KR101465626B1 - Target-specific anti-cancer prodrug comprising a biotin - Google Patents

Abstract

The present invention relates to a new drug delivering system which can easily monitor drug treatment effect and drug absorption by a two-photon fluorescence imaging in the subcellular level. Provided in the present invention is a target-specific prodrug represented by chemical formula 1. Provided in the present invention is a target-specific treatment-diagnostic anti-cancer prodrug which can effectively monitor drug distribution in vivo even target-specifically delivering drugs.

Description

TECHNICAL FIELD [0001] The present invention relates to a target-specific anticancer drug precursor comprising biotin,

The present invention relates to a novel drug delivery system capable of easily monitoring drug efficacy and drug absorption by two-photon fluorescence imaging at subcellular levels.

Target specific drug delivery systems are a very promising area in the modern pharmaceutical industry and are a field with a variety of advantages such as reduced drug dose by the patient, homeostasis of the drug levels in the body, and reduced side effects of the drug. Conventionally, various target-specific drug delivery systems such as gold nanoparticles, fluorescent quantum dots, polymers, porous silica, etc. exist, but no drug delivery system capable of effective drug delivery and excellent in vivo monitoring has been reported.

In recognition of the above-described problems, the present invention aims to provide a target-specific therapeutic diagnostic anti-cancer prodrug drug that can effectively deliver drugs in a target-specific manner while effectively monitoring drug distribution in the body.

The present invention provides a target specific drug precursor represented by the following Formula 1:

[Chemical Formula 1]

The present invention also provides a process for preparing a target specific drug precursor represented by the general formula (1). The preparation process according to the present invention can be illustrated by the following reaction formula (1).

[Reaction Scheme 1]

Specifically, a method for preparing a target specific drug precursor represented by Formula 1 of the present invention comprises the following steps:

Reacting a compound of formula (2) with hydroxyl ethyl disulfide to produce a compound of formula (3);

Preparing a compound of Formula 4 by reacting the compound of Formula 3 with acetic acid;

Reacting the compound of formula 4 with 4-nitrophenyl chloroformate to produce a compound of formula 5; And

Reacting the compound of formula (5) with a compound of formula (6) to produce a compound of formula (1).

(2)

(3)

상기 DMTr은 4,4'-디메톡시트리틸(dimethoxytrityl)을 의미한다.

DMTr means 4,4'-dimethoxytrityl.

≪ Formula 4 >

≪ Formula 5 >

(6)

According to the present invention, drug-monitoring through cell-level imaging is possible simultaneously with target-specific drug delivery.

Fig. 1 shows absorption and fluorescence spectrum of Compound 5. Fig. (a) is the absorption spectrum of Compound 5 in the presence or absence of GSH. (b) is the fluorescence spectrum of Compound 5 in the presence or absence of GSH. (C) is the fluorescence spectrum of 5 with increasing GSH concentration. (d) is a graph showing the change in fluorescence intensity at 476 nm as a function of GSH concentration.
Figure 2 is a confocal microscope image of A549 and W138 treated with 5. The photograph on the left is a confocal microscope photograph of A549 and W138 cells treated with 10.0 uM of chemical 5 in PBS buffer. The photo on the right is a non-focus contrast photo.
Figure 3 is a confocal microscope image of A549 cells treated with 5; Each image is pretreated cells in a medium containing 0, 0.2, and 0.5 mM N-ethylmaleimide (NEM).
Figure 4 is a confocal microscope image of colocalization experiments in A549 cells. (a) and (d) are fluorescence images of A549 cells containing Compound 5 (10.0 uM). (b) is a fluorescence image of A549 cells incubated with Lyso Tracker Blue DND-167 (0.05 uM) and compound 5. (C) is an image in which the images (a) and (b) are overlapped. (e) is a fluorescence image of A549 cells incubated with ER Tracker Red (0.05 uM) and Compound 5. (f) is an image in which the images (d) and (e) are overlapped.
5 is a graph of cell growth potential of 5 in A549 cell line.

