KR101597105B1 - Antibody-Linker-Drug Conjugates, Process for Preparing the Same and Anti-cancer Agents Comprising the Same - Google Patents

Abstract

The present invention provides an antibody-linker-drug conjugate in which an antibody and a cytotoxic drug are conjugated to each other through an enzyme-degrading peptide linker capable of directly binding to the lysine residue of an antibody, a method for producing the same, and an anticancer composition comprising the same as an active ingredient do.

Description

Antibody-Linker-Drug Conjugates, Process for Preparing the Same and Anti-cancer Agents Comprising the Same,

The present invention relates to an antibody-linker-drug conjugate having excellent anticancer activity, a process for producing the same, and an anticancer composition comprising the same as an active ingredient. More specifically, the present invention relates to an antibody-linker-drug conjugate in which an antibody and a cytotoxic drug are conjugated to each other through an enzyme-degrading peptide linker capable of directly binding to the lysine residue of the antibody, a method for producing the same, To an anticancer composition.

Antibodies are immunological proteins that bind to specific antigens, and many monoclonal antibodies are currently being developed as anticancer agents or used in the treatment of cancer. However, almost all antibodies inhibit the growth of cancer cells and inhibit cancer progression, but they are very limited for the treatment of cancer [Sharkey et al., Adv. Drug Del. Rev., 2008, 1407-1420]. As an alternative to overcome these limitations, antibody-drug conjugates (ADC) conjugated to antibodies have been developed as new types of antibody therapeutics [Chari, Acc. Chem. Res., 41 (2008), 98-107].

ADC means that a cytotoxic drug or toxin is conjugated to a monoclonal antibody (mAb) that selectively transfers them to the interior of a target tumor cell. When administered to a patient, the ADC binds to the target cell via its antibody portion and is delivered into the cell, thereby causing the cytotoxic drug or toxin to separate from the ADC and exhibit its own efficacy [Hamblett, et al., Clin . Cancer Res., 10: 7063-7070, October 15, 1999].

In order to effectively conjugate antibodies and cytotoxic drugs for the manufacture of ADCs, it is common to use a suitable type of linker. Typical examples of linkers used at this time include hydrazone, disulfide, and peptide linker. For an ADC to work effectively, all three components of the ADC, basically the mAb, the linker, and the cytotoxic drug have to perform at least a certain level [Chari, Acc. Chem. Res., 41 (2008), 98-107].

U.S. Patent No. 6,214,345 discloses an ADC that binds a cytotoxic drug such as doxorubicin or taxol to an antibody using an enzyme cleavable peptide linker that is degraded by the proteolytic enzyme cathepsin present in a specific lysosome Lt; / RTI > In addition, U.S. Patent No. 7,964,566 discloses that a cytotoxic drug such as MeVal-Val-Dil-Dap-Norephedrine (MMAE) and MeVal-Val-Dil-Dap-Phe (MMAF) Have been reported.

According to the conventional method of producing an ADC using an enzyme-degrading peptide linker, a linker is conjugated to an antibody to react with an antibody and a reducing agent such as dithiothreitol (DTT) to form an antibody After the generation of the thiol group, a technique of bonding the resulting thiol group to the linker is used. However, the use of a reducing agent to disrupt the intramolecular disulfide bond inevitably involves a modification of the antibody structure itself, which may adversely affect the affinity of the antibody for the antigen. In addition, partial reduction of the antibody may be accompanied by reduction in the reduction reaction, which may make it difficult to purify the final ADC. In addition, the antibody aggregates easily during the reaction, thereby lowering the yield of the final ADC.

Further, instead of generating a thiol group by reducing an intramolecular disulfide bond of an antibody, a thiol group generating reagent such as 2-iminothiolane (Traut's reagent) may be reacted with an amino group of a lysine residue of an antibody to generate a thiol group However, in this case, the number of the thiol group-forming reagents bound to the antibody varies from 0 to several, and when the resulting thiol group is conjugated with the linker, the linker-bound thiol group and the linker are not combined in the same antibody Resulting in a mixed thiol group. Therefore, the produced ADC has a problem in that the performance of the ADC, in particular, pharmacokinetics in vivo is poor due to the presence of an unnecessary thiol group to which the linker is not attached, residues derived from the thiol group generating reagent,

The inventors of the present invention have conducted intensive studies to develop an ADC in which a cytotoxic drug is conjugated to an antibody without structural modification of the above-mentioned non-desired antibody, and found that an enzyme-degrading peptide linker capable of directly binding to the lysine residue of the antibody And can overcome the problems of the conventional peptide linker, thereby completing the present invention.

Accordingly, one object of the present invention is to provide an antibody-linker-drug conjugate wherein an antibody and a cytotoxic drug are conjugated through an enzyme-degrading peptide linker capable of directly binding to the lysine residue of the antibody.

It is another object of the present invention to provide a method for producing the antibody-linker-drug conjugate in high yield.

It is another object of the present invention to provide an anticancer agent composition comprising the antibody-linker-drug conjugate.

The present invention relates to an antibody-linker-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof:

(I)

In this formula,

Ab is an antibody,

Y is a C ₁ -C ₈ alkyl group or a C ₃ -C ₆ cycloalkyl group,

R ₁ , R ₂ , R ₃ , R ₄ , R ₇ and R ₈ are each independently hydrogen or a C ₁ -C ₄ alkyl group,

R ₅ is hydrogen, hydroxy, C ₁ -C ₄ alkoxy, amino, oxo (═O) or hydroxyimino (═N-OH)

Ar is an aryl group,

X is nitrogen or oxygen,

R ₆ is hydrogen or a C ₁ -C ₄ alkyl group when X is nitrogen and is absent when X is oxygen,

n is 1 to 5;

In the present specification, the C ₁ -C ₈ alkyl group means a linear or branched hydrocarbon group having 1 to 8 carbon atoms, and examples thereof include methyl, ethyl, n-propyl, n-butyl, But are not limited thereto.

In the present specification, the C ₃ -C ₆ cycloalkyl group means a cyclic hydrocarbon having 3 to 6 carbon atoms, and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like, but is not limited thereto.

In the present specification, the C ₁ -C ₄ alkyl group means a linear or branched hydrocarbon group having 1 to 4 carbon atoms, and examples thereof include methyl, ethyl, n-propyl, i-propyl, n-butyl, , t-butyl, and the like.

In the present specification, the C ₁ -C ₄ alkoxy group means a straight or branched alkoxy group having 1 to 4 carbon atoms, and includes, but is not limited to, methoxy, ethoxy, n-propaneoxy, and the like.

As used herein, an aryl group includes both an aromatic group and a heteroaromatic group and a partially reduced derivative thereof. The arometric group is a simple or fused ring group of 5 to 15-ary, and the heteroaromatic group means an arometric group containing at least one of oxygen, sulfur or nitrogen. Exemplary aryl groups include, but are not limited to, phenyl, naphthyl, pyridinyl, furanyl, thiophenyl, indolyl, quinolinyl, imidazolinyl, But are not limited to, oxazolyl, thiazolyl, tetrahydronaphthyl, and the like.

The alkoxy group of C ₁ -C ₈ alkyl, C ₃ -C ₆ cycloalkyl group, C ₁ -C ₄ alkyl, C ₁ -C ₄ in the, and aryl groups have one or more hydrogen C ₁ -C ₄ C ₁ -C ₄ alkyl group, C ₂ -C ₄ alkenyl group, C ₂ -C ₄ alkynyl group, C ₃ -C ₁₀ cycloalkyl group, C ₁ -C ₄ haloalkyl group, C ₁ -C ₄ alkoxy group, C ₁ -C may be replaced by alkoxy groups of _four, an aryl group, an acyl group, a hydroxy, thio (thio), halogen, amino, alkoxycarbonyl, carboxy, carbamoyl, cyano, nitro and the like.

As used herein, antibodies are used in their broadest sense and specifically include intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e. G. Bispecific antibodies) formed from two or more intact antibodies, And antibody fragments exhibiting the desired biological activity. Antibodies are proteins produced by the immune system that can recognize and bind specific antigens. In its structural aspect, the antibody typically has a Y-shaped protein consisting of four amino acid chains (two heavy chains and two light chains). Each antibody has two regions, mainly a variable region and a constant region. The variable region located at the distal end of the arm of Y binds and interacts with the target antigen. The variable region comprises a complementarity determining region (CDR) that recognizes and binds to a specific binding site on a specific antigen. The constant region in the tail of Y is recognized and interacted by the immune system. Target antigens typically have multiple binding sites, also known as epitopes, that are recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, an antigen can have one or more corresponding antibodies.

In one embodiment of the present invention, the antibody may be an antibody that immunospecifically binds to a cancer cell antigen. Antibodies specific for cancer cell antigens may be obtained commercially or may be prepared by methods known in the art, such as recombinant expression techniques. Nucleotide sequences encoding antibodies specific for cancer cell antigens can be obtained, for example, from the Gene Bank database or literature, or by conventional cloning and sequencing. Examples of antibodies available for the treatment of cancer, wherein the humanized antibody -HER2 Herceptin ^® (Genentech's trastuzumab), for the treatment of non-metastatic breast cancer-chimeric anti -CD20 antibody, Rituxan for the treatment of Hodgkin lymphoma ^® humanized for (Genentech Inc. rituximab), a chimeric antibody, wherein -EGFR eobi Tuxtla ^® for the treatment of epidermal growth factor positive cancers such as cancer of the head and neck (not setuk's imkeulron Thank Inc.) and treated breeding anti-integrin αvβ3 antibody, including, non-Thaksin ^® (immune Med Inc.), but is not limited thereto.

In the antibody-linker-drug conjugate of the present invention, the linker-drug is directly bound to the lysine residue of the antibody. Thus, unnecessary deformation of the antibody protein structure, such as reduction of the intramolecular disulfide bond of the antibody and residue of the residue due to the thiol group forming reagent, does not occur and the occurrence of abnormal fragments or aggregation of the antibody is minimized, .

