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WO2015039587A1 - 一种免疫调节新型化合物、其用途和包含其的药物组合物 - Google Patents

一种免疫调节新型化合物、其用途和包含其的药物组合物 Download PDF

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WO2015039587A1
WO2015039587A1 PCT/CN2014/086538 CN2014086538W WO2015039587A1 WO 2015039587 A1 WO2015039587 A1 WO 2015039587A1 CN 2014086538 W CN2014086538 W CN 2014086538W WO 2015039587 A1 WO2015039587 A1 WO 2015039587A1
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fluoro
formula
isobutylphenyl
oxadiazol
reaction
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French (fr)
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郑伟
潘武宾
杨欣
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Suzhou Connect Biopharmaceuticals Ltd
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Suzhou Connect Biopharmaceuticals Ltd
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Priority to EP14846294.8A priority Critical patent/EP3048103B1/en
Priority to AU2014323822A priority patent/AU2014323822B2/en
Priority to US15/023,246 priority patent/US10280158B2/en
Priority to ES14846294T priority patent/ES2802977T3/es
Priority to KR1020167010722A priority patent/KR102372201B1/ko
Priority to JP2016543299A priority patent/JP6470757B2/ja
Priority to CA2928300A priority patent/CA2928300C/en
Priority to NZ719186A priority patent/NZ719186B2/en
Publication of WO2015039587A1 publication Critical patent/WO2015039587A1/zh
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    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/10—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing aromatic rings
    • A—HUMAN NECESSITIES
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4245—Oxadiazoles
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
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    • A61P25/00—Drugs for disorders of the nervous system
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the invention belongs to the field of medicine.
  • the present invention relates to novel compounds having S1P1 receptor agonist activity, pharmaceutical compositions comprising the same, and the compounds and pharmaceutical compositions for the preparation of a medicament for the treatment of diseases mediated by S1P1 receptor agonists Uses and uses in treatment-related diseases.
  • lymphocytes require the presence of a sphingosine-1-phosphate-like receptor (S1P1) to be derived from lymphoid tissues and enter the peripheral circulation, while S1P1 internalization prevents lymphocytes from being derived from lymphoid tissues and these important Immune cells are confined to lymphoid tissues.
  • S1P1 sphingosine-1-phosphate-like receptor
  • the S1P1 receptor agonist can reduce the ability of the human body to initiate an immune response by preventing the transport of lymphocytes, and thus can be used as an immunosuppressive agent for treating various autoimmune diseases.
  • FTY720 aka "Fingolimod”
  • Novartis is currently marketing FTY720 under the trade name Gilenya for the treatment of multiple sclerosis.
  • FTY720 has clinical efficacy, it is a non-selective S1P receptor agonist that activates several S1P receptors, such as S1P1, S1P2, S1P3, S1P4, and S1P5.
  • S1P1, S1P2, S1P3, S1P4, and S1P5 S1P receptor agonist
  • S1P1P3 the combination of FTY720 and S1P3 often leads to a series of important side effects, such as bradycardia. Therefore, in order to overcome the side effects of FTY720, many pharmaceutical companies and biotechnology companies have been looking for a more specific and safer second-generation S1P1 agonist.
  • the S1P1 receptor agonist In addition to increasing target specificity, shortening the half-life of the drug, the S1P1 receptor agonist, in vivo is also an important goal in screening second-generation S1P1 agonists (Pan et al., 2013, ACS Medicinal Chemistry Letters, 4, p333).
  • longer half-life small molecule drugs have been considered desirable because long half-lives can avoid frequent use of drugs.
  • an immunosuppressive drug a longer half-life can become a serious disadvantage because it will cause the lymphocyte transport to be continuously inhibited, and the number of peripheral blood lymphocytes will decrease, thereby making the patient's immune function low and increasing the virus. The risk of sexual infection.
  • the currently used S1P1 receptor agonist such as FTY720 has this defect.
  • the present inventors carried out medicinal chemical synthesis, and combined with pharmacokinetics and immune cell regulation in rats to screen a large number of synthesized compounds. It has been found that 1- ⁇ 4-[5-(4-isobutylphenyl)-1,2,4-oxadiazol-3-yl]-benzyl group has been described in the formula IA.
  • an object of the present invention to provide a novel compound which addresses the deficiencies in selectivity and half-life of existing S1P1 receptor agonists. Another object of the invention is to provide a pharmaceutical use of the compound. It is still another object of the present invention to provide a pharmaceutical composition comprising the compound as a main active ingredient. It is still another object of the present invention to provide a method of treating a disease using the compound or pharmaceutical composition. It is still another object of the present invention to provide a method of synthesizing the compound.
  • the invention provides a compound of formula I,
  • R is a halogen element or a C 1 -C 6 alkyl group.
  • R is F, Cl or Br; or R is a C 1 -C 3 alkyl group, preferably a methyl group.
  • the compounds provided by the present invention have agonist activity of the S1P1 receptor, and this activity is confirmed by detecting that each compound induces S1P1 receptor internalization and reduces the number of peripheral blood lymphocytes.
  • the compounds provided by the present invention also have the selective specificity of the S1P1 receptor, and in particular, each compound has no internalization-inducing effect on cells expressing the S1P3 receptor subtype.
  • pharmacokinetic experiments of the compounds of the present invention show that the half-life of some of the compounds is significantly shortened compared to the compounds of Formula IA, and much shorter than the current clinical drug FTY720.
  • the invention provides the use of a compound as described above for the manufacture of a medicament for the treatment of a disease or condition mediated by the SlP1 receptor.
  • the disease or condition is selected from the group consisting of rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, autoimmune disease, chronic inflammatory disease, asthma, inflammatory neuropathy, arthritis, transplantation, segmental gyrus Enteritis, ulcerative colitis, lupus erythematosus, psoriasis, ischemia-reperfusion injury, solid tumors, angiogenesis-related diseases, vascular diseases, pain conditions, acute viral diseases, inflammatory bowel diseases, insulin and non-insulin dependence Diabetes and other related immune diseases.
  • the disease or condition is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease and psoriasis.
  • treating in addition to curing a disease or condition mediated by the S1P1 receptor, "treating” as referred to herein also includes preventing the disease or condition as well as alleviating its symptoms and the like.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound provided herein and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutical composition described herein may be a pharmaceutical preparation itself, or may be formulated into a pharmaceutical preparation or a combination preparation with other adjuvants or drugs.
  • the pharmaceutical composition provided herein may be a tablet, a suppository, a dispersible tablet, an enteric coated tablet, a chewable tablet, an orally disintegrating tablet, a capsule, a sugar coating agent, a granule, a dry powder, an oral solution, or a small needle for injection. a lyophilized powder for injection or a large infusion solution; wherein the pharmaceutically acceptable adjuvant may be selected from the group consisting of a diluent, a solubilizer, a disintegrant, a suspending agent, a lubricant, a binder, a filler, and a flavoring agent. , sweeteners, antioxidants, surfactants, preservatives, encapsulants and pigments, to name a few.
  • the invention provides a method of treating a disease or condition mediated by an S1P1 receptor, The method comprises administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition provided herein.
  • the subject is a mammal.
  • the disease or condition is selected from the group consisting of rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, autoimmune disease, chronic inflammatory disease, asthma, inflammatory neuropathy, arthritis, transplantation, Crohn's disease , ulcerative colitis, lupus erythematosus, psoriasis, ischemia-reperfusion injury, solid tumors, angiogenesis-related diseases, vascular diseases, pain conditions, acute viral diseases, inflammatory bowel diseases, insulin and non-insulin dependent diabetes And other related immune diseases; preferably, the disease or condition is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, and psoriasis.
  • the compounds or pharmaceutical compositions provided herein can be co-administered with other therapies or therapeutic agents.
  • the dose of the compound or pharmaceutical composition required to effect a therapeutic, prophylactic or ameliorating effect will generally depend on the particular compound being administered, the patient, the particular disease or condition and its severity, the route and frequency of administration, and the like, and The division judges according to the specific circumstances.
  • the present invention provides a novel compound having S1P1 receptor agonist activity, wherein the compound is substituted at the 2-position of the compound of Formula IA with a halogen element, particularly fluorine, chlorine and bromine or Obtained by lower alkyl substitution.
  • a halogen element particularly fluorine, chlorine and bromine or Obtained by lower alkyl substitution.
  • the halogen-substituting compounds provided herein have a significantly reduced half-life compared to existing S1P1 receptor agonists and compounds of formula IA.
  • Pharmacokinetic experiments demonstrated that the half-life of these compounds can be significantly reduced from nearly 11 hours to less than 5.5 hours for the compound of formula IA.
  • both intravenous and oral administrations showed a significantly reduced half-life, consistent with a reduced mean residence time parameter.
  • substitution of specific substituents at specific positions is inventive.
  • the compound (Compound 5) obtained by substituting a lower alkyl group, particularly a methyl group, at the same position does not shorten the half-life, but has an effect on lymphocytes in vivo similar to a halogen-substituted compound.
  • the present invention also provides a synthesis method which is simple in reaction conditions, simple in post-treatment, high in yield, stable in process, and suitable for industrial production.
  • the present invention provides 1- ⁇ 2-fluoro-4-[5-(4-isobutylphenyl)-1,2,4-oxadiazol-3-yl]-benzyl as shown in Formula IB
  • the step (1) further comprises producing 2-fluoro-4-[5-(4-isobutylphenyl)-1,2,4- represented by the formula 1-5.
  • the crude product obtained is purified; preferably, purification is carried out by column chromatography or crystallization.
  • the crystallization solvent is selected from one or more of methanol, ethanol, acetone, dichloromethane, ethyl acetate and water, preferably a mixed solvent of methanol and water, further preferably a volume ratio of 3 A mixed solvent of methanol and water; preferably, the weight (in terms of g) of the crude product and the crystallization solvent is in a ratio of 1:3 to 20, preferably 1:5.
  • crystallization is carried out at 20 °C.
  • the reaction of the step (1) is carried out in a reaction solvent selected from one of acetonitrile, N-methylpyrrolidone and N,N-dimethylformamide or a variety of; reaction temperature of 80-140 ° C; 3-fluoro-N'-hydroxy-4-hydroxymethyl benzamidine represented by Formula 1-3 and 4-isobutylbenzoic acid of Formula 1-4
  • a reaction solvent selected from one of acetonitrile, N-methylpyrrolidone and N,N-dimethylformamide or a variety of
  • reaction temperature 80-140 ° C
  • the molar ratio is 1:1 to 2.0.
  • the reaction solvent of the step (1) is N,N-dimethylformamide
  • the reaction temperature is 130-140 ° C;
  • the molar ratio of 3-fluoro-N'-hydroxy-4-hydroxymethylbenzimidazole represented by Formula 1-3 to 4-isobutylbenzoic acid represented by Formula 1-4 is 1:1.
  • 1.5 is preferably 1:1 to 1.2.
  • the reaction of the step (2) is carried out in a reaction solvent selected from one or more of toluene, tetrahydrofuran and ethyl acetate;
  • a reaction solvent selected from one or more of toluene, tetrahydrofuran and ethyl acetate;
  • the ratio of ml is 1:10-30; the reaction temperature is 40-70 ° C; 2-fluoro-4-[5-(4-isobutylphenyl)-1,2,4 represented by formula 1-5
  • the molar ratio of -oxadiazol-3-yl]-benzyl alcohol to manganese dioxide is from 1:4 to 10.
  • the reaction solvent of the step (2) is ethyl acetate
  • 2-fluoro-4-[5-(4-isobutylphenyl)-1,2,4-oxadiazol-3-yl]-benzyl alcohol represented by Formula 1-5 is reacted with a reaction solvent
  • the weight (in g) volume (in ml) ratio is 1:10;
  • the reaction temperature is 60-70 ° C;
  • the molar ratio is 1:5 to 6, preferably 1:6.
  • the reaction of the step (3) is carried out in a reaction solvent of tetrahydrofuran and/or methanol; 2-fluoro-4-[5-( The molar ratio of 4-isobutylphenyl)-1,2,4-oxadiazol-3-yl]-benzaldehyde to 3-azetidinecarboxylic acid represented by Formula 1-7 is 1:0.5-6
  • the reaction temperature is 0-30 ° C; the reaction time is 1 to 16 hours.
  • the reaction solvent of the step (4) is methanol
  • 2-fluoro-4-[5-(4-isobutylphenyl)-1,2,4-oxadiazol-3-yl]-benzaldehyde represented by Formula 1-6 and Formula 1- 7 is a molar ratio of 3-azetidine carboxylic acid in a molar ratio of 1:1 to 1.1, preferably 1:1;
  • sodium cyanoborohydride is dissolved in methanol and added dropwise into the reaction system at a temperature of 0-20 ° C, preferably 15-20 ° C;
  • 2-fluoro-4-[ The molar ratio of 5-(4-isobutylphenyl)-1,2,4-oxadiazol-3-yl]-benzaldehyde to sodium cyanoborohydride is preferably 1:1;
  • the reaction temperature is 10-20 ° C, preferably 15-20 ° C;
  • the reaction time is from 4 to 16 hours.
  • the crude intermediate product may be purified in the step (1) preferably by crystallization purification instead of column chromatography.
  • the purification operation can be simplified and a large amount of solvent can be avoided.
  • the method of the present invention also adjusts the reactants, the solvent, the amount thereof, and the like of each reaction step.
  • step (2) the amount of manganese dioxide used can be reduced, thereby reducing the cost and using Ethyl acetate as a solvent also avoids the use of tetrahydrofuran, thereby avoiding the possible safety risks; using methanol as a solvent in step (3) can reduce by-products formed in the reaction, increase the reaction yield, and reduce the use in the reaction. The amount of solvent. Overall, the above improvements reduce costs and enable mass production of Formula IB (Compound 2) in an industrially low cost, high efficiency and safety.
  • FIG. 1 shows the results of pharmacokinetic experiments of the compound of the present invention in Example 6, wherein FIG. 1A is data of in vivo drug concentration changes with time after oral administration of the rats of Compounds 2, 3, and 4, and FIG. 1B is Compound 1 Data on the concentration of drug in vivo in rats after oral administration of 5 and 5 rats.
  • Fig. 2 shows the results of an experiment in which the compound 2 of the present invention in Example 8 lowers the number of peripheral blood lymphocytes.
  • Fig. 3 shows the results of experiments in which the compounds 3 and 4 of the present invention in Example 8 were used to reduce the number of peripheral blood lymphocytes.
  • Compounds 3 and 4 were administered at a dose of 0.1 mg/kg body weight.
  • Fig. 4 shows the results of an experiment in which the compound 5 of the present invention in Example 8 lowers the number of peripheral blood lymphocytes.
  • Compound 5 was administered at a dose of 0.1 mg/kg body weight.
  • Fig. 5 shows the results of an experiment in which the compound 2 of the present invention in Example 9 inhibits the development of joint swelling in arthritis.
  • Fig. 6 shows the results of an experiment in which the compound 2 of the present invention in Example 9 inhibits joint structure destruction in arthritis.
  • Figure 7 shows the results of an experiment in which the compound 2 of the present invention inhibits the development of EAE in Example 10.
  • the experimental animals used in this example and the following were male SD rats, aged between 7-9 weeks and weighing 186-231 g, all purchased from Beijing Vital Lihua Experimental Animal Technology Co., Ltd. After the animal was purchased, the veterinarian was responsible for quarantine (5 days quarantine period), after which the quarantined animals were selected and tested under SPF conditions with 3/test compounds of each group.
  • Oral administration group 2.74mg of compound 1-5 was weighed separately, and 9.13mL of 0.5% CMC-Na was used as a diluent to prepare a solution with a concentration of 0.3mg/mL. After mixing, vortex for 1-2 minutes, ultrasound 20 to 30 minutes until a uniform suspension was obtained, and the administration was carried out as an oral administration group, and the administration was carried out at 10 mL/kg body weight.
