WO2019223688A1

WO2019223688A1 - Application of compound fg-4592 in preparation of pharmaceutical preparation for treating thyroid hormone receptor-mediated diseases

Info

Publication number: WO2019223688A1
Application number: PCT/CN2019/087821
Authority: WO
Inventors: 李勇; 姚本强
Original assignee: 厦门大学
Priority date: 2018-05-22
Filing date: 2019-05-21
Publication date: 2019-11-28
Also published as: CN110507655B; CN110507655A

Abstract

Compound FG-4592, where application in preparation of a thyroid hormone receptor (THR) agonist and a pharmaceutical preparation for preventing or treating THR-mediated diseases is provided. The present invention relates to adjustment to the activity of a THR, in particular to selective activation of THRβ and lesion bodies thereof, and application in methods for preventing or treating THR-mediated diseases such as hypothyroidism, resistance to thyroid hormone, and depression.

Description

Application of compound FG-4592 in the preparation of thyroid hormone receptor-mediated diseases and pharmaceutical preparations

Technical field

The invention relates to the use of FG-4592 as a thyroid hormone receptor THR agonist, in particular to the application of the compound FG-4592 in the preparation of a pharmaceutical preparation for treating a thyroid hormone receptor-mediated disease.

Background technique

Compound FG-4592, also known as Roxadustat, CAS number is 808118-40-3, and its structural formula is as follows:

FG-4592 is a HIFαprolyl hydroxylase inhibitor that also induces EPO production and is used clinically to treat anemia associated with chronic kidney disease and end-stage renal disease. The oral small molecule stabilizes the heterodimer of HIF1α and HIFβ by inhibiting the hypoxia-inducible factor HIF1α hydroxylase, thereby regulating downstream target genes, promoting the production of erythropoietin (EPO), inhibiting inflammation, and down-regulating hepcidin levels (Free iron storage hormone) and up-regulate other iron-promoting genes to ensure effective mobilization and utilization of the body's own stored iron. Red blood cell production in the process of restoring the body's natural balance (Beuck, S., W. Schanzer, and M. Thevis, Drug Test Anal, 2012.4 (11): p.830-45).

A nuclear receptor is a ligand-activated transcription factor. Thyroid hormone receptors (THRs) are very important members of the 48 nuclear receptors in the human body. There are two types of THRα and THRβ. THRs promote the growth, differentiation and metabolism of the human body under the regulation of transcriptional activation of the natural ligand agonists thyroid hormones T3 and T4. The principle of THR-mediated pharmacological action is: Thyroid hormone ligands bind to the ligand binding domain (LBD) of THR and affect the recruitment of various co-activators (or co-inhibitors) by inducing THR receptor conformational changes ), Such as the co-activator SRC1, which in turn regulates the transcription of downstream target genes.

Thyroid production of T4 and T3 is under multiple negative feedback regulation. TSH (also called thyroid stimulating hormone) is responsible for normal thyroid function and regulation of thyroid hormone secretion. TSH is synthesized in the pituitary gland chiba, and its secretion is controlled by the thyroid stimulating hormone releasing hormone (TRH) synthesized in the hypothalamus. At normal levels, this neuro- and hormonal feedback regulation maintains body weight, metabolic rate, body temperature, mood, and affects serum low-density lipoprotein (LDL) levels. Therefore, there is weight gain, diabetes, hypertriglyceridemia, hypercholesterolemia, atherosclerosis, obesity, and endocrine system diseases when thyroid function is degraded or under thyroid hormone resistance.

Hypothyroidism or hypothyroidism, referred to as hypothyroidism, refers to a clinical syndrome caused by thyroid hormone deficiency due to different reasons, which causes the body's metabolism and the function of each system in the body to decline. According to its cause, it is divided into primary hypothyroidism, secondary hypothyroidism and peripheral hypothyroidism. Clinical manifestations include memory loss, mental retardation, drowsiness, ataxia; bradycardia, reduced cardiac output, and concurrent coronary heart disease; anorexia, bloating, constipation, which can lead to malignant anemia and iron deficiency anemia; muscle weakness, pain , Rigidity, may be accompanied by joint lesions such as chronic arthritis; women with more menstrual periods, chronic disease, amenorrhea, infertility; male impotence, hyposexuality; when the condition is severe, improper stress can induce coma, shock, and heart and kidney failure Waiting; minor illness; juvenile hypothyroidism such as short stature, low intelligence, delayed sexual development; and non-shiatsu edema (Myxedema). Early mild cases were dominated by oral thyroid hormones or thyroid hormone receptor (THR) agonists (Letrothyroxine, etc.) (China National Health and Family Planning Commission's authoritative medical science project communication network platform / Encyclopedia Medical Network).

Thyroid hormone resistance syndrome (Thyroid Hormone resistance syndrome) is also called thyroid hormone refractory syndrome or thyroid hormone insensitivity syndrome (THIS). There is currently a serious lack of treatment for this syndrome. According to its onset and clinical manifestations, it can be divided into 3 types: 1. Systemic thyroid hormone refractory disease, which involves the pituitary gland and surrounding tissues. This type can be divided into normal thyroid function compensatory normal type and hypothyroidism type; 2. Selective pituitary is refractory to thyroid hormones, which is characterized by a lot of pituitary involvement and does not respond to thyroid hormones, while the remaining peripheral tissues are not affected and can respond to thyroid hormones normally. Its clinical manifestations are hyperthyroidism; 3. Peripheral tissues should be refractory to thyroid hormones. This type is characterized by the surrounding tissues not responding or insensitive to thyroid hormones, while the pituitary gland is mostly unaffected and responds normally to thyroid hormones. Clinical manifestations of goiter, no deafness and epiphyseal changes. Although the thyroid hormone is normal and TSH is normal, the clinical manifestations of hypothyroidism, bradycardia, edema, fatigue, abdominal distension and constipation are abnormal. This type can be manifested as poor intelligence, lagging development, or having mature bones. Most thyroid hormone resistance syndromes are caused by mutations in the thyroid hormone receptor THRs gene, which causes changes in the amino acid sequence of the thyroid hormone receptor, leading to changes in the structure and function of the receptor, and resistance or insensitivity to the thyroid hormones T3 and T4. . Therefore, looking for thyroid hormone receptor agonists that are structurally different from the natural thyroid hormones T3 and T4 is expected to treat thyroid hormone resistance syndrome.

