CN110507655A

CN110507655A - Application of the compound FG-4592 in the disease mediated pharmaceutical preparation of preparation treatment Thyroid Hormone Receptors

Info

Publication number: CN110507655A
Application number: CN201810492095.1A
Authority: CN
Inventors: 李勇; 姚本强
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2018-05-22
Filing date: 2018-05-22
Publication date: 2019-11-29
Anticipated expiration: 2038-05-22
Also published as: WO2019223688A1; CN110507655B

Abstract

Application of the compound FG-4592 in the disease mediated pharmaceutical preparation of preparation treatment Thyroid Hormone Receptors, is related to using FG-4592 as Thyroid Hormone Receptors THR agonist.The compound FG-4592 or its pharmaceutically acceptable salt, can apply, the THR includes THR α and THR β, and the agonist includes full agonist or partial agonist in the agonist for preparing a kind of Thyroid Hormone Receptors.The compound FG-4592 or its pharmaceutically acceptable salt, can apply in the selective agonist for preparing a kind of THR β.FG-4592 is the agonist of nuclear receptor THR β, can be by combining function in terms of metabolism, inflammation, tumour, cirrhosis, the blood progenitor cells such as target THR β adjusts energetic supersession, lipid metaboli that THR β is participated in body, cholesterol-cholic acid is metabolized.

Description

Drug compound FG-4592 disease mediated in preparation treatment Thyroid Hormone Receptors Application in preparation

Technical field

The present invention relates to use FG-4592 as Thyroid Hormone Receptors THR agonist, more particularly, to compound FG- 4592 application in the disease mediated pharmaceutical preparation of preparation treatment Thyroid Hormone Receptors.

Background technique

Compound FG-4592, also known as Roxadustat, No. CAS is 808118-40-3, and structural formula is as follows:

FG-4592 is a kind of HIF α prolyl hydroxylase inhibitors, while EPO being induced to generate, and is used clinically for treating Anaemia relevant to chronic kidney disease and end-stage renal disease.The oral small molecule is by inhibiting hypoxia inducible factor HIF1 α hydroxylation Enzyme consolidates HIF1 α and HIF β heterodimer, to regulate and control downstream target gene, hematopoietin (EPO) is promoted to generate, Inhibit inflammation, lower hepcidin horizontal (hormone that free iron is sealed up for safekeeping) and raise other relevant genes of promotion iron, it is ensured that effectively Mobilize and utilize the storage iron of human body itself.Restore body natural equilibrium process RBC acceptor garland rate (Beuck, S., W.Schanzer,andM.Thevis,Drug TestAnal,2012.4(11):p.830-45)。

Nuclear receptor is a kind of transcription factor of ligand activation.Thyroid Hormone Receptors (THRs) is in 48 kinds of nuclear receptors of human body Very important a member has two kinds of hypotypes of THR α and THR β.THRs swashs in native ligand agonist thyroid hormone T3, T4 transcription Regulation living is lower to promote the various physiological activities such as growth, differentiation and the metabolism of human body.The ligand-mediated pharmacological action principle of THR is Thyroid hormone ligand is combined by the ligand binding domain (LBD) with THR, all kinds of co-activators of regulation THR recruitment (or auxiliary suppression The factor processed) adjust the transcription of downstream target gene.

The T4 and T3 that thyroid gland generates are under multiple reverse feedback regulation.TSH (also referred to as thyrotropic hormone) is responsible for Normal thyroid function and thyroid hormone secretion regulation, TSH are synthesized in pituitary gland Chiba, and its secretion is by hypothalamus The thyrotropin-releasing hormone (TRH) (TRH) of synthesis controls.In generally horizontal, this nerve, steroids feedback regulation holding people The weight of body, newly at metabolic rate, body temperature, mood and to influence serum low-density LP (LDL) horizontal.Therefore, in thyroid gland function When can fail or under thyroid hormone resistance disease, there is weight gain, diabetes, hypercholesterolemia, are moved hypertriglyceridemia Pulse atherosclerosis, obesity, endocrine system disease.

Hypothyroidism or low disease (hypothyroidism), abbreviation first subtract, and refer to and are drawn due to different Athyroxinosis is played, the metabolism of body and each system function decline of whole body, caused Comprehensive Clinical are made.Clinical manifestation Including failure of memory, feeblemindedness, incoordination；Bradycardia, cardiac output reduce, can and premature coronary heart disease；Anorexia, abdomen Swollen, constipation can lead to pernicious anaemia and hypoferric anemia；It is muscle weakness inability, pain, tetanic, it can be for example slow with arthropathy Property arthritis；Feminine menstrual is excessive, prolonged illness amenorrhoea, sterility；Impotence, sexual hypoesthesia；When being in a bad way, it is improper etc. stress Stupor can be induced, is suffered a shock, cardiorenal function failure etc..Light-duty case is in early days with thyroid glands such as oral thyroid piece or Levothyroxines (the national health State Family Planning Commission authority Medical popular science propagating items network platform/encyclopaedia name doctor based on the agonist of hormone receptor (THR) Net).

