Macrocyclic compounds and their use

Abstract

To provide novel macrocyclic compounds having tumor vascular remodeling effect and anti-CAF (Cancer Associated Fibroblasts) activity.SOLUTION: The present invention provides novel Compound (1) having tumor vascular remodeling effect and / or anti-CAF (Cancer Associated Fibroblasts) activity, or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier, and medical uses thereof.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Related Applications This application claims priority under 35 U.S.C. § 119(e) to Patent Document 1 filed on Apr. 5, 2017, Patent Document 2 filed on Jun. 29, 2017, and Patent Document 46 filed on Nov. 15, 2017, which are U.S. provisional patent applications, and under 35 U.S.C. § 120 to Patent Document 47 filed on Nov. 15, 2017. The entire contents of each of these are hereby incorporated by reference herein.

[0002] Technical Field The present invention provides novel macrocyclic compounds having a tumor blood vessel remodeling effect and anti-CAF (cancer-associated fibroblast) activity. The compounds can be used to treat cancer or inhibit tumor growth in a subject.

Background Art

[0003] Background Halichondrins such as halichondrin B were originally isolated from the sponge Halichondria okadai (see, e.g., Non-Patent Document 1), and subsequently found to be anti-cancer agents in Axinella sp., Phakellia carteri, and Lissondendryx sp. The total synthesis of halichondrin B was published in 1992 (see, e.g., Non-Patent Document 2). Halichondrin B has been shown to inhibit tubulin polymerization, microtubule assembly, beta5-tubulin cross-linking, GTP and vinblastine binding to tubulin, and tubulin-dependent GTP hydrolysis in vitro, and has shown anti-tumor activity in vitro and in vivo (see, e.g., Non-Patent Document 3; Non-Patent Document 4).

[0004] Eribulin mesylate (Halaven™) is developed based on halichondrin B (see, for example, Patent Document 3 published on December 23, 1999; Patent Document 4 published on December 15, 2005; and Non-Patent Document 5), and is currently clinically used in many countries for treating, for example, metastatic breast cancer and advanced liposarcoma.

[0005] Additional patent publications describing halichondrin include Patent Document 5 of Kishi et al. which became effective on July 25, 1995; Patent Document 6 of Kishi et al. which became effective on August 16, 1994; and Patent Document 7 filed by Kishi et al., all of which are assigned to the President and Fellows of Harvard College.

[0006] See also, for example, Patent Document 8; Patent Document 9; Patent Document 10; Patent Document 11; Patent Document 12; Patent Document 13; Patent Document 14; Patent Document 15; Patent Document 16; Patent Document 17; Patent Document 18; Patent Document 19; Patent Document 20; Patent Document 21; Patent Document 22; Patent Document 23; Patent Document 24; Patent Document 25; Patent Document 26; Patent Document 27; Patent Document 28; Patent Document 29; Patent Document 30; Patent Document 31; Patent Document 32; Patent Document 33; Patent Document 34; Patent Document 35; Patent Document 36; Patent Document 37; Patent Document 38; Patent Document 39; Patent Document 40; Patent Document 41; Patent Document 42.

[0007] Cancer Associated Fibroblast (CAF) is a stromal cell that is widely found in various solid tumors. CAF is well known to play important roles in angiogenesis, invasion, and metastasis. For example, it has been reported that there is a close correlation between the amount of CAF and clinical prognosis in invasive breast cancer (see, for example, Non-Patent Document 6) and esophageal adenocarcinoma (see, for example, Non-Patent Document 7). CAF has also been reported to be correlated with resistance in various tumors, such as breast cancer (see, for example, Non-Patent Document 8) and head and neck cancer (see, for example, Non-Patent Document 9; Non-Patent Document 10). Thus, it has been observed that the tumor vascular remodeling effect and anti-CAF activity bring about an improvement in the cancer microenvironment and assist in the treatment of tumors. Blood vessels are essential for tumor growth. The remodeled blood vessels within the tumor can deliver anticancer agents to the tumor in addition to achieving a reduction in hypoxia. Eribulin has been reported to remodel abnormal tumor vasculature into a more functional microenvironment and reduce the malignancy of tumors by reducing hypoxia within the tumor. Since the abnormal cancer microenvironment enhances drug resistance and metastasis, the apparent ability of eribulin to suppress these malignancies may contribute to its clinical benefits (see, for example, Non-Patent Document 11). An anticancer agent having a tumor vascular remodeling effect and anti-CAF activity has not been reported to date.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Patent Document 9

Patent Document 10

Patent Document 11

Patent Document 12

Patent Document 13

Patent Document 14

Patent Document 15

Patent Document 16

Patent Document 17

Patent Document 18

Patent Document 19

Patent Document 20

Patent Document 21

Patent Document 22

Patent Document 23

Patent Document 24

Patent Document 25

Patent Document 26

Patent Document 27

Patent Document 28

Patent Document 29

Patent Document 30

Patent Document 31

Patent Document 32

[0009] [Non-Patent Document 1] D. Uemura et al. "Norhalichondrin A: An Antitumor Polyether Macrolide from a Marine Sponge" J. Am. Chem. Soc., 107, 4796 (1985) [Non-Patent Document 2] Y. Kishi et al. "Total Synthesis of Halichondrin B and Norhalichondrin B" J. Am. Chem. Soc.,114, 3162 (1992) [Non-Patent Document 3] Y. Hirata et al. "Halichondrins - antitumor polyether macrolides from a marine sponge" Pure Appl. Chem., 58, 701 (1986) [Non-Patent Document 4] Fodstad et al. "Comparative antitumor activities of halichondrins and vinblastine against human tumor xenografts" J. of Experimental Therapeutics & Oncology 1996; 1: 119, 125 [Non-Patent Document 5] W. Zheng et al. "Macrocyclic ketone analogues of halichondrin B" Bioorganic & Medicinal Chemistry Letters 14, 5551 - 5554 (2004) [Non-Patent Document 6] M. Yamashita et al. "Role of stromal myofibroblasts in invasive breast cancer: stromal expression of alpha-smooth muscle actin correlates with worse clinical outcome" Breast Cancer 19, 170, 2012

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

[0010] Despite the progress made, further compounds are needed to advance tumor and cancer research and medicine. Summary of the Invention The present invention relates to macrocyclic compounds (e.g., compound (1)) having a tumor vascular remodeling effect and anti-CAF activity, pharmaceutically acceptable salts thereof, and isotope-labeled derivatives thereof, as well as pharmaceutical compositions thereof.

[0011] The present invention also includes methods of using compound (1) to treat cancer, methods of reversibly or irreversibly inhibiting mitosis in cells, and methods of inhibiting tumor growth in vitro, in vivo, or in a subject. In another aspect, the present invention provides a kit comprising compound (1), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0012] In one aspect, the present invention provides compound (1):

Chemical formula

[0013] In one aspect, the present invention provides a pharmaceutical composition comprising compound (1), or a pharmaceutically acceptable salt or isotope-labeled derivative thereof. The pharmaceutical composition may comprise one or more pharmaceutically acceptable excipients or carriers. The pharmaceutical composition may further comprise one or more additional therapeutic agents, in combination, alternately, or in other types of coordinated therapies, to achieve the desired goals of treatment.

[0014] The present invention also features a method for synthesizing compound (1) or an intermediate thereof. Synthetic intermediates are also provided herein as part of the present invention.

[0015] As demonstrated in the drawings and examples, compound (1) has been found to have an advantageous effect on tumor vessel remodeling and to have anti-CAF activity. Therefore, compound (1) has the potential to be used in the treatment of cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN), breast cancer, esophageal cancer, uterine cancer, ovarian cancer, colorectal cancer, endometrial cancer, gastric cancer, small bowel cancer, bladder cancer, sarcoma, rare cancers).

[0016] In another aspect, the present invention provides a method for inhibiting any tumor growth or cancer responsive to a compound having a tumor vessel remodeling effect and / or anti-CAF activity in a subject, typically a human, by compound (1) or a pharmaceutically acceptable salt, or an isotope-labeled derivative thereof.

[0017] Compound (1) or a pharmaceutically acceptable salt, or an isotope-labeled derivative thereof, or a composition thereof may be administered in combination with any other active agent that provides a beneficial result to a patient. In certain embodiments, Compound (1) is used in combination with an antibody (e.g., a monoclonal antibody). In one embodiment, Compound (1) is used in immunotherapy, e.g., in combination with, alternately, or in other coordinated therapies, with an anti-EGFR (epidermal growth factor receptor) antibody, an anti-HER2 (human epidermal growth factor receptor) antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody, etc., as described in more detail below.

[0018] For example, a method of treating head and neck squamous cell carcinoma (SCCHN) in a subject, typically a human, in need thereof, comprising administering to the subject an effective amount of Compound (1) or a pharmaceutically acceptable salt, or an isotope-labeled derivative thereof, or a composition thereof, in combination with an anti-EGFR (epidermal growth factor receptor) mAb therapy is provided. In certain embodiments, the anti-EGFR (epidermal growth factor receptor) monoclonal antibody (mAb) is cetuximab.

[0019] As another example, there is provided a method for treating breast cancer in a subject in need thereof, typically a human, the method comprising administering to the subject an effective amount of compound (1) or a pharmaceutically acceptable salt, or an isotope-labeled derivative thereof, or a composition thereof, in combination with HER2 (human epidermal growth factor receptor) mAb therapy. In certain embodiments, the HER2 (human epidermal growth factor receptor) mAb is trastuzumab. In other embodiments, compound (1) may be used in combination with traditional chemotherapy, such as adriamycin, cyclophosphamide, taxol, etc., or anti-estrogens, such as selective estrogen modulator (SERM), selective estrogen receptor downregulator (SERD), partial or complete estrogen inhibitor (such as fulvestrant), etc., or CDK4 / 6 inhibitors such as palbociclib (Pfizer), etc. to treat breast cancer.

[0020] Another aspect of the invention provides compound (1) or a pharmaceutically acceptable salt, or an isotope-labeled derivative thereof, which may be in the form of a hydrate, solvate, polymorph, or a composition thereof, in a kit which may be a dosage form package. The kits described herein may contain single or multiple doses of the compound or its pharmaceutical composition. The kits of the invention may include instructions for using the provided therapeutic dosage form (e.g., instructions for using the compound or pharmaceutical composition included in the kit).

[0021] Accordingly, the present invention includes at least the following features: (i) Compound (1), or a pharmaceutically acceptable salt or isotope-labeled derivative thereof, which may be in the form of a hydrate, solvate, or polymorph; (ii) A method of treatment for treating a subject such as a human with an effective amount of a compound (1) which may be in the form of a hydrate, solvate, or polymorph, or a pharmaceutically acceptable salt or isotope-labeled derivative thereof, for treating head and neck cancer (e.g., head and neck squamous cell carcinoma (SCCHN), adenoid cystic carcinoma), breast cancer (e.g., HER2-negative breast cancer, triple-negative breast cancer), esophageal cancer (e.g., esophageal adenocarcinoma), uterine cancer (e.g., uterine sarcoma), ovarian cancer, colorectal cancer, sarcoma (e.g., synovial sarcoma, angiosarcoma, soft tissue sarcoma, fibrosarcoma, uterine sarcoma), bladder cancer (e.g., urothelial cancer), small intestine cancer (e.g., small intestine adenocarcinoma), endometrial cancer, or rare cancer; (iii) A method of treatment for treating a subject such as a human with an effective amount of a compound (1) which may be in the form of a hydrate, solvate, or polymorph, or a pharmaceutically acceptable salt or isotope-labeled derivative thereof, for use in treating a medical disorder such as cancer or tumor that responds to a blood vessel remodeling effect and / or anti-CAF activity; (iv) A compound (1) which may be in the form of a hydrate, solvate, or polymorph, or a pharmaceutically acceptable salt or isotope-labeled derivative thereof, for use in treating head and neck squamous cell carcinoma (SCCHN), breast cancer, esophageal cancer, uterine cancer, ovarian cancer, colorectal cancer, sarcoma, bladder cancer, gastric cancer, small intestine cancer, endometrial cancer, or rare cancer; (v) A compound (1) which may be in the form of a hydrate, solvate, or polymorph, or a pharmaceutically acceptable salt or isotope-labeled derivative thereof, for use in treating a medical disorder such as cancer or tumor that responds to a blood vessel remodeling effect and / or anti-CAF activity; (vi) A deuterated derivative of compound (1); (vii) A process for manufacturing a medicament intended for therapeutic use for treating or preventing cancer or tumor etc. that responds to a blood vessel remodeling effect and / or anti-CAF activity, characterized in that a hydrate, solvate, or polymorph of the above-mentioned compound (1), or a pharmaceutically acceptable salt or isotope-labeled derivative thereof, or an embodiment of the active compound is used in the manufacture; (viii) Compound (1) in substantially pure form (e.g., at least 90 or 95%), or a pharmaceutically acceptable salt or isotopically labeled derivative thereof; (ix) A pharmaceutically acceptable composition of compound (1), or a pharmaceutically acceptable salt or isotopically labeled derivative thereof, which may be in the form of a hydrate, solvate, or polymorph in a pharmaceutically acceptable carrier or excipient; (x) A pharmaceutically acceptable dosage form of compound (1), or a pharmaceutically acceptable salt or isotopically labeled derivative thereof, which may be in the form of a hydrate, solvate, or polymorph and may be in a pharmaceutically acceptable carrier or excipient; (xi) Compound (1), or a pharmaceutically acceptable salt or isotopically labeled derivative thereof, for treating the disorders described herein that act by mechanisms other than the anti-CAF activity of the vascular remodeling effect and / or action; and (xii) A method for producing the compounds described herein and the intermediates in the synthesis.

Brief Description of the Drawings

[0022] The accompanying drawings are incorporated herein and form a part of this specification, illustrate some embodiments of the present invention, and together with this description, provide non-limiting examples of the present invention.

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Figure 18

Modes for Carrying Out the Invention

[0041] Definitions As used herein, the term "salt" means any and all salts, including pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" means a salt that is within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in Non-Patent Document 12, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed using inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, perchloric acid, etc., or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, etc., or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, bisulfate, boric acid, butyric acid, camphoric acid, camphorsulfonic acid, citric acid, cyclopentanepropionic acid, digluconic acid, dodecylsulfuric acid, ethanesulfonic acid, formic acid, fumaric acid, glucoheptonic acid, glycerophosphoric acid, gluconic acid, hemisulfate, heptanoic acid, hexanoic acid, hydroiodic acid, 2-hydroxy-ethanesulfonate, lactobionic acid, lactic acid, lauric acid, laurylsulfuric acid, malic acid, maleic acid, malonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, phosphoric acid, picric acid, pivalic acid, propionic acid, stearic acid, succinic acid, sulfuric acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecanoic acid, valeric acid salts, etc. Salts derived from suitable bases include alkali metals, alkaline earth metals, ammonium, N + (C 1~4 alkyl)4 -It contains salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Further pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonic acids, and aryl sulfonates, etc. Compound (1) is also provided as a free base and can be administered.

[0042] It should also be understood that compounds having the same molecular formula but different in the nature or sequence of the bonds of their atoms, or the arrangement of those atoms in space, are called "isomers". Isomers that differ in the arrangement of their atoms in space are called "stereoisomers".

[0043] The terms "composition" and "formulation" are used interchangeably.

[0044] The "subject" for which administration is contemplated means a human (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., an infant, a child, or an adolescent), or an adult subject (e.g., a young adult, a middle-aged adult, or an elderly adult)), or a non-human animal. In certain embodiments, the non-human animal is a mammal (e.g., a primate (e.g., a cynomolgus monkey or a rhesus monkey), a commercially relevant mammal (e.g., a cow, a pig, a horse, a sheep, a goat, a cat, or a dog), or a bird (e.g., a commercially relevant bird, e.g., a chicken, a duck, a goose, or a turkey, etc.)). In certain embodiments, the non-human animal is a fish, a reptile, or an amphibian. The non-human animal can be male or female at any stage of development. The non-human animal can be a transgenic animal or a genetically engineered animal. The term "patient" means a human subject in need of treatment for a disease.

[0045] The terms "administer", "administering", or "administration" mean embedding, absorbing, orally ingesting, injecting, inhaling, or otherwise introducing into or onto a subject the compounds or compositions described herein.

[0046] The terms "treat", "treating", and "treatment" mean restoring, alleviating, delaying the onset of, or inhibiting the progression of a disease described herein. In some embodiments, treatment may be administered after one or more signs or symptoms of the disease have occurred or been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms. Treatment may, for example, be continued after symptoms have resolved to delay or prevent recurrence.

[0047] An "effective amount" of a compound described herein means an amount sufficient to elicit a desired biological response. The effective amount of a compound described herein may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the conditions being treated, the mode of administration, the age and health of the subject, and the like. In certain embodiments, the effective amount is a therapeutically effective amount. Alternatively, in a separate method or use, the invention may be used as a prophylactic treatment if so indicated and found to be effective. In certain embodiments, the effective amount is the amount of the compound described herein in a single dose. In certain embodiments, the effective amount is the combined amount of the compounds described herein in multiple doses.

[0048] The "therapeutically effective amount" of a compound described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition, or to delay or minimize one or more symptoms associated with the condition. The therapeutically effective amount of a compound means the amount of the therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of a condition. The term "therapeutically effective amount" can include an amount that improves the overall therapy, reduces or avoids the symptoms, signs, or causes of a condition, and / or enhances the therapeutic effectiveness of another therapeutic agent. In certain embodiments, the therapeutically effective amount is an amount sufficient to treat any of the diseases or conditions described.

[0049] As used herein, terms such as "inhibit", "inhibition", "inhibiting", and "inhibitor" mean the ability of a compound to reduce, slow down, stop, or prevent the activity of a biological process (e.g., tumor growth). In certain embodiments, the inhibition is about 45% - 50%. In certain embodiments, the inhibition is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99.9%, or 100%.

[0050] The terms "neoplasm" and "tumor" are used interchangeably herein and refer to an abnormal mass of tissue that grows in excess of and uncoordinated with normal tissue growth. A neoplasm or tumor can be "benign" or "malignant" depending on the following characteristics: degree of cell differentiation (including morphology and functionality), rate of growth, local invasion, and metastasis. "Benign neoplasms" are generally well-differentiated, characteristically grow slower than malignant neoplasms, and remain localized to the site of origin. Furthermore, benign neoplasms do not have the ability to infiltrate, invade, or metastasize to distant sites. In contrast, "malignant neoplasms" are generally poorly differentiated (anaplastic), characteristically grow rapidly, accompanied by progressive infiltration, invasion, and destruction of the surrounding tissue. Furthermore, malignant neoplasms generally have the ability to metastasize to distant sites. The terms "metastasis", "metastatic", or "metastasize" mean the spread or migration of cancerous cells from a primary or original tumor to another organ or tissue, typically distinguishable by the presence of a "secondary tumor" or "secondary cell mass" that is of the tissue type of the primary or original tumor and not of the tissue type of the organ or tissue in which the metastatic (secondary) tumor is located.

