WO2015154716A1 - 菲罗啉膦酸类衍生物及其制备方法和应用 - Google Patents

菲罗啉膦酸类衍生物及其制备方法和应用 Download PDF

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WO2015154716A1
WO2015154716A1 PCT/CN2015/076273 CN2015076273W WO2015154716A1 WO 2015154716 A1 WO2015154716 A1 WO 2015154716A1 CN 2015076273 W CN2015076273 W CN 2015076273W WO 2015154716 A1 WO2015154716 A1 WO 2015154716A1
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compound
group
pharmaceutically acceptable
acceptable salt
formula
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PCT/CN2015/076273
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English (en)
French (fr)
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朱岳
廖玉珍
张莉
柏旭
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吉林省博创药业有限公司
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Priority to US15/302,568 priority Critical patent/US10072035B2/en
Priority to JP2017504230A priority patent/JP6545254B2/ja
Priority to EA201692045A priority patent/EA030709B1/ru
Priority to CA2944805A priority patent/CA2944805C/en
Priority to CN201580018088.2A priority patent/CN106661060B/zh
Priority to EP15777566.9A priority patent/EP3130593B1/en
Priority to SG11201608422VA priority patent/SG11201608422VA/en
Priority to KR1020167031228A priority patent/KR102369908B1/ko
Publication of WO2015154716A1 publication Critical patent/WO2015154716A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/40—Esters thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6564—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms
    • C07F9/6571—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms
    • C07F9/6574—Esters of oxyacids of phosphorus
    • C07F9/65742—Esters of oxyacids of phosphorus non-condensed with carbocyclic rings or heterocyclic rings or ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
    • C07F9/576—Six-membered rings
    • C07F9/60—Quinoline or hydrogenated quinoline ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65583—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system each of the hetero rings containing nitrogen as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6564—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms
    • C07F9/6571—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms
    • C07F9/657163—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms the ring phosphorus atom being bound to at least one carbon atom
    • C07F9/657181—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms the ring phosphorus atom being bound to at least one carbon atom the ring phosphorus atom and, at least, one ring oxygen atom being part of a (thio)phosphonic acid derivative

Definitions

  • the invention relates to the field of pharmacy, in particular to a novel phenanthroline phosphonate compound and a pharmaceutically acceptable salt thereof, a preparation method of the compound, the compound and a pharmaceutically acceptable salt thereof as collagen prolyl-4-hydroxylation
  • an enzyme inhibitor for the preparation of a medicament for preventing or treating a disease associated with collagen prolyl-4-hydroxylase.
  • the pathogenesis of liver fibrosis is based on the accumulation of excess collagen (especially type I collagen collagen I) by the liver and excessive secretion and deposition in extracellular mediators (ECM) (Clin. Sci. 1997, 92, 103).
  • the biological synthesis process of collagen includes a series of post-translational modifications of anterior collagen, in which the cell modification process requires the participation of five enzymes, including three collagen hydroxylases and two collagen glycosyltransferases.
  • prolyl-4-hydroxylase (P4H) is a tetramer having two ⁇ subunits (P4H ⁇ 1, P4H ⁇ 2) and two ⁇ subunits (P4H ⁇ 1, P4H ⁇ 2).
  • the ⁇ subunit is a disulfide isomerase, and the main part of the catalytic action exists in the ⁇ subunit, and the main role of the ⁇ subunit is to determine the activity of the enzyme.
  • P4H is an enzyme that plays a key role in all of the 21 known types of collagen synthesis (Critical Reviews in Biochemistry and Molecular Biology 2010, 45, 106) and is an important rate-limiting enzyme for collagen synthesis.
  • P4H is localized on the endoplasmic reticulum of cells, catalyzing the hydroxylation of proline residues on the X-Pro-Gly sequence of collagen, with the participation of iron ions, molecular oxygen, alpha-ketoglutarate and ascorbate. Thereby 4-hydroxyproline is formed.
  • P4H produces 4-hydroxyproline (4-HYP) by hydroxylating a specific site of proline in procollagen, thereby increasing the stability of the collagen triple helix under physiological conditions.
  • 4-HYP in the collagen structure is reduced, the collagen cannot be normally folded into a triple helix, which is unstable and degraded at physiological temperatures (Matrix Biol. 2003, 22, 15). Therefore, pharmacological intervention in P4H enzyme activity has been recognized as an effective way to inhibit excess collagen (ie, fibrosis) (Hepatol. 1998, 28, 404).
  • Chemically synthesized small molecule P4H inhibitors have been shown to prevent collagen synthesis in ex vivo and in vivo experiments (J. Hepatol.
  • the P4H inhibitor HOE077 inhibits procollagen mRNA expression and reduces hepatic stellate cell proliferation (Hepatol. Res. 2002, 23, 1; J. Hepatol. 1997, 27, 185) and inhibits hepatic stellate cell activation (Hepatol. 1996, 23, 755).
  • HOE077 has a dose-dependent effect on the inhibition of collagen gene and protein, but has no effect on the total protein synthesis of cells.
  • P4H is ubiquitous in the body. Therefore, targeting P4H inhibitors to diseased organs without affecting other normal organs is the key to developing safe and effective P4H inhibitors.
  • German Hermelo Company now Senofe, France
  • HOE077 for the treatment of cirrhosis (Hepatol. 1996, 23, 755; J. Hepatol. 1997, 27, 185), which was effective in preclinical experiments.
  • serious adverse reactions such as cataracts have occurred in clinical trials.
  • collagen inhibition can seriously affect important organ functions such as eyes and kidneys (J. Biol. Chem. 2010, 285, 42023).
  • Collagen synthesis is widely present in the cell matrix, so the inhibition of organ matrix collagen formation can directly lead to macromolecular exudation, resulting in changes in organ function. Therefore, how to deliver P4H inhibitors to designated organs has become the key to the development of P4H inhibitors for the treatment of organ fibrosis, such as liver fibrosis.
  • Precursor drug delivery therapy is widely used in targeted therapy (J. Pharmacol. Exp. Ther. 2005, 312, 554), and there are also reports on the use of phosphonates formed from 1,3-diols and phosphonic acids to achieve liver targets. Administration (J. Med. Chem. 2008, 51, 666).
  • liver precursor used in this patent refers to the modification of the active ingredient of the drug, and the drug precursor formed after the modification has almost no pharmacological effect.
  • the precursor can only form an active ingredient by metabolic cleavage catalyzed by a liver-specific enzyme (such as cytochrome P450, CYP), thereby exerting a pharmacological effect in the liver.
  • a liver-specific enzyme such as cytochrome P450, CYP
  • the invention provides a compound of formula I or formula II, and pharmaceutically acceptable salts thereof:
  • X is chlorine or an OR 3 group
  • R 3 is hydrogen, -C(O)-(alkyl group of 1 to 6 carbon atoms), -PO(OH) 2 or -CH 2 OPO ( OH) 2 ;
  • R 1 , R 2 are each independently selected from hydrogen, an alkyl group of 1 to 6 carbon atoms, -CH 2 OCO- (alkyl group of 1 to 6 carbon atoms), and -CH 2 OCOO- (1-6 carbons)
  • the alkyl group of the atom; R 1 and R 2 may also be a group of the following formula:
  • Y is an aryl or heteroaryl group
  • X can be selected from the group consisting of chlorine and an OR 3 group, R 3 is hydrogen, -C(O)-(alkyl of 1-6 carbon atoms), -PO(OH) 2 or -CH2OPO(OH) 2 .
  • R 1 and R 2 may each independently be selected from the group consisting of hydrogen, an alkyl group of 1 to 6 carbons, -CH 2 OCO- (alkyl group of 1 to 6 carbon atoms), and -CH 2 OCOO-(1)
  • R 1 and R 2 may also be a group bonded together as follows:
  • Y is an aryl group, a heteroaryl group.
  • Z is hydrogen or -CH2OPO(OH) 2 ;
  • R 1 and R 2 are each independently selected from hydrogen, an alkyl group of 1 to 6 carbon atoms, -CH 2 OCO- (1-6 An alkyl group of a carbon atom and -CH 2 OCOO- (an alkyl group of 1 to 6 carbon atoms); and R 1 and R 2 may also be a group bonded together as follows:
  • Y is an aryl or heteroaryl group.
  • Z can be selected from the group consisting of hydrogen and -CH2OPO(OH) 2 .
  • R 1 and R 2 may each independently be selected from the group consisting of hydrogen, an alkyl group of 1 to 6 carbons, -CH 2 OCO- (alkyl group of 1 to 6 carbon atoms), -CH 2 OCOO-(1 An alkyl group of 6 to 6 carbon atoms; R 1 and R 2 may also be a group bonded together as follows:
  • Y is an aryl group, a heteroaryl group.
  • the compound is a compound of the formula:
  • the compound is a compound of the formula:
  • the compound is a compound of the formula:
  • the present invention provides a process for the preparation of the above phenanthroline phosphonates and pharmaceutically acceptable salts thereof.
  • the present invention provides the above phenanthroline phosphonates and pharmaceutically acceptable salts thereof as collagen prolyl-4-hydroxylase inhibitors for the prophylaxis or treatment of collagen prolyl-4-hydroxylation The application of drugs for enzyme-related diseases.
  • the present invention provides the use of a compound of formula I or formula II or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prophylaxis or treatment of a disease associated with collagen prolyl-4-hydroxylase.
  • the present invention provides a compound of the formula I or formula II or a metabolite thereof or a pharmaceutically acceptable salt thereof as a gel Application of prolylyl-4-hydroxylase inhibitors.
  • the present invention provides a therapeutically effective amount of a compound of Formula I or Formula II or a pharmaceutically acceptable salt thereof to a subject suffering from chronic liver injury to protect liver function.
  • the present invention provides a therapeutically effective amount of a compound of Formula I or Formula II or a pharmaceutically acceptable salt thereof to a subject suffering from chronic liver injury to prevent and treat liver fibrosis.
  • the present invention provides for the use of a therapeutically effective amount of a compound of Formula I or Formula II, or a pharmaceutically acceptable salt thereof, for administration to a subject at risk of developing diabetes to prevent liver fibrosis.
  • Figure 1 is the 50% inhibitory concentration (IC50) of compound 9c against the P4H enzyme.
  • Figure 2 is a time-concentration curve of Compound 16c in plasma after oral administration of Compound 27 (39 mg/kg) and intravenous administration of Compound 16c (3 mg/kg).
  • Figure 3 is a HE staining of liver in sham-operated rats.
  • Figure 4 shows HE staining of liver (2 weeks) in a bile duct ligation (BDL) fibrosis model rat.
  • Figure 5 is a HE staining of liver (2 weeks) in a bile duct ligation (BDL) fibrosis model rat after oral administration of Compound 27 (30 mg/kg).
  • BDL bile duct ligation
  • alkyl refers to a saturated aliphatic hydrocarbon group comprising straight chain, branched chain, cyclic, up to and containing 20 carbon atoms. Suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl, and cyclopropyl groups and the like. The alkyl group may also have from 1 to 3 substituents.
  • aryl refers to an aromatic group containing from 5 to 14 ring atoms, at least one ring having a conjugated electron system, including an aromatic ring, a heteroaryl ring, and a aryl or biaryl ring having an all carbon atom, and With a substituent.
  • the aryl group may have 1 to 6 substituents.
  • a heteroaromatic ring or an aromatic heterocyclic ring refers to a group containing 5 to 14 ring atoms, wherein 1-4 heteroatoms are atoms on the aromatic ring, and the remaining ring atoms are carbon atoms.
  • Suitable heteroatoms are oxygen, sulfur, nitrogen and selenium atoms.
  • Suitable aromatic heterocycles are furan, thiophene, pyridine, pyrrolidine, pyrrolidine with a lower alkyl substituent on the nitrogen, pyridyl oxynitride, pyrimidine, pyrazine, imidazole and the like. With a substituent.
  • Substituted aryl and “substituted heteroaryl” refer to an aryl or heteroaryl ring group having 1-6 substituents. These substituents may be a lower alkyl group, a lower alkoxy group, a lower carbon polyhalogenated alkyl group, a halogen, a hydroxyl group or an amino group.
  • halogen means fluoro, chloro, bromo or iodo.
  • an effective therapeutic dose refers to a dose of a compound or composition that is required to improve, alleviate, remove or prevent, alter, delay one or more symptoms of a particular disease condition.
  • pharmaceutically acceptable salt refers to a salt of a compound of Formula I or Formula II and a prodrug thereof, mixed with an organic acid or base or an inorganic acid or base.
  • suitable acids include acetic acid, adipic acid, benzenesulfonic acid, camphorsulfonic acid, citric acid, 1,2-ethanedisulfonic acid, dodecylsulfonic acid, fumaric acid, gluconic acid, gluconic acid, glucuronic acid , hippuric acid, heptanic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, 2-hydroxyethanesulfonic acid, lactic acid, lactobionic acid, maleic acid, methanesulfonic acid, methylboronic acid, monomethyl sulfate, 2 -naphthalenesulfonic acid, nitric acid, oleic acid, palmitic acid, phosphoric acid, polygalacturonic acid,
  • subject refers to male and female mammals that are treated, such as dogs, cats, cows, horses, sheep, and humans.
  • prodrug refers to a substance that, when placed in a biological system, releases a biologically active compound via a spontaneous chemical reaction, an enzymatic chemical reaction, a metabolic chemical reaction, or a combination of these reactions.
  • a common prodrug is formed by the association of certain groups with functional groups of a drug molecule such as a hydroxyl group, a thiol group, a carboxyl group, and an amino group, and these linkages are broken in the living body.
