KR20140088828A - Antibody-Linker-Drug Conjugates, Process for Preparing the Same and Anti-cancer Agents Comprising the Same - Google Patents

Abstract

The present invention relates to an antibody-linker-drug conjugate having an excellent anti-cancer activity, a preparation method thereof, and an anti-cancer agent composition containing the same as an active ingredient. In particular, in the antibody-linker-drug conjugate, the antibody and the cytotoxic drug are conjugated by the catalyst-decomposition type peptide linker, which can be directly bound to a lysine residue of the antibody.

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 moiety 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,

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

R ₁ is hydrogen, hydroxy, a C ₁ -C ₄ alkoxy group or an oxo (═O)

R ₂ is hydrogen or a C ₁ -C ₄ alkyl group,

R ₃ is hydrogen, hydroxy, C ₁ -C ₄ alkoxy, amino, oxo (═O) or hydroxyimino (═N-OH)

R ₄ is an aryl group,

p 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 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.

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.

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 C ₁ -C ₈ alkyl group, the C ₃ -C ₆ cycloalkyl group, the C ₁ -C ₄ alkoxy group, the C ₁ -C ₄ alkyl group, and the aryl group may be substituted by one or more hydrogen atoms with 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 generating reagent does not occur, and abnormal fragmentation 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,

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

R ₁ is hydrogen, hydroxy or a C ₁ -C ₄ alkoxy group,

R ₂ is hydrogen or a C ₁ -C ₄ alkyl group,

R ₃ is hydrogen, oxo (= O) or hydroxyimino (= N-OH)

R ₄ is an optionally substituted phenyl with one or more substituents selected from the group consisting of an alkoxy group and a halogen alkyl group of C _{1 -C 4, C 1 -C 4} .

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

Ab is trastuzumab,

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

R < ₁ > is hydrogen, hydroxy or methoxy,

R < ₂ > is hydrogen or methyl,

R ₃ is hydrogen, oxo (= O) or hydroxyimino (= N-OH)

R < ₄ > is phenyl unsubstituted or substituted with one or more substituents selected from the group consisting of methyl, methoxy and halogen.

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, p 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,

Ab, X, R ₁ , R ₂ , R ₃ and R ₄ 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, and 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) and the like can be used, and the reaction temperature is preferably 20 to 25 占 폚.

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% 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 4 and Test Example 6).

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 agent 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-2) according to the present invention on BT-474 cells.
FIG. 2 is a graph showing the in vitro cell growth inhibitory effect of the antibody-linker-drug conjugate (I-6) according to the present invention on BT-474 cells.
3 is a graph showing the in vitro cell growth inhibitory effect of the antibody-linker-drug conjugate (I-7) according to the present invention on BT-474 cells.
FIG. 4 is a graph showing changes in tumor volume with time after administering the antibody-linker-drug conjugates (I-2), (I-6) and (I-7) .
5 is a graph showing the tumor weights after 56 days of administration of the antibody-linker-drug conjugates (I-2), (I-6) and (I-7) 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 - ((S) -1 - ((4- (Hydroxyphenyl) -6- (2,5-dioxo-2,5-dihydro- 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 under high vacuum, and the resulting residue was purified by silica gel column chromatography to give the title compound (3.65 g, 57%) as a off-white solid.

^{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)

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

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

Methoxy-2-methylpropanoic acid (0.63 g, 2.2 mmol) was added to a solution of (2R, 3R) -3 - ((S) -l- (t-butoxycarbonyl) pyrrolidin- 2- (2,6-difluorophenyl) ethanamine (0.38 g, 2.4 mmol) was added to the solution, and the reaction mixture was cooled to 0 ° C. Then, benzotriazole- (0.97 g, 2.2 mmol) and diisopropylethylamine (1.0 mL, 6.6 mmol) were successively added thereto at room temperature, 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 obtained residue was purified by silica gel column chromatography to obtain (R) -t-butyl 2 - ((1R, 2R) -3 - ((2,6- difluorophenethyl) -1-methoxy-2-methyl-3-oxopropyl) pyrrolidine-1-carboxylate (0.60 g, 64%).