[0051] The term "cancer" means a class of diseases characterized by the development of abnormal cells that grow uncontrollably and have the ability to infiltrate and destroy normal body tissues.

[0052] The term "rare cancer" means a cancer that occurs in a relatively small number of patients. Rare cancers include, but are not limited to, sarcomas (e.g., soft tissue sarcoma, liposarcoma, uterine sarcoma, leiomyosarcoma, myxofibrosarcoma, osteosarcoma, angiosarcoma, Ewing sarcoma, synovial sarcoma, rhabdomyosarcoma), malignant lymphomas, thymic cancers (e.g., thymoma), mesotheliomas, gastrointestinal stromal tumors (GIST), neuroendocrine cancers, eye cancers, brain tumors, bone soft tissue tumors, skin cancers, and germ cell tumors.

[0053] The term "anticancer agent" means a therapeutic agent useful for treating cancer (inhibiting cancer or tumor in a subject). Anticancer agents include biotherapeutic anti-cancer agents as well as chemotherapeutic agents.

[0054] Detailed description of certain embodiments The present invention will be described in detail below with reference to embodiments of the present invention and the like. The present invention provides a compound (for example, compound (1)), and a pharmaceutically acceptable salt or isotope-labeled derivative thereof, and a pharmaceutical composition thereof. The present invention also provides a method for inhibiting tumor growth and / or treating cancer in a subject, which comprises administering to the subject an effective amount of the compound or composition provided herein. The compound or composition may be administered as a monotherapy or in combination with another therapy as described herein. In yet another aspect, the present invention provides a method for synthesizing compound (1) and synthetic intermediates useful for this purpose.

[0055] The present invention relates to a compound having the structure:

Chemical formula

[0056] Compound (1) may exist as a crystalline polymorph, and the compounds of the present invention may be in any single crystalline form or a mixture of two or more crystalline forms. Compound (1) can exist in an amorphous form or can be a solvate such as an anhydrate or hydrate.

[0057] The present invention includes isotope-labeled derivatives of compound (1) and pharmaceutically acceptable salts thereof. The isotope-labeled compound is equivalent to compound (1) except that one or more of the atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, iodine, bromine, and chlorine, for example, 2 H, 3 H, 11 C, 13 C, 14 C, 18 F, 35 S, 123 I, and 125 I, etc. are included.

[0058] Isotope-labeled compounds, for example, 3 H and / or 14 C-radioisotope-incorporated compounds, etc. are useful for pharmaceutical and / or tissue distribution assays of substrates. Isotopes 3 H and 14 C are considered useful because these isotopes can be easily prepared and detected. Isotopes 11 C and 18 F are useful in PET (positron emission tomography). Isotope 125 I is considered useful in SPECT (single photon emission computed tomography) and may be useful in brain imaging. 2 Substitution with heavier isotopes such as

[0059] H results in certain therapeutic advantages such as an increase in in vivo half-life or a decrease in required dose due to higher metabolic stability, and is therefore considered useful in certain situations. Isotope-labeled compounds can be uniformly prepared by using readily available isotope-labeled reagents in place of non-isotope-labeled reagents and performing the procedures disclosed in the following schemes and / or examples.Compound (1) can be used as a chemical probe for capturing the target protein of a biologically active low molecular weight compound. Specifically, the compound of the present invention can be converted into an affinity chromatography probe, a photoaffinity probe, etc. by introducing a labeling group, a linker, etc. into a portion other than the structural portion essential for the expression of the activity of this compound by the method described in Non-Patent Document 13, Patent Document 43, etc.

[0060] Examples of the labeling group, linker, etc. used for such a chemical probe include groups belonging to the following groups (1) to (5). (1) Protein labeling groups such as photoaffinity labeling groups (for example, benzoyl group, benzophenone group, azide group, carbonyl azide group, diaziridine group, enone group, diazo group, nitro group, etc.) and chemical affinity groups (for example, a ketone group in which an alpha carbon atom is substituted with a halogen atom, carbamoyl group, ester group, alkylthio group, Michael acceptors such as alpha, beta-unsaturated ketone, ester, oxirane group, etc.), (2) Cleavable linkers such as -S-S-, -O-Si-O-, monosaccharides (glucose group, galactose group, etc.), disaccharides (lactose, etc.), and oligopeptide linkers cleavable by enzyme reactions, (3) Fishing tag groups such as biotin, 3-(4,4-difluoro-5,7-dimethyl-4H-3a,4a-diaza-4-bora-s-indacen-3-yl)propionyl group, (4) 125 I, 32 P, 3 H, 14 Radioactive labeling groups such as C, etc.; Fluorescent labeling groups such as fluorescein, rhodamine, dansyl, umbelliferone, 7-nitrofurazanyl, 3-(4,4-difluoro-5,7-dimethyl-4H-3a,4a-diaza-4-bora-s-indacen-3-yl)propionyl group, etc.; Chemiluminescent groups such as luciferin, luminol, etc.; Markers capable of detecting heavy metal ions such as lanthanoid metal ions, radium ions, etc., and (5) Groups for binding to solid-phase carriers such as glass beads, glass beds, microtiter plates, agarose beads, agarose beds, polystyrene beads, polystyrene beds, nylon beads, nylon beds, etc.

[0061] The probe prepared by introducing a labeling group or the like selected from the above groups (1) to (5) into the compound of the present invention according to the method or the like described in any of the above documents can be used as a chemical probe for identifying marker proteins useful for searching for new drug targets.

[0062] Examples of the "salt" used in the present specification include salts with inorganic acids, salts with organic acids, and salts with acidic amino acids. In particular, pharmaceutically acceptable salts are preferred. Further, the salts of the compounds of the present invention include solvates of pharmaceutically acceptable salts such as anhydrides and hydrates of the pharmaceutically acceptable salts. Preferred examples of the salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Preferred examples of the salts with organic acids include salts with acetic acid, succinic acid, fumaric acid, maleic acid, tartaric acid, citric acid, lactic acid, stearic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Preferred examples of the salts with acidic amino acids include salts with aspartic acid, glutamic acid, etc.

[0063] When the compound (1) according to the present invention is obtained as a salt or a hydrate of the compound (1), the salt and the hydrate can be converted into the free form of the compound (1) by a conventional method.

[0064] Pharmaceutical Compositions, Kits, and Administration The present invention provides a pharmaceutical composition comprising the compound (1), or a pharmaceutically acceptable salt or isotope-labeled derivative thereof, and a pharmaceutically acceptable excipient. In certain embodiments, the compounds described herein, or pharmaceutically acceptable salts or isotope-labeled derivatives thereof, are provided in an effective amount (e.g., a therapeutically effective amount) in the pharmaceutical composition.

[0065] The pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. Generally, such preparation methods involve combining the compound (1) (i.e., the "active ingredient") with a carrier or excipient, and / or one or more other accessory components, and then, if necessary and / or desirable, shaping and / or packaging the product into the desired single or multiple dose units. The pharmaceutical compositions of the present invention can be prepared by known methods such as those described in the general rules for the preparation of the Japanese Pharmacopoeia, 16th Edition, the United States Pharmacopoeia, and the European Pharmacopoeia, 9th Edition. The pharmaceutical compositions of the present invention can be appropriately administered to patients according to the dosage form.

[0066] The pharmaceutical compositions can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as multiple unit doses. A "unit dose" is an individual quantity of a pharmaceutical composition that contains a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient administered to the subject and / or a convenient fraction of such a dose, such as one-half or one-third of such a dose.

[0067] The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and / or any additional components in the pharmaceutical compositions described herein will vary depending on the individual differences, size, and / or condition of the subject being treated, and further depending on the route by which the composition is to be administered. The composition can contain from 0.1% to 100% (w / w) of the active ingredient.

[0068] Pharmaceutically acceptable excipients used in the manufacture of the provided pharmaceutical compositions include inert diluents, dispersing agents and / or granulating agents, surface active agents and / or emulsifying agents, disintegrants, binders, preservatives, buffering agents, lubricants, and / or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening agents, flavoring agents, and fragrances can also be present in the composition.

[0069] The compounds provided herein are typically formulated in unit dosage forms for ease of administration and uniformity of dosage. However, it will be understood that the total daily usage of the compositions described herein will be decided by the physician within the scope of sound medical judgment. The specific therapeutically effective dosage level for any particular subject or organism will depend upon a variety of factors including the disease and disorder being treated and its severity; the activity of the specific active ingredient being used; the specific composition being used; the age, body weight, general health, sex, diet of the subject; the time of administration, route of administration, rate of excretion of the specific active ingredient being used; the duration of the treatment; drugs used in combination with, or concurrently with, the specific active ingredient being used; and like factors well known in the medical arts.

[0070] The compounds of the invention (Compound (1)) and the compositions thereof provided herein can be administered by any route including enteral (e.g., oral), parenteral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical (such as powders, ointments, creams, and / or drops), mucosal, nasal, buccal, sublingual; by endotracheal instillation, bronchial instillation, and / or inhalation; and / or as oral sprays, nasal sprays, and / or aerosols. Specifically contemplated routes are oral administration, intravenous administration (e.g., bolus injection), local administration via the blood and / or lymph supply, and / or direct administration to the site of the disease. Generally, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the gastrointestinal tract environment), and / or the condition of the subject (e.g., whether the subject can tolerate oral administration).

[0071] The exact amount of compound (1) required to achieve an effective amount will vary depending on the subject, for example, the species, age, and general condition of the subject, the severity of side effects or disorders, the nature of the particular compound, the mode of administration, etc. The effective amount may be included in a single dose (e.g., a single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, any two doses of the multiple doses may contain different or substantially the same amount of the compounds described herein. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the frequency of administering multiple doses to the subject or applying multiple doses to the tissue or cell, by way of non-limiting example, is a dose three times a day, a dose two times a day, a dose once a day, a dose once every other day, a dose once every three days, a dose once a week, a dose once every two weeks, a dose once every three weeks, or a dose once every four weeks, or a slower controlled delivery over a time selected using a drug delivery device. In certain embodiments, the frequency of administering multiple doses to a subject or applying multiple doses to a tissue or cell is a dose once a day. In certain embodiments, the frequency of administering multiple doses to a subject or applying multiple doses to a tissue or cell is a dose two times a day. In certain embodiments, the frequency of administering multiple doses to a subject or applying multiple doses to a tissue or cell is a dose three times a day. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the duration between the first and last doses of the multiple doses is about or at least 1 day, 2 days, 4 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, 7 years, 10 years, 15 years, 20 years, or the lifespan of the subject, tissue, or cell. In certain embodiments, the duration between the first and last doses of the multiple doses is about or at least 3 months, 6 months, or 1 year. In certain embodiments, the duration between the first and last doses of the multiple doses is the lifespan of the subject, tissue, or cell.In certain embodiments, the dosages described herein (e.g., any dosage of a single dose or multiple doses) include, independently, between 0.001 mg / kg and 0.01 mg / kg, or between 0.01 mg / kg and 0.1 mg / kg, or between 0.1 mg / kg and 1 mg / kg (including both ends) of Compound (1). Examples are dosage forms having at least about 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 5, 20, 25, or 50 mg of the active compound, or a salt thereof, in the dosage form.

[0072] The dosage ranges described herein provide guidance for administering the provided pharmaceutical compositions to adults. For example, the amount administered to a child or adolescent can be determined by a physician or one of ordinary skill in the art and can be lower than or the same as the amount administered to an adult.

[0073] Kits (e.g., pharmaceutical packs) are also encompassed by the present disclosure. The provided kits may include a pharmaceutical composition or Compound (1) and a container (e.g., vial, ampoule, bottle, syringe, and / or dispensing container, or other suitable container). In some embodiments, the provided kits may further include a second container containing a pharmaceutical excipient for diluting or suspending the pharmaceutical composition or Compound (1). In some embodiments, the pharmaceutical composition or Compound (1) provided in the first container and the second container are combined to form one unit dosage form. The kits described herein may include one or more additional pharmaceutical agents described herein as separate compositions.

[0074] Methods of Treatment and Uses As shown herein, Compound (1) has a significant tumor vascular remodeling effect and anti-CAF activity, and thus it has the potential for use in the treatment of cancer and / or inhibition of tumor growth.

[0075] A method for treating cancer in a subject, which comprises administering to the subject an effective amount of compound (1), or a pharmaceutically acceptable salt or isotope-labeled derivative thereof, or a pharmaceutical composition thereof, is provided herein. The present invention also provides compound (1), or a pharmaceutically acceptable salt or isotope-labeled derivative thereof, or a pharmaceutical composition thereof for use in treating cancer in a subject. The present invention also provides the use of compound (1), or a pharmaceutically acceptable salt or isotope-labeled derivative thereof, or a pharmaceutical composition thereof for manufacturing a medicament for treating cancer.

[0076] A method for inhibiting tumor growth in a subject, which comprises administering to the subject compound (1), or a pharmaceutically acceptable salt or isotope-labeled derivative thereof, or a pharmaceutical composition thereof, is provided herein. Compound (1), or a pharmaceutically acceptable salt or isotope-labeled derivative thereof, or a pharmaceutical composition thereof for use in inhibiting tumor growth in a subject is also provided herein. The present invention also provides the use of compound (1), or a pharmaceutically acceptable salt or isotope-labeled derivative thereof, or a pharmaceutical composition thereof for manufacturing a medicament for inhibiting tumor growth.

[0077] In certain embodiments of the methods and uses provided herein, the cancer is head and neck cancer, breast cancer, esophageal cancer, uterine cancer, ovarian cancer, colorectal cancer, endometrial cancer, gastric cancer, small intestine cancer, bladder cancer, or sarcoma.

[0078] In certain embodiments of the methods and uses provided herein, the cancer is a head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer, pharyngeal cancer, salivary gland cancer, tongue cancer, adenoid cystic carcinoma). In certain embodiments, the cancer is head and neck squamous cell carcinoma (SCCHN). In certain embodiments, the cancer is adenoid cystic carcinoma. In certain embodiments, the cancer is breast cancer (e.g., HER2-positive breast cancer, triple-negative breast cancer). In certain embodiments, the cancer is HER2-positive breast cancer. In certain embodiments, the cancer is triple-negative breast cancer. In certain embodiments, the cancer is colorectal cancer (e.g., colon carcinoma). In certain embodiments, the cancer is colon carcinoma. In certain embodiments, the cancer is esophageal cancer (e.g., esophageal adenocarcinoma). In certain embodiments, the cancer is esophageal adenocarcinoma. In certain embodiments, the cancer is uterine cancer (e.g., uterine sarcoma). In certain embodiments, the cancer is uterine sarcoma. In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is a sarcoma (e.g., uterine sarcoma, fibrosarcoma, angiosarcoma, synovial sarcoma, soft tissue sarcoma). In certain embodiments, the cancer is fibrosarcoma. In certain embodiments, the cancer is angiosarcoma. In certain embodiments, the cancer is synovial sarcoma. In certain embodiments, the cancer is soft tissue sarcoma. In certain embodiments, the cancer is gastric cancer. In certain embodiments, the cancer is bowel cancer (e.g., small intestine cancer, small intestine adenocarcinoma). In certain embodiments, the cancer is small intestine cancer. In certain embodiments, the cancer is small intestine adenocarcinoma. In certain embodiments, the cancer is bladder cancer (e.g., urothelial cancer). In certain embodiments, the cancer is urothelial cancer. In certain embodiments, the cancer is endometrial cancer. In certain embodiments, the cancer is a rare cancer.

[0079] Combination therapy In addition to administration as a monotherapy, compound (1) can be administered in combination with other therapeutic agents or therapies. In certain embodiments, the additional therapeutic agent is an antibody. In certain embodiments, the additional therapeutic agent is a monoclonal antibody. The compounds of the present invention can be administered in combination with another therapeutic agent, such as an anti-EGFR therapy, an anti-HER2 therapy, an anti-PD-1 therapy, an anti-PD-L1 therapy, or radiation therapy, etc.

[0080] In certain embodiments, compound (1), or a pharmaceutically acceptable salt or isotope-labeled derivative thereof, or a pharmaceutical composition thereof, is administered in combination with an anti-EGFR therapy (e.g., an anti-EGFR monoclonal antibody (mAb) such as cetuximab). In certain embodiments, the anti-EGFR therapy is an anti-EGFR antibody. For example, provided herein is a method of treating squamous cell carcinoma of the head and neck (SCCHN) in a subject, comprising administering compound (1), or a pharmaceutically acceptable salt or isotope-labeled derivative thereof, or a pharmaceutical composition thereof, in combination with an anti-EGFR (epidermal growth factor receptor) mAb therapy to the subject. In certain embodiments, the anti-EGFR mAb is cetuximab (CTX).

[0081] In certain embodiments, compound (1), or a pharmaceutically acceptable salt or isotope-labeled derivative thereof, or a pharmaceutical composition thereof, is administered in combination with an anti-HER2 therapy (e.g., an anti-HER2 monoclonal antibody (mAb) such as trastuzumab). In certain embodiments, the anti-HER2 therapy is an anti-HER2 antibody. For example, provided herein is a method of treating breast cancer in a subject in need thereof, comprising administering compound (1), or a pharmaceutically acceptable salt or isotope-labeled derivative thereof, or a composition thereof, in combination with a HER2 (human epidermal growth factor receptor) mAb therapy to the subject. In certain embodiments, the anti-HER2 mAb is trastuzumab.

[0082] In certain embodiments, compound (1), or a pharmaceutically acceptable salt or isotopically labeled derivative thereof, or a pharmaceutical composition thereof, is administered in combination with an anti-PD-1 or anti-PD-L1 therapy (e.g., an anti-PD-1 or anti-PD-L1 monoclonal antibody). In certain embodiments, the anti-PD-1 or anti-PD-L1 therapy is an antibody. For example, provided herein is a method of treating colorectal cancer in a subject in need thereof, the method comprising administering to the subject compound (1), or a pharmaceutically acceptable salt or isotopically labeled derivative thereof, or a composition thereof, in combination with an anti-PD-1 or anti-PD-L1 therapy (e.g., mAb therapy).

[0083] In certain embodiments, compound (1), or a pharmaceutically acceptable salt or isotopically labeled derivative thereof, or a pharmaceutical composition thereof, is used in combination with radiation therapy (RT). In certain embodiments, the compound is administered in combination with surgery.

[0084] Example Synthesis of Compound (1) General Procedures and Methods The compounds according to the present invention can be produced by the methods described in the following examples. However, these examples are for illustrative purposes only, and the compounds according to the present invention are in no way limited to the specific examples described below.

