CN102627578A - Phenyl benzylamine compound and application thereof - Google Patents
Phenyl benzylamine compound and application thereof Download PDFInfo
- Publication number
- CN102627578A CN102627578A CN2012100816078A CN201210081607A CN102627578A CN 102627578 A CN102627578 A CN 102627578A CN 2012100816078 A CN2012100816078 A CN 2012100816078A CN 201210081607 A CN201210081607 A CN 201210081607A CN 102627578 A CN102627578 A CN 102627578A
- Authority
- CN
- China
- Prior art keywords
- compound
- compounds
- reaction
- preparation
- cell
- 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.)
- Granted
Links
- -1 Phenyl benzylamine compound Chemical class 0.000 title description 16
- 238000002360 preparation method Methods 0.000 claims abstract description 45
- GTWJETSWSUWSEJ-UHFFFAOYSA-N n-benzylaniline Chemical class C=1C=CC=CC=1CNC1=CC=CC=C1 GTWJETSWSUWSEJ-UHFFFAOYSA-N 0.000 claims abstract description 44
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 27
- 208000023275 Autoimmune disease Diseases 0.000 claims abstract description 23
- 239000003814 drug Substances 0.000 claims abstract description 12
- 201000006417 multiple sclerosis Diseases 0.000 claims abstract description 9
- 230000001363 autoimmune Effects 0.000 claims abstract description 7
- 201000008383 nephritis Diseases 0.000 claims abstract description 7
- 206010039073 rheumatoid arthritis Diseases 0.000 claims abstract description 7
- 230000002757 inflammatory effect Effects 0.000 claims description 6
- 206010025135 lupus erythematosus Diseases 0.000 claims description 6
- 230000009885 systemic effect Effects 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 abstract description 96
- 230000000694 effects Effects 0.000 abstract description 22
- 239000003018 immunosuppressive agent Substances 0.000 abstract description 5
- 231100000331 toxic Toxicity 0.000 abstract description 3
- 230000002588 toxic effect Effects 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 2
- 229960003444 immunosuppressant agent Drugs 0.000 abstract description 2
- 230000001861 immunosuppressant effect Effects 0.000 abstract description 2
- 230000007246 mechanism Effects 0.000 abstract description 2
- 208000022559 Inflammatory bowel disease Diseases 0.000 abstract 1
- 201000000596 systemic lupus erythematosus Diseases 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 description 57
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 36
- 239000003795 chemical substances by application Substances 0.000 description 29
- 241000699666 Mus <mouse, genus> Species 0.000 description 28
- 238000004440 column chromatography Methods 0.000 description 24
- 238000001514 detection method Methods 0.000 description 18
- 238000007867 post-reaction treatment Methods 0.000 description 16
- 238000005406 washing Methods 0.000 description 16
- 238000003756 stirring Methods 0.000 description 15
- 238000000034 method Methods 0.000 description 14
- 238000012360 testing method Methods 0.000 description 14
- 238000012546 transfer Methods 0.000 description 14
- 238000010792 warming Methods 0.000 description 14
- 108010074328 Interferon-gamma Proteins 0.000 description 13
- 239000002253 acid Substances 0.000 description 12
- 238000001816 cooling Methods 0.000 description 12
- 238000012545 processing Methods 0.000 description 12
- 238000000746 purification Methods 0.000 description 12
- 238000002347 injection Methods 0.000 description 11
- 239000007924 injection Substances 0.000 description 11
- 238000010172 mouse model Methods 0.000 description 11
- 230000003595 spectral effect Effects 0.000 description 11
- 230000003203 everyday effect Effects 0.000 description 10
- 230000005764 inhibitory process Effects 0.000 description 10
- 208000016192 Demyelinating disease Diseases 0.000 description 9
- 206010012305 Demyelination Diseases 0.000 description 9
- 238000002965 ELISA Methods 0.000 description 9
- 102000006386 Myelin Proteins Human genes 0.000 description 9
- 108010083674 Myelin Proteins Proteins 0.000 description 9
- 238000004043 dyeing Methods 0.000 description 9
- 210000004969 inflammatory cell Anatomy 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 239000006285 cell suspension Substances 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- 102100037850 Interferon gamma Human genes 0.000 description 7
- 206010070834 Sensitisation Diseases 0.000 description 7
- 239000002671 adjuvant Substances 0.000 description 7
- 230000000890 antigenic effect Effects 0.000 description 7
- 230000008595 infiltration Effects 0.000 description 7
- 238000001764 infiltration Methods 0.000 description 7
- 210000005012 myelin Anatomy 0.000 description 7
- 239000002994 raw material Substances 0.000 description 7
- 230000008313 sensitization Effects 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- YQYBUJYBXOVWQW-UHFFFAOYSA-N [3-[4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl]oxyphenyl]-(3,4-dihydro-1H-isoquinolin-2-yl)methanone Chemical compound NCC1=CC(=NC(=C1)C(F)(F)F)OC=1C=C(C=CC=1)C(=O)N1CC2=CC=CC=C2CC1 YQYBUJYBXOVWQW-UHFFFAOYSA-N 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 235000009508 confectionery Nutrition 0.000 description 6
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 6
- 230000028327 secretion Effects 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 238000000967 suction filtration Methods 0.000 description 6
- 230000004580 weight loss Effects 0.000 description 6
- 108700031361 Brachyury Proteins 0.000 description 5
- 230000003526 lymphopoietic effect Effects 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- YYYARFHFWYKNLF-UHFFFAOYSA-N 4-[(2,4-dimethylphenyl)diazenyl]-3-hydroxynaphthalene-2,7-disulfonic acid Chemical compound CC1=CC(C)=CC=C1N=NC1=C(O)C(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=C12 YYYARFHFWYKNLF-UHFFFAOYSA-N 0.000 description 4
- 239000012980 RPMI-1640 medium Substances 0.000 description 4
- 230000006044 T cell activation Effects 0.000 description 4
