ES2370655B1 - DENDRIMEROS AS NON-VIRAL VEHICLES FOR GENE THERAPY - Google Patents

Abstract

Dendrimers as non-viral vehicles for gene therapy. # The present invention relates to new compounds of general formula (I), (II) and (III) for use in gene therapy as non-viral vehicles and their use for the preparation of a medicine. In addition, the synthesis process of said compounds of general formula (I), (II) and (III) is described.

Description

Dendrimers as non-viral vehicles for gene therapy.

The present invention refers to new compounds of general formula (I), (II) and (III) for their genetic therapy as non-viral vehicles and their use for the preparation of a drug. In addition, the synthesis process of said compounds of general formula (I), (II) and (III) is described.

Prior art

The use of non-viral vectors in gene therapy is especially relevant, since the FDA has suspended, without die, clinical trials using viruses (adenoviruses, adeno-associated, etc.) because they generate immune reactions that have caused the death of some patients who participated in such trials. , such as, insecurity in its management, toxicity, provocation of an immune response that decreases its lack of cellular specificity. Together, these systems are quickly eliminated from the circulation, limiting the process of transfection by first step organs (lungs, liver and spleen).

It should also be borne in mind that recombination processes can cause a replicating virus although the danger is remote. However, the problems posed by viruses as gene therapy are serious and clinical trials of gene therapy in, for example, the United States, They have recently been interrupted by the FDA due to the death of several patients due to the multi-organ failure. This type of serious problems has led to the search and development of alternatives to the use of viruses as generators.

Non-viral vectors have a number of advantages over viral analogues: a) ease of preparation (including multigram scale) and modification, b) greater flexibility with respect to the size of the genetic material to be transfected, c) they are generally safe in vivo and d) they do not cause an immune response Specifi caypor can both be administered repeatedly.

Within non-viral vectors, dendrimers represent one of these alternatives, since they have a nanometric size, a globular structure, a low polydispersity and a high density that work on the surface with a small molecular volume.

Description of the invention

The present invention refers to new compounds of the general formula (I), (II) and (III) for use in gene therapy such as viral vehicles and used for the preparation of a medication or transfection kits. In addition, the synthesis process of said compounds of general formula (I), (II) and (III) is described.

Therefore, a first aspect of the present invention refers to a compound of general formula (I)

or a salt, prodrug or solvate thereof;

where:

X is selected from an unsaturated (C2-C12) alkyl or cycloalkyl (C3-C8), cycloalkenyl, aryl or heteroaryl group,

R1, R2, R3, R4 and R5 are the same or different and are selected from hydrogen, saturated or unsaturated alkyl (C2-C12), cycloalkyl (C3-C8), cycloalkenyl, aryloheteroaryl,

Z is selected from any anion chloride, tri-fluoroacetate, DNA, RNA, siRNA, miRNA, antagomir, drug, antibody, probe, (radioactive or not) for the diagnosis of diseases by imaging techniques (Nuclear Magnetic Resonance, Tomography by simple photon emission or Positron emission tomography ) or any combination thereof.

A preferred embodiment refers to compound of general formula (II):

or a salt, prodrug or solvate thereof;

where:

X is selected from a group of saturated or unsaturated alkyl (C2-C12) or cycloalkyl (C3-C8) or cycloalkenyl, aryl or heteroaryl,

R1, R2, R3, R4 and R5 are the same or different and are selected from hydrogen, saturated or unsaturated alkyl (C2-C12), cycloalkyl (C3-C8), cycloalkenyl, aryloheteroaryl,

Z is selected from any anion chloride, tri-fluoroacetate, DNA, RNA, siRNA, miRNA, antagomir, drug, antibody, probe, (radioactive or not) for the diagnosis of diseases by imaging techniques (Nuclear Magnetic Resonance, Tomography by simple photon emission or Positron emission tomography ) or any combination thereof.

According to another preferred embodiment, it refers to a compound of general formula (III):

or a salt, prodrug or solvate thereof; where: X is selected from an unsaturated (C2-C12) alkyl or cycloalkyl (C3-C8) or cycloalkenyl group,

aryl or heteroaryl,

R1, R2, R3, R4 and R5 are the same or different and are selected from hydrogen, saturated or unsaturated alkyl (C2-C12), cycloalkyl (C3-C8), cycloalkenyl, aryl or heteroaryl, Z is selected from any chloride anion, tri-fluoroacetate, DNA, RNA, siRNA, miRNA, antagomir or which

Any combination of them.

