Compounds as prolyl hydroxylase inhibitors and their preparation methods

Abstract

Provided is a compound as a prolyl hydroxylase inhibitor and a preparation method thereof, belonging to the technical field of medicine. The compound is shown as formula I and has prolyl hydroxylase inhibitory activity, and can be used for treating diseases mediated by prolyl hydroxylase activity, such as renal anemia and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and specifically discloses a compound as a prolyl hydroxylase inhibitor and a preparation method thereof. Background Art

[0002] In cases such as anemia, trauma, tissue necrosis and defects, tissues or cells are often in a hypoxic state. Hypoxia leads to the expression of a series of transcription-inducing factors, which are involved in angiogenesis, iron and sugar metabolism, and cell growth and proliferation. Among them, hypoxia-inducible factor (HIF) is a transcription factor initiated by somatic cells under hypoxic conditions, and mediates a series of gene regulations in biological cells to respond to the cell hypoxic state. HIF is a heterodimer containing an oxygen-regulated α-subunit (HIFα) and a constitutively expressed β-subunit (HIFβ / ARNT). In oxygen-containing (normoxic) cells, the HIFα subunit is rapidly degraded through the mechanism of ubiquitination by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. Under hypoxic conditions, HIFα is not degraded, and the active HIFα / β complex accumulates in the nucleus and activates the expression of various genes, including glycolytic enzymes, glucose transporters, erythropoietin (EPO), and vascular endothelial growth factor (VEGF).

[0003] Erythropoietin (EPO) is a naturally occurring hormone produced following HIFα, which stimulates the production of red blood cells (erythrocytes) that carry oxygen throughout the body. EPO is usually secreted by the kidneys, and endogenous EPO increases under conditions of reduced oxygen (hypoxia). All types of anemia are characterized by a reduced ability of the blood to carry oxygen and are thus accompanied by similar signs and symptoms, including pallor of the skin and mucous membranes, weakness, dizziness, easy fatigue, and lethargy, resulting in a decline in quality of life. Anemia is usually associated with conditions of blood deficiency in red blood cells or hemoglobin. Common causes of anemia include deficiencies in iron, vitamin B12, and folic acid, and it can also be associated with chronic diseases, such as inflammatory diseases, including those with secondary myelosuppression. Anemia is also associated with renal dysfunction, and most patients with renal failure who undergo regular dialysis suffer from chronic anemia.

[0004] Prolyl hydroxylase (prolyl hydroxylase domain, PHD) is a key factor in regulating HIF. There are three subtypes of PHD, namely PHD1, PHD2, and PHD3, and the active sites of all subtypes have a high degree of sequence homology. Under normoxic conditions, PHD can hydroxylate two key proline residues, Pro402 and Pro564, of HIFα, increasing its affinity for pVHL and accelerating the degradation process. Under hypoxic and other pathological conditions, the HIF reaction catalyzed by PHD is blocked, and the rate of protease degradation slows down, resulting in the accumulation of HIFα in cells, which in turn causes a series of adaptive responses of cells to hypoxia. Inhibiting PHD with a PHD inhibitor to prolong the action of HIF and thereby increase the expression of genes such as EPO can effectively treat and prevent HIF-related and / or EPO-related diseases, such as anemia, ischemia, and hypoxia-related diseases. Studies have shown that the deletion of the PHD2 gene can accelerate erythropoiesis.

[0005] Patent Application 200780030720.0 discloses a prolyl hydroxylase inhibitor and its use method. The prolyl hydroxylase inhibitor is a compound with the following general formula:

[0006]

[0007] Wherein, X is N or CH; R and R 1 are each independently a unit selected from the following atoms or groups: i) hydrogen; ii) substituted or unsubstituted phenyl; and iii) substituted or unsubstituted heteroaryl; the substituents are selected from: i) C 1 -C 4 linear, branched or cyclic alkyl; ii) C 1 -C 4 linear, branched or cyclic alkoxy; iii) C 1 -C 4 linear, branched or cyclic haloalkyl; iv) halogen; v) -CN; vi) -NHC(O)R 4 ; vii) -C(O)NR 5a R 5b ; viii) heteroaryl; or ix) two substituents can together form a fused ring having 5 to 7 atoms; R 4 is C 1 -C 4 linear, branched or cyclic alkyl; R 5a and R 5b are each independently: i) hydrogen; ii) C 1 -C 4 linear, branched or cyclic alkyl; or iii) R 5a and R 5b can together form a ring having 3 to 7 atoms; R2 Selected from: i) -OR 6 ; or ii) -NR 7a N 7b ; R 6 is hydrogen or C 1 -C 4 linear, branched or cyclic alkyl; R 7a and R 7b are each independently: i) hydrogen; ii) C 1 -C 4 linear, branched or cyclic alkyl; or iii) R 7a and R 7b may together form a ring having 3 to 7 ring atoms; Y is selected from: i) hydrogen; ii) -OR 3 ; R 3 is hydrogen, methyl or ethyl; L is a linking unit having the formula: -[C(R 8a R 8b )] n -R 8a and R 8b are each independently hydrogen, methyl or ethyl; the subscript n is selected from 1 to 3; R 9 is hydrogen or methyl.

[0008] Patent Application 201710653887.8 discloses a pyridine-derived compound acting on prolyl hydroxylase, having the following structure:

[0009]

[0010] Wherein: R1 is hydrogen or hydroxyl; R2 is independently selected from: hydrogen, halo, C 1-4 alkyl, phenyl, -O-phenyl, -O-benzyl, -O-C1-4 alkyl, phenyl optionally substituted with -C1-4 alkyl, benzyl optionally substituted with -C 1-4 substituted.

[0011] There is still a need for new and effective compounds for treating erythropoietin-related diseases targeting HIF. In view of this, the present invention provides a new compound as a prolyl hydroxylase inhibitor, its preparation method and use, expanding the types of drugs for treating erythropoietin-related diseases, and achieving good inhibitory activity against prolyl hydroxylase. Summary of the Invention

[0012] The object of the present invention is to provide a compound as a prolyl hydroxylase inhibitor, its preparation method and use, expanding the types of drugs for treating erythropoietin-related diseases, and achieving good inhibitory activity against prolyl hydroxylase.

