Branched linker for producing targeting substance-drug conjugate with improved hydrophilicity

Abstract

A linker-drug conjugate of the present invention has, as a basic structure, a structure in which, in a linkage structure of an attachment group structure for a targeting substance (for example, antibody) – a branched spacer – a drug, a peptide is linked as a branched chain to the branched spacer. A targeting substance-drug conjugate of the present invention has a structure in which a targeting substance is linked to the attachment group in the linker-drug conjugate structure.

Description

Branched linker for generating targeting substance-drug conjugates with improved hydrophilicity

[0001] The present invention relates to a branched linker for generating a targeting substance-drug conjugate with improved hydrophilicity. More specifically, the present invention relates to a linker technology capable of generating a targeting substance-drug conjugate (e.g., an antibody-drug conjugate (ADC)) with improved hydrophilicity and stability while maintaining the original functions of the targeting substance (e.g., an antibody) and the drug.

[0002] As a technology for delivering drugs (e.g., cytotoxic substances) to specific organs or cells of the body (e.g., cancer cells), a technology of conjugating drugs with targeting substances is being developed, and a representative example is the antibody-drug conjugate (ADC). For example, if an ADC is manufactured by successfully conjugating an antibody specific to a specific cancer cell with a cytotoxic drug capable of killing that cancer cell, it becomes possible to achieve effective anticancer treatment by using this ADC to specifically kill only the corresponding cancer cells.

[0003] Due to these advantages, various technologies have been studied to successfully manufacture ADCs; however, since many highly effective drugs exhibit high hydrophobicity, ADCs utilizing these drugs as payloads also exhibit hydrophobicity, which limits their use.

[0004] Accordingly, various attempts are being made to improve the hydrophilicity of ADCs; however, existing technologies often fail to sufficiently improve hydrophilicity to a satisfactory level, or even when hydrophilicity is improved, they frequently cause problems with ADC function, such as the cytotoxicity of the conjugated drug and the cell selectivity of the conjugated antibody.

[0005] Therefore, it is necessary to develop technology that can sufficiently improve the hydrophilicity of the ADC while maintaining the functions of the ADC's antibody and drug.

[0006] [Prior Art Literature]

[0007] [Patent Literature]

[0008] EP 4386006 A1

[0009] The objective of the present invention is to provide a linker-drug conjugate that enables the generation of a targeting substance-drug conjugate with improved hydrophilicity.

[0010] Another objective of the present invention is to provide a linker-drug conjugate that enables the generation of a targeting substance-drug conjugate (e.g., ADC) with improved hydrophilicity while maintaining the original functions of the targeting substance (e.g., antibody) and the drug.

[0011] Another objective of the present invention is to provide a targeting substance-drug conjugate with improved hydrophilicity while maintaining the original functions of the targeting substance and the drug by applying the linker-drug conjugate described above.

[0012] The above and other objectives of the present invention can all be achieved by the present invention described below.

[0013] In one aspect, the present invention provides a linker-drug conjugate according to the following item:

[0014] 1. A linker-drug conjugate represented by the following chemical formula 1, or a pharmaceutically acceptable salt thereof:

[0015] [Chemical Formula 1]

[0016]

[0017] In the above chemical formula 1, AG is an attachment group to the targeting substance; BS is a branching spacer; D is a drug; P is a terminal-modified or non-modified peptide; L1 and L3 are spacers; L2 is a spacer or a cleavable linker; and a, b and c are each independently 0 or 1.

[0018] 2. In Item 1, the BS is a linker-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein the amino acid is connected to the L1 or AG through the nitrogen of the amino group, connected to the L2 or D through the carbon of the carboxyl group, and connected to the L3 or P through the carbon or nitrogen of the side chain.

[0019] 3. In Item 1 or Item 2, the above BS is

[0020] The above BS is or Phosphorus, linker-drug conjugate, or pharmaceutically acceptable salt thereof.

[0021] 4. In any one of Items 1 to 3, the above BS is

[0022] or Phosphorus, linker-drug conjugate, or pharmaceutically acceptable salt thereof.

[0023] 5. In any one of items 1 to 4, the linker-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein P is a terminal-modified or non-modified chain of an oligopeptide in which two or more amino acids selected from glycine (G), serine (S), and threonine (T) are linked.

[0024] 6. In any one of Items 1 to 5, said P is a linker-drug conjugate, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

[0025]

[0026]

[0027] As mentioned above,

[0028] R1 is independently hydrogen, or C 1-4 It is a hydroxyalkyl, and

[0029] R2 is hydrogen, or (C 1-4 It is an alkyl)carbonyl, and

[0030] R3 is a hydroxyl or amino, and

[0031] n is a natural number from 1 to 10.

[0032] 7. In Item 6, R1 is a linker-drug conjugate, or a pharmaceutically acceptable salt thereof, which is each independently hydrogen, hydroxymethyl, or 1-hydroxyethyl.

[0033] 8. A linker-drug conjugate of item 6, wherein R2 is hydrogen or methyl carbonyl.

[0034] 9. In any one of items 1 to 8, said P is a linker-drug conjugate, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

[0035]

[0036]

[0037] In the above, n is a natural number from 1 to 10.

[0038] 10. A linker-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein L2 comprises one or more of a cleavable dipeptide, a para-aminobenzyl alcohol self-sacrificial group, and a glucuronide linkage in any one of items 1 to 9.

[0039] 11. In any one of Items 1 to 10, L2 is a linker-drug conjugate, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

[0040]

[0041] 12. A linker-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein in any one of items 1 to 11, the AG comprises a reactive group selected from the group consisting of a maleimide group, a thiol group, a cyclooctine group, an azido group, and a maleamic acid group.

[0042] 13. In any one of Items 1 to 12, the AG is a linker-drug conjugate, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

[0043]

[0044] As mentioned above,

[0045] L3 is a C that optionally contains phenylene, -CONH-, or -C(R4)- in the middle of the chain. 1-10 It is an alkylene, and

[0046] R4 is C 1-10 It is an aminoalkyl.

[0047] 14. In any one of Items 1 to 13, the AG is a linker-drug conjugate, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

[0048]

[0049]

[0050] In another aspect, the present invention provides a targeting substance-drug conjugate according to the following item or a pharmaceutically acceptable salt thereof:

[0051] 15. A targeting substance-drug conjugate represented by the following chemical formula 2 or a pharmaceutically acceptable salt thereof:

[0052] [Chemical Formula 2]

[0053]

[0054] In the above Chemical Formula 2, AG, BS, D, P, L1, L2, L3, a, b, and c are the same as defined in any one of items 1 to 14, and

[0055] TA is a targeting agent;

[0056] n' is an integer from 1 to 8.

[0057] 16. In Item 15, the TA is a targeting substance-drug conjugate or a pharmaceutically acceptable salt thereof, which is an antibody or an antigen-binding fragment thereof.

[0058] According to the present invention, a linker-drug conjugate is provided that enables the production of a targeting substance-drug conjugate with improved hydrophilicity and stability while maintaining the original functions of the targeting substance (e.g., antibody) and the drug. Furthermore, by using the linker-drug conjugate of the present invention, a targeting substance-drug conjugate with improved hydrophilicity and stability while maintaining the original functions of the targeting substance and the drug can be efficiently manufactured.

[0059] Figure 1 shows the results of analyzing the quality of a targeting substance-drug conjugate according to an embodiment of the present invention.

[0060] FIGS. 2 to 4 are the results of the thermal stability evaluation of a targeting substance-drug conjugate according to an embodiment of the present invention, where FIGS. 2 and 4 show the results analyzed under a formulation buffer, and FIG. 3 shows the results analyzed under PBS.

[0061] Figure 5 shows the pharmacokinetic (PK) analysis results as the results of an in vivo efficacy test of a targeting substance-drug conjugate according to an embodiment of the present invention.

[0062] Figures 6 to 8 show the results of an in vitro cytotoxicity evaluation according to the type of antibody of a targeting substance-drug conjugate according to an embodiment of the present invention.

[0063] Figure 9 shows the results of an in vivo efficacy test of a targeting substance-drug conjugate according to an embodiment of the present invention, and the results of an analysis of anticancer efficacy in an HCC1954 breast cancer model with high HER2 levels.

[0064] Figures 10 and 11 show the results of an in vivo efficacy test of a targeting substance-drug conjugate according to an embodiment of the present invention, illustrating the analysis of anticancer efficacy in a JIMT-1 breast cancer model with moderate HER2 levels. P-values ​​were calculated using a standard one-way analysis of variance (one-way ANOVA). *, P<0.05 vs. control; **, P<0.01 vs. control; ***, P<0.001 vs. control; #, P<0.05 vs. G3.

[0065] FIG. 12 shows an evaluation of the stability of a targeting substance-drug conjugate in the blood according to an embodiment of the present invention.

[0066] The linker-drug conjugate of the present invention is characterized by having the structure of Formula 1. The targeting substance-drug conjugate of the present invention is an application of the linker-drug conjugate of the present invention and is characterized by having the structure of Formula 2.

[0067]

[0068] The linker-drug conjugate of the present invention refers to a conjugate capable of binding to a targeting substance through the linker. When the targeting substance-drug conjugate formed thereby is administered in vivo, the antibody or its antigen-binding fragment binds to the targeting antigen and subsequently releases the drug, thereby enabling the drug to act on target cells and / or surrounding cells. Therefore, excellent efficacy and reduced side effects can be expected as a targeting drug.

[0069]

[0070] The linker-drug conjugate of the present invention may consist only of the mentioned components (e.g., AG, L1, BS, L2, D, L3, and P indicated in Formula 1 above), but may also be a structure in which other components, for example, other components typically applied to targeting substance-drug conjugates, for example ADCs, are additionally connected.

[0071]

[0072] The connecting line "-" between the components indicated in each formula of this specification, for example, in the case of Formula 1, AG and (L1) a between, (L1) a Between and BS, between AG and BS (when a is 0), BS and (L2) b between, (L2) b Between and D, between BS and D (when b is 0), between BS and (L3) c between, (L3) c The connecting lines between and P and between BS and P (when c is 0) can each independently mean not only direct connections but also indirect connections, for example, connections through other atoms or molecules.

[0073]

[0074] In the present invention, BS refers to a branching spacer, and is a molecule capable of forming a branched structure by having three or more arms based on a single atom, connecting AG or L1 and D or L2, and connecting P or L3. For example, it may be a structure in which amino acids are connected through the nitrogen of the amino group, the carbon of the carboxyl group, and elements of the side chain. In the case of such a structure, for the expected effects of the present invention, BS is preferably a structure in which it is connected to L1 or AG through the nitrogen of the amino group, connected to L2 or D through the carbon of the carboxyl group, and connected to L3 or P through the carbon or nitrogen of the side chain.

[0075] More preferably for the expected effects of the present invention, BS is

[0076] or As explained above, the structure of the above BS means that each of them represents the structure as follows in Chemical Formula 1 above.

[0077]

[0078] More preferably for the expected effects of the present invention, BS is a structure in which lysine or glutamic acid is linked, for example

[0079] or am.

[0080]

[0081] In the present invention, P is a peptide, and its terminals (N-terminal or C-terminal) may be modified or unmodified. This portion exists in a branched form by BS and plays an important role in imparting hydrophilicity to a linker-drug conjugate or a targeting substance-drug conjugate (e.g., ADC).

[0082] In the present invention, P is preferably a terminal-modified or non-modified peptide having a plurality of amino acids linked together for the expected effect of the present invention, and is connected to BS or L3 through the nitrogen at the N-terminus or the carbon at the C-terminus.

[0083] In the present invention, P is, more preferably for the expected effects of the present invention, a terminal-modified or non-modified peptide in which two or more amino acids selected from glycine (G), serine (S), and threonine (T) are linked; more preferably, a terminal-modified or non-modified peptide in which three or more amino acids selected from glycine, serine, and threonine are linked; and more preferably, a terminal-modified or non-modified peptide in which four or more amino acids selected from glycine, serine, and threonine are linked.

[0084] In the present invention, P is, more preferably for the expected effects of the present invention, a terminal-modified or non-modified peptide having four or more amino acids linked together, selected from glycine and serine.

[0085] In the present invention, P is, more preferably for the expected effects of the present invention, any one of the following:

[0086]

[0087]

[0088] As mentioned above,

[0089] R1 is independently hydrogen, or C 1-4 It is a hydroxyalkyl, and

[0090] R2 is hydrogen, or (C 1-4 It is an alkyl)carbonyl, and

[0091] R3 is a hydroxyl or amino, and

[0092] n is a natural number from 1 to 10.

[0093] More preferably for the expected effects of the present invention, R1 is each independently hydrogen, hydroxymethyl, or 1-hydroxyethyl. More preferably for the expected effects of the present invention, R2 is hydrogen or methyl carbonyl.

[0094] In the present invention, P is, more preferably for the expected effects of the present invention, any one selected from the group consisting of the following:

[0095]

[0096]

[0097] In the above, n is a natural number from 1 to 10.

[0098] More preferably for the expected effects of the present invention, n is a natural number from 1 to 4.

[0099] More preferably for the expected effects of the present invention, the BS In the case where, among the above P, the P whose connecting part is carbonyl combines to form an amide bond, and the above BS In this case, among the above P, the P whose connecting part is an amino group combines to form an amide bond.

[0100] In the present invention, the terminal-modified peptide may be one in which the hydrogen at the N-terminus of the peptide is substituted with an acetyl group, or one in which the carboxylic acid group at the C-terminus of the peptide is substituted with a carboxamide.

[0101]

[0102] In the present invention, L1 and L3 serve as spacers that exist between each component and help each component fully perform its inherent function by reducing mutual interference through securing distance between the components.

[0103] In the present invention, L1 may not exist, which corresponds to the case where a is 0. In the present invention, L3 may not exist, which corresponds to the case where c is 0.

[0104] In the present invention, L2 may be a spacer. If L2 is a spacer, it may perform the same role as L1 and L3.

[0105] In the present invention, L2 may be a cleavable linker. If L2 is a cleavable linker, it may be an enzymatically cleavable linker configured to release a drug portion under specific conditions, for example, when internalized into a target cell.

[0106] In the present invention, L2 preferably comprises one or more of a cleatable dipeptide, a para-aminobenzyl alcohol self-sacrificial group, and a glucuronide bond for the expected effects of the present invention.

[0107] In the present invention, L2 is preferably selected from the group consisting of the following for the expected effects of the present invention:

[0108]

[0109] In the present invention, L2 may not exist, which corresponds to the case where b is 0.

[0110] Preferably for the expected effects of the present invention, a is 0, b is 1 or 2, and c is 0.

[0111]

[0112] In the present invention, the targeting substance (TA) may be a molecule such as an antibody or its antigen-binding fragment; or an aptamer; but is not limited thereto, and may include various other molecules with known targeting capabilities.

[0113] In the present invention, where the targeting substance (TA) is an antibody or an antigen-binding fragment thereof, the antibody may be a monoclonal or polyclonal, single-chain or multi-chain immunoglobulin, and may be of natural origin or produced through recombination. It may also be an immunoglobulin of the same class as IgG, IgE, IgM, IgD, IgA, and IgY. The antigen-binding fragment refers to at least one part of the antibody that possesses the ability to specifically interact with an epitope of the antigen for said antibody. For example, the antigen-binding fragment may be, but is not limited to, a Fab, Fab', F(ab')2, Fv fragment, scFv antibody fragment, disulfide-linked Fv (sdFv), an Fd fragment consisting of VH and CH1 domains, or a linear antibody. In one embodiment, the antibody of the present invention is preferably, for the expected effect of the present invention, an antibody against HER2, and more preferably trastuzumab.

[0114]

[0115] The antibody of the present invention or its antigen-binding fragment may be connected to the AG of the present invention, for example, through the sulfhydryl group of cysteine.

[0116] In the present invention, AG refers to an attachment group for a targeting substance, meaning a molecule that causes a linker-drug conjugate to attach to a targeting substance.

[0117] In the present invention, AG is preferably composed of a reactive group selected from the group consisting of a maleimide group, a thiol group, a cyclooctine group, an azido group, and a maleamic acid group, as an attachment group to an antibody or its antigen-binding fragment, for the expected effect of the present invention.

[0118] In the present invention, AG is preferably selected from the group consisting of the following, as an attaching group to an antibody or its antigen-binding fragment, for the expected effects of the present invention:

[0119]

[0120] As mentioned above,

[0121] L3 is a C that optionally contains phenylene, -CONH-, or -C(R4)- in the middle of the chain. 1-10 It is an alkylene, and

[0122] R4 is C 1-10 It is an aminoalkyl.

[0123] For the expected effects of the present invention, R4 is preferably aminomethyl.

[0124] For the expected effects of the present invention, preferably, as an attaching group to an antibody or its antigen-binding fragment, it is any one selected from the group consisting of:

[0125]

[0126] As mentioned above,

[0127] n1 is an integer from 1 to 10, and

[0128] n2 is an integer from 0 to 9, and

[0129] R4 is as previously defined.

[0130] For the expected effects of the present invention, preferably, as an attaching group to an antibody or its antigen-binding fragment, it is any one selected from the group consisting of:

[0131]

[0132]

[0133] In the present invention, D signifies a drug, and said drug refers to an agent capable of modulating biological processes or having biological activity. For example, various drugs such as antitumor drugs, anticancer drugs, etc. may be included herein. According to one embodiment, the drug of the present invention is preferably a camptothecin-class drug to realize the expected effects of the present invention, and more preferably may be exatecan or an analog thereof. In another embodiment, the drug of the present invention is preferably monomethyl auristatin F (MMAF) or an analog thereof to realize the expected effects of the present invention. In yet another embodiment, the drug of the present invention is preferably duocarmycin or an analog thereof to realize the expected effects of the present invention. In yet another embodiment, the drug of the present invention is preferably monomethyl auristatin E (MMAE) or an analog thereof to realize the expected effects of the present invention. In another embodiment, the drug of the present invention is preferably a pyrrolobenzodiazepine (PBD) dimer or an analog thereof to realize the expected effects of the present invention. In another embodiment, the drug of the present invention is preferably a kinesin spindle protein (KSP) inhibitor, more preferably ispinesib or an analog thereof to realize the expected effects of the present invention. In another embodiment, the drug of the present invention is preferably a cyclopropylbenzoindole (CBI) dimer or an analog thereof to realize the expected effects of the present invention.

[0134]

[0135] The targeting substance-drug conjugate of the present invention may be in the form where one or more linker-drug conjugate molecules are connected to a single targeting substance molecule. For example, when an IgG immunoglobulin antibody is applied as the targeting substance, up to eight linker-drug conjugate molecules may be connected. Preferably, when an IgG immunoglobulin antibody is applied, seven or more linker-drug conjugate molecules are connected.

[0136]

[0137] In one embodiment, the linker-drug conjugate of the present invention has the structure shown in Table 1 below. When having such a structure, superior effects can be expected in terms of the expected effects of the present invention, namely, the ability to generate a targeting substance-drug conjugate with improved hydrophilicity and stability while maintaining the original functions of the targeting substance (e.g., antibody) and the drug.

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178] In the same context, in one embodiment, the targeting substance-drug conjugate of the present invention has a structure in which an antibody is bound to the maleimide group of Table 1. For example, the structure in which an antibody is bound to the LD-001 linker-drug conjugate of Table 1 is as follows and is designated as "TA-LD-001" in this specification. Targeting substance-drug conjugates in which antibodies are bound to the maleimide groups of the remaining targeting substance-drug conjugates of Table 1 are also designated in the same manner.

[0179]

[0180]

[0181] Furthermore, the linker-drug conjugate or targeting substance-drug conjugate of the present invention described above may exist as a salt, enantiomer, stereoisomer, solvate, polymorph, or isotopic derivative, and in particular, may exist in the form of a pharmaceutically acceptable salt. As for the salt, any salt commonly used in the art, such as an acid addition salt formed by a pharmaceutically acceptable free acid, may be used without limitation. The term "pharmaceutically acceptable salt" in the present invention refers to any organic or inorganic addition salt at a concentration having a relatively non-toxic and harmless active effect on the patient, wherein the side effects attributable to this salt do not impair the beneficial efficacy of the linker-drug conjugate or targeting substance-drug conjugate of the present invention.

[0182] The above free acid may be an organic acid or an inorganic acid. Inorganic acids may include hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc., and organic acids may include methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carboxylic acid, vanillic acid, hydroiodide, etc., but are not limited thereto.

[0183] In addition, pharmaceutically acceptable metal salts can be obtained by conventional methods using a base. For example, a pharmaceutically acceptable metal salt can be obtained by dissolving the targeting substance-drug conjugate of the present invention in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate. In this case, it is particularly preferable to prepare a sodium salt, a potassium salt, or a calcium salt as the metal salt.

[0184] Additionally, a salt or solvate of the linker-drug conjugate or targeting substance-drug conjugate of the present invention that is not pharmaceutically acceptable may be used as an intermediate in the preparation of the linker-drug conjugate or targeting substance-drug conjugate of the present invention, or a pharmaceutically acceptable salt or solvate thereof.

[0185]

[0186] In addition, the targeting substance-drug conjugate of the present invention described above, its pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate, polymorph, or isotope derivative may be provided as a pharmaceutical composition comprising as an active ingredient.

[0187] The above pharmaceutical composition may be formulated into oral or parenteral administration forms in accordance with standard pharmaceutical practices. These formulations may contain, in addition to the active ingredient, pharmaceutically acceptable additives such as carriers, adjuvants, or diluents.

[0188] Suitable carriers include, for example, physiological saline, polyethylene glycol, ethanol, vegetable oil, and isopropyl myristate, and diluents include, for example, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine, but are not limited thereto. In addition, the targeting substance-drug conjugate of the present invention described above can be dissolved in oil, propylene glycol, or other solvents commonly used in the preparation of injection solutions.

[0189] The targeting substance-drug conjugate of the present invention described above may be formulated as an injectable by dissolving, suspending, or emulsifying the compound in a water-soluble solvent such as ordinary saline or about 5% dextrose, or in a water-insoluble solvent such as synthetic fatty acid glycerides, higher fatty acid esters, or propylene glycol. The formulation of the present invention may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives.

[0190] The pharmaceutical administration forms of the targeting substance-drug conjugate of the present invention described above can be used alone, in combination with other pharmaceutically active compounds, as well as in a suitable combination.

[0191] The preferred dosage of the targeting substance-drug conjugate of the present invention described above may vary depending on the patient's condition and body weight, the severity of the disease, the form of the drug, the route of administration, and the duration, but can be appropriately selected by those skilled in the art. However, for a desirable effect, it is preferable to administer the targeting substance-drug conjugate of the present invention at about 0.0001 to 100 mg / kg (body weight) per day, preferably about 0.001 to 100 mg / kg (body weight). Administration may be administered once a day or in divided doses via oral or parenteral routes. Depending on the method of administration, the pharmaceutical composition may contain about 0.001 to 99 weight%, preferably about 0.01 to 60 weight%, of the targeting substance-drug conjugate of the present invention.

[0192] The targeting substance-drug conjugate of the present invention described above may be administered to mammals, including rats, mice, livestock, and humans, via various routes. Any mode of administration is expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intradural, or intracerebroventricular injection.

[0193]

[0194] The present invention will be described in more detail below through examples. These examples are merely illustrative of the present invention, and therefore the scope of the present invention should not be interpreted as being limited by these examples.

[0195]

[0196] Example 1. Synthesis of linker-drug conjugate

[0197] A linker-drug conjugate as shown in Table 1 was synthesized using the branched portion and drug portion as shown in Table 2 below as starting materials and the corresponding synthesis method.

[0198]

[0199]

[0200] The synthesis method of the starting materials in Table 2 above is as follows.

[0201]

[0202] E-(GGSG)1 synthesis

[0203] E-(GGSG)1, such as compound 11, was synthesized according to the reaction scheme below.

[0204]

[0205]

[0206]

[0207]

[0208]

[0209] (Step 1) CTC-resin (11.8 g, 11.23 mmol, 0.95 mmol / g) was added to DCM (200 mL) and swollen for 0.5 hours under nitrogen bubbling at 25°C. Subsequently, Compound 1 (2 g, 6.73 mmol) and DIPEA (7.26 g, 56.17 mmol, 9.78 mL) were dissolved in DCM (30 mL) and added, followed by a reaction for 1 hour under nitrogen bubbling at 25°C. The reaction was terminated by adding methanol (1.80 g, 56.17 mmol, 2.27 mL), and the mixture was treated again for 0.5 hours under nitrogen bubbling at 25°C. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 5) to obtain the resin. Compound 2 (1.99 g, crude) was obtained as a yellow solid in a quantitatively adsorbed state on the resin and was used in the next step. LCMS: RT = 0.514 min, m / z 320.1 [M+Na] + .

[0210] (Step 2) Compound 2 (1.99 g, 6.72 mmol, quantitatively adsorbed onto resin) was added to a piperidine / DMF mixed solution (5% in DMF, total 300 mL) and reacted for 1 hour at 25°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 5) to obtain the resin. Compound 3 (497 mg, crude) was obtained as a yellow solid, quantitatively adsorbed onto the resin.

[0211] (Step 3) Compound 3 (497 mg, 6.71 mmol, quantitatively adsorbed onto resin) was placed in DMF (250 mL), and (2,3,4,5,6-pentafluorophenyl)(4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyloxazolidine-4-carboxylate (5.56 g, 9.42 mmol) and DIPEA (3.47 g, 26.84 mmol, 4.68 mL) were added. The mixture was reacted for 12 hours at 25°C under nitrogen bubbling. Afterward, the reaction mixture was treated with HFIP (0.5 mL), filtered, and the resin was washed with DMF (100 mL x 5) to obtain the resin. Compound 4 (3.22 g, crude) was obtained as a yellow solid quantitatively adsorbed onto the resin. LCMS: RT = 0.516 min, m / z 504.2 [M+Na]+.

[0212] (Step 4) Compound 4 (3.22 g, 6.70 mmol, quantitatively adsorbed onto resin) was added to a piperidine / DMF (5% in DMF, 100 mL) mixed solution and reacted for 1 hour at 25°C under nitrogen bubbling. Afterward, the reaction mixture was treated with HFIP (0.5 mL), filtered, and the resin was washed with DMF (100 mL x 5) to obtain the resin. Compound 5 (1.73 g, crude) was obtained as a yellow solid in a quantitatively adsorbed state onto the resin.

[0213] (Step 5) Compound 5 (1.73 g, 6.70 mmol, quantitatively adsorbed onto resin) was placed in DMF (100 mL), and (2,3,4,5,6-pentafluorophenyl) 2-(9H-fluorene-9-ylmethoxycarbonylamino)acetate (4.36 g, 9.41 mmol) and DIPEA (1.73 g, 13.40 mmol, 2.33 mL) were added. The mixture was reacted at 25°C for 1.5 hours under nitrogen bubbling. Afterward, the reaction mixture was treated with HFIP (0.5 mL), filtered, and the resin was washed with DMF (100 mL x 10) to obtain the resin. Compound 6 (3.6 g, crude) was obtained as a yellow solid quantitatively adsorbed onto the resin. LCMS: RT = 0.501 min, m / z 539.2 [M+H] + .

[0214] (Step 6) Compound 6 (3.6 g, 6.70 mmol, quantitatively adsorbed onto resin) was added to a piperidine / DMF (5% in DMF, 100 mL) mixed solution and reacted for 1 hour at 25°C under nitrogen bubbling. Afterward, the reaction mixture was treated with HFIP (0.5 mL), filtered, and the resin was washed with DMF (100 mL x 5) to obtain the resin. Compound 7 (2.11 g, crude) was obtained as a yellow solid in a quantitatively adsorbed state onto the resin.

[0215] (Step 7) Compound 7 (2.11 g, 6.69 mmol, quantitatively adsorbed onto resin) was placed in DMF (100 mL), and O1-allyl O5-(2,3,4,5,6-pentafluorophenyl)(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)pentanedioate (5.62 g, 9.77 mmol) and DIPEA (3.46 g, 26.77 mmol, 4.66 mL) were added. The mixture was reacted for 1 hour at 25°C under nitrogen bubbling. Afterward, the reaction mixture was treated with HFIP (0.5 mL), filtered, and the resin was washed with DMF (100 mL x 10) to obtain the resin. Compound 8 (4.73 g, crude) was obtained as a yellow solid quantitatively adsorbed onto the resin. LCMS: RT = 0.525 min, m / z 708.3 [M+H] + .

[0216] (Step 8) Compound 8 (6 g, 8.49 mmol, quantitatively adsorbed onto resin) was added to a mixed solution of HFIP / DCM (20% of DCM, 100 mL) and reacted for 1 hour at 25°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DCM (50 mL x 3). The filtrate was concentrated under reduced pressure to obtain the residue. As a result, Compound 9 (6 g, purity 97%, crude) was obtained as a yellow solid. LCMS: RT = 0.803 min, m / z 730.2 [M+Na] + .

[0217] (Step 9) Compound 9 (5.9 g, 8.34 mmol) was added to THF (100 mL), and 2-tert-butyl-1,3-diisopropylisourea (6.68 g, 33.35 mmol) was added. The mixture was stirred at 25°C for 1 hour. After filtering the reaction mixture, the filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 1 to 0 / 1). Compound 10 (5.7 g, 6.57 mmol, yield 78.77%, purity 88%) was obtained as a white solid. LCMS: RT = 0.972 min, m / z 764.3 [M+H] + .

[0218] (Step 10) Compound 10 (2.8 g, 3.67 mmol) was added to DMF (30 mL), and Pd(PPh3)4 (1.27 g, 1.10 mmol) and phenylsilane (1.19 g, 11.00 mmol, 1.36 mL) were added. The mixture was stirred at 25°C for 1 hour under a nitrogen atmosphere. The reaction mixture was used as is in the next step without further purification. Compound 11 (2.65 g, crude) was used in the next step as a brown liquid. CTC-resin (7.7 g, 7.32 mmol) was added to DCM (50 mL) and swollen for 0.5 hours under nitrogen bubbling at 25°C. Next, Compound 11 (2.65 g, 3.66 mmol) and DIPEA (4.73 g, 36.61 mmol, 6.38 mL) were added, and the mixture was reacted for 0.5 hours at 25°C under nitrogen bubbling. Subsequently, methanol (1.17 g, 36.61 mmol, 1.48 mL) was added to terminate the reaction, after which the mixture was treated again for 0.5 hours at 25°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (30 mL x 10). Compound 11 (2.65 g, crude) was obtained as a yellow solid, quantitatively adsorbed onto the resin. Subsequently, Compound 11 (2.65 g, 3.67 mmol) was added to a mixed solution of HFIP / DCM (20% of DCM, total 60 mL) and reacted for 1 hour at 25°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DCM (100 mL x 3). The filtrate was concentrated under reduced pressure to obtain the residue. This residue was purified by prep-HPLC (Column: Phenomenex Luna C18 150 * 40 mm, 15 μm; Mobile phase: Water (FA) / ACN; Gradient: B 45% -> 75% over 15 min). As a result, Compound 11 (1.27 g, 1.75 mmol, yield 47.86%, purity 100%) was obtained as a white solid. LCMS: RT = 0.905 min, m / z 724.3 [M+H] + .

[0219]

[0220] E-(GGSG)2 synthesis

[0221] E-(GGSG)2, such as Compound 11, was synthesized according to the reaction scheme below. Here, Compound 1 was synthesized in the same manner as Compound 5 was synthesized in the synthesis of E-(GGSG)1 above.

[0222]

[0223]

[0224]

[0225]

[0226]

[0227] (Step 1) (2,3,4,5,6-pentafluorophenyl) 2-[[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]amino]acetate (8.52 g, 16.37 mmol, 1.21 eq) was added to the solution of Compound 1 (3.48 g, 13.48 mmol, 1 eq, synthesized in the same manner as Compound 5 of the above E-(GGSG)1 synthesis example, quantitatively adsorbed onto resin) in DMF (200 mL). The mixture was reacted for 12 hours at 30°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain Compound 2 (8.01 g, crude, quantitatively adsorbed onto resin) in the form of a yellow solid. LCMS: RT = 0.503 min, m / z 596.2 [M+H] + .

[0228] (Step 2) Compound 2 (8.01 g, 13.47 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (200 mL, 5% in DMF) was mixed and reacted for 1.5 hours at 30°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain dendritic compound 3 in the form of a yellow solid (5.02 g, crude, quantitatively adsorbed onto resin).

[0229] (Step 3) (2,3,4,5,6-pentafluorophenyl)(4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyl-oxazolidine-4-carboxylate (9.74 g, 16.49 mmol, 1.22 eq) and DIPEA (5.94 g, 45.93 mmol, 8 mL, 3.41 eq) were added to a solution of Compound 3 (5.02 g, 13.48 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (200 mL). The mixture was reacted for 12 hours under nitrogen bubbling at 30°C. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain Compound 4 (10.5 g, crude, quantitatively adsorbed onto resin) in the form of a yellow solid. LCMS: RT = 0.542 min, m / z 780.5 [M+H] + .

[0230] (Step 4) Compound 4 (10.5 g, 13.48 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (200 mL, 5% in DMF) was mixed and reacted for 1 hour at 30°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain Compound 5 (7.5 g, crude, quantitatively adsorbed onto resin) in the form of a yellow solid. LCMS: RT = 0.777 min, m / z 558.3 ​​[M+H] + .

[0231] (Step 5) (2,3,4,5,6-pentafluorophenyl) 2-(9H-fluorene-9-ylmethoxycarbonylamino)acetate (9.35 g, 20.18 mmol, 1.50 eq) and DIPEA (5.94 g, 45.93 mmol, 8 mL, 3.41 eq) were added to the solution of Compound 5 (7.5 g, 13.48 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (200 mL). The mixture was reacted for 12 hours at 30°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain Compound 6 (11.26 g, crude, quantitatively adsorbed onto resin) in the form of a yellow solid. LCMS: RT = 0.504 min, m / z 837.3 [M+H] + .

[0232] (Step 6) A mixture of Compound 6 (11.26 g, 13.47 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (200 mL, 5% in DMF) was reacted at 30°C under nitrogen bubbling for 1 hour. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain Compound 7 as a yellow solid (8.27 g, crude, quantitatively adsorbed onto resin). LCMS: RT = 0.349 min, m / z 615.4 [M+H] + .

[0233] (Step 7) O1-allyl O5-(2,3,4,5,6-pentafluorophenyl)(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)pentanedioate (8.43 g, 14.65 mmol, 1.09 eq) and DIPEA (5.94 g, 45.93 mmol, 8 mL, 3.41 eq) were added to a solution of Compound 7 (8.27 g, 13.48 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (200 mL). The mixture was reacted for 12 hours at 30°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain Compound 8 (13.55 g, crude, quantitatively adsorbed onto resin) in the form of a yellow solid. LCMS: RT = 0.526 min, m / z 1005.0 [M+H] + .

[0234] (Step 8) A mixture of Compound 8 (13.55 g, 13.48 mmol, 1 eq, quantitatively adsorbed onto resin) in HFIP / DCM (200 mL) was reacted at 30°C under nitrogen bubbling for 1 hour. The reaction mixture was filtered, and the resin was washed with DCM (50 mL x 3) to obtain Compound 9 (11.5 g, 11.43 mmol, yield 84.79%), a crude product in the form of a yellow gum, which was used in the next step without further purification. LCMS: RT = 0.514 min, m / z 1006.3 [M+H] + .

[0235] (Step 9) 2-tert-butyl-1,3-diisopropyl-isourea (13.4 g, 66.89 mmol, 5.85 eq) was added to a solution of Compound 9 (11.5 g, 11.43 mmol, 1 eq) in THF (100 mL). The mixture was stirred at 20°C for 2 hours. After filtering the reaction mixture, the solvent was removed under reduced pressure. The residue was purified by flash silica gel column chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0–10% Ethyl acetate / Petroleum ether gradient @ 80 mL / min) to obtain Compound 10 (8.3 g, 7.81 mmol, yield 68.36%) as a yellow solid. LCMS: RT = 0.590 min, m / z 1062.1 [M+H] + .

[0236] (Step 10) Compound 10 (8.3 g, 7.81 mmol, 1 eq), Pd(PPh3)4 (1.81 g, 1.56 mmol, 0.2 eq), and phenylsilane (2.54 g, 23.44 mmol, 2.89 mL, 3 eq) were mixed in DMF (50 mL), degassed, and purged with nitrogen three times. Subsequently, the mixture was stirred at 30 °C for 1 hour under a nitrogen atmosphere to obtain Compound 11 as the crude product. LCMS: RT = 0.544 min, m / z 1022.0 [M+H] + .

[0237]

[0238] E-(GGSG)3 synthesis

[0239] E-(GGSG)3, such as Compound 11, was synthesized according to the reaction scheme below. Here, Compound 1 was synthesized in the same manner as Compound 5 was synthesized in the synthesis of E-(GGSG)2 above.

