WO2016107536A1 - 一类Toll样受体7激动剂 - Google Patents

一类Toll样受体7激动剂 Download PDF

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WO2016107536A1
WO2016107536A1 PCT/CN2015/099375 CN2015099375W WO2016107536A1 WO 2016107536 A1 WO2016107536 A1 WO 2016107536A1 CN 2015099375 W CN2015099375 W CN 2015099375W WO 2016107536 A1 WO2016107536 A1 WO 2016107536A1
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amino
pyrrolo
butoxy
pyrimidine
methyl
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PCT/CN2015/099375
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English (en)
French (fr)
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丁照中
孙飞
吴立方
杜金华
勝康弘
胡国平
黎健
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南京明德新药研发股份有限公司
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Priority to EP15875212.1A priority Critical patent/EP3266785A4/en
Priority to US15/573,990 priority patent/US20180148452A1/en
Publication of WO2016107536A1 publication Critical patent/WO2016107536A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—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
    • A61K31/505—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
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • 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 relates to a novel pyrrolopyrimidine ring compound as a TLR7 agonist, a pharmaceutically acceptable salt, hydrate or prodrug thereof for use in the prevention or treatment of allergic rhinitis and asthma. Specifically, it relates to a compound of the formula (I), a pharmaceutically acceptable salt, hydrate or prodrug thereof.
  • TLRs Toll like receptors
  • TLR7 small molecule agonists of TLR7 are capable of inducing the production of interferon alpha in humans and animals. (Annu. Rev. Immunol; 2003: 21, 335-76) The response of different TLR agonists depends on the type of activated cells.
  • TLR7 and TLR8 are highly homologous. Activation of TLR7 induces the production of interferon alpha in humans and animals, and can treat allergic diseases such as allergic asthma and rhinitis and other inflammatory conditions, while activation of TLR8 mainly induces tumor necrosis factor alpha (TNF- ⁇ ) and chemotaxis. Proinflammatory cytokines such as factors may cause serious side effects. Therefore, increasing the selectivity between TLR7 and TLR8 is critical for the safety of TLR7 agonists.
  • Allergic diseases are mainly caused by dysregulation of cytokine secretion by antigen-specific Th2 cells, and the immune response of Th2 to allergens is closely related to the increase of the level of specific IgE. Among them, through its influence on mast cells, the hypersensitivity of allergens is caused, which further leads to visible symptoms. Healthy individuals have a more balanced immune response (Th2/Th1) to antigens. TLR7 ligands have been shown to reduce Th2 cytokines and promote the release of Th1 cytokines, and to improve the inflammatory response in Th2 type hypersensitive pneumonia in vivo models (J. All. Clin. Immunol., 2006: 118, 511-517; Am. J. Physiol. Lung Cell Mol. Physiol., 2006: 290, L987-995; Chin. Med. J., 2006: 119, 640-648). Therefore, the TLR7 ligand has the potential to improve the allergic response of an inflammatory individual and to heal the disease.
  • TLR7 agonists have repeatedly demonstrated that intranasal stimuli in patients with both allergic rhinitis and allergic asthma continue to reduce their response to allergens (Respiratory Research, 2012: 13, 53).
  • TLR7 agonists have been administered, such as imiquimod, resiquimod, and the like.
  • imiquimod imiquimod
  • resiquimod resiquimod
  • WO 2014/081645 reports a series of TLR7 agonists for the treatment of infectious diseases such as allergic rhinitis and asthma.
  • the series of compounds are represented by formula (II):
  • R 1 is selected from unsubstituted C 4-6 alkyl or C 1-2 alkoxy C 1-2 alkyl-;
  • R 2 is hydrogen or methyl;
  • R 3 is hydrogen, halogen or C 1
  • m represents an integer of 2 to 4;
  • n represents an integer of 0 to 3;
  • p represents an integer of 0 to 2.
  • GSK-2245035 is shown in formula (III):
  • R848 is shown in formula (IV):
  • the present invention provides a compound of the formula (I), a pharmaceutically acceptable salt, hydrate or prodrug thereof,
  • R is selected from C 3-8 alkyl optionally substituted
  • n is selected from 0, 1, 2, 3 or 4;
  • W is selected from the group consisting of C 3-8 heteroalkyl, 4 to 12 membered cycloalkyl or heterocycloalkyl or amino acid;
  • the number of heteroatoms or heteroatoms is independently selected from 0, 1, 2, 3 or 4.
  • the number of substituents is independently selected from 0, 1, 2 or 3.
  • the C 3-8 alkyl group is selected from the group consisting of
  • the C 3-8 heteroalkyl group is selected from the group consisting of: -N(C 1-4 alkyl)(C 1-4 alkyl).
  • the above 4- to 12-membered cycloalkyl or heterocycloalkyl group is selected from the group consisting of or D 1 is selected from N or C(R a ), and D 2-5 are independently selected from O, S, [C(R a )(R b )] 1-2 or N(R c ), D 3 and D, respectively.
  • R 4 can also be 1 or 2 substituents selected from a single bond
  • any two R a and R b are optionally bonded to the same atom to form a 4- to 6-membered cycloalkyl, oxa 4-6 cycloalkyl or aza 4-6 ring
  • the alkyl group, the 4- to 6-membered cycloalkyl group is optionally substituted by 1 to 3 C 1-4 alkyl groups.
  • the above W is selected from the group consisting of:
  • the above compound is selected from the group consisting of
  • the invention also provides two preparation methods of the compound of formula (I), as follows:
  • SEM stands for 2-(trimethylsilyl)ethoxymethyl group and is an amino protecting group.
  • the present invention also provides the use of the above compound, a pharmaceutically acceptable salt, hydrate or prodrug thereof for the preparation of a medicament for preventing and treating allergic diseases and other inflammatory diseases, infectious diseases or cancer.
  • the disease is selected from the group consisting of allergic rhinitis.
  • the above disease is selected from the group consisting of asthma.
  • treatment when used to treat a disease, slows or prevents the progression of the disease, or at least improves a symptom of the disease, more It has improved many symptoms of this disease.
  • pharmaceutically acceptable as used herein is intended to mean that those compounds, materials, compositions and/or dosage forms are within the scope of sound medical judgment and are suitable for use in contact with human and animal tissues. Without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salt refers to a salt of a compound of the invention prepared from a compound having a particular substituent found in the present invention and a relatively non-toxic acid or base.
  • a base addition salt can be obtained by contacting a neutral amount of such a compound with a sufficient amount of a base in a neat solution or a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonia or magnesium salts or similar salts.
  • an acid addition salt can be obtained by contacting a neutral form of such a compound with a sufficient amount of an acid in a neat solution or a suitable inert solvent.
  • pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogencarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and an organic acid salt, such as formic acid, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, bisphenol Acids, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methan
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing an acid group or a base by conventional chemical methods.
  • such salts are prepared by reacting these compounds with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture of the two via the free acid or base.
  • a nonaqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile is preferred.
  • the compounds provided herein also exist in the form of prodrugs.
  • Prodrugs of the compounds described herein are readily chemically altered under physiological conditions to convert to the compounds of the invention.
  • prodrugs can be converted to the compounds of the invention by chemical or biochemical methods in an in vivo setting.
  • Certain compounds of the invention may exist in unsolvated or solvated forms, including hydrated forms. In general, the solvated forms are equivalent to the unsolvated forms and are included within the scope of the invention. Certain compounds of the invention may exist in polycrystalline or amorphous form.
  • pharmaceutically acceptable carrier refers to any formulation or carrier medium that is capable of delivering an effective amount of an active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects to the host or patient, including water, oil, Vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, tackifiers, transdermal enhancers and the like.
  • excipient generally refers to the carrier, diluent and/or vehicle required to formulate an effective pharmaceutical composition.
  • an effective amount or “therapeutically effective amount” with respect to a pharmaceutical or pharmacologically active agent refers to a sufficient amount of a drug or agent that is non-toxic but that achieves the desired effect.
  • An "effective amount” of an active substance in a composition refers to the amount required to achieve the desired effect when used in combination with another active substance in the composition. The determination of the effective amount will vary from person to person, depending on the age and general condition of the recipient, and also on the particular active substance, and a suitable effective amount in a case can be determined by one skilled in the art based on routine experimentation.
  • active ingredient refers to a chemical entity that is effective in treating a target disorder, disease or condition.
  • substituted means that any one or more hydrogen atoms on a particular atom are replaced by a substituent.
  • optionally substituted means that it may or may not be substituted, and unless otherwise specified, the kind and number of substituents may be arbitrary on the basis of chemically achievable.
  • Alkyl refers to a saturated carbon-hydrogen chain containing a specific number of atoms. Contains linear and branched alkanes, except in special cases.
  • a C 1-6 alkyl group can be a saturated linear, branched hydrocarbon chain containing 1, 2, 3, 4, 5 or 6 carbons, such as ethyl, isopropyl; n-C 3-6 alkane
  • the base is a saturated linear hydrocarbon chain containing 3 to 6 carbons, such as n-butyl and n-butyl.
  • hetero denotes a hetero atom or a hetero atomic group (ie, a radical containing a hetero atom), including atoms other than carbon (C) and hydrogen (H), and radicals containing such heteroatoms, including, for example, oxygen (O).
  • a heteroalkyl group denotes a stable straight chain, branched or cyclic hydrocarbon group and combinations thereof, containing the number of carbon atoms and at least one hetero atom.
  • the hetero atom is selected from the group consisting of O, N, and S, wherein the N, S atom is likely to be in its oxidized form, and the N atom is likely to be in the form of a quaternary ammonium salt.
  • the O, N and S atoms may be at any position of the heteroalkyl group or between the alkyl group and the parent.
  • ring means substituted or unsubstituted cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, cycloalkynyl, heterocycloalkynyl, aryl or heteroaryl. So-called rings include single rings, interlocking rings, spiral rings, parallel rings or bridge rings. The number of atoms on the ring is generally defined as the number of elements of the ring. For example, “5 to 11 membered rings” means that 5, 6, 8, 8, 10 or 11 atoms are arranged in a circle. Unless otherwise specified, the ring optionally contains 1, 2 or 3 heteroatoms.
  • 5 to 11 membered ring includes, for example, phenylpyridine and piperidinyl; on the other hand, the term “5 to 11 membered heterocycloalkyl ring” includes pyridyl and piperidinyl, but does not include phenyl.
  • ring also includes ring systems containing at least one ring, each of which "ring” independently conforms to the above definition.
  • halo or halogen refers to fluoro, chloro, bromo and iodo.
  • protecting group includes, but is not limited to, "amino protecting group.”
  • amino protecting group refers to a protecting group suitable for preventing side reactions at the amino nitrogen position.
  • Representative amino protecting groups include, but are not limited to, formyl; acyl, such as alkanoyl, such as acetyl, etc; alkoxycarbonyl, such as t-butoxycarbonyl (Boc), etc; arylmethoxycarbonyl, such as Benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn); etc.; silyl, such as trimethylsilyl (TMS) and tert-butyl Methylsilyl (TBS) Wait.
  • TMS trimethylsilyl
  • TBS tert-butyl Methylsilyl
  • Certain compounds of the invention may include chiral atoms and multiple bonds, and thus one or more stereoisomeric forms may occur.
  • Compounds included in previous developments are either stereoisomers as individuals or mixtures in which the racemates are also included.
  • Certain compounds may exhibit all tautomeric forms in the present invention. Includes all tautomers in the previous study whether the compound is an individual or a mixture.
  • Polycrystals are properties in which a compound developed can have one or more crystal structures.
  • the developed compounds may exist in various crystalline or amorphous states, or in the form of solid plugs, powders, films due to the use of precipitation, crystallization, lyophilization, spray drying, evaporative drying, and the like. They should be present either alone or in combination with one or more compounds that have been developed, or in combination with one or more other drugs. Typically, they will be present in combination with one or more acceptable pharmaceutical excipients.
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth below, combinations thereof with other chemical synthetic methods, and those well known to those skilled in the art. Equivalent alternatives, preferred embodiments include, but are not limited to, embodiments of the invention.
  • the reaction is generally carried out under an inert nitrogen atmosphere in an anhydrous solvent.
  • Proton nuclear magnetic resonance data was recorded on a Bruker Avance III 400 (400 MHz) spectrometer with chemical shifts expressed in ppm at the low field of tetramethylsilane. Mass spectra were measured on an Agilent 1200 Series Plus 6110 (&1956A).
  • LC/MS or Shimadzu MS contains a DAD: SPD-M20A (LC) and Shimadzu Micromass 2020 detector.
  • the mass spectrometer is equipped with an electrospray ionization source (ESI) operating in either positive or negative mode.
  • ESI electrospray ionization source
  • the present invention employs the following abbreviations: aq for "aqueous"; eq for equivalent; SEM-Cl for (2-(chloromethoxy)ethyl)trimethylsilane; SEM for (2-(chloromethoxy) Alkyl)trimethylsilyl, a protecting group for amines; i-PrOH for isopropanol; DCM for methylene chloride; PE for petroleum ether; DMF for N,N-dimethylformamide; EtOAc or EA stands for ethyl acetate; EtOH stands for ethanol; MeOH stands for methanol; THF stands for tetrahydrofuran; DMSO stands for dimethyl sulfoxide; MeCN or ACN stands for acetonitrile; dioxane stands for 1,4-dioxane; Boc 2 O stands for Tert-butyl dicarbonate; BOC represents tert-butoxycarbonyl, a protecting group of amine; CuI
  • High performance liquid chromatography was performed using a Shimadzu LC20AB system equipped with a Shimadzu SIL-20A autosampler and a Shimadzu DAD: SPD-M20A detector using Xtimate C18 (3 m packing, size 2.1 x 300 mm) column.
  • 0-60AB_6 min method Apply a linear gradient, start elution with 100% A (A is 0.0675% TFA in water), and end the elution with 60% B (B is 0.0625% TFA in MeCN solution). The whole process is 4.2 minutes, then eluted with 60% B for 1 minute. The column was equilibrated for 0.8 minutes to reach 100:0 with a total run time of 6 minutes.
  • 10-80AB_6 min method Apply a linear gradient, start elution with 90% A (A is 0.0675% TFA in water), and end the elution with 80% B (B in 0.0625% TFA in acetonitrile). 4.2 minutes, then eluted with 80% B for 1 minute.
  • the column was equilibrated for 0.8 minutes to 90:10 with a total run time of 6 minutes.
  • the column temperature was 50 ° C and the flow rate was 0.8 mL/min.
  • the diode array detector has a scanning wavelength of 200-400 nm.
  • TLC Thin layer chromatography
  • a common solvent for flash column chromatography or thin layer chromatography is a mixture of dichloromethane/methanol, ethyl acetate/methanol and hexane/ethyl acetate.
  • hydrochloric acid method water (containing 0.05% by volume of hydrochloric acid (hydrochloric acid mass fraction w%: 33-37.5%)) - acetonitrile system;
  • Trifluoroacetic acid method water (containing trifluoroacetic acid in a volume ratio of 0.075%)-acetonitrile system;
  • Alkaline ammonia method water (containing 0.05% by volume of ammonia water (ammonia mass fraction w%: 25-28%)) - acetonitrile system.
  • the total running time is 8-15 minutes.
  • the compounds of the invention differ in that: (i) R 1 in WO 2014/081645 is an alkyl group, and the compound of the invention corresponds to an alkoxy group. The present inventors have found that alkoxy-substituted alkyl groups can significantly improve the efficacy and improve the pharmacokinetics. (b) R 2 in WO 2014/081645 is hydrogen or methyl, and the compound of the present invention corresponds to a cyano group. The present inventors have found that cyano groups are essential for improving drug efficacy; and the synthetic processes between the two are quite different and more challenging.
  • the compound of the present invention has a very potent effect of inducing interferon alpha, and can be used for preventing or treating allergic rhinitis and diseases such as asthma, viral infection, or cancer.
  • Formula (I) is prepared by the following general route.
  • Example 1 was prepared by the following synthetic route
  • Step A Synthesis of 2,4-dichloro-5-((2-(trimethylsilyl)ethoxy)methyl)pyrrolo[3,2-d]pyrimidine.
  • 2,4-Dichloro-5H-pyrrolo[3,2-d]pyrimidine (4.0 g, 21.39 mmol) was dissolved in 30 mL of dry tetrahydrofuran.
  • Sodium hydride (60% by mass, 1.03 g, 25.75 mmol) was added portionwise under nitrogen at 0 °C.
  • the reaction was stirred at room temperature for 30 minutes, cooled again to 0.degree. C. (2-(chloromethoxy)ethyl)trimethylsilane (3.9 g, 23.49 mmol). After the addition was completed, the reaction mixture was stirred at room temperature for 2 hr. The combined organic layers were washed with EtOAc EtOAc m.
  • the concentrate was purified by silica gel column chromatography (eluent gradient: ethyl acetate / petroleum ether 5% to 10%) to give a yellow solid 2,4-dichloro-5- ((2-(trimethylsilyl)) Ethoxy)methyl)pyrrolo[3,2-d]pyrimidine (5.8 g, yield: 85.55%).
  • Step B Synthesis of 2-chloro-5-((2-(trimethylsilyl)ethoxy)methyl)pyrrolo[3,2-d]pyrimidin-4-amine.
  • Step C Synthesis of 4-amino-2-butoxy-5-((2-(trimethylsilyl)ethoxy)methyl)pyrrolo[3,2-d]pyrimidine.
  • Step D Synthesis of 4-amino-2-butoxy-7-iodo-5-((2-(trimethylsilyl)ethoxy)methyl)pyrrolo[3,2-d]pyrimidine .
  • Step E 4-Amino-2-butoxy-7-[5-(1-piperidinyl)pent-1-ynyl]-5-((2-(trimethylsilyl)ethoxy) Synthesis of methyl)pyrrolo[3,2-d]pyrimidine.
  • Step F 4-Amino-2-butoxy-7-(5-(1-piperidinyl)pentyl)-5-((2-(trimethylsilyl)ethoxy)methyl) Synthesis of pyrrolo[3,2-d]pyrimidine.
  • Step G 4-(bis(tert-butoxycarbonyl)amino)-2-butoxy-7-(5-(1-piperidinyl)pentyl)-5-((2-(trimethylmethyl)) Synthesis of silyl)ethoxy)methyl)pyrrolo[3,2-d]pyrimidine.
  • the product was obtained as a yellow oil.
  • Step H 4-(bis(tert-butoxycarbonyl)amino)-2-butoxy-7-(5-(1-piperidinyl)pentyl)-5-((2-(trimethylmethyl)) Synthesis of silyl)ethoxy)methyl)pyrrolo[3,2-d]pyrimidine-6-carboxylic acid formate.
