KR102486721B1 - Novel mTOR inhibitor compounds and the use thereof - Google Patents

Abstract

The novel mTOR inhibitor compound of the present invention relates to a targeting compound in which a bone-directing ligand is connected to an mTOR-inhibiting bone disease therapeutic agent so that the drug can be selectively delivered to bone and slowly released from bone for a long period of time.

Description

Novel mTOR inhibitor compounds and the use thereof {Novel mTOR inhibitor compounds and the use thereof}

The present invention relates to novel mTOR inhibitor compounds and uses thereof.

Osteosarcoma (OS) is the most common primary malignant neoplasm in children and young adults and is characterized by high local aggressiveness and high metastasis. The cure rate for osteosarcoma is approximately 25% when accompanied by metastasis at diagnosis, which has been largely stagnant over the past 20 years.

Multiple myeloma (MM) is the second most common devastating clonal plasma cell malignancy with a 5-year survival rate of only 45% and arises in the bone marrow. Although many new pharmacological strategies have been developed over the past 30 years, including the introduction of new immunomodulatory drugs and proteasome inhibitors, multiple myeloma remains incurable.

Bone metastatic prostate cancer is the most common cancer and the second leading cause of cancer-related death in Western men, with 903,500 new diagnoses and 258,400 deaths in 2008. Although the prognosis for the majority of cases of prostate cancer is good, it is virtually incurable once it develops into a metastatic stage. Bone metastatic prostate cancer is classified as a major carcinoma that metastasizes to bone, and about 75% of prostate cancer patients experience bone metastasis.

Various studies have been conducted to treat bone diseases such as the osteosarcoma, multiple myeloma, and bone metastatic prostate cancer. Among them, mTOR inhibitors inhibit cell growth and proliferation, thereby exhibiting antitumor effects in osteosarcoma and improving the survival rate of osteosarcoma patients. point was revealed.

Various bone disease treatments have been studied and used, but drugs such as methotrexate, doxorubicin (adriamycin), cisplatin, epirubicin, ifosfamide, cyclophosphamide, itoposide, gemcitabine, and topotecan have poor bone penetration. It is used in high doses, which has side effects such as lowering the leukocyte level and causing secondary infection by affecting the bone marrow. VP-16), doxorubicin (Adriamycin), liposomal doxorubicin (Doxil), and bendamustine (Treanda) affect normal cells as well as cancer cells, resulting in hair loss, oral irritation, anorexia, vomiting, and nausea. , the side effects such as lowered immunity are very serious.

The inventors of the present invention felt the need for a drug with a new drug delivery platform that can be selectively delivered only to bone and is slowly released from bone for a long period of time, and developed a targeting compound that linked a bone-directing ligand to a mTOR-inhibiting bone disease treatment agent. Thus, the problems of existing bone disease treatments have been solved.

The present invention provides a compound represented by Formula 1, an optical isomer or a pharmaceutically acceptable salt thereof:

[Formula 1]

A-B-C-D

in the expression

,

,A is

,

,

및

으로 이루어진 군에서 선택되는 어느 하나이고,

and

Any one selected from the group consisting of

또는

이고,B is

or

ego,

또는

로서, C is

or

as,

l, m, n are each an integer of 1 to 5;

또는

이며, R은 H 또는 OH이다.D is

or

, and R is H or OH.

In addition, the present invention provides a pharmaceutical composition for preventing or treating bone disease comprising the compound, its optical isomer or pharmaceutically acceptable salt as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving bone disease comprising the compound, its optical isomer or pharmaceutically acceptable salt as an active ingredient.

In addition, the present invention provides a bone-targeting drug delivery composition comprising the compound, its optical isomer or pharmaceutically acceptable salt as an active ingredient.

Figure 1 shows a schematic diagram of the role played by the structure of the compound of the present invention.
FIG. 2 shows the results of Western blotting of phosphorylated S6 protein to confirm the extent to which the sirolimus compound of the present invention inhibits the endogenous mTORC pathway in RPMI8226 cells.
Figure 3 shows the absorbance of the bound FITC-conjugated alendronate after washing the plate wells.
Figure 4 shows a plot of the safety evaluation and safe administration dose determination study for the compound of formula 4 and the compound of formula 5.
Figure 5 shows the results of safety evaluation of the drug delivery system as changes in liver and kidney function values for mice administered with the compound of Formula 4 and the compound of Formula 5.
Figure 6 confirms the weight loss of the mice after administration of the compounds of Formula 4 and Formula 5.
Figure 7 shows the results of establishment of a transformed multiple myeloma cell line (RPMI8226).
8 shows multiple myeloma mouse models and drug treatment protocols.
Figure 9 confirms the tumor burden reduction effect according to the combined administration of the compound of Formula 4 and chloroquine.
10 shows the results of body weight changes in mice according to the combined administration of the compound of Formula 4 and chloroquine.
FIG. 11 shows the results of reducing the size of multiple myeloid tumors according to the combined administration of the compound of Formula 4 and chloroquine.
12 shows the results of identifying multiple myeloma cells in the tibia following the combined administration of the compound of Formula 4 and chloroquine.
13 shows the results of identification and quantification of multiple myeloma cells in the tibia following the combined administration of the compound of Formula 4 and chloroquine.
14 shows the results of mouse tumor size measurement according to the combined administration of the compound of Formula 4 and chloroquine.
FIG. 15 shows the results of reducing the size of multiple myeloid tumors according to the combined administration of the compound of Formula 4 and chloroquine.
16 confirms the correlation between IgL concentration and tumor size IVIS value.
Figure 17 confirms the concentration of IgL in the peripheral blood of mice according to the combined administration of the compound of Formula 4 and chloroquine.
FIG. 18 shows the results of IVIS image measurement after treatment with the compound of Formula 4 using mice with tibial osteosarcoma tumors (Osteosarcoma).
19 shows a sandwich ELISA methodology for pharmacokinetic experiments of the compound of Formula 4.
20 shows the result of verifying the sensitivity of the sandwich ELISA method.
21 shows the results of confirming the targeting properties of the compound of Formula 4.

The present invention relates to a compound represented by Formula 1 below, an optical isomer or a pharmaceutically acceptable salt thereof.

[Formula 1]

A-B-C-D

in the expression

,

,

및

으로 이루어진 군에서 선택되는 어느 하나이고,A is

,

,

and

Any one selected from the group consisting of

또는

이고,B is

or

ego,

또는

로서, C is

or

as,

l, m, n are each an integer of 1 to 5;

또는

이며, R은 H 또는 OH이다.D is

or

, and R is H or OH.

In the present invention, alkyl having 1 to 3 carbon atoms may be, for example, methyl, ethyl, or propyl, but is not limited thereto.

In the present invention, alkylene having 1 to 3 carbon atoms is a hydrocarbon having a double bond. For example, it may be ethylene, propylene, etc., but is not limited thereto.

In the present invention, A refers to a chemical structure corresponding to a therapeutic agent for bone diseases. The bone disease therapeutic agent may preferably be an mTOR inhibitor. The mTOR refers to a substance that serves as a sensor of nutrients and energy by transmitting both internal and external cell signals, and controls protein synthesis and a wide range of cell biological activities in both normal and cancer cells. The mTOR inhibitor of the present invention inhibits mTOR to prevent the proliferation of malignant cells that cause bone diseases.

,

,

및

으로 이루어진 군에서 선택되는 어느 하나일 수 있다. The A is preferably

,

,

and

It may be any one selected from the group consisting of.

또는

일 수 있다.In one specific embodiment, the A is

or

can be

The A may be a compound selected from the group consisting of sirolimus (=rapamycin), everolimus and temsirolimus, an optical isomer thereof, or a pharmaceutically acceptable salt thereof. The chemical structures corresponding to sirolimus, everolimus and temsirolimus are shown in Table 1 below.

Name of functional group chemical structure Sirolimus (=rapamycin)

everolimus

temsirolimus

In the present invention, B denotes a chemical structure corresponding to an acid-decomposable linker. An acid-cleavable linker refers to a linkage that can be cleaved by an acid to release the therapeutic agent at the target. When tumor metastasis to bone occurs, the acid-base balance of the tumor metastasis tissue is broken, and osteoclasts secrete protons and acid hydrolases to the bone resorption site to digest inorganic and organic substances in the bone matrix. At this time, the pH value decreases to 4.5 in the bone resorption microenvironment. The acid-cleavable linker is specially designed to be stably maintained at neutral pH of the blood, and has a technical feature in that it releases the therapeutic agent for bone diseases through hydrolysis in the acidic environment of the cell compartment.

또는

일 수 있으나, 이에 제한되는 것은 아니며 산성 조건에서 분해될 수 있는 임의의 적절한 산분해성 링커가 사용될 수 있다.The B is preferably

or

However, it is not limited thereto, and any suitable acid-decomposable linker that can be cleaved under acidic conditions can be used.

In the present invention, C means a chemical structure corresponding to a spacer. The spacer refers to a chemical structure designed to avoid steric hindrance by intercalating between a targeting ligand and a therapeutic drug, since the binding of the drug to the receptor may be hindered when the drug delivery compound attaches the therapeutic drug in direct proximity to the targeting ligand. . The spacer not only affects the binding of the conjugate to the receptor, but also affects non-specific adsorption to a non-target target. Since the spacer can affect pharmacokinetics and pharmacodynamics, such as penetration into tumor cells, metabolism, and excretion, the spacer design greatly affects the performance and success of the conjugate.

또는

로서, The C is preferably

or

as,

Each of l, m, and n may be an integer of 1 to 5.

In a specific embodiment, l and m may be 1 or 2, and n may be any one integer from 1 to 3.

In the present invention, D means a chemical structure corresponding to bisphosphonate. The chemical structure of the bisphosphonate is characterized by the fact that two PO ₃ functional groups are covalently linked to one carbon. Preferably, D may be H or OH. The bisphosphonate can bind to a mineralized bone matrix, inhibit tumor formation in the bone matrix, generate osteoclasts from progenitor cells, inhibit angiogenesis, and cause apoptosis of osteoclasts and tumor cells. can cause

Accordingly, the bisphosphonates can be used to treat myeloma, osteoporosis, bone metastasis and other bone-related diseases, can inhibit angiogenesis of tumor tissue, and exhibit strong binding force and affinity with bone. Accordingly, bisphosphonates can be used as excellent ligands for bone-targeted therapy.

또는

이며, R은 H 또는 OH이나, 이에 제한되는 것은 아니며 원하는 치료효과에 따라 적절한 임의의 비스포스포네이트가 사용될 수 있다.The D is preferably

or

And, R is H or OH, but is not limited thereto, and any suitable bisphosphonate may be used depending on the desired therapeutic effect.

The optional bisphosphonates include Alendronate (Alen), Pamidronate, Zoledronate, Neridronate, Olpadronate, It may be any one selected from the group consisting of ibandronate, risendronate, etidronate, clodronate and tiludronate, alendronate ( Alendronate) may be preferred, but is not limited thereto.