Hyperthyroidism, referred to as "hyperthyroidism," is a condition in which the thyroid gland releases excessive thyroid hormones, causing the body's metabolic metabolism and sympathetic nerve excitement to cause palpitations, sweating, eating, increased stool frequency, and weight loss. Most patients also have symptoms such as exophthalmos, eyelid edema, and vision loss.

THRs and cognitive impairment and depression:

Affective mental disorder caused by thyroid hormone homeostasis is one of the causes of individual illness. Although patients may show short-term thyroid function, in the subgroup of patients, the incidence of clinical hypothyroidism and hyperthyroidism is manifested. For a significant increase. Thyroid hormones are essential for the development of the central nervous system, especially the brain. Patients with mental illness, especially cognitive impairment and depression, are closely related to thyroid hormone metabolism and abnormal thyroid system immune function. Therefore, thyroid hormone is frequently used to accelerate and enhance the treatment of antidepressant drugs, support the maintenance treatment of bipolar disorder and affective disorders, even in patients with normal thyroid function (Bauer, M., et al., Mol Psychiatry, 2002.7 (2 ): p.140-56). The injection of thyroid hormone T4 in patients with hypothyroidism leads to improvement in mood and learning ability, and positive experimental results are positively correlated with increased free thyroid hormone concentrations (Bunevicius, R., CurrOpin Psychiatry, 2009.22 (4): p.391-5 ).

Thyroid hormones have an important effect on the central nervous system, and mental disorders, especially emotional disorders, are often associated with thyroid hormone metabolism disorders in the brain (Bauer, M., et al., J Neuroendocrinol, 2008.20 (10): p.1101 -14). For a long time, T3 has been used to treat patients with depression alone or in combination with other antidepressants to enhance or accelerate the effect of antidepressants on patients with thyroid depression. For example, the combined use of T3 and tricyclic antidepressants has significantly accelerated tricyclic antidepressants in clinical trials for the treatment of antidepressants, showing the obvious efficacy of this treatment method (Aronson, R., et al., Arch Gen Psychiatry, 1996.53 (9): p.842-8). In addition, the effectiveness and safety of T3 in combination with another mainstream antidepressant, selective serotonin transporter inhibitors, for the treatment of major depression have also been demonstrated (Cooper-Kazaz, R. and B. Lerer, Int J Neuropsychopharmacol, 2008.11 (5): p.685-99). These data indicate that to ensure the normal development and maturity of the central nervous system, specific enhancement of THRs activity is required to enhance metabolism, growth regulation and signaling in the nervous system, thereby increasing learning ability and avoiding cognitive disorders, as well as treating depression. And other emotional disorders.

THRs and high cholesterol series diseases:

Thyroid hormone regulates lipid and cholesterol metabolism through THRβ. However, the clinical use of natural thyroid hormone and thyroid receptor agonists to treat hypercholesterolemia is only used in patients with thyroid dysfunction. The reason is that non-specific agonists activate the THRα receptor, which has a negative impact on cardiovascular disease. Therefore, finding efficient and specific activation of THRβ instead of THRα will become a solid direction for the treatment of a series of diseases caused by abnormal lipid metabolism and high cholesterol. Therefore, it is still necessary to find effective and specific thyroid hormone receptor β agonists, regulate lipid metabolism, and lower cholesterol to treat diseases such as dyslipidemia, fatty liver, hypercholesterolemia, and atherosclerosis.

THRs and non-alcoholic steatitis:

Recent clinical reports indicate that thyroid hormone receptor beta-specific agonists can protect liver tissue, promote liver function, and treat non-alcoholic fatty liver (NAFLD). The research progress in the data published by Madrigal Pharmaceuticals is praised as a new and effective way to treat non-alcoholic steatohepatitis beyond FXR, PPARα / δ. Another scientist, Kim D, and others found that non-alcoholic steatohepatitis (NASH) and liver fibrosis in patients with subclinical hypothyroidism are directly related to their hypothyroidism. Non-alcoholic steatohepatitis and advanced fibrosis were higher in subjects with lower thyroid function. Patients with hypothyroidism have more severe hepatic steatosis, as well as more severe balloon degeneration and fibrosis (Kim, D., et al., Clin Gastroenterol Hepatol, 2018.16 (1): p.123-131e1). Similar to this, another group of scientists Miyake et al. Found that patients with hyperthyroidism have significantly improved the pathological state of non-alcoholic steatohepatitis. According to their research, this effect and liver enzyme levels increase with the increase of thyroid hormone levels related. Therefore, hyperthyroidism can improve the pathological state of non-alcoholic steatohepatitis (Miyake, T., et al., Intern Med, 2016.55 (15): p.2019-23). These data show that the prevention and treatment of non-alcoholic fatty liver disease, targeting thyroid hormone receptors and improving receptor activity is an effective way.