  • Step I 4-(Bis(tert-Butoxycarbonyl)amino)-2-butoxy-7-(5-(1-piperidinyl)pentyl)-5-((2-(trimethylmethyl) Synthesis of silyl)ethoxy)methyl)pyrrolo[3,2-d]pyrimidine-6-carboxamide.
  • Step J 4-(bis(tert-butoxycarbonyl)amino)-2-butoxy-7-(5-(1-piperidinyl)pentyl)-5-((2-(trimethylmethyl)) Synthesis of silyl)ethoxy)methyl)pyrrolo[3,2-d]pyrimidine-6-carbonitrile.
  • Step K Synthesis of 4-amino-2-butoxy-7-(5-(1-piperidinyl)pentyl)-5H-pyrrolo[3,2-d]pyrimidin-6-carbonitrilecarboxylate .
  • Step A 4-(Bis(tert-Butoxycarbonyl)amino)-2-((S)-1-methylbutoxy)-7-(5-(1-piperidinyl)pentyl)-5 Synthesis of ((2-(trimethylsilyl)ethoxy)methyl)pyrrolo[3,2-d]pyrimidine-6-carbonitrile.
  • Step B 4-Amino-2-((S)-1-methylbutoxy)-7-(5-(1-piperidinyl)pentyl)-5-((2-(trimethylmethyl)) Synthesis of silyl)ethoxy)methyl)pyrrolo[3,2-d]pyrimidine-6-carbonitrile.
  • Step C 4-Amino-2-((S)-1-methylbutoxy)-7-(5-(1-piperidinyl)pentyl)-5H-pyrrolo[3,2-d] Synthesis of pyrimidine-6-nitrilecarboxylate.
  • Example 4 was prepared by the following synthetic route
  • Step A 4-((bis-tert-Butoxycarbonyl)-amino)-2-butoxy-5-((2-(trimethylsilyl)ethoxy)methyl)pyrrolo[3, Synthesis of 2-d]pyrimidine.
  • reaction solution was continuously stirred at 20 ° C for 16 hours.
  • the reaction was completely converted by LC-MS.
  • the reaction was poured into aq.
  • the organic phase was washed successively with 500 ml of saturated aqueous ammonium chloride and 500 ml of water and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure. The remaining solid was placed in 300 ml of n-hexane, stirred vigorously for 1 hour and then filtered. The filter cake was dried under reduced pressure to give 4-((bis-tert-butoxycarbonyl)-amino)-2-butoxy-5-((2-(trimethylsilyl)ethoxy) as a white powder.
  • Step B 4-((Bis-tert-Butoxycarbonyl)-amino)-2-butoxy-5-((2-(trimethylsilyl)ethoxy)methyl)pyrrole And the synthesis of [3,2-d]pyrimidine-6-carboxylic acid.
  • the temperature of the reaction system was controlled to be about -60 ° C and stirring was continued for 1 hour. Then, similarly, the temperature of the reaction system is controlled within a range of not higher than -60 degrees Celsius, and the dry carbon dioxide gas is slowly introduced into the reaction liquid under continuous and effective stirring until the reaction is completed by LC-MS (about continuous gas introduction) 2 hours). The reaction was slowly quenched with 800 mL of saturated aqueous ammonium chloride and warmed to room temperature. It was then extracted with 800 ml of ethyl acetate.
  • Step C 4-((Bis-tert-Butoxycarbonyl)-amino)-2-butoxy-5-((2-(trimethylsilyl)ethoxy)methyl)pyrrolo[3, Synthesis of 2-d]pyrimidine-6-carboxamide.
  • EtOAc EtOAc
  • EtOAc EtOAc
  • Step D 4-((Bis-tert-Butoxycarbonyl)-amino)-2-butoxy-7-iodo-5-((2-(trimethylsilyl)ethoxy)methyl)- Synthesis of pyrrolo[3,2-d]pyrimidine-6-carboxamide.
  • reaction mixture was cooled to room temperature, and poured slowly with 1500 ml of saturated aqueous sodium thiosulfate, and extracted with ethyl acetate (500 ml).
  • the organic phase was washed twice with 500 ml of a 0.5 M aqueous sodium hydroxide solution, and then washed with 300 ml of a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate and filtered.
  • Step E 4-((Bis-tert-Butoxycarbonyl)-amino)-2-butoxy-7-iodo-5-((2-(trimethylsilyl)ethoxy)methyl)- Synthesis of pyrrolo[3,2-d]pyrimidine-6-carbonitrile.
  • Step F 4-Amino-2-butoxy-7-iodo-5-((2-(trimethylsilyl)ethoxy)methyl)-pyrrolo[3,2-d]pyrimidine- Synthesis of 6-nitrile.
  • Step G 4-Amino-2-butoxy-7-(5-chloro-1-pentynyl)-5-((2-(trimethylsilyl)ethoxy)methyl)-pyrrole And [3,2-d]pyrimidine-6-carbonitrile synthesis.
  • Step H 4-Amino-2-butoxy-7-(5-chloro-1-pentyl)-5-((2-(trimethylsilyl)ethoxy)methyl)-pyrrolo Synthesis of [3,2-d]pyrimidine-6-carbonitrile.
  • Step I 4-Amino-2-butoxy-7-(5-(morpholin-1-yl)-pentyl)-5-((2-(trimethylsilyl)ethoxy)
  • pyridyl[3,2-d]pyrimidine-6-carbonitrile
  • Step J Synthesis of 4-amino-2-butoxy-7-(5-(1-morpholinyl)-pentyl)-5H-pyrrolo[3,2-d]pyrimidine-6-carbonitrile.
  • Step A 4-Amino-2-butoxy-7-(5-(2-oxa-6-azaspiro[3.3]heptan-6-yl)pentyl)-5-((2- Synthesis of Trimethylsilyl)ethoxy)methyl)pyrrolo[3,2-d]pyrimidine-6-carbonitrile
  • Step B 4-Amino-2-butoxy-7-(5-(2-oxa-6-azaspiro[3.3]heptan-6-yl)pentyl)-5H-pyrrolo[3, Synthesis of 2-d]pyrimidine-6-nitrilecarboxylate
  • Example 11 was prepared by the following synthetic route
  • Step A 4-Amino-2-butoxy-7-(5-(4-benzyloxycarbonyl-2-oxo-piperazin-1-yl)pentyl)-5-((2-(tri-) Synthesis of silyl)ethoxy)methyl)pyrrolo[3,2-d]pyrimidine-6-carbonitrile
  • Step B 4-Amino-2-butoxy-7-(5-(2-oxopiperin-1-yl)pentyl)-5-((2-(trimethylsilyl)ethoxy) Synthesis of methyl)pyrrolo[3,2-d]pyrimidine-6-carbonitrile
  • Step C 4-Amino-2-butoxy-7-(5-(4-methyl-2-oxo-piperazin-1-yl)pentyl)-5-((2-(trimethyl) Silyl)ethoxy)methyl)pyrrolo[3,2-d]pyrimidine-6-carbonitrile
  • Step D 4-Amino-2-butoxy-7-(5-(4-methyl-2-oxo-piperazin-1-yl)pentyl)-5H-pyrrolo[3,2-d Synthesis of pyrimidine-6-nitrilecarboxylate
  • Step A 4-Amino-2-butoxy-7-(5-(2-(S)-methoxycarbonyl-pyrrolidin-1-yl)pentyl)-5-((2-(trimethyl) Synthesis of silyl)ethoxy)methyl)-pyrrolo[3,2-d]pyrimidine-6-carbonitrile.
  • Step B 4-Amino-2-butoxy-7-(5-(2-(S)-methoxycarbonyl-pyrrolidin-1-yl)pentyl)-5H-pyrrolo[3,2-d Synthesis of pyrimidine-6-nitrile.
  • Step C 4-Amino-2-butoxy-7-(5-(2-(S)-carboxylic acid-pyrrolidin-1-yl)pentyl)-5H-pyrrolo[3,2-d] Synthesis of pyrimidine-6-nitrile.
  • Step D 4-Amino-2-butoxy-7-(5-(2-(S)-carboxamido-pyrrolidin-1-yl)pentyl)-5H-pyrrolo[3,2-d Synthesis of pyrimidine-6-nitrile.
  • Step A 4-Amino-2-butoxy-7-(5-(2-carboxylic acid-pyrrolidin-1-yl)pentyl)-5H-pyrrolo[3,2-d]pyrimidine-6- Synthesis of nitriles.
  • Step B 4-Amino-2-butoxy-7-(5-(2-(N,N'-dimethylformamido)-pyrrolidin-1-yl)pentyl)-5H-pyrrole Synthesis of [3,2-d]pyrimidine-6-nitrilecarboxylate.
  • Step A 4-Amino-2-butoxy-7-(5-(2-carboxylic acid-pyrrolidin-1-yl)pentyl)-5H-pyrrolo[3,2-d]pyrimidine-6 - Synthesis of nitriles.
  • Step B 4-Amino-2-butoxy-7-(5-((2-(4-methylpiperazin-1-ylcarbonyl))-pyrrolidin-1-yl)pentyl)-5H- Synthesis of pyrrolo[3,2-d]pyrimidin-6-nitrilecarboxylate.
  • Step A 4-Amino-2-butoxy-7-(5-(2-carboxylic acid-pyrrolidin-1-yl)pentyl)-5H-pyrrolo[3,2-d]pyrimidine-6- Synthesis of nitriles.
  • Step B 4-Amino-2-butoxy-7-(5-((2-cyclopropylethylaminocarbonyl)-pyrrolidin-1-yl)pentyl)-5H-pyrrolo[3,2- d] Synthesis of pyrimidine-6-nitrilecarboxylate.
  • Step A 4-Amino-2-butoxy-7-(6-chloro-1-hexyl)-5-((2-(trimethylsilyl)ethoxy)methyl)-pyrrolo[ Synthesis of 3,2-d]pyrimidine-6-carbonitrile.
  • Step B 4-Amino-2-butoxy-7-(6-(tetrahydropyrrol-1-yl)hexyl)-5H-pyrrolo[3,2-d]pyrimidin-6-carbonitrile hydrochloride synthesis.
  • Example 20 was prepared by the following synthetic route:
  • Step A 4-Amino-2-butoxy-7-(5-hydroxypentyl-1-ynyl)-5-((2-(trimethylsilyl)ethoxy)methyl)- Synthesis of pyrrolo[3,2-d]pyrimidine-6-carbonitrile.
  • Step B 4-Amino-2-butoxy-7-(5-hydroxypentyl)-5-((2-(trimethylsilyl)ethoxy)methyl)-pyrrolo[3, Synthesis of 2-d]pyrimidine-6-carbonitrile.
  • Step C 4-Amino-2-butoxy-7-(5-oxopentyl)-5-((2-(trimethylsilyl)ethoxy)methyl)-pyrrolo[3 Synthesis of 2-d]pyrimidine-6-carbonitrile.
  • reaction mixture was stirred at 0 to 5 ° C for 1 hour, it was poured into 50 ml of saturated aqueous sodium thiosulfate solution and extracted with dichloromethane (50 ml ⁇ 2).
  • dichloromethane 50 ml ⁇ 2
  • the combined organic phases were washed once with 50 ml of a saturated aqueous solution of sodium thiosulfate, washed with a saturated aqueous solution of ammonium chloride three times (50 ml ⁇ 3), and washed once with aq.
  • Step D 4-Amino-2-butoxy-7-(5-(pyrrolidin-1-yl)pentyl)-5-((2-(trimethylsilyl)ethoxy)methyl Synthesis of pyrro[3,2-d]pyrimidine-6-carbonitrile.
  • Step E 4-Amino-2-butoxy-7-(5-(pyrrolidin-1-yl)pentyl)-5H-pyrrolo[3,2-d]pyrimidin-6-carbonitrile hydrochloride synthesis.
  • Example 21 was prepared by the following synthetic route:
  • Step A 4-Amino-2-butoxy-7-(5-(8-tert-butoxycarbonyl-3,8-diazabicyclo[3.2.1]oct-3-yl)pentyl)-5 Synthesis of ((2-(trimethylsilyl)ethoxy)methyl)-pyrrolo[3,2-d]pyrimidine-6-carbonitrile.
  • Step B 4-Amino-2-butoxy-7-(5-(3,8-diazabicyclo[3.2.1]oct-3-yl)pentyl)-5-((2-(3) Synthesis of methylsilyl)ethoxy)methyl)pyrrolo[3,2-d]pyrimidine-6-carbonitrile.
  • Step C 4-Amino-2-butoxy-7-(5-(3-methyl-3,8-diazabicyclo[3.2.1]oct-8-yl)pentyl)-5-( Synthesis of (2-(trimethylsilyl)ethoxy)methyl)pyrrolo[3,2-d]pyrimidine-6-carbonitrile.
  • Step D 4-Amino-2-butoxy-7-(5-(3-methyl-3,8-diazabicyclo[3.2.1]oct-8-yl)pentyl)-5H-pyrrole Synthesis of [3,2-d]pyrimidine-6-nitrilecarboxylate.
  • Step A 4-Amino-2-butoxy-7-(4-(2-(1-tert-butoxycarbonyl)-piperidinyl)-butyl)-5-((2-(trimethylmethyl)) Synthesis of silyl)ethoxy)methyl)-pyrrolo[3,2-d]pyrimidine-6-carbonitrile.
  • Step B 4-Amino-2-butoxy-7-(4-(piperidin-2-yl)butyl)-5-((2-(trimethylsilyl)ethoxy)methyl Synthesis of pyrro[3,2-d]pyrimidine-6-carbonitrile.
  • Step C 4-Amino-2-butoxy-7-(4-(piperidin-2-yl)butyl)-5H-pyrrolo[3,2-d]pyrimidin-6-carbonitrile hydrochloride synthesis.
  • Step A 4-Amino-2-butoxy-7-(4-(2-(1-nitromethylcarbonyl)-piperidinyl)-butyl)-5-((2-(trimethylmethyl)) Synthesis of silyl)ethoxy)methyl)-pyrrolo[3,2-d]pyrimidine-6-carbonitrile.
  • Step B 4-Amino-2-butoxy-7-(4-(1-(1-nitromethylcarbonyl)piperidin-2-yl)butyl)-5H-pyrrolo[3,2-d Synthesis of pyrimidine-6-nitrile.
  • Step A 4-Amino-2-butoxy-7-(3-(1-tert-butoxycarbonyl-4-piperidinyl)propyl)-5-((2-(trimethylsilyl)) Synthesis of oxy)methyl)-pyrrolo[3,2-d]pyrimidine-6-carbonitrile.
  • Step B 4-Amino-2-butoxy-7-(3-(1-methyl-4-piperidinyl)propyl)-5H-pyrrolo[3,2-d]pyrimidin-6-carbonitrile Synthesis of acid salts.
  • Step A Synthesis of 2-(1-tert-butoxycarbonyl-4-piperidinyl)ethanol.
  • Step B Synthesis of 2-(1-tert-butoxycarbonyl-4-piperidinyl)acetaldehyde.
  • Step C Synthesis of 3-(1-tert-butoxycarbonyl-4-piperidinyl)propyne.
  • Example 24 was prepared by the following synthetic route:
  • Step A 4-Amino-2-butoxy-7-(3-hydroxy-3-(1-tert-butoxycarbonyl-4-piperidyl)allyl)-5-((2-(tri-) Synthesis of silyl)ethoxy)methyl)pyrrolo[3,2-d]pyrimidine-6-carbonitrile.
  • reaction system was replaced with nitrogen three times, and under the continuous nitrogen protection, the temperature in the system was maintained at 130 ° C and stirred under microwave conditions for 30 minutes. After LCMS monitors the reaction, the reaction mixture is After cooling to room temperature, it was poured into 50 ml of saturated aqueous sodium hydrogen sulfate and extracted with ethyl acetate. The organic layer was washed with EtOAc (EtOAc m.
  • Step B 4-Amino-2-butoxy-7-(3-hydroxy-3-(1-tert-butoxycarbonyl-4-piperidyl)propyl)-5-((2-(trimethyl) Synthesis of silyl)ethoxy)methyl)pyrrolo[3,2-d]pyrimidine-6-carbonitrile.
  • Step C 4-Amino-2-butoxy-7-(3-hydroxy-(4-piperidinyl)propyl)-5-((2-(trimethylsilyl)ethoxy)
  • the yellow solid obtained after concentration of the organic phase under reduced pressure is 4-amino-2-butoxy-7-(3-hydroxy-(4-piperidinyl)propyl)-5-((2-(trimethylmethyl) Silyl)ethoxy)methyl)pyrrolo[3,2-d]pyrimidine-6-carbonitrile (80.00 mg, crude) was used directly in the next step.
  • Step D 4-Amino-2-butoxy-7-(3-hydroxy-(1-methyl-4-piperidyl)propyl)-5-((2-(trimethylsilyl)) Synthesis of ethoxy)methyl)pyrrolo[3,2-d]pyrimidine-6-carbonitrile.
  • Step E 4-Amino-2-butoxy-7-(3-hydroxy-(1-methyl-4-piperidinyl)propyl)-5H-pyrrolo[3,2-d]pyrimidine-6 - Synthesis of nitrile formate.
  • Preparation of 1.96-well compound plate Start the compound from a concentration of 10 mmol/L using a liquid workstation POD, and make a 3-fold gradient dilution with DMSO, and dilute a total of 10 points (from the 2nd column to the 11th column, 2 points per point) Repeat). Add 1 ⁇ l of 5 mmol/L positive compound R848 as a positive control in column 12, and add 1 ⁇ l of DMSO as a negative control in column 1. The volume of DMSO contained in each well is 1 ⁇ L.
  • the culture plate containing the cells and the compound was cultured in a CO 2 incubator for 24 hours under the conditions of 37 ° C, 5% CO 2 concentration.
  • the compounds of the present invention have the same level of in vitro receptor binding activity as GSK-2245035.
  • the aim of this protocol was to detect the expression levels of cytokines after 24 hours of stimulation of human peripheral blood mononuclear cells (PBMC) using existing compounds.
  • PBMC peripheral blood mononuclear cells
  • the cell supernatant was not diluted at the time of detection, and the levels of IFN- ⁇ and TNF- ⁇ were directly detected.
  • the compound was first formulated into a DMSO stock solution at a concentration of 20 mM, and a 10-fold gradient was diluted with cell culture medium for a total of 11 points.
  • Samples 1 and 2 have IFN- ⁇ values ⁇ 0.001 nM; TNF- ⁇ values ⁇ 1 nM
  • mice Female Balb/c mice (body weight 18-20 g) were anesthetized with isoflurane, and the compound was administered by nasal inhalation (20 ⁇ l drops between the nostrils), and the compound was predissolved in a solution containing 0.2% Tween 80. In saline. Two hours later, the mice were sacrificed with carbon dioxide and blood was collected from the heart. The blood sample is centrifuged and serum is collected. Serum samples were appropriately diluted and tested by ELISA. Refer to the instructions in the kit for specific methods of detection. In this model, the level of IFN- ⁇ /TNF- ⁇ induced by the compound was correlated with the amount of the compound administered, while the level of IFN- ⁇ /TNF- ⁇ was not detected in the group sample of the control solvent.