  • Intravenous administration group 1.61 mg of compound 1-5 was weighed, and 1.610 mL of 10% HP- ⁇ -CD was used as a diluent to prepare a solution having a concentration of 1 mg/mL, and after mixing, vortexing for 1 to 2 min, ultrasonic 28 ⁇ 30 min, as a bolus injection drug group. Administration was carried out at 1 mL/kg body weight.
  • Blood was collected at 0.0833 (5 min), 0.25 (15 min), 0.5, 1, 2, 4, 8, 24 h after administration, either orally or intravenously. At each time point, about 0.3 mL of whole blood was taken from the ocular venous plexus of the animal, and the blood was taken and anesthetized with isoflurane. After the end of all sample collection, the animals were euthanized.
  • the blood sample was collected, it was placed in an EP tube containing sodium heparin (about 10 ⁇ l, 1000 IU/mL), immediately placed in crushed ice, and then centrifuged at 4500 rpm for 5 minutes at a low temperature (4 ° C) to rapidly separate the plasma, and stored at -20 ° C or lower. analysis.
  • the concentration of each compound in the blood was analyzed by LC-MS/MS-001 (Q-trap-3200), and sulphate was used as an internal standard for analysis.
  • S1P1 small molecule agonists can prevent lymphocytes from entering the peripheral circulation by inducing internalization of the S1P1 receptor on the cell surface.
  • CHO-S cells expressing the human S1P1 receptor were used instead of lymphocytes as the S1P1 receptor internalization detection system.
  • a Myc tag sequence was fused at the N-terminus of the S1P1 receptor to first incubated the cells with a fluorescently labeled antibody against Myc tag, and then analyzed by flow cytometry. Receptor expression.
  • the compound 2 provided by the present invention was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution, and then a certain amount of the stock solution was further diluted with DMEM to various desired concentrations.
  • DMEM dimethyl sulfoxide
  • CHO-S cells expressing the human S1P1 receptor carrying the Myc tag were collected and adjusted to a density of one million cells per mL using Becco Modified Eagle Medium (DMEM) medium.
  • An equal volume of diluted Compound 2 was mixed with the cell suspension and incubated for one hour at 37 °C. After the end of the culture, the cells were harvested by centrifugation at 800 RPM for 5 minutes.
  • the cells were resuspended in FACS buffer (PBS containing 1% BSA), and fluorescein isothiocyanate (FITC)-labeled Myc antibody (Mexico Biotech, California) was placed and incubated on ice for one hour. .
  • FACS buffer PBS containing 1% BSA
  • FITC fluorescein isothiocyanate
  • the cells were washed and resuspended in pre-chilled FACS buffer and analyzed using a FACS Calibur flow cytometer.
  • Test data indicates that Compound 2 exhibited an activity of inducing S1P1 internalization in a dose-dependent manner (Table 3).
  • the S1P1 internalization inducing activity of the compounds 3 and 4 of the present invention was tested by the same method, and the results obtained were not significantly different from those of the compound 1, indicating that the substitutions of F, Cl and Br (compounds 2, 3 and 4) were all apparent. While shortening the half-life of the compound in vivo, it still has the activity of activating the S1P1 receptor.
  • the internalization detection experiment was carried out using CHO-S cells expressing the human S1P3 receptor subtype.
  • the experimental method was the same as the above S1P1 internalization detection method except for the cells.
  • the expression of the S1P1 receptor on the surface of lymphocytes is essential for lymphocytes to leave the secondary lymphoid tissue and then enter the peripheral circulation.
  • Small molecule agonists of the S1P1 receptor activate receptors and cause receptor internalization, a mechanism known to inhibit lymphocyte migration from secondary lymphoid tissue, which in turn affects the number of peripheral circulating lymphocytes.
  • in vivo lymphocyte influence experiments were performed.
  • Compound 2 was made into a suspension with carboxymethylcellulose (CMC-Na), and three Sprague-Dawley (SD) rats were orally administered. Blood samples (0.5 ml) were collected 30 minutes before dosing and at different time points after administration (collected in an appropriate amount of EDTA-2K solution EP tube), and blood samples were analyzed directly on an ADVIA 2120 hematology analyzer.
  • CMC-Na carboxymethylcellulose
  • SD Sprague-Dawley rats
  • the compounds 3, 4 and 5 of the present invention were tested in the same manner, and it was found that the effects of the compounds 3, 4 and 5 on lymphocytes in vivo were similar to those of the compound 2 (Fig. 3, Fig. 4).
  • Human rheumatoid arthritis is an autoimmune disease in which the patient's autoimmune system attacks joint tissue. Lymphocytes, including T cells and B cells, play an important role in the pathogenesis of this disease. It is known that the inhibition of T cell function by blocking T cell activation can effectively treat rheumatoid arthritis. Since Compound 2 blocks lymphocyte efflux, it is important to determine whether it is effective in inhibiting the development of arthritis in the CIA rat model. To this end, Lewis rats were first induced to develop the disease: rats were anesthetized with isoflurane, and then a total of 0.5 mL of CII/CFA emulsion was injected intradermally.
  • Emulsion injection was performed at three sites, one at the tail of the rat (0.1 mL) and the other two at the proximal end (0.2 mL/site). Seven days after the initial immunization, the same intensive immunization was performed intradermally, avoiding the previous injection site.
  • Compound 2 was prepared as a suspension in 0.5% CMC-Na and then orally administered to rats upon injection of CII/CFA.
  • the positive control group was treated with TOFACITINIB and orally administered on the 12th day after sensitization.
  • the severity of arthritis in the four-legged rats was scored twice a week starting 7 days after sensitization.
  • 0 is divided into no erythema and swelling
  • 1 is divided into erythema and slight swelling at the middle of the foot (tibia) or at the ankle joint
  • 2 is divided into erythema and slight swelling from the tendon to the middle of the foot
  • 4 is divided into erythema and severe swelling covering the ankle, foot and toe.
  • Joint swelling was measured by a paw volume meter after day 0 using a toe volume meter, and then measured twice a week from day 7 to day 28.
  • Joint destruction was measured by X-ray examination on the 28th day.
  • S1P1 antagonists have been found to be effective in human multiple sclerosis and MS animal models. Therefore assessment The efficacy of Compound 2 on experimental autoimmune encephalomyelitis (EAE), a mouse model of human multiple sclerosis. Eighty female C57BL/6 mice were randomly assigned to 8 groups according to body weight and then immunized. Each group consisted of 10 mice. To induce the disease, MOG 35-55 (MOG is a myeloid oligodendrocyte glycoprotein) was dissolved in saline to a concentration of 2 mg/mL and then emulsified in modified Complete Freund's adjuvant (CFA).
  • CFA Complete Freund's adjuvant
  • mice were anesthetized with isoflurane, and then 100 ⁇ L of the emulsion was subcutaneously injected into the back of the skin of the prepared skin, one along the midline of the back of the mouse, and the other two between the shoulders of the mouse. Both sides of the midline of the back.
  • all groups were intraperitoneally injected with pertussis toxin (200 ng in 200 ⁇ L of PBS).
  • the mice were clinically scored once daily from day 0 to day 30 after immunization to evaluate EAE development.
  • Compound 2 was made into a suspension of Na CMC, which was orally administered at the time of MOG immunization and continued to be administered throughout the study.
  • the data indicates that Compound 2 effectively inhibited the development of EAE at all three doses assessed (0.03, 0.1, and 1 mg/kg) (Figure 7).
  • FTY720 is a non-selective S1P1 antagonist that has been shown to cause a variety of cardiovascular effects, including bradycardia in humans.
  • a telemetry assay was performed in a conscious Beagle dog to evaluate the compound.
  • CMC-Na An appropriate amount of CMC-Na was weighed, and a 0.5% CMC-Na (w/v) solution was prepared using sterile water for injection. Prepared one day before administration.
  • the compound 2 (test sample) solution at a concentration of 2, 6, and 20 mg/mL was prepared on the day before the administration: an appropriate amount of the compound 2 was weighed, and an appropriate amount of a 0.5% sodium carboxymethylcellulose solution was added, and the dispersion emulsifier was used. The emulsion was homogenized, and the theoretical concentrations of the compound 2 in the test solution prepared were 2, 6, and 20 mg/mL.
  • This experiment shared 8 animals and was designed using a double Latin square experiment.
  • the dosing interval is 3 to 5 days.
  • the body weight of the animals was weighed 1 day before each round of administration and fasted overnight before the drug.
  • the telemetry system (Implanted Physiological Signal Telemetry System 1, Data Science International Inc., USA) was turned on, the test parameters were set, the implant was opened, and various physiological indexes of the animals were recorded. After the system is turned on for about 2 hours, it is administered in the order of the rounds of the table design. Blood pressure, ECG, body temperature and other indicators were collected within 24 hours after administration. In order to prevent possible system data overflow during the acquisition process, stop properly and then turn it back on, the switching process does not affect the value of the set data point.
  • Switching system time is recorded. After the next day's recording is completed, the telemetry system is turned off. Detection time point: within 1h before drug (-1h), 0.5h after administration ( ⁇ 5min), 1h ( ⁇ 10min), 1.5h ( ⁇ 10min), 2h ( ⁇ 15min), 3h ( ⁇ 15min), 4h ( ⁇ 15min), 8h ( ⁇ 45min), 24h ( ⁇ 1h). All data is automatically collected by PONEMAH Version 4.8 software. After the acquisition is completed, the analysis parameters are manually set, first analyzed automatically by PONEMAH Version 4.8 software, and then manually checked by point. The heart rate, blood pressure, respiration, body temperature index, etc.
  • the data at the detection time point is preferentially selected. If the noise is large at this point, or the heart rate is abnormal, or the waveform cannot be clearly recognized, the waveform with a clear signal within the given range is taken. If there is still no clear waveform available for analysis within the given range, then the analyzable data can be found before and after the point of the value, and is specified in the value table. Record the value time.
  • the data was processed by the statistical software SPSS13.0 used in this experiment. All statistical analyses were performed using two-tailed analysis and the statistical level was set at P ⁇ 0.05. Blood pressure, ECG, respiration, and body temperature indicators are expressed as “mean ⁇ standard deviation” and analyzed according to the following process: First, the data is homogenized by Levene Test. If the data is uniform (P>0.05), single factor is performed. Analysis of variance; if the analysis of variance was significant (P ⁇ 0.05), the difference between the vehicle group and the test group was compared by Dunnett's multiple comparisons. If the results of the Levene Test are significant (P ⁇ 0.05), the Kruskal-wallis nonparametric test is performed. If the Kruskal-wallis nonparametric test results are significant (P ⁇ 0.05), then the Mann-Whitney U test is used for the pairwise comparison.
  • the data of the time points with significant differences or significant changes are normalized to calculate the magnitude of the change.
  • b1 is the time value after the test drug
  • b0 is the pre-medication value of the test group
  • a1 is the value of the corresponding time point after the vehicle control drug
  • a0 is the pre-drug value of the vehicle
  • ⁇ % is the change range.
  • the crude compound of formula 1-5 was prepared and characterized according to Example 2 and found to have a purity of 77.25% by LCMS.
  • the obtained crude product was subjected to screening for crystallization purification conditions.
  • the crystallization operation is to dissolve the crude product with a crystallization solvent, and it is crystallized at 20 ° C, filtered, and dried in vacuo to give an off-white solid product as 2-fluoro-4-[5-(4-isobutyl) of formula 1-5. Phenyl)-1,2,4-oxadiazol-3-yl]-benzyl alcohol, purity by LCMS.
  • the above crystallization operation was carried out in accordance with the conditions listed in Table 6, to select a preferred amount of the mixed solvent.
  • the volume ratio of methanol to water in the mixed solvent was 3:1.
  • Step (2) of the method of the invention is carried out according to the following procedure:
  • the weight-to-volume ratio of the raw material solvent is 1 g: 10 ml, 1 g: 20 ml, and 1 g: 30 ml has little effect on the conversion and yield of the reaction, and in terms of cost, the ratio of using 1 g: 10 ml is selected as the reaction.
  • the amount of solvent used is 1 g: 10 ml, 1 g: 20 ml, and 1 g: 30 ml has little effect on the conversion and yield of the reaction, and in terms of cost, the ratio of using 1 g: 10 ml is selected as the reaction.
  • the amount of solvent used is 1 g: 10 ml, 1 g: 20 ml, and 1 g: 30 ml has little effect on the conversion and yield of the reaction, and in terms of cost, the ratio of using 1 g: 10 ml is selected as the reaction.
  • the amount of solvent used is 1 g: 10 ml, 1 g: 20 ml, and 1 g: 30 ml has
  • Step (3) of the method of the invention is carried out according to the following procedure:
  • the N,N-dimethylformamide was concentrated under reduced pressure.
  • the concentrate was dissolved in ethyl acetate (20L), and washed with water (15L ⁇ 2), saturated sodium hydrogen carbonate solution (15 L), and the organic phase was collected and used After drying over sodium sulfate, it was concentrated under reduced pressure to give 2.6 g of crude.