Hereinafter, the present invention will be described in more detail.

The target specific drug precursor according to the present invention has a biotin-coumarin-gemcitabine structure. The fluorescence reaction of the target specific drug precursor of formula (1) can be illustrated by the following reaction formula (2). The compound of formula (1) cleaves the SS bond in the presence of GSH to form a thiol, and the resulting thiol forms a 5-membered ring thiolactone by intramolecular nucleophilic substitution, and emits fluorescence while releasing a pharmaceutically active form of GMC molecule .

[Reaction Scheme 2]

Synthetic materials and methods

Silica gel 60 (Merck, 0.063-0.2 mm) was used in the column chromatography. Analytical thin-layer chromatography was performed using Merck 60 F254 silica gel (precoated sheet, 0.25 mm thick). H and C NMR spectra were measured in CDCl3, DMSO (Cambridge Isotope Laboratories, Cambridge, Mass.) Of a Varian 300 and 400 MHz spectrometer. Mass spectra were measured on an ion Spec HiRes mass spectrometer.

Spectroscopic measurement materials and methods

A storage solution of the biologically relevant analyte was prepared with a sump distillate water. 5 was also prepared as a sump distillation water. All spectrometer sides were measured under physiological conditions (DMSO 25% (v / v) in PBS buffer, pH 7.4, 36 degrees). Absorption spectra were measured with an S-3100 (Scinco) spectrometer and fluorescence spectra were measured with a RF-5301 PC fluorescence meter (Shimadzu) equipped with a xenon lamp. Samples for absorption and emission measurements were placed in a quartz cuvet (3 mL volume). This is done at 480 nm, and the excitation and emission slits have a width of 3 and 5 nm.

Synthesis of Compound (1)

(1) Synthesis of Compound (2)

Compounds of formula 2 were synthesized by known methods. Yield 50%; J = 6.6 Hz, 1H), 6.77 (d, J = 2.3, 8.5 Hz, 1H), 7.44 (d, J = 7.55 (s, 1H), 10.53 (s, phenolic OH), ppm. ≪ 13 > C NMR (DMSO-d6, 100 MHz): 102.7, 112.0, 114.5, 121.8, 128.5, 129.8, 153.4, 158.0, 161.0 ppm. HRMS: Calculated for C9H6O3N3 (M + 1); 204.0409; found 204.0409.

(2) Synthesis of Compound 3

A solution of phosgene (429 mg, 4.38 mmol, 2.14 ml) was added to a stirred solution of DMTr protected hydroxyl ethyl disulfide (500 mg, 1.10 mmol) and DIPEA (849 mg, 6.57 mmol) in dry dichloromethane Respectively. The reaction solution was stirred at 0 deg. C under argon atmosphere. After 2 h, excess phosgene was removed from the reaction mixture by argon purge. 3-azido-7-hydroxy coumarin (111 mg, 0.55 mmol) in dry dichloromethane was added to the reaction mixture and stirred overnight. After completion of the reaction, the reaction mixture was diluted with EtOAc, washed twice with brine, dried over sodium sulfate and filtered. The solvent was removed under reduced pressure. The crude product was purified by column chromatography on silica gel to give the compound of formula 3 as a yellow viscous liquid (460 mg, 61% yield). 1H NMR (CDCl3, 400 MHz) : δ 2.852.91 (m, 4H), 3.39 (t, J = 6.1 Hz, 2H), 3.77 (s, 6H), 4.46 (t, J = 6.6 Hz, 2H), 6.82 (d, J = 8.8 Hz , 4H), 7.11 (dd, J = 2.2, 8.5 Hz, 1H), 7.167.22 (m, 3H), 7.28 (t, J = 7.8 Hz, 2H), 7.34 (d J = 8.8 Hz, 4H), 7.38 (d, J = 8.6 Hz, 1H), 7.45 (d, J = 7.3 Hz, 2H), ppm. (CDCl3, 100 MHz): 36.8, 39.9, 55.5, 62.0, 67.1, 86.6, 109.8, 113.3, 113.3, 117.5, 118.6, 125.3, 126.3, 127.1, 128.1, 128.2, 128.4, 130.3, 136.2, 145.0, 152.2, 152.9, 157.3, 158.7 ppm. ESI-MS: Calculated for C35H32N3O8S2; 685.16; found 685.48.

