WO2018068769A1 - 二氢小檗碱型化合物在制备治疗stat3信号通路相关肿瘤疾病产品中的用途 - Google Patents

二氢小檗碱型化合物在制备治疗stat3信号通路相关肿瘤疾病产品中的用途 Download PDF

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WO2018068769A1
WO2018068769A1 PCT/CN2017/106227 CN2017106227W WO2018068769A1 WO 2018068769 A1 WO2018068769 A1 WO 2018068769A1 CN 2017106227 W CN2017106227 W CN 2017106227W WO 2018068769 A1 WO2018068769 A1 WO 2018068769A1
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compound
compounds
colorectal cancer
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present
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French (fr)
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秦海林
吴练秋
张志辉
张海婧
邓安珺
王文杰
李志宏
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中国医学科学院药物研究所
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Publication of WO2018068769A1 publication Critical patent/WO2018068769A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a series of medicinal chemistry and new pharmacological effects of a dihydroberberine-type compound having the same parent skeleton structure and a ( ⁇ )-8-acetonyldihydroberberine-type compound. Specifically, it relates to dihydroisoxanthine, dihydroclavine, dihydropalmatine, ( ⁇ )-8-acetonyl dihydroisoxanthine, ( ⁇ )-8-acetonyl dihydroclavinine and ( ⁇ )- The use of 8-acetonyl dihydropalmatine as a STAT3 inhibitor in the preparation of products for the prevention, alleviation and/or treatment of STAT3 signaling pathway-associated tumor diseases. It belongs to the field of medical technology.
  • cancers malignant tumors
  • cancers are all serious diseases that seriously endanger human health, causing tremendous physical and mental pain and economic pressure on patients and their families.
  • cancers malignant tumors
  • it is often clinically forced to combine the methods of surgery, radiation therapy and chemotherapy as a first-line clinical treatment to treat patients.
  • the prevention, alleviation and treatment of various human cancers is still a very difficult scientific research work in the field of medical science research. It is a systematic project that requires huge investment in capital and manpower.
  • Berberine-type compounds are generally referred to as the following three different types of compounds in the field of natural organic chemistry: (1) having 6,8-dihydro-5H-isoquino[3,2-a]isoquinoline ( 6,8-dihydro-5H-isoquinolino[3,2-a]isoquinoline) Basic structure of 7,8-imine salt type structure of berberine quaternary ammonium salt type compound (I), (2) having 6,8 - Dihydro-5H-isoquino[3,2-a]isoquinoline basic parent structure of dihydroberberine-type compounds (II) and (3) having 6,8,13,13a-tetrahydro- 5H-isoquinoline[3,2-a]isoquinoline (6,8,13,13a-tetrahydro-5H-isoquinolino[3,2-a]isoquinoline) basic parent structure of tetrahydroberberine ( The protoberberine-type compound and its various salts (III), the chemical formula of the three parent forms are shown in
  • the berberine quaternary ammonium salt type compound (I) and the dihydroberberine type compound (II) have a plurality of different resonance forms as shown in Formulas 2 and 3, respectively, but due to the difference in structure, The resonance structure and number of the two are not the same; their structural features and basic carbon framework structure with 6,8,13,13a-tetrahydro-5H-isoquino[3,2-a]isoquinoline type There is also a significant difference between the benzylisoquinoline type compound (i.e., the protoberberine type or the tetrahydroberberine type compound) and its various salts (III), that is, in the III, in addition to the asymmetric center of 13a.
  • the benzylisoquinoline type compound i.e., the protoberberine type or the tetrahydroberberine type compound
  • its various salts (III) that is, in the III, in addition to the asymmetric center of 13a.
  • the berberine quaternary ammonium salt type compound and the dihydroberberine type compound belong to different structural type compounds.
  • some berberine quaternary ammonium salt type compounds and tetrahydroberberine type compounds have relatively abundant natural resources, according to current research reports, the structure of the dihydroberberine type compound has only a specific environment. Stability (in the above three types of berberine-type compounds, the structure of the dihydro-type berberine compound is the most unique), so the existence in nature is greatly limited, or the raw material obtained from the scale of nature has Quite difficult.
  • the dihydroberberine type compound and the ( ⁇ )-8-acetonyldihydroberberine type compound are all synthesized by an organic chemical method.
  • berberine and palmatine have certain anti-colon cancer activity.
  • berberine can inhibit colon cancer cells by inactivating the Wnt/ ⁇ -catenin signaling pathway. Proliferation (see literature: Wu K, et. al.
  • Berberine inhibits the proliferation of colon cancer cells by inactivating Wnt/ ⁇ -catenin signaling, International Journal of Oncology, 2012, 41, 292-298), and has the effect of inhibiting human colon cancer cells
  • Pharmacological activity of growth in nude mice see patent document: CN1313093C
  • palmatine chloride is a photosensitizer and is phototoxic to colonic adenocarcinoma cell line HT-29 (see literature: Wu J, et al. Photodynamic action) Of palmatine hydrochloride on colon adenocarcinoma HT-29cells, Photodiagnosis and Photodynamic Therapy, 2016, 15, 53-58); we also evaluated the activity of copperine quaternary ammonium salt against colon cancer cell lines in vitro.
  • dihydroberberine-type compounds are known to be limited to the specific compound dihydroberberine showing synergistic anti-tumor effects in pharmaceutical compositions consisting of sunitinib, including colon, lung and liver cancer. Role (see patent document: CN 105434435 A), and so on.
  • the berberine-type compounds have structural diversity; from the perspective of the relationship between organic structure and properties, the three types of berberine-type compounds are not only structurally inconsistent, but also have significant physical and chemical properties.
  • the berberine quaternary ammonium salt type compound not only causes a poor solubility in various solvents due to the unique presence of a passivating functional group in the 7,8-imine salt type aromatic electrophilic substitution reaction, but also a conjugated system.
  • the isoproterenium quaternary ammonium salt, the berberine quaternary ammonium salt, the palmatine quaternary ammonium salt and the berberine quaternary ammonium salt in the berberine quaternary ammonium salt type compound are transformed into a series of fat-soluble dihydrogen by functional group change.
  • Berberine-type compounds including dihydroisoxanthine (1), dihydroclavidine (2), dihydropalmatine (3) and dihydroberberine, for solubility, bioavailability, activity and toxicity Evaluation, the results showed that these dihydroberberine-type compounds were significantly improved in solubility in various organic solvents such as dioxane, acetone or tetrahydrofuran, compared with the berberine quaternary ammonium salt type compound.
  • both compounds 1 and 2 are more than 5g / Kg, is a non-toxic compound;
  • Compound 3 has an LD 50 value of 800mg / Kg, is a low toxicity compound;
  • the LD 50 value of the tumor-acting dihydroberberine is only 160 mg/Kg, which is the most toxic compound in this series of compounds. This was confirmed in our animal experiments, that is, dihydroberberine was used in 50 mg. /Kg dose after experimental administration of experimental animals The phenomenon of rapid death, and 1, 2 and 3 did not appear the death of experimental animals, even 1 and 2 did not appear in the death of experimental animals when the dose was large.
  • the result of the inhibitory activity does not indicate its anticancer activity.
  • Compound 2 does not exhibit cytostatic activity against HCT cell lines, and the IC 50 value is greater than 5 ⁇ g/mL (data are described in Zhi-Hui Zhang, et al. Syntheses and structure – Activity relationships in cytotoxicities of 13-substituted quaternary coptisine derivatives, European Journal of Medicinal Chemistry, 2014, 86, 542-549; IC 50 values greater than 5 ⁇ g/mL, ie greater than 15.56 ⁇ M, considered to be inactive compounds; The results of the study showing anti-tumor activity gave us the opposite technical inspiration and led us to abandon the idea of this type of compound research.
  • the present invention synthesizes a ( ⁇ )-8-acetonyl substituted derivative of a dihydroberberine-type compound, including ( ⁇ )-8-acetonyldihydroisoxanthine, starting from a quaternary ammonium type berberine-type compound.
  • ( ⁇ )-8-acetonyl dihydroclavinine (5), ( ⁇ )-8-acetonyl dihydropalmatine (6) and ( ⁇ )-8-acetonyl dihydroberberine
  • the toxicity and antitumor activity were evaluated.
  • the results of the compound 5 showing no obvious cytotoxicity to IEC-6 intestinal epithelial cells also indicate that it is a low toxicity compound (the IEC-6 cytotoxicity experimental data of Compound 5 is shown in the literature: Zhi-Hui Zhang, et al., Synthesis and Structure-activity Relationships of Quaternary Coptisine Derivatives as Potential Anti-ulcerative Colitis Agents.Journal of Medicinal Chemistry.2015,58,7557-7571;Zhi-Hui Zhang,et al.Syntheses and structure-activity relationships in cytotoxicities of13-substituted Quaternary coptisine derivatives, European Journal of Medicinal Chemistry, 2014, 86, 542-549).
  • compounds 1, 2, 3 and 5 are used as examples to treat Azoxymethane (AOM)/Dextran sulfate sodium.
  • DSS Azoxymethane
  • STAT3 signal transduction and activator of transcription 3
  • STAT3 is closely related to the occurrence and development of various tumors.
  • STAT3 is over-activated and expressed at high levels in cells of various tumors.
  • Inhibition of overexpression of STAT3 is one of the means of treating tumors.
  • the results of the present study indicate that Compounds 1, 2, 3 and 5 all have significant STAT3 inhibition, further illustrating that the series of compounds of the present invention can prevent or treat STAT3 signaling pathway-associated tumor diseases by inhibiting STAT3.
  • the technical problem solved by the present invention is to provide a class of dihydroberberine-type compounds and ( ⁇ )-8-acetonyldihydroberberine-type compounds in the preparation of STAT3 inhibitors and preparation of products for preventing or treating STAT3 signaling pathway-related tumor diseases.
  • the application further, provides its use in the preparation of a product for the prevention or treatment of malignant colorectal cancer.
  • the present invention provides the following technical solutions:
  • a first aspect of the technical solution of the present invention provides a use of a dihydroberberine-type compound and a ( ⁇ )-8-acetonyldihydroberberine-type compound in the preparation of a STAT3 inhibitor, and a preparation thereof for prevention or For use in the treatment of STAT3 signaling pathway-associated tumor disease products
  • the dihydroberberine-type compounds include dihydroisoxantine, dihydroclavine, and dihydropalmatine, ( ⁇ )-8-acetonyl dihydrogen
  • the berberine-type compound includes ( ⁇ )-8-acetonyldihydroisoxanthine, ( ⁇ )-8-acetonyl dihydroclavinate, and ( ⁇ )-8-acetonyl dihydropalmatine.
  • Specific compounds are dihydroisoxanthine, dihydroclavonic base, dihydropalmatine, ( ⁇ )-8-acetonyldihydroisoxanthine, ( ⁇ )-8-acetone, as shown in Formulas 1-6, respectively.