The definitions of alkyl and alkylene have the same meaning as previously defined.

Specifically, the compound represented by Formula 1 according to the present invention may be any one selected from the group consisting of Formulas 2 to 7:

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

.

.

The compound represented by Formula 1 of the present invention may contain one or more asymmetric carbons, and thus may exist as racemates, racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. there is.

These isomers can be separated by conventional techniques, for example, the compound represented by Formula 1 can be separated by column chromatography or HPLC. Alternatively, each stereoisomer of the compound represented by Formula 1 can be stereospecifically synthesized using optically pure starting materials and/or reagents of known arrangement.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt in a form that can be used pharmaceutically among salts in which cations and anions are bonded by electrostatic attraction, and is usually a metal salt, an organic salt It can be a salt with a base, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid, and the like. Examples of metal salts include alkali metal salts (sodium salt, potassium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, barium salt, etc.), aluminum salts, and the like; Salts with organic bases include triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine salts with the like; Salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like; Salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like; Salts with basic amino acids include salts with arginine, lysine, ornithine, and the like; A salt with an acidic amino acid may be a salt with aspartic acid or glutamic acid.

Uses of Compounds of Formula 1

The present invention provides a pharmaceutical composition comprising the compound represented by Formula 1, an optical isomer or a pharmaceutically acceptable salt thereof.

In addition, the present invention relates to a compound represented by Formula 1, an optical isomer or a pharmaceutically acceptable salt thereof; And it provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

The present invention relates to a pharmaceutical composition for preventing or treating bone diseases comprising the compound of Formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient. Here, the bone disease consists of osteoporosis, osteomyelitis, osteomalacia, bone damage due to bone metastasis of cancer cells, osteosarcoma, multiple myeloma, bone metastatic prostate cancer, intervertebral disc herniation, fibrous osteodysplasia, Klippel-Pile syndrome, and cystic fibroosteitis. It may be one or more selected from the group, but is not limited thereto.

The composition according to the present invention can selectively deliver a therapeutic agent to malignant cells by designing a structure to attach a targeting moiety to a bone cytotoxic substance that exhibits strong toxicity, so that it not only exhibits excellent effects in the treatment of diseases but also has no side effects. Significantly less.

The pharmaceutical composition of the present invention may further contain at least one pharmaceutically acceptable excipient in addition to the compound of Formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof for administration. For example, a pharmaceutically acceptable carrier may be a mixture of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components, and, if necessary, an antioxidant , buffers, bacteriostatic agents and other conventional excipients may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets. Accordingly, the pharmaceutical composition of the present invention may be a patch, liquid, pill, capsule, granule, tablet, suppository or the like. These formulations may be prepared by a conventional method used for formulation in the art or a method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA, and formulated into various formulations depending on each disease or component It can be.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically applied) depending on the desired method, and the dosage is determined by the patient's body weight, age, sex, The range varies according to health status, diet, administration time, administration method, excretion rate, and severity of disease. The daily dose of the compound of Formula 1 of the present invention is about 0.01 to 1000 mg/kg, preferably 0.1 to 100 mg/kg, and may be administered once or several times a day.

The pharmaceutical composition of the present invention may further contain at least one active ingredient exhibiting the same or similar efficacy in addition to the derivative compound of Formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

In one embodiment of the present invention, a chloroquine derivative or a pharmaceutically acceptable salt thereof may be further included in addition to the derivative compound of Formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof. A chloroquine derivative or a pharmaceutically acceptable salt thereof may be administered in combination with a derivative compound of Formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof to treat bone diseases.

The chloroquine derivative used in combination is preferably chloroquine (7-chloro-4-(4-diethylamino-1-methylbutylamino)quinoline). It is a 4-aminokinoline derivative developed as an antimalarial drug and an antiave drug, and has a structure represented by Chemical Formula 8 below.

[Formula 8]

The chloroquine inhibits the formation of autolysosomes by inhibiting the fusion of autophagosomes and lysosomes. Chloroquine of the present invention blocks mTOR signaling pathway and autophagy signaling pathway at the same time when the compound represented by Formula 1, its optical isomer or pharmaceutically acceptable salt is concurrently treated, thereby preventing and preventing bone diseases such as multiple myeloma and osteosarcoma. It was confirmed that it exhibits excellent efficacy in treatment.

According to the above embodiment, in the present invention, a compound represented by Formula 1, an optical isomer or a pharmaceutically acceptable salt thereof; And it provides a pharmaceutical composition for preventing or treating bone diseases comprising a chloroquine derivative or a pharmaceutically acceptable salt thereof:

[Formula 1]

A-B-C-D

in the expression

,

,A is

,

,

및

으로 이루어진 군에서 선택되는 어느 하나이고,

and

Any one selected from the group consisting of

또는

이고,B is

or

ego,

또는

로서, C is

or

as,

l, m, n are each an integer of 1 to 5;

또는

이며, R은 H 또는 OH이다.D is

or

, and R is H or OH.

In the case of the combined administration, the daily dosage of the compound of Formula 1 of the present invention is about 0.01 to 1000 mg/kg, preferably 0.1 to 100 mg/kg, and may be administered once or several times a day. In addition, the daily dose of the chloroquine derivative co-administered is about 0.01 to 1000 mg/kg, preferably 0.1 to 100 mg/kg, and may be administered once or several times a day.

The concomitantly administered chloroquine derivative may be a compound containing at least one quinoline center selected from the following groups capable of exhibiting therapeutic and preventive effects on bone diseases:

7-hydroxy-4-(4-diethylamino-1-methylbutylamino)quinoline; chloroquine phosphate; 7-chloro-4-(4-diethylamino-1-butylamino)quinoline (desmethylchloroquine); 7-hydroxy-4-(4-diethylamino-1-butylamino)quinoline; 7-chloro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline; 7-hydroxy-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline; 7-chloro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline; 7-hydroxy-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline; 7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline (hydroxychloroquine); 7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; hydroxychloroquine phosphate; 7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline (desmethylhydroxychloroquine); 7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; 7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; 8-[(4-aminopentyl)amino]-6-methoxydihydrochloride quinoline; 1-acetyl-1,2,3,4-tetrahydroquinoline; 8-[4(-aminopentyl)amino]-6-methoxyquinoline dihydrochloride; 1-butyryl-1,2,3,4-tetrahydroquinoline; 7-chloro-2-( o -chlorostyryl)-4-[4-diethylamino-1-methylbutyl]aminoquinoline phosphate; 3-chloro-4-(4-hydroxy-α,α′-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline, 4-[(4-diethylamino)-1 -methylbutyl)amino]-6-methoxyquinoline; 3,4-dihydro-1-( 2H )-quinolinecarboxyaldehyde; 1,1'-pentamethylenediquinoleinium diiodide; and 8-quinolinol sulfate.,

Bone diseases that can be prevented or treated when a derivative compound of Formula 1, an optical isomer thereof or a pharmaceutically acceptable salt thereof and a chloroquine derivative or a pharmaceutically acceptable salt thereof are administered in combination are osteoporosis, osteomyelitis, osteomalacia, and cancer cells. It may be any one selected from the group consisting of bone damage due to bone metastasis, osteosarcoma, multiple myeloma, bone metastatic prostate cancer, intervertebral disc herniation, fibrous osteodysplasia, Klippel-Pile syndrome, and cystic fibroosteitis.

Specifically, in one embodiment, the bone disease may be any one selected from the group consisting of bone damage due to bone metastasis of cancer cells, osteosarcoma, and multiple myeloma.

The information regarding the above concomitant administration is equally applicable to the following bone disease treatment methods, food compositions, and uses, unless contradictory to each other.

The present invention provides a method for treating bone disease, comprising administering to a subject a therapeutically effective amount of the compound of Formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

The term "therapeutically effective amount" used in the present invention refers to an amount of the compound of Formula 1 effective for the treatment of bone diseases.

The treatment method of the present invention includes not only addressing the disease itself prior to the onset of symptoms, but also inhibiting or avoiding its symptoms, by administering the compound of Formula 1 above. In the management of disease, the prophylactic or therapeutic dose of a particular active ingredient will vary depending on the nature and severity of the disease or condition and the route by which the active ingredient is administered. Dosage and frequency of dosing will vary according to the age, weight and response of the individual patient. A suitable dosage regimen can be readily selected by those skilled in the art who take these factors into account. In addition, the treatment method of the present invention may further include the administration of a therapeutically effective amount of an additional active agent to help treat a disease together with the compound of Formula 1, the additional active agent together with the compound of Formula 1 They may exhibit synergistic or auxiliary effects.

The present invention provides a food composition for preventing or alleviating bone disease comprising the compound of Formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

The food composition of the present invention can be used as a health functional food. The term “health functional food” refers to food manufactured and processed using raw materials or ingredients having functional properties useful for the human body in accordance with the Health Functional Food Act No. 6727, and “functional” refers to the structure of the human body. And it refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients for functions or physiological functions.

The food composition of the present invention may contain conventional food additives, and the suitability as the "food additive" is determined in accordance with the General Rules of the Code of Food Additives and General Test Methods approved by the Korea Food and Drug Administration, unless otherwise specified. It is judged according to the standards and standards for

For the purpose of preventing and/or improving bone diseases, the food composition of the present invention may contain the compound of Formula 1 in an amount of 0.01 to 95%, preferably 1 to 80%, based on the total weight of the composition. In addition, for the purpose of preventing and/or improving bone diseases, it can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, beverages, and the like.

In addition, the present invention provides a pharmaceutical composition comprising the compound of Formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof for use in treating bone diseases.

In addition, the present invention is intended to provide a use of the compound of Formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof for the preparation of a drug for treating bone diseases. The compound of Formula 1 for the preparation of a drug may be mixed with acceptable adjuvants, diluents, carriers, etc., and may be prepared as a combined preparation with other active agents to have a synergistic action of the active ingredients.

Matters mentioned in the composition, use, and treatment method of the present invention are equally applied unless contradictory to each other.

The present invention also provides a bone-targeting drug delivery composition comprising the compound of Formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

The bone-targeting drug delivery composition refers to a drug delivery composition that specifically reaches bone tissue. In the present invention, the bisphosphonate is a compound of Formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof. It refers to a composition that can specifically reach bone tissue in the body due to the bone affinity of spornate. After the composition specifically reaches the bone tissue, the acid-decomposable linker is specifically decomposed by the acidified environment by osteoclasts to specifically release the therapeutic agent for bone diseases.

The bone-targeting drug delivery composition may be formulated and used in any form suitable for pharmaceutical preparations, but may be formulated for oral or parenteral administration, specifically, a sterilized aqueous solution, a non-aqueous solvent, a suspension, or an emulsion , It may be a lyophilized preparation. When formulated, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants commonly used in the art.

