Folic acid conjugates, their preparation methods and applications

Abstract

This invention relates to folic acid conjugates, their preparation methods, and applications. Specifically, this invention discloses a novel folic acid conjugate and its combination therapy strategy, and also provides a method for preparing the conjugate and its application in tumor treatment. The conjugate of this invention exhibits folic acid receptor expression dependence, can effectively inhibit the proliferation of ovarian cancer cells, and shows good efficacy when used in combination with DNA damaging agents.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular, to a folate conjugate and a preparation method and application thereof. BACKGROUND

[0002] Ovarian cancer is the second most common cause of gynecological cancer death in women worldwide. At present, the treatment of ovarian cancer mainly relies on tumor resection surgery and platinum-based chemotherapy, but the patient recurrence rate is high and drug resistance is easy to produce, and the survival prognosis has not been significantly improved for a long time, and the treatment strategy urgently needs a new breakthrough.

[0003] In recent years, PARP inhibitors (Poly(ADP-ribose) polymerase inhibitor, PARPi) have been approved for first-line treatment of recurrent ovarian cancer and BRCA1 / 2 mutant patients, but its use is limited by a series of side effects, and currently only BRCA gene mutant ovarian cancer patients can significantly benefit from PARP inhibitor first-line maintenance therapy.

[0004] Several clinical trials have shown that, based on the "synthetic lethality" effect, the combination of PARP inhibitors and DNA damaging agents can effectively enhance the activity of chemotherapy, but the adverse reactions caused by the targeting problem of the two drugs limit the combined efficacy, so it is urgent to improve the selectivity of the drugs to tumor cells.

[0005] The research of folate (Folic acid, FA) targeted antitumor drugs has made great progress, and some folate conjugate drugs have entered the clinical research stage. Studies have shown that folate receptors are highly expressed specifically on some cancer cells, while they are micro-expressed or not expressed in normal tissues. The use of this expression difference for selective drug delivery can greatly reduce the damage of antitumor drugs to normal cells and tissues, and improve the targeting and efficacy of the drugs.

[0006] Therefore, there is an urgent need in the art to develop a folate conjugate which has both folate binding properties and PARP enzyme inhibitory activity, and a novel structure, SUMMARY

[0007] The purpose of the present application is to provide a novel folate conjugate, which is connected by a non-cleavable linker to connect folate and PARP inhibitors, thereby forming a novel folate conjugate structure, and can effectively target folate and release drugs.

[0008] In a first aspect of the present application, a folate conjugate represented by formula I, or a tautomer thereof, or a pharmaceutically acceptable salt thereof is provided,

[0009]

[0010] wherein,

[0011] L is absent or L is a linking group;

[0012] Ola is a cytotoxic drug moiety.

[0013] In another preferred embodiment, L is absent or L is a divalent linking group.

[0014] In another preferred embodiment, L is absent or is selected from the group consisting of:

[0015]

[0016] wherein, n is selected from the group consisting of 1, 2, 3, 4, 5, or 6;

[0017] m is selected from the group consisting of 1, 2, or 3.

[0018] In another preferred embodiment, L has the structure:

[0019]

[0020] wherein,

[0021] m is selected from the group consisting of 1 or 2.

[0022] In another preferred embodiment, the cytotoxic drug is a PARP inhibitor.

[0023] In another preferred embodiment, the cytotoxic drug moiety Ola has the structure:

[0024]

[0025] represents the point of attachment between different moieties of the conjugate.

[0026] In another preferred embodiment, the conjugate is selected from the group consisting of:

[0027]

[0028] In a second aspect of the application, there is provided a structure of the formula L-Ola, wherein:

[0029]

[0030] wherein, L is as defined in the first aspect of the application.

[0031] In another preferred embodiment, the structure of L-Ola is selected from the group consisting of:

[0032]

[0033]

[0034] In a third aspect, the present application provides a method for preparing the folate conjugate according to the first aspect of the present application, comprising the steps of:

[0035] (a) providing a first reaction precursor folate FA and a second reaction precursor cytotoxic drug;

[0036] (b) reacting the first reaction precursor and the second reaction precursor with a linking molecule, thereby forming the folate conjugate according to the first aspect of the present application.

[0037] In another preferred embodiment, the first reaction precursor folate FA has the structure of:

[0038]

[0039] In another preferred embodiment, the second reaction precursor cytotoxic drug is a PARP inhibitor.

[0040] In another preferred embodiment, the second reaction precursor cytotoxic drug is olaparib, which has the structure of:

[0041]

[0042] In another preferred embodiment, the linking molecule has the structure of:

[0043]

[0044] wherein n and m are defined as above.

[0045] In another preferred embodiment, the second reaction precursor folate FA is first reacted with the linking molecule to form a first reaction intermediate; and the first reaction intermediate is reacted with the first reaction precursor cytotoxic drug to form the folate conjugate.

[0046] In another preferred embodiment, the first reaction intermediate has the structure of L-Ola as described above.

[0047] In another preferred embodiment, the reaction of the first reaction precursor and the second reaction precursor is carried out at a feed ratio of 1:2.

[0048] In a fourth aspect, the present application provides a pharmaceutical composition comprising:

[0049] (i) the folate conjugate according to the first aspect of the present application, or a tautomer thereof, or a pharmaceutically acceptable salt thereof; and

[0050] (ii) a pharmaceutically acceptable carrier.

[0051] In another preferred embodiment, the folate conjugate, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, can also be used in combination with other drugs.

[0052] In another preferred embodiment, the other drug is a DNA damaging agent.

[0053] In another preferred embodiment, the DNA damaging agent is selected from the group consisting of cisplatin, gemcitabine, paclitaxel, docetaxel, cabazitaxel, carboplatin, oxaliplatin, nedaplatin, lobaplatin, arsenic trioxide, doxorubicin, epirubicin, pirarubicin, amrubicin, apatinib, CDK12-IN-3, Onvansertib, chloroguanide, or a combination thereof.

[0054] In another preferred embodiment, the DNA damaging agent is selected from the group consisting of cisplatin, gemcitabine, paclitaxel, or a combination thereof.

[0055] In a fifth aspect of the present application, there is provided a use of the folate conjugate according to the first aspect of the present application in the manufacture of a medicament for treating a tumor.

[0056] In another preferred embodiment, the tumor is a folate receptor high expression dependent tumor, a PARP enzyme dependent tumor, or a DNA damage repair dependent tumor.

[0057] In another preferred embodiment, the tumor is selected from the group consisting of ovarian cancer, breast cancer, cervical cancer, bladder cancer, pleural mesothelioma, non-small cell lung cancer.

[0058] It should be understood that, within the scope of the present application, all combinations between the above technical features of the present application and the technical features specifically described hereinafter (e.g. in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1 The results of the colony formation experiment are shown. Among them, (A) colony staining scanning results of SKOV3 cells cultured with different concentrations of FA-002-D for 12 days; (B) number statistics of Hela cell colonies cultured with different concentrations of FA-002-D for 12 days. The experiments were repeated three times, and the data were expressed as mean ± SEM, * indicates P<0.05, **** indicates P<0.0001.

[0060] Figure 2 The fluorescence values of the apoptosis of SKOV3 cells cultured with different concentrations of FA-002-D for 48h are shown. The experiments were repeated three times, and the data were expressed as mean ± SEM, **** indicates P<0.0001.

[0061] Figure 3 The enzyme inhibition activity of the compound on PARP-1 is shown.

[0062] Figure 4 The folate receptor expression-dependent evaluation of the conjugate is shown. Among them, (A) the folate alpha receptor expression amount of each ovarian cancer cell line; (B) the proliferation inhibition of the compound on each strain of ovarian cancer cells.

[0063] Figure 5 The change of cell viability after treating SKOV3 cells with FA-002-D with or without FLOR1 antibody for 120h is shown.

[0064] Figure 6 The combination of FA-002-D and DNA damaging agents is shown.

[0065] Figure 7 The proportion of cells in S phase in each experimental group is shown. DETAILED DESCRIPTION

[0066] The present inventors have unexpectedly developed a new folate conjugate, in which the folate and the drug molecule are connected by an unbreakable linker, effectively avoiding premature drug release, and effectively improving drug targeting and reducing off-target effects, achieving the effect of efficiently treating tumors. On this basis, the present inventors completed the present application.

[0067] TERMS

[0068] In the present application, unless specifically indicated, the terms used have the general meanings known to those skilled in the art.

[0069] As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by a positively charged group on the compound of general formula (I) with an anion, or a salt formed by a negatively charged group on the compound of general formula (I) with a cation. Suitable anions include but are not limited to chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methylsulfonate, trifluoroacetate, acetate, malate, toluenesulfonate, tartrate, fumarate, glutamate, glucuronate, lactate, glutarate or maleate, etc. Suitable cations include but are not limited to sodium ion, potassium ion, magnesium ion, calcium ion, ammonium ion, etc.

[0070] In another preferred embodiment, the pharmaceutically acceptable salt of the present application refers to the salt of the compound of formula (I) with an acid such as, but not limited to, hydrofluoric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, oxalic acid, sulfuric acid, nitric acid, methanesulfonic acid, sulfamic acid, salicylic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, citric acid, acetic acid, lactic acid, tartaric acid, succinic acid, oxalic acid, pyruvic acid, malic acid, glutamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, malonic acid, fumaric acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, and isethionic acid, or the salt of the compound of formula (I) with an inorganic base such as, but not limited to, sodium salt, potassium salt, calcium salt, aluminum salt, or ammonium salt, or the salt of the compound of formula (I) with an organic base such as, but not limited to, methylamine salt, ethylamine salt, ethanolamine salt, TRIS ammonium salt, and the like.

[0071] Folate conjugate

[0072] The present application provides a novel folate conjugate. The structure of folate conjugate drug (FDC) generally includes three parts: the ligand FA of FR, the linker, and the cytotoxic drug.

