Therapeutic composition

Abstract

To provide a novel target protein degradation-inducing compound, a composition for cancer therapy that includes the compound, and in particular a composition for cancer therapy that does not cause serious teratogenicity.SOLUTION: A thalidomide derivative reduced in degradation of both SALL4 and PLZF proteins serves as an E3 ligase binder of a target protein degradation-inducing compound that is capable of avoiding serious teratogenicity. Using this knowledge, the present invention is completed by finding a thalidomide derivative that has a reduced ability of degradation of both SALL4 and PLZF proteins, and further confirming that a target protein degradation-inducing compound that includes the derivative has an anti-cancer effect.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to therapeutic compositions, particularly novel target protein degradation-inducing compounds, cancer treatment compositions containing said compounds, and more specifically, cancer treatment compositions that avoid teratogenicity. This application claims priority to Japanese application No. 2021-175996, as incorporated herein by reference. [Background technology]

[0002] (Thalidomide drug tragedy) Thalidomide was a drug used worldwide more than half a century ago as a sleep aid for pregnant women. However, it caused severe teratogenicity in the limbs of fetuses born to women who took it, leading to a global drug tragedy known as the thalidomide tragedy. However, thalidomide has been shown to be effective against leprosy and multiple myeloma, and currently, thalidomide and its derivatives (lenalidomide and pomalidomide) are used under strict safety controls as highly effective drugs for treating blood cancers such as multiple myeloma, with an annual cost of approximately 1 trillion yen. Thalidomide and thalidomide derivatives (Immunomodulatory drugs / IMiDs) have been shown to induce the degradation of various proteins by binding to cereblon (CRBN), a component of the proteolytic enzyme E3 ubiquitin ligase, resulting in a wide range of pharmacological effects and side effects.

[0003] It has been reported that SALL4 (Sal-like protein 4), a protein that plays a crucial role in fetal limb development, is degraded by CRBN in an IMiD-dependent manner. Furthermore, the present inventors have recently elucidated, through structural analysis, the mechanism by which hydroxylated thalidomide (5-hydroxylated thalidomide), produced when thalidomide is metabolized in the body, acts on SALL4 more efficiently than thalidomide itself (see Non-Patent Literature 1). However, many aspects of the teratogenicity caused by thalidomide and its derivatives remain unclear, and the existence of causative proteins other than SALL4 has been suggested.

[0004] (Target protein degradation-inducing compounds) PROTAC (Proteolysis Targeting Chimera) is a known small molecule compound that utilizes the ubiquitin-proteasome system (UPS) to induce targeted protein degradation (TPD) within cells (see Non-Patent Literature 2). Target protein degradation-inducing compounds (PROTAC compounds) bind E3 ubiquitin ligases to target proteins, forming a ternary complex. This formation leads to polyubiquitination of the target, followed by degradation by the proteasome. After the polyubiquitination reaction, the target protein degradation-inducing compound detaches from the complex and acts catalytically by repeatedly binding to new targets with E3 ligases. This catalytic action causes the target protein to be depleted by proteasomal digestion after ubiquitination within the cell, leading to cell death and proving effective against various diseases (e.g., cancer).

[0005] (Prior patents) Patent Document 1 discloses "a pharmaceutical composition comprising 6-hydroxylenalidomide, a salt thereof, or a solvate thereof." Patent document 2 discloses an "imide-based protein degradation modulator." However, these documents do not disclose or suggest the target protein degradation-inducing compounds of the present invention. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2021-20873 [Patent Document 2] Special Publication No. 2017-513862 [Non-patent literature]

[0007] [Non-Patent Document 1] (2020) Structural bases of IMiDselectivity that emerges by 5-hydroxythalidomide Nat Commun 11: 4578 [Non-Patent Document 2] https: / / www.funakoshi.co.jp / contents / 63923 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The inventors have identified PLZF (promyelocytic leukemia zinc finger) as a novel protein involved in thalidomide teratogenicity, and have clarified the molecular mechanism of thalidomide teratogenicity. Furthermore, they have revealed that PLZF is degraded in a CRBN-dependent manner by thalidomide, thalidomide derivatives, and 5-hydroxythalidomide. Next, the inventors conducted gene knockdown experiments on chicken embryos, a thalidomide-sensitive animal species, and confirmed that PLZF plays an important role in limb development in chicken embryos. They also confirmed that administering thalidomide or 5-hydroxythalidomide to chicken embryos reduced PLZF levels in the limb buds of the chicken embryos, while SALL4 levels did not decrease. In addition, they revealed that overexpression of PLZF in chicken embryos restores the decreased expression of fibroblast growth factors Fgf8 and Fgf10 caused by thalidomide administration. This study confirmed that thalidomide-dependent degradation of PLZF by CRBN is directly involved in thalidomide teratogenicity. It also confirmed that in thalidomide-sensitive species (mammals) such as humans, monkeys, and rabbits, thalidomide is metabolized to 5-hydroxythalidomide by metabolic enzymes in the body, and that severe teratogenicity is caused by the degradation of both SALL4 and PLZF proteins. Accordingly, the object of the present invention is to provide a novel target protein degradation-inducing compound, a cancer treatment composition containing the compound, and in particular a cancer treatment composition that does not cause serious teratogenicity. [Means for solving the problem]

[0009] Based on the above finding that thalidomide derivatives that reduce the degradation of both SALL4 and PLZF proteins can act as E3 ligase binders for target protein degradation-inducing compounds that can avoid severe teratogenicity, the present inventors have discovered thalidomide derivatives that reduce the degradation of both SALL4 and PLZF proteins, and have further confirmed that target protein degradation-inducing compounds containing these derivatives have anti-cancer activity, thereby completing the present invention. That is, the present invention is as follows.

[0010] 1. A target protein degradation-inducing compound having the following composition, "Cerebronchial binder" - "Linker" - "Target protein binder" Here, the cerebron binder is a target protein degradation-inducing compound selected from any one of the following: a compound represented by the general formula (1), a salt thereof, or a solvate thereof. [ka] (Here, R1 is hydrogen, F, Br, Cl, CH3, OCH 3、 I, CF3, or OCF3, R L1 and R L2 (It is either hydrogen, a functional group, or an atom, but at least one of them is either a functional group or an atom.) 2. A target protein degradation-inducing compound as described in item 1 above, having the following composition: The cerebron binder is selected from one of the following compounds represented by the general formula (1), their salts, or their solvates: [ka] (Here, R1 is hydrogen, F, Br, Cl, CH3, OCH 3、 I, CF3, or OCF3, R L1 and R L2 (It is either hydrogen, a functional group, or an atom, but at least one of them is either a functional group or an atom.) Furthermore, the target protein binder is JQ-1 or Birabresib. A compound that induces the degradation of target proteins. 3. The target protein degradation-inducing compound according to item 1 or 2 above, having the following configuration The cereblon binder is selected from any one of a compound represented by the following general formula (7), a salt thereof, or a solvate thereof

Chemical formula

Chemical formula

Chemical formula

[0011] Furthermore, the present invention includes the following: 1. A target protein degradation-inducing compound having the following composition, "Cerebronchial binder" - "Linker" - "Target protein binder" Here, the cerebron binder is a target protein degradation-inducing compound selected from one of the following: a compound represented by the general formula (10), a salt thereof, or a solvate thereof. [ka] (Here, X is CH2 or C=O, and R10 is hydrogen, a functional group, an atom, or NR) L1 R L2 And R L1 and R L2 R11 is hydrogen, a functional group, or an atom, but one of them is a functional group or an atom, and R11 is hydrogen, F, Br, Cl, CH3, or OCH 3、 I is either CF3 or OCF3, and R12 is either hydrogen, F, Br, CF3, I, Cl, OH, CH3, OCF3, or OCH3. 2. The target protein degradation-inducing compound described in item 1 above, wherein the target protein binder is JQ-1 or Birabresib. 3. A target protein degradation-inducing compound according to item 1 or 2 above, wherein X in the general formula (10) is CH2, R11 is CF3, and R12 is hydrogen. 4. A target protein degradation-inducing compound according to item 1 or 2 above, wherein X in the general formula (10) is CH2, R11 is F, and R12 is hydrogen. 5. The target protein degradation-inducing compound according to claim 1 or 2, wherein X in the general formula (10) is CH2, R11 is Cl, and R12 is hydrogen. [Effects of the Invention]

[0012] The present invention can provide novel target protein degradation-inducing compounds, cancer treatment compositions containing said compounds, and cancer treatment compositions with reduced teratogenicity. [Brief explanation of the drawing]

[0013] [Figure 1] An example of a linker. [Figure 2] An example of a linker. [Figure 3] Structures of each thalidomide derivative. [Figure 4] The results of the analysis of the affinity (degradation-inducing ability) of each thalidomide derivative for IKZF1, SALL4, and PLZF are shown (left column shows IKZF1, middle column shows SALL4, and right column shows PLZF). [Figure 5] Results of degradation analysis of the affinity (degradation-inducing ability) of each thalidomide derivative for IKZF1, SALL4, and CRBN. [Figure 6] The results of the analysis of the affinity (degradation-inducing ability) of each meta-substituted thalidomide derivative to IKZF1, SALL4, and PLZF are shown below {(1) shows the structure of each meta-substituted thalidomide derivative used, and (2) shows the affinity analysis results, with the left column showing IKZF1, the middle column showing SALL4, and the right column showing PLZF}. [Figure 7] The results of the analysis of the affinity (degradation-inducing ability) of meta-substituted lenalidomide derivatives for IKZF1, SALL4, and PLZF are shown below {(1) shows the structure of each meta-substituted thalidomide derivative used, and (2) shows the results of the affinity analysis, where X represents IKZF1, Y represents SALI4, and Z represents PLZF}. [Figure 8] The results of the affinity analysis of F3C-lenalidomide and F3CO-lenalidomide to SALL4 and PLZF are shown below {(1) shows the structures of F3C-lenalidomide and F3CO-lenalidomide, and (2) shows the affinity analysis results, with the left column showing IKZF1, the middle column showing SALL4, and the right column showing PLZF}. [Figure 9] (1) An overview of the method for measuring the binding ability to CRBN. (2) Results of the binding ability of each meta-substituted lenalidomide derivative to CRBN. [Figure 10] Results of cell culture endogenous protein resolution of lenalidomide derivatives with substituent insertions at each meta position. [Figure 11] Results of the anticancer activity of lenalidomide derivatives with substituent insertions at each meta position against cultured cells derived from multiple myeloma. [Figure 12] Results of the anticancer activity of lenalidomide derivatives with substituent insertions at each meta position against cultured cells derived from myelodysplastic syndrome with chromosome 5 defect. [Figure 13]Confirmation of neoprotein degradation ability of known PROTACs dBET1 and ARV-825 (1) and results of anticancer activity against cultured cells derived from multiple myeloma (2). [Figure 14] A schematic diagram illustrating the action of PROTAC in this invention. [Figure 15] Structural diagrams of each PROTAC. [Figure 16] Confirmation of the selectivity of the PROTAC of the present invention for cerebron binder neosubstrates (1) and results of its anticancer activity against cultured cells derived from multiple myeloma (2). [Figure 17] Results of the degradation-inducing ability of the PROTAC of the present invention on SALL4 and PLZF. [Figure 18] Confirmation of the anticancer effect of the PROTAC of the present invention. [Figure 19] Disassembly and evaluation of the CC-220 SALL4. [Figure 20] Examples of publicly known PROTACs. [Figure 21] Examples of publicly known PROTACs. [Figure 22] Examples of publicly known PROTACs. [Figure 23] Examples of publicly known PROTACs. [Figure 24] Examples of publicly known PROTACs. [Figure 25] Examples of publicly known PROTACs. [Figure 26] Examples of publicly known PROTACs. [Figure 27] Examples of publicly known PROTACs. [Figure 28] Examples of publicly known PROTACs. [Figure 29] Examples of publicly known PROTACs. [Figure 30] Examples of publicly known PROTACs. [Figure 31] Examples of publicly known PROTACs. [Figure 32] Examples of publicly known PROTACs. [Figure 33] Evaluation of the antitumor effect of PROTAC in a mouse xenograft model using IMR32 cells (neuroblastoma cells). [Figure 34] Results of the degradation-inducing ability of thalidomide, pomalidomide, and lenalidomide. [Figure 35] Results of the degradation-inducing ability of thalidomide derivatives modified at the 4-position. [Figure 36] Results showing the ability to induce degradation by substitutions at positions 5 and 6 of the pomalidomide skeleton. [Figure 37] Results of degradation induction ability by substitutions at positions 6 and 7 of the lenalimid skeleton. [Figure 38] Results of degradation induction activity with the 7-position modified pomalidomide-linker-JQ-1 derivative (PROTAC). [Modes for carrying out the invention]

[0014] (Target of this invention) The subject matter of this invention is as follows: A cancer treatment composition comprising a compound represented by the following general formula (1) or general formula (10), a salt thereof, or a solvate thereof (hereinafter sometimes referred to as "the cancer treatment composition of the present invention"). ○ A target protein degradation-inducing compound (PROTAC compound, hereafter sometimes referred to as "the target protein degradation-inducing compound of the present invention" or "the PROTAC compound of the present invention") having the following composition. "Cerebromide" - "Linker" - "Target Protein Binder"

[0015] [ka]

[0016] R1 is hydrogen, F, Br, Cl, CH3, OCH 3、 I, CF3, or OCF3, R L1 and R L2 is a hydrogen atom, a functional group, or an atom, but one of them is either a functional group or an atom.

[0017] [ka]

[0018] X is CH2 or C=O, and R10 is hydrogen, a functional group, an atom, or NR L1 R L2 And R L1 and R L2 R11 is hydrogen, a functional group, or an atom, but one of them is a functional group or an atom, and R11 is hydrogen, F, Br, Cl, CH3, or OCH 3、 I is CF3 or OCF3, and R12 is hydrogen, F, Br, CF3, I, Cl, OH, CH3, OCF3 or OCH3.

[0019] In the case of the target protein degradation-inducing compound of the present invention, R L1 and R L2 Either one or both of these elements are coupled to or part of the linker. The functional groups are not particularly limited as long as they can bond (especially covalently) or couple with the linker described below, but examples include alcohols, ketones, carboxylic acids, amines, ethers, etc. The atoms are not particularly limited as long as they can bond or couple with the linkers described below, but examples include oxygen, nitrogen, sulfur, phosphorus, halogens, heterocyclic amines (piperazine, piperidine), etc.

[0020] (The present invention's composition for cancer treatment) The cancer treatment composition of the present invention contains a compound represented by the above general formula (1) or general formula (10), a salt thereof, or a solvate thereof as an active ingredient. Examples of the compound represented by the above general formula (1) or general formula (10) are listed below. In addition, the active ingredient can function as a cerebron binder. The term "salt" is not particularly limited as long as it is pharmacologically acceptable, but specifically, acid addition salts, base addition salts, etc., are preferred. The solvates are not particularly limited as long as they are pharmacodynamically acceptable, but specifically, hydrates, ethanolates, etc., are preferred.

[0021] (F-Lenalidomide or its derivatives) As an example of general formula (1), the following general formula (3) can be given.

[0022] [ka]

[0023] R L1 and R L2 Each of these is independently H, a functional group, or an atom. F-Lenalidomide is a lenalidomide derivative in which fluorine is bonded to the 6th position of the aminoisoindolinone ring of lenalidomide. F-Lenalidomide can be synthesized, for example, by appropriately modifying the starting compounds and reaction steps in known lenalidomide synthesis methods to ensure that fluorine is present at the 6th position of the aminoisoindolinone ring. F-lenalidomide exists as S- and R-isomers, but this invention focuses on the S-isomer, the R-isomer, or mixtures thereof (such as racemic mixtures).

[0024] (Lenalidome or its derivatives) As an example of general formula (1), the following general formula (4) can be given.

[0025] [ka]

[0026] R L1 and R L2 Each of these is independently H, a functional group, or an atom. Lenalidomide is commercially available and can be manufactured by methods that are known to exist. Lenalidomide exists as S-isomers and R-isomers, but this invention focuses on the S-isomer, the R-isomer, or mixtures thereof (such as racemic mixtures).

[0027] (Cl-Lenalidomide or its derivatives) As an example of general formula (1), the following general formula (5) can be given.

[0028] [ka]

[0029] R L1 and R L2 Each of these is independently H, a functional group, or an atom. Cl-Lenalidamide is a lenalidomide derivative in which chlorine is bonded to the 6th position of the aminoisoindolinone ring of lenalidomide. Cl-Lenalidamide can be synthesized, for example, by appropriately modifying the starting compounds and reaction steps in known lenalidomide synthesis methods to have chlorine at the 6th position of the aminoisoindolinone ring. Cl-Lenalidomide exists as S-isomers and R-isomers, but this invention focuses on the S-isomer, the R-isomer, or mixtures thereof (such as racemic mixtures).

[0030] (Br-lenalidomide or its derivatives) As an example of general formula (1), the following general formula (6) can be given.

[0031] [ka]

[0032] R L1 and R L2 Each of these is independently H, a functional group, or an atom. Br-lenalidomide is a lenalidomide derivative in which a Br atom is bonded to the 6th position of the aminoisoindolone ring of lenalidomide. Br-lenalidomide can be synthesized, for example, by appropriately modifying the starting compounds and reaction steps in known lenalidomide synthesis methods to ensure that Br is present at the 6th position of the aminoisoindolone ring. Br-lenalidomide exists as S-isomers and R-isomers, but this invention focuses on the S-isomer, the R-isomer, or mixtures thereof (such as racemic mixtures).

[0033] (F3C-Lenalidomide or its derivatives) As an example of general formula (1), the following general formula (7) can be given.

[0034] [ka]

[0035] R L1 and R L2 Each of these is independently H, a functional group, or an atom. F3C-Lenalidomide is a lenalidomide derivative in which CF3 is bonded to the 6th position of the aminoisoindolone ring of lenalidomide. F3C-Lenalidomide can be synthesized, for example, by appropriately modifying the starting compounds and reaction steps in known lenalidomide synthesis methods to have CF3 at the 6th position of the aminoisoindolone ring. F3C-lenalidomide exists as S-isomers and R-isomers, but this invention focuses on the S-isomer, the R-isomer, or mixtures thereof (such as racemic mixtures).