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Abstract

本发明提供一种式I所示的化合物，其中R为卤族元素或C1-C6烷基。所述化合物具有S1P1受体激动剂活性和选择特异性，并具有显著缩短的体内半衰期，因此是优质第二代S1P1受体激动剂。本发明还提供了所述化合物在制备用于治疗由S1P1受体介导的疾病或病症的药物中的用途，包含其的药物组合物，以及采用所述化合物和药物组合物治疗由S1P1受体介导的疾病或病症中的用途。

Description

一种免疫调节新型化合物、其用途和包含其的药物组合物 技术领域
本发明属于医药领域。具体而言，本发明涉及具有S1P1受体激动剂活性的新型化合物、包含所述化合物的药物组合物、以及所述化合物和药物组合物在制备由S1P1受体激动剂介导的疾病治疗药物中的用途和治疗相关疾病中的用途。
背景技术
本领域公知，淋巴细胞需要鞘氨醇-1-磷酸一类受体(S1P1)的存在来从淋巴组织导出并进入外周循环，而S1P1内化会阻止淋巴细胞从淋巴组织导出并将这些重要的免疫细胞限制于淋巴组织中。
大量研究已表明，存在多种S1P1受体激动剂，它们能够结合到淋巴细胞上表达的同源受体并导致S1P1受体内化，继而阻止淋巴细胞导出。所以，S1P1受体激动剂可通过阻止淋巴细胞的运输来降低人体启动免疫反应的能力，因而可以作为免疫抑制剂用于治疗各种自身免疫性疾病。
许多S1P1受体激动剂已被描述过，其中最典型的化合物是FTY720(又名“芬戈莫德”)。目前诺华公司正以Gilenya的商品名推广销售FTY720，以用于治疗多发性硬化症。尽管FTY720具有临床功效，但是其是一个非选择性的S1P受体激动剂，能激活若干S1P受体，如S1P1、S1P2、S1P3、S1P4、S1P5。其中，FTY720与S1P3的结合往往会导致一系列重要的副作用，如心动过缓。因此，为了克服FTY720的副作用，许多制药公司和生物技术公司一直在寻找更专一、更安全的第二代S1P1激动剂。
除了提高靶点专一性外，缩短药物即S1P1受体激动剂在体内的半衰期也是筛选第二代S1P1激动剂的重要目标(Pan等人，2013，ACS Medicinal Chemistry Letters,4,p333)。传统上，较长半衰期的小分子药物被认为可取的，因为半衰期长可以避免药品的频繁使用。然而，作为免疫抑制剂药物，较长半衰期可变成一个严重劣势，其原因在于其将会导致淋巴细胞的运输被持续抑制，外周血淋巴细胞数减少，从而使得用药者免疫功能低下，增加病毒性感染的风险。目前临床采用的S1P1受体激动剂如FTY720即有这一缺陷。在发生感染的情况下，往往需通过停止药物使得外周血淋巴细胞数尽快恢复 到正常水平，以便能够快速恢复人体免疫功能。FTY720在人体内的半衰期长达6-9天，因此即便停止服用该药物，需要在很长时间内淋巴细胞数才能恢复正常(Budde等人,2002，Journal of the American Society of Nephrology,13:1073-83)。
因此，目前本领域仍然需要开发新型的具有S1P1受体选择性、且半衰期较短的S1P1受体激动剂，以克服现有疗法的缺陷。
发明内容
为了解决上述技术问题，本发明人开展了药物化学合成，并结合大鼠体内药代动力学及免疫细胞调节等研究对合成的大量化合物进行了筛选。研究发现，在以式IA所示的已被描述的化合物1-{4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(Li等人,2005,Journal of Medicinal Chemistry,48(20)6169-6173；本文又称为“化合物1”)的2-位置上添加卤族元素或烷基可以获得新的化合物，这些化合物经由静脉和口服后，不仅保持了体外及体内免疫调节的药效，并且卤族元素取代所得的化合物经两种给药方式的半衰期均可以得到明显降低。
Figure PCTCN2014086538-appb-000001
因此，本发明的一个目的在于提供一种新型化合物，以解决现有S1P1受体激动剂在选择性、半衰期方面的不足。本发明的另一个目的在于提供所述化合物在制药方面的用途。本发明的又一个目的在于提供包含所述化合物作为主要活性成分的药物组合物。本发明的还一个目的在于提供采用所述化合物或药物组合物的疾病治疗方法。本发明的再一个目的在于提供所述化合物的合成方法。
为了实现上述目的，本发明采取的技术方案如下：
一方面，本发明提供一种式I所示的化合物，
Figure PCTCN2014086538-appb-000002
其中，R为卤族元素或C1-C6烷基。
优选地，R为F、Cl或Br；或者R为C1-C3烷基，优选甲基。
在R为F时，该化合物为1-{2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(本文又称为“化合物2”)，由下式IB表示：
Figure PCTCN2014086538-appb-000003
在R为Cl时，该化合物为1-{2-氯-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(本文又称为“化合物3”)，由下式IC表示：
Figure PCTCN2014086538-appb-000004
在R为Br时，该化合物为1-{2-溴-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(本文又称为“化合物4”)，由下式ID表示：
Figure PCTCN2014086538-appb-000005
在R为甲基时，该化合物为1-{2-甲基-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(本文又称为“化合物5”)，由下式IE表示：
Figure PCTCN2014086538-appb-000006
大量实验表明，本发明提供的化合物具有S1P1受体的激动剂活性，这一活性通过检测到各个化合物诱导S1P1受体内化以及减少外周血淋巴细胞数目而得到证实。同时，本发明提供的化合物还具有S1P1受体的选择特异性，特别是各个化合物对表达S1P3受体亚型的细胞没有内化诱导作用。进一步地，本发明化合物的药代动力学实验表明，相比于式IA所示化合物，部分化合物的半衰期得到显著缩短，更远远短于目前的临床药物FTY720。
因此，另一方面，本发明提供了上述化合物在制备用于治疗由S1P1受体介导的疾病或病症的药物中的用途。特别地，所述疾病或病症选自类风湿性关节炎、多发性硬化症、炎症性肠炎、自身免疫性疾病、慢性炎性疾病、哮喘、炎性神经病、关节炎、移植、节段性回肠炎、溃疡性结肠炎、红斑狼疮、牛皮癣、缺血-再灌注损伤、实体肿瘤、与血管生成有关的疾病、血管疾病、疼痛病症、急性病毒病、炎性肠病、胰岛素和非胰岛素依赖性糖尿病以及其它相关免疫疾病。优选地，所述疾病或病症选自多发性硬化症、类风湿性关节炎、炎症性肠炎和牛皮癣。
除治愈由S1P1受体介导的疾病或病症之外，本文所述的“治疗”还包括预防所述疾病或病症以及减轻其症状等等。
又一方面，本发明提供一种药物组合物，其包含本文提供的化合物和任选的药学上可接受的载体。本文所述的药物组合物本身可以是一种药用制剂，也可以再与其它辅料或药物配制成药用制剂或联合用药制剂。
具体而言，本文提供的药物组合物可以为片剂、栓剂、分散片、肠溶片、咀嚼片、口崩片、胶囊、糖衣剂、颗粒剂、干粉剂、口服溶液剂、注射用小针、注射用冻干粉针或大输液；其中，所述药学上可接受的辅料可以选自稀释剂、增溶剂、崩解剂、悬浮剂、润滑剂、粘合剂、填充剂、矫味剂、甜味剂、抗氧化剂、表面活性剂、防腐剂、包裹剂和色素等等。
还一方面，本发明提供一种治疗由S1P1受体介导的疾病或病症的方法， 所述方法包括给受试者施用治疗有效量的本文提供的化合物或者药物组合物。优选地，所述受试者为哺乳动物。
其中，所述疾病或病症选自类风湿性关节炎、多发性硬化症、炎症性肠炎、自身免疫性疾病、慢性炎性疾病、哮喘、炎性神经病、关节炎、移植、节段性回肠炎、溃疡性结肠炎、红斑狼疮、牛皮癣、缺血-再灌注损伤、实体肿瘤、与血管生成有关的疾病、血管疾病、疼痛病症、急性病毒病、炎性肠病、胰岛素和非胰岛素依赖性糖尿病以及其它相关免疫疾病；优选地，所述疾病或病症选自多发性硬化症、类风湿性关节炎、炎症性肠炎和牛皮癣。
本发明提供的化合物或药物组合物可以与其它疗法或治疗剂共同施用。并且，实施治疗、预防或减轻等作用所需的化合物或药物组合物的剂量通常取决于施用的具体化合物、患者、具体疾病或病症及其严重程度、给药途径和频率等，并且需要由主治医师根据具体情况判定。
综上所述，本发明提供了一种新型的具有S1P1受体激动剂活性的化合物，所述化合物为式IA所示化合物的2-位置上进行卤族元素特别是氟、氯和溴取代或者进行低级烷基取代而得到的。通过进行S1P1受体内化以及外周血淋巴细胞数目减少的效应实验，可以证明这些化合物具有S1P1受体的激动剂活性；此外，利用表达S1P3受体亚型的细胞的内化诱导实验还证明了这些化合物具有S1P1受体的选择性。
特别是，相比于现有S1P1受体激动剂和式IA所示化合物，本文提供的进行卤族元素取代的化合物还具有显著缩短的半衰期。药代动力学实验证明，这些化合物的半衰期可以从式IA所示化合物的近11小时显著降低到小于5.5小时。并且，静脉和口服两种给药方式都显示了显著降低的半衰期，同时也与降低的平均滞留时间参数相一致。并且，这种采用特定取代基在特殊位置进行取代是创造性的。另外，在相同位置以低级烷基、特别是甲基取代而获得的化合物(化合物5)虽然没有缩短半衰期，但是对体内淋巴细胞的作用与卤族元素取代的化合物相似。这些表明，本文提供的化合物均是潜在的优质第二代S1P1受体激动剂。
再一方面，对于式IB所示化合物(化合物2)，本发明还提供一种反应条件简单、后处理简便易操作、收率高、工艺稳定、并宜于工业化生产的合成方法。
简言之，本发明的合成路线如下：
Figure PCTCN2014086538-appb-000007
具体而言，本发明提供式IB所示的1-{2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(化合物2)的合成方法，所述合成方法包括以下步骤：
(1)在缩合剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和1-羟基苯并三唑的存在下，使式1-3所示的3-氟-N'-羟基-4-羟甲基苯甲脒与式1-4所示的4-异丁基苯甲酸反应生成式1-5所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇，
Figure PCTCN2014086538-appb-000008
(2)使步骤(1)得到的式1-5所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇与二氧化锰生成式1-6所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛，
Figure PCTCN2014086538-appb-000009
(3)以醋酸为催化剂，氰基硼氢化钠为还原剂，使步骤(3)得到的式1-6所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛与式1-7所示的3-吖丁啶羧酸反应生成式IB化合物，
Figure PCTCN2014086538-appb-000010
根据本发明的优选技术方案，所述步骤(1)还包括在生成式1-5所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇之后，纯化制得的粗品的步骤；优选地，采用柱层析或结晶操作进行纯化。
在采用结晶操作进行纯化时，结晶溶剂选自甲醇、乙醇、丙酮、二氯甲烷、乙酸乙酯和水中的一种或多种，优选为甲醇和水的混合溶剂，进一步优选为体积比为3:1的甲醇和水的混合溶剂；优选地，粗品与结晶溶剂的重量(以g计)体积(以ml计)比为1:3-20，优选为1:5。优选地，在20℃下进行析晶。
根据本发明的优选技术方案，所述步骤(1)的反应在反应溶剂中进行，所述反应溶剂选自乙腈、N-甲基吡咯烷酮和N,N-二甲基甲酰胺中的一种或多种；反应温度为80-140℃；式1-3所示的3-氟-N'-羟基-4-羟甲基苯甲脒与式1-4所示的4-异丁基苯甲酸的摩尔比为1:1～2.0。
优选地，所述步骤(1)的反应溶剂为N,N-二甲基甲酰胺；
优选地，反应温度为130-140℃；
优选地，式1-3所示的3-氟-N'-羟基-4-羟甲基苯甲脒与式1-4所示的4-异丁基苯甲酸的摩尔比为1:1～1.5，优选为1:1～1.2。
根据本发明的优选技术方案，所述步骤(2)的反应在反应溶剂中进行，所述反应溶剂选自甲苯、四氢呋喃和乙酸乙酯中的一种或多种；式1-5所示 的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇与反应溶剂的重量(以g计)体积(以ml计)比为1:10-30；反应温度为40-70℃；式1-5所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇与二氧化锰的摩尔比为1:4～10。
优选地，所述步骤(2)的反应溶剂为乙酸乙酯；
优选地，式1-5所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇与反应溶剂的重量(以g计)体积(以ml计)比为1:10；
优选地，反应温度为60-70℃；
优选地，式1-5所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇与二氧化锰的摩尔比为1:5～6，优选为1:6。
根据本发明的优选技术方案，所述步骤(3)的反应在反应溶剂中进行，所述反应溶剂为四氢呋喃和/或甲醇；式1-6所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛与式1-7所示的3-吖丁啶羧酸的摩尔比为1:0.5～6；反应温度为0-30℃；反应时间为1～16小时。
优选地，步骤(4)的反应溶剂为甲醇；
优选地，式1-6所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛与式1-7所示的3-吖丁啶羧酸的摩尔比摩尔比为1:1～1.1，优选为1:1；
优选地，将氰基硼氢化钠溶解于甲醇后滴加进入反应体系中，滴加温度为0-20℃，优选为15-20℃；式1-6所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛与氰基硼氢化钠的摩尔比优选为1:1；
优选地，反应温度为10-20℃，优选为15-20℃；
优选地，反应时间为4～16小时。
在本发明的式IB(化合物2)的合成方法中，在步骤(1)中可以优选结晶纯化，而非柱层析的操作，来纯化中间产物粗品。通过弃用柱层析这种溶剂用量大、易造成环境污染并提高成本的操作步骤，可以简化纯化操作，避免大量溶剂的使用。此外，本发明的方法还对各反应步骤的反应物、溶剂及其用量等进行了调整，例如在步骤(2)中可以采用减少的二氧化锰的使用量，由此降低了成本，而使用乙酸乙酯作为溶剂，还避免使用四氢呋喃，从而规避了可能产生的安全风险；在步骤(3)中采用甲醇作为溶剂，可以降低反应中生成的副产物，增加反应收率，减少了反应中使用的溶剂量。整体而言，上述改进降低了成本，实现了式IB(化合物2)在工业上低成本、高效率和安全性的大规模生产。
在本说明书中所引用的所有出版物，包括但不局限于专利和专利申请，在此将它们引入作为参考，就像每个单独的出版物具体地和单独地在此被充分阐述地引用作为参考。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1显示了实施例6中本发明化合物的药代动力学实验结果，其中图1A为化合物2、3、4的大鼠口服给药后体内药物浓度随时间变化的数据，图1B为化合物1和5的大鼠口服给药后体内药物浓度随时间变化的数据。
图2显示了实施例8中本发明化合物2降低外周血淋巴细胞数目的实验结果。
图3显示了实施例8中本发明化合物3和4降低外周血淋巴细胞数目的实验结果。图中，化合物3和4的给药剂量为0.1毫克/公斤体重。
图4显示了实施例8中本发明化合物5降低外周血淋巴细胞数目的实验结果。图中化合物5的给药剂量为0.1毫克/公斤体重。
图5显示了实施例9中本发明化合物2抑制关节炎中的关节肿胀发展的实验结果。
图6显示了实施例9中本发明化合物2抑制关节炎中的关节结构破坏的实验结果。
图7显示了实施例10中本发明化合物2抑制EAE发展的实验结果。
图8A至8C显示了实施例11中本发明化合物2对心电图指标影响的实验结果。
实施发明的最佳方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的药材原料、试剂材料等，如无特殊说明，均为市售购买产品。
实施例1 1-{4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(化合物1)的合成
1.1 (Z)-N'-羟基-4-羟甲基苯甲脒(1-3)
Figure PCTCN2014086538-appb-000011
向4-羟甲基苯腈(1-1,20g,150.38mmol)的甲醇(250mL)溶液中依次加入盐酸羟胺(1-2,20.903g,300.76mmol)，碳酸氢钠(50.5g,601.5mmol)，加热回流所得悬浊液5小时，冷却至室温，过滤，以甲醇(100mL)洗涤滤饼，浓缩所得滤液得到白色粗品，(Z)-N'-羟基-4-羟甲基苯甲脒(1-3,粗品24.8g,得率99.3％)，直接用于下一步。液相质谱显示分子离子峰为MS(ESI):m/z167.3[M+H]+核磁1HNMR(400MHz,CD3OD)δ:7.64(d,J＝8.0Hz,2H),7.40(d,J＝8.0Hz,2H),4.65(s,2H).
1.2 4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5)
Figure PCTCN2014086538-appb-000012
室温下，将4-异丁基苯甲酸(1-4,26.6g,149.4mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI,28.685g,149.4mmol)和1-羟基苯并三唑(20.169g,149.4mmol)的N,N-二甲基甲酰胺(200mL)溶液搅拌30分钟后加入(Z)-N'-羟基-4-羟甲基苯甲脒(1-3,24.8g,149.4mmol)，所得混合体系在140℃油浴中加热两小时，液相质谱检测原料反应完全，冷却至室温，减压蒸去大量N,N-二甲基甲酰胺，以水和乙酸乙酯萃取，所得有机相分别经0.5N盐酸溶液、饱和碳酸氢钠溶液、水洗涤，无水硫酸钠干燥后过滤，浓缩至干，经柱层析纯化后(石油醚～乙酸乙酯＝10/1～4/1体系洗脱)得白色固体产物4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,34.5g,得率75％)。液相质谱显示分子离子峰为MS(ESI):m/z 309.0[M+H]+核磁1HNMR(400MHz,CDCl3)δ:8.16(d,J＝8.4Hz,2H),8.12(d,J＝8.4Hz,2H),7.51(d,J＝8.4Hz,2H),7.33(d,J＝8.4Hz,2H),4.79(d,J＝5.2Hz,2H),2.57(d,J＝7.2Hz,2H),1.95(m,1H),1.85(t,1H),0.97(d,J＝7.2Hz,6H).
1.3 4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6)
Figure PCTCN2014086538-appb-000013
60度下，4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,17.7g,57.5mmol)和二氧化锰(50g,575mmol)的四氢呋喃(330mL)悬浊体系搅拌两小时，冷却至室温，过滤浓缩至干，经柱层析纯化后(石油醚～乙酸乙酯＝20/1体系洗脱)得白色固体产品，4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,16.44g,得率93.5％)。液相质谱显示分子离子峰为MS(ESI):m/z 307.2[M+H]+.