[0073] The first part is artificial folate. Folic acid is composed of two parts, pterin and glutamate. The pterin part is mainly recognized by the folic acid receptor, and the glutamate part is not essential for this binding. Therefore, the glutamate part is usually connected to the drug, rather than the pterin. The carboxyl group in the glutamate structure can be divided into α-COOH and γ-COOH. It should be pointed out that the drug connected to γ-COOH has a greater affinity for the folic acid receptor than the drug connected to α-COOH. The reason may be that the drug connected to α-COOH is too close to the pterin part, which hinders the binding of the drug conjugate to the receptor.

[0074] As used herein, the folate has the following structure:

[0075]

[0076] The second part is a connecting chain, which is connected with folic acid through an unbreakable connecting chain, and the connecting mode comprises connecting the terminal carboxyl of folic acid directly with the PARP inhibitor, connecting the PARP inhibitor through a linker with different carbon chain lengths, and connecting the PARP inhibitor after introducing an O heteroatom in the carbon chain. Among them, the preferred one is to introduce an O heteroatom in the carbon chain with a length of 9, to protect the folic acid alpha-COOH, and then to remove the alpha-protecting group after the gamma position is connected with the PARP inhibitor. The optimized route solves the problem of selectivity when the alpha and gamma carboxyl groups of folic acid are activated, the composition of by-products is more single, the post-treatment operation is simplified, and it is beneficial to scale-up synthesis and purity improvement.

[0077] The third part is a cytotoxic drug, and a PARP inhibitor is selected, and the preferred one is olaparib.

[0078] In some embodiments, the tumor is preferably high in folic acid receptor expression and depends on PARP enzyme or DNA damage repair, for example, ovarian cancer, breast cancer, cervical cancer, bladder cancer, pleural mesothelioma and non-small cell lung cancer.

[0079] The application provides a conjugate of folic acid and olaparib, which is connected through an unbreakable bond, so that it has both PARP enzyme inhibition activity and folic acid binding characteristics, can effectively improve the targeting of the drug and reduce off-target effects, and provides a powerful tool for new drug combination therapy.

[0080] In the application, the conjugate has the following structure:

[0081]

[0082] wherein L and Ola are as defined above.

[0083] Preferably, the structure of L is:

[0084]

[0085] wherein,

[0086] m is selected from the group consisting of 1 or 2.

[0087] In another preferred example, in the compound, any one of L, Ola, m and n is independently the corresponding group in the specific compound described in the application.

[0088] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of Formula (I) formed from a positively charged group on the compound of Formula (I) and an anion, or a salt of a compound of Formula (I) formed from a negatively charged group on the compound of Formula (I) and a cation. Suitable anions include, but are not limited to, chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, acetate, malate, toluenesulfonate, tartrate, fumarate, glutamate, glucuronate, lactate, glutarate, or maleate, and the like. Suitable cations include, but are not limited to, sodium, potassium, magnesium, calcium, ammonium, and the like.

[0089] In another preferred embodiment, the pharmaceutically acceptable salt of the present application refers to a salt of a compound of Formula (I) with an acid, such as, but not limited to, hydrofluoric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, oxalic acid, sulfuric acid, nitric acid, methanesulfonic acid, sulfamic acid, salicylic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, citric acid, acetic acid, lactic acid, tartaric acid, succinic acid, oxalic acid, pyruvic acid, malic acid, glutamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, malonic acid, fumaric acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, and isethionic acid, and the like; or a salt of a compound of Formula (I) with an inorganic base, such as, but not limited to, a sodium salt, a potassium salt, a calcium salt, an aluminum salt, or an ammonium salt; or a salt of a compound of Formula (I) with an organic base, such as, but not limited to, a methylamine salt, an ethylamine salt, an ethanolamine salt, a TRIS ammonium salt, and the like.

[0090] Linker

[0091] The selection of the linker is critical in the design of folate conjugated drugs, as it determines whether the drug can be effectively targeted to the receptor site. A linker with the appropriate spacing distance and cleavage mode is the key to achieving this goal, which can effectively release the parent drug under specific enzymatic hydrolysis conditions or other suitable environment. The folate receptor-mediated drug release process is roughly as follows: first, the exogenous folate-drug conjugate binds to the folate receptor outside the membrane with high affinity (Kd≈10-9M); then, the cell membrane invaginates around the folate receptor and the conjugate to form an early endosome or "vesicle", endocytosis occurs, followed by the maturation of the endosome lumen acidification, the conformation of the receptor changes, and the folate-drug conjugate is released; finally, the folate receptor is transferred to the cell membrane and a new transport begins. The folate conjugated compounds reported so far are designed to be cleavable bonds such as disulfide bonds, ester bonds, etc. When the folate-drug conjugate enters the endosome, the cleavable bond can be hydrolyzed by specific enzymes and release the parent drug, in order to achieve the purpose of selective drug release.

[0092] To reduce the early release of drugs caused by the breakage of the connecting chain, the present application binds the drugs to folic acid through a non-breakable connecting chain, and selectively delivers the intact conjugate to cancer cells overexpressing FRa. Most of the folic acid conjugates currently in clinical trials or under research use breakable bonds. The advantage of designing non-breakable bonds in the present application is that: (1) it enhances the targeting of the compound to tumor cells, reduces the early release of cytotoxic drugs caused by the breakage of the connecting chain, and reduces the killing effect on normal cells, thereby reducing the incidence of adverse reactions; (2) it directly enters the cells by using folic acid receptor-mediated endocytosis, effectively avoiding the limitation of the compound with too large molecular weight to enter the cells.

[0093] On the basis of retaining the structural parts necessary for folic acid and olaparib receptor recognition, the present application designs connecting chains with different carbon chain lengths and hydrophilicity, and a compound with a folic acid and olaparib coupling ratio of 1:2 (double substitution of the terminal carboxyl group of folic acid), and the target synthesis is as follows: the folic acid and olaparib conjugates of the co-A, B, and C series shown below.

[0094] A series:

[0095]

[0096] B series:

[0097]

[0098] C series:

[0099]

[0100] Among them, the structure of Fa is as follows:

[0101]

[0102] Ola is defined as above.

[0103] Preparation method of folic acid conjugate

[0104] The embodiments of the present application specifically describe the preparation method of the compound of formula (I) of the present application, but these specific methods do not constitute any limitation to the present application. The compounds of the present application can also be conveniently prepared by optionally combining various synthetic methods described in the present specification or known in the art, and such combinations can be easily performed by those skilled in the art to which the present application belongs.

[0105] Typically, the preparation process of the compounds of the present application is as follows, wherein the raw materials and reagents used, if not specifically stated, can be purchased through commercial channels.

[0106] A series of compounds in order to investigate the addition of linker part on the activity of the compound, the terminal carboxyl of folic acid and the secondary amine on the piperazine ring of olaparib are connected, and the reverse synthesis reaction formula is as follows:

[0107]

[0108] B series of compounds is the secondary amine on the piperazine ring of olaparib connected with different carbon chain length of linker synthesis, so it needs to be condensed with the terminal carboxyl of linker and olaparib first, and then the amino group at the other end of the linker is substituted with folic acid nucleophilically.

[0109]

[0110] C series of compounds is to investigate the influence of linker composition on the solubility of the conjugate, the carbon chain of B series of compounds is introduced into O heteroatom. The synthesis method is similar to B series of compounds, and the reverse synthesis analysis is shown as follows:

[0111]

[0112] Among them, the structures of FA, Fa and Ola are defined as above.

[0113] Specifically, the condensation conditions of the double-substituted products of A, B and C series of compounds are carried out under the catalysis of EDCI, HOBT and DIPEA, the solvent is DMF, nitrogen protection and light protection. The reaction is carried out at room temperature for about 3h, after the reaction is completed, the reaction liquid is poured into methanol to precipitate the product, centrifuged, and the precipitate is dried. The double-substituted compounds of A and C series are dissolved in DMF, and then methanol is added to precipitate, which is repeated for 3 times, so that the double-substituted product with single component is obtained. Due to the existence of by-products, the dried solid of B series of double-substituted compounds is dissolved in as little DMF solvent as possible, 7% Na2CO3 solution is added, and the solid is precipitated by centrifugation, so that the double-substituted product with single component is obtained.

[0114] Pharmaceutical composition and administration method

[0115] The pharmaceutical composition of the present application comprises a safe and effective amount of the compound of the present application or its pharmacologically acceptable salt and a pharmacologically acceptable excipient or carrier. Among them, "safe and effective amount" refers to: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Generally, the pharmaceutical composition contains 1-2000 mg of the compound of the present application per dose, more preferably 5-1000 mg of the compound of the present application per dose. Preferably, the "dose" is a capsule or tablet.

[0116] "Pharmaceutically acceptable carrier" means one or more compatible solid or liquid filler or gel materials, which are suitable for human use, and which are nontoxic to the subject. "Compatible" means that the composition including the carrier, excipient, or diluent is tolerated by the subject and does not significantly reduce the efficacy of the compound of the application. Examples of pharmaceutically acceptable carriers are water, salt solutions, alcohols, gum arabic, glucose, carbohydrates, starch, calcium phosphate, various types of lubeilating agents, binders, preservatives, antioxidants, and antibacterial agents. Examples of such carriers are water, saline, phosphate buffered saline, and polyoxyethylene lauryl ether. wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

[0117] The pharmaceutical composition can be in the form of an injection, a capsule, a tablet, a pill, a powder, or a granule.

[0118] The mode of administration of the compound or pharmaceutical composition of the present application is not particularly limited, and representative modes of administration include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular, or subcutaneous), and topical administration.

[0119] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier) such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, such as hydroxymethylcellulose, alginic acid, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, such as glycerol; (d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solution retarders, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) absorbents, such as kaolin and bentonite clay; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage form can also comprise buffering agents.

[0120] Solid dosage forms, such as tablets, dragees, capsules, pills, and granules, can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the art. They can contain opacifying agents, and can also be of a composition that they release the active compound or compounds in a certain part of the digestive tract. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if desired, with one or more of the excipients described above.

[0121] Liquid dosage forms for oral administration include pharmaceutically-acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, dimethylformamide, and the like, or combinations thereof.

[0122] Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0123] Suspensions, in addition to the active compounds, can contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, sodium alginate, agar-agar, and the like.

[0124] Compositions for parenteral injection can contain physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyol, and suitable mixtures thereof.

