Patent Description:
Nitroxoline, a commercially available antibacterial drug, has long been used in the treatment of urinary tract infections. Recent discoveries have shown that nitroxoline is also very effective in inhibiting angiogenesis and inhibiting the growth and invasion of cancer cells, and is currently being developed for anti-tumor applications.

Human pharmacokinetic studies have shown that nitroxoline can be rapidly absorbed into the blood circulation. However, due to the severe first-pass effect of the liver on the drug, its biological half-life is very short (a single-arm, open-label, multi-center phase II clinical trial conducted by Jiangsu Yahong Meditech Co. in China has shown that its half-life is <NUM>-<NUM> hours), thus frequent administration is required. In order to maintain continuous drug exposure, nitroxoline drugs are generally prescribed to be administered three times a day (TID) or four times a day (QID), which not only brings economic losses, is not conducive to patient compliance, but increases the persistent damage of the drug to the body as a more severe consequence. Meanwhile, due to the very low water solubility of nitroxoline, it is often necessary to prepare it as an immediate-release formulation to improve the solubility, which virtually increases the production cost.

A prodrug is a compound obtained by chemical modification of an active drug, which is converted into the original drug in vivo by the action of enzymes to exert its efficacy. Prodrugs are widely used in drug research and development, and they have been successfully developed for many different drugs with good effects in application. The prodrug strategy can solve some defects of the active agent due to its own physical and chemical properties, for example: <NUM>) eliminating the bad odor of the drug; <NUM>) increasing the blood concentration of the drug; <NUM>) improving the lipid solubility or water solubility of the drug; <NUM>) prolonging the action time of the drug; <NUM>) changing the administration route of the drug, and the like.

The polymorphism of drugs have become an indispensable and important part in drug research and development and drug quality control. The research on drug polymorphism facilitates the selection of the biological activity of the drug compound, and helps to improve drug stability, solubility and other properties, which in turn is beneficial to the development of drug preparations, as well as the storage of drugs, the improvement of the quality of drug production, and the like. It can also improve the bioavailability of compounds and enhance the clinical efficacy.

Nitroxoline and nitroxoline analogues are disclosed in <CIT>). However, there is no relevant report of nitroxoline prodrugs and crystal forms thereof in the prior art.

The technical problem solved by the present invention is to provide a crystal form of a nitroxoline prodrug, a pharmaceutical composition containing same, and a preparation method therefor and an application thereof.

The inventor has studied a large number of nitroxoline prodrugs (especially the nitroxoline prodrug compounds described in the examples of <CIT>), and has found that ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, a nitroxoline prodrug, has better water solubility, gastrointestinal stability and pharmacokinetics than other compounds. Further, the inventor has found that the amorphous form of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate has poor stability, which is not conducive to the preparation of formulations. On this basis, through further research, the inventor has obtained the crystal forms of the present invention and the preparation method therefor.

The present invention provides crystal form A of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, wherein the X-ray powder diffraction pattern thereof, which is obtained by using Cu-Kα irradiation and expressed in 2θ angle, comprises characteristic peaks at <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>° and <NUM>±<NUM>°.

In some preferred embodiments, the X-ray powder diffraction pattern of the crystal form A of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, which is obtained by using Cu-Kα irradiation and expressed in 2θ angle, comprises characteristic peaks at <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>° and <NUM>±<NUM>°.

In some preferred embodiments, the X-ray powder diffraction pattern of the crystal form A of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, which is obtained by using Cu-Kα irradiation and expressed in 2θ angle, is as shown in <FIG>.

In some preferred embodiments, the differential scanning calorimetry of the crystal form A shows an endothermic peak at <NUM>.

In some preferred embodiments, the differential scanning calorimetry of the crystal form A of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate shows an endothermic peak at <NUM>.

The present invention also provides a method for preparing the aforementioned crystal form A of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, comprising the following steps of:
mixing Solution I containing ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate and a positive solvent with an anti-solvent to precipitate a solid, and performing solid-liquid separation to obtain the crystal form A.

In the above preparation method, the positive solvent can be a benign solvent capable of dissolving ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate. The positive solvent is preferably one or more of ester solvent, C<NUM>-C<NUM> alcohol solvent, ketone solvent, nitrile solvent, ether solvent and lower halogenated alkane solvent.

Wherein, the ester solvent is preferably ethyl acetate.

Wherein, the C<NUM>-C<NUM> alcohol solvent is preferably one or more of methanol, ethanol, isopropanol and isobutanol, more preferably methanol and/or ethanol, and further more preferably methanol or ethanol.

Wherein, 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 further more preferably acetone.

Wherein, the nitrile solvent is preferably acetonitrile.

Wherein, the ether solvent is preferably tetrahydrofuran and/or <NUM>,<NUM>-dioxane, and more preferably tetrahydrofuran.

Wherein, the lower halogenated alkane solvent is preferably dichloromethane.

Wherein, the positive solvent is more preferably an ester solvent, wherein the ester solvent is preferably a C<NUM>-C<NUM> ester solvent, and more preferably ethyl acetate.

In the above preparation method, preferably after filtration, Solution I is obtained.

In the above preparation method, the anti-solvent can be a poor solvent capable of promoting the crystallization or precipitation of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate in Solution I. The anti-solvent is preferably one or more of ether solvent, alcohol, lower alkane solvent and water, and more preferably one or more of ether solvent, lower alkane solvent and water.

Wherein, 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 tert-butyl ether, and further more preferably petroleum ether or methyl tert-butyl ether.

Wherein, the alcohol is preferably a C<NUM>-C<NUM> alcohol, and more preferably isopropanol.

Wherein, the lower alkane solvent is preferably one or more of n-heptane, n-hexane and n-octane, and more preferably n-heptane.

Wherein, the anti-solvent is more preferably an ether solvent, wherein the ether solvent is preferably petroleum ether.

In particular, in the above preparation method, the positive solvent is more preferably an ester solvent, wherein the ester solvent is preferably a C<NUM>-C<NUM> ester solvent, more preferably ethyl acetate; and the anti-solvent is more preferably an ether solvent, wherein the ether solvent is preferably petroleum ether.

