WO2022162693A1 - Benzodiazepine compounds having anti-cancer activity and a process for preparation thereof - Google Patents
Benzodiazepine compounds having anti-cancer activity and a process for preparation thereof Download PDFInfo
- Publication number
- WO2022162693A1 WO2022162693A1 PCT/IN2022/050063 IN2022050063W WO2022162693A1 WO 2022162693 A1 WO2022162693 A1 WO 2022162693A1 IN 2022050063 W IN2022050063 W IN 2022050063W WO 2022162693 A1 WO2022162693 A1 WO 2022162693A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- benzodiazepine
- predefined
- reaction mixture
- compound
- preparation
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 26
- 230000008569 process Effects 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 230000001093 anti-cancer Effects 0.000 title abstract description 15
- 125000003310 benzodiazepinyl group Chemical class N1N=C(C=CC2=C1C=CC=C2)* 0.000 title description 2
- 150000001875 compounds Chemical class 0.000 claims abstract description 37
- ZVAPWJGRRUHKGP-UHFFFAOYSA-N 1h-1,5-benzodiazepine Chemical class N1C=CC=NC2=CC=CC=C12 ZVAPWJGRRUHKGP-UHFFFAOYSA-N 0.000 claims abstract description 36
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 claims abstract description 14
- 239000002246 antineoplastic agent Substances 0.000 claims abstract description 14
- 229960000485 methotrexate Drugs 0.000 claims abstract description 14
- 102000004190 Enzymes Human genes 0.000 claims abstract description 11
- 108090000790 Enzymes Proteins 0.000 claims abstract description 11
- 239000011942 biocatalyst Substances 0.000 claims abstract description 8
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 claims abstract description 7
- 150000008062 acetophenones Chemical class 0.000 claims abstract description 6
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims abstract description 6
- 229960000344 thiamine hydrochloride Drugs 0.000 claims abstract description 5
- 235000019190 thiamine hydrochloride Nutrition 0.000 claims abstract description 5
- 239000011747 thiamine hydrochloride Substances 0.000 claims abstract description 5
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 20
- 239000011541 reaction mixture Substances 0.000 claims description 19
- -1 1,5-benzodiazepine compound Chemical class 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000012043 crude product Substances 0.000 claims description 9
- 125000001622 2-naphthyl group Chemical group [H]C1=C([H])C([H])=C2C([H])=C(*)C([H])=C([H])C2=C1[H] 0.000 claims description 6
- 125000003118 aryl group Chemical group 0.000 claims description 6
- 238000004440 column chromatography Methods 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 5
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 5
- 238000010791 quenching Methods 0.000 claims description 5
- 230000000171 quenching effect Effects 0.000 claims description 5
- 239000011877 solvent mixture Substances 0.000 claims description 5
- 230000002195 synergetic effect Effects 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 4
- 238000012544 monitoring process Methods 0.000 claims description 4
- 239000003208 petroleum Substances 0.000 claims description 4
- 125000000217 alkyl group Chemical group 0.000 claims description 3
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 claims description 3
- 229910052736 halogen Inorganic materials 0.000 claims description 3
- 125000005843 halogen group Chemical group 0.000 claims description 3
- 150000002367 halogens Chemical class 0.000 claims description 3
- 239000003230 hygroscopic agent Substances 0.000 claims description 3
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 abstract description 15
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 abstract description 15
- 150000001557 benzodiazepines Chemical class 0.000 abstract description 10
- 108010022394 Threonine synthase Proteins 0.000 abstract description 8
- 102000004419 dihydrofolate reductase Human genes 0.000 abstract description 8
- 229940041181 antineoplastic drug Drugs 0.000 abstract description 7
- 239000002904 solvent Substances 0.000 abstract description 4
- 239000000138 intercalating agent Substances 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 42
- 239000000243 solution Substances 0.000 description 16
- 206010028980 Neoplasm Diseases 0.000 description 14
- 201000011510 cancer Diseases 0.000 description 11
- 238000011282 treatment Methods 0.000 description 9
- 230000006907 apoptotic process Effects 0.000 description 8
- 229940079593 drug Drugs 0.000 description 8
- 239000003814 drug Substances 0.000 description 8
- 229940049706 benzodiazepine Drugs 0.000 description 7
- 230000005764 inhibitory process Effects 0.000 description 7
- 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 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- 230000001640 apoptogenic effect Effects 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 239000012091 fetal bovine serum Substances 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 238000003032 molecular docking Methods 0.000 description 6
- 238000006467 substitution reaction Methods 0.000 description 6
- 235000019439 ethyl acetate Nutrition 0.000 description 5
- 108010040476 FITC-annexin A5 Proteins 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- 239000008055 phosphate buffer solution Substances 0.000 description 4
- 208000010667 Carcinoma of liver and intrahepatic biliary tract Diseases 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 206010073069 Hepatic cancer Diseases 0.000 description 3
- 229930012538 Paclitaxel Natural products 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
- 238000004458 analytical method Methods 0.000 description 3
- 239000002543 antimycotic Substances 0.000 description 3
- 239000003054 catalyst Substances 0.000 description 3
- 238000002512 chemotherapy Methods 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 3
- 238000000021 kinase assay Methods 0.000 description 3
- 201000007270 liver cancer Diseases 0.000 description 3
- 201000002250 liver carcinoma Diseases 0.000 description 3
- 208000014018 liver neoplasm Diseases 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 229960001592 paclitaxel Drugs 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 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 3
- 238000012360 testing method Methods 0.000 description 3
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 208000019901 Anxiety disease Diseases 0.000 description 2
- 230000002112 DNA intercalation Effects 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 231100000002 MTT assay Toxicity 0.000 description 2
- 238000000134 MTT assay Methods 0.000 description 2
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 2
- 108091000080 Phosphotransferase Proteins 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000001857 anti-mycotic effect Effects 0.000 description 2
- 230000001028 anti-proliverative effect Effects 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 230000036506 anxiety Effects 0.000 description 2
- 229940053197 benzodiazepine derivative antiepileptics Drugs 0.000 description 2
- 239000012148 binding buffer Substances 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000003183 carcinogenic agent Substances 0.000 description 2
- 208000019065 cervical carcinoma Diseases 0.000 description 2
- AAOVKJBEBIDNHE-UHFFFAOYSA-N diazepam Chemical group N=1CC(=O)N(C)C2=CC=C(Cl)C=C2C=1C1=CC=CC=C1 AAOVKJBEBIDNHE-UHFFFAOYSA-N 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 229960003692 gamma aminobutyric acid Drugs 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000009830 intercalation Methods 0.000 description 2
- 230000002687 intercalation Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 150000002894 organic compounds Chemical class 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 102000020233 phosphotransferase Human genes 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 description 2
- 235000011152 sodium sulphate Nutrition 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 239000011534 wash buffer Substances 0.000 description 2
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 238000012287 DNA Binding Assay Methods 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 102000001301 EGF receptor Human genes 0.000 description 1