- YZCKVEUIGOORGS-NJFSPNSNSA-N Tritium Chemical compound [3H] YZCKVEUIGOORGS-NJFSPNSNSA-N 0.000 description 4
- 230000003187 abdominal effect Effects 0.000 description 4
- 210000004204 blood vessel Anatomy 0.000 description 4
- 239000000839 emulsion Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 230000000144 pharmacologic effect Effects 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 210000000278 spinal cord Anatomy 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 229910052722 tritium Inorganic materials 0.000 description 4
- 210000003462 vein Anatomy 0.000 description 4
- QFLWZFQWSBQYPS-AWRAUJHKSA-N (3S)-3-[[(2S)-2-[[(2S)-2-[5-[(3aS,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]-3-methylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-4-[1-bis(4-chlorophenoxy)phosphorylbutylamino]-4-oxobutanoic acid Chemical compound CCCC(NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@@H](NC(=O)CCCCC1SC[C@@H]2NC(=O)N[C@H]12)C(C)C)P(=O)(Oc1ccc(Cl)cc1)Oc1ccc(Cl)cc1 QFLWZFQWSBQYPS-AWRAUJHKSA-N 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 206010037714 Quadriplegia Diseases 0.000 description 3
- 229960000583 acetic acid Drugs 0.000 description 3
- 229940125904 compound 1 Drugs 0.000 description 3
- 229940125773 compound 10 Drugs 0.000 description 3
- 229940125782 compound 2 Drugs 0.000 description 3
- 229940126214 compound 3 Drugs 0.000 description 3
- 229940125898 compound 5 Drugs 0.000 description 3
- OMFXVFTZEKFJBZ-HJTSIMOOSA-N corticosterone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@H](CC4)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OMFXVFTZEKFJBZ-HJTSIMOOSA-N 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- FVIZARNDLVOMSU-UHFFFAOYSA-N ginsenoside K Natural products C1CC(C2(CCC3C(C)(C)C(O)CCC3(C)C2CC2O)C)(C)C2C1C(C)(CCC=C(C)C)OC1OC(CO)C(O)C(O)C1O FVIZARNDLVOMSU-UHFFFAOYSA-N 0.000 description 3
- 239000012362 glacial acetic acid Substances 0.000 description 3
- 229960000890 hydrocortisone Drugs 0.000 description 3
- 238000011835 investigation Methods 0.000 description 3
- ZLVXBBHTMQJRSX-VMGNSXQWSA-N jdtic Chemical compound C1([C@]2(C)CCN(C[C@@H]2C)C[C@H](C(C)C)NC(=O)[C@@H]2NCC3=CC(O)=CC=C3C2)=CC=CC(O)=C1 ZLVXBBHTMQJRSX-VMGNSXQWSA-N 0.000 description 3
- 230000001537 neural effect Effects 0.000 description 3
- 230000007170 pathology Effects 0.000 description 3
- LVTJOONKWUXEFR-FZRMHRINSA-N protoneodioscin Natural products O(C[C@@H](CC[C@]1(O)[C@H](C)[C@@H]2[C@]3(C)[C@H]([C@H]4[C@@H]([C@]5(C)C(=CC4)C[C@@H](O[C@@H]4[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@@H](O)[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@H](CO)O4)CC5)CC3)C[C@@H]2O1)C)[C@H]1[C@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1 LVTJOONKWUXEFR-FZRMHRINSA-N 0.000 description 3
- 238000012827 research and development Methods 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102100031547 HLA class II histocompatibility antigen, DO alpha chain Human genes 0.000 description 2
- 101000866278 Homo sapiens HLA class II histocompatibility antigen, DO alpha chain Proteins 0.000 description 2
- 101100077708 Mus musculus Mog gene Proteins 0.000 description 2
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 206010033799 Paralysis Diseases 0.000 description 2
- 201000005702 Pertussis Diseases 0.000 description 2
- 230000005867 T cell response Effects 0.000 description 2
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000005875 antibody response Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000006399 behavior Effects 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000001046 green dye Substances 0.000 description 2
- 208000026278 immune system disease Diseases 0.000 description 2
- 230000001506 immunosuppresive effect Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000009545 invasion Effects 0.000 description 2
- 210000003141 lower extremity Anatomy 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 239000008176 lyophilized powder Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 210000004248 oligodendroglia Anatomy 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 230000010412 perfusion Effects 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000007929 subcutaneous injection Substances 0.000 description 2
- 238000010254 subcutaneous injection Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000008961 swelling Effects 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 108700012359 toxins Proteins 0.000 description 2
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 2
- 210000000264 venule Anatomy 0.000 description 2
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010037211 Psychomotor hyperactivity Diseases 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 210000003050 axon Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- AGEZXYOZHKGVCM-UHFFFAOYSA-N benzyl bromide Chemical compound BrCC1=CC=CC=C1 AGEZXYOZHKGVCM-UHFFFAOYSA-N 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000005357 flat glass Substances 0.000 description 1
- 210000003194 forelimb Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000000527 lymphocytic effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 208000037922 refractory disease Diseases 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000000115 thoracic cavity Anatomy 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 one type of refractory disease, and its morbidity is how relevant with the lymphocytic overactivity of T, and many at present employing immunosuppressor are treated.Common autoimmune disorder comprises multiple sclerosis, systemic lupus erythematous, rheumatoid arthritis, autoimmune nephritis, inflammatory bowel etc.Autoimmune disorder does not have cure method at present, and mostly existing medicine is injection type, and curative effect is not good enough, and toxic side effect is big, and medical expense is very expensive, needs the novel therapeutic medicine that research and development meet pharmacoeconomics badly.
