CN118005709A - CDDO-Me/Ozanimod co-prodrug based on azo reductase activation as well as preparation method and application thereof - Google Patents
CDDO-Me/Ozanimod co-prodrug based on azo reductase activation as well as preparation method and application thereof Download PDFInfo
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- CN118005709A CN118005709A CN202410110694.8A CN202410110694A CN118005709A CN 118005709 A CN118005709 A CN 118005709A CN 202410110694 A CN202410110694 A CN 202410110694A CN 118005709 A CN118005709 A CN 118005709A
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- compound
- ozanimod
- cddo
- prodrug
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- XRVDGNKRPOAQTN-FQEVSTJZSA-N 5-[3-[(1s)-1-(2-hydroxyethylamino)-2,3-dihydro-1h-inden-4-yl]-1,2,4-oxadiazol-5-yl]-2-propan-2-yloxybenzonitrile Chemical compound C1=C(C#N)C(OC(C)C)=CC=C1C1=NC(C=2C=3CC[C@@H](C=3C=CC=2)NCCO)=NO1 XRVDGNKRPOAQTN-FQEVSTJZSA-N 0.000 title claims abstract description 49
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
- C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1011—Condensed systems
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
- C09K2211/1048—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms with oxygen
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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Abstract
The invention belongs to the technical field of biological medicines, and relates to an azo reductase activated CDDO-Me/Ozanimod co-prodrug, a preparation method and application thereof, wherein the co-prodrug has a structure shown in a general formula I: The CDDO-Me/Ozanimod co-prodrug of the invention can be specifically activated by azo reductase in intestinal inflammation, generates fluorescence and releases active raw drugs CDDO-Me and Ozanimod through 1,6 elimination reaction, and plays a synergistic effect for diagnosis and treatment of colonitis. The co-prodrug not only can provide accurate inflammation diagnosis based on fluorescence, but also can target and gather at the intestinal tract part, and increase the selective accumulation of two parent drugs in the intestinal tract, thereby playing the roles of selective fluorescent tracing and synergistic high-efficiency anti-colonitis treatment.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a CDDO-Me/Ozanimod co-prodrug based on azo reductase activation, a preparation method and application thereof.
Background
Inflammatory Bowel Disease (IBD) is a chronic, progressive, incurable intestinal disease consisting of crohn's disease and ulcerative colitis, resulting from an imbalance in the intestinal mucosal immune response of genetically susceptible individuals to environmental factors. Currently, immune-mediated IBD prevalence in young people steadily increases, which is associated with fast-paced life, high-fat food intake, irregular diet, and great stress in work or school for a long time that has caused damage to their physical and mental health. Thus, there is a great clinical need to investigate early diagnosis and treatment of IBD.
The oleanolic acid derivative 2-cyano-3, 12-dioxooleanolic acid-1, 9 (11) -diene-28-carboxylic acid methyl ester (CDDO-Me) is an anti-inflammatory and antioxidant, which has been used for inhibiting and treating chronic diseases such as gastrointestinal tract, cardiovascular, liver, neurodegenerative diseases, kidney diseases, etc., however, high doses of CDDO-Me significantly increase the risk of adverse reactions, especially heart failure. Currently, the a-ring α -cyano- α, β -unsaturated ketone fragment (CUK) of CDDO-Me is widely considered to have strong electrophilicity, which is responsible for cardiac side effects caused by CDDO-Me. Thus, the present invention contemplates masking the electrophilicity of CUK fragments by introducing nucleophilic groups, reducing their side effects. Ozanimod is an oral sphingosine 1-phosphate (S1P) receptor modulator that has been FDA approved for the treatment of active secondary progressive diseases, particularly moderate to severe active crohn' S disease and colitis. Although S1P is overexpressed in inflamed tissues, S1P is also expressed in other normal organs or tissues, including heart, brain, liver and stomach. In normal tissues, the adverse effects of Ozanimod on SIP are associated with adverse reactions such as hypertension, heart block, bradycardia and macular edema of Ozanimod. In view of this, prodrug strategies may be employed to enhance Ozanimod preferential distribution at the site of colitis, thereby reducing side effects.
