CN113527153A - Active compound for inhibiting intestinal inflammatory reaction - Google Patents
Active compound for inhibiting intestinal inflammatory reaction Download PDFInfo
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- CN113527153A CN113527153A CN202010312884.XA CN202010312884A CN113527153A CN 113527153 A CN113527153 A CN 113527153A CN 202010312884 A CN202010312884 A CN 202010312884A CN 113527153 A CN113527153 A CN 113527153A
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Abstract
The invention discloses an active compound for inhibiting intestinal inflammatory reaction, which has a structure shown in a formula I, wherein the definition of each substituent group is described in the specification and the claims. The compound of the invention can promote intestinal mucosa repair and barrier homeostasis maintenance, thereby inhibiting over-activated intestinal inflammatory reaction and immune response, and obviously reducing colon inflammation of IBD mice, thereby being used for treating inflammatory intestinal diseases.
Description
Technical Field
The invention belongs to the field of medicinal chemistry, and particularly relates to an active compound for inhibiting intestinal inflammatory reaction, a preparation method and application thereof.
Background
Inflammatory Bowel Disease (IBD) includes Ulcerative Colitis (UC) and Crohn's Disease (CD), characterized by recurrent episodes of inflammation of the digestive tract. In recent years, global incidence has seen a dramatic rise. The pathogenesis of IBD is not clear up to now and studies have shown to be associated with genetic, immunological, infectious and mental activities. Repeated attacks of disease can cause DNA damage and microsatellite instability of mucosal cells, leading to the development of colon cancer. It has been demonstrated that IBD patients are significantly more at risk of developing colon cancer than normal populations. The use of immunosuppressive agents and anti-cytokines improves the recurrence rate of IBD patients, however, there is still a lack of drugs that can cure IBD. Therefore, it is of great interest to find key molecules in the pathogenesis and/or disease progression of IBD and to develop new methods for treating IBD based on this.
Under physiological conditions, intestinal epithelial cells and mucus layers formed by secretion of the intestinal epithelial cells are combined with various specific and nonspecific protection mechanisms to jointly establish an effective intestinal mucosal barrier, which is of great importance in intestinal homeostasis maintenance and provides a first defense line of body defense. However, mucin secretion disorders, impaired epithelial integrity, and failure of the immune response by pathogenic microorganisms, alone or in combination, can lead to the development of inflammatory bowel disease. Of the inflammatory cytokines involved in the pathogenesis of IBD, members of the IL-12 family, particularly IL-23, are widely regarded as central factors in experimental models of enteritis and in the pathogenesis of human IBD. IL-23 can activate CD4+Memory cells, CD8+Cells, NK cells and a few mononuclear macrophages/dendritic cells. By binding to the IL-23 receptor (IL-23R), secretion of IL-10, IL-17, INF-gamma, and the like is induced, thereby promoting the development of inflammation. Recent human genetic studies have established that IL-23R variation is associated with inflammatory responses in the small intestine (ileum CD) and large intestine (UC). IL-23 was found to maintain and expand Th17 cell function and to exacerbate the inflammatory response of the intestinal tract. The P19 gene of IL-23 of the mouse is knocked out, so that the condition of T cell-dependent colitis of the mouse can be obviously relieved. Therefore, IL-23 is considered to be a promising new target for the treatment of IBD.
In recent years, the advent of biological therapies (such as anti-TNF therapy) has greatly improved the treatment of IBD. However, there are still a large number of patients with refractory ulcers or refractory epithelial isomerism, making "mucosal healing" difficult for these patients. Mucosal healing indicates that IBD patients have restored intestinal epithelial structure and function, while also revealing a long-term remission or low-risk prognosis of surgery. The importance of restoring the structure and function of the intestinal epithelium has been identified in the treatment of IBD at present; mucosal healing has also reached consensus among clinicians and researchers as a standard goal of treatment. However, suitable therapeutic means are not yet available. Intestinal stem cells located at the bottom of the intestinal crypts were found to be critical in maintaining intestinal epithelial cell self-renewal, structure and physical barrier function. Animal experiments show that the transplanted intestinal stem cells can remarkably improve the mucosal injury of a DSS-induced IBD mouse model, and the enhancement of the activity of the intestinal stem cells has the potential of repairing IBD refractory ulcer, so that the prognosis of an IBD patient is improved.
Therefore, it is of great interest to find key molecules in the pathogenesis and/or disease progression of IBD and to develop new methods for treating IBD based on this.
Disclosure of Invention
The invention aims to provide an active compound for inhibiting intestinal inflammatory reaction.
In a first aspect of the invention, there is provided a compound of formula (I), or an enantiomer, diastereomer, racemate, or mixture thereof, or a pharmaceutically acceptable salt thereof,
in the formula (I), the compound is shown in the specification,
x is CH, O or N;
y is CH, O or N;
m is 0 or 1;
R1、R2each independently is H, -NHCOO (C1-C4 alkyl), -NHCONH (C1-C4 alkyl), -NHCO (C1-C4 alkyl), -COO (C1-C4 alkyl), -NHCON (C1-C4 alkyl) (C1-C4 alkyl), C1-C4 alkyl, C1-C4 haloalkyl, CN, halogen, C6-C10 aryl, C1-C4 alkoxy, hydroxy or carboxy; or R1、R2Taken together with the attached carbon to form a substituted or unsubstituted 5-6 membered heterocyclyl; substituted means substituted with one or more substituents selected from the group consisting of: oxo, C1-C4 alkyl, C1-C4 haloalkyl, -NHCOO (C1-C4 alkyl), CN, halogen, C1-C4 alkoxy, hydroxy, carboxy;
W1is-SO2NH-、-CONH-、-SO2-, -NH-or-4 to 6 membered heteroaryl-NH-;
W2is absent, C1-C4Alkylene or-NH-;
W3is absent, C1-C4Alkylene, phenyl (C1-C4 alkylene), C3-C6 cycloalkyl, or 3-6 membered heterocycloalkyl;
R4is H, -OH, C1-C4 alkyl, -O (C1-C4 alkyl), -NH (C3-C6 cycloalkyl), -NH (C1-C4 alkyl), -NHSO2(C1-C4 alkyl) or-NH (3-6 membered heterocycloalkyl);
n is 0 or 1.
