WO2021197338A1 - 硝羟喹啉前药的晶型、含其的药物组合物及其制备方法和应用 - Google Patents

硝羟喹啉前药的晶型、含其的药物组合物及其制备方法和应用 Download PDF

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WO2021197338A1
WO2021197338A1 PCT/CN2021/084057 CN2021084057W WO2021197338A1 WO 2021197338 A1 WO2021197338 A1 WO 2021197338A1 CN 2021084057 W CN2021084057 W CN 2021084057W WO 2021197338 A1 WO2021197338 A1 WO 2021197338A1
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solvent
methyl
solvents
ether
oxy
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PCT/CN2021/084057
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English (en)
French (fr)
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吴亮
周琛
邓一军
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江苏亚虹医药科技股份有限公司
上海亚虹医药科技有限公司
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Priority to CA3171261A priority Critical patent/CA3171261A1/en
Priority to AU2021245884A priority patent/AU2021245884A1/en
Priority to JP2022557933A priority patent/JP2023519276A/ja
Priority to MX2022011768A priority patent/MX2022011768A/es
Priority to EP21781348.4A priority patent/EP4129983A4/en
Priority to CN202180024008.XA priority patent/CN115427397B/zh
Priority to US17/906,996 priority patent/US20230119296A1/en
Priority to BR112022017437A priority patent/BR112022017437A2/pt
Priority to KR1020227036720A priority patent/KR20230008047A/ko
Publication of WO2021197338A1 publication Critical patent/WO2021197338A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

Definitions

  • the invention relates to a crystal form of a nitroquinoline prodrug, a pharmaceutical composition containing the nitroquinoline prodrug, and a preparation method and application thereof.
  • Nitroxoline as an antibacterial drug that has been marketed, has been used for the treatment of urinary tract infections for a long time. Recent findings indicate that nitroquinoline is also very effective in inhibiting angiogenesis and inhibiting the growth and invasion of cancer cells. It is currently being developed for anti-tumor applications.
  • nitroquinoline can be quickly absorbed into the blood circulation, but due to the serious first-pass effect of the liver on the drug, its biological half-life is very short (according to the implementation of Jiangsu Yahong Pharmaceutical Technology Co., Ltd. in China A single-arm, open, multi-center clinical phase II trial showed that its half-life is 1.22-1.44 hours), and frequent dosing is required.
  • nitroquinoline drugs are generally prescribed to be taken three times a day (TID) or four times (QID), which not only brings economic losses, but is not conducive to patient compliance, and more serious is to increase drug exposure. Continuous damage to the normal body.
  • TID three times a day
  • QID four times
  • a prodrug is a compound obtained by chemical modification of an active drug, which is transformed into the original drug by the action of enzymes in the body to exert its efficacy.
  • Prodrugs have a wide range of applications in drug development, and they have been successfully studied in a variety of different drugs and have obtained good application effects.
  • the prodrug strategy can solve some of the defects of the active agent due to its own physical and chemical properties, such as: 1) Eliminate the bad smell of the drug; 2) Increase the blood concentration; 3) Improve the fat solubility or water solubility of the drug Sex; 4) Extend the action time of the drug; 5) Change the route of administration of the drug.
  • Polymorphic forms of drugs have become an indispensable and important part of drug development and drug quality control.
  • the research on drug polymorphs is helpful to the selection of the biological activity of drug compounds, helps to increase the stability, solubility and other properties of the drug, which in turn is beneficial to the development of drug preparations, the storage of drugs, and the improvement of the quality of drug production. It can improve the bioavailability of the compound and improve the clinical efficacy.
  • the technical problem to be solved by the present invention is to provide a crystal form of a nitroquinoline prodrug, a pharmaceutical composition containing the same, and a preparation method and application thereof.
  • the present inventors studied numerous prodrugs of nitroquinoline (especially the nitroquinoline prodrug compounds described in the example section of WO2020/063824) and found that ((5-nitroquinoline-8-yl) oxygen
  • the water solubility, gastrointestinal stability and pharmacokinetics of the nitroquinoline prodrug of methyl-isobutyryl-L-proline ester are superior to other compounds.
  • the present inventors found that the amorphous stability of ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester is poor, which is not conducive to dosage form preparation. Based on this, the inventor of the present invention, after further research, obtained the crystal form of the present invention and the preparation method thereof.
  • the present invention provides a crystal form A of ((5-nitroquinoline-8-yl)oxy)methyl-isobutyryl-L-proline ester, using Cu-K ⁇ radiation to obtain a 2 ⁇ angle
  • the X-ray powder diffraction pattern includes characteristic peaks at 5.74 ⁇ 0.2°, 6.78 ⁇ 0.2°, 10.86 ⁇ 0.2°, 13.54 ⁇ 0.2°, 16.70 ⁇ 0.2° and 22.65 ⁇ 0.2°.
  • the crystal form A of the ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester uses Cu-K ⁇ radiation
  • the obtained X-ray powder diffraction patterns expressed in 2 ⁇ angles include 5.74 ⁇ 0.2°, 6.78 ⁇ 0.2°, 8.25 ⁇ 0.2°, 10.86 ⁇ 0.2°, 13.54 ⁇ 0.2°, 14.92 ⁇ 0.2°, 16.70 ⁇ 0.2°, 17.23
  • the crystal form A of the ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester uses Cu-K ⁇ radiation, The obtained X-ray powder diffraction pattern expressed in 2 ⁇ angles is shown in Fig. 1.
  • the differential scanning calorimetry of the crystal form A shows that there is an endothermic peak at 101.4°C.
  • the differential scanning calorimetry of the crystal form A of the ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester shows There is an endothermic peak at 101.4°C.
  • the present invention also provides a method for preparing the aforementioned ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester crystal form A, which comprises the following steps :
  • the normal solvent may be a benign solvent capable of dissolving ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester.
  • the normal solvent is preferably one or more of ester solvents, C 1 -C 6 alcohol solvents, ketone solvents, cyanogen solvents, ether solvents and lower halogenated alkane solvents.
  • the ester solvent is preferably ethyl acetate.
  • the C 1 -C 6 alcohol solvent is preferably one or more of methanol, ethanol, isopropanol and isobutanol, more preferably methanol and/or ethanol, and still more preferably methanol or ethanol.
  • the ketone solvent is preferably one or more of acetone, methyl ethyl ketone and methyl isobutyl ketone, more preferably acetone or methyl isobutyl ketone, and still more preferably acetone.
  • the cyanogen solvent is preferably acetonitrile.
  • the ether solvent is preferably tetrahydrofuran and/or 1,4-dioxane, and more preferably tetrahydrofuran.
  • the lower halogenated alkane solvent is preferably dichloromethane.
  • the positive solvent is more preferably an ester solvent
  • the ester solvent is preferably a C1-C5 ester solvent, and more preferably ethyl acetate.
  • the anti-solvent may be capable of promoting the crystallization of ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline in the solution I or Poor solvent precipitated.
  • the anti-solvent is preferably one or more of ether solvents, alcohols, lower alkane solvents and water, and more preferably one or more of ether solvents, lower alkane solvents and water.
  • the ether solvent is preferably one or more of methyl tert-butyl ether, diethyl ether and petroleum ether, more preferably petroleum ether and/or methyl tertiary butyl ether, and still more preferably petroleum ether or methyl tertiary Butyl ether.
  • the alcohols are preferably C1-C6 alcohols, and more preferably isopropanol.
  • the lower alkane solvent is preferably one or more of n-heptane, n-hexane and n-octane, and more preferably n-heptane.
  • the anti-solvent is more preferably an ether solvent
  • the ether solvent is preferably petroleum ether.
  • the positive solvent is more preferably an ester solvent, wherein the ester solvent is preferably a C1-C5 ester solvent, more preferably ethyl acetate; and the antisolvent is more preferably an ether solvent, wherein The ether solvent is preferably petroleum ether.
  • the volume ratio of the positive solvent to the anti-solvent is preferably 1:20 to 2:1, more preferably 1:10 to 1:2, such as 1:5 or 1:9, more preferably 0.3-0.5 .
  • the mixing can be achieved by stirring.
  • the mixing temperature may be room temperature.
  • the temperature at which the solid precipitates may be room temperature.
  • the present invention also provides a second method for preparing the aforementioned ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester crystal form A method, which includes the following step:
  • Solution II containing ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester and solvent is mixed at room temperature to 50°C and centrifuged to obtain;
  • the solvent is a C 1 -C 6 alcohol solvent, ester solvent, ether solvent, lower alkane solvent, lower halogenated alkane solvent, ketone solvent, aromatic hydrocarbon solvent, cyanogen solvent, dimethyl sulfoxide And one or more of water, preferably C 1 -C 6 alcohol solvents, ester solvents, ether solvents, lower alkane solvents, ketone solvents, aromatic hydrocarbon solvents, cyanogen solvents, dimethyl sulfoxide One or more of and water.
  • the C 1 -C 6 alcohol solvent is preferably one or more of methanol, ethanol and isopropanol, and more preferably isopropanol and/or methanol.
  • the ester solvent is preferably one or more of methyl acetate, ethyl acetate and isopropyl acetate, and more preferably isopropyl acetate and/or ethyl acetate.
  • the ether solvent is preferably methyl ethyl ether, diethyl ether, methyl isopropyl ether, methyl tert-butyl ether, cyclopentyl methyl ether, anisole, tetrahydrofuran, 2-methyltetrahydrofuran and 1 ,
  • 4-dioxane more preferably methyl tert-butyl ether, cyclopentyl methyl ether, anisole, tetrahydrofuran, 2-methyltetrahydrofuran and 1,4-dioxane
  • 4-dioxane more preferably methyl tert-butyl ether, cyclopentyl methyl ether, anisole, tetrahydrofuran, 2-methyltetrahydrofuran and 1,4-dioxane
  • 4-dioxane more preferably methyl tert-butyl ether, cyclopentyl methyl ether, anisole, t
  • the lower alkane solvent is preferably n-heptane.
  • the lower halogenated alkane solvent is preferably dichloromethane.
  • the ketone solvent is preferably one or more of methyl ethyl ketone, methyl isobutyl ketone and acetone.
  • the aromatic hydrocarbon solvent is preferably toluene.
  • the cyanogen solvent is preferably acetonitrile.
  • the solvent is a mixed solvent of C 1 -C 6 alcohols and water, a mixed solvent of ethers and lower alkanes, ketones and Mixed solvents of lower alkanes, mixed solvents of ketones and ethers, mixed solvents of esters and C 1 -C 6 alcohols, mixed solvents of aromatic hydrocarbons and lower alkanes, ketones and C 1 -C 6 alcohols
  • a mixed solvent or a mixed solvent of ethers and esters preferably a mixed solvent of C 1 -C 6 alcohols and water, and more preferably a mixed solvent of isopropanol and water or methanol and water;
  • the volume ratio of the former to the latter in the mixed solvent is preferably 1:8-1:1, more preferably 1:4-1:2.
  • the solvent is one or more of C 1 -C 6 alcohol solvents, ester solvents, ketone solvents, ether solvents, lower alkane solvents, aromatic hydrocarbon solvents, cyanogen solvents and water, preferably two
  • the mixed solvent of the solvent is more preferably a mixed solvent of C 1 -C 6 alcohols and water, a mixed solvent of ketones and ethers, a mixed solvent of esters and C 1 -C 6 alcohols, ketones and C 1 -C 6 Mixed solvents of alcohols, mixed solvents of ethers and esters, mixed solvents of aromatic hydrocarbons and lower alkanes, mixed solvents of ethers and lower alkanes, mixed solvents of two ethers, or mixed solvents of cyanides and ethers
  • the mixed solvent is more preferably isopropanol/water, methyl isobutyl ketone/methyl tert-butyl ether, ethyl acetate/isopropanol, toluene/n
  • the volume ratio of the former to the latter in the mixed solvent is preferably 1:4 to 1:2.
  • the present invention also provides a third method for preparing the aforementioned ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester crystal form A, which includes the following step:
  • Solution III containing ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester and solvent is placed at 50°C ⁇ 5°C ⁇ 50°C for one to five cycles , Preferably in the three cycles, until the solids are precipitated and solid-liquid separation is obtained; or, after the solution III is heated to 50°C to dissolve, it is filtered hot, and the filtrate is cooled to 5°C to -20°C, and the solid-liquid separation is have to;
  • the solvent is one or more of C 1 -C 6 alcohol solvents, ester solvents, ketone solvents, ether solvents, lower alkane solvents, aromatic hydrocarbon solvents and water, preferably one or two.
  • the C 1 -C 6 alcohol solvent is preferably one or more of methanol, ethanol and isopropanol.
  • the ester solvent is preferably one or more of methyl acetate, ethyl acetate and isopropyl acetate.
  • the ketone solvent is preferably one or more of methyl ethyl ketone, methyl propyl ketone and acetone.
  • the ether solvent is preferably one or more of methyl ethyl ether, ethyl ether, methyl isopropyl ether, methyl tert-butyl ether, cyclopentyl methyl ether and anisole.
  • the lower alkane solvent is preferably n-heptane.
  • the aromatic hydrocarbon solvent is preferably toluene.
  • the solvent is a mixed solvent of C 1 -C 6 alcohols and water, and a mixed solvent of C 1 -C 6 alcohols and ethers.
  • the C 1 -C 6 alcohol solvent is preferably ethanol and/or isopropanol
  • the ketone solvent is preferably methyl ethyl ketone and/or methyl isobutyl ketone;
  • the ester solvent is preferably isopropyl acetate;
  • the ether solvent is preferably cyclopentyl methyl ether;
  • the aromatic hydrocarbon solvent is preferably toluene;
  • the lower alkane solvent is preferably n-heptane;
  • the volume ratio of the former to the latter in the mixed solvent is preferably 1:20 to 2:1, more preferably 1:10 to 1:2.
  • the present invention also provides a fourth method for preparing the aforementioned ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester crystal form A, which includes the following step:
  • the solvent is a C 1 -C 6 alcohol
  • solvents ether solvents, ketone solvents, ester solvents, aromatic hydrocarbon solvents, dimethyl sulfoxide and water.
  • the C 1 -C 6 alcohol solvent is preferably one or more of methanol, ethanol, isopropanol and isobutanol, and more preferably ethanol and/or isopropanol.
  • the ether solvent is preferably one or more of methyl tert-butyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane and anisole, and more preferably tetrahydrofuran.
  • the ketone solvent is preferably one or more of acetone, methyl ethyl ketone and methyl isobutyl ketone, and more preferably acetone.
  • the ester solvent is preferably ethyl acetate.
  • the aromatic hydrocarbon solvent is preferably toluene.
  • the present invention also provides a fifth method for preparing the aforementioned ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester crystal form A, which includes the following step:
  • the first container containing the solution IV containing ((5-nitroquinoline-8-yl)oxy)methyl-isobutyryl-L-proline ester and the positive solvent is placed in the open of the container.
