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    • 专利摘要:
    New boradiazaindacénic skeleton compounds and their use as teragnostic agents based on accumulation in lipid droplets. The present invention relates to novel dyes with BODIPY structure characterized by containing at least one (alkoxycarbonyl) alkyl moiety in its structure. specific accumulation capacity in lipid droplets and teragnostic activity, allowing dual use as fluorescent markers for fluorescence-based bioimaging diagnosis and as photocitotoxic agents in photodynamic therapy. In addition, it refers to its obtaining procedure and its dual application in fluorescence microscopy and photodynamic therapy; as well as a new strategy to achieve the desired teragnostic activity, consisting of the specific accumulation of the teragnostic agent in lipid drops. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2719000A1
    申请号:ES201900017
    申请日:2019-02-14
    公开日:2019-07-05
    发明作者:Garcia Ma Josefa Ortiz；La Moya Cerero Santiago De；Agarrabeitia Antonia Rodriguez；Castaneda Alejandro Prieto；Garrido Fernando Garcia；Oroquieta Angeles Villanueva；Truchado Andrea Tabero
    申请人:Universidad Complutense de Madrid；Universidad Autonoma de Madrid；
    IPC主号:
    专利说明:

    [0001]
    [0002] New compounds of boradiazaindacenic skeleton and its use as therapeutic agents based on accumulation in lipid droplets
    [0003]
    [0004] SECTOR OF THE TECHNIQUE
    [0005] The present invention is framed in the field of Precise Organic Chemistry, specifically in the field of chemical agents for bioimaging and photodynamic therapy for application in (bio) medicine.
    [0006]
    [0007] BACKGROUND OF THE INVENTION
    [0008] Teragnosis is a cutting-edge technology in the emerging field of "personalized precision medicine" that simultaneously allows diagnosis in the early stage of the disease and its efficient therapy, as well as real-time monitoring of terperatic efficacy (" Cancer Theranostics ", Eds ,: X. Chen, S. Wong, Elsevier, Amsterdam (Netherlands) 2014; R. Kumar, WS Shin, K. Sunwoo, WY Kim, S. Koo, S. Bhuniya, JS Kim, Chem. Soc. Rev. 2015, 44, 6670).
    [0009]
    [0010] The development of intelligent and advanced multimodal agents with objectives aimed at the treatment of cancer through early diagnosis by bioimaging and their efficient and simultaneous therapy constitute the cornerstone of teragnosis (E.-K. Lim, T. Kim, S. Paik, S Haam, Y.-M. Huh, K. Lee, Chem. Rev. 2015, 115 , 327; MH Lee, A. Sharma, MJ Chang, J. Lee, S. Son, JL Sessler, C. Kang, JS Kim, Chem. Soc. Rev. 2018, 47, 28.). In this sense, the development of dyes that combine the ability to act as photosensitizers for the generation of singlet, cytotoxic oxygen and, therefore, with the ability to be used as agents in photodynamic therapy (therapy consisting of producing selective cell death by light irradiation of the lesion treated with the photosensitizer), with fluorescent capacity to additionally allow diagnosis by fluorescence-based bioimaging, is a clear objective in the field of teragnosis since such dyes could be configured as interesting biomaterials for clinical use in precision medicine ( "Imaging in Photodynamic Therapy” in Series in Cellular and Clinical Imaging, Eds .: MR Hambling, Y. Huang, CRC Press, Boca Raton (USA) 2017; G. Duran-Sampedro, N. Epelde-Elezcano, V. Martínez- Martínez, I. Esnal, J. Bañuelos, I. Garcia-Moreno, AR Agarrabeitia, S. de la Moya, A. Tabero, A. Lazaro, A. Vilanueva, MJ Or tiz, I. López Arbeloa, Dyes Pigm. 2017, 142 , 77), However, the development of such dyes (capable of being used in photodynamic therapy and fluorescence image acquisition) is still at an early stage since its dual photoactivity requires combining in the same chemical structure two photophysically opposed processes: fluorescence vs. singlet oxygen photogeneration (A. Lamkaew, S, H. Lim, HB Lee, LV Kiew, LY Chung, K. Burgess, Chem. Soc. Rev. 2013, 42, 77; S. Zhen, S. Wang, S. Li, W. Luo, M. Gao, LG Ng, CC Goh, A. Qin, Z. Zhao, B. Liu, BZ Tang, Adv. Funt. Mater. 2018, 28, 1706945). Therefore, there is a need to develop dyes that act as teragnostic agents that have a fine balance between both capacities (fluorescence and singlet oxygen photogeneration) and are also stable enough to withstand the intense radiation required by this dual photoactivity.
    [0011]
    [0012] In the present invention there are new fluorescent dyes capable of efficiently marking the lipid droplets (GL) that allow advancing in the study of said organelles by studies based on fluorescence microscopy and modulating their photonic properties to achieve new bio-image based and bio-based theragnostic agents. photodynamic therapy activated by singlet oxygen generation in GL.