In another preferred embodiment, the heterocyclic group comprises a saturated or partially unsaturated cyclic hydrocarbon group, and heteroaryl groups are also included.
In another preferred embodiment, the compound has the structure shown in formula II below:
R1、R2、R3、R4、W1、W2、W3n is as defined above.
In another preferred embodiment, the compound has the structure shown in formula III below:
R1、R2、R3、R4、W1、W2、W3as defined above.
In another preferred embodiment, R1、R2Each independently is CN, halogen, -NHCOO (C1-C4 alkyl), -NHCONH (C1-C4 alkyl), -NHCO (C1-C4 alkyl), -COO (C1-C4 alkyl), -NHCON (C1-C4 alkyl) (C1-C4 alkyl), C1-C4 alkyl, C1-C4 haloalkyl, C6-C10 aryl, C1-C4 alkoxy, hydroxyl, carboxyl or hydrogen.
In another preferred embodiment, W1is-SO2NH-、-CONH-、-SO2-, -NH-or-4 to 6 membered heteroaryl-NH-;
W2is C1-C4Alkylene or-NH-;
W3is absent, C1-C4Alkylene, phenyl (C1-C4 alkylene), C3-C6 cycloalkyl, or 3-6 membered heterocycloalkyl.
In another preferred embodiment, R3Is H, halogen, C1-C4 haloalkyl, C1-C4 alkyl or C1-C4 alkoxy.
In another preferred embodiment, R4is-OH, -O (C1-C4 alkyl), -NH (C3-C6 cycloalkyl), -NH (C1-C4 alkyl) or-NHSO2(C1-C4 alkyl).
In another preferred embodiment, the compound has the following formula IV,
wherein the content of the first and second substances,
x 'and Y' are each independently carbon, nitrogen or oxygen;
R1' is selected from-Z-T, wherein Z is selected from-NHC (═ O) -, -OC (═ O) -, -NHS (═ O) -, -OS (═ O) -, -NHSO2-、-C(＝O)NH-、-C(＝O)O-、-S(＝O)NH-、-S(＝O)O-、-SO2NH-、-HNC(＝O)NH-、-OC(＝O)NH-、-NHS(＝O)NH-、-OS(＝O)NH-、-NHSO2NH-、-NHC(＝O)O-、-NHS(＝O)O-、-NHC(＝O)O-、-OC(＝O)O-、-NHS(＝O)O-、-OS(＝O)O-、-NHSO2O-、-OC(＝O)NH-、-OS(＝O)NH-、-OSO2NH-; t is selected from hydrogen and C2-C4Alkyl radical, C3-C8Cycloalkyl or C substituted by E1-C4Alkyl radical, C3-C8Cycloalkyl, phenyl or phenyl substituted by E, naphthyl or naphthyl substituted by E, tetrahydronaphthyl or tetrahydronaphthyl substituted by E, a fused heterocycle or a fused heterocycle substituted by E, pyridyl or pyridyl substituted by E, pyrimidinyl or pyrimidinyl substituted by E, or a five-or six-membered heteroaryl substituted by E containing 1 or 2N atoms, a five-or six-membered heteroaryl group substituted with E containing 1 or 2S atoms, a five-or six-membered heteroaryl group substituted with E containing 1 or 2O atoms, a five-or six-membered heteroaryl group substituted with E containing 1N atom and 1S atom, a five-or six-membered heteroaryl group substituted with E containing 1N atom and 1O atom, a five-or six-membered heteroaryl group substituted with E containing 2N atoms and 1S atom, a five-or six-membered heteroaryl group substituted with E containing 1N atom and 1O atom; or
R1’，R2' is independently selected from hydrogen, halogen, hydroxyl, amino, C1-C4Alkyl radical, C1-C4Alkoxy radical, C1-C4Alkylamino, hydroxymethyl, C1-C4A haloalkyl group;
w' is selected from-C (═ O) O-, -C (═ O) NHL1-、-S(＝O)O-、-S(＝O)NL1-、-(O＝)S(＝O)O-、-(O＝)S(＝O)NL1-；
R3' is selected from hydrogen, halogen, hydroxy, amino, C1-C4Alkyl radical, C1-C4Alkoxy radical, C1-C4Alkylamino, hydroxymethyl, C1-C4A haloalkyl group;
n' is selected from an integer between 0 and 5;
R4' selected from-COOL1、-CONL1L2、-S(＝O)OL1-、-S(＝O)NL1L2-、-(O＝)S(＝O)OL1-、-(O＝)S(＝O)NL1L2-；
L1、L2Independently selected from hydrogen, C1-C4Alkyl radical, C2-C4Alkenyl radical, C2-C4Alkynyl, C6-C10Aryl and 4-8 membered heteroaryl;
e is selected from halogen, nitro, cyano, C1-C6Alkyl radical, C1-C6Haloalkyl, C1-C6Alkoxy radical, C1-C6Haloalkoxy, C1-C6Alkylamino radical, C2-C6Alkenyl radical, C2-C6Haloalkenyl, C3-C6Cycloalkenyl radical, C2-C6Alkynyl, C2-C6Halogenated alkynyl, C3-C6Cycloalkynyl, phenyl, or 3-8 membered heterocycle (where phenyl and heterocycle may be substituted with halogen, nitro, cyano, alkyl, haloalkyl, haloalkoxy, alkoxy, alkylamino).
