WO2014016460A1 - Composés dendritiques carbosilanes homo et hétéro-fonctionnalisés - Google Patents

Composés dendritiques carbosilanes homo et hétéro-fonctionnalisés Download PDF

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WO2014016460A1
WO2014016460A1 PCT/ES2013/070529 ES2013070529W WO2014016460A1 WO 2014016460 A1 WO2014016460 A1 WO 2014016460A1 ES 2013070529 W ES2013070529 W ES 2013070529W WO 2014016460 A1 WO2014016460 A1 WO 2014016460A1
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sich
nme
yes
dendrimer
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Francisco Javier De La Mata De La Mata
Mª Ángeles MUÑOZ FERNÁNDEZ
Rafael GÓMEZ RAMÍREZ
José Luis JIMÉNEZ FUENTES
Javier SÁNCHEZ-NIEVES FERNÁNDEZ
Silvia FERNÁNDEZ SORIANO
Marta GALÁN HERRANZ
Raquel LORENTE RODRÍGUEZ
Elena FUENTES PANIAGUA
Javier SÁNCHEZ RODRÍGUEZ
Cornelia E. PEÑA GONZÁLEZ
Mª Jesús SERRAMÍA LOBERA
Rosa REGUERA
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Universidad De Alcalá (Uah)
Fundación Para La Investigación Biomédica Del Hospital Gregorio Marañón (Fibhgm)
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/002—Dendritic macromolecules
    • C08G83/003—Dendrimers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • the present invention relates to dendritic compounds, particularly dendrimers or dendrons, of carbosilane structure and functionalized at its periphery with functional groups that are preferably in anionic or cationic form.
  • the present invention also relates to its method of production carried out by thiol-eno or thiol-ino reaction.
  • the invention relates to the uses of said compounds in biomedicine. STATE OF THE PREVIOUS TECHNIQUE
  • Dendrimers are hyperbranched molecules of arborescent construction, of a well-defined size and three-dimensional structure and that have uniform chemical properties due in part to their low polydispersity.
  • the discovery that dendrimers by themselves may have a biological activity is quite recent, thus acting for example as antibacterial or antiviral agents. They can also act as transport agents for nucleic acids or drugs.
  • dendrimers described in the literature and potentially useful as antiviral agents may have different types of skeletons, but in what refers to the functional groups they present at their periphery, which are the real ones responsible for their antiviral activity, they can be grouped into Three types: carbohydrates, peptides and anions.
  • RNA molecules are not toxic in a range of concentrations between 1 and 5 ⁇ and are capable of electrostatically interacting with nucleic material such as oligonucleotides or small interfering RNAs (siRNA) forming bioconjugates called "dendriplexes.”
  • nucleic material such as oligonucleotides or small interfering RNAs (siRNA) forming bioconjugates called "dendriplexes.”
  • siRNA small interfering RNAs
  • carbosilane dendrimers have shown antimicrobial activity both against Gram positive bacteria and against Gram negative bacteria. This activity seems to be related to the multivalence of dendrimers, which allows the presence of a large number of functionalities on the same molecule.
  • carbosilane dendrimers with first, second or third generation terminal ammonium groups have proven effective as multivalent biocides, showing an antibacterial activity that is more than two orders of magnitude greater than that of monofunctional analog compounds (Beatriz Rasines, Carlos Manuel Hernández - Ros, Nativity of the Caves, Carlos Luis Copa-Pati ⁇ o, Juan Soliveri, Mar ⁇ a Angeles Mu ⁇ oz-Fernández, Rafael Gómez, F.
  • Carbosilane dendrimers are constructed by repeating hydrosilylation reactions with derivatives of the HSiMexCIp-x type) and subsequent alkenylation, thereby generating functionalized dendrimers with terminal olefins.
  • a hydrosilylation reaction with HSiMe 2 CI is carried out and subsequent replacement of the Si-CI system with another of the Si-H type with UAIH 4 , thus obtaining dendrimers with Si-H groups on the periphery .
  • terminal amines can be introduced by allylamine hydrosilylation reaction, which by subsequent quaternization allows to obtain cationic dendrimers.
  • neutral carbosilane dendrimers with -NHR groups obtained in this way have been used to obtain anionic carboxylate dendrimers by reaction with methyl acrylate and subsequent basic treatment and anionic dendrimers sulfonate by reaction with vinyl sulfonate (WO201 1/101520 A2).
  • This synthetic procedure is long with the consequent increase in the process and decrease in the overall yield to obtain the desired dendrimer.
  • the hydrosilylation of C3H5NH2 necessary to then obtain anionic dendrimers requires heating at 120 ° C for more than two days, significantly reducing the yield for higher generations.
  • the introduction of some of the terminal functional groups, such as sulfonate groups also requires high temperatures and long reaction times.
  • the present invention provides highly branched macromolecules, dendrimers or dendrons, of carbosilane structure and functionalized on its periphery with anionic groups (such as carboxylate, sulphonate or sulfates), which give the macromolecule a negative net charge, or cationic (ammonium), which endow the dendrimer with a net positive charge.
  • dendrimers have a preferably polyphenolic or silicon atom core.
  • a first aspect of the present invention relates to a carbosilane dendritic compound (hereinafter compound of the invention) comprising:
  • R 2 is an alkyl group (CrC 4 ), preferably R 2 is a methyl group;
  • p is an integer and varies between 1 and 3, preferably p is 2;
  • Ri is the following group - (CH 2 ), cS- (CI-l 2 ) and R 3 ;
  • x represents an integer that varies from 2 to 5; preferably x
  • R 3 is a group -OH, -S0 3 H, -OS0 3 H, -COOR 'or -NR “R”', where R ', R “and R'” independently represent an alkyl group (CrC 4 ) or a hydrogen;
  • R 3 is -NR “R”', preferably R “and R'", they independently represent a (C1-C4) alkyl or hydrogen group, more preferably an alkyl (CrC 2 ) or hydrogen group, even more preferably R 3 is a group -N (CH 3 ) 2 . Even more preferably x is 2 and even more preferably and is 2.
  • Ri is the group - (CH 2 ) 2 -S- (CH 2 ) 2 - N (CH 3 ) 2 .
  • R 3 is a group -C0 2 R ', preferably R' is H or CH 3 , more preferably x is 2 or 3, and even more preferably and is 1 or 2.
  • R 3 is a group -S0 3 H or -OS0 3 H, preferably x is 2 or 3, and more preferably and is 2 or 3.
  • alkyl refers in the present invention to aliphatic, linear or branched chains, having 1 to 4 carbon atoms, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, tert- butyl or sec-butyl, preferably has 1 to 2 carbon atoms, more preferably the alkyl group is a methyl.
  • dendritic compound refers in the present invention to a highly branched macromolecule where the growth units, branches or branches have carbosilane skeleton.
  • This carbosilane dendritic compound can be selected from dendrimer or dendron, also referred to as dendritic wedge.
  • dendrimer refers in the present invention to a highly branched macromolecule with a spherical shape, where the dendrimer's growth nucleus is polyfunctional, the growth units, branches or branches have carbosilane skeleton and the outer layer, surface or periphery of the dendrimer incorporates functional groups, R 3 groups. This surface or periphery would be the one corresponding to the extremities of the branches.
  • the skeleton of these carbosilane dendrimeros with different nuclei is widely known to one skilled in the art (ES226591; WO201 1101520).
  • polyfunctional core in the present invention a polyvalent element or compound covalently bonded with at least two branches, that is, at least it must be divalent.
  • the core is tetravalent and more preferably the core is silicon (i.e., a silyl group).
  • the core may be a polyphenol, and "polyphenol” means a benzene molecule substituted by at least two hydroxyl groups in any of its positions, for example 1,4-dihydroxybenzene, 1,2-dihydroxybenzene or 1, 3-dihydroxybenzene, more preferably it is hydroquinone (1,4-dihydroxybenzene), but it may have three, four, five or six hydroxyl groups. More preferably the polyphenol is trisubstituted, even more preferably 1,3,5-trihydroxybenzene.
