WO2013164513A1 - Dérivés du tamoxifène, procédé d'obtention, et leurs applications - Google Patents

Dérivés du tamoxifène, procédé d'obtention, et leurs applications Download PDF

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WO2013164513A1
WO2013164513A1 PCT/ES2013/070280 ES2013070280W WO2013164513A1 WO 2013164513 A1 WO2013164513 A1 WO 2013164513A1 ES 2013070280 W ES2013070280 W ES 2013070280W WO 2013164513 A1 WO2013164513 A1 WO 2013164513A1
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compound
group
alkyl
compound according
tamoxifen
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PCT/ES2013/070280
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Spanish (es)
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Mario DÍAZ GONZÁLEZ
Jorge Nicolas MARRERO ALONSO
Tomás GÓMEZ SOUTULLO
Raquel MARÍN CRUZADO
Fernando LAHOZ ZAMARRO
Alicia Boto Castro
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Universidad De La Laguna
Consejo Superior De Investigaciones Cientificas (Csic)
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D271/00—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms
    • C07D271/12—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/54—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton
    • C07C217/74—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton with rings other than six-membered aromatic rings being part of the carbon skeleton
    • 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
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/138—Aryloxyalkylamines, e.g. propranolol, tamoxifen, phenoxybenzamine

Definitions

  • the present invention relates to novel compounds, derivatives of tamoxifen, and their uses as a pharmaceutical composition in the treatment of diseases that occur with activation of estrogen signaling or as a tool for the identification of molecular targets of tamoxifen or as a molecular laser emitter .
  • the present invention relates to the process for obtaining these compounds and compositions comprising them.
  • Tamoxifen is the most commonly used drug in cancer chemotherapy in the treatment of estrogen-dependent breast cancer.
  • the introduction of the drug in clinical practice since the 1970s has allowed the accumulation of a large amount of epidemiological data that demonstrates its great efficacy in the treatment of this type of tumors.
  • tamoxifen acts, in estrogen-dependent breast tumors, as an antiestrogen, that is, as a molecule capable of antagonizing estrogen binding to its intracellular receptors, known as estrogens
  • estrogens Despite its well-accepted applicability, numerous evidences show that tamoxifen is capable of producing a considerable number of side effects, ranging from endometriosis (and endometrial cancer) to the generation of cataracts [MacGregor Jl, Jordan VC. "Basic guide to the mechanisms of antiestrogen action”. Pharmacol Rev. (1998) 50 (2): 151-196 and in Jordan VC. "Selective estrogen receptor modulation: concept and consequences in cancer”; Cancer cell. (2004) 5 (3): 207-213].
  • a first aspect of the present invention relates to the compound of general formula (I) (hereafter compound of the invention):
  • X represents a (C1-C10) alkyl or acyl group:
  • Y represents a group that is selected from the list comprising amino, ammonium, thiol, ether, (Ci-C3) alkyl, (C2-C3) alkenyl and (C2-C3) alkynyl;
  • R1 represents a fluoroforo, which can be attached to the Y group directly or by a (C1-C3) alkyl group; preferably R1 is directly linked to group Y.
  • R 2 , R 3 and R 4 independently represent a group that is selected from the list comprising H, OH, NH 2 , SH, O-alkyl, O-acyl, NH-alkyl, NH-acyl, S- alkyl, alkyl, acyl and aryl;
  • nym have, independently, a value between 1 and 5; Y p has a value between 1 to 4.
  • Another aspect of the invention relates to the process for obtaining the compound of the invention, when Y is an amino, thiol or ether group, which yields the reaction of a precursor of general formula (IV) with Ri-L:
  • a third aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one compound of general formula (I) and a pharmaceutically acceptable carrier.
  • said composition may comprise another active ingredient.
  • a fourth aspect of the present invention relates to the use of the compound of general formula (I), for the identification and evaluation of molecular targets of tamoxifen other than estrogen receptors, including transporters and ion channels, as well as other binding sites to antiestrogens (AEBS, antiestrogen-binding sites).
  • AEBS antiestrogen-binding sites
  • Another aspect of the present invention relates to the use of the compound of the invention, for the preparation of a pharmaceutical composition.