    [0013] The development of teragnostic agents using lipid droplets (GL) as therapeutic targets had not been previously tested, despite the enormous biological relevance of these organelles and their recognized involvement in a vast number of physiological processes (MA Welte, Curr. Biol . 2015, 25, R470; CC Scott, S. Vossio. F. Vacca, B. Snijder, J. Larios, O. Schaad, N. Guex, D. Kuznetsov, O. Martin, M. Chambón, G. Turcatti, L. Pelkmans, J. Gruenberg, EMBO reports 2015, 16, 741; H. Wang, MV Airóla, K. Reue, BBA-Mol. Cell Biol. Lipids 2017, 1862, 1131; AR Thiam, M. Beller, J. Cell Sci. 2017 , 130, 315; G. Gao, F.-J. Chen, L. Zhou, L. Su, D. Xu, L. Xu, P. Li, BBA-Mol. Cell Biol. Lipids 2017 , 1862, 1197) as cancer where GLs are involved as a generalized feature in the neoplastic process.
    [0014]
    [0015] The currently marketed GL fluorescent markers, such as Oil Red O, Nile Red, BODIPY 505/515 or BODIPY 493/503, exhibit significant limitations in the efficient marking of such related organelles, fundamentally, with low specificity, low photosensitivity and protocols of complex or tedious settling. Therefore, the current protocols for GL fluorescent marking imply the use of high concentrations of dye (which leads to increased cost, cytotoxicity, extinction of fluorescence by aggregation of the dye or loss of sharpness in the dizziness, among other inconveniences). The dyes described in this invention overcome such limitations.
    [0016]
    [0017] EXPLANATION OF THE INVENTION
    [0018] The present invention describes the structural design, synthetic development and activity of a new group of dyes belonging to the BODIPY family with the ability to efficiently signal GL by fluorescence, overcoming the limitations so far in the fluorescent marking of these organelles, and with additional capacity to produce efficient cell death by photodynamic therapy. These new dyes have potential for the development of new therapeutic agents for clinical use.
    [0019]
    [0020] Structurally, the dyes object of the present invention are characterized by having at least one (alkoxycarbonyl) alkyl moiety covalently bonded to its boradiazacenecentric skeleton, characteristic of all BODIPY dyes. This {alkoxycarbonyl) alkyl moiety is essential to achieve the specific accumulation of the dye in GL and that said specific accumulation in turn potentiates its phototoxic capacity in photodynamic therapy. These dyes are prepared following two general methodologies for the introduction of the (alkoxycarbonyl) alkyl moiety:
    [0021] - Pre-functionalization: direct obtaining from pyrrols and carboxylic acid derivatives based on (alkoxycarbonyl) alkyl
    [0022] - Post-functionalization in functionalized BODIPY dyes through:
    [0023] a) coupling of NEGISHI with organozine reagents in (alkoxycarbonyl) alkyl or,
    [0024] b) Friedel-Crafts acylation with a carboxylic acid derivative based on (alkoxycarbonyl) alkyl, followed by chemoselective reduction of the ketone group formed.
    [0025] These synthetic protocols are general in nature and can provide similar molecular biomaterials with advanced properties to be applied in the development of clinical use.
    [0026]
    [0027] More specifically, in a first aspect, the present invention relates to a set of compounds of general formula (I).
    [0028]
    [0029] Where at least one of R1 sustituyeles to R7 of boradiazaindacénico skeleton is a (alkoxycarbonyl) alkyl and different sustituyeles R1 to R7 of (alkoxycarbonyl) alkyl are each independently selected from hydrogen, C 1 -C 18 substituted or unsubstituted alkenyl C 2 -C 18 substituted or unsubstituted alkenyl, C 2 -C 18 substituted or unsubstituted aryl, substituted or unsubstituted, substituted or unsubstituted heteroaryl, halogen, hydroxyl (OH), alkoxy (oR ), amino (NH 2 ), substituted amino (NHR, NRR ', cyano (CN), carboxy (COOH), acyl (COR), alkoxycarbonyl (COOR), nitro (N02), mercapto (SH) or substituted mercapto (SR ) Two of these substituents can be connected to each other forming a fused cycle to the boradiazacenecentric skeleton.
    [0030]
    [0031] The term "alkyl" refers, in the present invention, to saturated, linear or branched hydrocarbon chains having 1 to 18 carbon atoms, for example, methyl, ethyl, n-propyl, i-propiium, n-butyl , tere-butyl , sec-butyl, n-pentyl, n-hexyl, etc. Preferably, the alkyl group has between 1 and 6 carbon atoms. The alkyl groups may be optionally substituted by one or more substituents such as aryl, heteroaryl, alkenyl, alkynyl, halogen, hydroxy, alkoxy, amino, substituted amino, cyano, carboxyl, acyl, alkoxycarbonyl, nitro, mercapto or mercapto substituted. Two of these substituents can be connected to each other forming a fused cycle to the boradiazacenecentric skeleton.
