JP5934986B2 - Apoptosis-inhibiting protein ligand-estrogen receptor ligand hybrid compound, estrogen receptor degradation inducer using the same, and preventive and therapeutic agent for breast cancer, cervical cancer or ovarian cancer - Google Patents

Description

  The present invention relates to apoptosis inhibiting protein (IAP) ligand-estrogen receptor (ER) ligand hybrid compounds. Specifically, the present invention relates to an IAP ligand-ER ligand hybrid compound that specifically degrades ER expressed in cancer cells such as breast cancer to induce cell death.

  Increased expression of ER is observed in many breast cancer patient tissues (see, for example, Non-Patent Document 1), and these cancer cells have been reported to exhibit estrogen-dependent proliferation (for example, Non-Patent Document 1). 2 and 3). Tamoxifen, which is frequently used as an anti-estrogen drug, competitively binds to estrogen for ER such as breast cancer tissue and suppresses ER-dependent gene expression, thereby suppressing cell growth and exerting an antitumor effect. (E.g., see Non-Patent Documents 4 and 5). However, tamoxifen is known to act as an agonist for ER in the uterine cancer tissue in the same manner as estrogen and to increase the risk of endometrial cancer (see, for example, Non-Patent Documents 6 and 7). Furthermore, tamoxifen has been reported to activate intracellular signal cascades such as Akt through binding to ER and inhibit apoptosis of cancer cells (see, for example, Non-Patent Documents 8 and 9).

  On the other hand, the present inventors previously developed a protein knockdown method for selectively degrading a target protein using the ubiquitin-proteasome system of the protein degradation mechanism (see Non-Patent Documents 10 to 13). That is, a protein in vivo is polyubiquitinated by the ubiquitin ligase (E3) that recognizes it, and is degraded by the proteasome, but uses a molecule in which the ligand of cIAP1 having ubiquitin ligase activity and the ligand of the target protein are linked. Thus, a population complex of cIAP1 and the target protein is formed, and the target protein is ubiquitinated in the same manner as the in vivo protein degradation mechanism to induce degradation by the proteasome. The present inventors synthesized a hybrid compound in which ubenimex and all-trans retinoic acid are actually bound by using the protein knockdown method, and the hybrid compound is a protein that binds to retinoic acid, CRABP (cellular recurrent acid). -Binding protein) was demonstrated to induce degradation by the proteasome by ubiquitination.

Frederik Holst, et al., "Estrogen receptor alpha (ESR1) gene amplification is ferquent in breast cancer", Nat Genet, 2007, 39, 655-660 S. F. Doisneau, et al., "Estrogen and antiestrogen regulation of cell cycle progression in breast cancer cells", Endocrine-Related Cancer, 2003, 10, 179-186 James S. Foster, et al., "Multifaceted Regulation of Cell Cycle Progression by Estrogen: Regulation of Cdk Inhibitors and Cdc25A Independent of Cyclin D1-Cdk4 Function", Mol. Cell. Biol., 2001, 21, 794-810 Bonnie J. Deroo, et al., "Estrogen receptors and human disease", J. Clin. Invest., 2006, 116 (3), 561-570 Early Breast Cancer Trialists' Collaborative Group, "Tamoxifen for early breast cancer: an overview of the randomised trials", Lancet, 1998, 351, 1451-1467 Leslie Bernstein, et al., "Tamoxifen Therapy for Breast Cancer and Endometrial Cancer Risk", J. Natl. Cancer Inst., 1999, 91, 1654-1662 Yongfeng Shang, et al., "Molecular Determinants for the Tissue Specificity of SERMs", Science, 2002, 295, 2465-2468 Grazia Arpino, et al., "Crosstalk between the Estrogen Receptor and the HER Tyrosine Kinase Recoptor Family: Molecular Mechanism and Clinical Implications for Endocrine Therapy Resistance", Endocr. Rev., 2008, 29, 217-233 Sheng-Li Lin, et al., "ER-α36, a Variant of ER-α, Promotes Tomxifen Agonist Action in Endometrial Cancer Cells via the MAPK / ERK and PI3K / Akt Pathways", PLoS One, 2010, 5, e9013 Keiko Sekine, et al., "Small Molecules Destabilize cIAP1 by Activating Auto-ubiquitylation", J. Biol. Chem., 2008, 283, 8961-8968 Yukihiro Itoh, et al., "Protein Knockdown Using Methyl Bestatin -Ligand Hybrid Molecules: Design and Synthesis of Inducers of Ubiquitination-Mediated Degradation of Cellular Retinoic Acid-Binding Proteins", J. Am. Chem. Soc., 2010, 132, 5820-5826 Keiichiro Okuhira, et al., "Specific degradation of CRABL-II via cIAP1-mediated ubiquitylation induced by hybrid molecules that crosslink cIAP1 and the target protein", FEBS Letters, 2011, 585, 1147-1152 Yukihiro Itoh, et al., "Development of target protein-selective degradation inducer for protein knockdown", Bioorg. Med. Chem., 2011, 19, 3229-3241

  The present invention has been made in view of the above-described problems of the prior art, and the object of the present invention is to specifically degrade ER expressed in cancer cells such as breast cancer by a novel mechanism of action to thereby cause cell death. It is to provide an IAP ligand-ER ligand hybrid compound to be induced, an ER degradation inducer using the compound, and a preventive and therapeutic agent for breast cancer, cervical cancer or ovarian cancer.

  The above object of the present invention is achieved by the following means.

(1) That is, the present invention relates to (S) -2-[(2S, 3R) -3-amino-2-hydroxy-4-phenylbutanamide] -N- {3-[(2- {4- [ (E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -3-oxoprovir} -4-methylpentanamide, (S) -2- [(2S, 3R) -3-amino-2-hydroxy-4-phenylbutanamide] -N- {5-[(2- {4-[(E / Z) -1- (4-hydroxyphenyl)- 2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -5-oxopentyl} -4-methylpentanamide, 7-{(S) -2-[(2S, 3R) -3-amino- 2-hydroxy-4-phenylbutanamide]- 4-methylpentanamide} -N- (2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) -N-methylheptanamide, (S) -2-[(2S, 3R) -3-Amino-2-hydroxy-4-phenylbutanamide] -N- (2- {2-[(2- {4-[(E / Z)- 1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -2-oxoethoxy} ethyl) -4-methylpentanamide or (R) -1-{(S ) -2-Cyclohexyl-2-[(S) -2- (methylamino) propanamide] acetyl} -N-((S) -1- {7-[(2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1 Enyl] phenoxy} ethyl) (methyl) amino] -7-oxo-heptyl amino} -1-oxo-3,3-diphenyl-2-yl) pyrrolidine -2-carboxamide, apoptosis inhibitory protein ligand - estrogen receptor Ligand hybrid compound.