In another preferred embodiment, the compound is:
in a second aspect of the present invention, there is provided a pharmaceutical composition comprising:
a compound of formula (I) according to the first aspect, or enantiomers, diastereomers, racemates thereof and mixtures thereof, or a pharmaceutically acceptable salt thereof; and
a pharmaceutically acceptable carrier.
In a third aspect of the present invention, there is provided a use of the compound represented by the general formula (I) in the first aspect or the pharmaceutical composition in the second aspect, for preparing a medicament for treating inflammatory bowel disease.
The present invention also provides a method of treating inflammatory bowel disease by administering to a subject in need thereof a compound of formula (I) according to the first aspect or a pharmaceutical composition according to the second aspect.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Not to be reiterated herein, but to the extent of space.
Drawings
Figure 1 shows the results of compound a1 treatment on a mouse colitis model.
Fig. 2 is a graph showing the results of the growth promoting effect of compound a1 on mouse-derived intestinal organoids.
FIG. 3 is a graph showing the results of the therapeutic effect of Compound A1 on a chronic spontaneous IL-10 knockout mouse model of IBD.
Detailed Description
The inventor of the invention finds a compound with a general formula I through extensive and intensive research and a mode of combining virtual screening and experimental verification. The compound can promote intestinal mucosa repair and barrier homeostasis maintenance, so that the over-activated intestinal inflammatory reaction and immune response are inhibited, and the colon inflammation of IBD mice is obviously reduced, so that the compound can be used for treating inflammatory intestinal diseases, and the invention is completed on the basis.
Term(s) for
In the present invention, halogen is fluorine, chlorine, bromine or iodine;
the alkyl, alkenyl and alkynyl are straight-chain or branched-chain alkyl; alkyl groups are selected by themselves or as part of other substituents from methyl, ethyl, propyl, butyl, pentyl, hexyl and isomers thereof; the isomer is selected from isopropyl, isobutyl, sec-butyl, tert-butyl, isoamyl or tert-amyl;
the halogenated alkyl is selected from the group consisting of-CH and the halogenated alkyl contains one or more halogen atoms which may be the same or different2Cl、-CHCl2、-CCl3、-CH2F、-CHF2、-CF3、-CH2Br、-CHBr2、-CBr3、-CH2I、-CHI2、-CI3、CF3CH2、CCl3CH2、CBr3CH2、CI3CH2、。
Said cycloalkyl group is selected, by itself or as part of another substituent, from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl;
said alkenyl group is selected as such or as part of another substituent from vinyl, allyl, 1-propenyl, buten-1-yl, buten-3-yl, penten-1-yl, penten-3-yl, hexen-1-yl or 4-methyl-3-pentenyl;
said alkynyl is selected by itself or as part of another substituent from ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-2-yl, 1-methyl-2-butynyl, hexyn-1-yl or 1-ethyl-2-butenyl;
the aryl is selected from monocyclic aromatic hydrocarbon or polycyclic aromatic hydrocarbon, wherein the polycyclic aromatic hydrocarbon comprises polyphenyl aliphatic hydrocarbon, biphenyl polycyclic aromatic hydrocarbon and polycyclic aromatic hydrocarbon;
the heteroaryl group is selected from substituted furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolyl, indazolyl, benzothienyl, benzofuryl, benzimidazolyl, benzotriazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, isomerized quinolyl, phthalazinyl, quinoxalyl, quinazolinyl, cinnolinyl or naphthyridinyl.
Metabolites of the compounds and pharmaceutically acceptable salts thereof to which this application relates, and prodrugs that can be converted in vivo to the structures of the compounds and pharmaceutically acceptable salts thereof to which this application relates, are also included in the claims of this application.
The invention provides a synthesis method of the compound, and specific examples are as follows:
for example, the compound IV shown in the invention can be prepared according to the route, and the synthesis of the target product of the invention comprises the following specific steps: commercially available or prepared acyl chloride, sulfonyl chloride or sulfinyl chloride compounds 1 are reacted with amino acid compounds 2 to obtain target products 3, and the compounds 3 are reacted with amines, alcohols or iodides to prepare compounds 4 with a general formula IV.
The pharmaceutically acceptable salts of the compounds of formula I can be synthesized by general chemical methods:
in general, salts can be prepared by reacting the free base or acid with a chemically equivalent or excess of the acid (inorganic or organic) or base (inorganic or organic) in a suitable solvent or solvent composition.
To further illustrate the present invention, a series of examples are given below, which are purely illustrative and are intended to be a detailed description of the invention only, and should not be taken as limiting the invention.