Development direction of thyroid hormone analogs:

At present, in view of the effectiveness and extensiveness of thyroid hormone receptors in treating diseases such as hypothyroidism, thyroid hormone resistance syndrome, and depression, the development of stronger thyroid hormone agonists has become the main direction to overcome. However, there are still two major problems to be solved in this research and development field. First, as mentioned above, the natural thyroid hormone T3 has not shown any selectivity in combining two THR subtypes (THRα and THRβ). Therefore, although administration of T3 can reduce plasma cholesterol, low-density lipoprotein (LDL), and triglyceride levels in animal models and humans, however, due to side effects of T3 on the heart (such as tachycardia, arrhythmia, and muscle Shrinking), limiting its application. Studies on knockout animals and the results of some selective ligands suggest that these cardiac side effects can be attributed to THRα activation. Therefore, the current research and development of thyroid hormone agonists is mainly to increase the selective activation of THRβ and reduce the activation of THRα. Another major problem is the adverse effects of genetic mutations. Thyroid hormone resistance (Refetoff syndrome) describes a rare symptom of elevated thyroid hormone levels, but thyroid stimulating hormone (TSH) levels are not suppressed or are not expected to be completely suppressed. In essence, this reduces the response of thyroid hormone effectors to thyroid hormones (Refetoff, S., et al., J Clin Endocrinol Metab, 1967.27 (2): p.279-94). Therefore, despite increasing serum thyroid hormone levels, individuals may experience no change in thyroid function. The most common symptoms are goiter and tachycardia. It is also related to some attention deficit hyperactivity disorder (ADHD) and depression (Hauser, P., et al., N. Engl Med, 1993.328 (14): p.997-1001; Fardella, CE, et al., Endocrine , 2007.31 (3): p.272-8). The most common cause of this syndrome is mutations in the THRβ gene. There are now more than 100 different cases of thyroid hormone receptor β site mutations leading to thyroid hormone resistance syndrome, and new mutation sites are still being reported ( Beato-Vibora, P., J. et al., Eur J. Obstet Gynecol Repeat Biol, 2013.167 (1): p.118-9). There is still no effective treatment for thyroid hormone resistance syndrome. Mutations of thyroid hormone receptor protein have rendered the original natural ligands such as T3 and a variety of THRβ selective agonists ineffective. New drugs suitable for polythyroxine resistance syndrome Urgent development.

Therefore, based on the above background, it is still urgent to develop more specific and selective thyroid hormone receptor ligands, especially THRβ selective agonists and new agonists that can activate THRs mutants. These new agonists can lead to specific prevention and treatment of various diseases such as hypothyroidism, thyroid hormone resistance syndrome and depression, while avoiding cardiovascular irritation and other toxicity of natural thyroid.

Summary of the Invention

The purpose of the present invention is to use the compound FG-4592 (or hydroxyquinine drugs) in the preparation of an thyroid hormone receptor (THR) agonist, and in the preparation of thyroid hormone receptor-mediated Application in pharmaceutical preparations.

The structural formula of the compound FG-4592 (or hydroxyquinine drugs) is as follows:

The compound FG-4592 or a pharmaceutically acceptable salt thereof can be used in preparing an agonist of thyroid hormone receptor (THR), the THR includes THRα and THRβ, and the agonist includes a full agonist Or partial agonist.

The compound FG-4592 or a pharmaceutically acceptable salt thereof can be used in preparing a selective agonist of THRβ.

The compound FG-4592 or a pharmaceutically acceptable salt thereof can be used in preparing a pharmaceutical preparation for preventing or treating a thyroid hormone receptor-mediated disease. The thyroid hormone receptor-mediated diseases include hypothyroidism, hypothyroidism, polythyroid hormone resistance syndrome, mental illness, non-alcoholic fatty liver, and the like. The mental illness includes, but is not limited to, attention deficit hyperactivity disorder (ADHD), depression, mental retardation, and cognitive dysfunction.

The polymorphic thyroid hormone resistance syndrome includes, but is not limited to, diseases caused by mutations in the THRβ receptor protein ligand binding domain known clinically. The THRβ site mutations include, but are not limited to, V264D, A268D , R282S, V283A, M310T, E311K, S314C, A317T, R320C, N331D, G332E, G332R, L346F, L346V, H435L, R438H, F459C, F459L, etc.

The compound FG-4592 or a pharmaceutically acceptable salt thereof can be used in the preparation of a pharmaceutical composition used in combination with other pharmaceutical preparations for preventing or treating thyroid hormone receptor-mediated diseases.

The compound FG-4592 is a pharmaceutical composition that includes an effective dose of FG-4592 or a pharmaceutically acceptable salt thereof.

The compound FG-4592 has a good THR agonistic effect. Experiments have shown that compound FG-4592 can bind to nuclear receptor thyroid hormone receptor (THR), induce THR to recruit co-regulatory factors, and activate the expression of target genes of THR. This result indicates that the compound is a small molecule "regulator" or "THR ligand" of the nuclear receptor THR, and therefore, it can be used as a THR agonist for preventing or treating related diseases mediated by THR. Wherein the THR agonist includes a full agonist or a partial agonist of THRα and THRβ.

AlphaScreen experiments show that FG-4592 can induce THRα and THRβ to recruit co-activators. Because coactivators can directly activate the THR downstream pathway, FG-4592 is an agonist of the nuclear receptor THR. Reporter gene experiments show that FG-4592 can activate the transcriptional activity of nuclear receptors THRα and THRβ on its target genes, indicating that FG-4592 is an agonist of nuclear receptor THR. The X-ray crystal diffraction method further elucidates the binding mode of THRβ and FG-4592 from the atomic level. Therefore, multiple methods have been used to prove that FG-4592 thyroid hormone receptor THR agonists can be used as thyroid hormone analogs to prevent or treat related diseases mediated by THR.

Reporter gene experiments have demonstrated that FG-4592 significantly activates the transcriptional activity of THRβ mutants with polymorphic thyroid hormone resistance syndrome and can be used to treat thyroid hormone resistance syndrome. X-ray crystal diffractometry showed that these mutants changed the conformation of THR ligand binding domain (LBD) and lost their strong binding ability to thyroid hormones T3 and T4. However, FG-4592 has significantly different structures from thyroid hormones T3 and T4. Therefore, FG-4592 can bind to these THRβ receptor protein site lesions and significantly activate their transcriptional activity. Therefore, FG-4592 can be used to treat thyroid hormone resistance synthesis Sign.