[0125] Dosage forms for topical administration of a compound of this application include ointments, powders, sprays, and inhalers. The active component is admixed with a carrier, which can be a sterile, pyrogen-free, aqueous or nonaqueous medium for transdermal, buccal, or sublingual administration or a pyrogen-free, sterile, water-free medium for injection or inhalation. The preparation can be a liquid solution, suspension, emulsion, or elixir, or it can be a solid, e.g., a powder, a tablet, or a capsule.

[0126] The compounds of the present application can be administered alone, or in combination with other pharmaceutically acceptable compounds (e.g., DNA damaging agents).

[0127] The therapeutic methods of the present application can be administered alone, or in combination with other therapeutic procedures or therapeutic agents.

[0128] Pharmaceutical compositions are used in a safe and effective amount of a compound of the present application for a mammal (e.g., a human) in need of treatment. When used in this manner, the compositions are administered in a pharmaceutically effective amount, which is typically in the range of 1 to 2000 mg, preferably 5 to 1000 mg, per day for a 60 kg body weight adult. The actual amount, however, will depend on the route of administration, the patient's health, and the like, and will be ultimately at the discretion of the attendant physician.

[0129] Compared with the prior art, the present application has the following main advantages:

[0130] 1. The folic acid conjugate of the present invention uses an unbreakable linker chain, which enhances the targeting of biotoxic drugs to tumor cells.

[0131] 2. The folic acid conjugate described in this invention can effectively prevent premature drug release and reduce the incidence of adverse drug reactions.

[0132] 3. The conjugates of this invention exhibit strong folic acid receptor expression dependence, proving the feasibility of the unbreakable linker design.

[0133] 4. The folic acid conjugate described in this invention exhibits strong cell inhibitory activity in ovarian cancer IGROWV1 cells.

[0134] 5. The conjugate of this invention has proliferation-inhibiting and apoptosis-promoting effects on SKOV3 cells, and has excellent PARP-1 binding inhibition ability.

[0135] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0136] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0137] Experimental methods

[0138] Cell proliferation toxicity assays (CTG and CCK-8 assays)

[0139] CTG method: Dilute cells in the logarithmic growth phase with culture medium and seed them at a density of 1400 cells / well in 96-well plates. Incubate at 37°C for 24 hours. After cell adhesion, remove the culture medium. Adjust the pH to approximately 10 by adding sodium hydroxide solution to the 10mM DMSO stock solution of each compound. Perform serial dilutions with the culture medium, adding 100 μL of the compound dilution to each well of the seeded plate, and incubate at 37°C for 120 hours. Add 50 μL / well of... Reagent Reagents, placed in a 37°C cell incubator for 2h. Use the orbital shaker to mix for 2 minutes to induce cell lysis. Place the plate in room temperature conditions for 10 minutes to stabilize the luminescent signal. Measure the full wavelength, IC50values were fitted by GraphPad Prism 9.

[0140] CCK-8 method: Cells in the logarithmic growth phase were diluted with culture medium and inoculated into a 96-well plate at a density of 5000 cells per well in a 37°C incubator for 24h. After the cells adhered and grew, the culture medium was removed. Sodium hydroxide solution was added to the 10mM DMSO stock solution of each compound to adjust the pH to about 10, and the medium was gradiently diluted. 100μL of compound diluent was added to each well of the inoculated plate and placed in a 37°C cell incubator for the corresponding time. 10μL of CCK-8 reagent was added to each well and incubated at 37°C for 2h to stabilize the luminescent signal. Then the absorbance at 450nm was measured by a microplate reader, 100% IC 50 values were fitted by GraphPad Prism 9.

[0141] Colony formation assay (CFA)

[0142] Logarithmic growth phase SKOV3 cells were collected and inoculated into a 6-well plate at a density of 1000 cells per well and cultured for 24h. Compound FA-002-D was diluted with culture medium to set concentrations of 1, 2, 4, and 8μM. The original culture medium was removed and 2mL of new culture medium containing different concentrations of compound was slowly added along the side wall of the well. The culture medium was replaced every 3 days, and the morphology and growth of the cells were observed during this period. After about 12 days of culture, the culture medium was discarded, the cells were washed twice with PBS, 10% formaldehyde solution was added to each well to fix for 20-30min, the fixing solution was discarded, the cells were washed twice with PBS, stained with crystal violet for 30min, the staining solution was discarded, the cells were washed with PBS until the background was clean, and then scanned after air-drying.

[0143] Apoptosis experiment

[0144] Logarithmic phase SKOV3 cells were collected, counted, and inoculated into a 6-well plate to control the concentration of each well to be about 3×10 5Cells in logarithmic growth phase were diluted with culture medium and seeded into 96-well plates at a density of 4000 cells / well, incubated at 37°C for 24h, and then 5, 2.5, 1.25 μM of FA-002-D was added respectively. The plates were incubated in the cell incubator for 72h. The cells were trypsinized for 2-5 min, collected and washed with PBS for 1-2 times. Binding Buffer and FITC / PI dye were added, and the mixture was incubated on ice for 15 min in the dark. The apoptosis of the cells was detected by Agilent flow cytometry, and the light was avoided. The data were processed by NovoExpress software and plotted, and the apoptosis rate was calculated.

[0145] Caspase-3 / 7 activity detection

[0146] Cells in logarithmic growth phase were diluted with culture medium and seeded into 96-well plates at a density of 4000 cells / well, incubated at 37°C for 24h, and then 5, 2.5, 1.25 μM of FA-002-D was added respectively. The plates were incubated in the cell incubator for 72h. The cells were trypsinized for 2-5 min, collected and washed with PBS for 1-2 times. Binding Buffer and FITC / PI dye were added, and the mixture was incubated on ice for 15 min in the dark. The apoptosis of the cells was detected by Agilent flow cytometry, and the light was avoided. The data were processed by NovoExpress software and plotted, and the apoptosis rate was calculated. 3 / 7 reagent was added to each well, and the plates were incubated at room temperature for 1h. The luminescence values of the samples were determined by using a microplate luminescence detector.

[0147] PARP inhibition activity determination

[0148] 50 μL of 1x PARP Buffer was added to each well, and the plates were incubated at room temperature for 30 min. The 1x PARP Buffer was aspirated, and a series of concentration gradients of inhibitors were added to the wells, a total of four drugs, namely FA-Ola-D, FA-C8-D, Olaparib and FA-C8-D. The concentration gradient of the drug groups was set as: 10, 2.5, 0.63, 0.156, 0.039, 0.0098, 0.002 nM. Background subtraction group: a negative control without PARP-HSA enzyme should be prepared to determine the background absorbance. Blank group: PARP-HSA enzyme 0.5 U / well without inhibitor. These wells provide a 100% activity reference value. Standard

[0149] Curve group: PARP-HSA enzyme was diluted in pre-chilled microtubes with 1×PARP Buffer to achieve total activities of 1 U / 25 μL, 0.5 U / 25 μL, 0.25 U / 25 μL, and 0.125 U / 25 μL, respectively. 25 μL of 1×PARP Cocktail was added to each well and incubated at room temperature for 60 min. The wells were washed twice with 1×PBS + 0.1% Triton X-100 (200 μL / well), followed by two washes with 1×PBS. 50 μL of Strep-HRP working solution was added to each well and incubated at room temperature for 60 min. The wells were washed twice with 1×PBS + 0.1% Triton X-100 (200 μL / well), followed by two washes with 1×PBS. 50 μL of TACS-Sapphire substrate was added to each well and incubated at room temperature in the dark for 15 min. Add 50 μL of 0.2 M HCl to each well to stop the reaction, and read the absorbance at 450 nm.

[0150] Western blotting assay

[0151] Cells were collected by centrifugation at 1500 rpm and washed three times with 1×PBS. RIPA universal protein lysis buffer was added, and cells were lysed on ice for 30 min. The supernatant was collected by centrifugation at 12000 rpm and 4°C for 30 min. The extracted protein supernatant was thoroughly mixed by pipetting, and 2 μL of protein sample and 18 μL of ddH2O were added to each well of a 96-well plate. Finally, 200 μL of detection solution was added to each well. The 96-well plate was incubated at 37°C for 30 min, and the absorbance at 562 nm was measured using a microplate reader. The results were then used to calculate the protein standard curve. Based on the previously determined sample protein concentration, the absorbance of 20 μg of protein was calculated.

[0152] Prepare the required sample volume and add ddH2O to bring the final loading volume to 20 μL. Add 5× Loading Buffer by volume, mix well, and then briefly centrifuge at low temperature. Perform denaturation in a 100℃ metal bath for 5 min, then briefly centrifuge again for later use. Run the gel electrophoresis and transfer the film. Use ImageJ software to perform grayscale analysis on the bands.

[0153] combination therapy

[0154] SKOV3 cells in logarithmic growth phase were collected at a density of 4 × 10⁻⁶ cells / cells. 3 FA-002-D, Cisplatin, Gemcitabine, and PTX were inoculated into 96-well plates and cultured for 24 hours until adherence. The single-drug groups were then diluted 50 / 50 with culture medium. The final concentrations of FA-002-D were as follows:

[0155] 512, 256, 128, 64, 32, 16, 8, 4, 2, 0 mM, set 2 wells. The final concentration of Cisplatin in the single-drug group was 384, 192, 96, 48, 24, 12, 6, 3, 1.5, 0 mM, the final concentration of Gemcitabine was 51.2, 25.6, 12.8, 6.4, 3.2, 1.6, 0.8, 0.4, 0.2, 0 mM, and the final concentration of PTX was 160, 80, 40, 20, 10, 5, 2.5, 1.25, 0.625, 0 mM. The concentration of FA-002-D combined with Cisplatin, Gemcitabine, and PTX was the sum of the corresponding concentrations, and two wells were set for each group (as shown in Table 4, taking the FA-002-D and Cisplatin group as an example). After adding the compounds at different concentrations, the cells were incubated for another 48 h.