The bone-targeting drug delivery composition of the present invention may use propylene glycol, polyethylene glycol, vegetable oil such as olive oil, or injectable ester such as ethyl oleate as a non-aqueous solvent or suspending agent.

The pharmaceutical dosage form of the bone-targeting drug delivery composition may be used in the form of a pharmaceutically acceptable salt thereof, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set. The salt is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, methane Sulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid and the like can be used.

The composition for bone-targeting drug delivery of the present invention can be administered parenterally as desired, and can be administered once or several times to be administered in an amount of 0.1 to 500 mg, preferably 1 to 100 mg per 1 kg of body weight per day. can be administered. The dosage for a specific patient may vary depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, severity of the disease, and the like.

Example

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are only provided to more easily understand the present invention, and the scope of the present invention is not limited by the examples.

synthesis example

Synthesis Example 1. Synthesis of Compound of Formula 2

The synthesis process of the compound of Formula 2 is shown in Schemes 1 and 2 below, and the specific synthesis process of the intermediate stage compound is described below.

[Scheme 1]

[Scheme 2]

Synthesis Example 1-1. Synthesis of 2-(Tritylthio)acetic acid (2-1)

Mercaptoacetic acid (0.87 mL, 12.4 mmol, 1 equivalent) and triphenylmethanol (3.25 g, 12.4 mmol, 1 equivalent) were dissolved in chloroform (12 mL), and trifluoroacetic acid (2.4 mL) was added dropwise to the mixture. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, the mixture was concentrated by rotary evaporation. The residue was recrystallized from a 2:1 mixed solution of Hexane/CH ₂ Cl ₂ to obtain compound 2-1 (3.0 g, 72%). ¹ H-NMR (400 MHz, CDCl ₃ ): δ 3.0 (s, 2H), 7.20-7.34 (m, 9H), 7.38-7.44 (m, 6H).

Synthesis Example 1-2. Synthesis of 2,5-Dioxopyrrolidin-1-yl 2-(tritylthio)acetate (2-2)

Tritylthioacetic acid 2-1 (500 mg, 1.5 mmol, 1 equivalent) and N-Hydroxysuccinimide (NHS) (172.5 mg, 1.5 mmol, 1 equivalent) prepared above were dissolved in anhydrous THF (5 mL) under argon gas. To the mixture, THF (5 mL) in which DCC was dissolved was added dropwise at 0 °C, raised to room temperature over 1 hour, and stirred at room temperature for 4 hours. After the reaction was completed, DCC urea was removed by filtration with CELITE, and the filtered solid was washed with THF (2 mL). The filtrate was evaporated to dryness under reduced pressure, and the residue was recrystallized from EtOAc to obtain compound 2-2 as a white solid (300 mg, 46%). 1H ^- NMR (400MHz, CDCl ₃ ): δ 2.80 (s, 4H), 3.18 (s, 2H), 7.21-7.28 (m, 3H), 7.28-7.35 (m, 6H), 7.39-7.45 (m, 6H).

Synthesis Example 1-3. tert -butyl 4,4-bis(dimethoxyphosphoryl)butylcarbamate (2-3)

t -BuOK (53 mg, 0.473 mmol, 1.1 equiv) was dissolved in anhydrous DMF (0.25 mL) under argon gas. The mixture was cooled to 0°C, and DMF (0.75 mL) in which tetramethyl methylene diphosphonate (100 mg, 0.43 mmol, 1 equivalent) was dissolved was added. The mixture was stirred at room temperature for 15 minutes and then cooled to 0 °C again. DMF (0.75 mL + 0.25 mL rinse) in which 3-(Boc-amino)propyl bromide (307.6 mg, 1.29 mmol, 3 equivalents) was dissolved was added, and the mixture was stirred at room temperature for 41 hours. After the reaction was complete, a saturated NH ₄ Cl aqueous solution was added and extracted with EtOAc (3 x 20 mL). The organic layer was washed with brine, dried over anhydrous Na ₂ SO ₄ and filtered. Concentrated by rotary evaporation, and the residue was purified by column chromatography (49:1 → 9:1 CH ₂ Cl ₂ /MeOH) to obtain a mixture of target compound 2-3 and tetramethyl methylene diphosphonate as a clear liquid (70 mg, 41 % ) was done.

Synthesis Example 1-4. Synthesis of 4,4-Bis(dimethoxyphosphoryl)butan-1-aminium 2,2,2-trifluoroacetate (2-4)

Compound 2-3 (44 mg, 0.113 mmol, 1 equivalent) prepared above was dissolved in anhydrous CH ₂ Cl ₂ (0.5 mL) under argon gas, and the mixture was cooled to 0°C. TFA (0.05mL, 0.678mmol 6.0 equiv) was added and the mixture was stirred at room temperature for 4 hours. After the reaction was completed, the mixture was concentrated by rotary evaporation. The residue was washed with ether (4 x 1 mL) and evaporated to dryness under reduced pressure to obtain the target compound 2-4 as a clear liquid (15 mg, 45%). ¹ H-NMR (400 MHz, CD ₃ OD): δ 1.85-2.03 (m, 4H), 2.72-3.00 (m, 3H), 3.75-3.90 (m, 12H).

Synthesis Example 1-5. Tetramethyl (4-(2-(tritylthio)acetamido)butane-1,1-diyl) bis Synthesis of (phosphonate) (2-5)

The prepared compound 2-4 (50 mg, 0.124 mmol, 1 equivalent) and the prepared compound NHS ester 2-2 (53.4 mg, 0.124 mmol, 1 equivalent) were dissolved in anhydrous DMF (1 mL) under argon gas. Triethylamine (Et ₃ N) (24 mL, 0.186 mmol, 1.5 equivalent) was added dropwise to the mixture, and the mixture was stirred at room temperature for 3 hours. After the reaction was completed, cooling water was added and stirred for 10 minutes. The reaction mixture was extracted with EtOAc (2 x 10 mL). The extracted organic layer was washed with brine, dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated by rotary evaporation. The residue was purified by column chromatography (95:5 CH ₂ Cl ₂ /MeOH) to obtain the desired compound 2-5 as a clear liquid (40 mg, 53%). R _f =0.3 (95:5 CH ₂ Cl ₂ /MeOH). ¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.56-1.68 (m, 2H), 1.74-1.90 (m, 2H), 2.31 (tt, J = 24.4, 6.4 Hz, 1H), 2.9-3.0 (m , 2H), 3.10 (s, 2H), 3.74–3.84 (m, 12 H), 7.18–7.34 (m, 9H), 7.36–7.44 (m, 6H).

Synthesis Example 1-6. Tetramethyl (4-(2-mercaptoacetamido)butane-1,1-diyl) bis Synthesis of (phosphonate) (2-6)

The prepared compound 2-5 (40 mg, 0.066 mmol, 1 equivalent) was dissolved in a mixture of trifluoroacetic acid (1 mL) and H ₂ O (0.3 mL), and triethylsilane (0.04 mL, 0.264 mmol, 4 equivalents) was added to the mixture. was added. The mixture was stirred at room temperature for 30 minutes. After the reaction was completed, the mixture was concentrated by rotary evaporation. The residue was washed with Hexane:EtOAC 9:1 mixed solution (3 x 3mL). The residue was evaporated to dryness under reduced pressure to obtain the desired compound 2-6 as a liquid (15 mg, 62%). ¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.70-1.87 (m, 2H), 1.87-2.08 (m, 2H), 2.39 (tt, J = 24.4, 6 Hz, 1H), 3.22 (d, J = 8.8 Hz, 2H), 3.25–3.38 (m, 2H), 3.7–3.9 (m, 12H), 7.00 (bs, 1H).

Synthesis Example 1-7. Synthesis of (1-Hydroxy-4-(2-(tritylthio)acetamido)butane-1,1-diyl)diphosphonic acid (3-1)

After dissolving alendronic acid (249 mg, 1 mmol, 1 equiv.) in H ₂ O (3 mL) and adding triethylamine (Et ₃ N) (0.83 mL, 6 mmol, 6 equiv.) to the mixture, 5 minutes Stir. The prepared NHS ester 2-2 (646 mg, 1.5 mmol, 1.5 equiv) was dissolved in anhydrous DMF (5 mL), added to the mixture, stirred at room temperature for 1 hour, and concentrated by rotary evaporation. The residue was dissolved in H ₂ O (5 mL) and EtOAc (10 mL), and the mixture was stirred for 30 minutes. After separating the mixture, the aqueous solution was rotary evaporated to obtain the target compound 3-1 as a white solid (600 mg, 76%). ¹ H-NMR (400 MHz, D ₂ O): δ 1.40-1.54 (m, 2H), 1.58-1.72 (m, 2H), 2.63-2.72 (m, 2H), 2.98 (S, 2H), 7.10- 7.22 (m, 9H), 7.26-7.32 (m, 6H).

Synthesis Example 1-8. Synthesis of (1-Hydroxy-4-(2-mercaptoacetamido)butane-1,1-diyl)diphosphonic acid (3-2)

The prepared compound 3-1 (100 mg, 0.130 mmol, 1 equivalent) was dissolved in a mixture of trifluoroacetic acid (1.5 mL) and H ₂ O (0.4 mL), and triethylsilane (0.08 mL, 0.52 mmol, 4 equivalents) was added to the mixture. added. The mixture was stirred at room temperature for 30 minutes. After the reaction was completed, the mixture was concentrated by rotary evaporation. The residue was washed with EtOAC (5 x 5 mL). The residue was evaporated to dryness under reduced pressure to obtain the target compound 3-2 as a colorless liquid (40 mg, 95%). ¹ H-NMR (400 MHz, D ₂ O): δ 1.60-1.72 (m, 2H), 1.74-1.90 (m, 2H), 3.06 (s, 2H), 3.02-3.10 (m, 2H). ³¹ P-NMR (162 MHz, D ₂ O): δ 18.95 ppm. ESI-HRMS m/z calcd for C ₆ H ₁₄ NO ₈ P ₂ S[MH] ^- : 321.9928, found: 321.9928.

Synthesis Example 1-9. Synthesis of 2-(Pyridin-2-yldisulfanyl)ethanol (2-7):

2,2-Ddipyridyl disulfide (1.33 g, 6.06 mmol, 2 equivalents) was dissolved in methanol (10 mL) under argon gas, and the mixture was degassed for 15 minutes using argon gas. 2-mercaptoethanol (0.2 mL, 3.03 mmol, 1 equivalent) was added dropwise under argon gas and stirred for 2 hours. After the reaction was completed, the mixture was concentrated by rotary evaporation. The residue was purified by column chromatography (7:3 Hexanes/EtOAc) to give the desired compound 2-7 as a clear solution (500 mg, 80%). R _f = 0.3 (95:5 hexanes/EtOAc). ¹ H-NMR (400 MHz, CDCl ₃ ): δ 2.94-2.99 (m, 2H), 3.76-3.85 (m, 2H), 5.65-5.75 (m, 1H), 7.12-7.18 (m, 1H), 7.38 -7.43 (m, 1H), 7.55-7.62 (m, 1H), 8.49-8.54 (m, 1H).