Example 1
Compound A1And A2The synthesis of (2):
the first step is as follows: 1.2g of Compound a2Adding into a bottle, and slowly adding 500mg of the compound a while stirring1And heating to 50 ℃ for reaction for 2 h. After the reaction was complete, the reaction mixture was poured into ice water with vigorous stirring, keeping the system temperature below 20 ℃. The precipitate was filtered off and washed with cold water until the filtrate was neutral. The solid was dried under vacuum at 60 ℃ overnight to give 425mg of Compound a2The yield was 52%.1H NMR(400MHz,CDCl3):δ7.99(d,J＝8.8Hz,2H),7.64(d,J＝8.8Hz,2H),7.00(s,1H),3.84(s,3H)。
5.0g of Compound a4Dissolving in 150mLCH3CH2Adding 500mg palladium carbon into OH, and introducing H2And reacting for 24 hours. After the reaction was completed, the reaction mixture was filtered through celite, and the filtrate was spin-dried to obtain 4.5g of Compound a5。1HNMR(400MHz,CDCl3) δ 3.52(m,1H),3.38(m,1H),2.55(m,2H),2.41(m,1H),2.13(m,1H),1.90(m, 1H). Then, use 140mLH2O9.0 mL of concentrated HCl was diluted and charged with 4.5g of Compound a5The reaction was refluxed overnight in the flask of (1). After the reaction was complete, the reaction was cooled to room temperature, the aqueous phase was washed 3 times with DCM and the water was spin-dried to give 4.5g of Compound a6The yield of the two steps is 72%.1H NMR(400MHz,DMSO-d6):δ8.31(s,2H),3.02(m,2H),2.82(m,1H),2.40(m,2H),1.80(m,2H)。
The second step is that: 371mg of Compound a6Dissolved in 10mL THF with stirring, 2.0mL aqueous NaOH (2M) was added. After the temperature of the system is reduced to 0 ℃, 500mL of the compound a is added2The reaction was maintained at 0 ℃ for 30 min. After the reaction was complete, water was added to quench the reaction, the aqueous phase was washed 3 times with DCM and then 2M aqueous HCl was slowly added dropwise to adjust the pH of the aqueous phase to 3-4. Adding DCM for extraction for 3 times, adding anhydrous sodium sulfate for drying, and spin-drying to obtain 480mg of compound A1The yield was 60%.1H NMR(400MHz,DMSO-d6):δ12.23(s,1H),10.13(s,1H),7.75(t,J＝6.5Hz,1H),7.72(d,J＝8.8Hz,2H),7.64(d,J＝8.8Hz,2H),3.70(s,3H),2.86(m,2H),2.44(m,1H),2.32(m,2H),1.75(m,2H)；13CNMR(125MHz,DMSO-d6):δ173.59,153.82,142.99,133.00,127.80,117.76,51.98,41.56,30.32,20.55；LRMS(ESI):421.0(M+Na)+,396.7(M-H)-；HRMS(ESI):calcd for C14H16F3N2O6S(M-H)-:397.0687,found:397.0683。
The third step: mixing 100mg of Compound A1Dissolved in 5mL CH3In OH, the temperature was reduced to 0 ℃. Slowly add 35 μ LSOCl dropwise to the reaction flask while stirring2After the dropwise addition, the temperature of the system was raised to room temperature for reaction overnight. After the reaction is finished, removing CH by rotation3OH and SOCl2Post-application Et2O wash the remaining solid and dry under vacuum at 60 deg.C overnight to yield 95mg of Compound A2The yield was 92%.1H NMR(400MHz,DMSO-d6):δ7.80(d,J＝8.8Hz,2H),7.56(d,J＝8.8Hz,2H),6.90(s,1H),4.91(t,J＝6.4Hz,1H),3.82(s,3H),3.69(s,3H),3.20(m,1H),3.05(m,1H),2.46(t,J＝7.2Hz,2H),2.38(m,1H),1.90(m,2H)。
The synthetic procedures for A3-A41 refer to the synthetic route of Compound A1 described above.
Synthesis of compound a 42:
135mg of Compound a7Dissolving in 8mL pyridine, and adding compound a6Then heating to 60 ℃ for reaction for 3 h. After the reaction, the concentration is reduced, DCM is added into the concentrated solution for dilution, the organic phase is washed for 3 times by dilute HCl aqueous solution, anhydrous sodium sulfate is added for drying, and concentration is carried out to obtain 135mg of compound A42The yield was 60%.
Compound A43-A45Reference to A42The synthetic route of (1).
Example 2
Animal and cell experiments
First, experimental material
Cells and animals
HCT116 cells and raw264.7 cells were provided by the cell bank of the chinese academy of sciences; intestinal organoids were derived from intestinal crypt cultures isolated from Lgr5-EGFP mice; the Lgr5-EGFP mouse is given by Hua national institute of intense research, the genetic background is C57/BL6, and the Lgr5-EGFP mouse is bred in the SPF animal room of second floor of the center of experimental animals of the pharmaceutical institute of the Compound Dan university.
Main instruments and equipment
Primary reagents and antibodies
Second, Experimental methods
A. Cell experiments
Screening the activity of the compound:
detection of IL-23 mRNA:
all compounds were made up in DMSO to a concentration of 50mM in stock solution and stored at-20 ℃ in a freezer. Raw264.7 cells are inoculated in a 12-well plate, the cell density is about 70-80% after 24 hours, compounds with different dilution ratios are added, after 1 hour of medicine addition, 20 mu l (namely 0.5 mu g/ml) of Lipopolysaccharide (LPS) is added into each well, and the culture plate is shaken gently and mixed evenly. Blank groups were not added. After 6 hours, the medium was discarded, washed 3 times with ice-cold PBS buffer, 1ml Trizol was added, and the mixture was allowed to stand for 10min, the lysate was collected, the supernatant was obtained by centrifugation, 1/5 volumes of chloroform were added, shaken 1min, and left to stand for 3min, and 12000g was centrifuged at 4 ℃. And (3) taking the supernatant, adding isopropanol with the same volume, uniformly mixing, standing for 10min, and centrifuging for 12000Xg for 10 min. Removing supernatant, washing precipitate with 70% ethanol, centrifuging at 7500g for 5min, drying precipitate, and adding 20 μ L DEPC water. The RNA concentration of the sample was measured and adjusted to 100-1000 ng/. mu.L, and the changes in the IL-23mRNA level were detected using a reverse transcription kit from an assist organism and a SYBR-qPCR kit.