  • the products include medicines and health care products.
  • the STAT3 signaling pathway-associated tumor disease is selected from the group consisting of colorectal cancer, and various types of malignant tumors, including colorectal cancer associated with ulcerative colitis and other types of colorectal cancer.
  • the colorectal cancer includes colon cancer and rectal cancer.
  • a second aspect of the present invention provides a dihydroisoxanthine, dihydropyrazine, dihydropalmatine, ( ⁇ )-8-acetonyldihydroisoxanthine as shown in Formula 1-6. , ( ⁇ )-8-acetonyl dihydroclavinine and Use of a pharmaceutical composition of a specific compound of one or more of ( ⁇ )-8-acetonyl dihydropalmatine for the preparation of a product for preventing or treating a STAT3 signaling pathway-associated tumor disease.
  • the STAT3 signaling pathway-associated tumor disease is selected from the group consisting of colorectal cancer, and various types of malignant tumors, and the products include medicines and health care products.
  • the colorectal cancer includes ulcerative colitis-associated colorectal cancer and other types of colorectal cancer.
  • the colorectal cancer includes colon cancer and rectal cancer.
  • the pharmaceutical composition can be prepared according to methods well known in the art. Any dosage form suitable for human or animal use may be prepared by combining a compound of the invention with one or more pharmaceutically acceptable solid or liquid excipients and/or adjuvants.
  • the preferred dosage form is ordinary or enteric. solid preparations.
  • the content of the compound of the present invention in its pharmaceutical composition is usually from 0.1 to 99.9% (w/w).
  • the compound of the present invention or a pharmaceutical composition containing the compound of the present invention can be administered in a unit dosage form, which can be enterally or parenterally, such as orally.
  • a unit dosage form which can be enterally or parenterally, such as orally.
  • intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eye, lung and respiratory, skin, vaginal, rectal administration, and the like can also be administered.
  • the dosage form can be a liquid dosage form, a solid dosage form or a semi-solid dosage form.
  • Liquid dosage forms can be solutions (including true and colloidal solutions), emulsions (including O/W type, W/O type and double emulsion), suspensions, injections (including water injections, powder injections and infusions), eye drops Agents, nasal drops, lotions, tinctures, etc.; solid dosage forms may be tablets (including ordinary tablets, enteric tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules ( Including hard capsules, soft capsules, enteric capsules), granules, powders, pellets, dropping pills, suppositories, films, patches, gas (powder) sprays, sprays, etc.; semi-solid dosage forms can be ointments, Gel, paste, etc. Preferred dosage forms of the dihydroberberine-type compound and the ( ⁇ )-8-acetonyldihydrober
  • the compounds of the present invention can be formulated into common preparations, sustained release preparations, controlled release preparations, targeted preparations, and various microparticle delivery systems.
  • the diluent may be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.;
  • the wetting agent may be water, ethanol, Isopropanol, etc.;
  • the binder may be starch syrup, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic, gelatin syrup, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl Methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; disintegrant can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linking Polyvinylpyrrolidone, croscarmellose sodium, sodium carboxymethyl starch, sodium hydrogencarbonate, polyoxyethylene
  • Tablets may also be further formed into coated tablets, such as sugar coated tablets, film coated tablets, enteric coated tablets, or bilayer tablets and multilayer tablets, preferably coated tablets are common or enteric coated sheet.
  • the active ingredient (the compound of the present invention) may be mixed with a diluent, a glidant, and the mixture may be directly placed in a hard capsule or a soft capsule.
  • the active ingredient (the compound of the present invention) may also be granulated or pelletized with a diluent, a binder or a disintegrating agent, and then placed in a hard capsule or a soft capsule.
  • the various diluents, binders, wetting agents, disintegrants, glidants of the formulations used to prepare the tablets of the present invention are also useful in the preparation of capsules of the compounds of the invention.
  • water, ethanol, isopropanol, propylene glycol or a mixture thereof may be used as a solvent, and an appropriate amount of a solubilizer, a solubilizer, a pH adjuster, an osmotic pressure adjusting agent which is commonly used in the art may be added and appropriately added.
  • a solubilizer e.g., a solubilizer, a solubilizer, a pH adjuster, an osmotic pressure adjusting agent which is commonly used in the art may be added and appropriately added.
  • the solubilizing agent or co-solvent may be poloxamer, lecithin, hydroxypropyl- ⁇ -cyclodextrin, etc.;
  • the pH adjusting agent may be sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, etc.;
  • the stabilizer may be Sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, etc.;
  • the osmotic pressure adjusting agent may be sodium chloride, mannitol, glucose, phosphate, acetate or the like.
  • mannitol, glucose or the like may also be added as a proppant.
  • coloring agents may also be added to the pharmaceutical preparations as needed.
  • the therapeutic effect can be enhanced, and the pharmaceutical or pharmaceutical composition of the present invention can be administered by any known administration method, and the preferred administration method is oral administration of a common or enteric preparation.
  • the pharmaceutical composition of the present invention can be administered in a wide range of dosages depending on the nature and severity of the colorectal cancer to be prevented or treated, the individual condition of the patient or animal, the route of administration and the dosage form, and the like.
  • a suitable daily dose of a compound of the invention will range from 0.001 to 150 mg/kg body weight, preferably 0.1-100 mg/Kg body weight, more preferably 1-60 mg/Kg body weight, and most preferably 2-30 mg/Kg body weight.
  • the above dosages may be administered in one dosage unit or in divided dose units depending on the clinical experience of the physician and the dosage regimen including the use of other therapeutic means.
  • the compounds or pharmaceutical compositions of this invention may be administered alone or in combination with other therapeutic or symptomatic agents.
  • the compound of the present invention synergizes with other therapeutic agents, its dosage should be adjusted according to the actual situation.
  • the present invention uses a PEG as a dispersing agent to prepare a dihydroberberine-type compound and a ( ⁇ )-8-acetonyldihydroberberine-type compound dispersant entity.
  • Oral administration or oral administration in the form of dispersion of CMC-Na/Tween-80 aqueous solution lays a foundation for subsequent formulation studies; the compound of the present invention can achieve therapeutic effects for colorectal cancer with remarkable effects.
  • the calculated tumor burden of coptisine (1), dihydroclavidine (2), dihydropalmatine (3) and ( ⁇ )-8-acetonyl dihydroisoxaine (4) at a dose of 50 mg /Kg reached 0.06, 0.13, 0.01, 0.08, respectively, even close to the value of 0.00 in the normal control group; compared with the 1.25 of the tumor control load of the positive control drug, the effect was very significant.
  • the calculated tumor burden of ( ⁇ )-8-acetonyl dihydroclavinine (5) and ( ⁇ )-8-acetonyl dihydropalmatine (6) reached 2.75 at a dose of 50 mg/kg, respectively. 0.40, compared with the same batch of positive control drug tumor load calculated value of 8.33, the effect is very significant.
  • the compound of the present invention has the advantages of no toxicity or low toxicity, and the LD 50 values of dihydroisoxantine and dihydroclavidine are all above 5.0 g/Kg, and dihydropalmatine
  • the LD 50 value is 0.8 g/Kg, ( ⁇ )-8-acetonyl dihydroisoxanthine, ( ⁇ )-8-acetonyl dihydroclavinine and ( ⁇ )-8-acetonyl dihydropalmatine
  • the LD 50 values were 5.0 g/Kg, 3.9 g/Kg, and 1.6 g/Kg, respectively, and were non-toxic or low-toxic specific anti-tumor compounds.
  • the compound of the present invention is STAT3 inhibitor; by pharmacodynamic experiments, it has been confirmed that the compound of the present invention has significant anti-colorectal cancer activity in vivo, and the activity is significantly higher than that of the positive control drug; the compound of the present invention is Non-toxic or low toxicity specific anti-tumor compounds, in prevention, slow It is a highly medicinal compound for the prevention, alleviation and/or treatment of colorectal cancer diseases by and/or treatment of STAT3 signaling pathway-associated tumor diseases.
  • FIG. 4 Histopathological examination of colorectal tumors of colorectal cancer model mice by compound 1-4 of the present invention (HE staining, 100-fold).
  • A is a normal control group
  • B is a colorectal cancer model group
  • C is a positive drug capecitabine administration group
  • D is a compound 1 administration group
  • E is a compound 2 administration group
  • F is a compound 3 administration group.
  • G is a compound 4 administration group.
  • FIG. 8 Histopathological examination of colorectal tumors of colorectal cancer model mice by compound 5 and 6 of the present invention (HE staining, 100-fold).
  • A is a normal control group
  • B is a colorectal cancer model group
  • C is a positive drug capecitabine administration group
  • D is a compound 5 administration group
  • E is a compound 6 administration group.
  • the compounds 1, 2, 3 and 4 of the present invention were each prepared as a PEG dispersing agent as a form of administration. When administered, The compound of the present invention is suspended by intragastric administration using water as a solvent.
  • C57BL/6J mice female were subjected to adaptive normal feeding for one week in a clean animal room, and then a single dose of AOM was administered to the experimental animals at a dose of 10 mg/Kg as the first day of the experiment, followed by normal feeding for 6 days. As the first week of the experiment.
  • the experimental animals were allowed to drink 2% of the DSS aqueous solution freely. After 7 days of induction, the normal drinking water was allowed to continue to allow the experimental animals to drink freely for two weeks (third week and fourth week) as the first induction.
  • the experimental animals were allowed to drink 2% of the DSS aqueous solution freely. After 7 days of induction, the normal drinking water was allowed to continue to allow the experimental animals to drink freely for two weeks (sixth and seventh weeks) as the second induction.
  • the experimental animals were allowed to drink 2% of the DSS aqueous solution freely. After 7 days of induction, the normal drinking water was allowed to continue to allow the experimental animals to drink freely for one week (ninth week) as the third induction.
  • each compound of the present invention was administered to each group of animals at a dose of 50 mg/kg according to the experimental design, once a day, once a day, the positive drug capecitabine was administered at a dose of 500 mg/Kg per day. once.
  • each group of animals was treated as usual, and the percentage change of body weight, spleen weight, colon length, colon tumor burden calculation, colon tumor histopathology, etc. after treatment were observed. Changes in indicators.
  • the experimental results show that the compounds of the present invention show significant anti-tumor activity in the experiments of treating colorectal cancer in vivo, and the curative effect is significantly better than that of the positive control drugs.
  • Figure 1 and Table 2 show that the model group is compared with the normal control spleen weight value (0.085 g). The spleen weight of the mice was significantly increased (0.131 g) (**, p ⁇ 0.01). The spleen weight of the mice in each administration group was reduced to different degrees compared with the mice in the model group.
  • the positive drug was 0.112 g, the compound 1 was 0.090 g, the compound 2 was 0.100 g, the compound 3 was 0.080 g, and the compound 4 was 0.100.
