WO2022021981A1 - Dérivé de di-(benzimidazole)-1, 2, 3-triazole, sa préparation et son utilisation - Google Patents

Dérivé de di-(benzimidazole)-1, 2, 3-triazole, sa préparation et son utilisation Download PDF

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WO2022021981A1
WO2022021981A1 PCT/CN2021/091140 CN2021091140W WO2022021981A1 WO 2022021981 A1 WO2022021981 A1 WO 2022021981A1 CN 2021091140 W CN2021091140 W CN 2021091140W WO 2022021981 A1 WO2022021981 A1 WO 2022021981A1
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compound
formula
benzimidazole
bis
mir
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PCT/CN2021/091140
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English (en)
Chinese (zh)
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陆前进
赵明
姜德建
李乾斌
武瑞芳
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中南大学湘雅二医院
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Priority claimed from CN202010748529.7A external-priority patent/CN113004253B/zh
Priority claimed from CN202010748184.5A external-priority patent/CN112999227B/zh
Application filed by 中南大学湘雅二医院 filed Critical 中南大学湘雅二医院
Publication of WO2022021981A1 publication Critical patent/WO2022021981A1/fr

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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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings

Definitions

  • the invention belongs to the technical field of medicine, and in particular relates to a small molecule with medicinal activity.
  • miR-210 is a classic hypoxia-inducible miRNA molecule, and its expression level is regulated by Hypoxia inducible factor-1 ⁇ (HIF-1 ⁇ ). Studies have confirmed that miR-210 is involved in the regulation of biological processes such as cell proliferation, apoptosis, mitochondrial metabolism, DNA damage repair and angiogenesis. Hypoxia is a common pathological condition that is common in various solid tumors, inflammatory reactions, and ischemic diseases. Current studies have shown that miR-210 is abnormally expressed in many solid tumors, cardiovascular and cerebrovascular diseases, inflammatory diseases, and bone metabolic diseases, and it is particularly closely related to inflammatory and immune responses.
  • HIF-1 ⁇ Hypoxia inducible factor-1 ⁇
  • CD4 + T cells including regulatory T cells and helper T cells, play a very important role in the initiation of immune responses and the maintenance of immune homeostasis. They can provide help to other cells and undertake a variety of effector functions.
  • TH1 and TH2 are the classic subtypes involved in immune response;
  • TH17 is a new cell lineage that secretes inflammatory factors such as IL-17 and IL-22 .
  • a large number of studies have shown that excessive activation of TH 17 and TH 1 is one of the important pathogenesis of many autoimmune diseases, inflammatory diseases and solid tumors. Abnormal pathways play a role in targeted therapy.
  • miRNA mimics In vivo administration of miRNA mimics, synthetic specific double-stranded small RNA molecules and miRNA overexpression vectors can restore the level of downregulated miRNAs in vivo; while the use of antimiRs, antisense, and antagomiRs can interfere with overexpressed miRNAs.
  • antimiRs, antisense, and antagomiRs can interfere with overexpressed miRNAs.
  • miRNA mimics synthetic specific double-stranded small RNA molecules and miRNA overexpression vectors can restore the level of downregulated miRNAs in vivo; while the use of antimiRs, antisense, and antagomiRs can interfere with overexpressed miRNAs.
  • antimiRs, antisense, and antagomiRs can interfere with overexpressed miRNAs.
  • the first object of the present invention is to provide a bis-(benzimidazole)-1,2,3-triazole derivative with a new structure.
  • the second object of the present invention is to provide a method for preparing the bis-(benzimidazole)-1,2,3-triazole derivative.
  • the third object of the present invention is to provide a use of the bis-(benzimidazole)-1,2,3-triazole derivative for preparing a drug for inhibiting the expression of miR-210.
  • the fourth object of the present invention is to provide a medicine comprising the bis-(benzimidazole)-1,2,3-triazole derivative.
  • a bis-(benzimidazole)-1,2,3-triazole derivative having the structural formula of formula 1;
  • n and m are independently integers from 1 to 6;
  • At least one substituent is an active substituent, and the active substituent is a hydroxyl group, an alkoxy group, a hydroxyalkyl group or an alkoxyalkyl group.