  • the second container of the solvent seal the second container and let it stand at room temperature. When it is observed that the solid becomes wet or precipitates out, collect the product to obtain;
  • the normal solvent is one or more of C 1 -C 6 alcohol solvents, ether solvents and ketone solvents;
  • the anti-solvent is lower alkane solvents, ether solvents, alcohol solvents and water One or more of.
  • the C 1 -C 6 alcohol solvent is preferably one or more of methanol, ethanol, isopropanol and isobutanol, more preferably ethanol.
  • the ether solvent is preferably one or more of tetrahydrofuran, 2-methyltetrahydrofuran and 1,4-dioxane, and more preferably 1,4-dioxane.
  • the ketone solvent is preferably one or more of acetone, methyl ethyl ketone and methyl isobutyl ketone, and more preferably methyl isobutyl ketone.
  • the lower alkane solvent is preferably n-heptane.
  • the ether solvent is preferably methyl tert-butyl ether.
  • the alcohol solvent is preferably isopropanol.
  • the volume ratio of the positive solvent to the anti-solvent is preferably 1:20 to 2:1, more preferably 1:10 to 1:2 , Such as 1:8 or 1:4.
  • the present invention also provides a sixth method for preparing the aforementioned crystal form A of ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester, which includes the following step:
  • the solvent is one of C 1 -C 6 alcohol solvents, ketone solvents, ester solvents, ether solvents, lower alkane solvents, aromatic hydrocarbon solvents, nitrile solvents, lower halogenated alkane solvents and water.
  • the C 1 -C 6 alcohol solvent is preferably one or more of methanol, ethanol and isopropanol, and more preferably isopropanol.
  • the ketone solvent is preferably one or more of methyl ethyl ketone, methyl isobutyl ketone and acetone, and more preferably methyl ethyl ketone and/or methyl isobutyl ketone.
  • the ester solvent is preferably one or more of methyl acetate, ethyl acetate and isopropyl acetate.
  • the ether solvent is preferably methyl ethyl ether, ethyl ether, methyl isopropyl ether, methyl tert-butyl ether, cyclopentyl methyl ether, anisole and 1,4-dioxane.
  • the ether solvent is preferably methyl ethyl ether, ethyl ether, methyl isopropyl ether, methyl tert-butyl ether, cyclopentyl methyl ether, anisole and 1,4-dioxane.
  • 1,4-dioxane 1,4-dioxane.
  • the lower alkane solvent is preferably n-heptane.
  • the aromatic hydrocarbon solvent is preferably toluene.
  • the nitrile solvent is preferably acetonitrile.
  • the lower halogenated alkane solvent is preferably dichloromethane.
  • the present invention also provides a crystal form B of ((5-nitroquinoline-8-yl)oxy)methyl-isobutyryl-L-proline ester, using Cu-K ⁇ radiation to obtain a 2 ⁇ angle
  • the indicated X-ray powder diffraction pattern includes characteristic peaks at 5.44 ⁇ 0.2°, 10.90 ⁇ 0.2°, 14.09 ⁇ 0.2°, 16.17 ⁇ 0.2°, 17.92 ⁇ 0.2°, 20.66 ⁇ 0.2° and 23.13 ⁇ 0.2°.
  • the crystal form B of ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester uses Cu-K ⁇ radiation
  • the obtained X-ray powder diffraction patterns expressed in 2 ⁇ angles include 5.44 ⁇ 0.2°, 6.19 ⁇ 0.2°, 10.90 ⁇ 0.2°, 14.09 ⁇ 0.2°, 14.88 ⁇ 0.2°, 16.17 ⁇ 0.2°, 17.92 ⁇ 0.2°, 20.66
  • the crystal form B of ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester uses Cu-K ⁇ radiation
  • the obtained X-ray powder diffraction patterns expressed in 2 ⁇ angles include 5.44 ⁇ 0.2°, 6.19 ⁇ 0.2°, 8.06 ⁇ 0.2°, 10.90 ⁇ 0.2°, 12.18 ⁇ 0.2°, 14.09 ⁇ 0.2°, 14.88 ⁇ 0.2°, 16.17
  • the crystal form B of ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester uses Cu-K ⁇ radiation, The obtained X-ray powder diffraction pattern expressed in 2 ⁇ angles is shown in Fig. 6.
  • the differential scanning calorimetry of the crystal form B of the ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester shows There is an endothermic peak at 101.5°C.
  • the present invention also provides a method for preparing the aforementioned ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester crystal form B, which comprises the following steps :
  • the positive solvent is an ester solvent
  • the ester solvent is preferably one or more of methyl acetate, ethyl acetate and isopropyl acetate, and more preferably ethyl acetate;
  • the anti-solvent is an alkane solvent
  • the alkane solvent is preferably one or more of n-hexane, n-heptane and n-octane, more preferably n-heptane.
  • the volume ratio of the positive solvent to the anti-solvent is 1:20 to 2:1, preferably 1:10 to 1:2.
  • the crystal form A of ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester is dissolved in a normal solvent to obtain solution A .
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester crystal form A or (( 5-Nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester crystal form B and auxiliary materials.
  • the adjuvant generally refers to a pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention also provides the aforementioned ((5-nitroquinoline-8-yl)oxy)methyl-isobutyryl-L-proline ester crystal form A or ((5-nitroquinoline-8 The application of the B crystal form of -yl)oxy)methyl-isobutyryl-L-proline ester or a pharmaceutical composition containing the same in the preparation of drugs for the treatment of infectious diseases or cancer.
  • infectious diseases are preferably systemic infections, reproductive system infections or urinary system infections.
  • the cancer is preferably bladder cancer or prostate cancer.
  • room temperature refers to 10-35°C, preferably 15-30°C.
  • alcohol solvent refers to a type of organic compound formed by replacing one or several hydrogens in a hydrocarbon molecule with a hydroxyl group, usually a linear or branched chain alcohol compound of 1-6 carbons, such as methanol, ethanol, One or more of n-propanol, isopropanol, n-butanol, sec-butanol, primary butanol and tert-butanol.
  • ketone solvent refers to a compound in which a carbonyl group is connected to two hydrocarbon groups, usually a linear or branched ketone compound with 1-6 carbons, such as acetone and methyl ethyl ketone (also named as methyl ethyl ketone) , One or more of methyl isopropyl ketone and methyl isobutyl ketone.
  • the ester solvent refers to a compound formed by the esterification reaction of an inorganic acid or organic acid with an alcohol to remove water, usually a linear or branched ester compound of 1-6 carbons, such as ethyl formate , One or more of methyl acetate, ethyl acetate, isopropyl acetate and isobutyl acetate.
  • ether solvent refers to the product in which the hydrogen in the hydroxyl group of alcohol or phenol is replaced by a hydrocarbon group, usually a linear, branched or cyclic ether compound of 1-6 carbons, such as diethyl ether, methyl tert-butyl One or more of ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, and cyclopentyl methyl ether.
  • lower alkane solvents refer to hydrocarbons that are liquid at room temperature, and their carbon atoms are usually 4-10 linear or branched alkanes or cycloalkane compounds, such as n-pentane, n-heptane , One or more of n-octane and cyclohexane.
  • the lower halogenated alkane solvent refers to a hydrocarbon compound containing one or more of fluorine, chlorine, bromine and iodine in a liquid state at room temperature, the number of carbon atoms is usually 1-10, preferably the number of carbon atoms is 1-6 linear or branched alkanes substituted by halogen, such as one or more of dichloromethane, dichloroethane, chloroform, bromoethane and bromobutane.
  • aromatic hydrocarbon solvents refer to hydrocarbons containing benzene ring structures in molecules that are liquid at room temperature, such as toluene and/or xylene.
  • the cyanogen solvent refers to a compound containing a cyano group in the molecule, and it generally refers to a linear or branched cyanogen compound of 1 to 6 carbons, preferably acetonitrile.
  • the amorphous ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester generally refers to the amorphous ((5-nitroquinoline-8-yl)oxy) methyl-isobutyryl-L-proline ester ((5-Nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester crude product, of course, can also be pure ((5-nitroquinolin-8-yl)oxy)methyl -Isobutyryl-L-proline ester.
  • the solution I can be made of any crystalline or amorphous ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester and a positive solvent Solution.
  • solution II can be made of any crystal form or amorphous ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester and solvent The solution.
  • solution III can be made of ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester and solvent in any crystalline or amorphous form Solution.
  • the solution IV can be made of any crystalline or amorphous ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester and a positive solvent The solution.
  • the solution V can be made of any crystalline or amorphous ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester and a solvent Solution.
  • solution A can be made of any crystalline or amorphous ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester and a positive solvent Solution.
  • the positive solvent refers to a benign solvent capable of dissolving ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester.
  • the normal solvent is preferably one or more of ester solvents, C 1 -C 6 alcohol solvents, ketone solvents, cyanogen solvents, ether solvents and lower halogenated alkane solvents.
  • the anti-solvent refers to a poor solvent that can promote the crystallization or precipitation of ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester in the solution.
  • the anti-solvent is preferably one or more of ether solvents, lower alkane solvents and water.
  • “Pharmaceutically acceptable” as described herein is useful for preparing a drug combination that is generally safe, has neither biological toxicity nor other unwanted toxicity, and is acceptable for veterinary use and human drug use. Things.
  • carrier refers to a diluent, adjuvant, or excipient with which the compound is administered.
  • Pharmaceutically acceptable carriers can be liquids, such as water and oils, including oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, rapeseed oil, and the like.
  • the pharmaceutically acceptable carrier may also be physiological saline, gum arabic, gelatin, starch paste, talc, keratin, silica gel, urea and the like.
  • auxiliary agents, stabilizers, thickeners, lubricants, and coloring agents can also be used.
  • compositions of the present invention can be formulated into various preparation forms well-known in the art, such as oral dosage forms (powder, tablet, capsule, soft capsule, liquid medicine, syrup, Elixirs, powders, sachets, granules), or topical preparations (creams, ointments, lotions, gels, balms, plasters, pastes, sprays, aerosols, etc.), or injection preparations (solutions , Suspension, emulsion).
  • oral dosage forms poowder, tablet, capsule, soft capsule, liquid medicine, syrup, Elixirs, powders, sachets, granules
  • topical preparations creams, ointments, lotions, gels, balms, plasters, pastes, sprays, aerosols, etc.
  • injection preparations solutions , Suspension, emulsion.
  • compositions of the present invention those suitable for oral, parenteral (intravenous or subcutaneous) or nasal administration may be mentioned in particular, for example, tablets or dragees, sublingual tablets, gelatin capsules , Lozenges, suppositories, creams, ointments, skin gels, injectable preparations, drinkable suspensions, etc.
  • the pharmaceutical composition according to the present invention may contain pharmaceutically acceptable carriers, adjuvants or diluents, such as fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, Preservatives, substrates, etc.
  • pharmaceutically acceptable carriers such as fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, Preservatives, substrates, etc.
  • Fillers such as starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc.; disintegrants such as starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, Cross-linked polyvinylpyrrole, low-substituted hydroxypropyl cellulose, croscarmellose sodium, etc.; lubricants such as magnesium stearate, sodium lauryl sulfate, talc, silicon dioxide, etc.; suspending agent For example: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropylmethylcellulose, etc.; binders such as starch slurry, polyvinylpyrrolidone, hydroxypropylmethylcellulose, etc.
  • the composition of the present invention can be prepared by using any method known in the art, so that the patient can provide fast, long-lasting or slow release of the active ingredient after taking the medicine.
  • the pharmaceutical composition of the present invention is administered to individual animals such as mammals (rats, mice, domesticated animals, or humans) through various routes. All modes of administration are expected.
  • the administration can be oral or topical. , Rectal administration or intravenous, intramuscular, transdermal, intrathecal, epidural or intracerebroventricular injection.
  • the dosage of the active ingredient of the present invention can be different according to the individual's condition and weight, the nature and severity of the disease, the drug form, the route of administration, and the period of administration, and it can also be selected by those skilled in the art.
  • the dosage can be varied between 1-1500 mg/day, and it can be administered as a single dose per day or divided into multiple doses per day.
  • the positive progress effect of the present invention lies in the fact that the color of the nitroquinoline API in the prior art is dark yellow, is easy to dye, has high requirements on industrial equipment during the production process, and is difficult to clean.
  • the crystal form A and crystal form B prepared by the present invention have stable properties, which is more conducive to quality control in industrial production and stability of drug efficacy.
  • crystal form A has more stable properties, which is more conducive to quality control in industrial production and stability of drug efficacy.
  • FIG. 1 is the XRPD pattern of the crystal form A prepared in Example 2.
  • FIG. 1 is the XRPD pattern of the crystal form A prepared in Example 2.
  • FIG. 2 is a TGA/DSC chart of crystal form A prepared in Example 2.
  • FIG. 2 is a TGA/DSC chart of crystal form A prepared in Example 2.
  • FIG. 3 is the DVS spectrum of crystal form A prepared in Example 2.
  • FIG. 4 is a comparison diagram of XRPD before and after the DVS test of the crystal form A prepared in Example 2.
  • FIG. 4 is a comparison diagram of XRPD before and after the DVS test of the crystal form A prepared in Example 2.
  • FIG. 5 is a PLM photograph of crystal form A prepared in Example 2.
  • Fig. 7 is a TGA/DSC chart of the crystal form B prepared in Example 11.
  • FIG. 8 is a PLM photograph of crystal form B prepared in Example 11.
  • the structure of the compound is determined by nuclear magnetic resonance (NMR) or/and mass spectrometry (MS).
  • NMR shift ( ⁇ ) is given in units of 10 -6 (ppm).
  • the NMR measurement was performed with a Bruker 400M nuclear magnetometer, and the measurement solvent was deuterated dimethyl sulfoxide (dimethyl sulfoxide-d 6 ).
  • MS is measured by liquid chromatography-mass spectrometer (Thermo, Ultimate3000/MSQ); LC/MS is measured by Agilent 6250 LC/MS; silica gel column chromatography generally uses Yantai Huanghai silica gel 200-300 Mesh silica gel is the carrier.
  • nitroquinoline and L-Boc proline were purchased from Shaoyuan Chemical Reagent Company.
  • Test time 5 minutes and 30 seconds
  • the DVS curve is collected on DVS Intrinsic of SMS (Surface Measurement Systems).
  • the relative humidity at 25°C is corrected by the deliquescent point of LiCl, Mg(NO 3 ) 2 and KCl.
  • the collection was performed at room temperature with an Axio Scope A1 microscope.
  • Step 2 Preparation of 1-(tert-butyl)2-(((5-nitroquinolin-8-yl)oxy)methyl)(S)-pyrrole-1,2-carbonic acid diester
  • the X-ray diffraction spectrum (XRPD) of the solid is shown in Figure 1, and the XRPD diffraction peak data is shown in Table 1 below.