  • a pharmaceutical composition Preferably for the treatment and / or prevention of diseases that occur through estrogenic activity.
  • another aspect of the present invention relates to a composition
  • a composition comprising at least one compound of general formula (I) and a non-polar organic solvent.
  • said composition as a laser pigment and / or for the manufacture of a laser device or as an optical resonator.
  • yet another aspect of the present invention relates to a laser or i device comprising the compound of the invention or the composition described above.
  • a new fluorescent derivative of tamoxifen is described, that is, the compound of the invention, the chemical process for obtaining it, and its different utilities or applications, both as a molecular tool and a pharmacological alternative to tamoxifen, are also described.
  • the study of biological effects also indicates that the
  • the compound of the invention has proved to be as powerful as tamoxifen itself in its antiestrogenic facet, but devoid of its side effects.
  • the present invention is based on the fact that the inventors have identified a new derivative of tamoxifen which surprisingly allows to identify in addition to the unconventional molecular target estrogen receptor of tamoxifen (Figure 1 B), that is, different from the estrogen receptor, both intracellular as cell surface and study its molecular pharmacology (Figure 1).
  • the compound of the invention prevents estrogen-dependent cell growth and proliferation in the MCF7 cell line, derived from a human breast carcinoma, similar to tamoxifen (Figure 2A), but does not show estrogenicity, that is, lacks the ability to induce estrogen receptor activation (Figure 3A).
  • the compound of the invention antagonizes the uterotrophic effect of ethinyl estradiol in a manner similar to tamoxifen but only at the highest doses.
  • the compound of the invention lacks the uterotrophic effects of tamoxifen or estradiol (Table 1), neither modifies nor the number of epithelial cells, nor their size, nor increases the number of Endometrial glands (Figure 4) does not modify the intestinal peristalsis (Figure 5A) or contractile responses of the uterus or aorta in the concentration range ( Figure 5B) where tamoxifen causes an acute spasmolytic response and is much less estrogenic than the Tamoxifen at any dose tested (Table 2).
  • the compound of the invention is well tolerated by experimental animals and does not exhibit toxicity in the concentration range tested (0.01 mg / kg / day - 10 mg / kg / day).
  • the compound of the invention could also be used as an anti-estrogen therapy in estrogen-dependent breast tumors, and in other diseases where estrogenic activity is required.
  • the first aspect of the present invention relates to the compound of general formula (I) (hereinafter compound of the invention):
  • X represents a (C1-C10) alkyl or acyl (-CO-) group; preferably it is an alkyl group;
  • Y represents a group that is selected from the list comprising amino, ammonium, thiol (-S-), ether (-O-), alkyl (Ci-C3), alkenyl (C2-C3) and alkynyl (C2-C3) ; preferably it is an amino or ammonium group;
  • R1 represents a fluorophore, which can be attached to the Y group directly or by a (C1-C3) alkyl group; preferably R1 binds directly to group Y;
  • R 2 , R 3 and R 4 independently represent a group selected from the list comprising H, OH, NH 2 , SH, O-alkyl (Ci-Cio), 0-acyl (-OCO-R ' ), NH-alkyl (Ci-Cio), NH-acyl (-NHCO-R "), S-alkyl (Ci-Cio), alkyl (Ci-Cio), acyl (-CO-R '") and aryl; preferably they are, independently, hydrogen, OH or O-alkyl;
  • n and m each have a value of between 1 and 5; Y
  • p has a value between 1 to 4.
  • R 2 and R 3 are H.
  • R is H or OH.
  • mop have the value of 1.
  • alkyl refers in the present invention to saturated, linear or branched hydrocarbon chains having 1 to 10 carbon atoms, for example, methyl, ethyl, n-propyl, / -propyl, n-butyl, ferc- butyl, sec-butyl, n-pentyl, n-hexyl, etc.
  • the alkyl group has between 1 and 6 carbon atoms, more preferably between 1 and 3 carbon atoms. Even more preferably when X is an alkyl group it is -CH 2 -CH 2 -.