  • the present invention provides a compound of formula 1 with a brand-new structure, and it is found that the compound with the brand-new structure can unexpectedly inhibit the expression of miR-210 (microRNA-210) based on the intramolecular action of its intramolecular fragments and groups.
  • miR-210 miR-210
  • it can inhibit the differentiation of autoimmune inflammatory TH 17 and TH 1 cells and the secretion of related cytokines, promote the differentiation of TH 2 cells and Secretion of IL-4; unexpected pharmacodynamic activity in autoimmune diseases, inflammatory diseases, infections and tumors mainly mediated by elevated TH17 and/or TH1 cells, and can be used in this type of Small molecule drug development for disease.
  • n is an integer of 1-6; preferably an integer of 2-4; more preferably 3.
  • the m is an integer of 1-6; preferably an integer of 2-4; more preferably 3.
  • the 1,2,3-triazole ring was innovatively modified at its end and controlled that the ring contained at least one of the bis-(benzimidazole) Oxygen-active groups, which can unexpectedly increase the inhibitory effect on miR-210 based on intramolecular as well as molecular steric interactions. For example, it can improve the inhibitory effect of autoimmune inflammatory TH17 and TH1 cell differentiation and the expression of related cytokines, promote the differentiation of TH2 cells and the secretion of IL-4, and strengthen the inhibition of keratinocyte proliferation. Effect.
  • the alkoxy group is C 1 -C 6 alkoxy group; for example, methoxy group, ethoxy group, propoxy group and the like.
  • the hydroxyalkyl group is a C 1 -C 6 alkyl group with one or more hydroxy substituents.
  • a C 1 -C 6 linear or branched alkyl group is modified on the 1,2,3-triazole ring, and a hydroxyl group is substituted with the linear or branched alkyl group.
  • the hydroxyalkyl group is hydroxymethyl, hydroxyethyl and the like.
  • the alkoxyalkyl group is a C 1 -C 6 alkyl group with one or more alkoxy substituents.
  • a C 1 -C 6 linear or branched alkyl group is modified on a 1,2,3-triazole ring, and an alkoxy group is substituted with the linear or branched alkyl group.
  • the structure of the alkoxyalkyl group is: The R is a straight-chain or straight-chain alkyl group, and the n1 is an integer from 1 to 10; when n1 is not 1, the alkoxyalkyl group is a polyether substituent.
  • R 1 and R 2 are a reactive substituent, and the other substituent is H, the reactive substituent, an alkyl group, a phenyl group, a benzyl group or a halogen.
  • the R 1 is hydrogen; the R 2 is a hydroxyalkyl group.
  • the hydroxyalkyl group is preferably hydroxymethyl, 1-hydroxyethyl, 1-hydroxypropyl, 2-hydroxyethyl, 2-hydroxypropyl or 3-hydroxypropyl.
  • the bis-(benzimidazole)-1,2,3-triazole derivative has the following structural formula (Formula 1-A);
  • the preferred novel compound has a better inhibitory effect on miR-210, for example, in autoimmune inflammatory TH17 , TH1 cell differentiation and related cytokine secretion .
  • Inhibition, as well as promotion of TH2 cell differentiation and IL-4 secretion, and inhibition of keratinocyte proliferation have unexpected efficacy and less toxic side effects.
  • the present invention also provides a method for preparing the bis-(benzimidazole)-1,2,3-triazole derivative, which is obtained by subjecting the compound of formula 2 and the compound of formula 3 to a cyclization reaction;
  • the solvent for the cyclization reaction is methanol.
  • a reaction aid is added to the cyclization reaction, preferably a Cu(I) source.
  • the Cu(I) source can be obtained based on the reaction between the Cu(II) source and the reducing agent in the reaction system.
  • the compound of formula 2 is obtained by amidation of the compound of formula 4 and the compound of formula 5;
  • Equation 4 The selection range of n and m in Equation 4 and Equation 5 is the same as that in Equation 1.
  • the amidation reaction can be realized based on existing means.
