EA040298B1 - MACROCYCLIC COMPOUND AND ITS APPLICATIONS FOR INHIBITION OF TUMOR OR CANCER GROWTH - Google Patents

Description

Related Applications

The present application claims priority under 35 U.S.C. § 119(e) pursuant to U.S. Provisional Applications Serial 62/482030 filed April 5, 2017, 62/526677 filed June 29, 2017, and Serial 62/586416 filed November 15, 2017 g., and in accordance with 35 U.S.C. § 120 pursuant to U.S. Patent Application Serial No. 15/814105, filed November 15, 2017; the entire contents of each are incorporated herein by reference.

The field of technology to which the present invention relates

The present invention relates to a novel macrocyclic compound having tumor vascular remodeling effects and activity against CAF (cancer associated fibroblast). The compound can be used to treat cancer or inhibit tumor growth in a subject.

Background of the Invention Halichondrins such as halichondrin B are anticancer agents originally isolated from the marine sponge Halichondria okadai (see, for example, D. Uemura et al. Norhalichondrin A: An Antitumor Polyether Macrolide from a Marine Sponge J. Am Chem. Soc, 107, 4796 (1985)) and subsequently found in Axinella sp., Phakellia carteri and Lissondendryx sp. A complete synthesis of halichondrin B was published in 1992 (see, for example, Y. Kishi et al. Total Synthesis of Halichondrin B and Norhalichondrin B J. Am. Chem. Soc. 114, 3162 (1992)). Halichondrin B has demonstrated in vitro inhibition of tubulin polymerization, microtubule assembly, cross-linking of beta-5-tubulin, binding of GTP and vinblastine to tubulin, and tubulin-dependent hydrolysis of GTP, and has shown anticancer properties in vitro and in vivo (see, e.g., Y. Fodstad et al, Comparative antitumor activities of halichondrins and vinblastine against human tumor xenografts J. of Experimental Therapeutics & Oncology 1996; 1:119, 125).

Eribulin mesylate (Halaven™), which was developed from halichondrin B (see, for example, International Patent Publication No. WO 1999/065894, published December 23, 1999; International Patent Publication No. WO 2005/118565, published December 15, 2005 ., and W. Zheng et al., Macrocyclic ketone analogues of halichondrin B Bioorganic & Medicinal Chemistry Letters 14, 5551-5554 (2004)), are now clinically used in many countries for the treatment of, for example, metastatic breast cancer and advanced liposarcoma. .

Additional patent publications that describe halichondrins include US Pat. No. 5,436,238 to Kishi et al. issued July 25, 1995; US patent No. 5338865, Kishi et al., issued August 16, 1994; and International Patent Publication WO 2016/003975 filed by Kishi et al., all assigned to the President and Fellows of the Harvard University Fellowship.

See also, for example, US patent No. 5786492; US patent No. 8598373; US patent No. 9206194; US patent No. 9469651; international patent publications WO/2009/124237A1;

WO/1993/01769OA1; WO/2012/147900A1; US patent No. 7982060; US patent No. 8618313; US patent No. 9303050; US patent No. 8093410; US patent No. 8350067; US patent No. 8975422; US patent No. 8987479; US patent No. 8203010; US patent No. 8445701; US patent No. 8884031; U.S. Patent No. RE45324; US patent No. 8927597; US patent No. 9382262; US patent No. 9303039; international patent publications WO/2009/046308A1; WO/2006/076100A3; WO/2006/076100A2;

WO/2015/085193A1; WO/2016/176560A1; US patent No. 9278979; US patent No. 9029573; international patent publications WO/2011/094339A1; WO/2016/179607A1; WO/2009/064029A1; WO/2013/142999A1; WO/2015/066729A1; WO/2016/038624A1 and WO/2015/000070A1.

Cancer-associated fibroblasts (CAF), which are widely found in various solid tumors, are stromal cells. It is well known that CAFs play an important role in angiogenesis, invasion and metastasis. It has been reported that there is a strong correlation between CAF count and clinical prognosis, for example, in invasive breast cancer (see, e.g., 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) and adenocarcinoma of the esophagus (see, for example, T. J. Underwood et al. Cancer-associated fibroblasts predict poor outcome and promote periostin-dependent invasion in esophageal adenocarcinoma Journal of Pathol., 235, 466, 2015). It has also been reported that CAFs correlate with resistance to neoadjuvant chemotherapy in breast cancer, such as, for example, breast cancer (see, e.g., P. Farmer et al. A stroma-related gene signature predicts resistance to neoadjuvant chemotherapy in breast cancer Nature Medicine., 15(1), 68, 2009) and head and neck cancer (see e.g. S. Schmitz et al. Cetuximab promotes epithelial to mesenchymal transition and cancer associated fibroblasts in patients with head and neck cancer Oncotarget, 6 (33), 34288 , 2015; Y. Matsuoka et al.. The tumor stromal features are associated with resistance to 5-FU-based chemoradiotherapy and a poor prognosis in patients with oral squamous cell carcinoma APMIS 123(3), 205, 2015).

Thus, the effects of tumor vascular remodeling and anti-CAF activity have been found to lead to an improvement in the cancer microenvironment, thus facilitating tumor treatment. Krove

- 1 040298 Nasal vessels are essential for the growth of tumors. Remodeled blood vessels in tumors can deliver anti-cancer agents to tumors, in addition to achieving relief from hypoxia. Eribulin-induced remodeling of the abnormal tumor vasculature is reported to result in a more functional microenvironment, which may reduce tumor aggressiveness due to elimination of hypoxia within the tumor. Since the abnormal tumor microenvironment enhances both drug resistance and metastasis, eribulin's apparent ability to reverse these aggressive characteristics may contribute to its clinical benefits (see, for example, Y. Funahashi et al. Eribulin mesylate reduces tumor microenvironment abnormality by vascular remodeling in preclinical human breast cancer models Cancer Sci. 105 (2014), 1334-1342). To date, no anti-cancer drugs having tumor vascular remodeling effects and anti-CAF activity have been reported.

Despite the progress made, additional compounds are needed to advance research and medical care in tumors and cancer.

Brief summary of the present invention

The present invention relates to a macrocyclic compound (eg, compound (1)) having tumor vascular remodeling effects and anti-CAF activity, and pharmaceutically acceptable salts thereof, and its radiolabelled derivatives, and pharmaceutical compositions thereof.

The present invention also provides methods for using Compound (1) for the treatment of cancer, methods for reversibly or irreversibly inhibiting mitosis in a cell, and methods for inhibiting tumor growth in vitro, in vivo, or in a subject. According to another aspect, the present invention relates to kits containing compound (1), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

According to one aspect, the present invention relates to a compound which is compound (1):

Compound (1) and pharmaceutically acceptable salts thereof; and its radiolabeled derivatives.

According to one aspect, the present invention relates to pharmaceutical compositions containing compound (1), or a pharmaceutically acceptable salt or radiolabelled derivative thereof. Pharmaceutical compositions may contain one or more pharmaceutically acceptable excipients or carriers. The pharmaceutical compositions may further comprise one or more additional therapeutic agents in combination, alternation, or other form of synchronized therapy to achieve the desired goal of treatment.

The present invention also relates to methods for producing compound (1) or intermediates thereof. Synthetic intermediates are also provided herein as part of the present invention.

Compound (1) was found to have a beneficial effect on tumor vascular remodeling and to have anti-CAF activity as demonstrated in the figures and examples. Accordingly, compound (1) has potential use in the treatment of cancer (e.g., head and neck squamous cell carcinoma (SCCHN), breast cancer, esophageal cancer, uterine cancer, ovarian cancer, colon and rectal cancer, endometrial cancer, gastric cancer, cancer small intestine, bladder cancer, sarcomas, rare forms of cancer).

According to another aspect, the present invention relates to methods for inhibiting the growth of any tumor or cancer that will respond to a compound with tumor vascular remodeling effects and/or anti-CAF activity, in a subject, typically a human, using compound (1), or a pharmaceutical formulation thereof. an acceptable salt, or a radiolabeled derivative thereof.

Compound (1), or a pharmaceutically acceptable salt thereof, or a radiolabelled derivative thereof, or a composition thereof, may be administered in combination with any other active agent that provides beneficial results for the patient. In certain embodiments, compound (1) is used in combination with an antibody (e.g., a monoclonal

- 2 040298 antibody). In one embodiment, compound (1) is used in combination, alternation, or other type of synchronized therapy with immunotherapy, such as 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, as described in more detail below.

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

According to another example, a method is provided for treating breast cancer in a subject in need, typically a human, comprising administering to said subject an effective amount of compound (1), or a pharmaceutically acceptable salt thereof, or a radiolabeled derivative thereof, or a composition thereof, in combination with therapy with mAb to HER2 (human epidermal growth factor receptor). In certain embodiments, the anti-HER2 (human epidermal growth factor receptor) mAb is trastuzumab. In other embodiments, compound (1) can be used to treat breast cancer in combination with conventional chemotherapy such as adriamycin, cyclophosphamide, taxol, etc., or an antiestrogen such as selective estrogen modulator (SERM), selective estrogen suppressor ( SERD), a partial or complete estrogen inhibitor (such as fulvestrant), or a CDK 4/6 inhibitor such as palbociclib (Pfizer).

Another aspect of the present invention relates to the compound (1), or a pharmaceutically acceptable salt thereof, or a radiolabeled derivative thereof, which may be in the form of a hydrate, solvate, polymorph, or composition thereof, in a kit, which may be a package with a dosage form. The kits described herein may include a single dose or multiple doses of a compound or pharmaceutical composition thereof. A kit according to the present invention may include instructions for use of the intended therapeutic dosage forms (eg, instructions for use of the compound or pharmaceutical composition included in the kit).

Thus, the present invention includes at least the following features:

(i) compound (1) or a pharmaceutically acceptable salt or radiolabeled derivative thereof, which may optionally be in the form of a hydrate, solvate or polymorph;

(ii) a method of treatment which comprises administering to a subject such as a human an effective amount of compound (1) or a pharmaceutically acceptable salt or radiolabelled derivative thereof, which may optionally be in the form of a hydrate, solvate or polymorph, for the treatment of the following: head cancer and neck (eg, squamous cell carcinoma of the head and neck (SCCHN), adenoid cystic carcinoma), breast cancer (eg, HER2-negative breast cancer, triple-negative breast cancer), esophageal cancer (eg, adenocarcinoma of the esophagus), uterine cancer ( eg, uterine sarcoma), ovarian cancer, colon and rectal cancer, sarcoma (eg, synovial sarcoma, angiosarcoma, soft tissue sarcoma, fibrosarcoma, uterine sarcoma), bladder cancer (eg, urothelial cancer), gastric cancer, small bowel cancer (eg, adenocarcinoma of the small intestine), endometrial cancer, or a rare form of cancer;

(iii) a method of treatment which comprises administering to a subject, such as a human, an effective amount of compound (1) or a pharmaceutically acceptable salt or radiolabeled derivative thereof, which may optionally be in the form of a hydrate, solvate or polymorph, for use in the treatment of a medical disorder such as a cancer or tumor that responds to vascular remodeling effects and/or anti-CAF activity;

(iv) a compound (1) or a pharmaceutically acceptable salt or radiolabeled derivative thereof, which may optionally be in the form of a hydrate, solvate or polymorph, for use in the treatment of the following: head and neck squamous cell carcinoma (SCCHN), breast cancer, cancer esophagus, uterine cancer, ovarian cancer, colon and rectal cancer, sarcoma, bladder cancer, stomach cancer, small intestine cancer, endometrial cancer, or a rare form of cancer;

(v) a compound (1) or a pharmaceutically acceptable salt or radiolabelled derivative thereof, which may optionally be in the form of a hydrate, solvate or polymorph, for use in the treatment of a medical disorder such as cancer or tumor that responds to the effects of vascular remodeling and /or activity against CAF;

(vi) a deuterated derivative of compound (1);

(vii) a process for the manufacture of a medicament intended for therapeutic use in the treatment or prevention of disorders such as cancer or tumor that respond to vascular remodeling effects and/or anti-CAF activity, characterized in that compound (1) or a pharmaceutically acceptable thereof salt or a radioactively labeled derivative that

- 3 040298 which may optionally be in the form of a hydrate, solvate or polymorph described above, or an embodiment of the active compound used in the manufacture;

(viii) compound (1) or a pharmaceutically acceptable salt or radiolabeled derivative thereof, in substantially pure form (eg, at least 90% or 95%);

(ix) a pharmaceutically acceptable composition of compound (1) or a pharmaceutically acceptable salt or radiolabeled derivative thereof, which may optionally 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 radiolabeled derivative thereof, which may optionally be in the form of a hydrate, solvate or polymorph, optionally in a pharmaceutically acceptable carrier or excipient;

(xi) a compound (1) or a pharmaceutically acceptable salt or radiolabelled derivative thereof, for the treatment of the disorder described herein, characterized in that it acts through a mechanism of action other than those of vascular remodeling and/or anti-CAF activity; and (xii) methods for preparing the compounds described herein and synthetic intermediates.

Brief description of graphic materials

The accompanying drawings, which are included in and form part of this specification, illustrate several embodiments of the present invention and, together with the description, provide non-limiting examples of the invention.

In FIG. 1 shows the antitumor effects of compound (1) in a subcutaneous xenograft model of FaDu (head and neck cancer) in mice as monotherapy as described in Pharmacological Test Example 4.

In FIG. 2 shows the antitumor activity of compound (1) against subcutaneous xenograft model OSC-19 (head and neck cancer) in mice as monotherapy as described in Pharmacological Test Example 5.

In FIG. 3 shows the antitumor activity of compound (1) against the subcutaneous xenograft model HCC-1806 (breast cancer) in mice as monotherapy as described in Pharmacological Test Example 6.

In FIG. 4 shows the antitumor effects of compound (1) in a subcutaneous FaDu xenograft model in combination with cetuximab in mice as described in Pharmacological Test Example 7.

In FIG. 5 shows the antitumor activity of compound (1) in a subcutaneous xenograft model of KPL-4 (breast cancer) in combination with trastuzumab in mice as described in Pharmacological Test Example 8.

In FIG. 6A-6B show the antitumor effect of compound (1) in an orthotopic transplantation model in HSC-2 mice. In FIG. 6A: Athymic mice were implanted with luciferase-transduced HSC-2 (1x106 cells per spot) in the tongue. The amount of luciferase-transduced HSC-2 was analyzed using an in vivo imaging system (IVIS). The data shows the levels of bioluminescence in the tongue of each mouse. In FIG. 6B: Representative bioluminescence image of 16 mice. CDDP, CTX, CDDP+CTX were used as comparators currently used in the treatment of patients with SCCHN cancer. CDDP=cisplatin, CTX=cetuximab.

In FIG. 7A-7B show the survival benefit of compound (1) in combination with cetuximab in an orthotopic HSC-2 transplantation model in mice. In FIG. 7A: Athymic mice were implanted with luciferase-transduced HSC-2 (1x106 cells per spot) in the tongue. The data show a survival curve up to 100 days after drug treatment (n=16). *P<0.0001 compared to Compound (1) or CTX alone (Log Rank Test (Mantel-Cox). Figure 7B: The amount of luciferase-transduced HSC-2 was analyzed using an in vivo imaging system (IVIS). Bioluminescence images 10 surviving mice per group treated with compound (1)+CTX combination on day 100. RBW=relative α body weight CDDP=cisplatin, CTX=cetuximab.

In FIG. 8A-8B show the antitumor effect of compound (1) in combination with radiation therapy in a xenograft model in FaDu mice. In FIG. 8A: Athymic mice were implanted subcutaneously with luciferase-transduced FaDu (5x106 cells per spot) in the right thigh. 13 days after inoculation, mice were randomized into groups (n=6) and intravenously injected with compound (1) at a dose of 90 μg/kg on day 1 and day 8 with or without RT (radiotherapy) of 18 Gy on day 4 and day 11. The amount of luciferase-transduced FaDu was analyzed using an in vivo imaging system (IVIS). The data show the average relative

- 4 040298 level of bioluminescence on day 1 and SEM (n=6). SEM=root mean square error of the mean.

*P<0.05 vs. untreated on day 29 (independent sample t-test). In FIG. 8B: Representative bioluminescence images of 6 mice from each group on day 29.

RT=radiotherapy.

In FIG. 9 shows the antitumor activities of compound (1) in combination with an anti-mPD-1 antibody. A subcutaneous syngeneic mouse model of CT26 (colon carcinoma) was treated with compound (1) and an anti-mPD-1 antibody in the Q7D and twice-weekly regimens, respectively, for 3 weeks. Results are shown as mean±SEM of tumor volumes (mm ³ ) (n=8).

In FIG. 10A shows a cell-free tubulin polymerization assay. Compound (1) has tubulin polymerization inhibitory activity.

In FIG. 10B shows an analysis of microtubule dynamics. Compound (1) also has microtubule dynamics inhibitory activity.

In FIG. 11 shows that compound (1) is a highly active antiproliferative agent against esophageal cancer cell lines (OE21, OE33 and TE-8) and uterine cancer (MES-SA, MES-SA/Dx5-Rx1).

In FIG. 12 shows that compound (1) has strong antitumor activity in subcutaneous xenograft models of breast cancer and ovarian cancer (KPL-4 and COLO-704, respectively) as monotherapy.

In FIG. 13 shows the effect of compound (1) on the tumor microenvironment. As shown, the compound (1) increases the density of the microvasculature. *P<0.05, **P<0.01, ****p<0.0001 compared with no treatment (Dunnett multiple comparison test).

In FIG. 14 shows the effect of compound (1) on the tumor microenvironment. As shown, compound (1) reduces the number of a-SMA-positive CAF.

In FIG. 15 shows that Compound (1) reduces CAF ECM proteins in a subcutaneous FaDu xenograft model. FaDu xenograft tumors were harvested on day 6 after a single administration of compound (1) 180 μg/kg + cetuximab on day 1.

In FIG. 16 shows that compound (1) exhibits a dose-dependent co-effect with cetuximab in a subcutaneous FaDu xenograft model. Single dose, n=6. Compound (1) and cetuximab (CTX) were administered on day 1 in a FaDu xenograft model.

In FIG. 17 shows antitumor effects in mice xenograft models of soft tissue sarcoma as monotherapy. MES-SA (human uterine sarcoma), HT-1080 (human fibrosarcoma) and CTG-2041 (human angiosarcoma) are shown.

In FIG. 18 shows antitumor effects in xenograft models of endometrial cancer in mice as monotherapy. HEC-108 and AN3CA (endometrial cancer) are shown.

Definitions.

As used herein, the term salt refers to any and all salts and encompasses pharmaceutically acceptable salts. The term pharmaceutically acceptable salt refers to those salts which, within the framework of sound medical judgment, are suitable for use in contact with tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe in detail pharmaceutically acceptable salts in J. Pharmaceutical Sciences, 1977, 66, 1-19, 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 amino salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate , heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate , phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts and the like. Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts and N ⁺ (C1_ ₄ alkyl) ₄ ^{- salts} . Exemplary alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Additional pharmaceutically acceptable salts include, when appropriate, non-toxic

- 5 040298 ammonium, quaternary ammonium and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkylsulfonate and arylsulfonate. In addition, compound (1) is provided as a free base and can be administered as a free base.

It should also be understood that compounds that have the same molecular formula but differ either in the nature of the binding sequence of their atoms or in the arrangement of their atoms in space are called isomers. Isomers that differ in the arrangement of their atoms in space are called stereoisomers.

