WO2014177593A1 - Amorfrutin analogs as ppargamma-modulators - Google Patents
Amorfrutin analogs as ppargamma-modulators Download PDFInfo
- Publication number
- WO2014177593A1 WO2014177593A1 PCT/EP2014/058769 EP2014058769W WO2014177593A1 WO 2014177593 A1 WO2014177593 A1 WO 2014177593A1 EP 2014058769 W EP2014058769 W EP 2014058769W WO 2014177593 A1 WO2014177593 A1 WO 2014177593A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cancer
- chr
- carcinoma
- amorfrutin
- cells
- Prior art date
Links
- 229930010828 amorfrutin Natural products 0.000 title abstract description 29
- 150000008030 amorfrutins Chemical class 0.000 title abstract description 28
- 150000001875 compounds Chemical class 0.000 claims abstract description 139
- 238000011282 treatment Methods 0.000 claims abstract description 111
- 102000003728 Peroxisome Proliferator-Activated Receptors Human genes 0.000 claims abstract description 43
- 108090000029 Peroxisome Proliferator-Activated Receptors Proteins 0.000 claims abstract description 43
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 36
- 201000011510 cancer Diseases 0.000 claims abstract description 26
- 150000003839 salts Chemical class 0.000 claims abstract description 20
- 208000030159 metabolic disease Diseases 0.000 claims abstract description 19
- 208000027866 inflammatory disease Diseases 0.000 claims abstract description 16
- 230000002265 prevention Effects 0.000 claims abstract description 14
- 239000003085 diluting agent Substances 0.000 claims abstract description 13
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 12
- 238000002360 preparation method Methods 0.000 claims abstract description 12
- 150000004677 hydrates Chemical class 0.000 claims abstract description 7
- 239000012453 solvate Substances 0.000 claims abstract description 7
- 239000003937 drug carrier Substances 0.000 claims abstract description 6
- 239000000546 pharmaceutical excipient Substances 0.000 claims abstract description 6
- 235000013376 functional food Nutrition 0.000 claims abstract description 5
- 210000004027 cell Anatomy 0.000 claims description 145
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 57
- 239000000203 mixture Substances 0.000 claims description 52
- 208000001072 type 2 diabetes mellitus Diseases 0.000 claims description 18
- 206010009944 Colon cancer Diseases 0.000 claims description 17
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 17
- 125000000654 isopropylidene group Chemical group C(C)(C)=* 0.000 claims description 17
- 206010022489 Insulin Resistance Diseases 0.000 claims description 16
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 16
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 15
- -1 -OH Chemical group 0.000 claims description 13
- 230000004060 metabolic process Effects 0.000 claims description 13
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 13
- 241001303601 Rosacea Species 0.000 claims description 12
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 12
- 201000004700 rosacea Diseases 0.000 claims description 12
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 11
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 claims description 10
- 201000009030 Carcinoma Diseases 0.000 claims description 10
- 201000010099 disease Diseases 0.000 claims description 10
- 125000001424 substituent group Chemical group 0.000 claims description 10
- 230000004054 inflammatory process Effects 0.000 claims description 9
- 206010061218 Inflammation Diseases 0.000 claims description 8
- 206010012601 diabetes mellitus Diseases 0.000 claims description 8
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 claims description 8
- 206010006187 Breast cancer Diseases 0.000 claims description 7
- 208000026310 Breast neoplasm Diseases 0.000 claims description 7
- 150000001720 carbohydrates Chemical class 0.000 claims description 7
- 208000017604 Hodgkin disease Diseases 0.000 claims description 6
- 208000008589 Obesity Diseases 0.000 claims description 6
- 206010060862 Prostate cancer Diseases 0.000 claims description 6
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 6
- 230000001086 cytosolic effect Effects 0.000 claims description 6
- 208000035475 disorder Diseases 0.000 claims description 6
- 230000002757 inflammatory effect Effects 0.000 claims description 6
- 201000001441 melanoma Diseases 0.000 claims description 6
- 235000020824 obesity Nutrition 0.000 claims description 6
- 208000011580 syndromic disease Diseases 0.000 claims description 6
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 claims description 5
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 5
- PHIQHXFUZVPYII-ZCFIWIBFSA-N (R)-carnitine Chemical compound C[N+](C)(C)C[C@H](O)CC([O-])=O PHIQHXFUZVPYII-ZCFIWIBFSA-N 0.000 claims description 4
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 claims description 4
- 206010003827 Autoimmune hepatitis Diseases 0.000 claims description 4
- 208000003174 Brain Neoplasms Diseases 0.000 claims description 4
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 4
- 208000007465 Giant cell arteritis Diseases 0.000 claims description 4
- 201000005569 Gout Diseases 0.000 claims description 4
- 206010019939 Herpes gestationis Diseases 0.000 claims description 4
- 208000031226 Hyperlipidaemia Diseases 0.000 claims description 4
- 206010021024 Hypolipidaemia Diseases 0.000 claims description 4
- 208000007976 Ketosis Diseases 0.000 claims description 4
- 206010025323 Lymphomas Diseases 0.000 claims description 4
- 206010030155 Oesophageal carcinoma Diseases 0.000 claims description 4
- 208000008223 Pemphigoid Gestationis Diseases 0.000 claims description 4
- 208000003251 Pruritus Diseases 0.000 claims description 4
- 206010039710 Scleroderma Diseases 0.000 claims description 4
- 208000000102 Squamous Cell Carcinoma of Head and Neck Diseases 0.000 claims description 4
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 4
- 208000031981 Thrombocytopenic Idiopathic Purpura Diseases 0.000 claims description 4
- 206010052779 Transplant rejections Diseases 0.000 claims description 4
- 206010046431 Urethral cancer Diseases 0.000 claims description 4
- 206010046458 Urethral neoplasms Diseases 0.000 claims description 4
- 206010047115 Vasculitis Diseases 0.000 claims description 4
- 208000010668 atopic eczema Diseases 0.000 claims description 4
- QHMBSVQNZZTUGM-ZWKOTPCHSA-N cannabidiol Chemical compound OC1=CC(CCCCC)=CC(O)=C1[C@H]1[C@H](C(C)=C)CCC(C)=C1 QHMBSVQNZZTUGM-ZWKOTPCHSA-N 0.000 claims description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 4
- 229960004203 carnitine Drugs 0.000 claims description 4
- 230000030833 cell death Effects 0.000 claims description 4
- 230000004064 dysfunction Effects 0.000 claims description 4
- 201000005206 focal segmental glomerulosclerosis Diseases 0.000 claims description 4
- 201000010175 gallbladder cancer Diseases 0.000 claims description 4
- 206010017758 gastric cancer Diseases 0.000 claims description 4
- 208000007345 glycogen storage disease Diseases 0.000 claims description 4
- 201000000459 head and neck squamous cell carcinoma Diseases 0.000 claims description 4
- 201000005962 mycosis fungoides Diseases 0.000 claims description 4
- 230000009826 neoplastic cell growth Effects 0.000 claims description 4
- 208000002154 non-small cell lung carcinoma Diseases 0.000 claims description 4
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 4
- 206010038038 rectal cancer Diseases 0.000 claims description 4
- 201000000306 sarcoidosis Diseases 0.000 claims description 4
- 208000017520 skin disease Diseases 0.000 claims description 4
- 201000011549 stomach cancer Diseases 0.000 claims description 4
- 201000000596 systemic lupus erythematosus Diseases 0.000 claims description 4
- 206010043207 temporal arteritis Diseases 0.000 claims description 4
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 claims description 4
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 claims description 3
- 239000004202 carbamide Substances 0.000 claims description 3
- 210000001035 gastrointestinal tract Anatomy 0.000 claims description 3
- 208000004104 gestational diabetes Diseases 0.000 claims description 3
- 230000000968 intestinal effect Effects 0.000 claims description 3
- 210000004153 islets of langerhan Anatomy 0.000 claims description 3
- 210000001550 testis Anatomy 0.000 claims description 3
- ZVLQJRXSGVAHOI-UHFFFAOYSA-N 2,4-Bis(3-methylbut-2-enyl)-5-pentylbenzene-1,3-diol Chemical compound CCCCCC1=CC(O)=C(CC=C(C)C)C(O)=C1CC=C(C)C ZVLQJRXSGVAHOI-UHFFFAOYSA-N 0.000 claims description 2
- PDNBFWVCEQNNBO-UHFFFAOYSA-N 2-(2-hydroxy-3-methylbut-3-enyl)-4-(3-methylbut-2-enyl)-5-pentylbenzene-1,3-diol Chemical compound CCCCCC1=CC(O)=C(CC(O)C(C)=C)C(O)=C1CC=C(C)C PDNBFWVCEQNNBO-UHFFFAOYSA-N 0.000 claims description 2
- ADDCNOCQPWDJSR-UHFFFAOYSA-N 3-methoxy-2-(3-methylbut-2-enyl)-5-(2-phenylethyl)phenol Chemical compound OC1=C(CC=C(C)C)C(OC)=CC(CCC=2C=CC=CC=2)=C1 ADDCNOCQPWDJSR-UHFFFAOYSA-N 0.000 claims description 2
- RSBOKJFJTXUTAI-UHFFFAOYSA-N 4-(2-hydroxy-3-methylbut-3-enyl)-2-(3-methylbut-2-enyl)-5-pentylbenzene-1,3-diol Chemical compound CCCCCC1=CC(O)=C(CC=C(C)C)C(O)=C1CC(O)C(C)=C RSBOKJFJTXUTAI-UHFFFAOYSA-N 0.000 claims description 2
- 208000002874 Acne Vulgaris Diseases 0.000 claims description 2
- 206010000748 Acute febrile neutrophilic dermatosis Diseases 0.000 claims description 2
- 206010000830 Acute leukaemia Diseases 0.000 claims description 2
- 206010001052 Acute respiratory distress syndrome Diseases 0.000 claims description 2
- 108700016155 Acyl transferases Proteins 0.000 claims description 2
- 102000057234 Acyl transferases Human genes 0.000 claims description 2
- 208000026872 Addison Disease Diseases 0.000 claims description 2
- 208000005676 Adrenogenital syndrome Diseases 0.000 claims description 2
- 206010061424 Anal cancer Diseases 0.000 claims description 2
- 206010002556 Ankylosing Spondylitis Diseases 0.000 claims description 2
- 206010003571 Astrocytoma Diseases 0.000 claims description 2
- 201000001320 Atherosclerosis Diseases 0.000 claims description 2
- 206010004146 Basal cell carcinoma Diseases 0.000 claims description 2
- 208000009137 Behcet syndrome Diseases 0.000 claims description 2
- 208000006373 Bell palsy Diseases 0.000 claims description 2
- 206010005003 Bladder cancer Diseases 0.000 claims description 2
- 206010005949 Bone cancer Diseases 0.000 claims description 2
- 208000018084 Bone neoplasm Diseases 0.000 claims description 2
- 201000006474 Brain Ischemia Diseases 0.000 claims description 2
- 206010006417 Bronchial carcinoma Diseases 0.000 claims description 2
- 208000011691 Burkitt lymphomas Diseases 0.000 claims description 2
- 206010006811 Bursitis Diseases 0.000 claims description 2
- 208000017897 Carcinoma of esophagus Diseases 0.000 claims description 2
- 206010008120 Cerebral ischaemia Diseases 0.000 claims description 2
- 206010008342 Cervix carcinoma Diseases 0.000 claims description 2
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 claims description 2
- 208000010007 Cogan syndrome Diseases 0.000 claims description 2
- 206010009900 Colitis ulcerative Diseases 0.000 claims description 2
- 208000009798 Craniopharyngioma Diseases 0.000 claims description 2
- 208000011231 Crohn disease Diseases 0.000 claims description 2
- 208000014311 Cushing syndrome Diseases 0.000 claims description 2
- 201000003883 Cystic fibrosis Diseases 0.000 claims description 2
- 201000004624 Dermatitis Diseases 0.000 claims description 2
- 206010012438 Dermatitis atopic Diseases 0.000 claims description 2
- 206010012442 Dermatitis contact Diseases 0.000 claims description 2
- 206010012455 Dermatitis exfoliative Diseases 0.000 claims description 2
- 206010059352 Desmoid tumour Diseases 0.000 claims description 2
- 208000002699 Digestive System Neoplasms Diseases 0.000 claims description 2
- 208000006926 Discoid Lupus Erythematosus Diseases 0.000 claims description 2
- 206010014733 Endometrial cancer Diseases 0.000 claims description 2
- 206010014759 Endometrial neoplasm Diseases 0.000 claims description 2
- 206010014954 Eosinophilic fasciitis Diseases 0.000 claims description 2
- 206010014967 Ependymoma Diseases 0.000 claims description 2
- 206010015226 Erythema nodosum Diseases 0.000 claims description 2
- 208000000461 Esophageal Neoplasms Diseases 0.000 claims description 2
- 208000006168 Ewing Sarcoma Diseases 0.000 claims description 2
- 208000004413 Eyelid Neoplasms Diseases 0.000 claims description 2
- 206010050497 Eyelid tumour Diseases 0.000 claims description 2
- 208000001640 Fibromyalgia Diseases 0.000 claims description 2
- 206010016654 Fibrosis Diseases 0.000 claims description 2
- 229930091371 Fructose Natural products 0.000 claims description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 2
- 239000005715 Fructose Substances 0.000 claims description 2
- 208000027472 Galactosemias Diseases 0.000 claims description 2
- 208000022072 Gallbladder Neoplasms Diseases 0.000 claims description 2
- 208000009796 Gangliosidoses Diseases 0.000 claims description 2
- 208000018522 Gastrointestinal disease Diseases 0.000 claims description 2
- 208000021309 Germ cell tumor Diseases 0.000 claims description 2
- 206010018338 Glioma Diseases 0.000 claims description 2
- 206010053250 Glycogen storage disease type III Diseases 0.000 claims description 2
- 206010018634 Gouty Arthritis Diseases 0.000 claims description 2
- 208000009329 Graft vs Host Disease Diseases 0.000 claims description 2
- 206010072579 Granulomatosis with polyangiitis Diseases 0.000 claims description 2
- 208000035186 Hemolytic Autoimmune Anemia Diseases 0.000 claims description 2
- 201000004331 Henoch-Schoenlein purpura Diseases 0.000 claims description 2
- 206010019617 Henoch-Schonlein purpura Diseases 0.000 claims description 2
- 208000007514 Herpes zoster Diseases 0.000 claims description 2
- 206010020112 Hirsutism Diseases 0.000 claims description 2
- 201000002563 Histoplasmosis Diseases 0.000 claims description 2
- 208000010747 Hodgkins lymphoma Diseases 0.000 claims description 2
- 206010020575 Hyperammonaemia Diseases 0.000 claims description 2
- 208000013016 Hypoglycemia Diseases 0.000 claims description 2
- 206010062767 Hypophysitis Diseases 0.000 claims description 2
- 201000009794 Idiopathic Pulmonary Fibrosis Diseases 0.000 claims description 2
- 206010021245 Idiopathic thrombocytopenic purpura Diseases 0.000 claims description 2
- 208000031814 IgA Vasculitis Diseases 0.000 claims description 2
- 208000028622 Immune thrombocytopenia Diseases 0.000 claims description 2
- 208000005016 Intestinal Neoplasms Diseases 0.000 claims description 2
- 206010059176 Juvenile idiopathic arthritis Diseases 0.000 claims description 2
- 208000007766 Kaposi sarcoma Diseases 0.000 claims description 2
- 206010023379 Ketoacidosis Diseases 0.000 claims description 2
- 208000008839 Kidney Neoplasms Diseases 0.000 claims description 2
- 206010023825 Laryngeal cancer Diseases 0.000 claims description 2
- 208000009625 Lesch-Nyhan syndrome Diseases 0.000 claims description 2
- 208000010557 Lipid storage disease Diseases 0.000 claims description 2
- 208000019693 Lung disease Diseases 0.000 claims description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 2
- 208000005777 Lupus Nephritis Diseases 0.000 claims description 2
- 208000033868 Lysosomal disease Diseases 0.000 claims description 2
- 208000015439 Lysosomal storage disease Diseases 0.000 claims description 2
- 208000001344 Macular Edema Diseases 0.000 claims description 2
- 206010025415 Macular oedema Diseases 0.000 claims description 2
- 206010025476 Malabsorption Diseases 0.000 claims description 2
- 208000004155 Malabsorption Syndromes Diseases 0.000 claims description 2
- 206010073059 Malignant neoplasm of unknown primary site Diseases 0.000 claims description 2
- 208000032271 Malignant tumor of penis Diseases 0.000 claims description 2
- 208000000172 Medulloblastoma Diseases 0.000 claims description 2
- 206010059282 Metastases to central nervous system Diseases 0.000 claims description 2
- 206010027457 Metastases to liver Diseases 0.000 claims description 2
- 208000003445 Mouth Neoplasms Diseases 0.000 claims description 2
- 208000002678 Mucopolysaccharidoses Diseases 0.000 claims description 2
- 206010028629 Myoglobinuria Diseases 0.000 claims description 2
- 201000002481 Myositis Diseases 0.000 claims description 2
- 208000034176 Neoplasms, Germ Cell and Embryonal Diseases 0.000 claims description 2
- 206010029260 Neuroblastoma Diseases 0.000 claims description 2
- 208000015914 Non-Hodgkin lymphomas Diseases 0.000 claims description 2
- 208000010505 Nose Neoplasms Diseases 0.000 claims description 2
- 201000010133 Oligodendroglioma Diseases 0.000 claims description 2
- 206010033128 Ovarian cancer Diseases 0.000 claims description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 2
- 208000002193 Pain Diseases 0.000 claims description 2
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 2
- 206010033645 Pancreatitis Diseases 0.000 claims description 2
- 206010034277 Pemphigoid Diseases 0.000 claims description 2
- 201000011152 Pemphigus Diseases 0.000 claims description 2
- 208000002471 Penile Neoplasms Diseases 0.000 claims description 2
- 206010034299 Penile cancer Diseases 0.000 claims description 2
- 208000009565 Pharyngeal Neoplasms Diseases 0.000 claims description 2
- 206010034811 Pharyngeal cancer Diseases 0.000 claims description 2
- 201000011252 Phenylketonuria Diseases 0.000 claims description 2
- 208000007452 Plasmacytoma Diseases 0.000 claims description 2
- 206010065159 Polychondritis Diseases 0.000 claims description 2
- 208000007048 Polymyalgia Rheumatica Diseases 0.000 claims description 2
- 241000097929 Porphyria Species 0.000 claims description 2
- 208000010642 Porphyrias Diseases 0.000 claims description 2
- 201000004681 Psoriasis Diseases 0.000 claims description 2
- 201000001263 Psoriatic Arthritis Diseases 0.000 claims description 2
- 208000036824 Psoriatic arthropathy Diseases 0.000 claims description 2
- 208000015634 Rectal Neoplasms Diseases 0.000 claims description 2
- 206010038389 Renal cancer Diseases 0.000 claims description 2
- 208000006265 Renal cell carcinoma Diseases 0.000 claims description 2
- 208000013616 Respiratory Distress Syndrome Diseases 0.000 claims description 2
- 201000000582 Retinoblastoma Diseases 0.000 claims description 2
- 206010039085 Rhinitis allergic Diseases 0.000 claims description 2
- 206010039491 Sarcoma Diseases 0.000 claims description 2
- 206010039705 Scleritis Diseases 0.000 claims description 2
- 206010040070 Septic Shock Diseases 0.000 claims description 2
- 208000021386 Sjogren Syndrome Diseases 0.000 claims description 2
- 208000000453 Skin Neoplasms Diseases 0.000 claims description 2
- 206010068771 Soft tissue neoplasm Diseases 0.000 claims description 2
- 208000021712 Soft tissue sarcoma Diseases 0.000 claims description 2
- 208000010346 Sphingolipidoses Diseases 0.000 claims description 2
- 201000001307 Sphingolipidosis Diseases 0.000 claims description 2
- 206010042033 Stevens-Johnson syndrome Diseases 0.000 claims description 2
- 208000006011 Stroke Diseases 0.000 claims description 2
- 208000005350 Sulfatidosis Diseases 0.000 claims description 2
- 208000010265 Sweet syndrome Diseases 0.000 claims description 2
- 201000009594 Systemic Scleroderma Diseases 0.000 claims description 2
- 206010042953 Systemic sclerosis Diseases 0.000 claims description 2
- 206010042971 T-cell lymphoma Diseases 0.000 claims description 2
- 208000027585 T-cell non-Hodgkin lymphoma Diseases 0.000 claims description 2
- 208000001106 Takayasu Arteritis Diseases 0.000 claims description 2
- 208000000491 Tendinopathy Diseases 0.000 claims description 2
- 206010043255 Tendonitis Diseases 0.000 claims description 2
- 208000024313 Testicular Neoplasms Diseases 0.000 claims description 2
- 206010057644 Testis cancer Diseases 0.000 claims description 2
- 208000033781 Thyroid carcinoma Diseases 0.000 claims description 2
- 208000024770 Thyroid neoplasm Diseases 0.000 claims description 2
- 206010062129 Tongue neoplasm Diseases 0.000 claims description 2
- 208000035317 Total hypoxanthine-guanine phosphoribosyl transferase deficiency Diseases 0.000 claims description 2
- 206010044223 Toxic epidermal necrolysis Diseases 0.000 claims description 2
- 231100000087 Toxic epidermal necrolysis Toxicity 0.000 claims description 2
- 206010044314 Tracheobronchitis Diseases 0.000 claims description 2
- 201000006704 Ulcerative Colitis Diseases 0.000 claims description 2
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 claims description 2
- 208000024780 Urticaria Diseases 0.000 claims description 2
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims description 2
- 208000002495 Uterine Neoplasms Diseases 0.000 claims description 2
- 206010046851 Uveitis Diseases 0.000 claims description 2
- 208000036826 VIIth nerve paralysis Diseases 0.000 claims description 2
- 208000014070 Vestibular schwannoma Diseases 0.000 claims description 2
- 208000004354 Vulvar Neoplasms Diseases 0.000 claims description 2
- 208000000260 Warts Diseases 0.000 claims description 2
- 208000008383 Wilms tumor Diseases 0.000 claims description 2
- 206010000496 acne Diseases 0.000 claims description 2
- 208000004064 acoustic neuroma Diseases 0.000 claims description 2
- 208000009956 adenocarcinoma Diseases 0.000 claims description 2
- 201000000028 adult respiratory distress syndrome Diseases 0.000 claims description 2
- 206010001689 alkaptonuria Diseases 0.000 claims description 2
- 230000000172 allergic effect Effects 0.000 claims description 2
- 201000010105 allergic rhinitis Diseases 0.000 claims description 2
- 201000007434 ampulla of Vater carcinoma Diseases 0.000 claims description 2
- 206010002022 amyloidosis Diseases 0.000 claims description 2
- 201000007538 anal carcinoma Diseases 0.000 claims description 2
- 206010003246 arthritis Diseases 0.000 claims description 2
- 208000006673 asthma Diseases 0.000 claims description 2
- 201000008937 atopic dermatitis Diseases 0.000 claims description 2
- 201000000448 autoimmune hemolytic anemia Diseases 0.000 claims description 2
- 201000003710 autoimmune thrombocytopenic purpura Diseases 0.000 claims description 2
- 210000004958 brain cell Anatomy 0.000 claims description 2
- 201000008275 breast carcinoma Diseases 0.000 claims description 2
- 208000003362 bronchogenic carcinoma Diseases 0.000 claims description 2
- 208000000594 bullous pemphigoid Diseases 0.000 claims description 2
- 230000034149 carbohydrate storage Effects 0.000 claims description 2
- 208000002458 carcinoid tumor Diseases 0.000 claims description 2
- 208000011825 carcinoma of the ampulla of vater Diseases 0.000 claims description 2
- 201000007455 central nervous system cancer Diseases 0.000 claims description 2
- 206010008118 cerebral infarction Diseases 0.000 claims description 2
- 201000010881 cervical cancer Diseases 0.000 claims description 2
- 208000019065 cervical carcinoma Diseases 0.000 claims description 2
- 210000003679 cervix uteri Anatomy 0.000 claims description 2
- 208000025302 chronic primary adrenal insufficiency Diseases 0.000 claims description 2
- 230000007882 cirrhosis Effects 0.000 claims description 2
- 208000019425 cirrhosis of liver Diseases 0.000 claims description 2
- 201000010989 colorectal carcinoma Diseases 0.000 claims description 2
- 235000009508 confectionery Nutrition 0.000 claims description 2
- 208000010247 contact dermatitis Diseases 0.000 claims description 2
- 208000004921 cutaneous lupus erythematosus Diseases 0.000 claims description 2
- 208000035250 cutaneous malignant susceptibility to 1 melanoma Diseases 0.000 claims description 2
- 230000007812 deficiency Effects 0.000 claims description 2
- 201000001981 dermatomyositis Diseases 0.000 claims description 2
- 201000006827 desmoid tumor Diseases 0.000 claims description 2
- 201000003914 endometrial carcinoma Diseases 0.000 claims description 2
- 201000004101 esophageal cancer Diseases 0.000 claims description 2
- 201000005619 esophageal carcinoma Diseases 0.000 claims description 2
- 208000004526 exfoliative dermatitis Diseases 0.000 claims description 2
- 208000021045 exocrine pancreatic carcinoma Diseases 0.000 claims description 2
- 208000024519 eye neoplasm Diseases 0.000 claims description 2
- 208000028149 female reproductive system neoplasm Diseases 0.000 claims description 2
- 231100000854 focal segmental glomerulosclerosis Toxicity 0.000 claims description 2
- 229930182830 galactose Natural products 0.000 claims description 2
- 201000007487 gallbladder carcinoma Diseases 0.000 claims description 2
- 208000005017 glioblastoma Diseases 0.000 claims description 2
- 206010061989 glomerulosclerosis Diseases 0.000 claims description 2
- 208000024908 graft versus host disease Diseases 0.000 claims description 2
- 201000009277 hairy cell leukemia Diseases 0.000 claims description 2
- 208000014829 head and neck neoplasm Diseases 0.000 claims description 2
- 230000002489 hematologic effect Effects 0.000 claims description 2
- 208000020346 hyperlipoproteinemia Diseases 0.000 claims description 2
- 230000002218 hypoglycaemic effect Effects 0.000 claims description 2
- 208000026621 hypolipoproteinemia Diseases 0.000 claims description 2
- 208000015446 immunoglobulin a vasculitis Diseases 0.000 claims description 2
- 208000036971 interstitial lung disease 2 Diseases 0.000 claims description 2
- 201000009019 intestinal benign neoplasm Diseases 0.000 claims description 2
- 201000002215 juvenile rheumatoid arthritis Diseases 0.000 claims description 2
- 230000004140 ketosis Effects 0.000 claims description 2
- 201000010982 kidney cancer Diseases 0.000 claims description 2
- 206010023841 laryngeal neoplasm Diseases 0.000 claims description 2
- 208000032839 leukemia Diseases 0.000 claims description 2
- 208000036546 leukodystrophy Diseases 0.000 claims description 2
- 201000011486 lichen planus Diseases 0.000 claims description 2
- 208000012987 lip and oral cavity carcinoma Diseases 0.000 claims description 2
- 201000007270 liver cancer Diseases 0.000 claims description 2
- 208000014018 liver neoplasm Diseases 0.000 claims description 2
- 201000005202 lung cancer Diseases 0.000 claims description 2
- 208000020816 lung neoplasm Diseases 0.000 claims description 2
- 201000010453 lymph node cancer Diseases 0.000 claims description 2
- 208000014416 lysosomal lipid storage disease Diseases 0.000 claims description 2
- 201000010230 macular retinal edema Diseases 0.000 claims description 2
- 230000003211 malignant effect Effects 0.000 claims description 2
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 2
- 206010027191 meningioma Diseases 0.000 claims description 2
- 206010028093 mucopolysaccharidosis Diseases 0.000 claims description 2
- 208000000690 mucopolysaccharidosis VI Diseases 0.000 claims description 2
- 201000006417 multiple sclerosis Diseases 0.000 claims description 2
- 208000029372 muscular lipidosis Diseases 0.000 claims description 2
- 206010028417 myasthenia gravis Diseases 0.000 claims description 2
- 208000037830 nasal cancer Diseases 0.000 claims description 2
- 210000005036 nerve Anatomy 0.000 claims description 2
- 210000000653 nervous system Anatomy 0.000 claims description 2
- 208000007538 neurilemmoma Diseases 0.000 claims description 2
- 201000008482 osteoarthritis Diseases 0.000 claims description 2
- 230000000010 osteolytic effect Effects 0.000 claims description 2
- 201000008968 osteosarcoma Diseases 0.000 claims description 2
- 201000002528 pancreatic cancer Diseases 0.000 claims description 2
- 210000002990 parathyroid gland Anatomy 0.000 claims description 2
- 201000001976 pemphigus vulgaris Diseases 0.000 claims description 2
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 claims description 2
- 201000001245 periodontitis Diseases 0.000 claims description 2
- 201000006292 polyarteritis nodosa Diseases 0.000 claims description 2
- 201000009395 primary hyperaldosteronism Diseases 0.000 claims description 2
- 230000002685 pulmonary effect Effects 0.000 claims description 2
- 230000004144 purine metabolism Effects 0.000 claims description 2
- 208000020615 rectal carcinoma Diseases 0.000 claims description 2
- 201000001275 rectum cancer Diseases 0.000 claims description 2
- 208000009169 relapsing polychondritis Diseases 0.000 claims description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 claims description 2
- 206010039667 schwannoma Diseases 0.000 claims description 2
- 230000036303 septic shock Effects 0.000 claims description 2
- 201000000849 skin cancer Diseases 0.000 claims description 2
- 201000010153 skin papilloma Diseases 0.000 claims description 2
- 201000002314 small intestine cancer Diseases 0.000 claims description 2
- 201000004415 tendinitis Diseases 0.000 claims description 2
- 201000003120 testicular cancer Diseases 0.000 claims description 2
- 208000008732 thymoma Diseases 0.000 claims description 2
- 201000002510 thyroid cancer Diseases 0.000 claims description 2
- 208000013077 thyroid gland carcinoma Diseases 0.000 claims description 2
- 201000006134 tongue cancer Diseases 0.000 claims description 2
- 206010044412 transitional cell carcinoma Diseases 0.000 claims description 2
- 201000008827 tuberculosis Diseases 0.000 claims description 2
- 201000005112 urinary bladder cancer Diseases 0.000 claims description 2
- 208000023747 urothelial carcinoma Diseases 0.000 claims description 2
- 206010046766 uterine cancer Diseases 0.000 claims description 2
- 206010046885 vaginal cancer Diseases 0.000 claims description 2
- 208000013139 vaginal neoplasm Diseases 0.000 claims description 2
- 201000005102 vulva cancer Diseases 0.000 claims description 2
- NHQPTPUMTXSDEV-UHFFFAOYSA-N 2-(2-hydroxy-3-methylbut-3-enyl)-4-(3-methylbut-2-enyl)-5-(2-phenylethyl)benzene-1,3-diol Chemical compound C1=C(O)C(CC(O)C(C)=C)=C(O)C(CC=C(C)C)=C1CCC1=CC=CC=C1 NHQPTPUMTXSDEV-UHFFFAOYSA-N 0.000 claims 1
- 125000002373 5 membered heterocyclic group Chemical group 0.000 claims 1
- 125000005605 benzo group Chemical group 0.000 claims 1
- 125000004432 carbon atom Chemical group C* 0.000 claims 1
- YASAKCUCGLMORW-UHFFFAOYSA-N Rosiglitazone Chemical compound C=1C=CC=NC=1N(C)CCOC(C=C1)=CC=C1CC1SC(=O)NC1=O YASAKCUCGLMORW-UHFFFAOYSA-N 0.000 description 190
- 229930010822 amorfrutin 4 Natural products 0.000 description 141
- UAMAHWUELDAAIA-LDADJPATSA-N amorfrutin 4 Chemical compound OC(=O)C1=C(O)C(C/C=C(C)/CCC=C(C)C)=C(O)C=C1CCC1=CC=CC=C1 UAMAHWUELDAAIA-LDADJPATSA-N 0.000 description 140
- BGYWMHFOMULQIA-UHFFFAOYSA-N amorfrutin B Natural products COc1cc(CCc2ccccc2)c(C(O)=O)c(O)c1CCC(C)CCC=C(C)C BGYWMHFOMULQIA-UHFFFAOYSA-N 0.000 description 139
- 229960004586 rosiglitazone Drugs 0.000 description 95
- 239000003981 vehicle Substances 0.000 description 51
- 230000014509 gene expression Effects 0.000 description 47
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical class CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 44
- 241000699670 Mus sp. Species 0.000 description 43
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 42
- 230000004913 activation Effects 0.000 description 39
- 230000000694 effects Effects 0.000 description 37
- 108090000623 proteins and genes Proteins 0.000 description 32
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 31
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 28
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 26
- 230000027455 binding Effects 0.000 description 26
- 210000002381 plasma Anatomy 0.000 description 24
- 239000000243 solution Substances 0.000 description 24
- 238000001514 detection method Methods 0.000 description 23
- 230000037396 body weight Effects 0.000 description 21
- 229930014626 natural product Natural products 0.000 description 21
- 238000003556 assay Methods 0.000 description 20
- 108090000765 processed proteins & peptides Proteins 0.000 description 20
- 102000005962 receptors Human genes 0.000 description 19
- 108020003175 receptors Proteins 0.000 description 19
- 235000002639 sodium chloride Nutrition 0.000 description 19
- 108090000376 Fibroblast growth factor 21 Proteins 0.000 description 18
- 241000674162 Glycyrrhiza foetida Species 0.000 description 18
- 210000001789 adipocyte Anatomy 0.000 description 18
- 239000000284 extract Substances 0.000 description 18
- 230000002829 reductive effect Effects 0.000 description 18
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 17
- 239000008103 glucose Substances 0.000 description 17
- 238000005160 1H NMR spectroscopy Methods 0.000 description 16
- 102000003973 Fibroblast growth factor 21 Human genes 0.000 description 16
- 238000004128 high performance liquid chromatography Methods 0.000 description 16
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 16
- 210000004185 liver Anatomy 0.000 description 16
- 230000002438 mitochondrial effect Effects 0.000 description 16
- 239000000523 sample Substances 0.000 description 16
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 15
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 15
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 15
- 210000004369 blood Anatomy 0.000 description 15
- 239000008280 blood Substances 0.000 description 15
- 238000004587 chromatography analysis Methods 0.000 description 15
- 238000002955 isolation Methods 0.000 description 15
- 239000002609 medium Substances 0.000 description 15
- 238000000034 method Methods 0.000 description 15
- 239000012071 phase Substances 0.000 description 15
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 14
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 14
- 230000005526 G1 to G0 transition Effects 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 14
- 235000019253 formic acid Nutrition 0.000 description 14
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 14
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 14
- 238000005084 2D-nuclear magnetic resonance Methods 0.000 description 13
- 102000011727 Caspases Human genes 0.000 description 13
- 108010076667 Caspases Proteins 0.000 description 13
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 13
- 238000005100 correlation spectroscopy Methods 0.000 description 13
- 238000005516 engineering process Methods 0.000 description 13
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 13
- 238000005570 heteronuclear single quantum coherence Methods 0.000 description 13
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 13
- 230000007115 recruitment Effects 0.000 description 13
- 238000003756 stirring Methods 0.000 description 13
- 101150014691 PPARA gene Proteins 0.000 description 12
- 239000004480 active ingredient Substances 0.000 description 12
- 230000029279 positive regulation of transcription, DNA-dependent Effects 0.000 description 12
- 239000003826 tablet Substances 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 238000011529 RT qPCR Methods 0.000 description 11
- 239000000975 dye Substances 0.000 description 11
- 230000009467 reduction Effects 0.000 description 11
- 238000003571 reporter gene assay Methods 0.000 description 11
- 238000003786 synthesis reaction Methods 0.000 description 11
- 210000001519 tissue Anatomy 0.000 description 11
- 238000013518 transcription Methods 0.000 description 11
- 230000035897 transcription Effects 0.000 description 11
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 10
- 230000015572 biosynthetic process Effects 0.000 description 10
- 239000003795 chemical substances by application Substances 0.000 description 10
- 230000001419 dependent effect Effects 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- 238000005259 measurement Methods 0.000 description 10
- 102000004196 processed proteins & peptides Human genes 0.000 description 10
- 239000007787 solid Substances 0.000 description 10
- 238000002877 time resolved fluorescence resonance energy transfer Methods 0.000 description 10
- FYNNIUVBDKICAX-UHFFFAOYSA-M 1,1',3,3'-tetraethyl-5,5',6,6'-tetrachloroimidacarbocyanine iodide Chemical compound [I-].CCN1C2=CC(Cl)=C(Cl)C=C2N(CC)C1=CC=CC1=[N+](CC)C2=CC(Cl)=C(Cl)C=C2N1CC FYNNIUVBDKICAX-UHFFFAOYSA-M 0.000 description 9
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 9
- 108010082126 Alanine transaminase Proteins 0.000 description 9
- 240000002066 Amorpha fruticosa Species 0.000 description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
- 229940123464 Thiazolidinedione Drugs 0.000 description 9
- 210000000593 adipose tissue white Anatomy 0.000 description 9
- 230000006907 apoptotic process Effects 0.000 description 9
- 239000002775 capsule Substances 0.000 description 9
- 239000012091 fetal bovine serum Substances 0.000 description 9
- 235000009200 high fat diet Nutrition 0.000 description 9
- 230000036470 plasma concentration Effects 0.000 description 9
- 239000000843 powder Substances 0.000 description 9
- 238000010186 staining Methods 0.000 description 9
- 150000001467 thiazolidinediones Chemical class 0.000 description 9
- 238000011740 C57BL/6 mouse Methods 0.000 description 8
- 102000004877 Insulin Human genes 0.000 description 8
- 108090001061 Insulin Proteins 0.000 description 8
- 108020004459 Small interfering RNA Proteins 0.000 description 8
- 229920002472 Starch Polymers 0.000 description 8
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 8
- 239000000556 agonist Substances 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 8
- 230000003178 anti-diabetic effect Effects 0.000 description 8
- 229940125396 insulin Drugs 0.000 description 8
- 150000002576 ketones Chemical class 0.000 description 8
- 239000000314 lubricant Substances 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 230000001105 regulatory effect Effects 0.000 description 8
- 239000000741 silica gel Substances 0.000 description 8
- 229910002027 silica gel Inorganic materials 0.000 description 8
- 239000004055 small Interfering RNA Substances 0.000 description 8
- 235000019698 starch Nutrition 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- 239000000725 suspension Substances 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 229930182555 Penicillin Natural products 0.000 description 7
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 7
- 239000011230 binding agent Substances 0.000 description 7
- 235000014113 dietary fatty acids Nutrition 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 230000006539 extracellular acidification Effects 0.000 description 7
- 239000000194 fatty acid Substances 0.000 description 7
- 229930195729 fatty acid Natural products 0.000 description 7
- 238000003818 flash chromatography Methods 0.000 description 7
- 239000003446 ligand Substances 0.000 description 7
- 108020001756 ligand binding domains Proteins 0.000 description 7
- 230000002503 metabolic effect Effects 0.000 description 7
- 210000000963 osteoblast Anatomy 0.000 description 7
- 229940049954 penicillin Drugs 0.000 description 7
- 230000026731 phosphorylation Effects 0.000 description 7
- 238000006366 phosphorylation reaction Methods 0.000 description 7
- 229960005322 streptomycin Drugs 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- ADPBTBPPIIKLEH-UHFFFAOYSA-N Altennsin Natural products COC1=CC(O)=C(C(O)=O)C(C=2C(=CC(O)=C(O)C=2)C)=C1 ADPBTBPPIIKLEH-UHFFFAOYSA-N 0.000 description 6
- 108090000672 Annexin A5 Proteins 0.000 description 6
- 102000004121 Annexin A5 Human genes 0.000 description 6
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 6
- 0 CCCC1C(C(CC)CC2CC(*C)CC2)C(C)C1C Chemical compound CCCC1C(C(CC)CC2CC(*C)CC2)C(C)C1C 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 6
- 101710198884 GATA-type zinc finger protein 1 Proteins 0.000 description 6
- 108010004460 Gastric Inhibitory Polypeptide Proteins 0.000 description 6
- 102100039994 Gastric inhibitory polypeptide Human genes 0.000 description 6
- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 description 6
- 108010067028 Mitochondrial Permeability Transition Pore Proteins 0.000 description 6
- 102100022929 Nuclear receptor coactivator 6 Human genes 0.000 description 6
- 102000004067 Osteocalcin Human genes 0.000 description 6
- 108090000573 Osteocalcin Proteins 0.000 description 6
- 102100040918 Pro-glucagon Human genes 0.000 description 6
- 230000009286 beneficial effect Effects 0.000 description 6
- 235000019577 caloric intake Nutrition 0.000 description 6
- 230000001413 cellular effect Effects 0.000 description 6
- 230000021615 conjugation Effects 0.000 description 6
- 230000007423 decrease Effects 0.000 description 6
- 150000004665 fatty acids Chemical class 0.000 description 6
- 238000000684 flow cytometry Methods 0.000 description 6
- 235000013305 food Nutrition 0.000 description 6
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 6
- 230000007246 mechanism Effects 0.000 description 6
- 210000004379 membrane Anatomy 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- HYAFETHFCAUJAY-UHFFFAOYSA-N pioglitazone Chemical compound N1=CC(CC)=CC=C1CCOC(C=C1)=CC=C1CC1C(=O)NC(=O)S1 HYAFETHFCAUJAY-UHFFFAOYSA-N 0.000 description 6
- 230000002250 progressing effect Effects 0.000 description 6
- 230000035755 proliferation Effects 0.000 description 6
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- AYEKOFBPNLCAJY-UHFFFAOYSA-O thiamine pyrophosphate Chemical compound CC1=C(CCOP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N AYEKOFBPNLCAJY-UHFFFAOYSA-O 0.000 description 6
- 230000002103 transcriptional effect Effects 0.000 description 6
- 235000019786 weight gain Nutrition 0.000 description 6
- DRCWOKJLSQUJPZ-DZGCQCFKSA-N (4ar,9as)-n-ethyl-1,4,9,9a-tetrahydrofluoren-4a-amine Chemical compound C1C2=CC=CC=C2[C@]2(NCC)[C@H]1CC=CC2 DRCWOKJLSQUJPZ-DZGCQCFKSA-N 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 5
- 101150018889 FABP4 gene Proteins 0.000 description 5
- PKNYXWMTHFMHKD-UHFFFAOYSA-N GW 7647 Chemical compound C1=CC(SC(C)(C)C(O)=O)=CC=C1CCN(C(=O)NC1CCCCC1)CCCCC1CCCCC1 PKNYXWMTHFMHKD-UHFFFAOYSA-N 0.000 description 5
- 101001008429 Homo sapiens Nucleobindin-2 Proteins 0.000 description 5
- 238000005481 NMR spectroscopy Methods 0.000 description 5
- 102100027441 Nucleobindin-2 Human genes 0.000 description 5
- 240000007594 Oryza sativa Species 0.000 description 5
- 235000007164 Oryza sativa Nutrition 0.000 description 5
- 210000000577 adipose tissue Anatomy 0.000 description 5
- CTNFTPUIYFUXBE-UHFFFAOYSA-N amorfrutin 1 Chemical compound OC1=C(CC=C(C)C)C(OC)=CC(CCC=2C=CC=CC=2)=C1C(O)=O CTNFTPUIYFUXBE-UHFFFAOYSA-N 0.000 description 5
- 230000001028 anti-proliverative effect Effects 0.000 description 5
- 230000001640 apoptogenic effect Effects 0.000 description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 230000033228 biological regulation Effects 0.000 description 5
- DEGAKNSWVGKMLS-UHFFFAOYSA-N calcein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(O)=O)CC(O)=O)=C(O)C=C1OC1=C2C=C(CN(CC(O)=O)CC(=O)O)C(O)=C1 DEGAKNSWVGKMLS-UHFFFAOYSA-N 0.000 description 5
- 230000002308 calcification Effects 0.000 description 5
- 235000014633 carbohydrates Nutrition 0.000 description 5
- 230000015556 catabolic process Effects 0.000 description 5
- 230000002860 competitive effect Effects 0.000 description 5
- 239000007884 disintegrant Substances 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 230000002538 fungal effect Effects 0.000 description 5
- 239000008187 granular material Substances 0.000 description 5
- 150000007857 hydrazones Chemical class 0.000 description 5
- 239000005457 ice water Substances 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 230000006698 induction Effects 0.000 description 5
- 210000002540 macrophage Anatomy 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- NIQQIJXGUZVEBB-UHFFFAOYSA-N methanol;propan-2-one Chemical compound OC.CC(C)=O NIQQIJXGUZVEBB-UHFFFAOYSA-N 0.000 description 5
- 229960002378 oftasceine Drugs 0.000 description 5
- 238000007410 oral glucose tolerance test Methods 0.000 description 5
- 239000012074 organic phase Substances 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- 230000036284 oxygen consumption Effects 0.000 description 5
- 239000003642 reactive oxygen metabolite Substances 0.000 description 5
- 235000009566 rice Nutrition 0.000 description 5
- 239000008107 starch Substances 0.000 description 5
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 description 4
- LTMRRSWNXVJMBA-UHFFFAOYSA-L 2,2-diethylpropanedioate Chemical compound CCC(CC)(C([O-])=O)C([O-])=O LTMRRSWNXVJMBA-UHFFFAOYSA-L 0.000 description 4
- LZENMJMJWQSSNJ-UHFFFAOYSA-N 3H-1,2-dithiole-3-thione Chemical compound S=C1C=CSS1 LZENMJMJWQSSNJ-UHFFFAOYSA-N 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- 235000004047 Amorpha fruticosa Nutrition 0.000 description 4
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 4
- 244000025254 Cannabis sativa Species 0.000 description 4
- 235000008697 Cannabis sativa Nutrition 0.000 description 4
- UGTJLJZQQFGTJD-UHFFFAOYSA-N Carbonylcyanide-3-chlorophenylhydrazone Chemical compound ClC1=CC=CC(NN=C(C#N)C#N)=C1 UGTJLJZQQFGTJD-UHFFFAOYSA-N 0.000 description 4
- 108010060434 Co-Repressor Proteins Proteins 0.000 description 4
- 102000008169 Co-Repressor Proteins Human genes 0.000 description 4
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- HWVNEWGKWRGSRK-UHFFFAOYSA-N GW 0742 Chemical compound CC=1N=C(C=2C=C(F)C(=CC=2)C(F)(F)F)SC=1CSC1=CC=C(OCC(O)=O)C(C)=C1 HWVNEWGKWRGSRK-UHFFFAOYSA-N 0.000 description 4
- 108010010803 Gelatin Proteins 0.000 description 4
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 4
- 229930006000 Sucrose Natural products 0.000 description 4
- CSCPPACGZOOCGX-WFGJKAKNSA-N acetone d6 Chemical compound [2H]C([2H])([2H])C(=O)C([2H])([2H])[2H] CSCPPACGZOOCGX-WFGJKAKNSA-N 0.000 description 4
- 229930010823 amorfrutin 1 Natural products 0.000 description 4
- 239000003963 antioxidant agent Substances 0.000 description 4
- 235000006708 antioxidants Nutrition 0.000 description 4
- 210000000988 bone and bone Anatomy 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000001768 carboxy methyl cellulose Substances 0.000 description 4
- 108091092356 cellular DNA Proteins 0.000 description 4
- 235000010980 cellulose Nutrition 0.000 description 4
- 229920002678 cellulose Polymers 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- 230000000875 corresponding effect Effects 0.000 description 4
- 239000002552 dosage form Substances 0.000 description 4
- 239000000839 emulsion Substances 0.000 description 4
- 150000002085 enols Chemical class 0.000 description 4
- 230000005284 excitation Effects 0.000 description 4
- 239000003925 fat Substances 0.000 description 4
- 235000019197 fats Nutrition 0.000 description 4
- 230000037406 food intake Effects 0.000 description 4
- 238000013467 fragmentation Methods 0.000 description 4
- 238000006062 fragmentation reaction Methods 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 229920000159 gelatin Polymers 0.000 description 4
- 235000019322 gelatine Nutrition 0.000 description 4
- 235000011852 gelatine desserts Nutrition 0.000 description 4
- 239000001257 hydrogen Substances 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- 230000010354 integration Effects 0.000 description 4
- 238000007912 intraperitoneal administration Methods 0.000 description 4
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 4
- 229920000609 methyl cellulose Polymers 0.000 description 4
- 239000001923 methylcellulose Substances 0.000 description 4
- 235000010981 methylcellulose Nutrition 0.000 description 4
- 230000010258 osteoblastogenesis Effects 0.000 description 4
- 230000036961 partial effect Effects 0.000 description 4
- 230000037361 pathway Effects 0.000 description 4
- 239000000813 peptide hormone Substances 0.000 description 4
- 238000000159 protein binding assay Methods 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 230000006950 reactive oxygen species formation Effects 0.000 description 4
- 238000003753 real-time PCR Methods 0.000 description 4
- 238000010992 reflux Methods 0.000 description 4
- 210000002027 skeletal muscle Anatomy 0.000 description 4
- RXCPGWSCILFWCH-UHFFFAOYSA-M sodium 3,4-dihydroxy-9,10-dioxoanthracene-2-sulfonate hydrate Chemical compound O.[Na+].O=C1C2=CC=CC=C2C(=O)C2=C1C(O)=C(O)C(S([O-])(=O)=O)=C2 RXCPGWSCILFWCH-UHFFFAOYSA-M 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 239000005720 sucrose Substances 0.000 description 4