(2) The present invention also provides ( S) -2-[(2S, 3R) -3-amino-2-hydroxy-4-phenylbutanamide] -N- {3-[(2- {4-[( E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -3-oxoprovir} -4-methylpentanamide, (S) -2- [ (2S, 3R) -3-Amino-2-hydroxy-4-phenylbutanamide] -N- {5-[(2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2 -Phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -5-oxopentyl} -4-methylpentanamide, 7-{(S) -2-[(2S, 3R) -3-amino-2 -Hydroxy-4-phenylbutanamide] -4- Chill pentanamide} -N- (2- {4 - [ (E / Z) -1- (4- hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) -N- methyl-heptanamide and (S ) -2-[(2S, 3R) -3-Amino-2-hydroxy-4-phenylbutanamide] -N- (2- {2-[(2- {4-[(E / Z) -1- (4-Hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -2-oxoethoxy} ethyl) -4-methylpentanamide selected from the group consisting of apoptosis-inhibiting protein ligands An estrogen receptor degradation inducer comprising an estrogen receptor ligand hybrid compound or a physiologically acceptable salt thereof as an active ingredient .

(3) The present invention is also a preventive and therapeutic agent for breast cancer, cervical cancer or ovarian cancer comprising the estrogen receptor degradation inducer according to (2) .

  Since the IAP ligand-ER ligand hybrid compound of the present invention has a structure in which an IAP ligand that specifically binds to IAP and an ER ligand that specifically binds to ER are bound via a linker, ubiquitin ligase is produced in the cell. The active cIAP1 and ER are cross-linked, and ER is specifically ubiquitinated to induce proteasome degradation. Therefore, when the IAP ligand-ER ligand hybrid compound of the present invention is administered to patients such as breast cancer, cervical cancer, ovarian cancer, etc., the expression itself of ER in cancer tissue can be decreased, and cancer cells can be led to self-destruction, It is extremely useful as an ER degradation inducer or a preventive and therapeutic agent for breast cancer and the like.

It is a figure which shows the evaluation result of ER degradation induction activity of the IAP ligand-ER ligand hybrid compound of this invention. It is a figure which shows the evaluation result of the apoptosis promoting activity of the IAP ligand-ER ligand hybrid compound of this invention. It is a figure which shows the evaluation result of the apoptosis promoting activity of the IAP ligand-ER ligand hybrid compound of this invention. It is a figure which shows the evaluation result of ER degradation induction activity of the IAP ligand-ER ligand hybrid compound of this invention.

  Hereinafter, embodiments of the present invention will be described in detail.

  The IAP ligand-ER ligand hybrid compound of the present invention is

The following general formula (I)
Wherein X represents an IAP ligand that specifically binds to IAP, Y represents an ER ligand that specifically binds to ER, and Z represents the specific binding properties of the IAP ligand and the ER ligand. It represents a low-molecular chain linker that binds both without inhibiting.

  In the general formula (I), the IAP ligand of X is not particularly limited as long as it is a monovalent group derived from a compound that specifically binds to IAP. Specific examples of such a compound that specifically binds to IAP include ubenimex ((2S) -2-[(2S, 3R) -3-amino-2-hydroxy-4-phenylbutanoylamino] -4-methylpentane. Acid), MV-1 ((R) -2-((R) -1-{(S) -2-cyclohexyl-2-[(S) -2- (methylamino) propanamide] acetyl} pyrrolidine-2- Carboxamide) -3,3-diphenylpropanoic acid) or AEG 40599 ((S) -N-((S) -3,3-dimethyl-1-oxo-1-{(R) -2-[(2,2, 2-trifluoro-N-phenethylacetamido) methyl] pyrrolidin-1-yl} butan-2-yl) -2- (methylamino) propanamide) or derivatives thereof, of which ubenimex is It is preferably used to.

  The ER ligand of Y is not particularly limited as long as it is a monovalent group derived from a compound that specifically binds to ER. Specific examples of such a compound that specifically binds to ER include tamoxifen ((Z) -2- [4- (1,2-diphenyl-1-butenyl) phenoxy] -N, N-dimethylethylamine), raloxifene ( [6-hydroxy-2- (4-hydroxyphenyl) benzo [b] thien-3-yl] [4- (2-piperidin-1-ylethoxy) phenyl] methanone), toremifene (2- [4-[(Z ) -4-chloro-1,2-diphenyl-1-butenyl] phenoxy] -N, N-dimethylethylamine), fulvestrant (7α- [9-[(4,4,5,5,5-pentafluoro) Pentylsulfinyl)] nonyl] estradi-1 (10), 2,4-triene-3,17β-diol), ICI 182780 (fluoropentylsulfinyl) noni Estrogen receptor inhibitors such as estra-1,3,5 (10) -triene-3,17β-diol) or derivatives thereof, among which tamoxifen is particularly preferably used.

Furthermore, the low molecular chain linker of Z is not particularly limited as long as it is a divalent group that binds both the IAP ligand and the ER ligand without inhibiting the specific binding property. Specific examples of such a low molecular chain linker include the following general formula (II)
(In the formula, R represents an alkylene group having 2 to 10 carbon atoms, a monoether group or polyether group having 2 to 10 carbon atoms, or an amide group having 3 to 10 carbon atoms, each of which may have a substituent. ) Is a divalent group. In the general formula (II), specific examples of the alkylene group having 2 to 10 carbon atoms include ethylene group, trimethylene group, tetramethylene group, pentamethylene group, hexamethylene group, propylene group, ethylethylene group, and methyltrimethylene group. Etc. Examples of the C2-C10 monoether group or polyether group include polyethylene glycol and polypropylene glycol. Furthermore, examples of the amide group having 3 to 10 carbon atoms include alkylamide and alkylsulfonylamide.