The antibody-linker-drug conjugate of the present invention is preferably an antibody-

Ab is an antibody that immunospecifically binds to a cancer cell antigen,

Y is a C ₁ -C ₈ alkyl group or a C ₃ -C ₆ cycloalkyl group,

R ₁ , R ₂ , R ₄ , R ₇ and R ₈ are each independently hydrogen or a C ₁ -C ₄ alkyl group,

R ₃ is a C ₁ -C ₄ alkyl group,

R ₅ is hydrogen, amino, oxo (= O) or hydroxyimino (= N-OH),

Ar is phenyl or naphthyl which is unsubstituted or substituted with one or more substituents selected from the group consisting of a C ₁ -C ₄ alkyl group, a C ₁ -C ₄ alkoxy group and a halogen,

X is nitrogen or oxygen,

R ₆ is hydrogen or a C ₁ -C ₄ alkyl group when X is nitrogen and is absent when X is oxygen.

More preferably, the antibody-linker-drug conjugate of the invention comprises

Ab is trastuzumab,

Y is methyl, n-propyl, n-pentyl, n-octyl or cyclopropyl,

R ₁ , R ₂ and R ₃ are methyl,

R ₄ , R ₇ and R ₈ are each independently hydrogen or methyl,

R ₅ is hydrogen, oxo (= O) or hydroxyimino (= N-OH)

Ar is phenyl or naphthyl which is unsubstituted or substituted with one or more substituents selected from the group consisting of methyl, methoxy and halogen,

X is nitrogen or oxygen,

R ₆ is hydrogen or methyl when X is nitrogen and is absent when X is oxygen.

Pharmaceutically acceptable salts herein include both non-toxic inorganic acid and organic acid salts and include, but are not limited to, hydrochlorides, sulfates, nitrates, phosphates, acetateates, benzenesulfonates, It is not.

Representative materials of the antibody-linker-drug conjugates of the present invention may be selected from the following groups.

In the above formula, n is 1 to 5.

In the meantime, the present invention relates to a method for producing an antibody-linker-drug conjugate of formula (I)

(i) condensing a compound of formula (II): < EMI ID = 27.1 >

(ii) subjecting a compound of the formula (IV) to the following reaction to obtain a compound of the formula (VI):

(iii) condensing N-hydroxysuccinimide with a compound of formula (VI) to obtain a compound of formula (VII) And

(iv) reacting an antibody with a compound of formula (VII)

≪ RTI ID = 0.0 &

(III)

(IV)

(V)

(VI)

(VII)

In this formula,

Y, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R _6, R ₇ , R ₈ , Ar and X are as defined in Formula I above.

Hereinafter, a method for preparing the antibody-linker-drug conjugate of Formula I according to the present invention will be described in detail with reference to the following Reaction Scheme I. The method described in the following Reaction Scheme I is illustrative of the method typically used, and the order of the unit operation, the reaction reagent, the reaction conditions, and the like may be changed as required.

[Reaction Scheme I]

In step (i) above, the compound of formula (II) is condensed with the dolastatin 10 derivative of formula (III) to give the compound of formula (IV).

The condensation reaction may be carried out in the presence of a condensing agent, and examples of the condensing agent include hydroxybenzotriazole (HOBt), hydroxyazabenzotriazole (HOAt) and the like, but are not limited thereto.

If necessary during the condensation reaction, organic bases such as pyridine, diisopropylethylamine can be used with the condensing agent.

The reaction solvent may be dimethylformamide (DMF), dimethylacetamide (DMA), dimethylsulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP) .

In the above step (ii), the compound of formula (IV) is reacted with the compound of formula (IV) by reaction with the compound of formula (IV).

As the reaction solvent, dimethylsulfoxide (DMSO), acetonitrile (MeCN), dimethylformamide (DMF) and the like can be used. The reaction temperature is preferably 20 to 25 ° C.

In the above step (iii), the compound of formula (VI) is condensed with N-hydroxysuccinimide to activate the carboxylic acid group of the compound of formula (VI).

The condensation reaction may be carried out in the presence of a condensing agent. Examples of the condensing agent include dicyclohexylcarbodiimide (DCC), (3-dimethylaminopropyl) carbodiimide (EDC) .

As the reaction solvent, dimethylformamide (DMF), dimethylacetamide (DMA), dichloromethane (DCM) and the like can be used. The reaction temperature is preferably 20 to 25 ° C.

In step (iv), the compound of formula (VII) is reacted with an antibody to obtain an end product, antibody-linker-drug conjugate of formula (I).

As the reaction solvent, a phosphate buffer having a pH of 6.0 to 8.0 can be used. Since the solubility of the linker-cytotoxic drug, which is a compound of the formula (VII), in the phosphate buffer varies depending on the kind of the cytotoxic drug, dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide (DMA), acetonitrile (MeCN) and 1,4-dioxane. At this time, the ratio of the organic solvent in the mixed solvent is preferably 50 vol% or less.

The compound of formula (VII) is preferably used in an amount of 3 to 25 equivalents based on the antibody. The drug-antibody ratio (DAR), which is the number of drugs bound to a molecule of the antibody in the antibody-linker-drug conjugate, is about 1 to 5.

The compound of formula (II) is known as an enzyme cleavable peptide linker and can be easily prepared according to the method described in U.S. Patent No. 6,214,345.

The dolastatin 10 derivative of formula (III) is a cytotoxic drug, as described in Korean Patent Application No. 10-2012-0104710 or U.S. Patent No. 5,599,902, filed on September 20, 2012 by the present applicant Can be manufactured. All of these patents are incorporated herein by reference.

For example, the dolastatin 10 derivative of formula III above may be prepared according to the process shown in Scheme II below.

[Reaction Scheme II]

The antibody-linker-drug conjugate of the present invention exhibits excellent antitumor activity (see Test Example 3 and Test Example 4).

Accordingly, the present invention relates to an anticancer composition, particularly a composition for treating breast cancer, comprising an antibody-linker-drug conjugate of the above formula (I) or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier.

The anticancer composition according to the present invention may be administered orally (e.g., by taking or inhalation) or parenterally (for example, by injection, sedation, implantation, suppository), and the injection may be, for example, Subcutaneous injection, intramuscular injection or intraperitoneal injection. The anticancer composition according to the present invention may be formulated into tablets, capsules, granules, fine subtilae, powders, sublingual tablets, suppositories, ointments, injections, emulsions, suspensions, syrups, . The above-described various forms of the anticancer composition according to the present invention can be prepared by a known technique using a pharmaceutically acceptable carrier commonly used in each formulation. Examples of pharmaceutically acceptable carriers are excipients such as excipients, binders, disintegrating agents, lubricants, preservatives, antioxidants, isotonic agents, buffers, encapsulating agents, sweeteners, solubilizers, bases, , Suspending agents, stabilizers, coloring agents and the like.

Although the anticancer composition according to the present invention varies depending on the form of the medicament, it contains about 0.01 to 95% by weight of the compound of the present invention or a pharmaceutically acceptable salt thereof.

The specific dose of the anticancer composition of the present invention may vary depending on the kind of the mammal including the treated person, the weight, the sex, the degree of the disease, the judgment of the doctor, and the like. Preferably, for oral administration, 0.01 to 50 mg of active ingredient per kilogram of body weight per day is administered, and in the case of parenteral administration, 0.01 to 10 mg of active ingredient per kilogram of body weight per day is administered. The total daily dose may be administered once or several times depending on the severity of the disease, the judgment of the physician, and the like.

The antibody-linker-drug conjugate according to the present invention is an antibody-linker-drug conjugate according to the present invention, in which the linker-drug is directly bound to the lysine residue of the antibody to prevent unnecessary deformation of the antibody protein structure and ensures effective and selective delivery of the cytotoxic drug Thereby enabling effective treatment of cancer, especially breast cancer. In addition, according to the preparation method of the present invention, an antibody-linker-drug conjugate can be produced with high yield.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the in vitro cell growth inhibitory effect of the antibody-linker-drug conjugate (I-1) according to the present invention on BT-474 cells.
FIG. 2 is a graph showing in vitro cell growth inhibitory activity of antibody-linker-drug conjugates (I-2) and (I-3) according to the present invention on BT-474 cells.
FIG. 3 is a graph showing the in vitro cell growth inhibitory effect of the antibody-linker-drug conjugates (I-4) and (I-5) according to the present invention on BT-474 cells.
FIG. 4 is a graph showing in vitro cell growth inhibitory activity of antibody-linker-drug conjugates (I-6) and (I-7) according to the present invention on BT-474 cells.
5 is a graph showing changes in tumor volume with time after administering the antibody-linker-drug conjugate (I-1) according to the present invention to a cancer-induced animal model.
6 is a graph showing tumor weights after 28 days of administration of the antibody-linker-drug conjugate (I-1) according to the present invention to a cancer-induced animal model.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are for illustrative purpose only and that the scope of the present invention is not limited to these embodiments.

Preparation Example 1: Preparation of the compound of formula (II)

Production Example 1-1: Preparation of Compound (II-1)

1 - ((S) -1 - ((4- (Hydroxy-phenoxy) Yl) amino) -3-methyl-1-oxobutan-2-yl) hexanamide (5.0 g, 8.7 mmol, Dubowchik et al. N, N-diisopropylethylamine (3.0 mL, 17.4 mmol) was added to the solution, which was then dissolved in anhydrous dimethylformamide Lt; 0 > C. To the reaction mixture, bis (4-nitrophenyl) carbonate (7.94 g, 26.1 mmol) was added in one portion, and the mixture was stirred at room temperature for 15 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography to obtain 4 - ((S) -2 - ((S) -2- (6- (2,5- Yl) hexanoamido) -3-methylbutanamido) -5-ureidopentanamido) benzyl (4-nitrophenyl) carbonate (3.65 g, 57%) as an off-white solid Lt; / RTI >

^{1 H NMR (400 MHz, DMSO-} d 6) δ 0.83 (d, J = 6.8 Hz, 3 H), 0.86 (d, J = 6.8 Hz, 3 H), 1.09 (t, J = 7.2 Hz, 1 H ), 1.19 (m, 2H), 1.37-1.76 (m, 1H), 1.96 (m, 1H) , 4.19 (s, 2H), 5.97 (brt, J = 5.6 Hz, 1H), 4.19 (t, J = 7.8 Hz, 1H) ), 7.00 (s, 2H) , 7.41 (d, J = 8.4 Hz, 2 H), 7.57 (d, J = 7.2 Hz, 2 H), 7.65 (d, J = 8.4 Hz, 2 H), 7.80 ( d, J = 8.4 Hz, 1 H), 8.09 (d, J = 7.2 Hz, 1 H), 8.31 (d, J = 7.2 Hz, 2 H), 10.05 (brs, 1 H)

(S) -2 - ((S) -2- (6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoamido) (4-nitrophenyl) carbonate (3.65 g, 4.95 mmol) was dissolved in 90 mL of anhydrous dimethylformamide under an argon atmosphere, and then t-butyl (2- Aminoethyl) (methyl) carbamate (0.86 g, 4.95 mmol) was added thereto, followed by stirring at 20-25 ° C for 2 hours. After completion of the reaction, the reaction solution was completely concentrated under high vacuum, and the resulting residue was purified by silica gel column chromatography to obtain a compound (3.8 g, 99%) in which the amino group of the compound (II-1) was protected.

