.04(d,J=8.4Hz,2H),6.90(s,1H),6.21(d,J=14.0Hz,2H),4.32(dd,J=13.6,8.4Hz,1H),4.13(brs,4H),2. 95-3.03(m,1H),2.85-2.93(m,1H),2.70-2.78(m,6H),2.61(t,J=6.8Hz,4H),1.91(t,J=6.0Hz,2H),1.76(brs ,8H),1.46-1.56(m,8H),1.19-1.30(m,18H),1.09-1.18(m,2H),1.01-1.08(m,2H); MS(ESI)m / z=1390.2[M+H] + 695.8 [M+2H] 2+ .

[0274] Example 6: Preparation of 4-{2-[(E)-2-((E)-2-[4-((S)-2-{4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)propyl]bicyclo[2.2.2]octane-1-carboxamido}-2-carboxyethyl)phenoxy]-3-{2-[(E)-3,3-dimethyl-5-sulfonyl-1-(4-sulfonylbutyl)indol-2-ylidene]ethenyl}cyclohex-1-en-1-yl)vinyl]-3,3-dimethyl-5-sulfonyl-3H-indol-1-on-1-yl}butyl-1-sulfonic acid inner salt (compound 6)

[0275] Step 1: Preparation of 4-{4-(methoxycarbonyl)bicyclo[2.2.2]octane-1-yl}but-3-enoic acid (intermediate 38)

[0276] THF (400 mL, 10 V) was added to solid 2-carboxyethyltriphenylphosphine bromide (CAS: 51114-94-4, 71.0 g, 1.1 eq.), stirred until homogeneous, and purged with nitrogen. The mixture was cooled to -10 °C, and NaHMDS (CAS: 1070-89-9, 216 mL, 2.1 eq.) was added dropwise over 30 min. The reaction mixture was then maintained at -10 °C for 1 h. The reaction system was cooled to -60 °C to -55 °C. Intermediate 29 (40.0 g, 1.0 eq.) was dissolved in THF (120 mL, 3 V), and the THF solution of intermediate 29 was added dropwise over 25 min. The reaction system was then heated to -10 °C and the reaction was continued for 1 h. TLC monitoring confirmed complete conversion of the starting material. The reaction was quenched by adding saturated NH4Cl solution (200 mL, 5 V), and the pH was adjusted to 6 by adding 1 N HCl. Extraction and separation were performed by adding EA (480 mL, 12 V). The aqueous phase was extracted once with EA (120 mL, 3 V). The organic phases were combined and washed once with saturated NaCl solution (320 mL, 8 V). The organic phase was dried over Na2SO4 and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (eluent PE / EA = 10:1 to 2:1, V / V) to give intermediate 38 as a colorless liquid (32.6 g, yield 63%).

[0277] 1 H NMR: (400MHz, DMSO-d6) δppm 12.19(brs,1H),5.36(dt,J=12.0,6.0Hz,1H),5.24(d,J=12.0Hz,1H),3. 56(s,3H),3.14(dd,J=6.0,1.2Hz,2H),1.67-1.75(m,6H),1.55-65(m,6H)

[0278] Step 2: Preparation of 4-{4-(methoxycarbonyl)bicyclo[2.2.2]octane-1-yl}butyric acid (intermediate 39)

[0279] Intermediate 38 (32.5 g, 1.0 eq.) and MeOH (320 mL, 10 V) were added sequentially to the reaction flask, stirred thoroughly, and purged with nitrogen three times. Wet Pd / C (CAS: 7440-05-3, 4.8 g, 15% W / W water content) with a loading of 10 wt% was added, and the mixture was purged with hydrogen three times. The reaction was carried out at 25 °C for 3–4 h. LC-MS monitoring showed complete conversion of the starting material. The reaction solution was filtered through diatomaceous earth, the filter cake was washed with methanol, and the combined filtrates were concentrated under reduced pressure to obtain a white solid intermediate 39 (29.6 g, molar yield: 90%). MS (ESI) m / z = 255 [M+H] + .

[0280] Step 3: Preparation of 4-{4-[(1-cyano-2-ethoxy-2-oxoethyl)amino]-4-oxobutyl}bicyclo[2.2.2]octane-1-carboxylic acid methyl ester (intermediate 40)

[0281] Intermediate 39 (13.5 g, 1.0 eq.), ethyl aminocyanoacetate p-toluenesulfonate (17.5 g, 1.1 eq.), ACN (135 mL, 10 V), and TCFH (CAS: 94790-35-9, 17.7 g, 1.2 eq.) were added sequentially to the reaction flask. The reaction system was cooled to 0-10 °C, and NMI (CAS: 616-47-7, 15.2 g, 3.5 eq.) was added dropwise over approximately 20 minutes. The temperature was then raised to 20-30 °C, and the reaction was continued for 3-4 hours. LC-MS monitoring confirmed complete conversion of the starting materials. H2O (1.0 L, 10 V) and EA (1.0 L, 10 V) were added to the reaction solution, and the mixture was stirred, extracted, and separated. The aqueous phase was extracted with EA (55 mL, 4 V). The EA phases were combined, washed twice with 1N HCl aqueous solution (135 mL * 2, 10 V), once with saturated NaHCO3 solution (68 mL, 5 V), and once with saturated NaCl solution (68 mL, 5 V). The organic phase was dried with Na2SO4, filtered, and the filtrate was concentrated under reduced pressure and purified by slurrying with petroleum ether to give intermediate 40 (white solid, 17.0 g, yield: 87.8%).

[0282] 1H NMR: (400MHz, CDCl3) δppm 6.26(d,J=8.0Hz,1H),5.53(d,J=8.0Hz,1H),4.36(m,2H),3.63(s,3H),2.25(t,J=8.0Hz,2H),1. 74-1.78(m,6H),1.55-1.59(m,2H),1.35-1.41(m,9H),1.11-1.15(m,2H); MS(ESI)m / z=365[M+H] + .

[0283] Step 4: Preparation of ethyl 5-amino-2-{3-[4-(methoxycarbonyl)bicyclo[2.2.2]octane-1-yl]propyl}thiazolyl-4-carboxylate (intermediate 41)

[0284] Intermediate 40 (16.5 g, 1.0 eq.), toluene (165 mL, 10 V), and Lawson's reagent (11.0 g, 0.6 eq.) were added to the reaction flask, stirred thoroughly, and purged with nitrogen. The reaction was heated to 65 °C and reacted for 16 h. LC-MS was used to monitor the reaction, and the starting material was completely converted. H2O (1.0 L, 10 V) was added to the reaction solution, and the mixture was extracted and separated. The solution was washed twice with saturated NaHCO3 solution (165 mL * 2, 10 V) and once with saturated NaCl solution (85 mL, 5 V). The organic phase was dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (eluent PE / EA = 20:1 to 1:1, V / V) to obtain intermediate 41 (7.5 g white solid, yield: 43%).

[0285] 1 H NMR: (400MHz, DMSO-d6) δppm 7.21(brs,2H),4.19(q,J=8.0Hz,2H),3.55(s,3H),2.64(t,J=8.0Hz,2H),1.65-1.69(m,6H),1. 43-1.55(m,2H),1.32-1.36(m,6H),1.23-1.27(m,5H),1.08-1.14(m,2H); MS(ESI)m / z=381[M+H] + .

[0286] Step 5: Preparation of methyl 4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)propyl]bicyclo[2.2.2]octane-1-carboxylate (intermediate 42)

[0287] Intermediate 41 (8.0 g, 1.0 eq), DME (80 mL, 10.0 V), and chloroformamidine hydrochloride (7.2 g, 3.0 eq.) were added sequentially to the reaction flask. Nitrogen was purged three times, and the mixture was heated to 160 °C for 3-4 h. LC-MS was used for monitoring, and the starting material was completely converted. The mixture was cooled to 20-30 °C, filtered, and the filter cake was collected. Water (240 mL, 30 V) was added to the mother liquor, precipitating a viscous solid. The liquid was decanted, and the solids were combined. After dissolving in DMF, the mixture was purified by HPLC (column: Welch XB-C18 5 μm 50*250 mm; mobile phase: [aqueous phase (TFA 0.1 v / v%) - acetonitrile]; ACN v / v%: 20%-60%, gradient time 16 min) to obtain intermediate 42 (1.7 g pale yellow solid, yield: 21%).

[0288] 1 H NMR: (400MHz, DMSO-d6) δppm 11.04(s,1H),6.65(brs,2H),2.82(t,J=7.6Hz,2H),1.61-1.69(m,8H),1.32-1.39(m,6H),1.10-1.18(m,2H); MS(ESI)m / z=377[M+H] + .

[0289] Step 6: Preparation of 4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)propyl]bicyclo[2.2.2]octane-1-carboxylic acid (intermediate 43)

[0290] Add intermediate 42 (1.7 g, 1.0 eq.), MeOH (10 mL, 5.0 V), water (17 mL, 10.0 V), and NaOH (720 mg, 4.0 eq.) sequentially to the reaction flask, and stir until dissolved. React at 20-30℃ for 3-4 h. Monitor the reaction mixture by LC-MS; the starting material conversion was complete. Concentrate the reaction solution under reduced pressure to 10 V. Maintain the temperature at 0-10℃, adjust the pH to 3 with 1N HCl, and a solid precipitates. Filter the solution, wash the filter cake with water, and concentrate under reduced pressure with methanol until no more liquid drips out, yielding intermediate 43 (1.6 g pale yellow solid, yield: 97%).

[0291] 1 H NMR: (400MHz, DMSO-d6) δppm 11.92(s,1H),11.07(s,1H),6.67(brs,2H),2.82(t,J=7.6Hz,2H),1.62- 1.70(m,8H),1.31-1.40(m,6H),1.06-1.14(m,2H); MS(ESI)m / z=363[M+H]+ .

[0292] Step 7: Preparation of (S)-2-{4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)propyl]bicyclo[2.2.2]octane-1-carbamate}-3-[4-(tert-butoxy)phenyl]propionate tert-butyl ester (intermediate 44)

[0293] Intermediate 43 (200 mg, 1.0 eq.), DMSO (2 mL), HATU (271.8 mg, 1.3 eq.), DIPEA (212.8 mg, 3.0 eq.), and O-tert-butyl-L-tyrosine tert-butyl hydrochloride (199.6 mg, 1.1 eq.) were added sequentially to the reaction flask. The mixture was purged with nitrogen three times and reacted at room temperature for 2 h. LC-MS was used for monitoring until the starting material was completely converted. The reaction solution was then added to H₂O (6 mL), stirred, and a solid precipitated. The solid was filtered to obtain intermediate 44 (300 mg, 85% yield), which was used directly in the next reaction without purification. MS (ESI) m / z = 638 [M+H] + .

[0294] Step 8: Preparation of 4-{2-[(E)-2-((E)-2-[4-((S)-2-{4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)propyl]bicyclo[2.2.2]octane-1-carboxamido}-2-carboxyethyl)phenoxy]-3-{2-[(E)-3,3-dimethyl-5-sulfonyl-1-(4-sulfonylbutyl)indol-2-ylidene]ethenyl}cyclohex-1-en-1-yl)vinyl]-3,3-dimethyl-5-sulfonyl-3H-indol-1-on-1-yl}butyl-1-sulfonic acid inner salt (compound 6)

[0295] Intermediate 44 (300 mg, 1.0 eq.), TFA (5 mL), and water (0.05 mL) were added sequentially to the reaction flask, and the reaction was carried out at 25 °C for 3 h. The reaction mixture was monitored by LC-MS until the starting material was completely converted. The reaction solution was concentrated under vacuum to remove TFA, and water (5 mL) was added directly. The pH was adjusted to 11 with 2 M sodium hydroxide.

[0296] SO456 was added to the reaction system, and the temperature was raised to 90℃ for 1 hour. LCMS monitoring showed incomplete conversion of the reactants, so SO456 was added again, the pH was adjusted to 11, and the temperature was raised to 90℃ for another hour. LCMS monitoring showed that the reactants had largely reacted. The reaction solution was filtered and purified by HPLC (column: Welch XBridge C18 250*21mm; mobile phase: [aqueous phase (TFA 0.1 v / v%) - acetonitrile]; ACN v / v%: 5%-40%, gradient time 13 min) to obtain compound 6 (208.0 mg, yield: 32%).

[0297] 1 H NMR: (400MHz, DMSO-d6) δppm 7.82(d,J=14.0Hz,2H),7.63(d,J=8.4Hz,4H),7.34(d,J=8.4Hz,2H),7.23(d,J=8.4Hz,2H),7.06( d,J=8.4Hz,2H),6.22(d,J=14.0Hz,2H),4.31(dd,J=14.0,8.0Hz,1H),4.13(brs,4H),2.94-3.03(m ,1H),2.88-2.93(m,1H),2.84(t,J=7.2Hz,2H),2.72(brs,4H),2.60(t,J=6.8Hz,4H),1.93(t,J=6. 8Hz,2H),1.76(s,8H),1.48-1.59(m,8H),1.21-1.32(m,18H),1.09(m,2H); MS(ESI)m / z=689[M+2H] 2+ .