  • alkenyl refers in the present invention to unsaturated, linear or branched hydrocarbon chains, having 2 to 3 carbon atoms, and containing a double carbon-carbon bond, for example, vinyl, 1-propenyl, allyl, isopropenyl, etc.
  • alkynyl refers to radicals of hydrocarbon chains, linear or branched, of 2 to 3 carbon atoms, and containing a triple carbon-carbon bond, for example, ethylino, propinyl, etc.
  • amino refers to -NR a -, where R a is a (C 1 -C 1 0) alkyl group, as described above. More preferably R a is a methyl group.
  • ammonium refers to the group - + NR to Rb-, where R a and R b independently represent a (C 1 -C 1 0) alkyl group, as described above. More preferably they are a methyl group.
  • O-alkyl refers to a group containing an oxygen atom attached to an alkyl group (Ci-Cio), defined above.
  • O-acyl refers to the group -OCO-R ', where R' is a hydrogen or a (C 1 -C 1 0) alkyl group, defined above.
  • NH-alkyl refers to a group -NH- linked to an alkyl group (Ci-Cio), as defined above.
  • NH-acyl refers to the group -NHCO-R ", where R” is a hydrogen or a (C 1 -C 1 0) alkyl group, as defined above.
  • S-alkyl refers to a group containing a sulfur atom attached to an alkyl group (Ci-Cio), as defined above.
  • acyl is meant the group -CO-R '", where R'" is a hydrogen or a (C 1 -C 1 0) alkyl group, as defined above.
  • aryl refers, in the present invention, to aromatic rings, single or multiple, having 5 to 18 carbon atoms in the ring part, such as but not limited to, phenyl, naphthyl, diphenyl, indenyl , phenanthryl, fluorenyl or anthracil.
  • the aryl group has 5 to 7 carbon atoms and more preferably the aryl group is a phenyl.
  • fluoroforo refers in the present invention to a fluorescent molecule.
  • this fluorophore is small in size, that is, it implies that said molecule is of small weight and volume, more preferably the fluorophore has a molecular weight equal to or less than 300 Da.
  • fluorophores are, but not limited to, AMCA (diethylamino cumarin carboxyl), NBD ((7-nitrobenzo [c] [1, 2,5] oxadiazol-4-yl), dansyl (5- (Dimethylamino) naphthalene-1 - sulfonyl) or 4- / V, A / -dimethylaminophthalimido (or 4- / V, A / -D ⁇ met ⁇ lamino-1, 8-naphthalimido).
  • AMCA diethylamino cumarin carboxyl
  • NBD ((7-nitrobenzo [c] [1, 2,5] oxadiazol-4-yl)
  • dansyl (5- (Dimethylamino) naphthalene-1 - sulfonyl) or 4- / V, A / -dimethylaminophthalimido (or 4- / V, A / -D ⁇ met ⁇ lamino-1, 8-
  • R 1 is NBD, giving rise to the general formula (la):
  • Y is an amino group and more preferably a group -N (CH 3 ) -, even more preferably R1 is NBD, more preferably X is -CH 2 -CH 2 - and still more preferably it is the compound of formula (II) called N- (7- nitrobenzo [c] [1, 2,5] oxadiazol-4-yl) demethyltamoxyphene (FLTX1 or FLUTAMOX):
  • Y is an ammonium group and is represented by the general formula (III):
  • R a and R b independently represent a (C1-C10) alkyl group, more preferably a (C1-C3) alkyl group, even more preferably a methyl group;
  • the anion may be, but not limited to a halide ion (fluoride, chloride, iodide or bromide), sulfate or any other anion.
  • the anion is a halide ion.
  • the compound of general formula (III) is a non-permeable derivative of tamoxifen, which allows to study its interactions at the membrane level.
  • a second aspect of the present invention relates to a process for obtaining the compound (I) or compound of the invention, when Y is an amino, thiol or ether group, which comprises the reaction of a precursor of general formula (IV) with R1-L:
  • leaving group is meant an atom or group of atoms that are easily separated from a molecule, in this case the fluorescent molecule R 1 .
