CN110551095B - Chromone compound and preparation method and application thereof - Google Patents

Abstract

The invention provides a chromone compound with novel structure, a preparation method and application thereof, wherein such compounds are prepared by fermentation of Aspergillus deflectus (Aspergillus deflectus). The preservation unit of the Aspergillus deflectus is the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is CGMCC No. 9095. The chromone compound prepared by the invention can be used for preparing a medicament for preventing or treating diseases mediated by inosine-5' -phosphate dehydrogenase (IMPDH).

Description

Chromone compound and preparation method and application thereof

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a chromone compound and a preparation method and application thereof.

Background

Inosine-5' -phosphate dehydrogenase (IMPDH; EC1.1.1.205) is an enzyme involved in the de novo synthesis of guanine nucleotides. IMPDH catalyzes the NAD-dependent oxidation of inosine-5 '-phosphate (IMP) to xanthosine-5' -phosphate (XMP) [ Jackson R.C. et al, Nature,256, pp.331-333, (1975) ].

IMPDH is ubiquitous in eukaryotes, bacteria and protozoa [ y.natsumeda & s.f.carr, ann.n.y.acad.,696, pp.88-93(1993) ]. Prokaryotic forms have 30-40% sequence homology with human enzymes. Two isoforms of human IMPDH, designated type I and type II, have been identified and their sequences determined [ f.r.collirt and e.huberman, j.biol.chem.,263, pp.15769-15772, (1988); y. natsumeda et al, j.biol. chem.,265, pp.5292-5295 (1990) ]. These two isoforms each have 514 amino acids and have 84% sequence homology. IMPDH types I and II form active tetramers in solution with subunits having a molecular weight of 56kDa [ Y.Yamada et al, Biochemistry,27, pp.2737-2745(1988) ].

Guanosine nucleotides of B and T-lymphocytes are synthesized de novo, and thus the activity of IMPDH is of particular importance. These cells rely on de novo synthesis rather than salvage synthesis to meet the nucleotides required for cell proliferation [ a.c. alison et al, Lancet ii, 1179, (1975) and a.c. alison et al, ciba found.symp.,48,207, (1977) ]. IMPDH is therefore an attractive target to selectively suppress the immune system without inhibiting the proliferation of other cells. U.S. Pat. Nos. 5,380,879 and 5,444,072, as well as PCT publications WO94/01105 and WO94/12184, describe mycophenolic acid (MPA) and certain derivatives thereof, which are potent, noncompetitive, reversible inhibitors of human IMPDH type I (Ki 33nM) and type II (Ki 9 nM). MPA blocks B and T-lymphocyte responses to mitogens or antigens [ a.c. allison et al, ann.n.y.acad.sci.,696,63, (1993) ]. The IMPDH inhibitor MPA has been a useful drug for the treatment of immune rejection and autoimmune diseases [ r.e. morris, Kidney int, 49, suppl.53, S-26, (1996) ]. Mycophenolate mofetil rapidly releases a prodrug of free MPA in vivo to prevent acute renal allograft rejection after renal transplantation [ l.m. shaw, et al, Therapeutic Drug Monitoring,17, pp.690-699 (1995); sollinger, Transplantation,60, pp.225-232 (1995).

Recently, different classes of IMPDH inhibitors have been described in PCT publications WO 97/40028 and WO 98/40381. IMPDH is also known to play a role in other metabolic events. An increase in IMPDH activity was also observed in rapidly proliferating tumor cell lines, indicating that IMPDH inhibitors are of anti-Cancer value [ m.nagai et al, Cancer res.,51, pp.3886-3890, (1991) ].

Studies have also shown a role for IMPDH in smooth muscle cell proliferation, suggesting that IMPDH inhibitors such as MPA or rapamycin may be useful in the prevention of restenosis or other hyperproliferative vascular diseases [ c.r. gregory et al, Transplantation,59, pp.655-61 (1995); PCT publication WO94/12184 and PCT publication WO94/01105 ].

Furthermore, the viral replication process is de novo synthesis of DNA and RNA, and IMPDH plays a crucial role in viral infectious replication [ s.f.ca, j.biol.chem.,268, pp.27286-27290(1993) ]. MPA and nucleoside analogs such as ribavirin have antiviral effects by inhibiting IMPDH [ L.Hedstrom, et al, Biochemistry,29, pp.849-854(1990) ].

Thus, IMPDH inhibitors have pharmaceutical value as immunosuppressive, anticancer, anti-vascular hyperproliferative, anti-inflammatory, antifungal, antipsoriatic and antiviral agents.

Disclosure of Invention

The invention aims to provide a chromone compound with IMPDH inhibition effect, and a preparation method and medical application of the compound.

In order to achieve the purpose of the invention, the invention provides chromone compounds, which have the following structures (1) to (5):

wherein R1 is H or COCH ₃ R2 is H or Cl

The chromone compound provided by the invention is a fermentation metabolite of Aspergillus elbow, the produced strain is Aspergillus elbow (NCC 0415), the preservation unit is China general microbiological culture Collection center, the preservation date is 4 months and 25 days in 2014, the preservation number is CGMCC No.9095, and the preservation address is as follows: the institute of microbiology, national academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, Beijing.

The invention provides a preparation method of the chromone compound, which is to prepare the chromone compound by fermenting Aspergillus elbow (Aspergillus deflectous). Specifically, the method comprises the following steps:

a. preparation of fermentation product of Aspergillus elbow (Aspergillus deflectous) NCC 0415:

inoculating Aspergillus elbow (Aspergillus deflectous) into fermentation culture medium, and culturing at 22-30 deg.C for 5-8 days to obtain fermentation liquid.

The preservation unit of the Aspergillus elbow (Aspergillus deflectous) is the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is CGMCC No. 9095.