  • Common prodrugs include, but are not limited to, esters, wherein the group may be an alkyl group, an aryl group, an aralkyl group, an acyloxyalkyl group attached to a carboxyl group.
  • the alkoxycarbonyloxyalkyl group may also be an acyl group, an alkoxycarbonyl group, an aminocarbonyl group, a phosphate ester or a sulfate ester bonded to a hydroxyl group, a mercapto group or an amino group.
  • These mentioned groups are merely examples, not all, and any other known type of prodrug can be prepared by any familiar person.
  • Prodrugs of the compounds of formula I or formula II are also within this scope. Prodrugs must be converted to biologically active compounds or precursors of the active compounds by some form of chemical reaction. In some cases, the prodrug is also biologically active, but it is usually less active than the drug itself.
  • the prodrug will improve the efficacy or safety of the drug by improving the bioavailability of the oral bioavailability or the half-life of the drug.
  • Sex. Prodrugs can be used to increase the bioavailability of the drug, and can increase the acceptability of the drug by masking or reducing unpleasant properties (such as bitter or gastrointestinal irritation), changing the solubility for intravenous injection, prolonging or retaining Release and deliver the drug, reduce the difficulty of the formula, or position the drug.
  • Prodrugs are described in detail in the following monographs: The Organic Chemistry of Drug Design and Drug Action, San Diego Academic Press, Richard B. Silverman, 1992. Chapter 8: "Prodrugs and Drug Delivery Systems” pp. 352- 401; Design of Prodrugs, by H.
  • [R] in the formula represents the amount of the R isomer
  • [S] represents the amount of the S isomer
  • treating includes the following conditions: preventing the occurrence of a disease (preventive therapy), stopping the disease (slowing or stopping the progression of the disease), alleviating the symptoms and side effects of the disease (including conservative treatment) and alleviating the disease (causing Disease recovery).
  • the compounds of the invention may be administered at a dose of from 0.01 to 2500 mg per day. On the one hand, this dosage range is from about 5 mg to about 500 mg. The dosage can be dispensed at will.
  • the compound of the present invention can be used together with other drugs, and can be used in a normal dose or in a dose. Appropriate ratio.
  • the compounds of the invention may be part of a combination therapy regimen, also known as co-administration or cocktail therapy, and the various drugs may be administered together, either simultaneously or in separate batches, or sequentially.
  • the compounds of the present invention can be used after a period of use of other drugs, can be used in combination with other drugs in one course of treatment, can be used as part of a combination treatment regimen, or taken before treatment with another drug in one treatment process. .
  • the compounds of the present invention can be administered in a variety of routes, including oral, parenteral, respiratory, topical or rectal administration, in association with pharmaceutically suitable carriers, excipients, and vehicles.
  • parenteral administration as used herein includes various injection methods such as subcutaneous injection, intravenous injection, intramuscular injection, and arterial injection. Both arterial and intravenous injections mentioned herein are administered via a catheter, and in general, intravenous injection is preferred.
  • Pharmaceutically acceptable salts of the compounds of the invention include sodium, potassium, calcium, magnesium, lithium, barium, amino acid, acetate, adipate, benzenesulfonate, hydrobromide, camphorsulfonate Acid salt, hydrochloride, citrate, ethanedisulfonate, propionate, lauryl sulfate, fumarate, glucoheptonate, gluconate, glucuronate, hippurate, sea Acid salt, bromide, chloride, iodide, isethionate, lactate, lactobionate, maleate, methanesulfonate, methyl bromide, methanesulfonate, Naphthalene sulfonate, nitrate, oleate, palmitate, phosphate, polygalacturonate, stearate, succinate, sulfate, sulfosalicylic acid, tannic acid, Tartrate, terephthalate
  • the active ingredients in the drug will have different states depending on the mode of administration. Orally, for example, it can be prepared into tablets, troches, lozenges, aqueous suspensions or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups and mashes. Agent.
  • the method for preparing an oral preparation can be referred to a production process of a known drug, and the preparation may also be a variety of other ingredients suitable for oral administration such as a sweetener, a flavoring agent, a coloring agent and a preservative.
  • non-toxic excipients that meet pharmaceutical standards can also be added to the tablets.
  • these excipients may be inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, and sodium phosphate; may be corn starch, pulverizing agents such as alginic acid, and granules; may be, for example, starch, coagulation A crosslinking agent such as a gum or a gum; or a lubricant such as magnesium stearate or stearic acid. Tablets may also be coated or stripped of the coating by known methods, including the use of microencapsulation techniques to extend the time during which the agent is broken down and absorbed in the gastrointestinal tract, thereby achieving a longer duration of action.
  • time extenders such as glyceryl monostearate or glyceryl distearate can be used alone or in combination with wax.
  • Oral preparations may be prepared by mixing the active ingredient with a solid inert diluent (such as calcium phosphate, kaolin) and encapsulating it in a hard gelatin capsule.
  • the active ingredient may be mixed with water or oil medium (such as peanut oil, liquid paraffin, olive). Oil) is mixed and packaged into soft gel capsules.
  • the active substance of the present invention can also be mixed with an excipient suitable for industrial production to prepare an aqueous suspension.
  • excipients include, for example, sodium cellulose methylcarboxylate, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinyl pyrrolidone, leaf gum, and A suspension of gum arabic; such as natural phospholipids (such as lecithin), condensation products of alkylene oxide with fatty acids (such as polyethylene glycol stearate), condensation products of ethylene oxide and long-chain fatty alcohols (such as ten Heptacycloalkane ethylene oxyethanol), a dispersing agent and a wetting agent for the condensation product of ethylene oxide with a hexitol which is partially esterified with a fatty acid, such as polyoxyethylene sorbitan monooleate.
  • natural phospholipids such as lecithin
  • the aqueous suspension may also have one or more preservatives such as ethyl p-hydroxybenzoate, n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, one or A variety of sweeteners such as sucrose and saccharin.
  • preservatives such as ethyl p-hydroxybenzoate, n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, one or A variety of sweeteners such as sucrose and saccharin.
  • an oil suspension preparation by placing the active ingredient in a vegetable oil (such as peanut oil, olive oil, sesame oil, coconut oil) or a mineral oil (such as liquid paraffin).
  • the oral suspension preparation may have a viscous agent (e.g., beeswax, hard paraffin, cetyl alcohol).
  • Sweeteners as described above, flavoring agents can also be added to the formulation to make it more suitable for administration. It can be preserved by adding an antioxidant such as ascorbic acid to the preparation.
  • the dispersible powders and granules of the present invention can be used in the preparation of aqueous suspensions, in which the active ingredient is added to water, together with dispersing agents, wetting agents, suspending agents, one or more preservatives. Suitable dispersing agents, wetting agents and suspending agents are disclosed above. Other excipients, such as sweeteners, flavoring agents and coloring agents, may also be added.
  • the pharmaceutical compositions of the invention may also be in the form of an oil-water emulsion.
  • the oil phase may be a vegetable oil (such as olive oil and peanut oil), a mineral oil (such as liquid paraffin), or a mixed oil.
  • Suitable emulsifiers include natural gums (such as gum arabic, tragacanth), natural phospholipids (such as soy lecithin), condensed esters of fatty acids with hexitols or partially condensed esters (such as sorbitan monooleate), and these A condensation product of a partially condensed ester with ethylene oxide (e.g., polyoxyethylene sorbitan monooleate).
  • Sweeteners and flavoring agents can also be added to the emulsion.
  • Syrups and elixirs can be formulated with sweeteners such as glycerol, sorbitol, sucrose. Such preparations may contain a demulcent, a preservative, a flavoring agent, and a coloring agent.
  • the pharmaceutical forms of the invention may also be formulated as sterile aqueous suspensions or oily suspensions for sterile injection.
  • the suspension can be prepared using existing methods using the appropriate dispersing or wetting agents and suspending agents mentioned above.
  • the sterile injectable solution may also be a solution or suspension of a diluent or solvent which is non-toxic for injection, for example, as an ice-dried powder, dissolved in 1,3-butanediol.
  • the carrier and solvent that can be used can be water, Ringer's solution, physiological saline.
  • aseptically treated oils can also be employed as a solvent or suspending medium. Any mild oil, including synthetic monoglycerides and diglycerides, can be used.
  • fatty acids such as oleic acid It can also be used for injection preparations.
  • a sustained release preparation for oral administration will contain from 20 to 2000 micromoles (about 10 to 1000 mg) of the active ingredient together with a suitable carrier (from 5% to 95% of the total ingredient). It is more appropriate to provide an accurate dosage form of the preparation.
  • an aqueous solution for intravenous injection is required to satisfy an injection amount of 30 ml per hour, and it is required to contain about 0.05 to 50 micromoles (about 0.025 to 25 mg) of active ingredient per ml.
  • oral preparations can be prepared as discrete units, such as capsules, cachets, tablets, each unit containing a fixed dose of active ingredient; may be in the form of a powder or granules; And suspensions; or oil-water emulsions or water-oil emulsions.
  • the active ingredient can also be administered in the form of a bolus, a dry syrup and a paste.
  • tablets can be made by tableting and casting.
  • Tableting is the separation of free-flowing active ingredients (powder or granules) with cross-linking agents (eg polyvinylpyridone, gel, hydroxypropyl methylcellulose), lubricants, inert diluents, preservatives, Decomposing agents (such as sodium starch glycolate, cross-linked polyvinylpyridone, cross-linked cellulose methylcarboxylate), surfactants or dispersants are mixed and compacted in a suitable machine.
  • the cast tablet is prepared by infusing a powdered compound with an inert liquid diluent and pouring it into a mold.
  • Tablets may also be selected for dressing and engraving at the time of formulation to slow or control the release of the active ingredient, for example, different ratios of hydroxypropyl methylcellulose may provide different release rates. Tablets can be added to the casing so that they are not in the stomach but are released in the intestines. In view of the fact that the compounds of formula I may be hydrolyzed in acid, this formulation has great advantages.
  • Suitable formulations for topical oral administration include lozenges in which the active ingredient is placed on a flavoring base (usually sucrose, acacia, tragacanth); the active ingredient is placed on an inert basis (gel, glycerin, sucrose and gum arabic) Lozenges; and mouthwashes in which the active ingredient is placed in a suitable liquid carrier.
  • a flavoring base usually sucrose, acacia, tragacanth
  • inert basis gel, glycerin, sucrose and gum arabic
  • mouthwashes in which the active ingredient is placed in a suitable liquid carrier.
  • the formulation for rectal administration may be a suppository in which the active compound is placed on a suitable base such as cocoa, butter, salicylate.
  • Formulations for vaginal administration may contain the active ingredient and other known carriers in a pessary, trocar, cream, gel, paste, foam or spray.
  • the preparation for administration by injection includes a water-sterilized injection solution or a non-aqueous osmotic sterile injection solvent containing an antioxidant, a buffer salt, a bactericide, and a solute for osmotic pressure of the preparation and the recipient's blood; water and non-aqueous A suspension of bacteria which may contain a suspending agent and a viscous agent.
  • the preparation may be in unit dose package or multi-dose package, for example, ampoules and vials, which can be stored in ice and added to the sterility before use.
  • Liquid carrier eg water for injection).
  • the solutions and suspensions for injection can be prepared aseptic powders, granules and tablets as described above.
  • the preparation for injection can be administered by continuous infusion by using an internal pump and a drug strip.
  • the injection can be by the Hickman method or the PICC method or any other means of administration suitable for injection and intravenous injection.
  • the dosage unit dose include the daily dose or unit, the amount of each dose, and the number of doses per day.
  • the compounds of the present invention can be prepared according to the procedures described below, or can be synthesized by an experienced person by a similar method in the published literature. It is to be understood that the following methods are merely illustrative of the preparation process, and the claims of the present invention are not limited thereto.
  • a typical synthetic method of Formula I can be summarized into five basic steps (from the back to the front): (1) preparation of the prodrug; (2) deprotection of the phosphonate; (3) quinoline ring Modification; (4) synthesis of quinoline ring; (5) synthesis of key intermediates.
  • Compounds of formula II can be obtained by reacting a compound of formula I with a suitable group. Specific operations for protection and deprotection in the route can be referred to known documents (for example, "Protecting Groups in Organic Synthesis” 3rd Edition, Wiley, 1999).
  • All stereoisomers of the compounds of the invention have a unique effect, whether the mixture is substantially pure or pure.
  • the compounds of the present invention may have a chiral center on the phosphorus atom and any carbon atom (including any R substituent).
  • the compounds of formula I may exist in enantiomeric or diastereomeric forms, or a mixture of the two.
  • the starting materials used in the preparation scheme may be racemates, enantiomers or diastereomers.
  • the resulting enantiomer or diastereomer can be isolated using conventional methods. For example, chromatography or fractional crystallization can be used to separate the diastereomers, and the enantiomer derivatives can be separated by chromatography.
  • Prodrugs can be introduced at different stages of the synthesis. Due to the instability of different prodrugs, they are usually introduced at the end of the synthesis, but there are also cases introduced at the initial stage of the synthesis for other reasons.
  • the compound of formula I may be a phosphonic acid (R 1 and R 2 are both hydrogen) and may also form a suitable protected state.
  • the phosphonic acid may be subjected to nucleophilic reaction with an electrophile such as an alkyl halide and an alkyl sulfate. A phosphonate is formed.
  • the compound of formula I may be a compound wherein R 1 and R 2 are hydrogen with an appropriate acyloxyalkyl halide (eg, chlorine, bromine, iodine; Phosphorus) Sulfur 1990, 54, 143; Synthesis 1988, 62) under the action of a suitable base (eg pyridine, triethylamine, diisopropylethylamine) in a suitable solvent (eg DMF, J. Med. Chem. 1994, 37) , 1875) prepared in the reaction.