Three, summary of the invention
The purpose of this invention is to provide one type of phenylbenzylamine compounds as the application of immunosuppressor in autoimmune disorder that preparation treatment T cell is participated in such as multiple sclerosis, systemic lupus erythematous, rheumatoid arthritis, autoimmune nephritis, inflammatory bowel medicine.
Technical scheme of the present invention:
The phenylbenzylamine compounds is characterized in that any one of following structural formula:
Wherein
The application of above-mentioned phenylbenzylamine compounds in preparation treatment autoimmune disorder medicine.
As the autoimmune disorder of the described autoimmune disorder of a kind of optimal way for the participation of T cell.
The autoimmune disorder of 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 of participating in as a kind of optimal way T cell is a multiple sclerosis.
The preparation method of phenylbenzylamine compounds:
Parent nucleus is synthetic: accomplish synthetic by reactions (), (two).
Reaction (one):
Raw material and injected volume:
Operation steps:
In the 2L four-necked bottle, add raw material (1), (2), (3), stir, behind the dropping raw material (4), be warming up to 190 ℃～200 ℃ by above-mentioned injected volume.(developping agent is PE: EA=1 to TLC detection reaction degree: 9).Post-reaction treatment: reaction product is poured in the zero(ppm) water, has solid to separate out.Suction filtration, filtrating is used CH
2Cl
2Extract 3 times, merge CH
2Cl
2Layer and suction filtration gained solid.Wash dry concentrating again 1 time; Last EA recrystallization gets pure article.
Reaction (two)
Raw material and injected volume:
Operation steps:
The three is added the 500ml eggplant-shape bottle, stir, refluxed 1 hour.(developping agent is PE: EA=5 to TLC detection reaction degree: 3).Post-reaction treatment: reaction back system is used NH
3H
2After O transfers PH to 9.0, add CH
2Cl
2500ml, suction filtration.Solid is used
CH
2Cl
2
Wash 1 time, merge CH
2Cl
2Layer is with washing 2 times, and is dry concentrated.
Compound (1): on the parent nucleus basis, accomplish synthetic by reactions (three), (four).
Reaction (three)
Raw material and injected volume:
Operation steps:
The 500ml three-necked bottle adds CDI, THF150ml.Cryosel is chilled to 0 ℃.Add Gly-Boc, 0 ℃ keeps stirring 15min.Be warming up to 60 ℃, reacted 1 hour.Be cooled to normal temperature, drip anils and THF50ml.Normal temperature spends the night, next day the column chromatography processing purified.
Reaction (four)
Raw material and injected volume:
Operation steps:
Add (1), (2) in the single neck bottle of 500ml, drip (3), reaction is spent the night, and (developping agent is PE: EA=5 to TLC detection reaction degree: 3).
Post-reaction treatment:
After the reaction system cooling, add 10% hydrochloric acid, transfer PH to acid, separatory, CHCl
3Layer with the washing of 10% hydrochloric acid saturated common salt once.Dry concentrating, column chromatography purification obtains compound (1).
Compound (2): on the parent nucleus basis, accomplish synthetic by reactions (five), (six).
Reaction (five)
Reaction (six)
Raw material injected volume, operation, post-reaction treatment with reference to reaction (three), (four), can get compound (2).
The residue compound is realized by following reaction:
R wherein
1=Me, R
2=C
aH
bN
cS
dO
eA=0-11 wherein, 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) TCR token stimulus agent anti-CD3/anti-CD28 activatory mouse T lymphocyte proliferation test
Get the inguinal region of mouse and 4 lymphoglandula of oxter, be prepared into single cell suspension.RPMI-1640 is made into 3 * 10 with perfect medium
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 (available from BD PharMingen company), hatches jointly with compound, and isotropic substance tritium mark method detects lymphopoietic situation.
2) MOG
35-55Cause the test of EAE model mice T lymphopoiesis and cytokine release
Get MOG
35-55(available from the Shanghai bio tech ltd of shining by force) and the inguinal region of 10 days the mouse of emulsion sensitization of complete Freund's adjuvant (available from sigma company) preparation and 4 lymphoglandula of oxter, and spleen are prepared into single cell suspension respectively.RPMI-1640 is made into 3 * 10 with perfect medium
6The cell suspension of/ml plants in 96 orifice plates 3 * 10
5The every hole of individual cell is with final concentration 10 μ g/ml MOG
35-55Stimulate 72h, hatch jointly with compound, isotropic substance tritium mark method detects the situation of lymphocyte antigen proliferated specifically, and supernatant is used to detect 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 (available from R&D company) is carried out.
4) EAE (EAE) model and pathology detection
Take by weighing MOG
35-55Lyophilized powder is dissolved in PBS (200 μ g/ only); Mycobacterium tuberculosis (available from Difco company) is dissolved in incomplete Freund's adjuvant (available from sigma company; Final concentration 6mg/ml); These two kinds of suspensions in 1: the 1 fully emulsified one-tenth water-in-oil of ratio state, are adopted the multi-point injection method, promptly 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 is available from sigma company).Tail vein injection PTX/PBS 0.2ml once more behind the 48h.From immunity same day, every day is to mouse observations of weighing, and carries out the scoring of neural function study of behaviour, and writes down the mouse invasion rate.Take out myeloid tissue in the mouse model onset peak period and fix, H&E dyeing shows the situation of inflammatory cell infiltration.Ponceau 2R-bright green dyeing shows the demyelination situation.
