Drug Linkers and Linker-Conjugate Compounds

Abstract

Drug linkers and linker conjugate compounds are provided. The drug linker provides adequate space, flexible synthetic sites, and a carboxylic acid moiety, and can be applied to link multiple pharmacophores to improve biological efficacy and drug-likeness. The linker-conjugated compounds also have excellent water solubility.

Description

[Technical Field]

[0001] The present invention relates to drug-linkers and linker-conjugate compounds in which the linker provides hydroxyl and carboxyl moieties as attachment points to the pharmacophore, and in particular to drug-linkers and linker-conjugate compounds utilizing carboxylic acid moieties. [Background technology]

[0002] In the field of drug development technology, small molecule drugs play an important 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 the therapeutic effect of single-function drugs insufficient. Therefore, new technologies are needed to solve these problems.

[0003] Linkers offer many advantages in drug therapy and can be used to combine multiple pharmacophores at the molecular level. Replacing multiple drugs with a single drug, whether they are small molecules or monoclonal antibodies, can simplify treatment. This not only improves patient convenience but also effectively reduces potential drug interactions. Linkers can also improve drug properties, providing flexibility in tailoring drug performance and achieving better therapeutic effects.

[0004] However, many lead compounds with excellent pharmacological activity often have deficiencies in their physical properties and pharmacokinetics, such as poor water solubility, low oral bioavailability, and rapid metabolism, limiting their direct clinical application. Carboxylic acid moieties affect pharmacological activity by generating intermediate acylphosphates 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 has been widely applied in the field of drug design.

[0005] As described above, developing drug linkers and linker-conjugated compounds that utilize carboxylic acid moieties to improve biological efficacy and drug mimicry, and have excellent water solubility, has become an urgent goal for those skilled in the art. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides a drug linker and a linker-conjugated compound in which the linker provides hydroxyl and carboxyl moieties as pharmacophore attachment points, and the carboxylic acid moiety can effectively improve biological effects and drug-likeness, while also having 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 formula (1), n is an integer from 1 to 10, X, Y, and Z each independently represent a halogen, carbon, oxygen, sulfur, or 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 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 the nitrogen; 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 a lactone or a substituted or unsubstituted heterocycle having one or more heteroatoms selected from N, O, and S formed together with the nitrogen; 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 is 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 5~10It is a lactone or a substituted or unsubstituted heterocycle having one or more heteroatoms selected from N, O, and S formed together with the 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 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 a lactone or a substituted or unsubstituted heterocycle having one or more heteroatoms selected from N, O, and S formed together with the nitrogen; 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 5~10 It is a lactone or a substituted or unsubstituted heterocycle having one or more heteroatoms selected from N, O, and S formed together with the nitrogen.

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

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

[0015] As described above, the present invention provides a drug linker and a linker-conjugated compound. The drug linker of the present invention can provide suitable space, flexible synthetic sites, and a carboxylic acid moiety, and can be used to link multiple pharmacophores to improve biological effects and drug similarity. In addition, the linker-conjugated compound has excellent water solubility. DETAILED DESCRIPTION OF THE INVENTION

[0016] Hereinafter, embodiments of the present invention will be described in detail. However, these embodiments are merely examples, and the disclosure of the present invention is not limited to these.

[0017] In this specification, ranges expressed as "one value to another value" are a summary method of avoiding the need to recite every single value within the range. Thus, the recitation of a particular numerical range includes any value within that range and smaller numerical ranges defined by any value within that range, as if those values ​​and smaller numerical ranges were expressly written 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 formula (1), n is an integer from 1 to 10, X, Y, and Z each independently represent a halogen, carbon, oxygen, sulfur, or NR 5 , or SiR 5 R 6 and R 1 is hydrogen, C(=O)R 5 , substituted or unsubstituted 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 the nitrogen; 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 a lactone or a substituted or unsubstituted heterocycle having one or more heteroatoms selected from N, O, and S formed together with the nitrogen; 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 is 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 5~10 It is a lactone or a substituted or unsubstituted heterocycle having one or more heteroatoms selected from N, O, and S formed together with the 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 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 a lactone or a substituted or unsubstituted heterocycle having one or more heteroatoms selected from N, O, and S formed together with the nitrogen; 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 5~10 It is a lactone or a substituted or unsubstituted heterocycle having one or more heteroatoms selected from N, O, and S formed together with the nitrogen.