LC-MS m / z : 773.5 [M + H] < ⁺ >

The compound (146 mg, 0.186 mmol) in which the amino group of the compound (II-1) was protected was dissolved in 5 mL of dichloromethane, 2 mL of trifluoroacetic acid was added dropwise thereto, and the mixture was stirred at 20-25 ° C for 2 hours. When the reaction was completed, the reaction solvent was removed by concentration under reduced pressure, and 5 mL of toluene was added twice to completely remove the trifluoroacetic acid to obtain a TFA salt concentrate of the title compound.

Production Example 1-2: Preparation of Compound (II-2)

(S) -2 - ((S) -2- (6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoamido) (4-nitrophenyl) carbonate (6.8 g, 9.22 mmol) was dissolved in 150 mL of anhydrous dimethylformamide under an argon atmosphere, and then t-butylmethyl (2 - (methylamino) ethyl) carbamate (1.82 g, 9.68 mmol) was added and the mixture was stirred at 20-25 ° C for 19 hours. After completion of the reaction, the reaction solution was completely concentrated under high vacuum, and the resulting residue was purified by silica gel column chromatography to obtain a compound (5.14 g, 71%) in which the amino group of the compound (II-2) was protected.

¹ H NMR (400 MHz, DMSO- d ₆ )? 0.78-0.8 (d, 3 H), 0.8-0.83 (d, 3 H), 1.09-1.99 (m, 10 H) , 1.92 (m, 1H), 2.07-2.19 (m, 2H), 2.66-2.73 (d, 3H), 2.8-2.81 2H), 5.94 (s, 2H), 5.94 (brt, 1H), 6.96 (s, 2H) (D, 2H), 7.55 (d, 2H), 7.77 (d, 1H), 8.04

LC-MS m / z : 787.5 [M + H] < ⁺ >

A TFA salt concentrate of the title compound was obtained in the same manner as in Preparation Example 1-1, using the compound in which the amino group of the compound (II-2) was protected.

Production Example 1-3: Preparation of Compound (II-3)

(S) -2 - ((S) -2- (6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoamido) (4-nitrophenyl) carbonate (6.8 g, 9.22 mmol) was dissolved in 80 mL of anhydrous dimethylformamide under an argon atmosphere, and then t-butyl (2- Aminoethyl) carbamate (1.63 g, 10.14 mmol) was added thereto, followed by stirring at 20-25 占 폚 for 15 hours. After completion of the reaction, the reaction solution was completely concentrated under high vacuum, and the resulting residue was purified by silica gel column chromatography to obtain a compound (4.13 g, 59%) in which the amino group of the compound (II-3) was protected.

¹ H NMR (400 MHz, DMSO- d ₆ )? 0.78-0.8 (d, 3 H), 0.8-0.83 (d, 3 H), 1.09-1.99 (m, 10 H) , 1.92 (m, 1H), 2.07-2.19 (m, 2H), 2.9-3.0 (m, 2H), 3.37 , 4.94 (s, 2H), 5.38 (s, 2H), 5.94 (brt, 1H), 6.76 , 7.55 (d, 2H), 7.77 (d, 1H), 8.04 (d, 1H), 9.95 (brs, 1H)

LC-MS m / z : 759.5 [M + H] < ^{+ &}

A TFA salt concentrate of the title compound was obtained in the same manner as in Preparation Example 1-1, using the compound in which the amino group of the compound (II-3) was protected.

Production Example 1-4: Preparation of Compound (II-4)

(S) -2 - ((S) -2- (6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoamido) (4-nitrophenyl) carbonate (0.74 g, 1 mmol) was dissolved in 30 mL of anhydrous dimethylformamide under an argon atmosphere, and then t-butyl (2- (Methylamino) ethyl) carbamate (0.25 g, 1.4 mmol) was added thereto, followed by stirring at 20-25 占 폚 for 15 hours. After completion of the reaction, the reaction solution was completely concentrated under high vacuum, and the resulting residue was purified by silica gel column chromatography to obtain 0.36 g (48%) of compound (II-4) in which the amino group was protected.

¹ H NMR (400 MHz, DMSO- d ₆ )? 0.79 (d, 3 H), 0.82 (d, 3 H), 1.13-1.7 2H), 2.79-3.02 (m, 5H), 3.14 (d, 1H), 3.2 (m, 1H) 1H), 4.15 (t, 1H), 4.34 (m, 1H), 4.9-4.94 (d, 2H), 5.37 , 7.24 (d, 2H), 7.54 (d, 2H), 7.76 (d, 1H), 8.04 (d, 1H), 9.95

LC-MS m / z : 773.5 [M + H] < ⁺ >

A TFA salt concentrate of the title compound was obtained in the same manner as in PREPARATION 1-1 by using the compound in which the amino group of the compound (II-4) was protected.

Preparation Example 2: Preparation of compound of formula (III)

Production Example 2-1: Preparation of compound (III-1)

(2S, 4S) -t-butyl 4 - ((t-butyldimethylsilyl) oxy) -2 - Pyrrolidine-1-carboxylate (0.87 g, 1.56 mmol) was dissolved in dichloromethane (10 mL) and trifluoroacetic acid 7 mL of loasetic acid was added dropwise, and the mixture was stirred at 20-25 DEG C for 3 hours. When the reaction was completed, the reaction solvent was removed by concentration under reduced pressure, and 5 ml of toluene was added twice to completely remove the trifluoroacetic acid, and the reaction was allowed to proceed.

The reaction mixture (TFA salt) and (3R, 4S, 5S) -4 - ((S) -2 - ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3- 5-methylheptanoic acid (0.67 g, 1.56 mmol) was dissolved in dimethylformamide (5 mL), and diethyl cyanophosphonate (DEPC) (0.26 mL, 1.56 mmol) And triethylamine (1.09 mL, 7.82 mmol) were added thereto, and the mixture was stirred at room temperature for 16 hours. After the reaction was completed, the reaction solvent was removed, and the residue was dissolved in 20 mL of ethyl acetate. The solution was extracted with 1 M potassium hydrogen sulfite, water, a saturated sodium hydrogencarbonate solution and brine, and then the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The concentrated residue was purified by silica gel column chromatography (ethyl acetate: hexane = 2: 1 - > ethyl acetate) to obtain a hydroxy-protected compound (1.14 g, 84%).

¹ H NMR (400 MHz, CDCl ₃ )? 0.04 (s, 6 H), 0.81-1.06 (m, 19 H), 1.19 H), 2.91-2.94 (m, 3H), 2.71-2.80 (m, 3H) 3H), 3.74 (m, 2H), 4.28-4.31 (m, 2H), 2.71-2.80 (m, (m, 2H), 7.18-7.82 (m, 2H), 6.18 (m, 1H), 6.84-6.89 )

The hydroxyl group protected compound (1.14 g, 1.31 mmol) was dissolved in 95 mL of tetrahydrofuran, 1.0 M tetrabutylammonium fluoride (4.9 mL, 4.09 mmol) was added, and the mixture was stirred for 5 hours. The reaction was terminated with a saturated aqueous ammonium chloride solution, extracted with 450 mL of ethyl acetate and 300 mL of water, and then concentrated under reduced pressure. The concentrated residue was purified by silica gel column chromatography (dichloromethane: methanol = 9: 1) to give (S) -2 - ((S) -2- (dimethylamino) 3R, 4S, 5S) -1 - ((2S, 4S) -2 - ((1R, 2R) -3- Yl) -3-methoxy-5-methyl-1-oxoheptan-4-yl ) -N, 3-dimethylbutanamide (0.45 g, 45%).

¹ H NMR (400 MHz, CDCl ₃ )? 0.79-1.05 (m, 19 H), 1.26 (m, 3 H), 1.45 (M, 2H), 2.71-2.80 (m, 3H), 2.71-2.80 (m, 3H), 2.81-2.89 3H), 3.74 (m, 2H), 4.28-4.31 (m, 2H), 4.75 (s, 3H) 7.18 (m, 1H), 7.18 (m, 1H), 6.84-6.89 (m, 2H)

LC-MS m / z : 754 [M + H] < ⁺ >

The compound (0.45 g, 0.59 mmol) was dissolved in 20 ml of dichloromethane, cooled to -50 to -60 캜, and pyridine (0.29 ml, 3.54 mmol) was added. P-Nitrophenyl chloroformate (0.71 g, 3.54 mmol) was dissolved in dichloromethane (10 ml), and the resulting reaction solution was slowly added dropwise thereto over 20 minutes, followed by stirring at -50 to -60 ° C for 3 hours. After the reaction was completed, 20 ml of dichloromethane was further added to the reaction solution, and the mixture was extracted with 0.5 M potassium hydrogen sulfite solution. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The concentrated residue was purified by silica gel column chromatography to give the title compound (0.4 g, 74%).

LC-MS m / z : 929.6 [M + H] < ^{+ &}

Production example 2-2: Preparation of compound (III-2)

(2S, 4S) -t-butyl 2 - ((1 R, 2R) -3 - ((2- (3- fluoro-4- methoxyphenyl) (1.28 g, 2.73 mmol) was dissolved in dichloromethane (30 mL), trifluoroacetic acid (10 mL) was added dropwise thereto, and a solution of 20-25 g Lt; 0 > C for 3 hours. When the reaction was completed, the reaction solvent was removed by concentration under reduced pressure, and 10 ml of toluene was added twice to completely remove the trifluoroacetic acid, and the reaction was allowed to proceed.

