KR102497821B1 - 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, Side effects such as nausea and reduced 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 -OZ이며, Z는 탄소 수 1 내지 3개의 알킬 또는 알킬렌이다.D is

or

, R is -OZ, and Z is an alkyl or alkylene having 1 to 3 carbon atoms.

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.

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

,

,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, l, m, n은 각각 1 내지 5 중 어느 하나의 정수이고, C is

or

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

또는

이며, R은 -OZ이며, Z는 탄소 수 1 내지 3개의 알킬 또는 알킬렌이다. D is

or

, R is -OZ, and Z is an alkyl or alkylene having 1 to 3 carbon atoms.

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. 구체적인 일 실시예에서, 상기 A는

또는

일 수 있다.The A is preferably A

,

,

and

It may be any one selected from the group consisting of. 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, l, m, n은 각각 1 내지 5 중 어느 하나의 정수일 수 있다.The C is preferably

or

As , l, m, n may each be any one integer from 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, in D, R is -H, -OH or -OZ, and Z may be methyl. 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 good ligands for bone-targeted therapy.

또는

이며, R은 -OZ이며, Z는 탄소 수 1 내지 3개의 알킬 또는 알킬렌이나, 이에 제한되는 것은 아니며 원하는 치료효과에 따라 적절한 임의의 비스포스포네이트가 사용될 수 있다.The D is preferably

or

, R is -OZ, and Z is alkyl or alkylene having 1 to 3 carbon atoms, 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 4:

[Formula 2]

[Formula 3]

[Formula 4]

.

.

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.

The compounds of Chemical Formulas 2, 3, and 4 may exhibit greater effects on bone diseases because of their superior targeting properties to bone targets compared to the other synthetic examples synthesized in the present invention.

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 typically 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 pharmaceutically acceptable excipients.

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.

For administration, the pharmaceutical composition of the present invention may further contain one or more pharmaceutically acceptable excipients in addition to the compound of Formula 1, its optical isomer or its pharmaceutically acceptable salt. 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, its optical isomer or its pharmaceutically acceptable salt.

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 diseases 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 the use of the compound of Formula 1, its optical isomer or its pharmaceutically acceptable salt for the preparation of a drug for the treatment of 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 compounds of Formulas 2 and 3 is shown in Reaction Schemes 1 and 2 below, and the specific synthesis process of intermediate compounds 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)

tBuOK (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 compound of Formula 2

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 compounds

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 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 compound of Formula 3

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 desired compound of Formula 3 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:

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 desired compound 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 Rapamycin-ligand conjugate compound

The synthesis process of the Rapamycin-ligand conjugate compound is shown in Scheme 4 below, and the specific synthesis process of intermediate compounds 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 compounds

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 target compound 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 Rapamycin diester conjugate compound

The synthetic process of the compound of Chemical Formula 7 is shown in Reaction Scheme 5, and the specific synthetic process of the intermediate stage compound 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) ( 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), 1.54-1.38 (m, 5H), 1.38-1.28 (m, 3H), 1.25-1.17 (m, 2H), 1.17-1.08 (m, 4H), 1.08-0.96 (m, 8H), 0.96-0.94 (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 compounds:

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 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 Rapamycin-ester conjugate compound

The synthetic process of the Rapamycin-ester conjugate compound is shown in Reaction Scheme 6 below, and the specific synthetic process of intermediate compounds is described below.