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

[0025] [ka]

[0026] Preparation method

[0027] All reactions in this study were carried out under a nitrogen atmosphere, using commercially available reagents without further purification. NMR analysis was performed using a Varian Unity 400 MHz spectrometer. Tetramethylsilane (TMS, Merck, Darmstadt, Germany) was used as the internal standard, and chloroform-d3 and methanol-d4 were used as solvents. Chemical shifts are expressed in ppm. Splitting patterns are as follows: s = singlet; d = doublet; t = triplet; q = quartet; dd = double doublet; m = multiplet. Analytical thin-layer chromatography (TLC) was performed using an Art. 5554 Kieselgel 60 GF254 column manufactured by E. Merck (Merck, Darmstadt, Germany). Compound spots were inspected using a UV indicator illuminated at 254 and 366 nm. Column chromatography was performed using E. Merck Art. 7734 Kieselgel 60 GF254 (70-400 mesh, Merck, Darmstadt, Germany). Mass spectra were recorded using a PuriFlash® MS (Interchim, Montlucon, France) liquid chromatography electrospray ionization mass spectrometer.

[0028] Abbreviations: Vol. (volume of solvent (mL) / limiting reactant (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 the linker is shown in the following reaction scheme (1).

[0031] [ka]

[0032] The synthesis of the linker began with 2,6-dimethylphenyl ester propionate (compound 1). 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 in THF, 1.1 eq) at −78 °C and stirred for 30 min at −78 °C. 2-(phenylthio)acetaldehyde (compound 2) (1.1 eq) and THF (40 vol.) were slowly added to the mixture and stirred for an additional 30 min at −78 °C. 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 in vacuo. Column chromatography (SiO, 1:9 EA / hexane) was used to obtain 2,6-dimethylphenyl 3-hydroxy-2-methyl-4-(phenylthio)butyrate (compound 3) as a yellow oil in 37% yield. 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 03S: 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, which was then stirred at −78 °C for 1 h. 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, which was then stirred at −78 °C for another 1 h. The mixture was quenched with saturated aqueous NH4Cl 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 step 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 stirred at -60 °C for 30 min. After the reaction was completed (monitored by TLC), the mixture was diluted with diethyl ether (50 Vol.) and saturated aqueous NH4Cl (130 Vol.) was slowly added. The organic layer was collected, dried over MgSO4, and concentrated in vacuo. Column chromatography (SiO2, 7:3 EA / hexane) provided (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(600MHz,CD3OD)δ5.39-5.33(m,1H), 5.21-5.15(m,1H), 4.08(d,J=6.8Hz,2H), 3.76-3.67(m,1H), 2.55-2.3 8(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). Chemical formula calculation: C 15 H 26 O4: m / z 270.2, measured value 291.2 (M + Na-2H - ), 305.2(M+Cl - ).

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

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

[0036] [ka]

[0037] Substitution of the carboxyl group: Ethyl iodide (1.1 eq) was added to a mixture of compound 5 (1.0 eq), K2CO3 (3.0 eq), and DMF (20 vol.) at room temperature and 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 layer was collected, dried over MgSO4, and concentrated in vacuo. Column chromatography (SiO2, 5:5 EA / hexane) provided (6E,10E)-3,12-dihydroxy-2,6,10-trimethyldodeca-6,10-dienoic acid ethyl ether (compound 6) as a colorless oil. The yield was 92%. 1 H NMR(400MHz,CDCl3)δ5.44-5.35(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=7.1Hz,3H), 1.22-1.15(m,3H). Chemical formula calculation: C 17 H 30 O4: m / z 298.2, observed value 321.2 (M+Na + ).