[0036] (F3CO-Lenalidomide or its derivatives) As an example of general formula (1), the following general formula (8) can be given.

[0037] [ka]

[0038] R L1 and R L2 Each of these is independently H, a functional group, or an atom. F3CO-lenalidomide is a lenalidomide derivative in which F3CO is bonded to the 6th position of the aminoisoindolone ring of lenalidomide. F3CO-lenalidomide can be synthesized, for example, by appropriately modifying the starting compounds and reaction steps in known lenalidomide synthesis methods to have F3CO at the 6th position of the aminoisoindolone ring. F3CO-Lenalidomide exists as S-isomers and R-isomers, but this invention focuses on the S-isomer, the R-isomer, or mixtures thereof (such as racemic mixtures).

[0039] In addition to the compounds mentioned above, derivatives of thalidomy, pomalidomide, and lenalidomide shown in Figures 35 to 38 are also included.

[0040] (Target protein degradation-inducing compound of the present invention) The target protein degradation-inducing compound of the present invention comprises a "cereblon binder" - "linker" - "target protein binder" configuration. The target protein degradation-inducing compound also includes the form of its salt or solvate. The salt is not particularly limited as long as it is pharmaceutically acceptable, but specifically, acid addition salts, base addition salts, etc., are preferred. The solvate is not particularly limited as long as it is pharmaceutically acceptable, but specifically, hydrates, ethanol hydrates, etc., are preferred. Here, the cerebron binder targets one or more compounds selected from the compounds represented by general formula (1) or general formula (10), general formula (3), general formula (4), general formula (5), general formula (6), general formula (7), and general formula (8) described above.

[0041] (Linker) The linker of the present invention can use any linker known in itself, as long as both ends of the linker can be linked (particularly chemically (covalently) linked or coupled) to the cerebron binder and the target protein binder. The linker can be linked to the cerebron binder and the target protein binder by known methods. For example, for a known method of linker linking in PROTAC, refer to the literature "Frontiers in Chemistry, 9, 707317 (2021)". Furthermore, for example, a linker can be expressed by the following formula:

[0042] [Chemical formula]

[0043] In the formula, q is an integer greater than 1; and L is independently a bond, CR M1 R M2 , O, S, SO, SO2, NR M3 , SO2NR M3 , SONR M3 , CONR M3 , NR M3 CONR M4 , NR M3 SO2NR M4 [[ID=2​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​3-11 Cycloalkyl, aryl, heteroaryl, C 3-11 Heterocycline, OC 1-8 Cycloalkyl, SC 1-8 Cycloalkyl, NHC 1-8 Cycloalkyl, N(C 1-8 Cycloalkyl)2, N(C 1-8 Cycloalkyl)(C 1-8 Alkyl), OH, NH2, SH, SO2C 1-8 alkyl, P(O)(OC 1-8 Alkyl)(C 1-8 alkyl), P(O)(OC 1-8 Alkyl)2, CC-C 1-8 Alkyl, CCH, CH=CH(C 1-8 Alkyl), C(C 1-8 Alkyl)=CH(C 1-8 Alkyl), C(C 1-8 Alkyl) = C(C 1-8 Alkyl)2, Si(OH)3, Si(C 1-8 Alkyl)3,Si(OH)(C 1-8 Alkyl)2, COC 1-8 Alkyl, CO2H, Halogen, CN, CF3, CHF2, CH2F, NO2, SF5, SO2NHC 1-8 Alkyl, SO2N(C 1-8 Alkyl) 2, SONHC 1-8 Alkyl, SON(C 1-8 Alkyl)2, CONHC 1-8 Alkyl, CON(C 1-8 Alkyl)2, N(C 1-8 Alkyl)CONH(C 1-8 Alkyl), N(C 1-8 Alkyl)CON(C 1-8 Alkyl)2, NHCONH(C 1-8 Alkyl), NHCON(C 1-8 Alkyl)2, NHCONH2, N(C 1-8 Alkyl)SO2NH(C 1-8 Alkyl), N(C 1-8 Alkyl)SO2N(C 1-8 Alkyl)2, NHSO2NH(C 1-8 Alkyl), NHSO2N(C 1-8It can be selected from the group consisting of alkyl)2 and NHSO2NH2. Also, if q is greater than 1, R M1 or R M2 However, each can independently connect to another L group, resulting in 0 to 4 R groups. M5 It is possible to form cycloalkyl and / or heterocyclyl moieties that can be further substituted with groups. Specifically, the linkers shown in Figures 1 and 2 can be used as examples.

[0044] (Target protein degradation-inducing compound of the present invention) The target protein degradation-inducing compounds of the present invention have reduced degradation-inducing ability for SALL4, PLZF, etc., as demonstrated in the following examples, and can therefore be applied to various diseases (especially cancers). The target protein degradation-inducing compound of the present invention, if it contains the cerebron binder of the present invention, can be used as an active ingredient in therapeutic agents for various diseases by including the known target protein binders shown in Figures 20-32. For example, based on the structure of ARV-825 No. 1 shown in Figure 20, if the composition of the target protein degradation-inducing compound of the present invention is "cereblon binder of the present invention" - "linker" - "JQ1 or OTX-015 (target protein binder)", then this compound can be used as an active ingredient in a treatment for multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), neuroblastoma, T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), or Burkitt's lymphoma. For example, based on the structure of SIAIS178 No. 5 shown in Figure 21, if the composition of the target protein degradation-inducing compound of the present invention is "cerebron binder of the present invention" - "linker" - "dasatinib (target protein binder)", then the compound can be used as an active ingredient in a CML (chronic myeloid leukemia) treatment agent. For example, based on the structure of UNC6852 No. 15 shown in Figure 25, if the composition of the target protein degradation-inducing compound of the present invention is "cereblon binder of the present invention" - "linker" - "EED226 (target protein binder)", then the compound can be used as an active ingredient in a treatment for diffuse large B-cell lymphoma (DLBCL). For example, based on the structure of DGY-08-097 No. 40 shown in Figure 31, if the structure of the target protein degradation-inducing compound of the present invention is "cerebron binder of the present invention" - "linker" - "telaprevir (target protein binder)", then the compound can be used as an active ingredient in a viral therapeutic agent. For example, based on the structure of GSK983 No. 41 shown in Figure 31, if the composition of the target protein degradation-inducing compound of the present invention is "cereblon binder of the present invention" - "linker" - "GSK4027 (target protein binder)", then the compound can be used as an active ingredient in an anti-inflammatory agent. For example, based on the structure of QC-01-175 No. 42 shown in Figure 32, if the composition of the target protein degradation-inducing compound of the present invention is "cereblon binder of the present invention" - "linker" - "AV-1451 (target protein binder)", then the compound can be used as an Alzheimer's disease treatment agent and an active ingredient for frontotemporal dementia. Those skilled in the art can design the target protein degradation-inducing compound of the present invention, including the cerebron binder, based on the above examples and with reference to the structures of known target protein degradation-inducing compounds shown in Figures 20-32.

[0045] (Target protein binder) The "target protein binder" of the present invention can utilize target protein binders used in known PROTACs, and the following are examples. JQ-1 (CAS No.: 1268524-70-4) OTX015 (CAS No.: 202590-98-5) Darolutamide (CASNo. :1297538-32-9) Ceritinib (CAS No. :1032900-25-6) Brigatinib(CAS No.:1197953-54-0) Crizotinib (CAS No.:877399-52-5) Alectinib(CAS No.:1256580-46-7) Venetoclax (CAS No. :1257044-40-8) Dasatinib (CAS No. :302962-49-8) Bosutinib (CAS No. :380843-75-4) RX-37(CAS No.:1627715-60-9) BI-882370 (CAS No. :1392429-79-6) ibrutinib (CAS No.:936563-96-1) CGI1746(CAS No.:910232-84-7) Palbociclib (CAS No.:571190-30-2) Ribociclib (CAS No.: 1211441-98-3) Abemaciclib(CAS No.:1231929-97-7) Apcin-A (CAS No. :1683617-62-0) Apcin(CAS No. :300815-04-7) UNC1999(CAS No.:1431612-23-5) EED226(CAS No.:2083627-02-3) Dacomitinib (CASNo. :1110813-31-4) Lapatinib (CAS No. :388082-78-8) Gefitinib (CAS No.:184475-35-2) afatinib (CAS No.:850140-72-6) Quizartinib (AC220) (CAS No.:950769-58-1) Gilteritinib(CASNo.:1254053-43-4.) MLN-518 (CAS No. :387867-13-2) Sunitinib (CAS No.:341031-54-7) Ponatinib (CAS No.:943319-70-8) ZYF0033 MI-1061(CAS No. :1410737-34-6) MI-77301(CAS No. :1303607-60-4) PD0325901(CAS No.:391210-10-9) EPZ015666(CAS No.:1616391-65-1) RA190(CAS No. :1617495-03-0) SI-109(CAS No. :2429877-30-3) CJ-887(CAS No. :1220952-08-8) GNF-8625(CAS No.:1196546-33-4) I-BRD9(CAS No. :1714146-59-4) BI-7273(CAS No.:1883429-21-7) cortistatin A (CAS No.: 882976-95-6) SNS-032(CAS No. :345627-80-7) wogonin(CAS No. :632-85-9) CX-4945(CAS No. :1009820-21-6) Defactinib(CAS No.:1073154-85-4) quizartinib(CAS No.: 950769-58-1) nexturastat A(CAS No.:1403783-31-2) EED226 (CAS No.: 2083627-02-3) UNC1999(CAS No. :1431612-23-5) Telaprevir (CAS No.:402957-28-2) GSK4027(CAS No. :2079896-25-4) AV-1451 (T807) (CASNo. :1415379-56-4)

[0046] (Target diseases) The target diseases may include, but are not limited to, the following. Multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), neuroblastoma, T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), Burkitt's lymphoma, castration-resistant prostate cancer (CRPC), non-small cell lung cancer (NSCLC), T-cell lymphoma (TCL), chronic myeloid leukemia (CML), triple-negative breast cancer (TNBC), malignant melanoma, mantle cell lymphoma (MCL), colorectal cancer (CRC), triple-negative breast cancer (TNBC), ovarian cancer, multiple myeloma (MM), acute T-cell lymphoma (Acute T-cell Lymphoma, breast cancer, ER (estrogen receptor)-positive breast cancer cells (ER+BC), liver cancer, prostate cancer, Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), viral diseases, inflammatory diseases, and Alzheimer's disease and frontotemporal dementia.

[0047] (Examples of target protein degradation-inducing compounds of the present invention) Preferred target protein degradation-inducing compounds of the present invention are exemplified below, but are not particularly limited. "Cerebromide" - "Linker" - "Target Protein Binder" "Lenalidomide" - "Linker" - "JQ-1 or Birabresib" "F-Lenalidomide" - "Linker" - "JQ-1 or Birabresib" "F3C-Lenalidomide" - "Linker" - "JQ-1 or Birabresib" "F3CO-Lenalidomide" - "Linker" - "JQ-1 or Birabresib" "Cl-Lenalidomide" - "Linker" - "JQ-1 or Birabresib" "Br-Lenalidomide" - "Linker" - "JQ-1 or Birabresib" "F-Pomalidomide" - "Linker" - "JQ-1 or Birabresib" "F3C-Pomalidomide" - "Linker" - "JQ-1 or Birabresib"

[0048] (Example of synthesis of the target protein degradation-inducing compound of the present invention) The synthesis of the target protein degradation-inducing compound of the present invention can be carried out by appropriately modifying known methods for synthesizing target protein degradation-inducing compounds. For example, "F-lenalidomide"-"linker"-"JQ-1 or Birabresib" can be synthesized by using "F-lenalidomide" instead of "lenalidomide" in the synthesis process of ARV-825 (CAS No.: 1818885-28-7), a known target protein degradation inducer.

[0049] (Indications for the cancer treatment composition of the present invention) Based on the results of the following examples, the preferred cancers for which the following compounds are included as active ingredients are hematological cancers (multiple myeloma, myelodysplastic syndrome, acute myeloid leukemia, etc.). 〇 Compound represented by general formula (4) (lenalidomide) 〇 Compound represented by general formula (5) (Cl-lenalidomide) 〇 Compound represented by general formula (3) (F-lenalidomide) ○ "Lenalidomide" - "Linker" - "JQ-1 or Birabresib"

[0050] (Indications for the cancer treatment composition of the present invention) The following compounds, which are included as active ingredients, are preferred for use in cancers, and based on the results of the following examples, their teratogenicity has been reduced. This includes not only hematological cancers but also the following types of cancer. 〇 Compound represented by general formula (5) (Cl-lenalidomide) 〇 Compound represented by general formula (3) (F-lenalidomide) 〇 Compound represented by general formula (6) (Br-lenalidomide) 〇 Compound represented by general formula (7) (F3C-lenalidomide) 〇 Compound represented by general formula (8) (F3CO-lenalidomide) ○ "F-Lenalidomide" - "Linker" - "JQ-1 or Birabresib" ○ "F3C-Lenalidomide" - "Linker" - "JQ-1 or Birabresib" ○ "F3CO-Lenalidomide" - "Linker" - "JQ-1 or Birabresib" ○ "Br-Lenalidomide" - "Linker" - "JQ-1 or Birabresib" ○ "Cl-Lenalidome" - "Linker" - "JQ-1 or Birabresib" carcinoma These include bladder cancer, intestinal cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, head cancer, kidney cancer, liver cancer, lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, leukemia, malignant lymphoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, hepatocellular carcinoma, renal cell carcinoma, malignant melanoma, myeloproliferative disorders, multiple myeloma, sarcoma, breast cancer, uterine cancer, lung cancer, testicular cancer, thyroid cancer, esophageal cancer, etc.

[0051] (Reduction of teratogenicity) Teratogenicity refers to the property or effect of certain substances, such as drugs, that inhibit the normal development of living organisms and cause birth defects. It is used synonymously with teratogenicity. In this invention, "reduced teratogenicity" can be expressed as a value of 100 when the protein resolution of SALL4, PLZF, CK1α, IKZF1, or IKZF3 by direct or indirect binding of lenalidomide, an E3 ligase (cereblon) binder in lenalidomide or a PROTAC compound, to cereblon is set to 100 (see Example 17). The resolution of F-Len's SALL4 is in the range of 1 to 70, preferably 5 to 60, and more preferably 10 to 50, compared to lenalidomide. The resolution of F-Len's PLZF is in the range of 0 to 30, preferably 0 to 20, and more preferably 0 to 10, compared to lenalidomide. The resolution of Cl-Len's SALL4 is in the range of 0.5 to 20, preferably 1.0 to 15, and more preferably 1.5 to 10, compared to lenalidomide. The resolution of Cl-Len's PLZF is in the range of 0 to 30, preferably 0 to 20, and more preferably 0 to 10, compared to lenalidomide. The resolution of Cl-Len's IKZF1 is in the range of 50 to 90, preferably 60 to 85, and more preferably 65 to 80, compared to lenalidomide. The resolution of Cl-Len's IKZF3 is in the range of 30 to 70, preferably 35 to 65, and more preferably 40 to 60, compared to lenalidomide. The resolution of Br-Len's SALL4 is in the range of 0.5 to 40, preferably 1.0 to 35, and more preferably 1.5 to 30, compared to lenalidomide. The resolution of Br-Len's PLZF is in the range of 0 to 30, preferably 0 to 20, and more preferably 0 to 10, compared to lenalidomide. The resolution of Br-Len's IKZF1 is in the range of 15 to 50, preferably 20 to 45, and more preferably 25 to 40, compared to lenalidomide. The resolution of Br-Len's IKZF3 is in the range of 0 to 30, preferably 0 to 20, and more preferably 0 to 10, compared to lenalidomide. The resolution of SALL4 in F3C-Len is in the range of 0 to 30, preferably 0 to 20, and more preferably 0 to 10, compared to lenalidomide. The resolution of CK1α in F3C-Len is in the range of 0 to 90, compared to lenalidomide. The resolution of IKZF1 in F3C-Len is in the range of 0 to 30, preferably 0 to 20, and more preferably 0 to 10, compared to lenalidomide. The resolution of IKZF3 in F3C-Len is in the range of 0 to 30, preferably 0 to 20, and more preferably 0 to 10, compared to lenalidomide. The resolution of SALL4 in F-Len-PROTAC is in the range of 30 to 90, preferably 40 to 80, and more preferably 50 to 70, compared to Len-PROTAC. The resolution of PLZF in F-Len-PROTAC is in the range of 0 to 60, preferably 5 to 50, compared to Len-PROTAC. The resolution of IKZF3 in F-Len-PROTAC is in the range of 60 to 90, compared to Len-PROTAC. The resolution of SALL4 in Cl-Len-PROTAC is in the range of 5 to 50, preferably 10 to 40, and more preferably 15 to 30, compared to Len-PROTAC. The resolution of PLZF in Cl-Len-PROTAC is in the range of 0 to 70, preferably 0 to 60, compared to Len-PROTAC. The resolution of IKZF1 in Cl-Len-PROTAC is in the range of 70 to 95, compared to Len-PROTAC. The resolution of IKZF3 in Cl-Len-PROTAC is in the range of 20 to 80, preferably 30 to 70, and more preferably 40 to 60, compared to Len-PROTAC. The resolution of SALL4 in F3C-Len-PROTAC is in the range of 0 to 30, preferably 0 to 20, and more preferably 0 to 10, compared to Len-PROTAC. The resolution of PLZF in F3C-Len-PROTAC is in the range of 0 to 30, preferably 0 to 15, compared to Len-PROTAC. The resolution of CK1α in F3C-Len-PROTAC is in the range of 0 to 30, preferably 0 to 20, and more preferably 0 to 10, compared to Len-PROTAC. The resolution of IKZF1 in F3C-Len-PROTAC is in the range of 5 to 50, preferably 10 to 40, and more preferably 15 to 35, compared to Len-PROTAC. The resolution of IKZF3 in F3C-Len-PROTAC is in the range of 10 to 70, preferably 20 to 60, and more preferably 30 to 50, compared to Len-PROTAC. The inventors have confirmed that thalidomide derivatives with reduced protein degradation capabilities of SALL4 or PLZF exhibit reduced teratogenicity (see EMBO J(2021)40:e105375).