  • dendron or “dendritic wedge” refers in the present invention to a very branched cone-shaped macromolecule that is defined by a focal point, the units, branches or branches of growth, which start from said focal point, have carbosilane skeleton and the outer layer, surface or periphery of said branches incorporates functional groups, R 3 groups.
  • the focal point can have a functional group, in its outer layer, equal to or different from the ramifications.
  • the skeleton of these carbosilane dendrons is widely known to one skilled in the art (ES226591; WO201 1 101520).
  • the focal point can be selected from the group or - (CH 2 ) Z -R4; where:
  • z is an integer that varies from 1 to 10, preferably z varies from 1 to 5 and more preferably z is 4;
  • R 4 is a group selected from the list comprising -OH, -SH, -Br, -COOR 4 '", -NR 4 ' R 4 ", phthalimide, -N 3 , - 0-CH 2 -CCH, -O- CCH, -NHR5, -R 5 , -SCOCH3 or -p-0-C 6 H 4 - (CH 2 ) -OH, where R 4 ', R 4 "and R 4 "' independently represent an alkyl group ( CrC) or a hydrogen, preferably they are hydrogen (-NH 2 or -COOH), x 'is an integer value ranging from 1 to 4, preferably x' is 1, and R 5 can be selected from a tag molecule, preferably a fluorophore, a steering group or an active substance.
  • the compound of the present invention can also be cationic, forming ammonium groups (for example NH 3 + or NMe 3 + ), that is, when R 3 is an amino, or anionic group, forming the carboxylate, sulfate or sulphonate groups, to the rest of R 3 groups described above.
  • ammonium groups for example NH 3 + or NMe 3 +
  • the present invention not only includes the compounds themselves, but any of their salts, for example, alkali metal or alkaline earth metal salts, for example they can be selecting from sodium, potassium or calcium salts, preferably the salts are sodium or halogen salts, which can be selected from chloride, bromide, iodide salts; or triflate, preferably the salts are iodide.
  • the compound of the invention in the outer layer, or optionally at the focal point, also comprises at least one functional group of different nature to the R 3 groups that are part of said outer layer or optionally of the focal point. , these R 3 groups can be any of those described above or of different nature to the R 3 described, such as a label molecule, a leader group or an active ingredient.
  • the compound of the invention contains at least one R 3 group in its outer layer, or optionally at the focal point, which is selected from a group R 3 different from the rest of the groups of the dendritic compound, a group -NHR 5 or -R 5 and where said R 5 can be selected from a tag molecule, preferably a fluorophore, a leader group or an active ingredient.
  • the present invention provides dendritic compounds, dendrimers or dendrons, which in addition to containing groups that provide them with a series of useful properties in biomedicine can also introduce into their outer layer at least one group with different functionality to give rise to a composed with multifunctionality and, therefore, with great versatility in its applications.
  • tag molecule refers in this description to any biorecognizable substance, chromophore, fluorophore or any other group detectable by spectrophotometric, fluorometric, optical microscopy, fluorescence or confocal, antibody and / or NMR techniques, and which easily allows detection of another molecule that alone is difficult to detect and / or quantify.
  • this tag molecule is a fluorophore capable of binding to an amine or containing an amino group by which it binds to the dendritic compound, or is capable of binding to the dendritic compound by the functional groups it contains or with a prior functionalization, for example and without limiting our the fluorophore is selected from the list comprising Cy5, fluorescein, rhodamine and dansyl.
  • leader group is meant a molecule or functional group capable of directing the dendritic compound specifically to a type of cells or to a specific area of a cell, for example, but not limited to, folic acid, trickle groups, signal signal or an antibody, among others known to any person skilled in the art. Said lead group can be previously functionalized to bind the dendritic compound.
  • active ingredient or “drug” is meant in the present invention any purified chemical substance used in the prevention, diagnosis, treatment, mitigation or cure of a disease; to avoid the appearance of an unwanted physiological process; or to modify physiological conditions for specific purposes.
  • said active ingredient is capable of binding to an amine or containing an amino group by which it binds to the dendritic compound, or is capable of binding to the dendritic compound by the functional groups it contains or with a prior functionalization, for example, without limited to penicillin, or where the active substance is capable of binding to an alkyne group through azide groups, for example AZT (zidovudine).
  • the compound of the invention can be a dendrimer or dendron of first, second, third, fourth or successive generations.
  • generation refers to the number of iterative branches that are necessary for the preparation of the compound.
  • Another aspect of the present invention relates to a process for obtaining the compounds of the invention, which comprises a thiol-ene or thiol-ino reaction, between a precursor of said compound with olefins or terminal alkynes, respectively, and the thiol group SH- (CH 2 ) and -R3, where R 3 e and are described above.
  • the reaction is carried out in the presence of a polar solvent and more preferably in the presence of a photoinitiator.
  • polar solvents for example MeOH or THF / MeOH or THF / MeOH / H 2 0 solvent mixtures
  • photoinitiators for example benzophenone, which can be used in this type of reaction are known to any person skilled in the art.
  • anionic compounds can be synthesized with terminal groups such as carboxylate, sulphonate, sulfate; or, from dendrimers with anionic precursor groups such as the ester or carboxylic acid that after their introduction to the compound are transformed into the corresponding anion by treatment with a base, such as NaOH, KOH, K 2 C0 3 or others that fulfill this same function.
  • a base such as NaOH, KOH, K 2 C0 3 or others that fulfill this same function.
  • the compounds of the invention obtained by this process are stable and soluble in water in their ionic forms and can also be isolated with good yields.
  • obtaining cationic compounds with terminal ammonium groups can be produced by a quaternization reaction of the corresponding amino group using an RX derivative, dialkyl sulfates (CrC 5 ), methyl triflate, or any of its combinations as a quaternizing agent (where R is selected from hydrogen, alkyl (CrC 24 ), alcohol (CrC 24 ) or an aryl, preferably benzyl; and X is a halogen, preferably Cl, Br or I), as per example methyl iodide (Mel), HCI, methyl chloride, methyl bromide, ethyl chloride, ethyl bromide, propyl chloride, hexyl chloride, dodecyl chloride, benzyl chloride, benzyl bromide, ethanol bromide, ethanol iodide (HO-CH 2 CH 2 -l) or any combination thereof.
  • RX dialkyl sulfates
  • compounds functionalized with ammonium groups of the NR 2 HCI type they are neutralized with basic medium and subsequently can be treated with other quaternizing agents such as those described above.
  • compounds with R 3 : NH 3 + , NMe 3 + are whitish solids stable to air and moisture, soluble in polar solvents (for example but not limited to DMSO, H 2 0) and can be stored without decomposition for long periods of time.
  • the compounds with R 3 : NH 2 , NMe 2 are colorless oils, also stable to air and moisture and soluble in halogenated, ethereal organic solvents, but not in aliphatic hydrocarbons.
  • the amino (-NH 2 ) or ammonium (-NH 3 CI) group serves as a linking group to other functions, such as those described as R 5 , so that compounds are prepared in this way, for example with fluorophores groups and anionic groups, soluble in water.
  • R 5 a linking group to other functions
  • the same products would also be obtained if the reaction was carried out with a thiol previously modified with the different group, for example R 5 .
  • the reaction conditions would be analogous to those described for homofunctionalized compounds, but using a combination of thiol derivatives suitable to introduce two functions in the proportion of interest.
  • the present invention also relates to the uses in biomedicine of the dendritic compounds described above that have cationic or anionic terminal groups, among them the use of cationic derivatives as non-viral transport agents for transfection or internalization of nucleic material in the inside different cell lines in gene therapy processes or also the use of these cationic or anionic compounds as "per se” therapeutic agents, for example as antibacterial, antiviral or antiprionic agents.
  • said dendritic compounds can be heterofunctional, with the advantage of being able to perform more than one function simultaneously.
  • anionic dendrimers in addition to having antiviral capacity due to their negative charge, may be marked to facilitate their monitoring or may also have lead groups that direct dendrimers specifically towards their place of action.