[0013] To achieve the above-mentioned invention object, the technical solution of the present invention is as follows:

[0014] First, the present invention provides a compound as a prolyl hydroxylase inhibitor shown in Formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate or prodrug thereof:

[0015]

[0016] Wherein, R is selected from any one of an aryl group or a heteroaryl group which is substituted or unsubstituted by a substituent;

[0017] The substituent is at least one of an alkyl group, an alkoxy group, a haloalkyl group, a haloalkyl ether, an aralkyl group, a halogen, a cyano group, a phenyl group, a substituted or unsubstituted phenoxy group.

[0018] Furthermore, the aryl group is selected from a phenyl group, and the heteroaryl group is selected from:

[0019]

[0020] Furthermore, the alkyl group is selected from C 1 -C 4 alkyl group, and is further selected from C 1 -C 2 alkyl group; the alkoxy group is selected from C 1 -C 4 alkoxy group, and is further selected from C 1 -C 2 alkoxy group; the haloalkyl group is selected from C 1 -C 4 haloalkyl group, and is further selected from C 1 -C 2 haloalkyl group; the haloalkyl ether is selected from C 1 -C 4 haloalkyl ether, and is further selected from C 1 -C 2 haloalkyl ether; the aralkyl group is selected from C 1 -C 4 a 5-6 membered aryl group substituted by an alkyl group, and is further selected from C 1 -C 2 phenyl group substituted by an alkyl group; the halogen is selected from any one of F, Cl, I and Br, and is further selected from F or Cl; the substituted or unsubstituted phenoxy group is selected from a phenoxy group substituted or unsubstituted by C 1 -C 4 alkyl group and / or halogen, and is further selected from a phenoxy group substituted or unsubstituted by at least one of C 1 -C 2 alkyl group, F, Cl, I and Br.

[0021] Further, the compound is selected from the following four:

[0022] (1) R is a phenoxyphenyl substituted by at least one substituent, and the substituent is independently selected from at least one of H, halogen, C 1 -C 4 alkyl, and preferably the phenoxy group is substituted at the meta position of benzene;

[0023] (2) R is an aryl substituted by at least one substituent, and the substituent is independently selected from at least one of H, C 1 -C 4 haloalkyl, C 1 -C 4 haloalkyl ether, halogen, aryl;

[0024] (3) R is a benzheterocycle substituted by at least one substituent, and the substituent is independently selected from at least one of H, C 1 -C 4 alkyl, and the heterocycle is a 5- or 6-membered heterocycle containing nitrogen or oxygen;

[0025] (4) R is a dibenzheterocycle, and the heterocycle is a 5- or 6-membered heterocycle containing sulfur or oxygen.

[0026] Even further, the compound is selected from the following four:

[0027] (1) R is a phenoxyphenyl substituted by at least one substituent, and the substituent is independently selected from at least one of H, F, Cl, I, Br, C 1 -C 2 alkyl;

[0028] (2) R is an aryl substituted by at least one substituent, and the substituent is independently selected from at least one of H, C 1 -C 2 haloalkyl, C 1 -C 2 haloalkyl ether, F, Cl, I, Br, phenyl;

[0029] (3) R is a benzheterocycle substituted by at least one substituent, and the substituent is independently selected from at least one of H, C 1 -C 2 alkyl, and the heterocycle is a 5- or 6-membered heterocycle containing nitrogen or oxygen; (12-16)

[0030] (4) R is a dibenzheterocycle, and the heterocycle is a 5- or 6-membered heterocycle containing sulfur or oxygen.

[0031] Still further, the compound is selected from the following four:

[0032] (1) R is a phenoxyphenyl substituted by at least one substituent, and the substituents are independently selected from any one of H, Cl, and methyl;

[0033] (2) R is a phenyl substituted by at least one substituent, and the substituents are independently selected from at least one of H, trifluoromethyl, trifluoromethyl ether, F, Cl, and phenyl;

[0034] (3) R is a benzheterocycle substituted by at least one substituent, and the substituents are independently selected from at least one of H and methyl, and the heterocycle is a 5- to 6-membered heterocycle containing nitrogen or oxygen, preferably selected from substituents A, B, C, D, and E;

[0035] (4) R is a dibenzheterocycle, and the heterocycle is a 5-membered heterocycle containing sulfur or oxygen, preferably selected from G and H.

[0036] Furthermore, the compound is selected from any one of the following compounds:

[0037]

[0038]

[0039]

[0040] Preferably, R is selected from a phenyl substituted by two substituents, and the substituents are independently selected from Cl, OCF 3 or CF 3 .

[0041] More preferably, R is selected from a phenyl substituted by two substituents, and the substituents are independently selected from Cl or CF 3 , and when both substituents are Cl, the two substituents are adjacent; when both substituents are CF 3 , the two substituents are separated; when the two substituents are Cl and CF respectively 3 , the two substituents are adjacent.

[0042] Most preferably, the compound is selected from any one of the following compounds:

[0043]

[0044] On the other hand, the present invention provides a pharmaceutical composition, comprising an effective dose of the above compound or its pharmaceutically acceptable salt, stereoisomer, tautomer, solvate or prodrug, and a pharmaceutical carrier.

[0045] On the other hand, the present invention provides the use of the above compound or its pharmaceutically acceptable salt, stereoisomer, tautomer, solvate or prodrug, or the above pharmaceutical composition in the preparation of a drug.

[0046] Furthermore, the drug is used for treating, preventing or pre-treating diseases mediated at least in part by hypoxia-inducing factor (HIF) and / or erythropoietin (EPO).

[0047] Furthermore, the diseases are selected from the group consisting of: anemia disorders, renal anemia, neurological disorders and / or injuries, peripheral vascular disorders, ulcers, burns, chronic wounds, and at least one of ischemia-reperfusion injuries.