    [0032]
    [0033] The term "alkenyl" refers, in the present invention, to unsaturated, linear or branched hydrocarbon chains, having 2 to 18 carbon atoms, preferably 2 to 6, and containing one or more double carbon-carbon bonds, for example, vinyl, 1-propenyl, allyl, isoprenyl, 2-buten-1-yl, 1,3-butadien-1-yl, 2-penten-1-yl, etc. These chains may include one or more substituents such as aryl, heteroaryl, alkyl, alkenyl, halogen, hydroxy, alkoxy, amino, substituted amino, carboxyl, cyano, aryl, carbonyl, acyl, alkoxycarbonyl, nitro, mercapto or substituted mercapto. Two of these substituents can be connected together forming an additional cycle.
    [0034]
    [0035] The term "alkynyl" refers to radicals of hydrocarbon chains, linear or branched, from 2 to 18 carbon atoms, preferably from 2 to 6, and containing one or more triple carbon-carbon bonds, and which may optionally contain some double carbon-carbon bond, for example, ethynyl, propynyl, 1-butin -1-ilo, etc. These chains may include one or more substituents such as aryl, heteroaryl, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy, carboxyl, cyano, aryl, ammo, substituted amino, carbonyl, acyl, alkoxycarbonyl, nitro, mercapto or substituted mercapto. Two of these substituents can be connected to each other forming a new cycle.
    [0036] The term "aryl" refers, in the present invention, to a monocyclic, bicyclic, tricyclic or tetracyclic aromatic hydrocarbon moiety, which comprises an aromatic structure formed between 5 and 18 carbon atoms, for example, phenyl, nañyl, indenyl, phenanthryl , anthracil or pyrenyl. Preferably, the aryl group has 5 to 7 carbon atoms and more preferably the aryl group is a phenyl. In turn, this aryl moiety may include one or more substituents such as alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy, carboxyl, cyano, aryl, amino, substituted amino, carbonyl, acyl, alkoxycarbonyl, nitro, mercapto or substituted mercapto . Two of these substituents can be connected to each other forming a new non-aromatic cycle fused with the aromatic moiety.
    [0037]
    [0038] The term "heteroaryl" refers to an aryl, as defined above, which additionally contains at least one non-carbon atom, such as S, N or O, forming part of the aromatic ring.
    [0039]
    [0040] By "halogen" is meant in the present invention a fluorine, chlorine, bromine or iodine atom.
    [0041]
    [0042] By "substituted amino " is meant an amino group of the type NHR or NRR ', where R and R' are selected, independently from aryl, heteroaryl, alkyl, alkenyl or alkynyl. In the case of NRR ', R and R' can be connected to each other forming a new cycle.
    [0043]
    [0044] In a second aspect, the invention describes four preferred structural options for the compound of the invention.
    [0045]
    [0046] The compound of the invention in its preferred option (a) is a compound of general formula (I) where R4 is an aryl group, R2, R3, R5 and R® are independently selected from the group consisting of hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl and heteroaryl; and at least one of the substituents R1 or R7 is a (alkoxycarbonyl) alkyl group and the other is independently selected from hydrogens, halogen, alkyl, alkenyl, aryl and heteroaryl.
    [0047]
    [0048] The compound of the invention in its preferred option (b) is a compound of general formula (I) where R4 is an aryl group, R1, R3, R5, R6 and R7 are independently selected from the group formed hydrogen, halogen, alkyl , alkenyl, alkynyl, aryl and heteroaryl; and R2 is a (alkoxycarbonyl) alkyl group.
    [0049]
    [0050] The compound of the invention in its preferred option (c) comprises a compound of general formula (I) wherein R4 is an alkyl group, R1, R2, R5, R6 and R7 are independently selected from the group formed hydrogen, halogen, alkyl , alkenyl, alkynyl, aryl and heteroaryl; and R3 is a (alkoxycarbonyl) alkyl group.
    [0051]
    [0052] The compound of the invention in its preferred option (d) comprises a compound of general formula (I) wherein R4 is a (alkoxycarbonyl) alkyl group, and R1, R2, R3, R5, R6 and R7 are independently selected from the group formed hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl and heterocycle.
    [0053]
    [0054] In a third aspect, the present invention describes preferred embodiments for the compound (I) of the invention.
    [0055]
    [0056] In a preferred embodiment, the compound of the invention refers to the compound of general formula (I) and structure (a), where R 4 is an optionally substituted aryl group, preferably it is an optionally substituted phenyl and, more preferably, it is a substituted phenyl by C 1 -C 6 alkyl and, even more preferably, it is mesityl (2,4,6 trimethylphenyl).