  • Outgoing groups are widely known to a person skilled in the art and can be, but not limited to, Cl, Br, I, mesyl (OMs), among others.
  • N-demethyltamoxyphene is obtained by demethylation of tamoxifen by treatment with alkyl chloroformates.
  • alkyl chloroformates refers to the compound CI-C (0) 0-alkyl, where the alkyl group (Ci-Cio) is defined above.
  • a particular process of the present invention, useful for obtaining compound (II), comprises:
  • the compound of the invention is applicable in the biotechnological field, specifically in the development of bioassays of estrogenic / antiestrogenic activity, as a molecular tool in the subcellular identification of molecular targets of tamoxifen outside estrogen receptors, in competition bioassays or in bioactivation assays under the control of the estrogen receptor. More particularly, as a probe in research laboratories to study physiological processes of little known clinical interest, since the usual interaction of tamoxifen is with estrogen receptors.
  • another aspect of the present invention relates to the use of the compound of the invention, for the identification and evaluation of molecular targets of tamoxifen other than estrogen receptors, these include ionic transporters and channels, as well as other antiestrogen binding sites (AEBS).
  • AEBS antiestrogen binding sites
  • the compound of the invention is applicable in the pharmaceutical field as an anti-estrogen, in particular in adjuvant treatments in estrogen-dependent breast cancer, lacking the uterotrophic, hyperplastic, hypertrophic and estrogenic agonism effects of tamoxifen. Therefore, another aspect of the present invention relates to a pharmaceutical composition comprising at least one compound of general formula (I) and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may comprise another active ingredient.
  • This active substance could be another antitumor agent, since a combination of active substances is used in a large part of the anticancer therapies applied to patients.
  • compositions suitable for oral administration include any solid composition (tablets, pills, capsules, granulated forms, etc.) or liquid (solutions, suspensions, emulsions, syrups, etc.) and may contain conventional excipients known in the art, such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, starch, corn, calcium phosphate, sorbitol or glycine, lubricants for the preparation of tablets, for example magnesium stearate, disaggregates such as starch, sodium polyvinyl pyrrolidone starch or microcrystalline cellulose, or agents Pharmaceutically acceptable humectants, such as sodium lauryl sulfate.
  • binding agents for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone
  • fillers for example lactose
  • Solid oral compositions may be prepared by conventional methods of mixing, filling or tabletting.
  • the Repeated mixing operations can be used to evenly distribute the active ingredient using large amounts of fillers. These operations are conventional in the art of this invention.
  • the tablets can be prepared, for example, through wet or dry granulation and can optionally be coated by methods well known in normal pharmaceutical practice, particularly with an enteric coating.
  • compositions can also be adapted for parenteral administration, such as sterile solutions, suspensions or lyophilized products in the appropriate pharmaceutical form.
  • Suitable excipients such as bulk agents, neutralizers or surfactants may be mentioned.
  • the compounds or compositions described in the present invention can be administered by any suitable method, such as intravenous infusion, oral preparations and intraperitoneal or intravenous administration.
  • suitable method such as intravenous infusion, oral preparations and intraperitoneal or intravenous administration.
  • the preferred route of administration will depend on the condition of the patient.
  • oral administration is preferred due to the
  • the pharmaceutical composition comprises an amount of between 0.01 mg / kg / day at 10 mg / kg / day of the compound of general formula (I).
  • Another aspect of the present invention relates to the use of the compound of the invention of general formula (I) for the preparation of a pharmaceutical composition, preferably for the treatment and / or prevention of diseases through its anti-estrogenic activity, therefore these diseases are treatable or preventable diseases through their Antiestrogenic activity and these can be selected from the list comprising: osteoporosis, male infertility (oligospermia), gynecomastia, ovulatory disorders (including infertility), mammary fibrocystic condition or hypercholesterolemia.
  • estrogen receptor positive cancer or ER positive cancer or estrogen dependent breast cancer.
  • a preferred embodiment of the invention comprises the use of the compound of the invention of general formula (I) for the preparation of a pharmaceutical composition for the treatment and / or prevention of a type of estrogen-dependent cancer, more preferably of cancer of estrogen dependent breast.
