A mono-substituted folic acid conjugate, and a preparation method and application thereof

Abstract

The application discloses a mono-substituted folic acid conjugate and a preparation method and application thereof. The mono-substituted folic acid conjugate has both PARP enzyme inhibiting activity and folic acid receptor expression dependence, can effectively improve drug targeting and reduce off-target effects, thereby greatly reducing damage of an antitumor drug to normal cells and tissues, and simultaneously efficiently inhibiting cancer cell proliferation.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a monosubstituted folic acid conjugate, its preparation method, and its application. Background Technology

[0002] Ovarian cancer is the second leading cause of gynecological cancer death among women worldwide. Currently, treatment for ovarian cancer mainly relies on surgical tumor resection and platinum-based chemotherapy. However, patients have a high recurrence rate and are prone to developing drug resistance, resulting in no significant improvement in long-term survival prognosis. New breakthroughs in treatment strategies are urgently needed.

[0003] In recent years, PARP inhibitors (Poly(ADP-ribose) polymerase inhibitors, or PARPi) have been approved for first-line treatment of recurrent ovarian cancer and BRCA1 / 2 mutation patients. 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 first-line maintenance therapy with PARP inhibitors.

[0004] Olaparib is a PARP inhibitor that inhibits PARP enzyme activity, blocking the repair pathway for single-strand DNA damage and thus inducing synthetic lethality in cancer cells with homologous recombination repair defects, thereby achieving the goal of cancer treatment. Olaparib has shown significant efficacy in the treatment of various cancers, especially those with BRCA1 / 2 gene mutations. Furthermore, studies have shown that olaparib, when used in combination with radiotherapy, can increase the radiosensitivity of cancer cells, further improving treatment efficacy. However, the targeting issues of olaparib lead to damage to normal cells and tissues, and the resulting adverse reactions limit its efficacy and its combination with other drugs. Therefore, there is an urgent need to improve the drug's selectivity for tumor cells. Summary of the Invention

[0005] To address the aforementioned technical problems, the primary objective of this invention is to provide a monosubstituted folic acid conjugate.

[0006] The second objective of this invention is to provide a method for preparing a monosubstituted folic acid conjugate.

[0007] A third objective of this invention is to provide the application of a monosubstituted folic acid conjugate in the preparation of PARP inhibitors.

[0008] A fourth objective of this invention is to provide the use of a monosubstituted folic acid conjugate in the preparation of an antitumor drug.

[0009] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0010] This invention claims protection for a monosubstituted folic acid conjugate having the structure shown in formula (I):

[0011]

[0012] Among them, R1 is selected from C 1-8 Alkylene, -R2-O-R3-;

[0013] R2 and R3 are each independently selected from C 1-6 Alkylene.

[0014] This invention provides a monosubstituted folic acid conjugate comprising three parts: artificial folic acid, an unbreakable linker, and a cytotoxic drug.

[0015] The first part is artificial folic acid. Folic acid is composed of two parts: pteroic acid and glutamate. The folic acid receptor mainly recognizes the pteroic acid part, while the glutamate part is not essential for this binding action. Therefore, the glutamate part is used to link the drug. The carboxyl group in the glutamate structure can be divided into α-COOH and γ-COOH. Drugs linked to γ-COOH have a greater affinity for the folic acid receptor than those linked to α-COOH. Therefore, in this invention, the cytotoxic drug is bonded to the γ-COOH terminal of the folic acid to achieve the best binding effect.

[0016] The second part of this invention concerns the linker chain. The selection of the linker chain is crucial, as it determines whether the drug can effectively target the receptor site. Linker chains with appropriate spacing and cleavage mechanisms are key to achieving this goal, enabling efficient release of the parent drug under specific enzymatic hydrolysis conditions or other suitable environments. The folic acid receptor-mediated drug release process is roughly as follows: First, the exogenous folic acid-drug conjugate exhibits high affinity (Kd≈10). -9 Folic acid conjugates bind to the extracellular folic acid receptor (FAR). Then, the cell membrane invaginates around the FAR and conjugate to form an early endosome or "vesicle," undergoing endocytosis. Subsequently, the mature endosome's lumen becomes acidic, the receptor conformation changes, and the folic acid-drug conjugate is released. Finally, the FAR is translocated to the cell membrane, initiating a new transport cycle. Currently reported folic acid conjugates are mostly designed with cleavable bonds such as disulfide bonds and ester bonds. When the folic acid-drug conjugate enters the endosome, the cleavable bonds can be hydrolyzed by specific enzymes, releasing the parent drug to achieve selective drug release.

[0017] In this invention, an unbreakable linker is used to bind artificial folic acid, and then a linker with different carbon chain lengths is used to connect it to a cytotoxic drug, or an O heteroatom is introduced into the carbon chain to bind it to the cytotoxic drug. The advantages of designing an unbreakable linker are: (1) it enhances the targeting of the compound to tumor cells, reduces the killing effect on normal cells caused by premature release of cytotoxic drugs due to the breakage of the linker, and reduces the incidence of adverse reactions; (2) it utilizes folic acid receptor-mediated endocytosis to directly take the intact conjugate into the cell, effectively avoiding the limitation of the compound being unable to enter the cell due to its large molecular weight. The third part is the cytotoxic drug, which is olaparib used in this invention.

[0018] This invention provides a monosubstituted folic acid conjugate structure that is linked by an unbreakable bond, giving it both PARP enzyme inhibitory activity and folic acid binding properties. This can effectively improve drug targeting and reduce off-target effects, providing a powerful tool for novel drug combination therapies.

[0019] More preferably, R1 is selected from C 1-6 Alkylene, -R2-O-R3-; R2 and R3 are each independently selected from C 1-4 Alkylene.

[0020] More preferably, R1 is selected from ethylene, n-propylene, isopropylene, n-butylene, isobutylene, tert-butylene, n-pentylene, isopentylene, neopentylene, hexylene, -R2-O-R3-; R2 and R3 are each independently selected from ethylene, n-propylene, isopropylene, n-butylene, isobutylene, tert-butylene.