Thyroid hormone resistance syndrome (Thyroidhormone resistance syndrome, SRTH) is also referred to as first shape Glandular hormone disorder or syndrome of thyroid hormone resistance (thyroid hormone insensitivity syndrome, THIS).At present for the therapeutic agent under the syndrome there is a serious shortage of.3 seed types can be divided into according to its morbidity and clinical manifestation: 1, systemic thyroid hormone disorder, hypophysis are involved with surrounding tissue, this type can be divided into thyroid function compensatory just again Normal type and hypothyroidism type；2, for selective hypophysis to thyroid hormone disorder, feature is that hypophysis has involvement more, right Thyroid hormone does not react, and remaining peripheral tissues is not involved, can react normal to thyroid hormone, clinical manifestation has first It is high；3, selective peripheral tissues are to thyroid hormone disorder, this type feature be surrounding tissue thyroid hormone is not reacted or It is insensitive, and hypophysis is mostly without involvement, to thyroid hormone normal reaction.Clinical manifestation Thyroid Gland Swell, no deaf and dumb and epiphysis become Change, though thyroid hormone is normal and TSH is normal, clinic has hypothyroidism performance, bradycardia, oedema, out of strength, abdomen Swollen and constipation etc. is abnormal.This type can behave as stupid, Development retardation, can have osseous maturation to fall behind performance.Most thyroid glands Hormonal resistance syndrome is to send out the amino acid sequence of Thyroid Hormone Receptors since Thyroid Hormone Receptors gene mutates Changing leads to the variation of receptor structure and function, resists to thyroid hormone T3, T4 generation or insensitive.Therefore, knot is found It is comprehensive that the agonist that structure is different from the Thyroid Hormone Receptors of native thyroid hormones T3, T4 is expected to treatment thyroid hormone resistance Sign.

Hyperthyroidism referred to as " hyperthyroidism ", is caused since thyroid gland synthesis discharges excessive thyroid hormone Organism metabolism is hyperfunction and sympathetic activation, and palpitaition, perspiration, feed and bowel frequency is caused to increase the illness with weight loss.It is most Patient also usually has the symptoms such as expophthalmos, palpebral edema, hypopsia simultaneously.

THRs and cognitive disorder and depression:

The variation of thyroid hormone stable state causes affective disorders to be one of the reason of causing Personal, although may It is normal that patient shows as thyroid function in short term.However, in the subgroup of patient, clinically subthyroidism and hyperfunction Incidence shows as obviously increasing trend.Thyroid hormone Central nervous systematic growth is vital, especially brain. Psychiatric patient especially cognitive disorder and depression, thyroid hormone metabolism and thyroid gland systemic immune function are abnormal to this disease Sick occurrence and development are vital.Thus thyroid hormone is frequently used in accelerating and enhancing the treatment of antidepressants drug, The maintenance therapy disturbance of emotion for supporting bipolar disorders, even euthyroid patient (Bauer, M., et al., Mol Psychiatry, 2002.7 (2): p.140-56) giving thyroid function deficiency patient injection thyroid hormone T4 leads to mood With the improvement of learning ability, and positive experimental result is positively correlated with increased free thyroid hormone concentration (Bunevicius,R.,CurrOpin Psychiatry,2009.22(4):p.391-5)。

Thyroid hormone Central nervous system has a major impact, and phrenoblabia, especially emotionally disturbed, it will usually with In brain thyroid hormone metabolism disorder it is related (Bauer, M., et al., JNeuroendocrinol, 2008.20 (10): p.1101-14).For a long time, T3 be used to be used individually always patients with depression or be used in combination with other antidepressants, increase Influence strong or that acceleration antidepressant is to thyroid gland depressive patient.For example, T3 and tricyclic antidepressant are used in combination to controlling Treat significantly accelerated tricyclic antidepressant in the clinical test of antidepression patient, it is shown that the obvious curative effects of this treatment method (Aronson,R.,et al.,Arch Gen Psychiatry,1996.53(9):p.842-8).In addition, T3 and another kind of master Flow the validity and safety of antidepressants selective serotonin transporter inhibitors drug combination treatment major depressive disorder It is proved (Cooper-Kazaz, R.and B.Lerer, Int JNeuropsychopharmacol, 2008.11 (5): p.685- 99).These statistics indicate that, it is ensured that the normal development of central nervous system and mature needs specificity enhancing THRs activity, thus Enhance metabolism, adjusting and controlling growth and the signal transduction in nervous system, to increase learning ability and avoid cognitive disorder, Yi Jizhi Treat the disturbances of emotion such as depression class.

THRs and high cholesterol series of disease:

Thyroid hormone adjusts lipid by THR β and cholesterol metabolic effect is obvious, but clinical use native thyroid swashs Element and thryoid receptor activator treatment hypercholesterolemia are only applied in the patient of dysthyreosis.Reason is that Nonspecific agonist can activate THR α receptor, thus the negative effect in terms of generating cardiovascular disease.Therefore it finds efficient And the activation THR β of specificity rather than THR α will become treatment abnormalities of sugar/lipid metabolism, series of disease caused by high cholesterol it is solid Direction.Therefore, effective and specific Thyroid hormone receptor β agonist is found, regulating lipid metabolism reduces cholesterol, from And dyslipidemia is treated, fatty liver, the diseases such as hypercholesterolemia, atherosclerosis are appointed so very necessary.