  • the compound of formula 4 and an activator are activated in advance, and then reacted with the compound of formula 5.
  • the activator is preferably carbodiimide and 1-hydroxybenzotriazole.
  • the compound of formula 4 is obtained by cyclization of compounds of formula 6 and formula 7, followed by ester hydrolysis;
  • the R 3 is a C 1 -C 6 alkyl group, and the selection range of n is the same as in formula 1.
  • the present invention also provides a bis-(benzimidazole)-1,2,3-triazole derivative and pharmaceutically acceptable salts, solvates, co-crystals, derivatized esters, derivatized amides thereof Use of at least one of the compounds (also referred to as an active ingredient in the present invention) for the manufacture of a medicament for inhibiting the expression of miR-210.
  • the compound of formula 1 described in the present invention can unexpectedly knock down the expression of miR-210, and can be used for the treatment of pathological miR-210 overexpression related indications.
  • the active ingredient is used to prepare a drug that specifically inhibits the expression of miR-210;
  • a further preferred application is to prepare a drug that specifically inhibits the expression of miR-210, thereby inhibiting the differentiation of TH 17 and TH 1 cells and their cytokine secretion, and promoting the differentiation of TH 2 cells and the secretion of IL-4.
  • a further preferred application is for the preparation of a drug for the treatment of autoimmune diseases, inflammatory diseases, infections and tumors mainly mediated by elevated expression of miR-210 and elevated TH17 and/or TH1 cells .
  • the autoimmune disease, inflammatory disease, and infection include but are not limited to inflammatory skin diseases (preferably cutaneous lupus erythematosus, psoriasis, parapsoriasis, pityriasis rosea, vitiligo, alopecia areata). at least one of), lichen planus, eczema, urticaria, rheumatoid arthritis, spondyloarthritis, inflammatory bowel disease, multiple sclerosis, type I diabetes, Hashimoto's thyroiditis, human immunodeficiency virus (HIV) infection, hepatitis B virus (HBV) infection, etc.
  • inflammatory skin diseases preferably cutaneous lupus erythematosus, psoriasis, parapsoriasis, pityriasis rosea, vitiligo, alopecia areata. at least one of), lichen planus, eczema, ur
  • a preferred application of the present invention is to combine the bis-(benzimidazole)-1,2,3-triazole derivatives and their pharmaceutically acceptable salts, solvates, co-crystals, esters, and amide derivatives into Use of at least one (also referred to as active ingredient in the present invention) for the preparation of a medicament for inflammatory skin diseases.
  • the pharmaceutically acceptable salt of the compound of formula 1 can be hydrochloride, acetate, mesylate, besylate, oxalate, citrate, sulfuric acid of the compound of formula 1 Salts and other pharmaceutically acceptable salts in any form.
  • the solvate of the compound of formula 1 can be, for example, other solvates such as the hydrate of the compound of formula 1.
  • the co-crystal of the compound of formula 1 is a co-crystal formed by the active compound of formula 1 and other pharmaceutical ligands.
  • the pharmaceutical ligands are, for example, polycarboxylic acid ligands.
  • the ester derived from the compound of formula 1 can be a compound modified with an ester group based on the hydroxyl group in the structure, in order to achieve the purpose of prodrug design, or to improve solubility and drug efficacy.
  • the amide compound derived from the compound of formula 1 can be a compound modified by amide group based on the primary amino group and secondary amino group in the structure, in order to achieve the purpose of prodrug design, or improving solubility and drug efficacy.
  • the bis-(benzimidazole)-1,2,3-triazole derivative is used as an active ingredient in combination with the existing pharmaceutical preparation technology to prepare any pharmaceutically acceptable pharmaceutical dosage form;
  • the existing pharmaceutical preparation technology for example, it is used to prepare skin transdermal external preparations, injection preparations, oral preparations and the like.
  • the inflammatory skin diseases include but are not limited to psoriasis, parapsoriasis, pityriasis rosea, and cutaneous lupus erythematosus.
