WO2023202501A1 - 取代的吡啶并嘧啶酮类化合物及包含该化合物的组合物及其用途 - Google Patents

取代的吡啶并嘧啶酮类化合物及包含该化合物的组合物及其用途 Download PDF

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WO2023202501A1
WO2023202501A1 PCT/CN2023/088563 CN2023088563W WO2023202501A1 WO 2023202501 A1 WO2023202501 A1 WO 2023202501A1 CN 2023088563 W CN2023088563 W CN 2023088563W WO 2023202501 A1 WO2023202501 A1 WO 2023202501A1
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compound
compounds
pharmaceutically acceptable
deuterium
solvent
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PCT/CN2023/088563
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English (en)
French (fr)
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王义汉
赵九洋
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深圳市塔吉瑞生物医药有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • the present invention belongs to the field of biomedicine technology, and in particular relates to a substituted pyridopyrimidinone compound and a composition containing the compound and its use. More specifically, the present invention relates to certain deuterium-substituted 7-(4,7-diazaspiro[2.5]oct-7-yl)-2-(2,8-dimethylimidazo[1,2 -b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one compounds and pharmaceutically acceptable salts, tautomers, stereoisomers, prodrugs, and crystal forms thereof , hydrates or solvent compounds.
  • SMN2 spinal muscular atrophy
  • SMA spinal muscular atrophy
  • SMA Spinal muscular atrophy
  • CNS central nervous system
  • SMA survival motor neuron
  • SMA is the second most common fatal autosomal recessive disease in infants and young children, with an incidence rate of 1/10 000 to 1/6000, and a population carrier rate of 1/72 to 1/47. It has racial differences, and its carrier rate The highest is among Caucasians, and the lowest among Hispanics and Africans.
  • the incidence rate in Taiwan is 1/9000.
  • the population carrier rate is 1/83 to 1/42.
  • the carrier rate is about 2.2% in Taiwan, 1.6% in Hong Kong, and 1.9% in Shanghai. Other regions and ethnic groups have not been reported.
  • SMA type 0 (SMA in utero) is the most severe form of the disease and begins before birth. Typically, the first symptom of SMA type 0 is decreased fetal movement that can first be observed between 30 and 36 weeks of pregnancy. After birth, these newborns move little and have difficulty swallowing and breathing.
  • Type I SMA infant SMA or Werdning-Hoffman disease
  • Type 2 SMA transitional SMA
  • Type 2 SMA has an onset age of 7-18 months. The patient is able to sit up without support, but is unable to stand or walk independently. The prognosis in this group depends largely on the degree of respiratory involvement.
  • SMA type 3 (juvenile or Kugelberg-Welander disease) is usually diagnosed after 18 months. Individuals with type 3 SMA are able to walk independently at some point during the course of the disease, but often rely on a wheelchair during youth or adulthood.
  • Type 4 SMA adult-onset SMA. Symptoms of weakness usually begin in late adolescence in the tongue, hands, or feet and then progress to other areas of the body. The course of adult SMA is slower and has little or no effect on life expectancy.
  • the SMN gene map has been obtained through linkage analysis of the complex region in chromosome 5q. In humans, this region contains about 500,000 base pairs (kb) of inverted duplication, resulting in two nearly identical copies of the SMN gene. SMA is caused by inactivating mutations or deletions of the telomeric copies of the gene (SMN1) on both chromosomes, resulting in loss of SMN1 gene function. However, all patients retained a centromeric copy of the gene (SMN2), and the copy number of the SMN2 gene in SMA patients often correlated inversely with disease severity; that is, patients with less severe SMA had more copies of SMN2.
  • SMN2 cannot fully compensate for the loss of SMN1 function due to alternative splicing of exon 7 caused by the translationally silent C to T mutation in exon 7. Therefore, the majority of transcripts produced by SMN2 lack exon 7 ( ⁇ 7SMN2) and encode a truncated SMN protein that has impaired function and is rapidly degraded.
  • SMN proteins are thought to play a role in RNA processing and metabolism, serving a well-characterized function in mediating the assembly of a specific class of RNA-protein complexes known as snRNPs.
  • snRNPs RNA-protein complexes
  • the SMN can have other functions, however, its role in preventing selective degeneration of motor neurons has not been well established.
  • SMA is diagnosed based on clinical symptoms and by testing for the presence of at least one copy of the SMN1 gene.
  • SMA is caused by mutations in genes other than inactivation of SMN1, some of which are known and others yet to be identified.
  • EMG electromyography
  • muscle tissue examination may be indicated.
  • Risdiplam (chemical name is 7-(4,7-diazaspiro[2.5]oct-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazine-6- Pyrido[1,2-a]pyrimidin-4-one, which has the following structural formula) is the world's first oral small molecule SMN2 gene splicing modulator for the treatment of SMA, which can increase functional SMN protein systemically.
  • ADME absorption, distribution, metabolism and/or excretion
  • the present invention discloses a new deuterium-substituted pyridopyrimidinone compound as a new and effective SMN2 gene splicing regulator, which can increase functional SMN protein and be used to treat SMA.
  • the compounds of the present invention also show better metabolic stability and/or pharmacokinetic properties.
  • a first aspect of the invention provides compounds of formula (I):
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 and R 16 each independently Selected from hydrogen, deuterium, halogen or trifluoromethyl;
  • X 1 and X 2 are each independently selected from CH 3 , CD 3 , CHD 2 or CH 2 D;
  • the invention provides compounds containing a compound of the invention or a pharmaceutically acceptable salt, tautomer, stereoisomer, prodrug, crystalline form, hydrate or solvent thereof and a pharmaceutically acceptable Excipients for pharmaceutical compositions.
  • a compound of the invention is provided in the pharmaceutical composition in an effective amount.
  • the compounds of the invention are provided in a therapeutically effective amount.
  • the compounds of the invention are provided in a prophylactically effective amount.
  • the present invention provides a preparation method of the pharmaceutical composition as described above, comprising the following steps: combining a pharmaceutically acceptable excipient with a compound of the present invention or a pharmaceutically acceptable salt thereof, tautomerization Isomers, stereoisomers, prodrugs, crystalline forms, hydrates or solvent compounds are mixed to form pharmaceutical compositions.
  • the invention provides a compound of the invention or a pharmaceutically acceptable salt, tautomer, stereoisomer, prodrug, crystalline form, hydrate or solvent compound thereof, or the above pharmaceutical composition in Use in the preparation of medicaments for the treatment and/or prevention of spinal muscular atrophy (SMA).
  • SMA spinal muscular atrophy
  • the present invention further provides a method for treating and/or preventing spinal muscular atrophy (SMA) disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the present invention or a pharmaceutical agent thereof.
  • SMA spinal muscular atrophy
  • deuterated means that one or more hydrogens in a compound or group are replaced by deuterium; deuterated can be mono-substituted, di-substituted, poly-substituted or fully-substituted.
  • deuterated can be mono-substituted, di-substituted, poly-substituted or fully-substituted.
  • deuterated can be mono-substituted, di-substituted, poly-substituted or fully-substituted.
  • non-deuterated compounds refer to compounds containing deuterium atoms in a proportion no higher than that of natural deuterium isotopes. content (0.015%) of the compound.
  • the term "subject” includes, but is not limited to: a human (i.e., male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent)) or an adult subject (e.g., Young adults, middle-aged adults, or older adults)) and/or non-human animals, e.g., mammals, e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses , sheep, goats, rodents, cats and/or dogs.
  • the subject is human.
  • the subject is a non-human animal.
  • treatment includes an action in a subject suffering from a specific disease, disorder or condition that reduces the severity of the disease, disorder or condition, or delays or slows down the disease, disorder or the development of a condition ("therapeutic treatment”), and also includes effects that occur before a subject begins to suffer from a specific disease, disorder or condition ("preventive treatment").
  • an "effective amount" of a compound is an amount sufficient to elicit a target biological response.
  • the effective amount of a compound of the present invention may vary depending on, for example, the biological target, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age of the subject. Health conditions and symptoms. Effective amounts include therapeutically and prophylactically effective amounts.
  • a "therapeutically effective amount" of a compound as used herein is an amount sufficient to provide a therapeutic benefit in treating a disease, disorder, or condition, or to render one or more of the symptoms associated with the disease, disorder, or condition Symptoms are delayed or minimized.
  • a therapeutically effective amount of a compound is that amount of therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of a disease, disorder, or condition.
  • the term "therapeutically effective amount” may include an amount that improves overall treatment, reduces or avoids symptoms or causes of a disease or disorder, or enhances the therapeutic efficacy of other therapeutic agents.
  • a prophylactically effective amount of a compound as used herein is an amount sufficient to prevent a disease, disorder or condition, or to prevent one or more symptoms associated with a disease, disorder or condition, or to prevent a disease , the number of recurrences of the disorder or condition.
  • a prophylactically effective amount of a compound refers to an amount of the therapeutic agent, alone or in combination with other agents, that provides a prophylactic benefit in preventing a disease, disorder, or condition.
  • the term “prophylactically effective amount” may include an amount that improves overall prophylaxis, or an amount that enhances the prophylactic efficacy of other prophylactic agents.
  • Combination and related terms refer to simultaneous or sequential administration of the therapeutic agents of the present invention.
  • a compound of the invention may be administered simultaneously or sequentially with another therapeutic agent in separate unit dosage forms, or with another therapeutic agent in a single unit dosage form.
  • compounds of the present invention refer to compounds of the following formula (I), or pharmaceutically acceptable salts, tautomers, stereoisomers, prodrugs, crystal forms, hydrates or solvent compounds thereof.
  • the invention relates to compounds of formula (I):
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 and R 16 each independently Selected from hydrogen, deuterium, halogen or trifluoromethyl;
  • X 1 and X 2 are each independently selected from CH 3 , CD 3 , CHD 2 or CH 2 D;
  • the deuterium isotope content of deuterium at the deuterated position is at least 0.015% greater than the natural deuterium isotope content, preferably greater than 30%, more preferably greater than 50%, even more preferably greater than 55%, even more preferably Greater than 60%, preferably greater than 65%, better than 70%, better than 75%, better than 80%, better than 85%, better than 90%, better Greater than 95%, preferably greater than 99%.
  • the deuterium isotope content of each deuterated position of R 15 , R 16 , X 1 and Better is greater than 15%, better is greater than 20%, better is greater than 25%, better is greater than 30%, better is greater than 35%, better is greater than 40%, better is greater than 45%, more Better is greater than 50%, better is greater than 55%, better is greater than 60%, better is greater than 65%, better than 70%, better than 75%, better than 80%, better than 85%, better than 90%, better than 95%, better than 99 %.
  • the compounds of the present invention contain at least one deuterium atom, more preferably two deuterium atoms, more preferably three deuterium atoms, more preferably four deuterium atoms, and more preferably five deuterium atoms.
  • deuterium atoms more preferably six deuterium atoms, more preferably seven deuterium atoms, more preferably eight deuterium atoms, more preferably nine deuterium atoms, more preferably ten deuterium atoms, more preferably Preferably it contains eleven deuterium atoms, more preferably it contains twelve deuterium atoms, more preferably it contains thirteen deuterium atoms, more preferably it contains fourteen deuterium atoms, more preferably it contains fifteen deuterium atoms, more preferably it contains Preferably it contains sixteen deuterium atoms, more preferably it contains seventeen deuterium atoms, more preferably it contains eighteen deuterium atoms, more preferably it contains nineteen deuterium atoms, more preferably it contains twenty deuterium atoms, and more preferably it contains Preferably it contains twenty-one deuterium atoms, more preferably twenty-two deuterium atoms.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 and R 16 are each independently selected from hydrogen, deuterium, halogen or trifluoromethyl" including R 1 is selected from hydrogen, deuterium, halogen or trifluoromethyl, R 2 is selected from hydrogen, deuterium, halogen or trifluoromethyl Methyl, R 3 is selected from hydrogen, deuterium, halogen or trifluoromethyl, and so on, until R 16 is selected from the technical solution of hydrogen, deuterium, halogen or trifluoromethyl.
  • R 1 is hydrogen, R 1 is deuterium, R 1 is halogen (F, Cl, Br or I) or R 1 is trifluoromethyl
  • R 2 is hydrogen, R 2 is deuterium, R 2 is Halogen (F, Cl, Br or I) or R 2 is trifluoromethyl
  • R 3 is hydrogen, R 3 is deuterium, R 3 is halogen (F, Cl, Br or I) or R 3 is trifluoromethyl , and so on, until R 16 is hydrogen, R 16 is deuterium, R 16 is halogen (F, Cl, Br or I) or R 16 is trifluoromethyl.