The above experiments show that FG-4592 is an agonist of nuclear receptor THRβ. It can regulate the functions of THRβ involved in endocrine metabolism such as energy metabolism, lipid metabolism, cholesterol-cholic acid metabolism, and nervous system functions in combination with the target THRβ. Adjustment.

FG-4592 is a hydroxyquinine drug that can be used to treat chronic kidney disease anemia (CKD) by oral administration. It is mainly used in the second generation of hypoxia-inducible factor proline hydroxylase inhibitor (HIF-PHI). Nephropathy and anemia associated with end-stage renal disease. So far, there have been no reports about its combination with THRs and its use in drugs that treat thyroid hormone receptor-mediated diseases such as thyroid hormone resistance, hypothyroidism, or hypothyroidism. Therefore, the FG-4592 given in the present invention has therapeutic functions in these diseases, and it is all about the new application method of FG-4592, which is creative.

Thyroid hormone receptor-mediated diseases such as thyroid hormone resistance, hypothyroidism, or hypothyroidism seriously affect human health and life. Therefore, the FG-4592 given in the present invention is useful in the preparation of pharmaceutical preparations for the treatment of these major diseases. Practical functions, which have important social value and huge economic value, are practical.

In some embodiments, the compounds in the methods described herein can be formulated into pharmaceutical compositions for use in some dosing regimens. The composition of the medicament of the present invention may contain the compound itself and a salt or a carrier for administration thereof. Such compositions may also optionally contain other therapeutic agents.

Certain agents or therapies may be administered in combination with the compounds described herein, such as antidepressants, various THR ligands, cytokine antagonists, immunosuppressants, cytokines, growth factors, immunomodulators, prostaglandins or Anti-hyperplasia compounds.

The term "combination" and its related terms used in the present invention sequentially correspond to that the therapeutic agent and the compound of the present invention are administered simultaneously or sequentially. For example, a certain compound may be administered simultaneously or sequentially with another therapeutic agent in a single unit dosage form. . Accordingly, the present invention provides a single unit dosage form comprising the compound, an additional therapeutic agent. In many dosing regimens, when a patient or individual is exposed to at least two agents at the same time, if the patient or individual simultaneously shows the relevance of the agent used in a particular target tissue or sample (e.g., in the brain, in serum, etc.) Therapeutic effects are generally considered to work in a "combination".

If these compounds are pharmaceutically acceptable salt preparations of the compounds of the present invention, the salts used should preferably be derived from inorganic or organic acids and bases. Acid salts include: acetate, adipate, alginate, aspartate, benzoate, besylate, bisulfate, butyrate, citrate, camphor, camphor Sulfonate, cyclopentane, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glyceryl phosphate, hemisulfate, heptanoate, hexanoate , Hydrochloride, hydrobromide, hydroiodate, 2-hydroxyethanesulfonate, lactate, maleate, mesylate, 2-naphthalenesulfonate, nicotinate, oxalic acid Salt, paraben, pectate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, toluene One of sulfonate and undecanoate. Alkali salts include ammonium, alkali metal, alkaline earth metal and organic base salts; the alkali metal salts include sodium and potassium salts, the alkaline earth metal salts include calcium and magnesium salts, and the organic base salts include bicyclic One of hexylamine salt, N-methyl-D-glucosamine salt, or salt with amino acid such as arginine, lysine and the like.

The pharmaceutical composition of the present invention can be formulated into solid or liquid forms, including the following suitable forms of administration: (1) oral administration, such as potions (aqueous or non-aqueous solutions or suspensions), tablets, cheeks, tongue Subcutaneous and systemic absorbents, boluses, powders, granules, sublingual pastes; (2) parenteral administration, prepared as sterile solutions or suspensions or sustained release agents, by subcutaneous, intramuscular, intravenous Or epidural injection; (3) topical applications such as creams, ointments, controlled release patches or sprays for the skin, lungs, or mouth; (4) intrarectal administration, such as as an emulsion or foam; ( 5) Others: sublingual, ophthalmic, percutaneous or nasal, pulmonary and other mucosal intake.

The method for preparing the pharmaceutical composition of the compound includes combining the compound FG-4592 with a carrier or a plurality of auxiliary ingredients in any process. Generally, compound FG-4592 can be prepared by uniformly and intimately combining with a carrier (liquid carrier, finely divided solid carrier, or both), and the product can be shaped as required.

In some cases, the effect can be prolonged by slowing the slow absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by preparing liquid suspensions of crystalline or amorphous substances with poor water solubility. The absorption rate of a drug depends on its dissolution rate, which in turn may depend on the crystal size and crystalline form. Alternatively, parenteral administration can be delayed by dissolving or suspending the drug in an oily carrier.

The injectable drug depot form can be made by combining the compound with a biodegradable polymer (such as polylactide-polyglycolide) in a microcapsule matrix. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Other biodegradable polymers include polyorthoesters and polyanhydrides. It is also possible to prepare injectable drug depot formulations compatible with body tissues by embedding the drug in liposomes or microemulsions.

The pharmaceutical ingredients of the present invention can be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. For oral tablets, the usual coatings are lactose and corn starch, and lubricants such as magnesium stearate are usually added. For oral capsule formulations, useful diluents include lactose and dried corn starch. When administered orally in aqueous suspensions and solutions and propylene glycol formulations, the pharmaceutically active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and / or flavoring and / or coloring agents may be added.

The pharmaceutical ingredients of the present invention can be administered as an aerosol or inhalant. Such agents can be prepared according to techniques widely used in the field of pharmaceutical formulation, or they can be prepared as saline solutions. Such prior art often uses benzyl alcohol or other suitable preservatives, fluorocarbons and / or other solubilizing or dispersing agents, absorption enhancers to increase their bioavailability.