- 108060006698 EGF receptor Proteins 0.000 description 1
- 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 1
- 206010027476 Metastases Diseases 0.000 description 1
- 239000007832 Na2SO4 Substances 0.000 description 1
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 description 1
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 229940100198 alkylating agent Drugs 0.000 description 1
- 239000002168 alkylating agent Substances 0.000 description 1
- 230000000340 anti-metabolite Effects 0.000 description 1
- 229940100197 antimetabolite Drugs 0.000 description 1
- 239000002256 antimetabolite Substances 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000001914 calming effect Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000009982 effect on human Effects 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 229940125532 enzyme inhibitor Drugs 0.000 description 1
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 1
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 238000000126 in silico method Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 210000004789 organ system Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 229910052763 palladium Inorganic materials 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 229910052703 rhodium Inorganic materials 0.000 description 1
- 239000010948 rhodium Substances 0.000 description 1
- MHOVAHRLVXNVSD-UHFFFAOYSA-N rhodium atom Chemical compound [Rh] MHOVAHRLVXNVSD-UHFFFAOYSA-N 0.000 description 1
- 229910052707 ruthenium Inorganic materials 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000001624 sedative effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000007761 synergistic anti-cancer Effects 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- 235000019157 thiamine Nutrition 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 229940121358 tyrosine kinase inhibitor Drugs 0.000 description 1
- 239000005483 tyrosine kinase inhibitor Substances 0.000 description 1
- 150000004917 tyrosine kinase inhibitor derivatives Chemical class 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
- A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D243/00—Heterocyclic compounds containing seven-membered rings having two nitrogen atoms as the only ring hetero atoms
- C07D243/06—Heterocyclic compounds containing seven-membered rings having two nitrogen atoms as the only ring hetero atoms having the nitrogen atoms in positions 1 and 4
- C07D243/10—Heterocyclic compounds containing seven-membered rings having two nitrogen atoms as the only ring hetero atoms having the nitrogen atoms in positions 1 and 4 condensed with carbocyclic rings or ring systems
- C07D243/12—1,5-Benzodiazepines; Hydrogenated 1,5-benzodiazepines
Definitions
- the present invention relates to benzodiazepine compounds used for anticancer activity and more particularly to 1,5 -benzodiazepine having specific substitution pattern.
- the present invention also relates to a process for preparation of 1, 5-benzodiazepine derivatives,
- Cancer is a chronic illness that has a high mortality rate. It is characterized by uncontrolled division of some cells in the body that leads to the formation of an abnormal mass of cells called as tumours. These cells can migrate to other parts of the body through metastasis and lead to the formation of new tumours. There are different types of cancers based on the tissues which they affect and organ systems where they occur.
- the incidence of cancer in an individual can be linked to age, exposure to carcinogenic agents, genetics, etc.
- Preventive measures for cancer include avoiding the risk factors and exposure to carcinogenic agents. Early diagnosis and early treatment of cancer can lead to high chances in the complete recovery of the patients.
- Chemotherapy involves the use of drugs for the treatment of cancer.
- the antineoplastic drugs are effective in the treatment of cancer as they target malignant or cancerous cells, and are not harmful to normal cells.
- Some of the anticancer drugs include alkylating agents, antimetabolites, natural products and hormones. Over the years, a number of compounds have been researched and developed for their anti-cancer activity. Compounds such as Methotrexate, Paclitaxel, Cis-platin are routinely used in chemotherapy for the treatment of cancer.
- Benzodiazepines are a class of compounds that are used for having a calming and sedative effect, treatment of fear, anxiety, tension, agitation and other states of mental disturbance. Benzodiazepines enhance the action of the neurotransmitter gamma-aminobutyric acid (GABA) helping in inhibition of anxiety by reducing nerve-impulse transmissions within the brain. Benzodiazepines are explored as potential anti-cancer agents for the treatment of cancer.
- GABA neurotransmitter gamma-aminobutyric acid
- the Chinese application CN112174901A by Henan Normal University discloses 1,3-benzodiazepine compound with anticancer activity.
- the application describes process of synthesis of the compound from the substrates N-aryl amidines and propargyl alcohol ester compounds using rhodium or ruthenium catalyst.
- the US patent US8426402B2 by Immunogen Inc describes benzodiazepine derivatives with antiproliferative activity, prepared using palladium catalyst.
- Chinese Patent CN106902123B by Guo Xunxiang et al. discloses the application of 5 -hydrogen- 1,4- benzodiazepine compound for the treatment of liver cancer.
- the 5 -hydrogen- 1,4- benzodiazepine compound has a good inhibition effect on human hepatocarcinoma (HCC), HepG2 and Huh7cell lines in seven tested 5-hydrogen-l,4-benzodiazepine compounds.
- HCC human hepatocarcinoma
- HepG2 HepG2
- Huh7cell lines seven tested 5-hydrogen-l,4-benzodiazepine compounds.
- 1.5-benzodiazepines Further they are costly, time consuming and are less environment friendly.
- 1,5-benzodiazepines are the class of compounds that have been rarely explored for their anti-cancer activity. There is a growing interest in the research and development of 1,5-benzodiazepines as anti-cancer agents. There is a need to develop
- the present invention relates to 1,5 -benzodiazepine derivatives used for anticancer activity and a process for preparation of 1,5-benzodiazepine derivatives.
- Benzodiazepines are bicyclic nitrogen heterocyclic scaffold having a ring complex including a benzene ring fused with a diazepam ring.
- the 1,5-benzodiazepine compounds have structural formula (I) as follows: wherein,
- R1 is an aryl group
- R2 is a methyl (-CH3) group
- R3 is H
- R4 is an alkyl group or -X (halogen).
- R1 is an aryl group selected from 3-BrPh, 3-NO2Ph, 3,4-(OCH3)2Ph, 4-NO2Ph, 4- BrPh, 4-ClPh, 2-Naphthyl, 2-Pyridinyl, 4-Pyridinyl, 2-Naphthyl and R4 is selected from a methyl (-CH3) group or halogen groups such as Cl or Br.
- the compound of Formula (I) and its derivatives exhibit their anti-cancer activity by tyrosine kinase inhibition, DNA intercalation leading to apoptotic cell death.
- the compound of Formula (I) and its derivatives act as both tyrosine kinase and DHFR inhibitor. Further the compounds of the present invention show synergistic effect with methotrexate, an anticancer drug, when combined in a ratio of 1:1.
- a process for preparation of 1,5 -benzodi azepine and its derivatives includes the steps of adding Immol of o-phenylenediamine and 2mmol of acetophenones to a round bottom flask followed by adding 5 mol% of a biocatalyst thiamine hydrochloride to the above reaction mixture and heating the reaction mixture on a water bath at a temperature of 70-80°C for a period of 2-4 hours; monitoring of reaction mixture till the completion of the reaction followed by quenching the reaction mixture in 15 ml of cold water followed by extracting using 30 ml of ethyl acetate and drying of final crude product using Na2SO4; purifying the crude product by column chromatography using a solvent mixture of petroleum ether- ethyl acetate in a ratio of 9: 1 to obtain the compound of Formula (I) and further analyzing the product purity using high performance liquid chromat
- the present invention relates to 1,5 -benzodiazepine derivatives used for anticancer activity and a process for preparation of 1,5-benzodiazepine derivatives.