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

[0039] [ka]

[0040] Displacement of primary alcohol: 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 completion of the reaction (monitored by TLC), the solvent was evaporated under vacuum. Column chromatography (SiO2, 1:9 EA / hexane) afforded (6E,10E)-3-hydroxy-2,6,10-trimethyl-12-(trityloxy)dodeca-6,10-dienoic acid (compound 7) as a colorless oil. 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 the active compound and the linker

[0042] The synthesis of the linker-conjugated compound is shown in the following reaction scheme (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 h, 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 h. Compound 8 (1.2 eq) was added to the mixture and stirred at room temperature overnight. After the reaction was completed (monitored by TLC), the mixture was quenched with saturated aqueous NH4Cl and extracted with EA (3 × 90 vol.). The organic layer was collected, dried over MgSO4, and concentrated under vacuum. Column chromatography (SiO, 1:9 EA / hexane) provided 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) as a pale yellow oil. 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] Lithium tri-sec-butylborohydride (1 M in THF, 8.0 eq) was added dropwise to a solution of compound 9 (1.0 eq) in THF (200 vol.) at -70 °C and stirred for 4 h. The reaction was diluted with DCM (500 vol.), silica gel was added, and the mixture was stirred for 10 min. After the reaction was completed (monitored by TLC), the silica gel was filtered, and the residue was concentrated in vacuo. Column chromatography (SiO2, 1:9 EA / hexane) provided 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) as a pale yellow oil. 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, observed 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 stirred at room temperature for 1 hour. After the reaction was completed (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 in vacuo to give compound 11 as a pale yellow oil. 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 the linker-conjugated amino acid derivative is shown in the following reaction scheme (5).

[0049] [ka]

[0050] To a stirred solution of compound 7 (1.0 eq) and NHS (2.0 eq) in DMF (20 vol.) at room temperature, EDCI (2.0 eq) was added, 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 at room temperature overnight. The mixture was extracted with DCM (2 × 100 vol.). The organic layer was dried over MgSO4 and concentrated under reduced pressure. Column chromatography (SiO2, 100 / 0 to 50 / 50 EA / MeOH) afforded compound 12 (23%) as a pale yellow oil. 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] Listed below are methods for testing various properties of the drug linkers and linker-conjugated compounds of the present invention.

[0052] Water Solubility Test

[0053] [Table 1]

[0054] Standard solution calibration curve preparation method

[0055] Five mg of the standard test compound was weighed and dissolved in 5 mL of IPA to prepare a stock solution (1 mg / mL). Further dilutions were made with double-distilled water to prepare standard solution calibration curves of 1000, 100, 50, 25, 12.5, 10, 5, 2.5, 1.25, 1, 0.5, 0.25, and 0.125 μg / mL. 60 μL of each standard solution was used for HPLC analysis. The peak areas were integrated to generate a calibration curve, and the accuracy of the analytical method was evaluated by the correlation coefficient (R-squared).

[0056] Water Solubility Test Method

[0057] 1 mL of double-distilled water was weighed into a microcentrifuge tube and an excess amount of test compound was dissolved. The mixture was vortexed for 5 minutes and sonicated for 20 minutes to ensure the presence of undissolved compound. After centrifugation, the supernatant was filtered through a 0.22 μm PTFE membrane. The residue was analyzed by HPLC, and the water solubility was calculated from the peak area.

[0058] Cytotoxicity Screening

[0059] cell culture

[0060] NOZ cells (human gallbladder carcinoma cells) were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (JCRB, Japan). 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, and 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 (1× penicillin, streptomycin, and 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% RPMI 1640 medium (Gibco, USA), 10% fetal bovine serum (FBS) (Gibco, USA), and 1% antibiotics (1x penicillin, streptomycin, and glutamine) (Gibco, USA).

[0063] AML12 cells (alpha mouse liver 12) were obtained from the American Type Culture Collection (ATCC, USA). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM):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 (1x penicillin, streptomycin, and glutamine) (Gibco, USA).

[0064] AsPC-1 cells (human pancreatic adenocarcinoma ascites metastasis cells) were obtained from the Bioresource Collection and Research Center (BCRC) (Taiwan). Cells were cultured in RPMI 1640 medium (Gibco, USA) containing 10% FBS (Gibco, USA) and 1% antibiotics (1x penicillin, streptomycin, and glutamine) (Gibco, USA).