Fluorescence imaging has become one of the research hotspots in the medical field, and generally combines selective imaging and treatment, so that diagnosis and treatment efficiency is improved, and pain of patients is relieved. Meanwhile, the visual distribution and release of the medicine at the target position are facilitated, and the real-time monitoring and the individual administration of the treatment effect are realized. Meanwhile, azo reductase is mainly produced by intestinal flora, and IBD is accompanied by abnormal increase of azo reductase level, so that the azo reductase is an ideal biomarker of colonitis. Therefore, the development of the azo reductase activated diagnosis and treatment co-prodrug has good application prospect.
Disclosure of Invention
The invention aims to provide CDDO-Me/Ozanimod co-pro-drugs based on azo reductase activation, and a preparation method and application thereof, so as to solve the problems in the background art.
In order to achieve the above purpose, the present invention provides the following technical solutions: an azo reductase-based activated CDDO-Me/Ozanimod co-prodrug having the structure of formula I:
The invention also provides a preparation method of the CDDO-Me/Ozanimod co-prodrug based on azo reductase activation, which comprises the following steps:
s1, carrying out condensation reaction on 1, 9-dimethyl-6-nitro-9H-pyrido [3,4-b ] indole-3-formaldehyde (1) and iodized 1, 4-dimethyl quinolinium (2) to obtain a compound 3, and then carrying out reduction on the compound 4 by iron powder;
S2.4-nitrobenzyl alcohol (7) is reduced by zinc powder, and FeCl 3·6H2 O is oxidized to obtain a compound 8;
S3, generating a compound 5 from the compound 4 and the compound 8 under the catalysis of acetic acid, and then carrying out an Appel reaction under the actions of CBr 4 and PPh 3 to obtain a compound 6;
S4. Reacting CDDO-Me with Ozanimod in DMF solution of/K 2CO3 to obtain intermediate 11, and then carrying out etherification reaction on intermediate 11 and compound 6 under the condition of DMF solution of K 2CO3 to obtain compound I.
The synthetic route is as follows:
The invention also provides application of the CDDO-Me/Ozanimod co-prodrug based on azo reductase activation in preparation of a colitis-selective near-infrared fluorescence imaging reagent with azo reductase activation response.
The invention also provides application of the CDDO-Me/Ozanimod co-prodrug based on azo reductase activation in preparation of a reagent with a therapeutic effect on colonitis.
The invention also provides application of the CDDO-Me/Ozanimod co-prodrug in preparing a reagent for monitoring in vivo release of the original drugs CDDO-Me and Ozanimod. The CDDO-Me/Ozanimod co-prodrug can realize the in vivo visual release and dynamic distribution monitoring of CDDO-Me and Ozanimod prodrugs.
The invention also provides application of the CDDO-Me/Ozanimod co-prodrug based on azo reductase activation in preparation of a colitis selective drug release reagent with azo reductase activation response.
The invention also provides application of the CDDO-Me/Ozanimod co-prodrug in preparation of a reagent with synergistic anti-inflammatory effect of the double original drugs CDDO-Me and Ozanimod.
The invention also provides application of the CDDO-Me/Ozanimod co-prodrug in preparing a reagent for monitoring the colonitis treatment effect in real time. The CDDO-Me/Ozanimod co-prodrug can realize the application of the real-time monitoring effect on the treatment effect of the colonitis in vivo.