  • the bis-(benzimidazole)-1,2,3-triazole derivatives and their pharmaceutically acceptable salts, solvates, co-crystals, derivatized esters, and derivatized amide compounds are added to At least one of them is used as an active ingredient in combination with the existing pharmaceutical preparation technology to prepare any pharmaceutically acceptable pharmaceutical dosage form; for example, for the preparation of skin transdermal absorption external preparations, injection preparations, oral preparations, etc.
  • the present invention also provides a medicament comprising a pharmaceutically effective amount of an active ingredient, wherein the active ingredient is the bis-(benzimidazole)-1,2,3-triazole derivative or a pharmaceutically acceptable ingredient thereof. At least one of acceptable salts, solvates, co-crystals, derivatized esters, derivatized amide compounds.
  • the active ingredient of the medicament of the present invention can be the compound of formula 1 (eg free state), the pharmaceutically acceptable salt of the compound of formula 1, the solvate of the compound of formula 1, the co-crystal of the compound of formula 1, the derivative ester, the derivative at least one of the amides.
  • the pharmaceutically acceptable salt of the compound of formula 1 can be hydrochloride, acetate, mesylate, besylate, oxalate, citrate, sulfate, etc. of the compound of formula 1 A pharmaceutically acceptable salt in any form.
  • the solvate of the compound of formula 1 can be, for example, other solvates such as hydrate of the compound of formula 1.
  • the co-crystal of the compound of formula 1 is a co-crystal formed by the active compound of formula 1 and other pharmaceutical ligands.
  • the pharmaceutical ligands are, for example, polycarboxylic acid ligands.
  • the ester derived from the compound of formula 1 can be a compound modified with an ester group based on the free hydroxyl group in the structure, in order to achieve the purpose of prodrug design, or to improve solubility and drug efficacy.
  • the amide compound derived from the compound of formula 1 can be a compound modified by amide group based on the free primary amino group and secondary amino group in the structure, in order to achieve the purpose of prodrug design, or improving solubility and drug efficacy.
  • the medicament further comprises pharmaceutically acceptable excipients.
  • the adjuvant can be any additive other than the active ingredient that is well known to those skilled in the industry, used to make the active ingredient into a formulation and facilitate the exertion of its medicinal effect, for example, it can be a dispersant, a cosolvent. , colorants, sweeteners, disintegrants, stabilizers, corrosion inhibitors, excipients, sprays, antioxidants, etc.
  • the medicament of the present invention has any pharmaceutically acceptable dosage form.
  • the active ingredients described in the present invention can be made into any pharmaceutically acceptable dosage form based on the existing formulation technology, equipment and theory.
  • the dosage form of the drug is external preparation, injection preparation or oral preparation.
  • the external preparation is a transdermal absorption preparation or a transmucosal absorption preparation.
  • a transdermal absorption preparation contains the active ingredient and any known adjuvants that can achieve its transdermal absorption and enhance its transdermal absorption.
  • the external preparation is a patch, dressing, external spray, gel, cream, ointment, external lotion, external oil solution, foam or suppository.
  • the injection preparations are preferably intravenous injection preparations, subcutaneous injection preparations, intradermal injection preparations, and intramuscular injection preparations; more preferably, injection powder preparations or injection solutions.
  • the oral preparation is a tablet, capsule, powder, oral solution or suspension.
  • the medicament of the present invention is a medicament for inhibiting the expression of miR-210.
  • the medicament of the present invention can be used to knock down pathological miR-210 overexpression, and can be used for the treatment of indications related to miR-210 overexpression in pathological conditions.
  • the drug is a drug that specifically inhibits the expression of miR-210, thereby inhibiting the differentiation of TH 17 and TH 1 cells and the secretion of cytokines, and promoting the differentiation of TH 2 cells and the secretion of IL-4.
  • the drug is a drug for the treatment of autoimmune diseases, inflammatory diseases, infections and tumors mainly mediated by elevated expression of miR-210 and elevated TH17 and/or TH1 cells .
  • the autoimmune disease, inflammatory disease, infection includes but is not limited to inflammatory skin disease (preferably at least one of cutaneous lupus erythematosus, psoriasis, parapsoriasis, pityriasis rosea, vitiligo, alopecia areata) a), lichen planus, eczema, urticaria, rheumatoid arthritis, spondyloarthritis, inflammatory bowel disease, multiple sclerosis, type I diabetes, Hashimoto’s thyroiditis, human immunodeficiency virus (HIV) infection, B At least one of hepatitis virus (HBV) infection.