    [0057]
    [0058] In a more preferred embodiment, the compound of the invention refers to a compound of formula (I) and structure (a), where R4 is mesityl and R2, R3, R5 and R6 are independently selected from the group consisting of hydrogen and CrCe alkyl and, more preferably, from the group consisting of hydrogen and methyl; and, even more preferably, R2, R3, R5 and R6 are hydrogen.
    [0059]
    [0060] In a more preferred embodiment, the compound of the invention refers to a compound of formula (I) and structure (a) where R4 is mesityl, R2, R3, R5 and R6 are hydrogen and where at least one of the substituents R1 or R7 is a group (alkoxycarbonyl) alkyl with a Ci-Ce alkyl group and a CrCe alkoxy group and the other of these substituents which is not (alkoxycarbonyl) alkyl is independently selected from the group consisting of halogen, alkyl, alkenyl, aryl and heteroaryl. More particularly preferably, at least one of the substituents R1 or R7 is a (alkoxycarbonyl) alkyl group with a C3 or Cs alkyl group and an ethoxycarbonyl group; and the other substituent is preferably independently selected from the group consisting of chlorine, methyl, 2-aryletenyl, aryl and thienyl.
    [0061]
    [0062] In a particularly preferred embodiment, the compound of the invention, as defined in structure (a) is selected from the group consisting of compounds 1 to 8.
    [0063]
    [0064]
    [0065]
    [0066]
    [0067] In another preferred embodiment, the compound of the invention refers to a compound of formula (I) and structure (b) where R 4 is an optionally substituted aryl, preferably an optionally substituted phenyl and, more preferably, is phenyl substituted by C 1 alkyl. Ce and, even more preferably R4 is mesityl.
    [0068] In a more preferred embodiment - the compound of the invention refers to a compound of formula (I) and structure (b) where R4 is mesityl, R1, R3, R5, R6 and R7 are independently selected from the group consisting of hydrogen and C r Ce alkyl and, more preferably, R1, R3, R5 and R7 are methyl and R6 is hydrogen.
    [0069]
    [0070] In an even more preferred embodiment, the compound of the invention refers to a compound of formula (I) and structure (b) where R4 is mesityl, R R3, R6, R6 and R7 are methyl, R6 is hydrogen and R2 is a (alkoxycarbonyl) alkyl group with a Ci-C6 alkyl group and a CrCe alkoxy group. Especially preferably, R2 is a (alkoxycarbonyl) alkyl group with a C 5 alkyl group and a methoxycarbonyl group.
    [0071]
    [0072] In a particularly preferred embodiment, the compound of the invention of structure (b) is compound 9.
    [0073]
    [0074]
    [0075]
    [0076] In another preferred embodiment, the compound of the invention refers to a compound of formula (I) and structure (c) wherein R 4 is a Cr-Ce alkyl group; preferably, it is a methyl group.
    [0077]
    [0078] In another more preferred embodiment, the compound of the invention refers to a compound of formula (I) and structure (c) where R4 is methyl and R R2, R5, R6 and R7 are independently selected from the group consisting of hydrogen , halogen or Ci-Cs alkyl; preferentially R1, R5, R7 are alkyl R6y Cr C 2 and R2 is chloro.
    [0079]
    [0080] In a further preferred embodiment, the compound of the invention relates to a compound of formula (I) structure (c) wherein R4 is methyl and R1, R2, R5, R6y R7 are C 1 -C 2 alkyl, R2 is chloro and R3 is a (alkoxycarbonyl) alkyl group with a C5 alkyl group and an ethoxycarbonyl group.
    [0081]
    [0082] In a particularly preferred embodiment, the compound of the invention of structure (c) is compound 10.
    [0083]
    [0084]
    [0085] In another preferred embodiment, the compound of the invention refers to a compound of formula (I) and structure (d) wherein R 4 is a (alkoxycarbonyl) alkyl group with a Ci-C 6 alkyl group and a Ci-C6 alkoxy group ; preferably R 4 is a (alkoxycarbonyl) alkyl group with a C 4 alkyl group and a methoxycarbonyl group.
    [0086]
    [0087] In another more preferred embodiment, the compound of the invention refers to a compound of formula (I) and structure (d) wherein R 4 is a (alkoxycarbonyl) alkyl group with a C 4 alkyl group and a methoxycarbonyl group and R2, R 3 , R5 and R 6 are independently selected from the group consisting of C 1 -C 6 alkyl and, more preferably, from the group consisting of C 1 -C 2 alkyl.
    [0088]
    [0089] In an even more preferred embodiment, the compound of the invention refers to a compound of formula (I) and structure (d) wherein R4 is a (alkoxycarbonyl) alkyl group with a C4 alkyl group and a methoxycarbonyl group and R2, R3, R5 and R6 are alkyl C rC 2 .and wherein R1 and R7 are independently selected from the group consisting of methyl and 2- (3,4-dimethoxyphenyl) ethenyl.