- 150000003626 triacylglycerols Chemical class 0.000 description 4
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 3
- WHBMMWSBFZVSSR-UHFFFAOYSA-N 3-hydroxybutyric acid Chemical compound CC(O)CC(O)=O WHBMMWSBFZVSSR-UHFFFAOYSA-N 0.000 description 3
- DTWGKAWUEOJXKI-UHFFFAOYSA-N 5-(2-carboxy-3-hydroxy-5-methoxyphenyl)-4-methyl-6-oxopyran-2-carboxylic acid Chemical compound COC1=CC(O)=C(C(O)=O)C(C=2C(OC(=CC=2C)C(O)=O)=O)=C1 DTWGKAWUEOJXKI-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 102000014777 Adipokines Human genes 0.000 description 3
- 108010078606 Adipokines Proteins 0.000 description 3
- 102100031786 Adiponectin Human genes 0.000 description 3
- 102100025672 Angiopoietin-related protein 2 Human genes 0.000 description 3
- 102100025674 Angiopoietin-related protein 4 Human genes 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000013948 Fatty acid-binding protein 4 Human genes 0.000 description 3
- 108050003772 Fatty acid-binding protein 4 Proteins 0.000 description 3
- 108010086246 Glucagon-Like Peptide-1 Receptor Proteins 0.000 description 3
- 102100032882 Glucagon-like peptide 1 receptor Human genes 0.000 description 3
- 101000693081 Homo sapiens Angiopoietin-related protein 2 Proteins 0.000 description 3
- 101000693076 Homo sapiens Angiopoietin-related protein 4 Proteins 0.000 description 3
- 101000974349 Homo sapiens Nuclear receptor coactivator 6 Proteins 0.000 description 3
- 101001123331 Homo sapiens Peroxisome proliferator-activated receptor gamma coactivator 1-alpha Proteins 0.000 description 3
- 101001132652 Homo sapiens Retinoic acid receptor responder protein 2 Proteins 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- 108050000123 Inactive phospholipase C-like protein 1 Proteins 0.000 description 3
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 3
- 108010062495 Mediator Complex Subunit 1 Proteins 0.000 description 3
- 102000010904 Mediator Complex Subunit 1 Human genes 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 3
- 241001529936 Murinae Species 0.000 description 3
- 241000612122 Myrsine Species 0.000 description 3
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 3
- 239000007832 Na2SO4 Substances 0.000 description 3
- 208000001132 Osteoporosis Diseases 0.000 description 3
- 102100038825 Peroxisome proliferator-activated receptor gamma Human genes 0.000 description 3
- 102100028960 Peroxisome proliferator-activated receptor gamma coactivator 1-alpha Human genes 0.000 description 3
- 241000212653 Picris rhagadioloides Species 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 102100033914 Retinoic acid receptor responder protein 2 Human genes 0.000 description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 3
- 244000062793 Sorghum vulgare Species 0.000 description 3
- AUYYCJSJGJYCDS-LBPRGKRZSA-N Thyrolar Chemical compound IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(I)=C1 AUYYCJSJGJYCDS-LBPRGKRZSA-N 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 102000010861 Type 3 Cyclic Nucleotide Phosphodiesterases Human genes 0.000 description 3
- 108010037543 Type 3 Cyclic Nucleotide Phosphodiesterases Proteins 0.000 description 3
- 101150022052 UCP1 gene Proteins 0.000 description 3
- 240000008042 Zea mays Species 0.000 description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 3
- 102100034825 [Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 4, mitochondrial Human genes 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 150000001263 acyl chlorides Chemical class 0.000 description 3
- 230000000996 additive effect Effects 0.000 description 3
- 239000000478 adipokine Substances 0.000 description 3
- 235000010443 alginic acid Nutrition 0.000 description 3
- 229920000615 alginic acid Polymers 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- UJSSSFNGQWZNEN-UHFFFAOYSA-N amorfrutin 2 Chemical compound CCCCCC1=CC(OC)=C(CC=C(C)C)C(O)=C1C(O)=O UJSSSFNGQWZNEN-UHFFFAOYSA-N 0.000 description 3
- 229960005070 ascorbic acid Drugs 0.000 description 3
- 239000002585 base Substances 0.000 description 3
- 210000002449 bone cell Anatomy 0.000 description 3
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 3
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 230000003081 coactivator Effects 0.000 description 3
- 239000003086 colorant Substances 0.000 description 3
- 235000005822 corn Nutrition 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 238000010494 dissociation reaction Methods 0.000 description 3
- 230000005593 dissociations Effects 0.000 description 3
- 230000008030 elimination Effects 0.000 description 3
- 238000003379 elimination reaction Methods 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 238000000105 evaporative light scattering detection Methods 0.000 description 3
- 235000012631 food intake Nutrition 0.000 description 3
- 235000003599 food sweetener Nutrition 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 108010036598 gastric inhibitory polypeptide receptor Proteins 0.000 description 3
- 229960003180 glutathione Drugs 0.000 description 3
- 238000005534 hematocrit Methods 0.000 description 3
- 230000002209 hydrophobic effect Effects 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 210000004969 inflammatory cell Anatomy 0.000 description 3
- 239000010410 layer Substances 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 3
- 235000019713 millet Nutrition 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 239000003921 oil Substances 0.000 description 3
- 229960005095 pioglitazone Drugs 0.000 description 3
- 229910052700 potassium Inorganic materials 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- PUIBPGHAXSCVRF-QHFGJBOXSA-N prostaglandin C1 Chemical compound CCCCC[C@H](O)\C=C\C1=CCC(=O)[C@@H]1CCCCCCC(O)=O PUIBPGHAXSCVRF-QHFGJBOXSA-N 0.000 description 3
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 3
- 108010083885 pyruvate dehydrogenase kinase 4 Proteins 0.000 description 3
- 239000011541 reaction mixture Substances 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 235000010413 sodium alginate Nutrition 0.000 description 3
- 239000000661 sodium alginate Substances 0.000 description 3
- 229940005550 sodium alginate Drugs 0.000 description 3
- 229910052938 sodium sulfate Inorganic materials 0.000 description 3
- 235000000346 sugar Nutrition 0.000 description 3
- 150000008163 sugars Chemical class 0.000 description 3
- 239000003765 sweetening agent Substances 0.000 description 3
- 238000004809 thin layer chromatography Methods 0.000 description 3
- GXPHKUHSUJUWKP-UHFFFAOYSA-N troglitazone Chemical compound C1CC=2C(C)=C(O)C(C)=C(C)C=2OC1(C)COC(C=C1)=CC=C1CC1SC(=O)NC1=O GXPHKUHSUJUWKP-UHFFFAOYSA-N 0.000 description 3
- 229960001641 troglitazone Drugs 0.000 description 3
- GXPHKUHSUJUWKP-NTKDMRAZSA-N troglitazone Natural products C([C@@]1(OC=2C(C)=C(C(=C(C)C=2CC1)O)C)C)OC(C=C1)=CC=C1C[C@H]1SC(=O)NC1=O GXPHKUHSUJUWKP-NTKDMRAZSA-N 0.000 description 3
- 238000007514 turning Methods 0.000 description 3
- 230000035899 viability Effects 0.000 description 3
- 239000001993 wax Substances 0.000 description 3
- 239000013585 weight reducing agent Substances 0.000 description 3
- LOYZVRIHVZEDMW-UHFFFAOYSA-N 1-bromo-3-methylbut-2-ene Chemical compound CC(C)=CCBr LOYZVRIHVZEDMW-UHFFFAOYSA-N 0.000 description 2
- AGJBKFAPBKOEGA-UHFFFAOYSA-M 2-methoxyethylmercury(1+);acetate Chemical compound COCC[Hg]OC(C)=O AGJBKFAPBKOEGA-UHFFFAOYSA-M 0.000 description 2
- WOVKYSAHUYNSMH-RRKCRQDMSA-N 5-bromodeoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-RRKCRQDMSA-N 0.000 description 2
- 230000002407 ATP formation Effects 0.000 description 2
- 108010076365 Adiponectin Proteins 0.000 description 2
- UIFFUZWRFRDZJC-UHFFFAOYSA-N Antimycin A1 Natural products CC1OC(=O)C(CCCCCC)C(OC(=O)CC(C)C)C(C)OC(=O)C1NC(=O)C1=CC=CC(NC=O)=C1O UIFFUZWRFRDZJC-UHFFFAOYSA-N 0.000 description 2
- NQWZLRAORXLWDN-UHFFFAOYSA-N Antimycin-A Natural products CCCCCCC(=O)OC1C(C)OC(=O)C(NC(=O)c2ccc(NC=O)cc2O)C(C)OC(=O)C1CCCC NQWZLRAORXLWDN-UHFFFAOYSA-N 0.000 description 2
- 241000416162 Astragalus gummifer Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 2
- 102100034808 CCAAT/enhancer-binding protein alpha Human genes 0.000 description 2
- 102100034798 CCAAT/enhancer-binding protein beta Human genes 0.000 description 2
- REOZWEGFPHTFEI-JKSUJKDBSA-N Cannabidivarin Chemical compound OC1=CC(CCC)=CC(O)=C1[C@H]1[C@H](C(C)=C)CCC(C)=C1 REOZWEGFPHTFEI-JKSUJKDBSA-N 0.000 description 2
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 2
- 241000699802 Cricetulus griseus Species 0.000 description 2
- 101100181494 Cricetulus griseus LDLR gene Proteins 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- 101710088194 Dehydrogenase Proteins 0.000 description 2
- 108010067722 Dipeptidyl Peptidase 4 Proteins 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000207934 Eriodictyon Species 0.000 description 2
- 235000006647 Eugenia jambos Nutrition 0.000 description 2
- 101150067853 Fgf21 gene Proteins 0.000 description 2
- 108010024636 Glutathione Proteins 0.000 description 2
- DHCLVCXQIBBOPH-UHFFFAOYSA-N Glycerol 2-phosphate Chemical compound OCC(CO)OP(O)(O)=O DHCLVCXQIBBOPH-UHFFFAOYSA-N 0.000 description 2
- 206010059484 Haemodilution Diseases 0.000 description 2
- 239000012981 Hank's balanced salt solution Substances 0.000 description 2
- 102000001554 Hemoglobins Human genes 0.000 description 2
- 108010054147 Hemoglobins Proteins 0.000 description 2
- 101000775469 Homo sapiens Adiponectin Proteins 0.000 description 2
- 101000945515 Homo sapiens CCAAT/enhancer-binding protein alpha Proteins 0.000 description 2
- 101000945963 Homo sapiens CCAAT/enhancer-binding protein beta Proteins 0.000 description 2
- 101150079986 Ibsp gene Proteins 0.000 description 2
- 238000008214 LDL Cholesterol Methods 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- 102100030874 Leptin Human genes 0.000 description 2
- 240000007472 Leucaena leucocephala Species 0.000 description 2
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 2
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 101100280998 Mus musculus Fgf21 gene Proteins 0.000 description 2
- 108020005497 Nuclear hormone receptor Proteins 0.000 description 2
- 206010030113 Oedema Diseases 0.000 description 2
- 108010016731 PPAR gamma Proteins 0.000 description 2
- 101150078768 Pdk4 gene Proteins 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 108700008625 Reporter Genes Proteins 0.000 description 2
- 102000034527 Retinoid X Receptors Human genes 0.000 description 2
- 108010038912 Retinoid X Receptors Proteins 0.000 description 2
- 108010086019 Secretin Proteins 0.000 description 2
- 102100037505 Secretin Human genes 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 229910000831 Steel Inorganic materials 0.000 description 2
- 244000087016 Syzygium jambos Species 0.000 description 2
- 229920001615 Tragacanth Polymers 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- 244000098338 Triticum aestivum Species 0.000 description 2
- 101150076688 UCP2 gene Proteins 0.000 description 2
- 101150016260 UCP3 gene Proteins 0.000 description 2
- 230000005856 abnormality Effects 0.000 description 2
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 2
- 229960004308 acetylcysteine Drugs 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 239000000443 aerosol Substances 0.000 description 2
- 239000000783 alginic acid Substances 0.000 description 2
- 229960001126 alginic acid Drugs 0.000 description 2
- 150000004781 alginic acids Chemical class 0.000 description 2
- 229930010825 amorfrutin 2 Natural products 0.000 description 2
- 229930010821 amorfrutin 3 Natural products 0.000 description 2
- BNAJQKIJVGVGFU-UHFFFAOYSA-N amorfrutin 3 Chemical compound OC1=C(CC(O)C(C)=C)C(OC)=CC(CCC=2C=CC=CC=2)=C1C(O)=O BNAJQKIJVGVGFU-UHFFFAOYSA-N 0.000 description 2
- UIFFUZWRFRDZJC-SBOOETFBSA-N antimycin A Chemical compound C[C@H]1OC(=O)[C@H](CCCCCC)[C@@H](OC(=O)CC(C)C)[C@H](C)OC(=O)[C@H]1NC(=O)C1=CC=CC(NC=O)=C1O UIFFUZWRFRDZJC-SBOOETFBSA-N 0.000 description 2
- PVEVXUMVNWSNIG-UHFFFAOYSA-N antimycin A3 Natural products CC1OC(=O)C(CCCC)C(OC(=O)CC(C)C)C(C)OC(=O)C1NC(=O)C1=CC=CC(NC=O)=C1O PVEVXUMVNWSNIG-UHFFFAOYSA-N 0.000 description 2
- 229910052786 argon Inorganic materials 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 239000012131 assay buffer Substances 0.000 description 2
- 238000003149 assay kit Methods 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 101150053306 bglap gene Proteins 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- CJZGTCYPCWQAJB-UHFFFAOYSA-L calcium stearate Chemical class [Ca+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O CJZGTCYPCWQAJB-UHFFFAOYSA-L 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 230000004611 cancer cell death Effects 0.000 description 2
- WVOLTBSCXRRQFR-DLBZAZTESA-N cannabidiolic acid Chemical compound OC1=C(C(O)=O)C(CCCCC)=CC(O)=C1[C@H]1[C@H](C(C)=C)CCC(C)=C1 WVOLTBSCXRRQFR-DLBZAZTESA-N 0.000 description 2
- RBHJBMIOOPYDBQ-UHFFFAOYSA-N carbon dioxide;propan-2-one Chemical compound O=C=O.CC(C)=O RBHJBMIOOPYDBQ-UHFFFAOYSA-N 0.000 description 2
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 2
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 230000004098 cellular respiration Effects 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 2
- 229960004316 cisplatin Drugs 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 229940126214 compound 3 Drugs 0.000 description 2
- 229940125898 compound 5 Drugs 0.000 description 2
- 238000007906 compression Methods 0.000 description 2
- 230000006835 compression Effects 0.000 description 2
- 238000013270 controlled release Methods 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 230000001186 cumulative effect Effects 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 238000002784 cytotoxicity assay Methods 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 238000007405 data analysis Methods 0.000 description 2
- 238000001212 derivatisation Methods 0.000 description 2
- 235000005911 diet Nutrition 0.000 description 2
- 230000037213 diet Effects 0.000 description 2
- PGHZHOJUTQCVBC-UHFFFAOYSA-N diethyl 2-(1-chloro-3-phenylpropylidene)propanedioate Chemical compound CCOC(=O)C(=C(Cl)CCc1ccccc1)C(=O)OCC PGHZHOJUTQCVBC-UHFFFAOYSA-N 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 230000002124 endocrine Effects 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- OPFXXQZYSOSLSR-UHFFFAOYSA-N ethyl 2,4-dihydroxy-3,5-bis(3-methylbut-2-enyl)-6-(2-phenylethyl)benzoate Chemical compound CCOC(=O)c1c(O)c(CC=C(C)C)c(O)c(CC=C(C)C)c1CCc1ccccc1 OPFXXQZYSOSLSR-UHFFFAOYSA-N 0.000 description 2
- 230000004129 fatty acid metabolism Effects 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 230000034659 glycolysis Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000003284 homeostatic effect Effects 0.000 description 2
- 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 2
- 210000000987 immune system Anatomy 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000003914 insulin secretion Effects 0.000 description 2
- 230000009545 invasion Effects 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 229960004768 irinotecan Drugs 0.000 description 2
- GURKHSYORGJETM-WAQYZQTGSA-N irinotecan hydrochloride (anhydrous) Chemical compound Cl.C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 GURKHSYORGJETM-WAQYZQTGSA-N 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 230000037356 lipid metabolism Effects 0.000 description 2
- 230000013190 lipid storage Effects 0.000 description 2
- ZCSHNCUQKCANBX-UHFFFAOYSA-N lithium diisopropylamide Chemical compound [Li+].CC(C)[N-]C(C)C ZCSHNCUQKCANBX-UHFFFAOYSA-N 0.000 description 2
- 210000005228 liver tissue Anatomy 0.000 description 2
- 239000011777 magnesium Substances 0.000 description 2
- 159000000003 magnesium salts Chemical class 0.000 description 2
- 235000019359 magnesium stearate Nutrition 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 150000002690 malonic acid derivatives Chemical class 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 210000003470 mitochondria Anatomy 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 102000006255 nuclear receptors Human genes 0.000 description 2
- 108020004017 nuclear receptors Proteins 0.000 description 2
- 238000013116 obese mouse model Methods 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 230000006461 physiological response Effects 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 210000000229 preadipocyte Anatomy 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000000770 proinflammatory effect Effects 0.000 description 2
- 230000009711 regulatory function Effects 0.000 description 2
- 229960002101 secretin Drugs 0.000 description 2
- OWMZNFCDEHGFEP-NFBCVYDUSA-N secretin human Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(N)=O)[C@@H](C)O)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)C1=CC=CC=C1 OWMZNFCDEHGFEP-NFBCVYDUSA-N 0.000 description 2
- 230000003584 silencer Effects 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 description 2
- 239000004299 sodium benzoate Substances 0.000 description 2
- 235000010234 sodium benzoate Nutrition 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 2
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 239000010959 steel Substances 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 210000000261 subcutaneous preadipocyte Anatomy 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000012730 sustained-release form Substances 0.000 description 2
- 239000000454 talc Substances 0.000 description 2
- 229910052623 talc Inorganic materials 0.000 description 2
- 235000012222 talc Nutrition 0.000 description 2
- 238000003419 tautomerization reaction Methods 0.000 description 2
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 description 2
- 229940034208 thyroxine Drugs 0.000 description 2
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 description 2
- 235000010487 tragacanth Nutrition 0.000 description 2
- 239000000196 tragacanth Substances 0.000 description 2
- 229940116362 tragacanth Drugs 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 230000009278 visceral effect Effects 0.000 description 2
- 230000004584 weight gain Effects 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 2
- LNAZSHAWQACDHT-XIYTZBAFSA-N (2r,3r,4s,5r,6s)-4,5-dimethoxy-2-(methoxymethyl)-3-[(2s,3r,4s,5r,6r)-3,4,5-trimethoxy-6-(methoxymethyl)oxan-2-yl]oxy-6-[(2r,3r,4s,5r,6r)-4,5,6-trimethoxy-2-(methoxymethyl)oxan-3-yl]oxyoxane Chemical compound CO[C@@H]1[C@@H](OC)[C@H](OC)[C@@H](COC)O[C@H]1O[C@H]1[C@H](OC)[C@@H](OC)[C@H](O[C@H]2[C@@H]([C@@H](OC)[C@H](OC)O[C@@H]2COC)OC)O[C@@H]1COC LNAZSHAWQACDHT-XIYTZBAFSA-N 0.000 description 1
- OFCWBJAYEIROGZ-KRWDZBQOSA-N (2s)-2-[3-[[1-(4-methoxybenzoyl)-2-methyl-5-(trifluoromethoxy)indol-3-yl]methyl]phenoxy]propanoic acid Chemical compound C1=CC(OC)=CC=C1C(=O)N1C2=CC=C(OC(F)(F)F)C=C2C(CC=2C=C(O[C@@H](C)C(O)=O)C=CC=2)=C1C OFCWBJAYEIROGZ-KRWDZBQOSA-N 0.000 description 1
- JSCUZAYKVZXKQE-YFHOEESVSA-N (2z)-1-bromo-3,7-dimethylocta-2,6-diene Chemical compound CC(C)=CCC\C(C)=C/CBr JSCUZAYKVZXKQE-YFHOEESVSA-N 0.000 description 1
- PKYCWFICOKSIHZ-UHFFFAOYSA-N 1-(3,7-dihydroxyphenoxazin-10-yl)ethanone Chemical compound OC1=CC=C2N(C(=O)C)C3=CC=C(O)C=C3OC2=C1 PKYCWFICOKSIHZ-UHFFFAOYSA-N 0.000 description 1
- FRJNKYGTHPUSJR-UHFFFAOYSA-N 1-benzothiophene 1,1-dioxide Chemical compound C1=CC=C2S(=O)(=O)C=CC2=C1 FRJNKYGTHPUSJR-UHFFFAOYSA-N 0.000 description 1
- KVUXYQHEESDGIJ-UHFFFAOYSA-N 10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthrene-3,16-diol Chemical compound C1CC2CC(O)CCC2(C)C2C1C1CC(O)CC1(C)CC2 KVUXYQHEESDGIJ-UHFFFAOYSA-N 0.000 description 1
- LTJOXDFGIGRGCK-UHFFFAOYSA-N 2,10-dimethylundeca-2,9-dien-6-one Chemical compound CC(C)=CCCC(=O)CCC=C(C)C LTJOXDFGIGRGCK-UHFFFAOYSA-N 0.000 description 1
- VNXWANZMRATDQM-UHFFFAOYSA-N 2-(2-phenylethyl)benzene-1,3-diol Chemical compound OC1=CC=CC(O)=C1CCC1=CC=CC=C1 VNXWANZMRATDQM-UHFFFAOYSA-N 0.000 description 1
- QUVGEKPNSCFQIR-AOSYACOCSA-N 2-Hydroxy-6-(8,11,14-pentadecatrienyl)benzoic acid Chemical compound OC(=O)C1=C(O)C=CC=C1CCCCCCC\C=C\C\C=C\CC=C QUVGEKPNSCFQIR-AOSYACOCSA-N 0.000 description 1
- VNDRRWBKNSHALL-UHFFFAOYSA-N 2-[(2,4-dichlorobenzoyl)amino]-5-(pyrimidin-2-yloxy)benzoic acid Chemical compound C=1C=C(NC(=O)C=2C(=CC(Cl)=CC=2)Cl)C(C(=O)O)=CC=1OC1=NC=CC=N1 VNDRRWBKNSHALL-UHFFFAOYSA-N 0.000 description 1
- 125000001431 2-aminoisobutyric acid group Chemical group [#6]C([#6])(N*)C(*)=O 0.000 description 1
- SZTKWWMTOKHUAW-UHFFFAOYSA-N 2-methoxy-1-(3-methylbut-2-enyl)-4-(2-phenylethyl)benzene Chemical compound COC=1C(=CC=C(C=1)CCC1=CC=CC=C1)CC=C(C)C SZTKWWMTOKHUAW-UHFFFAOYSA-N 0.000 description 1
- APIXJSLKIYYUKG-UHFFFAOYSA-N 3 Isobutyl 1 methylxanthine Chemical compound O=C1N(C)C(=O)N(CC(C)C)C2=C1N=CN2 APIXJSLKIYYUKG-UHFFFAOYSA-N 0.000 description 1
- WHNBKYRXAOMPAE-VSMNQWMWSA-N 3-[(2e)-3,7-dimethylocta-2,6-dienyl]-2,4-dihydroxy-6-[(e)-2-phenylethenyl]benzoic acid Chemical compound OC(=O)C1=C(O)C(C/C=C(C)/CCC=C(C)C)=C(O)C=C1\C=C\C1=CC=CC=C1 WHNBKYRXAOMPAE-VSMNQWMWSA-N 0.000 description 1
- MFEILWXBDBCWKF-UHFFFAOYSA-N 3-phenylpropanoyl chloride Chemical compound ClC(=O)CCC1=CC=CC=C1 MFEILWXBDBCWKF-UHFFFAOYSA-N 0.000 description 1
- 208000004611 Abdominal Obesity Diseases 0.000 description 1
- 101150054866 Acadl gene Proteins 0.000 description 1
- 101150113336 Acadm gene Proteins 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 101150073604 Adgre1 gene Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 241000212978 Amorpha <angiosperm> Species 0.000 description 1
- 244000226021 Anacardium occidentale Species 0.000 description 1
- 235000001274 Anacardium occidentale Nutrition 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- 101150031273 BDH1 gene Proteins 0.000 description 1
- 101150101337 Bdh2 gene Proteins 0.000 description 1
- 241001474374 Blennius Species 0.000 description 1
- 208000010392 Bone Fractures Diseases 0.000 description 1
- 102100031746 Bone sialoprotein 2 Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- FTNJQNQLEGKTGD-UHFFFAOYSA-N C1Oc2ccccc2O1 Chemical compound C1Oc2ccccc2O1 FTNJQNQLEGKTGD-UHFFFAOYSA-N 0.000 description 1
- HCMLNPZTRYNCMA-UHFFFAOYSA-N C1Sc2ccccc2S1 Chemical compound C1Sc2ccccc2S1 HCMLNPZTRYNCMA-UHFFFAOYSA-N 0.000 description 1
- 101710186200 CCAAT/enhancer-binding protein Proteins 0.000 description 1
- 101150111331 CCL5 gene Proteins 0.000 description 1
- 101150083327 CCR2 gene Proteins 0.000 description 1
- 101150017501 CCR5 gene Proteins 0.000 description 1
- 102000049320 CD36 Human genes 0.000 description 1
- 108010045374 CD36 Antigens Proteins 0.000 description 1
- 101100451537 Caenorhabditis elegans hsd-1 gene Proteins 0.000 description 1
- 108060001064 Calcitonin Proteins 0.000 description 1
- 102000055006 Calcitonin Human genes 0.000 description 1
- WVOLTBSCXRRQFR-SJORKVTESA-N Cannabidiolic acid Natural products OC1=C(C(O)=O)C(CCCCC)=CC(O)=C1[C@@H]1[C@@H](C(C)=C)CCC(C)=C1 WVOLTBSCXRRQFR-SJORKVTESA-N 0.000 description 1
- 241000218236 Cannabis Species 0.000 description 1
- 238000003731 Caspase Glo 3/7 Assay Methods 0.000 description 1
- LVILGAOSPDLNRM-UHFFFAOYSA-N Cc1ncncc1 Chemical compound Cc1ncncc1 LVILGAOSPDLNRM-UHFFFAOYSA-N 0.000 description 1
- 238000003734 CellTiter-Glo Luminescent Cell Viability Assay Methods 0.000 description 1
- 206010065941 Central obesity Diseases 0.000 description 1
- 240000008886 Ceratonia siliqua Species 0.000 description 1
- 235000013912 Ceratonia siliqua Nutrition 0.000 description 1
- 102000003706 Complement factor D Human genes 0.000 description 1
- 108090000059 Complement factor D Proteins 0.000 description 1
- 244000303965 Cyamopsis psoralioides Species 0.000 description 1
- 102000013717 Cyclin-Dependent Kinase 5 Human genes 0.000 description 1
- 108010025454 Cyclin-Dependent Kinase 5 Proteins 0.000 description 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 235000019739 Dicalciumphosphate Nutrition 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 102100025012 Dipeptidyl peptidase 4 Human genes 0.000 description 1
- 208000032928 Dyslipidaemia Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000005593 Endopeptidases Human genes 0.000 description 1
- 108010059378 Endopeptidases Proteins 0.000 description 1
- 235000002683 Eriodictyon californicum Nutrition 0.000 description 1
- 101150010122 FBP1 gene Proteins 0.000 description 1
- 206010017076 Fracture Diseases 0.000 description 1
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 description 1
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 description 1
- 101150023900 G6PC1 gene Proteins 0.000 description 1
- 101150022160 G6PC3 gene Proteins 0.000 description 1
- 238000002957 GeneBLAzer Methods 0.000 description 1
- 102000051325 Glucagon Human genes 0.000 description 1
- 108060003199 Glucagon Proteins 0.000 description 1
- 108010063919 Glucagon Receptors Proteins 0.000 description 1
- 102100040890 Glucagon receptor Human genes 0.000 description 1
- 208000002705 Glucose Intolerance Diseases 0.000 description 1
- 206010018429 Glucose tolerance impaired Diseases 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 229920002907 Guar gum Polymers 0.000 description 1
- 101150044169 HMGCL gene Proteins 0.000 description 1
- 101150032553 Hmgcs2 gene Proteins 0.000 description 1
- 101000839025 Homo sapiens Hydroxymethylglutaryl-CoA synthase, cytoplasmic Proteins 0.000 description 1
- 101000976075 Homo sapiens Insulin Proteins 0.000 description 1
- 101000974340 Homo sapiens Nuclear receptor corepressor 1 Proteins 0.000 description 1
- 101001086210 Homo sapiens Osteocalcin Proteins 0.000 description 1
- 101000741790 Homo sapiens Peroxisome proliferator-activated receptor gamma Proteins 0.000 description 1
- 101000734579 Homo sapiens Phosphoenolpyruvate carboxykinase [GTP], mitochondrial Proteins 0.000 description 1
- 241000029377 Hyalodendron Species 0.000 description 1
- 102100028888 Hydroxymethylglutaryl-CoA synthase, cytoplasmic Human genes 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 108010028750 Integrin-Binding Sialoprotein Proteins 0.000 description 1
- 229930194542 Keto Natural products 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 239000002211 L-ascorbic acid Substances 0.000 description 1
- 235000000069 L-ascorbic acid Nutrition 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 102000016267 Leptin Human genes 0.000 description 1
- 108010092277 Leptin Proteins 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Chemical compound CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 208000017170 Lipid metabolism disease Diseases 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 229920000881 Modified starch Polymers 0.000 description 1
- 101100161764 Mus musculus Acoxl gene Proteins 0.000 description 1
- 101100503236 Mus musculus Folr1 gene Proteins 0.000 description 1
- 101150107221 NCOR1 gene Proteins 0.000 description 1
- 102000034570 NR1 subfamily Human genes 0.000 description 1
- 108020001305 NR1 subfamily Proteins 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 101710202677 Non-specific lipid-transfer protein Proteins 0.000 description 1
- 102100022935 Nuclear receptor corepressor 1 Human genes 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102100031475 Osteocalcin Human genes 0.000 description 1
- 101150039326 PCK1 gene Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 101150000187 PTGS2 gene Proteins 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 241000282320 Panthera leo Species 0.000 description 1
- 102000003982 Parathyroid hormone Human genes 0.000 description 1
- 108090000445 Parathyroid hormone Proteins 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 102000003921 Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha Human genes 0.000 description 1
- 108090000310 Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha Proteins 0.000 description 1
- 102000018042 Peroxisome proliferator-activated receptor gamma coactivator 1-beta Human genes 0.000 description 1
- 108050007135 Peroxisome proliferator-activated receptor gamma coactivator 1-beta Proteins 0.000 description 1
- 229940122907 Phosphatase inhibitor Drugs 0.000 description 1
- 102100034792 Phosphoenolpyruvate carboxykinase [GTP], mitochondrial Human genes 0.000 description 1
- 102100022428 Phospholipid transfer protein Human genes 0.000 description 1
- 108010001441 Phosphopeptides Proteins 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 101150025052 Pklr gene Proteins 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 238000011530 RNeasy Mini Kit Methods 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 238000010818 SYBR green PCR Master Mix Methods 0.000 description 1
- 101150116689 Slc2a2 gene Proteins 0.000 description 1
- BCKXLBQYZLBQEK-KVVVOXFISA-M Sodium oleate Chemical compound [Na+].CCCCCCCC\C=C/CCCCCCCC([O-])=O BCKXLBQYZLBQEK-KVVVOXFISA-M 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 240000001058 Sterculia urens Species 0.000 description 1
- 235000015125 Sterculia urens Nutrition 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 102100036049 T-complex protein 1 subunit gamma Human genes 0.000 description 1
- QHMBSVQNZZTUGM-UHFFFAOYSA-N Trans-Cannabidiol Natural products OC1=CC(CCCCC)=CC(O)=C1C1C(C(C)=C)CCC(C)=C1 QHMBSVQNZZTUGM-UHFFFAOYSA-N 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000008200 Uncoupling Protein 3 Human genes 0.000 description 1
- 108010021098 Uncoupling Protein 3 Proteins 0.000 description 1
- 102000055135 Vasoactive Intestinal Peptide Human genes 0.000 description 1
- 108010003205 Vasoactive Intestinal Peptide Proteins 0.000 description 1
- 210000001766 X chromosome Anatomy 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- WETWJCDKMRHUPV-UHFFFAOYSA-N acetyl chloride Chemical compound CC(Cl)=O WETWJCDKMRHUPV-UHFFFAOYSA-N 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 230000011759 adipose tissue development Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 210000004404 adrenal cortex Anatomy 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000010419 agar Nutrition 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- SNAAJJQQZSMGQD-UHFFFAOYSA-N aluminum magnesium Chemical compound [Mg].[Al] SNAAJJQQZSMGQD-UHFFFAOYSA-N 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- KTBAIBUBSGVINZ-UHFFFAOYSA-N amorfrutin C Chemical compound COC1=C(CC=C(C)C)C(O)=C(C(O)=O)C(CCC=2C=CC=CC=2)=C1CC=C(C)C KTBAIBUBSGVINZ-UHFFFAOYSA-N 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000578 anorexic effect Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000001387 anti-histamine Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000003579 anti-obesity Effects 0.000 description 1
- 239000003472 antidiabetic agent Substances 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 230000005775 apoptotic pathway Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000012300 argon atmosphere Substances 0.000 description 1
- 230000003305 autocrine Effects 0.000 description 1
- 229940062310 avandia Drugs 0.000 description 1
- TZCXTZWJZNENPQ-UHFFFAOYSA-L barium sulfate Chemical compound [Ba+2].[O-]S([O-])(=O)=O TZCXTZWJZNENPQ-UHFFFAOYSA-L 0.000 description 1
- 239000003637 basic solution Substances 0.000 description 1
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 1
- 235000012216 bentonite Nutrition 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 238000012925 biological evaluation Methods 0.000 description 1
- 230000033558 biomineral tissue development Effects 0.000 description 1
- AZWXAPCAJCYGIA-UHFFFAOYSA-N bis(2-methylpropyl)alumane Chemical compound CC(C)C[AlH]CC(C)C AZWXAPCAJCYGIA-UHFFFAOYSA-N 0.000 description 1
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 1
- 210000001772 blood platelet Anatomy 0.000 description 1
- 230000006583 body weight regulation Effects 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 235000010338 boric acid Nutrition 0.000 description 1
- 239000012267 brine Substances 0.000 description 1
- 239000004067 bulking agent Substances 0.000 description 1
- DVECBJCOGJRVPX-UHFFFAOYSA-N butyryl chloride Chemical compound CCCC(Cl)=O DVECBJCOGJRVPX-UHFFFAOYSA-N 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 description 1
- 229960004015 calcitonin Drugs 0.000 description 1
- 235000010410 calcium alginate Nutrition 0.000 description 1
- 239000000648 calcium alginate Substances 0.000 description 1
- 229960002681 calcium alginate Drugs 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 235000013539 calcium stearate Nutrition 0.000 description 1
- 239000008116 calcium stearate Substances 0.000 description 1
- 235000011132 calcium sulphate Nutrition 0.000 description 1
- DIOLOCSXUMYFJN-UHFFFAOYSA-N calcium;azane Chemical compound N.[Ca+2] DIOLOCSXUMYFJN-UHFFFAOYSA-N 0.000 description 1
- ZTGXAWYVTLUPDT-UHFFFAOYSA-N cannabidiol Natural products OC1=CC(CCCCC)=CC(O)=C1C1C(C(C)=C)CC=C(C)C1 ZTGXAWYVTLUPDT-UHFFFAOYSA-N 0.000 description 1
- 229950011318 cannabidiol Drugs 0.000 description 1
- REOZWEGFPHTFEI-UHFFFAOYSA-N cannabidivarine Natural products OC1=CC(CCC)=CC(O)=C1C1C(C(C)=C)CCC(C)=C1 REOZWEGFPHTFEI-UHFFFAOYSA-N 0.000 description 1
- 230000023852 carbohydrate metabolic process Effects 0.000 description 1
- 125000002843 carboxylic acid group Chemical group 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000001364 causal effect Effects 0.000 description 1
- 101150062912 cct3 gene Proteins 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000002737 cell proliferation kit Methods 0.000 description 1
- 230000003822 cell turnover Effects 0.000 description 1
- 238000012054 celltiter-glo Methods 0.000 description 1
- 229920001429 chelating resin Polymers 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 150000001805 chlorine compounds Chemical class 0.000 description 1
- 239000003280 clastogen Substances 0.000 description 1
- 231100000506 clastogen Toxicity 0.000 description 1
- 239000004927 clay Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000011278 co-treatment Methods 0.000 description 1
- 229940110456 cocoa butter Drugs 0.000 description 1
- 235000019868 cocoa butter Nutrition 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 238000007398 colorimetric assay Methods 0.000 description 1
- 238000005056 compaction Methods 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 235000008504 concentrate Nutrition 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000013256 coordination polymer Substances 0.000 description 1
- 229940045031 corn grain extract Drugs 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 229960004397 cyclophosphamide Drugs 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- GVJHHUAWPYXKBD-UHFFFAOYSA-N d-alpha-tocopherol Natural products OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 1
- 235000021316 daily nutritional intake Nutrition 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000000326 densiometry Methods 0.000 description 1
- 238000010217 densitometric analysis Methods 0.000 description 1
- 230000002999 depolarising effect Effects 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- NEFBYIFKOOEVPA-UHFFFAOYSA-K dicalcium phosphate Chemical compound [Ca+2].[Ca+2].[O-]P([O-])([O-])=O NEFBYIFKOOEVPA-UHFFFAOYSA-K 0.000 description 1
- 229940038472 dicalcium phosphate Drugs 0.000 description 1
- 229910000390 dicalcium phosphate Inorganic materials 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- PCXRACLQFPRCBB-ZWKOTPCHSA-N dihydrocannabidiol Natural products OC1=CC(CCCCC)=CC(O)=C1[C@H]1[C@H](C(C)C)CCC(C)=C1 PCXRACLQFPRCBB-ZWKOTPCHSA-N 0.000 description 1
- SIPUZPBQZHNSDW-UHFFFAOYSA-N diisobutylaluminium hydride Substances CC(C)C[Al]CC(C)C SIPUZPBQZHNSDW-UHFFFAOYSA-N 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 238000007908 dry granulation Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 210000001198 duodenum Anatomy 0.000 description 1
- 230000008482 dysregulation Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 230000027721 electron transport chain Effects 0.000 description 1
- 229940066758 endopeptidases Drugs 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- ODQVUGCWWSDTMW-UHFFFAOYSA-N ethyl 2-hydroxy-4-methoxy-3,5-bis(3-methylbut-2-enyl)-6-(2-phenylethyl)benzoate Chemical compound CCOC(=O)c1c(O)c(CC=C(C)C)c(OC)c(CC=C(C)C)c1CCc1ccccc1 ODQVUGCWWSDTMW-UHFFFAOYSA-N 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- GFAUNYMRSKVDJL-UHFFFAOYSA-N formyl chloride Chemical compound ClC=O GFAUNYMRSKVDJL-UHFFFAOYSA-N 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 230000027119 gastric acid secretion Effects 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000007674 genetic toxicity Effects 0.000 description 1
- 231100000025 genetic toxicology Toxicity 0.000 description 1
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 description 1
- 229960004666 glucagon Drugs 0.000 description 1
- 230000004110 gluconeogenesis Effects 0.000 description 1
- 230000001890 gluconeogenic effect Effects 0.000 description 1
- 230000014101 glucose homeostasis Effects 0.000 description 1
- 230000010030 glucose lowering effect Effects 0.000 description 1
- 230000004153 glucose metabolism Effects 0.000 description 1
- 230000004190 glucose uptake Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 238000005469 granulation Methods 0.000 description 1
- 230000003179 granulation Effects 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 235000010417 guar gum Nutrition 0.000 description 1
- 239000000665 guar gum Substances 0.000 description 1
- 229960002154 guar gum Drugs 0.000 description 1
- 230000008821 health effect Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 230000002443 hepatoprotective effect Effects 0.000 description 1
- 231100000304 hepatotoxicity Toxicity 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 238000005734 heterodimerization reaction Methods 0.000 description 1
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 238000010842 high-capacity cDNA reverse transcription kit Methods 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 239000008240 homogeneous mixture Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229960000890 hydrocortisone Drugs 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 description 1
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 1
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 1
- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 239000000859 incretin Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 150000002484 inorganic compounds Chemical class 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 229940030980 inova Drugs 0.000 description 1
- PBGKTOXHQIOBKM-FHFVDXKLSA-N insulin (human) Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 PBGKTOXHQIOBKM-FHFVDXKLSA-N 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- VBUWHHLIZKOSMS-RIWXPGAOSA-N invicorp Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)C(C)C)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=C(O)C=C1 VBUWHHLIZKOSMS-RIWXPGAOSA-N 0.000 description 1
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 230000002361 ketogenic effect Effects 0.000 description 1
- 125000000468 ketone group Chemical group 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- NRYBAZVQPHGZNS-ZSOCWYAHSA-N leptin Chemical compound O=C([C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CC(C)C)CCSC)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CS)C(O)=O NRYBAZVQPHGZNS-ZSOCWYAHSA-N 0.000 description 1
- 229940039781 leptin Drugs 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 230000007056 liver toxicity Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 150000004702 methyl esters Chemical class 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 239000007758 minimum essential medium Substances 0.000 description 1
- 230000008437 mitochondrial biogenesis Effects 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 235000019426 modified starch Nutrition 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- 231100000707 mutagenic chemical Toxicity 0.000 description 1
- 229920001206 natural gum Polymers 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100001084 no genetic toxicology Toxicity 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 239000003605 opacifier Substances 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 230000004072 osteoblast differentiation Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000010627 oxidative phosphorylation Effects 0.000 description 1
- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 description 1
- 229940094443 oxytocics prostaglandins Drugs 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 229960001319 parathyroid hormone Drugs 0.000 description 1
- 239000000199 parathyroid hormone Substances 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000004031 partial agonist Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- XGISHOFUAFNYQF-UHFFFAOYSA-N pentanoyl chloride Chemical compound CCCCC(Cl)=O XGISHOFUAFNYQF-UHFFFAOYSA-N 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 238000011170 pharmaceutical development Methods 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 230000001817 pituitary effect Effects 0.000 description 1
- 210000004180 plasmocyte Anatomy 0.000 description 1
- 239000004014 plasticizer Substances 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 235000015277 pork Nutrition 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000023603 positive regulation of transcription initiation, DNA-dependent Effects 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 229940114930 potassium stearate Drugs 0.000 description 1
- ANBFRLKBEIFNQU-UHFFFAOYSA-M potassium;octadecanoate Chemical compound [K+].CCCCCCCCCCCCCCCCCC([O-])=O ANBFRLKBEIFNQU-UHFFFAOYSA-M 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- RZWZRACFZGVKFM-UHFFFAOYSA-N propanoyl chloride Chemical compound CCC(Cl)=O RZWZRACFZGVKFM-UHFFFAOYSA-N 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000010916 retrosynthetic analysis Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- SUFUKZSWUHZXAV-BTJKTKAUSA-N rosiglitazone maleate Chemical compound [H+].[H+].[O-]C(=O)\C=C/C([O-])=O.C=1C=CC=NC=1N(C)CCOC(C=C1)=CC=C1CC1SC(=O)NC1=O SUFUKZSWUHZXAV-BTJKTKAUSA-N 0.000 description 1
- 230000036186 satiety Effects 0.000 description 1
- 235000019627 satiety Nutrition 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 239000007909 solid dosage form Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000000528 statistical test Methods 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 230000004960 subcellular localization Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 235000010384 tocopherol Nutrition 0.000 description 1
- 229960001295 tocopherol Drugs 0.000 description 1
- 229930003799 tocopherol Natural products 0.000 description 1
- 239000011732 tocopherol Substances 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 210000003437 trachea Anatomy 0.000 description 1
- 108091006105 transcriptional corepressors Proteins 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 231100000747 viability assay Toxicity 0.000 description 1
- 238000003026 viability measurement method Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005550 wet granulation Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
- C07D307/79—Benzo [b] furans; Hydrogenated benzo [b] furans with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C39/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring
- C07C39/18—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring monocyclic with unsaturation outside the aromatic ring
- C07C39/19—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring monocyclic with unsaturation outside the aromatic ring containing carbon-to-carbon double bonds but no carbon-to-carbon triple bonds
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C39/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring
- C07C39/205—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring polycyclic, containing only six-membered aromatic rings as cyclic parts with unsaturation outside the rings
- C07C39/21—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring polycyclic, containing only six-membered aromatic rings as cyclic parts with unsaturation outside the rings with at least one hydroxy group on a non-condensed ring
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C39/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring
- C07C39/23—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring polycyclic, containing six-membered aromatic rings and other rings, with unsaturation outside the aromatic rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C43/00—Ethers; Compounds having groups, groups or groups
- C07C43/02—Ethers
- C07C43/20—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring
- C07C43/23—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring containing hydroxy or O-metal groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C47/00—Compounds having —CHO groups
- C07C47/52—Compounds having —CHO groups bound to carbon atoms of six—membered aromatic rings
- C07C47/56—Compounds having —CHO groups bound to carbon atoms of six—membered aromatic rings containing hydroxy groups
- C07C47/565—Compounds having —CHO groups bound to carbon atoms of six—membered aromatic rings containing hydroxy groups all hydroxy groups bound to the ring
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
- C07C49/76—Ketones containing a keto group bound to a six-membered aromatic ring
- C07C49/84—Ketones containing a keto group bound to a six-membered aromatic ring containing ether groups, groups, groups, or groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C65/00—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C65/01—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C65/00—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C65/01—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups
- C07C65/19—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups having unsaturation outside the aromatic ring
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C65/00—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C65/21—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing ether groups, groups, groups, or groups
- C07C65/24—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing ether groups, groups, groups, or groups polycyclic
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C65/00—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C65/21—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing ether groups, groups, groups, or groups
- C07C65/28—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing ether groups, groups, groups, or groups having unsaturation outside the aromatic rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C65/00—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C65/32—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing keto groups
- C07C65/40—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing keto groups containing singly bound oxygen-containing groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
- C07C69/02—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen
- C07C69/12—Acetic acid esters
- C07C69/18—Acetic acid esters of trihydroxylic compounds
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
- C07D307/79—Benzo [b] furans; Hydrogenated benzo [b] furans with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
- C07D307/80—Radicals substituted by oxygen atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
- C07D309/34—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
- C07D309/36—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms
- C07D309/38—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms one oxygen atom in position 2 or 4, e.g. pyrones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2601/00—Systems containing only non-condensed rings
- C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
- C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated
Definitions
- the present invention relates to Amorfrutin analogues and stereoisomeric forms, solvates, hydrates, conjugates and/or pharmaceutically acceptable salts of these compounds as well as pharmaceutical compositions containing at least one of these Amorfrutin analogues together with pharmaceutically acceptable carrier, excipient and/or diluents.