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246] (Step 1) (2,3,4,5,6-pentafluorophenyl) 2-[[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]amino]acetate (7.34 g, 14.10 mmol, 1.40 eq) and DIPEA (7.42 g, 57.41 mmol, 10 mL, 5.69 eq) were added to a solution of Compound 1 (5.62 g, 10.10 mmol, 1 eq; synthesized in the same manner as Compound 5 in the above E-(GGSG)2 synthesis, quantitatively adsorbed onto resin) in DMF (200 mL). The mixture was reacted for 12 hours under nitrogen bubbling at 20°C. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain the resin. Compound 2 in the form of a yellow solid (9.02 g, crude, quantitatively adsorbed onto resin) was obtained. LCMS: RT = 0.750 min, m / z 894.1 [M+H] + .

[0247] (Step 2) Compound 2 (9.02 g, 10.10 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (200 mL, 5% in DMF) was mixed and reacted for 1 hour under nitrogen bubbling at 20°C. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain the resin. Compound 3 (6.77 g, crude, quantitatively adsorbed onto resin) in the form of a yellow solid was obtained. LCMS: RT = 0.275 min, m / z 672.3 [M+H] + .

[0248] (Step 3) (2,3,4,5,6-pentafluorophenyl)(4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyl-oxazolidine-4-carboxylate (8.35 g, 14.14 mmol, 1.40 eq) was added to the solution of Compound 3 (6.77 g, 10.09 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (200 mL). The mixture was reacted for 12 hours at 20°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain the resin. Compound 4 (10.87 g, crude, quantitatively adsorbed onto resin) in the form of a yellow solid was obtained. LCMS: RT = 0.502 min, m / z 1078.2 [M+H] + .

[0249] (Step 4) Compound 4 (10.87 g, 10.09 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (200 mL, 5% in DMF) was mixed and reacted for 2 hours at 20°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain the resin. Compound 5 (8.63 g, crude, quantitatively adsorbed onto resin) in the form of a yellow solid was obtained. LCMS: RT = 0.380 min, m / z 856.3 [M+H] + .

[0250] (Step 5) (2,3,4,5,6-pentafluorophenyl) 2-(9H-fluorene-9-ylmethoxycarbonylamino)acetate (7.01 g, 15.13 mmol, 1.50 eq) and DIPEA (5.94 g, 45.93 mmol, 8 mL, 4.55 eq) were added to a solution of Compound 5 (8.63 g, 10.10 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (200 mL). The mixture was reacted for 2 hours under nitrogen bubbling at 20°C. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain the resin. Compound 6 (11.45 g, crude, quantitatively adsorbed onto resin) in the form of a yellow solid was obtained. LCMS: RT = 0.875 min, m / z 1135.6 [M+H] + .

[0251] (Step 6) Compound 6 (11.45 g, 10.10 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (200 mL, 5% in DMF) was mixed and reacted for 1 hour at 30°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain the resin. Compound 7 (9.21 g, crude, quantitatively adsorbed onto resin) in the form of a yellow solid was obtained. LCMS: RT = 0.422 min, m / z 913.4 [M+H] + .

[0252] (Step 7) O1-allyl O5-(2,3,4,5,6-pentafluorophenyl)(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)pentanedioate (8.43 g, 14.65 mmol, 1.45 eq) and DIPEA (5.94 g, 45.93 mmol, 8 mL, 4.55 eq) were added to a solution of Compound 7 (9.21 g, 10.10 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (200 mL). The mixture was reacted for 12 hours under nitrogen bubbling at 30°C. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain the resin. Compound 8 (13.16 g, crude, quantitatively adsorbed onto resin) in the form of a yellow solid was obtained. LCMS: RT = 0.514 min, m / z 1304.7 [M+H] + .

[0253] (Step 8) A solution of Compound 8 (13.16 g, 10.10 mmol, 1 eq, quantitatively adsorbed onto resin) in HFIP / DCM (200 mL) was reacted at 30°C under nitrogen bubbling for 1 hour. The reaction mixture was filtered, and the resin was washed with DCM (50 mL x 3) to obtain Compound 9 (13 g, 9.97 mmol, yield 98.71%), a crude product in the form of a yellow oil, which was used in the next step without further purification. LCMS: RT = 0.544 min, m / z 1304.6 [M+H] + .

[0254] (Step 9) 2-tert-butyl-1,3-diisopropyl-isourea (22 g, 109.82 mmol, 11.02 eq) was added to a solution of compound 9 (13 g, 9.97 mmol, 1 eq) in THF (100 mL). The mixture was stirred at 30°C for 1 hour. After filtering the reaction mixture, the solvent was removed under reduced pressure. The residue was purified by flash silica gel column chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0–15% Methanol / Ethyl acetate ether gradient @ 80 mL / min) to obtain the target compound 10 in the form of a yellow gum (5.7 g, 4.19 mmol, yield 42.04%, quantitatively adsorbed onto resin). LCMS: RT = 0.550 min, m / z 1360.8 [M+H] + .

[0255] (Step 10) Compound 10 (5.7 g, 4.19 mmol, 1 eq), Pd(PPh3)4 (968.33 mg, 837.98 μmol, 0.2 eq), and phenylsilane (1.36 g, 12.57 mmol, 1.55 mL, 3 eq) were mixed in DMF (50 mL), degassed, and purged with nitrogen three times. Subsequently, the mixture was stirred at 20°C for 1 hour under a nitrogen atmosphere to obtain Compound 11. LCMS analysis revealed that approximately 31% of the target mass was detected. LCMS: RT = 0.517 min, m / z 1320.8 [M+H] + .

[0256]

[0257] E-(GGSG)4 synthesis

[0258] E-(GGSG)4, such as Compound 11, was synthesized according to the reaction scheme below. Here, Compound 1 was synthesized in the same manner as Compound 5 was synthesized in the synthesis of E-(GGSG)3 above.

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265] (Step 1) Compound 1 (2.87 g, 3.36 mmol, 1 eq, synthesized in the same manner as the synthesis of Compound 5 in the synthesis of E-(GGSG)3 above, quantitatively adsorbed onto resin) in DMF (150 mL) was reacted for 0.5 hours under nitrogen bubbling at 20-25°C, then (2,3,4,5,6-pentafluorophenyl) 2-[[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]amino]acetate (2.27 g, 4.36 mmol, 1.3 eq) and DIPEA (433.90 mg, 3.36 mmol, 584.78 μL, 1 eq) in DMF (20 mL) were added, and the mixture was reacted for 1.5 hours under nitrogen bubbling at 20-25°C. Compound 2 (4 g, crude, quantitatively adsorbed onto resin), a crude product in the form of a yellow solid, was obtained and used in the next step without further purification. LCMS: RT = 0.775 min, m / z 1192.8 [M+H] + .

[0266] (Step 2) Compound 2 (4 g, 3.36 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (200 mL, 20% in DMF) was reacted for 0.5 hours at 20-25°C under nitrogen bubbling. Compound 3 (3.25 g, crude, quantitatively adsorbed onto resin), a crude product in the form of a yellow solid, was obtained and used in the next step without further purification.

[0267] (Step 3) Compound 3 (3.25 g, 3.35 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (150 mL) was reacted for 0.5 hours under nitrogen bubbling at 20-25°C, then (2,3,4,5,6-pentafluorophenyl) (4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyloxazolidine-4-carboxylate (2.5 g, 4.23 mmol, 1.26 eq) and DIPEA (1.30 g, 10.06 mmol, 1.75 mL, 3 eq) in DMF (20 mL) were added, and the mixture was reacted for 1.5 hours under nitrogen bubbling at 20-25°C. Compound 4 (4.61 g, crude, quantitatively adsorbed onto resin), a crude product in the form of a yellow solid, was obtained and used in the next step without further purification. LCMS: RT = 0.521 min, m / z 1377.1 [M+H] + .

[0268] (Step 4) Compound 4 (4.61 g, 3.35 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (200 mL, 20% in DMF) was reacted for 0.5 hours at 20-25°C under nitrogen bubbling. Compound 5 (3.87 g, crude, quantitatively adsorbed onto resin), a crude product in the form of a yellow solid, was obtained and used in the next step without further purification.

[0269] (Step 5) Compound 5 (3.87 g, 3.36 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (150 mL) was reacted for 0.5 hours under nitrogen bubbling at 20-25°C, then (2,3,4,5,6-pentafluorophenyl) 2-(9H-fluorene-9-ylmethoxycarbonylamino)acetate (2.07 g, 4.47 mmol, 1.33 eq) and DIPEA (1.30 g, 10.07 mmol, 1.75 mL, 3 eq) in DMF (20 mL) were added, and the mixture was reacted for 1.5 hours under nitrogen bubbling at 20-25°C. Compound 6 (4.81 g, crude, quantitatively adsorbed onto resin), a crude product in the form of a yellow solid, was obtained and used in the next step without further purification. LCMS: RT = 0.738 min, m / z 1432.5 [M+H] + .

[0270] (Step 6) Compound 6 (4.81 g, 3.36 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (200 mL, 20% in DMF) was reacted for 0.5 hours at 20-25°C under nitrogen bubbling. Compound 7 (4.06 g, crude, quantitatively adsorbed onto resin), a crude product in the form of a yellow solid, was obtained and used in the next step without further purification.

[0271] (Step 7) Compound 7 (4.06 g, 3.35 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (150 mL) was reacted for 0.5 hours under nitrogen bubbling at 20-25°C. Then, O1-benzyl O5-(2,3,4,5,6-pentafluorophenyl) (2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)pentanedioate (2.72 g, 4.35 mmol, 1.3 eq) and DIPEA (1.30 g, 10.06 mmol, 1.75 mL, 3 eq) dissolved in DMF (20 mL) were added, and the mixture was reacted for 1.5 hours under nitrogen bubbling at 20-25°C. Compound 8 (5.54 g, crude, quantitatively adsorbed onto resin), a crude product in the form of a yellow solid, was obtained and used in the next step without further purification. LCMS: RT = 0.873 min, m / z 1653.7 [M+H] + .

[0272] (Step 8) Compound 8 (5.54 g, 3.35 mmol, 1 eq, quantitatively adsorbed onto resin) in HFIP / DCM (300 mL) was bubbled with nitrogen at 20–25°C for 2 hours. The reaction mixture was concentrated under reduced pressure. As a result, Compound 9 (5.5 g, 3.06 mmol, yield 91.08%, purity 91.8%, quantitatively adsorbed onto resin), a crude product in the form of a yellow solid, was obtained and used in the next step without further purification. LCMS: RT = 0.565 min, m / z 1653.3 [M+H] + .

[0273] (Step 9) 2-tert-butyl-1,3-diisopropyl-isourea (2.45 g, 12.23 mmol, 10.11 eq) was added to a solution of Compound 9 (2 g, 1.21 mmol, 1 eq) in THF (10 mL). The reaction mixture was stirred at 20–25 °C for 2 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0–40% Ethyl acetate / MeOH @ 60 mL / min) to obtain Compound 10 (1.2 g, 554.78 μmol, yield 45.84%, purity 79%) in the form of a yellow solid. LCMS: RT = 0.576 min, m / z 1709.4 [M+H] + .

[0274] (Step 10) Pd / C (500 mg, 10% purity) was added to a solution of Compound 10 (1 g, 585.22 μmol, 1 eq) in MeOH (15 mL) under a nitrogen atmosphere. The suspension was degassed and purged three times with hydrogen. The mixture was stirred at 25°C for 4 hours under a hydrogen atmosphere (50 Psi). The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified by prep-HPLC (FA condition: Instrument: ACS-WH-GX-G; column: Phenomenex Luna C18 150*25mm, 10um; mobile phase: water (FA)-ACN; B%: 40%-70%, 10min; Flow rate: 25 mL / min; Column temperature: RT; Wavelength: 220nm) to obtain Compound 11 (400 mg, 241.19 μmol, yield 41.21%, purity 97.6%) as a white solid. LCMS: RT = 0.540 min, m / z 1620.3 [M+H] + .

[0275]

[0276] E-(GGTG)1 synthesis

[0277] E-(GGTG)1, such as Compound 10, was synthesized according to the reaction scheme below. Here, Compound 1 was synthesized in the same manner as Compound 3 was synthesized in the synthesis of E-(GGSG)1 above.

[0278]

[0279]

[0280]

[0281]

[0282]

[0283] (Step 1) (2,3,4,5,6-pentafluorophenyl)(2S,3S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-hydroxybutanoate (5.05 g, 9.95 mmol) was added to a mixture of Compound 1 (497 mg, 6.71 mmol, synthesized by the same method as Compound 3 in the above E-(GGSG)1 synthesis, quantitatively adsorbed onto resin) in DMF (30 mL). The reaction mixture was reacted at 25°C for 2 hours under nitrogen bubbling. The reaction mixture was filtered, and the resin was recovered by washing it with DMF (50 mL x 5). As a result, Compound 2 (2.67 g, crude, quantitatively adsorbed onto resin) in the form of a yellow solid was obtained. LCMS: RT = 0.523 min, m / z 399.2 [M+H] + .

[0284] (Step 2) TBSCl (6.3 g, 41.80 mmol, 5.14 mL) and imidazole (1.83 g, 26.84 mmol), dissolved in DMF (20 mL), were added to the mixture of Compound 2 (2.37 g, 5.96 mmol, quantitatively adsorbed onto resin) in DMF (80 mL). The reaction mixture was reacted at 25°C for 12 hours under nitrogen bubbling. After filtering the reaction mixture, the resin was recovered by washing it with DMF (50 mL x 5). As a result, Compound 3 (3.05 g, crude, quantitatively adsorbed onto resin) in the form of a yellow solid was obtained. LCMS: RT = 0.674 min, m / z 513.4 [M+H] + .

[0285] (Step 3) A mixture of Compound 3 (3.05 g, 5.96 mmol, quantitatively adsorbed onto resin) in piperidine / DMF (80 mL, 10% in DMF) was reacted at 25°C under nitrogen bubbling for 2 hours. After filtering the reaction mixture, the resin was recovered by washing it with DMF (50 mL x 5). As a result, Compound 4 (1.73 g, crude, quantitatively adsorbed onto resin) in the form of a yellow solid was obtained. LCMS: RT = 0.492 min, m / z 291.2 [M+H] + .

[0286] (Step 4) (2,3,4,5,6-pentafluorophenyl) 2-[[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]amino]acetate (4.67 g, 8.97 mmol) and DIPEA (1.55 g, 11.95 mmol, 2.08 mL) were added to a mixture of Compound 4 (1.73 g, 5.98 mmol, quantitatively adsorbed onto resin) in DMF (70 mL). The mixture was reacted for 1.5 hours at 25°C under nitrogen bubbling. After filtering the reaction mixture, the resin was washed with DMF (50 mL x 5) to recover the resin. As a result, Compound 5 (3.74 g, crude, quantitatively adsorbed onto resin), a yellow solid compound, was obtained and used in the next step without further purification. LCMS: RT = 0.610 min, m / z 627.5 [M+H] + .

[0287] (Step 5) The mixture of Compound 5 (3.74 g, 5.98 mmol, quantitatively adsorbed onto resin) in DMF / piperidine (80 mL, 10% in DMF) was reacted at 25°C under nitrogen bubbling for 1 hour. After filtering the reaction mixture, the resin was recovered by washing it with DMF (50 mL x 5). As a result, Compound 6 (2.41 g, crude, quantitatively adsorbed onto resin), a yellow solid compound, was obtained and used in the next step without further purification. LCMS: RT = 0.500 min, m / z 405.3 [M+H] + .

[0288] (Step 6) O1-benzyl O5-(2,3,4,5,6-pentafluorophenyl)(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)pentanedioate (5.58 g, 8.92 mmol) and DIPEA (2.32 g, 17.92 mmol, 3.12 mL) were added to a mixture of Compound 6 (2.41 g, 5.97 mmol) in DMF (80 mL). The mixture was reacted for 2 hours at 25°C under nitrogen bubbling. After filtering the reaction mixture, the resin was washed with DMF (50 mL x 5) to recover the resin. As a result, Compound 7 (5.05 g, crude, quantitatively adsorbed onto the resin), a yellow solid compound, was obtained and used in the next step without further purification. LCMS: RT = 0.643 min, m / z 846.6 [M+H] + .

[0289] (Step 7) A mixture of Compound 7 (5.05 g, 5.98 mmol) in HFIP / DCM (300 mL, 10% in DCM) was reacted at 25°C for 1 hour under nitrogen bubbling. The reaction mixture was filtered, and the filtrate was concentrated. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 250*50 mm*10 µm; mobile phase: [water(NH4HCO3)-ACN]; gradient: 20%-50% B over 26 min) to obtain Compound 8 (3 g, 3.02 mmol, yield 50.58%, purity 85.24%) as a white solid. LCMS: RT = 1.014 min, m / z 846.4 [M+H] + .

[0290] (Step 8) A mixture of Compound 8 (0.6 g, 604.53 μmol) and 2-tert-butyl-1,3-diisopropyl-isourea (1.21 g, 6.05 mmol) in THF (10 mL) was stirred at 25°C for 3.5 hours. After filtering the reaction mixture, the filtrate was concentrated. The residue was purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, Eluent of 0–100% Ethyl acetate / Petroleum ether gradient @ 40 mL / min) to obtain the crude product. The residue was purified by prep-HPLC (column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water(FA)-ACN]; gradient: 60%-90% B over 10 min) to obtain Compound 9 as a white solid (0.35 g, 376.61 μmol, yield 62.3%, purity 97.07%). LCMS: RT = 0.693 min, m / z 902.5 [M+H] + .

[0291] (Step 9) Compound 9 (350 mg, 387.98 μmol) was added to a Pd / C solution (100 mg, 93.97 μmol, purity 10%) in 5 mL of MeOH under an Ar atmosphere. The suspension was degassed and purged three times with hydrogen. Subsequently, the mixture was stirred at 25°C for 2 hours under hydrogen (50 Psi). The reaction mixture was filtered, and the filtrate was concentrated. The residue was purified by prep-HPLC (column: Phenomenex luna C18 150*25 mm*10 µm; mobile phase: [water(FA)-ACN]; gradient: 55%-85% B over 10 min) to obtain the compound. As a result, Compound 10 (210 mg, 258.62 μmol, yield 66.66%), a pale white solid compound, was obtained. LCMS: RT = 0.645 min, m / z 812.6 [M+H]+ .

[0292]

[0293] E-(GSSS)3 synthesis

[0294] E-(GSSS)3, such as compound 23, was synthesized according to the reaction scheme below.

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306] (Step 1) A mixture of CTC-RESIN (28 g, 0.93 mmol / g) in DCM (100 mL) was swollen for 0.5 hours at 20°C under nitrogen bubbling. Subsequently, Compound 1 (1 g, 2.61 mmol) and DIPEA (16.85 g, 130.40 mmol, 22.71 mL) were added, and the mixture was reacted for 1 hour at 20°C under nitrogen bubbling. Afterward, the reaction was terminated with DIPEA (16.85 g, 130.40 mmol, 22.71 mL), and the mixture was reacted for an additional 0.5 hours at 20°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was recovered by washing it with DMF (150 mL x 5). As a result, Compound 2 (997 mg, crude, quantitatively adsorbed onto the resin) in the form of a yellow solid was obtained. LCMS: RT = 0.612 min, m / z 406.1 [M+Na] + .

[0307] (Step 2) A mixture of Compound 2 (997 mg, 2.61 mmol, quantitatively adsorbed onto resin) in piperidine / DMF (150 mL, 5% in DMF) was reacted at 20°C for 1 hour under nitrogen bubbling. The reaction mixture was filtered, and the resin was recovered by washing it with DMF (150 mL x 5). As a result, Compound 3 (417 mg, crude, quantitatively adsorbed onto resin), a yellow solid compound, was obtained.

[0308] (Step 3) DCC (1.05 g, 5.09 mmol, 1.03 mL) and 2,3,4,5,6-pentafluorophenol (930 mg, 5.05 mmol) were added to a solution of (2S)-3-tertbutoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoic acid (1.3 g, 3.39 mmol) in DMF (15 mL). The mixture was stirred at 20°C for 1 hour. The reaction mixture was used as is without further processing in subsequent steps. As a result, the compound (2,3,4,5,6-pentafluorophenyl) (2S)-3-tertbutoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.86 g, crude, in the reaction mixture) was used in the next step as a white liquid. DIPEA (672.88 mg, 5.21 mmol, 906.84 μL) and (2,3,4,5,6-pentafluorophenyl) (2S)-3-tertbutoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.86 g, 3.38 mmol, in the reaction mixture) were added to a solution of Compound 3 (417 mg, 2.60 mmol, quantitatively adsorbed onto resin) in DMF (150 mL). The mixture was reacted for 12 hours at 20°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was recovered by washing it with DMF (150 mL x 5). As a result, Compound 4 (1.37 g, crude, quantitatively adsorbed onto resin), a yellow solid compound, was obtained. LCMS: RT = 0.635 min, m / z 527.5 [M+H] + .

[0309] (Step 4) A mixture of Compound 4 (1.37 g, 2.61 mmol, quantitatively adsorbed onto resin) in piperidine / DMF (150 mL, 5% in DMF) was reacted at 20°C for 1 hour under nitrogen bubbling. The reaction mixture was filtered, and the resin was recovered by washing it with DMF (150 mL x 5). As a result, Compound 5 (790 mg, crude, quantitatively adsorbed onto resin), a yellow solid compound, was obtained.

[0310] (Step 5) DCC (1.04 g, 5.03 mmol, 1.02 mL) and 2,3,4,5,6-pentafluorophenol (933.26 mg, 5.07 mmol) were added to a solution of (4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyl-oxazolidine-4-carboxylic acid (1.43 g, 3.37 mmol) in DMF (15 mL). The mixture was stirred at 20°C for 1 hour. LCMS analysis revealed approximately 94% of the target compound, (2,3,4,5,6-pentafluorophenyl) (4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyl-oxazolidine-4-carboxylate (1.99 g, crude, white liquid), and it was used in the next step. DIPEA (673.11 mg, 5.21 mmol, 907.15 μL) and (2,3,4,5,6-pentafluorophenyl) (4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyl-oxazolidine-4-carboxylate (1.99 g, 3.37 mmol, crude) were added to a solution of Compound 5 (790 mg, 2.60 mmol, quantitatively adsorbed onto resin) in DMF (150 mL). The mixture was reacted for 2 hours at 20°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was recovered by washing it with DMF (150 mL x 5). As a result, Compound 6 (1.86 g, crude, quantitatively adsorbed onto resin), a compound in the form of a yellow solid, was obtained. LCMS: RT = 0.622 min, m / z 711.6 [M+H] + .

[0311] (Step 6) A mixture of Compound 6 (1.85 g, 2.61 mmol, quantitatively adsorbed onto resin) in piperidine / DMF (150 mL, 5% in DMF) was reacted at 20°C under nitrogen bubbling for 1 hour. The reaction mixture was filtered, and the resin was recovered by washing it with DMF (150 mL x 5). As a result, Compound 7 (1.27 g, crude, quantitatively adsorbed onto resin), a yellow solid compound, was obtained. LCMS: RT = 0.489 min, m / z 489.4 [M+H] + .

[0312] (Step 7) DCC (1.05 g, 5.09 mmol, 1.03 mL) and 2,3,4,5,6-pentafluorophenol (930 mg, 5.05 mmol) were added to a solution of (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoic acid (1.3 g, 3.39 mmol) in DMF (15 mL). The mixture was stirred at 20°C for 1 hour. LCMS analysis revealed that the target compound, (2,3,4,5,6-pentafluorophenyl) (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.86 g, crude, white liquid), was detected at approximately 96% and was used in the next step without further purification. DIPEA (671.91 mg, 5.20 mmol, 905.53 μL) and (2,3,4,5,6-pentafluorophenyl) (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.86 g, 3.38 mmol, crude in the reaction mixture) were added to a mixture of Compound 7 (1.27 g, 2.60 mmol, quantitatively adsorbed onto resin) in a DMF (150 mL) solution. The mixture was reacted for 2 hours at 20°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was recovered by washing it with DMF (150 mL x 5). As a result, Compound 8 (2.22 g, crude, quantitatively adsorbed onto resin), a yellow solid compound, was obtained. LCMS: RT = 0.647 min, m / z 854.7 [M+H] + .

[0313] (Step 8) A mixture of Compound 8 (2.22 g, 2.60 mmol, quantitatively adsorbed onto resin) in piperidine / DMF (150 mL, 5% in DMF) was reacted at 20°C under nitrogen bubbling for 1 hour. The reaction mixture was filtered, and the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, Compound 9 (1.64 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.491 min, m / z 632.6 [M+H] + .

[0314] (Step 9) DCC (1.05 g, 5.09 mmol, 1.03 mL) and 2,3,4,5,6-pentafluorophenol (930 mg, 5.05 mmol) were added to a mixture of (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoic acid (1.3 g, 3.39 mmol) in DMF (15 mL). The mixture was stirred at 20°C for 1 hour. LCMS results showed that the target compound, (2,3,4,5,6-pentafluorophenyl) (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.86 g, crude, white liquid), was detected at approximately 95% and was used in the next step without further purification. DIPEA (672.08 mg, 5.20 mmol, 905.77 μL) and (2,3,4,5,6-pentafluorophenyl) (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.86 g, 3.38 mmol, crude in the reaction mixture) were added to a solution of Compound 9 (1.64 g, 2.60 mmol, quantitatively adsorbed onto resin) in DMF (150 mL). The mixture was reacted for 12 hours at 20°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, Compound 10 (2.59 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.673 min, m / z 997.9 [M+H] + .

[0315] (Step 10) A mixture of Compound 10 (2.59 g, 2.60 mmol, quantitatively adsorbed onto resin) in piperidine / DMF (150 mL, 5% in DMF) was reacted at 20°C under nitrogen bubbling for 1 hour. The reaction mixture was filtered, and the resin was washed with DMF (150 mL x 5) to obtain the resin. Compound 11 (2.01 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.484 min, m / z 775.8 [M+H] + .

[0316] (Step 11) DCC (1.04 g, 5.03 mmol, 1.02 mL) and 2,3,4,5,6-pentafluorophenol (933.26 mg, 5.07 mmol) were added to a solution of (4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyl-oxazolidine-4-carboxylic acid (1.43 g, 3.37 mmol) in DMF (20 mL). The mixture was stirred at 20°C for 1 hour. LCMS results showed that approximately 95% of the target compound, (2,3,4,5,6-pentafluorophenyl) (4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyl-oxazolidine-4-carboxylate (1.99 g, crude, white liquid, crude in reaction mixture), was detected and used in the next step without special purification. DIPEA (671.32 mg, 5.19 mmol, 904.74 μL) and (2,3,4,5,6-pentafluorophenyl) (4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyl-oxazolidine-4-carboxylate (1.99 g, 3.37 mmol, crude in the reaction mixture) were added to a solution of Compound 11 (2.01 g, 2.60 mmol, quantitatively adsorbed onto resin) in DMF (150 mL). The mixture was reacted for 2 hours at 20°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, Compound 12 (3.07 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.659 min, m / z 1182.1 [M+H] + .

[0317] (Step 12) A mixture of Compound 12 (3.07 g, 2.60 mmol, quantitatively adsorbed onto resin) in piperidine / DMF (150 mL, 5% in DMF) was reacted at 20°C under nitrogen bubbling for 1 hour. The reaction mixture was filtered, and the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, Compound 13 (2.49 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.488 min, m / z 959.9 [M+H] + .

[0318] (Step 13) DCC (1.05 g, 5.09 mmol, 1.03 mL) and 2,3,4,5,6-pentafluorophenol (930.00 mg, 5.05 mmol) were added to a solution of (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.3 g, 3.39 mmol) in DMF (15 mL). The mixture was stirred at 20°C for 1 hour. Approximately 95% of the target compound, (2,3,4,5,6-pentafluorophenyl) (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.86 g, crude, white liquid, crude in the reaction mixture), was detected by LCMS and was used in the next step without special purification. DIPEA (671.76 mg, 5.20 mmol, 905.33 μL) and (2,3,4,5,6-pentafluorophenyl) (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.86 g, 3.38 mmol, crude in the reaction mixture) were added to a solution of Compound 13 (2.49 g, 2.60 mmol, quantitatively adsorbed onto resin) in DMF (150 mL). The mixture was reacted for 12 hours at 20°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, Compound 14 (3.44 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.682 min, m / z 1325.2 [M+H] + .

[0319] (Step 14) A mixture of Compound 14 (3.44 g, 2.60 mmol, quantitatively adsorbed onto resin) in piperidine / DMF (150 mL, 5% in DMF) was reacted at 20°C under nitrogen bubbling for 1 hour. The reaction mixture was filtered, and the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, Compound 15 (2.86 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.565 min, m / z 1103.0 [M+H] + .

[0320] (Step 15) DCC (1.05 g, 5.09 mmol, 1.03 mL) and 2,3,4,5,6-pentafluorophenol (930.00 mg, 5.05 mmol) were added to a solution of (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.3 g, 3.39 mmol) in DMF (15 mL). The mixture was stirred at 20°C for 1 hour. LCMS results showed that approximately 89% of the target compound, (2,3,4,5,6-pentafluorophenyl) (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.86 g, crude, white liquid), was detected and used in the next step without special purification. DIPEA (671.26 mg, 5.19 mmol, 904.67 μL) and (2,3,4,5,6-pentafluorophenyl) (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.86 g, 3.38 mmol, crude in the reaction mixture) were added to a solution of Compound 15 (2.86 g, 2.60 mmol, quantitatively adsorbed onto resin) in DMF (150 mL). The mixture was reacted for 12 hours at 20°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, Compound 16 (3.81 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.695 min, m / z 1469.0 [M+H] + .

[0321] (Step 16) A mixture of Compound 16 (3.81 g, 2.60 mmol, quantitatively adsorbed onto resin) in piperidine / DMF (150 mL, 5% in DMF) was reacted at 20°C under nitrogen bubbling for 1 hour. The reaction mixture was filtered, and the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, Compound 17 (3.23 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.573 min, m / z 1245.9 [M+H] + .

[0322] (Step 17) DCC (1.04 g, 5.03 mmol, 1.02 mL) and 2,3,4,5,6-pentafluorophenol (933.26 mg, 5.07 mmol) were added to a solution of (4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyl-oxazolidine-4-carboxylic acid (1.43 g, 3.37 mmol) in DMF (15 mL). The mixture was stirred at 20°C for 1 hour. LCMS analysis revealed approximately 90% of the target compound, (2,3,4,5,6-pentafluorophenyl) (4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyl-oxazolidine-4-carboxylate (1.99 g, crude, white liquid), and it was used in the next step without special purification. DIPEA (670.34 mg, 5.19 mmol, 903.42 μL) and (2,3,4,5,6-pentafluorophenyl) (4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyloxazolidine-4-carboxylate (1.99 g, 3.37 mmol, crude in the reaction mixture) were added to a solution of Compound 17 (3.23 g, 2.59 mmol, quantitatively adsorbed onto resin) in DMF (150 mL). The mixture was reacted for 12 hours at 20°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, Compound 18 (4.28 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.682 min, m / z 1653.4 [M+H] + .

[0323] (Step 18) A mixture of Compound 18 (4.28 g, 2.59 mmol, quantitatively adsorbed onto resin) in piperidine / DMF (150 mL, 5% in DMF) was reacted at 20°C under nitrogen bubbling for 1 hour. The reaction mixture was filtered, and the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, Compound 19 (3.70 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.573 min, m / z 1430.3 [M+H] + .

[0324] (Step 19) DCC (1.04 g, 5.06 mmol, 1.02 mL) and 2,3,4,5,6-pentafluorophenol (931.29 mg, 5.06 mmol) were added to a solution of (4S)-5-benzyloxy-4-(9H-fluorene-9-ylmethoxycarbonylamino)-5-oxo-pentanoic acid (1.55 g, 3.37 mmol) in DMF (20 mL). The mixture was stirred at 20°C for 1 hour. LCMS analysis revealed approximately 78% of the target compound, O1-benzyl O5-(2,3,4,5,6-pentafluorophenyl) (2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)pentanedioate (2.11 g, crude, white liquid, reaction mixture), and it was used in the next step without special purification. DIPEA (669.42 mg, 5.18 mmol, 902.19 μL) and O1-benzyl O5-(2,3,4,5,6-pentafluorophenyl)(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)pentanedioate (2.11 g, 3.37 mmol, reaction mixture) were added to a solution of Compound 19 (3.7 g, 2.59 mmol, quantitatively adsorbed onto resin) in DMF (150 mL). The mixture was reacted for 12 hours at 20°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, Compound 20 (4.84 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.683 min, m / z 1872.3 [M+H] + .

[0325] (Step 20) A mixture of Compound 20 (2 g, 1.07 mmol) in TFA / DCM (200 mL) was reacted at 20°C under nitrogen bubbling for 0.5 hours. The reaction mixture was filtered, and the resin was washed with DCM (100 mL x 3). The filtrate was concentrated to obtain Compound 21 (2 g, crude), which was used in the next step without further purification. LCMS: RT = 0.696 min, m / z 1872.7 [M+H] + .

[0326] (Step 21) AcOH (288.84 mg, 4.81 mmol, 275.35 μL) and 2-tert-butyl-1,3-diisopropyl-isourea (3.85 g, 19.24 mmol) were added to a solution of compound 21 (1.8 g, 961.97 μmol) in THF (20 mL). The mixture was stirred at 20°C for 12 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was purified by prep-HPLC (column: Waters xbridge 150*25mm*10um; mobile phase: [water (ammonia hydroxide v / v)-ACN]; gradient: 80%-100% B over 10 min) to obtain Compound 22 (180 mg, 87.79 μmol, yield 9.13%, purity 94%) as a yellow oil. LCMS: RT = 0.747 min, m / z 1928.7 [M+H] + .

[0327] (Step 22) Compound 22 (130 mg, 67.45 μmol) was added to a Pd / C (13 mg, 10% purity) solution in MeOH (2 mL) under a nitrogen atmosphere. The suspension was degassed under vacuum and hydrogenated several times, then stirred at 25°C for 12 hours under a hydrogen (50 psi) atmosphere. The crude product was filtered through a Celite pad, and the cake was washed with MeOH (10 mL). The filtrate was concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Phenomenex luna C18 150*25 mm*10 µm; mobile phase: [water (FA)-ACN]; gradient: 59%-89% B over 10 min) to obtain Compound 23 (27 mg, 14.70 μmol, yield 21.79%) as a white solid. LCMS: RT = 0.701 min, m / z 1837.2 [M+H] + .

[0328]

[0329] K-(GGSG)1 synthesis

[0330] K-(GGSG)1, such as compound 9, was synthesized according to the reaction scheme below.

[0331]

[0332]

[0333]

[0334]

[0335] (Step 1) DIPEA (11.13 g, 86.12 mmol, 15 mL, 19.48 eq) and Compound 1 (2 g, 4.42 mmol, 1 eq) were added to a mixture of CTC-RESIN (23 g, 22.10 mmol, 5 eq) in DCM (200 mL). The mixture was reacted for 0.5 hours at 25°C under nitrogen bubbling. The reaction was then terminated with MeOH (3.54 g, 110.50 mmol, 4.47 mL, 25 eq), and the mixture was reacted for an additional 1 hour at 25°C under nitrogen bubbling. The mixture was filtered, and the resin was washed with DMF (200 mL x 3) to obtain the resin. As a result, Compound 2 (2.00 g, crude, quantitatively adsorbed onto the resin) was obtained as a yellow solid. LCMS: RT = 0.601 min, m / z 451.4 [M+H] + .

[0336] (Step 2) A mixture of Compound 2 (2.00 g, 4.43 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (150 mL) was reacted at 25°C under nitrogen bubbling for 0.5 hours. The reaction mixture was filtered, and the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, Compound 3 (1.02 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid.

[0337] (Step 3) (2,3,4,5,6-pentafluorophenyl) 2-[[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]amino]acetate (3.46 g, 6.65 mmol, 1.49 eq) was added to the solution of Compound 3 (1.02 g, 4.45 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (200 mL). The mixture was reacted for 1 hour at 25°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain the resin. As a result, Compound 4 (2.52 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.532 min, m / z 567.4 [M+H] + .

[0338] (Step 4) A mixture of Compound 4 (2.52 g, 4.45 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (200 mL) was reacted at 25°C under nitrogen bubbling for 0.5 hours. The reaction mixture was filtered, and the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, Compound 5 (1.53 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid.

[0339] (Step 5) (2,3,4,5,6-pentafluorophenyl)(4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyl-oxazolidine-4-carboxylate (3.92 g, 6.64 mmol, 1.49 eq) was added to the solution of Compound 5 (1.53 g, 4.46 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (200 mL). The mixture was reacted for 0.5 hours at 25°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain the resin. As a result, Compound 6 (3.34 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.542 min, m / z 751.6 [M+H]+ .

[0340] (Step 6) A mixture of Compound 6 (3.34 g, 4.45 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (200 mL) was reacted at 25°C under nitrogen bubbling for 0.5 hours. The reaction mixture was filtered, and the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, Compound 7 (2.35 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.381 min, m / z 529.4 [M+H] + .

[0341] (Step 7) Boc2O (1.94 g, 8.91 mmol, 2.05 mL, 2 eq) and DIPEA (2.88 g, 22.27 mmol, 3.88 mL, 5 eq) were added to the solution of Compound 7 (2.35 g, 4.45 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (200 mL). The mixture was reacted for 12 hours at 25°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, Compound 8 (2.80 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.468 min, m / z 629.5 [M+H] + .