The pharmacological tests of phenylbenzylamine compounds and analysis
1) the phenylbenzylamine compounds has suppressed the propagation of TCR token stimulus agent anti-CD3/anti-CD28 activatory mouse T cell
In order to estimate the external effect that whether has suppressor T cell propagation of a collection of phenylbenzylamine compounds of synthetic, 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) the phenylbenzylamine compounds has suppressed EAE mouse MOG
35-55T cells with antigenic specificity propagation and the sub-IFN-γ of secrete cytokines
Whether have the effect of improving EAE in order to estimate a collection of phenylbenzylamine compounds of synthetic, it is external to MOG that we have investigated this batch compound
35-55The influence 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 verified further whether this batch phenylbenzylamine compounds influences the secretion of IFN-γ.As shown in Figure 3, compound 1-11 has suppressed MOG to some extent
35-55The T cells with antigenic specificity secretion of gamma-IFN.Wherein with compound 5,10,11 inhibition IFN-γ excretory effect is the most remarkable.
3) the phenylbenzylamine compounds has improved the morbidity of EAE mouse
Next, we further investigate the Immunological diseases model that whether can suppress the Th1 mediation in this batch phenylbenzylamine compounds body.MOG
35-55Be a kind of transmembrane glycoprotein that is expressed in maincenter oligodendrocyte and myelin film surface, content seldom but has hyperimmunization originality in vivo, can cause that the demyelination antibody response can cause the cns myelin protein composition of t cell responses again.Pass through MOG
35-55Mix sensitization C57/BL6 mouse with adjuvant, can cause that mouse produces MOG
35-55Specific immunoreation, and then cause the reaction of neurone demyelination, show as weight loss, tetraplegia, tail is unable.From the 10th day beginning oral administration of modeling, the administration group gives the different phenylbenzylamine compounds of 10mg/kg respectively every day.CsA is dissolved in the sweet oil as positive control, every day abdominal injection 10mg/kg.Solvent control group gives sweet oil as contrast.Administration continues to experimental observation and finishes.As shown in Figure 4, in the time of the 23rd day, phenylbenzylamine compounds 1-11 all can improve the clinical score of EAE mouse to some extent in modeling.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 because the caused weight loss of morbidity.Onset peak period is got mouse waist section spinal cord, and its inflammatory cell infiltration is investigated in H&E dyeing, capillary blood vessel swelling, and blood vessel " oversleeve appearance " changes, and promptly little blood vessel has especially that a large amount of inflammatory cells are intensive to encompass " oversleeve appearance " around the Venule.As shown in Figure 6, compound 5,10,11 can suppress inflammatory cell infiltration significantly, and the capillary blood vessel perviousness.Through ponceau-bright green specific stain, can find that the myelin overwhelming majority of model mice comes off, and compound 5,10,11 can obviously suppress its demyelination effect (Fig. 6).
Beneficial effect
Phenylbenzylamine compounds 1-11 is a series of new compounds of synthetic; Can be used as autoimmune disorder that immunosuppressant treatment T cell participates in such 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 owing to the simple environmental protection of synthetic route, compares with the obvious advantage with existing medicine.
Description of drawings
Fig. 1. the The selection result of phenylbenzylamine compounds vitro inhibition anti-CD3/anti-CD28 activatory mouse T cell propagation.Adopt 10 μ g/ml anti-CD3 and 1 μ g/ml anti-CD28 to stimulate T cell activation propagation.Compound and the activating T cell waiting to sieve in the compound library are applied 72h altogether, through [H
3] mix each testing compound of method investigation for the lymphopoietic influence 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.With the compound and the MOG that wait to sieve in the compound library
35-55The activated T cell applies 72h altogether, through [H
3] mix each testing compound of method investigation for lymphopoietic influence.
Fig. 3. phenylbenzylamine compounds vitro inhibition MOG
35-55T lymphocyte specific discharges the The selection result of IFN-γ.MOG
35-55Can stimulate the T cell activation propagation in EAE model mice source.With the compound and the MOG that wait to sieve in the compound library
35-55The activated T cell is collected supernatant after applying 24h altogether.ELISA detects each compound for Th1 cytokine IFN-γ excretory restraining effect.
Fig. 4. the phenylbenzylamine compounds is to the effect of improving of 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 immunoreation of MOG, and then cause the reaction of neurone demyelination, show as weight loss, tetraplegia, tail is unable.The phenylbenzylamine compounds gives 10mg/kg every day from the 10th day beginning oral administration of modeling.CsA is dissolved in the sweet oil, CsA control group abdominal injection every day 10mg/kg.Administration continues to experimental observation and finishes.The phenylbenzylamine compounds can suppress the occurring degree of mouse to some extent, and is 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 the 10th day beginning oral administration of modeling, gives 10mg/kg respectively every day.CsA is dissolved in the 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 because morbidity caused weight loss (C).
Fig. 6. 5,10,11 pairs of EAE in mice model spinal cords of phenylbenzylamine compounds pathology level improve effect.Onset peak period is got mouse waist section spinal cord, and the visible compound 5,10,11 of H&E dyeing can be reduced inflammatory cell infiltration and capillary blood vessel perviousness, and the visible compound 5,10,11 of ponceau-bright green specific stain can suppress the 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) with benzyl bromine (200ml), DMF (1.2ml) slowly is added drop-wise in the reaction solution.Be warming up to 190 ℃～200 ℃.(developping agent is PE: EA=1 to TLC detection reaction degree: 9).Reaction product is poured in the zero(ppm) water, has solid to separate out.Suction filtration, filtrating is used CH
2Cl
2It is inferior to extract 3 (200ml*3), merges CH
2Cl
2Layer and suction filtration gained solid.Wash dry concentrating again 1 time.Get pure article with re-crystallizing in ethyl acetate at last, productive rate 58%.