  • Figure 2 and Table 3 show that the model group mice were compared with the normal control group (6.84 cm). Colon length was significantly reduced (5.52 cm) (**, p ⁇ 0.01).
  • the mice in the compound administration group of the present invention have different degrees of improvement in colonic contracture, and the colon length is increased compared with the model group, and the compound 1 colon
  • the length was 6.35 cm
  • the compound 2 was 6.30 cm
  • the compound 3 was 5.97 cm
  • the compound 4 was 5.90 cm (##, p ⁇ 0.01; #, p ⁇ 0.05).
  • the positive drug capecitabine had a statistically significant difference in the improvement of colonic contracture (5.80 cm) in mice compared with the model group.
  • Figure 3 and Table 4 show the effect of Compound 1-4 of the present invention on colorectal tumor burden in colorectal cancer model mice.
  • Figure 3 and Table 4 show that compared with the normal control group, the tumor load is calculated as a relative value of 0.00, colorectal cancer.
  • the tumor burden of the model group was significantly increased, and the relative calculated value was about 22.64 (**, p ⁇ 0.01).
  • the tumor burden of each group after continuous administration for 8 weeks, the tumor burden of each group showed a very significant improvement, which was manifested by a decrease in the number of tumors in the whole colon range and a decrease in tumor volume (including gland). Tumor and adenocarcinoma).
  • the tumor load was calculated to be 0.06 for compound 1, 0.13 for compound 2, 0.00 for compound 3, 0.08 for compound 4, and 1.25 for the positive drug group.
  • the statistical difference was significant between the drug-administered group and the colorectal cancer model group. (##, p ⁇ 0.01).
  • the compounds of the present invention have a very remarkable effect compared with the positive control drug.
  • the calculation method of tumor load calculation value is as follows:
  • Figure 4 is a histopathological examination (HE staining, 100-fold) of colorectal tumors of colorectal cancer model mice of each compound 1-4 of the present invention.
  • a normal control group normal structure of the colon, showing a clear layer of intestinal tissue, intact structure, no inflammatory changes.
  • Inflammatory lesions of intestinal tissue were significant; inflammation involved the entire mucosal epithelium and submucosal lamina intestinal (transmural inflammation), almost all crypts were destroyed, part of the mucosal epidermis was missing; a large number of inflammatory cells were infiltrated in the inflammatory lesions, and inflammatory cells were Lymphocytes, neutrophils, and a small number of monocytes; inflammatory ulcers can be seen in the area of local inflammation, and inflammatory fibrous tissue hyperplasia can be seen at the bottom of the ulcer, which is a moderately differentiated adenocarcinoma (++++).
  • C positive drug capecitabine group intestinal tissue inflammation lesions, inflammation involving 2 / 3 intestinal mucosa interstitial, local visible transmural inflammatory lesions, local crypt destruction, intestinal mucosa epidermis relatively intact; inflammatory lesions visible interstitial Infiltrated by inflammatory cells, inflammatory cells are mainly lymphocytes, neutrophils, with a small number of monocytes; local lamina basement inflammation is obvious, non-cancerous inflammation becomes the main (+++).
  • D compound 1 administration group local intestinal tissue interstitial showed scattered inflammatory cell infiltration, inflammation was confined to the mucosal layer, no obvious lamina basement membrane was observed, no obvious crypt destruction, intestinal mucosal epithelium intact; inflammatory cells were mainly lymphocytes With a small amount of neutrophils and monocytes, non-cancerous (+).
  • E compound 2 administration group local intestinal tissue interstitial showed scattered inflammatory cell infiltration, inflammation was confined to the mucosal layer, no obvious lamina basement membrane was observed, no obvious crypt destruction, intestinal mucosal epithelial integrity; inflammatory cells were mainly lymphocytes With a small amount of neutrophils and monocytes, non-cancerous (+).
  • F compound 3 administration group local intestinal tissue interstitial showed scattered inflammatory cell infiltration, inflammation was confined to the mucosal layer, no obvious lamina basement membrane was observed, no obvious crypt destruction, intestinal mucosal epithelium intact; inflammatory cells were drenched Ba cells are predominant, with a small number of neutrophils and monocytes, non-cancerous (+).
  • G compound 4 administration group local intestinal tissue interstitial showed scattered inflammatory cell infiltration, inflammation was mainly confined to the mucosal layer, local lamina basement membrane was slightly affected, no obvious crypt destruction, intestinal mucosal epithelium intact; inflammatory cells with lymphocytes Primary, with a small amount of neutrophils and monocytes, non-cancerous (+).
  • Compounds 5 and 6 of the present invention were each prepared as a PEG dispersant as a form of administration. At the time of administration, the compound of the present invention is suspended by using water as a solvent, followed by intragastric administration.
  • C57BL/6J mice female were subjected to adaptive normal feeding for one week in a clean animal room, and then a single dose of AOM was administered to the experimental animals at a dose of 10 mg/Kg as the first day of the experiment, followed by normal feeding for 6 days. As the first week of the experiment.
  • the experimental animals were allowed to drink 2% of the DSS aqueous solution freely. After 7 days of induction, the normal drinking water was allowed to continue to allow the experimental animals to drink freely for two weeks (third week and fourth week) as the first induction.
  • the experimental animals were allowed to drink 2% of the DSS aqueous solution freely. After 7 days of induction, the normal drinking water was allowed to continue to allow the experimental animals to drink freely for two weeks (sixth and seventh weeks) as the second induction.
  • the experimental animals were allowed to drink 2% of the DSS aqueous solution freely. After 7 days of induction, the normal drinking water was allowed to continue to allow the experimental animals to drink freely for one week (ninth week) as the third induction.
  • each compound of the present invention was administered to each group of animals at a dose of 50 mg/kg according to the experimental design, once a day, once a day, the positive drug capecitabine was administered at a dose of 500 mg/Kg per day. once.
  • each group of animals was treated as usual, and the percentage change of body weight, spleen weight, colon length, colon tumor burden calculation, colon tumor histopathology, etc. after treatment were observed. Changes in indicators.
  • the experimental results show that the compounds of the present invention show significant anti-tumor activity in the experiments of treating colorectal cancer in vivo, and the curative effect is significantly better than that of the positive control drugs.
  • the specific data are shown in Table 5, Figure 5, Table 6, Figure 6, respectively. Table 7, Figure 7, Table 8, and Figure 8.
  • Figure 5 and Table 6 show: model group compared with normal control spleen weight value (0.088 g) The spleen weight of the mice was significantly increased (0.106 g) (**, p ⁇ 0.01). The spleen weight of the mice in each administration group was reduced to some extent compared with the mice in the model group, the positive drug was 0.086 g, the compound 5 was 0.068 g, and the compound 6 was 0.060 g (##, p ⁇ 0.01). The results suggest that the compounds of the present invention have a significant improvement in the increase in spleen weight caused by model mice.
  • Figure 6 and Table 7 show that the model group has a significantly longer colon length than the normal control group (7.283 cm). Lower (6.000 cm) (**, p ⁇ 0.01).
  • the mice in the 5 and 6 administration groups of the present invention had different degrees of improvement in colonic contracture, which showed that the colon length was increased compared with the model group, and the compound 5 colon length was 6.333 cm. 6 is 6.517 cm (#, p ⁇ 0.05).
  • the positive drug capecitabine had a statistically significant difference in the improvement of colonic contracture (6.03 cm) in mice compared with the model group.
  • Figure 7 and Table 8 show the effect of compounds 5 and 6 of the present invention on colorectal tumor burden in colorectal cancer model mice.
  • Figure 7 and Table 8 show that colorectal cancer is compared with the normal control group tumor tumor relative to the calculated value of 0.00.
  • the tumor burden of the model group was significantly increased, and the relative calculated value was about 28.33 (**, p ⁇ 0.01).
  • the tumor burden of each group showed a very significant improvement effect, which was manifested by a decrease in the number of tumors in the whole colon range and a decrease in tumor volume. Small (including adenoma and adenocarcinoma).
  • the tumor load was calculated to be 2.75 for Compound 5, 0.40 for Compound 6, and 8.33 for the positive drug group.
  • the statistical difference was significant between the drug-administered group and the colorectal cancer model group (#, p ⁇ 0.05,##, p ⁇ 0.01).
  • the compounds 5 and 6 of the present invention are very effective in comparison with the positive control drug.
  • the calculation method of tumor load calculation value is as follows:
  • Figure 8 is a histopathological examination (HE staining, 100-fold) of colorectal tumors of colorectal cancer model mice of the present invention.
  • a normal control group normal structure of the colon, showing a clear layer of intestinal tissue, intact structure, no inflammatory changes.
  • Inflammatory lesions of intestinal tissue were significant; inflammation involved the entire mucosal epithelium and submucosal lamina intestinal (transmural inflammation), almost all crypts were destroyed, part of the mucosal epidermis was missing; a large number of inflammatory cells were infiltrated in the inflammatory lesions, and inflammatory cells were Lymphocytes, neutrophils, and a small number of monocytes; inflammatory ulcers can be seen in the area of local inflammation, and inflammatory fibrous tissue hyperplasia can be seen at the bottom of the ulcer, which is a moderately differentiated adenocarcinoma (++++).
  • C positive drug capecitabine group intestinal tissue inflammation lesions, inflammation involving 2 / 3 intestinal mucosa interstitial, local visible transmural inflammatory lesions, local crypt destruction, intestinal mucosa epidermis relatively intact; inflammatory lesions visible interstitial Infiltrated by inflammatory cells, inflammatory cells are mainly lymphocytes, neutrophils, with a small number of monocytes; local lamina basement inflammation is obvious, non-cancerous inflammation becomes the main (+++).
  • D compound 5 administration group local intestinal tissue interstitial showed scattered inflammatory cell infiltration, inflammation was confined to the mucosal layer, no obvious lamina basement membrane was observed, no obvious crypt destruction, intestinal mucosal epithelium intact; inflammatory cells were mainly lymphocytes With a small amount of neutrophils and monocytes, non-cancerous (+).
  • E compound 6 administration group local intestinal tissue interstitial showed scattered inflammatory cell infiltration, inflammation was confined to the mucosal layer, no obvious lamina basement membrane was observed, no obvious crypt destruction, intestinal mucosal epithelium intact; inflammatory cells were mainly lymphocytes With a small amount of neutrophils and monocytes, non-cancerous (+).
  • mice Eight weeks after administration of each group of animals, the mice were sacrificed in a conventional manner at the end of the experiment. Select 2 cm of colon tissue from the anus of the mouse. Weighed colon tissue was weighed and thawed twice in liquid nitrogen, and sonicated in RIPA lysate, then lysed at 4 degrees for 30 min, centrifuged at 13,000 rpm for 10 min. The supernatant was taken and the protein concentration was determined by the Brandford method. Western-blot (WB) detection was performed by taking the same amount of protein according to the protein concentration.
