Folic acid conjugate as well as preparation method and application thereof

Abstract

The invention relates to a folic acid conjugate as well as a preparation method and application thereof. Specifically, the invention discloses a novel folic acid conjugate and a drug combination strategy thereof, and also provides a preparation method of the conjugate and an application of the conjugate in tumor treatment. The conjugate provided by the invention has folate receptor expression dependence, can efficiently inhibit ovarian cancer cell proliferation, and shows a good action effect when being combined with a DNA (Deoxyribose Nucleic Acid) damaging agent.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and specifically, to folic acid conjugates, their preparation methods and applications. Background Art

[0002] Ovarian cancer is the second most common cause of death from gynecological cancers among women globally. Currently, the treatment of ovarian cancer mainly relies on tumor resection surgery and platinum-based chemotherapy, but patients have a high recurrence rate and are prone to drug resistance, and there has been no significant improvement in long-term survival prognosis. There is an urgent need for new breakthroughs in treatment strategies.

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

[0004] Several clinical trials have shown that based on the "synthetic lethality" effect, the combined use of PARP inhibitors and DNA-damaging agents can effectively enhance the chemotherapeutic activity. However, the adverse reactions caused by the targeting problems of the two drugs limit the combined efficacy. Therefore, there is an urgent need to improve the selectivity of drugs for tumor cells.

[0005] Great progress has been made in the research of folic acid (Folic acid, FA)-targeted anti-tumor drugs, and some folic acid-conjugated drugs have entered the clinical research stage. Research has shown that folate receptors are highly specifically expressed on certain cancer cells, while being minimally expressed or not expressed in normal tissues. By utilizing this expression difference for selective drug delivery, the damage of anti-tumor drugs to normal cells and tissues can be significantly reduced, and the targeting and efficacy of the drugs can be improved.

[0006] Therefore, there is an urgent need in this field to develop a folic acid conjugate that not only has folic acid-binding properties, but also has the inhibitory activity of PARP enzyme and has a novel structure. Summary of the Invention

[0007] The object of the present invention is to provide a novel folic acid conjugate, which is formed by connecting folic acid and a PARP inhibitor through an uncleavable linker, thereby forming a folic acid conjugate with a novel structure and being able to effectively target folic acid and release the drug.

[0008] In the first aspect of the present invention, there is provided a folic acid conjugate represented by Formula I, or a tautomer thereof, or a pharmaceutically acceptable salt thereof.

[0009]

[0010] Among them,

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

[0012] Ola is a cytotoxic drug moiety.

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

[0014] In another preferred example, L is absent or 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 example, the structure of L is:

[0019]

[0020] wherein,

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

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

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

[0024]

[0025] represents the linking position between different parts of the conjugate.

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

[0027]

[0028] In the second aspect of the present invention, there is provided the structure of L-Ola in the formula, and the structure is:

[0029]

[0030] wherein, the definition of L is as described in the first aspect of the present invention.

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

[0032]

[0033]

[0034] The third aspect of the present invention provides a method for preparing a folic acid conjugate as described in the first aspect of the present invention, comprising the following steps:

[0035] (a) Providing a first reaction precursor folic acid FA and a second reaction precursor cytotoxic drug;

[0036] (b) Reacting the first reaction precursor and the second reaction precursor with a linking molecule to form a folic acid conjugate as described in the first aspect of the present invention.

[0037] In another preferred example, the structure of the first reaction precursor folic acid FA is:

[0038]

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

[0040] In another preferred example, the second reaction precursor cytotoxic drug is olaparib, and its structure is as follows:

[0041]

[0042] In another preferred example, the linking molecule has the following structure:

[0043]

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

[0045] In another preferred example, the second reaction precursor folic acid FA first reacts with the linking molecule to form a first reaction intermediate; and the first reaction intermediate reacts with the first reaction precursor cytotoxic drug to form the folic acid conjugate.

[0046] In another preferred example, the first reaction intermediate has the above-mentioned structure of L-Ola.

[0047] In another preferred example, the reaction feed ratio of the first reaction precursor to the second reaction precursor is 1:2.

[0048] The fourth aspect of the present invention provides a pharmaceutical composition, comprising:

[0049] (i) A folic acid conjugate as described in the first aspect of the present invention, or a tautomer thereof, or a pharmaceutically acceptable salt thereof; and

[0050] (ii) A pharmaceutically acceptable carrier.

[0051] In another preferred embodiment, the folic acid conjugate, or its tautomer, or its pharmaceutically acceptable salt, 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, chloroquine, 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] The fifth aspect of the present invention provides a use of a folic acid conjugate as described in the first aspect of the present invention for preparing a drug for treating tumors.

[0056] In another preferred embodiment, the tumor is highly expressed with folate receptor and growth-dependent on PARP enzyme or DNA damage repair-related tumors.

[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 invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0062] Figure 4 Shows the folate receptor expression-dependent evaluation of the conjugate. Among them, (A) the expression levels of folate α receptor in each ovarian cancer cell line; (B) the proliferation inhibition of the compound on each strain of ovarian cancer cells.

[0063] Figure 5 Shows the change in cell viability after treating SKOV3 cells with FA-002-D with or without FLOR1 antibody for 120 h.

[0064] Figure 6 Shows the combination of FA-002-D and DNA-damaging agents.

[0065] Figure 7 Shows the proportion of cells in the S phase of each experimental group. Detailed implementation mode

[0066] Through extensive and in-depth research, the present inventors have unexpectedly developed a novel folate conjugate, in which the folate and the drug molecule in the conjugate are connected by an unbreakable bond (Linker), effectively avoiding the premature release of the drug, and effectively improving the targeting of the drug and reducing the off-target effect, achieving the effect of efficiently treating tumors. On this basis, the inventors have completed the present invention.