[0048] Furthermore, the diseases can also be selected from at least one of ischemia, such as myocardial infarction, pulmonary embolism, intestinal infarction, chronic renal failure, ischemic stroke, cardiac sclerosis, transient ischemic attack, macular degeneration, peripheral arterial disease, and congestive heart failure.

[0049] On the other hand, the present invention provides the use of the above-mentioned compound or pharmaceutical composition in the preparation of a drug for inhibiting the activity of prolyl hydroxylase.

[0050] Furthermore, the mode of action of the use is that the prolyl hydroxylase contacts the compound described herein or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate or prodrug thereof, or the pharmaceutical composition.

[0051] Finally, the present invention provides a preparation method of the above compound, and the synthetic route is as follows:

[0052] Wherein, R is selected from aryl or heteroaryl which is substituted or unsubstituted by substituents; the substituents are at least one of alkyl, alkoxy, haloalkyl, aralkyl, halogen, cyano, phenyl, and heteroaromatic rings containing oxygen or nitrogen.

[0053] It includes the following steps:

[0054] Step 1: React compound 1 with benzyl alcohol and sodium hydride to obtain compound 2;

[0055] Step 2: React compound 2 with an alkali metal hydroxide to obtain compound 3, and the alkali metal hydroxide is preferably sodium hydroxide;

[0056] Step 3: React compound 3 with glycine methyl ester hydrochloride in the presence of an amide coupling reagent to obtain compound 4;

[0057] Step 4: React compound 4 with palladium on carbon under hydrogen conditions to obtain compound 5;

[0058] Step 5: React compound 5 with (CF 3 SO 2 ) 2 NC6 H 5 React to obtain Compound 6;

[0059] Step Six: React Compound 6 with an arylboronic acid compound to obtain Compound 7;

[0060] Step Seven: React Compound 7 with an inorganic base to obtain Compound 8.

[0061] Furthermore, it includes the following steps:

[0062] Step One: React a mixture of benzyl alcohol and sodium hydride in an ice - water bath for half an hour, then add 1 and react overnight at room temperature to obtain Compound 2;

[0063] Step Two: Reflux Compound 2 with sodium hydroxide in a mixed solution of water and 1,4 - dioxane overnight to obtain Compound 3;

[0064] Step Three: Stir Compound 3 with glycine methyl ester hydrochloride and PyBOP in N,N - dimethylformamide or tetrahydrofuran at room temperature overnight to obtain Compound 4;

[0065] Step Four: Place Compound 4 and palladium - carbon in an alcohol solution or a tetrahydrofuran solution under a hydrogen atmosphere and stir at room temperature overnight to obtain Compound 5;

[0066] Step Five: React Compound 5 with (CF 3 SO 2 ) 2 NC 6 H 5 in an alcohol solution and stir at room temperature overnight to obtain Compound 6;

[0067] Step Six: React Compound 6 with an arylboronic acid compound in the presence of a palladium catalyst and an inorganic base, dissolve in 1,4 - dioxane, and stir and react overnight at 85 °C to obtain Compound 7;

[0068] Step Seven: Stir and react Compound 7 with an inorganic base in water and an organic solvent for 2 hours to obtain Compound 8;

[0069] Furthermore, in Step Six, the palladium catalyst can be selected from at least one of Pd(dppf)Cl 2 、Pd(PPh 3 ) 4 , and the inorganic base can be selected from at least one of cesium carbonate and sodium carbonate.

[0070] Furthermore, in Step Seven, the inorganic base can be selected from at least one of potassium hydroxide, sodium hydroxide, and lithium hydroxide, and the organic solvent can be selected from at least one of methanol, ethanol, and tetrahydrofuran.

[0071] As used herein, the term "pharmaceutically acceptable salt" refers to salts of the free acids or bases of the compounds represented by formula (I), which are non-toxic, biologically tolerable, or in other words, biologically suitable for administration to a subject. Generally, see G.S. Paulekuhn, et al., "Trends in Active Pharmaceutical Ingredient Salt Selection based Analysis of the Orange Book Database", J. Med. Chem., 2007, 50: 6665 - 72, S.M. Berge, et al., "Pharmaceutical Salts", J Pharm Sci, 1977, 66: 1 - 19, and Handbook of Pharmaceutical Salts, Properties, Selection, and Use, Stahl and Wermuth, Eds., Wiley-VC Hand VHCA, Zurich, 2002 (G.S. Paulekuhn et al., "Trends in Active Pharmaceutical Ingredient Salt Selection based Analysis of the Orange Book Database", Journal of Medicinal Chemistry, 2007, Vol. 50, pp. 6665 - 6672; S.M. Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Sciences, 1977, Vol. 66, pp. 1 - 19; and Handbook of Pharmaceutical Salts: Properties, Selection, and Use, edited by Stahl and Wermuth, Wiley-VCHandVHCA, Zurich, 2002). Examples of pharmaceutically acceptable salts are those salts that are pharmacologically effective and suitable for contact with the tissues of a patient without undue toxicity, irritation, or allergic response. The compounds of formula (I) can have groups that are sufficiently acidic, groups that are sufficiently basic, or both types of functional groups, and thus react with a variety of inorganic bases or organic bases, as well as inorganic acids and organic acids, to form pharmaceutically acceptable salts.

[0072] Examples of pharmaceutically acceptable salts include sulfates, bisulfates, hydrogen sulfates, sulfites, bisulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, caprates, octylates, acrylamides, formates, isobutyrates, hexanoates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates, tartrates, mesylates, propionates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, and mandelates.

[0073] The compounds of formula (I) contain basic nitrogen, and the required pharmaceutically acceptable salts can be prepared by any suitable method available in the art, for example, treating the free base with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, boric acid, phosphoric acid, etc., or treating the free base with an organic acid such as acetic acid, phenylacetic acid, propionic acid, stearic acid, lactic acid, ascorbic acid, maleic acid, hydroxymaleic acid, isethionic acid, succinic acid, valeric acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, oleic acid, palmitic acid, lauric acid, pyranosyl acids (such as glucuronic acid or galacturonic acid), α-hydroxy acids (such as mandelic acid, citric acid or tartaric acid), amino acids (such as aspartic acid, glutaric acid or glutamic acid), aromatic acids (such as benzoic acid, 2-acetoxybenzoic acid, naphthoic acid or cinnamic acid), sulfonic acids (such as laurylsulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid), any compatible mixture of acids such as those given as examples herein, and any other acids and mixtures thereof considered to be equivalents or acceptable substitutes according to the ordinary skill level in the art.