[0342] (Step 8) The mixture of Compound 8 (2.80 g, 4.46 mmol, 1 eq) in HFIP / DCM (30 mL, 25% in DCM) was reacted at 25°C under nitrogen bubbling for 0.5 hours. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified by prep-HPLC (FA condition: column: Phenomenex luna C18 150*40 mm*15 µm; mobile phase: [water(FA)-ACN]; gradient: 33%-63% B over 15 min) to obtain Compound 9 (1.28 g, 2.04 mmol, 45.64% yield) as a white solid. LCMS: RT = 0.464 min, m / z 629.5 [M+H] + .

[0343]

[0344] K-(GGSG)2 synthesis

[0345] Using compound 5 of the above K-(GGSG)1 synthesis reaction scheme as a starting material, the process from step 3 to step 6 was repeated once, and then the method from step 7 to step 8 was used to finally synthesize K-(GGSG)2 as follows.

[0346]

[0347]

[0348] K-(GGSG)3 synthesis

[0349] Using compound 5 of the above K-(GGSG)1 synthesis reaction scheme as a starting material, the process from step 3 to step 6 was repeated twice, and then the method from step 7 to step 8 was used to finally synthesize K-(GGSG)3 as follows.

[0350]

[0351]

[0352] K-(GGSS)3-acetyl synthesis

[0353] K-(GGSS)3-acetyl, such as Compound 13, was synthesized according to the reaction scheme below. Here, Compound 1 was synthesized in the same manner as Compound 5 was synthesized in the synthesis of K-(GGSG)1 above.

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361] (Step 1) (2,3,4,5,6-pentafluorophenyl) (4S)-3-[(2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoyl]-2,2-dimethyl-oxazolidine-4-carboxylate (1.05 g, 1.55 mmol, 1.42 eq) was added to a solution of Compound 1 (376 mg, 1.10 mmol, 1 eq; synthesized in the same manner as Compound 5 in the K-(GGSG)1 synthesis above, quantitatively adsorbed onto resin) in DMF (100 mL). The mixture was reacted for 12 hours at 20°C under nitrogen bubbling. After the reaction, the reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain the resin. As a result, Compound 2 (915 mg, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.569 min, m / z 837.4 [M+H] + .

[0362] (Step 2) A mixture of Compound 2 (915 mg, 1.09 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (100 mL, 5% in DMF) was reacted at 20°C under nitrogen bubbling for 1 hour. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain the resin. As a result, Compound 3 (671 mg, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.452 min, m / z 615.4 [M+H] + .

[0363] (Step 3) (2,3,4,5,6-pentafluorophenyl) 2-[[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]amino]acetate (881 mg, 1.69 mmol, 1.55 eq) was added to the solution of Compound 3 (671 mg, 1.09 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (100 mL). The mixture was reacted for 12 hours at 20°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain the resin. As a result, Compound 4 (1.04 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.553 min, m / z 951.6 [M+H] + .

[0364] (Step 4) The mixture of Compound 4 (1.04 g, 1.09 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (100 mL, 5% in DMF) was reacted at 20°C under nitrogen bubbling for 1 hour. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain the resin. As a result, Compound 5 (796 mg, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.421 min, m / z 729.4 [M+H] + .

[0365] (Step 5) (2,3,4,5,6-pentafluorophenyl) (4S)-3-[(2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoyl]-2,2-dimethyl-oxazolidine-4-carboxylate (1.06 g, 1.57 mmol, 1.43 eq) was added to the solution of Compound 5 (796 mg, 1.09 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (100 mL). The mixture was reacted for 12 hours at 20°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain the resin. As a result, Compound 6 (1.33 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.594 min, m / z 1221.8 [M+H] + .

[0366] (Step 6) A mixture of Compound 6 (1.33 g, 1.09 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (100 mL, 5% in DMF) was reacted at 20°C for 1 hour under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain the resin. As a result, Compound 7 (1.09 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid.

[0367] (Step 7) (2,3,4,5,6-pentafluorophenyl) 2-[[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]amino]acetate (881.00 mg, 1.69 mmol, 1.55 eq) was added to the solution of Compound 7 (1.09 g, 1.09 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (100 mL). The mixture was reacted for 4 hours at 20°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain the resin. As a result, Compound 8 (1.46 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.607 min, m / z 1336.1 [M+H] + .

[0368] (Step 8) A mixture of Compound 8 (1.46 g, 1.09 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (100 mL, 5% in DMF) was reacted at 20°C under nitrogen bubbling for 1 hour. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain the resin. As a result, Compound 9 (1.22 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.785 min, m / z 1113.7 [M+H] + .

[0369] (Step 9) (2,3,4,5,6-pentafluorophenyl) (4S)-3-[(2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoyl]-2,2-dimethyl-oxazolidine-4-carboxylate (1.06 g, 1.57 mmol, 1.43 eq) was added to the solution of Compound 9 (1.22 g, 1.10 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (100 mL). The mixture was reacted for 12 hours at 20°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain the resin. As a result, Compound 10 (1.76 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.615 min, m / z 1605.5 [M+H] + .

[0370] (Step 10) A mixture of Compound 10 (1.76 g, 1.10 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (100 mL, 5% in DMF) was reacted at 20°C under nitrogen bubbling for 1 hour. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain the resin. As a result, Compound 11 (1.52 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.500 min, m / z 1383.6 [M+H] + .

[0371] (Step 11) DIPEA (890.40 mg, 6.89 mmol, 1.2 mL, 6.27 eq) and (2,5-dioxopyrrolidine-1-yl)acetate (350 mg, 2.23 mmol, 2.03 eq) were added to a solution of Compound 11 (1.52 g, 1.10 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (100 mL). The mixture was reacted for 12 hours at 20°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain the resin. As a result, Compound 12 (1.57 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.535 min, m / z 1425.5 [M+H] + .

[0372] (Step 12) A mixture of Compound 12 (1.57 g, 1.10 mmol, 1 eq) in HFIP (100 mL) was reacted at 10°C under nitrogen bubbling for 1 hour. The resin was washed with DCM (50 mL x 3) to obtain the resin, and the reaction mixture was concentrated by removing the solvent under reduced pressure. The residue was purified by prep-HPLC (FA condition: Instrument: ACSWH-GX-AF; Column: Phenomenex luna C18 150*25mm*10um; Mobile phase: water (FA)-ACN; Gradient: B 34% - 64% in 10 min linearly; Flow rate: 25 mL / min; Column temperature: RT; Wavelength: 220nm / 254nm) to obtain Compound 13 (840 mg, 589.24 μmol, yield 53.47%) as a white solid. LCMS: RT = 0.567 min, m / z 1425.9 [M+H] + .

[0373]

[0374] K-(GSSS)3-acetyl synthesis

[0375] K-(GSSS)3-acetyl, such as Compound 23, was synthesized according to the reaction scheme below. Here, Compound 1 was synthesized in the same manner as Compound 3 was synthesized in the synthesis of K-(GGSG)1 above.

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388] (Step 1) DCC (888 mg, 4.30 mmol, 870.59 μL) and 2,3,4,5,6-pentafluorophenol (792 mg, 4.30 mmol) were added to a solution of 2-(9H-fluorene-9-ylmethoxycarbonylamino)acetic acid (853 mg, 2.87 mmol, synthesized in the same manner as Compound 3 in the K-(GGSG)1 synthesis above) in DMF (20 mL). The mixture was stirred at 20°C for 1 hour. LCMS results showed that the target compound, (2,3,4,5,6-pentafluorophenyl) 2-(9H-fluorene-9-ylmethoxycarbonylamino)acetate (1.33 g, crude, white liquid, crude state in the reaction mixture), was detected at approximately 81% and was used in the next step without special purification. (2,3,4,5,6-pentafluorophenyl) 2-(9H-fluorene-9-ylmethoxycarbonylamino)acetate (1.33 g, 2.87 mmol, crude state in the reaction mixture) and DIPEA (570.52 mg, 4.41 mmol, 768.90 μL) were added to a solution of Compound 1 (506 mg, 2.21 mmol, quantitatively adsorbed onto resin) in DMF (100 mL) and DCM (30 mL). The mixture was reacted for 12 hours under nitrogen bubbling at 20°C. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 10) to obtain the resin. As a result, Compound 2 (1.12 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.547 min, m / z 510.4 [M+H] + .

[0389] (Step 2) A mixture of Compound 2 (1.12 g, 2.20 mmol, quantitatively adsorbed onto resin) in piperidine / DMF (100 mL, 5% in DMF) was reacted at 20°C for 1 hour under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 10) to obtain the resin. As a result, Compound 3 (630 mg, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid.

[0390] (Step 3) DCC (888 mg, 4.30 mmol, 870.59 μL) and 2,3,4,5,6-pentafluorophenol (792 mg, 4.30 mmol) were added to a solution of (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.1 g, 2.87 mmol) in DMF (20 mL). The mixture was stirred at 20°C for 1 hour. Approximately 63% of the target compound, (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.58 g, crude, white liquid, crude in the reaction mixture), was detected by LCMS and was used in the next step without special purification. (2,3,4,5,6-pentafluorophenyl)(2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.58 g, 2.88 mmol, crude in the reaction mixture) and DIPEA (568.78 mg, 4.40 mmol, 766.54 μL) were added to a solution of Compound 3 (630 mg, 2.20 mmol, quantitatively adsorbed onto resin) in DMF (100 mL). The mixture was reacted for 2 hours at 20°C under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 10) to obtain the resin. As a result, Compound 4 (1.43 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.585 min, m / z 653.6 [M+H] + .

[0391] (Step 4) A mixture of Compound 4 (1.43 g, 2.19 mmol, quantitatively adsorbed onto resin) in piperidine / DMF (100 mL, containing 5% DMF) was reacted at 20°C under nitrogen bubbling for 1 hour. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 10) to obtain the resin. As a result, Compound 5 (942 mg, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.428 min, m / z 431.3 [M+H] + .

[0392] (Step 5) DCC (888.00 mg, 4.30 mmol, 870.59 μL) and 2,3,4,5,6-pentafluorophenol (792.00 mg, 4.30 mmol) were added to a solution of (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.1 g, 2.87 mmol) in DMF (20 mL). The mixture was stirred at 20°C for 1 hour. The target compound, (2,3,4,5,6-pentafluorophenyl) (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.58 g, crude, crude in the reaction mixture), was detected at approximately 82% by LCMS and was used in the next step without special purification. (2,3,4,5,6-pentafluorophenyl)(2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.58 g, 2.88 mmol, crude in the reaction mixture) and DIPEA (566.94 mg, 4.39 mmol, 764.07 μL) were added to a solution of Compound 5 (942 mg, 2.19 mmol, quantitatively adsorbed onto the resin) in DMF (100 mL). The mixture was reacted at 20°C for 12 hours under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 10) to obtain the resin. As a result, Compound 6 (1.74 g, crude, quantitatively adsorbed onto the resin) was obtained as a yellow solid. LCMS: RT = 0.623 min, m / z 796.7 [M+H] + .

[0393] (Step 6) A mixture of Compound 6 (1.74 g, 2.19 mmol, quantitatively adsorbed onto the resin) in piperidine / DMF (100 mL, 5% in DMF) was reacted at 20°C for 1 hour under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 10) to obtain the resin. As a result, Compound 7 (1.25 g, crude, quantitatively adsorbed onto the resin) was obtained as a yellow solid. LCMS: RT = 0.496 min, m / z 574.5 [M+H] + .

[0394] (Step 7) DCC (876 mg, 4.25 mmol, 858.82 μL) and 2,3,4,5,6-pentafluorophenol (782 mg, 4.25 mmol) were added to a solution of (4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyl-oxazolidine-4-carboxylic acid (1.2 g, 2.83 mmol) in DMF (20 mL). The mixture was stirred at 20°C for 1 hour. In LCMS, the target compound (2,3,4,5,6-pentafluorophenyl) (4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyl-oxazolidine-4-carboxylate (1.67 g, crude, white liquid) was detected at approximately 74% and was used in the next step without special purification. (2,3,4,5,6-pentafluorophenyl)(4S)-3-[2-(9H-flluoren-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyl-oxazolidine-4-carboxylate (1.67 g, 2.83 mmol, crude) and DIPEA (564.20 mg, 4.37 mmol, 760.38 μL) were added to Compound 7 (1.25 g, 2.18 mmol, quantitatively adsorbed onto resin) in DMF (100 mL). The mixture was reacted at 20°C for 2 hours under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 10) to obtain the resin. As a result, Compound 8 (2.14 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.613 min, m / z 980.8 [M+H] + .

[0395] (Step 8) A mixture of Compound 8 (2.14 g, 2.19 mmol, quantitatively adsorbed onto the resin) in piperidine / DMF (100 mL, 5% in DMF) was reacted at 20°C for 1 hour under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 10) to obtain the resin. As a result, Compound 9 (1.65 g, crude, quantitatively adsorbed onto the resin) was obtained as a yellow solid. LCMS: RT = 0.493 min, m / z 758.7 [M+H] + .

[0396] (Step 9) DCC (888.00 mg, 4.30 mmol, 870.59 μL) and 2,3,4,5,6-pentafluorophenol (792.00 mg, 4.30 mmol) were added to a solution of (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoic acid (1.1 g, 2.87 mmol) in DMF (20 mL). The mixture was stirred at 20°C for 1 hour. LCMS analysis revealed that the target compound, (2,3,4,5,6-pentafluorophenyl) (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoic acid ester (1.58 g, crude, white liquid, reaction mixture), was detected at approximately 82% and was used in the next step without special purification. (2,3,4,5,6-pentafluorophenyl)(2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoic acid ester (1.58 g, 2.88 mmol, crude in the reaction mixture) and DIPEA (563.50 mg, 4.36 mmol, 759.43 μL) were added to a solution of Compound 9 (1.65 g, 2.18 mmol, quantitatively adsorbed onto resin) in DMF (100 mL). The mixture was reacted at 20°C for 12 hours under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 10). As a result, Compound 10 (2.45 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.639 min, m / z 1124.1 [M+H] + .

[0397] (Step 10) A mixture of Compound 10 (2.45 g, 2.18 mmol, quantitatively adsorbed onto the resin) in piperidine / DMF (100 mL, 5% in DMF) was reacted at 20°C for 1 hour under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 10) to obtain the resin. As a result, Compound 11 (1.96 g, crude, quantitatively adsorbed onto the resin) was obtained as a yellow solid. LCMS: RT = 0.506 min, m / z 901.8 [M+H] + .

[0398] (Step 11) DCC (888.00 mg, 4.30 mmol, 870.59 μL) and 2,3,4,5,6-pentafluorophenol (792.00 mg, 4.30 mmol) were added to a solution of (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.1 g, 2.87 mmol) in DMF (20 mL). The mixture was stirred at 20°C for 1 hour. LCMS analysis revealed that the target compound, (2,3,4,5,6-pentafluorophenyl) (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.58 g, crude, white liquid, crude in the reaction mixture), was detected at approximately 78% and was used in the next step without special purification. (2,3,4,5,6-pentafluorophenyl)(2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.58 g, 2.88 mmol, crude in the reaction mixture) and DIPEA (562.88 mg, 4.36 mmol, 758.60 μL) were added to a solution of Compound 11 (1.96 g, 2.18 mmol, quantitatively adsorbed onto resin) in DMF (100 mL). The mixture was reacted at 20°C for 2 hours under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 10) to obtain the resin. As a result, Compound 12 (2.76 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.662 min, m / z 1267.1 [M+H] + .

[0399] (Step 12) A mixture of Compound 12 (2.76 g, 2.18 mmol, quantitatively adsorbed onto resin) in piperidine / DMF (100 mL, 5% in DMF) was reacted at 20°C for 1 hour under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 10) to obtain the resin. As a result, Compound 13 (2.28 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.495 min, m / z 1045.0 [M+H] + .

[0400] (Step 13) DCC (883 mg, 4.28 mmol, 865.69 μL) and 2,3,4,5,6-pentafluorophenol (788 mg, 4.28 mmol) were added to a solution of (4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyl-oxazolidine-4-carboxylic acid (1.21 g, 2.85 mmol) in DMF (20 mL). The mixture was stirred at 20°C for 1 hour. LCMS analysis revealed that the target compound, (2,3,4,5,6-pentafluorophenyl) (4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyl-oxazolidine-4-carboxylate (1.68 g, crude, white liquid, crude in the reaction mixture), was detected at approximately 86% and was used in the next step without special purification. (2,3,4,5,6-pentafluorophenyl)(4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyl-oxazolidine-4-carboxylate (1.68 g, 2.85 mmol, crude in the reaction mixture) and DIPEA (564.91 mg, 4.37 mmol, 761.34 μL) were added to a solution of Compound 13 (2.28 g, 2.19 mmol, quantitatively adsorbed onto resin) in DMF (100 mL). The mixture was reacted at 20°C for 12 hours under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 10). As a result, Compound 14 (3.17 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.654 min, m / z 1451.3 [M+H] + .

[0401] (Step 14) The mixture of Compound 14 (3.17 g, 2.19 mmol, quantitatively adsorbed onto the resin) in piperidine / DMF (100 mL, 5% in DMF) was reacted at 20°C for 1 hour under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 5). As a result, Compound 15 (2.68 g, crude, quantitatively adsorbed onto the resin) was obtained as a yellow solid. LCMS: RT = 0.541 min, m / z 1229.1 [M+H] + .

[0402] (Step 15) DCC (888.00 mg, 4.30 mmol, 870.59 μL) and 2,3,4,5,6-pentafluorophenol (792.00 mg, 4.30 mmol) were added to a solution of (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.1 g, 2.87 mmol) in DMF (20 mL). The mixture was stirred at 20°C for 1 hour. The target compound, (2,3,4,5,6-pentafluorophenyl) (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.58 g, crude, white liquid), was detected at approximately 85% by LCMS and was used in the next step without special purification. (2,3,4,5,6-pentafluorophenyl)(2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.58 g, 2.88 mmol) and DIPEA (564.38 mg, 4.37 mmol, 760.61 μL) were added to a solution of Compound 15 (2.68 g, 2.18 mmol, quantitatively adsorbed onto resin) in DMF (100 mL). The mixture was reacted at 20°C for 2 hours under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 10). As a result, Compound 16 (3.48 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.673 min, m / z 1594.4 [M+H] + .

[0403] (Step 16) A mixture of Compound 16 (3.48 g, 2.18 mmol, quantitatively adsorbed onto resin) in piperidine / DMF (100 mL, 5% in DMF) was reacted at 20°C for 1 hour under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 10). As a result, Compound 17 (2.99 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.551 min, m / z 1372.2 [M+H] + .

[0404] (Step 17) DCC (888.00 mg, 4.30 mmol, 870.59 μL) and 2,3,4,5,6-pentafluorophenol (792.00 mg, 4.30 mmol) were added to a solution of (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.1 g, 2.87 mmol) in DMF (20 mL). The mixture was stirred at 20°C for 1 hour. The target compound, (2,3,4,5,6-pentafluorophenyl) (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.58 g, crude, white liquid), was detected at approximately 77% by LCMS and was used in the next step without special purification. (2,3,4,5,6-pentafluorophenyl)(2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.58 g, 2.88 mmol, in the reaction mixture) and DIPEA (563.88 mg, 4.36 mmol, 759.95 μL) were added to a solution of Compound 17 (2.99 g, 2.18 mmol, quantitatively adsorbed onto resin) in DMF (100 mL). The mixture was reacted at 20°C for 12 hours under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 10) to obtain the resin. As a result, Compound 18 (3.79 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.686 min, m / z 1738.5 [M+H] + .

[0405] (Step 18) A mixture of Compound 18 (3.79 g, 2.18 mmol, quantitatively adsorbed onto resin) in piperidine / DMF (100 mL, 5% in 5% DMF solution) was reacted at 20°C for 1 hour under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 5) to obtain the resin. As a result, Compound 19 (3.3 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.563 min, m / z 1515.5 [M+H] + .

[0406] (Step 19) DCC (888.00 mg, 4.30 mmol, 870.59 μL) and 2,3,4,5,6-pentafluorophenol (792.00 mg, 4.30 mmol) were added to a solution of (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.1 g, 2.87 mmol) in DMF (20 mL). The mixture was stirred at 20°C for 1 hour. LCMS analysis revealed that the target compound, (2,3,4,5,6-pentafluorophenyl) (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.58 g, crude, white liquid), was detected at approximately 86% and was used in the next step without special purification. (2,3,4,5,6-pentafluorophenyl)(2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoate (1.58 g, 2.88 mmol, in the reaction mixture) and DIPEA (563.48 mg, 4.36 mmol, 759.40 μL) were added to a solution of Compound 19 (3.3 g, 2.18 mmol, quantitatively adsorbed onto resin) in DMF (100 mL). The mixture was stirred at 20°C for 2 hours. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 10) to obtain the resin. As a result, Compound 20 (4.1 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.705 min, m / z 1881.8 [M+H] + .

[0407] (Step 20) A mixture of Compound 20 (4.1 g, 2.18 mmol, quantitatively adsorbed onto resin) in piperidine / DMF (100 mL, 5% in 5% DMF) was reacted under nitrogen bubbling at 20°C for 1 hour. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 5) to obtain the resin. As a result, Compound 21 (3.62 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.570 min, m / z 1658.2 [M+H] + .

[0408] (Step 21) DIPEA (1.69 g, 13.11 mmol, 2.28 mL) and (2,5-dioxopyrrolidine-1-yl)acetate (514.91 mg, 3.28 mmol) were added to a solution of Compound 21 (3.62 g, 2.18 mmol, quantitatively adsorbed onto resin) in DMF (100 mL). The mixture was reacted at 20°C for 48 hours under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 10) to obtain the resin. As a result, Compound 22 (3.71 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.638 min, m / z 1701.6 [M+H] + .

[0409] (Step 22) A mixture of Compound 22 (3.71 g, 2.18 mmol, quantitatively adsorbed onto resin) in TFA / DCM (100 mL, 1% in DCM) was reacted at 20°C for 0.2 hours under nitrogen bubbling. NaHCO3 solution was added to the reaction mixture to adjust the pH to 8, and freeze-dried to obtain a pink solid. The obtained solid was dissolved in DCM (200 mL) and MeOH (50 mL) and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Waters xbridge 150*25mm*10um; mobile phase: [water(NH4HCO3)-ACN]; gradient: 30%-60% B over 10 min) and prep-HPLC (column: Waters xbridge 150*25mm*10um; mobile phase: [water(NH4HCO3)-ACN]; gradient: 28%-58% B over 10 min) to obtain Compound 23 (1.48 g, 870.58 μmol, yield 39.87%, purity 100%) as a white solid. LCMS: RT = 0.627 min, m / z 1700.6 [M+H] + .

[0410]

[0411] K-(SSSS)3-acetyl synthesis

[0412] K-(SSSS)3-acetyl, such as Compound 15, was synthesized according to the reaction scheme below. Here, Compound 1 was synthesized in the same manner as Compound 3 was synthesized in the synthesis of K-(GGSG)1 above.

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424] (Step 1) (2,3,4,5,6-pentafluorophenyl)(4S)-3-[(2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoyl]-2,2-dimethyl-oxazolidine-4-carboxylate (3.45 g, 5.10 mmol) and DIPEA (888.24 mg, 6.87 mmol, 1.20 mL) were added to a mixture of Compound 1 (787.8 mg, 3.44 mmol, quantitatively adsorbed onto resin) in DMF (100 mL). The mixture was reacted at 25°C for 2 hours under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (80 mL x 5). As a result, Compound 2 (2.48 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.629 min, m / z 723.6 [M+H] + .

[0425] (Step 2) The mixture of Compound 2 (2.48 g, 3.44 mmol, quantitatively adsorbed onto resin) in piperidine (220 mL, 10% in DMF) was reacted at 25°C for 1 hour under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (80 mL x 5). As a result, Compound 3 (1.72 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.482 min, m / z 501.4 [M+H] + .

[0426] (Step 3) (2,3,4,5,6-pentafluorophenyl)(4S)-3-[(2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoyl]-2,2-dimethyl-oxazolidine-4-carboxylate (3.45 g, 5.10 mmol) and DIPEA (889.94 mg, 6.89 mmol, 1.20 mL) were added to a mixture of Compound 3 (1.72 g, 3.44 mmol, quantitatively adsorbed onto resin) in DMF (100 mL). The mixture was reacted at 25°C for 12 hours under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (80 mL x 5). As a result, Compound 4 (3.42 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.666 min, m / z 935.8 [M+H] + .

[0427] (Step 4) A mixture of Compound 4 (3.42 g, 3.45 mmol, quantitatively adsorbed onto resin) in piperidine / DMF (220 mL, 10% in DMF) was reacted at 25°C for 1.5 hours under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 5). As a result, Compound 5 (2.65 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.521 min, m / z 771.7 [M+H] + .

[0428] (Step 5) (2,3,4,5,6-pentafluorophenyl) (4S)-3-[ (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoyl]-2,2-dimethyl-oxazolidine-4-carboxylate (3.45 g, 5.10 mmol) and DIPEA (889.71 mg, 6.88 mmol, 1.20 mL) were added to a mixture of Compound 5 (2.65 g, 3.44 mmol, quantitatively adsorbed onto resin) in DMF (100 mL). The mixture was reacted at 25°C for 12 hours under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (80 mL x 5). As a result, Compound 6 (4.35 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.716 min, m / z 1263.8 [M+H] + .

[0429] (Step 6) A mixture of Compound 6 (4.35 g, 3.45 mmol, quantitatively adsorbed onto resin) in piperidine / DMF (220 mL) was reacted at 25°C for 1 hour under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (90 mL x 5). As a result, Compound 7 (3.58 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.562 min, m / z 1041.7 [M+H] + .

[0430] (Step 7) (2,3,4,5,6-pentafluorophenyl)(4S)-3-[(2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoyl]-2,2-dimethyl-oxazolidine-4-carboxylate (3.45 g, 5.10 mmol) and DIPEA (889.58 mg, 6.88 mmol, 1.20 mL) were added to a mixture of Compound 7 (3.58 g, 3.44 mmol, quantitatively adsorbed onto resin) in DMF (200 mL). The mixture was reacted at 25°C for 62 hours under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (80 mL x 5). As a result, Compound 8 (5.28 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.583 min, m / z 1312.2 [M-Fmoc+H] + .

[0431] (Step 8) The mixture of Compound 8 (5.28 g, 3.44 mmol) in piperidine / DMF (220 mL, 10% in DMF) was reacted at 25°C for 1 hour under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (90 mL x 5). As a result, Compound 9 (4.51 g, crude, quantitatively adsorbed onto the resin) was obtained as a yellow solid. LCMS: RT = 0.581 min, m / z 1312.2 [M+H] + .

[0432] (Step 9) (2,3,4,5,6-pentafluorophenyl) (4S)-3-[(2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoyl]-2,2-dimethyl-oxazolidine-4-carboxylate (3.45 g, 5.10 mmol) and DIPEA (929.56 mg, 7.19 mmol, 1.25 mL) were added to a mixture of Compound 9 (4.51 g, 3.44 mmol, quantitatively adsorbed onto resin) in DMF (220 mL). The mixture was reacted at 25°C for 12 hours under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (80 mL x 5). As a result, Compound 10 (6.21 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid and used in the next step without further purification. LCMS: RT = 0.783 min, m / z 1805.1 [M+H] + .

[0433] (Step 10) A mixture of Compound 10 (6.21 g, 3.44 mmol, quantitatively adsorbed onto resin) in piperidine / DMF (220 mL, 10% in DMF) was reacted at 25°C for 1 hour under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (80 mL x 5). As a result, Compound 11 (5.44 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid and used in the next step without further purification. LCMS: RT = 0.636 min, m / z 1582.0 [M+H] + .

[0434] (Step 11) (2,3,4,5,6-pentafluorophenyl)(4S)-3-[(2S)-3-tertbutoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoyl]-2,2-dimethyl-oxazolidine-4-carboxylate (3.45 g, 5.10 mmol) and DIPEA (889.47 mg, 6.88 mmol, 1.20 mL) were added to a mixture of Compound 11 (5.44 g, 3.44 mmol, quantitatively adsorbed onto resin) in DMF (200 mL). The mixture was reacted at 25°C for 12 hours under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (90 mL x 5). As a result, Compound 12 (7.13 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.820 min, m / z 1038.4 [M / 2+H] + .

[0435] (Step 12) A mixture of Compound 12 (7.13 g, 3.44 mmol, quantitatively adsorbed onto resin) in piperidine / DMF (220 mL, 10% in DMF) was reacted at 25°C for 1.5 hours under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (80 mL x 5). As a result, Compound 13 (6.37 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.654 min, m / z 1853.8 [M+H] + .

[0436] (Step 13) (2,5-dioxopyrrolidine-1-yl)acetate (1.62 g, 10.32 mmol) and DIPEA (928.77 mg, 7.19 mmol, 1.25 mL) were added to the mixture of Compound 13 (6.37 g, 3.44 mmol, quantitatively adsorbed onto resin) in DMF (200 mL). The mixture was reacted at 25°C for 12 hours under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DMF (90 mL x 5). As a result, Compound 14 (6.51 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.738 min, m / z 1895.9 [M+H] + .

[0437] (Step 14) The mixture of Compound 14 (6.51 g, 3.44 mmol) in piperidine / DMF (220 mL, 20% in DMF) was reacted at 25°C for 1.5 hours under nitrogen bubbling. The mixture was concentrated, and the residue was purified by Prep-HPLC (column: Phenomenex luna C18 150*25 mm*10 um; mobile phase: [water(FA)-ACN]; gradient: 66%-96% B over 10 min) to obtain Compound 15 (2.2 g, 1.16 mmol, yield 33.78%, purity 100%) as a white solid. LCMS: RT = 0.738 min, m / z 1895.8 [M+H] + .

[0438]

[0439] K-(GGSG)1-acetyl synthesis

[0440] K-(GGSG)1-acetyl, such as Compound 4, was synthesized according to the reaction scheme below. Here, Compound 1 was synthesized in the same manner as Compound 7 in the synthesis of K-(GGSG)1.

[0441]

[0442] (Step 1) Compound 2 (7.18 g, 45.72 mmol, 2 eq) and N,N-diisopropylethylamine (5.94 g, 45.93 mmol, 2 eq) were added to a mixture of Compound 1 (12.06 g, 22.86 mmol, 1 eq, quantitatively adsorbed onto resin) in dimethylformamide (150 mL) and reacted for 12 hours under nitrogen bubbling at 25°C. The reaction mixture was filtered, and the resin was washed with dimethylformamide (200 mL x 5) to obtain the resin. As a result, Compound 3 (13.2 g, crude) was obtained as a yellow solid as a crude product (quantitatively adsorbed onto resin) and used in the next step without further purification. LCMS: RT = 0.376 min, m / z 571.2 [M+H] + .

[0443] (Step 2) The mixture of Compound 3 (13.2 g, 23.17 mmol, 1 eq) in hexafluoroisopropanol (150 mL) was reacted at 25°C under nitrogen bubbling for 1.5 hours. The reaction mixture was filtered, and the filtrate was concentrated. The residue was purified by prep-HPLC (column: Phenomenex luna c18 250 mm × 100 mm × 10 µm; mobile phase: [water (0.225% formic acid)-acetonitrile]; gradient: 10%–30% B over 20.0 min) to obtain Compound 4 (7 g, 12.27 mmol, yield 52%) as a yellow solid. LCMS: RT = 0.391 min, m / z 571.2 [M+H] + .

[0444]

[0445] K-(GGSG)2-acetyl synthesis

[0446] K-(GGSG)2-acetyl, such as Compound 7, was synthesized according to the reaction scheme below. Here, Compound 1 was synthesized in the same manner as Compound 7 in the synthesis of K-(GGSG)1 above.

[0447]

[0448]

[0449]

[0450]

[0451] (Step 1) A mixture of Compound 1 (2.31 g, 4.38 mmol, 1 eq, synthesized in the same manner as Compound 7 in the synthesis of K-(GGSG)1, quantitatively adsorbed onto resin) in DMF (150 mL) was reacted at 20-25°C for 0.5 hours under nitrogen bubbling, and (2,3,4,5,6-pentafluorophenyl) 2-[[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]amino]acetate (3 g, 5.76 mmol, 1.32 eq) and DIPEA (1.22 g, 9.47 mmol, 1.65 mL, 2.16 eq) in DMF (20 mL) was added, and the mixture was reacted at 20-25°C for 11.5 hours under nitrogen bubbling. As a result, Compound 2 (3.78 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid and used in the next step without special purification. LCMS: RT = 0.542 min, m / z 865.6 [M+H] + .

[0452] (Step 2) A mixture of Compound 2 (3.78 g, 4.38 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (150 mL, 10% in DMF) was reacted at 20-25°C for 0.5 hours under nitrogen bubbling. As a result, Compound 3 (2.81 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid and used in the next step without special purification.

[0453] (Step 3) A mixture of compound 3 (2.81 g, 4.38 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (150 mL) was reacted at 20-25°C for 0.5 hours under nitrogen bubbling, (2,3,4,5,6-pentafluorophenyl) (4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyl-oxazolidine-4-carboxylate (3.3 g, 5.59 mmol, 1.28 eq) and DIPEA (1.22 g, 9.47 mmol, 1.65 mL, 2.16 eq) in DMF (20 mL) were added, and the mixture was reacted at 20-25°C for 11.5 hours under nitrogen bubbling. As a result, Compound 4 (4.59 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid and used in the next step without special purification. LCMS: RT = 0.555 min, m / z 1049.8 [M+H] + .

[0454] (Step 4) A mixture of Compound 4 (4.59 g, 4.38 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (150 mL, 10% in DMF) was reacted at 20-25°C for 0.5 hours under nitrogen bubbling. As a result, Compound 5 (3.62 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid and used in the next step without special purification.

[0455] (Step 5) DIPEA (2.97 g, 22.99 mmol, 4 mL, 5.24 eq) and (2,5-dioxopyrrolidine-1-yl)acetate (1.03 g, 6.58 mmol, 1.5 eq) were added to the mixture of Compound 5 (3.62 g, 4.38 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (150 mL). The mixture was reacted at 20–25°C for 2 hours under nitrogen bubbling. As a result, Compound 6 (3.8 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid and used in the next step without special purification. LCMS: RT = 0.454 min, m / z 869.6 [M+H] + .

[0456] (Step 6) A mixture of Compound 6 (3.8 g, 4.38 mmol, 1 eq, quantitatively adsorbed onto resin) in HFIP / DCM (150 mL) was reacted at 20-25°C for 1 hour under nitrogen bubbling. The resin was treated with (DCM:HFIP = 1:1) and concentrated under reduced pressure. The residue was purified by prep-HPLC (FA condition: Instrument: ACS-WH-GX-G; column: Phenomenex Luna C18 150*25mm*10um; mobile phase: water (FA)-ACN; B%: 5%-35%, 10 min; Flow rate: 25 mL / min; Column temperature: RT; Wavelength: 220 nm) to obtain Compound 7 (2.2 g, 2.43 mmol, yield 55.51%, purity 96%) as a white solid. LCMS: RT = 0.407 min, m / z 869.7 [M+H] + .

[0457]

[0458] K-(GGSG)3-acetyl synthesis

[0459] Using compound 5 of the above K-(GGSG)2-acetyl synthesis reaction scheme as a starting material, K-(GGSG)3-acetyl as follows was finally synthesized through steps 1 to 6 of the above K-(GGSG)2-acetyl synthesis reaction scheme.

[0460]

[0461]

[0462] (GGSG)1 synthesis

[0463] (GGSG)1, which is the same as compound 11, was synthesized according to the reaction scheme below as a branched portion that binds to glutamic acid.

[0464]

[0465]

[0466]

[0467]

[0468] (Step 1) A mixture of CTC-RESIN (35.4 g, 33.64 mmol, 2.5 eq) in DCM (200 mL) was swollen by bubbling nitrogen at 30°C for 30 minutes, then DIPEA (21.74 g, 168.18 mmol, 29.29 mL, 12.5 eq) and Compound 1 (4 g, 13.45 mmol, 1 eq) dissolved in DCM (20 mL) were added. The resulting mixture was bubbling nitrogen at 30°C for 1 hour. Subsequently, the reaction was terminated with MeOH (5.39 g, 168.18 mmol, 6.81 mL, 12.5 eq), and nitrogen was bubbling was performed for an additional 0.5 hours at 30°C. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 3) to obtain Compound 2 (3.99 g, crude, quantitatively adsorbed onto the resin) as a yellow solid. LCMS: RT = 0.522 min, m / z 296.3 [M+H] + .

[0469] (Step 2) The mixture of Compound 2 (3.99 g, 13.47 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (200 mL, 5% in DMF) was nitrogen-bubbled at 30°C for 1 hour. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain Compound 3 (997 mg, crude, quantitatively adsorbed onto resin) as a yellow solid. LCMS: RT = 0.660 min, m / z 74.0 [M+H] + .

[0470] (Step 3) Compound 4 (12.52 g, 21.20 mmol, 1.57 eq) was added to a solution of Compound 3 (997 mg, 13.46 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (200 mL), and nitrogen was bubbled at 20°C for 12 hours. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain Compound 5 (6.47 g, crude, quantitatively adsorbed onto resin) as a yellow solid. LCMS: RT = 0.554 min, m / z 480.4 [M+H] + .