  • X 1 and _ _ and X 2 is selected from the technical solution of CH 3 , CD 3 , CHD 2 or CH 2 D. More specifically, X 1 is CH 3 , X 1 is CD 3 , X 1 is CHD 2 , or X 1 is CH 2 D, and X 2 is CH 3 , X 2 is CD 3 , 2 is the technical solution of CH 2 D.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 and R 16 are each independently selected from hydrogen or deuterium.
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are hydrogen.
  • R7 , R8 , R9 , R10 , R11 , R12 , R13 , R14 , R15 and R16 are hydrogen.
  • X1 is CD3 or CH3 .
  • X1 is CD3 .
  • X2 is CD3 or CH3 .
  • X2 is CD3 .
  • X1 and X2 are CD3 .
  • the present invention relates to the above-described compounds, or pharmaceutically acceptable salts, tautomers, stereoisomers, prodrugs, crystal forms, hydrates or Solvent compound , where _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ R 14 , R 15 , R 16 and X 2 are as defined above.
  • the present invention relates to the above-described compounds, or pharmaceutically acceptable salts, tautomers, stereoisomers, prodrugs, crystal forms, hydrates or Solvent compound , where _ _ _ _ _ _ _ _ _ _ _ _ _ R 14 , R 15 and R 16 are each independently selected from hydrogen or deuterium, and X 2 is selected from CH 3 , CD 3 , CHD 2 or CH 2 D.
  • the present invention relates to the above-described compounds, or pharmaceutically acceptable salts, tautomers, stereoisomers, prodrugs, crystal forms, hydrates or Solvent compound , wherein _ _ _ _ _ _ _ _ _ _ _ 13 , R14 , R15 and R16 are each independently selected from hydrogen or deuterium, and X2 is selected from CH3 , CD3 , CHD2 or CH2D .
  • the present invention relates to the above-described compounds, or pharmaceutically acceptable salts, tautomers, stereoisomers, prodrugs, crystal forms, hydrates or Solvent compound , where _ _ _ _ _ _ _ _ _ _ _ _ _ R 14 , R 15 and R 16 are hydrogen, and X 2 is selected from CH 3 , CD 3 , CHD 2 or CH 2 D.
  • the present invention relates to the above-described compounds, or pharmaceutically acceptable salts, tautomers, stereoisomers, prodrugs, crystal forms, hydrates or Solvent compound , where _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ R 14 , R 15 , R 16 and X 1 are as defined above.
  • the present invention relates to the above-described compounds, or pharmaceutically acceptable salts, tautomers, stereoisomers, prodrugs, crystal forms, hydrates or Solvent compound , where _ _ _ _ _ _ _ _ _ _ _ _ _ R 14 , R 15 and R 16 are each independently selected from hydrogen or deuterium, and X 1 is selected from CH 3 , CD 3 , CHD 2 or CH 2 D.
  • the present invention relates to the above-described compounds, or pharmaceutically acceptable salts, tautomers, stereoisomers, prodrugs, crystal forms, hydrates or Solvent compound , wherein _ _ _ _ _ _ _ _ _ _ _ 13 , R14 , R15 and R16 are each independently selected from hydrogen or deuterium, and X1 is selected from CH3 , CD3 , CHD2 or CH2D .
  • the present invention relates to the above-described compounds, or pharmaceutically acceptable salts, tautomers, stereoisomers, prodrugs, crystal forms, hydrates or Solvent compound , where _ _ _ _ _ _ _ _ _ _ _ _ _ R 14 , R 15 and R 16 are hydrogen, and X 1 is selected from CH 3 , CD 3 , CHD 2 or CH 2 D.
  • the present invention relates to the above-described compounds, or pharmaceutically acceptable salts, tautomers, stereoisomers, prodrugs, crystal forms, hydrates or Solvent compound , where X 1 and _ _ _ _ _ _ _ _ _ R 13 , R 14 , R 15 and R 16 are as defined above.
  • the present invention relates to the above-described compounds, or pharmaceutically acceptable salts, tautomers, stereoisomers, prodrugs, crystal forms, hydrates or Solvent compound , where X 1 and _ _ _ _ _ _ _ _ _ R 13 , R 14 , R 15 and R 16 are each independently selected from hydrogen or deuterium.
  • the present invention relates to the above-described compounds, or pharmaceutically acceptable salts, tautomers, stereoisomers, prodrugs, crystal forms, hydrates or Solvent compound , wherein X 1 and _ _ _ _ _ _ _ 12 , R13 , R14 , R15 and R16 are each independently selected from hydrogen or deuterium.
  • the present invention relates to the above-described compounds, or pharmaceutically acceptable salts, tautomers, stereoisomers, prodrugs, crystal forms, hydrates or Solvent compound , where X 1 and _ _ _ _ _ _ _ _ _ R 13 , R 14 , R 15 and R 16 are hydrogen.
  • the compound, or its pharmaceutically acceptable salt, tautomer, stereoisomer, prodrug, crystal form, hydrate or solvent compound is selected from any of the following compounds :
  • the compounds of the present invention may contain one or more asymmetric centers and thus may exist in multiple stereoisomeric forms, for example, enantiomeric and/or diastereomeric forms.
  • the compounds of the present invention may be individual enantiomers, diastereomers, or geometric isomers (e.g., cis and trans isomers), or may be in the form of mixtures of stereoisomers, Includes racemic mixtures and mixtures enriched in one or more stereoisomers.
  • the isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or the preferred isomers may be separated by Prepared by asymmetric synthesis.
  • HPLC high pressure liquid chromatography
  • solvate refers to a form of a compound or a salt thereof that is combined with a solvent, usually formed by a solvolysis reaction. This physical association may include hydrogen bonding.
  • solvents include water, methanol, ethanol, acetic acid, DMSO, THF, ether, etc.
  • Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-chemical solvates. Metric solvates. In some cases, the solvate will be capable of isolating, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid.
  • “Solvate” includes both solution solvates and isolable solvates. Representative solvates include hydrates, ethanolates, and methoxides.
  • hydrate refers to a compound combined with water. Typically, the ratio of the number of water molecules contained in a hydrate of a compound to the number of molecules of the compound in the hydrate is determined.
  • a hydrate of a compound may be represented, for example, by the general formula R.xH2O , where R is the compound and x is a number greater than zero.
  • a given compound may form more than one hydrate type, including, for example, monohydrate (x is 1), lower hydrate (x is a number greater than 0 and less than 1), for example, hemihydrate (R ⁇ 0.5H 2 O)) and polyhydrates (x is a number greater than 1, for example, dihydrate (R ⁇ 2H 2 O) and hexahydrate (R ⁇ 6H 2 O)).
  • monohydrate x is 1
  • lower hydrate x is a number greater than 0 and less than 1
  • hemihydrate R ⁇ 0.5H 2 O
  • polyhydrates x is a number greater than 1, for example, dihydrate (R ⁇ 2H 2 O) and hexahydrate (R ⁇ 6H 2 O)
  • the compounds of the invention may be in amorphous or crystalline forms (polymorphs). Furthermore, the compounds of the present invention may exist in one or more crystalline forms. Accordingly, the present invention includes within its scope all amorphous or crystalline forms of the compounds of the invention.
  • polymorph refers to a crystalline form of a compound (or a salt, hydrate or solvate thereof) in a specific crystal packing arrangement. All polymorphs have the same elemental composition. Different crystalline forms often have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optoelectronic properties, stability and solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors can lead to the dominance of one crystalline form. Various polymorphs of the compounds can be prepared by crystallization under different conditions.
  • the present invention also includes isotopically labeled compounds which are equivalent to those of the compounds of the present invention but in which one or more atoms are replaced by atoms having an atomic mass or mass number different from that found in nature.
  • isotopes that may be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as 2 H, 3 H, 13 C, 11 C, 14 C, 15 N, 18 respectively. O, 17 O, 31 P, 32 P, 35 S, 18 F and 36 Cl.
  • the isotope-labeled compounds of formula (I) of the present invention and their prodrugs can generally be prepared by replacing non-isotopes with readily available isotope-labeled reagents when performing the following processes and/or the processes disclosed in the Examples and Preparation Examples. Labeled reagents.
  • prodrugs are also included within the context of the present invention.
  • the term "prodrug” as used herein refers to a compound that is converted in the body to its active form having a medical effect, for example, by hydrolysis in the blood.
  • Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of A.C.S. Symposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, and D. Fleisher, S. Ramon and H. Barbra "Improved oral drug delivery: solubility limitations overcome by the use of prodrugs", Advanced Drug Delivery Reviews (1996) 19 (2) 115-130, each introduced This article serves as a reference.
  • a prodrug is any covalently bonded compound of the invention that releases the parent compound in the body when administered to a patient.
  • Prodrugs are typically prepared by modifying functional groups in a manner such that the modification can be cleaved by conventional manipulations or in vivo to yield the parent compound.
  • Prodrugs include, for example, compounds of the invention in which a hydroxyl, amino or thiol group is bonded to any group that can be cleaved to form a hydroxyl, amino or thiol group when administered to a patient.
  • representative examples of prodrugs include, but are not limited to, acetate/amide, formate/amide and benzoate/amide derivatives of the hydroxyl, thiol and amino functionality of compounds of formula (I).
  • esters such as methyl ester, ethyl ester, etc. can be used.
  • the ester itself may be reactive and/or hydrolyzable under human body conditions.
  • Suitable pharmaceutically acceptable in vivo hydrolyzable ester groups include those that readily break down in the human body to release the parent acid or salt thereof.
  • the compounds of the present invention can be prepared using known organic synthesis techniques and can be synthesized according to any of a variety of possible synthetic routes, such as those in the schemes below.
  • the reactions used to prepare the compounds of the present invention can be carried out in suitable solvents, which can be readily selected by those skilled in the art of organic synthesis. Suitable solvents may be substantially unreactive with the starting materials (reactants), intermediates, or products at the temperatures at which the reaction is conducted (eg, at temperatures ranging from the freezing temperature of the solvent to the boiling temperature of the solvent).
  • a given reaction can be carried out in one solvent or a mixture of more than one solvent. Skilled personnel can select solvents for specific reaction steps based on the specific reaction steps.
  • the preparation of the compounds of the present invention may involve the protection and deprotection of different chemical groups.
  • One skilled in the art can readily determine the need for and removal of protection and the selection of appropriate protecting groups.
  • the chemistry of the protecting groups can be found, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4th ed., John Wiley & Sons: New Jersey, (2006), which is incorporated herein by reference in its entirety.
  • the compound of the present invention can be prepared into a single stereoisomer by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomer compounds, separating the diastereoisomers and recovering optically pure enantiomers. isomer. Enantioresolution can be performed using diastereomeric derivatives of the compounds of the invention, preferably dissociable complexes (eg, crystalline diastereomeric salts). Diastereomers have significantly different physical properties (eg, melting point, boiling point, solubility, reactivity, etc.) and can be readily separated by taking advantage of these dissimilarities.
  • Diastereomers can be separated by chromatography, preferably by separation/resolution techniques based on differences in solubility. The optically pure enantiomers are then recovered, together with the resolving reagents, by any practical means that does not involve racemization.
  • a more detailed description of the techniques suitable for the resolution of stereoisomers of compounds starting from racemic mixtures can be found in Jean Jacques, Andre Collet, Samuel H. Wilen, "Enantiomers, Racemates and Resolutions” , Racemates and Resolutions", John Wiley And Sons, Inc., 1981.
  • the reaction can be monitored according to any suitable method known in the art.
  • spectroscopic means such as nuclear magnetic resonance (NMR) spectroscopy (e.g. 1 H or 13 C), infrared (IR) spectroscopy, spectrophotometry (e.g. UV-visible), mass spectrometry (MS)) or by chromatography Methods such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC) are used to monitor product formation.
  • NMR nuclear magnetic resonance
  • IR infrared
  • spectrophotometry e.g. UV-visible
  • MS mass spectrometry
  • HPLC high performance liquid chromatography
  • TLC thin layer chromatography
  • compositions preparations and kits
  • the invention provides pharmaceutical compositions comprising a compound of the invention (also referred to as an "active ingredient") and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition contains an effective amount of an active ingredient.
  • the pharmaceutical compositions comprise a therapeutically effective amount of an active ingredient.
  • the pharmaceutical compositions comprise a prophylactically effective amount of the active ingredient.
  • compositions of the present invention refer to non-toxic carriers, adjuvants or vehicles that do not destroy the pharmacological activity of the compounds with which they are formulated.
  • Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the compositions of the present invention include, but are not limited to, ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins (such as human serum albumin ), buffer substances (such as phosphate), glycine, sorbic acid, potassium sorbate, partial glyceride mixture of saturated vegetable fatty acids, water, salt or electrolyte (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, Sodium chloride, zinc salts, silica gel, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-embedded segment polymers, poly
  • kits eg, pharmaceutical packaging.
