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WO2013026947A1 - Internalisation et activation photocontrôlée de petites molécules pouvant se lier à l'adn à double brin - Google Patents

Internalisation et activation photocontrôlée de petites molécules pouvant se lier à l'adn à double brin Download PDF

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Publication number
WO2013026947A1
WO2013026947A1 PCT/ES2012/070632 ES2012070632W WO2013026947A1 WO 2013026947 A1 WO2013026947 A1 WO 2013026947A1 ES 2012070632 W ES2012070632 W ES 2012070632W WO 2013026947 A1 WO2013026947 A1 WO 2013026947A1
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compound
stranded dna
compound according
double stranded
binding molecule
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Mateo Isidro Sanchez Lopez
Olalla Vazquez Vazquez
Eugenio Vazquez Sentis
Jose Luis MASCAREÑAS CID
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Universidade de Santiago de Compostela
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
    • C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
    • C07D221/04—Ortho- or peri-condensed ring systems
    • C07D221/06—Ring systems of three rings
    • C07D221/10—Aza-phenanthrenes
    • C07D221/12—Phenanthridines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C257/00—Compounds containing carboxyl groups, the doubly-bound oxygen atom of a carboxyl group being replaced by a doubly-bound nitrogen atom, this nitrogen atom not being further bound to an oxygen atom, e.g. imino-ethers, amidines
    • C07C257/10—Compounds containing carboxyl groups, the doubly-bound oxygen atom of a carboxyl group being replaced by a doubly-bound nitrogen atom, this nitrogen atom not being further bound to an oxygen atom, e.g. imino-ethers, amidines with replacement of the other oxygen atom of the carboxyl group by nitrogen atoms, e.g. amidines
    • C07C257/18—Compounds containing carboxyl groups, the doubly-bound oxygen atom of a carboxyl group being replaced by a doubly-bound nitrogen atom, this nitrogen atom not being further bound to an oxygen atom, e.g. imino-ethers, amidines with replacement of the other oxygen atom of the carboxyl group by nitrogen atoms, e.g. amidines having carbon atoms of amidino groups bound to carbon atoms of six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/62—Compounds containing any of the groups, X being a hetero atom, Y being any atom, e.g. N-acylcarbamates
    • C07C271/64—Y being a hydrogen or a carbon atom, e.g. benzoylcarbamates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/14—Radicals substituted by nitrogen atoms, not forming part of a nitro radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/18—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
    • C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
    • C07D221/04—Ortho- or peri-condensed ring systems
    • C07D221/06—Ring systems of three rings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to compounds that allow the spatially and temporarily controlled activation of small double stranded DNA binding molecules, as well as their uses.
  • the DNA molecule is involved in numerous basic processes for cell life and is the therapeutic target with which numerous drugs interact.
  • DNA binding molecules can cause activation, modulation or inhibition of DNA function. They usually act causing the inhibition of basic processes for cell survival such as transcription or replication, causing cell death. Therefore, these types of molecules have been used in chemotherapy and for the treatment of bacterial, viral, fungal and parasitic diseases, among others. In addition, DNA is one of the most common targets of many of the anticancer drugs used in chemotherapy.
  • the inventors of the present invention have discovered that the use of photolabile protecting groups allows temporarily inactivating the activity of small molecules that selectively interact with double stranded DNA.
  • the resulting protected compound is not capable of binding to the double stranded DNA, whereby it is possible to have a non-active derivative that can be comfortably handled, and that is released at the precise time and place where it is desired to act by irradiation with light, thus inducing the interaction of the small molecule with the DNA in a controlled manner.
  • the invention is directed to a compound formed by a small double stranded DNA binding molecule, covalently conjugated to a photolabile protecting group, with the proviso that said compound is not itself a molecule double stranded DNA binding.
  • the invention is directed to the use of a compound formed by a small double-stranded DNA binding molecule covalently linked to a photolabile protecting group, for in vitro control of gene expression and / or protein synthesis.
  • small molecules that bind DNA cause a change in the structure and / or functionality of the double stranded DNA molecule when they interact with it, preventing basic processes for cell survival and proliferation, such as transcription and replication. Therefore, this type of small double stranded DNA binding molecules have important applications such as antitumor, antibacterial, antiviral, antifungal and antiparasitic, among others.