[0085] In the examples, unless specifically stated otherwise, the silica gel for purification used in silica gel column chromatography is a Hi-Flash (trademark) column (silica gel, 30 μm 60 Å or 40 μm 60 Å, Yamazen Corporation), and the silica gel for purification used in NH silica gel column chromatography is Chromatorex NH silica gel (Fuji Silysia Chemical Ltd.). Analytical thin layer chromatography (TLC) is TLC silica gel 60 F 254 , layer thickness 0.25 mm (Merck) or Chromatorex TLC NH silica gel F 254It was carried out using a layer thickness of 0.25 mm (Fuji Silysia Chemical Ltd.). The TLC plates were stained and visualized with p-anisaldehyde staining solution, phosphomolybdic acid staining solution, or Hanesian staining solution.

[0086] All reactions sensitive to moisture were carried out under an inert atmosphere. The reagents and solvents were of commercial grade and used as supplied unless otherwise noted.

[0087] NMR spectra were recorded on a JEOL ECZ500R (500 MHz), JEOL ECZ400S (400 MHz), Varian Inova 500 (500 MHz), Varian Mercury 400 (400 MHz), or Bruker Avance (600 MHz) spectrometer. Chemical shifts were reported in parts per million (ppm). 1 For 1H NMR spectra (CDCl3, C6D6, and / or CD3OD), the residual solvent peak was used as an internal standard (7.27 ppm in CDCl3; 7.16 ppm in C6D6; 3.31 ppm in CD3OD).

[0088] Analytical mass spectra (MS) results were obtained using a Waters Acquity UPLC equipped with a single quadrupole detector (SQ Detector2) or LTQ Orbitrap XL™ (Thermo Scientific).

[0089] High performance liquid chromatography (HPLC) was performed using a Shimadzu LC-10AD with a UV spectrophotometric detector (200 nm, Shimadzu SPD-10A).

[0090] The abbreviations used in this specification are as follows: AIBN: 2,2'-azobis(isobutyronitrile); 9-BBN: 9-borabicyclo[3.3.1]nonane; Bu3SnH: tri-n-butyltin hydride; (+)-CSA: (1S)-(+)-10-camphorsulfonic acid; DMAP: 4-dimethylaminopyridine; DCM: dichloromethane; DDQ: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; DIBAL: diisobutylaluminum hydride; DMF: N,N-dimethylformamide; DMSO: dimethyl sulfoxide; Et3N: triethylamine; EtOAc: ethyl acetate; HF-Py: hydrogen fluoride pyridine; HPLC: high performance liquid chromatography; IPA: isopropyl alcohol; MeCN: acetonitrile; MeOH: methanol; MPM: para-methoxybenzyl; PPh3: triphenylphosphine; t-BuOH: tert-butyl alcohol; tBuLi: tert-butyllithium; TBME: methyl-tert-butyl ether; TBAF: tetrabutylammonium fluoride; TBS: tert-butyldimethylsilyl; THF: tetrahydrofuran; TMS: trimethylsilyl; Ts: para-toluenesulfonyl.

[0091] The synthetic intermediates disclosed in this specification are considered to be part of the present invention.

Chemical formula

Chemical formula

[0092] Under a nitrogen atmosphere, to a solution of compound A-1: (4aR,5aS,6R,8aS,9aR)-2,2-di-tert-butyl-6-methylhexahydrofuro[2’,3’:5,6]pyrano[3,2-d][1,3,2]dioxasilin-7(8aH)-one (18.5 g, 54.0 mmol) (CAS number; 1095280-04-8), obtained by the method described in Non-Patent Document 14, in toluene (275 mL) at -78 °C was added DIBAL (70.2 mL, 70.2 mmol, 1.0 M toluene solution) over 30 minutes. The reaction mixture was then stirred at -78 °C. After 90 minutes, the reaction was carefully quenched with MeOH (4.37 mL) at -78 °C, and then the cooling bath was removed. An aqueous solution of saturated sodium potassium tartrate tetrahydrate (300 mL) was added to the reaction mixture, and stirring was continued at room temperature for 2 hours. The reaction mixture was poured into a separatory funnel, and then the layers were separated. The aqueous layer was extracted with EtOAc (300 mL). The combined organic extracts were washed with saturated brine (300 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude lactol was used in the next reaction without purification. Example 2 (4aR,6S,7S,8aR)-6-((S)-but-3-en-2-yl)-2,2-di-tert-butylhexahydropyrano[3,2-d][1,3,2]dioxasilin-7-ol (Compound A-2) [Chemical formula]

[0093] Under a nitrogen atmosphere, potassium tert-butoxide (17.27 g, 153.9 mmol) was added to a suspension of methyltriphenylphosphonium bromide (73.30 g, 205.2 mmol) in THF (200 mL) at -5 °C over 10 minutes, and then the mixture was stirred at -5 °C for 60 minutes. A solution of the crude lactol described in Example 1 in THF (40 mL) was transferred to the reaction mixture at -5 °C over 10 minutes, and then the mixture was stirred at -5 °C for 1 hour and at room temperature for 1 hour. The reaction mixture was quenched with ice water (400 mL), then diluted with TBME (400 mL), and then the layers were separated. The aqueous layer was extracted with TBME (400 mL). The combined organic extracts were washed with saturated brine (400 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was suspended in heptane / EtOAc = 1 / 1 (100 mL). The resulting suspension was filtered, washed with heptane / EtOAc = 1 / 1 (100 mL) to remove the triphenylphosphine-derived substance. Then the filtrate was concentrated under reduced pressure. The residue was subjected to silica gel (400 g, Silica Gel 60, spherical, 40 - 50 μm, Kanto Chemical) flash column chromatography using 0% - 20% EtOAc / heptane to obtain the title compound (Compound A-2, 16.7 g, 90% yield).

[0094] 1 H NMR (400 MHz, chloroform-d) δ ppm 1.03 (d, J = 6.8 Hz, 3 H) 1.05 (s, 9 H) 1.07 (s, 9 H) 1.75 (dt, J = 14.5, 3.0 Hz, 1 H) 2.37 (dt, J = 14.5, 2.9 Hz, 1 H) 2.65 - 2.76 (m, 1 H) 3.03 (dd, J = 9.8, 1.0 Hz, 1 H) 3.31 (m, 1 H) 3.69 (d, J = 15.0 Hz, 1 H) 3.75 - 3.79 (m, 1 H) 4.16 - 4.31 (m, 2 H) 4.41 (t, J = 2.9 Hz, 1 H) 4.95 - 5.09 (m, 2 H) 6.02 (ddd, J = 17.3, 10.5, 6.3 Hz, 1 H). Example 3 (4aR,6S,7S,8aR)-6-((S)-but-3-en-2-yl)-2,2-di-tert-butyl-7-((tert-butyldimethylsilyl)oxy)hexahydropyrano[3,2-d][1,3,2]dioxasilane (Compound A-3) [Chemical formula]

[0095] Under a nitrogen atmosphere, to a 0 °C solution of compound A-2: (4aR,6S,7S,8aR)-6-((S)-but-3-en-2-yl)-2,2-di-tert-butylhexahydropyrano[3,2-d][1,3,2]dioxasilin-7-ol (9.85 g, 28.8 mmol) in DCM (150 mL) were added 2,6-lutidine (6.68 mL, 57.5 mmol) and tert-butyldimethylsilyl trifluoromethanesulfonate (9.25 mL, 40.3 mmol). The reaction mixture was stirred at 0 °C for 30 minutes and then at room temperature for 2 hours. The reaction mixture was diluted with diethyl ether. The organic layer was washed with 0.5 N aqueous hydrochloric acid, saturated aqueous sodium hydrogen carbonate, and then saturated brine. The combined organic layers were dried over magnesium sulfate, filtered (using a small amount of SiO2), and concentrated under reduced pressure. The residue was subjected to silica gel flash column chromatography using 0% - 15% EtOAc / heptane to obtain the title compound (compound A-3, 12.0 g, 91% yield).

[0096] 1 H NMR (500 MHz, chloroform-d) δ ppm 0.10 (s, 3 H) 0.19 (s, 3 H) 0.91 (s, 9 H) 0.96 (d, J = 6.3 Hz, 3 H) 1.02 (s, 9 H) 1.06 (s, 9 H) 1.73 (dt, J = 15.0, 4.0 Hz, 1 H) 2.26 (dt, J = 15.0, 2.5 Hz, 1 H) 2.66 - 2.74 (m, 1 H) 2.95 (dd, J = 9.5, 2.2 Hz, 1 H) 3.17 (m, 1 H) 3.81 - 3.84 (m, 1 H) 4.12 - 4.22 (m, 2 H) 4.24 (t, J = 2.7 Hz, 1 H) 4.93 - 5.06 (m, 2 H) 6.08 (ddd, J = 17.3, 10.5, 6.3 Hz, 1 H). Example 4 (2R,3R,5S,6S)-6-((S)-but-3-en-2-yl)-5-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydro-2H-pyran-3-ol (Compound A-4)

Chem.

[0097] Under a nitrogen atmosphere, a solution of HF-pyridine (4.0 mL) and pyridine (20 mL) previously mixed in 20 mL of MeCN was added to a solution of compound A-3: (4aR,6S,7S,8aR)-6-((S)-but-3-en-2-yl)-2,2-di-tert-butyl-7-((tert-butyldimethylsilyl)oxy)hexahydropyrano[3,2-d][1,3,2]dioxasiline (12 g, 26.3 mmol) in MeCN (120 mL) and DCM (40 mL) at -10 °C. The reaction mixture was stirred at -10 °C for 15 minutes and then at room temperature for 1 hour. The reaction mixture was quenched with saturated aqueous sodium hydrogen carbonate at 0 °C, diluted with DCM, and then the layers were separated. The aqueous layer was extracted with DCM. The combined organic extracts were washed with saturated brine. The combined organic layers were dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel flash column chromatography using 15% - 60% EtOAc / heptane to obtain the title compound (compound A-4, 8.4 g, quantitative yield).

[0098] 11H NMR (500 MHz, chloroform-d) δ ppm 0.13 (s, 3 H) 0.19 (s, 3 H) 0.94 (s, 9 H) 0.96 (d, J=6.8 Hz, 3 H) 1.72 (dt, J=14.6, 2.9 Hz, 1 H) 2.15 (dd, J=9.8, 2.4 Hz, 1 H) 2.23 (dt, J=14.6, 2.9 Hz, 1 H) 2.55 - 2.65 (m, 1 H) 3.03 (d, J=9.8 Hz, 1 H) 3.41 - 3.46 (m, 1 H) 3.49 (d, J=11.7 Hz, 1 H) 3.62 - 3.72 (m, 2 H) 3.92 (ddd, J=11.7, 8.3, 2.4 Hz, 1 H) 4.02 (t, J=2.7 Hz, 1 H) 5.01 - 5.12 (m, 2 H) 5.93 (ddd, J=17.4, 10.4, 7.3 Hz, 1 H). Example 5 (((2S,3S,5R,6R)-2-((S)-but-3-en-2-yl)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3,5-diyl)bis(oxy))bis(tert-butyldimethylsilane) (Compound A-5)

Chemical formula

[0099] Under a nitrogen atmosphere, to a solution of compound A-4: (2R,3R,5S,6S)-6-((S)-but-3-en-2-yl)-5-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydro-2H-pyran-3-ol (997 mg, 3.15 mmol) in DCM (10 mL) at 5 °C were added 2,6-lutidine (1.83 mL, 15.8 mmol) and tert-butyldimethylsilyl trifluoromethanesulfonate (2.17 mL, 9.45 mmol). The reaction mixture was stirred at room temperature for 5 hours. The reaction mixture was diluted with diethyl ether, quenched with saturated aqueous sodium hydrogen carbonate, and then the layers were separated. The combined organic extracts were washed successively with 0.5 N aqueous hydrochloric acid, saturated aqueous sodium hydrogen carbonate, and then saturated brine. The organic layer was dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel flash column chromatography using 0% - 5% EtOAc / heptane (containing 1% Et3N) to give the title compound (compound A-5, 1.69 g, 98% yield).

[0100] 1 H NMR (500 MHz, chloroform-d) δ ppm 0.02 - 0.08 (m, 15 H) 0.11 (s, 3 H) 0.89 (s, 9 H) 0.90 - 0.92 (m, 18 H) 0.94 (d, J=6.8 Hz, 3 H) 1.82 (dt, J=14.9, 4.8 Hz, 1 H) 2.00 (dt, J=14.9, 2.9 Hz, 1 H) 2.62 - 2.72 (m, 1 H) 2.93 (dd, J=9.3, 2.0 Hz, 1 H) 3.27 - 3.34 (m, 1 H) 3.66 - 3.79 (m, 3 H) 3.83 - 3.87 (m, 1 H) 4.91 - 5.07 (m, 2 H) 6.11 (ddd, J=17.3, 10.7, 6.1Hz, 1 H). Example 6 (S)-3-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)butan-1-ol (Compound A-6)

Chemical Structure

[0101] Compound A-5 described in Example 5: To a solution of (((2S,3S,5R,6R)-2-((S)-but-3-en-2-yl)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3,5-diyl)bis(oxy))bis(tert-butyldimethylsilane) (1.32 g, 2.42 mmol) in THF (10 mL) at 0 °C was added 9-BBN (9.69 mL, 0.5 M THF solution, 4.84 mmol). The reaction mixture was stirred at 0 °C for 1 hour and then at room temperature for 1.5 hours. 3.0 M aqueous sodium hydroxide solution (3 mL, 9.00 mmol) and hydrogen peroxide (35% aqueous solution, 3 mL) were added to the reaction mixture at 0 °C. The reaction mixture was stirred at 0 °C for 30 minutes and then at room temperature for 1 hour. The reaction mixture was quenched with saturated aqueous sodium sulfite solution and then the layers were separated. The aqueous layer was extracted with EtOAc (3 times). The combined organic extracts were washed with saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel flash column chromatography using 0% - 20% EtOAc / heptane to obtain the title compound (Compound A-6, 1.36 g, 100% yield).

[0102] 1 H NMR (500 MHz, chloroform-d) δ ppm 0.03 (s, 3 H) 0.05 - 0.08 (m, 12 H) 0.10 (s, 3 H) 0.88 (d, J = 6.8 Hz, 3 H) 0.89 - 0.93 (m, 27 H) 1.55 - 1.65 (m, 1H) 1.82 (dt, J = 15.4, 4.4 Hz, 1 H) 1.87 - 1.96 (m, 1 H) 1.97 - 2.03 (m, 1 H) 2.17 - 2.26 (m, 1H) 2.67 (dd, J = 7.8, 3.9 Hz, 1 H) 2.98 - 3.10 (m, 1 H) 3.34 - 3.40 (m, 1 H) 3.59 - 3.86 (m, 6 H) ESI-MS (m / z): 563.64 [M+H] + , 585.62 [M+Na]+ Example 7 (S)-3-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)butanal (Compound A-7)

Chem.

[0103] Under a nitrogen atmosphere, to a solution of Compound A-6: (S)-3-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)butan-1-ol (1100 mg, 1.954 mmol) in DCM (30 mL) at 5 °C were added sodium hydrogen carbonate (41.0 mg, 0.49 mmol) and Dess-Martin periodinane (1077 mg, 2.54 mmol). The reaction mixture was stirred at room temperature. After 3 hours, the reaction mixture was diluted with DCM, quenched with saturated aqueous sodium hydrogen carbonate and saturated aqueous sodium bisulfite, and then the layers were separated. The aqueous layer was extracted with DCM. The combined organic extracts were washed with saturated brine, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel flash column chromatography using 0% - 25% EtOAc / heptane to give the title compound (Compound A-7, 950 mg, 87% yield).

[0104] 11H NMR (500 MHz, chloroform-d) δ ppm 0.00 (s, 3 H) 0.03 - 0.08 (m, 12 H) 0.11 (s, 3 H) 0.88 (s, 9 H) 0.91 - 0.92 (m, 21H) 1.82 (dt, J=15.0, 4.5 Hz, 1 H) 2.01 (dt, J=15.0, 2.5 Hz, 1 H) 2.28 (ddd, J=16.0, 7.3, 2.4 Hz, 1 H) 2.53 -2.58 (m, 1 H) 2.74 (ddd, J=16.0, 5.5, 2.0 Hz, 1 H) 2.94 (dd, J=9.0, 1.7 Hz, 1 H) 3.29 (td, J=5.9, 2.0 Hz, 1 H) 3.68 (d, J=5.9 Hz, 2 H) 3.75 - 3.82 (m, 1 H) 3.82 - 3.90 (m, 1 H) 9.73 (t, J=2.4 Hz, 1 H).

Chem.

Chem.

[0105] Under a nitrogen atmosphere, to a solution of compound B-1: (2S,3S)-3-((4-methoxybenzyl)oxy)-2-methyl-5-(trimethylsilyl)penta-4-yn-1-ol (11.08 g, 36.15 mmol) (CAS number; 157323-41-6) obtained by the method described in Patent Document 44 / Patent Document 45 in DCM (330 mL) were added Et3N (12.6 mL, 90.4 mmol) and para-toluenesulfonyl chloride (8.27 g, 43.4 mmol) at room temperature. The reaction mixture was stirred at room temperature overnight. The mixture was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over magnesium sulfate, filtered, and then concentrated under reduced pressure. The residue was subjected to silica gel (Silica Gel 60, spherical, 40 - 50 μm, Kanto Chemical) flash column chromatography using 0% - 10% EtOAc / heptane to obtain the title compound (Compound B-2, 17.7 g, 93% yield).

[0106] 1 H NMR (500 MHz, chloroform-d) δ ppm 0.17 (s, 9 H) 1.02 (d, J=6.8 Hz, 3 H) 2.10 - 2.18 (m, 1 H) 2.44 (s, 3 H) 3.82 (s, 3 H) 3.99 (d, J=6.8 Hz, 1 H) 4.04 - 4.07(m, 2 H) 4.33 (d, J=11.2 Hz, 1 H) 4.66 (d, J=11.2 Hz, 1 H) 6.87 (d, J=8.3 Hz, 2 H) 7.21 (d, J=8.3 Hz, 2 H) 7.33 (d, J=8.8 Hz, 2 H) 7.77 (d, J=8.8 Hz, 2 H). Example 9 ((3S,4R)-5-Iodo-3-((4-methoxybenzyl)oxy)-4-methylpent-1-yn-1-yl)trimethylsilane (Compound B-3)

Chemical Structure

[0107] Under a nitrogen atmosphere, sodium iodide (7.49 g, 50.0 mmol) was added to a solution of compound B-2: (2S,3S)-3-((4-methoxybenzyl)oxy)-2-methyl-5-(trimethylsilyl)penta-4-yn-1-yl 4-methylbenzenesulfonate (17.7 g, 38.4 mmol) in DMF (360 mL) at room temperature. The reaction mixture was stirred at 80 °C for 2 hours. An additional 2.0 g of sodium iodide was added to the reaction mixture. The reaction was stirred at 80 °C for 1.5 hours and then cooled to room temperature. The mixture was diluted with diethyl ether, washed with water and saturated brine, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was subjected to flash column chromatography on silica gel (Silica Gel 60, spherical, 40 - 50 μm, Kanto Chemical) using 10% - 20% EtOAc / heptane to obtain the title compound (compound B-3, 14.3 g, 89% yield).

