CN102627578B - Phenyl benzylamine compound and application thereof - Google Patents
Phenyl benzylamine compound and application thereof Download PDFInfo
- Publication number
- CN102627578B CN102627578B CN201210081607.8A CN201210081607A CN102627578B CN 102627578 B CN102627578 B CN 102627578B CN 201210081607 A CN201210081607 A CN 201210081607A CN 102627578 B CN102627578 B CN 102627578B
- Authority
- CN
- China
- Prior art keywords
- compound
- reaction
- compounds
- add
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 0 CC(C)(C)OC(*Nc(cc(C)cc1)c1NCc1ccccc1)=O Chemical compound CC(C)(C)OC(*Nc(cc(C)cc1)c1NCc1ccccc1)=O 0.000 description 3
- BXKFJRIHXLNJPC-UHFFFAOYSA-N Cc(cc1N)ccc1NCc1ccccc1 Chemical compound Cc(cc1N)ccc1NCc1ccccc1 BXKFJRIHXLNJPC-UHFFFAOYSA-N 0.000 description 3
- HSILOYXWDFISQM-UHFFFAOYSA-N CNc1ccccc1NCc1ccccc1 Chemical compound CNc1ccccc1NCc1ccccc1 HSILOYXWDFISQM-UHFFFAOYSA-N 0.000 description 1
Images
Abstract
The invention belongs to the technical field of biological pharmacy and provides application of phenyl benzylamine compounds as immunosuppressant in preparation of medicament for treating T cell participated autoimmune diseases, such as multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune nephritis and inflammatory bowel disease. A series of novel compounds synthesized from phenyl benzylamine compounds have significant efficacy, novel mechanism, little toxic or side effects; and as the synthetic route is simple and environment-friendly, the novel compounds have obvious advantages compared with existing ones.
Description
Technical field
The present invention belongs to phenylbenzylamine compounds and application thereof especially, belongs to biological pharmacy technical field.
Background technology
Autoimmune disorder is a class refractory disease, and its morbidity is how relevant with the lymphocytic overactivity of T, and the immunosuppressor that adopt are treated more at present.Common autoimmune disorder comprises multiple sclerosis, systemic lupus erythematous, rheumatoid arthritis, autoimmune nephritis, inflammatory bowel etc.Autoimmune disorder is at present without cure method, and existing medicine mostly is injection type, and curative effect is not good enough, and toxic side effect is large, and medical expense is very expensive, needs the novel therapeutic medicine that research and development meet pharmacoeconomics badly.
Summary of the invention
The object of this invention is to provide autoimmune disorder that a class phenylbenzylamine compounds participates at preparation treatment T cell as immunosuppressor as the application in multiple sclerosis, systemic lupus erythematous, rheumatoid arthritis, autoimmune nephritis, inflammatory bowel medicine.
Technical scheme of the present invention:
Phenylbenzylamine compounds, it is characterized in that following structural formula any one:
Wherein
The application of above-mentioned phenylbenzylamine compounds in preparation treatment autoimmune disorder medicine.
Be the autoimmune disorder that T cell participates in as the autoimmune disorder described in a kind of optimal way.
The autoimmune disorder participating in as a kind of optimal way T cell is multiple sclerosis, systemic lupus erythematous, rheumatoid arthritis, autoimmune nephritis or inflammatory bowel medicine.
The autoimmune disorder participating in as a kind of optimal way T cell is multiple sclerosis.
The preparation method of phenylbenzylamine compounds:
Parent nucleus is synthetic: complete synthetic by following reaction (), (two).
Reaction (one):
Raw material and injected volume:
Operation steps:
In 2L four-necked bottle, add raw material (1), (2), (3) by above-mentioned injected volume, stir, drip after raw material (4), be warming up to 190 DEG C～200 DEG C.(developping agent is PE: EA=1 to TLC detection reaction degree: 9).Post-reaction treatment: reaction product is poured in distilled water, has solid to separate out.Suction filtration, filtrate CH
2cl
2extract 3 times, merge CH
2cl
2layer and suction filtration gained solid.Wash again 1 time, dry concentrated; Last EA recrystallization obtains sterling.
Reaction (two)
Raw material and injected volume:
Operation steps:
Three is added to 500ml eggplant-shape bottle, stir, reflux 1 hour.(developping agent is PE: EA=5 to TLC detection reaction degree: 3).Post-reaction treatment: system NH after reaction
3h
2o adjusts after PH to 9.0, adds CH
2cl
2500ml, suction filtration.Solid is used
CH
2Cl
2
Wash 1 time, merge CH
2cl
2layer washes with water 2 times, dry concentrated.
Compound (1): on parent nucleus basis, complete synthetic by following reaction (three), (four).
Reaction (three)
Raw material and injected volume:
Operation steps:
500ml three-necked bottle, adds CDI, THF150ml.Cryosel is chilled to 0 DEG C.Add Gly-Boc, 0 DEG C keeps stirring 15min.Be warming up to 60 DEG C, react 1 hour.Be cooled to normal temperature, drip anils and THF50ml.Normal temperature spends the night, and next day, column chromatography was processed purifying.
Reaction (four)
Raw material and injected volume:
Operation steps:
In the mono-neck bottle of 500ml, add (1), (2), drip (3), reaction is spent the night, and (developping agent is PE: EA=5 to TLC detection reaction degree: 3).
Post-reaction treatment:
After reaction system is cooling, add 10% hydrochloric acid, adjust PH to acid, separatory, CHCl
3layer is washed once with 10% hydrochloric acid saturated common salt.Dry concentrated, column chromatography purification, obtains compound (1).
Compound (2): on parent nucleus basis, complete synthetic by following reaction (five), (six).
Reaction (five)
Reaction (six)
Raw material injected volume, operation, post-reaction treatment, with reference to reaction (three), (four), can obtain compound (2).
