WO2020233713A1 - 杂环化合物及其盐的应用 - Google Patents

杂环化合物及其盐的应用 Download PDF

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WO2020233713A1
WO2020233713A1 PCT/CN2020/091842 CN2020091842W WO2020233713A1 WO 2020233713 A1 WO2020233713 A1 WO 2020233713A1 CN 2020091842 W CN2020091842 W CN 2020091842W WO 2020233713 A1 WO2020233713 A1 WO 2020233713A1
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formula
hydrate
compound represented
ray powder
powder diffraction
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PCT/CN2020/091842
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English (en)
French (fr)
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刘喜宝
张红芬
李艳玲
马玉秀
吴希美
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石药集团中奇制药技术（石家庄）有限公司
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Priority to CN202080019479.7A priority Critical patent/CN114502164B/zh
Publication of WO2020233713A1 publication Critical patent/WO2020233713A1/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
    • A61K31/4353—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 ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—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 ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system

Definitions

  • the invention belongs to the technical field of medicine, and specifically relates to the application of heterocyclic compounds and their salts.
  • ALI/ARDS Acute lung injury/acute respiratory distress syndrome refers to acute and progressive hypoxic respiratory failure caused by various pathogenic factors inside and outside the lungs other than cardiogenic. Since Ashbaugh et al. reported adult respiratory distress syndrome (ARDS) in 1967, it has attracted great interest from domestic and foreign scholars and has done a lot of clinical and experimental research. my country has successively held ALI/ARDS seminars, which mainly discussed the definition, pathogenesis, diagnostic criteria and treatment of ALI/ARDS. The understanding of ALI/ARDS has been significantly improved. It is manifested in the gradual standardization of the naming and definition of ALI/ARDS, and a deeper understanding of its pathogenesis.
  • ALI/ARDS The diagnostic criteria of ALI/ARDS that are convenient for clinical use have been proposed, and some More mature treatment experience and measures have led to a downward trend in the incidence and mortality of ALI/ARDS.
  • the present invention provides the following technical solutions:
  • the acute lung injury is selected from cigarette smoke (CS) or lipopolysaccharide (LPS) induced acute lung injury.
  • CS cigarette smoke
  • LPS lipopolysaccharide
  • the pharmaceutically acceptable salt of the heterocyclic compound represented by formula A is selected from alkali metal salts.
  • the hydrate is selected from the hydrate of the heterocyclic compound represented by formula A, or the hydrate of the alkali metal salt of the heterocyclic compound represented by formula A.
  • the heterocyclic compound represented by formula A or a pharmaceutically acceptable salt thereof is in a crystalline form, such as the crystal form of the heterocyclic compound represented by formula A, the crystal form of its hydrate, and the pharmaceutically acceptable salt thereof.
  • the crystalline form of the heterocyclic compound represented by formula A or its hydrate is Cu-K ⁇ radiation, and the X-ray powder diffraction expressed in 2 ⁇ angles is 11.1 ⁇ 0.2°, 11.4 ⁇ 0.2°, 17.9 ⁇ 0.2 There are characteristic peaks at °, 22.6 ⁇ 0.2°, and 24.4 ⁇ 0.2°.
  • the crystal form uses Cu-K ⁇ radiation, and the X-ray powder diffraction expressed in 2 ⁇ angles is at 8.6 ⁇ 0.2°, 11.1 ⁇ 0.2°, 11.4 ⁇ 0.2°, 14.1 ⁇ 0.2°, 16.1 ⁇ 0.2°, 17.9 ⁇ There are characteristic peaks at 0.2°, 20.9 ⁇ 0.2°, 22.6 ⁇ 0.2°, 24.4 ⁇ 0.2°, and 25.8 ⁇ 0.2°.
  • the crystal form uses Cu-K ⁇ radiation, and the X-ray powder diffraction expressed in 2 ⁇ angles is at 8.6 ⁇ 0.2°, 11.1 ⁇ 0.2°, 11.4 ⁇ 0.2°, 14.1 ⁇ 0.2°, 15.6 ⁇ 0.2°, 16.1 There are characteristic peaks at ⁇ 0.2°, 17.9 ⁇ 0.2°, 18.3 ⁇ 0.2°, 20.9 ⁇ 0.2°, 22.6 ⁇ 0.2°, 24.4 ⁇ 0.2°, 25.8 ⁇ 0.2°, 26.5 ⁇ 0.2°, 28.9 ⁇ 0.2°.
  • the crystal form has an X-ray powder diffraction spectrum substantially as shown in FIG. 1.
  • the crystal form has a DSC-TGA spectrum substantially as shown in FIG. 2.
  • the crystal form of the heterocyclic compound hydrate represented by formula A is preferably a monohydrate. More preferably, the mass fraction of water in the hydrate is 4.2-5.2%, more preferably 4.5-5.0%.
  • the monohydrate is as follows:
  • the crystal form is a single crystal with the following single crystal parameters:
  • the present invention also provides a method for preparing the crystal form of the heterocyclic compound represented by formula A or its hydrate, which comprises the following steps:
  • the compound represented by formula A is placed in a mixed solvent composed of a ketone solvent and water, heated to dissolve and then cooled, stirred and crystallized to obtain the crystal form of the heterocyclic compound represented by formula A or its hydrate.
  • the preparation of the heterocyclic compound represented by formula A in the present invention can be prepared by referring to the methods described in Examples 3 and 4 in the patent document CN101896178B.
  • the racemate of the heterocyclic compound represented by formula A is eluted on a Chiralcel OJ-RH column (Chiralcel Technologies) with a methanol solution containing 0.05% trifluoroacetic acid to separate the heterocyclic compound represented by formula A.
  • the ketone solvent is selected from acetone or methyl ethyl ketone.
  • the volume ratio of the ketone solvent to water is (1-3):1, for example, 1:1.
  • the heating temperature is 30 to 80°C, preferably 40 to 60°C.
  • the pharmaceutically acceptable alkali metal salt of the heterocyclic compound represented by formula A is preferably a sodium salt, a lithium salt or a potassium salt; the hydrate of the alkali metal salt is selected from sodium salt, lithium salt Or potassium salt hydrate.
  • the crystal form of the alkali metal salt of the compound of formula A is in the form of a hydrate.
  • the crystalline hydrate of the alkali metal salt of the compound of formula A is preferably a monohydrate.
  • the crystalline hydrate of the alkali metal salt of the compound of formula A is selected from the compounds represented by the following formulas A-N, A-L or A-K:
  • the compound represented by the formula AN is a crystalline hydrate, which uses Cu-K ⁇ radiation, and X-ray powder diffraction expressed in 2 ⁇ angles is at 16.4 ⁇ 0.2°, 18.9 ⁇ 0.2°, 21.7 ⁇ 0.2°, 24.0 ⁇ 0.2° There are characteristic peaks.
  • the X-ray powder diffraction expressed in 2 ⁇ angles is at 11.8 ⁇ 0.2°, 16.4 ⁇ 0.2°, 16.7 ⁇ 0.2°, 16.9 ⁇ 0.2°, 17.1 ⁇ 0.2°, 17.8 ⁇ 0.2°, There are characteristic peaks at 18.6 ⁇ 0.2°, 18.9 ⁇ 0.2°, 21.7 ⁇ 0.2°, 23.7 ⁇ 0.2°, and 24.0 ⁇ 0.2°.
  • the crystalline hydrate uses Cu-K ⁇ radiation, and X-ray powder diffraction expressed in 2 ⁇ angles is at 5.6 ⁇ 0.2°, 11.8 ⁇ 0.2°, 14.0 ⁇ 0.2°, 15.8 ⁇ 0.2°, 16.4 ⁇ 0.2°, 16.7 ⁇ 0.2°, 16.9 ⁇ 0.2°, 17.1 ⁇ 0.2°, 17.8 ⁇ 0.2°, 18.6 ⁇ 0.2°, 18.9 ⁇ 0.2°, 20.3 ⁇ 0.2°, 21.7 ⁇ 0.2°, 23.7 ⁇ 0.2°, 24.0 ⁇ 0.2°, There are characteristic peaks at 26.1 ⁇ 0.2°, 28.1 ⁇ 0.2°, 28.5 ⁇ 0.2°, and 29.8 ⁇ 0.2°.
  • the crystalline hydrate has an X-ray powder diffraction spectrum (XRPD) substantially as shown in FIG. 4.
  • XRPD X-ray powder diffraction spectrum
  • the mass fraction of water in the crystalline hydrate of the compound represented by formula A-N is 3.4-4.4%, more preferably 3.6-4.2%.
  • the crystalline hydrate of the compound represented by formula A-N has a DSC-TGA spectrum substantially as shown in FIG. 5.
  • the potassium salt compound represented by the formula AK is a crystalline hydrate, which uses Cu-K ⁇ radiation, and the X-ray powder diffraction expressed at 2 ⁇ angles has the characteristics at 15.6 ⁇ 0.2°, 21.4 ⁇ 0.2°, 24.0 ⁇ 0.2° Characteristic peaks.
  • the potassium salt compound represented by formula AK is a crystalline hydrate, which uses Cu-K ⁇ radiation, and X-ray powder diffraction expressed in 2 ⁇ angles is at 11.7 ⁇ 0.2°, 15.6 ⁇ 0.2°, 16.6 ⁇ 0.2°, 17.9 ⁇ There are characteristic peaks at 0.2°, 18.5 ⁇ 0.2°, 21.4 ⁇ 0.2°, 24.0 ⁇ 0.2°, and 28.2 ⁇ 0.2°.
  • the potassium salt compound represented by formula AK is a crystalline hydrate, which uses Cu-K ⁇ radiation, and X-ray powder diffraction expressed in 2 ⁇ angles is at 11.7 ⁇ 0.2°, 15.6 ⁇ 0.2°, 15.9 ⁇ 0.2°, 16.6 ⁇ There are characteristic peaks at 0.2°, 17.4 ⁇ 0.2°, 17.9 ⁇ 0.2°, 18.5 ⁇ 0.2°, 21.4 ⁇ 0.2°, 23.5 ⁇ 0.2°, 24.0 ⁇ 0.2°, 27.7 ⁇ 0.2°, 28.2 ⁇ 0.2°.
  • the crystalline hydrate of the compound represented by the formula AK uses Cu-K ⁇ radiation, and the X-ray powder diffraction expressed in 2 ⁇ angles is at 11.7 ⁇ 0.2°, 14.0 ⁇ 0.2°, 15.6 ⁇ 0.2°, 15.9 ⁇ 0.2° , 16.6 ⁇ 0.2°, 17.4 ⁇ 0.2°, 17.9 ⁇ 0.2°, 18.5 ⁇ 0.2°, 20.1 ⁇ 0.2°, 21.4 ⁇ 0.2°, 23.5 ⁇ 0.2°, 24.0 ⁇ 0.2°, 27.5 ⁇ 0.2°, 27.7 ⁇ 0.2° , 28.2 ⁇ 0.2°, 28.6 ⁇ 0.2°, 29.3 ⁇ 0.2°, 29.6 ⁇ 0.2° have characteristic peaks.
  • the crystalline hydrate of the compound represented by formula A-K has an X-ray powder diffraction spectrum substantially as shown in FIG. 6.
  • the mass fraction of water in the crystal hydrate of the compound represented by formula A-K is 3.3-4.3%, more preferably 3.5-4.1%.
  • the crystalline hydrate of the compound represented by formula A-K has a DSC-TGA spectrum substantially as shown in FIG. 7.
  • the lithium salt compound represented by the formula AL is a crystalline hydrate, using Cu-K ⁇ radiation, the X-ray powder diffraction expressed at 2 ⁇ angles is 16.7 ⁇ 0.2°, 18.8 ⁇ 0.2°, 21.9 ⁇ 0.2°, 23.9 ⁇ There is a characteristic peak at 0.2°.
  • the crystalline hydrate of the compound represented by the formula AL uses Cu-K ⁇ radiation, and the X-ray powder diffraction represented by the angle of 2 ⁇ is at 5.6 ⁇ 0.2°, 8.8 ⁇ 0.2°, 11.8 ⁇ 0.2°, 14.0 ⁇ 0.2° , 16.3 ⁇ 0.2°, 16.7 ⁇ 0.2°, 16.9 ⁇ 0.2°, 17.0 ⁇ 0.2°, 17.7 ⁇ 0.2°, 18.5 ⁇ 0.2°, 18.8 ⁇ 0.2°, 21.9 ⁇ 0.2°, 23.9 ⁇ 0.2°, 28.2 ⁇ 0.2° There are characteristic peaks.
  • the crystalline hydrate of the compound represented by formula A-L has an X-ray powder diffraction spectrum substantially as shown in FIG. 8.
  • the mass fraction of water in the crystalline hydrate of the compound represented by formula A-L is 3.6-4.6%, more preferably 3.9-4.5%.
  • the crystalline hydrate of the compound represented by formula A-L has a DSC-TGA spectrum substantially as shown in FIG. 9.
  • the present invention also provides a method for preparing a pharmaceutically acceptable salt of the heterocyclic compound represented by formula A or a hydrate of the salt, which comprises the following steps:
  • the compound represented by formula A is dissolved in a ketone solvent, an aqueous solution of alkali metal hydroxide is added to the ketone solvent for reaction, and then filtered and dried to obtain it; wherein the alkali metal hydroxide is preferably sodium hydroxide or hydroxide Lithium or potassium hydroxide.
  • the preparation of the heterocyclic compound represented by formula A in the present invention can be prepared by referring to the methods described in Examples 3 and 4 in the patent document CN101896178B.
  • the racemate of the heterocyclic compound represented by formula A is eluted on a Chiralcel OJ-RH column (Chiralcel Technologies) with a methanol solution containing 0.05% trifluoroacetic acid to separate the heterocyclic compound represented by formula A.
  • heterocyclic compounds, pharmaceutically acceptable salts, hydrates and their preparation and performance related data of the heterocyclic compound represented by formula A of the present invention can refer to the application number 201910024238.0, and the application date is the invention patent application on January 10, 2019, which is incorporated in its entirety This article serves as a reference.
  • the present invention also provides a pharmaceutical composition for the treatment of acute lung injury or acute respiratory distress syndrome, which comprises a therapeutically effective amount of the heterocyclic compound represented by formula A of the present invention, its pharmaceutically acceptable salt or Its hydrate.
  • the acute lung injury is selected from acute lung injury induced by cigarette smoke CS or lipopolysaccharide LPS.
  • the pharmaceutically acceptable salt of the heterocyclic compound represented by formula A is selected from alkali metal salts.
  • the hydrate is selected from the hydrate of the heterocyclic compound represented by formula A, or the hydrate of the alkali metal salt of the heterocyclic compound represented by formula A.
  • the heterocyclic compound represented by formula A or a pharmaceutically acceptable salt thereof is in a crystalline form, for example selected from the crystal form of the heterocyclic compound represented by formula A, the crystal form of its hydrate, At least one of the pharmaceutically acceptable alkali metal salt crystal form or the alkali metal salt hydrate crystal form.
  • the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.
  • the adjuvant may be an inert, non-toxic excipient, carrier or diluent, for example, the adjuvant is selected from one, two or more of the following: disintegrant, glidant, lubricant, filler Agents, adhesives, coloring agents, effervescent agents, flavoring agents, preservatives, coating materials, etc.
  • the present invention also provides a method for the treatment of acute lung injury or acute respiratory distress syndrome, the method comprising administering an effective therapeutic amount of the heterocyclic compound represented by formula A, its pharmaceutically acceptable Salt or its hydrate.
  • the present invention also provides a method for treating acute lung injury or acute respiratory distress syndrome, the method comprising administering an effective therapeutic amount of the above-mentioned pharmaceutical composition to a mammal patient in need of such treatment.
  • the acute lung injury is selected from acute lung injury induced by CS or LPS.
  • the pharmaceutically acceptable salt of the heterocyclic compound represented by formula A is selected from alkali metal salts.
  • the hydrate is selected from the hydrate of the heterocyclic compound represented by formula A, or the hydrate of the alkali metal salt of the heterocyclic compound represented by formula A.
  • the heterocyclic compound represented by formula A or a pharmaceutically acceptable salt thereof is in a crystalline form, for example selected from the crystal form of the heterocyclic compound represented by formula A, the crystal form of its hydrate, At least one of the pharmaceutically acceptable alkali metal salt crystal form or the alkali metal salt hydrate crystal form.
  • the present invention provides the use of a heterocyclic compound represented by formula A, a hydrate, a pharmaceutically acceptable alkali metal salt thereof, or a hydrate of the alkali metal salt in the preparation of a medicine for treating acute lung injury or acute respiratory distress syndrome.
  • Figure 1 is the XRPD spectrum of the crystal form obtained in Preparation Example 2.
  • Figure 2 is a DSC-TGA spectrum of the crystal form obtained in Preparation Example 2.
  • Figure 5 is a DSC-TGA spectrum of the sodium salt hydrate obtained in Preparation Example 3.
  • Figure 7 is a DSC-TGA spectrum of the potassium salt hydrate obtained in Preparation Example 4.
  • Figure 8 is an XRPD spectrum of the lithium salt hydrate obtained in Preparation Example 5.
  • Figure 10 shows the effect of CT-NA on the number of inflammatory cells in BALF, partial pressure of oxygen (PO 2 ), lung weight coefficient and albumin content in BALF.
  • mice were given CT-NA (10 and 30 mg/kg), normal saline, and Dex (1 mg/kg) by gavage 1 hour before exposure to CS for seven consecutive days. BALF was collected 24 hours after the last CS exposure.
  • Figure 11 shows the effect of CT-NA on the pro-inflammatory cytokines (TNF- ⁇ , IL-1 ⁇ , IL-6), chemokines (KC) and anti-inflammatory cytokines (IL-10) in BALF of CS-induced ALI mice Express the influence.