Synthesis Example 1-10. H -Synthesis of Benzo[d][1,2,3]triazol-1-yl 2-(pyridin-2-yldisulfanyl)ethyl carbonate (2-8)

2-(pyridine-2-yl-disulfanyl) compound 2-7 (300 mg, 1.60 mmol, 1.0 equiv.), HOBt (490 mg, 3.20 mmol, 2 equiv.) and triethyl amine (0.22 mL, 1.60 mmol, 1 equivalent) was dissolved in CH ₃ CN (15 mL). Diphosgene (0.19 mL, 1.60 mmol, 1 eq) was added to the above mixture at 0 °C and stirred at 50 °C for 20 h. After the reaction was completed, the mixture was concentrated by rotary evaporation. The residue was dissolved in EtOAc (15 mL) and washed with a saturated aqueous NaHCO ₃ solution (2 x 10 mL). The combined organic extracts from the above were dried over anhydrous MgSO ₄ , filtered and concentrated by rotary evaporation. The residue was recrystallized from a 2:1 Hexane/CH ₂ Cl ₂ mixed solution to obtain the target compound 2-8 as a white solid (350 mg, 62%). ¹ H-NMR (400 MHz, CDCl ₃ ): δ 3.28 (t, J = 6.0 Hz, 2H), 4.82 (t, J = 6.0 Hz, 2H), 7.12-7.18 (m, 1H), 7.52-7.58 ( m, 1H), 7.70–7.82 (m, 3H), 7.98–8.04 (m, 1H), 8.21 (d, J = 8.4 Hz, 1H), 8.48 (d, J = 4.8 Hz 1H).

Synthesis Example 1-11. Compounds of Rapamycin-Carbonate conjugates (2-9):

Sirolimus (300 mg, 0.328 mmol, 1 equiv.) and Compound 2-8 (171 mg, 0.492 mmol, 1.5 equiv.) were dissolved in anhydrous CH ₂ Cl ₂ under argon gas. DMAP (60 mg, 0.492 mmol, 1.5 equiv) was added to the mixture and stirred at room temperature for 5 hours. After the reaction was completed, the mixture was concentrated by rotary evaporation and purified by column chromatography (7:3 → 2:3 Hexane/EtOAc) to obtain the target compound 2-9 as a white solid (250 mg, 67%). R _f = 0.4 (1:1 Hexane/EtOAc). ¹ H-NMR (400 MHz, CDCl ₃ ): δ 0.72-1.90 (m, 43H), 1.90-2.02 (m, 2H), 2.02-2.18 (m, 2H), 2.25-2.40 (m, 2H), 2.53 -2.63 (m, 1H), 2.67-2.79 (m, 1H), 3.05-3.24 (m, 6H), 3.24-3.74 (m, 11H), 3.80-3.93 (m, 1H), 4.14-4.20 (m, 1H), 4.31-4.42 (m, 2H), 4.42-4.55 (m, 1H), 4.74-4.82 (s, 1H), 5.12-5.20 (m, 1H), 5.24-5.32 (m, 1H), 5.38- 5.44 (m, 1H), 5.45-5.62 (m, 1H), 5.92-6.00 (m, 1H), 6.08-6.20 (m, 1H), 6.24-6.44 (m, 1H), 7.16-7.24 (m, 1H) ), 7.70–7.84 (m, 2H), 8.52 (d, J = 8.4 Hz, 1H).

Synthesis Example 1-12. Synthesis of ligand-rapamycin conjugate compounds

The prepared compound 2-9 (46 mg, 0.041 mmole, 1 equivalent) and the prepared 2-6 (15 mg, 0.041 mmole, 1 equivalent) were dissolved in anhydrous DMF (0.5 mL) under argon gas. The mixture was stirred at room temperature. After the reaction was completed, the mixture was concentrated by rotary evaporation. The residue was purified by column chromatography (49:1 → 9:1 CH ₂ Cl ₂ /MeOH) to obtain the compound of Formula 2 as a clear liquid (36 mg, 63%). R _f =0.3 (95:5 CH ₂ Cl ₂ /MeOH). ¹ H-NMR (400 MHz, CDCl ₃ ): δ 0.78-1.89 (m, 43H), 1.89-2.23 (m, 12H), 2.21-2.90 (m, 4H), 2.90-3.05 (m, 3H), 3.05 -3.23 (m, 5H), 3.22-3.45 (m, 8H), 3.5-3.75 (m, 3H), 3.75-3.90 (m, 12H), 4.12-4.30 (m, 1H), 4.30-4.44 (m, 3H), 4.44-4.56 (m, 1H), 4.76 (bs, 1H), 5.04-5.20 (m, 1H), 5.22-5.30 (m, 1H), 5.36-5.44 (m, 1H), 5.44-5.60 ( m, 1H), 5.80–6.04 (m, 1H), 6.06–6.20 (m, 1H), 6.24–6.44 (m, 2H), 6.72 (t, J = 5.2 Hz, 1H).

Synthesis Example 1-13. Synthesis of rapamycin-ligand conjugate compound of Formula 2

The prepared compound rapamycin-carbonate 2-9 (30 mg, 0.026 mmol, 1 equivalent) and the prepared compound thiol 3-2 (14 mg, 0.026 mmol, 1 equivalent) were mixed in anhydrous DMF (0.5 mL) under argon gas. After melting, the mixture was stirred at room temperature for 4 hours. After the reaction was completed, the mixture was concentrated by rotary evaporation. The residue was washed with a 1:1 mixture of Hexane/EtOAC (5 x 5 mL). The residue was evaporated to dryness under reduced pressure to obtain the desired compound of Formula 2 as a white solid (25 mg, 72%). ¹ H-NMR (400 MHz, CD ₃ OD): δ 0.75-1.55 (m, 27H), 1.55-2.22 (m, 24H), 2.22-2.38 (m, 2H), 2.55-2.65 (m, 1H), 2.75 -2.86 (m, 1H), 2.95-3.09 (m, 3H), 3.08-3.59 (m, 20H), 3.65-3.73 (m, 1H), 3.95-4.25 (m, 3H), 4.25-4.42 (m, 4H), 4.42-4.52 (m, 1H), 5.02-5.19 (m, 2H), 5.19-5.30 (m, 1H), 5.4-5.51 (m, 1H), 6.05-6.35 (m, 3H), 6.38- 6.51 (m, 1H). ³¹ P-NMR (162 MHz, CDCl ₃ ): δ 18.80 ppm. ESI-HRMS m/z calcd for C ₆₁ H ₉₇ N ₂ O ₂₂ P ₂ S ₂ [MH] ^- : 1337.5248, found: 1338.5245.

Synthesis Example 2. Synthesis of Compound of Formula 3

The synthesis process of the compound of Formula 3 is shown in Scheme 3 below, and the specific synthesis process of the intermediate stage compound is described below.

[Scheme 3]

Synthesis Example 2-1. Synthesis of 2,5-Dioxopyrrolidin-1-yl 3-(tritylthio)propanoate (16-10)

3-(tritylthio)propionic acid (500 mg, 1.436 mmol, 1 equiv) and NHS (165.2 mg, 1.436 mmol, 1 equiv) were dissolved in anhydrous THF (5 mL) under argon gas. THF (3 mL) in which DCC was dissolved was added dropwise to the above mixture at 0° C., warmed to room temperature over 1 hour, and stirred at room temperature for 3 hours. After the reaction was completed, DCC urea was removed by filtration with CELITE, and the filtered solid was washed with THF (2 mL). The filtrate was evaporated to dryness under reduced pressure, and the residue was recrystallized from EtOAc to obtain the target compound 2-10 (430 mg, 71%). ¹ H-NMR (400 MHz, CDCl ₃ ): δ 2.34-2.42 (m, 2H), 2.48-2.58 (m, 2H), 2.78 (s, 4H), 7.15-7.24 (m, 3H), 7.25-7.31 (m, 7H), 7.40–7.45 (m, 5H).

Synthesis Example 2-2. Synthesis of tetramethyl (4-(3-(tritylthio)propanamido)butane-1,1-diyl)bis(phosphonate) (4-1)

The prepared compound 2-4 (50 mg, 0.124 mmol, 1 equivalent) and the prepared compound NHS ester 2-10 (55.1 mg, 0.124 mmol, 1 equivalent) were dissolved in anhydrous DMF (1 mL) under argon gas. Triethylamine (Et ₃ N) (26 mL, 0.186 mmol, 1.5 equiv.) was added dropwise to the mixture, and the compound was stirred at room temperature for 3 hours. After the reaction was completed, the reaction mixture was added to a cooled saturated aqueous solution of NH ₄ Cl (10 mL) and stirred for 10 minutes. The reaction mixture was extracted with EtOAc (2 x 10 mL). The extracted organic layer was washed with brine, dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated by rotary evaporation. The residue was purified by column chromatography (95:5 CH ₂ Cl ₂ /MeOH) to obtain the desired compound 4-1 as a clear liquid (51 mg, 67%). R _f =0.3 (95:5 CH ₂ Cl ₂ /MeOH). ¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.65-1.79 (m, 2H), 1.82-2.0 (m, 2H), 2.05 (t, J = 6.4 Hz, 2H), 2.33 (tt, J = 24.0 , 6.0 Hz, 1H), 2.49 (t, J = 6.4 Hz, 2H), 3.15–3.25 (m, 2H), 3.70–3.85 (m, 12H), 7.16–7.24 (m, 3H), 7.24–7.32 ( m, 6H), 7.38–7.44 (m, 6H).

Synthesis Example 2-3. Synthesis of tetramethyl (4-(3-mercaptopropanamido)butane-1,1-diyl)bis(phosphonate) (4-2)

The prepared compound 4-1 (51 mg, 0.082 mmol, 1 equivalent) was dissolved in a mixture of trifluoroacetic acid (1 mL) and H ₂ O (0.3 mL), and triethylsilane (0.05 mL, 0.329 mmol, 4 equivalents) was added to the mixture. added. The mixture was stirred at room temperature for 30 minutes. After the reaction was completed, the mixture was concentrated by rotary evaporation. The residue was washed with a mixture of hexane (3 x 5 mL) and Hexane:EtOAC 95:1 (5 mL). The residue was evaporated to dryness under reduced pressure to obtain the target compound 4-2 as a clear liquid (25 mg, 80%). ¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.72-1.82 (m, 2H), 1.84-2.00 (m, 2H), 2.48 (t, J = 7.2 Hz, 2H), 2.66-2.98 (m, 3H) ), 3.21 (t, J = 7.2 Hz, 2H), 3.76–3.86 (m, 12H).