[0052] (Method for administering the cancer treatment composition of the present invention) The cancer treatment composition of the present invention can be administered orally or parenterally. For oral administration, known dosage forms such as tablets, capsules, coated tablets, lozenges, solutions, or suspensions can be used. Parenteral administration can be administered intravenously, intramuscularly, or subcutaneously by injection; transmucosal administration such as through the nasal cavity or oral cavity using sprays or aerosols; rectal administration using suppositories; or transdermal administration using patches, liniments, or gels. Preferably, oral administration, transnasal administration, or intravenous administration by injection are preferred.

[0053] (Carrier included in the cancer treatment composition of the present invention) The cancer treatment composition of the present invention may, in addition to the pharmaceutically active ingredient, optionally contain a suitable pharmaceutically acceptable carrier well known to those skilled in the art, depending on the dosage form and other factors. Examples of pharmaceutically acceptable carriers include antioxidants, stabilizers, preservatives, flavoring agents, colorants, solvents, solubilizers, surfactants, emulsifiers, defoaming agents, viscosity modifiers, gelling agents, absorption enhancers, dispersants, excipients, and pH adjusters.

[0054] (Method for formulating the cancer treatment composition of the present invention) When preparing the cancer treatment composition of the present invention as an injectable formulation, a solution or suspension formulation is preferred. For transmucosal administration, such as to the nasal cavity or oral cavity, a powder, drop, or aerosol formulation is preferred. For rectal administration, a semi-solid formulation such as a cream or suppository is preferred. All of these formulations can be prepared by any method known to those skilled in the art of pharmaceutical technology, such as as described in Remington's Pharmaceutical Sciences (Mac Publishing Company, Easton, PA, 1970). The injectable formulation can be used as a carrier, for example, by adding plasma-derived proteins such as albumin, amino acids such as glycine, and sugars such as mannitol. Furthermore, buffers, solubilizers, and isotonic agents may also be added. When used as a water-soluble or lyophilized formulation, it is preferable to add a surfactant such as Tween® 80 or Tween® 20 to prevent aggregation. Furthermore, parenteral dosage forms other than injectable formulations may contain distilled water or physiological saline, polyalkylene glycosides such as polyethylene glycosides, plant-derived oils, and hydrogenated naphthalene. For example, rectal formulations such as suppositories may contain polyalkylene glycosides, petrolatum, and cocoa oil as common excipients. Vaginal formulations may contain absorption enhancers such as bile salts, ethylenediamine salts, and citrates. Inhalation formulations may be solid and may contain lactose as an excipient, for example, and nasal drops may be water or oil solutions.

[0055] (Method for administering the cancer treatment composition of the present invention) The precise dosage and administration plan of the cancer treatment composition of the present invention can be adjusted depending on the required amount for each individual treatment target, the treatment method, the degree of the disease or need, etc. Specifically, the dosage can be determined according to age, weight, general health status, sex, diet, administration time, administration method, excretion rate, drug combination, and the patient's medical condition, and may also be determined by considering other factors. The daily dosage of the cancer treatment composition of the present invention varies depending on the patient's condition, weight, type of compound, route of administration, etc., but for example, in the case of parenteral administration, it is desirable to administer the active ingredient at a dose of about 0.01 to 1000 mg / person / day, preferably 0.1 to 500 mg / person / day, and in the case of oral administration, it is desirable to administer it at a dose of about 0.01 to 500 mg / person / day, preferably 0.1 to 100 mg / person / day.

[0056] The present invention will be described in more detail below with reference to examples, but the following examples should be considered as an aid to gaining a concrete understanding of the present invention, and the scope of the present invention is not limited in any way by the following examples. [Examples]

[0057] (Confirmation of the interaction ability of each thalidomide derivative with IKZF1, SALL4, and PLZF) In this example, the affinity (degradation-inducing ability) of each thalidomide derivative for IKZF1, SALL4, and PLZF was confirmed by biochemical interaction analysis. Details are as follows.

[0058] (Experimental method) FLAG-GST-IKZF1,-SALL4,-PLZF and N-terminally biotinylated bls-CRBN were synthesized as recombinant proteins using a wheat cell-free protein synthesis system. A 10 μl mixture containing 0.5 μl of the synthesized bls-CRBN was prepared in AlphaScreen buffer (100 mM Tris (pH 8.0), 0.01% Tween20, 100 mM NaCl, and 1 mg / mL BSA). A 5 μl mixture containing 0.8 μl of FLAG-GST-protein was prepared in AlphaScreen buffer. A 5 μl mixture containing DMSO (final concentration 0.5%) or the final concentration of the thalidomide derivative shown in Figure 4 was prepared in AlphaScreen buffer. Subsequently, the three mixtures were mixed in a 384-well AlphaPlate (PerkinElmer) and allowed to stand at 26°C for 1 hour. Next, a 5 μl detection mixture containing 0.2 μg / ml anti-DYKDDDDK antibody (FUJIFILM WakoPure Chemical), 0.08 μl streptavidin donor beads (PerkinElmer), and 0.08 μl protein A acceptor beads (PerkinElmer) was prepared under AlphaScreen buffer. Then, the 5 μl detection mixture was added to each well, and after standing at 26°C for 1 hour, the luminescence signal was detected using an Envision plate reader (PerkinElmer).

[0059] (result) The structures of each thalidomide derivative used in this example are shown in Figure 3. As is clear from the results in Figure 4, it was confirmed that the insertion of a substituent at the meta position of lenalidomide reduces its affinity (degradation-inducing ability) for the teratogenic proteins SALL4 and PLZF. [Examples]

[0060] (Confirmation of the degradation-inducing ability of each thalidomide derivative against IKZF1 and SALL4) In this example, the affinity (degradation-inducing ability) of each thalidomide derivative for IKZF1 and SALL4 was confirmed by analyzing the degradation of overexpressed neosubstrates (SALL4 and IKZF1) in cultured cells. Details are as follows.

[0061] (Experimental method) HEK293T cultured cells were seeded into 48-well cell culture plates (BD Falcon) and cultured overnight at 37°C under 5% CO2 conditions. Subsequently, AGIA-SALL4 and Myc-IKZF1 were transfected using polyethyleneimine (PEI)Max (MW 40,000) (PolyScience, Inc.). Six hours after transfection, DMSO (final concentration 0.1%) or thalidomide derivatives at the final concentrations shown in Figure 5 were administered. Eighteen hours after drug administration, the supernatant was removed using an aspirator, and the cultured cells were recovered and lysed using 1×SDS sample buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol). The recovered cell extracts were boiled at 95°C for 5 minutes and subjected to electrophoresis using a polyacrylamide gel. Next, immunoblotting was performed, and each protein was detected using the antibodies shown in Figure 5.

[0062] (result) As is clear from the results in Figure 5, the insertion of a substituent at the meta position of lenalidomide reduces the ability to induce degradation of SALL4, a teratogenic protein, similar to Example 1, but maintains the ability to induce degradation of IKZF1, and does not reduce the affinity for CRBN. [Examples]

[0063] (Confirmation of interaction-inducing ability of thalidomide derivatives with substituent insertions at each meta position to IKZF1, SALL4, and PLZF) In this example, the affinity (degradation-inducing ability) of each meta-substituted thalidomide derivative to IKZF1, SALL4, and PLZF was confirmed by biochemical interaction analysis. Details are as follows.

[0064] (Experimental method) FLAG-GST-IKZF1, -SALL4, -PLZF, and N-terminally biotinylated bls-CRBN were synthesized as recombinant proteins using a wheat cell-free protein synthesis system. A 10 μl mixture containing 0.5 μl of the synthesized bls-CRBN was prepared in AlphaScreen buffer (100 mM Tris (pH 8.0), 0.01% Tween20, 100 mM NaCl, and 1 mg / mL BSA). A 5 μl mixture containing 0.8 μl of FLAG-GST-protein was prepared in AlphaScreen buffer. A 5 μl mixture containing DMSO (final concentration 0.5%) or the final concentration of the thalidomide derivative shown in Figure 6 was prepared in AlphaScreen buffer. Subsequently, the three mixtures were mixed in a 384-well AlphaPlate (PerkinElmer) and allowed to stand at 26°C for 1 hour. Next, a 5 μl detection mixture containing 0.2 μg / ml anti-DYKDDDDK antibody (FUJIFILM WakoPure Chemical), 0.08 μl streptavidin donor beads (PerkinElmer), and 0.08 μl protein A acceptor beads (PerkinElmer) was prepared under AlphaScreen buffer. Then, the 5 μl detection mixture was added to each well, and after standing at 26°C for 1 hour, the luminescence signal was detected using an Envision plate reader (PerkinElmer).

[0065] (result) As is clear from the results in Figure 6, meta-substituted substituent insertion of lenalidomide reduced its affinity for the teratogenic proteins SALL4 and PLZF compared to meta-substituted substituent insertion of thalidomide and pomalidomide, while maintaining its affinity for IKZF1, a drug target protein for hematological cancers. Thereafter, in subsequent examples, lenalidomide and lenalidomide derivatives with substituents inserted at each meta-position were used. Although it is necessary to maintain the affinity for IKZF for blood cancers, generally, the affinity for IKZF is not required.

Example

[0066] (Synthesis of lenalidomide derivatives with substituents inserted at the meta-position) In this example, lenalidomide derivatives with substituents inserted at each meta-position were synthesized. Specifically, it is as follows. Lenalidomide was purchased from FUJIFILM Wako Pure Chemical. 5-hydroxythalidomide (5HT) was synthesized by the method disclosed in the literature Bioorg. Med Chem Lett 19:3973-3976.

[0067] (Synthesis of F-lenalidomide) It was synthesized according to the following procedure.

[0068]

Chemical formula

[0069] Methyl 2-(bromomethyl)-5-fluoro-3-nitrobenzoate (Reference: WO2005034939 A1) 438 mg (1.50 mmol) and 3-aminopiperidine-2,6-dione hydrochloride 247 mg (1.50 mmol) were dissolved in 1.5 mL of N,N-dimethylformamide, then 0.25 mL (1.8 mmol) of triethylamine was added, and the mixture was stirred at 50 °C for 12 hours. After cooling to room temperature, water was added and the mixture was extracted with ethyl acetate. After drying over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure. The obtained solid was purified by silica gel column chromatography to obtain 3-(6-fluoro-4-nitro-1-oxoisoindolin-2-yl)piperidine-2,6-dione in a yield of 150 mg and a yield of 33%. The compound data of 3-(6-fluoro-4-nitro-1-oxoisoindolin-2-yl)piperidine-2,6-dione (Reference: WO 2016065980 A1, WO 2019079701 A1) are shown below. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 11.05 (s, 1H), 8.38 (dd, J = 9.1, 1.9 Hz, 1H), 8.11 (dd, J = 6.8, 1.7 Hz, 1H), 5.22 - 5.11 (m, 1H), 4.80 (dd, J = 35.2, 18.9 Hz, 2H), 2.99 - 2.78 (m, 1H), 2.64 - 2.50 (m, 2H), 2.07 - 1.90 (m, 1H). 19F NMR (282 MHz, DMSO-d6) δ (ppm) 109.38 (dd, J = 7.7 Hz, 1F). ESIMS calculated for C13H9FN3O5 ([M-H]-) 306.05, found 306.05

[0070]

Chemical Structure

[0071] 100 mg (0.327 mmol) of 3-(6-fluoro-4-nitro-1-oxoisoindorin-2-yl)piperidine-2,6-dione was dissolved in 4.1 mL of 1,4-dioxane, and the reaction solution was degassed. Next, 22.9 mg of palladium-carbon hydroxide was added, and the reaction vessel was replaced with hydrogen. After stirring for 3 hours, the reaction solution was filtered, and the solvent was removed under reduced pressure to obtain 3-(4-amino-6-fluoro-1-oxoisoindorin-2-yl)piperidine-2,6-dione in a yield of 16.1 mg and 18%. The compound data for 3-(4-amino-6-fluoro-1-oxoisoindorin-2-yl)piperidine-2,6-dione is shown below. 1 H NMR(300 MHz, DMSO- d6) δ (ppm) 11.03 (s, 1H), 6.69 - 6.49 (m, 2H), 5.81(s, 2H), 5.15 - 5.04 (m, 1H), 4.13 (dd, J = 34.3, 17.0 Hz, 2H), 3.00 - 2.81 (m, 1H), 2.70 - 2.54 (m, 1H), 2.38 - 2.19 (m, 1H), 2.11 - 1.96 (m, 1H). 19 F NMR(282 MHz, DMSO- d6) δ (ppm) -113.63 (dd, J = 11.1, 8.3 Hz,1F). ESIMS calculated for C 13 H 11 FN3O([MH] - ) 276.08, found 276.05

[0072] (Synthesis of Cl-lenalidomide) It was synthesized using the following procedure.

[0073] [ka]

[0074] 2.1 g (6.7 mmol) of methyl 2-(bromomethyl)-5-chloro-3-nitrobenzoate (reference: Wang, Bing; Chu, Daniel, PCT Int. Appl. (2011), WO 2011130661 A1) and 1.3 g (8.1 mmol) of 3-aminopiperidine-2,6-dione hydrochloride were dissolved in 7.0 mL of N,N-dimethylformamide, and then 1.1 mL (8.1 mmol) of triethylamine was added. The mixture was stirred at 60 °C for 19 hours. After cooling to room temperature, water was added, and the precipitate was filtered off. The resulting solid was dried under reduced pressure to obtain 3-(6-chloro-4-nitro-1-oxoisoindorin-2-yl)piperidine-2,6-dione in a yield of 1.0 g and 49%. The following shows the compound data for 3-(6-chloro-4-nitro-1-oxoisoindorin-2-yl)piperidine-2,6-dione. mp: 253.3-254.1 ℃. 1 H NMR (300 MHz, DMSO-d6) δ:11.08 (s, 1H), 8.52 (d, J = 1.8 Hz, 1H), 8.28 (d, J = 1.6 Hz,1H), 5.20 (dd, J = 13.3, 5.1 Hz, 1H), 4.85 (dd, J = 34.6, 19.3Hz, 2H), 2.99-2.87 (m, 1H), 2.74-2.56 (m, 2H), 2.05-1.99 (m, 1H) ppm. 13 CNMR (176 MHz, DMSO-d6) δ: 172.8, 170.6, 164.8, 143.9, 136.2, 136.1, 134.3,129.3 (d, J = 26.8 Hz), 126.8 (d, J = 31.8 Hz), 52.0, 48.4, 31.2,22.2 ppm. ATR-FTIR (KBr): ν = 3087, 1691, 1537, 1450, 1353, 1322, 1192, 889, 731, 531 cm -1 HRMS (ESI + ): m / z calcd for C13H9ClN3O5[MH] -: 322.0236 found: 322.0234.

[0075] [ka]

[0076] 1.0 g (3.1 mmol) of 3-(6-chloro-4-nitro-1-oxoisoindorin-2-yl)piperidine-2,6-dione, 1.3 g (23.5 mmol) of iron, and 1.3 g (24.7 mmol) of ammonium chloride were dissolved in 39 mL of ethanol and 1.2 mL of water, and refluxed for 16 hours. After cooling to room temperature, the solid was removed by filtration, and the resulting filtrate was removed under reduced pressure. Purification by recrystallization with ethanol yielded 526 mg of 3-(4-amino-6-chloro-1-oxoisoindorin-2-yl)piperidine-2,6-dione in a yield of 58%. The compound data for 3-(4-amino-6-chloro-1-oxoisoindorin-2-yl)piperidine-2,6-dione is shown below. mp: 242.7-243.7 °C. 1 H NMR (500 MHz, DMSO-d6) δ:10.98 (s), 6.81 (d, J = 1.8 Hz), 6.77 (d, J = 1.8 Hz), 5.76 (s),5.05 (dd, J = 13.1, 5.2 Hz), 4.18-4.04 (m), 2.91-2.83 (m), 2.59-2.54(m), 2.29-2.20 (m), 2.04-1.97 (m) ppm. 13 ATR-FTIR (KBr): ν = 3455, 3347, 3036, 2847, 1685, 1671, 1487,1356, 1029, 957 cm -1 HRMS (ESI +): m / z calcd for C13H11ClN3O3 [MH] - : 292.0494 found: 292.0487.

[0077] (Synthesis of Br-lenalidomide) It was synthesized using the following procedure.

[0078] [ka]

[0079] 1.0 g (2.8 mmol) of methyl 5-bromo-2-(bromomethyl)-3-nitrobenzoate (Hayashi, Kazuya; Yamashiro, Yoshiko; Taya, Kyoko; Fukuyama, Hiroko; Todo, Yozo, PCT Int. Appl. (1999), WO9907682) and 560 mg (3.4 mmol) of 3-aminopiperidine-2,6-dione hydrochloride were dissolved in 3.9 mL of N,N-dimethylformamide. Then, 979 mg (7.1 mmol) of potassium carbonate was added, and the mixture was stirred at 50 °C for 2 hours. After cooling to room temperature, water was added, and the precipitate was filtered off. The resulting solid was dried under reduced pressure to obtain 3-(6-bromo-4-nitro-1-oxoisoindorin-2-yl)piperidine-2,6-dione in a yield of 564 mg and 54%. The following shows the compound data for 3-(6-bromo-4-nitro-1-oxoisoindorin-2-yl)piperidine-2,6-dione. mp: 226.6-227.6 °C. 1 H NMR (300 MHz, DMSO-d6) δ:11.06 (s, 1H), 8.59 (d, J = 1.5 Hz, 1H), 8.37 (d, J = 1.5 Hz,1H), 5.21-5.15 (m, 1H), 4.81 (dd, J = 34.3, 19.3 Hz, 2H), 2.97-2.87 (m,1H), 2.72-2.62 (m, 2H), 2.03-1.99 (m, 1H) ppm. 1313C NMR (176 MHz, DMSO-d6) δ: 172.8, 170.6, 164.7, 144.0, 136.4 (d, J = 20.1 Hz), 132.0, 129.4, 121.8, 52.0, 48.4, 31.1, 22.2 ppm. ATR-FTIR (KBr): ν = 3208, 3108, 3074, 1711, 1532, 1442, 1354, 1209, 1182, 720 cm -1 .HRMS (ESI + ): m / z calcd for C13H9BrN3O5 [M-H] - : 365.9731 found: 365.9723.

