EP3887380A1 - Anti-cancer activities of a new family of ethacrynic acid derivatives - Google Patents
Anti-cancer activities of a new family of ethacrynic acid derivativesInfo
- Publication number
- EP3887380A1 EP3887380A1 EP19839176.5A EP19839176A EP3887380A1 EP 3887380 A1 EP3887380 A1 EP 3887380A1 EP 19839176 A EP19839176 A EP 19839176A EP 3887380 A1 EP3887380 A1 EP 3887380A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- compound
- tested
- dose
- doses
- cancer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000001093 anti-cancer Effects 0.000 title abstract description 6
- AVOLMBLBETYQHX-UHFFFAOYSA-N etacrynic acid Chemical class CCC(=C)C(=O)C1=CC=C(OCC(O)=O)C(Cl)=C1Cl AVOLMBLBETYQHX-UHFFFAOYSA-N 0.000 title abstract description 4
- 238000000034 method Methods 0.000 claims abstract description 11
- 238000000338 in vitro Methods 0.000 claims abstract description 10
- 230000004083 survival effect Effects 0.000 claims abstract description 7
- 238000010172 mouse model Methods 0.000 claims abstract description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 36
- 206010028980 Neoplasm Diseases 0.000 claims description 29
- 241000699670 Mus sp. Species 0.000 claims description 22
- 150000001875 compounds Chemical class 0.000 claims description 20
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 10
- 201000011510 cancer Diseases 0.000 claims description 8
- 230000015572 biosynthetic process Effects 0.000 claims description 7
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 6
- 238000003786 synthesis reaction Methods 0.000 claims description 6
- 230000037396 body weight Effects 0.000 claims description 5
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 150000001412 amines Chemical class 0.000 claims description 3
- 230000002401 inhibitory effect Effects 0.000 claims description 3
- GPEOAEVZTOQXLG-UHFFFAOYSA-N 4-piperazin-1-ium-1-ylphenolate Chemical compound C1=CC(O)=CC=C1N1CCNCC1 GPEOAEVZTOQXLG-UHFFFAOYSA-N 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims description 2
- -1 amide derivatives of ethacrynic acid Chemical class 0.000 claims description 2
- LGTLXDJOAJDFLR-UHFFFAOYSA-N diethyl chlorophosphate Chemical compound CCOP(Cl)(=O)OCC LGTLXDJOAJDFLR-UHFFFAOYSA-N 0.000 claims description 2
- 150000003141 primary amines Chemical class 0.000 claims description 2
- 150000003335 secondary amines Chemical class 0.000 claims description 2
- INZYSGMVWBQNCK-UHFFFAOYSA-N P(=O)(OCC)(OCC)OC1=CC=C(C=C1)N1CCNCC1 Chemical compound P(=O)(OCC)(OCC)OC1=CC=C(C=C1)N1CCNCC1 INZYSGMVWBQNCK-UHFFFAOYSA-N 0.000 claims 1
- 150000001408 amides Chemical class 0.000 claims 1
- 239000002243 precursor Substances 0.000 claims 1
- 238000012360 testing method Methods 0.000 abstract description 16
- 230000001472 cytotoxic effect Effects 0.000 abstract description 10
- 238000001727 in vivo Methods 0.000 abstract description 9
- 241001465754 Metazoa Species 0.000 abstract description 3
- 230000003013 cytotoxicity Effects 0.000 abstract description 3
- 231100000135 cytotoxicity Toxicity 0.000 abstract description 3
- 229960003199 etacrynic acid Drugs 0.000 abstract description 2
- 210000004881 tumor cell Anatomy 0.000 abstract description 2
- 230000004580 weight loss Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 13
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 238000011282 treatment Methods 0.000 description 9
- 230000000259 anti-tumor effect Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 4
- 238000002512 chemotherapy Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 235000019439 ethyl acetate Nutrition 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 108010007355 Glutathione S-Transferase pi Proteins 0.000 description 3
- 102000007648 Glutathione S-Transferase pi Human genes 0.000 description 3
- 108010070675 Glutathione transferase Proteins 0.000 description 3
- 102000005720 Glutathione transferase Human genes 0.000 description 3
- 239000002246 antineoplastic agent Substances 0.000 description 3
- 231100000263 cytotoxicity test Toxicity 0.000 description 3
- 238000003304 gavage Methods 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 235000015112 vegetable and seed oil Nutrition 0.000 description 3
- 239000008158 vegetable oil Substances 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 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 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 239000012267 brine Substances 0.000 description 2
- 230000005907 cancer growth Effects 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- ITVPBBDAZKBMRP-UHFFFAOYSA-N chloro-dioxido-oxo-$l^{5}-phosphane;hydron Chemical group OP(O)(Cl)=O ITVPBBDAZKBMRP-UHFFFAOYSA-N 0.000 description 2
- 231100000433 cytotoxic Toxicity 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 229960004679 doxorubicin Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000003818 flash chromatography Methods 0.000 description 2
- 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 2
- 238000011534 incubation Methods 0.000 description 2
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- 239000013641 positive control Substances 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 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 2
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- UEJJHQNACJXSKW-UHFFFAOYSA-N 2-(2,6-dioxopiperidin-3-yl)-1H-isoindole-1,3(2H)-dione Chemical compound O=C1C2=CC=CC=C2C(=O)N1C1CCC(=O)NC1=O UEJJHQNACJXSKW-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
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 102000015735 Beta-catenin Human genes 0.000 description 1
- 108060000903 Beta-catenin Proteins 0.000 description 1
- 241000596110 Biosteres Species 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 206010007559 Cardiac failure congestive Diseases 0.000 description 1
- 108020005199 Dehydrogenases Proteins 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 102100030943 Glutathione S-transferase P Human genes 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 206010019663 Hepatic failure Diseases 0.000 description 1
- 101001010139 Homo sapiens Glutathione S-transferase P Proteins 0.000 description 1
- 101000632319 Homo sapiens Septin-7 Proteins 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 102100026741 Microsomal glutathione S-transferase 1 Human genes 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 206010061309 Neoplasm progression Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- 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 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 238000010165 Scheffé test Methods 0.000 description 1
- 102100027981 Septin-7 Human genes 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 238000010162 Tukey test Methods 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-M acrylate group Chemical group C(C=C)(=O)[O-] NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000011394 anticancer treatment Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- GXJABQQUPOEUTA-RDJZCZTQSA-N bortezomib Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)B(O)O)NC(=O)C=1N=CC=NC=1)C1=CC=CC=C1 GXJABQQUPOEUTA-RDJZCZTQSA-N 0.000 description 1
- 229960001467 bortezomib Drugs 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical group 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application 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
- 210000001072 colon Anatomy 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000002934 diuretic Substances 0.000 description 1
- 230000001882 diuretic effect Effects 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000005917 in vivo anti-tumor Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 229960004942 lenalidomide Drugs 0.000 description 1
- GOTYRUGSSMKFNF-UHFFFAOYSA-N lenalidomide Chemical compound C1C=2C(N)=CC=CC=2C(=O)N1C1CCC(=O)NC1=O GOTYRUGSSMKFNF-UHFFFAOYSA-N 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 208000007903 liver failure Diseases 0.000 description 1
- 231100000835 liver failure Toxicity 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 201000006512 mast cell neoplasm Diseases 0.000 description 1
- 208000006971 mastocytoma Diseases 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 108010074917 microsomal glutathione S-transferase-I Proteins 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000005445 natural material Substances 0.000 description 1
- 239000012038 nucleophile Substances 0.000 description 1
- 238000010534 nucleophilic substitution reaction Methods 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- MRDGZSKYFPGAKP-UHFFFAOYSA-N para-methoxyphenylpiperazine Chemical compound C1=CC(OC)=CC=C1N1CCNCC1 MRDGZSKYFPGAKP-UHFFFAOYSA-N 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000009703 regulation of cell differentiation Effects 0.000 description 1
- 230000025053 regulation of cell proliferation 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
- 230000011664 signaling Effects 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
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- 230000008961 swelling Effects 0.000 description 1
- 229960003433 thalidomide Drugs 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
- C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
- C07D295/18—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carboxylic acids, or sulfur or nitrogen analogues thereof
- C07D295/182—Radicals derived from carboxylic acids
- C07D295/185—Radicals derived from carboxylic acids from aliphatic carboxylic acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/645—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
- C07F9/6509—Six-membered rings
- C07F9/650952—Six-membered rings having the nitrogen atoms in the positions 1 and 4
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the present invention relates to a new class of small anticancer molecules derived from ethacrynic acid (symbolized by AE).