THRs and non-alcoholic fatty are scorching:

Nearest clinical report points out that Thyroid hormone receptor β specific agonist can protect hepatic tissue, promote liver function Can, and treatment nonalcoholic fatty liver (NAFLD).This progress quilt in the data that Madrigal drugmaker announces It is described as and treats the completely new effective way of nonalcoholic steatohepatitis more than FXR, PPAR α/δ.Independently of this it is found out that, Ling You section Scholar Kim D etc. has found that subclinical first subtracts the nonalcoholic steatohepatitis (NASH) that patient suffers from and liver fibrosis and theirs Hypothyroidism has direct relation.In the lower subject of thyroid function, nonalcoholic steatohepatitis and advanced stage are fine The ratio of dimensionization is higher.The patient of hypothyroidism has more serious fatty degeneration of liver, and the denaturation of more serious sacculus With fibre modification (Kim, D., et al., ClinGastroenterolHepatol, 2018.16 (1): p.123-131e1).In This similarly, the discovery hyperthyroid patient such as another group of scientist Miyake, which has, is obviously improved the disease of nonalcoholic fatty liver disease Reason state finds that this effect and liver enzyme levels have been improved with the raising of thyroid hormones level according to their research It closes, with the raising of thyroid hormones level, liver enzyme level is increased.Therefore, hyperthyroidism can improve non-alcoholic Pathological state (Miyake, T., et al., Intern Med, 2016.55 (15): p.2019-23) of fatty hepatitis.These numbers According to prevention and treatment nonalcoholic fatty liver disease is all shown, Thyroid Hormone Receptors is targeted, improves receptor active, being one has The mode of effect.

Thyroid hormone analogs developing direction:

Currently, in view of Thyroid Hormone Receptors treatment hypothyroidism, thyroid hormone resistance syndrome and depression The validity and popularity of the diseases such as disease, researching and developing stronger thyroid hormone agonist becomes the Main way captured.However mesh Up till now research and development field still had two large problems urgently to be resolved, and first, as previously mentioned, native thyroid hormones T3 is combining two Any selectivity is not shown in terms of THR hypotype (THR α and THR β).Therefore, although application T3 can drop in animal model and people Low plasma cholesterol, low-density lipoprotein (LDL), triglyceride levels index of correlation, however, the side effect due to T3 to heart (such as tachycardia, cardiac arrhythmia and muscular atrophy), limits its application.The research and some choosings that knock-out animal is carried out Selecting property ligand the result shows that these cardiac side effects are attributable to the activation of THR α.Therefore, existing thyroid hormone excitement Agent research and development, which mainly improve, to be activated THR beta selective and reduces the activation to THR α.Another main problem is exactly gene mutation Bring adverse effect.Thyroid hormone resistance (Refetoff syndrome) describes a kind of rare symptom, thyroid hormone Level increases, but thyroid-stimulating hormone (TSH) (TSH) is horizontal no suppressed, or is not expected complete inhibition.Substantially, this Thyroid hormone effector is reduction of to reaction (Refetoff, S., et al., the J ClinEndocrino of thyroid hormone lMetab,1967.27(2):p.279-94).Therefore, although improving the level of serum thyroid, individual may still go out Existing thyroid function is unchanged.The most common symptom is Thyroid Gland Swell and tachycardia.It also with some attention-deficit hyperactivities Obstacle (ADHD) and depression related (Hauser, P., et al., N Engl J Med, 1993.328 (14): p.997-1001； Fardella,C.E.,et al.,Endocrine,2007.31(3):p.272-8).The syndrome it is most common the reason is that base Because of the mutation of THR β gene, there is now the Thyroid hormone receptor β site mutation different more than 100 kinds causes thyroid hormone to support The case of anti-syndrome, but also thering is new mutational site to report (Beato-Vibora, P., J.et al., Eur J ObstetGynecolReprodBiol, 2013.167 (1): p.118-9).Thyroid hormone resistance syndrome still lacks at present to be had The treatment means of effect, the mutation of Thyroid Hormone Receptors albumen make script native ligand such as T3 and a variety of THR beta selective agonists Ineffective, drug that is completely new, adapting to many types of thyroxine resistance syndrome is urgently developed.

Therefore, based on the above background, the Thyroid Hormone Receptors ligand of exploitation more specificity and selectivity, especially THR beta selective agonists and the novel agonist that can activate THRs mutant, can lead to a variety of diseases, such as thyroid gland Hypofunction, thyroid hormone resistance syndrome and depression etc. carry out specific prevention and treatment, and avoid native thyroid It is still very urgent to the drug of cardiovascular stimulation and other toxicity.

Summary of the invention

Present invention aims to be swashed with compound FG-4592 (or hydroxyl quinine class drug) preparing a kind of thyroid gland Application in the agonist of plain receptor (THR), and the drug disease mediated in preparation prevention or treatment Thyroid Hormone Receptors Application in preparation.

The structural formula of the compound FG-4592 (or hydroxyl quinine class drug) is as follows:

The compound FG-4592 or its pharmaceutically acceptable salt, can prepare a kind of Thyroid Hormone Receptors (THR) it is applied in agonist, the THR includes THR α and THR β, and the agonist includes full agonist or partial agonist Agent.

The compound FG-4592 or its pharmaceutically acceptable salt, can be in the selectivity excitement for preparing a kind of THR β It is applied in agent.