[0298] Example 7: Preparation of 4-{2-[(E)-2-((E)-2-[4-((S)-2-{4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)propyl]-2-fluorobenzamido}-2-carboxyethyl)phenoxy]-3-{2-[(E)-3,3-dimethyl-5-sulfonyl-1-(4-sulfonylbutyl)indol-2-ylidene]ethenyl}cyclohex-1-en-1-yl)vinyl]-3,3-dimethyl-5-sulfonyl-3H-indol-1-on-1-yl}butyl-1-sulfonic acid inner salt (compound 7)

[0299] Step 1: Preparation of methyl 2-fluoro-4-(4-oxo-n-butyl)benzoate (intermediate 45)

[0300] At room temperature, weigh out 25 g (1.0 eq) of methyl 2-fluoro-4-bromobenzoate [CAS No.: 179232-29-2], 11.08 mL (1.2 eq) of but-3-en-1-ol [CAS No.: 627-27-0], 0.5 g (14.91 eq) of Bu4NCl, 0.5 eq of LiCl, 0.5 g (4.55 eq) of LiOAc, 2.5 g (17.70 g) of LiOAc, and 0.06 g (1.44 g) of palladium acetate, and add them to a 500 mL single-necked flask. Then add 130 mL of DMF. Replace with N2 and heat the reaction system to an internal temperature of 70 °C for 4 hours. The reaction was monitored by TLC until complete (PE:EA = 5:1). The reaction solution was cooled to room temperature and poured into a semi-saturated brine solution (1 L). It was extracted twice with ethyl acetate (1 L). The organic phases were combined and washed separately with semi-saturated brine (1 L) and saturated brine (1 L). The mixture was dried over anhydrous sodium sulfate, the drying agent was filtered off, and the product was concentrated to dryness by rotary evaporation. The crude product was purified by column chromatography (100-200 mesh silica gel, mobile phase PE / EA, from 20:1 to 15:1 to 10:1 V / V) to give 31.1 g of a yellow liquid product, yield 72%.

[0301] 1 H NMR: (400MHz, CDCl3) δppm 9.78(t,J=2.0Hz,1H),7.86(d,J=8.0Hz,1H),7.02(dd,J=8.0,2.8Hz,1H),6.96(dd,J=12.0,2 .8Hz,1H),3.92(s,3H),2.69(t,J=7.6Hz,2H),2.49(td,J=7.6,2.0Hz,2H),1.93-2.00(m,2H).

[0302] Step 2: Preparation of 4-(3-fluoro-4-methoxycarbonyl)phenylbutyric acid (intermediate 46)

[0303] At room temperature, intermediate 45 (31.1 g, 1.0 eq) was dissolved in a mixture of 250 mL tert-butanol and 65 mL water. Then, 2-methyl-2-butene (26 mL, 4.4 eq) and sodium dihydrogen phosphate monohydrate (19.14 g, 1.0 eq) were added sequentially. Finally, 42.65 g (3.4 eq) of sodium chlorite was weighed and added in 6 batches. The reaction was stirred at room temperature for 2 h. The reaction was monitored by TLC until the starting material was completely converted (PE / EA, 1:2 V / V). After the reaction was completed, the pH was adjusted to 2-3 with dilute hydrochloric acid (2 M). The aqueous phase was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered to remove the desiccant, and concentrated by rotary evaporation to obtain the crude product. The crude product was added to a sodium carbonate solution (2.0 N, 500 mL), stirred for 30 min, and then extracted with ethyl acetate (300 mL) to remove impurities. The aqueous phase was adjusted to pH 2-3 with dilute hydrochloric acid (2 M), and then extracted twice with dichloromethane (500 mL). The organic phases were collected and combined, washed with saturated brine (250 mL), dried with 100 g of anhydrous sodium sulfate, and evaporated to dryness to obtain 30 g of white solid (yield 90%, purity 99%).

[0304] 1 H NMR: (400MHz, DMSO-d6) δppm 7.81(d,J=8.0Hz,1H),7.20(dd,J=12.0,2.8Hz,1H),7.16(dd,J=8.0,2.8Hz,1H ), 3.83 (s, 3H), 2.66 (t, J = 7.6Hz, 2H), 2.22 (t, J = 7.6Hz, 2H), 1.77-1.85 (m, 2H).

[0305] Step 3: Preparation of methyl 4-{4-[(1-cyano-2-ethoxy-2-oxoethyl)amino]-4-oxobutyl}-2-fluorobenzoate (intermediate 47)

[0306] At room temperature, intermediate 46 (23 g, 1.0 eq) and p-toluenesulfonate of ethyl aminocyanoacetate (34.5 g, 1.2 eq) were dissolved in 300 mL of N,N-dimethylformamide, purged with nitrogen, and then cooled to 0 °C. N,N-diisopropylethylamine (133 mL, 8.0 eq) was measured and added dropwise to the reaction mixture using a constant pressure dropping funnel. Immediately afterwards, N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (72.76 g, 2.0 eq) was added in multiple portions, maintaining a nitrogen atmosphere and ensuring the internal temperature did not exceed 5 °C. After the addition was complete, the ice bath was removed, and the mixture was slowly brought to room temperature. The reaction was stirred for 30 min, and monitored by LC-MS (254 nm) until the starting material disappeared. The reaction solution was poured into 1 L of ice water and extracted three times with ethyl acetate (400 mL x 3). The organic phases were combined and washed twice with semi-saturated brine (500 mL x 2), followed by one wash with 750 mL of saturated brine. The mixture was dried over 100 g of anhydrous sodium sulfate, filtered to remove the drying agent, and concentrated by rotary evaporation to obtain the crude product. The crude product was purified by column chromatography (mobile phase PE / EA, 3:1 V / V) to give intermediate 47 (17 g off-white solid, yield 42%, purity 91.6%).

[0307] 1 H NMR: (400MHz, CDCl3) δppm 7.86(d,J=8.0Hz,1H),7.03(dd,J=8.0,2.8Hz,1H),6.97(dd,J=12.0,2.8Hz,1H),6.31(d,J=7.2Hz,1H),5.32(d,J=7.2Hz,1H ), 4.36 (q, J = 6.8Hz, 2H), 3.92 (s, 3H), 2.71 (t, J = 7.6Hz, 2H), 2.32 (t, J = 7.6Hz, 2H), 1.98-2.06 (m, 2H), 1.37 (t, J = 6.8Hz, 3H).

[0308] Step 4: Preparation of ethyl 5-amino-2-{3-[4-(methoxycarbonyl)-3-fluorophenyl]propyl}thiazole-4-carboxylate (intermediate 48)

[0309] Intermediate 47 (14 g, 1 eq) was dissolved in toluene (140 mL), and then Lawesson's reagent (8.89 g, 0.55 eq) was added. The mixture was heated to 80 °C and stirred overnight, monitored by TLC (DCM / EA, 5:1 V / V) until the starting material disappeared. After the reaction was complete, the reaction solution was evaporated to dryness. Water (200 mL) and ethyl acetate (100 mL) were added to the crude product, and the organic phase was collected after separation. The aqueous phase was extracted with 100 mL of ethyl acetate. The organic phases were combined, washed with 300 mL of saturated brine, dried over 20 g of anhydrous sodium sulfate, filtered to remove the drying agent, concentrated by rotary evaporation, and the resulting solid was purified by silica gel column chromatography (mobile phase DCM / EA, 40:1 to 10:1 V / V) to obtain the crude product. The crude product was purified by C18 reversed-phase column chromatography (acetonitrile, 0-51%) to obtain intermediate 48 (3.2 g off-white solid, yield 22%, purity 99.52%).

[0310] 1 H NMR: (400MHz, CDCl3) δppm 7.81(d,J=8.0Hz,1H),7.21-7.27(m,3H),7.19(dd,J=8.0,2.8Hz,1H),4.18(q,J=6.8Hz,2H),3.8 3(s,3H),2.68-2.74(m,4H),1.89-1.66(m,2H),1.24(t,J=6.8Hz,3H); MS(ESI)m / z=367.05[M+H] + .

[0311] Step 5: Preparation of ethyl 5-(3-benzoylthiourea)-2-{3-[3-fluoro-4-(methoxycarbonyl)phenyl]propyl}thiazole-4-carboxylate (intermediate 49)

[0312] Intermediate 48 (1.67 g, 1 eq) was dissolved in acetone (60 mL), and then benzoyl isothiocyanate (0.74 mL, 1.2 eq) was added. Nitrogen gas was purged three times, and the reaction was heated to reflux and stirred for 16 h. The reaction was monitored by LCMS (254 nm) until the starting material disappeared. The reaction solution was evaporated to dryness, purified by column chromatography (eluent PE:EA = 4:1 to 2:1, V / V), and concentrated to give intermediate 49 (4.26 g pale yellow solid, yield 93%).

[0313] 1H NMR: (400MHz, DMSO-d6)δppm 14.77(s,1H),12.16(s,1H),8.01(dd,J=6.8,1.6Hz,2H),7.81(t,J=6.8Hz,1 H),7.66-7.70(m,1H),7.55(t,J=6.8Hz,2H),7.25(dd,J=12.4,2.8Hz,1H),7. 20(dd,J=8.4,2.8Hz,1H),4.40(q,J=7.2Hz,2H),3.84(s,3H),2.93(t,J=7.6H z, 2H), 2.76 (t, J = 7.6Hz, 2H), 2.04 (qui, J = 7.6Hz, 2H), 1.34 (t, J = 7.2Hz, 3H).

[0314] Step 6: Preparation of ethyl 4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)propyl]-2-fluorobenzoate (intermediate 50)

[0315] At room temperature, intermediate 49 (4.26 g, 1 eq) was dissolved in DMF (42 mL), cooled to 5-10 °C, and then NaH (0.35 g, 1.1 eq) was added. Nitrogen gas was purged three times, and the mixture was stirred at room temperature for 30 minutes, during which bubbles were generated. The reaction system was cooled to an internal temperature of 5-10 °C, and then 0.55 mL of iodomethane (0.55 mL, 1.1 eq) was added. The mixture was stirred at room temperature for another 2 hours. As time progressed, a large amount of solid precipitated in the reaction system. The reaction was monitored by LCMS until complete. The reaction solution was poured into a saturated ammonium chloride aqueous solution (300 mL), extracted with ethyl acetate (150 mL x 2), the organic phases were combined, washed with saturated brine (150 mL x 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain 4.2 g of crude product.

[0316] Dissolve 4.2 g of the crude product in ammonia-methanol solution (30 mL), replace with nitrogen, stir at 60 °C for 16 h, monitor the reaction for completeness by LCMS, and after cooling the reaction solution to room temperature, a solid precipitates out. Filter, collect the solid, and dry to obtain intermediate 50 (2.1 g, white solid, yield 72%, purity 98%).

[0317] 1H NMR: (400MHz, DMSO-d6) δppm 7.81(d,J=8.0Hz,2H),7.23(d,J=12.4,1H),7.20(dd,J=8.0,3.2Hz,1H),6.66(brs,2H ), 3.83 (s, 3H), 2.90 (t, J = 7.6Hz, 2H), 2.74 (t, J = 7.6Hz, 2H), 2.02 (qui, J = 7.6Hz, 2H).

[0318] Step 7: Preparation of 4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)propyl]-2-fluorobenzoic acid (intermediate 51)

[0319] Intermediate 50 (2.1 g, 1 eq) was dissolved in a mixed solution of 1 M NaOH aqueous solution (16 mL) and methanol (30 mL), and the mixture was refluxed and stirred for 1 h. The disappearance of the starting material was monitored by LCMS (254 nm). After the reaction was completed, the reaction mixture was evaporated to dryness to remove methanol. Water (200 mL) and ethyl acetate (200 mL) were added to the obtained crude product, and the aqueous phase was separated and collected. The aqueous phase was adjusted to pH 4-5 with hydrochloric acid (2N), and a white solid precipitated out. The solid was collected by filtration and dried to obtain 1.7 g of white solid crude product, which showed the presence of acetic acid on NMR.

[0320] The crude product was added to a saturated sodium bicarbonate solution (20 mL) and stirred for 2 hours. After filtration, the filter cake was evaporated to obtain intermediate 51 (1.03 g, white solid, yield 51%, purity 99%).

[0321] 1 H NMR: (400MHz, DMSO-d6) δppm 12.03(brs,1H),7.54(d,J=8.0Hz,2H),7.16(brs,2H),6.89-6.94(m,2H),2.87(t,J= 7.6Hz,2H),2.64(t,J=7.6Hz,2H),1.99(qui,J=7.6Hz,2H); MS(ESI)m / z=349.10[M+H] + .

