WO2019001383A1 - 用于抑制Bcl-2蛋白的N-苯磺酰基苯甲酰胺类化合物及其组合物及应用 - Google Patents

用于抑制Bcl-2蛋白的N-苯磺酰基苯甲酰胺类化合物及其组合物及应用 Download PDF

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WO2019001383A1
WO2019001383A1 PCT/CN2018/092588 CN2018092588W WO2019001383A1 WO 2019001383 A1 WO2019001383 A1 WO 2019001383A1 CN 2018092588 W CN2018092588 W CN 2018092588W WO 2019001383 A1 WO2019001383 A1 WO 2019001383A1
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compound
bcl
mmol
cancer
hydrogen
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PCT/CN2018/092588
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English (en)
French (fr)
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王义汉
刘志强
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深圳市塔吉瑞生物医药有限公司
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Priority to EP18824477.6A priority Critical patent/EP3626241A4/en
Priority to US16/625,025 priority patent/US11718611B2/en
Priority to JP2020520706A priority patent/JP7374496B2/ja
Publication of WO2019001383A1 publication Critical patent/WO2019001383A1/zh
Priority to JP2022031100A priority patent/JP2022071077A/ja

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed 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
    • A61K31/33—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
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/002—Heterocyclic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/05—Isotopically modified compounds, e.g. labelled

Definitions

  • the present invention relates to the field of medical technology, and in particular, the present invention relates to N-benzenesulfonylbenzamides which have an excellent inhibitory effect on Bcl-2 protein, pharmaceutical compositions containing the same, and processes for their preparation and use.
  • apoptosis programmed death
  • Apoptosis is highly conserved, and similar molecular mechanisms of apoptosis exist in organisms of different species.
  • the known apoptotic signaling pathways include both endogenous and exogenous pathways, the mitochondrial apoptotic pathway is an endogenous pathway, and the apoptotic pathway mediated by death receptors is an exogenous pathway.
  • mitochondrial functional changes such as decreased mitochondrial membrane potential, mitochondrial membrane permeability "cavity” formation or “channel” opening, leading to the release of cytochrome c, which is apoptosis
  • mitochondrial functional changes such as decreased mitochondrial membrane potential, mitochondrial membrane permeability "cavity" formation or “channel” opening, leading to the release of cytochrome c, which is apoptosis
  • the core features.
  • B-cell lymphoma 2family of proteins are the apex and core factor of the mitochondrial pathway. According to the functional and structural characteristics, the Bcl-2 family proteins are divided into three subfamilies: subfamily 1 has anti-apoptotic functions, including Bcl-2 (B-cell lymphoma 2), Mcl-1 (Myeloid cell leukemia 1) and Bcl-x L (B-cell lymphoma x long), etc.; subfamily 2 has pro-apoptotic effects, mainly Bax (Bcl-2 related protein X) and Bck (Bcl-2antagonist/killer); the other class only contains BH3 region (BH3-only) pro-apoptotic subfamily 3, including Bad (Bcl-2antagonist of cell death), Bim (Bcl-2 interacting mediator) and the like.
  • subfamily 1 has anti-apoptotic functions, including Bcl-2 (B-cell lymphoma 2), Mcl-1 (Myeloid cell leukemia 1) and Bcl-x L (B-
  • Bcl-2 gene and / or protein Therefore, there is a need to develop Bcl-2 protein inhibitors in the field of cancer therapy.
  • Venetoclax is a breakthrough anticancer drug developed by AbbVie and Roche. It was approved by the US Food and Drug Administration on April 11, 2016 and was acquired on December 5, 2016. The European Medicines Agency is approved for the treatment of patients with a 17p gene deletion mutation and who have received at least one treatment for chronic lymphocytic leukemia (CLL).
  • CLL chronic lymphocytic leukemia
  • Venetoclax is the first FDA approved Bcl-2 protein inhibitor (chemical name 4-(4- ⁇ [2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-ene-1- Methyl]methyl ⁇ piperazin-1-yl)-N-( ⁇ 3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl ⁇ sulfonyl)-2 -(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide, which has the following structural formula), is a new treatment for CLL patients with a poor prognosis and limited treatment options; select. However, adverse reactions such as neutropenia, diarrhea, nausea, anemia, upper respiratory tract infection, thrombocytopenia, and fatigue can also be found.
  • ADME ulcerative co-oxidative desorption, distribution, metabolism, and/or excretion
  • Many of the drugs currently on the market also limit their range of applications due to poor ADME properties.
  • the rapid metabolism of drugs can lead to the inability of many drugs that could be effectively treated to treat diseases because they are too quickly removed from the body.
  • Frequent or high-dose medications may solve the problem of rapid drug clearance, but this approach can lead to problems such as poor patient compliance, side effects caused by high-dose medications, and increased treatment costs.
  • rapidly metabolizing drugs may also expose patients to undesirable toxic or reactive metabolites.
  • the present invention discloses a novel N-benzenesulfonylbenzamide Bcl-2 protein inhibitor, a pharmaceutical composition and use thereof. More specifically, the present invention relates to certain anthracene substituted 4-(4- ⁇ [2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl ⁇ piperazin-1-yl)-N-( ⁇ 3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl ⁇ sulfonyl)-2-(1H- Pyrrolo[2,3-b]pyridin-5-yloxy)benzamide, these oxime substituted compounds have better Bcl-2 kinase inhibitory activity and/or have better pharmacodynamics/pharmacokinetics performance.
  • the technical solution adopted by the present invention is:
  • a Bcl-2 protein inhibitor characterized by having an N-benzenesulfonylbenzamide compound represented by formula (I), or a pharmaceutically acceptable salt, prodrug, crystal form thereof, stereoisomer thereof , hydrate or solvate:
  • 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 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 And R 34 , R 35 , R 36 , R 37 , R 38 , R 39 and R 40 are each independently selected from hydrogen, deuterium, halogen or trifluoromethyl;
  • X 1 and X 2 are independently selected from the group consisting of "hydrogen (H), hydrazine (D), methyl, CH 2 D, CHD 2 , CD 3 , CH 2 CH 3 , CHDCH 3 , CHDCH 2 D, CHDCHD 2 , CHDCD 3, CD 2 CH 3, CD 2 CH 2 D, CD 2 CHD 2, CD 2 CD 3 " from the group consisting of;
  • Additional conditions are: 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 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , At least one of R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , X 1 and X 2 is deuterated or deuterated.
  • the shape and volume of the ruthenium in the drug molecule are substantially the same as those of the hydrogen. If the hydrogen in the drug molecule is selectively replaced with hydrazine, the deuterated drug generally retains the original biological activity and selectivity. At the same time, the inventors have confirmed through experiments that the binding of carbon-germanium bonds is more stable than the combination of carbon-hydrogen bonds, which can directly affect the absorption, distribution, metabolism and excretion of some drugs, thereby improving the efficacy, safety and tolerability of the drugs.
  • the strontium isotope content of the cerium in the deuterated position is at least greater than the natural strontium isotope content (0.015%), preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, and even more preferably greater than 95. %, more preferably greater than 99%.
  • 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 and R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 And ⁇ isotope content in each of the deuterated positions of R 32 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , X 1 and X 2 is at least 5%, preferably greater than 10%, more preferably more than 15%, more preferably more than 20%, more preferably more than 25%, more preferably more than 30%, more preferably more than 35%, more preferably more than 40%, more preferably more than 45 More preferably, more
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are each independently selected from hydrazine or hydrogen.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are ⁇ .
  • R 9 and R 10 are each independently selected from hydrazine or hydrogen.
  • R 9 and R 10 are deuterium.
  • R 11 , R 12 and R 13 are each independently selected from hydrazine or hydrogen.
  • R 11 , R 12 and R 13 are deuterium.
  • R 14 , R 15 , R 16 , R 17 , R 18 , R 19 and R 20 are each independently selected from hydrazine or hydrogen.
  • R 14 , R 15 , R 16 , R 17 , R 18 , R 19 and R 20 are ⁇ .
  • R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 are each independently selected from hydrazine or hydrogen.
  • R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 and R 28 are ⁇ .
  • R 29 and R 30 are each independently selected from hydrazine or hydrogen.
  • R 29 and R 30 are ⁇ .
  • R 31 , R 32 , R 33 , R 34 , R 35 , R 36 are each independently selected from hydrazine or hydrogen.
  • R 31 , R 32 , R 33 , R 34 , R 35 , and R 36 are ⁇ .
  • R 37 , R 38 , R 39 and R 40 are each independently selected from hydrazine or hydrogen.
  • R 37 , R 38 , R 39 , and R 40 are deuterium.
  • R 37 , R 38 , R 39 and R 40 are each independently selected from hydrazine or hydrogen.
  • R 37 , R 38 , R 39 , and R 40 are deuterium.
  • X 1 and X 2 are each independently selected from an alkyl group selected from one or more deuterations.
  • X 1 and X 2 are CD 3 .
  • the compound is selected from the group consisting of the following compounds or a pharmaceutically acceptable salt thereof:
  • the compound does not include a non-deuterated compound.
  • the invention also discloses a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a Bcl-2 protein inhibitor as described above, or a crystalline form, a pharmaceutically acceptable salt, a hydrate or a solvate thereof
  • a pharmaceutical composition of a stereoisomer, prodrug or isotopic variation comprising a pharmaceutically acceptable carrier and a Bcl-2 protein inhibitor as described above, or a crystalline form, a pharmaceutically acceptable salt, a hydrate or a solvate thereof.
  • the pharmaceutically acceptable carrier comprises a substance or an additive encapsulated in a capsule, a glidant, a sweetener, a diluent, a preservative, a dye/colorant, a flavor enhancer, a surfactant At least one of a wetting agent, a dispersing agent, a disintegrating agent, a suspending agent, a stabilizer, an isotonic agent, a solvent or an emulsifier.
  • the pharmaceutical composition is a tablet, a pill, a capsule, a powder, a granule, an ointment, an emulsion, a suspension, a solution, a suppository, an injection, an inhalant, a gel, a microsphere or Aerosol.
  • Typical routes of administration of the pharmaceutical compositions of the invention include, but are not limited to, oral, rectal, transmucosal, enteral, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal , intramuscular, subcutaneous, intravenous administration. Oral administration or injection administration is preferred.
  • the pharmaceutical composition of the present invention can be produced by a method known in the art, such as a conventional mixing method, a dissolution method, a granulation method, a sugar-coating method, a pulverization method, an emulsification method, a freeze-drying method, and the like.
  • the present invention also provides a method of preparing a pharmaceutical composition comprising the steps of: administering a pharmaceutically acceptable carrier to a Bcl-2 protein inhibitor as described above, or a crystalline form thereof, a pharmaceutically acceptable salt, or a hydrate thereof Or a pharmaceutical composition of a solvate, stereoisomer, prodrug or isotopic variation.
  • the present invention further comprises other active compounds, which may be selected from the group consisting of: alkylating agents, angiogenesis inhibitors, antibodies, metabolic antagonists, anti-mitotic agents, anti-proliferative agents, antiviral agents, Aurora kinase inhibitors, other apoptosis-inducing agents (eg, Bcl-xL, Bcl-w and Bfl-1 inhibitors), death receptor pathway activators, Bcr-Abl kinase inhibitors, BiTE (Bi-specific T cells) Adapters) antibodies, antibody drug conjugates, biological response modifiers, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DADs, leukemia virus oncogene homologs (ErbB2 Receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormone therapy, immune, inhibitors of apopto
  • active compounds may be selected
  • the active ingredients of the invention may also be used in combination with other active ingredients.
  • the choice of such combination is based on the condition of the treatment, the cross-reactivity of the ingredients, and the combined pharmaceutical properties. It is also possible to administer any of the compounds of the invention in combination with one or more other active ingredients in a single dosage form for simultaneous or sequential administration to a patient.
  • Combination therapies can be administered simultaneously or sequentially. When administered continuously, the combination can be administered in two or more administrations.
  • Combination therapy can provide "synergistic effects" or “synergistic effects”, in other words, when the active ingredients are used together, the effect obtained is greater than the sum of the effects obtained by using the compounds separately.
  • the active ingredient (1) is co-formulated and administered or delivered simultaneously in a combined formulation; (2) administered as a separate formulation or administered in parallel; or (3) obtained by some other dosage regimen Synergy.
  • synergistic effects can be obtained when the compounds are administered or released sequentially, for example, as separate tablets, pills or capsules, or by separate injections of separate syringes.
  • the effective dose of each active ingredient is administered sequentially, i.e., continuously, while in combination therapy, the effective dose of two or more active ingredients is administered together.
  • the invention also discloses the use of the Bcl-2 protein inhibitor or the pharmaceutical composition thereof as described above, which can be used for treating bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytes.
  • halogen means F, Cl, Br, and I unless otherwise specified. More preferably, the halogen atom is selected from the group consisting of F, Cl and Br.
  • deuterated means that one or more hydrogens in the compound or group are replaced by deuterium; deuteration may be monosubstituted, disubstituted, polysubstituted or fully substituted.
  • deuteration may be monosubstituted, disubstituted, polysubstituted or fully substituted.
  • deuterated is used interchangeably with “one or more deuterated”.
  • non-deuterated compound means a compound containing a proportion of germanium atoms not higher than the natural helium isotope content (0.015%).
  • the invention also includes isotopically labeled compounds (also referred to as "isotopic variants"), equivalent to the original compounds disclosed herein.
  • isotopes which may be listed as compounds of the present invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine isotopes such as 2 H, 3 H, 13 C, 14 C, 15 N, 17 O, 18 O, respectively. , 31 P, 32 P, 35 S, 18 F and 36 Cl.