[0021] More preferably, the monosubstituted folic acid conjugate is selected from any of the following structures:

[0022]

[0023]

[0024] Furthermore, the present invention also provides a method for preparing a monosubstituted folic acid conjugate, comprising the following preparation steps:

[0025] S1. Compounds of formula (I-1) and (I-2) undergo an amide condensation reaction, followed by the removal of the Boc group to prepare compound (I-3);

[0026] S2. Compound of formula (I) is prepared by amide condensation reaction of compound (I-3) and compound (I-4);

[0027] The reaction formula for the preparation method is shown below:

[0028]

[0029] More specifically, in some specific implementations, a method for preparing a monosubstituted folic acid conjugate includes the following preparation steps:

[0030] S1. Preparation of compound (I-1); Compound (I-1) and compound (I-2) undergo amide condensation reaction under the action of condensing agent and base, and then the Boc group is removed to prepare compound (I-3);

[0031] S2. Under a protective gas atmosphere, compounds of formula (I-3) and (I-4) undergo an amide condensation reaction in the dark with the aid of a condensing agent and a base to prepare compound (I).

[0032] More specifically, the compound of formula (I-1) can be prepared by reacting glutaric anhydride with the compound of formula (I-5) below;

[0033]

[0034] More specifically, the reaction is carried out at room temperature. More specifically, the solvent used in the reaction can be a solvent conventionally used in the art, including but not limited to dichloromethane.

[0035] More specifically, in step S1, the Boc group is removed under the action of trifluoroacetic acid.

[0036] More specifically, in steps S1 and S2, the condensing agent can be a condensing agent conventionally used in the art. More specifically, the condensing agent includes, but is not limited to, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU).

[0037] More specifically, in steps S1 and S2, the base can be a base conventionally used in amide condensation reactions in the art. More specifically, the base includes, but is not limited to, N,N-diisopropylethylamine (DIPEA).

[0038] More specifically, in step S1, the amide condensation reaction is carried out at room temperature. More specifically, the room temperature is 20-30°C.

[0039] More specifically, in steps S1 and S2, the solvent used for the amide condensation reaction can be a solvent commonly used in the art, including but not limited to N,N-dimethylformamide.

[0040] More specifically, in step S2, the protective gas atmosphere is nitrogen.

[0041] More specifically, in step S2, the temperature for the light-protected reaction is room temperature.

[0042] Furthermore, this invention claims protection for the use of monosubstituted folic acid conjugates in the preparation of PARP inhibitors or medicaments targeting folic acid receptors.

[0043] The monosubstituted folic acid conjugate provided by this invention has superior therapeutic effects in environments with high folic acid receptor expression or in cancer cells and / or in parp1-sensitive cancer cells.

[0044] Furthermore, this invention claims protection for the use of monosubstituted folic acid conjugates in the preparation of antitumor drugs.

[0045] Preferably, the tumor is at least one of ovarian cancer, breast cancer, cervical cancer, bladder cancer, pleural mesothelioma, or non-small cell lung cancer.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] This invention provides a monosubstituted folic acid conjugate that possesses both PARP enzyme inhibitory activity and folic acid receptor expression dependence, exhibiting folic acid binding properties. This effectively improves drug targeting and reduces off-target effects, thereby significantly reducing the damage of antitumor drugs to normal cells and tissues, while efficiently inhibiting cancer cell proliferation. This provides a powerful tool for novel drug combination therapy. Attached Figure Description

[0048] Figure 1 The expression levels of folate α receptors in various ovarian cancer cell lines. Detailed Implementation

[0049] The present invention will be further described below with reference to the specification and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0050] Example 1: Synthesis of Monosubstituted Folic Acid Couplings

[0051] (1) Synthesis of intermediate FA6-C5~C9

[0052] Synthesis of intermediate FA6-C5: Glutaric anhydride (25 mmol, 2.85 g) was dissolved in 15 mL of dichloromethane, and mono-Boc-ethylenediamine (25 mmol) was dissolved separately in 15 mL of dichloromethane. These solutions were slowly added dropwise to the dichloromethane solution of glutaric anhydride. After reacting at room temperature for 2 h, the solution was evaporated under reduced pressure to obtain a white solid or a colorless transparent oil. HATU (25 mmol, 9.5 g) was added and dissolved in 25 mL of DMF. The mixture was activated at room temperature for approximately 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. After DMF, the activated solution was slowly added dropwise. After reacting at room temperature for about 3 hours, saturated NaCl water 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 concentrate was separated by silica gel column chromatography (eluting agent: DCM / MeOH (v / v) = 20 / 1) to obtain a transparent oily substance.

[0053] A transparent oily substance 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 the mixture was concentrated under reduced pressure. The excess trifluoroacetic acid was evaporated three times. NaOH aqueous solution was added to adjust the pH to 10. This process generated a large number of bubbles and was exothermic. The resulting solution was directly evaporated to dryness. The solid was dissolved in a mixed solvent of dichloromethane and methanol, filtered, and the filtrate was concentrated under reduced pressure. Separation was carried out by silica gel column chromatography (eluent: DCM / MeOH (v / v) = 10 / 1) to obtain a colorless oily substance, which is the intermediate FA6-C5.

[0054] The structural formula of intermediate FA6-C5 is shown below.

[0055]

[0056] 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%.

[0057] 1H NMR(500MHz,DMSO-d6)δ8.26(d,J=7.8Hz,1H),7.96(d,J=8.0Hz,1H),7.92-7.87(m,1H),7.83(td,J= 7.5,1.2Hz,1H),7.76(t,J=5.7Hz,1H),7.44(ddd,J=7.6,5.0,2.3Hz,1H),7.39-7.33(m,1H),7.24(td ,J=9.0,2.0Hz,1H),4.33(s,2H),3.41-3.32(m,2H),3.16(dt,J=19.5,5.2Hz,2H),3.03(p,J=6.0Hz,2 H), 2.54 (q, J = 6.4Hz, 2H), 2.29 (dt, J = 33.8, 7.4Hz, 2H), 2.10 (q, J = 6.9Hz, 2H), 1.71 (h, J = 7.1Hz, 2H). 13 C NMR (126MHz, DMSO-d6) δ172.3,171.0,159.8,145.3,135.3,134.0,129.6,129.4,128. 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..

[0058] (2) The synthesis of intermediates FA6-C6~C9 is performed in the same way as in step (1) above, except that mono-Boc-ethylenediamine is replaced with N-Boc-1,3-propanediamine, (4-aminobutyl)carbamate, N-(5-aminopentyl)carbamate, and N-(6-aminohexyl)carbamate.

