Methods for Treating Cancer and Tumors

Abstract

Disclosed herein are methods for treating, preventing, or ameliorating a disease or condition associated with cancer or a tumor. Some embodiments relate to methods for inhibiting cancer cell proliferation. Some embodiments relate to methods for inducing apoptosis in cancer cells. In some embodiments, the methods comprise administering a compound of Formula (I) to a subject in need thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 253,332, filed October 7, 2021. All of the foregoing applications are hereby fully incorporated by reference in their entirety for all purposes.

[0002] Field of Disclosure The present disclosure relates to the fields of chemistry and medicine. More particularly, the present disclosure relates to compositions containing plinabulin and their use in therapy. [Background technology]

[0003] Gastric cancer is a disease in which malignant tumor cells form in the lining of the stomach. Stomach cancer, or gastric cancer, can develop in any part of the stomach and may metastasize throughout the stomach and to other organs, especially the esophagus, lungs, and liver. Gastric cancer is the fourth most common cancer in the world, with 930,000 cases diagnosed in 2002. In addition, it is a highly lethal disease (about 800,000 / year) and is the second most common cause of cancer deaths in the world after lung cancer.

[0004] Small cell lung cancer (SCLC) is named according to the size of the cancer cells when viewed under a microscope and must be differentiated from non-small cell lung cancer (NSCLC). SCLC accounts for approximately 10%–15% of all lung cancers (American Cancer Society, 2015a).

[0005] Both lung cancers (SCLC and NSCLC) are the second most common cancers in both men and women. Lung cancer is the leading cause of cancer deaths, accounting for approximately 25%. Thus, more people die from lung cancer each year than colon, breast, and prostate cancer combined. Furthermore, lung cancer accounts for approximately 13% (more than 1.8 million cases) of all new cancers. Lung cancer occurs primarily in older adults. The average age at diagnosis is approximately 70 years. Less than 2% of all cases are diagnosed in people under 45 years of age. The treatment and prognosis of SCLC largely depend on the stage of the cancer diagnosed. Staging of SCLC based on clinical results is more common than pathological staging. Clinical staging utilizes the results of physical examinations, various imaging tests, and biopsies. According to data introduced by the American Cancer Society, the 5-year relative survival rates are 31% for stage I, 19% for stage II, 8% for stage III, and 2% for stage IV.

[0006] Treatment of patients with triple-negative breast cancer (TNBC), which lacks estrogen receptor (ER) and progesterone receptor (PR) expression and human epidermal growth factor receptor 2 (HER2) amplification, has been challenging due to the heterogeneity of the disease and the lack of clear molecular targets (Pegram MD, et al. J Clin Oncol. 1998; 16(8):2659-2671; Wiggans RG, et al. Cancer Chemother Pharmacol. 1979;3(1):45-48; Carey LA, et al. Clin Cancer Res. 2007; 13(8):2329-2334). TNBC constitutes 10%-20% of all breast cancers, affects younger patients more frequently, and has a higher prevalence in women of African American descent (Morris GJ, et al. Cancer. 2007; 110(4):876-884). TNBC tumors are generally larger in size, higher grade, and more aggressive than non-TNBC tumors (Willis GJ, et al. Cancer. 2007; 110(4):876-884). have lymph node metastases at diagnosis and are biologically more aggressive (Haffty Haffty BG, et al. J Clin Oncol. 2006; 24(36):5652-5657). Despite having a higher rate of clinical response to neoadjuvant chemotherapy, TNBC patients have a higher rate of definite recurrence and a worse prognosis than women with other breast cancer subtypes (Haffty BG, et al. J Clin Oncol. 2006; 24(36):5652-5657; Dent R, et al. Clin Cancer Res. 2007; 13(15 pt 1):4429-4434). Fewer than 30% of women with metastatic TNBC survive 5 years, and nearly all of them die of their disease despite adjuvant chemotherapy, the mainstay of treatment (Dent R, et al. Clin Cancer Res. 2007; 13(15 pt 1):4429-4434).

[0007] Therefore, there remains a need to develop effective treatments for these and other aggressive cancers. Summary of the Invention [Means for solving the problem]

[0008] Some aspects relate to a method of treating cancer in a subject in need thereof. In some embodiments, the method comprises administering a compound of formula (I) as monotherapy. [ka] (I) or a pharma- ceutical acceptable salt thereof to a subject, wherein: R1, R4, and R6 are each independently a hydrogen atom, a deuterium atom, a halogen atom, or a saturated C1-C 24 Alkyl, unsaturated C1-C 24is selected from the group consisting of alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups, hydroxy, carboxy, -CO-O-R7, cyano, alkylthio, alkyl halides including polyhalogenated alkyls, carbonyl halides, and carbonyl -CH2CO-R7, where R7 is a hydrogen atom, a halogen atom, or a saturated C1-C 24 Alkyl, unsaturated C1-C 24 selected from alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups; R1' and R1'' are each independently a hydrogen atom, a deuterium atom, a halogen atom, or a saturated C1-C 24 Alkyl, unsaturated C1-C 24 alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups, hydroxy, carboxy, -CO-O-R7, cyano, alkylthio, alkyl halides including polyhalogenated alkyls, carbonyl halides, and carbonyl -CH2CO-R7, where R7 is a hydrogen atom, a halogen atom, or a saturated C1-C 24 Alkyl, unsaturated C1-C 24 selected from alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups; R, R', and R'' are either covalently bonded to each other or are not covalently bonded to each other; R2, R3 and R5 are each independently a hydrogen atom, a deuterium atom, a halogen atom, or a saturated C1-C12 Alkyl, unsaturated C1-C 12 selected from the group consisting of alkenyl, acyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro and substituted nitro groups, sulfonyl and substituted sulfonyl groups; m is an integer of 0, 1 or 2; X1 and X2 are independently selected from the group consisting of oxygen, nitrogen and sulfur atoms; Y is selected from the group consisting of NR5, oxygen, sulfur, oxidized sulfur, methylene and substituted methylene; Z for each individual n when not 0, and Z1, Z2, Z3, and Z4 are each independently selected from carbon atoms, sulfur atoms, nitrogen atoms, and oxygen atoms; The dashed bond may be either a single bond or a double bond, Here, the cancer is selected from gastric cancer, small cell lung cancer, osteosarcoma, bladder cancer, and triple-negative breast cancer.

[0009] In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is small cell lung cancer. In some embodiments, the cancer is triple negative breast cancer. In some embodiments, the compound of Formula (I) is administered at a dose of about 5 mg / m 2 ~150mg / m 2 In some embodiments, the compound of formula (I) is administered at a dose of 30 mg / m 2 In some embodiments, the compound of formula (I) is administered at a dose of greater than about 40 mg / m 2In some embodiments, the compound of formula (I) is administered on day 1 of a 14 day dosing cycle. In some embodiments, the compound of formula (I) is administered on day 1 of a 21 day dosing cycle. In some embodiments, the compound of formula (I) is administered on day 1 of a 21 day dosing cycle. In some embodiments, the compound of formula (I) is administered at a dose of plinabulin, (3Z,6Z)-3-(phenyl-2,3,4,5,6-d5)-methylene-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione; (3Z,6Z)-3-(phenyl-2,3,4,5,6-d5)-methylene-d-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione; (3Z,6Z)-3-(phenylmethylene-d)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione;(3Z,6Z)-3-(phenyl-2,3,4,5,6-d5)-methylene-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione;(3Z,6Z)-3-(phenylmethylene)-6- ((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione;(3Z,6Z)-3-(phenyl-2,3,4,5,6-d5)-methylene-d-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione;(3Z,6Z)-3-(4-fluoro-(phenyl-2,3,5,6-d4))-methylene-6-((5-(tert-butyl) )-1H-imidazol-4-yl)methylene)piperazine-2,5-dione;(3Z,6Z)-3-(4-fluoro-(phenyl-2,3,5,6-d4))-methylene-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione;(3Z,6Z)-3-(3-fluorobenzylidene)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione (3Z,6Z)-3-(3-benzoylbenzylidene)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione;(3Z,6Z)-3-(3-(4-fluorobenzoyl)benzylidene)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione;(3Z,6Z)-3-(3-(4-methoxybenzoyl)benzylidene)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione;(3Z,6Z)-3-(3-methoxybenzylidene)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione In some embodiments, the compound represented by Formula (I) is selected from (3Z,6Z)-3-(3-(trifluoromethyenzydene)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione; (3Z,6Z)-3-(3-(trifluoromethyenzydene)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione, and pharma- ceutically acceptable salts thereof. In some embodiments, the compound represented by Formula (I) is plinabulin or a pharma- ceutically acceptable salt thereof. In some embodiments, the cancer comprises a tumor and the tumor mass is reduced by about 50% to about 100%. In some embodiments, the tumor mass is reduced by about 50% to about 70%.