  • isotopically-labeled compounds of the present invention such as the radioisotopes of 3 H and 14 C, are also among them, useful in tissue distribution experiments of drugs and substrates. ⁇ , ie 3 H and carbon-14, ie 14 C, are easier to prepare and detect and are preferred in isotopes. In addition, heavier isotopic substitutions such as guanidine, or 2 H, are preferred in certain therapies due to their good metabolic stability, such as increased half-life or reduced dosage in vivo, and therefore may be preferred in certain circumstances. Isotopically labeled compounds can be prepared in a conventional manner by substituting a readily available isotopically labeled reagent with a non-isotopic reagent using the protocol of the examples.
  • the compound of the present invention having the formula (I) may exist in the form of an acid addition salt, a base addition salt or a zwitter ion. Salts of the compounds are prepared during or after isolation of the compound.
  • the acid addition salts of the compounds are those derived from the reaction of the compound with an acid.
  • the present invention includes acetates, adipates, alginates, bicarbonates, citrates, aspartates, benzoates, besylate, sulfuric acid of the compounds and prodrugs thereof.
  • Hydrogen salt butyrate, camphorate, camphorsulfonate, digluconate, formate, fumarate, glycerol phosphate, glutamate, hemisulfate, heptanoate, caproic acid Salt, hydrochloride, hydrobromide, hydroiodide, lactobionate, lactate, maleate, mesitylene sulfonate, methanesulfonate, naphthalene sulfonate, nicotinate, Oxalate, pamoate, pectate, persulfate, phosphate, picrate, propionate, succinate, tartrate, thiocyanate, trichloroacetate, Tosylate and undecanoate.
  • the base addition salts of the compounds are those derived from the reaction of a compound with a hydroxide, carbonate or bicarbonate of a cation of lithium, sodium, potassium, calcium and magnesium.
  • the compounds of the invention may include one or more asymmetric centers, and thus may exist in a variety of "stereoisomer" forms, for example, enantiomeric and/or diastereomeric forms.
  • the compounds of the invention may be in the form of individual enantiomers, diastereomers or geometric isomers (e.g., cis and trans isomers), or may be in the form of a mixture of stereoisomers, A racemic mixture and a mixture rich in one or more stereoisomers are included.
  • the isomers can 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 a chiral salt; or preferred isomers can be passed Prepared by asymmetric synthesis.
  • HPLC high pressure liquid chromatography
  • the compounds of the invention may also exist in the form of tautomers. Although only one type of non-localized resonant structure may be described, it is contemplated that all such forms fall within the scope of the present invention.
  • olefin-amine tautomers may be present, and all their possible tautomeric forms fall within the scope of the invention.
  • polymorph refers to a different arrangement of chemical drug molecules, generally expressed as the presence of a pharmaceutical material in a solid state.
  • a drug may exist in a plurality of crystalline forms, and different crystal forms of the same drug may have different dissolution and absorption in the body, thereby affecting the dissolution and release of the formulation.
  • prodrug refers to a compound that is converted in vivo to an active form having its medical effect by, for example, hydrolysis in blood.
  • a prodrug is any covalently bonded carrier which, when administered to a patient, releases the compound of the invention in vivo.
  • Prodrugs are typically prepared by modifying functional groups that cleave the prodrug in vivo to yield the parent compound.
  • Prodrugs include, for example, a compound of the invention wherein a hydroxy, amino or thiol group is bonded to any group which, when administered to a patient, can be cleaved to form a hydroxy, amino or thiol group.
  • prodrugs include, but are not limited to, covalent derivatives of the compounds of the invention formed by the hydroxyl, amino or thiol functional groups thereof with acetic acid, formic acid or benzoic acid.
  • acetic acid formic acid or benzoic acid.
  • an ester such as a methyl ester, an ethyl ester or the like can be used.
  • the ester itself may be active and/or may hydrolyze under conditions in humans.
  • Suitable pharmaceutically acceptable in vivo hydrolysable esters include those which readily decompose in the human body to release the parent acid or its salt.
  • solvate refers to a complex of a compound of the invention that is coordinated to a solvent molecule to form a specific ratio.
  • Hydrophilate means a complex formed by the coordination of a compound of the invention with water.
  • the beneficial effects of the present invention are: the compound of the present invention has excellent inhibition to Bcl-2 protein kinase; the technology of sputum is used to change the metabolism of the compound in the organism, so that the compound has better Pharmacokinetic parameter characteristics.
  • the dosage can be changed and a long-acting preparation can be formed to improve the applicability; the substitution of a hydrogen atom in the compound with hydrazine increases the drug concentration of the compound in the animal due to its strontium isotope effect, and improves the therapeutic effect of the drug; Substituting a hydrogen atom in a compound inhibits certain metabolites and increases the safety of the compound.
  • the present invention provides an N-benzenesulfonylbenzamide compound of the formula (I), or a pharmaceutically acceptable salt, prodrug, crystal form, stereoisomer, hydrate or solvate thereof:
  • 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 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 And R 34 , R 35 , R 36 , R 37 , R 38 , R 39 and R 40 are each independently selected from hydrogen, deuterium, halogen or trifluoromethyl;
  • X 1 and X 2 are independently selected from the group consisting of "hydrogen (H), hydrazine (D), methyl, CH 2 D, CHD 2 , CD 3 , CH 2 CH 3 , CHDCH 3 , CHDCH 2 D, CHDCHD 2 , CHDCD 3, CD 2 CH 3, CD 2 CH 2 D, CD 2 CHD 2, CD 2 CD 3 " from the group consisting of;
  • Additional conditions are: 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 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , At least one of R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , X 1 and X 2 is deuterated or deuterated.
  • the cerium isotope content of cerium in the deuterated position is at least 0.015%, preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, more preferably greater than the natural strontium isotope content.
  • the ground is greater than 95%, more preferably greater than 99%.
  • 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 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 and R 40 are each independently selected from hydrogen, deuterium, halogen or trifluoromethyl" including R 1 selected from hydrogen and deuterium.
  • R 2 is selected from hydrogen, deuterium, halogen or trifluoromethyl
  • R 3 is selected from hydrogen, deuterium, halogen or trifluoromethyl
  • R 40 is selected from hydrogen, deuterium , halogen or trifluoromethyl technical solutions.
  • R 1 is hydrogen, R 1 is ⁇ , R 1 is halogen (F, Cl, Br, I), R 1 is trifluoromethyl, R 2 is hydrogen, R 2 is ruthenium, and R 2 is Halogen (F, Cl, Br, I), R 2 is trifluoromethyl, R 3 is hydrogen, R 3 is deuterium, R 3 is halogen (F, Cl, Br, I), and R 3 is trifluoromethyl And so on, until R 40 is hydrogen, R 40 is ⁇ , R 40 is halogen (F, Cl, Br, I), and R 40 is a trifluoromethyl group.
  • X 1 , X 2 are independently selected from the group consisting of 'hydrogen (H), hydrazine (D), methyl, CH 2 D, CHD 2 , CD 3 , CH 2 CH 3 , CHDCH 3 , CHDCH 2 D, CHDCHD 2 , CHDCD 3 , CD 2 CH 3 , CD 2 CH 2 D, CD 2 CHD 2 , CD 2 CD 3 'group consisting of X 1 selected from hydrogen (H), ⁇ (D) , methyl, CH 2 D, CHD 2 , CD 3 , CH 2 CH 3 , CHDCH 3 , CHDCH 2 D, CHDCHD 2 , CHDCD 3 , CD 2 CH 3 , CD 2 CH 2 D, CD 2 CHD 2 , CD 2 CD 3 , X 2 is selected from the group consisting of hydrogen (H), hydrazine (D), methyl, CH 2 D, CHD 2 , CD 3 , CH 2 CH 3 , CHDCH 3 , CHDCH 2 D
  • X 1 is hydrogen, X 1 is ⁇ , X 1 is CH 2 D, X 1 is CHD 2 , X 1 is CD 3 , X 1 is CH 2 CH 3 , X 1 is CHDCH 3 , X 1 CHDCH 2 D, X 1 is CHDCHD 2 , X 1 is CHDCD 3 , X 1 is CD 2 CH 3 , X 1 is CD 2 CH 2 D, X 1 is CD 2 CHD 2 , and X 1 is CD 2 CD 3 .
  • X 2 is hydrogen, X 2 is ⁇ , X 2 is CH 2 D, X 2 is CHD 2 , X 2 is CD 3 , X 2 is CH 2 CH 3 , X 2 is CHDCH 3 , X 2 is CHDCH 2 D, X 2 is CHDCHD 2 , X 2 is CHDCD 3 , X 2 is CD 2 CH 3 , X 2 is CD 2 CH 2 D, X 2 is CD 2 CHD 2 , and X 2 is CD 2 CD 3 .
  • the invention relates to a compound of formula (I), or a pharmaceutically acceptable salt, prodrug, hydrate or solvent compound, crystal form, stereoisomer or isotopic variation thereof, wherein R 11 -R 20 and R 37 -R 40 are hydrogen, and X 1 and X 2 are independently selected from methyl, CH 2 D, CHD 2 , CD 3 , R 1 -R 10 , R 21 -R 30 , R 31 - R 36 are as defined above, with the proviso that the compound of formula (I) contains at least one deuterium atom.
  • the invention relates to a compound of formula (I), or a pharmaceutically acceptable salt, prodrug, hydrate or solvent compound, crystal form, stereoisomer or isotopic variation thereof, wherein R 11 -R 20 and R 37 -R 40 are hydrogen, and X 1 and X 2 are independently selected from the group consisting of methyl, CH 2 D, CHD 2 , CD 3 , R 1 -R 10 , R 21 -R 30 and R 31 - R 36 are independently selected from hydrogen or deuterium, with the proviso that the compound of formula (I) contains at least one deuterium atom.
  • the invention relates to a compound of formula (I), or a pharmaceutically acceptable salt, prodrug, hydrate or solvent compound, crystal form, stereoisomer or isotopic variation thereof, wherein R 11 -R 20 and R 37 -R 40 are hydrogen, X 1 and X 2 are independently selected from methyl or CD 3 , R 1 -R 8 and R 31 -R 36 are hydrogen, R 9 -R 10 And R 21 to R 30 are independently selected from hydrogen or deuterium, with the proviso that the compound of the formula (I) contains at least one deuterium atom.
  • the invention relates to a compound of formula (I), or a pharmaceutically acceptable salt, prodrug, hydrate or solvent compound, crystal form, stereoisomer or isotopic variation thereof, wherein R 11 -R 20 and R 37 -R 40 are hydrogen, X 1 and X 2 are independently selected from methyl, R 1 -R 8 and R 31 -R 36 are hydrogen, R 9 -R 10 and R 21 -R 30 is independently selected from hydrogen or deuterium, with the proviso that the compound of formula (I) contains at least one deuterium atom.
  • the invention relates to a compound of formula (I), or a pharmaceutically acceptable salt, prodrug, hydrate or solvent compound, crystal form, stereoisomer or isotopic variation thereof, wherein R 11 -R 20 and R 37 -R 40 are hydrogen, X 1 and X 2 are independently selected from methyl, R 1 -R 8 and R 31 -R 36 are hydrogen, and R 9 and R 10 are deuterium.
  • R 21 - R 30 are independently selected from hydrogen or hydrazine.
  • R 21 -R 28 is deuterium; in another specific embodiment, R 21 -R 28 are hydrogen; in another specific embodiment, R 29 -R 30 is deuterium; In a specific embodiment, R 29 -R 30 is hydrogen.
  • the invention relates to a compound of formula (I), or a pharmaceutically acceptable salt, prodrug, hydrate or solvent compound, crystal form, stereoisomer or isotopic variation thereof, wherein R 11 -R 20 and R 37 -R 40 are hydrogen, X 1 and X 2 are independently selected from methyl, R 1 -R 8 and R 31 -R 36 are hydrogen, and R 21 -R 28 are deuterium.
  • R 9 , R 10 , R 29 and R 30 are each independently selected from hydrogen or hydrazine. In another specific embodiment, R 9 and R 10 are deuterium; in another specific embodiment, R 29 and R 30 are hydrogen; in another specific embodiment, R 29 and R 30 are deuterium.
  • the invention relates to a compound of formula (I), or a pharmaceutically acceptable salt, prodrug, hydrate or solvent compound, crystal form, stereoisomer or isotopic variation thereof, wherein R 11 -R 20 and R 37 -R 40 are hydrogen, X 1 and X 2 are independently selected from methyl, R 1 -R 8 and R 31 -R 36 are hydrogen, and R 29 and R 30 are deuterium.
  • R 9 - R 10 and R 21 - R 28 are independently selected from hydrogen or hydrazine.
  • R 9 -R 10 is deuterium; in another specific embodiment, R 9 -R 10 hydrogen; in another specific embodiment, R 21 -R 28 is deuterium; In a particular embodiment, R 21 -R 28 are hydrogen.
  • the compound of formula (I) is selected from the group consisting of compounds of the formula:
  • the compounds of the 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 reaction for preparing the compound of the present invention can be carried out in a suitable solvent, and a solvent can be easily selected by those skilled in the art of organic synthesis. Suitable solvents can be substantially unreactive with the starting materials (reactants), intermediates or products at the temperature at which the reaction is carried out (for example, at temperatures ranging from solvent freezing temperatures to solvent boiling temperatures).
  • the given reaction can be carried out in one solvent or a mixture of more than one solvent.
  • the skilled person can select the solvent for the particular reaction step depending on the particular reaction step.
  • the preparation of the compounds of the invention may involve protection and removal protection of different chemical groups.
  • One skilled in the art can readily determine whether protection and removal of protection and the choice of appropriate protecting groups are desired.
  • the chemistry of the protecting group 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 reaction can be monitored according to any suitable method known in the art.
  • spectroscopic means such as nuclear magnetic resonance (NMR) spectroscopy (eg 1 H or 13 C), infrared (IR) spectroscopy, spectrophotometry (eg 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
  • MS mass spectrometry
  • HPLC high performance liquid chromatography
  • TLC thin layer chromatography
  • compositions, formulations and kits are provided.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of the invention (also referred to as "active ingredient") and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises an effective amount of the active component.
  • the pharmaceutical composition comprises a therapeutically effective amount of the active component.