(S) -2 - ((S) -2- (dimethylamino) -3-methylbutanamido) -N, 3-dimethylbutanamido) - 3-Methoxy-5-methylheptanoate (1.33 g, 2.73 mmol) was dissolved in 30 mL of dichloromethane, 10 mL of trifluoroacetic acid was added dropwise thereto, and the mixture was stirred at 20-25 ° C for 3 hours. When the reaction was completed, the reaction solvent was removed by concentration under reduced pressure, and 10 mL of toluene was added twice to completely remove the trifluoroacetic acid, and the reaction was allowed to proceed.

Two reaction concentrates (TFA salts) were dissolved in dichloromethane (60 mL) and diethylcyanophosphonate (DEPC) (0.52 mL, 3.28 mmol) and triethylamine (3.8 mL, 27.32 mmol) Stir for 16 hours. After the reaction was completed, the reaction solvent was removed, and the residue was dissolved in 50 mL of ethyl acetate. The solution was extracted with 1 M potassium hydrogen sulfite, water, saturated sodium hydrogencarbonate solution and brine, and then the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The concentrated residue was purified by silica gel column chromatography (ethyl acetate: hexane = 2: 1 to ethyl acetate) to obtain (S) -2 - ((S) -2- (dimethylamino) - ((3R, 4S, 5S) -1 - ((2S, 4S) -2 - ((1R, 2R) -3- Ethyl) amino) -1-methoxy-2-methyl-3-oxopropyl) -4-hydroxypyrrolidin- 1 -yl) -3-methoxy- ) -N, 3-dimethylbutanamide (1.3 g, 60%).

¹ H NMR (400 MHz, CDCl ₃ )? 0.78-0.88 (m, 3 H), 0.91-1.07 (m, 17 H), 1.31-1.37 , 2.28 (s, 6H), 2.43-2.62 (m, 6H), 3.06 (s, 3H), 3.11-3.16 ), 3.57-3.65 (m, 2H), 3.97 (s, 3H), 4.08-4.09 (brs, 2H), 4.7-4.72 (m, 2H), 4.76-4.8 (m, 2H), 4.85-4.91 (brs,

LC-MS m / z: 780.8 [M + H] +, 802.8 [M + Na] +

The compound (1.27 g, 1.63 mmol) was dissolved in 20 ml of dichloromethane, cooled to -50 to -60 캜, and pyridine (1.3 ml, 16.28 mmol) was added. P-Nitrophenyl chloroformate (16.28 g, 3.3 mmol) was dissolved in dichloromethane (10 ml), and the resulting reaction solution was slowly added dropwise over 20 minutes, followed by stirring at -50 to -60 캜 for 3 hours. After the reaction was completed, 20 ml of dichloromethane was further added to the reaction solution, and the mixture was extracted with 0.5 M potassium hydrogen sulfite solution. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The concentrated residue was purified by silica gel column chromatography to give the title compound (0.8 g, 54%).

LC-MS m / z: 945.7 [M + H] +, 967.7 [M + Na] +

Production Example 2-3: Preparation of Compound (III-3)

(2S, 4S) -t-butyl 2 - ((1 R, 2R) -3 - ((2- (3- fluoro-4- methoxyphenyl) (2S, 4S) -t-butyl 2 - ((1 R, 2R) -3 - ((2- ( Oxoethyl) amino) -1-methoxy-2-methyl-3-oxopropyl) -4-hydroxypyrrolidine-1-carboxylate instead of 2- -2 - ((S) -2- (dimethylamino) -3-methylbutanamido) -N - ((3R, 4S, 5S) ) -2 - ((1 R, 2R) -3 - ((2- (2,4-dimethylphenyl) -2- oxoethyl) amino) -1- (3-methoxy-5-methyl-1-oxoheptan-4-yl) -N, 3-dimethylbutanamide (3.2 g, 89%).

¹ H NMR (400 MHz, CDCl ₃ )? 0.79-0.85 (m, 3 H), 0.91-1.33 (m, 14 H), 1.15-1.19 2H), 2.53 (s, 3H), 2.55-2.62 (m, 1H), 2.39-2.48 (m, 3H) ), 2.87 (s, 6H), 2.89 (s, 1H), 3.02 (s, 3H), 3.07-3.17 (m, 2H), 3.32 (d, 2H), 4.69 (m, 2H), 4.11-4.12 (brs, 1H), 4.16-4.18 2H), 7.66 (d, 2H), 7.74 (m, 2H)

LC-MS m / z: 760.6 [M + H] +, 782.6 [M + Na] +

Using the above compound, the title compound (1.6 g, 43%) was obtained in the same manner as in Production Example 2-2.

LC-MS m / z: 925.6 [M + H] +, 947.6 [M + Na] +

Production example 2-4: Preparation of compound (III-4)

(2S, 4S) -t-butyl 2 - ((1 R, 2R) -3 - ((2- (3- fluoro-4- methoxyphenyl) (2S, 4S) -t-butyl 4-hydroxy-2 - ((1R, 2R) -1- Oxoethyl) amino) -2-methyl-3-oxopropyl) pyrrolidine-1-carboxylate as a starting material was used instead of 2- (S) -2 - ((S) -2- (dimethylamino) -3-methylbutanamido) -N - ((3R, 4S, 5S) (2S, 4S) -4-hydroxy-2 - ((1 R, 2R) -1-methoxy- 4-yl) -N, 3-dimethylbutanamide (0.17 g, 67.5%) was obtained as colorless crystals from 2- ).

¹ H NMR (400 MHz, CDCl ₃ )? 0.75-0.82 (m, 3H), 0.85-1.07 (m, 17H), 1.26-1.40 (m, 3H), 2.58-2.64 (m, 1H), 3.01 (s, 3H), 3.34 (s, 3H), 3.52-3.73 ), 4.19-4.21 (m, 2H), 4.87-4.94 (m, 3H), 6.91-6.94 (d, (d, IH), 8.42 (s, IH), 7.27-7.24 (m,

LC-MS m / z: 812.7 [M + H] +, 834.7 [M + Na] +

Using the above compound, the title compound (0.15 g, 87%) was obtained in the same manner as in Production Example 2-2.

LC-MS m / z: 977.9 [M + H] +, 999.9 [M + Na] +

Production example 2-5: Preparation of compound (III-5)

(2S, 4S) -t-butyl 4 - ((t-butyldimethylsilyl) oxy) -2 - (2S, 4S) -t-butyl 4 - ((t (4-methylpiperazin-1- - (butyldimethylsilyl) oxy) -2 - ((1 R, 2R) -3 - ((2,6- difluorophenethyl) amino) -1-methoxy- (2S, 4S) -1 - ((2S, 4S) -2- (4-fluorophenyl) ((1R, 2R) -3 - ((2,6-difluorophenethyl) amino) -1-methoxy-2-methyl- ) -3-methoxy-5-methyl-1-oxoheptan-4-yl) -2 - ((S) -2- (dimethylamino) -3-methylbutanamido) (0.45 g, 45%).

¹ H NMR (400 MHz, CDCl ₃ )? 0.79-1.05 (m, 19 H), 1.26 (m, 3 H), 1.45 , 2.91-2.94 (m, 2H), 2.71-2.80 (m, 3H), 2.81-2.89 3H), 3.74 (m, 2 H), 4.28-4.31 (m, 2 H), 4.75 7.18 (m, 1H), 6.18 (m, 1H), 6.84-6.89

LC-MS m / z : 754 [M ^< ⁺ &^gt;] ⁺

Using the above compound, the title compound (0.46 g, 83%) was obtained in the same manner as in Production Example 2-1.

LC-MS m / z : 920.1 [M + H] < ^{+ &}

Preparation 3: Preparation of the compound of formula (IV)

Production Example 3-1: Preparation of compound (IV-1)

To a TFA salt (88 mg, 0.11 mmol) of the compound (II-1) obtained in Preparation Example 1-1 was added 5 mL of anhydrous dimethylformamide and diisopropylethylamine (0.18 mL, 1.03 mmol) (75 mg, 0.08 mmol) obtained in Preparation Example 2-1 was added dropwise to a solution of 5 mg of anhydrous dimethylformamide in an argon atmosphere, followed by stirring at 20-25 ° C for 17 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure under high vacuum, and the obtained residue was purified by silica gel column chromatography to obtain the title compound (135 mg, 100%).

LC-MS m / z: 1462.9 [M + H] +, 1485.9 [M + Na] +

Production Example 3-2: Preparation of compound (IV-2)

TFA salt (0.17 g, 0.212 mmol) of the compound (II-2) obtained in Preparation Example 1-2 was used in place of the TFA salt of the compound (II-1) obtained in Production Example 1-1, (III-2) (0.2 g, 0.212 mmol) obtained in Preparation Example 2-2 was used instead of Compound (III-1) obtained in Step 2-1 g, 44%).

¹ H NMR (400 MHz, DMSO- d ₆ )? 0.69-0.78 (m, 6H), 0.81-0.90 (m, 14H), 1.11-1.64 (M, 3H), 1.91-1.99 (m, 3H), 2.11-2.26 (m, 7H), 2.35-2.42 (m, 1H), 1.51-1.62 2H), 2.87 (s, 6H), 2.92-3.05 (m, 3H), 3.13-3.18 (m, 4H), 3.32-3.38 2H), 5.96 (s, 2H), 5.96-5.99 (t, 1H), 6.99 (s, 1H), 4.07-4.09 (m, 1H), 4.15-4.84 , 7.22-7.3 (m, 2H), 7.53-7.65 (m, 2H), 7.77-7.80 (m, 2H), 7.84-7.89 m, 1 H), 9.97 (s, 1 H)

LC-MS m / z: 1492.8 [M + H] +, 1514.7 [M + Na] +

Production example 3-3: Preparation of compound (IV-3)

(0.16 g, 0.212 mmol) of the compound (II-3) obtained in Production Example 1-3 was used instead of the TFA salt of the compound (II-1) obtained in Production Example 1-1, (III-2) (0.2 g, 0.212 mmol) obtained in Preparation Example 2-2 was used instead of the compound (III-1) obtained in Step 2-1 g, 40%).