2. 1-{4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸
Figure PCTCN2014086538-appb-000014
室温下，将4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,10g,32.7mmol)、3-吖丁啶羧酸(1-7,3.63g,36mmol)和醋酸(15mL)的甲醇-四氢呋喃(200mL/200mL)溶液搅拌两小时，加入氰基硼氢化钠(1.03g,16.35mmol)的甲醇溶液(60mL)后室温继续搅拌16小时，过滤，以甲醇(90mL)洗涤滤饼，干燥得白色固体产品1-{4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(5.5g,回收1-6的还原产物1-5，再经氧化、还原胺化再得5g终产品，得率82％)。液相质谱显示分子离子峰为MS(ESI):m/z 392.2[M+H]+核磁1HNMR(400MHz,CD3OD)δ:8.23(d,J＝8.4Hz,2H),8.15(d,J＝8.0Hz,2H),7.64(d,J＝8.4Hz,2H),7.44(d,J＝8.0Hz,2H),4.34(s,2H),4.12(m,4H),3.42(m,1H),2.63(d,J＝7.2Hz,2H),1.97(m,1H),0.97(d,J＝7.2Hz,6H).
实施例2 1-{2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(化合物2)的合成
Figure PCTCN2014086538-appb-000015
1.1 2-氟-4溴苯甲醇(1-1)
Figure PCTCN2014086538-appb-000016
0度下，向盛有2-氟-4溴苯甲酸甲酯(4.66g,20mmol)的四氢呋喃(100mL)溶液中缓慢分批加入四氢锂铝(1.14g,30mmol)，滴完后撤去冰盐浴，室温搅拌1小时后反应完全(液相质谱和TLC板检测)，重新冷却至0度，分别以水(1.14mL)、10％氢氧化钠(11.4mL)淬灭反应，室温搅拌15分钟，过滤，分别以四氢呋喃(50mL×2)、乙酸乙酯EA(50mL×2)洗涤滤饼，所得滤液经无水硫酸钠干燥后。过滤浓缩得无色油状产物(3.4g,得率83％)。
1.2 3-氟-4-羟甲基苯腈(1-2)
Figure PCTCN2014086538-appb-000017
向2-氟-4溴苯甲醇(1-1,3.25g,15.85mmol)的DMF(35mL)溶液中加入氰化锌(1.85g,15.85mmol)、四(三苯基膦)钯(Pd(PPh3)4.0.916g,0.79mmol)，以氩气脱氧后在100度下加热反应16小时，冷却至室温，以乙酸乙酯(100mL)稀释，依次经水(100mL×3)、饱和盐水(100mL×3)洗涤之，经无水硫酸钠干燥后过滤，浓缩得粗品，再经柱层析纯化(石油醚～乙酸乙酯＝15/1～4/1体系洗脱)得到白色固体产物(0.72g,得率30％)。液相质谱显示分子离子峰为MS(ESI):m/z 152.1[M+H]+ 1HNMR(400MHz,CDCl3)δ:7.63(t,J＝7.6Hz,8.0Hz,1H),7.48(dd,J＝1.2Hz,8.0Hz,1H),7.48(dd,J＝1.2Hz,9.2Hz,1H),4.83(d,J＝10Hz,2H),2.00(t,J＝10Hz,1H).
1.3 (Z)-3-氟-N'-羟基-4-羟甲基苯甲脒(1-3)
Figure PCTCN2014086538-appb-000018
向3-氟-4-羟甲基苯腈(1-2,0.70g,4.64mmol)的甲醇(150mL)溶液中依次加入盐酸羟胺(0.645g,9.28mmol)、碳酸氢钠(1.56g,18.56mmol)，加热回流所得悬浊液5小时，冷却至室温，过滤，以甲醇(10mL)洗涤滤饼，浓缩所得滤液得到白色粗品，3-氟-N'-羟基-4-羟甲基苯甲脒(1-3,0.846g,得率99％)，直接用于下一步。液相质谱显示分子离子峰为MS(ESI):m/z 185.0[M+H]+ 1HNMR(400MHz,CD3OD)δ:7.51～7.45(m,2H),7.37～7.34(m,1H),4.67(s,2H).
1.4 2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5)
Figure PCTCN2014086538-appb-000019
室温下，将4-异丁基苯甲酸(1-4,0.819g,4.60mmol)、1-(3-二甲氨基丙 基)-3-乙基碳二亚胺盐酸盐(EDCI,0.882g,4.60mmol)和1-羟基苯并三唑(0.621g,4.60mmol)的N,N-二甲基甲酰胺(10mL)溶液搅拌30分钟后加入(Z)-3-氟-N'-羟基-4-羟甲基苯甲脒(1-3,0.846g,4.60mmol)，所得混合体系在140℃油浴中加热两小时，液相质谱检测原料反应完全，冷却至室温，减压蒸去大量N,N-二甲基甲酰胺，以水和乙酸乙酯萃取，所得有机相分别经0.5N盐酸溶液、饱和碳酸氢钠溶液、水洗涤，无水硫酸钠干燥后过滤，浓缩至干，经柱层析纯化后(石油醚～乙酸乙酯＝10/1～4/1体系洗脱)得白色固体产物2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,0.92g,得率61％)。液相质谱显示分子离子峰为MS(ESI):m/z 327.0[M+H]+ 1HNMR(400MHz,CDCl3)δ:8.11(d,J＝8.0Hz,2H),7.98(m,1H),7.86(m,1H),7.59(t,J＝8.0Hz,7.6Hz,1H),7.33(d,J＝8.0Hz,2H),4.85(s,2H),2.57(d,J＝6.8Hz,2H),1.93(m,1H),0.93(d,J＝6.8Hz,6H).
1.5 2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6)
Figure PCTCN2014086538-appb-000020
60度下，2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,0.91g,2.79mmol)和二氧化锰(2.43g,27.9mmol)的四氢呋喃(30mL)悬浊体系搅拌两小时，冷却至室温，过滤浓缩得白色固体产品，2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,0.90g,得率99.6％)。液相质谱显示分子离子峰为MS(ESI):m/z 325.0[M+H]+ 1HNMR(400MHz,CDCl3)δ:10.42(s,1H),8.12～7.99(m,5H),7.34(d,J＝7.2Hz,2H),2.58(d,J＝6.4Hz,2H),1.93(m,1H),0.93(d,J＝6.4Hz,6H).
1.6 1-{2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(化合物2)
Figure PCTCN2014086538-appb-000021
室温下，将2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,0.90g,2.78mmol)、3-吖丁啶羧酸(1-7,0.28g,2.78mmol)和醋酸(1mL)的甲醇-四氢呋喃(20mL/20mL)溶液搅拌两小时，加入氰基硼氢化钠(1.03g,16.35mmol)的甲醇溶液(60mL)后室温继续搅拌16小时，过滤，以甲醇(10mL)洗涤滤饼，干燥得白色固体产品1-{2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(化合物2)(0.20g,得率18％)。液相质谱显示分子离子峰为MS(ESI):m/z 410.0[M+H]+ 1HNMR(400MHz,CD3OD)δ:8.13(d,J＝8.4Hz,2H),8.05(m,1H),7.97(m,1H),7.68(t,J＝8.0Hz,7.6Hz,1H),7.42(d,J＝8.4Hz,2H),4.40(s,2H),4.15(m,4H),3.41(m,1H),2.61(d,J＝7.2Hz,2H),1.95(m,1H),0.94(d,J＝7.2Hz,6H).
实施例3 1-{2-氯-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(化合物3)的合成
1.1 2-氯-4溴苯甲酸甲酯(185312-82-7)
Figure PCTCN2014086538-appb-000022
0度下，向盛有2-氯-4溴苯甲酸(4.71g,20mmol)的甲醇(100mL)溶液中缓慢滴加氯化亚砜(3.57g,30mmol)，滴完后撤去冰盐浴，加热回流3小时,经TLC板和LCMS检测原料反应完全，旋蒸除去溶剂及过量的氯化亚砜，所得粗品经二氯甲烷(100mL)溶解，分别以饱和碳酸氢钠(100mL×2)，饱和盐水(100mL)洗之，经由无水硫酸钠干燥，过滤，旋蒸得黄色固体产物(4.79g,得率96％)。液相质谱显示分子离子峰为MS(ESI):m/z 248.9.8/250.8/252.8[M+H]+
1.2 2-氯-4溴苯甲醇(1-1)
Figure PCTCN2014086538-appb-000023
0度下，向盛有2-氯-4溴苯甲酸甲酯(4.78g,19.16mmol)的四氢呋喃(100mL)溶液中缓慢分批加入四氢锂铝(1.09g,30mmol)，滴完后撤去冰盐浴，室 温搅拌1小时后反应完全(液相质谱和TLC板检测)，重新冷却至0度，分别以水(1.09mL)、10％氢氧化钠(10.9mL)淬灭反应，室温搅拌15分钟，过滤，分别以四氢呋喃(50mL×2)、乙酸乙酯EA(50mL×2)洗涤滤饼，所得滤液经无水硫酸钠干燥后。过滤浓缩得无色油状产物(3.4g,得率80％)。液相质谱显示分子离子峰为MS(ESI):m/z 202.9/204.9[M-OH]+
1.3 3-氯-4-羟甲基苯腈(1-2)
Figure PCTCN2014086538-appb-000024
向2-氯-4溴苯甲醇(1-1,1.27g,5.73mmol)的DMF(15mL)溶液中加入氰化锌(0.67g,5.73mmol)、四(三苯基膦)钯(Pd(PPh3)4.0.33g,0.287mmol)，以氩气脱氧后在100度下加热反应16小时，冷却至室温，以乙酸乙酯(50mL)稀释，依次经水(50mL×3)、饱和盐水(50mL×3)洗涤之，经无水硫酸钠干燥后过滤，浓缩得粗品，再经柱层析纯化(石油醚～乙酸乙酯＝15/1～4/1体系洗脱)得到白色固体产物(0.387g,得率40％)。液相质谱显示分子离子峰为MS(ESI):m/z 168.0/170.1[M+H]+.
1.4 (Z)-3-氯-N'-羟基-4-羟甲基苯甲脒(1-3)
Figure PCTCN2014086538-appb-000025
向3-氯-4-羟甲基苯腈(1-2,0.387g,2.31mmol)的甲醇(80mL)溶液中依次加入盐酸羟胺(0.321g,4.62mmol)、碳酸氢钠(0.776g,9.24mmol)，加热回流所得悬浊液5小时，冷却至室温，过滤，以甲醇(10mL)洗涤滤饼，浓缩所得滤液得到白色粗品，3-氯-N'-羟基-4-羟甲基苯甲脒(1-3,0.324g,得率70％)，直接用于下一步。液相质谱显示分子离子峰为MS(ESI):m/z 201[M+H]+ 1HNMR(400MHz,DMSO-d6)δ:9.74(br,1H),7.68(s,1H),7.67(d,J＝8.4Hz,1H),7.54(d,J＝8.4Hz,1H),5.88(br,2H),5.49(br,1H),4.27(s,2H).
1.5 2-氯-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5)
Figure PCTCN2014086538-appb-000026
室温下，将4-异丁基苯甲酸(1-4,0.288g,1.62mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI,0.31g,1.62mmol)和1-羟基苯并三唑(0.219g,1.62mmol)的N,N-二甲基甲酰胺(8mL)溶液搅拌30分钟后加入(Z)-3-氯-N'-羟基-4-羟甲基苯甲脒(1-3,0.324g,1.21mmol)，所得混合体系在140℃油浴中加热两小时，液相质谱检测原料反应完全，冷却至室温，减压蒸去大量N,N-二甲基甲酰胺，以水和乙酸乙酯萃取，所得有机相分别经0.5N盐酸溶液、饱和碳酸氢钠溶液、水洗涤，无水硫酸钠干燥后过滤，浓缩至干，经柱层析纯化后(石油醚～乙酸乙酯＝10/1～4/1体系洗脱)得白色固体产物2-氯-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,0.36g,得率65％)。液相质谱显示分子离子峰为MS(ESI):m/z 343.0/345.0[M+H]+ 1HNMR(400MHz,CDCl3)δ:8.16(d,J＝1.2Hz,1H),8.10(d,J＝8.4Hz,2H),8.07(dd,J＝1.2Hz,8.0Hz,1H),7.66(d,J＝8.0Hz,1H),7.32(d,J＝8.4Hz,2H),4.85(s,2H),2.57(d,J＝7.2Hz,2H),0.94(d,J＝7.2Hz,6H).
1.6 2-氯-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6)
Figure PCTCN2014086538-appb-000027
40度下，2-氯-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,0.36g,1.05mmol)和二氧化锰(0.914g,10.5mmol)的四氢呋喃(30mL)悬浊体系搅拌两小时，冷却至室温，过滤浓缩得粗品，再经柱层析纯化(石油醚～乙酸乙酯＝20/1～10/1体系洗脱)得2-氯-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,0.34g,得率95％)。液相质谱显示分子离子峰为MS(ESI):m/z 341.1[M+H]+ 1HNMR(400MHz,CDCl3)δ:10.52(s,1H),8.28(d,J＝1.2Hz,1H),8.16(dd,J＝1.2Hz,8.4Hz,1H),8.10(d,J＝8.4Hz,2H),8.04(d,J＝8.4Hz,1H),7.33(d,J＝8.4Hz,2H),2.58(d,J＝7.6Hz,2H),0.94(d,J＝7.6Hz,6H).
1.7 1-{2-氯-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(化合物3)
Figure PCTCN2014086538-appb-000028
室温下，将2-氯-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,0.34g,1.0mmol)、3-吖丁啶羧酸(1-7,0.101g,1.0mmol)和醋酸(0.35mL)的甲醇-四氢呋喃(10mL/10mL)溶液搅拌两小时，加入氰基硼氢化钠(0.378g,6.0mmol)的甲醇溶液(20mL)后室温继续搅拌16小时，过滤，以甲醇(10mL)洗涤滤饼，干燥得白色固体产品1-{2-氯-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(化合物3)(0.109g,得率26％)。液相质谱显示分子离子峰为MS(ESI):m/z 426.1/428.3[M+H]+ 1HNMR(400MHz,CD3OD)δ:8.33(d,J＝1.6Hz,1H),8.22(dd,J＝1.6Hz,8.0Hz,1H),8.16(d,J＝8.0Hz,2H),7.76(d,J＝8.0Hz,1H),7.45(d,J＝8.0Hz,2H),4.72(s,2H),4.46(m,4H),3.74(m,1H),2.63(d,J＝7.2Hz,2H),1.97(m,1H),0.96(d,J＝7.2Hz,6H).