The compound (0.6 g, 1.4 mmol) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (1 mL) was added, and the mixture was stirred at room temperature for 3 hours. When the reaction was completed, the reaction solvent was concentrated under reduced pressure. (2R, 3R) -N- (2,6-difluorophenethyl) -3-methoxy-2-methyl- -Pyrrolidin-2-yl) propanamide. ≪ / RTI >

The above concentrate (TFA salt) and (5S, 8S, 11S, 12R) -11 - ((S) -sec- butyl) -5,8- diisopropyl-12-methoxy-4,10- Trioxatetradecan-14-acid (0.77 g, 1.4 mmol) was dissolved in 10 mL of dimethylformamide, and di Ethyl cyanophosphonate (DEPC) (0.23 mL, 1.54 mmol) and triethylamine (1.17 mL, 8.4 mmol) were added successively and the mixture was stirred at room temperature for 15 hours. After completion of the reaction, the solvent was removed, and the obtained residue was dissolved in 50 mL of ethyl acetate and washed with 30 mL of distilled water. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to obtain a compound (0.96 g, 80%) in which the N-terminal amino group of the compound (III-1) was protected.

¹ H NMR (400 MHz, CDCl ₃ )? 0.75-1.06 (m, 17 H), 1.18-1.38 (m, 5 H), 1.71-2.15 (M, 3 H), 3.87 (s, 3 H), 2.87 - 2.91 (m, 4 H) (M, 2H), 6.50-6.60 (m, 2H), 6.82-6.87 (m, 2H), 4.09-4.15 , 7.13-7.17 (m, 1H), 7.30-7.34 (m, 5H)

The compound (0.96 g, 1.1 mmol) protected with the N-terminal amino group of compound (III-1) was dissolved in 18 mL of t-butyl alcohol and 2 mL of water, 10% palladium carbon (0.1 g) And stirred for 3 hours. When the reaction was completed, the reaction mixture was filtered through celite, washed with methanol several times, and then the solvent was removed under reduced pressure to obtain the title compound (0.79 g, 99%).

¹ H NMR (400 MHz, CDCl ₃ )? 0.78-0.98 (m, 16 H), 1.06 (m, 1H), 1.16-1.27 (m, 2H), 1.89-2.02 (m, 4H), 2.31-2.38 (m, 5H), 2.71-2.75 , 3.29 (s, 3H), 3.41-3.48 (m, 3H), 3.79-3.82 (m, 1H), 4.08-4.15 (m, 2H), 4.72-4.86 (M, 1H), 6.56 (m, 1H), 6.79-6.85 (m, 2H), 7.10-7.15

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

Compound No. 2.36 (Compound No. III-2) in which the N-terminal amino group of the compound (III-2) is protected in the same manner as in Production Example 2-1 using 2-aminoacetophenone instead of 2- (2,6- difluorophenyl) g (81%).

¹ H NMR (400 MHz, CDCl ₃ )? 0.71-1.04 (m, 17H), 1.31-1.32 (m, 4H), 1.77-1.81 , 2.17-2.61 (m, 4H), 2.89-2.95 (m, 3H), 2.99 (s, 3H), 3.30 (M, 1H), 4.69-4.76 (m, 6H), 5.09-5.23 (m, 1H), 4.09-4.15 J = 7.6 Hz, 2 H), 7.62 (t, J = 7.2, 7.3 Hz, 2H), 6.46-6.49 (m, Hz, 1H), 7.97 (d, J = 7.6 Hz, 2H)

Using the compound in which the N-terminal amino group of the compound (III-2) was protected, 1.89 g (96%) of the title compound was obtained in the same manner as in Production Example 2-1.

¹ H NMR (400 MHz, CDCl ₃ )? 0.81-0.85 (m, 3 H), 0.92-1.44 (m, 13 H), 1.25-1.43 , 1.92-2.22 (m, 5H), 2.33 (s, 3H), 2.45-2.44 (m, 2H), 2.74 (d, J = 4.8 Hz, 1H) (M, 2 H), 3.92 (s, 3 H), 3.43 (d, J = 9.2 Hz, 3 H), 3.52-3.84 , 4.94-5.33 (m, 1H), 6.76-6.94 (m, 1H), 7.34-7.43 (m, 4H), 7.60

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

(2R, 3R) - (2R, 3R) -3 - ((S) -1- (tert- butoxycarbonyl) pyrrolidin- 2-yl) -3-methoxy-2-methylpropanoic acid was used, and 2- (2-methoxyphenyl) , 2-amino-1- (2-fluoro-4-methoxyphenyl) ethanone was used in the place of 2-amino-1- %).

