Compounds, compositions, methods of making and uses thereof targeting folate receptors

Abstract

The application discloses a kind of compound, composition, its preparation method and application of targeting folate receptor, belong to medical biotechnology field, the compound of targeting folate receptor is F-L-D, wherein, F is the structure of targeting folate receptor, D is near infrared dye, L is linker, structural formula is: Or its pharmaceutically acceptable salt.The compound of targeting folate receptor of the application is used as targeted near infrared fluorescent imaging agent, can be used with near infrared fluorescent camera system, can specifically target folate receptor FR-alpha, is used for the auxiliary diagnosis of intraoperative identification of malignant lesion of tumor patient.The application scene of the compound in lung cancer, ovarian cancer, can show higher TNR, can show tumor boundary in real time, to help identify intraoperative lesion, reduce positive margin rate and false positive rate, realize accurate resection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medical biotechnology, and particularly relates to a folate receptor-targeting compound, a composition, a preparation method and application thereof. BACKGROUND

[0002] Radical surgical resection of malignant tumors is the only reliable method to cure cancer. Therefore, the treatment of tumors urgently needs to introduce techniques that enhance the ability of tumor visualization. These techniques are expected to change the standard of surgical care by detecting the surgical margin, subclinical residual lesions, lymph node metastasis and synchronous / asynchronous tumor metastasis in real time.

[0003] Near-infrared (NIR) fluorescence-guided surgery (FGS) has shown great prospects as an intraoperative imaging mode. Intraoperative near-infrared (NIR) fluorescence imaging helps surgeons make judgments and reduces the positive rate of surgical margins, and has become an effective solution for detecting intraoperative tumor margins. Fluorescence imaging intraoperative navigation can accurately display information such as intraoperative blood vessels, lymph nodes, and tumor tissues based on the enrichment of fluorescein in specific tissues. At the same time, it can specifically visualize specific molecular targets overexpressed in tumor tissues, thereby achieving the effect of high signal-to-noise ratio tumor imaging and further reducing the positive rate of surgical margins. The wavelength range of near-infrared light is 650 nm to 900 nm, and its penetration depth in tissue can reach 10 mm, which is much higher than that of visible light, so it has a significant advantage in intraoperative imaging. Since normal tissues only show limited autofluorescence in the NIR spectrum, the use of NIR fluorescence imaging agents can maximize the contrast between fluorescent signals in tumors and healthy tissues. In addition, since near-infrared light is invisible to the human eye, near-infrared light will not affect the surgical field of view. The currently available imaging system combines white light illumination of the surgical area with NIR fluorescence images, providing surgeons with both visible light anatomical information and fluorescence information.

[0004] Folate receptor alpha (FR-α), also known as FOLR1 or folate-binding protein, is a glycoprotein anchored on the cell membrane by glycosylated phosphatidylinositol (GPI) and has high affinity for folic acid, which can transport folic acid through receptor-mediated endocytosis. FR-α acts as a signal molecule to promote the malignant growth of tumors. Similar to other glycosylated phosphatidylinositol family proteins, FR-α initiates an intracellular regulatory signal network after binding to folic acid. FR-α is mainly highly expressed in tumor tissues of epithelial origin and is not expressed or has very low expression in normal tissues, and has high affinity for folic acid and low affinity for reduced folic acid.

[0005] FR-α is known to be overexpressed on the entire tumor cell surface of various cancers, including ovarian cancer, triple-negative breast cancer (TNBC), endometrial cancer, mesothelioma, and lung cancer. FR-α is expressed at a low level in normal cells, but at a significantly higher level in some cancer cells, and has become an important target in cancer diagnosis and treatment in recent years. CYTALUX (Pafolacianine, code OTL38) is a new structure of targeted fluorescent imaging agent, which can specifically target folate receptor FR-α, and is used for intraoperative auxiliary diagnosis of adult ovarian cancer and lung cancer patients to identify malignant lesions. OTL38 has high binding affinity to FR-α, and has high fluorescence value accumulated in the tumor site in small animal live imaging test, but at the same time, it has high background fluorescence value, slow elimination rate, resulting in low tumor background ratio (TBR) and tumor tissue ratio (TNR).

[0006] Patent document CN119490506A discloses a small molecule compound targeting folate receptor, a preparation method, composition and application, wherein the compound represented by formula I is as follows:

[0007] The compound or its pharmaceutically acceptable salt can be used as a small molecule targeted fluorescent contrast agent for auxiliary surgical treatment, has novel structure, good solubility, small steric hindrance and strong targeting, and can significantly improve the visual field of tumor surgery, which is beneficial to efficient and precise implementation of resection of tumor tissue and boundary tissue and other lesion tissues. Although the present application improves the TBR compared with OTL38, there is still room for improvement in the TNR in lung cancer and ovarian cancer. Based on the clinical needs of precise diagnosis and treatment, how to develop a new type of targeted near-infrared fluorescent imaging agent that can specifically target folate receptor FR-α with higher TNR based on the compound represented by formula I has important clinical significance for the diagnosis and treatment of tumors. SUMMARY

[0008] To solve the above technical problems, the present application provides a compound targeting folate receptor, a composition, a preparation method and application thereof. The compound targeting folate receptor of the present application can exhibit higher TNR in the application scenario of lung cancer and ovarian cancer, display the tumor boundary in real time, help to identify intraoperative lesions, reduce the positive margin rate and false positive rate, and achieve precise resection.

[0009] In a first aspect, the present application provides a compound targeting folate receptor, wherein the compound targeting folate receptor is F-L-D, and

[0010] F is a structure targeting folate receptor, and the structural formula is:

[0011] ;

[0012] D is a near infrared dye having the structure:

[0013] or a pharmaceutically acceptable salt thereof.

[0014] L is a linker having the structure:

[0015] or a pharmaceutically acceptable salt thereof.

[0016] wherein R1is selected from any one of the following structures:

[0017] and

[0018] R3is selected from any one of the following structures:

[0019]

[0020] n is each independently an integer from 0 to 10.

[0021] R2is selected from any one of the following structures:

[0022] or a pharmaceutically acceptable salt thereof.

[0023] In some embodiments, the pharmaceutically acceptable salt of the present application is independently selected from a sodium salt, a potassium salt, or a lithium salt.

[0024] In some embodiments, n of the present application is an integer from 0 to 5; preferably 0, 1, 2, 3, 4, 5, or a range of values between any two of the aforementioned.

[0025] In some embodiments, R1of the present application is selected from any one of the following structures: or

[0026] In some embodiments, R1of the present application is Preferably, R3is selected from any one of the following structures: ,​​​​​​​​​​​​​​​​​ 、 、 n is an integer from 0 to 5.

[0027] In some embodiments, the folate receptor targeting compound of the present application has any one of the following structures:

[0028]

[0029] Preferably, the folate receptor targeting compound has any one of the following structures:

[0030]

[0031] Further preferably, the folate receptor targeting compound is:

[0032] In a second aspect, the present application provides a method for preparing the above-mentioned folate receptor targeting compound, comprising the following steps:

[0033] (1) first generating a compound V containing F and L structures by reaction, the compound V has the following structure:

[0034]

[0035] Compound V

[0036] wherein R1 and R2 have the same meanings as R1 and R2 described above, respectively.

[0037] (2) reacting the compound V with a compound W to obtain the folate receptor targeting compound, the compound W has the following structure:

[0038]

[0039] Compound W

[0040] wherein M 1 , M 2 , and M 3 are each independently H or a pharmaceutically acceptable cation.

[0041] In some embodiments, the cation is a monovalent cation, preferably a sodium ion, a potassium ion or a lithium ion.

[0042] In some embodiments, the reaction in step (2) is carried out in an aqueous carbonate solution. Preferably, the carbonate is sodium carbonate, potassium carbonate or lithium carbonate.

[0043] In some embodiments, the reaction conditions of the reaction in step (2) are 70-85°C for 0.5-2h. Preferably, 75-80°C for 0.5-2h.

[0044] Based on the above preparation method, the present application further provides a compound V, whose structural formula is shown as follows:

[0045]

[0046] wherein R1 and R2 have the same meanings as R1 and R2 described above, respectively.

[0047] In a third aspect, the present application provides a composition comprising the above-mentioned folate receptor-targeting compound and a pharmaceutically acceptable excipient.

[0048] The composition of the present application can be administered by parenteral routes. The composition of the present application can be administered by intravenous injection, intramuscular injection or subcutaneous injection. It is usually provided as a sterile aqueous solution or suspension or lyophilized powder, and adjusted to a suitable pH and isotonicity.

[0049] In a fourth aspect, the present application provides use of the above-mentioned folate receptor-targeting compound or the above-mentioned composition in the preparation of a near-infrared fluorescence imaging agent.

[0050] In a fifth aspect, the present application provides use of the above-mentioned folate receptor-targeting compound or the above-mentioned composition in the preparation of a diagnostic or therapeutic product for tumors.

[0051] The targeted folate receptor compound of the present application can achieve specific accumulation and retention in FR-α positive tumor tissues after administration, while being rapidly cleared in normal tissues, thereby achieving excellent tumor-to-background ratio (TBR), tumor tissue ratio (TNR) and imaging effect. FR-α is overexpressed in various epithelial-derived tumors, such as ovarian cancer, breast cancer, pleural cancer, lung cancer, cervical cancer, endometrial cancer, kidney cancer, bladder cancer, brain cancer, etc. The present application demonstrates its effectiveness in various solid tumors such as lung cancer, ovarian cancer, fallopian tube cancer, endometrial cancer, breast cancer or colorectal cancer through specific examples. Those skilled in the art should understand that the overexpression of FR-α in various malignant tumors is a recognized biological characteristic, so the imaging agent is also applicable to any other FR-α overexpressing tumor type without departing from the protected molecules of the present application. Preferably, the tumor is a tumor associated with overexpression of FR-α. Further preferably, the tumor includes at least one of lung cancer, ovarian cancer, fallopian tube cancer, endometrial cancer, breast cancer and colorectal cancer.

[0052] In some embodiments, the product described in the present application is a tumor diagnostic reagent or a tumor imaging reagent.

[0053] In a sixth aspect, the present application provides a kit for tumor targeting, comprising the aforementioned targeted folate receptor compound or the aforementioned composition.