  • the collected BALF was analyzed by ELISA kit to analyze the expression levels of TNF- ⁇ (A), IL-1 ⁇ (B), IL-6(C), KC(D) and IL-10(E).
  • ELISA kit to analyze the expression levels of TNF- ⁇ (A), IL-1 ⁇ (B), IL-6(C), KC(D) and IL-10(E).
  • Figure 12 shows the effect of CT-NA on the histopathological changes in lung tissue of ALI mice induced by CS.
  • the paraffin-embedded lung sections of each experimental group were stained with hematoxylin-eosin for histopathological evaluation.
  • A Representative image of lung tissue stained with H&E to demonstrate the infiltration of macrophages, neutrophils and inflammatory cells.
  • Figure 13 shows the effect of CT-NA on lung MPO activity and PGD 2 induced migration of neutrophils in vitro, evaluating CSE-induced PGD 2 secretion of primary macrophages.
  • FIG. 14 CT-NA effects CSE (4%)- and PGD 2 -induced pro-inflammatory cytokines (TNF- ⁇ , IL-1 ⁇ , IL-6), chemokines (KC) and RAW 264.7 macrophages Influence of the protein level of the produced anti-inflammatory cytokine (IL-10).
  • IL-1 ⁇ A and F was measured using an ELISA kit according to the instructions.
  • TNF- ⁇ B and G
  • IL-6 C and H
  • KC D and I
  • extracellular secreted IL-10 E and J protein levels.
  • *p ⁇ 0.05 and **p ⁇ 0.01 compared with PGD 2 All experiments were repeated three times in triplicate wells under each condition. Values are expressed as mean ⁇ SEM.
  • Figure 15 is CT-NA on CSE (4%)-and PGD 2 -induced pro-inflammatory cytokines (TNF- ⁇ , IL-1 ⁇ , IL-6), chemokine (KC) and RAW 264.7 macrophages The effect of the mRNA expression of the produced anti-inflammatory cytokine (IL-10).
  • RNAs isolated from RAW 264.7 macrophages were treated with CT-NA for 1 hour, and then treated with CSE/PGD 2 for 24 hours.
  • the IL-1 ⁇ (A and F) and TNF- ⁇ (B and G) were analyzed by RT-PCR.
  • IL-6 C and H
  • KC D and I
  • IL-10 E and J
  • Figure 16 shows the process of preparing LPS-induced ALI mouse model.
  • CT-NA (10 and 30 mg/kg) or Dex (positive control; 1 mg/kg) was given by gavage 1 hour before and 12 hours after the intratracheal instillation of LPS. 24 hours after LPS induction, the mice were sacrificed to prepare BALF and lung tissue samples.
  • Figure 17 shows the effects of CT-NA on the count and classification of BALF inflammatory cells, oxygen saturation (SO 2 ) and lung weight coefficient.
  • CT-NA 10 and 30 mg/kg or Dex (1 mg/kg) was administered intratracheally 1 hour before and 12 hours after LPS instillation.
  • Dex dexamethasone
  • Figure 18 shows the effect of CT-NA on the production of pro-inflammatory cytokines (TNF- ⁇ , IL-1 ⁇ , IL-6) and chemokines (KC) in BALF of LPS-induced ALI mice.
  • Figure 19 shows the effect of CT-NA on the pathological changes of lung tissue in ALI mice induced by LPS.
  • the paraffin-embedded lung sections of each experimental group were stained with H&E for histopathological analysis.
  • A Representative image of lung tissue stained with H&E showing edema, infiltration of neutrophils and inflammatory cells.
  • B Two pathologists with expertise in lungs were blindly selected to quantitatively analyze the pathology of lung injury.
  • Figure 20 shows the effect of CT-NA on LPS-induced pulmonary vascular permeability.
  • LPS-induced LPS-induced LPS-induced 24 hours (A) Use an albumin assay kit to measure albumin in BALF.
  • Evans blue dye (50 mg/kg) was injected into the tail vein of all mice, and euthanized after 1 hour. Pulmonary vascular permeability is determined by the accumulation of Evans blue dye in lung tissue.
  • FIG 21 is a CT-NA lung MPO activity, vitro PGD 2 induced neutrophil migration, and assessment of LPS-induced primary macrophages PGD 2 secretion.
  • A MPO activity of lung homogenate was measured using MPO kit.
  • B and C using the Boyden chamber assay kit (pore size 3 m), with or without assess the CT-NA where PGD 2 induced neutrophil migration.
  • Figure 22 shows the effect of CT-NA on pro-inflammatory cytokines (TNF- ⁇ , IL-1 ⁇ , IL-6) and chemokines (KC) secreted from LPS- or PGD 2 stimulated RAW264.7 macrophages.
  • RAW264.7 macrophages were pretreated with CT-NA for 1 hour, and further treated with CT-NA and LPS/PGD 2 for 24 hours, and then the medium was collected to measure IL-1 ⁇ (A and E) using corresponding ELISA kits, TNF- ⁇ (B and F), IL-6 (C and G) and KC (D and H) secretion levels.
  • Figure 23 shows the effect of CT-NA on the expression of chemokine (KC) mRNA secreted by RAW264.7 macrophages stimulated by pro-inflammatory cytokines (TNF- ⁇ , IL-1 ⁇ , IL-6) and LPS- or PGD 2 .
  • RAW264.7 macrophages were pretreated with CT-NA for 1 hour, and further treated with CT-NA and LPS/PGD 2 for 24 hours, then RNA was extracted, and RT-PCR was used to analyze IL-1 ⁇ (A and E), TNF- ⁇ (B and F), IL-6 (C and G) and KC (D and H) expression.
  • Control group ##P ⁇ 0.01; model group *P ⁇ 0.05, **P ⁇ 0.01.
  • Figure 24 shows the effect of CT-NA on the expression of chemokine (KC) mRNA secreted by primary macrophages stimulated by pro-inflammatory cytokines (TNF- ⁇ , IL-1 ⁇ , IL-6), LPS and PGD 2 .
  • the primary macrophages were pretreated with CT-NA for 1 hour, and further treated with CT-NA and LPS/PGD 2 for 24 hours, then total RNA was extracted, and RT-PCR was used to analyze IL-1 ⁇ (A and E), TNF -Expression of ⁇ (B and F), IL-6 (C and G) and KC (D and H).
  • Figure 25 shows the effect of CT-NA on the activation response of NF- ⁇ B in RAW 264.7 macrophages or LPS stimulation signaling pathway in the lung.
  • A RAW264.7 macrophages were pretreated with CT-NA (0.5, 1, 10 and 100 ⁇ M) for 1 hour 1 hour before LPS (100ng/ml) treatment.
  • B Homogenize the preserved lung tissue in RIPA buffer to extract total protein. Western blot analysis of proteins was performed with the designated antibodies. Use ⁇ -actin as an internal control. All experiments were repeated at least three times.
  • DSC-TGA test item instrument name and model Synchronous thermal analyzer (STA449F3) 20°C to 350°C.
  • the racemate of the heterocyclic compound of formula A (0.500g) was eluted with methanol containing 0.05% TFA on a Chiralcel OJ-RH column (Chiralcel Technologies), and collected The eluate was concentrated to dryness to obtain about 0.2 g of the product, which was amorphous. Acetone (2.5mL) and water (2.5mL) were added to the concentrate, heated at 40-50°C to dissolve and then dropped to 0 ⁇ 10°C, stirred and crystallized for 2 ⁇ 3h, filtered to obtain the heterocyclic compound crystalline form ( 0.156g), the yield was 31.2%.
  • the XRPD detection results of the crystal form are shown in Figure 1, and the DSC-TGA detection results are shown in Figure 2. From the DSC chart in Figure 2, it can be seen that the crystal form has endothermic peaks at 86.4°C and 130.4°C, respectively, and TGA thermal weight loss The graph shows that the weight loss is 4.29%.
  • the DSC-TGA pattern of the crystal form shows that the crystal form is a monohydrate.
  • Fig. 3 is a three-dimensional structure diagram and a unit cell diagram of the obtained crystal form.
  • the DSC chart in Figure 5 shows that there are endothermic peaks at 130.2°C and 176.6°C, respectively, and the TGA thermal weight loss chart shows a weight loss of 3.99%.
  • the DSC-TGA pattern of the crystal form shows that the crystal form is a monohydrate.
  • the hydrate and the compound of formula A obtained as above are tested for solubility in solutions of different pH.
  • the test method is:
  • pH1.0 medium take 9.0mL of hydrochloric acid, dilute to 1000mL with water, shake well, and get it.
  • pH4.5 medium Take 6.80g of potassium dihydrogen phosphate (KH 2 PO 4 ) and add an appropriate amount of water to dissolve and dilute to 1000mL, adjust the pH to 4.5 with phosphoric acid or sodium hydroxide, shake well, and get it.
  • KH 2 PO 4 potassium dihydrogen phosphate
  • pH6.8 medium take 55.38g of disodium hydrogen phosphate (Na 2 HPO 4 ⁇ 12H 2 O) and 4.77g of citric acid (C 6 H 8 O 7 ⁇ H 2 O), add an appropriate amount of water to dissolve and dilute to 1000 mL, Adjust the pH to 6.8 with phosphoric acid or sodium hydroxide, shake well, and get ready.
  • Test method Take a certain amount of test sample, add the corresponding pH medium gradually, and keep shaking until it reaches the saturation state, record the weighing amount of the test sample and the amount of solvent, and calculate the concentration when the sample is dissolved.
  • the test results are shown in Table 1:
  • sodium salt hydrate has the best water solubility at different pH
  • lithium salt hydrate is basically equivalent to sodium salt hydrate
  • potassium salt hydrate is combined with sodium salt hydrate and lithium salt at pH 4.5.
  • the compounds are comparable, but other pH and purified water are worse than sodium salt hydrate and lithium salt hydrate, and the free acid (compound A) has poor water solubility at different pH.
  • the solubility of sodium salt and lithium salt hydrate in particular is significantly better than that of compound A.
  • the monohydrate crystal form of Compound A of Preparation Example 2 has good solubility in different pH.
  • Step 1 Raw material pretreatment
  • Step 2 Mixing raw and auxiliary materials
  • Step 3 Capsule filling
  • the capsule filling plate to fill the capsules, lock the button, and control the filling volume difference: ⁇ 5%.
  • the lock should be in place and there should be no fork or concave top.
  • Dissolution medium aqueous solution
  • the three salt hydrates obtained in the present invention all show a good dissolution rate, and the sodium salt hydrate has the best dissolution rate.
  • Test process Take an appropriate amount of each test product and place it on a clean watch glass, place it in an open place, and place it under the conditions of light 4500lx+500lx, high temperature 60°C, high humidity 92.5%RH for 5 days and 10 days respectively, and determine the properties and related Substances, and compare with the results of 0 days to examine the stability.
  • the inspection methods for related substances are as follows:
  • test product solution Take about 10 mg of each test product, put it in a 10ml measuring flask, add 50% acetonitrile to dissolve and dilute to the mark, shake well, filter, and use as the test product solution. A precise amount of 10 ⁇ l of the test solution was taken, and the sample was injected according to the above chromatographic method, and the maximum single impurity and total impurity were calculated according to the area normalization method.
  • the stability test results of the crystal form of the compound of formula A monohydrate of Preparation Example 2 show that the crystal form of the compound of formula A has good stability at high temperature and high humidity.
  • the maximum single impurity does not exceed 0.1%, and the total impurity does not exceed 0.4%.
  • the inventors found that the stability of the crystal form of the compound of formula A under light conditions is worse than the stability under high temperature and high humidity, but it still has relatively good stability.
  • the test method is:
  • Dryer humidity 80% ⁇ 2%RH.
  • the sodium salt hydrate CT-NA of the compound of formula A prepared in Preparation Example 3 was used for the activity test.
  • the test method is as follows.
  • mice Female Balb/c mice (22-28 g; 8 weeks) were purchased from Shanghai SIPPR-BK Experimental Animal Co., Ltd. Mice are in an isolated ventilated cage (4-5 mice/cage), free to eat and drink in an environment of 40-60% humidity, 24 ⁇ 2°C, and 12 hours/12 hours dark-light alternating environment.
  • SPF Specific pathogen-free
  • mice were randomly divided into 5 groups (12 in each group), which were the control group (the mice were exposed to fresh air), the saline group (the mice were exposed to cigarette smoke), and dexamethasone (Dex) Group (1 mg/kg) (mice exposed to cigarette smoke), CT-NA 10 mg/kg group and CT-NA 30 mg/kg group (mice exposed to cigarette smoke).
  • the grouping situation mice were given saline, Dex and CT-NA by gavage. Afterwards, the mice were exposed to fresh air or cigarette smoke.
  • Cigarette smoke consists of 3R4F research-grade cigarettes (containing about 600mg TPM/m 3 and 29.9mg nicotine/m 3 ) in a square plastic box (65 ⁇ 50 ⁇ 45cm) according to 10 cigarettes per day. After each cigarette is burned, the next cigarette is lit. The frequency of burning was repeated continuously for 7 days.
  • the moor VMS-OXY TM measuring instrument was used to measure the partial pressure of oxygen (PO 2 ) of all mice for measuring oxygen/deoxyhemoglobin in the wavelength range of 500 to 650 nm The concentration and oxygen saturation (percentage) in the microcirculation. After that, all mice were euthanized to collect bronchoalveolar lavage fluid (BALF) for measuring the total number of inflammatory cells, cytokine levels and albumin concentration. And collect lung tissue to determine lung weight coefficient, tissue detection and MPO activity.
  • BALF bronchoalveolar lavage fluid
  • the trachea was surgically exposed, and then the right lung was lavaged three times with 0.4 mL/time of sterile normal saline containing 1% FBS and 5000 IU/L heparin, and passed through the tracheal tube to collect BALF. After measuring the total number of cells in BALF with a hemocytometer, the remaining BALF was centrifuged at 1000 ⁇ g at 4°C for 10 minutes. The supernatant was aliquoted and stored at -80°C for the next step to measure the cytokine or albumin concentration. Spread the obtained cell pellet on a glass slide. After that, according to the morphological standards of neutrophils, macrophages and lymphocytes, the smear was stained with Wright-Giemsa under an optical microscope to count 200 cells.
  • the lung weight ratio is the individual lung weight of each mouse after sucking the blood tissue from the lung surface, divided by the total weight.
  • the albumin concentration in the BALF supernatant was tested at 628nm using an albumin determination kit and a spectrophotometer.
  • the albumin concentration ratio measured by BALF not only represents the albumin level, but also represents the permeability of the pulmonary microvascular.
  • IL-1 ⁇ pro-inflammatory cytokines
  • KC chemokines
  • IL-10 anti-inflammatory cytokines
  • the left lower lobe of each mouse was stored in 10% neutral formalin for histopathological examination.
  • the preserved lower lobe of the left lung was taken out and embedded in paraffin, then sectioned (4 ⁇ m) to expose the largest longitudinal view of the bronchi in the main lung.
  • Hematoxylin and eosin (H&E) staining was performed using standard methods.
  • Each lung section is scored according to at least three different fields of view. Take the average of the scores of 12 mice.
  • mice were given 20ml/kg glycogen (1.5%) by gavage. After 4 hours, the mice were euthanized, and neutrophils were isolated from the peritoneal lavage. The effect of CT-NA on the migration of neutrophils was detected by Boyden chamber detection kit (3 ⁇ m pore size). PGD 2 was used as a chemoattractant because the activated PGD 2 /CRTH2 receptor promotes the migration of neutrophils . Initially, the isolated neutrophils (4 ⁇ 10 5 ) were diluted in 100 ⁇ L HBSS and allowed to migrate towards PGD 2 (0.1, 1 and 10 ⁇ M) for 4 hours to find the appropriate PGD 2 concentration.
  • the isolated neutrophils (4 ⁇ 10 5 ) were pretreated with CT-NA (1 and 10 ⁇ M), and their migration to PGD 2 (1 ⁇ M) was evaluated by counting the migrated neutrophils.
  • CT-NA CT-NA
  • PGD 2 1 ⁇ M
  • a vacuum pump is used to transport the cigarette smoke produced by 3R4F research grade cigarettes through 50ml PBS. Five cigarettes are used to make smoke passing 50ml PBS, and each cigarette is lit for 5 minutes. In the absence of cigarettes, a similar method was used to prepare the control solution. After the smoke extraction is complete, store the CSE at -80°C.
  • thioglycolate (4%) was injected into the abdominal cavity of mice at a dose of 20 ml/kg body weight for three consecutive days. On day 5 (48 hours after the last thioglycolate injection), the mice were euthanized to isolate primary macrophages from the peritoneal lavage.
  • the isolated primary macrophages (4 ⁇ 10 5 /well) were added to a 12-well plate and cultured at 37°C. After that, the medium of the 12-well plate was replaced with serum-free RPMI-1640 medium and incubated for 10-12 hours, and then exposed to different concentrations of CSE (2%, 4%, and 8%) for 24 hours. After the treatment is completed, the supernatant of the primary macrophages is collected, and the protein level of secreted extracellular PGD 2 is measured using an ELISA kit according to the method of the instructions.
  • RAW 264.7 macrophages mouse leukemia mononuclear macrophages, cell line purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, USA).
  • RAW 264.7 macrophages are cultured in RPMI-1640 medium, which contains penicillin (100U/ml), streptomycin (100 ⁇ g/ml) and 10% FBS.
  • Use CT-NA (0-100 ⁇ M) alone, and its combination with PGD 2 (0-100 ⁇ M) and CSE to determine the toxicity to RAW264.7 macrophages (1-10%).
  • MTT methylthiazole -Tetrazole
  • RAW 264.7 macrophages were seeded in a 96-well plate at a concentration of 4 ⁇ 10 5 cells/ml for 24 hours, and then exposed to CT-NA (0-100 ⁇ M) at 37° C. for 1 hour.