In another preferred embodiment, both for the compound of general formula (I), and for the compound of general formula (II)

or (III), X is selected without limiting meaning between:

According to another preferred embodiment, the radicals R1aR5 may be the same or different and are independently selected from H, any substituted or unsubstituted amine, basic amino acids such as lysine or arginine, cholesterol derivatives from cholesteryl chloroformate, folic acid, lactic acid, Dexamethasone, sugars such as, for example, in a limiting sense, lactose-freelayer to give off its derivative of detosyl, lysosomotropic agents such as chloroquine, nitrogen heterocycles such as uridine, piperidine or piperazine, hydrophilic chains derived from any poly-ethylene glycol, any basic structure:

In a preferred embodiment, said general formula (I), (II) or (III) compounds are selected, but not limited to, from the group comprising:

The term "alkyl" refers, in the present invention, to saturated, linear or branched, saturated, aliphatic chains.

or unsaturated, having 2 to 12 carbon atoms. For example, but not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, tert-butyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl etc. In addition, the term alkyl also refers to linear or branched aliphatic chains, saturated or unsaturated from 2 to 12 carbon atoms, which may be substituted by functional groups such as hydroxyl, carboxyl, carbonyl, amine, amide, and any heteroatom selected from nitrogen, oxygen and sulfur may be contained in its structure.

The term "cycloalkyl" refers to a stable monocyclic bicyclic radical of 3-8 members, which is saturated

or partially saturated, and that only consist of carbon and hydrogen atoms. For example, but not limited to, cyclobutane, cyclopentane, cyclohexane or cycloheptane. In addition, the term cycloalkyl also refers to a stable bicyclic monocyclic radical of 3 to 8 members, which is partially saturated or saturated, and which only consists of carbon and hydrogen atoms, which may be substituted by functional groups such as hydroxyl, carboxyl, carbonyl, amine, amide or which may contain in its structure any heteroatom selected from nitrogen, oxygen and sulfur.

The term "salts, solvates, prodrug pharmaceutically acceptable" refers to any salt, ester, solvatopharmaceutically acceptable, or any other compound that, when administered to a receptor, is capable of providing (directly or indirectly) a compound as described herein. . However, it will be appreciated that pharmaceutically acceptable salts are also within the scope of the invention and that these may be useful in the preparation of pharmaceutically acceptable salts. The preparation of salts, prodrugs and derivatives can be carried out by methods known in the art.

For example, pharmaceutically acceptable salts of compounds provided herein are synthesized by conventional chemical methods from an original compound containing an acidic basic moiety. Generally, such salts are prepared, for example, by reacting the free acid or base forms of the compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Examples of acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, iohydrate, sulfate, nitrate, phosphate and organic acid addition salts such as, for example, acetate, maleate, fumarate, citrate. , oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and p-toluenesulfonate. Examples of base addition salts include inorganic salts such as, for example, sodium, potassium, calcium, ammonium, magnesium, aluminum and lithium salts, and salts of organic bases such as, for example, ethylenediamine, ethanolamine, N, N- dialkylene ethanolamine, glucamine and basic amino acid salts.

Particularly favorite derivatives or prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a patient (for example, by making a compound administered orally more easily absorbed by blood), or that enhances the release of the original compound in a biological compartment (for example, the brain or lymphatic system) in relation to the original species.

Any compound that is a prodrug of a compound of formula (I) is within the scope of the invention. The term "prodrug" or "prodrug" is used in its broadest sense and encompasses those derivatives that become envy in the compounds of the invention. Such derivatives will be obvious to those experts in the technique, and include, depending on the functional groups present in the molecule and without limitation, the following derivatives of the compounds present esters, amino acid esters, phosphate esters, sulphonate esters of metal salts, carbamates, yamides.

The compounds of formula (I), (II) and (III) may be in crystalline form as free compounds or as solvates and both forms are intended to be within the scope of the present invention. Solvation methods are generally known within the art. Suitable solvates are pharmaceutically acceptable solvates. In a particular embodiment, elsolvate is a hydrate.

A second fundamental aspect of the present invention refers to a process for the preparation of a compound of general formula (I) comprising the following steps:

The values of R1-R5, X and Z as described above.

Another fundamental aspect of the present invention refers to a process for the preparation of a compound of general formula (II) which comprises the same stages for the preparation of the compound of general formula

(I)

starting from this last compound.

Another fundamental aspect of the present invention relates to a process for the preparation of a compound of general formula (III) comprising the same steps for the preparation of the compound of general formula

(I)

starting from the compound of general formula (II).