The terms composition and composition are used interchangeably.

The subject to whom administration is intended refers to a human (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., an infant, child, or adolescent) or an adult subject (e.g., a young adult, middle-aged adult, or elderly adult)) or non-human animal. In some embodiments, the non-human animal is a mammal (e.g., a primate (e.g., cynomolgus or rhesus monkey), commercially important mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog) or a bird (e.g. a commercially important bird such as a chicken, duck, goose or turkey)). In certain embodiments, the non-human animal is a fish, reptile, or amphibian. An animal other than a human may be male or female at any stage of development. The non-human animal may be a transgenic animal or a genetically engineered animal. The term patient refers to a person in need of treatment for a disease.

The term administer, administer, or administer refers to the implantation, absorption, ingestion, injection, inhalation, or other administration of a compound described herein, or a composition thereof, to a subject or onto a subject's body.

The terms treatment, treat, and treatment refer to reversing, alleviating, delaying the onset, or suppressing the progression of the disease described herein. In some embodiments, treatment may be administered after one or more signs or symptoms of a disease have developed or been detected. In other embodiments, treatment may be administered in the absence of signs or symptoms of a disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms. Treatment may also be continued after symptoms have resolved, for example to delay or prevent relapse.

An effective amount of a compound described herein refers to an amount sufficient to elicit the desired biological response. An effective amount of a compound described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the route of administration, and the age and health of the subject. In certain embodiments, the effective amount is a therapeutically effective amount. Alternatively, in a particular method or application, the present invention may be used, if indicated and effective, as a prophylactic treatment. In certain embodiments, an effective amount is the amount of a compound described herein in a single dose. In certain embodiments, an effective amount is the combined amounts of a compound described herein at multiple doses.

A therapeutically effective amount of a compound described herein is an amount sufficient to provide a therapeutic effect in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic effect in the treatment of a condition. The term "therapeutically effective amount" may encompass an amount that improves the overall therapy, reduces or eliminates the symptoms, signs, or causes of a condition, and/or enhances the therapeutic efficacy of another therapeutic agent. In certain embodiments, a therapeutically effective amount is an amount sufficient to treat any disease or condition described.

As used herein, the terms inhibition, inhibition, inhibit and inhibitor, and the like, refer to the ability of a compound to reduce, slow down, stop, or prevent the activity of a biological process (eg, tumor growth). In certain embodiments, the inhibition is about 45-50%. In certain embodiments, 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 %.

The terms neoplasm and tumor are used interchangeably herein and refer to an abnormal mass of tissue in which the growth of the mass is superior to and not coordinated with the growth of normal tissue. A neoplasm or tumor may be benign or malignant depending on the following characteristics: degree of cell differentiation (including morphology and functionality), growth rate, local invasion and metastasis. A benign neoplasm is usually well differentiated, characteristically slower growing than a malignant neoplasm, and remains localized at the site of origin. In addition, a benign neoplasm does not have the ability to infiltrate, invade, or metastasize to distant sites. In contrast, a malignant neoplasm is usually poorly differentiated (anaplasia) and is characterized by characteristically rapid growth accompanied by progressive infiltration, invasion, and destruction of surrounding tissues. In addition, a malignant neoplasm, as a rule, has the ability to metastasize to distant sites. The term metastasis, metastatic, or metastasize refers to the spread or migration of cancer cells from a primary or parent tumor to another organ or tissue and is generally defined by the presence of a secondary tumor or secondary cell mass of the tissue type of the primary or parent tumor rather than the tissue type of the organ or tissue. tissue in which a secondary (metastatic) tumor is located

The term cancer refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues.

The term rare cancer refers to forms of cancer that occur in a relatively small number of patients. Rare cancers include, without limitation, sarcomas (eg, soft tissue sarcoma, liposarcoma, uterine sarcomas, leiomyosarcoma, myxofibrosarcoma, osteosarcoma, angiosarcoma, Ewing's sarcoma, synovial sarcoma, rhabdomyosarcoma), malignant lymphomas, thymic cancer (eg, thymomas), mesothelioma , gastrointestinal stromal tumors (GIST), neuroendocrine cancer, eye cancer, brain tumors, bone tumors, soft tissue tumors, skin cancer and germ cell tumors.

The term anticancer agent refers to any therapeutic agent that is useful for treating cancer in a subject (eg, inhibiting cancer or tumor growth in a subject). Anticancer agents include biotherapeutic anticancer agents as well as chemotherapeutic agents.

Detailed Disclosure of Certain Embodiments The present invention is described in detail below with reference to embodiments of the present invention and the like. The present invention relates to compounds (eg compound (1)), and pharmaceutically acceptable salts or radiolabelled derivatives thereof, and pharmaceutical compositions thereof. The present invention also provides methods for inhibiting tumor growth and/or treating cancer in a subject, comprising administering to the subject an effective amount of a compound or composition provided herein. The compound or composition can be administered as monotherapy or in combination with other therapy as described herein. According to another aspect, the present invention relates to processes for the preparation of compound (1) and synthetic intermediates useful for this purpose.

The present invention includes a connection with the structure:

Compound (1) or a pharmaceutically acceptable salt or radiolabeled derivative thereof, which may optionally be in the form of a hydrate, solvate or polymorph, optionally in a pharmaceutically acceptable carrier or excipient.

Compound (1) may exist as a crystalline polymorph, and the compound of the present invention may be in any one of single crystal forms or a mixture of two or more crystalline forms. Compound (1) may be in an amorphous form, or may be an anhydride or a solvate such as a hydrate.

The present invention includes radiolabeled derivatives of compound (1) and pharmaceutically acceptable salts thereof. The radiolabeled compound is equivalent to the compound (1) except that one or more atoms are replaced by atom(s) which have an atomic mass or mass number different from those normally found in nature. Examples of isotopes that can be included in the compound of the present invention include the isotopes of hydrogen, carbon, nitrogen, oxygen,

- 7 040298 phosphorus, fluorine, iodine, bromine and chlorine, such as ² H, ³ H, ⁿ C, ¹³ C, ¹⁴ C, ¹⁸ F, ³⁵ S, ¹²³ I and ¹²⁵ I.

A radiolabelled compound, such as one in which a radioactive isotope, such as ³ H and/or ¹⁴ C, is incorporated, is useful for drug and/or matrix tissue distribution analysis. The ³ H and ¹⁴ C isotopes are considered useful because these isotopes can be easily obtained and detected. The isotopes 11C and ^18F are applicable in PET (positron emission tomography). The ¹²⁵ I isotope is considered applicable in SPECT (single photon emission computed tomography) and may be applicable for brain imaging. Substitution with a heavier isotope, such as ² H, causes, due to its higher metabolic stability, some processing advantages, such as an increase in in vivo half-life or a reduction in the required dose, and is therefore considered applicable under the given circumstances. A radiolabeled compound can be similarly prepared using a readily available radiolabelled reagent in place of a non-radiolabeled reagent and by following the methods disclosed in the Schemes and/or Examples described below.

Compound (1) can be used as a chemical probe to capture a target protein of a low molecular weight biologically active compound. In particular, the compound of the present invention can be transformed into an affinity chromatography probe, a photoaffinity probe, or the like by introducing a labeling group, a linker, or the like into a moiety other than the structural moiety necessary to express the activity of the compound by the method described in J. Mass Spectrum. soc. Jpn., Vol. 51, No. 5, 2003, p. 492-498, WO 2007/139149 or the like.

Examples of a labeling group, a linker or the like used in such a chemical probe include groups belonging to the following groups (1) to (5). (1) Protein labeling groups such as photoaffinity labeling groups (such as benzoyl group, benzophenone group, azide group, carbonyl azide group, diaziridine group, enone group, diazo group and nitro group) and chemical affinity groups (such as ketone group in which the alpha carbon atom is substituted with a halogen atom, a carbamoyl group, an ester group, an alkylthio group, a Michael acceptor of an α,P-unsaturated ketone, an ester or the like, and an oxirane group); (2) cleavable linkers such as SS, O-Si-O, a monosaccharide (such as a glucose or galactose group) and a disaccharide (such as lactose), and oligopeptide linkers that can be cleaved by an enzymatic reaction; (3) Fishhook label groups such as biotin and 3-(4,4difluoro-5,7-dimethyl-4H-3a,4a-diaza-4-bora-s-indacen-3-yl)propionyl group ; (4) radioactive labeling groups such as ¹²⁵ I, ³² P, ³ H and ¹⁴ C; fluorescent labeling groups such as fluorescein, rhodamine, dansyl, umbelliferone, 7-nitrofurazanil and 3-(4,4-difluoro-5,7-dimethyl-4H-3a,4a-diaza-4-boras-indacen-3-yl )propionylα group; chemiluminescent groups such as luciferin and luminol; and markers capable of detecting heavy metal ions such as lanthanide metal ions and radium ions; and (5) groups to be associated with a solid phase carrier such as glass beads, glass layer, microtiter plate, agarose beads, agarose layer, polystyrene beads, polystyrene layer, nylon beads and nylon layer.

A probe obtained by introducing a labeling group or the like selected from the above-described groups (1) to (5) into the compound of the present invention by the method described in any of the above references or the like can be used as a chemical probe for identifying a marker. protein applicable to the study of a new potential drug target.

Examples of the term salt as used herein include salts with inorganic acids, salts with organic acids, and salts with acidic amino acids, and pharmaceutically acceptable salts are particularly preferred. In addition, the salt of a compound of the present invention includes an anhydride of a pharmaceutically acceptable salt thereof and a solvate of a pharmaceutically acceptable salt such as a hydrate. Preferable examples of the inorganic acid salt include salts with hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and preferred examples of the organic acid salt 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 and the like. Preferred examples of the acidic amino acid salt include salts with aspartic acid and glutamic acid and the like.

When the compound (1) of the present invention is obtained as a salt of the compound (1) 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.

Pharmaceutical compositions, kits and administration.

The present invention relates to pharmaceutical compositions containing the compound (1),

- 8 040298 or its pharmaceutically acceptable salt or radioactively labeled derivative, and a pharmaceutically acceptable excipient. In certain embodiments, a compound described herein, or a pharmaceutically acceptable salt or radiolabeled derivative thereof, is provided in an effective amount in a pharmaceutical composition (eg, a therapeutically effective amount).

The pharmaceutical compositions described herein can be obtained by any method known in the field of pharmacology. Typically, such preparations involve bringing compound (1) (i.e., the active ingredient) into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, imparting the form and/or packaging of the product into the desired single or multi-dose unit. The pharmaceutical composition of the present invention can be prepared according to a known method, such as the method described in the General Rules for Preparations of Japanese Pharmacopoeia 16th Ed., USP and European Pharmacopoeia 9th Ed. The pharmaceutical composition according to the present invention can be administered to patients in an appropriate manner depending on the dosage form.

Pharmaceutical compositions can be prepared, packaged and/or sold in bulk, as a single unit dose and/or as a plurality of unit dose units. A unit dose is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of active ingredient is generally equal to the dosage of the active ingredient that is administered to the subject and/or a convenient fraction of such dosage, such as half or a third of such dosage.

The relative amounts of the active ingredient, pharmaceutically acceptable excipient and/or any additional ingredients in the pharmaceutical composition described herein will vary depending on the individual, size and/or condition of the subject being treated, and further depending on the route , which should enter the composition. The composition may contain from 0.1 to 100% w/w of the active ingredient.

Pharmaceutically acceptable excipients used in the manufacture of the intended pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surfactants and/or emulsifiers, disintegrating agents, binders, preservatives, buffering agents, lubricants and /or oils. Auxiliary substances such as cocoa butter and suppository waxes, coloring agents, coating agents, sweeteners, flavors and fragrances may also be present in the composition.

The compound provided herein is typically formulated in unit dosage form for ease of administration and dosage uniformity. However, it should be understood that the total daily use of the compositions described herein will be determined by the physician within the reasonable medical judgment. The specific level of therapeutically effective dose for any particular subject or organism will depend on a variety of factors, including the following: the disease being treated and the severity of the disorder; the activity of the particular active ingredient used; the particular composition used; age, body weight, general health, sex and diet of the subject; the time of administration, route of administration and rate of excretion of the particular active ingredient used; duration of treatment; medicinal products used in combination or simultaneously with the specific active ingredient used; and similar factors well known in the medical field.

The compound of the present invention (compound (1)) and its compositions provided herein can be administered by any route, including the following: enteral (e.g., oral), parenteral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (eg, via powders, ointments, creams and/or drops), transmucosal, nasal, buccal, sublingual; by intratracheal instillation, bronchial instillation and/or inhalation; and/or as an oral spray, nasal spray and/or aerosol. Specific routes contemplated are oral administration, intravenous administration (eg, systemic intravenous injection), regional administration via the bloodstream and/or lymphatics, and/or direct administration to the affected area. In general, the most appropriate route of administration will depend on a variety of factors, including the nature of the agent (eg, its stability in the gastrointestinal environment) and/or the condition of the subject (eg, whether the subject is able to tolerate oral administration).

The exact amount of Compound (1) required to achieve an effective amount will vary from subject to subject, depending on, for example, the species, age, and general condition of the subject, the severity of the side effects or disorder, the characteristics of the particular compound, the route of administration, and the like. . An effective amount may be included in a single dose (eg, a single oral dose) or multiple doses (eg, multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, any two of the multiple doses include different or substantially the same amounts of a compound 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 a subject or applying multiple doses to a tissue or cell can be, in non-limiting examples, the following: three doses per day, two doses per day, one dose per day, one dose every other day, one dose every three days, one dose every week, one dose every two weeks, one dose every three weeks or one dose every four weeks, or even slow controlled dose delivery over a selected time period using drug delivery devices. In certain embodiments, the frequency of administering multiple doses to a subject or applying multiple doses to a tissue or cell is one dose per day. In certain embodiments, the frequency of administering multiple doses to a subject or applying multiple doses to a tissue or cell is two doses per day. In certain embodiments, the frequency of administering multiple doses to a subject or applying multiple doses to a tissue or cell is three doses per day. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the duration between the first dose and the last dose of the multiple doses is about or at least one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years or the lifespan of the subject, tissue or cells. In certain embodiments, the duration between the first dose and the last dose of multiple doses is about or at least three months, six months, or one year. In certain embodiments, the duration between the first dose and the last dose of multiple doses is the lifetime of the subject, tissue, or cell. In certain embodiments, the dose (e.g., a single dose or any of multiple doses) described herein independently includes 0.001 to 0.01 mg/kg, 0.01 to 0.1 mg/kg, or 0.1 and 1 mg/kg inclusive of compound (1). Examples are dosage forms containing 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 salt thereof, in dosage form.

The dosage ranges described herein provide guidance for administering the intended pharmaceutical compositions to an adult human. The amount to be administered to, for example, a child or adolescent may be determined by the practitioner or one of skill in the art, and may be lower than or the same as the amount administered to an adult.

The disclosure of the present invention also covers kits (eg, pharmaceutical packages). Provided kits may contain a pharmaceutical composition or compound (1) and a container (eg vial, ampoule, bottle, syringe and/or dispenser pack or other suitable container). In some embodiments, provided kits may optionally further include a second container containing a pharmaceutical diluent or suspension of 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 kit described herein may include one or more of the additional pharmaceutical agents described herein as a separate composition.

Methods of treatment and application.

As shown herein, compound (1) has significant tumor vascular remodeling effects and anti-CAF activity, and therefore has potential use in cancer treatment and/or tumor growth inhibition.

Provided herein is a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of compound (1), or a pharmaceutically acceptable salt or radiolabeled derivative thereof, or a pharmaceutical composition thereof. The present invention also relates to compound (1), or a pharmaceutically acceptable salt or radiolabeled derivative thereof, or a pharmaceutical composition thereof, for use in the treatment of cancer in a subject. The present invention also relates to the use of compound (1), or a pharmaceutically acceptable salt or a radiolabeled derivative thereof, or a pharmaceutical composition thereof, for the manufacture of a medicament for the treatment of cancer.

Also provided herein is a method for inhibiting tumor growth in a subject, the method comprising administering compound (1), or a pharmaceutically acceptable salt or radiolabeled derivative thereof, or a pharmaceutical composition thereof, to the subject.

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Also provided herein is a compound (1), or a pharmaceutically acceptable salt or a radiolabeled derivative thereof, or a pharmaceutical composition thereof, for use in inhibiting tumor growth in a subject. The present invention also relates to the use of compound (1), or a pharmaceutically acceptable salt or a radiolabeled derivative thereof, or a pharmaceutical composition thereof, for the manufacture of a drug for inhibiting tumor growth.

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, colon and rectal cancer, endometrial cancer, gastric cancer, small intestine cancer, bladder cancer or sarcoma.

In certain embodiments of the methods and uses provided herein, the cancer is head and neck cancer (eg, head and neck squamous cell carcinoma, oral cancer, throat 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 (eg, 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 colon and rectal cancer (eg, colon carcinoma). In certain embodiments, the cancer is a colon carcinoma. In certain embodiments, the cancer is esophageal cancer (eg, esophageal adenocarcinoma). In certain embodiments, the cancer is esophageal adenocarcinoma. In certain embodiments, the cancer is uterine cancer (eg, uterine sarcoma). In certain embodiments, the cancer is a uterine sarcoma. In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is a sarcoma (eg, uterine sarcoma, fibrosarcoma, angiosarcoma, synovial sarcoma, soft tissue carcinoma). In certain embodiments, the cancer is a fibrosarcoma. In certain embodiments, the cancer is an angiosarcoma. In certain embodiments, the cancer is a synovial sarcoma. In certain embodiments, the cancer is a soft tissue carcinoma. In certain embodiments, the cancer is stomach cancer. In certain embodiments, the cancer is colon cancer (eg, small bowel cancer, small bowel adenocarcinoma). In certain embodiments, the cancer is small bowel cancer. In certain embodiments, the cancer is an adenocarcinoma of the small intestine. In certain embodiments, the cancer is bladder cancer (eg, 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 form of cancer.

Combined therapy.

In addition to administration as monotherapy, compound (1) may be administered in combination with other therapeutic agents or treatments. In certain embodiments, the additional therapeutic agent is an antibody. In certain embodiments, the additional therapeutic agent is a monoclonal antibody. The compound of the present invention may be administered in combination with another therapeutic agent such as anti-EGFR therapy, anti-HER2 therapy, anti-PD-1 therapy, anti-PD-LI therapy or radiation therapy.

In certain embodiments, compound (1), or a pharmaceutically acceptable salt or radiolabeled 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 for treating squamous cell carcinoma of the head and neck (SCCHN) in a subject, comprising administering to said subject compound (1), or a pharmaceutically acceptable salt or radiolabeled derivative thereof, or a pharmaceutical composition thereof, in combination with therapy with mAb to EGFR (epidermal growth factor receptor). In certain embodiments, the anti-EGFR mAb is cetuximab (CTX).

In certain embodiments, compound (1), or a pharmaceutically acceptable salt or radiolabeled derivative thereof, or a pharmaceutical composition thereof, is administered in combination with an anti-HER2 therapy (e.g., an anti-HER2 monoclonal antibody (mAb),

- 11 040298 such as trastuzumab). In certain embodiments, the aHTu-HER2 therapy is an anti-HER2 antibody. For example, provided herein is a method for treating breast cancer in a subject in need thereof, comprising administering to said subject compound (1), or a pharmaceutically acceptable salt or radiolabelled derivative thereof, or a composition thereof, in combination with therapy with an anti-HER2 mAb. (human epidermal growth factor receptor). In certain embodiments, the anti-HER2 mAb is trastuzumab.