The fermentation medium is prepared by the following method: 10.0 to 50.0 grams of glucose, 10.0 to 50.0 grams of soluble starch, 4.0 to 20.0 grams of hot-pressed soybean cake powder, 4.0 to 20.0 grams of cottonseed powder, 1.0 to 10.0 grams of potassium dihydrogen phosphate, 0.5 to 2.0 grams of sodium chloride, 0.5 to 2.0 grams of magnesium sulfate heptahydrate, adding tap water to a constant volume of 1000mL, keeping the pH value of 6.5 to 7.0, and sterilizing at 121 ℃ for 30 min.

b. Filtering or centrifuging the fermentation liquid, and removing supernatant to obtain mycelium;

c. extracting mycelium with organic solvent for 2-4 times, each for 2-4 hr, mixing extractive solutions, concentrating under reduced pressure, and removing organic solvent to obtain crude extract;

the organic solvent is methanol, ethanol, acetone or ethyl acetate;

d. and (3) performing silica gel column chromatography on the crude extract, wherein the chloroform: gradient eluting with methanol (10: 0-7: 3), collecting eluate, detecting by HPLC, mixing the components with similar peaks, evaporating to dryness under reduced pressure, and recording as S1-S9; s1, S3 and S9 have no target components and are discarded; fraction S2 was prepared by HPLC (mobile phase 65% acetonitrile, YMC C18 column, 21.2 × 250, 10 μm, flow rate 16mL/min) to give compounds 1(Rt ═ 10.0min) and 2(Rt ═ 15.6 min); the S4 fraction was subjected sequentially to Sephadex LH-20 column chromatography (column: 2.6 × 70cm, methanol as eluent) and HPLC preparation (mobile phase 55% acetonitrile, nano-micro C18 column 21.2 × 150,10 μm, flow rate 16mL/min) to yield compound 3(Rt ═ 9.2min) and 4(Rt ═ 11.5 min); the S5 fractions were subjected sequentially to Sephadex LH-20 column chromatography (column: 2.6 × 70cm, methanol as eluent), HPLC preparation (mobile phase 35% acetonitrile, nano-micro C18 column 21.2 × 150,10 μm, flow rate 16mL/min) to yield compounds 5(Rt ═ 11.6min), 6(Rt ═ 15.2min) and 7(Rt ═ 21.5 min); fraction S6 was prepared by HPLC (mobile phase: 50% acetonitrile, nano-micro C18 column, 21.2 × 150mm,10 μm,16ml/min) to yield compounds 8(Rt ═ 6.2min) and 9(Rt ═ 10.5 min); HPLC preparation of S7 fraction (30% acetonitrile, nano-micro chromatography column, 21.2 × 250, C18,10 μm,16ml/min) gave compound 10(Rt ═ 6.5 min);

in addition to the above preparation methods, the chromone compound of the invention can be obtained by synthesis, semisynthesis or biotransformation according to conventional methods.

The invention also provides a medical application of the chromone compound: the compound is used for preparing a medicament for preventing or treating IMPDH mediated diseases.

The IMPDH mediated diseases of the invention are specifically immune rejection, autoimmune diseases, viral infection, inflammation, tumor, parasitic infection, vascular hyperproliferation diseases and the like.

The methods of the invention are useful for suppressing an immune response in a mammal. Such methods are useful for treating or preventing diseases, including transplant rejection (e.g., kidney, liver, heart, lung, pancreas (islet cells), bone marrow, cornea, small intestine and skin allografts, and heart valve xenografts), graft-to-host disease, and autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, juvenile diabetes, asthma, Crohn's disease, ulcerative colitis, lupus, diabetes, myasthenia gravis, psoriasis, dermatitis, eczema, seborrhea, pneumouveitis, hepatitis, Graves ' disease, Hashimoto's thyroiditis, Behcet's or Sjogren's syndrome (ocular/oral dryness), malignant or immune hemorrhoidal anemia, idiopathic adrenal insufficiency, multiple gland autoimmune syndrome, and glomerulonephritis, scleroderma, Lichen planus, leukoplakia (skin depigmentation), autoimmune thyroiditis, and brussels disease.

The methods of the invention are useful for inhibiting viral replication in a mammal. Such methods are useful for treating or preventing diseases caused by infection with: orthomyxoviruses (influenza A and B viruses), paramyxoviruses (respiratory syncytial virus (RSV), Subacute Sclerosing Panencephalitis (SSPE) virus) measles and parainfluenza type 3), herpes viruses (HSV-1, HSV-2, HHV-6, HHV-7, HHV-8, EB virus (EBV), cytomegalovirus (HCMV) and Varicella Zoster Virus (VZV)), retroviruses (HIV-1, HIV-2, HTLV-1, HTLV-2), yellow and pestiviruses (yellow fever virus (YFV), Hepatitis C Virus (HCV), dengue fever virus, Bovine Viral Diarrhea Virus (BVDV), hepatotrophic virus (hepatitis A virus (HAV), Hepatitis B Virus (HBV), Hepatitis D Virus (HDV), Hepatitis E Virus (HEV), Hepatitis G Virus (HGV), Crimeria-Congo hemorrhagic fever (CCHF), Bunyavirus (Punta Toro virus, valley fever virus (RVFV), and phlebovirus sician virus), hantavirus, Caraparu virus), human papilloma virus, encephalitis virus (La cross virus), arenavirus (Junin and Tacaribe virus), reovirus, vesicular stomatitis virus, rhinovirus, enterovirus (poliovirus, coxsackievirus, encephalomyocarditis virus (EMC)), lassa fever virus, and togavirus (neopis and semliki (semlike) virus) forest and pox virus (bovine syndrome virus), adenovirus, rubiola, and rubella.

The methods of the invention are useful for inhibiting vascular cell hyperproliferation in a mammal. Such methods are useful for treating or preventing diseases, including restenosis, stenosis, atherosclerosis, and other hyperproliferative vascular diseases.