[0322] Step 8: Preparation of (S)-2-{4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)propyl]-2-fluorobenzamido}-3-[4-(tert-butoxy)phenyl]propionate tert-butyl ester (intermediate 52)

[0323] Intermediate 51 (190 mg, 1.0 eq), DMAc (4 mL), O-tert-butyl-L-tyrosine tert-butyl hydrochloride (185 mg, 1.03 eq), and TCFH (184 mg, 1.2 eq) were added sequentially to the reaction flask, followed by nitrogen purging three times. The temperature was lowered to 0-5℃, and NMI (157 mg, 3.5 eq) was added at 4℃. After the addition was complete, the reaction mixture was stirred at 20-25℃ for 2.5 h. The reaction was monitored by TLC (samples were taken, water and ethyl acetate were added, and the mixture was shaken; the upper organic phase was collected, with a DCM:MeOH ratio of 10:1) until the starting material was completely converted. Water and ethyl acetate were added to the reaction flask, and the mixture was extracted and separated. The upper organic phase was collected, and the aqueous phase was extracted again with ethyl acetate. The organic phases were combined, washed three times with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was initially purified by preparative thin-layer chromatography (DCM:MeOH = 8:1) to obtain intermediate 52 (350 mg of off-white solid, purity 92.36%, crude product yield 113%), which was directly used in the next reaction. MS (ESI) m / z = 624 [M+H] + .

[0324] Step 9: Preparation of (S)-2-{4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)propyl]-2-fluorobenzamido}-3-[4-hydroxyphenyl]propionic acid (intermediate 53)

[0325] Intermediate 52 (350 mg, 1.0 eq), water (175 mg), and 4N HCl / dioxane (1.75 mL, 5V) were added sequentially to the reaction flask. The temperature was controlled at 20-25℃, and the mixture was stirred for 2.5 h. The reaction was monitored by TLC (samples were added to dissolve the solid in ethanol, DCM:MeOH = 10:1) until the reaction was complete. Water was added to the reaction system, and a solid precipitated. 1N sodium hydroxide aqueous solution was added dropwise until the solution became clear. The clear reaction solution was purified by reverse-phase preparative HPLC [eluent H2O / CH3CN v / v% = 0-20-100%, gradient time 5-15-20 min] to obtain the target product component. The preparative solution was concentrated under reduced pressure at 35℃ to remove most of the water. The remaining solution was directly lyophilized to obtain intermediate 53 (260 mg of light yellow solid, purity 98.42%, yield 90.6%).

[0326] 1H NMR: (400MHz, D2O) δ7.51(t,J=8.0Hz,1H),7.06–7.03(m,3H),6.98(d,J=12.7Hz,1H),6.63(d,J=8.4Hz,2H),4.59– 4.55(m,1H),3.16–3.11(m,1H),2.99-2.91(m,3H),2.69(t,J=7.4Hz,2H),2.08–2.01(m,2H); MS(ESI)m / z=512[M+H] + .

[0327] Step 10: Preparation of 4-{2-[(E)-2-((E)-2-[4-((S)-2-{4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)propyl]-2-fluorobenzamido}-2-carboxyethyl)phenoxy]-3-{2-[(E)-3,3-dimethyl-5-sulfono-1-(4-sulfonobutyl)indol-2-ylidene]ethenyl}cyclohex-1-en-1-yl)vinyl]-3,3-dimethyl-5-sulfono-3H-indol-1-on-1-yl}butyl-1-sulfonic acid inner salt (compound 7)

[0328] Intermediate 53 (190 mg, 1.0 eq.) and water (6 mL) were added to the reaction flask. The pH was adjusted and controlled to 10.7-10.9 with 1 M sodium hydroxide solution. After stirring until dissolved, SO456 (80%, 370 mg, 0.9 eq.) was added, and nitrogen was purged three times. The temperature was raised to 65 °C, and the reaction was stirred for 3 h. The reaction was monitored by TLC (sample was taken and dissolved in water, DCM:MeOH = 5:1). The reaction proceeds were completely reacted. The reaction was cooled to room temperature and filtered. The target product fraction was obtained by reverse-phase preparative HPLC purification [eluent H2O (0.1% TFA v / v%) / CH3CN v / v% = 5-95%, gradient time 2-20 min]. The prepared solution was concentrated under reduced pressure at 35 °C to remove most of the water. The remaining solution was directly lyophilized to obtain compound 7 (220 mg green solid, yield 43.5%).

[0329] 1H NMR: (400MHz, DMSO-d6) δ8.09–7.98(m,1H),7.77(d,J=14.0Hz,2H),7.62–7.52(m,5H),7.30(d d,J=13.0,8.5Hz,4H),7.20–7.01(m,5H),6.20(d,J=14.2Hz,2H),4.65(dd,J=13.2,8.1Hz,1H), 4.11(s,4H),3.17(d,J=9.8Hz,1H),3.01(dd,J=13.7,9.3Hz,1H),2.86(t,J=7.6Hz,2H),2.78– 2.56(m,10H),1.92–1.86(m,4H),1.74(s,8H),1.19(d,J=15.2Hz,12H); MS(ESI)m / z=1363[M+H] + ,682[M+2H] 2+ .

[0330] Example 8: Preparation of 4-{2-[(E)-2-((E)-2-[4-((S)-2-{4-[4-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)butyl]-2-fluorobenzamido}-2-carboxyethyl)phenoxy]-3-{2-[(E)-3,3-dimethyl-5-sulfonyl-1-(4-sulfonylbutyl)indol-2-ylidene]ethenyl}cyclohex-1-en-1-yl)vinyl]-3,3-dimethyl-5-sulfonyl-3H-indol-1-on-1-yl}butyl-1-sulfonic acid inner salt (compound 8)

[0331] Step 1: Preparation of ethyl 5-amino-2-{4-[3-fluoro-4-(methoxycarbonyl)phenyl]but-3-en-1-yl}thiazolyl-4-carboxylate (intermediate 54)

[0332] Intermediate 20 (3.9 g, 1.0 eq) and 80 mL of DMF were added to a reaction flask. Methyl 2-fluoro-4-bromobenzoate (7.7 g, 2.0 eq), POT (1.0 g, 0.2 eq), DIPEA (11.0 g, 3.0 eq), and Pd(OAc)₂ (0.39 g, 0.1 eq) were added sequentially to the same flask at room temperature. The mixture was stirred, purged with nitrogen three times, and heated at 110 °C with stirring overnight. After the reaction was complete, the reaction solution was cooled to room temperature and extracted with ethyl acetate (150 mL x 2). The organic phases were combined. The organic phases were washed with 10% NaCl aqueous solution (180 mL x 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA, 5:1–3:1 V / V) to obtain intermediate 54 (4.5 g yellow solid, 69% yield).

[0333] 1 H NMR: (400MHz, CDCl3) δppm 7.86(d,J=8.0Hz,1H),6.96-7.10(m,2H),6.43(d,J=15.6Hz,1H),5.89-6.02(m,2H),4.40(q,J=7.2Hz,2H), 3.92(s,3H),3.03(t,J=6.8Hz,2H),2.65(t,J=6.8Hz,2H),1.40(t,J=7.2Hz,3H); MS(ESI)m / z=379.70[M+H] + .

[0334] Step 2: Preparation of ethyl 5-amino-2-{4-[3-fluoro-4-(methoxycarbonyl)phenyl]butyl}thiazole-4-carboxylate (intermediate 55)

[0335] Intermediate 54 (4.5 g, 1 eq) and DMF (80 mL) were added to a reaction flask, stirred, and purged with nitrogen. Pd(OH)₂ / C (0.5 g) was added, and the mixture was purged with hydrogen three times. The mixture was stirred at room temperature for 24 hours. HPLC analysis confirmed the reaction was complete. The mixture was filtered, and the filter cake was washed with DCM (30 mL x 2). The mixture was concentrated by rotary evaporation at 40 °C to remove low-boiling solvents. The resulting DMF solution was poured into 200 mL of water and extracted with ethyl acetate (150 mL x 2). The organic phases were combined. The organic phases were washed with 10% NaCl aqueous solution (160 mL x 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain intermediate 55 (4.5 g yellow solid, 100% yield).

[0336] 1H NMR: (400MHz, CDCl3) δppm 7.84(d,J=8.0Hz,1H),7.03(d,J=8.0Hz,1H),6.96(d,J=12.0Hz,1H),5.88(brs,2H),4.39(q,J=7.2Hz,2H),3.92(s,3H ),2.86(t,J=6.8Hz,2H),2.67(t,J=6.8Hz,2H),1.72(t,J=6.8Hz,4H),1.40(t,J=7.2Hz,3H); MS(ESI)m / z=381.65[M+H] + .

[0337] Step 3: Preparation of ethyl 5-(3-benzoylthiourea)-2-{4-[3-fluoro-4-(methoxycarbonyl)phenyl]butyl}thiazole-4-carboxylate (intermediate 56)

[0338] Acetone (50 mL), intermediate 55 (2.5 g, 1 eq), and benzoyl isothiocyanate (1.9 g, 1.2 eq) were added to a reaction flask, stirred, purged with nitrogen, and refluxed overnight. The reaction was monitored by LC-MS until complete. The solvent was removed by rotary evaporation at 35 °C. The crude product was purified by silica gel column chromatography (PE:EA, 5:1–3:1 V / V) to obtain intermediate 56 (3.0 g yellow solid, 84% yield). MS (ESI) m / z = 544.60 [M+H] + .

[0339] Step 4: Preparation of methyl 4-[4-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)butyl]-2-fluorobenzoate (intermediate 57)

[0340] Intermediate 56 (3.0 g, 1 eq) and DMF (30 mL) were added to the reaction flask, and the mixture was stirred and purged with nitrogen three times. The mixture was cooled to 0°C in an ice bath, and NaH (60%, 0.24 g, 1.1 eq) was added. Gas was generated, and the internal temperature rose to 5°C. The mixture was stirred at 0°C for 15 minutes, then the ice bath was removed, and the mixture was allowed to react at room temperature for 30 minutes. The reaction mixture was then cooled back to 0°C, and MeI (0.86 g, 1.1 eq) was added in one go. The mixture was reacted at 0°C for 15 minutes, then the ice bath was removed, and the mixture was allowed to react at room temperature for 1 hour. LC-MS was used to monitor the complete consumption of the starting materials until the reaction was finished. The reaction mixture was poured into a saturated ammonium chloride solution at 0°C (100 mL), diluted with 200 mL of water, and extracted with ethyl acetate (100 mL x 2). The organic phases were combined, washed with 10% NaCl aqueous solution (150 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation at 35°C to obtain a pale yellow solid.

[0341] The obtained solid was dissolved in 60 mL of NH3 / MeOH at room temperature and refluxed overnight. A white solid precipitated from the reaction. After the reaction solution was cooled, the solid was filtered, and the filter cake was washed with methanol (5 mL x 3). The obtained solid was dried at 50 °C to give intermediate 57 (1.5 g white solid, 75% overall yield of the two steps).

[0342] 1 H NMR: (400MHz, DMSO-d6) δppm 7.80(d,J=8.0Hz,1H),7.17-7.22(m,2H),6.64(brs,2H),3.83(s,3H),2.91(t,J=6.8Hz,2H), 2.67(t,J=6.8Hz,2H),1.69(t,J=6.8Hz,4H),1.40(t,J=7.2Hz,3H); MS(ESI)m / z=377.25[M+H] + .

[0343] Step 5: Preparation of 4-[4-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)butyl]-2-fluorobenzoic acid (intermediate 58)

[0344] Intermediate 58 (1.5 g, 1 eq) and 12 mL of MeOH were added to a reaction flask and stirred at room temperature. The starting material was a suspension in the solvent. NaOH aqueous solution (1 M, 12 mL, 3 eq) was added dropwise to the reaction solution at room temperature, and the mixture was stirred for 4 hours. LC-MS was used to monitor the complete consumption of the starting material until the reaction was complete. The MeOH was removed by rotary evaporation at 35 °C. 20 mL of water was added to the remaining solution, and impurities were removed by extraction with methyl tert-butyl ether (15 mL). The aqueous phase was collected. The pH of the aqueous phase was adjusted to 4 with hydrochloric acid (2 N). A white solid gradually precipitated with the addition of hydrochloric acid solution. The solid was collected by filtration, washed with water (10 mL x 2), and dried at 50 °C to obtain intermediate 58 (1.1 g of off-white solid, 76% yield).

[0345] 1 H NMR: (400MHz, DMSO-d6) δppm 11.44(br,1H),7.70(d,J=8.0Hz,1H),7.05-7.08(m,2H),6.87(brs,2H),2.91(t, J=6.8Hz,2H),2.66(t,J=6.8Hz,2H),1.61-1.76(m,4H); MS(ESI)m / z=363.15[M+H] + .