[0074] The compounds of formula (I) also contain a carboxylic acid, and the desired pharmaceutically acceptable salts can be prepared by any suitable method, for example, by treating the free acid with an inorganic or organic base, such as an amine (primary, secondary or tertiary amine), an alkali metal hydroxide, an alkaline earth metal hydroxide, any compatible mixture of bases such as those given as examples herein, and any other base and mixtures thereof considered to be equivalents or acceptable substitutes according to the ordinary skill level in the art. Exemplary examples of suitable salts include organic salts derived from the following substances: amino acids (such as N-methyl-D-glucosamine, lysine, choline, glycine and arginine), ammonia, carbonate, bicarbonate, primary amines, secondary amines, tertiary amines and cyclic amines (such as tromethamine, benzylamine, pyrrolidine, piperidine, morpholine and piperazine), and inorganic salts derived from the following substances: sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

[0075] The prodrugs described herein may include compounds having an amino acid residue or a polypeptide chain of two or more (such as two, three or four) amino acid residues covalently linked to the carboxylic acid group of formula (I) through an amide bond or an ester bond. Examples of amino acid residues include the twenty naturally occurring amino acids usually identified by three-letter symbols, as well as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, 3-methylhistidine, norvaline, β-alanine, γ-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine and methionine sulfone.

[0076] Other types of prodrugs can be prepared by derivatizing the free carboxylic acid of the structure of formula (I) into an amide or an alkyl ester. Examples of amides include those derived from ammonia, C 1-6 alkyl primary amines and di(C 1-6 alkyl) secondary amines. The secondary amines include 5-membered or 6-membered heterocycloalkyl or heteroaryl ring moieties. Examples of acid amides include those amides derived from ammonia.C 1-3 alkyl primary amines and di(C 1-2 alkyl) amines. Examples of the lipids of the present invention include C 1-7 alkyl esters, C 5-7 cycloalkyl esters, phenyl esters and (C 1-6alkyl)benzene esters. Preferably, the lipid includes methyl esters. Prodrugs can also be prepared by derivatizing free hydroxyl groups with groups including hemisuccinate esters, phosphate esters, dimethylaminoacetate esters, and phosphonyloxymethoxycarbonyl, according to methods such as those described in Fleisher et al., Adv. Drug Delivery Rev. 1996, 19, 115-130 (Fleisher et al., "Advances in Drug Delivery Reviews", 1996, Volume 19, Pages 115-130). Carbamate derivatives of hydroxyl groups and amino groups of amines can also produce prodrugs. Carbonate derivatives, sulfonate esters, and sulfate esters of hydroxyl groups can also provide prodrugs. Derivatizing the hydroxyl group to (acyloxy)methyl esters and (acyloxy)ethyl esters, where the acyl group can be an alkyl ester optionally substituted by one or more ether, amine, or carboxylic acid functional groups, or where the acyl group is an amino acid ester as described above, can also be used to produce prodrugs. This type of prodrug can be prepared as described in Robinson et al., J Med Chem. 1996, 39(1), 10-18 (Robinson et al., "Journal of Medicinal Chemistry", 1996, Volume 39, Issue 1, Pages 10-18). Free amines can also be derivatized to amides, sulfonamides, or phosphonamides. All of these prodrug moieties can be incorporated into groups including ether, amine, and carboxylic acid functional groups.

[0077] The beneficial effects of the present invention are as follows:

[0078] The present invention provides a new compound as a prolyl hydroxylase inhibitor, its preparation method, and uses, expanding the types of drugs for treating erythropoietin-related diseases and achieving good inhibitory activity against prolyl hydroxylase. The inhibitory effects of some compounds of the present invention on the PHD2 enzyme reach a level comparable to that of the marketed drug roxadustat (FG-4592), indicating that the compounds described in the present invention exhibit good biological activity against prolyl hydroxylase and can be used to prepare drugs for inhibiting the activity of prolyl hydroxylase. Detailed implementation manners

[0079] In order to make the technical means, creative features, achieved purposes, and functions of the present invention easy to understand, the following specific examples are used to further clarify the present invention. However, the following examples are only the preferred examples of the present invention and not all. Based on the examples in the implementation manners, other examples obtained by those skilled in the art without creative efforts all belong to the protection scope of the present invention.

[0080] In the following examples, unless otherwise specified, the operating methods used are conventional operating methods, and the equipment used is conventional equipment.

[0081] The following examples can further describe the present invention. Those skilled in the art will recognize that, to obtain the various compounds herein, the raw materials can be appropriately selected so that the ultimately required substituents will be carried with or without protection as needed throughout the reaction scheme to obtain the desired product. Alternatively, it may be necessary or desirable to use a suitable group to replace the ultimately required substituent, and the suitable group can be carried through the reaction scheme and then replaced with the required substituent under appropriate circumstances. Unless otherwise specified, the various variables are defined as above for formula (I). The reaction can be carried out between the melting point and the reflux temperature of the solvent, preferably between 0 °C and the reflux temperature of the solvent. The reaction can also be carried out above the normal reflux temperature of the solvent in a closed pressure vessel. However, these examples should not be construed as limiting the scope of the present invention.

[0082] Example 1

[0083] Preparation of {[5-(3-phenoxyphenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid

[0084]

[0085] Step 1) Preparation of 3,5-bis-benzyloxy-pyridine-2-carbonitrile (2):

[0086] DMF (500 mL) and 60% sodium hydride (26.4 g) were successively added to a reaction flask, and then the flask was placed in an ice-water bath. Benzyl alcohol (68.3 mL) was slowly added dropwise over about 20 minutes. After stirring for an additional ten minutes, 3,5-dichloro-2-cyanopyridine (51.6 g) was added to the reaction flask in one portion, and then the mixture was allowed to warm to room temperature and stirred overnight. After TLC detection showed that the raw materials had completely reacted, water was added to the reaction flask with stirring, and then the mixture was stirred for 1 hour, filtered, and dried to obtain 86.5 g of a yellow solid.