实施例4 1-{2-溴-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(化合物4)的合成
1.1 2,4-二溴苯甲酸甲酯(54335-33-0)
Figure PCTCN2014086538-appb-000029
0度下，向盛有2,4-二溴苯甲酸(5.60g,20mmol)的甲醇(100mL)溶液中缓慢滴加氯化亚砜(3.57g,30mmol)，滴完后撤去冰盐浴，加热回流3小时，经TLC板和LCMS检测原料反应完全，旋蒸除去溶剂及过量的氯化亚砜，所得粗品经二氯甲烷(100mL)溶解，分别以饱和碳酸氢钠(100mL×2)，饱和盐水(100mL)洗之，经由无水硫酸钠干燥，过滤，旋蒸得黄色固体产物(5.92g,得率100％)。液相质谱显示分子离子峰为MS(ESI):m/z 292.8/294.7/269.9[M+H]+
1.2 2,4-二溴苯甲醇(1-1)
Figure PCTCN2014086538-appb-000030
0度下，向盛有2,4-二溴苯甲酸甲酯(5.90g,20mmol)的四氢呋喃(120mL)溶液中缓慢分批加入四氢锂铝(1.14g,30mmol)，滴完后撤去冰盐浴，室温搅拌1小时后反应完全(液相质谱和TLC板检测)，重新冷却至0度，分别以水(1.14mL)、10％氢氧化钠(11.4mL)淬灭反应，室温搅拌15分钟，过滤，分别以四氢呋喃(60mL×2)，乙酸乙酯EA(60mL×2)洗涤滤饼，所得滤液经无水硫酸钠干燥后。过滤浓缩后经柱层析纯化(石油醚～乙酸乙酯＝10/1～4/1体系洗脱)得无色油状产物(2.3g,得率43％)。液相质谱显示分子离子峰为MS(ESI):m/z 246.9/248.9/250.9[M-OH]+
1.3 3-溴-4-羟甲基苯腈(1-2)
Figure PCTCN2014086538-appb-000031
向2,4-二溴苯甲醇(1-1,2.3g,8.65mmol)的DMF(20mL)溶液中加入氰化锌(1.01g,8.65mmol)、四(三苯基膦)钯(Pd(PPh3)4.0.50g,0.43mmol)，以氩气脱氧后在80度下加热反应5小时，冷却至室温，以乙酸乙酯(80mL)稀释，依次经水(80mL×3)、饱和盐水(80mL×3)洗涤之，经无水硫酸钠干燥后过滤，浓缩得粗品，再经柱层析纯化(石油醚～乙酸乙酯＝15/1～4/1体系洗脱)得到白色固体产物(0.81g,得率44％)。液相质谱显示分子离子峰为MS(ESI):m/z211.9/213.9[M+H]+ 1HNMR(400MHz,CDCl3)δ:7.82(s,1H),7.69(d,J＝8.0Hz,1H),7.65(d,J＝8.0Hz,1H),4.80(s,2H).
1.4 (Z)-3-溴-N'-羟基-4-羟甲基苯甲脒(1-3)
Figure PCTCN2014086538-appb-000032
向3-溴-4-羟甲基苯腈(1-2,0.80g,3.77mmol)的甲醇(120mL)溶液中依次加入盐酸羟胺(0.524g,7.54mmol)、碳酸氢钠(1.27g,15.08mmol)，加热回 流所得悬浊液5小时，冷却至室温，过滤，以甲醇(10mL)洗涤滤饼，浓缩所得滤液得到白色粗品，3-溴-N'-羟基-4-羟甲基苯甲脒(1-3,0.90g,得率97％)，直接用于下一步。液相质谱显示分子离子峰为MS(ESI):m/z 245/247[M+H]+.
1.5 2-溴-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5)
Figure PCTCN2014086538-appb-000033
室温下，将4-异丁基苯甲酸(1-4,0.653g,3.67mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI,0.704g,3.67mmol)和1-羟基苯并三唑(0.495g,3.77mmol)的N,N-二甲基甲酰胺(10mL)溶液搅拌30分钟后加入(Z)-3-溴-N'-羟基-4-羟甲基苯甲脒(1-3,0.90g,3.67mmol)，所得混合体系在140℃油浴中加热两小时，液相质谱检测原料反应完全，冷却至室温，减压蒸去大量N,N-二甲基甲酰胺，以水和乙酸乙酯萃取，所得有机相分别经0.5N盐酸溶液、饱和碳酸氢钠溶液、水洗涤，无水硫酸钠干燥后过滤，浓缩至干，经柱层析纯化后(石油醚～乙酸乙酯＝10/1～4/1体系洗脱)得白色固体产物2-氯-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,0.36g,得率36％)。液相质谱显示分子离子峰为MS(ESI):m/z 387.1/389.1[M+H]+.
1.6 2-溴-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6)
Figure PCTCN2014086538-appb-000034
50度下，2-溴-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,0.51g,1.32mmol)和二氧化锰(1.15g,13.2mmol)的四氢呋喃(30mL)悬浊体系搅拌两小时，冷却至室温，过滤浓缩得粗品，再经柱层析纯化(石油醚～乙酸乙酯＝20/1～10/1体系洗脱)得2-溴-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,0.34g,得率67％)。液相质谱显示分子离子峰为MS(ESI):m/z385.0/387.0[M+H]+.
1.7 1-{2-溴-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧 酸(化合物4)
Figure PCTCN2014086538-appb-000035
室温下，将2-溴-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,0.34g,0.88mmol)、3-吖丁啶羧酸(1-7,0.089g,0.88mmol)和醋酸(0.3mL)的甲醇-四氢呋喃(10mL/10mL)溶液搅拌两小时，加入氰基硼氢化钠(0.333g,5.28mmol)的甲醇溶液(20mL)后室温继续搅拌16小时，过滤，以甲醇(10mL)洗涤滤饼，干燥得白色固体产品1-{2-溴-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(化合物4)(0.1112g,得率27％)。液相质谱显示分子离子峰为.MS(ESI):m/z 469.9/471.8[M+H]+ 1HNMR(400MHz,CDCl3)δ:8.39(d,J＝1.2Hz,1H),8.12(dd,J＝1.2Hz,8.4Hz,1H),8.08(d,J＝8.4Hz,2H),7.70(d,J＝8.4Hz,1H),7.31(d,J＝8.4Hz,2H),4.23(s,2H),4.08(m,2H),3.99(m,2H),3.44(m,1H),2.56(d,J＝6.8Hz,2H),1.91(m,1H),0.93(d,J＝6.8Hz,6H).
实施例5 1-{2-甲基-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(化合物5)的合成
1.1 2-甲基-4溴苯甲醇(1-1)
Figure PCTCN2014086538-appb-000036
0度下，向盛有2-甲基-4溴苯甲酸甲酯(5.725g g,25mmol)的四氢呋喃(120mL)溶液中缓慢分批加入四氢锂铝(1.425g,37.5mmol)，滴完后撤去冰盐浴，室温搅拌1小时后反应完全(液相质谱和TLC板检测)，重新冷却至0度，分别以水(1.43mL)、10％氢氧化钠(14.3mL)淬灭反应，室温搅拌15分钟，过滤，分别以四氢呋喃(80mLx2)、乙酸乙酯EA(80mLx2)洗涤滤饼，所得滤液经无水硫酸钠干燥后，过滤浓缩得无色油状产物(4.535g,得率90％)。
1.2 3-甲基-4-羟甲基苯腈(1-2)
Figure PCTCN2014086538-appb-000037
向2-甲基-4溴苯甲醇(1-1,4.53g,22.5mmol)的DMF(50mL)溶液中加入氰化锌(2.63g,22.5mmol)、四(三苯基膦)钯(Pd(PPh3)4,1.31g,1.13mmol)，以氩气脱氧后在100度下加热反应16小时，冷却至室温，以乙酸乙酯(120mL)稀释，依次经水(120mL x3)、饱和盐水(120mL x3)洗涤之，经无水硫酸钠干燥后过滤，浓缩得粗品，再经柱层析纯化(石油醚～乙酸乙酯＝15/1～4/1体系洗脱)得到白色固体产物(2.80g,得率84％)。液相质谱显示分子离子峰为MS(ESI):m/z 148.1[M+H]+核磁1HNMR(400MHz,CDCl3)δ:7.57(d,J＝7.6Hz,1H),7.52(d,7.6Hz,1H),7.44(s,1H),4.76(d,J＝5.6Hz,2H),2.34(s,3H).
1.3 (Z)-3-甲基-N'-羟基-4-羟甲基苯甲脒(1-3)
Figure PCTCN2014086538-appb-000038
向3-甲基-4-羟甲基苯腈(1-2,2.8g,19mmol)的甲醇(500mL)溶液中依次加入盐酸羟胺(2.64g,38mmol)、碳酸氢钠(6.38g,76mmol)，加热回流所得悬浊液5小时，冷却至室温，过滤，以甲醇(100mLx2)洗涤滤饼，浓缩所得滤液得到白色粗品，3-甲基-N'-羟基-4-羟甲基苯甲脒(1-3,粗品3.425g,得率100％)，直接用于下一步。液相质谱显示分子离子峰为MS(ESI):m/z 181.0[M+H]+。
1.4 2-甲基-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5)
Figure PCTCN2014086538-appb-000039
室温下，将4-异丁基苯甲酸(1-4,3.382g,19mmol)、1-(3-二甲氨基丙 基)-3-乙基碳二亚胺盐酸盐(EDCI,3.642g,19mmol)和1-羟基苯并三唑(2.565g,19mmol)的N,N-二甲基甲酰胺(60mL)溶液搅拌30分钟后加入(Z)-3-甲基-N'-羟基-4-羟甲基苯甲脒(1-3,3.42g,19mmol)，所得混合体系在140℃油浴中加热两小时，液相质谱检测原料反应完全，冷却至室温，减压蒸去大量N,N-二甲基甲酰胺，以水和乙酸乙酯萃取，所得有机相分别经0.5N盐酸溶液、饱和碳酸氢钠溶液、水洗涤，无水硫酸钠干燥后过滤，浓缩至干，经柱层析纯化后(石油醚～乙酸乙酯＝10/1～4/1体系洗脱)得白色固体产物2-甲基-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,2.51g,得率41％)。液相质谱显示分子离子峰为MS(ESI):m/z 323.0[M+H]+核磁1HNMR(400MHz,CDCl3)δ:8.12(d,J＝8.4Hz,2H),7.98(m,2H),7.59(d,J＝8.0Hz,1H),7.32(d,J＝8.4Hz,2H),4.77(s,2H),2.57(d,J＝7.2Hz,2H),2.42(s,3H),1.93(m,1H),0.92(d,J＝7.2Hz,6H).
1.5 2-甲基-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6)
Figure PCTCN2014086538-appb-000040
60度下，2-甲基-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,2.5g,7.76mmol)和二氧化锰(6.75g,77.6mmol)的四氢呋喃(100mL)悬浊体系搅拌两小时，冷却至室温，过滤浓缩得白色固体产品，2-甲基-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,2.4g,得率97％)。液相质谱显示分子离子峰为MS(ESI):m/z 321.0[M+H]+核磁1HNMR(400MHz,CDCl3)δ:10.38(s,1H),8.20～8.13(m,4H),7.97(d,J＝8.4Hz,1H),7.37(d,J＝8.0Hz,2H),2.80(s,3H),2.61(d,J＝7.6Hz,2H),1.96(m,1H),0.96(d,J＝7.6Hz,6H)
1.6 1-{2-甲基-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(化合物5)
Figure PCTCN2014086538-appb-000041
室温下，将2-甲基-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6, 0.88g,2.75mmol)、3-吖丁啶羧酸(1-7,0.278g,2.75mmol)和醋酸(1mL)的甲醇-四氢呋喃(20mL/20mL)溶液搅拌两小时，加入氰基硼氢化钠(1.04g,16.5mmol)的甲醇溶液(60mL)后室温继续搅拌16小时，过滤，以甲醇(10mLx2)洗涤滤饼，干燥得白色固体产品1-{2-甲基-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(0.23g,得率21％)。液相质谱显示分子离子峰为MS(ESI):m/z 406.0[M+H]+核磁1HNMR(400MHz,CD3OD)δ:8.12(d,J＝8.0Hz,2H),8.08(s,1H),8.04(d,J＝7.6Hz,1H),7.53(d,J＝7.6Hz,1H),7.42(d,J＝8.0Hz,2H),4.47(s,2H),4.23(m,4H),3.44(m,1H),2.61(d,J＝7.2Hz,2H),2.52(s,3H),1.95(m,1H),0.94(d,J＝7.2Hz,6H).