  • the pharmaceutical composition comprises a prophylactically effective amount of the active component.
  • a pharmaceutically acceptable excipient for use in the present invention refers to a non-toxic carrier, adjuvant or vehicle which does not destroy the pharmacological activity of the compound formulated together.
  • 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, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin) ), buffer substances (such as phosphate), glycine, sorbic acid, potassium sorbate, a mixture of partial glycerides of saturated plant fatty acids, water, salt or electrolyte (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, Sodium chloride, zinc salt, silica gel, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based material, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, wax, polyethylene-polyoxypropylene-e
  • kits e.g., pharmaceutical packs.
  • Kits provided may include a compound of the invention, other therapeutic agents, and first and second containers (eg, vials, ampoules, bottles, syringes, and/or dispersible packages or other materials containing the compounds of the invention, other therapeutic agents) Suitable container).
  • first and second containers eg, vials, ampoules, bottles, syringes, and/or dispersible packages or other materials containing the compounds of the invention, other therapeutic agents
  • kits can also optionally include a third container containing a pharmaceutically acceptable excipient for diluting or suspending a compound of the invention and/or other therapeutic agent.
  • a compound of the invention provided in a first container and a second container is combined with other therapeutic agents to form a unit dosage form.
  • the pharmaceutical composition provided by the present invention can be administered by a variety of routes including, but not limited to, oral administration, parenteral administration, inhalation administration, topical administration, rectal administration, nasal administration, oral administration, vaginal administration.
  • parenteral administration as used herein includes subcutaneous administration, intradermal administration, intravenous administration, intramuscular administration, intra-articular administration, intra-arterial administration, intrasynovial administration, intrasternal administration. , intracerebroventricular administration, intralesional 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. .
  • the compound provided herein is administered to a subject at risk of developing the condition, typically based on a physician's recommendation and administered under the supervision of a physician, at the dosage level as described above.
  • Subjects at risk of developing a particular condition typically include subjects with a family history of the condition, or those genetically tested or screened to determine those subjects that are particularly susceptible to developing the condition.
  • long-term administration can also be administered chronically.
  • Long-term administration refers to administration of a compound or a pharmaceutical composition thereof for a long period of time, for example, 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or can be continuously administered indefinitely, For example, the rest of the subject.
  • chronic administration is intended to provide a constant level of the compound in the blood over a prolonged period of time, for example, within a therapeutic window.
  • a pharmaceutical composition of the present invention can be further delivered using various methods of administration.
  • a pharmaceutical composition can be administered by bolus injection, for example, to rapidly increase the concentration of a compound in the blood to an effective level.
  • the bolus dose will depend on the target systemic level of the active ingredient, for example, an intramuscular or subcutaneous bolus dose will allow the active component to be slowly released, while a bolus delivered directly to the vein (eg, via IV IV drip) can be more Delivered rapidly so that the concentration of the active ingredient in the blood is rapidly increased to an effective level.
  • the pharmaceutical composition can be administered in a continuous infusion form, for example, by IV intravenous drip to provide a steady state concentration of the active ingredient in the subject's body.
  • a bolus dose of the pharmaceutical composition can be administered first, followed by continued infusion.
  • Oral compositions can be in the form of a bulk liquid solution or suspension or bulk powder. More generally, however, the composition is provided in unit dosage form for ease of precise dosing.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human patients and other mammals, each unit containing a predetermined quantity of active ingredient suitable to produce the desired therapeutic effect with a suitable pharmaceutical excipient.
  • Typical unit dosage forms include prefilled, pre-measured ampoules or syringes of the liquid compositions, or pills, tablets, capsules and the like in the case of solid compositions.
  • the compound will generally be a minor component (about 0.1 to about 50% by weight, or preferably about 1 to about 40% by weight), with the remainder being useful for forming the desired form of administration.
  • a carrier or excipient and a processing aid is provided in unit dosage form for ease of precise dosing.
  • a representative 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, each preferably 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 from about 0.01 to about 20% by weight, preferably from 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.
  • the injection dose level ranges from about 1 mg/kg/hr to at least 10 mg/kg/hr from about 1 to about 120 hours, especially 24 to 96 hours.
  • a preload bolus of about 0.1 mg/kg to about 10 mg/kg or more can also be administered.
  • the maximum total dose cannot exceed about 2 g/day.
  • Liquid forms suitable for oral administration may include suitable aqueous or nonaqueous vehicles as well as buffers, suspending and dispersing agents, coloring agents, flavoring agents, and the like.
  • the solid form may include, for example, any of the following components, or a compound having similar properties: a binder, for example, microcrystalline cellulose, tragacanth or gelatin; an excipient such as starch or lactose, a disintegrant, For example, alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silica; a sweetener such as sucrose or saccharin; or a flavoring agent such as mint, water Methyl salicylate or orange flavoring.
  • a binder for example, microcrystalline cellulose, tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrant, For example, alginic acid, Primogel or corn star
  • Injectable compositions are typically based on injectable sterile saline or phosphate buffered saline, or other injectable excipients known in the art.
  • the active compound will typically be a minor component, often from 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 ingredient.
  • the active component When formulated as an ointment, the active component is typically combined with a paraffin or water miscible ointment base.
  • the active ingredient can be formulated as a cream with, for example, an oil-in-water cream base.
  • Such transdermal formulations are well known in the art and generally include other ingredients for enhancing stable skin penetration of the active ingredient or formulation. All such known transdermal formulations and components are included within the scope of the invention.
  • transdermal administration can be accomplished using a reservoir or a porous membrane type, or a patch of a plurality of solid matrices.
  • compositions for oral administration, injection or topical administration are merely representative.
  • Other materials and processing techniques, etc. are set forth in Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pennsylvania, part 8 of which is incorporated herein by reference.
  • the compounds of the invention may also be administered in sustained release form or from a sustained release delivery system.
  • sustained release materials can be found in Remington's Pharmaceutical Sciences.
  • the invention further relates to pharmaceutically acceptable formulations of the compounds of the invention.
  • the formulation comprises water.
  • the formulation comprises a cyclodextrin derivative.
  • the most common cyclodextrins are alpha-, beta- and gamma-cyclodextrins consisting of 6, 7 and 8 alpha-1,4-linked glucose units, respectively, optionally including one on the attached sugar moiety. Or a plurality of substituents including, but not limited to, methylated, hydroxyalkylated, acylated, and sulfoalkyl ether substituted.
  • the cyclodextrin is a sulfoalkyl ether beta-cyclodextrin, eg, sulfobutylether beta-cyclodextrin, also known as Captisol. See, for example, U.S. 5,376,645.
  • the formulation comprises hexapropyl- ⁇ -cyclodextrin (eg, 10-50% in water).
  • each reaction is usually carried out in an inert solvent at room temperature to reflux temperature (e.g., 0 ° C to 100 ° C, preferably 0 ° C to 80 ° C).
  • the reaction time is usually from 0.1 to 60 hours, preferably from 0.5 to 24 hours.
  • LiAlD 4 (0.2 g, 5.02 mmol) was slowly added dropwise to a solution of compound 7 (0.7 g, 2.51 mmol of tetrahydrofuran (20 ml) at 0 ° C, and the reaction was continued for 1 h after the addition was completed. 1 M hydrochloric acid (10 ml) was added. The reaction was quenched, extracted with dichloromethane (40 mL ⁇ 3), and then evaporated.
  • Bcl-2 (Cisbio 63ADK000CB01PEG); Bcl-xL (Cisbio 63ADK000CB04PEG);
  • test compound (10 mM stock solution) was diluted 500 times with DMSO, and diluted as a first concentration with a 3-fold gradient, a total of 10 concentration gradients, and the 11th concentration was DMSO control. .
  • the inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex in which a small molecule inhibits the binding of one protein to another protein or peptide. Therefore, a large Ki value indicates low binding affinity, and a small Ki value indicates high binding affinity.
  • the compounds of the invention and Venetoclax were tested by the above experimental procedure. Compared with non-deuterated Venetoclax, the compounds of the present invention have better binding affinity to anti-apoptotic Bcl-2 protein, and have poor binding affinity to Bcl-xL protein, showing high Bcl-2 protein. Selectivity.
  • Table 1 The specific experimental results are shown in Table 1 below.
  • ALL cell line RS4;11 was used as a primary human cell line to evaluate the in vitro cell viability of Bcl-2 selectors and their efficacy in vivo.
  • RS4; 11 was cultured in RPMI-1640 supplemented with 2 mM L-glutamine, 10% FBS, 1 mM sodium pyruvate, 2 mM HEPES, 1% penicillin/streptomycin, 4.5 g/L glucose, and contained 5% carbon dioxide The environment is maintained at 37 ° C.
  • cells were treated in a 96-well microtiter plate in a 96-well microtiter plate for 48 hours in the presence of 10% human serum in a 96-well microtiter plate. Cytotoxic EC 50 values were evaluated according to the manufacturer's recommendations using CellTier Glo (Promega). The EC 50 value is determined after the processing of viable cells as a percentage of the untreated control cells.
  • mice liver microsomes 0.5 mg/mL, Xenotech; coenzyme (NADPH/NADH): 1 mM, Sigma Life Science; magnesium chloride: 5 mM, 100 mM phosphate buffer (pH 7.4).
  • phosphate buffer 100 mM, pH 7.4.
  • the pH was adjusted to 7.4, diluted 5 times with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer (100 mM) containing 100 mM potassium phosphate, 3.3 mM magnesium chloride, and a pH of 7.4.
  • NADPH regeneration system containing 6.5 mM NADP, 16.5 mM G-6-P, 3 U/mL G-6-P D, 3.3 mM magnesium chloride was prepared and placed on wet ice before use.
  • Formulation stop solution acetonitrile solution containing 50 ng/mL propranolol hydrochloride and 200 ng/mL tolbutamide (internal standard). 25057.5 ⁇ L of phosphate buffer (pH 7.4) was taken into a 50 mL centrifuge tube, and 812.5 ⁇ L of mouse liver microsomes were added and mixed to obtain a liver microsome dilution having a protein concentration of 0.625 mg/mL.
  • the corresponding compound had a reaction concentration of 1 ⁇ M and a protein concentration of 0.5 mg/mL.
  • 100 ⁇ L of the reaction solution was taken at 10, 30, and 90 min, respectively, and added to the stopper, and the reaction was terminated by vortexing for 3 min.
  • the plate was centrifuged at 5000 x g for 10 min at 4 °C.
  • 100 ⁇ L of the supernatant was taken into a 96-well plate to which 100 ⁇ L of distilled water was previously added, mixed, and sample analysis was performed by LC-MS/MS.
  • the metabolic stability of human and rat liver microsomes was evaluated by simultaneously testing the compounds of the present invention and their compounds without deuteration.
  • the half-life and liver intrinsic clearance as indicators of metabolic stability are shown in Table 1.
  • the undeuterated compound Venetoclaxr was used as a control sample in Table 1.
  • the compound of the present invention can significantly improve metabolic stability by comparison with the undeuterated compound Venetoclax, and is thus more suitable as a hepatitis C virus inhibitor.
  • SD rat grade SPF grade
  • Weight range 180 ⁇ 220g (actual weight range is 187 ⁇ 197g)
  • the blood samples were centrifuged at 4000 rpm, 10 min, 4 ° C for 30 min, and all plasma was collected and stored at -20 ° C immediately. Plasma concentrations in plasma at each time point were determined after sample collection at all time points.
  • Winnonin software was used to calculate the drug of male SD rats administered with Venetoclax and representative compound (3 mg/kg) according to the non-compartmental statistical moment theory. Generational dynamics related parameters.