¹ H NMR (400 MHz, DMSO- d ₆ )? 0.69-0.76 (m, 6H), 0.81-0.92 (m, 14H), 1.10-1.20 (m, 4H), 1.25-1.26 (M, 2H), 1.51-1.55 (m, 1H), 1.50-1.55 (m, (M, 1H), 2.84 (m, 1H), 2.84 (m, 1H) 2H), 5.41 (s, 2H), 5.97-6.00 (t, 1H), 7.00 (s, 1H), 7.24-7.30 (m, 1H), 8.19 (t, IH), 7.32 (m, 3H), 7.58-7.60 (m, 2H), 7.77-7.80 8.44 (br s, 1 H), 9.97 (s, 1 H)

LC-MS m / z: 1464.8 [M + H] +, 1487.9 [M + Na] +

Production example 3-4: Preparation of compound (IV-4)

(0.26 g, 0.324 mmol) of the compound (II-2) obtained in Production Example 1-2 was used instead of the TFA salt of the compound (II-1) obtained in Production Example 1-1, (0.2 g, 0.216 mmol) obtained in Preparation Example 2-3 was used in place of the compound (III-1) obtained in Preparation Example 3-1 and the compound (III-3) g, 64%).

¹ H NMR (400 MHz, DMSO- d ₆ )? 0.71-0.75 (m, 6H), 0.80-0.91 (m, 15H), 1.18-1.19 (M, 3H), 2.98-2.40 (m, 7H), 2.83-2.86 (m, 4H), 2.96-3.01 (m, 4H), 3.15-3.19 (m, 3H), 3.26 (s, 3H), 3.35-3.38 (m, 16H), 3.85-3.96 2H), 7.42-7.69 (m, 2H), 5.42 (s, 2H), 5.97-5.99 (t, 2H), 7.81-7.83 (d, IH), 8.09-8.12 (d, IH), 8.29

LC-MS m / z: 1472.9 [M + H] +, 1494.9 [M + Na] +

Production example 3-5: Preparation of compound (IV-5)

(0.24 g, 0.324 mmol) of the compound (II-3) obtained in Production Example 1-3 was used in place of the TFA salt of the compound (II-1) obtained in Production Example 1-1, (0.2 g, 0.216 mmol) obtained in Preparation Example 2-3 was used in place of the compound (III-1) obtained in Preparation Example 3-1 and the compound (III-3) g, 67%).

^{1 H NMR (400 MHz, DMSO-} d 6) δ 0.71 - 0.75 (m, 7H), 0.81 - 0.92 (m, 16H), 1.03 - 1.04 (m, 2H), 1.11 - 1.12 (m, 1H), 1.18 (M, 3H), 2.09-2.33 (m, 6H), 2.28-2.38 (m, 7H), 2.96-3.03 (m, 9H), 3.15-3.17 (m, 4H), 3.28 (s, 3H), 3.35-3.38 (m, 14H), 3.62-3.65 2H), 7.27-7.29 (m, 2H), 4.94 (s, 2H), 5.43 (D, IH), 8.29 (brs, IH), 10.00 (s, IH)

LC-MS m / z: 1444.9 [M + H] +, 1466.9 [M + Na] +

Production example 3-6: Preparation of compound (IV-6)

(0.23 g, 0.307 mmol) of the compound (II-3) obtained in Preparation Example 1-3 was used in place of the TFA salt of the compound (II-1) obtained in Production Example 1-1, 4) (0.2 g, 0.205 mmol) obtained in Preparation Example 2-4 was used in place of the compound (III-1) obtained in the previous step g, 51%).

^{1 H NMR (400 MHz, DMSO-} d 6) δ 0.71 - 0.75 (m, 7H), 0.81 - 0.92 (m, 16H), 1.03 - 1.04 (m, 2H), 1.11 - 1.12 (m, 1H), 1.18 (M, 3H), 2.09-2.33 (m, 6H), 2.28-2.38 (m, 7H), 2.96-3.03 (m, 9H), 3.15-3.17 (m, 4H), 3.28 (s, 3H), 3.35-3.38 (m, 14H), 3.62-3.65 2H), 7.27-7.29 (m, 2H), 4.94 (s, 2H), 5.43 (D, IH), 8.29 (brs, IH), 10.00 (s, IH)

LC-MS m / z: 1497.6 [M + H] +, 1519.6 [M + Na] +

Production example 3-7: Preparation of compound (IV-7)

(TFA salt (1 g, 1.294 mmol) of the compound (II-1) obtained in Production Example 1-1 was used instead of the compound (III-1) The title compound (1.3 g, 72%) was obtained in the same manner as in Production Example 3-1, using the obtained compound (III-5) (1.2 g, 1.294 mmol).

^{1 H NMR (400 MHz, DMSO-} d 6) δ 0.48 - 0.49 (m, 1H), 0.58 - 0.59 (m, 2H), 0.64 - 0.65 (m, 2H), 0.70 - 0.76 (m, 5H), 0.81 (M, 3H), 1.22-1.28 (m, 9H), 1.44-1.49 (m, 5H), 1.90-1.98 (m, 2H), 1.16-1.20 3H), 2.09-2.33 (m, 7H), 2.96-3.04 (m, 9H), 3.13-3.18 (m, 4H), 3.33-3.38 2H), 5.42 (s, 2H), 5.86-5.99 (t, 1H), 7.22-7.29 (m, 4H), 7.38-7.98 (M, 2H), 8.29 (brs, 1H), 7.80-7.82 (m, 2H) 8.70 (s, (1H), 10.00 (s, 1 H)

LC-MS m / z: 1452.8 [M + H] +, 1474.7 [M + Na] +

Production example 3-8: Preparation of compound (IV-8)

(0.18 g, 0.23 mmol) of the compound (II-4) obtained in Preparation Example 1-4 was used in place of the TFA salt of the compound (II-1) obtained in Production Example 1-1, 2) (0.2 g, 0.212 mmol) obtained in Preparation Example 2-2 instead of the compound (III-1) obtained in Step 2-1), the title compound (0.16 g, 51%).

¹ H NMR (400 MHz, DMSO- d ₆ )? 0.68-0.76 (m, 6H), 0.81-0.90 (m, 14H), 1.08-1.20 (m, 4H), 1.23-1.27 (M, 3H), 1.91-1.99 (m, 3H), 2.13-2.20 (m, 6H), 2.35-2.42 (m, 2H), 2.85-2.87 (m, 4H), 2.99-3.01 (m, 4H), 3.14-3.17 (m, 4H), 3.28-3.38 (s, 2H), 5.98 (s, 2H), 5.98-6.00 (t, 1H), 7.01 (s, 2H) 1H), 7.22-7.36 (m, 3H), 7.54-7.64 (m, 2H), 7.77-7.97 (m, 2H), 8.08-8.11 s, 1 H)

LC-MS m / z: 1478.5 [M + H] +, 1500.5 [M + Na] +

Preparation Example 4: Preparation of compound of formula (VI)

Production Example 4-1: Preparation of compound (VI-1)

10 mL of methyl alcohol was added to the compound (IV-1) (137 mg, 0.093 mmol) obtained in Preparation Example 3-1, and 3 mL of anhydrous dimethylformamide was added thereto to dissolve the compound, and 2- (1- (mercaptomethyl) Propane) acetic acid (41 mg, 0.28 mmol) was added and the mixture was stirred at room temperature for 15 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give the title compound (25 mg, 17%) as a solid

LC-MS m / z : 1609.3 [M + H] < ⁺ >

Production Example 4-2: Preparation of compound (VI-2)

(IV-2) (0.19 g, 0.128 mmol) obtained in Preparation Example 3-2 was used in place of the compound (IV-1) obtained in Production Example 3-1, (0.085 g, 41%).

¹ H NMR (400 MHz, DMSO- d ₆ )? 0.42-0.55 (m, 6H), 0.66-0.75 (m, 8H), 0.81-0.87 1H), 2.38-2.42 (m, 3H), 1.91-1.98 (m, 7H), 2.27 (s, 2H), 2.75-2.86 (m, 6H), 2.93-2.99 (m, 4H), 3.12-3.24 (m, 4H), 3.31-3.35 (m, 2H), 5.97 (s, 2H), 5.97-5.98 (t, 1H), 7.29-7.30 (m, 2H), 8.46 (d, 2H), 8.46 (brs, 1H), 10.01 (m, 2H), 7.58-7.60 s, 1 H), 12.12 (brs, 1 H)

LC-MS m / z: 1639.0 [M + H] +, 1661.0 [M + Na] +

Production example 4-3: Preparation of compound (VI-3)

(IV-3) (0.18 g, 0.119 mmol) obtained in Preparation Example 3-3 was used in place of the compound (IV-1) obtained in Production Example 3-1, (0.12 g, 61%).

¹ H NMR (400 MHz, DMSO- d ₆ )? 0.44-0.52 (m, 5H), 0.66-0.75 (m, 7H), 0.82-0.92 1H), 2.38-2.46 (m, 4H), 2.10-2.22 (m, 7H), 2.25-2.26 (d, 2H), 2.86-2.91 (m, 2H), 2.96-3.04 (m, 5H), 3.10-3.24 (m, 4H) 2H), 4.94 (s, 2H), 3.94 (m, 2H), 3.35 (m, 2H) 1H), 7.85-8.00 (m, 2H), 7.84-7.81 (m, 2H) 2H), 8.47 (brs, 1H), 10.04 (s, 1H), 12.12 (brs,

LC-MS m / z: 1610.8 [M + H] +, 1632.8 [M + Na] +

Production Example 4-4: Preparation of compound (VI-4)

The compound (IV-4) (0.2 g, 0.138 mmol) obtained in Preparation Example 3-4 was used instead of the compound (IV-1) obtained in Production Example 3-1, To obtain a compound (0.17 g, 77%).