[0471] (Step 4) A mixture of Compound 5 (6.47 g, 13.47 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (200 mL, 5% in DMF) was nitrogen-bubbled at 30°C for 1 hour. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain Compound 6 (3.48 g, crude, quantitatively adsorbed onto resin) as a yellow solid. LCMS: RT = 0.658 min, m / z 258.2 [M+H] + .

[0472] (Step 5) Compound 7 (4.68 g, 10.10 mmol, 1.51 eq) was added to a solution of Compound 6 (1.73 g, 6.70 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (100 mL), and nitrogen was bubbled at 20°C for 2 hours. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 5) to obtain Compound 8 (3.6 g, crude, quantitatively adsorbed onto resin) as a yellow solid. LCMS: RT = 0.506 min, m / z 539.3 [M+H] + .

[0473] (Step 6) A solution of Compound 8 (3.6 g, 6.70 mmol, 1 eq, quantitatively adsorbed onto resin) in HFIP / DCM (100 mL) was bubbled with nitrogen at 30°C for 1 hour. The solvent was removed by concentrating the reaction mixture under reduced pressure. As a result, Compound 9 (3 g, 5.57 mmol, yield 83.18%) was obtained as a white solid. The resulting crude product was used in the next step without further purification. LCMS: RT = 0.541 min, m / z 539.3 [M+H] + .

[0474] (Step 7) 2-tert-butyl-1,3-diisopropyl-isourea (11 g, 54.91 mmol, 9.86 eq) was added to a solution of Compound 9 (3 g, 5.57 mmol, 1 eq) in THF (50 mL) and stirred at 20 °C for 2 hours. The reaction mixture was filtered and concentrated under reduced pressure to remove the solvent. The residue was purified by flash silica gel column chromatography (ISCO®; 10 g SepaFlash® Silica Flash Column, Eluent of 50–70% Ethyl acetate / Petroleum ether gradient @ 80 mL / min) to obtain Compound 10 (3.3 g, crude) as a white solid. LCMS: RT = 0.852 min, m / z 595.2 [M+H] + .

[0475] (Step 8) A solution of Compound 10 (3.3 g, 5.55 mmol, 1 eq) in piperidine / DMF (10 mL) was stirred at 20°C for 1 hour. After filtering the reaction mixture, the residue was purified by prep-HPLC (FA condition: Instrument: ACSWH-GX-H; Column: Phenomenex luna C18 150*25mm*10um; Mobile phase: water(FA)-ACN; Gradient: B 10% - 40% in 15 min linearly; Flow rate: 60 mL / min; Column temperature: RT; Wavelength: 220 nm / 254 nm) to obtain Compound 11 (1.1 g, 2.95 mmol, yield 53.22%) as a white solid. LCMS: RT = 0.453 min, m / z 373.2 [M+H] + .

[0476]

[0477] (GGSG)3 synthesis

[0478] (GGSG)3, such as Compound 4, was synthesized according to the reaction scheme below with a branched portion that binds to glutamic acid. Here, Compound 1 was synthesized in the same manner as Compound 6 in the synthesis of E-(GGSG)3 above.

[0479]

[0480]

[0481] (Step 1) A mixture of Compound 1 (7.63 g, 6.73 mmol, quantitatively adsorbed onto resin) in HFIP / DCM (150 mL, 25% in DCM) was reacted at 25°C for 1 hour under nitrogen bubbling. The reaction mixture was filtered, and the resin was washed with DCM (100 mL x 3). The filtrate was concentrated under reduced pressure to obtain the residue. As a result, Compound 2 (5.7 g, 5.02 mmol, yield 74.64%, purity 98%) was obtained as a yellow solid. LCMS: RT = 0.527 min, m / z 1135.9 [M+H] + .

[0482] (Step 2) 2-tert-butyl-1,3-diisopropyl-isourea (3.95 g, 19.73 mmol) was added to a solution of Compound 2 (5.6 g, 4.93 mmol) in THF (100 mL). The mixture was stirred at 25°C for 1 hour. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate = 1 / 1 to Ethyl acetate / MeOH = 4 / 1). As a result, Compound 3 (3.7 g, 3.11 mmol, yield 62.96%, purity 99%) was obtained as a yellow solid. LCMS: RT = 0.816 min, m / z 1191.7 [M+H] + .

[0483] (Step 3) The mixture of Compound 3 (3.6 g, 3.02 mmol) in piperidine / DMF (36 mL, 5% in DMF) was stirred at 25°C for 1 hour. FA was added to the reaction mixture to adjust the pH to 6. The residue was purified by prep-HPLC (column: Phenomenex luna C18 150*25 mm*10 µm; mobile phase: [water (FA)-ACN]; gradient: 1%-28% B over 10 min) to obtain Compound 4 (1.42 g, 1.47 mmol, yield 48.49%, purity 100%) as a white solid. LCMS: RT = 0.472 min, m / z 969.7 [M+H] + .

[0484]

[0485] (GGSG)2 synthesis

[0486] (GGSG)2 was synthesized in the same manner as the synthesis of (GGSG)3 above, but using compound 6 from the E-(GGSG)2 synthesis instead of compound 1.

[0487]

[0488] (GGSG)1-acetyl synthesis

[0489] (GGSG)1-acetyl, such as compound 7, was synthesized according to the reaction scheme below with the branched portion that binds to Lysine.

[0490]

[0491]

[0492]

[0493] (Step 1) A mixture of CTC-RESIN (26 g, 28.22 mmol, 5 eq) in DCM (150 mL) was swollen by bubbling nitrogen at 20-25°C for 30 minutes, then Compound 1 (2 g, 5.64 mmol, 1 eq) and DIPEA (18.55 g, 143.53 mmol, 25 mL, 25.43 eq) were added and bubbling nitrogen at 20-25°C for 1 hour. Subsequently, MeOH (4.75 g, 148.27 mmol, 6 mL, 26.27 eq) was added to terminate the reaction, and bubbling nitrogen at 20-25°C for an additional 0.5 hours. As a result, Compound 2 (1.99 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. The crude product was used in the next step without further purification. LCMS: RT = 0.527 min, m / z 355.1 [M+H] + .

[0494] (Step 2) The mixture of Compound 2 (1.99 g, 5.63 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (150 mL, 5% in DMF) was bubbling nitrogen at 20-25°C for 0.5 hours. As a result, Compound 3 (738.39 mg, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. The crude product was used in the next step without further purification.

[0495] (Step 3) After bubbling nitrogen at 20–25°C for 0.5 hours for a mixture of Compound 3 (738.39 mg, 5.63 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (100 mL), Compound 3b (4.29 g, 7.27 mmol, 1.29 eq) dissolved in DMF (20 mL) and DIPEA (1.11 g, 8.61 mmol, 1.5 mL, 1.53 eq) were added. The resulting mixture was bubbling nitrogen at 20–25°C for 11.5 hours. As a result, Compound 4 (3 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. The crude product was used in the next step without further purification. LCMS: RT = 0.753 min, m / z 539.3 [M+H] + .

[0496] (Step 4) A mixture of Compound 4 (3 g, 5.58 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (150 mL, 10% in DMF) was nitrogen-bubbled at 20-25°C for 0.5 hours. As a result, Compound 5 (1.76 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. The crude product was used in the next step without further purification.

[0497] (Step 5) (2,5-dioxopyrrolidine-1-yl)acetate (1.33 g, 8.47 mmol, 1.5 eq) and DIPEA (3.71 g, 28.71 mmol, 5 mL, 5.08 eq) were added to a mixture of Compound 5 (1.78 g, 5.65 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (50 mL), and nitrogen was bubbled at 20-25°C for 2 hours. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 2) and DCM (50 mL x 3) to obtain the resin. As a result, Compound 6 (2.02 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid.

[0498] (Step 6) A mixture of Compound 6 (2.02 g, 5.65 mmol, 1 eq, quantitatively adsorbed onto resin) in HFIP / DCM (50 mL, HFIP / DCM = 1 / 2) was nitrogen-bubbled at 20–25°C for 0.5 hours. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified by prep-HPLC (FA condition; column: Phenomenex luna C18 150*25 mm*10 µm; mobile phase: [water(FA)-ACN]; gradient: 1%–20% B over 10 min) to obtain Compound 7 (750 mg, 2.09 mmol, yield 37.02%, purity 100%) as a white solid. LCMS: RT = 0.405 min, m / z 359.2 [M+H] + .

[0499]

[0500] (GGSG)2-acetyl synthesis

[0501] (GGSG)2-acetyl, such as Compound 7, was synthesized according to the reaction scheme below with a branched portion that binds to Lysine. Here, Compound 1 was synthesized in the same manner as Compound 5 was synthesized in the synthesis of (GGSG)1-acetyl above.

[0502]

[0503]

[0504]

[0505] (Step 1) A mixture of Compound 1 (1.76 g, 5.58 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (100 mL) was nitrogen-bubbled at 20–25°C for 0.5 hours, after which Compound 1a (3.8 g, 7.30 mmol, 1.31 eq) and DIPEA (1.08 g, 8.37 mmol, 1.46 mL, 1.5 eq) dissolved in DMF (20 mL) were added. The resulting mixture was nitrogen-bubbled at 20–25°C for 11.5 hours. As a result, Compound 2 (3.64 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. The crude product was used in the next step without further purification. LCMS: RT = 0.485 min, m / z 653.4 [M+H] + .

[0506] (Step 2) The mixture of Compound 2 (3.64 g, 5.59 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (150 mL, 10% in DMF) was bubbling nitrogen at 20-25°C for 0.5 hours. As a result, Compound 3 (2.4 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. The crude product was used in the next step without further purification.

[0507] (Step 3) After bubbling nitrogen at 20–25°C for 0.5 hours with a mixture of Compound 3 (2.4 g, 5.59 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (120 mL), Compound 3b (4 g, 6.77 mmol, 1.21 eq) dissolved in DMF (20 mL) and DIPEA (1.48 g, 11.48 mmol, 2 mL, 2.05 eq) were added. The resulting mixture was bubbling nitrogen at 20–25°C for 1.5 hours. As a result, Compound 4 (4.67 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. The crude product was used in the next step without further purification. LCMS: RT = 0.536 min, m / z 837.6 [M+H] + .

[0508] (Step 4) A mixture of Compound 4 (4.67 g, 5.59 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (150 mL, 5% in DMF) was bubbling nitrogen at 20-25°C for 0.5 hours. As a result, Compound 5 (3.43 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. The crude product was used in the next step without further purification.

[0509] (Step 5) (2,5-dioxopyrrolidine-1-yl)acetate (1.33 g, 8.46 mmol, 1.5 eq) and DIPEA (3.64 g, 28.19 mmol, 4.91 mL, 5 eq) were added to a mixture of Compound 5 (3.46 g, 5.64 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (50 mL), and nitrogen was bubbled at 20-25°C for 1 hour. The reaction mixture was filtered, and the resin was washed with DMF (50 mL x 2) and DCM (50 mL x 3) to obtain the resin. As a result, Compound 6 (3.70 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid.

[0510] (Step 6) The mixture of Compound 6 (3.70 g, 5.65 mmol, 1 eq, quantitatively adsorbed onto resin) in HFIP / DCM (50 mL, HFIP / DCM = 1 / 2) was nitrogen-bubbled at 20–25°C for 0.5 hours. The product was purified by prep-HPLC (FA condition; column: Phenomenex luna C18 (250*70 mm, 10 µm); mobile phase: [water(FA)-ACN]; gradient: 5%–35% B over 17 min) to obtain Compound 7 (1.7 g, 2.59 mmol, yield 45.86%, purity 100%) as a white solid. LCMS: RT = 0.297 min, m / z 657.3 [M+H] + .

[0511]

[0512] (GGSG)3-acetyl synthesis

[0513] Compound 5 of the above (GGSG)2-acetyl synthesis reaction scheme was used as a starting material as the branching portion that binds to Lysine, and (GGSG)3-acetyl, such as Compound 7, was finally synthesized through the process from Step 1 to Step 6.

[0514]

[0515]

[0516]

[0517] (Step 1) A mixture of Compound 1 (3.43 g, 5.59 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (120 mL) was reacted at 20-25°C for 0.5 hours under nitrogen bubbling. (2,3,4,5,6-Pentafluorophenyl) 2-[[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]amino]acetate (3.8 g, 7.30 mmol, 1.31 eq) and DIPEA (1.48 g, 11.48 mmol, 2 mL, 2.05 eq) in DMF (20 mL) were added, and the mixture was reacted at 20-25°C for 11.5 hours under nitrogen bubbling. As a result, Compound 2 (5.31 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid and used in the next step without special purification. LCMS: RT = 0.509 min, m / z 951.6 [M+H] + .

[0518] (Step 2) A mixture of Compound 2 (5.31 g, 5.59 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (150 mL, 10% in DMF) was reacted at 20-25°C for 0.5 hours under nitrogen bubbling. As a result, Compound 3 (4.07 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid and used in the next step without special purification.

[0519] (Step 3) A mixture of Compound 3 (4.07 g, 5.59 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (150 mL) was reacted at 20-25°C for 0.5 hours under nitrogen bubbling. (2,3,4,5,6-Pentafluorophenyl) (4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyl-oxazolidine-4-carboxylate (4.3 g, 7.28 mmol, 1.3 eq) and DIPEA (1.48 g, 11.48 mmol, 2 mL, 2.05 eq) in DMF (20 mL) were added, and the mixture was reacted at 20-25°C for 11.5 hours under nitrogen bubbling. As a result, Compound 4 (6.34 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid and used in the next step without special purification. LCMS: RT = 0.526 min, m / z 1135.9 [M+H] + .

[0520] (Step 4) The mixture of Compound 4 (6.34 g, 5.59 mmol, 1 eq, quantitatively adsorbed onto resin) in piperidine / DMF (100 mL, 10% in DMF) was reacted at 20–25°C for 0.5 hours under nitrogen bubbling. As a result, Compound 5 (5.1 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid and used in the next step without special purification. LCMS: m / z 913.7 [M+H] + .

[0521] (Step 5) (2,5-dioxopyrrolidine-1-yl)acetate (1.3 g, 8.27 mmol, 1.48 eq) and DIPEA (3.61 g, 27.96 mmol, 4.87 mL, 5 eq) were added to a mixture of compound 5 (5.1 g, 5.59 mmol, 1 eq, quantitatively adsorbed onto resin) in DMF (100 mL). The mixture was reacted at 20-25°C for 2 hours under nitrogen bubbling. As a result, compound 6 (5.34 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid and used in the next step without special purification.

[0522] (Step 6) A solution of Compound 6 (5.34 g, 5.60 mmol, 1 eq, quantitatively adsorbed onto resin) in HFIP / DCM (200 mL) was reacted at 20-25°C for 1 hour under nitrogen bubbling. The mixture was concentrated under reduced pressure, and the residue was purified by prep-HPLC (FA condition: Instrument: ACS-WH-GX-G; column: Phenomenex Luna C18 150*25mm*10um; mobile phase: water (FA)-ACN; B%: 1%-26%, 10 min; Flow rate: 25 mL / min; Column temperature: RT; Wavelength: 220 nm) to obtain Compound 7 (2 g, 2.07 mmol, yield 37.05%, purity 78.64%) as a white solid. LCMS: RT = 0.559 min, m / z 956.2 [M+H] + .

[0523]

[0524] (GSSS)3-acetyl synthesis

[0525] (GSSS)3-acetyl, such as compound 10, was synthesized according to the reaction scheme below with the branched portion that binds to Lysine.

[0526]

[0527]

[0528]

[0529]

[0530]

[0531] (Step 1) A mixture of CTC-RESIN (36 g, 33.64 mmol, 0.93 mmol / g) in DCM (150 mL) was swollen by bubbling nitrogen at 20°C for 0.5 hours. Subsequently, Compound 1 (1 g, 3.36 mmol) and DIPEA (21.74 g, 168.18 mmol, 29.29 mL) dissolved in DCM (50 mL) were added, and nitrogen was bubbling was performed at 20°C for 1 hour. The reaction was terminated by adding MeOH (5.39 g, 168.18 mmol, 6.81 mL), followed by bubbling nitrogen at 20°C for an additional 0.5 hours. The reaction mixture was filtered, and the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, Compound 1 (996 mg, crude, quantitatively adsorbed onto the resin) was obtained as a yellow solid. A mixture of Compound 1 (996 mg, 3.36 mmol, quantitatively adsorbed onto resin) in piperidine / DMF (150 mL, 5% in DMF) was nitrogen-bubbled at 20°C for 1 hour. The reaction mixture was filtered, and the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, Compound 2 (248 mg, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.545 min, m / z 320.1 [M+Na] + .

[0532]

[0533] (Step 2) DCC (1.21 g, 5.87 mmol, 1.19 mL) and 2,3,4,5,6-pentafluorophenol (1.08 g, 5.87 mmol) were added to a mixture of compound SS (2 g, 3.92 mmol) in DMF (20 mL), and stirred at 20°C for 1 hour. The resulting (2,3,4,5,6-pentafluorophenyl) ester (2.65 g, crude, in the reaction mixture) was used as a white liquid in the next step.

[0534] After adding the above 2,3,4,5,6-pentafluorophenyl ester (2.65 g, 3.92 mmol, crude) and DIPEA (865.59 mg, 6.70 mmol, 1.17 mL) to a mixture of Compound 2 (248 mg, 3.35 mmol, quantitatively adsorbed onto resin) in DMF (150 mL), nitrogen was bubbled at 20°C for 12 hours. The reaction mixture was filtered, and the resin was washed with DMF (100 mL x 5) to obtain the resin. As a result, 2-[[(4S)-3-[(2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoyl]-2,2-dimethyl-oxazolidine-4-carbonyl]amino]acetic acid (1.9 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. The above product (1.90 g, 3.35 mmol, quantitatively adsorbed onto resin) was suspended in piperidine / DMF (150 mL, 5% in DMF) and nitrogen was bubbled at 20°C for 1 hour. The reaction mixture was filtered, and the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, compound 3 (1.15 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid.

[0535] (Step 3) DCC (1.23 g, 5.98 mmol, 1.21 mL) and 2,3,4,5,6-pentafluorophenol (1.11 g, 6.03 mmol) were added to a solution of compound SG (1.7 g, 4.01 mmol) in DMF (20 mL), and the mixture was stirred at 20°C for 1 hour. The resulting (2,3,4,5,6-pentafluorophenyl) ester (2.37 g, crude) was used in the next step as a white liquid in the reaction mixture state.

[0536] To a solution of Compound 3 (1.15 g, 3.34 mmol, quantitatively adsorbed onto resin) in DMF (150 mL), the above (2,3,4,5,6-pentafluorophenyl) ester (2.37 g, 4.01 mmol, crude product in the reaction mixture) and DIPEA (863.16 mg, 6.68 mmol, 1.16 mL) were added, and the mixture was reacted for 12 hours at 20°C while bubbling nitrogen. After filtering the reaction mixture, the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, 2-[[(4S)-3-[(2S)-3-tert-butoxy-2-[[(4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyl-oxazolidin-4-carbonyl]amino]propanoyl]-2,2-dimethyl-oxazolidin-4-carbonyl]amino]acetic acid (2.51 g, crude), and 2-[[(4S)-3-[(2S)-2-[[(4S)-3-(2-aminoacetyl)-2,2-dimethyl-oxazolidin-4-carbonyl]amino]-3-tert-butoxypropanoyl]-2,2-dimethyl-oxazolidin-4-carbonyl]amino]acetic acid (quantitatively adsorbed onto resin) were obtained as a yellow solid. This was added to piperidine / DMF (150 mL, 5% in DMF) and reacted for 1 hour at 20°C under nitrogen bubbling. After filtering the reaction mixture, the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, Compound 4 (1.66 g, crude, quantitatively adsorbed onto the resin) was obtained as a yellow solid. LCMS: RT = 0.424 min, m / z 530.3 [M+H] + .

[0537] (Step 4) DCC (1.45 g, 7.05 mmol, 1.43 mL) and 2,3,4,5,6-pentafluorophenol (1.30 g, 7.04 mmol) were added to a solution of compound SS (2.4 g, 4.70 mmol) in DMF (25 mL) and stirred at 20°C for 1 hour. The resulting (2,3,4,5,6-pentafluorophenyl) ester (3.18 g, crude) was used in the next step as a white liquid in the reaction mixture state.

[0538] DIPEA (811.78 mg, 6.28 mmol, 1.09 mL) and the (2,3,4,5,6-pentafluorophenyl) ester (3.18 g, 4.70 mmol, crude product in the reaction mixture state) were added to a solution of compound 4 (1.66 g, 3.14 mmol, quantitatively adsorbed onto resin) in DMF (150 mL), and the mixture was reacted for 12 hours at 20°C while bubbling nitrogen. After filtering the reaction mixture, the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, 2-[[(4S)-3-[(2S)-3-tert-butoxy-2-[[(4S)-3-[2-[[(4S)-3-[(2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoyl]-2,2-dimethyl-oxazolidin-4-carbonyl]amino]acetyl]-2,2-dimethyl-oxazolidin-4-carbonyl]amino]propanoyl]-2,2-dimethyl-oxazolidin-4-carbonyl]amino]acetic acid (3.21 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. This was added to piperidine / DMF (150 mL, 5% in DMF) and reacted for 1 hour at 20°C while bubbling nitrogen. After filtering the reaction mixture, the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, Compound 5 (2.51 g, crude, quantitatively adsorbed onto the resin) was obtained as a yellow solid quantitatively adsorbed onto the resin. LCMS: RT = 0.514 min, m / z 800.7 [M+H] + .

[0539] (Step 5) DCC (1.23 g, 5.98 mmol, 1.21 mL) and 2,3,4,5,6-pentafluorophenol (1.11 g, 6.03 mmol) were added to a solution of compound SG (1.7 g, 4.01 mmol) in DMF (20 mL), and the mixture was stirred at 20°C for 1 hour. The resulting (2,3,4,5,6-pentafluorophenyl) ester (2.37 g, crude) was used in the next step as a white liquid in the reaction mixture state.

[0540] DIPEA (812.11 mg, 6.28 mmol, 1.09 mL) and the above (2,3,4,5,6-pentafluorophenyl) ester (2.37 g, 4.01 mmol, crude product in the reaction mixture state) were added to a solution of compound 5 (2.51 g, 3.14 mmol, quantitatively adsorbed onto resin) in DMF (150 mL), and the mixture was reacted for 12 hours at 20°C while bubbling nitrogen. After filtering the reaction mixture, the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, compound 2-[[(4S)-3-[(2S)-3-tert-butoxy-2-[[(4S)-3-[2-[[(4S)-3-[(2S)-3-tert-butoxy-2-[[(4S)-3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]-2,2-dimethyl-oxazolidin-4-carbonyl]amino]propanoyl]-2,2-dimethyl-oxazolidin-4-carbonyl]amino]acetyl]-2,2-dimethyl-oxazolidin-4-carbonyl]amino]propanoyl]-2,2-dimethyl-oxazolidin-4-carbonyl]amino]acetic acid (3.79 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. This was added to piperidine / DMF (150 mL, 5% in DMF) and reacted for 1 hour at 20°C under nitrogen bubbling. After filtering the reaction mixture, the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, Compound 6 (3.09 g, crude, quantitatively adsorbed onto the resin) was obtained as a yellow solid. LCMS: RT = 0.512 min, m / z 984.6 [M+H] + .

[0541] (Step 6) DCC (1.16 g, 5.64 mmol, 1.14 mL) and 2,3,4,5,6-pentafluorophenol (1.04 g, 5.63 mmol) were added to a solution of compound SS (1.92 g, 3.76 mmol) in DMF (20 mL), and the mixture was stirred at 20°C for 1 hour. The resulting (2,3,4,5,6-pentafluorophenyl) ester (2.54 g, crude) was used in the next step as a white liquid in the reaction mixture state.

[0542] DIPEA (812.46 mg, 6.29 mmol, 1.09 mL) and the above (2,3,4,5,6-pentafluorophenyl) ester (2.54 g, 3.75 mmol, crude product in the reaction mixture) were added to a solution of Compound 6 (3.09 g, 3.14 mmol, quantitatively adsorbed onto resin) in DMF (150 mL), and the mixture was reacted for 12 hours at 20°C under nitrogen bubbling. After filtering the reaction mixture, the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, the compound 2-[[(4S)-3-[(2S)-3-tert-butoxy-2-[[(4S)-3-[2-[[(4S)-3-[(2S)-3-tert-butoxy-2-[[(4S)-3-[2-[[(4S)-3-[(2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoyl]-2,2-dimethyl-oxazoli [din-4-carbonyl]amino]acetyl]-2,2-dimethyl-oxazolidin-4-carbonyl]amino]propanoyl]-2,2-dimethyl-oxazolidin-4-carbonyl]amino]acetyl]-2,2-dimethyl-oxazolidin-4-carbonyl]amino]propanoyl]-2,2-dimethyl-oxazolidin-4-carbonyl]amino]acetic acid (4.64 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. This was added to piperidine / DMF (150 mL, 5% in DMF) and reacted for 1 hour at 20°C while bubbling nitrogen. After filtering the reaction mixture, the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, compound 7 (3.94 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.550 min, m / z 1255.1 [M+H] + .

[0543] (Step 7) DCC (1.45 g, 7.05 mmol, 1.43 mL) and 2,3,4,5,6-pentafluorophenol (1.29 g, 7.00 mmol) were added to a solution of (2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoic acid (1.8 g, 4.69 mmol) in DMF (20 mL), and stirred at 20°C for 1 hour. The resulting (2,3,4,5,6-pentafluorophenyl) ester (2.58 g, crude) was used in the next step as a white liquid in the reaction mixture.

[0544] The above (2,3,4,5,6-pentafluorophenyl) ester (2.58 g, 4.70 mmol, crude product in the reaction mixture) and DIPEA (812.51 mg, 6.29 mmol, 1.10 mL) were added to a solution of Compound 7 (3.94 g, 3.14 mmol, quantitatively adsorbed onto resin) in DMF (150 mL), and the reaction was carried out for 12 hours at 20°C under nitrogen bubbling. After filtering the reaction mixture, the resin was washed with DMF (100 mL x 5) to obtain the resin. As a result, the compound 2-[[(4S)-3-[(2S)-3-tert-butoxy-2-[[(4S)-3-[2-[[(4S)-3-[(2S)-3-tert-butoxy-2-[[(4S)-3-[2-[[(4S)-3-[(2S)-3-tert-butoxy-2-[[(2S)-3-tert-butoxy-2-(9H-fluorene-9-ylmethoxycarbonylamino)propanoyl]amino]propanoyl ]-2,2-dimethyl-oxazolidin-4-carbonyl]amino]acetyl]-2,2-dimethyl-oxazolidin-4-carbonyl]amino]propanoyl]-2,2-dimethyl-oxazolidin-4-carbonyl]amino]acetyl]-2,2-dimethyl-oxazolidin-4-carbonyl]amino]propanoyl]-2,2-dimethyl-oxazolidin-4-carbonyl]amino]acetic acid (5.09 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. This was added to piperidine / DMF (150 mL, 5% in DMF) and reacted for 1 hour at 20°C while bubbling nitrogen. After filtering the reaction mixture, the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, Compound 8 (4.39 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.552 min, m / z 1397.9 [M+H] + .

[0545] (Step 8) (2,5-dioxopyrrolidine-1-yl) acetate (980 mg, 6.24 mmol) and DIPEA (812.51 mg, 6.29 mmol, 1.10 mL) were added to the solution of Compound 8 (4.39 g, 3.14 mmol, quantitatively adsorbed onto resin) in DMF (150 mL), and nitrogen was bubbled at 20°C for 12 hours. The reaction mixture was filtered, and the resin was washed with DMF (150 mL x 5) to obtain the resin. As a result, Compound 9 (4.52 g, crude, quantitatively adsorbed onto resin) was obtained as a yellow solid. LCMS: RT = 0.639 min, m / z 1439.9 [M+H] + .

[0546] (Step 9) A mixture of Compound 9 (4.52 g, 3.14 mmol, quantitatively adsorbed onto resin) in HFIP / DCM (300 mL, 50% in DCM, 100 mL in DCM) was nitrogen-bubbled at 20°C for 1 hour. The reaction mixture was filtered, and the resin was washed with DCM (100 mL x 3). The filtrate was concentrated to obtain the product, which was purified by prep-HPLC (column: Phenomenex luna C18 150*40 mm* 15 µm; mobile phase: [water (FA)-ACN]; gradient: 60%-90% B over 10 min) to obtain Compound 10 (2.8 g, 1.94 mmol, yield 61.90%, purity 100%) as a white solid. LCMS: RT = 0.613 min, m / z 1439.9 [M+H] + .

[0547]

[0548] The compounds used in the above synthesis process were synthesized according to the following reaction scheme.

[0549]

[0550]

[0551]

[0552]

[0553]

[0554] VC-Exatecan Synthesis

[0555] VC-Exatecan, such as compound 5, was synthesized according to the reaction scheme below.

[0556]

[0557]

[0558] (Step 1) 2,3,4,5,6-pentafluorophenol (3.20 g, 17.36 mmol, 1.3 eq) and EDCI (2.56 g, 13.35 mmol, 1 eq) were added to a solution of Compound 1 (5 g, 13.35 mmol, 1 eq) in DMF (40 mL). The mixture was stirred at 20–25°C for 2 hours. As a result, Compound 2 (7.22 g, crude) was obtained as a colorless liquid and used in the next step without further purification. LCMS: RT = 0.868 min, m / z 541.3 [M+H] + .

[0559] (Step 2) Compound 2 (7.22 g, 13.36 mmol, 1 eq) was dissolved in DMF (50 mL), and methanesulfonic acid; Compound 3 (6 g, 11.29 mmol, 0.845 eq) and DIPEA (6.91 g, 53.43 mmol, 9.31 mL, 4 eq) were added. The mixture was stirred at 20-25°C for 2 hours. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate = 2 / 1 to Ethyl acetate / MeOH = 5 / 1) to obtain Compound 4 (10.5 g, 12.35 mmol, yield 92.46%, purity 93.15%) as a brown solid. LCMS: RT = 0.854 min, m / z 792.3 [M+H] + .

[0560] (Step 3) TFA (53.15 g, 466.16 mmol, 34.63 mL, 41.02 eq) was added to a solution of Compound 4 (9 g, 11.37 mmol, 1 eq) in DCM (5 mL) and MeOH (5 mL). The mixture was stirred at 20–25°C for 12 hours. The reaction was terminated by adding an aqueous NaHCO3 solution (100 mL), after which the residue was obtained by filtration and vacuum concentration. The residue was purified by prep-HPLC (FA condition; column: Phenomenex luna C18 150*40 mm*15 µm; mobile phase: [water(FA)-ACN]; gradient: 25%–55% B over 15 min) to obtain Compound 5 (10 g, crude) as a brown solid. LCMS: RT = 0.723 min, m / z 692.3 [M+H] + .

[0561]

[0562] VA-Exatecan Synthesis

[0563] VA-Exatecan, such as Compound 5, was synthesized by using Fmoc-Valine-Alanine instead of Boc-Valine-Citrulline of Compound 1 in the above VC-Exatecan synthesis reaction scheme.

[0564]

[0565]

[0566] (Step 1) EDCI (400 mg, 2.09 mmol, 1.71 eq) was added to a mixture of Compound 1 (500 mg, 1.22 mmol, 1 eq) and 2,3,4,5,6-Pentafluorophenol (300 mg, 1.63 mmol, 1.34 eq) in DCM (20 mL) and DMF (5 mL), and stirred at 25°C for 0.5 hours. Compound 2 (702.2 mg, crude) was obtained as a yellow oil in solution and used in the next step without special purification. LCMS: RT = 0.704 min, m / z 577.3 [M+H] + .

[0567] (Step 2) A mixture of Compound 2 (702.2 mg, 1.22 mmol, 1.29 eq), Compound 3 (500 mg, 940.64 μmol, 1 eq), and DIPEA (742.00 mg, 5.74 mmol, 1 mL, 6.10 eq) in DMF (2 mL) was stirred at 25°C for 0.5 hours. The resulting residue was purified by column chromatography (SiO2, PE:ethyl acetate = 1:1 to ethyl acetate:MeCN = 3:1) to obtain Compound 4 (0.76 g, 875.76 μmol, yield 93.10%, purity 95.4%) as a yellow solid. LCMS: RT = 0.645 min, m / z 828.5 [M+H] + .

[0568] (Step 3) Piperidine (1.72 g, 20.25 mmol, 2 mL, 22.06 eq) was added to a mixture of Compound 4 (0.76 g, 917.99 μmol, 1 eq) in MeCN (5 mL) and MeOH (5 mL), and the mixture was stirred at 25°C for 1 hour. The reaction mixture was concentrated to obtain the residue. The residue was purified by column chromatography (SiO2, PE:ethyl acetate = 1:1 to 0:1 to ethyl acetate:MeOH = 5:1) to obtain Compound 5 (0.4 g, 598.36 μmol, yield 65.18%, purity 90.6%) as a yellow solid. LCMS: RT = 0.466 and 0.487 min, m / z 606.5 [M+H] + .

[0569]

[0570] FG-Exatecan synthesis

[0571] FG-Exatecan, such as compound 5, was synthesized according to the reaction scheme below.

[0572]

[0573]

[0574] (Step 1) EDCI (3 g, 15.65 mmol, 1.01 eq) and 2,3,4,5,6-pentafluorophenol (3.7 g, 20.10 mmol, 1.3 eq) were added to a solution of Compound 1 (5 g, 15.51 mmol, 1 eq) in DMF (50 mL). The mixture was stirred at 20–25°C for 1 hour. As a result, Compound 2 was obtained as a white solid and used in the next step without special purification. LCMS: RT = 0.647 min, m / z 511.3 [M+Na] + .

[0575] (Step 2) DIPEA (12.03 g, 93.12 mmol, 16.22 mL, 6 eq) and Compound 3 (6.0 g, 13.78 mmol, 0.888 eq) were added to a solution of Compound 2 (7.58 g, 15.52 mmol, 1 eq) in DMF (50 mL). The mixture was stirred at 20–25°C for 1 hour. The mixture was concentrated under vacuum, and the residue was purified by column chromatography (SiO2, ethyl acetate / MeOH = 1 / 0 to 1 / 1) to obtain Compound 4 (8.5 g, 11.00 mmol, yield 70.85%, purity 95.7%) as a brown solid. LCMS: RT = 0.607 min, m / z 740.5 [M+H] + .

[0576] (Step 3) TFA (23.03 g, 201.93 mmol, 15 mL, 19.92 eq) was added to a solution of Compound 4 (7.5 g, 10.14 mmol, 1 eq) in DCM (3.5 mL) and MeOH (3.5 mL). The mixture was stirred at 20-25°C for 2 hours. The mixture was poured into an aqueous NaHCO3 solution (500 mL), filtered, and the filtrate was concentrated. The residue was purified by prep-HPLC (FA condition: Instrument: ACS-WH-GX-G; column: Phenomenex Luna C18 150*25mm*10um; mobile phase: water (FA)-ACN; B%: 10%-30%, 10 min; Flow rate: 25 mL / min; Column temperature: RT; Wavelength: 220 nm) to obtain Compound 5 (5.8 g, 8.89 mmol, yield 87.65%, purity 98%) as a white solid. LCMS: RT = 0.511 min, m / z 640.5 [M+H] + .

[0577]

[0578] β-glucuronide-Exatecan synthesis

[0579] β-glucuronide-Exatecan, such as compound 6, was synthesized according to the reaction scheme below.

[0580]

[0581]

[0582]

[0583] (Step 1) 3-(tert-butoxycarbonylamino)propanoic acid (920 mg, 4.86 mmol) and EEDQ (1.63 g, 6.59 mmol) were added to a solution of Compound 1 (2 g, 4.39 mmol) in DCM (10 mL) and MeOH (5 mL). The mixture was stirred at 25°C for 1 hour. The reaction mixture was concentrated by removing the solvent under reduced pressure. The residue was purified by flash silica gel column chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0–69% Ethyl acetate / Petroleum ether gradient @ 40 mL / min) to obtain Compound 2 (2.6 g, 3.98 mmol, yield 90.70%, purity 96%) as a yellow oil. LCMS: RT = 0.548 min, m / z 627.4 [M+H] + .

[0584] (Step 2) DIPEA (2.00 g, 15.47 mmol, 2.70 mL) and bis(4-nitrophenyl)carbonate (1.82 g, 5.98 mmol) were added to a solution of Compound 2 (2.5 g, 3.99 mmol) in DCM (30 mL). The mixture was stirred at 25°C for 1 hour. The solvent was removed by concentrating the reaction mixture under reduced pressure. The residue was purified by flash silica gel column chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0–50% Ethyl acetate / Petroleum ether gradient @ 45 mL / min) to obtain Compound 3 (2.7 g, 3.17 mmol, yield 79.49%, purity 93%) as a yellow oil. LCMS: RT = 0.629 min, m / z 814.5 [M+Na] + .