The preparation of N1-benzyl-4-methyl-O-Phenylene Diamine (III)
Adding N-benzyl-4-methyl-o-Nitraniline in the 500ml eggplant-shape bottle (10g, 0.04mol), SnCl
2.2H
2(95g, 0.44mol), ethanol (80ml) stirs O, refluxes 1 hour.(developping agent is PE: EA=5 to TLC detection reaction degree: 3).Post-reaction treatment: reaction back system is used NH
3H
2After O transfers pH to 9.0, add CH
2Cl
2500ml, suction filtration.Solid is used CH
2Cl
2Wash 1 time, merge CH
2Cl
2Layer is with washing 2 times, dry concentrating, compound N 1-benzyl-4-methyl-O-Phenylene Diamine productive rate 72%.
The preparation of compound 1
The preparation of 1 compd A
The 500ml three-necked bottle, and adding CDI (10mg, 0.06mmol), N1-benzyl-4-methyl-O-Phenylene Diamine (III) (6.69g, 0.03mol) THF (150ml).Cryosel is chilled to 0 ℃.(4g, 0.02mol), 0 ℃ keeps stirring 15min to add Gly-Boc.Be warming up to 60 ℃, reacted 1 hour.Be cooled to normal temperature, drip anils and THF 50ml.Normal temperature spends the night, and column chromatography processing purified next day (PE/EA=10/1) gets compd A, productive rate 82%.
The preparation of 2 compounds 1
Add in the single neck bottle of 500ml 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 the reaction system cooling, add 10% hydrochloric acid, transfer pH to acid, separatory, CHCl
3Layer with the washing of 10% hydrochloric acid saturated common salt once.Dry concentrating, (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
The 500ml three-necked bottle, and adding CDI (10mg, 0.06mmol), N1-benzyl-4-methyl-O-Phenylene Diamine (III) (6g, 0.028mol) THF (150ml).Cryosel is chilled to 0 ℃.(5g, 0.026mol), 0 ℃ keeps stirring 15min to add Ala-Boc.Be warming up to 60 ℃, reacted 1 hour.Be cooled to normal temperature, drip anils and THF 50ml.Normal temperature spends the night, and column chromatography processing purified next day (PE/EA=10/1) gets compd B, productive rate 80%.
The preparation of 2 compounds 2
Add in the single neck bottle of 500ml 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 the reaction system cooling, add 10% hydrochloric acid, transfer pH to acid, separatory, CHCl
3Layer with the washing of 10% hydrochloric acid saturated common salt once.Dry concentrating, (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
The 500ml three-necked bottle, and adding CDI (10mg, 0.06mmol), N1-benzyl-4-methyl-O-Phenylene Diamine (III) (6g, 0.028mol) THF (150ml).Cryosel is chilled to 0 ℃.(5g, 0.022mol), 0 ℃ keeps stirring 15min to add Leu-Boc.Be warming up to 60 ℃, reacted 1 hour.Be cooled to normal temperature, drip anils and THF 50ml.Normal temperature spends the night, and column chromatography processing purified next day (PE/EA=8/1) gets Compound C, productive rate 81%.
The preparation of 2 compounds 3
Add in the single neck bottle of 500ml 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 the reaction system cooling, add 10% hydrochloric acid, transfer pH to acid, separatory, CHCl
3Layer with the washing of 10% hydrochloric acid saturated common salt once.Dry concentrating, (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
The 500ml three-necked bottle, and adding CDI (10mg, 0.06mmol), N1-benzyl-4-methyl-O-Phenylene Diamine (III) (6g, 0.028mol) THF (150ml).Cryosel is chilled to 0 ℃.(5g, 0.022mol), 0 ℃ keeps stirring 15min to add Ile-Boc.Be warming up to 60 ℃, reacted 1 hour.Be cooled to normal temperature, drip anils and THF 50ml.Normal temperature spends the night, and column chromatography processing purified next day (PE/EA=9/1) gets compd E, productive rate 80%.
The preparation of 2 compounds 4
Add in the single neck bottle of 500ml 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 the reaction system cooling, add 10% hydrochloric acid, transfer pH to acid, separatory, CHCl
3Layer with the washing of 10% hydrochloric acid saturated common salt once.Dry concentrating, (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
The 500ml three-necked bottle, and adding CDI (10mg, 0.06mmol), N1-benzyl-4-methyl-O-Phenylene Diamine (III) (6g, 0.028mol) THF (150ml).Cryosel is chilled to 0 ℃.(5g, 0.02mol), 0 ℃ keeps stirring 15min to add compound f.
Be warming up to 60 ℃, reacted 1 hour.Be cooled to normal temperature, drip anils and THF 50ml.Normal temperature spends the night, and column chromatography processing purified next day (PE/EA=8/1) gets compound F 17-hydroxy-corticosterone, productive rate 78%.
The preparation of 2 compounds 5
Add in the single neck bottle of 500ml 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 the reaction system cooling, add 10% hydrochloric acid, transfer pH to acid, separatory, CHCl
3Layer with the washing of 10% hydrochloric acid saturated common salt once.Dry concentrating, (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
The 500ml three-necked bottle, and adding CDI (10mg, 0.06mmol), N1-benzyl-4-methyl-O-Phenylene Diamine (III) (6g, 0.028mol) THF (150ml).Cryosel is chilled to 0 ℃.(5g, 0.019mol), 0 ℃ keeps stirring 15min to add compound g.
Be warming up to 60 ℃, reacted 1 hour.Be cooled to normal temperature, drip anils and THF 50ml.Normal temperature spends the night, and column chromatography processing purified next day (PE/EA=8/1) gets compound G, productive rate 69%.