[0346] Step 6: Preparation of (S)-2-{5-[4-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)butyl]-2-fluorobenzamido}-3-[4-(tert-butoxy)phenyl]propionate tert-butyl ester (intermediate 59)

[0347] Intermediate 58 (190 mg, 1.0 eq), DMAc (3.8 mL), O-tert-butyl-L-tyrosine tert-butyl ester hydrochloride (178 mg, 1.03 eq), and TCFH (177 mg, 1.2 eq) were added sequentially to the reaction flask. The mixture was purged with nitrogen three times and the temperature was lowered to 0-5 °C. NMI (151 mg, 3.5 eq) was added at 4 °C. After the addition was complete, the mixture was stirred at 20-25 °C for 2.5 h. TLC was performed under controlled conditions (a sample was added and shaken with water and ethyl acetate; the upper organic phase was DCM:MeOH = 10:1) until the reactants were completely reacted. Water and ethyl acetate were added to the reaction flask, and the mixture was extracted and separated. The upper organic phase was collected, and the aqueous phase was extracted again with ethyl acetate. The combined organic phases were washed three times with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was preliminarily purified by preparative thin-layer chromatography (DCM:MeOH = 8:1) to obtain intermediate 59 (340 mg of off-white solid, crude product yield 102%), which was directly used in the next reaction.

[0348] Step 7: Preparation of (S)-2-{5-[4-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)butyl]-2-fluorobenzamido}-3-[4-hydroxyphenyl]propionic acid (intermediate 60)

[0349] Intermediate 59 (340 mg, 1.0 eq), water (170 mg), and 4N HCl / dioxane (1.7 mL, 5V) were added sequentially to the reaction flask. The temperature was controlled at 20-25℃, and the mixture was stirred for 2.5 h. The reaction was monitored by TLC (samples were added to dissolve the solid in ethanol, DCM:MeOH = 10:1) until the reaction was complete. Water was added to the reaction system, and a solid precipitated. 1N sodium hydroxide aqueous solution was added dropwise until the solution became clear. The clear reaction solution was purified by reverse-phase preparative HPLC [eluent H2O / CH3CN v / v% = 0-20-100%, gradient time 5-15-20 min] to obtain the target product component. The preparative solution was concentrated under reduced pressure at 35℃ to remove most of the water. The remaining solution was directly lyophilized to obtain intermediate 60 (white solid 245 mg, purity 99.6%, yield 87.5%).

[0350] 1H NMR: (400MHz, D2O) δ7.36(t,J=8.0Hz,1H),6.84–6.68(m,4H),6.38(d,J=8.3Hz,2H),4.37(t,J=6.1Hz,1H),2.93– 2.88(m,1H),2.81–2.75(m,1H),2.68(s,2H),2.34(d,J=5.2Hz,2H),1.40(d,J=34.1Hz,4H); MS(ESI)m / z=526[M+H] + .

[0351] Step 8: Preparation of the inner salt of 4-{2-[(E)-2-((E)-2-[4-((S)-2-{4-[4-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)butyl]-2-fluorobenzamido}-2-carboxyethyl)phenoxy]-3-{2-[(E)-3,3-dimethyl-5-sulfonyl-1-(4-sulfonyl)indol-2-ylidene]ethenyl}cyclohex-1-en-1-yl)vinyl]-3,3-dimethyl-5-sulfonyl-3H-indol-1-on-1-yl}butyl-1-sulfonic acid (compound 8)

[0352] Intermediate 53 (193 mg, 1.0 eq.) and water (6.2 mL) were added to the reaction flask. The pH was adjusted and controlled to 10.7-10.9 with 1 M sodium hydroxide solution. After stirring until dissolved, SO456 (80%, 367 mg, 0.9 eq.) was added, and nitrogen was purged three times. The temperature was raised to 65 °C, and the reaction was stirred for 3 h. The reaction was monitored by TLC (sample was taken and dissolved in water, DCM:MeOH = 5:1). The reaction proceeds were completely reacted. The reaction was cooled to room temperature and filtered. The target product fraction was obtained by reverse-phase preparative HPLC purification [eluent H2O (0.1% TFA v / v%) / CH3CN v / v% = 5-95%, gradient time 2-20 min]. The prepared solution was concentrated under reduced pressure at 35 °C to remove most of the water. The remaining solution was directly freeze-dried to obtain compound 8 (280 mg green solid, purity 98.7%, yield 54.4%).

[0353] 1H NMR: (400MHz, DMSO-d6) δppm 8.68(d,J=8.0Hz,1H),8.43(s,1H),7.93(d,J=8.0Hz,1H),7.89(d,J=8.0Hz,1H),7.72(d,J=14.0Hz,2H),7.62(d,J=8. 4Hz,2H),7.55(s,2H),7.32(d,J=8.4Hz,2H),7.26(d,J=8.4Hz,2H),7.01(d,J=8.4Hz,2H),6.18(d,J=14.0Hz,2H),4.6 9(dd,J=13.2,8.4Hz,1H),4.10(brs,4H),3.17-3.25(m,1H),3.08-3.16(m,1H),2.91(t,J=7.2Hz,2H),2.57-2.74(m,1 0H),1.91(t,J=6.0Hz,2H),1.60-1.80(m,10H),1.49-1.58(m,2H),1.16(s,6H),1.08(s,6H); MS(ESI)m / z=457.0[M+3H] 3+ 687.0 [M+2H] 2+ .

[0354] Example 9: Preparation of 4-{2-[(E)-2-((E)-2-[4-((S)-2-{4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[4,5-d]pyrimidin-2-yl)propyl]benzamido}-2-carboxyethyl)phenoxy]-3-{2-[(E)-3,3-dimethyl-5-sulfonyl-1-(4-sulfonylbutyl)indol-2-ylidene]ethenyl}cyclohex-1-en-1-yl)vinyl]-3,3-dimethyl-5-sulfonyl-3H-indol-1-on-1-yl}butyl-1-sulfonic acid inner salt (compound 9)

[0355] Step 1: Preparation of 2,6-diamino-5-thiocyanopyrimidine-4(3H)-one (intermediate 61)

[0356] Add 2,4-diamino-6-hydroxypyrimidine [CAS No.: 56-06-4] (1.0 g, 1.0 eq), acetic acid (55 mL, 55 V), and KSCN (3.09 g, 4.0 eq) sequentially to the reaction flask. Stir until dissolved. After purging with nitrogen three times, heat to 85 °C until dissolved, then cool to 15 °C-20 °C. Prepare liquid bromine (1.27 g, 1.0 eq.) dissolved in acetic acid (2 mL). Add the liquid bromoacetic acid solution dropwise to the reaction flask. The reaction solution turns red, and a solid begins to precipitate. Maintain the reaction temperature at 10-20 °C and react for 3 hours. Monitor the reaction with LCMS until complete. Cool the reaction system to 0-5 °C, quench the reaction by adding ammonia water (18 mL, 18 V), and stir overnight at 20-30 °C. The reaction solution was filtered and washed with methanol (10 mL) and water (10 mL). The filter cake was dried in a forced-air oven at 55 °C to give intermediate 61 (1.13 g of yellow solid, yield 89%).

[0357] 1 H NMR: (400MHz, DMSO-d6) δppm 10.36 (s, 1H), 7.02 (s, 2H), 6.65 (brs, 2H); MS (ESI) m / z = 184 [M+H] + 367[2M+H] + .

[0358] Step 2: Preparation of 2,5-diaminothiazo[4,5-d]pyrimidine-7(6H)-one (intermediate 62)

[0359] Take a 500 mL three-necked round-bottom flask and add DMF (100 mL) and intermediate 61 (10.0 g, 1.00 eq) sequentially. Add 2 M NaOH solution (25 mL) dropwise to the reaction system; the reaction mixture changes from a reddish-brown gel to a red solution. Under oil bath heating conditions, stir and reflux the reaction mixture at 120 °C for 16 h. Take a sample for further processing. 1 ¹H-NMR analysis showed that the starting material completely disappeared and the target compound was formed. The reaction mixture was filtered, and the filter cake was collected and washed successively with deionized water (100 mL × 3), acetonitrile (MeCN, 100 mL × 3), and n-hexane (100 mL × 3) to remove residual reagents and byproducts. The filter cake was placed in a vacuum drying oven and dried under reduced pressure to constant weight to give intermediate 62 (7.10 g of light yellow solid, yield 71.0%).

[0360] 1 H NMR: (400MHz, DMSO-d6) δppm 10.68 (brs, 1H), 7.92 (s, 2H), 6.40 (brs, 2H).

[0361] Step 3: Preparation of 2-bromo-5-aminothiazo[4,5-d]pyrimidin-7(6H)-one (intermediate 63)

[0362] Take a 1L three-necked round-bottom flask and add deionized water (140mL), intermediate 62 (7.00g, 1.00eq), and NaNO2 (6.99g, 2.65eq) sequentially. Add hydrobromic acid (48.0%, 138.33mL, 32.0eq) dropwise to the reaction system. After the addition is complete, stir and reflux the reaction mixture in an oil bath at 80℃ for 16h. Monitor the reaction by LCMS until the starting material is completely eliminated. Filter the reaction mixture, collect the filter cake, and wash the filter cake sequentially with deionized water (100mL×3), acetonitrile (MeCN, 100mL×3), and n-hexane (100mL×3). Grind the filter cake with acetonitrile (100mL) and deionized water (100mL), filter again, collect the filter cake, and wash the filter cake with the same solvent to finally obtain intermediate 63 (5.20g pink solid, purity 98.0%, yield 54.0%).

[0363] 1 H NMR: (400MHz, DMSO-d6) δppm 11.22-11.50 (m, 1H), 6.62-6.89 (m, 2H); MS (ESI) m / z = 246.8 [M+H] + .

[0364] Step 4: Preparation of methyl 4-(3-iodopropyl)benzoate (intermediate 63)

[0365] Solution 1: 1,3-Diiodopropane [CAS No.: 627-31-6] (41.3 g, 16.12 mL, 3.00 eq), methyl 4-bromobenzoate [CAS No.: 619-42-1] (10.0 g, 1.00 eq), bis[3,5-difluoro-2-[5-(trifluoromethyl)-2-pyridyl-κN]phenyl-κC][4,4'-bis(1,1-dimethylethyl)-2,2'-bipyridine-κN1,κN1′]iridium(I) complex hexafluorophosphate [CAS No.: 2173009-61- 3] (1.04 g, 0.02 eq), sodium carbonate (9.86 g, 2.00 eq), tris(trimethsilyl)silane [CAS No.: 1873-77-4] (17.3 g, 21.52 mL, 1.50 eq), [4,4'-bis(1,1-dimethylethyl)-2,2'-bipyridine-κN1,κN1′]nickel(II) dichloride [CAS No.: 1034901-50-2] (925 mg, 0.05 eq) dissolved in DME [CAS No.: 110-71-4] (500 mL).

[0366] Solution 1 was pumped into a flow reactor (PFA coil, inner diameter 3.175 mm, volume 30 mL, 40 °C) using a peristaltic pump (flow rate 30 mL / min) and circulated for 120 min under continuous irradiation with a 450 nm LED light source (200 W). All reaction solution was collected, and TLC (n-hexane:ethyl acetate = 5:1) was performed to monitor the disappearance of starting materials and confirm the completion of the reaction. The LED light source and peristaltic pump were turned off, the tubing was flushed with DME (50 mL), and the reaction mixtures were combined for post-processing.

[0367] The reaction mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate: n-hexane = 0:100 to 10:90) to give intermediate 64 (white solid 25.0 g, purity 92.0%, yield 32.5%).

[0368] 1 H NMR: (400MHz, CDCl3) δppm 7.98(d,J=8.2Hz,2H),7.27(s,2H),3.88-3.94(m,3H),3.17(t,J=6.8Hz,2 H),2.80(t,J=7.4Hz,2H),2.15(q,J=7.2Hz,2H); MS(ESI)m / z=304.9[M+H] + .

[0369] Step 5: Preparation of methyl 4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[4,5-d]pyrimidin-2-yl)propyl]benzoate (intermediate 65)

[0370] Solution 1: Intermediate 63 (5.20 g, 1.00 eq), Intermediate 64 (7.00 g, 1.09 eq), bis[3,5-difluoro-2-[5-(trifluoromethyl)-2-pyridyl-κN]phenyl-κC][4,4'-bis(1,1-dimethylethyl)-2,2'-bipyridine-κN1,κN1′]iridium(I) complex hexafluorophosphate (708 mg, 0.03 eq), 2,6-dimethyl Pyridine [CAS No.: 108-48-5] (4.51 g, 2.00 eq), tris(trimethsilyl)silane (7.85 g, 9.74 mL, 1.50 eq), and [4,4'-bis(1,1-dimethylethyl)-2,2'-bipyridine-κN1,κN1′]nickel(II) dichloride (503 mg, 0.06 eq) were dissolved in a mixed solvent of DMSO (50 mL) and DME (200 mL).