In another respect, the present invention refers to a compound of formula (I), (II) or (III) for the manufacture of a medicament.

In another aspect, the present invention relates to the use of the aforementioned medicament for the treatment and / or prevention of diseases related to the nervous system, strokes and diseases that occur with tumor processes.

Therefore, the compounds of the present invention could be used for the preparation of a medicament for the treatment of diseases that are selected from the list comprising: diseases of the nervous system such as neurodegenerative diseases (Parkinson's disease, dementia including Alzheimer's disease, Hungtinton's disease, demyelinating diseases such as multiple sclerosis and amyotrophic lateral sclerosis), strokes (including pathology derived from thrombosis and cerebral hemorrhage) .This includes also the treatment of tumors (especially prostate, lung and breast, without this list being limited). be exclusive of other types of tumor pathology).

In another aspect, the present invention refers to the compound of formula (I), (II) or (III) for the preparation of a siRNA transfection kit in primary cultures.

Preferably the primary cultures are selected from the group consisting of: nerve cells, glia, tumor cells and other primary cells such as hepatocytes.

In another aspect, the present invention relates to the use of the compound of formula (I), (II) or (III) in gene therapy as a non-viral vector.

In another aspect, the present invention refers to the compound of the formula (I), (II) or (III) for the elaboration of rounds, (radioactive sound) for the diagnosis of diseases by imaging techniques (Nuclear Magnetic Resonance, Tomography for simple photon emission Tomography by emission of samples).

For therapy, the compounds of formula (I) (II) and (III), their isomers, salts, prodrugs or solvates, will preferably be found in a pharmaceutically acceptable substance that is purely pure, that is, has a level of pharmaceutically acceptable purity excluding standard pharmaceutical additives such as diluents and carriers, including material considered toxic normal dosage levels.

The compounds described in the present invention, their pharmaceutically acceptable salts, prodrugs and / or solvates as well as the pharmaceutical compositions containing them can be used together with other additional drugs to provide a combination therapy.

Said additional drugs may be part of the same pharmaceutical composition or, alternatively, they may be provided in the form of a separate composition for simultaneous or non-simultaneous administration to the pharmaceutical composition comprising a compound of formula (I), (II) or (III) or prodrug solvate derivative

or a pharmaceutically acceptable salt thereof.

Another preferred embodiment of the present invention refers to a pharmaceutical composition useful for treating pathologies such as diseases of the nervous system such as neurodegenerative diseases (Parkinson's disease, dementia including Alzheimer's disease, Hungtinton's disease, demyelinating diseases such as multiple sclerosis, amyotrophic lateral sclerosis), cerebrovascular accidents (including derivative pathology) of thrombosis and cerebral haemorrhage) .Those treatment is also included in this section (especially of the prostate, the lung and the manma, without a statistically limited and is exclusive of other types of tumor pathology) or, in general, of diseases susceptible to benefiting from the biological activities shown by the compounds described in the present invention. , hereinafter, the pharmaceutical composition of the invention, comprising a compound, in therapeutically effective amount, of formula (I), (II) or (III), or mixtures thereof, a pharmaceutically acceptable salt, prodrug, pharmaceutically acceptable stereoisomer solvate or a carrier, pharmaceutically acceptable adjuvant, for administration to a patient.

The pharmaceutically acceptable adjuvants and vehicles that can be used in such compositions are the adjuvants and vehicles known to the technicians in the matter and usually used in the elaboration of therapeutic compositions.

In the sense used in this description, the expression “therapeutically effective amount” refers to the amount of the agent or compound capable of developing the therapeutic action determined by its pharmacological properties, calculated to produce the desired effect and, in general, will be determined, among other causes, by the characteristics of the compounds, including age, condition patient, the severity of the disorder or disorder, and the route and frequency of administration.

In another particular embodiment, said therapeutic composition is prepared in a solid or aqueous suspension form, in a pharmaceutically acceptable diluent. The therapeutic composition provided by this invention can be administered by any appropriate route of administration.

Throughout the description and the claims the word "comprises" and its variants is not intended to exclude other technical characteristics, additives, components or steps. For experts in the field, other objects, advantages and features of the invention will be derived in part from the description in part of the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

Brief description of the fi gures

Fig. 1, refers to the electrophoretic retardation analysis of siRNA by coupling to the compound of general formula (I). The numbers in (A) correspond to different N / P ratios (nitrogenous amines in compound of general formula (I) / phosphatosensiRNA) :( 1) 1: 0 (siRNA only), (2) 100: 1, (3) 200: 1, (4) 400: 1, (5) 800: 1, (6) 1600: 1 and (7) 3200: 1. Densitometric analysis of the results of the gel delay experiment are shown in (B).