[0010] Some aspects relate to methods of halting or reversing progressive cancer in a subject. In some embodiments, the methods include administering a compound of formula (I) as monotherapy. [ka] (I) or a pharma- ceutical acceptable salt thereof, wherein R1, R4, and R6 are each independently a hydrogen atom, a deuterium atom, a halogen atom, or a saturated C1-C 24 Alkyl, unsaturated C1-C 24is selected from the group consisting of alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups, hydroxy, carboxy, -CO-O-R7, cyano, alkylthio, alkyl halides including polyhalogenated alkyls, carbonyl halides, and carbonyl -CH2CO-R7, where R7 is a hydrogen atom, a halogen atom, or a saturated C1-C 24 Alkyl, unsaturated C1-C 24 selected from alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups; R1' and R1'' are each independently a hydrogen atom, a deuterium atom, a halogen atom, or a saturated C1-C 24 Alkyl, unsaturated C1-C 24 alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups, hydroxy, carboxy, -CO-O-R7, cyano, alkylthio, alkyl halides including polyhalogenated alkyls, carbonyl halides, and carbonyl -CH2CO-R7, where R7 is a hydrogen atom, a halogen atom, or a saturated C1-C 24 Alkyl, unsaturated C1-C 24 selected from alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups; R, R', and R'' are either covalently bonded to each other or are not covalently bonded to each other; R2, R3 and R5 are each independently a hydrogen atom, a deuterium atom, a halogen atom, or a saturated C1-C12 Alkyl, unsaturated C1-C 12 selected from the group consisting of alkenyl, acyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro and substituted nitro groups, sulfonyl and substituted sulfonyl groups; m is an integer of 0, 1 or 2; X1 and X2 are independently selected from the group consisting of oxygen, nitrogen and sulfur atoms; Y is selected from the group consisting of NR5, oxygen, sulfur, oxidized sulfur, methylene and substituted methylene; Z for each individual n when not 0, and Z1, Z2, Z3, and Z4 are each independently selected from carbon atoms, sulfur atoms, nitrogen atoms, and oxygen atoms; The dashed bond may be either a single bond or a double bond, Here, the cancer is selected from gastric cancer, small cell lung cancer, osteosarcoma, bladder cancer, and triple-negative breast cancer.

[0011] In some embodiments, the compound of Formula (I) is plinabulin or a pharma- ceutically acceptable salt thereof.

[0012] Some aspects relate to methods of inhibiting the proliferation of cancer cells, hi some embodiments, the methods include contacting cancer cells with an effective amount of plinabulin in the absence of other chemotherapeutic agents, the cancer cells being of a cancer selected from gastric cancer, small cell lung cancer, osteosarcoma, bladder cancer, and triple-negative breast cancer.

[0013] Some aspects relate to methods of inducing apoptosis in cancer cells, hi some embodiments, the methods include contacting cancer cells with an effective amount of plinabulin in the absence of other chemotherapeutic agents, wherein the cancer is selected from gastric cancer, small cell lung cancer, osteosarcoma, bladder cancer, and triple-negative breast cancer. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a graph showing the efficacy of plinabulin monohydrate in clonogenic assays. [Diagram 2] 1 is a heat map showing IC70 values ​​from the tests sorted by absolute IC70. [Diagram 3] 1 is a line graph showing the concentration-effect curve of plinabulin monohydrate and small cell lung cancer. [Figure 4] 1 is a line graph showing the concentration-effect curve of plinabulin monohydrate and gastric cancer. [Diagram 5] 1 is a line graph showing the concentration-effect curve of plinabulin monohydrate and triple-negative breast cancer. [Figure 6] 1 is a graph showing the efficacy of plinabulin monohydrate in the clonogenic assay as IC50 values. [Figure 7] 1 is a graph showing the efficacy of plinabulin monohydrate in the clonogenic assay as IC70 values. [Figure 8] 1 is a heat map showing the IC50 values ​​of the tests sorted by absolute IC50. [Figure 9] 1 is a heat map showing IC70 values ​​from the tests sorted by absolute IC70. [Figure 10] 1 is a line graph showing the concentration-effect curve of plinabulin monohydrate and small cell lung cancer. [Figure 11] 1 is a line graph showing the concentration-effect curve of plinabulin monohydrate and gastric cancer. [Figure 12] 1 is a line graph showing the concentration-effect curve of plinabulin monohydrate and triple-negative breast cancer. [Figure 13A] 1 is a heat map showing the IC50 / IC70 values ​​obtained with plinabulin. [Figure 13B] This is a continuation of the heat map in Figure 13A. [Figure 14A] 1 is a heat map showing the T / C values ​​obtained with plinabulin. [Figure 14B] This is a continuation of the heat map in Figure 14A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present disclosure provides a method for treating cancer or tumor. Some embodiments relate to the use of plinabulin to treat cancer or tumor, including but not limited to small cell lung cancer, gastric cancer, and triple negative breast cancer. In some embodiments, the methods provided herein are useful in treating, delaying the progression, preventing recurrence, or alleviating symptoms of cancer or tumor, including but not limited to small cell lung cancer, gastric cancer, and triple negative breast cancer. In some embodiments, the compound of formula (I) is plinabulin. Plinabulin's (3Z,6Z)-3-benzylidene-6-{[5-(2-methyl-2-propanyl)-1H-imidazol-4-yl]methylene}-2,5-piperazinedione is a synthetic analog of the natural compound phenylahistine. As described herein, it has been surprisingly discovered that plinabulin may be effective as a monotherapy against cancer or tumor, including but not limited to small cell lung cancer, gastric cancer, and triple negative breast cancer.

[0016] Before describing the present disclosure in detail, it is to be understood that the present disclosure is not limited to the particular embodiments described, as such may, of course, vary. Likewise, it is to be understood that the terminology used herein is for the purpose of describing only particular embodiments, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0017] Where a range of values ​​is provided, it is understood that each intervening value, up to ten times the unit of the lower limit unless otherwise clearly indicated by the context, between the upper and lower limits of that range, and any other stated or intervening value within that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both of its limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0018] Methods recited herein may be carried out in any order of the recited events which is logically possible, as well as the recited order of events.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are described herein.

[0020] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0021] It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional elements. As such, this statement shall serve as a predicate for the use of exclusionary terminology such as "solely," "only," and the like, or the use of a "negative" limit in connection with the recitation of claim elements.

[0022] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0023] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, applications, published applications, and other publications are incorporated by reference in their entirety. In the event that there are a plurality of definitions for terms herein, the definition in this paragraph prevails unless stated otherwise.

[0024] The term "agent" is used herein to denote a chemical compound, a mixture of compounds, a biopolymer, or an extract made from biological material.

[0025] As used herein, the term "ameliorate" refers to any decrease in the degree, severity, frequency, and / or probability of a symptom or clinical sign characteristic of a particular disease.

[0026] The terms "cancer," "neoplasm," and "carcinoma" are used interchangeably herein to refer to cells that exhibit relatively autonomous growth, such that they exhibit an abnormal growth phenotype characterized by a marked loss of control over cell proliferation. In general, cells of interest for detection or treatment in this application include pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells. Detection of cancer cells is of particular interest.

[0027] The term "pharmaceutical acceptable carrier" or "pharmaceutical acceptable excipient" includes any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. In addition, various adjuvants commonly used in the art may be included. Considerations for the inclusion of various ingredients in pharmaceutical compositions are described, for example, in Gilman et al. (Eds.) (1990); Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press, which is incorporated herein by reference in its entirety. The pharmaceutical acceptable excipient may be a monosaccharide or monosaccharide derivative.

[0028] As used herein, the term "subject" refers to a human or non-human mammal, such as a dog, cat, mouse, rat, cow, sheep, pig, goat, non-human primate, or bird, such as a chicken, and any other It means a vertebrate or an invertebrate.

[0029] The term "mammal" is used in its normal biological sense, and thus specifically includes, but is not limited to, monkeys (chimpanzees, apes, monkeys) and humans, cattle, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, or the like.

[0030] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a therapeutic agent that is effective to alleviate to some extent one or more of the symptoms of a disease or condition or to reduce the likelihood of its occurrence, which may include curing the disease or condition.

[0031] As used herein, the terms "treat", "treatment", or "treating" refer to the administration of a compound or pharmaceutical composition to a subject for preventative and / or therapeutic purposes. The term "prophylactic treatment" refers to treating a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to or otherwise at risk for a particular disease or condition, whereby the treatment reduces the patient's chance of developing the disease or condition. The term "therapeutic treatment" refers to administering treatment to a subject who is already suffering from a disease or condition.

[0032] The term "chemotherapeutic agent" as used herein refers to an agent that reduces, prevents, reduces, limits, and / or slows the growth of metastases or neoplasms, or that kills neoplastic cells directly, by neoplastic necrosis or apoptosis, or any other mechanism, or that may otherwise be used in a medicamentously effective amount to reduce, prevent, reduces, limits, and / or slow the growth of metastases or neoplasms in a subject with a neoplastic disease. Chemotherapeutic agents include, but are not limited to, for example, fluoropyrimidines; pyrimidine nucleosides; purine nucleosides; antifolates; platinum-based agents; anthracyclines / anthracenediones; epipodophyllotoxins; camptothecins; hormones; hormone conjugates; antihormones; enzymes; proteins; peptides and polyclonal and / or monoclonal antibodies; vinca alkaloids; taxanes; epothilones; microtubule inhibitors; alkylating agents; metabolic inhibitors; topoisomerase inhibitors; antiviral agents; and various other cytotoxic and cytostatic agents.