[0371] Solution 1 was pumped into a flow reactor (PFA coil, inner diameter 3.175 mm, volume 30 mL, 40 °C) using a peristaltic pump (flow rate 30 mL / min) and circulated for 120 min under continuous irradiation with a 450 nm LED light source (200 W). All reaction solution was collected, and samples were taken for LCMS monitoring to confirm the disappearance of starting materials and the completion of the reaction. The LED light source and peristaltic pump were turned off, the tubing was flushed with DME (50 mL), and the reaction mixtures were combined for post-processing.

[0372] The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, yielding intermediate 65 (8.00 g of yellow liquid, crude product yield 110%), which was used directly in the next reaction without purification. MS (ESI) m / z = 345.9 [M+H] + .

[0373] Step 6: Preparation of 4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[4,5-d]pyrimidin-2-yl)propyl]benzoic acid (intermediate 66)

[0374] Add intermediate 65 (8.00 g, 1.00 eq) to a 250 mL three-necked round-bottom flask, and slowly add 2 M sodium hydroxide solution (58.07 mL, 5.00 eq) dropwise. Add methanol (50 mL) to the reaction system and stir at 25 °C for 16 h. Monitor the reaction progress by LCMS until the starting material disappears. Evaporate and concentrate the reaction solution, and extract the aqueous phase with ethyl acetate (200 mL × 3 times). Combine all aqueous phases, add 2 M hydrochloric acid to the aqueous phase, adjust the pH to approximately 4, filter, and collect the filter cake. Dissolve the filter cake in 0.5 M sodium hydroxide solution (50 mL) and purify three times by preparative HPLC (column: Welch Xtimate C18 150 × 25 mm × 5 μm; mobile phase: [water (0.05% NH3H2O)-acetonitrile]; gradient: 0%-25% B% 9.0 min) to obtain the crude product solution. The crude product solution was adjusted to pH ≈ 4 with 2M hydrochloric acid, filtered, and the filter cake was collected. The filter cake was freeze-dried to obtain intermediate 66 (100 mg, purity 93.4%, yield 0.608%).

[0375] 1 H NMR: (400MHz, DMSO-d6) δppm 7.77-7.82(m,2H),7.09-7.16(m,2H),6.50-6.63(m,2H),2.95(br t,J=7.4Hz,2H),2.64-2.68(m,2H),1.98-2.07(m,2H); MS(ESI)m / z=331.3[M+H] + .

[0376] Step 7: Preparation of (S)-2-{4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[4,5-d]pyrimidin-2-yl)propyl]benzamido}-3-[4-(tert-butoxy)phenyl]propionate tert-butyl ester (intermediate 67)

[0377] The synthesis was performed using a method similar to step 8 of Example 7, except that the starting materials were sequentially replaced with intermediate 66 (90 mg, 1.0 eq), DMAc (3 mL), O-tert-butyl-L-tyrosine tert-butyl hydrochloride (93 mg, 1.03 eq), TCFH (92 mg, 1.2 eq), and NMI (78 mg, 3.5 eq.) to obtain intermediate 67 (150 mg of off-white solid, 90.9% yield), which was directly used in the next reaction. MS (ESI) m / z = 606 [M+H] + .

[0378] Step 9: Preparation of (S)-2-{4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[4,5-d]pyrimidin-2-yl)propyl]benzamido}-3-[4-hydroxyphenyl]propionic acid (intermediate 68)

[0379] The synthesis was performed using a method similar to step 9 of Example 7, except that the raw materials were changed sequentially to intermediate 67 (150 mg, 1.0 eq), water (70 mg), and 4N HCl / dioxane (0.7 mL, 5 V) to obtain intermediate 68 (120 mg of light yellow solid, purity 95.1%, yield 98.2%).

[0380] 1 H NMR: (400MHz, D2O) δ7.55(d,J=8.0Hz,2H),7.27(d,J=8.0Hz,2H),6.98(d,J=8.2Hz,2H),6.53(d,J=8.2Hz,2H),4.53(dd,J=7.5,5.1Hz,1H),3.09 (dd,J=14.0,4.9Hz,1H),3.00(t,J=7.3Hz,2H),2.92(dd,J=14.1,7.8Hz,1H),2.71(t,J=7.4Hz,2H),2.09(p,J=7.4Hz,2H); MS(ESI)m / z=494[M+H] + .

[0381] Step 10: Preparation of 4-{2-[(E)-2-((E)-2-[4-((S)-2-{4-[3-(5-amino-7-oxo-6,7-dihydrothiazo[4,5-d]pyrimidin-2-yl)propyl]benzamido}-2-carboxyethyl)phenoxy]-3-{2-[(E)-3,3-dimethyl-5-sulfonyl-1-(4-sulfonylbutyl)indol-2-ylidene]ethenyl}cyclohex-1-en-1-yl)vinyl]-3,3-dimethyl-5-sulfonyl-3H-indol-1-on-1-yl}butyl-1-sulfonic acid inner salt (compound 9)

[0382] The synthesis was performed using a method similar to step 10 of Example 7, except that the starting materials were replaced sequentially with intermediate 68 (80 mg, 1.0 eq.), water (4 mL), and SO456 (80%, 162 mg, 0.9 eq.) to obtain compound 9 (50 mg green solid, 95.1% purity, 22.9% yield).

[0383] 1 H NMR: (400MHz, DMSO-d6)δ8.46(d,J=8.2Hz,1H),8.25(s,2H),7.77–7.68(m,4H),7.59(d,J=7.7Hz,4H), 7.36(d,J=8.4Hz,2H),7.28(d,J=8.6Hz,2H),7.21(d,J=8.1Hz,2H),7.01(d,J=8.5Hz,2H),6.16(d,J=1 4.1Hz,2H),4.65(s,1H),4.09(s,4H),3.17(d,J=11.1Hz,1H),3.04–2.96(m,3H),2.72–2.60(m,8H),2. 55(t,J=7.3Hz,2H),1.90–1.80(m,4H),1.73(s,8H),1.14(s,6H),1.04(s,6H); MS(ESI)m / z=1345[M+H] + ,673[M+2H] 2+ .

[0384] Example 10: Preparation of 4-{2-[(E)-2-((E)-2-[4-((S)-2-{4-[4-(5-amino-7-oxo-6,7-dihydrothiazo[4,5-d]pyrimidin-2-yl)butyl]benzamido}-2-carboxyethyl)phenoxy]-3-{2-[(E)-3,3-dimethyl-5-sulfonyl-1-(4-sulfonyl)indol-2-ylidene]ethenyl}cyclohex-1-en-1-yl)vinyl]-3,3-dimethyl-5-sulfonyl-3H-indol-1-on-1-yl}butyl-1-sulfonic acid inner salt (compound 10)

[0385] Step 1: Preparation of methyl 4-(4-iodobutyl)benzoate (intermediate 70)

[0386] Solution 1: 1,4-Diiodobutane [CAS No.: 628-21-7] (28.8 g, 12.23 mL, 2.00 eq), methyl 4-bromobenzoate (10.0 g, 1.00 eq), bis[3,5-difluoro-2-[5-(trifluoromethyl)-2-pyridyl-κN]phenyl-κC][4,4'-bis(1,1-dimethylethyl)-2,2'-bipyridine-κN1,κN1′]iridium ( I) The complex hexafluorophosphate (1.04 g, 0.02 eq), sodium carbonate (9.86 g, 2.00 eq), tris(trimethylsilyl)silane (17.3 g, 21.52 mL, 1.50 eq), and [4,4'-bis(1,1-dimethylethyl)-2,2'-bipyridine-κN1,κN1′]nickel(II) chloride (925 mg, 0.05 eq) were dissolved in DME (500 mL).

[0387] Solution 1 was pumped into a flow reactor (PFA coil, inner diameter 3.175 mm, volume 30 mL, 40 °C) using a peristaltic pump (flow rate 30 mL / min) and circulated for 120 min under continuous irradiation with a 450 nm LED light source (200 W). All reaction solution was collected, and samples were taken for LCMS monitoring to confirm the disappearance of starting materials and the completion of the reaction. The LED light source and peristaltic pump were turned off, the tubing was flushed with DME (50 mL), and the reaction mixtures were combined for post-processing.

[0388] The reaction mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate: n-hexane = 0:100 to 3:97) to give intermediate 70 (4.90 g of colorless oily liquid, purity 93.0%, yield 30.8%).

[0389] 1H NMR: (400MHz, CDCl3) δppm 7.78(d,J=8.2Hz,2H),7.07(d,J=9.0Hz,2H),3.72(s,3H),3.01(t,J=6.8Hz,2H),2.51(t,J=7.4Hz,2H),1.62-1.70(m,2H),1.58(br d,J=7.0Hz,2H); MS(ESI)m / z=319.0[M+H] + .

[0390] Step 2: Preparation of methyl 4-[4-(5-amino-7-oxo-6,7-dihydrothiazo[4,5-d]pyrimidin-2-yl)butyl]benzoate (intermediate 71)

[0391] Solution 1: Intermediate 63 (2.50 g, 1.00 eq), Intermediate 70 (4.83 g, 1.50 eq), bis[3,5-difluoro-2-[5-(trifluoromethyl)-2-pyridyl-κN]phenyl-κC][4,4'-bis(1,1-dimethylethyl)-2,2'-bipyridine-κN1,κN1′]iridium(I) complex hexafluorophosphate (227 mg, 0.02 e q), sodium carbonate (2.14 g, 2.00 eq), tris(trimethsilyl)silane (3.77 g, 4.68 mL, 1.50 eq), and [4,4'-bis(1,1-dimethylethyl)-2,2'-bipyridine-κN1,κN1′]nickel(II) dichloride (201 mg, 0.05 eq) were dissolved in a mixed solvent of DMSO (125 mL) and DME (125 mL).

[0392] Solution 1 was pumped into a flow reactor (PFA coil, inner diameter 3.175 mm, volume 30 mL, 40 °C) using a peristaltic pump (flow rate 30 mL / min) and circulated for 120 min under continuous irradiation with a 450 nm LED light source (200 W). All reaction solution was collected, and samples were taken for LCMS monitoring to confirm the disappearance of starting materials and the completion of the reaction. The LED light source and peristaltic pump were turned off, the tubing was flushed with DME (50 mL), and the reaction mixtures were combined for post-processing.

[0393] The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, yielding intermediate 71 (4.00 g of yellow liquid, crude product yield 110%), which was used directly in the next reaction without purification. MS (ESI) m / z = 358.9 [M+H] + .

[0394] Step 3: Preparation of 4-[4-(5-amino-7-oxo-6,7-dihydrothiazo[4,5-d]pyrimidin-2-yl)butyl]benzoic acid (intermediate 72)

[0395] Add intermediate 71 (4.00 g, 1.00 eq) to a 500 mL three-necked round-bottom flask, followed by 12 mL of methanol. Slowly add 2 M sodium hydroxide solution (20.33 mL, 2.00 eq) dropwise to the reaction system, and stir the reaction at 25 °C for 16 h. Monitor the reaction progress by LC-MS until the starting material disappears. Pour the reaction mixture into 1 L of water and extract the aqueous phase with ethyl acetate (500 mL × 3 times). Combine all aqueous phases, add 2 M hydrochloric acid to adjust the pH to approximately 4, filter, and collect the filter cake. The filter cake was dissolved in 0.5M sodium hydroxide solution (50 mL) and purified by preparative HPLC (column: Welch Xtimate 75×40mm×3μm; mobile phase: [water (10mM NH4HCO3)-acetonitrile]; gradient: 0%-25% B% 20.0 min) to obtain intermediate 72 (105 mg, purity 97.91%, yield 2.67%).

[0396] 1 H NMR: (400MHz, DMSO-d6) δppm 12.80 (br s, 1H), 11.13 (s, 1H), 7.86 (m, J = 8.0Hz, 2H), 7.33 (m, J = 8.4Hz, 2H), 6.58 (br s,2H),3.03(t,J=7.2Hz,2H),2.66-2.75(m,2H),1.63-1.80(m,4H); MS(ESI)m / z=345.1[M+H] + .

[0397] Step 4: Preparation of (S)-2-{5-[4-(5-amino-7-oxo-6,7-dihydrothiazo[4,5-d]pyrimidin-2-yl)butyl]benzamido}-3-[4-(tert-butoxy)phenyl]propionate tert-butyl ester (intermediate 73)

[0398] The synthesis was performed using a method similar to step 6 of Example 8, except that the starting materials were changed sequentially to intermediate 58 (90 mg, 1.0 eq), DMAc (3 mL), O-tert-butyl-L-tyrosine tert-butyl ester hydrochloride (89 mg, 1.03 eq), TCFH (88 mg, 1.2 eq), and NMI (75 mg, 3.5 eq.) to obtain intermediate 59 (150 mg of off-white solid, yield 92.6%), which was directly used in the next reaction step.