Synthesis Example 2-4. Synthesis of rapamycin-ligand conjugate compounds

The prepared compound rapamycin-carbonate 2-9 (18 mg, 0.016 mmol, 1 equivalent) and the prepared 4-2 (7.2 mg, 0.019 mmol, 1 equivalent) were dissolved in anhydrous DMF (0.5 mL) under argon gas. The mixture was stirred at room temperature. After the reaction was completed, the mixture was concentrated by rotary evaporation. The residue was purified by column chromatography (49:1→9:1 CH ₂ Cl ₂ /MeOH) to obtain the target compound as a clear liquid (16 mg, 71%). R _f =0.3 (95:5 CH ₂ Cl ₂ :MeOH). 1H ^- NMR (400 MHz, CDCl ₃ ): δ 0.78-1.85 (m, 41H), 1.85-2.20 (m, 14H), 2.20-2.50 (m, 4H), 2.55-2.65 (m, 3H), 2.65 -2.80 (m, 2H), 2.88-3.05 (m, 4H), 3.05-3.50 (m, 10H), 3.50-3.72 (m, 2H), 3.72-3.90 (m, 12H), 4.10-4.32 (m, 1H), 4.32-4.45 (m, 2H), 4.45-4.60 (m, 1H), 4.72-5.0 (m, 1H), 5.05-5.2 (m, 1H), 5.20-5.35 (m, 1H), 5.35- 5.45 (m, 1H), 5.45–5.60 (m, 1H), 5.65–6.05 (m, 1H), 6.05–6.20 (m, 1H), 6.20–6.45 (m, 2H).

Synthesis Example 2-5. Synthesis of (1-Hydroxy-4-((3-(tritylthio)propanoyl)oxy)butane-1,1-diyl)diphosphonic acid (5-1)

Alendronic acid (18.6 mg, 0.074 mmol, 1 equiv) was dissolved in H ₂ O (0.5 mL), triethylamine (Et ₃ N) (0.06 mL, 0.444 mmol, 6 equiv) was added and stirred for 5 minutes. . NHS ester 2-2 (50 mg, 0.112 mmol, 1.5 eq) was dissolved in anhydrous DMF (0.5 mL) and added to the above mixture. When a white precipitate formed, additional triethylamine (Et ₃ N) (0.06 mL, 6 eq) was added and the mixture was stirred at room temperature for 1 hour and concentrated by rotary evaporation. The residue was washed with EtOAc (4 x 2 mL), and the remaining residue was rotary evaporated to obtain the target compound 5-1 as a white solid (60 mg, 62 % based on ¹ H-NMR). ¹ H-NMR (400 MHz, D ₂ O): δ 1.54-1.68 (m, 2H), 1.68-1.84 (m, 2H), 2.03 (t, J = 6.8 Hz, 2H), 2.27 (t, J = 6.8 Hz, 2H), 2.95-3.10 (m, 4H), 7.10-7.24 (m, 9H), 7.26-7.34 (m, 6H) ppm. ESI-HRMS m/z calcd for C ₂₆ H ₃₀ NO ₈ P ₂ S[MH] ^- : 578.1180, found: 578.1215.

Synthesis Example 2-6. (1-Hydroxy-4-(3-mercaptopropanamido)butane-1,1-diyl)diphosphonic acid (5-2)

The prepared compound 5-1 (60 mg, 0.076 mmol, 1 equivalent) was dissolved in a mixture of trifluoroacetic acid (1.5 mL) and H ₂ O (0.4 mL), and triethylsilane (0.05 mL, 0.52 mmol, 4 equivalents) was added thereto. The mixture was stirred at room temperature for 30 minutes. After the reaction was completed, the mixture was concentrated by rotary evaporation. The residue was washed with EtOAC (4 x 3 mL). The remaining residue was evaporated to dryness under reduced pressure to obtain the target compound 5-2 as a white solid (33 mg, 80%). ¹ H-NMR (400 MHz, D ₂ O): δ 1.58-1.70 (m, 2H), 1.74-1.88 (m, 2H), 2.36 (t, J = 6.8 Hz, 2H), 2.58 (t, J = 6.8 Hz, 2H), 3.06 (t, J = 6.8 Hz, 2H).

Synthesis Example 2-7. Rapamycin-ligand conjugate Synthesis of Compounds of Formula 3:

The above-prepared compound rapamycin-carbonate 2-9 (56 mg, 0.05 mmole, 1.3 equivalent) and the above-prepared thiol compound 5-2 (13 mg, 0.038 mmole, 1 equivalent) were mixed in anhydrous DMF (0.5 mL) under argon gas. After melting, the mixture was stirred at room temperature for 4 hours. After the reaction was completed, the mixture was concentrated by rotary evaporation. The residue was washed with a 1:1 mixture of Hexane/EtOAC (5 x 5 mL). The remaining residue was evaporated to dryness under reduced pressure to obtain the target compound of Formula 3 as a white solid (28 mg, 55%). ¹ H-NMR (400 MHz, CD ₃ OD): δ 0.75-1.54 (m, 25H), 1.54-2.35 (m, 23H), 2.55-2.65 (m, 4H), 2.9-3.05 (m, 6H), 3.05-3.50 (m, 16H), 3.5-3.75 (m, 2H), 3.96-4.2 (d, J = 5.2 Hz, 1H), 4.04-4.20 (m, 2H), 4.3-4.42 (m, 4H), 4.42-4.54 (m, 1H), 5.02-5.18 (m, 2H), 5.2-5.28 (m, 1H), 5.42-5.5 (m, 1H), 6.06-6.34 (m, 4H), 6.38-6.52 (m , 1H) ppm. ESI-HRMS m/z calcd for C ₆₁ H ₉₇ N ₂ O ₂₃ P ₂ S ₂ [MH] ^- : 1351.5404, found: 1351.5395.

Synthesis Example 3. Synthesis of Compound of Formula 4

The synthetic process of the compound of Chemical Formula 4 is shown in Reaction Scheme 4 below, and the specific synthetic process of the intermediate compound is described below.

[Scheme 4]

Synthesis Example 3-1. Synthesis of 3-(Pyridin-2-yldisulfanyl)propanoic acid (6-1)

2,2-dipyridyl disulfide (2 g, 9.09 mmol, 1.5 equivalent) was dissolved in methanol (20 mL) under argon gas, and degassed for 15 minutes with argon gas. 3-mercaptopropionic acid (0.52 mL, 6.06 mmol, 1 equivalent) was added dropwise to the mixture and stirred for 3 hours. After the reaction was completed, the residue was purified by column chromatography (85:15 → 70:30 Hexane/EtOAc) to obtain the target compound 23-1 as a white solid (912 mg, 70%). R _f =0.3 (50:50 Hexane:EtOAc). ¹ H-NMR (400 MHz, CDCl ₃ ): δ 2.80 (t, J = 8.0 Hz, 2H), 3.06 (t, J = 8.0 Hz, 2H), 7.10-7.20 (m, 1H), 7.63-7.72 ( m, 2H), 8.45–8.51 (m, 1H).

Synthesis Example 3-2. Synthesis of Rapamycin ester conjugate (6-2)

Sirolimus (100 mg, 0.109 mmol, 1 equiv), compound 6-1 prepared above (23.4 mg, 0.109 mmol, 1.0 equiv) and DCC (27 mg, 0.109 mmol, 1 equiv) were mixed at room temperature with anhydrous CH ₂ Cl ₂ (2 mL). DMAP (3 mg, 0.021 mmol, 0.2 equiv) was added to the mixture and stirred at room temperature for 2 hours. After the reaction was completed, DCC urea was removed by filtration with CELITE, and the remaining solid was washed with DCM (2 mL). The filtrate was evaporated to dryness under reduced pressure and purified by column chromatography (3:2 → 2:3 Hexane/EtOAc) to obtain the target compound 6-2 as a white solid (40 mg, 33%). R _f = 0.3 (1:1 Hexane/EtOAc). ¹ H-NMR (400 MHz, CDCl ₃ ): δ 0.72-1.90 (m, 44H), 1.90-2.15 (m, 4H), 2.24-2.40 (m, 2H), 2.50-2.90 (m, 5H), 2.95 -3.20 (m, 5H), 3.25-3.50 (m, 7H), 3.52-3.92 (m, 3H), 4.16-4.30 (m, 1H), 4.60-4.74 (m, 1H), 4.77 (bs, 1H) ( m, 1H), 6.22–6.44 (m, 2H), 7.08–7.18 (m, 1H), 7.64–7.78 (m, 2H), 8.44–8.54 (m, 1H). ESI-HRMS m/z calcd for C ₅₉ H ₈₆ N ₂ O ₁₄ NaS ₂ [M+Na] ⁺ : 1133.5413, found: 1133.5407.

Synthesis Example 3-3. Synthesis of rapamycin-ligand conjugate compound of Formula 4

Compound rapamycin-ester 6-2 (22 mg, 0.02 mmol, 1 equivalent) prepared above and compound 3-2 (6 mg, 0.02 mmol, 1 equivalent) prepared above were dissolved in anhydrous DMF (0.4 mL) under argon gas. After stirring at room temperature for 4 hours, the mixture was concentrated by rotary evaporation. The residue was washed with a 1:1 mixed solution of Hexane/EtOAC (5 x 4 mL). The remaining residue was evaporated to dryness under reduced pressure to obtain the desired compound of Formula 4 as a white solid (12 mg, 45%). ¹ H-NMR (400 MHz, CD ₃ OD): δ 0.72-1.88 (m, 45H), 1.88-2.52 (m, 7H), 2.72-2.85 (m, 4H), 2.92-3.05 (m, 4H), 3.05-3.37 (m, 15H), 3.37-3.80 (m, 5H), 3.95-4.40 (m, 2H), 4.60-4.72 (m, 1H), 5.05-5.15 (m, 2H), 5.15-5.40 (m , 2H), 5.4–5.6 (m, 1H), 6.05–6.22 (m, 2H), 6.22–6.36 (m, 1H), 6.40–6.52 (m, 1H). ESI-HRMS m/z calcd for C ₆₀ H ₉₅ N ₂ O ₂₂ P ₂ S ₂ [MH] ^- : 1321.5299, found: 1321.5298.

Synthesis Example 4. Synthesis of Compound of Formula 5

The synthetic process of the compound of Chemical Formula 5 is shown in Reaction Scheme 5 below, and the specific synthetic process of intermediate compounds is described below.