(1) Reverse transcription to synthesize cDNA
According to the instructions of the reverse transcription kit, the following mixture was prepared in an RNase free centrifuge tube and gently pipetted and mixed. Incubate at 42 ℃ for 2 min. The reverse transcription reaction procedure is as follows, stage 1:25 ℃,5 min; stage 2, 42 ℃ for 30 min; stage 3, 85 ℃ and 5 min; stage 4, keeping at 4 ℃, and storing the product at-20 ℃ or directly using the product in RT-qPCR.
(2) RT-PCR amplification
The primers involved in Real-Time PCR were designed using primer premier 5.0 software, and the primer sequences were as follows:
the two-step method is adopted for carrying out the programmed reaction, and the specific program is as follows:
Stage1:95℃，5min
Stage2:95℃，5s
60℃，30s
by using
Method for analyzing data and calculating
The value of (A) is the fold of the expression of the target gene in the experimental group relative to the control group.
Detection of CyclinD1 mRNA:
the colorectal cancer HCT116 cell strain is subjected to compound screening, and the culture medium is McCOY's 5A culture medium containing 10% FBS. Inoculating the cells into a 12-hole plate, and enabling the density of adherent cells to be 70% -80% on the next day. mu.L of stock solution (50mM) was added to 1ml of the medium to prepare 50. mu.M of the drug-containing medium (control group, 0.1% DMSO medium). Discarding the original culture medium, adding the medicated culture medium, and culturing at 37 deg.C for 6 hr. Discarding the culture medium, washing with ice-cold PBS buffer for 3 times, adding 1ml Trizol, standing for 10min, collecting lysate, centrifuging to obtain supernatant, adding 1/5 volume of chloroform, shaking for 1min, standing for 3min, and centrifuging at 4 deg.C for 12000 Xg. And (3) taking the supernatant, adding isopropanol with the same volume, uniformly mixing, standing for 10min, and centrifuging for 12000Xg for 10 min. Removing supernatant, washing precipitate with 70% ethanol, centrifuging at 7500Xg for 5min, drying precipitate, and adding 20 μ L DEPC water. The RNA concentration of the sample was measured and adjusted to 100-1000 ng/. mu.L, and the change in mRNA level of cyclinD1 was detected using a reverse transcription kit from an assist organism and a SYBR-qPCR kit.
(1) Reverse transcription to synthesize cDNA
According to the instructions of the reverse transcription kit, the following mixture was prepared in an RNase free centrifuge tube and gently pipetted and mixed. Incubate at 42 ℃ for 2 min. The reverse transcription reaction procedure is as follows, stage 1:25 ℃,5 min; stage 2, 42 ℃ for 30 min; stage 3, 85 ℃ and 5 min; stage 4, keeping at 4 ℃, and storing the product at-20 ℃ or directly using the product in RT-qPCR.
(2) RT-PCR amplification
The primers involved in Real-Time PCR were designed using primer premier 5.0 software, and the primer sequences were as follows:
the target fragment of cDNA was amplified using a CFX100 fluorescent quantitative PCR instrument from Bio-Rad, and the procedure was as follows:
collecting instrument data by
Method for analyzing data and calculating
The value of (A) is the fold of the expression of the target gene in the experimental group relative to the control group.
B. Animal experiments
Animal breeding and gene identification
Propagation of Lgr5-EGFP mice
Mouse genotype identification
Lgr5-EGFP mouse genotype identification
Gene identification sequence
| Primer and method for producing the same | Sequence of | SEQ ID NO.: |
| 8060 | 5’-CTGCTCTCTGCTCCCAGTCT-3’ | 9 |
| 8061 | 5’-ATACCCCATCCCTTTTGAGC-3’ | 10 |
| olMR9402 | 5’-GAACTTCAGGGTCAGCTTGC-3’ | 11 |
PCR was carried out as follows:
the reaction program was set up as indicated.
The reaction procedure was as follows:
1. establishment of mouse acute IBD model and chronic spontaneous IL-10 knockout IBD model and in-vivo efficacy experiment of compound A1
The experimental groups were set as a control group, a model group, a mesalazine administration group, an a1-1mg/kg administration group, and a compound a1-2mg/kg administration group, for five groups, each group of 10 8-week-old male C57/BL6 mice, except for the control group, were freely drunk with 2.5% dextran sulfate sodium salt (DSS) water, reconstituted every other day and replaced for 7 days. Compound A1Dissolving with DMSO to obtain mother solution, dissolving in ultrapure water to obtain 4% DMSO aqueous solutions with final concentrations of 0.1mg/ml and 0.2mg/ml, respectively, preparing mesalazine with 0.5% CMC-Na to obtain 5mg/ml suspension,the gavage was started at the same time as the molding, and the gavage dose was 10ml/kg (corresponding to administration doses of 1mg/kg, 2mg/kg and 50mg/kg) for 10 days. Mice weight changes were recorded daily and mice were sacrificed 10 days before sampling.