¹ H NMR (400 MHz, DMSO- d ₆ )? 0.00-0.57 (m, 6H), 0.69-0.76 (m, 8H), 0.82-0.87 1H), 1.81-2.05 (m, 4H), 2.20-2.23 (m, 2H), 1.20-1.24 (m, 2H), 2.82-2.86 (m, 7H), 2.96-3.00 (m, 5H), 3.15-2.68 (m, 3H), 4.14-4.25 (m, 2H), 4.26-4.82 (m, 8H), 3.32-3.34 (m, 5H) 2H), 7.59-7.69 (m, 3H), 7.28-7.30 (m, 2H), 5.43 (s, 2H) 1H), 8,04 (m, 1H), 8,04 (m, 1H), 8,04 (m,

LC-MS m / z: 1619.6 [M + H] +, 1641.6 [M + Na] +

Production Example 4-5: Preparation of compound (VI-5)

The compound (IV-5) (0.2 g, 0.137 mmol) obtained in Preparation Example 3-5 was used instead of the compound (IV-1) obtained in Production Example 3-1, (0.17 g, 76%).

¹ H NMR (400 MHz, DMSO- d ₆ )? 0.50-0.52 (m, 4H), 0.69-0.76 (m, 8H), 0.82-0.92 (M, 3H), 1.86 - 1.24 (m, 6H), 1.52 - 1.64 (m, 1H), 1.66 - 1.83 2H), 2.78-2.86 (m, 3H), 2.95-2.48 (m, 2H), 2.10-2.20 (m, 6H) 3H), 4.02-4.88 (m, 8H), 3.10-3.15 (m, 8H), 3.15-3.17 2H), 5.43 (s, 2H), 6.03 (t, 1H), 7.06-7.15 (m, 2H), 7.22-7.29 1H), 8.04 (d, IH), 8.09 (d, IH), 8.07 (d,

LC-MS m / z: 1591.7 [M + H] +, 1613.7 [M + Na] +

Production Example 4-6: Preparation of compound (VI-6)

(IV-6) (0.15 g, 0.099 mmol) obtained in Preparation Example 3-6 was used in place of the compound (IV-1) obtained in Production Example 3-1, To obtain a compound (0.11 g, 70%).

¹ H NMR (400 MHz, DMSO- d ₆ )? 0.41-0.54 (m, 6H), 0.58-0.65 (m, 2H), 0.68-0.76 (M, 3H), 1.87 - 2.04 (m, 3H), 2.09 - 2.23 (m, 3H), 2.62-2.68 (m, 1H), 2.74-3.25- (m, 15H), 3.33-3.50 (m, 14H), 3.90-3.97 (M, 2H), 7.31 (s, 3H), 6.90 (s, 2H) (Brs, 1H), 8.29 (brs, 1H), 7.41 (d, 2H), 7.55-7.85 (m, 2H), 7.86-8.01 8.59 (brs, 1 H), 8.84 (s, 1 H), 10.08 (s, 1 H)

LC-MS m / z: 1642.8 [M + H] +, 1664.8 [M + Na] +

Production example 4-7: Preparation of compound (VI-7)

(IV-7) (1.3 g, 0.895 mmol) obtained in Preparation Example 3-7 was used in place of the compound (IV-1) obtained in Production Example 3-1, To give the compound (1.1 g, 74%).

¹ H NMR (400 MHz, DMSO- d ₆ )? 0.40-0.50 (m, 7H), 0.70-0.77 (m, 8H), 0.82-0.99 (M, 4H), 1.89-1.94 (m, 5H), 2.10-2.23 (m, 12H), 2.32-2.44 (m, 3H), 3.20-3.33 (m, 10H), 3.90-4.00 (m, 4H), 4.16-3.00 (m, 2H), 5.41 (s, 2H), 6.01 - 6.02 (t, 1H), 7.01-7.05 (m, 2H), 7.28-7.29 1H), 8.23 (brs, 1H), 10.04 (s, 1H)

LC-MS m / z: 1598.9 [M + H] +, 1621.9 [M + Na] +

Production Example 4-8: Preparation of compound (VI-8)

(IV-8) (0.14 g, 0.095 mmol) obtained in Preparation Example 3-8 was used in place of the compound (IV-1) obtained in Production Example 3-1. (0.072 g, 47%).

¹ H NMR (400 MHz, DMSO- d ₆ )? 0.43-0.50 (m, 6H), 0.66-0.76 (m, 8H), 0.82-0.92 (M, 3H), 1.84-1.97 (m, 4H), 2.01-2.30 (m, 10H), 2.34-2.38 (m, 3H), 2.56-2.64 (m, 2H), 2.80-2.88 (m, 5H), 2.91-3.04 (m, 5H), 3.14-3.17 (m, 6H), 3.27-3.39 2H), 5.42 (s, 2H), 6.02-6.03 (t, 1H), 7.21-7.36 (m, 4H), 7.55-7.63 1H), 8.01 (m, 4H), 8.03-8.05 (d, 1H), 8.13 (brs,

LC-MS m / z: 1624.9 [M + H] +, 1646.9 [M + Na] +

Preparation Example 5: Preparation of compound of formula (VII)

Production Example 5-1: Preparation of compound (VII-1)

Compound (VI-1) (25 mg, 0.016 mmol) obtained in Preparation Example 4-1 was dissolved in 3 mL of anhydrous dimethylformamide under an argon atmosphere, and then N-hydroxysuccinimide (7.2 mg, 0.062 mmol) . The reaction mixture was cooled to 0 ° C, EDC / HCl (11.9 mg, 0.062 mmol) was added, and the mixture was stirred at room temperature for 15 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure under high vacuum, and the resulting residue was purified by silica gel column chromatography to give the title compound (20 mg, 76%).

LC-MS m / z : 1706.9 [M + H] < ⁺ & 1728.9 [M + Na] < ⁺ >

Production Example 5-2: Preparation of compound (VII-2)

(VI-2) (0.074 g, 0.0452 mmol) obtained in Preparation Example 4-2 was used in place of the compound (VI-1) obtained in Production Example 4-1, (0.049 g, 62.4%).

^{1 H NMR (400 MHz, DMSO-} d 6) δ 0.51 - 0.53 (m, 1H), 0.63 - 0.76 (m, 12H), 0.81 - 0.86 (m, 18H), 1.02 - 1.04 (d, 4H), 1.11 (M, 3H), 2.18-2.19 (m, 6H), 2.73-2.89 (m, 2H), 1.14-1.92 13H), 2.93-2.99 (m, 5H), 3.14-3.11 (m, 5H), 3.31-3.39 2H), 7.94-7.87 (m, 2H), 5.94 (s, 2H), 5.42 (M, 2H), 8.08-8.11 (d, 1H), 8.46 (br s, 1H), 10.01

LC-MS m / z: 1735.4 [M + H] +, 1758.4 [M + Na] +

Production Example 5-3: Preparation of compound (VII-3)

The compound (VI-3) (0.11 g, 0.0664 mmol) obtained in Preparation Example 4-3 was used in place of the compound (VI-1) obtained in Production Example 4-1, (0.079 g, 69.6%).

¹ H NMR (400 MHz, DMSO- d ₆ )? 0.51-0.53 (m, 1H), 0.63-0.76 (m, 7H), 0.82-0.92 (M, 4H), 2.11-2.19 (m, 8H), 2.81-2.83 (m, 10H), 2.99-3.14 (m, 8H), 1.91-1.99 4H), 3.97-4.04 (m, 2H), 4.13-4.85 (m, 8H), 4.94 (m, 2H) (m, 3H), 8.10-7.30 (m, 2H), 5.43 (s, 2H), 6.00-6.03 8.12 (m, 1 H), 8.47 (brs, 1 H), 10.02 (s, 1 H)

LC-MS m / z: 1708.4 [M + H] +, 1730.4 [M + Na] +

Production Example 5-4: Preparation of compound (VII-4)

(VI-4) (0.16 g, 0.096 mmol) obtained in Preparation Example 4-4 was used in place of the compound (VI-1) obtained in Production Example 4-1, (0.12 g, 71%).

¹ H NMR (400 MHz, DMSO- d ₆ )? 0.51-0.53 (m, 1H), 0.70-0.75 (m, 8H), 0.82-0.87 (M, 1H), 1.18-1.23 (m, 4H), 1.44-1.49 (m, 7H), 2.08-2.20 3H), 3.32-3.34 (m, 18H), 3.88-4.04 (m, 4H), 4.08-4.87 (m, 6H) (m, 2H), 4.97 (s, 2H), 5.42 (s, 2H), 5.99-6.00 (m, 2H), 7.80-7.83 (m, 1H), 7.97-8.05 (m, 1H), 8.10-8.11 s, 1 H)

LC-MS m / z: 1716.5 [M + H] +, 1740.5 [M + Na] +

Preparation Example 5-5: Preparation of compound (VII-5)

(VI-5) (0.15 g, 0.093 mmol) obtained in Preparation Example 4-5 was used in place of the compound (VI-1) obtained in Production Example 4-1, To obtain a compound (0.1 g, 67%).

¹ H NMR (400 MHz, DMSO- d ₆ )? 0.51-0.53 (m, 1H), 0.63-0.66 (m, 3H), 0.69-0.76 2H), 1.11-1.12 (m, 1H), 1.18-1.24 (m, 3H), 1.44-1.49 (m, 7H), 2.13-2.20 (m, 9H), 2.28-2.37 3H), 3.31-3.35 (m, 15H), 3.85-3.87 (m, 1H), 2.73-3.83 2H), 5.99 (s, 2H), 5.99-6.02 (t, 1H), 7.06-7.15 (m, 1H), 3.95-4.04 (m, 2H), 7.27-7.29 (m, 4H), 7.58-7.63 (m, 3H), 7.81-7.83 8.30 - 8.50 (m, 1 H), 10.02 (s, 1 H)

LC-MS m / z: 1687.6 [M + H] +, 1709.5 [M + Na] +

Production example 5-6: Preparation of compound (VII-6)

(VI-6) (0.12 g, 0.073 mmol) obtained in Preparative Example 4-6 was used in place of the compound (VI-1) obtained in Preparation Example 4-1, (0.058 g, 46%).