Compared with the prior art, the invention has the beneficial effects that: the invention adopts a co-prodrug strategy, combines the fluorescent fragment of enzyme response and the drug release characteristic, designs and synthesizes the CDDO-Me/Ozanimod diagnosis and treatment co-prodrug I based on azo reductase activation, utilizes the hydroxyl of Ozanimod to be coupled with electron-deficient CUK of CDDO-Me, can selectively degrade azo groups in the compound of the invention by utilizing azo reductase at a colonitis tissue target part, releases beta-carboline quinolinium fluorescent probes and double raw drugs CDDO-Me and Ozanimod through 1,6 elimination reaction, not only plays a role of the double raw drugs CDDO-Me and Ozanimod in synergistic high-efficiency anti-inflammatory effect, but also can reduce the side effect of the raw drugs on other tissues; on the other hand, the beta-carboline quinolinium fluorescent probe is utilized to play an imaging diagnosis role on the colonitis part, meanwhile, the release of the original drugs CDDO-Me and Ozanimod in the body is monitored, the colonitis treatment effect can be monitored in the later period, and the diagnosis, curative effect monitoring and double-drug synergistic treatment effects on the colonitis are realized.
Drawings
FIG. 1 is a graph of fluorescence spectrum data of the response of the compound of the present invention to azo reductase;
FIG. 2 is a graph of fluorescence data for specific responses of the azo reductase compounds of the present invention;
FIG. 3 is a graph showing in vitro release of CDDO-Me and Ozanimod prodrugs of the azo reductase responses of the compounds of the present invention;
FIG. 4 is a schematic representation of the imaging of a compound of the invention in a cell model of colitis;
FIG. 5 is a fluorescence imaging of a compound of the invention in an in vivo model of colitis;
FIGS. 6-7 are graphs of anti-colitis capacity of compounds of the present invention in vivo.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1:4- ((((E) -4- (E) -2- (6- (E) - (4- (((4 aR,6 aS) 6aS,6bR,8aS,12 bR,14 bS) -2-cyano-1- (2- (((S) -4- (5- (3-cyano-4-isopropoxyphenyl) -1,2, 4-oxadiazol-3-yl) -2, 4-oxadiazol-3 yl) -2, 3-dihydro-1H-inden-1-yl) ethoxy) -8 a-; methoxycarbonyl) -4, 6a,6ba,11, 14 b-heptylmethyl-methyl-1-oxo substituted-1, 4a,5, 6a,6b,7, 8a,9,10,11,12 a,12b,13,14 b-octadecan-3-yl oxy) methyl) phenyl) diazenyl-1, 9-dimethyl-9H-pyrido [3,4-b ] indol-3-yl) vinyl) -1-methylquinoline-1-iodonium salt (I)
S1 preparation of Compound 3
Compound 3: (E) -4- (2- (1, 9-dimethyl-6-nitro-9H-pyrido [3,4-b ] indol-3-yl) vinyl) -1-methylquinoline-1-iodonium salt
1, 9-Dimethyl-6-nitro-9H-pyrido [3,4-b ] indole-3-carbaldehyde (1) (533 mg,2.0 mmol) and 1, 4-dimethylquinolinium iodide (2) (587.56 mg,2.0 mmol) were added to a single vial, dissolved in absolute ethanol (10 mL) followed by 1-2 drops of piperidine and refluxed at 85℃for 12H. After TLC monitoring the end of the reaction, suction filtration, recrystallization and purification are carried out again to obtain the compound 3, the yield 79％.1H NMR(DMSO-d6,400MHz)δ9.36(d,J＝6.5Hz,1H,ArH),9.30(d,J＝2.3Hz,1H,ArH),8.92(d,J＝8.1Hz,1H,ArH),8.65(m,1H,ArH),8.49(dd,J＝9.3,2.5Hz,2H,2ArH),8.32(d,J＝7.8Hz,1H,ArH),8.13(d,J＝7.6Hz,1H,ArH),7.97(s,1H,ArH),7.71(dd,J＝7.7,1.6Hz,1H,CH＝C),7.48(m,1H,CH＝C),4.57(s,3H,CH3),4.30(s,3H,CH3),3.17(s,3H,CH3).