The methods of the invention are useful for inhibiting tumors and cancers in mammals. Such methods are useful for treating or preventing diseases, including tumors and malignancies such as solid tumors, lymphomas, leukemias, and other forms of cancer.

The methods of the invention are useful for inhibiting inflammation and inflammatory diseases in a mammal. Such methods may be useful for treating or preventing diseases including osteoarthritis, acute pancreatitis, chronic pancreatitis, asthma, and adult respiratory distress syndrome.

In the case of the above-mentioned pharmaceutical use, it is common general knowledge of those skilled in the art that not only the chromone compound of the present invention itself but also a pharmaceutically acceptable salt, isomer, ester or prodrug thereof may have the same or similar pharmaceutical use. The pharmaceutically acceptable salts include sodium, potassium, calcium salts, and amine salts; the amines include ammonia, alkylamines, anilines, and amino acids; the amino acids include arginine and lysine. The pharmaceutically acceptable salts can also form solvates and have the same or similar medical application; the solvate comprises hydrate and alcoholate. The prodrug can release the chromone compound after in vivo metabolic change.

The compositions of the present invention comprise a compound of the present invention or a salt thereof, and also include a compound or drug selected from other immunosuppressive agents, anticancer agents, antiviral agents, anti-inflammatory agents, antifungal agents, antibiotics, or anti-vascular hyperproliferative compounds or drugs. In addition, the composition of the invention comprises the compound or the salt thereof and a pharmaceutically acceptable carrier, auxiliary material or excipient. Such compositions may optionally comprise an additional active agent selected from an immunosuppressive agent, an anticancer agent, an antiviral agent, an anti-inflammatory agent, an antifungal agent, an antibiotic, or an anti-vascular hyperproliferative compound.

Immunosuppressants that may be included in the pharmaceutical compositions of the present invention include, but are not limited to, cyclosporin A, tacrolimus, rapamycin, leflunomide, deoxyspergualin, prednisone, azathioprine, mycophenolic acid, and mizoribine.

Anticancer agents that may be included in the pharmaceutical compositions of the present invention include, but are not limited to, cisplatin, actinomycin D, doxorubicin, vincristine, vinblastine, etoposide, amsacrine, mitoxantrone, teniposide, paclitaxel, colchicine, and interferons.

Antiviral agents that may be included in the pharmaceutical compositions of the present invention include, but are not limited to, ganciclovir, trisodium phosphonoformate, ribavirin, d4T, ddI, AZT, and acyclovir.

Anti-vascular hyperproliferative agents that may be included in the pharmaceutical compositions of the present invention include, but are not limited to, HMG CoA reductase inhibitors such as lovastatin, thromboxane A2 synthase inhibitors, eicosapentaenoic acid, cilaprost, trapidil, ACE inhibitors, low molecular weight heparin, mycophenolic acid, rapamycin, and 5- (3' -pyridylmethyl) benzofuran-2-carboxylic acid.

The medical application of the chromone compound is realized by the following method:

pharmaceutical compositions comprising the chromone compounds described above and a pharmaceutically acceptable carrier are prepared according to conventional methods. Wherein, the mass percentage of the chromone compound is 0.1-99%, preferably 5-75%. The pharmaceutical composition can be divided into oral preparation, sublingual or buccal preparation, inhalation preparation, injection, rectal preparation, transdermal preparation, etc. according to different administration routes. Among them, oral preparations are preferred.

When preparing an oral formulation, the content of the chromone compound in a unit dosage form is 1-500mg, preferably 1-250 mg. Alternative dosage forms include tablets, pills, troches, capsules, liquids, gels, syrups, slurries, suspensions and the like. Among them, tablets and capsules are preferable. Pharmaceutically acceptable carriers include lactose, sucrose, mannitol, starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, agar, alginic acid, sodium alginate, talc, magnesium stearate, aerosil, fatty oils, liquid paraffin, liquid polyethylene glycol, and the like.

When sublingual or buccal formulations are prepared, they may take the form of tablets or dragees, formulated according to conventional methods.

When preparing an inhalation formulation, it may take the form of a pressurized aerosol. Suitable propellants include dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide. The pharmaceutically acceptable carrier includes lactose and starch.

When preparing an injection, the content of the chromone compound in a unit dosage form is 0.1-100mg, preferably 0.1-25 mg. The optional dosage form comprises oily injection, aqueous injection or lyophilized powder for injection. The pharmaceutically acceptable carrier comprises fatty oil, ethyl oleate, triglyceride, liposome, sodium carboxymethylcellulose, dextran, cross-linked polyvinylpyrrolidone, agar, alginic acid, and sodium alginate.

When preparing formulations for rectal administration, they may take the form of suppositories or enemas. Pharmaceutically acceptable carriers include cocoa butter, glycerides.

When preparing a transdermal administration preparation, it may take the form of a patch. The medicinal carrier comprises sodium polyacrylate, polyvinylpyrrolidone and polyethylene glycol.

When the pharmaceutical composition is applied, an effective treatment dose is achieved, and the pharmaceutical composition is reasonably adjusted according to the nature of diseases and the age and the weight of a patient. Generally, the chromone compound is administered in an amount of 0.01-100mg/kg body weight, preferably 0.1-10mg/kg body weight, and more preferably 1-5mg/kg body weight. The dosage and frequency of administration should ultimately be at the discretion of the physician.

Detailed Description

The following examples are presented to further illustrate the invention but are not intended to limit the invention in any way. The methods used in the following examples are conventional methods unless otherwise specified.