  • Kits provided may include a compound of the invention, other therapeutic agents, and first and second containers (e.g., vials, ampoules, etc.) containing the compounds of the invention, other therapeutic agents. vials, bottles, syringes and/or dispersible packaging or other suitable containers).
  • provided kits may also optionally include a third container containing pharmaceutical excipients for diluting or suspending the compounds of the invention and/or other therapeutic agents.
  • the compound of the invention and the other therapeutic agent provided in the first container and the second container are combined to form a unit dosage form.
  • parenteral administration as used herein includes subcutaneous administration, intradermal administration, intravenous administration, intramuscular administration, intraarticular administration, intraarterial administration, intrasynovial administration, intrasternal administration , intracerebrospinal membrane drug administration, intralesional drug administration, and intracranial injection or infusion techniques.
  • an effective amount of a compound provided herein is administered.
  • the amount of compound actually administered can be determined by the physician depending on the circumstances, including the condition being treated, the route of administration chosen, the compound actually administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, etc. .
  • a compound provided herein is administered to a subject at risk of developing the condition, typically on the advice of and under the supervision of a physician, at dosage levels as described above.
  • Subjects at risk of developing a particular condition generally include subjects with a family history of the condition or those who have been determined by genetic testing or screening to be particularly susceptible to developing the condition.
  • compositions provided herein can also be administered over a long period of time ("chronic administration").
  • Long-term administration refers to the administration of a compound or pharmaceutical composition thereof over a long period of time, for example, 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or administration may be continued indefinitely, For example, the remainder of the subject's life.
  • chronic administration is intended to provide a constant level of the compound in the blood over an extended period of time, eg, within a therapeutic window.
  • a pharmaceutical composition may be administered as a bolus injection, eg, to rapidly increase the concentration of the compound in the blood to an effective level.
  • the bolus dose depends on the target systemic level of the active ingredient, for example, an intramuscular or subcutaneous bolus dose provides a slow release of the active ingredient, whereas a bolus dose delivered directly into the vein (e.g., via an IV drip) can provide a more rapid release of the active ingredient. Delivered rapidly, the concentration of the active ingredient in the blood quickly increases to effective levels.
  • the pharmaceutical composition may be administered as a continuous infusion, for example, by IV infusion, thereby providing a steady-state concentration of the active ingredient in the subject's body. Additionally, in other embodiments, a bolus dose of the pharmaceutical composition may be administered first, followed by a continuous infusion.
  • Oral compositions may take the form of bulk liquid solutions or suspensions, or bulk powders. However, more commonly, for To facilitate precise dosing, the compositions are provided in unit dosage form.
  • dosage unit form refers to physically discrete units suitable as unitary dosages for human patients and other mammals, each unit containing a predetermined quantity of active material suitable to produce the desired therapeutic effect in association with a suitable pharmaceutical excipient.
  • Typical unit dosage forms include prefilled, premeasured ampoules or syringes for liquid compositions, or pills, tablets, capsules, and the like in the case of solid compositions.
  • the compound will generally be a minor component (from about 0.1 to about 50% by weight, or preferably from about 1 to about 40% by weight), with the remainder being various components useful in forming the desired administration form. carriers or excipients and processing aids.
  • a typical regimen is one to five oral doses per day, especially two to four oral doses, typically three oral doses.
  • each dose provides from about 0.01 to about 20 mg/kg of a compound of the invention, with preferred doses each providing from about 0.1 to about 10 mg/kg, especially from about 1 to about 5 mg/kg.
  • a transdermal dose is generally selected in an amount of about 0.01 to about 20% by weight, preferably about 0.1 to about 20% by weight, preferably about 0.1 to about 10% by weight, and more preferably from about 0.5 to about 15% by weight.
  • Injectable dose levels range from about 0.1 mg/kg/hour to at least 10 mg/kg/hour from about 1 to about 120 hours, especially from 24 to 96 hours. To achieve adequate steady state levels, a preload bolus of about 0.1 mg/kg to about 10 mg/kg or more may also be given. For human patients weighing 40 to 80 kg, the maximum total dose should not exceed approximately 2 g/day.
  • Liquid forms suitable for oral administration may include suitable aqueous or non-aqueous carriers as well as buffering agents, suspending and dispersing agents, coloring agents, flavoring agents, and the like.
  • Solid forms may include, for example, any of the following components, or compounds of similar nature: binders, for example, microcrystalline cellulose, tragacanth, or gelatin; excipients, for example, starch or lactose, disintegrants, For example, alginic acid, Primogel or corn starch; lubricant, for example, magnesium stearate; glidant, for example, colloidal silicon dioxide; sweetener, for example, sucrose or saccharin; or flavoring agent, for example, mint, water Methyl glycolate or orange flavoring.
  • binders for example, microcrystalline cellulose, tragacanth, or gelatin
  • excipients for example, starch or lactose, disintegrants, For example, alginic acid, Primogel or corn starch
  • Injectable compositions are typically based on injectable sterile saline or phosphate buffered saline, or other injectable excipients known in the art. As stated previously, in such compositions the active compound is typically a minor component, often about 0.05 to 10% by weight, with the remainder being injectable excipients and the like.
  • Transdermal compositions are typically formulated as topical ointments or creams containing the active ingredients.
  • the active ingredients When formulated as an ointment, the active ingredients are typically combined with a paraffin or water-miscible ointment base. Alternatively, the active ingredient may be mixed with, for example For example, an oil-in-water cream base is formulated together into a cream.
  • Such transdermal formulations are well known in the art and often include other ingredients for promoting stable skin penetration of the active ingredient or formulation. All such known transdermal formulations and components are included within the scope provided by this invention.
  • transdermal administration may be achieved using reservoir or porous membrane types, or a variety of solid matrix patches.
  • compositions for oral administration, injection or topical administration are merely representative.
  • Other materials and processing techniques are described in Part 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pennsylvania, which article is incorporated by reference.
  • the compounds of the present invention may also be administered in sustained release form or from a sustained release drug delivery system.
  • sustained release materials can be found in Remington's Pharmaceutical Sciences.
  • the invention also relates to pharmaceutically acceptable formulations of the compounds of the invention.
  • the formulation includes water.
  • the formulation contains a cyclodextrin derivative.
  • the most common cyclodextrins are ⁇ -, ⁇ - and ⁇ -cyclodextrins consisting of 6, 7 and 8 ⁇ -1,4-linked glucose units respectively, optionally including a or multiple substituents including, but not limited to: methylated, hydroxyalkylated, acylated, and sulfoalkyl ether substitutions.
  • the cyclodextrin is a sulfoalkyl ether beta-cyclodextrin, for example, sulfobutyl ether beta-cyclodextrin, also known as Captisol. See, for example, U.S. 5,376,645.
  • the formulation includes hexapropyl-beta-cyclodextrin (eg, in water, 10-50%).
  • a compound of formula (I) disclosed herein (including all individual embodiments and generic subsets disclosed herein) or a tautomer, stereoisomer, prodrug, crystalline form thereof , pharmaceutically acceptable salts, hydrates or solvent compounds that have valuable pharmacological properties and are found to enhance the inclusion of exon 7 of SMN1 and/or SMN2 into the mRNA transcribed by the SMN1 and/or SMN2 genes, thereby increasing Expression of SMN protein in human subjects in need thereof.
  • the compounds of the present invention can be used alone or in combination with other drugs to treat or prevent diseases caused by inactivating mutations or deletions of the SMN1 gene and/or related to the loss or defect of the SMN1 gene function.
  • diseases include, but are not limited to, spinal muscular atrophy (SMA).
  • the compounds of the present invention can be used to treat or prevent spinal muscular atrophy (SMA) alone or in combination with other drugs. Treating SMA includes one or more of the following effects: (1) reducing or improving the severity of SMA; (2) Delay the onset of SMA; (3) inhibit the progression of SMA; (4) reduce the admission of subjects; (5) reduce the length of admission of subjects; (6) increase the survival of subjects; (7) improve the survival of subjects quality of life; (8) reduce the number of SMA-related symptoms; (9) reduce or improve the severity of one or more symptoms associated with SMA; (9) inhibit the development or onset of SMA symptoms; and/or (10 ) inhibits the progression of symptoms associated with SMA.
  • SMA spinal muscular atrophy
  • Symptoms of SMA include muscle weakness, low muscle tone, inability to cry, inability to cough, limp or tendency to fall, difficulty sucking or swallowing, difficulty breathing, accumulation of secretions in the lungs or throat, clenched fists with sweaty hands, trembling tongue/ Vibrations, head that often leans to one side (even when lying down), legs that tend to be weaker than arms, legs that often assume a "frog-leg" position, difficulty feeding, increased susceptibility to respiratory infections, intestinal/ Multiple congenital contractures (arthrogryposis) associated with bladder weakness, lower than normal weight, inability to sit without support, inability to walk, inability to crawl, hypotonia, areflexia, and loss of anterior horn cells.
  • the compounds of the present invention can be used to treat SMA alone or in combination with other drugs, and can achieve one or more of the following beneficial effects: (1) reduce muscle strength damage; (2) increase muscle strength; (3) reduce muscle strength damage. Muscle atrophy; (4) Reduce loss of motor function; (5) Increase motor neurons; (6) Reduce motor neuron loss; (7) Prevent SMA-deficient motor neurons from degeneration; (8) Improve motor function; ( 9) Improve lung function; and/or (10) Reduce loss of lung function.
  • Pd(dppf)Cl 2 [1,1'-bis(triphenylphosphine)ferrocene]palladium dichloride
  • Pd(OAc) 2 Palladium acetate
  • PCy 3 Tricyclohexylphosphonium
  • TsCl p-toluenesulfonyl chloride
  • KOAc potassium acetate
  • Isopropyl acetate Isopropyl acetate
  • TFA trifluoroacetic acid
  • DMSO dimethyl sulfoxide
  • DMF N,N-dimethylformamide
  • EA Ethyl acetate
  • DCM dichloromethane
  • Step 3 Synthesis of compound 6-chloro-8-methyl-2-(methyl-d 3 )imidazo[1,2-b]pyridazine
  • Step 1 Compound 7-(2-(2-methyl-8-(methyl-d 3 )imidazo[1,2-b]pyridazin-6-yl)-4-oxopyrido[1,2 -Synthesis of tert-butyl a]pyrimidin-7-yl)-4,7-diazaspiro[2.5]octyl-4-carboxylate
  • Step 1 Compound 7-(2-(2-(methyl-d 3 )-8-methylimidazo[1,2-b]pyridazin-6-yl)-4-oxopyrido[1,2 -Synthesis of tert-butyl a]pyrimidin-7-yl)-4,7-diazaspiro[2.5]octyl-4-carboxylate
  • Step 1 Compound 7-(2-(2,8-bis(methyl-d 3 )imidazo[1,2-b]pyridazin-6-yl)-4-oxopyrido[1,2-a Synthesis of ]pyrimidin-7-yl)-4,7-diazaspiro[2.5]octane-4-carboxylic acid tert-butyl ester
  • Metabolic stability is generally used to describe the speed and degree of metabolism of a compound, and is one of the main factors affecting pharmacokinetic properties. Many compounds are substrates of CYP450 enzymes and other drug metabolizing enzymes, and liver microsomes are a system rich in CYP450. The purpose of this experiment is to incubate and use the compounds of the present invention with human and SD rat liver microsomes respectively. LC-MS/MS detects the remaining proportion of the compound to conduct stability studies in vitro and in vivo.
  • Phosphate buffer saline Mix 150 mL of pre-prepared KH 2 PO 4 (0.5M) solution and 700 mL of K 2 HPO 4 (0.5M) solution, and then adjust the mixture with K 2 HPO 4 (0.5M) solution When the pH value is 7.4, use it as 5-fold concentration of PBS and store it at 4°C for later use. Before use, dilute 5 times with ultrapure water, add 3.3mM magnesium chloride, and obtain phosphate buffered saline PBS (100mM).
  • NADPH regeneration system solution Use 5mL of PBS to prepare a NADPH solution containing 6.5mM NADP, 16.5mM G-6-P, and 3U/mL G-6-P D.
  • Internal standard stop solution Use acetonitrile to prepare 50ng/mL propranolol hydrochloride and 200ng/mL tolbutamide as the internal standard working solution.
  • Human liver microsome solution Add 0.31mL human liver microsomes (25mg/mL) to 0.961mL PBS (pH7.4) and mix well to obtain a human liver microsome dilution with a protein concentration of 0.625mg/mL.
  • SD rat liver microsome solution Add 0.31mL SD rat liver microsomes (25mg/mL) to 0.961mL PBS (pH7.4) and mix well to obtain a dilute SD rat liver microsome with a protein concentration of 0.625mg/mL. liquid.