  • an appropriate acyloxyalkyl halide eg, chlorine, bromine, iodine; Phosphorus
  • the carboxylic acid ester moiety in the acyloxyalkyl halide can be, but is not limited to, acetate, propionate, isobutyric acid, trimethyl acetate, benzoate, carbonate and other carboxylic acid esters. .
  • the active phosphonic dichloride can be derived from the corresponding phosphonic acid chlorinating reagent (for example, dichlorosulfoxide, J. Med. Chem. 1994, 1857; oxalyl chloride, Tetrahedron Lett. 1990, 31, 3261; phosphorus pentachloride , Synthesis 1974, 490) Reaction formation. Further, the phosphonic dichloride may also be derived from the corresponding disilylphosphonate (Synth. Commu. 1987, 17, 1071) and the dialkylphosphonate (Tetrahedron Lett. 1983, 24, 4405; Bull. Soc. Chim .1993, 130, 485) Preparation.
  • phosphonic acid chlorinating reagent for example, dichlorosulfoxide, J. Med. Chem. 1994, 1857; oxalyl chloride, Tetrahedron Lett. 1990, 31, 3261; phosphorus pentachloride , Synthesis 1974, 490
  • the cyclic 1,3-propanediol phosphonate can be reacted with the corresponding phosphonic dichloride-substituted 1,3-propanediol (J. Med. Chem. 2008, 51, 666), or a suitable coupling reagent (for example, DCC, EDCI, PyBOP; Synthesis 1988, 62) performs a coupling reaction.
  • cyclic 1,3-propanediol phosphonate can also be prepared by coupling a phosphonic acid and a diol under cassite reaction conditions (Synthesis 1981, 1; J. Org. Chem. 1992, 52, 6331). And other acid coupling reagents including, but not limited to, carbonyl diimine (Collect. Czech. Chem. Commun. 1994, 59, 1853; Bioorg. Med. Chem. Lett. 1992, 2, 145; Tetrahedron Lett. 1988, 29, 1189), and PyBOP (Tetrahedron Lett. 1993, 34, 6743).
  • the invention discloses a method of synthesizing and isolating a phosphonate prodrug of formula I. Since the phosphorus atom is a chiral center, the prodrug prepared using the racemate in place of 1,3 propanediol will be a mixture of isomers. For example, the use of racemic 1-Y substituted 1,3-propanediol produces a racemic cis prodrug mixture and a racemic trans prodrug mixture. On the other hand, the use of the R enantiomerically enriched substituted 1,3-propanediol results in an enantiomerically enriched R-cis prodrug and an R-trans prodrug. These compounds can be separated by a combination of column chromatography and/or segmental crystallization.
  • the compound of formula I may have a different protecting group attached to the oxygen or nitrogen atom of the hydroxypyridine ring.
  • R 3 is an acyl group
  • the compound of formula I can be obtained by reacting a compound wherein R 3 is hydrogen with an appropriate acid halide under appropriate conditions (J. Org. Chem. 1989, 54, 166);
  • chlorine is present
  • the compound of formula I can be obtained by reacting a compound wherein X is OH with a chlorinating reagent under appropriate conditions (for example: phosphorus oxychloride, J. Org. Chem. 1950, 15, 1224; trichloroacetonitrile). , Tetrahedron Lett. 2012, 53, 674).
  • R 1 is hydrogen compounds of formula I can be used known conditions fracture phosphates and phosphonates prepared by reacting the phosphonate.
  • Halogenated silanes are often used to cleave different phosphonates, and the obtained silicophosphonates can be used to obtain the desired phosphonic acid via a mild hydrolysis process.
  • an acid scavenger e.g., hexamethyldisilazane, 2,6-lutidine
  • halosilanes include trimethylchlorosilane (J. Org. Chem. 1963, 28, 2975), trimethylbromosilane (Tetrahedron Lett.
  • the phosphonates can also be cleaved under strong acid conditions (for example, hydrobromic acid or hydrochloric acid, US Patent 3,524,846, 1970). These phosphonates can also be converted to phosphonic acid by conversion to phosphorus phosphide using a halogenating reagent such as phosphorus pentachloride, dichlorosulfoxide, boron tribromide; J. Chem. Soc. 1961, 238 to form a phosphonic acid.
  • a halogenating reagent such as phosphorus pentachloride, dichlorosulfoxide, boron tribromide
  • the aryl and benzylphosphonates may also be subjected to hydrogenolysis conditions (Synthesis 1982, 412; J. Med. Chem. 1985, 28, 1208; Nature 1953, 171, 76) or metal reduction conditions (J. Am. Chem. Soc). .1977, 99, 5118) fracture. Electrochemical (J. Org. Chem. 1979, 44, 4508) and high temperature pyrolysis (Synth. Commun. 1980, 10, 299) conditions can also be used to break different phosphonates.
  • the construction of the phenanthroline nucleus can be established using the conditions established in the literature. For example, the thermal shutdown strategy shown in the following scenario.
  • Aromatic amine 1 is produced under the conditions of sodium 3-nitrobenzenesulfonate, sulfuric acid and glycerol to produce quinoline. 2.
  • Quinoline 2 is brominated by NBS in acetic acid to obtain compound 3, and then reduced by ferric acid to obtain compound 4.
  • Compound 4 phosphine Acidification gives the phosphonate 5, which is then reacted with compound 17 to undergo a thermal ring closure reaction to give phenanthroline 7 wherein R is hydrogen (in the formula I, X is a hydroxyl group and R 1 and R 2 are ethyl groups).
  • Prodrugs are usually introduced at a later stage of the synthesis, but for other reasons, prodrugs can sometimes be introduced at an early stage.
  • a cyclic phosphonate diester prodrug can be synthesized as shown in the scheme below.
  • compound 16c can be reacted with a chlorophosphate ester under the action of a suitable base such as triethylamine and a catalyst such as 4-dimethylaminopyridine in a suitable solvent such as dichloromethane to give the ester.
  • a suitable base such as triethylamine and a catalyst such as 4-dimethylaminopyridine
  • a suitable solvent such as dichloromethane
  • Deprotection of diethyl phosphate can be achieved by using a common phosphate deprotection reagent.
  • compound 21 is treated with trimethylbromosilane to provide phosphate 22, which can be further converted to the desired salt.
  • compound 22 is treated with a mixed solution of sodium hydrogencarbonate in water and methanol to give the disodium salt 23.
  • compound 16c can be reacted with chloromethyl di-tert-butyl phosphate in a suitable solvent (e.g., dimethyl sulfoxide) with a suitable base (e.g., potassium carbonate) to afford phosphates 24 and 25.
  • a suitable solvent e.g., dimethyl sulfoxide
  • a suitable base e.g., potassium carbonate
  • Deprotection of di-tert-butyl phosphate can be achieved using a conventional t-butyl ester deprotecting reagent.
  • treatment of compounds 24 and 25 with trifluoroacetic acid in dichloromethane affords phosphoric acid 26 and 27, respectively, which can be further converted to the desired salt.
  • Recombinant human P4H expression was completed in the E. coli expression system.
  • the nucleotide sequence encoding the full-length P4H protein was cloned into the pET28_N-His_TEV plasmid expression system to obtain pET28_N-His_TEV_P4HA1/PDI, which was then transformed into E. coli Origami2 (DE3) for co-expression.
  • the enzyme protein was purified by MonoQ ion exchange column, identified by TEV digestion and mass spectrometry, and further purified by HistrapHP and gel filtration chromatography (Hiload 16/60 superdex 200).
  • the purified P4H enzyme protein was tested for enzyme activity in the following coupled enzyme reaction system and the effect of the compound on the enzyme activity was identified: 100 mM Tris (pH 7.0), 0.1 mM (NH4) 2 Fe(SO 4 ) 2 , 0.1 mM ascorbic acid. 0.2 mM CoA, 0.2 mM ATP, 0.5 uM succinyl-CoA synthetase, 100 uM 2-ketoglutarate, 100 uM (Pro-Pro-Gly) 10 peptide, 50 nM P4H protein, total 50 ul.
  • the reaction was carried out at 25 ° C for 45 min; 10 ul of MLG R1 was added for 10 min; 10 ul of MLG R2 was added and the reaction was carried out for 20 min.
  • P4H catalyzes the formation of the product succinic acid by ketoglutarate and the polypeptide substrate in a coenzyme and an appropriate enzymatic reaction environment.
  • Succinic acid then forms succinyl-Coenzyme and phosphoric acid (Pi) under the action of succinyl-Coenzyme synthetase.
  • the level of phosphoric acid produced can be determined using an MLG kit to reflect the level of P4H.
  • the resulting green product (MG+) H 2 PMo 12 O 40 ) was identified by absorption peak at an OD of 630 nm.
  • Wistar rats weighing 200 ⁇ 20g, were divided into two groups, 6 each, male and female, and free drinking water diet. Dosage: The first group was administered with 3 mg ⁇ kg -1 of the tail vein of the compound 16c, and the second group was administered with the disodium salt of the compound 27 for 39 mg ⁇ kg -1 . Before administration (0h) and 0.08h after administration, 0.17h, 0.33h, 0.5h, 0.75h, 1h, 1.5h, 2h, 3h, 5h, 7h, 0.3mL whole blood was taken through the posterior venous plexus. The pre-cooled heparinized EP tube was centrifuged (15,000 rpm) for 5 min at 4 ° C, and 100 ⁇ L of plasma was separated and immediately placed at -80 ° C for storage.
  • **t1/2 indicates the time when the blood concentration drops to half of the peak.
  • ***AUC0-t indicates the area under the blood concentration-time curve until the measurement time.
  • ****AUC0- ⁇ indicates the area under the blood concentration-time curve when all drugs are cleared.
  • Oral absorption utilization is calculated as Compound 16c in blood.
  • the calculation method is the area under the curve of the blood-medicine-time curve (AUC) after the oral administration (po) and the equivalent dose of the drug (AUC) and the equivalent dose of the drug (iv).
  • Miv represents the molar concentration of the intravenously administered drug
  • Mpo represents the molar concentration of the drug orally administered.
  • the AUC0-t of Compound 16c in the blood after intravenous injection of Compound 16c (3 mg/kg) and Compound 27 (39 mg/kg) was 2064.58 g ⁇ h/mL and 5235.70 g ⁇ h/mL, respectively. Therefore, based on blood The bioavailability (F) of Compound 27 calculated by Compound 16c was 25.4% (i.e., 5235.70 / (2064.58 x 10) x 100%).
  • Sham operation group 6 rats, after anesthesia, routine disinfection of the abdominal skin after shaving, aseptic operation, cut the abdominal cavity along the midline of the abdomen, free the common bile duct, suture the muscles and skin respectively;
  • Liver fibrosis model group 12 rats, after anesthesia, routine disinfection of the abdominal skin after shaving, aseptic operation to cut the abdominal cavity along the midline of the abdomen, free the common bile duct and ligation, respectively suture the muscles and skin;
  • Oral administration group 12 rats, after anesthesia, routine disinfection of the abdominal skin after shaving, aseptic operation cut the abdominal cavity along the midline of the abdomen, free the common bile duct and ligation, respectively suture the muscles and skin.
  • the disodium salt of Compound 27 was administered, 30 mg/kg, and dissolved in water for administration once a day.
  • ALT alanine aminotransferase
  • AST aspartate transaminase Enzyme
  • Rats were sacrificed 2 weeks after BDL, and liver tissues were stained with HE and Masson.
  • ALT and AST are the most commonly used indicators for detecting liver function in clinical practice. ALT is mainly present in hepatocyte cytoplasm. AST is mainly present in hepatocyte mitochondria. When liver cells are damaged, serum ALT and AST levels are elevated. Reflect the degree of damage to liver cells. After BDL, the ALT and AST of serum and liver tissue homogenate in the model group were significantly increased. After 14 days of compound 27 administration, the animals were sacrificed. The serum and liver tissue homogenate levels of ALT and AST decreased significantly, which was significantly different from the model group. (**P ⁇ 0.01), see Table 3; Table 4. It is indicated that compound 27 alleviates the degree of liver function damage after BDL and has a protective effect on liver damage caused by bile reflux.
  • hepatic lobule As shown in Fig. 3, the structure of hepatic lobule is normal.
  • the hepatocytes are arranged radially around the central vein.
  • the hepatocytes in the lobules are arranged neatly, the hepatocytes are uniform in size, and there is no degeneration or necrosis.
  • Model group see Figure 4, hepatic lobular structure disorder, hepatocyte swelling, cytoplasmic loose, fibrous connective tissue hyperplasia.
  • Drug administration group As shown in Figure 5, the treatment group changed the pathological changes of liver tissue.