In the above preparation method, the volume ratio of the positive solvent to the anti-solvent is preferably <NUM>:<NUM> to <NUM>:<NUM>, more preferably <NUM>:<NUM> to <NUM>:<NUM>, for example <NUM>:<NUM> or <NUM>:<NUM>, and even more preferably <NUM>-<NUM>.

In the above preparation method, the mixing can be achieved by stirring.

In the above preparation method, the temperature of mixing can be room temperature.

In the above preparation method, the temperature of solid precipitation can be room temperature.

The present invention also provides a second method for preparing the aforementioned crystal form A of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, comprising the following steps of:.

Wherein, the C<NUM>-C<NUM> alcohol solvent is preferably one or more of methanol, ethanol and isopropanol, and more preferably isopropanol and/or methanol.

Wherein, 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.

Wherein, the ether solvent is preferably one or more of methyl ethyl ether, diethyl ether, methyl isopropyl ether, methyl tert-butyl ether, cyclopentyl methyl ether, anisole, tetrahydrofuran, <NUM>-methyltetrahydrofuran and <NUM>,<NUM>-dioxane, and more preferably one or more of methyl tert-butyl ether, cyclopentyl methyl ether, anisole, tetrahydrofuran, <NUM>-methyltetrahydrofuran and <NUM>,<NUM>-dioxane.

Wherein, the lower alkane solvent is preferably n-heptane.

Wherein, the ketone solvent is preferably one or more of methyl ethyl ketone, methyl isobutyl ketone and acetone.

Wherein, the aromatic hydrocarbon solvent is preferably toluene.

In some preferred embodiments, in the second preparation method as described above, the solvent is a mixed solvent of C<NUM>-C<NUM> alcohol and water, a mixed solvent of ether and lower alkane, a mixed solvent of ketone and lower alkane, a mixed solvent of ketone and ether, a mixed solvent of ester and C<NUM>-C<NUM> alcohol, a mixed solvent of aromatic hydrocarbon and lower alkane, a mixed solvent of ketone and C<NUM>-C<NUM> alcohol or a mixed solvent of ether and ester, preferably a mixed solvent of C<NUM>-C<NUM> alcohol 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 <NUM> :<NUM>-<NUM>: <NUM>, and more preferably <NUM>:<NUM>-<NUM>:<NUM>.

In some preferred embodiments, in the second preparation method as described above, wherein:.

The present invention also provides a third method for preparing the aforementioned crystal form A of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, comprising the following steps of:.

Wherein, the C<NUM>-C<NUM> alcohol solvent is preferably one or more of methanol, ethanol and isopropanol.

Wherein, the ester solvent is preferably one or more of methyl acetate, ethyl acetate and isopropyl acetate.

Wherein, the ketone solvent is preferably one or more of methyl ethyl ketone, methyl propyl ketone and acetone.

Wherein, the ether solvent is preferably one or more of methyl ethyl ether, diethyl ether, methyl isopropyl ether, methyl tert-butyl ether, cyclopentyl methyl ether and anisole.

In some preferred embodiments, in the third preparation method as described above, wherein the solvent is a mixed solvent of C<NUM>-C<NUM> alcohol and water, a mixed solvent of C<NUM>-C<NUM> alcohol and ether, a mixed solvent of ketone and ester, a mixed solvent of aromatic hydrocarbon and ester or a mixed solvent of ketone and lower alkane; the C<NUM>-C<NUM> alcohol solvent is preferably ethanol and/or isopropanol, the ketone solvent is preferably butanone 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 <NUM>:<NUM> to <NUM>:<NUM>, and more preferably <NUM>:<NUM> to <NUM>:<NUM>.

The present invention also provides a fourth method for preparing the aforementioned crystal form A of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, comprising the following steps of:
placing an open first container with any crystal form or amorphous form of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate solid in a second container with a solvent, sealing the second container, leaving it to stand at room temperature, and collecting the product when it is observed that the solid becomes wet or there is solid precipitation to obtain the crystal form A; wherein, the solvent is one or more of C<NUM>-C<NUM> alcohol solvent, ether solvent, ketone solvent, ester solvent, aromatic hydrocarbon solvent, dimethyl sulfoxide and water.

Wherein, the C<NUM>-C<NUM> alcohol solvent is preferably one or more of methanol, ethanol, isopropanol and isobutanol, and more preferably ethanol and/or isopropanol.

Wherein, the ether solvent is preferably one or more of methyl tert-butyl ether, tetrahydrofuran, <NUM>-methyltetrahydrofuran, <NUM>,<NUM>-dioxane and anisole, and more preferably tetrahydrofuran.

Wherein, the ketone solvent is preferably one or more of acetone, methyl ethyl ketone and methyl isobutyl ketone, and more preferably acetone.

The present invention also provides a fifth method for preparing the aforementioned crystal form A of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, comprising the following steps of:.

As the positive solvent, the C<NUM>-C<NUM> alcohol solvent is preferably one or more of methanol, ethanol, isopropanol and isobutanol, and more preferably ethanol.

As the positive solvent, the ether solvent is preferably one or more of tetrahydrofuran, <NUM>-methyltetrahydrofuran and <NUM>,<NUM>-dioxane, and more preferably <NUM>,<NUM>-dioxane.

As the positive solvent, the ketone solvent is preferably one or more of acetone, methyl ethyl ketone and methyl isobutyl ketone, and more preferably methyl isobutyl ketone.

As the anti-solvent, the lower alkane solvent is preferably n-heptane.

As the anti-solvent, the ether solvent is preferably methyl tert-butyl ether.

As the anti-solvent, the alcohol solvent is preferably isopropanol.

In some preferred embodiments, in the fifth preparation method as described above, the volume ratio of the positive solvent to the anti-solvent is preferably <NUM>:<NUM> to <NUM>:<NUM>, and more preferably <NUM>: <NUM> to <NUM>:<NUM>, for example <NUM>:<NUM> or <NUM>:<NUM>.

The present invention also provides a sixth method for preparing the aforementioned crystal form A of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, comprising the following steps of:.

