Linker-conjugated compounds

Abstract

We provide drug linkers and linker-conjugated compounds. [Solution] The drug linker can provide suitable space, a flexible synthesis site, and a carboxylic acid moiety, and can be applied to link multiple pharmacophores to improve biological efficacy and drug similarity. In addition, the linker-conjugated compound has excellent water solubility.

Description

[Technical Field]

[0001] The present invention relates to drug linkers and linker-conjugated compounds in which the linker provides hydroxyl and carboxyl groups as pharmacophore bonding sites, and more particularly to drug linkers and linker-conjugated compounds utilizing carboxylic acid moieties. [Background technology]

[0002] In the field of drug development technology, small molecule drugs play a crucial role and can be used to design highly specific drugs for various diseases. However, problems such as drug resistance and side effects arise during the treatment process, making single-function drugs insufficient in terms of therapeutic effect. Therefore, new technologies are needed to solve these problems.

[0003] Linkers offer numerous advantages in drug therapy and can be used to combine multiple pharmacophores at the molecular level. Whether small molecules or monoclonal antibodies, they can simplify treatment by replacing multiple drugs with a single drug. This not only improves the convenience of treating patients but also effectively reduces potential drug interactions. Furthermore, linkers can improve drug properties, providing flexibility in drug performance and leading to better therapeutic outcomes.

[0004] On the other hand, many lead compounds with excellent pharmacological activity often have drawbacks in terms of physical properties and pharmacokinetics, such as low water solubility, low oral bioavailability, and rapid metabolism, which limits their direct clinical application. Carboxylic acid moieties influence pharmacological activity by generating intermediate acyl phosphates in various biosynthetic and metabolic pathways, and can improve solubility and bioavailability by forming carboxylate ions in solution. For these reasons, the introduction of carboxylic acids is widely applied in the field of drug design.

[0005] As described above, developing drug linkers and linker-conjugated compounds that utilize the carboxylic acid moiety to improve biological efficacy and drug similarity, while also possessing excellent water solubility, is a goal that engineers in this field urgently want to strengthen. [Overview of the initiative] [Problems that the invention aims to solve]

[0006] The present invention provides a drug linker and linker-conjugated compound in which the linker provides hydroxyl and carboxyl groups as pharmacophore bonding sites, and by utilizing the carboxylic acid portion, the biological effect and drug similarity can be effectively improved, and the linker has excellent water solubility. [Means for solving the problem]

[0007] The drug linker of the present invention has a chemical structure represented by the following formula (1).

[0008] [ka]

[0009] In equation (1), n is an integer between 1 and 10. X, Y, and Z are, independently, halogen, carbon, oxygen, sulfur, and NR. 5 , or SiR5 R 6 and, R 1 is hydrogen, C(=O)R 5 , substituted or unsubstituted C 1~8 alkyl, substituted or unsubstituted C 2~8 alkenyl, substituted or unsubstituted C 5~10 aryl, substituted or unsubstituted C 3~8 cycloalkyl, substituted or unsubstituted C 5~10 heteroaryl, substituted or unsubstituted C 5~10 lactone, substituted or unsubstituted cyclohexenone, substituted or unsubstituted quinone derivative, or a substituted or unsubstituted heterocyclic ring having one or more heteroatoms selected from N, O, and S formed together with the nitrogen atom, R 2 is hydrogen, substituted or unsubstituted C 1~8 alkyl, substituted or unsubstituted C 2~8 alkenyl, substituted or unsubstituted C 5~10 aryl, substituted or unsubstituted C 3~8 cycloalkyl, substituted or unsubstituted C 5~10 heteroaryl, substituted or unsubstituted C 5~10 lactone, or a substituted or unsubstituted heterocyclic ring having one or more heteroatoms selected from N, O, and S formed together with the nitrogen atom, R 3 , R 5 , and R 6 are each independently hydrogen, substituted or unsubstituted C 1~8 alkyl, or substituted or unsubstituted C 5~10 aryl, R 4 is hydrogen, substituted or unsubstituted C 1~8 alkyl, substituted or unsubstituted C 5~10 aryl, substituted or unsubstituted C 3~8 cycloalkyl, substituted or unsubstituted C 5~10 heteroaryl, substituted or unsubstituted C 5~10A lactone, or a substituted or unsubstituted heterocycle having one or more heteroatoms selected from N, O, and S, formed together with nitrogen.

[0010] The linker-conjugated compound of the present invention has a chemical structure represented by the following formula (1-1).