  • the invention relates to the use of a compound formed by a small double stranded DNA binding molecule covalently linked to a photolabile protecting group, for the preparation of a medicament.
  • This medication may be a controlled action medication, that is, a medication whose activity can be activated in a controlled manner at the desired time and / or place by irradiation with light.
  • the present invention relates to the use of a compound formed by a small double-stranded DNA binding molecule covalently linked to a photolabile protecting group, for the preparation of a medicament for the treatment of proliferative diseases or diseases.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising at least one compound formed by a small double-stranded DNA binding molecule covalently linked to a photolabile protecting group, and at least one pharmaceutically acceptable excipient.
  • the spectral characteristics of some small molecules that bind double-stranded DNA change when binding to DNA and, therefore, are useful as DNA markers or dyes.
  • the invention also relates to the use of a compound formed by a small double-stranded DNA binding molecule covalently linked to a photolabile protecting group, to mark in vitro specific areas of DNA and / or cells.
  • FIG. 1 Left: the solid line represents the HPLC spectrum of protected compound ⁇ 1 in 20 mM Tris-HCl buffer solution; 100 mM NaCl; pH 7.5; The dashed line represents the HPLC spectrum of the same solution after irradiating for 15 min with UV light.
  • fluorescence emission spectrum of a 0.8 ⁇ solution of ⁇ 2 in 50 mM Tris-HCl buffer, 100 mM NaCl, pH 7.5 with increasing irradiation time fluorescence emission microscopy of cells belonging to the Vero cell line (12.5 ⁇ ⁇ 2 after 15 min incubation); A: before irradiation and deprotection, B: after deprotection by irradiation with UV light for 10 min, C: control experiment with 12.5 ⁇ of 2 after 15 min. of incubation
  • FIG. 3 Fluorescence microscopy of cells belonging to the Vero line incubated with compound ⁇ 2, after increasing irradiation times 365 nm).
  • Figure 7 Control of the effect of UV irradiation on DAPI.
  • A DAPI 50 microM before being irradiated.
  • B after being irradiated no change is observed.
  • Figure 8. Control of the effect of UV irradiation on ⁇ 3 (Nvoc 2 -DAPI) 50 microM.
  • C after 15 minutes of irradiation. It is noted that ⁇ 3 is not visible with the microscope's optical cubes that span blue; Only after irradiation are the cells visible.
  • the present invention relates to a compound formed by a small double-stranded DNA binding molecule that is covalently bound to a photolabile protecting group, with the proviso that said compound is not in turn a double stranded DNA binding molecule.
  • the objective of the present invention is to control the ability of small molecules to bind double stranded DNA.
  • photolabile protective groups are used that inactivate the ability of the molecule to bind to DNA. Therefore, it is necessary that the compound of the invention, that is, the small double stranded DNA-binding molecule covalently linked to a photolabile protecting group, is not itself a molecule capable of binding to DNA. In this way it is possible to direct the inactive compound to the desired location for the subsequent release of the DNA binding molecule in a controlled manner.
  • small double stranded DNA binding molecule or "small molecule capable of binding double stranded DNA” refers, according to the present invention, to a molecule of molecular weight less than 300 Da which is capable of interacting with Double stranded DNA.
  • Such molecules are well known in the state of the art (eg, SM Nelson, LR Ferguson, WA Denny, Non-covalent link d / DNA interactions: Minor groove binding agents. Mutation Res. 2007, 623, 24-40; "Small molecule DNA andRNA binders", Wiley-VCH 2003).
  • covalent junction covalent type junctions between the molecule and DNA are formed
  • non-covalent such as hydrogen bonds or electrostatic interactions
  • the small double stranded DNA binding molecule is selected from molecules that bind to double stranded DNA covalently and molecules that bind noncovalently.
  • said molecule is selected from an alkylating agent, an intercalating agent and a groove binding molecule, preferably minor groove binding.