Wherein, the C<NUM>-C<NUM> alcohol solvent is preferably one or more of methanol, ethanol and isopropanol, and more preferably isopropanol.

Wherein, 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.

Wherein, the ether solvent is preferably one or more of methyl ethyl ether, diethyl ether, methyl isopropyl ether, methyl tert-butyl ether, cyclopentyl methyl ether, anisole and <NUM>,<NUM>-dioxane, and more preferably <NUM>,<NUM>-dioxane.

The present invention also provides crystal form B of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, wherein the X-ray powder diffraction pattern thereof, which is obtained by using Cu-Kα irradiation and expressed in <NUM> angle, comprises characteristic peaks at <NUM> ± <NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>° and <NUM>±<NUM>°.

In some preferred embodiments, the X-ray powder diffraction pattern of the crystal form B of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, which is obtained by using Cu-Kα irradiation and expressed in 2θ angle, comprises characteristic peaks at <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>° and <NUM>±<NUM>°.

In some preferred embodiments, the X-ray powder diffraction pattern of the crystal form B of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, which is obtained by using Cu-Kα irradiation and expressed in 2θ angle, comprises characteristic peaks at <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>° and <NUM>±<NUM>°.

In some preferred embodiments, the X-ray powder diffraction pattern of the crystal form B of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, which is obtained by using Cu-Kα irradiation and expressed in 2θ angle, is as shown in <FIG>.

In some preferred embodiments, the differential scanning calorimetry of the crystal form B of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate shows an endothermic peak at <NUM>.

The present invention also provides a method for preparing the aforementioned crystal form B of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, comprising the following steps of:.

In some preferred embodiments, the volume ratio of the positive solvent to the anti-solvent is <NUM>:<NUM> to <NUM>:<NUM>, and preferably <NUM>: <NUM> to <NUM>:<NUM>.

In some preferred embodiments, the crystal form A of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate is dissolved in a positive solvent to obtain Solution A.

The present invention also provides a pharmaceutical composition comprising the aforementioned crystal form A of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate or the crystal form B of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate and an auxiliary material.

In the aforementioned pharmaceutical composition, the auxiliary material generally refers to a pharmaceutically acceptable carrier, diluent or excipient.

The present invention also provides the aforementioned crystal form A of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate or the crystal form B of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate or a pharmaceutical composition containing same for use for treating an infectious disease or a cancer.

Wherein, the infectious disease is preferably systemic infection, reproductive system infection or urinary system infection.

Wherein, the cancer is preferably bladder cancer or prostate cancer.

Herein, the room temperature refers to <NUM>-<NUM>, and preferably <NUM>-<NUM>.

Herein, the alcohol solvent refers to a class of organic compounds formed by replacing one or several hydrogens in a hydrocarbon molecule with hydroxyl group(s), usually linear or branched chain alcohol compounds with <NUM>-<NUM> carbons, for example one or more of methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, primary butanol and tert-butanol.

Herein, the ketone solvent refers to a compound in which a carbonyl group is connected to two alkyl groups, usually a linear or branched chain ketone compound with <NUM>-<NUM> carbons, for example one or more of acetone, butanone (also known as methyl ethyl ketone), methyl isopropyl ketone and methyl isobutyl ketone.

Herein, the ester solvent refers to a compound formed by esterification of inorganic or organic acids and alcohols to remove water, usually a linear or branched chain ester compound with <NUM>-<NUM> carbons, for example one or more of ethyl formate, methyl acetate, ethyl acetate, isopropyl acetate and isobutyl acetate.

Herein, the ether solvent refers to a product in which the hydrogen in the hydroxyl group of alcohol or phenol is substituted by a alkyl group, usually a linear, branched chain or cyclic ether compound with <NUM>-<NUM> carbons, for example one or more of diethyl ether, methyl tert-butyl ether, tetrahydrofuran, <NUM>-methyltetrahydrofuran, <NUM>,<NUM>-dioxane and cyclopentyl methyl ether.

Herein, the lower alkane solvent refers to a hydrocarbon that is liquid at room temperature, usually a linear or branched chain alkane or cycloalkane compound with <NUM>-<NUM> carbon atoms, for example one or more of n-pentane, n-heptane, n-octane and cyclohexane.

Herein, the lower halogenated alkane solvent refers to a hydrocarbon compound containing one or more of fluorine, chlorine, bromine and iodine that is liquid at room temperature, usually with <NUM>-<NUM> carbon atoms, preferably a halogen-substituted linear or branched chain alkane compound with <NUM>-<NUM> carbon atoms, for example one or more of dichloromethane, dichloroethane, chloroform, bromoethane and bromobutane.

Herein, the aromatic hydrocarbon solvent refers to a hydrocarbon containing benzene ring structure in its molecule that is liquid at room temperature, for example toluene and/or xylene.

Herein, the nitrile solvent refers to a compound containing a cyano group in the molecule, which usually refers to a linear or branched chain nitrile compound with <NUM>-<NUM> carbons, and preferably acetonitrile.

Herein, amorphous form of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate generally refers to a crude product of amorphous form of ((<NUM>-nitroquinoline) olin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, of course, it can also be pure ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate.

Herein, Solution I can be a solution prepared from any crystal form or amorphous form of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, and a positive solvent.

Herein, Solution II can be a solution prepared from any crystal form or amorphous form of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, and a solvent.

Herein, Solution III can be a solution prepared from any crystal form or amorphous form of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, and a solvent.

Herein, Solution IV can be a solution prepared from any crystal form or amorphous form of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, and a positive solvent.

Herein, Solution V can be a solution prepared from any crystal form or amorphous form of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, and a solvent.

Herein, Solution A can be a solution prepared from any crystal form or amorphous form of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, and a positive solvent.

Herein, the positive solvent refers to a benign solvent capable of dissolving ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate. The positive solvent is preferably one or more of ester solvent, C<NUM>-C<NUM> alcohol solvent, ketone solvent, nitrile solvent, ether solvent and lower halogenated alkane solvent.

Herein, the anti-solvent refers to a poor solvent capable of promoting the crystallization or precipitation of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate in the solution. The anti-solvent is preferably one or more of ether solvent, lower alkane solvent and water.

