TWI535725B - Novel sesquiterpene derivatives and compositions comprising the same for treating inflammation and/or neoplasm related disorders - Google Patents

Description

a derivative of a novel sesquiterpenoid for treating inflammation and/or tumor and a composition comprising the derivative of the novel sesquiterpenoid

The present disclosure relates to the field of sesquiterpenoids, and in particular to derivatives of sesquiterpenoids and their use for the treatment or prevention of inflammatory and/or tumor-related diseases.

Sesquiterpenes are a class of terpenoids that are commonly found in plants and insects. Deoxyelephantopin (DET) is a sesquiterpene lactone isolated from the genus Lepidoptera, such as Elephantopus scaber . It is known to inhibit the growth of various cancer cells in vitro. The effect. In the present disclosure, the inventors of the present invention proposed a variety of different deoxycholestalin derivatives and studied their cell killing effects on different tumor cell lines, including cell lines resistant to chemotherapeutic drugs.

SUMMARY OF THE INVENTION The Summary of the Disclosure is intended to provide a basic understanding of the present disclosure. This Summary is not an extensive overview of the disclosure, and is not intended to be an

The present disclosure is based on the accidental discovery of specific deoxyelephantopin (DET) derivatives with similar or better anti-inflammatory and/or anti-tumor activity to DET; therefore, deoxycholicin derivatives are available To develop agents that treat or prevent inflammatory and/or tumor-related diseases.

Accordingly, the present disclosure provides, in one aspect, a novel synthetic sesquiterpenoid derivative having the structure of formula (I): Wherein: R is H or -(C=O)R ₁ ; and R ₁ is H, C _1-20 alkyl, C _2-20 alkenyl, aryl or heteroaryl, respectively: wherein: C _1-20 The alkyl group may be unsubstituted or have at least one substituent selected from the group consisting of halogen, hydroxy, alkoxy, alkoxy, phenyl, naphthyl, isobenzofuranyl, isophenylthiophenyl (isophenylthiophenyl) Isobenzothienyl) or isoquinolyl; the C _2-20 alkenyl group may be unsubstituted or have at least one substituent selected from the group consisting of phenyl, furanyl, thienyl or isobenzodioxole Isobenzodioxolyl; each phenyl and naphthyl group may be unsubstituted or have at least one substituent selected from the group consisting of halogen, alkyl, perfluoroalkyl, alkoxy or haloalkoxy; The aryl group may be unsubstituted or substituted with a halogen, a C _1-4 lower alkyl group, an alkoxy group or a haloalkoxy group.

In another aspect, the present disclosure provides a pharmaceutical composition for treating or preventing an individual having or suspected of having an inflammation and/or a tumor associated disease. The pharmaceutical composition comprises a therapeutically/prophylactically effective amount of a compound of formula (I), and a pharmaceutically acceptable carrier.

The compound of formula (I) is present in an amount from about 0.1% to about 99% by weight based on the total weight of the pharmaceutical composition. In certain embodiments, the compound of formula (I) comprises at least 1% by weight of the total pharmaceutical composition. In a particular embodiment, the compound of formula (I) comprises at least 5% by weight of the total pharmaceutical composition. In other embodiments, the compound of formula (I) comprises at least 10% by weight of the total pharmaceutical composition. In still other embodiments, The compound of formula (I) comprises at least 25% by weight of the total pharmaceutical composition.

According to certain preferred embodiments, the pharmaceutical composition further comprises a chemotherapeutic agent or a gluthathione biosynthesis blocker. In certain embodiments, the chemotherapeutic agent is DET, paclitaxel or PLX4032. In other embodiments, the glutathione biosynthesis blocker is buthionine sulfoximine (BSO) or sulfasalazine.

The inflammation suitable for treatment with the pharmaceutical composition is selected from the group consisting of inflammatory skin diseases, inflammatory bowel diseases, allergic lung diseases, asthma, allergic rhinitis, autoimmune diseases, acute and chronic inflammation, and repairing In a group consisting of syndromes, human immunodeficiency virus infections, and cancer.

A tumor-associated disease suitable for treatment with the pharmaceutical composition is a cancer selected from the group consisting of breast cancer, papilloma, melanoma, brain cancer, lung cancer, lymphoma, neuroepithelial neoplasia, renal cancer, prostate cancer, In a group consisting of gastric cancer, colorectal cancer, and uterine cancer.

According to some embodiments of the invention, the cancer is a drug resistant cancer. In one example, the cancer is a melanoma that is resistant to PLX4032.

According to a particular embodiment of the invention, the pharmaceutical composition reduces the side effects caused by administration of PLX4032 to the skin of a cancer-infected individual, for example, a skin papilloma that is induced by administration of PLX4032.

The present disclosure also encompasses a method of treating or preventing an individual having or suspected of having an inflammatory and/or tumor-related disease. The method comprises administering a therapeutically or prophylactically effective amount of a compound of formula (I) to the subject to slow, reduce and/or prevent inflammatory and/or tumor related diseases.

According to certain preferred embodiments, the method further comprises administering, prior to, or after administration of a compound of formula (I) according to the invention, other agents known to be useful in ameliorating inflammatory conditions and/or tumor-related diseases, such as a chemotherapeutic agent. Or a glutathione biosynthesis blocker to the individual.

The inflammation suitable for treatment by the method of the present invention is selected from the group consisting of inflammatory skin diseases, inflammatory bowel diseases, allergic lung diseases, asthma, allergic rhinitis, autoimmune diseases, acute and chronic inflammation, and Shergar's syndrome. Human immunodeficiency virus infection and cancer are grouped together.

A tumor-related disease suitable for treatment by the method of the present invention is a cancer selected from the group consisting of breast cancer, papilloma of the skin, melanoma, brain cancer, lung cancer, lymphoma, neuroepithelial neoplasia, renal cancer, prostate cancer, gastric cancer, In a group consisting of colorectal cancer and uterine cancer.

According to some embodiments, the cancer is a drug resistant cancer. In one example, the cancer is a melanoma that is resistant to PLX4032.

According to a particular embodiment, the method of the invention reduces the side effects of the administration of PLX4032 on the skin of a subject suffering from cancer, including reducing the number and size of cutaneous papillomas induced by PLX4032.

One or more embodiments of the present disclosure will be described in detail in the following embodiments. Features of the above disclosure will be better understood from the following detailed description and the appended claims. It is to be understood that the foregoing general descriptions

The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood. The description of the drawings is as follows: FIG. 1 shows DET, DETD-35 and according to an embodiment of the present disclosure. The effects of PTX on MDA-MB-231 cell migration and invasion were evaluated by (A) migration assay and (B) invasion assay, respectively; Figure 2 depicts MDA-MB-231 cells treated with DET and DETD-35, respectively. Dynamic characteristics of the degree of mobility; (A) Time-lapse photography series image; (B) Cell trajectory map of each cell after treatment; (C) Average migration rate of each cell after treatment, during observation period At 30-minute intervals (D) total migration distance of each cell for 12 hours after each treatment; data are expressed as mean ± standard deviation (n 12), when the difference mark is marked on the bar graph, it represents a significant difference ( P < 0.05, one-way ANOVA); Figure 3 shows the DETD-35 inhibits the in situ MDA-MB-231 of the female NOD/SCID mouse. Breast tumor growth effect; (A) time course of tumor size; (B) animal body weight; (C) tumor size on day 71; (D) organ index of mice in different test groups; Figure 4 shows DETD-35 Effects of lung metastasis on MDA-MB-231 triple-negative breast tumors in xenografted SCID mice; (A) calculation of the number of metastatic lung tumors in lung organs between different treatment groups; (B) different Analysis results of organ index, organ index (P<0.05; one-way ANOVA); Figure 5A shows DETD-35, DETD-39 and PTX individually for MDA-MB according to an embodiment of the present disclosure. The inhibitory effect of 231 cells; Figure 5B is based on an embodiment of the present disclosure, and uses DETD-35 and PTX, or a combination of DETD-39 and PTX to inhibit the MDA-MB-231 cells; Figure 5C is The equivalent line graph of Figure 5B analyzes the line graph; Figure 5D shows the combination index (CI) of the result of Figure 5B, where CI < 1 represents a synergistic effect; CI=1 represents the additive effect; and CI>1 represents the antagonistic effect; and FIG. 6 shows that DET and DETD-35 according to an embodiment of the present disclosure can effectively reduce the (A) butyl thiopurine imine And (B) survival rate of sulfasalazine pretreated MDA-MB-231 cells; Figures 7A, 7B are based on an embodiment of the present disclosure, and PLX4032 and DETD-35 inhibit proliferation of A375 melanoma cells A bar graph of the effect; Figure 7C is a bar graph of PTX and DETD-35 for inhibiting the proliferation of A375 melanoma cells according to an embodiment of the present disclosure; and Figure 7D shows the result of Figure 7C. Coincidence factor (CI), where CI < 1 represents a synergistic effect; CI = 1 represents an additive effect; and CI > 1 represents an antagonistic effect; and Figure 8A is depicted in accordance with an embodiment of the present disclosure, in vivo BRAF ^V600E Tumor size changes with time after treatment of mutant melanoma with PLX4032, DET, DETD-35, PLX4032+DET or PLX4032+DETD-35; Figure 8B is PLX4032, DET, DETD-35, PLX4032+DET or PLX4032+DETD -35 treatment on the weight of BRAF ^V600E mutant melanoma; Figure 9A is based on this According to an embodiment, the inhibitory effects of PLX4032 and DETD-35 on proliferation of non-resistant A375 cells and drug-resistant A375 cells (A375-R) cells; Figure 9B shows A375 cells treated with PLX4032 and drug-resistant A375 -R cells, the amount of key molecules in the MAPK signal path, the results of Western blot analysis; Figure 9C is shown in accordance with an embodiment of the present disclosure, treated with PLX4032 alone or in combination with PLX4032 and DETD- 35 (1 or 3 μM) treated A375-R cells, the amount of key molecules in the MAPK signal pathway, the results of Western blot analysis; 10A is based on an embodiment of the present disclosure, in vivo Tumor size changes with time after treatment of PLX4032 resistant BRAF ^V600E mutant melanoma with PLX4032, DET, DETD-35, PLX4032+DET or PLX4032+DETD-35; Figure 10B is PLX4032, DET, DETD-35 , PLX4032 + DET or PLX4032 + DETD-35 Effect of treatment with the drug-resistant mutant BRAF ^V600E PLX4032 melanoma by weight; based on FIG. 11A according to one embodiment of the present disclosure, to DMBA / TPA treated mice induced skin papilloma The experimental platform, after certification, the side effects of PLX4032 to promote skin papilloma and the combination of PLX4032 and DET or DETD-35 can reduce the photo of mouse skin papilloma; Figure 11B shows DET or DETD-35 Treatment delay PLX4032 promoted DMBA/TPA treatment induced time to skin papilloma in mice and the number of papilloma produced; and Figure 11C depicts DET or DETD-35 treatment induced PLX4032 induced DMBA/TPA treatment The effect of papilloma volume.

The various features and elements in the figures are not drawn to scale, and are in the In addition, similar elements/components are referred to by the same or similar element symbols throughout the different drawings.

In order to make the description of the present disclosure more detailed and complete, the following description of the embodiments of the present invention is intended to The features of various specific embodiments, as well as the method steps and sequences thereof, are constructed and manipulated in the embodiments. However, other specific embodiments may be utilized to achieve the same or equivalent function and sequence of steps.

The scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains, unless otherwise defined herein. In addition, the singular noun used in this specification covers the plural of the noun in the absence of conflict with the context. Type; the plural noun used also covers the singular form of the noun.

Noun definition

Unless the specification otherwise dictates, the term "alkyl" refers to a hydrocarbon group having from 1 to 20 carbon atoms and which is linear, branched and/or cyclic ("cycloalkyl") (ie, 1) -10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 or 1 carbon atom). A structure in which an alkyl group has 1 to 4 carbon atoms is referred to as a "lower alkyl group". For example, the alkyl group comprises methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, 2-isopropyl-3-methylbutyl, pentyl, pentyl -2-yl, hexyl, isohexyl, heptyl, hept-2-yl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, decyl, decyl, Undecyl and dodecyl. The cycloalkyl structure may be monocyclic or polycyclic, and examples thereof include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, and a cyclohexyl group. Unless otherwise defined, the monoalkyl group in each of the examples optionally contains an optional substituent, that is, an unsubstituted alkyl group or a substituted alkyl group having one or more substituents. . In a particular embodiment, the alkyl group is a C _2-10 alkyl group with a substituent.

Unless otherwise defined in the specification, the "alkenyl group" means a hydrocarbon group having 2 to 20 carbon atoms and which is linear, branched and/or cyclic (ie, 2-10, 2-9, 2- 8, 2-7, 2-6, 2-5, 2-4, 2-3 or 2 carbon atoms), wherein the alkenyl group contains one or more carbon-carbon double bonds. Examples of the alkenyl structure include a vinyl group, a propenyl group, a 1-butenyl group, a 2-butenyl group, an isobutenyl group, a 1-pentenyl group, a 2-pentenyl group, a 3-methyl-1-butenyl group, 2-methyl-2-butenyl, 2,3-dimethyl-2-butyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1-heptenyl, 2-heptyl Alkenyl, 3-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 1-decenyl, 2-nonenyl and 3-decenyl. One or more carbon-carbon double bonds may be internal to the hydrocarbyl group (e.g., 2-butenyl) or at the end (e.g., 1-butenyl). Unless otherwise defined, the monoalkenyl group in each of the examples individually contains an optional substituent, that is, an unsubstituted alkenyl group or an alkenyl group having one or more substituents (substituted alkenyl). . In a particular embodiment, the alkenyl group is a C _2-10 alkenyl group having a substituent.

Unless otherwise defined, the "alkoxy" refers to a mono-O-alkyl group. Examples of alkoxy groups include, but are not limited to, -OCH ₃ , -OCH ₂ CH ₃ , -O(CH ₂ ) ₂ CH ₃ , -O(CH ₂ ) ₃ CH ₃ , -O(CH ₂ ) ₄ CH ₃ and -O(CH ₂ ) ₅ CH ₃ . The "lower number alkoxy group" means -O-(lower alkyl group); for example, -OCH ₃ and -OCH ₂ CH ₃ .

Unless otherwise defined in the specification, the term "aryl" refers to an aromatic ring or a partial aromatic ring system consisting of carbon and hydrogen atoms. An aromatic group may comprise a plurality of rings bonded or fused to each other. Examples of the aromatic group include a naphthyl group and a phenyl group. Unless otherwise indicated, an aromatic group in each instance individually contains an optional substituent, i.e., an unsubstituted aryl group or a substituted aryl group having one or more substituents. In a particular embodiment, the fragrance The base is a phenyl group having a substituent. In another embodiment, the aryl group is a naphthyl group having a substituent.

Unless otherwise defined in the specification, the "heteroaryl" means that at least one carbon atom of an aromatic group carries a hetero atom (e.g., nitrogen, oxygen or sulfur). For example, including, but not limited to, benzofuranyl, benzothienyl, quinolyl, benzodioxolyl, furyl, and Thienyl.

Unless otherwise defined in the specification, "alkylaryl" or "alkyl-aryl" refers to a monoalkyl group bonded to an aromatic group.

Unless otherwise defined in the specification, "halogen (halo)" includes fluorine, chlorine, bromine and iodine.

Unless otherwise defined in the specification, the term "substituted" when referring to a chemical structure or chemical group refers to a derivative having one or more hydrogen atoms in its structure or group, by an atom, a chemical group. Substituted or functional group substituted; ie, but not limited to, -OH, -CHO, alkoxy, alkoxycarbonyl (eg, -OAc), alkenyl, alkyl (eg, methyl, ethyl, propyl, Tert-butyl), aryl, aryloxy, halogen or haloalkyl (for example, -CCl ₃ , -CF ₃ , -C(CF ₃ ) ₃ ).

In a particular embodiment, the "substituted", when describing a chemical structure or chemical group, refers to a The derivative has one or more hydrogen atoms in its structure or group substituted by one or more: alkoxy, alkoxy, alkyl, aryl, halogen, haloalkyl or hydroxy.

Unless otherwise defined in this specification, one or more adjectives are recited before a series of nouns and should be interpreted as the adjective applies to each noun. For example, "unsubstituted or substituted alkyl, alkenyl, aryl, or heteroaryl" and "unsubstituted or substituted" An alkyl group, an unsubstituted or substituted alkenyl group, an unsubstituted or substituted aryl group or an unsubstituted or substituted heteroaryl group (optionally substituted alky, optionally substituted alkenyl, optionally substituted) "aryl, or non-conductive substituted heteroaryl)" has the same meaning.

A "therapeutically effective amount" of a compound, unless otherwise defined in the specification, refers to an amount sufficient to provide a therapeutic effect in the treatment or management of a disease or condition, or to delay or reduce the symptoms associated with the disease or condition. A therapeutically effective amount of a compound refers to a therapeutic amount which, if used alone or in combination with other agents, provides a therapeutic effect in the treatment or management of a disease or condition. The term "therapeutically effective amount" can encompass improving the overall therapeutic effect, reducing or preventing the onset of a disease or condition-related symptom or enhancing the effectiveness of other therapeutic agents.