Specific examples of the IAP ligand-ER ligand hybrid compound of the present invention include the following formula (III)
(S) -2-[(2S, 3R) -3-amino-2-hydroxy-4-phenylbutanamide] -N- {3-[(2- {4-[(E / Z)- 1- (4-Hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -3-oxoprovir} -4-methylpentanamide, the following formula (IV)
(S) -2-[(2S, 3R) -3-amino-2-hydroxy-4-phenylbutanamide] -N- {5-[(2- {4-[(E / Z)- 1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -5-oxopentyl} -4-methylpentanamide, the following formula (V)
7-{(S) -2-[(2S, 3R) -3-amino-2-hydroxy-4-phenylbutanamide] -4-methylpentanamide} -N- (2- {4- [ (E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) -N-methylheptanamide, the following formula (VI)
(S) -2-[(2S, 3R) -3-amino-2-hydroxy-4-phenylbutanamide] -N- (2- {2-[(2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -2-oxoethoxy} ethyl) -4-methylpentanamide, the following formula (VII)
(R) -1-{(S) -2-cyclohexyl-2-[(S) -2- (methylamino) propanamide] acetyl} -N-((S) -1- {7- [ (2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -7-oxoheptylamino} -1-oxo -3,3-diphenylpropan-2-yl) pyrrolidine-2-carboxamide and the like.

Next, a method for producing the IAP ligand-ER ligand hybrid compound of the present invention will be described taking the case where the IAP ligand is ubenimex and the ER ligand is tamoxifen as an example. That is, the following reaction formula (VIII)
As shown in FIG. 2, the amino group of the ER ligand (tamoxifen) and the carboxyl group of the linker are subjected to a condensation reaction by a known method, and the linker is introduced onto the amino group of the IAP ligand. Subsequently, the amino group of the linker of the obtained reaction product and the hydroxyl group at the 1-position of the IAP ligand (Ubenimex) are subjected to a condensation reaction by a known method to obtain the target IAP ligand-ER ligand hybrid compound. In the reverse order, the linker may be first condensed with the IAP ligand and then condensed with the IAP ligand.

  The IAP ligand-ER ligand hybrid compound of the present invention has a structure in which an IAP ligand that specifically binds to IAP and an ER ligand that specifically binds to ER are bound via a linker. Therefore, cIAP1 having ubiquitin ligase activity and ER are cross-linked in the cell, and ER is specifically ubiquitinated to induce degradation by the proteasome. Therefore, if the IAP ligand-ER ligand hybrid compound of the present invention is administered to patients such as breast cancer, cervical cancer, ovarian cancer, etc., the expression of ER itself in the cancer tissue can be decreased, and cancer cells can be led to self-destruction, It is extremely useful as an ER degradation inducer or a preventive and therapeutic agent for breast cancer and the like.

  When the IAP ligand-ER ligand hybrid compound of the present invention is used as an ER degradation inducer or a prophylactic and therapeutic agent for breast cancer or the like, the IAP ligand-ER ligand hybrid compound alone or in the form of a tablet, pill, powder, powder, granule It can be orally administered to a patient as an agent, syrup, solution, suspension, emulsion, capsule or the like. Further, it can be administered as an injection intravenously, intramuscularly, intradermally, subcutaneously, intraperitoneally, intraarterially, intrathecally, or the like. Furthermore, it may be administered into the rectum as a suppository or can be implanted with a pellet. It can be administered directly to the affected area and its surroundings directly as eye drops, nasal drops, sprays, inhalants, etc. It may be administered dermally. Among the above-mentioned, a preferable formulation form, administration form, and the like are appropriately selected by a doctor according to the patient's age, sex, constitution, symptoms, treatment timing, and the like.

  When this preparation is used as a solid preparation such as a tablet, pill, powder, powder, granule, etc., the IAP ligand-ER ligand hybrid compound is mixed with an appropriate additive such as lactose, sucrose, man Knit, corn starch, synthetic or natural gum, excipients such as crystalline cellulose, starch, cellulose derivatives, gum arabic, gelatin, binders such as polyvinylpyrrolidone, carboxymethyl cellulose calcium, carboxymethyl cellulose sodium, starch Manufactured by mixing with disintegrants such as corn starch and sodium alginate, lubricants such as talc, magnesium stearate and sodium stearate, fillers and diluents such as calcium carbonate, sodium carbonate, calcium phosphate and sodium phosphate. can do. Tablets and the like may be coated with sugar coating, gelatin, enteric coating, film coating, etc. using an appropriate coating base as necessary.

  When the preparation is a liquid preparation such as an injection, eye drop, nasal drop, inhalant, spray, lotion, syrup, solution, suspension, emulsion, etc., the IAP ligand-ER ligand hybrid compound Purified water, suitable buffer solution such as phosphate buffer, physiological salt solution such as physiological saline, Ringer's solution, lock solution, vegetable oil such as cacao butter, sesame oil, olive oil, mineral oil, higher alcohol, higher fatty acid , Dissolved in an organic solvent such as ethanol, etc., if necessary, emulsifiers such as cholesterol, suspending agents such as gum arabic, dispersing aids, wetting agents, polyoxyethylene hydrogenated castor oil-based, polyethylene glycol-based interfaces, etc. Activators, solubilizers such as sodium phosphate, stabilizers such as sugar, sugar alcohol, albumin, preservatives such as paraben, sodium chloride, glucose, glycerin Isotonic agent, buffer, soothing agent, anti-adsorption agent, moisturizer, antioxidant, colorant, sweetener, flavor, fragrance, etc. It can be prepared as a suspension, liposome or emulsion. At this time, the injection preferably has a physiological pH, preferably in the range of 6-8.

  In order to make this preparation into a semi-solid preparation such as lotion, cream, ointment, etc., the IAP ligand-ER ligand hybrid compound is mixed with fat, fatty oil, lanolin, petrolatum, paraffin, wax, plaster, resin, plastic, It can be produced by appropriately mixing with glycols, higher alcohols, glycerin, water, emulsifiers, suspending agents and the like.

  The content of the IAP ligand-ER ligand hybrid compound contained in the ER degradation-inducing agent or the preventive and therapeutic agent for breast cancer of the present invention varies depending on the dosage form, severity, target dose, etc. Generally, it is 0.1 to 90% by weight, preferably 5 to 50% by weight, based on the total weight of the preparation.

  In addition, the dose of the ER degradation inducer or the preventive and therapeutic agent for breast cancer and the like of the present invention varies depending on the patient's age, body weight and symptoms, target dosage form and method, therapeutic effect, treatment period, etc. Although the amount is determined by a doctor, it is usually 10 to 1000 mg for oral administration and 5 for intravenous administration in terms of the dose of the IAP ligand-ER ligand hybrid compound per day for an adult. Administer ~ 500 mg in one or several divided doses.