[0011] [ka]

[0012] R 2 C is hydrogen, substituted or unsubstituted. 1~8 Alkyl, substituted or unsubstituted C 2~8 Alkenyl, substituted or unsubstituted C 5~10 Aryl, substituted, or unsubstituted C 3~8 Cycloalkyl, substituted or unsubstituted C 5~10 Heteroaryl, substituted or unsubstituted C 5~10 A lactone, or a substituted or unsubstituted heterocycle having one or more heteroatoms selected from N, O, and S formed together with nitrogen, R 4 C is hydrogen, substituted or unsubstituted. 1~8 Alkyl, substituted or unsubstituted C 5~10 Aryl, substituted, or unsubstituted C 3~8 Cycloalkyl, substituted or unsubstituted C 5~10 Heteroaryl, substituted or unsubstituted C 5~10 A lactone, or a substituted or unsubstituted heterocycle having one or more heteroatoms selected from N, O, and S, formed together with nitrogen.

[0013] The linker-conjugated compound of the present invention has a chemical structure represented by any of the following formulas (1-2) to (1-8).

[0014] [ka] [Effects of the Invention]

[0015] As described above, the present invention provides drug linkers and linker-conjugated compounds. The drug linkers of the present invention can provide suitable space, a flexible synthesis site, and a carboxylic acid moiety, and can be applied to link multiple pharmacophores to improve biological effects and drug similarity. Furthermore, the linker-conjugated compounds have excellent water solubility. [Modes for carrying out the invention]

[0016] Embodiments of the present invention will be described in detail below. However, these embodiments are exemplary, and the disclosure of the present invention is not limited thereto.

[0017] In this text, the range indicated as "from one number to another" is a general representation to avoid listing every single number within that range in the specification. Therefore, a description of a particular numerical range includes any number within that range and a relatively smaller numerical range limited by any number within that range, as if those arbitrary numbers and smaller numerical ranges were explicitly stated in the specification.

[0018] The drug linker of the present invention has a chemical structure represented by the following formula (1).

[0019] [ka]

[0020] In equation (1), n is an integer between 1 and 10. X, Y, and Z are, independently, halogen, carbon, oxygen, sulfur, and NR. 5 , or SiR 5 R 6 And, R 1 is hydrogen, C(=O)R 5 , substitution or non-substitution of C 1~8Alkyl, substituted or unsubstituted C 2~8 Alkenyl, substituted or unsubstituted C 5~10 Aryl, substituted, or unsubstituted C 3~8 Cycloalkyl, substituted or unsubstituted C 5~10 Heteroaryl, substituted or unsubstituted C 5~10 A lactone, a substituted or unsubstituted cyclohexenone, a substituted or unsubstituted quinone derivative, or a substituted or unsubstituted heterocycle having one or more heteroatoms selected from N, O, and S formed together with nitrogen, R 2 C is hydrogen, substituted or unsubstituted. 1~8 Alkyl, substituted or unsubstituted C 2~8 Alkenyl, substituted or unsubstituted C 5~10 Aryl, substituted, or unsubstituted C 3~8 Cycloalkyl, substituted or unsubstituted C 5~10 Heteroaryl, substituted or unsubstituted C 5~10 A lactone, or a substituted or unsubstituted heterocycle having one or more heteroatoms selected from N, O, and S formed together with nitrogen, R 3 , R 5 , and R 6 These are, independently, hydrogen, substituted or unsubstituted C 1~8 Alkyl, or substituted or unsubstituted C 5~10 It is Ariel, R 4 C is hydrogen, substituted or unsubstituted. 1~8 Alkyl, substituted or unsubstituted C 5~10 Aryl, substituted, or unsubstituted C 3~8 Cycloalkyl, substituted or unsubstituted C 5~10 Heteroaryl, substituted or unsubstituted C 5~10 A lactone, or a substituted or unsubstituted heterocycle having one or more heteroatoms selected from N, O, and S, formed together with nitrogen.

[0021] The linker-conjugated compound of the present invention has a chemical structure represented by the following formula (1-1).

[0022] [ka]

[0023] R 2 C is hydrogen, substituted or unsubstituted. 1~8 Alkyl, substituted or unsubstituted C 2~8 Alkenyl, substituted or unsubstituted C 5~10 Aryl, substituted, or unsubstituted C 3~8 Cycloalkyl, substituted or unsubstituted C 5~10 Heteroaryl, substituted or unsubstituted C 5~10 A lactone, or a substituted or unsubstituted heterocycle having one or more heteroatoms selected from N, O, and S formed together with nitrogen, R 4 C is hydrogen, substituted or unsubstituted. 1~8 Alkyl, substituted or unsubstituted C 5~10 Aryl, substituted, or unsubstituted C 3~8 Cycloalkyl, substituted or unsubstituted C 5~10 Heteroaryl, substituted or unsubstituted C 5~10 A lactone, or a substituted or unsubstituted heterocycle having one or more heteroatoms selected from N, O, and S, formed together with nitrogen.