[0033] compound In some embodiments, the compounds and therapeutic compositions for treating cancer or tumors described herein have formula (I): [ka] (I) In particular, the present invention includes compounds represented by the formula: R1, R4, and R6 are each independently a hydrogen atom, a deuterium atom, a halogen atom, or a saturated C1-C24 Alkyl, unsaturated C1-C 24 Alkenyl, cycloalkyl, cyclo is selected from the group consisting of alkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups, hydroxy, carboxy, -CO-O-R7, cyano, alkylthio, alkyl halides including polyhalogenated alkyls, carbonyl halides, and carbonyl -CH2CO-R7, where R7 is a hydrogen atom, a halogen atom, and a saturated C1-C 24 Alkyl, unsaturated C1-C 24 selected from alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups; R1' and R1'' are each independently a hydrogen atom, a deuterium atom, a halogen atom, or a saturated C1-C 24 Alkyl, unsaturated C1-C 24 is selected from the group consisting of alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups, hydroxy, carboxy, -CO-O-R7, cyano, alkylthio, alkyl halides including polyhalogenated alkyls, carbonyl halides, and carbonyl -CH2CO-R7, where R7 is a hydrogen atom, a halogen atom, or a saturated C1-C 24 Alkyl, unsaturated C1-C 24 selected from alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups; R, R', and R'' are either covalently bonded to each other or are not covalently bonded to each other; R2, R3 and R5 are each independently a hydrogen atom, a deuterium atom, a halogen atom, or a saturated C1-C 12 Alkyl, unsaturated C1-C 12 selected from the group consisting of alkenyl, acyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro and substituted nitro groups, sulfonyl and substituted sulfonyl groups; m is an integer of 0, 1 or 2; X1 and X2 are independently selected from the group consisting of oxygen, nitrogen and sulfur atoms; Y is selected from the group consisting of NR5, oxygen, sulfur, oxidized sulfur, methylene and substituted methylene; Z for each distinct n when not 0, and Z1, Z2, Z3, and Z4 are each independently selected from carbon atoms, sulfur atoms, nitrogen atoms, and oxygen atoms; the dashed bonds may be either single or double bonds.

[0034] Compounds of formula (I) may be readily prepared by the methods and procedures detailed in US Pat. Nos. 7,064,201 and 7,919,497, which are incorporated herein by reference in their entirety.

[0035] In some embodiments, the compounds described herein have the formula (II): [ka] (II) Dehydrophenylahistine represented by the formula: R2 and R3 each independently represent a hydrogen atom; a halogen atom; a mono-, poly- or non-substituted linear or branched chain variant of the following residues: C1-C 12 Alkyl, C1-C 12alkenyl, acyl, and alkoxy; and mono-, poly- or unsubstituted variants of the following residues: cycloalkyl, cycloalkoxy, aryl, heteroaryl, amino, nitro, and sulfonyl; or R2 is a bond to Ar; R4 and R6 are each independently hydrogen; halogen; hydroxyl; mono-, poly- or unsubstituted linear or branched versions of the following residues: C1-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 alkynyl, alkoxy, acyl, arylalkyl, heteroarylalkyl, alkyloxycarbonyloxy, ester, arylalkoxy, alkoxy, and alkylthio; mono-substituted, poly-substituted, or unsubstituted variants of the following residues: acyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, cycloalkoxy, aryl, heteroaryl, aryloxy, arylcarbonyl, heterocycloalkyl, carbonyl, amino, aminocarbonyl, amido, aminocarbonyloxy, nitro, azido, phenyl, hydroxyl, thio, alkylthio, arylthio, thiooxysulfonyl, thiophene, carboxy, and cyano; X1 and X2 are independently selected from the group consisting of oxygen, sulfur, and nitrogen atoms substituted with an R5 group; R5 is a hydrogen atom, a halogen atom, or a saturated C1-C 12 Alkyl, unsaturated C1-C 12 selected from the group consisting of alkenyl, acyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, and substituted nitro groups, sulfonyl, and substituted sulfonyl groups; Y is selected from the group consisting of NR5, oxygen, sulfur, oxidized sulfur, methylene, and substituted methylene; n is 0, 1, 2, 3, or 4; Ar is a cyclic or polycyclic aryl or heteroaryl ring system containing from one to three rings, each ring in the system is independently a 5-, 6-, 7-, or 8-membered ring; Each ring of the system contains 0, 1, 2, 3, or 4 heteroatoms selected from the group consisting of oxygen, sulfur, and nitrogen; Each ring of the system may be selected from the group consisting of hydrogen, halogen, hydroxyl, mono-, poly- or non-substituted linear or branched versions of the following residues: C1-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl, alkoxy, acyl, arylalkyl, heteroarylalkyl, alkyloxycarbonyloxy, ester, arylalkoxy, alkoxy, and alkylthio; mono-substituted, poly-substituted or unsubstituted variants of the following residues: acyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, cycloalkoxy, aryl, heteroaryl, aryloxy, arylcarbonyl, heterocycloalkyl, carbo Optionally substituted with one or more substituents selected from the group consisting of nyl, amino, aminocarbonyl, amido, aminocarbonyloxy, nitro, azido, phenyl, hydroxyl, thio, alkylthio, arylthio, thiophene, oxysulfonyl, sulfonyl, carboxy, and cyano; and optionally substituted fused rings selected from the group consisting of dioxole, dithiol, oxathiol, dioxine, dithiin, and oxathiin.

[0036] Compounds of formula (II) may be readily prepared by the methods and procedures detailed in US Pat. Nos. 7,064,201 and 7,919,497, which are incorporated herein by reference in their entirety.

[0037] In some embodiments, the compound of formula (I) is selected from the group consisting of plinabulin, (3Z,6Z)-3-(phenyl-2,3,4,5,6-d5)-methylene-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione; (3Z,6Z)-3-(phenyl-2,3,4,5,6-d5)-methylene-d-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione; (3Z,6Z)-3-(phenylmethylene-d)-6-((5-(tert -butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione;(3Z,6Z)-3-(phenyl-2,3,4,5,6-d5)-methylene-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione;(3Z,6Z)-3-(phenylmethylene)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione;(3Z,6Z)-3-(phenyl-2,3,4,5,6-d5)-methylene-d-6 -((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione;(3Z,6Z)-3-(4-fluoro-(phenyl-2,3,5,6-d4))-methylene-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione;(3Z,6Z)-3-(4-fluoro-(phenyl-2,3,5,6-d4))-methylene-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione;(3 Z,6Z)-3-(3-fluorobenzylidene)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione;(3Z,6Z)-3-(3-benzoylbenzylidene)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione;(3Z,6Z)-3-(3-(4-fluorobenzoyl)benzylidene)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione;(3Z,6Z)-3-(3-(4-methoxybenzoyl)benzylidene)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione; (3Z,6Z)-3-(3-methoxybenzylidene)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione; (3Z,6Z)-3-(3-(trifluoromethylenediene)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione; and pharma- ceutically acceptable salts thereof.

[0038] In some embodiments, the compound of formula (I) is plinabulin. In some embodiments, the compound of formula (I) is plinabulin monohydrate. In some embodiments, the compound of formula (I) is a salt form of plinabulin. Plinabulin can be readily prepared by the methods and procedures detailed in U.S. Patent Nos. 7,064,201 and 7,919,497, which are incorporated herein by reference in their entirety.

[0039] Methods of Use and Treatment In some aspects, the disclosure provides methods and therapeutic compositions for treating, preventing, or ameliorating cancer or tumor in a subject by administering a compound of Formula (I) (e.g., plinabulin) or a pharma- ceutically acceptable salt thereof as monotherapy. In some embodiments, the cancer or tumor is selected from gastric cancer, small cell lung cancer, osteosarcoma, bladder cancer, and triple-negative breast cancer. In some embodiments, the cancer or tumor is gastric cancer. In some embodiments, the cancer or tumor is small cell lung cancer. In some embodiments, the cancer or tumor is triple-negative breast cancer.

[0040] In some embodiments, the method includes administering a compound of Formula (I) (e.g., plinabulin) at a dose of about 5 mg / m 2 ~150mg / m 2In some embodiments, the method includes administering a compound of Formula (I) (e.g., plinabulin) at a dose of about 10 mg / m 2 ~50mg / m 2 In some embodiments, the method includes administering a compound of Formula (I) (e.g., plinabulin) at a dose of about 20 mg / m 2 ~30mg / m 2 In some embodiments, the compound of Formula (I) (e.g., plinabulin) is administered at a dose of 20 mg / m 2 In some embodiments, the compound of Formula (I) (e.g., plinabulin) is administered at a dose of greater than 30 mg / m 2 In some embodiments, the compound of Formula (I) (e.g., plinabulin) is administered at a dose of greater than about 40 mg / m 2 In some embodiments, plinabulin is administered at a dose of about 30 mg to about 40 mg. In some embodiments, plinabulin is administered at a dose of about 40 mg.

[0041] In some embodiments, a compound of Formula (I) (e.g., plinabulin) is administered on day 1 of a 14 day dosing cycle. In some embodiments, a compound of Formula (I) (e.g., plinabulin) is administered on day 1 of a 21 day dosing cycle.