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

[0066] BxPC-3 cells (human pancreatic adenocarcinoma cells) were obtained from the Bioresource Collection and Research Center (BCRC, Taiwan). Cells were cultured in RPMI 1640 medium (Gibco, USA) containing 10% FBS (Gibco, USA) and 1% antibiotics (1x penicillin, streptomycin, and 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, and glutamine) (Gibco, USA).

[0068] MCF7 cells (human breast cancer cells) were obtained from the Bioresource Collection and Research Center (BCRC, Taiwan). Cells were cultured in Eagle's 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, and 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 RPMI 1640 medium (Gibco, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA) and 1% antibiotics (1x penicillin, streptomycin, and 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 (1x penicillin, streptomycin, and glutamine) (Gibco, USA).

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

[0072] Cell viability assessment

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

[0074] Listed below are test results for various properties of the drug linkers and linker-conjugated compounds of the present invention.

[0075] Solubilizer action test

[0076] At physiological pH 7.4, ionization of the carboxyl group enhances its ability to form hydrogen bonds with adjacent water molecules, thereby improving overall water solubility. Water solubility tests were conducted to verify that the drug linkers of the present invention can effectively improve the water solubility of compounds (Table 2). Both the drug linkers of the present invention and farnesol contain a hydroxyl group at the 1'-position and 15 carbon atoms in a linear chain structure. However, compared to farnesol, the drug linkers of the present invention have an additional hydroxyl group at the 10'-position and a carboxyl group at the 12'-position, resulting in a dramatic increase in water solubility of nearly nine-fold, from 87.4 μg / mL to 780.1 μg / mL. These results demonstrate that compounds designed using the drug linkers of the present invention can effectively improve water solubility.

[0077] [Table 2]

[0078] Pharmacophore linker activity test

[0079] Bifunctional / multifunctional drugs achieve superior efficacy by linking two or more pharmacophores via a linker. The design of the linker is crucial for this technology, as it must maintain the activity of the original drug while avoiding introducing additional toxicity. Furthermore, a good linker must be synthetically flexible, allowing for easy coupling with the 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 3For example, this linker has significant advantages in terms of synthetic elastic properties. 1 The alcohol group on -X is a primary alcohol and is prone to substitution reactions (reaction scheme (3)). 1 -X and R 2 When the alcohol group of -Y is converted to a halide via the Appel reaction, this halide intermediate readily reacts with other nucleophiles, such as alcohols, organolithiums, enols, amines, and thiols (first step of reaction (4)). 4 The carboxyl group in -Z is highly reactive and can undergo acid / base catalysis for esterification (Reaction Scheme (2)) or react with the amino moiety on small molecules, amino acids, or protein residues via a crosslinker to form amides (Reaction Scheme (5)). These diverse conjugation methods significantly expand their application range. Structural flexibility is also a key feature of 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. According to Table 3 below, the linkers of the present invention have relatively high NRot values, demonstrating their great potential as linkers for bifunctional / multifunctional drugs. Non-cytotoxicity is a fundamental requirement for linkers, and cytotoxicity tests were conducted using various cell lines. Cell experiments demonstrated that the linkers of the present invention exhibit no cytotoxicity to either normal 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, flexible synthetic sites, and carboxylic acid moieties, and can be used to link multiple pharmacophores to improve biological effects and drug similarity. In addition, the linker-conjugated compounds have excellent water solubility. The drug linkers of the present invention can be used to improve solubility and are not cytotoxic.

Claims

1. A drug linker having a chemical structure represented by the following formula (1): 【Chemical 1】 In formula (1), n is an integer from 1 to 10, X, Y, and Z are each independently halogen, carbon, oxygen, sulfur, 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 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 the nitrogen; 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 a lactone or a substituted or unsubstituted heterocycle having, together with the nitrogen, one or more heteroatoms selected from N, O, and S; 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 is 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 ５～１０ 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 the nitrogen.

2. A linker-conjugated compound having a chemical structure represented by the following formula (1-1): 【Chemistry 2】 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 a lactone or a substituted or unsubstituted heterocycle having, together with the nitrogen, one or more heteroatoms selected from N, O, and S; 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 ５～１０ A linker-conjugate compound that is a lactone or a substituted or unsubstituted heterocycle having one or more heteroatoms selected from N, O, and S formed together with the nitrogen.

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

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