[0108] 1 H NMR (500 MHz, chloroform-d) δ ppm 0.21 (s, 9 H) 1.10 (d, J = 6.8 Hz, 3 H) 1.74 - 1.84 (m, 1 H) 3.30 - 3.37 (m, 2 H) 3.82 (s, 3 H) 3.96 (d, J = 7.3 Hz, 1 H) 4.44 (d, J = 11.2 Hz, 1 H) 4.73 (d, J = 11.2 Hz, 1 H) 6.89 (d, J = 8.8 Hz, 2 H) 7.30 (d, J = 8.8 Hz, 2 H).

Chemical Structure

Chemical Structure

[0109] Under an argon atmosphere, to a solution of the compound B-3 described in Example 9: ((3S,4R)-5-iodo-3-((4-methoxybenzyl)oxy)-4-methylpent-1-yn-1-yl)trimethylsilane (1408 mg, 3.382 mmol) in diethyl ether (25 mL) at -78 °C was added tert-butyllithium (1.61 M pentane solution, 4.11 mL, 6.62 mmol). The reaction mixture was stirred at -78 °C for 45 minutes. To the reaction mixture at -78 °C was added the compound A-7 described in Example 7: (S)-3-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)butanal (825 mg, 1.47 mmol) in 5.0 mL of diethyl ether. The reaction mixture was stirred at -78 °C for 60 minutes. The reaction mixture was quenched with a saturated aqueous ammonium chloride solution. The organic layer was washed with saturated brine, dried over sodium sulfate, and then concentrated under reduced pressure. The residue was subjected to silica gel flash column chromatography using 0% - 25% EtOAc / heptane to obtain the title compound (Compound C-1, 1167 mg, 93% yield).

[0110] 1 H NMR (500 MHz, chloroform-d) δ ppm 0.00 - 0.12 (m, 21 H) 0.15 - 0.24 (m, 6 H) 0.82 - 0.96 (m, 30 H) 1.03 (d, J=6.3 Hz, 3H) 1.38 - 1.55 (m, 1H) 1.68 - 1.99 (m, 4 H) 2.10 - 2.30 (m, 2 H) 2.76 - 2.87 (m, 1 H) 3.15 (d, J=9.75 Hz, 1 H) 3.33 - 3.38 (m, 1 H) 3.56 - 4.02 (m, 9 H) 4.37 - 4.50 (m, 1 H) 4.64 - 4.78 (m, 1 H) 6.83 - 6.88 (m, 2H) 7.23 - 7.35 (m, 2H). Example 11 (2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-Bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-7-((4-methoxybenzyl)oxy)-6-methyl-9-(tributylstannyl)nona-8-en-4-ol (Compound C-3)

Chemical Structure

[0111] Compound C-1 described in Example 10: (2S,6S,7S)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-7-((4-methoxybenzyl)oxy)-6-methyl-9-(trimethylsilyl)nona-8-yn-4-ol (1165 mg, 1.37 mmol) in MeOH (20 mL) at 20 °C was added potassium carbonate (189 mg, 1.37 mmol). The reaction mixture was stirred at 20 °C for 2 hours. The reaction mixture was diluted with EtOAc, quenched with saturated aqueous ammonium chloride, and then the layers were separated. The aqueous layer was extracted with EtOAc. The combined organic extracts were washed with saturated brine, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel flash column chromatography using 0% - 15% EtOAc / heptane to give Compound C-2: (2S,6S,7S)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-7-((4-methoxybenzyl)oxy)-6-methylnona-8-yn-4-ol (1050 mg, 98% yield). ESI-MS (m / z): 801.50 [M+Na] +

[0112] Under a nitrogen atmosphere, to a solution of the compound C-2 obtained above: (2S,6S,7S)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-7-((4-methoxybenzyl)oxy)-6-methylnona-8-yn-4-ol (780 mg, 1.00 mmol) in toluene (15 mL) at 20 °C were added tri-n-butyltin hydride (2.5 mL, 9.36 mmol) and 2,2'-azobis(isobutyronitrile) (82 mg, 0.50 mmol). The reaction mixture was stirred at 90 °C for 15 minutes. The reaction mixture was concentrated under reduced pressure. The residue was subjected to silica gel flash column chromatography using 0% - 15% EtOAc / heptane to obtain the title compound (Compound C-3, 970 mg, 91% yield).

[0113] 1 H NMR (500 MHz, chloroform-d) δ ppm 0.02 - 0.13 (m, 18 H) 0.84 - 0.96 (m, 48 H) 1.22 - 1.37 (m, 6 H) 1.47 - 1.56 (m, 7 H) 1.72 - 1.90 (m, 3 H) 1.95 - 2.03 (m, 1 H) 2.11 - 2.28 (m, 2 H) 2.82 - 2.86 (m, 1 H) 3.08 - 3.15 (m, 1 H) 3.33 - 3.40 (m, 1 H) 3.43 - 3.53 (m, 1 H) 3.58 - 3.87 (m, 8 H) 4.25 - 4.31 (m, 1 H) 4.49 - 4.54 (m, 1 H) 5.83 (dd, J=19.3, 7.6Hz, 1 H) 6.05 - 6.13 (m, 1 H) 6.83 - 6.90 (m, 2 H) 7.24 (d, J=8.8 Hz, 2 H). Example 12 (2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-Bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnona-8-en-4-one (Compound C-4)

Chemical Structure

[0114] Under a nitrogen atmosphere, to a solution of compound C-3: (2S,6S,7S)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-7-((4-methoxybenzyl)oxy)-6-methyl-9-(tributylstannyl)nona-8-en-4-ol (970 mg, 0.91 mmol) in 30 mL of DCM at 5 °C was added iodine (242 mg, 0.95 mmol) in DCM (6 mL) until the reaction mixture maintained an iodine color. The reaction mixture was quenched with saturated aqueous sodium bisulfite, and the layers were separated. The aqueous layer was extracted with DCM. The combined organic extracts were washed with saturated brine. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel flash column chromatography using 0% - 25% EtOAc / heptane to obtain (2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnona-8-en-4-ol (768 mg, 93% yield).

[0115] Under a nitrogen atmosphere, sodium hydrogen carbonate (17.8 mg, 0.21 mmol) and Dess-Martin periodinane (485 mg, 1.14 mmol) were added to a solution of (2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnona-8-en-4-ol (768 mg, 0.85 mmol) in DCM (25 mL) at room temperature. The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with DCM, quenched with saturated aqueous sodium hydrogen carbonate and saturated aqueous sodium sulfite, and then the layers were separated. The aqueous layer was extracted with DCM. The combined organic extracts were washed with saturated brine, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel flash column chromatography using 0% - 20% EtOAc / heptane to obtain the title compound (Compound C-4, 776 mg, quantitative yield).

[0116] 11H NMR (500 MHz, chloroform-d) δ ppm 0.00 (s, 3 H) 0.03 - 0.07 (m, 12 H) 0.10 (s, 3 H) 0.81 (d, J=6.3 Hz, 3 H) 0.84 (d, J=6.3 Hz, 3 H) 0.89 (s, 9 H) 0.91 (s, 9 H) 0.92 (s, 9 H) 1.80 (dt, J=15.0, 4.5 Hz, 1 H) 1.99 (dt, J=15.0, 2.5 Hz, 1 H) 2.17 (dd, J=16.6, 10.2 Hz, 1 H) 2.20 - 2.29 (m, 2 H) 2.43 - 2.48 (m, 1 H) 2.54 (d, J=12.7 Hz, 1 H) 2.87 (dd, J=9.0, 1.7 Hz, 1 H) 2.99 (dd, J=16.6, 2.9 Hz, 1 H) 3.27 (td, J=5.8, 2.4 Hz, 1 H) 3.50 - 3.56 (m, 1 H) 3.66 - 3.74 (m, 2H) 3.75 - 3.78 (m, 1 H) 3.80 (s, 3 H) 3.81 - 3.85 (m, 1 H) 4.26 (d, J=11.7 Hz, 1 H) 4.50 (d, J=11.7 Hz, 1 H) 6.26 (d, J=14.6 Hz, 1 H) 6.42 (dd, J=14.6, 7.8 Hz, 1 H) 6.87 (d, J=8.3 Hz, 2 H) 7.21 (d, J=8.3 Hz, 2 H). ESI-MS (m / z): 927.39 [M+Na] + Example 13 (2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-Bis((tert-butyldimethylsilyl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnona-8-en-4-one (Compound C-5) [Chemical formula]

[0117] Compound C-4 described in Example 12: (2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnona-8-en-4-one (600 mg, 0.66 mmol) was added to a solution of THF (5.0 mL), IPA (5.0 mL), and t-BuOH (5.0 mL) at 4 °C, and (1S)-(+)-10-camphorsulfonic acid (154 mg, 0.66 mmol) was added. The reaction mixture was stirred at 4 °C for 20 hours. The reaction mixture was diluted with EtOAc, quenched with saturated aqueous sodium bicarbonate, and then the layers were separated. The aqueous layer was extracted with EtOAc. The combined organic extracts were washed with saturated brine, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel flash column chromatography using 0% - 35% EtOAc / heptane to obtain the title compound (Compound C-5, 500 mg, 95% yield).

[0118] 11H NMR (500 MHz, chloroform-d) δ ppm 0.01 (s, 3 H) 0.04 (s, 3 H) 0.07 (s, 3 H) 0.11 (s, 3 H) 0.86 - 0.91 (m, 15 H) 0.93 (s, 9 H) 1.83 (dt, J=14.9, 4.8 Hz, 1 H) 1.93 - 2.00 (dt, J=14.9, 4.8 Hz, 1 H) 2.19 - 2.26 (m, 1 H) 2.29 (dd, J=14.9, 5.6 Hz, 1 H) 2.39 (dd, J=16.6, 8.3 Hz, 1 H) 2.44 - 2.66 (m, 4 H) 2.91 (dd, J=9.5, 1.7 Hz, 1 H) 3.36 - 3.41 (m, 1 H) 3.48 (td, J=11.3, 2.7 Hz, 1 H) 3.59 (t, J=7.1 Hz, 1 H) 3.74 - 3.78 (m, 2 H) 3.80 (s, 3 H) 3.85 (m, 1 H) 4.25 (d, J=11.2 Hz, 1 H) 4.46 (d, J=11.2 Hz, 1 H) 6.28 (d, J=14.6 Hz, 1 H) 6.43 (dd, J=14.6, 7.8 Hz, 1 H) 6.87 (d, J=8.8 Hz, 2 H) 7.21 (d, J=8.8 Hz, 2 H). ESI-MS (m / z): 813.30 [M+Na] + Example 14 ((2R,3R,5S,6S)-3,5-Bis((tert-butyldimethylsilyl)oxy)-6-((2S,6S,7S,E)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methyl-4-oxonona-8-en-2-yl)tetrahydro-2H-pyran-2-yl)methyl 4-methylbenzenesulfonate (Compound C-6)

Chem.

[0119] Under a nitrogen atmosphere, to a solution of the compound C-5: (2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnona-8-en-4-one (500 mg, 0.63 mmol) in DCM (10 mL) at 5 °C were added pyridine (2.54 mL, 31.6 mmol), para-toluenesulfonyl chloride (723 mg, 3.79 mmol), and 4-dimethylaminopyridine (77 mg, 0.63 mmol). The reaction mixture was stirred at room temperature for 24 h. Para-toluenesulfonyl chloride (150 mg, 0.79 mmol) was added to the reaction mixture at room temperature. The reaction mixture was then stirred at room temperature for 8 h. The reaction mixture was diluted with DCM, quenched with saturated aqueous sodium bicarbonate, and then the layers were separated. The aqueous layer was extracted with DCM. The combined organic extracts were washed with saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel flash column chromatography using 0% - 25% EtOAc / heptane to give the title compound (Compound C-6, 560 mg, 94% yield).

[0120] 11H NMR (500 MHz, chloroform-d) δ ppm 0.01 (s, 3 H) 0.04 (s, 3 H) 0.04 (s, 3 H) 0.08 (s, 3 H) 0.81 (d, J=6.8 Hz, 3 H) 0.83 (s, 9 H) 0.86 (d, J=6.8 Hz, 3 H) 0.89 (s, 9 H) 1.81 (dt, J=14.9, 4.5 Hz, 1 H) 1.91 - 1.96 (m, 1 H) 2.15 - 2.32 (m, 3 H) 2.36 - 2.42 (m, 1 H) 2.43 (s, 3 H) 2.57 (d, J=12.7 Hz, 1 H) 2.77 (dd, J=16.6, 3.4 Hz, 1 H) 2.87 (dd, J=9.0, 1.7 Hz, 1 H) 3.53 - 3.58 (m, 2 H) 3.70 - 3.75 (m, 1 H) 3.80 - 3.85 (m, 1H) 3.81 (s, 3 H) 4.06 (dd, J=10.0, 5.0 Hz, 1 H) 4.08 - 4.16 (m, 1 H) 4.28 (d, J=11.2 Hz, 1 H) 4.51 (d, J=11.2 Hz, 1 H) 6.30 (d, J=14.6 Hz, 1 H) 6.45 (dd, J=14.6, 7.8 Hz, 1 H) 6.88 (d, J=8.8 Hz, 2 H) 7.24 (d, J=8.8 Hz, 2 H) 7.31 (d, J=8.3 Hz, 2 H) 7.76 (d, J=8.3 Hz, 2 H). Example 15 (2S,6S,7S,E)-2-((2S,3S,5R,6R)-6-(azidomethyl)-3,5-bis((tert-butyldimethylsilyl)oxy)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnona-8-en-4-one (Compound C-7) [Chemical formula]

[0121] Under a nitrogen atmosphere, to a solution of compound C-6 described in Example 14: ((2R,3R,5S,6S)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-((2S,6S,7S,E)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methyl-4-oxonona-8-en-2-yl)tetrahydro-2H-pyran-2-yl)methyl 4-methylbenzenesulfonate (560 mg, 0.59 mmol) in DMSO (5.6 mL) at 20 °C was added sodium azide (385 mg, 5.92 mmol). The reaction mixture was stirred at 85 °C. After 2 hours, sodium azide (100 mg, 1.54 mmol) was added to the reaction mixture, and then the reaction mixture was stirred at 85 °C for 14 hours. The reaction mixture was diluted with EtOAc, quenched with water, and then the layers were separated. The organic extract was washed successively with water and saturated brine. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude residue. The residue was subjected to silica gel flash column chromatography using 0% - 15% EtOAc / heptane to obtain the title compound (compound C-7, 298 mg, 62% yield).

[0122] 11H NMR (500 MHz, chloroform-d) δ ppm 0.03 (s, 3 H) 0.06 (s, 3 H) 0.07 (s, 3 H) 0.10 (s, 3 H) 0.84 (d, J=6.8 Hz, 3 H) 0.85 (d, J=6.8 Hz, 3 H) 0.91 (s, 9 H) 0.92 (s, 9 H) 1.86 (dt, J=15.0, 4.7 Hz, 1 H) 1.98 (dt, J=15.0, 2.9 Hz, 1 H) 2.19 - 2.32 (m, 3 H) 2.41 - 2.49 (m, 1 H) 2.58 (d, J=12.7 Hz, 1 H) 2.94 (dd, J=16.6, 2.9 Hz, 1 H) 2.98 (dd, J=8.8, 2.0 Hz, 1 H) 3.02 (dd, J=12.7, 2.9 Hz, 1 H) 3.47 (dt, J=8.8, 2.7 Hz, 1 H) 3.49 - 3.54 (m, 1 H) 3.63 (dd, J=12.7, 8.8 Hz, 1 H) 3.69 - 3.73 (m, 1 H) 3.81 (s, 3H) 3.83 - 3.88 (m, 1 H) 4.26 (d, J=11.7 Hz, 1 H) 4.50 (d, J=11.7 Hz, 1 H) 6.26 (d, J=14.6 Hz, 1 H) 6.42 (dd, J=14.6, 7.8 Hz, 1 H) 6.87 (d, J=8.8 Hz, 2 H) 7.22 (d, J=8.8 Hz, 2 H). Example 16 (((2R,3R,5S,6S)-3,5-Bis((tert-butyldimethylsilyl)oxy)-6-((2S,6S,7S,E)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methyl-4-oxonona-8-en-2-yl)tetrahydro-2H-pyran-2-yl)methyl)carbamate (Compound C-8)

Chem.

[0123] Compound C-7 described in Example 15: (2S,6S,7S,E)-2-((2S,3S,5R,6R)-6-(azidomethyl)-3,5-bis((tert-butyldimethylsilyl)oxy)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnona-8-en-4-one (298 mg, 0.37 mmol) was added to a solution of triphenylphosphine (1437 mg, 5.478 mmol) in THF (10 mL) and water (1.0 mL) at 20 °C. The reaction mixture was stirred at 70 °C for 1.5 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude amine. To a solution of the crude amine obtained above in THF (10 mL) at 5 °C, Et3N (0.51 mL, 3.66 mmol) and diallyl dicarbonate (341 mg, 1.83 mmol) were added. The reaction mixture was stirred at room temperature for 60 minutes. The reaction mixture was concentrated under reduced pressure. The residue was subjected to silica gel flash column chromatography using 0% - 25% EtOAc / heptane to obtain the title compound (Compound C-8, 300 mg, 94% yield).