An additional advantage of transdermal patches is to achieve controlled delivery of the compounds of the invention to the body. This dosage form can be prepared by dissolving or dispersing the compound in an appropriate medium. Absorption enhancers can be used to increase the skin's absorption of the compound, or by using a rate-controlling film or dispersing the compound in a polymer matrix or gel, the rate of compound passing through the skin can be controlled.

According to the present invention, FG-4592 is a specific ligand for THR obtained through high-throughput screening. The effects of compounds inducing THR and co-activators or co-inhibitors were detected by AlphaScreen biochemical methods. Based on the analysis of luciferase reporter gene activity and molecular structure level in cell transfection experiments, the specific and selective recognition of this receptor and ligand was explained to further verify the compound's transcriptional activation effect on THR.

Polymorphisms of THRβ receptor protein loci that produce thyroid hormone resistance syndrome were selected to study the response to different ligands. Thyroid hormone T3 lost significant activation of the transcription of all the reporter genes in the table, but FG-4592 Significant activation of the transcriptional activity of these THRβ mutants.

The complex was prepared from THRβ / FG-4592, and the complex was crystallized, X-ray crystal diffracted, and structurally analyzed by X-ray crystal diffractometry. The bonding mode of THRβ and FG-4592 was clarified from the atomic level. Therefore, the present invention has discovered and verified that FG-4592 is a new type of THR agonist by combining various methods such as biochemistry, molecular biology and structural biology. Interestingly, FG-4592 can activate THR-mediated transcriptional activity as well as thyroid hormone T3, and also significantly activate THRβ mutant transcriptional activity that is resistant to thyroid hormone T3.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows that FG-4592 promotes the interaction of the SRC coactivator LXXLL motif with THRα and THRβ. The abscissa represents several biotin-labeled coregulatory polypeptides that contain the LXXLL motif that binds to THR.

FIG. 2 is a dose-response curve for measuring the relative activity of FG-4592 to regulate THRα and THRβ recruitment of co-activator SRC1LXXLL motifs by AlphaScreen assay.

FIG. 3 is a dose-response curve of cell-based luciferase assay for FG-4592 to regulate THRα and THRβ transcriptional activity. The mammalian two-hybrid method was used to clone THRLBD into a pBind expression vector, which can express a Gal4 tag fusion protein in the cell. This plasmid and pG5-luc luciferase reporter gene plasmid were co-transfected into 293T cells. After transfection, cells were treated with compounds, and the effects of reporter gene transcription activity were examined after treatment with various compounds. All experiments were performed in triplicate independently.

FIG. 4 is a three-dimensional crystal structure diagram of a complex in which FG-4592 and THRβ are combined. The band diagram shows the structure of the nuclear receptor THRβ protein, labeled with the name of the relevant alpha helix. The bar graph shows the structure of the ligand FG-4592 compound.

Figure 5 is a two-dimensional schematic diagram of the amino acid interaction between FG-4592 and the thyroid hormone receptor THRβ ligand binding domain. The hydrophobic phenyl end of the ligand forms a hydrophobic interaction with nearby nonpolar amino acids, while the carboxyl end of the ligand mainly forms hydrogen bonds directly with polar amino acids or forms a hydrogen bond network with polar amino acids through water. The gray arrows and black straight lines indicate that the ligand forms hydrogen bonds and hydrophobic interactions with amino acid residues, respectively.

Fig. 6 shows that the hydrophobic phenyl group of FG-4592 gives it the ability to selectively bind to THRβ. The structures of T3 / THRαLBD (gray) and FG-4592 / THRβLBD (black) are superimposed and compared. The arrows indicate the backbone and single residues between THRα and THRβ that help FG-4592 selectively bind to the receptor THRβ. Differential conformation of the basal side chain.

FIG. 7 is a comparison diagram of the conformational superposition of FG-4592 and other THR ligands in the THRβ pocket. FG-4592 shares a conserved carboxyl head (upper black arrow) but does not share a hydroxy tail (lower black arrow) with other THR ligands. The unique hydrophobic phenyl extension of FG-4592 is indicated by the gray arrow.

Figure 8 is the structural mechanism of FG-4592 activating THRβ mutants associated with thyroid hormone resistance. A superimposed display of T3-bound THRβ (light blue) and FG-4592-bound THRβ mutant (green), where T3 and FG-4592 are shown in cyan and yellow, respectively. Conformational changes in residues involved in thyroid hormone resistance caused by THRβ mutations are indicated by red arrows. Mutant residues are shown in salmon red. The key hydrogen bonds formed between the THRβH435 residue and the thyroid hormone T3 are marked with arrows.

Detailed ways

Example 1 demonstrates that FG-4592 enhances the ability of THRα and THRβ to recruit co-activators and is an agonist of thyroid hormone receptor THR.

Figure 1 shows that FG-4592 promotes the interaction of the SRC coactivator LXXLL motif with THRα and THRβ. The effect of FG-4592 with a final concentration of 1 μM on the interaction of LBD of THRα and ΤRβ with various co-regulators including the SRCLXXLL motif was detected using AlphaScreen technology.

The N-terminal biotinylated cofactor polypeptide sequence is:

SRC1-2: SPSSHSSLTERHKILHRLLQEGSP;

SRC2-3: QEPVSPKKKENALLRYLLDKDDTKD.