The preparation of 2 compounds 6
Adding compound G in the single neck bottle of 500ml (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 the reaction system cooling, add 10% hydrochloric acid, transfer pH to acid, separatory, CHCl
3Layer with the washing of 10% hydrochloric acid saturated common salt once.Dry concentrating, (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
The 500ml three-necked bottle, and adding CDI (10mg, 0.06mmol), N1-benzyl-4-methyl-O-Phenylene Diamine (III) (6g, 0.028mol) THF (150ml).Cryosel is chilled to 0 ℃.(5g, 0.018mol), 0 ℃ keeps stirring 15min to add compound h.
Be warming up to 60 ℃, reacted 1 hour.Be cooled to normal temperature, drip anils and THF 50ml.Normal temperature spends the night, and column chromatography processing purified next day (PE/EA=8/1) gets compound H, productive rate 72%.
The preparation of 2 compounds 7
Add in the single neck bottle of 500ml 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 the reaction system cooling, add 10% hydrochloric acid, transfer pH to acid, separatory, CHCl
3Layer with the washing of 10% hydrochloric acid saturated common salt once.Dry concentrating, (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
The 500ml three-necked bottle, and adding CDI (10mg, 0.06mmol), N1-benzyl-4-methyl-O-Phenylene Diamine (III) (6g, 0.028mol) THF (150ml).Cryosel is chilled to 0 ℃.(5g, 0.018mol), 0 ℃ keeps stirring 15min to add compound i.
Be warming up to 60 ℃, reacted 1 hour.Be cooled to normal temperature, drip anils and THF 50ml.Normal temperature spends the night, and column chromatography processing purified next day (PE/EA=8/1) gets compound I, productive rate 79%.
The preparation of 2 compounds 8
Add in the single neck bottle of 500ml 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 the reaction system cooling, add 10% hydrochloric acid, transfer pH to acid, separatory, CHCl
3Layer with the washing of 10% hydrochloric acid saturated common salt once.Dry concentrating, (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
The 500ml three-necked bottle, and adding CDI (10mg, 0.06mmol), N1-benzyl-4-methyl-O-Phenylene Diamine (III) (6g, 0.028mol) THF (150ml).Cryosel is chilled to 0 ℃.(5g, 0.018mol), 0 ℃ keeps stirring 15min to add compound j.
Be warming up to 60 ℃, reacted 1 hour.Be cooled to normal temperature, drip anils and THF 50ml.Normal temperature spends the night, and column chromatography processing purified next day (PE/EA=8/1) gets compound J, productive rate 77%.
The preparation of 2 compounds 9
Adding compound J in the single neck bottle of 500ml (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 the reaction system cooling, add 10% hydrochloric acid, transfer pH to acid, separatory, CHCl
3Layer with the washing of 10% hydrochloric acid saturated common salt once.Dry concentrating, (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
The 500ml three-necked bottle, and adding CDI (10mg, 0.06mmol), N1-benzyl-4-methyl-O-Phenylene Diamine (III) (6g, 0.028mol) THF (150ml).Cryosel is chilled to 0 ℃.(5g, 0.018mol), 0 ℃ keeps stirring 15min to add compound k.
Be warming up to 60 ℃, reacted 1 hour.Be cooled to normal temperature, drip anils and THF 50ml.Normal temperature spends the night, and column chromatography processing purified next day (PE/EA=4/1) gets compound K, productive rate 66%.
The preparation of 2 compounds 10
Add in the single neck bottle of 500ml 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 the reaction system cooling, add 10% hydrochloric acid, transfer pH to acid, separatory, CHCl
3Layer with the washing of 10% hydrochloric acid saturated common salt once.Dry concentrating, (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
The 500ml three-necked bottle, and adding CDI (10mg, 0.06mmol), N1-benzyl-4-methyl-O-Phenylene Diamine (III) (6g, 0.028mol) THF (150ml).Cryosel is chilled to 0 ℃.(5g, 0.017mol), 0 ℃ keeps stirring 15min to add compound l.
Be warming up to 60 ℃, reacted 1 hour.Be cooled to normal temperature, drip anils and THF 50ml.Normal temperature spends the night, and column chromatography processing purified next day (PE/EA=5/1) gets compound L, productive rate 63%.
The preparation of 2 compounds 11
Add in the single neck bottle of 500ml compound L (10g, 0.021mol), TFA (10g, 0.087mol), CHCl3 (80ml) reaction is spent the night, (developping agent is PE: EA=7 to TLC detection reaction degree: 4).Post-reaction treatment: after the reaction system cooling, add 10% hydrochloric acid, transfer pH to acid, separatory, CHCl
3Layer with the washing of 10% hydrochloric acid saturated common salt once.Dry concentrating, (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 anti-CD3/anti-CD28 activatory mouse T cell
Get the inguinal region of mouse and 4 lymphoglandula of oxter, be prepared into single cell suspension.RPMI-1640 is made into 3 * 10 with perfect medium
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 (available from BD PharMingen company), hatches jointly with compound, and isotropic substance tritium mark method detects lymphopoietic situation.