  • heterofunctional cationic dendrimers can simultaneously have, for example, positive charges for the transport of nucleic acids or anionic drugs and leader groups such as an antibody to direct these dendrimers to a specific place.
  • another aspect of the present invention relates to the use of the compounds of the invention, both cationic and anionic, for the manufacture of a medicament.
  • the medicament is used for the prevention and / or treatment of diseases caused by microorganisms, such as viruses, bacteria, protozoa or fungi. More preferably prevention and / or treatment are for diseases caused by HIV or by Leishmania, more preferably by the species Leishmania infantum. Therefore, in another preferred embodiment the compounds of the invention are used for the prevention and / or treatment of Leishmaniosis.
  • another aspect of the present invention relates to the use of these compounds as biocidal agents for non-therapeutic applications, such as for carrying out controls, such as those used with detergents to prevent the appearance of microorganisms on surfaces.
  • compositions comprising at least one dendritic compound as described above and a pharmaceutically acceptable carrier.
  • this pharmaceutical composition may comprise another active ingredient, preferably an antibiotic, antiviral or anti-inflammatory, the antibiotic may be from the beta-lactam group, such as penicillin.
  • the anti-inflammatory may be for example ibuprofen and the antiviral AZT.
  • compositions are the vehicles known to a person skilled in the art.
  • compositions examples include any solid composition (tablets, pills, capsules, granules, etc.) or liquid (gels, solutions, suspensions or emulsions) for oral, nasal, topical or parenteral administration.
  • the administration will be topical and even more preferably in the form of a gel.
  • the administration will be oral or parenteral (injectable).
  • the present invention relates to a method of treatment or prevention of diseases caused by microorganisms, such as viruses, bacteria, protozoa or fungi in a mammal, preferably a human, comprising the administration of a therapeutically effective amount of a composition comprising at least one dendritic compound of the invention.
  • the administration of the composition can be performed orally, nasally, topically or parenterally.
  • the administration will be topical and even more preferably in the form of a gel.
  • the administration will be oral or parenteral (injectable).
  • the term "therapeutically effective amount” refers to the amount of the composition calculated to produce the desired effect and, in general, will be determined, among other causes, by the characteristics of the composition itself, the age, condition and history of the patient, the severity of the disease, and the route and frequency of administration.
  • nucleic material refers in the present invention to a material, isolated and / or purified, which comprises a nucleotide sequence and can be selected from oligonucleotides, siRNA or DNA.
  • Another aspect of the present invention relates to a non-viral vector comprising at least one cationic compound of the present invention.
  • This vector can also comprise nucleic material, as described above.
  • Another aspect of the present invention relates to the use of the non-viral vector of the invention, for the preparation of a medicament. More preferably, for the preparation of a medicament for the treatment of HIV infection or cancer in gene therapy.
  • the greatest advantage of the complexes, dendritic compound / nucleic material formulated in the present invention is that they have a uniform and flexible structure allowing the possibility of versatilely modifying the skeleton and the surface thereof. It is also possible to use the compounds of the invention as vehicles for transporting molecules, preferably molecules with pharmacological activity (active ingredients), and more preferably anionic or cationic molecules, depending on whether the compound is cationic or anionic respectively, the active substance can be an antibiotic, an anti-inflammatory or an antiviral, including for example and not limited to an antibiotic of the beta-lactam group, such as penicillin, an anti-inflammatory such as ibuprofen or an antiviral such as AZT.
  • an antibiotic an anti-inflammatory or an antiviral
  • an antiviral including for example and not limited to an antibiotic of the beta-lactam group, such as penicillin, an anti-inflammatory such as ibuprofen or an antiviral such as AZT.
  • Fig. 1 It shows the viability of the four generations of BDEF031, BDEF032, BDEF033 and BDEF034 dendrimers, at concentrations of 1, 5, 10 and 20 ⁇ in PBMCs and the second generation of the BDEF023 dendrimer at concentrations of 0, 1, 0.3, 0, 5, 1, 5 and 10 ⁇ in PBMCs.
  • Fig. 1A MTT test 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazole bromide for the four generations of dendrimers BDEF031, BDEF032, BDEF033 and BDEF034. 10% DMSO represents 100% toxicity.
  • Dx (Dextran) is used as a safe molecule control.
  • Fig. 1 B MTT test of BDEF023 dendrimer. 10% DMSO represents 100% toxicity.
  • Fig. 2. Retention gels of the siRNA Nef./dendr ⁇ meros BDEF031, BDEF032, BDEF033 and BDEF034 complexes.
  • Fig. 2A Dendriplexes 31 and 32 after 2 hours of incubation at 37 ° C.
  • Fig. 2B Dendriplexes 31 and 32 after 24 hours of incubation at 37 ° C.
  • Fig. 2C Dendriplexes 33 and 34 after 2 hours of incubation at 37 ° C.
  • Fig. 2D Dendriplexes 33 and 34 after 24 hours of incubation at 37 ° C.
  • Fig. 3 It shows the viability using the MTT assay of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazole bromide of the dendrimers BDEF031, BDEF032, BDEF033 and BDEF034 alone and their siRNAs complexes / dendrimers at 1: 12 ratios with BDEF031, BDEF032 and 1: 8 with BDEF033 and BDEF034. 10% DMSO represents 100% toxicity.
  • Fig. 4A Heparin competition between siRNA and BDEF031 dendrimer.
  • Fig. 4B Heparin competition between siRNA and BDEF032 dendrimer;
  • Fig. 4C Heparin competition between siRNA and BDEF033 dendrimer.
  • Fig. 4D Heparin competition between siRNA and BDEF034 dendrimer.
  • Fig. 5 HIV replication inhibition assay. The results obtained after treatment of activated and infected HIV PBMCs with dendriplexes are shown. After treatment with dendriplexes, an inhibition of viral replication quantified by p24 ELISA is observed.
  • Fig. 6. Biodistribution test of BDEF023 dendrimer. The biodistribution of the BDEF023 dendrimer is presented in the various tissues / organs after injecting it into the tail vein into BALB / c mice.
  • Fig. 7- Test to determine the cytotoxicity by MTS of BDMG017 and BDMG018 in different cell lines.
  • DMSO was used as a toxicity control and dextran (Dxt) as a cell viability control.
  • Fig. 8 Internalization of HIV in human endometrial cells (HEC-1A).
  • Fig. 8A Pre-treatment with BDMG017 Dendrimer and infection with the viral isolate X4 HIVNL4.3.
  • Fig. 8B Pre-treatment with the BDMG018 dendrimer and infection with the viral isolate X4 HIVNL4.3.
  • Fig. 8C Pre-treatment with BDMG017 dendrimer and infection with the viral isolate R5 HIVBaL.
  • Fig. 8D Pre-treatment with BDMG018 Dendrimer and infection with the viral isolate R5 HIVBaL.
  • 9C-PBMC pre-treated with BDMG018 and after 1 h infected with the viral isolate X4 HIVNL4.3 or first infected with HIVNL4.3 and subsequently treated with BDMG018.
  • Example 1 Homofunctionalized dendrimers with cationic groups
  • the synthesis of these cationic compounds can be represented, in general, by the following scheme 1:
  • cationic or neutral dendrimers can be represented as GnXC x (F) or, where:
  • n indicates the number of generation G.
  • C x indicates the length of the carbon chain between the atom of Si and S.
  • Cx is C2
  • GnXAo is C3 and so on.
  • the compounds GnXVo and GnXAo of the following examples are described in J. Sánchez-Nieves et al., Tetrahedron 2010, 66, 9203. Made, AW vd; Leeuwen, PWNM v. J. Chem. Soc, Chem. Commun. 1992, 1400.
  • R 3 indicates the nature of the functional groups (R 3 ) located on the periphery of the dendrimer (NH 3 + , NMe 3 + , NH 2 , NMe 2 ) and the number of these functional groups, which will depend on the number of generations .