  • RAW 264.7 macrophages were further exposed to CSE (4%) and PGD 2 (10 ⁇ M) for 24 hours, and then treated with MTT (5 mg/ml) at 37° C. for 4 hours. Then, the supernatant of each well was replaced with DMSO (200 ⁇ l/well), and the absorbance at 570 nm was measured.
  • RNA samples from each processed plate were extracted with HiScript5 ⁇ QRTSuperMix and reverse transcribed into cDNA, and then subjected to RT-PCR.
  • RT-PCR was processed using BioRad CFX96 Touch TM real-time PCR detection system (BioRad, USA), which used qPCRSYBR Green Master Mix. The threshold cycle number is obtained using BioRad CFX Manager software.
  • the primers used in the RT-PCR reaction are shown in Table 1.
  • ⁇ -actin was used as an internal control.
  • the RT-PCR reaction was repeated three times. The relative expression of target mRNA is corrected by the respective ⁇ -actin.
  • the data are the mean ⁇ SEM. Statistics use SPSS (SPSS Inc., Chicago, IL) for calculation. The single-factor ANOVA method is used to compare the F value. If p>0.05, Dunnett multiple comparisons teat is used to calculate the difference in parameter data; if p ⁇ 0.05, the Mann-Whitney U non-parametric test is used to compare the difference. p ⁇ 0.05 and p ⁇ 0.01 were considered statistically significant.
  • CT-NA can significantly improve CS-induced lung inflammation through CRTH2 antagonism.
  • CS-induced hypoxemia, pulmonary edema, and lung permeability were evaluated by measuring partial pressure of oxygen (PO 2 ), lung weight coefficient, and BALF albumin content, respectively.
  • PO 2 partial pressure of oxygen
  • lung weight coefficient and BALF albumin content were significantly increased (p ⁇ 0.01), indicating that the CS-induced animal model was successful.
  • CT-NA (10 and 30 mg/kg) group PO 2 significantly increased (p ⁇ 0.01) ( Figure 10C)
  • the lung weight coefficient was partially reduced (p ⁇ 0.05)
  • BALF albumin content was significantly reduced (p ⁇ 0.01) ( Figure 10E).
  • ELISA kits were used to detect pro-inflammatory cytokines (TNF- ⁇ , IL-1 ⁇ , IL-6), chemokines (KC) and anti-inflammatory cytokines. (IL-10) expression level.
  • TNF- ⁇ , IL-1 ⁇ , IL-6 and KC pro-inflammatory cytokines
  • KC chemokines
  • IL-10 anti-inflammatory cytokines.
  • Figure 11A, B, C and D the expression levels of TNF- ⁇ , IL-1 ⁇ , IL-6 and KC in the CS exposure group were significantly increased compared with the control group (p ⁇ 0.01).
  • CS-induced overexpression of TNF- ⁇ , IL-1 ⁇ , IL-6 and KC can be effectively reduced by CT-NA treatment (10 and 30 mg/kg) (p ⁇ 0.01).
  • CT-NA (10 and 30 mg/kg) and Dex (1 mg/kg) significantly reduced the average pathological score (p ⁇ 0.01) in a dose-dependent manner (Figure 12B).
  • the test results show that CT-NA can significantly reduce the severity of lung injury caused by CS by blocking CRTH2 receptors.
  • MPO activity of lung tissue Due to the above-mentioned good test results, we further evaluated the MPO activity of lung tissue. MPO produced by activated neutrophils is an important marker of neutrophil infiltration and lung tissue damage. We found that the MPO activity of lung tissue was significantly increased when exposed to CS compared to exposure to fresh air (p ⁇ 0.01) ( Figure 13A). It is worth noting that CT-NA (10 and 30 mg/kg) and Dex (1 mg/kg) attenuate MPO activity (p ⁇ 0.01), indicating that CRTH2 receptor blockade can effectively inhibit the invasion of neutrophils into alveoli and spaces.
  • CT-NA treatment can inhibit CSE- and PGD 2 -stimulated RAW 264.7 macrophage cytokine secretion, because the PGD 2 /CRTH2 receptors on activated macrophages pass Increase the expression of proinflammatory cytokines to significantly increase disease activity.
  • MTT assay showed that PGD 2 (10 ⁇ M) plus CT-NA with a concentration of up to 100 ⁇ M, and CSE 4% plus CT-NA with a concentration of up to 100 ⁇ M were non-toxic to RAW 264.7 macrophages.
  • CRTH2 antagonism effectively improves the production of pro-inflammatory cytokines and chemokines, and promotes the production of anti-inflammatory cytokines from CSE- and PGD 2 -activated RAW 264.7 macrophages.
  • mice ⁇ / ⁇ ; 20-26 g; 8 weeks were purchased from Shanghai SIPPR-BK Experimental Animal Co., Ltd. Mice in an isolated ventilated cage, at 40-60% humidity, 24 ⁇ 2 °C, according to 12 hours / 12 hours dark-light alternate environment to eat and drink freely.
  • the method is simply described as follows: the mice are randomly divided into a control group (12 mice) and an LPS group (48 mice). The LPS group (48 mice) was further divided into four subgroups (each group contains 12 mice).
  • mice in the four subgroups of LPS were given normal saline (NS), 10 mg/kg CT-NA, 30 mg/kg CT-NA and 1 mg/kg Dex respectively.
  • NS normal saline
  • the mice were anesthetized with sodium pentobarbital (intraperitoneal injection of 40 mg/kg), and then intratracheal instillation of NS to the control group and LPS (4 mg/kg) to all LPS subgroups.
  • unanaesthetized mice in the LPS group were given NS, 10 mg/kg CT-NA, 30 mg/kg CT-NA, and 1 mg/kg Dex, respectively (Figure 16). Both physiological saline and LPS were administered at 10 ⁇ l/10 g body weight.
  • the moor VMS-OXY TM meter was used to measure the oxygen saturation of all mice by measuring the oxygen saturation (%) in the microcirculation in the wavelength range of 500 to 650 nm.
  • SO 2 represents the percentage of oxygenated hemoglobin to total hemoglobin in the blood.
  • the BALF of each mouse was collected for inflammatory cell count and classification, albumin concentration and pro-inflammatory cytokine/chemokine level determination.
  • the lung is also used for histological examination to determine the lung weight coefficient and MPO activity.
  • mice were euthanized to expose the trachea, and then the right lung was lavaged three times with 0.4 mL/time sterile saline containing bovine serum albumin (BSA) and 5000 IU/L heparin, and passed through the tracheal tube to collect BALF. After measuring the total number of cells in BALF with a hemocytometer, the remaining BALF was centrifuged at 1000 ⁇ g at 4°C for 10 minutes. The supernatant was aliquoted and stored at -80°C for the next step to measure the pro-inflammatory cytokine or albumin concentration. Spread the obtained cell pellet on a glass slide.
  • BSA bovine serum albumin
  • the lung weight coefficient is an indicator of pulmonary edema. It is calculated by dividing the individual lung weight of each mouse by the total weight of the individual lung weight after sucking the blood tissue from the lung surface.
  • the corresponding ELISA kits were used to determine the expression levels of pro-inflammatory cytokines (TNF- ⁇ , IL-1 ⁇ , IL-6) and chemokines (KC, mouse IL-8 homolog) in BALF according to the instructions. After measuring the optical density at 450 nm, the expression level was calculated from the standard curve.
  • the lower lobe of the left lung of each mouse was stored in 10% neutral formalin for histopathological examination, and formalin was added dropwise at room temperature under a constant pressure of 22 to 25 cm H 2 O for 48 hours.
  • the preserved lower lobe of the left lung was taken out and embedded in paraffin, then sectioned (4 ⁇ m) to expose the largest longitudinal view of the bronchi in the main lung.
  • H&E staining was used to assess pulmonary edema, and the infiltration of neutrophils and inflammatory cells was observed under a light microscope. Pulmonary edema, hemorrhage, alveolar wall thickening, neutrophil and inflammatory cell infiltration were counted and scored to evaluate the severity of lung injury.
  • the total lung injury score is the sum of the four criteria. Take the average of the scores of 12 mice.
  • Evans Blue is a dye that quickly binds to albumin and is confined in blood vessels because the endothelium is impermeable to albumin under normal physiological conditions. Pulmonary microvascular permeability is measured by measuring the extravasation of Evans blue dye in the lung. The method is briefly described as follows: The mice were randomly divided into a control group (12 mice) and an LPS group (48 mice). The LPS group (48 mice) was further divided into four subgroups (each group contains 12 mice). To measure pulmonary microvascular permeability, unanaesthetized mice in the four subgroups of LPS received NS, 10 mg/kg CT-NA, 30 mg/kg CT-NA, and 1 mg/kg Dex.
  • mice One hour later, the control group anesthetized mice were instilled intratracheally with NS (control group) and LPS (LPS subgroup), both of which were administered at 10 ⁇ l/10 g body weight.
  • NS control group
  • LPS LPS subgroup
  • Evans blue dye 50 mg/kg was injected into the tail vein of all mice, and euthanized one hour later.
  • NS is slowly injected into the right ventricle of the mouse to drain the blood from the lung tissue.
  • the right lung was carefully taken out, sliced and placed in formamide (3ml/100mg). After 24 hours of incubation, the samples were centrifuged at 500 ⁇ g for 10 minutes (4°C).
  • the formamide blank at 620 nm was measured by the standard curve method, and the absorbance of the Evans blue dye extracted in the supernatant was measured. The measured value is expressed as micrograms of dye per 100 mg of wet lung weight.
  • the albumin concentration in BALF was measured using a spectrophotometer and albumin measurement kit at 628 nm. The albumin concentration ratio measured by BALF not only represents the albumin level, but also represents the permeability of the pulmonary microvascular.
  • the MPO activity determination procedure is as follows: accurately weigh the left lung strip tissue and use the homogenization medium to prepare a 5% homogenate (the volume ratio of the left lung strip tissue and the homogenization medium is 1:19). Then mix the homogenate (0.9ml) and reaction buffer (0.1ml) thoroughly according to the ratio of 9:1 (if there is not enough homogenate, the volume fraction is 5%. The homogenate and reaction buffer can be in accordance with 9:1 The proportion is reduced accordingly), and then incubated at 37°C for 15 minutes. Then use a spectrophotometer to determine the activity of MPO by measuring the change in absorbance at 460nm according to the standard curve.
  • the method for isolating neutrophils and testing the effect of CT-NA on neutrophil migration is briefly described as follows. 1.5% glycogen was injected intragastrically at a dose of 20 ml/kg body weight into mice. Four hours later, the mice were euthanized and neutrophils were isolated from the peritoneal lavage. The effect of CT-NA on the migration of neutrophils was detected by Boyden chamber detection kit (3 ⁇ m pore size, Billerica, MA). PGD 2 was used as a chemoattractant because activated PGD 2 /CRTH2 receptors promote neutrophils Cell migration.
  • mice Thioglycolate (4%) was injected into the abdominal cavity of mice at a dose of 20 ml/kg body weight for three consecutive days. 48 hours after the last thioglycolate injection (on day 5), the mice were euthanized to isolate peritoneal macrophages from the peritoneal lavage. The isolated peritoneal macrophages were added to a 12-well plate (4 ⁇ 10 5 /well) and cultured at 37°C. Non-adherent cells were removed by gently washing three times with hot PBS.
  • macrophages which are cultured in DMEM/high glucose medium containing penicillin (100 U/ml), streptomycin (100 ⁇ g/ml) and 10% FBS at 37°C.
  • serum-free DMEM/high glucose was added to the 12-well plate for 10-12 hours, and then treated with different concentrations of LPS (0.01, 0.1, 1 and 10 ⁇ M) for 24 hours.
  • LPS low-density polypeptide
  • RAW 264.7 macrophages mouse leukemia mononuclear macrophages, cell line purchased from ATCC, (Manassas, Virginia), and cultured in RPMI-1640 medium containing penicillin (100U/ ml), streptomycin (100 ⁇ g/ml) and 10% fetal bovine serum.
  • RAW264.7 macrophages are an ideal model for screening anti-inflammatory drugs and evaluating inhibitor pathways that stimulate pro-inflammatory cytokines and enzymes. According to the standard method, MTT was used to determine the toxicity of CT-NA alone and its combination with PGD 2 and LPS to RAW264.7 macrophages and isolated peritoneal macrophages.
  • RAW 264.7 macrophages were seeded in a 96-well plate at a concentration of 4 ⁇ 10 5 cells/ml for 12 hours, and then exposed to CT-NA (0-200 ⁇ M) at 37° C. for 1 hour.
  • CT-NA CT-NA
  • the RAW 264.7 macrophages were further exposed to LPS (100ng/ml) and PGD 2 (10 ⁇ M) for 24 hours, and then treated with MTT (5 mg/ml) at 37°C for 4 hours. Then, the supernatant of each well was replaced with DMSO (200 ⁇ l/well), and the absorbance at 570 nm was measured.
  • RAW 264.7 macrophages were added to two 12-well plates at 70-80% confluence. After that, the medium of the 12-well plate was replaced with serum-free RPMI-1640 medium, incubated for 10-12 hours and then exposed to CT-NA (10 and 100 ⁇ M) for 1 hour. One hour later, one 12-well plate was treated with LPS (100ng/ml) for 24 hours, and the other with PGD 2 (10 ⁇ M) for 24 hours. After the treatment, the supernatant of the treated cells was collected, and the protein levels of TNF- ⁇ , IL-1 ⁇ , IL-6, and KC were measured using an ELISA kit according to the method of the instructions.
  • RNA samples from each processed plate were extracted with HiScript5 ⁇ QRTSuperMix and reverse transcribed into cDNA, and then subjected to RT-PCR.
  • RT-PCR was processed using the BioRad CFX96 Touch TM real-time PCR detection system (San Diego, California).
  • total RNA extracted from isolated peritoneal macrophages was used to analyze the mRNA levels of IL-1 ⁇ , TNF- ⁇ , IL-6 and KC.
  • the primers used in the RT-PCR reaction are shown in Table 2.
  • ⁇ -actin was used as an internal control. The RT-PCR reaction was repeated three times. The relative expression of target mRNA is corrected by the respective ⁇ -actin.
  • the method of total protein extraction and western blotting determination is as follows, homogenize lung tissue in RIPA buffer (0.5M Tris-HCl, pH 7.4, 1.5M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10mM EDTA) , The buffer also contains protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO). RAW264.7 macrophages were seeded into two 6-well plates at 70-80% confluence. After culturing in serum-free RPMI-1640 medium overnight and starving, RAW264.7 macrophages (0.5, 1, 10 and 100 ⁇ M) were pretreated with CT-NA for 1 hour.
  • RIPA buffer 0.5M Tris-HCl, pH 7.4, 1.5M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10mM EDTA
  • the buffer also contains protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO).
  • one 6-well plate was treated with LPS (100ng/ml) for 1 hour and the other with PGD 2 (10 ⁇ M) for 1 hour.
  • the cells were directly lysed for 30 minutes with shaking in RIPA buffer containing protease and phosphatase inhibitors in an ice environment. Then, the lysate was centrifuged at 12,300 ⁇ g for 15 minutes at 4°C, and the supernatant was collected. The Bradford protein assay (BCA) was performed to measure protein concentration. The same amount of protein (30 ⁇ g) was separated on 12% SDS-PAGE and transferred to a 0.45 ⁇ m polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA).
  • PVDF polyvinylidene fluoride
  • the data are the mean ⁇ SEM. Statistics use SPSS (SPSS Inc., Chicago, IL) for calculation. The single-factor ANOVA method is used to compare the F value. If p>0.05, Dunnett multiple comparisons teat is used to calculate the difference of parameter data; if p ⁇ 0.05, the Mann-Whitney U non-parametric test is used to compare the difference. p ⁇ 0.05 and p ⁇ 0.01 were considered statistically significant.
  • the LPS-induced hypoxemia and pulmonary edema were evaluated by measuring SO 2 and lung wet weight coefficient, respectively.
  • the results showed that compared with the control group, the LPS-induced group showed lower SO 2 and higher lung wet weight coefficient (P ⁇ 0.01), using CT-NA at doses of 10 and 30 mg/kg, significantly increasing SO 2 in a dose-dependent manner (P ⁇ 0.01) and significantly reducing lung wet weight coefficient (P ⁇ 0.01) ( Figure 17B and C).
  • CT-NA at 10 or 30 mg/kg and Dex at 1 mg/kg also enhance SO 2 and reduce lung wet weight coefficient, respectively.
  • the above results indicate that CRTH2 antagonism by CT-NA can significantly improve lung inflammation, hypoxemia and pulmonary edema in the LPS-induced ALI model.
  • CT-NA improves the production of pro-inflammatory cytokines and chemokines induced by LPS in BALF
  • ELISA kits were used to measure the expression levels of IL-1 ⁇ , TNF- ⁇ , IL-6 and KC in the collected BALF to determine the effect of CT-NA on the production of pro-inflammatory cytokines and chemokines.
  • LPS induced a significant increase in the expression of IL-1 ⁇ , TNF- ⁇ , IL-6 and KC (P ⁇ 0.01).
  • 10 and 30 mg/kg CT-NA and 1 mg/kg Dex were dose-dependent
  • the method effectively reduced the production of IL-1 ⁇ , TNF- ⁇ , IL-6 and KC (P ⁇ 0.05 or P ⁇ 0.01) ( Figure 18A-D).
  • the average pathology score in terms of bleeding and infiltration of inflammatory cells, and neutrophils entering peribronchioles and perivascular tissues, compared with carrier attack, LPS induction significantly increased (P ⁇ 0.01), but CT-NA 10 (P ⁇ 0.05) and 30mg/kg (P ⁇ 0.01) or Dex at a dose of 1 mg/kg (P ⁇ 0.01) significantly reduced the pathological score, and the CT-NA results again showed a dose-dependent ( Figure 19B). Therefore, CT-NA blocking CRTH2 receptor significantly reduced the severity of LPS-induced lung injury and reversed LPS-induced lung tissue injury.