Fig. 2 is a real study of the toxicity of the compound of general formula (I) in cortical neurons. Cells were treated with different concentrations of the compound of general formula (I) (75 to 2400 µM) for 48. Cell viability was assessed by quantifying the percentage of LDH released to the culture medium. Data are expressed as mean (% control) ± SEM, n = 12. * p <0.05, compared to the control.

Fig. 3 refers to the quantification of the transfection of the compound compound of general formula (I) - if fl uorescent RNA in granular neurons of the cerebellar rat (A, C) and of the toxicity produced by the complex (percentage of cells marked with propidium iodide) in this cell model (B, D) by means of the study using complex cytometry. different concentrations of the compound of general formula (I) and 100nM fluorescent fluorescent. The effects after 48 hours (A, B) or 72 hours (C, D) treatments were studied. Data are expressed as mean (% control) ± SEM, of a minimum of 3 different experiments. * P <0.05, compared to the control.

Fig. 4 describes the temporal course of quantification of the transfection of the complex compound of the general formula (I) - if fluorescent RNA in rat cortical neurons (A, C) and of the toxicity produced by the complex (percentage of cells marked with propidium iodide) in this same cell type (B, D) through its study by flow cytometry. The complexes were formed with different concentrations of the compound of general formula (I) and 100nM of fluorescent siRNA. The effects after 48 hours (A, B) or 72 hours (C, D) treatments were studied. Data are expressed as mean (% control) ± SEM, of a minimum of 3 different experiments. * P <0.05, compared to the control.

Fig. 5 describes the effect of dendriplejo of the compound of general formula (I) -siRNA on levels of mRNA and protein in rat granular neurons. Study of the transfection, during 48 hours, of rat granular neurons, with the dendipleiple of the compound of general formula (I) (300 µM) -siRNA (100 nM) against Colline 1. The effectiveness of the transfection was determined by real-time PCR (A) and Western blot (B). Real-time PCR (A) quantifies the RNAF of COFILINA1 and β-actin (endogenous control) and through Westernblot (B), the protein expression of COFILINA1 and GAPDH (endogenous control) was analyzed. of 3 different experiments. * p <0.05, compared to the control.

Fig. 6 describes the effect of dendriplejo of the compound of general formula (I) -siRNA on levels of mRNA and protein in rat cortical neurons. Study of the transfection, during 48 hours, of rat cortical neurons, with the dendriplejo of the compound of the general formula (I) (150 µM) -siRNA (100 nM) against Choline 1. The effectiveness of the transfection was determined by real-time PCR (A) and Western blot (B). Real-time PCR (A) quantifies the RNAF of COFILINA1 and β-actin (controlling halogen) and through Westernblot (B) the protein expression of COFILINA1 and GAPDH (endogenous control) was analyzed. different experiments. * p <0.05, compared to the control.

Examples of embodiment of the invention

The following examples illustrate the present invention. However, these examples are not limiting. They are informative and do not limit the methodologies used, which can be altered in order to achieve similar results.

In this specification the symbols and conventions used in these procedures, schemes and examples are consistent with those used in the International System and contemporary scientific literature, for example, the Journal of Medicinal Chemistry. Unless otherwise indicated, all starting materials were obtained from commercial suppliers and used without further purification. Specifically, the following abbreviations may be used in the examples and in the length of all descriptive memory: g (grams); mg (milligrams); Kg (kilograms); mL (milliliters); µL (microliters); mmol (millimoles); P.f. (melting point); Hz (hertz); MHz (megahertz); δ (chemical shift); ppm (parts per million); s (singlet); d (doublet); t (triplet); q (quartet); c (quintuplet); m (multiplet); J (coupling constant); NMR (nuclear magnetic resonance); MS (mass spectrometry); ES (electrospray); m / z (Mass / load ratio); Anal. (Elemental Analysis); Rto (Performance); ASD (triethylamine); CH2Cl2 (dichloromethane); CDCl3 (deuterated chloroform); CD3OD (deuterated methanol) DMSO (dimethylsulfoxide); i.p. (parental administration). All temperatures are expressed in ° C (degrees Celsius).