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Abstract

本发明涉及一种新的菲罗啉膦酸类化合物及其药用盐，所述化合物及其药用盐作为胶原脯氨酰羟化酶抑制剂在制备预防或治疗与胶原脯氨酰-4-羟化酶相关的疾病药物中的应用。

Description

菲罗啉膦酸类衍生物及其制备方法和应用 技术领域
本发明涉及制药领域，具体涉及一种新的菲罗啉膦酸类化合物及其药用盐，所述化合物的制备方法，所述化合物及其药用盐作为胶原脯氨酰-4-羟化酶抑制剂在制备预防或治疗与胶原脯氨酰-4-羟化酶相关的疾病的药物中的应用。
背景技术
下面关于与本发明相关的背景介绍用于帮助对本发明的理解，但不应被认为是本发明的已有技术。所有引用的出版物都被全文参考。
肝纤维化的发病基础是肝脏合成过量胶原(特别是I型胶原collagen I)并分泌过度而沉积在细胞外介质(ECM)(Clin.Sci.1997，92，103)。胶原蛋白的生物学合成过程包括一系列的前骨胶原的翻译后修饰，其中细胞修饰过程需要5种酶的参与，包括3种胶原羟化酶和2种胶原糖基转移酶。在这些羟化酶中，脯氨酰-4-羟化酶(P4H)是一个具有2个α亚基(P4Hα1,P4Hα2)和2个β亚基(P4Hβ1,P4Hβ2)的四聚体。其中β亚基是二硫化异构酶，起催化作用的主要部位存在于β亚基，而α亚基的主要作用是决定酶的活性。P4H是在所有的已知21种类型胶原合成过程中起关键作用的酶(Critical Reviews in Biochemistry and Molecular Biology2010，45，106)，是胶原合成的重要限速酶。P4H定位在细胞的内质网上，在铁离子，分子氧，α-酮戊二酸和抗坏血酸盐的参与下，通过催化胶原蛋白X-Pro-Gly序列上的脯氨酸残基的羟化，从而形成4-羟基脯氨酸。
P4H通过羟化前胶原中特定位点的脯氨酸生成4-羟基脯氨酸(4-HYP)，从而增加胶原三螺旋结构在生理条件下的稳定性。相反，如果胶原结构中4-HYP减少，胶原不能正常折叠成三螺旋，在机体生理温度下不稳定而降解(Matrix Biol.2003，22，15)。因此，药理性干预P4H酶活性已被公认为抑制过剩胶原(即纤维化)的有效途径(Hepatol.1998，28，404)。已经证实化学合成的小分子P4H抑制剂在离体和在体实验中具有阻止胶原合成的作用(J.Hepatol.1997，27，185；Hepatol.1996，23，755；Hepatol.1998，28，404；Biochem.J.1994，300，525；J.Hepatol.1991，13，S35)。例如,P4H抑制剂HOE077抑制前胶原mRNA表达和降低肝星状细胞增殖(Hepatol.Res.2002，23，1；J. Hepatol.1997，27，185)，并抑制肝星状细胞激活(Hepatol.1996，23，755)。HOE077对胶原基因和蛋白的抑制作用呈剂量关系，但对细胞的总蛋白合成无影响。其作用机制可能为通过阻止TIMP基因表达，从而促进胶原降解(J.Gastroenterol.1999，34，376)。而且多种P4H抑制剂在几种动物肝硬化模型(CCl4，TAA等)中有抑制肝纤维化的作用(Hepatol.1998，28，404；Hepatol.1996，23，755；J.Hepatol.1997，27，185)。还报道了另一种P4H抑制剂FG-041(1,4-二氢菲罗啉-4-酮-3-羧酸)在动物实验中能够抑制心肌梗死(Circulation2001，104，2216)。其他报道包括P4H抑制剂能够抑制膀胱阻滞等(Urology 2012，80，1390)。
P4H在机体内无处不在，因此，将P4H抑制剂靶向输送到疾病器官而不影响其他正常器官是开发安全有效P4H抑制剂的关键。九十年代德国赫美罗公司(现为法国塞诺菲)最先开发出HOE077治疗肝硬化(Hepatol.1996，23，755；J.Hepatol.1997，27，185)，前临床实验中效果显著，但在临床实验中出现严重不良反应如白内障等。还有文献报道了胶原抑制可严重影响眼、肾脏等重要器官功能(J.Biol.Chem.2010，285，42023)。胶原合成广泛存在于在细胞基质中，因此器官细胞基质胶原形成受到抑制后可直接导致大分子渗出，从而引起器官功能改变。因此，如何能将P4H抑制剂输送到指定的器官已成为是否能开发出用于治疗器官纤维化(如肝纤维化)的P4H抑制剂的关键。前体给药疗法在靶向治疗方面被广泛使用(J.Pharmacol.Exp.Ther.2005，312，554)，也有文献报道应用1,3-二醇与膦酸形成的膦酸酯实现肝脏靶向给药(J.Med.Chem.2008,51,666)。本专利采用的肝脏前体给药是指将药物的有效成分进行修饰，修饰后形成的药物前体几乎无药效作用。给药后前体只有在肝脏特有的酶(如细胞色素P450，CYP)的催化下代谢裂解后才能形成有效成分，从而在肝脏中发挥药效。
发明内容
本发明的目的在于提供一种新的菲罗啉膦酸类化合物及其药用盐。本发明的另一目的还在于提供所述化合物及其药用盐的制备方法。本发明的再一目的是提供所述化合物及其药用盐作为胶原脯氨酰-4-羟化酶抑制剂在制备预防或治疗与胶原脯氨酰-4-羟化酶相关的疾病的药物中的应用。
在第一方面，本发明提供了具有通式I或通式II的化合物及其药用盐：
Figure PCTCN2015076273-appb-000001
通式I通式II
其中在通式I中，X是氯或者OR3基；R3是氢、-C(O)-(1-6个碳原子的烷基)、-PO(OH)2或-CH2OPO(OH)2；
R1，R2分别独立地选自氢、1-6个碳原子的烷基、-CH2OCO-(1-6个碳原子的烷基)和-CH2OCOO-(1-6个碳原子的烷基)；R1和R2也可以是如下式的连接在一起的一个基团：
Figure PCTCN2015076273-appb-000002
其中Y是芳基或杂芳基；
一方面，X可以选自氯和OR3基团，R3是氢、-C(O)-(1-6个碳原子的烷基)、-PO(OH)2或-CH2OPO(OH)2。
另一方面，R1和R2可以分别独立地选自氢、1-6个碳的烷基、-CH2OCO-(1-6个碳原子的烷基)和-CH2OCOO-(1-6个碳原子的烷基)；R1和R2也可以是如下式的连接在一起的一个基团：
Figure PCTCN2015076273-appb-000003
其中Y是芳基，杂芳基。
其中在通式II中，Z是氢或-CH2OPO(OH)2；R1、R2分别独立地选自氢、1-6个碳原子的烷基、-CH2OCO-(1-6个碳原子的烷基)和-CH2OCOO-(1-6个碳原子的烷基)；R1和R2也可以是如下式的连接在一起的一个基团：
Figure PCTCN2015076273-appb-000004
其中Y是芳基或杂芳基。
一方面，Z可以选自氢和-CH2OPO(OH)2。
另一方面，R1和R2可以分别独立地选自氢、1-6个碳的烷基、-CH2OCO- (1-6个碳原子的烷基)、-CH2OCOO-(1-6个碳原子的烷基)；R1和R2也可以是如下式的连接在一起的一个基团：
Figure PCTCN2015076273-appb-000005
其中Y是芳基，杂芳基。
在一个优选的实施方案中，所述化合物为下式的化合物：
Figure PCTCN2015076273-appb-000006
在另一个优选的实施方案中，所述化合物为下式的化合物：
Figure PCTCN2015076273-appb-000007
在另一个优选的实施方案中，所述化合物为下式的化合物：
Figure PCTCN2015076273-appb-000008
在第二方面，本发明提供了制备上述菲罗啉膦酸类化合物及其药用盐的方法。
在第三方面，本发明提供了上述菲罗啉膦酸类化合物及其药用盐作为胶原脯氨酰-4-羟化酶抑制剂在制备预防或治疗与胶原脯氨酰-4-羟化酶相关的疾病的药物中的应用。
本发明提供了通式I或通式II化合物或其药用盐在制备预防或治疗与胶原脯氨酰-4-羟化酶相关的疾病药物中的应用。
本发明提供了通式I或通式II化合物或其体内代谢产物或其药用盐作为胶 原脯氨酰-4-羟化酶抑制剂的应用。
本发明将治疗有效剂量的通式I或通式II化合物或其药用盐给慢性肝损伤的受试者服用可保护肝脏功能。
本发明将治疗有效剂量的通式I或通式II化合物或其药用盐给慢性肝损伤的受试者服用可预防和治疗肝纤维化。
本发明提供了将治疗有效剂量的通式I或通式II化合物或其药用盐用于给有发展出糖尿病危险的受试者服用，可防止肝纤维化。
附图说明
图1为化合物9c对P4H酶的50％抑制浓度(IC50)。
图2为口服给药化合物27(39mg/kg)和静脉注射给药化合物16c(3mg/kg)后，血浆中化合物16c的时间-浓度曲线。
图3为假手术大鼠肝脏HE染色。
图4为胆管结扎(BDL)纤维化模型大鼠肝脏(2周)HE染色。
图5为口服给药化合物27(30mg/kg)后，胆管结扎(BDL)纤维化模型大鼠肝脏(2周)HE染色。
具体实施方式
术语定义
下面的术语应用于本发明中，除非特殊解释，否则将遵循下面的定义。
术语“烷基”指包含直链、支链、环状、上至并包含20个碳原子的饱和脂肪烃基团。合适的烷基基团包括有甲基、乙基、正丙基、异丙基、以及环丙基等。烷基也可以带有1-3个取代基。
术语“芳基”指含有5-14个环原子，至少一个环拥有共轭电子体系的芳香基团，包括有全碳原子的芳环、杂芳环和并芳环或联芳环，并可带有取代基。芳基可以带有1-6个取代基。
杂芳环或者芳杂环是指含有5-14个环上原子的基团，其中1-4个杂原子为芳香环上原子，其余环上原子为碳原子。适合的杂原子有氧、硫、氮和硒原子。合适的芳杂环有呋喃、噻吩、吡啶、吡咯烷、氮上带有低碳数烷基取代基的吡咯烷、吡啶氮氧化物、嘧啶、吡嗪、咪唑及其他类似杂环，并均可带有取代基。
术语“随意取代”或“取代”指基团带有1至4个不同的取代基，可以分别是低碳数烷基、低碳数芳基、低碳数芳烷基、低碳数环状烷基、低碳数杂环烷基、羟基、低碳数烷氧基、低碳数芳氧基、多卤代烷氧基、芳烷氧基、低碳数杂芳基、低碳数杂芳氧基、低碳数杂芳烷基、低碳数杂芳烷氧基、叠氮基、氨基、卤素、低碳数烷巯基、氧基、低碳数酰烷基、低碳数羧酸酯基、羧酸、酰胺基、硝基、低碳数酰氧基、低碳数胺烷基、低碳数烷胺芳基、低碳数烷芳基、低碳数烷胺烷基、低碳数烷氧芳基、低碳数芳胺基、低碳数芳烷胺基、磺酰基、低碳数酰胺烷芳基、低碳数酰胺芳基、低碳数羟烷基、低碳数卤代烷基、低碳数烷胺烷羧基、低碳数脲烷基、氰基、低碳数烷氧烷基、低碳数多卤代烷基或低碳数芳烷氧烷基。
“取代芳基”和“取代杂芳基”指芳环或者杂芳环基团上带有1-6个取代基。这些取代基可以是低碳数烷基、低碳数烷氧基、低碳数多卤代烷基、卤素、羟基或氨基。
术语“卤素”指氟、氯、溴或碘。
词组“有效治疗剂量”指的是改善、减轻、去除或者预防、改变、延迟某种特定疾病状况的一个或多个症状所需要的一种化合物或组合物的剂量。