  • CT-NA The protective effect of CT-NA on LPS-induced pulmonary vascular permeability was tested by measuring the albumin content in BALF in the lungs and the extraversion of Evans blue dye. Compared with the control group, the albumin content in BALF in the LPS-induced group was significantly increased (P ⁇ 0.01), while CT-NA was 10mg/kg (P ⁇ 0.05) and 30mg/kg (P ⁇ 0.01) or Dex was 1mg/kg (P ⁇ 0.01) At the dose, it significantly reduced the albumin content in BALF ( Figure 20A). Compared with the control group, the pulmonary vascular leakage and quantitative extravasation of Evans blue dye in the LPS-induced group were significantly increased (P ⁇ 0.01).
  • CT-NA administration was 10mg/kg (P ⁇ 0.05) and 30mg/kg (P ⁇ 0.01) or Dex at a dose of 1 mg/kg (P ⁇ 0.01) significantly reduced the pulmonary vascular leakage and extravasation of Evans blue dye induced by LPS ( Figure 20B). Therefore, these results indicate that CRTH2 antagonism can effectively improve the pulmonary vascular permeability of LPS-induced ALI mice.
  • CT-NA reduces lung MPO activity induced by LPS
  • MPO is produced by activated neutrophils and is an important marker of neutrophil infiltration and lung tissue damage.
  • the increase in MPO activity reflects the accumulation of activated neutrophils in the lung.
  • the MPO activity of mice in the LPS induction group was significantly higher than that of the control group (P ⁇ 0.01).
  • CT-NA significantly reduced MPO activity at 10 and 30 mg/kg (P ⁇ 0.01) or Dex 1 mg/kg (P ⁇ 0.01), and CT-NA again showed a dose-dependent manner ( Figure 21A). Therefore, CT-NA blocking CRTH2 receptor can effectively inhibit the infiltration of neutrophils into alveoli and interstices.
  • the transwell assay was used to evaluate the effect of CT-NA on the migration of neutrophils, because the main cause of ALI symptoms is the release of harmful inflammatory mediators from neutrophils.
  • Wright-Giemsa evaluated the characteristics of isolated neutrophils and performed cell viability assays.
  • the activated PGD 2 /CRTH2 receptor promotes the migration and function of neutrophils, so we use PGD 2 as a chemoattractant. After 4 hours of incubation, significant migration of neutrophils to PGD 2 was observed, with a concentration ranging from 1 to 10 ⁇ M (P ⁇ 0.05 or P ⁇ 0.01) (Figure 21B).
  • CT-NA reduces the secretion of proinflammatory cytokines and chemokines by LPS and PGD 2 induced RAW264.7 macrophages and isolated peritoneal macrophages
  • Pro-inflammatory cytokines are a key factor in the pathogenesis of ALI. Through over-expression of pro-inflammatory cytokines, the activation of PGD 2 /CRTH2 receptors on macrophages significantly worsens the disease condition. Therefore, the effect of CT-NA treatment on the production of pro-inflammatory factors induced by LPS- or PGD 2 was also tested in macrophages. MTT assay showed that LPS at a dose of 100ng/ml plus CT-NA at a dose of up to 100 ⁇ M, or PGD 2 at a dose of 10 ⁇ M plus CT-NA at a dose of up to 100 ⁇ M had no effect on RAW264.7 macrophages or isolated peritoneal macrophages. toxicity.
  • CT-NA The effect of CT-NA on the expression of pro-inflammatory cytokines and chemokines was measured by ELISA.
  • CT-NA was 10 and 100 ⁇ M, respectively, it inhibited IL-1 ⁇ , TNF- ⁇ , IL-6 and KC in a dose-dependent manner to stimulate the proteins in response to LPS ( Figure 22A-D) or PGD 2 ( Figure 22E-H) expression.
  • Quantitative RT-PCR proved that CT-NA reduced RAW264.7 macrophages stimulated by LPS or PGD 2 ( Figure 23A-H) and isolated peritoneal macrophages (Figure 23A-H) in a dose-dependent manner at doses of 10 and 100 ⁇ M.
  • CT-NA inhibits P65 activation in vitro and in vivo
  • CT-NA significantly inhibiting LPS-induced ALI was explored by Western blot analysis.
  • the focus of the study is the effect of CT-NA on the LPS-induced NF- ⁇ B activation pathway, because NF- ⁇ B is necessary for the activation of pro-inflammatory mediators, neutrophil infiltration and increased pulmonary vascular permeability.
  • the use of LPS-induced RAW264.7 macrophages and lung tissues strongly induced phosphorylation and degradation of I ⁇ B ⁇ , thereby increasing or decreasing phosphorylated P65 or P65 levels, respectively (Figure 25A and B).
  • the experimental results show that the present invention provides the heterocyclic compound represented by formula A, its hydrate, its pharmaceutically acceptable salt (such as alkali metal salt) or the hydrate of said salt (such as alkali metal salt) by inhibiting Improper transfer of pulmonary macrophages and neutrophils reduces pulmonary vascular permeability, improves the production of pro-inflammatory cytokines and cytokine chemokines, and enhances the production of IL-10, which greatly alleviates the Cigarette smoke CS-induced or LPS-induced acute lung injury.
  • the pharmaceutically acceptable salt such as alkali metal salt
  • the hydrate of said salt such as alkali metal salt
  • the present invention provides the crystal form of the heterocyclic compound represented by formula A, the crystal form of its hydrate, and its pharmaceutically acceptable alkali
  • the metal salt or the hydrate of the alkali metal salt has a better therapeutic or alleviating effect on acute respiratory distress syndrome.

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Abstract

式A所示杂环化合物、水合物、其药学上可接受的碱金属盐或者所述碱金属盐的水合物在制备治疗急性肺损伤或急性呼吸窘迫综合征药物中的用途。实验结果表明，上述化合物通过抑制巨噬细胞和嗜中性粒细胞向肺部的渗透，降低肺血管通透性，减少促炎性细胞因子和细胞因子趋化因子的产生和促进IL-10的产生，极大地缓解了由香烟烟雾CS诱导或LPS诱导的急性肺损伤或急性呼吸窘迫综合征。

Description

杂环化合物及其盐的应用
本申请要求2019年5月22日向中国国家知识产权局提交的，专利申请号为201910431026.4，发明名称为“杂环化合物及其盐的应用”在先申请的优先权。该申请的全文通过引用的方式结合于本申请中。
技术领域
本发明属于医药技术领域，具体涉及到杂环化合物及其盐的应用。
背景技术
急性肺损伤/急性呼吸窘迫综合征(ALI/ARDS)是指心源性以外的各种肺内外致病因素所导致的急性、进行性缺氧性呼吸衰竭。自1967年Ashbaugh等报道成人呼吸窘迫综合征(adult respiratory distress syndrome,ARDS)以来，引起了国内外广大学者的极大兴趣，并做了大量临床与实验研究工作。我国先后召开了ALI/ARDS专题研讨会，主要就ALI/ARDS定义、发病机制、诊断标准和治疗作了深入的探讨。对ALI/ARDS的认识有了明显提高，表现在对ALI/ARDS的命名、定义逐渐规范，对其发病机制有了较为深刻的认识，提出了便于临床使用的ALI/ARDS诊断标准，积累了一些较为成熟的治疗经验和措施，使ALI/ARDS的发病率和病死率均有下降趋势。但我们必须清楚地认识到，ALI/ARDS的病因及发病机制错综复杂，致病环节多，目前尚缺乏针对发病机制的有效治疗措施，ALI/ARDS的病死率仍较高。因此有必要开发可以治疗ALI/ARDS的药物。
发明内容
为解决上述问题，本发明提供如下技术方案：
式A所示杂环化合物、其药学上可接受的盐或其水合物中的至少一种在制备治疗急性肺损伤或急性呼吸窘迫综合征药物中的用途，
Figure PCTCN2020091842-appb-000001
根据本发明的实施方案，所述急性肺损伤选自香烟烟雾(cigarette smoke，CS)或脂多糖(lipopolysaccharide，LPS)诱导的急性肺损伤。
根据本发明的技术方案，所述式A所示杂环化合物药学上可接受的盐选自碱金属盐。
根据本发明的技术方案，所述水合物选自式A所示杂环化合物的水合物，或式A所示杂环化合物碱金属盐的水合物。
根据本发明的优选技术方案，所述式A所示杂环化合物或其药学上可接受的盐为结晶形式，如式A所示杂环化合物的晶型、其水合物的晶型、其药学上可接受的碱金属盐的晶型或者所述碱金属盐水合物的晶型。
根据本发明的实施方案，式A所示杂环化合物或其水合物的晶型使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在11.1±0.2°、11.4±0.2°、17.9±0.2°、22.6±0.2°、24.4±0.2°处具有特征峰。
优选地，所述晶型使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在8.6±0.2°、11.1±0.2°、11.4±0.2°、14.1±0.2°、16.1±0.2°、17.9±0.2°、20.9±0.2°、22.6±0.2°、24.4±0.2°、25.8±0.2°处具有特征峰。
还优选地，所述晶型使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在8.6±0.2°、11.1±0.2°、11.4±0.2°、14.1±0.2°、15.6±0.2°、16.1±0.2°、17.9±0.2°、18.3±0.2°、20.9±0.2°、22.6±0.2°、24.4±0.2°、25.8±0.2°、26.5±0.2°、28.9±0.2°处具有特征峰。
进一步优选地，所述晶型具有基本上如图1所示的X射线粉末衍射谱图。
更优选地，所述晶型具有基本如图2所示的DSC-TGA谱图。
根据本发明，式A所示杂环化合物水合物的晶型优选为一水合物。更优选，所述水合物中水的质量分数为4.2-5.2％，更优选为4.5～5.0％。
所述一水合物如下所示：
Figure PCTCN2020091842-appb-000002
还更优选地，所述晶型为单晶，具有下述的单晶参数：
Figure PCTCN2020091842-appb-000003
本发明还提供式A所示杂环化合物或其水合物的晶型的制备方法，包括如下步骤：
将式A所示化合物置于酮类溶剂和水构成的混合溶剂中，加热溶解后降温，搅拌析晶得到式A所示杂环化合物或其水合物的晶型，
Figure PCTCN2020091842-appb-000004
本发明中式A所示杂环化合物的制备可参考专利文献CN101896178B中实施例3和4所记载的方法制备。例如将式A所示杂环化合物消旋体在Chiralcel OJ-RH柱(Chiralcel Technologies)上用含0.05％三氟乙酸的甲醇溶液洗脱，分离得到式A所示杂环化合物。
根据本发明的制备方法，所述酮类溶剂选自丙酮或甲乙酮。
根据本发明的制备方法，所述酮类溶剂与水的体积比为(1-3):1，例如为1:1。
根据本发明的制备方法，所述加热的温度为30～80℃，优选为40～60℃。
根据本发明的实施方案，所述式A所示杂环化合物药学上可接受的碱金属盐优选为钠盐、锂盐或钾盐；所述碱金属盐的水合物选自钠盐、锂盐或钾盐的水合物。
优选地，所述式A化合物碱金属盐的晶型为水合物形式。
根据本发明，所述式A化合物碱金属盐的结晶水合物优选为一水合物。
作为实例，所述式A化合物碱金属盐的结晶水合物选自如下式A-N、A-L或A-K所示的化合物：
Figure PCTCN2020091842-appb-000005
Figure PCTCN2020091842-appb-000006
优选地，式A-N所示的化合物为结晶水合物，其使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在16.4±0.2°、18.9±0.2°、21.7±0.2°、24.0±0.2°处具有特征峰。
优选地，其使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在11.8±0.2°、16.4±0.2°、16.7±0.2°、16.9±0.2°、17.1±0.2°、17.8±0.2°、18.6±0.2°、18.9±0.2°、21.7±0.2°、23.7±0.2°、24.0±0.2°处具有特征峰。
还优选地，所述结晶水合物使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在5.6±0.2°、11.8±0.2°、14.0±0.2°、15.8±0.2°、16.4±0.2°、16.7±0.2°、16.9±0.2°、17.1±0.2°、17.8±0.2°、18.6±0.2°、18.9±0.2°、20.3±0.2°、21.7±0.2°、23.7±0.2°、24.0±0.2°、26.1±0.2°、28.1±0.2°、28.5±0.2°、29.8±0.2°处具有特征峰。
进一步优选地，所述结晶水合物具有基本上如图4所示的X射线粉末衍射谱图(XRPD)。
优选地，所述式A-N所示化合物的结晶水合物中水的质量分数为3.4～4.4％，更优选为3.6～4.2％。
更优选地，所述式A-N所示化合物的结晶水合物具有基本如图5所示的DSC-TGA谱图。
根据本发明，式A-K所示的钾盐化合物为结晶水合物，其使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在15.6±0.2°、21.4±0.2°、24.0±0.2°处具有特征峰。
优选地，式A-K所示的钾盐化合物为结晶水合物，其使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在11.7±0.2°、15.6±0.2°、16.6±0.2°、17.9±0.2°、18.5±0.2°、21.4±0.2°、24.0±0.2°、28.2±0.2°处具有特征峰。