  • WB Western-blot
  • 5% concentrated gel and 10% separating gel were prepared according to standard SDS-PAGE method.
  • Cell lysate supernatants containing the same protein concentration were mixed with 5 x SDS loading buffer and boiled for 5 min. Load after cooling.
  • the wet transfer method was transferred to the PVDF membrane.
  • Non-specific binding sites were blocked overnight at 4 °C with TBST (0.1% Tween-20; 10 mmol/L Tris-Cl, pH 7.5; 3% BSA; 150 mmol/L NaCl). The membrane was washed with TBST solution, 10 min/time x 3 times.
  • the membrane was incubated with diluted primary antibody (1:500) for 3 h at room temperature, and the membrane was washed with TBST solution for 10 min/time x 3 times.
  • the membrane was transferred to a secondary antibody (1:1000 dilution) and allowed to react at room temperature for 2 h.
  • the membrane was washed with TBST, 10 min/time ⁇ 3 times.
  • the film was placed flat, the luminescent liquid was added dropwise, and the chemiluminescence was imaged.
  • the results indicate that the compounds 1, 2, 3 and 5 of the present invention have a significant inhibitory effect on the signal molecule STAT3 which is closely related to the pathogenesis of colorectal cancer at the protein level.
  • the specific data is shown in Figure 9 and Figure 10.
  • the upper part is the WB hybrid development map, and the lower is the relative quantitative detection result of the protein.
  • Figure 9 is a graph showing the effects of compounds 1 and 2 on the expression of target STAT3 protein in colorectal tissues of mice with colorectal cancer.
  • the lanes are lane 1: normal control group; lane 2: AOM/DSS model group; lane 3: Compound 1 administration group; lane 4: Compound 2 administration group.
  • the semi-quantitative results of WB protein showed that compared with the relative quantitative calculation value of stat3/actin in the colon tissue of normal control mice, the expression of STAT3 in the intestinal tissue of the colorectal cancer model group was significantly increased, and the relative value was 1.82. The difference was very significant (**, p ⁇ 0.01).
  • the compounds 1 and 2 of the present invention all have a very significant improvement effect, and the expression of STAT3 protein is significantly decreased.
  • the relative ratio of stat3/actin is 0.25 for compound 1 and 0.33 for compound 2, statistics. The difference is very significant (##, p ⁇ 0.01).
  • Figure 10 is a graph showing the effects of compounds 1, 2, 3 and 5 of the present invention on the expression of target STAT3 protein in colorectal tissues of mice with colorectal cancer.
  • the lanes are lane 1: normal control group; lane 2: AOM/DSS Model group; lane 4: Compound 2 administration group; lane 5: Compound 5 administration group; lane 7: Compound 1 administration group; lane 8: Compound 3 administration group.
  • the semi-quantitative results of WB protein showed that compared with the normal quantitative calculation of stat3/actin in the colon tissue of normal control mice, the expression of STAT3 in the intestinal tissue of the colorectal cancer model group was significantly increased, the relative value was 0.43, statistically. The difference was very significant (**, p ⁇ 0.01).
  • the compounds of the present invention 1, 2, 3 and 5 all have a very significant inhibitory effect, showing a significant decrease in the expression of STAT3 protein, and the relative ratio of stat3/actin is 0.13, and compound 2 is 0.23, compound 3 was 0.19, and compound 5 was 0.20.
  • the statistical difference was very significant (p ⁇ 0.01).
  • the test results of the compounds 1-6 of the present invention showed LD 50 values of 5.0 g/kg, 5.0 g/kg, 0.8 g/kg, 5.0 g/kg, 3.9 g/kg and 1.6 g/kg, respectively. Further, the LD 50 value of dihydroberberine was 160 mg/Kg.

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Abstract

本发明提供了二氢小檗碱型化合物和(±)-8-丙酮基二氢小檗碱型化合物，包括二氢异黄连碱、二氢黄连碱和二氢巴马汀，(±)-8-丙酮基二氢小檗碱型化合物包括(±)-8-丙酮基二氢异黄连碱、(±)-8-丙酮基二氢黄连碱和(±)-8-丙酮基二氢巴马汀在制备预防、缓解和/或治疗STAT3信号通路相关肿瘤疾病产品中的应用。其中的STAT3信号通路相关肿瘤疾病选自各种肿瘤，尤其是结直肠癌。

Description

二氢小檗碱型化合物在制备治疗STAT3信号通路相关肿瘤疾病产品中的用途 技术领域
本发明涉及系列的具有相同母体骨架结构的二氢小檗碱型化合物和(±)-8-丙酮基二氢小檗碱型化合物的药物化学和新的药理作用。具体涉及二氢异黄连碱、二氢黄连碱、二氢巴马汀、(±)-8-丙酮基二氢异黄连碱、(±)-8-丙酮基二氢黄连碱和(±)-8-丙酮基二氢巴马汀作为STAT3抑制剂在制备预防、缓解和/或治疗STAT3信号通路相关肿瘤疾病产品中的应用。属医药技术领域。
背景技术