  • the ability of the compounds of the present invention to induce interferon IFN-[alpha] is significantly stronger than that of GSK2245035, and this advantage is particularly evident at relatively low dosing concentrations.

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Abstract

本发明公开了一类新颖的作为TLR7激动剂的吡咯并嘧啶环化合物或其药学上可接受的盐，用于过敏性鼻炎和哮喘的预防或治疗。具体公开了式(I)所示化合物或其药学上可接受的盐。

Description

一类Toll样受体7激动剂 技术领域
本发明涉及一类作为TLR7激动剂的新颖的吡咯并嘧啶环化合物、其药学上可接受的盐、水合物或前药，用于过敏性鼻炎和哮喘的预防或治疗。具体涉及式(I)所示化合物、其药学上可接受的盐、水合物或前药。
技术背景
Toll样受体(Toll like receptors，TLRs)在防御微生物感染的天然免疫中具有重要作用。Toll样受体的作用主要表现在通过监测到环境中的感染迹象，激活先天免疫细胞从而调动消除入侵的病原体的防御机制。这将导致多种细胞反应的发生，包括产生干扰素(IFNs)，介导促炎性细胞因子和效应细胞因子的适应性免疫应答。
小分子化合物能刺激先天免疫反应，其中通过Toll样受体激活I型干扰素和其它细胞因子，已成为人类疾病的治疗或预防的重要策略。已经有文献报导TLR7的小分子激动剂能够在人和动物体内诱导产生干扰素α。(Annu.Rev.Immunol；2003:21,335-76)不同TLR激动剂的响应情况取决于活化细胞的类型。
TLR7和TLR8高度同源。激活TLR7能够在人和动物体内诱导产生干扰素α，可以治疗如过敏性哮喘和鼻炎等过敏性疾病和其他炎性病症，而激活TLR8主要诱导产生肿瘤坏死因子α(TNF-α)和趋化因子等促炎细胞因子，可能导致严重的副作用。所以提高TLR7和TLR8之间的选择性对TLR7激动剂的安全性至关重要。
过敏性疾病，主要由抗原特异性Th2细胞产生的细胞因子分泌失调引起，而Th2对过敏原的免疫应答又与特异性IgE的水平的升高密切相关。其中，通过其对肥大细胞的影响，导致了过敏原的超敏性，从而进一步导致可见症状发生。健康个体对抗原的免疫应答(Th2/Th1)更加均衡，TLR7配体已经显示出有减少Th2细胞因子并促进Th1细胞因子释放的作用，能改善Th2型过敏性肺炎体内模型的炎症反应(J.All.Clin.Immunol.,2006:118,511-517；Am.J.Physiol.Lung Cell Mol.Physiol.,2006:290,L987-995；Chin.Med.J.,2006:119,640-648)。因此，TLR7配体拥有改善炎症个体的过敏应答的潜力，能够对疾病起到治愈的作用。
TLR7激动剂的临床研究已经多次证明，对同时患有过敏性鼻炎和过敏性哮喘患者的鼻内刺激能持续减少对变应原的响应(Respiratory Research,2012:13,53)。几个TLR7激动剂已被道，如咪喹莫特，瑞喹莫德等。但具有更好的选择性，治疗效用和安全性的TLR7新颖的激动剂的开发仍然有很大需求。
WO2014/081645报道了一系列用于治疗感染性疾病，如过敏性鼻炎和哮喘的TLR7激动剂，这一 系列化合物由式(II)表示：
Figure PCTCN2015099375-appb-000001
其中R1选自无取代的C4-6的烷基或C1-2的烷氧基C1-2的烷基-；R2是氢或甲基；R3是氢，卤素或C1-3的烷基；m代表2至4的整数；n代表0至3的整数；p代表0至2的整数。
GSK-2245035如式(Ⅲ)所示：
Figure PCTCN2015099375-appb-000002
R848的结构如式(Ⅳ)所示：
Figure PCTCN2015099375-appb-000003
发明内容
本发明提供了式(I)所示化合物、其药学上可接受的盐、水合物或前药，
Figure PCTCN2015099375-appb-000004
其中，
R选自任选被取代的C3-8烷基；
m选自0、1、2、3或4；
W选自任选被取代的：C3-8杂烷基、4～12元环烷基或杂环烷基或氨基酸；
上述“杂”代表杂原子或杂原子团，分别独立地选自O、S、N、C(＝O)、S(＝O)或S(＝O)2；
杂原子或杂原子团的数目分别独立地选自0、1、2、3或4。
本发明的一些方案中，上述C3-8烷基、C3-8杂烷基、4～12元环烷基或杂环烷基的取代基分别独立的选自F、Cl、Br、I、OH、CN、NH2、NH2C(＝O)、C1-4烷基或C1-4杂烷基、4～6元烷基或杂烷基、4～6元杂烷基-C(＝O)-，所述4～6元烷基或杂烷基任选被卤素、NH2、OH、CN或C1-4烷基取代，，“杂”如上述所定义。
本发明的一些方案中，上述C3-8烷基、C3-8杂烷基、4～12元环烷基或杂环烷基的取代基分别独立的选自F、Cl、Br、I、OH、CN、NH2、NH2C(＝O)、Me、Et、
Figure PCTCN2015099375-appb-000005
Figure PCTCN2015099375-appb-000006
“杂”如上述所定义。
本发明的一些方案中，取代基的数目独立地选自0、1、2或3。
本发明的一些方案中，上述的C3-8烷基选自：
Figure PCTCN2015099375-appb-000007
本发明的一些方案中，上述C3-8杂烷基选自：-N(C1-4烷基)(C1-4烷基)。
本发明的一些方案中，上述4～12元环烷基或杂环烷基选自
Figure PCTCN2015099375-appb-000008
或
Figure PCTCN2015099375-appb-000009
D1选自N或C(Ra)，D2-5分别独立地选自O、S、[C(Ra)(Rb)]1-2或N(Rc)，D3和D4中的1或2个还可以选自单键，Ra、Rb或Rc分别独立地选自H、C1-4烷基、C1-4杂烷基、卤素、OH、CN、NH2C(＝O)，任意两个Ra和Rb任选地连接到同一个原子上形成一个4～6元环烷基、氧杂4～6环烷基或氮杂4～6环烷基，所述4～6元环烷基任选地被1～3个C1-4烷基所取代。
本发明的一些方案中，上述W选自：
Figure PCTCN2015099375-appb-000010
本发明的一些方案中，上述化合物选自：
Figure PCTCN2015099375-appb-000011
Figure PCTCN2015099375-appb-000012
本发明还提供了式(I)化合物的两种制备方法，如下：
方法1：
Figure PCTCN2015099375-appb-000013
其中，SEM代表2-(三甲基硅)乙氧基甲基，是一种氨基保护基。
本发明还提供了上述化合物、其药学上可接受的盐、水合物或前药在制备预防和治疗过敏性疾病和其他炎性病症、感染性疾病或癌症的药物中的应用。
本发明的一些方案中，上述疾病选自过敏性鼻炎。
本发明的一些方案中，上述疾病选自哮喘。
有关定义：
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品 名时，意在指代其对应的商品或其活性成分。
这里所采用的术语“治疗”或者是其语法等效的词，当用于治疗一个疾病时，表示的减慢或者阻止了这个疾病的进程，或者至少改善了这个疾病的一个症状，更多的是改善了这个疾病的多个症状。
这里所采用的术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物的中性形式接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机氨或镁盐或类似的盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物的中性形式接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸，碳酸氢根，磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如甲酸、乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸；还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐(参见Berge et al.,"Pharmaceutical Salts",Journal of Pharmaceutical Science 66:1-19(1977))。本发明的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。
本发明的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱的形式，这些化合物与化学计量的适当的碱或酸反应来制备。一般地，优选醚、乙酸乙酯、乙醇、异丙醇或乙腈等非水介质。
除了盐的形式，本发明所提供的化合物还存在前药形式。本文所描述的化合物的前药容易地在生理条件下发生化学变化从而转化成本发明的化合物。此外，前体药物可以在体内环境中通过化学或生化方法被转换到本发明的化合物。
本发明的某些化合物可以以非溶剂化形式或者溶剂化形式存在，包括水合物形式。一般而言，溶剂化形式与非溶剂化的形式相当，都包含在本发明的范围之内。本发明的某些化合物可以以多晶或无定形形式存在。
术语“药学上可接受的载体”是指能够递送本发明有效量活性物质、不干扰活性物质的生物活性并且对宿主或者患者无毒副作用的任何制剂或载体介质代表性的载体包括水、油、蔬菜和矿物质、膏基、洗剂基质、软膏基质等。这些基质包括悬浮剂、增粘剂、透皮促进剂等。术语“赋形剂”通常是指配制有效的药物组合物所需要载体、稀释剂和/或介质。
针对药物或药理学活性剂而言，术语“有效量”或“治疗有效量”是指无毒的但能达到预期效果的药物或药剂的足够用量。组合物中一种活性物质的“有效量”是指与该组合物中另一种活性物质联用时为了达到预期效果所需要的用量。有效量的确定因人而异，取决于受体的年龄和一般情况，也取决于具体的活性物质，个案中合适的有效量可以由本领域技术人员根据常规试验确定。
术语“活性成分”、“治疗剂”，“活性物质”或“活性剂”是指一种化学实体，它可以有效地治疗目标紊乱、疾病或病症。
术语“被取代的”是指特定原子上的任意一个或多个氢原子被取代基取代。术语“任选被取代的”是指可以被取代，也可以不被取代，除非另有规定，取代基的种类和数目在化学上可以实现的基础上可以是任意的。
烷基指的是含有特定数目原子的饱和碳-氢链。除特殊情况外，包含直链以及支链烷烃。比如，C1-6烷基可以是含有1、2、3、4、5或6个碳的饱和直链、支链的碳氢链，如乙基、异丙基；正C3-6烷基则是含有3至6个碳的饱和直链碳氢链，如正炳基、正丁基。
除非另有规定，术语“杂”表示杂原子或杂原子团(即含有杂原子的原子团)，包括碳(C)和氢(H)以外的原子以及含有这些杂原子的原子团，例如包括氧(O)、氮(N)、硫(S)、硼(B)、-O-、-S-、＝O、＝S、-C(＝O)O-、-C(＝O)-、-C(＝S)-、-S(＝O)、-S(＝O)2-，以及任选被取代的-C(＝O)N(H)-、-N(H)-、-C(＝NH)-、-S(＝O)2N(H)-或-S(＝O)N(H)-。
除非另有规定，杂烷基单独或与其他词语连用时，表示的是稳定的直链、支链或环状的烃基及其组合物，含有所述碳原子数和至少一个杂原子。在其中一个示例方案中，杂原子选自O、N和S，其中N、S原子有可能是其氧化形式，N原子有可能是季铵盐形式。O、N和S原子可以处于杂烷基的任何位置或处于烷基与母体之间。
除非另有规定，“环”表示被取代或未被取代的环烷基、杂环烷基、环烯基、杂环烯基、环炔基、杂环炔基、芳基或杂芳基。所谓的环包括单环、联环、螺环、并环或桥环。环上原子的数目通常被定义为环的元数，例如，“5～11元环”是指环绕排列5、6、、8、9、10或11个原子。除非另有规定，该环任选地包含1、2或3个杂原子。因此，“5～11元环”包括例如苯基吡啶和哌啶基；另一方面，术语“5～11元杂环烷基环”包括吡啶基和哌啶基，但不包括苯基。术语“环”还包括含有至少一个环的环系，其中的每一个“环”均独立地符合上述定义。
术语“卤”或“卤素”是指氟、氯、溴和碘。
术语“保护基”包括但不限于“氨基保护基”。术语“氨基保护基”是指适合用于阻止氨基氮位上副反应的保护基团。代表性的氨基保护基包括但不限于：甲酰基；酰基，例如链烷酰基，如乙酰基基等；烷氧基羰基，如叔丁氧基羰基(Boc)等；芳基甲氧羰基，如苄氧羰基(Cbz)和9-芴甲氧羰基(Fmoc)等；芳基甲基，如苄基(Bn)等；甲硅烷基，如三甲基甲硅烷基(TMS)和叔丁基二甲基甲硅烷基(TBS) 等。
本发明中某些化合物可能包括手性原子和多重键，因此可能出现一种或多种立体异构的形式。包括在先前研发中的化合物无论是作为个体还是混合物的立体异构体，其中外消旋体也包括在内。
本发明中某些化合物可能出现所有的互变异构的形式。包括先前的研究中无论化合物是作为个体还是混合物的所有互变异构体。
多晶是研发的化合物可以存在一种或者多种晶体结构的性质。已开发的化合物可能以各种晶形或者不定形的状态存在，或者由于使用沉淀、结晶、冻干、喷雾干燥、蒸发干燥等方法以固体塞，粉末，薄膜的形式存在。他们应该单独的或者与已开发的一个或多个化合物结合的形式存在，抑或与其他一个或者多个药物结合的形式存在。通常情况下，他们将会以与一个或多个可接受药物赋形剂结合的形式存在。
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。
本发明具体实施方式的化学反应是在合适的溶剂中完成的，所述的溶剂须适合于本发明的化学变化及其所需的试剂和物料。为了获得本发明的化合物，有时需要本领域技术人员在已有实施方式的基础上对合成步骤或者反应流程进行修改或选择。
本发明所使用的所有溶剂可经市售获得。
反应一般是在惰性氮气下、无水溶剂中进行的。质子核磁共振数据记录在Bruker Avance III 400(400MHz)分光仪上，化学位移以四甲基硅烷低场处的(ppm)表示。质谱是在安捷伦1200系列加6110(&1956A)上测定。LC/MS或Shimadzu MS包含一个DAD：SPD-M20A(LC)和Shimadzu Micromass 2020检测器。质谱仪配备有一个正或负模式下操作的电喷雾离子源(ESI)。
本发明采用下述缩略词：aq代表“含水的”；eq表示当量；SEM-Cl代表(2-(氯甲氧基)乙基)三甲基硅烷；SEM代表(2-(氯甲氧基)乙基)三甲基硅基，是胺的一种保护基；i-PrOH代表异丙醇；DCM代表二氯甲烷；PE代表石油醚；DMF代表N,N-二甲基甲酰胺；EtOAc或EA代表乙酸乙酯；EtOH代表乙醇；MeOH代表甲醇；THF代表四氢呋喃；DMSO代表二甲基亚砜；MeCN或ACN代表乙腈；dioxane代表1,4-二氧六环；Boc2O代表二叔丁基二碳酸酯；BOC代表叔丁氧羰基，是胺的一种保护基；CuI代表碘化亚铜；Pd(OH)2/C代表氢氧化钯碳；Pd/C代表钯碳；(NH4)2CO3代表碳酸铵；NaH代表氢化钠；NH3·H2O代表氨水；Na代表金属钠；HATU代表O-(7-氮杂苯并三氮唑-1-YL)-N,N,N',N'-四甲基脲六氟膦酸盐；DMAP表示4-二甲氨基吡啶；TFA代表三氟乙酸；TFAA代表三氟乙酸酐；TEA代表三乙胺；DIEA或DIPEA代表二异丙基乙基胺；CO2代表二氧化碳气体，或固体二氧化碳(即干冰)；NIS代表N-碘代琥珀酰亚胺；Pd(PPh3P)2Cl2代表双(三苯基膦)二氯化钯；Pd(PPh3)4代表四(三苯基膦)钯；Pd(t-Bu3P)2代表双(三叔丁基膦)钯；LDA代表二异丙基氨基 锂；HCHO代表甲醛；NaBH(AcO)3代表三乙酰氧基硼氢化钠；HOBt代表1-羟基苯并三氮唑；EDCI代表1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐；DMP代表Dess-Martin氧化剂；m.w.或microwave代表微波反应；r.t.代表室温。
化合物经人工或者
Figure PCTCN2015099375-appb-000014
软件命名，市售化合物采用供应商目录名称。
用配有Shimadzu SIL-20A自动进样器和日本岛津DAD：SPD-M20A探测器的岛津LC20AB系统进行高效液相色谱分析，采用Xtimate C18(3m填料，规格为2.1x300mm)色谱柱。0-60AB_6分钟的方法：应用线性梯度，以100％A(A为0.0675％TFA的水溶液)开始洗脱，并以60％B(B为0.0625％TFA的MeCN溶液)结束洗脱，整个过程为4.2分钟，然后以60％B洗脱1分钟。将色谱柱再平衡0.8分钟达到100:0，总运行时间为6分钟。10-80AB_6分钟的方法：应用线性梯度，以90％A(A为0.0675％TFA的水溶液)开始洗脱，并以80％B(B为0.0625％TFA的乙腈溶液)结束洗脱，整个过程为4.2分钟，然后以80％B洗脱1分钟。将色谱柱再平衡0.8分钟达到90:10，总运行时间为6分钟。柱温为50℃，流速为0.8mL/min。二极管阵列检测器扫描波长为200-400nm。
在Sanpont-group的硅胶GF254上进行薄层色谱分析(TLC)，常用紫外光灯照射检出斑点，在某些情况下也采用其他方法检视斑点，在这些情况下，用碘(10g硅胶中加入约1g碘并彻底混合而成)、香草醛(溶解大约1g香草醛于100mL 10％H2SO4中制得)、茚三酮(从Aldrich购得)或特殊显色剂(彻底混合(NH4)6Mo7O24·4H2O、5g(NH4)2Ce(IV)(NO3)6、450mL H2O和50mL浓H2SO4而制得)展开薄层板，检视化合物。采用Still,W.C.；Kahn,M.；and Mitra,M.Journal of Organic Chemistry,1978,43,2923-2925.中所公开技术的类似方法，在Silicycle的40-63μm(230-400目)硅胶上进行快速柱色谱。快速柱色谱或薄层色谱的常用溶剂是二氯甲烷/甲醇、乙酸乙酯/甲醇和己烷/乙酸乙酯的混合物。
在Gilson-281Prep LC 322系统上采用吉尔森UV/VIS-156探测器进行制备色谱分析,所采用的色谱柱是Agella Venusi ASB Prep C18，5m，150x 21.2mm；Phenomenex Gemini C18，5m，150x30mm；Boston Symmetrix C18.5m，150x30mm；或者Phenomenex Synergi C18，4m，150x30mm。在流速约为25mL/min时，用低梯度的乙腈/水洗脱化合物，洗脱剂如下：
1)盐酸方法：水(含体积比0.05％的盐酸(盐酸质量分数w％：33-37.5％))-乙腈体系；
2)甲酸方法：水(含体积比0.2255％的甲酸)-乙腈体系；
3)三氟乙酸方法：水(含体积比0.075％的三氟乙酸)-乙腈体系；
4)碱性氨水方法：水(含体积比0.05％的氨水(氨水质量分数w％：25-28％))-乙腈体系。总运行时间为8-15分钟。
与WO2014/081645相对比，本发明化合物的区别在于：(一)WO2014/081645中的R1是烷基，而本发明的化合物与之对应的是烷氧基。本发明发现，烷氧基替换烷基可以显著提高药效，改善药代性。(二)WO2014/081645中的R2是氢或者甲基，而本发明的化合物与之对应的是氰基。本发明发现，氰 基对提高药效有至关重要的作用；而且两者之间合成工艺差别很大，挑战性也更大。
经验证，本发明化合物具有非常强劲的诱导产生干扰素α的药效，可用于预防或者治疗过敏性鼻炎和哮喘，病毒感染，或者癌症等疾病。
具体实施方式
为了更详细地说明本发明，给出下列实例，但本发明的范围并非限定于此。
式(I)由下面的通用路线来制备。
Figure PCTCN2015099375-appb-000015
通用路线1：
Figure PCTCN2015099375-appb-000016
Figure PCTCN2015099375-appb-000017
以可购得的2,4-二氯-5H-吡咯并[3,2-d]嘧啶(1-1)为起始原料，首先与SEM-Cl反应，用保护基SEM取代氮上裸露的氢，再与氨水在较高温度下(90-100℃)反应得到2-氯-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-5H-吡咯并[3,2-d]嘧啶-4-胺(1-2)；由各种不同的醇(用ROH表示)与金属钠制备醇钠，与化合物1-2在相应的醇中(例如与丁醇反应)制备醇取代的化合物1-3；再与NIS发生碘代反应得到化合物1-4；随后，该碘代物与各种末端炔烃发生钯催化下的偶联反应制备炔基取代的化合物(1-5)；接着在氢氧化钯碳条件下催化加氢还原，并用Boc保护氨基得到化合物(1-6)；高纯度的化合物1-6在-78℃下与LDA反应一段时间，比如1小时，然后用二氧化碳处理并适当酸化，得到由羧基取代的化合物(1-7)；再经由与碳酸铵的酸氨缩合反应而将羧酸转化为酰胺；在三氟乙酸酐/三乙胺的条件下脱水，得到化合物1-8；经简单的后处理，最后在三氟乙酸中室温脱去保护基SEM，得到最终产物(1-9).