实施例6 本发明化合物的体内药代动力学实验
在实施例中，在SD大鼠中通过静脉注射和口服给药进行了化合物1、2、3、4和5的药代动力学性质的评价。
本实施例以及下文使用的实验动物为雄性SD大鼠，年龄在7-9周之间，体重186-231克，均购自北京维通利华实验动物技术有限公司。在动物购入后，由兽医负责检疫(检疫期5天)，之后挑选检疫合格的动物在SPF条件下以各组的3只/测试化合物进行试验。
口服给药组：分别称取2.74mg的化合物1-5，以9.113mL的0.5％CMC-Na为稀释液，配制成浓度为0.3mg/mL的溶液，混匀后涡旋1～2min，超声20～30min直至得到均匀混悬液，作为口服给药组药液，给药按10mL/kg体重进行。
静脉给药组：称取1.61mg的化合物1-5，以1.610mL 10％HP-β-CD为稀释液，配制成浓度为1mg/mL的溶液，混匀后涡旋1～2min，超声28～30min，作为静脉推注给药组药液。给药按1mL/kg体重进行。
无论是口服还是静脉给药，在给药后0.0833(5min)、0.25(15min)、0.5、1、2、4、8、24h采血。每个时间点分别从动物眼框静脉丛取全血约0.3mL，取血时使用异氟烷麻醉。全部样品采集结束后，将动物实施安乐死。
血样采集后，放入含有肝素钠(约10μl,1000IU/mL)的EP管中，立即置于碎冰中，之后在4500rpm低温(4℃)离心5min，快速分离血浆，-20℃以下保存待分析。
血液中各个化合物的浓度采用LC-MS/MS-001(Q-trap-3200)进行分析，并以柳胺酚作为分析内标。6μL血浆试样中加入24μL空白血浆(稀释倍数 为5)，然后加入150μL的含内标(柳胺酚100μg/mL)的乙腈溶液。将该混合物振荡5分钟，并以14000rpm离心5分钟。取2μL样品注入LC-MS/MS分析。对于无稀释的血浆样本，取30μL样品加入150μ含内标(柳胺酚100μg/mL)的乙腈溶液。将该混合物振荡5分钟，并以14000rpm离心5分钟。取2μL样品注入LC-MS/MS分析。
数据分析使用WinNolin(V6.2)非房室模型(NCA)计算主要代谢动力学参数t1/2，AUC(0-t)，AUCinf，V，Cl，MRT等，并利用Microsoft Office EXCEL计算均值、标准差、变异系数。
下表1的数据清楚地表明，口服给药后，化合物2、3和4的终末半衰期都小于5.5小时。而化合物1的终末半衰期为近11小时。因此，三个不同的卤族元素取代物(化合物2、3和4)都分别比化合物1的半衰期减少了近50％。
静脉给药的药代研究中，半衰期也有类似的变化。下表2的数据清楚地表明，卤族元素取代的化合物2、3、4的终端半衰期及平均停留时间(MRT)都比化合物1显著减少。这些数据表明，化合物1在2-位置卤族元素取代加快了化合物在体内血液中的排除。而且清除率(CI)结果表明，终端半衰期及平均停留时间的缩短并非由化合物的清除率升高而引起。
化合物2、3和4在体内半衰期的显著减少是无法从现有常规理论上进行预测的，因为在化合物1的相同位置上其他的取代物并没有得到类似结果。比如，当R取代物是甲基时(化合物5)，半衰期不但没有缩短，反而延长了半衰期(见表1和表2)。并且，从口服给药后血液化合物浓度随时间变化的曲线看，与化合物1和5相比，化合物2、3、4在达到最高浓度后从血液中排除的速度明显加快(图1)。
表1：口服给药(3mg/kg)的代谢动力学
Figure PCTCN2014086538-appb-000042
表2：静脉给药(1mg/kg)的代谢动力学
Figure PCTCN2014086538-appb-000043
实施例7 本发明化合物的S1P1和S1P3受体的内化效应实验
(一)S1P1受体的内化效应实验
众所周知，S1P1小分子激动剂可以通过诱导细胞表面S1P1受体内化从而阻止淋巴细胞进入外周循环。为确定本发明提供的化合物是否具有诱导S1P1受体内化的活性，采用表达人S1P1受体的CHO-S细胞代替淋巴细胞来作为S1P1受体内化检测体系。为便于监测位于该细胞表面上的S1P1受体，在S1P1受体N-末端融合了一个Myc标签序列，从而首先让细胞与抗Myc标签的荧光标记抗体温育，然后采用流式细胞仪分析该受体的表达。
将本发明提供的化合物2溶解在二甲基亚砜(DMSO)中配成10mM的储液，然后一定量的储液再进一步用DMEM稀释成各种所需的不同浓度。收集表达带有Myc标签的人S1P1受体的CHO-S细胞，并用贝科改良Eagle培养基(DMEM)培养基调整到每mL含一百万细胞的密度。将等体积稀释的化合物2和细胞悬浮液相混合，并在37℃温育一小时。培养结束后，经800RPM离心5分钟收获细胞。将细胞重新悬浮于FACS缓冲液(含1％BSA的PBS)中，加入异硫氰酸荧光素(FITC)标记的Myc抗体(美国加州美天旎生物技术公司)并放置于冰上孵育一小时。洗涤细胞并重新悬浮在预冷的FACS缓冲液中，用FACS Calibur流式细胞仪进行分析。
试验数据表明，化合物2呈现出剂量依赖性地诱导S1P1内化的活性(表3)。采用同样方法测试了本发明化合物3和4的S1P1内化诱导活性，得到的结果与化合物1没有明显的差异，表明F、Cl和Br的取代物(化合物2、3和4)均能在明显缩短化合物体内半衰期的同时，仍具有激活S1P1受体的活性。
(二)S1P3受体内化试验
采用表达人S1P3受体亚型的CHO-S细胞进行内化检测实验，除细胞不同，实验方法和上述S1P1内化检测方法相同。
实验结果表明，与化合物1相似，化合物2、3和4对S1P受体的影响是专一的，即仅对S1P1受体有内化激活作用，而对S1P3受体亚型没有内化激活的效应(表3)。这表明，虽然F、Cl和Br取代的化合物明显缩短了化合物1的体内半衰期，但它们对S1P1靶点的选择性并没有改变。这与目前临床上使用的药物FTY720不同，后者是一个非选择性的S1P受体激动剂，能激活若干S1P受体，如S1P1、S1P2、S1P3、S1P4、S1P5，从而会导致一系列严重副作用，如心动过缓。
表3：化合物2、3、4保持受体的活性和专一性
Figure PCTCN2014086538-appb-000044
实施例8 本发明化合物对外周血淋巴细胞数目影响的实验
S1P1受体表达于淋巴细胞表面，对于淋巴细胞离开次级淋巴组织继而进入外周循环是必不可少的。S1P1受体的小分子激动剂可以激活受体并导致受体内化效应，该机制是目前已知的抑制淋巴细胞离开次级淋巴组织继而影响外周循环淋巴细胞数量降低的原因。为了确定本发明的化合物是否可以减少外周血中的淋巴细胞，进行了体内淋巴细胞影响实验。
用羧甲基纤维素(CMC-Na)将适量的化合物2制成混悬液，并使三只Sprague-Dawley(SD)大鼠口服。在给药前30分钟和给药后的不同时间点采集血液样本(0.5ml)(收集于适量EDTA-2K溶液EP管中)，血样直接在ADVIA2120血细胞分析仪上进行分析。
实验结果表明，化合物2非常有效地降低外周血淋巴细胞数目。外周血淋巴细胞数在给药30分钟后就有明显的降低，并在所有采样的时间点上(30，120，240，360，480分钟)进一步降低。化合物2在评估的所有三个剂量中 都具有活性，其中仅仅需要0.01mg/kg的剂量就可以观察到外周血淋巴细胞数目降低超过50％，1mg/kg剂量降低的最多(图2)。另外，化合物2的作用是淋巴细胞特异的，它并没有明显改变外周单核细胞和其他白细胞的数目。
采用同样方法对本发明的化合物3、4和5进行了测试，发现化合物3、4和5对体内淋巴细胞的作用与化合物2相似(图3，图4)。
实施例9 化合物2对Lewis大鼠中II型胶原诱导的关节炎的发展的作用
人的类风湿性关节炎为自身免疫疾病，在此病中，患者自体免疫系统会攻击关节组织。淋巴细胞，包括T细胞和B细胞，在此病的发病机制中起重要作用。已知通过阻断T细胞活化抑制T细胞功能，可以有效治疗类风湿性关节炎。由于化合物2阻断淋巴细胞外排，因此确定其是否有效抑制CIA大鼠模型中关节炎的发展非常重要。为此，首先诱导Lewis大鼠发展此疾病：用异氟醚麻醉大鼠，然后皮内注射总计0.5mL CII/CFA乳剂。在3个部位进行乳剂注射，其一为在大鼠尾根处(0.1mL)，其余两处为近尾根处的背部(0.2mL/部位)。初次免疫后7天，避开之前的注射部位，皮内进行相同的强化免疫。
将化合物2制成在0.5％CMC-Na中的悬液，然后在大鼠注射CII/CFA时口服给药。阳性对照组采用TOFACITINIB，在致敏后第12天口服给药。致敏后7天开始每周2次对大鼠四足中关节炎严重程度进行评分。标准如下：0分为未见红斑和肿胀；1分为足中部(跗骨)或踝关节处可见红斑和轻微肿胀；2分为从踝延伸至足中部的红斑和轻微肿胀；3分为从踝延伸至足中部的红斑和中度肿胀；4分为涵盖踝、足和足趾的红斑和严重肿胀。使用足趾容积测量仪，在第0天以后爪体积计测定关节肿胀，然后从第7天至第28天每周2次测定。在第28天通过X射线检查来测定关节破坏情况。结果表明，在1mg/kg，化合物2可以有效抑制关节炎中的关节肿胀发展(图5)和关节结构破坏(图6)。图5可以看出，化合物2与TOFACITINIB具有类似作用，但用量显著较少。
实施例10 化合物2对实验性自身免疫性脑炎(EAE)的发展的作用
已发现S1P1拮抗剂在人多发性硬化和MS动物模型中有效。因此评估 了化合物2对于实验性自身免疫性脑脊髓炎(EAE)的功效，该EAE为人多发性硬化的小鼠模型。根据体重将80只雌性C57BL/6小鼠随机分配成8组，然后免疫。每组由10只小鼠组成。为诱导该疾病，将MOG 35-55(MOG为髓少突胶质细胞糖蛋白)溶于盐水中达到2mg/mL的浓度，然后在经修饰的完全弗氏佐剂(CFA)中乳化。异氟醚麻醉小鼠，然后将100μL乳剂皮下注射到已备皮的小鼠背部3个部位处，其一沿着小鼠肩部之间背部中线，另两个部位在小鼠肩部之间背部中线两侧。在免疫当天和之后48小时，给所有组经腹膜内注射百日咳毒素(200μL PBS中200ng)。在免疫后第0天至第30天，每日一次给小鼠临床打分，评价EAE发展。
将化合物2制成Na CMC悬液，在MOG免疫之时开始口服给药，并且在研究整个过程中持续给药。数据表明，在评估的全部3个剂量下(0.03、0.1和1mg/kg)，化合物2有效抑制EAE的发展(图7)。
实施例11 化合物2对比格犬心血管功能的影响
FTY720为非选择性的S1P1拮抗剂，已显示造成多种心血管影响，包括在人中造成心动过缓。为了确定化合物2是否对心率和QT间期有影响，在清醒比格犬中进行遥测测定来评价该化合物。
称取适量的CMC-Na，采用灭菌注射用水配制浓度为0.5％CMC-Na(w/v)溶液。给药前一天配制。
浓度为2、6和20mg/mL的化合物2(供试品)溶液，于给药前一天进行配制：称取适量化合物2，加入适量0.5％羧甲基纤维素钠溶液，用分散乳化机进行乳化均质，所配制供试品溶液中化合物2的理论浓度为2、6和20mg/mL。
本试验共用8只动物，采用双拉丁方实验设计。给药轮次间隔3～5日。每轮次给药前1天称量动物体重，药前禁食过夜。给药当日开启遥测系统(植入式生理信号遥测系统1，Data Science International Inc.,USA)，设定试验参数，开启植入子，记录动物各项生理指标。系统开启约2小时后，按下表设计的轮次顺序给药。采集给药后24小时内动物血压、心电、体温等各项指标数据。采集过程中为防止可能的系统数据溢出，适当停机，然后重新开启，开关过程不影响设定数据点的取值。开关系统时间均做记录。次日记录结束后，关闭遥测系统。检测时间点：药前1h内(-1h)、给药后0.5h(±5min)、1h(±10min)、1.5h(±10min)、2h(±15min)、3h(±15min)、4h (±15min)、8h(±45min)、24h(±1h)。所有数据均由PONEMAH Version4.8软件自动采集。采集完成后，人工设定分析参数，先由PONEMAH Version4.8软件自动分析，后由人工逐点核对取值。心率，血压，呼吸，体温指标等取1分钟内连续波形的平均值，其余心电指标取10秒内连续波形的平均值。取值时优先选取检测时间点即刻数据，若此点噪音干扰大，或心率异常，或无法清晰辨认波形时则取给定范围内信号清晰的波形。若给定范围内仍无清晰波形可供分析则向取值点前后寻找可分析数据，并在取值表中特别说明。记录取值时间。
本试验采用的统计学软件SPSS13.0对数据进行处理。所有统计分析采用双尾分析，统计学水平设在P≤0.05。血压、心电、呼吸、体温指标均以“平均数±标准差”表示，并按下列过程分析：首先用Levene Test对数据进行均一性检验，如果数据均一(P﹥0.05)，则进行单因素方差分析；如果方差分析显著(P≤0.05)，则溶媒组与供试品组间的差异进行Dunnett’s多重比较。如果Levene Test的结果显著(P≤0.05)，则进行Kruskal-wallis非参数检验。如果Kruskal-wallis非参数检验结果显著(P≤0.05)，则进一步采用Mann-Whitney U检验进行两两比较。
对有显著差异或具明显改变趋势的时间点的数据采用归一化处理，计算变化幅度。归一化处理公式为：△％＝[(b1-b0)-(a1-a0)]/a1×100。其中，b1为供试品药后时间点值，b0为供试品组药前值，a1为溶媒对照药后相应时间点的值，a0为溶媒对照药前值，△％为改变幅度。
供试品各剂量下动物心率、QT间期、QTcF间期与溶媒对照相同时间点心电图指标比较均无统计学差异(P>0.05)，也未见趋势性改变(图8A至8C)。
下文将就1-{2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(化合物2)的合成方法，给出方法及其条件优化过程的实施例。
化合物2的合成将按照包括下述步骤的方法进行：
Figure PCTCN2014086538-appb-000045
实施例12 本发明合成方法的筛选——步骤(1)
按照实施例2制备和表征式1-5化合物粗品，经LCMS检测纯度为77.25％。
将制得的粗品进行结晶纯化条件的筛选。结晶操作为用结晶溶剂溶解粗品，并于20℃下析晶，过滤，真空干燥后得类白色固体产物为式1-5所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇，经LCMS检测纯度。
首先按照表4中所列进行上述结晶操作，以筛选出优选的结晶溶剂。
表4.结晶纯化的溶剂及其用量的筛选
Figure PCTCN2014086538-appb-000046
Figure PCTCN2014086538-appb-000047
从表4可知，在使用单一溶剂的结晶条件中，使用甲醇和乙醇作为溶剂对于产品的纯度提升比较明显。甲醇的收率相对乙醇高很多，但是也只有55.4％。使用纯水作为溶剂对产品的纯度基本没有提升，但是损失较少，所以在后续的研究中尝试将甲醇和水的混合溶剂作为结晶溶剂。
接下来，按照表5中所列进行上述结晶操作纯化，以筛选出甲醇和水的混合溶剂的优选比例。其中粗品与混合结晶溶剂的重量(以g计)体积(以ml计)比为1:5。
表5 混合溶剂比例的筛选
Figure PCTCN2014086538-appb-000048
从表5可知，提高混合溶剂中甲醇的量，可以提升产品的纯度，但是收率有所降低；提高水的量，可以提升纯化的收率，但是产品的纯度有所降低。综合考虑，选取甲醇和水体积比为3:1的条件作为结晶混合溶剂中比例。
然后，按照表6中所列进行上述结晶操作，以筛选出混合溶剂的优选用量。其中混合溶剂中甲醇和水体积比为3:1。
表6 溶剂用量的筛选
Figure PCTCN2014086538-appb-000049
Figure PCTCN2014086538-appb-000050
从表6可知，当使用粗品重量和混合溶剂体积比为1g:5ml，产品的收率和纯度较高；当使用粗品重量和混合溶剂体积比为1g:20ml，虽然产品的纯度相对1g:5ml较高，但损失了较多的收率，所以选取粗品重量和混合溶剂体积比为1g:5ml作为结晶的溶剂体系。
实施例13 本发明合成方法的筛选——步骤(2)
本发明方法的步骤(2)按照下述过程进行：
将实施例1纯化得到的式1-5所示2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇溶于反应溶剂中，再加入活性二氧化锰。将反应液加热至回流并继续反应后，降温至室温。过滤并收集浅黄色滤液，用无水硫酸钠干燥后，减压浓缩得白色固体产品式1-6所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛，经LCMS检测转化率。
首先按照表7中所列进行上述合成步骤，以筛选出优选的反应溶剂。其中式1-5所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇与二氧化锰的摩尔比为1:6。表7-9中的原料指式1-5所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇。
表7 反应溶剂的筛选
Figure PCTCN2014086538-appb-000051
从表7可知，使用四氢呋喃、乙酸乙酯、甲苯作为反应溶剂对反应的转化率和收率影响较小。但是四氢呋喃作为溶剂进行氧化反应有安全性的风险，而甲苯毒性较高，因此选择乙酸乙酯作为反应溶剂。
接下来，乙酸乙酯作为反应溶剂，按照表8中所列进行上述合成步骤，以筛选出优选的溶剂用量。其中式1-5所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇与二氧化锰的摩尔比为1:6。
表8 反应溶剂用量的筛选
溶剂用量 反应时间 转化率(LCMS) 收率
(原料:溶剂)      
1g:10ml 1h 97.76％ 93.3％
1g:20ml 1h 98.93％ 92.6％
1g:30ml 1h 98.83％ 93.1％
从表8可知，原料溶剂的重量体积比为1g:10ml、1g:20ml、1g:30ml对于反应的转化率和收率影响较小，以成本方面考虑，选用使用1g:10ml的比例作为反应的溶剂用量。
然后，按照表9中所列进行上述合成步骤，以筛选出优选的氧化剂二氧化锰用量。其中使用式1-5所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇与乙酸乙酯为1g:10ml的用量。
表9 氧化剂用量的筛选
Figure PCTCN2014086538-appb-000052
从表9可知，使用原料和二氧化锰的摩尔比为1:6时，反应转化率和收率较高，当二氧化锰的用量增加到二者摩尔比为1:10时，对于反应的转化率和收率影响较小，从成本和收率方面考虑，选用1:6作为二者的使用量。
实施例14 本发明合成方法的筛选——步骤(3)
本发明方法的步骤(3)按照下述过程进行：
在室温下，将式1-6所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛、式1-7所示的3-吖丁啶羧酸与冰醋酸加入到反应溶剂中，在20℃下搅拌2小时。将NaBH3CN溶于甲醇中，然后在1小时内将NaBH3CN的甲醇溶液滴加到反应体系中，滴加完毕后，在20℃下搅拌反应。反应液过滤，滤饼用甲醇淋洗后烘干，得到式IB(化合物2)即白色固体1-{2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸，经LCMS检测转化率。
首先按照表10中所列进行上述合成步骤，以筛选出优选的反应溶剂。 其中式1-6所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛与式1-7所示的3-吖丁啶羧酸的摩尔比为1:1.05，式1-6所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛与氰基硼氢化钠的摩尔比为1:1，NaBH3CN的甲醇溶液滴加温度为15～20℃。表10-12中的原料指式1-6所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛。
表10 反应溶剂的筛选
Figure PCTCN2014086538-appb-000053
从表10可知，使用四氢呋喃作为反应溶剂，反应转化率很低，而使用甲醇相对于乙醇作为反应溶剂，反应转化率和收率都较高。
接下来，按照表11中所列进行上述合成步骤，以筛选出优选的还原剂用量。其中式1-6所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛与式1-7所示的3-吖丁啶羧酸的摩尔比为1:1.05，反应溶剂为甲醇，NaBH3CN的甲醇溶液滴加温度为15～20℃。
表11 还原剂用量的筛选
Figure PCTCN2014086538-appb-000054
从表11可知，使用原料和氰基硼氢化钠摩尔比为1:1时，反应的转化率、收率和产品纯度都较高。
然后，按照表12中所列进行上述合成步骤，以筛选出优选的还原剂滴加温度。其中式1-6所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛与式1-7所示的3-吖丁啶羧酸的摩尔比为1:1.05，反应溶剂为甲醇。
表12 反应滴加温度的筛选
Figure PCTCN2014086538-appb-000055
从表12可知，使用15-20℃的氰基硼氢化钠滴加温度，反应的转化率和产品纯度较高。
实施例15 本发明的合成方法
(1)室温下，将4-异丁基苯甲酸(1-4,0.148Kg,0.83mol)溶于N,N-二甲基甲酰胺(1.7L)中，再加1-羟基苯并三唑(0.11Kg,0.83mol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.16Kg,0.83mol)，加热至30℃并继续搅拌30分钟后，加入3-氟-N'-羟基-4-羟甲基苯甲脒(1-3,0.153Kg,0.83mol)。将反应液加热至140℃并继续反应2小时后，降温至室温。减压浓缩去除N,N-二甲基甲酰胺，将浓缩物溶于乙酸乙酯(2.0L)，并分别用水(1.5L×2)，饱和碳酸氢钠溶液(1.5L)洗涤，收集有机相并用无水硫酸钠干燥后，减压浓缩得256g粗品。
上述粗品用1.28L甲醇和水混合溶剂(体积比3:1)重结晶，并于20℃析晶，过滤，真空干燥后得类白色固体产物2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,182g，收率71％)，经LCMS检测纯度为93.1％。
MS(ESI):m/z 327.0[M+H]+1HNMR(400MHz,CDCl3)δ:8.11(d,J＝8.0Hz,2H),7.98(m,1H),7.86(m,1H),7.59(t,J＝8.0Hz,7.6Hz,1H),7.33(d,J＝8.0Hz,2H),4.85(s,2H),2.57(d,J＝6.8Hz,2H),1.93(m,1H),0.93(d,J＝6.8Hz,6H).