"Pharmaceutically acceptable" as described herein is one which is useful for preparing a pharmaceutical composition that is generally safe, have neither biological toxicity nor other undesirable toxicity, and are acceptable for veterinary use and human pharmaceutical use.

"Carrier" as described herein refers to a diluent, adjuvant or excipient administered together with the compound. The pharmaceutically acceptable carrier can be a liquid, for example water and oil, including petroleum, oils of animal, plant or synthetic origin, for example peanut oil, soybean oil, mineral oil, rapeseed oil, and the like. The pharmaceutically acceptable carrier can also be physiological saline, gum arabic, gelatin, starch paste, talc, keratin, silica gel, urea and the like. In addition, an aid, stabilizer, thickener, lubricant, colorant, and the like can also be used.

Those skilled in the art are able to understand that the pharmaceutical composition of the present invention can be formulated according to the specific administration route into various formulations well-known in the art, for example oral formulations (powder, tablet, capsule, soft capsule, liquid medicine, syrup, elixir, pulvis, sachet, granule), or formulations for topical administration (cream, ointment, lotion, gel, balm, plaster, paste, spray, aerosol, and the like), or formulations for injection (solution, suspension, emulsion). In the pharmaceutical compositions according to the present invention, mention may notably be made of those suitable for oral, parenteral (intravenous or subcutaneous) or nasal administration, for example, tablet or dragee, sublingual tablet, gelatin capsule, lozenge, suppository, cream, ointment, skin gel, injection, drinkable suspension, and the like.

The pharmaceutical composition according to the present invention can comprise a pharmaceutically acceptable carrier, adjuvant or diluent, for example a filler, disintegrant, lubricant, suspending agent, binder, sweetener, flavoring agent, preservative, matrix, and the like. The filler is for example starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, and the like; the disintegrant is for example starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinylpyrrole, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, and the like; the lubricant is for example magnesium stearate, sodium lauryl sulfate, talc, silicon dioxide, and the like; the suspending agent is for example polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, and the like; the binder is for example starch slurry, polyvinylpyrrolidone, hydroxypropyl methylcellulose, and the like. The composition of the present invention can be prepared by any method known in the art, so as to provide rapid, sustained or slow release of the active ingredient after administration to a patient.

The pharmaceutical composition of the present invention is administered to an individual animal such as mammal (rat, mouse, domesticated animal or human) by various routes, all the administration routes are contemplated, for example, the administration route can be oral, topical, rectal administration or intravenous, intramuscular, transdermal, intrathecal, epidural or intraventricular injection.

The administration dosage of the active ingredient of the present invention can vary according to the condition and weight of the individual, the nature and severity of the disease, the form of drug, the administration route, and the administration period, and can also be selected by those skilled in the art. The dose can vary from <NUM> to <NUM>/day, and the drug can be administered daily in a single dose or in divided doses.

The positive improvement effect of the present invention is that: in prior art, the nitroxoline API is dark yellow in color, is prone to staining, has high requirements for industrial equipment in the production process and is difficult to clean. Compared with nitroxoline, the crystal form A of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate or the crystal form B of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate prepared in the present invention is not prone to staining, has low requirements for equipment, and is more suitable for industrial production. In addition, the crystal form A and crystal form B prepared in the present invention have stable properties, which are more conducive to quality control on industrial production and stability in drug efficacy. Further, compared with the crystal form B, the crystal form A has more stable properties, and is more conducive to quality control on industrial production and stability in drug efficacy.

The present invention will be described in more detail below with reference to the examples. The examples of the present invention are only used to illustrate the technical solutions of the present invention, but do not limit the essence and scope of the present invention.

In the following examples, the structures of the compounds were identified by nuclear magnetic resonance (NMR) or/and mass spectrometry (MS). The NMR shifts (δ) are given in the unit of <NUM>-<NUM> (ppm). The NMR determination was performed with a Bruker <NUM> nuclear magnetic resonance spectrometer, and the solvent for determination was deuterated dimethyl sulfoxide (dimethyl sulfoxide-d<NUM>).

In the following examples, liquid chromatography-mass spectrometer (Thermo, Ultimate3000/MSQ) was used for the MS determination; Agilent <NUM> liquid chromatography-mass spectrometer was used for the LC/MS determination; Yantai Huanghai silica gel of <NUM>-<NUM> mesh was generally used as carrier for the silica gel column chromatography.

In the following examples, nitroxoline and L-Boc proline were purchased from Accela Chemical Reagent Co.

An aqueous solution of sodium bicarbonate (<NUM>, <NUM> mol/L) and tetrabutylammonium hydrogen sulfate (<NUM>, <NUM> mmol) were added to a solution of nitroxoline (<NUM>, <NUM> mmol) in dichloromethane (<NUM>) at room temperature. The reaction system was stirred at room temperature for <NUM> minutes. Chloromethyl chlorosulfonate (<NUM>, <NUM> mmol) was added dropwise to the reaction system, which was then stirred at room temperature for <NUM> hours. The reaction solution was filtered, and the organic phase was separated, washed successively with a saturated solution of potassium carbonate and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane) to obtain <NUM>-nitro-<NUM>-chloromethoxyquinoline (<NUM>, <NUM>% yield).

<NUM>-Chloromethoxy-<NUM>-nitroquinoline (<NUM>, <NUM> mmol) and L-Boc proline (<NUM>, <NUM> mmol) were dissolved in <NUM> of DMF at room temperature, and potassium carbonate (<NUM>, <NUM> mmol) was added. The reaction solution was stirred at room temperature for <NUM> hours, <NUM> of water was added, and the reaction solution was extracted with ethyl acetate (<NUM> × <NUM>). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA=<NUM>:<NUM>) to obtain the product <NUM>-(tert-butyl) <NUM>-(((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl) (S)-pyrrolidine-<NUM>,<NUM>-dicarboxylate (<NUM>, <NUM>% yield).