[0067] Terms

[0068] In the present invention, unless otherwise specified, 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 represented by the general formula (I) and an anion, or a salt formed by a negatively charged group on the compound represented by the general formula (I) and a cation. Suitable anions include but are not limited to chloride ion, bromide ion, iodide ion, sulfate group, nitrate group, phosphate group, citrate group, methanesulfonate group, trifluoroacetate group, acetate group, malate group, tosylate group, tartrate group, fumarate group, glutamate group, glucuronate group, lactate group, glutarate group or maleate group, 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 salts of the present invention refer to salts formed by the compounds represented by the general formula (I) and acids in the following groups, such as but not limited to: hydrofluoric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, oxalic acid, sulfuric acid, nitric acid, methanesulfonic acid, aminosulfonic acid, salicylic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, citric acid, acetic acid, lactic acid, tartaric acid, succinic acid, oxalis 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 hydroxyethanesulfonic acid, etc.; or salts formed by the compounds represented by the general formula (I) and inorganic bases, such as but not limited to sodium salts, potassium salts, calcium salts, aluminum salts, or ammonium salts; or salts formed by the compounds represented by the general formula (I) and organic bases, such as but not limited to methylamine salts, ethylamine salts, ethanolamine salts, ammonium salts of hydroxymethylaminomethane (TRIS), etc.

[0071] Folic acid conjugate

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

[0073] The first part is artificial folic acid. Folic acid consists of two parts: pteroic acid and glutamic acid. The folic acid receptor mainly recognizes the pteroic acid part, and the glutamic acid part is not necessary for this binding effect. Therefore, usually, the glutamic acid part is connected to the drug instead of the pteroic acid. The carboxyl groups in its glutamic acid structure can be divided into α-COOH and γ-COOH. It should be noted that the affinity of the drug connected to γ-COOH for the folic acid receptor is greater than that of the drug connected to α-COOH. The reason may be that when the drug is connected to α-COOH, it is too close to the pteroic acid part, which will hinder the binding of the drug conjugate to the receptor.

[0074] As used in the present invention, the folic acid has the following structure:

[0075]

[0076] The second part is the linking chain, which is combined with folic acid through an unbreakable linking chain. The linking methods include directly connecting the terminal carboxyl group of folic acid with the PARP inhibitor, connecting the PARP inhibitor with linkers of different carbon chain lengths, and introducing an O heteroatom into the carbon chain and then connecting it with the PARP inhibitor. Preferably, an O heteroatom is introduced into a carbon chain with a length of 9. First, the α-COOH of folic acid is protected, and after the γ-position is connected with the PARP inhibitor, the α-position protecting group is removed. This optimized route solves the selectivity problem when the α and γ carboxyl groups of folic acid are activated, the by-product components are more single, simplifies the post-treatment operation, and is beneficial to scale-up synthesis and purity improvement.

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

[0078] In some embodiments, the tumor preferably has high expression of folate receptor and depends on PARP enzyme or DNA damage repair, such as ovarian cancer, breast cancer, cervical cancer, bladder cancer, pleural mesothelioma and non-small cell lung cancer.

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

[0080] In the present invention, 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 of the present invention.

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

[0089] In another preferred embodiment, the pharmaceutically acceptable salt of the present invention refers to salts formed by the compound represented by formula (I) and acids in the following group, such as, but not limited to: hydrofluoric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, oxalic acid, sulfuric acid, nitric acid, methanesulfonic acid, aminosulfonic acid, salicylic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, citric acid, acetic acid, lactic acid, tartaric acid, succinic acid, oxalacetic 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, etc.; or salts formed by the compound represented by formula (I) and inorganic bases, such as, but not limited to, sodium salts, potassium salts, calcium salts, aluminum salts or ammonium salts; or salts formed by the compound represented by formula (I) and organic bases, such as, but not limited to, methylamine salts, ethylamine salts, ethanolamine salts, ammonium salts of hydroxymethylaminomethane (TRIS), etc.

[0090] Linker

[0091] In the design of folic acid-conjugated drugs, the selection of the linker is crucial, which determines whether the drug can effectively target the receptor site. A linking bond with a suitable spacer distance and cleavage mode is the key to achieving this goal, which can effectively release the parent drug under specific enzyme hydrolysis conditions or other suitable environments. The process of folic acid receptor-mediated drug release is roughly as follows: First, the exogenous folic acid-drug conjugate binds to the folic acid receptor outside the membrane with high affinity (Kd≈10-9M); then, the cell membrane invaginates around the folic acid receptor and the conjugate to form early endosomes or "vesicles", and endocytosis occurs. Subsequently, the lumen of the mature endosome acidifies, the conformation of the receptor changes, and the folic acid-drug conjugate is released; finally, the folic acid receptor is transferred to the cell membrane and then starts a new round of transport. Most of the reported folic acid-conjugated compounds are designed with cleavable bonds such as disulfide bonds, ester bonds, etc. When the folic acid-drug conjugate enters the endosome, the cleavable bond can be hydrolyzed by specific enzymes to release the parent drug, so as to achieve the purpose of selective drug release.

[0092] To reduce the premature release of drugs caused by the breakage of the linking chain, the present invention binds the drug to folic acid through an unbreakable linking chain and selectively delivers the intact conjugate to cancer cells overexpressing FRα. Most of the folic acid conjugates currently in clinical trials or under research use cleavable bonds. The advantages of designing an unbreakable bond in the present invention are as follows: (1) enhancing the targeting of the compound to tumor cells, reducing the killing effect on normal cells caused by the premature release of cytotoxic drugs due to the breakage of the linking bond, and reducing the incidence of adverse reactions; (2) directly ingesting the intact conjugate into cells by folate receptor-mediated endocytosis, effectively avoiding the limitation that the compound cannot enter cells due to its too large molecular weight.

[0093] On the basis of retaining the structural parts essential for the recognition of folic acid and olaparib receptors, the present invention designs linking chains with different carbon chain lengths and hydrophilicities, as well as compounds with a folic acid to olaparib coupling ratio of 1:2 (double substitution of the terminal carboxyl group of folic acid), and aims to synthesize a total of three series of folic acid-olaparib conjugates, namely Series A, B, and C, as shown below.

[0094] Series A:

[0095]

[0096] Series B:

[0097]

[0098] Series C:

[0099]

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

[0101]

[0102] The structure of Ola is defined as above.

[0103] Preparation method of folic acid conjugate

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

[0105] Typically, the preparation process flow of the compounds of the present invention is as follows. Unless otherwise specified, the raw materials and reagents used can be purchased through commercial channels.

[0106] For the A series of compounds, in order to investigate the effect of adding the linker part on the activity of the compounds, the carboxyl group at the end of folic acid was directly connected to the secondary amine on the piperazine ring of olaparib. The retrosynthetic reaction formula is as follows:

[0107]

[0108] The B series of compounds were synthesized by connecting the secondary amine on the piperazine ring of olaparib with linkers of different carbon chain lengths. Therefore, after the carboxyl group at the end of the linker was first amide condensed with olaparib, the amino group at the other end of the linker could be obtained by nucleophilic substitution with folic acid.