- the invention relates to in vitro and in vivo anticancer activities and methods of preparing the new AE family.
- New EA analogs were synthesized and then their cytotoxic activities in vitro were evaluated on the tumor cell line P815 using the MTT test.
- the EA derivative which exhibited the best cytotoxicity in vitro, was then tested in vivo using the mouse model DBA2 / P815 (H 2 d). At 30 mg / kg, the effective dose, the animals showed general tolerance with a percentage of survival around 80%, and no significant weight loss was observed.
- Lung, liver, stomach, colon and breast cancers are the most common in the world.
- the development of new anticancer agents takes a very important place in the field of oncology.
- the development of specific molecules for the fight against this disease while circumventing the obstacle of cell resistance is necessary.
- Microsomal glutathione S-transferase 1 (mGSTl) and glutathione S-transferase pi (GSTpi) are often overexpressed in tumors, thereby conferring resistance to a number of chemotherapeutic agents, such as cisplatin and doxorubicin (DOX) [ (Johansson et al. 2011)]. These enzymes catalyze the conjugation of glutathione and act as detoxifying enzymes.
- EA or 2,3-dichloro-4- (2-methylenebutryl) -phenoxyacetic acid which is a well known diuretic, is used in the treatment of hypertension and swelling caused by diseases such as l congestive heart failure, liver failure and renal failure [(Borne, Levi, and Wilson 2002; Koechel 1981)]. It is also known as a good glutathione S-transferase pi-class inhibitor.
- EA has an acid function and an ⁇ , b-unsaturated carbonyl unit which reacts with nucleophiles, such as glutathione S-transferase Pl-1 thiol (GSTP1-1, GSTpi).
- EA inhibits the signaling of Wnt / beta catenin which plays an important role in the regulation of cell proliferation, differentiation and apoptosis [(Liu et al. 2006; Lu et al . 2009; Janovskâ and Bryja 2017)].
- the EA derivatives are prepared from commercially available EA ( Figure 1).
- Treatment of AE under the conditions of a peptide reaction with different amines in a DCM / DMF mixture in the presence of l-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDCI) and 4-dimethylaminopyridine (DMAP) at room temperature provides the desired products with moderate yields.
- EDCI l-ethyl-3- (3-dimethylaminopropyl) carbodiimide
- DMAP 4-dimethylaminopyridine
- the murine mastocytoma tumor line P815 (ATCC: TIB64) used in this study was kindly provided to our laboratory by Doctor Michel Lepoivre, UMR CNRS 9198, Bât. 430, University of Paris-saclay, France.
- This line is maintained in culture in the medium DMEM (Dulbecco's modified Eagle's medium) complete supplemented with 5% of SVF (Gibco BRL, CergyPontoise, France), 100 IU / mL of penicillin, 100 pg / mL streptomycin and 0.2% of sodium bicarbonate (Sigma) in a humid atmosphere at 37 ° C and 5% CO2.
- the viable cells are counted by exclusion with trypan blue.
- the aim being to obtain a suspension of 4 ⁇ 10 4 cells / ml to be incubated in 100 ⁇ l of complete culture medium per well with a flat bottom of the 96-well micro culture plates [(Bioster, Bastia di Rovolon, Italy)] .
- This micro-culture thus obtained is incubated 24 hours before carrying out the cytotoxicity tests.
- the latter is then carried out by applying decreasing doses of the molecules (P3, P4 and P5) obtained by half-to-half dilutions, in 100 ⁇ L of DMEM medium. Each test is carried out in duplicate and repeated three times with the positive and negative controls.
- the three molecules P3, P4 and P5 are first of all dissolved in DMSO, the final concentration of which, during the test, will not exceed 0.5% (this concentration having no effect on cell growth). These micro-cultures are incubated at 37 ° C. in a humid atmosphere containing 5% of CO 2 for 48 hours.
- the determination of the cytotoxic activity is carried out by evaluating the concentration of the molecules tested inhibiting 50% of cell growth (IC50) compared to a control cultivated under the same conditions in the absence of the compound studied. This simple and rapid test makes it possible to carry out a rapid selection of molecules exhibiting an activity liable to limit or stop the growth of cancer cells.
- the revelation of the cytotoxic action is carried out using the MTT test: 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide [(Mosmann 1983; Tilaoui et al. 2015)] . This test is carried out as described and modified by Mosmann, 1983.
- OD optical density corresponding to the cells treated with the molecules (P3, P4 and P5) and / or Methotrexate MTX (used as a positive control)
- DOcon optical density corresponding to the negative control (untreated cells).
- the model chosen for this study consists of the tumor line P815 and the syngeneic strain of mouse DBA2 (H2 d ) purchased from the breeding center of Orléans - France).
- P815 cells are capable of inducing solid tumors in DBA2 mice.
- DBA2 mice are reared in pet stores at a temperature of 25 ° C and a photoperiod of 12 hours. The animals are fed and watered ad-libitum.
- the mice used in our experiments are seven to eight weeks old with body weights between 20 and 24 g. Gender is not taken into account in our tests.
- the P815 cancer cells are collected by centrifugation at 1400 rpm for 10 min at room temperature. The pellets obtained are washed twice with PBS and resuspended in 1 ml of PBS and counted. About 10 7 living cells suspended in a volume of 100 ⁇ L of PBS are injected, under ether anesthesia, by subcutaneous route of the dorsolumbar region of each mouse. A small tumor appears in the injection site after one week to 10 days.
- mice When the tumors become palpable, the mice are divided into 4 lots at the rate of 6 mice per lot (days 0). Then, every two days, the mice are treated by gavage as follows:
- mice of batch A (control), B, C and D respectively receive 100 ⁇ L of vegetable oil alone, 10, 20 and 30 mg / kg of the molecule P4 (the most cytotoxic compared to other molecules tested) dissolved in 100 ⁇ L vegetable oil.
- the treatment is carried out in the order of a single oral administration every 48 hours for 14 days.
- the weight and survival of the mice, as well as the tumor volume are measured every other day for 28 days.
- the tumor volume on day n (TVn) is calculated as follows:
- the in vitro cytotoxic activity was measured by the MTT test against the P815 tumor line ( Figure 1).
- This cytotoxicity begins at low concentrations and increases in a dose-dependent manner for all of the molecules tested.
- the compound P4 has been evaluated for its anti-tumor effect.
- Antitumor activity preclinical studies In vivo tests represent an important step in the study of the antitumor activity of our molecule. The objective is to pass a test in conditions that are as close as possible to the reality of the disease. For this purpose, DBA-2 (H2 d ) mice carrying solid tumors P815 were used in order to test the anti-tumor effect in vivo of the molecule P4 which showed a very significant cytotoxic activity compared to the other molecules tested. PI and P3.
- mice were carried out by oral administration (gavage) of the molecules dissolved in a vegetable oil (table oil) to 6 to 8 week old mice every 48 hours for a period of 14 days.
- the results obtained are presented in FIG. 2.
- the treatment of mice with the P4 molecule induced a significant decrease in tumor volume.