尽管各种恶性肿瘤(癌症)都有其不同的发病特点，但都是严重危害人类健康的重大疾病，给患者及其家属造成巨大的身体上和精神上的痛苦以及经济上的压力。由于至今还没有特别有效的治疗癌症的方法，临床上常被迫将手术疗法、放射疗法和化学疗法的手段均作为临床一线治疗方案结合起来对患者进行治疗。目前对人类各种癌症的预防、缓解及治疗仍是医药科学研究领域必须面对的一项十分艰巨的科研工作，是一项需要在资金和人力方面做出巨大投入的***工程。将手术、放疗和化疗的治疗方法进行平行评价，与风险大又有痛苦的外科手术疗法，或者放射性疗法(它使用放射能，若稍有不慎，可能影响重要脏器的功能)相比，如果可以单独利用化疗法的各种药物抑制癌症的发展，或征服癌症，那么，毫无疑问，对患者而言是一巨大福音，也是医药科学进步的重大体现。显然能用高效且安全的药物治疗癌症是比较理想的手段之一。因此，寻找对包括结直肠癌在内的各种恶性肿瘤具有高效的治疗作用并具有低毒性甚至无毒性特点的特异性化学药物在医药技术领域具有重要的意义。目前科学家已发现了一些有一定疗效的抗肿瘤药物，正是这些药物使急性白血病患儿的平均生存期由过去的2～3个月延长到5年以上，使不少晚期肿瘤病人的生命明显延长。新抗肿瘤药物的深入研究已使肿瘤的化学治疗成为一个重要学科，并使肿瘤内科学得以诞生。Elion及Hitchings于1988年被授予诺贝尔医学奖，表明抗肿瘤药的历史性成就已举世公认。当然，这并不意味着抗肿瘤药物的研究不再需要发展，相反，目前它面临严峻的挑战，这就是多数常见实体瘤如肺癌、肝癌、胃癌、结直肠癌、乳腺癌及胰腺癌等还缺 乏有效的治疗药物，不少抗肿瘤药物在临床应用过程中产生耐药性和严重毒性。也就是说，截止目前，还没有特别高疗效低毒性的抗癌药物问世。因此，在目前恶性肿瘤发病呈明显上升趋势的情况下(见文献：杨玲，等.中国2000年至2005年恶性肿瘤发病死亡的估计与预测，中国卫生统计，2005，22(4)：218-221，231)寻找高疗效低毒性的抗癌新药一直是药物研究领域的重要课题。
小檗碱型化合物是泛指天然有机化学领域中的以下三类不同类型的化合物：(1)具有6,8-二氢-5H-异喹啉并[3,2-a]异喹啉(6,8-dihydro-5H-isoquinolino[3,2-a]isoquinoline)基本结构之7,8-亚胺盐型结构的小檗碱季铵盐型化合物(Ⅰ)、(2)具有6,8-二氢-5H-异喹啉并[3,2-a]异喹啉基本母体结构的二氢小檗碱型化合物(Ⅱ)和(3)具有6,8,13,13a-四氢-5H-异喹啉并[3,2-a]异喹啉(6,8,13,13a-tetrahydro-5H-isoquinolino[3,2-a]isoquinoline)基本母体结构的四氢小檗碱型(原小檗碱型)化合物及其各种盐(Ⅲ)，三者母体的化学式见式1。从结构上分析，小檗碱季铵盐型化合物(Ⅰ)和二氢小檗碱型化合物(Ⅱ)分别具有如式2和3所示的多个不同共振式，但由于结构的不同，二者的共振式结构和数目不可能相同；二者的结构特征与具有6,8,13,13a-四氢-5H-异喹啉并[3,2-a]异喹啉型基本碳架结构的苄基异喹啉型化合物(即原小檗碱型或四氢小檗碱型化合物)及其各种盐(Ⅲ)也存在显著的差别，即在Ⅲ中，除了含有13a位不对称中心外，同时由于C-13和C-13a之间的双键被打开，产物中只剩下两个完整的苯环(不再共轭)，氮原子是定域结构；或者可以认为Ⅲ为二苯基乙烷衍生物，因此与Ⅰ和Ⅱ更是显著不同。当然，Ⅰ和Ⅱ由于前者是季铵盐型化合物，而后者是叔胺型化合物，且二者的共轭体系存在明显差异，因此，二者的物理化学性质和其它性质也存在显著的差别，例如溶解性、化学反应性、生物活性等的差别；因此，从有机化学、药物化学和药学的角度，小檗碱季铵盐型化合物与二氢小檗碱型化合物分属不同结构类型化合物。虽然部分小檗碱季铵盐型化合物和四氢小檗碱型化合物具有比较丰富的天然资源，但根据目前的研究报道，由于二氢小檗碱型化合物的结构只有在特定的环境下才具有稳定性(在以上三种类型的小檗碱型化合物中，二氢型的小檗碱类化合物的结构最独特)，因此在自然界的存在受到很大限制，或从自然界规模化获得其原料具有相当的困难。本发明中二氢小檗碱型化合物和(±)-8-丙酮基二氢小檗碱型化合物全部是通过有机化学的方法合成获得的。
Figure PCTCN2017106227-appb-000001
式1.小檗碱季铵盐型(Ⅰ)、二氢小檗碱型(Ⅱ)和四氢小檗碱型(Ⅲ)化合物的基本结构
Figure PCTCN2017106227-appb-000002
式2.小檗碱季铵盐型(Ⅰ)化合物的共振式
Figure PCTCN2017106227-appb-000003
式3.二氢小檗碱型(Ⅱ)化合物的共振式
在我们以研制“高疗效低毒性的抗癌药物”为目标开展的抗结直肠癌(包括结肠癌和直肠癌)的创新药物设计和发现研究工作中，我们对小檗碱型化合物在化学和抗结直肠癌方面的研究进展进行了全面的文献查阅和分析，并根据药物化学的研究思路和方法开展了更深入细致的研究工作。现阶段已知的技术主要包括对小檗碱季铵盐型化合物的研究，具体化合物主要是氯化小檗碱季铵盐和氯化巴马汀季铵盐(文献也称为盐酸小檗碱和盐酸巴马汀，个别文献模糊地称为小檗碱和巴马汀)具有一定的抗结肠癌活性，例如，小檗碱通过使Wnt/β-catenin信号通路失活而能够抑制结肠癌细胞的增殖(见文献：Wu K,et.al.Berberine inhibits the proliferation of colon cancer cells by inactivating Wnt/β-catenin signaling,International Journal of Oncology,2012,41,292-298)，并具有抑制人结肠癌细胞在裸小鼠体内生长的药理活性(见专利文献：CN1313093C)；氯化巴马汀是一种光敏剂，对结肠腺癌细胞HT-29具有光毒性(见文献：Wu J,et al.Photodynamic action of palmatine hydrochloride on colon adenocarcinoma HT-29cells,Photodiagnosis and Photodynamic Therapy,2016,15,53-58)；我们也对黄连碱季铵盐进行了体外抗结肠癌细胞株的活性评价，考察将小檗碱季铵盐型化合物C-2和C-3以及C-9和C-10上的取代基均 替换成亚甲二氧基对活性的影响。结果表明黄连碱季铵盐也具有一定的体外活性，但作用强度较弱，对HCT细胞株的生长抑制活性IC50值为5.59μmol/L(见文献Zhi-Hui Zhang,et al.Syntheses and structure–activity relationships in cytotoxicities of13-substituted quaternary coptisine derivatives,European Journal of Medicinal Chemistry,2014,86,542-549)。对二氢小檗碱型化合物的研究已知的技术限于具体化合物二氢小檗碱在与苏尼替尼组成的药物组合物中显示具有协同抗肿瘤作用，包括对结肠癌、肺癌和肝癌的作用(见专利文献：CN 105434435 A)，等等。
正如前文所述，小檗碱型化合物具有结构多样性；从有机结构与性质的关系角度分析，3种类型的小檗碱型化合物不仅结构不一致，其物理和化学性质也显著不同。例如，小檗碱季铵盐型化合物由于独特地存在7,8-亚胺盐型芳香亲电取代反应中的钝化官能团，不仅导致其在各种溶剂中的溶解性很差、共轭体系增大，而且芳环上的芳香亲电取代反应等有机反应也难以进行；再例如，在小檗碱季铵盐型化合物的C-13位引入脂肪烃基的反应中，需要将其改变成二氢小檗碱型结构，而在C-12位的硝化反应方面，小檗碱季铵盐型化合物难以进行，但四氢小檗碱型化合物较易进行，等。根据药物化学的常规知识，一些活性化合物(在创新药物研究中称为先导化合物)往往存在一些缺陷，例如，各种小檗碱季铵盐型化合物不良的溶解性导致的其在体内难以被肌体吸收入血且生物利用度极差的问题(这一缺陷明显影响化合物的某些生物活性，比如需要通过吸收入血进行治疗的疾病受到很大的限制)，以及具体化合物小檗碱季铵盐和二氢小檗碱的严重毒性作用，其中，前者的LD50值是329mg/Kg(见专利文献：CN1313093C)，后者的LD50值是160mg/Kg(二氢小檗碱是本发明研究工作中发现的系列化合物中毒性最大的化合物)。这些成药性方面的严重不足本质上是由于其化学结构的特征不同所导致的；因此，需要进一步根据药物化学的研究思路和方法，对有关结构进行***分析、设计并合成溶解性能和药代性能更好、活性更高、选择性更强或毒副作用更小的结构优化化合物。当然，并不是这种研究仅通过理论上的结构设计即可达到目的，更重要的研究是需要通过切实可行的实验工作进行实际验证，因为在研究工作中经常遇到具有类似结构的化合物显示出不同或相反的生物活性；例如，在采用生物电子等排原理开展的结构改造研究中，有些研究获得活性类似但作用强度有增强或有降低的研究结果，有些研究获得毒性作用强度有增强或有降低的研究结果，还有一些研究获得了拮抗作用的研究结果等等(各种具体例证见文献：尤启冬主 编《药物化学》，化学工业出版社，第二版，2010年，北京，P29页)。
在充分了解上述信息的基础上，为了寻找抗癌创新药物候选物，我们对不同类型的小檗碱型化合物深入开展了衍生物的合成和生物性能评价研究。将小檗碱季铵盐型化合物中的异黄连碱季铵盐、黄连碱季铵盐、巴马汀季铵盐和小檗碱季铵盐通过官能团改变而改造为系列具有脂溶性的二氢小檗碱型化合物，包括二氢异黄连碱(1)、二氢黄连碱(2)、二氢巴马汀(3)和二氢小檗碱，进行溶解性、生物利用度、活性和毒性评价，结果表明，这些二氢小檗碱型化合物与小檗碱季铵盐型化合物相比，一方面在多种有机溶剂如二氧六环、丙酮或四氢呋喃等中的溶解性能均得到显著改善；另一方面，其急性毒性以LD50值计，化合物1和2均大于5g/Kg，为无毒性化合物；化合物3的LD50值为800mg/Kg，属低毒性化合物；而已知具有协同抗肿瘤作用的二氢小檗碱的LD50值仅为160mg/Kg，属本系列化合物中毒性最大的化合物，这一点在我们开展的动物实验中得到了证实，即二氢小檗碱在采用50mg/Kg的剂量经灌胃给药后出现实验动物较快死亡的现象，而1、2和3没有出现实验动物死亡的现象，甚至1和2在给药剂量较大时也未出现实验动物死亡的现象。化合物1和2对IEC-6肠上皮细胞不显示明显的细胞毒性的实验结果也说明了其是无毒性的化合物(化合物2的实验数据见文献：Zhi-Hui Zhang,et al.Syntheses and structure–activity relationships in cytotoxicities of13-substituted quaternary coptisine derivatives,European Journal of Medicinal Chemistry,2014,86,542-549)。此外，对于本发明系列化合物，由于其特殊的化学结构，特别是在特定条件下的特定性质(例如，溶解性特征、稳定性特征等)，采用MTT法在体外评价其对肿瘤细胞株的生长抑制活性的结果并不能说明其抗癌活性，例如，化合物2对HCT细胞株不显示细胞生长抑制活性，IC50值大于5μg/mL(数据见文献Zhi-Hui Zhang,et al.Syntheses and structure–activity relationships in cytotoxicities of 13-substituted quaternary coptisine derivatives,European Journal of Medicinal Chemistry,2014,86,542-549；IC50值大于5μg/mL，即大于15.56μM，被认为是无活性的化合物)；这种体外不显示抗肿瘤活性的研究结果给了我们相反的技术启示，并一度导致我们放弃这一类化合物的研究的想法。偶然机会，通过对二氢小檗碱型化合物的结构分析，我们开展了化合物1、2、3和二氢小檗碱的治疗结直肠癌的动物体内实验，以治疗后动物体重变化百分比、脾脏重量、结肠长度、结肠肿瘤负荷计算值、结肠肿瘤病理等为指标进行疗效评价。研究结果表明，除二氢小檗碱较快导致实验动物死亡的结果外(表明其毒性较大)，各指标结 果均表明化合物1、2、3均具有显著的抗结直肠癌活性，作用强度显著优于阳性对照药卡培他滨。