The compound FG-4592 or its pharmaceutically acceptable salt, can be in preparation prevention or treatment thyroid hormone It is applied in receptor-mediated disease pharmaceutical preparation.The disease mediated Thyroid Hormone Receptors includes the low disease of thyroid hormone, first Shape glandular hormone decline disease, many types of thyroid hormone resistance syndrome, mental disease, nonalcoholic fatty liver etc..The spirit disease Disease includes but is not limited to attention deficit hyperactivity disorder (ADHD), depression, mental retardation and cognition dysfunction.

The many types of thyroid hormone resistance syndrome includes but is not limited to clinically known THR beta receptor protein ligands The site lesion being mutated in binding domain, the site mutation include but is not limited to caused thyroid hormone resistance syndrome V264D、A268D、R282S、V283A、M310T、E311K、S314C、A317T、R320C、N331D、G332E、G332R、 L346F, L346V, H435L, R438H, F459C, F459L etc..

The compound FG-4592 or its pharmaceutically acceptable salt, can be in preparation and other preventions or treatment first shape The application in pharmaceutical composition that the disease mediated pharmaceutical preparation of hormone receptor is used in combination.

The compound FG-4592 be pharmaceutical composition, described pharmaceutical composition include effective dose FG-4592 or its Pharmaceutically acceptable salt.

The compound FG-4592 have good THR stirring effect, it is demonstrated experimentally that compound FG-4592 can with core by Body Thyroid Hormone Receptors (THR) combines, and induction THR raises counselor work, and activates the expression of the target gene of THR, shows The compound is the small molecule " adjusting control agent " or " THR ligand " of nuclear receptor THR, therefore, can be used as THR agonist and is used in advance The related disease that anti-or treatment THR is mediated.Wherein the THR agonist includes that the full agonist of THR α and THR β or part are swashed Dynamic agent.

AlphaScreen raises co-activator it is demonstrated experimentally that FG-4592 can induce THR α and THR β.It is activated because supplemented by The factor can directly activate THR downstream passages, show that FG-4592 is the agonist of nuclear receptor THR.Reporter Gene Experiments prove, FG-4592 can activate nuclear receptor THR α and THR β to the transcriptional activity of its target gene, show that FG-4592 is swashing for nuclear receptor THR Dynamic agent.Crystallography means further illustrate the binding pattern that THR β and FG-4592 be combined with each other from atomic level.Therefore it utilizes A variety of methods prove, the agonist of FG-4592 Thyroid Hormone Receptors THR can be used as thyroid hormone analogs preventing or The related disease for treating THR mediation.

It is demonstrated experimentally that transcriptional activity of the FG-4592 to the THR β mutant of many types of generation thyroid hormone resistance syndrome There is significant activation, can be used to treat thyroid hormone resistance syndrome.Since these mutant make THR ligand binding domain (LBD) conformation change loses the strong binding ability with thyroid hormone T3, T4.However FG-4592 and thyroid hormone T3, T4 structure is dramatically different, thus FG-4592 can be in conjunction with these THR beta receptor protein loci lesion bodies, and significantly activate theirs Transcriptional activity, so FG-4592 can be used to treat thyroid hormone resistance syndrome.

The above experiment shows that FG-4592 is the agonist of nuclear receptor THR β, can be by combining target THR β to adjust THR β Metabolism, inflammation, tumour, cirrhosis, the blood progenitor cells such as the energetic supersession, lipid metaboli, the cholesterol-cholic acid metabolism that are participated in body The function of differentiation etc..

FG-4592 is the hydroxyl quinine class drug of orally available small molecule therapy chronic kidney disease anaemia (CKD), is mainly used for Two generation hypoxia inducible factor prolyl hydroxylase inhibitors (HIF-PHI) are related to chronic kidney disease and end-stage renal disease to treat Anaemia.Have no so far it is any it is related its in treatment thyroid hormone resistance disease, thyroid hormone is low or the first shape such as decline disease It is applied in the disease mediated drug of hormone receptor.Therefore the FG-4592 that the present invention provides has treatment in terms of these diseases Function is all the new opplication method about FG-4592, creative.

Thyroid hormone resistance disease, thyroid hormone be low or the disease mediated serious shadow of Thyroid Hormone Receptors such as decline disease The health and lives of the mankind are rung, therefore, this FG-4592 that the present invention provides treats the drug system of these major diseases in preparation Utility function in terms of agent has important social value and huge economic value, has practicability.

In some embodiments, the compound in the method for the invention can be configured to pharmaceutical composition in some administrations It is applied in scheme.The composition of drug of the present invention may include the combound itself and available salt or drug administration carrier is administered in it. Such composition is also selectively included other therapeutic agents.

Certain medicaments or therapy can be administered in combination with compound of the present invention, as matrix metalloproteinase inhibits Agent, lipoxidase inhibitor, cytokine antagonist, immunosuppressor, cell factor, growth factor, immunomodulator, forefront Parathyrine or anti-angiogenic antihyperproliferative compound.

It with the compounds of this invention is same that term used herein " combination " and its relative words were corresponding in turn to, which is therapeutic agent, When or apply in order, for example, certain described compound can be simultaneously or sequentially with another therapeutic agent in single unit dosage forms Application.Therefore, the present invention provides a kind of single unit dosage forms, includes the compound, a kind of additional therapeutic agent.It is given in many In prescription case, when patient or individual simultaneously be exposed at least two medicaments when, if patient or individual in some specific target tissue Or sample (for example, in brain, in serum etc.) shows the associated treatment effect of agents useful for same simultaneously, it is generally recognized that they be with What the form of " combination " played a role.