[0585] (Step 3) DIPEA (1.32 g, 10.23 mmol, 1.78 mL) and Compound 4 (1.5 g, 2.82 mmol) were added to a solution of Compound 3 (2.7 g, 3.41 mmol) in DMF (30 mL). The mixture was stirred at 60 °C for 1 hour. The solvent was removed by concentrating the reaction mixture under reduced pressure. The residue was purified by flash silica gel column chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0–100% Ethyl acetate / Petroleum ether gradient @ 50 mL / min) to obtain Compound 5 (3.2 g, 2.71 mmol, yield 79.34%, purity 92%) as a yellow solid. LCMS: RT = 0.626 min, m / z 1088.7 [M+H] + .

[0586] (Step 4) Compound 5 (3 g, 2.76 mmol) was dissolved in a TFA / DCM solution (60 mL, TFA:DCM = 1:10) and stirred at 25°C for 1 hour. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by flash silica gel column chromatography (ISCO®; 60 g SepaFlash® Silica Flash Column, Eluent of 0–68% MeOH / Ethyl acetate gradient @ 50 mL / min) to obtain Compound 6 (2.2 g, 1.90 mmol, yield 68.79%, purity 95%, TFA form) as a yellow solid. LCMS: RT = 0.838 min, m / z 988.5 [M+H] + .

[0587]

[0588] FG-m-glucuronide-Exatecan synthesis

[0589] FG-m-glucuronide-Exatecan, such as compound 6, was synthesized according to the reaction scheme below.

[0590]

[0591]

[0592]

[0593] (Step 1) EEDQ (2.30 g, 9.31 mmol, 1.5 eq) was added to a solution of Compound 1 (2 g, 6.20 mmol, 1 eq) and methyl (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[2-amino-5-(hydroxymethyl)phenoxy]tetrahydropyran-2-carboxylate (2.83 g, 6.20 mmol, 1 eq) in DCM (20 mL) and MeOH (10 mL). The mixture was stirred at 25°C for 1 hour. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by flash silica gel column chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, eluent of 0–30% ethyl acetate / petroleum ether gradient @ 60 mL / min) to obtain Compound 2 (3.4 g, 4.16 mmol, yield 67.11%, purity 93.04%) as a white solid. LCMS: RT = 0.829 min, m / z 760.2 [M+H] + .

[0594] (Step 2) DIPEA (4.63 g, 35.80 mmol, 6.24 mL, 8 eq) was added to a solution of Compound 2 (3.40 g, 4.48 mmol, 1 eq) and Bis(4-Nitrophenyl)Carbonate (2.04 g, 6.71 mmol, 1.5 eq) in DMF (1 mL) and DCM (30 mL). The mixture was stirred at 25°C for 1 hour. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by flash silica gel column chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0–50% Ethyl acetate / Petroleum ether gradient @ 60 mL / min) to obtain Compound 3 (3.4 g, 3.68 mmol, yield 82.15%, purity 100%) as a white solid. LCMS: RT = 0.912 min, m / z 925.1 [M+H] + .

[0595] (Step 3) DIPEA (1.43 g, 11.03 mmol, 1.92 mL, 3 eq) was added to a solution of Compound 3 (3.4 g, 3.68 mmol, 1 eq) and Compound 4 in DMF (30 mL); methanesulfonic acid (1.9 g, 3.57 mmol, 0.972 eq). The mixture was stirred at 60°C for 0.5 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by flash silica gel column chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0–100% Ethyl acetate / Petroleum ether gradient @ 60 mL / min) to obtain Compound 5 (4 g, 3.25 mmol, yield 88.41%, purity 99.23%) as a brown solid. LCMS: RT = 0.979 min, m / z 1221.4 [M+H] + .

[0596] (Step 4) TFA (6.14 g, 53.85 mmol, 4 mL, 32.88 eq) was added to the solution of Compound 5 (2.00 g, 1.64 mmol, 1 eq) in DCM (20 mL). The mixture was stirred at 60°C for 0.5 hours. The solvent was removed from the reaction mixture using a nitrogen stream. The residue was purified by Prep-HPLC (TFA condition; column: Phenomenex luna C18 150*25 mm*10 um; mobile phase: [water (TFA)-ACN]; gradient: 24%-54% B over 10 min) to obtain Compound 6 (1.4 g, 1.25 mmol, yield 76.25%) as a green solid. LCMS: RT = 0.779 min, m / z 1121.4 [M+H] + .

[0597]

[0598] K-VA-Exatecan synthesis

[0599] K-VA-Exatecan, such as compound 4, was synthesized according to the reaction scheme below.

[0600]

[0601]

[0602] (Step 1) To a solution of Compound 1 (330 mg, 729.28 μmol, 1 eq) in DMF (4 mL), HATU (429.00 mg, 1.13 mmol, 1.55 eq) was added at 25°C for 0.5 hours, followed by the addition of Compound 2 (500.00 mg, 825.55 μmol, 1.13 eq) and DIPEA (282.76 mg, 2.19 mmol, 381.08 μL, 3 eq). The mixture was stirred at 25°C for 1 hour. The solvent was removed from the reaction mixture and concentrated under reduced pressure. Compound 3 was obtained as a yellow solid by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, Eluent of 27–90% Ethyl acetate / Petroleum ether gradient @ 80 mL / min). LCMS: RT = 0.631 min, m / z 1040.6 [M+1] + .

[0603] (Step 2) Piperidine (180.00 mg, 2.11 mmol, 208.77 μL, 2.44 eq) was added to a solution of Compound 3 (0.9 g, 865.27 μmol, 1 eq) in DMF (3 mL). The mixture was stirred at 20°C for 2 hours. The solvent was removed from the reaction mixture and concentrated under reduced pressure. The residue was purified by prep-HPLC (FA condition: Instrument: ACSWH-GX-R; Column: Phenomenex luna C18 150*25mm*10um; Mobile phase: water(FA)-ACN; Gradient: B 32% - 52% in 8 min linearly; Flow rate: 25 mL / min; Column temperature: RT; Wavelength: 220nm / 254nm) to obtain Compound 4 (240 mg, 293.43 μmol, yield 33.91%) as a pale yellow solid. LCMS: RT = 0.774 min, m / z 818.5 [M+1] + .

[0604]

[0605] K-MMAF synthesis

[0606] K-MMAF, such as Compound 4, was synthesized according to the reaction scheme below. Here, MMAF refers to monomethyl auristatin F.

[0607]

[0608]

[0609]

[0610] (Step 1) 2-tert-butyl-1,3-diisopropyl-isourea (750.00 mg, 3.74 mmol, 5.48 eq) was added to a solution of Compound 1 (500 mg, 683.10 μmol, 1 eq) in THF (5 mL). The mixture was stirred at 20-25°C for 2 hours. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified by prep-HPLC (FA condition; column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water(FA)-ACN]; gradient: 26%-56% B over 10 min) to obtain Compound 2 (350 mg, 444.13 μmol, yield 65.02%, purity 100%) as a white solid. LCMS: RT = 0.565 min, m / z 788.0 [M+H] + .

[0611] (Step 2) HATU (188 mg, 494.44 μmol, 1.49 eq) and DIPEA (214.21 mg, 1.66 mmol, 288.69 μL, 5 eq) were added to a solution of Compound 2 (200 mg, 253.79 μmol, 0.766 eq) in DMF (3 mL). The mixture was stirred at 20-25°C for 1 hour. Subsequently, (2S)-2-(allyloxycarbonylamino)-6-(9H-fluorene-9-ylmethoxycarbonylamino)hexanoic acid (150 mg, 331.49 μmol, 1 eq) was added, and the mixture was stirred at 20-25°C for an additional 2 hours. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified by flash silica gel column chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, eluent of 0–97% ethyl acetate / petroleum ether gradient @ 40 mL / min) to obtain Compound 3 (200 mg, 160.32 μmol, yield 48.36%, purity 98%) as a white solid. LCMS: RT = 1.088 min, m / z 1223.7 [M+H] + .

[0612] (Step 3) A mixture of Compound 3 (200 mg, 163.59 μmol, 1 eq) in piperidine / DMF (2 mL, 10% in DMF) was stirred at 20–25°C for 1 hour. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified by prep-HPLC (FA condition; column: Phenomenex luna C18 150*25 mm*10 µm; mobile phase: [water(FA)-ACN]; gradient: 29%–59% B over 10 min) to obtain Compound 4 (106 mg, 105.97 μmol, yield 64.77%, purity 100%) as a white solid. LCMS: RT = 0.868 min, m / z 1000.6 [M+H] + .

[0613]

[0614] E-MMAF synthesis

[0615] E-MMAF, such as compound 4, was synthesized according to the reaction scheme below.

[0616]

[0617]

[0618] (Step 1) HATU (620.63 mg, 1.63 mmol) was added to a solution of Compound 1 (500 mg, 1.09 mmol) in DMF (10 mL). After stirring the mixture at 25°C for 1 hour, Compound 2 (700 mg, 888.25 μmol) and DIPEA (703.18 mg, 5.44 mmol, 947.69 μL) were added and stirred at 25°C for 1 hour. Silica gel powder was added to the reaction mixture and concentrated under reduced pressure to obtain the residue. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, eluent of 0–100% ethyl acetate / petroleum ether gradient @ 40 mL / min) to obtain Compound 3 (855 mg, 695.38 μmol, yield 63.90%, purity 100%) as a white solid. LCMS: RT = 0.743 min, m / z 1230.0 [M+H] + .

[0619] (Step 2) Compound 3 (770 mg, 626.25 μmol) was added to a Pd / C (70 mg, 10% purity) solution in MeOH (10 mL) under an Ar atmosphere. The suspension was degassed under vacuum and purged several times with hydrogen. The mixture was stirred for 1 hour at 25°C under a hydrogen (50 psi) atmosphere. The crude product was filtered through a Celite pad. The filtrate cake was washed with MeOH (50 mL) and DCM (50 mL), and the filtrate was concentrated under reduced pressure to obtain Compound 4 (710 mg, 560.81 μmol, yield 89.55%, purity 90%) as a purple oil. LCMS: RT = 0.694 min, m / z 1140.1 [M+H] + .

[0620]

[0621] β-Glucuronide-MMAE Synthesis

[0622] β-Glucuronide-MMAE, such as Compound 4, was synthesized according to the reaction scheme below. Here, MMAE refers to monomethyl auristatin E.

[0623]

[0624]

[0625]

[0626] (Step 1) HOBT (40.96 mg, 303.14 μmol, 0.2 eq), TEA (306.75 mg, 3.03 mmol, 421.93 μL, 2 eq), and MMAE (1.09 g, 1.52 mmol, 1 eq) were added to a solution of Compound 1 (1.2 g, 1.52 mmol, 1 eq) in DMF (5 mL). The mixture was stirred at 25°C for 12 hours. The reaction mixture was concentrated under reduced pressure to obtain the residue. The residue was purified by prep-HPLC (FA condition: column: Phenomenex luna C18 150*40mm*15um; mobile phase: [water(FA)-ACN]; gradient: 56%-86% B over 10 min) to obtain Compound 2 (930 mg, 678.55 μmol, yield 44.77%) as a yellow solid. LCMS: RT = 1.053 min, m / z 1372.4 [M+H] + .

[0627] (Step 2) LiOH·H2O (42.86 mg, 1.02 mmol, 1.79 mL, 4 eq) was added to a solution of Compound 2 (350 mg, 255.37 μmol, 1 eq) in THF (10 mL). The mixture was stirred at 0°C for 1 hour. At 0°C, the pH of the reaction mixture was adjusted to 5 with TFA, then diluted with H2O (10 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic layer was dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by prep-HPLC (FA condition: column: Phenomenex luna C18 150*40mm*15um; mobile phase: [water(FA)-ACN]; gradient: 38%-68% B over 15 min) to obtain Compound 3 (120 mg, 97.53 μmol, yield 19.10%) as a white solid. LCMS: RT = 0.837 min, m / z 1230.9 [M+H] +.

[0628] (Step 3) TFA (1.54 g, 13.46 mmol, 1 mL, 138.04 eq) was added to the solution of Compound 3 (120 mg, 97.53 μmol, 1 eq) in DCM (3 mL). The mixture was stirred at 25°C for 1 hour. The reaction mixture was concentrated under reduced pressure to obtain the residue. The residue was purified by prep-HPLC (TFA conditional: column: Phenomenex luna C18 150*25 mm*10 µm; mobile phase: [water(TFA)-ACN]; gradient: 13%-43% B over 10 min) to obtain Compound 4 (60 mg, 53.08 μmol, yield 54.43%) as a yellow solid. LCMS: RT = 0.850 min, m / z 1130.7 [M+H] + .

[0629]

[0630] FG-Duocarmycin analog synthesis

[0631] An FG-Duocarmycin analog, such as compound 3, was synthesized according to the reaction scheme below.

[0632]

[0633]

[0634] (Step 1) EDCI (80 mg, 417.32 μmol) and Compound 1 (150 mg, 279.91 μmol, 2HCl) were added to a solution of 2-[[(2S)-2-(tert-butoxycarbonylamino)-3-phenylpropanoyl]amino]acetic acid (150.00 mg, 465.32 μmol) in pyridine (2 mL), and stirred at 20°C for 1 hour. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by prep-HPLC (column: Phenomenex luna C18 150*25mm* 10um; mobile phase: [water (FA)-ACN]; gradient: 26%-56% B over 10 min) to obtain Compound 2 (155 mg, 202.00 μmol, yield 72.17%, purity 100%) as a white solid. LCMS: RT = 0.533 min, m / z 767.4 [M+H] + .

[0635] (Step 2) 1 mL of HCl / dioxane solution (2 M) was added to the solution of Compound 2 (150 mg, 195.49 μmol) in DCM (3 mL), and the mixture was stirred at 20°C for 0.5 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent. As a result, Compound 3 (140 mg, crude, 2HCl) was obtained as an off-white solid. LCMS: RT = 0.450 min, m / z 667.3 [M+H] + .

[0636]

[0637] FG-Seco-DUBA analog synthesis

[0638] An FG-Seco-DUBA analog similar to compound 3 was synthesized according to the reaction scheme below.

[0639]

[0640]

[0641] (Step 1) EDCI (93.56 mg, 488.05 μmol, 5.49 eq) was added to a solution of 2-[[(2S)-2-(tert-butoxycarbonylamino)-3-phenylpropanoyl]amino]acetic acid (100.30 mg, 311.14 μmol, 3.5 eq) in pyridine (0.45 mL) and DMF (0.35 mL), and stirred at 25°C for 0.5 hours. Then, compound 1 (50 mg, 88.90 μmol, 1 eq, HCl salt) was added, and stirred at 25°C for an additional 1 hour. The reaction mixture was concentrated under reduced pressure, and the resulting crude product was triturated with MeCN (5 mL) and EtOAc (5 mL). The generated precipitated solid was recovered by filtration and dried under reduced pressure to obtain Compound 2 (65 mg, 66.54 μmol, yield 74.85%, purity 85%) as a brown solid. LCMS: RT = 0.585 min, m / z 830.6 [M+H] + .

[0642] (Step 2) An HCl / dioxane solution (2 M, 523.62 μL, 10.23 eq) was added to a solution of Compound 2 (100 mg, 102.37 μmol, 1 eq) in DCM (1 mL), and the mixture was stirred at 20°C for 0.5 hours. The resulting precipitate was recovered by filtration, washed with DCM (2 mL x 2), and dried under reduced pressure to obtain Compound 3 (71 mg, 92.61 μmol, yield 90%) in the hydrochloride (HCl) form as a yellow solid. LCMS: RT = 0.515 min, m / z 730.3 [M+H] + .

[0643]

[0644] VA-Ispinesib Synthesis

[0645] VA-Ispinesib, such as compound 3, was synthesized according to the reaction scheme below.

[0646]

[0647]

[0648] (Step 1) HATU (665 mg, 1.75 mmol) was added to a solution of Compound 1 (350 mg, 1.21 mmol) in DMF (6 mL). After stirring the mixture at 25°C for 0.5 hours, N-(3-aminopropyl)-N-[(1R)-1-(3-benzyl-7-chloro-4-oxo-quinazolin-2-yl)-2-methylpropyl]-4-methylbenzamide (Ispinesib, 625 mg, 1.21 mmol) and DIPEA (470.64 mg, 3.64 mmol, 634.29 μL, 3 eq) were added and stirred at 25°C for 0.5 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, eluent of 0–30% ethyl acetate / petroleum ether gradient @ 50 mL / min) to obtain Compound 2 (900 mg, 1.04 mmol, yield 85.33%, purity 90.62%) as a white solid. LCMS: RT = 0.732 min, m / z 787.3 [M+H] + .

[0649] (Step 2) The mixture of Compound 2 (900 mg, 1.14 mmol) in TFA / DCM (20 mL, 10% TFA in DCM) was stirred at 25°C for 6 hours. The solvent was removed by concentrating the reaction mixture under reduced pressure. The crude product was triturated in ethyl acetate:PE=1:1 (50 mL) at 25°C for 5 minutes to obtain Compound 3 (940 mg, 990.10 μmol, yield 86.62%, purity 96.41%, 2TFA) as a white solid. LCMS: RT = 0.580 min, m / z 687.3 [M+H] + .

[0650]

[0651] FG-PAB-Duocarmycin analog synthesis

[0652] An FG-PAB-Duocarmycin analog, such as compound 3, was synthesized according to the reaction scheme below.

[0653]

[0654]

[0655] (Step 1) TEA (196.71 mg, 1.94 mmol, 270.58 μL, 3 eq) and bis(trichloromethyl)carbonate (194.21 mg, 654.47 μmol, 1.01 eq) were added to a solution of Compound 1 (300 mg, 647.99 μmol, 1 eq) in DCM (20 mL) at 0-5°C. The mixture was stirred at 25°C for 30 minutes. Subsequently, tert-butyl N-[(1R)-1-benzyl-2-[[2-[4-(hydroxymethyl)anilino]-2-oxo-ethyl]amino]-2-oxo-ethyl]carbamate (600.00 mg, 1.40 mmol, 2.17 eq) and [acetoxy(dibutyl)stanyl]acetate (20 mg, 56.98 μmol, 15.29 μL, 0.088 eq) were added, and the mixture was stirred at 25°C for 72 hours. The reaction was terminated by adding an aqueous KF solution (10 mL) to the reaction mixture, and the mixture was extracted with DCM (40 mL, 20 mL x 2). The bound organic layer was washed with brine (40 mL, 20 mL x 2), dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by flash silica gel chromatography (ISCO®; 10 g SepaFlash® Silica Flash Column, eluent of 0–15% methanol / dichloromethane ether gradient @ 36 mL / min) to obtain Compound 2 (200 mg, 218.23 μmol, yield 33.68%) as a white solid. LCMS: RT = 0.856 min, m / z 916.5 [M+H] +.

[0656] (Step 2) TFA (1.54 g, 13.46 mmol, 1 mL, 61.69 eq) was added to the solution of Compound 2 (200 mg, 218.23 μmol, 1 eq) in DCM (5 mL). The mixture was stirred at 25°C for 1 hour. The reaction mixture was concentrated under reduced pressure to obtain the residue. The residue was purified by prep-HPLC (FA condition: column: Phenomenex luna C18 150*25 mm*10 um; mobile phase: [water(FA)-ACN]; gradient: 14%-44% B over 10 min) to obtain Compound 3 (100 mg, 122.50 μmol, yield 56.13%) as a yellow solid. LCMS: RT = 0.751 min, m / z 816.4 [M+H] + .

[0657]

[0658] FG-PAB-Dimethyl-ethanediamine-Duocarmycin DM Synthesis

[0659] FG-PAB-dimethyl-ethanediamine-Duocarmycin DM, similar to compound 6, was synthesized according to the reaction scheme below.

[0660]

[0661]

[0662]

[0663] (Step 1) DIPEA (196.29 mg, 1.52 mmol, 264.54 μL, 1 eq) and Compound 1 (900.00 mg, 1.52 mmol, 1 eq) were added to a solution of N,N'-dimethylethane-1,2-diamine (1.34 g, 15.19 mmol, 1.63 mL, 10 eq) in DCM (20 mL). The mixture was stirred at 0-5°C for 1 hour. The reaction was terminated by adding H2O (20 mL), and the organic layer was separated. The organic layer was dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by prep-HPLC (FA, column: Phenomenex luna C18 150*40mm*15um; mobile phase: [water(FA)-ACN]; gradient: 23%-53% B over 10 min) to obtain Compound 2 (650 mg, 1.05 mmol, yield 69.42%, purity 95.32%, containing FA) as a pale white solid. LCMS: RT = 0.747 min, m / z 542.3 [M+H] + .

[0664] (Step 2) DIPEA (165.29 mg, 1.28 mmol, 222.77 μL, 4 eq) and (4-nitrophenyl)carbonyl chloride (96.67 mg, 479.60 μmol, 1.5 eq) were added to a solution of Compound 3 (160 mg, 319.73 μmol, 1 eq, HCl) in DCM (5 mL). The mixture was stirred for 50 minutes under a nitrogen atmosphere at 20–25°C. The reaction was terminated by adding H2O (10 mL) and extracted with DCM (20 mL). The organic layer was dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. As a result, Compound 4 (200 mg, crude) was obtained as a yellow solid and used in the next step without special purification. LCMS: RT = 0.861 min, m / z 629.2 [M+H] + .

[0665] (Step 3) A mixture of Compound 4 (200 mg, 317.94 μmol, 1 eq), Compound 2 (242.89 mg, 413.32 μmol, 1.3 eq, FA), and DIPEA (123.27 mg, 953.81 μmol, 166.13 μL, 3 eq) in DCM (10 mL) was degassed after three gas exchanges with nitrogen. The mixture was then stirred for 1 hour at 20-25°C under a nitrogen atmosphere. The reaction was terminated by adding H2O (10 mL), and the mixture was extracted with DCM (20 mL). The organic layer was dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, DCM / MeOH = 20 / 1 to 15 / 1) to obtain Compound 5 (330 mg, 260.74 μmol, yield 82.01%, purity 81.51%) as a yellow solid. LCMS: RT = 0.867 min, m / z 1031.6 [M+H] + .

[0666] (Step 4) TFA (1.07 g, 9.42 mmol, 0.7 mL, 30.38 eq) was added to a solution of Compound 5 (320 mg, 310.20 μmol, 1 eq) in DCM (14 mL). The mixture was stirred at 20-25°C for 2 hours. After removing most of the DCM from the reaction mixture at 0-10°C under reduced pressure, it was diluted with i-Pr2O (50 mL). As some solid formed, the mixture was stirred for 10 minutes and then filtered to obtain the solid. The residue was purified by prep-HPLC (FA, column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water(FA)-ACN]; gradient: 15%-45% B over 10 min) to obtain Compound 6 (110 mg, 107.47 μmol, yield 34.65%, 2FA) as a pale white solid. LCMS: RT = 0.766 min, m / z 931.6 [M+H] + .

[0667]

[0668] FG-PAB-dimethyl-ethanediamine-Seco-DUBA synthesis

[0669] FG-PAB-dimethyl-ethanediamine-Seco-DUBA, similar to compound 4, was synthesized according to the reaction scheme below.

[0670]

[0671]

[0672] (Step 1) DIPEA (108.64 mg, 840.60 μmol, 146.42 μL, 4 eq) and (4-nitrophenyl)carbonyl chloride (84.72 mg, 420.30 μmol, 2 eq) were added to a solution of Compound 1 (120 mg, 210.15 μmol, 1 eq) in DCM (2 mL). The mixture was stirred at 20°C for 1 hour under a nitrogen atmosphere. The reaction was terminated by adding H2O (20 mL) and extracted with DCM (30 mL). The organic layer was dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. As a result, Compound 2 (140 mg, 190.19 μmol, yield 90.50%, crude) was obtained as a yellow solid and used in the next step without special purification. LCMS: RT = 0.638 min, m / z = 736.4 [M+H] + .

[0673] (Step 2) To a solution of Compound 2 (140 mg, 190.19 μmol, 1 eq) in DCM (3 mL), DIPEA (73.74 mg, 570.56 μmol, 99.38 μL, 3 eq) and [4-[[2-[[(2S)-2-(tert-butoxycarbonylamino)-3-phenylpropanoyl]amino]acetyl]amino]phenyl]methyl N-methyl-N-[2-(methylamino)ethyl]carbamate (134.12 mg, 228.22 μmol, 1.20 eq, FA) were added. The mixture was stirred at 20°C for 1 hour. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by prep-TLC (SiO2, PE:ethyl acetate = 1:1) to obtain Compound 3 (150 mg, 131.73 μmol, yield 69.27%) as a brown solid. LCMS: RT = 0.595 min, m / z = 1138.6 [M+H] + .

[0674] (Step 3) TFA (383.75 mg, 3.37 mmol, 0.25 mL, 29.48 eq) was added to the solution of Compound 3 (130.00 mg, 114.17 μmol, 1 eq) in DCM (2.5 mL). The mixture was stirred at 25°C for 1 hour. The solvent was removed by concentrating the reaction mixture under reduced pressure. The residue was purified by prep-HPLC (column: Phenomenex luna C18 150*25 mm*10 µm; mobile phase: [water (FA)-ACN]; gradient: 19%-49% B over 10 min) to obtain Compound 4 (70 mg, 70.39 μmol, yield 61.65%) as a white solid. LCMS: RT = 0.533 min, m / z = 994.7 [M+H] + .

[0675]

[0676] FG-PAB-PBD dimer analog 1 synthesis

[0677] FG-PAB-PBD dimer analog 1, similar to compound 12, was synthesized according to the reaction scheme below.

[0678]

[0679]

[0680]

[0681]

[0682]

[0683]

[0684]

[0685] (Step 1) NaBH(OAc)3 (547.50 mg, 2.58 mmol, 1.5 eq) was added to a solution of Compound 1 (600 mg, 1.72 mmol, 1 eq) in DCM (12 mL) and DMF (4 mL) at 0–5°C. The mixture was stirred at 0–25°C for 1 hour. The reaction was terminated by adding H2O (10 mL) and extracted with DCM (10 mL x 2). The bound organic layer was washed with brine (10 mL x 2), dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. As a result, Compound 2 (600 mg, crude) was obtained as a yellow solid and used in the next step without further purification. LCMS: RT = 0.814 min, m / z 351.2 [M+H] + .

[0686] (Step 2) Pyridine (372.46 mg, 4.71 mmol, 380.06 μL, 3 eq) and allyl carbonochloridate (283.78 mg, 2.35 mmol, 249.81 μL, 1.5 eq) were added to a solution of Compound 2 (550 mg, 1.57 mmol, 1 eq) in DCM (10 mL) at 0-5°C. The mixture was stirred at 20-25°C for 1 hour. The reaction was terminated by adding H2O (5 mL) and extracted with DCM (5 mL x 2). The bound organic layer was washed with brine (10 mL), dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate = 3 / 1 to 2 / 1) to obtain compound 3 (540 mg, 1.24 mmol, yield 79.18%) as a yellow solid.

[0687] (Step 3) MsOH (2.69 g, 27.99 mmol, 2 mL, 24.82 eq) was added to a solution of Compound 3 (490 mg, 1.13 mmol, 1 eq) in DCM (10 mL) at 0-5°C. The mixture was stirred at 20-25°C for 2 hours. The reaction was terminated by adding an aqueous solution of Na2CO3 (50 mL) and extracted with DCM (50 mL). The organic layer was washed with brine (20 mL), dried with Na2SO4, filtered, and then the solvent was removed under reduced pressure. As a result, Compound 4 (380 mg, crude) was obtained and used in the next step without special purification.

[0688] (Step 4) A mixture of compound 4 (380 mg, 1.10 mmol, 1 eq), methyl 4-(5-bromopentoxy)-2-(tert-butoxycarbonylamino)-5-methoxybenzoate (591.03 mg, 1.32 mmol, 1.2 eq) and K2CO3 (610.06 mg, 4.41 mmol, 4 eq) in DMF (6 mL) was degassed and purged with nitrogen three times, then stirred for 12 hours at 20-25°C under a nitrogen atmosphere. The reaction was terminated by adding H2O (20 mL) and extracted with ethyl acetate (10 mL x 2). The bound organic layer was washed with brine (10 mL x 2), dried with Na2SO4, filtered, and then the solvent was removed under reduced pressure. The remaining residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate = 2 / 1 to 1 / 1) to obtain compound 5 (450 mg, 634.00 μmol, yield 57.45%) as a yellow solid.

[0689] (Step 5) KOH (142.28 mg, 2.54 mmol, 4 eq) in H2O (4 mL) was added to a solution of compound 5 (450 mg, 634.00 μmol, 1 eq) in MeOH (6 mL) and THF (3 mL). The mixture was stirred at 20-25°C for 12 hours. An aqueous solution of HCl (0.5 N) was added to the reaction mixture to adjust the pH to < 5, and the mixture was extracted with ethyl acetate (25 mL x 2). The bound organic layer was washed with brine (10 mL), dried with Na2SO4, filtered, and then the solvent was removed under reduced pressure. The remaining residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate = 1 / 1 to 0 / 1) to obtain Compound 6 (380 mg, 444.26 μmol, yield 70.07%, purity 81.341%) as a yellow solid. LCMS: RT = 1.018 min, m / z 696.4 [M+H] + .

[0690] (Step 6) HCl / dioxane (2 M, 20 mL, 84.33 eq) was added to a solution of Compound 6 (330 mg, 474.31 μmol, 1 eq) in THF (3 mL). The mixture was stirred at 20–25°C for 12 hours. The solvent was removed from the reaction mixture under reduced pressure to obtain the residue. The residue was purified by prep-HPLC (HCl. column: Phenomenex luna C18 150*40 mm*15 µm; mobile phase: [water (HCl)-ACN]; gradient: 34%–64% B over 10 min) to obtain Compound 7 (200 mg, 316.41 μmol, yield 66.71%, HCl) as a yellow solid. LCMS: RT = 0.871 min, m / z 596.3 [M+H] + .

[0691] (Step 7) TBTU (121.29 mg, 377.75 μmol, 1.5 eq) was added to a solution of Compound 7 (150 mg, 251.83 μmol, 1 eq) in DCM (3 mL), and the mixture was stirred at 20-25°C for 0.5 hours. Then, TEA (76.45 mg, 755.49 μmol, 105.16 μL, 3 eq) and tert-butyl-dimethyl-[[(2S)-4-methylenepyrrolidin-2-yl]methoxy]silane (74.45 mg, 327.38 μmol, 1.3 eq) were added. The mixture was stirred at 20-25°C for an additional 1 hour. The reaction was terminated by adding H2O (2 mL), and the mixture was extracted with DCM (2 mL x 2). The bound organic layer was washed with brine (2 mL), dried with Na2SO4, filtered, and the solvent removed under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, DCM / MeOH = 100 / 1 to 50 / 1) and further purified by prep-TLC (SiO2, DCM:MeOH = 15:1) to obtain Compound 8 (200 mg, 204.22 μmol, yield 81.09%, purity 82.203%) as a yellow oil. LCMS: RT = 1.086 min, m / z 805.5 [M+H] + .

[0692] (Step 8) TEA (94.27 mg, 931.63 μmol, 129.67 μL, 5 eq) and Triphosgene (350 mg, 1.18 mmol, 6.33 eq) were added to a solution of Compound 8 (150 mg, 186.33 μmol, 1 eq) in DCM (3 mL) at 0-5°C under a nitrogen atmosphere. After stirring the mixture at 0-5°C for 0.5 hours, Tert-butyl N-[(1S)-1-benzyl-2-[[2-[4-(hydroxymethyl)anilino]-2-oxo-ethyl]amino]-2-oxoethyl]carbamate (159.31 mg, 372.65 μmol, 2 eq) was added. The mixture was stirred at 20-25°C for 1 hour under a nitrogen atmosphere. The reaction was terminated by adding H2O (5 mL), and the mixture was extracted with DCM (5 mL x 2). The bound organic layer was washed with brine (5 mL), dried with Na2SO4, filtered, and the solvent removed under reduced pressure to obtain the residue. The residue was purified by prep-TLC (SiO2, DCM:MeOH = 20:1) to obtain Compound 9 (170 mg, 135.08 μmol, yield 72.50%) as a gray solid. LCMS: RT = 1.181 min, m / z 1259.3 [M+H] + .

[0693] (Step 9) Compound 9 (170 mg, 135.08 μmol, 1 eq) was dissolved in THF (2 mL), H2O (1.5 mL), and HOAc (3 mL) and stirred at 20–25°C for 12 hours. The reaction was terminated by adding H2O (10 mL) and extracted with ethyl acetate (10 mL). The separated organic layer was washed with an aqueous Na2CO3 solution (10 mL), dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. As a result, Compound 10 (150 mg, crude) was obtained as a yellow solid. LCMS: RT = 1.002 min, m / z 1145.5 [M+H] + .

[0694] (Step 10) TEMPO (20.61 mg, 131.09 μmol, 1 eq) and PhI(OAc)2 (63.33 mg, 196.63 μmol, 1.5 eq) were added to a solution of Compound 10 (150 mg, 131.09 μmol, 1 eq) in DCM (3 mL) at 0-5°C. The mixture was stirred at 20-25°C for 12 hours. The reaction was terminated by adding an aqueous solution of Na2S2O3 (2 mL) and extracted with DCM (2 mL x 2). The bound organic layer was washed with brine (2 mL), dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. As a result, Compound 11 (150 mg, crude) was obtained as a yellow oil. LCMS: RT = 0.900 min, m / z 1142.9 [M+H] + .

[0695] (Step 11) TFA (1.54 g, 13.46 mmol, 1 mL, 102.52 eq) was added to a solution of Compound 11 (150 mg, 131.32 μmol, 1 eq) in DCM (5 mL). The mixture was stirred at 20–25°C for 1 hour. The reaction mixture was concentrated under reduced pressure to obtain the residue. The residue was purified by prep-HPLC (HCl. column: Phenomenex luna C18 150*40 mm*15 µm; mobile phase: [water (HCl)-ACN]; gradient: 23%–53% B over 10 min) to obtain Compound 12 (80 mg, 74.17 μmol, yield 56.48%, HCl) as a pale white solid. LCMS: RT = 0.839 min, m / z 1042.9 [M+H] + .

[0696]

[0697] FG-PAB-PBD dimer analog 2 synthesis

[0698] FG-PAB-PBD dimer analog 2, similar to compound 16, was synthesized according to the reaction scheme below.

[0699]

[0700]

[0701]

[0702]

[0703]

[0704]

[0705]

[0706]

[0707] (Step 1) NaOH (4.04 g, 100.91 mmol, 5 eq) was added to a solution of Compound 1 (6 g, 20.18 mmol, 1 eq) in THF (40 mL) and H2O (20 mL), and the mixture was stirred at 70°C for 5 hours. The reaction mixture was concentrated to obtain a residue. The residue was treated with citric acid to adjust the pH to 5, and then extracted with ethyl acetate (150 mL x 2). The combined organic layer was washed with brine (200 mL), dried with Na2SO4, and concentrated to obtain Compound 2 (5.3 g, 18.71 mmol, yield 92.71%) in the form of an off-white solid.

[0708] (Step 2) To a solution of Compound 2 (5.3 g, 18.71 mmol, 1 eq) in THF (80 mL), EDCI (7.17 g, 37.42 mmol, 7.03 mL, 2 eq), HOBt (5.06 g, 37.42 mmol, 2 eq), DIPEA (9.67 g, 74.84 mmol, 13.04 mL, 4 eq), and Compound 3 (3.99 g, 22.45 mmol, 1.2 eq, HCl) were added at 0°C. The mixture was stirred at 0-20°C for 12 hours. The reaction mixture was diluted with citric acid (100 mL) and then extracted with ethyl acetate (100 mL x 2). The combined organic layer was washed with a saturated NaHCO3 solution (200 mL) and brine (200 mL), dried with Na2SO4, and concentrated to obtain a brown oil. The oil was purified by prep-HPLC (column: Phenomenex luna C18 (250*70 mm, 10 µm); mobile phase: [water(FA)-ACN]; gradient: 38%-68% B over 18 min) to obtain Compound 4 (6.2 g, 15.25 mmol, yield 81.53%) in the form of a pale yellow oil. LCMS: RT = 0.475 min, m / z 407.1 [M+H] + .

[0709] (Step 3) HCl / MeOH (2 M, 38.14 mL, 5 eq) was added to a solution of Compound 4 (6.2 g, 15.25 mmol, 1 eq) in MeOH (60 mL), and the mixture was stirred at 40 °C for 15 hours. The pH was adjusted to 7 with TEA and then concentrated to obtain the residue. The residue was suspended in a 1 M HCl solution (50 mL), and the suspension was stirred for 1 hour. The suspension was then filtered. The filter cake was washed with water (20 mL) and concentrated to obtain Compound 5 (3.2 g, 11.53 mmol, yield 75.56%, purity 98.8%) as an off-white solid. LCMS: RT = 0.681 min, m / z 275.2 [M+H] + .

[0710] (Step 4) KI (605.25 mg, 3.65 mmol, 1 eq), Compound 6 (1.87 g, 4.19 mmol, 1.15 eq), and K2CO3 (806.24 mg, 5.83 mmol, 1.6 eq) were added to a solution of Compound 5 (1 g, 3.65 mmol, 1 eq) in DMF (20 mL) and stirred at 40°C for 15 hours. The reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (20 mL x 2). The combined organic layer was washed with water (40 mL) and brine (40 mL), dried with Na2SO4, and concentrated to obtain brown oil. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1) to obtain Compound 7 (2.15 g, 3.36 mmol, yield 92.18%) as a white solid. LCMS: RT = 0.993 min, m / z 640.4 [M+H] + .