[0087] HPLC-MS: m / z 317 [M+H]

[0088] Step 2) Preparation of 3,5-bis-benzyloxy-pyridine-2-carboxylic acid (3):

[0089] 3,5-Bis-benzyloxy-pyridine-2-carbonitrile (2) (43.3 g) was dissolved in ethanol (360 mL), and then 30% aqueous NaOH solution (690 mL) was added to the reaction flask. Subsequently, the temperature was raised to 90 °C and the reaction was carried out overnight. After TLC detection showed that the raw materials had completely reacted, the reaction was stopped and cooled to room temperature. The pH was adjusted to between 1 and 2 with 4N HCl, and a solid appeared. The solid was filtered and dried to obtain 40.2 g of a solid.

[0090] HPLC-MS: m / z 336 [M+H]

[0091] Step 3) Preparation of methyl [(3,5 - bis - benzyloxy - pyridine - 2 - carbonyl)-amino]acetate (4):

[0092] The reaction flask was operated under anhydrous and anaerobic conditions, and N 2 was replaced. Then, DMF (700 mL), 3,5 - bis - benzyloxy - pyridine - 2 - carboxylic acid (3) (80.4 g), EDCI (63.7 mL), HOBt (6.5 g), DIPEA (127 mL), and glycine methyl ester hydrochloride (45.2 g) were successively added to the reaction flask under an ice - water bath, and then stirred at room temperature for 3 days. After TLC detection showed that the raw materials had completely reacted, water was added to the reaction flask under stirring, and then stirred for 1 hour. After filtration and drying, 80 g of white solid was obtained.

[0093] HPLC - MS: m / z 407 [M + H]

[0094] Step 4) Preparation of methyl [(3,5 - dihydroxy - pyridine - 2 - carbonyl)-amino]acetate (5):

[0095] The reaction flask was operated under anhydrous and anaerobic conditions, and N 2 was replaced once, and then H 2 was replaced three times. Then, methyl [(3,5 - bis - benzyloxy - pyridine - 2 - carbonyl)-amino]acetate (4) (80.4 g) was added to the reaction flask and dissolved in MeOH (800 mL). Subsequently, 10% Pd / C (8.0 g) was added, and the mixture was stirred overnight at room temperature. After TLC detection showed that the raw materials had completely reacted, the reaction was stopped, and part of MeOH was removed by reduced - pressure concentration. Then, water was added to the reaction system until white solid appeared. After filtration and drying, 33.2 g of off - white solid was obtained.

[0096] HPLC - MS: m / z 227 [M + H]

[0097] Step 5) Preparation of methyl [(3 - hydroxy - 5 - trifluoromethanesulfonyloxy - pyridine - 2 - carbonyl)-amino]acetate (6):

[0098] Methyl [(3,5 - dihydroxy - pyridine - 2 - carbonyl)-amino]acetate (5) (34.1 g) and diisopropylethylamine (DIPEA) (32.1 mL) were dissolved in MeOH (300 mL). Under an ice - water bath, N - phenyltrifluoromethanesulfonimide (61.9 g) was added, and the mixture was slowly warmed to room temperature and stirred overnight. After TLC detection showed that the raw materials had completely reacted, the reaction was stopped, and 37.1 g of gray solid was obtained by filtration.

[0099] HPLC - MS: m / z 359 [M + H]

[0100] Step 6) Preparation of methyl {[5 - (3 - phenoxyphenyl)-3 - hydroxypyridine - 2 - carbonyl]amino}acetate (7):

[0101] The reaction flask was operated under anhydrous and anaerobic conditions. N 2 was replaced once, and then methyl [(3-hydroxy-5-trifluoromethanesulfonyloxypyridine-2-carbonyl)amino]acetate (6) (358 mg), 3-phenoxybenzeneboronic acid (323.1 mg), Pd(dppf)Cl 2 (73.2 mg), K3PO4 (636.9 mg), and 1,4-dioxane (10 mL) were successively added to the reaction flask. After sealing, the reaction was carried out at 85 °C overnight. After TLC detection showed that the raw materials had completely reacted, the reaction was stopped. After cooling to room temperature, the mixture was extracted with ethyl acetate, washed with water, the organic phase was analyzed, concentrated, and column chromatography was performed to obtain about 200 mg of a white solid.

[0102] Step 7) Preparation of {[5-(3-phenoxyphenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid (8):

[0103] Methyl {[5-(3-phenoxyphenyl)-3-hydroxypyridine-2-carbonyl]amino}acetate (7) (180 mg) was dissolved in MeOH (4 mL), and then 30% aqueous NaOH solution (4 mL) was added. The temperature was raised to 40 °C and stirred for two hours. After TLC detection showed that the raw materials had completely reacted, the reaction was stopped. After cooling to room temperature, the organic solvent was removed by rotary evaporation under reduced pressure, and then the pH was adjusted to between 1 and 2 with 4N HCl. A solid appeared, and it was filtered to obtain 104.3 mg of a white solid, which is the compound of the present invention as a prolyl hydroxylase inhibitor.

[0104] HPLC-MS: m / z 365.23 [M+H]

[0105] Example 2

[0106] Preparation of {[5-(3-(4-chlorophenoxy)phenyl-3-hydroxypyridine-2-carbonyl]amino}acetic acid

[0107]

[0108] According to the method of Example 1, in step 6), 3-(4-chlorophenoxy)benzeneboronic acid (363.7 mg) was used to replace 3-phenoxybenzeneboronic acid, and the rest were the same, to obtain 150.5 mg of a white solid, which is the compound of the present invention as a prolyl hydroxylase inhibitor.