    [0090]
    [0091] In a particularly preferred embodiment, the compound of the invention of structure (d) is selected from compounds 11 and 12.
    [0092]
    [0093]
    [0094]
    [0095] A fourth aspect of the present invention relates to a process for obtaining the compound of formula (I), hereafter "process of the invention ”, which includes two methodologies:
    [0096] - pre-functionalization by direct synthesis of (I) from conveniently functionalized pyrrols, and
    [0097] - post-functionalization, either in another compound of formula (I), or in a compound of formula (II).
    [0098]
    [0099]
    [0100]
    [0101] where, at least, one of the substituents R1 ', R2', R3 'or R7' is hydrogen, halogen or pseudohalogen (trifluoromethanesulfonate, acetate, etc.) and the remaining substituents are independently selected from the group consisting in hydrogen, halogen, alkyl, aryl, alkenyl, alkynyl and heteroaryl.
    [0102]
    [0103] A preferred embodiment of the process of the invention, directed to the preparation of the compound (I) of the invention as defined in (a) and (c), comprises the post-functionalization of a compound of formula (II) wherein at least one of the substituents R1 ', R3 or R7 is halogen or pseudohalogen, preferably halogen, and the remaining substituents are the same as those of the final compound of formula (I) in its option (a) or (c) to be synthesized.
    [0104]
    [0105] A more preferred embodiment of the process of the invention, directed to the preparation of the compound (I) of the invention as defined in (a) and (c), comprises the post-functionalization of a compound of formula (II), where at least one of the substituents R1, R3 'or R7' is halogen, and the remaining substituents are the same as those of the final compound of formula (!) in its option (a) or (c), by coupling, activated by a palladium catalyst, with an organozinic compound based on the R moiety and preferably selected from the group consisting of R2Zn and R2ZnX, where R is a (alkoxycarbonyl) alkyl group with a C 1 -C 6 alkyl group and a Ci alkoxy group Ce, as defined for the compound (I) of the invention in its options (a) or (c), and X a halogen. Preferably, the organozine reagent is 4-ethoxy-4-oxobutylzinc bromide or 6-ethoxy-6-oxohexylzinc bromide.
    [0106]
    [0107] An even more preferred embodiment of the process of the invention, directed to the Preparation of the compound (I) of the invention as defined in (a) and (c), comprises the post-functionalization of a compound of formula (II), wherein at least one of the substituents R1 ', R3' or R7 is halogen and the remaining substituents are the same as those of the final compound of formula (I) in its option (a) or (c), by its coupling reaction with 4-ethoxy-4-oxobutylzinc bromide or 6- bromide ethoxy-6-oxohexylzinc, in the presence of a catalyst based on palladium (O) or palladium (ll), supported or encapsulated in a solid matrix, and preferably selected from the group consisting of palladium (O), tetrakis (triphenylphosphine) palladium (0), tris (dibenzyldenacetone) dipaladium (0), bis (d! Benzylidenacetone) palladium (0), palladium acetate (ll), dichlorobis (triphenylphosphine) palladium (ll), bis (acetylacetonate) palladium (ll), dichlorobis (tricyclohexylphosphine) palladium (ll), chloro [(tricyclohexylphosphine) -2- (2'-amynobiphenyl)] palladium (ll). Preferably, the palladium catalyst is selected from the group consisting of dic! Orobis (triphenylphosphine) palladium (ll) (PdCI2 (PPh 3 ) 2 ) and chloro [(tricyclohexylphosphine) -2- (2-aminobiphenyl)] palladium (H ) ((PCy3) PdG2).
    [0108]
    [0109] In an even more preferred embodiment of the process of the invention, directed to the preparation of the compound (I) of the invention as defined in (a) and (c), it comprises the post-functionalization of a compound of formula (II ), where at least one of the substituents R1 ', R3' or R7 is halogen, and the remaining substituents are equal to those of the final compound of formula (I) in their option (a) or (c), by their reaction of coupling with 4-ethoxy-4-oxobutyl zinc bromide or 6-ethoxy-6-oxohexyl zinc bromide, catalyzed by PdCI2 (PPh 3) 2 or (PCy3) PdG2, and preferably using a compatible organic solvent selected from the group of solvents organic compatible with said coupling reaction. Preferably, the organic solvent is toluene.
    [0110]
    [0111] In a particularly preferred embodiment of the process of the invention, directed to the preparation of the compound (I) of the invention as defined in (a) and (c), it comprises the post-functionalization of a compound of formula (II) , where at least one of the substituents R1 ', R3' or R7 is halogen, and the remaining substituents are equal to those of the final compound of formula (I) in their option (a) or (c), by their reaction with bromide of 4-ethoxy-4-oxobutylzinc or 6-ethoxy-6-oxohexylzinc bromide, catalyzed by PdCI2 (PPh3) 2 or (PCy3) PdG2, using toluene as organic solvent, and carried out at a temperature between 0 and 140 ° C. Preferably, the reaction takes place at a temperature between 10 and 30 ° C, and more preferably in an inert atmosphere (eg argon).