  • the TGA/DSC spectrum is shown in Figure 2.
  • the TGA results show that the solid is heated to 150°C and loses 0.8% in weight; the DSC results show that the solid has an endothermic peak at 101.4°C (initial temperature).
  • the DVS spectrum is shown in Figure 3, which shows that the solid has a moisture adsorption of 0.17% at 25°C/80%RH, indicating that the sample has almost no hygroscopicity.
  • the XRPD comparison chart before and after the DVS test is shown in Fig. 4, which shows that the crystal form of the solid does not change before and after the DVS test.
  • the PLM results are shown in Figure 5, which indicates that the solid is irregular crystal particles. This crystal form is defined as A crystal form.
  • Example 2 Weigh about 30 mg of the solid prepared according to Example 2 into a glass bottle for each portion, add 0.5 mL of the solvents listed in Table 2 below, and place the resulting suspension under magnetic stirring ( ⁇ 1000 rpm) at room temperature for 6 days, then Centrifugal separation at 10,000 rpm to obtain a solid. It was detected by XRPD that it was the same as the crystal obtained in Example 2, and both were crystal form A.
  • Test number Solvent (v/v) result A1 Isopropanol Crystal Form A A2 Isopropanol/H 2 O (volume ratio between the two is 98:2) Crystal Form A A3 Isopropanol/H 2 O (volume ratio between the two is 96:4) Crystal Form A A4 Isopropanol/H 2 O (the volume ratio between the two is 92:8) Crystal Form A A5 Isopropanol/H 2 O (Volume ratio between the two is 85:15) Crystal Form A A6 H 2 O Crystal Form A A7 Isopropyl acetate Crystal Form A A8 Methyl tert-butyl ether Crystal Form A A9 N-heptane Crystal Form A A10 Dimethyl sulfoxide/cyclopentyl methyl ether, 1:2 Crystal Form A A11 Tetrahydrofuran/n-heptane, 1:2 Crystal Form A A12 Methanol/H 2 O, 1:4 Crystal Form A A13 Acetone/n-heptane
  • Example 2 Weigh about 30 mg of the solid prepared according to Example 2 into a glass bottle for each portion, add 0.5 mL of the solvents listed in Table 3 below, and place the resulting suspension at 50°C with magnetic stirring ( ⁇ 1000 rpm) for 3 days. Then centrifuged at 10,000 rpm to obtain a solid. It was detected by XRPD that it was the same as the crystal obtained in Example 2, and both were crystal form A.
  • Test number Solvent (v/v) result A14 Cyclopentyl methyl ether Crystal Form A A15 H 2 O Crystal Form A A16 Isopropyl acetate Crystal Form A A17 Isopropanol/H 2 O, 1:2 Crystal Form A A18 Methyl isobutyl ketone/methyl tert-butyl ether, 1:2 Crystal Form A A19 Ethyl acetate/isopropanol, 1:2 Crystal Form A A20 Toluene/n-heptane, 1:2 Crystal Form A A21 Dimethyltetrahydrofuran/n-heptane, 1:2 Crystal Form A A22 Methyl ethyl ketone/isopropanol, 1:2 Crystal Form A A23 Acetonitrile/cyclopentyl methyl ether, 1:4 Crystal Form A A24 Anisole/isopropyl acetate, 1:4 Crystal Form A A25 1,4-Dioxane/cyclopentyl methyl ether, 1:4
  • Test number Solvent (v/v) result A26 Isopropanol Crystal Form A A27 Methyl isobutyl ketone Crystal Form A A28 Isopropyl acetate Crystal Form A A29 Methyl tert-butyl ether Crystal Form A A30 Cyclopentyl methyl ether Crystal Form A A31 N-heptane Crystal Form A A32 H 2 O Crystal Form A A33 Ethanol/cyclopentyl methyl ether, 1:2 Crystal Form A A34 Toluene/isopropyl acetate, 1:4 Crystal Form A A35 Methyl ethyl ketone/isopropyl acetate, 1:4 Crystal Form A
  • Test number Solvent (v/v) result A36 Isopropanol/H 2 O, 9:1 Crystal Form A A37 Methyl isobutyl ketone/n-heptane, 1:1 Crystal Form A A38 Isopropyl acetate Crystal Form A A39 Methyl tert-butyl ether Crystal Form A A40 Cyclopentyl methyl ether Crystal Form A
  • Example 2 Each portion weighed about 30 mg of the solid prepared according to Example 2 into a 3 mL glass bottle, and added about 4 mL of the solvents listed in Table 7 below into a 20 mL glass bottle. After placing the 3 mL glass bottle open in the 20 mL glass bottle, the 20 mL glass bottle is sealed. Let it stand at room temperature until the solid surface becomes wet, or after 7 days, perform the XRPD test. It was detected by XRPD that it was the same as the crystal obtained in Example 2, and both were crystal form A.
  • Test number Solvent result A48 Methyl isobutyl ketone Crystal Form A A49 Dimethyl sulfoxide Crystal Form A A50 H 2 O Crystal Form A A51 Ethanol Crystal Form A A52 Tetrahydrofuran Crystal Form A A53 Methyl tert-butyl ether Crystal Form A A55 Toluene Crystal Form A A56 acetone Crystal Form A A57 Ethyl acetate Crystal Form A A58 1,4-Dioxane Crystal Form A A59 Isopropanol Crystal Form A A60 Anisole Crystal Form A
  • Test number Solvent (v/v) result A67 Isopropanol Crystal Form A A68 Methyl isobutyl ketone Crystal Form A A69 Methyl ethyl ketone Crystal Form A A70 Dichloromethane Crystal Form A A71 1,4-Dioxane Crystal Form A
  • Example 2 30.5 mg of the solid prepared in Example 2 was dissolved in 1 mL of ethyl acetate. After filtration, n-heptane was slowly added. After the addition to 7 mL, a solid precipitated out. The solid was obtained by filtration and dried.
  • the X-ray diffraction spectrum (XRPD) of the solid is shown in Figure 6, and the XRPD diffraction peak data is shown in Table 10 below.
  • the TGA/DSC spectrum is shown in Fig. 7, which has a weight loss of 1.2% when heated to 150°C, and has an endothermic peak at 101.5°C (initial temperature).
  • the PLM results show that the sample is needle-shaped with a length of about 100 ⁇ m (see Figure 8). This crystal form is defined as B crystal form.
  • Test Example 1 Determination of the water solubility of the compound ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester obtained in Example 1
  • Example 1 The compound obtained in Example 1 can slowly release the active ingredient nitroquinoline after entering the human body, and the latter can simultaneously inhibit the methionine aminopeptidase MetAP2 and silent information regulator 2 related enzymes in vascular endothelial cells, and can inhibit tumor blood vessels. Synergies of the newborn. At the same time, nitroquinoline also has an inhibitory effect on the proliferation of tumor cells. In addition, the released active ingredient nitroquinoline exerts a bacteriostatic effect by inhibiting the bacterial methionine aminopeptidase MetAP.
  • the inventors first conducted research on the water solubility of nitroquinoline and the compound obtained in Example 1.
  • Test Example 2 Determination of the liver microsome and plasma stability of the compound obtained in Example 1
  • Example 1 It is expected that the compound obtained in Example 1 is decomposed into nitroquinoline in the body, thereby exerting an anticancer effect.
  • Liver microsomal enzymes and plasma metabolizing enzymes are important ways of compound metabolism in vivo. Therefore, in vitro experiments were performed to determine the stability of the compound obtained in Example 1 in liver microsomes and plasma.
  • Example 1 The above data can show that the compound obtained in Example 1 can be quickly converted into nitroquinoline after entering the body, reducing the possibility of unnecessary biological toxicity, and it has advantages and characteristics as a drug development.
  • Test Example 3 Rat pharmacokinetic determination of the compound obtained in Example 1
  • This experiment studied the changes in the plasma concentration of the compound nitroquinoline in rats after a single intravenous or oral administration of nitroquinoline and the compound obtained in Example 1 to evaluate the nitroquinoline and the compound obtained in Example 1.
  • Tandem quadrupole mass spectrometer (API4000, American Applied Biosystems), liquid chromatography (1200, Agilent), autosampler (CTC A does not apply to lytics HTC PAL), American Applied Biosystems Analyst v1.6.2, low temperature Refrigerated centrifuge (1-15PK, Sigma), vortex oscillator (VX-III, Beijing Tin Technology Co., Ltd.).
  • Test compound Place the test compound in the EP tube, add 1.017 mL of dimethyl sulfoxide, 2.035mL and sterile water for injection (the volume ratio of the three is 1:2:17, v:v:v), ultrasonic for 20 minutes to make it fully dissolved (the compound concentration is 0.005mmol/mL).
  • the dose for intravenous administration is 0.01 mmol/kg, and the dose for oral administration is 0.1 mmol/kg.
  • Pre-dose (0 hour) and 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 24, 28, 32, 48 hours after administration (the sampling point is adjusted according to the situation) in the orbit 0.3ml of whole blood was collected from the venous plexus, placed in a centrifuge tube containing EDTA-K2 (Aldrich Reagent Company) anticoagulant, and placed in crushed ice after collection.
  • EDTA-K2 Aldrich Reagent Company
  • the preparation method of the stabilizing solution Dissolve 200 mg of vitamin C (Aldrich Reagent Company) in 8 mL of normal saline, then add 2 mL of formic acid, and mix well.
  • Standard curve configure a series of standard curve working solution, add 5 ⁇ L to 50 ⁇ L blank rat plasma, add 150 ⁇ L internal standard working solution (containing 2ng/mL diphenhydramine (Aldrich reagent company) in acetonitrile), vortex 1 minute. Centrifuge for 10 minutes at 4°C and 12000 rpm, take 100 ⁇ L of the supernatant into the sample tube, and inject 10 ⁇ L into the LC/MS system for measurement.
  • internal standard working solution containing 2ng/mL diphenhydramine (Aldrich reagent company) in acetonitrile
  • Sample to be tested 50 ⁇ L of plasma to be tested, add 5 ⁇ L of working solution diluent, then add 150 ⁇ L of internal standard working solution (2ng/mL diphenhydramine in acetonitrile), vortex for 1 minute. Centrifuge for 10 minutes at 4°C and 12000 rpm, take 100 ⁇ L of the supernatant into the sample tube, and inject 10 ⁇ L into the LC/MS system for measurement.
  • the WinNonlin V6.2 non-compartmental model was used to calculate the pharmacokinetic parameters.
  • Example 1 Compared with nitroquinoline, the compound obtained in Example 1 has a significant increase in absorption and half-life in rats. Therefore, the drug molecule has a good compliance improvement in reducing the dosage or the number of administrations.
  • Test Example 4 Canine pharmacokinetic determination of the compound obtained in Example 1
  • Nitroquinoline is mainly metabolized by the liver in two phases, with a fast metabolism rate, so it has a short half-life in the body.
  • This experiment studied the changes in the concentration of the compound nitroquinoline in dog plasma after a single intravenous or oral administration of nitroquinoline and the compound obtained in Example 1 to dogs to evaluate the effects of nitroquinoline and the compound obtained in Example 1 Pharmacokinetic behavior in the body.
  • Tandem quadrupole mass spectrometer (API5500, American Applied Biosystems), liquid chromatography (1200, Agilent), autosampler (CTC A does not apply to lytics HTC PAL), American Applied Biosystems A does not apply to lyst v1. 6.2.
  • Male beagle dogs (Beijing Max Biotechnology Co., Ltd., laboratory animal production license number: SCXK ( ⁇ )2016-0001, laboratory animal quality certificate number: 11400600001728), each group of 3, weight 10-13kg, 20- 22 months old, fasted the night before the administration, free access to water, and 4 hours after the administration.
  • Place the test compound in the EP tube add dimethyl sulfoxide, And sterile water for injection (the volume ratio of the three is 1:2:17, v:v:v), ultrasonic for 20 minutes to make it fully dissolved (the compound concentration is 0.005mmol/mL).
  • the dose for intravenous administration is 0.01 mmol/kg, and the dose for oral administration is 0.1 mmol/kg.
  • Example 1 It can be known from the data that, compared with nitroquinoline, the compound obtained in Example 1 has a good absorption in beagle dogs, which indicates that the drug molecule can be effectively reduced by the prodrug molecule.
  • test product Take about 1.0g of the test product, accurately weigh it, place it in the measuring cup, stir and dissolve, and titrate the water content of the test product;
  • V The volume of Karl Fischer titration solution consumed by the test sample, mL;
  • W The weighing amount of the test product, g.
  • test product Weigh about 25 mg of the test product, accurately weigh it, and place it in a 50 mL volumetric flask, add diluent to dissolve and dilute to the mark, and shake well.
  • a U the peak area of impurities in the test solution
  • test product Weigh about 20 mg of the test product, accurately weigh it, and place it in a 10 mL volumetric flask, add diluent to dissolve and dilute to the mark, and shake well. Prepare 2 copies in parallel and label them as S1/S2.
  • ——5 refers to the average peak area of photo solution 1;
  • M RS1 The weighed amount of related substance 2 in reference solution 1, mg;
  • M RS2 The weighed amount of related substance 2 in reference solution 2, mg;
  • test product Weigh about 20 mg of the test product, accurately weigh it, and place it in a 100 mL volumetric flask, add diluent to dissolve and dilute to the mark, and shake it well. Make 2 copies in parallel.