Unless otherwise defined in this specification, a "pre- Prophylactically effective amount means a condition that prevents a disease or condition, one or more symptoms associated with a disease/condition, or avoids a disease or condition recurrence. A prophylactically effective amount can be a therapeutic dose, The administration of the drug alone or in combination with other drugs has a preventive effect on the disease. The term "prophylactically effective amount" encompasses an amount which improves the overall preventive effect or enhances the effect of another preventive agent.

Unless otherwise defined in the specification, "treat, treating or treating" refers to the act of administering to a patient suffering from a particular disease or condition, wherein the behavior reduces the disease or condition of the patient, or one or more The severity of a symptom, or the process of slowing or delaying a disease or condition.

It should be noted that if there is no stereochemistry or a part of the structure (for example, a portion indicated by a thick line or a broken line) specifying a structure, the structure or part structure should be interpreted to cover all stereoisomers. . Similarly, when a compound contains one or more asymmetric centers, but the name does not specifically define the stereochemical structure that can be covered by the asymmetric centers, the range of the compound should be interpreted to cover its pure optics. An isomer and a mixture of the optical isomers. Furthermore, any atom having an unsaturated valence is shown in the figure, assuming that it can be connected to a sufficient number of hydrogen atoms to saturate the valence.

Although numerical ranges and parameters are used to define a wide range of the invention, the numerical values are approximated as much as possible. Relevant values in the example. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the term "about" means that the actual value falls within the acceptable standard error of the average, depending on the considerations of those of ordinary skill in the art to which the invention pertains. Except for the experimental examples, or unless otherwise explicitly stated, all ranges, quantities, values, and percentages used herein are understood (eg, to describe the amount of material used, the length of time, the temperature, the operating conditions, the quantity ratio, and the like. Are all modified by "about". Therefore, unless otherwise indicated to the contrary, the numerical parameters disclosed in the specification and the appended claims are intended to be At a minimum, these numerical parameters should be understood as the number of significant digits indicated and the values obtained by applying the general carry method.

The singular forms "a" and "the" are used in the plural unless otherwise defined.

2. Novel Deoxyelephantopin Derivatives (DETD)

The present invention comprises a compound as shown in formula (I), Wherein: R is H or -(C=O)R ₁ ; and R1 is H, C _1-20 alkyl, C _2-20 alkenyl, aryl or heteroaryl, respectively: wherein: C _1-20 alkane The group may be unsubstituted or have at least one substituent selected from the group consisting of halogen, hydroxy, alkoxy, alkoxy, phenyl, naphthyl, isobenzofuranyl, isophenylthiophenyl or isoquinolyl The C _2-20 alkenyl group may be unsubstituted or have at least one substituent selected from the group consisting of phenyl, furyl, thienyl or isobenzodioxolanyl, wherein each phenyl and naphthyl group It may be unsubstituted or have at least one substituent selected from the group consisting of halogen, alkyl, perfluoroalkyl, alkoxy or haloalkoxy; and the aryl group may be unsubstituted or halogen, C _1-4 a substituent of a lower alkyl, alkoxy or haloalkoxy group.

The compound of the present invention may be one or more stereocenters of the deoxycholestrin derivative; therefore, the compounds exist in different isomeric forms, for example, a mixture of racemic mirror image isomers and/or non-mirror a mixture of structures. The present invention encompasses mirror image isomers of such compounds, as well as mixtures of such mirror image isomers and/or non-image areomers. Mirror isomers can be prepared by asymmetric synthesis or by conventional techniques It is separated, for example, by crystallization, chromatography, and separation using a resolving agent. A preferred method for separating the enantiomers from the racemic mixture is by high performance liquid chromatography (HPLC). Alternatively, the two mirror image isomers may be separated from each other from the racemic isomer by reaction with an optically active form of a resolving agent in a solvent. Depending on the optical form of the separating agent used, one of the isomers of the second image isomer can be isolated as an insoluble salt, separated from the isomer remaining in the solution. The isomers have high yields and high optical purity.

The invention further comprises a mixture of stereoisomers of the compound. Furthermore, the invention also encompasses configurational isomers of the compounds (ie, trans and cis isomers, whether or not containing double bonds), including mixtures of isomers, pure isomers or substantially It is in the form of a pure isomer.

3. Synthesis method

The present invention comprises a process for the preparation of a compound of formula (I) using deoxycholin (DET) as a starting compound, wherein DET can be viewed from the lamp in accordance with the extraction step shown in the section "Materials and Methods" of the present disclosure. Separated from the decaying plant ( Elephantopus scaber L. ).

After the DET was isolated, the DET derivative was prepared according to the procedure shown in Scheme 1.

Briefly, DET is hydrolyzed by a strong base (e.g., NaOH or KOH); the resulting hydrolyzate is then acidified to provide the compound DETD-3, which is a novel C-8DET lactone. Further DET derivatives (abbreviated as DETDs) were produced in three different ways, wherein DETD-4 to DETD-61 were prepared by Method A or B, and DETD-62 was prepared by Method C.

In Method A, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and dimethylaminopyridine (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EDCI) In the presence of dimethylaminopyridine, DMAP), DETD-3 is reacted with a monocarboxylic acid (R ₁ COOH); and depending on the R ₁ group of the carboxylic acid reactant (R ₁ COOH), various forms of esters of the present disclosure can be obtained. Class of compounds, including DETD-4 to DETD-31, DETD-35, and DETD-39 to DETD-61. The R ₁ group may be an alkyl group, a branched alkyl group, a cycloalkyl group, a substituted alkyl group, an aryl group or a substituted aryl group, a heteroaryl group or a substituted heteroaryl group. , alkenyl, alkynyl and the like.

In Method B, DETD-3 is no longer reacted with a carboxylic acid (R ₁ COOH), but in the presence of triethylamine, it is reacted with a chloride of an acid (R ₁ COCl) to give DETD-32 to DETD-34, and ester compounds of DETD-36 to DETD-38.

In Method C, under standard Mitsunobu conditions (Calvert et al., Chemico-biological Interactions 1998, 111-112: 213-224.), in diethyl azodicarboxylate (DEAD) and triphenylphosphine In the presence of (triphenylphosphine, PPh ₃ ), DETD-3 is reacted with 1-naphthylacetic acid to give a DETD-62 ( epi- DETD-35) compound.

The side chain structure of the newly synthesized DETDs (i.e., the compound of formula (I)) is shown in Table 1.

4. Use

The invention encompasses a method of treating or preventing an individual having or suspected of having an inflammatory and/or tumor associated disease. The method comprises administering to a subject a therapeutically or prophylactically effective amount of a compound of formula (I) or DETDs to alleviate, alleviate and/or prevent the symptoms of the inflammatory and/or tumor-related disease. In certain embodiments, the method further comprises administering to the individual a chemotherapeutic agent or a glutathione biosynthesis blocker before, after or simultaneously with administering the compound of formula (I). The chemotherapeutic agent may be selected from the group consisting of PLX4032, docetaxel, paclitaxel, cisplatin, oxaliplatin, betulinic acid, 4-S-cysteamine 4-S-cysteaminyl catechol, 4-S-cysteaminyl phenol, everolimus, bortezomib, carboplatin , dacarbazine, celecoxib, temozolomide, sorafenib, thalidomide, lenalidomide, valproic acid , vinblastine, imatinib mesylate, bosentan, apomine, arsenic trioxide, carmustine, lat. Lambrolizuma, anti-CTLA-4 drug, anti-programmed death receptor-1 (PD-1) drug, Ipilimumab, Cui Mei Tremelimumab, doxorubicin, MEK inhibitor, capecitabine, poly(ADP-ribose) polymerase (PARP) inhibitor (poly(ADP-ribose) Polymerase (PARP) inhibitor, phosphoinositide 3-kinase (PI3K) inhibitor, mammalian target of rapamycin (mTOR) inhibitor and hemo In the group consisting of tamoxifen. According to certain preferred embodiments of the present disclosure, the chemotherapeutic agent and the glutathione biosynthesis blocker may enhance the effects of DET, DETDs, or a known anticancer drug (ie, paclitaxel), respectively. The chemotherapeutic agent may be DET, paclitaxel (PTX) or PLX4032; and the glutathione biosynthesis blocker may be butyl thiopurine imine (BSO) or sulfasalazine.

Inflammatory-related diseases are characterized by a local or systemic, acute or chronic inflammation. Examples of inflammation-related diseases include inflammatory skin diseases (eg, dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria ( Urticarial), necrotizing vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, eosinophilic myositis, polymyositis, derma Dermatomyositis, eosinophilic fasciitis, inflammatory bowel disease (eg, Crohn's disease) (Crohn's disease) and ulcerative colitis (allergic colitis), allergic lung disease (eg, hypersensitivity pneumonitis, eosinophilic pneumonia, delayed-type hypersensitivity (delayed-type hypersensitivity) ), interstitial lung disease (ILD), idiopathic pulmonary fibrosis, and ILD associated with rheumatoid arthritis, asthma, and allergic rhinitis. The examples also include autoimmune diseases (e.g., rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, myasthenia gravis, young Juvenile onset diabetes, glomerulonephritis, autoimmune thyroiditis, ankylosing spondylitis, systemic sclerosis, and multiple sclerosis (multiple sclerosis)), acute and chronic inflammation (eg, systemic anaphylaxia or hypersensitivity responses, drug allergies, insect sting allergies, autologous transplant rejection) (allograft rejection) and graft-versus-host disease, sedative syndrome, human immunodeficiency virus infection, cancer (eg, brain cancer, breast cancer, papilloma, melanoma, prostate cancer) , colorectal cancer, kidney cancer, ovary Cancer, thyroid cancer, lung cancer, and hematopoietic function-related cancers) and tumor metastasis.

Tumor-associated disease refers to uncontrolled autologous cell proliferation, including malignant and non-malignant cell proliferation. Examples of tumor-related diseases include cancers, such as malignant tumors, sarcomas, metastatic diseases, or hematopoietic neoplastic diseases. Metastatic tumors can be derived from a variety of primary tumor types including, but not limited to, prostate, large intestine, lung, breast, liver, thyroid, lymph, gastrointestinal, and genitourinary tract. Examples of malignant tumors include malignant tumors formed by the cervix, lung, prostate, breast, head and neck, large intestine, and ovarian tissue. Hematopoietic neoplastic diseases can occur in bone marrow, lymphoid or erythrocyte cell lines or their precursor cells. In a preferred embodiment, the disease can be produced by poorly differentiated acute leukemia, for example, erythroblastic leukemia and megakaryoblastic leukemia. Furthermore, examples of the bone marrow disease include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML), and chronic myelogenous leukemia (CML). Examples of such lymphoid malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), which comprises B cell line lymphoblastic leukemia (B-lineage ALL) and T cell line lymphoblasts Leukemia (T-lineage ALL), chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (prolymphocytic) Leukemia, PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). In addition, examples of other malignant lymphomas include, but are not limited to, non-Hodgkin lymphoma and its variants, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (adult T cell leukemia/lymphoma, ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia, Hodgkin's disease, and Reed - Reed-Sternberg disease.

The dosage of the compound of formula (I) will depend on the particular condition being treated, prevented or administered, as well as the age, sex and physiological condition of the patient. The importance of the above factors is well known in the art and, in addition, daily experimentation can be used to provide its importance.

5. Pharmaceutical formula

The invention comprises a pharmaceutical composition for treating/preventing an inflammatory and/or tumor-related disease comprising a therapeutically or prophylactically effective amount of a compound of formula (I).

The compound of the formula (I) of the present invention comprises from about 0.1% to about 99% by weight based on the total weight of the pharmaceutical composition, based on the total weight of the above pharmaceutical composition. In certain embodiments, the compounds of formula (I) of the invention The amount is at least about 1% by weight based on the total weight of the pharmaceutical composition. In a particular embodiment, the amount of the compound of formula (I) is at least about 5% by weight based on the total weight of the pharmaceutical composition. In other embodiments, the amount of the compound of formula (I) is at least about 10% of the total weight of the pharmaceutical combination. In another embodiment, the amount of the compound of formula (I) is at least about 25% by weight based on the total weight of the pharmaceutical composition.

In some preferred embodiments, the pharmaceutical composition further comprises a chemotherapeutic agent or a glutathione biosynthesis blocker. The chemotherapeutic agent may be selected from the group consisting of DET, PLX4032, docetaxel, paclitaxel, cisplatin, oxaliplatin, betulinic acid, 4-S-cysteine catechol, 4-S-cysteamine, Everolimus, bortezomib, carboplatin, dacarbazine, celecoxib, temozolomide, lesaza, salidome, lenalidomide, valproic acid, vinblastine, imatinib mesylate , bosentan, apramine, arsenic trioxide, carmustine, lapazide, anti-CTLA-4 drug, anti-programmed death receptor-1 (PD-1) drug, iprimim, Cui Mei Limma, leucomycin, MEK inhibitor, capecitabine, poly(ADP-ribose) polymerase (PARP) inhibitor, phosphoinositide 3-kinase (PI3K) inhibitor, mammalian rapamycin A group of substances consisting of a target (mTOR) inhibitor and tamoxifen. According to other preferred embodiments, the chemotherapeutic agent may be DET, paclitaxel or PLX4032; and the glutathione biosynthesis blocker may be butyl thiopurine imine (BSO) or sulfasalazine.

According to some particular embodiments of the invention, the inflammation suitable for treatment with the pharmaceutical composition is selected from the group consisting of an inflammatory skin disease, an inflammatory bowel disease, an allergic lung disease, asthma, allergic rhinitis, an autoimmune disease, In a group consisting of acute and chronic inflammation, Shering's syndrome, human immunodeficiency virus infection, and cancer.

According to other specific embodiments of the present invention, a tumor-related disease suitable for treatment with the pharmaceutical composition is a cancer selected from the group consisting of breast cancer, papilloma of the skin, melanoma, brain cancer, lung cancer, lymphoma, neuroepithelial In a group consisting of tumor, kidney cancer, prostate cancer, stomach cancer, colorectal cancer, and uterine cancer. According to some embodiments, the cancer is a drug resistant cancer. In one example, the cancer is a melanoma that is resistant to PLX4032.

According to a particular embodiment of the invention, the pharmaceutical composition reduces the side effects caused by administration of a chemotherapeutic agent (e.g., PLX4032) to the skin of a cancer-infected individual, such as reducing the number and size of dermal papillomas induced by PLX4032.

The pharmaceutical composition may be in a single dosage form via oral, mucosal (eg, nasal, sublingual, vaginal, buccal or rectal), parenteral (parenteral) (ie, subcutaneous, intravenous, bolus injection, intramuscular or intra-arterial) or transdermal (administered to transcutaneous) By. Examples of such dosage forms include, but are not limited to, tablets, caplets, capsules, for example, soft elastic gelatin capsules, cachets, buccal inclusions Troches, lozenges, dispersions, suppositories, ointments, cataplasms (also known as poultices), patches (pastes) ), powders, dressings, creams, plasters, solutions, patches, aerosols (eg, nasal sprays or inhalers) , gels (gels). The pharmaceutical composition of the present invention may be in a liquid dosage form suitable for oral or mucosal administration to a patient, including suspensions (e.g., water-soluble or water-insoluble liquid suspensions, oil-in-water emulsions or water-in-oil emulsions). And solutions and elixirs; furthermore, the pharmaceutical compositions of the invention may also be in a liquid dosage form suitable for parenteral administration; and the pharmaceutical composition may be a sterile solid (eg, crystalline or non-crystalline) Amorphous solids, which can be formulated to provide a suitable liquid dosage form for parenteral administration.

The compounds of the invention can be formulated for different routes of administration. For example, if administered orally, a casing film is applied to protect the compound of the invention to prevent degradation of the compound of the invention as it passes through the gastrointestinal tract. The formulation may also have other ingredients that allow it to deliver the active ingredient to the site of action. For example, a compound can utilize a liposome formulation to avoid degradation of the active ingredient by enzymes, in the circulation system. The system delivers and delivers the active ingredient through the cell membrane to the intracellular location.

Further, the poorly water-soluble compound may be formulated into a liquid dosage form (and a dosage form suitable for reconstitution) by adding a dissolving agent, an emulsifier, and a surfactant. The additive includes, but is not limited to, cyclodextrin (eg, alpha-cyclodextrin or beta-cyclodextrin) and non-aqueous solvent, including but not limited to, ethanol, isopropanol, ethyl carbonate, Ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, dimethyl sulfoxide (DMSO), biocompatible oils (eg, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil, glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, sorbitan fatty acid ester (fatty acid esters of A mixture of sorbitan) and the above compounds (eg, a mixture of DMSO and corn oil).

The type and form of the pharmaceutical composition formulation will vary depending on the application. For example, a dosage form for acute treatment will contain a greater amount of a single or multiple active ingredients than a dosage form for chronic treatment. In contrast, parenteral dosage forms may contain minor amounts of a single or multiple active ingredients in an amount less than the amount of active ingredient employed in the treatment of the same condition in an oral dosage form. The present invention also encompasses dosage forms for other routes of administration which will vary with the general skill of the art (see Remington's Pharmaceutical Sciences , 18th ed., Mack Publishing, Easton PA (1990)).