[0059] The structural formula of FA6-C6 is shown below.

[0060]

[0061] 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%.

[0062] 1H NMR(500MHz,DMSO-d6)δ8.26(d,J=7.9Hz,1H),7.98(dd,J=17.5,6.9Hz,2H),7.92-7.86(m,1H), 7.83(t,J=7.5Hz,1H),7.44(t,J=7.0Hz,1H),7.35(d,J=6.1Hz,1H),7.26-7.21(m,1H),4.33(s, 2H),3.51(d,J=5.2Hz,2H),3.36(dt,J=14.9,4.6Hz,2H),3.13(ddt,J=32.1,12.2,6.0Hz,4H),2 .63(q,J=6.4Hz,2H),2.30(dt,J=33.7,7.4Hz,2H),2.11(q,J=6.7Hz,2H),1.71(h,J=7.7Hz,2H). 13 C NMR (101MHz, DMSO) δ173.5,172.5,171.0,164.5,159.9,145.33,135.3,134.0,130.0,129.4,128. 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.

[0063] The structural formula of FA6-C7 is shown below.

[0064]

[0065] 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%.

[0066] 1H NMR (500MHz, DMSO-d6) δ12.61(s,1H),8.26(d,J=7.9Hz,1H),7.96(d,J=8.0Hz,1H),7.89(dt,J=8.6,5.5H z,2H),7.85-7.80(m,1H),7.47-7.42(m,1H),7.35(t,J=3.9Hz,1H),7.26-7.20(m,1H),4.33(s,2H),3.60( d,J=30.1Hz,2H),3.50(d,J=5.4Hz,2H),3.03(p,J=6.3Hz,2H),2.77(q,J=6.8Hz,2H),2.29(dt,J=32.0,7 .4Hz,2H),2.09(q,J=6.9Hz,2H),1.70(q,J=7.4Hz,2H),1.51(q,J=7.3,6.8Hz,2H),1.41(q,J=6.9Hz,2H). 13 C NMR(126MHz,DMSO-d6)δ172.3,171.1,159.9,158.8,158.6,145.4,134.0,129.4,128.3, 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..

[0067] The structural formula of FA6-C8 is shown below.

[0068]

[0069] 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%.

[0070] 1H NMR (500MHz, DMSO-d6) δ12.62(s,1H),8.27(d,J=7.8Hz,1H),7.97(d,J=8.1Hz,1H),7.92-7.80(m,5H),7. 46(d,J=8.4Hz,1H),7.35(d,J=6.7Hz,1H),7.24(t,J=8.9Hz,1H),4.34(s,2H),3.63(s,1H),3.57(s,1H), 3.50(d,J=5.7Hz,2H),3.01(q,J=6.5Hz,2H),2.75(d,J=9.8Hz,2H),2.29(dt,J=32.1,7.5Hz,2H),2.09(q ,J=6.7Hz,2H),1.70(q,J=7.4Hz,2H),1.52(d,J=7.4Hz,2H),1.38(q,J=7.2Hz,2H),1.28(d,J=6.6Hz,2H). 13 C NMR (126MHz, DMSO-d6) δ171.1,159.9,158.8,145.4,135.3,134.0,129.5,129. 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..

[0071] The structural formula of FA6-C9 is shown below.

[0072]

[0073] 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%.

[0074] 1H NMR (500MHz, DMSO-d6) δ12.63(s,1H),8.26(d,J=7.8Hz,1H),7.95(t,J=7.3Hz,1H),7.88(tt,J=9.8,4.5Hz,2H),7.82(t,J= 7.5Hz,1H),7.44(ddd,J=8.2,5.4,2.7Hz,1H),7.35(dd,J=6.5,2.2Hz,1H),7.22(td,J=9.0,1.8Hz,1H),4.33(s,2H),3.66- 3.54(m,2H),3.35(dt,J=15.9,5.1Hz,2H),3.21-3.11(m,2H),3.01(p,J=6.5Hz,2H),2.76(q,J=6.8Hz,2H),2.29(dt,J=32. 4,7.5Hz,2H),2.09(q,J=6.8Hz,2H),1.70(h,J=7.6Hz,2H),1.52(h,J=7.1Hz,2H),1.36(h,J=7.3Hz,2H),1.29-1.20(m,5H). 13 C NMR (126MHz, DMSO-d6) δ172.2,171.1,159.9,158.9,158.7,135.3,134.0,129.5,129.4,128.3 ,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..

[0075] (3) Synthesis of monosubstituted folic acid conjugates

[0076] Synthesis of monosubstituted folic acid conjugate FA-C5-S: Folic acid (0.45 mmol, 200 mg) and HATU (0.5 mmol, 190 mg) were placed in a three-necked flask, 25 mL of DMF was added, and the mixture was protected from light by nitrogen. DIPEA (1.8 mmol, 0.3 mL) was injected and activated at room temperature for 1 h. Subsequently, 12 mL of FA6-C5 (0.5 mmol) DMF solution was slowly added dropwise to the reaction system using a syringe pump at an injection rate of approximately 12 mL / h. The mixture was kept in an ice bath, and after the injection was completed, the reaction was allowed to return to room temperature for 3 h. After the reaction was completed, the reaction solution was transferred to a centrifuge tube. A mixture of dichloromethane and methanol was added as a poor solvent. 25 mL of poor solvent was added for every 25 mL of reaction solution. After precipitation, the solution was centrifuged and the supernatant was discarded. The precipitate was washed several times with dichloromethane, centrifuged again, and then dried in a vacuum drying oven. The resulting solid was crushed and dissolved in 5% Na2CO3 solution. After sonication and filtration of insoluble matter, the pH of the solution was slowly adjusted to 8-9 with dilute hydrochloric acid. During this process, solid precipitated. After centrifugation, the solid was washed with methanol and dried in a vacuum drying oven to obtain the monosubstituted folic acid conjugate FA-C5-S.

[0077] The structural formula of FA-C5-S is shown below.

[0078]

[0079] N 2 -(4-(((2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl)amino)benzoyl)-N5-(2-(5-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)-5-oxopentanamido)ethyl)-L-glutamine. Orange-yellow solid, yield: 36%, purity: 90%.