[0711] (Step 5) To a solution of Compound 7 (1.0 g, 1.56 mmol, 1 eq) in MeOH (8 mL) and THF (4 mL), a solution of KOH (219.80 mg, 3.92 mmol, 2.51 eq) in H2O (6 mL) was added and stirred at 40°C for 4.5 hours. The reaction mixture was adjusted to a pH of 6 with 1 M HCl, diluted with water (30 mL), and extracted with ethyl acetate (20 mL x 2). The combined organic layer was washed with water (40 mL) and brine (40 mL), dried with Na2SO4, and concentrated to obtain brown oil. The residue was purified by column chromatography (SiO2, DCM:MeOH = 50 / 1 to 10 / 1) to obtain Compound 8 (920 mg, 1.47 mmol, yield 94.06%) as a gray solid. LCMS: RT = 0.889 min, m / z 626.3 [M+H] + .

[0712] (Step 6) MsOH (706.59 mg, 7.35 mmol, 525.34 μL, 5 eq) was added to a solution of Compound 8 (920 mg, 1.47 mmol, 1 eq) in THF (12 mL), and the mixture was stirred at 60°C for 19 hours. After filtering the reaction mixture, the filter cake was concentrated to obtain Compound 9 (750 mg, 1.19 mmol, yield 80.96%, purity 98.68%, MsOH) as a gray solid. LCMS: RT = 0.773 min, m / z 526.3 [M+H] + .

[0713] (Step 7) TEA (529.01 mg, 5.23 mmol, 727.67 μL, 5 eq), TBDU (671.44 mg, 2.09 mmol, 2 eq), and Compound 10 (332.90 mg, 1.46 mmol, 1.4 eq) were added to a solution of Compound 9 (650 mg, 1.05 mmol, 1 eq, MsOH) in DCM (15 mL), and the mixture was stirred at 20°C for 0.5 hours. The reaction mixture was diluted with water (30 mL) and extracted with DCM (30 mL). The combined organic layer was washed with brine (30 mL), dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, DCM: MeOH = 50:1 to 10:1) to obtain Compound 11 (750 mg, 1.02 mmol, yield 97.60%) as a brown solid. LCMS: RT = 0.520 min, m / z 683.2 [M+Na] + .

[0714] (Step 8) To a solution of Compound 11 (670 mg, 911.62 μmol, 1 eq) in THF (12 mL), TEA (461.23 mg, 4.56 mmol, 634.43 μL, 5 eq) was added at 0°C, and then a solution of Triphosgene (220 mg, 741.37 μmol, 8.13 e-1 eq) in DCM (3 mL) was added to the reaction mixture. After stirring for 0.5 hours at 0°C, Compound 12 (513.67 mg, 1.20 mmol, 1.32 eq) was added at 0°C. After stirring for 5.5 hours at 0-20°C, water (50 mL) was added to terminate the reaction, and the mixture was extracted with ethyl acetate (50 mL). The organic layer was washed with water (40 mL x 2) and brine (40 mL), dried with Na2SO4, and concentrated to obtain the residue. The residue was purified by column chromatography (SiO2, DCM:MeOH = 100:1 to 10:1) to obtain Compound 13 (360 mg, 293.23 μmol, yield 32.17%, purity 96.8%) as a pale yellow solid. LCMS: RT = 1.076 min, m / z 1189.0 [M+H] + .

[0715] (Step 9) HOAc (4.20 g, 69.87 mmol, 4 mL, 267.87 eq) and H2O (3 mL) were added to a solution of Compound 13 (310.00 mg, 260.85 μmol, 1 eq) in THF (4 mL) at 20°C. After stirring for 12 hours at 20°C, the reaction was terminated with a saturated NaHCO3 solution (40 mL) and extracted with ethyl acetate (40 mL). The organic layer was washed with water (40 mL) and brine (40 mL), dried with Na2SO4, and concentrated to obtain Compound 14 (240 mg, 223.43 μmol, yield 85.65%) as a pale yellow oil. LCMS: RT = 0.952 min, m / z 1075.1 [M+H] + .

[0716] (Step 10) TEMPO (35.13 mg, 223.43 μmol, 33.49 μL, 1 eq) and PhI(OAc)2 (179.91 mg, 558.57 μmol, 2.5 eq) were added to a solution of Compound 14 (240.00 mg, 223.43 μmol, 1 eq) in DCM (8 mL) at 0°C. After stirring for 1 hour at 0-20°C, water (30 mL) was added to terminate the reaction, and the mixture was extracted with DCM (20 mL). The organic layer was washed with water (20 mL x 2) and brine (20 mL), dried with Na2SO4, and concentrated to obtain the residue. The residue was purified by prep-TLC (SiO2, DCM: MeOH = 10:1, Rf=0.3) to obtain Compound 15 (170 mg, 154.12 μmol, yield 68.98%, purity 97.2%) as a pale yellow solid. LCMS: RT = 0.958 min, m / z 1072.8 [M+H] + .

[0717] (Step 11) TFA (4.61 g, 40.39 mmol, 3 mL, 270.63 eq) was added to a solution of Compound 15 (160.00 mg, 149.23 μmol, 1 eq) in DCM (10 mL) at 20°C. After stirring for 15 minutes at 20°C, the residue was concentrated. The residue was purified by prep-HPLC (column: Waters xbridge 150*25 mm 10 µm; mobile phase: [water(NH4HCO3)-ACN]; gradient: 37%-57% B over 8 min) to obtain Compound 16 (120 mg, 123.45 μmol, yield 82.72%) as a white solid. LCMS: RT = 0.790 min, m / z 972.7 [M+H] + .

[0718]

[0719] VC-PAB-MMAE Synthesis

[0720] VC-PAB-MMAE, such as compound 6, was synthesized according to the reaction scheme below.

[0721]

[0722]

[0723]

[0724]

[0725]

[0726] (Step 1) A mixture of Compound 1 (5 g, 13.35 mmol, 1 eq), (4-aminophenyl)methanol (1.97 g, 16.02 mmol, 1.2 eq), and EEDQ (5 g, 20.22 mmol, 1.51 eq) in DCM (50 mL) and MeOH (25 mL) was stirred at 25°C for 1 hour. The reaction mixture was concentrated under reduced pressure to obtain the residue. The residue was triturated with an ethyl acetate:PE=1:1 (150 mL) mixture at 25°C for 30 minutes to obtain Compound 2 (5.2 g, 10.84 mmol, yield 81.20%) as a white solid. LCMS: RT = 0.492 min, m / z 480.4 [M+H] + .

[0727] (Step 2) Bis(4-nitrophenyl)carbonate (5 g, 16.44 mmol, 1.52 eq) and DIPEA (7.01 g, 54.22 mmol, 9.44 mL, 5 eq) were added to a solution of Compound 2 (5.2 g, 10.84 mmol, 1 eq) in DMF (20 mL). The mixture was stirred at 25°C for 1 hour. The reaction was terminated by adding H2O (200 mL), and the mixture was filtered to obtain a yellow solid. This was triturated with an ethyl acetate:PE=1:2 (200 mL) mixture at 25°C for 30 minutes to obtain Compound 3 (6 g, 9.31 mmol, yield 85.83%) as a yellow solid. LCMS: RT = 0.579 min, m / z 645.5 [M+H] + .

[0728] (Step 3) HOBt (90 mg, 666.06 μmol, 1.59 eq) and DIPEA (162.01 mg, 1.25 mmol, 218.34 μL, 3 eq) were added to a solution of compound 4 (300 mg, 417.84 μmol, 1 eq) and [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl(4-nitrophenyl)carbonate (300 mg, 465.35 μmol, 1.11 eq) in DMF (5 mL). The mixture was stirred at 25°C for 1 hour. The reaction mixture was concentrated under reduced pressure to obtain the residue, which was purified by prep-TLC (SiO2, DCM:MeOH=10:1) to obtain Compound 5 (470 mg, 384.13 μmol, yield 91.93%) as a white solid. LCMS: RT = 0.603 min, m / z 1224.3 [M+H] + .

[0729] (Step 4) TFA (2.77 g, 24.34 mmol, 1.81 mL, 63.35 eq) was added to a solution of Compound 5 (470 mg, 384.13 μmol, 1 eq) in DCM (5 mL). The mixture was stirred at 25°C for 1 hour. The reaction mixture was concentrated under reduced pressure to obtain the residue. The residue was purified by prep-HPLC (FA condition: column: Phenomenex luna C18 150*25 mm*10 um; mobile phase: [water(FA)-ACN]; gradient: 22%-52% B over 10 min) to obtain Compound 6 (180 mg, 160.22 μmol, yield 41.71%) as a white solid. LCMS: RT = 0.541 min, m / z 1124.2 [M+H] + .

[0730]

[0731] VA-PAB-PBD dimer analog 1 synthesis

[0732] According to the reaction scheme below, VA-PAB-PBD dimer analog 1, which is the same as compound 5, was synthesized by using VA-PAB instead of FG-PAB of compound 11 in the synthesis reaction scheme of FG-PAB-PBD dimer analog 1 above.

[0733]

[0734]

[0735]

[0736] (Step 1) TEA (314.24 mg, 3.11 mmol, 432.24 μL) and triphosgene (230 mg, 775.06 μmol) were added to a solution of Compound 1 (500 mg, 621.09 μmol) in DCM (20 mL) at 0-5°C under a nitrogen atmosphere. After stirring the mixture at 0-5°C for 0.5 hours, tert-butyl N-[(1S)-1-[[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxoethyl]carbamoyl]-2-methyl-propyl]carbamate (488.77 mg, 1.24 mmol) was added. After stirring the mixture at 25°C under a nitrogen atmosphere for 1 hour, water (10 mL) was added to terminate the reaction and the mixture was extracted with DCM (20 mL x 2). The combined organic layer was washed with brine (20 mL), dried with Na2SO4, and then filtered and concentrated under reduced pressure to obtain the residue. The residue was purified by prep-HPLC (FA condition: column: Phenomenex luna C18 150*40 mm*15 µm; mobile phase: [water(FA)-ACN]; gradient: 45%-75% B over 18 min) to obtain Compound 2 (400 mg, 326.66 μmol, yield 52.60%) as a white solid. LCMS: RT = 1.205 min, m / z 1224.7 [M+H] + .

[0737] (Step 2) H2O (3 mL) and HOAc (6 mL) were added to a solution of Compound 2 (400 mg, 326.66 μmol) in THF (4 mL), and the mixture was stirred at 25°C for 12 hours. The reaction was terminated by adding H2O (10 mL) to the reaction mixture, and the mixture was extracted with ethyl acetate (10 mL). The separated organic layer was washed with an aqueous Na2CO3 solution (10 mL), dried with Na2SO4, and then filtered and concentrated under reduced pressure to obtain the residue. As a result, Compound 3 (335 mg, 277.59 μmol, yield 84.98%, purity 92%) was obtained as a yellow solid. LCMS: RT = 0.624 min, m / z 1110.6 [M+H] + .

[0738] (Step 3) TEMPO (44.62 mg, 283.72 μmol) and PhI(OAc)2 (137.08 mg, 425.58 μmol) were added to a solution of Compound 3 (315 mg, 283.72 μmol) in DCM (5 mL) at 0 °C. The mixture was stirred at 25 °C for 12 hours. The reaction was terminated by adding an aqueous solution of Na2S2O3 (20 mL) to the reaction mixture, followed by extraction with DCM (20 mL x 2). The combined organic layer was washed with brine (20 mL), dried with Na2SO4, and then filtered and concentrated under reduced pressure to obtain the residue. As a result, Compound 4 (310 mg, crude) was obtained as a purple solid. LCMS: RT = 0.603 min, m / z 1108.5 [M+H] + .

[0739] (Step 4) TFA (460.50 mg, 4.04 mmol, 0.3 mL) was added to the solution of Compound 4 (310 mg, 279.72 μmol) in DCM (3 mL), and the mixture was stirred at 25°C for 1 hour. The solvent was removed by concentrating the reaction mixture under reduced pressure. The residue was purified by column chromatography (SiO2, Ethyl acetate / MeOH = 1 / 0 to 5 / 1) to obtain Compound 5 (170 mg, 168.63 μmol, yield 60.28%) as a yellow solid. LCMS: RT = 0.532 min, m / z 1008.5 [M+H] + .

[0740]

[0741] VA-PAB-PBD dimer analog 2 synthesis

[0742] VA-PAB-PBD dimer analog 2, similar to compound 5, was synthesized according to the reaction scheme below.

[0743]

[0744]

[0745]

[0746] (Step 1) Triphosgene (1 g, 3.37 mmol, 0.826 eq) and TEA (2.54 g, 25.15 mmol, 3.5 mL, 6.16 eq) were added to a solution of Compound 1 (3 g, 4.08 mmol, 1 eq) in THF (60 mL) at 0°C. The mixture was stirred at 0°C for 0.5 hours. Then, tert-butyl N-[(1S)-1-[[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate (2.01 g, 5.10 mmol, 1.25 eq) was added at 0°C. The mixture was stirred at 0–20°C for 12 hours. The reaction was terminated by adding H2O (60 mL) and extracted with ethyl acetate (80 mL). The organic layer was washed with H2O (80 mL x 2) and brine (80 mL), dried with Na2SO4, and concentrated to obtain the residue. The residue was purified by column chromatography (SiO2, DCM: MeOH = 100:1 to 10:1) to obtain Compound 2 (3.4 g, 2.62 mmol, yield 64.22%, purity 89%) as a pale yellow solid. LCMS: RT = 0.984 min, m / z 1153.9 [M+H] + .

[0747] (Step 2) To a solution of Compound 2 (3.4 g, 2.95 mmol, 1 eq) in THF (35 mL), a solution of HOAc (36.72 g, 611.39 mmol, 35 mL, 207.59 eq) and H2O (25 mL) was added at 20°C. The mixture was stirred at 20°C for 12 hours. The reaction was terminated by adding H2O (100 mL) and extracted with ethyl acetate (80 mL x 2). The bound organic layer was washed with H2O (80 mL x 2) and brine (80 mL), then dried with Na2SO4 and concentrated to obtain the residue. The residue was purified by column chromatography (SiO2, DCM:MeOH = 100:1 to 10:1) to obtain Compound 3 (2.8 g, 2.69 mmol, yield 91.40%) as a pale yellow solid. LCMS: RT = 0.515 min, m / z 1040.5 [M+H] + .

[0748] (Step 3) TEMPO (249.44 mg, 1.59 mmol, 237.79 μL, 1 eq) and PhI(OAc)2 (1.28 g, 3.97 mmol, 2.5 eq) were added to a solution of Compound 3 (1.65 g, 1.59 mmol, 1 eq) in DCM (20 mL) at 0°C. The mixture was stirred at 0–20°C for 1 hour. The reaction was terminated by adding H2O (50 mL) and extracted with DCM (30 mL). The organic layer was washed with H2O (40 mL x 2) and brine (40 mL), then dried with Na2SO4 and concentrated to obtain the residue. The residue was purified by column chromatography (SiO2, DCM:MeOH = 100:1 to 10:1) to obtain Compound 4 (2.6 g, 2.33 mmol, yield 91.77%, purity 93%) as a pale yellow solid. LCMS: RT = 0.510 min, m / z 1038.5 [M+H] + .

[0749] (Step 4) TFA (9.21 g, 80.77 mmol, 6 mL, 33.54 eq) was added to the solution of Compound 4 (2.5 g, 2.41 mmol, 1 eq) in DCM (30 mL) at 20°C. The mixture was stirred at 20°C for 0.5 hours. The residue was purified by prep-HPLC (column: Phenomenex luna C18 250*50 mm*10 um; mobile phase: [water(TFA)-ACN]; gradient: 15%-45% B over 20 min) to obtain Compound 5 (1.6 g, 1.52 mmol, yield 63.15%, TFA) as a pale yellow solid. LCMS: RT = 0.436 min, m / z 938.4 [M+H] + .

[0750]

[0751] VA-PAB-Ispinesib synthesis

[0752] VA-PAB-Ispinesib, such as compound 5, was synthesized according to the reaction scheme below.

[0753]

[0754]

[0755]

[0756]

[0757] (Step 1) A mixture of Compound 1 (4.9 g, 16.99 mmol), (4-aminophenyl)methanol (2.50 g, 20.29 mmol), and EEDQ (6.27 g, 25.36 mmol) in DCM (50 mL) and MeOH (25 mL) was stirred at 25°C for 1 hour. The reaction mixture was concentrated under reduced pressure to remove the solvent. The resulting crude product was triturated with ethyl acetate:PE=1:1 (150 mL) at 25°C for 30 minutes to obtain Compound 2 (5.56 g, 13.73 mmol, yield 80.78%, purity 97.15%) as a white solid. LCMS: RT = 0.514 min, m / z 416.3 [M+Na] + .

[0758] (Step 2) Bis(4-nitrophenyl)carbonate (6.26 g, 20.59 mmol) and DIPEA (8.87 g, 68.62 mmol, 11.95 mL) were added to a solution of Compound 2 (5.4 g, 13.72 mmol) in DMF (20 mL). The mixture was stirred at 25°C for 1 hour. The reaction mixture was diluted with H2O (200 mL) and filtered to obtain a yellow oil. The resulting residue was triturated with ethyl acetate:PE = 1:2 (200 mL) at 25°C for 30 minutes to obtain Compound 3 (6.3 g, 11.08 mmol, yield 80.72%, purity 98.22%) as a white solid. LCMS: RT = 0.632 min, m / z 559.2 [M+H] + .

[0759] (Step 3) DIPEA (299.86 mg, 2.32 mmol, 404.12 μL) and N-(3-aminopropyl)-N-[(1R)-1-(3-benzyl-7-chloro-4-oxo-quinazolin-2-yl)-2-methyl-propyl]-4-methyl-benzamide (400 mg, 773.60 μmol) were added to a solution of Compound 3 (432.00 mg, 773.39 μmol) in DMF (8 mL). The mixture was stirred at 60°C for 1 hour. The reaction mixture was concentrated by removing the solvent under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, eluent of 0–50% ethyl acetate / petroleum ether gradient @ 50 mL / min) to obtain Compound 4 (700 mg, 738.99 μmol, yield 95.55%, purity 98.87%) as a white solid. LCMS: RT = 0.733 min, m / z 936.3 [M+H] + .

[0760] (Step 4) The mixture of Compound 4 (780 mg, 832.86 μmol) in TFA / DCM (20 mL, 10% TFA in DCM) was stirred at 25°C for 4 hours. The reaction mixture was concentrated by removing the solvent under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0–10% MeOH / Ethyl acetate @ 45 mL / min) to obtain Compound 5 (616 mg, 504.74 μmol, yield 60.60%, purity 87.22%, 2TFA) as a white solid. LCMS: RT = 0.622 min, m / z 836.3 [M+H] + .

[0761]

[0762] VA-CBI dimer analog 1 synthesis

[0763] A VA-CBI dimer analog 1, similar to compound 12, was synthesized according to the reaction scheme below.

[0764]

[0765]

[0766]

[0767]

[0768]

[0769] (Step 1) A mixture of Compound 1 (2 g, 5.76 mmol, 1 eq) and LiOH·H2O (439.29 mg, 10.47 mmol, 1.82 eq) in EtOH (20 mL) and H2O (5 mL) was stirred at 25°C for 4 hours. When the reaction mixture was adjusted to pH 6 with 1 M HCl (1 mL), a solid precipitated from the reaction solution; the suspension was filtered, and the filter cake was concentrated. The residue was purified by prep-HPLC (column: Phenomenex luna C18 150*25 mm*10 um; mobile phase: [water(FA)-ACN]; gradient: 40%-70% B over 10 min) to obtain Compound 2 (0.65 g, 2.04 mmol, yield 35.36%) as a white solid. LCMS: RT = 0.575 min, m / z 264.1 [M-55] + .

[0770] (Step 2) A mixture of Compound 2 (195 mg, 610.69 μmol, 1 eq), Compound 3 (260.00 mg, 472.77 μmol, 7.74e-1 eq), and EDCI (175.50 mg, 915.49 μmol, 1.5 eq) in pyridine (1 mL) was stirred at 25°C for 1 hour. The reaction mixture was concentrated to remove the pyridine. The residue was diluted with H2O (10 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic layer was washed with brine (30 mL), dried with anhydrous Na2SO4, and concentrated under reduced pressure to obtain the residue. The residue was purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, eluent of 0–50% ethyl acetate / petroleum ether gradient @ 45 mL / min) to obtain Compound 4 (0.2 g, 234.95 μmol, yield 38.47%) as a yellow oil. LCMS: RT = 0.720 min, m / z 851.2 [M+H] + .

[0771] (Step 3) TFA (767.50 mg, 6.73 mmol, 500.00 μL, 28.65 eq) was added to a solution of Compound 4 (0.2 g, 234.95 μmol, 1 eq) in DCM (1.5 mL), and the mixture was stirred at 25°C for 1 hour. The reaction mixture was concentrated under reduced pressure and then freeze-dried with water. Compound 5 (0.17 g) was obtained as a yellow solid as the crude product and was used in the next step without further purification. LCMS: RT = 0.645 min, m / z 795.1 [M+H] + .

[0772] (Step 4) (COCl)2 (22.35 mg, 176.07 μmol, 15.41 μL, 2 eq) and DMF (643.48 μg, 8.80 μmol, 6.77e-1 μL, 0.1 eq) were added to a solution of Compound 5 (70 mg, 88.03 μmol, 1 eq) in DCM (2 mL), and the mixture was stirred for 1 hour at 20-25°C under a nitrogen atmosphere. The reaction mixture was concentrated under reduced pressure to obtain the residue. As a result, Compound 6 (70 mg, crude) was obtained as a brown solid and used in the next step without further purification.

[0773] (Step 5) Compound 7 (52 mg, 165.99 μmol, 1.93 eq, TFA) and DIPEA (44.48 mg, 344.16 μmol, 59.94 μL, 4 eq) were added to a solution of Compound 6 (70 mg, 86.04 μmol, 1 eq) in DMF (1.5 mL), and the mixture was stirred for 1 hour at 20-25°C under a nitrogen atmosphere. The reaction mixture was filtered to obtain the residue. The residue was purified by prep-HPLC (FA, column: Phenomenex Luna C18 150*25mm*10um; mobile phase: [H2O (0.225% FA)-ACN]; gradient: 65%-95% B over 10.0 min) to obtain Compound 8 (60 mg, 61.30 μmol, yield 71.25%, purity 99.756%) as a yellow solid. LCMS: RT = 1.115 min, m / z 976.3 [M+H] + .

[0774] (Step 6) Fe (34.32 mg, 614.52 μmol, 10 eq) and NH4Cl (32.87 mg, 614.52 μmol, 10 eq) were added to a solution of Compound 8 (60 mg, 61.45 μmol, 1 eq) dissolved in a mixed solvent of MeOH (4 mL), THF (4 mL), and H2O (1 mL), and the mixture was stirred at 75°C for 1 hour under a nitrogen atmosphere. The reaction mixture was extracted with DCM (30 mL x 2). The combined organic layer was washed with brine (10 mL), dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. As a result, Compound 9 (60 mg, crude) was obtained as a yellow solid. LCMS: RT = 1.090 min, m / z 946.3 [M+H] + .

[0775] (Step 7) DIPEA (81.94 mg, 633.99 μmol, 110.43 μL, 6 eq) and HATU (160.71 mg, 422.66 μmol, 4 eq) were added to a solution of Compound 10 (115.09 mg, 422.66 μmol, 4 eq) in DMF (2 mL), and the mixture was stirred for 0.5 hours at 20-25°C under a nitrogen atmosphere. Subsequently, Compound 9 (100 mg, 105.66 μmol, 1 eq) was added, and the mixture was stirred for 12 hours at 20-25°C under a nitrogen atmosphere. The reaction mixture was concentrated under reduced pressure to obtain the residue. The residue was purified by prep-HPLC (FA, column: Phenomenex Luna C18 150*25mm*10um; mobile phase: [H2O (0.225% FA)-ACN]; gradient: 57%-87% B over 10.0 min) to obtain Compound 11 (90 mg, 45.95 μmol, yield 43.49%, purity 61.301%) as a yellow solid. LCMS: RT = 1.116 min, m / z 1200.5 [M+H] + .

[0776] (Step 8) Phenylsilane (16.22 mg, 149.92 μmol, 18.50 μL, 2 eq) and palladium;triphenylphosphane (8.66 mg, 7.50 μmol, 0.1 eq) were added to a solution of Compound 11 (90 mg, 74.96 μmol, 1 eq) in DMF (2 mL), and the mixture was stirred for 2 hours at 20-25°C under a nitrogen atmosphere. The reaction mixture was filtered to obtain the residue. The residue was purified by prep-HPLC (FA, column: Phenomenex Luna C18 150*25mm*10um; mobile phase: [H2O (0.225% FA)-ACN]; gradient: 33%-63% B over 10.0 min) to obtain Compound 12 (50 mg, 43.92 μmol, yield 58.59%, purity 98.085%) as a yellow solid. LCMS: RT = 0.967 min, m / z 1117.9 [M+H] + .

[0777]

[0778] VA-CBI dimer analog 2 synthesis

[0779] A VA-CBI dimer analog 2, similar to compound 18, was synthesized according to the reaction scheme below.

[0780]

[0781]

[0782]

[0783]

[0784]

[0785]

[0786]

[0787]

[0788]

[0789]

[0790] (Step 1) TFA (10.75 g, 94.24 mmol, 7 mL, 33.29 eq) was added to a solution of Compound 1 (1.2 g, 2.83 mmol, 1 eq) in DCM (14 mL) and stirred for 1 hour at 20-25°C under a nitrogen atmosphere. The reaction mixture was concentrated under reduced pressure to obtain the residue. As a result, Compound 2 (1.24 g, crude, TFA) was obtained as a gray solid and used in the next step without further purification.

[0791] (Step 2) DIPEA (1.83 g, 14.16 mmol, 2.47 mL, 5 eq) was added to a solution of Compound 2 (1.24 g, 2.83 mmol, 1 eq, TFA) and Compound 3 (1.65 g, 7.98 mmol, 2.82 eq) in DCM (15 mL) at 0-5°C. The mixture was stirred for 0.5 hours at 0-25°C under a nitrogen atmosphere. The reaction was terminated by adding H2O (20 mL) to the reaction mixture, and the mixture was extracted with DCM (20 mL x 2). The combined organic layer was washed with brine (10 mL x 2), dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate = 10 / 1 to 5 / 1) to obtain Compound 4 (1.2 g, 2.33 mmol, yield 82.13%, purity 95.756%) as a yellow solid. LCMS: RT = 0.980 min, m / z 494.3 [M+H] + .

[0792] (Step 3) LiOH·H2O (305.79 mg, 7.29 mmol, 3 eq) was added to a solution of Compound 4 (1.2 g, 2.43 mmol, 1 eq) in THF (12 mL) and H2O (12 mL), and the mixture was stirred at 20–25°C for 12 hours. The reaction mixture was adjusted to pH < 4 with an aqueous HCl solution (1 N) and then extracted with ethyl acetate (30 mL x 2). The combined organic layer was washed with brine (10 mL), dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. As a result, Compound 5 (1.13 g, crude) was obtained as a yellow solid and used in the next step without further purification. LCMS: RT = 0.903 min, m / z 466.3 [M+H] + .

[0793] (Step 4) Ammonia;formic acid (1.53 g, 24.25 mmol, 10 eq) and Pd / C (0.5 g, 2.43 mmol, 10% purity, 1.00 eq) were added to a solution of compound 5 (1.13 g, 2.43 mmol, 1 eq) in THF (100 mL) and H2O (2 mL), and the mixture was stirred for 1 hour at 20-25°C under a nitrogen atmosphere. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The residue was extracted with ethyl acetate (50 mL), the organic layer was dried with Na2SO4, and then filtered and concentrated under reduced pressure. The residue was triturated with PE / ethyl acetate (30 mL, 10:1) and filtered to obtain Compound 6 (900 mg, 2.36 mmol, yield 97.49%, purity 98.734%) as a gray solid. LCMS: RT = 0.783 min, m / z 376.3 [M+H] + .

[0794] (Step 5) (COCl)2 (526.84 mg, 4.15 mmol, 363.34 μL, 2 eq) and DMF (15.17 mg, 207.53 μmol, 15.97 μL, 0.1 eq) were added to a solution of Compound 6 (780 mg, 2.08 mmol, 1 eq) in DCM (15 mL), and the mixture was stirred for 0.5 hours at 20-25°C under a nitrogen atmosphere. The reaction mixture was concentrated under reduced pressure to obtain the residue. As a result, Compound 7 (818 mg, crude) was obtained as a yellow solid and used in the next step without further purification.

[0795] (Step 6) DIPEA (1.07 g, 8.30 mmol, 1.45 mL, 4 eq) and Compound 8 (660.36 mg, 2.28 mmol, 1.1 eq) were added to a solution of Compound 7 (818 mg, 2.07 mmol, 1 eq) in DCM (20 mL) at 0-5°C, and stirred for 20 minutes at 20-25°C under a nitrogen atmosphere. After terminating the reaction by adding H2O (20 mL) to the reaction mixture, the mixture was extracted with DCM (20 mL x 2). The combined organic layer was washed with brine (20 mL), dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate / DCM = 4 / 2 / 1 to 2 / 2 / 1) to obtain Compound 9 (800 mg, 803.39 μmol, yield 38.72%, purity 64.994%) as a gray solid. LCMS: RT = 1.017 min, m / z 647.3 [M+H] + .

[0796] (Step 7) DIPEA (399.38 mg, 3.09 mmol, 538.25 μL, 2.5 eq) and Compound 10 (373.73 mg, 1.85 mmol, 1.5 eq) were added to a solution of Compound 9 (800 mg, 1.24 mmol, 1 eq) in DCM (20 mL), and the mixture was stirred for 1 hour at 20-25°C under a nitrogen atmosphere. After stopping the reaction by adding H2O (20 mL) to the reaction mixture, the mixture was extracted with DCM (20 mL x 2). The combined organic layer was washed with brine (40 mL), dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. As a result, Compound 11 (1 g, crude) was obtained as a yellow oil and used in the next step without further purification. LCMS: RT = 1.078 min, m / z 812.2 [M+H]+.

[0797] (Step 8) DIPEA (318.21 mg, 2.46 mmol, 428.86 μL, 2 eq) and Compound 12 (347.65 mg, 1.85 mmol, 1.5 eq) were added to a solution of Compound 11 (1 g, 1.23 mmol, 1 eq) in DCM (10 mL), and the mixture was stirred at 20-25°C for 0.5 hours. After terminating the reaction by adding H2O (20 mL) to the reaction mixture, the mixture was extracted with DCM (20 mL x 2). The combined organic layer was washed with brine (20 mL), dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate = 2 / 1 to 1 / 1) to obtain Compound 13 (800 mg, 744.72 μmol, yield 60.49%, purity 80.193%) as a yellow solid. LCMS: RT = 1.066 min, m / z 761.3 [M+H-100] + .

[0798] (Step 9) Pd / C (400 mg, 375.87 μmol, 10% purity, 4.05e-1 eq) and ammonia;formic acid (585.57 mg, 9.29 mmol, 10 eq) were added to a solution of Compound 13 (800 mg, 928.66 μmol, 1 eq) in THF (50 mL) and H2O (1 mL), and the mixture was stirred for 3 hours at 20-25°C under a nitrogen atmosphere. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The residue was extracted with ethyl acetate (50 mL), the organic layer was dried with Na2SO4, and then filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate = 2 / 1 to 0 / 1), then purified by prep-HPLC (FA, column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water (FA)-ACN]; gradient: 65%-95% B over 10 min), yielding Compound 14 (550 mg, 632.59 μmol, yield 68.12%, purity 88.716%) as a yellow solid. LCMS: RT = 0.971 min, m / z 771.3 [M+H] + .

[0799] (Step 10) TFA (3.07 g, 26.92 mmol, 2 mL, 46.15 eq) was added to a solution of Compound 14 (450 mg, 583.40 μmol, 1 eq) in DCM (10 mL) and stirred at 20-25°C for 1 hour. After adding H2O (10 mL) to the reaction mixture, the mixture was concentrated under reduced pressure to remove the DCM, and then freeze-dried to obtain the residue. As a result, Compound 15 (460 mg, crude, TFA) was obtained as a yellow solid and used in the next step without further purification.

[0800] (Step 11) DIPEA (302.84 mg, 2.34 mmol, 408.14 μL, 4 eq) was added to a solution of Compound 15 (460 mg, 585.80 μmol, 1 eq, TFA) and Compound 16 (476.73 mg, 878.71 μmol, 1.5 eq) in DMF (5 mL), and the mixture was stirred for 12 hours at 20-25°C under a nitrogen atmosphere. After terminating the reaction by adding H2O (20 mL) to the reaction mixture, the mixture was extracted with DCM (20 mL x 2). The combined organic layer was washed with brine (20 mL), dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by prep-HPLC (FA, column: Phenomenex luna C18 150*40mm*15um; mobile phase: [water (FA)-ACN]; gradient: 58%-88% B over 15 min) to obtain Compound 17 (300 mg, 272.29 μmol, yield 46.48%, purity 97.538%) as an off-white solid. LCMS: RT = 1.091 min, m / z 1074.5 [M+H] + .

[0801] (Step 12) A mixture of Compound 17 (250 mg, 232.63 μmol, 1 eq), Phenylsilane (37.76 mg, 348.95 μmol, 43.06 μL, 1.5 eq), and Pd(PPh3)4 (13.44 mg, 11.63 μmol, 0.05 eq) in DMF (3 mL) was degassed by nitrogen purging three times. Subsequently, the mixture was stirred at 20-25°C for 0.5 hours under a nitrogen atmosphere to obtain Compound 18 (230 mg, crude) as a brown oil in DMF solution. LCMS: RT = 0.931 min, m / z 990.4 [M+H] + .

[0802]

[0803] Synthesis of VA-Duocarmycin analog

[0804] A VA-Duocarmycin analog, such as compound 4, was synthesized according to the reaction scheme below.

[0805]

[0806] (Step 1) EDCI (2.55 g, 13.32 mmol, 3.5 eq) was added to a solution of Compound 1 (3.84 g, 13.32 mmol, 3.5 eq) in DMF (30 mL) and pyridine (902.77 mg, 11.41 mmol, 921.19 μL, 3 eq), and stirred at 0°C for 30 minutes under a nitrogen atmosphere. Subsequently, Compound 2 (1.9 g, 3.80 mmol, 1 eq, HCl) was added, and stirred at 0°C for 0.5 hours. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified by prep-HPLC (column: Phenomenex luna C18 (250*70 mm, 10 µm); mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 25%-48% B over 20.0 min) to obtain Compound 3 (1.3 g, 1.75 mmol, yield 46.08%, purity 98.880%) as a white solid. LCMS: RT = 0.869 min, m / z = 733.3 [M+H] + .

[0807] (Step 2) HCl / dioxane (2 M, 5.42 mL, 6.11 eq) was added to a solution of Compound 3 (1.3 g, 1.77 mmol, 1 eq) in MeOH (5 mL), and the mixture was stirred at 25°C for 1 hour. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The crude product was triturated with ethyl acetate at 25°C for 30 minutes to obtain Compound 4 (900 mg, 1.40 mmol, yield 78.81%, purity 98.293%) as an off-white solid. LCMS: RT = 0.403 min, m / z = 633.3 [M+H]+ .

[0808]

[0809] VA-PAB-MMAE Synthesis

[0810] VA-PAB-MMAE, such as compound 4, was synthesized according to the reaction scheme below.

[0811]

[0812]

[0813]

[0814] (Step 1) TEA (81.52 mg, 805.62 μmol, 112.13 μL, 1.5 eq) and HOBt (108.86 mg, 805.62 μmol, 1.5 eq) were added to a solution of Compound 1 (300 mg, 537.08 μmol, 1 eq) and Compound 2 (385.61 mg, 537.08 μmol, 1 eq) in DMF (5 mL), and the mixture was stirred at 25°C for 12 hours. After adding H2O (20 mL), the mixture was extracted with ethyl acetate (20 mL x 2). The combined organic layer was washed with brine (15 mL x 2), dried with Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (SiO2, Commercial PE: ethyl acetate = 0 / 1) to obtain Compound 3 (510 mg, 448.37 μmol, yield 83.48%) as a white solid. LCMS: RT = 1.027 min, m / z 1137.7 [M+H] + .

[0815] (Step 2) TFA (1.54 g, 13.46 mmol, 1.00 mL, 31.25 eq) was added to a solution of Compound 3 (490 mg, 430.79 μmol, 1 eq) in DCM (3 mL), and the mixture was stirred at 25°C for 0.5 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100*25 mm*10 µm; mobile phase: [H2O (0.225% FA)-ACN]; gradient: 15%-45% B over 35.0 min) to obtain Compound 4 (280 mg, 269.92 μmol, yield 62.66%) as a white solid. LCMS: RT = 2.814 min, m / z 1037.7 [M+H] + .