IL-10-/-Mice (Jackson laboratories, USA, C57BL/6 background). Mice of 8 weeks old were divided into a control group (WT), a model group (IL-10KO), a mesalazine group (IL-10KO + mesalazine 50mg/kg), a compound A1 low dose group (IL-10KO + compound A10.5mg/kg), a compound A1 medium dose group (IL-10KO + compound A11 mg/kg), and a compound A1 high dose group (IL-10KO + compound A12 mg/kg), and each group of mice had 6 mice each and were subjected to gastric lavage for 12 weeks.
2. Determination of serum inflammatory factors
After 10 days of the administration, the mice were bled by taking an eye-ball, and after collecting whole blood, the blood was allowed to clot undisturbed at room temperature for 15 to 30 minutes. Serum was obtained by centrifugation at 1,000 Xg for 10min at 4 ℃ in a centrifuge. Serum samples were tested for IL-1 β and IL-23 serum levels using IL-1 β and IL-23ELISA kits available from Dake, Inc.
3. Colon length measurement in mice
After blood was taken from the above eyeballs, the mice were sacrificed and the entire colon was photographed and length-measured.
4. In vivo intestinal permeability detection in mice
Intestinal permeability was measured using fluorescein isothiocyanate (FITC-dextran). After 4 hours, the eyeballs were bled by FITC-dextran (0.6mg/g mouse body weight, concentration 125mg/mL), absorbance was measured by a multi-functional microplate reader (in which excitation wavelength was set at 490nm and emission wavelength was set at 525nm) and the FITC-dextran content in the serum was calculated.
5. Detection of protein-related mRNA levels in colonic tissue
Repeatedly washing colon tissue with PBS, scraping mucus, separating colon mucosa and lamina propria of mouse, respectively adding Trizol for tissue homogenization, standing for 10min, centrifuging to obtain supernatant, adding 1/5 volume of chloroform, shaking for 1min, standing for 3min, and centrifuging at 4 deg.C for 12000 Xg. And (3) taking the supernatant, adding isopropanol with the same volume, uniformly mixing, standing for 10min, and centrifuging for 12000Xg for 10 min. Removing supernatant, washing precipitate with 70% ethanol, centrifuging at 7500Xg for 5min, drying precipitate, and adding appropriate amount of DEPC water. And detecting the RNA concentration of the sample, adjusting the RNA concentration to 100-1000 ng/. mu.L, and detecting the mRNA levels of the corresponding inflammatory factors and stem cell marker proteins by using a reverse transcription kit of an assist organism and a SYBR-qPCR kit.
6.RT-qPCR
According to the instructions of the reverse transcription kit, the following operations are carried out:
prepare the following mixture in RNase free centrifuge tube, gently blow and mix with a pipette. Incubate at 42 ℃ for 2 min.
Preparation of reverse transcription reaction System (20. mu.L System)
The reverse transcription reaction procedure is as follows
The cDNA was obtained and the working solution system was prepared according to the following table:
qPCR primers are shown in the following Table
The target fragment of cDNA was amplified using a CFX100 fluorescent quantitative PCR instrument from Bio-Rad, and the procedure was as follows
Collecting instrument data, analyzing the data by adopting a 2-delta Ct method, and calculating the numerical value of the 2-delta Ct, namely the multiple of the target gene expression of the experimental group relative to the control group.
Lgr-EGFP in vivo assay
In vivo pharmacodynamic experiments were repeated on Lgr5-EGFP transgenic mice and colon tissues were collected (N ═ 5).
8. Tissue specimen preparation and staining
Taking a colon of about 0.5cm in the middle section of a mouse, fixing the colon by paraformaldehyde at 4 ℃ for 16H, entrusting Wuhan Seville biology company to carry out paraffin/OCT embedding, slicing, carrying out hematoxylin-eosin (H & E)/beta-catenin antibody/Ki 67 antibody staining on the paraffin slice, and scanning the slice; the OCT embedded frozen sections were observed directly under a confocal laser microscope at 488 nm.
9. Isolation and culture of colonic organoids
The colon of a mouse of 6 to 8 weeks old is cut out from the middle, washed clean with precooled PBS, the small intestine is divided into segments of 5-8 cm in length, mucus on the surface of the colon is scraped off with a glass slide, and the scraped colon is placed in a 50mL centrifuge tube and placed on ice. The centrifuge tubes were transferred to a safety cabinet, washed 3-4 times with PBS containing the diabody, and the colon was transferred to 25ml of a solution of 2mM EDTA in a freezer at 4 ℃ for 25 minutes. Transferring the digested colon into a 50ml centrifuge tube, adding 25ml PBS containing the double antibody, properly shaking the colon under 50 ℃, filtering the suspension by using a 70um cell filter sieve, transferring the colon into a new centrifuge tube containing 25ml PBS containing the double antibody, continuously shaking the colon under 50, filtering the suspension by using a 70um cell filter sieve, transferring the suspension after twice filtration into the same 50ml centrifuge tube, and centrifuging the suspension at the room temperature under 900rpm for 5 minutes. The supernatant was discarded, the pellet was resuspended in 2mL of Advanced-DMEM/F12, and the appropriate amount of suspension was counted. The number of crypts is preferably 5-10/μ l. An appropriate volume of the suspension was added to a 1.5ml centrifuge tube and centrifuged at 900rpm for 5 minutes at room temperature. The pellet was resuspended with matrigel pre-cooled on ice. The 96-well plates were pre-warmed in an incubator before 5. mu.l of resuspension was plated per well. The inoculated plate is placed in an incubator for 10-15 minutes. Add 100. mu.l of medium to each well and then change the medium every 2-3 days. The WNER medium consists of:
10. detecting the Effect of Compounds on organoid growth
Prepare 50 μ M DMSO solution based on the Mw of Compound A1, add organoid medium, photograph the morphology, aspirate the medium on day 7, cool on ice for 10min to melt matrigel, and collect organoids by centrifugation in PBS. mRNA is extracted according to the above operation to detect the expression of the stem cell-related gene.