[0399] Step 5: Preparation of (S)-2-{5-[4-(5-amino-7-oxo-6,7-dihydrothiazo[4,5-d]pyrimidin-2-yl)butyl]benzamido}-3-[4-hydroxyphenyl]propionic acid (intermediate 74)

[0400] The synthesis was performed using a method similar to step 7 of Example 8, except that the starting materials were changed sequentially to intermediate 73 (150 mg, 1.0 eq), water (75 mg), and 4N HCl / dioxane (0.75 mL, 5V) to obtain intermediate 74 (90 mg white solid, purity 98.3%, yield 73.3%).

[0401] 1 H NMR: (400MHz, D2O) δ7.60(d,J=7.7Hz,2H),7.23(d,J=7.0Hz,2H),7.03(d,J=8.4Hz,2H),6.58(d,J=8.4Hz,2H),4.58(dd,J=7.7,5.1Hz, 1H),3.14(dd,J=14.0,5.0Hz,1H),3.00–2.95(m,3H),2.62(d,J=6.4Hz,2H),1.74(s,2H),1.61(d,J=6.1Hz,2H); MS(ESI)m / z=508[M+H] + .

[0402] Step 6: Preparation of 4-{2-[(E)-2-((E)-2-[4-((S)-2-{4-[4-(5-amino-7-oxo-6,7-dihydrothiazo[5,4-d]pyrimidin-2-yl)butyl]-2-fluorobenzamido}-2-carboxyethyl)phenoxy]-3-{2-[(E)-3,3-dimethyl-5-sulfonyl-1-(4-sulfonyl)indol-2-ylidene]ethenyl}cyclohex-1-en-1-yl)vinyl]-3,3-dimethyl-5-sulfonyl-3H-indol-1-on-1-yl}butyl-1-sulfonic acid inner salt (compound 8)

[0403] The synthesis was performed using a method similar to step 8 of Example 8, except that the starting materials were changed sequentially to intermediate 74 (70 mg, 1.0 eq.), water (4.2 mL), and SO456 (80%, 138 mg, 0.9 eq.) to obtain compound 10 (74 mg of green solid, purity 97.5%, yield 39.5%).

[0404] 1H NMR: (400MHz, DMSO-d6) δppm 8.48(d,J=8.3Hz,1H),8.32(s,2H),7.75(d,J=8.1Hz,2H),7.64(dd,J=15.8,11.7Hz, 4H),7.57(s,2H),7.38–7.24(m,6H),6.95(d,J=8.4Hz,2H),6.18(d,J=14.1Hz,2H),4 .57(t,J=7.8Hz,1H),4.12(s,4H),3.17–2.93(m,4H),2.77–2.61(m,8H),2.57(s,2H) ,1.91(s,2H),1.79–1.47(m,12H),1.13(s,6H),0.95(s,6H); MS(ESI)m / z=1359[M+H] + 680 [M+2H] 2+ .

[0405] The compounds used in the following test examples, the reference compounds OTL38 (CAS: 1628423-76-6) and ICG (CAS: 3599-32-4) were purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd., and PMX (structure below) was prepared according to the method of CN112010862B.

[0406] Test Example 1: Determination of the photophysical properties of compounds

[0407] The compounds were dissolved in DMSO (D5879, Sigma-Aldrich) to prepare a 50 mM stock solution. The stock solution was then diluted to 1 μM using PBS (B548117, Sangon Biotech (Shanghai) Co., Ltd.) and DMSO, respectively. Measurements were performed in a quartz cuvette using a METTLER UV5Bio UV spectrophotometer, with absorption peak values ​​controlled within the range of 0.2-0.8. The optimal absorption peak for each compound was determined. The emission spectra of each compound under maximum excitation were then measured using an Aglient Technologies Cary Eclipse fluorescence spectrometer, and the fluorescence emission intensity was calculated. ε was calculated using the molar absorptivity formula: A = ε * l * c. Here, A is the amount of light absorbed at a specific wavelength, ε is the molar absorptivity, l is the distance the light travels through the solution, and c is the concentration of the absorbing substance per unit volume. The units of c are mol / L and l are cm. The results showed that the fluorescence intensity of this series of compounds was at the same level as the marketed drug OTL38, with some compounds exhibiting superior fluorescence emission capabilities.

[0408] Table 3. Photophysical properties of the target compound in 1 μM DMSO solution.

[0409] Table 4. Photophysical properties of the target compound in 1 μM PBS solution.

[0410] Test Example 2: Determination of Fluorescence Quantum Yield of Compounds

[0411] The fluorescence quantum yield (Φ) of the compound of the present invention was determined using a reference method in PBS (pH 7.4). F Indocyanine Green (ICG) using the same buffer solution was used as a reference. At pH 7.4, the fluorescence quantum yield of ICG was 0.09. ICG and the compound of this invention were serially diluted to obtain eight concentration points, which were measured by transferring to a quartz cuvette. A METTLER UV5Bio UV spectrophotometer was used for full-spectrum scanning, with absorption peak values ​​controlled below 0.1. Further, the emission spectra at 780 nm excitation were measured using an Aglient Technologies Cary Eclipse fluorescence spectrometer, and the fluorescence emission intensity area was calculated and fitted with the absorbance values ​​at the corresponding concentrations to obtain the corresponding slopes. The fluorescence quantum yield calculation formula is as follows: QY S =((QY) R *K S *η S 2 ) / (K R *η R 2 *100%, QY represents the quantum yield, η represents the refractive index of the solvent, K represents the slope of the linear fitting curve of integrated fluorescence intensity and absorbance, and the subscripts R and S represent the reference and sample, respectively; the relative fluorescence quantum yield is the data obtained after normalization based on ICG. The results show that the fluorescence quantum yield of this series of compounds is significantly higher than that of the marketed drug ICG, better than or at the same level as the marketed drug OTL-038, and better than PMX.

[0412] Table 5. Data on the determination of fluorescence quantum yield of compounds.

[0413] Test Example 3: Determination of the affinity of compounds for the FRα target of folic acid

[0414] Biolayer interferometry (BLI) is a label-free technique based on the principle of optical interference. By monitoring the molecular binding process in real time, the system measures the binding and dissociation constants, and then calculates and analyzes the affinity (K0) through fitting. D Take one vial (25 μg) of biotinylated human FOLR1 recombinant protein, bring it to room temperature, add 125 μl of deionized water, mix well, and then dilute to 5 μg / ml with dilution buffer 1 (Qbuffer: 1×PBS with 0.02% Tween-20 (P1379, Sigma-Aldrich) and 0.2% BSA (B2064, Sigma-Aldrich), pH 7.4). Then, perform serial dilutions with dilution buffer 2 (PBST: 1×PBS with 0.05% Tween-20 + 1% DMSO, pH 7.4) to obtain 5 concentration points (100 uM-3.125 uM). The SMAP biosensor (probe) (160011, Gator) was pre-wetted in dilution buffer 1 for 300 seconds and a baseline was established for 60 seconds. The diluted recombinant protein was then immobilized onto the probe to a height of 4.2 nm. The probe was first subjected to another 60-second baseline establishment in dilution buffer 1, followed by a 30-second baseline establishment in dilution buffer 2. Finally, the binding and dissociation times of the probe were set for 60 seconds in different concentrations of compound dilution buffers. K was calculated and analyzed using the software included with Gator. D Value. In this embodiment, folic acid was used as a positive control. The results showed that the affinity of the compound of the present invention for the target FRα reached or exceeded the affinity level of its natural ligand, folic acid, and was superior to PMX. (A: Affinity <5 nM; B: Affinity 5 nM–50 nM; C: Affinity >50 nM)

[0415] Table 6. Affinity determination data for compound BLI

[0416] Test Example 4: Determination of the photostability of compounds

[0417] Compounds 1-3 were dissolved with OTL38 and SO456 in DMSO to prepare a 50 mM stock solution, which was then serially diluted with DPBS to 2 μM. The solutions were transferred to quartz cuvettes, and the xenon lamp power was adjusted to 780 nm (15 mW / cm²). The solutions in the cuvettes were irradiated with the xenon lamp light at 5-minute intervals for a total duration of 110 min. After each irradiation, the fluorescence spectra of the compounds were scanned using an Aglient Technologies Cary Eclipse fluorescence spectrometer with 780 nm as the excitation light. The fluorescence emission peak area was analyzed and calculated, and the solution not irradiated by the xenon lamp was simultaneously measured. As shown in Figure 1, the photostability of compounds 1-3 under xenon lamp irradiation was improved compared to OTL38.

[0418] Test Example 5: Determination of the binding ability of compounds to folic acid FRα target knockout

[0419] A stable transfection system for FRα knockdown of folate was constructed by infecting KB cells (Nanjing Kebai Biotechnology) with the lentivirus pLKO-Tet-On-puro-shFOLR1. Untreated KB cells served as the control group. 6*10 cells were seeded in 24-well plates. 4 Cells / well, incubated overnight at 37°C with 5% CO2 for cell adhesion, diluted the compound to the target concentration with fresh medium (L540KJ, BasalMedia) and added to 24-well plates, incubated at 37°C in the dark for 1 h, aspirated the medium and washed with PBS, digested with trypsin and then subjected to flow cytometry. NxT, Life Technologies (using APC-Cy7 channel) was used for detection. The results showed that the binding ability of compound 3 of the present invention to target cells was significantly reduced after FRα knockdown. FRα is the main receptor for its binding to target cells, and the FL part (S0456) in its structure did not make a major contribution to its binding ability to FRα.

[0420] Table 7. Inhibition rate of compound-target cell binding ability after FRα target knockdown.

[0421] In Table 7, the inhibition rate (%Inhibition) is calculated as follows:

[0422] Inhibition rate % = 100 * (1 - (mean of sample data - mean of sample solvent control data) / (mean of KB blast cell line control data - mean of KB blast cell line solvent control data))

[0423] Test Example 6: Study on the fluorescence distribution of tissues in mice

[0424] Human oral epidermoid carcinoma KB cell line (Nanjing Kebai Biotechnology) was cultured in MEM + 10% FBS + 1% P / S complete medium, and human non-small cell lung cancer A549 cell line (Chinese Academy of Sciences Type Culture Collection Committee Cell Bank) was cultured in RPMI-1640 + 10% FBS + 1% P / S complete medium, continuously in a CO2-containing incubator for up to ten passages. Seven-week-old female Balb / c Nude mice (Shanghai Yaokang Biotechnology Co., Ltd. / 20230007000774) were fed a low-fluorescence diet and used in this study after one week of acclimatization. The temperature of the experimental animal housing was 20-25℃, the humidity was 40-70%, and the day / night cycle was 12h / 12h. The mice were continuously provided with complete pelleted feed, which was allowed to be consumed freely without limit. They also had purified water available in water bottles for uninterrupted free access.

[0425] Mice were injected with 2×10 mice under the left axilla. 6 100 μL of KB tumor cells were injected into the right axilla. 6 One 100 μL of A549 tumor cells was grown for 3 weeks, resulting in a tumor volume of approximately 200 mm². 3 Animals were administered 200 μL each of OTL38, compound 1, compound 2, and compound 3 at a dose of 0.1 mg / kg via tail vein phosphate buffer. Nine hours after administration, animals were euthanized by CO2 asphyxiation. The animals were then dissected, and tissue imaging (tumor, spleen, lymph nodes, muscle, liver, and kidney) was performed using an infrared camera (ToupTek Photonics, E3ISPM08300KPD). Imaging settings were: excitation: 755 nm; filter: 850 nm; exposure: 100 ms. ImageJ 1.54 software was used to analyze the grayscale values ​​of each tissue.

[0426] Results: As shown in Table 8 and Figure 2, OTL38, compound 1, compound 2, and compound 3 all exhibited high tumor uptake in folate receptor-positive tumors but did not accumulate in folate receptor-negative tumors. They showed high uptake in the kidneys but virtually no fluorescent activity in other tissues.

[0427] Conclusion: OTL38, compound 1, compound 2, and compound 3 are highly specific for folate receptors and mainly accumulate in folate receptor-positive tumors and kidneys, while other normal tissues show low-level uptake or no uptake.

[0428] Table 8. Fluorescence gray values ​​of mouse tissues

[0429] Test Example 7: Time-of-Fluorescence Imaging Study in Mice

[0430] Human oral epidermoid carcinoma KB cell line (Nanjing Kebai Biotechnology) was cultured continuously in MEM + 10% FBS + 1% P / S complete medium at 37℃ with CO2 until passage 10. Seven-week-old female Balb / c Nude mice were fed a low-fluorescence diet and used in this study after one week of acclimatization. The temperature in the experimental animal housing was 20-25℃, the humidity was 40-70%, and the day / night cycle was 12h / 12h. The mice were continuously provided with complete pelleted feed, which was allowed to be consumed freely without restriction. They also had purified water available in water bottles for uninterrupted access.