  • Sample working solution Use DMSO to prepare the compound of the present invention and non-deuterated compound powder, positive control dextromethorphan powder and omeprazole powder to 10mM as a sample stock solution. Then dilute with 70% acetonitrile-water to obtain a 0.25mM sample working solution.
  • This experiment uses an LC-MS/MS system to detect the peak areas of the test compound, dextromethorphan, omeprazole and internal standard, and calculate the peak area ratio of the compound to the internal standard.
  • Rats were fed standard feed and given water. Fasting began 16 hours before the test.
  • the drug is dissolved in 10mM ascorbic acid/0.01mg/mL sodium thiosulfate pentahydrate. Blood was collected from the orbit at 0.083 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours and 24 hours after administration.
  • the rats were briefly anesthetized after inhaling ether, and 300 ⁇ L blood samples were collected from the orbits in test tubes. There are 30 ⁇ L of 1% heparin salt solution in the test tube. Before use, the test tubes were dried overnight at 60°C. After blood sample collection at the last time point, the rats were anesthetized with ether and sacrificed.

Abstract

一种取代的吡啶并嘧啶酮类化合物及包含该化合物的组合物及其用途，所述的化合物是如式(I)所示化合物或其互变异构体、立体异构体、前药、晶型、药学上可接受的盐、水合物或溶剂化合物。式(I)化合物可作为SMN2(Survival of motor neuron 2)基因剪接调节剂，用于治疗脊髓性肌萎缩(Spinal Muscular Atrophy，SMA)，具有高选择性和良好的药代动力学特性。

Description

取代的吡啶并嘧啶酮类化合物及包含该化合物的组合物及其用途 技术领域
本发明属于生物医药技术领域，尤其涉及一种取代的吡啶并嘧啶酮类化合物及包含该化合物的组合物及其用途。更具体而言，本发明涉及某些氘取代的7-(4,7-二氮杂螺[2.5]辛-7-基)-2-(2,8-二甲基咪唑并[1,2-b]哒嗪-6-基)吡啶并[1,2-a]嘧啶-4-酮化合物及其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物。这些氘取代的化合物及其组合物可用作SMN2(Survival of motor neuron 2)基因剪接调节剂，用于治疗脊髓性肌萎缩(Spinal Muscular Atrophy,SMA)症，具有高选择性和良好的药代动力学特性。
背景技术
脊髓性肌萎缩(SMA)是以脊髓和脑干中进行性运动神经元损伤为特征的多种遗传性和获得性中枢神经系统(CNS)疾病，其造成肌无力和肌萎缩。SMA最常见的形式是由运动神经元生存(SMN)基因中的突变所引起的，并且表现为严重影响从婴儿到成年人。
SMA为婴幼儿中第二常见的致死性常染色体隐性遗传病，发病率为1/10 000～1/6000，人群携带率为1/72～1/47，具有种族差异性，其携带率以高加索人种最高，最低为西班牙和非裔人种。中国台湾地区人群发病率为1/9000，大陆地区尚无大规模的研究数据，估计其人群携带率为1/83～1/42。台湾地区的携带率约为2.2％，香港地区为1.6％，上海地区为1.9％。其他地区和民族尚未见报道。
SMA病症的临床谱已被分成下列五组：
(1)0型SMA(子宫内SMA)是最严重的疾病形式并且在出生前开始。通常，0型SMA的首次症状是可以首先在妊娠30至36周之间观察到的胎儿运动减少。出生后，这些新生儿很少运动并且吞咽和呼吸困难。
(2)I型SMA(婴儿SMA或韦-霍二氏病(Werdning-Hoffman disease))在0至6个月出现症状，该类型SMA也是非常严重的。患者永远不能实现坐起，并且由于无呼吸支持，死亡通常发生在头2年。
(3)2型SMA(过渡SMA)的发病年龄在7-18个月。患者能够实现无支持坐起，但是无法独立站立或行走。该组的预后很大程度上依赖于呼吸相关的程度。
(4)3型SMA(青少年或库-韦二氏病(Kugelberg-Welander disease))通常在18个月后确诊。3型SMA个体在疾病过程期间在某些时候能够独立行走，但是在青年或成年期间通常会依靠轮椅。
(5)4型SMA(成年发病的SMA)。无力症状通常在青春期晚期在舌、手或足中开始，然后发展至身体的其他区域。成年SMA的过程更缓慢并且对预期寿命没有或几乎没有影响。
已通过对染色体5q中复杂区域的连锁分析获得SMN基因图谱。在人类中，该区域含有约50万个碱基对(kb)倒转复制，从而导致产生了两种几乎相同的SMN基因拷贝。SMA是由两个染色体上基因(SMN1)的端粒拷贝的失活突变或缺失，从而导致SMN1基因功能丧失所引起的。然而，所有患者保留了基因(SMN2)的着丝粒拷贝，并且SMA患者中SMN2基因的拷贝数通常与疾病严重性负相关；即SMA不太严重的患者具有更多的SMN2拷贝。尽管如此，由于外显子7中翻译沉默的C向T的突变所引起的外显子7的选择性剪接，SMN2不能完全补偿SMN1功能的损失。因此，由SMN2产生的大部分转录物缺失外显子7(△7SMN2)并且编码具有受损的功能并且被快速降解的截短的SMN蛋白。
SMN蛋白被认为在RNA加工和代谢中起作用，起具有良好鉴定的介导被称为snRNP的特定种类的RNA-蛋白质复合物组装的功能。在运动神经元中，SMN可以具有其他功能，然而，它在防止运动神经元选择性退化中的作用尚未得到很好的确认。
在大多数情况下，基于临床症状并且通过至少一个SMN1基因拷贝测试的存在来诊断SMA。然而，在约5％的病症中，SMA是由除SMN1失活之外的基因突变所引起的，这些基因中的一些是已知的而其他的尚未确定。在一些情况下，当SMN1基因测试不可行或者未显示任何异常时，其他测试如肌电描记术(EMG)或肌肉组织检查可以被指示。
Risdiplam(化学名称为7-(4,7-二氮杂螺[2.5]辛-7-基)-2-(2,8-二甲基咪唑并[1,2-b]哒嗪-6-基)吡啶并[1,2-a]嘧啶-4-酮，其具有以下结构式)是全球首个治疗SMA的口服小分子SMN2基因剪接调节剂，可以全身性地增加功能性SMN蛋白。
已知较差的吸收、分布、代谢和/或排泄(ADME)性质是导致许多候选药物临床试验失败的主要原因。当前上市的许多药物也由于较差的ADME性质限制了它们的应用范围。药物的快速代谢会导致许多本来可以高效治疗疾病的药物由于过快的从体内代谢清除掉而难以成药。频繁或高剂量服药虽然有可能解决药物快速清除的问题，但该方法会带来诸如病人依从性差、高剂量服药引起的副作用及治疗成本上升等问题。另外，快速代谢的药物也可能会使患者暴露于不良的毒性或反应性代谢物中。
发现具有很好的口服生物利用度且有成药性的新型有效的SMN2基因剪接调节剂还是具有挑战性的工作。因此，本领域仍需开发对适用作SMN2基因剪接调节剂具有更高选择性抑制活性和/或更好地药效学/药代动力学的化合物，本发明提供了这样的化合物。
发明内容
针对以上技术问题，本发明公开了一种新型的氘取代的吡啶并嘧啶酮类化合物作为新型、有效的SMN2基因剪接调节剂，可增加功能性SMN蛋白，用于治疗SMA。此外，本发明化合物还显示出更好的代谢稳定性和/或药代动力学性能。
对此，本发明采用以下技术方案：
本发明的第一方面，提供了式(I)化合物：
其中，
R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15和R16各自独立地选自氢、氘、卤素或三氟甲基；
X1和X2各自独立地选自CH3、CD3、CHD2或CH2D；
附加条件是，上述化合物至少含有一个氘原子；
或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物。
在另一方面，本发明提供了含有本发明化合物或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物和药学上可接受的赋形剂的药物组合物。在具体实施方案中，本发明化合物以有效量提供在所述药物组合物中。在具体实施方案中，本发明化合物以治疗有效量提供。在具体实施方案中，本发明化合物以预防有效量提供。
在另一方面，本发明提供了一种如上所述的药物组合物的制备方法，包括以下步骤：将药学上可接受的赋形剂与本发明化合物或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物进行混合，从而形成药物组合物。
在另一方面，本发明提供了本发明化合物或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物，或上述药物组合物在制备用于治疗和/或预防脊髓性肌萎缩(SMA)疾病的药物中的用途。
在另一方面，本发明提进一步提供了治疗和/或预防脊髓性肌萎缩(SMA)疾病的方法，该方法包括向由此需要的受试者施用治疗有效量的本发明化合物或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物，或本发明药物组合物。
由随后的具体实施方式、实施例和权利要求，本发明的其他目的和优点将对于本领域技术人员显而易见。
定义
本文中，如无特别说明，“氘代”指化合物或基团中的一个或多个氢被氘所取代；氘代可以是一取代、二取代、多取代或全取代。术语“一个或多个氘代的”与“一次或多次氘代”可互换使用。
本文中，如无特别说明，“非氘代的化合物”是指含氘原子比例不高于天然氘同位素 含量(0.015％)的化合物。
如本文所用，术语“受试者”包括但不限于：人(即，任何年龄组的男性或女性，例如，儿科受试者(例如，婴儿、儿童、青少年)或成人受试者(例如，年轻的成人、中年的成人或年长的成人))和/或非人的动物，例如，哺乳动物，例如，灵长类(例如，食蟹猴、恒河猴)、牛、猪、马、绵羊、山羊、啮齿动物、猫和/或狗。在一些实施方案中，受试者是人。在另一些实施方案中，受试者是非人动物。
“疾病”、“障碍”和“病症”在本文中可以互换地使用。
除非另作说明，否则，本文使用的术语“治疗”包括受试者患有具体疾病、障碍或病症时所发生的作用，它降低疾病、障碍或病症的严重程度，或延迟或减缓疾病、障碍或病症的发展(“治疗性治疗”)，还包括受试者开始患有具体疾病、障碍或疾病之前发生的作用(“预防性治疗”)。
通常，化合物的“有效量”是指足以引起目标生物反应的数量。正如本领域普通技术人员所理解的那样，本发明化合物的有效量可以根据下列因素而改变：例如，生物学目标、化合物的药物动力学、所治疗的疾病、给药模式以及受试者的年龄健康情况和症状。有效量包括治疗和预防性治疗有效量。
除非另作说明，否则，本文使用的化合物的“治疗有效量”是在治疗疾病、障碍或病症的过程中足以提供治疗有益处的数量，或使与疾病、障碍或病症有关的一或多种症状延迟或最小化。化合物的治疗有效量是指单独使用或与其他疗法联用的治疗剂的数量，它在治疗疾病、障碍或病症的过程中提供治疗益处。术语“治疗有效量”可以包括改善总体治疗、降低或避免疾病或病症的症状或病因、或增强其他治疗剂的治疗效能的数量。
除非另作说明，否则，本文使用的化合物的“预防有效量”是足以预防疾病、障碍或病症的数量，或足以预防与疾病、障碍或病症有关的一或多种症状的数量，或防止疾病、障碍或病症复发的数量。化合物的预防有效量是指单独使用或与其它药剂联用的治疗剂的数量，它在预防疾病、障碍或病症的过程中提供预防益处。术语“预防有效量”可以包括改善总体预防的数量，或增强其它预防药剂的预防效能的数量。
“组合”以及相关术语是指同时或依次给药本发明的治疗剂。例如，本发明化合物可以与另一治疗剂以分开的单位剂型同时或依次给药，或与另一治疗剂一起呈单一单位剂型同时给药。
具体实施方式
化合物