Residue compound is realized by following reaction:
Wherein R
1=Me, R
2=C
ah
bn
cs
do
ewherein a=0-11, b=1-16, c=1-3, d=0 or 1, e=0-2, R
3=H, R
4=H.
The pharmacological testing of phenylbenzylamine compounds
1) the mouse T lymphocyte proliferation test of TCR token stimulus agent anti-CD3/anti-CD28 activation
Get the inguinal region of mouse and 4 lymphoglandula of oxter, be prepared into single cell suspension.With perfect medium, RPMI-1640 is made into 3 × 10
6the cell suspension of/ml, plants in 96 orifice plates 3 × 10
5the every hole of individual cell, stimulates 72h with final concentration 10 μ g/ml anti-CD3 and 1 μ g/ml anti-CD28 (purchased from BD PharMingen company), jointly hatches with compound, and isotropic substance tritium mark method detects lymphopoietic situation.
2) MOG
35-55cause EAE model mice T lymphopoiesis and release of cytokines test
Get MOG
35-55the inguinal region of the emulsion sensitization mouse of 10 days and 4 lymphoglandula of oxter prepared by (purchased from Shanghai Qiangyao Biotechnology Co., Ltd.) and complete Freund's adjuvant (purchased from sigma company), and spleen, be prepared into respectively single cell suspension.With perfect medium, RPMI-1640 is made into 3 × 10
6the cell suspension of/ml, plants in 96 orifice plates 3 × 10
5the every hole of individual cell, with final concentration 10 μ g/ml MOG
35-55stimulate 72h, jointly hatch with compound, isotropic substance tritium mark method detects the situation of lymphocyte antigen proliferated specifically, and supernatant is for detection of the secretion of cytokine IFN-γ.
3) enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA)
The method that the mensuration of cytokine IFN-γ is described according to ELISA test kit (purchased from R & D company) is carried out.
4) experimental autoimmune encephalomyelitis (EAE) model and pathology detection
Take MOG
35-55lyophilized powder is dissolved in PBS (200 μ g/ only), mycobacterium tuberculosis (purchased from Difco company) is dissolved in incomplete Freund's adjuvant (purchased from sigma company, final concentration 6mg/ml), by these two kinds of suspensions in 1: the 1 fully emulsified one-tenth water-in-oil of ratio state, adopt multi-point injection method, select 3～4 subcutaneous injections, 300 μ l emulsions in neck and backbone both sides.Every mouse sensitization same day, the PBS solution 0.2ml (containing PTX 400ng) of tail vein injection Toxins, pertussis (PTX, purchased from sigma company).Tail vein injection PTX/PBS 0.2ml again after 48h.From immunity same day, every day is to the mouse observation of weighing, and carries out the scoring of neural function study of behaviour, and records mouse invasion rate.Take out myeloid tissue in mouse model onset peak period and fix, H & E dyeing shows the situation of inflammatory cell infiltration.Ponceau 2R-BG dyeing shows demyelination situation.
The pharmacological tests of phenylbenzylamine compounds and analysis
1) phenylbenzylamine compounds has suppressed the propagation of the mouse T cell of TCR token stimulus agent anti-CD3/anti-CD28 activation
In order to evaluate the external effect whether with suppressor T cell propagation of synthetic a collection of phenylbenzylamine compounds, we utilize [H
3] method of mixing carried out primary dcreening operation.As shown in Figure 1, compound 1-11 has suppressed the propagation of the T cell of anti-CD3/anti-CD28 stimulation to some extent.Wherein, with compound 5,10, the effect of 11 inhibition propagation is the most remarkable.
2) phenylbenzylamine compounds has suppressed EAE mouse MOG
35-55t cells with antigenic specificity propagation and the sub-IFN-γ of secrete cytokines
Whether have in order to evaluate synthetic a collection of phenylbenzylamine compounds the effect of improving EAE, it is external to MOG that we have investigated this batch of compound
35-55the impact of T cells with antigenic specificity propagation.As shown in Figure 2, compound 1-11 has suppressed MOG to some extent
35-55the propagation of T cells with antigenic specificity, wherein with compound 5,10, the effect of 11 inhibition propagation is the most remarkable.
EAE is the autoimmune disorder of Th1 mediation, and its important effector molecule is exactly IFN-γ.Therefore, we have further verified whether this batch of phenylbenzylamine compounds affects the secretion of IFN-γ.As shown in Figure 3, compound 1-11 has suppressed MOG to some extent
35-55t cells with antigenic specificity secretion of gamma-IFN.Wherein, with compound 5,10, the effect of 11 inhibition IFN-γ secretion is the most remarkable.
3) phenylbenzylamine compounds has improved the morbidity of EAE mouse
Next, we further investigate the Immunological diseases model that whether can suppress Th1 mediation in this batch of phenylbenzylamine compounds body.MOG
35-55be the transmembrane glycoprotein of a kind of expression on maincenter oligodendrocyte and myelin film surface, content seldom but has hyperimmunization originality in vivo, can cause that demyelination antibody response can cause again the central nervous system myelin protein composition of t cell responses.Pass through MOG
35-55mix sensitization C57/BL6 mouse with adjuvant, can cause that mouse produces MOG
35-55specific immune response, and then cause the reaction of neurone demyelination, show as weight loss, tetraplegia, tail is unable.Beginning oral administration from modeling the 10th day, administration group gives respectively the different phenylbenzylamine compounds of 10mg/kg every day.CsA, as positive control, is dissolved in sweet oil, every day abdominal injection 10mg/kg.Solvent control group gives sweet oil in contrast.Administration continues to experimental observation and finishes.As shown in Figure 4, in the time of modeling the 23rd day, phenylbenzylamine compounds 1-11 all can improve the clinical score of EAE mouse to some extent.Wherein with compound 5,10,11 to improve effect the most remarkable.Further, we find compound 5,10, and 11 can significantly suppress morbidity ratio and the severity (Fig. 5) of mouse, and can alleviate due to the caused weight loss of morbidity.Onset peak period is got Mouse Lumbar section spinal cord, and its inflammatory cell infiltration is investigated in H & E dyeing, capillary blood vessel swelling, and blood vessel " oversleeve sample " changes, and little blood vessel especially Venule has that a large amount of inflammatory cells are intensive encompasses " oversleeve sample " around.As shown in Figure 6, compound 5,10,11 can suppress inflammatory cell infiltration significantly, and capillary blood vessel perviousness.By the specific stain of ponceau-BG, can find, the myelin overwhelming majority of model mice comes off, and compound 5,10,11 can obviously suppress its demyelination effect (Fig. 6).