5.1 oral dosage form

The pharmaceutical compositions of the present invention are suitable for oral administration and may be in discrete dosage forms including, but not limited to, tablets (i.e., chewable tablets), lozenges, capsules, and liquids (i.e., flavored syrups). The above oral dosage forms can be prepared according to well-accepted pharmaceutical processes, such as those described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing, Easton PA (1990)). The oral dosage form comprises an amount of a predetermined active ingredient and can be prepared according to conventional methods of pharmacy. A typical oral dosage form can be prepared according to conventional pharmaceutical procedures and formulated to contain at least one excipient. The formulations of the present invention can be used in combination with a variety of different forms of excipients, depending on the dosage form desired for the particular mode of administration.

Based on the ease of administration of the tablets and capsules, the two dosage forms are the best unit form for oral dosage forms. The coating may be carried out by standard aqueous or non-aqueous techniques depending on the needs of use; the dosage form may also be prepared by conventional pharmaceutical procedures. In general, the pharmaceutical compositions and dosage forms are formulated into a predetermined form by mixing the active ingredient with a liquid carrier or ground solid carrier, or a mixture of the active ingredient and liquid, solid carrier. Further, the solid dosage form may contain a disintegrant to increase the dissolution rate; and the solid dosage form may also contain a lubricant to facilitate the manufacture of various dosage forms (e.g., tablets).

5.2 Intestinal dosage form

Routes for administration of parenteral dosage forms include, but are not limited to, subcutaneous injections, intravenous injections (including bolus injections), intramuscular injections, and arterial injections. When a parenteral dosage form is administered, it is necessary to avoid the patient being exposed to a contaminant infection, resulting in an immune response against the contaminant in the body, and therefore, the parenteral dosage form must be sterile. Parenteral dosage forms include, but are not limited to, liquid formulations for injection, powdered formulations and which can be dissolved or suspended in a pharmaceutically acceptable injectable vehicle, and in a creamy formulation.

Parenteral dosage forms can contain conventional carriers. For example, including, but not limited to, water and a water soluble carrier. The water-soluble carrier includes, but is not limited to, sodium chloride solution, Ringer's solution, and glucose solution; examples of water-miscible vehicles include, but are not limited to, ethanol, polyethylene glycol, and polypropylene glycol And the water-insoluble carrier includes, but is not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

5.3 Transdermal, topical and mucosal dosage forms

Transdermal, topical, and mucosal dosage forms include, but are not limited to, ophthalmic solutions, sprays, aerosols, creams, lotions, ointments, gels, solutions, emulsifiers, suspensions, or the like. A pattern known to the skilled person (see Remington's Pharmaceutical Sciences , 18th eds., Mack Publishing, Easton PA (1990)). The transdermal dosage form comprises a "reservoir type" and a "matrix type" patch which can be applied to the skin and which can penetrate the skin after a specific attachment period. In the human body.

Excipients (i.e., carriers and diluents) suitable for use in the present dosage forms, as well as other materials known in the art to be applied to transdermal, topical, and mucosal dosage forms, can be applied to the formulation, and pharmaceutical compositions for a particular tissue The dosage form or dosage form can also be applied to the formulation.

Additional ingredients may be administered at the same time or before/after administration of the active ingredients of the invention for a particular therapeutic tissue. For example, penetration enhancers may be additionally added to assist in delivering the active ingredient to the tissue.

The pH of the pharmaceutical composition or dosage form can be adjusted, or the pH of the tissue to which the pharmaceutical composition or dosage form is applied can be improved to improve the delivery efficiency of the single or multiple active ingredients. Furthermore, the transfer efficiency can be improved by adjusting the polarity, ionic strength or tension of the solvent carrier. In addition, the pharmaceutical compositions or dosage forms of the present invention may be added with stearates to alter the hydrophilicity or lipophilicity of one or more of the active ingredients, thereby improving the efficiency of delivery. Accordingly, stearates can be used as a lipid carrier, as an emulsifier or surfactant, or as a delivery enhancer or penetration enhancer. Further, the properties of the final composition can be further adjusted by using various salts, hydrates or solvates of the active ingredient.

The description of the embodiments of the present invention is intended to be illustrative and not restrictive. The features of various specific embodiments, as well as the method steps and sequences thereof, are constructed and manipulated in the embodiments. However, other specific embodiments may be utilized to achieve the same or equivalent function and sequence of steps.

Example Materials and Methods Cell culture

The cell material of the experiment is: mouse macrophage RAW 264.7, human normal epithelial cell line M10, melanocyte, melanoma cell line, including: mouse B16- F10 (N-RAS mutation), human MeWo (B-RAF and N-RAS wild type), human A375, A2058 (B-RAF ^V600E mutation), human A375-R (resistant to vemurafenib) Human A375 cells) and human SK-Mel-2 (N-RAS mutation). Breast cancer cell lines include: mouse TS/A (ER+), human MDA-MB-231 (ER-, Her2-, PR-), human MCF-7 (ER+, Her2-), human SKBR3 (ER-, Her2+) , human BT474 (ER+, Her2+). Brain cancer cell line U-87MG, colorectal cancer cell line HCT-116, kidney cancer cell line A498, lung cancer cell line PC6, lymphoma cell line U937, neuroepithelial cell line SK-N-MC, gastric cancer cell line KATO III and Uterine cancer cell line NES-SA. The above cell lines were obtained from ATCC (Manassas, VA) and cultured according to the manufacturer's recommended medium, and 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin were added to the medium. The cell culture conditions were 37 ° C and the inside was humidified with a 5% CO ₂ incubator.

animal

Female NOD/SCID mice (NOD.CB17-Prkdcscid/IcrCrlBltw), SCID mice (CB17-Prkdcscid/IcrCrlBltw) and FVB/N mice were purchased from the Taiwan Laboratory Animal Center (BioLASCO Taiwan Co., Ltd). The mice were given free access to the laboratory's standard mouse chow and distilled water, and the light in the rearing environment was 12 hours: 12 hours during the dark cycle and the feeding temperature was maintained at 22 ± 2 °C. All experimental procedures were reviewed and approved by the Academia Sinica Experimental Animal Care and Use Committee (Taiwan, Republic of China).

Cell viability analysis

Normal normal cells or cancer cells (2 x 10 ³ to 1 x 10 ⁴ cells/well) were seeded in 96-well plates for overnight culture; cells were individually treated with different compounds for 24 to 72 hours. The cell growth was measured by MTT colorimetric assay. Regarding the manner in which the test cell viability was calculated, the cell viability, which was treated with vehicle alone (0.5% DMSO), was defined as a survival rate of 100%. Cell viability by DETDs was calculated by the following formula: number of viable cells (%) = OD ₅₇₀ (treated cells) / OD ₅₇₀ (vector control group) x 100.

Cellular chamber assay

The migration resistance and anti-invasive efficacy of DET, DETD-35 and PTX were analyzed using a Boyden chamber. 10% fetal bovine serum (FBS) as chemokine, and DMEM medium (containing 0.05% DMSO, DET (2.5 and 4 μM), DETD-35 (1.25 and 2.5 μM) or PTX (2.5 and 4 μM) ) was added to the lower chamber of the Boyden chamber (diameter: 6.5 mm, aperture: 8 mm Costar, Cambridge, MA, USA). MDA-MB-231 cells (5 x 10 ⁴ cells/chamber) were placed in the upper chamber of the Boyden chamber and medium (containing 0.1% FBS) was added. The cells were cultured in a temperature condition of 37 ° C; in an atmosphere containing 5% CO ₂ , after 24 hours of incubation, the non-migrating cells on the upper surface of the membrane were scraped off with a cotton swab; and the migrated cells remaining under the membrane were treated with DAPI (1 μg). /ml 4,6-dimethylhydrazine-2-phenylindole) staining. The migrated cells were observed by an inverted fluorescence microscope, and the cell count was calculated by 20X original magnification, and the number of cells in the three regions was calculated. In order to test the anti-invasive efficacy of the compound, a filter membrane having a pore size of 8-μm was first coated with 30 μg of a gel matrix (Matrigel) and cultured at 37 ° C for 2 hours; then, a subsequent test was carried out by the migration analysis step.

Compound-drug binding analysis

Synergistic effects between DETDs and PLX4032 or between DETDs and PTX were determined with DETDs and PLX4032 in the specified concentration range or DETDs and PTX. Briefly, cells (1.5×10 ³ to 5×10 ³ cells/well) are seeded into a 96-well plate, and the compound or drug is administered separately by individual, or the cells are treated with the compound and the drug, and the treatment time is It is 24 to 72 hours. Cell proliferation was measured by MTT assay. The effects of the combination of the compound and the drug were determined by Isobologram analysis and Chou-Talalay method. The DETDs and PLX4032 were measured by the combination index (CI) or interacted with PTX, and the CI map was generated using the CompuSyn software. The binding potency of the two compounds can be summarized as follows: when CI = 1, it represents additive interaction, when CI < 1, it represents synergy (synergism) and when CI > 1, it represents antagonistic (antagonism).

Time-lapse microscope analysis

A 12-well cell culture plate was taken and 25 μg/ml fibronectin was applied thereto. After 1 hour, 5×10 ⁴ MDA-MB-231 cells were seeded into the culture plate and DMEM (containing 10). % serum) culture. Immediately 12 hours after inoculation, an instant microscopic experiment (images were recorded every 30 minutes) was performed with an inverted conjugate focusing microscope (LSM 510 META) equipped with an environmental chamber with optical phase difference. Using the object tracking application of the Metamorph software (Molecular Devices), the average of the nuclear displacements of 12 cells between successive images was assessed as the rate of cell migration. Overall, the cell trajectory needs to be recorded continuously for 24 hours. At the beginning of the instant photographic recording, cells were treated with vehicle (DMSO, 0.05%), DET (2.5, 4, and 10 μM) and DETD-35 (1.25, 2.5, 4 μM) to complete the above analysis.

Western blot analysis

Total cellular proteins in the test cells were prepared according to the experimental procedure described in the prior publication (Chiang et al., British J Pharmacol 2005, 146(3): 352-363.). Protein concentration was determined by the Bradford method (Bio-Rad). Protein samples were resolved on a 5% to 20% gradient SDS-PAGE followed by immunoblotting. Primary antibodies against ERK1/2 and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and MEK, phosphorylated ERK1/2 (phospho-ERK1/2) and phosphorylated MEK (phospho-MEK) were purchased from Cell Signaling Technology (Danvers, MA, USA; and selection of appropriate horseradish peroxidase-conjugated secondary antibodies. Enhanced chemical luminescence (Amersham) and exposure to chemically luminescent light (BioMax; Kodak Co.) Observe the protein band that reacts with a specific antibody.

Determination of the production of nitric oxide (NO)

Macrophages (2 x 10 ⁵ cells/well; 96-well plates) were treated with the compound for 1 hour, followed by medium (with/without addition of 100 ng/μL LPS) for 24 hours. The concentration of nitrous acid in the cell culture medium was determined by the Griess reaction method (see Green et al., Analytical Biochemistry 1982, 126(1): 131-138.). In order to compare the results measured by the Griess reaction method, the survival rate of macrophages was determined by MTT colorimetry.

Synergistic effect of glutathione biosynthesis blocker and DET and DETD-35 on cancer cell proliferation

The glutathione biosynthesis blocker, butyl thiopurine imine (BSO) or sulfasalazine, combined with DET or DETD-35, was confirmed to have a synergistic effect. Briefly, cells (5 x 10 ³ cells/well) were seeded in 96-well plates with BSO (concentrations: 100 μM, 45 μM, 18 μM, 7.2 μM, 2.8 μM, 1.15 μM, 461 nM, 184 nM, 74 nM, and 29 nM) Pretreatment with sulfasalazine (62.75 μM, 25.10 μM, 10 μM, 4 μM, 1.6 μM, 642 nM, 257 nM, 102 nM, 41 nM and 16.4 nM) for 30 hours, followed by the addition of 12 μM DET or 3 μM DETD-35 to the medium In the middle, after 24 hours, the analysis was performed by MTT.

Inhibition of melanoma growth in NOD/SCID mice

In this example, the effects of PLX4032, DET, DETD-35, DET+PLX4032 or DETD-35 + PLX4032 on melanoma were studied in NOD/SCID mice (6 weeks old) bearing A375 or A375-R melanoma cells. . 3x10 ⁶ of A375 or A375-R melanoma cells were inoculated subcutaneously on day 0 to the flanks of NOD/SCID mice. Next, the tumor in the mouse was allowed to grow for 7 days. Thereafter, the experimental animals were randomly assigned to seven groups (n=8/group): pseudo-control group (sham), tumor (vector) control group, PLX4032 treatment group (administered once a day), DET or DETD-35 (every 2 days of treatment), and the PLX4032+DET treatment group (administering DEX every 2 days for PLX4032+ every 4 days) or PLX4032+DETD-35 treatment group (administering PLX4032+ every 2 days) DETD-35 was administered every 4 days, and PLX4032, DET or DETD-35 was injected intraperitoneally (ip) into the experimental animals at a dose of 20 mg/kg in the A375-R experiment. The amount of PLX4032 is 75 mg/kg. The body weight of the mouse and the diameter of the tumor in the body were measured every 3 days. On the 33rd day, the test mice were sacrificed by cervical dislocation, the tumor was collected and the volume (V) of the tumor was measured with a vernier caliper, and The formula V = (L x W ² )/2 is calculated, where L is the length of the tumor and W is the width of the tumor.

Inhibition of NOD/SCID mouse mammary tumor growth

In this example, the effect of DETD-35 on breast tumors was investigated in NOD/SCID mice (6 weeks old) carrying MDA-MB-231 cancer cells. On day 0, 5 x 10 ⁶ MDA-MB-231 cells (dissolved in 100 μL of gel matrix (9.1 mg/mL) (Phenol Red-free)) were injected into the mouse mammary fat pad. Next, the tumor in the mouse was allowed to grow, and the growth period was 7 days. Experimental animals were randomly assigned to three groups (n=5/group): pseudo-control group (sham), tumor (vehicle) control group, and DETD-35 (10 mg/kg) treatment group. DETD-35 was injected intraperitoneally (ip) into experimental animals (injection frequency: once every three days). The body weight of the mouse and the diameter of the tumor in the body were measured every 3 days. On the 71st day, the test mice were sacrificed by cervical dislocation, the tumor was collected and the volume (V) of the tumor was measured with a vernier caliper, and The formula V = (L x W ² )/2 is calculated, where L is the length of the tumor and W is the width of the tumor.

Inhibition of lung metastasis of MDA-MB-231 cells in SCID mice

Female SCID mice (5 weeks old) were assigned to 8 groups: PTX group (5 mg/kg paclitaxel), DETD-35-2 group (2 mg/kg DETD-35), DETD-35- 10 groups (10 mg/kg DETD-35), PTX-5+DETD-35-2 group (5 mg/kg paclitaxel and 2 mg/kg DETD-35; 7 doses of PTX and 10 doses of DETD) -35), tumor (vehicle) group (5% DMSO) and pseudo control group (5% DMSO). Pre-DETD-35-10 group was injected with 1×10 ⁶ MDA-MB-231 cells into mice before iv, and 10 mg/kg DETD-35 was administered to mice every two days, one dose every two days. Three doses were administered. All other drugs or compounds were injected into the mice by ip injection every three days from the first day of the test. On the last day of the trial (Day 71), the mice were sacrificed by cervical dislocation. Next, the lungs, liver, kidneys, and spleen of the mice were removed and the weight of each organ was recorded, and the organ index (organ weight/weight%) was calculated. The number of tumor colonies in the lungs of the test mice was calculated.

Two-stage skin cancer animal model

One week old 6-week-old FVB/N female skin was treated with dimethylbenz[a]anthracene (DMBA) (concentration of 25 μg in 100 μl of acetone) for one week. It was then treated with DMBA by topical treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) (concentration of 4 μg in 100 μl of acetone). The TPA treatment frequency is 2 times a week. The size and number of new tumors are regularly assessed weekly. The diameter of the skin protrusions is more than 1 mm and is called a tumor.

Example 1 Preparation of a compound of formula (I) 1.1 Preparation of deoxycholicin (DET)

The dried lamp was immersed in three volumes of acetone ( Elephantopus scaber L. (Asteraceae)), the extraction period was 3 days, and repeated 2-3 times; then, the obtained crude extract was treated with ethyl acetate (ethyl acetate). , EA) performs fractional extraction to obtain an EA partitioned extract. The EA extract was purified by gel column chromatography, and the eluent was n-hexane (hexane, H) / EA eluent solvent (H: E / 3: 1, H: E / 1:1 and H: E/1: 2, v/v). The DET containing fraction was confirmed by thin layer chromatography or HPLC analysis. The HPLC conditions or DET purity of the DET-divided extract were determined using RP-18(R) rubber column chromatography (5 [mu], 150 x 4.6 mm). The eluent was 55% MeOH. The RP-C18 column in the MPLC system was used for chromatography and the DET fractionation extract was collected. The conditions of the eluent were: 30% methanol (MeOH), 2.5 column volume (CV), 30-55%. MeOH, 5 CV and 55% MeOH, 9.5 CV. A large amount of DET partitioned extract was collected and evaporated to dryness on a rotary evaporator. The dried chemical powder was dissolved in acetone to obtain DET crystals. The structure of DET was identified by electrospray free mass spectrometer (ThermoFinnigan LCQ, San Jose, CA, USA) and ¹ H and ¹³ C NMR (Brüker ADVANCE 500 AV) spectrometer, and spectral data disclosed in the prior publication (But PPH) Et al., Phytochemistry 1997, 44(1): 113-116.) Confirmation of the alignment.