[Scheme 5]

Synthesis Example 4-1. Synthesis of Rapamycin diester conjugate 7-1:

Sirolimus (100 mg, 0.109 mmol, 1 equiv), compound 6-1 prepared above (70.5 mg, 0.328 mmol, 3.0 equiv) and DCC (67.5 mg, 0.328 mmol, 3 equiv) were mixed with anhydrous EtOAc (3 equiv.) at room temperature. mL) was dissolved. DMAP (4 mg, 0.032 mmol, 0.3 equivalent) was added to the mixture and stirred at room temperature for 2 hours. After the reaction was completed, DCC urea was removed by filtration with CELITE, and the remaining solid was washed with EtOAc (2 mL). The filtrate was evaporated to dryness under reduced pressure and purified by column chromatography (3:2 → 2:3 Hexane/EtOAc) to obtain the target compound 7-1 as a white solid (82 mg, 68%). R _f = 0.3 (1:1 Hexane/EtOAc). ¹ H-NMR (400 MHz, CDCl ₃ ): δ 8.52-8.44 (m, 2H), 7.80-7.64 (m, 4H), 7.18-7.08 (m, 2H), 6.46-6.22 (m, 2H), 6.22 -6.08 (m, 1H), 6.05--9.96 (m, 1H), 5.62-5.50 (m, 1H), 5.46-5.30 (m, 2H), 5.30-5.23 (m, 1H), 5.22-5.12 (m , 1H), 3.90-3.80 (m, 2H), 3.67 (t, J = 7.6 Hz, 1H), 3.63-3.52 (m, 1H), 3.41-3.26 (m, 1H), 3.22-3.10 (m, 5H) ), 3.10-2.96 (m, 4H), 2.82-2.72 (m, 4H), 2.66-2.50 (m, 2H), 2.40-2.24 (m, 2H), 2.15-2.05 (m, 2H), 2.04-1.93 (m, 3H), 1.92-1.82 (m, 1H), 1.82-1.76 (m, 2H), 1.76-1.72 (m, 4H), 1.72-1.64 (m, 6H), 1.64-1.54 (m, 2H) ( m, 3H), 0.94–0.74 (m, 4H). ESI-HRMS m/z calcd for C ₆₇ H ₉₃ N ₃ O ₂₅ S ₄ Na [M+Na] ⁺ : 1330.5382, found: 1330.5388.

Synthesis Example 4-2. Synthesis of rapamycin diester conjugate compound of Formula 5:

Compound rapamycin-ester 7-1 (90 mg, 0.081 mmole, 1.2 equivalent) prepared above and compound 3-2 (22 mg, 0.067 mmole, 1 equivalent) prepared above were dissolved in anhydrous DMF (0.25 mL) under argon gas. It was stirred for 5 hours at room temperature. The reaction mixture was concentrated by rotary evaporation and washed with a 1:1 mixed solution of Hexane/EtOAC (5 x 5 mL). The residue was evaporated to dryness under reduced pressure to obtain the desired compound of Formula 5 as a white solid ( 13 mg ). ¹ H-NMR (400 MHz, CD ₃ OD): δ 6.50-6.38 (m, 1H), 6.36-6.24 (m, 1H), 6.24-6.16 (m, 1H), 6.16-6.08 (m, 1H), 5.56-5.42 (m, 1H), 5.38-5.28 (m, 1H), 5.14-5.01 (m, 2H), 4.74-4.59 (m, 1H), 4.29 (d, J = 4.0 Hz, 1H), 3.76- 3.64 (m, 1H), 3.60-3.52 (m, 1H), 3.52-3.32 (m, 11H), 3.30-3.18 (m, 14H), 3.18-3.10 (m, 4H), 3.06-2.90 (m, 4H) ), 2.88-2.62 (m, 5H), 2.50-2.04 (m, 10H), 1.99-1.57 (m, 19H), 1.57-1.27 (m, 17H), 1.18-1.02 (m, 6H), 1.02-0.78 (m, 9H).

Synthesis Example 5. Synthesis of Compound of Formula 6

The synthesis process of the compound of Formula 6 is shown in Scheme 6 below, and the specific synthesis process of the intermediate stage compound is described below.

[Scheme 6]

Synthesis Example 5-1. Synthesis of Compounds of Formula 6

Compound rapamycin-ester 2-9 (26 mg, 0.023 mmole, 1 equiv) and compound 5-2 (9.6 mg, 0.028 mmol, 1.2 equiv) prepared above were dissolved in anhydrous DMF (0.4 mL) under argon gas. It was stirred for 6 hours at room temperature. The reaction mixture was concentrated by rotary evaporation and washed with a 1:1 mixture of Hexane/EtOAC (5 x 5 mL). The residue was evaporated to dryness under reduced pressure to obtain the target compound , Compound 6, as a white solid (22 mg, 72%). ¹ H-NMR (400 MHz, CD ₃ OD): δ 6.51-6.40 (m, 1H), 6.38-6.20 (m, 2H), 6.20-6.05 (m, 1H), 5.47 (dd, J = 9.6, 14.8 Hz, 1H), 5.24 (d, J = 10 Hz, 1H), 5.18-5.0 (m, 2H), 4.74-4.58 (m, 2H), 4.20-4.16 (m, 1H), 3.99 (d, J = 5.2 Hz, 1H), 3.74-3.64 (m, 1H), 3.60-3.50 (m, 1H), 3.44-3.34 (m, 6H), 3.29-3.26 (m, 2H), 3.26-3.16 (m, 13H) , 3.14 (s, 3H), 3.02-2.89 (m, 9H), 2.82-2.70 (m, 5H), 2.66-2.54 (m, 6H), 2.14-1.96 (m, 7H), 1.96-1.85 (m, 5H), 1.85-1.78 (m, 3H), 1.78-1.68 (m, 5H), 1.68-1.56 (m, 3H), 1.56-1.37 (m, 5H), 1.37-1.28 (m, 9H), 1.21- 1.09 (m, 3H), 1.09–1.02 (m, 4H), 1.02–0.93 (m, 4H), 0.93–0.74 (m, 6H).

Synthesis Example 6. Synthesis of Compound of Formula 7

The synthetic process of the compound of Chemical Formula 7 is shown in Reaction Scheme 7 below, and the specific synthetic process of intermediate compounds is described below.

[Scheme 7]

Synthesis Example 6-1. Maleimidopropionyl Rapamycin (9-5)

Maleimidopropionic acid (72.1 mg, 426 μmol, 1.3 equiv), EDCI (94.3 mg, 492 μmol, 1.5 equiv) and DMAP (8.01 mg, 65.6 μmol, 0.2 equiv) were added to anhydrous CH ₂ Cl ₂ (5 mL) under argon gas. was dissolved in, and the mixture was stirred at room temperature for 15 minutes. A solution of sirolimus (300 mg, 328 μmol, 1.0 equiv) in anhydrous CH ₂ Cl ₂ (1 mL) was added at 0 °C and stirred at 0 °C for 1 hour. The reaction mixture was diluted in CH ₂ Cl ₂ (5 mL), saturated aqueous NH ₄ Cl solution (5 mL) was added, and then extracted with CH ₂ Cl ₂ (3 x 5 mL). The organic layer was dried over anhydrous MgSO ₄ , filtered and concentrated by rotary evaporation. The residue was purified by column chromatography (1:1 → 1:1.5 → 1:2 Hexane:EtOAc) to obtain the desired compound 9-5 as a yellow solid (197 mg, 185 μmol, 56%). TLC : R _f 0.63 (2:1 EtOAc/Hexane). ¹ H-NMR confirmed a mixture of rotamers. Major rotamer: ¹ H-NMR (400 MHz, benzene- d ₆ ): δ 6.48 (d, J = 10.4 Hz, 1H), 6.35 (d, J = 10.4 Hz, 1H), 6.19 (d, J = 10.4 Hz) , 1H), 6.02 (d, J = 10.4 Hz, 1H), 5.70 (s, 2H), 5.56–5.47 (m, 3H), 5.41 (m, 1H), 5.11 (s, 1H), 4.93 (m, 1H), 4.24 (dm, J = 5.6 Hz, 1H), 4.10 (m, 1H), 3.85 (m, 1H), 3.67-3.53 (m, 4H), 3.47 (m, 1H), 3.45 (d, J = 6.0 Hz, 1H), 3.26-3.04 (m, 3H), 3.23 (s, 3H), 3.18 (s, 3H), 3.13 (s, 3H), 2.77 (m, 1H), 2.73 (dd, J = 17.2, 6.4 Hz, 1H), 2.60 (d, J = 17.2, 6.4 Hz, 1H), 2.53-2.41 (m, 2H), 2.20-2.09 (m, 3H), 2.08-1.95 (m, 3H), 1.71 -1.47 (m, 4H), 1.70 (s, 3H), 1.62 (s, 3H), 1.42-1.26 (m, 10H), 1.23-0.73 (m, 5H), 1.17 (d, J = 6.8 Hz, 3H ), 1.10 (d, J = 6.8 Hz, 3H), 1.02 (d, J = 6.8 Hz, 3H), 0.93 (d, J = 6.8 Hz, 3H), 0.81 (d, J = 6.8 Hz, 3H). HRMS (ESI) m/z calculated for C ₅₈ H ₈₄ N ₂ NaO ₁₆ ⁺ [M+Na] ⁺ 1464.6540, found 1464.6523.

Synthesis Example 6-2. Synthesis of compound of formula 7 of rapamycin-succinimide-alendronate conjugate

The prepared compound rapamycin-ester 9-5 (28.9 mg, 27.2 μmol, 1.3 equivalent) and the prepared compound thiol 10-2 (8.1 mg, 20.9 μmol, 1 equivalent) were mixed in anhydrous DMF (0.25 mL) under argon gas. melted Triethylamine (Et ₃ N) (1.17 μL, 8.36 μmol, 0.4 equivalent) was added to the mixture and the mixture was stirred at room temperature for 5 hours. The reaction mixture was concentrated by rotary evaporation, and the residue was washed with diethyl ether (3 x 5 mL) and evaporated to dryness to obtain the desired compound of Formula 7 as a white solid (20 mg, 14.3 μmol, 68%). ¹ H-NMR confirmed a mixture of rotamers. Major rotamer: TLC : R _f 0.01 (10:1 CH ₂ Cl ₂ /MeOH). ¹ H-NMR (400 MHz, CD ₃ OD): δ6.47((d, J = 10.8 Hz, 1H), 6.45 (d, J = 10.8 Hz, 1H), 6.29 (d, J = 10.4 Hz, 1H ), 6.23 (d, J = 13.6 Hz, 1H), 6.16 (d, J = 10.4 Hz, 1H), 6.10 (d, J = 10.4 Hz, 1H), 5.49 (d, J = 10.0 Hz, 1H), 5.45 (d, J = 10.0 Hz, 1H), 5.24 (d, J = 10.4 Hz, 1H), 5.13-5.07 (m, 2H), 4.64 (m, 1H), 4.18 (d, J = 4.4 Hz, 1H) ), 3.98 (d, J = 5.2 Hz, 1H), 3.93 (dd, J = 8.8, 4.0 Hz, 1H), 3.79–3.72 (m, 2H), 3.69 (d, J = 10.8 Hz, 1H), 3.55 (d, J = 13.6 Hz, 2H), 3.46-3.14 (m, 9H), 3.39 (s, 3H), 3.28 (s, 3H), 3.15 (s, 3H), 3.00-2.93 (m, 2H), 2.83 (dm, J = 14.4 Hz, 1H), 2.68-2.60 (m, 3H), 2.58-2.53 (m, 3H), 2.52-2.44 (m, 3H), 2.32-2.11 (m, 5H), 2.06- 2.00 (m, 3H), 1.96-1.88 (m, 4H), 1.83 (s, 3H), 1.76-1.56 (m, 4H), 1.72 (s, 3H), 1.49-1.37 (m, 5H), 1.22- -80 (m, 4H), 1.05 (d, J = 6.8 Hz, 3H), 1.00 (d, J = 6.8 Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H), 0.89 (d, J = 6.8 Hz, 3H) 6.8 Hz, 3H), 0.85 (d, J = 6.8 Hz, 3H) HRMS (ESI) m/z calculated for C ₆₅ H ₁₀₀ N ₃ O ₂₄ P ₂ S ^- [MH] ^- 1400.5898, found 1400.5886.