[0080] 1H NMR (400MHz, DMSO-d6) δ12.59(s,1H),8.63(d,J=2.1Hz,1H),8.26(d,J=7.8Hz,1H),8.00–7.92(m,2H),7.85( dq,J=14.6,7.2Hz,4H),7.69–7.64(m,1H),7.62(d,J=8.1Hz,2H),7.43(d,J=7.9Hz,2H),7.36(dd,J=6.4,2.3 Hz,1H),7.22(t,J=9.0Hz,1H),6.90(q,J=6.1,5.2Hz,2H),6.63(s,2H),4.47(d,J=6.0Hz,4H),4.32(s,2H),3 .59(d,J=27.0Hz,5H),3.50(s,2H),3.11–3.02(m,5H),2.36–2.19(m,3H),2.08(d,J=7.0Hz,3H),1.69(s,2H). 13 C NMR (126MHz, DMSO-d6) δ172.5,172.4,171.1,164.5,159.9,154.7,151.1,149.0,148.8,145.3,134.0,129.5,129.4,129.2,128. 4,128.3,125.9,116.5,116.3,111.7,111.6,47.1,46.4,45.4,41.6,38.8,36.9,35.0,32.6,32.2,31.2,21.2.HRMS[ESI]:calcd for[M+H] + (C 46 H 49 N 13 O9F)requires m / z 946.3755,found 946.3779.

[0081] (4) Synthesis of monosubstituted folic acid conjugates FA6-C6~C9 The synthesis of FA6-C6~C9 is the same as the preparation method in step (3) above, except that FA6-C5 is replaced with FA6-C6, FA6-C7, FA6-C8 and FA6-C9 respectively, and FA-C6-S, FA-C7-S, FA-C8-S and FA-C9-S are prepared respectively.

[0082] The structural formula of FA-C6-S is shown below.

[0083]

[0084] N2-(4-(((2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl)amino)benzoyl)-N5-(3-(5-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)-5-oxopentanamido)propyl)-L-glutamine. Orange-yellow solid, yield: 26%, purity: 91%.

[0085] 1 H NMR(500MHz,DMSO-d6)δ12.62(s,1H),8.59(s,1H),8.25(d,J=7.6Hz,1H),7.95(dd,J=8.3,3.8Hz,1H),7.88(t,J=7.6Hz,2H),7.82(t,J=7.5Hz,1H),7.76(d,J=5.7Hz,1H),7.57(d,J=8.2Hz,2H),7.45–7.40(m,1H),7.35(d,J=6.7Hz,1H),7.22(t,J=9.0Hz,1H),6.89(t,J=6.1Hz,1H),6.64(d,J=8.5Hz,2H),6.59(d,J=8.5Hz,1H),4.51–4.40(m,2H),4.32(s,3H),3.62(s,1H),3.56(s,1H),3.49(q,J=7.7,4.5Hz,2H),3.17(s,2H),3.15–3.10(m,2H),3.00(h,J=6.7Hz,5H),2.28(dt,J=34.3,7.5Hz,2H),2.08(t,J=6.1Hz,4H),1.96–1.80(m,1H),1.70(p,J=7.4Hz,3H),1.47(h,J=7.6,6.6Hz,2H).13C NMR(126MHz,DMSO-d6)δ175.1,172.3,171.7,170.6,165.2,164.0,159.4,150.6,148.2,148.1,147.6,144.9,134.8,133.5,129.1,129.0,128.4,128.0,127.9,123.5,122.3,115.8,53.1,46.3,46.0,44.5,41.1,36.3,32.4,31.8,29.3,29.0,20.9.HRMS[ESI]:calcd for[M+H] + (C47 H 51 N 13 O9F)requires m / z 960.3911,found 960.3924.

[0086] The structural formula of FA-C7-S is shown below.

[0087]

[0088] N 2 -(4-(((2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl)amino)benzoyl)-N 5 -(4-(5-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)-5-oxopentanamido)butyl)-L-glutamine. Orange-yellow solid, yield: 23%, purity: 90%.

[0089] 1 H NMR (400MHz, DMSO-d6) δ12.58(s,1H),8.64(s,1H),8.26(d,J=7.8Hz,1H),7.96(d,J=8.4Hz ,1H),7.86(d,J=23.9Hz,3H),7.64(d,J=8.0Hz,3H),7.43(s,1H),7.35(s,1H),7.24(d,J=9. 3Hz,1H),6.92(s,2H),6.63(d,J=8.3Hz,3H),4.48(s,2H),4.32(s,2H),3.62(s,2H),3.56( s,1H),2.95(d,J=7.8Hz,3H),2.09(s,2H),1.69(s,2H),1.23(s,2H),1.12(t,J=7.3Hz,3H). 13C NMR (126MHz, DMSOd6) δ172.1,172.1,171.0,164.5,159.9,157.8,155.9,145.3,135.3,134.0,132.2,129.6,129.5,129.4,128. 4,128.4,126.6,125.9,124.1,116.3,111.7,46.8,44.9,41.6,41.0,38.7,38.6,36.9,35.1,32.2,27.1,21.4.HRMS[ESI]:calcd for[MH] - (C 48 H 51 N 13 O9F)requires m / z 972.3922,found972.3949.

[0090] The structural formula of FA-C8-S is shown below.

[0091]

[0092] N 2 -(4-(((2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl)amino)benzoyl)-N 5 -(5-(5-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)-5-oxopentanamido)pentyl)-L-glutamine. Orange-yellow solid, yield: 31%, purity: 92%.

[0093] 1H NMR (500MHz, DMSO-d6) δ12.70(d,J=16.7Hz,1H),8.48(s,1H),8.26(d,J=7.8Hz,1H),7.95(dt,J=9.1,4.4Hz,2H),7.89(t,J=7.8Hz,1H),7.82(t,J =7.3Hz,1H),7.62(d,J=8.5Hz,1H),7.58–7.50(m,2H),7.44(d,J=7.6Hz, 1H),7.38–7.33(m,1H),7.23(t,J=9.0Hz,1H),6.87(dq,J=17.7,11.4,8.8 Hz,2H),6.62(d,J=8.9Hz,2H),4.43(d,J=6.1Hz,3H),4.33(s,2H),3.65– 3.60(m,1H),3.52–3.45(m,2H),3.15–3.10(m,2H),2.97(dt,J=14.2,6.7 Hz,4H),2.28(dt,J=33.6,7.4Hz,2H),2.08(q,J=7.6,7.1Hz,3H),1.83(s ,1H),1.70–1.65(m,3H),1.38–1.28(m,5H),1.20(dd,J=12.7,5.7Hz,2H). 13 C NMR (126MHz, DMSO-d6) δ172.0,171.0,162.8,159.9,145.3,145.0,135.3,134.0,132.2,129.6,129.6,129.4,128.4,126.5,125.9,1 24.1,116.3,111.6,56.5,53.8,46.4,44.9,41.6,38.8,36.9,36.2,35.1,32.6,32.2,31.2,29.2,28.2,24.2,19.0.HRMS[ESI]:calcd for[MH] - (C 49 H 53 N 13 O9F)requires m / z 986.4079, found 986.4042.