¹ H NMR (400 MHz, CDCl ₃ )? 0.80-0.84 (m, 3 H), 0.88-1.03 (m, 17 H), 1.32 (d, J = 7.2 Hz, 2 H), 2.74 (m, 3 H), 3.02 (m, 3 H), 3.29 (m, s, 3 H), 3.34 ( s, 3 H), 3.45 (s, 3 H), 3.46 - 3.56 (m, 1 H), 3.87 (s, 3 H), 3.99 (dd, J = 7.6 Hz, 2.8 (M, 1H), 4.67 (m, 1H), 6.67 (m, 1H) 1 H), 6.78 (qd, J = 10.4 Hz, 2.8 Hz, 1H), 7.95 (m, 1H)

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

(2R, 3R) - (2R, 3R) -3 - ((S) -1- (tert- butoxycarbonyl) pyrrolidin- 2-yl) -3-methoxy-2-methylpropanoic acid was used, and 2- (2-methoxyphenyl) (S) -2-amino-1- (4-fluorophenyl) propan-1-one was used in the same manner as in Production Example 2-1, except for using 0.45 g (100%).

¹ H NMR (400 MHz, CDCl ₃ )? 0.71-1.03 (m, 20 H), 1.22 (d, 3 H), 1.33-1.37 3 H), 3.32 (s, 3 H), 3.32 (s, 3 H), 1.81 (m, (m, 2H), 5.23-5.30 (m, 2H), 4.06-4.15 (m, 2H), 4.22-4.27 (m, 2H), 5.50 (m, 1H), 7.18 (t, 2H), 8.01-8.05

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

2-amino-1- (3-methoxyphenyl) propan-1-one oxime was used instead of 2- (2,6-difluorophenyl) In the same manner, 0.17 g (94%) of the title compound was obtained.

¹ H NMR (400 MHz, CDCl ₃ )? 0.79-0.98 (m, 2H), 1.01 (m, 4H), 1.19-1.27 (M, 2H), 2.39 (s, 3H), 2.42 (s, 3H) , 4.15-4.37 (m, 3H), 4.70 (m, 1H), 5.21-5.23 (m, 2H), 5.36 H), 7.49 (m, 1H)

Production example 2-6: Preparation of compound (III-6)

(2R, 3R) - (2R, 3R) -3 - ((S) -1- (tert- butoxycarbonyl) pyrrolidin- 2-yl) -3-methoxy-2-methylpropane (2-ethylhexyl) (100%) of compound (III-6) in which the N-terminal amino group and the hydroxyl group of compound (III-6) were protected was obtained in the same manner as in Production Example 2-1.

The compound (1.0 g, 1.02 mmol) was dissolved in 10 mL of dimethylformamide, 1.0 M tetrabutylammonium fluoride (3.1 mL, 3.1 mmol) was added, and the mixture was stirred for 5 hours. After completion of the reaction, the solvent was removed, and the obtained residue was dissolved in ethyl acetate (50 mL), washed with saturated sodium hydrogencarbonate solution and brine, and then the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to obtain 0.75 g (84%) of compound (III-6) having an N-terminal amino group protected.

ES-MS m / z : 874 [M + H] < ⁺ >

0.64 g (100%) of the title compound was obtained in the same manner as in Production Example 2-1, using the compound in which the N-terminal amino group of the compound (III-6) was protected.

ES-MS m / z : 740 [M + H] < ^{+ &}

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

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

Compound (III-1) (0.36 g, 0.50 mmol) obtained in Preparation Example 2-1 was dissolved in 14 mL of anhydrous dimethylformamide under an argon stream, and then Compound (II) obtained in Preparation Example 1-1 , 0.50 mmol). The reaction mixture was cooled to 0 ° C, and then HOBt (81 mg, 0.60 mmol) and 3.5 mL of anhydrous pyridine were added successively, 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 (0.49 g, 74%) as an off-white solid.