- the administration by gavage of the molecule P4 at doses of 10, 20 and 30 mg / kg induced a significant reduction in the tumor volume after 28 days of the treatment compared to the control mice ( Figure 2).
- no significant difference was observed between the doses used.
- the mice treated with the 30 mg / kg dose showed tolerance towards this dose with a survival rate of approximately 80% (FIG. 4), and no significant impact.
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Abstract
The present invention relates to a new class of small anti-cancer molecules derived from ethacrynic acid (symbolized by AE). The invention relates to the in vitro and in vivo anti-cancer activities and to the methods for producing the new AE family. New AE analogues were synthesized and then the in vitro cytotoxic activities thereof were evaluated on the P815 tumour cell line using the MTT test. The AE derivative which exhibited the best in vitro cytotoxicity was then tested in vivo using the DBA2/P815 (H2d) mouse model. At 30 mg/kg, the effective dose, the animals showed general tolerance with a percentage survival of around 80%, and no significant weight loss was observed.
Description
Activités anticancéreuses d’une nouvelle famille des dérivés de l’acide ethacrynique Anticancer activities of a new family of ethacrynic acid derivatives
Saïd El Kazzouli1 , Abdelmajid Zyad2 , Nabil El Brahmi1 , Abdelmoula El Abbouchi1 , Khalid Saïd El Kazzouli 1 , Abdelmajid Zyad 2 , Nabil El Brahmi 1 , Abdelmoula El Abbouchi 1 , Khalid
Boujdi 1 , Mustapha Bousmina 1 , Elassan Ait Mouse2 , Mounir Tilaoui 2 Boujdi 1, Mustapha Bousmina 1, Elassan Ait Mouse2, Mounir Tilaoui 2
1 Euromed Research Center, Euro-Mediterranean University of Fes (UEMF) 1 Euromed Research Center, Euro-Mediterranean University of Fes (UEMF)
Fès-Shore, Route de Sidi FIrazem, 30070 Fès, Morocco Fès-Shore, Route de Sidi FIrazem, 30070 Fès, Morocco
2 Team of Experimental Oncology and Natural Substances Cellular & Molecular Immunopharmacology, Faculty of Sciences and Techniques, Sultan Moulay Slimane University, Béni Mellal, Morocco 2 Team of Experimental Oncology and Natural Substances Cellular & Molecular Immunopharmacology, Faculty of Sciences and Techniques, Sultan Moulay Slimane University, Béni Mellal, Morocco
Domaine de l’invention : Field of the invention:
La présente invention concerne une nouvelle classe de petites molécules anticancéreuses dérivées de l’acide éthacrynique (symbolisé par AE). L’invention concerne les activités anticancéreuses in vitro et in vivo et les procédés de préparation de la nouvelle famille de l’AE. De nouveaux analogues de l’AE ont été synthétisés puis, leurs activités cytotoxiques in vitro ont été évaluées sur la lignée cellulaire tumorale P815 en utilisant le test MTT. Le dérivé de l’AE, qui a présenté la meilleur cytotoxité in vitro, a été ensuite testé in vivo en utilisant le modèle de souries DBA2/P815 (H2d). A 30 mg/kg, la dose effective, les animaux ont montré une tolérance générale avec un pourcentage de survie aux alentours de 80%, et aucune perte de poids significative n’a été observée. The present invention relates to a new class of small anticancer molecules derived from ethacrynic acid (symbolized by AE). The invention relates to in vitro and in vivo anticancer activities and methods of preparing the new AE family. New EA analogs were synthesized and then their cytotoxic activities in vitro were evaluated on the tumor cell line P815 using the MTT test. The EA derivative, which exhibited the best cytotoxicity in vitro, was then tested in vivo using the mouse model DBA2 / P815 (H 2 d). At 30 mg / kg, the effective dose, the animals showed general tolerance with a percentage of survival around 80%, and no significant weight loss was observed.
Etat de la technique : State of the art:
Des avancés remarquables ont été réalisées dans le domaine de la chimiothérapie et ce, par l’introduction de nouvelles molécules telles que la thalidomide, le lénalidomide et le bortézomib. Malgré cela, le cancer reste toujours une maladie incurable. L’efficacité de la chimiothérapie reste à améliorer et ce, en réduisant la toxicité et les effets secondaires des traitements. De plus, la résistance intrinsèque ou acquise d’un grand nombre de tumeurs à la chimiothérapie est aussi un obstacle major devant l’efficacité des traitements anticancéreux. Plusieurs mécanismes de la résistance cellulaire à différentes substances actives ont été identifiés (Moscow and Cowan 1988). En prenant en considération ceci, la recherche de nouveaux agents efficaces en chimiothérapie capables de traiter les différents types de cancers est toujours indispensable.
D’après l’organisation mondiale de la santé, le cancer est l’une des principales causes de la mortalité dans le monde. Les cancers du poumon, du foie, de l’estomac, du colon et du sein sont les plus répondus dans le monde. Au vu de cette diversité des types de cancers, le développement de nouveaux agents anticancéreux prend une place très importante dans le domaine de l’oncologie. En plus, la mise au point de molécules spécifiques pour la lutte contre cette maladie tout en contournant l’obstacle de la résistance cellulaire est nécessaire. Remarkable advances have been made in the field of chemotherapy, through the introduction of new molecules such as thalidomide, lenalidomide and bortezomib. Despite this, cancer still remains an incurable disease. The effectiveness of chemotherapy remains to be improved, by reducing the toxicity and the side effects of the treatments. In addition, the intrinsic or acquired resistance of a large number of tumors to chemotherapy is also a major obstacle to the effectiveness of anticancer treatments. Several mechanisms of cell resistance to different active substances have been identified (Moscow and Cowan 1988). Taking this into consideration, the search for new agents effective in chemotherapy capable of treating the different types of cancer is always essential. According to the World Health Organization, cancer is one of the leading causes of death worldwide. Lung, liver, stomach, colon and breast cancers are the most common in the world. In view of this diversity of types of cancer, the development of new anticancer agents takes a very important place in the field of oncology. In addition, the development of specific molecules for the fight against this disease while circumventing the obstacle of cell resistance is necessary.
La glutathion S-transférase microsomale 1 (mGSTl) et la glutathion S-transférase pi (GSTpi) sont souvent surexprimées dans les tumeurs conférant ainsi une résistance à un certain nombre d’agents chimiothérapeutiques, tels que le cisplatine et la doxorubicine (DOX) [(Johansson et al. 2011)]. Ces enzymes catalysent la conjugaison de la glutathion et agissent comme des enzymes de détoxifications. Microsomal glutathione S-transferase 1 (mGSTl) and glutathione S-transferase pi (GSTpi) are often overexpressed in tumors, thereby conferring resistance to a number of chemotherapeutic agents, such as cisplatin and doxorubicin (DOX) [ (Johansson et al. 2011)]. These enzymes catalyze the conjugation of glutathione and act as detoxifying enzymes.
L’AE ou l’acide 2,3-dichloro-4-(2-méthylenebutryl)-phénoxyacétique, qui est un diurétique bien connu, est utilisé dans le traitement de l'hypertension et l'enflure causée par des maladies telles que l'insuffisance cardiaque congestive, l'insuffisance hépatique et l'insuffisance rénale [(Borne, Levi, and Wilson 2002; Koechel 1981)]. Il est également connu comme un bon inhibiteur de la glutathion S-transférase de classe pi. L’AE possède une fonction acide et une unité carbonyle a,b-insaturée qui réagit avec des nucléophiles, tels que le thiol de la glutathion S-transférase Pl-1 (GSTP1-1, GSTpi). De plus, il a été confirmé récemment que l’AE inhibe la signalisation de Wnt/béta caténine qui joue un rôle important dans la régulation de la prolifération cellulaire, la différentiation et l’apoptose [(Liu et al. 2006; Lu et al. 2009; Janovskâ and Bryja 2017)]. EA or 2,3-dichloro-4- (2-methylenebutryl) -phenoxyacetic acid, which is a well known diuretic, is used in the treatment of hypertension and swelling caused by diseases such as l congestive heart failure, liver failure and renal failure [(Borne, Levi, and Wilson 2002; Koechel 1981)]. It is also known as a good glutathione S-transferase pi-class inhibitor. EA has an acid function and an α, b-unsaturated carbonyl unit which reacts with nucleophiles, such as glutathione S-transferase Pl-1 thiol (GSTP1-1, GSTpi). In addition, it has recently been confirmed that EA inhibits the signaling of Wnt / beta catenin which plays an important role in the regulation of cell proliferation, differentiation and apoptosis [(Liu et al. 2006; Lu et al . 2009; Janovskâ and Bryja 2017)].