1.2 Synthesis of DET Derivatives (DETD) 1.2.1 DETD-1:

DETD-1 was synthesized and confirmed by the disclosure of the literature (Zhang et al., Phytochemistry 1986, 25(4): 899-904.) and the following method.

1.2.2 DETD-3 to DETD-62:

DETD-3 is derived from DET. And according to the above flow chart 1 The indicated methods A, B or C, DETD-4 to DETD-62 are derived from DETD-3.

1.2.2.1 DETD-3:

25 ml of 1 N aqueous NaOH solution was added to a DET solution (1.023 g of DET dissolved in 25 ml of dioxane) at 0 °C. The mixture was allowed to stand at room temperature and stirring was continued until overnight, followed by cooling to 0 °C. The mixture was acidified by the addition of 2N HCl solution and stirring was continued for 30 minutes. Extract twice with AcOEt and extract with 5% MeOH / AcOEt. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and finally concentrated in vacuo. The residue obtained was purified by SiO2 column chromatography to give a new C-8 lactone DETD-3 (C-8 lactone DETD-3) (0.744 g, 91%).

DETD-3 spectral data: High-Resolution Mass Spectroscopy: m/z calculated for C ₁₅ H ₁₆ O ₅ (M ⁺ +Na) 299.0890 was 299.0883. Proton Nuclear Magnetic Resonance ( ¹ H-NMR, 400 MHz, 2% CD ₃ OD/CDCl ₃ ) σ 6.94 (1H, s), 6.41 (1H, dd, J = 2.9 and 1.2 Hz), 6.32 ( 1H, d, J = 2.9 Hz), 5.47-5.43 (1H, m), 4.56 (1H, br d, J = 10.0 Hz), 4.27 (1H, t, J = 9.7 Hz), 4.10 (1H, ddd, J = 11.7, 6.4 and 1.6 Hz), 3.19 (1H, ddd, J = 13.1, 3.8 and 1.6 Hz), 2.89 - 2.82 (1H, m), 2.77 (1H, dd, J = 13.5 and 4.7 Hz), 2.67 (1H, dd, J = 13.1 and 10.7 Hz), 2.57 (1H, br d, J = 13.5 Hz), 1.71 (3H, d, J = 1.4 Hz). ¹³ C nuclear magnetic resonance ( ¹³ C-NMR, 400 MHz, DMSO-d ₆ ) σ 172.9, 169.4, 155.2, 140.3, 136.3, 127.2, 126.2, 125.7, 80.9, 78.5, 67.7, 50.9, 40.5, 31.8, 19.0.

The DETD-3 X-ray crystal structure analysis was performed at the X-ray Laboratory (Taiwan) of the Institute of Chemistry, Academia Sinica. Crystal data and structural optimization parameters are shown in Table 2.

1.2.2.2 Preparation of DETD-4 to DETD-62

DETD-4 to DETD-62 were prepared from DETD-3 according to the following methods A, B or C.

Method A: related carboxylic acid (R ₁ COOH), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrogen chloride (EDCI) and dimethylaminopyridine (dimethylaminopyridine, DMAP) was added to the DETD-3 solution (DETD-3 was dissolved in dichloromethane). The mixture was continuously stirred at room temperature until overnight. DETD-4 to DETD-31, DETD-35, and related esters of DETD-39 to DETD-61 were obtained by standard work-up. The R ₁ group may be an alkyl group, a branched alkyl group, a cycloalkyl group, a substituted alkyl group, an aromatic group or a substituted heteroaryl group, an alkenyl group, an alkynyl group or the like.

Method B: The related acid chloride (R ₁ COCl) and triethylamine were added to the DETD-3 solution (DETD-3 was dissolved in dichloromethane). The mixture was continuously stirred at room temperature until overnight. The relevant esters of DETD-32 to DETD-34, and DETD-36 to DETD-38 were obtained by standard operation.

Method C: at room temperature, according to standard Mitsunobu conditions (see Calvert et al., Chemico-biological Interactions 1998, 111-112: 213-224.), ie, diethyl azodicarboxylate (diethyl azodicarboxylate, In the presence of DEAD) and triphenylphosphine (PPh3), the DETD-3 solution (dissolved in tetrahydrofuran) was treated with 1-naphthylacetic acid to obtain DETD-62 (epi-DETD-35).

DETDs 4-62 spectral data

DETD-4 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 6.93 (1H, s), 6.40 (1H, d, J = 2.7 Hz), 7.516.10 (1H, d, J = 9.6 Hz), 5.94 ( 1H, d, J = 2.1 Hz), 5.65 (1H, s), 5.45 (1H, br s), 5.39 (1H, t, J = 10.0 Hz), 4.57 (1H, d, J = 10.0 Hz), 4.23 (1H, dd, J = 10.1 and 6.5 Hz), 3.28 (1H, d, J = 13.1 Hz), 3.15 (1H, ddd, J = 10.0, 6.5 and 3.3 Hz), 2.79 (1H, dd, J = =13.6) And 4.9 Hz), 2.68 (1H, dd, J = 13.1 and 10.8 Hz), 2.54 (1H, d, J = 13.6 Hz), 1.95 (3H, s), 1.87 (3H, s).

DETD-5 (Method A, containing 2% cis isomer):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 6.91 (1H, s), 6.75 (1H, dt, J = 7.4 and 1.2 Hz), 6.39 (1H, d, J = 2.9 Hz), 7.515. 93 (1H, d, J = 2.9 Hz), 5.48-5.40 (1H, m), 5.39 (1H, t, J = 10.0 Hz), 4.46 (1H, d, J = 10.0 Hz), 4.23 (1H, ddd , J = 11.7, 6.6 and 1.6 Hz), 3.32-3.22 (1H, m), 3.19-3.02 (2H, m), 2.78 (1H, dd, J = 13.5 and 4.9 Hz), 2.68 (1H, dd, J =13.1 and 10.8 Hz), 2.53 (1H, d, J = 13.5 Hz), 2.25-2.16 (2H, m), 1.86 (3H, d, J = 1.4 Hz), 1.83 (3H, s), 1.06 (3H) , t, J = 7.5 Hz).

DETD-6 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.71 (1H, d, J = 16.0 Hz), 7.55-7.50 (2H, m), 7.44-7.38 (3H, m), 6.94 (1H, s ), 6.41 (1H, d, J = 9.6 Hzs), 7.516.40 (1H, d, J = 16.0 Hz), 6.00 (1H, d, J = 2.5 Hz), 5.50-5.44 (2H, m), 4.51 (1H, d, J = 10.0 Hz), 4.25 (1H, ddd, J = 10.5, 6.5 and 1.6 Hz), 3.33 - 3.26 (1H, m), 3.19 - 3.11 (1H, m), 2.80 (1H, dd , J = 13.5 and 4.9 Hz), 2.69 (1H, dd, J = 13.1 and 10.7 Hz), 2.55 (1H, d, J = 13.5 Hz), 1.89 (3H, d, J = 1.4 Hz).

DETD-7 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.61 (1H, d, J = 15.8 Hz), 6.93 (1H, s), 6.74 (2H, d, J = 9.6 Hz), 7.516.41 ( 1H, d, J = 2.8 Hz), 6.31 (1H, s), 6.27 (1H, s), 6.00 (1H, d, J = 2.8 Hz), 5.52-5.43 (2H, m), 4.50 (1H, d , J = 10.1 Hz), 4.26 (1H, ddd, J = 10.1, 6.6 and 1.7 Hz), 3.89 (9H, s), 3.29 (1H, dd, J = 11.1 and 1.7 Hz), 3.14 (1H, ddd, J = 13.1,6.6 and 3.1Hz), 2.81 (1H, dd , J = 13.1 and 4.8Hz), 2.69 (1H, dd , J = 13.1 and 10.7Hz), 2.54 (1H, d , J = 13.1Hz), 1.89 (3H, d, J = 1.4 Hz).

DETD-8 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 6.92 (1H, s), 7.516.39 (1H, d, J = 2.9 Hz), 5.96 (1H, d, J = 2.9 Hz), 5.69 ( 1H, s), 5.47-5.41 (1H, m), 5.52-5.43 (2H, m), 5.34 (1H, t, J = 10.0 Hz), 4.45 (1H, d, J = 9.6 Hz), 4.25-4.17 (1H, m), 3.26 (1H, d, J = 13.1 Hz), 3.10-3.01 (1H, m), 2.78 (1H, dd, J = 11.1 and 4.9 Hz), 2.66 (1H, dd, J = 13.1) And 10.7 Hz), 2.53 (1H, d, J = 13.1 Hz), 2.16 (3H, s), 1.92 (3H, s), 1.86 (3H, s).

DETD-9 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 6.95 (1H, s), 6.50 (1H, d, J = 2.9 Hz), 6.19 (1H, d, J = 2.9 Hz), 5.93 (1H, s), 5.50-5.44 (1H, m), 5.32 (1H, t, J = 10.1 Hz), 4.20 (1H, ddd, J = 10.1, 6.5, and 1.5 Hz), 3.28 (1H, br d, J = 13.1 Hz), 3.21 (1H, ddd, J = 13.1, 6.3, and 3.1 Hz), 2.83 (1H, dd, J = 13.1 and 4.8 Hz), 2.69 (1H, dd, J = 13.1 and 10.7 Hz), 2.57 (1H, d, J = 13.1 Hz), 1.86 (3H, d, J = 1.2 Hz).

DETD-10 (Method A) :

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 6.92 (1H, s), 6.42 (1H, d, J = 2.8 Hz), 5.97 (1H, d, J = 2.8 Hz), 5.46-5.42 ( 1H, m), 5.28 (1H, t, J = 10.1 Hz), 4.42 (1H, d, J = 10.0 Hz), 4.16 (1H, ddd, J = 10.0, 6.5, and 1.6 Hz), 3.26 (1H, Dd, J = 13.1 and 1.6 Hz), 3.05 (1H, ddd, J = 13.0, 6.2, and 3.2 Hz), 2.79 (1H, dd, J = 11.1 and 4.8 Hz), 2.65 (1H, dd, J = 13.1) And 10.7 Hz), 2.53 (1H, d, J = 13.1 Hz), 2.24 - 2.14 (2H, m), 2.14 - 2.03 (1H, m), 1.84 (3H, d, J = 1.2 Hz), 0.93 (6H) , d, J = 6.6 Hz).

DETD-11 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 6.92 (1H, br s), 6.50-6.42 (1H, m), 6.17-5.95 (1H, m), 5.44 (1H, br s), 5.31 -5.11 (1H, m), 4.46-4.32 (1H, m), 4.24 - 4.15 (1H, m), 3.26 (1H, br d, J = 12.9 Hz), 3.14 - 3.02 (1H, m), 2.80 ( 1H, dd, J = 13.5 and 4.9 Hz), 2.70-2.61 (1H, m), 2.60-2.51 (1H, m), 2.06 (3H, s), 1.84 (3H, br s), 0.88 (3H, t , J = 7.6 Hz).

DETD-12 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 6.91 (1H, s), 6.40 (1H, d, J = 2.5 Hz), 5.96 (1H, d, J = 2.5 Hz), 5.63 (1H, s), 5.46-5.41 (1H, m), 5.34 (1H, t, J = 10.1 Hz), 4.46 (1H, d, J = 10.1 Hz), 4.24 - 4.16 (1H, m), 3.30 - 3.22 (1H , m), 3.10-3.02 (1H, m), 2.78 (1H, dd, J = 13.5 and 4.7 Hz), 2.66 (1H, dd, J = 13.1 and 10.7 Hz), 2.53 (1H, d, J = 13.5) Hz), 2.44-2.34 (1H, m), 2.12 (3H, d, J = 1.3 Hz), 1.86 (3H, d, J = 1.3 Hz), 1.07 (3H, t, J = 6.8 Hz).

DETD-13 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 6.92 (1H, s), 6.43 (1H, d, J = 2.8 Hz), 6.0 and 5.98 (1H, d, J = 2.8 Hz, 1:1 ), 5.46-5.41 (1H, m), 5.25 (1H, t, J = 10.1 Hz), 4.41 (1H, d, J = 10.3 Hz), 4.19 (1H, dd, J = 10.3 and 6.3 Hz), 3.07 (1H, ddd, J = 13.1, 6.4 and 3.3 Hz), 2.79 (1H, dd, J = 13.5 and 4.9 Hz), 2.66 (1H, dd, J = 13.1 and 10.5 Hz), 2.53 (1H, d, J) =13.5 Hz), 2.40-2.30 (1H, m), 1.84 (3H, brs), 1.72-1.58 (1H, m), 1.55-1.40 (1H, m), 1.13 and 1.12 (3H, d, J = 7.0 Hz, 1:1), 0.88 and 0.87 (3H, t, J = 7.4 Hz, 1:1).

DETD-14 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 6.93 (1H, s), 6.48-6.32 (1H, m), 6.06-5.88 (1H, m), 5.44 (1H, br s), 5.40- 5.20(1H,m), 5.16-5.01(1H,m), 4.51-4.39(1H,m),4.24-4.13(1H,m), 3.29-3.20(1H,m), 3.14-3.00(1H,m ), 2.84 - 2.50 (4H, m), 2.17 and 2.14 (3H, br s), 1.86 and 1.83 (3H, br s), 1.99-1.56 (10H, m).

DETD-16 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 6.92 (1H, s), 6.44 (1H, dd, J = 3.1 and 0.8 Hz), 6.02 (1H, dd, J = 2.6 and 0.8 Hz), 5.46-5.41 (1H, m), 5.26 (1H, t, J = 10.1 Hz), 4.40 (1H, br d, J = 10.1 Hz), 4.18 (1H, ddd, J = 10.1, 6.3 and 1.7 Hz), 3.26 (1H, dd, J = 13.0 and 2.2 Hz), 3.07 (1H, ddd, J = 13.0, 6.1 and 3.0 Hz), 2.79 (1H, dd, J = 13.5 and 4.6 Hz), 2.65 (1H, dd, J = 13.0 and 10.7 Hz), 2.53 (1H, br d, J = 13.5 Hz), 2.22 - 2.14 (1H, m), 1.85 (3H, d, J = 1.4 Hz), 1.66-1.44 (4H, m) , 0.86 (3H, t, J = 1.4 Hz), 0.85 (3H, t, J = 1.4 Hz).

DETD-17 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 6.93 (1H, s), 6.43 (1H, dd, J = 3.1 and 0.8 Hz), 5.96 (1H, dd, J = 2.7 and 0.8 Hz), 5.47-5.42 (1H, m), 5.28 (1H, t, J = 10.0 Hz), 4.44 (1H, br d, J = 10.0 Hz), 4.18 (1H, ddd, J = 10.7, 6.4 and 1.7 Hz), 3.27 (1H, ddd, J = 13.3, 3.8 and 1.7 Hz), 3.05 (1H, ddd, J = 13.0, 6.4 and 3.1 Hz), 2.79 (1H, dd, J = 13.5 and 4.9 Hz), 2.65 (1H, Dd, J = 13.0 and 10.7 Hz), 2.53 (1H, br d, J = 13.5 Hz), 2.06 (3H, s), 1.83 (3H, d, J = 1.4 Hz).

DETD-18 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 6.92 (1H, s), 6.43 (1H, d, J = 3.1 Hz), 5.97 (1H, d, J = 2.7 Hz), 5.46-5.41 ( 1H, m), 5.28 and 5.27 (1H, t, J = 10.0 Hz, 1:1), 4.42 (1H, d, J = 10.0 Hz), 4.18 (1H, dd, J = 10.6 and 6.1 Hz), 3.26 (1H, br d, J = 13.3 Hz), 3.09 - 3.01 (1H, m), 2.79 (1H, dd, J = 13.5 and 4.8 Hz), 2.65 (1H, dd, J = 13.1 and 10.7 Hz), 2.53 (1H, d, J = 13.5 Hz), 2.32 and 2.28 (1H, dd, J = 15.0 and 6.1 Hz, 1:1), 2.14 - 2.03 (1H, m), 1.92-1.80 (1H, m), 1.84 (3H, d, J = 1.2 Hz), 1.39-1.15 (4H, m), 0.92-0.85 (6H, m).

DETD-19 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 6.92 (1H, s), 6.40 (1H, d, J = 3.1 Hz), 6.12 (1H, d, J = 0.6 Hz), 5.93 (1H, d, J = 2.7 Hz), 5.61 (1H, d, J = 1.4 Hz), 5.47-5.42 (1H, m), 5.41 (1H, t, J = 10.0 Hz), 4.47 (1H, d, J = 10.0) Hz), 4.23 (1H, ddd, J = 10.5, 6.5 and 1.7 Hz), 3.28 (1H, dd, J = 13.3 and 1.8 Hz), 3.14 (1H, ddd, J = 12.8, 6.5, and 3.3 Hz), 2.79 (1H, dd, J = 13.5 and 4.9 Hz), 2.68 (1H, dd, J = 13.1 and 10.6 Hz), 2.53 (1H, d, J = 13.5 Hz), 2.38-2.26 (2H, m), 1.86 (3H, d, J = 1.4 Hz), 1.07 (3H, t, J = 7.4 Hz).