  • alkylating agent refers to molecules that bind to DNA covalently. This term includes both classic alkylating agents in which alkyl groups of these agents bind to the DNA molecule, such as compounds that do not have an alkyl group but also covalently bind to DNA, for example generating bonds between two chains of DNA (cross-link). In general, they are molecules with reactive electrophilic groups that covalently bind to DNA preferably through the nitrogen bases.
  • Alkylating agents are well known in the state of the art (eg, L. Kelland, The resurgence of platinum-based cancer chemotherapy, Nature Rev. Cancer 2007, 7, 573-584; T. Bando, H. Sugiyama, Synthesis and biological properties of sequence-specific DNA-alkylating pyrrole-imidazole polyamides, Acc. Chem. Res.
  • alkylating agents include, for example, nitrosoureas (eg carmustine, lomustine, semustine, fotemustine, streptozocin) , nitrogen mustards (bischloroethylamines, eg chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, uracil mustard, trophosphamide, estramustine), aziridines (eg thiotepa, mitomycin C), alkylsulfonates (eg busulfan, clomesone), hydrazines and triazines (eg altretamine, dacarbazine, procarbazine complexes, procarbazine, pharmozine complexes no (eg cisplatin, carboplatin, oxaliplatin, nedaplatin, saltraplatin, lobaplatin).
  • nitrogen mustards bischloroeth
  • the term "intercalating agent” refers to molecules that bind to double-stranded DNA in a non-covalent manner when intercalated between the nitrogen bases of the DNA. In general, they are flat polycyclic structures that interact with DNA through stacking or hydrophobic interactions.
  • Intercalating agents are well known in the state of the art (eg, L. Strekowski, B. Wilson, Noncovalent interactions with DNA: An overview. Mutation Res. 2007, 623, 3-13; JB Chaires, Drug-DNA Interactions, Curr. Opin. Struct. Biol.
  • Some typical intercalating agents include, for example, anthracyclines (eg doxorubicin, daunorubicin, rubidazone, epirubicin, aclarubicin), acridines (eg acridine orange, proflavin, amsacrine , quinacrine), anthraquinones (eg mitoxantrone), naphthalimides (eg amonafide, elinafide), ethidium bromide, propidium iodide, Actinomycin D, quinine, mefloquine, ellipticin and dithercalin.
  • anthracyclines eg doxorubicin, daunorubicin, rubidazone, epirubicin, aclarubicin
  • acridines eg acridine orange, proflavin, amsacrine , quinacrine
  • anthraquinones eg mitoxantron
  • the term "groove binding molecule” refers to a molecule that binds within the groove of a double stranded DNA, preferably within the minor groove, through non-covalent junctions. More preferably, it is a molecule that binds to the minor groove of the double-stranded DNA through sequences rich in A and T.
  • minor groove binding molecules are well known in the state of the art (eg, C. Bailly, JB Chaires, Sequence-specific DNA minor groove binders. Design and synthesis of netropsin and distamycin analogues, Bioconjugate Chem. 1998, 9, 513-538; DE Wemmer, Designed Sequence-Specific Minor Groove Ligands, Annu. Rev. Biophys. Biomol. Struct. 2000, 29, 439-461).
  • Some typical minor groove binding molecules include, for example, diarylamine dynes (eg pentamidine, propamidine, aza-propamidine, aza-pentamidine, furamidine, stilbamidine, berenyl, DAPI (4 ', 6- diamidino-2-phenylindole)), netropsin, distamycin, lexithropsin, mitramycin, cromomycin A 3 , olivomycin, antramycin, sibiromycin, bis-benzimidazoles (eg Hoechst 33258, Hoechst 33342).
  • diarylamine dynes eg pentamidine, propamidine, aza-propamidine, aza-pentamidine, furamidine, stilbamidine, berenyl
  • DAPI 4- ', 6- diamidino-2-phenylindole
  • netropsin distamycin
  • lexithropsin mitramycin
  • cromomycin A 3
  • the small double stranded DNA binding molecule is selected from a DNA dye, an antitumor agent and an antimicrobial agent.
  • the small double stranded DNA binding molecule is a DNA dye.
  • the DNA dye is selected from acridine orange, DAPI, ethidium bromide, propionium iodide Hoechst 33258 and Hoechst 33342.