Afin d'améliorer la capacité de l’AE à inhiber la croissance des cellules cancéreuses in vivo tout en conservant sa bonne activité d’inhibition de la glutathion S-transférase, nous proposons dans cette invention une nouvelle synthèse d’agents anticancéreux puissants et originaux. Ainsi, en se basant sur nos résultats très encourageants concernant les activités antitumorales de divers analogues de l’AE in vitro sur un panel de lignées cellulaires [(El Brahmi et al. Nanoscales, 2015; Mignani et al. Eur. J. Med. Chem. 2016)], nous proposons dans cette invention, la synthèse et l’évaluation in vivo des meilleurs analogues en effectuant des modifications structurelles, sur le squelette de base de la molécule d’AE. Ces transformations chimiques aboutissent à la formation de liaisons amides entre la fonction acide carboxylique de l’AE et des amines primaires et secondaires. La partie acrylate quant à elle est restée intacte.
Brève description des figures : In order to improve the ability of AE to inhibit the growth of cancer cells in vivo while retaining its good activity of inhibiting glutathione S-transferase, we propose in this invention a new synthesis of powerful and original anticancer agents . Thus, based on our very encouraging results concerning the antitumor activities of various EA analogues in vitro on a panel of cell lines [(El Brahmi et al. Nanoscales, 2015; Mignani et al. Eur. J. Med. Chem. 2016)], we propose in this invention, the synthesis and in vivo evaluation of the best analogues by carrying out structural modifications, on the basic skeleton of the EA molecule. These chemical transformations result in the formation of amide bonds between the carboxylic acid function of AE and of primary and secondary amines. The acrylate part, on the other hand, remained intact. Brief description of the figures:
Schéma 1. Synthèse des dérivés de l'AE : P3, P4 et P5. Diagram 1. Synthesis of EA derivatives: P3, P4 and P5.
Figure 1. Activité cytotoxique des dérivés de l’AE (P3, P4 et P5) contre la lignée tumorale P815. Figure 1. Cytotoxic activity of EA derivatives (P3, P4 and P5) against the tumor line P815.
Table 1 Valeurs des IC5o des composés P3, P4 et P5. Table 1 Values of IC 5 o of compounds P3, P4 and P5.
Figure 2. Effet de la molécule P4 sur l’évolution tumorale chez des souris DBA2 porteuses de tumeurs. Figure 2. Effect of the P4 molecule on tumor progression in DBA2 mice carrying tumors.
Figure 3. Evolution du poids corporel des souris DBA2 traitées par le composé P4. Figure 3. Evolution of the body weight of DBA2 mice treated with the compound P4.
Figure 4. Courbes de survie des souris traitées par le composé P4. Figure 4. Survival curves for mice treated with compound P4.
Description de Pinvention : Description of the invention:
Synthèse des molécules cibles Synthesis of target molecules
Les dérivés de l’AE sont préparés à partir de l’AE commercialement disponible (schéma 1). Le traitement de l’AE sous les conditions d’une réaction peptidique par différentes amines dans un mélange DCM/DMF en présence du l-éthyl-3-(3-diméthylaminopropyl)carbodiimide (EDCI) et de la 4-diméthylaminopyridine (DMAP) à température ambiante fournit les produits recherchés avec des rendements modérés. The EA derivatives are prepared from commercially available EA (Figure 1). Treatment of AE under the conditions of a peptide reaction with different amines in a DCM / DMF mixture in the presence of l-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDCI) and 4-dimethylaminopyridine (DMAP) at room temperature provides the desired products with moderate yields.
La présence de la fonction phénol sur l’AE modifié a été utilisée pour la substitution nucléophile avec des chlorophosphates. Ainsi, le traitement par différents chlorophosphates dans le dichlorométhane (DCM) en présence de la triméthylamine comme base organique permet d’obtenir les composés souhaités avec des rendements modérés. The presence of the phenol function on the modified AE has been used for nucleophilic substitution with chlorophosphates. Thus, treatment with different chlorophosphates in dichloromethane (DCM) in the presence of trimethylamine as an organic base makes it possible to obtain the desired compounds with moderate yields.
Méthode générale pour la préparation des molécules P3 et P4. A un mélange de EDCI (1.2 équiv), DMAP (en quantité catalytique) et 1 équivalent de AE dans DMF anhydride (5 mL), 1 équivalent des amines (4-hydroxyphenyl pipérazine ou 4-methoxyphenyl pipérazine) est ajouté à 0°C. Le mélange réactionnel est agité pendant une nuit à température ambiante, puis, l’acétate d’éthyle (100 mL) est ajouté et la phase organique est lavée par l’eau (2 x 50 mL) et l’eau salée (3 x 50 mL), séchée sur MgSO4 anhydre et concentrée à l’aide d’un évaporateur rotatif. Le résidu obtenu est purifié par chromatographie flash.
P3. Rendement = 60 %. (DCM/EtOAc (9: 1 à 8:2 (v/v))). RMN ' H (CDC13; 400 MHz), d (ppm): 7.16 (d, J = 8.6 Hz, 1H, Har), 7.00 (d, J = 8.6 Hz, 1H, Har), 6.84 (d, J = 9.0 Hz, 2H), 6.79 (d, J = 9.0 Hz, 2H), 5.95 (s, 1H), 5.61 (s, 1H), 5.54 (s, 1H), 4.88 (s, 2H), 3.76-3.84 (m, 4H), 3.06- 3.12 (m, 2H), 2.99-3.06 (m, 2H), 2.48 (q, J = 7.4 Hz, 2H), 1.16 (t , J = 7.4 Hz, 3H). RMN 13C (CDC13, 101 MHz); d (ppm): 195.8 (C, C(=0)), 165.3 (C, C(=0)N), 155.2 (C, Car), 150.7 (C, Car), 150.2 (C, Car), 144.9 (C, Car), 133.8 (C, Car), 131.4 (C, Car), 128.8 (C¾, C=C¾), 127.1 (2CH, CHar),General method for the preparation of molecules P3 and P4. To a mixture of EDCI (1.2 equiv), DMAP (in catalytic amount) and 1 equivalent of AE in DMF anhydride (5 mL), 1 equivalent of amines (4-hydroxyphenyl piperazine or 4-methoxyphenyl piperazine) is added at 0 ° C. . The reaction mixture is stirred overnight at room temperature, then the ethyl acetate (100 mL) is added and the organic phase is washed with water (2 x 50 mL) and brine (3 x 50 mL), dried over anhydrous MgSO 4 and concentrated using a rotary evaporator. The residue obtained is purified by flash chromatography. P3. Yield = 60%. (DCM / EtOAc (9: 1 to 8: 2 (v / v))). 1 H NMR (CDC1 3 ; 400 MHz), d (ppm): 7.16 (d, J = 8.6 Hz, 1H, H ar ), 7.00 (d, J = 8.6 Hz, 1H, H ar ), 6.84 (d, J = 9.0 Hz, 2H), 6.79 (d, J = 9.0 Hz, 2H), 5.95 (s, 1H), 5.61 (s, 1H), 5.54 (s, 1H), 4.88 (s, 2H), 3.76- 3.84 (m, 4H), 3.06-3.12 (m, 2H), 2.99-3.06 (m, 2H), 2.48 (q, J = 7.4 Hz, 2H), 1.16 (t, J = 7.4 Hz, 3H). 13 C NMR (CDC1 3 , 101 MHz); d (ppm): 195.8 (C, C (= 0)), 165.3 (C, C (= 0) N), 155.2 (C, C ar ), 150.7 (C, C ar ), 150.2 (C, C ar ), 144.9 (C, C ar ), 133.8 (C, C ar ), 131.4 (C, C ar ), 128.8 (C¾, C = C¾), 127.1 (2CH, CH ar ),
122.8 (C, Car), 119.3 (CH, CHar), 116.0 (2CH, CHar), 110.37 (CH, CHar), 68.7 (C¾), 51.5 (CH2),50.7 (CH2), 45.7 (C¾), 42.4 (C¾), 23.4 (C¾), 12.4 (C¾). HRMS (+ESI) m/z: [M+H]+ calculer pour C23H24C12N204: 463.1188, trouver, 463.1192. IR (neat): v = 3325 (OH), 1654 (C=0), 1645 (C=C) cm 1. 122.8 (C, C ar ), 119.3 (CH, CH ar ), 116.0 (2CH, CH ar ), 110.37 (CH, CH ar ), 68.7 (C¾), 51.5 (CH 2 ), 50.7 (CH 2 ), 45.7 (C¾), 42.4 (C¾), 23.4 (C¾), 12.4 (C¾). HRMS (+ ESI) m / z: [M + H] + calculate for C 23 H 24 C1 2 N 2 0 4 : 463.1188, find, 463.1192. IR (neat): v = 3325 (OH), 1654 (C = 0), 1645 (C = C) cm 1 .