MALDI-TOF MS m / z: 1345.6 [M + Na] +, 1361.6 [M + K] +

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

(III-2) (0.35 g, 0.50 mmol) obtained in Preparation Example 2-2 was used in place of the compound (III-1) obtained in Production Example 2-1, (0.42 g, 72%).

MALDI-TOF MS m / z: 1322.8 [M + Na] +, 1339.0 [M + K] +

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

(III-3) (79 mg, 0.10 mmol) obtained in Preparation Example 2-3 was used in place of the compound (III-1) obtained in Production Example 2-1, (97 mg, 78%).

MALDI-TOF MS m / z : 1400.3 [M + Na] < ^{+ &}

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

(III-4) (104 mg, 0.14 mmol) obtained in Preparation Example 2-4 was used in place of the compound (III-1) obtained in Production Example 2-1, (86 mg, 48%).

MALDI-TOF MS m / z : 1386.9 [M + Na] < ⁺ >, 1402.9 [M ⁺

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

(III-5) (65 mg, 0.085 mmol) obtained in Production Example 2-5 was used instead of the compound (III-1) obtained in Production Example 2-1, (43 mg, 41%).

MALDI-TOF MS m / z : 1381.6 [M + Na] < ^{+ &}

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

(III-6) (82 mg, 0.11 mmol) obtained in Preparation Example 2-6 was used in place of the compound (III-1) obtained in Production Example 2-1, (89 mg, 66%).

MALDI-TOF MS m / z: 1360.0 [M + Na] +, 1377.2 [M + K] +

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

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

5.0 mL of acetonitrile was added to and dissolved in the compound (IV-1) (274 mg, 0.21 mmol) obtained in Preparation Example 3-1, and 4-mercaptobutyric acid (V-1) (126 mg, And the mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated under reduced pressure to give the title compound (290 mg, 98%).

MALDI-TOF MS m / z : 1465.3 [M + Na] < ^{+ &}

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

2.5 mL of acetonitrile was added to dissolve the compound (IV-1) (70 mg, 0.053 mmol) obtained in Preparation Example 3-1 and 2- (1- (mercaptomethyl) cyclopropane) acetic acid (V- (44 mg, 0.3 mmol) and DMF (0.7 mL), and the mixture was stirred at room temperature for 4 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 (60 mg, 77%) as a solid.

MALDI-TOF MS m / z : 1492.0 [M + Na] < ^{+ &}

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

Mercaptohexanoic acid (V-3) was used in the place of 2- (1- (mercaptomethyl) cyclopropane) acetic acid (V-2) , 69%) as a solid.

MALDI-TOF MS m / z : 1493.2 [M + Na] < ^{+ &}

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

(V-4) was used in the place of 2- (1- (mercaptomethyl) cyclopropane) acetic acid (V-2) , 84%) as a solid.

MALDI-TOF MS m / z : 1522.0 [M + Na] < ^{+ &}

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

The title compound (71 mg, 0.38 mmol) was obtained as a colorless solid in the same manner as in Production Example 4-2 using 11-mercaptoundecanoic acid (V-5) instead of 2- (1- (mercaptomethyl) cyclopropane) , 86%) as a solid.

MALDI-TOF MS m / z : 1563.3 [M + Na] < ^{+ &}

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

(IV-2) (70 mg, 0.054 mmol) obtained in Preparation Example 3-2 was used in place of the compound (IV-1) obtained in Production Example 3-1, The compound (53 mg, 68%) was obtained as a solid.

MALDI-TOF MS m / z : 1469.5 [M + Na] < ^{+ &}

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

(IV-2) (70 mg, 0.054 mmol) obtained in Preparative Example 3-2 was used instead of the compound (IV-1) obtained in Production Example 3-1 and 2- (1- (mercaptomethyl ) Cyclopropane The title compound (46 mg, 59%) was solids obtained in the same manner as in Production Example 4-2, except that 6-mercaptohexanoic acid (V-3) was used in place of acetic acid (V-2).

MALDI-TOF MS m / z : 1471.6 [M + Na] < ^{+ &}

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

(IV-3) (88 mg, 0.064 mmol) obtained in Preparation Example 3-3 was used in place of the compound (IV-1) obtained in Production Example 3-1, The compound (48 mg, 49%) was obtained as a solid.