- Benzodiazepines are bicyclic nitrogen heterocyclic scaffold having a ring complex including a benzene ring fused with a diazepam ring.
- the invention discloses a compound and its derivatives represented by the Formula (I) given below: wherein,
- R1 is an aryl group
- R2 is a methyl (-CH3) group
- R3 is H and
- R4 is an alkyl group or -X (halogen).
- R1 is an aryl group selected from 3-BrPh, 3- NO 2 Ph, 3,4-(OCH 3 ) 2 Ph, 4-NO 2 Ph, 4-BrPh, 4-ClPh, 2-Naphthyl, 2-Pyridinyl, 4- Pyridinyl, 2-Naphthyl.
- R4 is selected from a methyl (-CH3) group or halogen groups such as Cl or Br.
- the compound of Formula (I) and its derivatives exhibit good cytotoxicity against cervical and liver carcinoma cells acting as anti-cancer agents.
- the compound of Formula (I) and its derivatives exhibit their anti-cancer activity by tyrosine kinase inhibition, DNA intercalation leading to apoptotic cell death.
- the compound of Formula (I) and its derivatives act as both tyrosine kinase and DHFR inhibitor. Further the compounds of the present invention show synergistic effect with methotrexate, an anticancer drug, when combined in a ratio of 1:1.
- the process of preparation of 1,5 -benzodiazepine and its derivatives includes the steps of: i. Adding a predefined quantity of o-phenylenediamine and a predefined quantity of acetophenones to a round bottom flask; ii. Adding a predefined quantity of a biocatalyst to the above reaction mixture and heating the reaction mixture on a water bath at a predefined temperature for a predefined period of time; iii. Monitoring of reaction mixture till the completion of the reaction; iv.
- the predefined quantity of o-phenylenediamine is Immol and the predefined quantity of acetophenones is 2mmol.
- the reaction proceeds with the addition of a predefined quantity of 5 mol% biocatalyst thiamine hydrochloride.
- the reaction mixture is heated on water bath for a predefined period of time ranging from 2 to 4 hours at a predefined temperature ranging from 70-80°C.
- the reaction was monitored at constant time intervals till the completion of the reaction.
- the quenching of the reaction takes place with a predefined quantity of 15ml of cold water.
- the predefined solvent used for extraction is ethyl acetate in a predefined quantity of 30 ml.
- the predefined hygroscopic agent used for drying is Na2SC>4.
- the predefined solvent mixture used for purifying the product by column chromatography is petroleum ether- ethyl acetate in a ratio of 9:1.
- the process of the present invention yields fourteen unique 1,5 benzodiazepine derivatives with varied substitution patterns.
- the present invention provides 1,5 benzodiazepine derivatives having varied substitution that possess anti-cancer activity.
- the process for the preparation of the compound of Formula (I) and its derivatives is simple, cost effective and eco-friendly. Further the catalyst is used in solvent free conditions.
- the invention explores the synergistic nature of the compound with a preexisting anti-cancer drug and studies their anticancer activity.
- Example 1 Process for preparation of compound of Formula (I) o-Phenylene diamine (Immol) and a substituted ketone derivative (2mmol) are added to a round bottom flask. 5 mol% of thiamine hydrochloride (Vitamin Bl) acts used as a biocatalyst is added and the reaction mixture is heated on a water bath at a temperature around 70-80 C for 2 to 4 hours. The reaction is monitored periodically till its completion. The reaction mixture is then quenched in cold water (15 ml) and is further extracted using ethyl acetate (30 ml) and is dried over anhydrous sodium sulphate (Na2SC>4).
- thiamine hydrochloride Vitamin Bl
- the final crude product is obtained and purified using column chromatography using the organic solvent petroleum ether (PE): ethyl acetate (EtOAc) in the ratio of 9:1.
- the purity of the product is further analyzed using high performance liquid chromatography (HPLC).
- HPLC high performance liquid chromatography
- Example 2 Studies demonstrating anti-cancer activity of 1,5-Benzodiazapiene i. In-vitro Tyrosine Kinase Inhibition
- FBS Fetal Bovine Serum
- the cells are seeded at a density of approximately lxl0 5 cells/well in a 96-well flat-bottom micro plate and maintained at 37°C in 95% humidity and 5% CO2 for overnight.
- the cells are treated with different concentration of test samples and the cells are incubated for another 24 hours.
- the cells in well are washed twice with phosphate buffer solution, and 1 ml of extraction buffer was added. By using cell scraper, the cells were recovered carefully and further the cells are centrifuged at 4°C for 10 min at 10,000 rpm. Collect the supernatant and store as further analysis.
- Tyrosine Kinase Assay The collected supernatant was diluted approximately 25 times with the kinase reacting solution provided along with kit. The diluted control and treated sample are added to each well in duplicates. Further 10 pl of 40 mM ATP-2Na solution is added into each well and mixed. The solution is incubated for 30 min at 37°C. The samples are removed and the wells are washed thrice with wash buffer. A lOOpl of blocking solution is added into each well and incubated for 30 min at 37°C. The blocking solution is then discarded and 50 pl of an anti-phospho tyrosine - HRP solution is added into each well and incubated for 30 min at 37°C.
- EFGR epidermal growth factor receptor
- the in vitro cytotoxicity of the derivatives of 1,5-benzodiazapene is determined by MTT [(3-(4,5-dimethyl-2- thiazolyl) 2,5-diphenyl-2H-tetrazolium bromide)] assay against a panel of three different human cancer cell lines namely; HEPG2 (human liver carcinoma), HeLa (human cervical) and HEK-293T (Human embryonic kidney cells) and the normal cell line.
- the cell lines as disclosed are seeded in a ninety-six (96) well flat-bottom micro plate containing Dulbecco’s Modified Eagle Media (DMEM) supplemented with 10% heat inactivated Fetal Bovine Serum (FBS) and 1% antibiotic- antimycotic 100X solution.
- DMEM Modified Eagle Media
- FBS Fetal Bovine Serum
- antibiotic- antimycotic 100X solution The cells are maintained overnight at 37°C at 95% humidity in 5% CO2 and were seeded in separate ninety-six (96 well plates.
- the compound concentration of 400, 200, 100, 50, 25, 12.5 pg/mL was treated and cells were incubated for 48 hours before the addition of MTT solution.