[0042] Some embodiments relate to a method of halting or reversing the progression of cancer in a subject. In some embodiments, the method comprises administering to the subject a compound of formula (I). In some embodiments, the cancer cells are from a cancer selected from gastric cancer, small cell lung cancer, osteosarcoma, bladder cancer, and triple-negative breast cancer. In some embodiments, the compound of formula (I) is plinabulin or a pharmaceutically acceptable salt thereof.

[0043] Some aspects relate to a method of inhibiting the proliferation of cancer cells. In some embodiments, the method comprises contacting cancer cells with a compound of formula (I) in a subject. In some embodiments, the cancer cells are from a cancer selected from gastric cancer, small cell lung cancer, osteosarcoma, bladder cancer, and triple-negative breast cancer. In some embodiments, the compound of formula (I) is plinabulin or a pharmaceutically acceptable salt thereof.

[0044] Some aspects relate to a method of inducing apoptosis in cancer cells. In some embodiments, the method comprises contacting a cancer cell with a compound of formula (I) in a subject. In some embodiments, the cancer cell is of a cancer selected from gastric cancer, small cell lung cancer, osteosarcoma, bladder cancer, and triple-negative breast cancer. In some embodiments, the compound of formula (I) is plinabulin or a pharmaceutically acceptable salt thereof.

[0045] Some embodiments relate to methods of inhibiting the progression of cancer, hi some embodiments, the methods comprise administering to a subject in need thereof an effective amount of plinabulin.

[0046] Administration Administration of the pharmaceutical compositions described herein may be via any of the accepted modes of administration for agents that serve similar utilities, including, but not limited to, oral, sublingual, buccal, subcutaneous, intravenous, intranasal, intratumoral, topical, transdermal, intradermal, intraperitoneal, intramuscular, intrapulmonary, intravaginal, rectal, or intraocular. Oral and parenteral administration are conventional for treating the indications covered by the preferred embodiments.

[0047] The compositions described herein may be provided in unit dosage form. As used herein, a "unit dosage form" is a composition containing an amount of a compound or composition suitable for administration in a single dose to an animal, preferably a mammalian subject, according to good medical practice. However, the preparation of a single or unit dosage form does not imply that the dosage form is to be administered once per day or once per course of treatment. Such dosage forms are contemplated to be administered once, twice, three or more times per day, may be administered as an infusion over a period of time (e.g., from about 30 minutes to about 2-6 hours) or may be administered as a continuous infusion, and may be given more than once during the course of treatment, although single administration is not specifically excluded. One of skill in the art will recognize that the formulation is not specifically contemplated for the entire course of treatment, and such a determination is left to those skilled in the art of treatment rather than formulation.

[0048] As described above, the useful compositions may be in any of a variety of suitable forms for various routes of administration, such as oral, sublingual, buccal, intranasal, rectal, topical (including transdermal and intradermal), intraocular, intracerebral, intracranial, intrathecal, intraarterial, intravenous, intramuscular, or other parenteral routes of administration. Those skilled in the art will recognize that oral and intranasal compositions include compositions administered by inhalation and made using available methodologies. Depending on the particular route of administration desired, a variety of pharmaceutically acceptable carriers well known in the art may be used. Pharmaceutically acceptable carriers include, for example, solid or liquid fillers, diluents, hydrotropes, surfactants, and encapsulating materials. Optional pharmaceutically active materials that do not substantially interfere with the inhibitory activity of the compound or composition may be included. The amount of carrier used in conjunction with the compound or composition is sufficient to provide a practical amount of material for administration per unit dose of the compound. Techniques and compositions for making dosage forms useful in the methods described herein are described in the following references, all of which are incorporated herein by reference: Modern Pharmaceutics, 4th Ed., Chapters 9 and 10 (Banker & Rhodes, editors, 2002); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1989); and Ansel, Introduction to Pharmaceutical Dosage Forms 8th Edition (2004).

[0049] A variety of oral dosage forms can be used, including solid dosage forms such as tablets, capsules (e.g., solid gel capsules and liquid gel capsules), granules, and bulk powders. Tablets can be compressed, tablet triturates, enteric coated, sugar coated, film coated, or multiple-compressed, and contain suitable binders, lubricants, diluents, disintegrants, colorants, flavorings, flow inducers, and melting agents. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules, containing suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, melting agents, colorants, and flavorings.

[0050] Pharmaceutically acceptable carriers suitable for preparing unit dosage forms for oral administration are well known in the art. Tablets typically contain inert diluents such as calcium carbonate, sodium carbonate, mannitol, lactose, and cellulose; starch, gelatin, and sucrose; The adjuvants include conventional pharma- ceutical acceptable adjuvants such as binders, disintegrants such as starch, alginic acid and croscarmellose, and lubricants such as magnesium stearate, stearic acid and talc. Lubricants such as silicon dioxide can be used to improve the flow properties of powder mixtures. Coloring agents such as FD&C dyes can be added for appearance. Sweeteners and flavoring agents such as aspartame, saccharin, menthol, peppermint, and fruit juice flavors are useful adjuvants for chewable tablets. Capsules typically contain one or more solid diluents as disclosed above. The selection of carrier components depends on secondary considerations such as non-critical flavor, cost, and storage stability, and can be readily made by those skilled in the art.

[0051] Oral compositions also include liquid solutions, emulsions, suspensions and the like. Pharmaceutically acceptable carriers suitable for preparing such compositions are well known in the art. Typical components of carriers for syrups, elixirs, emulsions and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water. For suspensions, typical suspending agents include methylcellulose, sodium carboxymethylcellulose, AVICEL RC-591, tragacanth and sodium alginate; typical wetting agents include lecithin and polysorbate 80; typical preservatives include methylparaben and sodium benzoate. Oral liquid compositions may also contain one or more components such as sweeteners, flavorings and colorings as previously disclosed.

[0052] Such compositions may also be coated by conventional methods, typically with a pH or time dependent coating, so that the subject composition is released in the digestive tract near the desired topical application or at various times to extend the desired effect. Such dosage forms typically include, but are not limited to, one or more of cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, ethylcellulose, Eudragit coatings, waxes and shellac.

[0053] The compositions described herein may optionally include additional pharmaceutical actives.

[0054] Other compositions useful for achieving systemic delivery of the target compound include sublingual, oral and nasal dosage forms.Such compositions typically include one or more of soluble filler materials such as sucrose, sorbitol and mannitol; and binders such as gum arabic, microcrystalline cellulose, carboxymethylcellulose and hydroxypropylmethylcellulose.The previously disclosed glidants, lubricants, sweeteners, colorants, antioxidants and flavorings may also be included.

[0055] Liquid compositions formulated for topical ophthalmic use are formulated so that they can be administered topically to the eye. Comfort may be maximized as much as possible, but sometimes formulation considerations (e.g., drug stability) may not require optimal comfort. When comfort cannot be maximized, the liquid may be formulated so that it is tolerable to patients for topical ophthalmic use. In addition, ophthalmically acceptable liquids may be packaged for single use or contain preservatives to prevent contamination with repeated use.

[0056] For ophthalmic applications, solutions or medicaments are often prepared using saline solution as the primary vehicle. Ophthalmic solutions may preferably be maintained at a comfortable pH with an appropriate buffer system. The formulations may also contain conventional pharma- ceutically acceptable preservatives, stabilizers, and surfactants.

[0057] Preservatives that may be used in the pharmaceutical compositions disclosed herein include benzalkonium chloride. Examples of useful surfactants include, but are not limited to, phenylmercuric acetate, PHMB, chlorobutanol, thimerosal, phenylmercuric acetate, and phenylmercuric nitrate. A useful surfactant is, for example, Tween 80. Similarly, a variety of useful vehicles may be used in the ophthalmic preparations disclosed herein. These vehicles include, but are not limited to, polyvinyl alcohol, povidone, hydroxypropylmethylcellulose, poloxamer, carboxymethylcellulose, hydroxyethylcellulose, and purified water.

[0058] If necessary or convenient, tonicity agents may be added, including, but not limited to, salts, particularly sodium chloride, potassium chloride, mannitol and glycerin, or any other suitable ophthalmically acceptable tonicity agent.

[0059] Various buffers and means for adjusting pH may be used, so long as the resulting preparation is ophthalmically acceptable. In many compositions, the pH will be between 4 and 9. Buffers thus include acetate buffers, citrate buffers, phosphate buffers and borate buffers. Acids or bases may be used to adjust the pH of these formulations as needed.

[0060] Ophthalmically acceptable antioxidants include, but are not limited to, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole, and butylated hydroxytoluene.

[0061] Another excipient component that may be included in the ophthalmic preparation is a chelating agent. A useful chelating agent is edetate disodium, although other chelating agents may be used instead or in conjunction therewith.

[0062] For topical use, creams, ointments, gels, solutions or suspensions, etc., containing the compositions disclosed herein are employed. Topical formulations may generally be comprised of a pharmaceutical carrier, cosolvent, emulsifier, penetration enhancer, preservative system, and emollient.