(2)室温下，将2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,0.14Kg,0.43mol)溶于乙酸乙酯(1.4L)中，再加入活性二氧化锰(0.21Kg,2.42mol)。将反应液加热至回流并继续反应3小时后，降温至室温。过滤并收集浅黄色滤液，用无水硫酸钠干燥后，减压浓缩得白色固体产品2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,139g,收率99.0％)，经LCMS 检测纯度97.6％。
MS(ESI):m/z 325.0[M+H]+1HNMR(400MHz,CDCl3)δ:10.42(s,1H),8.12～7.99(m,5H),7.34(d,J＝7.2Hz,2H),2.58(d,J＝6.4Hz,2H),1.93(m,1H),0.93(d,J＝6.4Hz,6H).
(3)室温下，将2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,60g,0.185mol)，3-吖丁啶羧酸(1-7,19.5g,0.193mol)与冰醋酸(360mL,0.63mol)加入到甲醇(1.6L)中，20℃下搅拌2小时。将NaBH3CN(11.5g,0.185mol)溶于甲醇(200mL)，然后在1小时内将NaBH3CN的甲醇溶液滴加到反应体系中，控制滴加温度在15-20℃之间，滴加完毕后，反应在20℃下搅拌16小时。反应液过滤，滤饼用300mL甲醇淋洗后烘干，得到白色固体1-{2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(化合物2,67g,收率89.0％)，经LC-MS检测的纯度为98.8％。
MS(ESI):m/z 410.2[M+H]+；1HNMR(400MHz,CDCl3)δ:8.13(d,J＝8.4Hz,2H),8.05(m,1H),7.97(m,1H),7.68(t,J＝8.0Hz,7.6Hz,1H),7.42(d,J＝8.4Hz,2H),4.40(s,2H),4.15(m,4H),3.41(m,1H),2.61(d,J＝7.2Hz,2H),1.95(m,1H),0.94(d,J＝7.2Hz,6H).
实施例16 本发明的合成方法
(1)室温下，将4-异丁基苯甲酸(1-4,1.477Kg,8.30mol)溶于N,N-二甲基甲酰胺(17L)中，再加1-羟基苯并三唑(1.12Kg,8.30mol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(1.58Kg,8.30mol)，加热至30℃并继续搅拌30分钟后，加入3-氟-N'-羟基-4-羟甲基苯甲脒(1-3,1.527Kg,8.30mol)。将反应液加热至140℃并继续反应2小时后，降温至室温。减压浓缩去除N,N-二甲基甲酰胺，将浓缩物溶于乙酸乙酯(20L)，并分别用水(15L×2)，饱和碳酸氢钠溶液(15L)洗涤，收集有机相并用无水硫酸钠干燥后，减压浓缩得2.6Kg粗品。
上述粗品用12.5L甲醇和水混合溶剂(体积比3:1)重结晶，并于20℃析晶，过滤，真空干燥后得类白色固体产物2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,1.9Kg，收率73％)，经LCMS检测纯度为93.89％。
MS(ESI):m/z 327.0[M+H]+1HNMR(400MHz,CDCl3)δ:8.11(d,J＝8.0Hz,2H),7.98(m,1H),7.86(m,1H),7.59(t,J＝8.0Hz,7.6Hz,1H),7.33(d,J＝8.0Hz,2H),4.85(s,2H),2.57(d,J＝6.8Hz,2H),1.93(m,1H),0.93(d,J ＝6.8Hz,6H).
(2)室温下，将2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,1.4Kg,4.30mol)溶于乙酸乙酯(14L)中，再加入活性二氧化锰(2.1Kg,24.15mol)。将反应液加热至回流并继续反应3小时后，降温至室温。过滤并收集浅黄色滤液，用无水硫酸钠干燥后，减压浓缩得白色固体产品2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,1.38kg,收率99.0％)，经LCMS检测纯度93.94％。
MS(ESI):m/z 325.0[M+H]+1HNMR(400MHz,CDCl3)δ:10.42(s,1H),8.12～7.99(m,5H),7.34(d,J＝7.2Hz,2H),2.58(d,J＝6.4Hz,2H),1.93(m,1H),0.93(d,J＝6.4Hz,6H).
(3)室温下，将2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,0.6kg,1.85mol)，3-吖丁啶羧酸(1-7,0.195kg,1.93mol)与冰醋酸(0.360L,6.3mol)加入到甲醇(16L)中，20℃下搅拌2小时。将NaBH3CN(0.115kg,1.85mol)溶于甲醇(2L)，然后在1小时内将NaBH3CN的甲醇溶液滴加到反应体系中，控制温度在15-20℃之间，滴加完毕后，反应在20℃下搅拌16小时。反应液过滤，滤饼用3L甲醇淋洗后烘干，得到白色固体1-{2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(化合物2,0.7kg,收率92.6％)，经LC-MS检测的纯度为97.6％。
MS(ESI):m/z 410.2[M+H]+；1HNMR(400MHz,CDCl3)δ:8.13(d,J＝8.4Hz,2H),8.05(m,1H),7.97(m,1H),7.68(t,J＝8.0Hz,7.6Hz,1H),7.42(d,J＝8.4Hz,2H),4.40(s,2H),4.15(m,4H),3.41(m,1H),2.61(d,J＝7.2Hz,2H),1.95(m,1H),0.94(d,J＝7.2Hz,6H).
实施例17 本发明的合成方法
(1)室温下，将4-异丁基苯甲酸(1-4,0.148Kg,0.83mol)溶于N,N-二甲基甲酰胺(1.7L)中，再加1-羟基苯并三唑(0.11Kg,0.83mol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.16Kg,0.83mol)，加热至30℃并继续搅拌30分钟后，加入3-氟-N'-羟基-4-羟甲基苯甲脒(1-3,0.153Kg,0.83mol)。将反应液加热至140℃并继续反应2小时后，降温至室温。减压浓缩去除N,N-二甲基甲酰胺，将浓缩物溶于乙酸乙酯(2.0L)，并分别用水(1.5L×2)，饱和碳酸氢钠溶液(1.5L)洗涤，收集有机相并用无水硫酸钠干燥后，减压浓缩得251g粗品。
上述粗品用1.28L甲醇和水混合溶剂(体积比1:1)重结晶，并于20℃析晶，过滤，真空干燥后得类白色固体产物2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,158g，收率63％)，经LCMS检测纯度为92.1％。
MS(ESI):m/z 327.0[M+H]+1HNMR(400MHz,CDCl3)δ:8.11(d,J＝8.0Hz,2H),7.98(m,1H),7.86(m,1H),7.59(t,J＝8.0Hz,7.6Hz,1H),7.33(d,J＝8.0Hz,2H),4.85(s,2H),2.57(d,J＝6.8Hz,2H),1.93(m,1H),0.93(d,J＝6.8Hz,6H).
(2)室温下，将2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,0.14Kg,0.43mol)溶于乙酸乙酯(1.4L)中，再加入活性二氧化锰(0.19Kg,2.15mol)。将反应液加热至回流并继续反应3小时后，降温至室温。过滤并收集浅黄色滤液，用无水硫酸钠干燥后，减压浓缩得白色固体产品2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,138g,收率99％)，经LCMS检测纯度98.5％。
MS(ESI):m/z 325.0[M+H]+1HNMR(400MHz,CDCl3)δ:10.42(s,1H),8.12～7.99(m,5H),7.34(d,J＝7.2Hz,2H),2.58(d,J＝6.4Hz,2H),1.93(m,1H),0.93(d,J＝6.4Hz,6H).
(3)室温下，将2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,60g,0.185mol)，3-吖丁啶羧酸(1-7,19.5g,0.193mol)与冰醋酸(360mL,0.63mol)加入到甲醇(1.6L)中，20℃下搅拌2小时。将NaBH3CN(5.8g,0.09mol)溶于甲醇(200mL)，然后在1小时内将NaBH3CN的甲醇溶液滴加到反应体系中，控制滴加温度在15-20℃之间，滴加完毕后，反应在20℃下搅拌16小时。反应液过滤，滤饼用300mL甲醇淋洗后烘干，得到白色固体1-{2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(化合物2,62g,收率81.9％)，经LC-MS检测的纯度为94.6％。
MS(ESI):m/z 410.2[M+H]+；1HNMR(400MHz,CDCl3)δ:8.13(d,J＝8.4Hz,2H),8.05(m,1H),7.97(m,1H),7.68(t,J＝8.0Hz,7.6Hz,1H),7.42(d,J＝8.4Hz,2H),4.40(s,2H),4.15(m,4H),3.41(m,1H),2.61(d,J＝7.2Hz,2H),1.95(m,1H),0.94(d,J＝7.2Hz,6H).
实施例18 本发明的合成方法
(1)室温下，将4-异丁基苯甲酸(1-4,0.148Kg,0.83mol)溶于N,N-二甲基甲酰胺(1.7L)中，再加1-羟基苯并三唑(0.11Kg,0.83mol)和1-(3-二甲氨基 丙基)-3-乙基碳二亚胺盐酸盐(0.16Kg,0.83mol)，加热至30℃并继续搅拌30分钟后，加入3-氟-N'-羟基-4-羟甲基苯甲脒(1-3,0.153Kg,0.83mol)。将反应液加热至140℃并继续反应2小时后，降温至室温。减压浓缩去除N,N-二甲基甲酰胺，将浓缩物溶于乙酸乙酯(2.0L)，并分别用水(1.5L×2)，饱和碳酸氢钠溶液(1.5L)洗涤，收集有机相并用无水硫酸钠干燥后，减压浓缩得260g粗品。
上述粗品用1.30L甲醇和水混合溶剂(体积比1:3)重结晶，并于20℃析晶，过滤，真空干燥后得类白色固体产物2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,196g，收率76％)，经LCMS检测纯度为88.7％。
MS(ESI):m/z 327.0[M+H]+1HNMR(400MHz,CDCl3)δ:8.11(d,J＝8.0Hz,2H),7.98(m,1H),7.86(m,1H),7.59(t,J＝8.0Hz,7.6Hz,1H),7.33(d,J＝8.0Hz,2H),4.85(s,2H),2.57(d,J＝6.8Hz,2H),1.93(m,1H),0.93(d,J＝6.8Hz,6H).
(2)室温下，将2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,0.14Kg,0.43mol)溶于乙酸乙酯(1.4L)中，再加入活性二氧化锰(0.21Kg,2.42mol)。将反应液加热至回流并继续反应3小时后，降温至室温。过滤并收集浅黄色滤液，用无水硫酸钠干燥后，减压浓缩得白色固体产品2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,139g,收率99％)，经LCMS检测纯度97.7％。
MS(ESI):m/z 325.0[M+H]+1HNMR(400MHz,CDCl3)δ:10.42(s,1H),8.12～7.99(m,5H),7.34(d,J＝7.2Hz,2H),2.58(d,J＝6.4Hz,2H),1.93(m,1H),0.93(d,J＝6.4Hz,6H).
(3)室温下，将2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,60g,0.185mol)，3-吖丁啶羧酸(1-7,19.5g,0.193mol)与冰醋酸(360mL,0.63mol)加入到甲醇(1.6L)中，20℃下搅拌2小时。将NaBH3CN(23.0g,0.37mol)溶于甲醇(200mL)，然后在1小时内将NaBH3CN的甲醇溶液滴加到反应体系中，控制滴加温度在15-20℃之间，滴加完毕后，反应在20℃下搅拌16小时。反应液过滤，滤饼用300mL甲醇淋洗后烘干，得到白色固体1-{2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(化合物2,60g,收率79.0％)，经LC-MS检测的纯度为94.2％。
MS(ESI):m/z 410.2[M+H]+；1HNMR(400MHz,CDCl3)δ:8.13(d,J＝8.4Hz,2H),8.05(m,1H),7.97(m,1H),7.68(t,J＝8.0Hz,7.6Hz,1H),7.42(d, J＝8.4Hz,2H),4.40(s,2H),4.15(m,4H),3.41(m,1H),2.61(d,J＝7.2Hz,2H),1.95(m,1H),0.94(d,J＝7.2Hz,6H).
实施例19 本发明的合成方法
(1)室温下，将4-异丁基苯甲酸(1-4,0.148Kg,0.83mol)溶于N,N-二甲基甲酰胺(1.7L)中，再加1-羟基苯并三唑(0.11Kg,0.83mol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.16Kg,0.83mol)，加热至30℃并继续搅拌30分钟后，加入3-氟-N'-羟基-4-羟甲基苯甲脒(1-3,0.153Kg,0.83mol)。将反应液加热至140℃并继续反应2小时后，降温至室温。减压浓缩去除N,N-二甲基甲酰胺，将浓缩物溶于乙酸乙酯(2.0L)，并分别用水(1.5L×2)，饱和碳酸氢钠溶液(1.5L)洗涤，收集有机相并用无水硫酸钠干燥后，减压浓缩得250g粗品。
上述粗品用1.25L甲醇和水混合溶剂(体积比2:1)重结晶，并于20℃析晶，过滤，真空干燥后得类白色固体产物2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,169g，收率68％)，经LCMS检测纯度为93.9％。
MS(ESI):m/z 327.0[M+H]+1HNMR(400MHz,CDCl3)δ:8.11(d,J＝8.0Hz,2H),7.98(m,1H),7.86(m,1H),7.59(t,J＝8.0Hz,7.6Hz,1H),7.33(d,J＝8.0Hz,2H),4.85(s,2H),2.57(d,J＝6.8Hz,2H),1.93(m,1H),0.93(d,J＝6.8Hz,6H).