- the wells are washed twice with phosphate buffer solution followed by addition of 20 p L of MTT solution and then they are incubated at 37°C. After 4 hours, formazan crystals are dissolved using 100 pL DMSO and absorbance was recorded at 570 nm
- IC50 values of the screened compounds and the reference drugs, Paclitaxel and Methotrexate show significant antitumor activities with IC50 values in the range of 0.067-1.65 pM and this indicated that the 1,5 -benzodiazepine group can play a significant role in intensifying the antitumor effects. All the screened compounds showed better activity (0.067-0.21 pM) against HeLa cell lines compared to one of the reference compounds Paclitaxel (IC50 values 0.23 pM). One of the derivatives of 1,5-benzodiazpene showed an IC50 value in close proximity to the other standard drug Methotrexate (0.057 pM and 0.083 pM). iii.
- the 1,5-benzodiazepine molecule was tested to check its activity as an intercalating agent between the DNA helix strands.
- the intercalation between the DNA helix strands is further also proved by DNA binding studies assay with good results for 1,5- benzodiazepine. iv. Apoptosis
- the mode of tumor cell death analysis was done by fluorescence activated cell sorter (FACS) caliber on HepG2 liver cancer cells treated with 20pM of selected compounds for 24 hours.
- FACS fluorescence activated cell sorter
- the flow cytometry analysis revealed that HepG2 cells treated with 1,5-benzodiazepine compounds showed apoptosis.
- the most prevailing technique used to study cell apoptosis/ necrosis is flow cytometry using propidium iodide (PI) and annexin V- FITC as dyes to spot viable and dead cells. A negative control was used for the analysis.
- This assay facilitates the detection of live cells (Ql-LL; AV-/PI-), early apoptotic cells (Ql-LR; AV+/PI-), late apoptotic cells (Ql-UR; AV+/PI+) and necrotic cells (Ql-UL; AV-/PI+).
- the apoptosis is carried out by a flow cytometer.
- the cell lines used for the assay are Hep G2 (Liver cancer) DMEM with low glucose, Fetal Bovine Serum (FBS) (Gibco, Invitrogen), Antibiotic - Antimycotic 100X solution (Thermo Fisher Scientific) and Annexin V-FITC Kit (Beckman Coulter) Tests Propidium Iodide (Sigma-Aldrich Methodology.
- the cells were seeded in a 24-well flat bottom micro plate containing cover slips and maintained at 37 °C in a CO2 incubator for overnight.
- the cells 20pg/ml of each sample compound were treated for 24 hrs.
- the cells were washed with phosphate buffer solution (PBS) and centrifuged for 5 minutes at 500 x g at 4°C. After centrifugation the supernatant was discarded and the cell pellets were resuspended in ice-cold IX Binding Buffer to 1 x 10 5 per mL. Tubes are kept on ice and then 1 pL of Annexin V-FITC solution and 5 L PI were added and mixed gently. Tubes are kept on ice and incubated for 15 minutes in the dark. 400 pL of ice-cold IX binding buffer is added and mixed gently. The cell preparations are analyzed within 30 minutes by flowcytometry.
- PBS phosphate buffer solution
- the synthesized 1,5-benzodiazepines derivatives can effectively function on targeted enzymes such as tyrosine kinase and dihydrofolate reductase (DHFR).
- DHFR dihydrofolate reductase
- the experiment studies the efficiency /bioactivity of 1,5 -benzodiazepine by checking the enhancement in the activity and reduction of toxicity of the drug Methotrexate.
- the combination of standard drug Methotrexate with the 1,5 -benzodiazepine resulted in improved activity with IC50 values of 0.046 ⁇ 0.002 against HeLa cell line and 0.057 ⁇ 0.002 against HEPG2 cell line.
- IC50 values 0.046 ⁇ 0.002 against HeLa cell line
- the IC50 values obtained for 1,5-benzodiazepine in synergism with drug Methotrexate show a difference of 0.015 micromolar as compared to the IC50 value of the drug Methotrexate alone.
- the percentage of late apoptotic cells induced by the combination was 67.6%.
- the molecular docking studies revealed that the compounds are good DHFR inhibitors.
- the obtained results from the studies were encouraging and 1,5- benzodiazepines and the derivatives were used with Methotrexate (acts as an DHFR enzyme inhibitor) synergistically.
- the docking results were screened thoroughly and screening of the best hits resulted in testing the compounds for in-vitro anti-cancer activity against two cancer cell lines and one human cell line.
- the organic compounds exhibited good cytotoxicity against cervical and liver carcinoma cells.
- the apoptotic cell pathway studies indicated late apoptotic cell death.
- the organic compound proves its potency as an anticancer agent.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Organic Chemistry (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention relates to a class of benzodiazepine compounds and a process for preparation thereof. The 1,5-benzodiazepine compounds effectively 5 function on targeted enzymes such as tyrosine kinase and dihydrofolate reductase (DHFR) and also acts as a DNA intercalating agent therefore demonstrating its anti-cancer activity. The 1,5-benzodiazepine compounds also act synergistically with a conventional anti-cancer drug such as Methotrexate. The process of preparation of 1,5-benzodiazepine and its derivatives includes the steps of reacting o-10 phenylenediamine and acetophenones in presence of a biocatalyst thiamine hydrochloride to obtain the compound of Formula (I). The process for preparation of the 1,5-benzodiazepine compounds is simple, cost effective and eco-friendly approach as it takes place in the absence of a solvent and uses a biocatalyst.
Description
“BENZODIAZEPINE COMPOUNDS HAVING ANTI-CANCER ACTIVITY AND A PROCESS FOR PREPARATION THEREOF”
FIELD OF THE INVENTION
The present invention relates to benzodiazepine compounds used for anticancer activity and more particularly to 1,5 -benzodiazepine having specific substitution pattern. The present invention also relates to a process for preparation of 1, 5-benzodiazepine derivatives,
BACKGROUND OF THE INVENTION
Cancer is a chronic illness that has a high mortality rate. It is characterized by uncontrolled division of some cells in the body that leads to the formation of an abnormal mass of cells called as tumours. These cells can migrate to other parts of the body through metastasis and lead to the formation of new tumours. There are different types of cancers based on the tissues which they affect and organ systems where they occur.
The incidence of cancer in an individual can be linked to age, exposure to carcinogenic agents, genetics, etc. Preventive measures for cancer include avoiding the risk factors and exposure to carcinogenic agents. Early diagnosis and early treatment of cancer can lead to high chances in the complete recovery of the patients.
Various new age treatments are available for cancer. These include radiation therapy, surgery, chemotherapy and immunotherapy. Chemotherapy involves the use of drugs for the treatment of cancer. The antineoplastic drugs are effective in the treatment of cancer as they target malignant or cancerous cells, and are not harmful to normal cells. Some of the anticancer drugs include alkylating agents,
antimetabolites, natural products and hormones. Over the years, a number of compounds have been researched and developed for their anti-cancer activity. Compounds such as Methotrexate, Paclitaxel, Cis-platin are routinely used in chemotherapy for the treatment of cancer.