[0109]

[0110] For the C series of compounds, in order to investigate the effect of the linker composition on the solubility of the conjugate, an O heteroatom was introduced into the carbon chain of the B series of compounds. The synthesis method was similar to that of the B series of compounds, and the retrosynthetic analysis was as follows:

[0111]

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

[0113] Specifically, the condensation conditions for the disubstituted products of the A, B, and C series of compounds were all carried out under the catalysis of EDCI, HOBT, and DIPEA. The solvent was DMF, protected by nitrogen, and shielded from light. The reaction was carried out at room temperature for about 3 h. After the reaction was completed, methanol was poured into the reaction solution to precipitate the product, centrifuged, and the precipitate was dried. The disubstituted compounds of the A and C series were dissolved in DMF, and then methanol was added to precipitate, repeated 3 times, and a disubstituted product with a single component was obtained. Due to the presence of by-products in the B series of disubstituted compounds, the dried solid was dissolved in as little DMF solvent as possible, and 7% Na2CO3 solution was added to centrifuge and precipitate the solid, and a disubstituted product with a single component was obtained.

[0114] Drug Compositions and Administration Methods

[0115] The pharmaceutical composition of the present invention comprises a compound of the present invention or a pharmaceutically acceptable salt thereof within a safe and effective amount range and a pharmaceutically acceptable excipient or carrier. The "safe and effective amount" herein refers to: an amount of the compound 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 invention per dose, more preferably, contains 5 - 1000 mg of the compound of the present invention per dose. Preferably, the "per dose" is a capsule or a tablet.

[0116] "Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be blended with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0117] The pharmaceutical composition is an injection, capsule, tablet, pill, powder or granule.

[0118] There is no particular limitation on the mode of administration of the compounds or pharmaceutical compositions of the present invention. 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 calcium phosphate, or is mixed with the following components: (a) fillers or bulking agents, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxypropylmethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; I slow solvents, for example, paraffin wax; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents.

[0120] Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shell materials, such as enteric coatings and other materials well known in the art. They can contain opacifying agents, and the release of the active compound or compounds in such compositions can be delayed in a manner such that they are released in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and wax-like substances. If necessary, the active compound can also be in the form of microcapsules with one or more of the above excipients.

[0121] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3 - butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, etc.

[0122] In addition to these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweetening agents, flavoring agents and perfumes.

[0123] In addition to the active compound, the suspension may contain suspending agents, for example, ethoxylated isooctadecanol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum monostearate and agar or mixtures of these substances, etc.

[0124] The compositions for parenteral injection may contain physiologically acceptable sterile aqueous or non - aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for re - dissolving into sterile injectable solutions or dispersions. Suitable aqueous and non - aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and their suitable mixtures.

[0125] The dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required when necessary.

[0126] The compounds of the present invention can be administered alone, or in combination with other pharmaceutically acceptable compounds (such as DNA - damaging agents).

[0127] The treatment method of the present invention can be administered alone, or in combination with other treatment means or therapeutic drugs.

[0128] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal in need of treatment (such as a human), wherein the dosage during administration is an effective dosage considered pharmaceutically. For a person weighing 60 kg, the daily dosage is usually 1 - 2000 mg, preferably 5 - 1000 mg. Of course, the specific dosage should also consider factors such as the route of administration and the health status of the patient, which are all within the scope of the skills of a skilled physician.

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

[0130] 1. The folate conjugate of the present invention uses an uncleavable linker chain to enhance the targeting of biotoxic drugs to tumor cells.

[0131] 2. The folate conjugate of the present invention can effectively avoid premature drug release and reduce the incidence of drug adverse reactions.

[0132] 3. The conjugate of the present invention exhibits strong folate receptor expression dependence, demonstrating the feasibility of the uncleavable linker chain design.

[0133] 4. The folate conjugate of the present invention exhibits strong cell inhibitory activity in ovarian cancer IGROV1 cells.

[0134] 5. The conjugate of the present invention has a proliferation inhibitory and pro-apoptotic effect on SKOV3 cells and has excellent binding inhibitory ability to PARP-1.

[0135] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0136] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the method of the present invention. The preferred implementation methods and materials described herein are for illustrative purposes only.

[0137] Experimental methods

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

[0139] CTG method: Cells in the logarithmic growth phase were diluted with medium and inoculated into 96-well plates at a density of 1400 cells / well. They were cultured in a 37°C incubator for 24 h. After the cells adhered and grew, the medium was aspirated. An aqueous sodium hydroxide solution was added to the 10 mM DMSO stock solution of each compound to adjust the pH to about 10. The medium was diluted in gradients, and 100 μL of the compound dilution solution was added to each well of the inoculated plate. It was placed in a 37°C cell incubator and cultured for 120 h. 50 μL / well of Reagent, incubated in a 37 °C cell culture incubator for 2 h. Mixed with an orbital shaker for 2 minutes to induce cell lysis. The plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal. The microplate reader measured the full wavelength, The IC50 value was fitted by GraphPad Prism 9.

[0140] CCK-8 method: Cells in the logarithmic growth phase were diluted with medium and seeded into a 96-well plate at a density of 5000 cells / well, and cultured in a 37 °C incubator for 24 h. After the cells adhered and grew, the medium was aspirated. The pH of the 10 mM DMSO stock solution of each compound was adjusted to about 10 with an aqueous sodium hydroxide solution, and then gradient diluted with the medium. 100 μL of the compound dilution was added to each well of the seeded plate and cultured in a 37 °C cell culture incubator for the corresponding time. 10 μL of CCK-8 reagent was added to each well and incubated at 37 °C for 2 h to stabilize the luminescence signal. Then, the absorbance at 450 nm was measured by a microplate reader, 100%. IC 50 value was fitted by GraphPad Prism 9.

[0141] Colony formation assay (CFA)

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

[0143] Cell apoptosis assay

[0144] Collect SKOV3 cells in the logarithmic phase, count and seed them in a 6-well plate, and control the concentration of each well to be about 3×10 5Cells were cultured in a density of [number] cells / well for 24 h. FA-002-D with final concentrations of 5, 2.5, and 1.25 μM were added. Then the cells were further cultured in an incubator for 72 h. Cells were digested with trypsin without EDTA for 2 - 5 min, collected, and washed with PBS for 1 - 2 times. BindingBuffer and FITC / PI dye were added and incubated in the dark on ice for 15 min. The apoptosis of cells was detected by an Agilent flow cytometer, with attention paid to avoiding light. The data was processed and graphed using NovoExpress software, and the apoptosis rate was calculated.