  • the small molecule binding to the DNA dye is selected from acridine orange, DAPI, ethidium bromide, propionium iodide Hoechst 33258 and Hoechst 33342.
  • the small molecule binding to the DNA dye is selected from acridine orange, DAPI, ethidium bromide, propionium iodide Hoechst 33258 and Hoechst 33342.
  • Double stranded DNA is an antitumor agent.
  • the antitumor agent is selected from among alkylating agents of DNA, such as nitrosoureas (eg carmustine, lomustine, semustine, photemustine, streptozocin), nitrogenous mustards (bischlorethylamines, eg chlorambucil, cyclophosphamide, ifosfamide, mechlorethatamine, melchlophthatamine, melchlophthatamine, melostataamide , estramustine), aziridines (eg thiotepa, mitomycin C), alkylsulfonates (eg busulfan, clomesone), hydrazines and triazines (eg altretamine, dacarbazine, procarbazine, temozolamide) and platinum complexes (eg cisplatin, carboplatin, oxaliplatin, neplaplatin,
  • doxorubicin doxorubicin, daunorubicin, rubidazone, epirubicin, aclarubicin
  • acridines e.g. acridine orange, proflavin, amsacrine, quinacrine
  • anthraquinones e. g. mitoxantrone
  • naphthalimides e.g. amonafide, elinafide
  • Actinomycin D ellipticin; ditercalinium; netropsin; and bis-benzimidazoles (e.g. Hoechst 33258, Hoechst 33342).
  • the small double strand DNA binding molecule is an antimicrobial agent.
  • the antimicrobial agent is selected from acridines (eg acridine orange, proflavin, amsacrine, quinacrine), quinine, mefloquine, diarylamidines (e.g., pentamidine, propamidine, aza-propamidine, aza-pentamidine, furamidine, strebamidine, berenyl , DAPI), netropsin.
  • the small double stranded DNA binding molecule is a compound of formula (la) or (Ib): HN NH
  • a and B are each independently selected from optionally substituted aryl and heteroaryl;
  • X may not exist, so that A and B are condensed, or it represents a single bond, -O-, -S-, -NH-, -OYO-, -SYS-, -NH-Y-NH-, or an optionally substituted Ci-C 6 alkyl, aryl or heteroaryl;
  • Y is selected from optionally substituted Ci-C 8 alkyl, aryl and heteroaryl, or a salt or solvate thereof.
  • a and B are independently selected from among phenyl, indole or together form a phenanthridine ring, optionally substituted, preferably by C 1 -C 3 alkyl or C 6 -C aryl .
  • a and B are independently selected from among phenyl, indole, or together represent a 5-ethyl-6- phenylphenanthridinium ring.
  • X is selected from a bond, -OYO- and -NH-Y-NH-; where Y preferably represents a Ci-C 8 alkyl group, more preferably a pentyl group.
  • the small double stranded DNA binding molecule is selected from pentamidine, aza-pentamidine, ethidium bromide, 4 ', 6- diamidino-2-phenylindole (DAPI), or a salt or solvate thereof .
  • Photolabile protective groups in the present invention, is defined as a protecting group whose binding to a molecule is broken or released by exposure to light of an appropriate wavelength.
  • Photolabile protecting groups as well as the conditions for their preparation and subsequent deprotection, are known in the prior art (eg PJ Kocienski, "Protecting Groups", Thieme 2005) and include, for example, o-nitrobenzyl derivatives, derivatives of benzoin, phenacil derivatives, etc.
  • the photolabile protecting group is deprotected by irradiation with UV light (preferably of a wavelength between 200 and 400 nm, more preferably> 365 nm), preferably with a power of between 5 and 10 W, more preferably 8 W.