DETD-20 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 6.92 (1H, br s), 6.44 (1H, d, J = 3.1 Hz), 6.02-5.96 (1H, m), 5.46-5.42 (1H, m), 5.24 and 5.23 (1H, t, J = 10.1 Hz, 1:1), 4.40 (1H, d, J = 9.8 Hz), 4.18 (1H, dd, J = 9.8 and 6.4 Hz), 3.27 (1H) , dd, J = 13.1 and 1.6 Hz), 3.12-3.04 (1H, m), 2.79 (1H, dd, J = 13.6 and 4.8 Hz), 2.66 (1H, dd, J = 11.1 and 10.7 Hz), 2.53 ( 1H, d, J = 13.5 Hz), 2.48-2.36 (1H, m), 1.84 (3H, br s), 1.68-1.55 (1H, m), 1.44-1.22 (3H, m), 1.13 and 1.12 (3H , d, J = 6.9 Hz, 1:1), 0.89 (3H, t, J = 7.2 Hz).

DETD-21 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 6.94 (1H, br s), 6.43 (1H, d, J = 2.9 Hz), 6.01 (1H, d, J = 2.5 Hz), 5.46-5.42 (1H, m), 5.26 (1H, t, J = 10.3 Hz), 4.40 (1H, d, J = 10.0 Hz), 4.22-4.14 (1H, m), 3.25 (1H, dd, J = 13.1 and 1.8) Hz), 3.12-3.03 (1H, m), 2.79 (1H, dd, J = 13.5 and 4.9 Hz), 2.66 (1H, dd, J = 13.1 and 10.7 Hz), 2.52 (1H, d, J = 13.5 Hz) ), 2.28-2.20 (1H, m), 1.85 (3H, d, J = 1.2 Hz), 1.69-1.38 (4H, m), 1.35-1.10 (4H, m), 0.92-0.81 (6H, m).

DETD-22 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 6.93 (1H, br s), 6.43 (1H, br d, J = 3.1 Hz), 6.00 (1H, br d, J = 2.5 Hz), 5.47 -5.42 (1H, m), 5.24 (1H, t, J = 10.0 Hz), 4.41 (1H, d, J = 10.0 Hz), 4.18 (1H, ddd, J = 10.7, 6.3 and 1.6 Hz), 3.25 ( 1H, dd, J = 11.1 and 2.0 Hz), 3.07 (1H, ddd, J = 12.7, 6.4, and 3.1 Hz), 2.79 (1H, dd, J = 13.5 and 4.8 Hz), 2.66 (1H, dd, J) =13.1 and 10.7 Hz), 2.53 (1H, d, J = 13.5 Hz), 2.45-2.34 (1H, m), 1.84 (3H, d, J = 1.4 Hz), 1.70-1.54 (2H, m), 143 -1.16 (4H, m), 1.13 (3H, d, J = 7.0 Hz), 0.88 (3H, t, J = 7.0 Hz).

DETD-23 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 6.92 (1H, s), 6.45 (1H, d, J = 3.1 Hz), 5.99 (1H, d, J = 2.5 Hz), 5.48-5.42 ( 1H, m), 5.29 (1H, t, J = 10.1 Hz), 4.41 (1H, br d, J = 10.1 Hz), 4.19 (1H, ddd, J = 10.1, 6.4 and 1.6 Hz), 3.76 (1H, Br s), 3.26 (1H, dd, J = 13.1 and 2.0 Hz), 3.12-3.04 (1H, m), 2.80 (1H, dd, J = 13.4 and 4.8 Hz), 2.66 (1H, dd, J = 13.1) And 10.0 Hz), 2.52 (1H, br d, J = 13.4 Hz), 2.40 (2H, s), 1.98-1.88 (2H, m), 1.83 (3H, d, J = 1.4 Hz), 0.91 (6H, t, J = 7.3 Hz), 0.89 - 0.82 (6H, m).

DETD-24 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.01 (1H, dq, J = 15.4 and 6.9 Hz), 6.93 (1H, s), 6.39 (1H, d, J = 3.1 Hz), 5.94 ( 1H, d, J = 2.7 Hz), 5.83 (1H, dd, J = 15.4 and 1.8 Hz), 5.48-5.42 (1H, m), 5.38 (1H, t, J = 10.1 Hz), 4.46 (1H, br d, J = 10.0Hz), 4.22 (1H, ddd, J = 10.7,6.6 and 1.6Hz), 3.26 (1H, dd , J = 13.1 and 2.0Hz), 3.10 (1H, ddd , J = 12.8,6.6 and 3.2 Hz), 2.78 (1H, dd, J = 13.4 and 4.9 Hz), 2.67 (1H, dd, J = 13.4 and 10.7 Hz), 2.53 (1H, br d, J = 13.4 Hz), 1.91 (3H, dd) , J = 6.9 and 1.8 Hz), 1.85 (3H, d, J = 1.4 Hz).

DETD-25 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.05 (1H, dt, J = 15.6 and 6.4 Hz), 6.93 (1H, s), 6.40 (1H, dd, J = 8.5 and 3.0 Hz), 5.96 (1H, dd, J = 14.6 and 2.5 Hz), 5.79 (1H, dt, J = 15.6 and 1.7 Hz), 5.48-5.42 (1H, m), 5.38 (1H, t, J = 10.1 Hz), 4.45 (1H, t, J = 10.0 Hz), 4.26-4.15 (1H, m), 3.31-3.20 (1H, m), 3.16-3.00 (2H, m), 2.78 (1H, dd, J = 13.7 and 4.9 Hz) ), 2.54 (1H, br d, J = 13.5 Hz), 2.30-2.20 (1H, m), 1.85 (3H, d, J = 1.4 Hz), 1.08 (3H, t, J = 7.4 Hz).

DETD-26 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.66 (1H, d, J = 15.9 Hz), 7.47 (2H, d, J = 8.9 Hz), 6.92 (1H, s), 6.91 (2H, d, J = 8.9 Hz), 6.41 (1H, d, J = 2.9 Hz), 6.26 (1H, d, J = 15.9 Hz), 6.01 (1H, d, J = 2.3 Hz), 5.46 (1H, t, J = 10.1 Hz), 5.47-5.43 (1H, m), 4.50 (1H, d, J = 10.1 Hz), 4.25 (1H, ddd, J = 10.6, 6.7 and 1.7 Hz), 3.85 (3H, s), 3.29 (1H, br d, J = 11.3 Hz), 3.14 (1H, ddd, J = 12.8, 6, 4 and 3.2 Hz), 2.80 (1H, dd, J = 13.4 and 4.9 Hz), 2.69 (1H, dd) , J = 13.4 and 10.6 Hz), 2.54 (1H, d, J = 13.4 Hz), 1.88 (3H, d, J = 1.4 Hz).

DETD-27 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.67 (1H, d, J = 16.0 Hz), 7.41 (2H, d, J = 8.0 Hz), 7.20 (2H, d, J = 8.0 Hz) , 6.93 (1H, s), 6.41 (1H, d, J = 2.9 Hz), 6.34 (1H, d, J = 16.0 Hz), 6.00 (1H, d, J = 2.3 Hz), 5.46 (1H, t, J =10.0 Hz), 5.48-5.43 (1H, m), 4.50 (1H, d, J = 10.0 Hz), 4.25 (1H, ddd, J = 10.6, 6.7 and 1.7 Hz), 3.29 (1H, dd, J) =13.2 and 2.0 Hz), 3.14 (1H, ddd, J = 12.7, 6.3 and 3.2 Hz), 2.80 (1H, dd, J = 13.4 and 4.8 Hz), 2.69 (1H, dd, J = 13.4 and 10.7 Hz) , 2.54 (1H, d, J = 13.4 Hz), 2.38 (3H, s), 1.88 (3H, d, J = 1.4 Hz).

DETD-28 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.65 (1H, d, J = 15.8 Hz), 7.45 (2H, d, J = 8.6 Hz), 7.37 (2H, d, J = 8.6 Hz) , 6.93 (1H, s), 6.41 (1H, d, J = 3.1 Hz), 6.37 (1H, d, J = 16.0 Hz), 5.98 (1H, d, J = 2.1 Hz), 5.46 (1H, t, J = 10.1 Hz), 5.48-5.43 (1H, m), 4.50 (1H, d, J = 10.0 Hz), 4.25 (1H, ddd, J = 10.7, 6.6 and 1.6 Hz), 3.29 (1H, br d, J = 11.2 Hz), 3.14 (1H, ddd, J = 12.7, 6.4 and 3.2 Hz), 2.80 (1H, dd, J = 13.4 and 4.9 Hz), 2.69 (1H, dd, J = 13.4 and 10.7 Hz), 2.54 (1H, d, J = 13.4 Hz), 2.38 (3H, s), 1.88 (3H, d, J = 1.4 Hz).

DETD-29 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, 3% CD ₃ OD in CDCl ₃ ) σ 7.51 (1H, s), 7.43 (1H, d, J = 16.0 Hz), 6.96 (1H, br s), 6.65 ( 1H, d, J = 3.1 Hz), 6.52-6.48 (1H, m), 6.41 (1H, d, J = 3.1 Hz), 6.28 (1H, d, J = 16.0 Hz), 6.01 (1H, d, J =2.5 Hz), 5.50-5.41 (2H, m), 5.48-5.44 (1H, m), 4.50 (1H, d, J = 10.1 Hz), 4.24 (1H, dd, J = 10.5 and 6.4 Hz), 3.27 (1H, d, J = 12.8 Hz), 3.19-3.10 (1H, m), 2.80 (1H, dd, J = 13.7 and 5.0 Hz), 2.70 (1H, dd, J = 12.8 and 11.0 Hz), 2.55 ( 1H, d, J = 13.7 Hz), 1.88 (3H, s).

DETD-30 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.81 (1H, dd, J = 3.8 and 1.2 Hz), 7.60 (1H, dd, J = 5.0 and 1.2 Hz), 7.14 (1H, dd, J =5.0 and 3.8 Hz), 6.94 (1H, br s), 6.41 (1H, d, J = 3.1 Hz), 6.08 (1H, d, J = 2.5 Hz), 5.51 (1H, t, J = 10.1 Hz) , 5.48-5.44 (1H, m), 4.55 (1H, br d, J = 10.0 Hz), 4.26 (1H, ddd, J = 10.7, 6.5 and 1.6 Hz), 3.30 (1H, br d, J = 13.2 Hz) ), 3.23 (1H, ddd, J = 12.8, 6.4 and 3.1 Hz), 2.80 (1H, dd, J = 13.5 and 4.9 Hz), 2.71 (1H, dd, J = 13.1 and 10.7 Hz), 2.54 (1H, d, J = 13.5 Hz), 1.90 (3H, d, J = 1.4 Hz).

DETD-31 (Method A):

Proton nuclear magnetic resonance (1H-NMR, 400MHz, CDCl3) σ 8.04-7.99 (2H, m), 7.63-7.58 (1H, m), 7.48 (1H, d, J = 8.0 Hz), 7.46 (1H, d, J = 7.4 Hz), 6.95 (1H, br s), 6.36 (1H, d, J = 3.3 Hz), 5.97 (1H, d, J = 2.7 Hz), 5.60 (1H, t, J = 10.0 Hz), 5.48 -5.44 (1H, m), 4.55 (1H, br d, J = 10.0 Hz), 4.26 (1H, ddd, J = 10.5, 6.5 and 1.6 Hz), 3.35 - 3.22 (2H, m), 2.80 (1H, Dd, J = 13.5 and 4.9 Hz), 2.72 (1H, dd, J = 13.3 and 10.7 Hz), 2.53 (1H, d, J = 13.3 Hz), 1.92 (3H, d, J = 1.4 Hz).

DETD-32 (method B):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.96 (2H, d, J = 8.0 Hz), 6.97-6.92 (3H, m), 6.35 (1H, d, J = 2.9 Hz), 5.96 ( 1H, d, J = 2.3 Hz), 5.58 (1H, t, J = 10.0 Hz), 5.48-5.43 (1H, m), 4.55 (1H, d, J = 10.0 Hz), 4.29 (1H, dd, J =9.4 and 7.1 Hz), 3.31 (1H, d, J = 12.7 Hz), 3.28-3.18 (1H, m), 2.80 (1H, dd, J = 13.5 and 4.9 Hz), 2.72 (1H, dd, J = 12.9 and 10.7 Hz), 2.54 (1H, d, J = 13.5 Hz), 1.91 (3H, br s).

DETD-33 (Method B):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.89 (2H, d, J = 8.2 Hz), 7.26 (2H, d, J = 8.0 Hz), 6.94 (1H, s), 6.35 (1H, d, J = 3.1 Hz), 5.96 (1H, d, J = 2.7 Hz), 5.58 (1H, t, J = 10.0 Hz), 5.49 - 5.43 (1H, m), 4.55 (1H, d, J = 10.0) Hz), 4.29 (1H, ddd, J = 10.6, 6.7, and 1.6 Hz), 3.36-3.27 (1H, m), 3.24 (1H, ddd, J = 12.8, 6.4, and 3.1 Hz), 2.80 (1H, Dd, J = 13.6 and 4.8 Hz), 2.72 (1H, dd, J = 11.1 and 10.7 Hz), 2.53 (1H, d, J = 13.5 Hz), 2.42 (3H, s), 1.91 (3H, d, J =1.4Hz).

DETD-34 (Method B):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.86 (2H, d, J = 8.6 Hz), 7.62 (2H, d, J = 8.4 Hz), 6.94 (1H, s), 6.35 (1H, d, J = 2.9 Hz), 5.89 (1H, d, J = 2.7 Hz), 5.59 (1H, t, J = 10.1 Hz), 5.48-5.44 (1H, m), 4.54 (1H, d, J = 10.1) Hz), 4.29 (1H, dd, J = 9.5 and 6.7 Hz), 3.31 (1H, br d, J = 13.5 Hz), 3.28-3.20 (1H, m), 2.81 (1H, dd, J = 13.5 and 4.7) Hz), 2.72 (1H, dd, J = 13.1 and 10.9 Hz), 2.53 (1H, d, J = 13.3 Hz), 1.91 (3H, d, J = 1.0 Hz).

DETD-35 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.91-7.84 (2H, m), 7.81 (1H, d, J = 8.0 Hz), 7.51 (1H, d, J = 9.6 Hz), 7.53 7.50 (1H, m), 7.44 (1H, t, J = 8.0 Hz), 7.37 (1H, d, J = 6.4 Hz), 6.85 (1H, s), 5.88 (1H, d, J = 3.1 Hz), 5.44-5.38 (1H, m), 5.20 (1H, t, J = 10.0 Hz), 5.06 (1H, d, J = 2.7 Hz), 4.33 (1H, d, J = 10.0 Hz), 4.13-4.00 (1H) , m), 4.07 (2H, d, J = 10.8 Hz), 3.18 (1H, br d, J = 12.9 Hz), 2.93 - 2.86 (1H, m), 2.77 (1H, dd, J = 13.4 and 4.8 Hz) ), 2.55 (1H, dd, J = 12.9 and 10.8 Hz), 2.50 (1H, d, J = 13.4 Hz), 1.81 (3H, d, J = 1.0 Hz).

DETD-36 (Method B):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 6.92 (1H, s), 6.44 (1H, d, J = 3.1 Hz), 6.04 (1H, d, J = 2.7 Hz), 5.49-5.41 ( 1H, m), 5.31 (1H, t, J = 10.0 Hz), 4.46 (1H, d, J = 10.0 Hz), 4.24 - 4.15 (1H, m), 3.26 (1H, br d, J = 11.7 Hz) , 3.14 - 3.03 (1H, m), 2.78 (1H, dd, J = 13.5 and 4.7 Hz), 2.66 (1H, dd, J = 13.1 and 10.7 Hz), 2.54 (1H, d, J = 13.5 Hz), 1.81 (3H, d, J = 1.4 Hz), 1.64-1.52 (1H, m), 1.05-0.86 (4H, m).

DETD-37 (Method B):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 6.92 (1H, s), 6.43 (1H, dd, J = 3.1 and 0.8 Hz), 6.00 (1H, dd, J = 2.7 and 0.8 Hz), 5.46-5.41 (1H, m), 5.24 (1H, t, J = 10.0 Hz), 4.42 (1H, d, J = 10.0 Hz), 4.18 (1H, ddd, J = 10.7, 6.4 and 1.6 Hz), 3.26 (1H, dd, J = 11.1 and 2.2 Hz), 3.10-3.02 (1H, m), 2.79 (1H, dd, J = 13.5 and 4.9 Hz), 2.74 - 2.66 (1H, m), 2.65 (1H, dd) , J = 13.1 and 10.6 Hz), 2.54 (1H, d, J = 13.1 Hz), 1.84 (3H, d, J = 1.6 Hz), 1.93-1.84 (1H, m), 1.80-1.63 (4H, m) , 1.63-1.55 (2H, m).

DETD-38 (Method B):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 6.91 (1H, s), 6.43 (1H, dd, J = 2.9 and 0.8 Hz), 5.98 (1H, dd, J = 2.5 and 0.8 Hz), 5.46-5.41 (1H, m), 5.25 (1H, t, J = 10.0 Hz), 4.40 (1H, d, J = 10.0 Hz), 4.18 (1H, ddd, J = 10.7, 6.4 and 1.6 Hz), 3.30 -3.22 (1H, m), 3.06 (1H, ddd, J = 13.1, 6.4 and 3.0 Hz), 2.78 (1H, dd, J = 13.5 and 4.7 Hz), 2.65 (1H, dd, J = 13.1 and 10.7 Hz) ), 2.53 (1H, d, J = 13.1 Hz), 2.32 - 2.21 (1H, m), 1.92-1.80 (2H, m), 1.83 (3H, d, J = 1.4 Hz), 1.80-1.71 (2H, m), 1.49-1.18 (6H, m).