  • a preferred embodiment of the invention comprises the use of the compound of the invention of general formula (I) for the preparation of a pharmaceutical composition for the treatment of ER + breast cancers in which there is a probable risk of uterine carcinomas due to tamoxifen as well as in the prevention and / or treatment of endometrial, ovarian and prostate cancers whose progress is associated with estrogenic activity .
  • treatment refers to eliminating, reducing or decreasing the cause or effects of the disease.
  • treatment includes, but is not limited to, alleviating, reducing or eliminating one or more symptoms of the disease; reduce the degree of disease, stabilize (i.e. no worsen) the state of the disease, delay or slow the progression of the disease, alleviate or improve the state of the disease and remit (either total or partial).
  • the compound of the invention - diluted in a suitable organic solvent, such as methanol, olive oil or acetone - behaves as a material medium with laser emission capacity (Figure 6). That is, the compound of the invention behaves like a "Laser Dye" or laser pigment and, therefore, can be applied in the field of nanotechnology, in particular or in optical or optofluidic. To ensure that this laser pigment emits laser light, it must be illuminated with pulsed light whose wavelength corresponds to the absorption band of the compound of the invention used (approx. 550 nm).
  • the present invention is a novelty since neither the tamoxifen molecule nor that of the NBD fluorophore produces, by themselves, laser emission. Be
  • the efficiency of conversion of pumping light to laser emission light is greater (approximately 30%) than the measurement of other commercial laser pigments of frequent use such as Rhodamine 6G ( less than 20%).
  • the compound of the invention used in the range of
  • the compound of the invention could be used for the development of lasers with applications in optoelectronics, optofluidics, optics, medicine, oncology, etc. Likewise, it could be used in methodologies based on FRET (Fluorescence Resonance Energy Transfer) as both a donor and an energy acceptor for microscopy imaging.
  • FRET Fluorescence Resonance Energy Transfer
  • another aspect of the present invention relates to a composition
  • a composition comprising at least one compound of general formula (I) or compound of the invention and a non-polar organic solvent.
  • non-polar or non-polar organic solvent is known to any person skilled in the art and as examples, but not limited to, alcohol, dimethyl sulfoxide, fatty lipid, ketone or any combination thereof, preferably methanol, dimethyl sulfoxide, olive oil, acetone, could be used. or any of its combinations.
  • the compound of formula (I) is in a concentration between 1 ⁇ to 50 mM with respect to the final composition.
  • compositions described above or of the compound of general formula (I), as a laser pigment, for the manufacture of a laser or as an optical resonator are useful for applications in optoelectronics, optofluidics, optics, medicine, oncology, in methodologies based on FRET (Fluorescence Resonance Energy Transfer), both as a donor and energy acceptor for imaging in microscopy, or photoactivation by laser means.
  • FRET Fluorescence Resonance Energy Transfer
  • another aspect of the present invention relates to a device laser comprising the compound of the invention or the composition described above.
  • the application of the laser emission of the compound of the invention or more preferably of the compound of formula (II) can be used to kill tumor cells, preferably ER + cells, in localized tumors.
  • tumor cells preferably ER + cells
  • these ER + tumor cells would be previously loaded with the compound by intratumoral, subcutaneous or parenteral injection and photoactivated by wavelengths i or within their excitation spectrum.
  • Another aspect of the present invention would be a method of treating these types of cancers, comprising:
  • Figure 1 Analysis by confocal microscopy of the fluorescent and cell-marking properties of the compound of the invention.
  • the upper panel shows cross sections of the uterine body.
  • the central panel shows the microscopic structure of the myometrium (myo), the stroma (stro) and the epithelium (epi).
  • the arrows point to the stroma glands.
  • the lower panel shows my crofotog raffias at 60 magnification of the epithelium. The double arrow marks the
  • FIG. 5 Effects of tamoxifen and N- (7-nitrobenzo [c] [1, 2,5] oxadiazol-4-yl) demethyltamoxyphene on spontaneous peristatic activity (A) and on uterine contractile response induced by depolarization by KCI 30 in the presence of CaCI 2 (B).