Beneficial effect
A series of new compounds that phenylbenzylamine compounds 1-11 is associated, can be used as the autoimmune disorder of immunosuppressant treatment T cell participation as multiple sclerosis, systemic lupus erythematous, rheumatoid arthritis, autoimmune nephritis, inflammatory bowel etc., drug effect is remarkable, mechanism is novel, toxic side effect is less, and due to the simple environmental protection of synthetic route, with the obvious advantage compared with existing medicine.
Brief description of the drawings
Fig. 1. the selection result of the proliferation of mouse T lymphocytes of phenylbenzylamine compounds vitro inhibition anti-CD3/anti-CD28 activation.Adopt 10 μ g/ml anti-CD3 and 1 μ g/ml anti-CD28 to stimulate T cell activation propagation.Compound in compound library to be sieved and activating T cell are applied to 72h altogether, by [H
3] method of mixing investigates each testing compound for the lymphopoietic impact of T.
Fig. 2. phenylbenzylamine compounds vitro inhibition MOG
35-55t lymphocyte specific proliferation experiment the selection result.MOG
35-55can stimulate the T cell activation propagation in EAE model mice source.By the compound in compound library to be sieved and MOG
35-55the T cell of activation applies 72h altogether, by [H
3] method of mixing investigates each testing compound for lymphopoietic impact.
Fig. 3. phenylbenzylamine compounds vitro inhibition MOG
35-55t lymphocyte specific discharges the selection result of IFN-γ.MOG
35-55can stimulate the T cell activation propagation in EAE model mice source.By the compound in compound library to be sieved and MOG
35-55the T cell of activation is collected supernatant after applying 24h altogether.ELISA detects the restraining effect of each compound for Th1 cytokine IFN-γ secretion.
Fig. 4. the improve effect of phenylbenzylamine compounds to EAE in mice model mice clinical score.Pass through MOG
35-55mix sensitization C57/BL6 mouse with adjuvant, can cause that mouse produces the specific immune response of MOG, and then cause the reaction of neurone demyelination, show as weight loss, tetraplegia, tail is unable.Phenylbenzylamine compounds is beginning oral administration from modeling the 10th day, gives 10mg/kg every day.CsA is dissolved in sweet oil, CsA control group abdominal injection every day 10mg/kg.Administration continues to experimental observation and finishes.Phenylbenzylamine compounds can suppress the occurring degree of mouse to some extent, wherein remarkable with compound 5,10 and 11 effects.
Fig. 5. 5,10,11 pairs of EAE in mice model body weight of phenylbenzylamine compounds and clinical score improve effect.Compound 5,10,11 from modeling the 10th day beginning oral administration, give respectively 10mg/kg every day.CsA is dissolved in sweet oil, CsA control group abdominal injection every day 10mg/kg.Administration continues to experimental observation and finishes.Compound 5,10,11 can significantly suppress morbidity ratio (A) and the severity (B) of mouse, and can alleviate due to the caused weight loss of morbidity (C).
Fig. 6. 5,10,11 pairs of EAE in mice model spinal cord pathology levels of phenylbenzylamine compounds improve effect.Onset peak period is got Mouse Lumbar section spinal cord, and the H & E visible compound 5,10,11 that dyes can be lowered inflammatory cell infiltration and capillary blood vessel perviousness, and the visible compound 5,10,11 of ponceau-BG specific stain can suppress demyelination effect.
Embodiment
The preparation of N-benzyl-4-methyl-o-Nitraniline (II)
In four-necked bottle, add people 4-methyl-o-Nitraniline (200g, 1.32mol), K
2cO
3(20g, 0.5mol) and benzyl bromine (200ml), DMF (1.2ml) is slowly added drop-wise in reaction solution.Be warming up to 190 DEG C～200 DEG C.(developping agent is PE: EA=1 to TLC detection reaction degree: 9).Reaction product is poured in distilled water, has solid to separate out.Suction filtration, filtrate CH
2cl
2extract 3 (200ml*3) inferior, merge CH
2cl
2layer and suction filtration gained solid.Wash again 1 time, dry concentrated.Finally obtain sterling by re-crystallizing in ethyl acetate, productive rate 58%.
The preparation of N1-benzyl-4-methyl-O-Phenylene Diamine (III)
In 500ml eggplant-shape bottle, add N-benzyl-4-methyl-o-Nitraniline (10g, 0.04mol), SnCl
2.2H
2o (95g, 0.44mol), ethanol (80ml), stirs, and refluxes 1 hour.(developping agent is PE: EA=5 to TLC detection reaction degree: 3).Post-reaction treatment: system NH after reaction
3h
2o adjusts after pH to 9.0, adds CH
2cl
2500ml, suction filtration.Solid CH
2cl
2wash 1 time, merge CH
2cl
2layer washes with water 2 times, dry concentrated, obtains compound N 1-benzyl-4-methyl-O-Phenylene Diamine productive rate 72%.
The preparation of compound 1
The preparation of 1 compd A
500ml three-necked bottle, adds CDI (10mg, 0.06mmol), N1-benzyl-4-methyl-O-Phenylene Diamine (III) (6.69g, 0.03mol) THF (150ml).Cryosel is chilled to 0 DEG C.Add Gly-Boc (4g, 0.02mol), 0 DEG C keeps stirring 15min.Be warming up to 60 DEG C, react 1 hour.Be cooled to normal temperature, drip anils and THF 50ml.Normal temperature spends the night, and next day, column chromatography was processed purifying (PE/EA=10/1), obtained compd A, productive rate 82%.