[0109] HPLC-MS: m / z 399.28 [M+H]

[0110] Example 3

[0111] Preparation of {[5-(3-(3-chlorophenoxy)phenyl-3-hydroxypyridine-2-carbonyl]amino}acetic acid

[0112]

[0113] According to the method of Example 1, in step 6), 3-(3-chlorophenoxy)phenylboronic acid (372.0 mg) was used to replace 3-phenoxyphenylboronic acid, and the rest were the same, obtaining 240.3 mg of a brown liquid, which is the compound of the present invention as a prolyl hydroxylase inhibitor.

[0114] HPLC-MS: m / z 397.21 [M-H]

[0115] Example 4

[0116] Preparation of {[5-(3-(2-chlorophenoxy)phenyl-3-hydroxypyridine-2-carbonyl]amino}acetic acid

[0117]

[0118] According to the method of Example 1, in step 6), 3-(2-chlorophenoxy)phenylboronic acid (372.0 mg) was used to replace 3-phenoxyphenylboronic acid, and the rest were the same, obtaining 241.3 mg of a white solid, which is the compound of the present invention as a prolyl hydroxylase inhibitor.

[0119] HPLC-MS: m / z 397.23 [M-H]

[0120] Example 5

[0121] Preparation of {[5-(3-(3,4-dichlorophenoxy)phenyl-3-hydroxypyridine-2-carbonyl]amino}acetic acid

[0122]

[0123] According to the method of Example 1, in step 6), 3-(3,4-dichlorophenoxy)phenylboronic acid (424.3 mg) was used to replace 3-phenoxyphenylboronic acid, and the rest were the same, obtaining 112.5 mg of a white solid, which is the compound of the present invention as a prolyl hydroxylase inhibitor.

[0124] HPLC-MS: m / z 433.13 [M+H]

[0125] Example 6

[0126] Preparation of {[5-(3-(4-methylphenoxy)phenyl-3-hydroxypyridine-2-carbonyl]amino}acetic acid

[0127]

[0128] According to the method of Example 1, in step 6), 3-(4-methylphenoxy)phenylboronic acid (342.7 mg) was used to replace 3-phenoxyphenylboronic acid, and the rest was the same. 123.5 mg of white solid was obtained, which is the compound of the present invention as a prolyl hydroxylase inhibitor.

[0129] HPLC-MS: m / z 379.24 [M+H]

[0130] Example 7

[0131] Preparation of {[5-(4-trifluoromethoxyphenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid

[0132]

[0133] According to the method of Example 1, in step 6), 4-trifluoromethoxyphenylboronic acid (308.9 mg) was used to replace 3-phenoxyphenylboronic acid, and the rest was the same. 150.5 mg of white solid was obtained, which is the compound of the present invention as a prolyl hydroxylase inhibitor.

[0134] HPLC-MS: m / z 357.18 [M+H]

[0135] Example 8

[0136] Preparation of {[5-(4-trifluoromethyl)phenyl-3-hydroxypyridine-2-carbonyl]amino}acetic acid

[0137]

[0138] According to the method of Example 1, in step 6), 4-trifluoromethylphenylboronic acid (284.9 mg) was used to replace 3-phenoxyphenylboronic acid, and the rest was the same. 75.3 mg of white solid was obtained, which is the compound of the present invention as a prolyl hydroxylase inhibitor.

[0139] HPLC-MS: m / z 341.07 [M+H]

[0140] Example 9

[0141] Preparation of {[5-(3,4-dichloro)phenyl-3-hydroxypyridine-2-carbonyl]amino}acetic acid

[0142]

[0143] According to the method of Example 1, in step 6), 3,4-dichlorophenylboronic acid (286.2 mg) was used to replace 3-phenoxyphenylboronic acid, and the rest was the same. 50.5 mg of white solid was obtained, which is the compound of the present invention as a prolyl hydroxylase inhibitor.

[0144] HPLC-MS: m / z 341.10 [M+H]

[0145] Example 10

[0146] Preparation of {[5-(3,5-bis(trifluoromethyl)phenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid

[0147]

[0148] According to the method of Example 1, in step 6), replace 3-phenoxybenzeneboronic acid with 3,5-bis(trifluoromethyl)benzeneboronic acid (386.9 mg), and the rest are the same, to obtain 120.5 mg of a white solid, which is the compound of the present invention as a prolyl hydroxylase inhibitor.

[0149] HPLC-MS: m / z 409.12 [M+H]

[0150] Example 11

[0151] Preparation of {[5-(4-phenyl)phenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid

[0152]

[0153] According to the method of Example 1, in step 6), replace 3-phenoxybenzeneboronic acid with 4-phenylbenzeneboronic acid (296.7 mg), and the rest are the same, to obtain 111.4 mg of a white solid, which is the compound of the present invention as a prolyl hydroxylase inhibitor.

[0154] HPLC-MS: m / z 349.21 [M+H]

[0155] Example 12

[0156] Preparation of {[5-(5-isoquinolinyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid

[0157]

[0158] According to the method of Example 1, in step 6), replace 3-phenoxybenzeneboronic acid with 5-isoquinolineboronic acid (259.4 mg), and the rest are the same, to obtain 167.5 mg of a white solid, which is the compound of the present invention as a prolyl hydroxylase inhibitor.

[0159] HPLC-MS: m / z 324.07 [M+H]

[0160] Example 13

[0161] Preparation of {[5-(2,3-dihydrobenzofuranyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid

[0162]

[0163] According to the method of Example 1, in step 6), 5-(2,3-dihydrobenzofuran)boronic acid (246.3 mg) was used to replace 3-phenoxybenzeneboronic acid, and the rest were the same. 143.5 mg of white solid was obtained, which was the compound of the present invention as a prolyl hydroxylase inhibitor.

[0164] HPLC-MS: m / z 315.11 [M+H]

[0165] Example 14

[0166] Preparation of {[5-(3-quinolyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid

[0167]

[0168] According to the method of Example 1, in step 6), 3-quinolineboronic acid (259.5 mg) was used to replace 3-phenoxybenzeneboronic acid, and the rest were the same. 30.1 mg of white solid was obtained, which was the compound of the present invention as a prolyl hydroxylase inhibitor.