[0145] Detection of Caspase-3 / 7 activity

[0146] Cells in the logarithmic growth phase were diluted with culture medium and seeded into 96-well plates at a density of 4000 cells / well. After culturing at 37 °C for 24 h, FA-002-D with final concentrations of 0, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 μM were added and cultured for 48 h. 100 μL of 3 / 7 reagent was added to each well and incubated at room temperature for 1 h. The luminescence values of each sample were measured using a microplate luminometer.

[0147] Determination of PARP inhibitory activity

[0148] 50 μL of 1×PARP Buffer was added to each well and incubated at room temperature for 30 min. Then the 1×PARP Buffer was aspirated, and a series of concentration gradients of inhibitors were added to the wells. There were four drugs in total, namely FA-Ola-D, FA-C8-D, Olaparib, and FA-C8-D. The concentration gradients of the drug treatment groups were set as follows: 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 measure the background absorbance. Blank group: 0.5 U / well of PARP-HSA enzyme without inhibitor. These wells provided a 100% activity reference value. Standard

[0149] Curve group: Dilute PARP-HSA enzyme in a pre-cooled microtube with 1×PARP Buffer to make the total activities 1U / 25μL, 0.5U / 25μL, 0.25U / 25μL, and 0.125U / 25μL respectively. Add 25μL of 1×PARP Cocktail to each well and incubate at room temperature for 60 min. Wash twice with 1×PBS + 0.1% TritonX-100 (200μL / well), and then wash twice with 1×PBS. Add 50μL of Strep-HRP working solution to each well and incubate at room temperature for 60 min. Wash twice with 1×PBS + 0.1% TritonX-100 (200μL / well), and then wash twice with 1×PBS. Add 50μL of TACS-Sapphire colorimetric substrate to each well and incubate in the dark at room temperature for 15 min. Add 50μL of 0.2M HCl to each well to stop the reaction, and read the absorbance at 450 nm.

[0150] Protein immunoblotting experiment (Western Blot)

[0151] Collect cells by centrifugation at 1500 rpm, wash them 3 times with 1×PBS, and then add RIPA universal protein lysate to lyse the cells on ice for 30 min; centrifuge at 12000 rpm and 4℃ for 30 min to collect the supernatant. After thoroughly pipetting and mixing the extracted protein supernatant with a pipette, add 2μL of the protein sample and 18μL of ddH2O to each well of a 96-well plate, and finally add 200μL of the detection solution to each well. Incubate the 96-well plate in a 37℃ cell culture incubator for 30 min, then use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance at a wavelength of 562 nm, and calculate by substituting into the protein standard curve. According to the previously measured sample protein concentration, calculate the sample volume required for 20μg of protein

[0152] loading amount, and add ddH2O to make the final loading volume 20μL. Add 5×LoadingBuffer according to the volume, mix well, centrifuge briefly at low temperature, heat and denature at 100℃ in a metal bath for 5 min, and centrifuge briefly for standby. Run the gel and transfer the membrane, and use ImageJ software to perform gray-scale analysis on the bands.

[0153] Combined drug use

[0154] Collect logarithmic-phase SKOV3 cells, inoculate and culture them in a 96-well plate at a density of 4×10 3 cells / well, culture for 24 h until they adhere to the wall, and dilute the single-drug groups of FA-002-D, Cisplatin, Gemcitabine, and PTX with the medium in a half-log dilution manner. The final concentrations of FA-002-D are

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

[0156] Cell cycle

[0157] Collect SKOV3-FUCCI cells in the logarithmic phase and seed them in 96-well plates at a density of 4×10 3 cells / well and culture for 24 h until they adhere. Dilute the single-drug groups of FA-002-D, Cisplatin, Gemcitabine, PTX in a medium by serial dilution. The final concentrations of FA-002-D were 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0 μM respectively, with 2 replicates. The final concentrations of Cisplatin in the single-drug groups were 6, 3, 1.5, 0.75, 0.375, 0.1875, 0.09375, 0.046875, 0 μM respectively, the final concentrations of Gemcitabine were 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625, 0 μM respectively, the final concentrations of PTX were 0.6, 0.3, 0.15, 0.075, 0.0375, 0.01875, 0.009375, 0.0046875, 0 μM respectively. The combined groups were the sum of the corresponding concentrations of FA-002-D and Cisplatin, Gemcitabine, PTX respectively, and all had 2 replicates (the experimental groups were set as shown in Table 5, taking the FA-002-D and Cisplatin group as an example). After adding compounds at different concentrations, the cells were cultured for another 48 h. Take live cell images with a high-content imaging analysis system, identify and analyze the images of each group with Cellprofiler software, calculate the proportion of the number of cells showing red fluorescence in each well to the total number of cells (cells showing red fluorescence + cells showing blue fluorescence), and export the data through R language statistics.

[0158] Table 5 Administration concentrations of each group of FA-002-D combined with Cisplatin

[0159]

[0160] Preparation of Compounds

[0161] Synthesis of Intermediate Series B

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

[0163] General Method for Intermediate Synthesis: Dissolve glutaric anhydride (25 mmol, 2.85 g) in 15 mL of dichloromethane. Dissolve mono-Boc-ethylenediamine, N-Boc-1,3-propanediamine, tert-butyl (4-aminobutyl)carbamate, tert-butyl N-(5-aminopentyl)carbamate, and tert-butyl N-(6-aminopentyl)carbamate (25 mmol) separately in 15 mL of dichloromethane, and slowly add them dropwise to the dichloromethane solution of glutaric anhydride. After reacting at room temperature for 2 h, evaporate the solvent under reduced pressure to obtain a white solid or a colorless transparent oily substance. Add HATU (25 mmol, 9.5 g), dissolve it in 25 mL of DMF, activate it at room temperature for about 10 min. Then dissolve 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (25 mmol, 9 g) and DIPEA (100 mmol, 17 mL) in 25 mL of DMF, and slowly add them dropwise to the activated solution. After reacting at room temperature for about 3 h, add saturated NaCl solution and ethyl acetate for extraction 3 - 4 times. Collect the upper organic phase, dry it over anhydrous Na2SO4, and then concentrate it under reduced pressure. The concentrated solution is separated by silica gel column chromatography (eluent: DCM / MeOH (v / v) = 20 / 1) to obtain a transparent oily substance.