通用路线2：
Figure PCTCN2015099375-appb-000018
Figure PCTCN2015099375-appb-000019
化合物2-1裸露的氨基由Boc保护，简单的打浆纯化后，得到带有两个Boc保护基的化合物2-2；该化合物在低温下与LDA反应，然后持续通入干燥的二氧化碳气体，酸化后得到羧酸取代的化合物2-3；粗品2-3经酰胺化得到化合物2-4，化合物2-4可由多次打浆纯化；然后用NIS碘代得到化合物2-5；在低温下用三乙胺和三氟乙酸酐条件脱水得到氰基化合物2-6；粗品2-6用较稀的三氟乙酸的二氯甲烷溶液处理，脱除保护基Boc后得到中间体2-7；该化合物与氯代的脂肪端炔经由Sonogashira交叉偶联反应得到化合物2-8；接着催化氢化得到中间体2-9；此中间体与各种氨发生烷基化反应，再用三氟乙酸脱除保护基SEM，经制备HPLC分离纯化得到最终产物2-11。
通用路线3：
Figure PCTCN2015099375-appb-000020
化合物3-1与端基脂肪炔醇发生Sonogashira交叉偶联反应得到化合物3-2；催化氢化得到相应的饱和醇(3-3)；化合物3-3经DMP氧化得到相应的醛3-4；接着与各种氨发生还原氨化反应得到化合物3-5；该化合物在三氟乙酸条件下脱除保护基SEM，经由制备HPLC分离纯化得到最终产物3-6。
通用路线4：
Figure PCTCN2015099375-appb-000021
4-氨基-2-丁氧基-7-碘-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(4-1)与端基炔发生交叉偶联反应得到7-炔基取代化合物(4-2)，然后依次经过催化氢化和脱除保护基团SEM两个步骤得到最终产物(4-4)。
本领域技术人员应该知道，为了制备本发明化合物，反应路线中反应步骤的顺序可以是不同的，这也属于本发明的范围。
为清楚起见，发明进一步用实施例来阐述。但是实施例不局限于定义或者指定发明范围。
实例1-3由通用路线1来制备
实例1
Figure PCTCN2015099375-appb-000022
4-氨基-2-丁氧基-7-(5-(哌啶-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐
实例1由以下合成路线制备
Figure PCTCN2015099375-appb-000023
步骤A：2,4-二氯-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶的合成。
2,4-二氯-5H-吡咯并[3,2-d]嘧啶(4.0克，21.39毫摩尔)溶解在30毫升无水四氢呋喃中。在氮气保护，0摄氏度下，分批加入氢化钠(60％质量含量，1.03克，25.75毫摩尔)。反应物室温下搅拌30分钟，再次冷却至0摄氏度，滴加(2-(氯甲氧基)乙基)三甲基硅烷(3.9克，23.49毫摩尔)。加完后，反应液在室温下搅拌2小时，加水120毫升淬灭，并用乙酸乙酯萃取(100毫升×2)。将合并的有机层用饱和碳酸钠水溶液和盐水洗涤，经无水硫酸钠干燥并在真空下浓缩。浓缩物通过硅胶柱色谱法(洗脱梯度：乙酸乙酯/石油醚5％至10％)纯化，得到黄色固体2,4-二氯-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶(5.8克，收率：85.55％)。MS(ESI)m/z:318[M+H+].
步骤B：2-氯-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-4-胺的合成。
在1000毫升高压釜中，依次加入2,4-二氯-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶(5克，15.77毫摩尔)，15毫升异丙醇和250毫升氨水。反应混合物在100-110摄氏度下搅拌3小时，随后自然冷却到室温，用250毫升水稀释后过滤，得到2-氯-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-4-胺(4.0克，粗品)。产物为白色固体，直接用于一步反应。MS(ESI)m/z:299[M+H+].
步骤C：4-氨基-2-丁氧基-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶的合成。
氮气保护下，向55毫升无水丁醇中加入金属钠(1.24克，53.91毫摩尔)，室温搅拌至完全溶解后，再加入4-氨基-2-氯-5-((2-(三甲基甲硅烷基)乙氧基)甲基吡咯并[3,2-d]嘧啶(4.0克，13.42毫摩尔)。该反应混合物加热至100摄氏度并持续搅拌8小时。自然冷却到室温后，反应混合物缓慢倒入200毫升水中，并用乙酸乙酯萃取(100毫升×3)。将合并的有机相用盐水洗涤，经无水硫酸钠干燥后过滤，滤液减压浓缩。残余物通过硅胶柱色谱纯化(洗脱梯度：乙酸乙酯/石油醚：15％至25％)，得到4-氨基-2-丁氧基-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶(4.1克，收率：90.91％)，产物为黄色固体。MS(ESI)m/z:337[M+H+].
步骤D：4-氨基-2-丁氧基-7-碘-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶的合成。
4-氨基-2-丁氧基-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶(40.00克，119.0毫摩尔)溶解在500毫升四氢呋喃中，在零摄氏度氮气保护下，分三批加入NIS(32.13克，142.8毫摩尔)。加完后，反应液在30摄氏度下搅拌2小时。TLC监测到原料反应完全。将反应液倒入1000毫升饱和硫代硫酸钠水溶液中并继续搅拌10分钟，随后用乙酸乙酯萃取(300毫升×2)。将合并的有机相用饱和食盐水洗涤(200毫升×2)，经无水硫酸钠干燥、过滤后，滤液减压浓缩，得到4-氨基-2-丁氧基-7-碘-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶(40.00克，收率：72.70％)，产物是黄色固体，直接用于下一步反应。1H NMR(400MHz,CHLOROFORM-d):δ7.22(s,1H),5.95(br.s.,2H),5.45-5.37(m,2H),4.48-4.36(m,2H),3.67-3.56(m,2H),1.86-1.75(m,2H),1.53(t,J＝7.4Hz,2H),1.06-0.92(m,5H),0.01(s,9H).
步骤E：4-氨基-2-丁氧基-7-[5-(1-哌啶基)戊-1-炔基]-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶的合成。
氮气保护下，向三口烧瓶中依次加入4-氨基-2-丁氧基-7-碘-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶(4.00克，8.65毫摩尔)，5-(1-哌啶基)-1-戊炔(1.57克，10.38毫摩尔)，二氯双(三苯基膦)钯(607毫克，865.00微摩尔)和三乙胺(2.63克，25.95毫摩尔)和60毫升乙腈。该反应体系用氮气置换3次后，在30摄氏度下搅拌12小时。将反应物过滤，滤液减压浓缩。残余物通过硅胶色谱法纯化(柱高度：250毫米，直径：100毫米，100-200目硅胶，石油醚/乙酸乙酯＝3/1，0/1)， 得到4-氨基-2-丁氧基-7-[5-(1-哌啶基)戊-1-炔基]-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶(2.00克，收率：47.60％)。MS(ESI)m/z:486[M+H+].
步骤F：4-氨基-2-丁氧基-7-(5-(1-哌啶基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶的合成。
4-氨基-2-丁氧基-7-(5-(1-哌啶基)戊-1-炔基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶(2.10克，4.32毫摩尔)溶解在30毫升甲醇中，氮气保护下，加入氢氧化钯碳(质量含量10％，0.20克)。反应体系用氢气置换5次，然后在30摄氏度，氢气压力50psi下搅拌在下48小时。LCMS检测反应完全。反应物过滤，滤饼用甲醇反复洗涤，滤液合并后减压浓缩，得到4-氨基-2-丁氧基-7-(5-(1-哌啶基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶(1.80克，收率：85.43％，产品为褐色油状物，直接用于下一步反应。MS(ESI)m/z:490[M+H+].
步骤G：4-(双(叔丁氧基羰基)氨基)-2-丁氧基-7-(5-(1-哌啶基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶的合成。
4-氨基-2-丁氧基-7-(5-(1-哌啶基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶(798.33毫克，1.63毫摩尔)和三乙胺(494.82毫克，4.89毫摩尔)溶于20毫升无水二氯甲烷中，分批加入BOC酸酐(1.07克，4.89毫摩尔)。加完后，反应液在30摄氏度下搅拌12小时。TLC监测反应完全后，将反应液减压浓缩。残余物经硅胶色谱法纯化(柱高度：250毫米，直径：20毫米，100-200目硅胶，二氯甲烷/甲醇＝100/1)，得到4-(双(叔丁氧基羰基)氨基)-2-丁氧基-7-(5-(1-哌啶基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶(600.00毫克，收率：53.35％)，产物为黄色油状物，直接用于下一步反应。
步骤H：4-(双(叔丁氧基羰基)氨基)-2-丁氧基-7-(5-(1-哌啶基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-羧酸甲酸盐的合成。
在氮气保护下，向三口烧瓶中加入4-(双(叔丁氧基羰基)氨基)-2-丁氧基-7-(5-(1-哌啶基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶(400.00毫克，579.71微摩尔)和10毫升四氢呋喃。将该溶液冷却至-70摄氏度，缓慢滴加LDA(1M的四氢呋喃溶液，1.74毫升)。反应混合物在-70℃下搅拌1小时后，迅速倒入干冰中，并用气球密封。反应混合物继续搅拌30分钟并自然升至室温，混合物用饱和氯化铵水溶液淬灭，随后用乙酸乙酯萃取(30毫升×3)。将有机相用30毫升饱和食盐水洗涤，经无水硫酸钠干燥过滤后，减压浓缩。粗产品经过制备HPLC(甲酸体系)纯化，得到4-(双(叔丁氧基羰基)氨基)-2-丁氧基-7-(5-(1-哌啶基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-羧酸甲酸盐(100.00毫克，收率：22.11％)。MS(ESI)m/z:734[M+H+].
步骤I：4-(双(叔丁氧基羰基)氨基)-2-丁氧基-7-(5-(1-哌啶基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-甲酰胺的合成。
向4-(双(叔丁氧基羰基)氨基)-2-丁氧基-7-(5-(1-哌啶基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-羧酸(80.00毫克，108.99微摩尔)的DMF(2毫升)溶液中，依次加入碳酸铵(104.73毫克，1.09毫摩尔)，三乙胺(27.57毫克，272.48微摩尔)和HATU(62.16毫克，163.49微摩尔)。反应混合物在氮气保护下，于30摄氏度搅拌12小时，然后倒入20毫升水中，并用乙酸乙酯萃取(20毫升×3)。将合并的有机相用20毫升饱和食盐水洗涤，用无水硫酸钠干燥，过滤后减压浓缩。残余物经制备TLC纯化(DCM/MeOH＝5/1)，得到4-(双(叔丁氧基羰基)氨基)-2-丁氧基-7-(5-(1-哌啶基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-甲酰胺(60毫克，粗品)。MS(ESI)m/z:733[M+H+].
步骤J：4-(双(叔丁氧基羰基)氨基)-2-丁氧基-7-(5-(1-哌啶基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈的合成。
在氮气保护，向三口烧瓶中依次加入4-(双(叔丁氧基羰基)氨基)-2-丁氧基-7-(5-(1-哌啶基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-甲酰胺(60.00毫克，81.85微摩尔)、三乙胺(372.72毫克，3.68毫摩尔)和2毫升二氯甲烷。0摄氏度下，逐滴加入三氟乙酸酐(343.83毫克，1.64毫摩尔)。滴加完毕后，反应物于30摄氏度搅拌12小时，然后用20毫升水稀释并用二氯甲烷萃取(10毫升×3)。将合并的有机相用10毫升饱和食盐水洗涤，经无水硫酸钠干燥、过滤，滤液减压浓缩，得到4-(双(叔丁氧基羰基)氨基)-2-丁氧基-7-(5-(1-哌啶基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(60.00毫克，粗品)。产物为淡黄色固体。MS(ESI)m/z:715[M+H+].
步骤K：4-氨基-2-丁氧基-7-(5-(1-哌啶基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐的合成。
4-(双(叔丁氧基羰基)氨基)-2-丁氧基-7-(5-(1-哌啶基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(60.00毫克，83.91微摩尔，)溶解于2毫升三氟乙酸中，反应物在氮气保护，30摄氏度下搅拌5小时。随后，减压下浓缩除去三氟乙酸。残余物用饱和碳酸氢钠水溶液碱化至pH＝8左右，并用乙酸乙酯萃取(10毫升×3)。有机相经无水硫酸钠干燥后减压浓缩，剩余物由制备HPLC(甲酸条件)分离纯化，得到4-氨基-2-丁氧基-7-(5-(1-哌啶基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐(19.00毫克)。产物为白色固体。1H NMR(400MHz,METHANOL-d4):δ8.18(s,1H),4.38(t,J＝6.5Hz,2H),3.65-3.42(m,3H),3.12-3.00(m,2H),2.85(t,J＝7.3Hz,3H),2.03-1.70(m,12H),1.63-1.37(m,4H),1.01(t,J＝7.4Hz,3H).MS(ESI)m/z:385[M+H+].
实例：1-(戊-1’-炔基)哌啶的制备
Figure PCTCN2015099375-appb-000024
将5-氯-1-戊炔(4.00克，39.00毫摩尔)和哌啶(16.60克，195.01毫摩尔)在60摄氏度下搅拌12小时。TLC监测显示反应完成。该反应物缓慢冷却至室温后，倒入30毫升水中并搅拌10分钟，再用乙酸乙酯萃取(30毫升×3)。将合并的有机相用水洗涤(30毫升×3)，经无水硫酸钠干燥、过滤，滤液减压浓缩，得到5-(1-哌啶基)-1-戊炔(5.20克，收率：88.15％)，产物为黄色液体。1H NMR(400MHz,CHLOROFORM-d):δ2.42-2.29(m,6H),2.19(dt,J＝2.6,7.2Hz,2H),1.92(t,J＝2.6Hz,1H),1.73-1.66(m,2H),1.58-1.53(m,4H),1.44-1.37(m,2H).
实例2
Figure PCTCN2015099375-appb-000025
4-氨基-((S)-1-甲基丁氧基)-7-(5-(哌啶-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐
步骤A：4-(双(叔丁氧基羰基)氨基)-2-((S)-1-甲基丁氧基)-7-(5-(1-哌啶基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈的合成。
4-(双(叔丁氧基羰基)氨基)-2-((S)-1-甲基丁氧基)-7-(5-(1-哌啶基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈根据实例1的制备中使用的方法，步骤C，D，E，F，G，H，I，J制备。其中将丁醇替换成(S)-1-甲基丁醇。
步骤B：4-氨基-2-((S)-1-甲基丁氧基)-7-(5-(1-哌啶基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈的合成。
将4-(双(叔丁氧基羰基)氨基)-2-((S)-1-甲基丁氧基)-7-(5-(1-哌啶基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(30.00毫克，54.87微摩尔)溶解于2毫升二氯甲烷中，随后加入0.5毫升三氟乙酸。反应混合物在室温下搅拌12小时，LC-MS检测反应完全后，将之缓慢倒入30毫升饱和碳酸氢钠水溶液中，并用二氯甲烷萃取(20毫升×3)。将合并的有机相用饱和食盐水洗涤(5毫升×2)，再经无水硫酸钠干燥、过滤，滤液减压浓缩，得到的4-氨基-2-((S)-1- 甲基丁氧基)-7-(5-(1-哌啶基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(30毫克，粗品)，直接用于下一步反应。MS(ESI)m/z:529[M+H+].
步骤C：4-氨基-2-((S)-1-甲基丁氧基)-7-(5-(1-哌啶基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐的合成。
将4-氨基-2-((S)-1-甲基丁氧基)-7-(5-(1-哌啶基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(30.00毫克，56.73微摩尔)溶于2毫升四氢呋喃中，一次性加入四丁基氟化铵(1M的四氢呋喃溶液，0.13毫升)。反应混合物在60摄氏度下搅拌12小时。LC-MS监测反应完全后，将反应混合物自然冷却至室温，随后缓慢倒入10毫升饱和碳酸氢钠水溶液中，然后用乙酸乙酯萃取(10毫升×3)。合并的有机相用饱和食盐水洗涤(5毫升×2)，经无水硫酸钠干燥、过滤，滤液减压浓缩。残余物经制备型HPLC(甲酸体系)纯化，得到4-氨基-2-((S)-1-甲基丁氧基)-7-(5-(1-哌啶基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐(3.80毫克，收率：16.81％)。产物为白色固体。1H NMR(METHANOL-d4,400MHz):δ8.56(br.s.,1H),5.23(dd,J＝6.3,12.6Hz,1H),2.89-3.22(m,6H),2.84(t,J＝7.3Hz,2H),1.38-1.95(m,16H),1.33(d,J＝6.0Hz,3H),0.97(t,J＝7.2Hz,3H).MS(ESI)m/z:399[M+H+].