Example 1

1.1. Synthesis of tert-butyl 2-aminoethylcarbamate (1)

A solution of ethylenediamine (26.97 g, 0.449 mol) (previously distilled over calcium oxide and potassium hydroxide) in 200 mL of dichloromethane is prepared in a 500 mL flask. Note: It has been carried out with the product without distillation with identical results.

The solution is cooled in ice bath at 0 ° C. On this solution, add a drop solution of di-tert-butyl carbonate (8.82 g, 0.040 mol) in 50 mL of dichloromethane. The addition takes 2-3 hours.

After the addition, the ice bath is removed and allowed to react overnight at room temperature under strong stirring (time approx. 12h). After this time the solvent is removed under reduced pressure forming a yellowish oil.

To the reaction crude, add 100mL of distilled water, to precipitate the disubstituted compound and filter.

Subsequently, the aqueous phase is extracted several times with ethyl acetate (4x25mL), adding to the aqueous phase a saturated solution of NaCl to facilitate extraction. The organic phase is dried over magnesium sulfate and filtered. The solvent is removed on a rotary evaporator under reduced pressure, forming a yellowish oil.

The product is finished purifying by column chromatography. (Stationary phase: silica gel; mobile phase: solvent gradient queuing from AcOEt: MeOH 9: 1a AcOEt: MeOH 1: 1) After the column, the product is pure by 1 H-NMR. Yield: 4.25 g, 66%. The spectroscopic data are consistent with those described in the literature (J. Am. Chem. Soc. 1993, 115, 10042-10055).

Note: This product is commercial, but its synthesis is cheaper. Its synthesis is described in the literature with quantitative performance (Eur. J. Org. Chem., 2009, 2675-2686).

1.2. Synthesis of 3,3 ’- (2- (tert-butoxycarbonylamino) ethylazadienyl) dimethyl dipropanoate

In a 500 mL flask, previously cooled to 0 ° C with an ice bath, a solution of 2-amino-ethylcarbamate of tert-butyl (1) (5.18g, 32.3mmol) in 250mL of methanol is prepared.On this solution a drop of a previous solution of methyl acrylate is added dropwise ( 8.34g, 96.9 mmol) in 80mL of methanol.

After addition, the mixture is allowed to stir at room temperature for 2 days. Note: Variations in the time between 2 and 6 days will not change in the process. This process can be accelerated by heating the methanol fl ow (66 ° C) for 2-3 hours.

After this time, the solvent is removed on a rotary evaporator under reduced pressure, forming a slightly yellow oil. The flask is coupled to the vacuum line for several hours, thus eliminating the methyl acrylate residues. Yield: 8.84g, 95%. The spectroscopic data coincide with those described in the literature (Bioconjugate Chem. 2006, 17, 3-5).

1.3. Synthesis of tert-butyl 2- (bis (3- (2-aminoethylamino) -3-oxopropyl) amino) ethylcarbamate (3) under microwave irradiation

In a microwave flask the ester2 (0.60 g, 2.08 mmol) dissolved in 3mL of methanolyla ethylenediamine (1.33 g, 20.8 mmol) is mixed. A reflux coolant is coupled to it and a CEM DISCOVERS-Classal microwave reactor is inserted into the microwave reactor, which selects a Cool-mate device whose mission is to increase the temperature due to the radiation and keep it constant. 40W power is radiated by controlling the temperature at 20 ° C.

The progress of the reaction is carried out by 1 H-NMR observing the progressive disappearance of the -OMe signal at 3.67ppmen CD3OD. After four cycles of 45min., In which intervals of 10 minutes of radiation do not alternate to avoid damaging the microwave reactor, the reaction is terminated.

After this time, the solvent is removed in a rotary evaporator under reduced pressure, without heating the bath over 40 ° C. Excess ethylenediamine is removed by using an azeotropic mixture of toluene: methanol (9: 1) (3 times) in the rotary evaporator and ending with methanol. The flask is coupled to the vacuum line to remove solvent residues.

A greater purification of the product is carried out by dissolving it in the minimum amount of dichloromethane and precipitating it in diethyl ether (3 times). It is centrifuged for 5,000 6500 rpm and a slightly yellowish oil is obtained. Yield: 0.44 g, 54%. The spectroscopic data coincide with those described in the literature (Bioconjugate Chem. 2006, 17, 3-5).

1.4. Synthesis of 3,3 ’- (2-aminoethylazadienyl) bis (N- (2-aminoethyl) propanamide) trichlorohydrate (4)

Amine3 (0.69g, 1.78 mmol) is dissolved in 30mL of methanol in a 50mL flask. Once dissolved, the flask is introduced in an ice bath and selebubujea hydrogen chloride, HCl (g) (2x3 minutes) leaving an interval of 5mins without bubbling HCl (g) to avoid overheating. After that time, and under a HCl atmosphere (g), the flask is closed with a septum and left stirring all night. The appearance of a white precipitate is observed.

After this time (approx. 12h), the solvent is eliminated at reduced pressure under pressure, without heating up to more than 40ºC. A yellowish solid is obtained. Yield: 0.656g, 93%. 1H-NMR (499.772 MHz, D2O) δ (ppm): 2.92 (wide t, 2H), 3.12 (wide t, 2H), 3.35 (wide s, 8H), 3.52 (wide t, 4H), 3.59 (wide t , 4H).

Example 2

Cell line cultures

Primary cultures of granular neurons of rat cerebellum

The cerebellar granular neuron culture was obtained according to previously described protocols (J Neurochem. 2007; 103: 1396-407), with small modifications. Briefly, 7-day-old offspring of the Spragle-Dawley strain were quickly decapitated and carefully removed from the brains. We separated the cerebellum aseptically, removed the meninges, and the cerebellum was cut into pieces of about 0.4 mm. Then, it was put to trypsin tissue and a culture-free medium was plated in a pre-cultivated culture medium. Cells were cultured in BME medium supplemented with 24.5mM potassium, 2mM glutamine, 10% FBS and 50 µg / mL gentamicin.After 24 hours, Ara-C (cytosine arabinoside) was added to the medium to obtain a final concentration of 10 µM to reduce astrocyte growth. Cells were used before 7 After cultivation, which is the time they need to finish differentiating.

Primary cultures of rat cortical neurons

The primary culture of cortical neurons was performed according to the methodology previously described (Eur. J. Neurosci. 2001. 13: 1469-1478). Frontolateral cortical lobes were dissected in 17-day fetuses of female rats of the Spragle-Dawleyy strain mechanically dissociated in HBSS. The cortical lobes were crushed by pipetting about ten times with a Pasteur pipette. After centrifuging 5 minutes 800 × g, the cells were resuspended in Neuobasal culture medium supplemented with B27.2 mM glutamine, 100 U / mL penicillin and 100 µg / mL streptomycin. cultivation, which is the time they need to finish differentiating.

Complex formation compound of general formula (I) -siRNA

The complexes composed of general formula (I) - if RNAs were formed by mixing equal quantities of the solution containing the compound of general formula (I) and containing the siRNA (Org Biomol Chem. 2007; 5: 1886-1893; Pharm. Res. 2009. 26,1181-1191), and mixing it under stirring for 30 minute ambient temperature. Both molecules were dissolved in DEPC water (RNAse free).

Gel delay experiments

The delay in the agarose was used to find the N / P ratio (nitrogenous amines in compound of general formula (I) / phosphates in siRNA) adequate to obtain the greatest possible binding effectiveness between both molecules (Brain Res Dev Brain Res. 1991. 63: 1-12 .; Neuropsychiatr Genet. 2008. 147: 769-777). The mixture of different concentrations of compound of general formula (I) and of 250ng of siRNA was tested. The mixture was run for 15 minute 60V in the aqueous agar at 1.2% with 0.017% of bromide of ethidium. The angels were photographed and the bands were quantified with an appropriate image analysis system (Quantity One). experiments for each of the tests described above.

Cytotoxicity Studies

Tests to assess the toxicity of the compound of general formula (I) were performed in the culture of rat cortical neurons, determining the activity of the enzyme lactate dehydrogenase (LDH) (Pharm. Res. 2009. 26,11811191) .To do this, the cells were seeded in 24-well plates seexpusierone solutions with different concentrations of compound of general formula (I) (5-80 µM) to perform concentration-dependent toxicity curves for 48 hours. Toxic effects were evaluated by measuring the rupture of the cell membrane and the consequent release of LDH to the supernatant through the CytoTox96® kit (Promega). The cells were mechanically detached, washed with PBS and centrifuged at 10,000 rpm for 10 minutes. The absorbance of the lysate and the cell supernatant was measured using a microplate photometer photometer at a length of 490 nm.

The toxicity of the treatments with the complex compounds of general formula (I) -siRNA, using different concentrations of compound of general formula (I) (75-2400 µM) in combination with 100 nM siRNA, was studied by flow cytometry. To do this, after the treatments, the cells were incubated with propidium iodide 0.5 mg / mL for at least 1 hour at 37 ° C in the dark. Then, the cells were trypsinized and analyzed in a flow cytometer (FACS Calibur, Becton-Dickinson, Franklin Lakes, NJ, USA). From the evaluation of

10,000 cells per experimental condition, the percentage of cells with the damaged cytoplasmic membrane (positive propidium iodide) was calculated (J Control Release. 2008. 132: 55-64; Biomaterials. 2008. 29: 3469-76).

Study of the percentage of translocation of the compound compound of general formula (I) -siRNA into the cell interior

After 48-72 hours with the cells in the presence of the complex compounds of general formula (I) -siRNA, using 100nM of fluorescent siRNA to perform them, the conditioned media were collected and the trypsinizarony cells were washed with PBS. Total cells - live and dead - present in the resulting suspension when the trypsinized cell is assembled and the medium conditioned is analyzed by a flow cytometer (FACS Calibur, Becton Dickinson, FranklinLakes, NJ, USA) .Applying the evaluation of 10,000 cells by experimental condition was calculated by the percentage of transfected cells. 55-64; Biomaterials. 2008. 29: 3469-76).

Translocation of the compound complex of general formula (I) -siRNA was also studied by con-focal microscopy. For this, the cells were seeded on coverslips and treated in the same manner as the previous samples. Cells treated with fluorescent siRNA, alone or forming complexes composed of the general formula (I) siRNA, were visualized and photographed in a confocal microscope (Nikon Eclipse TE200) using the appropriate wavelength for fluorophore excitation with which the siRNA is labeled (Pharm Res 2009. 26: 57786). The results were used to determine the percentage of cells positive for intracellular transfection of siRNA.

Study of gene silencing by polymerase chain reaction in real time (real-time PCR)

Total cell RNA was isolated by a standard method with guanidinium-phenol-chloroform thiocyanate (TriPure Isolation Reagent, Roche Applied Sciences, Indianapolis, IN). The RNA was transformed into cDNA and was used to make it real-timePCR. We use the real-timePCR to study the differentiation of different genes by means of 100nM of RNAs that have been made with different concentrations of compound of general formula (I). Beta beta-actin was used as a reference gene for all real-time PCR experiments. The reaction was performed using standard procedures for the StepOnePlus Real-Time PCR System (Applied Biosystems).

In each experiment, the mean cycle threshold [cycle threshold (CT)] of the triplicates of each of the genes studied and the gene used as a reference was calculated, being able to compare the gene expression after the different treatments (Pharm. Res. 2009. 26: 577-86; Cancer Res. 2007 67: 8156-63).

Evaluation of the degree of protein silencing by Western blot

Cellular extracts were obtained by lysate in Tris-HCl 50mM, pH7.4, NaCl150mM, EDTA1mM, Triton X-100al1%, sodium deoxycholate 1% and SDSal 0.1% and protease inhibitors (5 µg / mL aprotinin). Once lysed the cells were centrifuged at 13,000 r.p.m. during 15 minutes. The protein concentration present in the supernatant was determined by the method of Bradford (Pierce; Rockford, IL, USA), using bovine serum albumin as standard. Samples containing 40 µg of total protein were applied in each well of 10-15% polyacrylamide gels according to the protein to be studied. The gels were electrophoresed to nitrocellulose membranes using a semi-dry "blotter." Nitrocellulose-bound proteins were visualized with Poinceau, followed by blocking with TBS (50mMTris, pH7.5,200mMNaCl, 0.1% Tween) with 5% skimmed milk, and then incubated with the corresponding primary antibody overnight at 4 ° C. Transferred in TTBS, the secondary antibody was applied for 1 hour. room temperature.The detection was performed by chemiluminescence. The intensity of the bands was analyzed by gray levels with an appropriate image analysis system (Quantity One) (Pharm. Res. 2009. 26: 577-86; Cancer Res. 2007. 67: 8156-63).

Claims (19)