[0063] For intravenous administration, the compositions described herein may be dissolved or dispersed in a pharma- ceutically acceptable diluent, such as saline or dextrose solution. Suitable excipients, including but not limited to NaOH, sodium carbonate, sodium acetate, HCl, and citric acid, may be included to achieve the desired pH. In various embodiments, the pH of the final composition is in the range of 2-8, or preferably 4-7. Antioxidant excipients may include sodium bisulfite, acetone sodium bisulfite, sodium formaldehyde, sulfoxylates, thiourea, and EDTA. In some embodiments, excipients used for intravenous delivery may include Kolliphor HS15 (polyoxyl 15 hydroxystearate or Solutol HS-15), propylene glycol, and 5% dextrose in water (D5W). Other non-limiting examples of suitable excipients found in the final intravenous composition can include sodium or potassium phosphate, citric acid, tartaric acid, gelatin, and carbohydrates such as dextrol, mannitol, and dextran. Additional acceptable excipients are described in Powell, et al. al., Compendium of Excipients for Parenteral Formulations, PDA J Pharm Sci and Tech 1998, 52 238-311, and Nema et al., Excipients and Their Role in Approved Injectable Products: Current Usage and Future Directions, PDA J Pharm Sci and Tech 2011, 65 287-332, both of which are incorporated herein by reference in their entirety. Antimicrobial agents may also be included, including phenylmercuric acid, thimerosal, benzethonium chloride, benzalkonium chloride, phenol, cresol, and chlorobutanol. In some embodiments, plinabulin is added to a light-proof bag containing at least 200 ml of D5W to achieve 0.08 mg / ml to 0.2 mg / ml plinabulin. In some embodiments, 40 mg of plinabulin crude drug is removed and added to 6.0 g propylene glycol, which is then added to 200 ml of D5W. In some embodiments, plinabulin is a concentrated solution (4 mg / ml plinabulin in propylene glycol / polyoxy 15 hydroxystearate 60:40 (wt:wt)). In some embodiments, plinabulin in a concentrated solution is added to D5W injection (e.g., 1:10 dilution level or 1:20 dilution level).

[0064] Compositions for intravenous administration may be provided to the caregiver in another solid form that is reconstituted with a suitable diluent, such as sterile water, saline, or dextrose in water, for a short period of time prior to administration. In other embodiments, the compositions are provided in a solution that can be administered parenterally immediately. In yet other embodiments, the compositions are provided in a solution that is further diluted prior to administration. In embodiments involving administration of a combination of a compound described herein with another agent, the combination may be provided to the caregiver as a mixture, or the caregiver may mix the two agents prior to administration, or the two agents may be administered separately.

[0065] The actual dose of the active compounds described herein will depend on the specific compound and the condition being treated; selection of the appropriate dose is well within the knowledge of one of ordinary skill in the art. In some embodiments, the compound of formula (I) is administered at a dose of about 1 mg / m 2 ~about 50mg / m 2 In some embodiments, the compound of formula (I) may be administered at a dose ranging from about 1 to 50 mg / m 2In some embodiments, the compound of formula (I) is administered in a dose within the range of about 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 13.75, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 22.5, 1 to 25, 1 to 27.5, 1 to 30, 1.5 to 2, 1.5 to 3, 1.5 to 4, 1.5 to 5, 1.5 to 6, 1.5 to 7, 1.5 to 8, 1.5 to 9, 1.5 to 10, 1.5 to 11, 1.5 to 12, 1.5 to 13, 1.5 to 13.75, 1.5 to 14, 1.5 to 15, 1.5~16, 1.5~17, 1.5~18, 1.5~19, 1.5~20, 1.5~22.5, 1.5~25, 1.5~27.5, 1.5~30, 2.5~2, 2.5~3, 2.5~4, 2.5~5, 2.5~6, 2.5~7, 2.5~8, 2.5~9, 2.5~10, 2.5~11, 2.5~12, 2.5~13, 2.5~13.75, 2.5~14, 2.5~15, 2.5~16, 2.5~17, 2.5~18, 2.5~19, 2.5~20, 2.5~22.5, 2.5~25, 2.5~27.5, 2.5~30, 2.5~7.5 , 3~4, 3~5, 3~6, ​​3~7, 3~8, 3~9, 3~10, 3~11, 3~12, 3~13, 3~13.75, 3~14, 3~15, 3~16, 3~17, 3~18, 3~19, 3~20, 3~22.5, 3~25, 3~27.5, 3~30, 3.5~6.5, 3.5~13.75, 3.5~15, 2.5~17.5, 4~5, 4~6, 4~7, 4~8, 4~9, 4~10, 4~11, 4~12, 4~13, 4~13.75, 4~14, 4~15, 4~16, 4~17, 4~18, 4~19, 4~20, 4~22.5, 4~25, 4~2 7.5, 4-30, 5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 5-12, 5-13, 5-13.75, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 5-20, 5-22.5, 5-25, 5-27.5, 5-30, 6-7, 6-8, 6-9, 6-10, 6-11, 6-12, 6-13, 6-13.75, 6-14, 6-15, 6-16, 6-17, 6-18, 6-19, 6-20, 6-22.5, 6-25, 6-27.5, 6-30, 7-8, 7-9, 7-10, 7-11, 7-12, 7-13, 7-13.75, 7-14, 7-15, 7-16, 7-17, 7-18, 7-19, 7-20. , 7~22.5, 7~25, 7~27.5, 7~30, 7.5~12.5, 7.5~13.5, 7.5~15, 8~9, 8~10, 8~11, 8~12, 8~13, 8~13.75, 8~14, 8~15, 8~16, 8~17, 8~18, 8~19, 8~20, 8~22.5, 8 ~25, 8~27.5, 8~30, 9~10, 9~11, 9~12, 9~13, 9~13.75, 9~14, 9~15, 9~16, 9~17, 9~18, 9~19, 9~20, 9~22.5, 9~25, 9~27.5, 9~30, 10~11, 10~12, 10~13, 10~13 .75, 10~14, 10~15, 10~16, 10~17, 10~18, 10~19, 10~20, 10~22.5, 10~25, 10~27.5, 10~30, 11.5~15.5, 12.5~14.5, 7.5~22.5, 8.5~32.5, 9.5~15.5, 15.5~ 24.5, 5-35, 17.5-22.5, 22.5-32.5, 25-35, 25.5-34.5, 27.5-32.5, 2-20, 2.5-22.5, 9.5-21.5, 10-50, 15-50, 20-50, 25-50, 30-50, 35-50, or 40-50 mg / m 2 In some embodiments, the compound of formula (I) is administered in a dose range of about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 10 7.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26 .5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50mg / m 2In some embodiments, the compound of formula (I) is administered in a dose of about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102 7.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26 .5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50mg / m 2 In some embodiments, the compound of formula (I) is administered in a dose of about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5 , 29, 29.5, 30, 30.5, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50mg / m 2 In some embodiments, the compound of formula (I) is administered in a dose that is greater than or equal to about 40 mg / m 2 The dose is given per body surface area.