本发明从季铵型的小檗碱型化合物出发，合成了二氢小檗碱型化合物的(±)-8-丙酮基取代衍生物，包括(±)-8-丙酮基二氢异黄连碱(4)、(±)-8-丙酮基二氢黄连碱(5)、(±)-8-丙酮基二氢巴马汀(6)和(±)-8-丙酮基二氢小檗碱，并进行了毒性和抗肿瘤活性的评价，结果表明，化合物4-6以及(±)-8-丙酮基二氢小檗碱的急性毒性实验LD50值分别为5g/Kg、3.9g/Kg、1.6g/Kg和200mg/Kg，化合物4-6为无毒性或低毒性的化合物，而(±)-8-丙酮基二氢小檗碱具有显著的毒性。基于对化合物1-3同样的考虑，在进一步的模型动物体内实验中，化合物4-6也均显示出显著的抗结直肠癌作用，综合评价，作用强度均显著优于阳性对照药卡培他滨。化合物5对IEC-6肠上皮细胞不显示明显的细胞毒性的实验结果也说明了其是低毒性的化合物(化合物5的IEC-6细胞毒性实验数据见文献：Zhi-Hui Zhang,et al.,Synthesis and Structure-activity Relationships of Quaternary Coptisine Derivatives as Potential Anti-ulcerative Colitis Agents.Journal of Medicinal Chemistry.2015,58,7557-7571；Zhi-Hui Zhang,et al.Syntheses and structure–activity relationships in cytotoxicities of13-substituted quaternary coptisine derivatives,European Journal of Medicinal Chemistry,2014,86,542-549)。
为了阐明本发明系列化合物的治疗恶性结直肠癌的作用机制，以化合物1、2、3和5为例，对其对氧化偶氮甲烷(Azoxymethane,AOM)/葡聚糖硫酸钠(Dextran sulfate sodium,DSS)诱导的结直肠癌模型小鼠结肠组织中靶标分子STAT3(信号转导和转录激活因子3)的抑制效应进行了研究。STAT3与多种肿瘤的发生发展密切相关，在多种肿瘤的细胞中均可见STAT3过度激活且呈高水平表达。抑制STAT3的过度表达是***的手段之一。本发明研究结果表明化合物1、2、3和5均具有显著的STAT3抑制作用，进一步阐明了本发明系列化合物通过抑制STAT3，进而可以预防或治疗STAT3信号通路相关肿瘤疾病。
发明内容
本发明解决的技术问题是提供一类二氢小檗碱型化合物和(±)-8-丙酮基二氢小檗碱型化合物在制备STAT3抑制剂以及制备预防或治疗STAT3信号通路相关肿瘤疾病产品中的应用，进一步的，提供了其在制备预防或治疗恶性结直肠癌产品中的应用。
为解决上述技术问题，本发明提供了如下技术方案：
本发明技术方案的第一方面提供了一类二氢小檗碱型化合物和(±)-8-丙酮基二氢小檗碱型化合物在制备STAT3抑制剂中的应用，以及其在制备预防或治疗STAT3信号通路相关肿瘤疾病产品中的应用，所述的二氢小檗碱型化合物包括二氢异黄连碱、二氢黄连碱和二氢巴马汀，(±)-8-丙酮基二氢小檗碱型化合物包括(±)-8-丙酮基二氢异黄连碱、(±)-8-丙酮基二氢黄连碱和(±)-8-丙酮基二氢巴马汀。具体化合物分别是如式1-6所示的二氢异黄连碱、二氢黄连碱、二氢巴马汀、(±)-8-丙酮基二氢异黄连碱、(±)-8-丙酮基二氢黄连碱和(±)-8-丙酮基二氢巴马汀。其中，所述的产品包括药品和保健品。所述的STAT3信号通路相关肿瘤疾病选自结直肠癌、以及各类恶性肿瘤，结直肠癌包括溃疡性结肠炎相关结直肠癌以及其他类型的结直肠癌。所述的结直肠癌包括结肠癌、直肠癌。
Figure PCTCN2017106227-appb-000004
化合物4的合成路线如下：称取异黄连碱(384mg，1.08mmol)于反应瓶中，逐滴加入丙酮(1ml，13.56mmol)，搅拌2min后，再加入5N NaOH(10ml)水溶液，50℃搅拌4h，TLC监测原料反应完全，将反应液过滤，滤饼用水反复洗涤，至中性，然后用丙酮和水(丙酮:水＝3:1，v/v)的混合溶剂做重结晶，得黄色颗粒状结晶113mg，收率27.76％。1H-NMR(400MHz,DMSO-d6)δ：7.22(1H,s,ArH),6.77(1H,s,ArH),6.59(1H,s,ArH),6.57(1H,s,ArH),6.00,5.99(2H,2×d,J＝0.8Hz,OCH2 O),5.96(1H,s，ArCH＝C),5.914,5.909(2H，2×d,J＝0.8Hz,OCH2 O),4.80(1H,dd,J＝7.6,5.2Hz,CHCH2COCH3),3.23(2H,m,NCH2 CH2),2.79(2H,m,NCH2CH2 ),2.73–2.62(2H,m,CHCH2 COCH3),1.95(3H,s,COCH3 )。
本发明技术方案的第二方面是提供一种包含如式1-6所示二氢异黄连碱、二氢黄连碱、二氢巴马汀、(±)-8-丙酮基二氢异黄连碱、(±)-8-丙酮基二氢黄连碱和 (±)-8-丙酮基二氢巴马汀中的一个或多个的具体化合物的药物组合物在制备预防或治疗STAT3信号通路相关肿瘤疾病产品中的应用。其中，所述的STAT3信号通路相关肿瘤疾病选自结直肠癌、以及各类恶性肿瘤，所述的产品包括药品和保健品。所述的结直肠癌包括溃疡性结肠炎相关结直肠癌以及其他类型的结直肠癌。所述的结直肠癌包括结肠癌、直肠癌。
Figure PCTCN2017106227-appb-000005
该药物组合物可根据本领域公知的方法制备。可通过将本发明化合物与一种或多种药学上可接受的固体或液体赋形剂和/或辅剂结合，制成适于人或动物使用的任何剂型，优选的剂型是普通或肠溶固体制剂。本发明化合物在其药物组合物中的含量通常为0.1-99.9％(W/W)。
本发明化合物或含有本发明化合物的药物组合物可以单位剂量形式给药，给药途径可为肠道或非肠道，如口服。通过剂型研究，也可以进行静脉注射、肌肉注射、皮下注射、鼻腔、口腔粘膜、眼、肺和呼吸道、皮肤、***、直肠给药等。
给药剂型可以是液体剂型、固体剂型或半固体剂型。液体剂型可以是溶液剂(包括真溶液和胶体溶液)、乳剂(包括O/W型、W/O型和复乳)、混悬剂、注射剂(包括水针剂、粉针剂和输液)、滴眼剂、滴鼻剂、洗剂和搽剂等；固体剂型可以是片剂(包括普通片、肠溶片、含片、分散片、咀嚼片、泡腾片、口腔崩解片)、胶囊剂(包括硬胶囊、软胶囊、肠溶胶囊)、颗粒剂、散剂、微丸、滴丸、栓剂、膜剂、贴片、气(粉)雾剂、喷雾剂等；半固体剂型可以是软膏剂、凝胶剂、糊剂等。二氢小檗碱型化合物和(±)-8-丙酮基二氢小檗碱型化合物优选的剂型是普通或肠溶片剂和普通或肠溶胶囊剂
本发明化合物可以制成普通制剂、也可制成缓释制剂、控释制剂、靶向制剂及各种微粒给药***。
为了将本发明化合物制成普通片剂或肠溶片剂，可以广泛使用本领域公知的 各种赋形剂，包括稀释剂、黏合剂、润湿剂、崩解剂、润滑剂、助流剂。稀释剂可以是淀粉、糊精、蔗糖、葡萄糖、乳糖、甘露醇、山梨醇、木糖醇、微晶纤维素、硫酸钙、磷酸氢钙、碳酸钙等；润湿剂可以是水、乙醇、异丙醇等；粘合剂可以是淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、微晶纤维素、***胶浆、明胶浆、羧甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、乙基纤维素、丙烯酸树脂、卡波姆、聚乙烯吡咯烷酮、聚乙二醇等；崩解剂可以是干淀粉、微晶纤维素、低取代羟丙基纤维素、交联聚乙烯吡咯烷酮、交联羧甲基纤维素钠、羧甲基淀粉钠、碳酸氢钠、聚氧乙烯山梨糖醇脂肪酸酯、十二烷基磺酸钠等；润滑剂和助流剂可以是滑石粉、二氧化硅、硬脂酸盐、液体石蜡、聚乙二醇等。
还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片,优选的包衣片是普通或肠溶包衣片。
为了将给药单元制成胶囊剂，可以将有效成分(本发明化合物)与稀释剂、助流剂混合，将混合物直接置于硬胶囊或软胶囊中。也可将有效成分(本发明化合物)先与稀释剂、黏合剂、崩解剂制成颗粒或微丸，再置于硬胶囊或软胶囊中。用于制备本发明化合物片剂的各稀释剂、黏合剂、润湿剂、崩解剂、助流剂品种也可用于制备本发明化合物的胶囊剂。
为将本发明化合物制成注射剂，可以用水、乙醇、异丙醇、丙二醇或它们的混合物作溶剂并加入适量本领域常用的增溶剂、助溶剂、pH调节剂、渗透压调节剂，并加入适当的稳定剂。增溶剂或助溶剂可以是泊洛沙姆、卵磷脂、羟丙基-β-环糊精等；pH调节剂可以是氢氧化钠、氢氧化钾、碳酸钠、碳酸钾等；稳定剂可以是氢氧化钠、氢氧化钾、碳酸钠、碳酸钾等；渗透压调节剂可以是氯化钠、甘露醇、葡萄糖、磷酸盐、醋酸盐等。如制备冻干粉针剂，还可加入甘露醇、葡萄糖等作为支撑剂。
此外，如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂或其它添加剂。
为达到用药目的，增强治疗效果，本发明的药物或药物组合物可用任何公知的给药方法给药，优选的给药方法是普通或肠溶制剂口服给药。
本发明化合物药物组合物的给药剂量依照所要预防或治疗结直肠癌的性质和严重程度，患者或动物的个体情况，给药途径和剂型等可以有大范围的变化。一般来讲，本发明化合物的每天的合适剂量范围为0.001-150mg/Kg体重，优选为 0.1-100mg/Kg体重，更优选为1-60mg/Kg体重，最优选为2-30mg/Kg体重。上述剂量可以一个剂量单位或分成几个剂量单位给药，这取决于医生的临床经验以及包括运用其它治疗手段的给药方案。
本发明的化合物或药物组合物可单独服用，或与其他治疗药物或对症药物合并使用。当本发明的化合物与其它治疗药物存在协同作用时，应根据实际情况调整它的剂量。
有益技术效果