The specification and model numbers of the parts of raw materials related to the following examples are as follows:

nuclear magnetic resonance apparatus: bruker Avance III 500, TMS as internal standard

High-resolution mass spectrometry: LTQ Orbitrap Discovery (Thermo Scientific, Germany)

High pressure liquid phase system (analysis): waters corporation, 2 pumps model 515, 996 detector

High pressure liquid phase system (preparation): innovative constant P3000 double-pump UV3000 detector

An enzyme-labeling instrument: victor21420 Multilabel Counter from Perkin Elmer

Chromatography on silica gel: 300-400 mesh Qingdao ocean factory

Analytical chromatographic column: suzhou micro ODS column (4.6X 150mm,10 μm)

Chromatographic grade methanol and acetonitrile were purchased from Honeywell, other reagents were analytically pure and purchased from beijing chemicals.

EXAMPLE 1 preparation of chromone compounds

The chromone compound is prepared according to the following steps:

(a) preparing seed liquid of Aspergillus elbow (Aspergillus deflectous) NCC0415

Inoculating Aspergillus elbow (Aspergillus deflectous) fungus NCC0415 into a seed culture medium, and culturing at 25 deg.C and 200rpm for 72h to obtain seed solution. The preparation method of the seed culture medium comprises the following steps: 30.0 g of corn starch, 10.0 g of glucose, 4.0 g of hot-pressed soybean cake powder, 5.0 g of malt powder, 3.0 g of yeast powder, 2.0 g of sodium chloride and 1.0 g of magnesium sulfate heptahydrate, adding tap water to dissolve to a constant volume of 1000ml, keeping the pH value of the solution at 6.5, and sterilizing the solution at 121 ℃ for 30 min.

(b) Preparation of fermentation culture of Aspergillus elbow (Aspergillus deflectous) NCC 0415A seed solution was inoculated at 3% inoculum size into a 1000mL Erlenmeyer flask containing 150mL of liquid fermentation medium and cultured with shaking at 220rpm at 26 ℃ for 144h to obtain a fermentation culture. The fermentation medium is prepared by the following method: 30 g of glucose, 30 g of soluble starch, 10 g of hot-pressed soybean cake powder, 10 g of cottonseed powder, 5g of monopotassium phosphate, 1 g of sodium chloride, 0.5 g of magnesium sulfate heptahydrate, 2g of calcium carbonate, adding tap water to a constant volume of 1000mL, adjusting the pH value to 6.5-7.0, and sterilizing at 121 ℃ for 30 min.

(C) Preparation of chromone compounds

The fermentation culture was centrifuged at 3000rpm for 15min at 10L, and the cells were collected and the supernatant was discarded. Soaking the mycelia with equal volume of ethanol twice (2.0 hr/time), filtering, and concentrating the filtrate under reduced pressure to obtain crude extract 45 g; the crude extract was chromatographed on silica gel column (49 × 310mm), chloroform: eluting with methanol (10: 0-7: 3) at a flow rate of 80mL/min, and collecting the eluate at a flow rate of 50 mL/min. Performing HPLC detection, combining the components with similar peaks, and evaporating to dryness under reduced pressure to obtain S1-S9; s1, S3 and S9 have no target components and are discarded; fraction S2 was prepared by HPLC (mobile phase 65% acetonitrile, YMC C18 column, 21.2 × 250, 10 μm, flow rate 16mL/min) to give compounds 1(36mg, Rt ═ 10.0min) and 2(52mg, Rt ═ 15.6 min); fraction S4 was subjected sequentially to Sephadex LH-20 column chromatography (column: 2.6 × 70cm, methanol as eluent) and HPLC preparation (mobile phase 55% acetonitrile, nano-micro C18 column 21.2 × 150,10 μm, flow rate 16mL/min) to yield compounds 3(48.0mg, Rt ═ 9.2min) and 4(22.0mg, Rt ═ 11.5 min); the S5 fractions were subjected sequentially to Sephadex LH-20 column chromatography (column: 2.6 × 70cm, methanol as eluent), HPLC preparation (mobile phase 35% acetonitrile, nano-micro C18 column 21.2 × 150,10 μm, flow rate 16mL/min) to afford compounds 5(48.0mg, Rt ═ 11.6min), 6(69.0mg, Rt ═ 15.2min) and 7(21.0mg, Rt ═ 21.5 min); fraction S6 was prepared by HPLC (mobile phase: 50% acetonitrile, nano-micro C18 column, 21.2 × 150mm,10 μm,16mL/min) to give compounds 8(32.0mg, Rt ═ 6.2min) and 9(25.2mg, Rt ═ 10.5 min); HPLC preparation of S7 fraction (30% acetonitrile, nano-micro chromatography column, 21.2 × 250, C18,10 μm,16ml/min) gave compound 10(15.2mg, Rt ═ 6.5 min);

the chemical structures and nuclear magnetic data of the obtained compounds are as follows:

(1) compound 1:

pale yellow crystalline powder, HRMS gave M/z 359.1488[ M +1 ]] ⁺ The molecular formula is as follows: c ₂₀ H ₂₂ O ₆ ， ¹ H NMR(500MHz,CDCl ₃ )δ12.60(s,1H),7.59(t,J＝8.3Hz,1H),7.31(s,1H),6.91(dd,J＝8.4,1.0Hz,1H),6.80(dd,J＝8.3,0.9Hz,1H),5.07(d,J＝5.2Hz,2H),4.40(s,1H),4.13(t,J＝6.6Hz,2H),2.48(s,4H),2.11(t,J＝6.6Hz,2H),1.37(s,6H). ¹³ C NMR(126MHz,CDCl ₃ )δ184.39,161.85,155.65,154.09,152.76,142.33,136.89,134.06,119.66,118.05,110.57,109.13,106.56,72.95,70.24,56.97,42.61,29.78,29.78,17.78.