  • UV light preferably of a wavelength between 200 and 400 nm, more preferably> 365 nm
  • the photolabile protecting group has the following structure:
  • n is selected from 0, 1, 2, 3 and 4;
  • R 1 and each R 2 are independently selected from hydrogen, Ci-C 6 haloalkyl Ci-C 6, aryl C 6 -Ci5, 3- to 15 members and heterocycle 3-15 optionally substituted members, N0 2, CN , halogen, -OR ', -SR', -S (0) R ', -S (0) 2 R', -OS (0) 2 R ', -N (R') (R “), -C (0) R ', -C (0) OR', -C (0) N (R ') (R "), - OC (0) R' and -N (R ') C (0) R"; wherein each R 'and R "is independently selected from hydrogen, Ci-C 6 haloalkyl Ci-C6, C3-C7 aryl , C 6 -Ci5, 3- to 15 - membered heterocycle and 3-15 membered optionally substituted;
  • n is selected from 0, 1 and 2.
  • R 1 is selected from hydrogen, methyl, trifluoromethyl and carboxyl.
  • R 2 represents methoxy, or two contiguous R 2 radicals form a group -0-CH 2 -0-.
  • n is selected from 0, 1 and 2; R is selected from hydrogen and carboxyl; and R 2 represents methoxy.
  • the inventors have observed that the presence in the protective group of ionizable groups at physiological pH makes it possible to control even more effectively the inactivation of the small double stranded DNA binding molecule.
  • the carboxylic group is ionized as a carboxylate at physiological pH, causing electrostatic repulsions between the protected compound and the phosphate groups of the DNA, which makes it possible to control even more effectively than the Protected compound object of the present invention cannot bind to DNA.
  • the photolabile protecting group has an ionizable functional group at physiological pH.
  • at least one of R 1 and R 2 comprises an ionizable group at physiological pH. More preferably, at least one of
  • R 1 and R 2 is a carboxyl group.
  • the photolabile protecting group is selected from:
  • the compound of the invention is selected from a compound of formula (Illa) and (Illb):
  • A, B, X, n, R 1 and R 2 are as previously defined, or a salt or solvate thereof.
  • the compound of the invention is selected from:
  • protective groups allows not only to control the activation of the small double stranded DNA binding molecule, but also to modulate its physicochemical properties. It has been observed, for example, that the use of Nvoc as a photolabile protecting group improves the internalization properties in the ethidium bromide cell. It is important to note that ethidium bromide, due to its high polarity, crosses the cell membrane very difficult, which has made it difficult to use this molecule in in vivo experiments. The use of the protective groups mentioned above allows to solve this problem by increasing the hydrophobicity of the molecule.
  • alkyl refers to a straight or branched chain hydrocarbon radical consisting of carbon and hydrogen atoms, which does not contain any unsaturation, having one to eight (Ci-C 8 ), one to six ( Ci-C 6 ), or one to three (Ci- C 3 ) carbon atoms, and that is attached to the rest of the molecule by a single bond.
  • alkyl groups include, but are not limited to, alkyl groups such as methyl, ethyl, n-propyl, isopropyl, isobutyl, 3-methyl-l-pentyl, 4-methyl-l-pentyl, 3,3-dimethyl -l-butyl, t-butyl, n-pentyl, isopentyl and n-hexyl.
  • C3-C7 cycloalkyl means a non-aromatic monocyclic or polycyclic ring, comprising carbon and hydrogen atoms.
  • a cycloalkyl group may have one or more carbon-carbon double bonds in the ring as long as the ring is not aromatic.
  • cycloalkyl groups include, but are not limited to, fully saturated cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl, and cyclic and bicyclic terpenes, and cycloalkenyl groups, such as cyclopropenyl, cyclobutenyl, cyclohentenyl, cyclohexenyl cycloheptenyl, and unsaturated cyclic and bicyclic terpenes.
  • a cycloalkyl group may not be substituted or substituted with one or two suitable substituents.
  • the cycloalkyl group is a monocyclic ring or bicyclic ring.
  • heterocycle refers to a non-aromatic monocyclic or polycyclic ring comprising carbon and hydrogen atoms and at least one heteroatom, preferably, from 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur and which may include condensed ring systems .
  • a heterocycle group may be totally or partially saturated, or it may be aromatic (heteroaryl).
  • the heterocycle group is a monocyclic, bicyclic or tricyclic ring comprising from 3 to 15, preferably from 5 to 15, more preferably from 5 to 10 members and from 1 to 3 heteroatoms.