DETD-39 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.66 (1H, d, J = 15.9 Hz), 7.32 (1H, t, J = 8.0 Hz), 7.11 (1H, d, J = 7.8 Hz) , 7.05-7.01 (1H, m), 6.96 (1H, dd, J = 8.0 and 2.5 Hz), 6.93 (1H, br s), 6.41 (1H, d, J = 2.9 Hz), 6.38 (1H, d, J = 15.9 Hz), 6.00 (1H, d, J = 2.9 Hz), 5.45 (1H, t, J = 10.1 Hz), 4.50 (1H, d, J = 10.1 Hz), 4.25 (1H, ddd, J = 10.5, 6.6 and 1.6 Hz), 3.84 (3H, s), 3.29 (1H, br d, J = 13.0 Hz), 3.19-3.11 (1H, m), 2.80 (1H, dd, J = 13.3 and 4.9 Hz) , 2.69 (1H, dd, J = 13.3 and 10.5 Hz), 2.54 (1H, d, J = 13.3 Hz), 1.89 (3H, d, J = 1.2 Hz).

DETD-40 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.70 (1H, d, J = 7.8 Hz), 7.58 (1H, d, J = 8.4 Hz), 7.56 (1H, s), 7.49 (1H, Dd, J = 8.4 and 7.8 Hz), 7.34 (1H, t, J = 7.8 Hz), 6.95 (1H, s), 6.43 (1H, d, J = 3.1 Hz), 6.20 (1H, d, J = 2.7 Hz), 5.58 (1H, t, J = 10.1 Hz), 5.49-5.44 (1H, m), 4.59 (1H, d, J = 10.1 Hz), 4.33-4.24 (1H, m), 3.36-3.24 (2H , m), 2.82 (1H, dd, J = 13.5 and 4.9 Hz), 2.72 (1H, dd, J = 13.1 and 10.7 Hz), 2.56 (1H, d, J = 13.5 Hz), 1.93 (3H, br s ).

DETD-41 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 8.07 (1H, s), 7.89 (1H, d, J = 7.8 Hz), 7.87 (1H, d, J = 7.8 Hz), 7.50 (1H, Dt, J = 7.8 and 1.2 Hz), 7.42 (1H, dt, J = 7.8 and 1.2 Hz), 6.96 (1H, s), 6.43 (1H, d, J = 3.1 Hz), 6.14 (1H, d, J =2.5 Hz), 5.56 (1H, t, J = 10.0 Hz), 5.49-5.44 (1H, m), 4.58 (1H, d, J = 10.0 Hz), 4.27 (1H, ddd, J = 10.7, 6.4 and 1.5 Hz), 3.35-3.23 (2H, m), 2.82 (1H, dd, J = 13.7 and 4.9 Hz), 2.72 (1H, dd, J = 13.1 and 10.7 Hz), 2.56 (1H, d, J = 13.5) Hz), 1.92 (3H, d, J = 1.4 Hz).

DETD-42 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.79 (1H, d, J = 15.7 Hz), 7.41 (1H, d, J = 5.1 Hz), 7.27 (1H, d, J = 5.1 Hz) , 7.07 (1H, dd, J = 5.1 and 3.7 Hz), 6.93 (1H, br s), 6.41 (1H, d, J = 3.1 Hz), 6.18 (1H, d, J = 15.7 Hz), 5.99 (1H) , t, J = 2.7 Hz), 5.45 (1H, t, J = 10.0 Hz), 5.47-5.42 (1H, m), 4.48 (1H, d, J = 10.0 Hz), 4.24 (1H, dd, J = 9.3 and 6.5 Hz), 3.27 (1H, br d, J = 13.3 Hz), 3.17-3.08 (1H, m), 2.80 (1H, dd, J = 13.5 and 5.0 Hz), 2.69 (1H, dd, J = 13.3 and 10.7 Hz), 2.54 (1H, d, J = 13.5 Hz), 1.87 (3H, br s).

DETD-43 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.36-7.26 (3H, m), 7.24-7.20 (2H, m), 6.90 (1H, br s), 6.18 (1H, d, J = 2.9 Hz), 5.47 (1H, t, J = 2.4 Hz), 5.44 - 5.40 (1H, m), 5.24 (1H, t, J = 10.0 Hz), 4.40 (1H, d, J = 10.0 Hz), 4.14 ( 1H, ddd, J = 10.6, 6.3 and 1.6 Hz), 3.65 (1H, d, J = 15.2 Hz), 3.60 (1H, d, J = 15.2 Hz), 3.23 (1H, br d, J = 13.1 Hz) , 3.01 (1H, ddd, J = 12.8, 6.3 and 3.0 Hz), 2.78 (1H, dd, J = 13.5 and 4.9 Hz), 2.62 (1H, dd, J = 13.1 and 10.7 Hz), 2.52 (1H, d , J = 13.5 Hz), 1.83 (3H, d, J = 1.5 Hz).

DETD-44 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.13 (2H, d, J = 8.5 Hz), 6.90 (1H, br s), 6.86 (2H, d, J = 8.5 Hz), 6.22 (2H) , d, J = 3.1 Hz), 5.53 (1H, t, J = 2.4 Hz), 5.43 (1H, t, J = 2.4 Hz), 5.46-5.41 (1H, m), 5.24 (1H, t, J = 10.0 Hz), 4.40 (1H, d, J = 10.0 Hz), 4.18-4.11 (1H, m), 3.80 (3H, s), 3.58 (1H, d, J = 15.4 Hz), 3.53 (1H, d, J = 15.4 Hz), 3.23 (1H, br d, J = 13.1 Hz), 3.02 (1H, ddd, J = 12.6, 6.3 and 3.1 Hz), 2.77 (1H, dd, J = 13.4 and 4.8 Hz), 2.63 (1H, dd, J = 13.1 and 10.7 Hz), 2.52 (1H, d, J = 13.4 Hz), 1.82 (3H, br s).

DETD-45 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.78 (1H, d, J = 10.2 Hz), 7.70 (1H, d, J = 8.0 Hz), 7.39 (1H, t, J = 8.0 Hz) , 7.21 (1H, d, J = 7.0 Hz), 7.20-7.15 (2H, m), 6.86 (1H, br s), 5.90 (1H, d, J = 2.8 Hz), 5.44 - 5.39 (1H, m) , 5.19 (1H, t, J = 10.0 Hz), 5.04 (1H, d, J = 2.8 Hz), 4.33 (1H, d, J = 10.0 Hz), 4.15-3.99 (3H, m), 3.93 (3H, s), 3.18 (1H, br d, J = 12.5 Hz), 2.94 - 2.85 (1H, m), 2.77 (1H, dd, J = 13.5 and 4.8 Hz), 2.62 - 2.46 (2H, m), 1.81 ( 3H, br s).

DETD-46 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.71 (1H, d, J = 8.4 Hz), 7.68 (1H, d, J = 9.1 Hz), 7.60 (1H, br s), 7.29 (1H) , d, J = 8.4 Hz), 7.18-7.10 (2H, m), 6.89 (1H, br s), 6.09 (1H, d, J = 3.1 Hz), 5.46 (1H, d, J = 2.5 Hz), 5.45-5.40 (1H, m), 5.26 (1H, t, J = 10.0 Hz), 4.40 (1H, d, J = 10.0 Hz), 4.14 (1H, dd, J = 10.0 and 6.4 Hz), 3.92 (3H) , s), 3.77 (1H, d, J = 15.1 Hz), 3.72 (1H, d, J = 15.1 Hz), 3.22 (1H, d, J = 13.1 Hz), 3.05-2.95 (1H, m), 2.77 (1H, dd, J = 13.3 and 4.8 Hz), 2.60 (1H, dd, J = 13.1 and 10.8 Hz), 2.51 (1H, d, J = 13.3 Hz), 1.83 (3H, br s).

DETD-47 (method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.67 (1H, d, J = 16.0 Hz), 7.48-7.42 (2H, m), 7.36 (1H, s), 7.30-7.25 (1H, m ), 6.96 (1H, s), 6.42 (1H, d, J = 16.0 Hz), 6.42 (1H, d, J = 2.5 Hz), 5.99 (1H, d, J = 2.5 Hz), 5.49 (1H, t , J =10.1 Hz), 5.47-5.48 (1H, m), 4.51 (1H, d, J = 10.8 Hz), 4.25 (1H, dd, J = 10.5 and 6.7 Hz), 3.28 (1H, d, J = 13.1 Hz), 3.18-3.14 (1H, m), 2.81 (1H, dd, J = 13.7 and 4.8 Hz), 2.71 (1H, dd, J = 13.1 and 10.5 Hz), 2.56 (1H, d, J = =13.7) Hz), 1.90 (3H, s).

DETD-48 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.77 (1H, s), 7.72 (1H, d, J = 16.2 Hz), 7.66 (1H, d, J = 7.8 Hz), 7.54 (1H, t, J = 7.8 Hz), 6.95 (1H, s), 6.47 (1H, d, J = 16.2 Hz), 6.43 (1H, d, J = 2.8 Hz), 6.00 (1H, d, J = 2.8 Hz) , 5.48 (1H, t, J = 10.0 Hz), 5.48-5.45 (1H, m), 4.51 (1H, d, J = 10.0 Hz), 4.26 (1H, ddd, J = 10.5, 6.8 and 2.0 Hz), 3.30 (1H, dd, J = 13.2 and 2.0 Hz), 3.19-3.12 (1H, m), 2.82 (1H, dd, J = 13.7 and 4.8 Hz), 2.70 (1H, dd, J = 13.2 and 10.5 Hz) , 2.55 (1H, d, J = 13.3 Hz), 1.89 (3H, d, J = 1.4 Hz).

DETD-49 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.64 (1H, d, J = 15.6 Hz), 7.11 (1H, dd, J = 8.3 and 1.8 Hz), 7.03 (1H, d, J = 1.8) Hz), 6.94 (1H, s), 6.88 (1H, d, J = 8.3 Hz), 6.42 (1H, d, J = 2.6 Hz), 6.26 (1H, d, J = 15.6 Hz), 6.02 (1H, d, J = 2.6 Hz), 5.48 (1H, t, J = 10.1 Hz), 5.48-5.45 (1H, m), 4.50 (1H, d, J = 10.1 Hz), 4.26 (1H, ddd, J = =10.5) , 6.6 and 1.8 Hz), 3.93 (3H, s), 3.92 (3H, s), J = 1.3 Hz), 3.29 (1H, dd, J = 13.3 and 1.8 Hz), 3.18-3.10 (1H, m), 2.81 (1H, dd, J = 13.3 and 5.0 Hz), 2.70 (1H, dd, J = 13.3 and 10.5 Hz), 2.54 (1H, d, J = 13.3 Hz), 1.89 (3H, d, J = 1.4 Hz) ).

DETD-50 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.66 (1H, d, J = 16.0 Hz), 7.30 (1H, t, J = 7.7 Hz), 7.10 (1H, d, J = 7.7 Hz) , 7.04-7.02 (1H, m), 6.97-6.93 (1H, m), 6.94 (1H, s), 6.421H, d, J = 3.0 Hz), 6.37 (1H, d, J = 16.0 Hz), 6.00 (1H, d, J = 3.0 Hz), 5.46 (1H, t, J = 10.1 Hz), 5.45-5.44 (1H, m), 4.50 (1H, d, J = 10.1 Hz), 4.25 (1H, ddd, J = 10.8, 6.8 and 1.4 Hz), 4.06 (2H, q, J = 7.0 Hz), 3.29 (1H, dd, J = 13.3 and 2.1 Hz), 3.15 (1H, ddd, 12, 8, 6.9 and 3.5 Hz) ), 2.81 (1H, dd, J = 13.3 and 4.8 Hz), 2.70 (1H, dd, J = 13.3 and 10.8 Hz), 2.55 (1H, d, J = 13.3 Hz), 1.89 (3H, d, J = 1.4 Hz), 1.43 (3H, t, J = 7.0 Hz).

DETD-51 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.98 (1H, d, J = 7.8 Hz), 7.90-7.85 (1H, m), 7.75 (1H, d, J = 7.8 Hz), 7.54 ( 1H, dt, J = 6.8 and 1.3 Hz), 7.50 (1H, dt, J = 6.8 and 1.3 Hz), 7.38 (1H, t, J = 6.8 Hz), 7.31 (1H, d, J = 6.8 Hz), 6.87 (1H, s), 6.19 (1H, d, J = 2.8 Hz), 5.65 (1H, d, J = 2.8 Hz), 5.43 (1H, dd, J = 4.6 and 1.8 Hz), 5.26 (1H, t , J =10.1 Hz), 4.30 (1H, d, J = 10.1 Hz), 4.15 (1H, ddd, J = 10.5, 6.4 and 1.4 Hz), 3.40 (2H, t, J = 7.8 Hz), 3.26-3.19 (1H, m), 2.98-2.92 (1H, m), 2.82-2.73 (3H, m), 2.61 (1H, dd, J = 13.3 and 10.8 Hz), 2.50 (1H, d, J = 13.8 Hz), 1.85 (3H, d, J = 1.4 Hz).

DETD-52 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 8.84 (1H, dd, J = 4.1 and 1.8 Hz), 8.16 (1H, dd, J = 8.3 and 1.4 Hz), 7.79 (1H, dd, J =8.3 and 1.4 Hz), 7.62 (1H, d, J = 6.7 Hz), 7.51 (1H, dd, J = 8.3 and 6.7 Hz), 7.42 (1H, dd, J = 8.3 and 4.1 Hz), 6.89 (1H) , br s), 6.25 (1H, d, J = 2.7 Hz), 5.95 (1H, d, J = 2.7 Hz), 5.43 (1H, dd, J = 4.8 and 2.1 Hz), 5.28 (1H, t, J = 10.1 Hz), 4.43 (1H, d, J = 10.1 Hz), 4.36 (1H, d, J = 15.6 Hz), 4.17 (1H, ddd, J = 10.8, 6.7 and 1.5 Hz), 4.10 (1H, d , J = 15.6 Hz), 3.24 (1H, dd, J = 13.1 and 2.1 Hz), 3.04 (1H, ddd, J = 13.1, 6.7 and 3.3 Hz), 2.78H, dd, J = 13.2 and 4.8 Hz), 2.63 (1H, dd, J = 13.1 and 10.8 Hz), 2.53 (1H, d, J = 13.2 Hz), 1.80 (3H, d, J = 1.4 Hz).

DETD-53 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.70 (1H, d, J = 8.7 Hz), 7.69 (1H, d, J = 8.7 Hz), 7.57 (1H, br s), 7.29 (1H) , dd, J = 8.7 and 1.8 Hz), 7.16 (1H, dd, J = 8.7 and 2.5 Hz), 7.12 (1H, d, J = 2.3 Hz), 6.80 (1H, br s), 6.26 (1H, d , J = 2.8 Hz), 5.69 (1H, d, J = 2.5 Hz), 5.40-5.35 (1H, m), 5.23 (1H, t, J = 10.0 Hz), 4.21 (1H, d, J = 10.0 Hz) ), 4.18-4.11 (1H, m), 3.94 (3H, s), 3.84 (1H, q, J = 7.0 Hz), 3.21 (1H, br d, J = 13.3 Hz), 2.95 (1H, ddd, J =13.3, 6.5 and 3.1 Hz), 2.75 (1H, dd, J = =13.7 and 4.6 Hz), 2.57 (1H, dd, J = 13.3 and 10.7 Hz), 2.41 (1H, d, J = 13.3 Hz), 1.85 (3H, d, J = 1.4 Hz), 1.55 (3H, d, J = 7.0 Hz).

DETD-54 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.71 (1H, d, J = 8.3 Hz), 7.68 (1H, d, J = 9.0 Hz), 7.61 (1H, br s), 7.31-7.26 (1H,m), 7.16 (1H, dd, J = 9.0 and 2.3 Hz), 7.12 (1H, d, J = 2.3 Hz), 6.80 (1H, br s), 5.56 (1H, d, J = 2.7 Hz) ), 5.47-5.42 (1H, m), 5.20 (1H, t, J = 10.1Hz), 4.78 (1H, d, J = 2.7Hz), 4.41 (1H, d, J = 10.1Hz), 4.12-4.05 (1H, m), 3.94 (3H, s), 3.77 (1H, q, J = 7.0 Hz), 3.24 - 3.16 (1H, m), 2.94 (1H, ddd, J = 12.8, 6.1 and 3.1 Hz), 2.81 (1H, dd, J = 13.3 and 4.6 Hz), 2.63 - 2.52 (2H, m), 1.86 (3H, d, J = 1.4 Hz), 1.54 (3H, d, J = 7.0 Hz).

DETD-55 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 8.25 and 8.02 (1H, d, J = 8.7 Hz, 3:5), 7.90-7.82 (3H, m), 7.68 and 7.64 (1H, d, J = 6.9 Hz, 5:3), 7.54 - 7.36 (6H, m), 6.81 and 6.70 (1H, br s, 3: 5), 6.01 (5/8H, d, J = 2.7 Hz), 5.43-5.25 (27/8H, m), 5.13 and 5.11 (1H, t, J = 10.1 Hz, 3:5), 4.30 (3/8H, d, J = 2.7 Hz), 4.26-4.20 (5/8H, m) , 4.09-4.01 (1H, m), 3.91 (5/8H, d, J = 10.1 Hz), 3.17-3.12 (3/8H, m), 3.11 and 3.08 (3H, s, 5:3), 2.84 2.68 (2H, m), 2.54-2.25 (2H, m), 1.95 and 1.92 (3H, s, 5:3), 1.85 and 1.83 (3H, d, J = 1.4 Hz, 3:5).