  • inflammatory skin disease preferably at least one of cutaneous lupus erythematosus, psoriasis, parapsoriasis, pityriasis rosea, vitiligo, alopecia areata
  • a inflammatory skin disease
  • Described inflammatory dermatosis is at least one in cutaneous lupus erythematosus, psoriasis, parapsoriasis, pityriasis rosea, vitiligo, alopecia areata; further preferably including but not limited to psoriasis, parapsoriasis, Pityriasis rosea or cutaneous lupus erythematosus.
  • the drug in addition to the formula 1 and its related active ingredients described in the present invention, also includes other types of pharmaceutical active ingredients, preferably, other active ingredients that can be used to inhibit the expression of miR-210 .
  • the other active ingredients that can be used to inhibit the expression of miR-210 are the reported pharmaceutical active ingredients that can be used to inhibit the expression of miR-210, especially those that can be used to specifically inhibit the expression of miR-210.
  • the dosage form of the medicament of the present invention is an external preparation; more preferably, it is a transdermal absorption preparation or a transmucosal absorption preparation.
  • a transdermal absorption preparation contains the active ingredient and any known adjuvants that can achieve its transdermal absorption and enhance its transdermal absorption.
  • the external preparation is a patch, dressing, external spray, gel, cream, ointment, external lotion, external oil solution, foam or suppository.
  • the medicine is a medicine for the treatment of inflammatory skin diseases; further preferably, the inflammatory skin diseases include but are not limited to psoriasis, parapsoriasis, pityriasis rosea, cutaneous lupus erythematosus at least one of them.
  • a preferred medicine of the present invention is a topical preparation such as ointment, gel, liniment, cream, etc. for treating inflammatory skin diseases, preferably an ointment, preferably, it comprises an ointment base and the described ointment base dispersed in the ointment base. Active ingredient.
  • the ointment base can be an existing base, or a base that is prepared based on existing theories and is suitable for use.
  • the ointment base is an oily base, an emulsion type base or a water-soluble base; preferably an oily base.
  • the ointment base is at least one of white petrolatum, lanolin, and cetyl alcohol; preferably, it includes white petrolatum and lanolin.
  • the ratio of the white petrolatum and lanolin can be adjusted according to the needs of the paste, preferably 100-300:5-30; more preferably 250-300:5-15.
  • the weight percentage of the active ingredient is 0.001%-1%; preferably 0.008%-0.8%.
  • the present invention provides a novel compound, and it is found that the novel compound can inhibit the overexpression of pathological miR-210 and can be used for the treatment of diseases related to overexpression of miR-210.
  • the novel compounds and their derivatives can inhibit the differentiation of inflammatory TH17 and TH1 cells and the secretion of related cytokines, promote the differentiation of TH2 cells and the secretion of IL-4, and inhibit keratinocytes .
  • the brand-new compound of formula 1 described in the present invention not only has good effects in terms of drug efficacy, but also has advantages in terms of toxic and side effects, physical properties, etc.
  • the compound described in the present invention has good solubility and Stability, with lower toxic side effects.
  • the invention has good application prospects in the drug development of autoimmune diseases, inflammatory diseases and infectious diseases.
  • Fig. 1-1 is the HPLC chart of the formula 1-A (TD-02) synthesized in Example 1;
  • Figure 1-2 is the H-NMR chart of formula 1-A (TD-02) synthesized in Example 1;
  • Figures 1-3 to 1-9 are the H-NMR diagrams of TD-01, TD-03, TD-04, TD-05, TD-06, TD-07, and TD-08 prepared in Comparative Example 1, respectively;
  • FIG. 3-1 Topical application of TD-02 significantly improved imiquimod-induced psoriasis-like skin lesions in mice.