SRC3-3: PDAASKHKQLSELLRGGSG;

Protein purification: The human-derived THRαLBD (amino acid residues 148-410) and THRβLBD (amino acid residues 202-461) genes were constructed in the pET24a vector containing a 6-histidine fusion tag from Novagen. The constructed vector was transformed into E. coli BL21 (DE3) and cultured at 30 ° C until the OD600 was about 0.8. The temperature was lowered to 22 ° C, and 0.1 mM isopropyl 1-thio-β-D-galactoside (IPTG) was added. ) Induce target protein expression for 6h. The E. coli cells were subsequently centrifuged (4200 r.p.m.) for 30 min, resuspended in buffer (25 mM Tris pH 7.5, 25 mM imidazole, 150 m sodium chloride) on ice and frozen at -70 ° C for 2 h. The lysed cell solution was centrifuged (> 20,000 r.p.m.) at 4 ° C for 30 min, and the supernatant was taken and applied to a nickel ion exchange column (NiSO4-loaded HiTrap HP column, GE Healthcare) of GE. Subsequently, the nickel column loaded with the sample was eluted in a gradient of 25 to 500 mM imidazole in an AKTA pure protein purification instrument from GE. The eluted protein was further purified using an anion exchange column (Q-Sepharose column).

In the search for THR ligands, the LBD proteins of THRα and THRβ were used as "bait" to screen the compound library using AlphaScreen technology. Using AlphaScreen kit (Perkins-Elmer) experiments, the ability of THR and LBD proteins to bind to ligands to recruit various peptides with different motifs can be detected (Jin et al., Nature communication 2013, 4, 1937). The reaction system of this experiment is 20-80nM acceptor LBD protein, 20nM biotinylated cofactor polypeptide, 5μg / ml donor and acceptor glass beads, buffer solution (25mM Hepes, 100mM NaCl and 0.1mg / ml bovine seed serum , PH 7.0). This technology has been widely used in drug development based on nuclear receptor and ligand interactions. The results are shown in Figure 1. FG-4592 greatly enhanced the recruitment of THRα and THRβ to the co-activators SRC1, SRC2, and SRC3, indicating that FG-4592 is an agonist of THRα and THRβ, and that FG-4592 induced THRβ recruitment polytypes, respectively. The effects of co-activators are significantly stronger than THRα.

Further AlphaScreen detection with concentration gradient FG-4592 showed that the half-maximum effect concentration (EC50) of FG-4592 inducing THRβ to bind to the LXXLL motif was significantly lower than THRα (Figure 2 and Table 1), so FG-4592 had more effect on THRβ Strong selective activation. Relative to the control DMSO (dimethyl sulfoxide), the half-maximum effect concentration (EC50) of FG-4592-induced THRs binding to the LXXLL motif is shown in Figure 2 and Table 1.

Table 1

Example 2 demonstrates that FG-4592THR enhances the transcriptional activation of reporter genes in living cells and is an agonist of the thyroid hormone receptor THR.

In order to further show the activation of THR in cells by FG-4592, plasmids encoding Gal4 DNA binding domain, reporter gene, and THRLBD were co-transfected into African green monkey kidney fibroblasts Cos7 cells. Cos7 cell culture was performed using DMEM medium containing 10% fetal bovine serum, and transient transfection was performed using Lipofectamine 2000 (Invitrogen) kit. In a Gal-4 driven reporter experiment, 200 ng of Gal4-LBD was co-transfected with 200 ng of pG5Luc (Promega). Agonist compounds were added 5h after transfection. After 24 hours of treatment, cells were collected for luciferase detection experiments. The fluorescence detection experiment was performed by co-transfection of the Renilla reporter gene as an endogenous reference. The efficacy of the compound (5 μM) in inducing the transcription activity of THRα and THRβ wild type and mutant reporter genes is shown in Table 2. The experimental results are consistent with the results of AlphaScreen. FG-4592 can significantly enhance the transcriptional activation of THRα wild type and THRβ wild type reporter genes. Once again at the cellular level, FG-4592 is an agonist of thyroid hormone receptor THR and can activate THR. Mediated gene expression.

Table 2

Further detection with the reporter gene of the concentration gradient FG-4592 showed that the half-maximum effect concentration (EC50) of FG-4592 to activate the THRβ reporter gene was significantly lower than that of THRα (Figure 3), so once again, FG-4592 demonstrated that There is stronger selective activation.

Example 3 demonstrates that FG-4592 has a stronger selective activation of THRβ.

As mentioned above, thyroid hormone receptors (THRs) have two subtypes, THRα and THRβ, and the activation of THRα has certain side effects on the cardiovascular system. However, the natural thyroid hormone T3 has not shown any selectivity in binding the two THR subtypes (THRα and THRβ). Therefore, the development of existing thyroid hormone agonists is mainly to increase the selective activation of THRβ and reduce the activation of THRα. . The Gal-4 driven reporter gene experiment was performed on THRα and THRβ as in Example 2 to obtain the compound activation efficiency, and the potency EC50 was calculated using the full dose curve. The compounds induced the THRβ and THRα reporter genes, respectively. See Table 3 for the efficacy ratio and titer intensity EC50 ratio of transcription activity. Efficacy is a term in pharmaceutical pharmacology, which refers to the ability of a drug to produce the maximum effect, and is used to evaluate the maximum effect of different drugs. Potency: Also called potency strength, it refers to the dose or concentration required for a drug to produce a certain effect. EC50 is the half-effect concentration, that is, the dose of a drug that causes 50% of the subjects to produce a specific effect. A smaller EC50 indicates better activity and greater strength. As shown in Table 3, compared to T3, FG-4592 has stronger selective activation of THRβ in potency and potency, and is therefore a THRβ selective agonist.

table 3

Example 4 demonstrates the analysis of the crystal structure of the complex between FG-4592 and THRβ.