  Next, the IAP ligand-ER ligand hybrid compound of the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Step 1: (E / Z) -tert-butyl 3-[(2- {4- [1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -3 -Oxopropyl carbamate synthesis

Under a nitrogen atmosphere, (E / Z) -endoxifene (0.5 mmol), 3- (tert-butoxycarbonylamino) propanoic acid (0.6 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide Hydrochloride (EDC, 0.6 mmol), 1-hydroxybenzotriazole (HOBt, 0.6 mmol), and N, N-diisopropylamine (DIPEA, 1.2 mmol) were dissolved in dichloromethane (5 mL) and allowed to reach room temperature for 3 hours. Stir. After the reaction, saturated aqueous sodium hydrogen carbonate (10 mL) was added, and the mixture was extracted with chloroform (20 mL × 3). The organic layer was dried and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (developing solvent: ethyl acetate: n-hexane = 1: 2) to give (E / Z) -tert-butyl 3-[(2 -{4- [1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -3-oxopropylcarbamate was obtained in 88% yield.
¹ H NMR (400 MHz, CDCl ₃ ): δ = 7.06-7.24 (m, 7H), 6.71-6.83 (m, 4H), 6.47-6.50 (m, 2H) , 5.33 (brs, 1H), 4.10 (m, 1H), 3.95 (m, 1H), 3.38-3.76 (m, 5H), 3.02-3.39 (m , 3H), 2.44-2.95 (m, 4H), 1.42 (s, 9H), 1.24 (t, J = 8.0 Hz, 3H)
[ESI (+)-MS]: m / z 567 [M + Na] ⁺

Step 2: tert-butyl (2R, 3S) -3-hydroxy-4-((S) -1- {3-[(2- {4-[(E / Z) -1- (4-hydroxyphenyl)] -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -3-oxopropylamino} -4-methyl-1-oxopentan-2-ylamino) -4-oxo-1-phenylbutane-2 -Synthesis of ylcarbamate

(E / Z) -tert-butyl 3-[(2- {4- [1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino]-under nitrogen atmosphere 3-Oxopropylcarbamate (0.4 mmol) and trifluoroacetic acid (0.5 mL) were dissolved in dichloromethane (5 mL) and stirred at room temperature for 10 minutes. After the reaction solution was concentrated, the residue was dissolved in dichloromethane (5 mL), and (S) -2-((2S, 3R) -3- (tert-butoxycarbonylamino) -2-hydroxy-4-phenylbutanamide)- 4-methylpentanoic acid (0.5 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 0.5 mmol), 1-hydroxybenzotriazole (HOBt, 0.5 mmol), and N , N-diisopropylamine (DIPEA, 1.0 mmol) was added and stirred at room temperature for 12 hours. After the reaction, saturated aqueous sodium hydrogen carbonate (10 mL) was added, and the mixture was extracted with chloroform (20 mL × 3). The organic layer is dried and then concentrated under reduced pressure. The residue is purified by silica gel column chromatography (developing solvent: ethyl acetate) to obtain tert-butyl (2R, 3S) -3-hydroxy-4-((S) -1- {3-[(2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -3-oxopropylamino} -4-Methyl-1-oxopentan-2-ylamino) -4-oxo-1-phenylbutan-2-ylcarbamate was obtained in a yield of 82%.
¹ H NMR (400 MHz, CDCl ₃ ): δ = 7.00-7.52 (m, 15H), 6.67-6.83 (m, 4H), 6.45-6.49 (m, 2H) , 5.60 (brs, 1H), 5.22 (m, 1H), 4.40 (m, 1H), 3.88-4.13 (m, 4H), 3.43-3.70 (m , 4H), 2.87-3.07 (m, 5H), 2.41-2.50 (m, 4H), 1.56-1.62 (m, 2H), 1.24-1.36 (M, 10H), 0.87-0.94 (m, 9H)
[ESI (+)-MS]: m / z 857 [M + Na] ⁺

Step 3: (S) -2-[(2S, 3R) -3-Amino-2-hydroxy-4-phenylbutanamide] -N- {3-[(2- {4-[(E / Z)- Synthesis of 1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -3-oxopropyl} -4-methylpentanamide hydrochloride

tert-Butyl (2R, 3S) -3-hydroxy-4-((S) -1- {3-[(2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2- Phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -3-oxopropylamino} -4-methyl-1-oxopentan-2-ylamino) -4-oxo-1-phenylbutan-2-ylcarbamate (0.2 mmol) and 6M hydrochloric acid (0.1 mL) were dissolved in tetrahydrofuran (2 mL). After stirring at room temperature for 24 hours, the reaction solution was concentrated to give (S) -2-[(2S, 3R) -3-amino-2-hydroxy-4-phenylbutanamide] -N- {3- [ (2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -3-oxopropyl} -4-methylpenta Namide hydrochloride was obtained in 99% yield.
¹ H NMR (400 MHz, CD ₃ OD): δ = 7.00-7.34 (m, 12H), 6.90 (m, 1H), 6.74-6.76 (m, 2H), 6. 63 (d, J = 8.8 Hz, 1H), 6.54 (m, 1H), 6.39 (d, J = 8.4 Hz, 1H), 4.29 (m, 1H), 4.09− 4.12 (m, 2H), 3.94 (m, 1H), 3.40-3.79 (m, 5H), 2.90-3.12 (m, 5H), 2.44-2. 80 (m, 4H), 1.59-1.66 (m, 3H), 0.88-0.93 (m, 9H)
[ESI (+)-MS]: m / z 757 [M + Na] ⁺

Step 1: (E / Z) -tert-butyl 5-[(2- {4- [1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -5 -Synthesis of oxopentylcarbamate