(2)室温下，将2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,0.14Kg,0.43mol)溶于乙酸乙酯(1.4L)中，再加入活性二氧化锰(0.37Kg,4.3mol)。将反应液加热至回流并继续反应3小时后，降温至室温。过滤并收集浅黄色滤液，用无水硫酸钠干燥后，减压浓缩得白色固体产品2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,139g,收率99％)，经LCMS检测纯度99.2％。
MS(ESI):m/z 325.0[M+H]+1HNMR(400MHz,CDCl3)δ:10.42(s,1H),8.12～7.99(m,5H),7.34(d,J＝7.2Hz,2H),2.58(d,J＝6.4Hz,2H),1.93(m,1H),0.93(d,J＝6.4Hz,6H).
(3)室温下，将2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,60g,0.185mol)，3-吖丁啶羧酸(1-7,19.5g,0.193mol)与冰醋酸(360mL,0.63mol)加入到甲醇(1.6L)中，20℃下搅拌2小时。将NaBH3CN(69.0g,1.11mol)溶于甲醇(200mL)，然后在1小时内将NaBH3CN的甲醇溶液 滴加到反应体系中，控制滴加温度在15-20℃之间，滴加完毕后，反应在20℃下搅拌16小时。反应液过滤，滤饼用300mL甲醇淋洗后烘干，得到白色固体1-{2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(化合物2,54g,收率71.2％)，经LC-MS检测的纯度为94.4％。
MS(ESI):m/z 410.2[M+H]+；1HNMR(400MHz,CDCl3)δ:8.13(d,J＝8.4Hz,2H),8.05(m,1H),7.97(m,1H),7.68(t,J＝8.0Hz,7.6Hz,1H),7.42(d,J＝8.4Hz,2H),4.40(s,2H),4.15(m,4H),3.41(m,1H),2.61(d,J＝7.2Hz,2H),1.95(m,1H),0.94(d,J＝7.2Hz,6H).
实施例20 本发明的合成方法
(1)室温下，将4-异丁基苯甲酸(1-4,0.148Kg,0.83mol)溶于N,N-二甲基甲酰胺(1.7L)中，再加1-羟基苯并三唑(0.11Kg,0.83mol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.16Kg,0.83mol)，加热至30℃并继续搅拌30分钟后，加入3-氟-N'-羟基-4-羟甲基苯甲脒(1-3,0.153Kg,0.83mol)。将反应液加热至140℃并继续反应2小时后，降温至室温。减压浓缩去除N,N-二甲基甲酰胺，将浓缩物溶于乙酸乙酯(2.0L)，并分别用水(1.5L×2)，饱和碳酸氢钠溶液(1.5L)洗涤，收集有机相并用无水硫酸钠干燥后，减压浓缩得256g粗品。
上述粗品用1.28L甲醇和水混合溶剂(体积比1:2)重结晶，并于20℃析晶，过滤，真空干燥后得类白色固体产物2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,190g，收率74％)，经LCMS检测纯度为92.6％。
MS(ESI):m/z 327.0[M+H]+1HNMR(400MHz,CDCl3)δ:8.11(d,J＝8.0Hz,2H),7.98(m,1H),7.86(m,1H),7.59(t,J＝8.0Hz,7.6Hz,1H),7.33(d,J＝8.0Hz,2H),4.85(s,2H),2.57(d,J＝6.8Hz,2H),1.93(m,1H),0.93(d,J＝6.8Hz,6H).
(2)室温下，将2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,0.14Kg,0.43mol)溶于乙酸乙酯(1.4L)中，再加入活性二氧化锰(0.15Kg,1.72mol)。将反应液加热至回流并继续反应3小时后，降温至室温。过滤并收集浅黄色滤液，用无水硫酸钠干燥后，减压浓缩得白色固体产品2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,139g,收率99％)，经LCMS检测纯度96.9％。
MS(ESI):m/z 325.0[M+H]+1HNMR(400MHz,CDCl3)δ:10.42(s, 1H),8.12～7.99(m,5H),7.34(d,J＝7.2Hz,2H),2.58(d,J＝6.4Hz,2H),1.93(m,1H),0.93(d,J＝6.4Hz,6H).
(3)室温下，将2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,60g,0.185mol)，3-吖丁啶羧酸(1-7,19.5g,0.193mol)与冰醋酸(360mL,0.63mol)加入到甲醇(1.6L)中，20℃下搅拌2小时。将NaBH3CN(11.5g,0.185mol)溶于甲醇(200mL)，然后在1小时内将NaBH3CN的甲醇溶液滴加到反应体系中，控制滴加温度在15-20℃之间，滴加完毕后，反应在20℃下搅拌16小时。反应液过滤，滤饼用300mL甲醇淋洗后烘干，得到白色固体1-{2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(化合物2,64g,收率84.4％)，经LC-MS检测的纯度为95.5％。
MS(ESI):m/z 410.2[M+H]+；1HNMR(400MHz,CDCl3)δ:8.13(d,J＝8.4Hz,2H),8.05(m,1H),7.97(m,1H),7.68(t,J＝8.0Hz,7.6Hz,1H),7.42(d,J＝8.4Hz,2H),4.40(s,2H),4.15(m,4H),3.41(m,1H),2.61(d,J＝7.2Hz,2H),1.95(m,1H),0.94(d,J＝7.2Hz,6H).
实施例21 本发明的合成方法
(1)室温下，将4-异丁基苯甲酸(1-4,0.148Kg,0.83mol)溶于N,N-二甲基甲酰胺(1.7L)中，再加1-羟基苯并三唑(0.11Kg,0.83mol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.16Kg,0.83mol)，加热至30℃并继续搅拌30分钟后，加入3-氟-N'-羟基-4-羟甲基苯甲脒(1-3,0.153Kg,0.83mol)。将反应液加热至140℃并继续反应2小时后，降温至室温。减压浓缩去除N,N-二甲基甲酰胺，将浓缩物溶于乙酸乙酯(2.0L)，并分别用水(1.5L×2)，饱和碳酸氢钠溶液(1.5L)洗涤，收集有机相并用无水硫酸钠干燥后，减压浓缩得258g粗品。
上述粗品用1.29L甲醇和水混合溶剂(体积比3:1)重结晶，并于20℃析晶，过滤，真空干燥后得类白色固体产物2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5,186g，收率72％)，经LCMS检测纯度为93.8％。
MS(ESI):m/z 327.0[M+H]+1HNMR(400MHz,CDCl3)δ:8.11(d,J＝8.0Hz,2H),7.98(m,1H),7.86(m,1H),7.59(t,J＝8.0Hz,7.6Hz,1H),7.33(d,J＝8.0Hz,2H),4.85(s,2H),2.57(d,J＝6.8Hz,2H),1.93(m,1H),0.93(d,J＝6.8Hz,6H).
(2)室温下，将2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇(1-5, 0.14Kg,0.43mol)溶于乙酸乙酯(2.8L)中，再加入活性二氧化锰(0.21Kg,2.42mol)。将反应液加热至回流并继续反应3小时后，降温至室温。过滤并收集浅黄色滤液，用无水硫酸钠干燥后，减压浓缩得白色固体产品2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,138g,收率99％)，经LCMS检测纯度98.8％。
MS(ESI):m/z 325.0[M+H]+1HNMR(400MHz,CDCl3)δ:10.42(s,1H),8.12～7.99(m,5H),7.34(d,J＝7.2Hz,2H),2.58(d,J＝6.4Hz,2H),1.93(m,1H),0.93(d,J＝6.4Hz,6H).
(3)室温下，将2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛(1-6,60g,0.185mol)，3-吖丁啶羧酸(1-7,19.5g,0.193mol)与冰醋酸(360mL,0.63mol)加入到甲醇(1.6L)中，20℃下搅拌2小时。将NaBH3CN(11.5g,0.185mol)溶于甲醇(200mL)，然后在1小时内将NaBH3CN的甲醇溶液滴加到反应体系中，控制滴加温度在5-15℃之间，滴加完毕后，反应在20℃下搅拌16小时。反应液过滤，滤饼用300mL甲醇淋洗后烘干，得到白色固体1-{2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸(化合物2,64g,收率84.5％)，经LC-MS检测的纯度为96.9％。
MS(ESI):m/z 410.2[M+H]+；1HNMR(400MHz,CDCl3)δ:8.13(d,J＝8.4Hz,2H),8.05(m,1H),7.97(m,1H),7.68(t,J＝8.0Hz,7.6Hz,1H),7.42(d,J＝8.4Hz,2H),4.40(s,2H),4.15(m,4H),3.41(m,1H),2.61(d,J＝7.2Hz,2H),1.95(m,1H),0.94(d,J＝7.2Hz,6H).
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。

Claims (19)

  1. 一种式I所示的化合物，
    Figure PCTCN2014086538-appb-100001
    其中，R为卤族元素或C1-C6烷基。
  2. 根据权利要求1所示的化合物，其中，R为F、Cl或Br；或者R为C1-C3烷基，优选甲基。
  3. 根据权利要求1或2所述的化合物在制备用于治疗由S1P1受体介导的疾病或病症的药物中的用途。
  4. 根据权利要求3所述的用途，其中，所述疾病或病症选自类风湿性关节炎、多发性硬化症、炎症性肠炎、自身免疫性疾病、慢性炎性疾病、哮喘、炎性神经病、关节炎、移植、节段性回肠炎、溃疡性结肠炎、红斑狼疮、牛皮癣、缺血-再灌注损伤、实体肿瘤、与血管生成有关的疾病、血管疾病、疼痛病症、急性病毒病、炎性肠病、胰岛素和非胰岛素依赖性糖尿病以及其它相关免疫疾病。
  5. 根据权利要求4所述的用途，其中，所述疾病或病症选自多发性硬化症、类风湿性关节炎、炎症性肠炎和牛皮癣。
  6. 一种药物组合物，其包含根据权利要求1或2所述的化合物和任选的药学上可接受的载体。
  7. 根据权利要求6所述的药物组合物，其中，所述药物组合物为片剂、栓剂、分散片、肠溶片、咀嚼片、口崩片、胶囊、糖衣剂、颗粒剂、干粉剂、口服溶液剂、注射用小针、注射用冻干粉针或大输液；
    优选地，所述药学上可接受的辅料选自稀释剂、增溶剂、崩解剂、悬浮剂、润滑剂、粘合剂、填充剂、矫味剂、甜味剂、抗氧化剂、表面活性剂、防腐剂、包裹剂和色素。
  8. 一种治疗由S1P1受体介导的疾病或病症的方法，所述方法包括给受试者施用治疗有效量的根据权利要求1或2所述的化合物或者根据权利要求 6或7所述的药物组合物。
  9. 根据权利要求8所述的方法，其中，所述受试者为哺乳动物。
  10. 根据权利要求8或9所述的方法，其中，所述疾病或病症选自类风湿性关节炎、多发性硬化症、炎症性肠炎、自身免疫性疾病、慢性炎性疾病、哮喘、炎性神经病、关节炎、移植、节段性回肠炎、溃疡性结肠炎、红斑狼疮、牛皮癣、缺血-再灌注损伤、实体肿瘤、与血管生成有关的疾病、血管疾病、疼痛病症、急性病毒病、炎性肠病、胰岛素和非胰岛素依赖性糖尿病以及其它相关免疫疾病；
    优选地，所述疾病或病症选自多发性硬化症、类风湿性关节炎、炎症性肠炎和牛皮癣。
  11. 1-{2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苄基}-3-吖丁啶羧酸的合成方法，所述合成方法包括以下步骤：
    (1)在缩合剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和1-羟基苯并三唑的存在下，使式1-3所示的3-氟-N'-羟基-4-羟甲基苯甲脒与式1-4所示的4-异丁基苯甲酸反应生成式1-5所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇，
    Figure PCTCN2014086538-appb-100002
    (2)使步骤(1)得到的式1-5所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇与二氧化锰生成式1-6所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛，
    Figure PCTCN2014086538-appb-100003
    (3)以醋酸为催化剂，氰基硼氢化钠为还原剂，使步骤(3)得到的式1-6所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛与式1-7所示 的3-吖丁啶羧酸反应生成式IB化合物，
    Figure PCTCN2014086538-appb-100004
  12. 根据权利要求11所述的合成方法，其特征在于，所述步骤(1)还包括在生成式1-5所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇之后，纯化制得的粗品的步骤；
    优选地，采用柱层析或结晶操作进行纯化。
  13. 根据权利要求12所述的合成方法，其特征在于，所述结晶操作中，结晶溶剂选自甲醇、乙醇、丙酮、二氯甲烷、乙酸乙酯和水中的一种或多种，优选为甲醇和水的混合溶剂，进一步优选为体积比为3:1的甲醇和水的混合溶剂；
    优选地，粗品与结晶溶剂的重量(以g计)体积(以ml计)比为1:3-20，优选为1:5；
    优选地，在20℃下进行析晶。
  14. 根据权利要求11至13中任一项所述的合成方法，其特征在于，所述步骤(1)的反应在反应溶剂中进行，所述反应溶剂选自乙腈、N-甲基吡咯烷酮和N,N-二甲基甲酰胺中的一种或多种；反应温度为80-140℃；式1-3所示的3-氟-N'-羟基-4-羟甲基苯甲脒与式1-4所示的4-异丁基苯甲酸的摩尔比为1:1～2。
  15. 根据权利要求11至14中任一项所述的合成方法，其特征在于，所述步骤(1)的反应溶剂为N,N-二甲基甲酰胺；
    优选地，反应温度为130-140℃；
    优选地，式1-3所示的3-氟-N'-羟基-4-羟甲基苯甲脒与式1-4所示的4-异丁基苯甲酸的摩尔比为1:1～1.5，优选为1:1～1.2。
  16. 根据权利要求11至15中任一项所述的合成方法，其特征在于，所述步骤(2)的反应在反应溶剂中进行，所述反应溶剂选自甲苯、四氢呋喃和乙酸乙酯中的一种或多种；式1-5所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇与反应溶剂的重量(以g计)体积(以ml计)比为1:10-30；反应温度为40-70℃；式1-5所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3- 基]-苯甲醇与二氧化锰的摩尔比为1:4～10。
  17. 根据权利要求11至16中任一项所述的合成方法，其特征在于，所述步骤(2)的反应溶剂为乙酸乙酯；
    优选地，式1-5所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇与反应溶剂的重量(以g计)体积(以ml计)比为1:10；
    优选地，反应温度为60-70℃；
    优选地，式1-5所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醇与二氧化锰的摩尔比为1:5～6，优选为1:6。
  18. 根据权利要求11至17中任一项所述的合成方法，其特征在于，所述步骤(3)的反应在反应溶剂中进行，所述反应溶剂为四氢呋喃和/或甲醇；式1-6所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛与式1-7所示的3-吖丁啶羧酸的摩尔比为1:1～1.2；式1-6所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛与氰基硼氢化钠的摩尔比为1:0.5～6；反应温度为0-30℃；反应时间为1～16小时。
  19. 根据权利要求11至18中任一项所述的合成方法，其特征在于，步骤(3)的反应溶剂为甲醇；
    优选地，式1-6所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛与式1-7所示的3-吖丁啶羧酸的摩尔比为1:1～1.1，优选为1:1；
    优选地，将氰基硼氢化钠溶解于甲醇后滴加进入反应体系中，滴加温度为0-20℃，优选为15-20℃；式1-6所示的2-氟-4-[5-(4-异丁基苯基)-1,2,4-噁二唑-3-基]-苯甲醛与氰基硼氢化钠的摩尔比优选为1:1；
    反应温度为10-20℃，优选为15-20℃；
    优选地，反应时间为4～16小时。
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WO2025083549A1 (en) 2023-10-16 2025-04-24 Sun Pharma Advanced Research Company Limited Methods and combinations of inhibitors of il-23 pathway and modulators of s1p signaling pathway for the treatment of autoimmune disorders

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KR20170055445A (ko) 2017-05-19
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EP3048103A1 (en) 2016-07-27
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