- Said Amorfrutin analogues have been identified as modulators of the peroxisome proliferator-activated receptors (PPARs), especially PPARy and are useful for the prevention and treatment of metabolic diseases, inflammatory diseases, cancer and preparation of phytomedicals and/or functional food products for prevention of metabolic diseases.
- PPARs peroxisome proliferator-activated receptors
- the peroxisome proliferator-activated receptor gamma is a nuclear receptor that regulates transcription with two effector binding sites called activation function 1 (AF1 ) and activation function 2 (AF2). AF1 is localized within the N- terminal regulatory domain.
- the receptor's central DNA binding domain is followed by the C-terminal ligand binding domain (LBD), which comprises AF2.
- LBD C-terminal ligand binding domain
- PPARy is regulated by a phosphorylation site in the LBD at Ser273.
- the ligand-activated transcription factor PPARy acts in the nucleus as a heterodimer with the retinoid X receptor RXR. PPARy interacts with prostaglandins and fatty acids and their metabolites.
- PPARy acts as a sensor and regulator with a dominant role in glucose and lipid metabolism and adipose cell differentiation. Activation of the receptor improves insulin sensitivity through different metabolic actions, including regulation of adipokines.
- PPARy is a well-established drug target for type II diabetes. To reduce blood glucose levels recent pharmaceutical developments aimed to activate PPARy in peripheral metabolic target tissues such as adipose tissue, muscle, liver and macrophages. Recent publications indicate potential roles of PPARy - expressed in the central nervous system - in the regulation of weight balance ⁇ Nature Med. 2011 , 5, 618; Nature Med. 2011 , 5, 623.). This nuclear receptor further plays also a key role in inflammatory diseases and cancer (Nature Rev. Cancer 2012, 12, 181 ).
- PPARa and PPAR / ⁇ also bind fatty acids and are involved in fatty acid metabolism.
- PPARa and PPAR / ⁇ promote fatty acid catabolism in several tissues
- PPAR y regulates fatty acid storage in adipose tissues.
- Dual PPARa and PPAR / ⁇ agonists correcting glucose and lipid abnormalities in patients with type II diabetes have been already reported [Biochim. Biophys. Acta 2011 , 1812, 1007; Structure 2001 , 9, 699].
- the LBD of PPARy has several regulatory functions.
- the receptor determines the receptor's subcellular localization, initiates heterodimerization with RXR, and activates or represses transcription of target genes in a ligand-dependent manner.
- the ligand binding stabilizes the LBD and leads to a more compact and rigid conformation, which in turn causes recruitment of cell-specific coactivators like SRC1 to the LBD's AF2 effector binding site.
- PPARy bound to the promoter of a target gene activates transcription of that target gene upon coactivator recruitment.
- the synthetic agonist rosiglitazone induces coactivator recruitment and inhibits NCoR co-repressor-mediated CDK5-dependent phosphorylation of Ser273, which alters the expression of a subset of genes with regulatory functions in metabolism.
- Rosiglitazone and other glitazones strongly activate transcription of a large number of genes in various tissues. This unspecific action of glitazones is associated with severe side effects including weight gain, osteoporosis, cardiovascular complications, and edema and determined the withdrawn of these products from the market or the restriction of their prescription.
- Partial PPARy agonists which activate PPARy only weakly, are more selective PPARy modulators (SPPARyMs) and avoid side effects. [Structure 2007, 15, 1258] Dependent on the cellular context and transcriptional PPARy co-factors, partial PPARy agonists may activate or potentially repress transcription of target genes.
- the partial agonists BVT.13, MRL-24, nTZDpa and amorfrutin 1 block NCoR recruitment and Ser273 phosphorylation as effectively as rosiglitazone, but activate transcription of target genes only to a moderate level.
- Amorfrutins are a group of natural products that have recently been identified as PPARy modulators with the characteristics of SPPARyMs. They are binding the PPARy, having dissociation constants of around 300 nM.
- amorfrutin analogues of general formula I, conjugates and pharmaceutically acceptable salts thereof suitable to selectively modulate the PPARy with greatly increased specific activity with regard to the prior art and with less side effects.
- a further aspect of the invention is to provide amorfrutin analogues of general fornnula I, conjugates and pharnnaceutically acceptable salts thereof, which can be used as pharnnaceutically active agents, especially for the treatment of metabolic diseases, as well as compositions comprising at least one of those compounds and/or pharmaceutically acceptable salts thereof as pharmaceutically active ingredients.
- the compounds of the present invention can be used also as prophylactic dietary supplements.
- R 2 represents the following: -H, -OH, -OC q H( 2q+ i );
- R 4 represents the following: -H, -OH, -OCH 3 , -CH 3 or
- R 2 together with R 3 or R 3 together with R 4 form with the two carbons of the benzene ring to which R 2 and R 3 or R 3 and R 4 are attached one of the following moieties:
- R 15 represents one of the following -H, -CH 3 , -C2H 5 , -C3H 7 , -C 4 H 9 ;
- R 45 , R 46 , R 47 , R 48 , R 49 , R 52 and R 53 represent independently of each other -H, — CH3, — C2H 5 , — C3H 7 , — C 4 Hg i — OCH3, — OC2H 5 , — OC3H 7 , — F, —CI, — CF3, -CHF2, -CH 2 F.
- conjugates refers to a compound of general formula I covalently linked to a peptide or a peptide analogue that is recognized by a certain receptor expressed on the surface of the cell and involved in beneficial physiological responses, such as increased satiety.
- conjugates are able to specifically direct the compounds of general formula I to cells expressing the receptors that interact with the peptides or peptides analogues to which the compounds of general formula I are conjugated.
- Example of peptides suitable to be covalently linked to a compound of general formula I include, but are not restricted to metabolically active peptide hormones, such as incretin-derivatives.
- Glucagon-like peptide-1 (GLP-1 ) receptor or the glucose-dependent insulinotropic polypeptide (GIP) receptor constitutes examples of receptors expressed on the surface of the cell and involved in beneficial physiological responses.
- GLP-1 is one of the most potent incretins and it stimulates insulin secretion. GLP-1 is released by the gut into the circulation in response to carbohydrate or protein ingestion.
- the GLP-1 receptor is a 463 amino acid 7 transmembrane-spanning protein exhibiting 27% to 40% sequence homology to the receptors for secretin, calcitonin, and parathyroid hormone. As these receptors shared higher identity with each other than with other members of the G protein-coupled receptor superfamily, they were classified together into a new family of receptors now known as the type II receptor family.
- the GLP-1 receptor recognizes GLP-1 specifically, with no demonstrable binding by a number of related peptides, including secretin and vasoactive intestinal peptide.
- the mammalian GLP-1 receptor is mainly expressed in pancreatic cells, stomach and brain.
- Glucose-dependent insulinotropic polypeptide or gastric inhibitory polypeptide is a 42 amino acid peptide hormone secreted by K cells in the intestinal epithelium. The majority of intestinal K cells are located in the proximal duodenum. GIP secretion is primarily regulated by nutrients, especially fats. The GIP receptor is also a member of the glucagon receptor family. The GIP receptor is involved in glucose homeostasis via potentiation of glucose-dependent insulin secretion from the pancreatic islet ⁇ -cells. It also inhibits gastric acid secretion.
- the GIP receptor is expressed in the pancreas, stomach, small intestine, adipose tissue, adrenal cortex, pituitary, heart, testis, endothelial cells, bone, trachea, spleen, thymus, lung, kidney, thyroid, and several regions in the CNS.
- the receptors expressed on the surface of the cell and targeted by the conjugates of the current invention are not restricted to the above mentioned receptors, to the receptors of this family or to the receptors expressed only in brain; they further include receptors specifically expressed in any other tissues that are targeted by amorfrutin analogues, such as fibroblast growth factor 21 (FGF21 ) that is involved in the stimulation of glucose uptake in adipocytes, but not in other cell types.
- FGF21 fibroblast growth factor 21
- a preferred embodiment of the present invention relates to the so far unknown compounds of general formula (I)
- R 1 represents the following: -H
- R 2 represents the following: -H, -OH, -OC q H(2q+1 );
- R 4 represents the following: -H, -OH, -OCH3, -CH 3 or
- R 2 together with R 3 or R 3 together with R 4 form with the two carbons of the benzene ring to which R 2 and R 3 or R 3 and R 4 are attached one of the following moieties:
- tautomer is defined as an organic compound that is interconvertible by a chemical reaction called tautomerization. Tautomerization can be catalyzed preferably by bases or acids or other suitable compounds.
- Another aspect of the present invention is directed to novel compounds selected from the following group:
- NP-01221 1 2-[(1 R,6R)-3-methyl-6-prop-1 -en-2-yl-1 -cyclohex-2-enyl]-5- pentylbenzene-1 ,3-diol
- NP-016018 2-[(1 R,6R)-3-methyl-6-prop-1 -en-2-yl-1 -cyclohex-2-enyl]-5- propylbenzene-1 ,3-diol
- NP-015938 4-(2-hydroxy-3-methylbut-3-en-1 -yl)-2-(3-methyl-2-butenyl)-5- pentylbenzene-1 ,3-diol
- the present invention is also directed to a method of treatment comprising the step of administering to a patient a pharmaceutically effective amount of a compound of general formula (I)
- R 1 represents the following: -H, -CO 2 R 15 , -CHO, -C(O)-CH
- R 2 represents the following: -H, -OH, -OC q H( 2q+ i);
- R 4 represents the following: -H, -OH, -OCH 3 , -CH 3 or
- R 15 represents one of the following -H, -CH 3 , -C 2 H 5 , -C 3 H 7 , -C 4 H 9 ;
- R 45 , R 46 , R 47 , R 48 , R 49 , R 52 and R 53 represent independently of each other -H, — CH 3 , — ⁇ 2 ⁇ ⁇ , — C 3 H 7 , ,— C 4 Hg , — OCH 3 , — OC 2 Hs, — OC 3 H 7 , — F, —CI, — CF 3 , -CHF 2 , -CH 2 F ; m, n, p, o and q are integer numbers independently of each other selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13 and 14; or conjugates, enantiomers, stereoisomeric forms, mixtures of enantiomers, diastereoisomers, mixtures of diastereoisomers, hydrates, solvates, tautomers, racemates and pharmaceutically acceptable salts of the above mentioned compounds for the modulation of PPARs and/or for the treatment of PPAR
- R 2 together with R 3 , or R 3 together with R 4 form with the two carbons of the benzene ring to which R 2 and R 3 or R 3 and R 4 are attached one of the following moieties: wherein R 7 has the meaning above defined.
- R 1 substituent represents -H.
- R 3 , R 5 and R 6 are different from hydrogen.
- R represents preferably the following substituents: -OH or -OCH 3 and most preferably -OH.
- R 6 preferably contains a phenyl group, and more preferably a substituted phenyl group.
- R 3 and R 5 do preferably not contain a phenyl group, but preferably contain a substituent with one or two double bonds.
- R 16 is selected from
- R 4 , R 5 , R 15 , R 45 , R 46 , R 47 , R 48 , R 49 , R 52 and R 53 have the meanings disclosed above and at least one of the substituents R 45 , R 46 , R 47 , R 48 and R 49 is different of hydrogen.
- R 16 is selected from
- R 4 , R 5 , R 15 , R 45 , R 46 , R 47 , R 48 , R 49 , R 52 and R 53 have the meanings disclosed above and at least one of the substituents R 45 , R 46 , R 47 , R 48 and R 49 is different of hydrogen are also preferred.
- FT 5 , R 4b , FT , R , R 4y , R ⁇ and R M represent independently of each other: — CH3, — C2H 5 , — C3H 7 , — C 4 Hg, — OCH3, — OC2H 5 , — OC3H 7 , — F, —CI, — CF3, -CHF 2 , -CH 2 F.
- R 16 represents one of the following:
- R 4b , FT, R , R 4y , R ⁇ and R M represent independently of each other: — C2H5, — C3H 7 , ,— C 4 H9, — OCH3, — OC2H 5 ,— OC3H 7 , — F, —CI, — CF3, -CH 2 F are also preferred.
- the compound according to the general formula (I) is selected from the group of compounds depicted in the following Table 1 .
- the compounds of formula (I) are isolated from the roots of Glycyrrhiza foetida and from the fruits of Amorpha fructicosa. Indications
- novel compounds according to the general formula (I) are used as pharmaceutically active agent applicable in medicine.
- the above-mentioned compounds of general formula (I) as well as the pharmaceutical compositions comprising said compounds of general formula (I) are acting as modulators of PPARs, with great specificity for PPARy and and are useful for the prevention and/or treatment of metabolic diseases.
- modulator refers to a compound that modulates the transcriptional activity of PPAR involving specific activation or repression of a subgroup of genes regulated by PPAR, thus leading to a differential expression of PPAR target genes.
- a further aspect according to the present invention is directed to compounds of general formula (I) useful for prevention and/or treatment of inflammatory diseases and cancer.
- the compounds according to the general formula (I) are for the preparation of phytomedicals or functional food products for prevention of metabolic diseases.
- the term "functional food product” refers to a food purported or proven to have a beneficial health effect.
- the term “phytomedical” is defined as a pharmaceutical composition containing compounds isolated from plants for prevention and/or treatment of various health concerns.
- the compounds of general formula (I) have hepatoprotective properties.
- the compounds of general formula (I) and/or pharmaceutical acceptable salts thereof are useful for or can be used for the prevention and/or treatment of metabolic diseases.
- Metabolic diseases refer to diseases and conditions characterized by pathological disorders of the metabolism. They are mainly characterized by enzyme defects and abnormalities in the regulating system leading to a pathological enrichment of substrates, lack of metabolic products, failure of producing energy, of regeneration of cellular constituents, of elimination of metabolic products and of maintenance of homeostasis. They can be acquired or be a genetic disease. Metabolic disorders include, but are not limited to: obesity, type I diabetes, type II diabetes, maturity- onset diabetes of youth, gestational diabetes, hypoglycemia, amyloidosis, branched chain disease, hyperaminoacidemia, hyperaminoaciduria, disturbances of the metabolism of urea, hyperammonemia, mucopolysaccharidoses e.
- Maroteaux-Lamy syndrom glycogen storage diseases and lipid storage diseases, Cori's disease, intestinal carbohydrate malabsorption, maltase-, lactase-, sucrase- insufficiency, disorders of the metabolism of fructose, disorders of the metabolism of galactose, galactosaemia, disturbances of pyruvate metabolism, hypolipidemia, hypolipoproteinemia, hyperlipidemia, hyperlipoproteinemia, carnitine or carnitine acyltransferase deficiency, porphyrias, disturbances of the purine metabolism, lysosomal diseases, metabolic diseases of nerves and nervous systems like gangliosidoses, sphingolipidoses, sulfatidoses, leucodystrophies, Lesch-Nyhan syndrome, dysfunction of the parathyroid glands, pancreatic islet cell dysfunction, carbohydrate and lipid storage myopathies, glycogenoses, myoglobinuria, alka
- the compounds of general formula (I) and/or pharmaceutical acceptable salts thereof are useful for or can be used for the prevention and/or treatment of inflammatory diseases.
- Inflammatory diseases refer to diseases involving an inflammation process. Inflammation is the final common pathway of various insults, such as infection, trauma, and allergies to the human body. It is characterized by activation of the immune system with recruitment of inflammatory cells, production of proinflammatory cells and production of pro-inflammatory cytokines. Most inflammatory diseases and disorders are characterized by abnormal accumulation of inflammatory cells including monocytes/macrophages, granulocytes, plasma cells, lymphocytes and platelets. Along with tissue endothelial cells and fibroblasts, these inflammatory cells release a complex array of lipids, growth factors, cytokines and destructive enzymes that cause local tissue damage. 5
- inflammatory disease encompasses a variety of conditions, including autoimmune diseases, proliferative skin diseases, and inflammatory dermatoses. Inflammatory disorders result in the destruction of healthy tissue by an inflammatory process, dysregulation of the immune system, and unwanted proliferation of cells.
- inflammatory diseases are acne vulgaris, acute respiratory distress syndrome, Addison's disease, allergic rhinitis, allergic intraocular inflammatory diseases, antineutrophil cytoplasmic antibody (ANCA)- associated small-vessel vasculitis, ankylosing spondylitis, arthritis, asthma, atherosclerosis, atopic dermatitis, autoimmune hepatitis, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, Bell's palsy, bullous pemphigoid, cerebral ischemia, chronic obstructive pulmonary disease cirrhosis, Cogan's syndrome, contact dermatitis, Crohn's disease, Cushing's syndrome, dermatomyositis, diabetes mellitus, discoid lupus erythematosus, eosinophilic fasciitis, erythema nodosum, exfoliative dermatitis, fibromyalgia, focal glomerulosclerosis, focal
- the cancer type is preferably selected from the group comprising or consisting of: adenocarcinoma, choroidal melanoma, acute leukemia, acoustic neurinoma, ampullary carcinoma, anal carcinoma, astrocytoma, basal cell carcinoma, pancreatic cancer, desmoid tumor, bladder cancer, bronchial carcinoma, non-small cell lung cancer (NSCLC), breast cancer, Burkitt's lymphoma, corpus cancer, CUP- syndrome (carcinoma of unknown primary), colorectal cancer, small intestine cancer, small intestinal tumors, ovarian cancer, endometrial carcinoma, ependymoma, epithelial cancer types, Ewing's tumors, gastrointestinal tumors, gastric cancer, gallbladder cancer, gall bladder carcinomas, uterine cancer, cervical cancer, cervix
- compositions comprising at least one compound of the present invention as active ingredient, together with at least one pharmaceutically acceptable carrier, excipient and/or diluents.
- the pharmaceutical composition comprises at least one compound according to claim 1 or 6.
- the pharmaceutical compositions of the present invention can be prepared in a conventional solid or liquid carrier or 7 diluent and a conventional pharmaceutically-nnade adjuvant at suitable dosage level in a known way.
- the preferred preparations are adapted for oral application.
- These administration forms include, for example, pills, tablets, film tablets, coated tablets, capsules, powders and deposits.
- the present invention also includes pharmaceutical preparations for parenteral application, including dermal, intradermal, intragastral, intracutan, intravasal, intravenous, intramuscular, intraperitoneal, intranasal, intravaginal, intrabuccal, percutan, rectal, subcutaneous, sublingual, topical, or transdermal application, which preparations in addition to typical vehicles and/or diluents contain at least one compound according to the present invention and/or a pharmaceutical acceptable salt thereof as active ingredient.
- compositions according to the present invention containing at least one compound according to the present invention, and/or a pharmaceutical acceptable salt thereof as active ingredient will typically be administered together with suitable carrier materials selected with respect to the intended form of administration, i.e. for oral administration in the form of tablets, capsules (either solid filled, semi-solid filled or liquid filled), powders for constitution, extrudates, deposits, gels, elixirs, dispersable granules, syrups, suspensions, and the like, and consistent with conventional pharmaceutical practices.
- suitable carrier materials selected with respect to the intended form of administration, i.e. for oral administration in the form of tablets, capsules (either solid filled, semi-solid filled or liquid filled), powders for constitution, extrudates, deposits, gels, elixirs, dispersable granules, syrups, suspensions, and the like, and consistent with conventional pharmaceutical practices.
- the active drug component may be combined with any oral non-toxic pharmaceutically acceptable carrier, preferably with an inert carrier like lactose, starch, sucrose, cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, talc, mannitol, ethyl alcohol (liquid filled capsules) and the like.
- suitable binders, lubricants, disintegrating agents and coloring agents may also be incorporated into the tablet or capsule.
- Powders and tablets may contain about 5 to about 95 weight % of the benzothiophene-1 ,1 -dioxide derived compound and/or the respective pharmaceutically active salt as active ingredient.
- Suitable binders include starch, gelatin, natural sugars, corn sweeteners, natural and synthetic gums such as acacia, sodium alginate, carboxymethylcellulose, polyethylene glycol and waxes.
- suitable lubricants there may be mentioned boric acid, sodium benzoate, sodium acetate, sodium chloride, and the like.
- Suitable disintegrants include starch, methylcellulose, guar gum, and the like. Sweetening and flavoring agents as well as preservatives may also be included, where appropriate. The disintegrants, diluents, lubricants, binders etc. are discussed in more detail below.
- compositions of the present invention may be formulated in sustained release form to provide the rate controlled release of any one or more of the components or active ingredients to optimise the therapeutic effect(s), e.g. antihistaminic activity and the like.
- Suitable dosage forms for sustained release include tablets having layers of varying disintegration rates or controlled release polymeric matrices impregnated with the active components and shaped in tablet form or capsules containing such impregnated or encapsulated porous polymeric matrices.
- Liquid form preparations include solutions, suspensions, and emulsions. As an example, there may be mentioned water or water/propylene glycol solutions for parenteral injections or addition of sweeteners and opacifiers for oral solutions, suspensions, and emulsions. Liquid form preparations may also include solutions for intranasal administration. Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be present in combination with a pharmaceutically acceptable carrier such as an inert, compressed gas, e.g. nitrogen.
- a pharmaceutically acceptable carrier such as an inert, compressed gas, e.g. nitrogen.
- a low melting fat or wax such as a mixture of fatty acid glycerides like cocoa butter is melted first, and the active ingredient is then dispersed homogeneously therein e.g. by stirring. The molten, homogeneous mixture is then poured into conveniently sized moulds, allowed to cool, and thereby solidified.
- transdermal compositions may have the form of a cream, a lotion, an aerosol and/or an emulsion and may be included in a transdermal patch of the matrix or reservoir type as is known in the art for this purpose.
- capsule refers to a specific container or enclosure made e.g. of methyl cellulose, polyvinyl alcohols, or denatured gelatins or starch for holding or containing compositions comprising the active ingredient(s).
- Capsules with hard shells are typically made of blended of relatively high gel strength gelatins from bones or pork skin.
- the capsule itself may contain small amounts of dyes, opaquing agents, plasticisers and/or preservatives.
- Under tablet a compressed or moulded solid dosage form is understood which comprises the active ingredients with suitable diluents.
- the tablet may be prepared by compression of mixtures or granulations obtained by wet granulation, dry granulation, or by compaction well known to a person of ordinary skill in the art.
- Oral gels refer to the active ingredients dispersed or solubilised in a hydrophilic semi-solid matrix.
- Powders for constitution refers to powder blends containing the active ingredients and suitable diluents which can be suspended e.g. in water or in juice.
- Suitable diluents are substances that usually make up the major portion of the composition or dosage form. Suitable diluents include sugars such as lactose, sucrose, mannitol, and sorbitol, starches derived from wheat, corn rice, and potato, and celluloses such as microcrystalline cellulose.
- the amount of diluent in the composition can range from about 5 to about 95 % by weight of the total composition, preferably from about 25 to about 75 weight %, and more preferably from about 30 to about 60 weight %.
- disintegrants refers to materials added to the composition to support break apart (disintegrate) and release the pharmaceutically active ingredients of a medicament.
- Suitable disintegrants include starches, "cold water soluble" modified starches such as sodium carboxymethyl starch, natural and synthetic gums such as locust bean, karaya, guar, tragacanth and agar, cellulose derivatives such as methylcellulose and sodium carboxymethylcellulose, microcrystalline celluloses, and cross-linked microcrystalline celluloses such as sodium croscaramellose, alginates such as alginic acid and sodium alginate, clays such as bentonites, and effervescent mixtures.
- the amount of disintegrant in the composition may range from about 2 to about 20 weight % of the composition, more preferably from about 5 to about 10 weight %.
- Binders are substances which bind or "glue” together powder particles and make them cohesive by forming granules, thus serving as the "adhesive" in the formulation. Binders add cohesive strength already available in the diluent or bulking agent. Suitable binders include sugars such as sucrose, starches derived from wheat corn rice and potato, natural gums such as acacia, gelatin and tragacanth, derivatives of seaweed such as alginic acid, sodium alginate and ammonium calcium alginate, cellulose materials such as methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethylcellulose, polyvinylpyrrolidone, and inorganic compounds such as magnesium aluminum silicate. The amount of binder in the composition may range from about 2 to about 20 weight % of the composition, preferably from about 3 to about 10 weight %, and more preferably from about 3 to about 6 weight %.
- Lubricants refer to a class of substances which are added to the dosage form to enable the tablet granules etc. after being compressed to release from the mould or die by reducing friction or wear.
- Suitable lubricants include metallic stearates such as magnesium stearate, calcium stearate, or potassium stearate, stearic acid, high melting point waxes, and other water soluble lubricants such as sodium chloride, sodium benzoate, sodium acetate, sodium oleate, polyethylene glycols and D,L-leucine. Lubricants are usually added at the very last step before compression, since they must be present at the surface of the granules.
- the amount of lubricant in the composition may range from about 0.2 to about 5 weight % of the composition, preferably from about 0.5 to about 2 weight %, and more preferably from about 0.3 to about 1 .5 weight % of the composition.
- Glidents are materials that prevent caking of the components of the pharmaceutical composition and improve the flow characteristics of granulate so that flow is smooth and uniform.
- Suitable glidents include silicon dioxide and talc.
- the amount of glident in the composition may range from about 0.1 to about 5 weight % of the final composition, preferably from about 0.5 to about 2 weight %.
- Coloring agents are excipients that provide coloration to the composition or the dosage form. Such excipients can include food grade dyes adsorbed onto a suitable adsorbent such as clay or aluminum oxide.
- the amount of the coloring agent may vary from about 0.1 to about 5 weight % of the composition, preferably from about 0.1 to about 1 weight %.
- the compounds of general formula I according to the present invention can be obtained through isolation from natural sources and/or chemical synthesis.
- compounds of general formula I wherein R 1 is selected from -H, -CO 2 R 15 and -CHO, R 2 is selected from -OH and -OC q H( 2 q + i), R 4 is selected from -OH and -OCH 3 , and R 3 , R 5 and R 6 have the meanings defined above can be obtained starting from ketone 3 and a malonate derivative such as 4 (see Scheme 1 ).
- Ketones of general fornnula 3 are commercially available or can be accessed by reacting hydrazone 5 obtained from the corresponding methyl ketone with commercially available brominated derivative 6.
- Malonate derivative 4 can be synthesized starting from diethylmalonate 8 and acyl chloride 7.
- acid chlorides of general formula 7 are commercially available or can be easily prepared from the corresponding carboxylic acids by methods well known to the person skilled in the art.
- compounds of general formula 10, 11 and 12 can be synthesized according to the synthetic pathway described in Scheme 2.
- hydrazone 5 is reacted with brominated derivative 6 in presence of a strong base such as lithium diisopropylamide in anhydrous tetrahydrofurane.
- a strong base such as lithium diisopropylamide in anhydrous tetrahydrofurane.
- suitable brominated derivative 6 are 1 -bromo-3-methylbut-2-ene and (2Z)-1 - bromo-3,7-dimethylocta-2,6-diene.
- acyl chloride 7 After treatment of diethyl malonate 8 with Mg turnings in absolute ethanol and a catalytic amount of carbon tetrachloride, the intermediate magnesium salt is reacted with acyl chloride 7 to provide enol 9.
- Suitable acyl chlorides 7 are hydrocinamoyl chloride, pentanoyl chloride, butanoyl chloride, propanoyl chloride, ethanoyl chloride, formyl chloride.
- Enol 9 is converted to corresponding chloride 4 by treatment with phosphoryl chloride and triethylamine.
- Subsequent treatment of chloride 4 with ketone 3 provides amorfrutine analogue 10 that can be further methylated to provide the methylated analogue 11.
- amorfrutine 12 can be decarboxylated to afford amorfrutine analogues with R 1 being -H or submitted to esterification to provide amorfrutine analogues with R 1 being -CO 2 R 15 and R 15 being different of -H, or submitted to reduction with diisobutylaluminium hydride to provide amorfrutine analogues with R 1 being -CHO.
- the conjugates of the present invention can be constructed by covalently linking a compound of general compound I (amorfrutin) with a peptide or a peptide analogue using conjugation chemistries known to the skilled person.
- the inventive compounds can be covalently linked to the peptide via the carboxylic acid, the hydroxyl group or a suitable functionality introduced on the hydrophobic side chains by derivatisation.
- a stable conjugate which is resistant to proteolytic cleavage (by cellular proteases) can be generated via ether conjugation, by using a suitable functionality introduced on the hydrophobic side chains of the compounds of general formula I, or the carboxylic acid or the hydroxyl group.
- More labile, proteolytically cleavable conjugates can be generated via ester conjugation, by using a functionality present on the hydrophobic side chains of the compounds of general formula I, or the carboxylic acid or the hydroxyl group. Using this approach, the compounds of general formula I can be targeted to specific cell types, taken up and released intracellularly.
- Peptide conjugation shall take place using standard conjugation chemistry through carboxyl groups, free amines or thiol group on cysteine, or any other suitable methods, preferable C- or N-terminally. Peptide conjugation can benefit from introducing a (bio-) chemically inert (bifunctional) linker molecule. To improve the stability of the peptide, said peptide can be modified using appropriate derivatisation to avoid degradation in a cellular or physiological context. For example, the peptide hormones GLP-1 are rapidly inactivated by the enzyme dipeptidyl peptidase-4 (DPP-4).
- DPP-4 dipeptidyl peptidase-4
- amorfrutin NP-003520 can be linked via an ester bond achieved by reacting its carboxylic acid group to a FGF21 peptide derivative to produce a conjugate NP- 003520 / FGF21 peptide derivative that induces synergistic glucose-lowering effects in adipocytes via FGF21 pathways and PPARy-regulated pathways.
- Figure 1 shows the binding of PPARy by the compounds of general formula (I):
- Figure 2 shows the partial activation of PPARs by Amorfrutin B (NP-015142): (a) Chemical structure of amorfrutin B;
- Figure 3 shows the binding of PPARa by the compounds of general formula (I):
- Figure 4 shows the binding of PPAR / ⁇ by the compounds of general formula (I):
- FIG. 8 shows the gene expression profile in primary human adipocytes after treatment with amorfrutin B (AB) and rosiglitazone (RGZ). Cells were treated for 24 h with 10 ⁇ /L RGZ (grey bars) or 10 ⁇ /L AB (black bars) and expression of (a) known PPARy target genes and (b) adipokines was analyzed by qPCR.
- AB amorfrutin B
- RGZ rosiglitazone
- Figure 9 shows NCOR1 RNA expression after treatment of primary human adipocytes with amorfrutin B (AB) or rosiglitazone (RGZ).
- AB amorfrutin B
- RGZ rosiglitazone
- Figure 10 shows the effect of the compounds on proliferation of HT-29 colon carcinoma cells, PC3 prostate cancer cells and MCF-7 breast cancer cells, treated for 3 days with 20 ⁇ / ⁇ of the indicated compounds.
- Figure 11 shows the concentration-dependent effects of the compounds on proliferation of (a) HT-29 colon carcinoma cells, (b) T84 colon carcinoma cells and (c) PC3 prostate cancer cells.
- Figure 12 shows the effects of the compound on activation of caspases 2, 3, 6, 7,
- Figure 14 shows the effects of compound NP-015934 on DNA fragmentation in
- HT-29 colon cancer cells relative to DMSO treatment.
- Cells were treated with 30 or 100 ⁇ / ⁇ NP-015934 for 2 or 4 h.
- FIG. 15 shows the effect of amorfrutin B treatment in insulin-resistent DIO mice:
- Figure 16 shows the effect of amorfrutin B treatment of plasma lipid parameters in insulin-resistant DIO mice:
- Figure 17 shows the RNA expression of Ppargda, Ppargdb, Ucp1, Ucp2,
- Adipocq and Lep in the visceral white adipose tissues of DIO mice treated for 4 weeks with rosiglitazone (grey) or amorfrutin B (black) analysed by qPCR (n 6-9 per group). Data are expressed as mean ⁇ SEM. n.s. not significant, * p ⁇ 0.05, ** p ⁇ 0.01 , *** p ⁇ 0.001 vs. vehicle.
- Figure 18 shows the RNA expression of different genes in tissues of DIO mice treated for 4 weeks with rosiglitazone (grey) or amorfrutin B (black)
- liver tissues were analysed by qPCR for expression of genes associated with (a) glycolysis and gluconeogenesis, (b) fatty acid metabolism, or (c) ketogenesis;
- FIG 19 shows the FGF21 levels in DIO mice treated for 4 weeks with vehicle
- Figure 20 shows the phosphorylation of PPARy-Ser273 in WAT of DIO mice treated for 4 weeks with vehicle control (VEH, white), rosiglitazone (RGZ, grey) or amorfrutin B (AB, black):
- VH vehicle control
- RGZ rosiglitazone
- AB amorfrutin B
- AB amorfrutin B
- MMC clastogens mitomycin C
- CP cyclophosphamide
- VH vehicle
- RNA expression of liver Fabp4 was analyzed by qPCR;
- MC3T3-E1 preosteoblasts (pointed bars) were differentiated to osteoblasts in presence of vehicle only (white, set to 1 ), rosiglitazone (grey) or amorfrutin B (black). Expression of genes involved in osteoblastogenesis was determined by qPCR.
- Calcification of differentiated MC3T3-E1 osteoblasts and preosteoblasts was measured by Alizarin Red S staining. Data are expressed as mean ⁇ SEM. n.s. not significant, * p ⁇ 0.05, ** p ⁇ 0.01 vs. vehicle.