[0024] The linker-conjugated compound of the present invention has a chemical structure represented by any of the following formulas (1-2) to (1-8).

[0025] [ka]

[0026] Preparation method

[0027] All reactions in this invention were carried out under a nitrogen atmosphere using commercially available reagents, without any additional purification. NMR analysis was performed using a Varian Unity 400 MHz spectrometer, with tetramethylsilane (TMS, Merck, Darmstadt, Germany) as the internal standard and chloroform-d3 and methanol-d4 as solvents. Chemical shifts are expressed in ppm. The splitting patterns are as follows: s=singlet; d=doublet; t=triplet; q=quartet; dd=double doublet; m=multiplet. Thin-layer chromatography (TLC) was performed using an E. Merck Art.5554 Kieselgel 60 GF254 (Merck, Darmstadt, Germany). The compound spots were examined using ultraviolet indicators irradiated at 254 nm and 366 nm. Column chromatography was performed using E. Merck Art.7734 Kierselgel 60 GF254 (70-400 mesh, Merck, Darmstadt, Germany). Mass spectra were recorded using PuriFlash (registered trademark) MS (Interchim, Montlucon, France) liquid chromatography electrospray ionization mass spectrometer.

[0028] Abbreviations: Vol. (volume of solvent (mL) / limiting reaction substance (g)); THF (tetrahydrofuran); LDA (lithium diisopropylamide); EA (ethyl acetate); TMEDA (tetramethylethylenediamine); LHMDS (lithium bis(trimethylsilyl)amide); n-BuLi (n-butyllithium); DMF (dimethylformamide); DCM (dichloromethane); PPh3 (triphenylphosphine); NHS (N-hydroxysuccinimide); EDCI (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide); IPA (isopropyl alcohol).

[0029] Linker synthesis

[0030] The synthesis of linker is represented by the following reaction equation (1).

[0031] [ka]

[0032] The linker synthesis began with 2,6-dimethylphenyl ester propionate (compound 1). At -78°C, compound 1 (1.0 equivalent eq) and THF (40 Vol.) were slowly added to a stirred solution of THF (130 Vol.) and LDA (2 M, 1.1 eq in THF), and the mixture was stirred at -78°C for 30 minutes. 2-(phenylthio)acetaldehyde (compound 2) (1.1 eq) and THF (40 Vol.) were slowly added to the mixture, and the mixture was stirred at -78°C for another 30 minutes. After the reaction was complete (monitored by TLC), the mixture was quenched with 1 M HCl and extracted with EA (2 × 400 Vol.). The organic layer was collected, dried over MgSO4, and concentrated under vacuum. Column chromatography (SiO2, 1:9EA / hexane) was used to obtain a yellow, oily compound, 2,6-dimethylphenyl 3-hydroxy-2-methyl-4-(phenylthio)butyrate (compound 3). The yield was 37%. 1 H NMR(400MHz,CDCl3)δ7.45-7.40(m,2H), 7.34-7.21(m,3H), 7.06(s,3H), 3.96(ddd,J=8.0,6.4, 4.4Hz,1H), 3.30(dd,J=13.7,4.4Hz,1H), 3.14-3.06(m,2H), 2.14(s,6H), 1.43(d,J=7.3Hz,3H). Chemical formula calculation: C 19 H 22 O3S: m / z 330.1, measured value 69.0 (M+K + ).

[0033] Next, Compound 3 (1.1 eq) and TMEDA (1.1 eq) were stirred in THF (125 Vol.) at -78°C. n-BuLi (1.6 M, 1.2 eq) was added to the mixture, and the mixture was stirred at -78°C for 1 hour. A solution of (2E,6E)-8-bromo-3,7-dimethylocta-2,6-dien-1-yl acetate (Compound 4) (1.0 eq) in THF (25 Vol.) was added to the mixture, and the mixture was further stirred at -78°C for 1 hour. The mixture was quenched with saturated aqueous NH4Cl solution, and extracted with EA (2×250 Vol.). The organic layer was dried over MgSO4 and concentrated in vacuo. A yellow oily intermediate was obtained, which could be used in the next synthetic process without purification. The above intermediate dissolved in diethyl ether (20 Vol.) was added to a solution of Li (6.5 eq) in diethylamine (5.0 Vol.) and liquid NH3 (50 Vol.), and the mixture was stirred at -60°C for 30 minutes. After the reaction was completed (monitored by TLC), the mixture was diluted with diethyl ether (50 Vol.), and saturated aqueous NH4Cl solution (130 Vol.) was slowly added. The organic layer was collected, dried over MgSO4 and concentrated in vacuo. Column chromatography (SiO2, 7:3 EA / hexane) gave (6E,10E)-3,12-dihydroxy-2,6,10-trimethyldodeca-6,10-dienoic acid (Compound 5) as a colorless oil. The yield was 39%. 1 H NMR (600 MHz, CD3OD) δ 5.39-5.33 (m, 1H), 5.21-5.15 (m, 1H), 4.08 (d, J=6.8 Hz, 2H), 3.76-3.67 (m, 1H), 2.55-2.38 (m, 1H), 2.22-2.10 (m, 3H), 2.09-2.00 (m, 3H), 1.67 (s, 3H), 1.61 (s, 3H), 1.50-1.42 (m, 1H), 1.19-1.10 (m, 3H). Calculated chemical formula: C 15 H 26 O4: m / z 270.2, found 291.2 (M+Na-2H - ), 305.2 (M+Cl - ).