If the pharmacy that these compounds are the compounds of this invention can use salt product, the salt used should preferentially come from nothing Machine or organic bronsted lowry acids and bases bronsted lowry.Ackd salt includes: acetate, adipate, alginates, aspartate, benzoate, benzene sulfonic acid Salt, disulfate, butyrate, citrate, camphor hydrochlorate, camsilate, pentamethylene, digluconate, dodecyl sulphur Hydrochlorate, esilate, fumarate, gluceptate, glycerophosphate, Hemisulphate, heptan, caproate, hydrochloride, hydrobromic acid Salt, hydriodate, 2- isethionate, lactate, maleate, mesylate, 2- naphthalene sulfonate, nicotinate, oxalates, Embonate, pectate, persulfate, 3- phenylpropionic acid salt, picrate, Pivalate, propionate, succinate, One of tartrate, thiocyanic acid, toluene fulfonate and undecylate etc..Alkali salt includes ammonium salt, alkali metal salt, alkaline-earth metal Salt and organic alkali salt；The alkali metal salt includes sodium salt and sylvite, and the alkali salt includes calcium salt and magnesium salts, described organic Alkali salt includes dicyclohexyl amine salt, N- methyl-D-glucamine salt or with one in amino acid such as arginine, lysine salt Kind.

Pharmaceutical composition of the invention can be configured to the form of solid or liquid, including following suitable form of medication: (1) Oral administration, such as filled medicament (aqueous or non-aqueous solution or suspension), tablet, cheek, sublingual and systemic Absorption agent, bolus, Powder, granule, sublingual paste；(2) parenteral administration is prepared as sterile solution or suspension or sustained release agent, leads to Cross subcutaneous, intramuscular, intravenous or epidural injection；(3) topical application, such as emulsifiable paste, ointment, for releasing for skin, lung or oral cavity Put controlled patch or spray；(4) drop rectum with drug is such as used as emulsion or foam；(5) other: sublingual administration, it is ophthalmically acceptable, percutaneously Or intranasal, lung and the intake of other mucous membranes.

The preparation method of the pharmaceutical composition of the compound include with any process by compound FG-4592 and carrier or A variety of auxiliary element combinations.It under normal conditions, can be by compound FG-4592 and carrier (liquid-carrier, subdivided solids carrier or two Person has) uniformly and intimately combination preparation, and as needed by product figuration.

It, can be by slowing down drug from the speed of slow trapping is subcutaneously or intramuscularly infused to extend drug effect, Ke Yitong in certain situations The liquid suspension of the crystallization or amorphous substance that prepare poorly water-soluble is crossed to realize this purpose.The absorption rate of drug depends on In its solution rate, rate of dissolution is likely to be dependent on crystal size and crystal form again.Alternatively, by dissolving or hanging drug Float in oiliness carrier to postpone parenteral absorption.

It can be by by biodegradable polymer (such as polylactide-polyglycolide) in the compound and microencapsule matrices In conjunction with its injectable drug depot dosage form is made.It, can according to the property of the ratio and particular polymers used of drug and polymer The rate of Drug controlled release.Other biodegradable polymers include polyorthoester and polyanhydride.It can also be by by drug It is embedded in liposome or micro emulsion, to prepare the injectable drug depot dosage form compatible with bodily tissue.

Drug ingedient of the invention can arbitrarily take orally acceptable dosage form oral administration, and dosage form includes but is not limited to: capsule, Tablet and water slurry and solution.For oral tablet, common coating has lactose and cornstarch, usually can also Lubricant, such as magnesium stearate is added.For oral capsule preparation, available diluent includes that lactose and dry corn form sediment Powder.When with water slurry and the oral administration of solution and formulation propylene glycol, active pharmaceutical ingredient is in conjunction with emulsifier and suspending agent. If it is desired, certain sweeteners and/or flavoring agent and/or colorant can be added.

It can be by the way that aerosol or inhalant administration be made in drug ingedient of the invention.Such medicament can match according to pharmacy Widely applied technology preparation, can also be prepared as saline solution in square field.This kind of prior art be often used benzylalcohol or its Its preservative, fluorocarbon and/or other solubilizer or dispersing agent, sorbefacient appropriate are to improve its biological utilisation Rate.

The additional advantage of transdermal patch is to realize the compounds of this invention to the controlled delivery of body.The compound can be dissolved Or it is dispersed in medium appropriate and makes this dosage form.Absorption of the skin to compound can be increased with absorption enhancer.It can also It is scattered in the mode in polymer substrate or gel by using rate controlling membranes or by compound, realizes that compound passes through skin The control of skin rate.

The present invention obtains the ligands specific that FG-4592 is THR by high flux screening.Pass through AlphaScreen biology Chemical method detection compound induces the effect of THR and co-activator or Corepressors.

This receptor is illustrated based on luciferase reporter gene activity analysis in cell transfection assays and molecular structure level Special Selective recognition between ligand further verifies compound to the transcriptional activation of THR.