(2) Compound 2:

pale yellow crystalline powder, HRMS gave M/z 401.15945[ M +1 ]] ⁺ The molecular formula is as follows: c ₂₂ H ₂₄ O ₇ ， ¹ H NMR(500MHz,CDCl ₃ )δ12.55(s,1H),7.54(t,J＝8.3Hz,1H),7.20(s,1H),6.83(d,J＝8.3Hz,1H),6.74(d,J＝8.2Hz,1H),5.00(s,2H),3.97(t,J＝7.1Hz,2H),2.41(s,3H),2.39(t,J＝7.1Hz,2H),2.01(s,3H),1.57(s,6H). ¹³ C NMR(126MHz,CDCl ₃ )δ184.14,170.40,161.65,155.41,153.78,152.63,142.34,136.68,133.95,119.41,117.74,110.36,108.87,106.41,80.63,71.63,56.74,40.22,26.62,26.62,22.30,17.47.

(3) Compound 3:

pale yellow powder, HRMS gave M/z 719.2342[ M +1 ]] ⁺ The molecular formula is as follows: c ₃₈ H ₃₈ O ₁₄ ， ¹ H NMR(500MHz,DMSO-d6)δ12.01(1H,s),8.62(1H,s),6.96(1H,s),6.78(1H,t,J＝8.1Hz),6.61(1H,s),6.52(1H,s),6.50(1H,d,J＝8.1Hz),6.11(1H,s),6.04(1H,d,J＝8.1Hz),5.93(1H,d,J＝6.1Hz),5.49(1H,t,J＝5.7Hz),4.53(2H,m),4.17(1H,d,J＝8.0Hz),4.14(1H,m),4.12(1H,m),3.96(1H,m),3.68(3H,brs),3.53(1H,d,J＝10.4Hz),2.67(1H,dd,J＝6.0,8.0Hz),2.27(2H,t,J＝7.0Hz),2.16(3H,s),1.97(3H,s),1.52(3H,s),1.51(3H,s). ¹³ C NMR(126MHz,DMSO-d6)δ179.72,172.56,169.83,164.99,158.68,156.18,155.15,154.26,151.79,145.50,141.80,137.76,131.40,129.62,128.97,118.89,116.66,109.38,107.48,107.22,106.71,105.63,104.17,95.91,80.55,75.97,69.63,69.10,62.23,51.57,48.36,46.98,43.61,40.14,26.33,26.22,22.18,15.77.

(4) Compound 4:

pale yellow powder, HRMS gave m/z 753.1952, formula: c ₃₈ H ₃₇ ClO ₁₄ ， ¹ H NMR(500MHz,DMSO-d6)δ12.00(1H,s),11.45(1H,s),9.18(1H,s),8.76(1H,s),6.94(1H,s),6.94(1H,d,J＝8.8Hz),6.63(1H,s),6.49(1H,s),6.49(1H,d,J＝8.8Hz),6.11(1H,s),5.93(1H,brs),4.53(2H,dd),4.23(1H,dd,J＝8.0,0.8Hz),4.14(1H,m),4.13(1H,m,18-H),3.94(1H,m),3.68(3H,brs),3.54(1H,d,J＝10.2Hz),2.67(1H,dd,J＝6.0,8.0Hz),2.26(2H,t,J＝7.0Hz),2.15(3H,s),1.96(3H,s),1.51(3H,s),1.50(3H,s). ¹³ C NMR(126MHz,DMSO-d6)δ180.22,170.32,165.33,159.24,155.62,154.69,152.49,151.79,146.31,142.14,138.31,131.97,130.02,128.75,119.22,117.36,111.84,108.13,107.80,107.80,107.73,104.52,95.90,81.04,76.73,70.01,69.59,62.74,52.07,48.67,47.39,44.00,40.63,26.82,26.71,22.67,16.29

(5) Compound 5:

light yellowPowder, HRMS gave M/z 663.2075[ M +1 ]] ⁺ The molecular formula is as follows: c ₃₅ H ₃₄ O ₁₃ ， ¹ H NMR(500MHz,DMSO-d ₆ )δ12.22(1H,s),10.59(1H,s),8.55(1H,s),8.52(1H,s),7.03(1H,t,J＝8.1Hz),6.68(1H,brs),6.66(1H,brs),6.62(1H,d,J＝7.8Hz),6.45(1H,s),6.26(1H,s),6.16(1H,d,J＝7.8Hz),6.05(1H,s),5.48(1H,t,J＝5.6Hz),4.54(1H,d,J＝7.2Hz),4.51(2H,d,J＝5.6Hz),4.32(1H,s),4.00(2H,m),3.62(3H,s),2.95(1H,d,J＝7.2Hz),2.13(3H,s),1.86(2H,m),1.17(3H,s),1.15(3H,s). ¹³ C NMR(126MHz,DMSO-d6)δ179.31,173.24,168.74,159.71,156.75,156.64,156.22,152.16,145.97,142.42,136.09,131.17,129.51,129.33,123.77,118.60,108.82,108.49,107.94,106.41,106.27,104.15,90.40,76.80,76.56,70.37,67.82,62.02,55.18,52.62,52.18,43.06,29.76,29.53,15.92.

(6) Compound 6:

pale yellow powder, HRMS gave M/z 677.2230[ M +1 ]] ⁺ The molecular formula is as follows: c ₃₆ H ₃₆ O ₁₃ ， ¹ H NMR(500MHz,DMSO-d6)δ12.00(1H,s),11.58(1H,brs),8.84(1H,brs),8.61(1H,s),6.94(1H,s),6.76(1H,t,J＝8.1Hz),6.59(1H,s),6.50(1H,s),6.48(1H,d,J＝8.1Hz),6.09(1H,s),6.03(1H,d,J＝8.1Hz),5.85(1H,d,J＝5.8Hz),5.47(1H,t,J＝5.7Hz),4.52(2H,m),4.35(1H,s),4.15(1H,d,J＝8.0Hz),4.09(1H,m),4.09(1H,m),4.01(1H,m),3.66(3H,s),3.51(1H,d,J＝10.4Hz),2.65(1H,dd,J＝6.0,8.0Hz),2.15(3H,s),1.89(2H,t,J＝7.0Hz),1.19(3H,s),1.18(3H,s). ¹³ C NMR(126MHz,DMSO-d6)δ179.69,171.21,164.97,158.65,156.16,155.12,154.19,151.76,145.33,142.00,137.73,131.45,129.58,128.94,118.86,116.60,109.36,107.45,107.19,106.68,105.62,104.15,95.94,75.99,70.07,69.60,68.07,62.21,51.54,48.35,46.87,43.36,43.36,29.83,29.66,15.81.