In order to reveal the molecular mechanism of FG-4592 and THRβ mutual recognition and binding at the atomic level, the crystal structure of the complex formed by FG-4592 and THRβ was analyzed according to the conventional X-ray crystal diffraction method (Fig. 4). To obtain THRβ protein as described in Example 1, add FG4592 5 times the amount of THRβLBD protein and SRC2-3 polypeptide (ENALLRYLLDKD) which is twice the amount of THRβLBD protein. After incubating on ice for 1 h, concentrate to 10 mg / mL. Crystallization was performed using the Hampton Crystallization Screening Kit. The sample complex was mixed with the screening buffer 1: 1, and the crystals were screened by the hanging drop method. The crystals grow best at about 48 hours at room temperature. The best conditions are: 0.2M sodium citrate, 20% by volume polyethylene glycol 3350, and the crystals are directly frozen in liquid nitrogen after adding a cryoprotectant. The frozen crystals were collected at Shanghai National Synchrotron Radiation Center. After the data was processed and restored by HKL3000, the structure was determined using CCP4 tool software. Coot software is used for manual modification, and software such as REFMAC is used for modification. After the structural parameter evaluation is completed, the structural analysis is completed.

The structure shows that the binding of FG-4592 to the THRβ ligand binding domain conforms to the classic "sandwich" conformation. From the three-dimensional structure diagram, FG-4592 clearly exists in the ligand binding pocket of THRβ (Figure 4). In the complex structure, the THRβAF-2 helix bound to FG-4592 and helices H3, H4, and H5 together form a "pocket" and interact with the LXXLL motif of the co-activator SRC2. This is a typical pattern of nuclear receptors interacting with agonists. A detailed structure-activity relationship analysis showed that FG-4592 and amino acids in the thyroid hormone receptor THRβ ligand binding pocket form a series of hydrophobic, hydrogen bonding, and Van der Waals forces (Figure 5). These interactions regulate FG-4592 and Binding of thyroid hormone receptor THRβ. These structural analyses strongly demonstrate the typical mechanism by which FG-4592 regulates THR activation of the thyroid hormone receptor.

By comparing the structure of THRα-T3 and THRβ-T3 (Figure 6), it is found that ligands such as FG-4592 and T3 have a conservative hydrophilic hydroxyl group at one end. The difference is that its tail is uniquely large and hydrophobic. Phenyl group (Figure 7), which requires a larger ligand binding pocket space for conformational adaptation. After conformation analysis, it is found that the binding pocket of THRβ is larger than that of THRα, and its main chain H10 is obviously shifted outward (Figure 6). It is for this reason that FG-4592, which has a large hydrophobic phenyl group, is more suitable for large pockets such as THRβ. Similarly, the side chains of hydrophobic residues in the THRβ ligand binding pockets, such as F269 and F455, show a change that favors FG-4592 out-of-pocket offset relative to the corresponding residues in THRα (see Figure 6). In summary, the differential changes in THRβ compared to THRα in the main chain and individual residue side chains are conducive to FG-4592 with a larger hydrophobic phenyl group to selectively bind β subtypes, which highlights the residues within each THR subtype For the selective action of different ligands, the hydrophobic phenyl tail of FG-4592 is the key to its selective binding to THRβ.

Example 5 demonstrates that FG-4592 has significant activation of the THRβ mutant transcriptional activity and can be used to treat thyroid hormone resistance syndrome.

In addition to wild-type THRs, Table 2 also lists the THRβ receptor protein site lesions that select polytypes to produce thyroid hormone resistance syndrome (The Human Gene Gene Mutation Database, http://www.hgmd.cf.ac.uk) . Because these mutants change the conformation of the THR ligand binding domain (LBD), the strong binding ability of the THRβ receptor protein to the thyroid hormones T3 and T4 is affected, and therefore it is resistant or insensitive to the thyroid hormones T3 and T4. THRβ receptor mutations were performed using the Quick-Change site-directed mutagenesis kit (Stratagene), and Gal-4 driven reporter gene experiments were performed as in Example 2. The results are shown in Table 2. Thyroid hormone T3 lost or significantly reduced activation of the reporter gene transcription of all mutants in the table. In contrast, FG-4592, which has a significantly different structure from thyroid hormones T3 and T4, is able to bind to these THRβ receptor protein site lesions and significantly activate their transcriptional activity. Table 2 shows that FG-4592 can produce higher induced transcription activity on THRβ receptor protein lesions in the table than T3, so FG-4592 can be used to treat thyroid hormone resistance syndrome.

Example 6, FG-4592 activates the structural mechanism of THRβ mutants associated with thyroid hormone resistance.

We found that FG-4592 has the ability to treat thyroid hormone resistance caused by mutations in polymorphic receptor proteins. What surprised us, however, was that the sites of these mutations not only existed in the pockets of the receptor protein, such as THRβH435L, but also amino acid mutations outside the pocket, such as THRβV264D, R438H, R438W. In order to further understand the mechanism of mutations leading to traditional thyroid hormone ligand resistance, the mode of action of FG-4592 in mutant THRβ, and the mechanism of activation of difficult-to-understand out-of-pocket mutations, we performed co-crystallization of FG-4592 and ΤRβ muteins Experiment and try to interpret them from a structural perspective.

Similar to the analysis of the crystal structure of the complex of Example 4, we solved the three-dimensional crystal structure of FG-4592 and four THRβ mutant proteins, such as V264D, H435L, R438H, and R438W. Overlapping these mutant structures with T3-bound THRβ wild-type structures (Figure 8) shows that, although randomly distributed at different locations, even outside the ligand binding pocket, different THRβ mutants will eventually allosterically regulate the same key Switching the conformation of His435 residues disrupts the key hydrogen bonds required for thyroid hormone binding and leads to defects in thyroid hormone binding. Unlike thyroid hormones, FG-4592 does not rely on conformation with the correct His435 residue to form hydrogen bonds through hydrophobic interactions with THRβ, thus still effectively activating the activity of THRβ mutants, explaining at the atomic level Mechanism of FG-4592 in the treatment of thyroid hormone resistance syndrome.