Select the THR beta receptor protein loci lesion body of many types of generation thyroid hormone resistance syndrome, thyroid hormone T3 Significant activation is lost to the transcription of the reporter gene of mutant in all tables, but FG-4592 transcribes these THR β mutant Activity has significant activation.

THR β/FG-4592 is prepared into compound, by crystallography means, which is crystallized, X-ray crystal Diffraction and structure elucidation illustrate the binding pattern that THR β and FG-4592 be combined with each other from atomic level.Therefore, of the invention By combining biochemistry, a variety of methods such as molecular biology and structure biology find and verify FG-4592 to be a kind of novel The agonist of THR.Transcriptional activity the same with thyroid hormone T3, that FG-4592 can activate THR to mediate, also can significantly activate Thyroid hormone T3 has the THR β mutant transcriptional activity of resistance.

Detailed description of the invention

Fig. 1 is the interaction that FG-4592 promotes SRC co-activator LXXLL block and THR α and THR β.

Fig. 2 is complex three-dimensional crystalline structure figure of the FG-4592 in conjunction with THR β.Strip-chart indicates nuclear receptor THR β albumen The structure of matter is labeled with the name of related α spiral.The structure of bar graph expression ligand FG-4592 compound.

Fig. 3 is amino acid interaction two-dimensional representation in FG-4592 and Thyroid Hormone Receptors THR beta ligands binding domain. The hydrophobic alkyl end of ligand and neighbouring nonpolar amino acid form hydrophobic interaction, and ligand c-terminus is mainly directly and pole Acidic amino acid forms hydrogen bond or forms hydrogen bond network by water and polar amino acid.Wherein grey arrow and black line distinguish table Show that ligand and amino acid residue form hydrogen bond and hydrophobic interaction.

Specific embodiment

Embodiment 1, it was demonstrated that FG-4592 enhances THR α and THR β and raises co-activator, is Thyroid Hormone Receptors THR Agonist.

Fig. 1 is the interaction that FG-4592 promotes SRC co-activator LXXLL block and THR α and THR β.It utilizes AlphaScreen technology detects final concentration of 1 μM of FG-4592 to the LBD of THR α and Τ Η R β and including SRCLXXLL motif The influence of interior a variety of counselor works interaction.

The biotinylated co-factor polypeptide sequence in the end N- is:

SRC1-2:SPSSHSSLTERHKILHRLLQEGSP；

SRC2-3:QEPVSPKKKENALLRYLLDKDDTKD；

SRC3-3:PDAASKHKQLSELLRGGSG；

NCOR-1:GQVPRTHRLITLADHICQIITQDFARNQ；

NCOR-2:GHSFADPASNLGLEDIIRKALMGSF.

Protein purification: source of people THR α LBD (total number of atnino acid 148-410) and THR β LBD (total number of atnino acid 202- 461) gene is implemented in respectively in the pET24a carrier containing 6 polyhistidine fusion labels of Novagen company.Load after building Body converts in e. coli bl21 (DE3), and is cooled to 22 DEG C when it is about 0.8 that 30 DEG C of cultures are to OD600, and 0.1mM is added Thio-β-D- galactoside (IPTG) the induction target protein of isopropyl 1- expresses 6h.Bacillus coli cells are then centrifuged After (4200r.p.m.) 30min, it is resuspended in buffer (25mM Tris PH7.5,25mM imidazoles, 150m sodium chloride) on ice And sonicated cells after -70 DEG C of freezing 2h.The cell liquid of cracking takes supernatant after 4 DEG C of centrifugation (> 20000r.p.m.) 30min Upper prop is in the nickel ion exchange column (NiSO4-loaded HiTrap HP column, GE Healthcare) of GE company.Then, The nickel column for loading sample uses the elution buffer gradient elution of 25~500mM imidazoles in the AKTApure of GE company.Elution Albumen out further uses anion-exchange column (Q-Sepharose column) purifying.

During finding THR ligand, the LBD albumen of THR α and THR β is utilized into AlphaScreen as " bait " Technology screens compound library.THR is capable of detecting when with AlphaScreen kit (Perkins-Elmer) experiment Polypeptide (Jin et al., the Nature communications of LBD albumen and various different motifs needed for ligand binding 2013,4,1937).The reaction system of the experiment is the receptor LBD albumen of 20-80nM, the biotinylated co-factor polypeptide of 20nM, The donor and receptor bead of 5 μ g/ml, buffer (25mMHepes, 100mMNaCl and 0.1mg/ml bovine serum Albumin, pH7.0).This technology has been widely used for the medicament research and development based on nuclear receptor and ligand interaction at present In.

FG-4592 induces the half-maximal effect concentration (EC50) of THRs combination LXXLL block as shown in table 1, relative to right According to DMSO (dimethyl sulfoxide), FG-4592 significantly enhances THR α and THR β to co-activator SRC1, SRC2 and SRC3 It raises, but the recruitment of Corepressors is not influenced, illustrate that FG-4592 is the agonist of THR α Yu THR β.And FG- 4592 effects that induction THR β raises many types of co-activator respectively are obviously eager to excel than THR α.

Table 1

Further show that FG-4592 induces THR β combination LXXLL mould with concentration gradient FG-4592AlphaScreen detection The half-maximal effect concentration (EC50) of sequence is obviously lower than THR α, thus FG-4592 has stronger selective activation to THR β.