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Abstract

一种式(I)所示的N-苯磺酰基苯甲酰胺类化合物、或其晶型、前药、药学上可接受的盐、立体异构体、溶剂合物或水合物，含有其的药物组合物，及其作为Bcl-2蛋白抑制剂用于制备治疗白血病或癌症的药物的用途。

Description

用于抑制Bcl-2蛋白的N-苯磺酰基苯甲酰胺类化合物及其组合物及应用 技术领域
本发明属于医药技术领域，具体地，本发明涉及对Bcl-2蛋白具有优异的抑制作用的N-苯磺酰基苯甲酰胺类化合物，包含它们的药物组合物，以及它们的制备方法和用途。
背景技术
针对肿瘤特异性靶点设计、合成全新的抗肿瘤药物已经成为靶向抗肿瘤治疗的重点。近年来的研究表明，细胞凋亡(程序化死亡)机制参与了肿瘤发生、发展与消退。细胞凋亡具有高度保守的特点，相似的细胞凋亡分子机制存在于不同种属的生物体内。已知的细胞凋亡信号通路包括内源和外源两条通路，线粒体凋亡通路是内源通路，死亡受体所介导的凋亡通路属外源通路。在内源通路有细胞凋亡中，线粒体的功能变化如线粒体膜电位的降低、线粒体膜通透性“空洞”的形成或“通道”的开放，导致细胞色素C的释放，这是细胞凋亡的核心特征。
Bcl-2家族蛋白(B-cell lymphoma 2family of proteins)是线粒体通路的顶点和核心因子。根据功能和结构特征将Bcl-2家族蛋白分为3个亚族：亚族1具有抗凋亡功能，主要包括Bcl-2(B-cell lymphoma 2)、Mcl-1(Myeloid cell leukemia 1)和Bcl-x L(B-cell lymphoma x long)等；亚族2具有促凋亡作用，主要是Bax(Bcl-2related protein X)和Bck(Bcl-2antagonist/killer)；另外一类仅含有BH3区域(BH3-only)的促凋亡亚族3，包括Bad(Bcl-2antagonist of cell death)、Bim(Bcl-2interacting mediator)等。研究发现30％-60％的***癌、70％的乳腺癌、90％的结直肠癌、100％小细胞癌、以及淋巴细胞性、粒细胞性白血病细胞等高表达这些具有抗凋亡作用的Bcl-2基因和/或蛋白。因此，在癌症治疗领域需要研发Bcl-2蛋白抑制剂。
Bcl-2蛋白抑制剂类抗癌先导化合物的研发已经历时10余年，发表的小分子达到几十个。但是，目前国际市场只有一个获批的Bcl-2类蛋白抑制剂——Venetoclax(维奈妥拉)。Venetoclax由艾伯维(AbbVie)和罗氏(Roche)合作开发的一款突破性抗癌药，于2016年4月11日获美国食品药品监督管理局批准上市，又于2016年12月5日获得欧洲药物管理局批准上市，用于治疗带有17p基因缺失突变并且曾接收过至少一种治疗慢性淋细胞白血病(chronic lymphocytic leukemia，CLL)患者。
Venetoclax是FDA批准的首个Bcl-2蛋白抑制剂(化学名称为4-(4-{[2-(4-氯苯基)-4,4-二甲基环 己-1-烯-1-基]甲基}哌嗪-1-基)-N-({3-硝基-4-[(四氢-2H-吡喃-4-基甲基)氨基]苯基}磺酰基)-2-(1H-吡咯并[2,3-b]吡啶-5-基氧基)苯甲酰胺，其具有以下结构式)，是预后极差且治疗选择十分有限的17p基因缺失的CLL患者治疗的新选择。但是，也会发现中性粒细胞减少、腹泻、恶心、贫血、上呼吸道感染、血小板减少和疲乏等不良反应。
Figure PCTCN2018092588-appb-000001
已知较差的吸收、分布、代谢和/或***(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。
因此，本领域仍需要开发具有高特异性或更好药效学/药代动力学性能的Bcl-2小分子抑制剂，能选择性地抑制Bcl-2蛋白，从而恢复细胞凋亡过程，达到治疗癌症的效果。
发明内容
针对以上技术问题，本发明公开了一种新型N-苯磺酰基苯甲酰胺类Bcl-2蛋白抑制剂、药物组合物及其应用。更具体而言，本发明涉及某些氘取代的4-(4-{[2-(4-氯苯基)-4,4-二甲基环己-1-烯-1-基]甲基}哌嗪-1-基)-N-({3-硝基-4-[(四氢-2H-吡喃-4-基甲基)氨基]苯基}磺酰基)-2-(1H-吡咯并[2,3-b]吡啶-5-基氧基)苯甲酰胺，这些氘取代的化合物具有更好的Bcl-2激酶抑制活性和/或具有更好药效学/药代动力学性能。对此，本发明采用的技术方案为：
一种Bcl-2蛋白抑制剂，其特征在于：如式(I)所示的N-苯磺酰基苯甲酰胺化合物、或其药学上可接受的盐、前药、晶型、立体异构体、水合物或溶剂合物：
Figure PCTCN2018092588-appb-000002
其中，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、R 16、R 17、R 18、R 19、R 20、R 21、R 22、R 23、R 24、R 25、R 26、R 27、R 28、R 29、R 30、R 31、R 32、R 33、R 34、R 35、R 36、R 37、R 38、R 39和R 40相互独立地选自氢、氘、卤素或三氟甲基；
X 1、X 2相互独立地选自由“氢(H)、氘(D)、甲基、CH 2D、CHD 2、CD 3、CH 2CH 3、CHDCH 3、CHDCH 2D、CHDCHD 2、CHDCD 3、CD 2CH 3、CD 2CH 2D、CD 2CHD 2、CD 2CD 3”组成的组；
附加条件是，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、R 16、R 17、R 18、R 19、R 20、R 21、R 22、R 23、R 24、R 25、R 26、R 27、R 28、R 29、R 30、R 31、R 32、R 33、R 34、R 35、R 36、R 37、R 38、R 39、R 40、X 1和X 2中至少一个是氘代的或氘。
采用此技术方案，氘在药物分子中的形状和体积与氢基本上相同，如果药物分子中氢被选择性替换为氘，氘代药物一般还会保留原来的生物活性和选择性。同时发明人经过实验证实，碳氘键的结合比碳氢键的结合更稳定，可直接影响一些药物的吸收、分布、代谢和***等属性，从而提高药物的疗效、安全性和耐受性。
优选的，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75％，更佳地大于95％，更佳地大于99％。
具体地说，在本发明中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、R 16、R 17、R 18、R 19、R 20、R 21、R 22、R 23、R 24、R 25、R 26、R 27、R 28、R 29、R 30、R 31、R 32、R 33、R 34、R 35、R 36、R 37、R 38、R 39、R 40、X 1和X 2各氘代位置中氘同位素含量至少是5％，较佳地大于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％。
优选的，式(I)中化合物的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、R 16、R 17、R 18、R 19、R 20、R 21、R 22、R 23、R 24、R 25、R 26、R 27、R 28、R 29、R 30、R 31、R 32、R 33、R 34、R 35、R 36、R 37、R 38、R 39、R 40、X 1和X 2中，至少其中一个R/X含氘，更佳地两个R/X含氘，更佳地三个R/X含氘，更佳地四个R/X含氘，更佳地五个R/X含氘，更佳地六个R/X含氘，更佳地七个R/X含氘，更佳地八个R/X含氘，更佳地九个R/X含氘，更佳地十个R/X含氘，更佳地十一个R/X含氘，更佳地十二个R/X含氘，更佳地十三个R/X含氘，更佳地十四个R/X含氘，更佳地十五个R/X含氘，更佳地十六个R/X含氘，更佳地十七个R/X含氘，更佳地十八个R/X含氘，更佳地十九个R/X含氘，更佳地二十个R/X含氘，更佳地二十一个R/X含氘，更佳地二十二个R/X含氘，更佳地二十三个R/X含氘，更佳地二十四个R/X含氘，更佳地二十五个R/X含氘，更佳地二十六个R/X含氘，更佳地二十七个R/X含氘，更佳地二十八个R/X含氘，更佳地二十九个R/X含氘，更佳地三十个R/X含氘，更佳地三十一个R/X含氘，更佳地三十二个R/X含氘，更佳地三十三个R/X含氘，更佳地三十四个R/X含氘，更佳地三十五个R/X含氘，更佳地三十六个R/X含氘，更佳地三十七个R/X含氘，更佳地三十八个R/X含氘，更佳地三十九个R/X含氘，更佳地四十个R/X含氘，更佳地四十一个R/X含氘，更佳地四十二个R/X含氘。
作为本发明的进一步改进，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8各自独立地选自氘或氢。
在另一优选例中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8是氘。
作为本发明的进一步改进，R 9、R 10各自独立地选自氘或氢。
在另一优选例中，R 9、R 10是氘。
作为本发明的进一步改进，R 11、R 12、R 13各自独立地选自氘或氢。
在另一优选例中，R 11、R 12、R 13是氘。
作为本发明的进一步改进，R 14、R 15、R 16、R 17、R 18、R 19、R 20各自独立地选自氘或氢。
在另一优选例中，R 14、R 15、R 16、R 17、R 18、R 19、R 20是氘。
作为本发明的进一步改进，R 21、R 22、R 23、R 24、R 25、R 26、R 27、R 28各自独立地选自氘或氢。
在另一优选例中，R 21、R 22、R 23、R 24、R 25、R 26、R 27、R 28是氘。
作为本发明的进一步改进，R 29、R 30各自独立地选自氘或氢。
在另一优选例中，R 29、R 30是氘。
作为本发明的进一步改进，R 31、R 32、R 33、R 34、R 35、R 36各自独立地选自氘或氢。
在另一优选例中，R 31、R 32、R 33、R 34、R 35、R 36是氘。
作为本发明的进一步改进，R 37、R 38、R 39、R 40各自独立地选自氘或氢。
在另一优选例中，R 37、R 38、R 39、R 40是氘。
作为本发明的进一步改进，R 37、R 38、R 39、R 40各自独立地选自氘或氢。
在另一优选例中，R 37、R 38、R 39、R 40是氘。
作为本发明的进一步改进，X 1、X 2各自独立地选自选自一次或多次氘代的烷基。
在另一优选例中，X 1、X 2是CD 3。
作为本发明的进一步改进，所述化合物选自下述化合物或其药学上可接受的盐：
Figure PCTCN2018092588-appb-000003
Figure PCTCN2018092588-appb-000004
在另一优选例中，所述化合物不包括非氘代化合物。
本发明还公开了一种药物组合物，其含有药学上可接受的载体和如上述所述的Bcl-2蛋白抑制 剂，或其晶型、药学上可接受的盐、水合物或溶剂合物、立体异构体、前药或同位素变体的药物组合物。
作为本发明的进一步改进，所述药学上可接受的载体包括封入胶囊的物质或添加剂、助流剂、增甜剂、稀释剂、防腐剂、染料/着色剂、矫味增强剂、表面活性剂、润湿剂、分散剂、崩解剂、助悬剂、稳定剂、等渗剂、溶剂或乳化剂中的至少一种。
作为本发明的进一步改进，所述药物组合物为片剂、丸剂、胶囊剂、粉剂、颗粒剂、膏剂、乳剂、悬浮剂、溶液剂、栓剂、注射剂、吸入剂、凝胶剂、微球或气溶胶。
给予本发明药物组合物的典型途径包括但不限于口服、直肠、透黏膜、经肠给药，或者局部、经皮、吸入、肠胃外、舌下、***内、鼻内、眼内、腹膜内、肌内、皮下、静脉内给药。优选口服给药或注射给药。
本发明的药物组合物可以采用本领域周知的方法制造，如常规的混合法、溶解法、制粒法、制糖衣药丸法、磨细法、乳化法、冷冻干燥法等。
本发明还提供了一种制备药物组合物的方法，包括步骤：将药学上可接受的载体与如上所述的Bcl-2蛋白抑制剂，或其晶型、药学上可接受的盐、水合物或溶剂合物、立体异构体、前药或同位素变体的药物组合物。
作为本发明的进一步改进，其还包含其他活性化合物，所述活性化合物可选自：烷基化剂、血管生成抑制剂、抗体、代谢拮抗剂、抗有丝***剂、抗增殖剂、抗病毒剂、极光激酶抑制剂、其他细胞程序死亡促进剂(例如，Bcl-xL，Bcl-w和Bfl-1抑制剂)、死亡受体途径活化剂、Bcr-Abl激酶抑制剂、BiTE(Bi特异性T细胞接合器)抗体、抗体药物共轭物、生物反应改性剂、依赖细胞周期蛋白的激酶抑制剂、细胞周期抑制剂、环加氧酶-2抑制剂、DADs、白血病病毒癌基因同系物(ErbB2)受体抑制剂、生长因子抑制剂、热休克蛋白(HSP)-90抑制剂、组蛋白脱乙酰基酶(HDAC)抑制剂、激素疗法、免疫、细胞程序死亡蛋白的抑制剂(IAPs)、***抗生素、激酶抑制剂、驱动蛋白抑制剂、Jak2抑制剂、雷帕霉素抑制剂的温血动物靶向、微小RNA、***素-活化的细胞外信号调节激酶抑制剂、多价结合蛋白、NSAIDs、聚ADP(腺苷二磷酸)-核糖聚合酶(PARP)抑制剂、铂化学治疗、polo样激酶(Plk)抑制剂、磷酸肌醇-3激酶(PI3K)抑制剂、蛋白体抑制剂、嘌呤类似物、嘧啶类似物、受体酪氨酸激酶抑制剂、酒石酸麦角胺生物碱(etinoids)/三棱(deltoids)植物生物碱、小的抑制性核糖核酸(siRNAs)、局部异构酶抑制剂、泛素连接酶抑制剂、化疗剂等。
本发明的活性成分也可与其他活性成分联合使用。这种组合的选择基于治疗的情况、成份的交叉反应性和联合的药学性质。还可能使本发明的任意化合物联合一种或多种其他活性成分，以单 一剂型同时或连续对患者给药。联合治疗可以同时或连续给药方案给药。当连续给药时，联合可以两次或更多次给药施用。联合治疗可提供“增效作用”或“协同作用”，换言之，当活性成分一起使用获得的效果大于分开使用化合物所得效果之和。当活性成分：(1)被共同配制和给药或以组合制剂形式同时递送；(2)作为独立的制剂交替给药或平行给药；或(3)通过一些其他给药方案时，可获得协同作用。当以交替治疗递送时，当化合物序贯给药或释放，例如以独立的片剂、丸剂或胶囊剂，或通过单独注射器的不同注射，可获得协同作用。通常，在交替治疗期间，每种活性成分有效剂量被序贯，即连续地给予，而在联合治疗中，两种或更多种活性成分的有效剂量共同给予。
本发明还公开了一种如上所述的所述的Bcl-2蛋白抑制剂或其药物组合物的用途，即可用于治疗膀胱癌、脑癌、乳腺癌、骨髓癌、***、慢性淋巴细胞性白血病、结肠直肠癌、食道癌、肝细胞癌、原始淋巴细胞性白血病、滤泡性淋巴瘤、T细胞或B细胞源的淋巴恶性肿瘤、黑色瘤、粒细胞性白血病、骨髓瘤、口腔瘤、卵巢癌、非小细胞肺癌、***癌、小细胞癌或脾癌。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