[0066] In some embodiments, the dose of the compound of formula (I) is about 5 mg to 300 mg, 5 mg to 200 mg, 7.5 mg to 200 mg, 10 mg to 100 mg, 15 mg to 100 mg, 20 mg to 100 mg, 30 mg to 100 mg, 40 mg to 100 mg, 10 mg to 80 mg, 15 mg to 80 mg, 20 mg to 80 mg, 30 mg to 80 mg, 40 mg to 80 mg, 10 mg to 60 mg, 15 mg to 60 mg, 20 mg to 60 mg, 30 mg to 60 mg, or about 40 mg to 60 mg. In some embodiments, the compound of formula (I) administered is about 20 mg to 60 mg, 27 mg to 60 mg, 20 mg to 45 mg, or 27 mg to 45 mg. In some embodiments, the compound of formula (I) administered is about 5 mg to 7.5 mg, 5 mg to 9 mg, 5 mg to 10 mg, 5 mg to 12 mg, 5 mg to 14 mg, 5 mg to 15 mg, 5 mg to 16 mg, 5 mg to 18 mg, 5 mg to 20 mg, 5 mg to 22 mg, 5 mg to 24 mg, 5 mg to 26 mg, 5 mg to 28 mg, 5 mg to 29 mg, 5 mg to 30 mg, 5 mg to 31 mg, 5 mg to 32 mg, 5 mg to 33 mg, 5 mg to 34 mg, 5 mg to 35 mg, 5 mg to 36 mg, 5 mg to 37 mg, 5 mg to 38 mg, 5 mg to 39 mg, 5 mg to 40 mg, 5 mg to 41 mg, 5 mg to 42 g~18mg、5mg~20mg、5mg~22mg、5mg~24mg、5mg~26mg、5mg~28mg、5mg~30mg、5mg~32mg、5mg~34mg、5mg~36mg、5mg~38mg、5mg~40mg、5mg~42mg、5mg~44mg、5mg~46mg、5mg~48mg、5mg~50mg、5mg~52mg、5mg~54mg、5mg~56mg、5mg~58mg、5mg~60mg、7mg~7.7mg、7mg~9mg、7mg~10mg、7mg~12mg、7mg~14mg、7mg~15mg、7mg~16mg、7mg~18mg、7mg~20mg、7mg~22mg、7mg~24mg、7mg~26mg、7mg~28mg、7mg~30mg、7mg~32mg、7mg~34mg、7mg~36mg、7mg~38mg、7mg~40mg、7mg~42mg、7mg~44mg、7mg~46mg、7mg~48mg、7mg~50mg、7mg~52mg、7mg~54mg、7mg~56mg、7mg~58mg、7mg~60mg、9mg~10mg、9mg~12mg、9mg~14mg、9mg~15mg、9mg~16mg、9mg~18mg、9mg~20mg、9mg~22mg、9mg~24mg、9mg~26mg、9mg~28mg、9mg~30mg、9mg~32mg、9mg~34mg、9mg~36mg、9mg~38mg、9mg~40mg、9mg~42mg、9mg~44mg、9mg~46mg、9mg~48mg、9mg~50mg、9mg~52mg、9mg~54mg、9mg~56mg、9mg~58mg、9mg~60mg、10mg~12mg、10mg~14mg、10mg~15mg、10mg~16mg、10mg~18mg、10mg~20mg、10mg~22mg、10mg~24mg、10mg~26mg、10mg~28mg、10mg~30mg、10mg~32mg、10mg~34mg、10mg~36mg、10mg~38mg、10mg~40mg、10mg~42mg、10mg~44mg、10mg~46mg、10mg~48mg、10mg~50mg、10mg~52mg、10mg~54mg、10mg~56mg、10mg~58mg、10mg~60mg、12mg~14mg、12mg~15mg、12mg~16mg、12mg~18mg、12mg~20mg、12mg~22mg、12mg~24mg、12mg~26mg、12mg~28mg、12mg~30mg、12mg~32mg、12mg~34mg、12mg~36mg、12mg~38mg、12mg~40mg、12mg~42mg、12mg~44mg、12mg~46mg、12mg~48mg、12mg~50mg、12mg~52mg、12mg~54mg、12mg~56mg、12mg~58mg、12mg~60mg、15mg~16mg、15mg~18mg、15mg~20mg、15mg~22mg、15mg~24mg、15mg~26mg、15mg~28mg、15mg~30mg、15mg~32mg、15mg~34mg、15mg~36mg、15mg~38mg、15mg~40mg、15mg~42mg、15mg~44mg、15mg~46mg、15mg~48mg、15mg~50mg、15mg~52mg、15mg~54mg、15mg~56mg、15mg~58mg、15mg~60mg、17mg~18mg、17mg~20mg、17mg~22mg、17mg~24mg、17mg~26mg、17mg~28mg、17mg~30mg、17mg~32mg、17mg~34mg、17mg~36mg、17mg~38mg、17mg~40mg、17mg~42mg、17mg~44mg、17mg~46mg、17mg~48mg、17mg~50mg、17mg~52mg、17mg~54mg、17mg~56mg、17mg~58mg、17mg~60mg、20mg~22mg、20mg~24mg、20mg~26mg、20mg~28mg、20mg~30mg、20mg~32mg、20mg~34mg、20mg~36mg、20mg~38mg、20mg~40mg、20mg~42mg、20mg~44mg、20mg~46mg、20mg~48mg、20mg~50mg、20mg~52mg、20mg~54mg、20mg~56mg、20mg~58mg、20mg~60mg、22mg~24mg、22mg~26mg、22mg~28mg、22mg~30mg、22mg~32mg、22mg~34mg、22mg~36mg、22mg~38mg、22mg~40mg、22mg~42mg、22mg~44mg、22mg~46mg、22mg~48mg、22mg~50mg、22mg~52mg、22mg~54mg、22mg~56mg、22mg~58mg、22mg~60mg、25mg~26mg、25mg~28mg、25mg~30mg、25mg~32mg、25mg~34mg、25mg~36mg、25mg~38mg、25mg~40mg、25mg~42mg、25mg~44mg、25mg~46mg、25mg~48mg、25mg~50mg、25mg~52mg、25mg~54mg、25mg~56mg、25mg~58mg、25mg~60mg、27mg~28mg、27mg~30mg、27mg~32mg、27mg~34mg、27mg~36mg、27mg~38mg、27mg~40mg、27mg~42mg、27mg~44mg、27mg~46mg、27mg~48mg、27mg~50mg、27mg~52mg、27mg~54mg、27mg~56mg、27mg~58mg、27mg~60mg、30mg~32mg、30mg~34mg、30mg~36mg、30mg~38mg、30mg~40mg、30mg~42mg、30mg~44mg、30mg~46mg、30mg~48mg、30mg~50mg、30mg~52mg、30mg~54mg、30mg~56mg、30mg~58mg、30mg~60mg、33mg~34mg、33mg~36mg、33mg~38mg、33mg~40mg、33mg~42mg、33mg~44mg、33mg~46mg、33mg~48mg、33mg~50mg、33mg~52mg、33mg~54mg、33mg~56mg、33mg~58mg、33mg~60mg、36mg~38mg、36mg~40mg、36mg~42mg、36mg~44mg、36mg~46mg、36mg~48mg、36mg~50mg、36mg~52mg、36mg~54mg、36mg~56mg、36mg~58mg、36mg~60mg、40mg~42mg、40mg~44mg、40mg~46mg、40mg~48mg、40mg~50mg、40mg~52mg、40mg~54mg、40mg~56mg、40mg~58mg、40mg~60mg、43mg~46mg、43mg~48mg、43mg~50mg、43mg~52mg、43mg~54mg、43mg~56mg、43mg~58mg、42mg~60mg、45mg~48mg、45mg to 50mg, 45mg to 52mg, 45mg to 54mg, 45mg to 56mg, 45mg to 58mg, 45mg to 60mg, 48mg to 50mg, 48mg to 52mg, 48mg to 54mg, 48mg to 56mg, 48mg to 58mg, 48mg to 60mg, 50mg to 52mg, 50mg to 54mg, 50mg to 56mg, 50mg to 58mg, 50mg to 60mg, 52mg to 54mg, 52mg to 56mg, 52mg to 58mg, or 52mg to 60mg. In some embodiments, the dose of the compound of Formula (I) is greater than about 5 mg, about 10 mg, about 12.5 mg, about 13.5 mg, about 15 mg, about 17.5 mg, about 20 mg, about 22.5 mg, about 25 mg, about 27 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 125 mg, about 150 mg, or about 200 mg. In some embodiments, the dose of the compound of Formula (I) is less than about 5 mg, about 10 mg, about 12.5 mg, about 13.5 mg, about 15 mg, about 17.5 mg, about 20 mg, about 22.5 mg, about 25 mg, about 27 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 125 mg, about 150 mg, or about 200 mg.

[0067] In some embodiments, the treatment regimen includes administration of a compound of Formula (I) once per week, once per two weeks, once per three weeks, once per four weeks, once per five weeks, once per six weeks, once per seven weeks, or once per eight weeks. In some embodiments, the treatment regimen includes administration of a compound of Formula (I) twice per week, twice per two weeks, twice per three weeks, twice per four weeks, twice per five weeks, twice per six weeks, twice per seven weeks, or twice per eight weeks. In some embodiments, the treatment regimen includes administration of a compound of Formula (I) once per week for a one-week, two-week, three-week, four-week, five-week, six-week, seven-week, or eight-week treatment cycle. In some embodiments, the treatment regimen includes administration of a compound of Formula (I) twice per week for a one-week, two-week, three-week, four-week, five-week, six-week, seven-week, or eight-week treatment cycle. In some embodiments, the treatment regimen comprises administration of a compound of Formula (I) on days 1, 8, and 15 of a 21 day treatment cycle.

[0068] The treatment cycle may be repeated as long as the regimen is clinically tolerated. In some embodiments, the treatment cycle of the compound of formula (I) is repeated n times, where n is an integer in the range of 2 to 30. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the new treatment cycle may be performed immediately after the completion of the previous treatment cycle. In some embodiments, the new treatment cycle may be performed at a certain time after the completion of the previous treatment cycle. In some embodiments, the new treatment cycle may be performed 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or 7 weeks after the completion of the previous treatment cycle. EXAMPLES

[0069] The following examples are included to further illustrate the present disclosure. The examples should not be interpreted as specifically limiting the present disclosure. Variations of these examples within the scope of the claims are within the scope of the skilled artisan's competence and are considered to be included within the scope of the present disclosure as described herein and as set forth in the claims. The reader will recognize that a person skilled in the art who has access to this disclosure and the skills of the art may prepare and use the present disclosure without using the exhaustive examples.

[0070] Example 1 In this study, plinabulin is characterized for its ability to inhibit the adhesion-independent growth and ex vivo colony formation of tumor cells in semi-solid medium.The compound is examined in 87 of 93 originally planned tumor xenografts representing all major cancer types, utilizing 3D clonogenesis in 96-well format and colony counting based on image analysis as readout.By utilizing this assay, test compounds are evaluated for their ability to inhibit the adhesion-independent growth and ex vivo colony formation of tumor cells in semi-solid medium.

[0071] A master stock solution of plinabulin was prepared by dissolving the compound in DMSO at a concentration of 9.9 mM. A working stock solution was prepared by diluting the master stock solution to 0.99 mM (330 times the maximum test concentration) with DMSO. Small aliquots of the master stock and working stock were stored at -20°C. On each experimental day, aliquots of the working stock were used and stored at room temperature before and during treatment. All liquid handling steps were performed using a Tecan Freedom EVO 200 robotic platform. First, serial dilutions of the working stock solution were performed in DMSO. Then, the DMSO dilutions were diluted 1:22 in cell culture medium (IMDM supplemented with 20% (v / v) fetal bovine serum and 50 μg / ml gentamicin) in the intermediate dilution plate. Finally, 10 μl taken from the intermediate dilution plate was transferred to the final assay plate with 140 μl / well. The DMSO working stock was then diluted 1:330, resulting in a DMSO concentration in the assay of 0.3% v / v in all wells.