[0054] Compared with the prior art, the present application has the following beneficial effects:

[0055] (1) The present application obtains a compound with a specific spatial structure by optimizing the structure of the linker L, so that the target molecule has sufficient distribution volume, accumulates in the target tissue, and rapidly clears the background in a clinically convenient time, achieving high contrast between the tumor and the background, avoiding autoluminescence and the superposition of fluorescence after the drug enters the blood and the tissue, and causing possible false positives.

[0056] (2) The compound of the present application is a series of novel structure targeted folate receptor FR-α imaging agents, which can be used for intraoperative auxiliary diagnosis of various tumors. Compared with the prior art OTL38, the compound of the present application has more excellent pharmacokinetic characteristics, can provide higher TBR and TNR, and by being combined with a near-infrared fluorescence lens, can provide clear tumor boundaries for doctors during surgery, while discovering small or occult lesions, reducing the positive margin rate, and reducing the risk of disease recurrence. At the same time, compared with the compound disclosed in the prior art CN119490506A, the compound of the present application can exhibit higher TNR in the application scenarios of lung cancer and ovarian cancer, thereby presenting clearer lesion boundaries during surgery.

[0057] (3) Compared with the prior art CN119490506A, the compound C has higher TBR and TNR, and provides a better imaging scheme for the precise operation of FR-α related tumors. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 Tumor-to-background fluorescence ratio (TBR) at different time points in the KB cell model. DETAILED DESCRIPTION

[0059] The above features mentioned in the present application, or the features mentioned in the embodiments, can be combined arbitrarily. All the features explained in the specification can be used with any method form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.

[0060] The application will be further described with reference to the following examples. These examples are intended to be illustrative only and are not intended to limit the scope of the application. The following examples are presented for purposes of illustration and are not intended to limit the scope of the application. Unless otherwise indicated, all percentages and proportions are by weight.

[0061] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this application belongs. Also, any method and material similar or equivalent to those described herein can be used in the practice of the present application. The preferred methods and materials are described herein.

[0062] All ranges of values involving the same component or property are inclusive of the endpoints, which are combinable independently. Since these ranges are continuous, they include every value between the minimum and maximum values. It is also understood that any numerical range recited in this application is intended to include all sub-ranges of the same entire range.

[0063] TERMINOLOGY

[0064] The following terms and phrases, as used herein, have the following meanings, unless otherwise specified.

[0065] All numerical values or expressions representing numerals as used herein, in all instances, are to be understood as "about" the accurate value. When the term "about" is used in reference to a particular number or value, it is intended to convey that the end value(s) are not to be understood as a strict limitation. In the present application, the term "about" is intended to convey that the value is within 10% of the stated value or range, preferably within 5%. Unless otherwise stated, the percentages are mass percentages.

[0066] As used herein, "room temperature" refers to ambient temperature, which is from about 10 °C to about 40 °C. In some embodiments, "room temperature" refers to a temperature from about 20 °C to about 30 °C; in other embodiments, "room temperature" refers to a temperature from about 25 °C to about 30 °C; in yet other embodiments, "room temperature" refers to 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, etc.

[0067] The term "pharmaceutically acceptable" means that which the carrier, vehicle, adjuvant, diluent, and / or salt is generally biologically or otherwise physiologically compatible with the other ingredients of a pharmaceutical composition for its intended use.

[0068] The term "pharmaceutically acceptable adjuvant" means those carriers that do not have any significant detrimental effect on the biological activity and properties of the active compounds, and include, but are not limited to, any diluents, disintegrants, binders, glidants, wetting agents commonly used in the art for human or animal use.

[0069] The term "pharmaceutically acceptable salt" means a salt of a free acid or base of the specific compound that retains the biological effectiveness of the free acid or base and that does not impart undesired biological effects. For example, acid (including organic acids and inorganic acids) addition salts or base addition salts (including organic bases and inorganic bases) can be formed, and also include zwitterions, and also include quaternary ammonium salts, such as alkyl ammonium salts. Pharmaceutically acceptable salts of the present application can be synthesized from the parent compound that contains an acidic or basic moiety by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of the two.

[0070] The term "composition" means a composition comprising a folate receptor targeting compound of the present disclosure, and at least one pharmaceutically acceptable ingredient selected from the group consisting of carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweetening agents, flavoring agents, flavorants, antibacterial agents, antifungal agents, lubricants, dispersing agents, temperature-sensitive materials, temperature-regulating agents, adhesion agents, stabilizing agents, suspending agents, and the like, as determined by the mode of administration and the nature of the dosage form.

[0071] The "composition" of the present application can be prepared by any method known to the art of pharmacy. Generally, these methods of preparation include bringing the folate receptor targeting compound (hereinafter referred to as the active ingredient) into association with a carrier and / or one or more other accessory ingredients, and then, if necessary or desired, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0072] The "composition" of the present application can be prepared according to known methods, for example, the methods described in the general rules for the preparation of the Chinese Pharmacopoeia 2025 edition, Japanese Pharmacopoeia 16th edition, United States Pharmacopoeia, and European Pharmacopoeia 9th edition. The specific preparation method depends on the dosage form.

[0073] The active ingredient, pharmaceutically acceptable excipients in the "composition" described in the present application will vary depending on the identity, size and / or condition of the subject to be treated and further depending on the route of administration of the active ingredient. The composition can contain between 0.1% and 100% (w / w) of the active ingredient.

[0074] The "treatment" described in the present application, unless otherwise indicated, means reversing, alleviating the symptoms of the condition or disease to which the term applies, or one or more symptoms of such condition or disease, inhibiting the progress of the condition or disease or its one or more symptoms, or preventing the condition or disease or its one or more symptoms. The term "treatment" as used by the invention refers to the therapeutic act, as "treatment" is defined immediately above.

[0075] The term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of a drug or pharmaceutical composition to achieve the intended effect, without being toxic. The exact dose will vary depending on various factors, such as subject-dependent variables (e.g., age, immune system health, etc.), disease or illness, and the treatment administered.

[0076] DMSO is dimethyl sulfoxide, HATU is 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (380.24), HBTU is O-benzotriazol- N,N,N',N'-tetramethyluronium hexafluorophosphate (379.242), DIEA is N,N- diisopropylethylamine (129.25), TIPS is triisopropylsilane, MTBE is methyl tert- butyl ether, DMF is N,N-dimethylformamide, H-Tyr(tBu)-OtBu-HCl is O-tert- butyl-L-tyrosine tert-butyl ester hydrochloride (329.9), TIPS is triisopropylsilane, DCM is dichloromethane, TEA is triethylamine, CDI is 1,1'-carbonyldiimidazole, EA is ethyl acrylate, TFA is trifluoroacetic acid, THF is tetrahydrofuran, DBU is 1,8-diazabicyclo[5.4.0]undec-7-ene, BOP is Carter's condensing agent, PTEA is 2-phenylthioethanol propenoic acid, DIEA is N,N-diisopropylethylamine, HOBt is 1-hydroxybenzotriazole, DIC is N,N'-diisopropylcarbodiimide, Fmoc-Gly-OH is Fmoc-glycine, Fmoc-Tyr(tBu)-OH is Fmoc-O-tert-butyl-L-tyrosine, Fmoc-Glu-OtBu is Fmoc-L-glutamic acid-tert-butyl ester, TCFH is N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate, S0456 is Unless otherwise specified, the percentages in the present application are mass percentages.

[0077] Example 1 Compound A

[0078] Step 1: Synthesis of Compound A-2

[0079]

[0080] To a stirred solution of iodoquinolinic acid A-1 (10.0 g, 32.0 mmol, 1.0 eq) in DMSO (200 mL) was added HATU (14.6 g, 38.4 mmol, 1.2 eq) at 20-25 °C. This mixture was stirred at 20-25 °C for 2 h, then N-BOC-piperazine (6.3 g, 33.8 mmol, 1.05 eq) and DIEA (8.2 g, 64.0 mmol, 2.0 eq) were added. The solution was stirred at 20-25 °C for 0.5 h. The reaction was shown to be complete by in-process control (IPC). The mixture was precipitated with ethyl acetate (1000 mL) and filtered. The wet product was dried under vacuum for 2 h to give yellow solid A-2, 13.6 g, yield 88.3%. MS: [M+H] = 481.53. +

[0081] ​Step 2: Synthesis of compound A-3

[0082]

[0083] A-2 (13.6 g, 28.3 mmol, 1.0 eq) was added to trifluoroacetic acid (200 mL) and stirred at 20-25 °C for 1 h. The mixture was precipitated with ethyl acetate (2000 mL) and centrifuged. The wet product was dried under vacuum for 2 h to give yellow solid A-3, 12.2 g, yield 114.0%. MS: [M+H] + = 381.53.

[0084] Step 3: Synthesis of compound A-4

[0085]

[0086] To a stirred solution of A-3 (500 mg, 1.3 mmol, 1.0 eq) in DMSO (20 mL) was added HATU (594 mg, 1.56 mmol, 1.2 eq) at 20-25 °C. The mixture was stirred at 20-25 °C for 2 h, then 3-chloropropionic acid (148 mg, 1.37 mmol, 1.05 eq) and DIEA (336 mg, 2.6 mmol, 2.0 eq) were added. The solution was stirred at 20-25 °C for 0.5 h. The reaction was shown to be complete by in-process control (IPC). The mixture was precipitated with ethyl acetate (30 mL) and filtered. The wet product was dried under vacuum for 2 h to give yellow solid A-4, 500 mg, yield 80.9%. MS: [M+H] + = 471.92.

[0087] Step 4: Synthesis of A-5

[0088]

[0089] A-4 (500 mg, 1.06 mmol, 1.0 eq) was dissolved in DMF (20 mL), H-Tyr(tBu)-OtBu·HCl (419 mg, 1.27 mmol, 1.2 eq), K2CO3 (292.2 mg, 2.12 mmol, 2.0 eq) were added, stirred at 100 °C for 2 h, water (100 mL) and ethyl acetate (100 mL) were added, the solution was dried and rotary evaporated, post treatment: prep, lyophilized to give 82 mg white solid compound A-5. MS: [M+H] + = 728.52.

[0090] Step 5: Synthesis of A-6

[0091]

[0092] To a stirred solution of A-5 (100 mg, 0.14 mmol, 1.0 eq) in TFA (12 mL) was added TIPS (0.6 mL) and H2O (0.6 mL). The mixture was stirred at 20-25 °C for 1 h. The mixture was precipitated with MTBE (100 mL) and centrifuged. The wet product was purified by preparative HPLC (ACN / NH4HCO3, Daisogel C18, 10 µm 50mm*250mm) to give yellow solid A-6, 42 mg, yield 46.5%. MS: [M+H] + = 616.65.