The preparation of 2 compounds 1
In the mono-neck bottle of 500ml, add compd A (10g, 0.027mol), TFA (10g, 0.087mol), CHCl
3(80ml) reaction is spent the night, and (developping agent is PE: EA=5 to TLC detection reaction degree: 3).Post-reaction treatment: after reaction system is cooling, add 10% hydrochloric acid, adjust pH to acid, separatory, CHCl
3layer is washed once with 10% hydrochloric acid saturated common salt.Dry concentrated, (developping agent is PE: EA=5 to column chromatography purification: 3), obtain compound 1, productive rate 75%.
The spectral data of compound 1:
The preparation of compound 2
The preparation of 1 compd B
500ml three-necked bottle, adds CDI (10mg, 0.06mmol), N1-benzyl-4-methyl-O-Phenylene Diamine (III) (6g, 0.028mol) THF (150ml).Cryosel is chilled to 0 DEG C.Add Ala-Boc (5g, 0.026mol), 0 DEG C keeps stirring 15min.Be warming up to 60 DEG C, react 1 hour.Be cooled to normal temperature, drip anils and THF 50ml.Normal temperature spends the night, and next day, column chromatography was processed purifying (PE/EA=10/1), obtained compd B, productive rate 80%.
The preparation of 2 compounds 2
In the mono-neck bottle of 500ml, add compd B (10g, 0.026mol), TFA (10g, 0.087mol), CHCl
3(80ml) reaction is spent the night, and (developping agent is PE: EA=5 to TLC detection reaction degree: 3).Post-reaction treatment: after reaction system is cooling, add 10% hydrochloric acid, adjust pH to acid, separatory, CHCl
3layer is washed once with 10% hydrochloric acid saturated common salt.Dry concentrated, (developping agent is PE: EA=5 to column chromatography purification: 3), obtain compound 2, productive rate 72%.
The spectral data of compound 2:
The preparation of compound 3
The preparation of 1 Compound C
500ml three-necked bottle, adds CDI (10mg, 0.06mmol), N1-benzyl-4-methyl-O-Phenylene Diamine (III) (6g, 0.028mol) THF (150ml).Cryosel is chilled to 0 DEG C.Add Leu-Boc (5g, 0.022mol), 0 DEG C keeps stirring 15min.Be warming up to 60 DEG C, react 1 hour.Be cooled to normal temperature, drip anils and THF 50ml.Normal temperature spends the night, and next day, column chromatography was processed purifying (PE/EA=8/1), obtained Compound C, productive rate 81%.
The preparation of 2 compounds 3
In the mono-neck bottle of 500ml, add Compound C (10g, 0.024mol), TFA (10g, 0.087mol), CHCl
3(80ml) reaction is spent the night, and (developping agent is PE: EA=5 to TLC detection reaction degree: 3).Post-reaction treatment: after reaction system is cooling, add 10% hydrochloric acid, adjust pH to acid, separatory, CHCl
3layer is washed once with 10% hydrochloric acid saturated common salt.Dry concentrated, (developping agent is PE: EA=4 to column chromatography purification: 3), obtain compound 3, productive rate 74%.
The spectral data of compound 3 is:
The preparation of compound 4
The preparation of 1 compd E
500ml three-necked bottle, adds CDI (10mg, 0.06mmol), N1-benzyl-4-methyl-O-Phenylene Diamine (III) (6g, 0.028mol) THF (150ml).Cryosel is chilled to 0 DEG C.Add Ile-Boc (5g, 0.022mol), 0 DEG C keeps stirring 15min.Be warming up to 60 DEG C, react 1 hour.Be cooled to normal temperature, drip anils and THF 50ml.Normal temperature spends the night, and next day, column chromatography was processed purifying (PE/EA=9/1), obtained compd E, productive rate 80%.
The preparation of 2 compounds 4
In the mono-neck bottle of 500ml, add compd E (10g, 0.024mol), TFA (10g, 0.087mol), CHCl
3(80ml) reaction is spent the night, and (developping agent is PE: EA=5 to TLC detection reaction degree: 3).Post-reaction treatment: after reaction system is cooling, add 10% hydrochloric acid, adjust pH to acid, separatory, CHCl
3layer is washed once with 10% hydrochloric acid saturated common salt.Dry concentrated, (developping agent is PE: EA=4 to column chromatography purification: 3), obtain compound 4, productive rate 76%.
The spectral data of compound 4 is:
The preparation of compound 5
The preparation of 1 compound F 17-hydroxy-corticosterone
500ml three-necked bottle, adds CDI (10mg, 0.06mmol), N1-benzyl-4-methyl-O-Phenylene Diamine (III) (6g, 0.028mol) THF (150ml).Cryosel is chilled to 0 DEG C.Add compound f (5g, 0.02mol), 0 DEG C keeps stirring 15min.
Be warming up to 60 DEG C, react 1 hour.Be cooled to normal temperature, drip anils and THF 50ml.Normal temperature spends the night, and next day, column chromatography was processed purifying (PE/EA=8/1), obtained compound F 17-hydroxy-corticosterone, productive rate 78%.
The preparation of 2 compounds 5
In the mono-neck bottle of 500ml, add compound F 17-hydroxy-corticosterone (10g, 0.023mol), TFA (10g, 0.087mol), CHCl
3(80ml) reaction is spent the night, and (developping agent is PE: EA=5 to TLC detection reaction degree: 2).Post-reaction treatment: after reaction system is cooling, add 10% hydrochloric acid, adjust pH to acid, separatory, CHCl
3layer is washed once with 10% hydrochloric acid saturated common salt.Dry concentrated, (developping agent is PE: EA=5 to column chromatography purification: 2), obtain compound 5, productive rate 82%.