本文中，如无特别说明，“卤素”指F、Cl、Br、和I。更佳地，卤原子选自F、Cl和Br。
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。
本发明还包括同位素标记的化合物(也称为“同位素变体”)，等同于原始化合物在此公开。可以列为本发明的化合物同位素的例子包括氢，碳，氮，氧，磷，硫，氟和氯同位素，分别如 2H， 3H， 13C， 14C， 15N， 17O， 18O， 31P， 32P， 35S， 18F以及 36Cl。其中含有上述同位素或其他同位素原子的本发明的式(I)的化合物或其多晶型、药学上可接受的盐、前药、立体异构体、同位素变体、水合物或溶剂合物都在本发明的范围之内。本发明中某些同位素标记化合物，例如 3H和 14C的放射性同位素也在其中，在药物和底物的组织分布实验中是有用的。氚，即 3H和碳-14，即 14C，它们的制备和检测比较容易，是同位素中的首选。此外，较重同位素取代如氘，即 2H，由于其很好的代谢稳定性在某些疗法中有优势，例如在体内增加半衰期或减少用量，因此，在某些情况下可以优先考虑。同位素标记的化合物可以用一般的方法，通过用易得的同位素标记试剂替换为非同位素的试剂，用示例中的方案可以制备。
本发明具有式(I)的化合物可以以酸加成盐、碱加成盐或两性离子形态存在。化合物的盐是在化 合物的分离期间或纯化之后制备的。化合物的酸加成盐是衍生自化合物与酸反应的那些盐。例如，本发明包括化合物和其前体药物的乙酸盐、己二酸盐、藻酸盐、碳酸氢盐、柠檬酸盐、天冬氨酸盐、苯甲酸盐、苯磺酸盐、硫酸氢盐、丁酸盐、樟脑酸盐、樟脑磺酸盐、二葡糖酸盐、甲酸盐、富马酸盐、甘油磷酸盐、谷氨酸盐、半硫酸盐、庚酸盐、己酸盐、盐酸盐、氢溴酸盐、氢碘酸盐、乳糖酸盐、乳酸盐、马来酸盐、均三甲苯磺酸盐、甲磺酸盐、萘磺酸盐、烟酸盐、草酸盐、双羟萘酸盐、果胶酯酸盐、过硫酸盐、磷酸盐、苦味酸盐、丙酸盐、琥珀酸盐、酒石酸盐、硫氰酸盐、三氯乙酸盐、对甲苯磺酸盐和十一烷酸盐。化合物的碱加成盐是衍生自化合物与锂、钠、钾、钙和镁等的阳离子的氢氧化物、碳酸盐或碳酸氢盐反应的那些盐。
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。
在某些情况中，本发明化合物也可能以互变异构体的形式存在。尽管仅有一种非定域共振结构可能被描述，但设想所有的这类形式都落在本发明的保护范围内。例如，对于嘌呤、嘧啶、咪唑、胍、脒和四唑***而言，烯-胺互变异构体可能存在，并且所有它们可能的互变异构形式都落在本发明保护范围内。
术语“多晶型”是指化学药物分子的不同排列方式，一般表现为药物原料在固体状态下的存在形式。一种药物可以多种晶型物质状态存在，同一种药物的不同晶型，在体内的溶解和吸收可能不同，从而会对制剂的溶出和释放产生影响。
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。前药为任何共价键合的载体，当将这种前药给予患者时，其在体内释放本发明化合物。通常通过修饰官能团来制备前药，该修饰使得前药在体内裂解产生母体化合物。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，本发明化合物通过其中的羟基、氨基或巯基官能团与乙酸、甲酸或苯甲酸形成的共价衍生物。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包括容易在人体中分解而释放母体酸或其盐的那些。
术语“溶剂合物”指本发明化合物与溶剂分子配位形成特定比例的配合物。“水合物”是指本发明化合物与水进行配位形成的配合物。
与现有技术相比，本发明的有益效果为：本发明的化合物对Bcl-2蛋白激酶具有优异的抑制性；通过氘化这一技术改变化合物在生物体中的代谢，使化合物具有更好的药代动力学参数特性。在这种情况下，可以改变剂量并形成长效制剂，改善适用性；用氘取代化合物中的氢原子，由于其氘同位素效应，提高化合物在动物体内的药物浓度，提高了药物疗效；用氘取代化合物中的氢原子，可以抑制某些代谢产物，提高了化合物的安全性。
具体实施方式
化合物
本发明提供式(I)所示的N-苯磺酰基苯甲酰胺化合物、或其药学上可接受的盐、前药、晶型、立体异构体、水合物或溶剂合物：
Figure PCTCN2018092588-appb-000005
其中，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、R 16、R 17、R 18、R 19、R 20、R 21、R 22、R 23、R 24、R 25、R 26、R 27、R 28、R 29、R 30、R 31、R 32、R 33、R 34、R 35、R 36、R 37、R 38、R 39和R 40相互独立地选自氢、氘、卤素或三氟甲基；
X 1、X 2相互独立地选自由“氢(H)、氘(D)、甲基、CH 2D、CHD 2、CD 3、CH 2CH 3、CHDCH 3、CHDCH 2D、CHDCHD 2、CHDCD 3、CD 2CH 3、CD 2CH 2D、CD 2CHD 2、CD 2CD 3”组成的组；
附加条件是，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、R 16、R 17、R 18、R 19、R 20、R 21、R 22、R 23、R 24、R 25、R 26、R 27、R 28、R 29、R 30、R 31、R 32、R 33、R 34、R 35、R 36、R 37、R 38、R 39、R 40、X 1和X 2中至少一个是氘代的或氘。
作为本发明的优选实施方案，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75％，更佳地大于95％，更佳地大于99％。
在具体实施方案中，“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、R 16、R 17、R 18、R 19、R 20、R 21、R 22、R 23、R 24、R 25、R 26、R 27、R 28、R 29、R 30、R 31、R 32、R 33、R 34、R 35、R 36、R 37、R 38、R 39和R 40相互独立地选自氢、氘、卤素或三氟甲基”包括R 1选自氢、氘、卤素或三氟甲基，R 2选自氢、氘、卤素或三氟甲基，R 3选自氢、氘、卤素或三氟甲基，以此类推，直至R 40选自氢、氘、卤素或三氟甲基的技术方案。更具体地，包括R 1为氢、R 1为氘、R 1为卤素(F、Cl、Br、I)、R 1为三氟甲基，R 2为氢、R 2为氘、R 2为卤素(F、Cl、Br、I)、R 2为三氟甲基，R 3为氢、R 3为氘、R 3为卤素(F、Cl、Br、I)、R 3为三氟甲基，以此类推，直至R 40为氢、R 40为氘、R 40为卤素(F、Cl、Br、I)、R 40为三氟甲基的技术方案。
在另一具体实施方案中，“X 1、X 2相互独立地选自由‘氢(H)、氘(D)、甲基、CH 2D、CHD 2、CD 3、CH 2CH 3、CHDCH 3、CHDCH 2D、CHDCHD 2、CHDCD 3、CD 2CH 3、CD 2CH 2D、CD 2CHD 2、CD 2CD 3’组成的组”包括X 1选自氢(H)、氘(D)、甲基、CH 2D、CHD 2、CD 3、CH 2CH 3、CHDCH 3、CHDCH 2D、CHDCHD 2、CHDCD 3、CD 2CH 3、CD 2CH 2D、CD 2CHD 2、CD 2CD 3，X 2选自氢(H)、氘(D)、甲基、CH 2D、CHD 2、CD 3、CH 2CH 3、CHDCH 3、CHDCH 2D、CHDCHD 2、CHDCD 3、CD 2CH 3、CD 2CH 2D、CD 2CHD 2、CD 2CD 3的技术方案。更具体地，包括X 1为氢、X 1为氘、X 1为CH 2D、X 1为CHD 2、X 1为CD 3、X 1为CH 2CH 3、X 1为CHDCH 3、X 1为CHDCH 2D、X 1为CHDCHD 2、X 1为CHDCD 3、X 1为CD 2CH 3、X 1为CD 2CH 2D、X 1为CD 2CHD 2、X 1为CD 2CD 3，X 2为氢、X 2为氘、X 2为CH 2D、X 2为CHD 2、X 2为CD 3、X 2为CH 2CH 3、X 2为CHDCH 3、X 2为CHDCH 2D、X 2为CHDCHD 2、X 2为CHDCD 3、X 2为CD 2CH 3、X 2为CD 2CH 2D、X 2为CD 2CHD 2、X 2为CD 2CD 3的技术方案。
在优选地实施方案中，本发明涉及一种式(I)的化合物，或其药学上可接受的盐、前药、水合物或溶剂化合物、晶型、立体异构体或同位素变体，其中，R 11-R 20和R 37-R 40是氢，X 1和X 2相互独立地选自甲基、CH 2D、CHD 2、CD 3，R 1-R 10、R 21-R 30、R 31-R 36如上所定义，附加条件是，式(I)化合物至少含有一个氘原子。
在优选地实施方案中，本发明涉及一种式(I)的化合物，或其药学上可接受的盐、前药、水合物或溶剂化合物、晶型、立体异构体或同位素变体，其中，R 11-R 20和R 37-R 40是氢，X 1和X 2相互独立地选自甲基、CH 2D、CHD 2、CD 3，R 1-R 10、R 21-R 30和R 31-R 36相互独立地选自氢或氘，附加条件是，式(I)化合物至少含有一个氘原子。
在优选地实施方案中，本发明涉及一种式(I)的化合物，或其药学上可接受的盐、前药、水合物或溶剂化合物、晶型、立体异构体或同位素变体，其中，R 11-R 20和R 37-R 40是氢，X 1和X 2相互独立地选自甲基或CD 3，R 1-R 8和R 31-R 36为氢，R 9-R 10和R 21-R 30相互独立地选自氢或氘，附加条件是，式(I)化合物至少含有一个氘原子。
在优选地实施方案中，本发明涉及一种式(I)的化合物，或其药学上可接受的盐、前药、水合物或溶剂化合物、晶型、立体异构体或同位素变体，其中，R 11-R 20和R 37-R 40是氢，X 1和X 2相互独立地选自甲基，R 1-R 8和R 31-R 36为氢，R 9-R 10和R 21-R 30相互独立地选自氢或氘，附加条件是，式(I)化合物至少含有一个氘原子。
在优选地实施方案中，本发明涉及一种式(I)的化合物，或其药学上可接受的盐、前药、水合物或溶剂化合物、晶型、立体异构体或同位素变体，其中，R 11-R 20和R 37-R 40是氢，X 1和X 2相互独立地选自甲基，R 1-R 8和R 31-R 36为氢，R 9和R 10为氘，R 21-R 30相互独立地选自氢或氘。在另一具体实施方案中，R 21-R 28为氘；在另一具体实施方案中，R 21-R 28为氢；在另一具体实施方案中，R 29-R 30为氘；在另一具体实施方案中，R 29-R 30为氢。
在优选地实施方案中，本发明涉及一种式(I)的化合物，或其药学上可接受的盐、前药、水合物或溶剂化合物、晶型、立体异构体或同位素变体，其中，R 11-R 20和R 37-R 40是氢，X 1和X 2相互独立地选自甲基，R 1-R 8和R 31-R 36为氢，R 21-R 28为氘，R 9、R 10、R 29和R 30相互独立地选自氢或氘。在另一具体实施方案中，R 9和R 10为氘；在另一具体实施方案中，R 29和R 30为氢；在另一具体实施方案中，R 29和R 30为氘。
在优选地实施方案中，本发明涉及一种式(I)的化合物，或其药学上可接受的盐、前药、水合物或溶剂化合物、晶型、立体异构体或同位素变体，其中，R 11-R 20和R 37-R 40是氢，X 1和X 2相互独立地选自甲基，R 1-R 8和R 31-R 36为氢，R 29和R 30为氘，R 9-R 10和R 21-R 28相互独立地选自氢或氘。在另一具体实施方案中，R 9-R 10为氘；在另一具体实施方案中，R 9-R 10氢；在另一具体实施方案中，R 21-R 28为氘；在另一具体实施方案中，R 21-R 28为氢。
在优选实施方案中，式(I)化合物选自下式结构的化合物：
Figure PCTCN2018092588-appb-000006
Figure PCTCN2018092588-appb-000007
合成
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。
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、***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***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％)。
实施例
下面更具体地描述本发明式(I)结构化合物的制备方法，但这些具体方法不对本发明构成任何限制。本发明化合物还可以任选将在本说明书中描述的或本领域已知的各种合成方法组合起来而方便地制得，这样的组合可由本发明所属领域的技术人员容易地进行。
通常，在制备流程中，各反应通常在惰性溶剂中，在室温至回流温度(如0℃～100℃，优选0℃～80℃)下进行。反应时间通常为0.1小时-60小时，较佳地为0.5-24小时。
中间体化合物1：2-[(1H-吡咯并[2,3-b]吡啶-5-基)氧基]-4-氟苯甲酸甲酯的合成。
Figure PCTCN2018092588-appb-000008
氮气氛围下，依次将1H-吡咯并[2,3-b]吡啶-5-醇(1.0g，7.45mmol)、2,4-二氟苯甲酸甲酯(1.6g，9.31mmol)、磷酸钾(2.05g，9.69mmol)加入到20mL二甘醇二甲醚溶液中，该反应液在115℃下搅拌约10h，点板分析，直到原料反应完全。反应液冷却至室温，加水淬灭反应，用乙酸乙酯萃取，收集有机相，通过柱色谱分离纯化得到白色固体产物1.3g，收率62％。 1H NMR(400MHz,CDCl 3)δ10.66(s,1H),8.24(d,J＝2.5Hz,1H),7.99(dd,J＝8.8,6.6Hz,1H),7.69(d,J＝2.4Hz,1H),7.50–7.42(m,1H),6.83(ddd,J＝8.8,7.6,2.4Hz,1H),6.57–6.49(m,2H),3.93(s,3H).
中间体化合物2：4-氯苯硼酸频那醇酯的合成。
Figure PCTCN2018092588-appb-000009
氮气氛围下，依次将氯碘苯(6.0g，25.16mmol)、Pd(dppf)Cl 2·CH 2Cl 2(1.0g，1.26mmol)、醋酸钾(4.93g，50.3mmol)、联频哪醇硼酸酯(6.4g，26.4mmol)加入到50ml的DMSO溶液中，加热至95℃并反应10h。反应液冷却至室温，加水淬灭反应，用二氯甲烷萃取，收集有机相，通过柱色谱分离纯化得到黄色固体产物3.2g，收率50％。 1H NMR(400MHz,CDCl 3)δ7.73(d,J＝8.2Hz,2H),7.35(d,J＝8.3Hz,2H),1.34(s,12H).