S2 preparation of Compound 4
Compound 4: (E) -4- (2- (6-amino-1, 9-dimethyl-9H-pyrido [3,4-b ] indol-3-yl) vinyl) -1-methylquinoline-1-iodonium salt
After compound 3 (580 mg,1.7 mmol) was dissolved in ethanol, 3g of iron powder and 4g of ammonium chloride were added to the reaction system, and reacted at 80℃for 0.5h. Filtering the reaction solution while the reaction is hot after the reaction is finished, spin-drying to obtain the compound 4, and the yield is high 71.2％.1HNMR(DMSO-d6,400MHz)δ9.36(d,J＝6.5Hz,1H,ArH),9.30(d,J＝2.3Hz,1H,ArH),8.92(d,J＝8.1Hz,1H,ArH),8.65(m,1H,ArH),8.49(dd,J＝9.3,2.5Hz,2H,2ArH),8.32(d,J＝7.8Hz,1H,ArH),8.13(d,J＝7.6Hz,1H,ArH),7.97(s,1H,ArH),7.71(dd,J＝7.7,1.6Hz,1H,CH＝C),7.48(m,1H,CH＝C),4.57(s,3H,CH3),4.47(s,2H,NH2),4.30(s,3H,CH3),3.17(s,3H,CH3).
S3 preparation of Compound 8
Compound 8: (4-nitrosophenyl) methanol
To a solution of 4-nitrobenzyl alcohol (3.5 g,21.0 mmol) in 40mL ethanol was added ammonium chloride (1.6 g,29.4 mmol) and zinc powder (4.1 g,63.0 mmol). The reaction mixture was stirred at room temperature for 30 minutes. After the reaction, insoluble matter was filtered off. The remaining solution was added dropwise to a solution of ferric chloride hexahydrate (6.8 g,25.2 mmol) in 20mL of water and 8mL of ethanol at-5 ℃. The reaction mixture was stirred for an additional 30 minutes while maintaining the temperature below-5 ℃. The resulting solution was diluted with brine and extracted with DCM. The organic layer was washed with water, dried over magnesium sulfate, and the organic solvent was removed under reduced pressure. The crude product was obtained without purification as compound 8.
S4 preparation of Compound 5
Compound 5:4- ((E) -2- (6- ((E) - (4- (hydroxymethyl) phenyl) diazenyl) -1, 9-dimethyl-9H-pyrido [3,4-b ] indol-3-yl) vinyl) -1-methylquinoline-1-iodonium salt
Compound 4 (2 g,3.96 mmol) and compound 8 (0.96 g,7.92 mmol) were dissolved in 5mL of ethanol, followed by the addition of 5mL of acetic acid and reaction at room temperature for 12h. After the reaction was completed, it was concentrated under reduced pressure, and separated by column chromatography (DCM: meoh=50:1, v/v) to give compound 5 as a yellow solid in yield 65.5％.1H NMR(DMSO-d6,400MHz)δ9.34(m,1H,ArH),8.92(m,2H,2ArH),8.80(s,1H,ArH),8.59(m,1H,ArH),8.51(d,J＝15.4Hz,1H,ArH),8.44(d,J＝8.3Hz,1H,ArH),8.28(dd,J＝14.4,6.2Hz,2H,2ArH),8.22(m,1H,ArH),8.17(m,2H,2ArH),8.12(s,1H,CH＝C),7.99(m,2H,2ArH),7.93(d,J＝9.1Hz,1H,CH＝C),5.27(s,1H,OH),4.54(s,3H,CH3),4.27(s,2H,CH2),3.92(s,3H,CH3),3.15(s,3H,CH3).