The spectral data of compound 5:
The preparation of compound 6
The preparation of 1 compound G
500ml three-necked bottle, adds CDI (10mg, 0.06mmol), N1-benzyl-4-methyl-O-Phenylene Diamine (III) (6g, 0.028mol) THF (150ml).Cryosel is chilled to 0 DEG C.Add compound g (5g, 0.019mol), 0 DEG C keeps stirring 15min.
Be warming up to 60 DEG C, react 1 hour.Be cooled to normal temperature, drip anils and THF 50ml.Normal temperature spends the night, and next day, column chromatography was processed purifying (PE/EA=8/1), obtained compound G, productive rate 69%.
The preparation of 2 compounds 6
In the mono-neck bottle of 500ml, add compound G (10g, 0.022mol), TFA (10g, 0.087mol), CHCl
3(80ml) reaction is spent the night, and (developping agent is PE: EA=5 to TLC detection reaction degree: 4).Post-reaction treatment: after reaction system is cooling, add 10% hydrochloric acid, adjust pH to acid, separatory, CHCl
3layer is washed once with 10% hydrochloric acid saturated common salt.Dry concentrated, (developping agent is PE: EA=5 to column chromatography purification: 4), obtain compound 6, productive rate 81%.
The spectral data of compound 6:
The preparation of compound 7
The preparation of 1 compound H
500ml three-necked bottle, adds CDI (10mg, 0.06mmol), N1-benzyl-4-methyl-O-Phenylene Diamine (III) (6g, 0.028mol) THF (150ml).Cryosel is chilled to 0 DEG C.Add compound h (5g, 0.018mol), 0 DEG C keeps stirring 15min.
Be warming up to 60 DEG C, react 1 hour.Be cooled to normal temperature, drip anils and THF 50ml.Normal temperature spends the night, and next day, column chromatography was processed purifying (PE/EA=8/1), obtained compound H, productive rate 72%.
The preparation of 2 compounds 7
In the mono-neck bottle of 500ml, add compound H (10g, 0.023mol), TFA (10g, 0.087mol), CHCl
3(80ml) reaction is spent the night, and (developping agent is PE: EA=5 to TLC detection reaction degree: 4).Post-reaction treatment: after reaction system is cooling, add 10% hydrochloric acid, adjust pH to acid, separatory, CHCl
3layer is washed once with 10% hydrochloric acid saturated common salt.Dry concentrated, (developping agent is PE: EA=5 to column chromatography purification: 4), obtain compound 7, productive rate 86%.
The spectral data of compound 7:
The preparation of compound 8
The preparation of 1 Compound I
500ml three-necked bottle, adds CDI (10mg, 0.06mmol), N1-benzyl-4-methyl-O-Phenylene Diamine (III) (6g, 0.028mol) THF (150ml).Cryosel is chilled to 0 DEG C.Add compound i (5g, 0.018mol), 0 DEG C keeps stirring 15min.
Be warming up to 60 DEG C, react 1 hour.Be cooled to normal temperature, drip anils and THF 50ml.Normal temperature spends the night, and next day, column chromatography was processed purifying (PE/EA=8/1), obtained Compound I, productive rate 79%.
The preparation of 2 compounds 8
In the mono-neck bottle of 500ml, add Compound I (10g, 0.023mol), TFA (10g, 0.087mol), CHCl
3(80ml) reaction is spent the night, and (developping agent is PE: EA=5 to TLC detection reaction degree: 3).Post-reaction treatment: after reaction system is cooling, add 10% hydrochloric acid, adjust pH to acid, separatory, CHCl
3layer is washed once with 10% hydrochloric acid saturated common salt.Dry concentrated, (developping agent is PE: EA=5 to column chromatography purification: 3), obtain compound 8, productive rate 84%.
The spectral data of compound 8:
The preparation of compound 9
The preparation of 1 compound J
500ml three-necked bottle, adds CDI (10mg, 0.06mmol), N1-benzyl-4-methyl-O-Phenylene Diamine (III) (6g, 0.028mol) THF (150ml).Cryosel is chilled to 0 DEG C.Add compound j (5g, 0.018mol), 0 DEG C keeps stirring 15min.
Be warming up to 60 DEG C, react 1 hour.Be cooled to normal temperature, drip anils and THF 50ml.Normal temperature spends the night, and next day, column chromatography was processed purifying (PE/EA=8/1), obtained compound J, productive rate 77%.
The preparation of 2 compounds 9
In the mono-neck bottle of 500ml, add compound J (10g, 0.023mol), TFA (10g, 0.087mol), CHCl
3(80ml) reaction is spent the night, and (developping agent is PE: EA=5 to TLC detection reaction degree: 3).Post-reaction treatment: after reaction system is cooling, add 10% hydrochloric acid, adjust pH to acid, separatory, CHCl
3layer is washed once with 10% hydrochloric acid saturated common salt.Dry concentrated, (developping agent is PE: EA=5 to column chromatography purification: 3), obtain compound 9, productive rate 82%.
The spectral data of compound 9:
The preparation of compound 10
The preparation of 1 compound K
500ml three-necked bottle, adds CDI (10mg, 0.06mmol), N1-benzyl-4-methyl-O-Phenylene Diamine (III) (6g, 0.028mol) THF (150ml).Cryosel is chilled to 0 DEG C.Add compound k (5g, 0.018mol), 0 DEG C keeps stirring 15min.
Be warming up to 60 DEG C, react 1 hour.Be cooled to normal temperature, drip anils and THF 50ml.Normal temperature spends the night, and next day, column chromatography was processed purifying (PE/EA=4/1), obtained compound K, productive rate 66%.
The preparation of 2 compounds 10
In the mono-neck bottle of 500ml, add compound K (10g, 0.023mol), TFA (10g, 0.087mol), CHCl
3(80ml) reaction is spent the night, and (developping agent is PE: EA=1 to TLC detection reaction degree: 4).Post-reaction treatment: after reaction system is cooling, add 10% hydrochloric acid, adjust pH to acid, separatory, CHCl
3layer is washed once with 10% hydrochloric acid saturated common salt.Dry concentrated, (developping agent is PE: EA=1 to column chromatography purification: 4), obtain compound 10, productive rate 80%.