  • a RS2 The average peak area of reference solution 2;
  • ——5 refers to the average peak area of photo solution 1;
  • M RS1 The weighing amount of ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester reference substance in reference solution 1, mg;
  • M RS2 The weighing amount of ((5-nitroquinolin-8-yl)oxy)methyl-isobutyryl-L-proline ester reference substance in the reference substance solution 2, mg;

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Abstract

本发明涉及硝羟喹啉前药的晶型、含其的药物组合物及其制备方法和应用。A晶型的X-射线粉末衍射图谱包括位于5.74±0.2°、6.78±0.2°、10.86±0.2°、13.54±0.2°、16.70±0.2°和22.65±0.2°处的特征峰。B晶型的X-射线粉末衍射图谱包括位于5.44±0.2°、10.90±0.2°、14.09±0.2°、16.17±0.2°、17.92±0.2°、20.66±0.2°和23.13±0.2°处的特征峰。与硝羟喹啉相比，本发明的A晶型和B晶型，不易染色，对设备要求低，更利于工业生产化；性质稳定，更利于工业生产上的质量控制和药物药效的稳定性。

Description

硝羟喹啉前药的晶型、含其的药物组合物及其制备方法和应用 技术领域
本发明涉及硝羟喹啉前药的晶型、含其的药物组合物及其制备方法和应用。
背景技术
硝羟喹啉(Nitroxoline)作为一种已上市销售的抗菌药，长时间被用于治疗尿路感染。最近的发现表明，硝羟喹啉对抑制血管生成以及抑制癌细胞的生长和入侵也非常有效，目前正在被开发用于抗肿瘤。
人体药代动力学研究表明，硝羟喹啉能够迅速被吸收进入血液循环，但由于肝脏对药物的首过效应严重，致使其生物半衰期非常短(根据江苏亚虹医药科技有限公司在中国实施的一项单臂、开放、多中心临床二期实验表明其半衰期为1.22-1.44小时)，而需要频繁给药。为了维持连续的药物暴露量，硝羟喹啉药品一般处方要求每天服用三次(TID)或四次(QID)，这不仅带来经济损失，不利于患者依从，更严重的是加大了药物对正常机体的持续损害。同时，由于硝羟喹啉水溶性很低，往往需要将其制成速释制剂，提高溶解度，无形中增加了生产成本。
前药(prodrug)是活性药物经过化学修饰后得到的化合物，其在体内通过酶的作用转化为原来的药物而发挥药效。前药在药物研发中有广泛的应用，其已经在多种不同的药物中研究成功并得到良好的应用效果。通过前药策略可以解决母药(active agent)因其自身理化性质而产生的一些缺陷，例如：1)消除药物的不良臭味；2)提高血药浓度；3)提高药物的脂溶性或者水溶性；4)延长药物的作用时间；5)改变药物的给药途径等。
药物的多晶型已经成为药物研发和药品质量控制中必不可少的重要组成部分。对药物多晶型的研究有助于药物化合物生物活性的选择，有助于增加药物稳定性、溶解性等性质，进而有利于药物制剂的开发以及药品的储存，提高药品生产质量等，其还可提高化合物的生物利用度，增进临床疗效。
然而，现有技术中未见硝羟喹啉前药及其晶型的相关报道。
发明内容
本发明所要解决的技术问题是提供一种硝羟喹啉前药的晶型、含其的药物组合物及其制备方法和应用。
本发明人研究了众多的硝羟喹啉的前药(特别是WO2020/063824中实施例部分记载的硝羟喹啉前药化合物)，发现((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯这一硝羟喹啉前药的水溶性、肠胃液稳定性及药代优于其他化合物。进一步地，本发明人发现，((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的无定型的稳定性较差、不利于剂型的制备。基于此，本发明人，经过进一步地研究， 获得了本发明的晶型及其制备方法。
本发明提供一种((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型，使用Cu-Kα辐射，得到以2θ角表示的X-射线粉末衍射图谱包括位于5.74±0.2°、6.78±0.2°、10.86±0.2°、13.54±0.2°、16.70±0.2°和22.65±0.2°处的特征峰。
在一些优选的实施方案中，所述((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型，使用Cu-Kα辐射，得到以2θ角表示的X-射线粉末衍射图谱包括位于5.74±0.2°、6.78±0.2°、8.25±0.2°、10.86±0.2°、13.54±0.2°、14.92±0.2°、16.70±0.2°、17.23±0.2°、18.10±0.2°、19.56±0.2°、22.65±0.2°和27.22±0.2°处的特征峰。
在一些优选的实施方案中，所述((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型，使用Cu-Kα辐射，得到以2θ角表示的X-射线粉末衍射图谱如图1所示。
在一些优选的实施方案中，所述A晶型的差示扫描量热显示在101.4℃存在吸热峰。
在一些优选的实施方案中，所述((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型的差示扫描量热显示在101.4℃存在吸热峰。
本发明还提供一种制备前述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型的方法，其包括下述步骤：
含有((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯和正溶剂的溶液I与反溶剂混合，析出固体，固液分离，即得。
上述制备方法中，所述正溶剂可为能够溶解((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的良性溶剂。所述正溶剂优选酯类溶剂、C 1-C 6醇类溶剂、酮类溶剂、氰类溶剂、醚类溶剂和低级卤代烷烃类溶剂中的一种或多种。
其中，所述酯类溶剂优选乙酸乙酯。
其中，所述C 1-C 6醇类溶剂优选甲醇、乙醇、异丙醇和异丁醇中的一种或多种，更优选甲醇和/或乙醇，进一步更优选甲醇或乙醇。
其中，所述酮类溶剂优选丙酮、甲基乙基酮和甲基异丁基酮中的一种或多种，更优选丙酮或甲基异丁基酮，进一步更优选丙酮。
其中，所述氰类溶剂优选乙腈。
其中，所述醚类溶剂优选四氢呋喃和/或1,4-二氧六环，更优选为四氢呋喃。
其中，所述低级卤代烷烃类溶剂优选二氯甲烷。
其中，所述正溶剂更优选酯类溶剂，其中所述酯类溶剂优选C1-C5酯类溶剂，更优选乙酸乙酯。
在上述制备方法中，优选经过滤后，得所述溶液I。
上述制备方法中，所述反溶剂可为能够促进所述溶液I中的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯结晶或析出的不良溶剂。所述反溶剂优选醚类溶剂、醇类、低级烷烃类溶剂和水中的一种或多种，更优选为醚类溶剂、低级烷烃 类溶剂和水中的一种或多种。
其中，所述醚类溶剂优选甲基叔丁基醚、乙醚和石油醚中的一种或多种，更优选石油醚和/或甲基叔丁基醚，进一步更优选石油醚或甲基叔丁基醚。
其中，所述醇类优选C1-C6醇，更优选异丙醇。
其中，所述低级烷烃类溶剂优选正庚烷、正己烷和正辛烷中的一种或多种，更优选正庚烷。
其中，所述反溶剂更优选醚类溶剂，其中所述醚类溶剂优选石油醚。
特别地，上述制备方法中，所述正溶剂更优选酯类溶剂，其中所述酯类溶剂优选C1-C5酯类溶剂，更优选乙酸乙酯；且所述反溶剂更优选醚类溶剂，其中所述醚类溶剂优选石油醚。
上述制备方法中，所述正溶剂与所述反溶剂的体积比优选1:20至2:1，更优选1:10至1:2，例如1:5或1:9，更优选0.3-0.5。
上述制备方法中，所述混合可通过搅拌实现。
上述制备方法中，所述混合的温度可为室温。
上述制备方法中，所述固体析出的温度可为室温。
本发明还提供第二种制备前述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型的方法，其包括下述步骤：
含有((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯和溶剂的溶液II于室温至50℃混合，离心，即得；
所述溶剂为C 1-C 6醇类溶剂、酯类溶剂、醚类溶剂、低级烷烃类溶剂、低级卤代烷烃类溶剂、酮类溶剂、芳香烃类溶剂、氰类溶剂、二甲基亚砜和水的一种或多种，优选C 1-C 6醇类溶剂、酯类溶剂、醚类溶剂、低级烷烃类溶剂、酮类溶剂、芳香烃类溶剂、氰类溶剂、二甲基亚砜和水的一种或多种。
其中，所述C 1-C 6醇类溶剂优选甲醇、乙醇和异丙醇中的一种或多种，更优选为异丙醇和/或甲醇。
其中，所述酯类溶剂优选乙酸甲酯、乙酸乙酯和乙酸异丙酯中的一种或多种，更优选乙酸异丙酯和/或乙酸乙酯。
其中，所述醚类溶剂优选甲基乙基醚、乙醚、甲基异丙基醚、甲基叔丁基醚、环戊基甲基醚、苯甲醚、四氢呋喃、2-甲基四氢呋喃和1,4-二氧六环中的一种或多种，更优选甲基叔丁基醚、环戊基甲醚、苯甲醚、四氢呋喃、2-甲基四氢呋喃和1,4-二氧六环中的一种或多种。
其中，所述低级烷烃类溶剂优选正庚烷。
其中，所述低级卤代烷烃类溶剂优选二氯甲烷。
其中，所述酮类溶剂优选甲基乙基酮、甲基异丁基酮和丙酮中的一种或多种。
其中，所述芳香烃类溶剂优选甲苯。
其中，所述氰类溶剂优选乙腈。
在一些优选的实施方案中，在如上所述的第二种制备方法中，所述溶剂为C 1-C 6醇类与水的混合溶剂、醚类与低级烷烃类的混合溶剂、酮类与低级烷烃类的混合溶剂、酮类与醚类的混合溶剂、酯类与C 1-C 6醇类的混合溶剂、芳香烃类与低级烷烃类的混合溶剂、酮类与C 1-C 6醇类的混合溶剂或者醚类与酯类的混合溶剂，优选C 1-C 6醇类与水的混合溶剂，更优选异丙醇与水或甲醇与水的混合溶剂；
所述混合溶剂中前者与后者的体积比优选为1:8-1:1，更优选1:4-1:2。
在一些优选的实施方案中，在如上所述的第二种制备方法中，其中：
将任意晶型或无定型的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯于50℃的温度悬浮于溶剂中，搅拌，离心，即得；
所述溶剂为C 1-C 6醇类溶剂、酯类溶剂、酮类溶剂、醚类溶剂、低级烷烃类溶剂、芳香烃类溶剂、氰类溶剂和水的一种或多种，优选两种溶剂的混合溶剂，更优选C 1-C 6醇类与水的混合溶剂、酮类与醚类的混合溶剂、酯类与C 1-C 6醇类的混合溶剂、酮类与C 1-C 6醇类的混合溶剂、醚类与酯类的混合溶剂、芳香烃类与低级烷烃类的混合溶剂、醚类与低级烷烃类的混合溶剂、两种醚的混合溶剂或者氰类与醚类的混合溶剂，进一步优选异丙醇/水、甲基异丁基酮/甲基叔丁基醚、乙酸乙酯/异丙醇、甲苯/正庚烷、2-甲基四氢呋喃/正庚烷、丁酮/异丙醇、乙腈/环戊基甲醚、苯甲醚/异丙醇乙酸异丙酯或者1,4-二氧六环/环戊基甲醚混合溶剂；
所述混合溶剂中前者与后者的体积比优选1:4至1:2。
本发明还提供第三种制备前述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型的方法，其包括下述步骤：
含有((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯与溶剂的溶液III置于50℃→5℃→50℃的一至五次循环，优选三次循环中，至固体析出，固液分离，即得；或者，将所述溶液III加热至50℃溶解后，热过滤，将滤液降温至5℃至-20℃，固液分离，即得；
所述溶剂为C 1-C 6醇类溶剂、酯类溶剂、酮类溶剂、醚类溶剂、低级烷烃类溶剂、芳香烃类溶剂和水的一种或多种，优选一种或两种。
其中，所述C 1-C 6醇类溶剂优选甲醇、乙醇和异丙醇中的一种或多种。
其中，所述酯类溶剂优选乙酸甲酯、乙酸乙酯和乙酸异丙酯中的一种或多种。
其中，所述酮类溶剂优选甲基乙基酮、甲基丙基酮和丙酮中的一种或多种。
其中，所述醚类溶剂优选甲基乙基醚、乙醚、甲基异丙基醚、甲基叔丁基醚、环戊基甲基醚和苯甲醚中的一种或多种。
其中，所述低级烷烃类溶剂优选正庚烷。
其中，所述芳香烃类溶剂优选甲苯。
在一些优选的实施方案中，在如上所述的第三种制备方法中，其中所述溶剂为C 1-C 6醇类与水的混合溶剂、C 1-C 6醇类与醚类的混合溶剂、酮类与酯类的混合溶剂、芳香烃类与酯类的混合溶剂或者酮类与低级烷烃类的混合溶剂；所述C 1-C 6 醇类溶剂优选乙醇和/或异丙醇，所述酮类溶剂优选丁酮和/或甲基异丁基酮；所述酯类溶剂优选乙酸异丙酯；所述醚类溶剂优选环戊基甲醚；所述芳香烃类溶剂优选甲苯；所述低级烷烃类溶剂优选正庚烷；
所述混合溶剂中前者与后者的体积比优选1:20至2:1，更优选1:10至1:2。
本发明还提供第四种制备前述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型的方法，其包括下述步骤：
将盛有任意晶型或无定型的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯固体的第一容器敞口置于盛有溶剂的第二容器中，密封所述第二容器，于室温静置，当观察到固体变湿或有固体析出时，收集产物，即得；其中，所述溶剂为C 1-C 6醇类溶剂、醚类溶剂、酮类溶剂、酯类溶剂、芳烃类溶剂、二甲基亚砜和水中的一种或多种。
其中，所述C 1-C 6醇类溶剂优选甲醇、乙醇、异丙醇和异丁醇中的一种或多种，更优选乙醇和/或异丙醇。
其中，所述醚类溶剂优选甲基叔丁基醚、四氢呋喃、2-甲基四氢呋喃、1,4-二氧六环和苯甲醚中的一种或多种，更优选四氢呋喃。
其中，所述酮类溶剂优选丙酮、甲基乙基酮和甲基异丁基酮中的一种或多种，更优选丙酮。
其中，所述酯类溶剂优选乙酸乙酯。
其中，所述芳烃类溶剂优选甲苯。
本发明还提供第五种制备前述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型的方法，其包括下述步骤：
将盛有含有((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯与正溶剂的溶液IV的第一容器敞口置于盛有反溶剂的第二容器中，密封所述第二容器，于室温静置，当观察到固体变湿或有固体析出时，收集产物，即得；
其中，所述正溶剂为C 1-C 6醇类溶剂、醚类溶剂和酮类溶剂中的一种或多种；所述反溶剂为低级烷烃类溶剂、醚类溶剂、醇类溶剂和水中的一种或多种。
所述正溶剂中，所述C 1-C 6醇类溶剂优选甲醇、乙醇、异丙醇和异丁醇中的一种或多种，更优选乙醇。
所述正溶剂中，所述醚类溶剂优选四氢呋喃、2-甲基四氢呋喃和1,4-二氧六环中的一种或多种，更优选1,4-二氧六环。
所述正溶剂中，所述酮类溶剂优选丙酮、甲基乙基酮和甲基异丁基酮中的一种或多种，更优选甲基异丁基酮。
所述反溶剂中，所述低级烷烃类溶剂优选正庚烷。
所述反溶剂中，所述醚类溶剂优选甲基叔丁基醚。
所述反溶剂中，所述醇类溶剂优选异丙醇。