    [0112] In another preferred embodiment of the process of the invention, directed to the preparation of the compound (I) of the invention as defined in (b), it first comprises the post-functionalization of a compound of formula (II), wherein R2 is hydrogen, and the remaining substituents are the same as those of the final compound of formula (I) in its option (b) which is to be synthesized.
    [0113]
    [0114] A more preferred embodiment of the process of the invention, directed to the preparation of the compound (I) of the invention as defined in (b), first comprises the post-functionalization of a compound of formula (II), wherein R2 is hydrogen, and the remaining substituents are the same as those of the final compound of formula (I) in option (b) which is to be synthesized, by means of its condensation reaction with a carboxylic acid halide or anhydride based on RCO acyl radical , where R is a (alkoxycarbonyl) alkyl group with a Ci-C6 alkoxy group and a C 1 -C 5 alkyl group, and more preferably is 6-methoxy-6-oxohexanoyl chloride.
    [0115]
    [0116] An even more preferred embodiment of the process of the invention, directed to the preparation of the compound (I) of the invention as defined in (b), first comprises the post-functionalization of a compound of formula (II), where R2 'is hydrogen, and the remaining substituents are the same as those of the final compound of formula (I) in its option (b) which is to be synthesized, by its condensation reaction with activated 6-methoxy-6-oxohexanoyl chloride by Lewis acid, preferably selected from the group consisting of aluminum trichloride, iron tribromide, boron trifiuoride, boron trifuoride etherate, titanium tetrachloride and tin tetrachloride. Preferably, the Lewis acid is boron etherate trifiuoride.
    [0117]
    [0118] An even more preferred embodiment of the process of the invention, directed to the preparation of the compound (I) of the invention as defined in (b), first comprises the post-functionalization of a compound of formula (II), where R2 'is hydrogen, and the remaining substituents are the same as those of the final compound of formula (I) in option (b), which is intended to be synthesized, by reaction with trifiuoride activated 6-methoxy-6-oxohexanoyl chloride boron etherate and in the presence of an organic solvent compatible with said condensation reaction. Preferably, the organic solvent is dichloromethane.
    [0119]
    [0120] An especially preferred embodiment of the process of the invention, directed to the preparation of the compound (I) of the invention as defined in (b), first comprises the post-functionalization of a compound of formula (II), wherein R2 is hydrogen, and the remaining substituents are equal to those of the final compound of formula (I) in its option (b) which it is desired to synthesize, by means of its condensation reaction with 6-methoxy-6-oxohexanoyl chloride activated by boron trifluoride etherate, using dichloromethane as solvent at a temperature between 0 and 140 ° C . Preferably, the reaction takes place at a temperature between 0 and 30 ° C, and more preferably in an inert atmosphere (eg argon).
    [0121]
    [0122] The process of the invention, directed to the preparation of the compound (I) of the invention as defined in (b), comprises the subsequent selective reduction of a compound of formula (II) where R2 'is a group [(alkoxycarbonyl) ) alkyl] carbonyl with a Ci-Cs alkoxy group and a C 1 -C 5 alkyl group, as defined for the compound (I) of the invention at its option (b), and the remaining substituents are the same as of the final compound of formula (I) in its option (b) which is to be synthesized, by its reaction with a chemoselective reducing agent preferably selected from the group consisting of sodium borohydride, zinc (0) amalgamated with mercury in acidic medium, triethylsilane , sodium cyanoborohydride, nickel acetate (II) and copper (0) in acidic medium. Preferably, the reducing agent is sodium cyanoborohydride, and more preferably the reduction reaction is activated with a Lewis acid compatible with said reduction reaction. Preferably, the Lewis acid is zinc iodide.
    [0123]
    [0124] In a preferred embodiment of the process of the invention, directed to the preparation of the compound (I) of the invention as defined in (b), it comprises the subsequent selective reduction of a compound of formula (II) where R2 is a group [(alkoxycarbonll) alkyl] carbonyl with a Ci-Ce alkoxy group and a C 1 -C 5 alkyl group, and the remaining substituents are the same as those of the final compound of formula (I) in its option (b) which is intended synthesize, by means of its chemoselective reduction reaction with sodium cyanoborohydride in the presence of zinc iodide, preferably carried out in an organic solvent compatible with said reduction reaction. Preferably, the organic solvent is 1,2-dichloroethane.