In certain embodiments, compound (1), or a pharmaceutically acceptable salt or radiolabeled 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 monoclonal antibody or to PD-L1). In certain embodiments, the anti-PD-1 or anti-PD-L1 therapy is an antibody. For example, provided herein is a method for treating colon and rectal cancer in a subject in need thereof, comprising administering to said subject compound (1), or a pharmaceutically acceptable salt or radiolabeled derivative thereof, or a composition thereof, in combination with an anti-PD- 1 or anti-PD-L1 therapy (eg mAb therapy).

In certain embodiments, compound (1), or a pharmaceutically acceptable salt or radiolabeled derivative thereof, or a pharmaceutical composition thereof, is used in combination with radiotherapy (RT). In certain embodiments, the compound is administered in combination with an operation.

Examples

Synthesis of compound (1).

General procedures and methods.

The compound according to the present invention can be obtained using the methods described in the examples below. However, these examples are provided for illustrative purposes only, and the compound of the present invention is not limited in any way to the specific examples mentioned below.

In the examples, unless otherwise noted, the silica gel for purification using silica gel column chromatography was a Hi-Flash™ column (silica gel, 30 µm 60 A or 40 µm 60 A, Yamazen Corporation), silica gel for purification using silica gel column chromatography. silica gel NH was Chromatorex NH silica gel (Fuji Silysia Chemical LTD). Analytical thin layer chromatography (TLC) was performed using TLC 60 F ₂₅₄ silica gel with a layer thickness of 0.25 mm (Merck KGaA), or TLC NH Chromatorex F ₂₅₄ silica gel with a layer thickness of 0.25 mm (Fuji Silysia Chemical LTD). TLC plates were visualized by staining with p-anisaldehyde stain, phosphomolybdic acid stain, or Hanessian stain.

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

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 Avarice (600 MHz) spectrometer. Chemical shifts are presented in parts per million (ppm). For 1H NMR spectra (CDCl ₃ , C6D6 and/or CD3OD), the residual solvent peak was used as internal standard (7.27 ppm in CDCl ₃ ; 7.16 ppm in C6D6, 3.31 ppm . in CD3OD).

Analytical mass spectra (MS) results were obtained using a UPLC Waters Acquity equipped with a single quadrupole detector (SQ Detector 2) or LTQ Orbitrap XL™ (Thermoscientific).

High performance liquid chromatography (HPLC) was performed using a Shimadzu LC10AD on a UV spectrophotometric detector (200 nm, Shimadzu SPD-10A).

Abbreviations used herein: AIBN: 2,2'-azobis(isobutyronitrile); 9-BBN: 9-borabicyclo[3.3.1]nonane; Bu ₃ SnH: tri-normal-butyltin hydride; (+)-CSA: (1S)-(+)-10camphorsulfonic acid; DMAP: 4-dimethylaminopyridine; DCM: dichloromethane; DDQ: 2,3-dichloro5,6-dicyano-1,4-benzoquinone; DIBAL: diisobutylaluminum hydride; DMF: N,N-dimethylformamide; DMSO: dimethyl sulfoxide; Et ₃ N: triethylamine; EtOAc: ethyl acetate; HF-pyridine: pyridine hydrogen fluoride; HPLC: high performance liquid chromatography; IPA: isopropyl alcohol; MeCN: acetonitrile; MeOH: methanol; MPM: para-methoxybenzyl; PPh ₃ : triphenylphosphine; t-BuOH: tert-butyl alcohol; t-BuLi: t-butyllithium; TBME: methyl tert-butyl ether; TBAF: tetrabutylammonium fluoride; TBS: t-butyldimethylsilyl; THF: tetrahydrofuran; TMS: trimethylsilyl; Ts: p-toluenesulfonyl.

The synthetic intermediates disclosed herein are intended to be part of the present invention.

Scheme A. Preparation of compound A-7.

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Example 1

(4aR,5aS,6R,8aS,9aR)-2,2-di-tert-butyl-6-methyloctahydrofuro[2',3':5,6]pyrano[3,2-d][1,3,2]dioxasilin -7-ol.

In a nitrogen atmosphere to a solution of compound A-1: (4aR,5aS,6R,8aS,9aR)-2,2-di-tert-butyl-6methylhexahydrofuro[2',3':5,6]pyrano[3,2- d][1,3,2]dioxasilin-7(8aH)-one (A-1 18.5 g, 54.0 mmol) prepared by the method described in Organic Letters (2009), 11(2), 409-412 (CAS no: 1095280-048) in toluene (275 ml) at -78°C, DIBAL (70.2 ml, 70.2 mmol, 1.0 M toluene solution) was added over 30 minutes. Then the reaction mixture was stirred at -78°C. After 90 minutes, the reaction was carefully quenched with MeOH (4.37 ml) at -78°C, then the cooling bath was removed. A saturated solution of potassium sodium tartrate tetrahydrate (300 ml) was added to the reaction mixture, stirring was continued for 2 hours at room temperature. The reaction mixture was poured into a separating funnel, then the layers were separated. The aqueous layer was extracted with EtOAc (300 ml). The combined organic extracts were washed with brine (300 ml), dried over Na ₂ SO ₄ , filtered, concentrated under reduced pressure. The crude lactol was used for 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 ]dioxasilin7-ol (compound A-2).

- 13 040298

Under nitrogen atmosphere, to a suspension of methyltriphenylphosphonium bromide (73.30 g, 205.2 mmol) in THF (200 ml), potassium tert-butoxide (17.27 g, 153.9 mmol) was added at -5°C over 10 min, and then stirred for 60 min at -5°C. A solution of the crude lactol described in Example 1 in THF (40 ml) was transferred to the reaction mixture at -5°C for 10 min, then stirred at -5°C for 1 h, at room temperature for 1 h. the 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 brine (400 ml), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was suspended with heptane/EtOAc=1/1 (100 ml). The resulting suspension was filtered, washed with heptane/EtOAc=1/1 (100 ml) to remove the triphenylphosphine derived material. The filtrate was then concentrated under reduced pressure. Flash chromatography of the residue on silica gel (400 g, silica gel 60, spherical, 40-50 µm, Kanto Chemical) using 0-20% EtOAc/heptane gave the title compound (compound A-2, 16.7 g, 90 % exit.

¹ H NMR (400 MHz, chloroform-d) δ ppm 1.03 (d, J=6.8 Hz, 3H) 1.05 (s, 9H) 1.07 (s, 9H) 1.75 (dt, J=14.5, 3.0 Hz, 1H) 2.37 (dt, J=14.5, 2.9Hz, 1H) 2.65-2.76 (m, 1H) 3.03 (dd, J=9.8, 1.0Hz, 1H) 3.31 (m, 1H) 3.69 (d, J=15.0 Hz, 1H) 3.75-3.79 (m, 1H) 4.16-4.31 (m, 2H) 4.41 (t, J=2.9 Hz, 1H) 4.95-5.09 (m, 2H) 6.02 (ddd, J=17.3, 10.5, 6.3 Hz, 1H).

Example 3

(4aR,6S,7S,8aR)-6-((S)-but-3-en-2-yl)-2,2-di-tert-butyl-7-((tert-butyldimethylsilyl)oxy)hexahydropurano[ 3,2-d][1,3,2]dioxasilin (compound A-3).

A-2 A-3

In a nitrogen atmosphere to a solution of compound A-2: (4aR,6S,7S,8aR)-6-((S)-but-3-en-2-yl)-2,2-du-tert-butylhexahydropyrano[3,2- d][1,3,2]dioxasilin-7-ol (9.85 g, 28.8 mmol) described in Example 2, 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 min, then at room temperature for 2 h. The reaction mixture was diluted with diethyl ether. The organic layer was washed with 0.5 N. an aqueous solution of HCl, a saturated aqueous solution of NaHCO ₃ and then brine. The combined organic layers were dried over MgSO ₄ , filtered (small amount of SiO ₂ ) and concentrated under reduced pressure. Flash chromatography of the residue on silica gel using 0-15% EtOAc/heptane gave the title compound (compound A-3, 12.0 g, 91% yield).

¹ H NMR (500 MHz, chloroform-d) δ ppm 0.10 (s, 3H) 0.19 (s, 3H) 0.91 (s, 9H) 0.96 (d, J=6.3 Hz, 3H) 1.02 (s, 9H) 1.06 (s, 9H) 1.73 (dt, J=15.0, 4.0 Hz, 1H) 2.26 (dt, J=15.0, 2.5 Hz, 1H) 2.66-2.74 (m, 1H) 2.95 (dd, J=9.5, 2.2 Hz, 1H) 3.17 (m, 1H) 3.81-3.84 (m, 1H) 4.12-4, 22 (m, 2H) 4.24 (t, J=2.7 Hz, 1H) 4.93-5.06 (m, 2H) 6.08 (ddd, J=17.3, 10.5, 6 .3 Hz, 1H).

Example 4

(2R,3R,5S,6S)-6-((S)-but-3-en-2-yl)-5-((tert-butyldimethylsilyl)oxu)-2-(hydroxymethyl)tetrahydro-2H-pyran- 3-ol (compound A-4).

In a nitrogen atmosphere 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)oxu)hexahydropyrano[3,2-d][1,3,2]dioxaculine (12 g, 26.3 mmol) described in Example 3 in MeCN (120 ml) and DCM (40 ml) at -10°C was added a pre-mixed solution of HP-pyridine (4.0 ml) and pyridine (20 ml) in 20 ml MeCN. The reaction mixture was stirred at -10° C. for 15 min, then at room temperature for 1 h. The reaction mixture was quenched with saturated aqueous NaHCO ₃ at 0° C. and diluted with DCM, then the layers were separated. The aqueous layer was extracted with DCM. The combined organic extracts were washed with brine. The combined organic layer was dried over MgSO ₄ , filtered and concentrated under reduced pressure. Flash chromatography of the residue on silica gel using 15-60% EtOAc/heptane gave the title compound (compound A-4, 8.4 g, quant, yield).

1H NMR (500 MHz, chloroform-d) δ ppm 0.13 (s, 3H) 0.19 (s, 3H) 0.94 (s, 9H) 0.96 (d, J=6.8 Hz, 3H) 1.72 (dt, J=14.6 , 2.9 Hz, 1H) 2.15 (dd, J=9.8, 2.4 Hz, 1H) 2.23 (dt, J=14.6, 2.9 Hz, 1H) 2.55- 2.65 (m, 1H) 3.03 (d, J=9.8 Hz, 1H) 3.41-3.46 (m, 1H) 3.49 (d, J=11.7 Hz, 1 H ) 3.62-3.72 (m, 2H) 3.92 (ddd, J=11.7, 8.3, 2.4 Hz, 1H) 4.02 (t, J=2.7 Hz, 1H ) 5.01 - 5.12 (m, 2H) 5.93 (ddd, J=17.4, 10.4, 7.3 Hz, 1H).

Example 5

(((2S,3S,5R,6R)-2-((S)-but-3-en-2-yl)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2Hpyran-3,5 -diyl)bis(oxy))bis(tert-butyldimethylsilane) (compound A-5).

IBSOTf

A-4 A-5

In 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) described in Example 4 was 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 and quenched with saturated aqueous NaHCO ₃ , then the layers were separated. The combined organic extracts were washed successively with 0.5 N sodium hydroxide. an aqueous solution of HCl, a saturated aqueous solution of NaHCO ₃ and then brine. The organic layer was dried over MgSO ₄ , filtered and concentrated under reduced pressure. Flash chromatography of the residue on silica gel using 0-5% EtOAc/heptane (containing 1% Et ₃ N) gave the title compound (compound A-5, 1.69 g, 98% yield).

¹ H NMR (500 MHz, chloroform-d) δ ppm 0.02-0.08 (m, 15H) 0.11 (s, 3H) 0.89 (s, 9H) 0.90-0.92 (m, 18H) 0.94 (d, J=6 .8 Hz, 3H) 1.82 (dt, J=14.9, 4.8 Hz, 1H) 2.00 (dt, J=14.9, 2.9 Hz, 1H) 2.62-2, 72 (m, 1H) 2.93 (dd, J=9.3, 2.0 Hz, 1H) 3.27-3.34 (m, 1H) 3.66-3.79 (m, 3H) 3 .83 - 3.87 (m, 1H) 4.91 - 5.07 (m, 2H) 6.11 (ddd, J=17.3, 10.7, 6.1 Hz, 1H).

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).

A-5 A-6

To a solution of compound A-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) (1.32 g, 2.42 mmol) described in Example 5 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 h and at room temperature for 1.5 h. 3.0 M NaOH aqueous solution (3 ml, 9.00 mmol) and hydrogen peroxide (35% in water, 3 ml) was added to the reaction mixture at 0°C. The reaction mixture was stirred at 0° C. for 30 min, then at room temperature for 1 h. The reaction mixture was quenched with saturated aqueous Na ₂ SO ₃ and then the layers were separated. The aqueous layer was extracted with EtOAc (3 times). The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Flash chromatography of the residue on silica gel using 0-20% EtOAc/heptane gave the title compound (compound A-6, 1.36 g, 100% yield).

1H NMR (500 MHz, chloroform-d) δ ppm 0.03 (s, 3H) 0.05-0.08 (m, 12H) 0.10 (s, 3H) 0.88 (d, J=6.8 Hz, 3H) 0.89-0.93 (m, 27 H) 1.55-1.65 (m, 1H) 1.82 (dt, J=15.4, 4.4 Hz, 1H) 1.87-1.96 (m, 1H) 1 .97-2.03 (m, 1H) 2.17-2.26 (m, 1H) 2.67 (dd, J=7.8, 3.9 Hz, 1H) 2.98-3.10 ( m, 1H) 3.34-3.40 (m, 1H) 3.59-3.86 (m, 6H).

ESI-MS (m/z): 563.64 [M+H]+, 585.62 [M+Na]+.

- 15 040298

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).

Periodnnan

A-6 A-7

In 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-1ol (1100 mg, 1.954 mmol) described in Example 6 was added NaHCO ₃ (41.0 mg, 0.49 mmol) and Dess-Martin periodinan (1077 mg, 2.54 mmol). The reaction mixture was stirred at room temperature. After 3 hours, the reaction mixture was diluted with DCM and quenched with saturated aqueous NaHCO ₃ and saturated aqueous Na ₂ SO ₃ , then the layers were separated. The aqueous layer was extracted with DCM. The combined organic extracts were washed with brine, dried over MgSO ₄ , filtered and concentrated under reduced pressure. Flash chromatography of the residue on silica gel using 0-25% EtOAc/heptane gave the title compound (compound A-7, 950 mg, 87% yield).

¹ H NMR (500 MHz, chloroform-d) δ ppm 0.00 (s, 3H) 0.03-0.08 (m, 12H) 0.11 (s, 3H) 0.88 (s, 9H) 0.910.92 (m, 21H) 1.82 (dt, J=15.0, 4.5Hz, 1H) 2.01 (dt, J=15.0, 2.5Hz, 1H) 2.28 (ddd, J=16.0, 7.3, 2, 4 Hz, 1H) 2.53-2.58 (m, 1H) 2.74 (ddd, J=16.0, 5.5, 2.0 Hz, 1H) 2.94 (dd, J=9, 0, 1.7Hz, 1H) 3.29 (td, J=5.9, 2.0Hz, 1H) 3.68 (d, J=5.9Hz, 2H) 3.75-3.82 (m, 1H) 3.82-3.90 (m, 1H) 9.73 (t, J=2.4 Hz, 1H).

Scheme B. Preparation of compound B-3.

Example 8

(2S,3S)-3-((4-methoxybenzyl)oxy)-2-methyl-5-(trimethylsilyl)pent-4-yn-1-yl-4methylbenzenesulfonate (compound B-2).

Under nitrogen atmosphere to a solution of compound B-1: (2S,3S)-3-((4-methoxybenzyl)oxy)-2-methyl-5(trimethylsilyl)pent-4-yn-1-ol (11.08 g, 36.15 mmol) obtained using the method described in international patent publication WO 9317690 A1 / US 5436238 A (CAS no: 157323-41-6), Et ₃ N (12.6 ml, 90.4 mmol) and p-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 NaHCO ₃ solution and brine, dried over MgSO ₄ , filtered, then concentrated under reduced pressure. Flash chromatography of the residue on silica gel (silica gel 60, spherical, 40-50 µm, Kanto Chemical) using 0-10% EtOAc/heptane gave the title compound (compound B-2, 17.7 g, 93% yield) .

1H NMR (500 MHz, chloroform-d) δ ppm 0.17 (s, 9H) 1.02 (d, J=6.8 Hz, 3H) 2.10-2.18 (m, 1H) 2.44 (s, 3H) 3.82 (s, 3H ) 3.99 (d, J=6.8 Hz, 1H) 4.04-4.07(m, 2H) 4.33 (d, J=11.2 Hz, 1H) 4.66 (d, J =11.2Hz, 1H) 6.87 (d, J=8.3Hz, 2H) 7.21 (d, J=8.3Hz, 2H) 7.33 (d, J=8.8Hz , 2H) 7.77 (d, J=8.8 Hz, 2H).

Example 9

((3S,4R)-5-iodo-3-((4-methoxybenzyl)oxy)-4-methylnent-1-yn-1-yl)trimethylsilane (compound B-3).

- 16 040298

In a nitrogen atmosphere to a solution of compound B-2: (2S,3S)-3-((4-methoxybenzyl)oxy)-2-methyl-5(trimethylsilyl)pent-4-yn-1-yl-4-methylbenzenesulfonate (17 .7 g, 38.4 mmol) described in Example 8, NaI (7.49 g, 50.0 mmol) was added to DMF (360 ml) at room temperature. The reaction mixture was stirred at 80° C. for 2 hours. An additional 2.0 g of NaI was added to the reaction mixture. The reaction mixture was stirred for 1.5 h at 80°C, then cooled to room temperature. The mixture was diluted with diethyl ether, washed with water and brine, dried over MgSO ₄ , filtered and concentrated under reduced pressure. Flash chromatography of the residue on silica gel (silica gel 60, spherical, 40-50 µm, Kanto Chemical) using 10%-20% EtOAc/heptane gave the title compound (compound B-3, 14.3 g, 89% yield ).

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

Scheme C. Preparation of compound C-8.

TBSO

IMS tBuli ΤΒί

TBSO

Ether -78T

TMS

KjGOj

MeOH

IBSO

cat. Al BN ,ΟΜΡΜ

Bu ₃ S&H

Toluene SOX’

TBSO

CSA

DCM

Period Dess-Mart

DCM

THF-iPA-l-BuOH

HO

TsCI

Py DMAP DCM

I so

I'M

85°

PPh ₄

Dnallnl dncarbonate

THF-vsda

90°C 16 h

Et _; N TUI

- 17 040298

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)non-8-yn4-ol (compound C-1).

Under argon to a solution of compound B-3: ((3S,4R)-5-iodo-3-((4-methoxybenzyl)oxy)-4methylpent-1-yn-1-yl)trimethylsilane (1408 mg, 3.382 mmol) , described in example 9, in diethyl ether (25 ml) at -78°C was added tert-butyllithium (1.61 M in pentane, 4.11 ml, 6.62 mmol). The reaction mixture was stirred at -78°C for 45 min. Compound A-7: (S)-3-((2S,3S,5R,6R)-3,5bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H -pyran-2yl)butanal (825 mg, 1.47 mmol) described in Example 7 in 5.0 ml of diethyl ether was added to the reaction mixture at -78°C. The reaction mixture was stirred at -78°C for 60 min. The reaction mixture was quenched with saturated aqueous NH ₄ Cl. The organic layer was washed with brine, dried over Na ₂ SO ₄ then concentrated under reduced pressure. Flash chromatography of the residue on silica gel using 0-25% EtOAc/heptane gave the title compound (compound C-1, 1167 mg, 93% yield).