本文中，“本发明化合物”指的是以下式(I)化合物，或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物。
在一个实施方案中，本发明涉及式(I)化合物：
其中，
R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15和R16各自独立地选自氢、氘、卤素或三氟甲基；
X1和X2各自独立地选自CH3、CD3、CHD2或CH2D；
附加条件是，上述化合物至少含有一个氘原子；
或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物。
在一个具体的实施方案中，氘在氘代位置的氘同位素含量至少是大于天然氘同位素含量0.015％，较佳地大于30％，更佳地大于50％，更佳地大于55％，更佳地大于60％，更佳地大于65％，更佳地大于70％，更佳地大于75％，更佳地大于80％，更佳地大于85％，更佳地大于90％，更佳地大于95％，更佳地大于99％。
具体地说，在本发明中R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、X1和X2的各氘代位置的氘同位素含量至少是大于天然同位素含量0.015％，更佳地大于1％，更佳地大于5％，更佳地大于10％，更佳地大于15％，更佳地大于20％，更佳地大于25％，更佳地大于30％，更佳地大于35％，更佳地大于40％，更佳地大于45％，更佳地大于50％，更佳地大于55％，更佳地大于60％，更佳地大于 65％，更佳地大于70％，更佳地大于75％，更佳地大于80％，更佳地大于85％，更佳地大于90％，更佳地大于95％，更佳地大于99％。
在另一个具体的实施方案中，本发明化合物至少含有一个氘原子，更佳地含有二个氘原子，更佳地含有三个氘原子，更佳地含有四个氘原子，更佳地含有五个氘原子，更佳地含有六个氘原子，更佳地含有七个氘原子，更佳地含有八个氘原子，更佳地含有九个氘原子，更佳地含有十个氘原子，更佳地含有十一个氘原子，更佳地含有十二个氘原子，更佳地含有十三个氘原子，更佳地含有十四个氘原子，更佳地含有十五个氘原子，更佳地含有十六个氘原子，更佳地含有十七个氘原子，更佳地含有十八个氘原子，更佳地含有十九个氘原子，更佳地含有二十个氘原子，更佳地含有二十一个氘原子，更佳地含有二十二个氘原子。
在另一具体实施方案中，“R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15和R16各自独立地选自氢、氘、卤素或三氟甲基”包括R1选自氢、氘、卤素或三氟甲基，R2选自氢、氘、卤素或三氟甲基，R3选自氢、氘、卤素或三氟甲基，以此类推，直至R16选自氢、氘、卤素或三氟甲基的技术方案。更具体地，包括R1是氢、R1是氘、R1是卤素(F、Cl、Br或I)或R1是三氟甲基，R2是氢、R2是氘、R2是卤素(F、Cl、Br或I)或R2是三氟甲基，R3是氢、R3是氘、R3是卤素(F、Cl、Br或I)或R3是三氟甲基，以此类推，直至R16是氢、R16是氘、R16是卤素(F、Cl、Br或I)或R16是三氟甲基的技术方案。
在另一具体实施方案中，“X1和X2各自独立地选自CH3、CD3、CHD2或CH2D”包括X1选自CH3、CD3、CHD2或CH2D，和X2选自CH3、CD3、CHD2或CH2D的技术方案。更具体地，包括X1是CH3、X1是CD3、X1是CHD2或X1是CH2D，和X2是CH3、X2是CD3、X2是CHD2或X2是CH2D的技术方案。
在另一个具体实施方案中，R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15和R16各自独立地选自氢或氘。
在另一个具体实施方案中，R1、R2、R3、R4、R5和R6是氢。
在另一个具体实施方案中，R7、R8、R9、R10、R11、R12、R13、R14、R15和R16是氢。
在另一个具体实施方案中，X1是CD3或CH3。
在另一个具体实施方案中，X1是CD3。
在另一个具体实施方案中，X2是CD3或CH3。
在另一个具体实施方案中，X2是CD3。
在另一个具体实施方案中，X1和X2是CD3。
在式(I)化合物的一些实施方案中，优选地，本发明涉及上述化合物，或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物，其中，X1是CD3，R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16和X2如上文所定义。
在式(I)化合物的一些实施方案中，优选地，本发明涉及上述化合物，或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物，其中，X1是CD3，R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15和R16各自独立地选自氢或氘，X2选自CH3、CD3、CHD2或CH2D。
在式(I)化合物的一些实施方案中，优选地，本发明涉及上述化合物，或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物，其中，X1是CD3，R1、R2、R3、R4、R5和R6是氢，R7、R8、R9、R10、R11、R12、R13、R14、R15和R16各自独立地选自氢或氘，X2选自CH3、CD3、CHD2或CH2D。
在式(I)化合物的一些实施方案中，优选地，本发明涉及上述化合物，或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物，其中，X1是CD3，R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15和R16是氢，X2选自CH3、CD3、CHD2或CH2D。
在式(I)化合物的一些实施方案中，优选地，本发明涉及上述化合物，或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物，其中，X2是CD3，R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16和X1如上文所定义。
在式(I)化合物的一些实施方案中，优选地，本发明涉及上述化合物，或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物，其中，X2是CD3，R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15和R16各自独立地选自氢或氘，X1选自CH3、CD3、CHD2或CH2D。
在式(I)化合物的一些实施方案中，优选地，本发明涉及上述化合物，或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物，其中，X2是CD3，R1、R2、R3、R4、R5和R6是氢，R7、R8、R9、R10、R11、R12、R13、R14、R15和R16各自独立地选自氢或氘，X1选自CH3、CD3、CHD2或CH2D。
在式(I)化合物的一些实施方案中，优选地，本发明涉及上述化合物，或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物，其中，X2是CD3，R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15和R16是氢，X1选自CH3、CD3、CHD2或CH2D。
在式(I)化合物的一些实施方案中，优选地，本发明涉及上述化合物，或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物，其中，X1和X2是CD3，R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15和R16如上文所定义。
在式(I)化合物的一些实施方案中，优选地，本发明涉及上述化合物，或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物，其中，X1和X2是CD3，R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15和R16各自独立地选自氢或氘。
在式(I)化合物的一些实施方案中，优选地，本发明涉及上述化合物，或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物，其中，X1和X2是CD3，R1、R2、R3、R4、R5和R6是氢，R7、R8、R9、R10、R11、R12、R13、R14、R15和R16各自独立地选自氢或氘。
在式(I)化合物的一些实施方案中，优选地，本发明涉及上述化合物，或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物，其中，X1和X2是CD3，R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15和R16是氢。
作为本发明的优选实施方案，所述化合物，或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物，选自如下任一化合物：

本发明化合物可包括一个或多个不对称中心，且因此可以存在多种立体异构体形式，例如，对映异构体和/或非对映异构体形式。例如，本发明化合物可为单独的对映异构体、非对映异构体或几何异构体(例如顺式和反式异构体)，或者可为立体异构体的混合物的形式，包括外消旋体混合物和富含一种或多种立体异构体的混合物。异构体可通过本领域技术人员已知的方法从混合物中分离，所述方法包括：手性高压液相色谱法(HPLC)以及手性盐的形成和结晶；或者优选的异构体可通过不对称合成来制备。
本领域技术人员将理解，有机化合物可以与溶剂形成复合物，其在该溶剂中发生反应或从该溶剂中沉淀或结晶出来。这些复合物称为“溶剂合物”。当溶剂是水时，复合物称为“水合物”。本发明涵盖了本发明化合物的所有溶剂合物。
术语“溶剂合物”是指通常由溶剂分解反应形成的与溶剂相结合的化合物或其盐的形式。这个物理缔合可包括氢键键合。常规溶剂包括水、甲醇、乙醇、乙酸、DMSO、THF、乙醚等。本文所述的化合物可制备成，例如，结晶形式，且可被溶剂化。合适的溶剂合物包括药学上可接受的溶剂合物且进一步包括化学计量的溶剂合物和非化学 计量的溶剂合物。在一些情况下，所述溶剂合物将能够分离，例如，当一或多个溶剂分子掺入结晶固体的晶格中时。“溶剂合物”包括溶液状态的溶剂合物和可分离的溶剂合物。代表性的溶剂合物包括水合物、乙醇合物和甲醇合物。
术语“水合物”是指与水相结合的化合物。通常，包含在化合物的水合物中的水分子数与该水合物中该化合物分子数的比率确定。因此，化合物的水合物可用例如通式R·x H2O代表，其中R是该化合物，和x是大于0的数。给定化合物可形成超过一种水合物类型，包括，例如，单水合物(x为1)、低级水合物(x是大于0且小于1的数，例如，半水合物(R·0.5H2O))和多水合物(x为大于1的数，例如，二水合物(R·2H2O)和六水合物(R·6H2O))。
本发明化合物可以是无定形或结晶形式(多晶型)。此外，本发明化合物可以以一种或多种结晶形式存在。因此，本发明在其范围内包括本发明化合物的所有无定形或结晶形式。术语“多晶型物”是指特定晶体堆积排列的化合物的结晶形式(或其盐、水合物或溶剂合物)。所有的多晶型物具有相同的元素组成。不同的结晶形式通常具有不同的X射线衍射图、红外光谱、熔点、密度、硬度、晶体形状、光电性质、稳定性和溶解度。重结晶溶剂、结晶速率、贮存温度和其他因素可导致一种结晶形式占优。化合物的各种多晶型物可在不同的条件下通过结晶制备。
本发明还包括同位素标记的化合物，它们等同于本发明化合物的那些，但一个或多个原子被原子质量或质量数不同于自然界常见的原子质量或质量数的原子所代替。可以引入本发明化合物中的同位素的实例包括氢、碳、氮、氧、磷、硫、氟和氯的同位素，分别例如2H、3H、13C、11C、14C、15N、18O、17O、31P、32P、35S、18F和36Cl。含有上述同位素和/或其它原子的其它同位素的本发明化合物、其前体药物和所述化合物或所述前体药物的药学上可接受的盐都属于本发明的范围。某些同位素标记的本发明化合物、例如引入放射性同位素(例如3H和14C)的那些可用于药物和/或底物组织分布测定。氚、即3H和碳-14、即14C同位素是特别优选的，因为它们容易制备和检测。进而，被更重的同位素取代，例如氘、即2H，由于代谢稳定性更高可以提供治疗上的益处，例如延长体内半衰期或减少剂量需求，因而在有些情况下可能是优选的。同位素标记的本发明式(I)化合物及其前体药物一般可以这样制备，在进行下述流程和/或实施例与制备例所公开的工艺时，用容易得到的同位素标记的试剂代替非同位素标记的试剂。