[0034] Esterification of carboxyl group of linker and substitution of primary alcohol

[0035] The esterification reaction of the linker is represented by the following reaction formula (2).

[0036]

Chemical formula

[0037] Substitution of carboxyl group: At room temperature, ethyl iodide (1.1 eq) was added to a mixture of compound 5 (1.0 eq), K2CO3 (3.0 eq) and DMF (20 Vol.), and the mixture was stirred overnight. After the reaction was completed (monitored by TLC), the mixture was quenched with water and extracted with EA (2×130 Vol.). The organic layers were collected, dried over MgSO4, and concentrated in vacuo. Column chromatography (SiO2, 5:5 EA / hexane) afforded colorless oily ethyl (6E,10E)-3,12-dihydroxy-2,6,10-trimethyldodeca-6,10-dienoate (compound 6). The yield was 92%. 1 1H NMR (400 MHz, CDCl3) δ 5.44-5.35 (m, 1H), 5.19-5.12 (m, 1H), 4.25-4.07 (m, 4H), 3.90-3.57 (m, 1H), 2.67 (s, 1H), 2.56-2.45 (m, 2H), 2.21-2.00 (m, 6H), 1.66 (s, 3H), 1.60 (s, 3H), 1.54-1.44 (m, 2H), 1.27 (t, J=7.1 Hz, 3H), 1.22-1.15 (m, 3H). Calculated chemical formula: C 17 H 30 O4: m / z 298.2, found 321.2 (M+Na + ).

[0038] The substitution reaction of the linker is represented by the following reaction formula (3).

[0039]

Chemical formula

[0040] Substitution of primary alcohols: Triphenylmethyl chloride (1.1 eq), 4-dimethylaminopyridine (DMAP, 1.1 eq), triethylamine (1.1 eq), and compound 5 (1 eq) were dissolved in DCM (35 vol.) and stirred overnight at 0°C to room temperature. After the reaction was complete (monitored by TLC), the solvent was evaporated under vacuum. Column chromatography (SiO2, 1:9 EA / hexane) was used to obtain colorless oily (6E,10E)-3-hydroxy-2,6,10-trimethyl-12-(trityloxy)dodeca-6,10-dienoic acid (compound 7). The yield was 55%. 1 H NMR (600MHz, CDCl3) δ7.48-7.44(m,6H), 7.32-7.27(m,6H), 7.24-7.20(m,3H), 5.47-5.40(m,1H), 5.22-5.16(m,1H), 3.71-3.66(m ,1H), 3.61(d,J=6.4Hz,2H), 2.59-2.51(m,1H), 2.22-2.00(m,6H), 1.62(s,3H), 1.61-1.52(m,2H), 1.46(s,3H), 1.24-1.16(m,3H). Chemical formula calculation: C 34 H 40 O4: m / z 512.3, measured value 511.2 (MH) - ).

[0041] Conjugation of active compounds and linkers

[0042] The synthesis of linker-conjugated compounds is represented by the following reaction equation (4).

[0043] [ka]

[0044] To synthesize linker-conjugated compound 11, triphenylphosphine (1.1 eq), carbon tetrabromide (1.1 eq), and compound 6 (1 eq) were dissolved in DCM (35 Vol.) and stirred at 0°C. After 2 hours, the solvent was filtered and evaporated under vacuum. Next, LHMDS (1 M, in THF, 2 eq) was slowly added to the mixture containing THF (15 Vol.). The mixture was stirred at -78°C for 2 hours. Compound 8 (1.2 eq) was added to the mixture and stirred overnight at room temperature. After the reaction was complete (monitored by TLC), the mixture was quenched with saturated NH4Cl aqueous solution and extracted with EA (3 × 90 Vol.). The organic layer was collected, dried over MgSO4, and concentrated under vacuum. Column chromatography (SiO2, 1:9EA / hexane) yielded a pale yellow, oily ethyl (6E,10E)-3-hydroxy-2,6,10-trimethyl-12-((1S,6R)-3,4,5,5-tetramethoxy-6-methyl-2-oxocyclohex-3-en-1-yl)dodeca-6,10-dienoate (compound 9). The yield was 30%. 1 H NMR(600MHz,CDCl3)δ5.15(t,J=6.9Hz,1H), 5.07(t,J=6.9Hz,1H), 4.19-4.14( m,2H), 4.12(s,3H), 3.67(s,3H), 3.64(s,1H), 3.31(s,3H), 3.26(s,3H), 2.66-2 .55(m,2H), 2.54-2.46(m,1H), 2.38-2.28(m,2H), 2.11-1.96(m,6H), 1.63(s,3H) ), 1.59(s,3H), 1.27(t,J=7.1Hz,3H), 1.21-1.17(m,3H), 0.98(d,J=6.7Hz,3H). Chemical formula calculation: C 28 H 46 O8: m / z 510.3, measured value 533.4 (M+Na + ).