  • the second generation dendrimer G203C2 (NMe 3 l) i2 is prepared following a procedure similar to that described for the analog dendrimer G1, starting from G203C2 (NMe2) i2 (0.148 g, 0.06 mmol) and Mel (0.04 ml, 0.70 mmol). In this way, G203C2 (NMe 3 L) i2 is obtained as a white solid (0.233 g, 94%).
  • Ci 2 9H294li2Ni20 3 Si 2 YES9 (4222.20 g / mol): C, 36.70; H, 7.02; N, 3.98; S, 9.1 1; Exp .: C, 36.10; H, 6.80; N, 4.01; S, 7.31. Z potential: 72 ⁇ 1 mV. Synthesis of G303C2 (NMe 3 l) 24.
  • the third generation dendrimer G303C2 (NMe 3 l) 24 is prepared following a procedure similar to that described for analog dendrimer G1, starting from G303C2 (NMe2) 24 (0.186 g, 0.04 mmol) and Mel (0.05 ml, 0 , 87 mmol). In this way, G303C2 (NMe 3 L) 24 is obtained as a white solid (0.303 g, 98%).
  • the fourth generation dendrimer G403C2 (NMe 3 l) 48 is prepared following a procedure similar to that described for the analog dendrimer G1, starting from G403C2 (NMe2) 48 (0.176 g, 0.02 mmol) and Mel (0.06 ml, 0.90 mmol). In this way G403C2 (NMe 3 L) 48 is obtained as a white solid (0.229 g, 78%).
  • the G2SiC2 (NMe3l) i6 dendrimer is prepared following a procedure similar to that described for the G1 analog dendrimer, starting from G2SiC2 (NMe 2 ) i6 (0.134 g, 0.04 mmol) and Mel (0.05 mL, 0.80 mmol ). In this way, G2SiC2 (NMe3l) i6 is obtained as a white solid (0.165 g, 72%).
  • Example 2. Homofunctionalized dendrimers with ammonium groups.
  • anionic or neutral dendrimers can be represented as cationic by GnXC x (F) or; where, in this case, F is C0 2 " , SO3 " , OSO3 " , SO3H, OSO3H, C0 2 Me, C0 2 H or OH.
  • the above product is dissolved in methanol.
  • the solution is deoxygenated by passing a stream of argon through it and 4M HCI in excess dioxane is added.
  • the mixture is heated to 60 ° C and allowed to react for 15 h. Subsequently, the solvent is evaporated and the product HS (CH 2 ) 2 OS0 3 Na is obtained.
  • NMR-H (CDCI 3 ): ⁇ 3.69 (t, SCH 2 CH2OH), 2.70 (t, SCH2CH 2 OH), 2.52 (t, SiCH 2 CH 2 CH 2 S), 1.55 (m, SYCH 2 CH 2 CH 2 S), 1 .27 (m, SiCH 2 CH 2 CH 2 Si), 0.56 (m, SiCH 2 CH 2 CH 2 Si (Me) CH 2 CH 2 CH 2 S), -0.05 (s, Si e ).
  • NMR- 3 C (D 2 0): ⁇ 60.54 (SCH 2 CH 2 OH), 35.42 (SCH 2 CH 2 OH), 34.68 (SiCH 2 CH 2 CH 2 S), 24.27 (YES 2 CH 2 CH 2 S) , 18.46 (SiCH 2 CH 2 CH 2 Si), 18.30 (YES 2 CH 2 CH 2 S), 17.33 (SiCH 2 CH 2 CH 2 Si), 13.24 (SiCH 2 CH 2 CH 2 Si), -5.32 (SiMe) . CseH ⁇ OsSsSis.
  • NMR-H (CDCI 3 ): ⁇ 4.14 (t, SCH 2 CH 2 OS0 3 Na), 3.05 (t, SCH 2 CH 2 OS0 3 Na), 2.52 (t, YES 2 CH 2 CH 2 S), 1 .55 (m, YES 2 CH 2 CH 2 S), 1 .27 (m, SiCH 2 CH 2 CH 2 Si), 0.56 (m, SiCH 2 CH2CH2Si (Me) CH2CH2CH 2 S), -0.05 (s, SiMe).
  • RMN- 3 C (CDCI 3 ): ⁇ 170.98 (COOCH 3 ), 52.38 (COOCH3), 36.43 (SCH2CO), 33.42 (SiCH 2 CH 2 CH 2 S), 23.74 ( SiCH 2 CH 2 CH 2 S), 18.92-18.44 (SiCH 2 CH 2 CH 2 Si), 13.38 (SiCH 2 CH2CH2 Yes (Me) CH2CH2CH 2 S, SiCH 2 CH 2 CH 2 Si), -5.00 (SiMeCH 2 CH 2 CH 2 S), -5.17 (SiCH 2 SiMe 2 CH 2). C144H284O32S16YES13.
  • NMR-H (CDCI 3 ): ⁇ 3.74 (s, COOCH 3 ), 3.19 (s, SCH 2 CO), 2.61 (t, SiCH 2 CH 2 CH 2 S), 1 .55 (m, SiCH 2 CH 2 CH 2 S), 1 .24 (m, SiCH 2 CH 2 CH 2 Si), 0.57 (m, SiCH 2 CH 2 CH 2 Si (Me) CH 2 CH 2 CH 2 S), -0.06 (s, If eCH 2 CH 2 CH 2 S), - 0.10 (SiCH 2 CH 2 CH 2 If e) NMR- 3 C (D 2 0): ⁇ 170.90 (COOCH 3 ) , 52.29 (COOCH 3 ), 36.33 (SCH 2 CO), 3.32 (SiCH 2 CH 2 CH 2 S), 23.65 (SiCH 2 CH 2 CH 2 S), 18.83-18.38 (SiCH 2 CH 2 CH 2 Si), 13.29 (SiCH 2 CH 2 CH 2 Si (Me) CH 2 CH 2 S, SiCH 2 CH 2
  • NMR-H (D 2 0): ⁇ 3.03 (s, SCH 2 CO), 2.42 (t, SiCH 2 CH 2 CH 2 S), 1.44 (m, SiCH 2 CH 2 CH 2 S), 1 .25 (m, SiCH 2 CH 2 CH 2 Si), 0.48 (m, SiCH 2 CH 2 CH 2 Si (Me) CH 2 CH 2 CH 2 S), -0.14 (s, Si e).
  • NMR-H (D 2 0): ⁇ 3.15 (t, SCH 2 CH 2 CH 2 S0 3 Na), 2.81 (m, SiCH 2 CH 2 CH 2 S), 2.49 (m, SCH 2 CH 2 CH 2 S0 3 Na), 1.84 (m, SCH 2 CH 2 CH 2 S0 3 Na ), 1.44 (m, SiCH 2 CH 2 CH 2 S), 1 .23 (m, SiCH 2 CH 2 CH 2 Si), 0.58 (m, SiCH 2 CH 2 CH 2 Si (Me) CH 2 CH 2 CH 2 S), -0.14 (s, Si Me) NMR- 3 C (D 2 0): ⁇ 50.1 1 (SCH 2 CH 2 CH 2 S0 3 Na), 30.19 (SCH 2 CH 2 CH 2 S0 3 Na ), 27.05 (SiCH 2 CH 2 CH 2 S), 24.36 (SiCH 2 CH 2 CH 2 S, SCH 2 CH 2 CH 2 S0 3 Na), 18.42 (SiCH 2 CH 2 CH 2 Si), 14.38
  • NMR-H (D 2 0): ⁇ 2.82 (t, SCH 2 CH 2 CH 2 S0 3 Na), 2.49 (m, SiCH 2 CH 2 CH 2 S), 2.42 (m, SCH 2 CH 2 CH 2 S0 3 Na), 1.84 (m, SCH 2 CH 2 CH 2 S0 3 Na ), 1.44 (m, SiCH 2 CH 2 CH 2 S), 1 .24 (m, SiCH 2 CH 2 CH 2 Si), 0.48 (m, SiCH 2 CH 2 CH 2 Si (Me) CH 2 CH 2 CH 2 S), -0.16 (If e).