[0164] Dissolve the transparent oily substance in 15 mL of dichloromethane, add trifluoroacetic acid (100 mmol, 11.5 g), and react at room temperature for 3 h. After the reaction, add dichloromethane and concentrate it under reduced pressure. Rotate the flask to dry the excess trifluoroacetic acid three times. Add aqueous NaOH solution to adjust the pH to 10. A large amount of bubbles are generated and heat is released during this process. Directly evaporate the resulting solution to dryness. Dissolve the solid in a mixed solvent of dichloromethane and methanol, filter, concentrate the filtrate under reduced pressure, and separate it by silica gel column chromatography (eluent: DCM / MeOH (v / v) = 10 / 1) to obtain a colorless oily substance.

[0165] The structures of Intermediate 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 oily substance, yield 35%.

[0168] 1 1H 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 13C NMR (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] 11H 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 13C 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] 11H 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 13C 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] 11H 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 13C 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] 11H 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 13C 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 Intermediate of Series C

[0182] Synthesis of FA6-002

[0183] Glutaric anhydride (25 mmol, 2.85 g) was dissolved in 15 mL of dichloromethane. [2-(2-Aminoethoxy)ethyl] carbamic acid tert-butyl ester (25 mmol) was separately dissolved in 15 mL of dichloromethane and slowly added dropwise to the dichloromethane solution of glutaric anhydride. After reacting at room temperature for 2 h, the mixture was rotary evaporated under reduced pressure to obtain a white solid or a colorless transparent oil. Then, the condensing agent 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (25 mmol, 9.5 g) was added and dissolved in 25 mL of DMF. The terminal carboxyl group of the linker was activated at room temperature for about 10 min. Subsequently, 4-(4-Fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (25 mmol, 9 g) and DIPEA (100 mmol, 17 mL) were dissolved in 25 mL of N,N-dimethylformamide (DMF) and then slowly added dropwise to the activated solution. After reacting at room temperature for about 3 h, saturated NaCl aqueous solution and ethyl acetate were added for extraction 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.

[0184] 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, dichloromethane was added and the mixture was concentrated under reduced pressure. The excess trifluoroacetic acid was rotary evaporated 3 times. An aqueous NaOH solution was added to adjust the pH to neutral. A large amount of bubbles were generated and heat was released during this process. Saturated NaCl, chloroform, and a small amount of methanol (for solubilization) were added for extraction 7 - 8 times. The lower 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) = 10 / 1) to obtain a colorless transparent oil, namely the intermediate (FA6-002) with relatively 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] 11H 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 13C 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 Series A 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-(piperazine-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, shielded from light, DIPEA (3.15 mmol, 0.5 mL) was injected, and the reaction was carried out 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 the reaction solution, 25 mL of the poor solvent was added. After precipitation, centrifugation was carried out, and the supernatant was discarded. The precipitate was dissolved in DMF and then methanol solution was added again as a poor solvent to precipitate the product. This 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 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 Series B conjugates

[0194] General method for the synthesis of disubstituted end products

[0195] Folic acid (0.45 mmol, 200 mg), EDCI (1.8 mmol, 345 mg), HOBT (1.8 mmol, 243 mg), and 4-(4-fluoro-3-(piperazine-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, shielded from light, DIPEA (3.15 mmol, 0.5 mL) was injected, and the reaction was carried out 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 the reaction solution, 25 mL of the poor solvent was added. After precipitation, it was centrifuged, the supernatant was discarded, and it was dried in a vacuum drying oven. The dried solid was crushed and dissolved in as little DMF solvent as possible. 7% Na2CO3 solution was added and solid was precipitated by centrifugation. It was washed with methanol and centrifuged. The precipitate was dried in a vacuum drying oven to obtain the product.

[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] 11H 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 13C 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] 11H 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 / z 781.8682, 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), and FA6-002 (1 mmol) were placed in a three-necked flask, 25 mL of DMF was added, protected by nitrogen, shielded from light, DIPEA (3.15 mmol, 0.5 mL) was injected, and the reaction was carried out 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 the poor solvent was added to every 25 mL of the reaction solution, and after precipitation, centrifugation was performed. The supernatant was discarded, the precipitate was dissolved in DMF and then methanol solution was added again as a poor solvent to precipitate the product. This 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 to dry 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] 11H 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 13C 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] Verification experiment of double-substituted end product

[0209] The formation of the end product was verified using thin-layer chromatography. The developing solvent was n-propanol: n-butanol: ammonia water: water = 5:5:3:1. Under irradiation at a wavelength of 254 nm, the R values corresponding to substances with different polarities in the reaction system were observed fValue. Besides the raw folic acid spot in the reaction solution, a new product, namely FA-002-D, is formed. At the same time, liquid chromatography-mass spectrometry (LC-MS) is used to judge the reaction process by the target molecular weight. The folic acid and the product are dissolved in methanol, and ammonia water is added dropwise to assist dissolution. It is observed that the solid dissolves. The solution is analyzed by LC-MS again, and folic acid [M+H] is detected. + The molecular ion peak m / z 442.17 of folic acid, FA-002-D [M+H] + The molecular ion peak m / z 1538.78 (molecular weight: 1537.65), indicating that the disubstituted product is formed under this condition, which is consistent with the result of thin-layer chromatography detection.

[0210] Biological experiments

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

[0212] Experimental method: To detect the cytotoxicity of the compound, the CTG method and the CCK-8 method are used in the present invention, and the experiment is carried out using the cell proliferation toxicity test method described in the experimental method. CellTiter-Glo (CTG) is a rapid cell viability detection method based on ATP detection. Adenosine triphosphate (ATP) participates in various enzymatic reactions in organisms and is an index of the metabolism of living cells. Its content directly reflects the number and state of cells. During the experiment, the CTG reagent is added to the cell culture medium. The highly sensitive luciferase in it catalyzes the substrate luciferin to produce fluorescence, and this process requires the consumption of ATP. By measuring the fluorescence, the luminescence value is proportional to the amount of ATP, and ATP is positively correlated with the number of living cells. Therefore, the cell viability can be obtained by detecting the ATP content. CCK-8, full name Cell Counting Kit-8, can be used for simple and accurate cell proliferation and toxicity analysis. Its basic principle is as follows: The reagent contains the water-soluble tetrazolium salt 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8), which is reduced by dehydrogenases in cells to a highly water-soluble yellow formazan product under the action of the electron carrier 1-methoxy-5-methylphenazinium methyl sulfate. The number of formazan products generated is proportional to the number of living cells. Therefore, this property can be used to directly carry out cell proliferation and toxicity analysis.