[0093] Step 6: Compound A synthesis

[0094]

[0095] To a stirred solution of A-5 (100 mg, 0.14 mmol, 1.0 eq) in TFA (12 mL) was added TIPS (0.6 mL) and H2O (0.6 mL). The mixture was stirred at 20-25 °C for 1 h. The mixture was precipitated with MTBE (100 mL) and centrifuged. The wet product was purified by preparative HPLC (ACN / NH4HCO3, Daisogel C18, 10 µm 50mm*250mm) to give yellow solid A-6, 42 mg, yield 46.5%. MS: [M+H] 2+ = 734.34.

[0096] Example 2 Compound B

[0097] Step 1: Compound B-1 synthesis

[0098]

[0099] To a stirred solution of A-3 (500 mg, 1.3 mmol, 1.0 eq) in DMSO (20 mL) was added HATU (594 mg, 1.56 mmol, 1.2 eq) at 20-25 °C. This mixture was stirred at 20-25 °C for 2 h, then 3-chlorobutyric acid (159 mg, 1.36 mmol, 1.05 eq) and DIEA (335 mg, 2.6 mmol, 2.0 eq) were added. This solution was stirred at 20-25 °C for 0.5 h. The reaction was shown to be complete by in-process control (IPC). This mixture was precipitated with ethyl acetate (30 mL) and filtered. The wet product was dried under vacuum for 2 h to give yellow solid B-1, 400 mg, yield 62.9%. MS: [M+H]+ = 485.62.

[0100] Step 2: Synthesis of compound B-2

[0101]

[0102] Dissolve B-1 (400 mg, 0.82 mmol, 1.0 eq) in DMF (20 mL), add H-Tyr(tBu)-OtBu·HCl (327 mg, 0.99 mmol, 1.2 eq), K2CO3 (292.2 mg, 2.12 mmol), stir at 100 °C for 2 hours, add water (100 mL) and ethyl acetate (100 mL), separate, dry and rotary evaporate, post treatment. Preparation, freeze-drying to get 80 mg white solid compound B-2. MS: [M+H] + = 742.52.

[0103] Step 3: Synthesis of compound B-3

[0104]

[0105] To the stirring solution of B-2 (80 mg, 0.108 mmol, 1.0 eq) in TFA (12 mL) add TIPS (0.6 mL) and H2O (0.6 mL). Stir the mixture at 20-25 °C for 1 hour. Precipitate the mixture with MTBE (100 mL) and centrifuge. Purify the wet product by preparative HPLC (ACN / NH4HCO3, Daisogel C18, 10 µm 50mm*250mm) to get yellow solid B-3, 36 mg, yield 52.9%. MS: [M+H] + = 629.68.

[0106] Step 4: Synthesis of compound B

[0107]

[0108] Under nitrogen protection, add B-3 (36 mg, 0.057 mmol, 1.0 eq) and S0456 (55.7 mg, 0.057 mmol, 1.0 eq) to the solution of Na2CO3 (36.4 mg, 0.343 mmol, 6.0 eq) in H2O (4 mL). Heat the mixture to 70 °C and stir for 1 hour. Cool the mixture to 15-20 °C, and purify by preparative HPLC (ACN / CH3COONH4, Daisogel C18, 10 µm 30mm*250mm) to get green solid compound B, 18 mg, yield 20.2%. MS: [M-4Na+4H+2H] 2+= 741.52.

[0109] Example 3 Compound C

[0110] Step 1: Synthesis of compound C-1

[0111]

[0112] To a stirred solution of H-Tyr(tBu)-OtBu-HCl (1.05 g, 3.17 mmol, 1.0 eq) and DMAP (15.5 mg, 0.13 mmol, 0.04 eq) in DCM (10 mL) was added CDI (1.03 g, 6.34 mmol, 2.0 eq) at 0-5 °C. The mixture was stirred at 0-5 °C for 1 h. To the mixture was added H2O (5 mL) and the stirring was continued at 20-25 °C for 1 h. The DCM layer was separated and concentrated at 40 °C to give a white solid intermediate. To a stirred solution of A-3 (1.0 g, 2.63 mmol, 0.83 eq) and TEA (801.2 mg, 7.92 mmol, 2.5 eq) in DMSO (20 mL) was added all the above intermediate. The mixture was stirred at 20-25 °C for 12 h. The mixture was precipitated with EA (200 mL) and centrifuged. The wet product was dried under vacuum for 2 h to give yellow solid C-1, 720 mg, yield 39.1%. MS: [M+H] + = 700.41.

[0113] Step 2: Synthesis of compound C-2

[0114]

[0115] To a stirred solution of C-1 (600.0 mg, 0.86 mmol, 1.0 eq) in TFA (12 mL) was added TIPS (0.6 mL) and H2O (0.6 mL). The mixture was stirred at 20-25 °C for 1 h. The mixture was precipitated with MTBE (100 mL) and centrifuged. The wet product was purified by prep-HPLC (ACN / NH4HCO3, Daisogel C18, 10 µm 50 mm*250 mm) to give yellow solid C-2, 211 mg, yield 41.9%. MS: [M+H] + = 587.60.

[0116] Step 3: Synthesis of compound C

[0117]

[0118] To a solution of Na2CO3(71.7 mg, 0.68 mmol, 6.0 eq) in H2O (4 mL) was added C-2 (64.6 mg, 0.11 mmol, 1.0 eq) and S0456 (107.4 mg, 0.11 mmol, 1.0 eq) under nitrogen protection. The mixture was heated to 70 °C and stirred for 1 h. The mixture was cooled to 15-20 °C and purified by preparative HPLC (ACN / CH3COONH4, Daisogel C18, 10 µm 30 mm*250 mm) to give compound C as a green solid, 20 mg, yield 11.9%.

[0119] MS: [M-4Na+4H-2H] 2- : 718.1.

[0120] 1 H NMR: 1 H NMR (400 MHz, DMSO- d 6) δ 8.65 (s, 1H), 7.78 (d, 2H), 7.62 (s, 1H), 7.33 (s, 2H), 7.15 (d, 2H), 7.03 (d, 2H), 6.61 (d, 2H), 6.27 (s, 1H), 6.17 (d, 2H), 4.47 (s, 2H), 4.09 (m, 5H), 3.28 (m, 6H), 3.02 (s, 2H), 2.87 (d, 2H), 2.56 (m, 4H), 1.84 (s, 11H), 1.30 (s, 12H).

[0121] Example 4 Compound D

[0122] Step 1: synthesis of compound D-1

[0123]

[0124] To a solution of 4-formylpiperazine-1-carboxylic acid tert-butyl ester (685 mg, 3.2 mmol) in DCM (10 mL) was added TFA (2 mL) at 0-5 °C. The mixture was stirred at 0-5 °C for 1 h. The solution was concentrated and dried to give an intermediate. To a stirred solution of Butterfly A-1 (1.0 g, 3.2 mmol, 1.0 eq) in DMSO (20 mL) was added HATU (1.46 g, 3.84 mmol, 1.2 eq) at 20-25 °C. The mixture was stirred at 20-25 °C for 2 h, then all of the above intermediate and DIEA (0.82 g, 6.4 mmol, 2.0 eq) were added. The solution was stirred at 20-25 °C for 0.5 h. The reaction was shown to be complete by IPC. The mixture was precipitated with ethyl acetate (100 mL) and filtered. The wet product was dried under vacuum for 2 h to give yellow solid D-1, 826 mg, yield 63.5%. MS: [M+H] + = 409.41.

[0125] Step 2: Synthesis of compound D-2

[0126]

[0127] A solution of D-1 (800.0 mg, 1.96 mmol, 1.0 eq) and H-Tyr(tBu)-OtBu·HCl (647 mg, 1.96 mmol, 1.0 eq) in THF (10 mL) was stirred at 40 °C for 1 h, cooled to 0 °C, and NaBH4 (111 mg, 2.94 mmol, 1.5 eq) was added slowly. The mixture was stirred at room temperature for 1 h. The mixture was precipitated with MTBE (100 mL) and centrifuged. The wet product was purified by preparative HPLC (ACN / NH4HCO3, Daisogel C18, 10 µm 50mm*250mm) to give yellow solid D-2, 412 mg, yield 30.5%. MS: [M+H] + = 686.41.

[0128] Step 3: Synthesis of compound D-3

[0129]

[0130] To a solution of D-2 (400.0 mg, 0.58 mmol, 1.0 eq) in TFA (12 mL) was added TIPS (0.6 mL) and H2O (0.6 mL) with stirring. The mixture was stirred at 20-25 °C for 1 h. The mixture was precipitated with MTBE (100 mL) and centrifuged. The wet product was purified by preparative HPLC (ACN / NH4HCO3, Daisogel C18, 10 µm 50mm*250mm) to give yellow solid D-3, 180 mg, yield 54%. MS: [M+H] + = 573.41.

[0131] Step 4: Synthesis of compound D

[0132]

[0133] To a solution of Na2CO3 (108.1 mg, 1.02 mmol, 6.0 eq) in H2O (4 mL) was added D-3 (100.0 mg, 0.174 mmol, 1.0 eq) and S0456 (170.0 mg, 0.174 mmol, 1.0 eq) under nitrogen protection. The mixture was heated to 70 °C and stirred for 1 h. The mixture was cooled to 15-20 °C and purified by preparative HPLC (ACN / CH3COONH4, Daisogel C18, 10 µm 30mm*250mm) to give green solid compound D, 25 mg, yield 9.5%. MS: [M-4Na+4H-2H] 2- = 711.28.

[0134] Example 5 Compound E

[0135] Step 1: Synthesis of compound E-1

[0136]

[0137] To a solution of 4-(2-oxoethyl)piperazine-1 -carboxylic acid tert-butyl ester (730 mg, 3.2 mmol, 1.0 eq) in DCM (10 mL) was added TFA (2 mL) at 0-5 °C. The mixture was stirred at 0-5 °C for 1 h. The solution was concentrated and dried to give the intermediate. To a stirred solution of the intermediate in DMSO (20 mL) was added HATU (1.46 g, 3.84 mmol, 1.2 eq) at 20-25 °C. The mixture was stirred at 20-25 °C for 2 h, then added all of the above intermediate and DIEA (0.82 g, 6.4 mmol, 2.0 eq). The solution was stirred at 20-25 °C for 0.5 h. The reaction was shown to be complete by IPC. The mixture was precipitated with ethyl acetate (100 mL) and filtered. The wet product was dried under vacuum for 2 h to give yellow solid E-1, 830 mg, yield 61.5%. MS: [M+H] + = 422.45.