  • heterocycle groups include aziridinyl, pyrrolidinyl, pyrrolidino, piperidinyl, piperidino, piperazinyl, piperazino, morpholinyl, morpholino, thiomorpholinyl, thiomorpholino, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, benzipyridine, pyridine pyridine, pyridine, pyridine, pyridine, pyridine, pyridine pyridine pyrimidine, isothiazole, isoxazole, imidazole, indole, purine, quinoline, thiadiazole.
  • a heterocycloalkyl group isothiazole, isoxazole, imidazole, indole, purine, quinoline, thiadiazole.
  • halogen refers to chlorine, bromine, iodine or fluorine.
  • aryl refers to an aromatic group having between 6 and 15, preferably between 6 and 10 carbon atoms, comprising 1, 2 or 3 nuclei aromatic, optionally condensed, including the following non-limiting emplos: phenyl, naphthyl, diphenyl, indenyl, phenanthryl.
  • haloalkyl refers to an alkyl group as previously defined, where at least one hydrogen atom has been replaced by a halogen.
  • haloalkyl refers to a CF 3 group.
  • substituents include, for example and in a nonlimiting sense, Ci- 6 alkyl, C3-7 cicloal chyle, aryl, heterocycle, heteroaryl, halogen, cyano, nitro, trifluoromethyl, - N (R a) (R b), -OR a , -SR a , -C (0) R a , -C (0) OR a , -C (0) N (R a ) (R b ), -OC (0) R a ; wherein each R a and R b is independently selected from hydrogen, Ci-C 6 alkyl, aryl, heterocycle, heteroaryl and trifluoromethyl.
  • the compounds of the present invention may be in the form of salts, preferably pharmaceutically acceptable salts, or in the form of solvates.
  • pharmaceutically acceptable salts refers to any salt that upon administration to the recipient can provide (directly or indirectly) a compound as described herein.
  • solvate according to this invention is understood to mean any form of the active compound according to the invention that has another molecule (most likely a polar solvent) attached to it by non-covalent bonds. Examples of solvate sites include hydrates and alcoholates, eg methanolate. Preferably, the solvates are pharmaceutically acceptable solvates.
  • the preparation of salts and solvates can be carried out by methods known in the art.
  • 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, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and p-toluenesulfonate.
  • organic acid addition salts such as, for example, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and p-toluenesulfonate.
  • trifluoroacetate Preferably, trifluoroacetate.
  • compositions According to a further aspect, the invention relates to a pharmaceutical composition comprising at least one compound of the invention, as previously defined, and at least one pharmaceutically acceptable excipient.
  • pharmaceutically acceptable refers to molecular compositions and entities that are physiologically tolerable and normally do not produce an allergic reaction or a similar unfavorable reaction such as gastric disorders, dizziness and the like, when administered to a human being or an animal.
  • pharmaceutically acceptable means that it is approved by a regulatory agency of a state or federal government or is included in the US pharmacopoeia or other pharmacopoeia generally recognized for use in animals, and more particularly in humans.
  • excipient refers to a diluent, adjuvant, carrier or vehicle with which the active substance is administered.
  • Such pharmaceutical excipients may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • water or saline solutions of aqueous solution and aqueous solutions of dextrose and glycerol are used as carriers, particularly for injectable solutions.
  • Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
  • dispersing agents such as surfactants and / or polymers, can also be added.
  • the pharmaceutical composition of the invention can be administered in a plurality of pharmaceutical forms of administration, for example solid (tablets, pills, capsules, granules, etc.) or liquid (solutions, suspensions or emulsions). Administration may be carried out, for example, orally (sublingually, gastroenterically, rectally), parenterally (intravenously, intraarterially, intracardiacly, subcutaneously, transdermally, intraperitoneally or intramuscularly) or topically.
  • the interaction between small molecules and double stranded DNA causes changes in the structure and functionality of said DNA, which may allow modulating, by activation or inhibition, various processes in which DNA is involved, such as transcription or replication, and thereby regulate cellular activity.
  • photolabile protecting groups makes it possible to temporarily inactivate small double stranded DNA binding molecules. Subsequent irradiation with light allows the molecule to be released again and, thus, spatially and temporarily control the activity of the molecule, which could allow control over biological processes such as gene expression, protein synthesis and cell proliferation.