MALDI-TOF MS m / z : 1548.1 [M + Na] < ^{+ &}

Production example 4-9: Preparation of compound (VI-9)

(IV-4) (71 mg, 0.052 mmol) obtained in Preparation Example 3-4 was used in place of the compound (IV-1) obtained in Production Example 3-1, Compound (46 mg, 59%) was obtained as a solid.

MALDI-TOF MS m / z : 1529.4 [M + Na] < ^{+ &}

Production Example 4-10: Preparation of compound (VI-10)

The compound (IV-5) (35 mg, 0.026 mmol) obtained in Preparation Example 3-5 was used instead of the compound (IV-1) obtained in Production Example 3-1, The compound (29 mg, 74%) was obtained as a solid.

MALDI-TOF MS m / z : 1528.2 [M + Na] < ^{+ &}

Production Example 4-11: Preparation of compound (VI-11)

The compound (IV-6) (78 mg, 0.058 mmol) obtained in Preparation Example 3-6 was used instead of the compound (IV-1) obtained in Production Example 3-1, The compound (77 mg, 90%) was obtained as a solid.

MALDI-TOF MS m / z: 1508.1 [M + Na] +, 1524.8 [M + K] +

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

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

The compound (VI-1) (290 mg, 0.20 mmol) obtained in Preparation Example 4-1 was dissolved in 10 mL of anhydrous dimethylformamide under an argon atmosphere and then N-hydroxysuccinimide (35 mg, 0.30 mmol) . The reaction mixture was cooled to 0 C, EDC / HCl (58 mg, 0.30 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, and the residue was dissolved in 50 mL of ethyl acetate. 5 mL of 1M HCl was added to the residue, and the mixture was washed successively with 30 mL of distilled water and 30 mL of saturated brine, dried over anhydrous sodium sulfate, Respectively. The obtained residue was purified by silica gel column chromatography to obtain the title compound (226 mg, 73%).

MALDI-TOF MS m / z : 1562.9 [M + Na] < ^{+ &}

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

The compound (VI-2) (60 mg, 0.041 mmol) obtained in Preparation Example 4-2 was used instead of the compound (VI-1) obtained in Preparation Example 4-1, The compound (53 mg, 83%) was obtained as an off-white solid.

MALDI-TOF MS m / z : 1586.7 [M + Na] < ^{+ &}

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

(54 mg, 0.037 mmol) obtained in Preparative Example 4-3 was used instead of the compound (VI-1) obtained in Preparation Example 4-1. The compound (57 mg, 98%) was obtained as an off-white solid.

MALDI-TOF MS m / z : 1607.4 [M + K] < ^{+ &}

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

(VI-4) (66 mg, 0.044 mmol) obtained in Production Example 4-4 was used instead of the compound (VI-1) obtained in Production Example 4-1, The compound (59 mg, 84%) was obtained as an off-white solid.

MALDI-TOF MS m / z : 1617.0 [M + Na] < ^{+ &}

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

(VI-5) (71 mg, 0.046 mmol) obtained in Production Example 4-5 was used instead of the compound (VI-1) obtained in Production Example 4-1, The compound (66 mg, 88%) was obtained as an off-white solid.

MALDI-TOF MS m / z: 1660.9 [M + Na] +, 1677.1 [M + K] +

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

(VI-6) (53 mg, 0.037 mmol) obtained in Preparation Example 4-6 was used instead of the compound (VI-1) obtained in Preparation Example 4-1, The compound (38 mg, 67%) was obtained as an off-white solid.

MALDI-TOF MS m / z : 1565.5 [M + Na] < ^{+ &}

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

(VI-7) (46 mg, 0.032 mmol) obtained in Preparation Example 4-7 was used in place of the compound (VI-1) obtained in Production Example 4-1, The compound (30 mg, 61%) was obtained as an off-white solid.

MALDI-TOF MS m / z: 1568.9 [M + Na] +, 1585.0 [M + K] +

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

(VI-8) (38 mg, 0.025 mmol) obtained in Preparative Example 4-8 was used instead of the compound (VI-1) obtained in Preparation Example 4-1, The compound (31 mg, 78%) was obtained as an off-white solid.