<NUM>-(Tert-butyl) <NUM>-(((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl) (S)-pyrrolidine-<NUM>,<NUM>-dicarboxylate (<NUM>, <NUM> mmol) was put into HCl/dioxane (<NUM>) at <NUM> and stirred at room temperature for <NUM> minutes. The reaction solution was concentrated under reduced pressure to obtain the product ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl L-prolinate hydrochloride (<NUM>, <NUM>% yield).

((<NUM>-Nitroquinolin-<NUM>-yl)oxy)methyl L-prolinate hydrochloride (<NUM>, <NUM> mmol) was added to anhydrous dichloromethane (<NUM>) at room temperature. After cooling in an ice bath, isobutyryl chloride (<NUM>, <NUM> mmol) was added, triethylamine (<NUM>, <NUM> mmol) was slowly added dropwise between <NUM> and <NUM>, followed by stirring for <NUM> minutes. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (PE:EA=<NUM>:<NUM>-<NUM>:<NUM>) to obtain ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate (<NUM>, <NUM>% yield).

<NUM>H-NMR (<NUM>, dimethyl sulfoxide-d6): δ: <NUM> (d, J = <NUM>,<NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM>-<NUM> (dd, J = <NUM>,<NUM>,<NUM>),<NUM> (d, J=<NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>) , <NUM>-<NUM>(m, <NUM>) , <NUM>~<NUM>(m, <NUM>),<NUM>-<NUM>(m, <NUM>) , <NUM>-<NUM>(m, <NUM>) ,<NUM> (d, J=<NUM>. <NUM>), <NUM> (d, J=<NUM>.

Calculated MS: <NUM>; measured MS: <NUM> [M+H]+.

<NUM> of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate (<NUM>% purity) prepared according to Example <NUM> was added to and dissolved in ethyl acetate (<NUM>), and filtered through diatomaceous earth to obtain a clear filtrate. The filtrate was concentrated to <NUM> under reduced pressure, and petroleum ether (<NUM>) was added under stirring. Stirring was continued at <NUM>-<NUM> for <NUM> minutes until small particles were precipitated out of the solution. Petroleum ether (<NUM>) was slowly added dropwise, and a large amount of pale yellow solid was precipitated. Stirring was continued at <NUM>-<NUM> for <NUM> hours, and the reaction solution was filtered to obtain a wet product (<NUM>) as a pale yellow solid. The wet product was dried under reduced pressure at <NUM>-<NUM> to obtain a solid (<NUM>, <NUM>% yield, LCMS purity: <NUM>%).

The X-ray powder diffraction (XRPD) pattern of the solid is shown in <FIG>, and the XRPD diffraction peak data thereof is shown in Table <NUM> below. The TGA/DSC spectrum is shown in <FIG>. The TGA results show that the solid has a weight loss of <NUM>% when heated to <NUM>; the DSC results show that the solid has an endothermic peak at <NUM> (onset temperature). The DVS spectrum is shown in <FIG>, which shows that the vapor sorption of the solid at <NUM>/<NUM>%RH is <NUM>%, indicating that the sample has almost no hygroscopicity. The XRPD comparison pattern before and after the DVS test is shown in <FIG>, which shows that the crystal form of the solid is unchanged before and after the DVS test. The PLM results are shown in <FIG>, which indicate that the solid is irregular crystal particles. This crystal form is defined as crystal form A.

An aliquot of about <NUM> of the solid prepared according to Example <NUM> was weighed and put into a glass vial, and <NUM> of the solvents listed in Table <NUM> below were added respectively. The resulting suspension was placed under magnetic stirring (-<NUM> rpm) at room temperature for <NUM> days, and then centrifuged at <NUM>,<NUM> rpm to obtain a solid. The XRPD testing identified it as the same as the crystal prepared in Example <NUM>, both of which were crystal form A.

An aliquot of about <NUM> of the solid prepared according to Example <NUM> was weighed and put into a glass vial, and <NUM> of the solvents listed in Table <NUM> below were added respectively. The resulting suspension was placed under magnetic stirring (-<NUM> rpm) at <NUM> for <NUM> days, and then centrifuged at <NUM>,<NUM> rpm to obtain a solid. The XRPD testing identified it as the same as the crystal obtained in Example <NUM>, both of which were crystal form A.

An aliquot of about <NUM> of the solid prepared according to Example <NUM> was weighed and put into a glass vial, and <NUM> of the solvents listed in Table <NUM> below were added respectively. The resulting suspension was placed in a temperature cycle (<NUM>→<NUM>, <NUM>/min, <NUM>→<NUM>, <NUM>/min, for three cycles) under stirring, and then centrifuged at <NUM>,<NUM> rpm to obtain a solid. The XRPD testing identified it as the same as the crystal obtained in Example <NUM>, both of which were crystal form A.

An aliquot of about <NUM> of the solid prepared according to Example <NUM> was weighed and put into a glass vial, and <NUM> of the solvents listed in Table <NUM> below were added respectively. After the solution was stirred at <NUM> for <NUM> hours and filtered (PTFE filter membrane with pore size of <NUM>; manufacturer: Titan Chemical; model: syringe filter hydrophobic polytetrafluoroethylene (PTFE) <NUM> <NUM>), the resulting filtrate was placed in a biochemical incubator (manufacturer: Shanghai Yiheng Scientific Instrument Co. ; model: BPC-70F) in which the temperature was lowered from <NUM> to <NUM> at a cooling rate of <NUM>/min. If the solution was still clear, the clear sample was transferred to -<NUM> and left to stand overnight to obtain a solid, which was removed with a spatula. The XRPD testing identified it as the same as the crystal obtained in Example <NUM>, both of which were crystal form A.

An aliquot of about <NUM> of the solid prepared according to Example <NUM> was weighed and put into a glass vial, <NUM> of the positive solvents listed in Table <NUM> below were added respectively, and the solution was filtered. The anti-solvents listed in Table <NUM> below were added dropwise under stirring to the clear solution at room temperature until precipitation of solid. If there was no solid precipitation after adding about <NUM> of the anti-solvent, the dropwise addition was stopped, and the solution was centrifuged at <NUM>,<NUM> rpm to obtain a solid. The XRPD testing identified it as the same as the crystal obtained in Example <NUM>, both of which were crystal form A.