¹ H NMR (500 MHz, chloroform-d) δ ppm 0.00-0.12 (m, 21 N) 0.15-0.24 (m, 6N) 0.82-0.96 (m, 30 N) 1.03 (d, J=6.3 Hz , 3H) 1.38-1.55 (m, 1H) 1.68-1.99 (m, 4H) 2.10-2.30 (m, 2H) 2.76-2.87 (m, 1H) 3.15 (d, J=9.75 Hz, 1H) 3.33-3.38 (m, 1H) 3.56-4.02 (m, 9H) 4.37-4.50 (m , 1H) 4.64-4.78 (m, 1H) 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)non-8-en4-ol (compound C-3).

To a solution of compound C-1: (2S,6S,7S)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6(((tert-butyldimethylsilyl) hydroxy)methyl)tetrahydro-2H-pyran-2-yl)-7-((4-methoxybenzyl)oxy)-6-methyl9-(trimethylsilyl)non-8-yn-4-ol (1165 mg, 1.37 mmol ) described in example 10, in MeOH (20 ml) at 20°C was added K ₂ CO ₃ (189 mg, 1.37 mmol). The reaction mixture was stirred at 20° C. for 2 hours. The reaction mixture was diluted with EtOAc and quenched with saturated aqueous NH4Cl, then the layers were separated. The aqueous layer was extracted with EtOAc. The combined organic extracts were washed with brine, dried over MgSO ₄ , filtered and concentrated under reduced pressure. Flash chromatography of the residue on silica gel using 0-15% EtOAc/heptane gave 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)-6methylnon-8-yn-4-ol (1050 mg, 98% yield. ESI-MS (m/z): 801.50 [M+Na]+.

In a nitrogen atmosphere to a solution of compound С-2: (2S,6S,7S)-2-((2S,3S,5R,6R)-3,5-bis((daredabutyldimethylsilyl)oxy)-6-(((dareda- butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-7-((4methoxybenzyl)oxy)-6-methylnon-8-yn-4-ol (780 mg, 1.00 mmol) obtained above, in toluene (15 ml) at 20°C was added tri-n-butyltinhydride (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 min. The reaction mixture was concentrated under reduced pressure. Flash chromatography of the residue on silica gel using 0-18 040298

15% EtOAc/heptane gave the title compound (compound C-3, 970 mg, 91% yield).

1H NMR (500 MHz, chloroform-d) δ ppm 0.02 - 0.13 (m, 18 N) 0.84-0.96 (m, 48 N) 1.22-1.37 (m, 6N) 1.47-1.56 (m, 7 N ) 1.72-1.90 (m, 3H) 1.95-2.03 (m, 1H) 2.11-2.28 (m, 2H) 2.82-2.86 (m, 1H) 3 .08-3.15 (m, 1H) 3.33-3.40 (m, 1H) 3.43-3.53 (m, 1H) 3.58-3.87 (m, 8H) 4.25 -4.31 (m, 1H) 4.49-4.54 (m, 1H) 5.83 (dd, J=19.3, 7.6 Hz, 1H) 6.05-6.13 (m, 1H) 6.83-6.90 (m, 2H) 7.24 (d, J=8.8 Hz, 2H).

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-methylnon-8-en-4-one (compound C-4).

In a nitrogen atmosphere to a solution of compound С-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-((4methoxybenzyl)oxy)-6-methyl-9-(tributylstannyl)non-8-en-4-ol (970 mg, 0, 91 mmol) described in example 11 in 30 ml of DCM at 5°C was added iodine (242 mg, 0.95 mmol) in DCM (6 ml) until it retained the iodine color. The reaction mixture was quenched with saturated aqueous Na ₂ SO ₃ and the layers were separated. The aqueous layer was extracted with DCM. The combined organic extracts were washed with brine. The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Flash chromatography of the residue on silica gel using 0-25% EtOAc/heptane gave (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)-6methylnon-8-en-4-ol (768 mg, 93% yield).

In a nitrogen atmosphere to a solution of (2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6(((tert-butyldimethylsilyl) hydroxy)methyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6methylnon-8-en-4-ol (768 mg, 0.85 mmol) obtained above, NaHCO ₃ (17.8 mg, 0.21 mmol) and Dess-Martin periodinan (485 mg, 1.14 mmol) were added to DCM (25 ml) at room temperature. The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with DCM and quenched with saturated aqueous NaHCO3 and saturated aqueous Na ₂ SO ₃ , and then the layers were separated. The aqueous layer was extracted with DCM. The combined organic extracts were washed with brine, dried over MgSO ₄ , filtered and concentrated under reduced pressure. Flash chromatography of the residue on silica gel using 0-20% EtOAc/heptane gave the title compound (compound C-4, 776 mg, quant, yield).

1H NMR (500 MHz, chloroform-d) δ ppm 0.00 (s, 3H) 0.03-0.07 (m, 12H) 0.10 (s, 3H) 0.81 (d, J=6.3 Hz, 3H) 0.84 (d, J =6.3 Hz, 3H) 0.89 (s, 9H) 0.91 (s, 9H) 0.92 (s, 9H) 1.80 (dt, J=15.0, 4.5 Hz, 1H ) 1.99 (dt, J=15.0, 2.5 Hz, 1H) 2.17 (dd, J=16.6, 10.2 Hz, 1H) 2.20-2.29 (m, 2H ) 2.43-2.48 (m, 1H) 2.54 (d, J=12.7Hz, 1H) 2.87 (dd, J=9.0, 1.7Hz, 1H) 2.99 (dd, J=16.6, 2.9Hz, 1H) 3.27 (td, J=5.8, 2.4Hz, 1H) 3.50-3.56 (m, 1H) 3.66 -3.74 (m, 2H) 3.753.78 (m, 1H) 3.80 (s, 3H) 3.81-3.85 (m, 1H) 4.26 (d, J=11.7 Hz, 1H) 4.50 (d, J=11.7Hz, 1H) 6.26 (d, J=14.6Hz, 1H) 6.42 (dd, J=14.6, 7.8Hz, 1H ) 6.87 (d, J=8.3 Hz, 2H) 7.21 (d, J=8.3 Hz, 2H). 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-methylnon-8-en-4-one (compound C-5).

C-4 C-5

To a solution of compound C-4: (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)-6methylnon-8-en-4-one (600 mg, 0.66 mmol) , described in example 12, in THF (5.0 ml), IPA (5.0 ml) and tertBuOH (5.0 ml) at 4°C was added (1S)-(+)-10-camphorsulfonic acid (154 mg , 0.66 mmol). The reaction mixture was stirred at 4° C. for 20 hours. The reaction mixture was diluted with EtOAc and quenched with saturated aqueous NaHCO ₃ , then the layers were separated. The aqueous layer was extracted with EtOAc. The combined organic extracts were washed with brine, dried over

- 19 040298

MgSO ₄ , filtered and concentrated under reduced pressure. Flash chromatography of the residue on silica gel using 0-35% EtOAc/heptane gave the title compound (compound C-5, 500 mg, 95% yield).

1H NMR (500 MHz, chloroform-d) δ ppm 0.01 (s, 3H) 0.04 (s, 3H) 0.07 (s, 3H) 0.11 (s, 3H) 0.86-0.91 (m, 15 H) 0.93 (s , 9H) 1.83 (dt, J=14.9, 4.8 Hz, 1H) 1.93-2.00 (dt, J=14.9, 4.8 Hz, 1H) 2.19-2 .26 (m, 1H) 2.29 (dd, J=14.9, 5.6Hz, 1H) 2.39 (dd, J=16.6, 8.3Hz, 1H) 2.44-2 .66 (m, 4H) 2.91 (dd, J=9.5, 1.7Hz, 1H) 3.36-3.41 (m, 1H) 3.48 (td, J=11.3 , 2.7 Hz, 1H) 3.59 (t, J=7.1 Hz, 1H) 3.74-3.78 (m, 2H) 3.80 (s, 3H) 3.85 (m, 1H ) 4.25 (d, J=11.2Hz, 1H) 4.46 (d, J=11.2Hz, 1H) 6.28 (d, J=14.6Hz, 1H) 6.43 ( dd, J=14.6, 7.8 Hz, 1H) 6.87 (d, J=8.8 Hz, 2H) 7.21 (d, J=8.8 Hz, 2H). 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) hydroxy)-6-methyl-4-oxonon-8-en-2-yl)tetrahydro-2H-pyran-2-yl)methyl-4-methylbenzenesulfonate (compound C-6).

^P-5 p-6

In a nitrogen atmosphere to a solution of 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-methylnon-8-en-4-one (500 mg, 0.63 mmol) described in Example 13 , in DCM (10 ml) at 5°C were added pyridine (2.54 ml, 31.6 mmol), p-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. n-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 hours. The reaction mixture was diluted with DCM and quenched with saturated aqueous NaHCO ₃ , then the layers were separated. The aqueous layer was extracted with DCM. The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Flash chromatography of the residue on silica gel using 0-25% EtOAc/heptane gave the title compound (compound C-6, 560 mg, 94% yield).

1H NMR (500 MHz, chloroform-d) δ ppm 0.01 (s, 3H) 0.04 (s, 3H) 0.04 (s, 3H) 0.08 (s, 3H) 0.81 (d, J=6.8 Hz, 3H) 0.83 (s, 9H) 0.86 (d, J=6.8Hz, 3H) 0.89 (s, 9H) 1.81 (dt, J=14.9, 4.5Hz, 1H) 1.91 -1.96 (m, 1H) 2.152.32 (m, 3H) 2.36-2.42 (m, 1H) 2.43 (s, 3H) 2.57 (d, J=12.7 Hz, 1H) 2.77 (dd, J=16.6, 3.4 Hz, 1H) 2.87 (dd, J=9.0, 1.7 Hz, 1H) 3.53-3.58 (m, 2H) 3.70-3.75 (m, 1H) 3.80-3.85 (m, 1H) 3.81 (s, 3H) 4.06 (dd, J=10.0, 5.0 Hz , 1H) 4.08-4.16 (m, 1H) 4.28 (d, J=11.2 Hz, 1H) 4.51 (d, J=11.2 Hz, 1H) 6.30 (d , J=14.6 Hz, 1H) 6.45 (dd, J=14.6, 7.8 Hz, 1H) 6.88 (d, J=8.8 Hz, 2H) 7.24 (d, J=8.8 Hz, 2H) 7.31 (d, J=8.3 Hz, 2H) 7.76 (d, J=8.3 Hz, 2H).

Example 15

(2S,6S,7S,E)-2-((2S,3S,5R,6R)-6-(azidomethyl)-3,5-bis((tert-butyldimethylsilyl)oxy)tetrahydro2H-pyran-2-yl) -9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-one (compound C-7).

P-6 P-7

In a nitrogen atmosphere to a solution of compound C-6: ((2R,3R,5S,6S)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-((2S,6S,7S,E)-9-iodine- 7-((4-methoxybenzyl)oxy)-6-methyl-4-oxonon-8-en-2yl)tetrahydro-2H-pyran-2-yl)methyl-4-methylbenzenesulfonate (560 mg, 0.59 mmol), described in example 14, 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 h, sodium azide (100 mg, 1.54 mmol) was added to the reaction mixture, then the reaction mixture was stirred at 85°C for 14 h. The reaction mixture was diluted with EtOAc and quenched with H ₂ O, then the layers were separated. The organic extracts were washed successively with water and brine. The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a crude residue. Flash chromatography of the residue on silica gel using 0-15% EtOAc/heptane gave the title compound (compound C-7, 298 mg, 62% yield).

1H NMR (500 MHz, chloroform-d) δ ppm 0.03 (s, 3H) 0.06 (s, 3H) 0.07 (s, 3H) 0.10 (s, 3H) 0.84 (d, J=6.8 Hz, 3H) 0.85 (d, J=6.8 Hz, 3H) 0.91 (s, 9H) 0.92 (s, 9H) 1.86 (dt, J=15.0, 4.7 Hz, 1H) 1.98 (dt, J=15.0, 2.9 Hz,

- 20 040298

1H) 2.19-2.32 (m, 3H) 2.41-2.49 (m, 1H) 2.58 (d, J=12.7 Hz, 1H) 2.94 (dd, J=16 .6, 2.9 Hz, 1H) 2.98 (dd, J=8.8,

2.0Hz, 1H) 3.02 (dd, J=12.7, 2.9Hz, 1H) 3.47 (dt, J=8.8, 2.7Hz, 1H) 3.49-3 .54 (m, 1H) 3.63 (dd, J=12.7, 8.8

Hz, 1H) 3.69-3.73 (m, 1H) 3.81 (s, 3H) 3.83-3.88 (m, 1H) 4.26 (d, J=11.7 Hz, 1H ) 4.50 (d, J=11.7Hz, 1H) 6.26 (d, J=14.6Hz, 1H) 6.42 (dd, J=14.6, 7.8Hz, 1H) 6.87 (d, J=8.8 Hz, 2H) 7.22 (d, J=8.8 Hz, 2H).

Example 16

(((2R,3R,5S,6S)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-((2S,6s,7S,E)-9-iodo-7-((4-methoxybenzyl ) hydroxy)-6-methyl-4-oxonon-8-en-2-yl)tetrahydro-2H-pyran-2-yl)methyl)carbamate (compound C-8).

To a solution of compound C-7: (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-methylnon-8-en-4-one (298 mg, 0.37 mmol) described in Example 15 in THF ( 10 ml) and water (1.0 ml) at 20°C was added triphenylphosphine (1437 mg, 5.478 mmol). The reaction mixture was stirred at 70° C. for 1.5 hours. The reaction mixture was concentrated under reduced pressure to give crude amine. To a solution of the crude amine obtained above in THF (10 ml) at 5°C was added Et ₃ N (0.51 ml, 3.66 mmol) and diallyl dicarbonate (341 mg, 1.83 mmol). The reaction mixture was stirred at room temperature for 60 minutes. The reaction mixture was concentrated under reduced pressure. Flash chromatography of the residue on silica gel using 0-25% EtOAc/heptane gave the title compound (compound C-8, 300 mg, 94% yield).

¹ H NMR (500 MHz, chloroform-d) δ ppm 0.05-0.07 (m, 9H) 0.11 (s, 3H) 0.85 (d, J=6.3 Hz, 3H) 0.87 (d, J=6.3 Hz, 3H) 0.90 (s, 9H) 0.93 (s, 9H) 1.80 (dt, J=15.0, 4.4 Hz, 1H) 1.96 (dt, J=15.0, 2.8 Hz, 1H) 2.16-2.29 (m, 2H) 2.32-2.39 (m, 1H) 2.53-2.60 (m, 3H) 2.86 (d, J=7, 3 Hz, 1H) 3.04-3.11 (m, 1H) 3.30-3.34 (m, 1H) 3.38-3.48 (m, 1H) 3.58 (t, J=7 .1 Hz, 1H) 3.70-3.76 (m, 1H) 3.80 (s, 3H) 3.81-3.84 (m, 1H) 4.25 (d, J=11.2 Hz , 1H) 4.46 (d, J=11.2 Hz, 1H) 4.53-4.63 (m, 2H) 5.19 (dd, J=10.7, 1.5 Hz, 1H) 5 .32 (d, J=17.1 Hz, 1H) 5.47 (d, J=6.8 Hz, 1H) 5.88-5.99 (m, 1H) 6.28 (d, J=14 .6Hz, 1H) 6.43 (dd, J=14.6, 7.8Hz, 1H) 6.87 (d, J=8.8Hz, 2H) 7.21 (d, J=8 .8 Hz, 2 H). ESI-MS (m/z): 896.34 [M+Na]+.

- 21 040298

Scheme D Preparation of Compound D-7.

- 22 040298

Example 17.

Compound D-4.

In 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) prepared by the method described in Organic Letters (2002) , 4 (25), 4431-4434 (CAS no: 546141-34-8) and 1,8-bis(dimethylamino)naphthalene (107 mg, 0.497 mmol) in MeCN (0.75 ml) was added chromium(II) chloride (55.5 mg, 0.452 mmol) and then the resulting mixture was stirred in a glove box at room temperature for 1 hour. The resulting green solution was added to a mixture of compound C-8: allyl-(((2R,3R,5S,6S)- 3,5-bis((tert-butyldimethylsilyl)oxy)-6-((2S,6S,7S,E)-9-iodo-7-((4

- 23 040298 methoxybenzyl)oxy)-6-methyl-4-oxonon-8-en-2-yl)tetrahydro-2H-pyran-2-yl)methyl)carbamate (99.0 mg, 0.113 mmol) described in the example 16, compound D-l (80.0 mg, 0.09 mmol) prepared by the method described in Journal of the American Chemical Society (1992), 114(8), 3162-3164 (CAS no: 157322-23-1 ), compound D-3: dichloro(2,9-dimethyl-1,10-phenanthroline)nickel (0.46 mg, 1.36 µmol) prepared by the method described in the Journal of the American Chemical Society (2009) , 131(42), 15387-15393 (CAS no: 21361-04-6) and lithium chloride (3.83 mg, 0.09 mmol). The reaction mixture was stirred in a glovebox at room temperature for 60 minutes. The reaction mixture was then removed from the glove box, diluted with a mixture of diethyl ether-EtOAc (5.0 ml - 5.0 ml), then Florisil® (1600 mg, 15.94 mmol) (CAS no: 1343-88) was added to the mixture -0). The mixture was then stirred at room temperature for 30 minutes. The mixture was filtered (Celite®), washed with EtOAc/heptane = 2/1, then the filtrate was concentrated under reduced pressure. Flash chromatography of the residue on silica gel using 3-55% EtOAc/heptane gave the title compound (compound D-4, 140 mg, 95% yield).

Example 18.

Compound D-5.

In a nitrogen atmosphere, to a solution of compound D-4 (140 mg, 0.09 mmol) described in example 17 in DCM (5.0 ml) at 5°C was added NaHCO ₃ (28.8 mg, 0.34 mmol) and periodinan Dess-Martin (72.7 mg, 0.17 mmol). The reaction mixture was stirred at room temperature for 60 minutes. The reaction mixture was diluted with DCM and quenched with saturated aqueous NaHCO ₃ and saturated aqueous Na ₂ SO ₃ , and then the layers were separated. The aqueous layer was extracted with DCM. The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Flash chromatography of the residue on silica gel using 2-60% EtOAc/heptane gave the title compound (compound D-5, 120 mg, 86%).