[0080]

Chem.

[0081] 3-(6-Bromo-4-nitro-1-oxoisoindolin-2-yl)piperidine-2,6-dione (200 mg, 0.54 mmol), iron (231 mg, 4.1 mmol), and ammonium chloride (233 mg, 4.4 mmol) were dissolved in 0.8 mL of ethanol and 1.4 mL of water, and stirred at 60 °C for 2 hours. After cooling to room temperature, the solid was removed by filtration, and the resulting filtrate was evaporated under reduced pressure. Extraction was performed using ethyl acetate, the organic layer was washed with saturated brine, and dried over anhydrous sodium sulfate. Evaporation of the solvent under reduced pressure gave 3-(4-amino-6-bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione in a yield of 20% (36.6 mg). The compound data of 3-(4-amino-6-bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione are shown below. m.p.: 237.9 - 238.7 °C. 1H NMR (300 MHz, DMSO-d6) δ:11.03 (s, 1H), 6.97-6.95 (m, 2H), 5.80 (s, 2H), 5.12- 5.06 (m, 1H), 4.12 (dd, J= 35.5, 17.6 Hz, 2H), 2.95-2.87 (m, 1H), 2.73-2.63 (m, 2H), 2.05-1.99 (m, 1H)ppm. 13 C NMR (176 MHz, DMSO-d6) δ: 172.9, 171.1,167.5, 145.4, 134.2, 124.9, 121.7, 118.0, 112.4, 51.7, 45.6, 39.5, 31.2, 22.7ppm. ATR-FTIR (KBr): ν = 3425, 2186, 3083, 1681, 1605, 1481, 1462, 1335, 1199, 1026 cm -1 HRMS (ESI + ): m / z calcd for C13H11BrN3O3 [MH] - : 335.9989 found: 335.9974.

[0082] (Synthesis of F3C-lenalidomide) It was synthesized using the following procedure.

[0083] [ka]

[0084] 2-Methyl-3-nitro-5-(trifluoromethyl)benzoic acid (Reference: Kuntz, Kevin Wayne; Olhava, Edward James; Chesworth, Richard; Duncan, Kenneth William, WO 2012118812 A2) 1.1 g (4.4 mmol) was dissolved in 44 mL of methanol, and 1.0 mL (13.2 mmol) of thionyl chloride was slowly added dropwise. The reaction solution was then refluxed for 12 hours. After cooling to room temperature, water was added, followed by the addition of 1 mol / L aqueous sodium hydroxide solution, and the mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain methyl 2-methyl-3-nitro-5-(trifluoromethyl)benzoate in a yield of 939 mg and 78%. The compound data for methyl 2-methyl-3-nitro-5-(trifluoromethyl)benzoate is shown below. 1 H NMR (300 MHz, CDCl3)δ 8.27 (s, 1H), 8.11 (s, 1H), 3.98 (s, 3H), 2.70 (s, 3H) ppm. 19 F NMR (282 MHz, CDCl3)δ -63.46 (s, 3F) ppm.

[0085] [ka]

[0086] 646 mg (3.2 mmol) of methyl 2-methyl-3-nitro-5-(trifluoromethyl)benzoate and 1.1 g (6.3 mmol) of N-bromosuccinimide were dissolved in 4.5 mL of degassed carbon tetrachloride, and 102 mg (0.32 mmol) of 75% benzoyl peroxide was added. The reaction solution was then refluxed for 14 hours. After cooling to room temperature, by-products were removed by filtration, and the filtrate was removed under reduced pressure. The resulting mixture was purified by silica gel column chromatography to obtain 734 mg of methyl 2-(bromomethyl)-3-nitro-5-(trifluoromethyl)benzoate in 68% yield. The compound data for methyl 2-(bromomethyl)-3-nitro-5-(trifluoromethyl)benzoate is shown below. 1H NMR (300 MHz, CDCl3)δ 8.36 (s, 1H), 8.21 (s, 1H), 5.18 (s, 2H), 4.04 (s, 3H) ppm. 19 F NMR (282 MHz, CDCl3)δ -63.74 (s, 3F) ppm.

[0087] [ka]

[0088] 600 mg (1.8 mmol) of 2-(bromomethyl)-3-nitro-5-(trifluoromethyl)methyl benzoate and 346 mg (2.1 mmol) of 3-aminopiperidine-2,6-dione hydrochloride were dissolved in 1.8 mL of N,N-dimethylformamide, and then 0.5 mL (3.5 mmol) of triethylamine was added. The mixture was stirred at 50 °C for 14 hours. After cooling to room temperature, water was added, and the precipitate was filtered off. The resulting solid was washed with methanol and tetrahydrofuran and dried under reduced pressure to obtain 3-(4-nitro-1-oxo-6-(trifluoromethyl)isoindorin-2-yl)piperidine-2,6-dione in a yield of 197 mg and 31%. The compound data for 3-(4-nitro-1-oxo-6-(trifluoromethyl)isoindorin-2-yl)piperidine-2,6-dione is shown below. 1 H NMR (300 MHz, DMSO-d6)δ 11.08 (s, 1H), 8.71 (s, 1H), 8.52 (s, 1H), 5.23 (dd, J = 12.9, 5.0 Hz,1H), 4.96 (dd, J = 33.7, 19.9 Hz, 2H), 2.86-2.98 (m, 1H), 2.54-2.63 (m,2H), 2.03 (m, J = 5.1 Hz, 1H) ppm. 19 F NMR (282 MHz, DMSO-d6)δ -60.46 (s, 3F) ppm.

[0089] [ka]

[0090] 100 mg (0.29 mmol) of 3-(4-nitro-1-oxo-6-(trifluoromethyl)isoindorin-2-yl)piperidine-2,6-dione and 20 mg of palladium carbon were dissolved in 10 mL of tetrahydrofuran and 10 mL of methanol. The reaction solution was degassed, the reaction vessel was replaced with hydrogen, and the mixture was stirred at room temperature for 1 hour. The reaction solution was filtered, and the solvent was removed under reduced pressure to obtain 3-(4-amino-1-oxo-6-(trifluoromethyl)isoindorin-2-yl)piperidine-2,6-dione in a yield of 77 mg and 81%. The compound data for 3-(4-amino-1-oxo-6-(trifluoromethyl)isoindorin-2-yl)piperidine-2,6-dione is shown below. 1 H NMR (300 MHz, DMSO-d6)δ 11.22-10.84 (1H), 7.11 (s, 1H), 7.09 (s, 1H), 6.00 (s, 2H), 5.14 (dd, J= 13.2, 5.3 Hz, 1H), 4.24 (dd, J = 36.3, 17.7 Hz, 2H), 2.86-2.96 (m,1H), 2.60-2.73 (m, 1H), 2.25-2.39 (m, 1H), 2.01-2.08 (m, 1H) ppm. 19 F NMR (282 MHz, DMSO-d6)δ-60.64 (s,3F) ppm.

[0091] (Synthesis of F3CO-lenalidomide) It was synthesized using the following procedure.

[0092] [ka]

[0093] 500 mg (2.3 mmol) of 2-methyl-5-(trifluoromethoxy)benzoic acid (reference: WO 9730989 A1) was dissolved in 5.0 mL of concentrated sulfuric acid. The reaction solution was cooled to 0 °C, and 0.5 mL of fuming nitric acid was slowly added dropwise. The mixture was then stirred at room temperature for 4 hours. The reaction solution was poured into ice water, and the resulting precipitate was filtered off. The filtered precipitate was washed with water and dried under reduced pressure to obtain a mixture of positional isomers of 2-methyl-3-nitro-5-(trifluoromethoxy)benzoic acid. The resulting mixture was not further purified and was used in the next step.

[0094] [ka]

[0095] 2.0 g (7.5 mmol) of 2-methyl-3-nitro-5-(trifluoromethoxy)benzoic acid was dissolved in 15 mL of methanol, and 1.6 mL (22.6 mmol) of thionyl chloride was slowly added dropwise. The reaction solution was then refluxed for 12 hours. After cooling to room temperature, the solvent was removed under reduced pressure. The resulting mixture was purified by silica gel column chromatography to obtain methyl 2-methyl-3-nitro-5-(trifluoromethoxy)benzoate in a yield of 969 mg and 46%. The compound data for methyl 2-methyl-3-nitro-5-(trifluoromethoxy)benzoate is shown below. 1 H NMR (300 MHz, CDCl3)δ: 7.89 (s, 1H), 7.75 (s, 1H), 3.97 (s, 3H), 2.64 (s, 3H) ppm. 19 F NMR (282 MHz, CDCl3)δ: -58.7 (s, 1F) ppm. 13 C NMR (126 MHz, CDCl3)δ: 165.5, 152.3, 146.5 (d, J = 1.8 Hz), 134.9, 132.1, 126.3, 120.3 (q, J= 260.0 Hz), 119.6, 53.1, 16.0 ppm.

[0096] [ka]

[0097] 722 mg (2.5 mmol) of methyl 2-methyl-3-nitro-5-(trifluoromethoxy)benzoate and 893 mg (5.0 mmol) of N-bromosuccinimide were dissolved in 3.6 mL of degassed carbon tetrachloride, and 81 mg (0.25 mmol) of 75% benzoyl peroxide was added. The reaction solution was then refluxed for 14 hours. After cooling to room temperature, by-products were removed by filtration, and the filtrate was removed under reduced pressure. The resulting mixture was purified by silica gel column chromatography to obtain 734 mg of methyl 2-(bromomethyl)-3-nitro-5-(trifluoromethoxy)benzoate in 68% yield. The compound data for methyl 2-(bromomethyl)-3-nitro-5-(trifluoromethoxy)benzoate is shown below. 1 H NMR (300 MHz, CDCl3)δ 7.97 (s, 1H), 7.84 (s, 1H), 5.14 (s, 2H), 4.03 (s, 3H) ppm. 19 F NMR (282 MHz, CDCl3)δ: -58.5 (s, 3F) ppm. 13 C NMR (126 MHz, CDCl3)δ: 164.7, 151.2, 148.4, 134.3, 131.6, 127.0, 120.4, 120.2 (q, J = 260.9Hz), 53.7, 22.0 ppm.

[0098] [ka]

[0099] 104 mg (0.28 mmol) of methyl 2-(bromomethyl)-3-nitro-5-(trifluoromethoxy)benzoate and 55 mg (0.34 mmol) of 3-aminopiperidine-2,6-dione hydrochloride were dissolved in 0.28 mL of N,N-dimethylformamide, and then 0.08 mL (0.60 mmol) of triethylamine was added. The mixture was stirred at 50 °C for 14 hours. After cooling to room temperature, water was added, and the precipitate was filtered off. The resulting precipitate was washed with methanol and tetrahydrofuran and dried under reduced pressure to obtain 3-(4-nitro-1-oxo-6-(trifluoromethoxy)isoindorin-2-yl)piperidine-2,6-dione in a yield of 78 mg and 46%. The compound data for 3-(4-nitro-1-oxo-6-(trifluoromethoxy)isoindorin-2-yl)piperidine-2,6-dione is shown below. 1 H NMR (500 MHz, DMSO-d6)δ 11.06 (s, 1H), 8.46 (d, J = 1.5 Hz, 1H), 8.20 (d, J = 0.9 Hz,1H), 5.19 (dd, J = 13.1, 5.2 Hz, 1H), 4.88 (dd, J = 47.8, 19.4Hz, 2H), 2.95-2.87 (m, 1H), 2.73-2.53 (m, 2H), 2.05-1.99 (m, 1H) ppm. 19 F NMR (282 MHz, DMSO-d6)δ -57.0 (s, 3F) ppm. 13 C NMR (126 MHz, DMSO-d6)δ 172.8, 170.5, 164.8, 148.3, 144.1, 136.4, 122.3, 120.5, 119.9 (q, J =258.5 Hz), 52.1, 48.5, 31.1, 22.1 ppm.

[0100] [ka]

[0101] 50 mg (0.13 mmol) of 3-(4-nitro-1-oxo-6-(trifluoromethoxy)isoindorin-2-yl)piperidine-2,6-dione and 10 mg of palladium carbon were dissolved in 5.0 mL of tetrahydrofuran and 5.0 mL of methanol. The reaction solution was degassed, the reaction vessel was replaced with hydrogen, and the mixture was stirred at room temperature for 1 hour. The reaction solution was filtered, and the solvent was removed under reduced pressure to obtain 3-(4-amino-1-oxo-6-(trifluoromethoxy)isoindorin-2-yl)piperidine-2,6-dione in a yield of 16 mg and 34%. The compound data for 3-(4-amino-1-oxo-6-(trifluoromethoxy)isoindorin-2-yl)piperidine-2,6-dione is shown below. 1 H NMR (500 MHz, DMSO-d6)δ: 11.02 (s, 1H), 6.75 (d, J = 0.9 Hz, 1H), 6.72 (d, J = 0.9 Hz,1H), 5.92 (s, 2H), 5.11 (dd, J = 13.1, 5.2 Hz, 1H), 4.18 (dd, J =53.7, 17.1 Hz, 2H), 2.95-2.88 (m, 1H), 2.63-2.59 (m, 1H), 2.36-2.25 (m, 1H),2.06-2.02 (m, 1H) ppm. 19 F NMR (282 MHz, DMSO-d6)δ -56.4 (s, 3F) ppm. 13 C NMR (126 MHz, DMSO-d6)δ: 172.9, 171.1, 167.7, 149.5, 145.4, 133.7, 124.6, 120.1 (q, J = 255.8Hz), 108.0, 101.7, 51.8, 45.6, 31.2, 22.7 ppm. [Examples]

[0102] (Confirmation of the interaction ability of meta-substituted lenalidomide derivatives with IKZF1, SALL4, and PLZF) In this example, the affinity (degradation-inducing ability) of meta-substituted lenalidomide derivatives for IKZF1, SALL4, and PLZF was confirmed by biochemical interaction analysis.

[0103] (Experimental method) The affinity of lenalidomide, F-lenalidomide, Cl-lenalidomide, and Br-lenalidomide for IKZF1, SALL4, and PLZF was calculated as a relative value based on a signal value of 1 with DMSO. Details are as follows. FLAG-GST-IKZF1, -SALL4, -PLZF, and N-terminally biotinylated bls-CRBN were synthesized as recombinant proteins using a wheat cell-free protein synthesis system. A 10 μl mixture containing 0.5 μl of the synthesized bls-CRBN was prepared in AlphaScreen buffer (100 mM Tris (pH 8.0), 0.01% Tween20, 100 mM NaCl, and 1 mg / mL BSA). A 5 μl mixture containing 0.8 μl of FLAG-GST-protein was prepared in AlphaScreen buffer. A 5 μl mixture containing DMSO (final concentration 0.5%) or the final concentration of the thalidomide derivative shown in Figure 7 was prepared in AlphaScreen buffer. Subsequently, the three mixtures were mixed in a 384-well AlphaPlate (PerkinElmer) and allowed to stand at 26°C for 1 hour. Next, a 5 μl detection mixture containing 0.2 μg / ml anti-DYKDDDDK antibody (FUJIFILM WakoPure Chemical), 0.08 μl streptavidin donor beads (PerkinElmer), and 0.08 μl protein A acceptor beads (PerkinElmer) was prepared under AlphaScreen buffer. Then, the 5 μl detection mixture was added to each well, and after standing at 26°C for 1 hour, the luminescence signal was detected using an Envision plate reader (PerkinElmer).

[0104] The affinity of F3C-lenalidomide, F3CO-lenalidomide, pomalidomide, and lenalidomide for IKZF1, SALL4, and PLZF was calculated. Details are as follows. FLAG-GST-IKZF1, -SALL4, -PLZF and N-terminally biotinylated bls-CRBN were synthesized as recombinant proteins using a wheat cell-free protein synthesis system. A 10 μl mixture containing 0.5 μl of the synthesized bls-CRBN was prepared in AlphaScreen buffer (100 mM Tris (pH 8.0), 0.01% Tween20, 100 mM NaCl, and 1 mg / mL BSA). A 5 μl mixture containing 0.8 μl of FLAG-GST-protein was prepared in AlphaScreen buffer. A 5 μl mixture containing DMSO (final concentration 0.5%) or 10 μM thalidomide derivative was prepared in AlphaScreen buffer. Subsequently, the three mixtures were mixed in a 384-well AlphaPlate (PerkinElmer) and allowed to stand at 26°C for 1 hour. Next, a 5 μl detection mixture containing 0.2 μg / ml anti-DYKDDDDK antibody (FUJIFILM WakoPure Chemical), 0.08 μl streptavidin donor beads (PerkinElmer), and 0.08 μl protein A acceptor beads (PerkinElmer) was prepared under AlphaScreen buffer. Then, the 5 μl detection mixture was added to each well, and after standing at 26°C for 1 hour, the luminescence signal was detected using an Envision plate reader (PerkinElmer).

[0105] The results in Figure 7 confirm that lenalidomide, F-lenalidomide, Cl-lenalidomide, and Br-lenalidomide have low affinity for SALL4 and PLZF. Furthermore, it was confirmed that Cl-lenalidomide and Br-lenalidomide hardly interact with SALL4 or PLZF. The results in Figure 8 confirm that the affinity of F3C-lenalidomide and F3CO-lenalidomide for SALL4 and PLZF is significantly lower compared to the affinity of lenalidomide for SALL4 and PLZF. [Examples]

[0106] (Confirmation of the binding ability of lenalidomide derivatives with substituent insertions at each meta position to CRBN) In this example, the binding ability of each meta-substituted lenalidomide derivative to CRBN was confirmed using thalidomide-immobilized beads (see Figure 9(1)).