P4. Rendement = 47%. (DCM/EtOAc (8:2 (v/v))). RMN ' H (CDC13; 400 MHz) ; d (ppm): 7.15 (d, J = 8.5 Hz, 1H, Har), 6.99 (d, J = 8.5 Hz, 1H, Har), 6.93-6.82 (m, 4H, Har), 5.93 (s, 1H), 5.59 (s, 1H), 4.86 (s, 2H), 3.83-3.73 (m, 7H), 3.08 (t, J= 5.0 Hz, 2H), 3.03 (t, J= 5.0 Hz, 2H), 2.46 (q, J = 7.4 Hz, 2H), 1.14 (t, J = 7.4 Hz, 3H). RMN 13C (CDC13; 101 MHz); d (ppm): 195.9 (C, C(=0)), 165.3 (C, C(=0)N), 155.4 (C, Car), 154.7 (C, Car), 150.3 (C, Car), 145.2 (C, Car), 133.9 (C, Car), 131.7 (C, Car), 128.8 (C¾, C=C¾), 127.3 (2CH, CHar), 123.0 (C, Car), 119.3 (CH, CHar), 114.7 (2CH, CHar), 110.9 (CH, CHar), 68.9 (C¾), 55.7 (OCH3), 51.6 (C¾), 51.0 (C¾),P4. Yield = 47%. (DCM / EtOAc (8: 2 (v / v))). 1 H NMR (CDC1 3 ; 400 MHz); d (ppm): 7.15 (d, J = 8.5 Hz, 1H, H ar ), 6.99 (d, J = 8.5 Hz, 1H, H ar ), 6.93-6.82 (m, 4H, H ar ), 5.93 (s , 1H), 5.59 (s, 1H), 4.86 (s, 2H), 3.83-3.73 (m, 7H), 3.08 (t, J = 5.0 Hz, 2H), 3.03 (t, J = 5.0 Hz, 2H) , 2.46 (q, J = 7.4 Hz, 2H), 1.14 (t, J = 7.4 Hz, 3H). 13 C NMR (CDC1 3 ; 101 MHz); d (ppm): 195.9 (C, C (= 0)), 165.3 (C, C (= 0) N), 155.4 (C, C ar ), 154.7 (C, C ar ), 150.3 (C, C ar ), 145.2 (C, C ar ), 133.9 (C, C ar ), 131.7 (C, C ar ), 128.8 (C¾, C = C¾), 127.3 (2CH, CH ar ), 123.0 (C, C ar ) , 119.3 (CH, CH ar ), 114.7 (2CH, CH ar ), 110.9 (CH, CH ar ), 68.9 (C¾), 55.7 (OCH 3 ), 51.6 (C¾), 51.0 (C¾),
45.8 (C¾), 42.6 (C¾), 23.7 (C¾), 12.6 (CH3). 45.8 (C¾), 42.6 (C¾), 23.7 (C¾), 12.6 (CH 3 ).
HRMS (+ESI) m/z : [M+H]+ calculer pour C24H26C12N204: 477.1348, trouver, 477.1324. IR (neat): v = 1661 (C=0) cm 1. Analyses élémentaires pour C24H26C12N204; calculer : C, 60.38; H, 5.49; N, 5.85, trouver: C, 60.42; H, 5.24; N, 5.69. HRMS (+ ESI) m / z: [M + H] + calculate for C 24 H 26 C1 2 N 2 0 4 : 477.1348, find, 477.1324. IR (neat): v = 1661 (C = 0) cm 1 . Elementary analyzes for C 24 H 26 C1 2 N 2 0 4 ; calculate: C, 60.38; H, 5.49; N, 5.85, find: C, 60.42; H, 5.24; N, 5.69.