术语“药用盐”指通式I或通式II的化合物及其前体药与有机酸或碱或者无机酸或碱混合生成的盐。合适的酸包括乙酸、己二酸、苯磺酸、樟脑磺酸、柠檬酸、1,2-乙二磺酸、十二烷基磺酸、富马酸、葡萄糖甲酸、葡萄糖酸、葡萄糖醛酸、马尿酸、半乙醇酸盐酸、氢溴酸、盐酸、氢碘酸、2-羟基乙磺酸、乳酸、乳糖酸、马来酸、甲磺酸、甲基硼酸、硫酸单甲酯、2-萘磺酸、硝酸、油酸、帕莫酸、磷酸、多聚半乳糖醛酸、硬脂酸、琥珀酸、硫酸、磺基水杨酸、鞣酸、酒石酸、对苯二甲酸、对甲基苯磺酸。与合适的碱混合生成的盐包括钠盐、钾盐、钙盐、镁盐、锂盐、铯盐、氨基酸盐。
术语“受试者”指接受治疗的雄性及雌性哺乳动物，例如狗、猫、牛、马、羊以及人。
术语“前体药”指当被放入一个生物体系中，可以经由自发的化学反应、酶催化的化学反应、代谢化学反应或这几种反应的综合作用释放出生物活性化合物的物质。常见的前体药是由某些基团与药物分子的官能团如羟基、巯基、羧基和氨基等相连形成的，这些连接在生物体内会断裂。常见的前体药包括并不仅限于酯，其中的基团可以是连接在羧基上的烷基、芳基、芳烷基、酰氧烷 基、烷氧羰氧烷基，也可以是连接在羟基、巯基、氨基上的酰基、烷氧羰基、氨羰基、磷酸酯或硫酸酯。这些提到的基团仅仅是例子，并不是全部，任何熟悉的人都可以制备出其他已知的多种类型的前体药。通式I或通式II的化合物的前体药也在此范围内。前体药必须经过某种形式的化学反应转换为具有生物活性的化合物或活性化合物的前体。某些情况下，前体药也具有生物活性，但通常会比药物本身的活性低，这种情况下，前体药会通过提高口服生物利用度或者药动半衰期等性质提高药物的疗效或安全性。前体药可以用于提高药物的生物利用度，还可以通过遮盖或降低令人不快的特性(例如苦味或胃肠刺激)提高药物的可接受程度，改变溶解性用于静脉注射，延长或保留释放和送药，降低配方难度，或者是定位给药。前体药在以下几篇专著中有详细的介绍：The Organic Chemistry of Drug Design and Drug Action,圣地亚哥学术出版社Richard B.Silverman,1992.第8章:“Prodrugs and Drug delivery Systems”pp.352-401；Design of Prodrugs,由H.Bundgaard,Elsevier Science,Amsterdam,1985；Design of Biopharmaceutical Properties through Prodrugs and Analogs,由华盛顿美国医药协会E.B.Roche编辑,,1977；和Drug Delivery Systems,由牛津市牛津大学出版社R.L.Juliano编辑,1980。
术语“对映异构体过量百分值(％ee)”用于表述光学纯度。％ee值通过下式得到
Figure PCTCN2015076273-appb-000009
公式中的[R]代表R异构体的量，[S]代表S异构体的量。从上式可以计算出当R异构体为主要异构体时的％ee值。
术语“治疗”一种疾病，包括以下情况：预防疾病的发生(预防疗法)，制止疾病(减慢或停止疾病的发展)，使得疾病的症状及副作用减轻(包括保守疗法)和减轻疾病(导致疾病复原)。
本发明的化合物的制剂:
本发明的化合物可以以每天0.01到2500毫克的剂量服用。一方面，这个剂量区域是5毫克左右到500毫克左右。该服用剂量可以随意分配。
本发明化合物可以与其他的药物共同使用，可以使用正常剂量，也可以用 适当比例。本发明的化合物可以作为复合治疗方案中的一部分，也被称为联合给药或鸡尾酒疗法，多种药物可以一同服用，也可以同时或分批单独服用，或者顺序服用。本发明的化合物可以在使用其他药物一段时间后再使用，可以同其他药物在一个疗程内使用，可以作为复合治疗方案中的一部分，或者在一个治疗过程中在用另一种药物治疗前先服用。
为实现治疗目的，本发明中的化合物可以同药学适宜的载体、辅料和介质制成制剂通过多种途径给药，包括口服、肠胃外给药、呼吸道喷剂、局部或直肠给药。此处使用的术语肠胃外给药包括皮下注射、静脉注射、肌肉注射和动脉注射等多种注射方式。此处提到的动脉注射和静脉注射都是通过导管给药，一般说来，静脉注射更优先。
本发明化合物的药用盐包括钠盐、钾盐、钙盐、镁盐、锂盐、铯盐、氨基酸盐、醋酸盐、己二酸盐、苯磺酸盐、氢溴酸盐、樟脑磺酸盐、盐酸盐、柠檬酸盐、乙二磺酸盐、丙酸酯月桂硫酸盐、富马酸盐、葡庚酸盐、葡萄糖酸盐、葡糖醛酸盐、马尿酸盐、海克酸盐、溴化物、氯化物、碘化物、羟乙基磺酸盐、乳酸盐、乳糖醛酸盐、马来酸盐、甲磺酸盐、甲基溴化物、甲基磺酸盐、萘磺酸盐、硝酸盐、油酸盐、棕榈酸盐、磷酸盐、聚半乳糖醛酸盐、硬脂酸盐、琥珀酸盐、硫酸盐、磺基水杨酸盐、单宁酸盐、酒石酸盐、对苯二甲酸盐、对甲苯磺酸盐、以及三乙基碘化物。
药物中的有效成分会因为不同的给药方式具有不同的状态。以口服给药为例，可以制备成片剂、糖锭剂、菱形锭剂、水悬浊液或油悬浊液、可分散粉末或微粒、乳浊液、硬胶囊或软胶囊、糖浆及酏剂。制备成为口服药剂的方法可参照已知药物的生产流程,制剂中也可以为适于口服添加如甜味剂，调味剂，染色剂和防腐剂在内的多种其他成分。为适于生产，片剂中也可加入无毒符合药物标准的赋形剂。例如，这些赋形剂可以是如碳酸钙、碳酸钠、乳糖、磷酸钙、以及磷酸钠的惰性稀释药；可以是玉米淀粉、褐藻酸之类的粉碎剂和颗粒剂；可以是如淀粉、凝胶、树胶之类的交联剂；还可以是如硬脂酸镁、硬脂酸之类的润滑剂。片剂还可以用已知方法包裹或剥离糖衣，包括使用微封装技术来延长药剂在胃肠道中分解、吸收的时间，从而获得更长的作用时间。例如，像甘油单硬脂酸酯或甘油双硬脂酸酯这样的时间延长剂可以单独或跟蜡一起使用。
口服制剂可以将有效成分同固体惰性稀释剂(如磷酸钙，高岭土)混合后封装于硬凝胶胶囊内，也可以将有效成分同水或油介质(如花生油，液体石蜡，橄榄 油)混合封装成软凝胶胶囊。
本发明的活性物质也可以同适于工业生产的赋形剂混合制成水悬浮液。这类赋形剂包括如纤维素甲基羧酸钠、甲基纤维素、乙基纤维素、羟丙基纤维素、羟丙基甲基纤维素、海藻酸钠、聚乙烯吡咯烷酮、树叶胶和阿拉伯橡胶的悬浮剂；如天然磷脂(如卵磷脂)，环氧烷同脂肪酸的缩合产物(如聚乙二醇硬脂酸酯)，环氧乙烷和长链脂肪醇的缩合产物(如十七碳烷乙烯氧基乙醇)，环氧乙烷同与脂肪酸部分酯化的己糖醇的缩合产物(如聚氧乙烯山梨聚糖单油酸酯)的分散剂和湿润剂。水悬浮液中也可以有一种或多种如对羟基苯甲酸乙酯、对羟基苯甲酸正丙酯的防腐剂，一种或多种的染色剂，一种或多种调味剂，一种或多种甜味剂，如蔗糖和糖精。
也可以通过将活性成分置于植物油(如花生油，橄榄油，芝麻油，椰子油)或矿物油(如液体石蜡)中制成油悬浮液制剂。口服悬浮制剂可以有粘稠剂(如蜂蜡，硬石蜡，十六烷基醇)。甜味剂(如前文所述)，调味剂也可以加入制剂使之更适于服用。可以通过向制剂中加入抗坏血酸之类的抗氧化剂来保存。
本发明的分散粉剂和颗粒可以用于制备水悬浮液，将活性成分加入水中，并加入分散剂，湿润剂，悬浮剂，一种及多种防腐剂。前文已公开实例合适的分散剂，湿润剂及悬浮剂。其他的赋形剂，如甜味剂，调味剂和染色剂，也可加入。
本发明的药学成分也可制成油水乳浊液的形式。油相可以是植物油(如橄榄油和花生油)，矿物油(如液体石蜡)，或混合油。适合的乳化剂包括天然树胶(如阿拉伯树胶，黄芪胶)，天然磷脂(如大豆卵磷脂)，脂肪酸与己糖醇的缩合酯或部分缩合酯(如山梨聚糖单油酸酯)，以及这些部分缩合酯同环氧乙烷的缩合产物(如聚氧乙烯山梨聚糖单油酸酯)。乳化液也可加入甜味剂和调味剂。
糖浆及酏剂可与甜味剂(如甘油，山梨醇，蔗糖)一起制成制剂。这类制剂中可以包含缓和剂，防腐剂，调味剂及染色剂。
本发明的药剂形式还可以制成无菌水悬浮液或含油悬浮液，应用于无菌注射。可以应用现有方法，使用上面提到的合适的分散剂或湿润剂和悬浮剂制备悬浮液。无菌注射液还可以是无毒可用于注射的稀释剂或溶剂制成的溶液或悬浮液，例如制备成冰干粉末，溶于1,3-丁二醇。可使用的载体和溶剂可以是水，林格氏溶液，生理盐水。此外，经无菌处理的油也可用作溶剂或悬浮介质。任何温和油，包括合成单甘油酯和二甘油酯都可以使用。此外，如油酸的脂肪酸 也可用于注射制剂。
与运载物混合制成一份药剂的活性成分的剂量因接受治疗的主体和给药方式有变化。例如，给人口服的缓释制剂会含有20到2000微摩(大约10到1000毫克)的活性成分以及适当的载体(占总成分的5％到95％)。可以提供准确服用剂量的制剂方式更合适。例如，用于静脉注射的水溶液为满足每小时30毫升的注入量，每毫升需要含有大约0.05到50微摩(大约0.025到25毫克)的活性成分。
如上面注解，口服制剂可以制成分离的单元，如胶囊、扁囊剂、片剂，每个单元含有固定剂量的活性成分；可以制成粉末或颗粒；可以制成水或非水液体的溶液和悬浮液；或油水乳浊液或水油乳浊液。活性成分还可以以大丸药、干药糖剂和浆糊的形态服用。
选择性的与一种或多种附加成分混合，可以通过压片与灌铸制作片剂。压片是将自由流动的活性成分(粉末或颗粒)选择性的与交联剂(如聚乙烯吡啶酮，凝胶，羟丙基甲基纤维素)，润滑剂，惰性稀释剂，防腐剂，分解剂(如淀粉羟乙酸钠，交联聚乙烯吡啶酮，交联纤维素甲基羧酸钠)，表面活化剂或分散剂混合后在适当的机器中压紧制成的。灌铸片剂是粉末状化合物由惰性液体稀释剂湿润后灌注于模具中制成的。片剂也可以在制剂时选择加衣和刻纹，用于减慢或控制有效成分的释放，例如，不同比例的羟丙基甲基纤维素可以提供不同的释放速度。片剂可以加肠衣，使其不在胃中，而在肠中释放。鉴于通式I的化合物可能会在酸中水解，这种制剂方式有着很大的优势。
口腔局部给药的适合制剂包括将活性成分置于调味基(通常为蔗糖，阿拉伯胶，黄芪胶)上的锭剂；活性成分置于惰性基(凝胶，甘油，蔗糖和阿拉伯胶)上的锭剂；以及活性成分置于合适液体载体中的漱口液。
直肠给药的制剂可以是活性化合物置于适当的基础(如可可，黄油，水杨酸盐)上的栓剂。
阴道给药的制剂可以在子宫套，止血棉塞，乳霜，凝胶，浆糊，泡沫或喷剂中加入活性成分和其他已知的适合的载体。
注射给药的制剂包括水无菌注射溶液或非水等渗透压无菌注射溶剂，其中包含抗氧化剂，缓冲盐，杀菌剂和使制剂与受者血液等渗透压的溶质；水和非水无菌悬浮液，其中可能包含有悬浮剂和粘稠剂。制剂可以是单元剂量封装，也可以是多剂量封装，例如，安瓿和小瓶，可以冰干储存，在使用前加入无菌 液体载体(如注射用水)。注射用溶液和悬浮液可以如前所述，用无菌粉末，颗粒和片剂制成。
注射用制剂可以通过使用内泵和药带，采用连续注入的方式给药。注入可以采用Hickman方法或PICC方法或任何其他适用于注射及静脉注射的给药方式。
建议药剂单元剂量包含每天药量或单元，每次药量，以及每天用药次数。
需要明确的是，任何单独的患者的特定服药剂量有一系列因素决定，包括所使用的特定化合物的活性，年纪，体重，健康状况，性别以及营养状况；给药时间和途径；排泄速度；此前服用过的其他药物；患症的严重程度。这一点任何精通本领域的人都应能理解.