Under a nitrogen atmosphere, (E / Z) -endoxifene (0.5 mmol), 5- (tert-butoxycarbonylamino) pentanoic acid (0.6 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide Hydrochloric acid salt (EDC, 0.6 mmol), 1-hydroxybenzotriazole (HOBt, 0.6 mmol), and N, N-diisopropylamine (DIPEA, 1.2 mmol) were dissolved in dichloromethane (5 mL) for 24 hours at room temperature. Stir. After the reaction, saturated aqueous sodium hydrogen carbonate (10 mL) was added, and the mixture was extracted with chloroform (20 mL × 3). The organic layer was dried and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (developing solvent: ethyl acetate: n-hexane = 1: 2) to give (E / Z) -tert-butyl 5-[(2 -{4- [1- (4-Hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -5-oxopentylcarbamate was obtained in 90% yield.
¹ H NMR (400 MHz, CDCl ₃ ): δ = 7.40-7.10 (m, 7H), 6.67-6.85 (m, 4H), 6.49-6.51 (m, 2H) , 4.76 (brs, 1H), 4.13 (m, 1H), 3.96 (m, 1H), 3.60-3.76 (m, 2H), 2.94-3.15 (m , 5H), 2.26-2.52 (m, 4H), 1.48-1.67 (m, 5H), 1.43 (s, 9H), 0.92 (t, J = 6.4 Hz) , 3H)
[ESI (+)-MS]: m / z 595 [M + Na] ⁺

Step 2: tert-Butyl (2R, 3S) -3-hydroxy-4-((S) -1- {5-[(2- {4-[(E / Z) -1- (4-hydroxyphenyl)] -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -5-oxopentylamino} -4-methyl-1-oxopentan-2-ylamino) -4-oxo-1-phenylbutane-2 -Synthesis of ylcarbamate

(E / Z) -tert-butyl 5-[(2- {4- [1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino]-under nitrogen atmosphere 5-Oxopentylcarbamate (0.4 mmol) and trifluoroacetic acid (0.5 mL) were dissolved in dichloromethane (5 mL) and stirred at room temperature for 10 minutes. After the reaction solution was concentrated, the residue was dissolved in dichloromethane (5 mL), and (S) -2-((2S, 3R) -3- (tert-butoxycarbonylamino) -2-hydroxy-4-phenylbutanamide)- 4-methylpentanoic acid (0.5 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 0.5 mmol), 1-hydroxybenzotriazole (HOBt, 0.5 mmol), and N , N-diisopropylamine (DIPEA, 1.0 mmol) was added and stirred at room temperature for 12 hours. After the reaction, saturated aqueous sodium hydrogen carbonate (10 mL) was added, and the mixture was extracted with chloroform (20 mL × 3). The organic layer is dried and then concentrated under reduced pressure. The residue is purified by silica gel column chromatography (developing solvent: ethyl acetate) to obtain tert-butyl (2R, 3S) -3-hydroxy-4-((S) -1- {5-[(2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -5-oxopentylamino} -4-Methyl-1-oxopentan-2-ylamino) -4-oxo-1-phenylbutan-2-ylcarbamate was obtained in a yield of 86%.
¹ H NMR (400 MHz, CDCl 3): δ = 7.05-7.27 (m, 10H), 6.67-6.85 (m, 6H), 6.47-6.51 (m, 2H), 5.77 (brs, 1H), 5.07 (d, J = 9.6 Hz, 1H), 4.50 (m, 1H), 4.12-4.18 (m, 2H), 3.97 ( m, 1H), 3.70 (brs, 1H), 3.59 (brs, 1H), 3.31 (m, 1H), 2.90-3.14 (m, 5H), 2.17-2 .50 (m, 4H), 1.36-1.80 (m, 21H), 0.92-0.98 (m, 9H)
[ESI (+)-MS]: m / z 885 [M + Na] ⁺

Step 3: (S) -2-[(2S, 3R) -3-Amino-2-hydroxy-4-phenylbutanamide] -N- {5-[(2- {4-[(E / Z)- Synthesis of 1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -5-oxopentyl} -4-methylpentanamide hydrochloride

tert-Butyl (2R, 3S) -3-hydroxy-4-((S) -1- {5-[(2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2- Phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -5-oxopentylamino} -4-methyl-1-oxopentan-2-ylamino) -4-oxo-1-phenylbutan-2-ylcarbamate (0.2 mmol) and 6M hydrochloric acid (0.1 mL) were dissolved in tetrahydrofuran (2 mL). After stirring at room temperature for 24 hours, the reaction mixture was concentrated to give (S) -2-[(2S, 3R) -3-amino-2-hydroxy-4-phenylbutanamide] -N- {5- [ (2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -5-oxopentyl} -4-methylpenta Namide hydrochloride was obtained in 99% yield.
¹ H NMR (400 MHz, CD ₃ OD): δ = 7.30-7.35 (m, 6H), 7.09-7.31 (m, 5H), 7.00 (d, J = 8.4 Hz) , 1H), 6.95 (m, 1H), 6.74-6.76 (m, 2H), 6.63 (d, J = 8.4 Hz, 1H), 6.52 (m, 1H), 6.39 (d, J = 8.4 Hz, 1H), 4.33 (m, 1H), 3.77-4.17 (m, 3H), 3.30-3.80 (m, 3H), 2.90-3.17 (m, 7H), 2.32-2.47 (m, 4H), 1.52-1.86 (m, 7H), 0.90-0.98 (m, 9H) )
[ESI (+)-MS]: m / z 785 [M + Na] ⁺

Step 1: (E / Z) -tert-butyl 7-[(2- {4- [1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -7 -Oxoheptylcarbamate synthesis

Under a nitrogen atmosphere, (E / Z) -endoxifene (0.5 mmol), 7- (tert-butoxycarbonylamino) heptanoic acid (0.6 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide Hydrochloride (EDC, 0.6 mmol), 1-hydroxybenzotriazole (HOBt, 0.6 mmol), and N, N-diisopropylamine (DIPEA, 1.2 mmol) were dissolved in dichloromethane (5 mL) for 12 hours at room temperature. Stir. After the reaction, saturated aqueous sodium hydrogen carbonate (10 mL) was added, and the mixture was extracted with chloroform (20 mL × 3). The organic layer was dried and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (developing solvent: ethyl acetate: n-hexane = 1: 2) to give (E / Z) -tert-butyl 7-[(2 -{4- [1- (4-Hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -7-oxoheptylcarbamate was obtained in a yield of 76%.
¹ H NMR (400 MHz, CDCl ₃ ): δ = 7.04-7.17 (m, 7H), 6.70-6.86 (m, 4H), 6.48-6.52 (m, 2H) , 4.60 (brs, 1H), 4.13 (m, 1H), 3.96 (m, 1H), 3.61-3.75 (m, 2H), 2.95-3.15 (m , 5H), 2.23-2.50 (m, 4H), 1.42-1.63 (m, 18H), 0.94 (t, J = 7.2 Hz, 3H)
[ESI (+)-MS]: m / z 623 [M + Na] ⁺