  • A Expression of miR-210 in skin lesions of mice in each group;
  • B, C gross (B) and pathological (C) changes in inflammatory skin lesions of mice in each group;
  • D, E mice in each group Epidermal thickness (D) and inflammatory cell infiltration (E) in skin lesions.
  • FIG. 3-2 Topical application of TD-02 can improve the inflammatory disorder in psoriasis-like skin lesions in mice.
  • A, B Flow and statistical graphs of the proportion of TH 17 and TH 1 cell infiltration in the skin lesions of mice in each group;
  • CE mRNA expressions of il17a, ifng and il4 in the skin lesions of mice in each group. * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001
  • Figure 4 (such as 4-1 to 4-12) are respectively the microscopic pictures of organ tissue morphology (the scales are all 50 ⁇ m).
  • the solvents DMF and THF used in the reaction were anhydrous treated with CaH 2 , and EtOH was anhydrous treated with metallic Na.
  • the nuclear magnetic resonance apparatus used was Bruker DPX 400-MHz.
  • Step (2) Synthesis of 4-amino-3-nitrobenzylimide ethyl ester (7):
  • the peripheral blood of 4 patients with psoriasis vulgaris was collected, centrifuged by density gradient and used to separate CD4 + T cells with magnetic beads, and then treated with 2 ⁇ M compound 16 and TD-01 to TD-08 small molecule compound solutions and corresponding volumes of solvent, respectively, 48 hours later Cells were collected, total RNA was extracted, and the expression of miR-210 was detected by real-time PCR.
  • the results showed that compared with the solvent-treated control group, the expression of mature miR-210 in CD4 + T cells of all 4 patients with psoriasis was significantly decreased after compound 16, TD-02, TD-05, and TD-07 treatment.
  • TD-02 On miR-210 is the strongest among the four compounds, while the inhibitory effect of TD-05 and TD-07 on miR-210 is slightly weaker than that of compound 16; TD-01, TD-03, TD- 04, TD-06 and TD-08 had no significant effect on the expression of mature miR-210 in psoriatic CD4 + T cells (Fig. 2-1). Therefore, we selected TD-02, TD-05 and TD-07 for further functional screening.
  • CD4 + T cells were sorted by magnetic beads, activated with anti-CD3 antibody and anti-CD28 antibody for 24 hours (hrs), and 0 nM, 20 nM, 100 nM, 200 nM, 500 nM and 1 ⁇ M compound 16, TD-02 were added, respectively. , TD-05 and TD-07 solutions, and continue to incubate for 48hrs. Cells were collected, and flow cytometry was used to detect the proportion of different CD4 + T cell subsets ( TH 1, TH 2 and TH 17) in each group, and Real-time PCR was used to detect the corresponding cytokine IFN in cells of each group. - mRNA expression levels of ⁇ , IL-17A and IL-4.
  • Human keratinocyte line HaCaT cells were used in this experiment. Before treatment for 12hrs, HaCaT cells were seeded in 96-well plates and cultured in a 37°C, 5% CO2 incubator for 12h. When the cells grow to 80%-90% confluency, add OnM, 20nM, 100nM, 200nM, 500nM, 1 ⁇ M Compound 16, TD-02, TD-05 and TD-07 solutions to the keratinocytes, respectively, and continue to culture for 24hrs . 4 hrs before the end of the culture, 10 ⁇ L of CCK8 solution was added to each well to continue culturing for 4 hrs. Cell proliferation was detected at a wavelength of 450 nm. The results showed that compound 16 and TD-02 could significantly inhibit the proliferation of HaCaT cells, and the inhibitory effect was in a concentration-dependent manner. Cell proliferation was not significantly affected (Figure 2-3).
  • TD-02 shows better effects than other compounds: TD-02 unexpectedly effectively inhibits the expression of mature miR-210, thereby inhibiting the differentiation of TH 17 and TH 1 cells And cytokine secretion, promote TH 2 cell differentiation and IL-4 secretion; at the same time, TD-02 can also significantly inhibit the proliferation of keratinocytes.