P5. A un mélange du P3 (1 équiv) et triéthylamine (1.1 équiv) dans du DCM anhydre à 0 °C, diéthyl chlorophosphate est ajouté goutte à goutte (1 équiv). Le mélange réactionnel est agité à température ambiante pendant une nuit. Après, DCM (20 mL) est ajouté et la phase organique est lavée par l’eau (10 mL) et l’eau salée (10 mL), séchée sur MgS04 anhydre et puis concentrée à l’aide d’un évaporateur rotatif. Le produit brut est purifié par chromatographie flash. Rendement = 46%. (DCM/EtOAc (2: 1 à 1 : 1 (v/v))). RMN 31P (CDC13; 162 MHz), d (ppm): -5.8 (s, P). RMN CH (CDC13; 400 MHz), d (ppm): 7.18-7.09 (m, 3H, Har), 6.98 (d, J = 8.6 Hz, 1H, Har), 6.88-6.82 (m, 2H, Har), 5.93 (t, J= 1.5 Hz, 1H), 5.58 (s, 1H), 4.86 (s, 2H), 4.28-4.10 (m, 4H), 3.82-3.74 (m, 4H), 3.14 (t, J = 5.1 Hz, 2H), 3.09 (t, J = 5.1 Hz, 2H), 2.45 (q, J = 7.4 Hz, 2H), 1.34 (td, J = 7.1,
1.0 Hz, 6H), 1.13 (t, J = 7.4 Hz, 3H). RMN 13C (CDC¾; 101 MHz), d (ppm): 195.9 (C, C=0), 165.3 (C, C(=0)N), 155.3 (C, Car), 150.3 (C, Car), 148.2 (C, Car), 144.9 (d, J = 7.0 Hz, C, Car), 133.9 (C, Car), 131.6 (C, Car), 128.8 (C¾, C=CH2), 127.2 (CH, Car), 122.9 (C, Car), 120.8 (d, J = 4.7 Hz, 2CH, Car), 118.2 (2CH, Car), 110.8 (CH, Car), 68.9 (CH2, OC¾), 64.6 (d, J = 6.1 Hz, 2C¾), 50.7 (C¾), 50.0 (C¾), 45.6 (C¾), 42.3 (C¾), 23.5 (C¾), 16.2 (d, J = 6.7 Hz, 2C¾), 12.5 (CH3). HRMS (+ESI) m/z: [M+H]+ calculer pour C27H23C12N207P: 599.1481, trouver, 599.1486. IR (neat): v= 1663 (C=0) cm 1. P5. To a mixture of P3 (1 equiv) and triethylamine (1.1 equiv) in anhydrous DCM at 0 ° C, diethyl chlorophosphate is added dropwise (1 equiv). The reaction mixture is stirred at room temperature overnight. Afterwards, DCM (20 mL) is added and the organic phase is washed with water (10 mL) and brine (10 mL), dried over anhydrous MgS0 4 and then concentrated using a rotary evaporator . The crude product is purified by flash chromatography. Yield = 46%. (DCM / EtOAc (2: 1 to 1: 1 (v / v))). 31 P NMR (CDC1 3 ; 162 MHz), d (ppm): -5.8 (s, P). RMN C H (CDC1 3 ; 400 MHz), d (ppm): 7.18-7.09 (m, 3H, H ar ), 6.98 (d, J = 8.6 Hz, 1H, H ar ), 6.88-6.82 (m, 2H , H ar ), 5.93 (t, J = 1.5 Hz, 1H), 5.58 (s, 1H), 4.86 (s, 2H), 4.28-4.10 (m, 4H), 3.82-3.74 (m, 4H), 3.14 (t, J = 5.1 Hz, 2H), 3.09 (t, J = 5.1 Hz, 2H), 2.45 (q, J = 7.4 Hz, 2H), 1.34 (td, J = 7.1, 1.0 Hz, 6H), 1.13 (t, J = 7.4 Hz, 3H). 13 C NMR (CDC¾; 101 MHz), d (ppm): 195.9 (C, C = 0), 165.3 (C, C (= 0) N), 155.3 (C, C ar ), 150.3 (C, C ar ), 148.2 (C, C ar ), 144.9 (d, J = 7.0 Hz, C, C ar ), 133.9 (C, C ar ), 131.6 (C, C ar ), 128.8 (C¾, C = CH 2 ) , 127.2 (CH, C ar ), 122.9 (C, C ar ), 120.8 (d, J = 4.7 Hz, 2CH, C ar ), 118.2 (2CH, C ar ), 110.8 (CH, C ar ), 68.9 ( CH 2 , OC¾), 64.6 (d, J = 6.1 Hz, 2C¾), 50.7 (C¾), 50.0 (C¾), 45.6 (C¾), 42.3 (C¾), 23.5 (C¾), 16.2 (d, J = 6.7 Hz, 2C¾), 12.5 (CH 3 ). HRMS (+ ESI) m / z: [M + H] + calculate for C 27 H 23 C1 2 N 2 0 7 P: 599.1481, find, 599.1486. IR (neat): v = 1663 (C = 0) cm 1 .
Activité antitumorale Antitumor activity
La lignée tumorale P815 du mastocytome murin (ATCC: TIB64) utilisée dans cette étude a été gracieusement fournie à notre laboratoire par Docteur Michel Lepoivre, UMR CNRS 9198, Bât.430, Université de Paris-saclay, France. Cette lignée est maintenue en culture dans le milieu DMEM (Dulbecco’s modified Eagle’s medium) complet supplémenté avec 5% de SVF (Gibco BRL, CergyPontoise, France), 100 UI/mL de pénicilline, 100 pg/mL streptomycine et 0,2% de sodium bicarbonate (Sigma) dans une atmosphère humide à 37°C et à 5% de CO2. The murine mastocytoma tumor line P815 (ATCC: TIB64) used in this study was kindly provided to our laboratory by Doctor Michel Lepoivre, UMR CNRS 9198, Bât. 430, University of Paris-saclay, France. This line is maintained in culture in the medium DMEM (Dulbecco's modified Eagle's medium) complete supplemented with 5% of SVF (Gibco BRL, CergyPontoise, France), 100 IU / mL of penicillin, 100 pg / mL streptomycin and 0.2% of sodium bicarbonate (Sigma) in a humid atmosphere at 37 ° C and 5% CO2.
Test de cytotoxicité Cytotoxicity test
Avant de réaliser le test de cytotoxicité, on procède au comptage des cellules viables par exclusion au bleu de trypan. Le but étant d’obtenir une suspension de 4 x 104 cellules/mL à incuber dans 100 pL de milieu de culture complet par puits à fond plat des plaques de micro culture de 96 puits [(Bioster, Bastia di Rovolon, Italy)]. Cette micro-culture ainsi obtenue est mise en incubation 24 heures avant la réalisation des tests de cytotoxicité. Ce dernier est alors effectué en appliquant des doses décroissantes des molécules (P3, P4 et P5) obtenues par des dilutions de demi en demi, dans 100 pL de milieu DMEM. Chaque essai est réalisé en double et répété trois fois avec les témoins positifs et négatifs. Les trois molécules P3, P4 et P5 sont tout d’abord solubilisées dans du DMSO dont la concentration finale, lors du test, n’excédera pas 0,5% (cette concentration n’ayant pas d’effet sur la croissance cellulaire). Ces micro -cultures sont incubées à 37°C en atmosphère humide contenant 5% de C02 pendant 48h. Before carrying out the cytotoxicity test, the viable cells are counted by exclusion with trypan blue. The aim being to obtain a suspension of 4 × 10 4 cells / ml to be incubated in 100 μl of complete culture medium per well with a flat bottom of the 96-well micro culture plates [(Bioster, Bastia di Rovolon, Italy)] . This micro-culture thus obtained is incubated 24 hours before carrying out the cytotoxicity tests. The latter is then carried out by applying decreasing doses of the molecules (P3, P4 and P5) obtained by half-to-half dilutions, in 100 μL of DMEM medium. Each test is carried out in duplicate and repeated three times with the positive and negative controls. The three molecules P3, P4 and P5 are first of all dissolved in DMSO, the final concentration of which, during the test, will not exceed 0.5% (this concentration having no effect on cell growth). These micro-cultures are incubated at 37 ° C. in a humid atmosphere containing 5% of CO 2 for 48 hours.
La détermination de l’activité cytotoxique est réalisée en évaluant la concentration des molécules testées inhibant 50% de la croissance cellulaire (IC50) par rapport à un témoin cultivé dans les mêmes conditions en absence du composé étudié. Ce test simple et rapide permet de réaliser une sélection rapide des molécules présentant une activité susceptible de limiter ou stopper la
croissance des cellules cancéreuses. La révélation de l’action cytotoxique est effectuée à l’aide du test MTT : 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tétrazolium bromide [(Mosmann 1983; Tilaoui et al. 2015)]. Ce test est réalisé comme décrit et modifié par Mosmann, 1983. Après 48 d’incubation dans les conditions de culture citées ci-dessous, on ajoute 20 pL d’une solution de MTT (5mg/mL de PBS). Après 4 heures d’incubation dans les mêmes conditions de culture, les cristaux violets formés suite à la réduction du MTT par les déshydrogénases mitochondriales des cellules vivantes sont solubilisés en rajoutant 100 pL d’une solution de HCl / Isopropanol (24 :1). On procède ensuite à la lecture de la densité optique (DO) à deux longueurs d’ondes 540 nm et 630 nm en utilisant le lecteur spectrophotométrique de microplaques MultisKan EX. Ainsi, l’effet de P3, P4 et P5 sur la viabilité cellulaire peut être mesuré en utilisant la formule suivante : The determination of the cytotoxic activity is carried out by evaluating the concentration of the molecules tested inhibiting 50% of cell growth (IC50) compared to a control cultivated under the same conditions in the absence of the compound studied. This simple and rapid test makes it possible to carry out a rapid selection of molecules exhibiting an activity liable to limit or stop the growth of cancer cells. The revelation of the cytotoxic action is carried out using the MTT test: 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide [(Mosmann 1983; Tilaoui et al. 2015)] . This test is carried out as described and modified by Mosmann, 1983. After 48 hours of incubation under the culture conditions cited below, 20 μL of a solution of MTT (5 mg / ml of PBS) are added. After 4 hours of incubation under the same culture conditions, the purple crystals formed following the reduction of MTT by mitochondrial dehydrogenases of living cells are solubilized by adding 100 μl of a HCl / Isopropanol solution (24: 1). The optical density (OD) is then read at two wavelengths 540 nm and 630 nm using the MultisKan EX microplate spectrophotometric reader. Thus, the effect of P3, P4 and P5 on cell viability can be measured using the following formula:
Viabilité cellulaire (%) = (DO molécules/ DO contrôle) x 100 Cell viability (%) = (DO molecules / DO control) x 100
Avec : With:
DO : densité optique correspondant aux cellules traitées par les molécules (P3, P4 et P5) et/ou le Méthotrexate MTX (utilisé comme contrôle positif) OD: optical density corresponding to the cells treated with the molecules (P3, P4 and P5) and / or Methotrexate MTX (used as a positive control)
DOcon : densité optique correspondant au contrôle négatif (cellules non traitées). DOcon: optical density corresponding to the negative control (untreated cells).