[0213] Two tumor cell lines, IGROV1 (human ovarian cancer cells) and RKO (human colon cancer cells), were selected, and Olaparib was used as the positive control drug. Different concentration gradients of the compound were added to the cells in logarithmic growth, incubated for 120 h, the OD value was measured, and the IC50 value (the compound concentration corresponding to 50% cell death) was calculated. RKO cells served as the low folate receptor expression and low PARP1 dependence control strain for IGROV1 cells.

[0214] Table 1 Inhibitory activities of the compound against multiple tumor cell lines

[0215]

[0216] n = 2 (n belongs to biological replicates).

[0217] N.A.: Data not applicable (IC 50 > 150 μM); N.D.: Data not detected.

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

[0219] (1) The oxygen-containing heteroatom chain affects the activity and solubility of the conjugate to a certain extent. Due to the introduction of oxygen heteroatoms in the linker of FA-002-D, the solubility has been greatly improved, indicating that the overall solubility of the compound can be changed by introducing an oxygen-containing heteroatom chain, which is one of the strategies for subsequent optimization of compound solubility.

[0220] (2) Hydrophilicity is the key factor affecting the activity of the conjugate. In IGROV1 cells, the bis-substituted compound FA-002-D has better activity. Given that this compound also has a large molecular weight compared with the B series of bis-substituted compounds, it is speculated that the factor of hydrophilicity has a greater impact on the activity of the compound. In summary, it can be speculated that the molecular weight of the conjugate is a key influencing factor for cell proliferation inhibitory activity, and hydrophilicity is more critical.

[0221] (3) The coupling site of Olaparib and folic acid may not affect the activity of Olaparib. All compounds showed varying degrees of cell proliferation inhibitory activity. Among them, the activities of FA-Ola-D, FA-C8-D, and FA-002-D were similar or even better than the positive control Olaparib, preliminarily proving that the linker leading site of Olaparib determined previously may not affect the binding of the compound to the PARP protein pocket, pending further verification.

[0222] (4) The activity of the compound is affected by two factors: folate receptor and PARP1 expression levels. RKO is a cell with low PARP dependence and low folate receptor expression. Compared with IGROV1 cells, the IC50 value of Olaparib in RKO cells decreased by 2 times, and the inhibitory effect of all series of compounds on RKO cell proliferation also decreased significantly.

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

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

[0225] Experimental method: To evaluate the sensitivity of tumor cells to drugs, this experiment was carried out using the colony formation assay method described in the experimental method, using SKOV3 cells. The cell colony formation assay can be used to study the sensitivity of tumor cells to drugs by detecting the proliferation ability of cells. A small number of cells were seeded in a culture dish to disperse them into single cells. After the cells proliferated for more than 6 generations, their offspring could form cell colonies (also called clones). The proliferation ability of cells can be analyzed by observing the cell morphology and the number of formed cell colonies.

[0226] Experimental results: The more cell colonies formed, the stronger the proliferation ability of the cells. The results are as Figure 1 shown. Compared with the non-drug treatment 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. At a higher drug concentration, almost all cells died. The experiment shows that FA-002-D has a strong effect on inhibiting the proliferation of SKOV3 cells and has a concentration dependence.

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

[0228] Experimental method: To explore the action pathway of the compound, the 3 / 7 detection kit was used as a detection method to study the apoptosis effect of FA-002-D on SKOV3 cells. The experiment was carried out using the Caspase-3 / 7 activity detection method described in the experimental method. The 3 / 7 detection system is a homogeneous luminescence detection system used to detect the activities of Caspase-3 and Caspase-7. One of the reagents in this system is a caspase-3 / 7 luminescent substrate, and the substrate contains the tetrapeptide sequence DEVD. Only one With 3 / 7 reagent, cell lysis can be achieved. Subsequently, caspase cleaves the substrate, and under the action of luciferase, a luminescence signal is generated. The luminescence value is proportional to the activity of caspase. The Caspase-3 / 7 Activity Detection Kit can be used to detect the activity of Caspase-3 / 7 during apoptosis, and the catalytic activity of Caspase-3 / 7 is quantified by measuring the absorbance or fluorescence intensity, so as to understand the situation of apoptosis. Using the detection kit as a detection method for studying the apoptotic effect of FA-002-D on SKOV3 cells, inoculate at a concentration of 4×10 3 cells / well in a 96-well plate. After the cells adhere, add the medium with different concentrations of FA-002-D and continue to culture for 48 h. Add an equal volume of reagent, mix and incubate for 1 h, and read the fluorescence value (490 nm Ex , 510 - 570 nm Em ).

[0229] Experimental results: As Figure 2 shown, there was no significant difference in the fluorescence values between the low-dose treatment groups (0.25 μΜ, 0.5 μΜ, 1 μΜ, 2 μΜ) and the non-treatment group. The fluorescence values of the high-dose treatment groups (4 μΜ, 8 μΜ, 16 μΜ, 32 μΜ) increased with the increase of concentration. The apoptosis experiment further showed that FA-002-D had a significant pro-apoptotic effect on SKOV3 cells and was concentration-dependent.

[0230] Example 4 Evaluation of the PARP-1 Inhibitory Activity of Compound FA-002-D

[0231] Experimental method: To explore the inhibitory activity of compound FA-002-D on PARP-1, a commercially available HT Universal Colorimetric PARP Detection Kit was used for detection. The experiment was carried out using the PARP inhibitory activity determination method described in the experimental method. A series of concentration gradients of the compound, PARP-HSA enzyme, and PARP Cocktail were incubated in a 96-well plate with histone attachment for 1 h to cause a series of glycosylation reactions. After washing twice with the buffer system, add Strep-HRP and incubate for 1 h. Finally, add TACS-Sapphire into the wells to stain the system, and measure the absorbance OD value at 450 nm with an enzyme-labeled instrument. The content of biotinylated substrate attached to histone was measured by the catalytic color reaction of streptavidin-HRP, which can indirectly reflect the activity of PARP-1 at different concentrations of the compound.