[0107] (Experimental method) A thalidomide immobilization derivative (Thalidomide-O-COOH) was purchased from MedChemExpress, and magnetic beads (FG-beads) for immobilization were purchased from Tamagawa Seiki. 4 mM Thalidomide-O-COOH was immobilized onto the FG-beads using Tamagawa Seiki's standard protocol. FLAG-GST-CRBN was also synthesized using a wheat cell-free protein synthesis system. 50 μl of FLAG-GST-CRBN was added to 50 μl of 4 mM thalidomide immobilized beads, and the mixture was adjusted to 500 μl with IP Lysis buffer (Pierce) (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol). After rotation at room temperature for 2 hours, the mixture was washed four times with 800 μl of IP Lysis buffer (Pierce). Subsequently, the samples were dispensed in equivalent volumes into seven tubes, and 20 μl of IP Lysis buffer (Pierce) containing 200 μM of various lenalidomide derivatives was added. Competitive elution was performed by vortexing at room temperature for 30 minutes. Then, 20 μl of 2× SDS sample buffer was added to each eluted sample, and it was boiled. Next, electrophoresis was performed using a polyacrylamide gel, followed by immunoblotting, and each protein was detected using the antibodies shown in Figure 9.

[0108] (result) The results in Figure 9(2) confirm that F-lenalidomide, Cl-lenalidomide, Br-lenalidomide, F3C-lenalidomide, and F3CO-lenalidomide have similar levels of interaction with CRBN (binding ability to CRBN) as lenalidomide. [Examples]

[0109] (Confirmation of intracellular protein degradation of lenalidomide derivatives with substituent insertions at each meta position) In this example, the protein degradation capabilities of lenalidomide derivatives with substituent insertions at each meta position were confirmed within cultured cells. Details are as follows.

[0110] (Experimental method) MM1.S cultured cells were seeded into a 24-well cell culture plate (BD Falcon), administered with DMSO (0.1%) or the lenalidomide derivative concentration shown in Figure 10, and cultured for 24 hours at 37°C and 5% CO2. The cells were then collected by pipetting, centrifuged at 900×g for 3 minutes, and the supernatant was removed using an aspirator. Next, 500 μl of 1×PBS was added to the cell pellet for washing, followed by centrifugation again at 900×g for 3 minutes, and the supernatant was removed using an aspirator. Finally, 100 μl of RIPA buffer (25 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) containing a protease inhibitor cocktail (Sigma Aldrich) was added to the cell pellet to lyse the cells. Subsequently, the supernatant was obtained as a cell extract after centrifugation at 16,100 × g for 15 minutes. Next, protein quantification was performed using a BCA assay kit (Thermo Fisher Scientific), and the samples were adjusted to equal protein concentrations in 1 × SDS sample buffer and boiled at 95°C for 5 minutes. After electrophoresis using a polyacrylamide gel, immunoblotting was performed, and each protein was detected using the antibodies shown in Figure 10. HuH-7 and MCF7 cultured cells were seeded into 24-well cell culture plates (BD Falcon) and cultured for 24 hours at 37°C and 5% CO2. Next, drugs were administered at DMSO (0.1%) or the lenalidomide derivative concentrations shown in Figure 10, and the cells were cultured for 24 hours at 37°C and 5% CO2. Subsequently, the supernatant was removed using an aspirator, and the cultured cells were recovered and lysed using 1× SDS sample buffer. The recovered cell extracts were boiled at 95°C for 5 minutes, and electrophoresis was performed using a polyacrylamide gel. Next, immunoblotting was performed, and each protein was detected using the antibodies shown in Figure 10.

[0111] (result) The results shown in Figure 10 are as follows: We confirmed that F-lenalidomide and Cl-lenalidomide have the ability to induce degradation of the drug target (IKZF1 / 3·CK1α) in cell culture, but have low ability to induce degradation of the teratogenic proteins SALL4·PLZF. On the other hand, Br-Len was found to have a weak degradation ability against IKZF1 / 3 in cell culture, and to have virtually no ability to induce degradation of SALL4·PLZF, a teratogenic protein. [Examples]

[0112] (Confirmation of the anticancer activity of lenalidomide derivatives with substituent insertions at each meta position against cultured cells of multiple myeloma) In this example, the anticancer activity of lenalidomide derivatives with substituent insertions at each meta position against cultured cells derived from multiple myeloma was confirmed. Details are as follows.

[0113] (Experimental method) MM1.S or U266 cultured cells were seeded into 24-well cell culture plates (BD Falcon), administered with DMSO (0.1%) or lenalidomide derivative concentrations as shown in Figure 11, and cultured for 72 hours at 37°C and 5% CO2. One-third of the cells were collected by pipetting, centrifuged at 200×g for 3 minutes, and the supernatant was removed with an aspirator. The cell pellet was then resuspended in culture medium, administered with DMSO (0.1%) or lenalidomide derivative concentrations as shown in Figure 11, and cultured for 72 hours at 37°C and 5% CO2. As described above, the cells were cultured in fresh medium containing DMSO or a lenalidomide derivative every three days for a total of nine days. Afterward, the cells were suspended and 40 μl was added to each of the 96 wells of an opti-plate (PerkinElmer), and 40 μl of the Cell Titer-Glo kit (Promega) was added to lyse the cells. The luminescence signal was then detected using SpectraMax iD3 (Molecular Devise).

[0114] (result) The results shown in Figure 11 are as follows: We confirmed that F-lenalidomide's ability to inhibit the proliferation of multiple myeloma cultured cells (anti-cancer cell proliferation effect) is comparable to that of pomalidomide and lenalidomide, which are known active ingredients in multiple myeloma treatments. Furthermore, we confirmed that Cl-lenalidomide also possesses anti-proliferative activity, although it is weaker than that of pomalidomide and lenalidomide. [Examples]

[0115] (Confirmation of the anticancer activity of lenalidomide derivatives with substituent insertions at each meta position against chromosome 5-deficient myelodysplastic syndrome) In this example, the anticancer activity of lenalidomide derivatives with substituent insertions at each meta position against cultured cells derived from chromosome 5-deficient myelodysplastic syndrome was confirmed. Details are as follows.

[0116] (Experimental method) MDS-L cultured cells were seeded into 24-well cell culture plates (BD Falcon), administered with DMSO (0.1%) or lenalidomide derivative concentrations as shown in Figure 12, and cultured for 96 hours at 37°C and 5% CO2. Half of the cells were collected by pipetting, centrifuged at 200×g for 3 minutes, and the supernatant was removed with an aspirator. The cell pellet was then resuspended in culture medium, administered with DMSO (0.1%) or lenalidomide derivative concentrations as shown in Figure 12, and cultured for 96 hours at 37°C and 5% CO2. As described above, the cells were cultured in fresh medium containing DMSO or a lenalidomide derivative every four days for a total of 20 days. Afterward, the cells were suspended and 40 μl was added to each of the 96 wells of an opti-plate (PerkinElmer), and 40 μl of the Cell Titer-Glo kit (Promega) was added to lyse the cells. The luminescence signal was then detected using SpectraMax iD3 (Molecular Devise).

[0117] (result) The results shown in Figure 12 are as follows: We confirmed that F-lenalidomide's ability to inhibit the proliferation of cultured cells with chromosome 5 deficiency myelodysplastic syndrome (M5 deficiency) (anti-cancer cell proliferation effect) is stronger than that of lenalidomide, the active ingredient in known M5 deficiency myelodysplastic syndrome treatments. Furthermore, we confirmed that Cl-lenalidomide has an anti-proliferative effect comparable to that of lenalidomide. Therefore, the present invention applies to therapeutic agents for chromosome 5 deficiency myelodysplastic syndrome containing F-Len or Cl-Len. [Examples]

[0118] (Confirmation of neoprotein degradation of known protacs) In this example, we confirmed the neoprotein resolution capabilities of the known PROTACs dBET1 and ARV-825. Details are as follows.

[0119] (Experimental method) MM1.S cultured cells were seeded into 24-well cell culture plates (BD Falcon), and administered DMSO (0.1%), 20 μM thalidomide, 10 μM lenalidomide, 1 μM pomalidomide, 20 μM 5-hydroxythalidomide, 1 μM dBET1, or 1 μM ARV-825. The cells were cultured for 24 hours at 37°C under 5% CO2 conditions. The cells were then collected by pipetting, centrifuged at 900×g for 3 minutes, and the supernatant was removed using an aspirator. Next, 500 μl of 1×PBS was added to the cell pellet for washing, and the cells were centrifuged again at 900×g for 3 minutes, with the supernatant removed using an aspirator. Finally, 100 μl of RIPA buffer containing a protease inhibitor cocktail (Sigma Aldrich) was added to the cell pellet to lyse the cells. Subsequently, the supernatant was obtained as a cell extract after centrifugation at 16,100 × g for 15 minutes. Next, protein quantification was performed using a BCA assay kit (Thermo Fisher Scientific), and the samples were adjusted to equal protein concentrations in 1 × SDS sample buffer and boiled at 95°C for 5 minutes. After electrophoresis using a polyacrylamide gel, immunoblotting was performed, and each protein was detected using the antibodies shown in Figure 13. HuH-7 cultured cells were seeded into 24-well cell culture plates (BD Falcon) and cultured for 24 hours at 37°C and 5% CO2. Next, DMSO (0.1%), 20 μM thalidomide, 10 μM lenalidomide, 1 μM pomalidomide, 20 μM 5-hydroxythalidomide, 1 μM dBET1, or 1 μM ARV-825 were administered, and the cells were cultured for 24 hours at 37°C and 5% CO2. Subsequently, the supernatant was removed using an aspirator, and the cultured cells were recovered and lysed using 1× SDS sample buffer. The recovered cell extract was boiled at 95°C for 5 minutes, and electrophoresis was performed using a polyacrylamide gel. Next, immunoblotting was performed, and each protein was detected using the antibodies shown in Figure 13. MM1.S cultured cells were seeded into 24-well cell culture plates (BD Falcon), and administered DMSO (0.1%), DMSO (0.1%), 10 μM thalidomide, 1 μM lenalidomide, 0.1 μM pomalidomide, and 10 μM dBET1 or 0.1 μM ARV-825. The cells were cultured for 5 days at 37°C and 5% CO2. Subsequently, the cells were suspended and 40 μl were added to each of the 96-well opti-plates (PerkinElmer), and 40 μl of Cell Titer-Glo kit (Promega) was added to lyse the cells. The luminescence signal was then detected using SpectraMax iD3 (Moleculardevise).

[0120] (result) The results shown in Figure 13 are as follows: Regarding dBET1, we confirmed that it has low ability to induce the degradation of teratogenic proteins SALL4 and PLZF, as well as low ability to induce the degradation of drug targets IKZF1 and IKZF3. In other words, we confirmed the issue of low anti-cancer cell proliferation activity. Regarding ARV-825, we confirmed that it has high degradation-inducing ability for IKZF1 and IKZF3, but similarly, it also has high degradation-inducing ability for SALL4 and PLZF. In other words, we confirmed that it cannot be used in cancer treatments where teratogenicity is a concern. In other words, it was confirmed that known PROTACs induce degradation of not only the target protein but also the neosubstrate of the E3 binder, or that teratogenicity is a problem. [Examples]

[0121] (Target protein degradation-inducing compound of the present invention) The results from Examples 1 to 10 confirmed the following: Lenalidomide, F-lenalidomide, and Cl-lenalidomide, which degrade both IKZF1 / 3 / CK1α, the neosubstrate of the E3 binder, and the target protein, are thought to be cerebron binders that induce target protein degradation and are effective against hematological cancers (see Figure 14 left), and this was confirmed in the following examples. Cl-lenalidomide, which degrades only target proteins and has low neosubstrate degradation induction ability, and Br-lenalidomide, F3C-lenalidomide, and F3CO-lenalidomide, which have no neosubstrate degradation induction ability, have low or no degradation induction ability for IKZF1 / 3 / CK1α, the neosubstrate of the E3 binder. Therefore, they can be used for cancers other than specific cancers, and furthermore, they avoid or reduce teratogenicity, so they are considered to be cerebron binders that are target protein degradation induction compounds that can be used for various cancers (see Figure 14 right), and this was confirmed in the following examples. [Examples]

[0122] (Synthesis of the target protein degradation-inducing compound of the present invention) In this example, the target protein degradation-inducing compound of the present invention, as shown in Figure 15, was synthesized. Details are as follows. ARV-825 and dBET1 were commercially available products.

[0123] Synthesis of ("Lenalidomide" - "Linker" - "Birabresib") The Lenalidomide-Linker-JQ-1 derivative (1a) was synthesized using the following procedure.

[0124] [ka]

[0125] The synthesis of 3-(4-((2-(2-(2-(2-(4-nitrophenoxy)ethoxy)ethoxy)ethoxy)ethyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (3a) was carried out by the following procedure.

[0126] [ka]

[0127] Under a nitrogen atmosphere, lenalidomide (44.6 mg, 0.172 mmol, 1.0 equiv), 2 (87.7 mg, 0.206 mmol, 1.2 equiv), DMF (0.1 M, 2 mL), and N,N-Diisopropylethylamine (87.8 μL, 0.516 mmol, 3.0 equiv) were sequentially added to a 10 mL round-bottom flask and stirred at 100 °C for 12 hours. After cooling to room temperature, azeotropic chromatography with toluene was performed under reduced pressure, and the solvent was removed by distillation. The resulting crude compound was purified by silica gel chromatography (dichloromethane:methanol = 1 / 0 → 20 / 1). Due to the presence of multiple spots, further purification by silica gel chromatography (ethyl acetate) was performed to obtain compound 3a in 39.2 mg, with a yield of 41%. 1 H NMR(300MHz, CDCl3) δ: 8.51 (s, 1H), 8.18 (tlike, J = 9.1 Hz, 2H), 7.34 (t like, J = 7.8 Hz, 1H), 7.00-6.92(m, 2H), 6.78 (d, J = 7.9 Hz, 1H), 5.21 (dd, J = 13.0, 5.1 Hz,1H), 4.26-4.12 (m, 4H), 3.91-3.84 (m, 2H), 3.76-3.56 (m, 12H), 3.45-3.39 (m,2H), 2.94-2.73 (m, 1H), 2.35-2.12 (m, 2H)

[0128] The synthesis of 1a was carried out using the following procedure.

[0129] [ka]

[0130] The nitro group of the synthesized 3a was reduced. 3a (39.8 mg, 0.0715 mmol, 1.0 equiv) and iron (39.9 mg, 0.715 mmol, 10 equiv) were added to ethanol (1.0 ml, 0.072 M), and then ammonium chloride (38.3 mg, 0.715 mmol, 10 equiv) dissolved in water (0.5 ml, 0.14 M) was added. The mixture was then heated to 80 °C and stirred for 2 hours. After cooling to room temperature, the mixture was filtered and washed with ethyl acetate. Ethyl acetate and water were added to the filtrate, and the organic layer was washed with saturated brine and dehydrated with sodium sulfate. The solvent was then removed under reduced pressure to obtain the amine (4a below). This was used in the next step without further purification.

[0131] [ka]

[0132] Next, the amine compound was condensed with JQ-1. Under a nitrogen atmosphere, the amine compound (17.2 mg, 0.0327 mmol, 1.0 equiv), JQ-1 (13.1 mg, 0.0327 mmol, 1.0 equiv), and HATU (18.6 mg, 0.0491 mmol, 1.5 equiv) were dissolved in DMF (0.5 mL, 0.065 M), and then N,N-Diisopropylethylamine (31.1 μL, 0.183 mmol, 5.6 equiv) was added. The mixture was then stirred at room temperature for 12 hours. After filtering the reaction solution, isolation was performed by HPLC under the conditions described below. NOMURA CHEMICAL DevelosilODS-HG-5, 20 × 250 mm, CH3CN / H2O = 10 / 90 to 40 / 60 with 0.1% TFA, flow rate 5.0 mL / min, measurement wavelength = 220 nm, tR = 53.6 min. By removing the developing solvent under reduced pressure, 9.5 mg of the yellow solid 1a was obtained in 14% yield (2 steps). 1H NMR(300MHz, CD3OD) δ: 10.11 (s, 1H), 7.49 - 7.38(m, 6H), 7.32 (t-like, J = 7.8 Hz, 1H), 7.10 (d, J = 7.3 Hz, 1H),6.87 (d, J = 8.8 Hz, 2H), 5.01 - 4.85 (m, 2H), 5.12 (dd, J = 13.3, 4.8 Hz, 1H), 4.73 (dd, J = 8.5, 5.9 Hz, 1H), 4.29 (s, 2H), 4.08 - 4.05 (m, 2H), 3.80 - 3,77(m, 2H), 3.71 - 3.64 (m,10H), 3.44 - 3.37 (m, 2H),2.95 - 2.75 (m, 2H), 2.72 (s,3H), 2.45 (s, 3H), 2.40 - 2.34(m, 1H), 2.14 - 2.10 (m, 1H)1.69 (s, 3H) ppm. HRMS (ESI) calcd.for C 46 H 50 N8O8SCl [M + H] + :909.3160 ​​found 909.3141.

[0133] Synthesis of ("F-lenalidomide" - "linker" - "Birabresib") The synthesis of the Fluorolenalidomide-Linker-JQ-1 derivative (1b) was carried out using the following procedure.