Step 2: tert-butyl (2R, 3S) -3-hydroxy-4-((S) -1- {7-[(2- {4-[(E / Z) -1- (4-hydroxyphenyl)] -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -7-oxoheptylamino} -4-methyl-1-oxopentan-2-ylamino) -4-oxo-1-phenylbutane-2 -Synthesis of ylcarbamate

(E / Z) -tert-butyl 7-[(2- {4- [1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino]-under nitrogen atmosphere 7-oxoheptylcarbamate (0.3 mmol) and trifluoroacetic acid (0.5 mL) were dissolved in dichloromethane (5 mL) and stirred at room temperature for 10 minutes. After the reaction solution was concentrated, the residue was dissolved in dichloromethane (5 mL), and (S) -2-((2S, 3R) -3- (tert-butoxycarbonylamino) -2-hydroxy-4-phenylbutanamide)- 4-methylpentanoic acid (0.4 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 0.4 mmol), 1-hydroxybenzotriazole (HOBt, 0.4 mmol), and N , N-diisopropylamine (DIPEA, 1.0 mmol) was added and stirred at room temperature for 12 hours. After the reaction, saturated aqueous sodium hydrogen carbonate (10 mL) was added, and the mixture was extracted with chloroform (20 mL × 3). The organic layer is dried and then concentrated under reduced pressure. The residue is purified by silica gel column chromatography (developing solvent: ethyl acetate) to obtain tert-butyl (2R, 3S) -3-hydroxy-4-((S) -1- {7-[(2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -7-oxoheptylamino} -4-Methyl-1-oxopentan-2-ylamino) -4-oxo-1-phenylbutan-2-ylcarbamate was obtained in a yield of 77%.
¹ H NMR (400 MHz, CDCl ₃ ): δ = 7.03-7.36 (m, 12H), 6.66-6.83 (m, 5H), 6.46-6.49 (m, 2H) , 5.89 (brs, 1H), 5.06 (brs, 1H), 4.48 (m, 1H), 3.97-4.11 (m, 3H), 3.56-3.72 (m , 2H), 2.91-3.22 (m, 6H), 2.44-2.50 (m, 2H), 2.07-2.20 (m, 2H), 1.20-1.63 (M, 24H), 0.87-0.93 (m, 9H)
[ESI (+)-MS]: m / z 914 [M + Na] ⁺

Step 3: 7-{(S) -2-[(2S, 3R) -3-amino-2-hydroxy-4-phenylbutanamide] -4-methylpentanamide} -N- (2- {4- Synthesis of [(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) -N-methylheptanamide hydrochloride

tert-Butyl (2R, 3S) -3-hydroxy-4-((S) -1- {7-[(2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2- Phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -7-oxoheptylamino} -4-methyl-1-oxopentan-2-ylamino) -4-oxo-1-phenylbutan-2-ylcarbamate (0.2 mmol) and 6M hydrochloric acid (0.1 mL) were dissolved in tetrahydrofuran (2 mL). After stirring at room temperature for 12 hours, the reaction solution was concentrated to give 7-{(S) -2-[(2S, 3R) -3-amino-2-hydroxy-4-phenylbutanamide] -4-methyl. Pentanamide} -N- (2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) -N-methylheptanamide hydrochloride Was obtained in a yield of 99%.
¹ H NMR (400 MHz, CD ₃ OD): δ = 6.91-7.35 (m, 13H), 6.40-6.76 (m, 5H), 3.98-4.34 (m, 4H) ), 3.66-3.83 (m, 3H), 2.91-3.18 (m, 7H), 2.30-2.47 (m, 4H), 1.28-1.84 (m , 11H), 0.89-0.99 (m, 9H)
[ESI (+)-MS]: m / z 813 [M + Na] ⁺

Step 1: (E / Z) -tert-butyl 2- {2-[(2- {4- [1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino ] -2-Oxoethoxy} ethyl carbamate

Under a nitrogen atmosphere, (E / Z) -endoxifene (0.5 mmol), 2- [2- (tert-butoxycarbonylamino) ethoxy] acetic acid (0.6 mmol), 1-ethyl-3- (3-dimethyl) Aminopropyl) carbodiimide hydrochloride (EDC, 0.6 mmol), 1-hydroxybenzotriazole (HOBt, 0.6 mmol), and N, N-diisopropylamine (DIPEA, 1.2 mmol) are dissolved in dichloromethane (5 mL), Stir at room temperature for 12 hours. After the reaction, saturated aqueous sodium hydrogen carbonate (10 mL) was added, and the mixture was extracted with chloroform (20 mL × 3). The organic layer is dried and concentrated under reduced pressure. The residue is purified by silica gel column chromatography (developing solvent: ethyl acetate: n-hexane = 1: 1) to give (E / Z) -tert-butyl 2- {2- [(2- {4- [1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -2-oxoethoxy} ethylcarbamate was obtained in a yield of 87%. It was.
¹ H NMR (400 MHz, CDCl ₃ ): δ = 7.02-7.31 (m, 7H), 6.76-6.83 (m, 4H), 6.43-6.56 (m, 2H) , 5.08 (brs, 1H), 3.98-4.40 (m, 4H), 3.57-3.80 (m, 4H), 3.31-3.41 (m, 3H), 2 .82-3.12 (m, 3H), 2.43 (t, J = 7.6 Hz, 2H), 1.41 (s, 9H), 0.95 (t, J = 7.2 Hz, 3H)
[ESI (+)-MS]: m / z 597 [M + Na] ⁺

Step 2: tert-butyl (11S, 14S, 15R) -14-hydroxy-1- {4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy}- Synthesis of 11-isobutyl-3-methyl-4,10,13-trioxo-16-phenyl-6-oxa-3,9,12-triazahexadecan-15-ylcarbamate