[0138] Step 2: Synthesis of compound E-2

[0139]

[0140] A solution of E-1 (800.0 mg, 1.89 mmol, 1.0 eq) and H-Tyr(tBu)-OtBu HCI (623 mg, 1.89 mmol, 1.0 eq) in THF (10 mL) was stirred at 40 °C for 1 h, cooled to 0 °C, slowly added NaBH4 (107 mg, 2.84 mmol, 1.5 eq), stirred at room temperature for 1 h. The mixture was precipitated with MTBE (100 mL) and centrifuged. The wet product was purified by preparative HPLC (ACN / NH4HCO3, Daisogel C18, 10 pm 50 mm*250 mm) to give yellow solid E-2, 400 mg, yield 29.6%. MS: [M+H] + = 700.65.

[0141] Step 3: Synthesis of compound E-3

[0142]

[0143] To a solution of E-2 (400.0 mg, 0.58 mmol, 1.0 eq) in TFA (12 mL) was added TIPS (0.6 mL) and H2O (0.6 mL) with stirring. The mixture was stirred at 20-25 °C for 1 h. The mixture was precipitated with MTBE (100 mL) and centrifuged. The wet product was purified by preparative HPLC (ACN / NH4HCO3, Daisogel C18, 10 µm 50mm*250mm) to give yellow solid E-3, 182 mg, yield 54%. MS: [M+H] + = 588.21.

[0144] Step 4: Synthesis of compound E

[0145]

[0146] To a solution of Na2CO3 (108.1 mg, 1.02 mmol, 6.0 eq) in H2O (4 mL) was added E-3 (100.0 mg, 0.17 mmol, 1.0 eq) and S0456 (166.2 mg, 0.17 mmol, 1.0 eq) under nitrogen protection. The mixture was heated to 70 °C and stirred for 1 h, then cooled to 15-20 °C, and purified by preparative HPLC (ACN / CH3COONH4, Daisogel C18, 10 µm 30mm*250mm) to give green solid compound E, 25 mg, yield 9.6%, MS: [M-4Na+4H-2H] 2- = 718.53.

[0147] Example 6 Compound F

[0148] Step 1: Synthesis of compound F-1

[0149]

[0150] To a solution of 1-BOC-4-(4-formylphenyl)piperazine (1 g, 3.4 mmol) in DCM (10 mL) was added 2 mL TFA at 0-5 °C, the mixture was stirred at 0-5 °C for 1 h, the solution was concentrated and dried to give the intermediate.

[0151] At 20–25 °C, HATU (1.46 g, 3.8 mol) was added to a stirred solution of pteroic acid A-1 (1 g, 3.2 mmol, 1.0 eq) in DMSO (20 mL). The mixture was stirred at 20–25 °C for 2 hours, followed by the addition of all the above intermediates and DIEA (0.82 g, 6.4 mmol, 2.0 eq). This solution was stirred at 20–25 °C for 0.5 hours. The reaction was confirmed by instantaneous plasma concentration (IPC). The mixture was precipitated with ethyl acetate (100 mL) and filtered. The wet product was dried under vacuum for 2 hours to give a yellow solid F-1, 905 mg, in a yield of 61.6%. MS: [M+H] + =485.55.

[0152] Step 2: Synthesis of compound F-2

[0153]

[0154] A solution of F-1 (900.0 mg, 1.86 mmol, 1.0 eq) and H-Tyr(tBu)-OtBu·HCl (674 mg, 2.04 mmol, 1.1 eq) in THF (10 mL) was stirred at 40 °C for 1 h, cooled to 0 °C, and NaBH4 (106 mg, 2.79 mmol, 1.5 eq) was slowly added, with stirring at room temperature for 1 h. The mixture was precipitated with MTBE (100 mL) and centrifuged. The wet product was purified by preparative HPLC (ACN / NH4HCO3, Daisogel C18, 10 µm 50 mm * 250 mm) to give a yellow solid F-2, 455 mg, in 32.3% yield. MS: [M+H] + =763.55.

[0155] Step 3: Synthesis of compound F-3

[0156]

[0157] Add 0.6 mL of TIPS and 0.6 mL of H₂O to a stirred solution of F-2 (450.0 mg, 0.59 mmol) in 12 mL of TFA. Stir the mixture at 20–25 °C for 1 hour. Precipitate the mixture with 100 mL of MTBE and centrifuge. Purify the wet product by preparative HPLC (ACN / NH₄HCO₃, Daisogel C18, 10 µm 50 mm * 250 mm) to give a yellow solid F-3: 195 mg, yield 50.9%. MS: [M+H] + =650.21.

[0158] Step 4: Synthesis of compound F

[0159]

[0160] Under nitrogen protection, F-3 (100.0 mg, 0.154 mmol, 1.0 eq) and SO456 (150.3 mg, 0.154 mmol, 1.0 eq) were added to a solution of Na2CO3 (97.7 mg, 0.92 mmol, 6.0 eq) in H2O (4 mL). The mixture was heated to 70 °C and stirred for 1 hour. The mixture was then cooled to 15–20 °C and purified by preparative HPLC (ACN / CH3COONH4, Daisogel C18, 10 µm 30 mm × 250 mm) to give 18 mg of green solid compound F, in a yield of 4.9%. MS: [M-4Na+4H-2H] 2- =750.21.

[0161] Example 7 Compound G

[0162] Step 1: Synthesis of compound G-1

[0163]

[0164] TFA (2 mL) was added to a DCM (10 mL) solution of tert-butyl 4-(4-(2-bromoethoxy)phenyl)piperazine-1-carboxylic acid ester (1 g, 2.6 mmol) at 0-5 °C. The mixture was stirred at 0-5 °C for 1 hour, and the solution was concentrated and evaporated to dryness to obtain the intermediate.

[0165] HATU (1.17 g, 3.07 mmol) was added to a DMSO (20 mL) solution of pteroic acid A-1 (800 mg, 2.56 mmol) under stirring at 20–25 °C. This mixture was stirred at 20–25 °C for 2 hours, then all of the above intermediate and DIEA (660 mg, 5.12 mmol) were added. This solution was stirred at 20–25 °C for 0.5 hours. The reaction was confirmed to be complete by instantaneous plasma concentration (IPC). This mixture was precipitated with ethyl acetate (80 mL) and filtered. The wet product was dried under vacuum for 2 hours to give a yellow solid G-1, 504 mg, in a yield of 33.8%. MS: [M+H] + =580.45.

[0166] Step 2: Synthesis of compound G-2

[0167]

[0168] G-1 (500 mg, 0.86 mmol, 1.0 eq) was dissolved in DMF (20 mL), and H-Tyr(tBu)-OtBu·HCl (340 mg, 1.03 mmol, 1.2 eq) and K2CO3 (238 mg, 1.73 mmol) were added. The mixture was stirred at 100 °C for 2 hours, then water (100 mL) and ethyl acetate (100 mL) were added. The mixture was separated, dried, and evaporated to dryness. The final product was prepared by lyophilization to give a white solid compound G-2, 205 mg. MS: [M+H] + =792.95.

[0169] Step 3: Synthesis of compound G-3

[0170]

[0171] Add 0.6 mL of TIPS and 0.6 mL of H₂O to a 12 mL TFA solution of G-2 (200 mg, 0.25 mmol) under stirring. Stir the mixture at 20–25 °C for 1 hour. Precipitate the mixture with 100 mL of MTBE and centrifuge. Purify the wet product by preparative HPLC (ACN / NH₄HCO₃, Daisogel C18, 10 µm 50 mm * 250 mm) to give 100 mg of yellow solid G-3, yield 58.2%. MS: [M+H] + =679.74.

[0172] Step 4: Synthesis of Compound G

[0173]

[0174] Under nitrogen protection, G-3 (100.0 mg, 0.147 mmol, 1.0 eq) and SO456 (143.6 mg, 0.147 mmol, 1.0 eq) were added to a solution of Na2CO3 (97.7 mg, 0.92 mmol, 6.0 eq) in H2O (4 mL). The mixture was heated to 70 °C and stirred for 1 hour, then cooled to 15–20 °C and purified by preparative HPLC (ACN / CH3COONH4, Daisogel C18, 10 µm 30 mm * 250 mm) to give 24 mg of the green solid compound G. Yield 10%, MS: [M-4Na+4H-2H] 2- =764.81.

[0175] Example 8 Compound H

[0176] Step 1: Synthesis of compound H-1

[0177]

[0178] To a solution of S01 (1 g, 2.8 mmol) in DCM (10 mL) was added TFA (2 mL) at 0-5 °C. After stirring at 0-5 °C for 1 h, the solution was concentrated and dried to give the intermediate. To a stirred solution of pteroic acid A-1 (800 mg, 2.56 mmol) in DMSO (20 mL) was added HATU (1.17 g, 3.07 mmol) at 20-25 °C. After stirring at 20-25 °C for 2 h, all of the above intermediate and DIEA (660 mg, 5.12 mmol) were added. The solution was stirred at 20-25 °C for 0.5 h. The reaction was shown to be complete by IPC. The mixture was precipitated with ethyl acetate (80 mL) and filtered. The wet product was dried under vacuum for 2 h to give yellow solid H-1, 601 mg, yield 39.1%. MS: [M+H] + = 532.46.

[0179] Step 2: Synthesis of compound H-2

[0180]

[0181] H-1 (600 mg, 1.1 mmol, 1.0 eq) was dissolved in DMF (20 mL), H-Tyr(tBu)-OtBu·HCl (363 mg, 1.1 mmol, 1.0 eq) and K2CO3 (303 mg, 2.2 mmol) were added. Stirring at 100 °C for 2 h, water (100 mL) and ethyl acetate (100 mL) were added. The solution was dried, concentrated and rotary evaporated. The wet product was purified by preparative HPLC (ACN / NH4HCO3, Daisogel C18, 10 µm 50mm*250mm) and lyophilized to give white solid compound H-2, 252 mg, yield 30.1%, MS: [M+H] + = 744.46.