[0045] At -70°C, lithium tri-sec-butylbohydride (1 M, in THF, 8.0 eq) was added dropwise to a solution of compound 9 (1.0 eq) in THF (200 Vol.) and stirred for 4 hours. The reaction was diluted with DCM (500 Vol.), silica gel was added, and the mixture was stirred for 10 minutes. After the reaction was complete (monitored by TLC), the silica gel was filtered, and the residue was concentrated under vacuum. Column chromatography (SiO2, 1:9EA / hexane) yielded a pale yellow oily ethyl (6E,10E)-3-hydroxy-12-((1S,2S,6R)-2-hydroxy-3,4-dimethoxy-6-methyl-5-oxocyclohex-3-en-1-yl)-2,6,10-trimethyldodeca-6,10-dienoate (compound 10). The yield was 30%. 1 H NMR(400MHz,CDCl3)δ5.11-5.16(m,2H), 4.26-4.31(m,1H), 4.17(q,J=7.2Hz,2H), 4.12(s,3H), 3.67(s,3H), 3.62-3.64(m,1 H), 2.50-2.58(m,2H), 2.05-2.31(m,8H), 1.81-1.87(m,1H), 1.65(s,3H), 1.62-1.64(m,2H), 1.60(s,3H), 1.20-1.28(m,9H). Chemical formula calculation value: C 26 H 42 O7: m / z 466.3, measured value 489.4 (M+Na + ).

[0046] To a solution of compound 10 (1.0 eq) in MeOH (40 vol.), NaOH (10 wt% aqueous solution, 10 vol.) was added dropwise, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete (monitored by TLC), the reaction mixture was washed with DCM (2 × 200 vol.). The aqueous layer was acidified with 1 M HCl and extracted with DCM (2 × 200 vol.). The organic layer was collected, dried over MgSO4, and concentrated under vacuum to obtain a pale yellow oily compound 11. The yield was 61%. 1H NMR(400MHz,CDCl3)δ5.12-5.19(m,2H), 4.33(d,J=6.4Hz,3H), 4.08(s,3H), 3.59(s,3H), 3.44-3.48(m,1H), 2.37-2.46(m,1H) , 2.24-2.36(m,2H), 1.95-2.21(m,7H), 1.76-1.87(m,1H), 1.61(d,J=8.8Hz,6H), 1.23(d,J=7.2Hz,3H), 1.18(d,J=7.2Hz,3H). Chemical formula calculation: C 24 H 38 O7: m / z 438.3, measured value 437.4 (MH) - ).

[0047] Conjugation of amino acids and linkers

[0048] The synthesis of linker-conjugated amino acid derivatives is represented by the following reaction equation (5).

[0049] [ka]

[0050] At room temperature, EDCI (2.0 eq) was added to a stirred solution of compound 7 (1.0 eq) and NHS (2.0 eq) in DMF (20 Vol.), and the mixture was stirred at room temperature for 2 hours. The mixture was extracted with EA (3 × 130 Vol.). The organic layer was dried over MgSO4 and concentrated under reduced pressure. The crude compound was dissolved in DCM (20 Vol.), glycine (2.0 eq) was added, and the mixture was stirred overnight at room temperature. The mixture was extracted with DCM (2 × 100 Vol.). The organic layer was dried over MgSO4 and concentrated under reduced pressure. By column chromatography (SiO2, 100 / 0~50 / 50EA / MeOH), a pale yellow oily compound 12 (23%) was obtained. 1H NMR (600MHz, CDCl3) δ7.48-7.44(m,6H), 7.31-7.27(m,6H), 7.24-7.20(m,3H), 5.20-5.14(m,1 H), 5.14-5.07(m,1H), 5.01(dt,J=2.1,1.6Hz,1H), 4.98(dt,J=2.1,1.6Hz,1H), 4.94(dt,J=2.3 ,1.2Hz,1H), 4.92(dt,J=2.3,1.2Hz,1H), 3.86-3.80(m,1H), 3.60(d,J=6.3Hz,2H), 2.73-2.67 (m,1H), 2.12-1.99(m,6H), 1.76-1.69(m,2H), 1.46(s,3H), 1.43(s,3H), 1.30(d,J=7.1Hz,3H). Chemical formula calculation: C 38 H 46 N2O6: m / z 626.3, measured value 668.4 (M+ACN+H + ).