1H NMR (500 MHz, benzene^) δ ppm 0.01-0.05 (m, 9H) 0.10-0.12 (m, 6H) 0.15 (s, 3H) 0.76 (d, J=6.1 Hz, 3H) 0.96 (s, 9H) 1.02 (s, 9H) 1.04 (s, 9H) 0.95-1.10 (m, 7H) 1.20 (d, J=7.3 Hz, 3H) 1, 31-1.37 (m, 3H) 1.41 (dd, J=12.8, 4.9 Hz, 1H) 1.40-1.58 (m, 4H) 1.59-1.64 (m , 1H) 1.69-1.89 (m, 3H) 1.90-1.99 (m, 2H) 2.02-2.25 (m, 8H) 2.26-2.48 (m, 6H ) 2.49-2.70 (m, 6H) 2.71-2.84 (m, 2H) 3.00-3.07 (m, 1H) 3.12-3.30 (m, 4H) 3.36 (s, 3H) 3.40 (br.s, 1H) 3.44-3.53 (m, 2H) 3.65 (dd, J=6.4, 4.0 Hz, 1H) 3 .69-3.84 (m, 4H) 3.86-4.03 (m, 4H) 4.07-4.17 (m, 3H) 4.27-4.29 (m, 1H) 4.27 (d, J=11.0 Hz, 1H) 4.48-4.58 (m, 1H) 4.49 (d, J=11.0 Hz, 1H) 4.65-4.70 (m, 2 H) 4.68 (d, J=5.5Hz, 1H) 4.74-4.86 (m, 2H) 4.78 (s, 1H) 4.93 (s, 1H) 5.05 (d , J=10.4 Hz, 1H) 5.09 (br. s., 1H) 5.19 (br. s., 1H) 5.30 (dd, J=17.1, 1.2 Hz, 1H ) 5.82 (d, J=8.0 Hz, 1H) 5.86-5.96 (m, 1H) 6.46 (d, J=15.9 Hz, 1H) 6.846.92 (m, 3 H) 7.31 (d, J=8.6 Hz, 2H).

- 24 040298

Example 19.

Compound D-6.

Imidazole hydrochloride (155 mg, 1.48 mmol) was dissolved in DMF (2.9 ml) to give a 0.5 M solution of imidazole hydrochloride in DMF. 1.0 ml of this solution was mixed with 1.0 ml of TBAF (1.0 M, THF solution) to form a premixed solution of 0.5 M TBAF and 0.25 M imidazole hydrochloride in THF-DMF (1:1). Under nitrogen atmosphere, to a solution of compound D-5 (80.0 mg, 0.05 mmol) described in Example 18 in DMF (7.0 ml) at 20° C. was added 0.588 ml of a premixed solution of TBAF (0.5 M ) and imidazole hydrochloride (0.25 M) in THF-DMF (1:1) prepared above. The reaction mixture was stirred at room temperature for 14 hours. 1.6 g of CaCO ₃ 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. The mixture was then diluted with EtOAc, then filtered (Celite®), washed with EtOAc. The filtrate was concentrated under reduced pressure to give a crude residue. 1000 mg CaCO ₃ and 2.25 g 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. The mixture was then diluted with EtOAc, filtered (Celite®), washed with EtOAc. The filtrate was concentrated under reduced pressure to give a crude residue (63.0 mg). To a solution of the crude residue obtained above (63.0 mg) in DCM (6.0 ml), tert-BuOH (0.6 ml) and phosphate buffer pH 7 (0.6 ml, 1/15 M) at room temperature DDQ (111 mg, 0.49 mmol) was added. The reaction mixture was stirred at room temperature for 45 minutes. The reaction mixture was quenched with saturated aqueous NaHCO ₃ , 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 Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Flash chromatography of the residue on NH silica gel using 10-100% EtOAc/heptane followed by 10% MeOH/EtOAc gave the crudely purified title compound (compound D-6, 15.0 mg, 27%).

¹ H NMR (500 MHz, methanol^) δ ppm 0.97 (d, J=7.0Hz, 3H) 0.97 (d, J=7.0Hz, 3H) 1.00 - 1.02 (m, 1H) 1.05 (d, J= 7.3 Hz, 3H) 1.09 (d, J=6.3 Hz, 3H) 1.31-1.45 (m, 6H) 1.46-1.63 (m, 5H) 1.64- 1.75 (m, 3H) 1.80-1.86 (m, 2H) 1.87-1.93 (m, 2H) 1.94-2.11 (m, 9H) 2.13-2, 27 (m, 8H) 2.33 (d, J=2.4Hz, 2H) 2.39 (dd, J=13.4, 6.1Hz, 1H) 2.44 (dd, J=17, 6, 2.0 Hz, 1H) 2.55 (dd, J=17.6, 9.3 Hz, 1H) 2.75-2.84 (m, 1H) 2.97 (dd, J=9, 3, 2.0 Hz, 1H) 3.21 (dd, J=6.6, 4.6 Hz, 1H) 3.32 (m, 1H) 3.41-3.46 (m, 1H) 3, 57 (br. s., 1H) 3.60 (d, J=11.7 Hz, 1H) 3.67-3.74 (m, 2H) 3.78 (br. s., 1H) 3.86 -3.90 (m, 2H) 3.97 (d, J=2.4Hz, 1H) 4.02-4.11 (m, 4H) 4.17 (dd, J=6.6, 4 .6 Hz, 1H) 4.23 (dd, J=11.5, 2.2 Hz, 1H) 4.29 (br.s, 1H) 4.31 (td, J=9.3, 3.9 Hz, 1H) 4.44 (d, J=10.2Hz, 1H) 4.51 (d, J=5.4Hz, 2H) 4.59 (t, J=4.9Hz, 1H) 4 .61 (dd, J=7.3, 4.9 Hz, 1H) 4.69 (t, J=4.6 Hz, 1H) 4.80 (s, 1H) 4.85-4.87 (m , 1H) 5.01 (s, 1H) 5.05 (s, 1H) 5.16 (dd, J=10.7, 1.0 Hz, 1H) 5.28 (dd, J=17.1, 2.0 Hz, 1H) 5.92 (m, 1H). ESIMS (m/z): 1172.57 [M+Na]+.

- 25 040298

Example 20.

Compound D-7.

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. Flash chromatography of the residue on silica gel NH using 50% EtOAc/heptane followed by 0-20% MeOH/EtOAc gave a crude product. The resulting crude product was purified by HPLC to give the title compound (D-7, 7.0 mg, 47%, retention time=13.8 min).

HPLC conditions.

Column: YMC Pak Pro C18 (20 mmx250 mm).

Detection wavelength: 200 nm.

Column temperature: room temperature.

Mobile phase: MeCN-water (0.05% AcOH).

Flow rate: 8 ml/min.

Eluate:

MeCN/water 25% (iso, 2 min), then

MeCN/water 25% - 60% (gradient, 20 min).

Ή NMR (500 MHz, methanol-I4) δ ppm 0.99 (d, J=6.7 Hz, 3H) 1.00-1.03 (m, 1H) 1.04 (d, J=7.3 Hz, 3H) 1.06 (d, J= 7.3 Hz, 3H) 1.10 (d, J=6.1 Hz, 3H) 1.29-1.63 (m, 10 H) 1.65-1.78 (m, 3H) 1.79 -1.89 (m, 2H) 1.922.12 (m, 10H) 1.93 (s, 3H) 2.13-2.36 (m, 9H) 2.41 (dd, J=13.5, 6.1Hz, 1H) 2.45 (dd, J=17.6, 2.2Hz, 1H) 2.56 (dd, J=17.6, 9.8Hz, 1H) 2.75-2 .84 (m, 1H) 2.98 (dd, J=9.8, 1.8 Hz, 1H) 3.12 (dd, J=12.8, 3.7 Hz, 1H) 3.22 (dd , J=6.4, 4.6 Hz, 1H) 3.26 (dd, J=13.2, 7.8 Hz, 1H) 3.39 (d, J=1.8 Hz, 1H) 3, 61 (d, J=12.8 Hz, 1H) 3.63 -3.68 (m, 2H) 3.68-3.76 (m, 2H) 3.81-3.94 (m, 3H) 4 .00 (d, J=2.5 Hz, 1H) 4.03-4.15 (m, 4 H) 4.18 (dd, J=6.4, 4.6 Hz, 1H) 4.25 ( ddd, J=11.0, 4.3, 1.8 Hz, 1H) 4.27-4.36 (m, 2H) 4.46 (d, J=11.0 Hz, 1H) 4.57- 4.65 (m, 2H) 4.70 (t, J=4.6 Hz, 1H) 4.81 (d, J=1.2 Hz, 1H) 5.02 (br. s, 1H) 5, 06 (d, J=1.8Hz, 1H). ESI-MS (m/z): 1066.96 [M+H]+, 1090.19 [M+Na]+.

Compound (1) (salt-free form of compound D-7): ¹ H NMR (600 MHz, methanol-d ₄ ) δ ppm 0.98 (d, J=7.2Hz, 3H) 1.00 (d, J=6.8Hz, 3H) 1.02 (m, 1H) 1.05 (d, J=6.8Hz , 3H) 1.09 (d, J=6.4 Hz, 3H) 1.28 -1.45 (m, 5 H) 1.46-1.59 (m, 4 H) 1.57-1, 63 (m, 1H) 1.65-1.71 (m, 1H) 1.70-1.75 (m, 2H) 1.79-1.86 (m, 2H) 1.91 (dt, J= 14.9, 3.1 Hz, 1H) 1.94-2.11 (m, 8H) 2.14-2.34 (m, 9H) 2.39 (dd, J=13.2, 6.0 Hz, 1H) 2.44 (dd, J=17.4, 1.9Hz, 1H) 2.56 (dd, J=17.6, 9.6Hz, 1H) 2.69 (dd, J= 13.2, 4.2Hz, 1H) 2.79 (ddq, J=15.9, 7.6, 2.0Hz, 1H) 2.92 (dd, J=13.2, 8.3Hz , 1H) 2.97 (dd, J=9.6, 1.7 Hz, 1H) 3.21 (dd, J=6.4, 4.9 Hz, 1H) 3.29 (m, 1H) 3 .34 (dd, J=8.3, 4.15 Hz, 1H) 3.58 (br. s., 1H) 3.60 (br.d, J=11.3 Hz, 1H) 3.68- 3.73 (m, 2H) 3.80 (br. s., 1H) 3.84-3.90 (m, 2H) 3.98 (d, J=2.3 Hz, 1H) 4.03- 4.13 (m, 4 H) 4.17 (dd, J=6.4, 4.9 Hz, 1H) 4.24 (ddd, J=11.3, 4.5, 1.5 Hz, 1H ) 4.29 (dd, J=4.0, 1.9Hz, 1H) 4.32 (td, J=10.2, 4.2Hz, 1H) 4.44 (br. d, J=11 .0 Hz, 1H) 4.59 (t, J=4.5 Hz, 1H) 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).

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

Examples of pharmacological tests.

General information.

Natural compounds of halichondrin and their modified compounds are known from the literature

- 26 040298 (see, for example, D. Uemura et al. Norhalichondrin A: An Antitumor Polyether Macrolide from a Marine Sponge J. Am. Chem. Soc., 107, 4796 (1985); Marc Litaudon et al. Antitumor Polyether Macrolides : New and Hemisynthetic Halichondrins from the New Zealand Deep-Water Sponge Lissodendoryx sp. J. Org. Chem., 1997, 62, 1868-1871). However, most of them are not easily accessible. 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 up to 600 kg of Halichondria okadai Kadota (see e.g. D. Uemura et al. Norhalichondrin A: An Antitumor Polyether Macrolide from a Marine Sponge J. Am. Chem. Soc, 107, 4796 (1985)). Among natural halichondrin compounds, halichondrin B exhibits the greatest antitumor activity against B-16 melanoma cells in vitro and is very active against L-1210 leukemia in vivo (see, e.g., D. Uemura et al. Norhalichondrin A: An Antitumor Polyether Macrolide from a Marine Sponge J. Am. Chem. Soc, 107, 4796 (1985)). Halichondrin C is also active in various in vivo models, but is unstable in aqueous solution compared to halichondrin B. Norgalichondrin B is much weaker than halichondrin B, not only in vitro but also in vivo. See, for example, D. Uemura et al. Norhalichondrin A: An Antitumor Polyether Macrolide from a Marine Sponge J. Am. Chem. Soc, 107, 4796 (1985)). The following pharmacological tests use halichondrin B (Hali-B) as reference compounds, if necessary.

Pharmacological Test Example 1 FaDu Growth Inhibition Assay In this assay, the growth inhibitory activity of test compounds on the human head and neck squamous cell carcinoma (SCCHN) cell line FaDu was measured. FaDu cells were maintained in RPMI-1640 medium (Wako Pure Chemical Industries, Ltd., 187-02021) containing 10% fetal bovine serum (FBS: Nichirei, 12D168) and penicillin and streptomycin in a 5% CO2 incubator (37°C ). To each well of a 96-well plate (Becton, Dickinson and Company, 353219) was added 75 μl of FaDu cell suspension adjusted to a concentration of 4x104 cells/ml with culture medium, and the cells were incubated overnight in a 5% incubator. CO2 (37°C). The next day, 25 μl of compound (1) or halichondrin B in a 3-fold dilution series suspended in culture medium was added to each well, and the resulting product was incubated for 3 days in a 5% CO2 (37° C.) incubator. Cell viability was then determined using a CellTiter-Glo® fluorescent cell viability assay (Promega) using an EnVision 2103 multichannel reader (Perkin-Elmer, Wellesley, MA). The value of wells containing cells without addition of test compounds was defined as 100% and the value of wells containing no cells was defined as 0%. The concentration of test compound required to inhibit cell growth by 50% (ie, IC ₅₀ value) was calculated and is shown in Table 1. 1.

Table 1

FaDu

Test compound (ICso (nM))

Halichondrin B 0.124

Compound (1) 0.0714

Pharmacological Test Example 2: MDA-MB231 Growth Inhibition Assay.

This assay measured the growth inhibitory activity of test compounds on the human breast cancer cell line MDA-MB231. MDA-MB231 cells were maintained in Dulbecco's modified Eagle's medium (Wako Pure Chemical Industries, Ltd., 044-29765) containing 10% fetal bovine serum (FBS: Nichirei, 12D168) and penicillin and streptomycin in a 5% CO2 incubator. (37°C). To each well of a 96-well plate (Becton, Dickinson and Company, 353219) was added 75 μl of a suspension of MDA-MB231 cells adjusted to a concentration of 4x104 cells/ml with culture medium, and the cells were incubated overnight in an incubator with 5% CO2 (37°C). The next day, 25 µl of compound (1) or halichondrin B in a 3-fold dilution series suspended in culture medium was added to each well, and the resulting product was incubated for 3 days in a 5% CO2 (37°C) incubator. Cell viability was then determined using a CellTiter-Glo fluorescent cell viability assay (Promega) using an EnVision 2103 multichannel reader (Perkin-Elmer, Wellesley, MA). The value of wells containing cells without addition of test compounds was defined as 100% and the value of wells containing no cells was defined as 0%. The concentration of test compound required to inhibit cell growth by 50% (ie, IC ₅₀ value) was calculated and is shown in Table 1. 2.

table 2

MDA-MB231

Test compound (ICso (nM))

Halichondrin B 1,000

Compound (1) 0.109

- 27 040298

Pharmacological test example 3: HCC1954 growth inhibition assay.

This assay measured the growth inhibitory activity of test compounds on the human breast cancer cell line HCC1954. HCC1954 cells were maintained in RPMI-1640 medium modified to contain 2 mM L-glutamine, 10 MMHEPES, 1 mM sodium pyruvate, 4500 mg/l glucose and 1500 mg/l sodium bicarbonate (ATCC 30-2001) containing 10 % fetal bovine serum (FBS: Nichirei, 12D168), as well as penicillin and streptomycin in an incubator with 5% CO ₂ (37°C). To each well of a 96-well plate (Becton, Dickinson and Company, 353219) was added 75 μl of a suspension of HCC1954 cells adjusted to a concentration of 4x104 cells/ml with culture medium, and the cells were incubated overnight in an incubator with 5% CO ₂ (37°C). The next day, 25 μl of compound (1) or halichondrin B in a 3-fold dilution series suspended in culture medium was added to each well and the resulting product was incubated for 3 days in a 5% CO ₂ (37° C.) incubator. Cell viability was then determined using a CellTiter-Glo fluorescent cell viability assay (Promega) using an EnVision 2103 multichannel reader (Perkin-Elmer, Wellesley, MA). The value of wells containing cells without addition of test compounds was defined as 100% and the value of wells containing no cells was defined as 0%. The concentration of test compound required to inhibit cell growth by 50% (ie IC ₅₀ value) was calculated and is shown in Table 1. 3.

Table 3

NSS1954

Test compound (ICso (nM))

Halichondrin B 0.154

Compound (1) 0.0668

Pharmacological Test Example 4: Antitumor effects in a subcutaneous FaDu xenograft model in mice as monotherapy.

Human head and neck squamous cell carcinoma (SCCHN) cell line FaDu, which was cultured in RPMI-1640 medium containing 10% FBS, as well as penicillin and streptomycin, was adjusted to a concentration of 4.8x107 cells/ml using Hanks' balanced salt solution to obtain a cell suspension. The cell suspension was inoculated in a volume of 100 μl into the subcutaneous portion of the right flank of 7 week old nude mice (CAnN.Cg-Foxnlnu/CrlCrlj, female, Charles River Laboratories Japan Inc.). Nine days after cell inoculation, the shortest diameter and longest tumor diameter of each mouse were measured using an electronic digital caliper (Digimatic™ caliper, Mitutoyo Corporation) to calculate the tumor volume according to the following calculation formulas.

Tumor volume (mm ³ )=longest diameter (mm) x shortest diameter (mm) x shortest diameter (mm)/2.

Relative tumor volume (RTV)=tumor volume (day X)/tumor volume (first day).

Tumor regression (%)=(1 - minimum RTV)x100.

Based on the volumes of tumors obtained on the first day of administration, mice were divided into groups so that the average values of the tumor volumes were essentially the same among the groups. Each test compound was dissolved in DMSO and the solution was stored in a freezer until use. Immediately prior to injection, the mother liquor was diluted in an aqueous sodium chloride solution with 100 μM hydroxypropyl-P-cyclodextrin. Each evaluated sample was intravenously administered at the maximum tolerated dose (MTD). In this case, the experiment was carried out on groups, each of which consisted of 4 mice. Tumor regression (%) of each test compound is shown in Table 1. 4.

Table 4

Test compound Dose (mg/kg) Tumor regression (%) Galichondrin B 0.05 0 Connection (1) 0.2 43

Pharmacological Test Example 5: Antitumor activity against OSC-19 in a subcutaneous xenograft model in mice as monotherapy.

Human head and neck squamous cell carcinoma (SCCHN) cell line OSC-19, which was cultured in DMEM/Ham's F-12 (1:1) medium containing 10% FBS, as well as penicillin and streptomycin, was adjusted to a concentration of 1x108 cells /ml, using PBS to prepare a cell suspension, and the suspension was mixed with Matrigel™ (BD Bioscience, Cat # 366237) in a ratio of 1:1 to obtain a cell suspension at a concentration of 5x107 cells/ml. The cell suspension was inoculated in a volume of 100 μl into the subcutaneous

- 28 040298 right flank of 5 week old nude mice (CAnN.Cg-Foxnlnu/CrlCrlj, female, Charles River

Laboratories Japan, Inc.). Six days after cell inoculation, the shortest diameter and longest tumor diameter of each mouse were measured using an electronic digital caliper (Digimatic™ caliper, Mitutoyo Corporation) to calculate the tumor volume according to the following calculation formulas.

Tumor volume (mm ³ )=longest diameter (mm) x shortest diameter (mm) x shortest diameter (mm)/2.

Relative tumor volume (RTV)=tumor volume (day X)/tumor volume (first day).

Tumor regression (%)=(1 - minimum RTV)x100.

Based on the volumes of tumors obtained on the first day of administration, mice were divided into groups so that the average values of the tumor volumes were essentially the same among the groups. The experiment was carried out on groups, each of which consisted of 6 mice. The test compound was dissolved in aqueous sodium chloride and administered intravenously at doses ranging from 0.06 mg/kg to 0.18 mg/kg once a week for 2 weeks (schedule Q7Dx2). Tumor regression (%) of each dose studied is shown in Table 1. 5.