11. Data analysis
The data are analyzed by using SPSS 13.0, the matching t test is adopted for comparison between two groups, the one-factor analysis of variance is adopted for comparison between multiple groups, and the difference of the test results is considered to be when P is less than 0.05. Data are expressed as mean ± standard deviation (mean ± SD). Making bar chart and line chart by GraphPad Prism 7.0 software; photoshop CC2015 performs picture merging and text addition.
Third, experimental results
1. Cell test compound activity table
The inhibition rate calculation formula is as follows:
as shown in the table above, screening shows that 38 compounds have an up-regulation effect on cyclinD1 mRNA exceeding 1.5, 12 compounds have an inhibition rate on IL-23mRNA exceeding 50%, and 10 compounds have both the above two effects, while the positive control drug mesalazine can only inhibit the expression of IL-23mRNA and has no obvious up-regulation effect on cyclinD1 mRNA.
2. Results of animal experiments
Figure 1 shows the therapeutic effect of compound a1 on a mouse colitis model. The a is the survival profile (N ═ 10) of C57 mice during Dextran Sodium Sulfate (DSS) molding for 7 days and simultaneous oral administration (1mg/kg and 2mg/kg of compound a1 and 50mg/kg mesalamine), and the results indicate that the compound a1 high dose group can significantly reduce the mortality of acute IBD mice. B is the percent change in mouse body weight (N ═ 5-8), and the results show that compound a1 can improve body weight loss in mice with acute IBD dose-dependently, and the potency is superior to that of the positive drug mesalazine. C is a photograph of the colon of a mouse and colon length statistics (N is 5-8) after 7 days of DSS modeling and 10 days of synchronous administration, and the result shows that the compound A1 can remarkably improve colon contracture caused by inflammation in a dose-dependent manner, and the drug effect is better than that of the positive drug mesalazine. D is the ELISA detection of the level of the inflammatory factor in the serum of the mice, and the result shows that the compound A1 can obviously reduce the levels of proinflammatory cytokines IL-1 beta, IL-12/23, IL-6 and TNF alpha in the serum in a dose-dependent manner, and the drug effect is superior to that of the positive drug mesalazine. And E is detection of mRNA expression of fluorescence quantitative PCR detection of intestinal stem cell related factors and proinflammatory cytokines after the colon tissue of a mouse is separated, and results show that the compound A1 can remarkably up-regulate mRNA expression of the intestinal stem cell related factors cyclin D1, Lgr5 and Axin2 in a dose-dependent manner, simultaneously down-regulate the expression of mRNA of L-1 beta, IL-23, IL-6 and TNF alpha, and has better drug effect than the positive drug mesalazine. F is a mouse colon specimen LGR5-EGFP immunofluorescence photograph, and shows that the compound A1 can remarkably promote the expression of an intestinal stem cell marker protein LGR5 in a dose-dependent manner, and the positive drug mesalazine has no obvious effect on LGR 5. G is the H & E staining result of a mouse colon specimen, and the result shows that the compound A1 can obviously improve crypt destruction and inflammatory infiltration in a dose-dependent manner, and the drug effect is superior to that of the positive drug mesalazine. H and I are beta-catenin and Ki67 immunohistochemical staining of colon specimens, respectively, and the results showed that Compound A1 significantly enhanced the proliferative capacity of mouse colon epithelial cells dose-dependently (# p < 0.05; # p < 0.01; # p < 0.001).
Figure 2 shows the growth promoting effect of compound a1 on mouse-derived intestinal organoids. Where A is the growth of mouse colon crypt isolates in Matrigel (Matrigel) coated with organoid (WNER) medium, and the concentration of compound A1 was 50 μ M and was photographed using Zeiss 710 live cell imager. B is the sprouting rate of the organoid, C is the organoid surface area, and Image analysis was performed using Image J software (N ═ 5). D is day 7 (168h) after organoids were collected, RNA was extracted for intestinal stem cell marker gene detection (N-5). P <0.05, P <0.01, P < 0.001. The results show that the compound A1 has obvious promotion effect on the growth of the intestinal organoid of the mouse source.
Figure 3 shows the therapeutic effect of compound a1 on a chronic spontaneous IL-10 knockout mouse model of IBD. Wherein A is weight change; after 12 weeks of administration, the body weights of mice in the low, medium and high dose groups and the positive control mesalazine group of compound a1 were significantly increased compared with those in the model group. B is colon length change; after 12 weeks of administration, the colon length of mice in the low, medium and high dose groups and the positive control mesalazine group of compound a1 was significantly increased compared to the model group. C is HE staining result; the colonic mucosa of the model mice had slight inflammatory cell infiltration, slight goblet cell loss and slight hyperplasia compared to the control group. Inflammatory cell infiltration was reduced in colonic mucosa in low, medium and high dose groups of compound a1 and mesalazine groups of mice. Meanwhile, in the compound A1, the goblet cells in the colonic mucosa of the mice in the high dose group and the mesalazine group are increased, and the proliferation is reduced. D is the mouse intestinal permeability assay; the intestinal permeability of mice in the low, medium and high dose groups and mesalazine group of compound a1 was significantly decreased compared to the model group (#. p <0.001 vs. normal group; # p <0.01, # p <0.001 vs. model group).