DETD-56 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.87-7.80 (4H, m), 7.55-7.49 (2H, m), 7.42 (1H, dd, J = 8.5 and 1.4 Hz), 6.81 (1H) , br s), 6.22 (1H, d, J = 3.2 Hz), 5.78 (1H, d, J = 2.7 Hz), 5.40-5.35 (1H, m), 5.28 (1H, t, J = 10.1 Hz), 4.92 (1H, s), 4.24 (1H, d, J = 10.1 Hz), 4.17-4.10 (1H, m), 3.43 (3H, s), 3.20 (1H, dd, J = 13.3 and 1.8 Hz), 3.03 (1H, ddd, J = 12.9, 6.5 and 3.2 Hz), 2.72 (1H, dd, J = 13.3 and 4.7 Hz), 2.58 (1H, dd, J = 12.9 and 10.8 Hz), 2.38 (1H, d, J) =13.3 Hz), 1.81 (1H, d, J = 1.4 Hz).

DETD-57 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.90-7.81 (4H, m), 7.58-7.50 (2H, m), 7.46 (1H, dd, J = 8.5 and 1.6 Hz), 6.87 (1H) , br s), 5.70 (1H, d, J = 3.2 Hz), 5.45-5.40 (1H, m), 5.27 (1H, t, J = 10.1 Hz), 5.03 (1H, d, J = 2.3 Hz), 4.86 (1H, s), 4.36 (1H, d, J = 10.1 Hz), 4.15-4.07 (1H, m), 3.37 (3H, s), 3.20 (1H, br d, J = 12.8 Hz), 3.03- 2.95 (1H, m), 2.76 (1H, dd, J = 13.3 and 4.8 Hz), 2.59 (1H, dd, J = 12.8 and 10.8 Hz), 2.48 (1H, d, J = 13.3 Hz), 1.84 (1H) , d, J = 1.4Hz).

DETD-58 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.90-7.85 (1H, m), 7.68-7.64 (1H, m), 7.44-7.36 (2H, m), 7.32 (1H, s), 6.89 (1H, br s), 6.07 (1H, d, J = 2.7 Hz), 5.45-5.41 (1H, m), 5.37 (1H, d, J = 2.3 Hz), 5.26 (1H, t, J = 10.1 Hz) ), 4.37 (1H, d, J = 10.1 Hz), 4.16 - 4.08 (1H, m), 3.88 (2H, s), 3.24 - 3.17 (1H, m), 2.97 (1H, ddd, J = 13.0, 6.0 And 3.2 Hz), 2.78 (1H, dd, J = 13.3 and 4.7 Hz), 2.60 (1H, d, J = 13.0 Hz), 2.51 (1H, d, J = 13.3 Hz), 1.84 (1H, d, J) =1.4Hz).

DETD-59 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.59 (1H, s), 7.47 (2H, t, J = 8.7 Hz), 7.34-7.28 (1H, m), 7.25 (1H, m), 6.90 (1H, br s), 6.22 (1H, d, J = 3.2 Hz), 5.65 (1H, d, J = 2.3 Hz), 5.45-5.41 (1H, m), 5.30 (1H, t, J = 10.1) Hz), 4.39 (1H, d, J = 10.1 Hz), 4.20-4.09 (1H, m), 3.70 (2H, s), 3.22 (1H, br d, J = 12.8 Hz), 3.02 (1H, ddd, J = 12.8, 6.4 and 3.2 Hz), 2.79 (1H, dd, J = 13.3 and 4.7 Hz), 2.63 (1H, dd, J = 13.3 and 10.7 Hz), 2.51 (1H, d, J = 13.3 Hz), 1.84 (1H, d, J = 1.4 Hz).

DETD-60 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.65 (1H, d, J = 15.6 Hz), 7.46 (2H, d, J = 8.7 Hz), 6.94 (1H, br s), 6.89 (2H) , d, J = 8.7 Hz), 6.41 (1H, d, J = 2.5 Hz), 6.25 (1H, d, J = 15.6 Hz), 6.01 (1H, d, J = 2.5 Hz), 5.46 (1H, t , J =10.1 Hz), 5.48-5.42 (1H, m), 4.50 (1H, d, J = 9.6 and 6.5 Hz), 4.07 (2H, q, J = 6.9 Hz), 3.28 (1H, br d, J =11.9z), 3.19-3.10 (1H, m), 2.80 (1H, dd, J = 13.3 and 5.0 Hz), 2.69 (1H, dd, J = 13.3 and 10.3 Hz), 2.54 (1H, d, J = 13.3 Hz), 1.89 (1H, br s), 1.43 (3H, t, J = 6.9 Hz).

DETD-61 (Method A):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.60 (1H, d, J = 16.0 Hz), 7.06-6.97 (2H, m), 6.94 (1H, br s), 6.82 (1H, d, J = 7.7 Hz), 6.41 (1H, d, J = 3.2 Hz), 6.21 (1H, d, J = 16.0 Hz), 6.02 (2H, s), 6.00 (1H, d, J = 2.7 Hz), 5.45 (1H, t, J = 10.1 Hz), 5.49-5.43 (1H, m), 4.50 (1H, d, J = 10.1 Hz), 4.28 - 4.22 (1H, m), 3.28 (1H, br d, J = 11.6 Hz), 3.02 (1H, ddd, J = 13.0, 6.4 and 3.2 Hz), 2.80 (1H, dd, J = 13.3 and 4.8 Hz), 2.69 (1H, dd, J = 13.0 and 10.7 Hz), 2.54 ( 1H, d, J = 13.3 Hz), 1.88 (1H, d, J = 1.4 Hz).

DETD-62 (Method C):

Proton nuclear magnetic resonance ( ¹ H-NMR, 400 MHz, CDCl ₃ ) σ 7.96-7.91 (1H, m), 7.91-7.85 (1H, m), 7.80 (1H, d, J = 8.3 Hz), 7.57-7.47 (2H , m), 7.43 (1H, dd, J = 8.3 and 6.9 Hz), 7.37 (1H, d, J = 6.9 Hz), 7.03 (1H, br s), 6.20 (1H, d, J = 3.4 Hz), 5.84 (1H, d, J = 16.0 Hz), 5.45 (1H, d, J = 3.4 Hz), 5.27 (1H, dd, J = 16.0 and 9.6 Hz), 5.24 - 5.17 (1H, m), 4.44 - 4.35 (1H, m), 3.99 (2H, s), 3.57-3.48 (1H, m), 3.21 (1H, br d, J = 13.0 Hz), 2.83 (1H, t, J = 12.2 Hz), 2.56 (1H) , dd, J = 14.6 and 6.4 Hz), 2.32 (1H, d, J = 14.6 Hz), 2.51 (1H, d, J = 13.0 Hz), 1.71 (1H, s).

Example 2 Inhibition effect of DETD shown in Example 1.2.2 on proliferation of breast cancer cells

This example is to test the activity of DETDs shown in Example 1.2.2 against breast cancer cells, and the results are shown in Table 2. The IC ₅₀ values of DET and DETDs were determined by the MTT method (inhibition of cell proliferation rate by 50%). This experimental example evaluates mouse breast cancer cells TS/A (ER+, Her2-), human breast cancer cells MCF-7 (ER+, Her2-) and MDA-MB-231 (ER-, Her2-, PR-). The inhibitory effect of the compound after 24 hours of treatment. The inhibitory activity (IC ₅₀ ) of DET on TS/A and MCF-7 cells was 2.3 and 5.0 μM, respectively; however, DET had a limited inhibitory effect on MDA-MB-231 cells with an IC _{50 of} about 14.0 μM. This example tested about 60 DETDs, with DETD-39 (0.8 μM), DETD-26 (1.5 μM) and DETD-35 (1.7 μM) improving the effect of DET on the anti-proliferative activity of TS/A cells. Furthermore, DETD-6, DETD-39 and DETD-7 have the best anti-proliferative activity against MCF-7 cells with IC50 of approximately 1.6 μM, 2.1 μM and 2.3 μM, respectively; and DETD-39, DETD-34 and DETD-35 was the best for anti-proliferative activity of MDA-MB-231 cells with IC50 of approximately 1.9 μM, 2.2 μM and 3.5 μM, respectively. In addition, DETD-6, DETD-26 and DETD-39 are partially toxic to normal mammary epithelial M10 cells (see Table 3).

Example 3 DET and DETD-35 inhibit MDA-MB-231 cell migration and invasion

The effect of DET and DETD-35 on MDA-MB-231 cell migration and invasion was studied by Boyden chamber analysis; PTX was used as a reference control group in this experimental example. The experimental results are shown in Figure 1. DET (2.5 and 4 μM), DETD-35 (1.25 and 2.5 μM) and PTX (2.5 and 4 μM) significantly inhibited cell migration, and its inhibitory effect increased as the concentration of the compound increased (Fig. 1A). Compared with the vehicle-controlled group of each of the compounds, administration of higher concentrations of DET (4 μM), DETD-35 (2.5 μM), and PTX (4 μM) resulted in cytostatic effects of approximately 77%, 84%, and 88%, respectively. . However, when lower concentrations of DET (2.5 μM), DETD-35 (1.25 μM), and PTX (2.5 μM) were administered, the results of cell migration inhibition were 16%, 10%, and 87%, respectively (P<0.05). ). The results of Trans-well invasion assay showed that DET and DETD-35 inhibited the invasion of MDA-MB-231 cells, making it impossible for MDA-MB-231 cells to penetrate a thin layer of Matrigel matrix. Furthermore, the experimental results showed that the application of relatively high concentrations of DET (4 μM), DETD-35 (2.5 μM) and PTX (4 μM) significantly inhibited cell invasion with inhibition rates of 76%, 80% and 90%, respectively; The anti-invasive efficacies of low concentrations of DET (2.5 μM), DETD-35 (1.25 μM) and PTX (2.5 μM) were 57%, 64.6% and 85%, respectively (P<0.05). Taken together, the above results show that DET and its derivative DETD-35 can reduce cell migration and invasion of the basement membrane barrier. Force (Fig. 1B).

Example 4 Compared with DET, DETD-35 has a better inhibitory effect on the motility of breast cancer cells.

This example investigated the effect of DET and DETD-35 on the motility of MDA-MB-231 cells using time-lapse microscopy. The migration trajectories of MDA-MB-231 cells within 24 hours after treatment under different conditions were observed and analyzed. The results showed that the cell motility was good in the vector-controlled cells, and the cells were observed to have a membrane protrusion in continuous motion (0-24 hours), and were also recorded in general cells within 24 hours. Proliferation (Fig. 2A). As shown in Fig. 2B, in the vector control group, a significant cell-dispersed region which was outwardly diffused from the origin was observed; in contrast, the diffusion region of the cells treated with 2.5 μM DET was relatively small, and cell movement was restricted. Cells treated with 10 μM DET or 1.25, 2.5 and 4 μM DETD-35, respectively, showed a relatively large reduction in cell trajectories. Cells treated with DETD-35 (1.25, 2.5, and 4 μM) or at higher concentrations of DET (4 and 10 μM) showed a significant decrease in mean rate of movement compared to vehicle-controlled cells and cells treated with 2.5 μMDET. The effect of the movement is more pronounced (Fig. 2C). Furthermore, cells treated with DET (2.5, 4, 10 μM) and DETD-35 (1.25, 2.5, 4 μM) compared to the control group (186 μM) (144 vs. 15 vs. 9 and 70 vs. 23 vs .3 μM), the total distance of movement of cells in each group was significantly shorter. The above results show that compared to DET, the novel synthesis Sesquiterpene lactone DETD-35 has better biological activity in inhibiting the motility of MDA-MB-231 cells.

Example 5 DETD-35 reduces the growth of in situ breast tumors in NOD/SCID mice

This experimental study investigated the therapeutic effect of DETD-35 on xenograft NOD/SCID mice bearing MDA-MB-231 cancer cells. On day 56, compared with the control group, mice injected intraperitoneally with 10 mg/kg DETD-35 had significantly delayed cell growth (P < 0.05) (Fig. 3A), and the mice weighed There is no impact (Figure 3B). On day 71, the tumor volume of the mice in the DETD-35 treated group was reduced by a factor of 1.8 (1268 vs. 712 mm ³ ) compared to the control group (Fig. 3C). The organ index (organ weight/body weight, %) of the lung, liver, kidney and spleen of the mice was calculated, and the results are shown in Fig. 3D. The data of the figure showed that the kidney index of the control group was higher than that of the pseudo control. Group (sham group) and DETD-35 treatment group.

Example 6 DETD-35 has the potential to inhibit lung metastasis of MDA-MB-231 cells

The effects of DETD-35 pretreatment and post treatment on triple negative breast cancer MDA-MB-231 xenograft mice compared to administration of the chemotherapy drug PTX were compared. Compared with the tumor control group, the number of tumor colonies in the lungs of the mice in the DETD-35 pretreatment group (expressed as pre-DETD-35-10 in the figure) was significantly reduced by 83%, while the DETD-35 post-treatment group ( In the figure The inhibitory effects of DETD-35-10 and PTX-5 were 50% and 62%, respectively. Treatment with DETD-35-2 inhibited lung metastasis by 9%, and this data had no statistically significant effect. Compared with the single treatment group, mice in the PTX-5+DETD-35-2 cross-treatment group (represented by PTX-5+DETD-35-2 in the figure) had a 71% reduction in lung tumor colonies (P< 0.05, Figure 4A). Compared to the other groups, the mice in the DETD-35-2 treated group had a slight decrease in body weight, but there was no statistical difference in the data (data not shown). In addition, there was no significant difference in organ index of lung, liver, kidney and spleen among all the test animals (Fig. 4B).

Example 7 DETD-35 and DETD-39 can enhance the anti-proliferative effect of PTX against MDA-MB-231 cells, respectively.

The effects of DETD-35 and DETD-39 on the synergistic effect of synergistic PTX against MDA-MB-231 cell activity were investigated, and the IC ₆₀ values of each compound were determined by MTT assay. The results showed that the IC ₆₀ values of PTX, DETD-35 and DETD-39 for MDA-MB-231 were approximately 23.8 nM, 3.32 μM and 3.79 μM, respectively (Fig. 5A). Furthermore, 1 μM DETD-35 or DETD-39 was administered as compared to the PTX group alone, and both PTX and DET derivative-treated cells were co-treated (PTX concentrations: 0, 2, 5, 10, 15, 20 and 25 nM) have better effects of inhibiting cancer cell proliferation (Fig. 5B). Based on the IC ₆₀ value of each compound, DETD-35 (0-4 μM) or DETD-39 (0-4 μM) and PTX (0-25 μM) were used for the compound-drug combination study. Synergistic effects of compounds and drugs were studied using the Classical isobologram analysis and the Chou-Talalay method. The coefficient (CI) was calculated using CalcuSyn software (Version 2.0, Biosoft) and expressed as CI vs. Fa (fraction affected). CI < 1 represents synergy (synergy); CI = 1 represents additive effect; CI > 1 represents antagonistic effect (antagonism). Based on the experimental results, it was found that PTX and DETD-35 or DETD-39 were used in combination with the effect of synergistically inhibiting the growth of MDA-MB-231 cells (Fig. 5C and 5D).

Example 8 DET and DETD-35 enhance the anti-proliferative effect of glutathione synthesis blockers against MDA-MB-231 cells, respectively.

DET can induce transient ROS production, which is an upstream factor that triggers a series of reactions leading to cancer cell death (Lee and Shyur, Free Radical Biology & Medicine 2012, 52(8): 1423-1436.). The pharmacological properties of DET make DET and its derivatives a potential agent as an emerging "redox-directed cancer therapeutics" (Wondrak GT, Antioxidants & Redox Signaling 2009, 11(12): 3013 -3069.). r - amine bran acyl homocysteine synthase inhibitor (Gamma-glutamylcysteine synthetase inhibitor): "buthionine sulfur (PEI) (BSO)" and cystine / glutamate transporters (cystine / glutamate transporter) (xCT) Inhibitor: The function of "sulfasalazine" is to weaken the defense mechanism of glutathione antioxidants. The present invention selects these two drugs to study the effects of the combined use of drugs. The synergistic effect between DET or its derivative (DETD-35) and BSO or sulfasalazine was tested. If the combination of the above compounds and drugs has a synergistic effect, that is, the combination of the compound and the drug has great potential for prevention or treatment, and is resistant to DET/DETD-35 and the second phase drug BSO or the clinical drug sulfasalazine, Or increase its sensitivity to DET/DETD-35 and Phase II drug BSO or clinical drug sulfasalazine.