实例3
Figure PCTCN2015099375-appb-000026
4-氨基-2-丁氧基-7-(4-(哌啶-1-基)丁基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐
4-氨基-2-丁氧基-7-(4-(哌啶-1-基)丁基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐根据实例1的制备中使用的方法，步骤E，F，G，H，I，J，K制备。其中将5-(1-哌啶基)-1-戊炔替换成4-(1-哌啶基)-1-丁炔。1H NMR(400MHz,METHANOL-d4):δ8.49(brs.,1H),4.34(t,J＝6.5Hz,2H),3.32-2.65(m,8H),1.96-1.45(m,14H),1.01(t,J＝7.4Hz,3H).
实例：4-(1-哌啶基)-1-丁炔的制备
Figure PCTCN2015099375-appb-000027
4-溴-1-丁炔(2.00克,15.04毫摩尔)溶解于2毫升丙酮中，冷却至0摄氏度后，再依次加入哌啶(1.28克,15.04毫摩尔)和碳酸铯(4.90克,15.04毫摩尔)。反应混合物在0摄氏度下搅拌5分钟后缓慢升至室温，并继续搅拌2小时。将混合物倒入20毫升水中，然后用二氯甲烷萃取(20毫升×2)。合并的有机相用水洗涤(30毫升×3)，经无水硫酸钠干燥、过滤，滤液减压浓缩后得到4-(1-哌啶基)-1-丁炔(600.00毫克,4.37毫摩尔,收率：29.07％)。产品为棕黑色液体。1H NMR(400MHz,CHLOROFORM-d):δ2.63-2.56(m,2H),2.49-2.37(m,6H),1.99(t,J＝2.7Hz,1H),1.61(m,4H),1.50-1.41(m,2H).
实例4-19由下面的通用路线2来制备。
实例4
Figure PCTCN2015099375-appb-000028
4-氨基-2-丁氧基-7-(5-(吗啉-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐
实例4由以下合成路线制备
Figure PCTCN2015099375-appb-000029
Figure PCTCN2015099375-appb-000030
步骤A：4-((双-叔丁氧羰基)-氨基)-2-丁氧基-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶的合成。
4-氨基-2-丁氧基-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶(50克，148.59毫摩尔)溶解于300毫升干燥的四氢呋喃中，随后在搅拌下依次加入三乙胺(16.54克，163.45毫摩尔)和4-N,N’-二甲氨基吡啶(907.65毫克，7.43毫摩尔)。将Boc2O(113.50克，0.52摩尔)溶解在另外200毫升的干燥四氢呋喃中，控制温度在20摄氏度左右，将该溶液缓慢滴加至前述反应液中。滴加完成后，反应液在20摄氏度下持续搅拌16小时。LC-MS监测反应转化完全。将该反应室温下液倒500毫升饱和的碳酸氢钠水溶液中，并用500毫升乙酸乙酯萃取。有机相依次用500毫升饱和氯化铵水溶液和500毫升水洗涤，并用无水硫酸钠干燥。过滤后减压蒸馏除去溶剂。将余下的固体置于300毫升正己烷中，强搅拌1小时后过滤。滤饼减压干燥后得到白色粉末状固体4-((双-叔丁氧羰基)-氨基)-2-丁氧基-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶(67.00克，124.83毫摩尔，收率：84.01％)。1H NMR(400MHz,CHLOROFORM-d)δ:7.46(d,J＝3.1Hz,1H),6.55(d,J＝3.3Hz,1H),5.38(s,2H),4.38(t,J＝6.6Hz,2H),3.34-3.46(m,2H),1.75-1.89(m,2H),1.47-1.58(m,2H),1.41(s,18H),0.97(t,J＝7.4Hz,3H),0.88(dd,J＝9.0,7.7Hz,2H),0.00(s,9H).
步骤B：4-((双-叔丁氧羰基)-氨基)-2-丁氧基-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯 并[3,2-d]嘧啶-6-羧酸的合成。
将4-((双-叔丁氧羰基)-氨基)-2-丁氧基-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶(87.00克，162.09毫摩尔)在溶解在870毫升干燥的四氢呋喃中，氮气保护下冷却-65摄氏度。随后控制反应体系内温度不高于-60摄氏度，缓慢滴加2M的(二异丙基)-胺基锂(LDA)的正己烷溶液(121.57毫升，243.14毫摩尔)。滴加完成后，控制反应体系温度在-60摄氏度左右，并持续搅拌1小时。然后，同样地，反应体系温度控制在不高于-60摄氏度范围内，持续有效地搅拌下，将干燥的二氧化碳气体缓慢通入反应液中，直至LC-MS监测反应完全(约持续通入气体2小时)。反应液用800毫升饱和氯化铵水溶液缓慢淬灭，并升至室温。随后用800毫升乙酸乙酯萃取。有机相用800毫升氯化铵水溶液洗涤两次，并用500毫升饱和氯化钠水溶液洗涤一次，经无水硫酸钠干燥，过滤后减压蒸馏除去溶剂，得到的红色油状物为4-((双-叔丁氧羰基)-氨基)-2-丁氧基-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-羧酸(94.13克，粗品)，并直接用于下一步反应。MS(m/z):581[M+H+].
步骤C：4-((双-叔丁氧羰基)-氨基)-2-丁氧基-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-甲酰胺的合成。
将上一步得到的94.13克粗品4-((双-叔丁氧羰基)-氨基)-2-丁氧基-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-羧酸溶解在600毫升四氢呋喃中，室温下依次加入HOBt(21.90克，162.09毫摩尔)和EDCI(31.07克，162.09毫摩尔)。该混合物在30-40摄氏度下搅拌1小时。0摄氏度下，将氨气通入的四氢呋喃溶液中至饱和，并在30-40摄氏度下，将250毫升该氨溶液缓慢滴加至前述混合物中。滴加完成后，反应液控制内温在30-40摄氏度范围内，持续搅拌16小时，至LC-MS监测反应已经完全。然后用800毫升饱和氯化铵水溶液淬灭反应，并用800毫升乙酸乙酯萃取。有机相用饱和氯化铵水溶液洗涤(800毫升×2)，用500毫升饱和氯化钠水溶液洗涤一次，经无水硫酸钠干燥后过滤，减压蒸馏除去溶剂得到褐黄色固体剩余物。该固体剩余物置于150毫升体积比正己烷:乙酸乙酯＝10:1的混合溶液中，强搅拌1小时后过滤。滤饼经减压干燥后得到4-((双-叔丁氧羰基)-氨基)-2-丁氧基-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-甲酰胺(55.80克，96.25毫摩尔，两步收率：59.38％)。产品为黄色固体粉末。1H NMR(400MHz,CHLOROFORM-d)δ:6.94(s,1H),5.81(s,2H),4.39(t,J＝6.6Hz,2H),3.43-3.56(m,2H),1.79-1.88(m,2H),1.48-1.57(m,2H),1.42(s,18H),0.98(t,J＝7.4Hz,3H),0.85-0.92(m,2H),0.00(s,9H).
步骤D：4-((双-叔丁氧羰基)-氨基)-2-丁氧基-7-碘-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-甲酰胺的合成。
将4-((双-叔丁氧羰基)-氨基)-2-丁氧基-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-甲酰胺(55.80克，96.25毫摩尔)溶解于560毫升DMF中，室温搅拌下分批加入NIS(43.31克，192.50毫摩尔)。加入完成后，该反应混合物升温至60-70摄氏度，并在该温度下持续搅拌72小 时。LC-MS监测反应完全后，将反应液冷却至室温，搅拌下缓慢倾倒入1500毫升饱和的硫代硫酸钠水溶液中，并用500毫升乙酸乙酯萃取。有机相依次用500毫升0.5M的氢氧化钠水溶液洗涤两次，300毫升饱和氯化钠水溶液洗涤一次，经无水硫酸钠干燥后过滤，减压蒸馏除去溶剂后得到褐色固体剩余物。该固体剩余物置于100毫升体积比正己烷:乙酸乙酯＝15:1的混合溶液中，强搅拌1小时后过滤。滤饼经减压干燥后得到4-((双-叔丁氧羰基)-氨基)-2-丁氧基-7-碘-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-甲酰胺(51.00克，72.27毫摩尔，收率：75.09％)。产物未浅黄褐色粉末。1H NMR(400MHz,CHLOROFORM-d)δ:6.60(br.s.,1H),6.14(br.s.,1H),5.71(s,2H),4.46(t,J＝6.6Hz,2H),3.43-3.54(m,2H),1.78-1.90(m,2H),1.53(d,J＝7.5Hz,2H),1.41(s,18H),0.99(t,J＝7.3Hz,3H),0.81-0.93(m,2H),0.00(s,9H).
步骤E：4-((双-叔丁氧羰基)-氨基)-2-丁氧基-7-碘-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈的合成。
持续搅拌下，将4-((双-叔丁氧羰基)-氨基)-2-丁氧基-7-碘-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-甲酰胺(51.00克，72.27毫摩尔)溶解至500毫升干燥的二氯甲烷中，并一次性加入三乙胺(36.57克，361.35毫摩尔)。反应体系在氮气保护下，维持反应体系内温在0摄氏度，缓慢滴加三氟乙酸酐(37.95克，180.68毫摩尔)，约需滴加半小时。滴加完成后，将反应液缓慢升温至20摄氏度，并继续搅拌16小时。LC-MS监测反应完全后，将该反应液用依次500毫升饱和碳酸氢钠水溶液洗涤两次，500毫升饱和氯化铵水溶液洗涤两次，饱和氯化钠水溶液洗涤一次，经无水硫酸钠干燥后过滤。减压蒸馏除去溶剂后得到黄色油状物为4-((双-叔丁氧羰基)-氨基)-2-丁氧基-7-碘-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈(49.70克，粗品)，并直接用于下一步反应。MS(m/z):688[M+H+].
步骤F：4-氨基-2-丁氧基-7-碘-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈的合成。
将49.70克4-((双-叔丁氧羰基)-氨基)-2-丁氧基-7-碘-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈粗品溶解于440毫升无水二氯甲烷中，在20-25摄氏度下缓慢加入84.15克三氟乙酸。反应混合物维持在20-25摄氏度下持续搅拌20小时。TLC监测反应完全后，减压蒸馏除去溶剂。剩余物溶解在600毫升二氯甲烷中，用饱和碳酸氢钠水溶液将混合物的pH值调节至8-9。分离有机相，并用300毫升饱和的氯化钠水溶液洗涤，经无水硫酸钠干燥后过滤，减压蒸馏至干燥。剩余物置于250毫升体积比正己烷:乙酸乙酯＝10:1的混合溶液中，强搅拌1小时后过滤。滤饼经减压干燥后得到4-氨基-2-丁氧基-7-碘-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈(30.80克，58.77毫摩尔，两步收率：81.31％)。产物为黄色固体。1H NMR(400MHz,CHLOROFORM-d)δ:6.08(br.s.,2H),5.64(s,2H),4.39(t,J＝6.6Hz,2H),3.67-3.76(m,2H),1.73-1.86(m,2H),1.48-1.56(m,2H),0.95-1.04(m, 5H),0.00(m,9H).
步骤G：4-氨基-2-丁氧基-7-(5-氯-1-戊炔基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈的合成。
干燥的三口烧瓶经氮气反复置换后，依次加入125毫升干燥的乙腈、4-氨基-2-丁氧基-7-碘-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈(10克，20.52毫摩尔)、三乙胺(6.23克，61.56毫摩尔)、碘化亚铜(390.74毫克，2.05毫摩尔)和Pd(PPh3)2Cl2(1.44克，2.05毫摩尔)。反应体系经氮气置换3次，并在持续的氮气保护下，室温(25摄氏度)搅拌16小时。LC-MS监测反应完全后，将反应混合物过滤。滤饼重新溶解在120毫升二氯甲烷中。随后加入约5克活性炭，室温下搅拌1小时后过滤。滤饼经二氯甲烷反复洗涤至TLC监测无荧光物。所有滤液合并后用稀氨水洗涤(200毫升×3)。有机相经无水硫酸钠干燥后过滤，滤液减压蒸馏除去溶剂。剩余物置于80毫升体积比正己烷:乙酸乙酯＝20:1的混合溶液中，强搅拌1小时后过滤。滤饼经减压干燥后得到4-氨基-2-丁氧基-7-(5-氯-1-戊炔基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈(8.20克，14.73毫摩尔，收率：71.78％)。产物为灰白色固体。1H NMR(400MHz,CHLOROFORM-d)δ:5.59(s,2H),4.38(t,J＝6.6Hz,2H),3.81(t,J＝6.3Hz,2H),3.64-3.72(m,2H),2.75(t,J＝6.8Hz,2H),2.13(t,J＝6.5Hz,2H),1.74-1.84(m,2H),1.51(d,J＝7.5Hz,2H),0.94-1.04(m,5H),0.00(s,9H).
步骤H：4-氨基-2-丁氧基-7-(5-氯-1-戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈的合成。
将4-氨基-2-丁氧基-7-(5-氯-1-戊炔基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈(8.00克，17.16毫摩尔)置于400毫升乙醇中，氮气置换3次后，一次性加入10％的干钯碳(6.00克)。然后氢气置换3次后，该反应混合物在氢气压力50psi环境，40摄氏度下持续搅拌16小时。TLC监测反应完全后，反应混合物冷却至室温，经硅藻土过滤，滤饼用二氯甲烷洗涤(200毫升×3)。合并的滤液减压蒸馏至干燥，得到4-氨基-2-丁氧基-7-(5-氯-1-戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈(8.00克，17.16毫摩尔)。产物为灰白色固体。1H NMR(400MHz,CHLOROFORM-d)δ:5.57(s,2H),4.33(t,J＝6.6Hz,2H),3.64-3.71(m,2H),3.54(t,J＝6.7Hz,2H),2.85(t,J＝7.5Hz,2H),1.74-1.90(m,6H),1.46-1.55(m,4H),0.94-1.03(m,5H),0.00(s,9H).
步骤I：4-氨基-2-丁氧基-7-(5-(吗啉-1-基)-戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈的合成：
将4-氨基-2-丁氧基-7-(5-氯-1-戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(100.00毫克,214.55微摩尔)溶解于5毫升乙腈中，依次加入吗啡林(56.07毫克,643.65微摩尔)，三乙胺(21.71毫克,214.55微摩尔)和碘化钠(3.22毫克,21.46微摩尔)。反应混合物升温至75-85摄氏度，并持续搅拌14小时。反应液冷却至室温后，用20毫升水稀释，并用乙酸乙酯萃取(20 毫升×2)。合并的有机相用30毫升饱和食盐水洗涤，经无水硫酸钠干燥，过滤后减压浓缩，得到的4-氨基-2-丁氧基-7-(5-(吗啉-1-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(102.00毫克，粗品)，直接用于下一步反应。MS(ESI)m/z:517[M+H+].
步骤J：4-氨基-2-丁氧基-7-(5-(1-吗啉基)-戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈的合成。
4-氨基-2-丁氧基-7-(5-(1-吗啉基)-戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈(102毫克，粗品)溶解于2毫升三氟乙酸中。该反应混合物在15-20摄氏度下搅拌14小时。LC-MS监测反应完全后，减压蒸馏除去溶剂。剩余物由制备HPLC(甲酸方法)纯化，冷冻干燥后得到47.89毫克4-氨基-2-丁氧基-7-(5-(1-吗啉基)-戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐。1H NMR(METHANOL-d4,400MHz):δ8.32(br.s.,2H),4.33(t,J＝6.6Hz,2H),3.88(d,J＝4.3Hz,4H),3.21(br.s.,4H),3.08-3.02(m,2H),2.82(t,J＝7.3Hz,2H),1.83-1.72(m,6H),1.55-1.39(m,4H),0.99(t,J＝7.4Hz,3H).MS(ESI)m/z:387[M+H+].
实例5
Figure PCTCN2015099375-appb-000031
7-(5-(2-氧杂-6-氮杂螺[3.3]庚烷-6-基)戊基)-4-氨基-2-丁氧基-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐
步骤A：4-氨基-2-丁氧基-7-(5-(2-氧杂-6-氮杂螺[3.3]庚烷-6-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈的合成
4-氨基-2-丁氧基-7-(5-(2-氧杂-6-氮杂螺[3.3]庚烷-6-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈根据实例4的制备中使用的方法，步骤I制备。其中将吗啡啉替换成2-氧杂-6-氮杂螺[3.3]庚烷。MS(ESI)m/z:529[M+H+].
步骤B：4-氨基-2-丁氧基-7-(5-(2-氧杂-6-氮杂螺[3.3]庚烷-6-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐的合成
向4-氨基-2-丁氧基-7-(5-(2-氧杂-6-氮杂螺[3.3]庚烷-6-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(49.00毫克，92.67微摩尔)的四氢呋喃(2.00毫升)溶液中加入四丁基氟化铵的四氢呋喃溶液(1M,185.34微升)。反应混合物加热至60摄氏度，并持续搅拌14小时。冷却至室温后，将反应混合物倒入20毫升水中，并用二氯甲烷/甲醇(10/1，20毫升×2)萃 取。合并的有机相用30毫升饱和食盐水洗涤，经无水硫酸钠干燥，过滤后减压浓缩。剩余物溶于4毫升甲醇中，并加入70毫克碳酸钾，在40摄氏度下搅拌1小时，冷却后过滤；滤液减压浓缩后，剩余物经制备HPLC(甲酸条件)分离纯化，冷冻干燥后得到4-氨基-2-丁氧基-7-(5-(2-氧杂-6-氮杂螺[3.3]庚烷-6-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐(15.59毫克，收率37.84％)，产物为白色固体。1H NMR(METHANOL-d4,400MHz):δ8.33(br.s.,2H),4.78(s,4H),4.46-4.17(m,6H),3.16-3.05(m,2H),2.81(t,J＝7.3Hz,2H),1.82-1.69(m,4H),1.63-1.36(m,6H),0.99(t,J＝7.4Hz,3H).MS(ESI)m/z:399[M+H+].
实例6
Figure PCTCN2015099375-appb-000032
4-氨基-2-丁氧基-7-(5-(4-甲基哌嗪-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐
4-氨基-2-丁氧基-7-(5-(4-甲基哌嗪-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐根据实例4的制备中使用的方法，步骤I，J制备。其中将吗啡啉替换成4-甲基哌嗪。1H NMR(METHANOL-d4,400MHz):δ8.23(d,J＝14.4Hz,2H),4.41-4.26(m,2H),3.29-2.83(m,8H),2.83-2.70(m,4H),2.62(s,3H),1.82-1.70(m,4H),1.70-1.60(m,2H),1.56-1.46(m,2H),1.45-1.36(m,2H),0.99(t,J＝7.4Hz,3H).MS(ESI)m/z:400[M+H+].