中间体化合物3：3-硝基-4-[(四氢-2H-吡喃-4-基甲基)氨基]苯磺酰胺的合成。
Figure PCTCN2018092588-appb-000010
依次将4-氟-3-硝基苯磺酰胺(1.0g，4.54mmol)、(四氢-2H-吡喃-4-基)甲氨(0.6g，4.49mmol)、三乙胺(1.3g，6.81mmol)加入到10ml的四氢呋喃溶液中，室温搅拌5h后，移除溶剂，加入20ml甲醇打浆，干燥后得到产物1.4g，收率97％。 1H NMR(400MHz,DMSO-d 6)δ8.59(s,1H),8.47(d,J＝2.2Hz,1H),7.83(dd,J＝9.2,2.1Hz,1H),7.35(s,2H),7.30(d,J＝9.3Hz,1H),3.85(dd,J＝11.0,3.3Hz,2H),3.37(s,2H),3.26(t,J＝11.6Hz,2H),1.91(ddd,J＝11.2,7.6,4.0Hz,1H),1.61(d,J＝12.7Hz,2H),1.33–1.21(m,2H).
实施例1 制备4-(4-{[2-(4-氯苯基)-4,4-二甲基环己-1-烯-1-基]甲基-d 2}哌嗪-1-基)-N-({3-硝基-4-[(四氢-2H-吡喃-4-基甲基)氨基]苯基}磺酰基)-2-(1H-吡咯并[2,3-b]吡啶-5-基氧基)苯甲酰胺，即化合物T-1，分子式如下：
Figure PCTCN2018092588-appb-000011
采用以下路线进行合成：
Figure PCTCN2018092588-appb-000012
步骤1 化合物5的合成。
氮气保护下将氢化钠(1.9g，79.3mmol)和碳酸二甲酯(14.3g，158.6mmol)加入到无水THF(15ml)溶液中加热，回流时滴加3,3-二甲基环己酮(5.0g，39.6mmol)的THF溶液，滴加完毕后，继续回流4h。冷却至室温，加入甲醇淬灭反应，再加入水和二氯甲烷萃取，收集有机相，通过柱色谱分离纯化得到无色液体产品4.2g，收率70％。
步骤2 化合物6的合成。
在0℃下，将化合物5(4.2g，22.8mmol)缓慢滴加到氢化钠(1.0g，45.6mmol)的二氯甲烷(80mL)溶液中，反应0.5h，降温至-25℃，再缓慢滴加三氟甲磺酸酐(Tf 2O，7.7g，27.4mmol)，滴加完毕后，反应液在室温下反应过夜。加入2M的氯化铵(50ml)溶液萃取，收集有机相，水(60ml×2)洗涤，无水硫酸钠干燥，除去溶剂，浓缩液进行柱分离(洗脱剂：石油醚/乙酸乙酯(v/v)＝100:1)，得到淡黄色液体产品5.0g，收率69％。 1H NMR(400MHz,CDCl 3)δ3.81(s,3H),2.55–2.47(m,2H),2.18(t,J＝2.4Hz,2H),1.44(t,J＝6.4Hz,2H),1.01(s,6H).
步骤3 化合物7的合成。
氮气保护下依次将化合物2(0.70g，2.93mmol)、化合物6(0.928g，2.93mmol)、四三苯基磷钯(0.300g，0.29mmol)和氟化钾(0.42g，7.33mmol)加入到二氯甲烷(20ml)和甲醇(10ml)的混合液中，加热至65℃反应6h。冷却至室温，加入水(20ml)淬灭反应，二氯甲烷(30ml×3)萃取，合并有机相，无水硫酸钠干燥，除去溶剂，浓缩液进行柱分离(洗脱剂：石油醚/乙酸乙酯(v/v)＝40:1)，得到无色液体产品0.54g，收率67％。 1H NMR(400MHz,CDCl 3)δ7.33–7.27(m,2H),7.06(d,J＝8.5Hz,2H),3.49(s,3H),2.53–2.45(m,2H),2.15(t,J＝2.3Hz,2H),1.51(t,J＝6.5Hz,2H),1.02(s,6H).
步骤4 化合物8的合成。
在0℃条件下，将LiAlD 4(0.2g，5.02mmol)缓慢滴加到化合物7(0.7g，2.51mmol的四氢呋喃(20ml)溶液中，滴加完毕后继续反应1h。加入1M的盐酸(10ml)淬灭反应，二氯甲烷(40ml×3)萃取，合并有机相，无水硫酸钠干燥，除去溶剂得到淡黄色液体产品0.6g，收率96％。
步骤5 化合物9的合成。
将含有化合物8(1.2g，4.78mmol)的乙腈(15ml)和***(15ml)的混合溶液中，冷却至-5℃，加入咪唑(0.72g，10.53mmol)和三苯基磷(2.5g，9.57mmol)，待完全溶解后再缓慢地加入碘(2.92g，11.51mmol)，继续反应1h，加水(20ml)淬灭反应，乙酸乙酯(40ml×2)萃取，合并有机相，无水硫酸钠干燥，除去溶剂，浓缩液进行柱分离(洗脱剂：石油醚/乙酸乙酯(v/v)＝50:1)，得到无色液体产品1.3g，收率77％。
步骤6 化合物10的合成。
依次将化合物9(1.2g，3.33mmol)，N-叔丁氧羰基-哌嗪(0.74g，4.00mmol)，N,N-二异丙基乙胺(DIPEA，0.86g，6.66mmol)加入至N,N-二甲基甲酰胺(DMF，10ml)溶液中，加热至80℃反应1h，冷却至室温，加水(20ml)淬灭反应，乙酸乙酯(20ml×3)萃取，合并有机相， 无水硫酸钠干燥，除去溶剂，浓缩液进行柱分离(洗脱剂：石油醚/乙酸乙酯(v/v)＝30:1)，得到产品1.1g，收率79％。
步骤7 化合物11的合成。
在0℃下，缓慢地将4M盐酸的乙酸乙酯(6ml)溶液加入至化合物10(1.0g，2.40mmol)的二氯甲烷(15ml)溶液中，升至室温继续反应3h。抽滤，用水溶解滤饼，用磷酸钾调至中性，乙酸乙酯(30ml×2)萃取，合并有机相，无水硫酸钠干燥，除去溶剂得到产品0.7g，收率92％。LC-MS(APCI):m/z＝322.13(M+1) +。
步骤8 化合物12的合成。
氮气保护下，依次将化合物11(0.9g，2.83mmol)、化合物1(0.8g，2.83mmol)和磷酸氢二钾(0.98g，5.66mmol)加入至二甲亚砜(DMSO，25ml)溶液中，该反应液在140℃反应12h，冷却至室温，加水(50ml)淬灭反应，乙酸乙酯(40ml×3)萃取，合并有机相，无水硫酸钠干燥，除去溶剂，浓缩液进行柱分离(洗脱剂：石油醚/乙酸乙酯(v/v)＝3:1)，得到等黄色油状物1.3g，收率78％。LC-MS(APCI):m/z＝588.16(M+1) +。
步骤9 化合物13的合成。
依次将氢氧化钠(0.27g，6.85mmol)、水(2ml)加入至化合物12(0.8g，1.37mmol)的四氢呋喃(10ml)和甲醇(3ml)的混合溶液中，反应液在45℃搅拌10h后，冷却至室温，移除大部分溶剂，残留液用2M盐酸调至PH值为2，用二氯甲烷(30ml×3)萃取，合并有机相，无水硫酸钠干燥，除去溶剂得到白色固体0.65g，收率83％。 1H NMR(400MHz,DMSO)δ11.72(s,1H),10.87(s,1H),8.00(d,J＝2.6Hz,1H),7.78(d,J＝8.9Hz,1H),7.51–7.47(m,1H),7.45(d,J＝2.5Hz,1H),7.40(d,J＝8.5Hz,2H),7.11(d,J＝8.4Hz,2H),6.77(dd,J＝9.0,2.4Hz,1H),6.45–6.37(m,2H),3.69(d,J＝13.2Hz,2H),3.55(d,J＝4.1Hz,2H),3.28(d,J＝12.3Hz,2H),2.72(d,J＝11.6Hz,2H),2.38(s,2H),2.02(s,2H),1.44(t,J＝6.1Hz,2H),0.94(s,6H).
步骤10 化合物T-1的合成。
依次将化合物13(0.10g，0.18mmol)、化合物3(0.055g，0.18mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC，0.052g，0.27mmol)和4-二甲氨基吡啶(DMAP，0.044mg，0.36mmol)加入至二氯甲烷(20ml)溶液中，室温搅拌10h。加水(10ml)淬灭反应，二氯甲烷(15ml×3)萃取，合并有机相，无水硫酸钠干燥，除去溶剂，浓缩液进行柱分离(洗脱剂：乙酸乙酯/甲醇(v/v)＝30:1)，得到等黄色固体80mg，收率53％。LC-MS(APCI):m/z＝871.36(M+1) +。 1H NMR(400MHz,DMSO-d 6)δ11.71(s,1H),11.48(s,1H),8.60–8.50(m,2H),8.00(dd,J＝15.2,5.6Hz,2H),7.76(d,J＝9.0Hz,1H),7.50(t,J＝6.1Hz,3H),7.34(d,J＝8.4Hz,2H),7.26(d,J＝8.6Hz,1H),7.04 (d,J＝8.3Hz,3H),6.66(d,J＝8.9Hz,1H),3.84(dd,J＝11.2,2.9Hz,2H),3.26(dd,J＝14.6,8.7Hz,4H),3.10(s,4H),2.81(s,2H),2.21(d,J＝34.6Hz,6H),1.61(d,J＝11.5Hz,2H),1.38(t,J＝6.2Hz,2H),1.23(s,3H),0.92(s,6H).
实施例2 制备4-(4-{[2-(4-氯苯基)-4,4-二甲基环己-1-烯-1-基]甲基-d 2}哌嗪-1-基)-N-({3-硝基-4-[(四氢-2H-吡喃-4-基甲基-d 2)氨基]苯基}磺酰基)-2-(1H-吡咯并[2,3-b]吡啶-5-基氧基)苯甲酰胺，即化合物T-2，分子式如下：
Figure PCTCN2018092588-appb-000013
采用以下合成路线：
Figure PCTCN2018092588-appb-000014
步骤1 化合物15的合成。
在0℃条件下，将LiAlD 4(0.4g，10.08mmol)缓慢滴加至化合物14(1.0g，9.00mmol)的四氢呋喃(20ml)溶液中，滴加完毕后继续反应1h。加入1M的盐酸(10ml)淬灭反应，二氯甲烷(40ml×3)萃取，合并有机相，无水硫酸钠干燥，除去溶剂得到淡黄色固体0.72g，收率68.6％。LC-MS(APCI):m/z＝118.29(M+1) +.
步骤2 化合物16的合成。
依次将4-氟-3-硝基本磺酰胺(1.12g，5.12mmol)、化合物15(0.6g，5.12mmol)、三乙胺(0.78g，7.68mmol)加入至四氢呋喃(10ml)溶液中，反应液室温搅拌5h，移除溶剂，加入甲醇(10ml)打浆，干燥后得到产物0.8g，收率50％。LC-MS(APCI):m/z＝318.20(M+1) +.
步骤3 化合物T-2的合成。
依次将化合物16(0.056g，0.18mmol)、化合物13(0.10g，0.18mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC，0.052g，0.27mmol)和4-二甲氨基吡啶(DMAP，0.044mg，0.36mmol)加入至二氯甲烷(20ml)溶液中，室温下搅拌10h。加水(10ml)淬灭反应，二氯甲烷(15ml×3)萃取，合并有机相，无水硫酸钠干燥，除去溶剂，浓缩液进行柱分离(洗脱剂：乙酸乙酯/甲醇(v/v)＝30:1)，得到等黄色固体45mg，收率30％。LC-MS(APCI):m/z＝873.47(M+1) +。 1H NMR(400MHz,DMSO-d 6)δ11.71(s,1H),11.49(s,1H),8.56(m,2H),8.00(dd,J＝5.2Hz,2H),7.76(d,J＝9.0Hz,1H),7.51(t,J＝6.1Hz,3H),7.36(d,J＝8.4Hz,2H),7.26(d,J＝8.2Hz,1H),7.14(d,J＝8.3Hz,3H),6.60(d,J＝8.9Hz,1H),3.45-3.26(dd,4H),3.10(s,4H),2.81(s,2H),2.56-2.21(d,J＝4.6Hz,6H),1.61(d,J＝11.5Hz,2H),1.38(t,J＝6.2Hz,2H),1.23(s,3H),0.92(s,6H).