2) MOG
35-55Cause the test of EAE model mice T lymphopoiesis and cytokine release
Get MOG
35-55(available from the Shanghai bio tech ltd of shining by force) and the inguinal region of 10 days the mouse of emulsion sensitization of complete Freund's adjuvant (available from sigma company) preparation and 4 lymphoglandula of oxter are prepared into single cell suspension.RPMI-1640 is made into 3 * 10 with perfect medium
6The cell suspension of/ml plants in 96 orifice plates 3 * 10
5The every hole of individual cell is with final concentration 10 μ g/ml MOG
35-55Stimulate 72h, hatch jointly with compound, isotropic substance tritium mark method detects the situation of lymphocyte antigen proliferated specifically, and supernatant is used to detect 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 following: from the sealing bag of balance to room temperature, take out required lath, other lath sealing is put back to 4 ℃.Except that blank well, respectively sample or different concns standard substance (100 μ l/ hole) are added in the respective aperture, seal reacting hole with the shrouding gummed paper, 37 ℃ of incubators were hatched 90 minutes.Wash plate 4 times.Except that blank well, add biotinylated antibody working fluid (100 μ l/ hole).Seal reacting hole with the shrouding gummed paper, 37 ℃ of incubators were hatched 60 minutes.Wash plate 4 times.Except that blank well, add enzyme conjugates working fluid (100 μ l/ hole).Seal reacting hole with the shrouding gummed paper, 37 ℃ of incubators were hatched 30 minutes.Wash plate 4 times.Add developer 100 μ l/ holes, 37 ℃ of incubators of lucifuge were hatched 10-15 minute.Add stop buffer 100 μ l/ holes, measure OD450 value (in 5 minutes) behind the mixing at once.The OD value of each standard substance and sample should deduct the OD value in zero hole.Make X-coordinate with standard substance concentration, the OD value is made ordinate zou, connects the coordinate point of each standard substance with sweep, and the OD value through sample can calculate its concentration on typical curve.If sample OD value is higher than the typical curve upper limit, resurvey after should suitably diluting, should multiply by extension rate during calculating concentration.
4) EAE (EAE) model and pathology detection
Take by weighing MOG
35-55Lyophilized powder is dissolved in PBS (200 μ g/ only); Mycobacterium tuberculosis (available from Difco company) is dissolved in incomplete Freund's adjuvant (available from sigma company; Final concentration 6mg/ml); These two kinds of suspensions in 1: the 1 fully emulsified one-tenth water-in-oil of ratio state, are adopted the multi-point injection method, promptly 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 is available from sigma company).Tail vein injection PTX/PBS 0.2ml once more behind the 48h.From the immune same day, weigh to mouse every day, and observe, 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 minute, do not fall ill; 1 minute, afterbody was unable; 2 minutes, the unable and incomplete hind leg of afterbody (1～2 hind leg) paralysis; 3 minutes, two backs acroparalysis; 4 minutes, two hind legs and arbitrary forelimb were all paralysed, and can not reset after passive the standing up; 5 minutes, moribund condition or death.
With mouse anesthesia, open the thoracic cavity in the mouse model onset peak period, through PBS heart perfusion 20ml, with 4% Paraformaldehyde 96 perfusion internal fixing.Take out myeloid tissue then, put into behind the pipe that 4% Paraformaldehyde 96 is housed fixing.Gradient is dewatered actually, and YLENE is transparent, and transparent good tissue is put into the paraffin embedding of thawing, and the tissue block that embedding is good is cut into the thin slice of 5 μ m, on exhibition sheet and the sheet glass, copies sheet, H&E dyeing, and mounting, observation is taken pictures.H&E dyeing can show the situation of inflammatory cell infiltration.
Ponceau 2R-bright green dyeing can through ponceau 2R and myelin phospholipid composition combine make myelin present pink, and axon, lamellar sheath and neural in clothing green, the myelin of sex change is because so phosphatide disappears not painted.The section routine dewaxes to zero(ppm) water, with ponceau 2R dye liquor dyeing 5min, distillation washing; And then 2.5% act on 1min in the phosphotungstic acid aqueous solution, redyes 4min with bright green dye liquor, again with 1% glacial acetic acid aqueous solution differentiation 10s; Washing from the beginning; Gradient alcohol dehydration, YLENE is transparent, the neutral gum sealing.Wherein ponceau 2R staining fluid is by ponceau 2R 1g, Glacial acetic acid min. 99.5 1ml, zero(ppm) water 99ml configuration; Bright green dye liquor is by bright green 1.0g, Glacial acetic acid min. 99.5 1ml, zero(ppm) water 99ml configuration.
2. the pharmacological tests of phenylbenzylamine compounds and analysis
1) propagation of phenylbenzylamine compounds vitro inhibition anti-CD3/anti-CD28 activatory mouse T cell
Whether has immunosuppressive activity in order to estimate embodiment 1 described phenylbenzylamine compounds 1-11; At first adopt 10 μ g/ml anti-CD3 and 1 μ g/ml anti-CD28 to stimulate T cell activation propagation; Compound and the activating T cell waiting to sieve in the compound library are applied 72h altogether, through [H
3] mix each testing compound of method investigation for the lymphopoietic influence of T.As shown in Figure 1, compound 1-11 has suppressed the propagation of anti-CD3/anti-CD28 activated T cell 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) the phenylbenzylamine compounds suppresses EAE mouse MOG
35-55T cells with antigenic specificity propagation and secrete cytokines IFN-γ
Whether have the effect of improving EAE in order to estimate a collection of phenylbenzylamine compounds of synthetic, we at first utilize external MOG
35-55The antigen-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 verified further whether this batch phenylbenzylamine compounds influences the secretion of IFN-γ.As shown in Figure 3, compound 1-11 has suppressed MOG to some extent
35-55The T cells with antigenic specificity secretion of gamma-IFN.Wherein with compound 5,10,11 inhibition IFN-γ excretory effect is the most remarkable.