    [0125]
    [0126] In a more preferred embodiment of the process of the invention, directed to the preparation of the compound (I) of the invention as defined in (b), it comprises the subsequent selective reduction of a compound of formula (II) where R2 is a [(a! coxicarbonll) alkyl] carbonyl group with a C 1 -C 6 alkoxy group and a C 1-C 5 alkyl group, and the remaining substituents are the same as those of the final compound of formula (I) at its option (b ) that it is desired to synthesize, by means of its chemoselective reduction reaction with sodium cyanoborohydride in the presence of zinc iodide, in 1,2-dichloroethane as solvent, and preferably at a temperature between 0 and 140 ° C. Preferably, the reaction takes place at a temperature of 25 ° C, and more preferably in an inert atmosphere (eg argon).
    [0127]
    [0128] In another preferred embodiment of the process of the invention, directed to the preparation of the compound (I) of the invention as defined in (d), it comprises pre-functionalization, by the synthesis of a compound of formula (I) from pyrroles of general formula (III),
    [0129]
    [0130]
    [0131]
    [0132] Where U is independently selected from the group formed by R1 and R7, as defined for the compound (I) of the invention in its option (d), V is independently selected from the group formed by R2 and R6, as defined for the compound (I) of the invention in its option (d) and, W is independently selected from the group consisting of R3 and R5, as defined for the compound (I) of the invention in its option (d) , through the prior obtaining of a dipyrromethene derivative, conveniently functionalized, by the condensation of two pyroles (III), the same or different, with a carboxylic acid derivative based on organic radical R4, where R4 is a (alkoxycarbonyl) alkyl group with a C 1 -C 6 alkyl group and a CrC6 alkoxy group, as defined for the compound (I) of the invention at its option (d), said carboxylic acid derivative being able to promote acylation processes in compounds aromatic by substitution reaction aromatic electrophile. Preferably, the two equivalents of pyrrole (III) involved in the condensation are the same, and even more preferably pyrrole (III) is 3-ethyl-2,4-dimethylpyrrole. On the other hand, preferably the carboxylic acid derivative is a carboxylic acid halide, R4COX, where X is a halogen atom, and more preferably it is 6-methoxy-6-oxohexanoyl chloride.
    [0133]
    [0134] In a more preferred embodiment of the process of the invention, directed to the preparation of the compound (I) of the invention as defined in (d), it comprises the previous condensation of 3-ethyl-2,4-dimethylpyrrole with 6-methoxy-6-oxohexanoyl in an organic solvent compatible with the condensation reaction. Preferably, the organic solvent is dichloromethane.
    [0135] In an even more preferred embodiment of the process of the Invention, directed to the preparation of the compound (I) of the invention as defined in (d), it comprises the previous condensation of 3-ethyl-2,4-dimethylpyrrole with chloride of 6-methoxy-6-oxohexane in an organic solvent compatible with the condensation reaction. Preferably, the organic solvent is dichloromethane, and preferably at a temperature between 0 and 140 ° C. Preferably, the reaction takes place at a temperature of 50 ° C, and more preferably in an inert atmosphere (eg argon).
    [0136] The process of the invention, directed to the preparation of the compound (I) of the invention as defined in (d), comprises the subsequent complexation of the dipyrrometic derivative previously obtained with boron etherate trifluoride, preferably in the presence of a low base nucleophilic Preferably, this base is a tertiary amine selected from the group consisting of trimethylamine, triethylamine, disopropylmethylamine and ethyl (disopropyl) amine, and more preferably, the amine is triethylamine.
    [0137]
    [0138] In a more preferred embodiment of the process of the invention, directed to the preparation of the compound (I) of the invention as defined in (d), it comprises the subsequent complexation of the dipyrrometic derivative with boron trifluoride etherate in the presence of triethylamine, and in an organic solvent, compatible with the complexation reaction, at a temperature between 0 and 30 ° C. Preferably, the reaction is carried out in dichloromethane as a solvent and at room temperature, and more preferably in an inert atmosphere (eg argon).
    [0139]
    [0140] In another preferred embodiment of the process of the invention, directed to the preparation of the compound (I) of the invention as defined in (d), where at least one of the residues R1 and R7 is a 2-arylethenyl group, it comprises the post-functionalization of a compound of formula (I), where at least one of the residues R1 and R7 is a methyl group, by means of its condensation reaction with an aromatic aldehyde, preferably chosen from the group consisting of benzaldehyde or substituted benzaldehyde , preferably the aldehyde is 3,4-dimethoxybenzaldehyde. Preferably, this condensation is catalyzed by an acid, preferably selected from the group consisting of formic acid, acetic acid and propanoic acid; or by a base, preferably selected from the group consisting of triethylamine, ethyl (diisopropyl) amine, piperidine, pyrrolidine and morpholine; or by a mixture of acid and base. Preferably, the catalyst is a mixture formed by piperidine and acetic acid.