An aliquot of about <NUM> of the solid prepared according to Example <NUM> was weighed and put into a <NUM> glass vial, and about <NUM> of the solvents listed in Table <NUM> below were added in another <NUM> glass vial. The open <NUM> glass vial was placed in the <NUM> glass vial, and then the <NUM> glass vial was sealed. The <NUM> glass vial was left to stand at room temperature until the solid surface became wet, or after being left to stand for <NUM> days, and then the XRPD testing was carried out. The XRPD testing identified it as the same as the crystal obtained in Example <NUM>, both of which were crystal form A.

An aliquot of about <NUM> of the solid prepared according to Example <NUM> was weighed and dissolved in <NUM>-<NUM> of the positive solvents listed in Table <NUM> below, and the solution was filtered. The resulting filtrate was transferred to a <NUM> glass vial, and about <NUM> of the anti-solvents listed in Table <NUM> below were added to another <NUM> glass vial. The open <NUM> glass vial with the filtrate was placed in the <NUM> glass vial, and then the <NUM> glass vial was sealed and left to stand at room temperature. When solid precipitation was observed, the solid was collected, and the XRPD testing was carried out. The XRPD testing identified it as the same as the crystal obtained in Example <NUM>, both of which were crystal form A.

An aliquot of about <NUM> of the solid prepared according to Example <NUM> was weighed and put into a <NUM> glass vial, and <NUM>-<NUM> of the solvents listed in Table <NUM> below were added respectively. After the solution was shaken and filtered (PTFE filter membrane with pore size of <NUM>; manufacturer: Titan Chemical; model: syringe filter hydrophobic polytetrafluoroethylene (PTFE) <NUM> <NUM>), the filtrate was collected. The glass vial with the clear solution was sealed with parafilm, on which several small holes was poked, and the glass vial was left to stand at room temperature for slow evaporation. When there was solid precipitation, the resulting solid was collected, and the XRPD testing was carried out. The XRPD testing identified it as the same as the crystal obtained in Example <NUM>, both of which were crystal form A.

<NUM> of the solid prepared in Example <NUM> was dissolved in <NUM> of ethyl acetate. After filtration, n-heptane was slowly added, and a solid was precipitated when <NUM> was added. The solid was obtained by filtration and dried.

The X-ray powder diffraction (XRPD) pattern of the solid is shown in <FIG>, and the XRPD diffraction peak data are shown in Table <NUM> below. The TGA/DSC spectrum is shown in <FIG>, showing that the solid has a weight loss of <NUM>% when heated to <NUM> and has an endothermic peak at <NUM> (onset temperature). The PLM results show that the sample is needle-like with a length of about <NUM> (see <FIG>). This crystal form is defined as crystal form B.

The compound obtained in Example <NUM> can slowly release the active ingredient nitroxoline after entering the human body, and the latter can simultaneously inhibit the methionine aminopeptidase MetAP2 and the silent mating-type information regulation <NUM> homolog in vascular endothelial cells, exerting a synergistic inhibitory effect on tumor angiogenesis. Meanwhile, nitroxoline also has an inhibitory effect on the proliferation of tumor cells. In addition, the released active ingredient nitroxoline exerts a bacteriostatic effect by inhibiting the methionine aminopeptidase MetAP in bacteria.

The inventor first conducted a research on the water solubility of nitroxoline and the compound obtained in Example <NUM>.

Experimental instruments: <NUM>-well filter plate (MSHVN4510 or MSHVN4550, Millipore); electronic digital vortex (MS3 Digital, IKA); circulating water-type multipurpose vacuum pump (SHB-III, Zhengzhou Greatwall Science, Industry and Trade Co. ); balance (XSLT05, METTLER TOLEDO); ThermoMixer comfort (Eppendorf AG <NUM> Hamburg); liquid chromatography (LC-30AD, Shimadzu); mass spectrometer (API4000, Applied); sampler (CTC Anylytics AG System). Nitroxoline was synthesized by Wisdom Pharmaceutical Co. according to the method disclosed in<NPL>.

Experimental procedures: <NUM>µL of phosphate buffer (pH=<NUM>, <NUM>, <NUM> or <NUM>) was added into a glass vial, <NUM> of compound powder was added. The vial was sealed with a cap and placed on a vortex (VORTEX-GENIE2) to mix well at room temperature for <NUM> hours. The solution was then subjected to vacuum filtration, the filtrate was processed, and the concentration of the compound was determined by LC/MS/MS.

The solubility results of the compound obtained in Example <NUM> are shown in Table <NUM> below.

Conclusion: Compared with nitroxoline (<NUM>-nitro-<NUM>-hydroxyquinoline), the water solubility of the compound obtained in Example <NUM> is several times higher in the buffer solution with pH <NUM>, and its water solubility varies little at different pH, which can be regarded as basically unchanged. This feature is notably important in the development of drug formulations.

It is expected that the compound obtained in Example <NUM> is decomposed into nitroxoline in vivo, thereby exerting an anticancer effect. Liver microsomal enzymes and plasma metabolizing enzymes are important ways of compound metabolism in vivo. Thus, in vitro experiments were carried out to determine the stability of the compound obtained in Example <NUM> in liver microsome and plasma.

Experimental instruments: thermostatic oscillator (SHA-B, Guohua Instrument); centrifuge (5810R, Eppendorf), mass spectrometer (API4000, Applied), liquid chromatography (LC-30AD, Shimadzu); sampler (CTC Analytics AG System, CTC).

Experimental procedures: to <NUM> phosphate buffer was added <NUM>µg/mL alamethicin (Aldrich Reagents), <NUM> magnesium chloride and <NUM>/mL microsomes (XENOTECH) to prepare a reaction solution without coenzymes. To a portion of the reaction solution was added <NUM> reduced nicotinamide adenine dinucleotide phosphate (Aldrich Reagents) and <NUM> uridine diphosphate glucuronic acid (Aldrich Reagents) to prepare a reaction solution with coenzymes. Then, to the two reaction solutions was added the working solution of the compound obtained in Example <NUM>, so that the final concentration of the compound was <NUM>. Immediately after mixing well, <NUM>µL of the solution was collected as the <NUM>-minute sample, and another <NUM>µL was collected after <NUM> minutes of incubation of the remaining sample at <NUM>. The proteins in all collected samples were immediately precipitated, and the supernatants were collected by centrifugation, in which the compound concentrations were determined by LC/MS/MS.