通式I和通式II的化合物的合成方法：
本发明的化合物可以按照下述的过程制备，也可由有经验的人通过公开发表文献中的类似方法合成。需要被明确的一点是，下述的方法仅仅是用来说明制备过程，本发明的权利要求不被局限于此。通式I的典型合成方法可以被归纳为基本的五步(从后到前的顺序)：(1)前体药的制备；(2)膦酸酯的去保护；(3)喹啉环的修饰；(4)喹啉环的合成；(5)关键中间体的合成。通式II化合物可由通式I化合物与适当基团反应获得。路线中保护和去保护的具体操作可参考已知文献(例如，“Protecting Groups in Organic Synthesis”3rd Edition，Wiley，1999)。
本发明化合物的所有立体异构体都有独特的作用，无论是混合物还是大体纯还是纯品。本发明的化合物可以在磷原子及任何碳原子(包含任何R取代基)上有手性中心。因此，通式I的化合物可以以对映异构体或非对映异构体的形式存在，或者是二者的混合物。制备流程中使用的起始物可以是消旋体，对映异构体或非对映异构体。可以使用常规方法分离生成的对映异构体或非对映异构体。例如，色谱法或分段结晶可以用来分离非对映异构体，可以用色谱法分离对映异构体的衍生物。
1)前体药的制备
前体药可以在合成的不同阶段引入。由于不同前体药的不稳定性，通常都是在合成的临近结束阶段引入，但也有因为其他原因在合成的初始阶段引入的情况。
通式I的化合物可以是膦酸(R1和R2都是氢)，也可以形成合适的被保护状态，膦酸可以与亲电子体(如卤代烷和硫酸烷酯)在亲核反应的条件下生成膦酸酯。例如，当R1和R2是酰氧烷基时，通式I的化合物可以由R1和R2是氢时的化合物同适当的酰氧烷基卤素(例如，氯、溴、碘；Phosphorus Sulfur1990，54，143；Synthesis1988，62)在适当的碱(例如吡啶，三乙胺，二异丙基乙基胺)作用下，在适当的溶剂(如DMF，J.Med.Chem.1994，37，1875)中反应制得。酰氧烷基卤素中的羧酸酯部分可以是但不局限于乙酸酯，丙酸酯，异丁酸值，三甲基乙酸酯，苯甲酸酯，碳酸酯及其他的羧酸酯。
活泼的膦酰二氯可以由对应的膦酸同氯代试剂(例如，二氯亚砜，J.Med.Chem.1994，1857；草酰氯，Tetrahedron Lett.1990，31，3261；五氯化磷，Synthesis 1974，490)反应生成。此外，膦酰二氯还可以由对应的二硅基膦酸酯(Synth.Commu.1987，17，1071)和二烷基膦酸酯(Tetrahedron Lett.1983，24，4405；Bull.Soc.Chim.1993，130，485)制备。
环状的膦酸1,3-丙二醇酯既可以由对应的膦酰二氯同取代的1,3-丙二醇反应(J.Med.Chem.2008,51,666)，也可以使用适当的偶联试剂(例如，DCC，EDCI，PyBOP；Synthesis 1988，62)进行偶联反应。
此外，环状的膦酸1,3-丙二醇酯还可以由膦酸和二醇在光岩反应条件下偶联制得(Synthesis 1981，1；J.Org.Chem.1992，52，6331)，以及其他的酸偶联试剂，包括但不局限于，羰基二亚胺(Collect.Czech.Chem.Commun.1994，59，1853；Bioorg.Med.Chem.Lett.1992，2，145；Tetrahedron Lett.1988，29，1189)，还有PyBOP(Tetrahedron Lett.1993，34，6743)。
一方面，本发明公开了合成以及分离通式I膦酸前药单体的方法。由于磷原子是一个手性中心，使用取代1,3丙二醇的消旋体制备的前药会是一个异构体的混合物。例如，使用消旋的1-Y取代的1,3-丙二醇会生成一个消旋的顺式前药混合物和消旋的反式前药混合物。另一方面，使用R对映异构体富集的取代1,3-丙二醇会得到对映异构体富集的R-顺式前体药和R-反式前体药。这些化合物可以通过结合使用色谱柱层析和(或)分段结晶的方式分离。
还可以通过引入第二个前体药基团来获得期望的性质。通式I的化合物(X是OH时)可以在羟基吡啶环的氧原子或氮原子上连接不同的保护基团。例如，当R3是酰基时，通式I的化合物可以由R3是氢时的化合物在适当的条件下同适当的酰卤反应得到(J.Org.Chem.1989,54,166)；当X是氯时，通式I的化 合物可以由X是OH时的化合物在适当的条件下同氯代试剂反应得到(例如：三氯氧磷，J.Org.Chem.1950,15,1224；三氯乙腈，Tetrahedron Lett.2012，53，674)。
2)膦酸酯的去保护
R1是氢的通式I化合物可以使用已知的磷酸酯和膦酸酯的断裂条件将膦酸酯反应制备。卤代硅烷经常被用于切断不同的膦酸酯，获得的硅膦酸酯可以经由温和的水解过程得到目标膦酸。如有需要，在合成酸断裂的化合物时，可以使用酸清除剂(如，六甲基二硅氮烷，2,6-二甲基吡啶)。这些卤代硅烷包括三甲基氯硅烷(J.Org.Chem.1963，28，2975)，三甲基溴硅烷(Tetrahedron Lett.1977，155)，三甲基碘硅烷(J.Chem.Soc.,Chem.Commun.1978,870)。此外，膦酸酯还可以在强酸条件下断裂(例如，氢溴酸或盐酸，U.S.Patent 3,524,846,1970)。这些膦酸酯还可以通过使用卤代试剂(如五氯化磷，二氯亚砜，三溴化硼；J.Chem.Soc.1961，238)转化为磷酰二氯后水解生成膦酸。芳香基及苄基膦酸酯还可以通过氢解条件(Synthesis1982，412；J.Med.Chem.1985，28，1208；Nature 1953，171，76)或金属还原条件(J.Am.Chem.Soc.1977，99，5118)断裂。电化学(J.Org.Chem.1979，44，4508)和高温热解(Synth.Commun.1980，10，299)条件也可用于断裂不同的膦酸酯。
3)菲罗啉膦酸的合成
菲罗啉母核的构建可以用文献确立的条件。例如下面方案所示的热关环策略。
Figure PCTCN2015076273-appb-000010
芳胺1在3-硝基苯磺酸钠，硫酸和甘油的条件下生成喹啉2.喹啉2在醋酸中被NBS溴代得到化合物3，再使用铁酸还原得到化合物4.化合物4膦酸化得到膦酸酯5，再同化合物17反应后经历热关环反应得到R为氢的菲罗啉7(在通式I中，则是X为羟基，R1和R2是乙基)。化合物7同氢氧化钠反应得到R是氢的化合物8(通式I中，X是羟基，R1是氢，R2是乙基)；另外，化合物7同48％氢溴酸反应得到R是氢的化合物9(通式I中，X是羟基，R1和R2是氢)。在某些情况下，想要的取代基并不适于接下来的反应，这时可以设想对已有的菲罗啉环使用常规化学进行修饰(Larock,Comprehensive organic transformations,VCH,New York,1989；Trost,Comprehensive organic synthesis；Pergamon press,New York,1991)。
前体药通常在合成的靠后阶段引入，但出于其他考虑，有时也可将前药在早期阶段引入。例如，环状膦酸二酯前药可以按照下面方案所示合成。
Figure PCTCN2015076273-appb-000011
化合物3经膦酸化得到膦酸酯10，膦酸酯10用48％氢溴酸去保护后得到膦酸11.膦酸11同三氯氧磷反应得到活泼磷酰二氯12，并迅速与二醇20偶联(J.Am.Chem.Soc.2004，5154)得到化合物13。化合物13上的硝基还原后与化合物17反应，接下来的热关环反应得到R是氢的菲罗啉16(通式I中，X是羟基，R1和R2一同构成环状基团)。
还可以通过引入第二个前体药基团来获得期望的性质。例如，化合物16c与氯磷酸酯在合适的碱(如三乙胺)和催化剂(如4-二甲胺基吡啶)作用下在适当的溶剂(如二氯甲烷)中反应可以得到磷酸酯21。二乙基磷酸酯的脱保护可以通过使用常见的磷酸酯脱保护试剂实现。例如，化合物21经三甲基溴硅烷处理可得到磷酸酯22，其后还可以进一步转化为期望的盐。例如，化合物22经碳酸氢钠的水和甲醇混合溶液处理可以得到二钠盐23。
Figure PCTCN2015076273-appb-000012
另一方面，还可以引入其他类型的前体药来获得期望的性质。例如，化合物16c在合适的溶剂(如二甲亚砜)中经适当的碱(如碳酸钾)处理后与二叔丁基磷酸氯甲酯反应可以得到磷酸酯24和25。使用常见的叔丁酯去保护试剂可以实现二叔丁基磷酸酯的脱保护。例如，化合物24和25在二氯甲烷中经三氟乙酸处理可分别得到磷酸26和27，其后可进一步转化为预期的盐。
Figure PCTCN2015076273-appb-000013
实施例
本发明中的化合物及制备可以通过下面的实例更好地说明。这些实例不应理解为是对本发明的限制，现在已知的或将来开发的这些化合物的变化体也应被认为属于本发明的范畴并申请保护。
实施例1.化合物的制备
8-硝基喹啉(2c)的制备
Figure PCTCN2015076273-appb-000014
依次将20毫升水、47克浓硫酸、23.4克3-硝基苯磺酸钠、22毫升甘油加入到反应瓶中。缓慢加热至形成溶液(85℃)，分批加入11克2-硝基苯胺。回流反应5小时，冷却至室温，冰浴下缓慢倒入600毫升水中，用氨水调至pH 6–7，抽滤，滤饼干燥后柱层析(乙酸乙酯：石油醚＝1:5)得6.177g黄色固体2c，收率44％。
1H NMR(300MHz,CDCl3)δ9.09(dd,J＝1.8Hz,4.5Hz,1H),8.28(dd,J＝1.8Hz,8.4Hz,1H),8.05(d,J＝9Hz,2H),7.66-7.55(m,2H)
3-溴-8-硝基喹啉(3c)的制备
Figure PCTCN2015076273-appb-000015
将化合物2c 6.177克加入到110毫升冰醋酸中，再加入6.651克N-溴代丁二酰亚胺。50℃反应2小时，反应液冷却后搅拌下倒入600毫升水中，抽滤，滤饼干燥后柱层析(乙酸乙酯：石油醚＝1:15)得2.625克黄色固体3c，收率29％。
1H NMR(300MHz,CDCl3)δ9.06(d,J＝2.1Hz,1H),8.44(d,J＝2.4Hz,1H),8.06(d,J＝7.5Hz,1H),7.98(d,J＝8.1Hz,1H),7.67(t,J＝7.8Hz,1H).
3-溴喹啉-8-胺(4c)的制备
Figure PCTCN2015076273-appb-000016
将化合物13.0克3c加入到150毫升乙醇中，再依次加入11.6克铁粉，11.0克氯化铵，回流反应过夜。反应液冷却后硅藻土过滤，蒸干滤液柱层析(乙酸乙酯：石油醚＝1:5)得8.23克黄色固体4c，收率72％。
1H NMR(300MHz,CDCl3)δ8.72(d,J＝2.1Hz 1H),8.21(d,J＝2.1Hz,1H),7.35(t,J＝7.8Hz,1H),7.05(dd,J＝1.2Hz,8.1Hz,1H),7.61(dd,J＝1.2Hz,7.5Hz,1H),4.98(s,2H).
8-氨基喹啉-3-膦酸二乙酯(5c)的制备
Figure PCTCN2015076273-appb-000017
氮气保护下将4.0克化合物4c加入到53毫升乙醇中，再依次加入3.0毫升亚磷酸二乙酯、3.7毫升三乙胺、1.27克三苯基膦、800毫克醋酸钯。回流反应过夜，反应液降至室温后加入100毫升水，乙酸乙酯萃取，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥。有机相浓缩后柱层析(乙酸乙酯：石油醚＝1:1)，得1.4克黄色油5c，收率25％。
1H NMR(300MHz,CDCl3)δ8.98(dd,J＝1.8Hz,4.2Hz,1H),8.59(dd,J＝2.1Hz,15.3Hz,1H),7.38(d,J＝7.8Hz,1H),7.21(d,J＝7.5Hz,1H),7.01(t,J＝7.5Hz,1H),4.20–4.07(m,4H),1.35(t,J＝6.9Hz,6H).
8-((2,2-二甲基-1,3-二氧环己烷-4,6-二酮)-5-亚甲氨基)喹啉-3-膦酸二乙 酯(6c)的制备
Figure PCTCN2015076273-appb-000018
氮气氛围下将1.4克化合物5c加入到40毫升乙醇中，再加入1.3克化合物17。回流反应过夜，反应液降至室温，蒸出溶剂，柱层析(乙酸乙酯:石油醚＝1:1)得1.125克黄色固体6c，收率52％。
1H NMR(300MHz,CDCl3)δ12.8(d,J＝15Hz,1H),9.20(dd,J＝1.8Hz,4.2Hz,1H),8.91(d,J＝14.7Hz,1H),8.74(dd,J＝1.8Hz,15.3Hz 1H),7.80-7.76(m,2H),7.67(t,J＝7.8Hz,1H),4.30–4.09(m,4H),1.81(s,6H),1.35(t,J＝6.9Hz,6H).
7-羟基-1,10-菲罗啉-3-膦酸二乙酯(7c)的制备
Figure PCTCN2015076273-appb-000019
将1.1克化合物6c快速加入到沸腾的二苯醚中，回流搅拌2分钟，反应液 降温至100℃后搅拌下倒入640毫升石油醚中，抽滤，滤饼柱层析(二氯甲烷:甲醇＝20:1)得650毫克黄色固体7c，收率77％。
1H NMR(300MHz,CDCl3)δ10.8(s,1H),9.31(dd,J＝1.8Hz,5.1Hz,1H),8.72(dd,J＝1.8Hz,14.7Hz,1H),8.49(d,J＝8.7Hz,1H),7.94(d,J＝7.5Hz,1H),7.71(d,J＝9Hz,1H),6.62(d,J＝7.5Hz,1H),4.35–4.14(m,4H),1.39(t,J＝6.9Hz,6H).
7-羟基-1,10-菲罗啉-3-膦酸(9c)的制备
Figure PCTCN2015076273-appb-000020
将650毫克化合物7c加入到48％氢溴酸中，回流反应过夜。反应液降至室温，蒸干溶剂，加少量水搅拌，抽滤，干燥得513毫克灰色固体9c，收率95％。
1H NMR(300MHz,D2O)δ8.99(dd,J＝4.5Hz,1.8Hz,1H),8.30(dd,J＝12.6Hz,1.8Hz,1H),7.62(d,J＝6.9Hz,1H),740(d,J＝8.7Hz,1H),7.26(d,J＝8.7Hz,1H),6.19(d,J＝7.2Hz,1H)
8-硝基喹啉-3-膦酸二乙酯(10c)的制备
Figure PCTCN2015076273-appb-000021
N2氛围下，依次向反应瓶中加入30克化合物3c、23.4克乙酸钾、18.4毫升亚磷酸二乙酯、300毫升甲苯、1克[1,1’-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物，回流反应3小时，加入乙酸乙酯稀释，硅胶过滤，滤液蒸干得46克10c。
1H NMR(300MHz,CDCl3)δ9.28(dd,J＝1.8Hz,4.2Hz,1H),8.82(dd,J＝1.8Hz,15Hz,1H),8.16(t,J＝6Hz,2H),7.21(d,J＝7.5Hz,1H),7.74(t,J＝8.1Hz,1H),4.33–4.11(m,4H),1.37(t,J＝6.9Hz,6H).