[0182] Step 3: Synthesis of compound H-3

[0183]

[0184] To a stirred solution of H-2 (250 mg, 0.33 mmol) in TFA (12 mL) was added TIPS (0.6 mL) and H2O (0.6 mL). The mixture was stirred at 20-25 °C for 1 h. The mixture was precipitated with MTBE (100 mL) and centrifuged. The wet product was purified by preparative HPLC (ACN / NH4HCO3, Daisogel C18, 10 µm 50mm*250mm) to give yellow solid H-3, 85 mg, yield 40.1%. MS: [M+H] + = 632.46.

[0185] Step 4: Synthesis of compound H

[0186]

[0187] Under nitrogen protection, H⁻³ (80 mg, 0.124 mmol, 1.0 eq) and SO₄⁻⁶ (121.0 mg, 0.124 mmol, 1.0 eq) were added to a solution of Na₂CO₃ (78.7 mg, 0.74 mmol, 6.0 eq) in H₂O (4 mL). The mixture was heated to 70 °C and stirred for 1 hour. The mixture was then cooled to 15–20 °C and purified by preparative HPLC (ACN / CH₃COONH₄, Daisogel C18, 10 µm 30 mm × 250 mm) to give 24 mg of green solid compound H, in a yield of 12.2%. MS: [M⁻⁴Na + 4H⁻²H⁺] 2- =740.84.

[0188] Example 9 Compound I

[0189] Step 1: Synthesis of Compound I-1

[0190]

[0191] At 0–5 °C, TFA (2 mL) was added to a solution of I-0 (1 g, 2.7 mmol) in 10 mL of DCM. The mixture was stirred at 0–5 °C for 1 hour, and the solution was concentrated and evaporated to dryness to obtain the intermediate. At 20–25 °C, HATU (1.17 g, 3.07 mmol) was added to a solution of pteroic acid A-1 (800 mg, 2.56 mmol) in 20 mL of DMSO under stirring. The mixture was stirred at 20–25 °C for 2 hours, and then all of the above intermediate and DIEA (660 mg, 5.12 mmol) were added. The solution was stirred at 20–25 °C for 0.5 hours. The reaction was confirmed to be complete by instantaneous plasma concentration (IPC). The mixture was precipitated with ethyl acetate (80 mL) and filtered. The wet product was dried under vacuum for 2 hours to give a yellow solid I-1, 575 mg, yield 40%, MS: [M+H]. + =564.71.

[0192] Step 2: Synthesis of Compound I-2

[0193]

[0194] Compound I-2 was prepared by dissolving I-1 (575 mg, 1.02 mmol, 1.0 eq) in DMF (20 mL), adding H-Tyr(tBu)-OtBu-HCl (363 mg, 1.1 mmol, 1.1 eq) and K2CO3 (303 mg, 2.2 mmol), stirring at 100 °C for 2 hours, adding water (100 mL) and ethyl acetate (100 mL), separating, drying, and rotary evaporation to give a white solid, which was lyophilized to give compound I-2, 284 mg, yield 36.13%, MS: [M+H] + = 774.94.

[0195] Step 3: Synthesis of compound I-3

[0196]

[0197] To a stirred solution of I-2 (283 mg, 0.37 mmol) in TFA (12 mL) was added TIPS (0.6 mL) and H2O (0.6 mL) and stirred at 20-25 °C for 1 hour. The mixture was precipitated with MTBE (100 mL) and centrifuged. The wet product was purified by preparative HPLC (ACN / NH4HCO3, Daisogel C18, 10 µm 50mm*250mm) to give yellow solid I-3, 65 mg, yield 26.6%. MS: [M+H] + = 663.26.

[0198] Step 4: Synthesis of compound I

[0199]

[0200] To a solution of Na2CO3 (62.5 mg, 0.59 mmol, 6.0 eq) in H2O (4 mL) was added I-3 (65 mg, 0.098 mmol, 1.0 eq) and S0456 (95.7 mg, 0.098 mmol, 1.0 eq) under nitrogen protection. The mixture was heated to 70 °C and stirred for 1 hour, cooled to 15-20 °C, and purified by preparative HPLC (ACN / CH3COONH4, Daisogel C18, 10 µm 30mm*250mm) to give green solid compound I (18 mg). Yield 11.6%, MS: [M-4Na+4H-2H] 2- = 755.35.

[0201] Example 10 Compound J

[0202] Step 1: Synthesis of compound J-3

[0203]

[0204] To a solution of J-1 (1.00 g, 3.75 mmol, 1.0 eq) and J-2 (1.36 g, 4.13 mmol, 1.1 eq) in THF (20 mL) was added sodium cyanoborohydride (NaBH3CN, 283.5 mg, 4.5 mmol, 1.2 eq). The mixture was stirred at 20-25 °C for 1 h. TLC test showed the reaction was substantially complete. The reaction mixture was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1 / 4) to give the product J-3 as colorless oil, 1.5 g, yield 73.2%, MS: [M+H] + = 545.69.

[0205] Step 2: Synthesis of compound J-4

[0206]

[0207] To a solution of J-3 (600.0 mg, 1.1 mmol, 1.0 eq) in DMSO (6 mL) was added DBU (83 mg, 0.55 mmol, 0.5 eq) at 20-25 °C and stirred for 0.5 h. HPLC test showed the reaction was complete to give 300 mg of compound J-4. The reaction mixture was used in the next step without further purification. MS: [M+H] + = 323.45.

[0208] Step 3: Synthesis of compound J-5

[0209]

[0210] To a solution of J-4 (300 mg, 0.93 mmol, 1.0 eq) and DIEA (N,N-diisopropylethylamine, 240 mg, 1.86 mmol, 2.0 eq) in dimethyl sulfoxide (DMSO, 8 mL) was added BOP (412 mg, 0.93 mmol, 1.0 eq) and stirred for 0.5 h, then chloroacetic acid (0.93 mmol, 1.0 eq) was added. HPLC test showed the reaction was substantially complete. The reaction mixture was added into MTBE (300 mL) to give the wet product; after drying, the product J-5 was obtained as white solid, 250 mg, yield 67.4%, MS: [M+H] + = 399.93.

[0211] Step 4: Synthesis of compound J-6

[0212]

[0213] Dissolve J-5 (250 mg, 0.62 mmol) in DMF (20 mL), add A-3 (238 mg, 0.62 mmol), K2CO3 (171.2 mg, 1.24 mmol), stir at 100 °C for 2 hours, add water (10 mL) and ethyl acetate (10 mL), separate, dry and rotary evaporate, post-treatment: preparation, freeze-drying to obtain compound J-6 as a white solid, 80 mg, yield 17.3%, MS: [M+H] + = 743.88.

[0214] Step 5: synthesis of compound J-7

[0215]

[0216] To a solution of J-6 (80 mg, 0.11 mmol, 1.0 eq) in TFA (2 mL), add TIPS (100 µL, 0.18 mmol, 1.0 eq), stir at 20-25 °C for 1 hour. HPLC detection shows that the reaction is basically complete. Purify the reaction mixture by preparative high performance liquid chromatography to obtain compound J-7 as a yellow solid, 35.0 mg, yield 10.47%. MS: [M+H] + = 631.61.

[0217] Step 6: synthesis of compound J

[0218]

[0219] Under nitrogen protection, add J-7 (35 mg, 0.055 mmol, 1.0 eq) and S0456 (53.7 mg, 0.055 mmol, 1.0 eq) to a solution of Na2CO3 (34.3 mg, 0.32 mmol, 6.0 eq) in H2O (4 mL), heat to 70 °C and stir for 1 hour. Cool the mixture to 15-20 °C, and purify by preparative HPLC (ACN / CH3COONH4, Daisogel C18, 10 µm 30 mm*250 mm) to obtain compound J as a green solid, 22 mg, yield 26.0%, MS: [M-4Na+4H-2H] 2- = 739.81.

[0220] Example 11 Compound K

[0221] Step 1: synthesis of compound K-1

[0222]

[0223] M-4 (500 mg, 1.49 mmol, 1.0 eq) was dissolved in DMF (20 mL), chloroethylamine (118 mg, 1.49 mmol, 1.0 eq), K2CO3 (411.2 mg, 2.98 mmol, 2.0 eq) were added, stirred at room temperature for 2 hours, added water (10 mL) and ethyl acetate (10 mL), separated, dried and rotary evaporated, post treatment: prep, lyophilized to get compound K-1 as white solid, 200 mg, yield 35.2%, MS: [M+H] + = 380.55.

[0224] Step 2: synthesis of compound K-2

[0225]

[0226] To a stirred solution of theophylline A-1 (164.5 mg, 0.53 mmol, 1.0 eq) in DMSO (20 mL) was added HATU (242 mg, 0.64 mmol, 1.2 eq) at 20-25 °C and stirred for 2 hours at 20-25 °C, then K-1 (200 mg, 0.53 mmol, 1.0 eq) and DIEA (136.7 mg, 1.06 mmol, 2.0 eq) were added. This solution was stirred at 20-25 °C for 0.5 hours. The reaction was shown to be complete by in-process control (IPC). This mixture was precipitated with ethyl acetate (50 mL) and filtered. The wet product was dried under vacuum for 2 hours to get K-2 as yellow solid, 250 mg, yield 70.7 %. MS: [M+H] + = 674.82.

[0227] Step 3: synthesis of compound K-3

[0228]

[0229] K-2 (250 mg, 0.37 mmol) was added to TFA (2 mL) and stirred at 20-25 °C for 1 hour. This mixture was precipitated with ethyl acetate (20 mL) and centrifuged. The wet product was dried under vacuum for 2 hours to get K-3 as yellow solid, 80 mg, crude was directly used for next step. MS: [M+H] + = 562.82.

[0230] Step 4: synthesis of compound K

[0231]

[0232] To a solution of Na2CO3(89.4 mg, 0.84 mmol, 6.0 eq) in H2O (4 mL) was added K-3 (80 mg, 0.142 mmol, 1.0 eq) and S0456 (138.7 mg, 0.142 mmol, 1.0 eq) under nitrogen protection, heated to 70 °C and stirred for 1 hour, cooled to 15-20 °C, and purified by preparative HPLC (ACN / CH3COONH4, Daisogel C18, 10 µm 30 mm*250 mm) to give compound K as a green solid, 34 mg, yield 16.2%, MS: [M-4Na+4H-2H] = 705.26. 2- = 705.26.

[0233] Example 12 Compound L

[0234] Step 1: Synthesis of compound L-1

[0235]

[0236] Resin preparation: A reaction vessel was charged with 2-chlorotrityl chloride resin (degree of substitution: 1.08 mmol / g, 1.08 mmol, 1.0 g) and DMF (10 mL) was added and swelled for 0.5 h.