[0094] The structural formula of FA-C9-S is shown below.

[0095]

[0096] N 2-(4-(((2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl)amino)benzoyl)-N 5 -(6-(5-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)-5-oxopentanamido)hexyl)-L-glutamine. Orange-yellow solid, yield: 43%, purity: 93%.

[0097] 1 H NMR(500MHz, DMSO-d6) δ 12.65(s,1H), 8.56(q,J=2.3Hz,1H), 8.26(dd,J=7.9,1.4Hz,1H), 7.96(d,J=8.2Hz,1H), 7.89(t,J=7.6Hz,1H), 7.85–7.80(m,1H), 7.78(p,J=4.9,4.1Hz,1H), 7.67(t,J=9.6Hz,1H), 7.57(d,J=8.3Hz,2H), 7.44(p,J=5.0,4.0Hz,1H), 7.37(d,J=6.6Hz,1H), 7.23(t,J=9.0Hz,1H), 6.88(t,J=5.7Hz,1H), 6.67–6.63(m,2H), 6.59(d,J=8.5Hz,1H), 4.46(s,2H), 4.33(s,3H), 3.67–3.55(m,4H), 3.54–3.47(m,2H), 3.36(d,J=18.3Hz,4H), 3.16–3.12(m,1H), 3.00(dt,J=14.4,8.5Hz,4H), 2.29(dt,J=34.0,7.5Hz,2H), 2.08(p,J=7.6,6.9Hz,3H), 1.90–1.80(m,1H), 1.70(q,J=7.3Hz,2H), 1.34(s,5H), 1.22(d,J=10.6Hz,4H). 13C NMR (126MHz, DMSO-d6) δ175.4,172.6,172.0,171.1,166.5,165.6,159.9,151.0,145.3,134.0,129.5,129.4,128.8,128.6,128 .4,126.5,125.9,116.3,111.6,54.4,49.0,47.1,46.4,44.9,36.9,35.0,32.9,32.2,29.6,26.6,26.4,21.4.HRMS[ESI]:calcd for[MH]-(C50H55N13O9F)requires m / z 1000.4235,found 1000.4266.

[0098] Example 2 Synthesis of monosubstituted folic acid conjugates

[0099] (1) Synthesis of intermediate FA6-002

[0100] Glutaric anhydride (25 mmol, 2.85 g) was dissolved in 15 mL of dichloromethane. [2-(2-aminoethoxy)ethyl]carbamate tert-butyl ester (25 mmol) was also dissolved in 15 mL of dichloromethane. These solutions were slowly added dropwise to the dichloromethane solution of glutaric anhydride. After reacting at room temperature for 2 h, the mixture was evaporated under reduced pressure to obtain a white solid or a colorless, transparent oil. A condensing agent, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (25 mmol, 9.5 g), was added and dissolved in 25 mL of DMF. The linker-terminal carboxyl group was activated at room temperature for approximately 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. After N,N-dimethylformamide (DMF), it was slowly added dropwise to the activated solution. After reacting at room temperature for about 3 hours, saturated NaCl water 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 concentrate was separated by silica gel column chromatography (eluting agent: DCM / MeOH (v / v) = 20 / 1) to obtain a transparent oily substance.

[0101] A transparent oily substance 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 the mixture was concentrated under reduced pressure. The excess trifluoroacetic acid was evaporated three times. NaOH aqueous solution was added to adjust the pH to neutral. This process generated a large number of bubbles and was exothermic. Saturated NaCl, chloroform, and a small amount of methanol (as a co-solvent) were added and the mixture was extracted 7-8 times. The lower organic phase was collected, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The concentrate was separated by silica gel column chromatography (eluent: DCM / MeOH (v / v) = 10 / 1) to obtain a colorless and transparent oily substance, which is the intermediate FA6-002 with high purity.

[0102] The structural formula of intermediate FA6-002 is shown below.

[0103]

[0104] O-(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%.

[0105] 1 H NMR (500MHz, DMSO-d6) δ12.63(s,1H),8.26(d,J=7.8Hz,1H),7.95(t,J=7.4Hz,3H),7.88(td,J=7.7,3.2Hz,1H),7.8 2(t,J=7.5Hz,1H),7.44(ddd,J=8.1,5.3,2.5Hz,1H),7.35(dd,J=6.4,2.3Hz,1H),7.22(td,J=9.0,2.0Hz,1H),4.32( s,2H),3.65-3.54(m,4H),3.42(q,J=5.9Hz,2H),3.36(dq,J=14.1,4.9,4.3Hz,2H),3.23(q,J=5.5Hz,2H),3.16(dt,J =22.1,5.1Hz,3H),2.98(q,J=4.9Hz,2H),2.29(dt,J=32.1,7.5Hz,2H),2.13(q,J=6.8Hz,2H),1.70(h,J=7.6Hz,2H). 13C NMR(101MHz,DMSO-d6)δ172.6,159.9,159.0,145.4,135.3,129.5,129.3,128.3,1 25.9,122.0,119.1,116.1,113.2,69.5,66.7,44.9,41.6,38.6,36.2,32.2,21.3..