在一些优选的实施方案中，在如上所述的第五种制备方法中，所述正溶剂与 所述反溶剂的体积比优选1:20至2:1，更优选1:10至1:2，例如1:8或1:4。
本发明还提供第六种制备前述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型的方法，其包括下述步骤：
含有((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯和溶剂的溶液V，于室温挥发，收集析出的固体，即得；
所述溶剂为C 1-C 6醇类溶剂、酮类溶剂、酯类溶剂、醚类溶剂、低级烷烃类溶剂、芳香烃类溶剂、腈类溶剂、低级卤代烷烃类溶剂和水的一种或多种，优选C 1-C 6醇类溶剂、酮类溶剂、醚类溶剂和低级卤代烷烃类溶剂中的一种或多种。
其中，所述C 1-C 6醇类溶剂优选甲醇、乙醇和异丙醇中的一种或多种，更有选异丙醇。
其中，所述酮类溶剂优选甲基乙基酮、甲基异丁基酮和丙酮中的一种或多种，更优选甲基乙基酮和/或甲基异丁基酮。
其中，所述酯类溶剂优选乙酸甲酯、乙酸乙酯和乙酸异丙酯中的一种或多种。
其中，所述醚类溶剂优选甲基乙基醚、乙醚、甲基异丙基醚、甲基叔丁基醚、环戊基甲基醚、苯甲醚和1,4-二氧六环中的一种或多种，更优选1,4-二氧六环。
其中，所述低级烷烃类溶剂优选正庚烷。
其中，所述芳香烃类溶剂优选甲苯。
其中，所述腈类溶剂优选乙腈。
其中，所述低级卤代烷烃类溶剂优选二氯甲烷。
本发明还提供一种((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的B晶型，使用Cu-Kα辐射，得到以2θ角表示的X-射线粉末衍射图谱包括位于5.44±0.2°、10.90±0.2°、14.09±0.2°、16.17±0.2°、17.92±0.2°、20.66±0.2°和23.13±0.2°处的特征峰。
在一些优选的实施方案中，所述((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的B晶型，使用Cu-Kα辐射，得到以2θ角表示的X-射线粉末衍射图谱包括位于5.44±0.2°、6.19±0.2°、10.90±0.2°、14.09±0.2°、14.88±0.2°、16.17±0.2°、17.92±0.2°、20.66±0.2°、21.69±0.2°和23.13±0.2°处的特征峰。
在一些优选的实施方案中，所述((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的B晶型，使用Cu-Kα辐射，得到以2θ角表示的X-射线粉末衍射图谱包括位于5.44±0.2°、6.19±0.2°、8.06±0.2°、10.90±0.2°、12.18±0.2°、14.09±0.2°、14.88±0.2°、16.17±0.2°、17.92±0.2°、20.66±0.2°、21.69±0.2°、23.13±0.2°、24.42±0.2°和26.03±0.2°处的特征峰。
在一些优选的实施方案中，所述((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的B晶型，使用Cu-Kα辐射，得到以2θ角表示的X-射线粉末衍射图谱如图6所示。
在一些优选的实施方案中，所述((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯 氨酸酯的B晶型的差示扫描量热显示在101.5℃存在吸热峰。
本发明还提供一种制备前述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的B晶型的方法，其包括下述步骤：
含有((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯和正溶剂的溶液A与反溶剂混合，析出固体，固液分离，即得；
所述正溶剂为酯类溶剂，所述酯类溶剂优选乙酸甲酯、乙酸乙酯和乙酸异丙酯中的一种或多种，更优选乙酸乙酯；
所述反溶剂为烷烃类溶剂，所述烷烃类溶剂优选正己烷、正庚烷和正辛烷中的一种或多种，更优选正庚烷。
在一些优选的实施方案中，所述正溶剂与所述反溶剂的体积比为1:20至2:1，优选1:10至1:2。
在一些优选的实施方案中，将((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型溶解于正溶剂中得到溶液A。
本发明还提供一种药物组合物，其包含前述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型或((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的B晶型和辅料。
上述药物组合物中，所述辅料一般指的是药学上可接受的载体、稀释剂或赋形剂。
本发明还提供前述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型或((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的B晶型或含有其的药物组合物在制备治疗感染类疾病或癌症的药物中的应用。
其中，所述感染类疾病优选全身性感染、生殖系统感染或泌尿系统感染。
其中，所述癌症优选膀胱癌或前列腺癌。
在本文中，室温指的是10-35℃，优选15-30℃。
在本文中，醇类溶剂是指烃分子中一个或几个氢被羟基取代而生成的一类有机化合物，通常为1-6个碳的直链或支链醇类化合物，例如甲醇、乙醇、正丙醇、异丙醇、正丁醇、仲丁醇、伯丁醇和叔丁醇中的一种或多种。
在本文中，酮类溶剂是指羰基与两个烃基相连的化合物，通常为1-6个碳的直链或支链酮类化合物，例如丙酮、丁酮(又命名为甲基乙基酮)、甲基异丙基酮和甲基异丁基酮中的一种或多种。
在本文中，酯类溶剂是指由无机酸或有机酸与醇进行酯化反应脱去水而形成的化合物，通常为1-6个碳的直链或支链酯类化合物，例如甲酸乙酯、乙酸甲酯、乙酸乙酯、乙酸异丙酯和乙酸异丁酯中的一种或多种。
在本文中，醚类溶剂是指醇或酚的羟基中的氢被烃基取代的产物，通常为1-6个碳的直链、支链或环醚类化合物，例如乙醚、甲基叔丁基醚、四氢呋喃、2-甲基四氢呋喃、1,4-二氧六环和环戊基甲基醚中的一种或多种。
在本文中，低级烷烃类溶剂是指常温下为液态的碳氢化合物，其碳原子数通常为4-10的直链或支链烷烃类或环烷烃类化合物，例如正戊烷、正庚烷、正辛烷和环己烷中的一种或多种。
在本文中，低级卤代烷烃类溶剂是指常温下位液态的含氟、氯、溴和碘中一种或多种的碳氢化合物化合物，其碳原子数通常为1-10，优选碳原子数为1-6的被卤素取代的直链或支链烷烃类化合物，例如二氯甲烷、二氯乙烷、氯仿、溴乙烷和溴丁烷中的一种或多种。
在本文中，芳香烃类溶剂是指常温下为液态的分子中含有苯环结构的碳氢化合物，例如甲苯和/或二甲苯。
在本文中，氰类溶剂是指分子中含有氰基的化合物，其通常指的是1-6个碳的直链或支链氰类化合物，优选乙腈。
在本文中，无定型的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯通常指的是无定型的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的粗品，当然，也可为纯的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯。
在本文中，溶液I可为由任意晶型或无定型的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯和正溶剂制成的溶液。
在本文中，溶液II可为由任意晶型或无定型的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯与溶剂的制成的溶液。
在本文中，溶液III可为由任意晶型或无定型的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯与溶剂制成的溶液。
在本文中，溶液IV可为由任意晶型或无定型的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯与正溶剂制成的溶液。
在本文中，溶液V可为由任意晶型或无定型的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯和溶剂制成的溶液。
在本文中，溶液A可为由任意晶型或无定型的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯和正溶剂制成的溶液。
在本文中，正溶剂是指能够溶解((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的良性溶剂。所述正溶剂优选酯类溶剂、C 1-C 6醇类溶剂、酮类溶剂、氰类溶剂、醚类溶剂和低级卤代烷烃类溶剂中的一种或多种。
在本文中，反溶剂是指能够促进溶液中的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯结晶或析出的不良溶剂。所述反溶剂优选醚类溶剂、低级烷烃类溶剂和水中的一种或多种。
本文所述的“药学上可接受的”是其有用于制备通常是安全、既无生物学上的毒性又无其它不需要的毒性的，并且对于兽医使用和人类药物使用是可接受的药物组合物。
本文所述的“载体”是指与化合物一起施用的稀释剂、佐剂或赋形剂。药学上可 接受的载体可以是液体，例如水和油，包括石油、动物、植物或合成来源的油，例如花生油、大豆油、矿物油、菜籽油等。药学上可接受的载体也可以是生理盐水、阿拉伯树胶、明胶、淀粉糊、滑石粉、角蛋白、硅胶、尿素等。另外，还可以使用辅助剂、稳定剂、增稠剂、润滑剂和着色剂等。
本领域技术人员能够理解，本发明的药物组合物可以根据具体的施用方式被配制成各种本领域熟知的制剂形式，例如口服剂型(粉剂、片剂、胶囊、软胶囊、液体药物、糖浆、酏丸、散剂、囊剂、粒剂)，或局部施用制剂(乳膏、软膏、洗剂、凝胶、香脂、膏药、糊剂、喷雾剂、气雾剂等等)，或注射制剂(溶液、悬浮剂、乳剂)。在本发明的药物组合物中，尤其可以提及的是适合于口服、胃肠外(静脉内或皮下)或鼻部给药的那些，例如，片剂或糖衣丸、舌下片、明胶胶囊、锭剂、栓剂、霜剂、软膏剂、皮肤凝胶、可注射制剂、可饮用的混悬液等。
根据本发明的药物组合物可以包含药学上可接受的载体、佐剂或稀释剂，例如：填充剂、崩解剂、润滑剂、助悬剂、粘合剂、甜味剂、矫味剂、防腐剂、基质等。填充剂例如：淀粉、预胶化淀粉、乳糖、甘露醇、甲壳素、微晶纤维素、蔗糖等；崩解剂例如：淀粉、预胶化淀粉、微晶纤维素、羧甲基淀粉钠、交联聚乙烯吡咯、低取代羟丙纤维素、交联羧甲基纤维素钠等；润滑剂例如：硬脂酸镁、十二烷基硫酸钠、滑石粉、二氧化硅等；助悬剂例如：聚乙烯吡咯烷酮、微晶纤维素、蔗糖、琼脂、羟丙基甲基纤维素等；粘合剂例如，淀粉浆、聚乙烯吡咯烷酮、羟丙基甲基纤维素等。本发明的组合物可以通过利用本领域任何已知方法制成，以使病人用药后能提供快速、持久或缓慢释放的活性成分。
本发明的药物组合物通过各种途径给药至个体动物如哺乳动物(大鼠、小鼠、驯化动物或人类)，所有的给药方式均是预期的，例如，给药可以是口服、局部、直肠给药或经静脉、肌肉内、经皮、鞘膜内、硬膜外或脑室内注射。
本发明活性成分的给药剂量可以根据个体的情况和重量、病情的性质和严重程度、药物形式、给药途径以及给药周期的不同而不同，其也可以由本领域技术人员进行选择。剂量可以在1-1500mg/天之间改变，可以每天单次给药或每天分多次给药。
本发明的积极进步效果在于：现有技术中的硝羟喹啉API的颜色为深黄色，易染色，在生产过程中对工业设备要求都很高，难清洗。本发明制得的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型或((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的B晶型相对于硝羟喹啉，不易染色，对设备要求低，更利于工业生产化。此外，本发明制得的A晶型和B晶型性质稳定，更利于工业生产上的质量控制和药物药效的稳定性。进一步地，与B晶型相比，A晶型的性质更稳定，更利于工业生产上的质量控制和药物药效的稳定性。
附图说明
图1为实施例2制得的A晶型的XRPD图谱。
图2为实施例2制得的A晶型的TGA/DSC图谱。
图3为实施例2制得的A晶型的DVS图谱。
图4为实施例2制得的A晶型的在DVS测试前后的XRPD对比图。
图5为实施例2制得的A晶型的PLM照片。
图6为实施例11制得的B晶型的XRPD图谱。
图7为实施例11制得的B晶型的TGA/DSC图谱。
图8为实施例11制得的B晶型的PLM照片。
具体实施方式
以下将结合实施例更详细地阐述本发明，本发明的实施例仅用于说明本发明的技术方案，并非限定本发明的实质和范围。
下述实施例中，化合物的结构是通过核磁共振(NMR)或/和质谱(MS)来确定的。NMR位移(δ)以10 -6(ppm)的单位给出。NMR的测定是用Bruker 400M核磁仪，测定溶剂为氘代二甲基亚砜(二甲基亚砜-d 6)。
下述实施例中，MS的测定用液相色谱质谱联用仪(Thermo，Ultimate3000/MSQ)；LC/MS的测定用安捷伦6250液质联用；硅胶柱色谱法一般使用烟台黄海硅胶200-300目硅胶为载体。
下述实施例中，硝羟喹啉、L-Boc脯氨酸购自韶远化学试剂公司。
实验仪器
1、X-射线粉末衍射(XRPD)
仪器型号：Empyrean；
X射线：Cu，kα，Kα1
Figure PCTCN2021084057-appb-000001
：1.540598；Kα2
Figure PCTCN2021084057-appb-000002
：1.544426；Kα2/Kα1强度比例：0.50；
X射线光管设定：电压：45kV，电流：40mA
扫描模式：连续
扫描范围(°2Theta)：3-40
每步扫描时间(s)：17.8
扫描步长(°2TH)：0.0167
测试时间：5分30秒
2、热重分析(TGA)
仪器型号：TA Q5000/Discovery 5500
方法：线性升温
温度范围：室温-350℃
扫描速率：10℃/min
保护气体：氮气
样品量：1-3mg
3、差示扫描量热(DSC)
仪器型号：TA Q2000/Discovery 2500
方法：线性升温
温度范围：25℃-260℃
扫描速率：10℃/min
保护气体：氮气
样品量：1-3mg
4、调制差示扫描量热(mDSC)
测试模式：常规mDSC
振幅(℃)：1.0
调制周期(秒)：60
扫描速率(℃/分钟)：3.0
保护气体：氮气
样品量：1-3mg
5、动态水分吸附(DVS)
DVS曲线在SMS(Surface Measurement Systems)的DVS Intrinsic上采集。在25℃时的相对湿度用LiCl、Mg(NO 3) 2和KCl的潮解点校正。
测试温度：25℃
样品量：10-20mg
保护气体及流量：氮气，200mL/min
dm/dt：0.002％/min；
最小dm/dt平衡时间：10min
最大平衡时间：180min
RH范围：0％RH-95％RH-0％RH
RH梯度：10％
6、偏光显微镜(PLM)
通过Axio Scope A1显微镜在室温进行采集。
实施例1((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的制备
Figure PCTCN2021084057-appb-000003
步骤1：5-硝基-8-氯甲氧基喹啉的制备