(3) Synthesis of Compound 4

Compound (3) (450 mg, 0.65 mmol) was dissolved in a dichloromethane solvent and 80% cold acetic acid was slowly added. The reaction mixture was completed by TLCdp with stirring for 24 hours. The cold acetic acid was neutralized by saturated aqueous sodium bicarbonate solution. EtOAc was added and the organic phase was dried over sodium sulfate and filtered. The solvent was removed under reduced pressure. The crude product was purified by column chromatography on silica gel to give the compound of formula 4 as a yellow viscous liquid (180 mg, 71% yield). 1H NMR (CDCl3, 400 MHz) : δ 2.87 (t, J = 5.8 Hz, 2H), 3.00 (t, J = 6.6 Hz, 2H), 3.86 (t, J = 5.8 Hz, 2H), 4.51 (t, J = 6.6 Hz, 2H), 7.11 (dd, J = 2.2, 8.6 Hz, 1H), 7.177.20 (m, 2H), 7.40 (d, J = 8.6 Hz, 13C NMR (CDCl3, 100 MHz): 36.7, 41.7, 60.5, 67.0, 109.8, 117.5, 118.6, 125.4, 126.3, 128.3, 151.7, 152.1, 152.9, 157.3 ppm. HRMS: Calculated for C14H14N3O6S2 (M + 1) 384.0324; found 384.0326.

(4) Synthesis of Compound 5

A catalytic amount of 4-nitrophenyl chloroformate (157.7 mg, 0.78 mmol), DIPEA (134 mg, 1.04 mmol) and pyridine was added to a solution of compound 3 (100 mg, 0.26 mmol) in DCM Lt; / RTI > The reaction mixture was concentrated in vacuo. The crude product was dissolved in 5 mL of DMF. To this solution was added gemcitabine (205 mg, 0.78 mmol) in DMF (2 mL) and TEA (0.5 mL) and stirred for 24 hours. After completion of the reaction, the reaction mixture was diluted with water. The compound was extracted with EA. The organic layer was dried over anhydrous sodium sulfate. The crude compound was passed through silica column chromatography using DCM / MeOH (9: 1) as eluent to give the compound of formula 5 as a yellow viscous liquid (30 mg, 17% yield). 4H), 5.30 (s, 1H, < RTI ID = 0.0 > ), 5.82 (d, J = 6.00 Hz, 1 H), 6.23 (br s, 1 H), 7.14 (d, J = 6.5 Hz, 1 H), 7.227.23 ), 7.55 (br s, 1 H), 8.26 (d, J = 7.2 Hz, 1 H), ppm. (CDCl3, 100 MHz): 36.6, 36.9, 55.1, 59.7, 65.6, 66.9, 79.0, 95.9, 109.6, 117.4, 118.5, 121.9, 125.5, 125.4, 128.2, 141.6, 151.6, 152.8, 153.8, 155.6, 156.0 , 165.9, 166.1 ppm. HRMS: Calculated for C24H23F2N6O11S2 (M + 1) 673.0834; found 673.033.

(5) Synthesis of Compound (1)