[0232] Experimental results: As Figure 3 shown, FA-002-D (IC 50 = 0.70 nM) showed good PARP inhibitory activity, better than the positive control Olaparib (IC 50(more preferably, = 0.79 nM). When both carboxyl groups at the end of the folic acid molecule carry positive drugs, the surface can exert a comparable or even better effect than the parent drug. The PARP-1 inhibitory activity evaluation experiment shows that the compounds of the present invention can all exert inhibitory activity, and they have the potential to increase the inhibitory activity against PARP protein and improve the drug targeting on the basis of the parent drug Olaparib. They can be further studied as preferred compounds. The difference in cytotoxicity is due to the influence of different coupling methods on artificial folic acid and folate receptors.

[0233] Example 5 Evaluation of the folate receptor expression dependence of the conjugate of folic acid and olaparib

[0234] Experimental method: Further investigate the ovarian cancer cell targeting and folate receptor expression dependence of the conjugate. Studies have reported that folate receptors are highly specifically expressed on a series of solid tumors, but not expressed or slightly expressed in normal tissues. Therefore, it is necessary to prove that the synthesized compounds have targeting to folic acid and the activity is related to the folate receptor expression level.

[0235] Experimental results: Six ovarian cancer cell lines with different folate α receptor expression levels (sorted according to the RNA sequencing results of the cell lines in the TCGA database) were selected, and their corresponding folate α receptor expression levels are shown in Table 2. Since there is a correlation but not necessarily a complete correlation between the transcriptional level and the protein level, the folate α receptor expression levels of each ovarian cancer cell line were verified by Western blot experiment (such as Figure 4 (A)), which is consistent with the results in Table 2.

[0236] Combining the evaluation results of the compound's dependence on folate receptor expression in Table 1 and Table 2, in the case of a huge difference in folate receptor expression, the compound activity is mainly affected by the folate receptor; when the folate receptor expressions are similar, for example, in the low folate α receptor expression cells ES2 and RKO (Table 1), due to the difference in PARP1 dependence, the IC50 values of the compounds FA-C8-D and FA-002-D are also slightly different in the two cell lines, indicating that the compound activity is also affected by the PARP1 dependence of the cell line.

[0237] According to the results of the proliferation inhibitory activity in IGROV1 cells, FA-002-D is the conjugate with the best activity. Therefore, FA-002-D was first determined to be included in the evaluation of the folate receptor expression dependence. In order to investigate the influence of the linker, the present invention selected FA-C8-D with a similar molecular weight and a carbon chain linker as a comparison, and olaparib was still used as the positive control drug.

[0238] The fitting curve results of the cell proliferation inhibitory activity of the compound are as Figure 4(B), the IC50 values in each ovarian cancer cell line are shown in Table 3. It can be analyzed from this that generally speaking, the cell proliferation inhibitory activity of the compound increases with the increase in the expression level of folate receptor in the ovarian cancer cell line. Although the activities of FA-C8-D and FA-002-D in cell lines with high folate receptor expression levels (IGROV1, SKOV3) are slightly lower or similar to those in cell lines with medium expression levels (CAOV3, OVCAR3), it may be because CAOV3 and OVCAR3 are Olaparib-sensitive strains (the IC50 value of Olaparib is smaller). In the cell lines ES2 and TOV-21G with low folate receptor expression levels, which are also Olaparib-sensitive strains, the activities of FA-C8-D and FA-002-D decrease significantly, proving that the compound activity is dependent on the folate receptor expression level.

[0239] In cell lines with similar sensitivities to Olaparib, due to different folate receptor expression levels, there are significant differences in the compound activities. In SKOV3 and CAOV3 cells, the IC50 values of Olaparib are similar. When conjugated with folic acid, the activity of FA-C8-D in SKOV3 is about 2 times that in CAOV3, and the activity of FA-002-D in SKOV3 is about 3 times that in CAOV3; in OVCAR3 and ES2 cells, the activity of FA-C8-D in OVCAR3 is about 36 times that in ES2, and the activity of FA-002-D in OVCAR3 is about 85 times that in ES2 (Table 3). Thus, it can be seen that the compound activity is greatly affected by the cell folate receptor expression level, suggesting the existence of a drug transmembrane mechanism mediated by active transport of folate receptor, and reflecting the ability of folic acid to carry cytotoxic drugs to target tumor tissues with high expression of folate receptor.

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

[0241]

[0242] Table 3 IC of the compound against each ovarian cancer cell line 50 value

[0243]

[0244] Example 6 Folic acid competition assay

[0245] The entry of folic acid conjugate into cells depends to a large extent on its receptor FOLR1 on the cancer cell membrane, and the drug entry mediated by FOLR1 can be antagonized by FOLR1 antibody. To further determine the key role of FOLR1 in determining the activity of folic acid conjugate, SKOV3 cells were pretreated with FOLR1 antibody, and then the IC of the preferred compound FA-002-D was measured 50。

[0246] Experimental results: As Figure 5 , 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 level of cell folate receptor and is folate receptor-dependent.

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

[0248] Olaparib can be used regardless of the BRCA status of ovarian cancer patients. However, patients with germline or somatic BRCA gene mutations in ovarian cancer can significantly benefit from first-line maintenance with PARP inhibitors. However, this part of patients only accounts for 30% of the total ovarian cancer population in China. Based on the "synthetic lethality" effect, when the degree of cell DNA damage is increased under the action of DNA-damaging agents and PARP inhibitors are used simultaneously, the therapeutic effect will theoretically be enhanced.

[0249] The combination of PARP inhibitors and various DNA-damaging agents has been preliminarily tested in clinical trials, and the chemotherapy activity has been significantly improved. However, 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 invention uses the conjugate of folic acid and the PARP inhibitor olaparib to investigate whether there is a synergistic effect in the combination of DNA-damaging agents in ovarian cancer cells.

[0250] Experimental method: Select FA-002-D as the preferred compound to explore its combined drug effect with three DNA-damaging agents, cisplatin (Cis), gemcitabine (Gem), and paclitaxel (PTX), in the ovarian cancer cell line SKOV3 with high folate receptor expression. The experiment was carried out using the combined drug method described in the experimental method.