Benzodiazepines are a class of compounds that are used for having a calming and sedative effect, treatment of fear, anxiety, tension, agitation and other states of mental disturbance. Benzodiazepines enhance the action of the neurotransmitter gamma-aminobutyric acid (GABA) helping in inhibition of anxiety by reducing nerve-impulse transmissions within the brain. Benzodiazepines are explored as potential anti-cancer agents for the treatment of cancer.
The Chinese application CN112174901A by Henan Normal University discloses 1,3-benzodiazepine compound with anticancer activity. The application describes process of synthesis of the compound from the substrates N-aryl amidines and propargyl alcohol ester compounds using rhodium or ruthenium catalyst. The US patent US8426402B2 by Immunogen Inc describes benzodiazepine derivatives with antiproliferative activity, prepared using palladium catalyst. Chinese Patent CN106902123B by Guo Xunxiang et al. discloses the application of 5 -hydrogen- 1,4- benzodiazepine compound for the treatment of liver cancer. The 5 -hydrogen- 1,4- benzodiazepine compound has a good inhibition effect on human hepatocarcinoma (HCC), HepG2 and Huh7cell lines in seven tested 5-hydrogen-l,4-benzodiazepine compounds. The processes disclosed in the prior art result in end products other than
1.5-benzodiazepines. Further they are costly, time consuming and are less environment friendly.
1,5-benzodiazepines are the class of compounds that have been rarely explored for their anti-cancer activity. There is a growing interest in the research and development of 1,5-benzodiazepines as anti-cancer agents. There is a need to develop
1.5-benzodiazepines of varied substitution pattern and a process of synthesis of
benzodiazepine derivatives that is cost effective, time saving, environment friendly and causes minimum hazard to the living beings. SUMMARY OF THE INVENTION
The present invention relates to 1,5 -benzodiazepine derivatives used for anticancer activity and a process for preparation of 1,5-benzodiazepine derivatives. Benzodiazepines are bicyclic nitrogen heterocyclic scaffold having a ring complex including a benzene ring fused with a diazepam ring. The 1,5-benzodiazepine compounds have structural formula (I) as follows:
wherein,
R1 is an aryl group;
R2 is a methyl (-CH3) group; R3 is H and
R4 is an alkyl group or -X (halogen).
R1 is an aryl group selected from 3-BrPh, 3-NO2Ph, 3,4-(OCH3)2Ph, 4-NO2Ph, 4- BrPh, 4-ClPh, 2-Naphthyl, 2-Pyridinyl, 4-Pyridinyl, 2-Naphthyl and R4 is selected from a methyl (-CH3) group or halogen groups such as Cl or Br. The compound of
Formula (I) and its derivatives exhibit their anti-cancer activity by tyrosine kinase inhibition, DNA intercalation leading to apoptotic cell death. The compound of Formula (I) and its derivatives act as both tyrosine kinase and DHFR inhibitor. Further the compounds of the present invention show synergistic effect with methotrexate, an anticancer drug, when combined in a ratio of 1:1.
In a preferred embodiment, a process for preparation of 1,5 -benzodi azepine and its derivatives is disclosed. The process of preparation of 1,5 -benzodiazepine and its derivatives includes the steps of adding Immol of o-phenylenediamine and 2mmol of acetophenones to a round bottom flask followed by adding 5 mol% of a biocatalyst thiamine hydrochloride to the above reaction mixture and heating the reaction mixture on a water bath at a temperature of 70-80°C for a period of 2-4 hours; monitoring of reaction mixture till the completion of the reaction followed by quenching the reaction mixture in 15 ml of cold water followed by extracting using 30 ml of ethyl acetate and drying of final crude product using Na2SO4; purifying the crude product by column chromatography using a solvent mixture of petroleum ether- ethyl acetate in a ratio of 9: 1 to obtain the compound of Formula (I) and further analyzing the product purity using high performance liquid chromatography (HPLC). The process of the present invention yields fourteen unique 1,5 benzodiazepine derivatives with varied substitution patterns.
DETAILED DESCRIPTION OF THE INVENTION
The invention described herein is explained using specific exemplary details for better understanding. However, the invention disclosed can be worked on by a person skilled in the art without the use of these specific details.
References in the specification to "one embodiment" or "an embodiment"means that particular feature, structure, characteristic, or function
described in connection with the embodiment is included in at least one embodiment of the invention. The appearances of the phrase “in one embodiment” in various places in the specification are not necessarily all referring to the same embodiment.
References in the specification to “preferred embodiment” means that a particular feature, structure, characteristic, or function described in detail thereby omitting known constructions and functions for clear description of the present invention.
The foregoing description of specific embodiments of the present invention has been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the present invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching.
The present invention relates to 1,5 -benzodiazepine derivatives used for anticancer activity and a process for preparation of 1,5-benzodiazepine derivatives. Benzodiazepines are bicyclic nitrogen heterocyclic scaffold having a ring complex including a benzene ring fused with a diazepam ring.
In an embodiment of the present invention, the invention discloses a compound and its derivatives represented by the Formula (I) given below:
wherein,
R1 is an aryl group;
R2 is a methyl (-CH3) group;
R3 is H and
R4 is an alkyl group or -X (halogen).
In this embodiment, R1 is an aryl group selected from 3-BrPh, 3- NO2Ph, 3,4-(OCH3)2Ph, 4-NO2Ph, 4-BrPh, 4-ClPh, 2-Naphthyl, 2-Pyridinyl, 4- Pyridinyl, 2-Naphthyl. R4 is selected from a methyl (-CH3) group or halogen groups such as Cl or Br.
In accordance with the embodiment of the present invention, the compound of Formula (I) and its derivatives exhibit good cytotoxicity against cervical and liver carcinoma cells acting as anti-cancer agents. The compound of Formula (I) and its derivatives exhibit their anti-cancer activity by tyrosine kinase inhibition, DNA intercalation leading to apoptotic cell death.
The compound of Formula (I) and its derivatives act as both tyrosine kinase and DHFR inhibitor. Further the compounds of the present invention show synergistic effect with methotrexate, an anticancer drug, when combined in a ratio of 1:1.