[0237] Coupling reaction: DIEA (3.0 eq) and Fmoc-O-tert-butyl-L-tyrosine (3.0 eq) were dissolved in DMF (7 mL, 7 volumes, 7V) to prepare a solution, which was then added to the resin; the mixture was stirred under nitrogen at 15-20 °C for 1.5 h. The resin was washed with DMF (7 mL x 3 times), followed by dissolving methanol (6.0 eq) and DIEA (10.0 eq) in DMF (7 mL, 7V) to prepare a solution, which was then added to the resin; the mixture was stirred under nitrogen at 15-20 °C for 1 h, and finally the resin was washed with DMF (7 mL x 6 times).

[0238] Deprotection reaction: A 20% piperidine / DMF solution (7 mL, 10 volumes, 10V) was added, and the resin was stirred under nitrogen at 15-20 °C for 20 min; the resin was washed with DMF (7 mL x 6 times), and after filtration, the deprotected resin was obtained.

[0239] Coupling reaction: HOBt (3.0 eq), DIC (3.8 eq) and Fmoc-Gly-OH (3.0 eq) were dissolved in DMF (7 mL, 10V) to prepare a solution, which was then added to the resin; the mixture was stirred under nitrogen at 15-20 °C for 30 min, and the resin was washed with DMF (7 mL x 3 times).

[0240] Deprotection reaction: 20% piperidine / DMF solution (7 mL, 10V) was added, and the resin was stirred under nitrogen at 15-20 °C for 20 minutes; the resin was washed with DMF (7 mL x 6 times), and after filtration, the deprotected resin was obtained.

[0241] Coupling reaction: HOBt (1.5 eq), DIEA (4.5 eq), HBTU (3.0 eq) and the pterin A-1 (3.0 eq) were dissolved in DMSO (14 mL, 20V) to prepare a solution, which was then added to the resin; the mixture was stirred under nitrogen at 15-20 °C for 30 minutes, and the resin was washed with DMF (7 mL x 3 times).

[0242] Peptide cleavage and purification: after completion of synthesis, the peptide-resin complex was washed with methanol (7 mL x 3 times), and then dried by bubbling nitrogen overnight to obtain 1.53 g of resin.

[0243] Take 432 mg of the above resin and place it in a flask, then add the cleavage solution (15 mL, composition: 95% TFA, 2.5% TIPS, 2.5% H2O) at 15-20 °C, and stir for 2.5-3 hours.

[0244] Filter the reaction mixture and collect the filtrate; add pre-cooled MTBE (40 mL) to the filtrate to precipitate, then centrifuge at 3000 rpm for 3 minutes to obtain the crude peptide.

[0245] The crude peptide was purified by preparative high performance liquid chromatography to obtain compound L-1 as a white solid, 42.0 mg, yield 7.3%, purity 99.99%. MS: [M-H] - = 530.96.

[0246] Step 2: Synthesis of compound L

[0247]

[0248] To a stirred solution of L-1 (30.0 mg, 56.34 µmol, 1.0 eq), S0456 (55.0 mg, 56.34 µmol, 1.0 eq) in water (1.2 mL), Na2CO3 (35.8 mg, 338.01 µmol, 6.0 eq) was added, and stirred at 80 °C for 0.5 hours. HPLC detection showed that the reaction was complete. The reaction mixture was purified by preparative high performance liquid chromatography to obtain green solid product compound L, 47.2 mg, purity 99.70%, yield 57.84%.

[0249] MS: [M-4Na+4H-2H] 2- = 690.16.

[0250] 1 H NMR: 1 H NMR (400 MHz, d6-DMSO) δ 8.64 (s, 1H), 8.47 (t, J = 5.6 Hz,1H), 7.72 – 7.58 (m, 10H), 7.32 (t, J = 9.2 Hz, 3H), 7.12 (d, J = 8.4 Hz, 2H),7.05 (dd, J = 14.8, 8.8 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 6.66 (d, J = 8.8 Hz,2H), 6.16 (d, J = 14.4 Hz, 2H), 4.48 (d, J = 6.0 Hz, 2H), 4.30 (dd, J = 11.6,4.8 Hz, 1H), 4.10 (s, 4H), 3.75 (ddd, J = 36.4, 16.4, 5.6 Hz, 2H), 2.88 (d, J =4.4 Hz, 2H), 2.67 (s, 4H), 2.54 (t, J = 6.8 Hz, 4H), 1.88 (dd, J = 8.4, 4.0 Hz,1H), 1.75 (s, 8H), 1.21 (t, J = 10.4 Hz, 13H)。

[0251] Example 13 Compound M

[0252] Step 1: Synthesis of Compound M-3

[0253]

[0254] To a solution of M-1 (1.00 g, 3.55 mmol, 1.0 eq), M-2 (1.29 g, 3.91 mmol, 1.1 eq) in THF (20 mL), sodium cyanoborohydride (NaBH3CN, 268.0 mg, 4.26 mmol, 1.2 eq) was added and stirred at 20-25 °C for 1 h. TLC test showed the reaction was substantially complete. The reaction mixture was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1 / 4, by volume) to give the product M-3 as colorless oil, 1.45 g, yield 73.2%, purity 79.57%. MS: [M+H] + = 559.61.

[0255] Step two: synthesis of compound M-4

[0256]

[0257] To a solution of M-3 (600.0 mg, 1.07 mmol, 1.0 eq) in DMSO (6 mL), DBU (81.7 mg, 0.54 mmol, 0.5 eq) was added and stirred at 20-25 °C for 0.5 h. HPLC test showed the reaction was complete. MS: [M+H] + = 337.48. The reaction mixture was used in the next step without further purification.

[0258] Step three: synthesis of compound M-5

[0259]

[0260] To a solution of the indole acid A-1 (335.3 mg, 1.074 mmol, 1.0 eq) and DIEA (278 mg, 2.148 mmol, 2.0 eq) in DMSO (8 mL), BOP (474.9 mg, 1.07 mmol, 1.0 eq) was added and stirred at 20-25 °C for 0.5 h, then M-4 (1.07 mmol, 1.0 eq) was added and the reaction was allowed to proceed for 1-2 h. HPLC monitoring showed the reaction was substantially complete. The reaction mixture was added to MTBE (300 mL) to give the wet product; after drying, the product M-5 was obtained as yellow solid, 406.3 mg, yield 60.01%, purity 71.00%. MS: [M-H] - = 629.11.

[0261] Step four: synthesis of compound M-6

[0262]

[0263] To a solution of M-5 (406.3 mg, 0.64 mmol, 1.0 eq) in TFA (2 mL), was added triisopropylsilane (100 µL, 0.18 mmol) and stirred at 20-25 °C for 1 h. HPLC detection showed the reaction was substantially complete. The reaction mixture was purified by preparative high-performance liquid chromatography to give the product M-6 as a yellow solid, 35.0 mg, purity 93.72%, yield 10.47%. MS: [M+H] + = 518.83.

[0264] Step five: synthesis of compound M

[0265]

[0266] To a stirred solution of M-6 (30.0 mg, 57.86 µmol, 1.0 eq) and S0456 (62.1 mg, 63.65 µmol, 1.1 eq) in water (1.2 mL), was added Na2CO3 (36.7 mg, 347.13 µmol, 6.0 eq). The mixture was stirred at 80-85 °C for 0.5 h. HPLC detection showed the reaction was complete. The reaction mixture was purified by preparative high-performance liquid chromatography to give the product M as a green solid, 35.9 mg, purity 98.11%, yield 43.3%.

[0267] MS: [M-4Na+4H-2H] 2- = 683.18.

[0268] 1 H NMR: 1 H NMR (400 MHz, d6-DMSO) δ 8.63 (s, 1H), 8.08 (s, 1H), 7.76(d, J = 14.0 Hz, 2H), 7.69 – 7.50 (m, 7H), 7.32 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 7.15 – 6.88 (m, 6H), 6.62 (d, J = 8.8 Hz, 2H), 6.19 (d, J = 14.4Hz, 2H), 4.48 (d, J= 6.0 Hz, 2H), 4.10 (s, 4H), 3.82 – 3.56 (m, 2H), 2.98 –2.52 (m, 13H), 1.90 – 1.85 (m, 1H), 1.73 (s, 8H), 1.55 (d, J = 4.4 Hz, 1H),1.25 (s, 12H)。

[0269] Example 14 Compound N

[0270] Step 1: Synthesis of compound N-1

[0271]

[0272] Resin preparation: To a vessel containing 2-chlorotrityl chloride resin (degree of substitution: 1.09 mmol / g, 1.09 mmol, 1.00 g) was added DMF (10 mL) and swelled for 0.5 h.

[0273] Coupling reaction: DIEA (6.0 eq) and Fmoc-Tyr (tBu)-OH (3.0 eq) in DMF (7 mL, 7x volume) were added to the resin and stirred at 15-20 °C for 1.5 h under a stream of nitrogen. The resin was then washed with DMF (10 mL x 3 times). DIEA (10.0 eq) and methanol (MeOH, 10.0 eq) in DMF (10 mL, 10x volume) were added to the resin and stirred at 15-20 °C for 40 min under a stream of nitrogen, after which the resin was washed with DMF (10 mL x 3 times).

[0274] Deprotection: 20% piperidine in DMF (10 mL) was added and the resin was stirred at 15-20 °C for 20 min under a stream of nitrogen. The resin was then washed with DMF (10 mL x 6 times) and filtered to obtain the resin.

[0275] Coupling reaction: HBTU (3.0 eq), HOBt (1.5 eq), DIEA (4.5 eq) and Fmoc-Glu-OtBu (3.0 eq) in DMF (7 mL) were added to the resin and stirred at 15-20 °C for 30 min under a stream of nitrogen. The resin was then washed with DMF (10 mL x 3 times).

[0276] The above procedure was repeated to couple the subsequent amino acids.

[0277] To a flask containing resin (1.5769 g) was added cleavage solution (11.0 mL TFA, 0.275 mL water, 0.275 mL triisopropylsilane) at 15-20 °C and stirred for 2 hours. The reaction was filtered and the filtrate was precipitated with cold methyl tert-butyl ether (110 mL), centrifuged (3000 rpm, 3 minutes) to get wet product. The precipitate was washed with methyl tert-butyl ether (50 mL) for three times and dried under vacuum for 2 hours to get yellow solid crude N-1, 541.3 mg, purity 92.38%, yield 75.1%. MS: [M+Na] + = 684.29.