(E / Z) -tert-butyl 2- {2-[(2- {4- [1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) under nitrogen atmosphere Amino] -2-oxoethoxy} ethyl carbamate (0.4 mmol) and trifluoroacetic acid (0.5 mL) were dissolved in dichloromethane (5 mL) and stirred at room temperature for 1 hour. After the reaction solution was concentrated, the residue was dissolved in dichloromethane (5 mL), and (S) -2-((2S, 3R) -3- (tert-butoxycarbonylamino) -2-hydroxy-4-phenylbutanamide)- 4-methylpentanoic acid (0.5 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 0.5 mmol), 1-hydroxybenzotriazole (HOBt, 0.5 mmol), and N , N-diisopropylamine (DIPEA, 1.0 mmol) was added and stirred at room temperature for 12 hours. After the reaction, saturated aqueous sodium hydrogen carbonate (10 mL) was added, and the mixture was extracted with chloroform (20 mL × 3). The organic layer is dried and concentrated under reduced pressure, and the residue is purified by silica gel column chromatography (developing solvent: ethyl acetate: methanol = 20: 1) to give tert-butyl (11S, 14S, 15R) -14-hydroxy-1 -{4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} -11-isobutyl-3-methyl-4,10,13-trioxo-16-phenyl -6-oxa-3,9,12-triazahexadecan-15-ylcarbamate was obtained in a yield of 71%.
¹ H NMR (400 MHz, CDCl ₃ ): δ = 7.76 (m, 1H), 6.95-7.26 (m, 13H), 6.66-6.82 (m, 4H), 6.45 -6.48 (m, 2H), 5.67 (m, 1H), 5.13 (d, J = 8.0 Hz, 1H), 4.62 (m, 1H), 3.95-4.43 (M, 7H), 3.26-3.70 (m, 6H), 2.84-3.04 (m, 5H), 2.46-2.73 (m, 2H), 1.28-1 .88 (m, 12H), 0.92-0.98 (m, 9H)
[ESI (+)-MS]: m / z 887 [M + Na] ⁺

Step 3: (S) -2-[(2S, 3R) -3-Amino-2-hydroxy-4-phenylbutanamide] -N- (2- {2-[(2- {4-[(E / Z) -1- (4-Hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -2-oxoethoxy} ethyl) -4-methylpentanamide hydrochloride

tert-Butyl (11S, 14S, 15R) -14-hydroxy-1- {4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} -11-isobutyl -3-methyl-4,10,13-trioxo-16-phenyl-6-oxa-3,9,12-triazahexadecan-15-ylcarbamate (0.2 mmol), and 6M hydrochloric acid (0.1 mL). Dissolved in tetrahydrofuran (2 mL). After stirring at room temperature for 24 hours, the reaction solution was concentrated to give (S) -2-[(2S, 3R) -3-amino-2-hydroxy-4-phenylbutanamide] -N- (2- { 2-[(2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -2-oxoethoxy} ethyl) -4-Methylpentanamide hydrochloride was obtained with a yield of 99%.
¹ H NMR (400 MHz, CD ₃ OD): δ = 7.25-7.38 (m, 7H), 6.91-7.16 (m, 6H), 6.75-6.77 (m, 2H) ), 6.55-6.63 (m, 2H), 6.40 (d, J = 8.0 Hz, 1H), 3.92-4.37 (m, 6H), 3.51-3.83 (M, 5H), 3.36-3.40 (m, 2H), 2.92-3.13 (m, 5H), 2.43-2.54 (m, 2H), 1.59-1 .84 (m, 3H), 0.90-0.98 (m, 9H)
[ESI (+)-MS]: m / z 787 [M + Na] ⁺

Step 1: tert-butyl (S) -1-{(S) -1-cyclohexyl-2-[(R) -2-((S) -1- {7-[(2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -7-oxoheptylami} -1-oxo-3,3-diphenylpropane-2 Synthesis of -ylcarbamoyl) pyrrolidin-1-yl] -2-oxoethylamino} -1-oxopropan-2-yl (methyl) carbamate

Under a nitrogen atmosphere, tert-butyl (2R, 3S) -3-hydroxy-4-((S) -1- {7-[(2- {4-[(E / Z) -1- (4-hydroxyphenyl) ) -2-Phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -7-oxoheptylamino} -4-methyl-1-oxopentan-2-ylamino) -4-oxo-1-phenylbutane- 2-ylcarbamate (0.4 mmol) and trifluoroacetic acid (0.5 mL) were dissolved in dichloromethane (5 mL) and stirred at room temperature for 1 hour. After the reaction solution was concentrated, the residue was dissolved in dichloromethane (5 mL), and (R) -2-[(R) -1-((S) -2-{(S) -2- [tert-butoxycarbonyl (methyl) ) Amino] propanamide} -2-cyclohexylacetyl) pyrrolidine-2-carboxamide] -3,3-diphenylpropanoic acid (0.5 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 0.5 mmol), 1-hydroxybenzotriazole (HOBt, 0.5 mmol), and N, N-diisopropylamine (DIPEA, 1.0 mmol) were added, and the mixture was stirred at room temperature for 2 hours. After the reaction, saturated aqueous sodium hydrogen carbonate (10 mL) was added, and the mixture was extracted with chloroform (20 mL × 3). The organic layer is dried and concentrated under reduced pressure, and the residue is purified by silica gel column chromatography (developing solvent: ethyl acetate: methanol = 50: 1) to give tert-butyl (S) -1-{(S) -1- Cyclohexyl-2-[(R) -2-((S) -1- {7-[(2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1- Enyl] phenoxy} ethyl) (methyl) amino] -7-oxoheptylamino} -1-oxo-3,3-diphenylpropan-2-ylcarbamoyl) pyrrolidin-1-yl] -2-oxoethylamino} -1 -Oxopropan-2-yl (methyl) carbamate was obtained in 33% yield.
¹ H NMR (400 MHz, CDCl ₃ ): δ = 7.05-7.30 (m, 17H), 6.70-6.84 (m, 4H), 6.31-6.52 (m, 3H) , 6.00 (brs, 1H), 5.20 (brs, 1H), 4.62-4.76 (m, 2H), 4.39-4.41 (m, 2H), 3.62-4 .14 (m, 5H), 2.77-3.31 (m, 9H), 2.24-2.54 (m, 4H), 0.91-1.95 (m, 40H)
[ESI (+)-MS]: m / z 1168 [M + Na] ⁺