The spectral data of compound 10:
The preparation of compound 11
The preparation of 1 compound L
500ml three-necked bottle, adds CDI (10mg, 0.06mmol), N1-benzyl-4-methyl-O-Phenylene Diamine (III) (6g, 0.028mol) THF (150ml).Cryosel is chilled to 0 DEG C.Add compound l (5g, 0.017mol), 0 DEG C keeps stirring 15min.
Be warming up to 60 DEG C, react 1 hour.Be cooled to normal temperature, drip anils and THF 50ml.Normal temperature spends the night, and next day, column chromatography was processed purifying (PE/EA=5/1), obtained compound L, productive rate 63%.
The preparation of 2 compounds 11
In the mono-neck bottle of 500ml, add compound L (10g, 0.021mol), TFA (10g, 0.087mol), CHCl3 (80ml) reaction is spent the night, and (developping agent is PE: EA=7 to TLC detection reaction degree: 4).Post-reaction treatment: after reaction system is cooling, add 10% hydrochloric acid, adjust pH to acid, separatory, CHCl
3layer is washed once with 10% hydrochloric acid saturated common salt.Dry concentrated, (developping agent is PE: EA=7 to column chromatography purification: 4), obtain compound 11, productive rate 77%.
The spectral data of compound 11
1. the pharmacological testing of phenylbenzylamine compounds
1) proliferation test of the mouse T cell of anti-CD3/anti-CD28 activation
Get the inguinal region of mouse and 4 lymphoglandula of oxter, be prepared into single cell suspension.With perfect medium, RPMI-1640 is made into 3 × 10
6the cell suspension of/ml, plants in 96 orifice plates 3 × 10
5the every hole of individual cell, stimulates 72h with final concentration 10 μ g/ml anti-CD3 and 1 μ g/ml anti-CD28 (purchased from BD PharMingen company), jointly hatches with compound, and isotropic substance tritium mark method detects lymphopoietic situation.
2) MOG
35-55cause EAE model mice T lymphopoiesis and release of cytokines test
Get MOG
35-55the inguinal region of the emulsion sensitization mouse of 10 days and 4 lymphoglandula of oxter prepared by (purchased from Shanghai Qiangyao Biotechnology Co., Ltd.) and complete Freund's adjuvant (purchased from sigma company), be prepared into single cell suspension.With perfect medium, RPMI-1640 is made into 3 × 10
6the cell suspension of/ml, plants in 96 orifice plates 3 × 10
5the every hole of individual cell, with final concentration 10 μ g/ml MOG
35-55stimulate 72h, jointly hatch with compound, isotropic substance tritium mark method detects the situation of lymphocyte antigen proliferated specifically, and supernatant is for detection of the secretion of cytokine IFN-γ.
3) enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA)
The method that the mensuration of cytokine IFN-γ is described according to ELISA test kit (R & D company) is carried out.Roughly flow process is as follows: to the sealing bag of room temperature, take out required lath from balance, other lath sealing is put back to 4 DEG C.Except blank well, respectively sample or different concns standard substance (100 μ l/ hole) are added in respective aperture, seal reacting hole with shrouding gummed paper, 37 DEG C of incubators are hatched 90 minutes.Wash plate 4 times.Except blank well, add biotinylated antibody working fluid (100 μ l/ hole).Seal reacting hole with shrouding gummed paper, 37 DEG C of incubators are hatched 60 minutes.Wash plate 4 times.Except blank well, add enzyme conjugates working fluid (100 μ l/ hole).Seal reacting hole with shrouding gummed paper, 37 DEG C of incubators are hatched 30 minutes.Wash plate 4 times.Add developer 100 μ l/ holes, 37 DEG C of incubators of lucifuge are hatched 10-15 minute.Add stop buffer 100 μ l/ holes, after mixing, at once measure OD450 value (in 5 minutes).The OD value of each standard substance and sample should deduct the OD value in zero hole.Make X-coordinate with standard substance concentration, OD value is made ordinate zou, connects the coordinate point of each standard substance with sweep, can on typical curve, calculate its concentration by the OD value of sample.If sample OD value is resurveyed after higher than the typical curve upper limit, should suitably diluting, when calculating concentration, should be multiplied by extension rate.
4) experimental autoimmune encephalomyelitis (EAE) model and pathology detection
Take MOG
35-55lyophilized powder is dissolved in PBS (200 μ g/ only), mycobacterium tuberculosis (purchased from Difco company) is dissolved in incomplete Freund's adjuvant (purchased from sigma company, final concentration 6mg/ml), by these two kinds of suspensions in 1: the 1 fully emulsified one-tenth water-in-oil of ratio state, adopt multi-point injection method, select 3～4 subcutaneous injections, 300 μ l emulsions in neck and backbone both sides.Every mouse sensitization same day, the PBS solution 0.2ml (containing PTX 400ng) of tail vein injection Toxins, pertussis (PTX, purchased from sigma company).Tail vein injection PTX/PBS 0.2ml again after 48h.From the immune same day, weigh every day to mouse, observes, and carry out the scoring of neural function study of behaviour, and record mouse invasion rate.Scoring is labeled as 5 grades of general point systems: 0 point, do not fall ill; 1 point, afterbody is unable; 2 points, the unable and incomplete hind leg of afterbody (1～2 hind leg) paralysis; 3 points, two rear acroparalysis; 4 points, two hind legs and arbitrary forelimb are all paralysed, and after passive standing up, can not reset; 5 points, moribund condition or death.