Experimental Example 1: mTORC1 pathway inhibition test in multiple myeloma RPMI8226 cells

In order to confirm the extent to which the synthesized sirolimus-conjugate inhibits endogenous mTOR activity, the phosphorylated S6 protein was measured by western blot after treatment with the sirolimus-conjugate.

Multiple myeloma RPI8226 cells were purchased from Korean Cell Line Bank (KCLB, Seoul, Korea). Multiple myeloma _RPI8226 cells were cultured in RPMI medium (RPMI-1640, 10% fetal bovine serum (heat-inactivated) plus 50 U/ml penicillin, and 50 μg /ml streptomycin) and maintained for growth. Growth medium components and other chemicals were purchased from ThermoFisher (Waltham, MA, USA) and used. For quality control, mycoplasma infection was regularly checked using the Universal Mycoplasma Detection Kit (American Type Culture Collection (ATCC), Manassas, VA, USA) (ATCC30-1012K).

The sirolimus conjugates mimicked the bone tumor cell environment in which cleavage was induced, and were treated in a weakly acidic buffer (citrate buffer at pH 4.8) at a concentration of 50 uM at 37° C. for 1 hour, and then neutralized to pH 7. The final target pH was adjusted by mixing 0.2M Na ₂ HPO ₄ and 0.1 M citric acid. A sirolimus conjugate treated with PBS was used as a negative control for uncleaved compounds. In RPMI medium, RPMI8226 cells (40,000 cells per sample in 96-well plate) were treated with 100 uL of 90 nM cleaved/uncleaved sirolimus conjugate for 2 hours, followed by lysis buffer (150 mM NaCl, cocktail of 1% NP-40, 50 mM Tris-Cl pH 8, 2 mM EDTA + protease inhibitors (Roche, Basel, Switzerland)).

Cell lysates of 10,000 cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% reducing gel) and separated by polyvinylidene difluoride membranes. ) (Millipore, Bedford, MA, USA). After blocking the membrane in 5% skim milk (Difco/Becton Dickinson, Franklin Lakes, NJ, USA), phospho-S6 ribosomal protein (Ser240/244) mAb (Cell Signaling Technology, Beverly, MA) , USA) overnight at 4 °C and washed with TBST (10 mM Tris, 140 mM NaCl, 0.1% Tween-20, pH 7.6).

Membranes were incubated with the appropriate secondary antibody for 3 hours at room temperature and washed again with TBST. Western blot bands were visualized by enhanced chemiluminescence (ECL) (Luminata Forte; Millipore). Subsequently, the membrane was injected again with β-actin Ab to compare that the amount of the cell lysate sample was the same, and the results are shown in FIG. 2 .

In FIG. 2, * is a comparison at low concentration and high concentration as follows after treating the compound at 37 ^o C, pH 4.8 for 10 minutes. Compound concentration: L (15 nM) / H (90 nM) IM9 cell line (MM) Parental rapamycin was used as a positive control, and untreated cells were used as a negative control.

As a result of the above experiment, the compound of Formula 4 showed a better effect at a lower pH than neutral pH and a high dose, and three conjugates (compounds of Synthesis Example 1-12, compound of Synthesis Example 2-4 and Synthesis Example 6-2) compound) showed effects at all concentrations, including neutral pH, low concentration pH, and test.

Experimental Example 2: Hydroxyapatite Bead Binding Test

In order to quantify the degree of binding of alendronate-sirolimus conjugates to hydroxyapatite beads, an experiment was performed as follows. The degree to which the test compound competitively inhibits the binding of fluorescein isothiocyanate (FITC)-conjugated alendronate to the hydroxyapatite matrix was quantified by enzyme-linked immunosorbent assay (ELISA). 0.5 g of dry hydroxyapatite (HAP) beads (average diameter 40 μm) (cat # 1584000, Bio-Rad, Hercules, CA, USA) were reconstituted in 1 ml of phosphate buffered saline (PBS) for 3 h at room temperature. Then, they were washed three times with PBS and resuspended in PBS containing 1% bovine serum albumin (BSA). Reconstituted beads were stored at 4 °C. 50 μL of the bead slurry was dispensed into a round bottom 96-well plate and incubated with 150 μL of the test compound in the presence of 10 nM FITC-conjugated alendronate. Binding to the hydroxyapatite beads was allowed to proceed on a shaking platform for 30 minutes at room temperature in the dark. The plate was centrifuged and the captured HAP beads were washed 3 times with 1% BSA in PBS. Anti-FITC antibody directly conjugated with horse-radish peroxidase (HRP) (cat # ab6656, Abcam, Cambridge, UK) was dissolved at a concentration of 0.2 μg/mL in PBS containing 1% BSA, followed by incubation with HAP beads. It was added to the plate wells and incubated for 30 minutes at room temperature on the plate-shaking-platform. Plate wells were then washed and the amount of bound FITC-conjugated alendronate was measured using a microtiter plate reader (Molecular Devices, Sunnyvale, CA) using the Ultra-TMB Substrate Solution Kit (cat # 34028, Thermo Fisher). , USA) was determined as absorbance reading. The results are shown in Figure 3.

Experimental Example 3: In vivo efficacy evaluation and safety evaluation of drug delivery system

(1) Determination of safe dose of drug delivery system

The compound of Formula 4 and the compound of Formula 5 were injected intraperitoneally (IP) in athymic nude mice to evaluate the safety of the compound of Formula 4 and the compound of Formula 5 and to determine the safe administration dose. And the study plot is shown in Figure 4.

0.25, 0.5, 1, 2 mg/kg of the drug in the presence of 0.8 mg/kg bortezomib (BTZ) twice weekly by I.P. The study was conducted by administering 2 mg/kg for mouse experiments.

(2) Evaluation of stability and toxicity of drug delivery system

When the compound of Formula 4 and the compound of Formula 5 were administered to nude mice in which the thymus was removed for 3 weeks, liver and kidney functions did not change. After administering the compound of Formula 4 and the compound of Formula 5 for a period of 3 weeks, the mice were sacrificed and blood was collected for liver and kidney function analysis. When compared to PBS (vehicle) treated mice, stability was confirmed at all drug doses and the results are shown in FIG. 5 .

As a result, as a result of comparing the drug-treated mice with the PBS-treated mice, the liver function markers for ALT and AST did not change. Compared to PBS-treated mice, renal function markers such as CRE (creatinine) and BUN (blood urea nitrogen) also showed normal functional ranges in all drug-treated groups. Therefore, it was confirmed that the drug delivery system developed in the present invention is safe within the range of use.

In addition, the weight of the mice was measured once a week to determine whether there was weight loss, and the results are shown in FIG. 6 . The compound of Formula 4 and the compound of Formula 5 did not affect body weight over 3 weeks of dosing, and there was no significant weight loss in all drug-treated experimental groups compared to PBS (vehicle)-treated mice.

(3) NOG (NOD/scid/IL-2R γ null) mouse experimental design

After injecting the multiple myeloma cell line (RPMI8226) into a mouse, a transformed multiple myeloma cell line (RPMI8226) into which the luciferase gene was inserted was secured in order to quantitatively measure the size change of cancer tumors in IVIS (In Vivo Imaging System). The confirmed results are shown in Figure 7,

Specifically, the experiment was conducted by receiving female NOG (NOD/scid/IL-2R γ null) mice at 4 weeks of age. On day 11 after mice received, 2x10 ⁶ RPMI-8226 luc + multiple myeloma (MM) cells per mouse were transplanted by intra-tibia injection. Animal experiments were performed according to IACUC-approved protocols. For tumor identification and randomization of mice, luminol signals were injected I, P, 10 min before imaging, and imaging of bone-resident tumors was performed with an in vivo imaging system (IVIS). IVIS imaging was performed weekly from day 0 to day 31 after treatment. Signal intensity was plotted and analyzed, and the experimental groups were classified as follows: control group, PBS (n = 5), chloroquine (CQ) (60 mg/kg IP, 5 times a week; 10 mg/kg) (n = 5), compound of formula 4 (4 mg/kg IV, once a week; continuously 8 mg/kg) (n = 5), CQ and compound of formula 5 (n = 5). Details of the multiple myeloma mouse model and drug treatment protocol are shown in FIG. 8 .

(4) Measurement of tumor size

An experiment to confirm the therapeutic effect of the compound of Formula 4 on primary multiple myeloma (MM) was performed on female NOG (NOD / scid / IL-2Rγ null) mice of the compound of Formula 4. Single administration or combined administration with chloroquine (CQ) was performed in parallel. As shown in FIG. 9, after 4 weeks of co-administration of the combination of the compound of Formula 4 and chloroquine, the tumor burden reduction effect was confirmed (p<0.05). In order to block autophagy and mTOR-dependent protein synthesis in RPMI8228 tumors present in the bone cavity, respectively, chloroquine and the compound of Formula 4 were used in combination.

The strategy used by the present inventors to maximize the therapeutic effect of multiple myeloma was to simultaneously block the mTOR signaling pathway (compound of Formula 4) and the autophagy (chloroquine, CQ) signaling pathway. That is, based on the dual-pathway co-blocking strategy that simultaneously targets the mTOR and autophagy pathways, the multiple myeloma therapeutic effect of the candidate material developed in the present invention could be maximized.

The combination of chloroquine and rapamycin/rapalog showed promise in clinical trials in patients with relapsed/refractory multiple myeloma, but was not fully tolerated for use in patients due to systemic side effects (clinical trial number: NCT01689987). Similar trials have confirmed that using a bone-directed rapalog in the form of a compound of Formula 4 has a high possibility of providing a better and more effective treatment and minimizing systemic side effects in patients.

(5) Mouse body weight change by combined treatment of chloroquine and the compound of Formula 4

The combination therapy of chloroquine and the compound of Formula 4 did not adversely affect the body weight of mice for 8 weeks after tumor transplantation and drug treatment. All four experimental mice (NOD/scid/IL-2R γ null) maintained a normal body weight for 8 weeks. That is, it was proved that the combination therapy of chloroquine/compound of Formula 4 was safe without weight loss toxicity within the treatment dose range.