Now a preferred process for the preparation of compound of Formula (I) and its derivatives in accordance with the present invention is disclosed. The process of preparation of 1,5 -benzodiazepine and its derivatives includes the steps of: i. Adding a predefined quantity of o-phenylenediamine and a predefined quantity of acetophenones to a round bottom flask; ii. Adding a predefined quantity of a biocatalyst to the above reaction mixture and heating the reaction mixture on a water bath at a predefined temperature for a predefined period of time; iii. Monitoring of reaction mixture till the completion of the reaction;
iv. Quenching the reaction mixture in a predefined quantity of cold water followed by extracting using a predefined quantity of a predefined solvent and drying of final crude product with the use of a predefined hygroscopic agent; v. Purifying the crude product by column chromatography using a predefined solvent mixture to obtain the compound of Formula (I); and vi. Further analyzing the product purity using high performance liquid chromatography (HPLC)
In accordance with this embodiment of the present invention the predefined quantity of o-phenylenediamine is Immol and the predefined quantity of acetophenones is 2mmol. The reaction proceeds with the addition of a predefined quantity of 5 mol% biocatalyst thiamine hydrochloride. The reaction mixture is heated on water bath for a predefined period of time ranging from 2 to 4 hours at a predefined temperature ranging from 70-80°C. The reaction was monitored at constant time intervals till the completion of the reaction. The quenching of the reaction takes place with a predefined quantity of 15ml of cold water. The predefined solvent used for extraction is ethyl acetate in a predefined quantity of 30 ml. The predefined hygroscopic agent used for drying is Na2SC>4. The predefined solvent
mixture used for purifying the product by column chromatography is petroleum ether- ethyl acetate in a ratio of 9:1.
The process of the present invention yields fourteen unique 1,5 benzodiazepine derivatives with varied substitution patterns.
Advantageously, the present invention provides 1,5 benzodiazepine derivatives having varied substitution that possess anti-cancer activity. The process for the preparation of the compound of Formula (I) and its derivatives is simple, cost effective and eco-friendly. Further the catalyst is used in solvent free conditions.
The invention explores the synergistic nature of the compound with a preexisting anti-cancer drug and studies their anticancer activity.
EXAMPLES
Only a few examples and implementations are disclosed. Variations, modifications and enhancements to the described examples and implementations and other implementations can be made based on what is disclosed.
Examples are set forth herein below and are illustrative of different amounts and types of reactants and reaction conditions that can be utilized in practicing the disclosure. It will be apparent, however, that the disclosure can be practiced with other amounts and types of reactants and reaction conditions than those used in the examples, and the resulting devices various different properties and uses in accordance with the disclosure above and as pointed out hereinafter.
Example 1: Process for preparation of compound of Formula (I) o-Phenylene diamine (Immol) and a substituted ketone derivative (2mmol) are added to a round bottom flask. 5 mol% of thiamine hydrochloride (Vitamin Bl) acts used as a biocatalyst is added and the reaction mixture is heated on a water bath
at a temperature around 70-80 C for 2 to 4 hours. The reaction is monitored periodically till its completion. The reaction mixture is then quenched in cold water (15 ml) and is further extracted using ethyl acetate (30 ml) and is dried over anhydrous sodium sulphate (Na2SC>4). The final crude product is obtained and purified using column chromatography using the organic solvent petroleum ether (PE): ethyl acetate (EtOAc) in the ratio of 9:1. The purity of the product is further analyzed using high performance liquid chromatography (HPLC). The reaction provides fourteen (14) derivatives of 1,5 -benzodiazepine (BZ) with different substituents in them.
Example 2: Studies demonstrating anti-cancer activity of 1,5-Benzodiazapiene i. In-vitro Tyrosine Kinase Inhibition
In silico molecular docking study was performed on the compounds with Molegro Virtual Docker (MVD-2007, 6.0). The molecular docking study reveals that 1,5-benzodiazepines act as good inhibitors of tyrosine kinase due to characteristic features. This was validated by in-vitro EGFR tyrosine kinase assay.
Materials used for the in-vitro Tyrosine Kinase Inhibition
1. Fetal Bovine Serum (FBS)
2. Antibiotic-Antimycotic 100X solution
3. Well plates
4. Universal Tyrosine Kinase Assay kit
Methodology
Cell Treatment: The cells are seeded at a density of approximately lxl05cells/well in a 96-well flat-bottom micro plate and maintained at 37°C in 95% humidity and 5% CO2 for overnight. The cells are treated with different concentration of test samples and the cells are incubated for another 24 hours. The cells in well are washed twice
with phosphate buffer solution, and 1 ml of extraction buffer was added. By using cell scraper, the cells were recovered carefully and further the cells are centrifuged at 4°C for 10 min at 10,000 rpm. Collect the supernatant and store as further analysis.
Tyrosine Kinase Assay: The collected supernatant was diluted approximately 25 times with the kinase reacting solution provided along with kit. The diluted control and treated sample are added to each well in duplicates. Further 10 pl of 40 mM ATP-2Na solution is added into each well and mixed. The solution is incubated for 30 min at 37°C. The samples are removed and the wells are washed thrice with wash buffer. A lOOpl of blocking solution is added into each well and incubated for 30 min at 37°C. The blocking solution is then discarded and 50 pl of an anti-phospho tyrosine - HRP solution is added into each well and incubated for 30 min at 37°C. The antibody solution is discarded and each well is then washed 4 times with washing buffer. 100 pl of HRP substrate solution (TMBZ) is added into each well. The solution is incubated for 30 min at 37°C. Lastly, a 100 pl of stop solution is added into each well in the same order as HRP substrate solution. Absorbance is measured at 450 nm with a plate reader.
The docking studies of the compounds against EFGR-Tyrosine kinase proved the action of the compounds by inhibiting protein tyrosine kinase. The enzyme inhibition assay was also performed against epidermal growth factor receptor (EFGR) tyrosine kinase and led to promising results for the molecules.
Results: The IC50 values of the synthesized compounds ranged from 0.156 to 0.514 pM, which further proved that 1,5-benzodiazepines have the potential to be good anticancer agents with tyrosine kinase inhibitory action. ii. In-vitro MTT assay:
The in vitro cytotoxicity of the derivatives of 1,5-benzodiazapene is determined by MTT [(3-(4,5-dimethyl-2- thiazolyl) 2,5-diphenyl-2H-tetrazolium
bromide)] assay against a panel of three different human cancer cell lines namely; HEPG2 (human liver carcinoma), HeLa (human cervical) and HEK-293T (Human embryonic kidney cells) and the normal cell line.
Procedure for the MTT assay:
The cell lines as disclosed are seeded in a ninety-six (96) well flat-bottom micro plate containing Dulbecco’s Modified Eagle Media (DMEM) supplemented with 10% heat inactivated Fetal Bovine Serum (FBS) and 1% antibiotic- antimycotic 100X solution. The cells are maintained overnight at 37°C at 95% humidity in 5% CO2 and were seeded in separate ninety-six (96 well plates. The compound concentration of 400, 200, 100, 50, 25, 12.5 pg/mL was treated and cells were incubated for 48 hours before the addition of MTT solution. The wells are washed twice with phosphate buffer solution followed by addition of 20 p L of MTT solution and then they are incubated at 37°C. After 4 hours, formazan crystals are dissolved using 100 pL DMSO and absorbance was recorded at 570 nm using microplate reader. The calculation of the IC50 values was carried out using graph pad prism version 5.1.