[0124] 11H NMR (500 MHz, chloroform-d) δ ppm 0.05 - 0.07 (m, 9 H) 0.11 (s, 3 H) 0.85 (d, J = 6.3 Hz, 3 H) 0.87 (d, J = 6.3 Hz, 3 H) 0.90 (s, 9 H) 0.93 (s, 9 H) 1.80 (dt, J = 15.0, 4.4 Hz, 1 H) 1.96 (dt, J = 15.0, 2.8 Hz, 1 H) 2.16 - 2.29 (m, 2 H) 2.32 - 2.39 (m, 1 H) 2.53 - 2.60 (m, 3 H) 2.86 (d, J = 7.3 Hz, 1 H) 3.04 - 3.11 (m, 1 H) 3.30 - 3.34 (m, 1 H) 3.38 - 3.48 (m, 1 H) 3.58 (t, J = 7.1 Hz, 1 H) 3.70 - 3.76 (m, 1 H) 3.80 (s, 3 H) 3.81 - 3.84 (m, 1 H) 4.25 (d, J = 11.2 Hz, 1 H) 4.46 (d, J = 11.2 Hz, 1 H) 4.53 - 4.63 (m, 2 H) 5.19 (dd, J = 10.7, 1.5 Hz, 1 H) 5.32 (d, J = 17.1 Hz, 1 H) 5.47 (d, J = 6.8 Hz, 1 H) 5.88 - 5.99 (m, 1 H) 6.28 (d, J = 14.6 Hz, 1 H) 6.43 (dd, J = 14.6, 7.8 Hz, 1 H) 6.87 (d, J = 8.8 Hz, 2 H) 7.21 (d, J = 8.8 Hz, 2 H). ESI-MS (m / z): 896.34 [M+Na] + [Chemical formula] [Chemical formula] Example 17 Compound D-4 [Chemical formula]

[0125] Under a nitrogen atmosphere (in a glove box), to a solution of compound D-2: (S)-N-(2-(4-isopropyl-4,5-dihydrooxazol-2-yl)-6-methoxyphenyl)methanesulfonamide (155 mg, 0.497 mmol) (CAS number; 546141-34-8), and 1,8-bis(dimethylamino)naphthalene (107 mg, 0.497 mmol) in MeCN (0.75 mL), chromium(II) chloride (55.5 mg, 0.452 mmol) was added, and then the resulting mixture was stirred at room temperature for 1 hour in the glove box. The resulting green solution was added to a mixture of compound C-8 described in Example 16: allyl (((2R,3R,5S,6S)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-((2S,6S,7S,E)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methyl-4-oxonona-8-en-2-yl)tetrahydro-2H-pyran-2-yl)methyl)carbamate (99.0 mg, 0.113 mmol), compound D-1 obtained by the method described in Non-Patent Document 16 (80.0 mg, 0.09 mmol) (CAS number; 157322-23-1), compound D-3: dichloro(2,9-dimethyl-1,10-phenanthroline)nickel (0.46 mg, 1.36 μmol) (CAS number; 21361-04-6), and lithium chloride (3.83 mg, 0.09 mmol). Then the reaction mixture was stirred at room temperature for 60 minutes in the glove box. Then the reaction mixture was taken out of the glove box, diluted with diethyl ether-EtOAc (5.0 mL - 5.0 mL), and then Florisil® (1600 mg, 15.94 mmol) (CAS number; 1343-88-0) was added to the mixture. Then the mixture was stirred at room temperature for 30 minutes. The mixture was filtered (Celite®), washed with EtOAc / heptane = 2 / 1, and then the filtrate was concentrated under reduced pressure. The residue was subjected to silica gel flash column chromatography using 3% - 55% EtOAc / heptane to obtain the title compound (compound D-4, 140 mg, 95% yield). Example 18 Compound D-5 [Chem.]

[0126] Under a nitrogen atmosphere, sodium hydrogen carbonate (28.8 mg, 0.34 mmol) and Dess-Martin periodinane (72.7 mg, 0.17 mmol) were added to a solution of Compound D-4 (140 mg, 0.09 mmol) in DCM (5.0 mL) at 5 °C described in Example 17. The reaction mixture was stirred at room temperature for 60 minutes. The reaction mixture was diluted with DCM, quenched with saturated aqueous sodium hydrogen carbonate solution and saturated aqueous sodium sulfite solution, and then the layers were separated. The aqueous layer was extracted with DCM. The combined organic extracts were washed with saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel flash column chromatography using 2% - 60% EtOAc / heptane to obtain the title compound (Compound D-5, 120 mg, 86%).

[0127] 11H NMR (500 MHz, benzene-d6) δ ppm 0.01 - 0.05 (m, 9 H) 0.10 - 0.12 (m, 6 H) 0.15 (s, 3 H) 0.76 (d, J=6.1 Hz, 3 H) 0.96 (s, 9 H) 1.02 (s, 9 H) 1.04 (s, 9 H) 0.95 - 1.10 (m, 7H) 1.20 (d, J=7.3 Hz, 3 H) 1.31 - 1.37 (m, 3 H) 1.41 (dd, J=12.8, 4.9 Hz, 1 H) 1.40 - 1.58 (m, 4 H) 1.59 - 1.64 (m, 1 H) 1.69 - 1.89 (m, 3H) 1.90 - 1.99 (m, 2 H) 2.02 - 2.25 (m, 8 H) 2.26 - 2.48 (m, 6 H) 2.49 - 2.70 (m, 6 H) 2.71 - 2.84 (m, 2 H) 3.00 - 3.07 (m, 1 H) 3.12 - 3.30 (m, 4 H) 3.36 (s, 3 H) 3.40 (br.s, 1 H) 3.44 - 3.53 (m, 2 H) 3.65 (dd, J=6.4, 4.0 Hz, 1 H) 3.69 - 3.84 (m, 4H) 3.86 - 4.03 (m, 4H) 4.07 - 4.17 (m, 3 H) 4.27 - 4.29 (m, 1H) 4.27 (d, J=11.0 Hz, 1H) 4.48 - 4.58 (m, 1 H) 4.49 (d, J=11.0 Hz, 1H) 4.65 - 4.70 (m, 2 H) 4.68 (d, J=5.5 Hz, 1H) 4.74 - 4.86 (m, 2H) 4.78 (s, 1H) 4.93 (s, 1 H) 5.05 (d, J=10.4 Hz, 1 H) 5.09 (br. s., 1 H) 5.19 (br. s., 1 H) 5.30 (dd, J=17.1, 1.2 Hz, 1 H) 5.82 (d, J=8.0 Hz, 1 H) 5.86 - 5.96 (m, 1 H) 6.46 (d, J=15.9 Hz, 1 H) 6.84 - 6.92 (m, 3 H) 7.31 (d, J=8.6 Hz, 2 H). Example 19 Compound D-6 [Chem.]

[0128] Imidazole hydrochloride (155 mg, 1.48 mmol) was dissolved in DMF (2.9 mL) to obtain a 0.5 M imidazole hydrochloride solution in DMF. 1.0 mL of this solution was mixed with 1.0 mL of TBAF (1.0 M, THF solution) to obtain a pre-mixed solution of 0.5 M TBAF and 0.25 M imidazole hydrochloride in THF-DMF (1:1). Under a nitrogen atmosphere, 0.588 mL of the pre-mixed solution of TBAF (0.5 M) and imidazole hydrochloride (0.25 M) in THF-DMF (1:1) prepared above was added to a solution of Compound D-5 (80.0 mg, 0.05 mmol) in DMF (7.0 mL) at 20 °C described in Example 18. The reaction mixture was stirred at room temperature for 14 hours. 1.6 g of calcium carbonate and 4.0 g of Dowex® 50WX8 (hydrogen form, 200 - 400 mesh, SIGMA-ALDRICH) were added to the reaction mixture. The mixture was stirred at room temperature for 2 hours. Then the mixture was diluted with EtOAc, then filtered (Celite®), and washed with EtOAc. The filtrate was concentrated under reduced pressure to obtain a crude residue. 1000 mg of calcium carbonate and 2.25 g of Dowex® 50WX8 were added to a solution of the crude residue in EtOAc (6.0 mL). The mixture was stirred at room temperature for 2.5 hours. Then the mixture was diluted with EtOAc, filtered (Celite®), and washed with EtOAc. The filtrate was concentrated under reduced pressure to obtain a crude residue (63.0 mg). DDQ (111 mg, 0.49 mmol) was added to a solution of the crude residue (63.0 mg) obtained above in DCM (6.0 mL), t-BuOH (0.6 mL), and pH 7 phosphate buffer (0.6 mL, 1 / 15 M) at room temperature. The reaction mixture was stirred at room temperature for 45 minutes. The reaction mixture was quenched with saturated aqueous sodium bicarbonate solution, then diluted with DCM, and the layers were separated. The aqueous layer was extracted with DCM (3 times). The combined organic extracts were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was subjected to NH silica gel flash column chromatography using 10% - 100% EtOAc / heptane, and then 10% MeOH / EtOAc to obtain the crudely purified title compound (Compound D-6, 15.0 mg, 27%).

[0129] 11H NMR (500 MHz, methanol-d4) δ ppm 0.97 (d, J = 7.0 Hz, 3 H) 0.97 (d, J = 7.0 Hz, 3 H) 1.00 - 1.02 (m, 1 H) 1.05 (d, J = 7.3 Hz, 3 H) 1.09 (d, J = 6.3 Hz, 3 H) 1.31 - 1.45 (m, 6H) 1.46 - 1.63 (m, 5H) 1.64 - 1.75 (m, 3 H) 1.80 - 1.86 (m, 2 H) 1.87 - 1.93 (m, 2 H) 1.94 - 2.11 (m, 9H) 2.13 - 2.27 (m, 8H) 2.33 (d, J = 2.4 Hz, 2 H) 2.39 (dd, J = 13.4, 6.1 Hz, 1 H) 2.44 (dd, J = 17.6, 2.0 Hz, 1 H) 2.55 (dd, J = 17.6, 9.3 Hz, 1 H) 2.75 - 2.84 (m, 1 H) 2.97 (dd, J = 9.3, 2.0 Hz, 1 H) 3.21 (dd, J = 6.6, 4.6 Hz, 1 H) 3.32 (m, 1 H) 3.41 - 3.46 (m, 1 H) 3.57 (br.s., 1 H) 3.60 (d, J = 11.7 Hz, 1 H) 3.67 - 3.74 (m, 2 H) 3.78 (br.s., 1 H) 3.86 - 3.90 (m, 2 H) 3.97 (d, J = 2.4 Hz, 1 H) 4.02 - 4.11 (m, 4 H) 4.17 (dd, J = 6.6, 4.6 Hz, 1 H) 4.23 (dd, J = 11.5, 2.2 Hz, 1 H) 4.29 (br.s, 1 H) 4.31 (td, J = 9.3, 3.9 Hz, 1 H) 4.44 (d, J = 10.2 Hz, 1 H) 4.51 (d, J = 5.4 Hz, 2 H) 4.59 (t, J = 4.9 Hz, 1 H) 4.61 (dd, J = 7.3, 4.9 Hz, 1 H) 4.69 (t, J = 4.6 Hz, 1 H) 4.80 (s, 1 H) 4.85 - 4.87 (m, 1 H) 5.01 (s, 1 H) 5.05 (s, 1 H) 5.16 (dd, J = 10.7, 1.0 Hz, 1 H) 5.28 (dd, J=17.1, 2.0 Hz, 1 H) 5.92 (m, 1 H). ESI-MS (m / z): 1172.57 [M+Na]. + Example 20 Compound D-7

Chem.

[0130] Under a nitrogen atmosphere, to a solution of the compound D-6 (15.0 mg, 0.013 mmol) described in Example 19 and pyrrolidine (10.8 μL, 0.13 mmol) in DCM (2.0 mL) at room temperature was added tetrakis(triphenylphosphine)palladium(0) (7.53 mg, 6.52 μmol). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure. The residue was subjected to NH silica gel flash column chromatography using 50% EtOAc / heptane and then 0% - 20% MeOH / EtOAc to obtain a crudely purified product. The obtained crudely purified product was purified by HPLC to obtain the title compound (D-7, 7.0 mg, 47%, retention time = 13.8 minutes).

[0131] HPLC conditions: Column: YMC Pack Pro C18 (20 mm × 250 mm) Detection wavelength: 200 nm Column temperature: Room temperature Mobile phase: MeCN - water (0.05% AcOH) Flow rate: 8 mL / min Eluent: MeCN / water 25% (isocratic, 2 minutes), then MeCN / water 25% - 60% (gradient, 20 minutes)

[0132] 11H NMR (500 MHz, methanol-d4) δ ppm 0.99 (d, J = 6.7 Hz, 3 H) 1.00 - 1.03 (m, 1 H) 1.04 (d, J = 7.3 Hz, 3 H) 1.06 (d, J = 7.3 Hz, 3 H) 1.10 (d, J = 6.1 Hz, 3 H) 1.29 - 1.63 (m, 10 H) 1.65 - 1.78 (m, 3 H) 1.79 - 1.89 (m, 2 H) 1.92 - 2.12 (m, 10 H) 1.93 (s, 3 H) 2.13 - 2.36 (m, 9 H) 2.41 (dd, J = 13.5, 6.1 Hz, 1 H) 2.45 (dd, J = 17.6, 2.2 Hz, 1 H) 2.56 (dd, J = 17.6, 9.8 Hz, 1 H) 2.75 - 2.84 (m, 1 H) 2.98 (dd, J = 9.8, 1.8 Hz, 1 H) 3.12 (dd, J = 12.8, 3.7 Hz, 1 H) 3.22 (dd, J = 6.4, 4.6 Hz, 1 H) 3.26 (dd, J = 13.2, 7.8 Hz, 1 H) 3.39 (d, J = 1.8 Hz, 1 H) 3.61 (d, J = 12.8 Hz, 1 H) 3.63 - 3.68 (m, 2 H) 3.68 - 3.76 (m, 2 H) 3.81 - 3.94 (m, 3 H) 4.00 (d, J = 2.5 Hz, 1 H) 4.03 - 4.15 (m, 4 H) 4.18 (dd, J = 6.4, 4.6 Hz, 1 H) 4.25 (ddd, J = 11.0, 4.3, 1.8 Hz, 1 H) 4.27 - 4.36 (m, 2 H) 4.46 (d, J = 11.0 Hz, 1 H) 4.57 - 4.65 (m, 2 H) 4.70 (t, J = 4.6 Hz, 1 H) 4.81 (d, J = 1.2 Hz, 1 H) 5.02 (br. s, 1 H) 5.06 (d, J = 1.8 Hz, 1 H). ESI-MS (m / z): 1066.96 [M+H] + , 1090.19 [M+Na] +

[0133] Compound (1) (free form of Compound D-7): 11H NMR (600 MHz, methanol-d4) δ ppm 0.98 (d, J = 7.2 Hz, 3 H) 1.00 (d, J = 6.8 Hz, 3 H) 1.02 (m, 1 H) 1.05 (d, J = 6.8 Hz, 3 H) 1.09 (d, J = 6.4 Hz, 3 H) 1.28 - 1.45 (m, 5 H) 1.46 - 1.59 (m, 4 H) 1.57 - 1.63 (m, 1 H) 1.65 - 1.71 (m, 1 H) 1.70 - 1.75 (m, 2 H) 1.79 - 1.86 (m, 2 H) 1.91 (dt, J = 14.9, 3.1 Hz, 1 H) 1.94 - 2.11 (m, 8 H) 2.14 - 2.34 (m, 9 H) 2.39 (dd, J = 13.2, 6.0 Hz, 1 H) 2.44 (dd, J = 17.4, 1.9 Hz, 1 H) 2.56 (dd, J = 17.6, 9.6 Hz, 1 H) 2.69 (dd, J = 13.2, 4.2 Hz, 1 H) 2.79 (ddq, J = 15.9, 7.6, 2.0 Hz, 1 H) 2.92 (dd, J = 13.2, 8.3 Hz, 1 H) 2.97 (dd, J = 9.6, 1.7 Hz, 1 H) 3.21 (dd, J = 6.4, 4.9 Hz, 1 H) 3.29 (m, 1 H) 3.34 (dd, J = 8.3, 4.15 Hz, 1 H) 3.58 (br.s., 1 H) 3.60 (br.d, J = 11.3 Hz, 1 H) 3.68 - 3.73 (m, 2 H) 3.80 (br.s., 1 H) 3.84 - 3.90 (m, 2 H) 3.98 (d, J = 2.3 Hz, 1 H) 4.03 - 4.13 (m, 4 H) 4.17 (dd, J = 6.4, 4.9 Hz, 1 H) 4.24 (ddd, J = 11.3, 4.5, 1.5 Hz, 1 H) 4.29 (dd, J = 4.0, 1.9 Hz, 1 H) 4.32 (td, J = 10.2, 4.2 Hz, 1 H) 4.44 (br.d, J = 11.0 Hz, 1 H) 4.59 (t, J = 4.5 Hz, 1 H) 4.62 (dd, J = 7.4, 4.7 Hz, 1H), 4.69 (t, J = 4.7 Hz, 1H), 4.80 (br. s., 1H), 4.87 (s, 1H), 5.00 (br. s., 1H), 5.05 (br. d, J = 1.1 Hz, 1H).

[0134] ESI-MS (m / z): 1066.57 [M + H] + , 1088.55 [M + Na] +

[0135] Pharmacological test examples General information Natural halichondrin compounds and their modified compounds are known in the literature (see, for example, Non-Patent Document 1; Non-Patent Document 18). However, most of these are not readily available. For example, Dr. Uemura et al. isolated 12.5 mg of halichondrin B, 35.0 mg of norhalichondrin A, and 17.2 mg of homohalichondrin A from 600 kg of Halichondria okadai Kadota (see, for example, Non-Patent Document 1). Among the natural halichondrin compounds, halichondrin B shows the strongest antitumor activity against B-16 melanoma cells in vitro and is very active against L-1210 leukemia in vivo (see, for example, Non-Patent Document 1). Halichondrin C is also active in various in vivo models but is unstable in aqueous solution compared to halichondrin B. Norhalichondrin B is much weaker than halichondrin B not only in vitro but also in vivo (see, for example, Non-Patent Document 1). In the following pharmacological tests, halichondrin B (Hali-B) is used as a control compound as needed.

[0136] Pharmacological Test Example 1. FaDu Proliferation Inhibition Assay In this assay, the growth inhibitory activity of the test compound in the human head and neck squamous cell carcinoma (SCCHN) cell line FaDu was measured. FaDu cells were cultured in RPMI-1640 (Wako Pure Chemical Industries, Ltd., 187-02021) medium containing 10% fetal bovine serum (FBS: Nichirei, 12D168), penicillin / streptomycin in a 5% CO2 incubator (37°C). 75 μL of the FaDu cell suspension adjusted to a concentration of 4×10 4 cells / mL with medium was added to each well of a 96-well plate (Becton Dickinson, 353219), and the cells were cultured overnight in a 5% CO2 incubator (37°C). The next day, 25 μL of the compound (1) or halicondrin B in a three-fold dilution series suspended in the medium was added to each well, and the cells were cultured for 3 days in a 5% CO2 incubator (37°C). Then, cell viability was determined using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) with an EnVision 2103 multilabel reader (PerkinElmer, Wellesley, MA). The value of the well with cells without the test compound added was defined as 100%, and the value of the well without cells was defined as 0%. The concentration of the test compound required to inhibit cell growth by 50% (IC 50 value) was calculated and shown in Table 1.