[0072] Plinabulin was studied in 87 PDX models representing multiple cancer types. Tumors were passaged as subcutaneous xenografts in NMRI nu / nu mice. Tumor volumes ranged from 400 to 1000 mm. 3At that time, tumor-bearing mice were euthanized and tumors were rapidly harvested under sterile conditions in accordance with the relevant SOPs and relevant animal welfare guidelines issued by FELASA and GV-SOLAS. Tumors were mechanically disaggregated and then incubated at 37°C for 60-12 h with an enzyme cocktail consisting of collagenase type IV (41 U / ml), DNase I (125 U / ml), hyaluronidase type III (100 U / ml) and dispase II (1 U / ml) in RPMI 1640 medium (Life Technologies). The cells were incubated for 10 min. The cells were passed through 100 μm and 40 μm mesh size sieves (cell strainer, BD Falcon™) and washed with RPMI1640 medium (Biochrom). The percentage of viable cells was determined in a Neubauer hemocytometer using the trypan blue dye exclusion method. An aliquot of cells was frozen and stored in the vapor phase of liquid nitrogen. On each experimental day, a frozen aliquot of tumor cells was thawed and used to prepare assay plates.

[0073] Clonogenic assays were performed in a 96-well plate format using ultra-low attachment plates. For each test, cells were prepared as previously described and assay plates were prepared as follows: each test well contained a semi-solid medium layer with tumor cells (50 μl) and a second medium supernatant layer with or without test compound (100 μl). Cell layers ranged from 2.5 to 12.5 × 10 cells per well. 3The wells consisted of tumor cells, which were seeded on day 0 (d0) in 50 μl / well cell culture medium (IMDM supplemented with 20% (v / v) fetal bovine serum, 50 μg / ml gentamicin and 0.4% (w / v) agar). After 24 h, the soft agar layer was covered with 90 μl of the same culture medium without agar. Seven days after seeding, 10 μl of test compound or control medium was added and left on the cells for 24 h. Each 96-well plate contained six DMSO-treated control wells and nine drug-treated wells in duplicate at different concentrations. After the 24-h incubation period, the supernatant (90 μl medium without agar + 10 μl compound / control) was replaced with drug-free medium and incubated until the end of the study. The cultures were incubated for a total of 13 days at 37°C and 7.5% CO2 in a humidified atmosphere and the growth of colonies was closely monitored using an inverted microscope. Within this time period, ex vivo tumors grew to diameters of more than 50 μm (2000 μm 2 At the time of maximum colony formation in the Max vehicle-treated wells, viable colonies were stained with a sterile aqueous solution of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT, 1 mg / ml, 25 μl / well) for 48 h, and colony counts were performed with an automated image analysis system (Bioreader 5000 V-alpha, BIO-SYS GmbH).

[0074] The ability of plinabulin to inhibit ex vivo colony formation of cells with anchorage-independent growth potential in semi-solid medium was investigated in 87 PDX models of various tissue types using a 3D clonogenic assay. The results are shown in Figure 1 and the absolute and relative IC 70 The concentration-effect curves for small cell lung cancer, gastric cancer, and triple-negative breast cancer are shown in Figures 3-5.

[0075] Plinabulin inhibited tumor colony formation in almost all cell lines in a concentration-dependent manner, yielding sigmoidal concentration-effect curves as shown in Figures 3 to 5, with geometric mean absolute IC 70Of all tumor models tested, 40 out of 87 were sensitive to plinabulin, with a 6.8-fold difference in absolute IC values ​​in the sensitive models compared to all models. 70 The result was (Table 1).

[0076] [Table 1]

[0077] Geometric mean absolute IC 70 The histological types with the greatest sensitivity based on values ​​were small cell lung cancer (geometric mean absolute IC 70 = 0.035 μM; n = 7), bladder cancer (geometric mean absolute IC 70 = 0.038 μM; n = 9), and soft tissue sarcoma (geometric mean absolute IC 70 = 0.057 μM; n = 10).

[0078] In summary, the most responsive models are BXF1258, BXF2211, BXF2775, and SXFS627, with absolute IC 70 was 3 nM, with the melanoma model being the most resistant model, and the geometric mean absolute IC 70 was observed to be 1.105 μM (n=19).

[0079] Example 2 The study examined plinabulin for ex vivo anti-cancer activity in 68 of 71 originally planned patient-derived xenograft (PDX) models representing major cancer types. Experiments were performed using 3D clonogenesis assays in a 96-well format with image analysis-based colony counting as a readout. Using this assay, test compounds were evaluated for their ability to inhibit the anchorage-independent growth of tumor cells in semi-solid medium and ex vivo colony formation.

[0080] A master stock solution of plinabulin was prepared by dissolving the compound in DMSO at a concentration of 9.9 mM. A working stock solution was prepared by diluting the master stock solution with DMSO to a concentration of 0.99 mM (330 times the maximum test concentration). Small aliquots of the master stock and working stock were stored at -20°C. On each experimental day, aliquots of the working stock were used and stored at room temperature before and during treatment. All liquid handling steps were performed using a Tecan Freedom EVO 200 robotic platform. First, serial dilutions of the working stock solution were performed in DMSO. Then, the DMSO dilutions were diluted 1:22 in cell culture medium (IMDM supplemented with 20% (v / v) fetal bovine serum and 50 μg / ml gentamicin) in the intermediate dilution plate. Finally, 10 μl taken from the intermediate dilution plate was transferred to the final assay plate with 140 μl / well. The DMSO working stock was then diluted 1:330, resulting in a DMSO concentration in the assay of 0.3% v / v in all wells.

[0081] Plinabulin was studied in 68 PDX models representing multiple cancer types. Tumors were passaged as subcutaneous xenografts in NMRI nu / nu mice. Tumor volumes ranged from 400 to 1000 mm. 3 At that time, tumor-bearing mice were euthanized and tumors were rapidly harvested under sterile conditions in accordance with the relevant SOPs and relevant animal welfare guidelines issued by FELASA and GV-SOLAS. The tumors were mechanically disaggregated and then incubated at 37°C for 60–120 min with an enzyme cocktail consisting of collagenase type IV (41 U / ml), DNase I (125 U / ml), hyaluronidase type III (100 U / ml) and dispase II (1 U / ml) in RPMI 1640 medium (Life Technologies). The cells were then incubated for 3 min. The cells were passed through 100 μm and 40 μm mesh size sieves (cell strainer, BD Falcon™) and washed with RPMI1640 medium (Biochrom). The percentage of viable cells was determined using a Neubauer hemocytometer using the trypan blue dye exclusion method. An aliquot of cells was frozen and stored in the vapor phase of liquid nitrogen. On each experimental day, a frozen aliquot of tumor cells was thawed and used to prepare assay plates.

[0082] Clonogenic assays were performed in a 96-well plate format using ultra-low attachment plates. For each test, cells were prepared as previously described (section 6.6) and assay plates were prepared as follows: each test well contained a semi-solid medium layer with tumor cells (50 μl) and a second medium supernatant layer with or without test compound (100 μl). Cell layers ranged from 2.5 to 12.5 × 10 cells per well. 3 The wells consisted of tumor cells, which were seeded on day 0 (d0) in 50 μl / well cell culture medium (IMDM supplemented with 20% (v / v) fetal bovine serum, 50 μg / ml gentamicin and 0.4% (w / v) agar). After 24 h, the soft agar layer was covered with 90 μl of the same culture medium without agar. Seven days after seeding, 10 μl of test compound or control medium was added and left on the cells for 24 h. Each 96-well plate contained six DMSO-treated control wells and nine drug-treated wells in duplicate at different concentrations. After the 24-h incubation period, the supernatant (90 μl medium without agar + 10 μl compound / control) was replaced with drug-free medium and incubated until the end of the study. The cultures were incubated for a total of 13 days at 37°C and 7.5% CO2 in a humidified atmosphere and the growth of colonies was closely monitored using an inverted microscope. Within this time period, ex vivo tumors grew to diameters of more than 50 μm (2000 μm 2At the time of maximum colony formation in the maximum vehicle-treated wells, viable colonies were stained with a sterile aqueous solution of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT, 1 mg / ml, 25 μl / well) for 48 h, and colony counts were performed with an automated image analysis system (Bioreader 5000 V-alpha, BIO-SYS GmbH).

[0083] The ability of plinabulin to inhibit ex vivo colony formation of cells with anchorage-independent growth potential in semi-solid medium was investigated in 68 PDX models of various tissue types using a 3D clonogenic assay. Results are shown as IC 50 and IC 70 (Figures 6 and 7, respectively) and absolute and relative IC values ​​(Figures 8 and 9, respectively). Individual results, i.e., relative and absolute IC 50 / I C 70 Concentration-response tables containing the T / C values ​​(FIGS. 13A and 13B) and T / C values ​​were determined (FIGS. 14A and 14B). The concentration-response curves for small cell lung cancer, gastric cancer, and triple-negative breast cancer are shown in FIGS. 10-12, respectively.