- PRE preosteoblasts
- VEH vehicle
- RGZ rosiglitazone
- PGZ pioglitazone
- TGZ troglitazone
- AB amorfrutin B (NP-015142); A1 , amorfrutin 1 (NP-003520); A2, amorfrutin 2 (NP-003521 ); A3, amorfrutin 3 (NP-006430); A4, amorfrutin 4 (NP-009525).
- re 24 shows:
- RGZ rosiglitazone
- AB amorfrutin B.
- Figure 26 shows additive effects of compound NP-015934 and anticancer reference drugs on proliferation of HT-29 colon carcinoma cells.
- Cells were treated for 3 days with different mixtures of the indicated compounds, and proliferation was quantified by fluorometric detection of the cellular DNA content.
- IC'70 data were plotted as isobologram:
- Figure 27 shows staining of HT-29 colon carcinoma cells with annexin-V-FLUOS and propidium iodide after detection by flow cytometry.
- Cells were treated with 20 ⁇ of NP-015934 for 2 days before staining:
- Figure 30 shows opening of the mitochondrial permeability transition pore (MPTP) in HT-29 cells treated with NP-015934 or NP-015934met for 30 min.
- Cells were stained with calcein and CoCI 2 and were quantified by flow cytometry:
- Figure 31 shows the effects of NP-015934 and NP-015934met on oxygen consumption and extracellular acidification of HT-29 cells by use of phosphorescent oxygen- and pH-sensitive probes:
- AB Amorfrutin B, NP- 015142
- ALT Alanine transaminase
- DIO Diet-induced obesity
- HFD High-fat diet
- PPAR Peroxisome proliferator-activated receptor
- RGZ Rosiglitazone
- siRNA Small interfering RNA
- SPPARyM selective Peroxisome proliferator- activated receptor ⁇ modulator
- TZDs thiazolidinediones
- VEH Vehicle
- vWAT Viscerale white adipose tissue.
- rosiglitazone (Cayman, Biozol, Eching, Germany), pioglitazone, troglitazone, GW7647, GW0742, N- acetylcysteine, glutathione, 3H-1 ,2-dithiole-3-thione, a-tocopherol, ascorbic acid, carbonyl cyanide m-chlorophenyl hydrazone (Sigma Aldrich, Taufkirchen, Germany), Antimycin A (Biomol GmbH). All natural products (NPs) described in this study were provided by AnalytiCon Discovery (Potsdam, Germany) and isolated by standard procedures from natural sources. The compounds were isolated using standard chromatography procedures from natural sources (isolated microbial strains (terrestrial or marine origin) or plants (several partitions with different chemical profiles)). Identity of each isolated natural product was confirmed using LC/MS and NMR.
- Example A.2. Isolation of NP-003521 , NP-006431 , NP-015934, NP-015935, NP- 015936, NP-006430, NP-006427, NP-015933, NP-015953, NP-015954, NP- 015937, NP-015938, NP-015939 327g of roots of Giycyrrhiza foetida (provided by Friedrich Nature Discovery Gmbh; Eusmün, Germany) were extracted twice with MeOH-MTB-ether and yielded 33g raw extract.
- Rapanaea melanophloeos provided by Kenya National Academy of Science, Washington, Kenya
- MeOH-MTB-ether MeOH-MTB-ether
- Example A.6 Isolation of NP-002329 and NP-01 1855
- An undetermined fungal strain (isolated at AnalytiCon, strain No. 01458fxxx000010) was fermented in a nutrient medium containing mainly sucrose, glutamic acid, salts and Amberlite XAD 1 180 for 5 days at 30°C in a stirred vessel with 10 litres working volume.
- the lyophilized biomass was extracted twice with MeOH-Acetone and yielded 40g raw extract.
- a fungal strain (Hyalodendron sp., isolated for AnalytiCon, strain No. 05048febs006260) was fermented in a nutrient medium containing mainly corn meal and malt extract for 7 days at 21 °C in a stirred vessel with 10 litres working volume.
- NP-000420 was isolated in a purity (HPLC, ELSD-detection, and H-NMR) of >70%. Structure was elucidated by interpretation of LCMS, 1 D and 2D NMR (HSQC, HMBC, HH-COSY) data.
- An undetermined fungal strain (isolated at AnalytiCon, strain No. 02465fxxx000012) was cultivated on 1 .5 kg of a solid substrate containing mainly rice, millet and a solution of salts for 19 days at 25°C.
- the culture was extracted twice with MeOH-Acetone and yielded 70g raw extract.
- repeated chromatography stationary phase RP-8 and RP-18, mobile phase methanol-water and acetonitrile-water (buffered with ammonium formate/formic acid) natural product NP-012584 was isolated in a purity (HPLC, ELSD-detection, and H-NMR) of >70%. Structure was elucidated by interpretation of LCMS, 1 D and 2D NMR (HSQC, HMBC, HH-COSY) data.
- the culture was extracted twice with MeOH-Acetone and yielded 50g raw extract.
- repeated chromatography stationary phase RP-8 and RP-18, mobile phase methanol-water and acetonitrile-water (buffered with ammonium formate/formic acid) natural product NP-001782 and NP-001787 were isolated in a purity (HPLC, 7
- An undetermined fungal strain (isolated at AnalytiCon, strain No. 00410fxxx000005) was cultivated on 0.5 kg of a solid substrate containing mainly rice, millet and a solution of salts for 19 days at 25°C.
- the culture was extracted twice with MeOH-Acetone and yielded 10g raw extract.
- By repeated chromatography stationary phase RP-8 and RP-18, mobile phase methanol-water and acetonitrile-water (buffered with ammonium formate/formic acid) natural product NP-001269 was isolated in a purity (HPLC, ELSD-detection, and H-NMR) of >70%. Structure was elucidated by interpretation of LCMS, 1 D and 2D NMR (HSQC, HMBC, HH-COSY) data. Purity assessment of isolated compounds
- 1 H and 13 C NMR spectra were obtained on 500 and 600 MHz Varian INOVA NMR spectrometers using CDCI3 and acetone-d6 (Cambridge Isotope Laboratories) as solvents. Spectra were calibrated to the solvent, 7.26 ppm for 1 H NMR spectra in CDCI3, 2.05 ppm for 1 H NMR spectra in acetone-d6, 77.16 ppm for 13 C NMR spectra in CDCI3, and 29.8 ppm for 13 C NMR spectra in acetone-d6.
- the reaction was heated under reflux at 80 °C overnight to achieve complete dissolution of the magnesium turnings and subsequently cooled to room temperature.
- the resulting magnesium salt was concentrated under reduced pressure and then resuspended in anhydrous diethyl ether (50 ml_).
- the resulting suspension was heated under reflux at 50°C for 30 minutes and then recooied to room temperature.
- Hydrocinnamoyl chloride (10 g, 59.3 mmol, 1 equiv) was added cautiously over a period of 15 minutes at room temperature, and the reaction was stirred overnight.
- the reaction was then heated to 80 °C under reflux and stirred at this temperature for an additional 4 h.
- the reaction was then cooled to 0 °C using an ice-water bath and diluted with 200 mL anhydrous dichloromethane (DCM).
- DCM anhydrous dichloromethane
- the diluted solution was then added drop wise cautiously over a period of 15 min to an ice- cooled suspension of NaHCO3 (124 mmol) in H 2 O (600 mL).
- the pH of the aqueous mixture was adjusted to 7 using 1 M aqueous HCI.
- the organic phase was then isolated, and the aqueous layer was washed with DCM (2 x 100 mL). The organic collections were combined, dried over Na 2 SO 4 and concentrated under reduced pressure.
- the mixture was then concentrated under reduced pressure 5 and the concentrate was dissolved in a 1 :1 :1 mixture (v/v/v) of ice water, hexanes and diethyl ether.
- the solution was neutralized with 1 M aqueous HCI.
- the organic phase was isolated and the aqueous phase was washed with a 1 :1 mixture of hexanes and diethyl ether (2 x 50 mL).
- the collected organic phases were combined, dried over a 2 S0 and concentrated under reduced pressure.
- the chilled solution was transferred to a 1 L Erlenmeyer flask containing 1 M aqueous HCI (265 mL), hexanes (200 mL), and diethyl ether (200 mL), being vigorously stirred at 0°C.
- Binding of natural products to PPARs was quantified by use of a competitive time- resolved fluorescence resonance energy transfer (TR-FRET) assay according to the manufacturer's protocol (Lanthascreen PPAR competitive binding assay, Life Technologies, CA, USA) as described recently (Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 7257). Briefly, terbium-tagged PPAR ligand binding domain was titrated with varying ligand concentrations in the presence of constant concentrations of the fluorescein-labelled PPAR ligand fluormone. Increasing the concentration of unknown PPAR ligands results in a displacement of the labelled PPAR ligand and hence in a decrease of the TR-FRET signal.
- TR-FRET time- resolved fluorescence resonance energy transfer
- Binding of transcriptional cofactors was measured by a peptide-based TR-FRET assay according to the manufacturer's instruction (Lanthascreen PPARy coactivator assay, Life Technologies). Efficacy is the maximal association (for coactivators) or dissociation (for corepressors) normalized to the full PPARy agonist rosiglitazone (set to 100%). Transcriptional activation of PPARs was assessed in cellular reporter gene assays according to the manufacturer's protocols (GeneBLAzer PPAR Assay, Life Technologies). Briefly, HEK 293 cells were stably expressing a GAL4-PPAR-LBD fusion protein and an UAS-beta- lactamase reporter gene.
- preadipocytes Primary subcutaneous preadipocytes isolated from human patients were provided by Zen-Bio (BioCat, Heidelberg, Germany) and treated as described previously. [Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 7257] Briefly, preadipocytes were maintained in preadipocyte medium (PM-1 , Zen-Bio) and then differentiated using PPARy agonist-free adipocyte medium (AM-1 , Zen-Bio) supplemented with 500 ⁇ /L 3-isobutyl-1 -methylxanthine (Sigma Aldrich, Taufkirchen, Germany) for 7 days. Subsequently, medium was changed to pure AM-1 for additional 7 days. Mature adipocytes were treated with 10 ⁇ /L amorfrutin B or 10 ⁇ /L rosiglitazone diluted in AM-1 for 24 hours, whereas 0.1 % DMSO was used as vehicle control.
- Mouse MC3T3-E1 preosteoblast cells (subclone 4, CRL-2593, ATCC, LGC Promochem, Wesel, Germany) were cultured in Minimum Essential Medium a medium (MEMa, A1049001 , Gibco, Life Technologies) with 10% fetal bovine serum (FBS, Biochrom, Berlin, Germany) and 1 % penicillin/streptomycin (Biochrom) at 37 °C and 5% CO2.
- MEMa Minimum Essential Medium a medium
- FBS fetal bovine serum
- Biochrom penicillin/streptomycin
- One-day post-confluent cells were differentiated to osteoblasts in MEMa additionally supplemented with 200 ⁇ /L ascorbic acid and 10 mmol/L ⁇ -glycerophosphate (all Sigma-Aldrich).
- the preosteoblasts were treated with amorfrutin B (10 ⁇ ), amorfrutins 1 -4 (each 10 ⁇ ), rosiglitazone (10 ⁇ ), pioglitazone (10 ⁇ ), troglitazone (10 ⁇ ) or vehicle (0.1 % DMSO) during the whole differentiation.
- the cells were collected after 7 days for gene expression analyses and after 24 days for staining. Calcification was determined by staining of osteoblasts with Alizarin red S (Sigma) according to [Anal. Biochem. 2004, 329, 77].
- Viability and cytotoxicity were assessed in human HepG2 cells (ATCC) cultured in DMEM (Gibco, Life Technologies) supplemented with 10% FBS treated with amorfrutin B for 24 h using the CellTiter-Glo Luminescent Cell Viability Assay and the CytoTox-Glo Cytotoxicity Assay (both Promega, Mannheim, Germany) respectively, according to the manufacturer's protocols.
- In vitro micronucleus assays were performed in CHO-K1 cells at Cerep, Inc. (Redmond, WA, USA) according to Mutat. Res. 2007 630, 1 .
- HT-29 and T84 cells were cultured in DMEM/F-12 (ATCC) supplemented with 5% FBS (Biochrom) and 1 % penicillin/streptomycin (Biochrom), PC3 cells were cultivated in RPMI 1640 (Biochrom) supplemented with 10% FBS (Biochrom) and 1 % penicillin/streptomycin (Biochrom), MCF-7 cells were cultured in DMEM GlutaMAX (Gibco, Life Technologies) supplemented with 10% FBS (Biochrom), 10 g/ml human insulin (Sigma Aldrich) and 1 % penicillin/streptomycin (Biochrom), all at 37 °C and 5% CO2.
- Additive effects were determined by treatment with compound mixtures with following ratios: 7:0, 6:1 , 5:2, 4:3, 3:4, 2:5, 1 :6, 0:7.
- HT-29 cells were treated with different concentration series of these compound mixtures.
- compound IC'70 values which gives the concentration needed to inhibit cancer cell growth by 70%, were calculated and plotted as isobologram according the Loewe additivity model (Klin Klischr. 1927, 6, 1077).
- Phosphatidylserine external ization of HT-29 cells treated with NP-015934 was determined by staining with annexin-V-FLUOS and propidium iodide (Roche Life Science) and subsequent flow cytometry (Accuri C6, BD Biosciences) according to manufacturer's instructions. Analysis was performed using FlowJo 7.6 (Tree Star).
- ROS reactive oxygen species
- the JC-1 assay (Cayman Chemicals) was performed according to the manual. This assay makes use of a lipophilic cationic dye (5,5',6,6'-Tetrachloro-1 ,1 ',3,3'- tetraethylbenzimidazolylcarbocyanine iodide), which selectively enters into mitochondria and changes reversibly its color from red to green as the membrane potential decreases.
- HT-29 cells were seeded in 96 well plates (TPP) with a density of 40000 cells/well.
- the MitoProbe Transition Pore Assay Kit (Life Technologies) was used according to the manufacturer's instructions.
- Cells were loaded with a calcein dye that accumulates in cytosolic compartments, including the mitochondria.
- the fluorescence of cytosolic calcein was quenched by addition of C0CI2, while mitochondrial fluorescence is maintained. Opening of the MPTP leads to loss of mitochondrial calcein fluorescence.
- HT-29 cells were suspended in HBSS/Ca buffer with a density of 10 6 cells/ml, and labeled with 10 nM calcein and 400 ⁇ CoCI 2 .
- NP-015934 or NP-015934met was added as indicated.
- Cells were incubated at 37 °C for 30 min and subsequently washed with HBSS/Ca buffer before counting by flow cytometry (Accuri C6, BD Biosciences) according to manufacturer's instructions. Analysis was performed using FlowJo 7.6 (Tree Star) and Prism 5.0 (GraphPad).
- Oxygen consumption was determined by time-resolved fluorescence of an oxygen-sensitive probe (MitoXpress-Xtra HS, Luxcel Biosciences). Probe fluorescence is quenched by molecular oxygen, so that fluorescence lifetime increases with reduction in extracellular oxygen concentration.
- HT-29 cells were seeded in 96 well plates (TPP) with a density of 80000 cells/well in DMEM/F-12 supplemented with 5% FBS and 1 % penicillin/streptomycin and incubated at 37 °C and 5% CO2.
- the medium was removed and cells were incubated with 140 ⁇ of pre-warmed probe diluted in phenol red-free DMEM/F- 12/FBS/penicillin/streptomycin. After incubation for 10 min at 37 °C, cells were then treated with the indicated compound concentrations by adding 20 ⁇ _ of a 8 times stock concentration. Finally, cells were sealed with 100 ⁇ of pre-warmed HS mineral oil (MitoXpress-Xtra HS) to prevent back diffusion of ambient oxygen.
- pre-warmed HS mineral oil Mitsubishi Xpress-Xtra HS
- Extracellular acidification was determined by time-resolved fluorescence of a pH- sensitive probe (pH Xtra, Luxcel Biosciences). Fluorescence lifetime of this probe increases with decrease in pH, so that it allows measurement of extracellular acidification.
- HT-29 cells were seeded in 96 well plates (TPP) with a density of 80000 cells/well in DMEM/F-12 supplemented with 5% FBS and 1 % penicillin/streptomycin and incubated at 37 °C in a CO 2 -free incubator.
- the following low-buffering aspiration medium was used according to the manufacturer's instructions: 1 mM PBS (pH 7.4), 20 mM glucose, 75 mM NaCI, 54 mM KCI, 2.4 mM CaCI 2 and 0.8 mM MgSO 4 .
- cells were washed twice with 200 ⁇ aspiration buffer, and incubated with 140 ⁇ of pre-warmed probe diluted in aspiration medium. After incubation for 10 min at 37 °C, cells were then treated with the indicated compound concentrations by adding 20 ⁇ _ of a 8 times stock concentration.
- PPARy modulation was investigated in siRNA-mediated PPARy- knockdown in adipocytes with subsequent real-time PCR detection as described in Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 7257. Briefly, differentiated human adipocytes were seeded in 24-well-plates (Nunc) at a confluence of 30 to 60%. Cells were transfected with 10 nmol/L PPARy Silencer Select Validated siRNA (ID S10888) or 10 nmol/L Silencer Select Negative Control #1 siRNA (all Life Technologies) using DeliverX Plus siRNA Transfection Kit (Panomics, BioCat).
- Transfection was carried out in serum- and antibiotic-free AM-1 medium (AM-1 - PRF-SF, Zen-Bio) for 4 h and continued for 3 days in standard AM-1 medium. Afterwards, cells were additionally treated with 10 ⁇ /L amorfrutin B, 10 ⁇ /L RGZ or vehicle control for 24 hours prior to RNA collection.
- RNA purification RNA purification and cDNA synthesis and quantitative real-time PCR (qPCR)
- Quantitative PCR was carried out on the ABI Prism 7900HT Sequence Detection System using the SYBR Green PCR Master Mix (all Life Technologies). After an initial denaturation at 95 °C for 10 min, the cDNA was amplified by 40 cycles of PCR (95 °C, 15 s; 60 °C, 60 s). The relative gene expression levels were normalized using ⁇ -actin gene and quantified by the 2 AACt method [Method. Methods San Diego, Calif, 2001 , 25, 402]. Primer sequences are summarized in Table 4.
- Activation of caspases 3 and 7 were investigated using the luminometric Caspase- Glo 3/7 Assay (Promega) according to the manufacturer's instructions. Briefly, one day before treatment HT-29 and MCF-7 cells were seeded in black 384 well plates (Corning, #3712) with a density of 2000 cells/well in a final volume of 20 L/well. Cells were treated for the indicated time with the given compound concentrations by adding 5 ⁇ of a 5 times stock concentration. Luminescence was measured with the POLARstar Omega (BMG LABTECH).
- Activation of caspases 2, 3, 6, 7, 8, 9 and 10 were investigated using the fluorimetric Homogeneous Caspases Assay, (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. Briefly, one day before treatment HT-29 and PC-3 cells were seeded in black 384 well plates (Corning, #3712) with a density of 1300 and 1000 cells/well, respectively, in a final volume of 20 L/well. Cells were treated for the indicated time with the given compound concentrations by adding 5 ⁇ of a 5 times stock concentration. Fluorescence was measured with the POLARstar Omega (BMG LABTECH).
- HT-29 cells were seeded in 96 well plates (TPP) with a density of 13000 cells/well in a final volume of 200 L/well with 10 ⁇ /L BrdU and incubated for 2 days at 37 °C. Supernatant was removed and cells were incubated in 100 ⁇ _ medium containing the compound at given concentrations. After the indicated treatment time, the cytosolic fractions were harvested and analysed on a 96 well half-area clear high-binding microplate (Corning, #3690) according to the manual. Absorbance was measured at 450 and 690 nm. Cell-free samples were used as background control for subtraction.
- RGZ rosiglitazone
- AB amorfrutin B
- IPIST intraperitoneal insulin sensitivity test
- OGTT oral glucose tolerance test
- mice After 27 days of dosing, fasted mice were sacrificed by cervical dislocation. Hematocrit was measured by weighting of blood samples before and after plasma separation. Plasma and tissues were collected and stored at -80 °C before use. Metabolic parameters measurements
- Plasma glucose was analysed in a Hemocue B-Glucose analyzer. Plasma glucose was measured using the Amplex Red Glucose Assay Kit (Life Technologies). Plasma triacylglycerols, NEFA, HDL and LDL cholesterol and plasma alanine transaminase (ALT) were determined with colorimetric quantification kits (Biovision, BioCat). Plasma ⁇ -hydroxybutyrate was analyzed colorimetrically (Cayman, Biomol, Hamburg, Germany).
- Insulin Insulin Ultrasensitive EIA, ALPCO, Immundiagnostik, Bensheim, Germany
- triiodothyronine T3
- thyroxin T4
- osteocalcin BGLAP bone gamma- carboxyglutamate (gla) protein
- ABIN415574 antibodies-online, Aachen, Germany
- FGF21 BioVendor, Heidelberg, Germany
- Visceral white adipose tissue of treated mice was lysed in UEES lysis buffer (9 M Urea, 100 mM EDTA EGTA, 4% SDS with protease and phosphatase inhibitors) using 5 mm steel beads at 20 Hz for 8 min (TissueLyser). After centrifugation for 10 min at 10000 g, the supernatants were stored at -80 °C until use. Samples were denatured and separated using a NuPAGE Novex 4-12% Bis-Tris gel (Invitrogen) and blotted onto nitrocellulose membranes.
- Membrane was blocked with a solution containing 1 .5% milk powder, 1 .5% BSA in PBS-T (0.1 %) and 0.5x phosphatase inhibitor overnight at 4 °C. Membranes were washed in PBS-T (0.1 %).
- a rabbit polyclonal phospho-specific antibody against PPARy Ser 273 was produced by Eurogentech (Seraing, Belgium) with the phosphopeptide Ac- KTTDKpSPFVIYDC-amide [Nature 2010, 466, 451].
- PPARy-pSer273 and 0.5 pg/mL PPARy (E-8, Santa Cruz, Heidelberg, Germany) antibody were diluted in PBS-T (0.1 %) with 1 .5% milk powder and 1 .5% BSA.
- Membranes were shaken overnight at 4 °C and subsequently incubated with anti-rabbit IgG-HRP (Santa Cruz, #sc-2004) and anti-mouse IgG- HRP (Santa Cruz, #sc-2005), respectively, prior to detection with Western Lightning ECL solution (Perkin Elmer).
- Membranes were stripped with Restore Plus Western Blot Stripping Buffer (Thermo Scientific) for 10 min. Densitometry was performed with GelAnalyzer 2010. The rate of PPARy phosphorylation was normalized to total PPARy protein and plotted on logarithmic scale.
- ANCOVA works by plotting individual data for two phenotypes (e.g. HOMA-IR index vs. body weight) and fitting them with a linear regression curve. Comparison in that regression line of two different treatments (e.g. treated vs. untreated) allow for effectively testing if both phenotypes are strongly dependent on each other (same slope and interception of regression lines) or if both phenotypes are fully or partly independent from each other (different slope and/or interception of regression lines).
- Table 4 Primer sequences used for quantitative real-time PCR.
- ANGPTL2 23452 GGGAGACGTACAAGCAAGGG CGGAAACTGGCGTATTCTGC
- ANGPTL4 51 129 GATCCCCACGGCGAGTTC CCGTGATGCTATGCACCTTCT
- CD36 948 GTTGATTTGTGAATAAGAACCAGAGC TGTTAAGCACCTGTTTCTTGCAA
- CEBPA 1050 CTAACTCCCCCATGGAGTCGG GTCGATGGACGTCTCGTGC
- CEBPB 1051 ACTTTAG CG AGTCAG AG CCG GATTTAAAGGCAGGCGGCG
- FGF21 26291 TGGATCGCTCCACTTTGACC GGGCTTCGGACTGGTAAACA
- HSD1 1 B1 3290 GGCCTCATAGACACAGAAACAGC TGATCTCCAGGGCACATTCC
- PCK2 5106 CTGAGGAGGAGAATGGGCG AG AG CCAACC AG CAGTTGTCA
- PDE3B 5140 TCGAGACATTCCTTATCACAATCG GGAACTGGCCGTGTTGTCA
- PDK4 5166 CTGGACTTTGGTTCAGAAAATGC CCTTCAGAATGTTGGCGAGTCT
- PPARG 5468 CATGGCAATTGAATGTCGTGTC CCGGAAGAAACCCTTGCAT
- RARRES2 5919 CAGGCCCAATGGGAGGAAAC GGCCCAGAACTTTGTCCTCA
- Acoxl 1 1430 CAGCACTGGTCTCCGTCATG CTCCGGACTACCATCCAAGATG
- Ccl5 20304 CTCACTGCAGCCGCCCTCTG CCG AG CCATATG GTG AGG CAGG
- Emr1 13733 ACCCTCCAGCACATCCAGCCAA TC AC AG CCCG AGG GTGTCCA
- Fbp1 14121 GCATCGCACAGCTCTATGGT ACAGGTAGCGTAGGACGACT
- Fgf21 56636 AGACAGCCTTAGTGTCTTCTCA CCAAGGCAGCTGGAATTGTG
- G6pc 14377 GCTGGAGTCTTGTCAGGCAT ATCCAAGCGCGAAACCAAAC
- G6pc3 68401 TATGGGTTGACTGCTCTGGC CCAGGTTGATGGACCAGGAAA
- Hmgcl 15356 TGTACCCACCCCAGTGAAGA GAGTGGTCAGCCATCTGTGG
- Taldol 21351 CAACGAAGACCAAATGGCCG CATTCGTTCCGTGAGCATCC
- Ucp1 22227 CACGGGGACCTACAATGCTT TAGGGGTCGTCCCTTTCCAA
- Example C.1 Binding affinity assays.
- NP-001269 913690-90-1 F n.d. n.d. n.d. n.d. n.d. > 100 n.d. n.d. n.d. n.d.
- NP-001782 Altenusin 31 186-12-6 F n.d. 60 n.d. n.d. 15 3.2 n.d. n.d. n.d. n.d. n.d.
- NP-001787 Alternarian acid 91868-93-8 F n.d. > 100 n.d. n.d. > 100 > 100 n.d. n.d. n.d. n.d.
- NP-004978 none P Myrsine capitellata 11 n.d. n.d. 6.7 3.2 n.d. n.d. n.d. n.d. n.d.
- NP-01221 1 Cannabidiol 13956-29-1 P Cannabis sativa n.d. n.d. n.d. n.d. 4.7 n.d. n.d. n.d. n.d. n.d.
- NP-016018 Cannabidivarin 24274-48-4 P Cannabis sativa n.d. n.d. n.d. n.d. 6.0 n.d. n.d. n.d. n.d. n.d.
- NP-016020 25555-57-1 P Cannabis sativa 7.0 1.4 24 2.7 0.280 0.093 12 n.d. n.d.
- Binding and activation of PPARs by amorfrutin B (NP-015142): Binding affinity (Ki) values were obtained by using competitive TR-FRET assays, effective concentrations (EC50) and efficacy values were determined from reporter gene assays. Efficacy is the maximum activation relative to the reference agonist, n.d., not determined.
- Example C.2 Transcriptional activation assays.
- amorfrutin B (NP-015142) only partially induced recruitment of important transcriptional cofactors including CBP, PGC1 a, TRAP220/DRIP and PRIP/RAP250 to PPARy (see Figure 2e-i, Table 7 and Figure 7).
- amorfrutin B (NP-015142) reduced binding of the corepressor NCoR with IC50 value similar to rosiglitazone (AB, 60 nmol/L vs. RGZ, 23 nM/L), but with lower maximal dissociation efficacy (61 % vs. RGZ, see Figure 2h, Table 7). This was similar for other compounds of this invention (see Figure 7 and Table 5).
- Table 7 Cofactor recruitment profile of amorfrutin B (NP-015142) bound to PPARy.
- Example C.3 PPARy activation assay in primary human adipocytes.
- Amorfrutin B (NP-015142) induced expression of adipogenesis-related genes such as CCAAT/enhancer binding protein a and ⁇ (CEBPA and CEBPB) and the fatty acid binding protein 4 (FABP4) much less strongly than RGZ (see Figure 8a) in human primary adipocytes. These results indicate alleviated adipocyte differentiation. In contrast to RGZ, AB further showed reduced RNA expression of the Cortisol generating hydroxysteroid (1 1 -beta) dehydrogenase 1 (HSD11B1), which is linked to central obesity [Science 2001 , 294, 2166].
- HSD11B1 Cortisol generating hydroxysteroid (1 1 -beta) dehydrogenase 1
- AB treatment led to decreased transcription of the pyruvate dehydrogenase kinase 4 (PDK4), a glycerogenesis-activating enzyme that is linked to excess lipid storage in adipocyte [Diabetes 2008, 57, 2272].
- PDK4 pyruvate dehydrogenase kinase 4
- PDE3B phosphodiesterase 3B
- Example C.4 Antiproliferative and apoptotic effects in cancer cells
- HT-29 colon carcinoma cells, PC3 prostate cancer cells and MCF-7 breast cancer cells Treatment of HT-29 colon carcinoma cells, PC3 prostate cancer cells and MCF-7 breast cancer cells with 20 ⁇ /L of amorfrutins for 3 days clearly showed antiproliferative effects, especially for NP-01221 1 , NP-016018, NP-015135, NP- 015142, NP-015933, NP-015934, NP-015935, NP-015936, NP-015937 and NP- 015939 (see Figure 10).
- IC 5 o inhibitory concentrations ranging from 8.1 pmol/L (NP-015934, HT-29 cells) to 57.3 pmol/L (NP-015135, PC3 cells) and with efficacies of up to 100% cancer cell death induction (see Figure 11 and Table 8).
- HT-29 colon carcinoma cells were further treated with different mixtures of compound NP-015934 and cisplatin or irinotecan, which are commonly used in treatment of various cancer diseases.
- Combinative treatment showed additive effects on proliferation inhibition, with a combination index (CI) of approx. 1 (see Figure 26).
- NP-015934-treated cells showed striking increase in oxygen consumption, similar to the known mitochondrial uncoupler CCCP (see Figure 31 a,b), probably as consequence of disturbed ATP production without inhibiting the electron transport chain.
- treatment with NP-015934met did not show any effects (see Figure 31 a,b).
- Example B.5 Pharmacokinetic profile of amorfrutin B (NP-015142) in C57BL/6 mice.
- amorfrutin B C57BL/6 mice were orally challenged once with a loading dose of 100 mg/kg amorfrutin B (see Figure 15a, Table 9).
- Administration of amorfrutin B led to a fast plasma concentration peak, indicating a high bioavailability of that natural product.
- Amorfrutin B was almost completely eliminated after 24 h of dosage with an elimination half-life of about 2 h (see Table 9).
- Amorfrutin B and amorfrutin A1 showed similar pharmacokinetic properties (see Table 9).
- Table 9 Pharmacokinetics of amorfrutin B (NP-015142) and amorfrutin 1 (NP- 003520) after single oral administration in male C57BL/6 mice.
- Amorfrutin B has strong antidiabetic effects and considerably improves dyslipidemia in diet-induced obesity mice.
- HFD-fed C57BL/6 mice were treated for 4 weeks with 100 mg/kg/d amorfrutin B, 4 mg/kg/d rosiglitazone or vehicle only.
- amorfrutin B-treated mice had strikingly reduced concentrations of fasting blood glucose (see Figure 15b) and fasting plasma insulin (see Figure 15c) equally to or even better than RGZ-treated mice.
- Amorfrutin B and RGZ-treated mice both showed equal reduction of insulin resistance as determined by homeostatic modelling (see Figure 15d).
- OGTT oral glucose tolerance tests
- IPIST intraperitoneal insulin sensitivity tests
- Amorfrutin B strongly decreased concentrations of triacylglycerols and NEFA in plasma of HFD-fed mice comparable to RGZ (see Figure 16). After 4 weeks of treatment, amorfrutin B and RGZ both reduced fasting plasma levels of triacylglycerols by 25% (see Figure 16a). Fasting concentrations of deleterious plasma NEFA were decreased by 29% with amorfrutin B and 40% with RGZ treatment (see Figure 16b).
- AB significantly increased expression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha and beta (2-fold each, see Figure 17), indicating improved mitochondrial biogenesis and fatty acid breakdown.
- AB and RGZ equally increased expression of adiponectin (Adipoq) and uncoupling protein 3 (Ucp3, approx. 2-fold each), but only RGZ clearly boosted Ucp1 expression (20-fold vs. 1 .6-fold, see Figure 17).
- liver in contrast to RGZ, AB only partly regulated gluconeogenic genes (see Figure 18).
- FGF21 peptide hormone fibroblast growth factor 21
- Example B.6 Investigation of the side-effects associated with TZDs treatment on the compounds of the present invention.
- amorfrutin B Potential cancerogenity of amorfrutin B was evaluated using a cellular micronudeus assay. In this assay, formation of micronudei during cell division of Chinese hamster ovary (CHO) cells is observed microscopically after treatment with potential mutagens. Amorfrutin B was tested up to a concentration of 32 ⁇ /L and showed no genetic toxicity either in the presence (+S9) or absence (-S9) of metabolic activation by rat liver homogenate extracts ( Figure 22a). Noteworthy, amorfrutin B significantly reduced the basal formation of micronudei in the presence of S9 extract in a concentration-dependent manner, thus -,
- livers of RGZ-treated HFD- fed obese mice showed strongly increased expression of inflammation markers indicating macrophage infiltration and potential local inflammation.
- AB treatment led to reduced gene expression of these markers (see Figure 22b), suggesting anti-inflammatory effects of AB-treatment in the liver of HFD-fed mice.
- RGZ treatment increased Fabp4 expression by a factor of 31 , indicating increased lipid storage in the mouse liver, whereas AB did not show any increase in Fabp4 expression (see Figure 22c).
- RGZ elevated plasma alanine transaminase (ALT) levels compared to untreated HFD-fed mice, indicating liver toxicity.
- amorfrutin B treatment led to reduction of plasma ALT levels (see Figure 22d), proving liver- protective effects of the compounds of the present invention.
- thiazolidinediones Another well-known side-effect of thiazolidinediones is the impairment of osteoblastogenesis leading to osteoporosis and increased fracture risk.
- Treatment of murine MC3T3-E1 preosteoblasts with rosiglitazone or related TZDs resulted in reduced expression of genes involved in osteoblastogenesis such as phosphate regulating gene with homologies to endopeptidases on the X chromosome (Phex), integrin binding sialoprotein ⁇ Ibsp) and osteocalcin ⁇ Bglap) (see Figure 22e).
- RGZ- treatment led to impaired calcification of bone cells in vitro as assessed by Alizarin red S staining (see Figure 22f).
- Adverse body weight gain is a frequent side effect of PPARy activation due to increased fat storage in white adipose tissue.
- amorfrutin B treatment did not lead to increased adiposity, but instead to a beneficial reduction of body weight gain by approximately 15% compared to HFD- fed mice treated with vehicle control (see Figure 22h). Given the inconspicuous results from the various described assays, this observation cannot be explained by potential compound toxicity.
- RGZ has recently been reported to decrease HDL cholesterol [Diabet. Med. 2007, 24, 94]. After 4 weeks of treatment, RGZ-treated DIO mice showed a reduction in plasma HDL cholesterol by 24%, whereas amorfrutin B neither changed plasma HDL nor LDLA/LDL levels (see Figure 25a).
- RGZ administration is further associated with the development of hemodilution and edema as a result of fluid retention.
- Amorfrutin B treatment did not impair hematocrit (see Figure 25b) or levels of whole blood haemoglobin (see Figure 25c) in DIO mice.
Abstract
The present invention relates to Amorfrutin analogues and stereoisomeric forms, solvates, hydrates, conjugates and/or pharmaceutically acceptable salts of these compounds as well as pharmaceutical compositions containing at least one of these Amorfrutin analogues together with pharmaceutically acceptable carrier, excipient and/or diluents. Said Amorfrutin analogues have been identified as modulators of the peroxisome proliferator-activated receptors (PPARs), especially PPARϒ and are useful for the prevention and treatment of metabolic diseases, inflammatory diseases, cancer and preparation of phytomedicals and/or functional food products for prevention of metabolic diseases.
Description
AMORFRUTIN ANALOGS AS PPARGAMMA-MODULATORS
Specification The present invention relates to Amorfrutin analogues and stereoisomeric forms, solvates, hydrates, conjugates and/or pharmaceutically acceptable salts of these compounds as well as pharmaceutical compositions containing at least one of these Amorfrutin analogues together with pharmaceutically acceptable carrier, excipient and/or diluents. Said Amorfrutin analogues have been identified as modulators of the peroxisome proliferator-activated receptors (PPARs), especially PPARy and are useful for the prevention and treatment of metabolic diseases, inflammatory diseases, cancer and preparation of phytomedicals and/or functional food products for prevention of metabolic diseases. Background of the invention
The peroxisome proliferator-activated receptor gamma (PPARy) is a nuclear receptor that regulates transcription with two effector binding sites called activation function 1 (AF1 ) and activation function 2 (AF2). AF1 is localized within the N- terminal regulatory domain. The receptor's central DNA binding domain is followed by the C-terminal ligand binding domain (LBD), which comprises AF2. PPARy is regulated by a phosphorylation site in the LBD at Ser273. The ligand-activated transcription factor PPARy acts in the nucleus as a heterodimer with the retinoid X receptor RXR. PPARy interacts with prostaglandins and fatty acids and their metabolites. It acts as a sensor and regulator with a dominant role in glucose and lipid metabolism and adipose cell differentiation. Activation of the receptor improves insulin sensitivity through different metabolic actions, including regulation of adipokines. PPARy is a well-established drug target for type II diabetes. To reduce blood glucose levels recent pharmaceutical developments aimed to activate PPARy in peripheral metabolic target tissues such as adipose tissue, muscle, liver and macrophages. Recent publications indicate potential roles of PPARy - expressed in the central nervous system - in the regulation of weight balance {Nature Med. 2011 , 5, 618; Nature Med. 2011 , 5, 623.). This nuclear receptor further plays also a key role in inflammatory diseases and cancer (Nature Rev. Cancer 2012, 12, 181 ).
The two other PPAR family members, PPARa and PPAR /δ, also bind fatty acids and are involved in fatty acid metabolism. In general, PPARa and PPAR /δ promote fatty acid catabolism in several tissues, whereas PPAR y regulates fatty
acid storage in adipose tissues. Dual PPARa and PPAR /δ agonists correcting glucose and lipid abnormalities in patients with type II diabetes have been already reported [Biochim. Biophys. Acta 2011 , 1812, 1007; Structure 2001 , 9, 699]. The LBD of PPARy has several regulatory functions. It determines the receptor's subcellular localization, initiates heterodimerization with RXR, and activates or represses transcription of target genes in a ligand-dependent manner. The ligand binding stabilizes the LBD and leads to a more compact and rigid conformation, which in turn causes recruitment of cell-specific coactivators like SRC1 to the LBD's AF2 effector binding site. PPARy bound to the promoter of a target gene activates transcription of that target gene upon coactivator recruitment. The synthetic agonist rosiglitazone induces coactivator recruitment and inhibits NCoR co-repressor-mediated CDK5-dependent phosphorylation of Ser273, which alters the expression of a subset of genes with regulatory functions in metabolism.
Rosiglitazone and other glitazones (thiazolidinediones, TZDs) strongly activate transcription of a large number of genes in various tissues. This unspecific action of glitazones is associated with severe side effects including weight gain, osteoporosis, cardiovascular complications, and edema and determined the withdrawn of these products from the market or the restriction of their prescription. Partial PPARy agonists, which activate PPARy only weakly, are more selective PPARy modulators (SPPARyMs) and avoid side effects. [Structure 2007, 15, 1258] Dependent on the cellular context and transcriptional PPARy co-factors, partial PPARy agonists may activate or potentially repress transcription of target genes. The partial agonists BVT.13, MRL-24, nTZDpa and amorfrutin 1 block NCoR recruitment and Ser273 phosphorylation as effectively as rosiglitazone, but activate transcription of target genes only to a moderate level. [Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 7257; Nature 2010, 466, 451 .] Amorfrutins are a group of natural products that have recently been identified as PPARy modulators with the characteristics of SPPARyMs. They are binding the PPARy, having dissociation constants of around 300 nM.
It is the object of the present invention to provide amorfrutin analogues of general formula I, conjugates and pharmaceutically acceptable salts thereof suitable to selectively modulate the PPARy with greatly increased specific activity with regard to the prior art and with less side effects.
A further aspect of the invention is to provide amorfrutin analogues of general fornnula I, conjugates and pharnnaceutically acceptable salts thereof, which can be used as pharnnaceutically active agents, especially for the treatment of metabolic diseases, as well as compositions comprising at least one of those compounds and/or pharmaceutically acceptable salts thereof as pharmaceutically active ingredients. The compounds of the present invention can be used also as prophylactic dietary supplements. The objective of the present invention is solved by the teaching of the independent claims. Further advantageous features, aspects and details of the invention are evident from the dependent claims, the description, the figures, and the examples of the present application.
Description of the invention
Thus, the present invention relates to a compound of general formula (I)
(I)
wherein
R1 represents the following: -H, -CO2R15, -CHO, -C(O)-CH=CH-Ph;
R2 represents the following: -H, -OH, -OCqH(2q+i );
R4 represents the following: -H, -OH, -OCH3, -CH3 or
R2 together with R3 or R3 together with R4 form with the two carbons of the benzene ring to which R2 and R3 or R3 and R4 are attached one of the following moieties:
R3, R5, R6 and R7 represent independently of each other: -R8, -R9, -R10, -R16 -H, -OH, _(CH2)n-R8, -(CH2)m-R9, -(CH2)P-R10, -(CH2)0-R16 -CH=CH-R8, -CH=CH-R9, -CH=CH-R10, -CH=CH-R16, -(CH2)n-CHR8R9 -(CH2)m-CHR17R18, -(CH2)p-CHR19R20, -(CH2)0-CHR21R22, -OCH3, -OC2H5 -CH=CH-CH(CH3)2, -CH2-CH=CR8R9, -CH2-CH=CR17R18 :
-CH2-CH=CR19R20, -CH2-CH=CR21R22 -CH2-CH(R1 1)-C(CH3)=CH2;
-CH2-CH(R23)-C(CH3)=CH2j -CH2-CH(R24)-C(CH3)=CH2; -CH2-CH(R25)-C(CH3)=CH2j -CH2-CH(OH)-R8, -CH2-CH(OH)-R9
-CH2-CH(OH)-R10, -CH2-CH(OH)-R16 : -CH2-CH=C(R8)-CH2-CH2-CH=CR9R10, -CR8=CH2) -CR9=CH2:
-CR10=CH2) -CR16=CH2j -CH2-CH=C(R26)-CH2-CH2-CH=CR27R28 :
-CH2-CH=C(R29)-CH2-CH2-CH=CR30R31, -CH2-CH2-CHR50R51, -CH2-CH=C(R32)-CH2-CH2-CH=CR33R34,
R50 and R51represent independently of each other: -H, -CH3, -C2H5, -C3H7, -C4H9, -C5Hi i, -C6H13, -CH2OH, -CH(OH)(CH3), -C(OH)(CH3)2, -CH(CH3)-O-CO-CH3j -C(CH3)=CH2, -CH(OH)-CH2-CH=C(CH3)2,
-CH2-CH2-CH=C(CH3)2j -CH(OH)-CH(OH)-CO-CH=CH-CH=CHR11, -CH(OH)-CH(OH)-CO-CH=CH-CH=CHR42,
-CH(OH)-CH(OH)-CO-CH=CH-CH=CHR43,
-CH(OH)-CH(OH)-CO-CH=CH-CH=CHR44, -CH=CH-CH2-CH=CHR11 , -CH=CH-CH2-CH=CHR42, -CH=CH-CH2-CH=CHR43, -CH=CH-CH2-CH=CHR44, -CH(OH)-CH3, -C(CH3)=CH-CH2-CH=C(CH3)2,
-CH=CH-CH=CHR1 1, -CH=CH-CH=CHR ,42
R11, R12, R13, R14, R23, R24, R25, R35, R36, R37, R38, R39, R40, R41, R42, R43 and R44 represent independently of each other: -H, -CH3, -C2H5, -C3H7, -C4H9, -C5H11 , -OH, -OCH3, -CO2H, -CH2-CH=CH2, -C(CH3)=CH2;
R15 represents one of the following -H, -CH3, -C2H5, -C3H7, -C4H9;
R45, R46, R47, R48, R49, R52 and R53 represent independently of each other -H, — CH3, — C2H5, — C3H7, — C4Hgi — OCH3, — OC2H5, — OC3H7, — F, —CI, — CF3, -CHF2, -CH2F. m, n, p, o and q are integer numbers independently of each other selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13 and 14; and conjugates, enantiomers, stereoisomeric forms, mixtures of enantiomers, diastereomers, mixtures of diastereomers, hydrates, solvates, tautomers, racemates and pharmaceutically acceptable salts of the above mentioned compounds for use as modulator of PPARs. As used herein the term "conjugate" refers to a compound of general formula I covalently linked to a peptide or a peptide analogue that is recognized by a certain receptor expressed on the surface of the cell and involved in beneficial physiological responses, such as increased satiety. Hence, such conjugates are able to specifically direct the compounds of general formula I to cells expressing the receptors that interact with the peptides or peptides analogues to which the compounds of general formula I are conjugated. Example of peptides suitable to
be covalently linked to a compound of general formula I include, but are not restricted to metabolically active peptide hormones, such as incretin-derivatives.