[0156] Cell cycle

[0157] The logarithmic phase SKOV3-FUCCI cells were collected and seeded in a 96-well plate at a density of 4 x 10 3 cells / well. After 24 h of culture, the cells were diluted with the culture medium in a half-dilution manner. The final concentration of FA-002-D in the single-drug group was 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0 mM, and two wells were set for each group. The final concentration of Cisplatin in the single-drug group was 6, 3, 1.5, 0.75, 0.375, 0.1875, 0.09375, 0.046875, 0 mM, the final concentration of Gemcitabine was 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625, 0 mM, and the final concentration of PTX was 0.6, 0.3, 0.15, 0.075, 0.0375, 0.01875, 0.009375, 0.0046875, 0 mM. The concentration of FA-002-D combined with Cisplatin, Gemcitabine, and PTX was the sum of the corresponding concentrations, and two wells were set for each group (as shown in Table 5, taking the FA-002-D and Cisplatin group as an example). After adding the compounds at different concentrations, the cells were incubated for another 48 h. The live cell images were taken by the high-content imaging analysis system, and the images of each group were analyzed by Cellprofiler software to calculate the proportion of red fluorescent cells in the total cell count (red fluorescent cells + blue fluorescent cells). The data were statistically derived by R language.

[0158] Table 5 Drug concentrations of each group in the combination of FA-002-D and Cisplatin

[0159]

[0160] Preparation of compounds

[0161] Synthesis of intermediates in series B

[0162] Synthesis of FA6-C5 to FA6-C9

[0163] General procedure for the synthesis of intermediates: glutaric anhydride (25 mmol, 2.85 g) was dissolved in 15 mL of dichloromethane, and mono-Boc-ethylenediamine, N-Boc-1,3-propanediamine, tert-butyl (4-aminobutyl)carbamate, tert-butyl N-(5-aminopentyl)carbamate, tert-butyl N-(6-aminopentyl)carbamate (25 mmol) were dissolved in 15 mL of dichloromethane, respectively, and slowly added dropwise to the glutaric anhydride solution in dichloromethane. After 2 h of reaction at room temperature, the white solid or colorless transparent oil was obtained by rotary evaporation under reduced pressure. HATU (25 mmol, 9.5 g) was dissolved in 25 mL of DMF and activated at room temperature for about 10 min. Then 4-(4-fluoro-3-(piperazin-1- carbonyl)benzyl)phthalazin-1(2H)-one (25 mmol, 9 g) and DIPEA (100 mmol, 17 mL) were dissolved in 25 mL of DMF and slowly added dropwise to the activated solution. After about 3 h of reaction at room temperature, saturated NaCl aqueous solution was added and the mixture was extracted with ethyl acetate for 3-4 times. The upper organic phase was collected, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The concentrated solution was separated by silica gel column chromatography (eluent: DCM / MeOH (v / v) = 20 / 1) to obtain a transparent oil.

[0164] The transparent oil was dissolved in 15 mL of dichloromethane, and trifluoroacetic acid (100 mmol, 11.5 g) was added dropwise. The reaction was carried out at room temperature for 3 h. After the reaction was completed, dichloromethane was added and concentrated under reduced pressure. The excess trifluoroacetic acid was removed by rotary evaporation for 3 times. NaOH aqueous solution was added to adjust the pH to 10, which generated a large amount of bubbles and heat. The obtained solution was directly rotary evaporated to dryness. The solid was dissolved in a mixture of dichloromethane and methanol, filtered, and the filtrate was concentrated under reduced pressure. The concentrated solution was separated by silica gel column chromatography (eluent: DCM / MeOH (v / v) = 10 / 1) to obtain a colorless oil.

[0165] The structures of intermediates in series B are as follows:

[0166]

[0167] N-(2-aminoethyl)-5-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)-5-oxopentanamide. Colorless oil, yield 35%.

[0168] 1 H NMR (500 MHz, DMSO-d6) δ 8.26 (d, J = 7.8 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.92 - 7.87 (m, 1H), 7.83 (td, J = 7.5, 1.2 Hz, 1H), 7.76 (t, J = 5.7 Hz, 1H), 7.44 (ddd, J = 7.6, 5.0, 2.3 Hz, 1H), 7.39 - 7.33 (m, 1H), 7.24 (td, J = 9.0, 2.0 Hz, 1H), 4.33 (s, 2H), 3.41 - 3.32 (m, 2H), 3.16 (dt, J = 19.5, 5.2 Hz, 2H), 3.03 (p, J = 6.0 Hz, 2H), 2.54 (q, J = 6.4 Hz, 2H), 2.29 (dt, J = 33.8, 7.4 Hz, 2H), 2.10 (q, J = 6.9 Hz, 2H), 1.71 (h, J = 7.1 Hz, 2H). 13 CNMR (126 MHz, DMSO-d6) δ 172.3, 171.0, 159.8, 145.3, 135.3, 134.0, 129.6, 129.4, 128.4, 126.6, 125.9, 116.5, 116.3, 60.2, 47.1, 46.8, 42.5, 41.7, 41.0, 36.9, 35.0, 21.3.

[0169]

[0170] N-(3-aminopropyl)-5-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)-5-oxopentanamide. Colorless oil, yield 25%.

[0171] 1H NMR (500 MHz, DMSO-d6) δ 8.26 (d, J = 7.9 Hz, 1H), 7.98 (dd, J = 17.5, 6.9 Hz, 2H), 7.92 - 7.86 (m, 1H), 7.83 (t, J = 7.5 Hz, 1H), 7.44 (t, J = 7.0 Hz, 1H), 7.35 (d, J = 6.1 Hz, 1H), 7.26 - 7.21 (m, 1H), 4.33 (s, 2H), 3.51 (d, J = 5.2 Hz, 2H), 3.36 (dt, J = 14.9, 4.6 Hz, 2H), 3.13 (ddt, J = 32.1, 12.2, 6.0 Hz, 4H), 2.63 (q, J = 6.4 Hz, 2H), 2.30 (dt, J = 33.7, 7.4 Hz, 2H), 2.11 (q, J = 6.7 Hz, 2H), 1.71 (h, J = 7.7 Hz, 2H). 13 C NMR (101 MHz, DMSO) δ 173.5, 172.5, 171.0, 164.5, 159.9, 145.33, 135.3, 134.0, 130.0, 129.4, 128.3, 126.6, 125.9, 116.5, 116.3, 45.4, 44.9, 42.1, 41.6, 40.9, 40.6, 36.9, 35.0, 32.2, 22.7, 21.3.

[0172]

[0173] N-(4-aminobutyl)-5-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)-5-oxopentanamide. Colorless oil, yield 40%.

[0174] 1H NMR (500 MHz, DMSO-d6) δ 12.61 (s, 1H), 8.26 (d, J = 7.9 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.89 (dt, J = 8.6, 5.5 Hz, 2H), 7.85 - 7.80 (m, 1H), 7.47 - 7.42 (m, 1H), 7.35 (t, J = 3.9 Hz, 1H), 7.26 - 7.20 (m, 1H), 4.33 (s, 2H), 3.60 (d, J = 30.1 Hz, 2H), 3.50 (d, J = 5.4 Hz, 2H), 3.03 (p, J = 6.3 Hz, 2H), 2.77 (q, J = 6.8 Hz, 2H), 2.29 (dt, J = 32.0, 7.4 Hz, 2H), 2.09 (q, J = 6.9 Hz, 2H), 1.70 (q, J = 7.4 Hz, 2H), 1.51 (q, J = 7.3, 6.8 Hz, 2H), 1.41 (q, J = 6.9 Hz, 2H). 13 C NMR (126 MHz, DMSO-d6) δ 172.3, 171.1, 159.9, 158.8, 158.6, 145.4, 134.0, 129.4, 128.3, 126.5, 125.9, 116.4, 114.1, 49.0, 41.6, 39.0, 38.2, 36.9, 35.0, 32.2, 26.6, 24.9, 21.4.

[0175]

[0176] N-(5-aminopentyl)-5-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)-5-oxopentanamide. Colorless oil, yield 46%.

[0177] 1H NMR (500 MHz, DMSO-d6) δ 12.62 (s, 1H), 8.27 (d, J = 7.8 Hz, 1H), 7.97 (d, J = 8.1 Hz, 1H), 7.92 - 7.80 (m, 5H), 7.46 (d, J = 8.4 Hz, 1H), 7.35 (d, J = 6.7 Hz, 1H), 7.24 (t, J = 8.9 Hz, 1H), 4.34 (s, 2H), 3.63 (s, 1H), 3.57 (s, 1H), 3.50 (d, J = 5.7 Hz, 2H), 3.01 (q, J = 6.5 Hz, 2H), 2.75 (d, J = 9.8 Hz, 2H), 2.29 (dt, J = 32.1, 7.5 Hz, 2H), 2.09 (q, J = 6.7 Hz, 2H), 1.70 (q, J = 7.4 Hz, 2H), 1.52 (d, J = 7.4 Hz, 2H), 1.38 (q, J = 7.2 Hz, 2H), 1.28 (d, J = 6.6 Hz, 2H). 13 C NMR (126 MHz, DMSO-d6) δ 171.1, 159.9, 158.8, 145.4, 135.3, 134.0, 129.5, 129.4, 126.5, 125.9, 116.5, 49.0, 39.1, 38.6, 35.0, 32.2, 29.0, 27.1, 23.7, 21.4.

[0178]

[0179] N-(6-aminohexyl)-5-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)-5-oxopentanamide. Colorless oil, yield 32%.