实例7
Figure PCTCN2015099375-appb-000033
4-氨基-2-丁氧基-7-(5-(5-甲基-2,5-二氮杂双环[2.2.1]庚烷-2-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐
4-氨基-2-丁氧基-7-(5-(5-甲基-2,5-二氮杂双环[2.2.1]庚烷-2-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐根据实例4的制备中使用的方法，步骤I，J制备。其中将吗啡啉替换成5-甲基-2,5-二氮杂双环[2.2.1]庚烷。1H NMR(METHANOL-d4,400MHz):δ8.26(br.s.,2H),4.34(t,J＝6.5Hz,2H),3.88(d,J＝18.3Hz,2H),3.22-2.77(m,7H),2.68(s,3H),2.17-2.02(m,2H),1.82-1.71(m,4H),1.70-1.14(m,7H),0.99(t,J＝7.4Hz,3H).MS(ESI)m/z:412[M+H+].
实例8
Figure PCTCN2015099375-appb-000034
4-氨基-2-丁氧基-7-(5-(二乙胺基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐
4-氨基-2-丁氧基-7-(5-(二乙胺基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐根据实例4的制备中使用的方法，步骤I，J制备。其中将吗啡啉替换成二乙胺盐酸盐，并将三乙胺的用量增加至3当量。1H NMR(METHANOL-d4,400MHz):δ8.37(br.s.,2H),4.32(t,J＝6.6Hz,2H),3.20(q,J＝7.3Hz,4H),3.14-3.05(m,2H),2.84(t,J＝7.3Hz,2H),1.86-1.67(m,6H),1.56-1.39(m,4H),1.29(t,J＝7.3Hz,6H),0.99(t,J＝7.3Hz,3H).MS(ESI)m/z:373[M+H+].
实例9
Figure PCTCN2015099375-appb-000035
4-氨基-2-丁氧基-7-(5-(4,4’-二氟-1-哌啶基)-戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐
4-氨基-2-丁氧基-7-(5-(4,4’-二氟-1-哌啶基)-戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐根据实例4的制备中使用的方法，步骤I，J制备。其中将吗啡啉替换成4,4’-二氟哌啶。1H NMR(400MHz,METHANOL-d4)δ8.21(s,1H),4.37(t,J＝6.5Hz,2H),3.37(t,J＝5.5Hz,4H),3.15-3.07(m,2H),2.82(t,J＝7.4Hz,2H),2.31(tt,J＝6.1,12.7Hz,4H),1.83-1.72(m,6H),1.55-1.40(m,4H),0.99(t,J＝7.4Hz,3H).MS(ESI)m/z:421[M+H+].
实例10
Figure PCTCN2015099375-appb-000036
4-氨基-2-丁氧基-7-(5-(1-四氢喹啉基)-戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐
4-氨基-2-丁氧基-7-(5-(1-四氢喹啉基)-戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐根据实例4的制备中使用的方法，步骤I，J制备。其中将吗啡啉替换成四氢喹啉。1H NMR(400MHz,METHANOL-d4)δ8.45(br.s.,1H),6.89-6.97(m,1H),6.84(d,J＝7.4Hz,1H),6.53(d,J＝8.3Hz,1H),6.46(t,J＝7.3Hz,1H),4.33(t,J＝6.6Hz,2H),3.18-3.29(m,4H),2.84(t,J＝7.3Hz,2H),2.69(t,J＝6.3Hz,2H),1.84-1.93(m,2H),1.72-1.83(m,4H),1.64(t,J＝7.5Hz,2H),1.47-1.54(m,2H),1.40(t,J＝7.5Hz,2H),0.99(t,J＝7.4Hz,3H).MS(ESI)m/z:433[M+H+].
实例11
Figure PCTCN2015099375-appb-000037
4-氨基-2-丁氧基-7-(5-(4-甲基-2-氧代哌嗪-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐
实例11由以下合成路线制备
Figure PCTCN2015099375-appb-000038
步骤A：4-氨基-2-丁氧基-7-(5-(4-苄氧羰基-2-氧代-哌嗪-1-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈的合成
4-苄氧羰基-2-哌嗪酮(190.00毫克，811.97微摩尔)溶解在5毫升无水四氢呋喃中，在氮气保护下，0摄氏度时一次性加入氢化钠(60％质量含量，35.73毫克，893.16微摩尔)。反应液在保持在0摄氏度搅拌1小时，随后加入4-氨基-2-丁氧基-7-[5-(1-氯)戊基]-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(250毫克，537.63微摩尔)。反应液自然升至室温后持续搅拌24小时，随后用5毫升饱和氯化铵水溶液淬灭，并加入20毫升水稀释，然后用乙酸乙酯萃取(20毫升×2)。合并的反应液用饱和食盐水洗涤，经无水硫酸钠干燥，过滤并减压浓缩滤液。剩余物用制备TLC(二氯甲烷：甲醇＝15:1)纯化，得到4-氨基-2-丁氧基-7-(5-(4-苄氧羰基-2-氧代-哌嗪-1-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(220mg，收率：61.62％)。MS(ESI)m/z:663[M+H+].
步骤B：4-氨基-2-丁氧基-7-(5-(2-氧代哌嗪-1-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈的合成
将4-氨基-2-丁氧基-7-(5-(4-苄氧羰基-2-氧代-哌嗪-1-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(200.00毫克,303.10微摩尔)溶于10毫升乙醇和5毫升甲醇的混合溶剂中。氩气保护下加入干钯碳(100毫克)。反应混合物在氢气压力50psi，10-15摄氏度下持续搅拌36小时，然后经硅藻土过滤，滤液减压浓缩后得到的黄色液体为4-氨基-2-丁氧基-7-(5-(2-氧代哌嗪-1-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(130.00mg，粗 品)，直接用于下一步反应。MS(ESI)m/z:530[M+H+].
步骤C：4-氨基-2-丁氧基-7-(5-(4-甲基-2-氧代-哌嗪-1-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈
4-氨基-2-丁氧基-7-(5-(2-氧代哌嗪-1-基)戊)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(130.00毫克,245.40微摩尔)溶解在3毫升四氢呋喃中，依次加入甲醛(22.11毫克,736.20微摩尔)，醋酸硼氢化钠(130.03毫克,613.50微摩尔)和催化量的醋酸。反应混合物在15-20摄氏度下搅拌3小时后，倒入10毫升饱和碳酸氢钠的水溶液中，并用10毫升乙酸乙酯萃取。有机相经无水硫酸钠干燥，过滤减压浓缩得到4-氨基-2-丁氧基-7-(5-(4-甲基-2-氧代-哌嗪-1-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(150.00mg,粗品)，直接用于下一步反应。MS(ESI)m/z:544[M+H+].
步骤D：4-氨基-2-丁氧基-7-(5-(4-甲基-2-氧代-哌嗪-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐的合成
4-氨基-2-丁氧基-7-(5-(4-甲基-2-氧代-哌嗪-1-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(150.00毫克,275.85微摩尔)溶解于1.5毫升三氟乙酸中。该溶液在15-20摄氏度下搅拌12小时后，减压浓缩除去三氟乙酸后，加入30毫升饱和碳酸氢钠水溶液中和，随后用30毫升乙酸乙酯萃取。有机相经无水硫酸钠干燥后，过滤减压浓缩得到褐色固体。将该固体溶于10毫升甲醇中，随后加入120毫克碳酸钾，该混合物在40-50摄氏度下搅拌一小时后，过滤。滤液减压浓缩，剩余物经制备HPLC(甲酸方法)纯化后得到4-氨基-2-丁氧基-7-(5-(4-甲基-2-氧代-哌嗪-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐(23.50毫克,46.74微摩尔,收率：16.94％)。1H NMR(400MHz,METHANOL-d4):δ8.21(br.s.,1H),4.39(t,J＝6.6Hz,2H),3.45-3.36(m,4H),3.14(s,2H),2.87-2.72(m,4H),2.40(s,3H),1.83-1.72(m,4H),1.64-1.51(m,4H),1.38-1.32(m,2H),1.01(t,J＝7.4Hz,3H).MS(ESI)m/z:414[M+H+].
实例12
Figure PCTCN2015099375-appb-000039
4-氨基-2-丁氧基-7-(5-(2-(S)-甲酰胺基-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈
步骤A：4-氨基-2-丁氧基-7-(5-(2-(S)-甲氧羰基-吡咯烷-1-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈的合成。
将4-氨基-2-丁氧基-7-(5-氯-1-戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈(100毫克，214.55微摩尔)加入至3毫升乙腈中，随后依次加入2-(S)-脯氨酸甲酯盐酸盐(71.07毫克，429.10微摩尔)、碳酸钾(88.96毫克，644.64微摩尔)和碘化钠(3.22毫克，21.46微摩尔)。反应混合物在75-85摄氏度下搅拌14小时后，LC-MS监测氯代物尚未反应完全。另外补加碳酸钾(88.96毫克，644.64微摩尔)后，反应混合物在75-85摄氏度下继续搅拌14小时。LC-MS监测氯代物的转化率达到72％。反应液冷却至室温后，倒入20毫升水中，并用乙酸乙酯萃取(20毫升×2)。合并的有机相用30毫升饱和氯化钠水溶液洗涤后，经无水硫酸钠干燥并过滤。滤液减压蒸馏除去溶剂，干燥后得到4-氨基-2-丁氧基-7-(5-(2-(S)-甲氧羰基-吡咯烷-1-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈(115.00毫克，粗品)，直接用于下一步反应。MS(ESI)m/z:559[M+H+]
步骤B：4-氨基-2-丁氧基-7-(5-(2-(S)-甲氧羰基-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈的合成。
将4-氨基-2-丁氧基-7-(5-(2-(S)-甲氧羰基-吡咯烷-1-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈(115.00毫克，粗品)溶解于2毫升三氟乙酸中，在15-20摄氏度下搅拌14小时。LC-MS监测反应完成。减压蒸馏除去溶剂后，剩余物倒入30毫升饱和碳酸氢钠水溶液中，并用二氯甲烷萃取(20毫升×2)。合并的有机相经无水硫酸钠干燥并过滤，滤液减压蒸馏除去溶剂，干燥后得到88.00毫克4-氨基-2-丁氧基-7-(5-(2-(S)-甲氧羰基-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈的粗品，直接用于下一步反应。MS(ESI)m/z:429[M+H+]。
步骤C：4-氨基-2-丁氧基-7-(5-(2-(S)-羧酸-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈的合成。
室温下，4-氨基-2-丁氧基-7-(5-(2-(S)-甲氧羰基-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈(88.00毫克，粗品)溶解于5毫升甲醇中，一次性加入一水合氢氧化锂(17.25毫克，410.70微摩尔)。反应混合物在室温下搅拌14小时。LC-MS监测反应完成。反应液倒入20毫升饱和氯化铵水溶液中，并用体积比10/1的二氯甲烷和甲醇的混合溶液萃取(20毫升×3)。合并的有机相由无水硫酸钠干燥，过滤后减压蒸馏除去溶剂。剩余物由制备HPLC纯化(甲酸方法)，冻干后得到4-氨基-2-丁氧基-7-(5-(2-(S)-羧酸-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈(22.07毫克，纯度：97％)。产物为白色固体。1H NMR(400MHz,METHANOL-d4)δ8.22(s,1H),4.34(t,J＝6.6Hz,2H),3.85(dd,J＝6.2,9.2Hz,1H),3.72(ddd,J＝3.8,7.1,11.1Hz,1H),3.28-3.19(m,1H),3.15-3.04(m,2H),2.80(t,J＝7.4Hz,2H), 2.42(qd,J＝8.7,13.5Hz,1H),2.17-2.04(m,2H),2.00-1.90(m,1H),1.83-1.69(m,6H),1.55-1.39(m,4H),0.99(t,J＝7.4Hz,3H).
步骤D：4-氨基-2-丁氧基-7-(5-(2-(S)-甲酰胺基-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈的合成。
室温下，4-氨基-2-丁氧基-7-(5-(2-(S)-羧酸-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈(50.00毫克，120.63微摩尔)溶解于3毫升DMF中，依次加入碳酸铵(115.91毫克，1.21毫摩尔)、三乙胺(24.41毫克，241.25微摩尔)和HATU(68.76毫克，180.95微摩尔)，随后密封。反应混合物在40-50摄氏度下搅拌16小时。LC-MS监测反应完成。反应液用20毫升水稀释，并用乙酸乙酯萃取(20毫升×2)。有机相合并，经无水硫酸钠干燥，过滤后滤液减压蒸馏除去溶剂。剩余物经制备HPLC分离纯化(甲酸方法)，冷冻干燥后得到20毫克4-氨基-2-丁氧基-7-(5-(2-(S)-甲酰胺基-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈，产物为白色固体。1H NMR(400MHz,METHANOL-d4)δ4.50(t,J＝6.5Hz,2H),4.18(dd,J＝7.0,9.2Hz,1H),3.77(dd,J＝4.0,7.2,11.3Hz,1H),3.29-3.12(m,3H),2.83(t,J＝7.4Hz,2H),2.66-2.49(m,1H),2.26-1.96(m,3H),1.87-1.67(m,6H),1.59-1.40(m,4H),1.02(t,J＝7.4Hz,3H).MS(ESI)m/z:414[M+H+].
实例13
Figure PCTCN2015099375-appb-000040
4-氨基-2-丁氧基-7-(5-(2-(R)-甲酰胺基-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈
4-氨基-2-丁氧基-7-(5-(2-(R)-甲酰胺基-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈根据实例12的制备方法，步骤A，B，C，D合成。其中将2-(S)-脯氨酸甲酯盐酸盐替换成2-(R)-脯氨酸甲酯盐酸盐。1H NMR(400MHz,METHANOL-d4)δ4.53(t,J＝6.53Hz,2H),4.17(dd,J＝7.03,9.03Hz,1H),3.72-3.82(m,1H),3.12-3.28(m,3H),2.83(t,J＝7.40Hz,2H),2.51-2.63(m,1H),1.97-2.26(m,3H),1.67-1.88(m,6H),1.40-1.59(m,4H),1.02(t,J＝7.40Hz,3H).MS(ESI)m/z:414[M+H+].
实例14
Figure PCTCN2015099375-appb-000041
4-氨基-2-丁氧基-7-(5-(2-(N,N’-二甲基甲酰胺基)-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐
步骤A：4-氨基-2-丁氧基-7-(5-(2-羧酸-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈的合成。
4-氨基-2-丁氧基-7-(5-(2--羧酸-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈根据实例12的制备方法，步骤A，B，C合成。其中将2-(S)-脯氨酸甲酯替换成2-(R,S)-脯氨酸甲酯。
步骤B：4-氨基-2-丁氧基-7-(5-(2-(N,N’-二甲基甲酰胺基)-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐的合成。
4-氨基-2-丁氧基-7-(5-(2-(N,N’-二甲基甲酰胺基)-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐根据实例12的制备方法，步骤D合成。其中将碳酸铵替换成二甲胺盐酸盐。1H NMR(400MHz,METHANOL-d4)δ8.41(br.s.,2H),4.59(dd,J＝6.6,9.5Hz,1H),4.34(t,J＝6.5Hz,2H),3.80-3.78(m,1H),3.25-3.10(m,3H),3.07(s,3H),3.03(s,3H),2.83(t,J＝7.3Hz,2H),2.69-2.55(m,1H),2.26-2.14(m,1H),2.04-1.93(m,2H),1.86-1.67(m,6H),1.58-1.38(m,4H),1.01(t,J＝7.4Hz,3H).MS(ESI)m/z:442[M+H+].
实例15
Figure PCTCN2015099375-appb-000042
4-氨基-2-丁氧基-7-(5-((2-(4-甲基哌嗪-1-基羰基))-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐
步骤A：4-氨基-2-丁氧基-7-(5-(2--羧酸-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈的合成。
4-氨基-2-丁氧基-7-(5-(2--羧酸-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈根据实例12的制 备方法，步骤A，B，C合成。其中将2-(S)-脯氨酸甲酯替换成2-(R,S)-脯氨酸甲酯。
步骤B：4-氨基-2-丁氧基-7-(5-((2-(4-甲基哌嗪-1-基羰基))-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐的合成。
4-氨基-2-丁氧基-7-(5-((2-(4-甲基哌嗪-1-基羰基))-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐根据实例12的制备方法，步骤D合成。其中将碳酸铵替换成4-甲基哌嗪。1H NMR(400MHz,METHANOL-d4)δ8.29(br.s.,3H),4.61(dd,J＝6.6,9.0Hz,1H),4.35(t,J＝6.6Hz,2H),3.87-3.49(m,5H),3.27-3.08(m,3H),2.84(t,J＝7.3Hz,2H),2.74-2.54(m,5H),2.46(s,3H),2.28-2.15(m,1H),2.10-1.95(m,2H),1.86-1.68(m,6H),1.59-1.40(m,4H),1.01(t,J＝7.4Hz,3H).MS(ESI)m/z:497[M+H+].
实例16
Figure PCTCN2015099375-appb-000043
4-氨基-2-丁氧基-7-(5-((2-环丙乙胺基羰基)-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐
步骤A：4-氨基-2-丁氧基-7-(5-(2-羧酸-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈的合成。
4-氨基-2-丁氧基-7-(5-(2-羧酸-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈根据实例12的制备方法，步骤A，B，C合成。其中将2-(S)-脯氨酸甲酯替换成2-(R,S)-脯氨酸甲酯。
步骤B：4-氨基-2-丁氧基-7-(5-((2-环丙乙胺基羰基)-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐的合成。
4-氨基-2-丁氧基-7-(5-((2-环丙乙胺基羰基)-吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐根据实例12的制备方法，步骤D合成。其中将碳酸铵替换成环丙基甲胺。1H NMR(400MHz,METHANOL-d4)δ8.66(br.s.,1H),4.59(t,J＝6.5Hz,2H),4.18(br.s.,1H),3.79(br.s.,1H),3.28-3.12(m,5H),2.85-2.72(m,2H),2.63-2.53(m,1H),2.24-2.00(m,3H),1.89-1.68(m,6H),1.59-1.43(m,4H),1.06-0.94(m,4H),0.57-0.51(m,2H),0.28-0.22(m,2H).MS(ESI)m/z:468[M+H+].
实例17
Figure PCTCN2015099375-appb-000044
4-氨基-2-丁氧基-7-(5-(2-甲酰胺基-哌啶-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈盐酸盐
4-氨基-2-丁氧基-7-(5-(2-甲酰胺基-哌啶-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈盐酸盐根据实例12的制备方法，步骤A，B，C，D合成。其中将2-(S)-脯氨酸甲酯盐酸盐替换成2-(R，S)-哌啶甲酸甲酯盐酸盐。并由制备HPLC分离纯化(盐酸方法)，冷冻干燥后得到产物。1H NMR(400MHz,METHANOL-d4)δ4.59(t,J＝6.5Hz,2H),3.91(dd,J＝2.9,12.1Hz,1H),3.72-3.65(m,1H),3.18-3.01(m,3H),2.87-2.80(m,3H),2.21(d,J＝14.6Hz,1H),1.95-1.45(m,14H),1.03(t,J＝7.4Hz,3H).MS(ESI)m/z:428[M+H+].