By mouse anesthesia, open thoracic cavity in mouse model onset peak period, through PBS heart perfusion 20ml, with fixing in 4% paraformaldehyde perfusion.Then take out myeloid tissue, put into after the pipe of the paraformaldehyde that is equipped with 4% fixing.Gradient is dewatered actually, and dimethylbenzene is transparent, transparent good tissue is put into the paraffin embedding of thawing, embedded tissue block is cut into the thin slice of 5 μ m, on exhibition sheet and sheet glass, copies sheet, H & E dyeing, and mounting, observes, and takes pictures.H & E dyeing can show the situation of inflammatory cell infiltration.
Ponceau 2R-BG dyeing can make myelin present pink with combination myelin phospholipid composition by ponceau 2R, and axon, lamellar sheath and neural interior clothing are green, therefore the myelin of sex change is because phosphatide disappears not painted.Section routine dewaxes to distilled water, with ponceau 2R dye liquor dyeing 5min, distillation washing, and then 2.5% act on 1min in phosphotungstic acid aqueous solution, redye 4min with BG dye liquor, then with 1% glacial acetic acid aqueous solution differentiation 10s, washing from the beginning, gradient alcohol dehydration, dimethylbenzene is transparent, neutral gum sealing.Wherein ponceau 2R staining fluid is by ponceau 2R 1g, Glacial acetic acid 1ml, distilled water 99ml configuration; BG dye liquor is by BG 1.0g, Glacial acetic acid 1ml, distilled water 99ml configuration.
2. the pharmacological tests of phenylbenzylamine compounds and analysis
1) propagation of the mouse T cell of phenylbenzylamine compounds vitro inhibition anti-CD3/anti-CD28 activation
For whether the phenylbenzylamine compounds 1-11 described in Evaluation operation example 1 has immunosuppressive activity, first adopt 10 μ g/ml anti-CD3 and 1 μ g/ml anti-CD28 to stimulate T cell activation propagation, compound in compound library to be sieved and activating T cell are applied to 72h altogether, by [H
3] method of mixing investigates each testing compound for the lymphopoietic impact of T.As shown in Figure 1, compound 1-11 has suppressed the propagation of the T cell of anti-CD3/anti-CD28 activation to some extent, and wherein with compound 5,10, the effect of 11 inhibition propagation is the most remarkable.These phenylbenzylamine compounds of above results suggest have immunosuppressive activity.
2) phenylbenzylamine compounds suppresses EAE mouse MOG
35-55t cells with antigenic specificity propagation and secrete cytokines IFN-γ
Whether have in order to evaluate synthetic a collection of phenylbenzylamine compounds the effect of improving EAE, first we utilize external MOG
35-55antigen-specific proliferation test has carried out primary dcreening operation.As shown in Figure 2, the compound 1-11 of 10 μ M has suppressed MOG to some extent
35-55the propagation of T cells with antigenic specificity, wherein with compound 5,10, the effect of 11 inhibition propagation is the most remarkable.
EAE is the autoimmune disorder of Th1 mediation, and its important effector molecule is exactly IFN-γ.Therefore, we have further verified whether this batch of phenylbenzylamine compounds affects the secretion of IFN-γ.As shown in Figure 3, compound 1-11 has suppressed MOG to some extent
35-55t cells with antigenic specificity secretion of gamma-IFN.Wherein, with compound 5,10, the effect of 11 inhibition IFN-γ secretion is the most remarkable.
3) phenylbenzylamine compounds has improved the morbidity of EAE mouse
Next, we further investigate the Immunological diseases model that whether can suppress Th1 mediation in this batch of phenylbenzylamine compounds body.MOG
35-55be the transmembrane glycoprotein of a kind of expression on maincenter oligodendrocyte and myelin film surface, content seldom but has hyperimmunization originality in vivo, can cause that demyelination antibody response can cause again the central nervous system myelin protein composition of t cell responses.Pass through MOG
35-55mix sensitization C57/BL6 mouse with adjuvant, can cause that mouse produces MOG
35-55specific immune response, and then cause the reaction of neurone demyelination, show as weight loss, tetraplegia, tail is unable.Beginning oral administration from modeling the 10th day, administration group gives respectively the different phenylbenzylamine compounds of 10mg/kg every day.CsA, as positive control, is dissolved in sweet oil, every day abdominal injection 10mg/kg.Solvent control group gives sweet oil in contrast.Administration continues to experimental observation and finishes.As shown in Figure 4, in the time of modeling the 23rd day, phenylbenzylamine compounds 1-11 all can improve the clinical score of EAE mouse to some extent.Wherein with compound 5,10,11 to improve effect the most remarkable.Further, we find compound 5,10, and 11 can significantly suppress morbidity ratio and the severity (Fig. 5) of mouse, and can alleviate due to the caused weight loss of morbidity.Onset peak period is got Mouse Lumbar section spinal cord, and its inflammatory cell infiltration is investigated in H & E dyeing, capillary blood vessel swelling, and blood vessel " oversleeve sample " changes, and little blood vessel especially Venule has that a large amount of inflammatory cells are intensive encompasses " oversleeve sample " around.As shown in Figure 6, compound 5,10,11 can suppress inflammatory cell infiltration significantly, and capillary blood vessel perviousness.By the specific stain of ponceau-BG, can find, the myelin overwhelming majority of model mice comes off, and compound 5,10,11 can obviously suppress its demyelination effect (Fig. 6).
Above result is comprehensively pointed out, phenylbenzylamine compounds can be used as immunosuppressor in autoimmune disorder, particularly treats the autoimmune disorder of T cell participation as multiple sclerosis, systemic lupus erythematous, rheumatoid arthritis, autoimmune nephritis, inflammatory bowel.
Claims (3)
1. phenylbenzylamine compounds, it is characterized in that for following structural formula any one:
2. the application of phenylbenzylamine compounds according to claim 1 in preparation treatment autoimmune disorder medicine; Described autoimmune disorder is the autoimmune disorder that T cell participates in; The autoimmune disorder that described T cell participates in is multiple sclerosis, systemic lupus erythematous, rheumatoid arthritis, autoimmune nephritis or inflammatory bowel.