[0106] (2) Synthesis of monosubstituted folic acid conjugate FA-002-S

[0107] Folic acid (0.45 mmol, 200 mg) and HATU (0.5 mmol, 190 mg) were placed in a three-necked flask, and 25 mL of DMF was added. The flask was protected from light by nitrogen and then DIPEA (1.8 mmol, 0.3 mL) was injected. The flask was activated at room temperature for 1 h. Then, 12 mL of FA6-002 (0.5 mmol) DMF solution was slowly added to the reaction system using a syringe under a syringe pump at an injection rate of about 12 mL / h. The flask was kept in an ice bath. After the injection was completed, the flask was allowed to return to room temperature for 3 h. After the reaction was completed, the reaction solution was transferred to a centrifuge tube. A mixture of dichloromethane and methanol was added as a poor solvent. 25 mL of the poor solvent was added for every 25 mL of reaction solution. After precipitation, the mixture was centrifuged, and the supernatant was discarded. The precipitate was washed several times with dichloromethane, centrifuged again, and then dried in a vacuum drying oven. The resulting solid was crushed and dissolved in 5% Na2CO3 solution. The solution was sonicated, and the insoluble matter was filtered out. The pH of the solution was slowly adjusted to neutral with dilute hydrochloric acid. During this process, solid precipitated. After centrifugation, the precipitate was washed with methanol and centrifuged again to obtain the monosubstituted folic acid conjugate FA-002-S.

[0108] Thin-layer chromatography (TLC) was used to verify the formation of the final product. The developing solvent was n-propanol:n-butanol:ammonia:water = 5:5:3:1. Under irradiation at 254 nm, the Rf values ​​corresponding to different polar substances in the reaction system were observed. In addition to the folic acid spot, two new product spots, FA-002-S, were generated in the reaction solution. Liquid chromatography-mass spectrometry (LC-MS) was used to determine the reaction progress by measuring the target molecular weight. Folic acid and the product were dissolved in methanol, and ammonia was added dropwise to aid dissolution. Solid dissolution was observed. LC-MS analysis of the solution was performed again, detecting a folic acid [M+H]+ molecular ion peak at m / z 442.17 and a FA-002-S [M+H]+ molecular ion peak at m / z 990.63 (molecular weight: 989.39). This indicates that the terminal carboxyl group of FA and the terminal amino group of FA6-002 underwent an amide condensation reaction, indicating the simultaneous formation of a monosubstituted product under these conditions, consistent with the TLC results.

[0109] The structural formula of the monosubstituted folic acid conjugate FA-002-S is shown below.

[0110]

[0111] N 2 -(4-(((2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl)amino)benzoyl)-N 5 -(2-(2-(5-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)-5-oxopentanamido)ethoxy)ethyl)-L-glutamine. Orange-yellow solid, yield: 29%, purity: 94%.

[0112] 1 H NMR (500 MHz, DMSO-d6) δ 12.64–12.60 (m, 1H), 8.62 (d, J=3.2 Hz, 1H), 8.25 (dd, J=7.8, 1.4 Hz, 1H), 7.96 (d, J=10.2 Hz, 2H), 7.89 (q, J=7.8, 7.4 Hz, 3H), 7.82 (t, J=7.5 Hz, 1H), 7.66 (d, J=8.1 Hz, 1H), 7.63–7.59 (m, 1H), 7.43 (t, J=6.9 Hz, 1H), 7.36 (dd, J=6.4, 2.2 Hz, 1H), 7.29 (s, 1H), 7.23 (t, J=9.0 Hz, 1H), 6.95 (q, J=5.8 Hz, 1H), 6.64 (dd, J=6.6, 2.9 Hz, 2H), 4.47 (d, J=5.9 Hz, 2H), 4.32 (s, 2H), 4.18 (d, J=6.8 Hz, 1H), 3.65–3.46 (m, 4H), 3.24–3.11 (m, 6H), 2.34–2.27 (m, 1H), 2.24 (t, J=7.5 Hz, 2H), 2.19–2.14 (m, 1H), 2.13–2.07 (m, 2H), 2.06–1.99 (m, 1H), 1.88 (t, J=11.7 Hz, 1H), 1.68 (q, J=7.3 Hz, 2H). 13CNMR(126MHz,DMSO-d6)δ171.9,170.6,164.1,159.4,154.3,150.7,148.4,144.9,134.8,133.5,131.8,129.1,129.0,128.7,128.0 ,127.9,126.1,116.0,111.3,68.9,59.8,56.0,56.0,45.9,41.1,38.4,36.4,34.4,31.7,20.9,20.8,18.6,14.1.HRMS[ESI]:calcd for[MH] - (C 48 H 51 N 13 O 10 F)requires m / z 988.3871, found 988.3854.

[0113] Comparative Example 1: Synthesis of Folic Acid-Olaparib Conjugate

[0114] (1) Synthesis of folic acid and olaparib conjugate FA-Ola-S

[0115] Folic acid (0.45 mmol, 200 mg) and HATU (0.5 mmol, 190 mg) were placed in a three-necked flask, and 25 mL of DMF was added. The flask was protected from light by nitrogen and then injected with DIPEA (1.8 mmol, 0.3 mL). The flask was activated at room temperature for 1 h. Subsequently, 12 mL of DMF solution of 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazine-1(2H)-one (0.5 mmol, 185 mg) was slowly added dropwise to the reaction system using a syringe pump at an injection rate of approximately 12 mL / h. The flask was kept in an ice bath. After the injection was completed, the reaction was allowed to return to room temperature for 3 h. After the reaction was completed, the reaction solution was transferred to a centrifuge tube. A mixture of dichloromethane and methanol was added as a poor solvent, with 25 mL of poor solvent added for every 25 mL of reaction solution. After precipitation, the solution was centrifuged, and the supernatant was discarded. The precipitate was washed several times with dichloromethane, centrifuged again, and then dried in a vacuum drying oven. The resulting solid was crushed and dissolved in a 5% Na2CO3 solution, sonicated, and the insoluble matter was filtered out. The pH of the solution was slowly adjusted to neutral with dilute hydrochloric acid. During this process, solid precipitated. After centrifugation, the precipitate was washed with methanol, centrifuged again, and then dried in a vacuum drying oven to obtain FA-Ola-S.

[0116] The structural formula of FA-Ola-S is shown below.

[0117]

[0118] (S)-2-(4-(((2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl)amino)benzamido)-5-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)-5-oxopentanoic acid. Orange-yellow solid, yield: 36%, purity: 91%.