S5 preparation of Compound 6
Compound 6:4- ((E) -2- (6- ((E) - (4- (bromomethyl) phenyl) diazenyl) -1, 9-dimethyl-9H-pyrido [3,4-b ] indol-3-yl) vinyl) -1-methylquinoline-1-iodonium salt
Compound 5 (1.25 g,2.0 mmol) and CBr 4 (1.35 g,4 mmol) were dissolved in anhydrous (10 mL) THF, protected with N 2, stirred at room temperature for 1h, then PPh 3 (1.1 g,4 mmol) was added and stirring continued for 5h. After the reaction was completed, the reaction solution was diluted with DCM (10 mL), the solution was washed with brine (3×10 mL), and the organic layer was dried over Na 2SO4 and concentrated, and the residue was purified by silica gel column chromatography (DCM: meoh=100:1, v/v) to give compound 6 in yield 71.0％.1H NMR(DMSO-d6,400MHz)δ9.34(m,1H,ArH),8.92(m,2H,2ArH),8.80(s,1H,ArH),8.59(m,1H,ArH),8.51(d,J＝15.4Hz,1H,ArH),8.44(d,J＝8.3Hz,1H,ArH),8.28(dd,J＝14.4,6.2Hz,2H,2ArH),8.22(m,1H,ArH),8.17(m,2H,2ArH),8.12(s,1H,CH＝C),7.99(m,2H,2ArH),7.93(d,J＝9.1Hz,1H,CH＝C),4.54(s,3H,CH3),4.27(s,2H,CH2),3.92(s,3H,CH3),3.15(s,3H,CH3).ESI-MS(m/z):calcd for C32H27N5Br+:560.1444,found 560.1450.
S6 preparation of Compound I
4- ((((E) -4- (E) -2- (6- (E) - (4- (((4 aR,6 aS) 6aS,6bR,8aS,12 bR,14 bS) -2-cyano-1- (2- (((S) -4- (5- (3-cyano-4-isopropoxyphenyl) -1,2, 4-oxadiazol-3-yl) -2, 4-oxadiazol-3 yl) -2, 3-dihydro-1H-inden-1-yl) ethoxy) -8 a-; methoxycarbonyl) -4, 6a,6ba,11, 14 b-heptylmethyl-methyl-1-oxo substituted-1, 4a,5, 6a,6b,7, 8a,9,10,11,12 a,12b,13,14 b-octadecan-3-yl oxy) methyl) phenyl) diazenyl-1, 9-dimethyl-9H-pyrido [3,4-b ] indol-3-yl) vinyl) -1-methylquinoline-1-iodonium salt (I)
CDDO-Me (100 mg,0.198 mmol) was dissolved in anhydrous DMF (4 mL) and K 2CO3 (54.7 mg, 0.3996 mmol) and Ozanimod (80.1 mg,0.198 mmol) were added and reacted at N 2 under protection at 0deg.C for 12h. Intermediate 6 (187.4 mg,0.30 mmol) was then dissolved in DMF with vigorous stirring and added dropwise to the mixture for 2h. After completion of the reaction, extracted with CH 2Cl2 (60 mL), and the organic layer was dried over Na 2SO4 and concentrated, purified by column chromatography using dichloromethane/methanol (20:1, v/v) as eluent to give yellow solid i, yield 48.0％.1H NMR(CDCl3,400MHz)δ8.57(d,J＝8.1Hz,1H,ArH),8.36(m,2H,2ArH),8.27(s,1H,ArH),8.18(s,1H,ArH),8.05(s,1H,ArH),8.01(m,2H,2ArH),7.71(q,J＝4.6Hz,3H,3ArH),7.55(d,J＝8.3Hz,2H,2ArH),7.33(d,J＝8.2Hz,2H,2ArH),7.09(m,4H,4ArH),6.84(m,2H,2ArH),6.72(dd,J＝11.1,5.3Hz,2H,2ArH),6.50(s,1H,NH),5.77(s,1H,CH＝C),5.49(d,J＝11.0Hz,1H,CH),5.42(d,J＝11.1Hz,1H,CH),4.97(d,J＝8.1Hz,1H,CH),4.80(s,2H,CH2),4.47(s,3H,CH3),4.01(s,1H,CH),3.80(s,3H,OCH3),3.69(s,3H,CH3),3.47(s,3H,CH3),3.10(m,1H,CH),2.92(d,J＝4.7Hz,1H,CH),1.99(dd,J＝9.9,4.4Hz,1H,CH),1.88(dt,J＝13.6,4.3Hz,2H,CH2),1.78(m,1H,CH),1.70(m,9H,4CH2,CH),1.44(m,1H,CH),1.34(m,1H,CH),1.25(d,J＝7.1Hz,6H,2CH3),1.13(d,J＝10.1Hz,7H,2CH3,CH),1.07(m,21H,CH3,9CH2).13C NMR(CDCl3,101MHz)δ199.9,178.4,176.8,173.8,173.0,149.5,147.4,146.6,138.2,131.0,129.1,124.4,121.2,119.3,83.5,83.2,74.0,60.8,58.2,53.9,51.9,49.8,47.4,45.4,44.2,43.82,42.56,41.58,40.65,36.0,34.5,33.3,32.9,31.6,30.7,28.3,25.0,23.9,23.7,23.4,23.1,22.7,21.4,21.3,21.3,21.0,20.7,19.3,18.5.ESI-MS(m/z):calcd for C87H93N10O7 +:1389.7223,found 1389.7276.