[0134] [ka]

[0135] Synthesis of 3-(6-fluoro-4-((2-(2-(2-(2-(4-nitrophenoxy)ethoxy)ethoxy)ethoxy)ethyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione(3b)

[0136] [ka]

[0137] Under a nitrogen atmosphere, 6-fluorolenalidomide (50 mg, 0.180 mmol, 1.0 equiv), 2 (92.0 mg, 0.216 mmol, 1.2 equiv), DMF (0.1 M, 2 mL), and N,N-Diisopropylethylamine (92 μL, 0.540 mmol, 3.0 equiv) were sequentially added to a 10 mL round-bottom flask and stirred at 100 °C for 12 hours. After cooling to room temperature, azeotropic distillation with toluene was performed under reduced pressure, and the solvent was removed by distillation. The resulting crude compound was purified by silica gel chromatography (ethyl acetate:methanol = 1 / 0 → 20 / 1). Compound 3b was obtained in 36.9 mg, yielding 36%, by distillation under reduced pressure. 1 H NMR (300MHz, CDCl3) δ: 8.24-8.13 (m, 3H), 7.03-6.92 (m, 3H), 6.50(dd, J = 11.4, 2.1 Hz, 1H), 5.23 (dd, J = 13.0, 5.4 Hz, 1H),4.33-4.12 (m, 4H), 3.94-3.88 (m, 2H), 3.77-3.63 (m, 10H), 3.39-3.35 (m, 2H),2.99-2.84 (m, 2H), 2.54-2.54 (m, 1H), 2.37-2.25 (m, 2H), 1.79-1.79 (m, 1H) 19 F NMR(282MHz, CDCl3) δ: -111.1 (t like, J = 9.4 Hz, 1F)

[0138] Synthesis of 1b

[0139] [ka]

[0140] The nitro group of the synthesized 3b was reduced. 3b (36.9 mg, 0.0642 mmol, 1.0 equiv) and iron (35.9 mg, 0.642 mmol, 10 equiv) were added to ethanol (1.0 ml, 0.064 M), followed by the addition of ammonium chloride (34.4 mg, 0.642 mmol, 10 equiv) dissolved in water (0.5 ml, 0.13 M). The mixture was then heated to 80 °C and stirred for 2 hours. After cooling to room temperature, the mixture was filtered and washed with ethyl acetate. Ethyl acetate and water were added to the filtrate, and the organic layer was washed with saturated brine and dehydrated with sodium sulfate. The solvent was then removed under reduced pressure to obtain the amine (25.8 mg, 74% yield, crude). This amine was used in the next step without further purification. Next, the amine compound was condensed with JQ-1. Under a nitrogen atmosphere, the amine compound (17.4 mg, 0.0314 mmol, 1.0 equiv), JQ-1 (12.6 mg, 0.0314 mmol, 1.0 equiv), and HATU (17.9 mg, 0.0471 mmol, 1.5 equiv) were dissolved in DMF (0.5 mL, 0.063 M), and then N,N-Diisopropylethylamine (16.0 μL, 0.0942 mmol, 3.0 equiv) was added. The mixture was then stirred at room temperature for 15 hours. After filtering the reaction solution, isolation was performed by HPLC under the conditions described below. NOMURA CHEMICAL DevelosilODS-HG-5, 20 × 250 mm, CH3CN / H2O = 10 / 90 to 40 / 60 with 0.1% TFA, flow rate 5.0 mL / min, measurement wavelength = 220 nm. Compound 1b was obtained in 9.5 mg in 33% yield by removing the developing solvent under reduced pressure. 1H NMR (300 MHz, CD3OD) δ: 10.11(s, 1H), 7.98 (s, 1H), 7.49 - 7.39(m, 6H), 6.87 (d, J = 9.1 Hz, 2H), 6.70 (dd, J = 7.6, 2.1 Hz,1H), 6.58 (dd, J = 12.3, 2.1 Hz, 1H), 5.10 (dd, J = 13.3, 5.4 Hz,1H), 4.79 - 4.71 (m, 1H), 4.24 (s, 2H), 4.07 (t, J = 4.5 Hz, 2H), 3.79 (t, J = 4.5 Hz, 2H),3.70 - 3.64 (m, 10H), 3.37 - 3.34 (m, 2H), 2.72 (s, 3H), 2.45(s, 3H), 1.70 (s, 3H) ppm. 19 F NMR (282 MHz, CD3OD) δ -107.8 (dd, J = 11.9, 7.9 Hz, 1F) ppm. HRMS(ESI) calcd. for C 46 H 49 N8O8FSCl [M+ H] + : 927.3067 found 927.3024.

[0141] ("Cl-レナリドミド"-"リンカー"-"Birabresib") synthesis Synthesis of Chloro lenalidomide-Linker-JQ-1 inducer (4b), following instructions.

[0142]

change

[0143] 3-(6-chloro-4-((2-(2-(2-(2-(4-nitrophenoxy)ethoxy)ethoxy)ethoxy)ethyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione(3b) Under a nitrogen atmosphere, lenalidomide 1b (58.7 mg, 0.200 mmol, 1.0 equiv), 2 (340.2 mg, 0.800 mmol, 4.0 equiv), DMF (0.2 M, 1.0 mL), and N,N-Diisopropylethylamine (0.10 mL, 0.600 mmol, 3.0 equiv) were sequentially added to a 10 mL round-bottom flask and stirred at 110 °C for 24 hours. The solvent was then removed under reduced pressure, and the resulting crude product was purified by silica gel chromatography (ethyl acetate:methanol = 20 / 1) to obtain compound 3b in 58.5 mg, with a yield of 49%. 1 H NMR (300MHz, CDCl3) δ: 8.55(s, 1H), 8.15 (d, J = 12.1 Hz, 2H), 7.16 (s, 1H), 6.92 (d, J =12.2 Hz, 2H), 6.69 (s, 1H), 5.15 (dd, J = 12.9, 5.3 Hz, 1H), 4.33 (s,1H), 4.07-4.26 (m, 4H), 3.84 (t, J = 4.6 Hz, 2H), 3.54-3.74 (m, 10H),3.33 (d like, J = 4.1 Hz, 2H), 2.71-2.87 (m, 2H), 2.10-2.30 (m, 2H)

[0144] 3-(4-((2-(2-(2-(2-(4-aminophenoxy)ethoxy)ethoxy)ethoxy)ethyl)amino)-6-chloro-1-oxoisoindolin-2-yl)piperidine-2,6-dione 3b (58.5 mg, 0.099 mmol, 1.0 equiv) and iron (55.2 mg, 0.990 mmol, 10 equiv) were added to ethanol (1.7 ml), followed by the addition of ammonium chloride (53.0 mg, 0.990 mmol, 10 equiv) dissolved in water (0.4 ml). The mixture was then heated to 80 °C and stirred for 1 hour. After cooling to room temperature, the mixture was filtered and washed with ethyl acetate. Ethyl acetate and water were added to the filtrate, and the organic layer was washed with saturated brine and dehydrated with sodium sulfate. The solvent was then removed under reduced pressure to obtain the reduced product. This product was used in the next step without further purification. Under a nitrogen atmosphere, at 0 °C, the above reduced product (46.8 mg, 0.0834 mmol, 1.0 equiv), JQ-1 (33.3 mg, 0.0834 mmol, 1.0 equiv), and HATU (63.1 mg, 0.166 mmol, 2.0 equiv) were dissolved in DMF (0.8 mL, 0.1 M), and then N,N-Diisopropylethylamine (43.0 μL, 0.250 mmol, 3.0 equiv) was added, and the mixture was stirred at room temperature for 1 hour. Water was added to the reaction solution, and it was extracted with ethyl acetate. The organic layer was washed with saturated saline solution and dried over sodium sulfate. Isolation was performed by HPLC under the conditions described below. NOMURA CHEMICAL Develosil ODS-HG-5, 20 × 250mm, CH3CN / H2O = 10 / 90 to 60 / 40 with 0.1% TFA, flow rate 5.0 mL / min, measurement wavelength = 220 nm, t R = 69.6 min. By removing the developing solvent under reduced pressure, 64.0 mg of yellow solid 4b was obtained in 81% yield (2 steps). 1H NMR (300 MHz, CD3OD) δ: 7.30-7.59(m, 6H), 6.93-7.02 (m, 1H), 6.78-6.85 (m, 3H), 5.06 (dd, J = 13.2, 5.0Hz, 1H), 4.75-4.84 (m, 1H), 4.15-4.28 (m, 2H), 4.02-4.09 (m, 2H), 3.75-3.85 (m,2H), 3.48-3.68 (m, 12H), 3.19-3.35 (m, 4H), 2.67-2.89 (m, 5H), 2.22-2.48 (m,4H), 2.01-2.11 (m, 1H), 1.59-1.74 (m, 3H)

[0145] Synthesis of ("F3C-lenalidomide" - "linker" - "Birabresib") The synthesis of the trifluoromethyl lenalidomide-Linker-JQ-1 derivative (4a) was carried out by the following procedure.

[0146] [ka]

[0147] 3-(4-((2-(2-(2-(2-(2-(4-nitrophenoxy)ethoxy)ethoxy)ethoxy)ethyl)amino)-1-oxo-6-(trifluoromethyl)isoindolin-2-yl)piperidine-2,6-dione(3a) Under a nitrogen atmosphere, lenalidomide 1a (65.5 mg, 0.200 mmol, 1.0 equiv), 2 (255.1 mg, 0.600 mmol, 3.0 equiv), NMP (0.2 M, 1.0 mL), and N,N-Diisopropylethylamine (0.10 mL, 0.600 mmol, 3.0 equiv) were sequentially added to a 10 mL round-bottom flask and stirred at 110 °C for 12 hours. The solvent was then removed by distillation at 50 °C under reduced pressure. The resulting crude product was purified by silica gel chromatography (ethyl acetate:methanol = 1 / 0 → 5 / 1) to obtain compound 3a in 46.3 mg in yield of 41%. 1 H NMR (300MHz, CDCl3) δ: 8.71 (s, 1H), 8.10-8.15 (m, 2H), 7.44 (s, 1H), 6.90-6.98 (m, 3H),5.18 (dd, J = 12.9, 5.3 Hz, 1H), 4.49 (s, 1H), 4.15-4.35 (m, 4H), 3.84(t, J = 4.6 Hz, 2H), 3.74 (t, J = 4.9 Hz, 2H), 3.54-3.68 (m, 8H),3.37 (m, 2H), 2.71-2.85 (m, 2H), 1.98-2.39 (m, 2H) 19 F NMR(282MHz, CDCl3) δ: -62.84 (s, 3F)

[0148] 3-(4-((2-(2-(2-(2-(4-aminophenoxy)ethoxy)ethoxy)ethoxy)ethyl)amino)-1-oxo-6-(trifluoromethyl)isoindolin-2-yl)piperidine-2,6-dione 3a (46.3 mg, 0.074 mmol, 1.0 equiv) and iron (41.3 mg, 0.740 mmol, 10 equiv) were added to ethanol (1.2 ml), and then ammonium chloride (40.0 mg, 0.740 mmol, 10 equiv) dissolved in water (0.3 ml) was added. The mixture was then heated to 80 °C and stirred for 1 hour. After cooling to room temperature, the mixture was filtered and washed with ethyl acetate. Ethyl acetate and water were added to the filtrate, and the organic layer was washed with saturated saline solution and dehydrated with sodium sulfate. The solvent was then removed under reduced pressure to obtain the reduced product. This product was used in the next step without further purification.

[0149] Under a nitrogen atmosphere, at 0 °C, the above amine compounds (reduced form) (37.5 mg, 0.0630 mmol, 1.0 equiv), JQ-1 (25.3 mg, 0.0630 mmol, 1.0 equiv), and HATU (47.9 mg, 0.126 mmol, 2.0 equiv) were dissolved in DMF (0.6 mL, 0.1 M). Then, N,N-Diisopropylethylamine (32.5 μL, 0.189 mmol, 3.0 equiv) was added, and the mixture was stirred at room temperature for 2 hours. Water was added to the reaction solution, and it was extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over sodium sulfate. Isolation was performed by HPLC under the conditions described below. NOMURA CHEMICAL Develosil ODS-HG-5, 20 × 250mm, CH3CN / H2O = 10 / 90 to 90 / 10 with 0.1% TFA, flow rate 5.0 mL / min, measurement wavelength = 220 nm, t R = 39.5 min. By removing the developing solvent under reduced pressure, 41.0 mg of yellow solid 4a was obtained in 67% yield (2 steps). 1H NMR (300MHz, CD3OD) δ:7.39-7.48 (m, 6H), 7.27 (s, 1H), 7.03 (s, 1H), 6.84 (d, J = 8.8 Hz, 2H),5.09-5.14 (m, 1H), 4.79 (d, J = 5.6 Hz, 1H), 4.33 (s, 2H), 4.04 (t, J= 4.4 Hz, 2H), 3.76-3.79 (m, 2H), 3.53-3.71 (m, 13H), 3.42 (t, J = 5.0Hz, 2H), 2.70-2.86 (m, 5H), 2.37-2.45 (m, 5H), 2.14 (s, 1H), 1.69 (s, 3H) 19 F NMR(282MHz, CDCl3) δ: -62.89 (s, 3F) [Examples]

[0150] (Confirmation of the selectivity of the cerebron binder of the target protein degradation-inducing compound of the present invention for the neosubstrate) In this example, the selectivity of the cerebron binder for the neosubstrate of the following target protein degradation-inducing compounds was confirmed. "Cerebromide" - "Linker" - "Target Protein Binder" Le-PROTAC: "Lenalidomide" - "Linker (see Figure 15)" - "Birabresib" NE-005-PROTAC: "F-Lenalidomide" - "Linker (see Figure 15)" - "Birabresib" NE-013-PROTAC: "Cl-Lenalidome" - "Linker (see Figure 15)" - "Birabresib" NE-015-PROTAC: "F3C-Lenalidomide" - "Linker (see Figure 15)" - "Birabresib"

[0151] (Experimental method) MM1.S or H929 cultured cells were seeded into 24-well cell culture plates (BD Falcon), administered with DMSO (0.1%) or the drug at the concentrations shown in Figure 16, and cultured for 24 hours at 37°C and 5% CO2. The cells were then collected by pipetting, centrifuged at 900×g for 3 minutes, and the supernatant was removed using an aspirator. Next, 500 μl of 1×PBS was added to the cell pellet for washing, and the cells were centrifuged again at 900×g for 3 minutes, with the supernatant removed using an aspirator. Next, 100 μl of RIPA buffer containing a protease inhibitor cocktail (Sigma Aldrich) was added to the cell pellet to lyse the cells. The cells were then centrifuged at 16,100×g for 15 minutes, and the supernatant was obtained as the cell extract. Next, protein quantification was performed using a BCA assay kit (Thermo Fisher Scientific), and the samples were adjusted to equal protein concentrations in 1×SDS sample buffer and boiled at 95°C for 5 minutes. After electrophoresis using a polyacrylamide gel, immunoblotting was performed, and each protein was detected using the antibodies shown in Figure 16. MM1.S cultured cells were seeded into 24-well cell culture plates (BD Falcon), administered with DMSO (0.1%) or a drug (0.1 μM), and cultured for 5 days at 37°C and 5% CO2. The cells were then suspended and 40 μl were added to each of the 96-well opti-plates (PerkinElmer), and 40 μl of Cell Titer-Glo kit (Promega) was added to lyse the cells. The luminescence signal was then detected using SpectraMax iD3 (Molecular Devise).

[0152] (result) The results are shown in Figure 16. Unlike the known PROTAC ARV-825, the target protein degradation-inducing compound of the present invention does not induce degradation of IKZF1 / 3 / CK1α, thus exhibiting neosubstrate selectivity for cerebron binders. Furthermore, it has been confirmed to have anticancer activity (particularly inhibitory activity against multiple myeloma proliferation). In addition, the target protein degradation-inducing compound of the present invention can induce degradation of the target proteins BRD2, BRD3, and BRD4. BRD2-4 have been reported to be involved in the proliferation of various cancer types, and in fact, JQ1 has been reported to be effective against various cancer types, including lung cancer, prostate cancer, and NUT midline adenocarcinoma, in addition to hematological cancers such as AML, DLBCL, MM, and ALL. That is, the target protein degradation-inducing compound of the present invention has not only an inhibitory effect on the proliferation of multiple myeloma, but also an inhibitory effect on the proliferation of various cancer cells such as prostate cancer, Burkitt lymphoma, NUT midline adenocarcinoma, and lung cancer. [Examples]

[0153] (Confirmation of the degradation-inducing ability of the target protein degradation-inducing compound of the present invention on SALL4 and PLZF) In this example, the degradation-inducing ability of the following target protein degradation-inducing compounds against SALL4 and PLZF was confirmed. "Cerebromide" - "Linker" - "Target Protein Binder" Le-PROTAC: "Lenalidomide" - "Linker (see Figure 15)" - "Birabresib" F-PROTAC: "F-Lenalidomide" - "Linker (see Figure 15)" - "Birabresib" Cl-PROTAC: "Cl-Lenalidomide" - "Linker (see Figure 15)" - "Birabresib" F3C-PROTAC: "F3C-Lenalidomide" - "Linker (see Figure 15)" - "Birabresib"

[0154] (Experimental method) NTERA cultured cells were seeded into a 24-well cell culture plate (BD Falcon) and cultured for 24 hours at 37°C and 5% CO2. Next, DMSO (0.1%) or the drug at the concentration shown in Figure 17 was administered, and the cells were cultured for 24 hours at 37°C and 5% CO2. Subsequently, the supernatant was removed using an aspirator, 500 μl of 1×PBS was added to the cell pellet for washing, and the supernatant was removed using an aspirator. Next, 100 μl of RIPA buffer containing a protease inhibitor cocktail (Sigma Aldrich) was added to the cells to lyse them. After centrifugation at 16,100×g for 15 minutes, the supernatant was obtained as the cell extract. Next, protein quantification was performed using a BCA assay kit (Thermo Fisher Scientific), and the samples were adjusted to equal protein concentrations in 1× SDS sample buffer and boiled at 95°C for 5 minutes. After electrophoresis using a polyacrylamide gel, immunoblotting was performed, and each protein was detected using the antibodies shown in Figure 17. MDS-L cultured cells were seeded into a 24-well cell culture plate (BD Falcon), administered with DMSO (0.1%) or the concentration shown in Figure 17, and cultured for 24 hours at 37°C and 5% CO2. The cells were then collected by pipetting, centrifuged at 900×g for 3 minutes, and the supernatant was removed using an aspirator. Next, 500 μl of 1×PBS was added to the cell pellet for washing, and the cells were centrifuged again at 900×g for 3 minutes, with the supernatant removed using an aspirator. Next, 100 μl of RIPA buffer containing a protease inhibitor cocktail (Sigma Aldrich) was added to the cell pellet to lyse the cells. The cells were then centrifuged at 16,100×g for 15 minutes, and the supernatant was obtained as the cell extract. Next, protein quantification was performed using a BCA assay kit (Thermo Fisher Scientific), and the samples were adjusted to equal protein concentrations in 1×SDS sample buffer and boiled at 95°C for 5 minutes. After electrophoresis using a polyacrylamide gel, immunoblotting was performed, and each protein was detected using the antibodies shown in Figure 17.