Results: The IC50 values of the screened compounds and the reference drugs, Paclitaxel and Methotrexate show significant antitumor activities with IC50 values in the range of 0.067-1.65 pM and this indicated that the 1,5 -benzodiazepine group can play a significant role in intensifying the antitumor effects. All the screened compounds showed better activity (0.067-0.21 pM) against HeLa cell lines compared to one of the reference compounds Paclitaxel (IC50 values 0.23 pM). One of the derivatives of 1,5-benzodiazpene showed an IC50 value in close proximity to the other standard drug Methotrexate (0.057 pM and 0.083 pM). iii. Intercalation of 1,5-benzodiazepine between the DNA helix strands
The molecular docking studies revealed that the ligands get intercalated in the minor grove of the DNA fragment. N 1 atom of benzodiazepine forms hydrogen bond with Thiamine while N atom of 4-pyridine ring forms hydrogen bond with adenine of the chain B of the DNA fragment. The 2-pyridine ring exhibits pi-stacked in-between two adenine rings of the DNA chain A. DNA binding assay also proved the same.
After achieving promising results and increase in the late apoptotic cells, the 1,5-benzodiazepine molecule was tested to check its activity as an intercalating agent between the DNA helix strands. The intercalation between the DNA helix strands is further also proved by DNA binding studies assay with good results for 1,5- benzodiazepine. iv. Apoptosis
For further investigative studies relating to the mechanism underlying the antiproliferative effect of these potent compounds, the mode of tumor cell death analysis was done by fluorescence activated cell sorter (FACS) caliber on HepG2 liver cancer cells treated with 20pM of selected compounds for 24 hours. The flow cytometry analysis revealed that HepG2 cells treated with 1,5-benzodiazepine compounds showed apoptosis. The most prevailing technique used to study cell apoptosis/ necrosis is flow cytometry using propidium iodide (PI) and annexin V- FITC as dyes to spot viable and dead cells. A negative control was used for the analysis. This assay facilitates the detection of live cells (Ql-LL; AV-/PI-), early apoptotic cells (Ql-LR; AV+/PI-), late apoptotic cells (Ql-UR; AV+/PI+) and necrotic cells (Ql-UL; AV-/PI+).
Procedure for Apoptosis:
Annexin V- FITC/ Propidium iodide dual staining assay
The apoptosis is carried out by a flow cytometer. The cell lines used for the assay are Hep G2 (Liver cancer) DMEM with low glucose, Fetal Bovine Serum
(FBS) (Gibco, Invitrogen), Antibiotic - Antimycotic 100X solution (Thermo Fisher Scientific) and Annexin V-FITC Kit (Beckman Coulter) Tests Propidium Iodide (Sigma-Aldrich Methodology. The cells were seeded in a 24-well flat bottom micro plate containing cover slips and maintained at 37 °C in a CO2 incubator for overnight. The cells 20pg/ml of each sample compound were treated for 24 hrs. After the incubation, the cells were washed with phosphate buffer solution (PBS) and centrifuged for 5 minutes at 500 x g at 4°C. After centrifugation the supernatant was discarded and the cell pellets were resuspended in ice-cold IX Binding Buffer to 1 x 105 per mL. Tubes are kept on ice and then 1 pL of Annexin V-FITC solution and 5 L PI were added and mixed gently. Tubes are kept on ice and incubated for 15 minutes in the dark. 400 pL of ice-cold IX binding buffer is added and mixed gently. The cell preparations are analyzed within 30 minutes by flowcytometry.
Results: The percentage of late apoptotic cells induced by compounds was in the range (10-65%).
Example 3: Studies demonstrating synergism with other anti-cancer drugs
The synthesized 1,5-benzodiazepines derivatives can effectively function on targeted enzymes such as tyrosine kinase and dihydrofolate reductase (DHFR). The experiment studies the efficiency /bioactivity of 1,5 -benzodiazepine by checking the enhancement in the activity and reduction of toxicity of the drug Methotrexate. The combination of standard drug Methotrexate with the 1,5 -benzodiazepine resulted in improved activity with IC50 values of 0.046 ± 0.002 against HeLa cell line and 0.057 ± 0.002 against HEPG2 cell line. Thus, proving the potency of the compound to be good leads for synergistic anticancer activity.
The IC50 values obtained for 1,5-benzodiazepine in synergism with drug Methotrexate show a difference of 0.015 micromolar as compared to the IC50 value of the drug Methotrexate alone. The percentage of late apoptotic cells induced by the combination was 67.6%.
The molecular docking studies revealed that the compounds are good DHFR inhibitors. The obtained results from the studies were encouraging and 1,5- benzodiazepines and the derivatives were used with Methotrexate (acts as an DHFR enzyme inhibitor) synergistically. The docking results were screened thoroughly and screening of the best hits resulted in testing the compounds for in-vitro anti-cancer activity against two cancer cell lines and one human cell line. The organic compounds exhibited good cytotoxicity against cervical and liver carcinoma cells. The apoptotic cell pathway studies indicated late apoptotic cell death. The organic compound proves its potency as an anticancer agent.
Inference: The above studies demonstrate that 1,5-benzodiazepine act as a tyrosine kinase inhibitor, DNA intercalating agent and also acts synergistically with standard drug Methotrexate.
The embodiments were chosen and described in order to best explain the principles of the present invention and its practical application, to thereby enable others, skilled in the art to best utilize the present invention and various embodiments with various modifications as are suited to the particular use contemplated.
It is understood that various omission and substitutions of equivalents are contemplated as circumstance may suggest or render expedient, but such are intended to cover the application or implementation without departing from the spirit or scope of the present invention.
Claims
R1 is an aryl group;
R2 is a methyl (-CH3) group;
R3 is H and
R4 is an alkyl group or -X (halogen).
2. A 1,5-benzodiazepine compound as claimed in Claim 1, wherein R1 is an aryl group selected from 3-BrPh, 3-NO2Ph, 3,4-(OCH3)2Ph, 4-NO2Ph, 4-BrPh, 4-ClPh, 2-Naphthyl, 2-Pyridinyl, 4-Pyridinyl, 2-Naphthyl.
3. A 1,5-benzodiazepine compound as claimed in Claim 1, wherein R4 is selected from a methyl (-CH3) group or halogen groups such as Cl or Br.
4. A process for the preparation of 1,5-benzodiazepine compound as claimed in Claim 1, comprising the steps of: a) adding predefined quantity of o-phenylenediamine and predefined quantity of acetophenones to a round bottom flask; b) adding predefined quantity of a biocatalyst to the above reaction mixture and heating the reaction mixture on a water bath at a predefined temperature for a predefined period of time;
c) monitoring of reaction mixture till the completion of the reaction; d) quenching the reaction mixture in predefined quantity of cold water followed by extracting using predefined quantity of ethyl acetate and drying of final crude product using a predefined hygroscopic agent; e) purifying the crude product by column chromatography using a predefined solvent mixture in a predefined ratio to obtain the compound of Formula (I); and f) further analyzing the product purity using high performance liquid chromatography (HPLC). A process for the preparation of 1,5-benzodiazepine compound as claimed in Claim 4, comprising the steps of: a) adding Immol of o-phenylenediamine and 2mmol of acetophenones to a round bottom flask; b) adding 5 mol% of a biocatalyst thiamine hydrochloride to the above reaction mixture and heating the reaction mixture on a water bath at a temperature of 70-80°C for a period of 2-4 hours; c) monitoring of reaction mixture till the completion of the reaction; d) quenching the reaction mixture in 15 ml of cold water followed by extracting using 30 ml of ethyl acetate and drying of final crude product using Na2SC>4; e) purifying the crude product by column chromatography using a solvent mixture of petroleum ether- ethyl acetate in a ratio of 9:1 to obtain the compound of Formula (I); and f) further analyzing the product purity using high performance liquid chromatography (HPLC).