  • Embodiment 3 TD-02 ointment small test recipe technology:
  • Preferred Composition 1 Preferred Composition 2
  • White Vaseline 18.0g 19.0g
  • Lanolin 1.0g 1.0g cetyl alcohol 1.0g /
  • Imiquimod (IMQ) applied to the back skin of mice can induce a psoriasis-like mouse model, and psoriasis-like pathological changes appear on the back skin of mice. Therefore, the IMQ-induced psoriasis model has been widely recognized for the study of psoriasis pathogenesis and therapeutic drugs.
  • mice in each group were photographed every other day and scored for PASI of psoriatic skin lesions. They were sacrificed on the 7th day, and their back skin lesions were removed, and HE staining was performed to observe the pathological changes. At the same time, RNA was extracted to detect the changes of miR-210 and inflammatory cytokines. , the dermal single cell suspension was separated and flow cytometry was used to detect the proportion of TH 17 and TH 1 cells. The results showed that: on the 7th day of modeling, compared with normal mice, the expression of miR-210 in the skin lesions of the IMQ group and the blank matrix group was significantly increased (Fig.
  • Topical TD-02 ointment can significantly improve IMQ-induced inflammatory skin lesions in mice (including gross and pathological changes), and the 0.08% TD-02 group was the most significant ( Figure 3-1B-E).
  • topical TD-02 ointment can significantly inhibit the infiltration of TH 17 and TH 1 cells in IMQ-induced inflammatory skin lesions in mice ( Figure 3-2A, B), inhibit the expression of IL-17A and IFN- ⁇ mRNA, and promote IL-4 mRNA expression ( Figure 3-2C-E).
  • test product in this study is TD-02 ointment (prepared in Example 3), and the main indication is the topical treatment of inflammatory skin diseases.
  • the intended clinical route of administration is skin administration, and the intended clinical dosage should not exceed 100g per week.
  • Non-clinical trials of TD-02 ointment carried out toxicological studies Rodents (SD rats) were used as the experimental system for the toxicological studies of TD-02 ointment to investigate the long-term toxic effects of the test substance after transdermal administration, and the results were obtained in The long-term toxicity test is accompanied by the assessment of plasma and skin exposure to determine the toxic reaction, toxic target organ or target tissue of repeated administration of the test substance.
  • the toxicology study of TD-02 ointment used rodents (SD rats) as the experimental system to investigate the long-term toxic effects of the test substance after administration through the skin, and the drug exposure in plasma and skin was evaluated in the long-term toxicity test. , to determine the toxic reaction, toxic target organ or target tissue of repeated administration of the test substance.
  • SPF SD rats were selected as the experimental system, and a 14-day repeated administration toxicity study was carried out in rats according to the proposed clinical route of TD-02 ointment.
  • 40 male SD rats were selected and randomly divided into 4 groups according to body weight, with 10 animals in each group.
  • TD-02 ointment low-dose damaged skin group TD-02 ointment medium-dose damaged skin group
  • TD-02 ointment high-dose damaged skin group drug content: 0.000%, 0.080%, 0.240%, 0.800%, respectively
  • each The animals in the group were given 10% body surface area, and the coating dose was about 0.03 g/cm 2 , once a day, 7 days a week, for a total of 14 days (2 weeks).
  • toxicokinetic blood collection was performed for the first administration, and toxicokinetic blood collection and skin collection were performed for the last administration. /2 etc.
  • the lower limit of skin quantification was 5mg/L, and the linear range was 5-320mg/L. No TD-02 was detected in the skin of the blank group for 30 minutes. g; 3h skin concentration was 19.82 ⁇ 13.65mg/L, and the content was 79.26 ⁇ 54.62mg/g; 24h skin concentration was lower than the lower limit of quantification. According to the characteristics of local administration of the drug, it is suggested that the drug can penetrate the skin, and the clearance time is relatively fast, and it will not accumulate in the skin within 24 hours.
  • test product in this study is TD-02 ointment, and its main indication is the topical treatment of inflammatory skin diseases.
  • the intended clinical route of administration is skin administration, and the intended clinical dosage should not exceed 100g per week.