[0816]

[0817] VA-MMAF Synthesis

[0818] VA-MMAF, such as compound 4, was synthesized according to the reaction scheme below.

[0819]

[0820]

[0821]

[0822] (Step 1) A mixture of Compound 2 (0.35 g, 852.70 μmol, 1.92 eq) and HATU (253.30 mg, 666.19 μmol, 1.5 eq) in DMF (5 mL) was stirred at 25°C for 0.5 hours. Subsequently, Compound 1 (0.35 g, 444.13 μmol, 1 eq) and DIPEA (114.80 mg, 888.25 μmol, 154.72 μL, 2 eq) were added, and the mixture was stirred at 25°C for 1.5 hours. The reaction mixture was diluted with H2O (10 mL) and then extracted with ethyl acetate (10 mL x 3). The combined organic layer was washed with brine (30 mL), dried with anhydrous Na2SO4, and concentrated under reduced pressure to obtain the residue. The residue was purified by flash silica gel column chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, Eluent of 0–100% Ethyl acetate / Commercial hexanes gradient @ 30 mL / min) to obtain Compound 3 (0.43 g, 349.50 μmol, yield 78.69%, purity 95.95%) as a yellow oil. LCMS: RT = 1.151 min, m / z 1180.7 [M+H] + .

[0823] (Step 2) Piperidine (31.01 mg, 364.25 μmol, 35.97 μL, 1 eq) was added to a solution of Compound 3 (0.43 g, 364.25 μmol, 1 eq) in DMF (3 mL), and the mixture was stirred at 25°C for 1 hour. Afterward, the pH was adjusted to 5 using AcOH and concentrated. The resulting residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100*25mm*10um; mobile phase: [H2O(0.225% FA)-ACN]; gradient: 22%-52% B over 35.0 min) to obtain Compound 4 (0.16 g, 166.97 μmol, yield 45.84%) as a white solid. LCMS: RT = 0.515 min, m / z 958.7 [M+H] + .

[0824]

[0825] Synthesis methods 1, 1a, 2, and 3 of Table 2 above (refer to the synthesis method classification item) are as follows.

[0826]

[0827] Synthesis Method 1

[0828] Synthesis method 1 is performed in the order of (1) synthesizing the corresponding branch linker moiety, (2) connecting the corresponding drug moiety (a form including or not including a cleavable linker) to it, (3) connecting the corresponding attacher to it, and (4) deprotecting.

[0829]

[0830] As a representative example of Synthesis Method 1, the specific synthesis process of compound LD-010 is as follows, and the remaining compounds were also prepared in the same way by changing the starting materials.

[0831]

[0832] LD-010 Synthesis

[0833]

[0834]

[0835]

[0836] (Step 1) A mixture of Compound 1 (150 mg, 113.61 μmol) and HATU (56.16 mg, 147.69 μmol) in DMF (2 mL) was stirred at 25°C for 0.5 hours. Compound 2 (69.00 mg, 113.93 μmol) and DIPEA (73.41 mg, 568.03 μmol, 98.94 μL) were added to the mixture and stirred at 25°C for 12 hours. The residue was purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, Eluent of 0–10% Methanol / Ethyl acetate @ 45 mL / min). As a result, the crude product Compound 3 (0.22 g, crude) was obtained in the form of a yellow oil and was used in the next step without further purification. LCMS: RT = 0.592 min, m / z 1909.4 [M+H] + .

[0837] (Step 2) The mixture of Compound 3 (215 mg, 112.68 μmol) in piperidine / DMF (3 mL, 5% in DMF) was stirred at 25°C for 1 hour. The reaction mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Phenomenex luna C18 150*25 mm*10 µm; mobile phase: [water(FA)-ACN]; gradient: 19%-49% B over 10 min) to obtain Compound 4 (70 mg, 40.32 μmol, yield 35.78%, purity 97.1%) as a white solid. LCMS: RT = 0.520 min, m / z 1687.2 [M+H] + .

[0838] (Step 3) A mixture of Compound 4 (65 mg, 38.56 μmol) and (2,5-dioxopyrrolidine-1-yl)6-(2,5-dioxopyrrol-1-yl)hexanoate (12 mg, 38.92 μmol) in DMF (2 mL) was stirred at 25°C for 3 hours. The reaction mixture was concentrated. The residue was purified by prep-HPLC (column: Phenomenex luna C18 150*25 mm*10 µm; mobile phase: [water(FA)ACN]; gradient: 33%-63% B over 10 min) to obtain Compound 5 (70 mg, 37.25 μmol, yield 96.62%) as a white solid. LCMS: RT = 0.635 min, m / z 1880.8 [M+H] + .

[0839] (Step 4) A mixture of Compound 5 (30 mg, 15.97 μmol) in TFA / DCM (1 mL, DCM:TFA=3:1) was stirred at 25°C for 2 hours. The reaction mixture was concentrated at low temperature to remove CH2Cl2. DMF (1 mL) and H2O (0.5 mL) were added to the reaction mixture to precipitate a solid from the reaction solution, and the resulting suspension was filtered. After concentrating the filtrate, it was purified by prep-HPLC (column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water(FA)-ACN]; gradient: 19%-39% B over 11 min) to obtain Compound LD-010 (7.43 mg, 4.08 μmol, yield 25.54%, purity 95.98%, FA) as an off-white solid. LCMS: RT = 0.815 min, m / z 1703.7 [M+H] + .

[0840]

[0841] Synthesis method 1a

[0842] Synthesis method 1a is performed in the order of (1) synthesizing the corresponding branch linker moiety, (2) attaching the corresponding drug moiety (a form including or not including a cleavable linker) to it, (3) hydrolyzing, (4) attaching the corresponding attacher to it, and (5) deprotecting.

[0843]

[0844] As a representative example of synthesis method 1a, the specific synthesis process of compound LD-028 is as follows, and the remaining compounds were also prepared in the same way by changing the starting materials.

[0845]

[0846] LD-028 Synthesis

[0847]

[0848]

[0849]

[0850]

[0851] (Step 1) HATU (225.00 mg, 591.75 μmol, 2.17 eq) was added to a solution of Compound 1 (375.00 mg, 284.01 μmol, 1.04 eq) in DMF (6 mL) and stirred at 25°C for 0.5 hours. Then, Compound 2 (300 mg, 272.24 μmol, 1 eq, TFA) and DIPEA (35.19 mg, 272.24 μmol, 47.42 μL, 1 eq) were added and stirred at 25°C for an additional 0.5 hours. After filtering the reaction mixture, the filtrate was purified by prep-HPLC (FA condition: column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water(FA)-ACN]; gradient: 42%-72% B over 10 min) to obtain Compound 3 (400 mg, 174.65 μmol, yield 64.15%) as a yellow solid. LCMS: RT = 0.591 min, m / z 1146.6 [M / 2+H] + .

[0852] (Step 2) Piperidine (14.87 mg, 174.65 μmol, 1.49 mL, 1 eq) was added to a solution of Compound 3 (400 mg, 174.65 μmol, 1 eq) in DMF (3 mL), and the mixture was stirred at 20°C for 1 hour. After filtering the reaction mixture, the residue was purified by prep-HPLC (FA condition: column: Phenomenex luna C18 150*25 mm* 10 µm; mobile phase: [water (FA)-ACN]; gradient: 22%-52% B over 10 min) to obtain Compound 4 (320 mg, 154.74 μmol, yield 88.60%) as a white solid. LCMS: RT = 0.543 min, m / z 1035.3 [M / 2+H] + .

[0853] (Step 3) LiOH·H2O (0.05 M, 14.51 mL, 10 eq) was added to a solution of Compound 4 (150 mg, 72.53 μmol, 1 eq) in MeOH (14 mL) at 0°C and stirred for 10 minutes at 0°C. TFA was added to the reaction mixture to adjust the pH to 5, and the mixture was freeze-dried to obtain the residue. The residue was purified by prep-HPLC (FA condition: column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water (FA)-ACN]; gradient: 18%-48% B over 10 min) to obtain Compound 5 (90 mg, 46.68 μmol, yield 64.36%) as a white solid. LCMS: RT = 0.513 min, m / z 965.2 [M / 2+H] + .

[0854] (Step 4) (2,5-dioxopyrrolidine-1-yl)6-(2,5-dioxopyrrol-1-yl)hexanoate (6.40 mg, 20.75 μmol, 319.82 μL, 1 eq) and DIPEA (2.68 mg, 20.75 μmol, 26.82 μL, 1 eq) were added to a solution of compound 5 (40 mg, 20.75 μmol, 1 eq) in DMF (1 mL), and stirred at 20°C for 1 hour. After filtering the reaction mixture, the residue was purified by prep-HPLC (FA condition: column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water (FA)-ACN]; gradient: 30%-60% B over 10 min) to obtain Compound 6 (30 mg, 14.14 μmol, yield 68.17%) as a white solid. LCMS: RT = 0.510 min, m / z 1061.9 [M / 2+H] +

[0855] (Step 5) TFA (4.61 g, 40.39 mmol, 3.00 mL, 1616.34 eq) was added to a solution of Compound 6 (53 mg, 24.99 μmol, 1 eq) in DCM (3 mL), and the mixture was stirred at 25°C for 45 minutes. The reaction mixture was concentrated under reduced pressure to obtain the residue. The crude product was triturated with a DMF:MeOH = 1:4 mixed solvent at 20°C for 30 minutes, and the resulting yellow solid was washed with MeOH. As a result, Compound LD-028 (45 mg, 20.80 μmol, yield 83.26%, purity 95.185%, TFA) was obtained as a yellow solid. LCMS: RT = 0.490 min, m / z 1946.6 [M+H] + .

[0856]

[0857] Synthesis Method 2

[0858] Synthesis method 2 is performed in the order of (1) synthesizing the corresponding branched spacer moiety, (2) connecting the corresponding drug moiety (a form including or not including a cleavable linker) to it, (3) connecting the branched linker moiety to it, (4) connecting the corresponding attacher to it, and (5) deprotecting.

[0859]

[0860] As a representative example of synthesis method 2, the specific synthesis process of compound LD-036 is as follows, and the remaining compounds were also prepared in the same way by changing the starting materials.

[0861]

[0862] LD-036 Synthesis

[0863]

[0864]

[0865]

[0866]

[0867] (Step 1) To a solution of Compound 1 (270 mg, 269.92 μmol, 0.921 eq) and Compound 2 (280 mg, 293.21 μmol, 1 eq) in DMF (3 mL), HATU (170 mg, 447.10 μmol, 1.52 eq) and DIPEA (222.60 mg, 1.72 mmol, 0.3 mL, 5.87 eq) were added and stirred at 20-25°C for 2 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC (FA condition: Instrument: ACS-WH-GX-G; column: Phenomenex Luna C18 150*25mm*10um; mobile phase: water (FA)-ACN; B%: 40%-70%, 10 min; Flow rate: 25 ml / min; Column temperature: RT; Wavelength: 220 nm) to obtain Compound 3 (278 mg, 143.50 μmol, yield 48.94%, purity 100%) as a white solid. LCMS: RT = 0.870 min, m / z 1935.1 [M+H] + .

[0868] (Step 2) Pd(PPh3)4 (16 mg, 13.85 μmol, 0.0993 eq) and phenylsilane (22 mg, 203.31 μmol, 25.09 μL, 1.46 eq) were added to a solution of compound 3 (270 mg, 139.37 μmol, 1 eq) in DMF (3 mL), and the mixture was stirred at 20-25°C for 1 hour. The reaction mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC (FA condition: Instrument: ACS-WH-GX-G; column: Phenomenex Luna C18 150*25mm*10um; mobile phase: water (FA)-ACN; B%: 22%-52%, 10 min; Flow rate: 25 ml / min; Column temperature: RT; Wavelength: 220 nm) to obtain Compound 4 (250 mg, 134.90 μmol, yield 96.79%, purity 100%) as a white solid. LCMS: RT = 0.865 min, m / z 1854.0 [M+H] + .

[0869] (Step 3) (2,5-dioxopyrrolidine-1-yl)6-(2,5-dioxopyrrol-1-yl)hexanoate (38.40 mg, 124.56 μmol, 0.962 eq) was added to a solution of compound 4 (240 mg, 129.51 μmol, 1 eq) in DMF (3 mL) and stirred at 20-25°C for 1 hour. The residue was purified by prep-HPLC (FA condition: Instrument: ACS-WH-GX-G; column: Phenomenex Luna C18 150*25mm*10um; mobile phase: water (FA)-ACN; B%: 40%-70%, 10 min; Flow rate: 25 ml / min; Column temperature: RT; Wavelength: 220 nm) to obtain Compound 5 (200 mg, 97.73 μmol, yield 75.47%, purity 100%) as a white solid. LCMS: RT = 0.918 min, m / z 1024.0 [M / 2+H] + .

[0870] (Step 4) TFA (10.03 mg, 87.96 μmol, 6.53 μL, 1 eq) was added to a solution of compound 5 (180 mg, 87.96 μmol, 1 eq) in DCM (1 mL), and stirred at 20-25°C for 1 hour. The reaction mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC (FA condition: Instrument: ACS-WH-GX-G; column: Phenomenex Luna C18 150*25mm*10um; mobile phase: water (FA)-ACN; B%: 20%-50%, 10 min; Flow rate: 25 ml / min; Column temperature: RT; Wavelength: 220 nm) to obtain compound LD-036 (95.54 mg, 49.77 μmol, yield 56.58%, purity 99.82%, FA) as a white solid. LCMS: RT = 0.808 min, m / z 1871.1 [M+H] + .

[0871]

[0872] Synthesis Method 3

[0873] Synthesis method 3 is performed in the order of (1) preparing a compound in which the corresponding branch linker moiety and the corresponding drug moiety (a form including or not including a cleavable linker) are connected based on the synthesis method above, (2) attaching an open type attacher thereto, and (3) deprotecting.

[0874] Here, the specific synthesis process for the open-type attacher connection is as follows:

[0875]

[0876]

[0877]

[0878]

[0879] Among the attachment groups, aminomethyl maleimide was synthesized as follows.

[0880]

[0881] Aminomethyl maleimide synthesis

[0882] Aminomethyl maleimide was synthesized according to the reaction scheme below.

[0883]

[0884]

[0885] (Step 1) Furan-2,5-dione (960.29 mg, 9.79 mmol, 1 eq) was added to a solution of Compound 1 (2 g, 9.79 mmol, 1 eq) in AcOH (30 mL) and stirred at 25 °C for 3 hours. The reaction mixture was concentrated under reduced pressure. The crude product was triturated with DCM (25 mL). The resulting precipitate was recovered by filtration and dried under reduced pressure to obtain Compound 2 (2.94 g, 9.73 mmol, yield 99.31%) as a white solid. LCMS: RT = 0.372 min, m / z 325.0 [M+Na] + .

[0886] (Step 2) TEA (4.36 g, 43.11 mmol, 6 mL, 4.43 eq) and DMA (4 mL) were added to a solution of Compound 2 (2.94 g, 9.73 mmol, 1 eq) and 4A molecular sieve (4A MS, 2 g, 1.00 eq) in toluene (50 mL), and the mixture was stirred at 125°C for 16 hours under a nitrogen atmosphere. The reaction mixture was concentrated under reduced pressure. Subsequently, the reaction mixture was diluted by adding a 10% aqueous citric acid solution (30 mL) and extracted with EtOAc (15 mL x 3). The combined organic layer was washed with brine (10 mL), dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Phenomenex luna C18 (250*70mm, 10 um); mobile phase: [water(FA)-ACN]; gradient: 15%-45% B over 15 min) to obtain compound 3 (600 mg, 2.11 mmol, yield 21.70%) in the form of a yellow solid.

[0887] (Step 3) DCC (54.44 mg, 263.84 μmol, 53.37 μL, 1.5 eq) was added to a solution of Compound 3 (50 mg, 175.89 μmol, 1 eq) and 2,3,4,5,6-Pentafluorophenol (48.56 mg, 263.84 μmol, 1.5 eq) in DCM (0.5 mL), and the mixture was stirred at 25°C for 0.5 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative TLC (Petroleum ether / Ethyl acetate = 4 / 1) using silica gel to obtain Compound 4 (70 mg, 152.34 μmol, yield 87%, purity 98%) as a yellow solid. LCMS: RT = 0.602 min, m / z 473.1 [M+Na] + .

[0888]

[0889] As a representative example of synthesis method 3, the specific synthesis process of compound LD-063 is as follows, and the remaining compounds were also prepared in the same way by changing the starting materials.

[0890]

[0891] LD-063 Synthesis

[0892]

[0893]

[0894]

[0895]

[0896] (Step 1) HATU (5.02 g, 13.21 mmol, 1.2 eq) was added to a solution of Compound 1 (3.27 g, 11.01 mmol, 1 eq) in DMF (50 mL) and stirred at 25°C for 0.5 hours. Then, tert-butyl 3-aminopropanoate hydrochloride (2 g, 11.01 mmol, 1 eq) and DIPEA (2.85 g, 22.02 mmol, 3.84 mL, 2 eq) were added and stirred further at 25°C for 0.5 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, eluent of 0–50% ethyl acetate / petroleum ether gradient @ 60 mL / min) to obtain Compound 2 (2.5 g, 5.89 mmol, yield 53.49%) as a colorless oil. LCMS: RT = 0.823 min, m / z 369.3 [M-tBu+H] + .

[0897] (Step 2) TFA (7.68 g, 67.31 mmol, 5 mL, 11.43 eq) was added to the solution of Compound 2 (2.5 g, 5.89 mmol, 1 eq) in DCM (25 mL), and the mixture was stirred at 25°C for 12 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by prep-HPLC (FA condition; column: Phenomenex luna C18 (250*70 mm, 10 µm); mobile phase: [water(FA)-ACN]; gradient: 35%-65% B over 20 min) to obtain Compound 3 (2 g, 5.43 mmol, yield 92.18%) as a white solid. LCMS: RT = 0.717 min, m / z 369.4 [M+H] + .

[0898] (Step 3) DCC (252.04 mg, 1.22 mmol, 247.10 μL, 1.5 eq) was added to a solution of Compound 3 (300 mg, 814.37 μmol, 1 eq) and 2,3,4,5,6-Pentafluorophenol (224.84 mg, 1.22 mmol, 1.5 eq) in DCM (4 mL) and DMF (1 mL), and the mixture was stirred at 25°C for 0.5 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent. The crude product was triturated with an EA:MeOH = 1:1 (1 mL) mixed solvent at 25°C for 5 minutes. As a result, Compound 4 (280 mg, 523.92 μmol, yield 64.33%) was obtained as a white solid. LCMS: RT = 0.876 min, m / z 535.2 [M+H] + .

[0899] (Step 4) DIPEA (34.50 mg, 266.94 μmol, 46.50 μL, 3 eq) was added to a solution of Compound E-(GGSG)3 (150 mg, 88.98 μmol, 1 eq) and Compound 4 (57 mg, 106.66 μmol, 1.2 eq) in DMF (1.5 mL) and stirred at 25°C for 1 hour. After filtering the reaction mixture, the residue was purified by prep-HPLC (TFA condition; column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water (TFA)-ACN]; gradient: 30%-60% B over 10 min) to obtain Compound 5 (155 mg, 76.12 μmol, yield 85.55%) as a yellow solid. LCMS: RT = 0.840 min, m / z 1018.3 [M / 2+H] + .

[0900] (Step 5) Piperidine (7.50 mg, 88.08 μmol, 8.70 μL, 1.2 eq) was added to a solution of Compound 5 (150 mg, 73.67 μmol, 1 eq) in DMF (1 mL), and the mixture was stirred at 25°C for 2 hours. After filtering the reaction mixture, the residue was purified by prep-HPLC (TFA condition; column: Phenomenex luna C18 150*25 mm*10 µm; mobile phase: [water (TFA)-ACN]; gradient: 10%-40% B over 10 min) to obtain Compound 6 (75 mg, 27.70 μmol, yield 37.60%, purity 67%) as a yellow solid. LCMS: RT = 0.724 min, m / z 1813.5 [M+H] + .

[0901] (Step 6) A mixture of Compound 6 (48 mg, 26.46 μmol, 1 eq) and Furan-2,5-Dione (4.80 mg, 48.95 μmol, 1.85 eq) in AcOH (0.5 mL) was stirred at 25°C for 12 hours. The solvent was removed from the reaction mixture by nitrogen purging. The residue was purified by prep-HPLC (TFA condition; column: Phenomenex C18 75*30 mm*3 µm; mobile phase: [water (TFA)-ACN]; gradient: 15%-45% B over 40 min) to obtain Compound 7 (20 mg, 10.46 μmol, yield 39.53%) as a yellow solid. LCMS: RT = 0.755 min, m / z 1910.2 [M+H] + .

[0902] (Step 7) TFA (767.50 mg, 6.73 mmol, 500.00 μL, 514.78 eq) was added to a solution of Compound 7 (25 mg, 13.08 μmol, 1 eq) in DCM (0.5 mL) and stirred at 25°C for 0.5 hours. The solvent was removed from the reaction mixture by nitrogen purging. The residue was purified by prep-HPLC (TFA condition; column: UniSil 3-100 C18 UItra (150*25mm*3um); mobile phase: [water (TFA)-ACN]; gradient: 8%-38% B over 40 min) to obtain Compound LD-063 (3.61 mg, 1.84 μmol, yield 14.07%, purity 88.47%) in the form of a yellow gum. LCMS: RT = 0.677 min, m / z 1734.7 [M+H] + .

[0903]

[0904] Example 2: Preparation of a Targeting Substance-Drug Conjugate

[0905] An antibody-drug conjugate (ADC) was prepared as a targeting substance drug conjugate by conjugating the linker-drug conjugate of Example 1 with an antibody. The method for preparing the antibody-drug conjugate involved reducing the cysteine-cysteine ​​bond of the antibody, site-specifically conjugating the linker-drug conjugate of Example 1 to the cysteine ​​residue in an optimized conjugation environment, purifying by ion exchange, and then replacing it with a formulation buffer. The specific preparation method is as follows.

[0906]

[0907] 2-1. Antibody Reduction

[0908] The antibodies used were trastuzumab, cetuximab, rituximab, sacituzumab, polatuzumab, tisotumab, and izalontamab.

[0909] A. Each antibody was diluted using PBS 6.0 / EDTA buffer (10 mM phosphate buffer pH 6.0, 137 mM NaCl, 5 mM EDTA). To minimize agglutination during the reduction reaction and ensure reduction efficiency, the antibodies were diluted to 10 mg / mL or less.

[0910] B. 1 M dipotassium hydrogen-phosphate was added so that the final concentration of dipotassium hydrogen-phosphate was 0.46 M.

[0911] C. The concentration of the diluted antibody was measured, and 10 mM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) was added to reach 7.8 to 10 times the antibody molar concentration. Depending on the antibody, 10 mM TCEP was added to reach a molar concentration of up to 20 times the antibody molar concentration.

[0912] D. The mixture was thoroughly mixed by gentle pipetting, and the reaction was allowed to stand at 25°C or room temperature for 2 to 15 hours.

[0913]

[0914] 2-2. TCEP Removal and Buffer Swap

[0915] A. During the reduction reaction, 15 mL of triple-distilled water was added to an Amicon tube 10 kDa, 15 mL (10 kDa MWCO, 15 mL sample volume, centrifugal concentrator), and the membrane was activated by centrifuging at 3,000xg for 10 minutes. After centrifugation, the remaining triple-distilled water was removed, and 15 mL of PBS 6.0 / EDTA buffer was added. After centrifugation at 3,000xg for 10 minutes, the remaining PBS 6.0 / EDTA buffer was removed to create the same buffer environment as the antibody being reduced.

[0916] B. The antibody after the reduction reaction was completed was loaded into an Amicon tube 10 kDa, 15 mL, and the sample was concentrated to 1 mL or less by centrifugation at 3,000xg. At this time, the number of Amicon tubes used was set so that the total amount of antibody loaded per unit volume for an Amicon tube 10 kDa, 15 mL did not exceed 40 mg, and the concentration step may be omitted when the amount of antibody after the reduction reaction was completed was 1 mL or less.

[0917] C. 14 times the volume of PBS 6.0 / EDTA buffer was added to an antibody concentrated to 1 mL or less, gently mixed with a pipette, and centrifuged at 3,000xg for 30 minutes. This process was repeated 3 times to perform buffer replacement with a dilution factor of more than 3,000 times.

[0918]

[0919] 2-3. Joining

[0920] A. The linker-drug conjugate of Example 1 was dissolved in DMSO so that the concentration of the linker-drug conjugate was 10 mM.

[0921] B. The antibody, after TCEP removal and buffer replacement were completed in Item 2-2, was recovered, and its concentration was measured. If the concentration was greater than 9.5 mg / mL and less than 10.5 mg / mL, conjugation was performed; if it was 9.5 mg / mL or less, further concentration was performed; and if it was 10.5 mg / mL or more, it was diluted to 10 mg / mL (concentration range allowed by 5%) using PBS 6.0 / EDTA buffer. If significant aggregation occurred during the conjugation process, the concentration was lowered to 1 mg / mL.

[0922] C. The molar concentration of the linker-drug conjugate can be 10 to 40 times that of the antibody, depending on the degree of conjugation reaction with the antibody, and the corresponding optimal reaction ratio was determined experimentally. DMSO was added to the antibody with the concentration adjusted in B and gently mixed with a pipette. At this time, the total amount of DMSO was set to fall within the final 10% to 15% (v / v) range of the total reaction volume, including the amount of DMSO in the linker-drug conjugate prepared in B. After thoroughly mixing the antibody and DMSO, 10 mM linker-drug conjugate was added and gently mixed with a pipette. At this stage, some precipitation may occur depending on the solubility of the linker-drug conjugate, but since it dissolves as the reaction proceeds, the process was moved to the next step.

[0923] D. When the reaction volume was less than 1 mL, the mixture was mixed in a microcentrifuge and stirred at 25°C or room temperature for 2 hours; when the volume was 1 mL or more, the mixture was mixed in a 15 mL conical tube, a Biotage magnetic stir bar was added, and the tube was mounted on a magnetic hot-plate stirrer, MR Hei-Tec, and the reaction was carried out at 700 rpm for 2 hours.

[0924]

[0925] 2-4. Quenching and Removal of Residual Linker-Drug Conjugate

[0926] A. Once conjugation was complete, 10 mM NAC (N-acetyl-L-cysteine), which is twice the molar equivalent of the linker-drug conjugate added in item 2-3, was added and the reaction was carried out by stirring for 20 minutes under the same conditions as conjugation.

[0927] B. Preparations were made to perform cation ion exchange (CEX) to remove the free linker-drug conjugate while the quenching reaction was in progress. For the cation ion exchange (CEX), Pierce™ strong cation exchange spin columns were used; Mini was used when the antibody amount was 4 mg or less, and Maxi was used when the amount was greater than 4 mg and 80 mg or less.

[0928] Depending on the type used, either C. Maxi or Mini, 400 μL (Mini) or 5 mL (Maxi) of 25 mM sodium acetate buffer, pH 5.5 was added to the Pierce™ strong cation exchange spin columns, and centrifuged at 2,000xg or 500xg for 5 minutes, respectively. The permeated buffer was removed.

[0929] D. For each sample after quenching was completed, 7 times the volume of 25 mM sodium acetate buffer, pH 5.5 was added to the sample to adjust the pH to 5.5.

[0930] Samples prepared in E.D. were added to the corresponding column (Mini: max. 400 μL, Maxi: max. 15 mL) and centrifuged at 500xg or 2,000xg for 5 minutes, respectively. If the sample volume exceeded the maximum allowable volume, the centrifugation process was repeated until all samples were processed.

[0931] F. Once sample loading was complete, 400 μL (Mini) or 5 mL (Maxi) of 25 mM sodium acetate buffer, pH 5.5 was added to remove any remaining glass linker-drug conjugate in the column, and the column was centrifuged at 500xg or 2,000xg for 5 minutes, respectively.

[0932] H. Mounted in a new collection tube, 400 μL (Mini) or 5 mL (Maxi) of 25 mM sodium acetate buffer, pH 5.5 with 400 mM NaCl was added depending on the type used (Mini or Maxi), and the ADC adsorbed on the column was eluted by centrifuging at 500xg or 2,000xg for 5 minutes, respectively.

[0933]

[0934] 2-5. Substitution with Formulation Buffer

[0935] A. Depending on the allowable volume of the Amicon tube (0.5 mL or 15 mL), 0.5 mL or 15 mL of triple-distilled water was added, respectively, and the mixture was centrifuged at 2,999 xg or 8,000 xg for 10 minutes. After removing the triple-distilled water, 20 mM histidine acetate, 240 mM sucrose, and pH 5.5 were added, and the mixture was centrifuged under the same conditions. The inside of the membrane filter and the permeated buffer were removed.

[0936] B. The ADC eluted in item 2-4 was loaded into an Amicon tube and concentrated by centrifugation at 8,000xg (Amicon tube 10 kDa, 0.5 mL) or 2,999xg (Amicon tube 10 kDa, 15 mL) for 10 minutes. At this time, the number of Amicon tubes used was set so that the total amount of antibody loaded per unit volume did not exceed 40 mg for an Amicon tube 10 kDa, 15 mL.

[0937] C. Concentration was carried out under the same conditions until the volume reached 50 μL (Amicon tube 10 kDa, 0.5 mL) or 1.5 mL (Amicon tube 10 kDa, 15 mL).

[0938] D. Add 9 times the sample volume of histidine acetate, 240 mM sucrose, and pH 5.5 to the concentrated ADC and gently mix the ADC adsorbed on the membrane by pipetting, then centrifuge at 8,000xg (Amicon tube 10 kDa, 0.5 mL) or 2,999xg (Amicon tube 10 kDa, 15 mL) for 30 minutes to replace the buffer.

[0939] E. The substitution process was repeated a total of three times to ensure that the concentration of NaCl remained at 1 mM or less, and if the volume of the sample exceeded 1.5 mL or 50 μL after centrifugation, additional centrifugation was performed to concentrate the sample to that volume.

[0940] F. The concentration of the substituted sample was measured, and to reach the target concentration suitable for the application, it was diluted with histidine acetate, 240 mM sucrose, and pH 5.5 and filtered under sterile conditions through a 0.2 μm filter. The completed sample was stored in a -80℃ freezer.

[0941]

[0942] 2-6. ADC Quality Evaluation

[0943] The quality of the ADC was analyzed for DAR, purity, and HMW (high molecular weight species) content using RP-LC / MS (reversed-phase liquid chromatography and mass spectrometry) and SE-HPLC (Size exclusion-high-performance liquid chromatography) analysis methods, respectively.

[0944] A. To evaluate the DAR of the ADC, RP-LC / MS (reversed-phase liquid chromatography and mass spectrometry) analysis was performed using a Waters BioResolve RP mAb polyphenyl column. 3 μL of a sample diluent, prepared by mixing Mobile Phase A and Mobile Phase B at a ratio of 70:30 (v / v), was injected into the column. The flow rate was 0.3 mL / min, and 0.05% TFA water was used for Mobile Phase A and 0.05% TFA acetonitrile for Mobile Phase B. Concentration gradients of Mobile Phase B at 30, 32, 34, 37, 50, and 80% were applied from 0 to 21 minutes, and the ratio of Mobile Phase B was reduced to 30% from 21 to 25 minutes to revert to the initial analysis conditions. For MS analysis, light and heavy chains were analyzed using HMR mode Full MS - SIM analysis. For light chains, a scan range of 1,000–4,000 [m / z], a Max Inject Time of 200 ms, and a Resolution of 140,000 were applied, while for heavy chains, a scan range of 1,500–4,500 [m / z], a Max Inject Time of 100 ms, and a Resolution of 17,500 were applied. The Mass TIC (total ion chromatogram) profiles analyzed under these conditions were deconvolved using Thermo Bio Pharma Finder 5.2 to determine the DAR values ​​of the ADC substances and the molecular weights of the light and heavy chains. For cross-validation, DAR values ​​were calculated using UV profiles.

[0945] B. SE-HPLC (Size exclusion-high-performance liquid chromatography) analysis (Waters, XBridge Protein BEH SEC column) was used to analyze the purity and high molecular weight species (HMW) of the ADC. 10 μL of the Mobile Phase was mixed with the ADC sample (approx. 2 mg / mL) at a 1:1 ratio and injected into the column. The Mobile Phase was flowed isocratically under UV 280 nm for 30 minutes. The flow rate was 0.6 mL / min, and 30 mM sodium phosphate, 150 mM NaCl, and 10% isopropyl alcohol were used as the Mobile Phase.

[0946]

[0947] The analysis results are as shown in Table 3 (conjugation results using Trastuzumab) and Table 4 (conjugation results for various antibodies) below, and the ADC quality evaluation indicators are as follows. Table 3 shows the results using Trastuzumab as the antibody, and Table 4 shows the results using LD-010 and various antibodies other than Trastuzumab.

[0948] - RP-LC / MS DAR target level ≥ 7

[0949] - RP-LC / MS Mass(Da): Information only

[0950] - SE-HPLC Analysis Results Target Level HMWs (%) < 5%

[0951]

[0952] Meanwhile, in Tables 3 and 4 below, ppm means the following.

[0953] ppm = (Measured (Da) - Theoretical (Da)) / Theoretical (Da) * 1,000,000

[0954] [Correction pursuant to Rule 26 05.02.2026]

[0955] [Correction pursuant to Rule 26 05.02.2026]

[0956] [Correction pursuant to Rule 26 05.02.2026]

[0957] [Correction pursuant to Rule 26 05.02.2026]

[0958]

[0959] [Correction pursuant to Rule 26 05.02.2026]

[0960]

[0961] In addition, for comparison with the ADC according to the embodiment of the present invention, an ADC without the branched spacer according to the present invention and an ADC with the branched spacer according to the present invention but with PEG applied instead of the hydrophilicity-imparting group according to the present invention were manufactured. The former was named using "C" as in TA-LD-CO1, and the latter was named using "P" as in TA-LD-P01, and their structures are shown in Table 5 below.

[0962]

[0963] For an ADC without the branch spacer according to the present invention, the quality of the ADC was evaluated in the same manner as above and compared with the ADC according to an embodiment of the present invention, and the results are shown in FIG. 1. As shown in FIG. 1, it was confirmed that when the branch spacer according to the present invention is applied, the purity increases and the HMW decreases.

[0964]

[0965] Experimental Example 1: Evaluation of Hydrophilicity

[0966] The hydrophilicity of the ADC according to an embodiment of the present invention was evaluated as follows.

[0967]

[0968] HI-HPLC (Hydrophobic Interaction High Performance Liquid Chromatography) analysis was performed to compare hydrophilicity (Thermo Fisher, MAbPac TM (using HIC-butyl). HI-HPLC is a technique that separates substances based on differences in hydrophobicity; substances with higher hydrophilicity are characterized by faster elution. The retention time of each ADC was determined by measuring absorbance at UV 280 nm, and the relative retention time (RRT) was calculated by comparing it to the control, trastuzumab (Herceptin). Hydrophilicity was compared using the RRT values ​​of each ADC. An ADC was evaluated as having superior hydrophilicity as its RRT relative to trastuzumab was closer to 1. The evaluation results are shown in Table 5 below, where "-CA" indicates "-Carboxylic acid" and "PEG n " means that n ethylene glycol units are connected.