Glucagon-like peptide-1 (GLP-1 ) receptor or the glucose-dependent insulinotropic polypeptide (GIP) receptor constitutes examples of receptors expressed on the surface of the cell and involved in beneficial physiological responses.
GLP-1 is one of the most potent incretins and it stimulates insulin secretion. GLP-1 is released by the gut into the circulation in response to carbohydrate or protein ingestion. The GLP-1 receptor is a 463 amino acid 7 transmembrane-spanning protein exhibiting 27% to 40% sequence homology to the receptors for secretin, calcitonin, and parathyroid hormone. As these receptors shared higher identity with each other than with other members of the G protein-coupled receptor superfamily, they were classified together into a new family of receptors now known as the type II receptor family. However, despite the strong sequence homology between GLP-1 and the other members of the glucagon related family of peptides, the GLP-1 receptor recognizes GLP-1 specifically, with no demonstrable binding by a number of related peptides, including secretin and vasoactive intestinal peptide. The mammalian GLP-1 receptor is mainly expressed in pancreatic cells, stomach and brain.
Glucose-dependent insulinotropic polypeptide or gastric inhibitory polypeptide (GIP) is a 42 amino acid peptide hormone secreted by K cells in the intestinal epithelium. The majority of intestinal K cells are located in the proximal duodenum. GIP secretion is primarily regulated by nutrients, especially fats. The GIP receptor is also a member of the glucagon receptor family. The GIP receptor is involved in glucose homeostasis via potentiation of glucose-dependent insulin secretion from the pancreatic islet β-cells. It also inhibits gastric acid secretion. The GIP receptor is expressed in the pancreas, stomach, small intestine, adipose tissue, adrenal cortex, pituitary, heart, testis, endothelial cells, bone, trachea, spleen, thymus, lung, kidney, thyroid, and several regions in the CNS.
However, the receptors expressed on the surface of the cell and targeted by the conjugates of the current invention are not restricted to the above mentioned receptors, to the receptors of this family or to the receptors expressed only in brain; they further include receptors specifically expressed in any other tissues that are targeted by amorfrutin analogues, such as fibroblast growth factor 21 (FGF21 )
that is involved in the stimulation of glucose uptake in adipocytes, but not in other cell types.
A preferred embodiment of the present invention relates to the so far unknown compounds of general formula (I)
wherein
R1 represents the following: -H;
R2 represents the following: -H, -OH, -OCqH(2q+1 );
R4 represents the following: -H, -OH, -OCH3, -CH3 or
R2 together with R3 or R3 together with R4 form with the two carbons of the benzene ring to which R2 and R3 or R3 and R4 are attached one of the following moieties:
R3, R5, R6 and R7 represent independently of each other: -R8, -R9, -R10, -R16, -H, -OH, -(CH2)n-R8, -(CH2)m-R9, -(CH2)P-R10, -(CH2)0-R16, -C2H4-Ph, -CH2-Ph, -CH=CH-Ph, -OCH3, -OC2H5, -CH=CH-CH(CH3)2, -CH2- CH=C(CH3)2, -CH2-CH=CR8R9, -CH2-CH=CR10R16,
-CH2-CH(R11)-C(CH3)=CH2, -CH2-CH(OH)-R8, -CH2-CH(OH)-R9,
-CH2-CH(OH)-R10, -CH2-CH(OH)-R 16 -CH2-CH=C(R°)-CH2-CH2-
CH=CR9R10 8=CH2, -CR9=CH2, CR10=CH2, CR16=CH2,
R8, R9, R10 and R16 represent independently of each other: -H, -CH3, -C2H5, -C3H7, -C4H9, -C5H 1 1 , -C6H i 3, -CH2OH, -CH(OH)(CH3), -C(OH)(CH3)2,
-CH(CH3)-O-CO-CH3, -C(CH3)=CH2, -CH(OH)-CH2-CH=C(CH3)2, — CH2— CH2— CH=C(CH3)2, -CH(OH)-CH(OH)-CO-CH=CH-CH=CHR1 1 , -CH=CH-CH2-CH=CHR1 1 , ■CH(OH)-CH3, -C(CH3)=CH-CH2-CH=C(CH3)2, -CH=CH-CH=CHR1 1 ;
R1 1 , R12, R13 and R14 represent independently of each other: -H, -CH3, -C2H5, — C3H7, — C4Hg, — C5H11 , —OH, — CO2H, — CH;?— CH=CH2; m, n, p, o and q are integer numbers independently of each other selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13 and 14; and conjugates, enantiomers, stereoisomeric forms, mixtures of enantiomers, diastereomers, mixtures of diastereomers, hydrates, solvates, tautomers, racemates and pharmaceutically acceptable salts of the above mentioned compounds.
The expression tautomer is defined as an organic compound that is interconvertible by a chemical reaction called tautomerization. Tautomerization can be catalyzed preferably by bases or acids or other suitable compounds. Another aspect of the present invention is directed to novel compounds selected from the following group:
Compound Name
NP-01221 1 2-[(1 R,6R)-3-methyl-6-prop-1 -en-2-yl-1 -cyclohex-2-enyl]-5- pentylbenzene-1 ,3-diol
NP-016018 2-[(1 R,6R)-3-methyl-6-prop-1 -en-2-yl-1 -cyclohex-2-enyl]-5- propylbenzene-1 ,3-diol
NP-015933 2,6-di-(3-methyl-2-butenyl)-5-pentylbenzene-1 ,3-diol
NP-015939 2-(2-hydroxy-3-methylbut-3-en-1 -yl)-6-(3-methyl-2-butenyl)-5- pentylbenzene-1 ,3-diol
NP-015137 3-methoxy-2-(3-methyl-2-butenyl)-5-(2-phenylethyl)-benzene-
1 -ol
NP-015938 4-(2-hydroxy-3-methylbut-3-en-1 -yl)-2-(3-methyl-2-butenyl)-5- pentylbenzene-1 ,3-diol
NP-015937 2-(2-hydroxy-3-methylbut-3-en-1 -yl)-6-(3-methyl-2-butenyl)-5-
(2-phenylethyl)-benzene-1 ,3-diol
The present invention is also directed to a method of treatment comprising the step of administering to a patient a pharmaceutically effective amount of a compound of general formula (I)
wherein
R1 represents the following: -H, -CO2R15, -CHO, -C(O)-CH
R2 represents the following: -H, -OH, -OCqH(2q+i);
R4 represents the following: -H, -OH, -OCH3, -CH3 or
R2 together with R3 or R3 together with R4 form with the two carbons of the benzene r
R3, R5, R6 and R7 represent independently of each other: -R8, -R9, -R10, -R16, -H, -OH, -(CH2)n-R8, -(CH2)m-R9, -(CH2)P-R10, -(CH2)0-R16, -CH=CH-R8, -CH=CH-R9, -CH=CH-R10, -CH=CH-R16, -(CH2)n-CHR8R9, -(CH2)m-CHR17R18, -(CH2)p-CHR19R20, -(CH2)0-CHR21R22, -OCH3, -OC2H5, -CH=CH-CH(CH3)2, -CH2-CH=CR8R9, -CH2-CH=CR17R18, -CH2-CH=CR19R20, -CH2-CH=CR21R22, -CH2-CH(R1 1)-C(CH3)=CH2,
-CH2-CH(R23)-C(CH3)=CH2, -CH2-CH(R24)-C(CH3)=CH2, -CH2-CH(R25)-C(CH3)=CH2, -CH2-CH(OH)-R8, -CH2-CH(OH)-R9,
-CH2-CH(OH)-R10, -CH2-CH(OH)-R16, -CH2-CH=C(R8)-CH2-CH2-
CH=CR9R10, -CR8=CH2, -CR9=CH2, -CR10=CH2, -CR16=CH2, -CH2- CH=C(R26)-CH2-CH2-CH=CR27R28, -CH2-CH=C(R29)-CH2-CH2-CH=CR30R31 , -CH -CH=C(R32)-CH2-CH2-CH=CR33R34, - -CH2-CHR50R51
D8 D9 D10 D16 D17 D18 D19 D20 D21 D22 D26 D27 D28 D29 D30 D31 D32 D33 D34 r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ ,
R50 and R51 represent independently of each other: -H, -CH3, -C2H5, -C3H7j -C4H9j -C5Hi i, -C6H i 3, -CH2OH, -CH(OH)(CH3), -C(OH)(CH3)2, -CH(CH3)-O-CO-CH3j -C(CH3)=CH2, -CH(OH)-CH2-CH=C(CH3)2j
-CH2-CH2-CH=C(CH3)2j -CH(OH)-CH(OH)-CO-CH=CH-CH=CHR11, -CH(OH)-CH(OH)-CO-CH=CH-CH=CHR42,
-CH(OH)-CH(OH)-CO-CH=CH-CH=CHR43,
-CH(OH)-CH(OH)-CO-CH=CH-CH=CHR44,
-CH=CH-CH2-CH=CHR11, -CH=CH-CH2-CH=CHR42, -CH=CH-CH2-CH=CHR43, -CH=CH-CH2-CH=CHR44, -CH(OH)-CH3, -C(CH3)=CH-CH2-CH=C(CH3)2j -CH=CH-CH=CHR1 1, -CH=CH-CH=CHR42, -CH=CH-CH=CHR43, -CH=CH-CH=CHR44,
R11, R12, R13, R14, R23, R24, R25, R35, R36, R37, R38, R39, R40, R41, R42, R43 and R44 represent independently of each other: -H, -CH3, -C2H5, -C3H7, -C4H9, -C5Hii, -OH, -OCH3j -CO2H, -CH2-CH=CH2, -C(CH3)=CH2;
R15 represents one of the following -H, -CH3, -C2H5, -C3H7, -C4H9;
R45, R46, R47, R48, R49, R52 and R53 represent independently of each other -H, — CH3, — Ο2Ηδ, — C3H7, ,— C4Hg, — OCH3, — OC2Hs, — OC3H7, — F, —CI, — CF3, -CHF2, -CH2F;
m, n, p, o and q are integer numbers independently of each other selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13 and 14; or conjugates, enantiomers, stereoisomeric forms, mixtures of enantiomers, diastereoisomers, mixtures of diastereoisomers, hydrates, solvates, tautomers, racemates and pharmaceutically acceptable salts of the above mentioned compounds for the modulation of PPARs and/or for the treatment of PPAR associated diseases. The PPAR associated diseases are discussed further below in detail.
In a preferred embodiment of the present application the substituents R3 and R5 are the same substituent selected from: -R8, -(CH2)n-R8, -(CH2)n-CHR8R9, -CH=CH-CH(CH3)2, -CH2-CH=CR8R9, -C2H4-Ph, -CH=CH-Ph,
-CH2-CH(OH)-R8, -CR8=CH2, -CH2-CH=C(R8)-CH2-CH2-CH=CR9R10, -CH2-CH(R11)-C(CH3)=CH2;
wherein
R8, R9 and R10 represent independently of each other: -H, -CH3, -C2H5, -C3H7, — C4Hg, — C5H 11 , — CeHi 3, — C(CH3)=CH2, — C(CH3)=CH— CH2— CH=C(CH3)2, -CH2-CH2-CH=C(CH3)2, -CH=CH-CH=CHR11, -CH=CH-CH2-CH=CHR11; and
R11 represents: -H, -CH3, -C2H5, -C3H7, -C4H9, -C5Hn, -CH2-CH=CH2. The wording "the same substituent" simply means R3 = R5.
In another preferred embodiment R2 together with R3, or R3 together with R4 form with the two carbons of the benzene ring to which R2 and R3 or R3 and R4 are attached one of the following moieties:
wherein R7 has the meaning above defined.
Further preferred compounds of general formula (I) are these compounds, wherein R1 substituent represents -H. Moreover compounds are preferred wherein R3, R5 and R6 are different from hydrogen.
R represents preferably the following substituents: -OH or -OCH3 and most preferably -OH.
R6 preferably contains a phenyl group, and more preferably a substituted phenyl group. R3 and R5 do preferably not contain a phenyl group, but preferably contain a substituent with one or two double bonds.
R3 and R5 are preferably independently of each other selected from the following substituents: -CH=CH-CH(CH3)2, -CH2-CH=CR8R9, -CH2-CH=CR > 1"RD 1f
23
-CH2-CH(R11)-C(CH3)=CH2, -CH2-CH(R^)-C(CH3)=CH2, -CH2-CH(OH)-R°,
59D 10
-CH2-CH(OH)-R16, -CH2-CH=C(R°)-CH2-CH2-CH=CRaR lu,
-CH2-CH=C(R26)-CH2-CH2-CH=CR27R28, -CR16=CH2, -CR8=CH2,
-(CH2)p-CHR19R20 or -(CH2)o-CHR21R22. Compounds of the following general formula (II) - (VII) are also preferred:
Another preferred embodiment according to the current invention is directed to compounds of general formula VIII
and R4, R5, R15, R45, R46, R47, R48, R49, R52 and R53 have the meanings disclosed above and at least one of the substituents R45, R46, R47, R48 and R49 is different of hydrogen.
Compounds of general formula IX
and R4, R5, R15, R45, R46, R47, R48, R49, R52 and R53 have the meanings disclosed above and at least one of the substituents R45, R46, R47, R48 and R49 is different of hydrogen are also preferred.
Even more preferred compounds are compounds of general formula VIII or IX wherein R16 represents one of the followin :
and FT5, R4b, FT , R , R4y, R^ and RM represent independently of each other: — CH3, — C2H5, — C3H7, — C4Hg, — OCH3, — OC2H5, — OC3H7, — F, —CI, — CF3, -CHF2, -CH2F. Connpounds of general fornnula X and XI
X
wherein R16 represents one of the following:
R4b, FT, R , R4y, R^ and RM represent independently of each other: — C2H5, — C3H7, ,— C4H9, — OCH3, — OC2H5,— OC3H7, — F, —CI, — CF3, -CH2F are also preferred. In yet another preferred embodiment of the present invention, the compound according to the general formula (I) is selected from the group of compounds depicted in the following Table 1 .
Table 1. Compounds according to the present invention.
Compound Structure
In another preferred embodiment the compounds of formula (I) are isolated from the roots of Glycyrrhiza foetida and from the fruits of Amorpha fructicosa. Indications
In a further aspect of the present invention, the novel compounds according to the general formula (I) are used as pharmaceutically active agent applicable in medicine. Surprisingly it was found that the above-mentioned compounds of general formula (I) as well as the pharmaceutical compositions comprising said compounds of general formula (I) are acting as modulators of PPARs, with great specificity for
PPARy and and are useful for the prevention and/or treatment of metabolic diseases.
The term modulator refers to a compound that modulates the transcriptional activity of PPAR involving specific activation or repression of a subgroup of genes regulated by PPAR, thus leading to a differential expression of PPAR target genes.
As PPARy is involved in inflammatory and cancer processes, a further aspect according to the present invention is directed to compounds of general formula (I) useful for prevention and/or treatment of inflammatory diseases and cancer.
Further aspects of the present invention relate to the use of the compounds of general formula (I) for the preparation of a pharmaceutical composition useful for prevention and/or treatment of metabolic diseases, inflammatory diseases and cancer.
In yet another aspect of the present invention, the compounds according to the general formula (I) are for the preparation of phytomedicals or functional food products for prevention of metabolic diseases.
The term "functional food product" refers to a food purported or proven to have a beneficial health effect. The term "phytomedical" is defined as a pharmaceutical composition containing compounds isolated from plants for prevention and/or treatment of various health concerns.
Furthermore, the compounds of general formula (I) have hepatoprotective properties.
Preferably, the compounds of general formula (I) and/or pharmaceutical acceptable salts thereof are useful for or can be used for the prevention and/or treatment of metabolic diseases.
Metabolic diseases refer to diseases and conditions characterized by pathological disorders of the metabolism. They are mainly characterized by enzyme defects and abnormalities in the regulating system leading to a pathological enrichment of
substrates, lack of metabolic products, failure of producing energy, of regeneration of cellular constituents, of elimination of metabolic products and of maintenance of homeostasis. They can be acquired or be a genetic disease. Metabolic disorders include, but are not limited to: obesity, type I diabetes, type II diabetes, maturity- onset diabetes of youth, gestational diabetes, hypoglycemia, amyloidosis, branched chain disease, hyperaminoacidemia, hyperaminoaciduria, disturbances of the metabolism of urea, hyperammonemia, mucopolysaccharidoses e. g. Maroteaux-Lamy syndrom, glycogen storage diseases and lipid storage diseases, Cori's disease, intestinal carbohydrate malabsorption, maltase-, lactase-, sucrase- insufficiency, disorders of the metabolism of fructose, disorders of the metabolism of galactose, galactosaemia, disturbances of pyruvate metabolism, hypolipidemia, hypolipoproteinemia, hyperlipidemia, hyperlipoproteinemia, carnitine or carnitine acyltransferase deficiency, porphyrias, disturbances of the purine metabolism, lysosomal diseases, metabolic diseases of nerves and nervous systems like gangliosidoses, sphingolipidoses, sulfatidoses, leucodystrophies, Lesch-Nyhan syndrome, dysfunction of the parathyroid glands, pancreatic islet cell dysfunction, carbohydrate and lipid storage myopathies, glycogenoses, myoglobinuria, alkaptonuria, adrenogenital syndrome, ketosis, ketoacidosis, methylmalonaciduria, Morbus Addison, Morbus Conn, Morbus Cushing, Morbus Fabry, Morbus Gaucher, Morbus Hunter, cystic fibrosis, phenylketonuria, thesaurismosis, uricopathia, insulin resistance.
In another preferred embodiment, the compounds of general formula (I) and/or pharmaceutical acceptable salts thereof are useful for or can be used for the prevention and/or treatment of inflammatory diseases.
Inflammatory diseases refer to diseases involving an inflammation process. Inflammation is the final common pathway of various insults, such as infection, trauma, and allergies to the human body. It is characterized by activation of the immune system with recruitment of inflammatory cells, production of proinflammatory cells and production of pro-inflammatory cytokines. Most inflammatory diseases and disorders are characterized by abnormal accumulation of inflammatory cells including monocytes/macrophages, granulocytes, plasma cells, lymphocytes and platelets. Along with tissue endothelial cells and fibroblasts, these inflammatory cells release a complex array of lipids, growth factors, cytokines and destructive enzymes that cause local tissue damage.
5
The term "inflammatory disease" encompasses a variety of conditions, including autoimmune diseases, proliferative skin diseases, and inflammatory dermatoses. Inflammatory disorders result in the destruction of healthy tissue by an inflammatory process, dysregulation of the immune system, and unwanted proliferation of cells. Examples of inflammatory diseases are acne vulgaris, acute respiratory distress syndrome, Addison's disease, allergic rhinitis, allergic intraocular inflammatory diseases, antineutrophil cytoplasmic antibody (ANCA)- associated small-vessel vasculitis, ankylosing spondylitis, arthritis, asthma, atherosclerosis, atopic dermatitis, autoimmune hepatitis, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, Bell's palsy, bullous pemphigoid, cerebral ischemia, chronic obstructive pulmonary disease cirrhosis, Cogan's syndrome, contact dermatitis, Crohn's disease, Cushing's syndrome, dermatomyositis, diabetes mellitus, discoid lupus erythematosus, eosinophilic fasciitis, erythema nodosum, exfoliative dermatitis, fibromyalgia, focal glomerulosclerosis, focal segmental glomerulosclerosis, giant cell arteritis, gout, gouty arthritis, graft versus host disease, hand eczema, Henoch-Schonlein purpura, herpes gestationis, hirsutism, idiopathic cerato-scleritis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, immune thrombocytopenic purpura inflammatory bowel or gastrointestinal disorders, inflammatory dermatoses, lichen planus, lupus nephritis, lymphomatous tracheobronchitis, macular edema, multiple sclerosis, myasthenia gravis, myositis, nonspecific fibrosing lung disease, osteoarthritis, pancreatitis, pemphigoid gestationis, pemphigus vulgaris, periodontitis, polyarteritis nodosa, polymyalgia rheumatica, pruritus scroti, pruritis/inflammation, psoriasis, psoriatic arthritis, pulmonary histoplasmosis, rheumatoid arthritis, relapsing polychondritis, rosacea caused by sarcoidosis, rosacea caused by scleroderma, rosacea caused by Sweet's syndrome, rosacea caused by systemic lupus erythematosus, rosacea caused by urticaria, rosacea caused by zoster-associated pain, sarcoidosis, scleroderma, segmental glomerulosclerosis, septic shock syndrome, shoulder tendinitis or bursitis, Sjogren's syndrome, Still's disease, stroke-induced brain cell death, Sweet's disease, systemic lupus erythematosus, systemic sclerosis, Takayasu's arteritis, temporal arteritis, toxic epidermal necrolysis, transplant- rejection and transplant-rejection-related syndromes, tuberculosis, type-1 diabetes, ulcerative colitis, uveitis, vasculitis, and Wegener's granulomatosis.
Another aspect of the present invention is directed to the use of compound of general formula (I) and/or pharmaceutically acceptable salts thereof for prevention and/or treatment of cancer.
The cancer type is preferably selected from the group comprising or consisting of: adenocarcinoma, choroidal melanoma, acute leukemia, acoustic neurinoma, ampullary carcinoma, anal carcinoma, astrocytoma, basal cell carcinoma, pancreatic cancer, desmoid tumor, bladder cancer, bronchial carcinoma, non-small cell lung cancer (NSCLC), breast cancer, Burkitt's lymphoma, corpus cancer, CUP- syndrome (carcinoma of unknown primary), colorectal cancer, small intestine cancer, small intestinal tumors, ovarian cancer, endometrial carcinoma, ependymoma, epithelial cancer types, Ewing's tumors, gastrointestinal tumors, gastric cancer, gallbladder cancer, gall bladder carcinomas, uterine cancer, cervical cancer, cervix, glioblastomas, gynecologic tumors, ear, nose and throat tumors, hematologic neoplasias, hairy cell leukemia, urethral cancer, skin cancer, skin testis cancer, brain tumors (gliomas), brain metastases, testicle cancer, hypophysis tumor, carcinoids, Kaposi's sarcoma, laryngeal cancer, germ cell tumor, bone cancer, colorectal carcinoma, head and neck tumors (tumors of the ear, nose and throat area), colon carcinoma, craniopharyngiomas, oral cancer (cancer in the mouth area and on lips), cancer of the central nervous system, liver cancer, liver metastases, leukemia, eyelid tumor, lung cancer, lymph node cancer (Hodgkin's/Non-Hodgkin's), lymphomas, stomach cancer, malignant melanoma, malignant neoplasia, malignant tumors gastrointestinal tract, breast carcinoma, rectal cancer, medulloblastomas, melanoma, meningiomas, Hodgkin's disease, mycosis fungoides, nasal cancer, neurinoma, neuroblastoma, kidney cancer, renal cell carcinomas, non-Hodgkin's lymphomas, oligodendroglioma, esophageal carcinoma, osteolytic carcinomas and osteoplastic carcinomas, osteosarcomas, ovarial carcinoma, pancreatic carcinoma, penile cancer, plasmocytoma, squamous cell carcinoma of the head and neck (SCCHN), prostate cancer, pharyngeal cancer, rectal carcinoma, retinoblastoma, vaginal cancer, thyroid carcinoma, Schneeberger disease, esophageal cancer, spinalioms, T-cell lymphoma (mycosis fungoides), thymoma, tube carcinoma, eye tumors, urethral cancer, urologic tumors, urothelial carcinoma, vulva cancer, wart appearance, soft tissue tumors, soft tissue sarcoma, Wilm's tumor, cervical carcinoma and tongue cancer.
Therefore, another aspect of the present invention is directed to pharmaceutical compositions comprising at least one compound of the present invention as active ingredient, together with at least one pharmaceutically acceptable carrier, excipient and/or diluents. Preferably, the pharmaceutical composition comprises at least one compound according to claim 1 or 6. The pharmaceutical compositions of the present invention can be prepared in a conventional solid or liquid carrier or
7 diluent and a conventional pharmaceutically-nnade adjuvant at suitable dosage level in a known way. The preferred preparations are adapted for oral application. These administration forms include, for example, pills, tablets, film tablets, coated tablets, capsules, powders and deposits.
Furthermore, the present invention also includes pharmaceutical preparations for parenteral application, including dermal, intradermal, intragastral, intracutan, intravasal, intravenous, intramuscular, intraperitoneal, intranasal, intravaginal, intrabuccal, percutan, rectal, subcutaneous, sublingual, topical, or transdermal application, which preparations in addition to typical vehicles and/or diluents contain at least one compound according to the present invention and/or a pharmaceutical acceptable salt thereof as active ingredient.
The pharmaceutical compositions according to the present invention containing at least one compound according to the present invention, and/or a pharmaceutical acceptable salt thereof as active ingredient will typically be administered together with suitable carrier materials selected with respect to the intended form of administration, i.e. for oral administration in the form of tablets, capsules (either solid filled, semi-solid filled or liquid filled), powders for constitution, extrudates, deposits, gels, elixirs, dispersable granules, syrups, suspensions, and the like, and consistent with conventional pharmaceutical practices. For example, for oral administration in the form of tablets or capsules, the active drug component may be combined with any oral non-toxic pharmaceutically acceptable carrier, preferably with an inert carrier like lactose, starch, sucrose, cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, talc, mannitol, ethyl alcohol (liquid filled capsules) and the like. Moreover, suitable binders, lubricants, disintegrating agents and coloring agents may also be incorporated into the tablet or capsule. Powders and tablets may contain about 5 to about 95 weight % of the benzothiophene-1 ,1 -dioxide derived compound and/or the respective pharmaceutically active salt as active ingredient.
Suitable binders include starch, gelatin, natural sugars, corn sweeteners, natural and synthetic gums such as acacia, sodium alginate, carboxymethylcellulose, polyethylene glycol and waxes. Among suitable lubricants there may be mentioned boric acid, sodium benzoate, sodium acetate, sodium chloride, and the like. Suitable disintegrants include starch, methylcellulose, guar gum, and the like. Sweetening and flavoring agents as well as preservatives may also be included,
where appropriate. The disintegrants, diluents, lubricants, binders etc. are discussed in more detail below.
Moreover, the pharmaceutical compositions of the present invention may be formulated in sustained release form to provide the rate controlled release of any one or more of the components or active ingredients to optimise the therapeutic effect(s), e.g. antihistaminic activity and the like. Suitable dosage forms for sustained release include tablets having layers of varying disintegration rates or controlled release polymeric matrices impregnated with the active components and shaped in tablet form or capsules containing such impregnated or encapsulated porous polymeric matrices.
Liquid form preparations include solutions, suspensions, and emulsions. As an example, there may be mentioned water or water/propylene glycol solutions for parenteral injections or addition of sweeteners and opacifiers for oral solutions, suspensions, and emulsions. Liquid form preparations may also include solutions for intranasal administration. Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be present in combination with a pharmaceutically acceptable carrier such as an inert, compressed gas, e.g. nitrogen. For preparing suppositories, a low melting fat or wax, such as a mixture of fatty acid glycerides like cocoa butter is melted first, and the active ingredient is then dispersed homogeneously therein e.g. by stirring. The molten, homogeneous mixture is then poured into conveniently sized moulds, allowed to cool, and thereby solidified.
Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration. Such liquid forms include solutions, suspensions, and emulsions. The compounds according to the present invention may also be delivered transdermally. The transdermal compositions may have the form of a cream, a lotion, an aerosol and/or an emulsion and may be included in a transdermal patch of the matrix or reservoir type as is known in the art for this purpose.
The term capsule as recited herein refers to a specific container or enclosure made e.g. of methyl cellulose, polyvinyl alcohols, or denatured gelatins or starch for holding or containing compositions comprising the active ingredient(s). Capsules with hard shells are typically made of blended of relatively high gel strength gelatins from bones or pork skin. The capsule itself may contain small
amounts of dyes, opaquing agents, plasticisers and/or preservatives. Under tablet a compressed or moulded solid dosage form is understood which comprises the active ingredients with suitable diluents. The tablet may be prepared by compression of mixtures or granulations obtained by wet granulation, dry granulation, or by compaction well known to a person of ordinary skill in the art.
Oral gels refer to the active ingredients dispersed or solubilised in a hydrophilic semi-solid matrix. Powders for constitution refers to powder blends containing the active ingredients and suitable diluents which can be suspended e.g. in water or in juice.
Suitable diluents are substances that usually make up the major portion of the composition or dosage form. Suitable diluents include sugars such as lactose, sucrose, mannitol, and sorbitol, starches derived from wheat, corn rice, and potato, and celluloses such as microcrystalline cellulose. The amount of diluent in the composition can range from about 5 to about 95 % by weight of the total composition, preferably from about 25 to about 75 weight %, and more preferably from about 30 to about 60 weight %.
The term disintegrants refers to materials added to the composition to support break apart (disintegrate) and release the pharmaceutically active ingredients of a medicament. Suitable disintegrants include starches, "cold water soluble" modified starches such as sodium carboxymethyl starch, natural and synthetic gums such as locust bean, karaya, guar, tragacanth and agar, cellulose derivatives such as methylcellulose and sodium carboxymethylcellulose, microcrystalline celluloses, and cross-linked microcrystalline celluloses such as sodium croscaramellose, alginates such as alginic acid and sodium alginate, clays such as bentonites, and effervescent mixtures. The amount of disintegrant in the composition may range from about 2 to about 20 weight % of the composition, more preferably from about 5 to about 10 weight %.
Binders are substances which bind or "glue" together powder particles and make them cohesive by forming granules, thus serving as the "adhesive" in the formulation. Binders add cohesive strength already available in the diluent or bulking agent. Suitable binders include sugars such as sucrose, starches derived from wheat corn rice and potato, natural gums such as acacia, gelatin and tragacanth, derivatives of seaweed such as alginic acid, sodium alginate and ammonium calcium alginate, cellulose materials such as methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethylcellulose, polyvinylpyrrolidone,
and inorganic compounds such as magnesium aluminum silicate. The amount of binder in the composition may range from about 2 to about 20 weight % of the composition, preferably from about 3 to about 10 weight %, and more preferably from about 3 to about 6 weight %.
Lubricants refer to a class of substances which are added to the dosage form to enable the tablet granules etc. after being compressed to release from the mould or die by reducing friction or wear. Suitable lubricants include metallic stearates such as magnesium stearate, calcium stearate, or potassium stearate, stearic acid, high melting point waxes, and other water soluble lubricants such as sodium chloride, sodium benzoate, sodium acetate, sodium oleate, polyethylene glycols and D,L-leucine. Lubricants are usually added at the very last step before compression, since they must be present at the surface of the granules. The amount of lubricant in the composition may range from about 0.2 to about 5 weight % of the composition, preferably from about 0.5 to about 2 weight %, and more preferably from about 0.3 to about 1 .5 weight % of the composition.
Glidents are materials that prevent caking of the components of the pharmaceutical composition and improve the flow characteristics of granulate so that flow is smooth and uniform. Suitable glidents include silicon dioxide and talc. The amount of glident in the composition may range from about 0.1 to about 5 weight % of the final composition, preferably from about 0.5 to about 2 weight %.
Coloring agents are excipients that provide coloration to the composition or the dosage form. Such excipients can include food grade dyes adsorbed onto a suitable adsorbent such as clay or aluminum oxide. The amount of the coloring agent may vary from about 0.1 to about 5 weight % of the composition, preferably from about 0.1 to about 1 weight %.
Chemical synthesis
The compounds of general formula I according to the present invention can be obtained through isolation from natural sources and/or chemical synthesis. For example, compounds of general formula I, wherein R1 is selected from -H, -CO2R15 and -CHO, R2 is selected from -OH and -OCqH(2q+i), R4 is selected from -OH and -OCH3, and R3, R5 and R6 have the meanings defined above can be obtained starting from ketone 3 and a malonate derivative such as 4
(see Scheme 1 ). Ketones of general fornnula 3 are commercially available or can be accessed by reacting hydrazone 5 obtained from the corresponding methyl ketone with commercially available brominated derivative 6. Malonate derivative 4 can be synthesized starting from diethylmalonate 8 and acyl chloride 7. Conveniently, acid chlorides of general formula 7 are commercially available or can be easily prepared from the corresponding carboxylic acids by methods well known to the person skilled in the art.
Scheme 1. Retrosynthetic analysis.
For example, compounds of general formula 10, 11 and 12 can be synthesized according to the synthetic pathway described in Scheme 2. To access ketone 3, hydrazone 5 is reacted with brominated derivative 6 in presence of a strong base such as lithium diisopropylamide in anhydrous tetrahydrofurane. Example of suitable brominated derivative 6 are 1 -bromo-3-methylbut-2-ene and (2Z)-1 - bromo-3,7-dimethylocta-2,6-diene.
After treatment of diethyl malonate 8 with Mg turnings in absolute ethanol and a catalytic amount of carbon tetrachloride, the intermediate magnesium salt is reacted with acyl chloride 7 to provide enol 9. Suitable acyl chlorides 7 are hydrocinamoyl chloride, pentanoyl chloride, butanoyl chloride, propanoyl chloride, ethanoyl chloride, formyl chloride. Enol 9 is converted to corresponding chloride 4 by treatment with phosphoryl chloride and triethylamine. Subsequent treatment of chloride 4 with ketone 3 provides amorfrutine analogue 10 that can be further methylated to provide the methylated analogue 11. Cleavage of the methyl ester on methylated analogue 11 furnishes amorfrutine 12. It is obvious for the person skilled in the art that amorfrutine 12 can be decarboxylated to afford amorfrutine analogues with R1 being -H or submitted to esterification to provide amorfrutine analogues with R1 being -CO2R15 and R15
being different of -H, or submitted to reduction with diisobutylaluminium hydride to provide amorfrutine analogues with R1 being -CHO.
10 11 12
Scheme 2. Synthesis of amorfrutine derivatives of general formula 10, 11 and 12: a. LDA, THF; b. Mg turnings, EtOH, CCI4; c. POCI3, Et3N; d. LDA, THF;
e. Mel, NaHCO3; f. KOH, DMSO. The conjugates of the present invention can be constructed by covalently linking a compound of general compound I (amorfrutin) with a peptide or a peptide analogue using conjugation chemistries known to the skilled person. The inventive compounds can be covalently linked to the peptide via the carboxylic acid, the hydroxyl group or a suitable functionality introduced on the hydrophobic side chains by derivatisation.
For example, a stable conjugate, which is resistant to proteolytic cleavage (by cellular proteases) can be generated via ether conjugation, by using a suitable functionality introduced on the hydrophobic side chains of the compounds of general formula I, or the carboxylic acid or the hydroxyl group.
More labile, proteolytically cleavable conjugates can be generated via ester conjugation, by using a functionality present on the hydrophobic side chains of the compounds of general formula I, or the carboxylic acid or the hydroxyl group.
Using this approach, the compounds of general formula I can be targeted to specific cell types, taken up and released intracellularly.
Peptide conjugation shall take place using standard conjugation chemistry through carboxyl groups, free amines or thiol group on cysteine, or any other suitable methods, preferable C- or N-terminally. Peptide conjugation can benefit from introducing a (bio-) chemically inert (bifunctional) linker molecule. To improve the stability of the peptide, said peptide can be modified using appropriate derivatisation to avoid degradation in a cellular or physiological context. For example, the peptide hormones GLP-1 are rapidly inactivated by the enzyme dipeptidyl peptidase-4 (DPP-4). To prevent this physiologically common enzymatic degradation, the regular amino acid alanine at the second position of GLP-1 can be replaced with 2-aminoisobutyric acid. Thus, amorfrutin NP-003520 can be linked via an ester bond achieved by reacting its carboxylic acid group to a FGF21 peptide derivative to produce a conjugate NP- 003520 / FGF21 peptide derivative that induces synergistic glucose-lowering effects in adipocytes via FGF21 pathways and PPARy-regulated pathways.
Description of the Figures
Figure 1 shows the binding of PPARy by the compounds of general formula (I):
Binding of different compounds was determined in a competitive time- resolved fluorescence resonance energy transfer-based binding assay. Data are expressed as mean (n=2-3).
Figure 2 shows the partial activation of PPARs by Amorfrutin B (NP-015142): (a) Chemical structure of amorfrutin B;
(b) Transcriptional activation of PPARy by amorfrutin B (black triangles) or rosiglitazone (grey squares) in a reporter gene assay;
(c) Transcriptional activation of PPARa by amorfrutin B (black triangles) or
GW7647 (grey circles) in a reporter gene assay;
(d) Transcriptional activation of PPAR /δ by amorfrutin B (black triangles) or
GW0742 (grey diamonds) in a reporter gene assay;
(e-i) Recruitment of transcriptional cofactor peptides to PPARy-LBD titrated with amorfrutin B (black triangles) or rosiglitazone (grey squares). Binding of cofactor peptides was measured by time-resolved
fluorescence resonance energy transfer. Recruitment is represented relative to rosiglitazone. Peptides are derived from coactivators CBP; (e), PGC1 a (f), TRAP220/DRIP (g), PRIP/RAP250 (h) or corepressor NCoR (i). Data are expressed as mean ± SD (n=3).
Figure 3 shows the binding of PPARa by the compounds of general formula (I):
Binding of different compounds was determined in a competitive time- resolved fluorescence resonance energy transfer-based binding assay. Data are expressed as mean (n=2-3).
Figure 4 shows the binding of PPAR /δ by the compounds of general formula (I):
Binding of different compounds was determined in a competitive time- resolved fluorescence resonance energy transfer-based binding assay. Data are expressed as mean (n=2-3).
Figure 5 shows the transcriptional activation of PPARy: PPARy activation of different compound concentrations was determined in a reporter gene assay and is normalized to rosiglitazone-induced activation. Axes of ordinates were differentially scaled according to individual activation efficacies. Data are expressed as mean (n=2-3).
Figure 6 shows the transcriptional activation of PPARa: PPARa activation of different compound concentrations was determined in a reporter gene assay and is normalized to GW7647-induced activation. Axes of ordinates were differentially scaled according to individual activation efficacies. Data are expressed as mean (n=2-3).
Figure 7 shows:
(a) The recruitment of transcriptional cofactor peptides to PPARy-LBD titrated with amorfrutin B (black) or rosiglitazone (grey). Data are expressed as mean ± SD (n=3). ***p<0.001 amorfrutin B vs. Rosiglitazone;
(b, c, d) The recruitment of the transcriptional corepressor NCoR to PPARy titrated with the indicated compounds. Data are expressed as mean (n=2-3). Binding of cofactor peptides was measured by time-resolved fluorescence resonance energy transfer. Recruitment is represented relative to rosiglitazone.
Figure 8 shows the gene expression profile in primary human adipocytes after treatment with amorfrutin B (AB) and rosiglitazone (RGZ). Cells were treated for 24 h with 10 μηηοΙ/L RGZ (grey bars) or 10 μηηοΙ/L AB (black bars) and expression of (a) known PPARy target genes and (b) adipokines was analyzed by qPCR. (c) Primary human adipocytes were transfected with PPARy siRNA (hatched bars) or control siRNA (unhatched bars) and were treated with either 10 μηηοΙ/L RGZ (grey bars), 10 μηηοΙ/L AB (black bars) or vehicle only (white bar) for 24 h. RNA expression was analyzed by quantitative PCR. Data are expressed as mean ± SEM (n=3-4/group). n.s. not significant, *p<0.05, **p<0.01 , ***p<0.001 vs. vehicle. AB, amorfrutin B; RGZ, rosiglitazone.
Figure 9 shows NCOR1 RNA expression after treatment of primary human adipocytes with amorfrutin B (AB) or rosiglitazone (RGZ). Cells were treated for 24 h with 10 μηηοΙ/L RGZ (grey bars) or 10 μηηοΙ/L AB (black bars) and RNA expression was analyzed by quantitative PCR. Data are expressed as mean ± SEM (n=3-4/group). n.s. not significant vs. vehicle. AB, amorfrutin B; RGZ, rosiglitazone.
Figure 10 shows the effect of the compounds on proliferation of HT-29 colon carcinoma cells, PC3 prostate cancer cells and MCF-7 breast cancer cells, treated for 3 days with 20 μηηοΙ/Ι of the indicated compounds. Cells were quantified by fluorometric detection of the cellular DNA content. Data are normalised to DMSO-treated cells. Data are expressed as mean (n=2-3/group).
Figure 11 shows the concentration-dependent effects of the compounds on proliferation of (a) HT-29 colon carcinoma cells, (b) T84 colon carcinoma cells and (c) PC3 prostate cancer cells. Cells were treated for 3 days (HT-29, PC3) and 4 days (T84) with various concentrations of the indicated compounds, and cells were quantified by fluorometric detection of the cellular DNA content. Data are expressed as mean (n=3-4/group).
Figure 12 shows the effects of the compound on activation of caspases 2, 3, 6, 7,
8, 9 and 10 in HT-29 and PC3 cancer cells, treated for 24 h with 20 μηηοΙ/Ι of the indicated amorfrutin analogue or 100 nmol/l paclitaxel. Data are normalised to DMSO-treated cells and are expressed as mean ± SEM (n=2-3/group).