3) the phenylbenzylamine compounds has improved the morbidity of EAE mouse
Next, we further investigate the Immunological diseases model that whether can suppress the Th1 mediation in this batch phenylbenzylamine compounds body.MOG
35-55Be a kind of transmembrane glycoprotein that is expressed in maincenter oligodendrocyte and myelin film surface, content seldom but has hyperimmunization originality in vivo, can cause that the demyelination antibody response can cause the cns myelin protein composition of t cell responses again.Pass through MOG
35-55Mix sensitization C57/BL6 mouse with adjuvant, can cause that mouse produces MOG
35-55Specific immunoreation, and then cause the reaction of neurone demyelination, show as weight loss, tetraplegia, tail is unable.From the 10th day beginning oral administration of modeling, the administration group gives the different phenylbenzylamine compounds of 10mg/kg respectively every day.CsA is dissolved in the sweet oil as positive control, every day abdominal injection 10mg/kg.Solvent control group gives sweet oil as contrast.Administration continues to experimental observation and finishes.As shown in Figure 4, in the time of the 23rd day, phenylbenzylamine compounds 1-11 all can improve the clinical score of EAE mouse to some extent in modeling.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 because the caused weight loss of morbidity.Onset peak period is got mouse waist section spinal cord, and its inflammatory cell infiltration is investigated in H&E dyeing, capillary blood vessel swelling, and blood vessel " oversleeve appearance " changes, and promptly little blood vessel has especially that a large amount of inflammatory cells are intensive to encompass " oversleeve appearance " around the Venule.As shown in Figure 6, compound 5,10,11 can suppress inflammatory cell infiltration significantly, and the capillary blood vessel perviousness.Through ponceau-bright green specific stain, can find that the myelin overwhelming majority of model mice comes off, and compound 5,10,11 can obviously suppress its demyelination effect (Fig. 6).
Above result comprehensively points out; The phenylbenzylamine compounds can be used as immunosuppressor in autoimmune disorder, particularly treats autoimmune disorder that the T cell participates in such as multiple sclerosis, systemic lupus erythematous, rheumatoid arthritis, autoimmune nephritis, inflammatory bowel.
Claims (5)
1. phenylbenzylamine compounds is characterized in that being any one of following structural formula:
2. the application of phenylbenzylamine compounds according to claim 1 in preparation treatment autoimmune disorder medicine.
3. the application of phenylbenzylamine compounds according to claim 2 in preparation treatment autoimmune disorder medicine is characterized in that the autoimmune disorder that described autoimmune disorder is participated in for the T cell.
4. the application of phenylbenzylamine compounds according to claim 3 in preparation treatment autoimmune disorder medicine is characterized in that the autoimmune disorder that the T cell is participated in is multiple sclerosis, systemic lupus erythematous, rheumatoid arthritis, autoimmune nephritis or inflammatory bowel medicine.
5. the application of phenylbenzylamine compounds according to claim 4 in preparation treatment autoimmune disorder medicine, the autoimmune disorder that its characteristic is participated at the T cell is a 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 |
|---|---|---|---|
| CN201110319721.5 | 2011-10-20 | ||
| CN201110319721 | 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 true CN102627578A (en) | 2012-08-08 |
| CN102627578B 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 (1)
| Title |
|---|
| 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》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102627578B (en) | 2014-06-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Roesler et al. | Molecular mechanisms mediating gastrin-releasing peptide receptor modulation of memory consolidation in the hippocampus | |
| CN104825479B (en) | Icariside class compound, its preparation method and its people's cell is being promoted to generate gamma interferon effect and application in disease treatment | |
| Song et al. | Fundamentals of psychoneuroimmunology | |
| CN102112131A (en) | Methods of treating thalassemia | |
| MX2010012358A (en) | Multiple myeloma treatments. | |
| Grando et al. | A historic perspective on the current progress in elucidation of the biologic significance of non-neuronal acetylcholine | |
| 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 | |
| Chen et al. | Schisandrin B inhibits Th1/Th17 differentiation and promotes regulatory T cell expansion in mouse lymphocytes | |
| CN112972652A (en) | Application of insulin-like growth factor binding protein 2 in preparation of medicine for resisting mental disorder caused by early stress | |
| Liu et al. | Dexmedetomidine alleviated neuropathic pain in dorsal root ganglion neurons by inhibition of anaerobic glycolysis activity and enhancement of ROS tolerance | |
| CN102627578B (en) | Phenyl benzylamine compound and application thereof | |
| CN103977013A (en) | Application of catechol glycoside compounds to early-stage Alzhemier's disease | |
| CN104224796B (en) | Application of oleanane triterpene ester derivative in preparation for anti-neurodegeneration medicine | |
| CN106749228A (en) | A kind of jamaicin medicine and preparation method and application | |
| Zhao et al. | Edaravone mitigates cognitive impairment and hippocampal injury in juvenile rats with obstructive sleep apnea hypopnea syndrome via regulation of cAMP/PKACREB pathway | |
| Hess | Relation of the ground substance of the central nervous system to the blood-brain barrier | |
| CN101653607B (en) | Pharmaceutical composition containing hepatocyte growth factor receptor inhibitor and mitogen extracellular kinase inhibitor and application thereof | |
| Khan et al. | ANTIDIABETIC AND ANTIOXIDANT POTENTIAL OF COPRINUS COMATUS IN ALLOXAN-INDUCED DIABETIC RATS MODELS | |
| CN111544426A (en) | Propargyl cysteine and application thereof in preparing novel target spot medicament | |
| Shilliam et al. | Gradient of dopamine responsiveness to dopamine receptor agonists in subregions of the rat nucleus accumbens | |
| Xu et al. | Propionate alleviates itch in murine models of atopic dermatitis by modulating sensory TRP channels of dorsal root ganglion | |
| Wybran | Interregulations between the immune system and the central nervous system: physiological and pathological consequences | |
| CN112294819B (en) | Inflammatory corpuscle inhibitor and application thereof | |
| Miller et al. | The induction of suppressor T cells by lipopolysaccharide in human peripheral blood lymphocyte cultures in the presence of fetal calf serum | |
| Yamashita et al. | Specific stimulatory effects of Graves’ IgG on the release of triiodothyronine from the patients’ own thyroids |
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 |