1. Compound of general formula (I) or a salt, prodrug or solvate thereof; where: X is selected from an unsaturated (C2-C12) alkyl or cycloalkyl (C3-C8), cycloalkenyl, aryl or heteroaryl group, R1, R2, R3, R4 and R5 are the same or different and are selected from hydrogen, saturated or unsaturated alkyl (C2-C12), cycloalkyl (C3-C8), cycloalkenyl, aryl or heteroaryl, Z is selected from any anion chloride, tri fl uoroacetate, DNA, RNA, siRNA, miRNA, antagomir, any drug, antibody, probes for disease diagnosis through technical techniques or any combination thereof 2. Compound of general formula (II) according to claims 1 a salt, prodrug or solvate thereof; where: X is selected from a group of saturated or unsaturated alkyl (C2-C12) or cycloalkyl (C3-C8) or cycloalkenyl, aryl or heteroaryl, R1, R2, R3, R4 and R5 are the same or different and are selected from hydrogen, saturated or unsaturated alkyl (C2-C12), cycloalkyl (C3-C8), cycloalkenyl, aryloheteroaryl, Z is selected from any anion chloride, tri fl uoroacetate, DNA, RNA, siRNA, miRNA, antagomir, any drug, antibody, probe, for the diagnosis of diseases by means of imaging techniques or any combination thereof 3. Compound of general formula (III) according to any of claims 1 or 2 or a salt, prodrug or solvate thereof; where: X is selected from a group of saturated or unsaturated alkyl (C2-C12) or cycloalkyl (C3-C8) or cycloalkenyl, aryl or heteroaryl, R1, R2, R3, R4 and R5 are the same or different and are selected from hydrogen, saturated or unsaturated alkyl (C2-C12), cycloalkyl (C3-C8), cycloalkenyl, aryloheteroaryl, Z is selected from any anion chloride, tri fl uoroacetate, DNA, RNA, siRNA, miRNA, antagomir, any drug, antibody, probe for the diagnosis of diseases by means of techniques of any combination thereof.

Four.

Compound of general formula (I), (II) or (III) according to any of claims 1 to 3, wherein X are selected from:

5.

Compound of formula (I), (II) or (III) according to any of claims 1 to 4, wherein R 1 to R 5 may be the same or different and are independently selected from H, any substituted or unsubstituted amine, basic amino acids, cholesterol derivatives, folic acid, lactic acid, dexamethasone, sugars, lysomotropic agents, nitrogen heterocycles, hydrophilic chains derived from poly-ethylene glycol, any diacrylate, any basic amino acid and the following structures:

6.

Compound of general formula (I), (II) or (III) according to any of claims 1 to 5 selected from the list comprising:

7.

Pharmaceutical composition characterized in that it comprises a compound of formula (I) according to any one of claims 1,4 and 5, together with one or more pharmaceutically acceptable ingredients.

8.

Pharmaceutical composition characterized in that it comprises a compound of formula (II) according to any one of claims 2,4 and 5, together with one or more pharmaceutically acceptable ingredients.

9.

Pharmaceutical composition characterized in that it comprises a compound of formula (III) according to any one of claims 3 to 5, together with one or more pharmaceutically acceptable ingredients.

10.

Composition according to any one of claims 7,8 or 9 characterized in that it further comprises one or more active ingredients.

eleven.

Synthesis process of the compounds of general formula (I) characterized in that it comprises the following steps:

12.

Process for obtaining a compound of the general formula (II), where the compound of the general formula (I) is started and all of them are repeated as claimed in claim 11.

13.

Process for obtaining a compound of the general formula (III), where the compound of the general formula (II) is started and all steps a-d according to claim 11 are repeated.

14.

Procedure for obtaining a compound of the general formula (I), (II) and (III) where X is selected from a group of saturated unsaturated alkyl (C2-C12) or cycloalkyl (C3-C8), cycloalkenyl, aryl or heteroaryl, R1, R2, R3, R4 and R5 they are the same or different and are selected from hydrogen, saturated or unsaturated alkyl (C2-C12), cycloalkyl (C3-C8), cycloalkenyl, aryloheteroaryl and Z is selected from any chloride anion, tri-fluoroacetate, DNA, RNA, siRNA, miRNA, antagonize any drug, antibody, probe , for the diagnosis of diseases by imaging techniques or any combination thereof.

15.Use of a compound of general formula (I), (II) or (III) according to any of claims 1 to 7 for the preparation of a medicament. 16.Use of the compound of formula (I), (II) or (III) according to claim 15 for the preparation of a medicament for the prevention and / or treatment of pathologies such as diseases of the nervous system, such as neurodegenerative diseases, strokes and any disease that may occur with the appearance of tumors 17.Use of the compound of formula (I), (II) or (III) according to any of claims 1 to 6 for the elaboration of non-RNA transfection in primary cultures of nerve cells, glia, tumor cells and any type of primary cells. 18. Use of the compound of formula (I), (II) or (III) according to any of claims 1 to 6 as a non-viral vector. 19.Use of the compound of formula (I), (II) or (III) according to any of claims 1 to 6 for the preparation of probes and for diagnosis of diseases by imaging techniques.