The results show that the compound A1 can improve intestinal mucosa destruction and inflammatory infiltration of DSS-induced acute IBD and chronic spontaneous IL-10 knockout mouse IBD models dose-dependently, promote the recovery of body weight and colon length of mice, reduce the levels of TNF-alpha, IL-1 beta, IL-6 and IL-23 in peripheral blood and the mRNA expression level of inflammatory cytokines in intestinal tracts, improve the mRNA expression level of stem cell related genes such as cyclin D1, Lgr5 and Axin2 and improve the colon stem cell number and epithelial cell proliferation capacity of IBD mice. The data show that the compound A1 has a remarkable improvement effect on the condition of acute and chronic IBD in mice.
Sequence listing
<110> Shanghai pharmaceutical research institute of Chinese academy of sciences
FUDAN University
<120> active compounds for inhibiting intestinal inflammatory reaction
<130> P2020-0591
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Claims (10)
1. A compound shown in a general formula (I), or enantiomer, diastereoisomer, racemate and mixture thereof, or pharmaceutically acceptable salt thereof,
in the formula (I), the compound is shown in the specification,
x is CH, O or N;
y is CH, O or N;
m is 0 or 1;
R1、R2each independently is H, -NHCOO (C1-C4 alkyl), -NHCONH (C1-C4 alkyl), -NHCO (C1-C4 alkyl), -COO (C1-C4 alkyl), -NHCON (C1-C4 alkyl) (C1-C4 alkyl), C1-C4 alkyl, C1-C4 haloalkyl, CN, halogen, C6-C10 aryl, C1-C4 alkoxy, hydroxy or carboxy; or R1、R2Taken together with the attached carbon to form a substituted or unsubstituted 5-6 membered heterocyclyl; substituted means substituted with one or more substituents selected from the group consisting of: oxo, C1-C4 alkyl, C1-C4 haloalkyl, -NHCOO (C1-C4 alkyl), CN, halogen, C1-C4 alkoxy, hydroxy, carboxy;
W1is-SO2NH-、-CONH-、-SO2-, -NH-or-4 to 6 membered heteroaryl-NH-;
W2is absent, C1-C4Alkylene or-NH-;
W3is absent, C1-C4Alkylene, phenyl (C1-C4 alkylene), C3-C6 cycloalkyl, or 3-6 membered heterocycloalkyl;
R4is H, -OH, C1-C4 alkyl, -O (C1-C4 alkyl), -NH (C3-C6 cycloalkyl), -NH (C1-C4 alkyl), -NHSO2(C1-C4 alkyl) or-NH (3-6 membered heterocycloalkyl);
n is 0 or 1.
2. The compound of claim 1, wherein the compound has the structure of formula II:
R1、R2、R3、R4、W1、W2、W3n is as defined in claim 1.
3. The compound of claim 1, wherein the compound has the structure shown in formula III below:
R1、R2、R3、R4、W1、W2、W3is as defined in claim 1.
4. A compound according to any one of claims 1 to 3 wherein R is1、R2Each independently is CN, halogen, -NHCOO (C1-C4 alkyl), -NHCONH (C1-C4 alkyl), -NHCO (C1-C4 alkyl), -COO (C1-C4 alkyl), -NHCON (C1-C4 alkyl) (C1-C4 alkyl), C1-C4 alkyl, C1-C4 haloalkyl, C6-C10 aryl, C1-C4 alkoxy, hydroxyl, carboxyl or hydrogen.
5. A compound according to any one of claims 1 to 3 wherein W is1is-SO2NH-、-CONH-、-SO2-, -NH-or-4 to 6 membered heteroaryl-NH-;
W2is C1-C4Alkylene or-NH-;
W3is absent, C1-C4Alkylene, phenyl (C1-C4 alkylene), C3-C6Cycloalkyl or 3-6 membered heterocycloalkyl.
6. A compound according to any one of claims 1 to 3 wherein R is3Is H, halogen, C1-C4 haloalkyl, C1-C4 alkyl or C1-C4 alkoxy.
7. A compound according to any one of claims 1 to 3 wherein R is4is-OH, -O (C1-C4 alkyl), -NH (C3-C6 cycloalkyl), -NH (C1-C4 alkyl) or-NHSO2(C1-C4 alkyl).
8. The compound of claim 1, wherein said compound is:
9. a pharmaceutical composition, comprising:
a compound of formula (I) according to claim 1, or enantiomers, diastereomers, racemates thereof and mixtures thereof, or pharmaceutically acceptable salts thereof; and
a pharmaceutically acceptable carrier.
10. Use of a compound of general formula (I) according to claim 1 or a pharmaceutical composition according to claim 9 for the preparation of a medicament for the treatment of inflammatory bowel diseases.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103588697A (en) * | 2012-08-14 | 2014-02-19 | 中国科学院上海药物研究所 | 2,5-disubstituted benzsulfamide compounds, preparation method thereof and applications thereof |
| CN112778156A (en) * | 2019-11-08 | 2021-05-11 | 中国科学院上海药物研究所 | Bishydrazide structure compound, preparation method and application thereof |
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| CN103588697A (en) * | 2012-08-14 | 2014-02-19 | 中国科学院上海药物研究所 | 2,5-disubstituted benzsulfamide compounds, preparation method thereof and applications thereof |
| CN112778156A (en) * | 2019-11-08 | 2021-05-11 | 中国科学院上海药物研究所 | Bishydrazide structure compound, preparation method and application thereof |
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| BURMISTROV, S. I.等: "Derivatives of 2-piperidone and 5-aminopentanoic acid", 《TRUDY DNEPROPETROVSKOGO KHIMIKO-TEKHNOLOGICHESKOGO INSTITUTA》 * |
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