[0051] The following lists methods for testing various properties of the drug linker and linker-conjugated compound of the present invention.

[0052] Water solubility test

[0053] [Table 1]

[0054] Method for preparing standard solution calibration curves

[0055] 5 mg of the standard test compound was weighed and dissolved in 5 mL of IPA to prepare a storage solution (1 mg / mL). Further dilution with redistilled water was used to prepare calibration curves for standard solutions at concentrations of 1000, 100, 50, 25, 12.5, 10, 5, 2.5, 1.25, 1, 0.5, 0.25, and 0.125 μg / mL. HPLC analysis was performed using 60 μL of each standard solution. The peak areas were integrated to create a calibration curve, and the accuracy of the analytical method was evaluated using the correlation coefficient (R-squared).

[0056] Water solubility test method

[0057] 1 mL of redistilled water was weighed into a microcentrifuge tube, and the excess amount of the test compound was dissolved. Vortexing was performed for 5 minutes, followed by sonication for 20 minutes, to ensure that undissolved compound remained. After centrifugation, the supernatant was filtered through a 0.22 μm PTEF membrane. The residue was analyzed by HPLC, and solubility was calculated from the peak area.

[0058] Cytotoxicity screening

[0059] cell culture

[0060] NOZ cells (human gallbladder cancer cells) were obtained from the JCRB (Japanese Collection of Research Bioresources) cell bank (JCRB, Japan). The cells were cultured in a medium containing 90% William's E medium (Gibco, USA), 10% fetal bovine serum (FBS) (Gibco, USA), and 1% antibiotics (1x penicillin, streptomycin, glutamine) (Gibco, USA).

[0061] HuCCT1 cells (human cholangiocarcinoma cells) were obtained from iCell Bioscience (iCell, China). The cells were cultured in a medium containing 90% RPMI1640 medium (Gibco, USA), 10% fetal bovine serum (FBS) (Gibco, USA), and 1% antibiotics (1x penicillin, streptomycin, glutamine) (Gibco, USA).

[0062] HuH28 cells (human liver, cholangiocarcinoma cells) were obtained from the JCRB Cell Bank (JCRB, Japan). The cells were cultured in a medium containing 90% RPMI1640 medium (Gibco, USA), 10% fetal bovine serum (FBS) (Gibco, USA), and 1% antibiotics (1x penicillin, streptomycin, glutamine) (Gibco, USA).

[0063] AML12 cells (α mouse liver 12) were obtained from the American Type Culture Collection (ATCC) (ATCC, USA). The cells were cultured in DMEM (Dulbecco's Modified Eagle Medium):F12 medium (Gibco, USA), containing 10% fetal bovine serum (FBS) (Gibco, USA), 10 μg / mL insulin, 5.5 μg / mL transferrin, 5 ng / mL selenium, 40 ng / mL dexamethasone (Gibco, USA), and 1% antibiotics (1× penicillin, streptomycin, glutamine) (Gibco, USA).

[0064] AsPC-1 cells (human pancreatic adenocarcinoma ascites metastasis cells) were obtained from the Bioresource Collection and Research Center (BCRC) (BCRC, Taiwan). The cells were cultured in RPMI1640 medium (Gibco, USA) containing 10% FBS (Gibco, USA) and 1% antibiotics (1x penicillin, streptomycin, glutamine) (Gibco, USA).

[0065] PANC-1 cells (human pancreatic epithelial-like carcinoma) were obtained from the American Type Culture Collection (ATCC, USA). The cells were cultured in DMEM medium (Gibco, USA) containing 10% FBS (Gibco, USA) and 1% antibiotics (1x penicillin, streptomycin, glutamine) (Gibco, USA).

[0066] BxPC-3 cells (human pancreatic adenocarcinoma cells) were obtained from the Bioresource Collection and Research Center (BCRC, Taiwan). The cells were cultured in RPMI1640 medium (Gibco, USA) containing 10% FBS (Gibco, USA) and 1% antibiotics (1x penicillin, streptomycin, glutamine) (Gibco, USA).