¹ H NMR (400 MHz, DMSO- d ₆ )? 0.48-0.53 (m, 1H), 0.58-0.59 (m, 1H), 0.64-0.76 (D, 2H), 1.11-1.22 (m, 6H), 1.42-1.49 (m, 6H), 1.86-1.97 (m, 3H), 2.11-2.20 (m, 9H), 2.73-2.95- (M, 2H), 3.14 (m, 2H), 3.14-3.14 (m, 2H) 2H), 5.42 (s, 2H), 6.00-6.01 (t, 1H), 7.22-7.29 (m, 4H), 7.38-7.42 8.01 (d, 1H), 8.28-8.51 (m, 2H), 7.81-7.83 (d, 1H), 8.64 (s, 1H), 10.02 (s, 1H)

LC-MS m / z: 1740.4 [M + H] +, 1762.3 [M + Na] +

Production Example 5-7: Preparation of compound (VII-7)

The compound (VI-7) (0.1 g, 0.063 mmol) obtained in Preparation Example 4-7 was used in place of the compound (VI-1) obtained in Production Example 4-1, (0.072 g, 68%).

¹ H NMR (400 MHz, DMSO- d ₆ )? 0.51-0.53 (m, 1H), 0.62-0.69 (m, 2H), 0.70-0.77 2H), 2.74-3.00 (m, 3H), 1.94-1.47 (m, 6H), 2.19-2.21 (m, 4H), 4.95 (s, 2H), 5.40 (s, 1H), 3.17-3.18 (m, 7H), 3.25-3.33 2H), 7.91-7.82 (m, 1H), 7.94-7.60 (m, 2H), 6.01-6.02 7.99 (m, 2H), 8.08-8.10 (m, 1 H), 10.00 (s, 1 H)

LC-MS m / z: 1697.2 [M + H] +, 1719.1 [M + Na] +

Production Example 5-8: Preparation of compound (VII-8)

(VI-8) (0.062 g, 0.038 mmol) obtained in Production Example 4-8 was used instead of the compound (VI-1) obtained in Production Example 4-1, To obtain a compound (0.037 g, 56%).

^{1 H NMR (400 MHz, DMSO-} d 6) δ 0.51 - 0.53 (m, 1H), 0.63 - 0.77 (m, 13H), 0.81 - 0.91 (m, 17H), 1.03 - 1.04 (d, 2H), 1.09 3H), 2.11-2.19 (m, 7H), 2.77-2.89 (m, 13H), 3.13-3.17 (m, 6H), 1.42-1.49 2H), 4.42 (s, 2H), 4.42 (s, 2H), 4.42 (s, 2H) 2H), 5.88-6.00 (t, 1H), 7.28-7.41 (m, 4H), 7.58-7.65 (d, 2H), 7.77-8.06 (m, 4H), 8.10-8.11 8.45 (m, 1 H), 10.00 (s, 1 H)

LC-MS m / z: 1722.5 [M + H] +, 1744.3 [M + Na] +

Examples: Preparation of antibody-linker-drug conjugates of formula (I)

Example 1: Preparation of the conjugate (I-1)

To 5 mL (5.4 g / mL, 36 μM) of Trastuzumab (Herceptin) solution that had been exchanged with pH 7.5 of PBS (containing 10 mM phosphate, 137 mM NaCl, 2.7 mM KCl) 8.5 mL of 0.05 M sodium borate buffer (7.5 mL) was added and the compound (VII-1) (5.7 mg, 0.0036 mmol, 20 equivalent) obtained in Preparation Example 5-1 dissolved in 3.75 mL of 20% DMSO was added thereto. Lt; / RTI > for 18 hours. After completion of the reaction, the reaction mixture was filtered through a 0.45 μm filter, and the filtrate was transferred to a centrifugal filter (Amicon Ultra-15 30K) and the pH was adjusted to 7.5 with PBS until the organic solvent and unreacted material were removed. And concentrated three times. The concentrate was separated and purified by MPLC (280 nm, UV range 0.08, flow rate 10 mL / min) using a Sephadex G-25 resin packing column (30 x 300 mm) equilibrated in PBS of pH 7.5 to give 26.4 mg 2.20 mg / mL, total volume 12 mL, 98%). The concentration of the obtained compound was determined by measuring the absorbance of each of the three wavelengths (280 nm, 320 nm, and 350 nm) with a UV spectrometer.

Example 2: Preparation of the conjugate (I-2)

(VII-2) (4.2 mg, 0.0024 mmol, 15 equivalent) obtained in Production Example 5-2 was used instead of the compound (VII-1) obtained in Production Example 5-1 in the same manner as in Example 1 14.5 mg (1.21 mg / mL, total volume 12 mL, 61%) of the title compound was obtained.

Example 3: Preparation of the conjugate (I-3)

(VII-3) (4.1 mg, 0.0024 mmol, 15 equivalent) obtained in Production Example 5-3 was used instead of the compound (VII-1) obtained in Production Example 5-1 in the same manner as in Example 1 17.9 mg (1.63 mg / mL, total volume 11 mL, 75%) of the title compound were obtained.

Example 4: Preparation of the conjugate (I-4)

(VII-4) (4.1 mg, 0.0024 mmol, 15 equivalent) obtained in Production Example 5-4 was used instead of the compound (VII-1) obtained in Production Example 5-1 in the same manner as in Example 1 11.8 mg (1.48 mg / mL, total volume 8 mL, 50%) of the title compound was obtained

Example 5: Preparation of the conjugate (I-5)

(VII-5) (4.0 mg, 0.0024 mmol, 15 equivalent) obtained in Production Example 5-5 was used instead of the compound (VII-1) obtained in Production Example 5-1 in the same manner as in Example 1 15.8 mg (1.21 mg / mL, total volume 13 mL, 66%) of the title compound was obtained

Example 6: Preparation of the conjugate (I-6)

(VII-6) (4.2 mg, 0.0024 mmol, 15 equivalent) obtained in Production Example 5-6 was used instead of the compound (VII-1) obtained in Production Example 5-1 in the same manner as in Example 1 18.3 mg (1.52 mg / mL, total volume 12 mL, 77%) of the title compound were obtained.

Example 7: Preparation of the conjugate (I-7)

(VII-7) (4.6 mg, 0.0027 mmol, 15 equivalent) obtained in Production Example 5-7 was used instead of the compound (VII-1) obtained in Production Example 5-1 in the same manner as in Example 1 12.7 mg (1.15 mg / mL, total volume 11 mL, 47%) of the title compound was obtained.

Example 8: Preparation of the conjugate (I-8)

(VII-8) (4.1 mg, 0.0024 mmol, 15 equivalent) obtained in Production Example 5-8 was used instead of the compound (VII-1) obtained in Production Example 5-1 in the same manner as in Example 1 16.1 mg (1.24 mg / mL, total volume 13 mL, 67%) of the title compound were obtained.

Test Example 1: DAR determination using protein mass spectrometry

Each of the antibody-linker-drug conjugates prepared in the Examples was treated with PNGase F enzyme at 37 캜 for 15 3 hours to remove the N-linked sugar. The sugar-removed antibody-linker-drug conjugate was analyzed using LC-ESI-MS consisting of Agilent 1200 HPLC and Agilent 6530 Q-TOF mass spectrometer. The antibody-linker-drug conjugate was isolated using an Agilent Zorbax column (300SB-C18, 2.1 mm x 75 mm, 5 mu m) and a gradient elution of 0.1% aqueous formic acid / 0.1% acetonitrile in formic acid, (D0, D1, < / RTI > < RTI ID = 0.0 > D1) < / RTI > integrated with the molecular weight after acquiring mass / charge data at a scan rate of 1 spectra / D2 .. Dn) was calculated using the abundance information.

The relative abundance of each peak was calculated using the following equation. The abundance ratio of each peak was divided by the sum of the abundance ratios of all the peaks, and the% relative abundance ratio of each peak was calculated.

The DAR of the antibody-linker-drug conjugate was calculated using the% relative abundance ratio and the following equation, and the results are shown in Table 1 below.

 Conjugate (I-1) D0 D1 D2 D3 D4 D5 D6 D7 D8 D9 Sum Mass (Da) 145177.7 146777.9 148378.3 149972.4 151563 153163.7 Area 5864 10420 8579 5125 1867 849 32704 % Area 17.9 31.9 26.2 15.7 5.7 2.6 100.0 DAR 1.67 Conjugate (I-2) D0 D1 D2 D3 D4 D5 D6 D7 D8 D9 Sum Mass (Da) 145180 146805.8 148427.5 150051.7 151669 153291.4 154912.5 156539.5 158132.1 159784.1 Area 0 1791 7950 11577 11722 9767 7214 4651 1852 949 57473 % Area 0.0 3.1 13.8 20.1 20.4 17.0 12.6 8.1 3.2 1.7 100.0 DAR 4.30 Conjugate (I-3) D0 D1 D2 D3 D4 D5 D6 D7 D8 D9 Sum Mass (Da) 145180 146784.9 148374.9 149964.2 151552.3 153144 154742.7 156334.3 157940.4 159524.4 Area 0 232 2467 5116 6483 4829 3222 1457 716 356 24878 % Area 0.0 0.9 9.9 20.6 26.1 19.4 13.0 5.9 2.9 1.4 100.0 DAR 4.38 The conjugate (I-4) D0 D1 D2 D3 D4 D5 D6 D7 D8 D9 Sum Mass (Da) 145180 146787.7 148387.9 149985.8 151588.4 153191.5 154798.6 156392.1 157995.3 Area 0 3495 7843 10015 8988 6940 4126 1274 389 43070 % Area 0.0 8.1 18.2 23.3 20.9 16.1 9.6 3.0 0.9 100.0 DAR 3.64  Conjugate (I-5) D0 D1 D2 D3 D4 D5 D6 D7 D8 D9 Sum Mass (Da) 145180 146759 148331.1 149903 151476.4 153055.9 154631 156203.6 Area 0 2221 7557 9075 7933 5512 3044 968 % Area 0.0 3.1 20.8 25.0 21.8 15.2 8.4 2.7 DAR 3.55 Conjugate (I-6) D0 D1 D2 D3 D4 D5 D6 D7 D8 D9 Sum Mass (Da) 145187.1 146810.7 148434.4 150059.8 151687.1 153320.3 154948.4 Area 1463 5565 11616 11593 6224 2476 710 39647 % Area 3.7 14.0 29.3 29.2 15.7 6.2 1.8 100.0 DAR 2.65 Conjugate (I-7) D0 D1 D2 D3 D4 D5 D6 D7 D8 D9 Sum Mass (Da) 145180 146770.5 148351.3 149928.2 151506.8 153095 154671.9 156259.1 157840.3 159429.5 Area 0 1848 6545 10020 11504 9447 6600 4246 2260 661 53131 % Area 0.0 3.5 12.3 18.9 21.6 17.8 12.4 7.99 4.25 1.26 100.0 DAR 4.36 Conjugate (I-8) D0 D1 D2 D3 D4 D5 D6 D7 D8 D9 Sum Mass (Da) 145180 146792.1 148401 150007 151601.6 153213.6 154823.1 Area 0 236 1671 3110 3322 2200 1234 11773 % Area 0.0 2.0 14.2 26.4 28.2 18.7 10.5 100.0 DAR 3.79

Test Example 2: SEC-HPLC analysis

Size exclusion chromatography (SEC-HPLC) was used to evaluate the occurrence of abnormal fragments or aggregation of antibodies during binding of antibody and drug. This abnormal protein structure indirectly affects the superiority of the antibody-linker-drug conjugate produced, since it affects the antigen-specific binding ability inherent in the antibody and in vivo pharmacokinetics.