Activité antitumorale in vivo In vivo anti-tumor activity
Le modèle choisi pour cette étude est constitué de la lignée tumorale P815 et la souche de souris DBA2 (H2d) syngénique achetées du centre d’élevage d’Orléans - France). Les cellules P815 sont capables d’induire des tumeurs solides chez les souris DBA2. Les souris DBA2 sont élevées en animalerie à une température de 25°C et une photopériode de 12 heures. L’alimentation et l’abreuvage des animaux sont fournis ad-libitum. Les souris utilisées dans nos expérimentations sont âgées de sept à huit semaines avec des poids corporels compris entre 20 et 24 g. Le sexe n’est pas pris en considération dans nos essais. The model chosen for this study consists of the tumor line P815 and the syngeneic strain of mouse DBA2 (H2 d ) purchased from the breeding center of Orléans - France). P815 cells are capable of inducing solid tumors in DBA2 mice. DBA2 mice are reared in pet stores at a temperature of 25 ° C and a photoperiod of 12 hours. The animals are fed and watered ad-libitum. The mice used in our experiments are seven to eight weeks old with body weights between 20 and 24 g. Gender is not taken into account in our tests.
Induction des tumeurs primaires Induction of primary tumors
Les cellules cancéreuses P815 sont recueillies par centrifugation à 1400 rpm pendant 10 mn à température ambiante. Les culots obtenus sont lavés deux fois avec du PBS et remis en suspension dans 1 mL de PBS et comptées. Environ 107 cellules vivantes en suspension dans un volume de 100 pL de PBS sont injectées, sous anesthésie à l’éther, par voie sous cutanée de la région dorsolombaire de chaque souris. Une petite tumeur apparaît dans le lieu de l’injection au bout d’une semaine à 10 jours.
Traitement des souris The P815 cancer cells are collected by centrifugation at 1400 rpm for 10 min at room temperature. The pellets obtained are washed twice with PBS and resuspended in 1 ml of PBS and counted. About 10 7 living cells suspended in a volume of 100 μL of PBS are injected, under ether anesthesia, by subcutaneous route of the dorsolumbar region of each mouse. A small tumor appears in the injection site after one week to 10 days. Mouse treatment
Lorsque les tumeurs deviennent palpables, les souris sont réparties en 4 lots à raison de 6 souris par lot (jours 0). Ensuite, chaque deux jours, les souris sont traitées par gavage de la façon suivante: When the tumors become palpable, the mice are divided into 4 lots at the rate of 6 mice per lot (days 0). Then, every two days, the mice are treated by gavage as follows:
Les souris du lot A (témoin), B, C et D reçoivent respectivement 100 pL d’huile végétale seule, 10, 20 and 30 mg/kg de la molécule P4 (la plus cytotoxiques par rapport autres molécules testées) dissoute dans 100 pL d’huile végétale. Le traitement est effectué à l’ordre d’une seule administration orale toutes les 48h pendant 14 jours. Le poids et la survie des souris, ainsi que le volume tumoral sont mesurés tous les deux jours pendant 28 jours. The mice of batch A (control), B, C and D respectively receive 100 μL of vegetable oil alone, 10, 20 and 30 mg / kg of the molecule P4 (the most cytotoxic compared to other molecules tested) dissolved in 100 μL vegetable oil. The treatment is carried out in the order of a single oral administration every 48 hours for 14 days. The weight and survival of the mice, as well as the tumor volume are measured every other day for 28 days.
Le volume tumoral au jour n (TVn) est calculé de la façon suivante : The tumor volume on day n (TVn) is calculated as follows:
TV= (L* W)/2 où L et W représentent la longueur et la largeur de la tumeur, comme décrit par Yoshikawa [(Yoshikawa et al. 1995)]. TV = (L * W) / 2 where L and W represent the length and width of the tumor, as described by Yoshikawa [(Yoshikawa et al. 1995)].
Analyses statistiques Statistical analyzes
Les expériences réalisées in vitro ont été effectuées en triplicate. Les données reflètent la moyenne de trois expériences différentes. L’analyse statistique pour ces études utilise un test de Student « Student Test ». Les données sont considérées comme statistiquement significatives pour un p<0,05. Concernant l’étude in vivo, chaque condition comprend 6 souris, n=3, et l’analyse statistique a été réalisée en utilisant le test ANOVA à un facteur (one-way Anova) suivie du test post hoc de Tukey et de Scheffé. Les données sont considérées comme statistiquement significatives pour un p<0,05. The experiments carried out in vitro were carried out in triplicate. The data reflect the average of three different experiences. The statistical analysis for these studies uses a Student test "Student Test". The data are considered to be statistically significant for a p <0.05. Regarding the in vivo study, each condition includes 6 mice, n = 3, and the statistical analysis was performed using the one-way ANOVA test (one-way Anova) followed by the post hoc Tukey and Scheffé test. The data are considered to be statistically significant for a p <0.05.
RESULTATS RESULTS
L’activité cytotoxique in vitro a été mesurée par le test MTT contre la lignée tumorale P815 (Figure 1). Cette cytotoxicité commence à de faibles concentrations et augmente d’une manière dose dépendante pour toutes les molécules testées. Ces dernières présentent une activité cytotoxique très importante avec une IC5o comprise entre 0,15 et 9,2 pM (Table 1), et c’est la molécule P4 qui a montrée un effet cytotoxique très fort par rapport aux autres molécules P3 et P4 avec une IC5o=0,15pM. Ainsi, le composé P4 a été évalué pour son effet antitumoral. The in vitro cytotoxic activity was measured by the MTT test against the P815 tumor line (Figure 1). This cytotoxicity begins at low concentrations and increases in a dose-dependent manner for all of the molecules tested. The latter have a very significant cytotoxic activity with an IC 5 o of between 0.15 and 9.2 pM (Table 1), and it is the molecule P4 which has shown a very strong cytotoxic effect compared to the other molecules P3 and P4 with a CI 5 o = 0.15 pM. Thus, the compound P4 has been evaluated for its anti-tumor effect.
Activité antitumorale: études précliniques
Les tests in vivo représentent une étape importante de l’étude de l’activité antitumorale de notre molécule. L’objectif est de passer à un test dans des conditions se rapprochant au mieux de la réalité de la maladie. A cet effet, des souris DBA-2 (H2d) porteuses de tumeurs solides P815 ont été utilisées dans le but de tester l’effet antitumoral in vivo de la molécule P4 qui a montré une activité cytotoxique très importante par rapport aux autres molécules testées PI et P3. Antitumor activity: preclinical studies In vivo tests represent an important step in the study of the antitumor activity of our molecule. The objective is to pass a test in conditions that are as close as possible to the reality of the disease. For this purpose, DBA-2 (H2 d ) mice carrying solid tumors P815 were used in order to test the anti-tumor effect in vivo of the molecule P4 which showed a very significant cytotoxic activity compared to the other molecules tested. PI and P3.