[0067] A549 cells (human non-small cell lung cancer cells) were obtained from the Bioresource Collection and Research Center (BCRC, Taiwan). The cells were cultured in F12K medium (Gibco, USA) containing 10% FBS (Gibco, USA) and 1% antibiotics (1x penicillin, streptomycin, glutamine) (Gibco, USA).

[0068] MCF7 cells (human breast cancer cells) were obtained from the Bioresource Collection and Research Center (BCRC, Taiwan). The cells were cultured in Eagle Minimum Essential Medium (Gibco, USA) containing 2 mM L-glutamic acid, 1.5 mg / mL sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate (Gibco, USA), 10% fetal bovine serum (FBS) (Gibco, USA), and 1% antibiotics (1× penicillin, streptomycin, glutamine) (Gibco, USA).

[0069] HCT-15 cells (colorectal adenocarcinoma cells) were obtained from the Bioresource Collection and Research Center (BCRC, Taiwan). The cells were cultured in RPMI1640 medium (Gibco, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA) and 1% antibiotics (1x penicillin, streptomycin, glutamine) (Gibco, USA).

[0070] Caco-2 cells (colorectal adenocarcinoma cells) were obtained from the Bioresource Collection and Research Center (Elabscience, USA). The cells were cultured in Eagle's Minimum Essential Medium (Gibco, USA) containing 2 mM L-glutamine, 1.5 mg / mL sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate (Gibco, USA), 20% fetal bovine serum (FBS) (Gibco, USA), and 1% antibiotics (1× penicillin, streptomycin, glutamine) (Gibco, USA).

[0071] Vero E6 cells (African green monkey (cercopithecus aethiops) renal epithelial cells) were obtained from the American Type Culture Collection (ATCC, USA). The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, USA) containing 10% FBS (Gibco, USA), 100 U / mL penicillin, and 100 μg / mL streptomycin (Gibco, USA).

[0072] Cell viability evaluation

[0073] Cell viability was evaluated using the PrestoBlue assay (Invitrogen, USA). Cells were seeded in 96-well plates and cultured with the test compound according to the manufacturer's protocol. After 48 hours of exposure to the test compound, the medium was removed and the plates were washed with PBS. Then, 10 μL of PrestoBlue reagent containing 90 μL of medium was added to the cells, and the cell plates were cultured in the dark at 37°C for 120 minutes. Absorbance was measured at the experimental wavelength of 570 nm and the standardized wavelength of 600 nm using a SpectraMax iD3 microplate reader. The results were corrected by subtracting blank values, and cell viability was expressed as a percentage relative to the control, calculated using the following formula: Cell viability (%) = [(Absorbance of test well at 570 nm - Absorbance of medium-only well at 570 nm - Absorbance of test well at 600 nm + Absorbance of medium-only well at 600 nm) / (Absorbance of control well at 570 nm - Absorbance of medium-only well at 570 nm - Absorbance of control well at 600 nm + Absorbance of medium-only well at 600 nm)] × 100. The data are displayed as the mean ± standard deviation shown by GraphPad Prism.

[0074] The following lists the test results for various properties of the drug linker and linker-conjugated compound of the present invention.

[0075] Solubilizer action test

[0076] At a physiological pH of 7.4, the ionization of carboxyl groups enhances their ability to form hydrogen bonds with adjacent water molecules, thereby improving overall water solubility. To verify that the drug linker of the present invention can effectively improve the water solubility of compounds, water solubility tests were conducted (Table 2). Both the drug linker of the present invention and farnesol contain a hydroxyl group at the 1' position and 15 carbon atoms in their linear structure. However, compared to farnesol, the drug linker of the present invention, by adding a hydroxyl group at the 10' position and a carboxyl group at the 12' position, shows a dramatic increase in water solubility of nearly nine times, from 87.4 μg / mL to 780.1 μg / mL. This result demonstrates that compounds designed using the drug linker of the present invention can effectively improve water solubility.

[0077] [Table 2]

[0078] Pharmacophorelinker action test

[0079] Bifunctional / multifunctional drugs achieve superior efficacy compared to their use alone by linking two or more pharmacophores via a linker. In this technique, the design of the linker is crucial, as it must maintain the activity of the original drug while avoiding the introduction of additional toxicity. Furthermore, a good linker must be flexibly synthesized to readily bind to pharmacophores.

[0080] The drug linker of the present invention has a chemical structure represented by the following formula (1).