[0180] 1H NMR (500 MHz, DMSO-d6) δ 12.63 (s, 1H), 8.26 (d, J = 7.8 Hz, 1H), 7.95 (t, J = 7.3 Hz, 1H), 7.88 (tt, J = 9.8, 4.5 Hz, 2H), 7.82 (t, J = 7.5 Hz, 1H), 7.44 (ddd, J = 8.2, 5.4, 2.7 Hz, 1H), 7.35 (dd, J = 6.5, 2.2 Hz, 1H), 7.22 (td, J = 9.0, 1.8 Hz, 1H), 4.33 (s, 2H), 3.66 - 3.54 (m, 2H), 3.35 (dt, J = 15.9, 5.1 Hz, 2H), 3.21 - 3.11 (m, 2H), 3.01 (p, J = 6.5 Hz, 2H), 2.76 (q, J = 6.8 Hz, 2H), 2.29 (dt, J = 32.4, 7.5 Hz, 2H), 2.09 (q, J = 6.8 Hz, 2H), 1.70 (h, J = 7.6 Hz, 2H), 1.52 (h, J = 7.1 Hz, 2H), 1.36 (h, J = 7.3 Hz, 2H), 1.29 - 1.20 (m, 5H). 13 C NMR (126 MHz, DMSO-d6) δ 172.2, 171.1, 159.9, 158.9, 158.7, 135.3, 134.0, 129.5, 129.4, 128.3, 125.9, 116.4, 114.0, 56.5, 41.6, 41.0, 38.7, 36.9, 36.2, 32.2, 29.4, 27.4, 26.0, 21.4, 18.9.

[0181] Synthesis of intermediates in series C

[0182] Synthesis of FA6-002

[0183] Dissolve glutaric anhydride (25 mmol, 2.85 g) in 15 mL dichloromethane, dissolve [2-(2-aminoethoxy)ethyl]carbamic acid tert-butyl ester (25 mmol) in 15 mL dichloromethane, slowly drop the glutaric anhydride solution into the dichloromethane solution, react at room temperature for 2 h, then dry under reduced pressure to obtain white solid or colorless transparent oil, add condensing agent 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (25 mmol, 9.5 g) dissolved in 25 mL DMF, activate the terminal carboxyl group of the linker at room temperature for about 10 min, then slowly drop 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (25 mmol, 9 g) and DIPEA (100 mmol, 17 mL) dissolved in 25 mL N,N-dimethylformamide (DMF) into the activated solution, react at room temperature for about 3 h, then add saturated NaCl, extract with ethyl acetate for 3-4 times, collect the upper organic phase, dry with anhydrous Na2SO4, then concentrate under reduced pressure, and separate the concentrated solution by silica gel column chromatography (eluent: DCM / MeOH (v / v) = 20 / 1) to obtain transparent oil.

[0184] Dissolve the transparent oil in 15 mL dichloromethane, drop trifluoroacetic acid (100 mmol, 11.5 g), react at room temperature for 3 h, then add dichloromethane and concentrate under reduced pressure, repeat 3 times to dry the excess trifluoroacetic acid, add NaOH aqueous solution to adjust the pH to neutral, which generates a large amount of bubbles and heat. Add saturated NaCl, chloroform and a small amount of methanol (to assist dissolution), extract repeatedly for 7-8 times, collect the lower organic phase, dry with anhydrous Na2SO4, then concentrate under reduced pressure, and separate the concentrated solution by silica gel column chromatography (eluent: DCM / MeOH (v / v) = 10 / 1) to obtain colorless transparent oil, which is the intermediate (FA6-002) with high purity.

[0185]

[0186] N-(2-(2-aminoethoxy)ethyl)-5-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)-5-oxopentanamide. Colorless transparent oil, yield 63%.

[0187] 1H NMR (500 MHz, DMSO-d6) δ 12.63 (s, 1H), 8.26 (d, J = 7.8 Hz, 1H), 7.95 (t, J = 7.4 Hz, 3H), 7.88 (td, J = 7.7, 3.2 Hz, 1H), 7.82 (t, J = 7.5 Hz, 1H), 7.44 (ddd, J = 8.1, 5.3, 2.5 Hz, 1H), 7.35 (dd, J = 6.4, 2.3 Hz, 1H), 7.22 (td, J = 9.0, 2.0 Hz, 1H), 4.32 (s, 2H), 3.65 - 3.54 (m, 4H), 3.42 (q, J = 5.9 Hz, 2H), 3.36 (dq, J = 14.1, 4.9, 4.3 Hz, 2H), 3.23 (q, J = 5.5 Hz, 2H), 3.16 (dt, J = 22.1, 5.1 Hz, 3H), 2.98 (q, J = 4.9 Hz, 2H), 2.29 (dt, J = 32.1, 7.5 Hz, 2H), 2.13 (q, J = 6.8 Hz, 2H), 1.70 (h, J = 7.6 Hz, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 172.6, 159.9, 159.0, 145.4, 135.3, 129.5, 129.3, 128.3, 125.9, 122.0, 119.1, 116.1, 113.2, 69.5, 66.7, 44.9, 41.6, 38.6, 36.2, 32.2, 21.3.

[0188] Synthesis of a series of conjugates

[0189] Synthesis of FA-Ola-D

[0190] Folic acid (0.45 mmol, 200 mg), EDCI (1.8 mmol, 345 mg), HOBT (1.8 mmol, 243 mg), 4-(4-fluoro-3-(piperazin-1-ylcarbonyl)benzyl)phthalazin-1(2H)-one (1 mmol, 367 mg) were placed in a three-necked flask, 25 mL of DMF was added, protected by nitrogen, avoid light, injected DIPEA (3.15 mmol, 0.5 mL), reacted at room temperature for 3 h. After the reaction was completed, the reaction solution was transferred to a centrifuge tube, methanol solution was added as a poor solvent, 25 mL of poor solvent was added per 25 mL of reaction solution, the precipitate was separated after centrifugation, the supernatant was discarded, the precipitate was dissolved in DMF and then methanol solution was added as a poor solvent to precipitate the product, which was repeated 3-4 times, the precipitate was washed with methanol after centrifugation, and the solid was placed in a vacuum drying oven to dry to obtain the product.

[0191]

[0192] (S)-4-(((2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl)amino)-N-(1,5-bis(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)-1,5-dioxopentan-2-yl)benzamide. Yellow solid, yield: 33%, purity: 92%. 1 H NMR (500 MHz, DMSO-d6) δ 12.60 (s, 2H), 8.64 (s, 1H), 8.25 (d, J = 7.3 Hz, 2H), 8.14 (q, J = 8.5 Hz, 1H), 7.98 - 7.76 (m, 6H), 7.66 (q, J = 6.6 Hz, 2H), 7.47 - 7.32 (m, 4H), 7.22 (t, J = 9.0 Hz, 2H), 6.96 (q, J = 5.1 Hz, 1H), 6.63 (t, J = 7.1 Hz, 2H), 4.87 (d, J = 32.0 Hz, 1H), 4.48 (t, J = 5.2 Hz, 2H), 4.32 (s, 4H), 3.57 (d, J = 20.2 Hz, 8H), 3.17 (dd, J = 28.0, 11.7 Hz, 4H), 2.47 - 2.25 (m, 2H), 1.98 - 1.91 (m, 1H), 1.83 (s, 1H). 13 C NMR (126 MHz, DMSO-d6) δ 170.6, 164.2, 164.1, 159.5, 155.4, 150.9, 145.0, 134.9, 133.6, 131.6, 129.1, 129.0, 128.0, 127.9, 126.1, 125.5, 121.2, 115.9, 111.2, 48.7, 45.9, 44.4, 41.8, 41.3, 36.5, 28.6, 26.8. HRMS [ESI]: calcd for [M-H] - (C 59 H 52 N 15 O8F2) requires m / z 1136.4097, found 1136.4103.

[0193] Synthesis of B series conjugates

[0194] General method for synthesis of disubstituted final products

[0195] Folate (0.45 mmol, 200 mg), EDCI (1.8 mmol, 345 mg), HOBT (1.8 mmol, 243 mg), 4-(4-fluoro-3-(piperazin-1-carbonyl)benzyl)phthalazin-1(2H)-one (1 mmol, 367 mg) were placed in a three-necked flask, 25 mL of DMF was added, protected by nitrogen, and light-protected. DIPEA (3.15 mmol, 0.5 mL) was injected, and the reaction was allowed to proceed at room temperature for 3 h. After the reaction was completed, the reaction solution was transferred to a centrifuge tube, and methanol solution was added as a poor solvent. For every 25 mL of reaction solution, 25 mL of poor solvent was added, and the precipitate was separated by centrifugation. The supernatant was discarded, and the dried solid was ground. The solid was dissolved in as little DMF as possible, and 7% Na2CO3 solution was added. The solid was separated by centrifugation, washed with methanol, and dried in a vacuum drying oven. The product was obtained.

[0196]

[0197] (S)-2-(4-(((2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl)amino)benzamido)-N 1 ,N 5 -bis(5-(5-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)-5-oxopentanamido)pentyl)pentanediamide. Yellow solid, yield: 29%, purity: 96%.

[0198] 1H NMR (500 MHz, DMSO-d6) δ 12.65 (s, 2H), 8.42 (s, 1H), 8.26 (d, J = 7.8 Hz, 2H), 7.97 (s, 1H), 7.95 (s, 2H), 7.89 (t, J = 7.7 Hz, 2H), 7.83 (t, J = 7.6 Hz, 2H), 7.79 (s, 3H), 7.64 (d, J = 8.4 Hz, 2H), 7.44 (dd, J = 9.1, 4.6 Hz, 2H), 7.37 (dd, J = 6.4, 2.4 Hz, 2H), 7.23 (t, J = 9.0 Hz, 2H), 6.86 (t, J = 6.0 Hz, 1H), 6.64 (d, J = 8.3 Hz, 2H), 5.98 (s, 2H), 4.42 (d, J = 5.9 Hz, 2H), 4.33 (s, 4H), 4.28 (q, J = 7.8 Hz, 1H), 3.63 (s, 3H), 3.57 (s, 6H), 3.53 - 3.47 (m, 4H), 3.15 (dd, J = 15.1, 10.0 Hz, 4H), 2.99 (dd, J = 13.0, 6.7 Hz, 9H), 2.28 (dt, J = 33.1, 7.4 Hz, 4H), 2.08 (q, J = 7.2, 6.7 Hz, 6H), 1.95 (d, J = 7.8 Hz, 1H), 1.85 (s, 1H), 1.70 (h, J = 7.2 Hz, 4H), 1.34 (dp, J = 16.0, 8.2, 7.7 Hz, 9H), 1.25 - 1.14 (m, 5H). 13 C NMR (126 MHz, DMSO d6) δ 171.7, 171.6, 170.6, 164.0, 159.4, 144.8, 144.6, 134.8, 133.5, 129.1, 128.9, 127.9, 126.1, 125.5, 115.8, 111.2, 56.0, 53.3, 46.6, 46.3, 41.6, 41.3, 38.4, 36.4, 35.8, 34.6, 31.8, 30.8, 23.8, 18.6. HRMS [ESI]: calcd for [M+2H] 2+ (C 79 H 91 N 19 O 12 F2)requires m / z 767.8526,found 767.8548.