MALDI-TOF MS m / z: 1643.7 [M + Na] +, 1660.2 [M + K] +

Production Example 5-9 : Preparation of Compound (VII-9)

The compound (VI-9) (34 mg, 0.023 mmol) obtained in Preparation Example 4-9 was used instead of the compound (VI-1) obtained in Production Example 4-1, The compound (27 mg, 75%) was obtained as an off-white solid.

MALDI-TOF MS m / z: 1627.7 [M + Na] +, 1644.8 [M + K] +

Production Example 5-10: Preparation of compound (VII-10)

(VI-10) (29 mg, 0.019 mmol) obtained in Production Example 4-10 was used instead of the compound (VI-1) obtained in Production Example 4-1, The compound (8 mg, 27%) was obtained as an off-white solid.

MALDI-TOF MS m / z: 1625.4 [M + Na] +, 1642.0 [M + K] +

Production Example 5-11: Preparation of compound (VII-11)

The compound (VI-11) (77 mg, 0.052 mmol) obtained in Preparation Example 4-11 was used instead of the compound (VI-1) obtained in Production Example 4-1, The compound (34 mg, 41%) was obtained as an off-white solid.

MALDI-TOF MS m / z: 1603.8 [M + Na] +, 1621.2 [M + K] +

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

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

To 5 mL (4.0 g / mL, 27 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) (2 mg, 0.001 mmol, 10 equivalent) obtained in Preparation Example 5-1 dissolved in 3.75 ml of 20% DMSO was added thereto. Lt; / RTI > for 4 h. 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 yield 13.5 mg 1.28 mg / mL, total volume 10.5 mL, 68%). 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, 0.003 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 12.1 mg (1.1 mg / mL, total volume 11 mL, 45%) of the title compound were obtained.

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

(VII-3) (3.12 mg, 0.002 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 13.7 mg (1.71 mg / mL, total volume 8 mL, 68%) of the title compound was obtained.

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

(VII-4) (3.20 mg, 0.002 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 13.9 mg (1.54 mg / mL, total volume 9 mL, 68%) of the title compound were obtained.

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

(VII-5) (3.27 mg, 0.002 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 15.3 mg (1.53 mg / mL, total volume 10 mL, 77%) of the title compound were obtained.

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

(VII-6) (4.17 mg, 0.003 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 14.2 mg (1.29 mg / mL, total volume 11 mL, 53%) of the title compound were obtained.

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

(VII-7) (3.08 mg, 0.002 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 9.5 mg (1.19 mg / mL, total volume 8 mL, 48%) of the title compound was obtained.

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

(VII-8) (5.4 mg, 0.003 mmol, 10 eq.) 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 50 mg (2.5 mg / mL, total volume 20 mL, 100%) of the title compound was obtained.

Example 9: Preparation of the conjugate (I-9)

(VII-9) (5.7 mg, 0.004 mmol, 20 equivalent) obtained in Production Example 5-9 was used instead of the compound (VII-1) obtained in Production Example 5-1 in the same manner as in Example 1 15.2 mg (1.01 mg / mL, total volume 15 mL, 56%) of the title compound was obtained.

Example 10: Preparation of the conjugate (I-10)

(VII-10) (4.3 mg, 0.003 mmol, 15 equivalent) obtained in Production Example 5-10 was used instead of the compound (VII-1) obtained in Production Example 5-1 in the same manner as in Example 1 19.4 mg (1.55 mg / mL, total volume 12.5 mL, 72%) of the title compound were obtained.

Example 11: Preparation of the conjugate (I-11)

(VII-11) (2.3 mg, 0.0015 mmol, 10 equivalents) obtained in Preparation Example 5-11 was used instead of the compound (VII-1) obtained in Production Example 5-1 in the same manner as in Example 1 20.1 mg (1.6 mg / mL, total volume 12.5 mL, 92%) of the title compound were obtained.

Test Example 1: DAR determination using protein mass spectrometry

PNGase F enzyme was treated at 37 ° C for 15 ± 3 hours in each of the antibody-linker-drug conjugates (I-2), (I-6) and (I-7) prepared in Examples 2, -Bonded sugar was removed. 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.

 The results are shown in Table 1 below.