优选地，式A-K所示的钾盐化合物为结晶水合物，其使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在11.7±0.2°、15.6±0.2°、15.9±0.2°、16.6±0.2°、17.4±0.2°、17.9±0.2°、18.5±0.2°、21.4±0.2°、23.5±0.2°、24.0±0.2°、27.7±0.2°、28.2±0.2°处具有特征峰。
还优选地，所述式A-K所示化合物的结晶水合物使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在11.7±0.2°、14.0±0.2°、15.6±0.2°、15.9±0.2°、16.6±0.2°、17.4±0.2°、17.9±0.2°、18.5±0.2°、20.1±0.2°、21.4±0.2°、23.5±0.2°、24.0±0.2°、27.5±0.2°、27.7±0.2°、28.2±0.2°、28.6±0.2°、29.3±0.2°、29.6±0.2°处具有特征峰。
进一步优选地，所述式A-K所示化合物的结晶水合物具有基本上如图6所示的X射线粉末衍射谱图。
优选地，所述式A-K所示化合物的结晶水合物中水的质量分数为3.3～4.3％，更优选为3.5～4.1％。
进一步优选地，所述式A-K所示化合物的结晶水合物具有基本上如图7所示的DSC-TGA谱图。
根据本发明，式A-L所示的锂盐化合物为结晶水合物，使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在16.7±0.2°、18.8±0.2°、21.9±0.2°、23.9±0.2°处具有特征峰。
还优选地，所述式A-L所示化合物的结晶水合物使用Cu-Kα辐射，以2θ角度表示的X射 线粉末衍射在5.6±0.2°、8.8±0.2°、11.8±0.2°、14.0±0.2°、16.3±0.2°、16.7±0.2°、16.9±0.2°、17.0±0.2°、17.7±0.2°、18.5±0.2°、18.8±0.2°、21.9±0.2°、23.9±0.2°、28.2±0.2°处具有特征峰。
进一步优选地，所述式A-L所示化合物的结晶水合物具有基本上如图8所示的X射线粉末衍射谱图。
优选地，所述式A-L所示化合物的结晶水合物中水的质量分数为3.6～4.6％，更优选为3.9～4.5％。
更优选地，所述式A-L所示化合物的结晶水合物具有基本如图9所示的DSC-TGA谱图。
本发明还提供式A所示杂环化合物药学上可接受的盐或者所述盐的水合物的制备方法，包括如下步骤：
将式A所示化合物溶于酮类溶剂中，向酮类溶剂中加入碱金属氢氧化物的水溶液进行反应，过滤干燥即得；其中所述碱金属氢氧化物优选为氢氧化钠、氢氧化锂或氢氧化钾。
本发明中式A所示杂环化合物的制备可参考专利文献CN101896178B中实施例3和4所记载的方法制备。例如将式A所示杂环化合物消旋体在Chiralcel OJ-RH柱(Chiralcel Technologies)上用含0.05％三氟乙酸的甲醇溶液洗脱，分离得到式A所示杂环化合物。
本发明式A所示杂环化合物、药学上可接受的盐、水合物及其制备以及性能相关数据可以参考申请号为201910024238.0，申请日为2019年1月10日的发明专利申请，其全文引入本文作为参考。
本发明式A所示杂环化合物、药学上可接受的盐、水合物的制备以及性能数据可以参考申请号为201910024247.X，申请日为2019年1月10日的发明专利申请，其全文引入本文作为参考。
本发明还提供一种用于治疗急性肺损伤或急性呼吸窘迫综合征的药物组合物，其包括治疗有效量的本发明所述的式A所示杂环化合物、其药学上可接受的盐或其水合物。
根据本发明的实施方案，所述急性肺损伤选自香烟烟雾CS或脂多糖LPS诱导的急性肺损伤。
根据本发明的技术方案，所述式A所示杂环化合物药学上可接受的盐选自碱金属盐。
根据本发明的技术方案，所述水合物选自式A所示杂环化合物的水合物，或式A所示杂环化合物碱金属盐的水合物。
根据本发明的优选技术方案，所述式A所示杂环化合物或其药学上可接受的盐为结晶形式，例如选自式A所示杂环化合物的晶型、其水合物的晶型、其药学上可接受的碱金属盐的晶型或者所述碱金属盐水合物的晶型中的至少一种。
根据本发明的实施方案，所述药物组合物还包含至少一种药学上可接受的辅料。所述辅料可以为惰性的、无毒的赋形剂、载体或稀释剂，例如所述辅料选自下列中的一种、两种或多种：崩解剂、助流剂、润滑剂、填充剂、粘合剂、着色剂、泡腾剂、矫味剂、防腐剂、包衣材料等。
本发明还提供一种治疗急性肺损伤或急性呼吸窘迫综合征的方法，该方法包括向需要此治疗的哺乳动物患者施用有效治疗量的上述式A所示杂环化合物、其药学上可接受的盐或其水合物。
本发明还提供一种治疗急性肺损伤或急性呼吸窘迫综合征的方法，该方法包括向需要此治疗的哺乳动物患者施用有效治疗量的上述药物组合物。
根据本发明的实施方案，所述急性肺损伤选自CS或LPS诱导的急性肺损伤。
根据本发明的技术方案，所述式A所示杂环化合物药学上可接受的盐选自碱金属盐。
根据本发明的技术方案，所述水合物选自式A所示杂环化合物的水合物，或式A所示杂环化合物碱金属盐的水合物。
根据本发明的优选技术方案，所述式A所示杂环化合物或其药学上可接受的盐为结晶形式，例如选自式A所示杂环化合物的晶型、其水合物的晶型、其药学上可接受的碱金属盐的晶型或者所述碱金属盐水合物的晶型中的至少一种。
有益效果
本发明提供式A所示杂环化合物、水合物、其药学上可接受的碱金属盐或者所述碱金属盐的水合物在制备治疗急性肺损伤或急性呼吸窘迫综合征药物中的用途。实验结果表明，上述化合物通过抑制巨噬细胞和嗜中性粒细胞向肺部的渗透，降低肺血管通透性，减少促炎性细胞因子和细胞因子趋化因子的产生和促进IL-10的产生，极大地缓解了由香烟烟雾CS诱导或LPS诱导的急性肺损伤或急性呼吸窘迫综合征，并且由于所述化合物可中止或减缓炎症性肺损伤、减轻肺水肿、保证组织供氧，表明其对于急性呼吸窘迫综合症具有较好的治疗或缓解作用。
附图说明
图1为制备例2所得晶型的XRPD谱图。
图2为制备例2所得晶型的DSC-TGA谱图。
图3为制备例2所得晶型的三维结构图和晶胞图。
图4为制备例3所得钠盐水合物的XRPD谱图。
图5为制备例3所得钠盐水合物的DSC-TGA谱图。
图6为制备例4所得钾盐水合物的XRPD谱图。
图7为制备例4所得钾盐水合物的DSC-TGA谱图。
图8为制备例5所得锂盐水合物的XRPD谱图。
图9为制备例5所得锂盐水合物的DSC-TGA谱图。
图10为CT-NA对BALF中炎症细胞数量，氧分压(PO 2)，肺重量系数和BALF中白蛋白含量的影响。
小鼠连续七天在暴露于CS前1小时灌胃给予CT-NA(10和30mg/kg)、生理盐水、Dex(1mg/kg)。BALF是在最后一次CS暴露后24小时收集。
(A)收集的BALF中的嗜中性粒细胞(黑色箭头)和巨噬细胞(灰色箭头)的图像。
(B)BALF中所有细胞，巨噬细胞，嗜中性粒细胞和淋巴细胞的浸润特征(从左到右分别为载体生理盐水组、载体CS暴露组CT-NA 10mg/kgCS暴露组、CT-NA 30mg/kgCS暴露组及地塞米松(Dex)1mg/kg CS暴露组)。
(C)在CS暴露后24小时使用moor VMS-OXY TM监测仪(Moor Instruments，United Kingdom)测量所有小鼠的氧分压(PO 2)。
(D)从解剖的肺组织吸出表面血液后，肺重量比通过将每只小鼠的个体肺重量除以其总体重来测量肺重量比。
(E)使用白蛋白测定试剂盒测量BALF中的白蛋白浓度。与对照(Ctrl)组相比，##p<0.01；*p<0.05和**p<0.01与模型组(model)相比。数值表示为平均值±SEM；n＝12(每组)。
图11为CT-NA对CS诱导的ALI小鼠的BALF中促炎细胞因子(TNF-α，IL-1β，IL-6)，趋化因子(KC)和抗炎细胞因子(IL-10)表达的影响。
收集的BALF分别通过ELISA试剂盒分析TNF-α(A)，IL-1β(B)，IL-6(C)，KC(D)和IL-10(E)的表达水平。与对照(Ctrl)组相比，##p<0.01；*p<0.05和**p<0.01与模型组(model)相比。数值表示为平均值±SEM；n＝12(每组)。
图12为CT-NA对CS诱导的ALI小鼠肺组织的组织病理学变化的影响。
将每个实验组的石蜡包埋肺切片用苏木精-伊红染色进行组织病理学评估。(A)用H&E染色的肺组织的代表性图像，以证明巨噬细胞，嗜中性粒细胞和炎症细胞的浸润。(B)定量分析炎症细胞的浸润和根据肺切片判断炎症的严重程度。与对照(Ctrl)组相比，##p<0.01；*p<0.05和**p<0.01与模型组(model)相比。数值表示为平均值±SEM；n＝12(每组)。
图13为CT-NA对肺MPO活性和PGD 2诱导的体外嗜中性粒细胞迁移的影响，评估CSE诱导的PGD 2分泌的原代巨噬细胞。
(A)使用MPO试剂盒测试肺匀浆中MPO的活性。数值表示为平均值±SEM；n＝12(每组)。在不使用CT-NA(B)和使用CT-NA(C)下通过Boyden室检测试剂盒(3μm孔尺寸)检测PGD 2诱导的中性粒细胞迁移。(D)使用不同浓度的CSE(2％，4％和8％)处理分离的原代巨噬细胞24小时，然后通过PGD 2 ELISA试剂盒评估分泌细胞外PGD 2的蛋白质水平。##与对照(Ctrl)组相比p<0.01；*p<0.05且**p<0.01与模型组(model)相比。所有实验在每个条件下，在三份孔中进行三次重复实验。数值表示为平均值±SEM。
图14为CT-NA对CSE(4％)-和PGD 2-诱导的促炎性细胞因子(TNF-α，IL-1β，IL-6)，趋化因子(KC)和RAW 264.7巨噬细胞产生的抗炎细胞因子(IL-10)的蛋白质水平的影响。
从RAW 264.7巨噬细胞中分离出的上清液用CT-NA预处理1小时，并用CSE/PGD 2处理24小时，然后根据说明书的方法分别使用ELISA试剂盒测量IL-1β(A和F)，TNF-α(B和G)，IL-6(C和H)，KC(D和I)和细胞外分泌的IL-10(E和J)的蛋白质水平。##p<0.01，与对照(Ctrl)组相比；*p<0.05且**p<0.01与PGD 2相比。所有实验在每个条件下，在三份孔中进行三次重复实验。数值表示为平均值±SEM。
图15为CT-NA对CSE(4％)-和PGD 2-诱导的促炎性细胞因子(TNF-α，IL-1β，IL-6)，趋化因子(KC)和RAW 264.7巨噬细胞产生的抗炎细胞因子(IL-10)的mRNA表达的影响。
从RAW 264.7巨噬细胞分离出的RNAs使用CT-NA处理1小时，然后用CSE/PGD 2处理24小时，分别通过RT-PCR分析IL-1β(A和F)，TNF-α(B和G)，IL-6(C和H)，KC(D和I)和IL-10(E和J)的mRNA表达水平。##p<0.01，与对照(Ctrl)组相比；*p<0.05且**p<0.01与PGD 2相比。所有实验在每个条件下，在三份孔中进行三次重复实验。数值表示为平均值±SEM。
图16为制备LPS诱导的ALI小鼠模型过程。在气管内滴注LPS前1小时和12小时后灌胃给予CT-NA(10和30mg/kg)或Dex(阳性对照；1mg/kg)。在LPS诱导后24小时，处死小鼠以制备BALF和肺组织样本。
图17为CT-NA对BALF炎症细胞计数和分类，氧饱和度(SO 2)和肺重量系数的影响。
对于急性肺损伤，(A)CT-NA(10和30mg/kg)或Dex(1mg/kg)在气管内滴注LPS1小时前和12小时后灌胃给予。在LPS诱导后24小时收集BALF以计算细胞总数，嗜中性粒细胞，巨噬细胞和淋巴细胞的数量(细胞总数中从左到右分别为载体生理盐水组、载体LPS诱导组、LPS诱导+CT-NA 10mg/kg组、LPS诱导+CT-NA30mg/kg组及LPS诱导+地塞米松(Dex)1mg/kg组，其他几种检测结果的表述与细胞总数中相同)。(B)在LPS诱导后24小时，使用moor VMS-OXY TM监测仪测量所有小鼠的氧饱和度(SO 2)。(C)LPS诱导后24小时，肺重量系数通过将每只小鼠的个体肺重量除以其体重来测量。载体生理盐水组##P<0.01；载体LPS诱导组，*P<0.05，**P<0.01。数值表示为平均值±S.E.M.，每组n＝12。
图18为CT-NA对LPS诱导的ALI小鼠的BALF中促炎细胞因子(TNF-α，IL-1β，IL-6)和趋化因子(KC)产生的影响。收集BALF并分别使用对应的ELISA试剂盒分析TNF-α(A)，IL-1β(B)，IL-6(C)和KC(D)水平。载体+载体组##P<0.01；载体LPS诱导组*P<0.05，**P<0.01。数值表示为平均值±S.E.M.,每组n＝12。
图19为CT-NA对LPS诱导的ALI小鼠肺组织病理学改变的影响。每个实验组石蜡包埋的肺切片用H&E染色用于组织病理学分析。(A)用H&E染色的肺组织的代表性图像显示出水肿、嗜中性粒细胞和炎性细胞的浸润。(B)由盲选的两位具有肺部专业知识的病理学家对肺损伤进行定量分析病理。载体+载体组##P<0.01；载体LPS诱导组*P<0.05，**P<0.01。数值表示为平均值±S.E.M.，每组的n＝12。
图20为CT-NA对LPS诱导的肺血管通透性的影响。LPS诱导LPS诱导LPS诱导后24小时，(A)使用白蛋白测定试剂盒测量BALF中的白蛋白。(B)将伊文思蓝染料(50mg/kg)注入所有小鼠的尾静脉，1小时后安乐死。通过伊文思蓝染料在肺组织中的积累确定肺血管通透性。载体+载体组##P<0.01；载体LPS诱导组*P<0.05，**P<0.01。数值表示为平均值±S.E.M.，每组的n＝12。
图21为CT-NA对肺MPO活性、体外PGD 2诱导的嗜中性粒细胞迁移的影响，和评估LPS诱导的原代巨噬细胞中PGD 2的分泌。(A)使用MPO试剂盒测量肺匀浆的MPO活性。载体+载体组##P<0.01；载体LPS诱导组**P<0.01。数值表示为平均值±S.E.M.，每组n＝12。(B和C)使用Boyden室检测试剂盒(3μm孔径)评估使用或不使用CT-NA情况下PGD 2诱导的 嗜中性粒细胞迁移。载体+载体组##P<0.01；载体组或PGD 2+载体组**P<0.05，**P<0.01。数值表示为平均值±S.E.M.，每组n＝3。(D)分离的原代巨噬细胞用不同浓度的LPS(0.01,0.1,1和10μM)预处理24h后收集上清液，用PGD 2ELISA试剂盒测定PGD 2蛋白水平。载体组##P<0.01。数值表示为平均值±S.E.M.，每组n＝3。
图22为CT-NA对促炎细胞因子(TNF-α，IL-1β，IL-6)和从LPS-或PGD 2刺激的RAW264.7巨噬细胞分泌的趋化因子(KC)的影响。用CT-NA预处理RAW264.7巨噬细胞1小时，并进一步用CT-NA和LPS/PGD 2处理24小时，然后收集培养基分别使用对应ELISA试剂盒测量IL-1β(A和E)，TNF-α(B和F)，IL-6(C和G)和KC(D和H)分泌水平。载体+载体组##P<0.01；LPS/PGD 2+载体组，*P<0.05，**P<0.01。数值表示为平均值±S.E.M.，每组n＝3。
图23为CT-NA对促炎细胞因子(TNF-α，IL-1β，IL-6)和LPS-或PGD 2刺激的RAW264.7巨噬细胞分泌的趋化因子(KC)mRNA表达的影响。用CT-NA预处理RAW264.7巨噬细胞1小时，并进一步用CT-NA和LPS/PGD 2处理24小时，然后提取RNA，使用RT-PCR分析IL-1β(A和E)，TNF-α(B和F)，IL-6(C和G)和KC(D和H)的表达。对照组##P<0.01；模型组*P<0.05，**P<0.01。载体+载体组##P<0.01；LPS/PGD 2+载体组，*P<0.05，**P<0.01。数值表示为平均值±S.E.M.，每组n＝3。
图24为CT-NA对促炎细胞因子(TNF-α，IL-1β，IL-6)、LPS和PGD 2刺激的原代巨噬细胞分泌的趋化因子(KC)mRNA表达的影响。将原代巨噬细胞用CT-NA预处理1小时，并进一步用CT-NA和LPS/PGD 2处理24小时，然后提取总RNA，使用RT-PCR分析IL-1β(A和E)，TNF-α(B和F)，IL-6(C和G)和KC(D和H)的表达。载体+载体组##P<0.01；LPS/PGD 2+载体组*P<0.05，**P<0.01。数值表示为平均值±S.E.M.，每组n＝3。
图25为CT-NA对NF-κB在RAW 264.7巨噬细胞或肺中的LPS刺激信号通路激活响应的影响。(A)RAW264.7巨噬细胞在LPS(100ng/ml)处理前1小时用CT-NA(0.5,1,10和100μM)预处理1小时。(B)将保存的肺组织在RIPA缓冲液中匀浆以提取总蛋白。用指定的抗体对蛋白质进行蛋白质印迹分析。使用β-肌动蛋白作为内部控制。所有实验至少重复三次。
DSC-TGA检测项目仪器名称及型号：同步热分析仪(STA449F3)20℃升至350℃。
制备例2中XRPD检测项目仪器名称及型号：AFC10/Saturn724+Rigaku型X射线衍射分析仪。
制备例3-5中XRPD检测项目仪器名称及型号：D2PHASER X射线衍射仪(德国布鲁克)。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。
制备例1、式A化合物的制备
Figure PCTCN2020091842-appb-000007
参考专利文献CN101896178B中实施例3和4所记载的方法，将式A所示杂环化合物消旋体(20g)在Chiralcel OJ-RH柱(Chiralcel Technologies)上用含0.05％TFA的甲醇洗脱，分离得到式A所示杂环化合物(6.24g，收率为31.2％)。
1H NMR(600MHz,CD 3OD):8.14-8.15(m,1H),8.00-8.03(m,2H),7.92-7.94(m,1H),7.38-7.42(m,2H),7.10-7.13(m,1H),4.50-4.54(m,1H),4.38-4.40(m,1H),3.92-3.96(m,1H),3.63-3.72(m,2H),3.17-3.22(m,1H),2.85-2.95(m,4H),1.99-2.02(m,1H),1.76-1.79(m,1H).
制备例2、式A化合物一水合物晶型的制备
Figure PCTCN2020091842-appb-000008
参考专利文献CN101896178B中实施例3和4所记载的方法，将式A杂环化合物消旋体(0.500g)在Chiralcel OJ-RH柱(Chiralcel Technologies)上用含0.05％TFA的甲醇洗脱，收集洗脱液浓缩至干，得到产物约0.2g，其为无定型态。向浓缩物中加入丙酮(2.5mL)和水(2.5mL)，40-50℃加热溶解后降至0～10℃，搅拌析晶2～3h，过滤得到式A所示杂环化合物晶型(0.156g)，收率为31.2％。所述晶型的XRPD检测结果如图1所示，其DSC-TGA检测结果如图2所示，由图2的DSC图可知，其分别在86.4℃和130.4℃出现吸热峰，TGA热失重图上显示失重为4.29％。由该晶型的DSC-TGA图谱表明，该晶型为一水合物。图3为所得晶型的三维结构图和晶胞图。
1H NMR(600MHz,CD 3OD):8.13-8.15(m,1H),8.00-8.04(m,2H),7.92-7.95(m,1H),7.37-7.42(m,2H),7.09-7.14(m,1H),4.50-4.55(m,1H),4.38-4.42(m,1H),3.91-3.97(m,1H),3.61-3.71(m,2H),3.17-3.23(m,1H),2.85-2.95(m,4H),1.97-2.02(m,1H),1.75-1.79(m,1H).
制备例3、式A化合物钠盐水合物(CT-NA)的制备
Figure PCTCN2020091842-appb-000009
将化合物A(17.6g)用丙酮(210mL)溶解，升温至50℃搅拌至溶解，向该溶液中滴加氢氧 化钠水溶液(将1.5g氢氧化钠溶于4mL纯化水中配制)，滴加完毕后，50℃保温搅拌4h。过滤，将滤饼在50℃真空减压干燥6h，得化合物A的钠盐水合物(15.8g，收率82％)。所得钠盐呈现良好的结晶性，其XRPD表征数据如图4所示。DSC-TGA检测结果如谱图5所示，由图5的DSC图可知，其分别在130.2℃和176.6℃出现吸热峰，TGA热失重图上显示失重为3.99％。由该晶型的DSC-TGA图谱表明，该晶型为一水合物。
1H NMR(600MHz,CD 3OD):8.08-8.10(m,1H),7.96-8.02(m,3H),7.37-7.41(m,2H),7.05-7.08(m,1H),4.50-4.52(m,1H),4.37-4.41(m,1H),3.89-3.93(m,1H),3.46-3.55(m,2H),3.17-3.22(m,1H),2.93(s,3H),2.90-2.92(m,1H),1.96-2.00(m,1H),1.70-1.73(m,1H).