于室温，将碳酸氢钠水溶液(60mL,3.5mol/L)、四丁基硫酸氢铵(1.78g,5.24mmol)加入到硝羟喹啉(10.00g,52.59mmol)的二氯甲烷(100mL)溶液中。于室温搅拌20分钟。向反应体系中滴加氯磺酸氯甲酯(10.42g,63.15mmol)，于室温搅拌反应16小时。过滤反应液，分离有机相，将有机相依次用碳酸钾饱和溶液和饱和食盐水洗涤，无水硫酸钠干燥，过滤后减压浓缩。残余物通过硅胶柱层析色谱法(洗脱剂：二氯甲烷)纯化，得到5-硝基-8-氯甲氧基喹啉(2.5g,产率20％)。
步骤2：1-(叔丁基)2-(((5-硝基喹啉-8-基)氧基)甲基)(S)-吡咯-1,2-碳酸二酯的制备
于室温，将8-氯甲氧基-5-硝基喹啉(1.5g，6.3mmol)和L-Boc脯氨酸(2.02g，9.4mmol)溶于15mL的DMF中，加入碳酸钾(1.73g，12.6mmol)。室温搅拌反应3小时，加入70mL水，用乙酸乙酯(50mL x 2)萃取，有机相用饱和食盐水洗涤，无水硫酸钠干燥，过滤后减压浓缩，得到粗品。粗品通过硅胶柱层析色谱法(PE:EA＝1:1)纯化，得到产品1-(叔丁基)2-(((5-硝基喹啉-8-基)氧基)甲基)(S)-吡咯-1,2-碳酸二酯(2.6g，产率98％)。
步骤3：((5-硝基喹啉-8-基)氧基)甲基L-脯氨酸酯盐酸盐的制备
于0℃，将1-(叔丁基)2-(((5-硝基喹啉-8-基)氧基)甲基)(S)-吡咯-1,2-碳酸二酯(2.6g，6.4mmol)置于HCl/二氧六环(30mL)中，室温搅拌20分钟。反应液减压浓缩，得到产品((5-硝基喹啉-8-基)氧基)甲基L-脯氨酸酯盐酸盐(2.3g，产率97％)。
步骤4：((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的制备
于室温，将((5-硝基喹啉-8-基)氧基)甲基L-脯氨酸酯盐酸盐(150mg，0.43mmol)加到无水二氯甲烷(5mL)中。冰浴冷却，加入异丁酰氯(103.7mg,0.90mmol)， 在0-10℃之间慢慢滴入三乙胺(180mg，1.72mmol)，搅拌20分钟。将反应液减压浓缩，残余物通过硅胶柱层析色谱法纯化(PE:EA＝1:1-0:1)，得到((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯(85mg，收率49.3％)。
1H-NMR(400MHz,二甲基亚砜-d6):δ：9.05(d,J＝4.0Hz,1H),9.00(d,J＝8.8Hz,1H),8.56(d,J＝8.8Hz,1H),7.89-7.86(dd,J＝4.0Hz,8.8Hz,1H),7.55(d,J＝8.8Hz,1H),6.24-6.11(m,2H),4.36-4.33(m,1H),3.59-3.68(m,2H),2.51-2.66(m,1H),2.14～2.19(m，1H),1.92-1.85(m,2H),1.83-1.78(m,1H),0.95(d,J＝6.8Hz.3H),0.89(d,J＝6.8Hz.3H)。
MS计算:387.3；MS实测:388.2[M+H] +。
实施例2((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型的制备例一
将260g根据实施例1制得的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯(纯度98.5％)，加入乙酸乙酯(800mL)溶解，用硅藻土过滤，得澄清滤液。将滤液减压浓缩至400mL，搅拌下加入石油醚(100mL)。于20-30℃，继续搅拌20分钟至溶液有小颗粒析出。缓慢滴加石油醚(900mL)，有大量淡黄色固体析出。于20-30℃，继续搅拌2小时，过滤，得淡黄色固体湿品(300g)。于30-40℃减压干燥，得固体(240g，收率92.3％，LCMS纯度：99.6％)。
该固体的X-射线衍射谱图(XRPD)见图1，其XRPD衍射峰数据见下表1。TGA/DSC谱图见图2，TGA结果表明，该固体加热至150℃失重0.8％；DSC结果表明，该固体在101.4℃(起始温度)处有一个吸热峰。DVS谱图见图3，表明该固体在25℃/80％RH的水分吸附为0.17％，表明样品几乎无引湿性。DVS测试前后的XRPD对比图见图4，其显示该固体在DVS测试前后晶型不变。PLM结果见图5，其表明该固体为不规则的晶体颗粒。将此晶型定义为A晶型。
表1 A晶型的XRPD衍射峰数据
Figure PCTCN2021084057-appb-000004
Figure PCTCN2021084057-appb-000005
实施例3((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯A晶型的制备例二
每份称取约30mg根据实施例2制得的固体至玻璃瓶中，分别加入0.5mL下表2中所列的溶剂，得到的悬浊液置于室温磁力搅拌(～1000rpm)6天，然后在10000rpm下离心分离，得固体。经XRPD检测其与实施例2制得晶体相同，均为A晶型。
表2
试验编号 溶剂(v/v) 结果
A1 异丙醇 A晶型
A2 异丙醇/H 2O(两者体积比98:2) A晶型
A3 异丙醇/H 2O(两者体积比96:4) A晶型
A4 异丙醇/H 2O(两者体积比92:8) A晶型
A5 异丙醇/H 2O(两者体积比85:15) A晶型
A6 H 2O A晶型
A7 乙酸异丙酯 A晶型
A8 甲基叔丁基醚 A晶型
A9 正庚烷 A晶型
A10 二甲基亚砜/环戊基甲基醚，1:2 A晶型
A11 四氢呋喃/正庚烷，1:2 A晶型
A12 甲醇/H 2O，1:4 A晶型
A13 丙酮/正庚烷，1:4 A晶型
实施例4((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯A晶型的制备三
每份称取约30mg根据实施例2制得的固体至玻璃瓶中，分别加入0.5mL下表3中所列的溶剂，得到的悬浊液置于50℃磁力搅拌(～1000rpm)3天，然后在10000rpm下离心分离，得固体。经XRPD检测其与实施例2制得晶体相同，均为A晶型。
表3
试验编号 溶剂(v/v) 结果
A14 环戊基甲基醚 A晶型
A15 H 2O A晶型
A16 乙酸异丙酯 A晶型
A17 异丙醇/H 2O，1:2 A晶型
A18 甲基异丁基酮/甲基叔丁基醚，1:2 A晶型
A19 乙酸乙酯/异丙醇，1:2 A晶型
A20 甲苯/正庚烷，1:2 A晶型
A21 二甲基四氢呋喃/正庚烷，1:2 A晶型
A22 甲基乙基酮/异丙醇，1:2 A晶型
A23 乙腈/环戊基甲基醚，1:4 A晶型
A24 苯甲醚/乙酸异丙酯，1:4 A晶型
A25 1,4-二氧六环/环戊基甲基醚，1:4 A晶型
实施例5((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯A晶型的制备四
每份称取约30mg根据实施例2制得的固体至玻璃瓶中，分别加入0.5mL下表4中所列的溶剂，得到的悬浊液置于温度循环(50→5℃，0.1℃/min，5→50℃，0.375℃/min，三个循环)搅拌，然后在10000rpm下离心分离，得固体。经XRPD 检测其与实施例2制得晶体相同，均为A晶型。
表4
试验编号 溶剂(v/v) 结果
A26 异丙醇 A晶型
A27 甲基异丁基酮 A晶型
A28 乙酸异丙酯 A晶型
A29 甲基叔丁基醚 A晶型
A30 环戊基甲基醚 A晶型
A31 正庚烷 A晶型
A32 H 2O A晶型
A33 乙醇/环戊基甲基醚，1:2 A晶型
A34 甲苯/乙酸异丙酯，1:4 A晶型
A35 甲基乙基酮/乙酸异丙酯，1:4 A晶型
实施例6((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯A晶型的制备五
每份称取约30mg根据实施例2制得的固体至玻璃瓶中，分别加入0.5mL下表5中所列的溶剂，在50℃搅拌2小时后过滤(孔径0.45μm的PTFE滤膜；其厂商：泰坦化学；其型号：针头式滤器疏水性聚四氟乙烯(PTFE)0.45μm 13mm)，将所得滤液放置在生化培养箱(其厂商：上海一恒科学仪器有限公司；其型号：BPC-70F)中，以0.1℃/分钟的降温速度从50℃降温至5℃。若溶液仍然澄清，将澄清的样品转移至-20℃放置过夜，得到固体，用药匙将其取出。经XRPD检测其与实施例2制得晶体相同，均为A晶型。
表5
试验编号 溶剂(v/v) 结果
A36 异丙醇/H 2O，9:1 A晶型
A37 甲基异丁基酮/正庚烷，1:1 A晶型
A38 乙酸异丙酯 A晶型
A39 甲基叔丁基醚 A晶型
A40 环戊基甲基醚 A晶型
实施例7((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯A晶型的制备六
每份称取约30mg根据实施例2制得的固体至玻璃瓶中，分别加入1.0mL下表6所列的正溶剂溶解，过滤。于室温，向澄清溶液中加入下表6所列的反溶剂，边滴加边搅拌至有固体析出，若加入9mL左右的反溶剂后仍无固体析出则停止滴加，然后在10000rpm下离心分离，得固体。经XRPD检测其与实施例2制得晶体相同，均为A晶型。
表6
Figure PCTCN2021084057-appb-000006
实施例8((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯A晶型的制备七
每份称取约30mg根据实施例2制得的固体于3mL玻璃瓶中，另在20mL玻璃瓶中加入约4mL下表7所列的溶剂。将3mL玻璃瓶敞口置于20mL玻璃瓶中后，将20mL玻璃瓶密封。于室温静置至固体表面变湿，或放置7天后进行XRPD测试。经XRPD检测其与实施例2制得晶体相同，均为A晶型。
表7
试验编号 溶剂 结果
A48 甲基异丁基酮 A晶型
A49 二甲基亚砜 A晶型
A50 H 2O A晶型
A51 乙醇 A晶型
A52 四氢呋喃 A晶型
A53 甲基叔丁基醚 A晶型
A55 甲苯 A晶型
A56 丙酮 A晶型
A57 乙酸乙酯 A晶型
A58 1,4-二氧六环 A晶型
A59 异丙醇 A晶型
A60 苯甲醚 A晶型
实施例9((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯A晶型的制备八
每份称取约30mg根据实施例2制得的固体，溶于0.5-1.0mL下表8所列的正溶剂中，过滤得滤液转移至3mL玻璃瓶，另取20mL的玻璃瓶向其中加入约4mL的下表8所列的反溶剂。将装有滤液的3mL玻璃瓶敞口置于20mL玻璃瓶后，密封20mL的玻璃瓶，并于室温静置。当观察到有固体析出时，则收集固体并进行XRPD测试。经XRPD检测其与实施例2制得晶体相同，均为A晶型。
表8
Figure PCTCN2021084057-appb-000007
实施例10((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯A晶型的制备九
每份称取约30mg根据实施例2制得的固体至3mL玻璃瓶中，分别加入1.0-2.0mL下表9所列的溶剂，经震荡过滤(孔径0.45μm的PTFE滤膜；其厂商：泰坦化学；其型号：针头式滤器疏水性聚四氟乙烯(PTFE)0.45μm 13mm)后取其滤液。用封口膜封住装有澄清溶液的玻璃瓶并在上面扎数个小孔，放置在室温缓慢挥发。当有固体析出时，收集所得固体并进行XRPD测试。经XRPD检测其与实施例2制得晶体相同，均为A晶型。
表9
试验编号 溶剂(v/v) 结果
A67 异丙醇 A晶型
A68 甲基异丁基酮 A晶型
A69 甲基乙基酮 A晶型
A70 二氯甲烷 A晶型
A71 1,4-二氧六环 A晶型
实施例11((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯B晶型的制备
将30.5mg实施例2制得的固体溶于1mL乙酸乙酯中，过滤后缓慢加入正庚烷，加至7mL后有固体析出，过滤得到固体并干燥。
该固体的X-射线衍射谱图(XRPD)见图6，其XRPD衍射峰数据见下表10。TGA/DSC谱图见图7，其加热至150℃失重1.2％，并在101.5℃(起始温度)处有一个吸热峰。PLM结果显示样品为针状，长度100μm左右(见图8)。将此晶型定义为B晶型。
表10 B晶型的XRPD衍射峰数据
Figure PCTCN2021084057-appb-000008
Figure PCTCN2021084057-appb-000009
测试例1：实施例1所得化合物((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的水溶性的测定
实施例1所得化合物进入人体后可以缓慢释放活性成分硝羟喹啉，后者可以同时抑制血管内皮细胞中的甲硫氨酸氨基肽酶MetAP2和沉默信息调节因子2相关酶类，发挥抑制肿瘤血管新生的协同效应。同时硝羟喹啉还对肿瘤细胞的增殖有抑制作用。除此之外，释放出的有效成分硝羟喹啉通过抑制细菌的甲硫氨酸氨基肽酶MetAP发挥抑菌作用。
本发明人首先对硝羟喹啉以及实施例1所得化合物进行了水溶性的研究。
实验仪器：96孔过滤板(MSHVN4510或MSHVN4550，密理博)；电子数显涡旋(MS3Digital，IKA)；循环水式多用真空泵(SHB-Ⅲ，郑州长城科工贸易有限公司)；天平(XSLT05，梅特勒-托利多)；舒适型混匀仪(Eppendorf AG 22331 Hamburg，艾本德)；液相色谱(LC-30AD，岛津)；质谱(API4000，美国应用)进样器(Anylytics AG System，CTC)。硝羟喹啉委托海门汇聚根据Journal of Heterocyclic Chemistry，1971,vol.8,p821公开的方法合成。
实验过程：取500μL磷酸缓冲液(pH＝1.2、4.5、6.8或7.4)加入玻璃瓶中，加入2mg化合物粉末，加瓶塞，至于混匀仪(VORTEX-GENIE2)上，室温混匀24小时。然后真空抽滤，滤液经处理后，用LC/MS/MS测定化合物的浓度。
实施例1所得化合物的溶解度结果如下表11所示。
表11实施例1所得化合物的溶解度
Figure PCTCN2021084057-appb-000010
结论：与硝羟喹啉(5-硝基-8羟基喹啉)相比，实施例1所得化合物的水溶性在pH为7.4的缓冲溶液中有几倍的提高，且其水溶性在不同pH下变化很小，基本可看作不变化，该特点在药物的制剂开发中尤为重要。
测试例2：实施例1所得化合物的肝微粒体和血浆稳定性的测定
预期实施例1所得化合物在体内分解成为硝羟喹啉，从而发挥抗癌的作用。肝微粒体酶及血浆代谢酶是化合物体内代谢的重要方式，因此进行了体外实验，测定实施例1所得化合物在肝微粒体及血浆中的稳定性。
1.肝微粒体稳定性测定
实验仪器：恒温振荡器(SHA-B，国华企业)；离心机(5810R，艾本德)，质谱(API4000，美国应用)，液相色谱(LC-30AD，岛津)；进样器(CTC A不适用lytics AG System，CTC)。
实验过程：在100mM磷酸盐缓冲液中加入25μg/mL的丙甲甘肽(Aldrich试剂公司)、5mM氯化镁和0.5mg/mL微粒体(XENOTECH)配制成不含辅酶的反应溶液。然后取一部分加入1mM还原性烟酰胺腺嘌呤二核苷酸磷酸(Aldrich试剂公司)和5mM尿苷二磷酸葡萄糖醛酸(Aldrich试剂公司)配制成含有辅酶的反应溶液。然后在两种反应溶液中加入实施例1所得化合物的工作溶液，使化合物终浓度为2μM。混匀后立即取出50μL溶液作为0分钟样品，剩余样品在37℃孵育30分钟后取出50μL。所有取出的样品立即沉淀蛋白，离心取上清后经LC/MS/MS测定化合物浓度。