Step 2: (R) -1-{(S) -2-cyclohexyl-2-[(S) -2- (methylamino) propanamide] acetyl} -N-((S) -1- {7-[( 2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -7-oxoheptylamino} -1-oxo- Synthesis of 3,3-diphenylpropan-2-yl) pyrrolidine-2-carboxamide hydrochloride

tert-Butyl (S) -1-{(S) -1-cyclohexyl-2-[(R) -2-((S) -1- {7-[(2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -7-oxoheptylamino} -1-oxo-3,3-diphenylpropan-2-ylcarbamoyl ) Pyrrolidin-1-yl] -2-oxoethylamino} -1-oxopropan-2-yl (methyl) carbamate (0.1 mmol) and 6M hydrochloric acid (0.05 mL) were dissolved in tetrahydrofuran (2 mL). After stirring at room temperature for 12 hours, the reaction solution was concentrated to give (R) -1-{(S) -2-cyclohexyl-2-[(S) -2- (methylamino) propanamide] acetyl} -N. -((S) -1- {7-[(2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino ] -7-oxoheptylamino} -1-oxo-3,3-diphenylpropan-2-yl) pyrrolidine-2-carboxamide hydrochloride was obtained in a yield of 99%.
¹ H NMR (400 MHz, CD ₃ OD): δ = 7.00-7.44 (m, 17H), 6.41-6.92 (m, 6H), 5.12 (d, J = 9.2 Hz) , 1H), 3.95-4.47 (m, 5H), 3.30-3.87 (m, 5H), 2.65-3.18 (m, 6H), 2.14-2.50. (M, 4H), 0.90-1.98 (m, 31H)
[ESI (+)-MS]: m / z 1068 [M + Na] ⁺

  ER degradation-inducing activity and apoptosis promoting activity of the IAP ligand-ER ligand hybrid compound obtained in Example 3 were evaluated.

  The breast cancer cell line MCF-7 was cultured in 10% FBS / RPMI + 60 μg / ml kanmycin. Cells were seeded and the compound added the next day, and 6 hours later, a picture of the cells was taken. For the evaluation of expression levels of various proteins including ER, the cells after the compound reaction were dissolved in 1% SDS, 0.1 M Tris-HCl (pH 7) and 10% Glycerol and heat-treated, and then protein quantification was performed by the BCA method. 10 μg equivalent was separated by SDS-PAGE. The protein was transferred to a PVDF membrane, and the protein was detected using the corresponding antibody and secondary antibody, respectively (FIGS. 1 and 4). For the evaluation of apoptosis, after the compound reaction, the cells were observed with a phase-contrast microscope (FIG. 2), and the buffer and cells containing AnnexinV-FITC were incubated at room temperature for 5 minutes, the cells were fixed with methanol, and AnnexinV was examined with a fluorescence microscope. Binding to the cells was observed (FIG. 3).

  The IAP ligand-ER ligand hybrid compound of the present invention induced degradation of ERα and ERβ and promoted cell death by apoptosis in breast cancer cell line MCF-7 expressing ER (FIGS. 2 and 3). ). In cell U2OS that does not express ER, almost no induction of cell death was observed. Treatment with the proteasome inhibitor MG132 inhibited ER degradation by the compound of the present invention (FIG. 1) and suppressed cell death (FIG. 2). The activation of Akt and Erk observed by tamoxifen stimulation was suppressed by SNIPER (ER) stimulation (FIG. 4). It was suggested that the degradation of ER by the IAP ligand-ER ligand hybrid compound of the present invention inhibits these anti-apoptotic signals and promotes cell death.

  As described above, the IAP ligand-ER ligand hybrid compound of the present invention crosslinks cIAP1 having ubiquitin ligase activity and ER in the cell, specifically ubiquitinates ER, and induces degradation by the proteasome. Therefore, it is extremely useful when the IAP ligand-ER ligand hybrid compound of the present invention is used as an ER degradation inducer or a preventive and therapeutic agent for breast cancer, cervical cancer, ovarian cancer and the like.

Claims (3)

(S) -2-[(2S, 3R) -3-Amino-2-hydroxy-4-phenylbutanamide] -N- {3-[(2- {4-[(E / Z) -1- ( 4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -3-oxoprovir} -4-methylpentanamide, (S) -2-[(2S, 3R) -3- Amino-2-hydroxy-4-phenylbutanamide] -N- {5-[(2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy } Ethyl) (methyl) amino] -5-oxopentyl} -4-methylpentanamide, 7-{(S) -2-[(2S, 3R) -3-amino-2-hydroxy-4-phenylbutanamide ] -4-methylpentanamide} N- (2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) -N-methylheptanamide, (S) -2- [ (2S, 3R) -3-Amino-2-hydroxy-4-phenylbutanamide] -N- (2- {2-[(2- {4-[(E / Z) -1- (4-hydroxyphenyl) ) -2-Phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -2-oxoethoxy} ethyl) -4-methylpentanamide or (R) -1-{(S) -2-cyclohexyl-2 -[(S) -2- (methylamino) propanamido] acetyl} -N-((S) -1- {7-[(2- {4-[(E / Z) -1- (4-hydroxy Phenyl) -2-phenylbut-1-enyl] phenoxy} eth ) (Methyl) amino] -7-oxo-heptyl amino} -1-oxo-3,3-diphenyl-2-yl) pyrrolidine -2-carboxamide, apoptosis inhibitory protein ligand - estrogen receptor ligands hybrid compound. ( S) -2-[(2S, 3R) -3-Amino-2-hydroxy-4-phenylbutanamide] -N- {3-[(2- {4-[(E / Z) -1- ( 4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -3-oxoprovir} -4-methylpentanamide, (S) -2-[(2S, 3R) -3- Amino-2-hydroxy-4-phenylbutanamide] -N- {5-[(2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy } Ethyl) (methyl) amino] -5-oxopentyl} -4-methylpentanamide, 7-{(S) -2-[(2S, 3R) -3-amino-2-hydroxy-4-phenylbutanamide ] -4-methylpentanamide} -N- (2- {4-[(E / Z) -1- (4-hydroxyphenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) -N-methylheptanamide and (S) -2- [(2S, 3R) -3-amino-2-hydroxy-4-phenylbutanamide] -N- (2- {2-[(2- {4-[(E / Z) -1- (4-hydroxy Phenyl) -2-phenylbut-1-enyl] phenoxy} ethyl) (methyl) amino] -2-oxoethoxy} ethyl) -4-methylpentanamide selected from the group consisting of apoptosis inhibitor protein ligand and estrogen receptor ligand An estrogen receptor degradation inducer comprising a hybrid compound or a physiologically acceptable salt thereof as an active ingredient . A preventive and therapeutic agent for breast cancer, cervical cancer or ovarian cancer comprising the estrogen receptor degradation inducer according to claim 2 .