The stability results of the compound obtained in Example <NUM> in microsome are shown in Table <NUM> below.

In this experiment, after a single intravenous or oral administration of nitroxoline and the compound obtained in Example <NUM> to rats, the changes in the concentration of the compound nitroxoline in rat plasma were studied, so as to evaluate the in vivo pharmacokinetic behaviors of nitroxoline and the compound obtained in Example <NUM> in rats.

Tandem quadrupole mass spectrometer (API4000, Applied Biosystems, USA), liquid chromatography (<NUM>, Agilent), autosampler (CTC Analytics HTC PAL), Analyst v1. <NUM> by Applied Biosystems, USA, refrigerated centrifuge (<NUM>-15PK, Sigma), vortex (VX-III, Beijing Targin Technology Co.

Male SD rats (Beijing Vital River Laboratory Animal Technology Co. , laboratory animal production license No.: SCXK (Beijing) <NUM>-<NUM>, laboratory animal certificate No.: <NUM>), <NUM> rats per group, weight <NUM>-<NUM>, <NUM>-<NUM> weeks old, were fasted overnight before drug administration with free access to water, and food was given <NUM> hours after drug administration. The compound to be tested was put into an EP tube, <NUM> dimethyl sulfoxide, <NUM> Solutol® and sterile water for injection (the volume ratio of the three was <NUM>:<NUM>:<NUM>, v:v:v) were added, and the EP tube was sonicated for <NUM> minutes to fully dissolve the compound (formulation concentration of the compound: <NUM> mmol/mL). The intravenous dose was <NUM> mmol/kg, and the oral dose was <NUM> mmol/kg. <NUM> of whole blood was collected from the orbital venous plexus before drug administration (<NUM> hour) and <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> hours after drug administration (the sampling points were adjusted according to the situation) and placed in a centrifuge tube containing EDTA-K2 (Aldrich Reagents) for anticoagulation, which was placed in crushed ice after sample collection. The tube was centrifuged at <NUM> rpm for <NUM> minutes within <NUM> hours of sample collection. All clean plasma were isolated and placed in another clean centrifuge tube, the stabilizing solution was added at a ratio of <NUM>:<NUM> (plasma/stabilizing solution, v/v), and the tube was placed in a -<NUM> refrigerator until testing.

The preparation method of the stabilizing solution: <NUM> of vitamin C (Aldrich Reagents) was dissolved in <NUM> of physiological saline, and then <NUM> of formic acid was added and mixed well.

Standard curve: A series of working solutions for standard curve was prepared, <NUM>µL of which was added to <NUM>µL blank rat plasma. <NUM>µL of internal standard working solution (a solution of <NUM> ng/mL diphenhydramine (Aldrich Reagents) in acetonitrile) was added, and the resulting solution was vortexed for <NUM> minute. After centrifuging at <NUM>, <NUM>,<NUM> rpm for <NUM> minutes, <NUM>µL of the supernatant was collected into a sample tube, and <NUM>µL was injected into the LC/MS system for determination.

Sample to be tested: to <NUM>µL of plasma sample to be tested was added <NUM>µL of diluted working solution, then <NUM>µL of internal standard working solution (a solution of <NUM> ng/mL diphenhydramine in acetonitrile), and the resulting solution was vortexed for <NUM> minute. After centrifuging at <NUM>, <NUM>,<NUM> rpm for <NUM> minutes, <NUM>µL of the supernatant was collected into a sample tube, and <NUM>µL was injected into the LC/MS system for determination. Pharmacokinetic parameters were calculated using WinNonlin V6. <NUM> non-compartmental model.

The testing results are shown in Table <NUM> to Table <NUM> below.

Conclusion: Compared with nitroxoline, the compound obtained in Example <NUM> has significantly improved absorption or half-life in rats. As a result, the drug molecule has a good compliance improvement in reducing the dosage or the frequency of administration.

Nitroxoline is mainly metabolized by Phase II metabolism in the liver with a high metabolism rate, thus the in vivo half-life is short. In this experiment, after a single intravenous or oral administration of nitroxoline and the compound obtained in Example <NUM> to dogs, the changes in the concentration of the compound nitroxoline in dog plasma were studied, so as to evaluate the in vivo pharmacokinetic behaviors of nitroxoline and the compound obtained in Example <NUM>.

Tandem quadrupole mass spectrometer (API5500, Applied Biosystems, USA), liquid chromatography (<NUM>, Agilent), autosampler (CTC Analytics HTC PAL), Analyst v1. <NUM> by Applied Biosystems, USA.

Male beagles (Beijing Marshall Bioresources Co. , laboratory animal production license No.: SCXK (Beijing) <NUM>-<NUM>, laboratory animal certificate No.: <NUM>), <NUM> beagles per group, weight <NUM>-<NUM>, <NUM>-<NUM> months old, were fasted overnight before drug administration with free access to water, and food was given <NUM> hours after drug administration. The compound to be tested was put into an EP tube, dimethyl sulfoxide, Solutol® and sterile water for injection (the volume ratio of the three was <NUM>:<NUM>:<NUM>, v:v:v) were added, and the EP tube was sonicated for <NUM> minutes to fully dissolve the compound (formulation concentration of the compound: <NUM> mmol/mL). The intravenous dose was <NUM> mmol/kg, and the oral dose was <NUM> mmol/kg. <NUM> of whole blood was collected from the jugular vein before drug administration (<NUM> hour) and <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> hours after drug administration (the sampling points were adjusted according to the situation) and placed in a centrifuge tube containing EDTA-K2 (Aldrich Reagents) for anticoagulation, which was placed in crushed ice after sample collection. The tube was centrifuged at <NUM> for <NUM> minutes within <NUM> hours of sample collection. All clean plasma were isolated and placed in another clean centrifuge tube, which was placed in a -<NUM> refrigerator until testing.