实施例1所得化合物的微粒体稳定性结果如下表12所示。
表12实施例1所得化合物的微粒体稳定性
Figure PCTCN2021084057-appb-000011
结论：上述数据可以表明实施例1所得化合物在进入体内后可以快速转化成硝羟喹啉，减小了不必要得生物体毒性的可能性，其具有作为药物开发的优势和特点。
测试例3：实施例1所得化合物的大鼠药代动力学测定
本试验研究了大鼠分别单次静脉或口服给予硝羟喹啉及实施例1所得化合物后，大鼠血浆中化合物硝羟喹啉的浓度变化，以此评价硝羟喹啉及实施例1所得化合物在大鼠体内的药代动力学行为。
1.实验仪器
串联四极杆质谱仪(API4000，美国应用生物系统公司)，液相色谱(1200，安捷伦)，自动进样器(CTC A不适用lytics HTC PAL)，美国应用生物系统公司 Analyst v1.6.2，低温冷冻离心机(1-15PK，Sigma)，涡旋振荡器(VX-Ⅲ，北京踏锦科技有限公司)。
2.药代动力学实验
雄性SD大鼠(北京维通利华实验动物技术有限公司，实验动物生产许可证号：SCXK(京)2016-0006，实验动物合格证号：11400700325643)，每组3只，体重180-250g，6-7周龄，给药前一晚禁食，自由进水，给药4小时后进食。将待测化合物置于EP管中，加入二甲基亚砜1.017mL、
Figure PCTCN2021084057-appb-000012
2.035mL和灭菌注射用水(三者体积比为1：2：17，v:v:v)，超声20分钟使其充分溶解(化合物的配制浓度为：0.005mmol/mL)。静脉给药剂量为0.01mmol/kg，口服给药剂量为0.1mmol/kg。分别于给药前(0小时)和给药后0.0833、0.25、0.5、1、2、4、6、8、10、24、28、32、48小时(取样点根据情况有所调整)于眼眶静脉丛采集全血0.3ml，置于含有EDTA-K2(Aldrich试剂公司)抗凝的离心管中，采集后放置于碎冰中。0.5小时内在5000rpm条件下离心5分钟，分离全部干净血浆，置于另一干净离心管内，按照100:3(血浆/稳定液，v/v)比例加入稳定液，放置于-20℃冰箱中待测。
稳定液的配制方法：将200mg维生素C(Aldrich试剂公司)溶解在8mL生理盐水中，之后加入2mL甲酸，充分混匀。
3.样品浓度测定
标准曲线：配置一系列标准曲线的工作液，取5μL加入到50μL空白大鼠血浆中，加150μL内标工作液(含2ng/mL苯海拉明(Aldrich试剂公司)的乙腈溶液)，涡旋1分钟。4℃，12000转/分钟条件下离心10分钟，取上清液100μL到进样管中，进样10μL到液质联用系统进行测定。
待测样品：50μL待测样品血浆，加5μL工作液的稀释液，然后加入150μL内标工作液(含2ng/mL苯海拉明的乙腈溶液)，涡旋1分钟。4℃，12000转/分钟条件下离心10分钟，取上清液100μL到进样管中，进样10μL到液质联用系统进行测定。采用WinNonlin V6.2非房室模型计算药代动力学参数。
测定结果见下表13至表14。
表13 SD大鼠口服给予硝羟喹啉后硝羟喹啉的血浆浓度
Figure PCTCN2021084057-appb-000013
Figure PCTCN2021084057-appb-000014
表14 SD大鼠口服给予实施例1所得化合物后硝羟喹啉的血浆浓度
Figure PCTCN2021084057-appb-000015
结论：相比硝羟喹啉，实施例1所得化合物在大鼠上，吸收或半衰期都有显著提高。从而对药物分子在降低服用剂量或给药次数上都有很好的依从性提高。
测试例4：实施例1所得化合物的犬药代动力学测定
硝羟喹啉主要经肝二相代谢，代谢速率快，因此在体内半衰期短。本试验研究了犬分别单次静脉或口服给予硝羟喹啉及实施例1所得化合物后，犬血浆中化合物硝羟喹啉的浓度变化，以此评价硝羟喹啉及实施例1所得化合物在体内的药代动力学行为。
1.实验仪器
串联四极杆质谱仪(API5500，美国应用生物系统公司)，液相色谱(1200，安捷伦)，自动进样器(CTC A不适用lytics HTC PAL)，美国应用生物系统公司A不适用lyst v1.6.2。
2.药代动力学实验
雄性比格犬(北京玛斯生物技术有限公司，实验动物生产许可证号：SCXK(京)2016-0001，实验动物质量合格证号：11400600001728)，每组3只，体重10-13kg， 20-22月龄，给药前一晚禁食，自由进水，给药4小时后进食。将待测化合物置于EP管中，加入二甲基亚砜、
Figure PCTCN2021084057-appb-000016
和灭菌注射用水(三者体积比为1：2：17，v:v:v)，超声20分钟使其充分溶解(化合物的配制浓度为：0.005mmol/mL)。静脉给药剂量为0.01mmol/kg，口服给药剂量为0.1mmol/kg。分别于给药前(0小时)和给药后0.0833、0.25、0.5、0.75、1、2、4、6、8、10、12小时(取样点根据情况有所调整)于颈静脉采集全血0.3ml，置于含有EDTA-K2(Aldrich试剂公司)抗凝的离心管中，采集后放置于碎冰中。0.5小时内在1530g条件下离心10分钟，分离全部干净血浆，置于另一干净离心管内，放置于-20℃冰箱中待测。
3.样品浓度测定
配置一系列标准曲线的溶液。取10μL标准曲线溶液和样品分别加1000μL内标工作液(含5ng/mL的维拉帕米(Aldrich试剂公司)，50ng/mL的格列本脲(Aldrich试剂公司)和50ng/mL的双氯芬酸(Aldrich试剂公司)的乙腈溶液)，涡旋5分钟。4℃，3700转/分钟条件下离心10分钟，取上清液60μL到进样管中与120μL水混匀，进样10μL到液质联用系统进行测定。采用WinNonlin V6.2非房室模型计算药代动力学参数。
测定结果见下表15至18。
表15比格犬静脉注射给予硝羟喹啉后硝羟喹啉的血浆浓度
Figure PCTCN2021084057-appb-000017
表16比格犬口服给予硝羟喹啉后硝羟喹啉的血浆浓度
Figure PCTCN2021084057-appb-000018
Figure PCTCN2021084057-appb-000019
表17比格犬静脉注射给予实施例1所得化合物后硝羟喹啉的血浆浓度
Figure PCTCN2021084057-appb-000020
表18比格犬口服给予实施例1所得化合物后硝羟喹啉的血浆浓度
Figure PCTCN2021084057-appb-000021
Figure PCTCN2021084057-appb-000022
结论：通过数据可知，相比硝羟喹啉，实施例1所得化合物在比格犬上有着很好的吸收，预示着通过前药分子可以有效的降低药物分子的给药剂量。
测试例5本发明A晶型的稳定性试验
将实施例2-10所得的晶型，包装于双层PE袋+铝箔袋+纸板桶中，于温度40℃，相对湿度75％；温度25℃，相对湿度60％；5±3℃密闭保存6个月，对考察项目(外观、水分、有关物质1、有关物质2、含量)进行检测，具体测试方法如下。结果如表19所示。从表19中可以看出，A晶型杂质含量较少，且在各条件下杂质含量都基本不增加，表明A晶型在长期的条件下稳定性良好。
1水分
1.1仪器设备
水分测定仪
电子天平
1.2试剂
卡尔费休滴定液：分析纯
无水甲醇：分析纯
1.3检测方法
在测量杯中泵入甲醇约50mL，进行预滴定；
取供试品约1.0g，精密称定，置测量杯中，搅拌溶解后滴定供试品的水分；
分别记录加入供试品的重量、测定时卡尔费休滴定液的浓度和消耗的卡尔费休滴定液的体积；同法测定第2份供试品的水分。
1.4计算公式
Figure PCTCN2021084057-appb-000023
V——供试品消耗卡尔费休滴定液体积，mL；
F——每1mL卡尔费休滴定液相当于水的质量，mg/mL；
W——供试品称样量，g。
2有关物质1
2.1色谱条件：高效液相色谱仪(HPLC)
色谱柱：Waters XBridge C18 4.6×150mm，5μm
检测器：UV或等同的检测器
波长：210nm
柱温：35℃
流速：1.0mL/min
进样体积：10μL
洗针液：乙腈
流动相梯度：
时间(min) 流动相A：0.1％磷酸水溶液 流动相B：乙腈
0.0 90 10
10.0 55 45
15.0 40 60
20.0 5 95
21.0 90 10
30.0 90 10
2.2试剂及对照品
乙腈(色谱级)
磷酸(色谱纯)
二氯甲烷(色谱纯)
5-硝基-8-羟基喹啉对照品；结构式同5-硝基-8-羟基喹啉
((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯对照品：结构式同((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯
杂质1对照品：
Figure PCTCN2021084057-appb-000024
杂质2对照品：
Figure PCTCN2021084057-appb-000025
2.3溶液配制
1)流动相A：0.1％磷酸水溶液
精密量取1.0mL的磷酸，置1000mL超纯水中，混合均匀。
2)流动相B：乙腈
3)稀释剂(空白溶液)：乙腈
4)系统适用性溶液：
称取5-硝基-8-羟基喹啉对照品约25mg、杂质2对照品约25mg，精密称定，置50mL容量瓶中，加稀释剂溶解并稀释至刻度，摇匀，标记为溶液1。
称取杂质1对照品约5mg，称定，置50mL容量瓶中，用己在40℃水浴加热后的二氯甲烷溶解完全并稀释至刻度，摇匀，标记为溶液2。
称取((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯对照品约50mg，精密称定，置100mL容量瓶中，加适量稀释剂溶解，再精密加入1.0mL的溶液1和5.0mL的溶液2，加稀释剂稀释至刻度，摇匀。标记为系统适用性溶液。
5)灵敏度溶液
精密量取1.0mL的系统适用性溶液，置100mL容量瓶中，加稀释剂溶解并稀释至刻度，摇匀。精密量取上述溶液1.0mL，置20mL容量瓶中，加稀释剂溶解并稀释至刻度，摇匀。
6)供试品溶液
称取供试品约25mg，精密称定，置50mL容量瓶中，加稀释剂溶解并稀释至刻度，摇匀。
2.4计算：扣除空白
1)
Figure PCTCN2021084057-appb-000026
A U——供试品溶液中杂质的峰面积；
A T——供试品溶液中总峰面积。
2)总杂(％)＝∑单杂
Figure PCTCN2021084057-appb-000027
Figure PCTCN2021084057-appb-000028
3有关物质2(D-异丁酰脯氨酸)
3.1色谱条件：高效液相色谱仪(HPLC)
色谱柱：Synergi Hydro RP 4.6×250mm,4μm
检测器：UV或等同的检测器
波长：210nm
柱温：35℃
流速：1.0mL/min
进样体积：5μL
洗针液：乙腈
流动相梯度：
时间(min) 流动相A：0.1％磷酸水溶液 流动相B：乙腈
0.0 75 25
8.0 65 35
10.0 20 80
10.1 75 25
20.0 75 25
3.2试剂及对照品
乙腈(色谱级)
磷酸(色谱纯)
超纯水
有关物质2对照品：D-异丁酰脯氨酸
3.3溶液配制
1)流动相A：0.1％磷酸水溶液
2)流动相B：乙腈
3)稀释剂(空白溶液)：50％乙腈水溶液
4)对照品溶液
称取有关物质2对照品约40mg，精密称定，置100mL容量瓶中，加稀释剂溶解并稀释至刻度，摇匀；精密量取上述溶液1.0mL，置100mL容量瓶中，加稀释剂稀释至刻度，摇匀。平行配制2份溶液，分别标记为RS1/RS2。
5)供试品溶液
称取供试品约20mg，精密称定，置10mL容量瓶中，加稀释剂溶解并稀释至刻度，摇匀。平行配制2份，分别标记为S1/S2。
3.4计算：只积分有关物质2峰
Figure PCTCN2021084057-appb-000029
A RS2——对照品溶液2的平均峰面积:
Figure PCTCN2021084057-appb-000030
——5针对照品溶液1的平均峰面积；
As——供试品溶液中有关物质2的峰面积；
M RS1——对照品溶液1中有关物质2的称样量，mg；
Ms——供试品溶液中供试品的称样量，mg；
M RS2——对照品溶液2中有关物质2的称样量，mg；
名称 保留时间(RT)/min
有关物质2 ～4.8
4含量
4.1色谱条件：高效液相色谱仪(HPLC)
色谱柱：Waters XBridge C18 4.6×150mm，5μm
检测器：UV或等同的检测器
波长：210nm
柱温：35℃
流速：1.0mL/min
进样体积：5μL
洗针液：乙腈
流动相：10mM KH 2PO 4水溶液(pH2.8)：ACN＝57:43(V/V)
运行时间：10min
4.2试剂及对照品
磷酸二氢钾(色谱级)
乙腈(色谱级)
磷酸(色谱纯)
((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯对照品：结构式同((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯
4.3溶液配制
1)流动相：10mM KH 2PO 4水溶液(pH2.8)：ACN＝57:43(V/V)
称取1.36g的KH 2PO 4置1000mL超纯水中，溶解完全，用磷酸调节pH至2.8，用0.45μm滤膜过滤。
2)稀释剂(空白溶液)：乙腈：超纯水＝43:57(v/v)
量取430mL的乙腈和570mL的超纯水，混合均匀，超声。
3)对照品溶液
称取((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯对照品约20mg，精密称定，置100mL容量瓶中，加稀释剂溶解并稀释至刻度，摇匀。平行配制2份。
4)供试品溶液
称取供试品约20mg，精密称定，置100mL容量瓶中，加稀释剂熔解并稀释至刻度，摇匀。平行配制2份。
4.4计算
Figure PCTCN2021084057-appb-000031
A RS2——对照品溶液2的平均峰面积；
Figure PCTCN2021084057-appb-000032
——5针对照品溶液1的平均峰面积；
As——供试品溶液中((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的峰面积；
M RS1——对照品溶液1中((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯对照品的称样量，mg；
Ms——供试品溶液中供试品的称样量，mg；
M RS2——对照品溶液2中((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯对照品的称样量，mg；
P——((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯对照品的含量，％。
名称 保留时间(RT)/min
((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯 ～4.0
Figure PCTCN2021084057-appb-000033

Claims (19)