[0119] 1 H NMR(500MHz,DMSO-d6)δ12.62(s,1H),8.67–8.65(m,1H),8.24(dd,J=7.8,5.0Hz,1H),8.19(dt,J=12.3,6.6Hz,1H),7.99–7.92(m,1H),7.88(dd,J=10.4,7.2Hz,1H),7.81(dt,J=15.6,8.1Hz,1H),7.65(td,J=5.9,2.8Hz,2H),7.55(s,2H),7.47–7.41(m,1H),7.34(ddd,J=12.5,6.4,2.2Hz,1H),7.23(t,J=9.0Hz,1H),6.66–6.63(m,2H),4.49(d,J=3.9Hz,3H),4.32(s,2H),3.29(s,2H),3.13(s,2H),2.44(d,J=7.6Hz,1H),2.37(dt,J=8.6,4.3Hz,1H),2.09–2.00(m,1H),2.00–1.91(m,1H). 13C NMR(126MHz,DMSO-d6)δ191.7,174.3,170.8,166.9,164.5,159.9,154.6,151.3,149.0,145.3,135.3,134.0,132.2,132.0,129.5,129.5,128.4,128.4,126.5,126.0,124.1,121.8,116.5,113.1,111.6,52.6,46.3,41.7,36.9,29.6,26.7. HRMS[ESI]: calcd for [M-H]-(C 39 H 35 N 11 O7F) requires m / z 788.2710, found 788.2688.

[0120] Test Example 1: Preliminary Evaluation of the Anticancer Activity of Folic Acid and Olaparib Conjugate

[0121] To detect the cytotoxicity of the compounds, this invention employed the CTG and CCK-8 assays, selecting two tumor cell lines: IGROWV1 (human ovarian cancer cells) and RKO (human colon cancer cells), with olaparib as a positive control. Different concentration gradients of the compounds were added to cells undergoing logarithmic growth, incubated for 120 hours, and the OD values ​​were measured to calculate the IC50. 50 Value (compound concentration at 50% cell death). RKO cells served as a low folate receptor expression, low PARP1-dependent control line for IGROWV1 cells.

[0122] The specific experimental procedures are as follows: IGROWV1 and RKO cells in the logarithmic growth phase were diluted with culture medium and seeded into 96-well plates at a density of 1400 cells / well. The cells were incubated at 37°C for 24 hours. After cell adhesion, the culture medium was removed. The pH of each compound was adjusted to approximately 10 by adding sodium hydroxide solution to the 10mM DMSO stock solution. The culture medium was serially diluted, and 100 μL of the compound dilution was added to each well of the seeded plate. The plates were then incubated at 37°C for 120 hours. 50 μL of CellTiter-1 was added to each well. Reagent reagent was used, and the plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal. Then, the full wavelength was measured using a microplate reader. The cell proliferation inhibition rate was calculated as (ODg). control -OD 给药 / OD control -OD 空白 )×100%. IC 50 The values ​​were fitted using GraphPad Prism9. The test results are shown in Table 1 below.

[0123] Table 1. Inhibitory activity of compounds against multiple tumor cell lines.

[0124]

[0125] a: The compound has poor solubility and precipitates; abbreviations: ND, Not Done; NA, Not Available.

[0126] The experimental results are shown in the table below, from which the following conclusions can be drawn:

[0127] (1) The length of the linker carbon chain and the presence of oxygen heteroatoms in the linker chain affect the activity and solubility of the conjugate to a certain extent. In IGROWV1 cells, the activity of FA-C5-S to FA-C9-S and FA-002-S generally increases with the increase of the linker carbon chain length. The solubility of FA-002-S is significantly improved due to the introduction of oxygen heteroatoms into the linker chain.

[0128] (2) Hydrophilicity is the key factor affecting the activity of the conjugate. In IGROV1 cells, FA-002-S has high activity, possibly because the oxygen heteroatoms in the linker chain can form hydrogen bonds with the solvent, increasing the hydrophilicity of the compound, thereby regulating the oil-water partition coefficient of the compound and significantly increasing the solubility of the compound.

[0129] (3) The coupling site between olaparib and folic acid may not have affected the activity of olaparib. Except for FA-Ola-S (precipitated in the culture medium), all other compounds in the series showed varying degrees of cell proliferation inhibition activity. Among them, the activities of FA-C6-S, FA-C8-S, and FA-002-S were similar to or even better than those of the positive control olaparib, which preliminarily proves that the previously identified olaparib linker extraction site does not affect the binding of the compound to the PARP protein pocket.

[0130] (4) The activity of the compound is affected by two factors: the expression level of folate receptor and PARP1. RKO cells are PARP-independent and folate receptor-expressing cells. Compared with IGROWV1 cells, olaparib showed a lower IC50 concentration in RKO cells. 50 The value decreased by 2-fold, and the inhibitory effect of all series of compounds on RKO cell proliferation also decreased significantly. In cases of significant differences in folate receptor expression, compound activity is mainly influenced by the folate receptor itself; when folate receptor expression is similar, for example, in ES2 cells (Table 2) and RKO cells (Table 3) with low folate α receptor expression, the IC50 of compounds differs due to differences in PARP1 dependence. 50 The values ​​also differed slightly between the two cell lines, indicating that the activity of the compound was also affected by the cell line's dependence on PARP1.

[0131] In summary, the type and length of the linker chain, the molecular size, and hydrophilicity of the conjugate are all key factors affecting its activity. The synthesized folic acid-olaparib conjugate exhibits targeted activity and high inhibitory activity against IGROV1 ovarian cancer cells.

[0132] Test Example 2: Evaluation of Folic Acid Receptor Expression Dependence of Folic Acid and Olaparib Conjugate

[0133] (1) The specific procedures for Western blotting are as follows: Centrifuge at 1500 rpm to collect cells, wash three times with 1×PBS, add RIPA universal protein lysis buffer and lyse cells on ice for 30 min; centrifuge at 12000 rpm and 4℃ for 30 min to collect the supernatant. Use a pipette to thoroughly mix the extracted protein supernatant, add 2 μL of protein sample and 18 μL of ddH2O to each well of a 96-well plate, and finally add 200 μL of detection solution to each well. Incubate the 96-well plate in a 37℃ cell culture incubator for 30 min, and then use a microplate reader to detect the absorbance at 562 nm wavelength, and calculate using the protein standard curve. Calculate the sample volume required for loading 20 μg of protein based on the previously determined sample protein concentration, and add ddH2O to make the final loading volume 20 μL. Add 5×Loading Buffer according to the volume, mix well, briefly centrifuge at low temperature, heat in a 100℃ metal bath for denaturation for 5 min, and then briefly centrifuge for later use. The gel transfer was performed, and the grayscale analysis of the strips was conducted using ImageJ software.