Embodiment 2, it was demonstrated that FG-4592 enhances the transcriptional activation of THR reporter gene, is Thyroid Hormone Receptors THR Agonist.

In order to further provide FG-4592 to the activation of THR, will coding Gal4DNA binding domain, reporter gene, The plasmid co-transfection of THRLBD is into Cos7 cell.Cos-7 cell is carried out using the DMEM culture medium containing 10% fetal calf serum Culture, is transiently transfected using Lipofectamine 2000 (Invitrogen) kit.In the report base of Gal-4 driving Because carrying out cotransfection using the pG5Luc (Promega) of the Gal4-LBD and 200ng of 200ng in experiment.5h, which is added, after transfection swashs Dynamic immunomodulator compounds.After processing for 24 hours, collects cell and be used for luciferase test experience.Fluorescence detection experiment passes through cotransfection Renilla reporter gene does interior source reference.Experimental result is consistent with AlphaScreen testing result, and FG-4592 can be significant Enhance the transcriptional activation of the reporter gene of THR α and THR β, compound (5 μM) induces THR α and THR β wild type and mutation respectively The active efficiency of body reporter gene transcription (efficacy) illustrates FG-4592 thyroid gland in cellular level again referring to table 2 The agonist of hormone receptor THR, the gene expression that THR can be activated to mediate.

Table 2

Embodiment 3, it was demonstrated that FG-4592 has significant activation to the transcriptional activity of THR β mutant, can be used to treat first shape Glandular hormone resists syndrome.

Table 2 lists the THR beta receptor protein loci lesion body (The for selecting many types of generation thyroid hormone resistance syndrome HumanGene Mutation Database, http://www.hgmd.cf.ac.uk).Since these mutant make THR ligand Binding domain (LBD) conformation change loses the strong binding ability with thyroid hormone T3, T4.Use Quick-Change Site-directedmutagenesis kit (Stratagene) carries out THR β plasmid encoding mutant, carries out such as embodiment 2 The Reporter Gene Experiments of Gal-4 driving.As shown in table 2, turn of the thyroid hormone T3 to the reporter gene of mutant in all tables Record loses significant activation.But because FG-4592 and thyroid hormone T3, T4 structure are dramatically different, thus FG-4592 can be tied These THR beta receptor protein loci lesion bodies are closed, and significantly activate their transcriptional activity, thus FG-4592 can be used to treat Thyroid hormone resistance syndrome.

Embodiment 4, it was demonstrated that FG-4592 has stronger selective activation to THR β.

As described above, Thyroid Hormone Receptors (THRs) has two kinds of hypotypes of THR α and THR β, and the activation of THR α is to painstaking effort Guard system etc. has certain side effect.Therefore, native thyroid hormones T3 is in terms of combining two THR hypotypes (THR α and THR β) Any selectivity is not shown, and existing thyroid hormone agonist research and development, which are mainly improved, to be activated and reduce to THR beta selective Activation to THR α.The Reporter Gene Experiments driven such as the Gal-4 of embodiment 2 are carried out for THR α and THR β to obtain compound It activates efficiency (efficacy), and calculates separately out potency (potency) EC with full dose curve₅₀, compound lures respectively The active efficiency ratio of THR β and THR α reporter gene transcription and potency EC50 ratio are led referring to table 3.Efficiency is drug medicine A term in Neo-Confucianism refers to the ability that drug generates ceiling effect, and the ceiling effect for evaluating different pharmaceutical is strong and weak.Effect Valence (potency): also known as potency refers to dosage or concentration needed for drug generates certain effect.EC₅₀It is that half effect is dense Degree, that is, cause 50% individual of study subject to generate a kind of drug dose of specific effect.The active better, intensity of the smaller expression of EC50 It is bigger.As shown in table 3, compared with T3, FG-4592 has stronger selective activation to THR β in efficiency and potency, It is thus THR beta selective agonists.

Table 3

Embodiment 5, it was demonstrated that the crystallographic structural analysis of compound of the FG-4592 in conjunction with THR β.

In order to disclose the molecular mechanism that FG-4592 and THR β is mutually distinguishable and combines from atomic level, solve according to a conventional method The crystal structure (Fig. 2) that FG-4592 and THR β forms compound is analysed.If method obtains THR β albumen in embodiment 1, it is added 5 The co-activator SRC2-3 of FG4592 and 2 times of THR β LBD protein content of THR β LBD protein content times after purification is more Peptide (ENALLRYLLDKD) is concentrated into 10mg/mL after this is incubated for 1h on ice.Hampton company crystallization screening is used when crystallization Kit, sessile drop method crystallization screening after sample composites are mixed with screening 1 ︰ 1 of buffer.Crystal at room temperature about 48h long to most It is good, best conditions are as follows: 0.2M sodium citrate, 20% volume ratio polyethylene glycol 3350, crystal are direct after cryoprotector is added Liquid nitrogen cryopreservation.The crystal frozen in Shanghai country synchrotron radiation central data data, data after by HKL3000 processing reduction, Structure determination uses CCP4 tool software.Modification is assessed using the modification such as REFMAC after Coot software to structural parameters manually Structure elucidation is completed after the completion.