[0199]

[0200] (S)-2-(4-(((2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl)amino)benzamido)-N 1 ,N 5 - bis(6-(5-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)-5-oxopentanamido)hexyl)pentanediamide. Yellow solid, yield: 25%, purity: 97%.

[0201] 1H NMR (500 MHz, DMSO-d6) δ 12.62 (s, 2H), 8.53 (s, 1H), 8.26 (dd, J = 7.8, 1.4 Hz, 2H), 7.96 (s, 4H), 7.89 (t, J = 7.7 Hz, 2H), 7.85 - 7.80 (m, 2H), 7.78 - 7.72 (m, 3H), 7.65 (d, J = 8.3 Hz, 2H), 7.44 (dd, J = 8.8, 4.9 Hz, 2H), 7.37 (dd, J = 6.6, 2.3 Hz, 2H), 7.23 (t, J = 9.0 Hz, 2H), 7.01 (s, 1H), 6.88 (d, J = 6.1 Hz, 1H), 6.65 (d, J = 8.3 Hz, 2H), 4.45 (d, J = 5.8 Hz, 2H), 4.33 (s, 4H), 4.28 (q, J = 7.9 Hz, 1H), 3.63 (s, 2H), 3.57 (s, 2H), 3.50 (d, J = 5.7 Hz, 4H), 3.45 - 3.26 (m, 7H), 3.16 (dt, J = 19.7, 5.0 Hz, 4H), 2.99 (hept, J = 6.0 Hz, 8H), 2.29 (dt, J = 33.2, 7.4 Hz, 4H), 2.08 (q, J = 6.9 Hz, 5H), 2.00 - 1.91 (m, 1H), 1.85 (t, J = 7.5 Hz, 1H), 1.70 (q, J = 7.3 Hz, 4H), 1.33 (s, 8H), 1.21 (d, J = 13.5 Hz, 9H).13C NMR (126 MHz, DMSO-d6) δ 171.5, 170.6, 164.0, 162.3, 159.4, 144.9, 134.8, 133.5, 131.7, 129.1, 129.0, 127.9, 125.5, 115.8, 111.2, 53.3, 44.9, 41.6, 41.1, 40.6, 38.3, 36.4, 35.8, 34.6, 32.2, 31.8, 30.8, 29.1, 26.1, 21.0. HRMS [ESI]: calcd for [M+2H] 2+ (C 81 H 95 N 19 O 12 F2)requires m / z781.8682,found 781.8663.

[0202] Synthesis of C series conjugates

[0203] Synthesis of FA-002-D

[0204] Folic acid (0.45 mmol, 200 mg), EDCI (1.8 mmol, 345 mg), HOBT (1.8 mmol, 243 mg), FA6-002 (1 mmol) were placed in a three-necked flask, 25 mL of DMF was added, and the mixture was protected by nitrogen and kept away from light. DIPEA (3.15 mmol, 0.5 mL) was injected, and the mixture was reacted at room temperature for 3 h. After the reaction was completed, the reaction solution was transferred to a centrifuge tube, and methanol solution was added as a poor solvent. For every 25 mL of reaction solution, 25 mL of poor solvent was added. After the product was precipitated and centrifuged, the supernatant was discarded. The precipitate was dissolved in DMF, and methanol solution was added as a poor solvent to precipitate the product. The operation was repeated 3-4 times. After centrifugation, the precipitate was washed with methanol. After centrifugal washing, the solid was placed in a vacuum drying oven for drying to obtain the product.

[0205]

[0206] (S)-2-(4-(((2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl)amino)benzamido)-N 1 ,N 5 -bis(2-(2-(5-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)-5-oxopentanamido)ethoxy)ethyl)pentanediamide. Yellow solid, yield: 21%, purity: 93%.

[0207] 1H NMR (500 MHz, DMSO-d6) δ 12.60 (s, 2H), 8.63 (d, J = 7.0 Hz, 1H), 8.26 (dd, J = 7.8, 1.4 Hz, 2H), 8.02 (d, J = 7.8 Hz, 1H), 7.98 - 7.93 (m, 2H), 7.92 - 7.79 (m, 6H), 7.69 - 7.61 (m, 3H), 7.43 (t, J = 7.1 Hz, 2H), 7.36 (dd, J = 6.5, 2.3 Hz, 2H), 7.23 (t, J = 9.0 Hz, 2H), 6.93 (t, J = 6.2 Hz, 1H), 6.69 - 6.59 (m, 3H), 4.49 (d, J = 6.1 Hz, 2H), 4.32 (s, 5H), 3.62 (d, J = 5.5 Hz, 2H), 3.56 (d, J = 4.8 Hz, 1H), 3.49 (s, 5H), 3.17 (tt, J = 12.6, 7.5 Hz, 14H), 2.28 (dt, J = 31.8, 7.3 Hz, 3H), 2.19 - 2.06 (m, 6H), 1.94 (q, J = 5.5, 3.7 Hz, 1H), 1.86 (d, J = 10.1 Hz, 1H), 1.69 (q, J = 7.2 Hz, 5H). 13 C NMR (126 MHz, DMSO-d6) δ 172.0, 171.9, 170.6, 170.4, 164.1, 159.4, 154.0, 144.9, 134.8, 133.5, 131.8, 129.1, 129.0, 127.9, 126.1, 125.5, 115.9, 111.3, 111.2, 68.9, 68.8, 59.8, 46.3, 45.9, 41.6, 41.1, 40.4, 38.5, 36.4, 34.5, 31.7, 20.8, 14.1. HRMS [ESI]: calcd for [M+2H] 2+ (C 77 H 87 N 19 O 14 F2) requires m / z 769.8318, found 769.8285.

[0208] Disubstituted end product verification experiment

[0209] The generation of end product was verified by thin layer chromatography, the developing agent was n-propanol:n-butanol:ammonia water:water = 5:5:3:1, under the irradiation of wavelength 254 nm, the Rf values of different polar substances in the reaction system were observed fThe value, the new product FA-002-D is generated in the reaction solution except the raw material folic acid. At the same time, liquid chromatography-mass spectrometry (LC-MS) is used to judge the reaction process by target molecular weight. Folic acid and product are dissolved in methanol, and ammonia water is added to assist dissolution. It is observed that the solid is dissolved. The solution is analyzed again by LC-MS, and folic acid [M+H] + Molecular ion peak m / z 442.17, FA-002-D [M+H] + Molecular ion peak m / z 1538.78 (molecular weight: 1537.65), indicating that a double-substituted product is generated under this condition, which is consistent with the thin layer chromatography detection result.

[0210] Biological experiments

[0211] Example 1: Preliminary evaluation of the anticancer activity of folic acid and olaparib conjugate

[0212] Experimental method: In order to detect the cytotoxicity of the compound, the present application adopts CTG method and CCK-8 method, and uses the cell proliferation toxicity test method described in the experimental method to carry out the experiment. CellTiter-Glo (CTG) is a rapid cell viability detection method based on ATP detection. ATP adenine nucleotide triphosphate (abbreviated as adenosine triphosphate) is involved in various enzyme reactions in vivo, and is an indicator of cell metabolism. Its content directly reflects the number and state of cells. During the experiment, CTG reagent is added to the cell culture medium. The high-sensitivity luciferase in the reagent catalyzes the substrate luciferin to produce fluorescence. This process consumes ATP, and the fluorescence is measured. The luminescence value is proportional to the amount of ATP, and the ATP is positively correlated with the number of living cells. Therefore, the cell viability can be obtained by detecting the ATP content. CCK-8, which stands for Cell Counting Kit-8, can be used for simple and accurate cell proliferation and toxicity analysis. The basic principle is as follows: the reagent contains water-soluble tetrazolium salt 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid phenyl)-2H-tetrazole monosodium salt (WST-8), which is reduced to highly water-soluble yellow formazan product under the action of electron carrier 1-methoxy-5-methyl phenylzolium sulfate dimethyl. The amount of generated formazan product is proportional to the number of living cells. Therefore, this property can be used for direct cell proliferation and toxicity analysis.

[0213] Two tumor cell lines, IGROWV1 (human ovarian cancer cells) and RKO (human colon cancer cells), were selected, with olaparib as a positive control. Different concentration gradients of the compound were added to cells undergoing logarithmic growth, incubated for 120 h, and the OD value was measured. The IC50 value (the compound concentration at which 50% cell death occurs) was calculated. RKO cells served as a low folate receptor expression, low PARP1-dependent control line for IGROWV1 cells.

[0214] Table 1. Inhibitory activity of compounds against multiple tumor cell lines.

[0215]

[0216] n = 2 (n represents biological repeats).

[0217] NA: Data not applicable (IC) 50 >150μM); ND: Data not detected.

[0218] Experimental results: As shown in Table 1, the following conclusions can be drawn:

[0219] (1) The presence of oxygen heteroatom chains affects the activity and solubility of the coupling compound to a certain extent. The solubility of FA-002-D is significantly improved due to the introduction of oxygen heteroatoms in the linker, indicating that the overall solubility of the compound can be changed by introducing oxygen heteroatom chains, which is one of the strategies for optimizing the solubility of the compound.

[0220] (2) Hydrophilicity is a key factor affecting the activity of the conjugate. In IGROV1 cells, the disubstituted compound FA-002-D showed better activity. Given that this compound also has a large molecular weight compared to the B-series disubstituted compounds, it is speculated that hydrophilicity has a significant impact on the compound's activity. In summary, it can be inferred that the molecular weight of the conjugate is a key factor affecting the cell proliferation inhibitory activity, with hydrophilicity being the most critical.