[0431] Mice were injected with 2×10⁻⁶ cells in each axilla. 6 One KB tumor cell, 100 μL, grew for 3 weeks and the tumor volume was approximately 200 mm. 3 OTL38, compound 1, compound 2, compound 3, and compound 5 were administered via tail vein in 200 μL of phosphate buffer at a concentration of 0.1 mg / kg. At 3 h, 6 h, 9 h, and 24 h, two nude mice from each group were randomly selected and euthanized by cervical dislocation. Tumors and adjacent tissues were then removed and in vitro imaging was performed using a 4K open-cell fluorescence imaging system (OPTO-CAM214K). Image J 1.54 was used to analyze the fluorescence grayscale values ​​of the tumor and background areas, and the TBR (tumor-background ratio) was calculated as the ratio of tumor grayscale value to adjacent normal tissue grayscale value. Detailed results are shown in Table 9.

[0432] Table 9. Results of the fluorescence imaging time screening test of compounds in mice.

[0433] Results: As shown in Figures 3, 4, and 5, under the 4K open-circuit fluorescence imaging system, high tumor uptake was observed at folate receptor-positive tumor sites at all time points, with no obvious fluorescence in the surrounding tissues, except at 24 h, where the tumors and background fluorescence of compounds 1, 2, and 5 were weak. Compound 3 showed the brightest tumor brightness, with visible background fluorescence; compound 5 showed the darkest tumor brightness. Software analysis of the TBR values ​​of tumor and background fluorescence in each group showed that at 24 h, compound 3 had the highest TBR (3.55), and OTL38 had a TBR of 3.20. At 9 h, the overall TBR for each group was: compound 5 > compound 2 > 3.0.

[0434] Conclusion: All compounds in vivo within 24 hours showed specificity for folate receptors. Background tissues around folate receptors exhibited low-level uptake or no uptake, resulting in a high fluorescence ratio between tumor and background tissues.

[0435] Test Example 8: In vivo fluorescence imaging intensity study in mice

[0436] KB cells (Nanjing Kebai Biotechnology, folate receptor positive) were cultured in MEM + 10% FBS + 1% P / S complete medium, A549 cells (Chinese Academy of Sciences Type Culture Collection Committee Cell Bank, folate receptor negative), and LNCaP cells (Nanjing Kebai Biotechnology, folate receptor negative) were cultured in RPMI-1640 + 10% FBS + 1% P / S complete medium, respectively, in a CO2-containing incubator at 37℃ for up to ten passages. Seven-week-old female Balb / c Nude mice were fed a low-fluorescence diet and used in this study after one week of acclimatization. The temperature in the experimental animal housing was 20-25℃, the humidity was 40-70%, and the day / night cycle was 12h / 12h. Mice were continuously provided with complete pelleted feed, which was allowed to be consumed freely without restriction. Purified water was provided continuously from water bottles for free consumption.

[0437] Three groups of mice were injected with 2×10⁻⁶ mice under both armpits. 6 100 μL of KB tumor cells, 4 × 10 6 100 μL of A549 tumor cells, 5 × 10 6 One 100 μL of LNCaP tumor cells was incubated for 3 weeks. The tumor volume was approximately 200 mm². 3 At the same time, OTL38, compound 1, compound 2, compound 3, compound 4, compound 5, compound 7, and compound 8 were injected intravenously in 200 μL of phosphate buffer at a concentration of 0.1 mg / kg. Six hours after administration, nude mice were euthanized by cervical dislocation, and tumors and surrounding tissues were removed. In vitro imaging experiments were performed using a 4K open-circuit fluorescence imaging system. The fluorescence grayscale values ​​of the tumor and background areas were scored using Image J 1.54, and the TBR values ​​were calculated. See Table 10 for details.

[0438] Table 10 Fluorescence imaging intensity data of the compounds in KB tumor-bearing mice.

[0439] Results: As shown in Figures 6 and 7, under the 4K open-circuit fluorescence imaging system, all compounds showed significant fluorescence at folate receptor-positive tumor sites, while no significant fluorescence was observed in tissues near the tumor. Compound 3 showed the brightest tumor fluorescence, with visible background fluorescence, and its brightness was significantly higher than that of OTL038 tumors; compound 5 showed the darkest tumor fluorescence intensity. All compounds were enriched in tumor tissues of the KB cell mouse model but did not accumulate in folate receptor-negative tumors such as A549 cells and LNCaP cells, indicating the specificity of the compounds of this invention for folate receptors.

[0440] Conclusion: All compounds have an affinity for the folic acid receptor.

[0441] Test Example 9: Hepatocyte Metabolic Stability

[0442] Experimental method: Take 0.5*106 99 μL of human, monkey, dog, rat, and mouse hepatocyte suspensions (cells / mL) were transferred to 48-well plates, and 1 μL of the compound drug solution was added to the aliquoted hepatocyte suspension. The plates were incubated in a CO2 incubator at 37°C, 5% CO2, and 500 rpm. At the reaction time point, 600 μL of internal standard working solution was added to terminate the reaction. The mixture was vortexed, centrifuged at 5500g for 10 minutes at 6°C, and 150 μL of the supernatant was transferred to a 96-well plate containing 150 μL of ultrapure water. The mixture was vortexed, and the sample was analyzed by LC-MS / MS. The half-life (t) was calculated using first-order kinetics. 1 / 2 ) and clearance rate (CL).

[0443] The test results are shown in Table 11.

[0444] Table 11 Results of hepatocyte metabolic stability

[0445] Conclusion: Under the conditions of this experiment, the residual rate of the series of compounds of this invention after incubation with hepatocytes for 4 hours was ≥83.8%, and the metabolic rate of hepatocytes was very low. The residual rate of OTL-038 after incubation with hepatocytes for 4 hours was in the range of 32-84%. MULTI-DISCIPLINE REVIEW (Application number: 214907Orig1s000:38). The compounds in this invention exhibit better hepatocyte metabolic stability than OTL-038.

[0446] Test Example 10: CYP Enzyme Metabolic Phenotypic Study

[0447] Experimental Methods: A certain amount of phosphate buffer solution was added to a centrifuge tube, followed by a certain amount of MgCl2 solution, and then each subtype of recombinant enzyme was added separately. The mixture was vortexed and aliquoted into 89 μL / tube. 1 μL of compound 1 / compound 2 working solution was added and pre-incubated in a 37°C water bath for 5 min. At the same time, NADPH was pre-incubated in a 37°C water bath for 5 min. 10 μL / well of NADPH working solution was added to start the reaction and incubated for 60 min. Then, 600 μL / well of internal standard working solution was added and vortexed. For the control sample without NADPH, 10 μL / well of phosphate buffer solution was added to start the reaction and incubated for 60 min. Then, 600 μL / well of internal standard working solution was added and vortexed. After vortexing, the sample was centrifuged at 6°C and 5500g for 10 min. 150 μL of the supernatant was added to 150 μL of ultrapure water, vortexed, and analyzed by LC-MS / MS.

[0448] The test results are shown in Table 12.

[0449] Table 12 Results of CYP enzyme metabolic phenotype study

[0450] Note: Data on the percentage of remaining raw material in Table 12 exceeding 100% are due to experimental measurement error.

[0451] Conclusion: Under the experimental conditions, the compounds of this invention are not metabolized by CYP enzymes.

[0452] Test Example 11: Identification Study of S9 Metabolites

[0453] Experimental Method: Compound 1 / Compound 2 working solutions were added to the S9 working solutions of liver from five different species and pre-incubated at 37°C for 3 min. For the 0 min sample: 200 μL of internal standard working precipitant was added, followed by 30 μL of NADPH solution. For other samples: 135 μL of NADPH solution was added to initiate the reaction. After incubation at 37°C for 120 min, 60 μL of the incubated sample was removed and 200 μL of internal standard precipitant was added. All samples were vortexed and centrifuged. 150 μL of the supernatant was added to 150 μL of water, vortexed, and analyzed by LC-MS / MS.

[0454] The experimental results are shown in Tables 13 and 14.

[0455] Table 13 Information on Compound 1 and its metabolites

[0456] Table 14 Information on Compound 2 and its metabolites

[0457] Conclusion: The compounds in this series are mainly metabolized unchanged in the liver S9 cells of the five species, and no specific metabolites were formed in the human liver S9 cells.

[0458] Test Example 12: Hepatic Microsomal Metabolic Stability

[0459] Experimental Methods: 15 μL of compound 3 working solution was mixed with 1185 μL of mouse, rat, dog, monkey, and human liver microsomal working solutions, respectively. 200 μL of each solution (n=3) was then transferred to a 96-well plate and pre-incubated at 37°C for 5 minutes. 50 μL of NADPH cofactor solution was added to the 200 μL incubation sample to initiate the reaction. After mixing, 30 μL of the sample was quickly added to an organic solvent solution containing the internal standard to terminate the reaction; this was the 0-minute sample. At 5, 15, 30, 45, and 60 minutes, 30 μL of the sample was added to the organic solvent solution containing the internal standard to terminate the reaction. The precipitated sample was vortexed and centrifuged, and the supernatant was collected for LC-MS / MS analysis. The peak area ratio of the analyte to the internal standard was used to calculate the half-life T. 1 / 2 Including inherent clearance rate, etc.

[0460] The test results are shown in Table 15.

[0461] Table 15 Information on Compound 3 and its hepatic microsomal metabolic stability

[0462] Conclusion: After incubation with liver microsomes of different species for 60 min, the residual amount of compound 3 of the present invention was ≥72.7%, with almost no metabolic trend observed in the human species. Compound 3 was slowly metabolized in the liver microsomes of mice, rats, monkeys, and humans, and moderately metabolized in dogs.

[0463] Test Example 13: Liver S9 metabolic stability

[0464] Experimental Method: 15 μL of compound 3 working solution was mixed with 1185 μL of mouse, rat, dog, monkey, and human liver S9 working solutions, respectively. 200 μL of each solution (n=3) was transferred to a 96-well plate and pre-incubated at 37°C for 5 minutes. 50 μL of cofactor solution was added to 200 μL of the incubated sample to initiate the reaction. After mixing, 30 μL of the sample was quickly added to an organic solvent solution containing the internal standard to terminate the reaction; this was the 0-minute sample. At 5, 15, 30, 45, and 60 minutes, 30 μL of the sample was added to an organic solvent solution containing the internal standard to terminate the reaction. The precipitated sample was vortexed and centrifuged, and the supernatant was collected for LC-MS analysis. The peak area ratio of the analyte to the internal standard was used to calculate the half-life T. 1 / 2 Including inherent clearance rate, etc.

[0465] The test results are shown in Table 16.

[0466] Table 16 Information on Compound 3 and its metabolic stability in liver S9.

[0467] Conclusion: After incubation with liver S9 of different species for 60 min, the residual amount of compound 3 of the present invention was ≥90.7%, and it was slow metabolized in liver S9 of mice, rats, dogs, monkeys and humans.

[0468] Test Example 14: CYP Metabolic Enzyme Phenotypic Study

[0469] Human CYP recombinase: Add 5 μL of Compound 3 working solution to 495 μL of different isotype human recombinase working solutions and mix well (n=1). Take 60 μL of the solution (n=3) and pre-incubate at 37°C for 5 minutes. Then add 60 μL of 2 mM NADPH solution (for samples containing NADPH) or phosphate buffer solution (for samples without NADPH) and mix well. Quickly take 30 μL of the sample and add it to 200 μL of organic solution containing internal standard to precipitate the protein, as the 0-minute sample (sample placed on ice), and start timing simultaneously. At 60 minutes, take 30 μL of the sample and add it to acetonitrile containing 60 μL of 5 mM sodium acetate solution and 200 μL of 100 ng / mL internal standard to terminate the reaction. Vortex and centrifuge at 4000 rpm for 10 minutes. Dilute the supernatant with 2 volumes of 0.01% ammonia and analyze by liquid chromatography-mass spectrometry.

[0470] Human liver microsomes: Add 318 μL of liver microsome working solution containing compound 3 to an incubation tube (n=3). Add 2 μL of inhibitor working solution to the corresponding tube and pre-incubate at 37°C for 5 minutes. For samples without inhibitor, add 2 μL of inhibitor solvent as a substitute. Add 80 μL of 5 mM NADPH solution to start the reaction. For samples without NADPH, add 80 μL of phosphate buffer as a substitute. Quickly remove 30 μL of sample and add it to 200 μL of organic solution containing internal standard to precipitate the protein, as the 0-minute sample (sample placed on ice), and start timing simultaneously. At the 60-minute time point, remove 30 μL of sample and add it to acetonitrile containing 60 μL of 5 mM sodium acetate solution and 200 μL of 100 ng / mL internal standard, respectively, to terminate the reaction. Vortex and centrifuge at 4000 rpm for 10 minutes. Dilute the supernatant with 2 volumes of 0.01% ammonia and analyze using liquid chromatography-mass spectrometry.

[0471] The experimental results are shown in Tables 17 and 18.

[0472] Table 17 Results of the metabolic enzyme phenotype study of compound 3 in recombinant enzymes

[0473] Table 18 Results of the metabolic enzyme phenotype study of compound 3 in human liver microsomes

[0474] Note: In Tables 17 and 18, if the remaining amount of FZ-P001 is ≥94.1%, then the metabolic amount treatment is 0.0.