  • NMR-H (D 2 0): ⁇ 5.79 (s, Ar), 3.06 (s, SCH 2 CO), 2.45 (t, S ⁇ CH2CH2S), 1.52 (m, OCH2CH2CH2CH2YES), 1.20 (m, SiCH 2 CH 2 S, OCH2CH2CH2YES), 0.76 (m, SiCH 2 CH 2 CH 2 Si), 0.45 (m, SiCH 2 CH2CH2Yes (Me) CH2CH 2 S, OCH2CH2CH2CH2YES), -0.1 1 (s, SiMe CH2CH2S), -0.21 ( s, SiMe).
  • NMR-H (D 2 0) dendrimer is obtained: ⁇ 3.06 (s, SCH 2 CO), 2.46 (t, SiCH 2 CH 2 S), 1 .59 ( m, OCH2CH2CH2CH2YES), 1, 22 (m, SiCH 2 CH 2 S, OCH2CH2CH2YES), 0.76 (m, SiCH 2 CH 2 CH 2 Si), 0.45 (m, SiCH 2 CH2CH2Yes (Me) CH2CH 2 S, OCH2CH2CH2CH2YES), -0.10 (s, SiMe CH 2 CH 2 S), -0.18 (s, SiMe).
  • NMR-H (D 2 0): ⁇ 5.87 (s, Ar-H), 3.50 (t, CH 2 0-Ar), 2.86 (m, SCH 2 CH 2 CH 2 S0 3 Na), 2.56 (m, SCH 2 CH 2 CH 2 S0 3 Na), 1.85 (m, SCH 2 CH 2 CH 2 S0 3 Na), 1.71 (m, SiCH 2 CH 2 S), 1.25 (m, OCH2CH2CH2CH2YES), 0.73 (m, SiCH 2 CH 2 S), 0.40 (m, OCH2CH2CH2CH2YES), -0.15 (s, SiMe).
  • NMR-H (D 2 0): ⁇ 3.58 (t, CH 2 0-Ar), 2.88 (m, SCH 2 CH 2 CH 2 S0 3 Na), 2.79 (m, SCH 2 CH 2 CH 2 S0 3 Na) , 1.88 (m, SCH 2 CH 2 CH 2 S0 3 Na), 1 .71 (m, SiCH 2 CH 2 S), 1.21 (m, SiCH 2 CH 2 CH 2 Si, OCH 2 CH 2 CH 2 CH 2 Si), 0.76 (m, SiCH 2 CH 2 S), 0.46 (m, OCH 2 CH 2 CH 2 CH 2 SiCH 2 CH 2 CH 2 Si), -0.1 1 (s, Si eCH 2 CH 2 S ), -0.20 (s, Si e).
  • the heterofunctionalized dendrimers described below can be represented as GnXCx (F ') q (F ") p, where: n, G and X have been described in the previous examples; Cx indicates the length of the carbon chain between the Si atom and the S atom; F 'and F "indicate the nature of the functional groups located on the periphery of the dendrimer: F' corresponds to the groups F described in the previous examples and F" represents a fluorophore group (Flu ), in the following examples FITC is fluorescein, Rho is Rhodamine and DNS is dansyl; q and p are the number of these functions, respectively, where q + p is o.
  • the dendrimer G203C2 (NH2) (S03Na) n is obtained.
  • R 4 , R 3 e and are defined above, M is a metal, Pht is phthalimide and represents a group R 5 , for example:
  • the dendrons described in this section can be represented as XGnC and (F) or, where: n indicates the number of generation G; X, indicates the nature of the focal point, in the case of the following examples these functional groups are linked to Si by an alkyl group (CH 2 ) 4-, Cx, indicates the length of the carbon chain between the atom of S and he does; F indicates the nature of the functional groups located on the periphery of the dendron (corresponds to the R 3 groups described in the description) and I is the number of these functions and will depend on the number of generations.
  • the third generation dendron FITCNHG3C2 (NMe3l) is prepared following a procedure similar to that described for the corresponding dendron G2. Starting from NH2G3C2 (NMe3l) e (0.084 g, 0.05 mmol) and FITC (0.024 g, 0.06 mmol) in EtOH (5 mL) and subsequent addition of excess Mel (0.03 mL, 0.48 mmol) FITCNHG 3 C2 (NMe3l) 8 is obtained as a yellow solid (0.132 g, 82%).
  • the fourth generation dendron FITCNHG4C2 (NMe3l) i6 is prepared following a procedure similar to that described for the corresponding dendron G2. Starting from NH2G4C2 (NMe3l) i6 (0, 120 g, 0.04 mmol) and FITC (0.017 g, 0.04 mmol) in EtOH (5 mL) and subsequent addition of excess Mel (0.04 mL, 0 , 64 mmol) FITCG 4 C2 (NMe3l) i6 is obtained as a yellow solid (0.178 g, 83%).
  • HOArG 3 C2 (NMe 2 l-ICI) 8 (0.349 g, 0.36 mmol) in a mixture of H2O / CHCl3 (1: 1, 20 ml) an aqueous solution of NaOH is added (0,150 g, 3.75 mmol) finally obtaining HOArG 3 C2 (NMe2) 8 as a yellowish oil (0.330, 99%).
  • PhtG 2 C2 (NMe 2 HCI) 4 (1, 202 g, 1, 10 mmol) in a mixture of H 2 0 / CHCI 3 (1: 1, 20 ml) is added an aqueous solution of Na 2 C0 3 (0.467 g, 4.41 mmol).
  • the organic phase is separated and after drying with Na 2 S0 PhtG 2 C2 (NMe 2) is obtained as a yellowish oil (0.943, 90%).
  • a solution of PhtG 2 C2 (NMe 2 ) 4 (0.896 g, 0.95 mmol) in Et 2 0 (20 mL) is then taken and an excess of Mel (0.28 mL, 4.48 mmol) is added .
  • PhtG 2 C2 (NMe 3 L) 4 is obtained as a white solid (1.199 g, 70%).
  • PhtG 2 C2 (NMe 3 L) 4 (1, 300 g, 0.86 mmol) in MeOH (10 mL) is added in excess N 2 H 4 (0.07 mL, 2.15 mmol) and the mixture
  • the reaction is heated at 80 ° C in a vacuum ampoule for 16 h. Then we bring the reaction mixture to dryness and dissolve in water. Hl (7.6 M, 0.25 ml) is added and filtered.
  • PhtG C2 (NMe 2) 8 (0.927 g, 0.51 mmol) in Et 2 0 (20 ml) and excess Mel (0.32 mi, 5, 10 mmol) is added .
  • the reaction mixture is allowed to stir at room temperature for 16 hours.
  • the volatiles are then evaporated and washed with hexane.
  • PhtG 3 C2 (NMe 3 L) 8 is obtained as a white solid (1.068 g, 71%).
  • PhtG 3 C2 (NMe 3 L) 8 (0.932 g, 0.32 mmol) in MeOH (10 mL) is added in excess N 2 H 4 (0.02 mL, 0.50 mmol) and the reaction mixture it is heated at 80 ° C in a vacuum ampoule for 16 h. Then we bring the reaction mixture to dryness and dissolve in water. Hl (7.6 M, 0.08 ml) is added and filtered. The solvent is evaporated and the residue is washed with Et 2 0 to obtain INH3G3C2 (NMe3l) and after drying as an oil (0.821 g, 87%).
  • N3G2V4 is obtained as a colorless oil.
  • N3G3Ve is obtained as a colorless oil. Synthesis of N3G1 C2 (NMe 2 -HCI) 2 .
  • N3G3C2 (NMe 2 HCI) 8 is obtained as a white solid.
  • HSG2C2 (NMe-HCI). Following the procedure for analogous dendrons, from Me (CO) SG2C2 (NMe2-HCI) 4 and HCI (4 M, dioxane) HSG2C2 (NMe 2 HCI) 4 is obtained .
  • HSG3C2 (NMe 2 HCI) 8 is obtained as a white solid.