  1. 一种((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型，其特征在于，使用Cu-Kα辐射，得到以2θ角表示的X-射线粉末衍射图谱包括位于5.74±0.2°、6.78±0.2°、10.86±0.2°、13.54±0.2°、16.70±0.2°和22.65±0.2°处的特征峰。α
  2. 根据权利要求1所述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型，其特征在于，使用Cu-Kα辐射，得到以2θ角表示的X-射线粉末衍射图谱包括位于5.74±0.2°、6.78±0.2°、8.25±0.2°、10.86±0.2°、13.54±0.2°、14.92±0.2°、16.70±0.2°、17.23±0.2°、18.10±0.2°、19.56±0.2°、22.65±0.2°和27.22±0.2°处的特征峰。
  3. 根据权利要求1所述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型，其特征在于，使用Cu-Kα辐射，得到以2θ角表示的X-射线粉末衍射图谱如图1所示。
  4. 根据权利要求1至3中任一项所述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型，其特征在于，所述A晶型的差示扫描量热显示在101.4℃存在吸热峰。
  5. 一种制备根据权利要求1至4中任一项所述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型的方法，其特征在于，其包括下述步骤：
    含有((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯和正溶剂的溶液I与反溶剂混合，析出固体，固液分离，即得；
    所述正溶剂优选酯类溶剂、C 1-C 6醇类溶剂、酮类溶剂、氰类溶剂、醚类溶剂和低级卤代烷烃类溶剂中的一种或多种；其中，所述酯类溶剂优选乙酸乙酯；所述C 1-C 6醇类溶剂优选甲醇、乙醇、异丙醇和异丁醇中的一种或多种，更优选甲醇和/或乙醇，进一步更优选甲醇或乙醇；所述酮类溶剂优选丙酮、甲基乙基酮和甲基异丁基酮中的一种或多种，更优选丙酮或甲基异丁基酮，进一步更优选丙酮；所述氰类溶剂优选乙腈；所述醚类溶剂优选四氢呋喃和/或1,4-二氧六环，更优选为四氢呋喃；所述低级卤代烷烃类溶剂优选二氯甲烷；所述正溶剂更优选酯类溶剂，其中所述酯类溶剂优选C1-C5酯类溶剂，更优选乙酸乙酯；
    所述反溶剂优选醚类溶剂、醇类、低级烷烃类溶剂和水中的一种或多种，更优选为醚类溶剂、低级烷烃类溶剂和水中的一种或多种；其中，所述醚类溶剂优选甲基叔丁基醚、乙醚和石油醚中的一种或多种，更优选石油醚和/或甲基叔丁基醚，进一步更优选石油醚或甲基叔丁基醚；所述醇类优选C1-C6醇，更优选异丙 醇；所述低级烷烃类溶剂优选正庚烷、正己烷和正辛烷中的一种或多种，更优选正庚烷；所述反溶剂更优选醚类溶剂，其中所述醚类溶剂优选石油醚；
    特别地，所述正溶剂更优选酯类溶剂，其中所述酯类溶剂优选C1-C5酯类溶剂，更优选乙酸乙酯；且所述反溶剂更优选醚类溶剂，其中所述醚类溶剂优选石油醚；
    更特别地，所述正溶剂与所述反溶剂的体积比为1:20至2:1，优选1:10至1:2，更优选0.3-0.5。
  6. 一种制备根据权利要求1至4中任一项所述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型的方法，其特征在于，其包括下述步骤：
    含有((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯与溶剂的溶液II于室温至50℃的温度下，混合，离心，即得；
    所述溶剂为C 1-C 6醇类溶剂、酯类溶剂、醚类溶剂、低级烷烃类溶剂、低级卤代烷烃类溶剂、酮类溶剂、芳香烃类溶剂、氰类溶剂、二甲基亚砜和水的一种或多种；
    其中，所述C 1-C 6醇类溶剂优选甲醇、乙醇和异丙醇中的一种或多种，更优选为异丙醇和/或甲醇；所述酯类溶剂优选乙酸甲酯、乙酸乙酯和乙酸异丙酯中的一种或多种，更优选乙酸异丙酯和/或乙酸乙酯；所述醚类溶剂优选甲基乙基醚、乙醚、甲基异丙基醚、甲基叔丁基醚、环戊基甲基醚、苯甲醚、四氢呋喃、2-甲基四氢呋喃和1,4-二氧六环中的一种或多种，更优选甲基叔丁基醚、环戊基甲醚、苯甲醚、四氢呋喃、2-甲基四氢呋喃和1,4-二氧六环中的一种或多种；所述低级烷烃类溶剂优选正庚烷；所述低级卤代烷烃类溶剂优选二氯甲烷；所述酮类溶剂优选甲基乙基酮、甲基异丁基酮和丙酮中的一种或多种；所述芳香烃类溶剂优选甲苯；所述氰类溶剂优选乙腈。
  7. 根据权利要求6所述的方法，其特征在于，所述溶剂为C 1-C 6醇类与水的混合溶剂、醚类与低级烷烃类的混合溶剂、酮类与低级烷烃类的混合溶剂、酮类与醚类的混合溶剂、酯类与C 1-C 6醇类的混合溶剂、芳香烃类与低级烷烃类的混合溶剂、酮类与C 1-C 6醇类的混合溶剂或者醚类与酯类的混合溶剂，优选C 1-C 6醇类与水的混合溶剂，更优选异丙醇与水或甲醇与水的混合溶剂；
    所述混合溶剂中前者与后者的体积比优选为1:8-1:1，更优选1:4-1:2。
  8. 根据权利要求6所述的方法，其特征在于，其包括如下步骤：
    将任意晶型或无定型的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯于50℃的温度悬浮于溶剂中，搅拌，离心，即得；
    所述溶剂为C 1-C 6醇类溶剂、酯类溶剂、酮类溶剂、醚类溶剂、低级烷烃类溶 剂、芳香烃类溶剂、氰类溶剂和水的一种或多种，优选两种溶剂的混合溶剂，更优选C 1-C 6醇类与水的混合溶剂、酮类与醚类的混合溶剂、酯类与C 1-C 6醇类的混合溶剂、酮类与C 1-C 6醇类的混合溶剂、醚类与酯类的混合溶剂、芳香烃类与低级烷烃类的混合溶剂、醚类与低级烷烃类的混合溶剂、两种醚类的混合溶剂或者氰类与醚类的混合溶剂，进一步优选异丙醇/水、甲基异丁基酮/甲基叔丁基醚、乙酸乙酯/异丙醇、甲苯/正庚烷、2-甲基四氢呋喃/正庚烷、丁酮/异丙醇、乙腈/环戊基甲醚、苯甲醚/异丙醇乙酸异丙酯或者1,4-二氧六环/环戊基甲醚混合溶剂；
    所述混合溶剂中前者与后者的体积比优选1:4至1:2。
  9. 一种制备根据权利要求1至4中任一项所述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型的方法，其特征在于，其包括下述步骤：
    含有((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯与溶剂的溶液III置于50℃→5℃→50℃的一至五次循环，优选三次循环中，至固体析出，固液分离，即得；或者，将所述溶液III加热至50℃溶解后，热过滤，将滤液降温至5℃至-20℃，固液分离，即得；
    所述溶剂为C 1-C 6醇类溶剂、酯类溶剂、酮类溶剂、醚类溶剂、低级烷烃类溶剂、芳香烃类溶剂和水的一种或多种，优选一种或两种；
    其中，所述C 1-C 6醇类溶剂优选甲醇、乙醇和异丙醇中的一种或多种；所述酯类溶剂优选乙酸甲酯、乙酸乙酯和乙酸异丙酯中的一种或多种；所述酮类溶剂优选甲基乙基酮、甲基丙基酮和丙酮中的一种或多种；所述醚类溶剂优选甲基乙基醚、乙醚、甲基异丙基醚、甲基叔丁基醚、环戊基甲基醚和苯甲醚中的一种或多种；所述低级烷烃类溶剂优选正庚烷；所述芳香烃类溶剂优选甲苯。
  10. 根据权利要求9所述的方法，其特征在于，所述溶剂为C 1-C 6醇类与水的混合溶剂、C 1-C 6醇类与醚类的混合溶剂、酮类与酯类的混合溶剂、芳香烃类与酯类的混合溶剂或者酮类与低级烷烃类的混合溶剂；其中，所述C 1-C 6醇类溶剂优选乙醇和/或异丙醇；所述酮类溶剂优选丁酮和/或甲基异丁基酮；所述酯类溶剂优选乙酸异丙酯；所述醚类溶剂优选环戊基甲醚；所述芳香烃类溶剂优选甲苯；所述低级烷烃类溶剂优选正庚烷；
    所述混合溶剂中前者与后者的体积比优选1:20至2:1，更优选1:10至1:2。
  11. 一种制备根据权利要求1至4中任一项所述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型的方法，其特征在于，其包括下述步骤：
    将盛有任意晶型或无定型的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯固体的第一容器敞口置于盛有溶剂的第二容器中，密封所述第二容器，于室温静置，当观察到固体变湿或有固体析出时，收集产物，即得；
    所述溶剂为C 1-C 6醇类溶剂、醚类溶剂、酮类溶剂、酯类溶剂、芳烃类溶剂、二甲基亚砜和水中的一种或多种；所述C 1-C 6醇类溶剂优选甲醇、乙醇、异丙醇和异丁醇中的一种或多种，更优选乙醇和/或异丙醇；所述醚类溶剂优选甲基叔丁基醚、四氢呋喃、2-甲基四氢呋喃、1,4-二氧六环和苯甲醚中的一种或多种，更优选四氢呋喃；所述酮类溶剂优选丙酮、甲基乙基酮和甲基异丁基酮中的一种或多种，更优选丙酮；所述酯类溶剂优选乙酸乙酯；所述芳烃类溶剂优选甲苯。
  12. 一种制备根据权利要求1至4中任一项所述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型的方法，其特征在于，其包括下述步骤：
    将盛有含有((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯与正溶剂的溶液IV的第一容器敞口置于盛有反溶剂的第二容器中，密封所述第二容器，于室温静置，当观察到固体变湿或有固体析出时，收集产物，即得；
    所述正溶剂为C 1-C 6醇类溶剂、醚类溶剂和酮类溶剂中的一种或多种；所述反溶剂为低级烷烃类溶剂、醚类溶剂、醇类溶剂和水中的一种或多种；所述正溶剂中，所述C 1-C 6醇类溶剂优选甲醇、乙醇、异丙醇和异丁醇中的一种或多种，更优选乙醇；所述正溶剂中，所述醚类溶剂优选四氢呋喃、2-甲基四氢呋喃和1,4-二氧六环中的一种或多种，更优选1,4-二氧六环；所述正溶剂中，所述酮类溶剂优选丙酮、甲基乙基酮和甲基异丁基酮中的一种或多种，更优选甲基异丁基酮；所述反溶剂中，所述低级烷烃类溶剂优选正庚烷；所述反溶剂中，所述醚类溶剂优选甲基叔丁基醚；所述反溶剂中，所述醇类溶剂优选异丙醇；
    所述正溶剂与所述反溶剂的体积比优选1:20至2:1，更优选1:10至1:2。
  13. 一种制备根据权利要求1至4中任一项所述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型的方法，其特征在于，其包括下述步骤：
    含有((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯和溶剂的溶液V，于室温挥发，收集析出的固体，即得；
    所述溶剂为C 1-C 6醇类溶剂、酮类溶剂、酯类溶剂、醚类溶剂、低级烷烃类溶剂、芳香烃类溶剂、腈类溶剂、低级卤代烷烃类溶剂和水的一种或多种，优选C 1-C 6醇类溶剂、酮类溶剂、醚类溶剂和低级卤代烷烃类溶剂中的一种或多种；
    其中，所述C 1-C 6醇类溶剂优选甲醇、乙醇和异丙醇中的一种或多种，更有选异丙醇；所述酮类溶剂优选甲基乙基酮、甲基异丁基酮和丙酮中的一种或多种，更优选甲基乙基酮和/或甲基异丁基酮；所述酯类溶剂优选乙酸甲酯、乙酸乙酯和乙酸异丙酯中的一种或多种；所述醚类溶剂优选甲基乙基醚、乙醚、甲基异丙基醚、甲基叔丁基醚、环戊基甲基醚、苯甲醚和1,4-二氧六环中的一种或多种，更优选1,4-二氧六环；所述低级烷烃类溶剂优选正庚烷；所述芳香烃类溶剂优选甲苯；所述腈类溶剂优选乙腈；所述低级卤代烷烃类溶剂优选二氯甲烷。
  14. 一种((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的B晶型，其特征在于，使用Cu-Kα辐射，得到以2θ角表示的X-射线粉末衍射图谱包括位于5.44±0.2°、10.90±0.2°、14.09±0.2°、16.17±0.2°、17.92±0.2°、20.66±0.2°和23.13±0.2°处的特征峰。
  15. 根据权利要求14所述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的B晶型，其特征在于，使用Cu-Kα辐射，得到以2θ角表示的X-射线粉末衍射图谱包括位于5.44±0.2°、6.19±0.2°、10.90±0.2°、14.09±0.2°、14.88±0.2°、16.17±0.2°、17.92±0.2°、20.66±0.2°、21.69±0.2°和23.13±0.2°处的特征峰；
    特别地，使用Cu-Kα辐射，得到以2θ角表示的X-射线粉末衍射图谱包括位于5.44±0.2°、6.19±0.2°、8.06±0.2°、10.90±0.2°、12.18±0.2°、14.09±0.2°、14.88±0.2°、16.17±0.2°、17.92±0.2°、20.66±0.2°、21.69±0.2°、23.13±0.2°、24.42±0.2°和26.03±0.2°处的特征峰。
  16. 根据权利要求14所述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的B晶型，其特征在于，使用Cu-Kα射线，得到以2θ角表示的X-射线粉末衍射图谱如图6所示。
  17. 一种制备根据权利要求14至16中任一项所述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的B晶型的方法，其特征在于，其包括下述步骤：
    含有((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯和正溶剂的溶液A与反溶剂混合，析出固体，固液分离，即得；
    所述正溶剂为酯类中的一种或多种，所述酯类溶剂优选乙酸甲酯、乙酸乙酯和乙酸异丙酯中的一种或多种，更优选乙酸乙酯；
    所述反溶剂为烷烃类，所述烷烃类溶剂优选正己烷、正庚烷和正辛烷中的一种或多种，更优选正庚烷；
    特别地，所述正溶剂与所述反溶剂的体积比为1:20至2:1，优选1:10至1:2；
    尤其地，将权利要求1-4任一项中所定义的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型溶解于正溶剂中得到溶液A。
  18. 一种药物组合物，其特征在于，其包含根据权利要求1至4中任一项所述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的A晶型或者根据权利要求14至16中任一项所述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的B晶型和辅料；所述辅料优选药学上可接受的载体、稀释剂或赋形剂。
  19. 根据权利要求1至4中任一项所述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰 基-L-脯氨酸酯的A晶型或者根据权利要求14至16中任一项所述的((5-硝基喹啉-8-基)氧基)甲基-异丁酰基-L-脯氨酸酯的B晶型或者根据权利要求18所述的药物组合物在制备治疗感染类疾病或癌症的药物中的应用；其中，所述感染类疾病优选全身性感染、生殖系统感染或泌尿系统感染；所述癌症优选膀胱癌或前列腺癌。
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