为了克服二氢小檗碱型化合物的结构不稳定性，本发明采用以PEG为分散剂制备二氢小檗碱型化合物和(±)-8-丙酮基二氢小檗碱型化合物分散剂实体的形式口服给药或采用CMC-Na/吐温-80水溶液分散的形式口服给药，为后续的制剂研究奠定基础；本发明化合物可以达到治疗结直肠癌的疗效，且作用显著。在通过建模并采用结直肠癌动物模型进行的动物体内实验中，以治疗后实验动物体重变化百分比、脾脏重量、结肠长度、结肠肿瘤负荷计算值、结肠肿瘤组织病理等为考察指标进行疗效评价，各指标结果均表明本发明化合物具有显著的抗结直肠癌活性。在50mg/Kg的剂量下，本发明化合物抗恶性结直肠癌的作用强度明显高于阳性对照药物卡培他滨500mg/Kg剂量的治疗效果，其中，在实验结束观察疗效中发现，二氢异黄连碱(1)、二氢黄连碱(2)、二氢巴马汀(3)和(±)-8-丙酮基二氢异黄连碱(4)的肿瘤负荷计算值在给药剂量为50mg/Kg时分别达到0.06、0.13、0.01、0.08，甚至与正常对照组的0.00值接近一致；与阳性对照药的肿瘤负荷计算值1.25相比，疗效非常显著。(±)-8-丙酮基二氢黄连碱(5)和(±)-8-丙酮基二氢巴马汀(6)的肿瘤负荷计算值分别在给药剂量为50mg/Kg时达到2.75和0.40，与同批次的阳性对照药的肿瘤负荷计算值8.33相比，疗效非常显著。根据对药理作用特异性的研究，本发明化合物同时具有无毒性或低毒性的优点，二氢异黄连碱和二氢黄连碱的LD50值均达5.0g/Kg以上，二氢巴马汀的LD50值为0.8g/Kg，(±)-8-丙酮基二氢异黄连碱、(±)-8-丙酮基二氢黄连碱和(±)-8-丙酮基二氢巴马汀的LD50值分别为5.0g/Kg、3.9g/Kg和1.6g/Kg，属无毒性或低毒性的特异性抗肿瘤化合物。
本发明重要特征在于，阐明了本发明化合物为STAT3抑制剂；通过药效学实验，证实了本发明化合物在体内具有显著的抗结直肠癌活性，活性明显高于阳性对照药物；本发明化合物是无毒性或低毒性的特异性抗肿瘤化合物，在预防、缓 解和/或治疗STAT3信号通路相关肿瘤疾病，进一步的在预防、缓解和/或治疗结直肠癌疾病方面是极有药用价值的化合物。
附图说明
图1、本发明化合物1-4对结直肠癌模型小鼠脾脏重量的影响(n＝10)
图2、本发明化合物1-4对结直肠癌模型小鼠结肠长度的影响(n＝10)
图3、本发明化合物1-4对结直肠癌模型小鼠结直肠肿瘤负荷的影响
图4、本发明化合物1-4对结直肠癌模型小鼠结直肠肿瘤的组织病理检测(HE染色，100倍)。A为正常对照组，B为结直肠癌模型组，C为阳性药卡培他滨给药组，D为化合物1给药组，E为化合物2给药组，F为化合物3给药组，G为化合物4给药组。
图5、本发明化合物5和6对结直肠癌模型小鼠脾脏重量的影响(n＝10)
图6、本发明化合物5和6对结直肠癌模型小鼠结肠长度的影响(n＝10)
图7、本发明化合物5和6对结直肠癌模型小鼠结直肠肿瘤负荷的影响
图8、本发明化合物5和6对结直肠癌模型小鼠结直肠肿瘤的组织病理检测(HE染色，100倍)。A为正常对照组，B为结直肠癌模型组，C为阳性药卡培他滨给药组，D为化合物5给药组，E为化合物6给药组。
图9、本发明化合物1和2对结直肠癌模型小鼠结直肠组织中靶点分子STAT3蛋白表达的影响
图10、本发明化合物1、2、3和5对结直肠癌模型小鼠结直肠组织中靶点分子STAT3蛋白表达的影响
图11、(±)-8-丙酮基二氢异黄连碱的1H NMR图谱
具体实施方式
本发明的具体实施方式不以任意方式限制本发明。
实验例1：本发明化合物1-4对氧化偶氮甲烷(Azoxymethane,AOM)/葡聚糖硫酸钠(Dextran sulfate sodium,DSS)诱导的结直肠癌模型小鼠肿瘤形成及生长的抑制作用
将本发明化合物1、2、3和4分别制成PEG分散剂，作为给药形式。给药时， 以水作为溶剂，将本发明化合物混悬后灌胃给药。
将C57BL/6J小鼠(雌性)在清洁级动物房进行适应性正常饲养一周，然后按10mg/Kg的剂量单次给予实验动物腹腔注射AOM，作为实验起始第一天，随后正常饲养6天，作为实验的第一周。
实验第二周，允许实验动物自由饮用2％的DSS水溶液，诱导7天后换正常饮用水继续允许实验动物自由饮用两周(第三周及第四周)，作为第一次诱导。
实验第五周，允许实验动物自由饮用2％的DSS水溶液，诱导7天后换正常饮用水继续允许实验动物自由饮用两周(第六周及第七周)，作为第二次诱导。
实验第八周，允许实验动物自由饮用2％的DSS水溶液，诱导7天后换正常饮用水继续允许实验动物自由饮用一周(第九周)，作为第三次诱导。
从第二周至第九周，将本发明各化合物按实验设计剂量50mg/Kg分别予每组动物进行连续灌胃给药，每天一次，阳性药卡培他滨按500mg/Kg剂量给药，每天一次。于第九周实验结束时(共给药八周)按常规操作处理各组动物，并观察治疗后动物体重变化百分比、脾脏重量、结肠长度、结肠肿瘤负荷计算值、结肠肿瘤组织病理等各项指标的变化。实验结果表明，本发明各化合物在治疗结直肠癌的动物体内实验中显示出显著的抗肿瘤活性，疗效显著优于阳性对照药，具体数据分别见表一、图1、表二、图2、表三、图3、表四、图4。
表一、本发明化合物1-4对结直肠癌模型小鼠体重的影响
Figure PCTCN2017106227-appb-000006
表一注：在AOM/DSS交替诱导C57BL/6J小鼠结直肠癌达9周后，正常对照组小鼠体重增加百分比为25.9％，结直肠癌模型组小鼠体重增加17.6％，而阳性药卡培他滨、化合物1、化合物2、化合物3和化合物4给药组结直肠癌模型小鼠的体重变化百分比分别为8.9％、19.5％、18.4％、19.9％、20.9％。本发明各化合物给药组对模型小鼠的体重没有明显的抑制作用，而阳性药卡陪他滨显著降低模型小鼠的体重，统计学上差异显著(*，p<0.05，与正常对照组相比；#，p<0.05，与模型组相比)。
图1和表二为本发明化合物1-4对结直肠癌模型小鼠脾脏重量的影响(n＝10)图1和表二显示：与正常对照组脾脏重量值(0.085g)比较，模型组小鼠脾脏重量显著增加(0.131g)(**，p<0.01)。而各给药组小鼠脾脏重量与模型组小鼠相比有不同程度的降低，阳性药为0.112g、化合物1为0.090g、化合物2为0.100g、化合物3为0.080g、化合物4为0.100g(##，p<0.01；#，p<0.05)。结果提示本发明各化合物对模型小鼠所造成的脾脏重量增加有较为明显的改善作用。
表二、本发明化合物1-4对结直肠癌模型小鼠脾脏重量的影响(n＝10)
Figure PCTCN2017106227-appb-000007
图2和表三为本发明化合物1-4对结直肠癌模型小鼠结肠长度的影响(n＝10)图2和表三显示：与正常对照组(6.84cm)相比，模型组小鼠结肠长度明显降低(5.52cm)(**，p<0.01)。而本发明各化合物给药组小鼠与模型组小鼠相比，结肠挛缩均有不同程度的改善，表现为结肠长度较模型组小鼠增加，化合物1结肠 长度为6.35cm，化合物2为6.30cm，化合物3为5.97cm，化合物4为5.90cm(##，p<0.01；#，p<0.05)。而阳性药卡培他滨对小鼠结肠挛缩(5.80cm)的改善作用与模型组相比，在统计学上未见显著性差异。
表三、本发明化合物1-4对结直肠癌模型小鼠结肠长度的影响(n＝10)
Figure PCTCN2017106227-appb-000008
图3和表四为本发明化合物1-4对结直肠癌模型小鼠结直肠肿瘤负荷的影响图3和表四显示：与正常对照组小鼠肿瘤负荷相对计算值0.00相比，结直肠癌模型组小鼠肿瘤负荷明显增加，相对计算值约为22.64(**，p<0.01)。而各本发明化合物给药组在经过8周连续灌胃给药后，各组肿瘤负荷均显示非常显著的改善，表现为整体结肠范围内肿瘤发生数目的降低及肿瘤体积的减小(含腺瘤及腺癌)。肿瘤负荷相对计算值化合物1为0.06、化合物2为0.13、化合物3为0.00、化合物4为0.08，而阳性药组为1.25，各给药组与结直肠癌模型组相比，统计学差异非常显著(##，p<0.01)。本发明各化合物与阳性对照药相比，疗效非常显著。肿瘤负荷计算值的计算方法如下：
Figure PCTCN2017106227-appb-000009
表四、本发明化合物1-4对结直肠癌模型小鼠结直肠肿瘤负荷的影响
Figure PCTCN2017106227-appb-000010
Figure PCTCN2017106227-appb-000011
图4为本发明各化合物1-4对结直肠癌模型小鼠结直肠肿瘤的组织病理检测(HE染色，100倍)。
图4显示：
A正常对照组：结肠正常结构，表现为肠组织层次清晰，结构完整，无炎性变。
B模型组：肠组织炎症病变显著；炎症累及整个粘膜上皮及粘膜下固有层(透壁性炎症)，几乎所有隐窝破坏，部分粘膜表皮缺失；炎症病变部位可见大量炎症细胞浸润，炎症细胞以淋巴细胞、中性粒细胞为主、伴少量单核细胞；局部炎症显著区域可见炎症溃疡形成，溃疡灶底部可见炎性纤维组织增生，为中分化腺癌(++++)。
C阳性药卡培他滨组：肠组织炎症病变明显，炎症累及2/3肠粘膜间质，局部可见透壁性炎症病灶，局部隐窝破坏，肠粘膜表皮相对完整；炎症病变部位间质可见散在炎症细胞浸润，炎症细胞以淋巴细胞、中性粒细胞为主、伴少量单核细胞；局部固有层炎症明显，为非癌炎性变为主(+++)。
D化合物1给药组：局部肠组织间质可见散在炎症细胞浸润，炎症局限于粘膜层，未见固有层明显受累，未见明显隐窝破坏，肠粘膜上皮完整；炎症细胞以淋巴细胞为主，伴少量中性粒细胞和单核细胞，非癌(+)。
E化合物2给药组：局部肠组织间质可见散在炎症细胞浸润，炎症局限于粘膜层，未见固有层明显受累，未见明显隐窝破坏，肠粘膜上皮完整；炎症细胞以淋巴细胞为主，伴少量中性粒细胞和单核细胞，非癌(+)。
F化合物3给药组：局部肠组织间质可见散在炎症细胞浸润，炎症局限于粘膜层，未见固有层明显受累，未见明显隐窝破坏，肠粘膜上皮完整；炎症细胞以淋 巴细胞为主，伴少量中性粒细胞和单核细胞，非癌(+)。
G化合物4给药组：局部肠组织间质可见散在炎症细胞浸润，炎症主要局限于粘膜层，局部固有层轻度受累，未见明显隐窝破坏，肠粘膜上皮完整；炎症细胞以淋巴细胞为主，伴少量中性粒细胞和单核细胞，非癌(+)。
实验例2：本发明化合物5和6对氧化偶氮甲烷(Azoxymethane,AOM)/葡聚糖硫酸钠(Dextran sulfate sodium,DSS)诱导的结直肠癌模型小鼠肿瘤形成及生长的抑制作用
将本发明化合物5和6分别制成PEG分散剂，作为给药形式。给药时，以水作为溶剂，将本发明化合物混悬后灌胃给药。
将C57BL/6J小鼠(雌性)在清洁级动物房进行适应性正常饲养一周，然后按10mg/Kg的剂量单次给予实验动物腹腔注射AOM，作为实验起始第一天，随后正常饲养6天，作为实验的第一周。
实验第二周，允许实验动物自由饮用2％的DSS水溶液，诱导7天后换正常饮用水继续允许实验动物自由饮用两周(第三周及第四周)，作为第一次诱导。
实验第五周，允许实验动物自由饮用2％的DSS水溶液，诱导7天后换正常饮用水继续允许实验动物自由饮用两周(第六周及第七周)，作为第二次诱导。