[0155] (result) The results are shown in Figure 17. The target protein degradation-inducing compounds of the present invention (particularly Cl-PROTAC and F3C-PROTAC) differ from the known PROTAC ARV-825 in that they do not induce degradation of SALL4 and PLZF, and have been confirmed to possess neosubstrate selectivity for cerebron binders. In other words, the target protein degradation-inducing compounds of the present invention (particularly Cl-PROTAC and F3C-PROTAC) can be used for various types of cancer by using various target protein binders. [Examples]

[0156] (Confirmation of the anticancer effect of the target protein degradation-inducing compound of the present invention) In this example, the anticancer effects of the following target protein degradation-inducing compounds were confirmed. "Cerebromide" - "Linker" - "Target Protein Binder" Le-PROTAC: "Lenalidomide" - "Linker (see Figure 15)" - "Birabresib" F-PROTAC: "F-Lenalidomide" - "Linker (see Figure 15)" - "Birabresib" Cl-PROTAC: "Cl-Lenalidomide" - "Linker (see Figure 15)" - "Birabresib" F3C-PROTAC: "F3C-Lenalidomide" - "Linker (see Figure 15)" - "Birabresib"

[0157] (Experimental method) HCT116 or NTERA cultured cells were seeded into 96-well cell culture plates (BD Falcon), administered with DMSO (0.1%) or the drug at the concentrations shown in Figure 18, and cultured for 2 days at 37°C and 5% CO2. The supernatant was then removed using an aspirator, and 40 μl of culture medium was added. Next, 40 μl of Cell Titer-Glo kit (Promega) was added to each well to lyse the cells, and 40 μl of this mixture was added to 384-well opti-plates (PerkinElmer). The luminescence signal was detected using SpectraMax iD3 (Molecular Devise).

[0158] (result) The results are shown in Figure 18. The target protein degradation-inducing compound of the present invention has been confirmed to be effective against solid tumors (particularly embryonic cancer and colorectal cancer). [Examples]

[0159] (Disassembly and evaluation of CC-220 SALL4) In this example, the SALL4 resolution of the compound CC-220 was determined by referring to the measurement method of the above example. We confirmed that CC-220 exhibits weak degradation of SALL4 at concentrations capable of degrading IKZF1 and IKZF3 (see Figure 19). In other words, we confirmed that CC-220 can be an active ingredient in a composition for the treatment of hematological cancers. Furthermore, the following fluorinated CC-220, CC-92480, and fluorinated CC-92480, which have similar chemical structures and properties to CC-220, can also be used as active ingredients in compositions for the treatment of hematological cancers. In addition, CC-220, fluorinated CC-220, CC-92480, and fluorinated CC-92480 can serve as cerebron binders for the target protein degradation-inducing compounds of the present invention.

[0160] [ka]

[0161] [ka]

[0162] [ka]

[0163] [ka] [Examples]

[0164] (Resolution of the target protein degradation-inducing compound and cancer treatment composition of the present invention) The resolution of each protein for which the target protein degradation-inducing compound was determined was calculated using the Len-PROTAC resolution as a baseline of 100, as shown in the table below. ○ Degradation of each protein in cancer treatment compositions 1) The value was converted to a number where Len is set to 100. Furthermore, if the value was less than 0, it was set to 0. 2) The results for HuH7 cells in MM1.S cells (1 μmol / l, 10 μmol / l) shown in Figure 10 were adopted, and for F3C-Len, which was not measured in Figure 10, the results from Figures 16 and 17 were adopted. 3) For each numerical value, the range was defined as the largest and smallest values. ○ Degradation level of each protein of the target protein degradation-inducing compound 1) The values ​​were converted to a number where Len-PROTAC is set to 100. Furthermore, if the value was lower than 0, the value was set to 0. 2) For each numerical value, the range was defined as the largest and smallest values.

[0165] [Table 1]

[0166] [Table 2] [Examples]

[0167] (Experimental method) To evaluate the antitumor effect of PROTAC in animal organisms, we performed an analysis in a mouse Xenograft model using neuroblastoma cultured cells (IMR32 cells), for which BRD2-4 has been reported as a therapeutic target in multiple studies. Details are as follows.

[0168] (Experimental method) IMR32 cultured cells were seeded into a 10 cm cell culture dish (BD Falcon) and cultured at 37°C and 5% CO2. Subsequently, 5 × 10⁶ cells were cultured per nude mouse. 6 IMR32 cells were resuspended in 50 μl of PBS and mixed with 50 μl of Matrigel (Corning). 100 μl of the mixed cell solution was transplanted subcutaneously into the flank of nude mice. One week after transplantation, DMSO or PROTAC was administered intraperitoneally once daily (weekdays only). The administration solution was diluted in PBS to 10% DMSO, 5 mg / kg PROTAC, and 1% Tween 80, and 200 μl of each was administered intraperitoneally. Five weeks after administration, the tumor volume was measured using electronic calipers, and the tumor was removed and photographed.

[0169] (result) The results shown in Figure 33 are as follows: PROTACs using lenalidomide or lenalidomide derivatives were confirmed to suppress tumor growth in mice. In particular, PROTACs using F3C-lenalidomide showed an equivalent or better tumor growth inhibitory effect compared to PROTACs using lenalidomide.

[0170] Cereblon is a component of ubiquitin ligase, which is responsible for protein degradation. When thalidomide derivative compounds bind to cereblon, the substrate specificity of cereblon changes, and proteins that are not normally degraded (neo-substrates) are degraded, resulting in either drug effects or side effects. As previously mentioned, the inventors discovered that the binding of SALL4 and PLZF to the cereblon-thalidomide derivative compound complex induces degradation, which is a key event in the expression of teratogenicity. In other words, thalidomide-derived inducers (thalidomide, pomalidomide, lenalidomide), or targeted degradation inducers utilizing the thalidomide skeleton, will also degrade these proteins and exhibit teratogenicity if they have binding activity with SALL4 and PLZF. On the other hand, Kroenke and Lu have reported on the antitumor effect of lenalidomide (inhibitory effect on cell proliferation in multiple myeloma), and it has been revealed that the antitumor effect is expressed through a series of mechanisms, from substrate recognition by cereblon to ubiquitination and degradation by the proteasome of IKZF1 (IKZF1) and IKZF3 (IKZF3), which are essential factors for the differentiation of B cells and T cells. (Kroenke J, et al., Science 343,301-305(20014)). Therefore, in the following examples, we investigated functional group modifications of the thalidomide skeleton with the aim of developing thalidomide-based drugs that do not have teratogenic effects and targeted degradation inducers utilizing the thalidomide skeleton. [Examples]

[0171] Among thalidomide-derived agents (including thalidomide, pomalidomide, and lenalidomide), the glutarimide ring is primarily involved in binding to cereblon (see: Nature 2014, 512, 49-53, Nature Structural & Molecular Biology 2014, 21, 803-809). Therefore, various functional group substitutions were performed on the benzene ring from position 4 to 7 of the phthalimide ring, and the binding ability to IKZF1, IKZF3, SALL4, and PLZF, as well as the degree of degradation of IKZF1, IKZF3, SALL4, and PLZF in cells due to this binding ability, were investigated. Furthermore, as is clear from the results of the above examples, by attaching a function to bind to a specific protein via a linker (target protein binder) at position 4, it is possible to give the protein the function of inducing the degradation of the target protein. In this example, BRD was selected as the target protein to be degraded, and JQ-1 was used as the target protein binder. Furthermore, since this linker and target protein binder portion is thought to exhibit high steric exclusion volume effects, we similarly investigated its binding ability to IKZF1, IKZF3, SALL4, and PLZF, as well as the degree of degradation of these proteins in cells.

[0172] The binding ability of thalidomide, pomalidomide, and lenalidomide to each molecule and the degree of degradation within cells were evaluated. Binding ability was analyzed using biochemical interactions (AlphaScreen method), and the degradation of each molecule within cells was determined by the following methods.

[0173] (1) Selection of cell tumors H929 cells were used for IKZF1, IKZF3, and BRD (BRD2, BRD3, BRD4), while Huh7 cells were used for SALL4 and PLZF. (Culture conditions) Each cell is 50-60 x 10 4 The solution was prepared at cells / mL and dispensed into 24-well plates, with 1 ml dispensed into each well. The incubation temperature was 37°C for 16 to 20 hours, and the carbon dioxide concentration was 5%. (Addition of compounds) A 1 μL solution of the compound, adjusted to 10 mM using DMSO as the solvent, was added to the culture medium to achieve a final compound concentration of 10 μM. This solution was incubated at 37°C for 16 to 20 hours under a 5% carbon dioxide concentration. Simultaneously, cells containing the same amount of DMSO solvent without the dissolved compound were prepared as an evaluation control.

[0174] (Cell harvesting and sample preparation for Western blotting evaluation) 1)H929 cells H929 cell saturation was collected in any 1.5 mL tube. Since H929 cells are suspension cells, no detachment procedure is necessary. To ensure the same number of cells in each collected sample, quantification was performed using the BCA method, and the sample was diluted to match the least concentrated sample. SDS sample buffer containing mercaptoethanol was added to the diluted H929 cell saturation, and the mixture was heated at 98°C for 5 minutes. 2)Huh cells The culture medium for HuH7 cells was aspirated. Since HuH7 cells are adherent cells, they adhere to the bottom of the plate. 100 μL of SDS sample buffer containing mercaptoethanol was added to each 24-well plate. The HuH7 cells were detached from the bottom of the 24-well plate by scraping with a wide-mouthed tip and collected in 1.5 mL tubes. As with H929 cells, the cells were quantified by the BCA method, the cell counts were matched, and then the cells were heated at 98°C for 5 minutes.

[0175] (Quantitative determination of IKZF1, IKZF3, SALL4, and PLZF by Western blotting) The levels of IKZF1, IKZF3, SALL4, and PLZF remaining in each cell after degradation were quantified. (Quantification of each protein by Western blotting method) Detection (imaging of chemiluminescence) was performed using an Amercham ImageQuant 800 / Cytiva. After detection, the membrane was stripped of the antibody using a stripping solution, and the next antibody reaction was performed. Image data acquired with an Amercham ImageQuant 800 was loaded and analyzed using Quantity One / Bio-Rad software. The target protein band was quantified in Quantity One, and the amount of each molecule remaining after adding DMSO (a control sample without the compound) was set to 100%. The degree of degradation was evaluated by using the relative ratio of the number of remaining molecules after compound addition as the percentage of remaining molecules after degradation (%). The antibodies used for detection are shown in Table 3 below. We also checked for any increase or decrease in CRBN expression in cells due to the addition of the compounds using Anti-CRBN-Ab, but no significant fluctuations were observed with the addition of the compounds, confirming uniform expression. Each antibody is commercially available from Thermo Fisher, SANTA Cruz, Cell Signaling, and MBL.

[0176] [Table 3]

[0177] (Synthesis of 7-CF3-pomaridomide) It was synthesized using the following procedure. 30 mg (0.1 mmol) of ((trifluoromethyl)sulfonyl)benzene, 10 mg (0.06 mmol) of copper(I) iodide, and 10 mg (0.1 mmol) of potassium t-butoxy were dissolved in 1 mL of dry N,N-dimethylformamide and stirred at room temperature for 30 minutes. Then, 7 mg (0.02 mmol) of 7-Br-pomalidomide was added, and after 12 hours, the solvent was removed under reduced pressure. The mixture was then redissolved in ethyl acetate and purified by silica gel column chromatography. 4-amino-2-(2,6-diociopiperidine-3-yl)-7-trifluoromethylisoindorin-1,3-dione was obtained in a yield of 2.0 mg and 30%. 1 H-NMR(500 MHz, CDCl3) δ 2.11-2.18 ppm (m, 1 H), 2.69-2.95 ppm (m, 3 H), 4.90-4.97 ppm (m, 1 H), 5.67-5.87 ppm (m, 2 H), 5.26 ppm (brs, 2 H), 7.30 (m, 1H), 7.48 (m, 1H), 7.9 (brs, 1H); 19 F-NMR (470 MHz, CDCl3): -121.9. (Synthesis of 5,7-Br-pomaridomide) It was synthesized using the following procedure. 100 mg (0.366 mmol) of pomalidomide and 390 mg (2.2 mmol) of N-bromosuximide were dissolved in 4 mL of acetonitrile and stirred at room temperature for 24 hours. After removing the solvent under reduced pressure, the mixture was redissolved in ethyl acetate and purified by silica gel column chromatography. 4-amino-7-bromo-2-(2,6-diociopiperidine-3-yl)isoindorin-1,3-dione was obtained in a yield of 42 mg and 33%. 1 H-NMR(500 MHz, CDCl3) δ 2.11-2.18 ppm (m, 1 H), 2.69-2.95 ppm (m, 3 H), 4.90-4.97 ppm (m, 1 H), 5.67-5.87 ppm (m, 2 H), 5.26 ppm (brs, 2 H), 7.46 (m, 1H), 7.54 (m, 1H), 7.68 (brs, 1H) As a by-product of the above reaction, 2.1 mg of 4-amino-5,7-bromo-2-(2,6-diociopiperidine-3-yl)isoindorin-1,3-dione was obtained. 1 H-NMR(500 MHz, CDCl3) δ 2.11-2.18 ppm (m, 1 H), 2.69-2.95 ppm (m, 3 H), 4.90-4.97 ppm (m, 1 H), 5.67-5.87 ppm (m, 2 H), 5.26 ppm (brs, 2 H), 8.1 (s, 1H) 7.81 (brs, 1H)

[0178] Similar trends were observed with thalidomide, pomalidomide, and lenalidomide, all of which led to the degradation of IKZF1 and IKZF3, which contribute to the antitumor effect in multiple myeloma (see Figure 34). On the other hand, it was found that degradation of SALL4 and PLZF, which are the cause of teratogenicity, was also observed simultaneously. Furthermore, the binding ability of each IKZF1, IKZF3, SALL4, and PLZF compound to CRBN was strengthened in the presence of these compounds, which enhanced the degradation of these molecules in cells (see Figure 34).

[0179] As a target protein degradation inducer, we used compounds in which a sterically hindrance-heavy linker and a protein target binder were attached to the 4-position to investigate the effect of the 4-position modification on the binding of each IKZF1, IKZF3, SALL4, and PLZF, and consequently on the degradation of each IKZF1, IKZF3, SALL4, and PLZF in expressed cells (see Figure 35). As a hydrogen substitution of the amine at position 4, we used a model compound in which polyethylene glycol was used as a linker, and a BET bromodomain inhibitor compound JQ-1, which targets BRD, was attached to the end of the linker. This compound is an extremely flexible substitution due to the extremely bulky nature of JQ-1 and the use of n=3 polyethylene glycol as a linker. The results are shown in Figure 35. Regarding the biochemical interactions of BRD, the interaction between Flag-GST-BRD4 and biotinylated CRBN was evaluated using the AlphaScreen method, similar to the evaluation for IKZF, etc. Furthermore, the degradation of BRD in cells was assessed using the BRD antibodies shown in Table 3, with the average degradation values ​​for BRD2, BRD3, and BRD4 being used. The amount of each molecule remaining after adding DMSO (a control without the compound) was set to 100%, and the degree of degradation was evaluated by using the relative ratio of remaining molecules after compound addition as the percentage of remaining molecules after degradation (%). In both the pomalidomide and lenalidomide basic skeletons, the binding ability to IKZF1, IKZF3, SALL4, and PLZF showed a nearly identical trend. Surprisingly, when substitution was made at the 4-position using a "linker" and a "target binder" (in this experiment, n=3 polyethylene glycol was used as the linker, and JQ-1 targeting BRD was used as the target protein binder as a model compound), it was confirmed that this substituent did not affect the binding ability to IKZF1, IKZF3, SALL4, and PLZF. Furthermore, a comparison of lenalidomide and tetrafluorinated lenalidomide revealed that modifications other than the 4th position of the benzene ring have a dominant influence on the binding tendency and degradation tendency of each molecule, compared to the influence of 4-position modifications. In other words, when a protein binder (protein target function) is added via a linker at the 4th position, the target protein binder can be arbitrarily selected. More specifically, functional group modifications at the 6th and 7th positions of the benzene ring in pomalidomide and lenalidomide compounds are important for suppressing their binding ability to SALL4 and PLZF. We have also confirmed that modifications at the 5th position can suppress the binding ability to SALL4 and PLZF.

[0180] We confirmed substituent effects on the pomalidomide skeleton, mainly at positions 5 and 6 (see Figure 36). Regarding the results of 6-position modification, when halogen substitution was performed, the binding to SALL4 and PLZF was clearly reduced for all of F, Cl, and Br compared to pomalidomide, and this effect increased as the van der Waals radius increased. In terms of the degree of degradation in cells, Cl and Br were also the most effective. On the other hand, since the ability to bind to IKZF1 and IKZF3 was not completely lost, it was confirmed that the teratogenic effect was attenuated while retaining the antitumor effect against multiple myeloma. On the other hand, regarding the effect of halogen modification at position 7, F shows a significant decrease in its binding ability to IKZF1 and IKZF3, while simultaneously losing its binding ability to SALL4 and PLZF. As a result, when used as the core of a targeted degradation inducer, not only is the teratogenic toxicity reduced, but the degradation of IKZF1 and IKZF3 is also suppressed. Therefore, it can be used as a targeted degradation inducer that can recruit only cereblon in a neutral manner (without biological effects) by utilizing only the targeting ability of the target protein binder (molecular glue function). On the other hand, when Cl and Br are substituted with halogens that have a large van der Waals radius, Cl's binding ability to both SALL4 and PLZF increases compared to fluorine substitution, and intracellular degradation also increases. When Br, which has a larger van der Waals radius than Cl, is substituted, the degradation of SALL4 and PLZF is further suppressed. Furthermore, when the substituent is a methyl trifluoride group, the binding to SALL4 and PLZF is significantly reduced, and the degradation of SALL4 and PLZF is further suppressed. It was also found that the binding to IKZF1 and IKZF3 is considerably suppressed. This trend is clearly different from that of the 6-position modification. In addition, when comparing pomalidomide and lenalidomide systems with fluorine substitution at position 7, it can be seen that they both show almost the same trend. When Br substitution was performed at positions 5 and 7, the binding ability to IKZF1, IKZF3, SALL4, and PLZF was lost compared to when only position 7 was substituted, and intracellular degradation was almost suppressed. When a hydroxyl group substitution was performed at position 7, the molecular binder function was extremely high, similar to that of a trimethyl fluoride group substitution. The binding ability to IKZF1, IKZF3, SALL4, and PLZF was lost, and intracellular degradation was almost suppressed. Therefore, it was confirmed that it is optimal as the core of a targeted degradation inducer with linker and target binder function at position 4.