  • spectral characteristics of these small molecules change when they bind to DNA, so those that absorb in UV are usually used as markers or dyes of DNA and cells (eg acridine orange, DAPI, ethidium bromide, propinium iodide Hoechst 33258 and Hoechst 33342).
  • markers or dyes of DNA and cells eg acridine orange, DAPI, ethidium bromide, propinium iodide Hoechst 33258 and Hoechst 33342.
  • the invention is directed to an in vitro method for marking DNA sequences or cells, comprising (a) interacting the DNA sequence or the cell with a small double stranded DNA binding molecule, and ( b) irradiate the sample with UV light.
  • a further aspect refers to the use of the compounds of the invention, as previously described, for the preparation of a medicament.
  • Another additional aspect relates to the use of the compounds of the invention, as previously described, for the preparation of a medicament for the treatment of proliferative diseases and microbial diseases.
  • the invention is directed to a therapeutic method, which comprises the administration of the compounds of the invention and subsequent irradiation with UV light.
  • the therapeutic method is directed to the treatment of proliferative diseases and microbial diseases.
  • proliferative diseases refers to abnormal cell growth or abnormal cell growth without physiological control.
  • Proliferative diseases can be benign or malignant tumors (cancers).
  • Non-cancerous proliferative diseases include lipomas, adenomas, hemangiomas, lymphanginomas, nevus, teratomas, fibromas, myxomas, chondromas, osteomas, meningiomas, gliómicos tumors, leiomyomas, rhabdomyomas, papillomas, angiomas and myomas.
  • cancers include, but are not limited to, spleen, colorectal and / or colon cancer, colon carcinomas, ovarian carcinomas, ovarian cancer, breast cancer, uterine carcinomas, lung cancer, stomach cancer, cancer of the esophagus, liver cancer, carcinomas of the pancreas, kidney cancer, bladder cancer, prostate cancer, testicular cancer, bone cancer, skin cancer, sarcoma, Kaposi sarcomas, brain tumors, myosarcomas, neuroblastomas, lymphomas and leukemia, melanoma, glioma, medulloblastoma, head and neck carcinoma.
  • the proliferative disease is cancer.
  • microbial diseases includes diseases caused by bacteria, viruses, fungi and parasites. Therefore, a particular embodiment refers to the use of the compounds of the invention, as previously defined, in the preparation of a medicament for the treatment of microbial diseases, preferably bacterial, viral, fungal and parasitic diseases.
  • the compounds of the present invention can be prepared by reacting the small double stranded DNA binding molecule and the photlabile protecting group, by synthetic methods common in the state of the art and known to the subject matter expert (eg PJ Kocienski, "Protecting Groups”, Thieme 2005; “March 's Advanced Organic Chemistry”, Wiley-Interscience 2001; TW Greene, “Protective Groups in Organic Chemistry”, Wiley 2007).
  • the isolated product containing the tere-butyl protecting group was dissolved in 4.5 mL of CH 2 CI 2 and cooled to 0 ° C. Then, 4.4 mL of TFA was added slowly. It was allowed to react with magnetic stirring for 2 h and subsequently the solvent was removed under reduced pressure. Finally, the reaction crude was purified in the reverse phase (Water / MeCN) and the fractions containing the desired product were lyophilized to obtain a yellowish solid (63 mg, 0.079 mmol, 89% in total).
  • DAPI (12 mg, 0.034 mmol) was dissolved in 0.7 mL of DMSO in an eppendorf. Then, Et 3 N (30 mg, 41 ⁇ ., 0.296 mmol, 8 equiv) and finally nitroveratril chloride (56 mg, 0.204 mmol, 6 equiv) was added. The resulting mixture was allowed to react with magnetic stirring and in the absence of light for 16 hours. The reaction crude was purified in reverse phase (Water / MeCN) and the fractions containing the desired product were lyophilized to obtain the product as a yellowish solid (14 mg, 0.018 mmol, 54%).
  • the hairpin oligonucleotide containing the AAATTT target sequence (SEQ ID NO: 1) was supplied by Thermo Fischer Scientific GmbH.