[0169] HPLC-MS: m / z 324.12 [M+H]

[0170] Example 15

[0171] Preparation of {[5-(2-benzofuranyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid

[0172]

[0173] According to the method of Example 1, in step 6), 2-benzofuranboronic acid (243.7 mg) was used to replace 3-phenoxybenzeneboronic acid, and the rest were the same. 195.5 mg of white solid was obtained, which was the compound of the present invention as a prolyl hydroxylase inhibitor.

[0174] HPLC-MS: m / z 313.10 [M+H]

[0175] Example 16

[0176] Preparation of {[5-(6-N-methylindole)-3-hydroxypyridine-2-carbonyl]amino}acetic acid

[0177]

[0178] According to the method of Example 1, in step 6), 6-N-methylindoleboronic acid (262.5 mg) was used to replace 3-phenoxybenzeneboronic acid, and the rest were the same. 130.2 mg of white solid was obtained, which is the compound of the present invention as a prolyl hydroxylase inhibitor.

[0179] HPLC-MS: m / z 326.13 [M+H]

[0180] Example 17

[0181] Preparation of {[5-(4-[b,d]-dibenzothiophene)-3-hydroxypyridine-2-carbonyl]amino}acetic acid

[0182]

[0183] According to the method of Example 1, in step 6), 4-[b,d]-dibenzothiopheneboronic acid (342.2 mg) was used to replace 3-phenoxybenzeneboronic acid, and the rest were the same. 207.9 mg of white solid was obtained, which is the compound of the present invention as a prolyl hydroxylase inhibitor.

[0184] HPLC-MS: m / z 379.20 [M+H]

[0185] Example 18

[0186] Preparation of {[5-(4-[b,d]-dibenzofuran)-3-hydroxypyridine-2-carbonyl]amino}acetic acid

[0187]

[0188] According to the method of Example 1, in step 6), 4-[b,d]-dibenzofuranboronic acid (318.7 mg) was used to replace 3-phenoxybenzeneboronic acid, and the rest were the same. 134.7 mg of white solid was obtained, which is the compound of the present invention as a prolyl hydroxylase inhibitor.

[0189] HPLC-MS: m / z 363.20 [M+H]

[0190] Example 19

[0191] Preparation of {[5-(3-trifluoromethyl-5-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid

[0192]

[0193] According to the method of Example 1, in step 6), 3-trifluoromethyl-4-chlorobenzeneboronic acid (336.6 mg) was used to replace 3-phenoxybenzeneboronic acid, and the rest were the same. 54.7 mg of white solid was obtained, which is the compound of the present invention as a prolyl hydroxylase inhibitor.

[0194] HPLC-MS: m / z 375.16 [M+H]

[0195] Example 20

[0196] Preparation of {[5-(3,5-dichlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid

[0197]

[0198] According to the method of Example 1, in step 6), replace 3-phenoxybenzeneboronic acid with 3,5-dichlorobenzeneboronic acid (286.1 mg), and the rest are the same, to obtain 130.1 mg of a white solid, which is the compound of the present invention as a prolyl hydroxylase inhibitor.

[0199] HPLC-MS: m / z 341.04 [M+H]

[0200] Example 21

[0201] Preparation of {[5-(2,5-dichlorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid

[0202]

[0203] According to the method of Example 1, in step 6), replace 3-phenoxybenzeneboronic acid with 2,5-dichlorobenzeneboronic acid (286.1 mg), and the rest are the same, to obtain 90.5 mg of a white solid, which is the compound of the present invention as a prolyl hydroxylase inhibitor.

[0204] HPLC-MS: m / z 341.06 [M+H]

[0205] Example 22

[0206] Preparation of {[5-(3-chloro-4-fluorophenyl)-3-hydroxypyridine-2-carbonyl]amino}acetic acid

[0207]

[0208] According to the method of Example 1, in step 6), replace 3-phenoxybenzeneboronic acid with 3-chloro-4-fluorobenzeneboronic acid (261.5 mg), and the rest are the same, to obtain 103.5 mg of a white solid, which is the compound of the present invention as a prolyl hydroxylase inhibitor.

[0209] HPLC-MS: m / z 339.08 [M+H]

[0210] Biological tests

[0211] Test Example 1: Detection of PHD2 enzyme activity inhibition

[0212] Reagents and consumables:

[0213] PHD2 enzyme: purchased from Activemotif; Sodium α - ketoglutarate: purchased from Sigma; FITC - HIF1α: purchased from GL; Multi - well plate Nunc TM 384: purchased from ThermoScientific.

[0214] Test methods

[0215] (1) Prepare 1× Assay buffer detection solution (10 mM HEPES, 150 mM NaCl, 0.05% Tween 20, pH 7.4); (2) Preparation of compound concentration gradient: The test concentration of the test compound starts from 10 μM, is diluted 3 - fold, with 10 concentrations, and duplicate wells are tested; Dilute it into a solution with a final concentration of 100 - fold in a 384 - well plate, and then transfer 100 nL to a 384 - well reaction plate for standby using Echo550. Add 100 nL of 100% DMSO to the negative control well and the positive control well respectively; (3) Prepare an enzyme solution with a 2 - fold final concentration using 1× Assay buffer; (4) Add 5 μL of the enzyme solution with a 2 - fold final concentration to the compound well and the positive control well respectively; (5) Add 5 μL of 1× Assay buffer to the negative control well; (6) Centrifuge at 1000 rpm for 30 seconds, mix well by oscillation and incubate for 15 min; (7) Prepare a tracer solution with a 2 - fold final concentration using 1× Assay buffer. Add 5.0 μL of the tracer solution with a 2 - fold final concentration to initiate the reaction; (8) Centrifuge the 384 - well plate at 1000 rpm for 30 seconds, mix well by oscillation for 60 min, read the mP value from Envision (Multimode Plate Reader, PerkinElmer), export the data and process it to obtain the inhibition rate of the test compound. The results are shown in the following table:

[0216] Table 1.