The antibody-linker-drug conjugate prepared in Example 1 was analyzed for the ratio of the normal antibody structure using SEC-HPLC. The results are shown in Table 2 below.

sample Main peak ^One)  (%) HMW ²⁾  (%) LMW ³⁾  (%) Antibody before conjugation 99.48 0.27 0.25 Conjugate (I-1) 98.63 1.02 0.35 Conjugate (I-2) 98.93 0.77 0.29 Conjugate (I-3) 98.33 1.43 0.24 The conjugate (I-4) 98.85 0.87 0.28 Conjugate (I-5) 98.31 1.44 0.25 Conjugate (I-6) 97.00 2.75 0.24 Conjugate (I-7) 98.15 1.49 0.36 Conjugate (I-8) 97.61 2.15 0.24 ¹⁾ Main peak: the ratio of the substance to the normal antibody size
²⁾ HMW (High molecular weight): The proportion of substances larger than the normal antibody due to aggregation phenomenon
³⁾ LMW (Low molecular weight): The ratio of the substance smaller than the normal antibody due to the fragmentation phenomenon

The antibody-linker-drug conjugates prepared in the examples showed normal antibody structure ratios ranging from 97.00% to 98.93% compared to the antibody (99.48%) prior to drug conjugation.

Test Example 3: In vitro cytotoxicity test

The anticancer potency of the antibody-linker-drug conjugate was assessed by in vitro assay using HER-2 overexpressing breast cancer cell line (BT-474) for cell proliferation activity. As a control, trastuzumab used in the preparation of antibody-linker-drug conjugate was used. Trastuzumab is a monoclonal antibody against the HER-2 antigen that is specifically expressed in breast cancer cells.

1 X 10 ⁴ BT-474 cell lines were prepared per well in a 96-well microplate, and the control group, Trastuzumab, and the antibody-linker-drug conjugate prepared in Example 1 were individually treated at the concentration, Live cell counts were indirectly determined by measuring the absorbance after treatment with the chromogenic reagent (CCK-8). The relationship between concentration-efficacy was determined by quadratic curve function, and the relative efficacy was evaluated by comparing EC ₅₀ (effective concentration of 50%, half-effect concentration). The results are shown in Figs. 1 to 4 and Table 3.

As shown in Table 3, the antibody-linker-drug conjugates prepared in the examples showed a lower EC ₅₀ value, 8.9 to 22.6 times, as compared to the control Trastuzumab, And the effect of reducing side effects can be expected.

In addition, as shown in Figs. 1 to 4, the antibody-linker-drug conjugates prepared in the examples reach lower absorbance in the quadratic curve function as compared with the control group, trastuzumab, It means that it is better than Stu. That is, the tumor cells are further reduced by the mechanism of the cytotoxic drug conjugated to the original tumor cell inhibiting effect of the antibody.

sample EC ₅₀ (ng / ml) Relative efficacy (times) Trastuzumab 104 One Conjugate (I-1) 11.3 9.2 Trastuzumab 104 One Conjugate (I-2) 10.3 10.1 Trastuzumab 104 One Conjugate (I-3) 5.74 18.1 Trastuzumab 116 One The conjugate (I-4) 13 8.9 Trastuzumab 116 One Conjugate (I-5) 5.13 22.6 Trastuzumab 94.7 One Conjugate (I-6) 7 13.5 Trastuzumab 94.7 One Conjugate (I-7) 4.42 21.4

Test Example 4: Animal model efficacy test

Animal model efficacy tests were performed in nude mice xenografted with HER-2 overexpressing breast cancer cell line (BT-474) in three doses (0.05) with antibody-linker-drug conjugate (I-1) prepared in Example 1 , 0.5, 5 mg / kg), followed by comparative evaluation of the volume and final weight of the tumor for 28 days. As a negative control, saline (PBS) was administered, and as a positive control, trastuzumab (Herceptin) was administered at 5 mg / kg. The results are shown in Fig. 5 and Fig.

5 and 6, the positive control group treated with trastuzumab (Herceptin) significantly decreased tumors as compared with the negative control group and the groups administered with the antibody-linker-drug conjugate (I-1) And the tumor was more markedly decreased.

Claims (15)

An antibody-linker-drug conjugate of formula (I): < EMI ID =
(I)

In this formula,
Ab is an antibody,
Y is a C ₁ -C ₈ alkyl group or a C ₃ -C ₆ cycloalkyl group,
R ₁ , R ₂ , R ₃ , R ₄ , R ₇ and R ₈ are each independently hydrogen or a C ₁ -C ₄ alkyl group,
R ₅ is hydrogen, hydroxy, C ₁ -C ₄ alkoxy, amino, oxo (═O) or hydroxyimino (═N-OH)
Ar is an aryl group,
X is nitrogen or oxygen,
R ₆ is hydrogen or a C ₁ -C ₄ alkyl group when X is nitrogen and is absent when X is oxygen,
n is 1 to 5; The method according to claim 1,
Ab is an antibody that immunospecifically binds to a cancer cell antigen,
Y is a C ₁ -C ₈ alkyl group or a C ₃ -C ₆ cycloalkyl group,
R ₁ , R ₂ , R ₄ , R ₇ and R ₈ are each independently hydrogen or a C ₁ -C ₄ alkyl group,
R ₃ is a C ₁ -C ₄ alkyl group,
R ₅ is hydrogen, amino, oxo (= O) or hydroxyimino (= N-OH),
Ar is phenyl or naphthyl which is unsubstituted or substituted with one or more substituents selected from the group consisting of a C ₁ -C ₄ alkyl group, a C ₁ -C ₄ alkoxy group and a halogen,
X is nitrogen or oxygen,
R ₆ is hydrogen or a C ₁ -C ₄ alkyl group when X is nitrogen and is absent when X is oxygen, or a pharmaceutically acceptable salt thereof. The method according to claim 1,
Ab is trastuzumab,
Y is methyl, n-propyl, n-pentyl, n-octyl or cyclopropyl,
R ₁ , R ₂ and R ₃ are methyl,
R ₄ , R ₇ and R ₈ are each independently hydrogen or methyl,
R ₅ is hydrogen, oxo (= O) or hydroxyimino (= N-OH)
Ar is phenyl or naphthyl which is unsubstituted or substituted with one or more substituents selected from the group consisting of methyl, methoxy and halogen,
X is nitrogen or oxygen,
R ₆ is hydrogen or methyl when X is nitrogen and is absent when X is oxygen. The antibody-linker-drug conjugate or a pharmaceutically acceptable salt thereof. The antibody-linker-drug conjugate according to claim 1, which is selected from the following compounds or a pharmaceutically acceptable salt thereof:

In the above formula, n is 1 to 5. 5. An antibody-linker-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, characterized in that the linker-drug is directly bound to the lysine residue of the antibody. 5. An antibody-linker-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein the antibody has a normal antibody structural ratio of 97% or more. (i) condensing a compound of formula (II): < EMI ID = 27.1 >
(ii) subjecting a compound of the formula (IV) to the following reaction to obtain a compound of the formula (VI):
(iii) condensing N-hydroxysuccinimide with a compound of formula (VI) to obtain a compound of formula (VII) And
(iv) reacting an antibody with a compound of formula (VII): < EMI ID =
≪ RTI ID = 0.0 &

(III)

(IV)

(V)

(VI)

(VII)

(I)

In this formula,
Ab is an antibody,
Y is a C ₁ -C ₈ alkyl group or a C ₃ -C ₆ cycloalkyl group,
R ₁ , R ₂ , R ₃ , R ₄ , R ₇ and R ₈ are each independently hydrogen or a C ₁ -C ₄ alkyl group,
R ₅ is hydrogen, hydroxy, C ₁ -C ₄ alkoxy, amino, oxo (═O) or hydroxyimino (═N-OH)
Ar is an aryl group,
X is nitrogen or oxygen,
R ₆ is hydrogen or a C ₁ -C ₄ alkyl group when X is nitrogen and is absent when X is oxygen,
n is 1 to 5; The process according to claim 7, wherein the condensation reaction of step (i) is carried out in the presence of a condensing agent selected from the group consisting of hydroxybenzotriazole (HOBt) and hydroxyazabenzotriazole (HOAt) and a group consisting of pyridine and diisopropylethylamine ≪ / RTI > is carried out in the presence of an organic base selected from < RTI ID = 0.0 > The process according to claim 7 wherein the condensation reaction of step (iii) is carried out in the presence of a condensing agent selected from the group consisting of dicyclohexylcarbodiimide (DCC) and (3-dimethylaminopropyl) carbodiimide (EDC) . The process according to claim 7, wherein the reaction solvent of step (iv) is a phosphate buffer having a pH of 6.0 to 8.0. 8. The process according to claim 7, wherein the reaction solvent of step (iv) is a mixed solvent of a phosphate buffer having a pH of 6.0 to 8.0 and an organic solvent. 12. The method according to claim 11, wherein the ratio of the organic solvent in the mixed solvent is 50 vol% or less. 8. A process according to claim 7 wherein in step (iv) the compound of formula (VII) is used in an amount of from 3 to 25 equivalents based on the antibody. An anticancer composition comprising an antibody-linker-drug conjugate according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier. 15. The anticancer composition according to claim 14, wherein the anticancer agent is applied to the treatment of breast cancer.

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