[Scheme 6]

Synthesis Example 5-1. Synthesis of rapamycin-ester conjugate compounds

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 desired compound 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 4

The synthetic process of the compound of Chemical Formula 4 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 dissolved in anhydrous CH ₂ Cl ₂ (5 mL) under argon gas. After melting, the mixture was stirred at room temperature for 15 minutes. A solution of sirolimus (300 mg, 328 μmol, 1.0 eq) 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 rapamycin-succinimide-bisphosphonate conjugate compound of Formula 4

The prepared compound rapamycin-ester 9-5 (25.0 mg, 23.5 μmol, 1 equivalent) and the prepared compound thiol 9-4 (8.9 mg, 23.5 μmol, 1 equivalent) were dissolved in anhydrous DMF (0.24 mL) at room temperature. Stir for 2 hours. Triethylamine (Et ₃ N) (1.3 μL, 9.4 μmol, 0.4 equivalent) was added to the mixture and stirred at room temperature for 19 hours. The reaction mixture was concentrated by rotary evaporation, and the residue was purified by column chromatography (40:1 → 20:1 CH ₂ Cl ₂ /MeOH) to obtain the desired compound of formula 4 as a white solid (16.2 mg, 11.5 μmol , 48%). TLC : R _f 0.55 (7:1 EtOAc/MeOH). ¹ H-NMR confirmed a mixture of rotamers. Major rotamer: ¹ H-NMR (400 MHz, benzene- d ₆ ): δ 6.42-6.34 (m, 2H), 6.33 (d, J = 10.0 Hz, 1H), 6.16 (d, J = 10.0 Hz, 1H) , 6.12 (d, J = 10.0 Hz, 1H), 5.97 (d, J = 10.0 Hz, 1H), 5.57 (d, J = 8.8 Hz, 1H), 5.53 (d, J = 8.8 Hz, 1H), 5.42 (d, J = 10.0 Hz, 1H), 5.28 (m, 1H), 5.17 (m, 1H), 4.18 (d, J = 6.4 Hz, 1H), 3.92–3.72 (m, 4H), 3.84 (s, 3H), 3.82 (s, 3H), 3.81 (s, 3H), 3.79 (s, 3H), 3.68-3.64 (m, 2H), 3.56 (d, J = 13.2 Hz, 2H), 3.45-3.37 (m , 5H), 3.36 (s, 3H), 3.33 (s, 3H), 3.22-3.06 (m, 4H), 3.14 (s, 3H), 3.05 (m, 1H), 2.74-2.64 (m, 4H), 2.61-2.51 (m, 3H), 2.37 (tt, J = 24.4, 6.0 Hz, 1H), 2.36-2.30 (m, 2H), 2.10-1.91 (m, 6H), 1.87-1.63 (m, 7H), 1.75 (s, 3H), 1.65 (s, 3H), 1.62-1.47 (m, 4H), 1.42-1.06 (m, 8H), 1.10 (d, J = 6.8 Hz, 3H), 1.05 (d, J = 6.8 Hz, 3H), 0.99 (d, J = 6.8 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3H), 0.91 (d, J = 6.8 Hz, 3H), 0.88–0.79 (m, 2H) . HRMS (ESI) m/z calculated for C ₆₉ H 10 ₉ N ₃ NaO ₂₃ P ₂ S ⁺ [M+Na] ⁺ 1464.6540, found 1464.6523.

Synthesis Example 6-3. Synthesis of rapamycin-succinimide-alendronate conjugate compound

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 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- 0.80 (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), 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 were treated in a weakly acidic buffer (citrate buffer at pH 4.8) at a concentration of 50 uM for 1 hour at 37° C., mimicking the bone tumor cell environment in which cleavage is induced, and then neutralized with 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 Synthesis Example 3-3 showed a better effect at a lower pH and higher dose than at neutral pH, and the three conjugates (compounds of Formulas 2, 3 and 4) were neutral pH, low concentration pH, test, etc. The effect was shown at the concentration.

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.

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 (10)

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

, R is OZ, and Z is an alkyl having 1 to 3 carbon atoms. 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 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 the following Formulas 2 to 4, an optical isomer or a pharmaceutically acceptable salt thereof:
[Formula 2]

[Formula 3]

[Formula 4]

. A pharmaceutical composition for preventing or treating 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,
The bone disease is in the group consisting 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 Whichever one is selected, a pharmaceutical composition for preventing or treating bone 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,
The bone disease is in the group consisting 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 Whichever one is selected, a health functional food composition for preventing or improving bone disease. delete

Images