(7) Compound 7:

pale yellow powder, HRMS gave m/z 711.1840, formula: c ₃₆ H ₃₅ ClO ₁₃ ， ¹ H NMR(500MHz,DMSO-d6)δ12.00(1H,s),11.44(1H,s),9.18(1H,s),8.71(1H,s),6.94(1H,s),6.94(1H,d,J＝8.7Hz),6.62(1H,s),6.49(1H,s),6.49(1H,d,J＝8.7Hz),6.10(1H,s),5.86(1H,d,J＝5.5Hz),5.47(1H,brs),4.53(2H,dd),4.22(1H,d,J＝8.2Hz),4.11(1H,m),4.11(1H,m,18-H),4.01(1H,m),3.67(3H,br),3.53(1H,d,J＝10.1Hz),2.66(1H,dd,J＝6.0,8.0Hz),2.15(3H,s),1.89(2H,t,J＝7.3Hz),1.20(3H,s),1.19(3H,s). ¹³ C NMR(126MHz,DMSO-d6)δ180.22,170.88,165.22,159.24,155.62,154.66,152.49,151.79,146.16,142.36,138.28,132.04,130.01,128.75,119.21,117.33,111.85,108.13,107.82,107.81,107.73,104.52,95.94,76.79,70.57,70.03,68.59,62.74,52.07,48.69,47.31,43.87,43.87,30.35,30.18,16.31.

(8) Compound 8:

pale yellow powder, HRMS gave M/z 609.1241[ M +1 ]] ⁺ The molecular formula is as follows: c ₃₀ H ₂₄ O ₁₄ ， ¹ H NMR(500MHz,DMSO-d6)δ12.54(1H,s),12.20(1H,s),7.78(1H,s),7.01(1H,s),6.89(1H,s),6.82(1H,s),6.76(1H,s),5.50(1H,t,J＝5.5Hz),5.42(1H,q,J＝5.5Hz),5.01(1H,d,J＝9.0Hz),4.58(2H,t,J＝5.5Hz),4.51(2H,t,J＝5.5Hz),3.80(1H,t,J＝9.0Hz),3.75(3H,s),3.71(3H,s),3.13(1H,m),2.70(2H,m). ¹³ C NMR(126MHz,DMSO-d6)δ179.58,178.93,170.79,169.47,169.38,168.11,159.98,159.58,156.84,155.59,152.33,152.33,119.24,111.74,109.03,108.78,108.42,108.31,105.70,104.88,104.23,88.23,62.18,62.02,52.76,52.27,46.80,42.56,36.93,26.22.

(9) Compound 9:

pale yellow powder, HRMS given M/z 705.2185[ M +1] ⁺ The molecular formula is as follows: c ₃₇ H ₃₆ O ₁₄ ， ¹ H NMR(500MHz,DMSO-d6)δ12.22(1H,s),10.57(1H,s),8.56(1H,s),8.51(1H,s),7.03(1H,t,J＝8.1Hz),6.68(1H,brs),6.66(1H,brs),6.62(1H,d,J＝8.0Hz),6.46(1H,s),6.27(1H,s),6.16(1H,d,J＝8.0Hz),6.06(1H,s),5.47(1H,dd,J＝4.7,6.3Hz),4.54(1H,d,J＝7.2Hz),4.51(2H,d,J＝4.7Hz),4.00(2H,m),3.62(3H,s),2.95(1H,d,J＝7.2Hz),2.25(2H,m),2.13(3H,s),1.96(3H,s),1.48(6H,s). ¹³ C NMR(126MHz,DMSO-d6)δ179.32,173.24,169.80,168.71,159.72,156.76,156.64,156.23,152.15,146.11,142.19,136.01,131.05,129.50,129.29,123.76,118.66,108.82,108.50,107.95,106.41,106.27,104.16,90.36,80.33,76.78,76.51,69.24,62.04,55.13,52.58,52.24,39.95,26.15,26.14,22.09,15.83.

(10) Compound 10:

white powder, HRMS gave M/z 247.0599[ M +1 ]] ⁺ The molecular formula is as follows: c ₁₃ H ₁₀ O ₅ ， ¹ H NMR(500MHz,DMSO-d6)δ12.41(1H,s),7.11(1H,s),6.85(1H,s),5.54(1H,t,J＝5.8Hz),4.59(2H,d,J＝5.6Hz),3.22–3.07(2H,m),2.64–2.57(2H,m). ¹³ C NMR(126MHz,DMSO-d6)δ198.13,192.20,178.51,161.47,156.61,153.46,117.63,110.37,109.40,105.33,62.58,34.39,26.45.

EXAMPLE 2 fermentation of NCC0415 and preparation of crude extracts containing chromone compounds

(a) Seed solutions of Aspergillus elbow (Aspergillus deflectous) NCC0415 were prepared as in example 1.