(6) Confirmation of reduction in the size of multiple myeloid tumors induced by treatment with chloroquine and the compound of Formula 4 (IVIS system)

In order to quantify the reduction in tumor size by treatment with chloroquine and the compound of Formula 4, the IVIS system was utilized. IVIS images were measured and the results are shown in FIG.

(7) Results of identification of multiple myeloma cells in the tibia (tibial cell immunohistochemistry test using luciferase antibody)

After the mice were sacrificed on the 3rd day after the final drug treatment, the tibia region into which the multiple myeloid tumor cells were injected was cut and an immunohistochemistry (IHC) experiment was performed using an anti-luciferase antibody. and multiple myeloid tumor cells were identified in the tibia (Isotype, negative control group). A detailed image is shown in FIG. 12 .

To measure phospho-S6 (p-S6) protein, bones and bone cavities were demineralized and fixed with paraformaldehyde, and confirmed by IHC, with a compound of Formula 4. In the treated samples, it was confirmed that the level of p-S6 was reduced compared to the control group and the group treated with chloroquine alone (FIG. 13(A)).

Antibodies were treated on the same slide to minimize variation between samples, and microscope images were obtained under the same microscope parameter settings. The staining intensity by the p-S6 antibody was quantified using ImageJ software, and the results are shown in FIG. 13(B).

(8) Mouse tumor size measurement

To establish the optimal conditions for the treatment of multiple myeloma by the compound of Formula 4 and chloroquine combination therapy, and to lower the potential toxicity of chloroquine, a second experiment was conducted under conditions where the dose was reduced up to 6 times, and the results are shown in FIG. 14 was

In female NOG (NOD/scid/IL-2Rγ null) mice, the compound of Chemical Formula 4 was administered alone or in combination with chloroquine to determine whether tumor burden was reduced. In the second experiment (1 x 10 ⁶ cells), a smaller number of tumor cells were transplanted compared to the first experiment (2 x 10 ⁶ cells), which resulted in various tumor sizes at the beginning of administration.

All groups co-administered with the combination of the compound of Formula 4 and chloroquine showed a greater sensitivity of the tumor burden ( showed efficacy in reducing tumor burden.

In this second experiment, a lower level of chloroquine was used than in the first experiment by lowering the dose of chloroquine in order to reduce the masking effect of the compound of Formula 4 by chloroquine (CQ, CQ during the first week of treatment, 10 mg/kg vs. 60 mg/kg).

Since larger tumors showed higher sensitivity to the combination of chloroquine and compound 4, it is assumed that the environment of the larger tumor provides the low pH environment required for cleavage and activation of compound 4.

(9) Confirmation of reduction in the size of multiple myeloid tumors by combination therapy of low-dose chloroquine and the compound of Formula 4 (IVIS system)

Tibial RPMI8226 tumors were transplanted into second MM NOG (NOD/scid/IL-2R γ null) mice, treated with drugs, and IVIS images were measured and shown in FIG. 15 before sacrificing the mice.

When administered in combination with low-dose chloroquine and the compound of Formula 4, the effect of reducing the size of multiple myeloid tumors was effective enough to correspond to the results of the first experiment (FIG. 15, IVIS system). Therefore, from the results of the second experiment, it was found that the size of multiple myeloid tumors could be reduced even if the dose of chloroquine, which could exhibit potential toxicity, was lowered up to 6 times.

(10) Confirmation of tumor burden reduction effect through measurement of immunoglobulin lambda (IgL) concentration in blood

Since tumors formed by the multiple myeloma cell line (RPMI8226) secrete immunoglobulin lambda (IgL) into the blood, the concentration of IgL in the blood is a secondary indicator that can quantify the tumor burden. Therefore, in this experiment, after IVIS was measured and mice were sacrificed, peripheral blood plasma was measured by ELISA, and it was confirmed that the IgL concentration correlated with the tumor size IVIS value (FIG. 16).

As shown in FIG. 17, it was confirmed that the IgL concentration in the peripheral blood of mice when the compound of Formula 4 and chloroquine were co-administered was lower than the IgL concentration when chloroquine alone or PBS alone was administered. thus, It was confirmed that the effect of reducing the size of multiple myeloid tumors was maximized when the compound of Formula 4 and chloroquine were co-administered.

(11) Confirmation of efficacy in osteosarcoma

18 shows the result of IVIS image measurement after treatment with the compound of Formula 4 using a mouse with tibial osteosarcoma tumor (Osteosarcoma).

Osteosarcoma cells were a HOS cell line stably transfected with luciferase suitable for IVIS analysis, and IVIS intensities quantified according to parameters were recorded and displayed.

The tibial osteosarcoma mouse model was first established by the present inventors by injecting the HOS cell line, which is an osteosarcoma cell line, into the tibia.

(12) Securing sandwich ELISA methodology for pharmacokinetic experiments of the compound of Formula 4

For pharmacokinetic experiments, the present inventors secured a sandwich ELISA methodology as shown in FIG. 19 . In order to confirm the body distribution (eg, blood, urine and various organs) and concentration of the administered compound of Formula 4, tissue samples of mice administered with the compound of Formula 4 were collected, and then hydroxyapatite beads , HB).

In order to select the compound of formula 4 bound to hydroxyapatite beads, a 0.45 um filter was used to remove unbound molecules, the HB-compound complex of formula 4 was recovered, and HRP (horse radish peroxidase) By mixing with the bound antibody and rapamycin antibody, an HB and anti-Rapamycin-HRP sandwich was previously obtained.

The compound of Formula 4 with a targeting ligand was captured using hydroxyapatite beads, an in vitro bone tissue mimicking bone tissue, and then rapa-conjugated rapa using a commercially available anti-rapamycin antibody. The mycin moiety was detected.

Hydroxyapatite beads were incubated with the compound of Formula 4 for 1 hour at room temperature, and after blocking the beads with PBS, they were incubated with Rapamycin Sheep Ab for 30 minutes at room temperature. After washing the beads again, they were incubated with Sheep Ab HRP for 30 minutes at room temperature. After another round of washing, the beads were incubated with the TMB reagent at room temperature with periodic mixing for 30 minutes. It was measured by reading the absorbance at 450 nm in a spectrophotometer.

A dose-response curve contrasted with a negative control group (unconjugated rapamycin/no primary antibody) is shown in FIG. 20 . The assay method was sensitive and sufficient for detection in 100% serum. It was confirmed that the ELISA detection limit of the full-length compound of Formula 4 was 10 nM.

In order to verify the sensitivity of the sandwich ELISA method, after mixing rapamycin alone, secondary antibody alone (anti-sheep only), and the compound of Formula 4 with hydroxyapatite, the degree of binding to hydroxyapatite was measured by absorbance. As a result (Y-axis), it was confirmed that the compound of Formula 4 was compatible with bone within a sufficient sensitivity range (FIG. 20).

(13) Confirmation of the targeting property of the compound of Formula 4

The compound of Formula 4 and hydroxyapatite beads, a bone tissue mimetic, were incubated together at room temperature for 1 hour, the beads were blocked with PBS, and then incubated with rapamycin sheep Ab for 30 minutes at room temperature to quantify As a result, it was confirmed that the compound of Formula 4 binds to hydroxyapatite beads. On the other hand, it was confirmed that the sirolimus drug to which the targeting ligand was not attached did not bind to the hydroxyapatite beads (FIG. 21). The above experimental results show that the compound of Formula 4 developed in the present invention binds to bone tissue, and it was confirmed that the compound of Formula 4 has a targeting property.

As described above, the present invention has been described through examples. Those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing its technical spirit or essential features. Therefore, the above-described embodiments should be understood as illustrative in all respects and not limiting. The scope of the present invention is indicated by the following claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be construed as being included in the scope of the present invention.

Claims (13)

A compound represented by Formula 1, an optical isomer or a pharmaceutically acceptable salt thereof:
[Formula 1]
ABCD
in the expression
A is

,

,

and

Any one selected from the group consisting of
B is

or

ego,
C is

or

as,
l, m, n are each an integer of 1 to 5;
D is

or

, and R is H or OH. The method of claim 1, wherein A is

A compound, an optical isomer or a pharmaceutically acceptable salt thereof. The compound according to claim 1, wherein A is any one selected from the group consisting of sirolimus, everolimus and temsirolimus, an optical isomer or a pharmaceutically acceptable salt thereof. The compound according to claim 1, wherein B is a linker that is cleaved by an acid, an optical isomer or a pharmaceutically acceptable salt thereof. According to claim 1,
l and m are each 1 or 2;
n is an integer of any one of 1 to 3, a compound, an optical isomer or a pharmaceutically acceptable salt thereof. The compound of claim 1, wherein the compound represented by Formula 1 is any one selected from the group consisting of Formulas 2 to 7, an optical isomer or a pharmaceutically acceptable salt thereof:
[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

. A pharmaceutical composition for preventing or treating bone diseases comprising the compound according to any one of claims 1 to 6, an optical isomer or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the bone disease is osteoporosis, osteomyelitis, or osteomalacia. , Bone damage due to bone metastasis of cancer cells, osteosarcoma, multiple myeloma, bone metastatic prostate cancer, intervertebral disc herniation, fibrous osteodysplasia, Klippel-Pile syndrome, and cystic fibroosteitis, which is at least one member selected from the group consisting of bone A pharmaceutical composition for preventing or treating a disease. delete A health functional food composition for preventing or improving bone disease, comprising the compound according to any one of claims 1 to 6, an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the bone disease is osteoporosis or osteomyelitis. , osteomalacia, bone damage caused by bone metastasis of cancer cells, osteosarcoma, multiple myeloma, bone metastatic prostate cancer, intervertebral disc herniation, fibrous osteodysplasia, Klippel-Pile syndrome, and cystic fibroosteitis. , Health functional food composition for preventing or improving bone disease. delete A compound represented by Formula 1, an optical isomer or a pharmaceutically acceptable salt thereof; And chloroquine or a pharmaceutical composition for preventing or treating bone diseases comprising a pharmaceutically acceptable salt thereof, wherein the bone diseases include osteoporosis, osteomyelitis, osteomalacia, bone damage due to bone metastasis of cancer cells, osteosarcoma, multiple myeloma, bone metastatic prostate A pharmaceutical composition for preventing or treating bone diseases, which is at least one member selected from the group consisting of cancer, intervertebral disc herniation, fibrous osteodysplasia, Klipel-Pile syndrome and cystic fibroosteitis:
[Formula 1]
ABCD
in the expression
A is

,

,

and

Any one selected from the group consisting of
B is

or

ego,
C is

or

as,
l, m, n are each an integer of 1 to 5;
D is

or

, and R is H or OH. delete The pharmaceutical composition for preventing or treating bone diseases according to claim 11, wherein the bone disease is any one selected from the group consisting of bone damage caused by bone metastasis of cancer cells, osteosarcoma, and multiple myeloma.

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