[0278] Step 2: Synthesis of compound N

[0279]

[0280] To a solution of N-1 (31.5 mg, 0.0476 mmol, 1.0 eq) in aqueous sodium carbonate (1.25 mL) (containing 29.7 mg, 6.0 eq sodium carbonate, dissolved in water) was added S0456 (46.5 mg, 0.0476 mmol, 1.0 eq) with stirring at 70-75 °C for 1.5 hours. High performance liquid chromatography showed that the reaction was completed. The mixture was purified by preparative high performance liquid chromatography (column: Gemini-C18 250 x 21.2 mm, 10 pm; mobile phase: acetonitrile-water (containing 10 mM ammonium bicarbonate); gradient: 5%-95%) to give green solid compound N, 45.1 mg, purity 95.23%, yield 61.3%. MS: [M-5Na+5H+2H] 2+ = 740.5.

[0281] Example 15 Compound P

[0282] Step 1: Synthesis of compound P-1

[0283]

[0284] Resin preparation: To a vessel containing 2-chlorotrityl chloride resin (degree of substitution: 1.09 mmol / g, 1.09 mmol, 1.0 g) was added N, N-dimethylformamide (DMF) and swelled for 0.5 hours.

[0285] Coupling reaction: A DMF solution (7 mL, 7 times the volume) of N,N-diisopropylethylamine (DIEA, 6.0 eq) and Fmoc-Tyr (tBu)-OH (3.0 eq) was added to the resin, and the mixture was stirred under nitrogen at 15-20°C for 1.5 hours. The resin was then washed with DMF (10 mL × 3 times). Next, a DMF solution (10 mL, 10 times the volume) of DIEA (10.0 eq) and methanol (MeOH, 10.0 eq) was added to the resin, and the mixture was stirred under nitrogen at 15-20°C for 40 minutes. The resin was then washed with DMF (10 mL × 3 times).

[0286] Deprotection: Add 10 mL of 20% piperidine DMF solution and stir the resin under nitrogen at 15-20°C for 20 minutes. Then wash the resin with DMF (10 mL × 6 times) and filter to obtain the resin.

[0287] Coupling reaction: A DMF solution (7 mL) of 1-hydroxybenzotriazole (HOBt, 3.0 eq), N,N'-diisopropylcarbodiimide (DIC, 3.8 eq) and Fmoc-L-phenylalanine (Fmoc-Phe-OH, 3.0 eq) was added to the resin and stirred under nitrogen at 15-20 °C for 30 minutes. The resin was then washed with DMF (10 mL × 3 times).

[0288] Repeat the above steps to couple subsequent amino acids.

[0289] At 15-20°C, cutting fluid (11.0 mL TFA, 0.275 mL water, and 0.275 mL triisopropylsilane) was added to a flask containing 1.6352 g of resin, and the mixture was stirred for 2 hours. After filtration, the filtrate was precipitated with cold methyl tert-butyl ether (110 mL), centrifuged (3000 rpm, 3 min) to obtain a wet product. The precipitate was washed three times with methyl tert-butyl ether (50 mL) and dried under vacuum for 2 hours to obtain a yellow solid crude product P-1, 548.8 mg, with a purity of 90.21% and a yield of 64.4%. MS: [M+H] + =782.21.

[0290] Step 2: Synthesis of compound P

[0291]

[0292] To a 2.0 mL aqueous solution of sodium carbonate (containing 43.1 mg, 6.0 eq of sodium carbonate dissolved in water) of P-1 (52.1 mg, 0.0666 mmol, 1.0 eq), SO456 (65.1 mg, 0.0666 mmol, 1.0 eq) was added with stirring, and the mixture was stirred at 70-75 °C for 2 hours. High-performance liquid chromatography (HPLC) showed that the reaction was complete. The mixture was purified by preparative HPLC (column: Gemini-C18 250 × 21.2 mm, 10 μm; mobile phase: acetonitrile-water (containing 10 mM ammonium bicarbonate); gradient: 5%-95%) to give 43.9 mg of the green solid compound P with a purity of 95.51% and a yield of 38.8%. MS: [M-4Na+4H+2H] 2+ =805.1.

[0293] Example 16 Compound Q

[0294] Step 1: Synthesis of compound Q-2

[0295]

[0296] In a solution of Q-1 (10.00 g, 47.10 mmol, 1.0 eq) in 150 mL of dichloromethane, (Boc)₂O (10.30 g, 47.10 mmol, 1.0 eq) and N,N-diisopropylethylamine (6.10 g, 47.10 mmol, 1.0 eq) were added, and the mixture was stirred at 15–20 °C for 24 hours. Thin-layer chromatography (TLC, eluent: EA:PE = 1:3, Rf = 0.4) showed that the reaction was essentially complete. The reaction mixture was purified by silica gel column chromatography (eluent: ethyl acetate: petroleum ether = 1:5, v / v) to give 8.5 g of yellow solid Q-2, in 57.8% yield.

[0297] 1 H NMR: 1 H NMR (400 MHz, CDCl3) δ 7.78 (d, J = 8.0 Hz, 1H), 7.37 – 7.32(m, 1H), 7.30 (d, J = 1.6 Hz, 1H), 7.24 (t, J = 5.2 Hz, 1H), 6.76 (d, J = 8.0Hz, 1H), 6.26 (s, 1H), 2.25 (d, J = 16.8 Hz, 6H), 1.52 (s, 9H).

[0298] Step 2: Synthesis of compound Q-4

[0299]

[0300] To a solution of Q-2 (1.00 g, 3.2 mmol, 1.0 eq) in acetonitrile (16 g) was added hydrochloric acid (24 mL, 0.4 mol / L) at 0 °C, labeled as flask A. Sodium nitrite (265 mg) was dissolved in water (8 g) and cooled to 0 °C, labeled as flask B. The mixture in flask B was added dropwise to flask A, labeled as flask D, at 0 °C. The mixture was stirred at 0 °C for 1 h. To a solution of Q-3 (1.37 g, 4.0 mmol, 1.25 eq) in water (32 g) was added sodium bicarbonate (1.10 g) at 0 °C, labeled as flask C. The mixture in flask D was added dropwise to flask C at 0 °C. The mixture was then stirred at 10-15 °C for 2 h. High performance liquid chromatography detection showed that the raw material was not detected, and the target product accounted for 96.88%, indicating that the reaction was completed. The reaction mixture was purified by preparative high performance liquid chromatography (Prep-HPLC) to obtain purple compound Q-4, 1.78 g, yield 86.4%. MS: [M+H] + = 640.61.

[0301] Step 3: Synthesis of compound Q-5

[0302]

[0303] To a solution of Q-4 (1.00 g, 1.56 mmol) in acetonitrile (ACN, 20 mL) was added hydrochloric acid (HCl, 20 mL, 0.5 mol). The mixture was stirred at 15-20 °C for 3 h. High performance liquid chromatography detection showed that the ratio of raw material to target product was 1.30 / 85.40, indicating that the reaction was basically completed. The mixture was treated by freeze-drying to obtain purple solid compound Q-5, 0.92 g, yield 109.5%. MS: [M+H] + = 540.61.

[0304] Step 4: Synthesis of compound Q-7

[0305]

[0306] In a solution of Q-5 (100 mg, 0.18 mmol, 1.0 eq) in N,N-dimethylformamide (2 mL), Q-6 (101 mg, 0.18 mmol, 1.0 eq), TCFH (55.6 mg, 0.20 mmol, 1.1 eq) and NMI (31 mg, 0.37 mmol, 2.1 eq) were added. The mixture was stirred at 15-20 °C for 1 h. HPLC detection showed that the ratio of raw material to target product Q-7 was 5.76 / 69.48, indicating that the reaction was basically completed. The reaction mixture was purified by preparative high performance liquid chromatography to obtain purple compound Q-7, 156 mg, purity 93.26%, yield 80.8%. MS: [M+H] + = 1086.01.

[0307] Step 5: Synthesis of compound Q

[0308]

[0309] In a solution of Q-7 (50 mg, 46.04 μmol, 1.0 eq) in water (2 mL), S0456 (44.9 mg, 46.04 μmol, 1.0 eq) was added. The pH value was adjusted to 13 with Cs2CO3. The mixture was stirred at 70 °C for 1 h. HPLC detection showed that the ratio of raw material to target product compound Q was 18.31 / 68.70, indicating that the reaction was basically completed. The reaction mixture was purified by preparative high performance liquid chromatography to obtain blue solid compound Q, 30.8 mg, yield 33.4%, purity 98.14%.

[0310] MS: [M-5Na+5H-4H] 4- = 483.3.

[0311] 1 H-NMR: 1 H NMR (400 MHz, DMSO- d6) δ 15.96 (s, 1H), 9.43 (s, 1H), 8.63 (s, 1H), 8.37 (s, 1H), 8.02 (d, J = 10.0 Hz, 2H), 7.87 (d, J = 9.2 Hz, 1H), 7.79 (d, J = 14.0 Hz, 2H), 7.65-7.59 (m, 8H), 7.55 (s, 2H), 7.47 (d, J = 8.4Hz, 1H), 7.31 (m, 5H), 7.03 (m, 3H), 6.88 (m, 1H), 6.60 (m, 2H), 6.39 (m,1H), 6.19 (m, 3H), 4.53 (s, 1H), 4.44 (d, J = 5.2 Hz, 2H), 4.09 (s, 4H), 3.05(s, 4H), 2.91 (s, 1H), 2.81-2.66 (m, 5H), 2.54 (s, 2H), 2.24 (s, 3H), 1.95-1.87 (m, 4H), 1.73 (s, 8H), 1.29-1.19 (m, 15H).

[0312] Effect Test Case

[0313] 1. Compound AN, compound PT, in vivo activity imaging: KB cell model

[0314] A subcutaneous tumor-bearing mouse model of KB (ATCC-CRL-17) was constructed in BALB / c nude mice (athymic nude mice). KB (ATCC-CRL-17) cells were cultured in a monolayer in vitro under the following conditions: EMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution, and incubated at 37°C in a 5% CO2 incubator. Cells were passaged twice a week using trypsin-EDTA digestion. When the desired cell count was reached, cells from different culture flasks were collected, mixed thoroughly, counted, and seeded.

[0315] Put 0.2 mL of 10×10 6 22Rv1 cells (with matrix gel, volume ratio 1:1) were subcutaneously seeded into the nape of the neck of each mouse. The average tumor volume reached 300-400 mm. 3 Dosing will begin in groups at that time.