(b) Preparation of fermentation culture of Aspergillus elbow (Aspergillus deflectous) NCC 0415A seed solution was inoculated at 3% inoculum size into a 1000mL Erlenmeyer flask containing 150mL of liquid fermentation medium and cultured with shaking at 220rpm at 22 ℃ for 192 hours to obtain a fermentation culture. The fermentation medium is prepared by the following method: 10 g of glucose, 50 g of soluble starch, 20 g of hot-pressed soybean cake powder, 4 g of cottonseed powder, 10 g of monopotassium phosphate, 2.0 g of sodium chloride, 1.0 g of magnesium sulfate heptahydrate and 2g of calcium carbonate, adding tap water to a constant volume of 1000mL, adjusting the pH value to 6.5-7.0, and sterilizing at 121 ℃ for 30 min.

(c) Preparation of crude extract containing chromone compounds

The fermentation culture was centrifuged at 3000rpm for 15min at 10L, and the cells were collected and the supernatant was discarded. The mycelia were soaked in an equal volume of acetone three times (2.0 hours/time), filtered, and the filtrate was concentrated under reduced pressure to obtain 42g of crude extract.

EXAMPLE 3 fermentation of NCC0415 and preparation of crude extracts containing chromone compounds

(a) Seed solutions of Aspergillus elbow (Aspergillus deflectous) NCC0415 were prepared as in example 1.

(b) Preparation of fermentation culture of Aspergillus elbow (Aspergillus deflectous) NCC 0415A seed solution was inoculated at 3% inoculum size into a 1000mL Erlenmeyer flask containing 150mL of liquid fermentation medium and shake-cultured at 30 ℃ and 220rpm for 120h to obtain a fermentation culture. The fermentation medium is prepared by the following method: 50 g of glucose, 10 g of soluble starch, 4 g of hot-pressed soybean cake powder, 20 g of cottonseed powder, 1.0 g of monopotassium phosphate, 0.5 g of sodium chloride, 2.0 g of magnesium sulfate heptahydrate and 2g of calcium carbonate, adding tap water to a constant volume of 1000mL, adjusting the pH value to 6.5-7.0, and sterilizing at 121 ℃ for 30 min.

(c) Preparation of crude extract containing chromone compounds

The fermentation culture was centrifuged at 3000rpm for 15min at 10L, and the cells were collected and the supernatant was discarded. The mycelia were soaked with ethyl acetate of equal volume for four times (2.0 hr/time), filtered, and the filtrate was concentrated under reduced pressure to give 48g of crude extract.

EXAMPLE 4 inhibitory Activity of Compounds 1-10 on IMPDH

Human IMPDH type II was expressed recombinantly in vitro and the enzyme activity was measured spectrophotometrically by the modified IMPDH enzyme assay reported by Magasanik (Magasanik et al, J.Bio1.chem.,226, p.339(1957) by monitoring the increase in absorbance due to NADH formation at 340 nM. the reaction mixture contained 0.1M Tris, 0.1M Kcl, 3mM EDTA, 5mM IMP and enzyme at 15-50nM concentration (human IMPDH type II). the solution was incubated at 37 ℃ for 15 minutes. the reaction was started by the addition of NAD at a final concentration of 5mM and the initial rate was determined by a linear increase in absorbance at 340nM (30 minutes).

Determination of the inhibitory Activity of Compounds: the test compounds were dissolved in DMSO and added to a 96-well plate at 1-2. mu.l, followed by the enzyme-containing reaction mixture and preincubated for 15 minutes. Then, NAD was added to start the reaction, and 30 minutes later, the light absorption at 340nm was measured, and the inhibition rate was calculated. IC (integrated circuit) ₅₀ The value is the concentration of inhibitor at which half of the enzyme activity is inhibited.

TABLE 1 Compounds 1-10 inhibitory Activity on IMPDH

Example 5 anti-HBV viral replication Activity of Compounds 1-10

Experimental materials cell: HepG22.15 cells, HepG2, DMEM medium containing 10% FBS and 1% double antibody, DMEM medium containing 2% FBS and 1% double antibody, 96-well plate cell culture plate, ELISA kit of HBsAg and HBeAg.

Cell culture and activity assay: HepG2.2.15 cells are cultured in a T25 culture bottle, after the cells are overgrown, pancreatin is digested, 10% serum DMEM culture medium is paved into a 96-well plate, the cell amount of each well is 100ul, when the cells are completely adhered to the wall, 100ul of 2% FBS culture medium containing the medicine is used for replacing, the medicine is replaced once every other day, in 8-12 days, the supernatant is collected, an ELISA kit is adopted to detect the content of HBsAg and HBeAg in the cell supernatant, and the inhibition condition of the medicine on the HBsAg and the HBeAg is calculated.

TABLE 2 IC of Compounds 1-10 against HBV proliferation ₅₀ (μg/mL)

EXAMPLE 6 antitumor cell proliferation Activity of Compounds 1-10

The tumor cells were cultured in T25 culture flasks to full length, and then trypsinized and counted at about 1-2X 10 per well ⁴ The cells were plated in 96-well plates and allowed to thinAfter the cells are attached to the wall, test samples with different dilution concentrations are added, after incubation for 48 hours at 37 ℃ in 5% CO2, 20 mu L (5mg/L) of MTT solution is added into each well, incubation is continued for 6 hours, the absorption culture medium is taken out, 100 mu L of DMSO is added into each well, oscillation is carried out for 5min, the absorbance value is measured under the condition of 570nm wavelength, and the cell proliferation inhibition rate is calculated according to the following formula: the inhibition rate of cell growth (1-average absorbance in experimental group/average absorbance in control group) × 100%.