[0475] Compound 3 (1 μM) was co-incubated with seven human recombinant enzymes of CYP enzyme isotypes (100 pmol / mL) for 60 minutes with or without NADPH, and the remaining percentage of compound 3 was determined. Compared with 0 minutes, the average remaining percentage of compound 3 in the seven human recombinant enzyme isotypes ≥101.7% under NADPH-added conditions, indicating that none of these seven enzymes are the major metabolic enzymes of compound 3.

[0476] Compound 3 (1 μM) was co-incubated with human liver microsomes (0.5 mg / mL) for 60 minutes with or without the addition of CYP enzyme-specific inhibitors. The remaining percentage of compound 3 was then determined, and the metabolic rate and inhibition rate were further calculated. The remaining amount of compound 3 was ≥94.1% with or without the addition of the specific inhibitors for each of the seven enzymes, indicating no inhibitory effect. These results suggest that none of the seven enzymes are the major metabolic enzymes of FZ-P001.

[0477] Conclusion: Under the conditions of this experiment, compound 3 was not metabolized by CYP enzymes.

[0478] Test Example 15: Identification of Liver Microsomal Metabolites

[0479] Add 178 μL of liver microsomal working solution to a tube (n = 2). For T 120 For each sample, add 2 μL of Compound 3 working solution to 178 μL of liver microsome working solution and mix gently (n=2). Pre-incubate all samples with the cofactor (NADPH) solution in a 37°C water bath for 5 min. Add 20 μL of the cofactor (NADPH) solution to the pre-incubation system to start the reaction. Immediately transfer the incubation system to a 37°C water bath and incubate for 120 min. For T0 samples, first add 200 μL of 5 mM sodium acetate, then add 400 μL of acetonitrile to terminate the reaction, followed by 2 μL of Compound 3 working solution and 20 μL of cofactor (NADPH) solution (n=2). After incubation for 120 min, immediately add 200 μL of 5 mM sodium acetate, then add 400 μL of acetonitrile to terminate the reaction. Centrifuge all samples at 12,000 rpm and 4°C for 10 min. Combine 150 μL of supernatant from each replicate. Take 100 μL of the combined supernatant and add 200 μL of a 2 mM ammonium acetate aqueous solution containing 0.1% ammonia. Mix the solution directly for injection analysis.

[0480] The results are shown in Table 19.

[0481] Table 19. Relative abundance of compound 3 and its metabolites in liver microparticles of mice, rats, dogs, monkeys, and humans.

[0482] +: Detected only by mass spectrometry, not by ultraviolet light.

[0483] Conclusion: After incubation for 120 min in mouse, rat, dog, monkey, and human liver microsomes, two possible metabolites were detected for compound 3. Compound 3 is metabolized in mouse, rat, dog, monkey, and human liver microsomes via monooxidation and dealkylation pathways. In all species, the parent compound 3 was the predominant form of exposure, with a relative abundance of over 95% in the UV absorption peak of the parent compound. No metabolites specific to human microsomes were found.

Claims

1. A compound of formula (I), a pharmaceutically acceptable salt thereof, or a tautomer thereof; in, X is CH or N, Y is S; or Y is CH or N, X is S; n is 2, 3, 4, 5 or 6; It is a C / C single or double bond, and a ring. It has aromatic properties; AR stands for "5-10 membered heteroaryl groups having 1-4 heteroatoms independently selected from N and O", C6-C 10 Aryl, "a 5- to 10-membered heterocyclic alkenyl group having 1 to 3 heteroatoms independently selected from N, O, and S" or C5-C 10 Cycloalkyl, and AR is formed by the ring carbon atom with a C=O group and (CH2). n Group linkage; AR optionally linked by 1 to 3 R groups 0 Replace; the R mentioned above 0 It is a halogen; AA is The a-terminus is connected to the carbonyl group; L 1 It is a C1-C6 alkylene or a C1-C4 alkylene-phenyl; L 2 is O, S or NH; * indicates that the chirality of the carbon atom is S, R, or a mixture of S and R; FL is a fluorescent dye.

2. The compound of formula (I) as claimed in claim 1, its pharmaceutically acceptable salt, or its tautomer, characterized in that, One or more of the following conditions must be met: (1) The 5-10 heteroaryl group having 1-4 heteroatoms independently selected from N and O is a 5-6 heteroaryl group having 1 heteroatom independently selected from N and O, such as pyridyl, indolyl, indazole, furanyl, benzofuranyl, oxazolyl, tetrazolyl, and pyridine, for example. (2) The C6-C 10 The aryl group is phenyl or naphthyl, preferably phenyl; (3) The 5- to 10-membered heterocyclic alkenyl group having 1 to 3 heteroatoms independently selected from N, O and S is a 5- to 6-membered heterocyclic alkenyl group having 1 heteroatom independently selected from N, O and S; (4) The 5- to 10-membered heterocyclic alkenyl group contains one or two unsaturated double bonds, and the 5- to 10-membered heterocyclic alkenyl group is not aromatic; (5) The C5-C 10 The cycloalkyl group can be a monocyclic, fused, spirocyclic, or bridged ring, preferably a bridged ring; (6) The C5-C 10 The cycloalkyl group is a C6-C8 bridged cycloalkyl group, for example... (7) The halogen is Cl, F, Br, or I, preferably F; (8) The C1-C6 alkylene group is a C1-C4 alkylene group, for example... Preferred More preferably (9) said C1-C4alkylene-phenyl is And (10)AR is either not replaced or replaced by 1 R 0 Replacement; preferably, AR is not replaced.

3. The compound as shown in formula (I), a pharmaceutically acceptable salt thereof or a tautomer thereof according to claim 1, wherein, One or more of the following conditions must be met: ​ (1) n is 3 or 4; (2)R 0 It can be Cl, F, Br or I independently, preferably F; (3) AR is a 5-6 membered heteroaryl group with one heteroatom independently selected from N and O, surrounded by 1-3 R atoms. 0 The substituted phenyl, phenyl, or C6-C8 bridged cycloalkyl group is preferably a 5-6 membered heteroaryl group containing one nitrogen atom and surrounded by 1-3 R atoms. 0 Substituted phenyl, phenyl or C6-C8 bridged cycloalkyl; Preferably, AR is phenyl, F-substituted phenyl, pyridyl or bicyclooctane; More preferably, AR is Further preferably, AR is (4) When L 1 When it is a C1-C4 alkylene-phenyl, L 1 phenyl and L 2 Connected; (5)L 1 It is a C1-C4 alkylene or a C1-C3 alkylene-phenylene, preferably methylene, n-butylene or methylene-phenylene, more preferably methylene-phenylene; (6) L 2 is O; (7) *The chirality of the carbon atom is marked as S or R, preferably S; (8) AA is Preferably Preferably, AA is Preferably (9) FL is a dye that has fluorescence excitation and emission spectra in the near-infrared range; Preferably, FL is Among them, R 1a and R 1b It can be independently O, S, -NH- or -CH2-, preferably -CH2-; R 2 It is -CH2- or -CH2CH2-, preferably -CH2-; R 3 and R 4 It is -(CH2)3SO3H; R 5 and R 6 Independently, it is a halogen, hydroxyl, amino, -NH (C1-C6 alkyl), -N (C1-C6 alkyl)2, C1-C6 perfluoroalkyl, C1-C6 alkyl, C1-C6 alkoxy or SO3H, preferably SO3H; X 1 and X 2 Independent for CR 7 2. NR 8 O or S, R 7 and R 8 Independently selected from C1-C4 alkyl groups; preferably, X 1 and X 2 For CR 7 2, R 7 It is methyl; More preferably, FL is and (10) structural fragments For Preferably 4. The compound of formula (I) as claimed in any one of claims 1-3, its pharmaceutically acceptable salt, or its tautomer, characterized in that, The structure of the compound of formula (I) is shown as formula (II) or formula (III): Where X is CH or N; Y is either CH or N; The definitions of n, AR, AA and FL are as described in any one of claims 1-3; Preferably, X is N; Y is N; AR is 5. The compound as shown in formula (I), a pharmaceutically acceptable salt thereof or a tautomer thereof according to any one of claims 1-3, wherein Satisfy one of the following options: Option 1, Option 2, and Option 3: Option 1: The structure of the compound of formula (I) is shown as formula (IV); wherein X, Y, n, AR, *, L 1 and L 2 The definition is as described in any one of claims 1-3; Option 2: The structure of the compound of formula (I) is shown as formula (II); in, X is CH or N, preferably N; n is 3 or 4; AR is a 5- to 6-membered heteroaryl, phenyl, or C6-C8 bridged cycloalkyl group that contains one nitrogen atom; AA is The a-terminus is connected to the carbonyl group; L 1 It is a C1-C6 alkylene or a C1-C4 alkylene-phenyl; L 2 For O, S, or NH; FL is Option 3: The structure of the compound represented by formula (I) is shown in formula (III); in, Y can be CH or N, preferably N; n is 3 or 4; AR is a 5- to 6-membered heteroaryl, phenyl, or C6-C8 bridged cycloalkyl group that contains one nitrogen atom; AA is The a-terminus is connected to the carbonyl group; L 1 It is a C1-C6 alkylene or a C1-C4 alkylene-phenyl; L 2 For O, S, or NH; FL is 6. The compound of formula (I) as claimed in claim 1, its pharmaceutically acceptable salt, or its tautomer, characterized in that, The compound represented by formula (I) is any one of the following compounds: Preferably, the compound represented by formula (I) above is any of the following compounds:

7. A method for preparing a compound of formula (IV) as described in any one of claims 1-6, comprising the following steps: (1) The compound shown in formula (VI) undergoes a deprotection reaction in the presence of acid, and the pH is adjusted to alkaline after the reaction is complete. (2) In an alkaline aqueous solution, the product obtained in step (1) reacts with SO456 to obtain the compound shown in formula (IV); Among them, R 1 It is a C1-C6 alkyl protecting group, preferably tert-butyl; X, Y n, AR, *, L 1 and L 2 The definition is as described in any one of claims 1-6; Preferably, the preparation method satisfies one or more of the following conditions: (1) In step (1), the deprotection reaction is carried out in the presence of a haloalkane solvent (e.g., DCM) or water, or directly in an acid. (2) In step (1), the acid is a strong protic acid, such as trifluoroacetic acid or hydrochloric acid, preferably trifluoroacetic acid; (3) In step (1), the alkaline pH value is 9-12, for example 11; (4) Step (1) includes the following steps: in a haloalkane solvent (e.g., DCM), the compound shown in (VI) undergoes a deprotection reaction in the presence of trifluoroacetic acid. After the deprotection reaction is completed, the acid is removed under reduced pressure and then adjusted to alkalinity with sodium hydroxide solution. (5) Step (1) includes the following steps: in a haloalkane solvent (e.g., DCM), the compound shown in (VI) undergoes a deprotection reaction in the presence of hydrochloric acid, and after the deprotection reaction is completed, it is directly adjusted to alkalinity with sodium hydroxide solution. (6) Step (1) includes the following steps: the compound shown in (VI) undergoes a deprotection reaction in the presence of water and trifluoroacetic acid. After the deprotection reaction is completed, the reaction solution is added to methyl tert-butyl ether and then adjusted to alkalinity with sodium hydroxide solution. (7) In step (2), the pH value of the alkaline aqueous solution is 9-12, for example 11; (8) In step (2), the temperature of the reaction is 70-100℃, for example 90℃.

8. A pharmaceutical composition comprising a compound of formula (I) as described in any one of claims 1-6, a pharmaceutically acceptable salt thereof, or a tautomer thereof, and further comprising at least one pharmaceutically acceptable carrier or excipient.

9. Use of a compound of formula (I) as described in any one of claims 1-6, a pharmaceutically acceptable salt thereof, or a tautomer thereof, or a pharmaceutical composition as described in claim 8, said use comprising: (1) Use in the preparation of tumor diagnostic reagents for tumor-targeted imaging; Alternatively, (2) the use of an image-guided reagent in the preparation of a surgical procedure for a subject with a disease, wherein the reagent is visualized by irradiating a designated surgical site with infrared light, the disease being a tumor; Preferably, the tumor is breast cancer, lung cancer, ovarian cancer, endometrial cancer, bladder cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, head and neck cancer, or mesothelioma, and is more preferably ovarian cancer or lung cancer.

10. A compound represented by formula (VI), in, R 1 The protecting group is a C1-C6 alkyl group or hydrogen, and the C1-C6 alkyl protecting group is preferably tert-butyl; X, Y n, AR, *, L 1 and L 2 The definition is as described in any one of claims 1-6; Preferably, the compound represented by formula (VI) is any of the following compounds:

11. A compound represented by formula (VII), in, X, Y The definitions of n and AR are as described in any one of claims 1-6; preferably, the compound represented by formula (VII) is any one of the following compounds:

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