 Conjugate (I-2) D0 D1 D2 D3 D4 D5 D6 D7 D8 Sum Mass (Da) 145179.9 146631.2 148080.8 149535.2 150983.3 152436.5 153888.3 Area 3080 5170 6730 5764 4659 2430 1059 28892 % Area 10.7 17.9 23.3 20.0 16.1 8.4 3.7 100.0 DAR 2.53 Conjugate (I-6) D0 D1 D2 D3 D4 D5 D6 D7 D8 Sum Mass (Da) 145170.3 146616.1 148043.7 149473.9 150904.2 152340 153771.2 155201.9 156631.2 Area 474 2570 5670 6155 6765 5567 3940 2430 655 34226 % Area 1.4 7.5 16.6 18.0 19.8 16.3 11.5 7.1 1.9 100.0 DAR 3.89 Conjugate (I-7) D0 D1 D2 D3 D4 D5 D6 D7 D8 Sum Mass (Da) 145173.9 146631.36 148048.1 149481 150912.6 152348 153781.7 155215.2 156641.7 Area 330 1788 4947 6111 6769 7133 4975 3005 784 35842 % Area 0.9 5.0 13.8 17.0 18.9 19.9 13.9 8.4 2.2 100.0 DAR 4.18

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 excellence of the antibody-linker-drug conjugate produced, since it affects the antigen-specific binding ability and in vivo pharmacokinetics inherent in the antibody.

The antibody-linker-drug conjugates (I-2), (I-6) and (I-7) prepared in Examples 2, 6 and 7 were analyzed for SEC- The results are shown in Table 2 below.

sample Main peak ^One)  (%) HMW ²⁾  (%) LMW ³⁾  (%) Antibody before conjugation 99.16 0.65 0.19 Conjugate (I-2) 98.55 1.07 0.38 Conjugate (I-6) 98.21 1.40 0.39 Conjugate (I-7) 98.42 1.16 0.42 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

All of the antibody-linker-drug conjugates (I-2), (I-6) and (I-7) showed a normal antibody structure ratio of 98.21% or more compared to the antibody (99.16% , Which is equivalent to a commercially available antibody.

Test Example 3: IEF analysis

(I-2), (I-4), (I-6) and (I-4) prepared in Examples 2, 4, 6 and 7, using isoelectric focusing (I-7) were analyzed, and the results are shown in Table 3 below.

Lane 2 3 4 5 6 7 8 9 sample Antibody before conjugation concrete
(I-4) concrete
(I-2) concrete
(I-7) concrete
(I-7) concrete
(I-6) concrete
(I-6) Antibody before conjugation Band 1 8.96 8.87 8.90 8.78 8.80 8.78 8.78 8.98 Band 2 8.85 8.78 8.82 8.71 8.72 8.70 8.69 8.86 Band 3 8.76 8.69 8.74 8.62 8.62 8.6 8.59 8.77 Band 4 8.68 8.59 8.63 8.51 8.51 8.49 8.48 8.68 Band 5 - 8.48 8.52 8.39 8.4 8.38 8.37 - Band 6 - - 8.41 8.29 8.3 8.27 8.26 - Band 7 - - - 8.21 8.2 8.18 8.17 - * Lane 1 and 10 are PI markers.

As shown in Table 3, a lower pI band was found in the conjugates (I-6) and (I-7) in which more drugs were bound than in the DAR results, from which the number of bound drugs increased .

Test Example 4: 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 (Herceptin) 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 per well were prepared in 96-well microplates, and the control group, Trastuzumab (Herceptin) and the antibody-linker-drug conjugates prepared in Examples 2, 6 and 7, After 5 days of incubation, viable cells were treated with a chromogenic reagent (CCK-8), and the absorbance was measured indirectly. 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 3 and Table 4.

As shown in Table 4, the antibody-linker-drug conjugates (I-2), (I-6) and (I-7) prepared in Examples 2, 6 and 7, compared to the control Trastuzumab, showed a low EC ₅₀ value of 17.8 to fold, indicating that the do showed equivalent efficacy with less side effects, a reduced amount can be expected effect.