Table 5

Test compound Dose (mg/kg) Tumor regression (%) Connection (1) 0.06 59 Connection (1) 0.18 90

Pharmacological Test Example 6: Antitumor activity against HCC1806 in a subcutaneous xenograft model in mice as monotherapy.

The human breast cancer cell line HCC1806, which was cultured in RPMI-1640 medium containing 10% FBS, as well as penicillin and streptomycin, was adjusted to a concentration of 1 x 10 ⁸ cells/ml with PBS to obtain a cell suspension, and the suspension mixed with Matrigel™ (BD Bioscience, Cat # 366237) in a ratio of 1:1 to obtain a cell suspension at a concentration of 5 x 10 ⁷ cells/ml. The cell suspension was inoculated in a volume of 100 μl into the subcutaneous portion of the right flank of 5 week old nude mice (CAnN.Cg-Foxnlnu/CrlCrlj, female, Charles River Laboratories Japan, Inc.).

12 days after cell inoculation, the shortest diameter and longest tumor diameter of each mouse were measured using an electronic digital caliper (Digimatic™ caliper, Mitutoyo Corporation) to calculate tumor volume according to the following calculation formulas.

Tumor volume (mm ³ )=longest diameter (mm) x shortest diameter (mm) x shortest diameter (mm)/2.

Relative tumor volume (RTV)=tumor volume (day Xj/tumor volume (first day).

Tumor regression (%)=(1 - minimum RTV)χ100.

Based on the volumes of tumors obtained on the first day of administration, mice were divided into groups so that the average values of the tumor volumes were essentially the same among the groups. The experiment was carried out on groups, each of which consisted of 6 mice. The test compound was dissolved in an aqueous solution of sodium chloride and administered intravenously at a dose of 0.18 mg/kg once a week for 2 weeks (scheme Q7Dχ2). Tumor regression (%) for compound (1) is shown in table. 6.

Table 6

Study Dose (mg/kg) Tumor regression (%) Compound

Compound (1) 0.18 90

Pharmacological Test Example 7: Antitumor effects in a subcutaneous FaDu xenograft model in combination with cetuximab in mice.

The human head and neck squamous cell carcinoma (SCCHN) cell line FaDu, which was cultured in RPMI-1640 medium containing 10% FBS, as well as penicillin and streptomycin, was adjusted to a concentration of 5 x 10 ⁷ cells/ml using Hanks' balanced salt solution for obtaining a cell suspension. The cell suspension was inoculated in a volume of 100 μl into the subcutaneous portion of the right flank of 7 week old nude mice (CAnN.CgFoxnlnu/CrlCrlj, female, Charles River Laboratories Japan Inc.). 10 days after cell inoculation, the shortest diameter and longest tumor diameter of each mouse were measured using an electronic digital caliper (Digimatic™ caliper, Mitutoyo Corporation) to

- 29 040298 calculate the tumor volume according to the following calculation formulas.

Tumor volume (mm ³ )=longest diameter (mm) x shortest diameter (mm) x shortest diameter (mm)/2.

Relative tumor volume (RTV)=tumor volume (Day X)/tumor volume (Day 1).

Tumor regression on day 35 (%)=(1-RTV on day 35)x100.

Based on the volumes of tumors obtained on the first day of administration, mice were divided into groups so that the average values of the tumor volumes were essentially the same among the groups. Each test compound was dissolved in DMSO and the solution was stored in a freezer until use. Immediately prior to injection, the mother liquor was diluted in an aqueous sodium chloride solution with 100 μM hydroxypropyl-P-cyclodextrin. Each test compound was administered intravenously at doses ranging from 1/4 MTD to 1/2 MTD in combination with cetuximab (Erbitux, Merck Serono Co., Ltd.). In this case, the experiment was carried out on groups, each of which consisted of 4 mice. Tumor regression at day 35 (%) of each test compound is shown in Table 1. 7.

Table 7

Test compound Dose (mg/kg) Cetuximab (mg/kg) Tumor regression at day 35 (%) - - 20 -242 Galichondrin B 0.0125 20 -38 0.025 20 -2 Connection (1) 0.05 20 38 0.1 20 60

Pharmacological Test Example 8: Antitumor activity in a subcutaneous xenograft model of KPL-4 in combination with trastuzumab in mice.

The HER-2 positive human breast cancer cell line KPL-4, which was cultured in RPMI-1640 medium containing 10% FBS, as well as penicillin and streptomycin, was adjusted to a concentration of 1x10 ⁸ cells/ml using balanced salt solution Hanks to obtain a cell suspension. The cell suspension was inoculated in a volume of 100 μl into the subcutaneous portion of the right flank of 7 week old nude mice (CAnN.CgFoxnlnu/CrlCrlj, female, Charles River Laboratories Japan, Inc.). 16 days after cell inoculation, the shortest diameter and longest tumor diameter of each mouse were measured using an electronic digital caliper (Digimatic™ caliper, Mitutoyo Corporation) to calculate tumor volume according to the following calculation formulas.

Tumor volume (mm ³ )=longest diameter (mm) x shortest diameter (mm) x shortest diameter (mm)/2.

Relative tumor volume (RTV)=o6beM tumor (day X)/tumor volume (first day).

Tumor regression (%)=(1 - minimum RTV)xl00.

Based on the volumes of tumors obtained on the first day of administration, mice were divided into groups so that the average values of the tumor volumes were essentially the same among the groups. The experiment was carried out on groups, each of which consisted of 6 mice. Each test compound was dissolved in DMSO and the solution was stored in a freezer until use. Immediately prior to administration, the mother liquor was diluted in an aqueous sodium chloride solution. The test compound was intravenously administered at a dose of 0.09 mg/kg or 0.18 mg/kg in combination with trastuzumab (Herceptin, Genentech, Inc.). Tumor regression for compound (1) is shown in table. 8.

Table 8

Test compound Dose (mg/kg) Trastuzumab (mg/kg) Tumor regression (%) - - 10 0 Connection (1) 0.09 - 43 0.09 10 83 0.18 - 87 0.18 10 100

Pharmacological Test Example 9. Effect on CD31-positive vessel in a subcutaneous FaDu mouse model.

The FaDu human head and neck squamous cell carcinoma (SCCHN) cell line, which was cultured in RPMI-1640 medium containing 10% FBS, as well as penicillin and streptomycin, was

-30040298 was diluted to a concentration of 5x10 ⁷ cells/ml with PBS to obtain a cell suspension. The cell suspension was inoculated in a volume of 100 μl into the subcutaneous portion of the right flank of 7 week old nude mice (CAnN.Cg-Foxnlnu/CrlCrlj, female, Charles River Laboratories Japan, Inc.). Ten days after cell inoculation, test compound in aqueous sodium chloride solution with 100 μM hydroxypropyl-3-cyclodextrin was intravenously administered at doses ranging from 1/2 MTD to MTD. The experiment was carried out on groups, each of which consisted of 3 mice. Tumor samples were collected 5 days after injection and fixed with Zinc Fixative (BD Pharmingen) at 4°C for 24 h. and air dried. Immunohistochemical staining for CD31 was performed using a Ventana model Discover XT autostainer (Roche Diagnostics) according to the manufacturer's protocol. Sections were deparaffinized, conditioned and antigens were extracted with CC1 (Ventana Medical Systems). The slides were blocked with Blocker A and Blocker B (endogenous biotin blocking kit, Roche Diagnostics). Rat anti-mouse CD31 IgG (Dianova GmbH) was used at a concentration of 2 μg/ml. Sections were incubated with antibody for 6 hours followed by a 32 minute incubation with biotinylated anti-rat IgG antibody (Jackson ImmunoResearch Laboratories) at a concentration of 2.2 μg/ml. Detection was performed with streptavidin-HRP D for 16 minutes followed by incubation with DAB D and DAB H ₂ O ₂ D (DABMap kit, Ventana Medical Systems, Inc) for 8 minutes. Slides were stained with hematoxylin II (Roche Diagnostics) for 16 minutes followed by incubation with Bluing's reagent for 4 minutes. Sections were dehydrated in graded ethanols, degreased when replacing xylene, and coated with DPX (Merck KGaA).

Immunostained slides were scanned using a Vectra 2 Automated Slide Imaging System (Perkin Elmer Inc.). The number of blood vessels in the entire tumor was quantified by counting CD31 positive subjects using inForm 2 software (PerkinElmer Inc.). The area of the tumor area was measured by assessing the area of hematoxylin staining using inForm 2 software (PerkinElmer Inc.). The number of blood vessels was normalized according to the area of the tumor area. The rate of increase in the number of blood vessels in the test compound group was calculated by the formula below and is shown in Table 1. 9.

The ratio of increase in the number of blood vessels (%)=((the number of blood vessels in the group receiving the test compound - the number of blood vessels in the control group)/the number of blood vessels in the control group)x100.

Table 9

Test compound Dose (mg/kg) Increase ratio of blood vessels (%) Galichondrin B 0.025 31 0.05 39 Connection (1) 0.10 69 0.20 154

Pharmacological Test Example 10. Effect on a-SMA-positive CAFs in a subcutaneous FaDu model.

The human head and neck squamous cell carcinoma (SCCHN) cell line FaDu, which was cultured in RPMI-1640 medium containing 10% FBS, as well as penicillin and streptomycin, was adjusted to a concentration of 5x10 ⁷ cells/ml using PBS to obtain a cell suspension . The cell suspension was inoculated in a volume of 100 μl into the subcutaneous portion of the right flank of 5-6 week old nude mice (CAnN.Cg-Foxnlnu/CrlCrlj, female, Charles River Laboratories Japan, Inc.). 10 days after cell inoculation, the test compound in aqueous sodium chloride solution with 100 μM hydroxypropyl-P-cyclodextrin was administered intravenously at a dose of 1/2 MTD and MTD. The experiment was carried out on groups, each of which consisted of 3 mice. Two days after injection, tumor samples were collected and fixed with IHC Zinc Fixative (BD Pharmingen) at 4°C for 24 h. and air dried for 6 hours. Immunohistochemical staining for α-SMA was performed using a Ventana model Discover XT autostainer (Roche Diagnostics). Sections were deparaffinized, conditioned and antigens were extracted using proprietary buffers, EZPrep and CC1 (Ventana Medical Systems). Alkaline phosphatase-conjugated anti-α-SMA monoclonal mouse antibody (clone 1A4, Sigma) was used at a concentration of 5 μg/ml. Sections were incubated with antibody

-31 040298 for 6 hours. Detection was performed using the RedMap kit (Ventana Medical Systems, Inc.). Sections were dehydrated in graded ethanols, degreased when replacing xylene, and coated with DPX (Merck

KGaA). Serial sections of the tumor were deparaffinized and stained with Mayer's hematoxylin (Muto

Pure Chemicals) for 1 min. Sections were dehydrated in graded ethanols, degreased when replacing xylene, and coated with DPX (Merck KGaA).

Immunostained slides were scanned using a Vectra 2 Automated Slide Imaging System (Perkin Elmer Inc.). The area of the a-SMA positive region in the entire tumor was quantified by counting the αSMA positive objects using inForm 2 software (PerkinElmer Inc.). The area of the tumor area was measured by assessing the area of hematoxylin staining using inForm 2 software (PerkinElmer Inc.). The area of the a-SMA positive region was normalized to the area of the tumor region. The suppression ratio of the a-SMA-positive region in the group receiving the test compound was calculated by the formula below, and it is shown in Table 1. 10.

Table 10

Test compound Dose (mg/kg) α-SMA positive suppression ratio (%) Galichondrin B 0.025 7 0.05 3 Connection (1) 0.10 21 0.20 28

α-SMA-positive region suppression ratio (%)=((a-SMA-positive region of the test compound group - α-SMA-positive region of the control group)/aSMA-positive region of the control group)x100.

Pharmacological Test Example 11 Mouse Orthotopic Transplantation Model of HSC-2.

Luciferase-transduced HSC-2-Luc cells were generated by retrovirus-mediated gene transfer. First, a DNA fragment encoding firefly luciferase was obtained from the pGL3 enhancer plasmid (GenBank no: U47297) and subcloned into the pCX4pur retroviral vector (GenBank no: AB086386). Helperless recombinant retroviruses were then obtained by transfection of the above retroviral expression vector together with pGP and pE-Ampho plasmids (Takara Bio; Shiga, Japan) into 293T cells (ATCC; Manassas, USA). The HSC-2 cells were then infected with recombinant retroviruses and cultured for two weeks in the presence of puromycin (2 μg/ml). Infected cells were selected from a polyclonal proliferative culture population.

Under anesthesia, the human SCCHN cell line, HSC-2-Luc, was inoculated into the tongue of female athymic mice (1 x 10 ⁶ cells in 50 μl PBS) 6 weeks of age (CAnN.Cg-Foxnlnu/CrlCrlj mice; Charles River, Inc.; Shizuoka, Japan). 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 administered intraperitoneally to nude mice under inhalation of isoflurane anesthesia at 1 to 2%. The bioluminescence signal was monitored using the IVIS SPECTRUM series (PerkinElmer, Waltham, MA) consisting of a highly sensitive CCD cooled chamber. Living Image software (PerkinElmer, Waltham, MA) was used to grid the image data and integrate the total bioluminescence signal in each region of interest (ROI). All bioluminescent images were obtained with an exposure of 1 s. The data was analyzed using the total emission of the photon flux (photons per second) in the ROI.

Based on the total photon flux irradiance obtained on the first day of administration, the mice were divided into groups so that the average values of the total photon flux irradiance were essentially the same among the groups. Compound (1) or cisplatin was administered intravenously with or without cetuximab (Erbitux, Merck Serono Co., Ltd.) once a week for 3 weeks (scheme Q7Dx3). Two experiments were performed using an identical procedure and all data was collected from the experiments. Each group consisted of 16 mice.

Imaging data showed that compound (1) treatment with cetuximab alone clearly reduced the bioluminescence signal in all mice after day 14 (FIGS. 6A-6B). Mean survival time (MST) was calculated for each treatment group as the median days of death. The increase in lifespan (ILS) was calculated by the following formula: ILS (%)=(MST of animals treated with the test compound - MST of control animals)/MST of control animals x 100. The ILS (%) of each test compound is shown in Table 1. eleven.

- 32 040298

Table 11

Test compound Dose (mg/kg) Cetuximab (mg/kg) ILS (%) - - 5 231 Cisplatin 5 - 0 5 5 150 Connection (1) 0.09 - 238 0.09 5 >1150

Pharmacological Test Example 12 Subcutaneous FaDu Xenograft Model in Combination with Radiation Therapy.

Luciferase-transduced FaDu-Luc cells were generated by retrovirus-mediated gene transfer. First, a DNA fragment encoding firefly luciferase was obtained from the pGL3 enhancer plasmid (GenBank no: U47297) and subcloned into the pCX4pur retroviral vector (GenBank no: AB086386). Helperless recombinant retroviruses were then obtained by transfection of the above retroviral expression vector together with pGP and pE-Ampho plasmids (Takara Bio; Shiga, Japan) into 293T cells (ATCC; Manassas, USA). FaDu cells were then infected with recombinant retroviruses and cultured for two weeks in the presence of puromycin (2 μg/ml). Infected cells were selected from a polyclonal proliferative culture population.

Luciferase-transduced human cell line FaDu-Luc SCCHN, which was cultured in RPMI-1640 medium containing 10% FBS, as well as penicillin and streptomycin, was adjusted to a concentration of 5 x 10 ⁷ cells/ml using Hank's balanced salt solution to obtain a cell suspension . The cell suspension was inoculated in a volume of 100 μl into the subcutaneous portion of the right thigh of 6 week old nude mice (CAnN.CgFoxnlnu/CrlCrlj, female, Charles River Laboratories Japan, Inc.). 13 days after cell inoculation, 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 administered intraperitoneally to nude mice under inhalation of isoflurane anesthesia at 1 to 2%. The bioluminescence signal was monitored using the IVIS SPECTRUM series (PerkinElmer, Waltham, MA) consisting of a highly sensitive CCD cooled chamber. Living Image software (PerkinElmer, Waltham, MA) was used to grid the image data and integrate the total bioluminescence signal in each region of interest (ROI). All bioluminescent images were obtained with an exposure of 1 s. The data was analyzed using the total emission of the photon flux (photons per second) in the ROI. The total radiation of the photon flux was calculated in accordance with the following calculation formulas.

Relative bioluminescence level=total photon flux irradiance (day X)/total photon flux irradiance (first day).

Tumor regression (%)=(1 - minimum relative level of bioluminescence)x100.

Based on the total photon flux irradiance obtained on the first day of administration, the mice were divided into groups so that the average values of the total photon flux irradiance were essentially the same among the groups. The experiment was carried out on groups, each of which consisted of 6 mice. Compound (1) was administered by tail vein injection on days 1 and 8. Irradiation was performed at a dose of 18 Gy on days 4 and day 11. Tumor regression for compound (1) is shown in Table 1. 12.

Table 12

Test compound Dose (mg/kg) Irradiation (Gy) Tumor regression (%) - - 18 16 Connection (1) 0.09 - 0 0.09 18 97

Pharmacological Test Example 13 Antitumor activity in a subcutaneous syngeneic model of CT26 in combination with an anti-mPD-1 antibody in mice.

The murine undifferentiated colon carcinoma cell line CT26, which was cultured in RPMI-1640 medium containing 10% FBS, as well as penicillin and streptomycin, was adjusted to a concentration of 2 x 10 ⁷ cells/ml using Hank's balanced salt solution to obtain a cell suspension. On day 1, the cell suspension was inoculated in a volume of 100 μl into the right flank subcutaneous of 6 week old BALB/c mice.

- 33 040298 (BALB/cAnNCrlCrlj, females, Charles River Laboratories Japan, Inc.). 2 days after cell inoculation, mice were randomly divided into 4 groups, and each group consisted of 8 mice. The shortest diameter and the longest tumor diameter of each mouse were measured using an electronic digital caliper (Digimatic™ caliper, Mitutoyo Corporation) to calculate the tumor volume according to the following calculation formula.

Tumor volume (mm ³ )=longest diameter (mm) x shortest diameter (mm) x shortest diameter (mm)/2.

T/C=(mean tumor volume of treated group)/mean tumor volume of control group).

Tumor growth inhibition (%)=(1-T/C)x100.

Study compound was administered intravenously at a dose of 0.09 mg/kg on days 3 and 11. Anti-mPD-1 antibody (BE0146, Bio X Cell) was administered intravenously at a dose of 10 mg/kg on days 3, 7, 11 and 15. Tumor growth inhibition at day 15 (%) of each test compound is shown in Table 1. 13.

Table 13

Test compound Dose (mg/kg) Antibody to mPD- 1 (mg/kg) Tumor growth inhibition at day 15 (%) - - 10 32 Connection (1) 0.09 - thirty 0.09 10 62

Pharmacological test example 14 Effect on in vitro tubulin polymerization (FIG. 10A).