[0181] Substituent effects were observed on the lenalimid skeleton, mainly at positions 6 and 7 (see Figure 37). We confirmed that when halogen substitution was performed at the 6th position, the binding ability to SALL4 and PLZF was significantly reduced while maintaining the binding ability to IKZF1 and IKZF3, thereby greatly reducing teratogenicity. This trend was similar to that observed in the pomalidomide skeleton. Furthermore, it was found that a larger van der Waals radius of the functional group reduced the binding ability to SALL4 and PLZF, resulting in a greater suppression of degradation in cells. Similarly, this trend was the same as that of the pomalidomide skeleton. In particular, we confirmed that substitution with bulky functional groups such as methyl trifluoride and methoxy trifluoride was especially effective.

[0182] As shown in Figure 36, a 7-modified pomalidomide-linker-JQ-1 derivative was synthesized using the same chemical reaction as in Example 12 on a 7-modified pomalidomide skeleton that did not induce degradation of IKZF1, IKZF3, SALL4, or PLZF. The biochemical interactions (AlphaScreen method) between IKZF1, IKZF3, SALL4, PLZF, BRD4 and CRBN, and the degree of degradation of each protein in cells were evaluated (see Figure 38). Both the 7-position fluoride pomalidomide-linker-target binder (JQ-1) and the 7-position trifluoropomalidomide-linker-target binder (JQ-1) exhibited low binding ability to SALL4 and PLZF. As a result, the degree of degradation of SALL4 and PLZF in cells was significantly reduced compared to that of the pomalidomide-linker-target binder (JQ-1). This trend confirmed that the binding ability to the basic skeleton IKZF1, IKZF3, SALL4, and PLZF, prior to the substitution of 'linker' and 'target binder' in the 4th position, was inherited as a property. This allows us to create target protein degradation inducers that, when used as CRBN binders, induce only the degradation of the target protein while maintaining other biologically neutral properties.

[0183] (Overall assessment) Regarding hematological cancers, since the therapeutic targets are both the neosubstrates IKZF1 / 3 and CK1α and the target proteins BRD2 / 3 / 4, compounds with high decomposition capabilities for IKZF1 / 3 are considered to have high therapeutic efficacy. On the other hand, in cancers other than blood cancers, IKZF1 / 3 and CK1α are not considered targets for drug efficacy and are thought to lead to blood-related side effects. More specifically, IKZF1 / 3 is involved in the proliferation of hematological cancers (especially multiple myeloma), but as it is a protein expressed in blood cells (especially white blood cells), it is thought to be not significantly involved in cancers other than hematological cancers. On the other hand, IKZF1 / 3 is an extremely important transcription factor in the differentiation and function of hematopoietic stem cells into white blood cells (especially lymphocytes such as T cells and B cells). In fact, IKZF1 / 3 knockout mice become immunodeficient and lack T cells and B cells. In addition, it has been reported that the degradation of IKZF1 and IKZF3 by lenalidomide administration in mice causes a decrease in white blood cells and platelets. Furthermore, there have been reports of a decrease in white blood cells as a side effect of administering thalidomide derivatives to patients. In other words, significant degradation of IKZF1 / 3 in cancers other than blood cancers is thought to have no contribution to drug efficacy and to lead to side effects.

[0184] Genetically, it has been shown that even heterozygous mutations in SALL4 can cause a congenital disorder called Okihiro syndrome (Duane-radial ray syndrome). In addition, this disorder exhibits teratogenicity in various parts of the body, including the limbs, and its phenotype has been reported to be very similar to that of thalidomide-induced fetal disease. Furthermore, considering that thalidomide and thalidomide derivatives have a very high ability to induce degradation of SALL4, and that Okihiro syndrome is caused even when the SALL4 mutation is heterozygous, it is highly likely that teratogenicity is caused by the degradation of SALL4. It has also been confirmed that administering thalidomide to rabbits reduces SALL4 levels in the fetuses. PLZF has been reported to cause teratogenicity in humans and mice through mutation. Furthermore, the inventors have reported that PLZF is degraded in a system using chicken embryos in which SALL4 degradation is not induced. In addition, they have experimentally demonstrated that overexpression of PLZF in chicken embryos reduces thalidomide-induced teratogenicity. In other words, it is thought that severe teratogenicity is caused by the significant degradation of both SALL4 and PLZF in humans. Furthermore, it has been reported that double knockout of SALL4 and PLZF in mice results in a more pronounced teratogenic phenotype, closely resembling thalidomide fetal disease. Therefore, reducing the degradation of SALL4 and PLZF can lead to a reduction in teratogenicity.

[0185] Based on the above findings and the results of the above examples, the following was confirmed. Compared to the existing drug lenalidomide, F-Len exhibits higher activity against multiple myeloma and significantly higher activity against 5qMDS. On the other hand, its ability to induce the degradation of SALL4 and PLZF is lower than that of lenalidomide. In short, F-Len is a thalidomide derivative with reduced teratogenicity and higher efficacy than the existing drug lenalidomide. Compared to the existing drug lenalidomide, Cl-Len has weaker activity against multiple myeloma and 5qMDS, but it does not induce the degradation of SALL4 or PLZF. In other words, Cl-Len is less potent than the existing drug lenalidomide, but it significantly reduces teratogenicity. Compared to the existing drug lenalidomide, Br-Len has a weaker efficacy against multiple myeloma, but it does not induce the degradation of SALL4 or PLZF. In other words, Br-Len has a weaker efficacy than the existing drug lenalidomide, but it significantly reduces teratogenicity. F3C-Len does not induce degradation of representative neosubstrates such as IKZF1 / 3 and CK1α, which are drug targets for hematological cancers, or SALL4 and PLZF, which are involved in teratogenicity. However, it can induce high protein degradation via CRBN. Therefore, F3C-Len is expected to be applied to various cancer types as a proteacologic agent with fewer side effects. PROTACs using F-Len exhibit similar efficacy to existing PROTACs against hematological cancers, while reducing their ability to induce the degradation of SALL4 and PLZF. In other words, F-Len-PROTACs are highly effective against hematological cancers while reducing teratogenicity. Furthermore, they are also effective against other types of cancer. Cl-Len-based PROTACs are less effective against hematological cancers than existing PROTACs, but they do not induce the degradation of SALL4 or PLZF. In other words, Cl-Len-PROTACs are effective against hematological cancers while significantly reducing teratogenicity. Furthermore, they are also effective against other types of cancer. F3C-LenPROTAC is effective against various types of cancer and is considered to have no teratogenic or hematological side effects. Based on its similar chemical structure and properties to F3C-Len-PROTAC, as well as the results from the examples, F3CO-Len-PROTAC is considered effective against various types of cancer and is thought to have no teratogenic or hematological side effects. Based on the results of the examples, Br-Len-PROTAC, like F3C-Len-PROTAC, is considered effective against various types of cancer and is thought to have no teratogenic or hematological side effects.

[0186] Based on the above findings and the results of Example 18, the following was confirmed. 1) We confirmed that the functional groups of thalidomide derivatives (especially at positions 6 and 7) have a significant impact on teratogenicity. 2) Strong biochemical binding of thalidomide derivatives to IKZF1, IKZF3, SALL4, and PLZF indicates high decomposition of IKZF1, IKZF3, SALL4, and PLZF in cells by thalidomide derivatives. 3) When a linker is extended to the 4-position of a thalidomide derivative (particularly the amino group at the 4-position) and a target molecule (POI: protein of interest: bulky) is attached, it was confirmed that the linker and the POI have almost no effect on the degradation to SALL4 and PLZF. In other words, attaching a linker to the 4-position, and further attaching a POI to the linker, does not inhibit or enhance the degrading ability of SALL4 and PLZF. In addition, the modification status of the 6- and 7-positions of the thalidomide derivative is extremely important for its degradability to SALL4 and PLZF. 4) Substitution of the 6-position modifying group weakens the binding ability to SALL4 and PLZF. This suggests the possibility of steric hindrance from the modifying group. On the other hand, the binding ability to IKZF1 and IKZF3 remains. 5) Modification at position 7 weakens the binding ability to IKZF1,3, SALL4, and PLZF. 6) The 7th position is occupied by a functional group with a large van der Waals radius, which reduces the binding selectivity to SALL4 and PLZF in particular. 7) Even without steric hindrance, highly electronegative functional groups (F) or hydrogen bonding functional groups (OH) strongly inhibit binding to SALL4 and PLZF. This is thought to be because, when thalidomide derivatives interact with CRBN, the structural presence of His359 in the vicinity causes interaction with the His imidazole group, reducing the space available for SALL4 and PLZF to enter. [Industrial applicability]

[0187] This invention provides novel target protein degradation-inducing compounds, cancer treatment compositions containing such compounds, and cancer treatment compositions with reduced teratogenicity.

[0188] The present invention is as follows: 1. A target protein degradation-inducing compound having the following composition, "Cerebronchial binder" - "Linker" - "Target protein binder" Here, the cerebron binder is a target protein degradation-inducing compound selected from one of the following: a compound represented by the general formula (10), a salt thereof, or a solvate thereof. [ka] (Here, X is CH2 or C=O, and R10 is hydrogen, a functional group, an atom, or NR) L1 R L2 And R L1 and R L2 R11 is hydrogen, a functional group, or an atom, but one of them is a functional group or an atom, and R11 is hydrogen, F, Br, Cl, CH3, or OCH 3、 I is CF3 or OCF3, and R12 is hydrogen, F, Br, CF3, I, OH, Cl, CH3, OCF3, or OCH3. 2. The target protein degradation-inducing compound described in item 1 above, wherein the target protein binder is JQ-1 or Birabresib. 3. A target protein degradation-inducing compound according to item 1 or 2 above, wherein X in the general formula (10) is CH2, R11 is CF3, and R12 is hydrogen. 4. A target protein degradation-inducing compound according to item 1 or 2 above, wherein X in the general formula (10) is CH2, R11 is F, and R12 is hydrogen. 5. A target protein degradation-inducing compound according to item 1 or 2 above, wherein X in the general formula (10) is CH2, R11 is Cl, and R12 is hydrogen. 6. A target protein degradation-inducing compound according to item 1 or 2 above, having the following composition: The cerebron binder is selected from one of the compounds represented by the following general formula (7), their salts, or their solvates. [ka] (Here, R L1 and R L2 (It is either hydrogen, a functional group, or an atom, but at least one of them is either a functional group or an atom.) Furthermore, the target protein binder is Birabresib or JQ-1. A compound that induces the degradation of target proteins. 7. A target protein degradation-inducing compound according to item 1 or 2 above, having the following composition: The cerebron binder is selected from one of the following compounds represented by the general formula (3), their salts, or their solvates: [ka] (Here, R L1 and R L2 (It is either hydrogen, a functional group, or an atom, but at least one of them is either a functional group or an atom.) Furthermore, the target protein binder is Birabresib or JQ-1. A compound that induces the degradation of target proteins. 8. A target protein degradation-inducing compound according to item 1 or 2 above, having the following composition: The cerebron binder is selected from one of the compounds represented by the following general formula (8), their salts, or their solvates. [ka] (Here, R L1 and R L2 (It is either hydrogen, a functional group, or an atom, but at least one of them is either a functional group or an atom.) Furthermore, the target protein binder is Birabresib or JQ-1. A compound that induces the degradation of target proteins. 9. A target protein degradation-inducing compound according to item 1 or 2 above, having the following composition: The cerebron binder is selected from one of the following compounds represented by the general formula (6), their salts, or their solvates: [ka] (Here, R L1 and R L2 (It is either hydrogen, a functional group, or an atom, but at least one of them is either a functional group or an atom.) Furthermore, the target protein binder is Birabresib or JQ-1. A compound that induces the degradation of target proteins. 10. A target protein degradation-inducing compound according to item 1 or 2 above, having the following composition: The cerebron binder is selected from one of the following compounds represented by the general formula (5), salts thereof, or solvates thereof. [ka] (Here, R L1 and R L2 (It is either hydrogen, a functional group, or an atom, but at least one of them is either a functional group or an atom.) Furthermore, the target protein binder is Birabresib or JQ-1. A compound that induces the degradation of target proteins. 11. A target protein degradation-inducing compound according to item 1 or 2 above, having the following composition: The cerebron binder is selected from one of the following compounds represented by the general formula (4), their salts, or their solvates: [ka] (Here, R L1 and R L2 (It is either hydrogen, a functional group, or an atom, but at least one of them is either a functional group or an atom.) Furthermore, the target protein binder is a target protein degradation-inducing compound, which is Birabresib or JQ-1. 12. A target protein degradation-inducing compound as described in item 1 or 2 above, which reduces the protein degradation ability of SALL4 or PLZF. 13. A target protein degradation-inducing compound as described in item 1 or 2 above, wherein teratogenicity is reduced. 14. A cancer treatment composition comprising one or more target protein degradation-inducing compounds as specified in item 1 or 2 above. 15. A cancer treatment composition comprising a compound represented by the following general formula (1), a salt thereof, or a solvate thereof. [ka] (Here, R1 is hydrogen, F, Br, Cl, CH3, OCH 3、 I, CF3, or OCF3, R L1 and R L2 Each of these is independently a hydrogen atom, a functional group, or an atom. 16. The cancer treatment composition according to item 15 above, wherein the compound represented by the following general formula (1) is the compound represented by general formula (7), a salt thereof, or a solvate thereof. [ka] (Here, R L1 and R L2 Each of these is independently a hydrogen atom, a functional group, or an atom. 17. The cancer treatment composition described in paragraph 15, wherein the compound represented by the following general formula (1) is the compound represented by general formula (8), a salt thereof, or a solvate thereof. [ka] (Here, R L1 and R L2 Each of these is independently a hydrogen atom, a functional group, or an atom. 18. The cancer treatment composition according to item 15 above, wherein the compound represented by the following general formula (1) is the compound represented by general formula (6), a salt thereof, or a solvate thereof. [ka] (Here, R L1 and R L2 Each of these is independently a hydrogen atom, a functional group, or an atom. 19. The cancer treatment composition according to item 15 above, wherein the compound represented by the following general formula (1) is the compound represented by general formula (3), a salt thereof, or a solvate thereof. [ka] (Here, R L1 and R L2 Each of these is independently a hydrogen atom, a functional group, or an atom. 20. The cancer treatment composition according to paragraph 15, wherein the compound represented by the following general formula (1) is the compound represented by general formula (5), a salt thereof, or a solvate thereof. [ka] (Here, R L1 and R L2 Each of these is independently a hydrogen atom, a functional group, or an atom. 21. A therapeutic agent for chromosome 5-deficient myelodysplastic syndrome comprising a compound represented by the following general formula (5), a salt thereof, or a solvate thereof. [ka] (Here, R L1 and R L2Each of these is independently a hydrogen atom, a functional group, or an atom. 22. A therapeutic agent for chromosome 5-deficient myelodysplastic syndrome comprising a compound represented by the following general formula (3), a salt thereof, or a solvate thereof. [ka] (Here, R L1 and R L2 Each of these is independently a hydrogen atom, a functional group, or an atom.

Claims

1. A target protein degradation-inducing compound having the following composition, "Cerebronchial binder" - "Linker" - "Target protein binder" Here, the cerebron binder is selected from one of the following compounds: general formula (5), general formula (7), general formula (8), general formula (6), 7-trifluoromethyl-pomalidomide or 7-hydroxy-pomalidomide, a salt thereof, or a solvate thereof. A compound that induces the degradation of target proteins. 【Chemistry 1】 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 【Transformation 5】 【Transformation 6】 (Here, R Ｌ1 and R Ｌ2 One of the atoms is hydrogen, and the other is bound to the "linker" – the "target protein binder."

2. The target protein degradation-inducing compound according to claim 1, wherein the cerebron binder is a compound represented by general formula (5).

3. The target protein degradation-inducing compound according to claim 1, wherein the cerebron binder is a compound represented by general formula (7).

4. The target protein degradation-inducing compound according to claim 1, wherein the cerebron binder is a compound represented by general formula (8).

5. The target protein degradation-inducing compound according to claim 1, wherein the cerebron binder is a compound represented by general formula (6).

6. The target protein degradation-inducing compound according to claim 1, wherein the cerebron binder is 7-trifluoromethyl-pomalidomide.

7. The target protein degradation-inducing compound according to claim 1, wherein the cerebron binder is 7-hydroxypomalidomide.

8. A multiple myeloma growth inhibitor comprising the target protein degradation-inducing compound described in claim 1.

9. A therapeutic agent for embryonic carcinoma or colorectal cancer comprising the target protein degradation-inducing compound described in claim 1.

10. A neuroblastoma growth inhibitor comprising the target protein degradation-inducing compound described in claim 1.

11. A multiple myeloma growth inhibitor comprising the target protein degradation-inducing compound described in claim 3.

12. A therapeutic agent for embryonic carcinoma or colorectal cancer comprising the target protein degradation-inducing compound described in claim 3.

13. A neuroblastoma growth inhibitor comprising the target protein degradation-inducing compound described in claim 3.

14. The multiple myeloma growth inhibitor according to claim 8 or 11, wherein teratogenic toxicity is reduced due to a reduced ability to break down SALL4 and PLZF.

15. The embryonic carcinoma or colorectal cancer therapeutic agent according to claim 9 or 12, wherein teratogenic toxicity is reduced due to a reduction in the decomposition ability of SALL4 and PLZF.

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