实施例3 制备4-(4-{[2-(4-氯苯基)-4,4-二甲基环己-1-烯-1-基]甲基}哌嗪-1-基)-N-({3-硝基-4-[(四氢-2H-吡喃-4-基甲基-d 2)氨基]苯基}磺酰基)-2-(1H-吡咯并[2,3-b]吡啶-5-基氧基)苯甲酰胺，即化合物T-3，分子式如下：
Figure PCTCN2018092588-appb-000015
采用以下合成路线：
Figure PCTCN2018092588-appb-000016
Figure PCTCN2018092588-appb-000017
步骤1 化合物17的合成。
在0℃条件下，将LiAlH 4(0.14g，3.59mmol)缓慢滴加到化合物7(1.0g，3.59mmol)的四氢呋喃(20ml)溶液中，滴加完毕后继续反应1h。加入1M的盐酸(10ml)淬灭反应，二氯甲烷(40ml×3)萃取，合并有机相，无水硫酸钠干燥，除去溶剂得到淡黄色液体产品0.8g，收率89％。
步骤2 化合物18的合成。
将化合物17(0.8g，3.19mmol)加入乙腈(15ml)和***(15ml)的混合溶液中，冷却至-5℃，加入咪唑(0.48g，7.01mmol)和三苯基磷(1.67g，6.38mmol)，待完全溶解后再缓慢地加入碘(1.95g，7.65mmol)，继续反应1h，加水(20ml)淬灭反应，乙酸乙酯(40ml×2)萃取，合并有机相，无水硫酸钠干燥，除去溶剂，浓缩液进行柱分离(洗脱剂：石油醚/乙酸乙酯(v/v)＝50:1)，得到无色液体产品0.9g，收率78％。
步骤3 化合物19的合成。
依次将化合物18(0.6g，1.67mmol)，N-叔丁氧羰基-哌嗪(0.37g，2.00mmol)，N,N-二异丙基乙胺(DIPEA，0.43g，3.33mmol)加入至N,N-二甲基甲酰胺(DMF，10ml)溶液中，加热至80℃反应1h，冷却至室温，加水(20ml)淬灭反应，乙酸乙酯(20ml×3)萃取，合并有机相，无水硫酸钠干燥，除去溶剂，浓缩液进行柱分离(洗脱剂：石油醚/乙酸乙酯(v/v)＝30:1)，得到产品0.6g，收率85％。
步骤4 化合物20的合成。
在0℃下，缓慢地将4M盐酸的乙酸乙酯(4ml)溶液加入至化合物19(0.6g，1.44mmol)的二氯甲烷(15ml)溶液中，升至室温继续反应3h。抽滤，用水溶解滤饼，用磷酸钾调至中性， 乙酸乙酯(30ml×2)萃取，合并有机相，无水硫酸钠干燥，除去溶剂得到产品0.4g，收率87％。LC-MS(APCI):m/z＝319.87(M+1) +。
步骤5 化合物21的合成。
氮气保护下，依次将化合物20(1.0g，3.13mmol)、化合物1(0.9g，3.13mmol)和磷酸氢二钾(1.10g，6.26mmol)加入至二甲亚砜(DMSO，25ml)溶液中，该反应液在140℃反应12h，冷却至室温，加水(50ml)淬灭反应，乙酸乙酯(50ml×3)萃取，合并有机相，无水硫酸钠干燥，除去溶剂，浓缩液进行柱分离(洗脱剂：石油醚/乙酸乙酯(v/v)＝3:1)，得到等黄色油状物1.4g，收率76％。LC-MS(APCI):m/z＝585.91(M+1) +。
步骤6 化合物22的合成。
依次将氢氧化钠(0.20g，5.12mmol)、水(2ml)加入至化合物21(0.6g，1.03mmol)的四氢呋喃(10ml)和甲醇(3ml)的混合溶液中，反应液在45℃搅拌10h后，冷却至室温，移除大部分溶剂，残留液用2M盐酸调至PH值为2，用二氯甲烷(30ml×3)萃取，合并有机相，无水硫酸钠干燥，除去溶剂得到白色固体0.45g，收率85％。LC-MS(APCI):m/z＝572.11(M+1) +。
步骤8 化合物T-3的合成。
依次将化合物22(0.10g，0.18mmol)、化合物16(0.055g，0.18mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC，0.052g，0.27mmol)和4-二甲氨基吡啶(DMAP，0.044mg，0.36mmol)加入至二氯甲烷(20ml)溶液中，室温搅拌10h。加水(10ml)淬灭反应，二氯甲烷(15ml×3)萃取，合并有机相，无水硫酸钠干燥，除去溶剂，浓缩液进行柱分离(洗脱剂：乙酸乙酯/甲醇(v/v)＝30:1)，得到等黄色固体50mg，收率33％。 1H NMR(400MHz,DMSO-d 6)δ11.70(s,1H),11.45(s,1H),8.62–8.51(m,2H),8.00(dd,J＝18.9,5.6Hz,2H),7.78(d,J＝9.4Hz,1H),7.54–7.45(m,3H),7.34(d,J＝8.4Hz,2H),7.26(d,J＝8.6Hz,1H),7.06(dd,J＝17.2,8.7Hz,3H),6.67(d,J＝7.2Hz,1H),6.38(dd,J＝3.2,1.8Hz,1H),6.20(s,1H),3.84(dd,J＝11.2,3.2Hz,2H),3.09(s,4H),2.79(s,2H),2.20(d,J＝3.7Hz,5H),1.95(s,2H),1.61(d,J＝12.3Hz,2H),1.38(t,J＝6.2Hz,2H),1.23(s,2H),0.92(s,6H).
实施例4 制备4-(4-{[2-(4-氯苯基)-4,4-二甲基环己-1-烯-1-基]-甲基-d 2}哌嗪-1-基2,2,3,3,5,5,6,6-d 8)-N-({3-硝基-4-[(四氢-2H-吡喃-4-基甲基)氨基]苯基}磺酰基)-2-(1H-吡咯并[2,3-b]吡啶-5-基氧基)苯甲酰胺，即化合物T-4，分子式如下：
Figure PCTCN2018092588-appb-000018
采用以下合成路线：
Figure PCTCN2018092588-appb-000019
步骤1 化合物23的合成。
依次将化合物9(1.0g，2.76mmol)，N-叔丁氧羰基哌嗪-2,2,3,3,5,5,6,6-d 8(0.64g，3.31mmol)，N,N-二异丙基乙胺(DIPEA，0.54g，4.13mmol)加入至N,N-二甲基甲酰胺(DMF，10ml)溶液中，加热至80℃反应1h，冷却至室温，加水(20ml)淬灭反应，乙酸乙酯(20ml×3)萃取，合并有机相，无水硫酸钠干燥，除去溶剂，浓缩液进行柱分离(洗脱剂：石油醚/乙酸乙酯(v/v)＝30:1)，得到产品0.8g，收率68％。
步骤2 化合物24的合成。
在0℃下，缓慢地将4M盐酸的乙酸乙酯(6ml)溶液加入至化合物23(0.8g，1.86mmol)的二氯甲烷(15ml)溶液中，升至室温继续反应3h。抽滤，用水溶解滤饼，用磷酸钾调至中性，乙酸乙酯(30ml×2)萃取，合并有机相，无水硫酸钠干燥，除去溶剂得到产品0.7g，收率92％。 LC-MS(APCI):m/z＝329.41(M+1) +。
步骤3 化合物25的合成。
氮气保护下，依次将化合物24(0.5g，1.52mmol)、化合物1(0.44g，1.52mmol)和磷酸氢二钾(0.53g，3.04mmol)加入至二甲亚砜(DMSO，15ml)溶液中，该反应液在140℃反应12h，冷却至室温，加水(30ml)淬灭反应，乙酸乙酯(40ml×3)萃取，合并有机相，无水硫酸钠干燥，除去溶剂，浓缩液进行柱分离(洗脱剂：石油醚/乙酸乙酯(v/v)＝3:1)，得到等黄色油状物0.62g，收率69％。LC-MS(APCI):m/z＝596.39(M+1) +。
步骤4 化合物26的合成。
依次将氢氧化钠(0.21g，5.04mmol)、水(2ml)加入至化合物25(0.6g，1.01mmol)的四氢呋喃(10ml)和甲醇(3ml)的混合溶液中，反应液在45℃搅拌10h后，冷却至室温，移除大部分溶剂，残留液用2M盐酸调至PH值为2，用二氯甲烷(30ml×3)萃取，合并有机相，无水硫酸钠干燥，除去溶剂得到白色固体0.48g，收率82％。
步骤5 化合物T-4的合成。
依次将化合物26(0.10g，0.18mmol)、化合物3(0.055g，0.18mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC，0.052g，0.27mmol)和4-二甲氨基吡啶(DMAP，0.044mg，0.36mmol)加入至二氯甲烷(20ml)溶液中，室温搅拌10h。加水(10ml)淬灭反应，二氯甲烷(15ml×3)萃取，合并有机相，无水硫酸钠干燥，除去溶剂，浓缩液进行柱分离(洗脱剂：乙酸乙酯/甲醇(v/v)＝30:1)，得到等黄色固体90mg，收率59％。LC-MS(APCI):m/z＝878.92(M+1) +。 1H NMR(400MHz,DMSO-d 6)δ11.72(s,1H),11.43(s,1H),8.71–8.50(m,2H),8.10(dd,J＝15.6,5.6Hz,2H),7.76(d,J＝9.0Hz,1H),7.60(t,J＝6.1Hz,3H),7.34(d,J＝8.4Hz,2H),7.16(d,J＝8.6Hz,1H),7.04(d,J＝8.3Hz,3H),6.51(d,J＝8.9Hz,2H),3.84(dd,2H),3.26(dd,J＝8.7Hz,2H),3.10(s,2),2.81(s,2H),2.35(d,2H),1.61(d,J＝11.5Hz,2H),1.38(t,J＝6.2Hz,2H),1.21(s,2H),0.94(s,6H).
实施例5 制备4-(4-{[2-(4-氯苯基)-4,4-二甲基环己-1-烯-1-基]甲基}哌嗪-1-基-2,2,3,3,5,5,6,6-d 8)-N-({3-硝基-4-[(四氢-2H-吡喃-4-基甲基)氨基]苯基}磺酰基)-2-(1H-吡咯并[2,3-b]吡啶-5-基氧基)苯甲酰胺，即化合物T-5，分子式如下：
Figure PCTCN2018092588-appb-000020
采用以下合成路线：
Figure PCTCN2018092588-appb-000021
步骤1 化合物27的合成。
依次将化合物18(1.3g，3.60mmol)，N-叔丁氧羰基哌嗪-2,2,3,3,5,5,6,6-d 8(0.70g，3.60mmol)，N,N-二异丙基乙胺(DIPEA，0.70g，5.40mmol)加入至N,N-二甲基甲酰胺(DMF，10ml)溶液中，加热至80℃反应1h，冷却至室温，加水(20ml)淬灭反应，乙酸乙酯(30ml×3)萃取，合并有机相，无水硫酸钠干燥，除去溶剂，浓缩液进行柱分离(洗脱剂：石油醚/乙酸乙酯(v/v)＝30:1)，得到产品1.2g，收率78％。
步骤2 化合物28的合成。
在0℃下，缓慢地将4M盐酸的乙酸乙酯(6ml)溶液加入至化合物27(1.2g，2.81mmol)的二氯甲烷(15ml)溶液中，升至室温继续反应3h。抽滤，用水溶解滤饼，用磷酸钾调至中性，乙酸乙酯(30ml×2)萃取，合并有机相，无水硫酸钠干燥，除去溶剂得到产品0.8g，收率87％。 LC-MS(APCI):m/z＝327.26(M+1) +。
步骤3 化合物29的合成。
氮气保护下，依次将化合物28(0.8g，2.45mmol)、化合物1(0.70g，2.45mmol)和磷酸氢二钾(0.64g，3.67mmol)加入至二甲亚砜(DMSO，15ml)溶液中，该反应液在140℃反应12h，冷却至室温，加水(30ml)淬灭反应，乙酸乙酯(50ml×3)萃取，合并有机相，无水硫酸钠干燥，除去溶剂，浓缩液进行柱分离(洗脱剂：石油醚/乙酸乙酯(v/v)＝3:1)，得到等黄色油状物1.21g，收率83％。LC-MS(APCI):m/z＝594.09(M+1) +。
步骤4 化合物30的合成。
依次将氢氧化钠(0.34g，8.43mmol)、水(2ml)加入至化合物29(1.0g，1.69mmol)的四氢呋喃(10ml)和甲醇(3ml)的混合溶液中，反应液在45℃搅拌10h后，冷却至室温，移除大部分溶剂，残留液用2M盐酸调至PH值为2，用二氯甲烷(30ml×3)萃取，合并有机相，无水硫酸钠干燥，除去溶剂得到白色固体0.70g，收率71％。
步骤5 化合物T-5的合成。
依次将化合物30(0.10g，0.18mmol)、化合物3(0.055g，0.18mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC，0.052g，0.27mmol)和4-二甲氨基吡啶(DMAP，0.044mg，0.36mmol)加入至二氯甲烷(20ml)溶液中，室温搅拌10h。加水(10ml)淬灭反应，二氯甲烷(15ml×3)萃取，合并有机相，无水硫酸钠干燥，除去溶剂，浓缩液进行柱分离(洗脱剂：乙酸乙酯/甲醇(v/v)＝30:1)，得到等黄色固体55mg，收率36％。LC-MS(APCI):m/z＝876.85(M+1) +。 1H NMR(400MHz,DMSO-d 6)δ11.72(s,1H),11.43(s,1H),8.76–8.50(m,2H),8.17(dd,J＝5.6Hz,2H),7.76(d,J＝9.0Hz,1H),7.60(t,J＝6.1Hz,3H),7.34(d,J＝8.4Hz,2H),7.16(d,J＝8.6Hz,1H),7.04(d,J＝8.3Hz,3H),6.51(d,J＝8.9Hz,2H),3.85(dd,2H),3.57(dd,2H),3.26(dd,J＝8.7Hz,2H),3.10(s,2),2.81(s,2H),2.35(d,2H),1.63(d,2H),1.38-1.19(m,4H),0.94(s,6H).
实施例6 制备4-(4-{[2-(4-氯苯基)-4,4-二甲基环己-1-烯-1-基]甲基}哌嗪-1-基-2,2,3,3,5,5,6,6-d 8)-N-({3-硝基-4-[(四氢-2H-吡喃-4-基甲基-d 2)氨基]苯基}磺酰基)-2-(1H-吡咯并[2,3-b]吡啶-5-基氧基)苯甲酰胺，即化合物T-6，分子式如下：
Figure PCTCN2018092588-appb-000022
采用以下合成路线：
Figure PCTCN2018092588-appb-000023
依次将化合物30(0.10g，0.18mmol)、化合物16(0.055g，0.18mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC，0.052g，0.27mmol)和4-二甲氨基吡啶(DMAP，0.044mg，0.36mmol)加入至二氯甲烷(20ml)溶液中，室温搅拌10h。加水(10ml)淬灭反应，二氯甲烷(15ml×3)萃取，合并有机相，无水硫酸钠干燥，除去溶剂，浓缩液进行柱分离(洗脱剂：乙酸乙酯/甲醇(v/v)＝30:1)，得到等黄色固体60mg，收率39％。LC-MS(APCI):m/z＝878.79(M+1) +。 1H NMR(400MHz,DMSO)δ11.73(s,1H),11.40(s,1H),8.76–8.60(m,2H),8.17(dd,J＝5.8Hz,2H),7.82(d,J＝9.0Hz,1H),7.60(t,J＝6.1Hz,3H),7.34(d,J＝8.4Hz,2H),7.16(d,J＝8.6Hz,2H),7.04(d,J＝8.3Hz,2H),6.51(d,J＝8.9Hz,2H),3.87(m,2H),3.57(dd,2H),3.10(s,2),2.81(s,2H),2.35(m,2H),1.63(d,2H),1.38-1.16(m,4H),0.96(s,6H).