  • the dendritic wedge NH2G2C3 (C02Me) 4 is added in excess by weight and left under stirring for 12 h. After this time the solvent is evaporated and dissolved in water.
  • the dendritic wedge NH2G3C3 (C02Me) e in MeOH NaOH is added in excess by weight and left with stirring for 12 h. After this time the solvent is evaporated and dissolved in water.
  • NMR-H (D 2 0): ⁇ 2.70 (t, SCh ⁇ ChkCA ⁇ SOsNa), 2.49 (m, SiCH 2 CH 2 CH 2 S), 2.38 (m, SCH 2 CH 2 CH 2 S0 3 Na), 1.84 (m, SChkCA ⁇ ChkSOsNa), 1.59 (m, NH 2 CH 2 CH2CH 2 CH 2 YES), 1.44 (m, YES 2 CH 2 CH 2 S), 1.25 (m, SiCH 2 CH 2 CH 2 Si), 0.58 (m, NH 2 CH 2 CH 2 CH2 YES, SiCH 2 CH 2 CH 2 Si (Me) CH 2 CH 2 CH 2 S), 0.14 (s, SiMe) NMR- 3 C (D 2 0): ⁇ 50.10 (SChkChkCh ⁇ SOsNa), 39.42 (NH 2 CH 2 CH 2 CH 2 CH 2 YES), 35.34 (SChkChkCh ⁇ SOsNa), 31.12 (NH 2 CH 2 CH 2 CH
  • NMR-H (D 2 0): ⁇ 2.81 (t, SCH 2 CH 2 CH 2 S0 3 Na), 2.52 (m, SiCH 2 CH 2 CH 2 S), 2.43 (m, SCA ⁇ ChkChkSOsNa), 1.85 (m , SCH 2 CH 2 CH 2 S0 3 Na), 1.45 (m, SiCH 2 CH 2 CH 2 S), 1.24 (m, SiCH 2 CH 2 CH 2 Si, NH 2 CH 2 CH 2 CH2CH 2 YES), 0.49 ( m, NH 2 CH 2 CH 2 CH2S ⁇ , SiCH 2 CH 2 CH 2 Si (me) CH 2 CH 2 CH 2 S), - 0.14 (s, SiMe).
  • NMR-H (D 2 0): ⁇ 2.80 (t, SCH 2 CH 2 CH 2 S0 3 Na), 2.48 (m, SiCH 2 CH 2 CH 2 S), 2.41 (m, SCH 2 CH 2 CH 2 S0 3 Na), 1.84 (m, SCH 2 CH 2 CH 2 S0 3 Na ), 1 .44 (m, NH 2 CH 2 CH 2 CH 2 Si, SiCH 2 CH 2 CH 2 S), 1 .23 (m, SiCH 2 CH 2 CH 2 Si), 0.47 (m, NH 2 CH 2 CH 2 CH 2 Yes, SiCH 2 CH 2 CH 2 Si (Me) CH 2 CH 2 CH 2 S), - 0.15 (s, Si e) NMR- 3 C (D 2 0): ⁇ 50.06 (SCH 2 CH 2 CH 2 S0 3 Na), 35.99 (NH 2 CH 2 CH 2 CH 2 Si), 35.26 (SCH 2 CH 2 CH 2 S0 3 Na), 31.30 (NH 2 CH 2 CH 2 CH 2 Si), 30.30 (SiCH
  • Me (CO) SG1V2 and HSCH 2 C0 2 CH 3 Me (CO) SG1 C2 (C0 2 Me) 2 is obtained as a yellowish oil.
  • Me (CO) SG2C2 (C0 2 Me) is obtained as a yellowish oil.
  • HSG2C2 (C0 2 Na) 4 is obtained as a white solid.
  • HSG3C2 (C0 2 Me) 8 and NaOH under Ar atmosphere
  • HSG3C2 (C0 2 Na) s is obtained as a white solid.
  • NMR-H (D 2 0): 2.79 (t, CH 2 S0 3 Na), 2.49 (m, CH 2 S), 2.42 (t, HSCH2CH2CH2CH2YES), 1.84 (m, SCH 2 CH 2 CH 2 S0 3 Na), 1 .65 (m, CH2CH2CH2CH2YES), 1 .38 (m, CH2CH2CH2CH2YES and HS), 0.76 (m, SiCH 2 CH 2 S), 0.38 (m, CH2CH2CH2CH2YES), - 0.23 (s, SiMe) .
  • HSG2C2 (S0 3 Na) is obtained as a white solid.
  • N 3 G1 C3 (C0 2 Me) 2 is obtained as a yellowish oil.
  • N 3 G2C3 (C0 2 Me) is obtained as a yellowish oil.
  • NMR-H (CDCI 3 ): 3.72 (s, COOCH 3 ), 3.24 (s, 2 H, N 3 CH 2 ), 3.22 (s, SCH 2 CO), 2.48 (t, SiCH 2 CH 2 CH 2 S) , 1 .61 (m, 2H, N-CH2-CH2-CH2-CH2-YES), 1 .42 (m, S ⁇ CH2CH2CH2S, S ⁇ CH2CH2CH2S), 1 .38 (m, SiCH 2 CH 2 CH 2 Si, N 3 CH2CH 2 CH2CH 2 Si), 0.80 (t, S1CH2 CH2CH2S), 0.48 (m, SiCH 2 CH2CH2Si (Me) CH2CH2CH 2 S, SiCH 2 CH 2 CH 2 Yes, N3CH2CH2CH2CH2YES), -
  • N3G3C3 (C0 2 Me) 8 is obtained as a yellowish oil.
  • N3G2C3 (C02Me) 4 and NaOH or from BrG2C3 (C0 2 Na) 4 and NaN 3 , N3G2C3 (C0 2 Na) 4 is obtained as a white solid.
  • N3G1 C2 (S0 3 Na) 2 is obtained Like a white solid NMR-H (D 2 0): 3.24 (m, 2 H, N 3 CH 2 ), 2.79 (t, CH 2 S0 3 Na), 2.49 (m, CH 2 S), 1.84 (m, SCH 2 CH 2 CH 2 S0 3 Na), 1.65 (m, CH2CH2CH2CH2YES), 1.38 (m, CH2CH2CH2CH2YES), 0.76 (m, SiCH 2 CH 2 CH 2 S), 0.38 (m, CH2CH2CH2CH2YES), - 0.23 (s, SiMe).
  • N3G2C3 (S0 3 Na) is obtained as a white solid .
  • N3G3C3 (S0 3 Na) 4 is obtained as a white solid.
  • PBMCs PERIPHERAL BLOOD MONONUCLEAR CELLS
  • the blood from buffy coats from healthy donors, is diluted twice with 6.7 mM phosphate buffered saline (PBS, Bio-Whittaker®) and centrifuged in density gradient (Ficoll-lsopaque ® ). After said centrifugation, the halo containing the CMSPs is recovered and It proceeds to two subsequent wash-spin cycles with PBS (10 minutes at 1, 500 rpm). The resulting CMSPs are resuspended in complete medium and under culture conditions.
  • PBS phosphate buffered saline
  • the dendrimers G103C2 (NMe 3 l) 6 , G203C2 (NMe 3 l) and 2 , G303C2 (NMe 3 l) 2 4, G403C2 (NMe 3 l) 4 8 correspond to compounds BDEF031, BDEF032, BDEF033 and BDEF034 respectively, while the G203C2 (NMe 3 L) n (NHFITC) dendrimer refers to the BDEF023 compound.
  • a screening system was used to determine the biocompatible concentrations of the 4 generations of dendrimers.
  • the dendrimers were dissolved at concentrations of 3 mM, 2 mM and 1 mM, the latter being the one with the best solubility without the help of additional physical factors for perfect solubilization (vortex, heat, etc.).
  • concentrations of 3 mM, 2 mM and 1 mM were established.
  • the dendrimers were dissolved at concentrations of 3 mM, 2 mM and 1 mM, the latter being the one with the best solubility without the help of additional physical factors for perfect solubilization (vortex, heat, etc.).