[0969] Evaluation TargetSpacer Branch Cleavable Linker DrugHI-HPLC RRTT-DxdN / AN / AGly-Gly-Phe-GlyDeruxtecan2.212HerceptinN / AN / AN / AN / A1.000TA-LD-C01N / AN / APhe-GlyExatecan2.317TA-LD-029EGGTG1-CAPhe-GlyExate can1.596TA-LD-011EGGSG1-CAPhe-GlyExatecan1.519TA-LD-012EGGSG2-CAPhe-GlyExatecan1.336TA-LD-013EGGSG3-CAPhe-GlyExatecan1.249TA-LD-P01EPEG 12-CAPhe-GlyExatecan1.887TA-LD-025KGGSG3-AcetylPhe-GlyExatecan1.394TA-LD-022KGGSS3-AcetylPhe-GlyExatecan1.459TA-LD-023KGSSS3-AcetylPhe-GlyExatecan1.333TA-LD-P02KPEG 12 -AcetylPhe-GlyExatecan2.302TA-LD-C02N / AN / AVal-AlaExatecan1.731TA-LD-008EGGSG1-CAVal-AlaExatecan1.281TA-LD-009EGGSG2-CAVal-AlaExatecan1.243TA-LD-010EGGSG3-CAVal-AlaExatecan1.177TA-LD-050EGSSS3-CAVal-AlaExatecan1.132TA-LD-056EGGSG4-CAVal-AlaExatecan1.111TA-LD-P03EPEG 12 -CAVal-AlaExatecan1.618TA-LD-P04EPEG 24 -CAVal-AlaExatecan1.832TA-LD-045KGGSG3-AcetylVal-AlaExatecan1.218TA-LD-047KGGSS3-AcetylVal-AlaExatecan1.176TA-LD-049KGSSS3-AcetylVal-AlaExatecan1.187TA-LD-P05KPEG 24 -AcetylVal-AlaExatecan2.102TA-LD-C03N / AN / AVal-CitExatecan1.512TA-LD-005EGGSG1-CAVal-CitExatecan1.237TA-LD-006EGGSG2-CAVal-CitExatecan1.176TA-LD-007EGGSG3-CAVal-CitExatecan1.132TA-LD-044KGGSG3-AcetylVal-CitExatecan1.177TA-LD-046KGGSS3-AcetylVal-CitExatecan1.144TA-LD-P06KPEG 24-AcetylVal-CitExatecan1.976TA-LD-C04N / AN / Aβ-GlucuronideExatecan1.181TA-LD-027EGGSG2-CAβ-GlucuronideExatecan1.074TA-LD-028EGGSG3-CAβ-GlucuronideExatecan1.047TA-LD-055EGSSS3-CAβ-GlucuronideExatecan1.014TA-LD-037KGGSG1-Acetylβ-GlucuronideExatecan1.166TA-LD-039KGGSG3-Acetylβ-GlucuronideExatecan1.091TA-LD-054KGSSS3-Acetylβ-GlucuronideExatecan1.07TA-LD-C05N / AN / AN / AMMAF2.834TA-LD-017EGGSG1-CAN / AMMAF2.041TA-LD-018EGGSG2-CAN / AMMAF1.840TA-LD-019EGGSG3-CAN / AMMAF1.688TA-LD-034KGGSG1-AcetylN / AMMAF2.162TA-LD-035KGGSG2-AcetylN / AMMAF1.933TA-LD-P07EPEG 24 -CAN / AMMAF2.216TA-LD-P08KPEG 24-AcetylN / AMMAF2.352TA-LD-057EGGSG3-CAPhe-GlyDuocarmycin analog0.981TA-LD-058EGGSG3-CAPhe-GlySeco-DUBA analog1.928TA-LD-059KGGSG3-Acetylβ-GlucuronideMMAE2.937TA-LD-060EGGSG3-CAβ-GlucuronideMMAE1.463TA-LD-061EGGSG3-CAVal-AlaIspinesib3.365TA-LD-073KGGSG3-AcetylPhe-Gly-PABPBD dimer analog 13.090TA-LD-074KGGSG3-AcetylPhe-Gly-PABPBD dimer analog 22.521TA-LD-075EGGSG3-CAPhe-Gly-PABPBD dimer analog 22.629TA-LD-077EGGSG3-CAVal-Ala-PABPBD dimer analog 13.045TA-LD-078EGGSG3-CAVal-Ala-PABPBD dimer analog 21.403TA-LD-066KGGSG3-AcetylVal-AlaExatecan1.166TA-LD-069KGGSG3-AcetylVal-AlaExatecan1.157TA-LD-C08N / AN / AVal-AlaMMAF1.783TA-LD-084KGGSG3-AcetylVal-AlaMMAF1.369TA-LD-C06N / AN / AVal-Ala-PABMMAE2.834TA-LD-083EGGSG3-CAVal-Ala-PABMMAE1.884TA-LD-C07N / AN / Aβ-GlucuronideMMAE2.097TA-LD-060EGGSG3-CAβ-GlucuronideMMAE1.463TA-LD-C09N / AN / AVal-Ala-PABPBD dimer analog 22.332TA-LD-078EGGSG3-CAVal-Ala-PABPBD dimer analog 21.403TA-LD-C10N / AN / AVal-AlaDuocarmycin analog2.423TA-LD-082EGGSG3-CAVal-AlaDuocarmycin analog0.956.

[0970]

[0971] When Exatecan was used as the drug, the ADC according to the embodiment of the present invention exhibited improved hydrophilicity compared to the existing ADC T-Dxd, and also exhibited improved hydrophilicity under equivalent conditions, that is, under conditions where equivalent antibodies and cleavable linkers were applied, compared with the control group without branching (TA-LD-C01, TA-LD-C02, TA-LD-C03, TA-LD-C04) and the control group with non-peptide PEG branching (TA-LD-P01, TA-LD-P02, TA-LD-P03, TA-LD-P04, TA-LD-P05, TA-LD-P06).

[0972]

[0973] In addition, when MMAF was used as a drug, the ADC according to the embodiment of the present invention exhibited improved hydrophilicity compared to the control group without branching (TA-LD-C05, TA-LD-C08) and the control group with non-peptide PEG branching (TA-LD-P07, TA-LD-P08).

[0974]

[0975] In addition, when a PBD dimer analog was used as a drug, the ADC according to the embodiment of the present invention exhibited improved hydrophilicity compared to a control group (TA-LD-C09) without branching.

[0976]

[0977] In addition, when a Duocarmycin analog was used as the drug, the ADC according to the embodiment of the present invention exhibited improved hydrophilicity compared to the control group (TA-LD-C10) without branching.

[0978]

[0979] In addition, HI-HPLC of ADC substances conjugated to various antibodies and bispecific antibodies was analyzed using the same method as above, and the results are shown in Table 6 below. In Table 6 below, "-CA" means "-Carboxylic acid".

[0980] Evaluation Target AntibodySpacer Branch Cleavable Linker DrugHI-HPLC Retention Time (min)TA-LD-C02-1CetuximabN / AN / AVal-AlaExatecan13.0TA-LD-010-1CetuximabEGGSG3-CAVal-AlaExatecan10.7TA-LD-C02-2RituximabN / AN / AVal-AlaExatecan15.0 TA-LD-010-2RituximabEGGSG3-CAVal-AlaExatecan13.8TA-LD-C02-3SacituzumabN / AN / AVal-AlaExatecan26.0TA-LD-010-3SacituzumabEGGSG3-CAVal-AlaExatecan24. 2TA-LD-C02-4TisotumabN / AN / AVal-AlaExatecan13.0TA-LD-010-4TisotumabEGGSG3-CAVal-AlaExatecan11.2TA-LD-C02-5IzalontamabN / AN / AVal-AlaExatecan13.5TA -LD-010-5IzalontamabEGGSG3-CAVal-AlaExatecan11.7TA-LD-C02-6PolatuzumabN / AN / AVal-AlaExatecan12.6TA-LD-010-6PolatuzumabEGGSG3-CAVal-AlaExatecan9.8

[0981]

[0982] These results demonstrate that using the linker-drug conjugate of the present invention allows for obtaining an ADC with improved hydrophilicity, and that the ADC of the present invention can exhibit improved hydrophilicity compared to conventional ADCs.

[0983]

[0984] Experimental Example 2. Stability Evaluation

[0985] The stability of the ADC according to an embodiment of the present invention was evaluated as follows.

[0986]

[0987] The thermal stability of each ADC was evaluated by comparing it with a control group using the SE-HPLC analysis method. The method involved analyzing samples subjected to heat stress at 40°C for two weeks after substituting the ADC with a formulation buffer or PBS, and samples without heat stress, using SE-HPLC (Waters, XBridge Protein BEH SEC column) to compare changes in HMW, LMW, and Main content. Stability was evaluated as excellent when the decrease in SE-HPLC Main content was small and the increases in HMW and LMW were small.

[0988]

[0989] The evaluation results are shown in Table 7 (Thermal stability of ADC materials conjugated to Trastuzumab in formulation buffers), Table 8 (Thermal stability of ADC materials conjugated to Trastuzumab in PBS buffers), and Table 9 (Thermal stability of ADC materials conjugated to various antibodies and bispecific antibodies in formulation buffers), as well as Figures 2, 3, and 4 below. The values ​​in Table 7 were applied as the average values ​​for three batches, except for TA-LD-049, where the results from one batch were applied. The values ​​in Tables 8 and 9 were applied as the results from one batch. In Tables 7 to 9 below, "-CA" refers to "-Carboxylic acid".

[0990] Subject to Evaluation Spacer Branch-cleavable Linker Drug Thermal Stability Delta Main (%) Thermal Stability Delta HMW (%) Thermal Stability Delta LMW (%) T-DxdN / AN / AGly-Gly-Phe-GlyDeruxtecan-3.44 1.6 1.8 2TA-LD-C02N / AN / AVal-AlaExatecan-2.43 1.04 1.3 9TA-LD-01 0EGGSG3-CAVal-A laExatecan-1.670.311.35TA-LD-050EGSSS3-CAVal-AlaExatecan-1.330.191.15TA-LD-045KGGSG3- AcetylVal-AlaExatecan-1.540.091.45TA-LD-049KGSSS3-AcetylVal-AlaExatecan-0.82-0.561.38

[0991]

[0992] Subject to Evaluation Spacer Branch-cleavable Linker Drug Thermal Stability Delta Main(%) Thermal Stability Delta HMW(%) Thermal Stability Delta LMW(%) T-DxdN / AN / AGly-Gly-Phe-GlyDeruxtecan-4.452.921.53TA-LD-C02N / AN / AVal-AlaExatecan-5.974.701.27TA-LD-010EGGSG3-CAVal- AlaExatecan-1.690.441.25TA-LD-050EGSSS3-CAVal-AlaExatecan-1.620.441.18TA-LD-045KGGSG3 -AcetylVal-AlaExatecan-2.150.821.33TA-LD-049KGSSS3-AcetylVal-AlaExatecan-1.820.441.38

[0993]

[0994] Antibody Subject to Evaluation Spacer Branch-cleavable Linker Drug Thermal Stability Delta Main (%) Thermal Stability Delta HMW (%) Thermal Stability Delta LMW (%) TA-LD-C02-1CetuximabN / AN / AVal-AlaExatecan-3.3 1.02.3 TA-LD-010-1CetuximabEGGSG3-CAVal-AlaExatecan-1.7 0.6 2.2 TA-LD-C02-2RituximabN / AN / AVal-AlaExatecan-8.4 0.8 7.7 TA-LD-010-2RituximabEGGSG3-CAVal-AlaExatecan-5.6 0.0 5.6 TA-LD-C02-3SacituzumabN / AN / AVal-AlaExatecan-9.6 0.0 9.6 TA -LD-010-3SacituzumabEGGSG3-CAVal-AlaExatecan-3.7-0.64.3TA-LD-C02-4TisotumabN / AN / AVal-AlaExatecan-3.61.91.7TA-LD-010-4TisotumabEGGSG3-C AVal-AlaExatecan-0.80.80.0TA-LD-C02-5IzalontamabN / AN / AVal-AlaExatecan-1.91.90.0TA-LD-010-5IzalontamabEGGSG3-CAVal-AlaExatecan-1.51.50.0

[0995]

[0996] The ADC according to an embodiment of the present invention exhibited improved thermal stability compared to the conventional ADC T-Dxd, and also showed improved thermal stability compared to a control group (TA-LD-C02) without branching. These results demonstrate that using the linker-drug conjugate of the present invention allows for obtaining an ADC with improved thermal stability, and that the ADC of the present invention can exhibit improved hydrophilicity compared to conventional ADCs while also demonstrating improved thermal stability.

[0997]

[0998] Experimental Example 3. In vitro efficacy evaluation

[0999] The in vitro efficacy of the ADC according to an embodiment of the present invention was evaluated as follows.

[1000]

[1001] ADC-induced cytotoxicity was evaluated in HER2-overexpressing breast cancer cell lines SK-BR-3 and HCC1954 through cell viability analysis. To evaluate ADC-induced cytotoxicity, CellTiter-Glo ® The Luminescent Cell Viability Assay was used, and the materials used in the experiment are as shown in Table 10.

[1002] Item Product Name Supplier Catalog Number Cell Line SK-BR-3 Korea Cell Line Bank 300 30HCC 1954 Korea Cell Line Bank 9S 1954 Culture Medium RPMI-16 40Gibco A10 49 101 PBS, pH 7.4 Gibco 100 100 23FB SG Gibco 100 8 21 47 Penicillin-Streptomycin Gibco 15 14 01 22 0.25% Trypsin Gibco 25 20 0 0 56 Analysis Plate Cell culture plate, 96 wells, White SPL 30 196 Reagent CellTiter-Glo(r) Luminescent Cell Viability Assay Promega G75 73 Device Varioskan(tm) LUX multimode microplate reader Thermo Scientific VLBLAT 0 Positive Control Inhibitor ENHERTU AstraZeneca - Daiichi Sankyo / Famots AZ-A7 120388HERCEPTINLoche / Novamedics-

[1003]

[1004] The specific procedure is as follows:

[1005] (1) Breast cancer cell lines SK-BR-3 and HCC1954 were cultured in 100 mm culture dishes in RPMI-1640 medium containing 1% PS (Penicillin-Streptomycin) and 10% FBS (fetal bovine serum) at 5% CO2, 37°C, and 95% humidity, and subcultured every 3 days;

[1006] (2) 6.5 x 10⁶ SK-BR-3 and HCC1954 cells, respectively 3 Cells / well and 3x10 3 Cells were dispensed into a 96-well plate.

[1007] (3) Cell-sampled 96-well plates were cultured for 12 hours in an incubator maintained at 37°C, 5% CO2, and 95% humidity to stabilize the cells;

[1008] (4) An ADC solution five times the final experimental concentration was prepared by diluting the ADC stock solution, and then serially diluted three times to prepare a total of nine different concentrations of ADC solutions;

[1009] (5) 20 μL of serially diluted ADC solution (100 μL of final culture) was added to each 96-well plate containing cells, and the cells were cultured for 6 days in an incubator maintained at 37°C, 5% CO2, and 95% humidity;

[1010] (6) CellTiter-Glo to evaluate cell viability ® Buffer and freeze-dried CellTiter-Glo ® The substrate was thawed and left at room temperature for 1 hour;

[1011] (7) CellTiter-Glo ® CellTiter-Glo ® Mixed with the substrate to form an enzyme / substrate mixture and vortexed for 1 to 2 minutes to obtain a homogeneous solution;

[1012] (8) 100 µl of CellTiter-Glo in each well ® Cell lysis was induced by adding reagents, covering the plate with aluminum foil to protect it from light, and mixing in an orbital shaker for 2 minutes;

[1013] (9) The 96-well plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal, and the luminescence was measured using a microplate reader;

[1014] (10) Using GraphPad Prism data analysis software, a non-linear regression analysis model was created using the log (inhibitor) vs. response-variable slope (4 parameters) model to generate a curve graph and IC 50 The value was calculated.

[1015] At this time, the ADC's IC 50 When the value was similar to T-Dxd (same digit ng / mL), it was evaluated as having suitable efficacy.

[1016]

[1017] In addition, antigen-specific binding of the ADC was confirmed to evaluate whether the ADC binds to cells or tissues other than its intended target.

[1018] Cytotoxicity induced by non-targeted ADCs was evaluated and compared by producing ADCs in which rituximab, an antibody targeting CD20 (cluster of differentiation 20), was conjugated to the antibody site of the ADC instead of trastuzumab, an antibody targeting HER2. For the evaluation of cytotoxicity, the CellTiter-Glo mentioned above ® The Luminescent Cell Viability Assay was used, and for the rituximab-introduced ADC, the spacer, branch, cleavable linker, and drug were applied identically to their respective paired trastuzumab-introduced ADCs, excluding the antibody site. Additionally, to ensure accurate evaluation, each paired ADC was evaluated on the same plate.

[1019] At this time, the IC of the targeted ADC conjugated with trastuzumab, an antibody targeting HER2, 50 The IC value of a non-targeted ADC conjugated with rituximab, an antibody targeting CD20. 50 If it appears lower than the value, specifically [non-targeted ADC IC 50 Value / IC of Targeted ADC 50 If the value exceeds 50, it was determined that the targeted ADC performs antigen-specific binding.

[1020]

[1021] The evaluation results are as shown in Tables 11 and 12 below. IC 50 The value is the median, and the selectivity is the result of a single experiment. IC 50 Information on the number of runs: T-Dxd (n=6), Herceptin (n=12), TA-LD-C02 (n=2), TA-LD-010 (n=3), TA-LD-050 (n=3), TA-LD-045 (n=2), TA-LD-049 (n=1), and others n=1 or more. In Tables 11 and 12 below, "-CA" means "-Carboxylic acid".

[1022] Spacer, branch-cutting linker, drug IC under evaluation 50 (ng / mL)SK-BR-3IC 50(ng / mL)HCC-1954Selectivity (> fold)SK-BR-3Selectivity (> fold)HCC-1954T-DxdN / AN / AGly-Gly-Phe-GlyDeruxtecan23.6533.9196150HerceptinN / AN / AN / AN / A> 15,000> 15,000N / AN / ATA-LD-C02N / AN / AVal-AlaExatecan> 5,000> 5,000N / AN / ATA-LD-010EGGSG3-CAVal-AlaExatecan19.9229.56323237TA-LD-050EGSSS3-CAVal-AlaExatecan12.6623.863452 28TA-LD-045KGGSG3-AcetylVal-AlaExatecan14.4522.86350235TA-LD-049KGSSS3-AcetylVal-AlaExatecan13.7818.95363264

[1023]

[1024] All subjects evaluated in Table 12 below have Trastuzumab as the antibody and Exatecan as the drug.

[1025] Spacer under evaluation branch-cutting capable linker SK-BR-3HCC1954IC 50 (nM)Max inhibition (%)IC 50 (nM)Max inhibition (%)TA-LD-C01N / AN / APhe-Gly0.54368.111.00262.68TA-LD-011EGGSG1-CAPhe-Gly0.09892.600.16681.08TA-LD-012EGGSG2-CAPhe-Gly0.1039 3.280.15881.56TA-LD-025KGGSG3-AcetylPhe-Gly0.05993.110.10785.40TA-LD-013EGGSG3-CAPhe-Gly0.09192.290.15481.18TA-LD-P01EPEG 12 -CAPhe-Gly0.09093.060.16183.30TA-LD-029EGGTG1-CAPhe-Gly0.07892.920.16981.29TA-LD-P02KPEG 12-AcetylPhe-Gly0.03492.090.07479.55TA-LD-023KGSSS3-AcetylPhe-Gly0.04393.450.08682.14TA-LD-022KGGSS3-AcetylPhe-Gly0.05292.210.10679.58TA-LD-051KGGSS3-AcetylPhe-Gly-m-Glucuronide0.09789.960.23981.84TA-LD-052KGSSS3-AcetylPhe-Gly-m-Glucuronide0.10490.050.24382.81TA-LD-C03N / AN / AVal-Cit0.89052.6232.2309.82TA-LD-006EGGSG2-CAVal-Cit0.07392.810.26384.22TA-LD-005EGGSG1-CAVal-Cit0.06693.390.26480.05TA-LD-007EGGSG3-CAVal-Cit0.08491.940.26079.75TA-LD-044KGGSG3-AcetylVal-Cit0.06891.960.25575.00TA-LD-P06KPEG 24 -AcetylVal-Cit0.05090.480.22675.56TA-LD-046KGGSS3-AcetylVal-Cit0.05992.030.17974.30TA-LD-048KGSSS3-AcetylVal-Cit0.07091.550.22076.34TA-LD-C02N / AN / AVal-Ala32.37038.2132.37010.47TA-LD-008EGGSG1-CAVal-Ala0.16089.220.21179.31TA-LD-009EGGSG2-CAVal-Ala0.13990.470.21281.39TA-LD-010EGGSG3-CAVal-Ala0.10292.070.15584.11TA-LD-045KGGSG3-AcetylVal-Ala0.09091.620.15182.88TA-LD-P05KPEG 24 -AcetylVal-Ala0.09192.300.14081.08TA-LD-P03EPEG 12- CAVal-Ala0.09492.580.12981.19TA-LD-P04EPEG24 -CAVal-Ala0.09492.690.14883.29TA-LD-049KGSSS3-AcetylVal-Ala0.08492.550.11680.94TA-LD-047KGG SS3-AcetylVal-Ala0.08391.900.10884.02TA-LD-056EGGSG4-CAVal-Ala0.09191.480.14783.59TA-LD-050 EGSSS3-CAVal-Ala0.07493.470.15685.65TA-LD-066KGGSG3-AcetylVal-Ala0.10791.760.16985.79TA-LD- 062EGGSG3-CAVal-Ala0.13190.170.24284.72TA-LD-069KGGSG3-AcetylVal-Ala0.09091.550.15883.68TA-L D-085EGGSG3-CarboxamideVal-Ala0.07887.620.11690.13TA-LD-C04N / AN / Aβ-Glucuronide0.10591.390.2 4085.48TA-LD-039KGGSG3-Acetylβ-Glucuronide0.11890.680.24587.23TA-LD-028EGGSG3-CAβ-Glucuroni de0.10390.610.25678.90TA-LD-054KGSSS3-Acetylβ-Glucuronide0.08890.420.22975.96TA-LD-053KGGSS 3-Acetylβ-Glucuronide0.08890.160.21681.61TA-LD-055EGSSS3-CAβ-Glucuronide0.09491.380.21084.68

[1026]

[1027] As shown in Table 11, the control group consisting only of the antibody (Herceptin) and the control group without branching (TA-LD-C02) showed less than 50% cytotoxicity in both SK-BR-3 and HCC1954 cells, indicating an IC50. 50While the value was not measured, the ADC according to the embodiment of the present invention has an IC level similar to that of the conventional ADC T-Dxd in SK-BR-3 and HCC1954 cells. 50 It exhibited high cytotoxicity and high values. In addition, the ADC according to the embodiment of the present invention exhibited high antigen specificity. Furthermore, as shown in Table 12, it was confirmed that the ADC according to the embodiment of the present invention significantly improved cytotoxicity efficacy. These results demonstrate that using the linker-drug conjugate of the present invention allows for obtaining an ADC with improved hydrophilicity and stability while maintaining the inherent efficacy of each antibody and drug, and that the ADC of the present invention can fully exhibit the inherent efficacy of each antibody and drug while displaying improved hydrophilicity and stability.

[1028]

[1029] Experimental Example 4: Evaluation of Antigen Specificity

[1030] A non-targeted antibody-drug conjugate in which only the antibody portion is modified to Rituximab, an antibody targeting CD20 (cluster of differentiate 20), and IC in SK-BR-3 and HCC1954 cells in the same manner as in Experimental Example 3 above 50 The values ​​were measured, and the results are shown in Table 13 below.

[1031] Antibody IC for evaluation 50(nM)Ratio (Non-Targeting / Targeting)SK-BR-3HCC1954SK-BR-3HCC1954TA-LD-P05Trastuzumab0.0910.140>342>222TA-LD-P05NRituximab>30.983>30.983TA-LD-P04Trastuzumab0.0940.148>330>210TA-LD-P04NRituximab>31.046>31.046TA-LD-P03Trastuzumab0.0940.129>338>248TA-LD-P03NRituximab>31.884>31.884TA-LD-C04Trastuzumab0.1070.224245>146TA-LD-C04NRituximab26.261>32.695TA-LD-039Trastuzumab0.1200.240257>130TA-LD-039NRituximab30.860>31.155TA-LD-054Trastuzumab0.0880.229>351>135TA-LD-054NRituximab>30.878>30.878TA-LD-028Trastuzumab0.1510.294>206>106TA-LD-028NRituximab>31.219>31.219TA-LD-055Trastuzumab0.1010.231>308>134TA-LD-055NRituximab>30.941>30.941TA-LD-044Trastuzumab0.0680.255>461>123TA-LD-044NRituximab>31.400>31.400TA-LD-007Trastuzumab0.0680.260>464>121TA-LD-007NRituximab>31.465>31.465TA-LD-025Trastuzumab0.0760.127>414>248TA-LD-025NRituximab>31.482>31.482TA-LD-013Trastuzumab0.0910.154>346>205TA-LD-013NRituximab>31.547>31.547

[1032]

[1033] As mentioned above, the non-targeted antibody-drug conjugate modified with Rituximab is IC 50 The value was found to be 30 nM or higher, confirming that the antibody-drug conjugate according to the present invention maintains antigen specificity.

[1034]

[1035] Experimental Example 5: Evaluation of In Vitro Cytotoxicity According to Antibody Type

[1036] Experimental Example 3 was the same, except the cell line was changed as shown in Table 14 below, and in vitro cytotoxicity according to the type of antibody was evaluated, and the results are shown in Figures 6 to 8 and Table 15 below.

[1037] Product Name Supplier Catalog No. Remarks HCC827 Korea Cell Line Bank 70827 Non-small cell lung cancer cell line A4 31 ATCCCRL-1555 Skin cancer cell line BxPC3 ATCCCRL-1687 Pancreatic cancer cell line MDA-MB-231 Korea Cell Line Bank 30026 Breast cancer cell line Caski Korea Cell Line Bank 21550 Cervical cancer cell line Ramos ATCCCRL-1596 Lymphoma cell line

[1038]

[1039] Cell lines IC with or without hydrophilic linkers under evaluation 50 (nM)Maximum cell growth inhibitory activity (at 5 ug / mL, %) Cell line IC 50(nM)Maximum cell growth inhibitory activity (at 5 µg / mL, %) CetuximabXA43 13.37 44 0.00 HCC82 70.6 40 83.07 TA-LD-C02-1X3.6 24 47.8 60.0 80 72.95 TA-LD-010-1O 0.8 00 64.1 30.0 37 70.73 SacituzumabXBxPC3>10 3.0 79 -0.29 HCC82 7>10 3.0 7 9-1.77TA-LD-C02-3X4.06250.690.51358.76TA-LD-010-3O0.15486.320.15967.42Tiso tumabXMDA-MB-231>103.6910.97Caski>103.691-5.44TA-LD-C02-4X0.16276.670.10081 .30TA-LD-010-4O0.03985.780.04184.83IzalontamabXBxPC3>253.57537.45HCC8270.8 9567.06TA-LD-C02-5X101.04683.070.13661.23TA-LD-010-5O12.95179.720.03466.90P olatuzumabXRamos>103.093-23.60TA-LD-C02-6X>32.9185.08TA-LD-010-6O3.07699.1 4RituximabXRamos>138.889-10.80TA-LD-C02-2X90.03279.31TA-LD-010-2O3.27699.53

[1040]

[1041] As shown in Table 15 above, it can be confirmed that the antibody-drug conjugate with the hydrophilic linker of the present invention improves cytotoxic activity compared to the conjugate without the hydrophilic linker.

[1042]

[1043] Experimental Example 4. Evaluation of In vivo Exposure

[1044] The in vivo efficacy of the ADC according to an embodiment of the present invention was evaluated as follows.

[1045]

[1046] The experiment was conducted on fasted male CD-1 (ICR) mice. Each ADC was formulated at 0.50 mg / mL in 20 mM His-Acetate / 240 mM Sucrose / pH 5.5 and administered intravenously at a dose of 1 mg / kg. Subsequently, blood samples were collected at 0.083, 7, 24, 72, 168, 336, and 504 hours, and the concentrations were quantified using ELISA.

[1047]

[1048] As a result of the evaluation, as shown in Fig. 5, the ADC according to the embodiment of the present invention showed better exposure than T-DXd.

[1049]

[1050] Experimental Example 5. Evaluation of In vivo efficacy

[1051] The in vivo efficacy of the ADC according to an embodiment of the present invention was evaluated as follows.

[1052]

[1053] To evaluate anticancer efficacy, HER2-overexpressing breast cancer cell lines HCC1954 or JIMT-1 were implanted on the left dorsal side of BALB / c-nude mice, 5 x 10 cells per mouse 6 After confirming tumor formation in an animal model prepared by subcutaneously implanting canine tumor cells, the tumor growth inhibitory ability was evaluated by administering the evaluation substance as a single intravenous injection into the mouse caudate vein. Tumor size (shortening, mm) 2 It was calculated using the formula x (major axis, mm) x 0.5.

[1054]

[1055] As a result of the evaluation, as shown in FIGS. 9 to 11, the ADC according to the embodiment of the present invention exhibited excellent anticancer efficacy without weight loss in both the HCC1954 breast cancer model with high HER2 expression levels and the JIMT-1 breast cancer model with intermediate HER2 expression levels. In particular, in the case of the JIMT-1 breast cancer model, the ADC according to the embodiment of the present invention exhibited significantly superior anticancer efficacy compared to the existing ADC T-Dxd.

[1056]

[1057] These results demonstrate that using the linker-drug conjugate of the present invention allows for obtaining an ADC with improved PK exposure and efficacy in actual vivo, and that the ADC of the present invention thereby can be obtained with improved in vivo PK exposure and efficacy while exhibiting improved hydrophilicity and stability.

[1058]

[1059] Experimental Example 6: Evaluation of the in-sebum stability of an antibody-drug conjugate

[1060] The in vitro serum stability of the linker-drug conjugate of the present invention according to whether a hydrophilic branch was introduced was evaluated as follows.

[1061]

[1062] For comparative evaluation, TA-LD-010 according to an embodiment of the present invention, in which a hydrophilic branch was introduced, and TA-LD-C02, in which a hydrophilic branch was not introduced as a control substance, were each prepared by spiking them into IgG-removed human serum (Human Serum; Innovative Research, USA) to a final concentration of 500 μg / mL. Each prepared mixture was incubated at 37°C, and samples were collected at 0, 4, and 7 days, respectively, and used for total antibody concentration analysis and antibody-conjugated drug (acDrug) concentration analysis.

[1063] Total antibody concentration was analyzed using the ELISA method. Specifically, a 96-well microplate was coated with Goat Anti-Human IgG-UNLB (Southern Biotech, Country: USA), diluted serum culture samples and samples for standard curve construction were added, and the mixture was reacted at 37°C for 1 hour. After removing unbound components with a wash buffer, the absorbance was measured at 450 nm using HRP-conjugated anti-Human IgG Fc secondary antibody (Supplier: Invitrogen, Country: USA) and 3,3',5,5'-tetramethylbenzidine (TMB, Supplier: R&D Systems, Country: USA), and the results were applied to the standard curve to calculate the total antibody concentration in the samples.

[1064] To quantify the concentration of antibody-bound drugs (acDrug), serum culture samples and samples for constructing a standard curve were prepared. For each sample, magnetic beads bound to anti-human IgG were treated to recover ADCs from the serum via immunoprecipitation (IP), after which the ADCs were eluted from the beads. Papain was added to the eluted ADC solution and stirred at 37°C for 24 hours to release the drug from the ADCs; the concentration of the released drug was measured using HPLC analysis. To measure acDrug concentrations, samples were separated at 70°C using an Agilent AdvanceBio Peptide Mapping Column and a mobile phase of water and acetonitrile containing 0.1% TFA. After injecting 65 μL of the sample, the gradient elution method was applied at a flow rate of 1.0 mL / min for 30 minutes and analyzed at a wavelength of 370 nm. The acDrug concentration of each sample was calculated by comparing the intensity of the signal detected at 370 nm with the intensity of the standard substance. Using the measured total antibody concentration and acDrug concentration, the Drug-to-antibody Ratio (DAR) was calculated using the following formula, and the results are shown in Figure 12.

[1065]

[1066]

[1067] As shown in Figure 12, TA-LD-010 with introduced hydrophilic branches exhibited significantly improved DAR stability compared to the control substance, TA-LD-C02. Specifically, at the 7th day of serum culture, the DAR of the control substance, TA-LD-C02, decreased to approximately 4.5 and drug shedding was observed, whereas the DAR of TA-LD-010 with introduced hydrophilic branches remained above approximately 6.5, demonstrating high structural stability. This suggests that the linker structure with introduced hydrophilic branches contributes to improved serum stability by protecting the ADC from proteolytic enzymes or other destabilizing factors in serum, or by inhibiting linker cleavage through structural steric hindrance.

[1068]

[1069] Experimental Example 7: Concentration experiment for the development of a high-concentration liquid SC formulation

[1070] To confirm the feasibility of high-concentration liquid formulations, purity analysis and viscosity measurement using SE-HPLC were performed on ADC materials obtained through a conjugation reaction following concentration experiments. For this purpose, LD-010 linker-drug conjugates were conjugated to trastuzumab and cetuximab, and concentrations were performed at targeted concentrations of 30 mg / mL, 50 mg / mL, 100 mg / mL, 150 mg / mL, and 200 mg / mL. Samples were taken at each concentration and subjected to SE-HPLC to analyze purity and HMW content, while viscosity was measured using mVROC. SE-HPLC (Size exclusion-high-performance liquid chromatography) analysis (Waters, XBridge Protein BEH SEC column) was used to analyze the purity and HMW (high molecular weight species) of the ADC at each concentration. After diluting each ADC sample to 2 mg / mL, 10 μL of the Mobile Phase was mixed at a 1:1 ratio and injected into the column. The Mobile Phase was flowed isocratically under UV 280 nm for 30 minutes to perform analysis. The flow rate was 0.6 mL / min, and 30 mM sodium phosphate, 150 mM NaCl, and 10% isopropyl alcohol were used as the Mobile Phase. To measure the viscosity at each concentration, bubble-removed samples were filled into syringes and mounted on an m-VROC chip; the pressure drop generated as the samples passed through the microchannels was measured using a sensor.

[1071]

[1072] The results are shown in Table 16 below. Depending on the type of antibody, it was confirmed that TA-LD-010 is suitable as an injectable SC formulation up to 152.7 mg / mL. It was confirmed that TA-LD-010-1 is suitable as an injectable SC formulation up to 97.0 mg / mL.

[1073] Antibody to be evaluatedADC Conc.(mg / mL)DAR by RP-LC / MSTIC%HMW (SE-HPLC)UV280%Main (SE-HPLC)UV280%LMW (SE-HPLC)UV280Viscosity (cP)TA-LD-010Trastuzumab31.07.91.298.8N / A1.5851.07.91.398.7N / A2.02101.17.91.498.6N / A4.82152.77.91.498.6N / A12.47201.47.91 .498.6N / A79.99TA-LD-010-1Cetuximab30.38.00.799.4N / A2.0553.78.00.699.4N / A3.8297.08.00.698.11.315.40135.68.00.798.11.390.81

Claims

1. A linker-drug conjugate represented by the following chemical formula 1, or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] In the above chemical formula 1, AG is an attachment group to the target substance; BS is a branching spacer; D is a drug; P is a terminal-modified or non-modified peptide; L1 and L3 are spacers; L2 is a spacer or a cuttable linker; a, b, and c are each independently 0 or 1.

2. In Paragraph 1, The above BS is a structure in which an amino acid is connected to the above L1 or AG through the nitrogen of the amino group, connected to the above L2 or D through the carbon of the carboxyl group, and connected to the above L3 or P through the carbon or nitrogen of the side chain, A linker-drug conjugate, or a pharmaceutically acceptable salt thereof.

3. In Paragraph 2, The above BS is or person, A linker-drug conjugate, or a pharmaceutically acceptable salt thereof.

4. In Paragraph 2, The above BS is or person, A linker-drug conjugate, or a pharmaceutically acceptable salt thereof.

5. In Paragraph 1, The above P is a terminal-modified or non-modified chain of an oligopeptide in which two or more amino acids selected from glycine (G), serine (S), and threonine (T) are linked together. A linker-drug conjugate, or a pharmaceutically acceptable salt thereof.

6. In Paragraph 1, The above P is any one selected from the group consisting of the following, Linker-drug conjugate, or a pharmaceutically acceptable salt thereof: As mentioned above, R1 is independently hydrogen, or C 1-4 It is a hydroxyalkyl, and R2 is hydrogen, or (C 1-4 It is an alkyl)carbonyl, and R3 is a hydroxyl or amino, and n is a natural number from 1 to 10.

7. In Paragraph 6, R1 is each independently hydrogen, hydroxymethyl, or 1-hydroxyethyl, A linker-drug conjugate, or a pharmaceutically acceptable salt thereof.

8. In Paragraph 6, R2 is hydrogen, or methyl carbonyl, A linker-drug conjugate, or a pharmaceutically acceptable salt thereof.

9. In Paragraph 1, The above P is any one selected from the group consisting of the following, Linker-drug conjugate, or a pharmaceutically acceptable salt thereof: As mentioned above, n is a natural number from 1 to 10.

10. In Paragraph 1, The above L2 comprises one or more of a cleavable dipeptide, a para-aminobenzyl alcohol self-sacrificial group, and a glucuronide bond, A linker-drug conjugate, or a pharmaceutically acceptable salt thereof.

11. In Paragraph 10, The above L2 is any one selected from the group consisting of the following, Linker-drug conjugate, or a pharmaceutically acceptable salt thereof:

12. In Paragraph 1, The above AG comprises a reactive group selected from the group consisting of a maleimide group, a thiol group, a cyclooctine group, an azido group, and a maleamic acid group, A linker-drug conjugate, or a pharmaceutically acceptable salt thereof.

13. In Paragraph 12, The above AG is any one selected from the group consisting of the following, Linker-drug conjugate, or a pharmaceutically acceptable salt thereof: As mentioned above, L3 is a C that optionally contains phenylene, -CONH-, or -C(R4)- in the middle of the chain. 1-10 It is an alkylene, and R4 is C 1-10 It is an aminoalkyl.

14. In Paragraph 12, The above AG is any one selected from the group consisting of the following, Linker-drug conjugate, or a pharmaceutically acceptable salt thereof:

15. A targeting substance-drug conjugate represented by the following chemical formula 2, or a pharmaceutically acceptable salt thereof: [Chemical Formula 2] In the above chemical formula 2, AG, BS, D, P, L1, L2, L3, a, b, and c are identical to those defined in any one of paragraphs 1 through 14, and TA is a targeting agent; n' is an integer from 1 to 8.

16. In Paragraph 15, The above TA is an antibody or an antigen-binding fragment thereof, Targeting substance-drug conjugate, or a pharmaceutically acceptable salt thereof.

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