Figure 13 shows the effects of compound NP-015934 (20 μηηοΙ/Ι) on caspase activation in different cancer cells relative to DMSO treatment, (a) Activation of caspases 2, 3, 6, 7, 8, 9 and 10 after 24 h treatment of HT- 29 cells, (b) Activation of caspases 3 and 7 after 24 h treatment of HT- 29 cells, (c) Activation of caspases 3 and 7 after 3 h treatment of MCF-7 cells, (d) Activation of caspases 2, 3, 6, 7, 8, 9 and 10 after 3 h treatment of PC3 cells. Data are expressed as mean ± SEM (n=1 - 2/group). *p<0.05, **p<0.01 vs. DMSO control.
Figure 14 shows the effects of compound NP-015934 on DNA fragmentation in
HT-29 colon cancer cells relative to DMSO treatment. Cells were treated with 30 or 100 μηηοΙ/Ι NP-015934 for 2 or 4 h. Data are expressed as mean ± SEM (n=4/group). **p<0.01 , ***p<0.001 vs. DMSO control.
Figure 15 shows the effect of amorfrutin B treatment in insulin-resistent DIO mice:
(a) Pharmacokinetic profile of amorfrutin B after oral administration in
C57BL/6 mice. Data are expressed as mean ± SEM (n=3);
(b) Fasting blood glucose of DIO mice after 15 days of treatment with vehicle (BW= 41 .7 g ± 1 .1 g), rosiglitazone (BW= 41 .2 g ± 1 .5 g) or amorfrutin B (BW= 35.7 g ± 1 .4 g);
(c) Fasting plasma insulin of DIO mice after 15 days of treatment; (d) Effect of treatment for 15 days on insulin resistance determined by homeostatic model assessment of insulin resistance (HOMA-IR);
(e, f) Glucose and insulin concentrations during oral glucose tolerance test
(OGTT) after 22 days of treatment with vehicle (white circles, BW= 39.9 g ± 1 .0 g), rosiglitazone (grey squares, BW= 39.9 g ± 1 .5 g) or amorfrutin B (black triangles, BW= 34.4 g ± 1 .3 g). AUC, area under the curve. AUC, area under the curve;
(g) Glucose levels during intraperitoneal insulin sensitivity test (IPIST) after
15 days of treatment. AUCi, inverse area under the curve. Data are expressed as mean ± SEM. *p<0.05, **p<0.01 , ***p<0.001 vs. vehicle. VEH, vehicle (white circles, n=13); RGZ, rosiglitazone (grey squares, n=8-10); AB, amorfrutin B (black triangles, n=13); BW, body weight (mean ± SEM).
7
Figure 16 shows the effect of amorfrutin B treatment of plasma lipid parameters in insulin-resistant DIO mice:
(a) Fasting plasma triacylglycerols after 27 days of treatment;
(b) Fasting plasma NEFA after 27 days of treatment. Data are expressed as mean + SEM. *p<0.05, **p<0.01 , vs. vehicle. VEH, vehicle (n=13); RGZ, rosiglitazone (n=10) ; AB, amorfrutin B (n=12-13).
Figure 17 shows the RNA expression of Ppargda, Ppargdb, Ucp1, Ucp2,
Adipocq and Lep in the visceral white adipose tissues of DIO mice treated for 4 weeks with rosiglitazone (grey) or amorfrutin B (black) analysed by qPCR (n=6-9 per group). Data are expressed as mean ± SEM. n.s. not significant, *p<0.05, **p<0.01 , ***p<0.001 vs. vehicle.
Figure 18 shows the RNA expression of different genes in tissues of DIO mice treated for 4 weeks with rosiglitazone (grey) or amorfrutin B (black)
(n=6-9 per group):
(a-c) Liver tissues were analysed by qPCR for expression of genes associated with (a) glycolysis and gluconeogenesis, (b) fatty acid metabolism, or (c) ketogenesis;
(d) Plasma levels of β-hydroxybutyrate determined by colorimetry;
(e) Skeletal muscle tissues were analysed by qPCR (n=6-9 per group).
Data are expressed as mean ± SEM. n.s. not significant, *p<0.05, **p<0.01 , ***p<0.001 vs. vehicle. VEH, vehicle; RGZ, rosiglitazone; AB, amorfrutin B.
Figure 19 shows the FGF21 levels in DIO mice treated for 4 weeks with vehicle
(white), rosiglitazone (grey) or amorfrutin B (black) (n=6-9 per group):
(a) Fgf21 gene expression in white adipose tissue (WAT) and liver;
(b) Plasma levels of FGF21 determined by ELISA. Data are expressed as mean ± SEM. n.s. not significant, *p<0.05, **p<0.01 , ***p<0.001 vs. vehicle. VEH, vehicle; RGZ, rosiglitazone; AB, amorfrutin B.
Figure 20 shows the phosphorylation of PPARy-Ser273 in WAT of DIO mice treated for 4 weeks with vehicle control (VEH, white), rosiglitazone (RGZ, grey) or amorfrutin B (AB, black):
(a) Western Blot of representative samples (n=4 per group);
Densitometric analysis (n=10-13 per group). Data are expressed as mean ± SEM. re 21 shows the effect of amorfrutin B on viability (white triangles) and cytotoxicity (black triangles) in human HepG2 cells after treatment for 24 h (n=3, mean ± SD). re 22 shows:
the analysis of genetic toxicity in the in vitro micronucleus assay in Chinese hamster ovary (CHO) cells in absence (-S9) or presence (+S9) of rat liver homogenate extract: CHO cells were treated with different concentrations of amorfrutin B (AB, n=2), the clastogens mitomycin C (MMC, n=4) or cyclophosphamide (CP, n=4), or vehicle (VEH), and micronuclei were stained with Hoechst dye and counted by fluorescence microscopy;
the expression of genes related with macrophage invasion and inflammation in murine livers of DIO mice treated 4 weeks with rosiglitazone or amorfrutin B: Murine livers of DIO mice treated for 4 weeks with rosiglitazone (grey) or amorfrutin B (black) were analysed by qPCR for expression of genes associated with macrophage invasion and inflammation (n=6-9 per group);
RNA expression of liver Fabp4: RNA expression of liver Fabp4 was analyzed by qPCR;
the effect of amorfrutin B (n=13) and rosiglitazone (n=10) or vehicle (n=1 1 ) on liver damage-indicating plasma levels of alanine transaminase (ALT) after treatment for 4 weeks in DIO mice;
the expression of genes involved in osteoblagenesis: MC3T3-E1 preosteoblasts (pointed bars) were differentiated to osteoblasts in presence of vehicle only (white, set to 1 ), rosiglitazone (grey) or amorfrutin B (black). Expression of genes involved in osteoblastogenesis was determined by qPCR.
the calcification of differentiated MC3T3-E1 osteoblasts: the calcification of differentiated MC3T3-E1 osteoblasts treated with amorfrutin B (AB, n=6), rosiglitazone (RGZ, n=4) or vehicle (VEH, n=4) was measured by Alizarin Red S staining;
the effect of amorfrutin B on plasma osteocalcin concentration after treatment for 4 weeks in DIO mice. Osteocalcin was measured by ELISA;
the body weight gain of obese mice after treatment with amorfrutin B (black triangles, n=13), rosiglitazone (grey squares, n=10) or vehicle only (white circles, n=13). Data are expressed as mean ± SEM. n.s. not significant, *p<0.05, **p<0.01 , ***p<0.001 vs. vehicle. VEH, vehicle; RGZ, rosiglitazone; AB, amorfrutin B; PRE, preosteoblasts. re 23 shows the effect of the compounds A1 , A2, A3, A4 and thiazolidinediones on osteoblast differentiation:
Expression of genes involved in osteoblastogenesis was determined by qPCR;
Calcification of differentiated MC3T3-E1 osteoblasts and preosteoblasts was measured by Alizarin Red S staining. Data are expressed as mean ± SEM. n.s. not significant, *p<0.05, **p<0.01 vs. vehicle. PRE, preosteoblasts; VEH, vehicle; RGZ, rosiglitazone; PGZ, pioglitazone; TGZ, troglitazone; AB, amorfrutin B (NP-015142); A1 , amorfrutin 1 (NP-003520); A2, amorfrutin 2 (NP-003521 ); A3, amorfrutin 3 (NP-006430); A4, amorfrutin 4 (NP-009525). re 24 shows:
the effect of amorfrutin B (NP-015142) treatment on daily food intake in DIO mice. Data are expressed as mean ± SEM. ***p<0.001 vs. vehicle; variation of body weight with cumulative energy intake: Analysis of co- variance (ANCOVA) was performed on cumulative energy intake and body weight after 14 days of treatment. In both treatment groups body weight moderately correlates with energy intake (VEH, p=0.07; AB, p=0.09), but AB-treated mice show a shift towards lower body weight additional to the decrease in energy intake (p=0.02 for elevation of regression curve), indicating that anti-obesity effects of AB are not exclusively attributed to less energy intake during the first 3 days;
the effect of amorfrutin B and rosiglitazone on plasma triiodothyronine (T3) and thyroxine (T4) concentration after 27 days of treatment (n=5- 13). Data are expressed as mean + SEM. n.s. not significant vs. vehicle; variation of HOMA-IR index with body weight: ANCOVA was performed on HOMA-IR index and body weight data after 14 days of treatment. As expected, in both treatment groups insulin resistance correlates with total body weight (VEH, p=0.01 ; AB, p=0.002), but AB-treated mice clearly show an additional shift towards lower insulin resistance irrespective of decrease in body weight (p=0.01 for elevation of
regression curve), indicating that anti-diabetic effects of AB are not exclusively attributed to body weight reduction. VEH, vehicle; AB, amorfrutin B. Figure 25 shows the effect of amorfrutin B (NP-015142) and rosiglitazone on blood parameters after treatment for 4 weeks in DIO mice (n=9-13):
(a) Plasma HDL and LDLA/LDL cholesterol levels;
(b) Hematocrit after treatment for 4 weeks in DIO mice;
(c) Colorimetric analysis of whole blood hemoglobin. Data are expressed as mean + SEM. n.s. not significant, **p<0.01 , vs. vehicle. VEH, vehicle;
RGZ, rosiglitazone; AB, amorfrutin B.
Figure 26 shows additive effects of compound NP-015934 and anticancer reference drugs on proliferation of HT-29 colon carcinoma cells. Cells were treated for 3 days with different mixtures of the indicated compounds, and proliferation was quantified by fluorometric detection of the cellular DNA content. IC'70 data were plotted as isobologram:
(a) NP-015934 and cisplatin;
(b) NP-015934 and irinotecan;
Data are expressed as mean ± SD (n=4/group). CI, combination index.
Figure 27 shows staining of HT-29 colon carcinoma cells with annexin-V-FLUOS and propidium iodide after detection by flow cytometry. Cells were treated with 20 μΜ of NP-015934 for 2 days before staining:
(a) Microscopical image and annexin-V/propidium iodide scatter plot of treated HT- 29 cells.
(b) Percentages of apoptotic cells (annexin-V-positive) after treatment. Data are expressed as mean ± SEM (n=3/group). ***p<0.001 vs. control. Figure 28 shows the formation of reactive oxygen species (ROS) in HT-29 cells treated with NP-015934 and its role in apoptosis:
(a) Fluorescence intensity of the ROS-sensitive dye H2-DCFDA during treatment of HT-29 cells with 30 μΜ of NP-015934 in the absence or presence of antioxidants.
(b) Area under the curve (AUC) of H2-DCFDA fluorescence (a). Data are expressed as mean ± SEM (n=7/group). ***p<0.001 vs. control, ###p<0.001 vs. NP-015934.
(c) Percentages of apoptotic cells (annexin-V-positive) after treatment. Data are expressed as mean ± SEM (n=3/group). ***p≤0.001 vs. control only, n.s. not significant vs. NP-015934 only. Ctrl, Control; NAC, N-acetylcysteine (1 mM); GSH, glutathione (5 mM); D3T, 3H-1 ,2-Dithiole-3-thione (50 μΜ); ATOC, o Tocopherol (50 μΜ), AA, ascorbic acid (1 mM).
(d) Annexin-V/propidium iodide scatter plot of HT-29 cells. Cells were pre-treated for 1 h with indicated antioxidants, and 30 μΜ NP-015934 was added for additional 24 h of co-treatment. Figure 29 shows the effect of NP-015934 on the essential mitochondrial transmembrane potential (ΔΨητι) and the role of the 2-hydroxyl residue:
(a) Chemical structure of NP-015934 and NP-015934met.
(b) Concentration-dependent effect of NP-015934 and NP-015934met on the mitochondrial transmembrane potential, determined in the fluorometric JC-1 assay. HT-29 cells were treated with indicated compound concentrations for
15 min. Data are expressed as mean ± SD (n=4/group).
Figure 30 shows opening of the mitochondrial permeability transition pore (MPTP) in HT-29 cells treated with NP-015934 or NP-015934met for 30 min. Cells were stained with calcein and CoCI2 and were quantified by flow cytometry:
(a) Fluorescence intensity histogram of treated HT-29 cells.
(b) Mean fluorescence intensities of indicated treatments. Data are expressed as mean ± SEM (n=3/group). ***p<0.001 vs. control, ###p<0.001 vs. NP-015934, n.s. not significant
Figure 31 shows the effects of NP-015934 and NP-015934met on oxygen consumption and extracellular acidification of HT-29 cells by use of phosphorescent oxygen- and pH-sensitive probes:
(a) Fluorescence lifetime of an oxygen-sensitive probe in HT-29 cells during treatment with indicated compounds. Fluorescence lifetime increases with reduction in extracellular oxygen concentration.
(b) The rate of probe fluorescence lifetime was determined between 20 and 60 min of treatment (a) and plotted relative to untreated cells.
(c) Extracellular acidification in HT-29 cells during treatment with indicated compounds, measured by fluorescence of a pH-sensitive probe.
(d) The rate of extracellular acidification was determined between 20 and 150 min of treatment (c) and plotted as hydrogen ions per minute. Data are expressed
as mean ± SEM (n=8/group). *p<0.05, **p<0.01 , ***p<0.001 vs. control, ###p<0.001 vs. NP-015934, n.s. not significant. CCCP, carbonyl cyanide m- chlorophenyl hydrazone (2 μΜ); AMA, antimycin A (2 μΜ). Figure 32 shows staining of HT-29 colon carcinoma cells with annexin-V-FLUOS and propidium iodide after detection by flow cytometry. Cells were treated for 1 day with indicated concentrations of NP-015934 or NP-015934met or both:
(a) Annexin-V/propidium iodide scatter plot of treated HT-29 cells.
(b) Percentages of apoptotic cells (annexin-V-positive) after treatment. Data are expressed as mean ± SEM (n=3/group). ***p<0.001 vs. control, n.s. not significant.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.
EXAMPLES
Abbreviations used in the Examples that follow are: AB (Amorfrutin B, NP- 015142), ALT (Alanine transaminase), DIO (Diet-induced obesity), HFD (High-fat diet), PPAR (Peroxisome proliferator-activated receptor), RGZ (Rosiglitazone), siRNA (Small interfering RNA), SPPARyM (selective Peroxisome proliferator- activated receptor γ modulator), TZDs (thiazolidinediones), VEH (Vehicle), vWAT (Viscerale white adipose tissue).
A. Materials
Compounds were purchased from the following: rosiglitazone (Cayman, Biozol, Eching, Germany), pioglitazone, troglitazone, GW7647, GW0742, N- acetylcysteine, glutathione, 3H-1 ,2-dithiole-3-thione, a-tocopherol, ascorbic acid, carbonyl cyanide m-chlorophenyl hydrazone (Sigma Aldrich, Taufkirchen, Germany), Antimycin A (Biomol GmbH). All natural products (NPs) described in this study were provided by AnalytiCon Discovery (Potsdam, Germany) and isolated by standard procedures from natural sources. The compounds were isolated using standard chromatography procedures from natural sources (isolated microbial strains (terrestrial or marine origin) or plants (several partitions with different chemical profiles)). Identity of each isolated natural product was confirmed using LC/MS and NMR.
Example A.1. Isolation of NP-003520, NP-015136, NP-009525, NP-015142, NP- 015135, NP-015137
530g of dried seeds of Amorpha fruticosa (provided by Friedrich Nature Discovery Gmbh; Euskirchen, Germany) were extracted twice with MeOH-MTB-ether and yielded 73g raw extract. By repeated chromatography (stationary phase RP-8 and RP-18, mobile phase methanol-water and acetonitrile-water (buffered with ammonium formate) natural products NP-003520, NP-015136, NP-009525, NP- 015142, NP-015135, NP-015137 were isolated in a purity (HPLC, ELSD-detection, and H-NMR) of >70%. Structures were elucidated by interpretation of LCMS, 1 D and 2D NMR (HSQC, HMBC, HH-COSY) data. Example A.2. Isolation of NP-003521 , NP-006431 , NP-015934, NP-015935, NP- 015936, NP-006430, NP-006427, NP-015933, NP-015953, NP-015954, NP- 015937, NP-015938, NP-015939
327g of roots of Giycyrrhiza foetida (provided by Friedrich Nature Discovery Gmbh; Euskirchen, Germany) were extracted twice with MeOH-MTB-ether and yielded 33g raw extract. By repeated chromatography (stationary phase RP-8 and RP-18, mobile phase methanol-water and acetonitrile-water (buffered with ammonium formate/formic acid) natural products NP-003521 , NP-006431 , NP- 015934, NP-015935, NP-015936, NP-006430, NP-006427, NP-015933, NP- 015953, NP-015954, NP-015937, NP-015938, NP-015939 were isolated in a purity (HPLC, ELSD-detection, and H-NMR) of >70%. Structures were elucidated by interpretation of LCMS, 1 D and 2D NMR (HSQC, HMBC, HH-COSY) data.
Example A.3. Isolation of NP-01221 1 , NP-016018, NP-016020, NP-016021 570g of aerial parts of Cannabis sp. (collected in Michendorf, Germany, by AnalytiCon Discovery) were extracted twice with MeOH-MTB-ether and yielded 66g raw extract. By repeated chromatography (stationary phase RP-8 and RP-18, mobile phase methanol-water and acetonitrile-water (buffered with ammonium formate /formic acid) natural products NP-01221 1 , NP-016018, NP-016020, NP- 016021 were isolated in a purity (HPLC, ELSD-detection, and H-NMR) of >70%. Structures were elucidated by interpretation of LCMS, 1 D and 2D NMR (HSQC, HMBC, HH-COSY) data.
Example A.4. Isolation of NP-01241 1 , NP-012412, NP-012626
552g of aerial parts of Eriodictyon sp. (Syn. Yerba santa, provided by Alfred Galke Gmbh, Gittelde, Germany) were extracted twice with MeOH-MTB-ether and yielded 154g raw extract. By repeated chromatography (stationary phase RP-8 and RP-18, mobile phase methanol-water and acetonitrile-water (buffered with ammonium formate/formic acid) natural products NP-01241 1 , NP-012412, NP- 012626 were isolated in a purity (HPLC, ELSD-detection, and H-NMR) of >70%. Structures were elucidated by interpretation of LCMS, 1 D and 2D NMR (HSQC, HMBC, HH-COSY) data.
Example A.5. Isolation of NP-009326, NP-004976, NP-004977, NP-004978 560g of aerial parts of Rapanaea melanophloeos (provided by Kenya National Academy of Science, Nairobi, Kenya) were extracted twice with MeOH-MTB-ether and yielded 50g raw extract. By repeated chromatography (stationary phase RP-8 and RP-18, mobile phase methanol-water and acetonitrile-water (buffered with ammonium formate/formic acid) natural products NP-009326, NP-004976, NP- 004977, and NP-004978 were isolated in a purity (HPLC, ELSD-detection, and H-
5
NMR) of >70%. Structures were elucidated by interpretation of LCMS, 1 D and 2D NMR (HSQC, HMBC, HH-COSY) data.
Example A.6. Isolation of NP-002329 and NP-01 1855
525g of seeds of Anacardium occidentale (provided by Friedrich Nature discovery, Euskirchen, Gernnany) were extracted twice with MeOH-MTB-ether and yielded 50g raw extract. By repeated chromatography (stationary phase RP-8 and RP-18, mobile phase methanol-water and acetonitrile-water (buffered with ammonium formiate/formic acid) natural products NP-002329 and NP-01 1855 were isolated in a purity (HPLC, ELSD-detection, and H-NMR) of >70%. Structures were elucidated by interpretation of LCMS, 1 D and 2D NMR (HSQC, HMBC, HH-COSY) data.
Example A.7. Isolation of NP-001727 and NP-001728
331 g of aerial parts of Picris altissima (provided by the Botanical Garden of Berlin, Germany) were extracted twice with MeOH-MTB-ether and yielded 50g raw extract. By repeated chromatography (stationary phase RP-8 and RP-18, mobile phase methanol-water and acetonitrile-water (buffered with ammonium formate/formic acid) natural products NP-001727 and NP-001728 were isolated in a purity (HPLC, ELSD-detection, and H-NMR) of >70%. Structures were elucidated by interpretation of LCMS, 1 D and 2D NMR (HSQC, HMBC, HH-COSY) data.
Example A.8. Isolation of NP-014467
331 g of aerial parts of Syzygium jambos (provided by Friedrich Nature Discovery, Euskirchen, Germany) were extracted twice with MeOH-MTB-ether and yielded 50g raw extract. By repeated chromatography (stationary phase RP-8 and RP-18, mobile phase methanol-water and acetonitrile-water (buffered with ammonium formate/formic acid) natural product NP-014467 was isolated in a purity (HPLC, ELSD-detection, and H-NMR) of >70%. Structure was elucidated by interpretation of LCMS, 1 D and 2D NMR (HSQC, HMBC, HH-COSY) data.
Example A.9. Isolation of NP-006243
An undetermined fungal strain (isolated at AnalytiCon, strain No. 01458fxxx000010) was fermented in a nutrient medium containing mainly sucrose, glutamic acid, salts and Amberlite XAD 1 180 for 5 days at 30°C in a stirred vessel with 10 litres working volume.
The lyophilized biomass was extracted twice with MeOH-Acetone and yielded 40g raw extract. By repeated chromatography (stationary phase RP-8 and RP-18, mobile phase methanol-water and acetonitrile-water (buffered with ammonium formate/formic acid) natural product NP-006243 was isolated in a purity (HPLC, ELSD-detection, and H-NMR) of >70%. Structure was elucidated by interpretation of LCMS, 1 D and 2D NMR (HSQC, HMBC, HH-COSY) data.
Example A.10. Isolation of NP-000420
A fungal strain (Hyalodendron sp., isolated for AnalytiCon, strain No. 05048febs006260) was fermented in a nutrient medium containing mainly corn meal and malt extract for 7 days at 21 °C in a stirred vessel with 10 litres working volume.
The lyophilized biomass was extracted twice with MeOH-Acetone and yielded 40g raw extract. By repeated chromatography (stationary phase RP-8 and RP-18, mobile phase methanol-water and acetonitrile-water (buffered with ammonium formate/formic acid) natural product NP-000420 was isolated in a purity (HPLC, ELSD-detection, and H-NMR) of >70%. Structure was elucidated by interpretation of LCMS, 1 D and 2D NMR (HSQC, HMBC, HH-COSY) data. Example A.11. Isolation of NP-012584
An undetermined fungal strain (isolated at AnalytiCon, strain No. 02465fxxx000012) was cultivated on 1 .5 kg of a solid substrate containing mainly rice, millet and a solution of salts for 19 days at 25°C.
The culture was extracted twice with MeOH-Acetone and yielded 70g raw extract. By repeated chromatography (stationary phase RP-8 and RP-18, mobile phase methanol-water and acetonitrile-water (buffered with ammonium formate/formic acid) natural product NP-012584 was isolated in a purity (HPLC, ELSD-detection, and H-NMR) of >70%. Structure was elucidated by interpretation of LCMS, 1 D and 2D NMR (HSQC, HMBC, HH-COSY) data.
Example A.12. Isolation of NP-001782 and NP-001787
An fungal strain allocated to the genus Penicillium (isolated at AnalytiCon, strain No. 01672fxxx000012) was cultivated on 1 .1 kg of a solid substrate containing mainly rice, millet and a solution of salts for 19 days at 25°C.
The culture was extracted twice with MeOH-Acetone and yielded 50g raw extract. By repeated chromatography (stationary phase RP-8 and RP-18, mobile phase methanol-water and acetonitrile-water (buffered with ammonium formate/formic acid) natural product NP-001782 and NP-001787 were isolated in a purity (HPLC,
7
ELSD-detection, and H-NMR) of >70%. Structure was elucidated by interpretation of LCMS, 1 D and 2D NMR (HSQC, HMBC, HH-COSY) data.
Example A.13. Isolation of NP-001269
An undetermined fungal strain (isolated at AnalytiCon, strain No. 00410fxxx000005) was cultivated on 0.5 kg of a solid substrate containing mainly rice, millet and a solution of salts for 19 days at 25°C.
The culture was extracted twice with MeOH-Acetone and yielded 10g raw extract. By repeated chromatography (stationary phase RP-8 and RP-18, mobile phase methanol-water and acetonitrile-water (buffered with ammonium formate/formic acid) natural product NP-001269 was isolated in a purity (HPLC, ELSD-detection, and H-NMR) of >70%. Structure was elucidated by interpretation of LCMS, 1 D and 2D NMR (HSQC, HMBC, HH-COSY) data. Purity assessment of isolated compounds
All isolated natural products were analysed by HPLC/MS/ELSD to get the molecular weights and the purity. HPLC-conditions are listed in Table 2. The results of the purity assessment together with the retention time Rt are listed in Table 3.
Table 2: Conditions of the HPLC/MS/ELSD analysis
HPLC System PE Series 200
MS System Applied Biosystems API 150, 165 or 365
Data System Analyst 1 .3 or Masschrom 1 .2.1 .
Stationary Phase Merck Select B 250x4 mm, 5 μιτι
Flow Rate 1 ml/min
Detection (+/(-)-ESI, Fast-Switching-Mode
ELSD (Sedex 75)
UV (Merck, 254 nm)
Sample Concentration 10 mg/ml in DMSO
Injection Volumen 30 μΙ
Mobile Phase: A: 5 mM ammonium formate and 0.1 % formic acid
B: acetonitrile/methanol = 1 :1 , 5 mM ammonium formate and 0.1 % formic acid (pH 3)
Gradient Time [min] % A % B
00.0 85 15
30.0 0 100
35.0 0 100
Table 3: Purity [%] and retention times (Rt [min]) of the isolated natural products
Compound Rt [min] Purity [%]
NP-000420 19.19 99.1
NP-001269 16.49 81 .0
NP-001727 22.55 70.3
NP-001728 25.35 80.6
NP-001782 16.7 99.9
NP-001787 .6 86.3
NP-002329 29.77 88.3
NP-003520 27.79 99.9
NP-003521 28.69 99.6
NP-004976 25.52 86.2
NP-004977 25.8 91 .8
NP-004978 26.39 90.5
NP-006243 24.33 97.7
NP-006427 21 .78 93.3
NP-006430 24.36 99.6
NP-006431 25.33 94.0
NP-009326 26.45 83.8
NP-009525 27.98 88.4
NP-01 1855 30.32 98.6
NP-01221 1 27.6 100.0
NP-01241 1 20.19 95.7
NP-012412 20.91 87.1
NP-012584 30.27 99.4
NP-012626 22.72 93.4
NP-014467 26.25 99.2
NP-015135 25.96 85.9
NP-015136 27.56 97.8
NP-015137 27.1 98.7
NP-015142 30.19 100.0
NP-015933 28.52 98.9
NP-015934 29.82 99.9
NP-015935 30.51 95.5
NP-015936 28.94 96.0
NP-015937 26.58 96.5
NP-015938 27.01 96.2
NP-015939 27.44 97.9
NP-015953 23.24 93.5
NP-015954 23.45 85.8
NP-015955 25.06 97.5
NP-016018 25.79 85.9
NP-016020 28.51 94.7
NP-016021 27.87 96.8
B. Chemical synthesis
General procedures. Starting materials were synthesized as described by Tu (Synthesis, 2000, 13, 1956) or purchased from Sigma-Aldrich and used without further purification. Dry THF was distilled from CaH2 and all other anhydrous solvents were prepared using 3 A molecular sieves. All reactions were performed under argon atmosphere with a continuous argon flow. Silica gel flash column chromatography was performed on Combiflash RF (Teledyne ISCO) chromatography systems using normal-phase silica gel and ethyl acetate/hexanes mixtures as solvents. 1H and 13C NMR spectra were obtained on 500 and 600 MHz Varian INOVA NMR spectrometers using CDCI3 and acetone-d6 (Cambridge Isotope Laboratories) as solvents. Spectra were calibrated to the solvent, 7.26 ppm for 1H NMR spectra in CDCI3, 2.05 ppm for 1 H NMR spectra in acetone-d6, 77.16 ppm for 13C NMR spectra in CDCI3, and 29.8 ppm for 13C NMR spectra in acetone-d6.
2* 1*
To a solution of lithium diisopropyi amide (LDA) (19.4 mmol, 1 .3 equiv) in anhydrous THF (40 mL) contained within a 500 mL Schlenk flask cooled to -78 °C using a dry ice-acetone bath was added drop wise hydrazone 2* (3.1 g, 14.9 mmol, 1 equiv) (prepared according to Synthesis, 2000, 13, 1956). The mixture was allowed to warm to -30 °C while stirring over a period of 60 minutes and then subsequently recooled to -78 °C. To the deprotonated hydrazone was added a solution under argon of 3,3-dimethylallylbromide in THF (5 mL) very slowly as to maintain a uniform temperature. The reaction mixture was allowed to warm to room temperature while stirring overnight. The reaction was first diluted with THF (100 mL) and was proceeded by the addition of 1 M HCI (150 mL, 10 equiv) to quench excess base and hydrolyze the hydrazone. The hydrolysis was monitored using thin layer chromatography (hexanes: ethyl acetate = 10:1 ). After 30 minutes of stirring, the product was extracted with a 1 :1 mixture of hexanes and ethyl acetate (3 x 50 mL) and the combined organic extracts were dried over Na2S04, decanted into a 1 L round bottom flask and concentrated under reduced pressure. Purification of the crude product using flash column chromatography (silica gel, 100% hexanes progressing to 2:1 hexanes, ethyl acetate) afforded the ketone 1*
5
(1 .4 g, 48% yield) as a clear liquid. 1H NMR (600 MHz, CDCI3): δ 5.06 (m, 2H, CH=C(CH3)2), 2.41 (t, 4H, CH2C=O), 2.24 (dt, 4H, CH2CH2C=O), 1 .67 (s, 6H), 1 .61 (s, 6H) ppm. Example B.2. Synthesis of diethyl 2-(1 -hydroxy-3-phenylpropylidene) malonate
(3*)
To a flame-dried 500 ml_ Schlenk flask was added magnesium turnings (1 .59 g, 65.3 mmol, 1 .10 equiv) and absolute ethanol (5 ml_). A portion (1 ml.) of a 1 :1 solution by volume of diethyl malonate (9.5 g, 59.3 mmol, 1 equiv) and absolute ethanol (9 mi_) was added to the turnings suspension and stirred vigorously, followed by the addition of a catalytic amount of CCI4 (91 .2 mg, 0.593 mmol, 0.01 equiv). Once the reaction was initiated (usually requiring 5 min stirring), the remainder of the diethyl malonate solution was added slowly with continuous stirring. The reaction was heated under reflux at 80 °C overnight to achieve complete dissolution of the magnesium turnings and subsequently cooled to room temperature. The resulting magnesium salt was concentrated under reduced pressure and then resuspended in anhydrous diethyl ether (50 ml_). The resulting suspension was heated under reflux at 50°C for 30 minutes and then recooied to room temperature. Hydrocinnamoyl chloride (10 g, 59.3 mmol, 1 equiv) was added cautiously over a period of 15 minutes at room temperature, and the reaction was stirred overnight. The mixture was cooled to 0 °C and added to an ice-cold aqueous solution (100 mL) of H2SO4 (30 mmol, 0.5 equiv), and the resulting mixture was extracted with diethyl ether (3 x 100 mL) in a 1 L separatory funnel. The combined organic layers were dried over Na2SO4, decanted into a 1000 mL round bottom flask and concentrated under reduced pressure. Purification using flash column chromatography (silica gel, 20:1 hexanes : ethyl acetate progressing to 1 :1 hexanes : ethyl acetate) afforded compound 3* (14.4 g, 83% yield) as a viscous, clear liquid. 1 H NMR (600 MHz, CDCI3): (enol form) δ 13.49 (s, 1 H, enol- OH), 4.27 (q, 2H), 4.22 (q, 2H), 2.96 (m, 2H), 2.77 (m, 2H), 1 .31 (t, 3H), 1 .27 (t, 3H) ppm.
*Note: The title compound exists as an keto:enol ratio of ca 1 :3.
5
Example B.3. S nthesis of diethyl 2-(1 -chloro-3-phenylpropylidene)malonate (4*)
A 250 mL Schlenk flask was charged with compound 3* (12.0 g, 41 mmol, 1 .0 equiv). POCI3 (37.8 g, 246 mmol, 6.0 equiv) was subsequently added and the mixture was stirred for 5 min at room temperature. Following the 5 minute period, the reaction was cooled to -10 °C using an ice-salt bath. At -10 °C was added drop wise triethylamine (4.16 g, 41 mmol, 1 .0 equiv) to the vigorously stirred solution over the course of 10 min, yielding an off-white suspension. The ice bath was removed and the mixture was allowed to stir at room temperature for 22 h. The reaction was then heated to 80 °C under reflux and stirred at this temperature for an additional 4 h. The reaction was then cooled to 0 °C using an ice-water bath and diluted with 200 mL anhydrous dichloromethane (DCM). The diluted solution was then added drop wise cautiously over a period of 15 min to an ice- cooled suspension of NaHCO3 (124 mmol) in H2O (600 mL). Once the gas evolution ceased, the pH of the aqueous mixture was adjusted to 7 using 1 M aqueous HCI. The organic phase was then isolated, and the aqueous layer was washed with DCM (2 x 100 mL). The organic collections were combined, dried over Na2SO4 and concentrated under reduced pressure. The residue was purified using flash column chromatography (silica gel, 10:1 hexanes : ethyl acetate progressing to 1 :1 hexanes : ethyl acetate) yielding chloride 4* as a faintly yellow oil. H NMR (600 MHz, CDCI3): δ 7.32-7.28 (m, 2H), 7.26-7.15 (m, 3H), 4.32 (q, 2H), 4.20 (q, 2H), 3.21 (m, 2H), 2.96 (m, 2H), 1 .34 (t, 3H), 1 .26 (t, 3H) ppm.
Example B.4. Synthesis of ethyl 2,4-dihydroxy-3,5-bis(3-methyl-2-butenyl)-6- phenethylbenzoate (5*)
4* 1 * 5*
To a solution of LDA (17.0 mmol, 2.2 equiv) in anhydrous THF (34 mL) cooled to -78 °C using a dry ice-acetone bath was added ketone 1* (3.0 g, 15.5 mmol, 2 equiv). The reaction mixture was allowed to warm to -30 °C while stirring over a
5 period of 30 min. Subsequently, the reaction was recooled to -78 °C, followed by the addition of a solution of chloride 4* (2.24 g, 7.73 mmol,1 equiv) in THF (4 mL) (Note: chloride 4* was dried via azeotropic evaporation of toluene (50 mL) in vacuo, prior to use in this reaction). The reaction was allowed to slowly warm to room temperature and was monitored by thin layer chromatography (hexanes : ethyl acetate = 10:1 ). After 16 h of stirring at room temperature, the reaction was cooled to 0°C using an ice-water bath, diluted with hexanes (80 mL) and acidified using 1 M aqueous HCI (15 mL). The mixture was extracted using diethyl ether (3 x 100 mL) and the organic phases were combined. The organic collection was washed with brine solution (1 x 100 mL) an H2O (1 x 100 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified using flash column chromatography (silica gel, 100% hexanes progressing to hexanes : ethyl acetate = 2:1 ) yielding compound 5* (1 .82g, 56% yield ) as a clear, viscous liquid. 1H NMR (600 MHz, CDCI3): δ 1 1 .67 (s, 1 H, OH), 7.26-7.22 (m, 2H, phenyl), 7.17-7.12 (m, 3H, phenyl), 6.01 (s, 1 H, OH), 5.26 (m, 1 H), 5.06 (m, 1 H), 4.44 (q, 2H, COOCH2CH3), 3.45 (d, 2H), 3.36 (d, 2H), 3.23 (m, 2H), 2.84 (m, 2H), 1 .83 (s, 3H), 1 .75 (broad s, 6H), 1 .71 (s, 3H), 1 .36 (t, 3H, COOCH2CHs) ppm; 13C NMR (126 MHz, CDCI3): δ 172.14, 160.17, 158.46, 142.31 , 141 .27, 135.07, 133.40, 128.55, 128.19, 126.08, 123.04, 121 .83, 1 19.45, 1 12.50, 105.98, 61 .61 , 37.31 , 32.74, 26.01 , 25.89, 25.28, 22.48, 18.18, 18.08 14.52 ppm.
Example B.5. Synthesis of ethyl 2-hydroxy-4-methoxy-3,5-bis(3-methyl-2- butenyl)-6-phenethylbenzoate (6*)
A solution of compound 5* (1 .68 g, 3.98 mmol, 1 .0 equiv) in anhydrous acetone (75 mL) was cooled to 0 °C using an ice-water bath and K2CO3 (3.30 g, 23.9 mmol, 6.0 equiv) was added. After 5 min of stirring at 0 °C, methyl iodide (0.58 g, 4.09 mmol, 1 .03 equiv) was slowly added to the vigorously stirring solution. The ice bath was then removed and the reaction was allowed to stir at room temperature for 6 h. The mixture was then concentrated under reduced pressure
5 and the concentrate was dissolved in a 1 :1 :1 mixture (v/v/v) of ice water, hexanes and diethyl ether. The solution was neutralized with 1 M aqueous HCI. The organic phase was isolated and the aqueous phase was washed with a 1 :1 mixture of hexanes and diethyl ether (2 x 50 mL). The collected organic phases were combined, dried over a2S0 and concentrated under reduced pressure. The residue was purified using flash column chromatography (silica gel, 100% hexanes progressing to 2:1 hexanes : ethyl acetate) yielding amorf rutin 6* (1 .3 g, 75% yield) as soft clear crystals. 1H NMR (600 MHz, CDCI3): δ 1 1 .16 (s, 1 H, OH), 7.32-7.28 (m, 2H, phenyl), 7.23-7.17 (m, 3H, phenyl), 5.25 (m, 1 H), 5.03 (m, 1 H), 4.45 (q, 2H, COOCH2CH3 ), 3.71 (s, 3H, OCH3), 3.39 (d, 2H), 3.34 (d, 2H,), 3.23 (m, 2H), 2.82 (m, 2H), 1 .79 (s, 3H), 1 .71 (s, 3H), 1 .70 (s, 3H), 1 .66 (s, 3H) ppm; 13C NMR (125 MHz, CDCI3): δ 171 .80, 161 .61 , 160.27, 142.34, 141 .63, 131 .93, 131 .64, 128.55, 128.25, 126.32, 126.1 1 , 124.38, 123.02, 121 .37, 109.96, 61 .87, 61 .67, 37.57, 32.59, 25.93, 25.83, 25.52, 23.72, 18.26, 18.14, 14.49 ppm.
Example B.6. Synthesis of 2-Hydroxy-4-methoxy-3,5-bis(3-methyl-2-butenyl)-6- phenethylbenzoic acid (7*) corresponding to isolated product NP_015934
A 500 mL Schlenk flask was charged with KOH (13.1 g, 234 mmol, 20 equiv) pellets, which were subsequently dissolved using H2O (10 mL) at room temperature. Once the KOH was completely dissolved and all excess heat dissipated, dimethylsulfoxide (DMSO) (100 mL) was added. Amorfrutin 6* (5.1 g, 1 1 .7 mmol, 1 .0 equiv) was transferred to the basic solution with a minimal amount of DMSO (5 mL). The reaction mixture was heated to 120 °C under reflux for 6 h, while monitoring with thin layer chromatography (hexanes : ethyl acetate = 2:1 ) the disappearance of the spot representing starting material, and then cooled to 0°C using an ice-water bath. The chilled solution was transferred to a 1 L Erlenmeyer flask containing 1 M aqueous HCI (265 mL), hexanes (200 mL), and diethyl ether (200 mL), being vigorously stirred at 0°C. After 5 min stirring, the organic phase was isolated using a 1 L separatory funnel, and the aqueous layer was washed with 1 :1 hexanes : diethyl ether (2 x 100 mL). The organic extracts were
5 combined, washed with H2O (2 x 100 ml_), dried over Na2SO and concentrated under reduced pressure. Purification using flash column chromatography (silica gel, 6:1 hexanes : ethyl acetate progressing to 1 :1 hexanes : ethyl acetate) afforded amorfrutin 7* (4.0 g, 84% yield) as a crystalline white solid. 1H NMR (600 MHz, acetone-de): δ 7.32-7.25 (m, 4H), 7.18-7.14 (m, 1 H), 5.28 (m, 1 H), 5.07 (m,1 H), 3.71 (s, 3H, OCH3), 3.41 -3.33 (m, 6H), 2.85 (m, 2H), 1 .78 (s, 3H), 1 .73 (s, 3H), 1 .66 (bs, 3H) ppm; 13C NMR (125 MHz, acetone-d6): δ 174.27, 162.22, 161 .74, 143.45, 143.19, 131 .58, 131 .38, 129.14, 129.07, 126.64, 126.41 , 125.51 , 124.07, 121 .46, 1 10.41 , 61 .75, 38.30, 34.02, 25.93, 25.85, 25.76, 24.03, 18.20, 17.99 ppm.
C. Biological evaluation Methods used in the following examples:
PPAR binding, cofactor recruitment and transcriptional activation assays
Binding of natural products to PPARs was quantified by use of a competitive time- resolved fluorescence resonance energy transfer (TR-FRET) assay according to the manufacturer's protocol (Lanthascreen PPAR competitive binding assay, Life Technologies, CA, USA) as described recently (Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 7257). Briefly, terbium-tagged PPAR ligand binding domain was titrated with varying ligand concentrations in the presence of constant concentrations of the fluorescein-labelled PPAR ligand fluormone. Increasing the concentration of unknown PPAR ligands results in a displacement of the labelled PPAR ligand and hence in a decrease of the TR-FRET signal. Fluorescence intensity was measured with the POLARstar Omega (BMG LABTECH, Offenburg, Germany). Data were fitted using GraphPad Prism 5.0 according equation: Y=Bottom + (Top- Bottom)/(1 +10A((LoglC50-X)*HillSlope)) with variable Hill slope, and affinity constants (K,) were calculated according to the manufacturer's protocol.
Binding of transcriptional cofactors was measured by a peptide-based TR-FRET assay according to the manufacturer's instruction (Lanthascreen PPARy coactivator assay, Life Technologies). Efficacy is the maximal association (for coactivators) or dissociation (for corepressors) normalized to the full PPARy agonist rosiglitazone (set to 100%). Transcriptional activation of PPARs was assessed in cellular reporter gene assays according to the manufacturer's protocols (GeneBLAzer PPAR Assay, Life Technologies). Briefly, HEK 293 cells
were stably expressing a GAL4-PPAR-LBD fusion protein and an UAS-beta- lactamase reporter gene. Cells were incubated with different concentrations of compounds resulting in differential expression of the reporter gene. In reporter gene assays efficacy is the maximal transcriptional activation normalized to the full PPAR agonists RGZ, GW7647 or GW0742 (set to 100%). Fluorescence of all assays was measured with the POLARstar Omega (BMG LABTECH, Offenburg, Germany). Data were fitted using GraphPad Prism 5.0 according equation: Y=Bottom + (Top-Bottom)/(1 +10A((LogEC50-X)*HillSlope)) with variable Hill slope.