The tubulin polymerization assay kit was purchased from Cytoskeleton, Inc. (Cat No.: VK011R). The kit contained 1 vial of lyophilized porcine brain purified tubulin protein, 3 vials of lyophilized GTP, 2 vials of lyophilized assay buffer, and 1 vial of glycerol-based tubulin buffer. The assay buffer was prepared by dissolving the contents in 10 ml of deionized and sterilized water. This solution contained 80 mmol/l sodium sesquisalt and piperazine-N,N'-bis[2-ethanesulfonic acid], 2.0 mmol/l magnesium chloride, 0.5 mmol/l ethylene glycol bis(2-aminoethyl ether) N, N,N',N'-tetraacetic acid, pH 6.9 and 10 µmol/L fluorescent reporter. The buffer was stored at -70°C until use. Glycerol-based tubulin buffer consisted of 80 mmol/L sodium sesquisalt and piperazine-N,N'-bis[2-ethanesulfonic acid], 2.0 mmol/L magnesium chloride, 0.5 mmol/L ethylene glycol bis(2 -aminoethyl ether) N,N,N',N'-tetraacetic acid and 60% v/v. glycerol, pH 6.9. It was stored at 4°C until use. A stock solution of GTP was prepared by dissolving the contents of each tube in 100 μl of deionized and sterilized water to reach a concentration of 100 mmol/l GTP. Aliquots of this stock solution were stored at -70°C until use. Tubulin stock solution (10 mg/ml) was prepared by dissolving tubulin powder with the addition of 1.1 ml of a mixture of assay buffer and GTP stock solution (100:1, v/v). Aliquots were frozen in liquid nitrogen and then stored at -70°C until use.

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 glycerol-based tubulin buffer. The reaction mixture (1015 μl) was combined with 240 μl of tubulin stock solution. This solution was called the tubulin reaction mixture and was used for measurement in test and control wells. The reaction mixture without tubulin was prepared by mixing 89.85 μl of the reaction mixture and 21.25 μl of assay buffer to measure blank wells. A solution of compound (1) (6.25-100 µmol/L; final concentrations 0.625-10 µmol/L) or vehicle was added in a volume of 5 µl to individual wells of a half-well 96-well microtiter plate. The tubulin reaction mixture or the reaction mixture without tubulin was added at 45 μl to each well of the plate. Fluorescence emission at 460 nm (excitation wavelength at 360 nm) was measured every 2 minutes for 90 minutes using a SpectraMax® M5e microplate reader (Molecular Devices). Polymerization of tubulin was accompanied by an increase in fluorescence due to incorporation of a fluorescence reporter into microtubules as polymerization proceeded. The analysis was performed in two parallels. The analysis showed that compound (1) inhibited tubulin polymerization in a concentration-dependent manner. The fluorescence intensity at each time point was calculated using the following formulas.

Fluorescence intensity=average fluorescence measurement in test or control wells - average fluorescence measurement in blank wells; blank well: with carrier medium without tubulin; control well: with carrier medium and tubulin; test well: with compounds and tubulin.

Pharmacological test example 15. Cellular microtubule dynamics analysis (FIG. 10B).

Cellular analysis of microtubule (MT) dynamics was performed with the U2OS-EB3-AG osteosarcoma cell line, which stably expressed the EB3 fusion protein (microtubule together with the terminal binding protein) and Azami-Green (EB3-AG). U2OS-EB3-AG cells were cultured in RPMI-1640 medium containing 10% FBS and penicillin-streptomycin at 37° C. in a humidified atmosphere with 5% CO ₂ . The dynamics of MT in living cells can be visualized as the movement of the comet-like structure EB3-AG. U2OS-EB3-AG cells plated on glass-based culture plates (EZVIEW plate, AGC Techno Glass, Japan) were treated with compound (1) at the indicated concentration, and microtubule dynamics were monitored by frame-by-frame imaging using an oil-oil fluorescence microscope. lens with 60x magnification (BZX710, KEYENCE, Japan). Still images at the indicated time points are shown in FIG. 10V. Views with higher magnification of the framed areas are shown in the insets. When treated with compound (1) at a concentration of 0.5 nM (IC ₅₀ value for anti-proliferative activity in U2OS-EB3-AG cells), comet-like structures became difficult to observe approximately 60 minutes after addition of the compound. These results clearly demonstrated that the compound (1) has the ability to suppress the dynamics of MT.

Pharmacological Test Example 16 In vitro antiproliferative activity (FIG. 11).

In vitro antiproliferative assays for compound (1) were 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-Dx5Rxl). All cell lines were cultured in RPMI-1640 medium containing 10% FBS as well as penicillin-streptomycin (culture medium) in a 5% CO2 incubator (37°C). To each well of a 96-well plate (Becton, Dickinson and Company, 353219) was added 75 μl of cell suspension adjusted to a concentration of 4x10 ⁴ cells/ml with culture medium, and the cells were incubated overnight in a 5% CO ₂ incubator (37°C). The next day, 25 μl of compound (1) in a 3-fold dilution series suspended in culture medium was added to each well, and the resulting product was incubated for 72 hours in a 5% CO2 (37°C) incubator. Cell viability was then determined using a CellTiter-Glo® (Promega) fluorescent cell viability assay using a 2013 EnVision™ multichannel reader (Perkin-Elmer, Wellesley, MA). The value of wells containing cells without addition of test compounds was defined as 100% and the value of wells containing no cells was defined as 0%. The concentration of compound (1) required to inhibit cell growth by 50% (ie, IC ₅₀ value) was calculated and is shown in FIG. 11. P-gp responsiveness was calculated as the ratio of the IC ₅₀ value in MES-SA-Dx5-Rxl cells that overexpress P-gp to the IC ₅₀ b value in MES-SA cells.

Pharmacological Test Example 17: Antitumor effects in mice xenograft models of KPL-4 as monotherapy; antitumor effects in COLO-704 xenograft models in mice as monotherapy (Fig. 12).

The HER-2 positive human breast cancer cell line KPL-4, which was cultured in DMEM containing 10% FBS as well as penicillin-streptomycin, was adjusted to a concentration of 1x108 cells/mL with Hank's balanced salt solution to obtain cell suspension. The cell suspension was inoculated in a volume of 100 μl into the subcutaneous portion of the right flank of 8 week old nude mice (CAnN.CgFoxnlnu/CrlCrlj, female, Charles River Laboratories Japan Inc.). 11 days after cell inoculation (Day 1), the shortest diameter and longest tumor diameter of each mouse were measured using an electronic digital caliper (Digimatic™ caliper, Mitutoyo Corporation) to calculate tumor volume according to the following calculation formulas.

Tumor volume (mm ³ )=longest diameter (mm) x shortest diameter (mm) x shortest 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).

Based on the volumes of tumors obtained on day 1, mice were divided into groups so that the average values of the tumor volumes were essentially the same among the groups. The experiment was carried out on groups, each of which consisted of six mice. The test compound was dissolved in DMSO and the solution was stored in a freezer prior to use. Immediately prior to administration, the mother liquor was diluted with an aqueous sodium chloride solution. The test compound in aqueous sodium chloride solution was administered intravenously once a week at a dose of 20 μg/kg, 60 μg/kg or 180 μg/kg for 2 weeks (day 1 and day 8). Tumor regression was observed in the 60 μg/kg and 180 μg/kg treated groups, and administration at 180 μg/kg completely abolished xenograft tumors in all mice by day 15.

The human ovarian cancer cell line COLO-704, which was cultured in RPMI-1640 medium containing 10% FBS as well as penicillin-streptomycin, was adjusted to a concentration of 1x108 cells/ml with Hank's balanced salt solution to obtain a cell suspension. The cell suspension was inoculated in a volume of 100 μl into the subcutaneous portion of the right flank of athymic mice 5 weeks old (CAnN.Cg-Foxnlnu/CrlCrlj, female, Charles River

- 35 040298

Laboratories Japan Inc.). Nine days after cell inoculation (day 1), the shortest diameter and longest tumor diameter of each mouse were measured using an electronic digital caliper (Digimatic™ caliper, Mitutoyo Corporation) to calculate tumor volume according to the following calculation formulas.

Tumor volume (mm ³ )=longest diameter (mm) x shortest diameter (mm) x shortest 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).

Based on the volumes of tumors obtained on day 1, mice were divided into groups so that the average values of the tumor volumes were essentially the same among the groups. The experiment was carried out on groups, each of which consisted of six mice. The test compound was dissolved in DMSO and the solution was stored in a freezer prior to use. Immediately prior to administration, the mother liquor was diluted with an aqueous sodium chloride solution. The test compound in aqueous sodium chloride solution was administered intravenously once a week at a dose of 20 μg/kg, 60 μg/kg or 180 μg/kg for 2 weeks (day 1 and day 8). Treatment with the compound induced tumor regression at the 180 μg/kg dose and tumor growth retardation at the 60 μg/kg dose. Administration at a dose of 180 μg/kg completely abolished xenograft tumors in all mice on day 22.

Pharmacological Test Example 18. Effect on CD31-positive vessel in subcutaneous FaDu mouse model (FIG. 13).

The FaDu human head and neck squamous cell carcinoma (SCCHN) cell line, which was cultured in RPMI-1640 medium containing 10% FBS as well as penicillin-streptomycin (culture medium), was adjusted to a concentration of 5 x 10 ⁷ cells/ml with culture media to obtain a cell suspension. The cell suspension was inoculated in a volume of 100 μl into the subcutaneous portion of the right flank of 6 week old nude mice (CAnN.CgFoxnlnu/CrlCrlj, female, Charles River Laboratories Japan, Inc.). Ten days after cell inoculation, mice were grouped such that mean tumor volumes were substantially the same across groups. The experiment was carried out on groups, each of which consisted of 6 mice. Each test compound was dissolved in DMSO and the solution was stored in a freezer prior to use. Immediately prior to administration, the mother liquor was diluted with an aqueous sodium chloride solution. The test compound in aqueous sodium chloride was intravenously administered at a dose of 20 μg/kg, 60 μg/kg, or 180 μg/kg. Five days after a single injection, tumor samples were collected and fixed with IHC Zinc Fixative (BD Pharmingen) at 4°C for 24 h. glass and air dried. Immunohistochemical staining for CD31 was performed using a Ventana model Discover XT autostainer (Roche Diagnostics) according to the manufacturer's protocol. Sections were deparaffinized, conditioned and antigens were extracted with CC1 (Ventana Medical Systems). The slides were blocked with Blocker A and Blocker B (endogenous biotin blocking kit, Roche Diagnostics). Rat anti-mouse CD31 IgG (Dianova GmbH) was used at a concentration of 2 μg/ml. Sections were incubated with antibody for 6 hours followed by a 32-minute incubation with biotinylated anti-rat IgG antibody (Jackson ImmunoResearch Laboratories) at a concentration of 2.2 μg/ml. Detection was performed with streptavidin-HRP D for 16 minutes followed by incubation with DAB D and DAB H ₂ O ₂ D (DABMap kit, Ventana Medical Systems, Inc.) for 8 minutes. Slides were stained with hematoxylin II (Roche Diagnostics) for 16 min followed by incubation with Bluing's reagent for 4 min. Sections were dehydrated in graded ethanols, degreased when replacing xylene, and coated with DPX® (Merck KGaA). Immunostained slides were scanned using the Vectra® 2 Automated Slide Imaging System (Perkin Elmer Inc.). The number of blood vessels in the entire tumor was quantified by counting CD31-positive subjects using inForm 2 software (PerkinElmer Inc.). The area of the tumor area was measured by assessing the area of hematoxylin staining using inForm 2 software (PerkinElmer Inc.). The number of blood vessels was normalized according to the area of the tumor area. Single administration of the test compound at doses of 20, 60 and 180 μg/kg increased the number of tumor blood vessels. The ratios of the number of blood vessels in the test compound treated groups compared to the untreated group were calculated using the formula below.

Tumor vessel ratio=number of blood vessels in test compound group/number of blood vessels in untreated group).

Pharmacological Test Example 19. Effect on a-SMA-positive CAFs in a subcutaneous FaDu mouse model (FIG. 14).

The FaDu human head and neck squamous cell carcinoma (SCCHN) cell line, which

- 36 040298 were cultured in RPMI-1640 medium containing 10% FBS as well as penicillin-streptomycin (culture medium) adjusted to a concentration of 5×10 ⁷ cells/ml using the culture medium to obtain a cell suspension. The cell suspension was inoculated in a volume of 100 μl into the subcutaneous portion of the right flank of 6 week old nude mice (CAnN.Cg-Foxnlnu/CrlCrlj, female, Charles River Laboratories Japan, Inc.). Ten days after cell inoculation, mice were grouped such that mean tumor volumes were substantially the same across groups. The experiment was carried out on groups, each of which consisted of 5 mice. Each test compound was dissolved in DMSO and the solution was stored in a freezer prior to use. Immediately prior to administration, the mother liquor was diluted with an aqueous sodium chloride solution. The test compound in aqueous sodium chloride was intravenously administered at doses of 20 μg/kg, 60 μg/kg, or 180 μg/kg. Two days or five days after a single injection, tumor samples were collected and fixed with IHC Zinc Fixative (BD Pharmingen) at 4°C for 24 h. positively charged glass slides and dried in air. Sections were deparaffinized, conditioned and antigens were extracted using a microwave oven with 1 mM EDTA at pH 6.0. Sections were blocked with 1% BSA in TBS. Alkaline phosphatase-conjugated anti-α-SMA monoclonal mouse antibody (clone 1A4, Sigma) was used at a concentration of 5 μg/ml. Sections were incubated with antibody for 2.5 hours. Detection was performed using the Fast red II substrate kit (Nichirei Bioscience Inc.). Sections were counterstained with Mayer's hematoxylin (Muto Pure Chemicals) for 50 seconds. Sections were dehydrated in graded ethanols, degreased when replacing xylene, and coated with DPX (Merck KGaA). Immunostained slides were scanned using a Vectra 2 Automated Slide Imaging System (Perkin Elmer Inc.). The area of a-SMA-positive region in the entire tumor was quantified by counting a-SMA-positive objects using inForm 2 software (PerkinElmer Inc.). The area of the tumor area was measured by assessing the area stained with hematoxylin using inForm 2 software (PerkinElmer Inc.). The area of the a-SMA positive region was normalized to the area of the tumor region. Single administration of study compound significantly reduced the a-SMA positive area at doses of 60 and 180 µg/kg on day 3 and at 180 µg/kg on day 6. group compound was calculated by the formula below.

The ratio a-SMA=a-SMA-positive area of the group that received the test compound/a-SMA-positive area of the group that did not receive treatment.

Pharmacological test example 20 Effects on tenascin-C and EDA-fibronectin in a subcutaneous FaDu mouse model (FIG. 15).

The FaDu human head and neck squamous cell carcinoma (SCCHN) cell line, which was cultured in RPMI-1640 medium containing 10% FBS as well as penicillin-streptomycin (culture medium), was adjusted to a concentration of 5 x 10 ⁷ cells/ml with culture media to obtain a cell suspension. The cell suspension was inoculated in a volume of 100 μl into the subcutaneous portion of the right flank of 6 week old nude mice (CAnN.Cg-Foxnlnu/CrlCrlj, female, Charles River Laboratories Japan, Inc.). 10 days after cell inoculation, mice were divided into groups so that the average values of tumor volumes were essentially the same among the groups. The experiment was carried out on groups, each of which consisted of 5 mice. Compound (1) was dissolved in DMSO and the solution was stored in a freezer before use. Compound (1) (180 μg/kg) and cetuximab (CTX, Erbitux®, Merck Serono Co. Ltd.) (10 mg/kg) were diluted with aqueous sodium chloride solution and intravenously injected on day 1. Five days after single injection, tumor samples were collected and fixed with IHC Zinc Fixative (BD Pharmingen) at 4°C for 24 h. Sections were deparaffinized, conditioned and antigens were extracted using a microwave oven with 1 mM EDTA pH 6.0 for tenascin-C. For EDA-fibronectin, the antigen extraction procedure was not necessary. Sections were incubated with BLOXALL blocking solution (Vector Labs) for 10 minutes to block endogenous peroxidase and Mouse on Mouse Ig Blocking Reagent (Vector Labs) for 1 hour and then with 2.5% normal horse serum for 30 minutes. . For immunohistochemical staining of tenascin-C, a mouse monoclonal antibody to tenascin-C (clone 4C8MS, IBL) was used at a concentration of 5 μg/ml. Sections were incubated with antibody overnight at 4°C. For immunohistochemical staining of EDA fibronectin, a mouse monoclonal antibody to EDA fibronectin (clone IST-9, Abcam) was used at a concentration of 1.5 μg/ml. Sections were incubated with antibody for 1 h at room temperature. Detection was performed using the Mouse On Mouse ImmPRESS™ Peroxidase Polymer Kit (Vector Labs). Sections were counterstained with Mayer's hematoxylin (Muto Pure Chemicals) in

- 37 040298 for 50 s. Sections were dehydrated in graded ethanols, degreased when replacing xylene, and coated with DPX (Merck KGaA). Immunostained slides were scanned using a Vectra 2 Automated Slide Imaging System (Perkin Elmer Inc.). Expression levels of both tenascin-C and ED-A fibronectin were reduced in tumors treated with compound (1) and CTX compared to control tumors.

Pharmacological Test Example 21 Antitumor effects in a subcutaneous FaDu xenograft model in combination with cetuximab in mice (Fig. 16).

The FaDu human head and neck squamous cell carcinoma (SCCHN) cell line, which was cultured in RPMI-1640 medium containing 10% FBS and penicillin-streptomycin, was adjusted to a concentration of 5 x 10 ⁷ cells/ml with Hank's balanced salt solution to obtain cell suspension. The cell suspension was inoculated in a volume of 100 μl into the subcutaneous portion of the right flank of nude mice (CAnN.Cg-Foxnlnu/CrlCrlj, female, 7 weeks old, Charles River Japan Inc.). Ten days after cell inoculation (Day 1), the length and width of the tumor in each mouse was measured using an electronic digital caliper (Mitutoyo Corporation) to calculate the tumor volume according to the following calculation formula.

Tumor volume (mm ³ )=longest diameter (mm) x shortest diameter (mm) x shortest diameter (mm)/2.

Relative tumor volume (RTV)=tumor volume (day X)/tumor volume (first day).

Based on TV, mice were randomized into groups (Day 1). Each group consisted of six mice. Compound (1) was dissolved in DMSO and the solution was stored in a freezer before use. Compound (1) (20, 60, or 180 μg/kg) and cetuximab (CTX, Erbitux®, Merck Serono Co., Ltd.) (10 mg/kg) were diluted with aqueous sodium chloride and injected intravenously on day 1 The RTV changes of each group are shown in FIG. 16. At doses of 180 μg/kg and 60 μg/kg, the antitumor efficacy of compound (1) with CTX was stronger than that of CTX monotherapy, with tumor regression. The antitumor efficacy of Compound (1) at doses of 20 μg/kg in combination with CTX was generally stronger than that of CTX alone.

Pharmacological Test Example 22 Antitumor Effects in Mouse Xenograft Models of Soft Tissue Sarcoma as Monotherapy (FIG. 17).

MES SA.

The human uterine sarcoma cell line MES-SA, which was cultured in RPMI-1640 medium containing 10% FBS and penicillin-streptomycin, was adjusted to a concentration of 2 x 10 ⁸ cells/ml with Hank's balanced salt solution to obtain a cell suspension, and the suspension was mixed with Geltrex® (Thermo Fisher Scientific Inc., Cat # A1413202) at a ratio of 1:1 to obtain a cell suspension at a concentration of 1 x 10 ⁸ cells/ml. The cell suspension was inoculated in a volume of 100 μl into the subcutaneous portion of the right flank of 6 week old nude mice (CAnN.Cg-Foxnlnu/CrlCrlj, female, Charles River Laboratories Japan Inc.). 6 days after cell inoculation (Day 1), the shortest diameter and longest tumor diameter of each mouse were measured using an electronic digital caliper (Digimatic™ caliper, Mitutoyo Corporation) to calculate the tumor volume according to the following calculation formulas.