实例18
Figure PCTCN2015099375-appb-000045
4-氨基-2-丁氧基-7-(5-((1-甲酰胺基-1’-异丙基)-胺基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈盐酸盐
4-氨基-2-丁氧基-7-(5-((1-甲酰胺基-1’-异丙基)-胺基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈盐酸盐根据实例12的制备方法，步骤A，B，C，D合成。其中将2-(S)-脯氨酸甲酯盐酸盐替换成2-氨基-3-甲基-丁酸甲酯。并由制备HPLC分离纯化(盐酸方法)，冷冻干燥后得到产物。1H NMR(400MHz,METHANOL-d4)δ4.56(t,J＝6.6Hz,2H),3.74(d,J＝5.0Hz,1H),3.05-2.92(m,2H),2.86-2.83(m,2H),2.31-2.22(m,1H),1.88-1.69(m,6H),1.59-1.43(m,4H),1.14(d,J＝6.9Hz,3H),1.09(d,J＝6.9Hz,3H),1.03(t,J＝7.4Hz,3H).MS(ESI)m/z:416[M+H+].
实例19
Figure PCTCN2015099375-appb-000046
4-氨基-2-丁氧基-7-(6-(四氢吡咯-1-基)己基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐
步骤A：4-氨基-2-丁氧基-7-(6-氯-1-己基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈的合成。
4-氨基-2-丁氧基-7-(6-氯-1-己基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈根据实例4的制备方法，步骤G，H合成。其中将5-氯-1-戊炔替换成6-氯-1-己炔。
步骤B：4-氨基-2-丁氧基-7-(6-(四氢吡咯-1-基)己基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐的合成。
4-氨基-2-丁氧基-7-(6-(四氢吡咯-1-基)己基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐根据实例4的制备方法，步骤I，J合成。其中将4-氨基-2-丁氧基-7-(5-氯-1-戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈替换成4-氨基-2-丁氧基-7-(6-氯-1-己基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈，同时将吗啡啉替换成四氢吡咯。1H NMR(400MHz,METHANOL-d4)δ8.40(s,2H),4.34(t,J＝6.6Hz,2H),3.33(td,J＝1.6,3.3Hz,4H),3.21-3.11(m,2H),2.82(t,J＝7.3Hz,2H),2.08(br.s.,4H),1.86-1.67(m,6H),1.59-1.39(m,6H),1.01(t,J＝7.3Hz,3H).MS(ESI)m/z:385[M+H+].
实例20-21由通用路线3制备
实例20
Figure PCTCN2015099375-appb-000047
4-氨基-2-丁氧基-7-(5-(四氢吡咯-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈盐酸盐
实例20由以下合成路线制备:
Figure PCTCN2015099375-appb-000048
步骤A：4-氨基-2-丁氧基-7-(5-羟戊基-1-炔基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈的合成。
4-氨基-2-丁氧基-7-碘-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(1.5克，3.08毫摩尔)，4-戊炔-1-醇(0.52克，6.15毫摩尔)，二氯双(三苯基膦)钯(43.2毫克，61.55微摩尔)，碘化亚铜(58.61毫克，307.75微摩尔)和三乙胺(0.93克，9.23毫摩尔)置于25毫升乙腈中。反应体系用氮气置换后，在15-20摄氏度下搅拌14小时。将反应混合物倒入50毫升水中，用50毫升乙酸乙酯萃取两次。合并的有机相用50毫升饱和氯化铵水溶液洗涤一次，经无水硫酸钠干燥，过滤后减压浓缩。残余物通过硅胶色谱法纯化(柱高度：250毫米，直径：100毫米，100-200目硅胶，石油醚/乙酸乙酯＝3/1，1/1)，得到4-氨基-2-丁氧基-7-(5-羟戊基-1-炔基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈(1.20克，收率：87.83％)。MS(ESI)m/z:444[M+H+]。
步骤B：4-氨基-2-丁氧基-7-(5-羟基戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈的合成。
4-氨基-2-丁氧基-7-(5-羟戊基-1-炔基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(1.20克，2.71毫摩尔)溶解在40毫升乙醇中，氮气保护下，一次性加入50毫克10％的干 钯碳。反应体系用氢气置换5次，然后在氢气压力15psi，15-20摄氏度下搅拌14小时。TLC监测反应完全。反应混合物经硅藻土过滤，滤饼用二氯甲烷反复洗涤5次，滤液合并后减压浓缩，得到4-氨基-2-丁氧基-7-(5-羟基戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈(1.10克，收率：90.77％)。产品为黄色固体，直接用于下一步反。MS(ESI)m/z:448[M+H+].
步骤C：4-氨基-2-丁氧基-7-(5-氧代戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈的合成。
4-氨基-2-丁氧基-7-(5-羟基戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈(0.85克，1.90毫摩尔)溶解在50毫升二氯甲烷中，降温至0～5摄氏度，依次加入饱和碳酸氢钠(34毫升)，TEMPO(597.19毫克，3.80毫摩尔)，溴化钾(384.14毫克，3.23毫摩尔)和次氯酸钠水溶液(1.3M，4.38毫升)。反应混合物在0-5摄氏度下搅拌1小时后，倒入50毫升饱和硫代硫酸钠水溶液中，并用二氯甲烷萃取(50毫升×2)。合并的有机相依次用50毫升饱和硫代硫酸钠水溶液洗涤一次，饱和氯化铵水溶液洗涤3次(50毫升×3)，并用75毫升饱和氯化钠水溶液洗涤一次，有机相经无水硫酸钠干燥，过滤后减压浓缩，得到4-氨基-2-丁氧基-7-(5-氧代戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈(600毫克，粗品)。产品为黄色固体，直接用于下一步反应。MS(ESI)m/z:446[M+H+].
步骤D:4-氨基-2-丁氧基-7-(5-(吡咯烷-1-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈的合成。
将4-氨基-2-丁氧基-7-(5-氧代戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈(130毫克，291.72微摩尔)与吡咯烷(41.49毫克，583.44微摩尔)溶于5毫升四氢呋喃中，依次加入三乙酰氧基硼氢化钠(154.57毫克,729.30微摩尔)和醋酸(2-3滴)。该反应混合物在15-20摄氏度下搅拌14小时后，倒入30毫升饱和碳酸氢钠水溶液中，然后用乙酸乙酯萃取(20毫升×2)。合并的有机相用30毫升饱和氯化钠水溶液洗涤，经无水硫酸钠干燥，过滤后减压浓缩，得到4-氨基-2-丁氧基-7-(5-(吡咯烷-1-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈(146毫克，粗品)，直接用于下一步反应。MS(ESI)m/z:501[M+H+]。
步骤E：4-氨基-2-丁氧基-7-(5-(吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈盐酸盐的合成。
将4-氨基-2-丁氧基-7-(5-(吡咯烷-1-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈(146.00毫克，291.56微摩尔)溶解于2毫升三氟乙酸中，在15-20摄氏度下搅拌14小时后，缓慢倒入30毫升饱和碳酸氢钠水溶液中，并用乙酸乙酯萃取(20毫升×2)。合并的有机相用30毫升饱和氯化钠水溶液洗涤后，经无水硫酸钠干燥，过滤并减压浓缩。剩余物经制备HPLC(盐酸方法)分离纯化，冷冻干燥后得到的白色固体为4-氨基-2-丁氧基-7-(5-(吡咯烷-1-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈盐酸盐(53.03毫克，收率：49.09％)。1H NMR(METHANOL-d4,400MHz): δ4.57(t,J＝6.5Hz,2H),3.65(br.s.,2H),3.23-3.16(m,2H),3.08(d,J＝7.3Hz,2H),2.82(t,J＝7.4Hz,2H),2.19-2.10(m,2H),2.08-1.99(m,2H),1.86-1.69(m,6H),1.50(dt,J＝7.7,15.3Hz,4H),1.01(t,J＝7.4Hz,3H).MS(ESI)m/z:371[M+H+].
实例21
Figure PCTCN2015099375-appb-000049
4-氨基-2-丁氧基-7-(5-(8-甲基-3,8-二氮杂双环[3.2.1]辛-3-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐
实例21由以下合成路线制备:
Figure PCTCN2015099375-appb-000050
步骤A：4-氨基-2-丁氧基-7-(5-(8-叔丁氧羰基-3,8-二氮杂双环[3.2.1]辛-3-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈的合成。
4-氨基-2-丁氧基-7-(5-(8-叔丁氧羰基-3,8-二氮杂双环[3.2.1]辛-3-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈根据实例20的制备方法，步骤D合成。其中将吡咯烷替换成8-叔丁氧羰基-3,8-二氮杂双环[3.2.1]辛烷。
步骤B：4-氨基-2-丁氧基-7-(5-(3,8-二氮杂双环[3.2.1]辛-3-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈的合成。
将4-氨基-2-丁氧基-7-(5-(8-叔丁氧基羰基-3,8-二氮杂双环[3.2.1]辛-3-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(140.00毫克，218.1微摩尔)溶于8毫升二氯甲烷与1毫升三氟乙酸的混合溶剂中，在10-20摄氏度下搅拌14小时后，缓慢倒入30毫升饱和碳酸氢钠水溶液中，并用二氯甲烷萃取(20毫升×2)。合并的有机相用30毫升饱和氯化钠水溶液洗涤，经无水硫酸钠干燥，过滤后减压浓缩，得到4-氨基-2-丁氧基-7-(5-(3,8-二氮杂双环[3.2.1]辛-3-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(110毫克，粗品)，直接用于下一步反应。MS(ESI)m/z:542[M+H+].
步骤C：4-氨基-2-丁氧基-7-(5-(3-甲基-3,8-二氮杂双环[3.2.1]辛-8-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈的合成。
将4-氨基-2-丁氧基-7-(5-(3,8-二氮杂双环[3.2.1]辛-3-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(110毫克，203.03微摩尔)溶于5毫升四氢呋喃中，依次加入三乙酰氧基硼氢化钠(154.57毫克,729.30微摩尔)，37％甲醛水溶液(12.19毫克，406.06微摩尔)和催化量的醋酸。反应混合物在15-20摄氏度下搅拌14小时后，倒入30毫升饱和碳酸氢钠水溶液中，然后用乙酸乙酯萃取(25毫升×2)。合并的有机相用30毫升饱和氯化钠水溶液洗涤，经无水硫酸钠干燥，过滤后减压浓缩，得到4-氨基-2-丁氧基-7-(5-(3-甲基-3,8-二氮杂双环[3.2.1]辛-8-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(110毫克，粗品)，直接用于下一步反应。MS(ESI)m/z:556[M+H+].
步骤D：4-氨基-2-丁氧基-7-(5-(3-甲基-3,8-二氮杂双环[3.2.1]辛-8-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐的合成。
将4-氨基-2-丁氧基-7-(5-(3-甲基-3,8-二氮杂双环[3.2.1]辛-8-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(110.00毫克，197.90微摩尔)溶解于2毫升三氟乙酸中。反应混合物在15-20摄氏度下搅拌14小时。反应液减压浓缩，剩余物倒入30毫升饱和碳酸氢钠水溶液中，用体积比10/1的二氯甲烷和甲醇的混合溶液萃取3次(20毫升×3)。合并的有机相用无水硫酸钠干燥，过滤后减压浓缩。剩余物经制备HPLC(甲酸方法)分离纯化，冷冻干燥后得到的黄色固体为4-氨基-2-丁氧基-7-(5-(3-甲基-3,8-二氮杂双环[3.2.1]辛-8-基)戊基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐(24.80毫克，收率：26.57％)。1H NMR(METHANOL-d4,400MHz):δ8.33(s,2H),4.34(t,J＝6.6Hz,2H),3.80(br.s.,2H),2.88-2.78(m,4H),2.76(s,3H),2.48-2.38(m,4H),2.14-1.93(m,4H),1.79-1.70(m,4H),1.55-1.46(m,4H),1.42-1.34(m,2H),0.99(t,J＝7.4Hz,3H).MS(ESI)m/z:426[M+H+].
实例22-25由通用路线4来制备
实例22
Figure PCTCN2015099375-appb-000051
4-氨基-2-丁氧基-7-(4-(哌啶-2-基)丁基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐
步骤A：4-氨基-2-丁氧基-7-(4-(2-(1-叔丁氧羰基)-哌啶基)-丁基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈的合成。
4-氨基-2-丁氧基-7-(4-(2-(1-叔丁氧羰基)-哌啶基)-丁基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈根据实例1的合成方法，步骤E，F。其中将4-氨基-2-丁氧基-7-碘-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶替换成4-氨基-2-丁氧基-7-碘-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈，将1-(戊-1’-炔基)哌啶替换成4-(2-(1-叔丁氧羰基)-哌啶基)-1-丁炔。
步骤B：4-氨基-2-丁氧基-7-(4-(哌啶-2-基)丁基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈的合成。
4-氨基-2-丁氧基-7-(4-(2-(1-叔丁氧羰基)-哌啶基)-丁基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈(900.00毫克，1.50毫摩尔)溶解在2毫升三氟乙酸和20毫升二氯甲烷的混合液中，并在室温下搅拌12小时。LC-MS监测反应转化完全。反应液用20毫升饱和碳酸氢钠水溶液中和后，用二氯甲烷萃取(30毫升×3)。合并的有机相经无水硫酸钠干燥后，过滤。滤液减压蒸馏除去溶剂，得到4-氨基-2-丁氧基-7-(4-(哌啶-2-基)丁基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈(700.00毫克，粗品)。产品为白色固体，直接用于下一步反应。
步骤C：4-氨基-2-丁氧基-7-(4-(哌啶-2-基)丁基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐的合成。
4-氨基-2-丁氧基-7-(4-(哌啶-2-基)丁基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐根据实例4的合成方法，步骤J。其中将4-氨基-2-丁氧基-7-(5-(1-吗啉基)-戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈替换成4-氨基-2-丁氧基-7-(4-(哌啶-2-基)丁基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈。1H NMR(400MHz,METHANOL-d4)δ8.42(br.s.,1H),4.34(t,J＝6.53Hz,2H),3.38(br.s.,1H),2.91-3.14(m,2H),2.84(t,J＝7.15Hz,2H),1.34-2.10(m,16H),1.01(t,J＝7.40Hz,3H).MS(ESI)m/z:371[M+H+].
实例：4-(2-(1-叔丁氧羰基)-哌啶基)-1-丁炔的制备：
Figure PCTCN2015099375-appb-000052
4-(2-(1-叔丁氧羰基)-哌啶基)-1-丁炔
将4-(2-(1-叔丁氧羰基)-哌啶基)-1-丁醛(1.70克，7.04毫摩尔)溶解在30毫升无水甲醇中，室温下加入1-重氮基-1-二甲氧基磷酰基-丙酮(1.35克，7.04毫摩尔)。反应混合物在室温下搅拌12小时。TLC监测反应完全。反应混合物用30毫升水淬灭，并用的氯甲烷萃取(30毫升×3)。合并的有机相用无水硫酸钠干燥，过滤后滤液减压蒸馏除去溶剂，得到4-(2-(1-叔丁氧羰基)-哌啶基)-1-丁炔。1H NMR(400MHz,CHLOROFORM-d)δ4.32(d,J＝4.02Hz,1H),4.00(br.s.,1H),3.69(s,1H),2.77(t,J＝12.67Hz,1H),2.17(td,J＝3.14,7.53Hz,1H),1.94-2.04(m,1H),1.24-1.83(m,17H).
实例23
Figure PCTCN2015099375-appb-000053
4-氨基-2-丁氧基-7-(4-(1-(1-氮甲基羰基)哌啶-2-基)丁基)-5H-吡咯并[3,2-d]嘧啶-6-腈
步骤A：4-氨基-2-丁氧基-7-(4-(2-(1-氮甲基羰基)-哌啶基)-丁基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈的合成。
氮气保护下，将4-氨基-2-丁氧基-7-(4-(哌啶-2-基)丁基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈(200.00毫克，399.40微摩尔)和三乙胺(60.62毫克，599.10微摩尔)溶解于10毫升无水二氯甲烷中，逐滴加入氮甲基甲酰氯(44.82毫克，479.28微摩尔)。滴加完成后，反应混合物在室温下搅拌1小时。LC-MS监测反应完全。反应混合物用20毫升饱和碳酸氢钠水溶液淬灭，并用二氯甲烷萃取(30毫升×3)。合并的有机相用无水硫酸钠干燥，并过滤。滤液经减压蒸馏除去溶剂后，得到4-氨基-2-丁氧基-7-(4-(2-(1-氮甲基羰基)-哌啶基)-丁基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈(200毫克，粗品)，直接用于下一步反应。
步骤B：4-氨基-2-丁氧基-7-(4-(1-(1-氮甲基羰基)哌啶-2-基)丁基)-5H-吡咯并[3,2-d]嘧啶-6-腈的合成。
4-氨基-2-丁氧基-7-(4-(1-(1-氮甲基羰基)哌啶-2-基)丁基)-5H-吡咯并[3,2-d]嘧啶-6-腈根据实例4的合成方法，步骤J。其中将4-氨基-2-丁氧基-7-(5-(1-吗啉基)-戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈替换成4-氨基-2-丁氧基-7-(4-(2-(1-氮甲基羰基)-哌啶基)-丁基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈。1H NMR(300MHz,METHANOL-d4)δ8.20(s,1H),4.39(t,J＝6.53Hz,2H),4.12(br.s.,1H),3.77(d,J＝12.80Hz,1H),2.70-2.95(m,6H),1.46-1.88(m,12H),1.19-1.44(m,3H),1.01(t,J＝7.40Hz,3H).MS(ESI)m/z:428[M+H+].