Table 1

[0137] Pharmacological Test Example 2. MDA-MB231 Proliferation Inhibition Assay In this assay, the growth inhibitory activity of the test compound in the human breast cancer cell line MDA-MB231 was measured. MDA-MB231 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Wako Pure Chemical Industries, Ltd., 044-29765) containing 10% fetal bovine serum (FBS: Nichirei, 12D168), penicillin / streptomycin in a 5% CO2 incubator (37°C). 75 μL of the cell suspension adjusted to a concentration of 4×10 475 μL of an MDA-MB231 cell suspension adjusted to a concentration of cells / mL was added, and the cells were cultured overnight in a 5% CO2 incubator (37 °C). The next day, 25 μL of compound (1) or halicondrin B in a three-fold dilution series suspended in the medium was added to each well and cultured in a 5% CO2 incubator (37 °C) for 3 days. Then, cell viability was determined using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) with an EnVision 2103 multilabel reader (PerkinElmer, Wellesley, MA). The value of the well with cells to which no test compound was added was defined as 100%, and the value of the well without cells was defined as 0%. The concentration of the test compound required to inhibit cell growth by 50% (IC 50 value) was calculated and shown in Table 2.

Table 2

[0138] Pharmacological Test Example 3. HCC1954 Proliferation Inhibition Assay In this assay, the growth inhibitory activity of the test compound in the human breast cancer cell line HCC1954 was measured. HCC1954 cells were cultured in RPMI-1640 medium (ATCC 30-2001) containing 10% fetal bovine serum (FBS: Nichirei, 12D168), penicillin / streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg / L glucose, and 1500 mg / L sodium bicarbonate in a 5% CO2 incubator (37 °C). To each well of a 96-well plate (Becton Dickinson, 353219), 4 × 10 475 μL of HCC1954 cell suspension adjusted to a concentration of cells / mL was added, and the cells were cultured overnight in a 5% CO2 incubator (37 °C). The next day, 25 μL of compound (1) or halicondrin B in a three-fold dilution series suspended in the medium was added to each well, and the resulting mixture was cultured in a 5% CO2 incubator (37 °C) for 3 days. Then, cell viability was determined using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) with an EnVision 2103 multilabel reader (PerkinElmer, Wellesley, MA). The value of the well with cells without the test compound added was defined as 100%, and the value of the well without cells was defined as 0%. The concentration of the test compound required to inhibit cell growth by 50% (IC 50 value) was calculated and shown in Table 3.

Table 3

[0139] Pharmacological Test Example 4. Antitumor Effect in FaDu Subcutaneous Xenograft Mouse Model as Monotherapy The human head and neck squamous cell carcinoma (SCCHN) cell line FaDu cultured in RPMI-1640 medium containing 10% FBS and penicillin / streptomycin was adjusted to a concentration of 4.8×10 7 cells / mL in Hanks’ Balanced Salt Solution to prepare a cell suspension. The cell suspension was transplanted subcutaneously into the right flank of 7-week-old nude mice (CAnN.Cg-Foxn1nu / CrlCrlj, female, Charles River Laboratories Japan, Inc.) at a volume of 100 μL. Nine days after cell transplantation, the short diameter and long diameter of the tumor of each mouse were measured using a digital electronic caliper (Digimatic® Caliper, Mitutoyo Corporation), and the tumor volume was calculated using the following formula: Tumor volume (mm 3 ) = long diameter (mm) × short diameter (mm) × short diameter (mm) / 2 Relative tumor volume (RTV) = tumor volume (day X) / tumor volume (first day) Tumor regression (%) = (1 - minimum RTV) × 100

[0140] Mice were grouped so that the average tumor volume was equal among groups based on the tumor volume obtained on the first day of administration. Each test compound was dissolved in DMSO and the solution was stored frozen until use. Immediately before administration, the stored solution was diluted with saline containing 100 μM hydroxypropyl-β-cyclodextrin. Each evaluation sample was intravenously administered at the maximum tolerable dose (MTD). The experiment was conducted with 4 mice per group. The tumor regression (%) of each test compound is shown in Table 4.

Table 4

[0141] Pharmacological Test Example 5. Antitumor Activity against OSC-19 in Mouse Subcutaneous Xenograft Model as Monotherapy The human head and neck squamous cell carcinoma (SCCHN) cell line OSC-19 cultured in Dulbecco's modified Eagle's medium (DMEM) / Ham's F-12 (1:1) medium containing 10% FBS and penicillin / streptomycin was adjusted to a concentration of 1×10 8 cells / ml with PBS to prepare a cell suspension. The suspension was mixed with Matrigel (trademark) (BD Biosciences, #366237) at a ratio of 1:1 to prepare a cell suspension with a concentration of 5×10 7 cells / mL. The cell suspension was transplanted subcutaneously into the right flank of 5-week-old nude mice (CAnN.Cg-Foxn1nu / CrlCrlj, female, Charles River Laboratories Japan) at a volume of 100 μL. Six days after cell transplantation, the minor and major diameters of the tumors of each mouse were measured using an electronic digital caliper (Digimatic (trademark) Caliper, Mitutoyo Corporation), and the tumor volume was calculated using the following formula: Tumor volume (mm 3 ) = major diameter (mm) × minor diameter (mm) × minor diameter (mm) / 2 Relative tumor volume (RTV) = tumor volume (day X) / tumor volume (first day) Tumor regression (%) = (1 - minimum RTV) × 100

[0142] Mice were grouped so that the average tumor volume was equal among groups based on the tumor volume obtained on the first administration day. The experiment was conducted with 6 mice per group. The test compound was dissolved in physiological saline and intravenously administered at a dose of 0.06 mg / kg to 0.18 mg / kg once a week for 2 weeks (Q7D×2 schedule). The tumor regression (%) for each test dose is shown in Table 5.

Table 5

[0143] Pharmacological Test Example 6. Antitumor Activity against HCC1806 in Mouse Subcutaneous Xenograft Model as Monotherapy The human breast cancer cell line HCC1806 cultured in RPMI-1640 medium containing 10% FBS and penicillin / streptomycin was adjusted to a concentration of 1×10 8 cells / mL with PBS to prepare a cell suspension. The suspension was mixed with Matrigel (trademark) (BD Biosciences, #366237) at a ratio of 1:1 to prepare a cell suspension with a concentration of 5×10 7 cells / mL. The cell suspension was transplanted at a volume of 100 μL into the subcutaneous tissue of the right flank of 5-week-old nude mice (CAnN.Cg-Foxn1nu / CrlCrlj, female, Charles River Laboratories Japan). Twelve days after cell transplantation, the short and long diameters of the tumors of each mouse were measured using an electronic digital caliper (Digimatic (trademark) Caliper, Mitutoyo Corporation), and the tumor volume was calculated using the following formula: Tumor volume (mm 3 ) = long diameter (mm) × short diameter (mm) × short diameter (mm) / 2 Relative tumor volume (RTV) = tumor volume (day X) / tumor volume (first day) Tumor regression (%) = (1 - minimum RTV) × 100

[0144] Mice were grouped so that the average tumor volume was equal among groups based on the tumor volume obtained on the first administration day. The experiment was conducted with 6 mice per group. The test compound was dissolved in physiological saline and intravenously administered at a dose of 0.18 mg / kg once a week for 2 weeks (Q7D×2 schedule). The tumor regression (%) of compound (1) is shown in Table 6.

Table 6

[0145] Pharmacological Test Example 7. Antitumor Effect in FaDu Subcutaneous Xenograft Mouse Model in Combination with Cetuximab The human head and neck squamous cell carcinoma (SCCHN) cell line FaDu cultured in RPMI-1640 medium containing 10% FBS and penicillin / streptomycin was adjusted to a concentration of 5×10 7 cells / mL with Hanks’ Balanced Salt Solution to prepare a cell suspension. The cell suspension was transplanted at a volume of 100 μL into the subcutaneous tissue of the right flank of 7-week-old nude mice (CAnN.Cg-Foxn1nu / CrlCrlj, female, Charles River Laboratories Japan, Inc.). Ten days after cell transplantation, the short and long diameters of the tumors in each mouse were measured using an electronic digital caliper (Digimatic (trademark) Caliper, Mitutoyo Corporation), and the tumor volume was calculated using the following formula: Tumor volume (mm 3 ) = long diameter (mm) × short diameter (mm) × short diameter (mm) / 2 Relative tumor volume (RTV) = tumor volume (day X) / tumor volume (first day) Tumor regression on day 35 (%) = (1 - RTV on day 35) × 100

[0146] The mice were grouped so that the average value of the tumor volume was equal among the groups based on the tumor volume obtained on the first day of administration. Each test compound was dissolved in DMSO and the solution was stored frozen until use. Immediately before administration, the stock solution was diluted with physiological saline containing 100 μM hydroxypropyl-β-cyclodextrin. Each test compound was intravenously administered in combination with cetuximab (Erbitux, Merck Serono) at a dose of 1 / 4 MTD to 1 / 2 MTD. The experiment was conducted with 4 mice per group. The tumor regression on day 35 (%) of each test compound is shown in Table 7.

Table 7

[0147] Pharmacological Test Example 8. Antitumor Activity in KPL-4 Subcutaneous Xenograft Mouse Model in Combination with Trastuzumab The human HER-2 positive breast cancer cell line KPL-4 cultured in RPMI-1640 medium containing 10% FBS and penicillin / streptomycin was adjusted to a concentration of 1×10 8 cells / mL with Hanks’ Balanced Salt Solution to prepare a cell suspension. The cell suspension was transplanted at a volume of 100 μL into the subcutaneous area of the right flank of 7-week-old nude mice (CAnN.Cg-Foxn1nu / CrlCrlj, female, Charles River Laboratories Japan, Inc.). Sixteen days after cell transplantation, the short diameter and long diameter of the tumor of each mouse were measured using a digital electronic caliper (Digimatic (trademark) Caliper, Mitutoyo Corporation), and the tumor volume was calculated using the following formula: Tumor volume (mm 3 ) = long diameter (mm) × short diameter (mm) × short diameter (mm) / 2 Relative tumor volume (RTV) = tumor volume (day X) / tumor volume (first day) Tumor regression (%) = (1 - minimum RTV) × 100

[0148] The mice were grouped so that the average value of the tumor volume was equal among groups based on the tumor volume obtained on the first day of administration. The experiment was conducted with 6 mice per group. Each test compound was dissolved in DMSO and the solution was stored frozen until use. Immediately before administration, the stock solution was diluted with physiological saline. The test compound was intravenously administered at 0.09 mg / kg or 0.18 mg / kg in combination with trastuzumab (Herceptin, Genentech). The tumor regression for compound (1) is shown in Table 8.

Table 8

[0149] Pharmacological Test Example 9. Effect on CD31-Positive Blood Vessels in FaDu Subcutaneous Mouse Model The human head and neck squamous cell carcinoma (SCCHN) cell line FaDu cultured in RPMI-1640 medium containing 10% FBS and penicillin / streptomycin was adjusted to 5×10 7A cell suspension was prepared by adjusting the concentration to cells / mL. The cell suspension was transplanted at a volume of 100 μL into the subcutaneous tissue of the right flank of 7-week-old nude mice (CAnN.Cg-Foxn1nu / CrlCrlj, female, Charles River Laboratories Japan). Ten days after cell transplantation, the test compound in saline containing 100 μM of hydroxypropyl-β-cyclodextrin was intravenously administered at a dose of 1 / 2MTD to MTD. The experiment was conducted with 3 mice per group. Five days after administration, tumor samples were collected and fixed at 4°C for 24 hours with IHC zinc fixative (BD Pharmingen). Paraffin-embedded tissues were sectioned (3 μm), placed on positively charged slide glasses, and air-dried. Immunohistochemical staining of CD31 was performed using a Ventana Autostainer model Discover XT (Roche Diagnostics) according to the manufacturer's protocol. The sections were deparaffinized, rehydrated, and antigen activation was performed with CC1 (Ventana Medical Systems). The slides were blocked with Blocking Agent A and Blocking Agent B (Endogenous Biotin Blocking Kit, Roche Diagnostics). The sections were incubated for 6 hours with a rat anti-mouse IgG CD31 antibody (Dianova) at 2 μg / mL, and then incubated for 32 minutes with a biotinylated anti-rat IgG antibody (Jackson ImmunoResearch Laboratories) at 2.2 μg / mL. Detection was performed for 16 minutes with streptavidin-HRP D, and then incubated for 8 minutes with DAB D and DAB H2O2D (DABMap Kit, Ventana Medical Systems). The slides were counterstained with hematoxylin II (Roche Diagnostics) for 16 minutes, and then incubated for 4 minutes with a bluing reagent. The sections were dehydrated with graded ethanol, defatted by xylene exchange, and covered with DPX (Merck).

[0150] The immunostained slides were scanned using a Vectra(™) 2 Automated Slide Imaging System (PerkinElmer). The number of blood vessels in the whole tumor was quantified by counting CD31-positive objects using inForm2 software (PerkinElmer). Also, the hematoxylin staining area was measured using inForm2 software (PerkinElmer) and used as the tumor area. The number of blood vessels was normalized by the tumor area. The increase rate of the number of blood vessels in the test compound dosing group was calculated by the following formula and shown in Table 9. Increase rate of the number of blood vessels (%) = ((Number of blood vessels in the test compound dosing group - Number of blood vessels in the control group) / Number of blood vessels in the control group) × 100

Table 9

[0151] Pharmacological Test Example 10. Effect on α-SMA-Positive CAF in FaDu Subcutaneous Model The human head and neck squamous cell carcinoma (SCCHN) cell line FaDu cultured in RPMI-1640 medium containing 10% FBS and penicillin / streptomycin was resuspended in PBS at 5×10 7A cell suspension was prepared by adjusting the concentration to cells / mL. The cell suspension was transplanted at a volume of 100 μL into the lower right abdominal skin of nude mice (CAnN.Cg-Foxn1nu / CrlCrlj, female, Charles River Laboratories Japan) at 5 - 6 weeks of age. Ten days after cell transplantation, the test compound in physiological saline containing 100 μM hydroxypropyl-β-cyclodextrin was intravenously administered at 1 / 2 MTD and MTD. The experiment was conducted with 3 mice per group. Two days after administration, tumor samples were collected and fixed at 4°C for 24 hours with IHC zinc fixative (BD Pharmingen). Paraffin-embedded tissues were sectioned (3 μm), placed on positively charged slide glasses, and air-dried for 6 hours. Immunohistochemical staining of α-SMA was performed using a Ventana autostainer model Discover XT (Roche Diagnostics). The sections were deparaffinized, rehydrated, and antigen activation was carried out with a proprietary buffer, EZPrep, and CC1 (Ventana Medical Systems). The sections were incubated for 6 hours with a mouse anti-α-SMA monoclonal antibody (clone 1A4, Sigma) conjugated with alkaline phosphatase at 5 μg / mL. Detection was performed with a RedMap kit (Ventana Medical Systems). The sections were dehydrated with graded ethanol, defatted by xylene exchange, and covered with DPX (Merck). Serial tumor slices were deparaffinized and stained with Mayer's hematoxylin (Muto Chemical) for 1 minute. The sections were dehydrated with graded ethanol, defatted by xylene exchange, and covered with DPX (Merck).

[0152] The immunostained slides were scanned using a Vectra(™) 2 Automated Slide Imaging System (PerkinElmer). The area of the α-SMA positive region in the whole tumor was quantified by counting α-SMA positive objects using inForm2 software (PerkinElmer). Also, the hematoxylin staining area was measured using inForm2 software (PerkinElmer) and used as the tumor area. The area of the α-SMA positive region was normalized by the tumor area. The inhibition rate of the α-SMA positive area in the test compound dosing group was calculated by the following formula and shown in Table 10. [Table 10]

[0153] Pharmacological Test Example 11. HSC-2 Orthotopic Transplantation Mouse Model HSC-2-Luc cells transfected with the luciferase gene were established by gene transfer using a retrovirus. First, a DNA fragment encoding firefly luciferase was obtained from the pGL3-enhancer plasmid (GenBank#: U47297) and subcloned into the retrovirus vector pCX4pur (GenBank#: AB086386). Next, the above retroviral expression vector was transfected together with the pGP and pE-Ampho plasmids (Takara Bio; Shiga, Japan) into 293T cells (ATCC; Manassas, USA) to generate helper-free recombinant retrovirus. Next, HSC-2 cells were infected with the recombinant retrovirus. Infected cells were selected from the polyclonal proliferative population by culturing for 2 weeks in the presence of puromycin (2 μg / mL).

[0154] Under anesthesia, the human SCCHN cell line, HSC-2-Luc, was transplanted into the tongues of female nude mice, 6 weeks old (CAnN.Cg-Foxn1nu / CrlCrlj mice; Charles River; Shizuoka, Japan) (1×10 in 50 μL of PBS 6Cells). Seven days after transplantation, tumor volume was analyzed using the bioluminescence signal from HSC-2-Luc cells. For bioluminescence imaging, 0.1 mL of 15 mg / mL D-luciferin (Promega, Madison, WI) was injected intraperitoneally into nude mice under 1% - 2% inhaled isoflurane anesthesia. The bioluminescence signal was monitored using an IVIS SPECTRUM series consisting of a high-sensitivity cooled charge-coupled device camera (PerkinElmer, Waltham, MA). Imaging data were gridded using Living Image software (PerkinElmer, Waltham, MA), and the total bioluminescence signal in each region of interest (ROI) was integrated. All bioluminescence images were acquired with a 1-second exposure. Data were analyzed using the total number of emitted photons (photons / second) in the ROI.

[0155] Mice were grouped so that the average value of the total number of emitted photons was equal among groups based on the total number of emitted photons obtained on the first day of administration. Compound (1) or cisplatin was administered intravenously once a week for 3 weeks (Q7D×3 schedule), with or without cetuximab (Erbitux, Merck Serono). Two experiments were conducted using the same procedure, and all data were collected from the experiments. Each group consisted of 16 mice.

[0156] Imaging data showed that only the treatment with compound (1) with cetuximab significantly reduced the bioluminescence signal in all mice after day 14 (Figures 6A - 6B). The median survival time (MST) was calculated for each treatment group as the median of the death days. The Increase Life Span (ILS) was calculated using the following formula: ILS (%) = (MST of animals treated with the test compound - MST of control animals) / MST of control animals × 100. The ILS (%) of each test compound is shown in Table 11.

Table 11

[0157] Pharmacological Test Example 12. FaDu Subcutaneous Transplant Model in Combination with Radiation FaDu-Luc cells transfected with the luciferase gene were established by gene transfer using a retrovirus. First, a DNA fragment encoding firefly luciferase was obtained from the pGL3-enhancer plasmid (GenBank#: U47297) and subcloned into the retrovirus vector pCX4pur (GenBank#: AB086386). Next, the retrovirus expression vector was transfected into 293T cells (ATCC; Manassas, USA) together with the pGP and pE-Ampho plasmids (Takara Bio; Shiga, Japan) to generate helper-free recombinant retrovirus. Then, FaDu cells were infected with the recombinant retrovirus. Infected cells were selected from the polyclonal proliferating population by culturing for 2 weeks in the presence of puromycin (2 μg / mL).