Example 7: Analyze the complex key binding sites and modes of action of FG-4592 and THRβ, and design FHR-4592 series of THRβ selective and targeted THRβ mutants, with a view to applying them in the preparation or prevention of thyroid hormones. Body-mediated diseases in pharmaceutical preparations.

Structural analysis revealed that, unlike THR ligands such as T3, which have a conservative hydrophilic hydroxyl group at one end, FG-4592 uniquely has a large hydrophobic phenyl group at the tail (Figure 7). In the binding mode, the hydrophobic interaction of the hydrophobic phenyl tail of FG-4592 with the THRβ near the position of His435 does not depend on the conformation of the His435 residue induced by the mutant to undergo a heterogeneous change to form hydrogen bonds, which is still effective. Activated the activity of the THRβ mutant.

In the design of derivative templates with FG-4592 as the lead compound, we will provide choices based on the unique binding mode of FG-4592 and THRβ to retain the conservative carboxyl group (head end) and the other end (tail end) that contribute hydrogen bonds at one end. The key hydrophobic base is the mother nucleus. The remaining groups on the backbone are optimized to achieve two significant pharmaceutical advantages of THRβ selectivity and activation of THRβ mutants. For the general formula shown below, the R1 or R2 group can be O, C, F, Cl, Br or I, R3 can be C or N, R4 can be O, C or H, and R5 can be C, O or N, R6 can be phenyl, heterocyclic compounds (such as five-membered heterocyclic compounds and six-membered heterocyclic compounds

(Such as five-membered heterocyclic compounds and six-membered heterocyclic compounds), ethyl, or propyl (n-propyl, isopropyl, and cyclopropyl) and other hydrophobic groups.

Industrial applicability

The present invention relates to a FG-4592 compound, which is given in the preparation of an thyroid hormone receptor (THR) agonist and in the preparation of a pharmaceutical preparation for preventing or treating a thyroid hormone receptor-mediated disease. The present invention relates to regulating the activity of thyroid hormone receptors, particularly the selective activation of thyroid hormone receptor β (THRβ) and its lesions, and is used for preventing or treating thyroid hormone receptor-mediated diseases such as hypothyroidism, thyroid hormone The application in resistance and depression methods has good industrial applicability.

Claims

The use of compound FG-4592 or a pharmaceutically acceptable salt thereof in the preparation of a thyroid hormone receptor agonist, the structural formula of the compound FG-4592 is as follows:
The use according to claim 1, wherein the thyroid hormone receptor agonist is a selective agonist of THRβ.
Compound FG-4592 or a pharmaceutically acceptable salt thereof for use in preparing a pharmaceutical preparation for preventing or treating a thyroid hormone receptor-mediated disease; the thyroid hormone receptor-mediated disease includes hypothyroidism, hypothyroidism Disease, polythyroid thyroid hormone resistance syndrome, mental illness, high cholesterol series, non-alcoholic fatty liver disease;
The use of claim 3, wherein the mental illness includes attention deficit hyperactivity disorder, depression, mental retardation, and cognitive dysfunction.
The use according to claim 3 or 4, wherein the hypothyroidism includes primary hypothyroidism, pituitary hypothyroidism, hypothalamic hypothyroidism, and peripheral hypothyroidism.
The application according to claim 3 or 4, characterized in that the clinical manifestations of hypothyroidism include memory loss, mental retardation, drowsiness, ataxia; bradycardia, decreased cardiac output, and concurrent coronary heart disease; anorexia, Abdominal distension, constipation, can lead to pernicious anemia and iron deficiency anemia; muscle weakness, pain, rigidity, and joint disease such as chronic arthritis; women with more menstrual periods, chronic amenorrhea, infertility; male impotence, hyposexuality In severe cases, improper stress can induce coma, shock, heart and kidney failure, etc .; minor illnesses; juvenile hypothyroidism such as short stature, low intelligence, delayed sexual development; and non-shiatsu edema.
The use according to claim 3 or 4, characterized in that the polymorphic thyroid hormone resistance syndrome comprises a lesion caused by a mutation in the THRβ receptor protein site.
The application according to claim 7, characterized in that the site mutation comprises V264D, A268D, R282S, V283A, M310T, E311K, S314C, A317T, R320C, N331D, G332E, G332R, L346F, L346V, H435L, R438H, F459C and F459L.
The use according to claim 3 or 4, characterized in that the clinical manifestations of the polytype thyroid hormone resistance syndrome include goiter; autoimmune thyroiditis; bradycardia, edema, fatigue, abdominal distension and constipation abnormality; poor intelligence , Backward development, or bones mature backward performance.
The use according to claim 3 or 4, wherein the thyroid hormone receptor-mediated diseases include mental retardation and developmental delay.
The use according to claim 3 or 4, wherein the thyroid hormone receptor-mediated diseases include attention deficit hyperactivity disorder, depression, mental retardation and cognitive dysfunction.
The use according to claim 3 or 4, wherein the thyroid hormone receptor-mediated diseases include weight gain, diabetes, hypertriglyceridemia, hypercholesterolemia, non-alcoholic fatty liver, atherosclerosis , Obesity, diseases of the endocrine system.
A method for designing a FG-4592 derivative based on a binding mode of FG-4592 and THR, characterized in that the FG-4592 derivative does not form hydrogen bonds with residues of THRβHis435, and has hydrophobic interaction with amino acids near THRβHis435 to activate THRβ active.
The method according to claim 13, characterized in that the method is to design a compound represented by the following general formula 1:

Among them, R1 or R2 group is O, C or I, R3 is C or N, R4 can be O, C or H, R5 is C, O or N, R6 is phenyl, heterocyclic compounds include five-membered heterocyclic ring Compounds and six-membered heterocyclic compounds, ethyl, or propyl or n-propyl or isopropyl or cyclopropyl or other hydrophobic groups.