TABLE 3 IC of Compounds 1-10 against tumor cell proliferation ₅₀ (μg/mL)

Example 7 inhibitory Activity of Compounds 1-10 on proliferation of splenic lymphocytes in mice

Preparing spleen lymphocyte suspension by conventional method, resuspending with RPMI-1640 culture medium containing 10% newborn calf serum, adjusting cell concentration to 1 × 10 ⁶ Adding 100 mul/well of 96-well cell culture plate, and simultaneously adding the liquid medicine to be tested according to the experimental design. ConA is used for inducing the proliferation of mouse spleen T lymphocytes, negative control is replaced by corresponding culture solution, and positive control mycophenolic acid (MYC) is parallelly provided with 3 multiple holes. Put 5% CO ₂ After incubation in a 37 ℃ incubator for 48 hours, the cells were stained by the MTT method, wet-packed overnight at 37 ℃, and the absorbance (OD) of each well was measured using a Victor21420 Multimarker instrument at a wavelength λ 570nm and a reference wavelength λ 630 nm. And calculating the inhibition rate IC of the proliferation of the spleen lymphocytes ₅₀ (μg/mL)。

TABLE 4 inhibition of proliferation of mouse splenocytes by Compounds 1-10 IC ₅₀ (μg/mL)

Claims (8)

1. Chromone compounds with the structures shown in formulas (1) to (3) and (5):

formula (1) formula (2)

Formula (3) formula (5)

Wherein the content of the first and second substances,

when R1 is H, the compound of formula (1) is compound 1;

when R1 is COCH3, the compound of formula (1) is compound 2;

when R1 is COCH3 and R2 is H, the compound of formula (2) is compound 3;

when R1 is COCH3 and R2 is Cl, the compound of formula (2) is compound 4;

when R1 is H and R2 is H, the compound of formula (2) is compound 6;

when R1 is H and R2 is Cl, the compound of formula (2) is compound 7;

when R1 is H, the compound of formula (3) is compound 5;

when R1 is COCH3, the compound of formula (3) is compound 9;

the compound of formula (5) is compound 10.

2. A process for the preparation of a compound according to claim 1, characterized in that it comprises the following steps:

a. preparing fermentation liquor of the elbow aspergillus NCC0415, wherein the preservation unit of the elbow aspergillus is the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is CGMCC number 9095;

b. filtering or centrifuging the fermentation liquor, and removing supernatant to obtain mycelium;

c. extracting mycelium with organic solvent, collecting extractive solution, concentrating under reduced pressure, and removing organic solvent to obtain crude extract;

d. and (3) performing silica gel column chromatography on the crude extract, wherein the chloroform: gradient elution is carried out on methanol at a ratio of 10: 0-7: 3, eluent is collected, HPLC detection is carried out, components with similar peaks are combined, reduced pressure evaporation is carried out, and the mixture is recorded as S1-S9;

1) fraction S2 was prepared by HPLC to give compounds 1 and 2;

2) the S4 part is sequentially subjected to Sephadex LH-20 column chromatography and HPLC preparation to obtain compounds 3 and 4;

3) the S5 part is sequentially subjected to Sephadex LH-20 column chromatography and HPLC preparation to obtain compounds 5, 6 and 7;

4) portion S6 was prepared by HPLC to afford compound 9;

5) HPLC preparation of portion S7 afforded compound 10.

3. A process for preparing a compound according to claim 2, characterized in that:

in the step 1), the mobile phase is 65% acetonitrile, a YMC 18 chromatographic column, 21.2 × 250 and 10 μm, the flow rate is 16mL/min, and the compounds 1 and 2 are respectively Rt =10.0min and Rt =15.6 min;

2) the Sephadex LH-20 column chromatography described in (1): column: 2.6 × 70cm, methanol as eluent; the HPLC is as follows: the mobile phase is 55% acetonitrile, the nanometer C18 chromatographic column 21.2 × 150,10 μm, the flow rate is 16 mL/min; compounds 3 and 4 are Rt =9.2min, Rt =11.5min, respectively;

3) the Sephadex LH-20 column chromatography described in (1): column: 2.6 × 70cm, methanol as eluent; the HPLC is as follows: the mobile phase is 35% acetonitrile, the nanometer C18 chromatographic column 21.2 × 150,10 μm, the flow rate is 16 mL/min; compounds 5, 6 and 7 are Rt =11.6min, Rt =15.2min, Rt =21.5min, respectively;

in the 4), the mobile phase: 50% acetonitrile, nano-micro C18 chromatography column, 21.2 x 150mm,10 μm,16mL/min, compound 9, Rt =10.5 min;

in the 5), the mobile phase is 30% acetonitrile, a nano-micro chromatographic column, 21.2 × 250, C18,10 μm and 16mL/min, the compound is 10, and Rt =6.5 min.

4. A process for preparing a compound according to claim 2, characterized in that:

the step a is specifically as follows: inoculating Aspergillus elbow into fermentation culture medium, and culturing at 22-30 deg.C for 5-8 days to obtain fermentation liquid;

the fermentation medium is prepared by the following method: 10.0-50.0 g of glucose, 10.0-50.0 g of soluble starch, 4.0-20.0 g of hot-pressed soybean cake powder, 4.0-20.0 g of cottonseed powder, 1.0-10.0 g of potassium dihydrogen phosphate, 0.5-2.0 g of sodium chloride, 0.5-2.0 g of magnesium sulfate heptahydrate, adding tap water to a constant volume of 1000mL, keeping the pH value at 6.5-7.0, and sterilizing at 121 ℃ for 30 min;

in the step c, the organic solvent is methanol, ethanol, acetone or ethyl acetate; leaching for 2-4 times, each time for 2-4 hours, and mixing the leaching solutions.

5. The use of a compound of claim 1 for the manufacture of a medicament for treating or preventing an IMPDH mediated disease in a mammal.

6. The use of claim 5, wherein: the disease includes immune rejection, autoimmune disease, viral infection, inflammation, tumor, parasitic infection or vascular hyperproliferation disease.

7. The use of claim 6, wherein: the disease includes immune rejection, autoimmune disease, viral infection, inflammation or tumor.

8. A pharmaceutical composition comprising a compound of any one of claim 1 and a pharmaceutically acceptable carrier.