  • the MTT assay was used to assess mitochondrial activity to study the toxicity of generations of dendrimers in primary cultures of peripheral blood mononuclear cells since they are the first target of HIV and the most physiological (PBMCs). This technique was used to show deleterious effects on cell metabolism.
  • Opti-MEM® 20 ⁇ of previously filtered MTT was added to achieve sterility (Tiazolyl Blue, Sigma®) in PBS at a concentration of 5 mg / ml (final well concentration of 0.5 mg MTT / ml). After 4 hours of incubation under culture conditions, the plate was centrifuged at 2,000 rpm. 10 minutes and after removal of the supernatant with the excess MTT that did not react. Formazan crystals were observed under the phase contrast microscope and subsequently dissolved with 200 ⁇ of DMSO. The plate was stirred at 700 rpm. in an Eppendorf ® stirrer to ensure the correct dissolution of said crystals.
  • Formazan concentration was determined by spectrophotometry using a plate reader at a wavelength of 570 nm with a reference of 690 nm.
  • the spectrophotometer was calibrated using Opti-MEM® without cells.
  • the relative cell viability (%) with respect to the control (untreated cells) was calculated based on the formula: [A] test / [A] control x 100.
  • Each dendrimer concentration was tested in triplicate, following the guidelines of the ATCC. It was used as lysis control of 0.2% Triton x-100 cells.
  • siRNA small interfering RNAs
  • siNEF siRNA 5'- GUGCCUGGCUAGAAGCACAdTdT-3 ', labeled with cyanine 3 (cy3) was used at the 5' end of the sense strand and the antisense siNEF: 3'-UGUGCUUCUAGCCAGGCACdTdT-5 '. in sterile H 2 0 and stored at 4 ° C.
  • concentration depended in each case on the desired load ratio (from 1: 1 to 1: 12 ratio, see table 1).
  • the ratios SiRNA Nef / dendrimer 1: 8 and 1: 12 were selected and an MTT toxicity test was carried out to verify that the dendriplexes were not toxic to those ratios following the same procedure that in the toxicity evaluation section of dendrimers described above. The study of dendrimer alone and complexes was carried out in parallel.
  • heparin exclusion competition trial was performed to analyze the junctions between dendrimers and siRNA. They were carried out with mixtures of dendriplexes (+/- ratio 1: 8 and 1: 12) of the dendrimers BDEF031, BDEF032, BDEF033, BDEF034 with varying concentrations of heparin (0, 1, 0.2, 0.3 and 0.6 U / ⁇ IQ siRNA). The mixture was run on a 2% agarose gel for 2 and 16 hours.
  • the viral isolate X4 HIV-1 N L4-3 is an established laboratory viral strain and MT-2 cells (T-cell leukemia cell line with integrated HTLV-1 DNA were used, obtained from the American Type Culture Collection ( ATCC) for virus expansion 2x10 6 MT-2 cells were washed twice with complete medium (RPMI 1640 (Gibco) supplemented with 10% Fetal Calf Serum (SFT), 2mM L-glutamine and antibiotics (1% cloxacycline, 1% ampicillin and 0.32% gentamicin)] in 24 or 96 well plates, under culture conditions (37 ° C in an atmosphere of 5% C0 2 and 95% relative humidity) and transferred to a tube 15 ml conical at a concentration of 2x10 6 cells / ml in complete medium Subsequently, HIV-1 N L4-3 was added at a concentration of 1 particle per cell or what is the same, 1 MOI.
  • the MT-2 were cultured with the virus for 2 hours under culture conditions, shaking the culture every 15-30 minutes.
  • the cultures (virus cells) were washed twice to remove the virus not integrated into the cell genome.
  • the cells were transferred to a six-well plate well in a volume of 3-4 ml. It was left in culture for 2-3 days and the presence of syncytia was observed in the well. When the presence of syncytia reached 80-90% of production, 12 ml of complete medium with 20x10 6 MT-2 was added and dispensed in petri dish. At 2-3 days, the entire volume was centrifuged and the supernatant was collected.
  • the HIV-1 N L4-3 viral isolate was titrated in the MT-2 cell line. 2x10 4 MT-2 cells were cultured with complete medium in 96-well plates and 40 ⁇ of the viral preparation was added at different concentrations, for which the corresponding dilutions were made. The dilutions were placed in octuplicate and kept under culture conditions for one week. After this time, the titration was read by visualization of the cytopathic effect. The title was calculated applying the Spearman-Karber formula. It was also titrated by quantification of p24 protein by an enzymatic immunoassay (ELISA p24.
  • ELISA p24 enzymatic immunoassay
  • the HIV-1 Ba -L viral isolate was titrated by quantification of p24 protein by ELISA. To ensure virus purity, thawed aliquots were filtered through 0.22 ⁇ filters before quantification.
  • PBMCs peripheral blood mononuclear cell cultures
  • PBMCs peripheral blood mononuclear cells
  • PHA phytohemagglutinin
  • Ul of IL-2 interleukin 2
  • the previously infected PBMCs are placed in a 96-well plate (2x10 5 cells per well) in complete medium (200 ⁇ per well) and the different complexes are added at a 1: 8 ratio (in this case they are placed as Examples are siNef / BDEF33 and siNEf / BDEF34
  • AZT drugs are used (Zidovudine®), nucleoside analogue retrotranscriptase inhibitor and T20 (Fuzeon®), viral entry inhibitor, in particular of fusion.
  • After the addition of the dendrimers it is incubated at 37 ° C and 5% C0 2 for 24 hours. After the time the supernatant is collected to quantify p24 antigen by ELISA.
  • mice of the BALB / c (H-2d) mouse strain of 4 to 8 weeks of age were used. Mice were injected into the tail vein with the BDEF023 dendrimer at concentrations of 1 mg / kg, 5 mg / kg, 40 mg / kg and 80 mg / kg for 30 min and 1 hour, BDEF023 at a concentration of 20 mg / kg , Cy5.5 labeled Nef siRNA at a concentration of 2 ⁇ and the Nef./BDEF023 siRNA complex. The animals were subsequently sacrificed and fluorescence emission in spleen, liver, kidney and brain was studied in the IVIS Lumina (Xenogen).
  • the ratios 1: 12 were selected for the siRNA-Nef -BDEF31 and siRNA-Nef-BDEF32 complexes and 1: 8 ratio for the siRNA-Nef-BDEF33 and siRNA complexes -Nef-BDEF34. At lower ratios, less union is observed characterized by a lower band expression at the level of the siRNA from the 1: 2 ratio (Fig. 2). As shown in Fig. 3 after 24 hours of treatment with the different dendriplexes, PBMCs were viable, using dextran (Dx) and cell death or DMSO toxicity as a control of viability.
  • Dx dextran
  • DMSO toxicity a control of viability.
  • PBMCs previously stimulated with PHA were infected with 20ng / 1x106 cells with HIV-NL4.3 for 2 h.
  • the siNef / BDEF33 and siNEf / BDEF34 dendriplexes and the AZT and T20 antiretrovirals were added as positive controls for HIV inhibition for 24 h and the culture supernatant was collected to quantify by ELISA the production of Agp24 by ELISA.
  • BDEF33 and BDEF34 showed an HIV inhibition capacity of 85% and 65%, respectively. This data would confirm that they could have a therapeutic application against HIV.
  • the inhibition observed due to dendriplexes was greater when the fourth generation siNEF / BDEF34 was used with 35% than when the SYRNA / BDEF33 was used, the inhibition was 15%. (Fig. 5).
  • Figure 6 shows the results obtained in spleen after injecting in the tail of BALB / c mice 40 mg / kg of the dendrimer alone.
  • the mice were treated for 30 min with the dendrimer no fluorescence was observed at concentrations of 1 and 5 mg / kg, an increase from 40 mg / kg was observed, the maximum expression being at 80 mg / kg.
  • the presence of the dendrimer, siRNA and dendriplex in the spleen of a mouse was observed in the IVIS Lumina (Xenogen) after 1 and 24 hours of treatment. Increased tide was observed when using dendriplex.