In this example, the experimental results show that BSO or sulfasalazine is not cytotoxic to MDA-MB-231 cells in a concentration range, but DET and DETD-35 can increase cancer cells for BSO or sulfasalazine. Drug susceptibility. MDA-MB-231 cells treated with BSO or sulfasalazine for 30 minutes were treated with DET (12 μM) and DETD-35 (3 μM) for 24 hours, and the cell viability was reduced by about 70%, 90% to 1% or Unable to detect (Figures 6A, 6B). It is worth noting that both DET and DETD-35 synergistically enhance the anti-cancer proliferation effect of BSO when BSO is applied at concentrations of 0.5 μM and 3 μM, respectively. Similar results were also observed when DET or DETD-35 was combined with sulfasalazine, and the concentration of the sulfasalazine was 25 μM and 62 μM (Fig. 6B). In summary, when DET and DETD-35 are combined with the clinical drug sulfasalazine or the phase II clinical trial drug BSO, The effect of cancer cells on compound drugs is sensitized to inhibit the activity of triple-negative breast cancer cells.

Example 9 The inhibitory effect of DET and DETDs of Example 1.2.2 against melanoma cell proliferation

Several genes are known to be involved in the development and pathology of melanoma; specifically, in malignant melanoma, V-raf murine sarcoma viral oncogene homolog B1, B- The incidence of mutations in RAF is about 50-60%, and the incidence of neuroblastoma RAS viral oncogene homolog (N-RAS) is about 30% (Yeh et al., Oncogene 2006, 25). (50): 6574-6581.). This example uses wild-type or mutant melanoma cell lines, including B16-F10, MeWo, A375, A2058, HTB-68, and melanocytes to study the original plant compound (parental phytocompound). The effect of DET and newly synthesized DETDs on the proliferation of melanoma cells. Table 4 shows the IC ₅₀ values of DET and DETDs, which were analyzed by MTT reagent for the anti-melanoma cells (treated 24 hours). The results showed that the susceptibility of each cell to DET and DETDs was different between different cells. For DET B16-F10 and A375 cells IC ₅₀ value of about 6μM, but other melanoma cell line is not active (administration of the test to a concentration of about 10μM). Among the other test results of about 60 DETDs (DETD-1 to DETD-62), of which DETD-6, -28, -30, -33, -34, -35, -39, -45, -53-54 , -55, -56, -58, -60 and -61 all inhibit melanoma cell types, an IC ₅₀ value of about 1.6 to 9.9μM. DETD-35 (IC ₅₀ =2.5 to 6.0 μM) and DETD-39 (IC ₅₀ =1.6 to 3.5 μM) are most effective against melanoma cell proliferation, but DETD-39 is partially toxic to normal human melanocytes, IC ₅₀ was 9 μM (Table 4).

Example 10 Synergistic effect of DETD-35 and PLX4032 against A375 melanoma cells

To investigate whether DETD-35 and PLX4032 have synergistic effects on A375 melanoma cells; first, in this example, anti-proliferation analysis of each compound was carried out using MTT reagent to obtain IC ₅₀ values of each compound. The results indicate PLX4032 DETD-35 and IC A375 cells for ₅₀ values are about 0.07 and 0.85 m (of FIG. 7A and 7B). The compound-drug combination test was carried out based on the IC ₅₀ value of each compound in DETD-35 (concentration range 0.1 to 1 μM) and PLX4032 (concentration range 10 to 100 nM). CI analysis was performed using CalcuSyn software (Version 2.0, Biosoft) to quantify the synergistic effect of the compound-drug, expressed as CI vs. Fa (partially affected). CI < 1 indicates a synergistic effect; CI = 1 indicates an additive effect; and CI > 1 indicates an antagonistic effect. The experimental results indicate that the combination of DETD-35 and PLX4032 has a synergistic effect on inhibiting the growth of A375 melanoma cells (Figs. 7C and 7D).

The inventors further implanted A375 human melanoma cells in NOD/SCID mice in the manner described in the aforementioned "Materials and Methods", and then administered PLX4032, DET, DETD-35, PLX4032+DET to the mice at the indicated time points, respectively. Or PLX4032+DETD-35, according to the evaluation of the effect of inhibiting A375 melanoma in vivo using PLX4032, DET, DETD-35, PLX4032+DET or PLX4032+ DETD-35 alone, the results are shown in Figures 8A and 8B.

Figure 8A shows the change in tumor volume over time, and Figure 8B shows the effect of each treatment on tumor weight. The results showed that both DET and DETD-35 inhibited tumor growth, and the melanoma weight was reduced by about 47.5% and 70.5%, respectively, compared with the vector control group, especially DETD-35, which inhibited tumors in a manner comparable to PLX40332. (about 71.9%). In addition, when PLX4032 is used in combination with DETD-35 (as described in Materials and Methods, in the combined test, the number of applications of PLX4032 and DETD-35 is only the number of times when PLX4032 or DETD-35 is used alone. Half), its tumor suppressing effect is not far from PLX40332 or DETD-35 alone (about 72.3%), indicating that PLX4032 and DETD-35 also have synergistic effects in inhibiting melanoma in mice.

Example 11 DETD-35 overcomes the resistance of A375 human melanoma cells to PLX4032

In order to explore how DETD-35 overcomes the drug resistance mechanism of BRAF inhibitors, A375 cells (A375-R) resistant to PLX4032 were used as test subjects. Briefly, A375 melanoma was inoculated into T75 dish, followed by PLX4032 (20, 50, 100, 250, 500, 1000, 2000 nM), and 20 subcultures were developed for about 2 months during the experiment. A375-R acquired drug resistant cell line. The A375-R cell strain was less sensitive to PLX4032, and its drug administration concentration was 100 times that of the original cell strain (IC ₅₀ : 8.3 vs. 0.07 μM); whereas the cell survival rate of DETD-35 for A375 and A375-R was With a consistent inhibitory effect, DETD-35 had an IC ₅₀ value of 0.8 μM for both A375 and A375-R cells (Fig. 9A). Furthermore, PLX4032 inhibits MAPK phosphorylation-MEK (phospho-MEK) and phosphorylation-ERK (phospho-ERK) signaling molecules in A375 cells, but does not inhibit the effects of these signaling molecules on A375-R cells (Article 9B). Figure). Based on the results of the MAP kinase reaction in A375-R cells treated with PLX4032, it was further confirmed that the cell line did develop resistance to PLX4032. Western blot analysis showed that DETD-35 has an effect of inhibiting the MAP kinase response in A375-R cells (Fig. 9C). Furthermore, when DETD-35 and PLX40332 are used in combination, the MAPK signal molecule can also be inhibited. Therefore, DETD-35 can overcome the resistance of cancer cells to PLX4032.

The inventors further tested the results of the above-mentioned DETD-35 inhibition of PLX4032-resistant cancer cells (i.e., A375-R cells) using a live animal test. Similarly, according to the "Materials and Methods" section and the method described in Example 10, different treatments for drug resistance to PLX4032 were implanted into NOD/SCID mice, and PLX4032 was administered to mice at the indicated time points. DET, DET-35, PLX4032+DET, or PLX4032+DET-35, the results are shown in Figures 10A and 10B.

Figure 10A shows the change in tumor volume over time after different treatments, and Figure 10B shows the effect of different treatments on tumor weight. As expected, the experimental mice did not respond to PLX4032 (75 mg/kg) treatment because A375-R cells were resistant to PLX4032. Tumor volume and weight are also not far from the control group. In contrast, both DET and DET-35 significantly inhibited the growth of A375-R tumor cells, which were reduced by approximately 32% and 46.9%, respectively, compared to the vehicle-controlled group. More specifically, the combined application of PLX4032 and DET or PLX4032 and DET-35 is much better than DET or DET-35 alone. After combining PLX4032 and DET-35, the tumor weight was reduced by about 65.3%, which was the best among all groups, indicating the synergistic effect of DET-35 and PLX4032. Our results show that DET and DETD-35 alone or in combination with PLX4032 can overcome the resistance of melanoma to PLX4032.

Example 12 DET-35 delays the production and number of papilloma induced by DMBA/TPA treatment in vivo by PLX4032

This example employs a conventional animal model of DMBA/TPA-induced skin cancer to simulate the side effects of skin tissue administration of PLX4032 to assess whether administration of DETD-35 is effective in improving the skin of patients due to administration of PLX4032. side effect. First, treat the abdominal skin of mice with DMBA according to the method described in “Materials and Methods”. After one week, the same site should be given TPA, TPA+PLX4032, TPA+DET or TPA+DETD-35 twice a week. The results are shown in Figures 11A-11C.

As can be seen from the results of Figures 11A and 11B, DMBA+TPA treatment induces papilloma in mice, and PLX4032 promotes this. The phenomenon of papilloma formation induced by DMBA+TPA can be confirmed by an increase in the number of papillomas. However, DET or DETD-35 can effectively alleviate the phenomenon of papilloma-promoting induced by PLX4032. The number of papilloma on the skin can be reduced from 25.6 in the control group to 11,7 and 4.8, respectively. The tumor size is also controlled by the control group. 77.6 cubic millimeters were reduced to 26.3 and 8.9 cubic millimeters respectively (Fig. 11A-11C). Furthermore, neither DET nor DETD-35 affected the body weight of the experimental animals (results not shown).

In summary, it can be seen that DET and DETD-35 can effectively reduce the side effects of PLX4032 on the skin, and the effect of DETD-35 is most significant.

Example 13 The relationship between the structural activity of DETDs of Example 1.2.2 and the proliferation of various cancer cell lines

Based on the toxicity results of DET and DETDs of Example 1.2.2 for different breast cancer and melanoma cell lines, the present invention selects DETD-6, DETD-32, DETD-35 and DETD-39 for further experiments and is structurally active. The inhibitory effects of these compounds against other cancer cell lines were evaluated under the consideration of the correlation, and the results are shown in Table 5. In addition to effectively inhibiting the growth of breast cancer and melanoma cells, the four DETDs compounds can also effectively inhibit brain cancer, lung cancer, lymphoma, neuroepithelial neoplasia, renal cancer, prostate cancer, gastric cancer, colon cancer, and uterine cancer cell proliferation. In conclusion, DETD-35 has the most significant inhibitory effect in different cancer cell types, and for normal cell lines M10, melanocytes and giants. The phagocytes were non-cytotoxic (Tables 3 and 4). Therefore, DETD-35 has the potential to develop into an anticancer drug. DETD-6 and -39 also have novel activities for different types of cancer, but in vitro, the two drugs are partially toxic to normal cells.

Example 14 Inhibition of nitric oxide (NO) by LPS-stimulated macrophages

Lipopolysaccharide (LPS) is located in the outer membrane of Gram-negative bacteria and promotes the expression of inducible nitric oxide synthase (iNOS). The nitric oxide synthase can catalyze the oxidative deamination of L-arginine to produce nitric oxide (NO). When a large amount of NO is produced, it interacts with superoxide anion to produce highly reactive oxides, causing cellular inflammation, damage to DNA, proteins and tissues, carcinogenesis and anti-apoptosis. In this example, the present invention utilizes in vitro LPS to stimulate the RAW264.7 cell system to produce an inflammatory response, assessing the effect of DET and its derivative DETDs on NO production. After the test cells were treated with LPS for 24 hours, the concentration of nitrite in the medium (corresponding to the concentration of NO) was measured. The compound is a known plant DET NO inhibitor, IC ₅₀ values of 2.9μM. Furthermore, most DETDs have the effect of inhibiting LPS-induced NO production in macrophages, with the best inhibition by DETD-35 and DETD-39, and the IC ₅₀ value is reduced by 2-3 fold (1.5 and 1.0 μM). DETD-39 also shows that for a normal mouse macrophage RAW 264.7 cells are toxic, IC ₅₀ of 3.5 m (Table 4).

Although the embodiments of the present invention are disclosed in the above embodiments, the present invention is not intended to limit the invention, and the present invention may be practiced without departing from the spirit and scope of the invention. Various changes and modifications may be made thereto, and the scope of the invention is defined by the scope of the appended claims.

Claims (31)

A compound of formula (I): Wherein: R is H or -(C=O)R ₁ ; and R ₁ is H, C _1-20 alkyl, C _2-20 alkenyl, C _6-10 aryl or C _6-10 heteroaryl, respectively. Wherein the C _1-20 alkyl group may be unsubstituted or have at least one substituent selected from the group consisting of halogen, hydroxy, C _1-20 alkoxy, C _1-20 alkoxy, phenyl, naphthyl , isobenzofuranyl, isophenylthienyl or isoquinolyl; the C _2-20 alkenyl group may be unsubstituted or have at least one substituent selected from the group consisting of phenyl, furyl, thienyl or isophthalene And dioxolanyl; each phenyl and naphthyl group may be unsubstituted or have at least one substituent selected from the group consisting of halogen, C _1-20 alkyl, perfluorinated C _1-20 alkyl, C _{1 -20} alkoxy or halogenated C _1-20 alkoxy; and the aryl group may be unsubstituted or carry halogen, C _1-4 alkyl, C _1-20 alkoxy or halogenated C _1-20 alkane Oxy substituent. The compound of claim 1, wherein the C _6-10 heteroaryl group is a thienyl group, an isobenzofuranyl group or an isophenylthiophenyl group. The compound of claim 1, wherein the C _6-10 aryl group is phenyl or naphthyl. The compound of claim 3, wherein the phenyl group is unsubstituted or carries a halogen, C _1-20 alkyl or C _1-20 alkoxy substituent. The compound of claim 1, wherein the C _1-20 alkyl group is a cyclopropyl group, a cyclopentyl group or a cyclohexyl group. The compound of claim 1, wherein R ₁ is the C _2-20 alkenyl group. The compound of claim 6 wherein R ₁ is a propylene group. The compound of claim 7, wherein the propylene group further has a phenyl substituent. The compound of claim 8 wherein the phenyl group further has an -OCH ₃ substituent. The compound of claim 1, wherein the R ₁ is a monomethyl group. The compound of claim 10, wherein the methyl group further has a naphthyl substituent. A pharmaceutical composition for treating an individual having/suspiciously having an inflammation and/or a tumor-related disease, comprising: a therapeutically effective amount of a compound of formula (I) as described in claim 1; and a pharmaceutically acceptable Accepted carrier. The pharmaceutical composition according to claim 12, wherein in the compound of the formula (I), the C _6-10 heteroaryl group is a thienyl group, an isobenzofuranyl group or an isophenylthiophenyl group. The pharmaceutical composition according to claim 12, wherein in the compound of the formula (I), the aryl group is a phenyl group or a naphthyl group. The pharmaceutical composition of claim 14, wherein the phenyl group is unsubstituted or carries a halogen, C _1-20 alkyl or C _1-20 alkoxy substituent. The pharmaceutical composition according to claim 12, wherein in the compound of the formula (I), the C _1-20 alkyl group is a cyclopropyl group, a cyclopentyl group or a cyclohexyl group. The pharmaceutical composition according to claim 12, wherein in the compound of the formula (I), R ₁ is a C _2-20 alkenyl group. The pharmaceutical composition according to claim 17, wherein the R ₁ is a propylene group. The pharmaceutical composition of claim 18, wherein the propylene group further has a phenyl substituent. The pharmaceutical composition of claim 19, wherein the phenyl group further has an -OCH ₃ substituent. The pharmaceutical composition according to claim 12, wherein in the compound of the formula (I), R ₁ is the C _1-20 alkyl group. The pharmaceutical composition according to claim 21, wherein the R ₁ is a monomethyl group and may be unsubstituted or have a naphthyl substituent. The pharmaceutical composition according to claim 12, further comprising a chemotherapeutic agent or a glutathione biosynthesis blocker. The pharmaceutical composition according to claim 23, wherein the chemotherapeutic agent is selected from the group consisting of Deoxyelephantopin (DET), PLX4032, docetaxel, paclitaxel, cisplatin, oxaliplatin, betulinic acid, 4 -S-cysteamine naphthol, 4-S-cysteamine, everolimus, bortezomib, carboplatin, dacarbazine, celecoxib, temozolomide, leisawa, salidome , lenalidomide, valproic acid, vinblastine, imatinib mesylate, bosentan, apolamine, arsenic trioxide, carmustine, laboplatin Lizma, anti-CTLA-4 drug, anti-programmed death receptor-1 (PD-1) drug, ipilimumma, Cui Mim Limma, erythromycin, MEK inhibitor, capecitabine, poly ( ADP-ribose) polymerase (PARP) inhibitor, phosphoinositide 3-kinase (PI3K) inhibitor, mammalian target of rapamycin (mTOR) inhibitor and tamoxifen. The pharmaceutical composition according to claim 24, wherein the glutathione biosynthesis blocker is butyl thiopurine imine (BSO) or sulfasalazine. The pharmaceutical composition according to claim 12, wherein the tumor-related disease is a cancer selected from the group consisting of breast cancer, papilloma, melanoma, brain cancer, lung cancer, lymphoma, neuroepithelial neoplasia, renal cancer, prostate cancer , in the group consisting of gastric cancer, colorectal cancer and uterine cancer. The pharmaceutical composition according to claim 26, wherein the cancer is a drug-resistant cancer. The pharmaceutical composition according to claim 27, wherein the cancer is a melanoma resistant to PLX4032. The pharmaceutical composition of claim 28 which reduces the side effects caused by PLX4032 on the individual. The pharmaceutical composition according to claim 29, wherein the side effect refers to a papilloma induced by PLX4032 on the skin of the individual. The pharmaceutical composition according to claim 12, wherein the inflammatory related disease is selected from the group consisting of an inflammatory skin disease, an inflammatory bowel disease, an allergic lung disease, asthma, allergic rhinitis, an autoimmune disease, acute and chronic In a group consisting of inflammation, repair syndrome, human immunodeficiency virus infection, and cancer.