3. the application of phenylbenzylamine compounds according to claim 2 in preparation treatment autoimmune disorder medicine, the autoimmune disorder that its feature participates at T cell is multiple sclerosis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210081607.8A CN102627578B (en) | 2011-10-20 | 2012-03-23 | Phenyl benzylamine compound and application thereof |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110319721 | 2011-10-20 | ||
| CN201110319721.5 | 2011-10-20 | ||
| CN201210081607.8A CN102627578B (en) | 2011-10-20 | 2012-03-23 | Phenyl benzylamine compound and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102627578A CN102627578A (en) | 2012-08-08 |
| CN102627578B true CN102627578B (en) | 2014-06-11 |
Family
ID=46586004
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210081607.8A Expired - Fee Related CN102627578B (en) | 2011-10-20 | 2012-03-23 | Phenyl benzylamine compound and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102627578B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1729175A (en) * | 2002-12-20 | 2006-02-01 | 阿克佐诺贝尔公司 | Tetrahydroquinoline derivatives |
| WO2006049835A2 (en) * | 2004-10-19 | 2006-05-11 | Novartis Vaccines And Diagnostics Inc. | Indole and benzimidazole derivatives |
| US7662581B1 (en) * | 2003-12-18 | 2010-02-16 | Novartis Vaccines And Diagnostics, Inc. | Eg5 co-crystals |
| EP2193123A1 (en) * | 2007-08-09 | 2010-06-09 | EMC microcollections GmbH | Novel benzimidazol-2-yl-alkylamines and the use thereof as microbicidal agents |
-
2012
- 2012-03-23 CN CN201210081607.8A patent/CN102627578B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1729175A (en) * | 2002-12-20 | 2006-02-01 | 阿克佐诺贝尔公司 | Tetrahydroquinoline derivatives |
| US7662581B1 (en) * | 2003-12-18 | 2010-02-16 | Novartis Vaccines And Diagnostics, Inc. | Eg5 co-crystals |
| WO2006049835A2 (en) * | 2004-10-19 | 2006-05-11 | Novartis Vaccines And Diagnostics Inc. | Indole and benzimidazole derivatives |
| EP2193123A1 (en) * | 2007-08-09 | 2010-06-09 | EMC microcollections GmbH | Novel benzimidazol-2-yl-alkylamines and the use thereof as microbicidal agents |
Non-Patent Citations (2)
| Title |
|---|
| Anti-inflammatory benzene diamine compound inhibited toll-like receptor 4-mediated inducible nitric oxide synthase expression and nuclear factor-kappaB activation;Byung Hak Kim等;《Biol.Pharm.Bull》;20050531;第28卷(第5期);908-911 * |
| Byung Hak Kim等.Anti-inflammatory benzene diamine compound inhibited toll-like receptor 4-mediated inducible nitric oxide synthase expression and nuclear factor-kappaB activation.《Biol.Pharm.Bull》.2005,第28卷(第5期),908-911. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102627578A (en) | 2012-08-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jiang et al. | Antidepressant‐like effects of fenofibrate in mice via the hippocampal brain‐derived neurotrophic factor signalling pathway | |
| Liu et al. | MicroRNA-129-5p inhibits the development of autoimmune encephalomyelitis-related epilepsy by targeting HMGB1 through the TLR4/NF-kB signaling pathway | |
| Carlini et al. | Ghrelin and memory: differential effects on acquisition and retrieval | |
| Roesler et al. | Molecular mechanisms mediating gastrin-releasing peptide receptor modulation of memory consolidation in the hippocampus | |
| CN102112131A (en) | Methods of treating thalassemia | |
| CN104619691A (en) | Heterocycles capable of modulating T-cell responses, and methods of using same | |
| WO2016183905A1 (en) | Icarisid compound, preparation method therefor, and application thereof | |
| EA006556B1 (en) | Methods, compounds, and compositions for reducing body fat and modulating fatty acid metabolism | |
| MX2010012358A (en) | Multiple myeloma treatments. | |
| CN103270022A (en) | Agonists of neurotrophin receptors and their use as medicaments | |
| Dhuria et al. | Food consumption and activity levels increase in rats following intranasal Hypocretin-1 | |
| Lee et al. | Acid-sensing by airway afferent nerves | |
| CN104619687A (en) | Bicyclic heterocycles capable of modulating t-cell responses, and methods of using same | |
| Mesbah Moosavi et al. | The unfolded protein response in relation to mitochondrial biogenesis in skeletal muscle cells | |
| Yuan et al. | Frontline Science: Two flavonoid compounds attenuate allergic asthma by regulating epithelial barrier via G protein-coupled estrogen receptor: Probing a possible target for allergic inflammation | |
| CN102824400A (en) | Composition containing eucommia ulmoides and teasel roots and application of composition for treating osteoporosis | |
| CN102627578B (en) | Phenyl benzylamine compound and application thereof | |
| CN104603133B (en) | For treating cancer and immunosuppressant combination treatment | |
| Zyuzkov et al. | Cerebroprotective and regenerative effects of alkaloid Z77 under conditions of brain ischemia | |
| BRPI0706865A2 (en) | Method for preparing shinyleaf yellowhorn extract and shinyleaf yellowhorn extract | |
| CN103405787B (en) | Molecular targeted nucleic acid nano-medicament based on miR-141 (micro ribonucleic acid-141), preparation and application thereof | |
| CN102247347B (en) | Application of taurine in preparing medicament for preventing nerve cell damage caused by dependence producing medicaments | |
| CN105085308A (en) | Calamus amide compound as well as preparation method and application of calamus amide compound | |
| CN104224796B (en) | Application of oleanane triterpene ester derivative in preparation for anti-neurodegeneration medicine | |
| CN104788391B (en) | Cinnamoyl dimethyl diaminophenazine chloride amide (CA PZ) and its preparation and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140611 Termination date: 20180323 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |