WO2013164513A1

WO2013164513A1 - Tamoxifen derivatives, method for obtaining same and use thereof

Info

Publication number: WO2013164513A1
Application number: PCT/ES2013/070280
Authority: WO
Inventors: Mario DÍAZ GONZÁLEZ; Jorge Nicolas MARRERO ALONSO; Tomás GÓMEZ SOUTULLO; Raquel MARÍN CRUZADO; Fernando LAHOZ ZAMARRO; Alicia Boto Castro
Original assignee: Universidad De La Laguna; Consejo Superior De Investigaciones Cientificas (Csic)
Priority date: 2012-05-04
Filing date: 2013-05-06
Publication date: 2013-11-07
Also published as: ES2428464B1; ES2428464A1

Abstract

The invention relates to derivatives of tamoxifen and to the uses thereof as a pharmaceutical composition in the treatment of diseases associated with activation of estrogen signaling, as a tool for the identification of target molecules of tamoxifen, or as a molecular laser emitter. In addition, the invention relates to the method for the production of said compounds and to compositions comprising same.

Description

DERIVATIVES OF TAMOXIFEN, PROCEDURE OF OBTAINING AND ITS

APPLICATIONS

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 . In addition, the present invention relates to the process for obtaining these compounds and compositions comprising them.

STATE OF THE TECHNIQUE

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. The fundamental paradigm that explains the beneficial effects of tamoxifen is that it 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 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 good part of its unwanted effects are related to its ability to modulate cellular responses independent of the receptors of estrogens, some of which start in the cell membrane. The preparation of non-permeable derivatives of tamoxifen, with ammonium groups in the side chain, have allowed us to know some responses at the membrane level as described in Marrero-Alonso J., Garcia-Marrero B., Gómez 5 T. and Díaz M. Functional inhibition of intestinal and uterine muscles by non-permeant triphenyl ethylene derivatives, Eur. J. Pharmacol. (2006), 532, 1 15-127.

Despite these advances, the molecular basis of the actions and collaterals are largely unknown, due to the lack of molecular tools to identify the cell targets on which they occur. Among the possible tools would be the fluorescent derivatives of the drug. Although for other compounds different fluorescent derivatives have been developed [R. W. Sinkeldam, NJ Greco, Y. Tor, "Fluorescent 15 Analogs of Biomolecular Building Blocks: Design, Properties, and Applications", Chemical Reviews, (2010), 1 10, 2579-2619], there are very few works on derivatives of Tamoxifen, and none of them is commercial.

The synthesis and characterization of fluorescent derivatives of 4-hydroxy-Tamoxifen (4-OH-Tx) [Rickert EL, Oriana S, Hartman-Frey C, Long X, Webb TT, Nephew KP, Weatherman RV (2010) has recently been reported. ) Synthesis and characterization of fluorescent 4-hydroxytamoxifen conjugates with unique antiestrogenic properties. Bioconjugate Chemistry. 21: 903-910]. These modifications consist of the conjugation of 4-OH-Tx with different fluorophores, namely BODIPY, carboxyfluorescein and AlexaFluor 546, through a linker of 6 carbon atoms (1, 6-diaminohexane) on the side chain of the tamoxifen Both the incorporation of the linker (which lengthens the side chain) and the size of the fluorophore (which is very bulky), interfere with the binding of tamoxifen to estrogen receptors.

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Therefore, there is a need to synthesize compounds with activity similar to tamoxifen and that have a minimal effect on the affinity of the derivative for estrogen receptor

DESCRIPTION OF THE INVENTION

Brief Description of the Invention

A first aspect of the present invention relates to the compound of general formula (I) (hereafter compound of the invention):

^R 1

(i)

or any of its salts, where:

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:

(IV) (I)

where: X, Ri, R ₂ , R3, R ₄ , n, myp have been described above; Y 'is an amino group, OH or SH and L is a leaving group.

A third aspect of the present invention relates to a pharmaceutical composition comprising at least one compound of general formula (I) and a pharmaceutically acceptable carrier. Optionally 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).

Another aspect of the present invention relates to the use of the compound of the invention, for the preparation of a pharmaceutical composition. Preferably for the treatment and / or prevention of diseases that occur through estrogenic activity.

In addition, another aspect of the present invention relates to a composition comprising at least one compound of general formula (I) and a non-polar organic solvent. And the use of said composition as a laser pigment and / or for the manufacture of a laser device or as an optical resonator.

Therefore, 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.

Detailed description of the invention

In the present invention 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). In addition, 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). In immature rats, where tamoxifen behaves as a partial antagonist, the compound of the invention antagonizes the uterotrophic effect of ethinyl estradiol in a manner similar to tamoxifen but only at the highest doses.

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On the contrary, and of great biological and pharmaceutical importance, 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).

With respect to its biological activity, the compound of the invention, like tamoxifen, 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).

Given the biological properties demonstrated, 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.

Therefore, the first aspect of the present invention relates to the compound of general formula (I) (hereinafter compound of the invention):

or any of its salts, where:

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.

In a preferred embodiment of the compound of the invention, R ₂ and R 3 are H.

In another preferred embodiment of the compound of the invention, R is H or OH. In a more preferred embodiment n, mop have the value of 1.

By "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. Preferably 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 -.

By "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.

The term "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.

By "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.

By "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.

By "O-alkyl" refers to a group containing an oxygen atom attached to an alkyl group (Ci-Cio), defined above.

By "O-acyl" refers to the group -OCO-R ', where R' is a hydrogen or a (C ₁ -C ₁ 0) alkyl group, defined above. By "NH-alkyl" refers to a group -NH- linked to an alkyl group (Ci-Cio), as defined above.

By "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.

By "S-alkyl" refers to a group containing a sulfur atom attached to an alkyl group (Ci-Cio), as defined above. By "acyl" is meant the group -CO-R '", where R'" is a hydrogen or a (C ₁ -C ₁ 0) alkyl group, as defined above.

By "aryl", it 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. Preferably the aryl group has 5 to 7 carbon atoms and more preferably the aryl group is a phenyl.

By "fluoroforo" refers in the present invention to a fluorescent molecule. Preferably 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. Examples of 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).

In a more preferred embodiment of the compound of the invention, R ₁ is NBD, giving rise to the general formula (la):

(the)

Where: X, Y, R ₂ , R3, R ₄ , n, myp, are as described above. In an even more preferred embodiment of the compound of the invention, 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):

(II) In another preferred embodiment of the compound of the invention, Y is an ammonium group and is represented by the general formula (III):

where: X, Ri, R ₂ , R3, R ₄ , n, myp, are as described above;

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; Y

A ^" represents an anion, the anion may be, but not limited to a halide ion (fluoride, chloride, iodide or bromide), sulfate or any other anion. Preferably 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:

(IV) (I)

where: X, Ri, R ₂ , R3, R ₄ , n, myp have been described above; Y 'is an amino group (NH), OH or SH and L is a leaving group.

By "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.

In a preferred embodiment of the process of the invention, when X is a CH ₂ -CH _{2 group} , Y is an amino group and R ₂ , R ₃ , R are hydrogen of the compound of formula (I) that is obtained, or more preferably It is the compound of formula (II), the precursor is N-desmethyltamoxyphene:

In another preferred embodiment, N-demethyltamoxyphene is obtained by demethylation of tamoxifen by treatment with alkyl chloroformates. By "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:

a) the demethylation of tamoxifen 2 by treatment with alkyl chloroformats to obtain / V-Desmethyltamoxyphene 3, and

b a treatment of / V-Desmethyltamoxyphene 3 with NBD-CI.

(2) (3) (II)

On the other hand, 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.

Therefore, 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).

In addition, 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. Optionally, the pharmaceutical composition may comprise another active ingredient.

15 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.

Pharmaceutical forms 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.

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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.

The pharmaceutical 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. However, the preferred route of administration will depend on the condition of the patient. In particular, oral administration is preferred due to the

20 comfort for the patient and the chronic nature of the diseases that should be treated, although administration by intratumoral, subcutaneous or parenteral injection is also preferred.

In a preferred embodiment, 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.

Many breast cancers are sensitive to estrogens, which means that estrogen causes the breast cancer tumor to grow. This type of cancer is called estrogen receptor positive cancer or ER positive cancer or estrogen dependent breast cancer.

Therefore, 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.

In addition to breast cancer, estrogens and their receptors have been associated with a higher incidence of uterine and endometrial cancers, ovarian cancer and prostate cancer. Therefore, since FLTX1 lacks the hyperplastic and hypertrophic effects of tamoxifen in the uterus and endometrium in the tests performed, 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 .

Throughout the present description, the term "treatment" refers to eliminating, reducing or decreasing the cause or effects of the disease. For the purposes of this invention, 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).

In addition, 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

15 is a relevant discovery considering that 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%). Preferably, the compound of the invention used, in the range of

20 concentrations between 1 μΜ and 50 mM and dissolved in a suitable solvent, it behaves as a high-efficiency light material amplifier medium (laser pigment) in the range of 540-570 nm. It should be noted that:

to. It produces laser emission in any of those wavelengths even without the need for mirrors that form a laser cavity when pressed with

A minimum energy within the fluorophore absorption band;

b. When excited with a pulsed laser in nanoseconds, it produces high-energy laser emission pulses (of the order of milli joules) with a temporary duration of the nanosecond pulses; and c. It produces laser emission in any of those wavelengths when a film of said medium is adhered by capillary around a cylindrical or circular symmetry structure. In addition, 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.

Therefore, another aspect of the present invention relates to a composition comprising at least one compound of general formula (I) or compound of the invention and a non-polar organic solvent.

Any 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.

In a preferred embodiment of the composition, the compound of formula (I) is in a concentration between 1 μΜ to 50 mM with respect to the final composition.

Additionally, another aspect of the present invention relates to the use of the composition 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. In a preferred embodiment, both the composition and the compound of formula (I) 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.

Taking into account the application of said composition or compound of formula (I), another aspect of the present invention relates to a device laser comprising the compound of the invention or the composition described above.

In another preferred embodiment, 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. Within this application, 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.

Therefore, another aspect of the present invention would be a method of treating these types of cancers, comprising:

- administration of the compound of the invention in tumor cells by intratumoral, subcutaneous or parenteral injection; Y

- irradiation at a wavelength suitable for photoactivation of the compound and whose laser emission allows localized destruction of ER + tumor cells.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are

25 provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Figure 1.- Analysis by confocal microscopy of the fluorescent and cell-marking properties of the compound of the invention.

Excitation and emission spectra of the compound of formula II (Figure 1A). This compound of the invention allows to mark intracellular targets in permeabilized cells of different cell lines (MCF7, SN56 and HT22, Figure 1 B) and cell membrane targets (MCF7, Figure 1 C).

5 Figure 2.- Estrogenic / cytotoxic (A) and antiproliferative (B) effects of tamoxifen and N- (7-nitrobenzo [c] [1, 2,5] oxadiazol-4-yl) demethyltamoxyphene on the MCF7 cell line. Asterisks indicate significant differences of p <0.05 with respect to the vehicle. i o Figure 3.- Transcriptional activity of tamoxifen and N- (7- nitrobenzo [c] [1, 2,5] oxadiazol-4-yl) demethyltamoxyphene in MCF7 and T47D-KBIuc cells. Both cell lines were transfected with the luciferase gene under the control of the estrogen receptor. The results of the antiestrogenic (A) and estrogenic (B) approach are shown. Asterisks

15 indicate significant differences of p <0.05 with respect to the vehicle.

Figure 4.- Effects of tamoxifen, estradiol and N- (7- nitrobenzo [c] [1, 2,5] oxadiazol-4-yl) demethyltamoxyphene on the histological structure of the uterus of CD1 mice. Representative examples are shown

20 of the hematoxylin-eosin stains under the four treatments. 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

25 limits in height of the epithelium.

Figure 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). In 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. In 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.

5

Figure 6.- Laser emission energy of N- (7-nitrobenzo [c] [1, 2,5] oxadiazol-4-yl) demethyltamoxyphene as a function of the incident pumping energy. In the center a scheme of the system with the trajectories of the incident light and emitted from the cuvette is shown. In the photograph, the arrow indicates the zone of laser emission at 550 nm. A concentrated solution of N- (7- nitrobenzo [c] [1,2,5] oxadiazol-4-yl) demethyltamoxyphene (10 mM) in acetone was used.

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. A concentrated solution of N- (7- nitrobenzo [c] [1,2,5] oxadiazol-4-yl) demethyltamoxyphene (10 mM) in acetone was used.

EXAMPLES OF REALIZATION

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Example 1.- Chemical obtaining of the compound of the invention of general formula (Ib).

1 .1 .- First stage of obtaining / V-Desmethyltamoxyphene (3).

Tamoxifen (2) / V-Desmethyl tamoxifen (3)

25 To a solution of tamoxifen (2) (1.85 g, 5.0 mmol) in dry 1,2-dichloroethane (25 mL) was added ethyl doroformate (0.7 mL, 7.5 mmol) and the solution resulting heated at reflux for 7 h. Then more ethyl doroformate (0.7 mL, 7.5 mmol) was added and reflux was continued for 8 h. The reaction mixture was cooled to 26 ° C and poured onto a dilute aqueous NaOH solution and extracted with ethyl acetate. The organic layer was dried with sodium sulfate, filtered and the solvent was evaporated under vacuum.

The residue was purified by silica gel column chromatography io (hexane / EtOAc 1: 1), obtaining / V-desmethyltamoxyphene 3 (1.26 g, 71%) as a crystalline solid: mp 75-76 ° C (a from hexane); ¹ H NMR (500 MHz, CD ₃ OD) δ _Η 0.89 (3H, dd, J = 7.4, 7.5 Hz), 2.40 (3H, s), 2.44 (2H, ddd, J = 7.4, 7.4, 7.5 Hz), 2.87 (2H, dd, J = 5.2, 5.3 Hz), 3.94 (2H, dd, J = 5.0, 5.3 Hz), 6.58 (2H, d, J = 8.8 Hz), 6.76 (2H, d, J = 8.8 Hz), 7.08 (1 H, dd, J = 7 , 0, 7.2 Hz), 7, 1 1 15 (2H, d, J = 7.2 Hz), 7, 14 (2H, dd, J = 7.2, 7.3 Hz), 7.20 (2H, d, J = 8.2 Hz), 7.25 (1 H, dd, J = 7.2, 7.5 Hz), 7.33 (2H, dd, J = 7.3, 7, 7 Hz); MS m / z (relative intensity) 357 (M ⁺ , 64), 300 (M ⁺ + H - MeNHCH ₂ CH ₂ , 100); HRMS m / z caled for C ₂₅ H ₂₇ NO, 357,2093; found, 357,2063; caled for C ₂₂ H ₂₀ O, 300, 1514; Found, 300, 1491. Anal. Caled for C ₂₅ H ₂₇ NO: C, 83.99; H, 7.61; N, 3.92. Found: C, 83.99; H, 7.79; N, 3.91.

1 .2.- Second stage of obtaining the compound of the invention N- (7- nitrobenzo [c] [1, 2,5] oxadiazol-4-yl) desmethylmethanophene (Formula II).

25 (3) (II) To a stirred solution of desmethyl tamoxifen (3) (200 mg, 0.56 mmol) in dry methanol (15 ml_) was added 4-chloro-7-nitro-1, 2,3-benzoxadiazole (NBD-CI, 244 mg, 1.2 mmol) and the solution was stirred at 26 ° C for 5 h. It was then poured onto a saturated aqueous solution of NaHC0 ₃ and extracted with EtOAc. The organic layer was dried, filtered and evaporated as in the previous experiment, and the residue was purified by column chromatography on silica gel (hexanes-EtOAc, from 95: 5 to 1: 1), obtaining the compound of formula II as a crystalline orange solid (182 mg, 63%): mp 141 ^~ -142 ° C (pentane-EtOAc); ¹ H NMR (500 MHz, acetone-d) δ _Η 0.85 (3H, dd, J = 7.4, 7.4 Hz), 2.39 (2H, ddd, J = 7.4, 7.4 , 7.5 Hz), 3.57 (3H, br s), 4.27 (2H, dd, J = 6.3, 6.3 Hz), 4.52 (1 H, m), 6.41 (1 H, d, J = 9.3 Hz), 6.52 (2H, d, J = 8.2 Hz), 6.73 (2H, d, J = 7.9 Hz), 7.057, 16 ( 5H, m), 7, 18 (2H, d, J = 7.5 Hz), 7.23 (1 H, dd, J = 7, 1, 7.3 Hz), 7.32 (2H, dd, J = 7.0, 7.2 Hz), 8.42 (1 H, d, J = 8.9 Hz); ¹³ C NMR (125.7 MHz, acetone-d) 5 _C 12.8 (CH ₃ ), 29.0 (CH ₂ ), 42.0 (CH ₃ ), 54.6 (CH ₂ ), 65.7 (CH ₂ ), 102.2 (CH), 1 13.4 (2 CH), 126, 1 (CH), 126.6 (CH), 127.8 (2 CH), 128.2 (2 x CH ), 129.2 (2 x CH), 129.6 (2 x CH), 131, 7 (2 χ CH), 135.0 (C), 135.5 (CH), 136.0 (C), 138.4 (C), 141, 4 (C), 142.4 (C), 143.7 (C), 145.0 (2 χ C), 146, 1 (C), 156.5 (C) ; MS m / z (relative intensity) 520 (M ⁺ , 100); HRMS m / z caled for C ₃ i H ₂₈ N ₄ 0 ₄ , 520.21 1 1; found, 520,2099; Anal. Caled for C ₃ i H ₂₈ N ₄ 0 ₄ : C, 71, 52; H, 5.42; N, 10.76. Found: C, 71, 72; H, 5.49; N, 10.76.

Example 2.- Study of the fluorescent properties and cellular dizziness of the compound N- (7-nitrobenzo [c] [1, 2,5] oxadiazol-4-yl) demethyltamoxyphene) (FLTX1)

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. Subsequently, the fixed cells were labeled with 50 μΜ 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. In 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

15 1 B). 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

20 is not modified by excess fluorophore (NBD). A good part of the total tide, around 20%, is displaced by estradiol, indicating that in addition to the estrogen receptor, FLTX1, allows marking other binding sites to tamoxifen. A part of this residual tide corresponds to the calcium channels of type L.

25

Example 3.- Trials of inhibition of cell proliferation of FLTX1,

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. For estrogenicity and toxicity tests, increasing concentrations of the compound of the invention (10 nM-10 μΜ) are they were added to the cultures 24 hours after the start of the culture and they were kept for 6 days. For the antiestrogenic assays, 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).

Thus, it was found that the compound of the invention prevents estrogen-dependent cell growth and proliferation in the MCF7 cell line similar to tamoxifen (Figure 2A). By themselves, neither tamoxifen nor the compound of the invention have proliferative effects on this cell line (Figure 2B).

15 Example 4.- Analysis of the antiestrogenic effects of N- (7- nitrobenzo [c] [1, 2,5] oxadiazol-4-yl) demethyltamoxyphene (FLTX1)

The analysis of the transcriptional effects of FLTX1 was performed in cotransfected cells by electroporation with the reporter gene 3x-Vit-ERE-

20 TATA-luciferase and with the β-galactosidase gene. In parallel, 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

25 of FLTX1 at the indicated concentrations (10 nM - 10 μΜ) for 15 hours (in the estrogenic approach). In the study of antiestrogenic effects, the cells were exposed to these same concentrations of tamoxifen or FLTX1 for 8 hours, and then incubated with 100 pM 17 -estradiol in the presence of tamoxifen or FLTX1 for another 15 hours. One time

After the experiments were completed, the cells were collected, destroyed in lysis buffer, centrifuged at 12000 g for 2 minutes, and luciferase activity in the supernatant was determined by luminometry. The Luminescence was normalized to the amount of protein in the same samples. (Figure 3)

In the analysis of the modification of the transcriptional activity in 5 MCF7 and T47D-KBIuc cells transfected with the luciferase gene under the control of the estrogen receptor, it is observed that unlike tamoxifen, the compound of the invention does not show estrogenicity, that is, it lacks the ability to induce estrogen receptor activation (Figure 3A). The anti-estrogenic approaches show that the compound of the invention is i or as powerful as tamoxifen inhibiting transcriptional activity (Figure 3B), with IC50 values of about 1 μΜ.

Example 5.- Uterotrophic activity of N- (7-nitrobenzo [c] [1, 2,5] oxadiazol-4-yl) demethyltamoxyphene (FLTX1).

fifteen

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

20 FLTX1 (in olive oil) at doses of 0.01, 0, 1 and 1 mg / kg / day (in the case of rats, doses of 10 mg / kg / day were also administered) for 3 days. As positive controls 17-estradiol (1 Mg / kg / day) was used, in the case of mice, or ethinyl estradiol (0.5 Mg / kg / day) in the case of rats, also administered subcutaneously for 3 days. In the case of rats, where they were determined

25 the anti-estrogenic effects, the administration of tamoxifen and FLTX1, at the indicated doses, was also carried out jointly with that of ethinyl estradiol (0.5 Mg / kg / day). After this period the animals were sacrificed by anesthesia with sodium pentobarbital and the different organs and tissues were removed and weighed. In some CD-1 mice, the extracted uterine tissue was divided

30 in two portions (horns) of which one was fixed in Bouin solution for morphometric tests, and the other was embedded in a PBS solution containing 4% paraformaldehyde for immunohistochemical tests. (Tables 1 and 2). Table 1. Relative weights of different organs and tissues in pubertal CD-1 mice treated with tamoxifen and FLUTAMOX

Uterus Treatment

Wet Dry Horns Cervix Vagina Liver Spleen

155.3 12.85 140.7 12.00 106.5 34¿ 13.69 0 \

Vehicle ο ± ^Δ 7 ± ^Δ 4 ± 9.06 ^Δ 3 ± 4.13 ^Δ 98.63 ± ^Δ 5.07 ± 8 736.86 ± 79.58

17p-estradiol

572.1 78.15 469.6 41.63 373.7 38.39 95.9 215.5 23.10 0.1

(1 Mg / kg / day) 1 ± * 9 ± * 5 ± * 4 ± 4.35 * 0 ± * 4.76 ± 9 571, 44 ± 98.41

Tamoxifen

(mg / kg / day)

220.3 33.12 219.8 31.19 176.3 24.42 43.4 159.3 0.5

0.01 7 ± ^Δ 4 ± ^Δ 8 ± ^Δ 7 ± 6.95 ^Δ 8 ± 19.03 5.04 ± 5 593.12 ± 65.16

546.1 62.84 433.8 27.05 373.8 18.92 66.3 7.04 * 213.6 24.13 0.1

0.1 1 ± * 5 ± * 4 ± * 5 ± ^Δ 3 ± * 4.52 ± 2 610.67 ± 73.43

1 474.0 63.06 373.1 17.91 328.6 17.69 44.5 132.7 16.41 0.1

2 ± * 6 ± * 6 ± * 0 ± 5.37 ^Δ 2 ± ^Δ 4.57 ± 2 579.02 ± 38.16

FLUTAMOX

(mg / kg / day)

116, 25.7 113, 27.9 91.9 21.4 21.7 100, 21.6 0.1 531.2

35 ± 5 ^Δ 72 ± 1 ^Δ 8 ± 9 ^Δ 4 ± 6.55 ^Δ 43 ± 3 ^Δ 4.54 ± 7 7 +82.18

110, 13.9 104, 16.7 88.1 12.0 15.9 89.6 18.4 0.2 520.3

70 ± 7 ^Δ 09 ± 1 ^Δ 9 ± 6 ^Δ 0 ± 5.08 ^Δ 1 ± 9 ^Δ 4.44 ± 1 6 + 94.29

126, 20.6 119, 25.1 99.5 20.5 19.7 127, 28.8 0.1 614.9

35 ± 5 ^Δ 30 ± 1 ^Δ 6 ± 5 ^Δ 4 ± 4.93 ^Δ 90 ± 6 ^Δ 4.55 ± 4 1 +69.39

Values expressed as mean ± standard error, corrected to 100 g body weight. Absolute weights are indicated in mg. Liver values are expressed as a percentage of body weight. * Different from the vehicle with statistical significance of p <0.05. ^Δ different from estradiol with statistical significance of p <0.05.

Table 2. Relative weights of different organs and tissues in prepubertal Sprague-Dawley rats treated with tamoxifen and FLUTAMOX

Uterus Treatment

Wet Dry Horns Cervix Vagina Liver Spleen

6/18 332.2 30.2

Vehicle 55.57 ± 3.71 ^Δ 48.73 ± 4.45 ^Δ 41.32 ± 4.10 ^Δ 7.41 ± 1.06 ^Δ 55.30 ± ^Δ 4.47 ± 0.17 7 ± 5

Ethinyl

estradiol

(ΕΕ)

(0.5 313.4 229.6 185.7 106.2 6.58 325.9 21.7

Mg / kg / day) 3 ± 27.41 * 7 ± 12.92 * 3 ± 11.09 * 43.94 ± 4.47 * 7 ± * 3.55 ± 0.64 3 ± 8

Tamoxifen

(mg / kg / day)

6.23 366.9 33.5

0.01 83.56 ± 6.03 ^Δ 75.17 ± 4.60 ^Δ 67.27 ± 4.72 ^Δ 7.90 ± 0.44 ^Δ 61.67 ± ^Δ 4.01 ± 0.13 2 ± 6

148.3 16.63 * 135.5 14.75 * 118.2 12.51 * 2.48 * 7.32 351.3 37.4

0.1 1 ± ^Δ 3 ± ^Δ 4 ± ^Δ 17.29 ± ^Δ 73.78 ± ^Δ 4.27 ± 0.25 5 ± 0

172.9 10.95 * 159.2 130.9 5.45 * ^Δ 2.31 * 6.42 314.0 25.8

1 ± ^Δ 1 ± 7.62 * ^Δ 9 ± 28.23 ± ^Δ 92.66 ± * 4.05 ± 0.10 2 ± 3

208.0 198.6 156.0 3.07 295.2

5 ± 4.10 * ^Δ 9 ± 3.96 * 0 ± 4.78 * 42.69 ± 1.15 * 97.83 ± * 4.26 ± 0.09 1 ± 7.16

1.67 341.1 13.4

59.94 ± 2.93 ^Δ 57.51 ± 4.09 ^Δ 50.21 ± 3.60 ^Δ 7.30 ± 0.92 ^Δ 54.06 ± ^Δ 4.29 ± 0.14 7 ± 4

6.15 329.2 10.7

60.69 ± 2.45 ^Δ 56.95 ± 3.32 ^Δ 50.84 ± 3, 13 ^Δ 6.11 ± 0.43 ^Δ 59.89 ± ^Δ 4.29 ± 0.20 1 ± 2

5.36 308.7 23.7

65.60 ± 3.40 ^Δ 60.66 ± 4.02 ^Δ 52.90 ± 4.04 ^Δ 7.76 ± 0.12 ^Δ 53.59 ± ^Δ 4.15 ± 0.11 8 ± 4

151.0 138.1 120.2 1.69 * 9.95 0.14 353.8

0 ± 8.80 * ^Δ 3 ± 8.37 * ^Δ 1 ± 7.18 * ^Δ 17.92 ± ^Δ 84.45 ± * 3.86 ± * 6 ± 2.99

238.1 190.5 157.8 350.8 22.2

6 ± 8.40 † 1 ± 6.73 † 0 ± 4.52 32.71 ± 2.83 98.63 ± 8.55 4.02 ± 0.05 0 ± 3 207.5 19.78 173.9 11.59 139.9 11.9 334.5 32.4

7 ± † 8 ± † 7 ± 6.28 † 34.01 ± 5.65 97.97 ± 6 3.93 ± 0.26 8 ± 4 176.2 165.8 133.5 10.4 338.6 17.3

8 ± 4.23 † 6 ± 3.07 † 2 ± 2.89 † 32.34 ± 3.46 78.72 ± 1 4.20 ± 0.19 6 ± 1 196.0 11.71 186.3 11 , 51 149.1 328.8 28.5

0 ± † 8 ± † 8 ± 9.39 † 37.20 ± 3.58 94.16 ± 7.84 4.04 ± 0.04 8 ± 0

FLUTAMOX +

EE (0.5

g / kg / day)

(mg / kg / day)

314.9 255.7 208.0 116.7 348.3 10.3

0.01 4 ± 18.07 0 ± 17.95 1 +14.04 47.69 ± 4.65 6 ± 7.83 4.06 ± 0.10 1 ± 3

290.9 232.6 194.3 104.1 279.0 84.2

0.1 5 ± 21, 56 4 ± 16.02 8 ± 11.23 38.26 ± 4.98 9 ± 7.01 4.37 ± 0.10 2 ± 7

235.5 203.6 167.7 323.8 21.5

1 2 ± 34.40 8 ± 28.32 1 ± 21.29 35.97 ± 7.19 99.28 ± 5.94 4.09 ± 0.08 8 ± 5

216.2 15.02 190.6 157.1 0.15 337.1 32.4 10 4 ± † 5 ± 11.50 8 ± 11.02 33.47 ± 1.74 98.62 ± 4.99 3 , 86 ± * 5 ± 9

Values expressed as mean ± standard error, corrected to 100 g body weight. Absolute weights are indicated in mg. Liver values are expressed as a percentage of body weight. * Different from the vehicle with statistical significance of p <0.05. ^Δ Different from ethinyl estradiol with statistical significance of p <0.05. † Different from ethinyl estradiol with statistical significance of p <0.05 for antagonism.

Uterotrophic tests in mice, where tamoxifen acts as an estrogenic agonist and causes the increase in uterine size and weight, as well as the cervix, show that FLTX1 lacks the uterotrophic effects of tamoxifen or estradiol (Table 1).

5

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).

Histological and immunohistochemical studies were performed on fixed uterine samples as indicated above. The uterine horns were embedded in paraffin and serial cuts of 10 μηι were made,

15 dewaxed and stained with hematoxylin-eosin. Using the Leica QWinV3 morphometry software, the number of cells, epithelial heights and the number of stroma glands on consecutive areas of 300 μηΊ ² were analyzed. Immunohistochemical studies were performed on samples fixed in paraformaldehyde, using an anti-PCNA monoclonal antibody.

20 (proliferating cell nuclear antigen). The reaction was revealed by a biotinylated secondary antibody coupled to peroxidase using diaminobenzidine as a substrate. The number of PCNA positive cells in areas of 300 μηι ² in consecutive sections of the middle portion of the uterine horn was determined. (Figure 4).

25

In immature mice, 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) . However, the values observed for the compound of the invention were similar (even slightly lower) to those of the vehicle (-14%). In summary, 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.

5

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). One time

After the animals were sacrificed, the corresponding organs were removed and kept in physiological, oxygenated and glucosed saline solutions, specific for each organ, at 37 ° C for 30 minutes. The baseline tension was adjusted and the contractile range was determined for each tissue at the beginning of each experiment. Contractile responses of uterus and aorta were induced

20 by depolarization with 60 mM KCI. Both tamoxifen and FLTX1 (in the range 0.1-30 μΜ) were added directly to organ baths, while the contractile signal was monitored using isometric transducers. (Figure 5).

In summary, the study of the effects on the contractile activity of the smooth musculature of the digestive tract and of the uterus, indicate that the compound of the invention does not modify neither the intestinal peristalsis (Figure 5A) nor the contractile responses of the uterus in the range of concentrations (Figure 5B) where tamoxifen causes an acute spasmolytic response. However, FLTX1,

30 caused a mild relaxing effect, similar to that exerted by tamoxifen, on the contractile activity of the smooth aortic musculature. 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 μΜ 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).

fifteen

Finally, if it is used in an environment with cylindrical, spherical or circular geometry, and dimensions of tens to hundreds of microns, 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. Thus, 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.

25

Claims

one . Compound of general formula (I):

Ri

(i)

or any of its salts, where:

X represents a (C1-C10) alkyl or acyl group:

Y represents a group 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;

n and m have, independently, a value between 1 and 5; Y

p has a value between 1 to 4.

2. Compound according to claim 1, wherein R ₂ and R3 are H.

3. Compound according to any of claims 1 or 2, wherein R is H or OH.

4. Compound according to any of claims 1 to 3, wherein n, m or p have the value of 1.

5. A compound according to any one of claims 1 to 4, wherein the fluorophore is selected from the list comprising AMCA, NBD, dansyl or 4- Λ / JV-dimethylaminophthalimido.

6. Compound according to any one of claims 1 to 5, wherein X is a (C1-C3) alkyl group.

7. Compound according to any of claims 1 to 6, wherein X is a C2 alkyl.

8. Compound according to any of claims 1 to 7 wherein Y is an amino group.

9. Compound according to any of claims 1 to 8, wherein said compound is of formula (II):

(II)

10. Compound according to any of claims 1 to 7, wherein Y is an ammonium group and is represented by the general formula (III):

(III) wherein: X, Ri, R ₂ , R3, R ₄ , n, myp have been described according to any one of claims 1 to 7;

R _a and R _b independently represent a (C1-) alkyl group

A ^" represents an anion.

eleven . Compound according to claim 10, wherein the anion is a halide or sulfate ion.

12. Compound according to any of claims 10 or 1, wherein R _a or R _b independently represent a (C1-C3) alkyl group.

13. Compound according to the preceding claim, wherein R _a or R _b is a methyl group.

14. A compound according to any of claims 10 to 13, wherein R1 is a fluorophore linked to the ammonium group by means of a (C1-C3) alkyl group.

15. Method of obtaining the compound (I) according to any one of claims 1 to 9, comprising the reaction of a precursor of general formula (IV) with Ri-L:

(IV) (I)

wherein: X, Ri, R ₂ , R3, R ₄ , n, myp have been described according to any one of claims 1 to 14; Y is an amino, thiol or ether group, Y 'is an NH, OH or SH group and L is a leaving group.

16. The method according to claim 15, wherein L is Cl, Br or OMs.

17. Method according to any of claims 15 or 16, characterized in that, when X is a CH ₂ -CH _{2 group} , Y is an amino group, and R2, R3 and R4 are hydrogen, the precursor is N-demethyltamoxyphene.

18. Method according to the preceding claim, wherein N-demethyltamoxyphene is obtained by demethylation of tamoxifen by treatment with alkyl chloroformates.

19. Pharmaceutical composition comprising at least one compound of general formula (I) described according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

20. Pharmaceutical composition according to claim 19, further comprising another antitumor agent.

twenty-one . Pharmaceutical composition according to any of claims 19 or 20, wherein the compound of general formula (I) is in an amount of between 0.01 mg / kg / day to 10 mg / kg / day.

5

22. Use of the compound according to any of claims 1 to 14, for the identification and evaluation of molecular targets of tamoxifen other than estrogen receptors. i o

23. Use of the compound according to any of claims 1 to 14, for the preparation of a pharmaceutical composition.

24. Use of the compound according to any of claims 1 to 14, for the preparation of a pharmaceutical composition for the treatment and / or prevention of treatable or preventable diseases through their anti-estrogenic activity that are selected from the list comprising: cancer , osteoporosis, male infertility (oligospermia), gynecomastia, female infertility, fibrocystic breast condition or hypercholesterolemia.

Use according to claim 24, wherein the cancer is a breast cancer.

26. Composition comprising at least one compound of general formula (I) described according to any one of claims 1 to 14 and a non-polar organic solvent.

25

27. Composition according to the preceding claim, wherein the organic solvent is an alcohol, dimethyl sulfoxide, fatty lipid, ketone or any combination thereof.

Composition according to any one of claims 26 or 27, wherein the compound of formula (I) is in a concentration of between 1 DlVl at 50 mM with respect to the final composition.

29. Use of the composition according to any of claims 26 to 28 or of the compound according to any of claims 1 to 14, as a laser pigment.

30. Use of the composition according to any of claims 26 to 28 or of the compound according to any of claims 1 to 14, for the manufacture of a laser.

31. Use of the composition according to any of claims 26 to 28 or of the compound according to any of claims 1 to 14, as an optical resonator.

32. Use according to any of the preceding claims for applications in optoelectronics, optofluidics, optics, medicine or oncology.

33. Use according to any of claims 29 to 31, for applications in methodologies based on FRET or photoactivation by laser means.

34. Laser device comprising the compound described in any one of claims 1 to 14 or the composition described according to any of claims 26 to 28.