此外，前药也包括在本发明的上下文内。本文所用的术语“前药”是指在体内通过例如在血液中水解转变成其具有医学效应的活性形式的化合物。药学上可接受的前药描述于T.Higuchi和V.Stella，Prodrugs as Novel Delivery Systems，A.C.S.Symposium Series的Vol.14，Edward B.Roche，ed.，Bioreversible Carriers in Drug Design，American Pharmaceutical Association and Pergamon Press，1987，以及D.Fleisher、S.Ramon和H.Barbra“Improved oral drug delivery：solubility limitations overcome by the use of prodrugs”，Advanced Drug Delivery Reviews(1996)19(2)115-130，每篇引入本文作为参考。
前药为任何共价键合的本发明化合物，当将这种前药给予患者时，其在体内释放母体化合物。通常通过修饰官能团来制备前药，修饰是以使得该修饰可以通过常规操作或在体内裂解产生母体化合物的方式进行的。前药包括，例如，其中羟基、氨基或巯基与任意基团键合的本发明化合物，当将其给予患者时，可以裂解形成羟基、氨基或巯基。因此，前药的代表性实例包括(但不限于)式(I)化合物的羟基、巯基和氨基官能团的乙酸酯/酰胺、甲酸酯/酰胺和苯甲酸酯/酰胺衍生物。另外，在羧酸(-COOH)的情况下，可以使用酯，例如甲酯、乙酯等。酯本身可以是有活性的和/或可以在人体体内条件下水解。合适的药学上可接受的体内可水解的酯基包括容易在人体中分解而释放母体酸或其盐的那些基团。
制备本发明化合物的方法
本发明化合物(包括其盐)可使用已知有机合成技术来制备，且可按照多种可能合成途径中的任一种(诸如下文方案中的那些)来合成。用于制备本发明化合物的反应可在合适的溶剂中进行，有机合成领域的技术人员可容易地选择溶剂。合适的溶剂可在进行反应的温度(例如，在溶剂结冻温度至溶剂沸点温度范围内的温度)下与起始物质(反应物)、中间体或产物实质上不反应。既定反应可在一种溶剂或一种以上溶剂的混合物中进行。技术人员可依据具体反应步骤来选择用于具体反应步骤的溶剂。
本发明化合物的制备可涉及不同化学基团的保护和去除保护。本领域技术人员可容易地判定是否需要保护和去除保护以及适当保护基的选择。保护基的化学性质可参见例如Wuts和Greene，Protective Groups in Organic Synthesis，第4版，John Wiley&Sons：New Jersey，(2006)，其通过引用整体并入本文中。
本发明化合物可通过化合物的消旋混合物与光学活性的拆分剂反应形成一对非对映异构体化合物、分离非对映异构体并回收光学纯度的対映体，制备成其单个立体异构体。对映体拆分时可使用本发明化合物的非对映体衍生物进行，优先可解离的复合物(例如，结晶非对映体盐)。非对映体具有显著不同的物理性质(例如，熔点、沸点、溶解度、反应性等)，并可通过这些不相似性的优势容易地得到分离。非对映体可通过色谱，优选通过基于溶解度的差异的分离/拆分技术进行分离。然后通过不会消旋化的任何实际手段，回收光学纯对映体，连同拆分试剂。适用于从消旋混合物开始拆分得到化合物立体异构体的技术的更详细的描述可见于Jean Jacques，Andre Collet，Samue1 H.Wilen，“对映体、消旋体和拆分”(“Enantiomers，Racemates and Resolutions”)，John Wiley And Sons，Inc.,1981。
可按照本领域已知任何合适的方法来监测反应。例如，可通过光谱手段(诸如核磁共振(NMR)光谱法(例如1H或13C)、红外(IR)光谱法、分光光度法(例如，UV-可见光)、质谱(MS))或通过色谱方法(诸如高效液相色谱法(HPLC)或薄层色谱法(TLC))来监测产物形成。
药物组合物、制剂和试剂盒
在另一方面，本发明提供了药物组合物，其包含本发明化合物(还称为“活性组分”)和药学上可接受的赋形剂。在一些实施方案中，所述药物组合物包含有效量的活性组分。在一些实施方案中，所述药物组合物包含治疗有效量的活性组分。在一些实施方案中，所述药物组合物包含预防有效量的活性组分。
用于本发明的药学上可接受的赋形剂是指不会破坏一起配制的化合物的药理学活性的无毒载剂、佐剂或媒剂。可以用于本发明组合物中的药学上可接受的载剂、佐剂或媒剂包括但不限于，离子交换剂、氧化铝、硬脂酸铝、卵磷脂、血清蛋白(如人类血清白蛋白)、缓冲物质(如磷酸盐)、甘氨酸、山梨酸、山梨酸钾、饱和植物脂肪酸的偏甘油酯混合物、水、盐或电解质(如硫酸鱼精蛋白)、磷酸氢二钠、磷酸氢钾、氯化钠、锌盐、硅胶、三硅酸镁、聚乙烯吡咯烷酮、基于纤维素的物质、聚乙二醇、羧甲基纤维素钠、聚丙烯酸酯、蜡、聚乙烯-聚氧丙烯-嵌段聚合物、聚乙二醇以及羊毛脂。
本发明还包括试剂盒(例如，药物包装)。所提供的试剂盒可以包括本发明化合物、其它治疗剂，以及含有本发明化合物、其它治疗剂的第一和第二容器(例如，小瓶、安 瓿瓶、瓶、注射器和/或可分散包装或其它合适的容器)。在一些实施方案中，提供的试剂盒还可以任选包括第三容器，其含有用于稀释或悬浮本发明化合物和/或其它治疗剂的药用赋形剂。在一些实施方案中，提供在第一容器和第二容器中的本发明化合物和其它治疗剂组合形成一个单位剂型。
本发明提供的药物组合物可以通过许多途径给药，包括但不限于：口服给药、肠胃外给药、吸入给药、局部给药、直肠给药、鼻腔给药、口腔给药、阴道给药、通过植入剂给药或其它给药方式。例如，本文使用的肠胃外给药包括皮下给药、皮内给药、静脉内给药、肌肉内给药、关节内给药、动脉内给药、滑膜腔内给药、胸骨内给药、脑脊髓膜内给药、病灶内给药、和颅内的注射或输液技术。
通常，给予有效量的本文所提供的化合物。按照有关情况，包括所治疗的病症、选择的给药途径、实际给予的化合物、个体患者的年龄、体重和响应、患者症状的严重程度，等等，可以由医生确定实际上给予的化合物的量。
当用于预防本发明所述病症时，给予处于形成所述病症危险之中的受试者本文所提供的化合物，典型地基于医生的建议并在医生监督下给药，剂量水平如上所述。处于形成具体病症的危险之中的受试者，通常包括具有所述病症的家族史的受试者，或通过遗传试验或筛选确定尤其对形成所述病症敏感的那些受试者。
还可以长期给予本文所提供的药物组合物(“长期给药”)。长期给药是指在长时间内给予化合物或其药物组合物，例如，3个月、6个月、1年、2年、3年、5年等等，或者可无限期地持续给药，例如，受试者的余生。在一些实施方案中，长期给药意欲在长时间内在血液中提供所述化合物的恒定水平，例如，在治疗窗内。
可以使用各种给药方法，进一步递送本发明的药物组合物。例如，在一些实施方案中，可以推注给药药物组合物，例如，为了使化合物在血液中的浓度快速提高至有效水平。推注剂量取决于活性组分的目标全身性水平，例如，肌内或皮下的推注剂量使活性组分缓慢释放，而直接递送至静脉的推注(例如，通过IV静脉滴注)能够更加快速地递送，使得活性组分在血液中的浓度快速升高至有效水平。在其它实施方案中，可以以持续输液形式给予药物组合物，例如，通过IV静脉滴注，从而在受试者身体中提供稳态浓度的活性组分。此外，在其它实施方案中，可以首先给予推注剂量的药物组合物，而后持续输液。
口服组合物可以采用散装液体溶液或混悬剂或散装粉剂形式。然而，更通常，为 了便于精确地剂量给药，以单位剂量形式提供所述组合物。术语“单位剂型”是指适合作为人类患者及其它哺乳动物的单元剂量的物理离散单位，每个单位包含预定数量的、适于产生所需要的治疗效果的活性物质与合适药学赋形剂。典型的单位剂量形式包括液体组合物的预装填的、预先测量的安瓿或注射器，或者在固体组合物情况下的丸剂、片剂、胶囊剂等。在这种组合物中，所述化合物通常为较少的组分(约0.1至约50重量％，或优选约1至约40重量％)，剩余部分为对于形成所需给药形式有用的各种载体或赋形剂以及加工助剂。
对于口服剂量，代表性的方案是，每天一个至五个口服剂量，尤其是两个至四个口服剂量，典型地是三个口服剂量。使用这些剂量给药模式，每个剂量提供大约0.01至大约20mg/kg的本发明化合物，优选的剂量各自提供大约0.1至大约10mg/kg，尤其是大约1至大约5mg/kg。
为了提供与使用注射剂量类似的血液水平，或比使用注射剂量更低的血液水平，通常选择透皮剂量，数量为大约0.01至大约20％重量，优选大约0.1至大约20％重量，优选大约0.1至大约10％重量，且更优选大约0.5至大约15％重量。
从大约1至大约120小时，尤其是24至96小时，注射剂量水平在大约0.1mg/kg/小时至至少10mg/kg/小时的范围。为了获得足够的稳定状态水平，还可以给予大约0.1mg/kg至大约10mg/kg或更多的预载推注。对于40至80kg的人类患者来说，最大总剂量不能超过大约2g/天。
适于口服给药的液体形式可包括合适的水性或非水载体以及缓冲剂、悬浮剂和分散剂、着色剂、调味剂，等等。固体形式可包括，例如，任何下列组份，或具有类似性质的化合物：粘合剂，例如，微晶纤维素、黄蓍胶或明胶；赋形剂，例如，淀粉或乳糖，崩解剂，例如，褐藻酸、Primogel或玉米淀粉；润滑剂，例如，硬脂酸镁；助流剂，例如，胶体二氧化硅；甜味剂，例如，蔗糖或糖精；或调味剂，例如，薄荷、水杨酸甲酯或橙味调味剂。
可注射的组合物典型地基于可注射用的无菌盐水或磷酸盐缓冲盐水，或本领域中已知的其它可注射的赋形剂。如前所述，在这种组合物中，活性化合物典型地为较少的组分，经常为约0.05至10％重量，剩余部分为可注射的赋形剂等。
典型地将透皮组合物配制为含有活性组分的局部软膏剂或乳膏剂。当配制为软膏剂时，活性组分典型地与石蜡或可与水混溶的软膏基质组合。或者，活性组分可与例 如水包油型乳膏基质一起配制为乳膏剂。这种透皮制剂是本领域中公知的，且通常包括用于提升活性组分或制剂的稳定的皮肤渗透的其它组份。所有这种已知的透皮制剂和组份包括在本发明提供的范围内。
本发明化合物还可通过经皮装置给予。因此，经皮给药可使用贮存器(reservoir)或多孔膜类型、或者多种固体基质的贴剂实现。
用于口服给予、注射或局部给予的组合物的上述组份仅仅是代表性的。其它材料以及加工技术等阐述于Remington's Pharmaceutical Sciences,17th edition,1985,Mack Publishing Company,Easton,Pennsylvania的第8部分中，本文以引用的方式引入该文献。
本发明化合物还可以以持续释放形式给予，或从持续释放给药系统中给予。代表性的持续释放材料的描述可在Remington's Pharmaceutical Sciences中找到。
本发明还涉及本发明化合物的药学上可接受的制剂。在一个实施方案中，所述制剂包含水。在另一个实施方案中，所述制剂包含环糊精衍生物。最常见的环糊精为分别由6、7和8个α-1,4-连接的葡萄糖单元组成的α-、β-和γ-环糊精，其在连接的糖部分上任选包括一个或多个取代基，其包括但不限于：甲基化的、羟基烷基化的、酰化的和磺烷基醚取代。在一些实施方案中，所述环糊精为磺烷基醚β-环糊精，例如，磺丁基醚β-环糊精，也称作Captisol。参见，例如，U.S.5,376,645。在一些实施方案中，所述制剂包括六丙基-β-环糊精(例如，在水中，10-50％)。
适应症
在另一方面，提供了本发明公开的式(I)化合物(包括本文公开的所有单独的实施方案及类属子集)或其互变异构体、立体异构体、前药、晶型、药学上可接受的盐、水合物或溶剂化合物具有价值的药理学性质并且被发现增强SMN1和/或SMN2的外显子7包含到由SMN1和/或SMN2基因转录的mRNA中，由此增加SMN蛋白在有此需要的人受试者中的表达。
本发明的化合物可以单独地或与其他药物组合地用于治疗或预防由SMN1基因的失活突变或缺失引起的和/或与SMN1基因功能丧失或缺陷相关的疾病。这些疾病包括但不限于脊髓性肌萎缩(SMA)。
本发明的化合物可以单独地或与其他药物组合地用于治疗或预防脊髓性肌萎缩(SMA)。治疗SMA包括以下效果中的一个或多个：(1)减少或改善SMA的严重度；(2) 延迟SMA的发作；(3)抑制SMA的进展；(4)减少受试者入院；(5)减少受试者的入院时长；(6)增加受试者的存活；(7)提高受试者的生活质量；(8)减少SMA相关症状的数目；(9)减少或改善与SMA相关的一种或多种症状的严重度；(9)抑制SMA症状的发展或发作；和/或(10)抑制与SMA相关的症状的进展。
SMA的症状包括肌无力、肌张力低下、哭泣无力、咳嗽无力、跛行或摔倒倾向、吮吸或吞咽困难、呼吸困难、肺或咽喉中分泌物积累、用汗手紧握的拳头、舌头颤动/振动、常常倾向一侧的头部(即使在躺下时)、倾向于弱于臂部的腿部、经常呈“蛙腿”位置的腿部、摄食困难、对呼吸道感染敏感度提高、肠/膀胱无力、低于正常的体重、不能无支撑坐立、不能行走、不能爬行、张力减退、反射消失以及前角细胞丧失相关的多发性先天性挛缩(关节挛缩)。
本发明的化合物可以单独地或与其他药物组合地用于治疗SMA，可以达到以下有益效果中的一种或多种：(1)减少肌力损伤；(2)增加肌力；(3)减少肌萎缩；(4)减少运动功能丧失；(5)增加运动神经元；(6)减少运动神经元损失；(7)使SMA不足的运动神经元免于退化；(8)提高运动功能；(9)提高肺功能；和/或(10)减少肺功能丧失。
实施例
下面结合具体实施例，作进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则份数和百分比为重量份和重量百分比。
缩略语：
Pd(dppf)Cl2：[1,1'-双(三苯基膦)二茂铁]二氯化钯
Pd(OAc)2:乙酸钯
PCy3：三环己基磷
TsCl：对甲苯磺酰氯
KOAc：醋酸钾
Isopropyl acetate：乙酸异丙酯
TFA：三氟乙酸
DMSO：二甲基亚砜
DMF：N,N-二甲基甲酰胺
EA：乙酸乙酯
Xylene：二甲苯
MeOH：甲醇
Dioxide：1,4-二氧六环
DCM：二氯甲烷
中间体化合物A-1 7-(4-氧代-2-(对甲苯磺酰氧基)-4H-吡啶并[1,2-a]嘧啶-7-基)-4,7- 二氮杂螺[2.5]辛-4-甲酸叔丁酯制备
采用以下合成路线
步骤1化合物7-(6-硝基吡啶-3-基)-4,7-二氮杂螺[2.5]辛-4-甲酸叔丁酯的合成
将5-溴-2-硝基吡啶(2.0g，10mmol)和4,7-二氮杂螺[2.5]辛-4-甲酸叔丁酯(2.23g，10.5mmol)溶于10ml二甲基亚砜中，依次加入氯化锂(1.47g，35mmol)和四甲基胍(4.4ml，35mmol)，氮气保护下将反应液加热至80℃搅拌过夜，TLC监测反应完毕，冷却至室温，将反应液缓慢滴加入冰水中，析出淡黄色固体，过滤，滤饼真空干燥后得到黄色固体2.76g，收率82.6％。LC-MS(APCI):m/z＝335.1(M+1)+。
步骤2化合物7-(6-氨基吡啶-3-基)-4,7-二氮杂螺[2.5]辛-4-甲酸叔丁酯的合成
将上步所得7-(6-硝基吡啶-3-基)-4,7-二氮杂螺[2.5]辛-4-甲酸叔丁酯(2.76g，8.26mmol)置于100ml烧瓶中，加入10ml乙酸乙酯和10ml甲醇溶解，氮气保护下加入10％wt的Pd/C(300mg)，用氢气置换三次，室温下搅拌反应2-4小时，TLC监测反 应完毕，硅藻土助滤，滤液浓缩至干。得到2.55g粗品，无需纯化直接投入到下一步反应。LC-MS(APCI):m/z＝305.4(M+1)+。
步骤3化合物7-(2-羟基-4-氧代-4H-吡啶并[1,2-a]嘧啶-7-基)-4,7-二氮杂螺[2.5]辛-4-甲酸叔丁酯的合成
在反应瓶中加入7-(6-氨基吡啶-3-基)-4,7-二氮杂螺[2.5]辛-4-甲酸叔丁酯(2.0g，6.58mmol)和双(2,4,6-三氯苯基)丙二酸酯(3.66g，7.89mmol)，加入25ml二甲苯，氮气保护下将反应液加热至150℃搅拌1-2小时，TLC监测反应完毕，冷却至室温析出浅黄色固体，过滤，滤饼用少量乙酸乙酯洗涤，真空干燥后得到1.92g固体，收率78.4％。LC-MS(APCI):m/z＝373.4(M+1)+。
步骤4中间体化合物A-1的合成
将7-(2-羟基-4-氧代-4H-吡啶并[1,2-a]嘧啶-7-基)-4,7-二氮杂螺[2.5]辛-4-甲酸叔丁酯(1.12g，3.0mmol)溶于40ml无水二氯甲烷中，加入三乙胺(0.54ml，3.9mmol)，氮气保护下分批加入TsCl(0.63g，3.3mmol)，加毕，室温下搅拌反应2-4小时，TLC监测反应完毕。加入30ml水淬灭反应，用二氯甲烷萃取3-4次，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，过滤，滤液浓缩后硅胶柱层析纯化得到1.4g产物，收率：88.7％。LC-MS(APCI):m/z＝527.8(M+1)+。
中间体B-1 2-甲基-8-(甲基-d3)-6-(4,4,5,5-四甲基-1,3,2-二氧杂硼杂环戊烷-2-基)咪 唑并[1,2-b]哒嗪制备
采用以下合成路线
步骤1化合物4-溴-6-氯哒嗪-3-氨的合成
将6-氯哒嗪-3-氨(4.0g，31mmol)、乙酸钠(2.03g，24.8mmol)和乙酸(0.5g，8.27mmol)置于100ml烧瓶中，加入40ml甲醇，分批加入1,3-二溴-5,5-二甲基海因(5.3g，18.5mmol)，室温下搅拌过夜，TLC监测反应完毕，降温至0℃，缓慢加入10ml亚硫酸钠饱和水溶液淬灭反应，浓缩除去甲醇，加入少量水稀释，用1N的氢氧化钠调PH至7-8，析出淡黄色固体，过滤，滤饼用水洗涤，真空干燥后得到4.5g目标产物，收率：70.1％，无需纯化直接投入到下一步反应。LC-MS(APCI):m/z＝208.1(M+1)+。
步骤2化合物6-氯-4-(甲基-d3)哒嗪-3-氨的合成
将上步所得4-溴-6-氯哒嗪-3-氨(2.07g，10mmol)溶于100ml无水THF中，氮气保护下，缓慢滴加氘代甲基碘化镁(27ml，27mmol)，加毕，依次加入氯化锌的2-甲基四氢呋喃溶液(2.5ml，5mmol)和四(三苯基膦)钯(116mg，0.1mmol),升温至50℃搅拌反应4-6小时，TLC监测反应完毕。将反应液冷却至室温，加入50ml饱和氯化铵淬灭反应，用乙酸乙酯萃取3-4次，合并有机相，浓缩后硅胶柱层析纯化得到1.34g产物，收率：44.3％。LC-MS(APCI):m/z＝147.5(M+1)+。
步骤3化合物6-氯-2-甲基-8-(甲基-d3)咪唑并[1,2-b]哒嗪的合成
将6-氯-4-(甲基-d3)哒嗪-3-氨(194mg，1.33mmol)溶于10ml无水DMF中，加热至100℃，缓慢滴加溴丙酮(200mg，1.46mmol)的3ml DMF溶液，氮气保护下升温至110℃搅拌反应2-3小时，TLC监测反应完毕。降至室温后加入10ml饱和碳酸氢钠水溶液淬灭反应，用乙酸乙酯萃取3-4次，合并有机相，用饱和食盐水洗涤，无水硫酸钠干燥，浓缩，经硅胶柱分离得固体140mg，收率57.1％。LC-MS(APCI): m/z＝185.5(M+1)+。
步骤4中间体化合物B-1的合成
将6-氯-2-甲基-8-(甲基-d3)咪唑并[1,2-b]哒嗪(90mg，0.5mmol)、联硼酸频哪醇酯(380mg，1.5mmol)、Pd(OAc)2(6mg，0.025mmol)、PCy3(14mg，0.05mmol)和乙酸钾(100mg，1.0mmol)加入到20ml微波管中，氮气保护下加入8ml乙酸异丙酯，密封后微波加热至80℃反应1小时，TLC监测反应完毕，降至室温后浓缩除去溶剂，硅胶柱层析纯化得到108mg固体，收率：79.1％。LC-MS(APCI):m/z＝277.2(M+1)+。
中间体B-2 8-甲基-2-(甲基-d3)-6-(4,4,5,5-四甲基-1,3,2-二氧杂硼杂环戊烷-2-基)咪 唑并[1,2-b]哒嗪的制备
采用以下合成路线
步骤1化合物1-溴丙烷-2-酮-1,1,3,3,3-d5的合成
将氘代丙酮(5g，78.1mmol)加入到100ml甲醇中，降温至0℃，分批加入溴的二氧六环络合物(19.4g，78.1mmol)，冰浴下搅拌反应3-4小时，GC监测反应完毕，加入80ml水淬灭反应，用80ml乙醚萃取3-4次，合并有机相，无水硫酸钠干燥，过滤后，滤液在低温下浓缩除去溶剂得到产物粗品，不需纯化直接投入到下一步反应。
步骤2：化合物6-氯-4-甲基哒嗪-3-氨的合成
将1-溴丙烷-2-酮-1,1,3,3,3-d5(2.07g，10mmol)溶于100ml无水THF中，氮气保护下，缓慢滴加甲基碘化镁(27ml，27mmol)，加毕，依次加入氯化锌的2-甲基四氢呋喃溶液(2.5ml，5mmol)和四(三苯基膦)钯(116mg，0.1mmol),升温至50℃ 搅拌反应4-6小时，TLC监测反应完毕。将反应液冷却至室温，加入50ml饱和氯化铵淬灭反应，用乙酸乙酯萃取3-4次，合并有机相，浓缩后硅胶柱层析纯化得到1.18g产物，收率：39.0％。LC-MS(APCI):m/z＝144.2(M+1)+。
步骤3：化合物6-氯-8-甲基-2-(甲基-d3)咪唑并[1,2-b]哒嗪的合成
将6-氯-4-甲基哒嗪-3-氨(194mg，1.34mmol)溶于10ml无水DMF中，加热至100℃，缓慢滴加1-溴丙烷-2-酮-1,1,3,3,3-d5(200mg，1.40mmol)的3ml DMF溶液，氮气保护下升温至110℃搅拌反应2-3小时，TLC监测反应完毕。降至室温后加入10ml饱和碳酸氢钠水溶液淬灭反应，用乙酸乙酯萃取3-4次，合并有机相，用饱和食盐水洗涤，无水硫酸钠干燥，浓缩，经硅胶柱分离得固体152mg，收率61.9％。LC-MS(APCI):m/z＝185.5(M+1)+。
步骤4：中间体化合物B-2的合成
将6-氯-8-甲基-2-(甲基-d3)咪唑并[1,2-b]哒嗪(90mg，0.5mmol)、联硼酸频哪醇酯(380mg，1.5mmol)、Pd(OAc)2(6mg，0.025mmol)、PCy3(14mg，0.05mmol)和乙酸钾(100mg，1.0mmol)加入到20ml微波管中，氮气保护下加入8ml乙酸异丙酯，密封后微波加热至80℃反应1小时，TLC监测反应完毕，降至室温后浓缩除去溶剂，硅胶柱层析纯化得到111mg固体，收率：81.3％。LC-MS(APCI):m/z＝277.2(M+1)+。
中间体化合物B-2 2,8-双(甲基-d3)-6-(4,4,5,5-四甲基-1,3,2-二氧杂硼杂环戊烷-2-基) 咪唑并[1,2-b]哒嗪的制备
采用以下合成路线
步骤1化合物6-氯-4-(甲基-d3)哒嗪-3-氨的合成
将4-溴-6-氯哒嗪-3-氨(2.07g，10mmol)溶于100ml无水THF中，氮气保护下，缓慢滴加氘代甲基碘化镁(27ml，27mmol)，加毕，依次加入氯化锌的2-甲基四氢呋喃溶液(2.5ml，5mmol)和四(三苯基膦)钯(116mg，0.1mmol),升温至50℃搅拌反应4-6小时，TLC监测反应完毕。将反应液冷却至室温，加入50ml饱和氯化铵淬灭反应，用乙酸乙酯萃取3-4次，合并有机相，浓缩后硅胶柱层析纯化得到1.32g产物，收率：43.6％。LC-MS(APCI):m/z＝144.2(M+1)+。
步骤2化合物6-氯-2,8-双(甲基-d3)咪唑并[1,2-b]哒嗪的合成
将6-氯-4-(甲基-d3)哒嗪-3-氨(194mg，1.32mmol)溶于10ml无水DMF中，加热至100℃，缓慢滴加1-溴丙烷-2-酮-1,1,3,3,3-d5(200mg，1.40mmol)的3ml DMF溶液，氮气保护下升温至110℃搅拌反应2-3小时，TLC监测反应完毕。降至室温后加入10ml饱和碳酸氢钠水溶液淬灭反应，用乙酸乙酯萃取3-4次，合并有机相，用饱和食盐水洗涤，无水硫酸钠干燥，浓缩，经硅胶柱分离得固体163mg，收率66.4％。LC-MS(APCI):m/z＝188.5(M+1)+。
步骤3中间体化合物B-3的合成
将6-氯-2,8-双(甲基-d3)咪唑并[1,2-b]哒嗪(90mg，0.5mmol)、联硼酸频哪醇酯(380mg，1.5mmol)、Pd(OAc)2(6mg，0.025mmol)、PCy3(14mg，0.05mmol)和乙酸钾(100mg，1.0mmol)加入到20ml微波管中，氮气保护下加入8ml乙酸异丙酯，密封后微波加热至80℃反应1小时，TLC监测反应完毕，降至室温后浓缩除去溶剂，硅胶柱层析纯化得到95mg固体，收率：69.6％。LC-MS(APCI):m/z＝280.2(M+1)+。
实施例1 7-(4,7-二氮杂螺[2.5]辛-7-基)-2-(2-甲基-8-(甲基-d3)咪唑并[1,2-b]哒嗪-6- 基)吡啶并[1,2-a]嘧啶-4-酮(化合物T-1)制备
采用以下合成路线
步骤1化合物7-(2-(2-甲基-8-(甲基-d3)咪唑并[1,2-b]哒嗪-6-基)-4-氧代吡啶并[1,2-a]嘧啶-7-基)-4,7-二氮杂螺[2.5]辛-4-甲酸叔丁酯的合成
将中间体化合物A-1(90mg，0.17mmol)和中间体化合物B-1(52mg，0.19mmol)加入到10ml乙腈和2ml水中，氮气保护下加入Pd(dppf)Cl2(12mg，0.017mmol)和碳酸钾(47mg，0.34mmol)，加热至80℃搅拌反应过夜，TLC监测反应完毕后，降至室温后浓缩除去溶剂，硅胶柱层析纯化得到69mg固体，收率：81.1％。LC-MS(APCI):m/z＝505.2(M+1)+。
步骤2化合物T-1的合成
将上步所得7-(2-(2-甲基-8-(甲基-d3)咪唑并[1,2-b]哒嗪-6-基)-4-氧代吡啶并[1,2-a]嘧啶-7-基)-4,7-二氮杂螺[2.5]辛-4-甲酸叔丁酯(69mg，0.14mmol)加入到5ml二氯甲烷中，加入TFA(0.2ml，2.8mmol)，室温下搅拌反应0.5-1小时，TLC监测反应完毕，加入10ml二氯甲烷稀释，依次用20ml饱和碳酸氢钠水溶液和饱和食盐水洗涤2-3次，有机相浓缩后硅胶柱层析纯化得到48mg类白色固体，收率：87.2％。LC-MS(APCI):m/z＝405.5(M+1)+。1H NMR(400MHz,CDCl3)δ8.45(d,J＝2.4Hz,1H),7.92(d,J＝1.0Hz,1H),7.73(d,J＝9.6Hz,1H),7.80(s,1H),7.70(dd,J＝9.7,2.5Hz,1H),7.38(s,1H),3.31-3.22(m,2H),3.20-3.16(m,2H),3.08(s,2H),2.55(s,3H),1.68(br s,1H),0.77-0.75(m,2H),0.67-0.64(m,2H).