In addition, antibody-linker-drug conjugates (I-2), (I-6) and (I-7) reach lower absorbance in quadratic curve function as shown in FIGS. 1 to 3, Which is better than the control, Trastuzumab (Herceptin). 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 (One ^st ) (ng / ml) EC ₅₀ (2 ^nd ) (ng / ml) EC ₅₀ (Mean) (ng / ml) Relative efficacy (times) Trastuzumab 125 - 125 One Conjugate (I-2) 9.47 - 9.47 13.2 Trastuzumab 88.2 - 88.2 One Conjugate (I-6) 6.28 6.28 6.28 14.0 Trastuzumab 85.1 - 85.1 One Conjugate (I-7) 4.66 4.88 4.77 17.8

Test Example 5: Pharmacokinetic test

The antibody-linker-drug conjugates (I-2) and (I-4) prepared in Examples 2 and 4 and the control trastuzumab were intravenously administered to Balb / c Slc-nu mice at a dose of 20 mg / After one dose, pharmacokinetics (PK) tests were performed for one month.

The half-lives of antibody-linker-drug conjugates (I-4) and (I-2) were 8.3 days and 7.9 days, respectively, which were not statistically different from those of the control group Trastuzumab 8.6 days, The site (FcRn) that affects the PK of the antibody itself indicates that the cytotoxic drug is not conjugated.

parameter The conjugate (I-4) Conjugate (I-2) Trastuzumab AUC _all (hr * μg / mL) 507280 575495 607582 C ₀ (μg / mL) 375.2 403.7 379.9 T _1/2 (day) ¹⁾ 8.3 7.9 8.6 ¹⁾ User specified lambda-z range (4m ~ 27day, 9 points)

Test Example 6: Animal model efficacy test

Animal model efficacy studies were performed on nude mice xenografted with HER-2 overexpressing breast cancer cell lines (BT-474), antibody-linker-drug conjugates (I-2) 6) and (I-7) were administered by single intravenous injection with three doses (1.25, 5, 20 mg / kg) respectively. The tumor volume and final weight were compared and evaluated for 56 days. As a negative control, physiological saline (PBS) was administered, and as a positive control, trastuzumab (Herceptin) was administered at 20 mg / kg. The results are shown in Fig. 4 and Fig.

4 and 5, the positive control treated with trastuzumab (Herceptin) significantly decreased the tumor as compared with the negative control, and the antibody-linker-drug conjugates (I-2), I-7) was more markedly reduced than the positive control group, and after 28 days, the transplanted tumor completely disappeared and did not recur.

Claims (15)

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

In this formula,
Ab is an antibody,
X is a C ₁ -C ₈ alkyl group or a C ₃ -C ₆ cycloalkyl group,
R ₁ is hydrogen, hydroxy, a C ₁ -C ₄ alkoxy group or an oxo (═O)
R ₂ is hydrogen or a C ₁ -C ₄ alkyl group,
R ₃ is hydrogen, hydroxy, C ₁ -C ₄ alkoxy, amino, oxo (═O) or hydroxyimino (═N-OH)
R ₄ is an aryl group,
p is 1 to 5; The method according to claim 1,
Ab is an antibody that immunospecifically binds to a cancer cell antigen,
X is a C ₁ -C ₈ alkyl group or a C ₃ -C ₆ cycloalkyl group,
R ₁ is hydrogen, hydroxy or a C ₁ -C ₄ alkoxy group,
R ₂ is hydrogen or a C ₁ -C ₄ alkyl group,
R ₃ is hydrogen, oxo (= O) or hydroxyimino (= N-OH)
R ₄ is phenyl optionally 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, or an antibody-linker- Acceptable salt. The method according to claim 1,
Ab is trastuzumab,
X is methyl, n-propyl, n-pentyl, n-octyl or cyclopropyl,
R < ₁ > is hydrogen, hydroxy or methoxy,
R < ₂ > is hydrogen or methyl,
R ₃ is hydrogen, oxo (= O) or hydroxyimino (= N-OH)
R ₄ is phenyl which is unsubstituted or substituted with one or more substituents selected from the group consisting of methyl, methoxy and halogen, 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, p 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 98% 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,
X is a C ₁ -C ₈ alkyl group or a C ₃ -C ₆ cycloalkyl group,
R ₁ is hydrogen, hydroxy, a C ₁ -C ₄ alkoxy group or an oxo (═O)
R ₂ is hydrogen or a C ₁ -C ₄ alkyl group,
R ₃ is hydrogen, hydroxy, C ₁ -C ₄ alkoxy, amino, oxo (═O) or hydroxyimino (═N-OH)
R ₄ is an aryl group,
p 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% 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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