A combination including 1,5-benzodiazepine compound of Claim 1 along with an anticancer agent in a predefined ratio, wherein the 1,5-benzodiazepine compounds show synergistic effect with the anticancer agent. A combination including 1,5-benzodiazepine compound of Claim 1 along with Methotrexate in a ratio of 1:1, wherein the 1,5-benzodiazepine compounds show synergistic effect with methotrexate.
17
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IN202121003650 | 2021-01-27 | ||
IN202121003650 | 2021-01-27 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2022162693A1 true WO2022162693A1 (en) | 2022-08-04 |
Family
ID=82654250
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/IN2022/050063 WO2022162693A1 (en) | 2021-01-27 | 2022-01-27 | Benzodiazepine compounds having anti-cancer activity and a process for preparation thereof |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2022162693A1 (en) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
MX2007008753A (en) * | 2005-01-19 | 2007-09-26 | Zeria Pharm Co Ltd | Antitumor agent. |
WO2014128283A1 (en) * | 2013-02-22 | 2014-08-28 | Centre National De La Recherche Scientifique | Use of compositions obtained by calcining particular metal-accumulating plants for implementing catalytical reactions |
IN202021007824A (en) * | 2020-02-24 | 2021-08-27 | A. Peerzade Nargisbano |
-
2022
- 2022-01-27 WO PCT/IN2022/050063 patent/WO2022162693A1/en active Application Filing
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
MX2007008753A (en) * | 2005-01-19 | 2007-09-26 | Zeria Pharm Co Ltd | Antitumor agent. |
WO2014128283A1 (en) * | 2013-02-22 | 2014-08-28 | Centre National De La Recherche Scientifique | Use of compositions obtained by calcining particular metal-accumulating plants for implementing catalytical reactions |
IN202021007824A (en) * | 2020-02-24 | 2021-08-27 | A. Peerzade Nargisbano |
Non-Patent Citations (2)
Title |
---|
NEOCHORITIS CONSTANTINOS G., NEOCHORITIS CONSTANTINOS, TSOLERIDIS CONSTANTINOS, STEPHANIDOU STEPHANATOU JULIA, KONTOGIORGIS CHRIST: "1,5-Benzoxazepines vs 1,5-Benzodiazepines. One-Pot Microwave-Assisted Synthesis and Evaluation for Antioxidant Activity and Lipid Peroxidation Inhibition", JOURNAL OF MEDICINAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 53, no. 23, 9 December 2010 (2010-12-09), US , pages 8409 - 8420, XP055960497, ISSN: 0022-2623, DOI: 10.1021/jm100739n * |
ORLOV V. D., ORLOV, DESENKO, KIROGA: "Aromatic derivatives of 2,3-dihydro-1H-1,5-benzodiazepine", CHEMISTRY OF HETEROCYCLIC COMPOUNDS, SPRINGER US, NEW YORK, vol. 23, no. 3, 1 March 1987 (1987-03-01), New York , pages 311 - 315, XP055960496, ISSN: 0009-3122, DOI: 10.1007/BF00761991 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN112912077B (en) | Combination therapy for the treatment of triple negative breast cancer | |
Abbass et al. | Design, efficient synthesis, docking studies, and anticancer evaluation of new quinoxalines as potential intercalative Topo II inhibitors and apoptosis inducers | |
JP6730321B2 (en) | Methods and compositions for inhibiting the interaction of menin with MLL proteins | |
EP3517526B1 (en) | Pyridinones as rearranged during transfection (ret) inhibitors | |
CN107531665B (en) | Small molecule inhibitors of EGFR and PI3K | |
CN103354809B (en) | A kind of aryl urea compounds, its intermediate and application thereof | |
WO2017145050A1 (en) | Pyridylpyridone derivative useful as a ret kinase inhibitor in the treatment of ibs and cancer | |
Luo et al. | Structure-activity relationships of 2, 4-disubstituted pyrimidines as dual ERα/VEGFR-2 ligands with anti-breast cancer activity | |
CN101595084A (en) | Be used to adjust the biaryl compositions and the method for kinase cascade | |
CN105503827A (en) | EGFR (Epidermal growth factor receptor) inhibitor and preparation method and use thereof | |
Čimbora-Zovko et al. | Synthesis and biological evaluation of 4-nitro-substituted 1, 3-diaryltriazenes as a novel class of potent antitumor agents | |
CN105669564A (en) | Urea compound and preparation method, medicine composition, intermediate and application thereof | |
Kahriman et al. | Synthesis and biological evaluation of new 2, 4, 6-trisubstituted pyrimidines and their N-alkyl derivatives | |
Ramisetti et al. | Design and synthesis of novel thiobarbituric acid derivatives targeting both wild-type and BRAF-mutated melanoma cells | |
Yu et al. | Design, synthesis and antitumor activity of 4-aminoquinazoline derivatives targeting VEGFR-2 tyrosine kinase | |
Wang et al. | Impact of STAT3 inhibition on survival of osteosarcoma cell lines | |
Gawandi et al. | Assessment of elementary derivatives of 1, 5-benzodiazepine as anticancer agents with synergy potential | |
Yan et al. | Design, synthesis, and anticancer properties of isocorydine derivatives | |
JP6941900B2 (en) | Aldehyde group pyridine derivative showing FGFR4 inhibitory activity, its production method and application | |
Osman et al. | New pyrimidine-5-carbonitrile derivatives as EGFR inhibitors with anticancer and apoptotic activities: design, molecular modeling and synthesis | |
Li et al. | Design, synthesis and biological evaluation of novel plumbagin derivatives as potent antitumor agents with STAT3 inhibition | |
Mettu et al. | Mechanistic investigations on substituted benzene sulphonamides as apoptosis inducing anticancer agents | |
WO2022162693A1 (en) | Benzodiazepine compounds having anti-cancer activity and a process for preparation thereof | |
CN103113274B (en) | Ras and HDAC dual inhibitor as well as preparation method and application thereof | |
Bae et al. | Antitumor effect of 3-(quinolin-2-ylmethylene)-4, 6-dimethyl-5-hydroxy-7-azaoxindole down-regulating the Gas6-Axl axis |
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: 22745522 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: 22745522 Country of ref document: EP Kind code of ref document: A1 |