Cell culture
Primary subcutaneous preadipocytes isolated from human patients were provided by Zen-Bio (BioCat, Heidelberg, Germany) and treated as described previously. [Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 7257] Briefly, preadipocytes were maintained in preadipocyte medium (PM-1 , Zen-Bio) and then differentiated using PPARy agonist-free adipocyte medium (AM-1 , Zen-Bio) supplemented with 500 μηηοΙ/L 3-isobutyl-1 -methylxanthine (Sigma Aldrich, Taufkirchen, Germany) for 7 days. Subsequently, medium was changed to pure AM-1 for additional 7 days. Mature adipocytes were treated with 10 μηηοΙ/L amorfrutin B or 10 μηηοΙ/L rosiglitazone diluted in AM-1 for 24 hours, whereas 0.1 % DMSO was used as vehicle control.
Mouse MC3T3-E1 preosteoblast cells (subclone 4, CRL-2593, ATCC, LGC Promochem, Wesel, Germany) were cultured in Minimum Essential Medium a medium (MEMa, A1049001 , Gibco, Life Technologies) with 10% fetal bovine serum (FBS, Biochrom, Berlin, Germany) and 1 % penicillin/streptomycin (Biochrom) at 37 °C and 5% CO2. One-day post-confluent cells were differentiated to osteoblasts in MEMa additionally supplemented with 200 μηηοΙ/L ascorbic acid and 10 mmol/L β-glycerophosphate (all Sigma-Aldrich). The preosteoblasts were treated with amorfrutin B (10 μΜ), amorfrutins 1 -4 (each 10 μΜ), rosiglitazone (10 μΜ), pioglitazone (10 μΜ), troglitazone (10 μΜ) or vehicle (0.1 % DMSO) during the whole differentiation. The cells were collected after 7 days for gene expression analyses and after 24 days for staining. Calcification was determined by staining of osteoblasts with Alizarin red S (Sigma) according to [Anal. Biochem. 2004, 329, 77].
Primary subcutaneous preadipocytes (Zen-Bio, BioCat, Heidelberg, Germany) were differentiated and treated as described in Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 7257.
5
Viability and cytotoxicity were assessed in human HepG2 cells (ATCC) cultured in DMEM (Gibco, Life Technologies) supplemented with 10% FBS treated with amorfrutin B for 24 h using the CellTiter-Glo Luminescent Cell Viability Assay and the CytoTox-Glo Cytotoxicity Assay (both Promega, Mannheim, Germany) respectively, according to the manufacturer's protocols. In vitro micronucleus assays were performed in CHO-K1 cells at Cerep, Inc. (Redmond, WA, USA) according to Mutat. Res. 2007 630, 1 .
Antiproliferative effects were investigated in human HT-29 (DSZM, Braunschweig, Germany) and T84 (ATCC) colon carcinoma cells, in PC3 (ATCC) prostate cancer cells and MCF-7 (ATCC) breast cancer cells. HT-29 and T84 cells were cultured in DMEM/F-12 (ATCC) supplemented with 5% FBS (Biochrom) and 1 % penicillin/streptomycin (Biochrom), PC3 cells were cultivated in RPMI 1640 (Biochrom) supplemented with 10% FBS (Biochrom) and 1 % penicillin/streptomycin (Biochrom), MCF-7 cells were cultured in DMEM GlutaMAX (Gibco, Life Technologies) supplemented with 10% FBS (Biochrom), 10 g/ml human insulin (Sigma Aldrich) and 1 % penicillin/streptomycin (Biochrom), all at 37 °C and 5% CO2. For one-concentration screening studies, one day before treatment cells were seeded in 96 well plates (TPP) with a density of 4000 cells/well (HT-29), 5000 cells/well (MCF-7) and 1500 cells/well (PC3), respectively, in a final volume of 200 L/well. Cells were then treated with the indicated compound concentrations by adding 50 μί of a 5 times stock concentration. For concentration series, cells were seeded in black 384 well plates (Corning #3712) with a density of 750 cells/well (HT-29), 900 cells/well (T84), 250 cells/well (PC3), respectively, in a final volume of 50 L/well. Cells were then treated with the indicated compound concentrations by adding 10 μί of a 6 times stock concentration. After 3 days (HT-29, PC-3, MCF-7) and 4 days (T84) of treatment, cells were quantified using the CyQUANT NF Cell Proliferation Assay Kit (Life Technologies) according to the manufacturer's instructions. Fluorescence intensity was measured with the POLARstar Omega (BMG LABTECH). For dilution series, data were fitted using GraphPad Prism 5.0 according equation: Y=Bottom + (Top- Bottom)/(1 +10A((LoglC50-X)*HillSlope)) with variable Hill slope. Efficiency is the maximal observed induction of cell death after treatment relative to nontreated cells. Additive effects were determined by treatment with compound mixtures with following ratios: 7:0, 6:1 , 5:2, 4:3, 3:4, 2:5, 1 :6, 0:7. HT-29 cells were treated with different concentration series of these compound mixtures. For each mixture, compound IC'70 values, which gives the concentration needed to inhibit cancer
cell growth by 70%, were calculated and plotted as isobologram according the Loewe additivity model (Klin Wochenschr. 1927, 6, 1077). The combination index (CI) for compounds 1 and 2 in each mixture (M) was calculated as follows: CI = IC∞(M1 )/IC50(1 ) + IC5o(M2)/IC5o(2).
Phosphatidylserine external ization of HT-29 cells treated with NP-015934 was determined by staining with annexin-V-FLUOS and propidium iodide (Roche Life Science) and subsequent flow cytometry (Accuri C6, BD Biosciences) according to manufacturer's instructions. Analysis was performed using FlowJo 7.6 (Tree Star).
Formation of reactive oxygen species (ROS) was measured by use of the ROS- sensitive dye H2-DCFDA (Life Technologies) according to the manufacturer's instruction. Briefly, one day before treatment HT-29 cells were seeded in 96 well plates (TPP) with a density of 15000 cells/well. Before treatment, adherent cells were washed once with pre-warmed PBS and loaded with 50 μΜ dye diluted in PBS. Cells were then incubated for 30 min at 37°C to allow incorporation and activation of H2-DCFDA, followed by removal of free dye and washing with pre- warmed PBS. Phenol-Red-free medium was added and cells were again incubated at 37 °C for 60 min. Compounds were added as indicated, and fluorescence (485/530 nm) was measured with the POLARstar Omega (BMG Labtech) at 37 °C for 22 h of treatment using orbital averaging and bottom optics.
For investigating the mitochondrial transmembrane potential (ΔΨιτι) the JC-1 assay (Cayman Chemicals) was performed according to the manual. This assay makes use of a lipophilic cationic dye (5,5',6,6'-Tetrachloro-1 ,1 ',3,3'- tetraethylbenzimidazolylcarbocyanine iodide), which selectively enters into mitochondria and changes reversibly its color from red to green as the membrane potential decreases. For this assay, one day before treatment HT-29 cells were seeded in 96 well plates (TPP) with a density of 40000 cells/well. One day later, cells were treated with indicated compounds for 5 min at 37 °C, followed by addition of JC-1 dye for additional 10 min. Cells were then washed twice with pre- warmed JC-1 assay buffer to remove free JC-1 dye. Fluorescence measurement was performed in JC-1 assay buffer in the POLARstar Omega (BMG Labtech) with following filter settings: JC-1 -aggregates (excitation 560/10 nm, emission 590/30 nm), JC-1 monomers (excitation 485/30 nm, emission 520/10 nm). The ratio of JC- 1 aggregates to monomers fluorescence was used as indicator of mitochondrial transmembrane potential. Data were fitted using GraphPad Prism 5.0 according
5 equation: Y=Bottom + (Top- Bottom)/(1 +10A((LoglC50-X)*HillSlope)) with variable Hill slope.
To explore the effects on the mitochondrial permeability transition pore (MPTP) the MitoProbe Transition Pore Assay Kit (Life Technologies) was used according to the manufacturer's instructions. Cells were loaded with a calcein dye that accumulates in cytosolic compartments, including the mitochondria. The fluorescence of cytosolic calcein was quenched by addition of C0CI2, while mitochondrial fluorescence is maintained. Opening of the MPTP leads to loss of mitochondrial calcein fluorescence. For that purpose, HT-29 cells were suspended in HBSS/Ca buffer with a density of 106 cells/ml, and labeled with 10 nM calcein and 400 μΜ CoCI2. Subsequently, NP-015934 or NP-015934met was added as indicated. Cells were incubated at 37 °C for 30 min and subsequently washed with HBSS/Ca buffer before counting by flow cytometry (Accuri C6, BD Biosciences) according to manufacturer's instructions. Analysis was performed using FlowJo 7.6 (Tree Star) and Prism 5.0 (GraphPad).
Oxygen consumption was determined by time-resolved fluorescence of an oxygen-sensitive probe (MitoXpress-Xtra HS, Luxcel Biosciences). Probe fluorescence is quenched by molecular oxygen, so that fluorescence lifetime increases with reduction in extracellular oxygen concentration. One day before treatment, HT-29 cells were seeded in 96 well plates (TPP) with a density of 80000 cells/well in DMEM/F-12 supplemented with 5% FBS and 1 % penicillin/streptomycin and incubated at 37 °C and 5% CO2. For subsequent oxygen consumption measurements, the medium was removed and cells were incubated with 140 μΙ of pre-warmed probe diluted in phenol red-free DMEM/F- 12/FBS/penicillin/streptomycin. After incubation for 10 min at 37 °C, cells were then treated with the indicated compound concentrations by adding 20 μΙ_ of a 8 times stock concentration. Finally, cells were sealed with 100 μΙ of pre-warmed HS mineral oil (MitoXpress-Xtra HS) to prevent back diffusion of ambient oxygen. Time-resolved fluorescence was measured in the POLARstar Omega (BMG Labtech) with following settings: temperature = 37 °C; TRF optic Z height = 6 mm; excitation = 380/20 nm; emission = 655/50 nm; window 1 (w1 ): 30 με delay and 30 με integration time; window 2 (w2): 70 με delay and 30 με integration time; interval time = 90 s; measurement time = 150 min. Background fluorescence was measured in wells with medium and oil, but without cells and probe. For data analysis, background fluorescence was subtracted individually for both measurement windows. Fluorescence lifetime (τ) for each sample was then
5 calculated by τ = 40/(ln(w1/w2)), and plotted over treatment time. For comparing oxygen consumption between treatments, the rate of probe fluorescence lifetime was determined between 20 and 60 min and expressed relative to untreated cells. Data were analyzed using Prism 5.0 (GraphPad).
Extracellular acidification was determined by time-resolved fluorescence of a pH- sensitive probe (pH Xtra, Luxcel Biosciences). Fluorescence lifetime of this probe increases with decrease in pH, so that it allows measurement of extracellular acidification. One day before treatment, HT-29 cells were seeded in 96 well plates (TPP) with a density of 80000 cells/well in DMEM/F-12 supplemented with 5% FBS and 1 % penicillin/streptomycin and incubated at 37 °C in a CO2-free incubator. For subsequent measurements the following low-buffering aspiration medium was used according to the manufacturer's instructions: 1 mM PBS (pH 7.4), 20 mM glucose, 75 mM NaCI, 54 mM KCI, 2.4 mM CaCI2 and 0.8 mM MgSO4. Before treatment, cells were washed twice with 200 μΙ aspiration buffer, and incubated with 140 μΙ of pre-warmed probe diluted in aspiration medium. After incubation for 10 min at 37 °C, cells were then treated with the indicated compound concentrations by adding 20 μΙ_ of a 8 times stock concentration. Time- resolved fluorescence was measured in the POLARstar Omega (BMG Labtech) with following settings: temperature = 37°C; TRF optic Z height = 6 mm; excitation = 380/20 nm; emission = 615/50 nm; window 1 (w1 ): 100 s delay and 30 s integration time; window 2 (w2): 300 s delay and 30 s integration time; interval time = 100 s; measurement time = 200 min. Background fluorescence was measured in wells with medium and oil, but without cells. For data analysis, background fluorescence was subtracted individually for both measurement windows. Fluorescence lifetime (τ) for each sample was then calculated by T = 200/(ln(w1/w2)), transformed to absolute pH values according pH = (1687.2- lifetime)/199.12 (Anal. Biochem. 2009, 390, 21 ), and plotted over treatment time. For comparing extracellular acidification between treatments, the acidification rate was determined between 20 and 150 min. Data were analyzed using Prism 5.0 (GraphPad).
PPARy knockdown
Specificity of PPARy modulation was investigated in siRNA-mediated PPARy- knockdown in adipocytes with subsequent real-time PCR detection as described in Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 7257. Briefly, differentiated human adipocytes were seeded in 24-well-plates (Nunc) at a confluence of 30 to 60%. Cells were transfected with 10 nmol/L PPARy Silencer Select Validated siRNA (ID
S10888) or 10 nmol/L Silencer Select Negative Control #1 siRNA (all Life Technologies) using DeliverX Plus siRNA Transfection Kit (Panomics, BioCat). Transfection was carried out in serum- and antibiotic-free AM-1 medium (AM-1 - PRF-SF, Zen-Bio) for 4 h and continued for 3 days in standard AM-1 medium. Afterwards, cells were additionally treated with 10 μηηοΙ/L amorfrutin B, 10 μηηοΙ/L RGZ or vehicle control for 24 hours prior to RNA collection.
RNA purification, cDNA synthesis and quantitative real-time PCR (qPCR)
Animal tissues were first lysed and homogenized in TRIzol (Invitrogen) with 5 mm steel beads at 20 Hz for 8 min (TissueLyser, QIAGEN), and isolation of total RNA was done according to the manufacturer's instruction. Subsequent RNA purification was performed using the RNeasy Mini Kit (QIAGEN) and genomic DNA digestion (DNase-Set, QIAGEN) according to the manual. The concentration of extracted RNA was measured using the Nanodrop ND-1000 Spectrophotometer (Fisher Scientific). RNA was reversely transcribed into cDNA applying the High Capacity cDNA Reverse Transcription Kit (Life Technologies) with random primers. Quantitative PCR was carried out on the ABI Prism 7900HT Sequence Detection System using the SYBR Green PCR Master Mix (all Life Technologies). After an initial denaturation at 95 °C for 10 min, the cDNA was amplified by 40 cycles of PCR (95 °C, 15 s; 60 °C, 60 s). The relative gene expression levels were normalized using β-actin gene and quantified by the 2 AACt method [Method. Methods San Diego, Calif, 2001 , 25, 402]. Primer sequences are summarized in Table 4.
Caspase activation
Activation of caspases 3 and 7 were investigated using the luminometric Caspase- Glo 3/7 Assay (Promega) according to the manufacturer's instructions. Briefly, one day before treatment HT-29 and MCF-7 cells were seeded in black 384 well plates (Corning, #3712) with a density of 2000 cells/well in a final volume of 20 L/well. Cells were treated for the indicated time with the given compound concentrations by adding 5 μί of a 5 times stock concentration. Luminescence was measured with the POLARstar Omega (BMG LABTECH). Activation of caspases 2, 3, 6, 7, 8, 9 and 10 were investigated using the fluorimetric Homogeneous Caspases Assay, (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. Briefly, one day before treatment HT-29 and PC-3 cells were seeded in black 384 well plates (Corning, #3712) with a density of 1300 and 1000 cells/well, respectively, in a final volume of 20 L/well. Cells were treated for the indicated time with the given compound concentrations by adding 5 μί of a 5 times
stock concentration. Fluorescence was measured with the POLARstar Omega (BMG LABTECH).
DNA fragmentation assay
BrdU-labeled DNA fragments in the cytoplasm of treated HT-29 cells were quantified with the Cellular DNA Fragmentation ELISA (Roche Diagnostics) according to the instructions for use. Briefly, HT-29 cells were seeded in 96 well plates (TPP) with a density of 13000 cells/well in a final volume of 200 L/well with 10 μηηοΙ/L BrdU and incubated for 2 days at 37 °C. Supernatant was removed and cells were incubated in 100 μΙ_ medium containing the compound at given concentrations. After the indicated treatment time, the cytosolic fractions were harvested and analysed on a 96 well half-area clear high-binding microplate (Corning, #3690) according to the manual. Absorbance was measured at 450 and 690 nm. Cell-free samples were used as background control for subtraction.
Pharmacokinetics Pharmacokinetic experiments were performed at Charles River (Edinburgh, UK). Briefly, 18 male C57BL/6 mice were orally administered with 100 mg/kg body weight amorfrutin B in 1 % carboxymethylcellulose. Terminal blood samples were collected after 0.5, 1 , 2, 4, 8, and 24 h after dosing and analyzed using LC- MS/MS. Animal studies
Animal studies have been approved by the State Office of Health and Social Affairs Berlin and were carried out according to internationally approved guidelines. All animals were singly housed under temperature-, humidity- and light- controlled conditions (22 °C, 50% humidity, 12 hours light/12 hours dark-cycle). Mice had ad libitum access to food and water. Mice and food were weighed in a regularly manner to determine changes in body weight and food intake. Male C57BL/6 mice at age of 6 weeks were fed for 12 weeks with high-fat diet (HFD, D12492, kJ composition: 60% fat, 19% protein, 21 % carbohydrate, 25.3 MJ/kg, ssniff, Soest, Germany) to induce obesity and insulin resistance. Prior to treatment, mice were weighed and distributed uniformly to 3 groups (n = 10-13 each). Mice were fed over 4 weeks with HFD without compound (vehicle), with 4 mg/kg/d rosiglitazone (RGZ) or with 100 mg/kg/d amorfrutin B (AB) incorporated in the food.
After 15 days of treatment an intraperitoneal insulin sensitivity test (IPIST) was performed. Mice were fasted overnight and then had ad libitum access to food for
1 h before the test. 1 .5 U/kg body weight of insulin (Sigma-Aldrich) was injected intraperitoneally. Blood was taken from tail vein at the indicated time points and blood glucose was analysed in a Hemocue B-Glucose analyser (Hemocue, Grc^ostheim, Germany). Whole blood was further collected using Microvette lithium-heparin coated capillary tubes (CB300, Sarstedt, Nurnbrecht, Germany). After centrifugation for 5 min at 2000 g and 4 °C, plasma was collected and stored at -80 °C for subsequent measurements.
After 22 days of feeding an oral glucose tolerance test (OGTT) was carried out. Mice were fasted overnight before being subjected to an oral dose of 2 g/kg body weight of glucose (Sigma-Aldrich). Blood was taken from tail vein at the indicated time points and examined as described above.
After 27 days of dosing, fasted mice were sacrificed by cervical dislocation. Hematocrit was measured by weighting of blood samples before and after plasma separation. Plasma and tissues were collected and stored at -80 °C before use. Metabolic parameters measurements
Blood glucose was analysed in a Hemocue B-Glucose analyzer. Plasma glucose was measured using the Amplex Red Glucose Assay Kit (Life Technologies). Plasma triacylglycerols, NEFA, HDL and LDL cholesterol and plasma alanine transaminase (ALT) were determined with colorimetric quantification kits (Biovision, BioCat). Plasma β-hydroxybutyrate was analyzed colorimetrically (Cayman, Biomol, Hamburg, Germany). Insulin (Insulin Ultrasensitive EIA, ALPCO, Immundiagnostik, Bensheim, Germany), triiodothyronine (T3), thyroxin (T4) (Calbiotech, San Diego, CA, USA), osteocalcin (BGLAP bone gamma- carboxyglutamate (gla) protein, ABIN415574, antibodies-online, Aachen, Germany) and FGF21 (BioVendor, Heidelberg, Germany) were determined in plasma samples using ELISA. Whole blood haemoglobin (Hemoglobin Colorimetric Assay Kit, Cayman, Biomol) was analysed for investigation of hemodilution. All assays were performed according to the manufacturer's instructions. HOMA-IR was determined according to HOMA-IR = fasting blood glucose (mg/dL) χ fasting insulin ( U/mL)/405.
Immunoblottinq
Visceral white adipose tissue of treated mice was lysed in UEES lysis buffer (9 M Urea, 100 mM EDTA EGTA, 4% SDS with protease and phosphatase inhibitors) using 5 mm steel beads at 20 Hz for 8 min (TissueLyser). After centrifugation for
10 min at 10000 g, the supernatants were stored at -80 °C until use. Samples were denatured and separated using a NuPAGE Novex 4-12% Bis-Tris gel (Invitrogen) and blotted onto nitrocellulose membranes. Membrane was blocked with a solution containing 1 .5% milk powder, 1 .5% BSA in PBS-T (0.1 %) and 0.5x phosphatase inhibitor overnight at 4 °C. Membranes were washed in PBS-T (0.1 %). A rabbit polyclonal phospho-specific antibody against PPARy Ser 273 was produced by Eurogentech (Seraing, Belgium) with the phosphopeptide Ac- KTTDKpSPFVIYDC-amide [Nature 2010, 466, 451]. For detection, 0.8 pg/mL PPARy-pSer273 and 0.5 pg/mL PPARy (E-8, Santa Cruz, Heidelberg, Germany) antibody, respectively, were diluted in PBS-T (0.1 %) with 1 .5% milk powder and 1 .5% BSA. Membranes were shaken overnight at 4 °C and subsequently incubated with anti-rabbit IgG-HRP (Santa Cruz, #sc-2004) and anti-mouse IgG- HRP (Santa Cruz, #sc-2005), respectively, prior to detection with Western Lightning ECL solution (Perkin Elmer). Membranes were stripped with Restore Plus Western Blot Stripping Buffer (Thermo Scientific) for 10 min. Densitometry was performed with GelAnalyzer 2010. The rate of PPARy phosphorylation was normalized to total PPARy protein and plotted on logarithmic scale.
Statistical analyses
Data are presented as mean ± standard error of mean (SEM) if not otherwise denoted. Statistical tests were performed in GraphPad Prism 5.0. For comparison of two groups statistical significance was examined by unpaired two-tailed Student's t-test if not otherwise stated. For multiple comparisons data were analysed by one-way ANOVA with subsequent Dunnett's post test. A p value < 0.05 was defined as statistically significant. To exclude that observed reduction in adiposity is not exclusively attributed to a change in caloric intake (see Figure 24b), and to determine that the anti-diabetic effects are not exclusively attributed to body weight reduction (see Figure 24d), we performed analysis of covariance (ANCOVA) tests as explained in [Nat. Methods 2012, 9, 57]. Briefly, ANCOVA works by plotting individual data for two phenotypes (e.g. HOMA-IR index vs. body weight) and fitting them with a linear regression curve. Comparison in that regression line of two different treatments (e.g. treated vs. untreated) allow for effectively testing if both phenotypes are strongly dependent on each other (same slope and interception of regression lines) or if both phenotypes are fully or partly independent from each other (different slope and/or interception of regression lines).
Table 4: Primer sequences used for quantitative real-time PCR.
Gene
Symbol Forward primer Reverse primer
ID
ACTB 60 CAGCCATGTACGTTGCTATCCAGG AGGTCCAGACGCAGGATGGCATG
ADIPOQ 9370 GGTGAGAAGGGTGAGAAAGG TCCTTTCCTGCCTTGGATT
ANGPTL2 23452 GGGAGACGTACAAGCAAGGG CGGAAACTGGCGTATTCTGC
ANGPTL4 51 129 GATCCCCACGGCGAGTTC CCGTGATGCTATGCACCTTCT
CD36 948 GTTGATTTGTGAATAAGAACCAGAGC TGTTAAGCACCTGTTTCTTGCAA
CEBPA 1050 CTAACTCCCCCATGGAGTCGG GTCGATGGACGTCTCGTGC
CEBPB 1051 ACTTTAG CG AGTCAG AG CCG GATTTAAAGGCAGGCGGCG
CFD 1675 TGAAGGTCAGGGTCACCCAA GACCAACCAGATGCAGGAGT
FABP4 2167 GGTGGTGGAATGCGTCATG CAACGTCCCTTGGCTTATGC
FGF21 26291 TGGATCGCTCCACTTTGACC GGGCTTCGGACTGGTAAACA
HSD1 1 B1 3290 GGCCTCATAGACACAGAAACAGC TGATCTCCAGGGCACATTCC
LEP 3952 TGTGCGGATTCTTGTGGCTT GGAGACTGACTGCGTGTGT
LPL 4023 ACAGAATTACTGGCCTCGATCC CTGCATCATCAGGAGAAAGACG
NR1 H3 10062 CACCTACATGCGTCGCAAGT G ACAG G ACACACTCCTCCCG
PCK2 5106 CTGAGGAGGAGAATGGGCG AG AG CCAACC AG CAGTTGTCA
PDE3B 5140 TCGAGACATTCCTTATCACAATCG GGAACTGGCCGTGTTGTCA
PDK4 5166 CTGGACTTTGGTTCAGAAAATGC CCTTCAGAATGTTGGCGAGTCT
PLTP 5360 G ACACCGTG CCTGTG CG GGTGGAAGCCACAGGATCCT
PPARG 5468 CATGGCAATTGAATGTCGTGTC CCGGAAGAAACCCTTGCAT
RARRES2 5919 CAGGCCCAATGGGAGGAAAC GGCCCAGAACTTTGTCCTCA
Acadl 1 1363 AGCCTGGGGCTGGAAGTGACTTA CACGGTTGGTGACGGCCACG
Acadm 1 1364 ACGGGGGAAAGGCCAACTGGTAT AGGATCTGGGTTAGAACGTGCCA
Acoxl 1 1430 CAGCACTGGTCTCCGTCATG CTCCGGACTACCATCCAAGATG
Actb 1 1461 TGTCCACCTTCCAGCAGATGT AGCTCAGTAACAGTCCGCCTAGA
Adipoq 1 1450 AGGAAAGGAGAGCCTGGAGA CGAATGGGTACATTGGGAAC
Bdh1 7191 1 GTGTGTGTAAGGCACATCCG CTTCTGGCTTGGAGTTGGCT
5
Bdh2 69772 GTCTTTGGATTGTGCCGCAG GCAAAAGCTAATGCGGATGC
Bglap 12096 GTCCCACACAGCAGCTTGGCCC ACAGAGCGCAGCCAGGGTCAG
Ccl3 20302 GCTCCCAGCCAGGTGTCATTTTCC GGGGTTCCTCGCTGCCTCCA
Ccl5 20304 CTCACTGCAGCCGCCCTCTG CCG AG CCATATG GTG AGG CAGG
Ccr2 12772 TC AG CTG CCTG CAAAG ACCAG A CGGTGTGGTGGCCCCTTCAT
Ccr5 12774 AGACTCTGGCTCTTGCAGGATGGA GGCAGGAGCTGAGCCGCAAT
Cptla 12894 TCTGCAGACTCGGTCACCACTCAAG GGCTCAGGCGGAGATCGATGC
Cptl b 12895 GAGATCAAGCCGGTCATGGCAC AGGCGCGAGCCCTCATAGAGC
Cpt2 12896 AAGCAGCGATGGGCCAG GAGCTCAGGCAGGGTGACC
CxcM 14825 GGCCCCACTGCACCCAAACC CAAGGCAAGCCTCGCGACCA
Emr1 13733 ACCCTCCAGCACATCCAGCCAA TC AC AG CCCG AGG GTGTCCA
Fabp4 1 1770 CATGGCCAAGCCCAACAT CG CCCAGTTTG AAG G AAATC
Fbp1 14121 GCATCGCACAGCTCTATGGT ACAGGTAGCGTAGGACGACT
Fgf21 56636 AGACAGCCTTAGTGTCTTCTCA CCAAGGCAGCTGGAATTGTG
G6pc 14377 GCTGGAGTCTTGTCAGGCAT ATCCAAGCGCGAAACCAAAC
G6pc3 68401 TATGGGTTGACTGCTCTGGC CCAGGTTGATGGACCAGGAAA
Hmgcl 15356 TGTACCCACCCCAGTGAAGA GAGTGGTCAGCCATCTGTGG
Hmgcsl 208715 GTCCTGGCACAGTACTCACC TGTGGCGTCTTGTGTGACTT
Hmgcs2 15360 AGAGGCCTTCAGGGGTCTAA TTGAACATGTCCAGGGAGGC
Ibsp 15891 CG GTTTCCAGTCCAGG G AGG CA TGGGGTGTGGTCTCTGCTCCG
116 16193 TCTGCAAGAGACTTCCATCCAGTTGC AGGCCGTGGTTGTCACCAGC
Lep 16846 CCCTGTGTCGGTTCCTGTGGC TGGCGGATACCGACTGCGTG
Pck1 18534 CCTAGTGCCTGTGGGAAGAC AGCCCTTAAGTTGCCTTGGG
Pck2 74551 GTACCACTG GTGTACG AG GC GTCTTTCCTTTGTGCTCCGC
Pcx 18563 GGGCGGAGCTAACATCTACC TATACTCCAGACGCCGGACA
Pdk4 27273 ATATTCAGTGACTCAAAGACGGGAA ACACTCAAAGGCATCTTGGACTACT
Phex 18675 GTGGCACCCTGGTGTTGGGC ATGGCAGCAGCGGCTTCTATGC
Pklr 18770 AGTCTGTG GTTCTTG CAG CC CTGG AG CCCCACTTAAAG CA
Ppargda 19017 TCCCATACACAACCGCAGTCGC GGGGTCATTTGGTGACTCTGGGGT
Ppargd b 170826 GGGAAAAGGCCATCGGTGAA CAGCACCTGGCACTCTACAA
Ptgs2 19225 CCCTGCTGCCCGACACCTTC CCAGCAACCCGGCCAGCAAT
Slc2a2 20526 CCAGGTCCAATCCCTTGGTT CCCAAGGAAGTCCGCAATGT
Taldol 21351 CAACGAAGACCAAATGGCCG CATTCGTTCCGTGAGCATCC
Tnf 21926 AGCCCACGTCGTAGCAAACCA CATGCCGTTGGCCAGGAGGG
Ucp1 22227 CACGGGGACCTACAATGCTT TAGGGGTCGTCCCTTTCCAA
Ucp2 22228 CGCCTTCTACAAGGGGTTCA CGAGATTGGTAGGCAGCCAT
Ucp3 22229 ACAAAGGATTTGTGCCCTCC TCAAAACGGAGATTCCCGCA
Example C.1 : Binding affinity assays.
The binding affinity of the compounds displayed in Table 1 for PPARs was analyzed using in vitro binding assays.
The binding affinities for PPARy ranging from 19 nmol/L to 6 μηηοΙ/L are summarized in Figure 1 and Table 5.
Table 5: Compound data and PPAR binding/activation constants
Source PPARa PPARv
Compound Additional name CAS Ki EC50 Efficacy Ki Ki EC50 Efficacy EC50 Efficacy
Genus and Species
(μιηοΙ/Ι) (μιηοΙ/Ι) (%) (μιηοΙ/Ι) (pmol/l) (μιηοΙ/Ι) (%) (μιηοΙ/Ι) (%)
NP-000420 1083197-79-8 # F n.d. 48 n.d. n.d. 4.4 0.708 n.d. n.d. 8.5 74
NP-001269 913690-90-1 F n.d. n.d. n.d. n.d. n.d. > 100 n.d. n.d. n.d. n.d.
NP-001727 none P Picris altissima 38 n.d. n.d. 38 1.3 n.d. n.d. 9.6 81
NP-001728 none P Picris altissima 9.6 4.3 49 n.d. 0.860 n.d. n.d. 2.9 94
NP-001782 Altenusin 31 186-12-6 F n.d. 60 n.d. n.d. 15 3.2 n.d. n.d. n.d. n.d.
NP-001787 Alternarian acid 91868-93-8 F n.d. > 100 n.d. n.d. > 100 > 100 n.d. n.d. n.d. n.d.
NP-002329 103904-73-0 P 2.8 0.173 60 2.2 0.264 5.4 54 0.928 124
NP-003520 Amorfrutin 1 80489-90-3 P G.foetida, A. fruticosa 27* 0.699 23 27* 0.236* n.d. n.d. 0.051* 75
NP-003521 Amorfrutin 2 80489-91-4 P G.foetida, A. fruticosa 25* 0.390 18 17* 0.287* n.d. n.d. 0.318* 85
NP-004976 none P Myrsine capitellata 24 n.d. n.d. 12 1.7 n.d. n.d. n.d. n.d.
NP-004977 none P Myrsine capitellata n.d. n.d. n.d. n.d. 2.9 n.d. n.d. n.d. n.d.
NP-004978 none P Myrsine capitellata 11 n.d. n.d. 6.7 3.2 n.d. n.d. n.d. n.d.
NP-006243 none F n.d. 12 4.7 15 3.8 0.305 0.066 12 1.1 73
NP-006427 1083192-64-6 P Glycyrrhiza foetida n.d. n.d. n.d. n.d. 3.2 n.d. n.d. n.d. n.d.
NP-006430 Amorfrutin 3 946570-99-6 P G.foetida, A. fruticosa 115* 0.807 13 68* 0.352* n.d. n.d. 2.8* 92
NP-006431 70610-12-7 P Glycyrrhiza foetida 7.8 0.903 28 2.3 0.093 1.3 24 0.413 97
NP-009326 none P n.d. n.d. n.d. n.d. > 100 n.d. n.d. n.d. n.d.
NP-009525 Amorfrutin 4 none P G.foetida, A. fruticosa 8.0* 0.660 14 6.0* 0.278* n.d. n.d. 0.335* 78
NP-01 1855 103904-74-1 P 1.6 0.224 68 3.8 0.167 5.8 70 0.770 125
NP-01221 1 Cannabidiol 13956-29-1 P Cannabis sativa n.d. n.d. n.d. n.d. 4.7 n.d. n.d. n.d. n.d.
GAR-P03598WO07 Application (without Figures).doc
MP-01241 1 1083200-69-4 # P 128 n.d. n.d. 40 1.6 n.d. n.d. 9.4
MP-012412 none P 32 n.d. n.d. 5.4 1.0 n.d. n.d. 4.9 81
MP-012584 llicicolin B 22581-07-3 F n.d. 3.2 n.d. n.d. 8.9 1.1 n.d. n.d. n.d. n.d.
MP-012626 Eriolic acid A 1207435-63-9 P 86 n.d. n.d. 39 0.871 n.d. n.d. 2.2 74
MP-014467 143522-31-0 P Syzygium jambos 25 n.d. n.d. 30 2.3 n.d. n.d. n.d. n.d.
MP-015135 1 174387-93-9 P Amorpha fruticosa 7.2 n.d. n.d. 3.6 1.2 n.d. n.d. 0.660 108
MP-015136 73436-07-4 P Amorpha fruticosa 5.0 1.7 22 1.4 0.134 1.0 36 n.d. n.d.
MP-015137 70610-10-5 P Amorpha fruticosa 6.8 1.5 20 8.9 0.969 0.215 6.0 6.1 1 18
MP-015142 Amorfrutin B 78916-42-4 P G.foetida, A. fruticosa 2.6" 0.906 61 1.8 0.019* 0.073 25 0.060 61
MP-015933 1 189096-44-3 P Glycyrrhiza foetida 1.6 0.157 16 3.0 0.41 1 0.298 27 0.349 73
MP-015934 1 189096-45-4 P Glycyrrhiza foetida 9.1 n.d. n.d. 5.2 0.723 1.2 7.0 n.d. n.d. iMP-015935 1189096-22-7 P Glycyrrhiza foetida 8.2 n.d. n.d. 5.8 0.524 0.439 5.0 0.581 69
NP-015936 1 189096-23-8 P Glycyrrhiza foetida 4.4 0.480 14 7.0 0.613 0.423 16 0.169 60
NP-015937 1 189096-25-0 P Glycyrrhiza foetida 6.0 4.4 22 5.0 0.413 0.379 7.0 0.023 60
NP-015938 1189096-26-1 P Glycyrrhiza foetida 6.6 n.d. n.d. 5.6 1.3 n.d. n.d. n.d. n.d.
NP-015939 1 189096-29-4 P Glycyrrhiza foetida 6.2 n.d. n.d. 6.8 0.626 0.796 12 1.7 67
NP-015953 1234832-83-7 P Glycyrrhiza foetida 7.8 2.8 29 4.8 0.492 0.025 5.0 0.135 58
NP-015954 1235317-27-7 P Glycyrrhiza foetida 1 1 1.9 48 4.5 0.508 6.0 1 1 2.2 74
NP-015955 1235159-42-8 P Glycyrrhiza foetida 18 n.d. n.d. 24 0.454 6.9 79 0.150 104
NP-016018 Cannabidivarin 24274-48-4 P Cannabis sativa n.d. n.d. n.d. n.d. 6.0 n.d. n.d. n.d. n.d.
NP-016020 25555-57-1 P Cannabis sativa 7.0 1.4 24 2.7 0.280 0.093 12 n.d. n.d.
NP-016021 Cannabidiolic acid 1244-58-2 P Cannabis sativa n.d. n.d. n.d. n.d. > 100 n.d. n.d. n.d. n.d.
Amorfrutin B (NP-015142) (Figure 2a) showed the lowest, nanomolar binding affinity constant to PPARy (Ki = 19 nM, Table 6) similar to the standard PPARy- targeting drug rosiglitazone (Avandia, Ki = 7 nM, Table 6), and 12-times lower than the initially described amorfrutin A1 .
Table 6. Binding and activation of PPARs by amorfrutin B (NP-015142): Binding affinity (Ki) values were obtained by using competitive TR-FRET assays, effective concentrations (EC50) and efficacy values were determined from reporter gene assays. Efficacy is the maximum activation relative to the reference agonist, n.d., not determined.
Receptor Amorfrutin B Rosiglitazone GW7647 GW0742
PPARa
Ki(mmol/L) 2624 n.d. 1 n.d.
EC50(mmol/L) 906 n.d. 0.3 n.d.
Efficacy(%) 61 n.d. 100 n.d.
PPARp/y
Ki(mmol/L) 1782 n.d. n.d. 0.4
EC50(mmol/L) 740 n.d. n.d. 0.2
Efficacy(%) 3 n.d. n.d. 100
PPARy
Ki(mmol/L) 19 7 n.d. n.d.
EC50(mmol/L) 73 4 n.d. n.d.
Efficacy(%) 25 100 n.d. n.d.
Additionally, the compounds bound to the isotypes PPARa and PPAR /δ. The results are summarized in Figure 3, Figure 4 and Table 5. Amorfrutin B (NP- 015142) revealed potent binding to PPARa (Ki = 2624 nM, Table 6) and PPAR /δ (Ki = 1782 nM, Table 6) with low-micromolar affinity, suggesting that this compound may also modulate the activity of these PPARs. Such partial pan-PPAR modulation may enable to concomitantly treat diabetes-associated disorders, but is difficult to trace mechanistically due to the involvement and potential cross-talk of various tissues.
Example C.2: Transcriptional activation assays.
The transcriptional activation potential of the compounds displayed in Table 1 using reporter gene assays was evaluated. PPARy activation with EC50 values ranging from 25 nmol/L to 6.9 μηηοΙ/L and transactivation efficacies from 5% to
7
79% relative to RGZ (see Figure 5 and Table 5) was observed. Amorfrutin B (NP-015142) showed nanomolar effective concentrations (EC50 = 73 nM) and reduced maximal PPARy activation (efficacy = 25%, see Figure 2b and Table 5). In contrast to the full PPARy agonist RGZ, amorfrutins only partially induced transcriptional activation of PPARy, suggesting that the compounds of this invention represent a new chemical class of selective PPAR modulators (SPPARMs).
Transcriptional activation of the PPARa isotype was additionally tested in reporter gene assays, with amorfrutin B (NP-015142) revealing EC50 and efficacy values of 906 nmol/L and 61 %, respectively (see Figure 2c and Table 6) and a range from 157 nmol/L to 4.7 μηηοΙ/L and 13% to 68%, respectively, for the rest of tested compounds (see Figure 6 and Table 6). In spite of activation of the PPARp/δ isotype by amorfrutin B (NP-015142) at nanomolar concentrations (EC50 = 740 nM), efficacy of PPARp/δ activation was low (3%, see Figure 2d and Table 7).
Furthermore, amorfrutin B (NP-015142) only partially induced recruitment of important transcriptional cofactors including CBP, PGC1 a, TRAP220/DRIP and PRIP/RAP250 to PPARy (see Figure 2e-i, Table 7 and Figure 7). In contrast, amorfrutin B (NP-015142) reduced binding of the corepressor NCoR with IC50 value similar to rosiglitazone (AB, 60 nmol/L vs. RGZ, 23 nM/L), but with lower maximal dissociation efficacy (61 % vs. RGZ, see Figure 2h, Table 7). This was similar for other compounds of this invention (see Figure 7 and Table 5). These results indicate that amorfrutin B (NP-015142) is a high-affinity SPPARM with potential to exhibit strong antidiabetic properties without provoking side effects associated with full PPARy activation.
Table 7: Cofactor recruitment profile of amorfrutin B (NP-015142) bound to PPARy.
Compound Rosiglitazone Amorfrutin B
EC50 EC50
Cofactor (nmol/l) Efficacy (%) (nmol/l) Efficacy (%)
CBP 12 100 1 1 1 26
PGC1 a 57 100 126 30
TRAP220/DRIP 36 100 273 8
PRIP/RAP250 41 100 14 27
NCoR 23 100 60 61
7
Example C.3: PPARy activation assay in primary human adipocytes.
Amorfrutin B (NP-015142) induced expression of adipogenesis-related genes such as CCAAT/enhancer binding protein a and β (CEBPA and CEBPB) and the fatty acid binding protein 4 (FABP4) much less strongly than RGZ (see Figure 8a) in human primary adipocytes. These results indicate alleviated adipocyte differentiation. In contrast to RGZ, AB further showed reduced RNA expression of the Cortisol generating hydroxysteroid (1 1 -beta) dehydrogenase 1 (HSD11B1), which is linked to central obesity [Science 2001 , 294, 2166]. Additionally, AB treatment led to decreased transcription of the pyruvate dehydrogenase kinase 4 (PDK4), a glycerogenesis-activating enzyme that is linked to excess lipid storage in adipocyte [Diabetes 2008, 57, 2272]. However, AB treatment also resulted in increased phosphodiesterase 3B (PDE3B) expression that is responsible for beneficial NEFA release of TZD-treated mice [Diabetes 1999, 48, 1830]. No treatment had an effect of NCOR1 gene expression (see Figure 9).
Since secretion of endocrine factors by adipose tissue play a pivotal role in systemic metabolism, the expression of important adipokines in treated human adipocytes was investigated. Amorfrutin B (NP-015142) treatment led to increasing transcription of the beneficial adipokines adiponectin (ADIPOQ), fibroblast growth factor 21 (FGF21) and angiopoietin-like 4 (ANGPTL4) similar to RGZ, but we observed no regulation of leptin {LEP) transcription (see Figure 8b). Furthermore, AB resulted in reduced expression of pro-inflammaotory angiopoietin-like 2 (ANGPTL2), retinoic acid receptor responder 2 (RARRES2) and adipsin (complement factor 2, CFD) (see Figure 8b). In summary, these data indicate potential antidiabetic effects with presumably concomitant less susceptibility to adipose-derived body weight gain in vivo upon amorfrutin B (NP- 015142) treatment.