A series of solutions for standard curve was prepared. To <NUM>µL of solutions for standard curve and samples was added <NUM>µL of internal standard working solution (a solution of <NUM> ng/mL verapamil (Aldrich Reagents), <NUM> ng/mL glibenclamide (Aldrich Reagents) and <NUM> ng/mL diclofenac (Aldrich Reagents) in acetonitrile) was added, and the resulting solution was vortexed for <NUM> minutes. After centrifuging at <NUM>, <NUM> rpm for <NUM> minutes, <NUM>µL of the supernatant was collected into a sample tube and mixed well with <NUM>µL of water, and <NUM>µL of the mixed solution was injected into the LC/MS system for determination. Pharmacokinetic parameters were calculated using WinNonlin V6. <NUM> non-compartmental model.

The crystal forms obtained in Examples <NUM>-<NUM> were packaged in double-layer PE bags + aluminum foil bags + cardboard barrels, sealed and stored at a temperature of <NUM> and a relative humidity of <NUM>%; a temperature of <NUM> and a relative humidity of <NUM>%; at <NUM>±<NUM> for <NUM> months. The inspection items (appearance, moisture, related substance <NUM>, related substance <NUM>, content) were tested, and the specific test methods were as follows. The results are shown in Table <NUM>. It can be seen from Table <NUM> that the impurity content of crystal form A is relatively low, and the impurity content does not substantially increase under each condition, indicating that crystal form A has good stability under long-term conditions.

<NUM> Related substance <NUM>
<NUM> Chromatographic conditions: high performance liquid chromatography (HPLC).

Column: Waters XBridge C18 <NUM>×<NUM>, <NUM>
Detector: UV or equivalent detector
Wavelength: <NUM>
Column temperature: <NUM>
Flow rate: <NUM>/min
Injection volume: <NUM>µL
Needle Wash: Acetonitrile
Mobile phase gradient:.

About <NUM> of reference substance of impurity <NUM> was weighed and put into a <NUM> volumetric flask. Dichloromethane that was already heated in a water bath at <NUM> was used to completely dissolve the substance and dilute the solution to the mark. The solution was shaken well and labeled as Solution <NUM>.

About <NUM> of reference substance of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate was accurately weighed and put into a <NUM> volumetric flask. An appropriate amount of diluent was added to dissolve the substance, and then <NUM> of Solution <NUM> and <NUM> of Solution <NUM> were accurately added, and the diluent was added to dilute the solution to the mark. The solution was shaken well and labeled as the system suitability solution.

<NUM> of the system suitability solution was accurately measured and put into a <NUM> volumetric flask. The diluent was added to dilute the solution to the mark, and the solution was shaken well. <NUM> of the above solution was accurately measured and put into a <NUM> volumetric flask. The diluent was added to dilute the solution to the mark, and the solution was shaken well.

About <NUM> of the test sample was accurately weighed and put into a <NUM> volumetric flask. The diluent was added to dissolve the substance and dilute the solution to the mark, and the solution was shaken well.

<NUM> Calculation: The blank was subtracted.

<NUM>) Total impurity (%) = Σ single impurity.

<NUM> Related substance <NUM> (D-isobutyrylproline)
<NUM> Chromatographic conditions: high performance liquid chromatography (HPLC) Column: Synergi Hydro RP <NUM>×<NUM>, <NUM>.

Detector: UV or equivalent detector
Wave length: <NUM>
Column temperature: <NUM>
Flow rate: <NUM>/min
Injection volume: <NUM>µL
Needle Wash: Acetonitrile
Mobile phase gradient:.

About <NUM> of reference substance of related substance <NUM> was accurately weighed and put into a <NUM> volumetric flask. The diluent was added to dissolve the substance and dilute the solution to the mark, and the solution was shaken well. <NUM> of the above solution was accurately measured and put into a <NUM> volumetric flask. The diluent was added to dilute the solution to the mark, and the solution was shaken well. Two solutions were prepared in parallel and labeled as RS1/RS2 respectively.

About <NUM> of the test sample was accurately weighed and put into a <NUM> volumetric flask. The diluent was added to dissolve the test sample and dilute the solution to the mark, and the solution was shaken well. Two solutions were prepared in parallel and labeled as S1/S2 respectively.

<NUM> Calculation: Only the peak of related substance <NUM> was integrated <MAT> <MAT>.

ARS2-Average peak area of reference solution <NUM>;
Average peak area of <NUM> injections of reference solution <NUM>;
As-Peak area of related substance <NUM> in the test sample solution;
MRS1-Weighed sample amount of related substance <NUM> in reference solution <NUM>, mg;
Ms-Weighed sample amount of the test sample in the test sample solution, mg;
MRS2-Weighed sample amount of related substance <NUM> in reference solution <NUM>, mg;.

<NUM> Content
<NUM> Chromatographic conditions: high performance liquid chromatography (HPLC).

About <NUM> of the test sample was accurately weighed and put into a <NUM> volumetric flask. The diluent was added to dissolve the test sample and dilute the solution to the mark, and the solution was shaken well. Two solutions were prepared in parallel. <NUM> Calculation <MAT> <MAT>.

ARS2-Average peak area of reference solution <NUM>;
ARS<NUM>-Average peak area of <NUM> injections of reference solution <NUM>;
As-Peak area of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate in the test sample solution;
MRS1-Weighed sample amount of reference substance of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate in reference solution <NUM>, mg;
Ms-Weighed sample amount of the test sample in the test sample solution, mg;
MRS2-Weighed sample amount of reference substance of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate in reference solution <NUM>, mg;
P-Content of reference substance of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, %.

Claim 1:
Crystal form A of ((<NUM>-nitroquinolin-<NUM>-yl)oxy)methyl-isobutyryl-L-prolinate, characterized in that, the X-ray powder diffraction pattern thereof, which is obtained by using Cu-Kα irradiation and expressed in 2θ angle, comprises characteristic peaks at <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>°, <NUM>±<NUM>° and <NUM>±<NUM>°.