Tumor volume (mm ³ )=longest diameter (mm) x shortest diameter (mm) x shortest diameter (mm)/2.

Relative tumor volume (RTV)=tumor volume (day X)/tumor volume (first day).

Based on the volumes of tumors obtained on day 1, mice were divided into groups so that the average values of the tumor volumes were essentially the same among the groups. The experiment was carried out on groups, each of which consisted of 6 mice. The test compound was dissolved in DMSO and the solution was stored in a freezer prior to use. Immediately prior to administration, the mother liquor was diluted with an aqueous sodium chloride solution. The test compound in aqueous sodium chloride solution was administered intravenously once a week at a dose of 180 μg/kg for 2 weeks (day 1 and day 8). Antitumor activity was observed with delayed tumor growth in the treated group.

NT-1080.

The human fibrosarcoma cell line HT-1080, which was cultured in E-MEM medium containing 10% FBS, NEAA and antibiotics, was adjusted to a concentration of 3x10 ⁷ cells/ml using a cell suspension medium. The cell suspension was inoculated in a volume of 100 μl into the subcutaneous part of the right flank of nude mice (CAnN.Cg-Foxnlnu/CrlCrlj, female, 6 weeks old, Charles River Japan Inc.). 6 days after cell inoculation (day 1), the length and width of the tumor in each mouse was measured using an electronic digital caliper (Mitutoyo Corporation) to calculate the tumor volume according to the following calculation formula.

- 38 040298

Tumor volume (mm ³ )=longest diameter (mm) x shortest diameter (mm) x shortest diameter (mm)/2.

Relative tumor volume (RTV)=tumor volume (day X)/tumor volume (first day).

Based on TV, mice were randomized into groups (Day 1). Each group consisted of six mice. Compound (1) was dissolved in DMSO and the solution was stored in a freezer before use. Compound (1) (180 μg/kg) was diluted with aqueous sodium chloride and administered intravenously by injection on day 1 and day 8. The change in RTV of each group is shown in FIG. 17. Antitumor activity was observed with tumor regression in the treated group.

CTG-2041.

Fragments of human angiosarcoma tumor CTG-2041 were implanted subcutaneously in the left flank of female mice. Tumor growth was monitored twice a week using a digital caliper to calculate the tumor volume according to the following calculation formula.

Tumor volume (mm ³ )=longest diameter (mm) x shortest diameter (mm) x shortest diameter (mm)/2.

Relative tumor volume (RTV)=tumor volume (day X)/tumor volume (first day).

When the volume of the tumors reached approximately 200 mm ³ , the animals were assigned by tumor volume to the treatment or control groups and dosed on day 1. Each group consisted of five mice. Compound (1) was dissolved in DMSO and the solution was stored in a freezer before use. Compound (1) (100 μg/kg) was diluted in aqueous sodium chloride solution and intravenously administered by injection on day 1 and day 8. Changes in RTV of each group are shown in FIG. 17. Antitumor activity was observed with tumor regression in the treated group.

Pharmacological Test Example 23 Antitumor Effects in Mouse Endometrial Sarcoma Xenograft Models as Monotherapy (FIG. 18).

NES-108.

The human endometrial cancer cell line HEC-108, which was cultured in E-MEM medium containing 15% FBS and antibiotics, was adjusted to a concentration of 7.14 x 10 ⁷ cells/ml using a cell suspension medium. The cell suspension was inoculated in a volume of 150 μl into the subcutaneous portion of the right flank of nude mice (CAnN.Cg-Foxnlnu/CrlCrlj, female, 6 weeks old, Charles River Japan Inc.). 13 days after cell inoculation (Day 1), the length and width of the tumor in each mouse was measured using an electronic digital caliper (Mitutoyo Corporation) to calculate the tumor volume according to the following calculation formula.

Tumor volume (mm ³ )=longest diameter (mm) x shortest diameter (mm) x shortest diameter (mm)/2.

Relative tumor volume (RTV)=tumor volume (day X)/tumor volume (first day).

Based on TV, mice were randomized into groups (Day 1). Each group consisted of six mice. Compound (1) was dissolved in DMSO and the solution was stored in a freezer before use. Compound (1) (180 μg/kg) was diluted in aqueous sodium chloride solution and administered intravenously by injection on day 1 and day 8. Changes in RTV of each group are shown in FIG. 18. Antitumor activity was observed with tumor growth inhibition in the treated group.

AN3CA.

The human endometrial cancer cell line AN3CA, which was cultured in E-MEM medium containing 10% FBS and penicillin-streptomycin, was adjusted to a concentration of 1.4 x 10 ⁸ cells/ml with Hank's balanced salt solution to obtain a cell suspension, and the suspension was mixed with Geltrex® (Thermo Fisher Scientific Inc., cat no: A1413202) in a ratio of 1:1 to obtain a cell suspension at a concentration of 7x10 ⁷ cells/ml. The cell suspension was inoculated in a volume of 100 μl into the subcutaneous portion of the right flank of athymic mice 6 weeks old (CAnN.Cg-Foxnlnu/CrlCrlj, female, Charles River Laboratories Japan Inc.). 12 days after cell inoculation (day 1), the shortest diameter and longest tumor diameter of each mouse were measured using an electronic digital caliper (Digimatic™ caliper, Mitutoyo Corporation) to calculate tumor volume according to the following calculation formulas.

Tumor volume (mm ³ )=longest diameter (mm) x shortest diameter (mm) x shortest diameter (mm)/2.

Relative tumor volume (RTV)=tumor volume (day X)/tumor volume (first day).

Based on the volumes of tumors obtained on day 1, mice were divided into groups so that the average values of the tumor volumes were essentially the same among the groups. The experiment was carried out on groups, each of which consisted of five mice. The test compound was dissolved in DMSO and the solution was stored in a freezer prior to use. Directly

- 39 040298 before the introduction of the mother liquor was diluted with an aqueous solution of sodium chloride. The test compound in aqueous sodium chloride solution was intravenously administered once a week at a dose of 180 μg/kg for 2 weeks (Day 1 and Day 8). Antitumor activity was observed with tumor regression in the treated group.

Pharmacological test example 24: NCI-N87 and MKN-28 growth inhibition assay.

This assay measured the growth inhibitory activities of test compounds in human gastric cancer cell lines NCI-N87 and MKN-28, respectively. NCI-N87 and MKN-28 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) was added 100 μl of a suspension of NCI-N87 or MKN-28 cells adjusted to a concentration of 3x10 ⁴ cells/ml using culture medium, and the cells were incubated in overnight in an incubator with 5% CO ₂ (37°C). The next day, 100 μl of compound (1) or halichondrin B in a 3-fold dilution series suspended in culture medium was added to each well, and the resulting product was incubated for 3 days in a 5% CO ₂ (37° C.) incubator. Cell viability was then determined using a CellTiter-Glo® fluorescent cell viability assay (Promega) using an EnVision 2103 multichannel reader (Perkin-Elmer, Wellesley, MA). The value of wells containing cells without addition of test compounds was defined as 100% and the value of wells containing no cells was defined as 0%. The concentrations of test compound required to inhibit cell growth by 50% (ie, 1C ₅₀ value) were calculated and are shown in Table 1. 14.

Table 14

Test compound NCI-N87 (IC50 (nM)) MKN-28 (IC50 (nM)) Galichondrin B 0.007 0.017 Connection (1) 0.002 0.015

Pharmacological test example 25 Growth inhibition assay.

HuTu 80 This assay measured the growth inhibitory activities of test compounds in the human small intestine cancer cell line HuTu 80, which was isolated from duodenal tissue. HuTu 80 cells were maintained in EMEM medium containing 10% FBS, penicillin and streptomycin in a 5% CO ₂ incubator (37°C). To each well of a 96-well plate (Becton, Dickinson and Company, 353219) was added 100 μl of a suspension of HuTu80 cells adjusted to a concentration of 3x10 ⁴ cells/ml with culture medium, and the cells were incubated overnight in an incubator with 5 % CO ₂ (37°C). The next day, 100 μl of compound (1) or halichondrin B in a 3-fold dilution series suspended in culture medium was added to each well, and the resulting product was incubated for 3 days in a 5% CO ₂ (37° C.) incubator. Cell viability was then determined using a CellTiter-Glo® fluorescent cell viability assay (Promega) using an EnVision 2103 multichannel reader (Perkin-Elmer, Wellesley, MA). The value of wells containing cells without addition of test compounds was defined as 100% and the value of wells containing no cells was defined as 0%. The concentrations of test compounds required to inhibit cell growth by 50% (ie 1C ₅₀ value) were calculated and are shown in Table 1. 15.

Table 15

Test compound HuTu 80 (1C ₅ o (nM)) Galichondrin B 0.031 Connection (1) 0.019

Pharmacological Test Example 26 SW780 Growth Inhibition Assay.

This assay measured the growth inhibitory activities of test compounds in the SW780 human urothelial cancer cell line. SW780 cells were maintained in RPMI-1640 medium containing 10% FBS, penicillin and streptomycin in a 5% CO ₂ incubator (37°C). To each well of a 96-well plate (Becton, Dickinson and Company, 353219), 100 μl of a suspension of SW780 cells adjusted to a concentration of 3x10 ⁴ cells/ml with culture medium was added and the cells were incubated overnight in an incubator with 5% CO ₂ (37°C). The next day, 100 μl of compound (1) or halichondrin B in a 3-fold dilution series suspended in culture medium was added to each well, and the resulting product was incubated for 3 days in a 5% CO ₂ (37° C.) incubator. Cell viability was then determined using a CellTiter-Glo fluorescent cell viability assay (Promega using a multi-channel reader).

-40040298

EnVision 2103 (Perkin-Elmer, Wellesley, MA). The value of wells containing cells without addition of test compounds was defined as 100% and the value of wells containing no cells was defined as 0%.

The concentrations of the studied compounds necessary for the inhibition of cell growth on

50% (ie the value of IC ₅₀ ), and they are shown in table. 16.

Table 16

SW780

Test compound (ICso (nM))

Halichondrin B 0.032

Compound (1) 0.017

Pharmacological Test Example 27: HS-SY-II Growth Inhibition Assay.

This assay measured the growth inhibitory activities of test compounds in the human synovial sarcoma cell line HS-SY-II. SH-SY-II cells were maintained in DMEM 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) was added 100 μl of a suspension of HSSY-II cells adjusted to a concentration of 3 x 10 ⁴ cells/ml with culture medium, and the cells were incubated overnight in a 5 % CO2 (37°C). The next day, 100 μl of compound (1) or halichondrin B in a 3-fold dilution series suspended in culture medium was added to each well, and the resulting product was incubated for 3 days in a 5% CO2 (37° C.) incubator. Cell viability was then determined using a CellTiter-Glo fluorescent cell viability assay (Promega) using an EnVision 2103 multichannel reader (Perkin-Elmer, Wellesley, MA). The value of wells containing cells without addition of test compounds was defined as 100% and the value of wells containing no cells was defined as 0%. The concentrations of test compounds required to inhibit cell growth by 50% (ie IC ₅₀ value) were calculated and are shown in Table 1. 17.

Table 17

HS-SY-II

Test compound (ICso (nM))

Halichondrin B 0.010

Compound (1) 0.002

Equivalents and volume.

In the claims, singular forms may mean one or more than one, unless otherwise indicated or otherwise evident from the context. Claims or descriptions that include or between one or more group members are considered to be executable if one, more than one, or all of the group members are present, used, or otherwise related to a given product or process, unless otherwise noted or otherwise obvious from the context. The invention includes embodiments in which exactly one member of the group is present, used, or otherwise related to a given product or process. The present invention includes embodiments in which more than one or all members of the group are present, used, or otherwise related to a given product or process.

In addition, the invention covers all variations, combinations and permutations in which one or more of the features, elements, parts of the claims and descriptive expressions from one or more of the listed claims are introduced in another claim. For example, any claim that depends on another claim can be modified to include one or more of the features found in any other claim that depends on the same main claim. When elements are presented as lists, such as in the Markush group format, each subgroup of elements is also disclosed and any element(s) may be removed from the group. It should be understood that, in general, when the present invention or aspects of the present invention are referred to as including specific elements and/or features, certain embodiments of the present invention or aspects of the present invention consist or consist essentially of such elements and/or features. For the sake of simplicity, these embodiments have not been specifically set forth in the same terms herein. It is also noted that the terms including and containing are intended to be open-ended and allow inclusion of additional elements or steps. Where ranges are specified, endpoints are also included. In addition, unless otherwise indicated or obvious from the context and understanding of a person skilled in the art, values that are expressed as ranges can take on any particular value or subrange within the specified ranges in various embodiments of the present invention to the nearest tenth of the lower

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Claims (65)

range limits, unless the context clearly dictates otherwise. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited by the above description, but is as set forth in the appended claims. Those skilled in the art will appreciate that various changes and modifications to this disclosure may be made without departing from the spirit or scope of the present invention as defined in the following claims. CLAIM 1. Connection of the following structure: or a pharmaceutically acceptable salt thereof. 2. A pharmaceutical composition containing a compound according to claim 1, or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier. 3. The use of a compound or a pharmaceutically acceptable salt thereof according to claim 1 for the treatment of a tumor or cancer. 4. The use of a compound or a pharmaceutically acceptable salt thereof according to claim 1 for inhibiting the growth of a tumor or cancer. 5. The use of a compound or a pharmaceutically acceptable salt thereof according to claim 1 for inhibiting the growth of a tumor or cancer characterized by angiogenesis, invasion or metastasis. 6. The use of a compound or a pharmaceutically acceptable salt thereof according to claim 1 for the treatment of a tumor or cancer characterized by angiogenesis, invasion or metastasis. 7. Use according to any one of claims 3 to 6, wherein the cancer or tumor is head and neck cancer, breast cancer, esophageal cancer, uterine cancer, ovarian cancer, colon and rectal cancer, endometrial cancer, gastric cancer, small cancer bowel, bladder cancer, or sarcoma. 8. Use according to claim 7, wherein the cancer or tumor is head and neck cancer. 9. Use according to claim 8, wherein the head and neck cancer is head and neck squamous cell carcinoma (SCCHN). 10. Use according to claim 8, wherein the head and neck cancer is adenoid cystic carcinoma. 11. Use according to claim 7, wherein the cancer or tumor is breast cancer. 12. Use according to claim 11, wherein the breast cancer is HER2 positive breast cancer. 13. Use according to claim 11, wherein the breast cancer is HER2-negative breast cancer. 14. Use according to claim 11, wherein the breast cancer is triple negative breast cancer. 15. Use according to claim 7, wherein the cancer or tumor is colon and rectal cancer. 16. Use according to claim 7, wherein the cancer or tumor is cancer of the esophagus. 17. Use according to claim 16, wherein the esophageal cancer is esophageal adenocarcinoma. 18. Use according to claim 7, wherein the cancer or tumor is uterine cancer. 19. Use according to claim 7, wherein the cancer or tumor is ovarian cancer. 20. Use according to claim 7, wherein the cancer or tumor is a sarcoma. 21. Use according to claim 20, wherein the sarcoma is uterine sarcoma, fibrosarcoma, synovial sarcoma, soft tissue sarcoma or angiosarcoma. 22. Use according to claim 7, wherein the cancer or tumor is gastric cancer. 23. Use according to claim 7, wherein the cancer or tumor is small bowel cancer. 24. Use according to claim 23, wherein the small bowel cancer is small bowel adenocarcinoma. 25. Use according to claim 7, wherein the cancer or tumor is bladder cancer. 26. Use according to claim 7, wherein the cancer or tumor is urothelial cancer. - 42 040298 27. Use according to claim 7, wherein the cancer or tumor is endometrial cancer. 28. Use according to any one of claims 3-27 in combination with an antibody to programmed death protein 1 (PD-1). 29. Use according to any one of claims 3-27 in combination with an antibody to programmed death L1 protein (PD-L1). 30. Use according to any one of claims 3 to 27 in combination with an antibody to EGFR (epidermal growth factor receptor). 31. Use according to claim 30, wherein the anti-EGFR antibody is an anti-EGFR monoclonal antibody. 32. Use according to claim 30 or 31, wherein the cancer or tumor is head and neck cancer. 33. Use according to claim 32, wherein the head and neck cancer is head and neck squamous cell carcinoma (SCCHN). 34. Use according to any one of claims 31-33, wherein the anti-EGFR monoclonal antibody is cetuximab. 35. Use according to any one of claims 3-27 in combination with an antibody to HER2 (human epidermal growth factor receptor). 36. Use according to claim 35, wherein the anti-HER2 antibody is an anti-HER2 monoclonal antibody. 37. Use according to claim 35 or 36, wherein the cancer or tumor is breast cancer. 38. Use according to claim 36 or 37, wherein the anti-HER2 monoclonal antibody is trastuzumab. 39. Use according to any one of claims 3 to 38 in combination with radiation therapy. 40. Use according to any of claims 3 to 39 in combination with surgery. 41. A method of treating a cancer or tumor in a subject, comprising administering to the subject a compound of claim 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 2. 42. A method of inhibiting the growth of a tumor or cancer in a subject, comprising administering to the subject a compound of claim 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 2. 43. A method of treating a tumor or cancer characterized by angiogenesis, invasion or metastasis in a subject, comprising administering to the subject a compound of claim 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 2. 44. A method for inhibiting the growth of a tumor or cancer characterized by angiogenesis, invasion, or metastasis in a subject, comprising administering to the subject a compound of claim 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 2. 45. The method according to any one of claims 41 to 44, wherein the cancer or tumor is head and neck cancer, breast cancer, esophageal cancer, uterine cancer, ovarian cancer, colon and rectal cancer, endometrial cancer, gastric cancer, cancer small intestine, bladder cancer, or sarcoma. 46. The method of claim 45 wherein the cancer or tumor is head and neck cancer. 47. The method of claim 46 wherein the head and neck cancer is head and neck squamous cell carcinoma (SCCHN). 48. The method of claim 46 wherein the head and neck cancer is adenoid cystic carcinoma. 49. The method of claim 45 wherein the cancer or tumor is breast cancer. 50. The method of claim 49, wherein the breast cancer is HLR2-positive breast cancer. 51. The method of claim 49, wherein the breast cancer is HER2-negative breast cancer. 52. The method of claim 49, wherein the breast cancer is triple negative breast cancer. 53. The method of claim 45, wherein the cancer or tumor is colon and rectal cancer. 54. The method of claim 45, wherein the cancer or tumor is esophageal cancer. 55. The method of claim 54, wherein the esophageal cancer is esophageal adenocarcinoma. 56. The method of claim 45 wherein the cancer or tumor is uterine cancer. 57. The method of claim 45 wherein the cancer or tumor is ovarian cancer. 58. The method of claim 45 wherein the cancer or tumor is a sarcoma. 59. The method of claim 58, wherein the sarcoma is uterine sarcoma, fibrosarcoma, synovial sarcoma, soft tissue sarcoma, or angiosarcoma. 60. The method of claim 45 wherein the cancer or tumor is gastric cancer. 61. The method of claim 45 wherein the cancer or tumor is small bowel cancer. 62. The method of claim 61, wherein the small bowel cancer is small bowel adenocarcinoma. 63. The method of claim 45, wherein the cancer or tumor is bladder cancer. 64. The method of claim 45 wherein the cancer or tumor is urothelial cancer. 65. The method of claim 45 wherein the cancer or tumor is endometrial cancer. -