Example 2: fluorescence spectrum test of the inventive compound based on azo reductase activation response
Compound I of the present invention was dissolved in deionized water containing 5% (v/v) DMSO, various concentrations of azo reductase (0-0.40. Mu.g/mL) were added, and incubated at 37 ℃. The emission spectrum was recorded with a fluorescence spectrometer (RF-5301 PC).
The results show that the inventive compound i shows a relatively weak fluorescence upon excitation at 650 nm. However, immediately after treatment with azo reductase, up to 11-fold fluorescence response was observed at 650 nm. Furthermore, fluorescence titration analysis showed a dose-dependent increase in fluorescence intensity, reaching a maximum when 0.4 μg/mL azo reductase was used (fig. 1A). In this case, the fluorescence intensity had a good linear correlation with the azo reductase concentration between 0 and 0.4. Mu.g/mL (FIG. 1B). The compounds of the invention thus find use in detecting and quantifying the presence of azo reductases at low concentrations.
Example 3: fluorescence test of azo reductase specific response of the Compounds of the invention
The compound I of the present invention was dissolved in deoxyDMSO. In a test tube, 4mL of PBS (pH 7.4) was mixed, followed by the addition of azo reductase or a solution containing various biological analytes (K +、Zn2+, LAP, L-phenylalanine, L-serine, NQO1, vcNa, H 2O2 and azo reductase (AzoR)). The final volume was adjusted to 5mL with PBS. After incubation at 37 ℃ for 90 minutes, the configured solution was transferred to a 1cm long quartz cell for measurement.
As shown in FIG. 2, the compound I of the present invention has excellent selectivity for the diazo reductase as compared to other competitive analytes (K +、Zn2+, LAP, L-phenylalanine, L-serine, NQO1, vcNa, H 2O2, azoR).
Example 4: in vitro azo reductase response CDDO-Me and Ozanimod original drug release test of the compound
The compound I of the present invention was dissolved in deionized water containing 5% (v/v) DMSO, and then incubated with azo reductase at 37℃in the absence of light. CDDO-Me and Ozanimod drug release were monitored by high performance liquid chromatography at various time points (0, 1, 2, 4, 6, 8 and 12 h).
The results show that the compound I of the invention itself has a signal peak at 20.9 min. After the reaction with azo reductase, the signal peak of the compound I of the present invention was reduced, and a new peak was present at 19.2min and 7.8min, respectively, wherein the peak at 7.8min was identical to the retention time of CDDO-Me, and the peak at 19.2min was identical to the retention time of Ozanimod (FIG. 3). Therefore, the compound I can effectively release drug molecules at target sites and exert therapeutic and diagnostic functions on the colon inflammation.