Structure shows that the combination of FG-4592 and THR beta ligands binding domain meets classical " sandwich " conformation.It is tied from three-dimensional Composition sees that FG-4592 is clearly present in the ligand binding pocket (Fig. 2) of THR β.In composite structure, tied with FG-4592 The THR β AF-2 spiral and spiral H3, H4 and H5 of conjunction collectively form one " pocket ", the LXXLL motif with co-activator SRC2 It interacts.This is the typical module of a kind of nuclear receptor and agonist interaction.Detailed Structure-activity analysis shows, FG-4592 forms a series of hydrophobic, hydrogen bonds and Van der Waals force etc. with amino acid in Thyroid Hormone Receptors THR beta ligands binding domain It interacts (Fig. 3), these interactions regulate and control the combination of FG-4592 and Thyroid Hormone Receptors THR β.These structures point Analysis has convincingly demonstrated the typical mechanism of FG-4592 regulation Thyroid Hormone Receptors THR activation.

The present invention relates to a kind of hydroxyl quinine, it is given at the excitement for preparing a kind of Thyroid Hormone Receptors (THR) In agent, and the application in the disease mediated pharmaceutical preparation of preparation prevention or treatment Thyroid Hormone Receptors.The present invention relates to The activity of Thyroid Hormone Receptors, the especially activity of Thyroid hormone receptor β (THR β) are adjusted, for preventing or treating first shape Application in hormone receptor disease mediated such as hypothyroidism, thyroid hormone resistance and depression.

Sequence table

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Claims

1. compound FG-4592 or its pharmaceutically acceptable salt, in the agonist for preparing a kind of Thyroid Hormone Receptors Using the Thyroid Hormone Receptors is denoted as THR, and the structural formula of the compound FG-4592 is as follows:

2. application as described in claim 1, it is characterised in that the THR includes THR α and THR β.

3. application as described in claim 1, it is characterised in that the agonist includes full agonist or partial agonist.

4. application as claimed in claim 2, it is characterised in that the THR β is the selective agonist of THR β.

5. compound FG-4592 or its pharmaceutically acceptable salt are mediated in preparation prevention or treatment Thyroid Hormone Receptors It is applied in disease medicament preparation；It may include that the low disease of thyroid hormone, thyroid gland swash that the Thyroid Hormone Receptors is disease mediated One of element decline disease, many types of thyroid hormone resistance syndrome, mental disease, nonalcoholic fatty liver；The spirit disease Disease may include but be not limited to attention deficit hyperactivity disorder, depression, mental retardation and cognition dysfunction.

6. application as claimed in claim 5, it is characterised in that many types of thyroid hormone resistance syndrome includes but is not limited to The site lesion being clinically mutated in known THR beta receptor protein ligands binding domain, the site mutation includes but is not limited to institute Caused thyroid hormone resistance syndrome V264D, A268D, R282S, V283A, M310T, E311K, S314C, A317T, R320C、N331D、G332E、G332R、L346F、L346V、H435L、R438H、F459C、F459L。

7. compound FG-4592 or its pharmaceutically acceptable salt, in preparation and other preventions or treatment thyroid hormone receptor The application in pharmaceutical composition that the disease mediated pharmaceutical preparation of body is used in combination.

8. as claimed in claim 7 application, it is characterised in that described pharmaceutical composition include effective dose FG-4592 or its Pharmaceutically acceptable salt.

9. application as claimed in claim 8, it is characterised in that the pharmaceutically acceptable salt is inorganic or organic bronsted lowry acids and bases bronsted lowry, The ackd salt include: acetate, adipate, alginates, aspartate, benzoate, benzene sulfonate, disulfate, Butyrate, citrate, camphor hydrochlorate, camsilate, pentamethylene, digluconate, lauryl sulfate, ethanesulfonic acid Salt, fumarate, gluceptate, glycerophosphate, Hemisulphate, heptan, caproate, hydrochloride, hydrobromate, hydroiodic acid Salt, 2- isethionate, lactate, maleate, mesylate, 2- naphthalene sulfonate, nicotinate, oxalates, pamoic acid Salt, pectate, persulfate, 3- phenylpropionic acid salt, picrate, Pivalate, propionate, succinate, tartrate, One of thiocyanic acid, toluene fulfonate and undecylate；Alkali salt includes ammonium salt, alkali metal salt, alkali salt and organic base Salt；The alkali metal salt includes sodium salt and sylvite, and the alkali salt includes calcium salt and magnesium salts, and organic alkali salt includes two Cyclohexyl amine salt, N- methyl-D-glucamine salt or one of with amino-acid salt, it is described with amino-acid salt be arginine, One of lysine salt.

10. application as claimed in claim 8, it is characterised in that described pharmaceutical composition is configured to the form of solid or liquid, packet It includes following suitable form of medication: (1) being administered orally, the oral administration includes filled medicament, tablet, cheek absorbent, sublingual suction Agent, systemic Absorption agent, bolus, powder, granule, sublingual paste are received, the filled medicament includes aqueous filled medicament, non-aqueous Property solution filled medicament, suspension filled medicament；(2) parenteral administration；(3) topical application, the topical application includes emulsifiable paste, soft Cream, for skin, lung or the controlled release in oral cavity patch or spray；(4) drop rectum with drug, the drop rectum with drug are emulsion Or foam；(5) other: ophthalmically acceptable including sublingual administration, percutaneous or intranasal, lung and the intake of other mucous membranes.