实施例7 制备4-(4-{[2-(4-氯苯基)-4,4-二甲基环己-1-烯-1-基]甲基-d2}哌嗪-1-基-2,2,3,3,5,5,6,6-d 8))-N-({3-硝基-4-[(四氢-2H-吡喃-4-基甲基-d 2)氨基]苯基}磺酰基)-2-(1H-吡咯并[2,3-b]吡啶-5-基氧基)苯甲酰胺，即化合物T-7，分子式如下：
Figure PCTCN2018092588-appb-000024
采用以下合成路线：
Figure PCTCN2018092588-appb-000025
依次将化合物26(0.10g，0.18mmol)、化合物16(0.055g，0.18mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC，0.052g，0.27mmol)和4-二甲氨基吡啶(DMAP，0.044mg，0.36mmol)加入至二氯甲烷(20ml)溶液中，室温搅拌10h。加水(10ml)淬灭反应，二氯甲烷(15ml×3)萃取，合并有机相，无水硫酸钠干燥，除去溶剂，浓缩液进行柱分离(洗脱剂：乙酸乙酯/甲醇(v/v)＝30:1)，得到等黄色固体40mg，收率26％。LC-MS(APCI):m/z＝880.52(M+1) +。 1H NMR(400MHz,DMSO)δ11.73(s,1H),11.42(s,1H),8.76–8.63(m,2H),8.17(dd,J＝5.8Hz,2H),7.82(d,J＝9.0Hz,1H),7.60(t,J＝6.1Hz,3H),7.34(d,J＝8.4Hz,2H),7.16(d,J＝8.6Hz,2H),7.04(d,J＝8.3Hz,2H),3.87(m,2H),3.57(dd,2H),3.10(s,2),2.81(s,2H),2.35(m,4H),1.63(m,2H),1.38-1.16(m,2H),0.95(s,6H).
生物活性测试
(1)Bcl-2蛋白活力测试
蛋白：Bcl-2(Cisbio 63ADK000CB01PEG)；Bcl-xL(Cisbio 63ADK000CB04PEG)；
实验步骤：a)DMSO中稀释化合物：待测化合物(10mM储液)用DMSO稀释500倍，,作为第一个浓度进行3倍的梯度稀释，共10个浓度梯度，第11个浓度为DMSO对照。b)用ECHO向384反应板(6008280,PerkinElmer)中加入100nL/孔的化合物(在步骤a中制备)，1000rpm离心1分钟备用。c)向上述加有化合物的384反应板中分别加入5μL/孔BCL-2溶液(2nM)、BCL-Xl(5nM)、制备)，1000rpm离心1分钟，加入5μL/孔BAK(80nM)制备)，1000rpm离心1分钟， 25℃孵育15分钟。化合物的浓度依次为100nM起始浓度，3倍稀释共10+0个点。DMSO终浓度为0.5％。d)向上述384反应板中加入10μL/孔Anti-Tag1-Eu3+&Anti-Tag2-XL665溶液(cisbio)中制备)，1000rpm离心1分钟，25℃孵育180分钟。e)使用Envision多功能读板机读取665/615nm比值。按此方法测定本发明化合物的抑制常数(Ki)，其中化合物的Ki作为“<”(小于)特定数值来表现的，是指结合亲和力值低于所使用试验的检测限。使用Wang’s Competitive Binding of Two Different Ligands to a Protein Molecule.FEBS Lett.1995,360:111-4).
抑制常数(Ki)是酶-抑制剂复合物或蛋白/小分子复合物的离解常数，其中小分子抑制一种蛋白与另一种蛋白或肽的结合。因此Ki值大表明低的结合亲和性，Ki值小表明高的结合亲和性。
通过上述实验步骤测试了本发明化合物和Venetoclax。与非氘代的Venetoclax相比，本发明化合物对于抗凋亡Bcl-2蛋白具有更好地结合亲和性，且对Bcl-xL蛋白的结合亲和性差，表现出对Bcl-2蛋白的高选择性。具体实验结果见下表1。
表1：
实施例化合物 Bcl-2(KinM) Bcl-xL(KinM)
Venetoclax 0.90 >100
T-1 1.20 >100
T-2 1.03 >100
T-3 0.86 >100
T-4 0.88 >100
T-5 0.18 >100
T-6 1.71 >100
T-7 0.64 >100
(2)细胞活力检测
急性淋细胞白血病(ALL)细胞系RS4；11被用作初代人细胞系，以评价Bcl-2选择剂在体外的细胞活性，以及它们在体内的功效。
RS4；11在添加有2mM L-谷酰胺，10％FBS，1mM丙酮酸钠，2mM HEPES，1％青霉素/链霉素，4.5g/L葡萄糖的RPMI-1640中培养，并在含有5％二氧化碳的环境于37℃保持。为了实验在体外化合物的细胞活性，在具有5％二氧化碳的潮湿的室内，将细胞在96-孔微量滴定板中以每孔50000细胞、在10％人血清的存在下处理48小时。细胞毒性EC 50值按照生产商的推荐使用CellTier  Glo(Promega)进行评价。该EC 50值由处理之后有活力的细胞相对于未处理的对照细胞的百分比来确定。
通过实验结果表明，本发明的化合物在抑制RS4；11细胞方面是非常有效的,具体实验结果见下表2
表2
实施例化合物 RS4；11(KinM)
Venetoclax 3.07
T-1 4.13
T-2 4.29
T-3 3.66
T-4 2.93
T-5 2.86
T-6 6.74
T-7 3.15
(3)肝微粒代谢实验
微粒体实验：小鼠肝微粒体：0.5mg/mL，Xenotech；辅酶(NADPH/NADH)：1mM，Sigma Life Science；氯化镁：5mM，100mM磷酸盐缓冲剂(pH为7.4)。
储备液的配制：精密称取一定量的化合物的粉末，并用DMSO分别溶解至5mM。
磷酸盐缓冲液(100mM，pH7.4)的配制：取预先配好的0.5M磷酸二氢钾150mL和700mL的0.5M磷酸氢二钾溶液混合，再用0.5M磷酸氢二钾溶液调节混合液pH值至7.4，使用前用超纯水稀释5倍，加入氯化镁，得到磷酸盐缓冲液(100mM)，其中含100mM磷酸钾，3.3mM氯化镁，pH为7.4。
配制NADPH再生***溶液(含有6.5mM NADP，16.5mM G-6-P，3U/mL G-6-P D，3.3mM氯化镁)，使用前置于湿冰上。
配制终止液：含有50ng/mL盐酸***和200ng/mL甲苯磺丁脲(内标)的乙腈溶液。取25057.5μL磷酸盐缓冲液(pH7.4)至50mL离心管中，分别加入812.5μL小鼠肝微粒体，混匀，得到蛋白浓度为0.625mg/mL的肝微粒体稀释液。样品的孵育：用含70％乙腈的水溶液将相应化合物的储备液分别稀释至0.25mM，作为工作液，备用。分别取398μL的人肝微粒体或者大鼠肝微粒 体稀释液加入96孔孵育板中(N＝2)，分别加入2μL 0.25mM的的工作液中，混匀。
代谢稳定性的测定：在96孔深孔板的每孔中加入300μL预冷的终止液，并置于冰上，作为终止板。将96孔孵育板和NADPH再生***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***溶液，作为0min样品。再向孵育板每孔加入80μL的NADPH再生***溶液，启动反应，开始计时。相应化合物的反应浓度为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5000×g，4℃条件下离心10min。取100μL上清液至预先加入100μL蒸馏水的96孔板中，混匀，采用LC-MS/MS进行样品分析。
数据分析：通过LC-MS/MS***检测相应化合物及内标的峰面积，计算化合物与内标峰面积比值。通过化合物剩余量的百分率的自然对数与时间作图测得斜率，并根据以下公式计算t 1/2和CL int，其中V/M即等于1/蛋白浓度。
Figure PCTCN2018092588-appb-000026
对本发明化合物及其没有氘代的化合物同时测验比较，评价其在人和大鼠肝微粒体的代谢稳定性。作为代谢稳定性的指标的半衰期及肝固有清除率如表1所示。表1中采用未经氘代的化合物Venetoclaxr作为对照样品。如表1所示，通过与未经氘代的化合物Venetoclax对照，本发明化合物可以显著改善代谢稳定性，进而更适于作为丙型肝炎病毒抑制剂。
表1 实施例1-7(即化合物T-1至T-7)与Venetoclaxr对照样的代谢稳定性对比表
Figure PCTCN2018092588-appb-000027
(4)大鼠药代动力学实验
实验目的：研究大鼠给予实验的化合物后，考察本发明化合物的药代动力学行为。
实验动物：
种类及品系：SD大鼠等级：SPF级
性别及数量：雄性，6只
体重范围：180～220g(实际体重范围为187～197g)
来源：上海西普尔必凯实验动物有限公司
实验及动物合格证号：SCXK(沪)2013-0016
实验过程：
在血样采集之前，预先在EDTA-K2抗凝管中加入20L的2M氟化钠溶液(酯酶抑制剂)，于80度烘箱内烘干后，置于4度冰箱存放。
大鼠，雄性，体重187～197g，随机分为2组，于实验前一天下午开始禁食过夜但可自由饮水，给药后4h给食物。A组给予Venetoclaxr(3mg/kg)，B组给予实施例1-7化合物(3mg/kg)，分别于给药后15min、30min、1、2、3、5、8、10h从大鼠眼眶静脉取血100-200L左右，置于经EDTA-K2抗凝的0.5mL的Eppendorf管中，立即混匀，抗凝后，尽快将试管轻轻颠倒混匀5-6次后,血取好后放置在冰盒中，30min内把血样本在4000rpm，10min，4℃条件下离心分离血浆，收集全部血浆后立即于-20℃保存。所有时间点样品采集后测定每个时间点的血浆中的血药浓度。
根据上述所得的给药后平均血药浓度-时间数据，采用Winnonin软件，按非房室统计矩理论求算雄性SD大鼠分别i.g给予Venetoclax、代表性实施例化合物(3mg/kg)后的药代动力学相关参数。
实验表明，与未氘代的化合物相比，本发明化合物具有更优异的药代动力学性质。具体实验结果如下：
Figure PCTCN2018092588-appb-000028
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，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。
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 (8)

  1. 一种Bcl-2蛋白抑制剂，其特征在于：如式(I)所示的N-苯磺酰基苯甲酰胺化合物、或其药学上可接受的盐、前药、晶型、立体异构体、水合物或溶剂合物：
    Figure PCTCN2018092588-appb-100001
    其中，
    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、R 16、R 17、R 18、R 19、R 20、R 21、R 22、R 23、R 24、R 25、R 26、R 27、R 28、R 29、R 30、R 31、R 32、R 33、R 34、R 35、R 36、R 37、R 38、R 39和R 40相互独立地选自氢、氘、卤素或三氟甲基；
    X 1、X 2相互独立地选自由“氢(H)、氘(D)、甲基、CH 2D、CHD 2、CD 3、CH 2CH 3、CHDCH 3、CHDCH 2D、CHDCHD 2、CHDCD 3、CD 2CH 3、CD 2CH 2D、CD 2CHD 2、CD 2CD 3”组成的组；
    附加条件是，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、R 16、R 17、R 18、R 19、R 20、R 21、R 22、R 23、R 24、R 25、R 26、R 27、R 28、R 29、R 30、R 31、R 32、R 33、R 34、R 35、R 36、R 37、R 38、R 39、R 40、X 1和X 2中至少一个是氘代的或氘。
  2. 根据权利要求1所述的Bcl-2蛋白抑制剂，其特征在于：R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 31、R 32、R 33、R 34、R 35和R 36为氢，X 1和X 2为甲基。
  3. 根据权利要求2所述的Bcl-2蛋白抑制剂，其特征在于：R 9和R 10为氘。
  4. 根据权利要求2所述的Bcl-2蛋白抑制剂，其特征在于：R 21、R 22、R 23、R 24、R 25、R 26、R 27和R 28为氘。
  5. 根据权利要求2-4任意一项所述的Bcl-2蛋白抑制剂，其特征在于：R 29和R 30为氘。
  6. 根据权利要求1所述的Bcl-2蛋白抑制剂，其可选自下式化合物：
    Figure PCTCN2018092588-appb-100002
    Figure PCTCN2018092588-appb-100003
  7. 一种药物组合物，其特征在于：其含有药学上可接受的载体和如权利要求1-6任意一项所述的Bcl-2蛋白抑制剂，或其晶型、药学上可接受的盐、水合物或溶剂合物、立体异构体、前药或同位素变体的药物组合物。
  8. 一种如权利要求1-6任意一项所述的Bcl-2蛋白抑制剂的用途，其特征在于：用于制备治疗膀胱癌、脑癌、乳腺癌、骨髓癌、***、慢性淋巴细胞性白血病、结肠直肠癌、食道癌、肝细胞癌、原始淋巴细胞性白血病、滤泡性淋巴瘤、T细胞或B细胞源的淋巴恶性肿瘤、黑色瘤、粒细胞性白血病、骨髓瘤、口腔瘤、卵巢癌、非小细胞肺癌、***癌、小细胞癌或脾癌。
PCT/CN2018/092588 2017-06-26 2018-06-25 用于抑制Bcl-2蛋白的N-苯磺酰基苯甲酰胺类化合物及其组合物及应用 WO2019001383A1 (zh)

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