[0134] Table 1. Expression levels of FAα and PARP1 in various ovarian cancer cell lines (Data source: CCLE and TCGA databases)

[0135]

[0136] Further investigation was conducted into the targeting properties of the conjugate to ovarian cancer cells and its dependence on folate receptor expression. Studies have reported that folate receptors are specifically highly expressed in a range of solid tumors, but not expressed or minimally expressed in normal tissues. Therefore, it is necessary to demonstrate that the synthesized compound targets folate and that its activity is correlated with folate receptor expression levels.

[0137] Six ovarian cancer cell lines with different folate α receptor expression levels were selected (sorted according to RNA sequencing results from the TCGA database), and their corresponding folate α receptor expression levels are shown in Table 2. Since transcriptional and protein levels are correlated but not necessarily completely correlated, Western blot experiments were used to verify the folate α receptor expression levels of each ovarian cancer cell line. Figure 1 The results are consistent with those in Table 2.

[0138] Based on the proliferation inhibition activity results in IGROV1 cells, FA-002S was one of the most active conjugates. Therefore, FA-002S was initially included in the evaluation of folate receptor expression dependence. To investigate the effect of the linker chain, FA-C8-S, with a similar molecular weight and a carbon chain linker chain, was selected as a control. Olaparib was used as a positive control. IC50 in various ovarian cancer cell lines... 50 The values ​​are shown in Table 2.

[0139] Table 2 shows the IC50 of the compounds against various ovarian cancer cell lines.50 value

[0140]

[0141] Analysis reveals that, generally speaking, the cell proliferation inhibitory activity of the compounds increases with increasing folate receptor expression levels in ovarian cancer cell lines. Although the activities of FA-C8-S and FA-002-S in cell lines with high folate receptor expression (IGROV1, SKOV3) are slightly lower or similar to those in cell lines with moderate expression (CAOV3, OVCAR3), this may be because CAOV3 and OVCAR3 are olaparib-sensitive (olaparib's IC50 value is lower than that of folate receptor 1). 50 (The values ​​are relatively small). In the cell lines ES2 and TOV-21G, which have low expression levels of the folate receptor, the activities of FA-C8-S and FA-002-S, which are also olaparib-sensitive, decreased significantly, proving that the activity of the compound depends on the expression level of the folate receptor.

[0142] In cell lines with similar sensitivity to olaparib, the compound's activity varied considerably due to differences in folate receptor expression levels. In SKOV3 and CAOV3 cells, olaparib IC50 showed higher activity levels. 50 With similar values, when conjugated with folic acid, FA-C8-S and FA-002-S showed approximately twice the activity in SKOV3 cells compared to CAOV3 cells; in OVCAR3 and ES2 cells, FA-C8-S showed approximately 26 times the activity in OVCAR3 cells compared to ES2 cells, and FA-002-S showed approximately 73 times the activity in OVCAR3 cells compared to ES2 cells. This indicates that the activity of the compounds is significantly affected by the expression level of the folate receptor in cells, suggesting the existence of a folate receptor-mediated active drug transmembrane transport mechanism, reflecting the ability of folic acid to carry cytotoxic drugs to target tumor tissues with specifically high folate receptor expression.

[0143] The foregoing examples are merely illustrative, used to explain some features of the method described in this invention. The appended claims are intended to claim the broadest possible scope, and the embodiments presented herein are demonstrated by the applicant's actual experimental results. Therefore, the applicant intends that the appended claims are not limited by the selection of examples illustrating the features of the invention. Some numerical ranges used in the claims also include sub-ranges within them, and variations within these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. A monosubstituted folic acid conjugate, characterized in that, The monosubstituted folic acid conjugate has the structure shown in formula (I): Among them, R1 is selected from C 1-8 Alkylene, -R2-O-R3-; R2 and R3 are each independently selected from C 1-6 Alkylene.

2. The monosubstituted folic acid conjugate according to claim 1, characterized in that, R1 is selected from C 1-6 Alkylene, -R2-O-R3-; R2 and R3 are each independently selected from C 1-4 Alkylene.

3. The monosubstituted folic acid conjugate according to claim 2, characterized in that, R1 is selected from ethylidene, n-propylidene, isopropylidene, n-butylidene, isobutylidene, tert-butylidene, n-pentylidene, isopentylidene, neopentylidene, hexylidene, -R2-O-R3-; R2 and R3 are each independently selected from ethylidene, n-propylidene, isopropylidene, n-butylidene, isobutylidene, and tert-butylidene.

4. The monosubstituted folic acid conjugate according to claim 3, characterized in that, The monosubstituted folic acid conjugate is selected from any of the following structures:

5. The method for preparing the monosubstituted folic acid conjugate according to any one of claims 1-4, characterized in that, The preparation steps include the following: S1. Compounds of formula (I-1) and (I-2) undergo an amide condensation reaction, followed by the removal of the Boc group to prepare compound (I-3); S2. Compound of formula (I) is prepared by amide condensation reaction of compound (I-3) and compound (I-4); The reaction formula for the preparation method is shown below:

6. The preparation method according to claim 5, characterized in that, The preparation steps include the following: S1. Preparation of compound (I-1); Compound (I-1) and compound (I-2) undergo amide condensation reaction under the action of condensing agent and base, and then the Boc group is removed to prepare compound (I-3); S2. Under a protective gas atmosphere, compounds of formula (I-3) and (I-4) undergo an amide condensation reaction in the dark with the aid of a condensing agent and a base to prepare compound (I).

7. The preparation method according to claim 6, characterized in that, The compound of formula (I-1) is prepared by reacting glutaric anhydride with the compound of formula (I-5) below; 8. Use of the monosubstituted folic acid conjugate according to any one of claims 1-4 in the preparation of PARP inhibitors or medicaments targeting folic acid receptors.

9. The use of the monosubstituted folic acid conjugate according to any one of claims 1-4 in the preparation of an antitumor medicament.

10. The application according to claim 9, characterized in that, The tumor is at least one of ovarian cancer, breast cancer, cervical cancer, bladder cancer, pleural mesothelioma, or non-small cell lung cancer.

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