  • the deprotection of the photolabile protecting groups was carried out by irradiation with a transilluminator with an 8 watt lamp (max. Approx. 360 nm).
  • the cells in which the experiments were performed in vivo belong to the Vero cell line, the culture medium of these is DMEM buffer (Dulbecco Modified Eagle Medium) containing 10% FBS (Fetal Bovine Serum). The day before the experiment, they were transferred to wells containing their corresponding 15 mm coverslips, then washed and stored with serum-free PBS.
  • DMEM buffer Dulbecco Modified Eagle Medium
  • FBS Fetal Bovine Serum
  • the cells were incubated for 30 min at room temperature and in the dark, after that time they were washed 3 times with PBS, once washed the coverslips were transferred with the cells to the slides where they were mounted with Mowiol 4-88 ® [100 mg / mL in 100 mM Tris-HCl pH 8.5, 25% glycerol and 0.1% DABCO (as an antifungal agenta)] for irradiation and subsequent visualization with fluorescence microscopy.
  • the deprotection was performed with a UV transilluminator for gels during different times before being observed in the fluorescence microscope.
  • the selected images were taken with an Olympus DP-50 digital camera, then these were processed with the Adobe Photoshop (Adobe Systems) software.
  • Figures 5 to 8 show the images taken by fluorescence microscopy of the cells incubated with compounds 2, ⁇ 2, 3 and ⁇ 3, before and after being irradiated with UV light.
  • the antimicrobial activity of compound ⁇ 4 was measured by the conventional method of disk diffusion with Candida albicans, following the procedure of the Clinical and Laboratory Standards Institute (CLSI, 2006).
  • the antimicrobial solution will be prepared using water or DMSO as solvent.
  • the sterile 6 mm diameter discs (Liofilchem, Italy) embedded in the drug were kept on the surface of Mueller Hinton Agar (Cultimed, Spain) inoculated with the yeast.

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Abstract

L'invention concerne des composés formés par une petite molécule capable de se lier à l'ADN à double brin conjuguée de manière covalente à un groupe protecteur photolabile, et à leurs utilisations. La déprotection de ces composés par irradiation à la lumière permet de contrôler de manière spatio-temporelle l'activation de ces molécules de liaison à l'ADN.
PCT/ES2012/070632 2011-08-24 2012-08-22 Internalisation et activation photocontrôlée de petites molécules pouvant se lier à l'adn à double brin Ceased WO2013026947A1 (fr)

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ES201131415A ES2396076B2 (es) 2011-08-24 2011-08-24 Internalización y activación fotocontrolada de moléculas pequeñas capaces de unirse al adn de doble hebra.
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WO2014202814A1 (fr) * 2013-06-20 2014-12-24 Universidade De Santiago De Compostela Composés fluorescents
WO2016086060A1 (fr) * 2014-11-26 2016-06-02 The J. David Gladstone Institutes Procédés pour traiter une infection à cytomégalovirus
CN111362834A (zh) * 2020-02-26 2020-07-03 湖南大学 一种具有抗耐药性的抗菌脒类低聚物及其制作方法和用途
WO2023054288A1 (fr) * 2021-09-28 2023-04-06 学校法人東京理科大学 Générateur de photobases, composé, composition photoréactive et produit de réaction

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014202814A1 (fr) * 2013-06-20 2014-12-24 Universidade De Santiago De Compostela Composés fluorescents
WO2016086060A1 (fr) * 2014-11-26 2016-06-02 The J. David Gladstone Institutes Procédés pour traiter une infection à cytomégalovirus
US10478431B2 (en) 2014-11-26 2019-11-19 The J. David Gladstone Institutes Methods for treating a cytomegalovirus infection
CN111362834A (zh) * 2020-02-26 2020-07-03 湖南大学 一种具有抗耐药性的抗菌脒类低聚物及其制作方法和用途
CN111362834B (zh) * 2020-02-26 2021-04-02 湖南大学 一种具有抗耐药性的抗菌脒类低聚物及其制作方法和用途
WO2023054288A1 (fr) * 2021-09-28 2023-04-06 学校法人東京理科大学 Générateur de photobases, composé, composition photoréactive et produit de réaction

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