[0221] (3) The olaparib-folate coupling site may not have affected the activity of olaparib. All compounds showed varying degrees of cell proliferation inhibition activity, among which FA-Ola-D, FA-C8-D, and FA-002-D had similar or even better activities than the positive control olaparib. This preliminarily proves that the previously identified olaparib linker extraction site may not affect the binding of the compound to the PARP protein pocket, which needs further verification.

[0222] (4) The activity of compounds is affected by two factors: the expression level of folate receptor and PARP1. RKO cells are low-dependent PARP cells and low-expressing folate receptor cells. Compared with IGROWV1 cells, the IC50 value of olaparib in RKO cells was reduced by 2 times, and the inhibitory effect of all series of compounds on RKO cell proliferation was also significantly reduced.

[0223] In summary, the molecular size and hydrophilicity of the conjugate are key factors affecting the activity of the conjugate. The synthesized folate and olaparib conjugate has targeting ability and high inhibitory activity on IGROV1 ovarian cancer cells, and can be further evaluated in subsequent experiments.

[0224] Evaluation of the ability of the preferred compound FA-002-D of Example 2 to inhibit the proliferation of ovarian cancer cells

[0225] Experimental method: In order to evaluate the sensitivity of tumor cells to drugs, the clonogenic assay method described in the experimental method was used to carry out the experiment, and SKOV3 cells were used. The cell clonogenic assay can be used to study the sensitivity of tumor cells to drugs by detecting the proliferation ability of cells. A small amount of cells was inoculated in a culture dish to disperse into single cells, and the progeny of the cells proliferated for more than 6 generations, after which they formed cell colonies (also known as clones). The cell morphology and the number of cell colonies formed can be used to analyze the cell proliferation ability.

[0226] Experimental results: The more cell colonies formed, the stronger the cell proliferation ability. The results are shown in Figure 1 Compared with the non-drug group, the number of cell colonies in the compound FA-002-D treatment group was significantly reduced, and the higher the drug concentration, the fewer the number of colonies, and the cells were almost all dead at a higher drug concentration. The experiment showed that FA-002-D had a strong effect on inhibiting the proliferation of SKOV3 cells, and had a concentration-dependent effect.

[0227] Example 3: Effect of preferred compound FA-002-D on the apoptosis of SKOV3 cells

[0228] Experimental method: In order to explore the action pathway of the compound, the 3 / 7 detection kit was used as a detection method to study the effect of FA-002-D on the apoptosis of SKOV3 cells. The Caspase-3 / 7 activity detection method described in the experimental method was used to carry out the experiment, The 3 / 7 detection system is a homogeneous luminescence detection system for detecting the activity of Caspase-3 and Caspase-7. The reagents contained in the system contain a caspase-3 / 7 luminescent substrate, which contains a tetrapeptide sequence DEVD. Only one The 3 / 7 reagent enables cell lysis. Caspase then cleaves the substrate and, under the action of luciferase, produces a luminescent signal; the luminescence value is directly proportional to the caspase activity. A Caspase-3 / 7 activity assay kit can be used to detect the activity of caspase-3 / 7 during apoptosis. The catalytic activity of caspase-3 / 7 is quantified by measuring absorbance or fluorescence intensity, thereby understanding the status of apoptosis. The detection kit is used as a detection method to study the effect of FA-002-D on SKOV3 cell apoptosis, with 4×10 3 Cells were seeded at a concentration of [number] cells / well in 96-well plates. After cell adhesion, different concentrations of FA-002-D medium were added and the cells were cultured for 48 hours. An equal volume of [unspecified medium] was then added. The reagents were mixed and incubated for 1 hour, and the fluorescence value was read (490 nm). Ex 510-570nm Em ).

[0229] Experimental results: such as Figure 2 As shown, there was no significant difference in fluorescence values ​​between the low-concentration drug group (0.25 μM, 0.5 μM, 1 μM, 2 μM) and the no-drug group. The fluorescence values ​​of the high-concentration drug group (4 μM, 8 μM, 16 μM, 32 μM) increased with increasing concentration. The apoptosis experiment further showed that FA-002-D has a significant pro-apoptotic effect on SKOV3 cells in a concentration-dependent manner.

[0230] Example 4 Evaluation of PARP-1 inhibitory activity of compound FA-002-D

[0231] Experimental Methods: To investigate the inhibitory activity of compound FA-002-D on PARP-1, a commercially available HT universal colorimetric PARP assay kit was used. The PARP inhibitory activity assay method described in the experimental procedures was employed. A series of concentration gradients of the compound, PARP-HSA enzyme, and PARP cocktail were incubated for 1 h in a 96-well plate with histone attachment to induce a series of glycosylation reactions. After washing twice with buffer, Strep-HRP was added and incubated for 1 h. Finally, TACS-Sapphire was added to the wells for staining, and the absorbance (OD) value was measured at 450 nm using a microplate reader. The content of histone-attached biotinylated substrate was determined using a streptavidin-HRP-catalyzed colorimetric reaction, which can indirectly reflect the activity of PARP-1 at different compound concentrations.

[0232] Experimental results: such as Figure 3 As shown, FA-002-D(IC) 50 =0.70 nM) showed better PARP inhibitory activity than the positive control Olaparib (IC50). 50= 0.79 nM) is more optimal, and the two carboxyl groups at the end of the folate molecule can carry positive drugs to achieve a comparable or even more optimal effect than the parent drug. The PARP-1 inhibition activity evaluation experiment shows that the compounds of the present application can all exhibit inhibitory activity, and they have the potential to increase the inhibitory activity of PARP protein and improve drug targeting based on the parent drug Olaparib, and can be used as preferred compounds for further study. The difference in cytotoxicity is due to the influence of different coupling methods on artificial folate and folate receptors.

[0233] Example 5 Folate and Olaparib conjugate folate receptor expression-dependent evaluation

[0234] Experimental method: The ovarian cancer cell targeting and folate receptor expression-dependent of the conjugate were further investigated. It has been reported that folate receptors are highly specifically expressed on a series of solid tumors, and are not expressed or minimally expressed in normal tissues, so it is necessary to prove that the synthesized compounds have targeting activity on folate and the activity is related to the expression amount of folate receptors.

[0235] Experimental results: Six ovarian cancer cell lines with different expression amounts of folate alpha receptors were selected (sorted according to the RNA sequencing results of the cell lines in the TCGA database), and the corresponding expression amounts of folate alpha receptors are shown in Table 2. Since the transcription level is related to the protein level, but not necessarily completely related, the expression amount of folate alpha receptor in each ovarian cancer cell line was verified by Western blot experiment (as shown in Figure 4 (A)), which is consistent with the results in Table 2.

[0236] Table 1 in combination with the results of folate receptor expression-dependent evaluation of the compounds in Table 2, in the case of a large difference in the expression of folate receptors, the activity of the compounds is mainly affected by the folate receptor; when the expression of folate receptors is similar, for example, ES2 and RKO (Table 1) which are both low folate alpha receptor expression cells, due to the difference in PARP1 dependence, the IC50 values of compounds FA-C8-D and FA-002-D are slightly different in the two cell lines, indicating that the activity of the compounds is also affected by the dependence of the cell lines on PARP1.

[0237] According to the results of the proliferation inhibition activity in IGROV1 cells, FA-002-D is the most active conjugate, so FA-002-D is first determined to be included in the evaluation of folate receptor expression-dependent, in order to investigate the influence of linker, the present application selects FA-C8-D with similar molecular weight and carbon chain linker as a comparison, and Olaparib is still used as a positive control drug.

[0238] The fitting curve results of the cell proliferation inhibition activity of the compounds are as follows Figure 4(B), IC50 values in each ovarian cancer cell line are shown in Table 3. From the analysis, it can be seen that, in general, the cell proliferation inhibitory activity of the compounds increased with the expression level of folate receptor in the ovarian cancer cell lines. Although the activities of FA-C8-D and FA-002-D in the high folate receptor expression cell lines (IGROV1, SKOV3) were slightly lower or similar to those in the cell lines with moderate expression (CAOV3, OVCAR3), it was possible that CAOV3 and OVCAR3 were sensitive to olaparib (smaller IC50 value of olaparib). In the low folate receptor expression cell lines ES2 and TOV-21G, which were also sensitive to olaparib, the activities of FA-C8-D and FA-002-D decreased significantly, proving that the activities of the compounds were dependent on the expression level of folate receptor.

[0239] In cell lines with similar sensitivity to olaparib, the activities of the compounds differed greatly due to the different expression levels of folate receptor. In SKOV3 and CAOV3 cells, the IC50 values of olaparib were similar, but after coupling with folate, the activity of FA-C8-D in SKOV3 was about 2 times that in CAOV3, and the activity of FA-002-D in SKOV3 was about 3 times that in CAOV3; in OVCAR3 and ES2 cells, the activity of FA-C8-D in OVCAR3 was about 36 times that in ES2, and the activity of FA-002-D in OVCAR3 was about 85 times that in ES2 (Table 3). It can be seen that the activity of the compound was greatly affected by the expression level of folate receptor in the cells, suggesting that there was a mechanism of active transport of folate receptor-mediated drug penetration into the membrane, reflecting the ability of folate to carry cytotoxic drugs to target tumor tissues with high expression of folate receptor.

[0240] Table 2 Expression levels of FAa and PARP1 in each ovarian cancer cell line (data from CCLE and TCGA databases)

[0241]

[0242] Table 3 IC50 values of the compounds for each ovarian cancer cell line 50 values

[0243]

[0244] Example 6 Folate competition test

[0245] The entry of folate conjugates into cells depends largely on their receptor FOLR1 on the membrane of cancer cells, and FOLR1-mediated drug entry can be antagonized by FOLR1 antibodies. In order to further determine the key role of FOLR1 in determining the activity of folate conjugates, SKOV3 cells were pretreated with FOLR1 antibodies, and then the IC50 value of the preferred compound FA-002-D was measured. 50.

[0246] Experimental results: as Figure 5 shown in Table 1, the FOLR1 antibody pretreatment group significantly increased the IC50 of FA-002-D from 2.2 μM to 8.6 μM. Consistent with previous research results, the activity of the conjugate is greatly affected by the expression of folate receptor on cells, which has folate receptor dependence.