[0081] [ka]

[0082] The linker of the present invention (n=2; X,Y,Z=O;R 1 ,R 2 ,R 4 =H;R 3Taking CH3 as an example, this linker has significant advantages in terms of the elastic properties of the composite. 1 - The alcohol group on X is a primary alcohol and readily undergoes substitution reactions (reaction equation (3)). Furthermore, R 1 -X and R 2 When the alcohol group of -Y is converted to a halide by the Appel reaction, this halide intermediate readily reacts with other nucleophiles, such as alcohols, organolithium, enols, amines, and thiols (first step of reaction equation (4)). At the same time, R 4 The carboxyl group in -Z exhibits very high reactivity and can undergo esterification under acid / base catalysis (reaction formula (2)), or react with small molecules, amino acids, or amino moieties on protein residues via a crosslinking agent to form amides (reaction formula (5)). These diverse binding methods greatly expand its range of applications. Structural flexibility is also a key point for linkers, allowing the entire drug molecule to move freely after binding to the target. A common indicator of molecular flexibility is the number of rotatable bonds (NRot), with higher values ​​indicating greater flexibility. As shown in Table 3 below, the linker of the present invention has a relatively high NRot value, indicating great potential as a linker for bifunctional / multifunctional drugs. On the other hand, non-cytotoxicity is a fundamental requirement for linkers, and cytotoxicity tests were conducted using various cell lines. Cell experiments demonstrated that the linker of the present invention does not exhibit cytotoxicity to either normal cells or cancer cells (Table 4).

[0083] [Table 3]

[0084] [Table 4]

[0085] As described above, the present invention provides drug linkers and linker-conjugated compounds. The drug linkers of the present invention can provide suitable space, a flexible synthesis site, and a carboxylic acid moiety, and can be applied to link multiple pharmacophores to improve biological effects and drug similarity. Furthermore, the linker-conjugated compounds have excellent water solubility. The drug linkers of the present invention can be used to improve solubility and are non-cytotoxic.

Claims

1. A drug linker having a chemical structure represented by the following formula (1), 【Chemistry 1】 In equation (1), n is an integer between 1 and 10. X, Y, and Z are, independently, halogen, carbon, oxygen, sulfur, and NR. ５ , or SiR ５ R ６ And, R １ is hydrogen, C(=O)R ５ , substituted or unsubstituted C １～８ alkyl, substituted or unsubstituted C ２～８ alkenyl, substituted or unsubstituted C ５～１０ aryl, substituted or unsubstituted C ３～８ cycloalkyl, substituted or unsubstituted C ５～１０ heteroaryl, substituted or unsubstituted C ５～１０ lactone, substituted or unsubstituted cyclohexenone, a substituted or unsubstituted quinone derivative, or a substituted or unsubstituted heterocycle having one or more heteroatoms selected from N, O and S formed together with nitrogen, R ２ However, hydrogen, substituted or unsubstituted C １～８ Alkyl, substituted, or unsubstituted C ２～８ Alkenyl, substituted or unsubstituted C ５～１０ Aryl, substituted, or unsubstituted C ３～８ Cycloalkyl, substituted or unsubstituted C ５～１０ Heteroaryl, substituted or unsubstituted C ５～１０ A lactone, or a substituted or unsubstituted heterocycle having one or more heteroatoms selected from N, O, and S formed together with nitrogen, R ３ , R ５ , and R ６ However, each is independent of hydrogen, substituted or unsubstituted C １～８ Alkyl, or substituted or unsubstituted C ５～１０ It is Ariel, R ４ However, hydrogen, substituted or unsubstituted C １～８ Alkyl, substituted, or unsubstituted C ５～１０ Aryl, substituted, or unsubstituted C ３～８ Cycloalkyl, substituted or unsubstituted C ５～１０ Heteroaryl, substituted or unsubstituted C ５～１０ A drug linker that is a lactone, or a substituted or unsubstituted heterocycle having one or more heteroatoms selected from N, O, and S formed together with nitrogen.

2. A linker-conjugated compound having the chemical structure represented by the following formula (1-1), 【Chemistry 2】 R ２ However, hydrogen, substituted or unsubstituted C １～８ Alkyl, substituted, or unsubstituted C ２～８ Alkenyl, substituted or unsubstituted C ５～１０ Aryl, substituted, or unsubstituted C ３～８ Cycloalkyl, substituted or unsubstituted C ５～１０ Heteroaryl, substituted or unsubstituted C ５～１０ A lactone, or a substituted or unsubstituted heterocycle having one or more heteroatoms selected from N, O, and S formed together with nitrogen, R ４ However, hydrogen, substituted or unsubstituted C １～８ Alkyl, substituted, or unsubstituted C ５～１０ Aryl, substituted, or unsubstituted C ３～８ Cycloalkyl, substituted or unsubstituted C ５～１０ Heteroaryl, substituted or unsubstituted C ５～１０ A linker-conjugated compound which is a lactone, or a substituted or unsubstituted heterocycle having one or more heteroatoms selected from N, O, and S formed together with nitrogen.

3. A linker-conjugated compound having a chemical structure represented by any of the following formulas (1-2) to (1-8). 【Transformation 3】

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