  • A representative examples of the responses to tamoxifen (left panel) and N- (7-nitrobenzo [c] [1, 2,5] oxadiazol-4-l) demethyltamoxyphene (right panel) on duodenal peristalsis are shown in CD1 female mice.
  • B typical response of uteri of ovariectomized mice in which a tonic contraction is induced by CaC + KCI. Tamoxifen and N- (7-nitrobenzo [c] [1,2,5] oxadiazol-4-yl) demethyltamoxyphene are applied at the indicated times.
  • Figure 7 Resonance spectrum of the laser emission energy of N- (7- nitrobenzo [c] [1, 2.5] oxadiazol-4-yl) demethyltamoxyphene.
  • the spectrum 15 corresponds to the energy above the pumping threshold in the beam of light with vertical polarization.
  • Example 1 Chemical obtaining of the compound of the invention of general formula (Ib).
  • the normalized fluorescence spectra of the FLTX1 compound in methanolic solution are shown in Figure 1A.
  • the compound has a maximum excitation at 428 nm and a maximum emission at 530 nm (shown in Figure 1A for a solution of FLTX1 at 10 mM in methanol).
  • the study of the fluorescent properties of FLTX1 cell dyeing was performed on HT22, SN56 and MCF7 cells, fixed in 2% paraformaldehyde 0.1% glutaraldehyde and 150 mM sucrose, at room temperature and under both conditions permeabilizers (0.5% nonidet P-40 2 minutes) and non-permeabilizers.
  • the fixed cells were labeled with 50 ⁇ FLTX1 for 2 hours, washed in PBS and mounted on glass supports in PBS / glycerol (1: 1). Fluorescent marking was analyzed using confocal microscopy 5 using an argon line with excitation at 415 nm and recording the fluorescence emitted at 560 nm.
  • the competition assays prior to the FLTX1 addition, the cells were exposed to different concentrations of estradiol, tamoxifen or NBD, for 30 minutes at room temperature. Estrogen receptor colocalization assays were performed by first determining the immunosensal of the anti-ER alpha antibody, and then the FLTX1 fluorescent signal.
  • the FLTX1 fluorescent derivative allows intracellular targets to be marked in permeabilized cells of different cell lines (MCF7, SN56 and HT22, Figure
  • Fluorescent marking of the cell membrane is also possible, in line with the existence of membrane binding sites for antiestrogens under non-permeabilizing conditions (shown in Figure 1C for MCF7 cells, similar results are obtained in the other two cell lines) .
  • the marking is specific for tamoxifen: it is fully competed by it and
  • NBD excess fluorophore
  • Cell proliferation assays were performed on MCF7 cells, derived from a human breast carcinoma, positive for the 30 estrogen receptor cultured in phenol red free medium and in the presence of carbon-inactivated fetal serum.
  • increasing concentrations of the compound of the invention (10 nM-10 ⁇ ) are they were added to the cultures 24 hours after the start of the culture and they were kept for 6 days.
  • the same concentrations of the compound of the invention were used as in the estrogenic assays, but were maintained for 4 days, after which 5 100 pM 17 -estradiol was incorporated.
  • Cell viability was quantified using the WST-1 reagent (Cell Proliferation Reagent, Roche), which determines mitochondrial activity in functionally viable cells. ( Figure 2).
  • the compound of the invention prevents estrogen-dependent cell growth and proliferation in the MCF7 cell line similar to tamoxifen ( Figure 2A).
  • tamoxifen nor the compound of the invention have proliferative effects on this cell line ( Figure 2B).
  • T47D-KBIuc cells were used, stably transfected with the pGL2.TATA gene. lnr.luc. neo. After transfected, the cells were incubated in phenol red free medium in the presence of inactivated serum for 24 hours. After this period they were incubated in the presence of tamoxifen or
  • Example 5 Uterotrophic activity of N- (7-nitrobenzo [c] [1, 2,5] oxadiazol-4-yl) demethyltamoxyphene (FLTX1).