[0084] Plinabulin concentration-dependently inhibited tumor colony formation in almost all cell lines, resulting in a sigmoidal concentration-effect curve, as shown in Figures 10, 11, and 12. Compared to the concentration-effect curves obtained in Example 1, where cells were treated on days 2-3, treatment on days 7-8 in this study showed a higher minimum plateau and an IC 50 and IC 70 However, the small cell lung cancer model was observed to be the most sensitive model in both the Example 1 and Example 2 studies.

[0085] Plinabulin inhibits tumor colony formation and the geometric mean absolute IC 50 The absolute IC value was 0.6 μM (Figures 6 and 9). 50Based on the results, 24 of 68 tumor models were sensitive to plinabulin, with a 10.7-fold difference in absolute IC in the sensitive models compared to the overall models. 50 (Table 2).

[0086] [Table 2]

[0087] Geometric Mean Absolute IC 50 The most sensitive histological types based on values ​​(Figure 6) were small cell lung cancer (geometric mean absolute IC 50 = 0.074 μM; n = 7), osteosarcoma (geometric mean absolute IC 50 = 0.117 μM; n = 3), gastric cancer (Asian, geometric mean absolute IC 50 = 0.166 μM, n = 3), and central nervous system cancer (geometric mean absolute IC 50 = 0.426 μM; n = 6). In summary, the absolute IC 50 The maximum response model based on (Figure 8) was optimized for small cell lung cancer model LXFS2156 (absolute IC 50 = 0.007 μM) and LXFS650 (absolute IC 50 =0.022 μM), osteosarcoma model SXFO1186 (absolute IC 50 =0.019 μM), and the (Asian) gastric cancer model GXA3067 (absolute IC 50 = 0.026 μM). Melanoma model (geometric mean absolute IC 50 = 1.367 μM; n = 9), Her2-positive breast cancer model (geometric mean absolute IC 50 = 2.252 μM; n = 6), and soft tissue sarcoma models (geometric mean absolute IC 50 = 2.584 μM; n = 8) was observed to be the most resistant tumor type.

[0088] Plinabulin inhibits tumor colony formation and the geometric mean absolute IC 70 The absolute IC value was 1.78 μM (Figures 7 and 9). 70Based on the results, 9 of 68 tumor models were sensitive to plinabulin, with a 27.1-fold difference in absolute IC in the sensitive models compared to the overall models. 70 (Table 3).

[0089] [Table 3]

[0090] Geometric Mean Absolute IC 70 The most sensitive histological types based on values ​​(Figure 7) were gastric cancer (Asian, geometric mean absolute IC 70 = 0.319 μM; n = 3), small cell lung cancer (geometric mean absolute IC 70 = 0.385 μM; n = 7), osteosarcoma (geometric mean absolute IC 70 = 0.624 μM; n = 3), and central nervous system cancer (geometric mean absolute IC 70 = 1.521 μM; n = 6). In summary, the absolute IC 70 The maximum response model based on (Figure 9) was optimized for small cell lung cancer model LXFS2156 (absolute IC 70 =0.015μM), LXFS1129 (absolute IC 70 =0.032μM) and LXFS650 (absolute IC 70 =0.032 μM), as well as the osteosarcoma model SXFO1186 (absolute IC 70 Triple-negative breast cancer (MAXFTN) was also observed to be sensitive to plinabulin (see Figure 7). Soft tissue sarcoma model (n=8), Her2-positive breast cancer model (n=6), and melanoma model (n=1). Ranoma (n=9), bladder cancer (n=6), and gastric cancer (Caucasian, n=3) were the most resistant tumor types, with geometric mean absolute IC 70 Values ​​above 3 μM were observed.

Claims

1. A pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of cancer as monotherapy, wherein the compound of formula (I) is: 【Chemical 1】 (I) During the ceremony, R １ , R ４ , and R ６ are respectively hydrogen atoms, deuterium atoms, halogen atoms, and saturated C １ ~C ２４ Alkyl, unsaturated C １ ~C ２４ Alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups, hydroxy, carboxy, -CO-O-R ７ , cyano, alkylthio, alkyl halides including polyhalogenated alkyls, carbonyl halides, and carbonyl-CH ２ CO-R ７ where R ７ is a hydrogen atom, a halogen atom, and a saturated C １ ~C ２４ Alkyl, unsaturated C １ ~C ２４ selected from alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups; R １ ' and R １ '' are each independently a hydrogen atom, a deuterium atom, a halogen atom, or a saturated C １ ~C ２４ Alkyl, unsaturated C １ ~C ２４ Alkenyl, cycloalkyl, cyclo Alkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups, hydroxy, carboxy, —CO—O—R ７ , cyano, alkylthio, alkyl halides including polyhalogenated alkyls, carbonyl halides, and carbonyl-CH ２ CO-R ７ where R ７ is a hydrogen atom, a halogen atom, and a saturated C １ ~C ２４ Alkyl, unsaturated C １ ~C ２４ selected from alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups; R, R １ ' and R １ " are either covalently bonded to each other or are not covalently bonded to each other; R ２ , R ３ and R ５ are respectively hydrogen atoms, deuterium atoms, halogen atoms, and saturated C １ ~C １２ Alkyl, unsaturated C １ ~C １２ selected from the group consisting of alkenyl, acyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro and substituted nitro groups, sulfonyl and substituted sulfonyl groups; m is an integer of 0, 1 or 2; X １ and X ２ are independently selected from the group consisting of oxygen, nitrogen, and sulfur atoms; Y is NR ５ , an oxygen atom, a sulfur atom, an oxidized sulfur atom, a methylene group, and a substituted methylene group; Z for each distinct n when not 0, and Z １ , Z ２ , Z ３ and Z ４ are each independently selected from carbon, sulfur, nitrogen, and oxygen atoms; The dashed bond is a compound that can be either a single bond or a double bond, wherein the cancer is selected from gastric cancer, small cell lung cancer, osteosarcoma, bladder cancer, and triple-negative breast cancer.

2. The pharmaceutical composition of claim 1 , wherein the cancer is gastric cancer.

3. The pharmaceutical composition of claim 1 , wherein the cancer is small cell lung cancer.

4. The pharmaceutical composition of claim 1, wherein the cancer is triple-negative breast cancer.

5. The pharmaceutical composition according to claim 1, wherein the compound represented by formula (I) is plinabulin or a pharmaceutically acceptable salt thereof.

6. The compound represented by formula (I) is administered at a concentration of 10 mg / m ２ ~50 mg / m ２ The pharmaceutical composition of claim 5, wherein the composition is administered at a dose of

7. The compound represented by formula (I) is administered at a concentration of 20 mg / m ２ ~30 mg / m ２ The pharmaceutical composition of claim 6, wherein the composition is administered at a dose of

8. The compound represented by formula (I) is administered at a concentration of 40 mg / m ２ The pharmaceutical composition of claim 7, wherein the composition is administered at a dose of

9. 6. The pharmaceutical composition of claim 5, wherein the compound of formula (I) is administered on day 1 of a 14-day dosing cycle.

10. 10. The pharmaceutical composition of claim 1, wherein the compound of formula (I) is administered on day 1 of a 21-day dosing cycle.

11. The compound represented by formula (I) is plinabulin, (3Z,6Z)-3-(phenyl-2,3,4,5,6-d ５ )-methylene-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione; (3Z,6Z)-3-(phenyl-2,3,4,5,6-d ５ )-methylene-d-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione; (3Z,6Z)-3-(phenylmethylene-d)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione; (3Z,6Z)-3-(phenyl-2,3,4,5,6-d ５ )-methylene-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione; (3Z,6Z)-3-(phenylmethylene)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione; (3Z,6Z)-3-(phenyl-2,3,4,5,6-d ５ )-methylene-d-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione; (3Z,6Z)-3-(4-fluoro-(phenyl-2,3,5,6-d ４ ))-methylene-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione; (3Z,6Z)-3-(4-fluoro-(phenyl-2,3,5,6-d ４ ))-methylene-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione; (3Z,6Z)-3-(3-fluorobenzylidene)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione; (3Z,6Z)-3-(3-benzyl (3Z,6Z)-3-(3-(4-fluorobenzoyl)benzylidene)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione; ...

10. The pharmaceutical composition of claim 1, wherein the compound is selected from (3Z,6Z)-3-(3-(4-methoxybenzoyl)benzylidene)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione; (3Z,6Z)-3-(3-methoxybenzylidene)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione; (3Z,6Z)-3-(3-(trifluoromethyenzydene)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene-d)piperazine-2,5-dione, and pharmaceutically acceptable salts thereof.

12. 12. The pharmaceutical composition of claim 11, wherein the cancer comprises a tumor and the mass of the tumor is reduced by 50% to 100%.

13. The pharmaceutical composition described in claim 12, wherein the treatment of cancer comprises administering an effective amount of plinabulin and not administering other chemotherapeutic agents.

14. 14. The pharmaceutical composition of claim 13, wherein the tumor mass is reduced by 50% to 70%.

15. The pharmaceutical composition of claim 12, wherein the compound represented by formula (I) is plinabulin monohydrate or a pharmaceutically acceptable salt thereof.

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