Example 5: selective fluorescent imaging test of compound of the invention on colon inflammatory cell model
Intracellular fluorescence activation and cell selectivity of RAW264.7 were assessed by confocal microscopy images. RAW264.7 cells (2X 10 5 cells/well) were seeded into 35mm glass-bottomed cell culture dishes, after 12h growth in a cell incubator, RAW264.7 cells were washed and treated with lipopolysaccharide (LPS, 10. Mu.g/mL) in fresh medium at 37℃for 12h. All cells were then washed twice with PBS solution and incubated with Compound I (20. Mu.M) in DMEM medium for 1, 2 and 4h. Thereafter, fluorescence images were recorded using a LEICA TCS SP LSM confocal microscope with a 40X objective.
The results indicate that fluorescence was found in the red channel when RAW 264.7 cells were incubated with compound i of the invention, whereas red fluorescence was very weak when incubated with PBS (fig. 4A). Over time, the fluorescence intensity gradually increased and reached a maximum at 4h, which increased by about 9-fold compared to the PBS group (fig. 4B). This means that the compounds of the invention can detect azo reductase in colitis cells and release fluorescence for diagnosis of colitis.
Example 6: in vivo fluorescence imaging test of the Compounds of the invention
To evaluate in vivo fluorescence imaging of compound i of the present invention, female BALB/c mice were divided into 2 groups (n=3). For dextran sulfate sodium salt (DSS) groups, mice were fed 3.5% DSS solution for 7 days, and control groups were drinking distilled water. On day 7, all mice were given intravenous injection of compound i of the invention (200 μΜ,50 μl). Whole body fluorescence images were captured using the IVIS luminea imaging system. Finally, mice were euthanized and immediately their hearts, livers, spleens, kidneys and intestines were collected and subjected to ex vivo fluorescence imaging on the IVIS lumine system.
Experimental results indicate that the fluorescence intensity of IBD mice increases gradually over time. As shown in figures 5A and 5B, DSS-induced IBD mice exhibited strong NIR fluorescence 1h post injection and remained at high intensity levels for at least 12h. In addition, other major organs (heart, liver, spleen, lung and kidney) were collected from the mice. Other organs besides the colon had no apparent fluorescent signal (fig. 5C and 5D). These results show that azo reductase is mainly distributed in colon inflammation, and can realize the real-time monitoring effect on the treatment effect of colonitis in vivo, and can be used as a diagnostic reagent for researching colonitis diseases.
Example 7: evaluation of the in vivo anti-colitis Capacity of the Compounds of the invention
To assess the therapeutic effect of compound i of the present invention on the mouse colitis model, this was achieved by measuring body weight, assessing colon length and tissue H & E staining. The experiment is divided into seven groups (a.PBS;b.DSS;c.DSS+Ozanimod;d.DSS+CDDO-Me;e.DSS+Ozanimod+CDDO-Me;f.DSS+0.74μmol/LⅠ;g.DSS+1.48μmol/LⅠ).
First, the DSS group showed a significant decrease in body weight from day two, peaking at day 7, indicating successful construction of the mouse DSS colitis model (fig. 6A). Second, there were significant differences in disease occurrence index, mouse body weight, colon length for positive control, drug and blank groups. The colon length (4.5.+ -. 0.23 cm) of the DSS group was significantly shorter than that of the PBS group (8.2.+ -. 0.41 cm) after the colon injury of the model group, whereas the weight gain (8.1.+ -. 0.2 g) and the colon length gain (2.2.+ -. 0.25 cm) of the mice treated with the compound I of the present invention were significantly increased (FIGS. 6B-6D), and the therapeutic effect was more significant with the increase of the concentration of the compound I of the present invention. The colon tissue of each group was then assessed by hematoxylin and eosin (H & E) staining. As shown in fig. 6E, the PBS group has intact colonic mucosa and the cells are aligned. The colonic mucosa of the DSS group was damaged and inflammatory cells were visible in the lamina propria. The symptoms in the treatment group disappear, and the tissue morphology is restored to be normal. These results strongly demonstrate that compound i of the present invention has a significant anti-colitis capacity in IBD mice.