[0316] Sixty mice with well-developed and regularly shaped tumors were selected for the study. The 60 mice were randomly divided into 20 groups of 3 mice each. G1-G19 were the compound test groups in the corresponding order, and G20 was the solvent control group. The scanning times were 1, 2, 4, 8, and 24 hours.

[0317] Different groups of mice bearing KB tumor xenografts were intravenously injected with 10 nmol / each of different compounds (5% glucose solution, same below), and the blank control group was injected with 5% glucose solution (see Table 1 below), and then whole body imaging was performed using a small animal live imaging system (IVIS Lumina LT serier III), which contains a near-infrared fluorescence imaging system. Imaging was performed at 1 h, 2 h, 4 h, 8 h, and 24 h (the blank control group was imaged at 1 h and 24 h), and the same diameter circles were used to take tumor and background sites to obtain fluorescence values, calculate the tumor-to-background ratio (TBR), and calculate the average value. The larger the value, the more likely it is to provide clearer tumor boundaries and better surgical field in surgery. The TBR values are shown in Table 2.

[0318] Table 1 In vivo live imaging grouping

[0319]

[0320] Note: Compound R is compound 1 disclosed in patent document CN119490506A, compound S is compound 2 disclosed in patent document CN119490506A, and compound T is marketed drug OTL38.

[0321] Table 2 Comparison of live imaging compound tumor-to-background ratio data

[0322]

[0323] The 19 compound administration groups can provide certain tumor-to-background ratios at 1 h and thereafter (there is a significant difference compared with the blank control group), and the degree of advantage exhibited at different time points is different, as shown in Table 2:

[0324] ① All compounds show an upward trend in TBR within 1-24 h, and the TBR peak appears at 24 h;

[0325] ② All compounds can be clearly distinguished from the solvent control (except that reference drug T is not clearly distinguished at 1 h, and can be clearly distinguished from the solvent control at 24 h);

[0326] ③ Compared with reference drug T, 18 compounds have obvious TBR improvement, among which compound C is the most obvious (the TBR value reaches 2.7 at 1 h after administration, and shows an upward trend at subsequent time points, and the TBR value at 2-24 h is greater than that of all other compounds), and compounds R and S also perform very outstandingly;

[0327] ④ Different compounds have advantages at different time points, as shown in detail in Table 2. Figure 1 .

[0328] 2. Ex vivo tissue imaging - tissue distribution studies

[0329] It is worth noting that the tumor-to-background ratio (TBR) is less accurate than the ex vivo tumor-to-healthy tissue ratio due to its way of being taken (tumor and pelt skin are taken together on live animals). Therefore, we calculated the TBR value at different time points to get the trend of the tumor-to-background difference and to observe the best time for surgery. The more accurate tumor differentiation potential was obtained by dissecting the animals at the TBR peak, taking their naked tumors and healthy target tissues, and calculating the ex vivo tumor-to-healthy tissue ratio (tumor-to-tissue ratio), which is called Tumor / Normal Ratio (TNR).

[0330] BALB / c nude (athymic nude mice) were used to construct KB (ATCC-CRL-17) subcutaneous tumor-bearing mouse models. KB (ATCC-CRL-17) cells were cultured in monolayer in vitro. The culture conditions were as follows: EMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution, 37°C, 5% CO2 incubator. Routine digestion was performed twice a week using trypsin-EDTA for subculture. When the cell number reached the required amount, the cells from different culture bottles were collected, mixed evenly, counted, and inoculated.

[0331] 0.2 mL 10×10 6 22Rv1 cells (plus Matrigel, volume ratio 1:1) were subcutaneously inoculated on the back of each mouse. When the average tumor volume reached 300-400 mm 3 , the mice were grouped and administered.

[0332] Sixty mice with good tumor growth and regular shape were selected for the study. The 60 mice were randomly divided into 20 groups, with 3 mice in each group. G1-G19 were the corresponding compound test groups, and G20 was the vehicle control group.

[0333] The KB tumor-bearing xenograft mice were intravenously injected with 10 nmol of different compounds per mouse (5% glucose solution, same below), and the blank control group was injected with 5% glucose solution (see Table 1). After euthanasia, the organs (tumor, heart, liver, spleen, lung, kidney, stomach, muscle, skin, pancreas, small intestine, colorectum, the rest of the large intestine except colorectum, esophagus, bronchus, ovary, bladder, uterus, fallopian tube, breast, fat) of the mice were collected for IVIS Lumina LT imaging system measurement of fluorescence signals (organ tissues were stored at 4 ºC).

[0334] The ratio of tumor to each tissue fluorescence value was calculated. The larger the value, the greater the potential for providing clearer tumor boundaries during surgery. The tumor-to-tissue ratio is shown in Table 3 below.

[0335] Table 3 Average fluorescence ratio of subcutaneous tumor to normal tissue in dissected tissue of tumor-bearing mice 24 h after injection of compound and 5% glucose

[0336]

[0337] KB cells are FR-a high expression cells. According to existing research data, FR-a is overexpressed in various solid tumors, but the frequency and degree differ. Based on this principle, to explore the application prospect of the fluorescence probe targeting FR-a receptor of the application in various solid tumors, the tumor / healthy tissue ratios of various solid tumors in the KB cell xenotransplantation model were calculated to clarify the potential of the fluorescence probe in providing clear tumor boundaries in multiple cancers. As shown in Table 3, the ratios of tumor / lung, tumor / ovary, tumor / fallopian tube, tumor / uterus, tumor / breast, and tumor / colorectum in the compound group and the blank solvent group were significantly different, indicating that the 19 compounds have great potential as drugs for intraoperative navigation of lung cancer, ovarian cancer, fallopian tube cancer, endometrial cancer, breast cancer, and colorectal cancer. Specifically, as follows:

[0338] 1) Among the 6 cancers, the 18 compounds provided by the application are significantly better than the reference compound T;

[0339] 2) Tumor / lung: C > I > A > M > L > J > G > Q > F > E = H > N > K > D > B > P > R > S > T;

[0340] 3) Tumor / ovary: C > K > M > L > G > A > F > Q > J > P = B > I > D = N > H > E > R > S > T;

[0341] 4) Tumor / fallopian tube: C > G > Q > M > J > D = S > R > A > E = K > H > P > I > L > B > F > N > T;

[0342] 5) Tumor / uterus: R > C > P > G > Q > M > F > K > H > L > S > E > A = D > I > J > B > N > T;

[0343] 6) Tumor / breast: M > C > S > R > F > Q > L > A > K > P > H > E > D > I > J > N > B > G > T;

[0344] 7) Tumor / colorectum: C > Q > L > H > J > P > B > R > G > D > F > M > N > E > K > A > I > S > T.

[0345] The above is a further description of the present application in combination with specific embodiments, but these embodiments are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements all fall within the protection scope of the present application.

Claims

1. A folate receptor-targeting compound, characterized in that, The folate receptor-targeting compound is F-L-D, wherein, F is a folate receptor-targeting structure, and has the following structure: ； D is a near-infrared dye, and has the following structure: or a pharmaceutically acceptable salt thereof; L is a linker, and has the following structure: or a pharmaceutically acceptable salt thereof; In the formula, R1 is selected from any one of the following structures: , , , , and ; R3 is selected from any one of the following structures: 、 、 、 、 、 、 ； n is independently an integer from 0 to 10; R2 is selected from any one of the following structures: 、 or a pharmaceutically acceptable salt thereof.

2. The folate receptor-targeting compound of claim 1, wherein, The pharmaceutically acceptable salt is independently selected from a sodium salt, a potassium salt, or a lithium salt.

3. The folate receptor-targeted compound of claim 1, wherein, The n is independently an integer from 0 to 5.

4. The folate receptor-targeted compound of claim 1, wherein, The R1 is selected from any one of the following structures: , , or .

5. The folate receptor-targeted compound of claim 1, wherein, The R1 is ; R3is selected from any one of the following structures: , , , n is an integer from 0 to 5.

6. The folate receptor-targeted compound of claim 1, wherein, L is a linker, and has the following structure: or a pharmaceutically acceptable salt thereof; wherein R1is , R2is .

7. The folate receptor-targeted compound of claim 1, wherein, The folate receptor-targeting compound has any one of the following structures:

8. The folate receptor-targeted compound of claim 7, wherein, The folate receptor-targeting compound has any one of the following structures:

9. A process for the preparation of the folate receptor targeting compound according to any one of claims 1 to 8, characterized in that, The method comprises the following steps: (1) first generating a compound V comprising the structures of F and L by a reaction, and the compound V has the following structure: Compound V wherein R1 and R2 have the same meanings as R1 and R2 in the folate receptor-targeting compound of any one of claims 1-8, respectively; (2) reacting the compound V with a compound W to obtain the folate receptor-targeting compound, and the compound W has the following structure: Compound W wherein M 1 , M 2 , M 3 each independently is H or a pharmaceutically acceptable cation, the cation being a monovalent cation, the monovalent cation being a sodium ion, a potassium ion, or a lithium ion.

10. The method of claim 9, wherein, The preparation method satisfies one or more of the following conditions: 1) the reaction in step (2) is carried out in an aqueous carbonate solution; 2) the reaction conditions in step (2) are 70-85°C, and the reaction is carried out for 0.5-2h.

11. The method of claim 10, wherein, The carbonate is selected from sodium carbonate, potassium carbonate, or lithium carbonate.

12. A composition characterized in that, The composition comprises the folate receptor-targeting compound of any one of claims 1-8 and a pharmaceutically acceptable excipient.

13. A kit for tumor targeting, comprising the folate receptor-targeting compound of any one of claims 1-8 or the compound obtained by the preparation method of any one of claims 9-11.

14. Use of the folate receptor-targeting compound of any one of claims 1-8 or the composition of claim 12 in the preparation of a near-infrared fluorescence imaging agent.

15. Use of the folate receptor-targeting compound of any one of claims 1-8 or the composition of claim 12 in the preparation of a product for diagnosing or treating a tumor.

16. The use according to claim 15, characterized in that, The tumor is a tumor associated with overexpression of FR-α.

17. Use according to claim 16, characterized in that, The tumor is at least one of lung cancer, ovarian cancer, fallopian tube cancer, endometrial cancer, breast cancer, and colorectal cancer.

18. The use according to claim 15, characterized in that, The product is a tumor diagnostic reagent or a tumor imaging reagent.

Images