[0158] The human SCCHN cell line FaDu-Luc transfected with the luciferase gene, which was cultured in RPMI-1640 medium containing 10% FBS and penicillin / streptomycin, was adjusted to 5×10 in Hanks’ Balanced Salt Solution 7The cell suspension was prepared by adjusting the concentration to cells / mL. The cell suspension was transplanted subcutaneously into the right thigh of 6-week-old nude mice (CAnN.Cg-Foxn1nu / CrlCrlj, female, Charles River Laboratories Japan, Inc.) at a volume of 100 μL. Thirteen days after cell transplantation, the tumor volume was analyzed using the bioluminescence signal from FaDu-Luc cells. For bioluminescence imaging, 0.1 mL of 15 mg / mL D-luciferin (Promega, Madison, WI) was injected intraperitoneally into the nude mice under 1% - 2% inhaled isoflurane anesthesia. The bioluminescence signal was monitored using an IVIS SPECTRUM series (PerkinElmer, Waltham, MA) consisting of a high-sensitivity cooled charge-coupled device camera. Imaging data was gridded using Living Image software (PerkinElmer, Waltham, MA), and the total bioluminescence signal in each region of interest (ROI) was integrated. All bioluminescence images were acquired with an exposure time of 1 second. The data was analyzed using the total number of emitted photons (photons / second) in the ROI. The total number of emitted photons was calculated using the following formula: Relative bioluminescence level = Total number of emitted photons (day X) / Total number of emitted photons (day 0) Tumor regression (%) = (1 - Minimum relative bioluminescence level) × 100

[0159] The mice were grouped so that the average value of the total number of emitted photons was equal among the groups based on the total number of emitted photons obtained on the first day of administration. The experiment was conducted with 6 mice per group. Compound (1) was administered by tail vein injection on days 1 and 8. Irradiation was performed at 18 Gy on days 4 and 11. The tumor regression for compound (1) is shown in Table 12.

Table 12

[0160] Pharmacological Test Example 13. Antitumor Activity in CT26 Subcutaneous Xenograft Mouse Model in Combination with Anti-mPD-1 Antibody The mouse undifferentiated colorectal cancer cell line CT26 cultured in RPMI-1640 medium containing 10% FBS and penicillin / streptomycin was adjusted to a concentration of 2×10 7 cells / mL with Hanks’ Balanced Salt Solution to prepare a cell suspension. On day 1, the cell suspension was transplanted subcutaneously into the right lower abdominal skin of 6-week-old BALB / c mice (BALB / cAnNCrlCrlj, female, Charles River Laboratories Japan, Inc.) at a volume of 100 μL. Two days after cell transplantation, the mice were randomly divided into four groups. Each group consisted of 8 mice. The short diameter and long diameter of the tumor of each mouse were measured using an electronic digital caliper (Digimatic (trademark) Caliper, Mitutoyo Corporation), and the tumor volume was calculated using the following formula: Tumor volume (mm 3 ) = long diameter (mm) × short diameter (mm) × short diameter (mm) / 2 T / C = (average tumor volume of the treatment group) / (average tumor volume of the control group) Inhibition of tumor growth (%) = (1 - T / C) × 100

[0161] The test compound was intravenously administered at 0.09 mg / kg on days 3 and 11. The anti-mPD-1 antibody (BE0146, Bio X Cell) was intravenously administered at 10 mg / kg on days 3, 7, 11, and 15. The tumor growth inhibition (%) of each test compound on day 15 is shown in Table 13.

Table 13

[0162] Pharmacological Test Example 14. Effect on Tubulin Polymerization in Vitro (Figure 10A) A tubulin polymerization assay kit was purchased from Cytoskeleton, Inc. (Catalog No. BK011P). The kit contained one vial of freeze-dried tubulin protein purified from porcine brain, three tubes of freeze-dried GTP, two vials of freeze-dried assay buffer, and one vial of tubulin glycerol buffer. The assay buffer was prepared by dissolving the contents in 10 mL of sterile deionized water. This solution contained 80 mmol / L piperazine-N,N'-bis[2-ethanesulfonic acid] sesquisodium salt, 2.0 mmol / L magnesium chloride, 0.5 mmol / L ethylene glycol-bis(2-amino-ethyl ether)N,N,N',N'-tetra-acetic acid, pH 6.9, and 10 μmol / L fluorescent reporter. The buffer was stored at -70 °C until use. The tubulin glycerol buffer consisted of 80 mmol / L piperazine-N,N'-bis[2-ethanesulfonic acid] sesquisodium salt, 2.0 mmol / L magnesium chloride, 0.5 mmol / L ethylene glycol-bis(2-amino-ethyl ether)N,N,N',N'-tetra-acetic acid, 60% v / v glycerol, and pH 6.9. This was stored at 4 °C until use. The GTP stock solution was prepared by dissolving the contents of each tube in 100 μL of sterile deionized water to a concentration of 100 mmol / L GTP. This solution was aliquoted and stored at -70 °C until use. The tubulin stock solution (10 mg / mL) was prepared by adding 1.1 mL of a mixture of assay buffer and GTP stock solution (100:1, v / v) to dissolve the tubulin powder. This solution was aliquoted, frozen in liquid nitrogen, and stored at -70 °C until use.

[0163] In the tubulin polymerization assay, a reaction mixture was prepared by mixing 820 μL of assay buffer, 17.6 μL of GTP stock solution, and 600 μL of tubulin glycerol buffer. This reaction mixture (1015 μL) was mixed with 240 μL of tubulin stock solution. This solution was called the tubulin reaction mixture and was used for the measurement of test and control wells. A reaction mixture without tubulin was prepared by mixing 89.85 μL of the reaction mixture and 21.25 μL of assay buffer, and the blank well was measured. A compound (1) solution (6.25 - 100 μmol / L; final concentration 0.625 - 10 μmol / L) or vehicle was added at 5 μL per individual well of a 96-well half-area microtiter plate. The tubulin reaction mixture or the reaction mixture without tubulin was added at 45 μL per well of the plate. Fluorescence emission at 460 nm (excitation wavelength of 360 nm) was measured every 2 minutes over 90 minutes using a SpectraMax® M5e microplate reader (Molecular Devices). Since the fluorescent reporter is incorporated into microtubules by tubulin polymerization, fluorescence enhancement occurs after tubulin polymerization. The assay was performed with two-point measurement. The assay demonstrated that compound (1) inhibits tubulin polymerization in a concentration-dependent manner. The fluorescence intensity at each time point was calculated by the following formula: Fluorescence intensity = average fluorescence measurement value of the test well or control well - average fluorescence measurement value of the blank well; Blank well: contains vehicle without tubulin; Control well: contains vehicle and tubulin; Test well: contains compound and tubulin.

[0164] Pharmacological Test Example 15. Cell-Based Microtubule Dynamics Assay (Figure 10B) The microtubule dynamics assay in cells was performed using the U2OS-EB3-AG osteosarcoma cell line that stably expresses a fusion protein of EB3 (microtubule plus-end binding protein) and Azami-Green (EB3-AG). The U2OS-EB3-AG cells were cultured in RPMI-1640 medium containing 10% FBS and penicillin-streptomycin at 37 °C in a humidified 5% CO2 incubator. Microtubule dynamics in living cells can be visualized as the movement of the comet-like structures of EB3-AG. The U2OS-EB3-AG cells on a glass base culture plate (EZVIEW plate, AGC Techno Glass, Japan) were treated with the compound (1) at the concentrations shown in the figure, and the microtubule dynamics were monitored by time-lapse imaging using a fluorescence microscope equipped with a 60x oil immersion objective lens (BZ-X710, Keyence, Japan). The still images at each time point are presented in Fig. 10B. The high-magnification images of the areas surrounded by squares are shown in the inset figure. When the cells were treated with compound (1) at 0.5 nM (50% growth inhibitory concentration in U2OS-EB3-AG cells), the comet-like structures became difficult to observe approximately 60 minutes after the addition of the compound. These results clearly demonstrated that compound (1) has the ability to suppress microtubule dynamics.

[0165] Pharmacological Test Example 16. In Vitro Proliferation Inhibition Activity (Figure 11) The in vitro growth inhibition assay of compound (1) was performed using a small panel of cancer cell lines including human esophageal squamous cell carcinoma (OE21, TE-8), human esophageal adenocarcinoma (OE33), and human uterine sarcoma (MES-SA, MES-SA-Dx5-Rx1). All cell lines were cultured in RPMI-1640 medium containing 10% FBS and penicillin-streptomycin in a 5% CO2 incubator at 37 °C. In each well of a 96-well plate (Becton Dickinson, 353219), 4×10 475 μL of the cell suspension adjusted to a concentration of cells / mL was added, and the cells were incubated overnight in a 5% CO2 incubator (37 °C). The next day, 25 μL of compound (1) in a three-fold dilution series suspended in the medium was added to each well and incubated in a 5% CO2 incubator (37 °C) for 72 hours. Subsequently, cell viability was determined using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) with a 2013 EnVision™ Multilabel Reader (PerkinElmer, Wellesley, MA). The value of the well of cells to which the test compound was not added was defined as 100%, and the value of the well without cells was defined as 0%. The concentration of compound (1) required to inhibit cell growth by 50% (IC 50 value) was calculated and is shown in Figure 11. P-gp sensitivity was calculated as the ratio of the IC 50 value in MES-SA-Dx5-Rx1 cells overexpressing P-gp to the IC 50 value in MES-SA cells.

[0166] Pharmacological Test Example 17. Antitumor Effect in KPL-4 Transplant Mouse Model as Monotherapy; Antitumor Effect in Mouse COLO-704 Transplant Model as Monotherapy (Figure 12) The human HER-2 positive breast cancer cell line KPL-4 cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS and penicillin-streptomycin was adjusted to a concentration of 1×10 8 cells / mL with Hanks’ Balanced Salt Solution to prepare a cell suspension. The cell suspension was transplanted at a volume of 100 μL subcutaneously into the right flank of 8-week-old nude mice (CAnN.Cg-Foxn1nu / CrlCrlj, female, Charles River Laboratories Japan, Inc.). Eleven days after cell transplantation (day 1), the short diameter and long diameter of the tumor of each mouse were measured using a digital electronic caliper (Digimatic® Caliper, Mitutoyo Corporation), and the tumor volume was calculated using the following formula: Tumor volume (mm 3 ) = long diameter (mm) × short diameter (mm) × short diameter (mm) / 2 Relative tumor volume (RTV) = tumor volume (day X) / tumor volume (day 1) Relative body weight (RBW) = body weight (day X) / body weight (day 1)

[0167] Based on the tumor volumes obtained on the first day, the mice were grouped so that the average tumor volume was equal among the groups. The experiment was conducted with 6 mice per group. The test compound was dissolved in DMSO and the solution was stored frozen until use. Immediately before administration, the stored solution was diluted with physiological saline. The test compound in physiological saline was intravenously administered once a week at 20 μg / kg, 60 μg / kg, or 180 μg / kg for 2 weeks (on the first and eighth days). Tumor regression was observed in the treatment groups at 60 μg / kg and 180 μg / kg, and administration at 180 μg / kg completely eradicated the transplanted tumors in all mice on the 15th day.

[0168] The human ovarian cancer cell line COLO-704 cultured in RPMI-1640 containing 10% FBS and penicillin-streptomycin was adjusted to a concentration of 1 × 10 8 cells / mL with Hanks’ Balanced Salt Solution to prepare a cell suspension. The cell suspension was transplanted subcutaneously into the right flank of 5-week-old nude mice (CAnN.Cg-Foxn1nu / CrlCrlj, female, Charles River Laboratories Japan, Inc.) at a volume of 100 μL. Nine days after cell transplantation (the first day), the short and long diameters of the tumors in each mouse were measured using a digital vernier caliper (Digimatic (trademark) Caliper, Mitutoyo Corporation), and the tumor volume was calculated using the following formula: Tumor volume (mm 3 ) = long diameter (mm) × short diameter (mm) × short diameter (mm) / 2 Relative tumor volume (RTV) = tumor volume (day X) / tumor volume (first day) Relative body weight (RBW) = body weight (day X) / body weight (first day)

[0169] Based on the tumor volumes obtained on the first day, the mice were grouped so that the average tumor volume was equal among the groups. The experiment was conducted with 6 mice per group. The test compound was dissolved in DMSO and the solution was stored frozen until use. Immediately before administration, the stock solution was diluted with physiological saline. The test compound in physiological saline was intravenously administered once a week at 20 μg / kg, 60 μg / kg, or 180 μg / kg over a two-week period (on the first and eighth days). Treatment with the compound induced tumor regression at 180 μg / kg and delayed tumor growth at 60 μg / kg. Administration at 180 μg / kg completely eradicated the transplanted tumors in all mice on day 22.

[0170] Pharmacological Test Example 18. Effect on CD31-Positive Blood Vessels in FaDu Subcutaneous Xenograft Mouse Model (Figure 13) The human head and neck squamous cell carcinoma (SCCHN) cell line FaDu cultured in RPMI-1640 medium containing 10% FBS and penicillin-streptomycin was adjusted to 5×10 7The cell suspension was prepared by adjusting the concentration to cells / mL. The cell suspension was transplanted at a volume of 100 μL into the subcutaneous tissue of the right flank of 6-week-old nude mice (CAnN.Cg-Foxn1nu / CrlCrlj, female, Charles River Laboratories Japan, Inc.). Ten days after cell transplantation, the mice were grouped so that the average tumor volume was equal among the groups. The experiment was conducted with 6 mice per group. Each test compound was dissolved in DMSO, and the solution was stored frozen until use. Immediately before administration, the stock solution was diluted with physiological saline. The test compound in physiological saline was administered intravenously at 20 μg / kg, 60 μg / kg, or 180 μg / kg. Five days after a single administration, tumor samples were collected and fixed with IHC zinc fixative (BD Pharmingen) at 4°C for 24 hours. Paraffin-embedded tissues were sectioned (3 μm), placed on positively charged slide glasses, and air-dried. Immunohistochemical staining for CD31 was performed using a Ventana Autostainer model Discover XT (Roche Diagnostics) according to the manufacturer's protocol. The sections were deparaffinized, rehydrated, and antigen-activated with CC1 (Ventana Medical Systems). The slides were blocked with Blocking Agent A and Blocking Agent B (Endogenous Biotin Blocking Kit, Roche Diagnostics). The sections were incubated with a rat anti-mouse IgG CD31 antibody (Dianova) at 2 μg / mL for 6 hours, and then incubated with a biotinylated anti-rat IgG antibody (Jackson ImmunoResearch Laboratories) at 2.2 μg / mL for 32 minutes. Detection was performed with streptavidin-HRPD for 16 minutes, and then incubated with DAB D and DAB H2O2D (DABMap Kit, Ventana Medical Systems) for 8 minutes. The slides were counterstained with hematoxylin II (Roche Diagnostics) for 16 minutes, and then incubated with a bluing reagent for 4 minutes. The sections were dehydrated with graded ethanol, defatted by xylene exchange, and covered with DPX (registered trademark) (Merck). The immunostained slides were scanned using a Vectra (registered trademark) 2 Automated Slide Imaging System (PerkinElmer).The number of blood vessels in the whole tumor was quantified by counting CD31-positive objects using inForm2 software (PerkinElmer). Also, the hematoxylin staining area was measured using inForm2 software (PerkinElmer) and used as the tumor area. The number of blood vessels was normalized by the tumor area. When the test compound was administered once at doses of 20, 60, and 180 μg / kg, the number of tumor blood vessels increased. The ratio of the number of blood vessels in the test compound-administered group compared to the untreated group was calculated by the following formula:. Tumor blood vessel ratio = number of blood vessels in the test compound-administered group / number of blood vessels in the untreated group

[0171] Pharmacological Test Example 19. Effect on α-SMA-Positive CAF in FaDu Subcutaneous Xenograft Mouse Model (Figure 14) The human head and neck squamous cell carcinoma (SCCHN) cell line FaDu cultured in RPMI-1640 medium containing 10% FBS and penicillin-streptomycin was adjusted to 5×10 7The cell suspension was prepared by adjusting the concentration to cells / mL. The cell suspension was transplanted at a volume of 100 μL into the subcutaneous area of the right flank of 6-week-old nude mice (CAnN.Cg-Foxn1nu / CrlCrlj, female, Charles River Laboratories Japan, Inc.). Ten days after cell transplantation, the mice were grouped so that the average tumor volume was equal among the groups. The experiment was conducted with 5 mice per group. Each test compound was dissolved in DMSO and the solution was stored frozen until use. Immediately before administration, the stored solution was diluted with physiological saline. The test compound in physiological saline was administered intravenously at 20 μg / kg, 60 μg / kg, or 180 μg / kg. Two days or five days after a single administration, tumor samples were collected and fixed with IHC zinc fixative (BD Pharmingen) at 4°C for 24 hours. Paraffin-embedded tissues were sectioned (3 μm), placed on positively charged glass slides, and air-dried. The sections were deparaffinized, rehydrated, and antigen activation was performed using a microwave with 1 mM EDTA at pH 6.0. The sections were blocked with 1% BSA in TBS. The sections were incubated for 2.5 hours with a mouse anti-α-SMA monoclonal antibody (clone 1A4, Sigma) conjugated with alkaline phosphatase at 5 μg / mL. Detection was performed using a Fast Red II substrate kit (Nichirei Bioscience). The sections were counterstained with Mayer's hematoxylin (Muto Chemical) for 50 seconds. The sections were dehydrated with graded ethanol, defatted by xylene exchange, and covered with DPX (Merck). The immunostained slides were scanned using a Vectra (registered trademark) 2 Automated Slide Imaging System (PerkinElmer). The area of the α-SMA-positive region in the whole tumor was quantified by counting the α-SMA-positive objects using inForm2 software (PerkinElmer). Also, the hematoxylin-stained area was measured using inForm2 software (PerkinElmer) and used as the tumor area. The area of the α-SMA-positive region was normalized by the tumor area. When the test compound was administered as a single dose, the α-SMA-positive area was significantly reduced at doses of 60 and 180 μg / kg on day 3 and at a dose of 180 μg / kg on day 6. The inhibition rate of the α-SMA-positive area in the test compound dosing group was calculated by the following formula: α-SMA ratio = α-SMA area of the test compound dosing group / α-SMA area of the untreated group

[0172] Pharmacological Test Example 20. Effect on Tenascin-C and EDA-Fibronectin in FaDu Subcutaneous Xenograft Mouse Model (Figure 15) The human head and neck squamous cell carcinoma (SCCHN) cell line FaDu cultured in RPMI-1640 medium containing 10% FBS and penicillin-streptomycin was treated with 5×10 7The cell suspension was prepared by adjusting the concentration to cells / mL. The cell suspension was transplanted at a volume of 100 μL into the subcutaneous area of the right flank of 6-week-old nude mice (CAnN.Cg-Foxn1nu / CrlCrlj, female, Charles River Laboratories Japan). Ten days after cell transplantation, the mice were grouped so that the average tumor volume was equal among the groups. The experiment was conducted with 5 mice per group. Compound (1) was dissolved in DMSO and the solution was stored frozen until use. Compound (1) (180 μg / kg) and cetuximab (CTX, Erbitux®, Merck Serono) (10 mg / kg) were diluted with physiological saline on day 1 and administered by intravenous injection. Five days after a single administration, tumor samples were collected and fixed with IHC zinc fixative (BD Pharmingen) at 4 °C for 24 hours. Paraffin-embedded tissues were sectioned (3 μm), placed on positively charged glass slides, and air-dried. The sections were deparaffinized and rehydrated, and for tenascin-C, antigen retrieval was performed using a microwave with 1 mM EDTA at pH 6.0 to recover the antigen. For EDA-fibronectin, the antigen retrieval procedure was not necessary. The sections were incubated with BLOXALL blocking solution (Vector Laboratories) for 10 minutes to block endogenous peroxidase, with mouse-on-mouse Ig blocking reagent (Vector Laboratories) for 1 hour, and then with 2.5% normal horse serum for 30 minutes. For immunohistochemical staining of tenascin-C, the sections were incubated overnight at 4 °C with mouse anti-tenascin-C monoclonal antibody (clone 4C8MS, IBL) at 5 μg / mL. For immunohistochemical staining of EDA-fibronectin, the sections were incubated at room temperature for 1 hour with mouse anti-EDA-fibronectin monoclonal antibody (clone IST-9, Abcam) at 1.5 μg / mL. Detection was performed using the mouse-on-mouse ImmPRESS™ peroxidase polymer kit (Vector Laboratories). The sections were counterstained with Mayer's hematoxylin (Muto Chemical) for 50 seconds. The sections were dehydrated with graded ethanol, defatted by xylene exchange, and covered with DPX (Merck).The immunostained slides were scanned using a Vectra™ 2 Automated Slide Imaging System (PerkinElmer). The expression levels of both tenascin-C and EDA-fibronectin were reduced in tumors treated with compound (1) and CTX compared to control tumors.