制备例4、式A化合物钾盐水合物的制备
Figure PCTCN2020091842-appb-000010
将化合物A(50.0g)用丙酮(600mL)溶解，室温下向反应液中缓慢滴加氢氧化钾水溶液(6.4g氢氧化钾加入到12mL纯化水中配制而成)，室温下搅拌反应3h，抽滤，50℃真空减压干燥6h，得化合物A的钾盐水合物(36.4g，收率64％)。所得钾盐呈现良好的结晶性，其XRPD谱图如图6所示，其DSC-TGA如图7所示。由图7的DSC图可知，其在64.1℃和270.4℃分别出现吸热峰，TGA热失重图上显示失重为3.82％。由该晶型的DSC-TGA图谱表明，该晶型为一水合物。
1H NMR(600MHz,DMSO-d 6):7.98-8.05(m,3H),7.80-7.83(m,1H),7.48-7.52(m,2H),6.94-6.97(m,1H),4.30-4.33(m,1H),4.16-4.20(m,1H),3.81-3.86(m,1H),3.06-3.17(m,3H),2.83(s,3H),2.76-2.82(m,1H),1.82-1.90(m,1H),1.55-1.58(m,1H).
制备例5、式A化合物锂盐水合物的制备
Figure PCTCN2020091842-appb-000011
将化合物A(50.0g)用丙酮(900mL)溶解，室温下向反应液中缓慢滴加氢氧化锂(2.75g氢氧化锂加入到18mL纯化水中配制而成)水溶液，室温下搅拌反应3h，抽滤，将滤饼在50℃真空减压干燥6h，得化合物A的锂盐水合物(26.0g，收率49％)。所得锂盐呈现良好的结晶性，其XRPD谱图如图8所示，其DSC-TGA如图9所示。由图9的DSC图可知，其在138.8℃和180.7℃分别出现吸热峰，TGA热失重图上显示失重为4.47％。由该晶型的DSC-TGA图谱表明，该晶型为一水合物。
1H NMR(600MHz,DMSO-d 6):7.98-8.06(m,3H),7.81-7.83(m,1H),7.47-7.52(m,2H),6.91-6.94(m,1H),4.28-4.34(m,1H),4.15-4.19(m,1H),3.80-3.84(m,1H),3.18-3.26(m,2H),3.05-3.08(m,1H),2.82(s,3H),2.77-2.81(m,1H),1.80-1.87(m,1H),1.53-1.56(m,1H).
测试例1、溶解度测试
将如上所得水合物与式A化合物在不同pH的溶液中进行溶解度测试，测试方法为：
1.1、不同pH介质的配制
pH1.0介质：取盐酸9.0mL，加水稀释至1000mL，摇匀，即得。
pH4.5介质：取磷酸二氢钾(KH 2PO 4)6.80g加水适量使溶解并稀释至1000mL，用磷酸或氢氧化钠调节pH值至4.5，摇匀，即得。
pH6.8介质：取磷酸氢二钠(Na 2HPO 4·12H 2O)55.38g与枸橼酸(C 6H 8O 7·H 2O)4.77g，加水适量使溶解并稀释至1000mL，用磷酸或氢氧化钠调节pH值至6.8，摇匀，即得。
纯水介质：纯化水
1.2、试验方法：取一定量的供试样品，分别逐量加入相应pH的介质，不断震摇，直至达到饱和状态，记录供试品称样量及溶剂用量，记算溶解样品时的浓度，测试结果如表1所示：
表1 化合物A、晶型及其盐在水中的溶解度(单位：mg/mL)
Figure PCTCN2020091842-appb-000012
由表1结果可知，钠盐水合物在不同pH下的水溶性最好，锂盐水合物与钠盐水合物基本相当，钾盐水合物在pH4.5时与钠盐水合物和锂盐水合物相当，但其它pH和纯化水中均差于钠盐水合物和锂盐水合物，游离酸(化合物A)在不同pH下的水溶性均较差。所得三种盐的水合物中，特别是钠盐和锂盐水合物的溶解性能明显优于化合物A。
同时制备例2的化合物A的一水合物晶型在不同的pH中均具有良好的溶解度。
测试例2、溶出度测试
将如上所得水合物与式A化合物进行溶出度测试，测试方法为：
步骤1：原料预处理
将样品过100目筛。
步骤2：原辅料混合
按处方量称取原料和乳糖，等量递加混合，然后再过筛混合。
Figure PCTCN2020091842-appb-000013
步骤3：胶囊灌装
用胶囊填充板灌装胶囊，锁扣，控制装量差异为：±5％。进行外观检测，应锁口到位、无叉口或凹顶现 象。
检查方法：溶出度测定法(中国药典2015年版四部通则0931第二法)
溶出介质：水溶液
转速：50rpm
取样时间：30min
具体试验方法：取本品，照溶出度测定法(中国药典2015年版四部通则0931第二法)，以水溶液900ml为溶出介质，转速为50rpm，依法操作，经30min时，取溶液约10ml，滤过，精密量取续滤液适量，用溶出介质定量稀释制成每1ml中约含样品10μg的溶液，照紫外-可见分光光度法(中国药典2015年版四部通则0401)，于231nm波长处测定吸光度；另取样品对照品适量，精密称定，加溶出介质溶解并定量稀释制成每1ml中约含样品10μg的溶液，同法测定，计算每粒的溶出量。
结果见表2-表4。
表2 制备例3化合物A钠盐水合物在水中的溶出度
Figure PCTCN2020091842-appb-000014
表3 制备例4化合物A钾盐水合物在水中的溶出度
Figure PCTCN2020091842-appb-000015
表4 制备例5化合物A锂盐水合物在水中的溶出度
Figure PCTCN2020091842-appb-000016
采用上述相同的方法测试化合物A的溶出度，因化合物A水溶性较差，其溶出度基本为零。
基于上述内容可知，本发明所得三种盐的水合物均表现出较好的溶出度，其中钠盐水合物的溶出度最好。
测试例3、稳定性测试
试验过程：取各供试品适量置于干净的表面皿上，敞口放置，于光照4500lx+500lx、高温60℃、高湿92.5％RH条件下分别放置5天、10天，测定性状、有关物质，并与0天结果对比，考察稳定性。
有关物质检查方法如下：
供试品溶液的制备：取各供试品约10mg，置10ml量瓶中，加50％乙腈溶解并稀释至刻度，摇匀，滤过，作为供试品溶液。精密量取供试品溶液10μl，按上述色谱方法进样，按面积归一化法计算最大单杂和总杂。
试验结果：
制备例1式A化合物影响因素试验结果
Figure PCTCN2020091842-appb-000017
Figure PCTCN2020091842-appb-000018
制备例4钾盐水合物影响因素试验结果
Figure PCTCN2020091842-appb-000019
制备例5锂盐水合物影响因素试验结果
Figure PCTCN2020091842-appb-000020
Figure PCTCN2020091842-appb-000021
由上述结果可知，在高温、高湿条件下，式A化合物、本申请所得钠盐、钾盐和锂盐水合物均较为稳定；而在光照条件下，式A化合物降解明显，而其成盐后得到的化合物对光照的稳定性显著增强。由此可见，钠盐、锂盐、钾盐的稳定性明显优于式A化合物。
同时对制备例2的式A化合物一水合物的晶型的稳定性测试结果表明，表明式A化合物的晶型在高温、高湿下具有良好的稳定性。最大单杂不超过0.1％，而总杂不超过0.4％。并且发明人发现，所述式A化合物的晶型在光照条件下的稳定性虽差于高温、高湿下的稳定性，但仍具有相对良好的稳定性。
测试例4引湿性测试
将制备例2所得式A化合物的一水合物晶型进行引湿性测试；
测试方法为：
检查方法：中国药典方法(中国药典2015年版四部通则9103)
试验条件：
操作温度：25℃±1℃
干燥器湿度：80％±2％RH。
具体试验操作：将干燥的扁形称量瓶在恒温干燥器(下部放置氯化铵或硫酸铵饱和溶液)中放置24h后，取实施例1所得晶型1g平铺于扁形称量瓶中，敞口并与瓶盖同置于干燥器中24h。
样品名称 增重(％) 结论
晶型 0.23 略有引湿性
由上述结果可知，式A化合物的一水合物晶型在高湿条件下，略有引湿性，说明式A化合物的一水合物晶型稳定性较高。
实施例1
将制备例3制备的式A化合物钠盐水合物CT-NA用于活性测试。测试方法如下。
1.1小鼠处理
特异性无病原体(SPF)雌性Balb/c小鼠(22-28克；8周)购自上海SIPPR-BK实验动物有限公司。小鼠在隔离的通风笼中(4-5只小鼠/笼)，在40-60％湿度，24±2℃，按照12小时/12小时暗-光交替的环境中自由进食和饮水。
1.2香烟烟雾(CS)诱导的肺损伤模型的制备和测量氧分压
模型制备：小鼠随机分为5组(每组12只)，分别为对照组(小鼠暴露在新鲜空气中)、生理盐水组(小鼠暴露在香烟烟雾中)、地塞米松(Dex)组(1mg/kg)(小鼠暴露在香烟烟雾中)、CT-NA 10mg/kg组和CT-NA30mg/kg组(小鼠暴露在香烟烟雾中)。根据分组情况分别于小鼠灌胃给予生理盐水、Dex和CT-NA。之后，将小鼠暴露于在新鲜空气中或香烟烟雾中。香烟烟雾由3R4F研究级卷烟(含约600mg TPM/m 3和29.9mg尼古丁/m 3)在方形塑料箱(65×50×45cm)中按照每天10支，每支燃烧完成后点燃下一支的频率燃烧，共连续重复7天。检查小鼠每天的体重和常规状况。在完成最后一次暴露于香烟气氛中后24小时，使用moor VMS-OXY TM测量仪测量所有小鼠的氧分压(PO 2)，用于测量在500至650nm的波长范围内含氧/脱氧血红蛋白的微循环中的浓度和氧饱和度(百分比)。之后，所有老鼠被安乐死以收集支气管肺泡灌洗液(BALF)用于测量炎症细胞总数，细胞因子水平和白蛋白浓度。并收集肺部组织以测定肺重量系数，组织检测和MPO活性。
1.2.1炎症细胞计数
将小鼠安乐死后手术暴露气管，然后对右肺用0.4mL/次含有1％FBS和5000IU/L肝素的无菌生理盐水灌洗三次，通过气管导管以收集BALF。用血细胞计数器测量BALF中的细胞总数后，将剩余的BALF在1000×g，4℃下离心10分钟。将上清液等分并且储存在-80℃用于下一步测量细胞因子或白蛋白浓度。将获得的细胞沉淀涂在载玻片上。之后，根据中性粒细胞，巨噬细胞和淋巴细胞的形态学标准，在光学显微镜下对涂片进行Wright-Giemsa染色以计数200个细胞。
1.2.2肺重量比
作为肺水肿的指标，肺重量比为每只小鼠吸去肺表面血液组织后的个体肺重量，除以总体重。
1.2.3白蛋白测定
使用白蛋白测定试剂盒及分光光度计在628nm处测试BALF上清液中的白蛋白浓度。使用BALF测定的白蛋白浓度比率不仅代表白蛋白水平，也代表肺微血管通透性。
1.2.4通过ELISA进行体内细胞因子测定
根据使用说明书分别采用ELISA测试套装测定BALF上清液中促炎性细胞因子(TNF-α，IL-1β，IL-6)，趋化因子(KC)和抗炎细胞因子(IL-10)的表达水平。测量在450nm处的光密度后，通过标准曲线计算细胞因子的表达。
1.2.5肺组织病理学
将每只小鼠左肺下叶保存在10％中性福尔马林中用于组织病理学检查。将保存的左肺下叶取出包埋在石蜡中，然后切片(4μm)以暴露主要肺内支气管的最大纵向视图。使用标准方法进行苏木精和伊红(H&E)染色。使用5点评分系统来评价肺水肿，炎症的严重程度和炎症细 胞的浸润程度。简单来说，得分系统是，0＝正常；1＝非常轻微；2＝轻微；3＝中等；4＝显著；5＝严重。每个肺截面按照至少三个不同的视野进行评分。取12只小鼠得分的平均值。
1.2.6 MPO测定
为了评估MPO活性，取50mg左肺条状组织进行清洗，然后用生理盐水匀浆。使用MPO测定试剂盒，根据检测标准通过测量吸光度在460nm处的变化来确定MPO的活性。
1.2.7嗜中性粒细胞的分离和测试CT-NA对PGD 2诱导的嗜中性粒细胞迁移的影响
小鼠灌胃给予20ml/kg的糖原(1.5％)。4小时后对小鼠实施安乐死，从腹膜灌洗中分离嗜中性粒细胞。CT-NA对嗜中性粒细胞迁移的影响使用Boyden腔室检测试剂盒(3μm孔径)检测，PGD 2用作化学引诱剂，因为活化的PGD 2/CRTH2受体促进嗜中性粒细胞的迁移。最初，分离的嗜中性粒细胞(4×10 5)稀释在100μLHBSS中使其向PGD 2迁移(0.1,1和10μM)4小时以找出合适的PGD 2浓度。然后，使用CT-NA(1和10μM)预处理分离的嗜中性粒细胞(4×10 5)，它们向PGD 2(1μM)的迁移通过计算迁移的嗜中性粒细胞评估。而且，我们还使用了另一个有效的CRTH2抑制剂OC459，以复查CT-NA的检测结果。
1.2.8香烟烟雾提取物(CSE)的制备
使用真空泵输送3R4F研究级香烟产生的香烟烟雾通过50ml PBS。用五支香烟来制造通过50ml PBS的烟雾，每支香烟点燃5分钟。在没有香烟的情况下采用类似的方法制备对照溶液。烟雾萃取后完成后将CSE储存在-80℃下。
1.2.9原代巨噬细胞的分离和评估CSE诱导的从原代巨噬细胞分泌PGD 2
通过腹膜腔分离原代巨噬细胞，方法简单说明如下：巯基乙酸盐(4％)按照20ml/kg体重的剂量连续三天注入小鼠腹腔。在第5天(最后一次巯基乙酸盐注射后48小时)，小鼠安乐死以便从腹膜灌洗中分离出原代巨噬细胞。将分离的原代巨噬细胞(4×10 5/孔)加入12孔板并在37℃下培养。之后，将12孔板的培养基用无血清RPMI-1640培养基替换，并培育10-12小时，然后暴露于不同浓度的CSE(2％，4％，和8％)下24小时。处理完成后，收集原代巨噬细胞的上清液，根据说明书的方法，使用ELISA试剂盒测量分泌的细胞外PGD 2的蛋白质水平。
1.2.10细胞活性测试
RAW 264.7巨噬细胞，小鼠白血病单核巨噬细胞，细胞系购自美国典型培养物保藏中心(ATCC，马纳萨斯，弗吉尼亚州，美国)。RAW 264.7巨噬细胞在RPMI-1640培养基中培养，该培养基中含有青霉素(100U/ml)、链霉素(100μg/ml)及10％FBS。单独使用CT-NA(0-100μM)，及其与PGD 2(0-100μM)、CSE组合后测定对RAW264.7巨噬细胞(1-10％)的毒性，根据标准方法，使用甲基噻唑-四唑(MTT)测定评估。简而言之，将RAW 264.7巨噬细胞以4×10 5个细胞/ml的浓度接种在96孔板中24小时，随后在37℃下暴露于CT-NA(0-100μM)1小时。接下来，将RAW 264.7巨噬细胞进一步暴露于CSE(4％)和PGD 2(10μM)24小时，随后用MTT(5mg/ml)在37℃下处理4小时。然后，用DMSO(200μl/孔)更换每个孔的上清液，测定在570nm处的吸光度。
1.2.11通过ELISA和实时聚合酶链反应(RT-PCR)进行体外细胞因子测定
将RAW 264.7巨噬细胞加入到两个12孔板。之后，12孔板的培养基用无血清RPMI-1640培养基替换，培育10-12小时后暴露于CT-NA(10和100μM)1小时。1小时后，一个12孔板用CSE(4％)处理，另一个用PGD 2(10μM)处理24小时。处理后，收集处理细胞的上清液，根据说明书的方法，使用ELISA试剂盒测量TNF-α，IL-1β，IL-6，KC和细胞外分泌的IL-10的蛋白质水平。随后，来自每个处理过的板的RNA样品用HiScript5×QRTSuperMix提取并逆转录成cDNA，然后进行RT-PCR。RT-PCR使用BioRad CFX96 Touch TM实时PCR检测系统(BioRad，美国)进行处理，这个系统使用
Figure PCTCN2020091842-appb-000022
qPCRSYBR Green Master Mix。而阈值循环数是使用BioRad CFX Manager软件获得的。用于RT-PCR反应的引物如表1所示。β-actin用作内部对照。RT-PCR反应重复三次。靶mRNA的相对表达通过各自的β-actin矫正。
表1
Figure PCTCN2020091842-appb-000023
1.2.12统计分析
数据为平均值±SEM。统计学使用SPSS(SPSS Inc.，Chicago，IL)进行计算。采用单因素ANOVA方法比较F值，如果p>0.05，则采用Dunnett multiple comparisons teat计算参数数据的差异；如果p<0.05，则用Mann-Whitney U non-parametric test比较差异。p<0.05和p<0.01被认为是具有统计学意义。
2.1 CT-NA在BALF中对CS诱导的炎症细胞数量的影响
在最后一次暴露于CS后24小时，CT-NA对BALF中总细胞和不同细胞的浸润，尤其是嗜中性粒细胞和巨噬细胞的影响通过Wright-Giemsa染色方法分析。如图10A和B所示，CS暴露后，总细胞数，巨噬细胞数和嗜中性粒细胞数显著增加(p<0.01)。同时，用CT-NA(10和30mg/kg)和Dex(1mg/kg)预处理显著降低总细胞数，巨噬细胞数和嗜中性粒细胞数(p<0.01)。这些显著的效果证明CT-NA可以通过CRTH2拮抗作用显著改善CS诱导的肺部炎症。
2.2 CT-NA对CS诱导的低氧血症，肺水肿和肺通透性的影响