Les essais ont été réalisés par administration orale (gavage) des molécules dissoute dans une huile végétale (huile de table) à des souris de 6-8 semaines toutes les 48 heures pendant une durée de 14 jours. Les résultats obtenus sont présentés sur la figure 2. A la lecture de cette figure, le traitement des souris par la molécule P4 a induit une diminution significative du volume tumoral. L'administration par gavage de la molécule P4 à des doses de 10, 20 et 30 mg / kg a induit une réduction significative du volume tumoral après 28 jours du traitement par rapport au souris témoins (Figure 2). En outre, aucune différence significative n'a été observée entre les doses utilisées. Cependant, on a noté que les souris traitées par la dose 30 mg / kg, ont montré une tolérance vis-à-vis de cette dose avec un taux de survie d’environ 80% (Figure 4), et pas d'impact significatif sur la perte de poids corporel (Figure 3) par rapport aux souris traitées par les doses 10 et 20 mg / kg (P < 0,05). Aussi, on ne note aucune différence significative dans la diminution du volume tumoral après traitement par les trois doses (10, 20 et 30 mg / kg) (P < 0,05). Ainsi, nous pourrions considérer de point de vue efficacité que la dose 30 mg/kg du composé P4 est plus efficace par rapport autres doses testées.
The tests were carried out by oral administration (gavage) of the molecules dissolved in a vegetable oil (table oil) to 6 to 8 week old mice every 48 hours for a period of 14 days. The results obtained are presented in FIG. 2. On reading this figure, the treatment of mice with the P4 molecule induced a significant decrease in tumor volume. The administration by gavage of the molecule P4 at doses of 10, 20 and 30 mg / kg induced a significant reduction in the tumor volume after 28 days of the treatment compared to the control mice (Figure 2). In addition, no significant difference was observed between the doses used. However, it was noted that the mice treated with the 30 mg / kg dose showed tolerance towards this dose with a survival rate of approximately 80% (FIG. 4), and no significant impact. on the loss of body weight (FIG. 3) compared with the mice treated with the doses 10 and 20 mg / kg (P <0.05). Also, there was no significant difference in the decrease in tumor volume after treatment with the three doses (10, 20 and 30 mg / kg) (P <0.05). Thus, we could consider from an efficacy point of view that the 30 mg / kg dose of compound P4 is more effective compared to other doses tested.
Claims
1. Procédé de synthèse de nouveaux dérivés amides de GAE à partir de GAE commercial et différentes amines primaires et secondaires comme précurseurs de départ, comprenant les étapes suivantes : a) réaction de l’acide éthhacrynique AE avec les amines dans un mélange de dichlorométhane (DCM) et N, V-di m éth y 1 form am i de (DMF) en présence de l-éthyl-3-(3- diméthylaminopropyl)carbodiimide (EDCI) et de la 4-diméthylaminopyridine (DMAP) pour obtenir des composés P de formule R= OH ou OMe : 1. Process for the synthesis of new amide derivatives of GAE from commercial GAE and various primary and secondary amines as starting precursors, comprising the following stages: a) reaction of ethhacrynic acid AE with the amines in a mixture of dichloromethane ( DCM) and N, V-di m eth y 1 form am i of (DMF) in the presence of l-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDCI) and 4-dimethylaminopyridine (DMAP) to obtain compounds P of formula R = OH or OMe:
b) la réaction entre le diéthyl chlorophosphate et le composé P3 (R= OH), formé à l’étape (a) dans le dichlorométhane (DCM) à température ambiante en présence de la triéthylamine abouti au composé de la formule P5 b) the reaction between diethyl chlorophosphate and the compound P3 (R = OH), formed in step (a) in dichloromethane (DCM) at room temperature in the presence of triethylamine resulting in the compound of formula P5
2. Procédé de synthèse selon la revendication 1, abouti aux dérivés amides de l’acide ethacrynique avec des groupements 4-(piperazin-l-yl)phenol pour le composé P3, l-(4- methoxyphenyl)piperazine pour le composé P4 et diethyl (4-(piperazin-l-yl)phenyl) phosphate pour le composé P5. 2. Synthesis process according to claim 1, resulting in the amide derivatives of ethacrynic acid with groups 4- (piperazin-1-yl) phenol for the compound P3, l- (4-methoxyphenyl) piperazine for the compound P4 and diethyl (4- (piperazin-l-yl) phenyl) phosphate for compound P5.
3. Procédé pour inhiber ou traiter le cancer in vitro des composés de la revendication 2. 3. A method for inhibiting or treating cancer in vitro of the compounds of claim 2.
4. Procédé selon la revendication 3, dans lequel le cancer est une lignée cancéreuse P815 4. The method of claim 3, wherein the cancer is a P815 cancer line.
5. Le composé P4 selon la revendication 4, a été testé en études précliniques sur un modèle de sourie. Le modèle choisi est constitué de la lignée tumorale P815 et la souche de souris DBA2 (H2d) syngénique. 5. The compound P4 according to claim 4, has been tested in preclinical studies on a mouse model. The model chosen consists of the tumor line P815 and the syngeneic strain of mouse DBA2 (H2 d ).
6. Selon la revendication 5, au moins trois doses du composé P4 à savoir : 10 mg/Kg, 20 mg/Kg et 30 mg/Kg ont été testées pour évaluer l’évolution du volume tumoral chez des souris DBA2 porteuses de tumeurs.
6. According to claim 5, at least three doses of compound P4, namely: 10 mg / Kg, 20 mg / Kg and 30 mg / Kg were tested to evaluate the evolution of the tumor volume in DBA2 mice carrying tumors.
7. Procédé selon les revendications 5, et 6, au moins trois doses à savoir : 10 mg/Kg, 20 mg/Kg et 30 mg/Kg ont été testées pour évaluer l’évolution du poids corporel des souris DBA2 traitées par le composé P4. 7. Method according to claims 5, and 6, at least three doses, namely: 10 mg / Kg, 20 mg / Kg and 30 mg / Kg were tested to evaluate the evolution of the body weight of the DBA2 mice treated with the compound. P4.
8. Procédé selon les revendications 5, 6 et 7, au moins trois doses à savoir : 10 mg/Kg, 20 mg/Kg et 30 mg/Kg ont été testées pour évaluer la survie des souris traitées par le composé8. Method according to claims 5, 6 and 7, at least three doses, namely: 10 mg / Kg, 20 mg / Kg and 30 mg / Kg were tested to assess the survival of mice treated with the compound
P4. P4.
9. Procédé selon les revendications 5, 6, 7 et 8, au moins trois doses à savoir : 10 mg/Kg, 20 mg/Kg et 30 mg/Kg ont été testées pour évaluer la dose du composé P4. 9. Method according to claims 5, 6, 7 and 8, at least three doses, namely: 10 mg / Kg, 20 mg / Kg and 30 mg / Kg were tested to evaluate the dose of compound P4.
10. Procédé selon la revendication 9, les souris traitées par la dose 30 mg / kg du composé P4 ont montré une tolérance vis-à-vis de cette dose avec un taux de survie d'environ 80% et pas d'impact significatif sur la perte de poids corporel. La dose 30 mg/kg, est considérée comme la dose effective.
10. Method according to claim 9, the mice treated with the 30 mg / kg dose of compound P4 showed tolerance towards this dose with a survival rate of approximately 80% and no significant impact on loss of body weight. The dose 30 mg / kg is considered to be the effective dose.
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