Compound (6) (29 mg, 0.01 mmol) and ascorbate (10 mol%) were added to a solution of compound 4 (70 mg, 0.01 mmol) in MeOH (3.0 mL). The reaction mixture was degassed by argon gas purge for 15 minutes. 2 mg (0.002 mmol) CuSo4 in 0.5 mL water was added to the reaction mixture and stirred for 3 hours. The crude reaction mixture was directly passed through silica column chromatography using DCM / MeOH (8.5: 1.5) as eluent to give the compound of formula 1 as a yellow viscous liquid (25 mg, 25% yield). J = 7.3 Hz, 2H), 2.532 (m, 2H), 2.50 (d, ), 2.772.83 (m, 2H), 2.993.10 (m, 5H), 3.68 (br s, 2H), 3.793.82 (m, 3H), 4.374.41 (m, 2H), 5.255.27 (m, 1H), 5.79 (d, J = 7.4 Hz, 1H), 6.186.25 3H), 7.437.45 (m, 2H), 7.64 (d, J = 7.5 Hz, 1H), 8.218.24 (m, 2H), 13C NMR (CDCl3, 100 MHz): 25.8, 28.3, 28.7, 28.8, 128.9, 128.1, 130.5, 140.7, 116.9, 116.9, 118.9, 119.5, 121.7, 124.7, 126.9, 127.0, 128.1, 130.5, 140.7, 142.1, 147.7, 153.7, 155.6, 163.3, 163.4, 166.3, 172.5 ppm. ESI-MS: Calculated for C37H42F2N9O13S3 (M + 1) 954.20; found 954.21

Cell culture preparation

Human lung adenocarcinoma epithelial cells (KCLB, Seoul, Korea) were cultured in RPMI supplemented with 10% FBS (Welgene), penicillin (100 units / ml) and streptomycin (100 ug / ml) Respectively. Two days before photographing, cells were subcultured and plated on glass bottom plates.

All cells were maintained in a humidified atmosphere of CO2 / air at a rate of 5/95 (v / v) at 37 degrees. For labeling, the growth medium was removed and replaced with RPMI without FBS. The cells were treated with ligand at 37 ° C under 5% CO2 for 15 min and incubated. The cells were washed three times with phosphate buffered saline (PBS, Gibco) and further incubated for 15 minutes in colorless serum-free medium.

Microscopic image capture of cells

Cells from Example 2 were planted in 24-well plates and allowed to stabilize overnight. Compound 5 was applied to the cells to monitor the absorption and release of the drug. Cells were incubated with Lyso- or ERtracker-containing media prior to treatment with 5. The cells were simply washed with 1 ml of PBS and treated with PBS 5. After the incubation, the remaining compound 5 which was not absorbed by the cells was removed. Cells were transferred to 1 ml of PBS solution. Fluorescence images were taken using a confocal laser scanning microscope (Zeiss LSM 510, Zeiss, Oberko, Germany).

Analysis

As can be seen in Figure 2, the biotin moiety preferentially directs the drug precursor of the invention to biotin receptor positive cancer cells. A549 cells are cells with biotin receptors and W138 cells are cells without biotin receptor. A549 cells show strong fluorescence, while W138 cells show almost no fluorescence. From these results, it can be confirmed that the drug precursor of formula (1) was introduced into cells by the interaction of the biotin receptor and the biotin moiety of the cancer cell.

It can be seen from FIG. 4 that GMC was released into the cells of the GMC after cell entry. Cleavage of the thiol-derived disulfide bond in the drug precursor of formula (1) occurs in lysosomes, releasing GMCs and entering the cell nucleus. These GMCs replace cytidine and nucleic acid units in DNA replication, leading to apoptosis.

As shown in FIG. 5, the anti-cancer effect of the drug precursor having biotin molecules was confirmed in A549 cells. In Fig. 5, 4 is a compound having no biotin and 5 is a compound having biotin. When the compound having biotin was administered, the cell viability was higher. That is, biotin has an effect of targeting anticancer cells in the drug precursor.

Claims (2)

A target-specific anticancer drug precursor represented by the following formula (1).
≪ Formula 1 >

Reacting a compound of formula (2) with hydroxyl ethyl disulfide to produce a compound of formula (3);
Preparing a compound of Formula 4 by reacting the compound of Formula 3 with acetic acid;
Reacting the compound of formula 4 with 4-nitrophenyl chloroformate to produce a compound of formula 5; And
Reacting the compound of formula 5 with a compound of formula 6 to produce a compound of formula 1
(1), wherein the target specific anti-cancer drug precursor is represented by Formula (1).
(2)

(3)

≪ Formula 4 >

≪ Formula 5 >

(6)

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