CS诱导的低氧血症，肺水肿，肺通透性分别通过测量氧分压(PO 2)，肺重量系数、和BALF白蛋白含量来评估。在CS处理组，PO 2明显降低(p<0.01)，然而与对照组相比，肺重量系数和BALF白蛋白含量显著增加(p<0.01)，表明CS诱导的动物模型成功。但是，CT-NA(10和30mg/kg)组PO 2显著升高(p<0.01)(图10C)，肺重量系数部分降低(p<0.05)(图10D)，并显著减弱BALF白蛋白含量(p<0.01)(图10E)。这些优异的结果表明的CRTH2抗体CT-NA通 过减轻低氧血症，肺通透性和水肿，可以有效地保护小鼠免受CS诱导的肺损伤。
2.3 CT-NA对BALF中CS诱导的细胞因子分泌的影响
为了确定CT-NA是否可以影响BALF中细胞因子的分泌，分别使用ELISA试剂盒检测促炎细胞因子(TNF-α，IL-1β，IL-6)，趋化因子(KC)和抗炎细胞因子(IL-10)的表达水平。如图11A，B，C和D所示，CS暴露组的TNF-α，IL-1β，IL-6和KC的表达水平与对照组相比显著增加(p<0.01)。同时，CS诱导的TNF-α，IL-1β，IL-6和KC的过度表达通过CT-NA处理可以有效地减少(10和30mg/kg)(p<0.01)。相比之下，CS暴露组中IL-10的表达水平显著降低(p<0.01)，而CT-NA处理逆转了CS诱导的IL-10抑制作用(p<0.01)(图11E)。这些结果表明CRTH2受体被CT-NA阻断，从而通过抑制促炎性细胞因子、嗜中性粒细胞趋化因子的产生，和刺激抗炎细胞因子(IL-10)的产生来保护CS诱导的ALI小鼠免于进一步的肺部炎症。
2.4 CT-NA对CS诱导的肺组织病理学改变的影响
为了评估CT-NA对CS诱导的肺组织病理学变化的保护作用，进行了H&E染色实验。如图12A所示，对照组的肺组织显示正常肺组织学，而CS暴露组的肺组织显示明显组织病理学变化，如炎症细胞和巨噬细胞的浸润，及嗜中性粒细胞进入肺泡腔和间质浮肿。相反，通过使用CT-NA(10和30mg/kg)或Dex(1mg/kg)预处理，这些变化显著改善。此外，还进行了病理评分以确定炎症，炎症细胞浸润和肺水肿的严重程度。肺部炎症评分结果显示，暴露在CS下显著增加平均病理评分(p<0.01)。而CT-NA(10和30mg/kg)和Dex(1mg/kg)以剂量依赖的方式显著降低平均病理评分(p<0.01)(图12B)。检测结果显示CT-NA通过阻断CRTH2受体显著减轻CS引起的肺损伤的严重程度。
2.5 CT-NA对CS诱导的MPO活性的影响
由于上述良好的检测结果，我们进一步评估了肺组织的MPO活性。由活化的嗜中性粒细胞产生的MPO，是嗜中性粒细胞浸润和肺组织损伤的重要标志物。我们发现肺组织的MPO活性暴露在CS中与暴露在新鲜空气中相比显著增加(p<0.01)(图13A)。值得注意的是，CT-NA(10和30mg/kg)和Dex(1mg/kg)减弱MPO活性(p<0.01)，表明CRTH2受体阻滞可有效抑制嗜中性粒细胞侵入肺泡和间隙。
2.6.CT-NA对PGD 2诱导的体外嗜中性粒细胞迁移的影响
基于MPO的检测结果，我们进一步使用Boyden室检测试剂盒评估了CT-NA对PGD 2诱导的嗜中性粒细胞迁移的直接影响，因为激活的PGD 2/CRTH2受体促进嗜中性粒细胞的迁移及其功能。此外，嗜中性粒细胞释放的炎症介质极可能加剧肺损伤。从小鼠中腹腔分离出的嗜中性粒细胞用1.5％糖原攻击，然后进行Wright-Giemsa染色和细胞活力测定以检查嗜中性粒细胞的特征。孵育4小时显示出显著的嗜中性粒细胞向PGD 2的迁移(p<0.01)(1和10μM)(图13B)。同时，用CT-NA预处理(1和10μM)显著减弱PGD 2诱导的嗜中性粒细胞迁移(p<0.01)(图13C)。同样，另一种CRTH2拮抗剂OC459也抑制PGD 2诱导的嗜中性粒细胞迁移(图13C)。总之，这些数据清楚地体现CRTH2拮抗剂明显减少PGD 2诱导的嗜中性粒细胞的迁移。
2.7 CSE促进原代巨噬细胞分泌PGD 2
为了测试CSE是否影响PGD 2的分泌，我们使用不同浓度的CSE(2％，4％和8％)处理分离的原代巨噬细胞24小时，然后通过PGD 2ELISA试剂盒评估分泌细胞外PGD 2的蛋白质水平。我们发现与对照组相比使用CSE(4％)处理显著促进了PGD 2的分泌(p<0.01)(图13D)。
2.8 CT-NA对CSE-和PGD 2-诱导的RAW 264.7巨噬细胞因子分泌的影响
基于显著的体内测试结果，我们进一步考虑CT-NA处理是否能抑制CSE-和PGD 2-刺激的RAW 264.7巨噬细胞细胞因子的分泌，因为激活的巨噬细胞上的PGD 2/CRTH2受体通过增加促炎细胞因子的表达来显著增加疾病活性。MTT测定显示PGD 2(10μM)加浓度高达100μM的CT-NA，和CSE 4％加浓度高达100μM的CT-NA对RAW 264.7巨噬细胞无毒性。此外，ELISA(图14)和RT-PCR(图15)结果证明CT-NA(10和100μM)处理不仅可以抑制由CSE-及PGD 2-刺激的RAW 264.7巨噬细胞产生的IL-1β，IL-6，TNF-α和KC的蛋白和mRNA水平，(p<0.01)，而且以剂量依赖性的方式逆转CSE-和PGD 2-诱导的IL-10抑制(p<0.01)。因此，体外获得的检测结果(图14和15)与体内获得的检测结果相似(图11)的检测结果。总的来说，这些数据表明CRTH2拮抗作用有效地改善了促炎性细胞因子和趋化因子的产生，促进来自CSE-和PGD 2-激活的RAW 264.7巨噬细胞产生抗炎细胞因子。
实施例2
2.1.1小鼠处理、制备LPS诱导的ALI模型和测量氧饱和度
特异性无病原体(SPF)Balb/c小鼠(♂/♀；20-26克；8周)购自上海SIPPR-BK实验动物有限公司。小鼠在隔离的通风笼中，在40-60％湿度，24±2℃，按照12小时/12小时暗-光交替的环境中自由进食和饮水。制备LPS诱导的ALI模型时，方法简单说明如下：将小鼠随机分为对照组(12只小鼠)和LPS组(48只小鼠)。LPS组(48只小鼠)进一步分为四个亚组(每组包含12只小鼠)。LPS四个亚组的未麻醉小鼠分别灌胃给予生理盐水(NS)，10mg/kg CT-NA、30mg/kg CT-NA和1mg/kg Dex。1小时后，小鼠用戊巴比妥钠麻醉(腹腔注射40mg/kg)，然后气管内滴注NS至对照组和LPS(4mg/kg)至所有LPS亚组。12小时后，LPS组的未麻醉小鼠分别给予NS、10mg/kgCT-NA、30mg/kg CT-NA和1mg/kg Dex(如图16)。生理盐水和LPS均以10μl/10克体重的方式施用。LPS诱导后24小时，使用moor VMS-OXY TM测量仪通过测量在500至650nm波长范围内微循环中氧饱和度(％)来测量所有小鼠的氧饱和度。SO 2代表血液中的氧合血红蛋白对总血红蛋白的百分比。在测量完SO 2后，收集每只小鼠的BALF用于炎症细胞计数和分类以及白蛋白浓度和促炎细胞因子/趋化因子水平的测定。此外，肺也用于组织学检查，测定肺重量系数和MPO活性。
2.1.2细胞计数和肺重量系数
将小鼠安乐死以暴露气管，然后对右肺用0.4mL/次含有牛血清白蛋白(BSA)和5000IU/L肝素的无菌生理盐水灌洗三次，通过气管导管以收集BALF。用血细胞计数器测量BALF中的细胞总数后，将剩余的BALF在1000×g，4℃下离心10分钟。将上清液等分并且储存在-80℃用于下一步测量促炎细胞因子或白蛋白浓度。将获得的细胞沉淀涂在载玻片上。之后，根据嗜中性粒 细胞，巨噬细胞和淋巴细胞的形态学标准，在光学显微镜下对涂片进行Wright-Giemsa染色以计数200个细胞。取出的肺组织吸出表面血液后称重，计算肺重量系数。肺重量系数为肺水肿的指标，其计算方式为每只小鼠的个体肺重量吸去肺表面血液组织后的个体肺重量除以总体重。
2.1.3通过ELISA评估体内促炎细胞因子和趋化因子
根据使用说明书分别采用对应的ELISA试剂盒测定BALF中促炎细胞因子(TNF-α，IL-1β，IL-6)，趋化因子(KC，小鼠IL-8同源物)的表达水平。测量在450nm处的光密度后，通过标准曲线计算表达水平。
2.1.4病理学测试
将每只小鼠左肺下叶保存在10％中性福尔马林中用于组织病理学检查，福尔马林按照在室温下在22至25cm H 2O的恒定压力下滴加48小时。将保存的左肺下叶取出包埋在石蜡中，然后切片(4μm)以暴露主要肺内支气管的最大纵向视图。然后使用H&E染色以评估肺水肿，和光学显微镜下观察嗜中性粒细胞和炎症细胞的浸润。对肺部水肿，出血，肺泡壁增厚、嗜中性粒细胞和炎症细胞的浸润进行计数和评分以评价肺损伤的严重程度。得分系统是，0＝正常；1＝非常轻微；2＝轻微；3＝中等；4＝显著；5＝严重。总肺损伤评分为四个标准的总和。取12只小鼠得分的平均值。
2.1.5体内肺血管通透性测试
伊文思蓝是一种快速与白蛋白结合并被限制在血管内的染料，因为内皮在正常生理条件下对白蛋白是不可渗透的。通过测量伊文思蓝染料在肺中的外渗来测量肺微血管通透性。方法简单描述如下：将小鼠随机分为对照组(12只小鼠)和LPS组(48只小鼠)。LPS组(48只小鼠)进一步分为四个亚组(每组包含12只小鼠)。为了测量肺微血管通透性，LPS四个亚组的未麻醉小鼠分别接受NS，10mg/kg CT-NA、30mg/kg CT-NA和1mg/kg Dex灌胃给药。1小时后对照组麻醉小鼠气管内滴注NS(对照组)和LPS(LPS亚组)，两者均以10μl/10克体重的方式施用。LPS诱导后24小时，将伊文思蓝染料(50毫克/千克)注入所有小鼠的尾静脉，1小时后安乐死。小鼠杀死后，NS缓慢注入到小鼠右心室，以排干肺部组织的血液。在室温下，小心地取出右肺，切片并置于甲酰胺(3ml/100mg)中。孵育24小时后，将样品以500×g离心10分钟(4℃)。通过标准曲线法测定620nm处的甲酰胺空白，测定在上清液中提取的伊文思蓝染料的吸光度。测量值表示为微克染料每100毫克湿肺重量。另外，在628nm使用分光光度计和白蛋白测定试剂盒进行测量BALF中的白蛋白浓度。使用BALF测定的白蛋白浓度比率不仅代表白蛋白水平，也代表肺微血管通透性。
2.1.6 MPO活性测定
MPO活性测定步骤如下：准确称取左肺条状组织使用匀浆培养基制备体积分数为5％匀浆(左肺条状组织和匀浆培养基的体积比为1:19)。然后将匀浆(0.9ml)和反应缓冲液(0.1ml)按照9:1的比例充分混合(如果没有足够的匀浆，则体积分数为5％匀浆和反应缓冲液可以按照9：1的比例相应减少)，然后在37℃培育15分钟。之后根据标准曲线使用分光光度计通过测量吸光度在460nm处的变化来测定MPO的活性。
2.1.7分离嗜中性粒细胞和评估PGD 2诱导的体外嗜中性粒细胞迁移
分离嗜中性粒细胞和测试CT-NA对嗜中性粒细胞迁移的影响的方法简单描述如下，将1.5％糖原按照20ml/kg体重的剂量胃内注射给小鼠。四小时后对小鼠实施安乐死，从腹膜灌洗中分离嗜中性粒细胞。CT-NA对嗜中性粒细胞迁移的影响使用Boyden室检测试剂盒(3μm孔径，Billerica,MA)检测，PGD 2用作化学引诱剂，因为活化的PGD 2/CRTH2受体促进嗜中性粒细胞的迁移。首先，将分离的嗜中性粒细胞按照4×10 5细胞/ml接种在Boyden室检测试剂盒孔的上侧，而下室含有不同浓度的PGD 2(0.1,1和10μM)，让嗜中性粒细胞37℃下向PGD 2迁移4小时，以便于找到出合适的PGD 2浓度。在测试出合适的PGD 2浓度后，测试使用CT-NA(1和10μM)和另一种有效的CRTH2抑制剂OC459(10μM)预处理的嗜中性粒细胞4小时向PGD 2(1μM)的迁移。
2.1.8分离腹腔巨噬细胞和评估LPS诱导的腹腔巨噬细胞分泌PGD 2
方法简单说明如下：巯基乙酸盐(4％)按照20ml/kg体重的剂量连续三天注入小鼠腹腔。最后一次巯基乙酸盐注射后48小时(在第5天)，小鼠安乐死以便从腹膜灌洗中分离出腹腔巨噬细胞。将分离的腹腔巨噬细胞加入12孔板(4×10 5/孔)并在37℃下培养。通过用热的PBS轻轻洗涤三次除去非粘附细胞。此时，超过90％的细胞是巨噬细胞，将其在37℃下，含有青霉素(100U/ml)、链霉素(100μg/ml)及10％FBS的DMEM/高葡萄糖培养基中培养。适应后，将无血清DMEM/高葡萄糖加入12孔板中10-12小时，然后用不同浓度的LPS(0.01,0.1,1和10μM)处理24小时。处理完成后，收集腹腔巨噬细胞的上清液，根据说明书的方法，使用ELISA试剂盒测量PGD 2的蛋白质水平。
2.1.9细胞培养和细胞活性测定
RAW 264.7巨噬细胞，小鼠白血病单核巨噬细胞，细胞系购自ATCC，(马纳萨斯，弗吉尼亚州)，并在RPMI-1640培养基中培养，该培养基中含有青霉素(100U/ml)、链霉素(100μg/ml)及10％胎牛血清。RAW264.7巨噬细胞是筛选抗炎药物和评估刺激促炎细胞因子和酶的产生抑制剂途径的理想模型。根据标准方法，使用MTT测定单独使用CT-NA，及其与PGD 2、LPS组合后对RAW264.7巨噬细胞和分离的腹腔巨噬细胞的毒性。简而言之，将RAW 264.7巨噬细胞以4×10 5个细胞/ml的浓度接种在96孔板中12小时，随后在37℃下暴露于CT-NA(0-200μM)1小时。接下来，将RAW 264.7巨噬细胞进一步暴露于LPS(100ng/ml)和PGD 2(10μM)24小时，随后用MTT(5mg/ml)在37℃下处理4小时。然后，用DMSO(200μl/孔)更换每个孔的上清液，测定在570nm处的吸光度。
2.1.10体外ELISA测定和实时聚合酶链反应(RT-PCR)分析
对于ELISA和RT-PCR，将RAW 264.7巨噬细胞在70-80％汇合度时加入到两个12孔板。之后，12孔板的培养基用无血清RPMI-1640培养基替换，培育10-12小时后暴露于CT-NA(10和100μM)1小时。一个小时后，一个12孔板用LPS(100ng/ml)处理24小时，另一个用PGD 2(10μM)处理24小时。处理后，收集处理细胞的上清液，根据说明书的方法，使用ELISA试剂盒测量TNF-α，IL-1β，IL-6，KC的蛋白质水平。随后，来自每个处理过的板的RNA样品用HiScript5×QRTSuperMix提取并逆转录成cDNA，然后进行RT-PCR。RT-PCR使用BioRad CFX96  Touch TM实时PCR检测系统(加利福尼亚州圣地亚哥)进行处理。同样地，使用从分离的腹膜巨噬细胞中提取的总RNA分析IL-1β，TNF-α，IL-6和KC的mRNA水平。用于RT-PCR反应的引物如表2所示。β-actin用作内部对照。RT-PCR反应重复三次。靶mRNA的相对表达通过各自的β-actin矫正。
表2
Figure PCTCN2020091842-appb-000024
2.1.11蛋白质印迹分析
进行总蛋白质提取和蛋白质印迹测定方法如下，将肺组织在RIPA缓冲液(0.5M Tris-HCl，pH 7.4,1.5M NaCl，2.5％脱氧胆酸，10％NP-40,10mM EDTA)中匀浆，缓冲液中还含有蛋白酶和磷酸酶抑制剂(Sigma-Aldrich，St.Louis，MO)。将RAW264.7巨噬细胞以70-80％汇合度接种到两个6孔板中。用无血清RPMI-1640培养基培养过夜饥饿后，用CT-NA预处理RAW264.7巨噬细胞(0.5,1,10和100μM)1小时。之后，一个6孔板用LPS(100ng/ml)处理1小时，另一个用PGD 2(10μM)处理1小时。用PBS洗涤三次后，细胞在冰环境中用含有蛋白酶和磷酸酶抑制剂的RIPA缓冲液振荡直接裂解30分钟。然后，将裂解物在4℃环境下以12,300×g离心15分钟，收集上清液。进行Bradford蛋白质测定(BCA)以测量蛋白质浓度。将等量的蛋白质(30μg)在12％SDS-PAGE上分离，转移至0.45μm聚偏二氟乙烯(PVDF)膜(Millipore，Bedford，MA)。在室温下用5％(wt/vol)脱脂奶粉将膜封闭1-2小时减少非特异性结合。然后将膜与IκBα(1：1000)，磷酸-IκBα(1：1000)，NF-κBP65(1：1000)，磷酸化NF-κBP65特异性的原代抗体在4℃下一起孵育过夜，与第二抗体IRDye680和800在室温下孵育1小时，然后用TBST洗涤三次。用Odyssey红外成像系统(LI-COR Biosciences Lincoln，NE)显现免疫反应信号。Western印迹分析重复三次，β-actin用作内标。将肺匀浆后进行相同的操作。
2.1.12统计分析
数据为平均值±SEM。统计学使用SPSS(SPSS Inc.，Chicago，IL)进行计算。采用单因素ANOVA方法比较F值，如果p>0.05，则采用Dunnett multiple comparisons teat计算参数数据的差 异；如果p<0.05，则用Mann-Whitney U non-parametric test比较差异。p<0.05和p<0.01被认为是具有统计学意义。