[0217] Compound Number <![CDATA[IC 50 (nM)]]> FG-4592 65.6 1 61.6 2 50.5 3 86.9 4 59.6 5 138.8 6 76.7 7 104.2 8 90.8 9 25.3 10 26.4 11 96.6 12 118.0 13 87.9 14 54.2 15 99.7 16 106.2 17 142.4 18 87.9 19 53.0 22 23.0

[0218] As can be seen from the above table, the compounds in the embodiments of the present invention have good HIF - prolyl hydroxylase inhibitory activity.

[0219] Test Example 2: Cell experiment

[0220] ① Digest and collect Hep - 3B cells, use a complete culture medium (MEM basal medium containing NEAA, Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd., ZQ - 300) containing 10% FBS (Gibco, 10270 - 106) to adjust the cell density to 4×10 4cells / mL, inoculate 100 μL per well into 2 96-well plates pretreated with PDL (final concentration 10 μg / mL) for 1 h, and place them in a normoxic incubator (5% CO 2 , 21% O 2 , 37 °C) for 6 h to allow cell attachment. One of the plates is used to examine the effect of two concentrations of the compound on EPO production under normoxic conditions for 24 h, and at the same time, a normoxic solvent control well (DMSO, final concentration 0.1%) is set up. The other plate is used for the hypoxic solvent control well (DMSO, final concentration 0.1%) under hypoxic conditions (5% CO 2 , 1% O 2 , 37 °C) for 24 h.

[0221] ② After the cells are cultured under normoxia for 6 hours, aspirate and discard the original culture medium, rinse 3 times with PBS, and add DMSO (final concentration 0.1%) and the test compound (final concentration 10 μM) respectively, 200 μL per well, and culture under normoxia in serum-free medium for 24 h. The cells induced under hypoxia are not treated with drugs and are cultured in serum-free medium with DMSO (final concentration 0.1%) for 24 h. Aspirate the supernatant (about 200 μL) into a 0.2 mL EP tube and store it at -80 °C in the refrigerator for later ELISA detection. Add MTT solution (final concentration 0.5 mg / mL) to the lower-layer cells and continue to incubate in the normoxic incubator for about 4 h. When purple-blue crystals appear in the cells, stop the incubation, discard the supernatant, add 150 μL DMSO per well, shake and dissolve at 200 rpm for 10 min, and measure the absorbance at 570 nm, with 650 nm as the reference wavelength. Evaluate the cell viability changes of each experimental group with the cell viability of the solvent control well as 100%.

[0222] ③ For Abcam ELISA (Abcam, ab119522), use 50 μL of the cell supernatant sample, and the detailed procedure is carried out according to the kit instructions. Finally, calculate the EPO expression content (mIU / mL) of the test substance through the absorbance value (OD 450nm -OD 620nm ) of the sample and the standard curve. The results are shown in the following table:

[0223] Table 2.

[0224] Compound Number <![CDATA[IC 50 (nM)]]> FG-4592 65.6 1 61.6 2 50.5 3 86.9 4 59.6 5 138.8 6 76.7 7 104.2 8 90.8 9 25.3 10 26.4 11 96.6 12 118.0 13 87.9 14 54.2 15 99.7 16 106.2 17 142.4 18 87.9 19 23.0

[0225] The results of the biological activity test show that the compound of the present invention has a more significant promoting effect on the intracellular EPO expression compared with the hypoxic condition.

[0226] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt thereof: Wherein, R is selected from phenyl substituted by two substituents, said substituents being independently selected from Cl, OCF 3 or CF 3 .

2. The compound according to claim 1, characterized in that, the compound is selected from any one of the following compounds:

3. The compound according to claim 1, characterized in that, The substituents are independently selected from Cl or CF 3 , and when both substituents are Cl, the two substituents are adjacent; when both substituents are CF 3 , the two substituents are spaced apart; when the two substituents are Cl and CF respectively 3 , the two substituents are adjacent.

4. The compound according to claim 3, characterized in that, the compound is selected from any one of the following compounds:

5. A compound, characterized in that, the compound is selected from any one of the following compounds:

6. A compound, characterized in that, the compound is selected from any one of the following compounds:

7. A method for preparing the compound according to any one of claims 1-6, characterized in that, the synthetic route is as follows: including the following steps: Step 1: React compound 1 with benzyl alcohol and sodium hydride to obtain compound 2; Step 2: React compound 2 with an alkali metal hydroxide to obtain compound 3; Step 3: React compound 3 with glycine methyl ester hydrochloride in the presence of an amide coupling reagent to obtain compound Step 4: React compound 4 with palladium on carbon under hydrogen conditions to obtain compound 5; Step 5: React compound 5 with (CF 3 SO 2 ) 2 NC 6 H 5 to obtain compound 6; Step 6: React compound 6 with an arylboronic acid compound to obtain compound 7; Step 7: React compound 7 with an inorganic base to obtain compound 8.

8. The preparation method according to claim 7, characterized in that, specifically including the following steps: Step 1: React a mixture of benzyl alcohol and sodium hydride in an ice-water bath for half an hour, then add 1, and react overnight at room temperature to obtain compound 2; Step 2: Reflux compound 2 with sodium hydroxide in a mixed solution of water and 1,4-dioxane overnight to obtain compound 3; Step 3: Stir compound 3 with glycine methyl ester hydrochloride and PyBOP in N,N-dimethylformamide or tetrahydrofuran at room temperature overnight to obtain compound 4; Step 4: Place compound 4 and palladium on carbon in an alcohol solution or a tetrahydrofuran solution under a hydrogen atmosphere, and stir overnight at room temperature to obtain compound 5; Step 5: Compound 5 and (CF 3 SO 2 ) 2 NC 6 H 5 were stirred at room temperature overnight in an alcohol solution to obtain Compound 6; Step 6: React compound 6 with an arylboronic acid compound in the presence of a palladium catalyst and an inorganic base, dissolve it in 1,4-dioxane, under the condition of 85 °C, stir and react overnight to obtain compound 7; Step 7: Stir and react compound 7 with an inorganic base in water and an organic solvent for 2 hours to obtain compound 8.

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