实施例2 7-(4,7-二氮杂螺[2.5]辛-7-基)-2-(8-甲基-2-(甲基-d3)咪唑并[1,2-b]哒嗪-6- 基)吡啶并[1,2-a]嘧啶-4-酮(化合物T-2)制备
采用以下合成路线
步骤1化合物7-(2-(2-(甲基-d3)-8-甲基咪唑并[1,2-b]哒嗪-6-基)-4-氧代吡啶并[1,2-a]嘧啶-7-基)-4,7-二氮杂螺[2.5]辛-4-甲酸叔丁酯的合成
将中间体化合物A-1(90mg，0.17mmol)和中间体化合物B-2(52mg，0.19mmol)加入到10ml乙腈和2ml水中，氮气保护下加入Pd(dppf)Cl2(12mg，0.017mmol)和碳酸钾(47mg，0.34mmol)，加热至80℃搅拌反应过夜，TLC监测反应完毕后，降至室温后浓缩除去溶剂，硅胶柱层析纯化得到74mg固体，收率：87.0％。LC-MS(APCI):m/z＝505.2(M+1)+。
步骤2化合物T-2的合成
将上步所得7-(2-(2-(甲基-d3)-8-甲基咪唑并[1,2-b]哒嗪-6-基)-4-氧代吡啶并[1,2-a]嘧啶-7-基)-4,7-二氮杂螺[2.5]辛-4-甲酸叔丁酯(74mg，0.15mmol)加入到5ml二氯甲烷中，加入TFA(0.2ml，2.8mmol)，室温下搅拌反应0.5-1小时，TLC监测反应完毕，加入10ml二氯甲烷稀释，依次用20ml饱和碳酸氢钠水溶液和饱和食盐水洗涤2-3次，有机相浓缩后硅胶柱层析纯化得到51mg类白色固体，收率：92.6％。LC-MS(APCI):m/z＝405.5(M+1)+。1H NMR(400MHz,CDCl3)δ8.46(d,J＝2.4Hz,1H),7.94(d,J＝1.0Hz,1H),7.73(d,J＝9.6Hz,1H),7.81(s,1H),7.73(dd,J＝9.7,2.5Hz,1H),7.38(s,1H),3.31-3.22(m,2H),3.20-3.16(m,2H),3.09(s,2H),2.74(d,J＝0.9Hz,3H),1.69(br s,1H),0.77-0.75(m,2H),0.68-0.64(m,2H).
实施例3 7-(4,7-二氮杂螺[2.5]辛-7-基)-2-(2,8-双(甲基-d3)咪唑并[1,2-b]哒嗪-6-基)吡 啶并[1,2-a]嘧啶-4-酮(化合物T-3)制备
采用以下合成路线
步骤1化合物7-(2-(2,8-双(甲基-d3)咪唑并[1,2-b]哒嗪-6-基)-4-氧代吡啶并[1,2-a]嘧啶-7-基)-4,7-二氮杂螺[2.5]辛-4-甲酸叔丁酯的合成
将中间体化合物A-1(90mg，0.17mmol)和中间体化合物B-2(52mg，0.19mmol)加入到10ml乙腈和2ml水中，氮气保护下加入Pd(dppf)Cl2(12mg，0.017mmol)和碳酸钾(47mg，0.34mmol)，加热至80℃搅拌反应过夜，TLC监测反应完毕后，降至室温后浓缩除去溶剂，硅胶柱层析纯化得到58mg固体，收率：68.2％。LC-MS(APCI):m/z＝508.2(M+1)+。
步骤2化合物T-3的合成
将上步所得7-(2-(2,8-双(甲基-d3)咪唑并[1,2-b]哒嗪-6-基)-4-氧代吡啶并[1,2-a]嘧啶-7-基)-4,7-二氮杂螺[2.5]辛-4-甲酸叔丁酯(58mg，0.12mmol)加入到5ml二氯甲烷中，加入TFA(0.2ml，2.8mmol)，室温下搅拌反应0.5-1小时，TLC监测反应完毕，加入10ml二氯甲烷稀释，依次用20ml饱和碳酸氢钠水溶液和饱和食盐水洗涤2-3次，有机相浓缩后硅胶柱层析纯化得到35mg类白色固体，收率：63.5％。LC-MS(APCI):m/z＝408.5(M+1)+。1H NMR(400MHz,CDCl3):δ8.45(d,J＝2.4Hz,1H),7.93(d,J＝1.0Hz,1H),7.74(d,J＝9.6Hz,1H),7.81(s,1H),7.70(dd,J＝9.7,2.5Hz,1H),7.39(s,1H),3.30-3.22(m,2H),3.20-3.16(m,2H),3.08(s,2H),1.69(br s,1H),0.78-0.75(m,2H),0.68-0.62(m,2H).
生物活性测试。
(1)代谢稳定性评价
代谢稳定性一般用来描述化合物被代谢的速度和程度，是影响药代动力学性质的主要因素之一。很多化合物都是CYP450酶和其它的药物代谢酶的底物，而肝微粒体是富含CYP450的体系，本实验的目的是通过将本发明化合物与人和SD大鼠肝微粒体分别孵育并运用LC-MS/MS检测化合物的剩余比例来进行体代谢外稳定性的研究。
①溶液的配制
磷酸盐缓冲液(PBS)：取预先配好的KH2PO4(0.5M)溶液150mL和K2HPO4(0.5M)溶液700mL混合，再用K2HPO4(0.5M)溶液调节混合液pH值至7.4，作为5倍浓度PBS，存于4℃备用。使用前用超纯水稀释5倍，加入3.3mM氯化镁，得到磷酸盐缓冲液PBS(100mM)。
NADPH再生系统溶液：用5mL的PBS配制含有6.5mM NADP，16.5mM G-6-P，3U/mL G-6-P D的NADPH溶液。
内标终止液：用乙腈配制50ng/mL盐酸普萘洛尔和200ng/mL甲苯磺丁脲作为内标工作液。
人肝微粒体溶液：取0.31mL人肝微粒体(25mg/mL)加入0.961mL PBS(pH7.4)中混匀，得到蛋白浓度为0.625mg/mL的人肝微粒体稀释液。
SD大鼠肝微粒体溶液：取0.31mLSD大鼠肝微粒体(25mg/mL)加入0.961mL PBS(pH7.4)中混匀，得到蛋白浓度为0.625mg/mL的SD大鼠肝微粒体稀释液。
样品工作液：用DMSO配制本发明化合物和非氘代化合物粉末、阳性对照右美沙芬粉末和奥美拉唑粉末至10mM，作为样品储备液。再用70％乙腈-水稀释得到0.25mM样品工作液。
②样品孵育
取398μL的人肝微粒体稀释液加入96孔孵育板中(N＝2)，分别加入2μL 0.25mM的待测化合物、右美沙芬，混匀。
取398μL的SD大鼠肝微粒体稀释液加入96孔孵育板中(N＝2)，分别加入2μL0.25mM的待测化合物、奥美拉唑，混匀。
每孔加入300μL预冷的终止液至96孔深孔板中，置于冰上，作为终止板。
将96孔孵育板和NADPH再生系统置于37℃水浴箱中，100转/分钟震荡，预孵5min。从孵育板每孔取出80μL孵育液加入终止板，混匀，补充20μL NADPH再生系统溶液，作为0min样品。再向孵育板每孔加入80μL的NADPH再生系统溶液，启动反应，开始计时。待测化合物反应浓度为1μM，蛋白浓度为0.5mg/mL。
分别于反应10、30、90min时，各取100μL反应液，加入终止板中，涡旋3min终止反应。
将终止板于5000rpm，4℃条件下离心15min。取200μL上清液至预先加入200μL超纯水的96孔板中，混匀，采用LC-MS/MS进行样品分析，进样10uL。
③样品分析方法
本实验采用LC-MS/MS系统检测待测化合物、右美沙芬、奥美拉唑及内标的峰面积，计算化合物与内标峰面积比值。
④数据处理
样品以及内标的峰面积由质谱仪和Analyst软件获得，利用Graphpad prism7.0软件单指数式降解模型对化合物剩余量(R％)与时间作图可得底物消除速率常数K
Ct/C0＝exp(-K*t)
并根据以下公式计算半衰期T1/2和内在清除率CLint，其中V/M即等于1/C(蛋白)。
t1/2(min)；CLint(μL/min/mg)。
实验结果：对本发明化合物及其非氘代的化合物同时测验比较，评价其在人和SD大鼠肝微粒体的代谢稳定性。与非氘代的化合物相比，本发明化合物具有更长的半衰期T1/2和更低的清除率CLint，可以明显改善代谢稳定性。表性实施例化合物的结果归纳于下表1中。
表1
(2)大鼠药代动力学实验
6只雄性Sprague-Dawley大鼠，7-8周龄，体重约210g，分成2组，每组3只，经静脉或口服单个剂量的化合物(口服10mg/kg)，比较其药代动力学差异。
大鼠采用标准饲料饲养，给予水。试验前16小时开始禁食。药物用10mM抗坏血酸/0.01mg/mL硫代硫酸钠五水合物溶解。眼眶采血，采血的时间点为给药后0.083小时，0.25小时、0.5小时、1小时、2小时、4小时、6小时、8小时、12小时和24小时。
大鼠吸入乙醚后短暂麻醉，眼眶采集300μL血样于试管。试管内有30μL 1％肝素盐溶液。使用前，试管于60℃烘干过夜。在最后一个时间点血样采集完成之后，大鼠乙醚麻醉后处死。
血样采集后，立即温和地颠倒试管至少5次，保证混合充分后放置于冰上。血样在4℃ 5000rpm离心5分钟，将血浆与红细胞分离。用移液器吸出100μL血浆到干净的塑料离心管中，标明化合物的名称和时间点。血浆在进行分析前保存在-80℃。用LC-MS/MS测定血浆中本发明化合物的浓度。药代动力学参数基于每只动物在不同时间点的血药浓度进计算。
实验表明，本发明化合物在动物体内具有更好的药代动力学性质，因此具有更好的药效学和治疗效果。表性实施例化合物的结果归纳于下表2中。
表2
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。

Claims (11)

  1. 式(I)化合物，或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物：
    其中，
    R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15和R16各自独立地选自氢、氘、卤素或三氟甲基；
    X1和X2各自独立地选自CH3、CD3、CHD2或CH2D；
    附加条件是，上述化合物至少含有一个氘原子。
  2. 根据权利要求1所述的化合物，其中，R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15和R16各自独立地选自氢或氘。
  3. 根据权利要求1或2所述的化合物，其中，R1、R2、R3、R4、R5和R6是氢。
  4. 根据权利要求1-3中任一项所述的化合物，其中，R7、R8、R9、R10、R11、R12、R13、R14、R15和R16是氢。
  5. 根据权利要求1-4中任一项所述的化合物，其中，X1是CD3或CH3。
  6. 根据权利要求1-5中任一项所述的化合物，其中，X1是CD3。
  7. 根据权利要求1-6中任一项所述的化合物，其中，X2是CD3或CH3。
  8. 根据权利要求1-7中任一项所述的化合物，其中，X2是CD3。
  9. 根据权利要求1所述的化合物，或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物，其中，所述化合物选自：

  10. 一种药物组合物，其含有药学上可接受的赋形剂和权利要求1-9中任一项所述的化合物，或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物。
  11. 权利要求1-9中任一项所述的化合物，或其药学上可接受的盐、互变异构体、立体异构体、前药、晶型、水合物或溶剂化合物，或权利要求10所述的药物组合物在制备用于治疗和/或预防脊髓性肌萎缩(SMA)疾病的药物中的用途。
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CN106459092A (zh) * 2014-05-15 2017-02-22 豪夫迈·罗氏有限公司 用于治疗脊髓性肌萎缩的化合物
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