  • mice In the "in vivo" bioassays of uterotrophic activity immature CD-1 mice (17 days) and immature Sprague-Dawley rats (19 days) were used, maintained between 22 ° C and 24 ° C under 12-hour light-dark cycles. The mice were injected subcutaneously - and daily with solutions of tamoxifen or
  • FLTX1 In immature rats, where tamoxifen behaves as a partial antagonist, FLTX1 antagonizes the uterotrophic effect of ethinyl estradiol similarly to tamoxifen but only at higher doses. In contrast, FLTX1 is much less estrogenic than tamoxifen at any dose i or tested (Table 2).
  • both tamoxifen and 17-pestradiol cause an increase in cell proliferation determined as immunoreactivity to the nuclear proliferation antigen (PCNA) (+1 10% and + 79% with respect to the vehicle, for tamoxifen and estradiol, respectively) .
  • PCNA nuclear proliferation antigen
  • the values observed for the compound of the invention were similar (even slightly lower) to those of the vehicle (-14%).
  • the compound of the invention unlike tamoxifen, which causes hyperplasia and hypertrophy of the uterine endomethne, the compound of the invention neither modifies the number of epithelial cells, nor their size, nor increases the number of endomethales glands.
  • Example 6. The influences of the compound of the invention on contractile activities associated with intestinal peristalsis and mechanical responses of the uterus and aorta.
  • io Contractile activities associated with intestinal peristalsis and mechanical responses of the uterus and aorta were performed in rodent models appropriate to the type of tissue tested (male CD-1 mice for the study of duodenal peristalsis, ovariectomized mice for uterine tests , and male Sprague-Dawley rats for aorta studies).
  • rodent models appropriate to the type of tissue tested male CD-1 mice for the study of duodenal peristalsis, ovariectomized mice for uterine tests , and male Sprague-Dawley rats for aorta studies.
  • Example 7 Characterization of FLTX1 as a laser emission material.
  • the laser emission of FLTX1 is achieved by illuminating the material (a solution of FLTX1 between 1 ⁇ and 50 mM dissolved in a non-polar organic solvent 5 suitable as acetone, methanol or vegetable oil) with pulsed light of a wavelength between 425 nm and 520 nm, corresponding to the absorption band of FLTX1.
  • the density of pumping energy must be above a minimum value. If a pulsed light source with a pulse duration of 10 ns at 470 nm is used, the minimum energy density io to obtain laser emission is 0.6 mJ / cm 2
  • the material medium that produces the laser emission is a fluid that can be housed in a transparent quartz cuvette. Said cuvette does not need mirrored coatings or be inside an outer laser cavity. Laser emission occurs in the material medium in the absence of additional mirrors. ( Figure 6A).
  • an optical resonator can be built that amplifies the emission of the FLTX1 obtaining very narrow laser emission peaks corresponding to the modes 20 of the resonant cavity ( Figure 7).
  • the position of these peaks is sensitive to changes in the physical or chemical properties of the medium.
  • variations of these laser emission peaks can be used as a sensor to determine interactions between FLTX1 and other molecules, such as estrogen receptors or other molecular targets.

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Abstract

L'invention concerne des dérivés du tamoxifène, et leurs utilisations comme composition pharmaceutique dans le traitement de maladies qui se traduisent par l'activation de la signalisation oestrogène ou comme outil d'identification de cibles moléculaires du tamoxifène ou comme émetteur laser moléculaire. En outre, la présente invention concerne le procédé d'obtention de ces composés et des compositions les contenant.
PCT/ES2013/070280 2012-05-04 2013-05-06 Dérivés du tamoxifène, procédé d'obtention, et leurs applications WO2013164513A1 (fr)

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ESP201230670 2012-05-04
ES201230670A ES2428464B1 (es) 2012-05-04 2012-05-04 Derivados del tamoxifeno, procedimiento de obtención y sus aplicaciones.

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EMILY L. RICKERT ET AL: "Synthesis and Characterization of Fluorescent 4-Hydroxytamoxifen Conjugates with Unique Antiestrogenic Properties", BIOCONJUGATE CHEMISTRY, vol. 21, no. 5, 19 May 2010 (2010-05-19), pages 903 - 910, XP055083672, ISSN: 1043-1802, DOI: 10.1021/bc900461h *

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