8-硝基喹啉-3-膦酸(11c)的制备
Figure PCTCN2015076273-appb-000022
将44.5克化合物10c加入到230毫升48％氢溴酸中，回流反应4小时，降温蒸干溶剂，固体用乙醇和乙酸乙酯混合洗涤2小时，抽滤，得31.5克黄色固体11c，收率79％(两步)。
1H NMR(300MHz,D2O)δ9.18(dd,J＝1.8Hz,6Hz,1H),8.98(dd,J＝1.8Hz,13.2Hz,1H),8.54(d,J＝7.8Hz,1H),8.36(d,J＝8.4Hz,1H),7.81(t,J＝7.8Hz,1H).
8-硝基喹啉-3-磷酰二氯(12c)的制备
Figure PCTCN2015076273-appb-000023
将50.3克化合物11c加入到650毫升二氯乙烷中，加入3.6毫升N,N-二甲基甲酰胺，冰浴下滴加42毫升草酰氯，滴毕回流反应过夜。降温蒸干，所得12c直接用于下一步反应。
(4S)-4-(3-氯苯基)-2-(8-硝基喹啉-3-基)-1,3-二氧-2-磷环己烷磷氧化 物(13c)的制备
Figure PCTCN2015076273-appb-000024
将36.95克(s)-1-(3-氯苯基)丙基-1,3-二醇20加入到540毫升二氯甲烷中，-78℃下滴加22毫升四氯化钛，滴毕搅拌5分钟，移至冰浴下搅拌5分钟，滴加110毫升三乙胺。将上述溶液滴加到化合物12c的二氯甲烷溶液中，滴毕，室温反应过夜。加入700毫升二氯甲烷稀释后，加入210毫升10％酒石酸搅拌2分钟。硅藻土过滤，二氯甲烷萃取，无水硫酸钠干燥，蒸干后用乙腈重结晶两次，得35.5克黄色固体13c，收率44％。
m/z:405.1[M+1]；
(4S)-4-(3-氯苯基)-2-(8-氨基喹啉-3-基)-1,3-二氧-2-磷环己烷磷氧化 物(14c)的制备
Figure PCTCN2015076273-appb-000025
将62.9克化合物13c加入到160毫升/160毫升乙醇/冰乙酸中，再加入43.6克铁粉，40℃反应10分钟，降温，饱和碳酸钠溶液调PH6，二氯甲烷萃取，无水硫酸钠干燥，蒸干得50克黄色固体14c，收率86％。
m/z:375.0[M+1]；
(4S)-4-(3-氯苯基)-2-(8-((2,2-二甲基-1,3-二氧环己烷-4,6-二酮)-5- 亚甲氨基)喹啉-3-基)-1,3-二氧-2-磷环己烷磷氧化物(15c)的制备
Figure PCTCN2015076273-appb-000026
将49克化合物14c加入到320毫升乙醇中，再加入31.4克化合物5-(乙氧基亚甲基)-2,2-二甲基-1,3-二氧环己烷-4,6-二酮17，回流反应2小时，降温，抽滤，得60克黄色固体15c，收率87％。
m/z:529.0[M+1],找到471.0；
(4S)-4-(3-氯苯基)-2-(7-羟基-1,10-菲罗啉-3-基)-1,3-二氧-2-磷环己 烷磷氧化物(16c)的制备
Figure PCTCN2015076273-appb-000027
将3克化合物15c快速加入到沸腾的二苯醚中，回流50秒，降至100℃，倒入500毫升石油醚中，抽滤，滤饼进行柱层析(二氯甲烷:甲醇＝30:1)，得黄色固体16c 1.676克，收率70％。
1H NMR(300MHz,DMSO)δ12.53(s,1H),9.34(dd,J＝2.1Hz,5.1Hz,1H),9.15(dd,J＝1.8Hz,15.3Hz,1H),8.27(d,J＝8.7Hz,1H),8.12-7.98(m,2H), 7.56(s,1H),7.47-7.43(m,3H),6.36(d,J＝7.2Hz,1H),5.96(d,J＝11.1Hz,1H),4.88-4.76(m,1H),4.65–4.55(m,1H),2.68-2.54(m,1H),2.34-2.22(m,1H).
3-氯苯甲酰乙酸甲酯(18)的制备
Figure PCTCN2015076273-appb-000028
氮气氛围下将15克叔丁醇钾加入到50毫升四氢呋喃中，室温搅拌15分钟,冰浴下10克1–(3-氯苯基)乙酮和11毫升碳酸二甲酯缓慢滴加到反应瓶中，室温搅拌1.5小时。向反应液中加入40毫升水及1.3毫升稀盐酸溶液，搅拌15分钟，分离有机相，水相用甲苯萃取，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，过滤蒸干得13.22克棕色油18收率96％。
3S-3-羟基-3-(3-氯苯基)丙酸甲酯(19)的制备
Figure PCTCN2015076273-appb-000029
氮气氛围冰浴下将5.38克三乙胺缓慢滴加到9.8克甲酸中，滴毕搅拌20分钟，后室温反应1小时。将11.3克化合物18及45毫升N,N-二甲基甲酰胺和68毫克(S,S)-N-(对甲苯磺酰)-1,2-二苯乙烷二胺(对异丙基苯)氯化钌(II)加入到反应瓶中，60℃反应过夜。冷却至室温，加入100毫升水，乙酸乙酯萃取，有机相经饱和食盐水洗涤，无水硫酸钠干燥，过滤后蒸干，柱层析(乙酸乙酯:石油醚＝1:10)得10.434克橘红色油19，收率91％.
1H NMR(300MHz,CDCl3)δ7.45(s,1H),7.37-7.27(m,3H),5.16(t,J＝6.9Hz,1H),3.78(s,3H),2.78(d,J＝1.8Hz,1H),2.76(s,1H).
1S-1-(3-氯苯基)-1,3-丙二醇(20)的制备
Figure PCTCN2015076273-appb-000030
氮气氛围下将1.84克硼氢化钠及0.62毫升水加入到37.5毫升正丁醇中，冰浴下滴加10.4克化合物19的正丁醇溶液，滴毕搅拌0.5小时，后90℃反应4小时。冷却至室温，加入碳酸钾溶液(10％,23毫升)，搅拌10分钟。分离有机相，有机相用碳酸钾溶液(10％,8毫升)洗涤，饱和食盐水洗涤，无水硫酸钠干燥，过滤，蒸干柱层析(二氯甲烷：甲醇＝30：1)得7.75克黄色油20，收率 85.5％.
1H NMR(300MHz,CDCl3)δ7.36(s,1H),7.30-7.20(m,3H),4.92(q,J＝4.5Hz,7.8Hz,1H),3.90-3.79(m,2H),2.82(s,2H),2.03-1.85(m,2H).
3-(4S-4-(3-氯苯基)-1,3-二氧-2-磷磷氧环己烷-2-基)-1,10-菲罗啉-7-磷 酸(22)的制备
Figure PCTCN2015076273-appb-000031
将2克化合物16c溶于100毫升二氯甲烷中，再将2毫升三乙胺与57毫克4-二甲胺基吡啶加入到反应液中，将反应放置在冰浴中。将2毫升氯磷酸二乙酯溶于20毫升二氯甲烷中缓慢滴加到反应液中，冰浴下反应一个小时之后升至室温反应2个小时。然后将反应液倒入200毫升饱和氯化钠溶液中，分离有机相，水相使用二氯甲烷萃取，合并有机相，用无水硫酸钠干燥，旋干后柱层析(二氯甲烷：甲醇＝100：1)得到21共计1.7克。再将1.7克21溶解在2毫升二氯甲烷中，冰浴下一次性加入三甲基溴硅烷4毫升，保持冰浴下反应1个小时，将50毫升乙醚加入到反应瓶中，析出的固体抽滤，收集滤饼，将滤饼溶解在20毫升甲醇中，搅拌十分钟。将反应液旋干进行柱层析(二氯甲烷：甲醇：乙酸＝20:1:0.05～二氯甲烷：甲醇＝4:1)得到白色固体22共计600mg，收率25％。
m/z:507.0[M+1]；
1H NMR(300MHz,dmso)
Figure PCTCN2015076273-appb-000032
13.84(m,1H),9.27(dd,J＝4.8,1.8Hz,1H),8.99(dd,J＝14.3,1.8Hz,1H),8.39(d,J＝7.1Hz,1H),8.30(d,J＝8.9Hz,1H),8.13(d,J＝9.0Hz,1H),7.50(s,1H),7.45–7.38(m,1H),7.35–7.25(m,2H),6.85(d,J＝7.1Hz,1H),5.35(dd,J＝9.0,5.9Hz,1H),4.11–3.98(m,2H),2.68–2.55(m,1H),2.50–2.34(m,1H).
3-(4S-4-(3-氯苯基)-1,3-二氧-2-磷磷氧环己烷-2-基)-1,10-菲罗啉-7-磷 酸二钠(23)的制备
Figure PCTCN2015076273-appb-000033
将500毫克化合物额22悬浮在10毫升甲醇中，在室温下将1N碳酸氢钠溶液2毫升缓慢滴加到反应液中，搅拌20分钟后将反应液减压抽干(保持温度在30摄氏度以下)，得到白色固体23共计540mg，收率100％。
m/z:550.0[M+1]，找到507；
1H NMR(300MHz,dmso)δ9.27(dd,J＝4.8,1.8Hz,1H),8.99(dd,J＝14.3,1.8Hz,1H),8.39(d,J＝7.1Hz,1H),8.30(d,J＝8.9Hz,1H),8.13(d,J＝9.0Hz,1H),7.50(s,1H),7.45–7.38(m,1H),7.35–7.25(m,2H),6.85(d,J＝7.1Hz,1H),5.35(dd,J＝9.0,5.9Hz,1H),4.11–3.98(m,2H),2.68–2.55(m,1H),2.50–2.34(m,1H).
(3-(4S-4-(3-氯苯基)-1,3-二氧-2-磷磷氧环己烷-2-基)-1,10-菲罗啉-7- 氧基)-7-甲基磷酸二叔丁酯(24)和(3-(4S-4-(3-氯苯基)-1,3-二氧-2-磷磷 氧环己烷-2-基)-7-氧基-1,10-菲罗啉)-10(7氢)-甲基磷酸二叔丁酯(25)的 制备
Figure PCTCN2015076273-appb-000034
将化合物16c(200mg，0.47mmol)溶于2毫升二甲基亚砜中，再将碳酸钾(195mg,1.41mmol)加入到反应液中，将反应加热至30℃搅拌15分钟，再将二叔丁基氯甲基磷酸酯(146mg，0.56mmol)加到反应液中，保持30℃下反应过夜。然后将反应液倒入20毫升饱和氯化钠溶液中，分离有机相，水相使用二氯甲烷萃取，合并有机相，用无水硫酸钠干燥，旋干后进行柱层析(乙酸乙酯)得到中间体24和25。
m/z：649.2[M+1]；
化合物24：
1H NMR(300MHz,dmso)δ9.48(dd,J＝4.9,1.9Hz,1H),9.14(dd,J＝15.4,1.9Hz,1H),9.10(d,J＝5.3Hz,1H),8.31(d,J＝9.1Hz,1H),7.63–7.58(m,2H),7.55–7.41(m,3H),6.11–5.91(m,3H),4.92–4.75(m,1H),4.71–4.53(m,1H),2.73–2.55(m,1H),2.36–2.19(m,1H),1.37(s,18H)
13C NMR(75MHz,dmso)δ158.59,151.37,150.71,150.54,147.15,146.18, 142.12,142.01,141.71,133.29,130.57,128.41,126.26,125.79,124.59,121.53,120.83,106.97,87.59,82.98,77.56,66.24,33.30,29.38
化合物25：
1H NMR(300MHz,dmso)δ9.39(dd,J＝4.6,2.1Hz,1H),9.19(dd,J＝15.6,2.0Hz,1H),8.43(d,J＝8.7Hz,1H),8.22(d,J＝8.1Hz,1H),8.18(d,J＝8.4Hz,1H),7.58(s,1H),7.50–7.44(m,3H),7.18–6.99(m,2H),6.49(d,J＝7.9Hz,1H),5.98(d,J＝11.2Hz,1H),4.93–4.72(m,1H),4.69–4.49(m,1H),2.72–2.52(m,1H),2.34–2.19(m,1H),1.21(s,9H),1.19(s,9H).
13C NMR(75MHz,dmso)δ176.22,148.52,147.50,142.91,141.99,141.88,136.23,133.28,130.54,129.08,128.91,128.46,125.72,125.00,124.60,124.08,121.52,112.53,82.30,80.38,77.54,66.52,33.21,29.16.
(3-(4S-4-(3-氯苯基)-1,3-二氧-2-磷磷氧环己烷-2-基)-1,10-菲罗啉-7- 氧基)-7-甲基磷酸(26)的制备
Figure PCTCN2015076273-appb-000035
将化合物24(50mg，0.08mmol)溶解在3毫升二氯甲烷中，然后在室温下向反应液中加入1毫升三氟乙酸，室温搅拌30分钟后，将反应液旋干，然后加入1毫升甲醇，抽滤，得到化合物26。
1H NMR(300MHz,dmso)δ9.46(dd,J＝5.0,1.8Hz,1H),9.23–9.05(m,2H),8.37–8.21(m,2H),7.68(d,J＝5.6Hz,1H),7.56(s,1H),7.51–7.36(m,3H),6.12–5.90(m,3H),4.90–4.74(m,1H),4.71–4.50(m,1H),2.71–2.54(m,1H),2.33–2.21(m,1H)
(3-(4S-4-(3-氯苯基)-1,3-二氧-2-磷磷氧环己烷-2-基)-7-氧基-1,10-菲 罗啉)-10(7氢)-甲基磷酸(27)的制备
Figure PCTCN2015076273-appb-000036
将化合物25(50mg，0.08mmol)溶解在3毫升二氯甲烷中，然后在室温下向反应液中加入1毫升三氟乙酸，室温搅拌30分钟后，将反应液旋干，然后加入1毫升甲醇，抽滤，得到化合物27。
1H NMR(300MHz,dmso)δ9.38(dd,J＝4.5,2.0Hz,1H),9.13(dd,J＝15.6,2.0Hz,1H),8.40(d,J＝8.7Hz,1H),8.19(d,J＝8.3Hz,1H),8.13(d,J＝8.7Hz,1H),7.58(s,1H),7.54–7.35(m,3H),7.04(t,J＝9.9Hz,1H),6.91(t,J＝9.8Hz,1H),6.43(d,J＝7.8Hz,1H),5.95(d,J＝10.8Hz,1H),4.88–4.76(m,1H),4.69–4.45(m,1H),2.74–2.56(m,1H),2.31–2.22(m,1H).