[0247] Example 7 Combination of preferred compound FA-002-D and DNA damaging agents

[0248] Olaparib can be used regardless of the BRCA status of ovarian cancer patients, but patients with germline or systemic BRCA gene mutations can significantly benefit from the first-line maintenance of PARP inhibitors, however, this part of patients only accounts for 30% of the total population of ovarian cancer in China. Based on the "synthetic lethal" effect, increasing the degree of DNA damage in cells under the action of DNA damaging agents while using PARP inhibitors, the theoretical treatment effect will be enhanced.

[0249] The combination of PARP inhibitors and various DNA damaging agents has been preliminarily tested in clinical trials, and the activity of chemotherapy has been significantly improved, but due to the killing effect of off-target effects on normal cells, the incidence of adverse reactions in patients is high, based on this, the present application uses the conjugate compound of folate and PARP inhibitor olaparib to investigate whether the combination of DNA damaging agents has a synergistic effect in ovarian cancer cells.

[0250] Experimental method: FA-002-D was selected as the preferred compound to explore its combination with three DNA damaging agents, cisplatin (Cis), gemcitabine (Gem) and paclitaxel (PTX) in the combination effect in ovarian cancer cells SKOV3 with high expression of folate receptor. The experimental method described in the combination method was used to carry out the experiment.

[0251] Data processing was performed using CompuSyn, which is based on the classic Chou-Talalay method, by analyzing the dose-effect curve of the drug, calculating the affected fraction (Fraction affected, Fa = 1-cell survival rate) and the combination index CI (0.2≤CI<0.4, strong synergistic effect; 0.4≤CI<0.6, high synergistic effect; 0.6≤CI<0.8, moderate synergistic effect; 0.8≤CI<0.9, low synergistic effect; 0.9≤CI≤1.1, additive effect).

[0252] Experimental results: as Figure 6 shown in Table 1, the dose-effect relationship fitting curve shows that the cell growth inhibition curve of the combination of FA-002-D and Cisplatin is upward compared with the single drug group (Figure 6 , A), indicating that the combination of FA-002-D and Cisplatin enhances the inhibition of ovarian cancer cell growth, and the synergistic effect is better; the combination of FA-002-D and Gemcitabine enhances the inhibition of ovarian cancer cell growth compared with the FA-002-D single-drug group, and is slightly weaker than the Gemcitabine single-drug group Figure 6 , C); the combination of FA-002-D and PTX has a stronger inhibition of ovarian cancer cell growth than the FA-002-D and PTX single-drug groups Figure 6 , E). The Fa-CI graph shows that the three combination groups maintain a high synergistic effect in the lower effect interval to the higher effect interval, and only enter antagonism in the very high effect interval, which indicates a good overall synergistic effect Figure 6 , B, D, F; Table 4). In summary, the combination of FA-002-D and the three DNA-damaging agents all exhibit good synergistic effects within a certain concentration range.

[0253] The results show that all the combination groups exhibit super-strong synergistic effects (synergy index CI value <0.1) within a certain effect interval range, and the synergistic effect of cisplatin is the best.

[0254] These results also show that the combination of the folate and olaparib conjugate of the application and the DNA-damaging agent has significant potential, effectively reduces the incidence of adverse reactions by improving the distribution selectivity of the PARP inhibitor, and provides a new strategy for targeted drug treatment of ovarian cancer.

[0255] Table 4 Dosing concentrations of FA-002-D combined with Cisplatin in each group

[0256]

[0257] Example 8 Effect of combination drug on cell cycle

[0258] Cell cycle is a basic process of cell life activities, and accurate operation of the cell cycle is the basis for survival, development and reproduction of eukaryotes. In order to explore the effect of the combination of FA-002-D single drug and DNA-damaging agents cisplatin (Cis), gemcitabine (Gem) and paclitaxel (PTX) on the cell cycle, SKOV3-FUCCI human ovarian cancer cells with a reporter gene were constructed to explore the mechanism of FA-002-D. The experiment was carried out using the cell cycle method described in the experimental method.

[0259] Experimental results: as Figure 7 , the G1 phase cell ratio of each combination group does not increase compared with the single-drug group. This statistical result shows that the mechanism of the combination of FA-002-D and the three DNA-damaging agents is not achieved by inducing cell cycle arrest.

[0260] The present application evaluates the cell proliferation inhibition activity of the synthesized compounds in cancer cells (mainly ovarian cancer cells IGROV1). The analysis of the structure-activity relationship shows that the activity of the double-substituted series of compounds is better than that of the single-substituted series, which preliminarily proves that the introduction of folic acid into the cyclopropyl part of olaparib and the connecting chain does not affect the binding of it to PARP1, and a series of folic acid-coupled compounds with good ovarian cancer cell proliferation inhibition activity are screened.

[0261] According to the activity evaluation results in IGROV1, the FA-002-D with the highest inhibition activity is selected for a series of pharmacodynamic tests, and it is found that FA-002-D has a significant effect of inhibiting proliferation and promoting apoptosis on SKOV3 cells. The inhibition activity evaluation experiment of FA-002-D on PARP-1 protein shows that the coupling of folic acid and olaparib may hinder the binding with PARP to some extent, but FA-002-D shows the potential of increasing the inhibition activity on PARP protein and improving the drug targeting on the basis of the parent drug olaparib. Then, FA-002-D and the control compound FA-C8-D with a carbon chain as the linker are further evaluated for folic acid receptor expression dependence in ovarian cancer cell lines. It is found that both of them obviously show a decreasing trend in activity in ovarian cancer cells with high, medium and low expression of folic acid receptor, which proves that the compounds have folic acid receptor expression dependence, and provides a strong basis for the compounds to target tumor tissues with high expression of folic acid receptor. The folic acid competition test again proves that FA-002-D has strong folic acid receptor dependence. It is proved that folic acid can carry cytotoxic drugs into cells through folic acid receptor-mediated endocytosis, and the design of non-cleavable connecting chain is reasonable and feasible.

[0262] In the combination drug experiment, FA-002-D combined with three DNA damaging agents cisplatin, gemcitabine and paclitaxel all show good cell inhibition effect, among which the combination index CI value of FA-002-D combined with cisplatin reaches 0.068, showing a strong synergistic effect, and the inhibition effect is greatly improved compared with that of FA-002-D and cisplatin alone. The folic acid and olaparib series of conjugates show good effect when combined with DNA damaging agents while targeting folic acid receptors, which to some extent shows that the folic acid conjugated compounds have the potential to improve the targeting of PARP inhibitors, and are expected to overcome the problem of narrow therapeutic window in the treatment of ovarian cancer caused by the combination of PARP inhibitors and DNA damaging agents.

[0263] All the documents mentioned in the present application are cited as references in the present application, as if each document is cited as a reference individually. In addition, it should be understood that those skilled in the art can make various modifications or changes to the present application after reading the above teaching of the present application, and these equivalent forms also fall within the scope defined by the claims attached to the present application.

Claims

1. A folate conjugate of Formula I or a pharmaceutically acceptable salt thereof, ###0001### Formula I wherein, n is selected from the group consisting of 1, 2, 3, 4 or 5; m is selected from the group consisting of 1, 2 or 3; Ola is a PARP inhibitor.

2. The folate conjugate of claim 1, wherein n is 1. L is absent or 3. The folate conjugate of claim 1, wherein n is 2.

4. The folate conjugate of claim 1, wherein n is 3.

5. The folate conjugate of claim 1, wherein n is 4.

2. The folate conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein 6. The folate conjugate of claim 1, wherein n is 5.

7. The folate conjugate of claim 1, wherein m is 1.

8. The folate conjugate of claim 1, wherein m is 2.

3. The folate conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein 9. The folate conjugate of claim 1, wherein m is 3.

4. The folate conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein 10. The folate conjugate of claim 1, wherein Ola is olaparib.

5. A compound as shown in L-Ola, characterized in that, 11. The folate conjugate of claim 1, wherein the conjugate is selected from any one of the group consisting of: ###0002### ###0003### ###0004### ###0005### ###0006### ###0007### ###0008### ###0009### ###0010### ###0011### ###0012### ###0013### ###0014### ###0015### ###0016### ###0017### ###0018### ###0019### ###0020### ###0021### ###0022### ###0023### ###0024### ###0025### ###0026### ###0027### ###0028### ###0029### ###0030### ###0031### ###0032### ###0033### ###0034### ###0035### ###0036### ###0037### ###0038### ###0039### ###0040### ###0041### ###0042### ###0043### ###0044### ###0045### ###0046### ###0047### ###0048### ###0049### ###0050### ###0051### ###0052### ###0053### ###0054### ###0055### ###0056### ###0057### ###0058### ###0059### ###0060### ###0061### ###0062### ###0063### ###0064### ###0065### ###0066### ###0067### ###0068### ###0069### ###0070### ###0071### ###0072### ###0073### ###0074### ###0075### ###0076### ###0077### ###0078### ###0079### ###0080### ###0081### ###0082### ###0083### ###0084### ###0085### ###0086### ###0087### ###0088### ###0089### ###0090### ###0091### ###0092### ###0093### ###0094### ###0095### ###0096### ###0097### ###0098### ###0099### ###0100### ###0101### ###0102### ###0103### ###0104### ###0105### ###0106### ###0107### ###0108### ###0109### ###0110### ###0111### ###0112### ###0113### ###0114### ###0115### ###0116### ###0117### ###0118### ###0119### ###0120### ###0121### ###0122### ###0123### ###0124### ###0125 wherein L is ​ ​ 6. The compound of claim 5, wherein ​ 7. The method for preparing the folate conjugate according to claim 1, wherein the method comprises the steps of: (a) synthesizing the folate conjugate according to claim 1; and (b) purifying the folate conjugate synthesized in step (a). ​ ​ ​ 8. A pharmaceutical composition, characterized by, ​ ​ ​ 9. Use of the folate conjugate or pharmaceutically acceptable salt thereof according to claim 1, characterized in that, ​ 10. The use of the folate conjugate or pharmaceutically acceptable salt thereof according to claim 9, wherein ​ ​

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