实例24
Figure PCTCN2015099375-appb-000054
4-氨基-2-丁氧基-7-(3-(1-甲基-4-哌啶)丙基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐
步骤A：4-氨基-2-丁氧基-7-(3-(1-叔丁氧羰基-4-哌啶)丙基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈的合成。
4-氨基-2-丁氧基-7-(3-(1-叔丁氧羰基-4-哌啶)丙基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈根据实例1的合成方法，步骤E，F。其中将4-氨基-2-丁氧基-7-碘-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶替换成4-氨基-2-丁氧基-7-碘-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈，将1-(戊-1’-炔基)哌啶替换成3-(1-叔丁氧羰基-4-哌啶基)丙炔。
步骤B：4-氨基-2-丁氧基-7-(3-(1-甲基-4-哌啶)丙基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐的合成。
4-氨基-2-丁氧基-7-(3-(1-甲基-4-哌啶)丙基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐根据实例21的合成方法，步骤B，C，D。其中4-氨基-2-丁氧基-7-(5-(8-叔丁氧基羰基-3,8-二氮杂双环[3.2.1]辛-3-基)戊基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈替换成4-氨基-2-丁氧基-7-(3-(1-叔丁氧羰基-4-哌啶)丙基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)-吡咯并[3,2-d]嘧啶-6-腈。1H NMR(400MHz,METHANOL-d4)δ8.51(br.s.,1H),4.31(t,J＝6.5Hz,2H),3.43(d,J＝12.3Hz,2H),2.93(t,J＝12.2Hz,2H),2.83-2.77(m,5H),1.97(d,J＝14.3Hz,2H),1.81-1.34(m,11H),0.99(t,J＝7.4Hz,3H).MS m/z:371[M+H+].
实例：3-(1-叔丁氧羰基-4-哌啶基)丙炔的制备
Figure PCTCN2015099375-appb-000055
3-(1-叔丁氧羰基-4-哌啶基)丙炔
3-(1-叔丁氧羰基-4-哌啶基)丙炔由下面的合成路线制备：
Figure PCTCN2015099375-appb-000056
步骤A：2-(1-叔丁氧羰基-4-哌啶基)乙醇的合成。
将2-(4-哌啶基)乙醇(3.00克,23.22毫摩尔)溶解于30毫升二氯甲烷中。15-20摄氏度下，向此溶液中缓慢分批加入(Boc)2O(5.22克,23.92毫摩尔)。加完后，反应混合物在室温下搅拌20小时。TLC检测反应完成后，将此反应液缓慢倒入50毫升水中，并用二氯甲烷萃取(50毫升×2)。合并的有机相用无水硫酸钠干燥，过滤后减压浓缩。残余物经硅胶柱层析纯化(柱高度：250毫米，直径：20毫米，100-200目硅胶，石油醚/乙酸乙酯＝3/1，1/1)，得到2-(1-叔丁氧羰基-4-哌啶基)乙醇(4.89克，产率91.83％)，产物为无色液体。
步骤B：2-(1-叔丁氧羰基-4-哌啶基)乙醛的合成。
2-(1-叔丁氧羰基-4-哌啶基)乙醇(4.89克,21.32毫摩尔)溶解在50毫升二氯甲烷中，随后一次性加入Dess-Martin氧化剂(9.95克,23.46毫摩尔)。反应混合物在室温下搅拌2小时，随后反应液倒入250毫升10％的硫代硫酸钠水溶液和150毫升饱和碳酸氢钠水溶液的混合溶液中，并持续搅拌45分钟，然后用二氯甲烷萃取(100毫升×3)。合并的有机相依次用150毫升饱和碳酸氢钠水溶液和150毫升饱和氯化钠水溶液洗涤，经无水硫酸钠干燥，过滤后减压浓缩。得到的2-(1-叔丁氧羰基-4-哌啶基)乙醛(4.85克,粗品)，直接用于下一步反应。
步骤C：3-(1-叔丁氧羰基-4-哌啶基)丙炔的合成。
2-(1-叔丁氧羰基-4-哌啶基)乙醛(4.85克,21.34毫摩尔)溶解在50毫升甲醇中，并加入碳酸钾(5.90克,42.68毫摩尔)。该混合物在室温下搅拌5分钟后，逐滴加入(1-重氮基-2-氧代丙基)膦酸二甲酯(4.10克,21.34毫摩尔)。滴加完成后，反应混合物在室温下持续搅拌17小时，然后倒入250毫升二氯甲烷和50毫升饱和碳酸氢钠水溶液的混合液中。分离出的有机相经硅藻土垫过滤后浓缩。剩余物用50毫升乙酸乙酯溶解，溶液通过硅胶垫过滤，滤饼用乙酸乙酯洗涤(30毫升×3)。合并的滤液 减压浓缩干后得到3.92克3-(1-叔丁氧羰基-4-哌啶基)丙炔。1H NMR(400MHz,CHLOROFORM-d)δ4.12(d,J＝3.5Hz,2H),2.69(t,J＝11.8Hz,2H),2.15(dd,J＝2.6,6.7Hz,2H),1.99(t,J＝2.6Hz,1H),1.76(d,J＝13.3Hz,3H),1.46(s,9H),1.20(dq,J＝4.4,12.3Hz,2H).
实例25
Figure PCTCN2015099375-appb-000057
4-氨基-2-丁氧基-7-(3-(1-甲基-4-哌啶)-3’-羟基-丙基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐
实例24由下面的合成路线制备：
Figure PCTCN2015099375-appb-000058
步骤A:4-氨基-2-丁氧基-7-(3-羟基-3-(1-叔丁氧羰基-4-哌啶基)烯丙基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈的合成。
将3-(1-叔丁氧羰基-4-羟基-4-哌啶基)-1-丙烯(400.00毫克，1.66毫摩尔)和4-氨基-2-丁氧基-7-碘-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(734.79毫克，1.51毫摩尔)溶于15毫升干燥二氧六环中，然后一次性加入N，N-二异丙基乙胺(386.17毫克，2.99毫摩尔)和二(三叔丁基磷)钯(16.97毫克，33.20微摩尔)。反应体系经氮气置换3次，并在持续的氮气保护下，维持体系内温度为130摄氏度，微波条件下搅拌30分钟。LCMS监测反应完成后，将反应混合物 冷却至室温，随后倒入50毫升饱和碳酸氢钠水溶液中，并用60毫升乙酸乙酯萃取。有机相经80毫升饱和氯化钠水溶液洗涤，无水硫酸钠干燥，过滤后减压浓缩。剩余物经制备HPLC(三氟乙酸体系)分离纯化，冷冻干燥后得到的黄色固体为4-氨基-2-丁氧基-7-(3-羟基-3-(1-叔丁氧羰基-4-哌啶基)烯丙基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(160.00毫克，266.30微摩尔，收率：16.04％)。
步骤B:4-氨基-2-丁氧基-7-(3-羟基-3-(1-叔丁氧羰基-4-哌啶基)丙基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈的合成。
将4-氨基-2-丁氧基-7-(3-羟基-3-(1-叔丁氧羰基-4-哌啶基)烯丙基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(230.0毫克，382.81微摩尔)溶解于25毫升四氢呋喃中，氮气置换3次后一次性加入10％干钯碳(100毫克)。然后体系再用氢气置换3次，反应混合物在氢气压力15psi，10-15摄氏度下持续搅拌14小时。LCMS监测反应完成。反应混合物经硅藻土过滤，滤液减压蒸馏除去溶剂，得到的黄色固体为4-氨基-2-丁氧基-7-(3-羟基-3-(1-叔丁氧羰基-4-哌啶基)丙基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(190.00毫克，粗品)，并直接用于下一步反应。
步骤C:4-氨基-2-丁氧基-7-(3-羟基-(4-哌啶基)丙基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈的合成。
将4-氨基-2-丁氧基-7-(3-羟基-3-(1-叔丁氧羰基-4-哌啶基)丙基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(100.00毫克，165.88微摩尔)溶解于500微升三氟乙酸和4毫升二氯甲烷的混合溶剂中。反应液维持在10-15摄氏度下持续搅拌4.5小时。LCMS监测反应完成。将反应液用20毫升饱和碳酸氢钠水溶液淬灭，并用15毫升二氯甲烷萃取。有机相减压浓缩后得到的黄色固体为4-氨基-2-丁氧基-7-(3-羟基-(4-哌啶基)丙基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(80.00毫克，粗品)，并直接用于下一步反应。
步骤D:4-氨基-2-丁氧基-7-(3-羟基-(1-甲基-4-哌啶基)丙基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈的合成。
将4-氨基-2-丁氧基-7-(3-羟基-(4-哌啶基)丙基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(80.00毫克，159.13微摩尔)溶解于5毫升四氢呋喃中，依次加入甲醛(14.34毫克，477.39微摩尔)，乙酸(9.56毫克，159.13微摩尔)和醋酸硼氢化钠(84.32毫克，397.83微摩尔)。反应液维持在10-15摄氏度下持续搅拌2小时。LCMS监测反应完成。将反应液用15毫升水淬灭，并用20毫升二氯甲烷萃取。有机相经无水硫酸钠干燥，过滤并减压浓缩后得到的黄色固体为4-氨基-2-丁氧基-7-(3-羟基-(1-甲基-4-哌啶基)丙基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(90.00毫克，粗品)，并直接用于下一步反应。
步骤E:4-氨基-2-丁氧基-7-(3-羟基-(1-甲基-4-哌啶基)丙基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐的合成。
将4-氨基-2-丁氧基-7-(3-羟基-(1-甲基-4-哌啶基)丙基)-5-((2-(三甲基甲硅烷基)乙氧基)甲基)吡咯并[3,2-d]嘧啶-6-腈(90.00毫克，174.17微摩尔)溶解于1.5毫升三氟乙酸中，反应液维持在10-15摄氏度下持续搅拌5小时。LCMS监测反应完成。残余物减压浓缩去除大部分三氟乙酸后，经制备HPLC(甲酸方法)分离纯化，冷冻干燥后得到28.27毫克4-氨基-2-丁氧基-7-(3-羟基-(1-甲基-4-哌啶基)丙基)-5H-吡咯并[3,2-d]嘧啶-6-腈甲酸盐。1H NMR(400MHz,METHANOL-d4)δ8.24(br.s.,1H),4.36(t,J＝6.5Hz,2H),3.33-3.22(m,4H),2.91-2.80(m,5H),1.90-1.72(m,8H),1.64-1.47(m,4H),1.01(t,J＝8.0Hz,3H)。MS(ESI)m/z:387[M+H+].
实例：3-羟基-3-(1-叔丁氧羰基-4-哌啶基)-1-丙烯的合成
Figure PCTCN2015099375-appb-000059
3-羟基-3-(1-叔丁氧羰基-4-哌啶基)-1-丙烯
将1-叔丁氧羰基-4-哌啶酮(2.50克，12.55毫摩尔)溶解于25毫升干燥四氢呋喃中，反应体系在氮气保护下，维持反应体系内温在0摄氏度，缓慢滴加丙烯基溴化镁(1M，16.30毫升)，约在1小时左右滴加完。随后反应混合物在0摄氏度下持续搅拌2小时至TLC板监测反应转化完全。反应液用80毫升饱和氯化铵水溶液淬灭后自由升至室温，随后用100毫升乙酸乙酯萃取。有机相用100毫升饱和食盐水洗涤一次，经无水硫酸钠干燥，过滤后减压浓缩。剩余物通过硅胶柱色谱法纯化(柱高度：100毫米，直径：50毫米，100-200目硅胶，石油醚/乙酸乙酯＝4/1,2/1),得到的3-羟基-3-(1-叔丁氧羰基-4-哌啶基)-1-丙烯(750.00毫克，3.11毫摩尔，收率：19.08％)。产物为黄色液体。1H NMR(400MHz,CHLOROFORM-d)δ5.93-5.83(m,1H),5.27-5.14(m,2H),3.83(br.s.,2H),3.18(br.s.,2H),2.25(d,J＝7.5Hz,2H),1.61-1.50(m,4H),1.48(s,9H).
生物实验
实验1：Toll样受体7体外受体结合活性筛选方案
试剂:
HEK-blue hTLR7细胞
DMEM培养基
热灭活胎牛血清
抗支原体试剂Normocin
博来霉素
杀稻瘟菌素
方案：
1.96孔化合物板的准备:利用液体工作站POD将化合物从10毫摩尔/升浓度起始,用DMSO做3倍梯度稀释,共稀释10个点(从第2列到底11列,每个点2个重复).在第12列加入1微升5毫摩尔/升的阳性化合物R848作为阳性对照,在第1列加入1微升DMSO作为阴性对照.每孔中含有的DMSO体积都是1微升.
2.收取细胞培养瓶中的细胞,将细胞密度稀释成250,000个细胞/毫升.
3.加入200微升(50,000个细胞/孔)细胞悬液至准备好的化合物板中.每孔中DMSO终浓度为0.5％.
4.将含有细胞和化合物的培养板放入CO2培养箱中培养24小时,培养条件为37℃,5％CO2浓度.
5.培养24小时后,从细胞培养板中每孔取出20微升上清液转移到一块96孔透明检测板中.然后往检测板中每孔加入180微升Quanti-Blue试剂,并置于37度,5％CO2培养箱孵育1小时.
6.1小时后,用酶标仪OD650读板检测20微升上清液中碱性磷酸酶的含量.
7.利用Prism软件分析数据,得出各化合物的EC50.
Figure PCTCN2015099375-appb-000060
实验结果如表1所示：
表1
样品 EC50(nM) 样品 EC50(nM) 样品 EC50(nM)
GSK-2245035 A 9 B 18 A
1 A 10 D 19 A
2 A 11 B 20 A
3 A 12 A 21 B
4 A 13 A 22 B
5 A 14 A 23 B
6 A 15 B 24 A
7 C 16 B 25 D
8 A 17 A    
【注】EC50值的范围如下所示：100nM≥A≥1nM；250nM≥B>100nM；500nM≥C>250nM；500nM>D。
从上表的实验结果来看，本发明化合物与GSK-2245035具有同一水平的体外受体结合活性。
实验2：外周血单个核细胞试验方案
本方案的目的在于检测利用现有化合物物刺激人外周血单核细胞(PBMC)24小时后细胞因子的表达水平。检测时细胞上清液不稀释，直接检测IFN-α和TNF-α的水平。实验过程中首先将化合物配制成20毫摩浓度的DMSO储存液，用细胞培养基做10倍梯度稀释，总共稀释11个点。取其中9个稀释点的化合物(化合物的最高浓度为200微摩/升)加入96孔板中，每孔50微升，然后接种新鲜的人外周血单核细胞，每孔接种150微升体系，其中含有450,000个细胞。将细胞培养板置于37℃和5％的CO2培养箱中培养24小时，培养结束后将培养板以1200rpm的速度离心5分钟，随后收集上层血清，并将其储存于零下20℃以待检测。细胞因子的检测利用BD公司的流式液相多重蛋白定量技术(CBA)，在流式细胞仪上完成检测。利用上述检测方法，我们将刺激产生最低检测限至少3倍以上细胞因子水平的最低药物浓度，定义为该化合物在该细胞因子刺激实验上的MEC值。
实验结果：
样品1和2的IFN-α的值≤0.001nM；TNF-α的值≥1nM
实验3：鼻腔给药后小鼠体内干扰素-α(IFN-α)与肿瘤坏死因子-α(TNF-α)的诱导实验
将雌性Balb/c小鼠(体重18-20g)用异氟烷麻醉后，将化合物通过鼻腔吸入的方式进行给药(20微升滴在鼻孔间)，化合物预先溶解在含有0.2％吐温80的生理盐水中。2小时后，将小鼠用二氧化碳处死并进行心脏采血。将血样进行离心然后收取血清。血清样本进行适当稀释之后用ELISA进行检测。检测具体方法参照试剂盒里的说明书。在这个模型里，化合物诱导IFN-α/TNF-α的水平与化合物的给药量相关，同时给对照溶剂的组别样品中没有检测到IFN-α/TNF-α的水平。
Figure PCTCN2015099375-appb-000061
实验结果如表2所示：
表2
Figure PCTCN2015099375-appb-000062
从上表可以看出，本发明的化合物对干扰素IFN-α的诱导能力明显强于GSK2245035，并且在相对较低的给药浓度下，这一优势体现得尤为明显。

Claims (10)

  1. 式(I)所示化合物、其药学上可接受的盐、水合物或前药，
    Figure PCTCN2015099375-appb-100001
    其中，
    R选自任选被取代的C3-8烷基；
    m选自0、1、2、3或4；
    W选自任选被取代的：C3-8杂烷基、4～12元环烷基或杂环烷基或氨基酸；
    所述“杂”代表杂原子或杂原子团，分别独立地选自O、S、N、C(＝O)、S(＝O)或S(＝O)2；
    杂原子或杂原子团的数目分别独立地选自0、1、2、3或4。
  2. 根据权利要求1所述的化合物，其中所述C3-8烷基、C3-8杂烷基、4～12元环烷基或杂环烷基的取代基分别独立的选自F、Cl、Br、I、OH、CN、NH2、NH2C(＝O)、C1-4烷基或C1-4杂烷基、4～6元烷基或杂烷基、4～6元杂烷基-C(＝O)-，所述4～6元烷基或杂烷基任选被卤素、NH2、OH、CN或C1-4烷基取代；具体地，所述C3-8烷基、C3-8杂烷基、4～12元环烷基或杂环烷基的取代基分别独立的选自F、Cl、Br、I、OH、CN、NH2、NH2C(＝O)、Me、Et、
    Figure PCTCN2015099375-appb-100002
    “杂”如权利要求1所定义；
    取代基的数目独立地选自0、1、2或3。
  3. 根据权利要求1或2所述的化合物，其中所述的C3-8烷基选自：
    Figure PCTCN2015099375-appb-100003
  4. 根据权利要求1或2所述的化合物，其中所述C3-8杂烷基选自：-N(C1-4烷基)(C1-4烷基)。
  5. 根据权利要求1或2所述的化合物，其中所述4～12元环烷基或杂环烷基选自
    Figure PCTCN2015099375-appb-100004
    Figure PCTCN2015099375-appb-100005
    D1选自N或C(Ra)，D2-5分别独立地选自O、S、[C(Ra)(Rb)]1-2或N(Rc)，D3和D4中的1或2个还可以选自单键，Ra、Rb或Rc分别独立地选自H、C1-4烷基、C1-4杂烷基、卤素、OH、CN、NH2C(＝O)，任意两个Ra和Rb任选地连接到同一个原子上形成一个4～6元环烷基、氧杂4～6环烷基或氮杂4～6环烷基，所述4～6元环烷基任选地被1～3个C1-4烷基所取代。
  6. 根据权利要求1所述的化合物，其中W选自：
    Figure PCTCN2015099375-appb-100006
  7. 根据权利要求1所述的化合物，其选自：
    Figure PCTCN2015099375-appb-100007
    Figure PCTCN2015099375-appb-100008
  8. 根据权利要求1所述的化合物，其制备方法如下：
    方法1：
    Figure PCTCN2015099375-appb-100009
    方法2：
    Figure PCTCN2015099375-appb-100010
    其中，SEM代表2-(三甲基硅)乙氧基甲基，是一种氨基保护基。
  9. 根据权利要求1～7任意一项所述化合物、其药学上可接受的盐、水合物或前药在制备预防和治疗过敏性疾病和其他炎性病症、感染性疾病或癌症的药物中的应用。
  10. 根据权利要求9所述的应用，其中所述疾病选自过敏性鼻炎或哮喘。
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