Knockdown of PPARy in these cells led to a significant reduction of amorfrutin B- induced gene expression (see Figure 8c), indicating specific modulation of PPARy-derived transcription in human adipocytes.
Example C.4: Antiproliferative and apoptotic effects in cancer cells
Treatment of HT-29 colon carcinoma cells, PC3 prostate cancer cells and MCF-7 breast cancer cells with 20 μηηοΙ/L of amorfrutins for 3 days clearly showed antiproliferative effects, especially for NP-01221 1 , NP-016018, NP-015135, NP- 015142, NP-015933, NP-015934, NP-015935, NP-015936, NP-015937 and NP- 015939 (see Figure 10). Additional studies revealed concentration-dependent
7 antiproliferative effects with inhibitory concentrations (IC5o) ranging from 8.1 pmol/L (NP-015934, HT-29 cells) to 57.3 pmol/L (NP-015135, PC3 cells) and with efficacies of up to 100% cancer cell death induction (see Figure 11 and Table 8). HT-29 colon carcinoma cells were further treated with different mixtures of compound NP-015934 and cisplatin or irinotecan, which are commonly used in treatment of various cancer diseases. Combinative treatment showed additive effects on proliferation inhibition, with a combination index (CI) of approx. 1 (see Figure 26).
To further investigate the mechanism of cell death, the activation of various caspases in the treated cells was determined. Noteworthy, at 20 μηηοΙ/L most of the tested compounds significantly induced caspase activation in at least one tested cell line (see Figure 12), and NP-015142 treatment particularly resulted in considerable caspase activation in all tested cancer cells during 24 h of treatment (see Figure 13). These results suggest the involvement of apoptosis as mechanism of compound-induced cancer cell death. Consistently, treatment of HT-29 cells with 30 and 100 μηηοΙ/L of NP-015934 led to striking DNA fragmentation during the first hours, which is a hallmark of apoptosis (see Figure 14).
Table 8: Antiproliferative profile of amorfrutins in HT-29, T84 and PC3 cancer cells
HT-29 Τ84 PC3
Compound
IC50 Efficacy IC50 Efficacy IC50 Efficacy
NP-015135 40.0 μΜ 100% 41 .8 μΜ 99% 57.3 μΜ 89%
NP-015142 20.3 μΜ 98% 12.6 μΜ 99% 32.2 μΜ 95%
NP-015933 21 .8 μΜ 98% 20.3 μΜ 99% 21 .8 μΜ 99%
NP-015934 8.1 μΜ 95% 1 1 .2 μΜ 99% 15.9 μΜ 95%
NP-015935 18.1 μΜ 96% 12.8 μΜ 100% 37.5 μΜ 97%
NP-015936 48.5 μΜ 100% 29.7 μΜ 100% 50.6 μΜ 96%
NP-015937 21 .1 μΜ 100% 40.5 μΜ 99% 31 .4 μΜ 95%
NP-015939 26.7 μΜ 100% 24.1 μΜ 99% 45.1 μΜ 93%
In line with apoptosis induction, treatment of HT-29 colon carcinoma cells with 20 μΜ of compound NP-015934 further led to substantial phosphatidylserine external ization (see Figure 27). To explore the underlying mechanism of apoptosis, NP-015934- induced generation of reactive oxygen species (ROS) was measured. Treatment with NP-015934 resulted in significant formation of ROS that could completely be quenched by antioxidants (see Figure 28 a,b). However, antioxidants did not reduce the NP-015934- induced phosphatidylserine external ization, proving that the ROS formation is not causal for apoptosis (see Figure 28 c,d).
7
Degradation of the essential mitochondrial transmembrane potential (ΔΨιτι) is a common mechanism of mitochondria-targeting anticancer drugs (Nat. Rev. Drug Discov. 2010, 9, 447). Indeed, NP-015934 abolished the mitochondrial transmembrane potential already after 15 min of treatment (see Figure 29), indicating an early uncoupling effect on the mitochondrial electrochemical gradient (IC5o = 625 nM). Strikingly, this depolarizing effect was completely abolished by methylation of the 2-hydroxyl residue (NP-015934met), showing an essential role of the 2-hydroxyl residue during apoptosis induction (see Figure 29). Degradation of the mitochondrial transmembrane potential can lead to irreversible opening of the mitochondrial permeability transition pore (MPTP), resulting in loss of mitochondrial integrity and subsequent induction of cytosolic apoptosis pathways (Front. Oncol. 2013, 3, 41 ). Consistently, HT-29 cells treated with NP- 015934 for 30 min showed opening of the MPTP as assessed by flow cytometry. However, treatment with NP-015934met only slightly led to MPTP opening (see Figure 30).
The mitochondrial transmembrane potential is required for oxidative phosphorylation, and hence, mitochondrial uncoupler can affect cellular respiration (Anal. Biochem. 2009, 390, 21 ). For that purpose, cellular respiration of HT-29 cells that were treated with NP-015934 or NP-015934met was assessed using phosphorescent oxygen- and pH-sensitive probes. NP-015934-treated cells showed striking increase in oxygen consumption, similar to the known mitochondrial uncoupler CCCP (see Figure 31 a,b), probably as consequence of disturbed ATP production without inhibiting the electron transport chain. However, treatment with NP-015934met did not show any effects (see Figure 31 a,b). Cells with affected mitochondrial ATP synthesis might activate their glycolysis and lactate production, which accounts for the majority of intracellular pH changes (Anal. Biochem. 2009, 390, 21 ). Consistently, NP-015934-treated cells considerably showed extracellular acidification, whereas NP-015934met only had minor effects (see Figure 31 c,d).
In agreement with these mitochondrial studies, treatment with 30 μΜ NP-015934met for 24 h did not induce apoptotic phosphatidylserine external ization (see Figure 32). Noteworthy, co-treated NP-015934met could not reduce the NP- 015934- induced apoptosis, even when added in excess (see Figure 32). This indicates a passive rather than a receptor-driven mechanism of apoptosis.
Example B.5: Pharmacokinetic profile of amorfrutin B (NP-015142) in C57BL/6 mice.
To initially explore the pharmacokinetic profile of amorfrutin B, C57BL/6 mice were orally challenged once with a loading dose of 100 mg/kg amorfrutin B (see Figure 15a, Table 9). Administration of amorfrutin B led to a fast plasma concentration peak, indicating a high bioavailability of that natural product. Amorfrutin B was almost completely eliminated after 24 h of dosage with an elimination half-life of about 2 h (see Table 9). Amorfrutin B and amorfrutin A1 showed similar pharmacokinetic properties (see Table 9).
Table 9: Pharmacokinetics of amorfrutin B (NP-015142) and amorfrutin 1 (NP- 003520) after single oral administration in male C57BL/6 mice.
Amorfrutin B Amorfrutin A1
Parameter
(100 mg/kg) (100 mg/kg)
Cmax (mg/l) 30.4 42.4
Cmin (mg/l) 0.015 0.015
AUC (mg/l x h) 85.4 1 1 1 .1
t1/2 (h) 2.3 2.2
ke (h-1 ) 0.307 0.318
CL (ml/h) 30.0 26.0 Cmax, maximal concentration; Cmin, minimal concentration; AUC, area under the curve; t1/2, half-life; ke, elimination rate constant; CL, clearance.
Amorfrutin B has strong antidiabetic effects and considerably improves dyslipidemia in diet-induced obesity mice. To characterize amorfrutin B in a diabetic obese animal model, HFD-fed C57BL/6 mice were treated for 4 weeks with 100 mg/kg/d amorfrutin B, 4 mg/kg/d rosiglitazone or vehicle only. After 2 weeks amorfrutin B-treated mice had strikingly reduced concentrations of fasting blood glucose (see Figure 15b) and fasting plasma insulin (see Figure 15c) equally to or even better than RGZ-treated mice. Amorfrutin B and RGZ-treated mice both showed equal reduction of insulin resistance as determined by homeostatic modelling (see Figure 15d). In oral glucose tolerance tests (OGTT, see Figure 15e and 15f) and intraperitoneal insulin sensitivity tests (IPIST) amorfrutin B reduced insulin resistance and glucose intolerance similar to RGZ (see Figure 15g).
7
Amorfrutin B strongly decreased concentrations of triacylglycerols and NEFA in plasma of HFD-fed mice comparable to RGZ (see Figure 16). After 4 weeks of treatment, amorfrutin B and RGZ both reduced fasting plasma levels of triacylglycerols by 25% (see Figure 16a). Fasting concentrations of deleterious plasma NEFA were decreased by 29% with amorfrutin B and 40% with RGZ treatment (see Figure 16b).
Based on the above described results, the physiological effects of amorfrutin B treatment seemed to be largely controlled via nanomolar activation of PPARy and differential expression of its target genes in the adipocytes. In vWAT, AB significantly increased expression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha and beta (Ppargda and b) (2-fold each, see Figure 17), indicating improved mitochondrial biogenesis and fatty acid breakdown. AB and RGZ equally increased expression of adiponectin (Adipoq) and uncoupling protein 3 (Ucp3, approx. 2-fold each), but only RGZ clearly boosted Ucp1 expression (20-fold vs. 1 .6-fold, see Figure 17).
Additionally, the in vivo gene expression was investigated in order to explore the potential additional physiological effects derived from regulation via the liver and skeletal muscle. In liver, in contrast to RGZ, AB only partly regulated gluconeogenic genes (see Figure 18).
Both RGZ and AB showed a 25% increase in transcription of Ppargda, but in general minor expression changes of fatty acid oxidation genes (see Figure 18b). The most prominent difference between RGZ and AB-treated livers was pyruvate dehydrogenase 4 (Pdk4), which was highly activated only in RGZ-treated mice (+96% vs. vehicle, see Figure 18b). These data confirm the effects observed in human primary adipocytes (Figure 2a), suggesting a switch between carbohydrate and lipid metabolism that may play a role in the different mechanisms between rosiglitazone and amorfrutin.
Since ketone body metabolism is involved in diabetes and obesity, the expression of ketogenic genes in liver of the C57BL/6 mice was further analyzed. Both RGZ and AB activated genes of ketone metabolism with striking increase in expression of 3-hydroxy-3-methylglutaryl-CoA synthase 1 (Hmgcsl) in AB-treated mice (+123%, see Figure 18c). However, plasma levels of β-hydroxybutyrate (β-ΗΒ) were not significantly changed after treatment (see Figure 18d). To further explore potential PPAR /5-induced insulin-sensitizing effects in skeletal muscle, the gene
expression of various target genes was analysed, but no amorfrutin-induced differential regulation could be detected (see Figure 18e). These data suggest a minor role of the skeletal muscle in the observed antidiabetic profile of amorfrutins, confirming the low PPAR /δ activation observed in reporter gene assays (see Figure 2d).
To investigate the role of the peptide hormone fibroblast growth factor 21 (FGF21 ) in the observed metabolic improvement, the Fgf21 expression in WAT and liver and circulating FGF21 plasma levels was analyzed. As described for TZDs [Mol. Pharmacol. 2008, 74, 403], RGZ induced Fgf21 gene expression in liver and adipose tissue (see Figure 19) and strikingly increased FGF21 plasma levels (see Figure 19). However, AB completely failed to induce expression and secretion of FGF21 , suggesting alternative PPARy activation to the described TZD-FGF21 autocrine loop.
Furthermore, it was investigated if the phosphorylation of PPARy on Ser273 in WAT [Nature 2010, 466, 451 ] mediate the antidiabetic effects of AB, but no significant change at this phosphorylation site after RSG or AB treatment was observed (see Figure 20).
Example B.6: Investigation of the side-effects associated with TZDs treatment on the compounds of the present invention.
As discussed above, PPARy-targeting drugs such as RGZ provoked undesirable side effects. Therefore, the potential side-effects of the compounds of the present invention was tested.
Viability and cytotoxicity assays in HepG2 cells did not reveal any adverse effects up to 32 μηηοΙ/L amorfrutin B (see Figure 21 ).
Potential cancerogenity of amorfrutin B was evaluated using a cellular micronudeus assay. In this assay, formation of micronudei during cell division of Chinese hamster ovary (CHO) cells is observed microscopically after treatment with potential mutagens. Amorfrutin B was tested up to a concentration of 32 μηηοΙ/L and showed no genetic toxicity either in the presence (+S9) or absence (-S9) of metabolic activation by rat liver homogenate extracts (Figure 22a). Noteworthy, amorfrutin B significantly reduced the basal formation of micronudei in the presence of S9 extract in a concentration-dependent manner, thus
-,
10 improving genetic integrity. After 4 weeks of treatment, livers of RGZ-treated HFD- fed obese mice showed strongly increased expression of inflammation markers indicating macrophage infiltration and potential local inflammation. In contrast, AB treatment led to reduced gene expression of these markers (see Figure 22b), suggesting anti-inflammatory effects of AB-treatment in the liver of HFD-fed mice. In addition, RGZ treatment increased Fabp4 expression by a factor of 31 , indicating increased lipid storage in the mouse liver, whereas AB did not show any increase in Fabp4 expression (see Figure 22c). Consistently, RGZ elevated plasma alanine transaminase (ALT) levels compared to untreated HFD-fed mice, indicating liver toxicity. In contrast, amorfrutin B treatment led to reduction of plasma ALT levels (see Figure 22d), proving liver- protective effects of the compounds of the present invention.
Another well-known side-effect of thiazolidinediones is the impairment of osteoblastogenesis leading to osteoporosis and increased fracture risk. Treatment of murine MC3T3-E1 preosteoblasts with rosiglitazone or related TZDs resulted in reduced expression of genes involved in osteoblastogenesis such as phosphate regulating gene with homologies to endopeptidases on the X chromosome (Phex), integrin binding sialoprotein {Ibsp) and osteocalcin {Bglap) (see Figure 22e). RGZ- treatment led to impaired calcification of bone cells in vitro as assessed by Alizarin red S staining (see Figure 22f). In contrast, treatment with compounds of the present invention did not show any reductions in expression of this osteoblast gene set (see Figure 22e and Figure 23a) or on mineralization of bone cells (see Figure 22f and Figure 23b) in vitro. In HFD-fed mice, rosiglitazone substantially increased plasma bone osteocalcin levels by 50% (see Figure 22g), indicating elevated bone cell turnover. However, amorfrutin B-treated DIO mice did not show any effect of plasma bone osteocalcin levels (see Figure 22g). Taken together, these results indicate that the compounds of the present invention do not provoke osteoporosis in mice.
Adverse body weight gain is a frequent side effect of PPARy activation due to increased fat storage in white adipose tissue. However, in HFD-fed mice amorfrutin B treatment did not lead to increased adiposity, but instead to a beneficial reduction of body weight gain by approximately 15% compared to HFD- fed mice treated with vehicle control (see Figure 22h). Given the inconspicuous results from the various described assays, this observation cannot be explained by potential compound toxicity. Feeding of amorfrutin B only had a slight impact on
7 food intake during the first three days (see Figure 24a), and correction for food intake using analysis of covariance (ANCOVA, see Figure 24b) revealed that anoretic effects cannot exclusively explain the observed reduction in weight gain after amorfrutin B treatment. To investigate the potential contribution of the hypothalamic-pituitary-thyroid axis, the plasma levels of triiodothyronine (T3) and thyroxine (T4) were measured. Amorfrutin B-treated mice did not show significant changes on these endocrine hormones (see Figure 24c). In general, weight reduction may result in improved insulin sensitivity. Although amorfrutin B induced effects on body weight correlated (in part) with improved insulin sensitivity, analysis of covariance (ANCOVA) clearly showed antidiabetic effects, which were independent from weight regulation (see Figure 24d).
RGZ has recently been reported to decrease HDL cholesterol [Diabet. Med. 2007, 24, 94]. After 4 weeks of treatment, RGZ-treated DIO mice showed a reduction in plasma HDL cholesterol by 24%, whereas amorfrutin B neither changed plasma HDL nor LDLA/LDL levels (see Figure 25a).
RGZ administration is further associated with the development of hemodilution and edema as a result of fluid retention. Amorfrutin B treatment did not impair hematocrit (see Figure 25b) or levels of whole blood haemoglobin (see Figure 25c) in DIO mice.
Claims
Claims A compound of general formu
R1 represents the following: -H;
R2 represents the following: -H, -OH, -OCqH(2q+i );
R4 represents the following: -H, -OH, -OCH3, -CH3 or
R2 together with R3 or R3 together with R4 form with the two carbons of the benzene ring to which R2 and R3 or R3 and R4 are attached one of the following moieties:
R3, R5, R6 and R7 represent independently of each other: -R8, -R9, -R10, -R16 -H, -OH, -(CH2)n-R8, -(CH2)m-R9, -(CH2)P-R10, -(CH2)0-R16 -C2H4-Ph, -CH2-Ph, -CH=CH-Ph, -OCH3, -OC2H5 -CH=CH-CH(CH3)2, -CH2-CH=C(CH3)2, -CH2-CH=CR8R9
-CH2-CH=CR10R16, -CH2-CH(R11)-C(CH3)=CH2, -CH2-CH(OH)-R8
-CH2-CH(OH)-R9, -CH2-CH(OH)-R 10 -CH2-CH(OH)-R 16 -CH2-CH=C(R8)-CH2-CH2-CH=CR9R10, -CR8=CH2, -CR9=CH; CR10=CH 16=CH2,
R8, R9, R10 and R16 represent independently of each other: -H, -CH3, -C2H5, -C3H7, -C4H9, -C5H 11 , -CeHi s, -CH2OH , -CH(OH)(CH3), -C(OH)(CH3)2, -CH(CH3)-O-CO-CH3, -C(CH3)=CH2, -CH(OH)-CH2-CH=C(CH3)2, -CH2-CH2-CH=C(CH3)2, -CH(OH)-CH(OH)-CO-CH=CH-CH=CHR1 1 , -CH=CH-CH2-CH=CHR1 1 , -CH(OH)-CH3, -C(CH3)=CH-CH2-CH=C(CH3)2, -CH=CH-CH=CHR1 1 ;
R1 1 , R12, R13 and R14 represent independently of each other: -H, -CH3, -C2H5, — C3H7, — C4Hg, — C5H11 , —OH, — CO2H, — CH;?— CH=CH2; m, n, p, o and q are integer numbers independently of each other selected from selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13 and 14; and conjugates, enantiomers, stereoisomeric forms, mixtures of enantiomers, diastereomers, mixtures of diastereomers, hydrates, solvates, tautomers, racemates and pharmaceutically acceptable salts of the above mentioned compounds.
2. A compound of general formu
I
wherein
R1 represents the following: -H, -CO2R15, -CHO, -C(O)-CH=CH-Ph;
R2 represents the following: -H, -OH, -OCqH(2q+i);
R4 represents the following: -H, -OH, -OCH3, -CH3 or
R2 together with R3 or R3 together with R4 form with the two carbons of the benzene ring to which R2 and R3 or R3 and R4 are attached one of the following moieties:
R3, R5, R6 and R7 represent independently of each other: -R8, -R9, -R10, -R16, -H, -OH, -(CH2)n-R8, -(CH2)m-R9, -(CH2)P-R10, -(CH2)0-R16, -CH=CH-R8, -CH=CH-R9, -CH=CH-R10, -CH=CH-R16, -(CH2)n-CHR8R9, -(CH2)m-CHR17R18, -(CH2)P-CHR19R20,
-(CH2)o-CHR21R22, -OCH3j -OC2H5, -CH=CH-CH(CH3)2,
-CH2-CH=CR8R9, -CH2-CH=CR17R18, -CH2-CH=CR19R20,
-CH2-CH=CR21 R22, -CH2-CH(R11)-C(CH3)=CH2j -CH2-CH(R23)-C(CH3)=CH2j -CH2-CH(R24)-C(CH3)=CH2, -CH2-CH(R25)-C(CH3)=CH2j -CH2-CH(OH)-R8, -CH2-CH(OH)-R9, -CH2-CH(OH)-R10, -CH2-CH(OH)-R16, -CH2-CH=C(R8)-CH2-CH2-CH=CR9R10,
-CH2-CH=C(R26)-CH2-CH2-CH=CR27R28,
-CH2-CH=C(R29)-CH2-CH2-CH=CR30R31,
-CH2-CH=C(R32)-CH2-CH2-CH=CR33R34, -CR8=CH2, -CR9=CH2, -CR10=CH2, -CR16=CH2, -CH2-CH2-CHR50R51
D8 D9 D10 D16 D17 D18 D19 D20 D21 D22 D26 D27 D28 D29 D30 D31 D32 D33 r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ , r\ , R34, R50 and R51 represent independently of each other: -H, -CH3, — Ο2Ηδ, — C3H7, — C4Hg, — C5H 11 , — ΟβΗι3, — CH2OH, — CH(OH)(CH3)i -C(OH)(CH3)2j -CH(CH3)-O-CO-CH3j -C(CH3)=CH2,
-CH(OH)-CH2-CH=C(CH3)2j -CH2-CH2-CH=C(CH3)2, -CH(OH)-CH(OH)-CO-CH=CH-CH=CHR11 ,
-CH(OH)-CH(OH)-CO-CH=CH-CH=CHR42,
-CH(OH)-CH(OH)-CO-CH=CH-CH=CHR43,
-CH(OH)-CH(OH)-CO-CH=CH-CH=CHR44, -CH=CH-CH2-CH=CHR11 , -CH=CH-CH2-CH=CHR42, -CH=CH-CH2-CH=CHR43, -CH=CH-CH2-CH=CHR44, -CH(OH)-CH3, -C(CH3)=CH-CH2-CH=C(CH3)2,
-CH=CH-CH=CHR1 1 , -CH=CH-CH=CHR42, -CH=CH-CH=CHR43, -CH=CH-CH=CHR44;
R1 1 , R12, R13, R14, R23, R24, R25, R35, R36, R37, R38, R39, R40, R41 , R42, R43 and R44 represent independently of each other: -H, -CH3, -C2H5, -C3H7, -C4H9, -C5H11 , -OH, -OCH3, -CO2H, -CH2-CH=CH2, -C(CH3)=CH2;
R15 represent independently of each other: -H, -CH3, -C2H5, -C3H7, -C4H9;
R45, R46, R47, R48 , R49, R52 and R53 represent independently of each other -H, — CH3, — C2H5, — C3H7, — C4Hgi — OCH3, — OC2H5, — OC3H7, — F, —CI, -CF3, -CHF2, -CH2F; m, n, p, o and q are integer numbers independently of each other selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13 and 14; and conjugates, enantiomers, stereoisomeric forms, mixtures of enantiomers, diastereomers, mixtures of diastereomers, hydrates, solvates, tautomers, racemates and pharmaceutically acceptable salts of the above mentioned compounds for use as modulator of PPARs.
3. The compound according to claim 1 or 2, wherein R3 and R5 are the same substituent selected from: -R8, -(CH2)n-R8, -(CH2)n-CHR8R9, -CH=CH-CH(CH3)2, -CH2-CH=CR8R9, -C2H4-Ph, -CH=CH-Ph, -CH2-CH(OH)-R8, -CR8=CH2, -CH2-CH=C(R8)-CH2-CH2-CH=CR9R10, -CH2-CH(R1 1)-C(CH3)=CH2;
R8, R9 and R10 represent independently of each other: -H, -CH3, -C2H5, -C3H7, — C4Hg, — C5H11 , — C6H 13, — C(CH3)=CH2, — C(CH3)=CH— CH2— CH=C(CH3)2, -CH2-CH2-CH=C(CH3)2, -CH=CH-CH=CHR1 1 , -CH=CH-CH2-CH=CHR1 1 ; and
R1 1 represents: -H, -CH3, -C2H5, -C3H7, -C4H9, -C5Hn , — CH2- CH=CH2.
The compound according to claim 2, wherein R1 represents
6. The compound according to claim 1 , wherein the compound is selected from the following group:
Compound Name
NP-01221 1 2-[(1 R,6R)-3-methyl-6-prop-1 -en-2-yl-1 -cyclohex-2-enyl]-5- pentylbenzene-1 ,3-diol
NP-016018 2-[(1 R,6R)-3-methyl-6-prop-1 -en-2-yl-1 -cyclohex-2-enyl]-5- propylbenzene-1 ,3-diol
NP-015933 2,6-di-(3-methyl-2-butenyl)-5-pentylbenzene-1 ,3-diol
NP-015939 2-(2-hydroxy-3-methylbut-3-en-1 -yl)-6-(3-methyl-2-butenyl)-5- pentylbenzene-1 ,3-diol
NP-015137 3-methoxy-2-(3-methyl-2-butenyl)-5-(2-phenylethyl)-benzene-
1 -ol
NP-015938 4-(2-hydroxy-3-methylbut-3-en-1 -yl)-2-(3-methyl-2-butenyl)-5- pentylbenzene-1 ,3-diol
NP-015937 2-(2-hydroxy-3-methylbut-3-en-1 -yl)-6-(3-methyl-2-butenyl)-5-
(2-phenylethyl)-benzene-1 ,3-diol
7. The compound according to claim 1 or 6 for medical use.
8. The compound according to any one of claims 1 - 7 for use as a modulator of PPARs.
9. The compound according to any one of claims 1 - 7 for use as modulator of PPARy.
10. The compound according to any one of the claims 1 - 9 for use in the prevention or treatment of metabolic diseases, inflammatory diseases, cancer and preparation of phytomedicals and/or functional food products for prevention of metabolic diseases.
1 1 . The compound according to claim 10, wherein the metabolic diseases are selected from the group comprising or consisting of: obesity, type I diabetes, type II diabetes, maturity-onset diabetes of youth, gestational diabetes, hypoglycemia, amyloidosis, branched chain disease, hyperaminoacidemia, hyperaminoaciduria, disturbances of the metabolism of urea, hyperammonemia, mucopolysaccharidoses e. g. Maroteaux-Lamy syndrom, glycogen storage diseases and lipid storage diseases, Cori's disease, intestinal carbohydrate malabsorption, maltase-, lactase-, sucrase-insufficiency, disorders of the metabolism of fructose, disorders of the metabolism of galactose, galactosaemia, disturbances of pyruvate metabolism, hypolipidemia, hypolipoproteinemia, hyperlipidemia, hyperlipoproteinemia, carnitine or carnitine acyltransferase deficiency, porphyrias, disturbances of the purine metabolism, lysosomal diseases, metabolic diseases of nerves and nervous systems like gangliosidoses, sphingolipidoses, sulfatidoses, leucodystrophies, Lesch-Nyhan syndrome, dysfunction of the parathyroid glands, pancreatic islet cell dysfunction, carbohydrate and lipid storage myopathies, glycogenoses, myoglobinuria, alkaptonuria, adrenogenital syndrome, ketosis, ketoacidosis, methyl malonaciduria, Morbus Addison, Morbus Conn, Morbus Cushing, Morbus Fabry, Morbus Gaucher, Morbus Hunter, cystic fibrosis, phenylketonuria, thesaurismosis, uricopathia, insulin resistance.
12. The compound according to claim 1 1 wherein the metabolic diseases are insulin resistance, type I diabetes, type II diabetes, maturity-onset diabetes of youth, gestational diabetes.
13. The compound according to claim 10, wherein the inflammatory diseases selected from the group comprising or consisting of: acne vulgaris, acute respiratory distress syndrome, Addison's disease, allergic rhinitis, allergic intraocular inflammatory diseases, antineutrophil cytoplasmic antibody (ANCA)- associated small-vessel vasculitis, ankylosing spondylitis, arthritis, asthma, atherosclerosis, atopic dermatitis, autoimmune hepatitis, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, Bell's palsy, bullous pemphigoid, cerebral ischemia, chronic obstructive pulmonary disease cirrhosis, Cogan's syndrome, contact dermatitis, Crohn's disease, Cushing's syndrome, dermatomyositis, diabetes mellitus, discoid lupus erythematosus, eosinophilic fasciitis, erythema nodosum, exfoliative dermatitis, fibromyalgia, focal glomerulosclerosis, focal segmental glomerulosclerosis, giant cell arteritis, gout, gouty arthritis, graft versus host disease, hand eczema, Henoch- Schonlein purpura, herpes gestationis, hirsutism, idiopathic cerato-scleritis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, immune thrombocytopenic purpura inflammatory bowel or gastrointestinal disorders, inflammatory dermatoses, lichen planus, lupus nephritis, lymphomatous tracheobronchitis, macular edema, multiple sclerosis, myasthenia gravis, myositis, nonspecific fibrosing lung disease, osteoarthritis, pancreatitis, pemphigoid gestationis, pemphigus vulgaris, periodontitis, polyarteritis nodosa, polymyalgia rheumatica, pruritus scroti, pruritis/inflammation, psoriasis, psoriatic arthritis, pulmonary histoplasmosis, rheumatoid arthritis, relapsing polychondritis, rosacea caused by sarcoidosis, rosacea caused by scleroderma, rosacea caused by Sweet's syndrome, rosacea caused by systemic lupus erythematosus, rosacea caused by urticaria, rosacea caused by zoster-associated pain, sarcoidosis, scleroderma, segmental glomerulosclerosis, septic shock syndrome, shoulder tendinitis or bursitis, Sjogren's syndrome, Still's disease, stroke-induced brain cell death, Sweet's disease, systemic lupus erythematosus, systemic sclerosis, Takayasu's arteritis, temporal arteritis, toxic epidermal necrolysis, transplant-rejection and transplant-rejection-related syndromes, tuberculosis, type-1 diabetes, ulcerative colitis, uveitis, vasculitis, and Wegener's granulomatosis.
14. The compound according to claim 10, wherein the cancer type is selected from the group comprising or consisting of: adenocarcinoma, choroidal melanoma, acute leukemia, acoustic neurinoma, ampullary carcinoma, anal carcinoma, astrocytoma, basal cell carcinoma, pancreatic cancer, desmoid tumor, bladder cancer, bronchial carcinoma, non-small cell lung cancer (NSCLC), breast cancer,
Burkitt's lymphoma, corpus cancer, CUP-syndrome (carcinoma of unknown primary), colorectal cancer, small intestine cancer, small intestinal tumors, ovarian cancer, endometrial carcinoma, ependymoma, epithelial cancer types, Ewing's tumors, gastrointestinal tumors, gastric cancer, gallbladder cancer, gall bladder carcinomas, uterine cancer, cervical cancer, cervix, glioblastomas, gynecologic tumors, ear, nose and throat tumors, hematologic neoplasias, hairy cell leukemia, urethral cancer, skin cancer, skin testis cancer, brain tumors (gliomas), brain metastases, testicle cancer, hypophysis tumor, carcinoids, Kaposi's sarcoma, laryngeal cancer, germ cell tumor, bone cancer, colorectal carcinoma, head and neck tumors (tumors of the ear, nose and throat area), colon carcinoma, craniopharyngiomas, oral cancer (cancer in the mouth area and on lips), cancer of the central nervous system, liver cancer, liver metastases, leukemia, eyelid tumor, lung cancer, lymph node cancer (Hodgkin's/Non-Hodgkin's), lymphomas, stomach cancer, malignant melanoma, malignant neoplasia, malignant tumors gastrointestinal tract, breast carcinoma, rectal cancer, medulloblastomas, melanoma, meningiomas, Hodgkin's disease, mycosis fungoides, nasal cancer, neurinoma, neuroblastoma, kidney cancer, renal cell carcinomas, non-Hodgkin's lymphomas, oligodendroglioma, esophageal carcinoma, osteolytic carcinomas and osteoplastic carcinomas, osteosarcomas, ovarial carcinoma, pancreatic carcinoma, penile cancer, plasmocytoma, squamous cell carcinoma of the head and neck (SCCHN), prostate cancer, pharyngeal cancer, rectal carcinoma, retinoblastoma, vaginal cancer, thyroid carcinoma, Schneeberger disease, esophageal cancer, spinalioms, T-cell lymphoma (mycosis fungoides), thymoma, tube carcinoma, eye tumors, urethral cancer, urologic tumors, urothelial carcinoma, vulva cancer, wart appearance, soft tissue tumors, soft tissue sarcoma, Wilm's tumor, cervical carcinoma and tongue cancer.
15. A pharmaceutical composition comprising at least one compound according to claim 1 together with at least one pharmaceutically acceptable carrier, excipient and/or diluent.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP13165716 | 2013-04-29 | ||
EP13165716.5 | 2013-04-29 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2014177593A1 true WO2014177593A1 (en) | 2014-11-06 |
Family
ID=48236687
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2014/058769 WO2014177593A1 (en) | 2013-04-29 | 2014-04-29 | Amorfrutin analogs as ppargamma-modulators |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2014177593A1 (en) |
Cited By (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015176153A1 (en) * | 2014-05-23 | 2015-11-26 | The Governing Council Of The University Of Toronto | Ppar modulators |
CN106748778A (en) * | 2016-12-01 | 2017-05-31 | 南昌大学 | A kind of new bibenzyl natural drug with hypoglycemic effect and its production and use |
KR101761767B1 (en) | 2015-03-04 | 2017-07-26 | 한국과학기술연구원 | Composition for decreasing nephrotoxicity diseases comprising chalcone derivatives |
US10004753B2 (en) | 2015-01-16 | 2018-06-26 | The J. David Gladstone Institutes | Methods for treating tauopathy |
WO2018206719A1 (en) * | 2017-05-10 | 2018-11-15 | Evolva Sa | Compounds for upregulating ucp1 expression and promoting transdifferentiation into brown adipose tissue |
CN108911974A (en) * | 2018-08-21 | 2018-11-30 | 南京师范大学常州创新发展研究院 | A kind of synthesis technology of the natural products Amorfrutin C with anticancer activity |
CN109232231A (en) * | 2018-10-31 | 2019-01-18 | 榆林学院 | A method of extracting Amorfrutin from false indigo fruit |
WO2019159168A1 (en) * | 2018-02-13 | 2019-08-22 | Beetlebung Pharma Ltd. | Cannabinoid derivatives and conjugates and uses thereof |
US10927075B2 (en) * | 2014-12-12 | 2021-02-23 | The Jackson Laboratory | Compositions and methods relating to the treatment of cancer, autoimmune disease, and neurodegenerative disease |
WO2021198692A1 (en) | 2020-03-31 | 2021-10-07 | Phytotherapeutix Ltd | Terpenophenolic compounds and their use |
CN115490661A (en) * | 2022-08-09 | 2022-12-20 | 海南师范大学 | Antioxidant active compound in mangrove-derived fungi and preparation method thereof |
WO2023063585A1 (en) * | 2021-10-13 | 2023-04-20 | 한국해양과학기술원 | Composition for preventing or treating inflammation disease, comprising metabolites derived from antarctic fungal strain pleosporales sp. sf-7343 |
WO2023177194A1 (en) * | 2022-03-15 | 2023-09-21 | 중앙대학교 산학협력단 | Novel dimethylchalcone derivatives |
WO2023177191A1 (en) * | 2022-03-15 | 2023-09-21 | 서울대학교 산학협력단 | Composition for preventing or treating fibrotic diseases, comprising dimethylchalcone derivative as active ingredient |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007093387A1 (en) * | 2006-02-16 | 2007-08-23 | Dsm Ip Assets B.V. | Novel nutraceutical and pharmaceutical compositions and use thereof for the treatment, co-treatment or prevention of inflammatory disorders |
-
2014
- 2014-04-29 WO PCT/EP2014/058769 patent/WO2014177593A1/en active Application Filing
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007093387A1 (en) * | 2006-02-16 | 2007-08-23 | Dsm Ip Assets B.V. | Novel nutraceutical and pharmaceutical compositions and use thereof for the treatment, co-treatment or prevention of inflammatory disorders |
Non-Patent Citations (25)
Title |
---|
ANAL. BIOCHEM., vol. 329, 2004, pages 77 |
ANAL. BIOCHEM., vol. 390, 2009, pages 21 |
BIOCHIM. BIOPHYS. ACTA, vol. 1812, 2011, pages 1007 |
DIABET. MED., vol. 24, 2007, pages 94 |
DIABETES, vol. 48, 1999, pages 1830 |
DIABETES, vol. 57, 2008, pages 2272 |
FRONT. ONCOL., vol. 3, 2013, pages 41 |
KLIN WOCHENSCHR., vol. 6, 1927, pages 1077 |
METHOD. METHODS SAN DIEGO, CALIF, vol. 25, 2001, pages 402 |
MOL. PHARMACOL., vol. 74, 2008, pages 403 |
MUTAT. RES., vol. 630, 2007, pages 1 |
NAT. METHODS, vol. 9, 2012, pages 57 |
NAT. REV. DRUG DISCOV., vol. 9, 2010, pages 447 |
NATURE MED., vol. 5, 2011, pages 618 |
NATURE MED., vol. 5, 2011, pages 623 |
NATURE REV. CANCER, vol. 12, 2012, pages 181 |
NATURE, vol. 466, 2010, pages 451 |
PROC. NATL. ACAD. SCI. U.S.A., vol. 109, 2012, pages 7257 |
RAMER ET AL.: "COX-2- and PPAR-gamma Confer Cannabidiol-Induced Apoptosis", MOLECULAR CANCER THERAPEUTICS, vol. 12, 7 December 2012 (2012-12-07), pages 69 - 82, XP002728150 * |
SCIENCE, vol. 294, 2001, pages 2166 |
STRUCTURE, vol. 15, 2007, pages 1258 |
STRUCTURE, vol. 9, 2001, pages 699 |
SYNTHESIS, vol. 13, 2000, pages 1956 |
TU, SYNTHESIS, vol. 13, 2000, pages 1956 |
WEIDNER ET AL.: "Amorfrutins are potent antidiabetic dietary natural products", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 109, 8 May 2012 (2012-05-08), pages 7257 - 7262, XP002728149, DOI: 10.1073/pnas.1116971109 * |
Cited By (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015176153A1 (en) * | 2014-05-23 | 2015-11-26 | The Governing Council Of The University Of Toronto | Ppar modulators |
US10927075B2 (en) * | 2014-12-12 | 2021-02-23 | The Jackson Laboratory | Compositions and methods relating to the treatment of cancer, autoimmune disease, and neurodegenerative disease |
US10004753B2 (en) | 2015-01-16 | 2018-06-26 | The J. David Gladstone Institutes | Methods for treating tauopathy |
KR101761767B1 (en) | 2015-03-04 | 2017-07-26 | 한국과학기술연구원 | Composition for decreasing nephrotoxicity diseases comprising chalcone derivatives |
CN106748778B (en) * | 2016-12-01 | 2019-12-20 | 南昌大学 | New bibenzyl natural medicine with hypoglycemic effect and its prepn and use |
CN106748778A (en) * | 2016-12-01 | 2017-05-31 | 南昌大学 | A kind of new bibenzyl natural drug with hypoglycemic effect and its production and use |
WO2018206719A1 (en) * | 2017-05-10 | 2018-11-15 | Evolva Sa | Compounds for upregulating ucp1 expression and promoting transdifferentiation into brown adipose tissue |
WO2019159168A1 (en) * | 2018-02-13 | 2019-08-22 | Beetlebung Pharma Ltd. | Cannabinoid derivatives and conjugates and uses thereof |
CN108911974A (en) * | 2018-08-21 | 2018-11-30 | 南京师范大学常州创新发展研究院 | A kind of synthesis technology of the natural products Amorfrutin C with anticancer activity |
CN109232231A (en) * | 2018-10-31 | 2019-01-18 | 榆林学院 | A method of extracting Amorfrutin from false indigo fruit |
WO2021198692A1 (en) | 2020-03-31 | 2021-10-07 | Phytotherapeutix Ltd | Terpenophenolic compounds and their use |
WO2023063585A1 (en) * | 2021-10-13 | 2023-04-20 | 한국해양과학기술원 | Composition for preventing or treating inflammation disease, comprising metabolites derived from antarctic fungal strain pleosporales sp. sf-7343 |
WO2023177194A1 (en) * | 2022-03-15 | 2023-09-21 | 중앙대학교 산학협력단 | Novel dimethylchalcone derivatives |
WO2023177191A1 (en) * | 2022-03-15 | 2023-09-21 | 서울대학교 산학협력단 | Composition for preventing or treating fibrotic diseases, comprising dimethylchalcone derivative as active ingredient |
CN115490661A (en) * | 2022-08-09 | 2022-12-20 | 海南师范大学 | Antioxidant active compound in mangrove-derived fungi and preparation method thereof |
CN115490661B (en) * | 2022-08-09 | 2023-09-08 | 海南师范大学 | Antioxidant active compound in mangrove-derived fungi and preparation method thereof |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2014177593A1 (en) | Amorfrutin analogs as ppargamma-modulators | |
US7157492B2 (en) | Dibenzo chromene derivatives and their use as ERβ selective ligands | |
DE602004011358T2 (en) | (4-HYDROXYPHENYL) -1H-INDOL-3-CARBALDEHYDOXIM DERIVATIVES AS ESTROGEN AGENTS | |
KR101864426B1 (en) | 1,2-Naphthoquinone-based Derivatives and and Methods for Preparing them | |
EP2803665B1 (en) | Synthesis of polyhydroxy benzopyran ketone compound and anti-tumor effect thereof | |
KR20130041115A (en) | Heterocyclic compound, and p27 kip1 degradation inhibitor | |
WO2008147713A1 (en) | Wnt signaling inhibitors, and methods for making and using them | |
CN105431148A (en) | Mixed lineage kinase inhibitors and method of treatments | |
CA2953472C (en) | Halogen-substituted heterocyclic compound salt | |
KR102005068B1 (en) | 1,2-Naphthoquinone-based Derivatives and Methods for Preparing them | |
WO2016066134A1 (en) | Six-membered ring benzo derivatives as dpp-4 inhibitor and use thereof | |
JP5476587B2 (en) | Condensed compounds having activity against estrogen receptors | |
KR102372599B1 (en) | Substituted bicyclic heteroaryl compounds as rxr agonists | |
WO2013157926A1 (en) | Geranyl geranyl acetone analogs and uses thereof | |
KR20150028130A (en) | Indolizine derivatives, pharmaceutically acceptable salt thereof, preparation method thereof and pharmaceutical composition containing the same as an active ingredient | |
US11370766B2 (en) | Sulfonyl amidine as indoleamine-2,3-dioxygenase inhibitor, and preparation method therefor and use thereof | |
JP6734294B2 (en) | Compositions for the treatment of fibrosis and conditions associated with fibrosis | |
KR19980701690A (en) | Substituted phenyl compounds as endothelin antagonists | |
JP6608605B2 (en) | Sirtuin expression enhancer | |
Barsoum | Synthesis and vasodilation activity of some novel bis (3-pyridinecarbonitrile) derivatives | |
US10125073B2 (en) | 2-methoxy-4-(3-(4-methoxyphenyl)prop-1-en-1-yl)phenol and preparation method therefor | |
KR100962581B1 (en) | Novel cinnamaldehyde derivatives having improved solubility in water, a method for preparing the same and a pharmaceutical composition comprising the same | |
KR101690665B1 (en) | Novel 2-hydroxy curcuminoid derivatives, a method for preparing the same and pharmaceutical compositions for anticancer property comprising the same | |
KR20210080378A (en) | treatment of obesity | |
JP4176150B2 (en) | Glutamate release inhibitors and novel compounds |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 14726540 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 14726540 Country of ref document: EP Kind code of ref document: A1 |