3.11 CT-NA缓解了LPS诱导的肺损伤
在LPS诱导后24小时，收集BALF通过Wright-Giemsa染色法分析CT-NA对细胞浸润的影响。与NS攻击组相比，LPS诱导加强了总细胞，嗜中性粒细胞和巨噬细胞的浸润(P<0.01)。然而，与载体处理的对照组相比，10和30mg/kg剂量的CT-NA以剂量依赖性的方式显著减弱LPS诱导的总细胞，嗜中性粒细胞和巨噬细胞的增加，但不包括BALF中淋巴细胞(P<0.01)，CT-NA剂量为30mg/kg时与1mg/kg的Dex表现基本相同(图17A)。分别通过测量SO 2和肺湿重系数来评估LPS诱导的低氧血症和肺水肿，结果表明与对照组相比，LPS诱导组显示较低的SO 2和较高的肺湿重系数(P<0.01)，使用CT-NA按照10和30mg/kg剂量处理，以剂量依赖的方式显著提高SO 2(P<0.01)并显著降低肺湿重系数(P<0.01)(图17B和C)。10或30mg/kg的CT-NA、以及1mg/kg的Dex同样分别增强SO 2和降低肺湿重系数。上述结果表明通过CT-NA的CRTH2拮抗作用可以显著改善LPS诱导的ALI模型中肺部炎症，低氧血症和肺水肿。
3.2 CT-NA改善BALF中LPS诱导的促炎细胞因子和趋化因子的产生
分别使用ELISA试剂盒测量收集的BALF中IL-1β，TNF-α，IL-6和KC的表达水平，以此来确定CT-NA对促炎细胞因子和趋化因子产生的影响。与载体攻击组相比，LPS诱导显著增加IL-1β，TNF-α，IL-6和KC的表达(P<0.01)，相反，10和30mg/kgCT-NA，1mg/kgDex以剂量依赖性的方式有效减少IL-1β，TNF-α，IL-6和KC的产生(P<0.05或P<0.01)(图18A-D)。这些数据意味着CT-NA对CRTH2的拮抗作用可以进一步保护LPS诱导的ALI小鼠避免促炎细胞因子和嗜中性粒细胞趋化因子引起的肺部炎症。
3.3 CT-NA减轻了LPS诱导的肺组织病理学变化
CT-NA对LPS诱导的肺组织病理学的保护作用是在H&E染色和石蜡包埋的肺切片中检查。受LPS诱导的小鼠与对照小鼠相比有显著的组织病理学变化(图19A)。相比之下，用10和30mg/kg的CT-NA或以1mg/kg的Dex处理LPS诱导的小鼠表现出显著减轻的这些组织病理学变化，使用CT-NA引起的组织病理学变化显示出剂量依赖性(图19A)。平均病理学评分，在炎症细胞的出血和浸润、嗜中性粒细胞进入细支气管周围和血管周围组织方面，与载体攻击相比，LPS诱导后显著增加(P<0.01)，然而CT-NA在10(P<0.05)和30mg/kg(P<0.01)或Dex为1mg/kg剂量(P<0.01)时显著降低了病理评分，CT-NA的结果再次显示出剂量依赖性(图19B)。因此，CT-NA阻断CRTH2受体显著降低了LPS诱导的肺损伤的严重程度，并逆转了LPS诱导的肺组织损伤。
3.4 CT-NA使肺血管通透性最小化
通过测定肺中的BALF中白蛋白含量和伊文思蓝染料的外转来测试CT-NA对LPS诱导的肺血管通透性的保护作用。与对照组相比，LPS诱导组BALF中白蛋白含量显著增加(P<0.01)，而CT-NA为10mg/kg(P<0.05)和30mg/kg(P<0.01)或Dex为1mg/kg(P<0.01)剂量时，显著降低了BALF中白蛋白含量(图20A)。与对照组相比，LPS诱导组伊文思蓝染料的肺血管渗漏和定 量外渗明显增高(P<0.01)，CT-NA给药10mg/kg(P<0.05)和30mg/kg(P<0.01)或Dex以1mg/kg(P<0.01)剂量给药时显著降低LPS诱导的伊文思蓝染料的肺血管渗漏和外渗(图20B)。因此，这些结果表明CRTH2拮抗能有效改善LPS诱导的ALI小鼠的肺血管通透性。
3.5 CT-NA降低了LPS诱导的肺部MPO活性
MPO由活化的嗜中性粒细胞产生，是嗜中性粒细胞浸润和肺组织损伤的重要标志物。MPO活性的增加反映了活化的嗜中性粒细胞在肺中的积累。LPS诱导组小鼠的MPO活性明显高于对照组(P<0.01)。CT-NA在10和30mg/kg(P均<0.01)或Dex 1mg/kg(P<0.01)剂量处理时显著减弱MPO活性，CT-NA再次表现出剂量依赖性(图21A)。因此，CT-NA阻断CRTH2受体可有效抑制嗜中性粒细胞浸润肺泡和间隙。
3.6 CT-NA减弱了体外PGD 2诱导的嗜中性粒细胞迁移
使用transwell测定评价CT-NA对嗜中性粒细胞迁移的影响，因为ALI症状的主要原因是嗜中性粒细胞释放出有害的炎症介质。Wright-Giemsa评估了分离的嗜中性粒细胞的特征，并进行了细胞活性测定。活化的PGD 2/CRTH2受体促进嗜中性粒细胞迁移及其功能，因此我们使用PGD 2作为化学引诱物。孵育4小时后，观察到了明显的嗜中性粒细胞向PGD 2的迁移，浓度范围为1至10μM(P<0.05或P<0.01)(图21B)。用1和10μM CT-NA(P均<0.01)或10μM的OC459(P<0.01)预处理嗜中性粒细胞，显著减弱1μM的PGD 2诱导的嗜中性粒细胞迁移(图21C)。总的来说，这些数据表明CRTH2拮抗剂显著减弱嗜中性粒细胞向PGD 2的迁移。
3.7 LPS促进分离的腹膜巨噬细胞分泌PGD 2
用不同浓度的LPS诱导分离的腹膜巨噬细胞，采用ELISA试剂盒测定细胞外分泌的PGD 2蛋白的量。与载体处理相比，使用浓度为0.01～10μM的LPS(P均<0.01)处理显著促进PGD 2的分泌(图21D)。
3.8 CT-NA减少LPS和PGD 2诱导的RAW264.7巨噬细胞和分离的腹膜巨噬细胞分泌促炎细胞因子和趋化因子
促炎细胞因子是ALI发病机制的关键因素，通过过度表达的促炎细胞因子，巨噬细胞上PGD 2/CRTH2受体的活化显著恶化了疾病状况。因此，还在巨噬细胞中测试了CT-NA处理对LPS-或PGD 2诱导的促炎因子产生的影响。MTT测定表明，以100ng/ml剂量的LPS加高达100μM剂量的CT-NA，或以10μM剂量的PGD 2加高达100μM剂量的CT-NA对RAW264.7巨噬细胞或分离的腹膜巨噬细胞无毒性。CT-NA对促炎细胞因子和趋化因子表达的影响通过ELISA测量。CT-NA分别为10和100μM时以剂量依赖性的方式抑制IL-1β，TNF-α，IL-6和KC对LPS(图22A-D)或PGD 2(图22E-H)刺激响应的蛋白表达。定量RT-PCR证明CT-NA在10和100μM剂量时以剂量依赖性的方式降低了在LPS或PGD 2刺激的RAW264.7巨噬细胞(图23A-H)和分离的腹膜巨噬细胞(图24A-H)中IL-1β，TNF-α，IL-6和KC的mRNA表达(P均<0.05或P均<0.01)。因此，这些体外结果与图19所示的体内结果一致，表明CT-NA的CRTH2拮抗作用有效地改善了来源于LPS-或PGD 2-诱导的来自RAW264.7巨噬细胞及分离的腹膜巨噬细胞的促炎细胞因子和趋化因子的产生。
3.9 CT-NA在体外和体内抑制P65活化
CT-NA显著抑制LPS诱导的ALI的潜在机制通过蛋白质印迹分析进行探索。研究的重点是CT-NA对LPS诱导的NF-κB活化通路的影响，因为NF-κB是促炎介质活化，嗜中性粒细胞浸润和肺血管通透性增加所必需的。与载体处理组相比，使用LPS诱导RAW264.7巨噬细胞和肺组织强烈诱导IκBα的磷酸化和降解，从而分别增加或减少磷酸化P65或P65水平(图25A和B)。用0.5,1.0,10和100μM的CT-NA处理RAW264.7巨噬细胞，或以10mg/kg或30mg/kg剂量处理ALI小鼠，以剂量依赖性的方式降低LPS诱导的IκBα和P65活性，并强烈逆转LPS诱导的IκBα和P65的降解(图25A和B)。因此，CRTH2受体拮抗剂CT-NA最有可能通过抑制NF-κB信号传导的方式保护小鼠免受LPS诱导的ALI。
综上，实验结果表明，本发明提供式A所示杂环化合物、其水合物、其药学上可接受的盐(例如碱金属盐)或者所述盐(例如碱金属盐)的水合物通过抑制肺部巨噬细胞和嗜中性粒细胞不恰当的转移，降低肺血管通透性，改善促炎性细胞因子和细胞因子趋化因子的产生和增强IL-10的产生，极大地缓解了由香烟烟雾CS诱导或LPS诱导的诱导的急性肺损伤。并且，由于可中止或减缓炎症性肺损伤、减轻肺水肿、保证组织供氧，表明本发明提供式A所示杂环化合物的晶型、其水合物的晶型、其药学上可接受的碱金属盐或者所述碱金属盐的水合物对于急性呼吸窘迫综合症具有较好的治疗或缓解作用。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 式A所示杂环化合物、其药学上可接受的盐或其水合物中的至少一种在制备治疗急性肺损伤或急性呼吸窘迫综合征药物中的用途，
    Figure PCTCN2020091842-appb-100001
  2. 根据权利要求1所述的用途，其特征在于，所述急性肺损伤选自香烟烟雾(cigarette smoke，CS)或脂多糖(lipopolysaccharide，LPS)诱导的急性肺损伤。
  3. 根据权利要求1或2所述的用途，其特征在于，所述式A所示杂环化合物药学上可接受的盐选自碱金属盐；
    优选地，所述水合物选自式A所示杂环化合物的水合物，或式A所示杂环化合物碱金属盐的水合物；
    优选地，所述式A所示杂环化合物或其药学上可接受的盐为结晶形式，如式A所示杂环化合物的晶型、其水合物的晶型、其药学上可接受的碱金属盐的晶型或者所述碱金属盐水合物的晶型。
  4. 根据权利要求1-3任一项所述的用途，其特征在于，式A所示杂环化合物或其水合物的晶型使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在11.1±0.2°、11.4±0.2°、17.9±0.2°、22.6±0.2°、24.4±0.2°处具有特征峰；
    优选地，所述晶型使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在8.6±0.2°、11.1±0.2°、11.4±0.2°、14.1±0.2°、16.1±0.2°、17.9±0.2°、20.9±0.2°、22.6±0.2°、24.4±0.2°、25.8±0.2°处具有特征峰；
    还优选地，所述晶型使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在8.6±0.2°、11.1±0.2°、11.4±0.2°、14.1±0.2°、15.6±0.2°、16.1±0.2°、17.9±0.2°、18.3±0.2°、20.9±0.2°、22.6±0.2°、24.4±0.2°、25.8±0.2°、26.5±0.2°、28.9±0.2°处具有特征峰；
    进一步优选地，所述晶型具有基本上如图1所示的X射线粉末衍射谱图；
    优选地，式A所示杂环化合物水合物的晶型为一水合物；
    还优选地，所述晶型为单晶，具有下述的单晶参数：
    Figure PCTCN2020091842-appb-100002
  5. 根据权利要求1-3任一项所述的用途，其特征在于，所述式A所示杂环化合物药学上可接受的碱金属盐为钠盐、锂盐或钾盐；所述碱金属盐的水合物选自钠盐、锂盐或钾盐的水合物；
    优选地，所述式A化合物碱金属盐的晶型为水合物形式；
    还优选地，所述式A化合物碱金属盐的结晶水合物为一水合物；
    优选地，所述式A化合物碱金属盐的结晶水合物选自如下式A-N、A-L或A-K所示的化合物：
    Figure PCTCN2020091842-appb-100003
  6. 根据权利要求5所述的用途，其特征在于，式A-N所示的化合物为结晶水合物，其使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在16.4±0.2°、18.9±0.2°、21.7±0.2°、24.0±0.2°处具有特征峰；
    优选地，其使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在11.8±0.2°、16.4±0.2°、16.7±0.2°、16.9±0.2°、17.1±0.2°、17.8±0.2°、18.6±0.2°、18.9±0.2°、21.7±0.2°、23.7±0.2°、24.0±0.2°处具有特征峰；
    还优选地，所述结晶水合物使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在5.6±0.2°、11.8±0.2°、14.0±0.2°、15.8±0.2°、16.4±0.2°、16.7±0.2°、16.9±0.2°、17.1±0.2°、17.8±0.2°、18.6±0.2°、18.9±0.2°、20.3±0.2°、21.7±0.2°、23.7±0.2°、24.0±0.2°、26.1±0.2°、28.1±0.2°、28.5±0.2°、29.8±0.2°处具有特征峰；
    进一步优选地，所述结晶水合物具有基本上如图4所示的X射线粉末衍射谱图(XRPD)；
    优选地，所述式A-N所示化合物的结晶水合物中水的质量分数为3.4～4.4％。
  7. 根据权利要求5所述的用途，其特征在于，式A-K所示的钾盐化合物为结晶水合物，其 使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在15.6±0.2°、21.4±0.2°、24.0±0.2°处具有特征峰；
    优选地，式A-K所示的钾盐化合物为结晶水合物，其使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在11.7±0.2°、15.6±0.2°、16.6±0.2°、17.9±0.2°、18.5±0.2°、21.4±0.2°、24.0±0.2°、28.2±0.2°处具有特征峰；
    优选地，式A-K所示的钾盐化合物为结晶水合物，其使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在11.7±0.2°、15.6±0.2°、15.9±0.2°、16.6±0.2°、17.4±0.2°、17.9±0.2°、18.5±0.2°、21.4±0.2°、23.5±0.2°、24.0±0.2°、27.7±0.2°、28.2±0.2°处具有特征峰；
    还优选地，所述式A-K所示化合物的结晶水合物使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在11.7±0.2°、14.0±0.2°、15.6±0.2°、15.9±0.2°、16.6±0.2°、17.4±0.2°、17.9±0.2°、18.5±0.2°、20.1±0.2°、21.4±0.2°、23.5±0.2°、24.0±0.2°、27.5±0.2°、27.7±0.2°、28.2±0.2°、28.6±0.2°、29.3±0.2°、29.6±0.2°处具有特征峰；
    进一步优选地，所述式A-K所示化合物的结晶水合物具有基本上如图6所示的X射线粉末衍射谱图；
    优选地，所述式A-K所示化合物的结晶水合物中水的质量分数为3.3～4.3％。
  8. 根据权利要求5所述的用途，其特征在于，式A-L所示的锂盐化合物为结晶水合物，使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在16.7±0.2°、18.8±0.2°、21.9±0.2°、23.9±0.2°处具有特征峰；
    还优选地，所述式A-L所示化合物的结晶水合物使用Cu-Kα辐射，以2θ角度表示的X射线粉末衍射在5.6±0.2°、8.8±0.2°、11.8±0.2°、14.0±0.2°、16.3±0.2°、16.7±0.2°、16.9±0.2°、17.0±0.2°、17.7±0.2°、18.5±0.2°、18.8±0.2°、21.9±0.2°、23.9±0.2°、28.2±0.2°处具有特征峰；
    进一步优选地，所述式A-L所示化合物的结晶水合物具有基本上如图8所示的X射线粉末衍射谱图；
    优选地，所述式A-L所示化合物的结晶水合物中水的质量分数为3.6～4.6％。
  9. 一种用于治疗急性肺损伤或急性呼吸窘迫综合征的药物组合物，其包括治疗有效量的权利要求1-7任一项所述的式A所示杂环化合物、其药学上可接受的盐或其水合物。
  10. 根据权利要求9所述的药物组合物，其特征在于，所述药物组合物还包含至少一种药学上可接受的辅料；
    优选地，所述辅料为惰性的、无毒的赋形剂、载体或稀释剂，例如所述辅料选自下列中的一种、两种或多种：崩解剂、助流剂、润滑剂、填充剂、粘合剂、着色剂、泡腾剂、矫味剂、防腐剂、包衣材料。
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