实施例2.P4H的表达和纯化
重组人源性P4H表达在大肠杆菌表达系统完成。将编码全长P4H蛋白的核苷酸序列克隆到pET28_N-His_TEV质粒表达系统中，获得pET28_N-His_TEV_P4HA1/PDI，然后转化到大肠杆菌Origami2(DE3)进行共表达。用MonoQ离子交换柱进行酶蛋白纯化后，用TEV消化和质谱鉴定，并用HistrapHP和凝胶过滤色谱法(Hiload 16/60superdex200)进一步洗脱纯化鉴定。
实施例3.P4H酶活性鉴定和本发明的化合物对酶活性的影响
纯化的P4H酶蛋白在如下偶联酶反应体系中进行酶活性测试和鉴定化合物对酶活性的影响：100mM Tris(pH7.0),0.1mM(NH4)2Fe(SO4)2,0.1mM抗坏血酸,0.2mM CoA,0.2mM ATP,0.5uM琥珀酰辅酶A合成酶,100uM 2-酮戊二酸,100uM(Pro-Pro-Gly)10肽,50nM P4H蛋白,总计50ul。25℃下反应45min；加入10ul MLG R1,反应10min；加入10ul MLG R2,反应20min。P4H在辅酶和适当的酶反应环境中催化酮戊二酸和多肽底物形成产物琥珀酸，琥珀酸然后在琥珀酰辅酶合成酶作用下生成琥珀酰辅酶和磷酸(Pi)。生成磷酸的水平可用MLG试剂盒测定，从而反应P4H的水平。生成的绿色产物(MG+)(H2PMo12O40)在OD为630nm进行吸收峰鉴定。
化合物对P4H酶抑制的评价在96孔板中完成。每个浓度有两个平行样(n＝2)。将化合物9c按以下浓度加入酶反应体系中(按顺序在P4H酶蛋白前加入)：0.01，0.03，0.1，0.3，1，3，30，100，300nM。数据分析和统计用Prism软件完成。分析化合物对酶50％的抑制浓度(IC50)为8.1μM (图1)。图1表明，化合物9c对人P4H酶活性的抑制作用随9c浓度的升高而增强。
实施例4.体内药代动力学的研究
Wistar大鼠，体重200±20g，分为两组，各6只，雌雄各半，自由饮水饮食。给药剂量：第一组用化合物16c尾静脉给药3mg·kg-1，第二组用化合物27的二钠盐灌胃给药39mg·kg-1。于给药前(0h)和给药后0.08h，0.17h，0.33h，0.5h，0.75h，1h，1.5h，2h，3h，5h，7h经眼球后静脉丛取全血0.3mL，置预冷的肝素化EP管中，4℃离心(15000rpm)5min，分离血浆100μL，立即置入-80℃保存待测。
分别以地西泮和米屈肼为内标，建立了大鼠血浆中化合物9c、前体药16c的LC-MS/MS定量分析方法(前体药27在实验条件下未能从血浆中检测到)，并对血浆样品进行测试分析(结果见表1、图2)。
表1：化合物16c静脉给药和化合物27口服给药后血浆中化合物16c的浓度
Figure PCTCN2015076273-appb-000037
＊Cmax表示血药浓度峰值。
＊＊t1/2表示血药浓度降为峰值一半的时间。
＊＊＊AUC0-t表示至测量时间止，血药浓度-时间曲线下面积。
＊＊＊＊AUC0-∞表示至所有药物清除时，血药浓度-时间曲线下面积。
口服吸收利用度：
口服吸收利用度以血中的化合物16c计算。计算方法即口服给药(po)后血中药-时曲线下面积(area under concentration-time curve ，简称AUC)与该药等同剂量静脉注射(iv)后血中药-时曲线下面积(AUC)的比值，以吸收百分率表示：生物利用度(F)＝AUCpo·Miv/AUCiv·Mpo×100％。其中，Miv表示静脉注射的药物的摩尔浓度，Mpo表示口服给药的药物的摩尔浓度。
化合物16c静脉注射(3mg/kg)和化合物27口服(39mg/kg)后血中的化合物16c的AUC0-t分别为2064.58g·h/mL和5235.70g·h/mL。因此，基于血中 化合物16c计算的化合物27的生物利用度(F)为25.4％(即5235.70/(2064.58×10)×100％)。
肝脏中化合物16c和9c的浓度：
16只Wistar大鼠，体重200±20g，随机分为4组，雌雄各半，实验前自由饮水饮食，灌胃给药化合物27(39mg·kg-1)后，于给药后4个时间点(15min，45min，8h，24h)处死，立即解剖采集肝组织。用生理盐水冲净血液及内容物。先用剪刀将组织剪成小块并搅拌均匀，称取1g，加入1mL甲醇/水溶液；匀浆后再加1mL甲醇/水溶液，超声15s，再离心(4500rpm)10min，取上清液，采用LC-MS/MS法测定大鼠给药后不同时间肝脏组织中的化合物16c和9c的药物浓度(结果见表2)。结果表明，前体药27经口服后，可在体内转化为化合物16c，16c可在肝脏中转化为化合物9c。
表2：化合物27口服给药后不同时间点肝组织中化合物16c和9c的浓度(ng·g-1)
  0.25h 0.75h 8h 24h
化合物16c 521.50 662.5 584.50 50.88
化合物9c 16.13 64.25 44.68 3.55
实施例5.体内药效研究
本实验采用胆管结扎(BDL)的方法诱导大鼠肝纤维化病理模型，观察口服前体药化合物27对BDL型肝纤维化的治疗作用。
取Wistar大鼠，体重200±20g，分为三组，雌雄各半:
假手术组(SHAM组)：6只大鼠，麻醉后，腹部皮肤剃毛后常规消毒，无菌操作沿腹部正中线剪开腹腔，游离胆总管，分别缝合肌肉和皮肤；
肝纤维化模型组(MODEL组)：12只大鼠，麻醉后，腹部皮肤剃毛后常规消毒，无菌操作沿腹部正中线剪开腹腔，游离胆总管并结扎，分别缝合肌肉和皮肤；
口服给药组：12只大鼠，麻醉后，腹部皮肤剃毛后常规消毒，无菌操作沿腹部正中线剪开腹腔，游离胆总管并结扎，分别缝合肌肉和皮肤。术后回笼开始给药化合物27的二钠盐，30mg/kg，溶于水中灌胃给药，每日一次。
检测指标：
BDL 2周后，血清及肝匀浆测定标本测定：谷丙转氨酶(ALT)、谷草转氨 酶(AST)。
BDL 2周后处死大鼠，肝组织行HE染色、Masson染色。
口服化合物27对BDL肝纤维化大鼠血清和肝脏匀浆中的谷丙转氨酶 (ALT)、谷草转氨酶(AST)的影响：
ALT与AST是目前临床上最常用的检测肝功能指标，ALT主要存在于肝细胞浆内，AST主要存在于肝细胞线粒体内，当肝细胞受损时，血清中ALT、AST水平升高，能够反映肝细胞的损伤程度。经BDL后模型组大鼠血清和肝组织匀浆ALT和AST明显升高，化合物27给药14天后处死动物，血清和肝组织匀浆ALT和AST水平明显下降，与模型组比较有显著性差异(＊＊P<0.01)，见表3；表4。说明化合物27减轻了BDL后肝功能的损伤程度，对胆汁回流导致的肝损伤具有保护作用。
表3：化合物27对BDL肝纤维化大鼠血清ALT(IU/L)的影响
Figure PCTCN2015076273-appb-000038
＊＊P<0.01,与MODEL组比较
表4：化合物27对BDL肝纤维化大鼠血清AST(IU/L)的影响
组别 血清AST 肝脏AST
SHAM 67.7±4.67 67.40±5.28
MODEL 207.2±30.96 198.75±27.70
27(30mg/kg) 87.2±12.51＊＊ 91.42±8.79＊＊
＊＊P<0.01,与MODEL组比较
口服化合物27对BDL肝纤维化大鼠HE染色的影响：
HE染色：
Sham组：见图3，肝小叶结构正常，肝细胞以中央静脉为中心呈放射状排列，小叶内肝细胞索排列较整齐，肝细胞大小均匀，无变性、坏死。
Model组：见图4，肝小叶结构紊乱，肝细胞肿胀，胞质疏松，纤维结缔组织增生。
给药组：见图5，治疗组改变了肝组织的病理变化。

Claims (24)

  1. 一种由通式(I)或通式(II)表示的化合物或其药用盐：
    Figure PCTCN2015076273-appb-100001
    其中：
    X是氯或OR3,其中R3选自氢、-C(O)-(1-6个碳原子的烷基)、-PO(OH)2或-CH2OPO(OH)2,
    R1，R2各自独立的选自氢、1-6个碳原子的烷基、-CH2OCO-(1-6个碳原子的烷基)或-CH2OCOO-(1-6个碳原子的烷基)；或R1和R2是如下式连接在一起的一个基团：
    Figure PCTCN2015076273-appb-100002
    其中Y是芳基或杂芳基；
    Figure PCTCN2015076273-appb-100003
    其中：
    Z选自氢或-CH2OPO(OH)2；
    R1，R2各自独立的选自氢、1-6个碳原子的烷基、-CH2OCO-(1-6个碳原子的烷基)或-CH2OCOO-(1-6个碳原子的烷基)；或R1和R2是如下式连接在一起的一个基团：
    Figure PCTCN2015076273-appb-100004
    其中Y是芳基或杂芳基。
  2. 权利要求1所述的化合物或其药用盐，其中在通式I中X是羟基。
  3. 权利要求1所述的化合物或其药用盐，其中在通式I中X是-OPO(OH)2。
  4. 权利要求1所述的化合物或其药用盐，其中在通式I中X是 -OCH2OPO(OH)2。
  5. 权利要求1所述的化合物或其药用盐，其中在通式I中R1和R2均为氢。
  6. 权利要求1所述的化合物或其药用盐，其中在通式I中R1和R2是如下式连接在一起的基团：
    Figure PCTCN2015076273-appb-100005
    其中Y是芳基。
  7. 权利要求1所述的化合物或其药用盐，其中在通式I中R1和R2是如下式连接在一起的基团：
    Figure PCTCN2015076273-appb-100006
    其中Y是杂芳基。
  8. 如权利要求1所述的化合物或其药用盐，其中所述化合物选自以下结构：
    Figure PCTCN2015076273-appb-100007
  9. 如权利要求8所述的化合物或其药用盐，其中所述化合物具有以下结构：
    Figure PCTCN2015076273-appb-100008
  10. 如权利要求9所述的化合物或其药用盐，其中所述化合物选自以下结构：
    Figure PCTCN2015076273-appb-100009
  11. 如权利要求8所述的化合物或其药用盐，其中所述化合物选自以下结构：
    Figure PCTCN2015076273-appb-100010
  12. 如权利要求11所述的化合物或其药用盐，其中所述化合物选自以下结构：
    Figure PCTCN2015076273-appb-100011
  13. 如权利要求11所述的化合物或其药用盐，其中所述化合物选自以下结构：
    Figure PCTCN2015076273-appb-100012
  14. 权利要求1所述的化合物，其中在通式II中Z是-OCH2OPO(OH)2。
  15. 权利要求1所述的化合物，其中在通式II中R1和R2均为氢。
  16. 权利要求1所述的化合物，其中在通式II中R1和R2是如下式连接在一起的基团：
    Figure PCTCN2015076273-appb-100013
    其中Y是芳基。
  17. 权利要求1所述的化合物，其中在通式II中R1和R2是如下式连接在一起的基团：
    Figure PCTCN2015076273-appb-100014
    其中Y是杂芳基。
  18. 权利要求1所述的化合物或其药用盐，其中所述化合物有以下结构：
    Figure PCTCN2015076273-appb-100015
  19. 权利要求18所述的化合物或其药用盐，其中所述化合物选自以下结构：
    Figure PCTCN2015076273-appb-100016
  20. 权利要求1-19任一项所述的化合物或其药用盐作为胶原脯氨酰-4-羟化酶抑制剂的应用。
  21. 权利要求1-19任一项所述的化合物或其药用盐在制备预防或治疗与胶原脯氨酰-4-羟化酶相关的疾病的药物中的应用。
  22. 权利要求1-19任一项所述的化合物或其药用盐在制备预防或治疗纤维化疾病的药物中的应用。
  23. 权利要求1-19任一项所述的化合物或其药用盐在制备预防或治疗肝纤维化疾病的药物中的应用。
  24. 权利要求1-19任一项所述的化合物或其药用盐在制备保护肝脏功能的药物中的应用。
PCT/CN2015/076273 2014-04-10 2015-04-10 菲罗啉膦酸类衍生物及其制备方法和应用 WO2015154716A1 (zh)

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