实验第八周，允许实验动物自由饮用2％的DSS水溶液，诱导7天后换正常饮用水继续允许实验动物自由饮用一周(第九周)，作为第三次诱导。
从第二周至第九周，将本发明各化合物按实验设计剂量50mg/Kg分别予每组动物进行连续灌胃给药，每天一次，阳性药卡培他滨按500mg/Kg剂量给药，每天一次。于第九周实验结束时(共给药八周)按常规操作处理各组动物，并观察治疗后动物体重变化百分比、脾脏重量、结肠长度、结肠肿瘤负荷计算值、结肠肿瘤组织病理等各项指标的变化。实验结果表明，本发明各化合物在治疗结直肠癌的动物体内实验中显示出显著的抗肿瘤活性，疗效显著优于阳性对照药，具体数据分别见表五、图5、表六、图6、表七、图7、表八、图8。
表五、本发明化合物5和6对结直肠癌模型小鼠体重的影响
Figure PCTCN2017106227-appb-000012
Figure PCTCN2017106227-appb-000013
表五注：在AOM/DSS交替诱导C57BL/6J小鼠结直肠癌9周后，正常对照组小鼠体重增加百分比为18.32％，结直肠癌模型组小鼠体重增加15.43％，而阳性药卡培他滨、化合物5和化合物6给药组结直肠癌模型小鼠的体重变化百分比分别为8.52％、14.29％、14.15％。本发明各化合物5和6给药组对结直肠癌模型小鼠的体重没有明显抑制作用，而阳性药卡培他滨会显著降低模型小鼠的体重，统计学上差异显著(*，p<0.05，与正常对照组相比；#，p<0.05，与模型组相比)。
图5和表六为本发明化合物5和6对结直肠癌模型小鼠脾脏重量的影响(n＝10)图5和表六显示：与正常对照组脾脏重量值(0.088g)比较，模型组小鼠脾脏重量显著增加(0.106g)(**，p<0.01)。而各给药组小鼠脾脏重量与模型组小鼠相比有不同程度的降低，阳性药为0.086g、化合物5为0.068g、化合物6为0.060g(##，p<0.01)。结果提示本发明各化合物对模型小鼠所造成的脾脏重量增加有较为明显的改善作用。
表六、本发明化合物5和6对结直肠癌模型小鼠脾脏重量的影响(n＝10)
Figure PCTCN2017106227-appb-000014
图6和表七为本发明化合物对结直肠癌模型小鼠结肠长度的影响(n＝10)图6和表七显示：与正常对照组(7.283cm)相比，模型组小鼠结肠长度明显降低 (6.000cm)(**，p<0.01)。而本发明各化合物5和6给药组小鼠与模型组小鼠相比，结肠挛缩均有不同程度的改善，表现为结肠长度较模型组小鼠增加，化合物5结肠长度为6.333cm，化合物6为6.517cm(#，p<0.05)。而阳性药卡培他滨对小鼠结肠挛缩(6.03cm)的改善作用与模型组相比，在统计学上未见显著性差异。
表七、本发明化合物对结直肠癌模型小鼠结肠长度的影响(n＝10)
Figure PCTCN2017106227-appb-000015
图7和表八为本发明化合物5和6对结直肠癌模型小鼠结直肠肿瘤负荷的影响图7和表八显示：与正常对照组小鼠肿瘤负荷相对计算值0.00相比，结直肠癌模型组小鼠肿瘤负荷明显增加，相对计算值约为28.33(**，p<0.01)。而各本发明化合物5和6给药组在经过8周连续灌胃给药后，各组肿瘤负荷均显示非常显著的改善作用，表现为整体结肠范围内肿瘤发生数目的降低及肿瘤体积的减小(含腺瘤及腺癌)。肿瘤负荷相对计算值化合物5为2.75、化合物6为0.40，而阳性药组为8.33，各给药组与结直肠癌模型组相比，统计学差异非常显著(#，p<0.05，##，p<0.01)。本发明化合物5和6与阳性对照药相比，疗效非常显著。肿瘤负荷计算值的计算方法如下：
Figure PCTCN2017106227-appb-000016
表八、本发明化合物5和6对结直肠癌模型小鼠结直肠肿瘤负荷的影响
Figure PCTCN2017106227-appb-000017
Figure PCTCN2017106227-appb-000018
图8为本发明各化合物对结直肠癌模型小鼠结直肠肿瘤的组织病理检测(HE染色，100倍)。
图8显示：
A正常对照组：结肠正常结构，表现为肠组织层次清晰，结构完整，无炎性变。
B模型组：肠组织炎症病变显著；炎症累及整个粘膜上皮及粘膜下固有层(透壁性炎症)，几乎所有隐窝破坏，部分粘膜表皮缺失；炎症病变部位可见大量炎症细胞浸润，炎症细胞以淋巴细胞、中性粒细胞为主、伴少量单核细胞；局部炎症显著区域可见炎症溃疡形成，溃疡灶底部可见炎性纤维组织增生，为中分化腺癌(++++)。
C阳性药卡培他滨组：肠组织炎症病变明显，炎症累及2/3肠粘膜间质，局部可见透壁性炎症病灶，局部隐窝破坏，肠粘膜表皮相对完整；炎症病变部位间质可见散在炎症细胞浸润，炎症细胞以淋巴细胞、中性粒细胞为主、伴少量单核细胞；局部固有层炎症明显，为非癌炎性变为主(+++)。
D化合物5给药组：局部肠组织间质可见散在炎症细胞浸润，炎症局限于粘膜层，未见固有层明显受累，未见明显隐窝破坏，肠粘膜上皮完整；炎症细胞以淋巴细胞为主，伴少量中性粒细胞和单核细胞，非癌(+)。
E化合物6给药组：局部肠组织间质可见散在炎症细胞浸润，炎症局限于粘膜层，未见固有层明显受累，未见明显隐窝破坏，肠粘膜上皮完整；炎症细胞以淋巴细胞为主，伴少量中性粒细胞和单核细胞，非癌(+)。
实验例3：本发明化合物1、2、3和5对氧化偶氮甲烷(Azoxymethane,AOM)/葡聚糖硫酸钠(Dextran sulfate sodium,DSS)诱导的结直肠癌模型小鼠结直肠组织靶标分子STAT3的表达具有显著的抑制作用。
动物实验造模及给药方案同实验例1和2。
各组动物给药八周后，于实验终点按常规方法处死小鼠。选择距小鼠***上1cm处剪取2cm结肠组织。以天平称取等重的结肠组织在液氮中反复冻融两次，于RIPA裂解液中进行超声破碎，之后4度裂解30min，13000rpm离心10min后 取上清液，采用Brandford法测定蛋白浓度。根据蛋白浓度取等量蛋白进行Western-blot(WB)检测。
WB实验检测，按标准SDS-PAGE方法配制5％浓缩胶及10％分离胶。各取含相同蛋白浓度的细胞裂解上清液与5×SDS上样缓冲液混合后，煮沸5min。冷却后上样。电泳结束后湿转法转移至PVDF膜上。用TBST(0.1％Tween-20；10mmol/L Tris-Cl，pH7.5；3％BSA；150mmol/L NaCl)4℃封闭非特异结合位点过夜。用TBST液洗膜，10min/次×3次。将膜与稀释的一抗(1:500)室温孵育3h，TBST液洗膜，10min/次×3次。将膜移入二抗(1:1000稀释)，室温反应2h。TBST液洗膜，10min/次×3次。将膜平放，滴加发光液，化学发光仪呈像。结果表明，本发明化合物1、2、3和5，在蛋白水平对与结直肠癌发病密切相关的信号分子STAT3具有显著的抑制作用。具体数据见图9和图10。图9和图10中，上为WB杂交显影图，下为蛋白相对定量检测结果。
图9为本发明化合物1和2对结直肠癌模型小鼠结直肠组织中靶点分子STAT3蛋白表达的影响图中泳道分别为lane 1：正常对照组；lane 2：AOM/DSS模型组；lane 3：化合物1给药组；lane 4：化合物2给药组。WB蛋白半定量结果显示，与正常对照组小鼠结肠组织中stat3/actin相对定量计算值0.50相比，结直肠癌模型组小鼠肠组织中STAT3表达明显增加，相对值为1.82，统计学上差异非常显著(**，p<0.01)。与AOM/DSS模型组相比，本发明化合物1和2均具有非常显著的改善作用，表现为STAT3蛋白的表达明显降低，stat3/actin的相对比值化合物1为0.25，化合物2为0.33，统计学上差异非常显著(##，p<0.01)。
图10为本发明化合物1，2，3和5对结直肠癌模型小鼠结直肠组织中靶点分子STAT3蛋白表达的影响图中泳道分别为lane 1：正常对照组；lane 2：AOM/DSS模型组；lane 4：化合物2给药组；lane 5：化合物5给药组；lane 7：化合物1给药组；lane 8：化合物3给药组。WB蛋白半定量结果显示，与正常对照组小鼠结肠组织中stat3/actin相对定量计算值0.07相比，结直肠癌模型组小鼠肠组织中STAT3表达明显增加，相对值为0.43，统计学上差异非常显著(**，p<0.01)。与AOM/DSS模型组相比，本发明化合物1，2，3和5均具有非常显著的抑制作用，表现为STAT3蛋白的表达显著降低，stat3/actin的相对比值化合物1为0.13，化合物2为0.23，化合物3为0.19，化合物5为0.20。统计学上差异非常显著(p<0.01)。
实验例4：本发明化合物的急性毒性试验结果
取昆明种小鼠(18-22g)分组，每组10只，雌雄各半，共设8个剂量组，从最高剂量(5g/Kg)按等比级数增减，相邻两剂量比值为1:0.8。灌胃给药。给药前一夜，动物禁食不禁水。动物给药后4h恢复正常饮食。单次给药后，连续观察14天动物的体重、饮食、行为、分泌物、***物、死亡及中毒反应。本发明化合物1-6的试验结果显示LD50值分别为5.0g/Kg、5.0g/Kg、0.8g/Kg、5.0g/Kg、3.9g/kg和1.6g/Kg。此外，二氢小檗碱的LD50值为160mg/Kg。

Claims (6)

  1. 如式1-6所示的化合物二氢异黄连碱、二氢黄连碱、二氢巴马汀、(±)-8-丙酮基二氢异黄连碱、(±)-8-丙酮基二氢黄连碱和(±)-8-丙酮基二氢巴马汀及其药学上可接受的盐在制备STAT3抑制剂中的应用，
    Figure PCTCN2017106227-appb-100001
  2. 如式1-6所示的化合物二氢异黄连碱、二氢黄连碱、二氢巴马汀、(±)-8-丙酮基二氢异黄连碱、(±)-8-丙酮基二氢黄连碱和(±)-8-丙酮基二氢巴马汀及其药学上可接受的盐在制备预防、缓解或/和治疗STAT3信号通路相关肿瘤疾病产品中的应用，
    Figure PCTCN2017106227-appb-100002
  3. 根据权利要求2的应用，其特征在于，所述的STAT3信号通路相关肿瘤疾病选自结肠癌或直肠癌。
  4. 一种药物组合物在制备预防、缓解或/和治疗STAT3信号通路相关肿瘤疾病产品中的应用，其特征在于，所述的药物组合物包含如式1-6所示的二氢异黄连碱、二氢黄连碱、二氢巴马汀、(±)-8-丙酮基二氢异黄连碱、(±)-8-丙酮基二氢黄连碱和(±)-8-丙酮基二氢巴马汀中的一个或多个化合物以及药学上可接受的载体 或赋形剂，
    Figure PCTCN2017106227-appb-100003
  5. 根据权利要求4的应用，其特征在于，所述的结直肠癌包括结肠癌或直肠癌。
  6. 根据权利要求1-5任一项的应用，其特征在于，所述的产品选自药品、保健品。
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