  • This study was evaluated by the preclinical preliminary pharmacology and toxicology study of TD-02 ointment, including:
  • Fig. 4-1 Liver of blank matrix damaged skin group (1M02 animal). At the end of administration, HE staining, ⁇ 200. Hepatocyte cords were arranged in an orderly manner, no degeneration or necrosis of hepatocytes, and no congestion of hepatic sinusoids.
  • Figure 4-3 The heart of the blank matrix damaged skin group (1M02 animal). At the end of administration, HE staining, ⁇ 200. Myocardial fibers were evenly stained, with clear horizontal stripes, no degeneration, necrosis, interstitial hemorrhage, or infiltration of inflammatory cells.
  • Fig. 4-4 Lungs of blank matrix damaged skin group (1M02 animal). At the end of administration, HE staining, ⁇ 200. The structure of the alveolar wall was normal, there was no inflammatory cell infiltration in the interstitium, and no obvious exudate was found in the alveolar cavity.
  • FIG 4-5 Spleen of blank matrix damaged skin group (1M02 animal). At the end of administration, HE staining, ⁇ 200. The structure of the white pulp and red pulp of the spleen was clear, and there was no congestion or fibrous tissue hyperplasia.
  • Fig. 4-6 Damaged skin of blank matrix damaged skin group (1M02 animal). At the end of administration, HE staining, ⁇ 100. Epidermal parakeratosis, thickened acanthus, blisters and crusts can be seen.
  • FIG. 4-7 Liver of TD-02 ointment high-dose damaged skin group (4M01 animal). At the end of administration, HE staining, ⁇ 200. Hepatocyte cords were arranged in an orderly manner, no degeneration or necrosis of hepatocytes, and no congestion of hepatic sinusoids.
  • Figure 4-9 Heart of TD-02 ointment high-dose damaged skin group (4M01 animal). At the end of administration, HE staining, ⁇ 200. Myocardial fibers were evenly stained, with clear horizontal stripes, no degeneration, necrosis, interstitial hemorrhage, or infiltration of inflammatory cells.
  • Fig. 4-10 Lungs of TD-02 ointment high-dose damaged skin group (4M01 animal). At the end of administration, HE staining, ⁇ 200. The structure of the alveolar wall was normal, there was no inflammatory cell infiltration in the interstitium, and no obvious exudate was found in the alveolar cavity.
  • FIG. 4-11 Spleen of TD-02 ointment high-dose damaged skin group (4M01 animal). At the end of administration, HE staining, ⁇ 200. The structure of the white pulp and red pulp of the spleen was clear, and there was no congestion or fibrous tissue hyperplasia.
  • FIG. 4-12 Damaged skin of TD-02 ointment high-dose damaged skin group (4M01 animal). At the end of administration, HE staining, ⁇ 100. Focal ulceration of the skin epidermis, parakeratosis, thickening of the acanthus, and crusts can be seen.
  • the compound of formula 1-A described in the present invention has excellent efficacy and lower toxic and side effects, and can be used for drug development of inflammatory skin diseases.

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Abstract

La présente invention appartient au domaine technique des micro-molécules médicales. L'invention concerne spécifiquement un nouveau dérivé de di-(benzimidazole)-1, 2, 3-triazole, sa préparation, son utilisation et une préparation contenant le principe actif. Selon la présente invention, les recherches montrent que le nouveau composé de marque a un effet inattendu en termes d'inhibition de l'expression de miR-210, et peut être utilisé pour développer des médicaments pour des maladies associées à la surexpression de miR-210 pathologique.
PCT/CN2021/091140 2020-07-30 2021-04-29 Dérivé de di-(benzimidazole)-1, 2, 3-triazole, sa préparation et son utilisation WO2022021981A1 (fr)

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CN202010748645.9 2020-07-30
CN202010748529.7A CN113004253B (zh) 2020-07-30 2020-07-30 二-(苯并咪唑)-1,2,3-三唑衍生物及其制备和在炎症性皮肤病中的应用
CN202010748645 2020-07-30
CN202010748184.5 2020-07-30
CN202010748184.5A CN112999227B (zh) 2020-07-30 2020-07-30 一种包含二-(苯并咪唑)-1,2,3-三唑衍生物的药物
CN202010748529.7 2020-07-30

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