  • the data indicate that it may be possible to use dendriplexes in vivo and therefore could have application in different therapies. BIOLOGICAL ACTIVITY OF ANIONIC DENDR ⁇ MEROS AS ANTIVIRAL AGENTS AGAINST HIV.
  • HEC-1A Human Endometrial Carcinoma cells
  • PBMC Peripheral Blood Mononuclear Cells
  • VK2 / E6E7 Human vaginal epithelial cell line. Obtained from the American Type Culture Collection (ATCC), they were grown in Keratinocyte-Serum Free medium (Gibco) with 0.1 ng / ml human EGF, 0.05 mg / ml bovine pituitary extract and 44.1 mg / l calcium chloride (final concentration 0.4 mM), in different plate formats (6, 24 or 96 wells, transwells) and under culture conditions.
  • ATCC American Type Culture Collection
  • TZM.bl Expresses the CD4 and CCR5 markers, and the luciferase and ⁇ -galactosidase genes under the control of the HIV-1 promoter. It is very sensitive to infection by HIV-1 isolates. Its culture medium is DMEM (90%), 10% FBS, 100 u. of penicillin and 0.1 mg / ml streptomycin.
  • HeLa human epithelial cell line, from a cervical adenocarcinoma. They were obtained through the NIH AIDS Research and Reference Reagent Program. Grown in "Dulbecco ' s Modified Eagle Medium” supplemented with 5% SFT, 1% penicillin / streptomycin, and 2 mM L-glutamine at 37 ° C in an atmosphere of 5% C0 2 .
  • the dendrimers described in the previous section are used. And also the dendrimers G2SiC3 (S0 3 Na) i6, and, G2SiC3 (C0 2 Na) i 6 corresponding to the compounds BDMG017 and BDMG018, respectively.
  • the technique used to study the concentrations at which BDMG017 and BDMG018 dendrimers were viable was the MTS that measures cytotoxicity in relation to mitochondrial activity. This method was applied to study the toxicity of the second generation of dendrimers (from 1 to 1000 ⁇ ) in PBMCs and different cell lines: HEC-1A, HeLa, TZM.bl and VK2.
  • This technique was used to demonstrate deleterious effects of dendrimers on cell metabolism. It is a colorimetric assay based on the selective ability of viable cells to reduce (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H - tetrazolium) (Promega) in insoluble crystals of formazan. After the desired incubation time of the different cell populations with different concentrations of 96-well plate dendrimers, and with 3 wells as a positive control of cell inactivity [10% dimethyl sulfoxide (DMSO, Sigma).
  • DMSO dimethyl sulfoxide
  • HEC-1A cells were grown in 12-well plates in culture medium. To study the internalization of HIV in HEC-1A, the cells were pre-treated with the BDMG017 and BDMG018 dendrimers at doses of 10 ⁇ and 100 ⁇ for 1 hour, before carrying out the infection. Once the time was up, the HEC-1A was infected with the viral isolates X4 HIV-1 NL . 3 and R5 VI H- 1 at 100 ng of HIV / 10 6 cells for two hours. After that period of time the cells were washed with sterile PBS and the cell lysis was carried out using Triton x-100 0.2% for 40 minutes at 37 ° C. Agp24 of the cell lysate was quantified by ELISA according to the test instructions.
  • PBMC Peripheral blood mononuclear cells
  • PHA PHA
  • IL-2 Peripheral blood mononuclear cells
  • the cells were treated at the concentrations of 10 ⁇ and 100 ⁇ of BDMG017 and BDMG018 dendrimers for 1 hour before infection with 10 ng of the HIV-1 -3 viral isolate per million cells for 2 hours in growing conditions After this time, the plate was washed three times with PBS and incubated under culture conditions. The concentration of HIV in the supernatant of the PBMC cultures at 3 days was quantified by p24 ELISA according to the kit instructions.
  • PBMCs were first infected with viral isolates X4 HIVNL4.3 and R5 HIVBaL for 2 hours, using the same experimental conditions as in the pre-treatment experiment. After this time, the culture plate was washed three times with PBS to remove excess virus and the cells were treated with the dendrimers BDMG017 and BDMG018. After 3 days the culture supernatant was collected and the viral infection was quantified by a p24 ELISA.
  • the ability of the dendrimers BDMG017 (SIG2SS03) and BDMG018 (SIG2SCBX) to interact with the viral particles in the process of adhesion of these to the surface of the cell membrane was evaluated.
  • the dendrimers would act as a physical barrier in preventing HIV infection of the endometrial cells, blocking the passage of the virus through the mucous membranes and the infection of other target cells such as CMSP.
  • the antiretroviral T20 (an inhibitor of HIV fusion to cells due to its binding to HIV gp41) was used; a specific CXCR4 antagonist that also blocks the T-tropic and dual-tropic variants (R5 X4) which need the CXCR4 to enter the cells, the AMD3100 or Biciclamo; a non-peptide compound that interacts with CCR5 is a quaternary ammonium derivative called TAK-779; a CCR5 co-receptor antagonist used as a drug in HAART, Maraviroc.
  • Figure 8 shows the effect that dendrimers have on the entry of the virus into vaginal epithelial cells.
  • transwell devices were used to recreate the phenomenon of transcytosis ( transport of macromolecules from one extracellular space to another through the cytoplasm of a cell by means of an endocytic vesicle) due to the possibility of forming a perfect monolayer of adherent cells inside and collecting information from the supernatants of the apical and basolateral face of the monolayer.
  • R5 viruses were used as they are the strains that appear in the first moments of infection (type X4 virus was also used).

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Abstract

L'invention concerne des macromolécules hautement ramifiées dénommées dendrimères ou dendrons, présentant une structure carbosilane et fonctionnalisées à leur périphérie avec des groupes anioniques ou cationiques conférant à la macromolécule une charge nette négative ou positive, respectivement. Ces molécules ont été synthétisées à partir d'un noyau polyvalent, en particulier un noyau polyphénolique ou d'atome de silicium, de dendrimères sphériques, ou elles ont été obtenues sous la forme de matrices dendritiques présentant un point de croissance connu en tant que point focal. La fonctionnalisation de la périphérie de ces dendrimères est obtenue par réaction thiol-ène ou thiol-ine entre un thiol qui contient le groupe anionique ou cationique, ou son précurseur correspondant, et un dendrimère fonctionnalisé avec des oléfines ou alcynes terminales. En outre, l'invention concerne un procédé permettant de les obtenir ainsi que leurs utilisations en biomédecine.
PCT/ES2013/070529 2012-07-25 2013-07-19 Composés dendritiques carbosilanes homo et hétéro-fonctionnalisés WO2014016460A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2543640A1 (es) * 2014-02-19 2015-08-20 Universidad De Alcalá Dendrímeros carbosilano que presentan en su superficie grupos tiol terminales, su preparación y sus usos

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ES2657282B1 (es) * 2016-09-02 2018-12-17 Universidad De Alcalá Dendrones carbosilano funcionalizados con ácidos grasos: formación de micelas y usos
ES2677242B1 (es) * 2017-01-31 2019-03-27 Univ Alcala Henares Nanoconjugados formados por moléculas dendríticas y péptidos como agentes antitumorales frente al cáncer

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
B RASINES ET AL.: "Synthesis and fluorescent properties of cationic carbosilane dendrimers containing augenol linkers for their use in biomedical applications", NEW JOURNAL OF CHEMISTRY 2012, vol. 36, February 2012 (2012-02-01), pages 360 - 370 *
CHUN-FANG LI ET AL.: "Optical-gain enhancement of carbosilane dendrimer containing fluorescein groups in the periphery", JOURNAL OF LUMINISCENCE 2010, vol. 130, April 2010 (2010-04-01), pages 544 - 548 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2543640A1 (es) * 2014-02-19 2015-08-20 Universidad De Alcalá Dendrímeros carbosilano que presentan en su superficie grupos tiol terminales, su preparación y sus usos

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