Finally, TNF- α, IL-6 and calfaecal protein levels were determined in colon tissue of all groups of mice by ELISA. Frozen distal colon specimens were homogenized with potassium phosphate buffer (pH 6.0) containing a protease inhibitor cocktail and centrifuged at 2500rpm and 4℃for 5 minutes. The supernatant was collected and centrifuged at 10000rpm and 4℃for 10 minutes. The final samples were analyzed by ELISA to quantify TNF- α, IL-6 and calfaecal protein levels. ELISA kit Total protein concentration was determined by the Lowery method using the BCA kit (FISHER SCIENTIFIC, waltham, mass., USA) according to the manufacturer's instructions.
As shown in FIG. 7, significant increases in TNF- α and IL-6 and calprotectin levels were observed in the DSS group, while the increase in levels was substantially negligible in the control group. Compared to DSS groups, the drug-treated groups all significantly reduced cytokine levels in the serum of mice. Wherein the treatment group with compound I showed a dose-dependent decrease in TNF-alpha and IL-6 and calprotectin production in mouse serum and a better therapeutic effect than CDDO-Me alone and Ozanimod groups.
In conclusion, the CDDO-Me/Ozanimod co-prodrug based on the azo reductase specific response obtains near infrared fluorescence imaging of the azo reductase response in the intestinal tract, selectively releases the original drug in colonitis tissues, and plays a role in the selective fluorescence imaging tracing of the intestinal tract part and the original drug and the synergistic high-efficiency colonitis treatment.
Claims (7)
1. An azo reductase-based activated CDDO-Me/Ozanimod co-prodrug characterized in that the co-prodrug has the structure of formula I:
2. A method for preparing a CDDO-Me/Ozanimod co-prodrug based on azo reductase activation, characterized by: the method comprises the following steps:
s1, carrying out condensation reaction on a compound 1 and a compound 2 to obtain a compound 3, and then reducing the compound 3 by iron powder to obtain a compound 4; wherein, the compound 1 is 1, 9-dimethyl-6-nitro-9H-pyrido [3,4-b ] indole-3-carbaldehyde; compound 2 is 1, 4-dimethylquinolinium iodide;
S2, reducing the compound 7 by zinc powder, and oxidizing FeCl 3·6H2 O to obtain a compound 8; wherein, the compound 7 is 4-nitrobenzyl alcohol;
S3, generating a compound 5 from the compound 4 and the compound 8 under the catalysis of acetic acid, and then carrying out an Appel reaction under the actions of CBr 4 and PPh 3 to obtain a compound 6;
s4, reacting CDDO-Me with Ozanimod in a DMF solution of K 2CO3 to obtain an intermediate 11, and then carrying out etherification reaction on the intermediate 11 and a compound 6 in a DMF solution of K 2CO3 to obtain a compound I;
The synthetic route is as follows:
3. Use of a CDDO-Me/Ozanimod co-prodrug of claim 1 in the preparation of a colitis-selective near-infrared fluorescence imaging agent with azo reductase activation response.
4. Use of a CDDO-Me/Ozanimod co-prodrug of claim 1 in the preparation of an agent having a therapeutic effect on colitis.
5. Use of a CDDO-Me/Ozanimod co-prodrug of claim 1 in the preparation of a reagent for monitoring the release of the prodrugs CDDO-Me and Ozanimod in vivo.
6. Use of a CDDO-Me/Ozanimod co-prodrug of claim 1 in the preparation of a reagent having synergistic anti-inflammatory effects against the dual prodrugs CDDO-Me and Ozanimod.
7. Use of a CDDO-Me/Ozanimod co-prodrug of claim 1 in the preparation of a reagent for monitoring the effect of treatment of colitis in real time.
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