[0173] Pharmacological Test Example 21. Antitumor Effect in FaDu Subcutaneous Transplant Mouse Model Combined with Cetuximab (Figure 16) The human head and neck squamous cell carcinoma (SCCHN) cell line FaDu cultured in RPMI-1640 medium containing 10% FBS and penicillin-streptomycin was adjusted to a concentration of 5×10 7 cells / mL with Hanks’ Balanced Salt Solution to prepare a cell suspension. The cell suspension was transplanted subcutaneously into the right flank of nude mice (CAnN.Cg-Foxn1nu / CrlCrlj, female, 7 weeks old, Charles River Laboratories Japan) at a volume of 100 μL. Ten days after cell transplantation (day 1), the length and width of the tumor in each mouse were measured using an electronic digital caliper (Mitutoyo Corporation), and the tumor volume was calculated using the following formula: Tumor volume (mm 3 ) = major axis (mm) × minor axis (mm) × minor axis (mm) / 2 Relative tumor volume (RTV) = tumor volume (day X) / tumor volume (first day)

[0174] Based on the TV, the mice were randomly grouped on day 1. Each group consisted of 6 mice. Compound (1) was dissolved in DMSO and the solution was stored frozen until use. Compound (1) (20, 60, or 180 μg / kg) and cetuximab (CTX, Erbitux®, Merck Serono) (10 mg / kg) were diluted with saline and injected intravenously on day 1. The changes in RTV of each group are shown in Figure 16. At doses of 180 μg / kg and 60 μg / kg, the antitumor efficacy of compound (1) in combination with CTX was stronger than that of CTX monotherapy, and there was tumor regression. The antitumor efficacy of compound (1) at a dose of 20 μg / kg in combination with CTX tended to be stronger than that of CTX monotherapy.

[0175] Pharmacological Test Example 22. Antitumor Effect in Soft Tissue Sarcoma Transplant Mouse Model as Monotherapy (Figure 17) MES-SA The human uterine sarcoma cell line MES-SA cultured in RPMI-1640 containing 10% FBS and penicillin-streptomycin was adjusted to a concentration of 2×10 8 cells / mL with Hanks’ Balanced Salt Solution to prepare a cell suspension. The suspension was mixed with Geltrex (trademark) (Thermo Fisher Scientific, #A1413202) at a ratio of 1:1 to prepare a cell suspension with a concentration of 1×10 8 cells / mL. The cell suspension was transplanted subcutaneously into the right flank of 6-week-old nude mice (CAnN.Cg-Foxn1nu / CrlCrlj, female, Charles River Laboratories Japan, Inc.) at a volume of 100 μL. Six days after cell transplantation (day 1), the short and long diameters of the tumors in each mouse were measured using a Digimatic (trademark) caliper (Mitutoyo Corporation), and the tumor volume was calculated using the following formula: Tumor volume (mm 3 ) = long diameter (mm) × short diameter (mm) × short diameter (mm) / 2 Relative tumor volume (RTV) = tumor volume (day X) / tumor volume (first day)

[0176] Based on the tumor volume obtained on day 1, the mice were grouped so that the average value of the tumor volume was equal among groups. The experiment was conducted with 6 mice per group. The test compound was dissolved in DMSO and the solution was stored frozen until use. Immediately before administration, the stock solution was diluted with physiological saline. The test compound in physiological saline was intravenously administered once a week at 180 μg / kg for 2 weeks (on days 1 and 8). Antitumor activity was observed with tumor growth delay in the treatment group.

[0177] HT-1080 The human fibrosarcoma cell line HT-1080 cultured in E-MEM containing 10% FBS, NEAA, and antibiotics was adjusted to 3×10 7The cell suspension was prepared by adjusting the concentration to cells / mL. The cell suspension was transplanted at a volume of 100 μL subcutaneously into the right lower abdominal skin of thymus-deficient mice (CAnN.Cg-Foxn1nu / CrlCrlj, female, 6 weeks old, Charles River Japan). Six days after cell transplantation (day 1), the length and width of the tumor of each mouse were measured using an electronic digital caliper (Mitutoyo Corporation), and the tumor volume was calculated using the following formula: Tumor volume (mm 3 ) = major axis (mm) × minor axis (mm) × minor axis (mm) / 2 Relative tumor volume (RTV) = tumor volume (day X) / tumor volume (first day)

[0178] Based on the TV, the mice were randomly grouped (day 1). Each group consisted of 6 mice. Compound (1) was dissolved in DMSO, and the solution was stored frozen until use. Compound (1) (180 μg / kg) was diluted with physiological saline and injected intravenously on day 1 and day 8. The changes in RTV of each group are shown in Figure 17. Antitumor activity was observed with tumor regression in the treatment group.

[0179] CTG-2041 Tumor fragments of human angiosarcoma CTG-2041 were transplanted subcutaneously into the left lower abdomen of female mice. Tumor growth was monitored twice a week using a digital caliper, and the tumor volume was calculated using the following formula: Tumor volume (mm 3 ) = major axis (mm) × minor axis (mm) × minor axis (mm) / 2 Relative tumor volume (RTV) = tumor volume (day X) / tumor volume (first day)

[0180] When the tumor volume reached approximately 200 mm 3 the animals were grouped into a treatment group or a control group based on the tumor volume, and dosing was started on day 1. Each group consisted of 5 mice. Compound (1) was dissolved in DMSO, and the solution was stored frozen until use. Compound (1) (100 μg / kg) was diluted with physiological saline and injected intravenously on day 1 and day 8. The changes in RTV of each group are shown in Figure 17. Antitumor activity was observed with tumor regression in the treatment group.

[0181] Pharmacological Test Example 23. Antitumor Effect in Endometrial Cancer Transplant Mouse Model as Monotherapy (Figure 18) HEC-108 The human endometrial cancer cell line HEC-108 cultured in E-MEM containing 15% FBS and antibiotics was adjusted to a concentration of 7.14×10 7 cells / mL in the medium to prepare a cell suspension. The cell suspension was transplanted subcutaneously into the right lower abdominal skin of thymus-deficient mice (CAnN.Cg-Foxn1nu / CrlCrlj, female, 6 weeks old, Charles River Japan) at a volume of 150 μL. Thirteen days (day 1) after cell transplantation, the length and width of the tumor of each mouse were measured using an electronic digital caliper (Mitutoyo Corporation), and the tumor volume was calculated using the following formula: Tumor volume (mm 3 ) = major axis (mm) × minor axis (mm) × minor axis (mm) / 2 Relative tumor volume (RTV) = tumor volume (day X) / tumor volume (first day)

[0182] Based on the TV, the mice were randomly grouped (day 1). Each group consisted of 6 mice. Compound (1) was dissolved in DMSO, and the solution was stored frozen until use. Compound (1) (180 μg / kg) was diluted with physiological saline and intravenously injected on day 1 and day 8. The changes in RTV of each group are shown in Figure 18. Antitumor activity was observed with tumor growth delay in the treatment group.

[0183] AN3CA The human endometrial cancer cell line AN3CA cultured in E-MEM containing 10% FBS and penicillin-streptomycin was adjusted to a concentration of 1.4×10 8 cells / mL in Hanks’ Balanced Salt Solution to prepare a cell suspension, and the suspension was mixed with Geltrex (trademark) (Thermo Fisher Scientific, #A1413202) at a ratio of 1:1 to 7×10 7A cell suspension with a concentration of cells / mL was prepared. The cell suspension was transplanted at a volume of 100 μL into the lower right abdominal skin of 6-week-old nude mice (CAnN.Cg-Foxn1nu / CrlCrlj, female, Charles River Laboratories Japan, Inc.). Twelve days after cell transplantation (day 1), the minor axis and major axis of the tumor of each mouse were measured using an electronic digital caliper (Digimatic (trademark) Caliper, Mitutoyo Corporation), and the tumor volume was calculated using the following formula: Tumor volume (mm 3 ) = major axis (mm) × minor axis (mm) × minor axis (mm) / 2 Relative tumor volume (RTV) = tumor volume (day X) / tumor volume (first day)

[0184] Based on the tumor volume obtained on day 1, the mice were grouped so that the average value of the tumor volume was equal among groups. The experiment was conducted with 5 mice per group. The test compound was dissolved in DMSO and the solution was stored frozen until use. Immediately before administration, the stock solution was diluted with physiological saline. The test compound in physiological saline was intravenously administered once a week at 180 μg / kg for 2 weeks (on day 1 and day 8). Anti-tumor activity was observed with tumor regression in the treatment group.

[0185] Pharmacological Test Example 24. Proliferation Inhibition Assay of NCI-N87 and MKN-28 In this assay, the growth inhibitory activity of the test compound in the human gastric cancer cell lines NCI-N87 and MKN-28 was measured respectively. NCI-N87 cells and MKN-28 cells were maintained in RPMI-1640 medium containing 10% FBS, penicillin and streptomycin in a 5% CO2 incubator (37 °C). Into each well of a 96-well plate (Becton, Dickinson and Company, 353219), 3×10 4100 μL of a cell suspension of NCI-N87 or MKN-28 adjusted to a concentration of cells / mL was added, and the cells were incubated overnight in a 5% CO2 incubator (37 °C). The next day, 100 μL of compound (1) or halicondrin B in a three-fold dilution series suspended in the medium was added to each well, and the resultant was incubated in a 5% CO2 incubator (37 °C) for 3 days. Then, cell viability was determined by the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) using an EnVision 2103 Multilabel Reader (Perkin-Elmer, Wellesley, MA). The value of the cell-containing well without the test compound added was defined as 100%, and the value of the well without cells was defined as 0%. The concentration of the test compound required to inhibit cell growth up to 50% (i.e., the IC 50 value) was calculated and shown in Table 14.

Table 14

[0186] Pharmacological Test Example 25. Proliferation Inhibition Assay of HuTu80 In this assay, the growth inhibitory activity of the test compound in the human small intestine cancer cell line HuTu80 isolated from duodenal tissue was measured. HuTu80 cells were maintained in EMEM medium containing 10% FBS, penicillin, and streptomycin in a 5% CO2 incubator (37 °C). To each well of a 96-well plate (Becton, Dickinson and Company, 353219), 3 × 10 4100 μL of HuTu80 cell suspension adjusted to a concentration of cells / mL was added, and the cells were incubated overnight in a 5% CO2 incubator (37 °C). The next day, 100 μL of compound (1) or halicondrin B in a three-fold dilution series suspended in the medium was added to each well, and the resultant was incubated in a 5% CO2 incubator (37 °C) for 3 days. Subsequently, cell viability was determined by the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) using an EnVision 2103 Multilabel Reader (Perkin-Elmer, Wellesley, MA). The value of the cell-containing well without the test compound added was defined as 100%, and the value of the well without cells was defined as 0%. The concentration of the test compound required to inhibit cell growth up to 50% (i.e., the IC 50 value) was calculated and shown in Table 15.

Table 15

[0187] Pharmacological Test Example 26. Proliferation Inhibition Assay of SW780 In this assay, the growth inhibitory activity of the test compound in the human urothelial cancer cell line SW780 was measured. SW780 cells were maintained in RPMI-1640 medium containing 10% FBS, penicillin, and streptomycin in a 5% CO2 incubator (37 °C). To each well of a 96-well plate (Becton, Dickinson and Company, 353219), 3 × 10 4100 μL of SW780 cell suspension adjusted to a concentration of cells / mL was added, and the cells were incubated overnight in a 5% CO2 incubator (37 °C). The next day, 100 μL of compound (1) or halicondrin B in a three-fold dilution series suspended in the medium was added to each well, and the resulting product was incubated in a 5% CO2 incubator (37 °C) for 3 days. Then, cell viability was determined by the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) using an EnVision 2103 Multilabel Reader (Perkin-Elmer, Wellesley, MA). The value of the cell-containing well without the test compound added was defined as 100%, and the value of the well without cells was defined as 0%. The concentration of the test compound required to inhibit cell growth by up to 50% (i.e., the IC 50 value) was calculated and shown in Table 16.

Table 16

[0188] Pharmacological Test Example 27. Proliferation Inhibition Assay of HS-SY-II In this assay, the growth inhibitory activity of the test compound in the human synovial sarcoma cell line HS-SY-II was measured. HS-SY-II cells were maintained in DMEM medium containing 10% FBS, penicillin, and streptomycin in a 5% CO2 incubator (37 °C). To each well of a 96-well plate (Becton, Dickinson and Company, 353219), 3 × 10 4100 μL of HS-SY-II cell suspension adjusted to a concentration of cells / mL was added, and the cells were incubated overnight in a 5% CO2 incubator (37 °C). The next day, 100 μL of compound (1) or halicondrin B in a three-fold dilution series suspended in the medium was added to each well, and the resulting product was incubated in a 5% CO2 incubator (37 °C) for 3 days. Subsequently, cell viability was determined by the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) using an EnVision 2103 Multilabel Reader (Perkin-Elmer, Wellesley, MA). The value of the cell-containing well without the test compound added was defined as 100%, and the value of the well without cells was defined as 0%. The concentration of the test compound required to inhibit cell growth up to 50% (i.e., the IC 50 value) was calculated and shown in Table 17.

Table 17

[0189] Equivalents and ranges In the claims, articles such as "a", "an", "the", etc. can mean one or more than one, unless indicated to the contrary or otherwise clear from the context. A claim or specification that includes "or" between one or more elements of a group is considered satisfied, unless indicated to the contrary or otherwise clear from the context, if one, more than one, or all of the elements of the group are present in, used in, or otherwise related to a certain product or process. The present invention includes embodiments in which exactly one element of the group is present in, used in, or otherwise related to a certain product or process. The present invention includes embodiments in which more than one or all of the group elements are present in, used in, or otherwise related to a certain product or process.

[0190] Furthermore, the present invention encompasses all modifications, combinations, permutations in which one or more limitations, elements, clauses, descriptive terms from one or more of the recited claims are introduced into another claim. For example, any claim that depends on another claim can be modified to include one or more limitations found in any other claim that depends on the same basic claim. If elements are presented as a list, for example, in Markush group form, each subgroup of those elements is also disclosed and any element can be removed from the group. Generally, when the present invention or an aspect of the present invention is referred to as including a particular element and / or feature, it should be understood that a particular embodiment of the present invention or an aspect of the present invention consists of, or consists essentially of, such element and / or feature. For the sake of simplicity, such embodiments are not specifically shown herein in these exact words. It is also noted that the terms "comprising" and "containing" are intended to be open and allow the inclusion of additional elements or steps. When a range is given, the endpoints are included. Further, unless otherwise indicated or otherwise obvious from the context and the understanding of one of ordinary skill in the art, values expressed as ranges can assume any specific value or subrange within the stated range in different embodiments of the present invention, down to one-tenth of the unit of the lower limit of the range, unless the context clearly requires otherwise.

[0191] One of ordinary skill in the art will recognize many equivalents to the specific embodiments described herein and can verify these, at most, using routine experimentation. The scope of the embodiments described herein is not intended to be limited as described above, but rather as set forth in the appended claims. One of ordinary skill in the art will appreciate that various changes and modifications to this description can be made without departing from the spirit or scope of the invention as defined in the following claims.

Claims

1. as follows: 【Chemical Formula 1】 A compound selected from the group consisting of the following and salts thereof.

2. The compound according to claim 1, wherein the compound is represented by formula (C-8) or a salt thereof.

3. The compound according to claim 1, wherein the compound is represented by the following formula: 【Chemical Formula 2】 or a salt thereof.

4. The compound according to claim 1, wherein the compound is represented by formula (C-7) or a salt thereof.

5. The compound according to claim 1, wherein the compound is represented by formula (C-6) or a salt thereof.

6. The compound according to claim 1, wherein the compound is represented by formula (C-5) or a salt thereof.

7. The compound according to claim 1, wherein the compound is represented by formula (C-4) or a salt thereof.

8. The compound according to claim 1, wherein the compound is represented by formula (C-3) or a salt thereof.

9. as follows: 【Chemical Formula 3】 A compound selected from the group consisting of the following and salts thereof.

10. The compound according to claim 9, wherein the compound is represented by formula (C-2) or a salt thereof.

11. The compound according to claim 9, wherein the compound is represented by formula (C-1) or a salt thereof.

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