[0251] CompuSyn was used for data processing. Based on the classical Chou-Talalay method, by analyzing the dose-effect curve of the drug, the affected fraction (Fa = 1 - cell survival rate) and the combination index CI (0.2 ≤ CI < 0.4, strong synergistic effect; 0.4 ≤ CI < 0.6, highly synergistic effect; 0.6 ≤ CI < 0.8, moderately synergistic effect; 0.8 ≤ CI < 0.9, low synergistic effect; 0.9 ≤ CI ≤ 1.1, additive effect) were calculated.

[0252] Experimental results: As Figure 6 shown, the dose-effect relationship fitting curve shows that the cell growth inhibition curve of the combination of FA-002-D and cisplatin is shifted upward compared with the single drug group (Figure 6 A) showed that the inhibitory effect on the growth of ovarian cancer cells was enhanced after combined treatment with Cisplatin, and the synergistic effect was relatively good; the inhibitory effect on cell growth in the FA-002-D and Gemcitabine combination group was enhanced compared with the FA-002-D single drug group, and was slightly weaker than the Gemcitabine single drug group ( Figure 6 , C); the inhibitory effect on cell growth in the FA-002-D and PTX combination group was stronger than that in the FA-002-D and PTX single drug groups ( Figure 6 , E). The Fa-CI graph showed that the three combination groups maintained a high synergistic effect in the lower to higher effect intervals and entered antagonism only in the extremely high effect interval, indicating 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 showed good synergistic effects within a certain concentration range.

[0253] The results showed that all combination groups exhibited a super strong synergistic effect (synergy index CI value < 0.1) within a certain effect interval range, and the synergistic effect of Cisplatin was the best.

[0254] These results also indicated that the combination drug strategy of the folic acid and olaparib conjugate of the present invention with DNA-damaging agents has significant potential. By improving the distribution selectivity of PARP inhibitors, the incidence of adverse reactions can be effectively reduced, providing a new strategy for targeted drug treatment of ovarian cancer.

[0255] Table 4 Administration concentrations of each group of FA-002-D and Cisplatin in combination

[0256]

[0257] Example 8 Effect of combined drug use on cell cycle

[0258] The cell cycle is a basic process of cell life activities, and the precise operation of the cell cycle is the basis for the survival, development, and reproduction of eukaryotes. 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 ratio of G1-phase cells in each combination group did not increase compared with the single drug group. This statistical result indicated that the combination mechanism of FA-002-D and the three DNA-damaging agents was not achieved by inducing cell cycle arrest.

[0260] The present invention evaluated the cell proliferation inhibitory activity of the synthesized compounds in cancer cells (mainly ovarian cancer cell line IGROV1). By analyzing the structure-activity relationship, it was found that the activity of the disubstituted series compounds was better than that of the monosubstituted series, which preliminarily proved that the introduction of folic acid and the linker into the cyclopropyl moiety of olaparib did not affect its binding to PARP1, and a series of folic acid-conjugated compounds with good ovarian cancer cell proliferation inhibitory activity were screened out.

[0261] According to the results of the activity evaluation in IGROV1, a series of pharmacodynamic experiments were carried out on FA-002-D with the highest inhibitory activity. The results showed that FA-002-D had a significant effect on inhibiting the proliferation and promoting apoptosis of SKOV3 cells. The evaluation experiment of the inhibitory activity of FA-002-D on PARP-1 protein showed that the conjugation of folic acid and olaparib might hinder the binding to PARP to a certain extent, but FA-002-D showed the potential to increase the inhibitory activity on PARP protein and improve the drug targeting ability based on the parent drug Olaparib. Then, the folate receptor expression dependence of FA-002-D and its control compound FA-C8-D with a carbon chain linker in ovarian cancer cell lines was further evaluated. The results showed that the activities of both compounds decreased significantly in ovarian cancer cells with high, medium, and low folate receptor expression levels, which proved the folate receptor expression dependence of the compounds and provided strong evidence that the compounds could target tumor tissues with high-specificity folate receptor expression. The folate competition experiment further proved that FA-002-D had strong folate receptor dependence, indicating that folic acid could carry cytotoxic drugs into cells through folate receptor-mediated endocytosis, and the design of the non-cleavable linker was reasonable and feasible.

[0262] In the combination therapy experiments, the combination of FA-002-D with three DNA-damaging agents, cisplatin, gemcitabine, and paclitaxel, all showed good cell inhibitory effects. Among them, the combination index (CI) value with cisplatin reached 0.068, indicating a strong synergistic effect, and the inhibitory effect was significantly improved compared with the single-agent treatment of FA-002-D and cisplatin. The folic acid-conjugated olaparib series showed good effects when combined with DNA-damaging agents while targeting folate receptors. This result demonstrated to a certain extent that folic acid-conjugated compounds had the potential to improve the targeting ability of PARP inhibitors and were expected to overcome the narrow therapeutic window of the combined lethal effect with DNA-damaging agents in the treatment of ovarian cancer.

[0263] All the documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A folic acid conjugate of formula I, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, wherein, L is absent or L is a linking group; Ola is a cytotoxic drug moiety.

2. The folic acid conjugate according to claim 1, characterized in that, L is absent or selected from the group consisting of: wherein, n is selected from the group consisting of: 1, 2, 3, 4, 5 or 6; m is selected from the group consisting of: 1, 2 or 3.

3. The folic acid conjugate according to claim 1, wherein, The structure of L is: wherein, m is selected from the group consisting of: 1 or 2.

4. The folic acid conjugate according to claim 1, wherein The cytotoxic drug is a PARP inhibitor.

5. The folic acid conjugate according to claim 1, wherein, The conjugate is selected from the group consisting of:

6. The folic acid conjugate according to claim 1, wherein, The structure of L-Ola is: wherein, the definition of L is as described in claim 1.

7. The folic acid conjugate according to claim 1, wherein, The structure of the L-Ola is selected from the group consisting of:

8. A method for preparing the folic acid conjugate according to claim 1, characterized in that, Comprising the steps of: (a) Providing a first reaction precursor folic acid FA and a second reaction precursor cytotoxic drug; (b) Reacting the first reaction precursor and the second reaction precursor with a linking molecule to form the folic acid conjugate as described in claim 1.

9. A pharmaceutical composition, characterized in that, Comprising: (i) The folic acid conjugate as described in claim 1, or a tautomer thereof, or a pharmaceutically acceptable salt thereof; and (ii) A pharmaceutically acceptable carrier.

10. Use of a folic acid conjugate as described in claim 1, characterized in that, For the preparation of a medicament for the treatment of tumors.

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