    [0141] In another more preferred embodiment of the process of the invention, directed to the preparation of the compound (I) of the invention as defined in (d), where at least one of the residues R1 and R 'is a 2-aryletenyl group , comprises the postfunctionalization of a compound of formula (I), where at least one of the residues R1 and R7 is a methyl group, by means of its condensation reaction with 3,4-dimethoxybenzaldehyde catalyzed by piperidine / acetic acid mixture, and up carried out in an organic solvent compatible with said condensation reaction at a temperature between 20 and 140 ° C, Preferably, the reaction is carried out in / V, A / -dimethylformamide as solvent, more preferably at 120 ° C, even more preferably under microwave irradiation, and especially preferably in an inert atmosphere (eg argon).
    [0142]
    [0143] Finally, after obtaining it, the compound (I) of the invention can be purified by the usual techniques of purification of organic molecular compounds. Preferably, the purification technique is column elution chromatography, and more preferably the adsorbent in this chromatography is silica gel.
    [0144]
    [0145] A fifth aspect of the present invention relates to the use of the compound (I) of the invention as a fluorescent biomarker and / or as a photocitotoxic agent, preferably for cellular mapping, and even more preferably for GL specific cellular mapping, preferably of living cells. , and even more preferably in living mammalian cells. Especially preferably, the cells to be labeled or treated by photodynamic therapy are human, preferably of a cancerous nature.
    [0146]
    [0147] Thus, a sixth aspect of the present invention comprises the use of the compound (I) of the invention for the development and manufacture of markers for the diagnosis of diseases related to GL abnormalities, such as hepatitis C virus proliferation, diabetes type-2, obesity or cancer.
    [0148]
    [0149] In a preferred use, the compounds of formula (I) can be used directly, or covalently, or supramolecularly bound, to a chemical system that improves their biocompatibility and biodistribution; preferably this system is a biomolecule, and more preferably selected from the group consisting of amino acid, peptide, protein, lipid, carbohydrate, nucleic acid and toxin. In preferred use of the compound (I) of the invention, it is used directly and is preferably photoactivated by irradiation with visible light, preferably from the green-red region of the electromagnetic spectrum, and more preferably the light is light To be.
    [0150]
    [0151] A final aspect of the present invention thus comprises a new strategy to enhance the activity in teragnostic agents based on photodynamic therapy and in diagnosis by fluorescent bioimage, consisting of the specific accumulation of the teragnostic agent in GL.
    [0152]
    [0153] Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will be partially detached. of the description and in part of the practice of the invention The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    [0154]
    [0155] BRIEF DESCRIPTION OF THE DRAWINGS
    [0156] Figure 1 shows the labeling of HeLa cells with compounds 2, 3 (0.1 j M and 24 h incubation) and 11 (5 pM and 24 h incubation) and with the commercial marker lipid droplets BODIPY497 / 503 (40 pM and 10 min incubation). The signal is shown under green and blue excitation. Scale bar: 10 pm.
    [0157]
    [0158] Figure 2 shows the percentage of viability of HeLa cells after treatments with compounds 2, 3 and 11 at different concentrations in dark conditions and after 10.3 J / cm2 of green irradiation. The average viability value of six experiments is represented ± standard deviation.
    [0159]
    [0160] PREFERRED EMBODIMENT OF THE INVENTION
    [0161]
    [0162] The invention is illustrated below by examples of obtaining various dyes of the invention, as well as their evaluation as fluorescent GL markers in living cells of tumor origin, and as photocitotoxic agents for the efficient destruction of the cells so labeled.
    [0163]
    [0164] SYNTHESIS OF FORMULA COMPOUNDS (I)
    [0165]
    [0166] All reagents and starting compounds have been obtained commercially, unless otherwise indicated, and have been used without further purifications. Common solvents have been dried and distilled by standard procedures. The products have been purified by flash column chromatography, using gel Merck 60 silica (230-400 mesh), the eluents used are specified in each case and the proportions indicate the volume: volume ratio. All isolated products have been identified according to their spectroscopic and / or analytical data. The 1 H and 13 C NMR spectra have been recorded at 20 ° C, and the residual solvent peak has been used as an internal standard. The multiplicities of chemical shifts are indicated as s = singlet, d = doublet, t = triplet, c = quadruplet, q = quintuplet and m = multiplet. FTIR spectra have been obtained from pure samples using the ATR technique. High Resolution Mass Spectrometry (HRMS) has been performed using the EL technique
    [0167]
    [0168] Example 1. Synthesis of compounds 1, 2 and 3
    [0169]
    [0170] The preparation of compounds 1,2 and 3 is carried out through the synthetic routes shown in scheme 1, illustrating the procedure based on the post-functionalization methodology described in this invention.
    [0171]
    [0172]
    [0173]
    [0174]
    [0175] Thus, the introduction of the (ethoxycarbonyl) alkyl group at position 3 of the BODIPY is carried out by post-functionalization in a starting chloroBODIPY by Negishi coupling reactions catalyzed by palladium. For this, the methodology described by our research group is used for this type of coupling in haloBODIPY (E. Palao, G. Duran-Sampedro, S. de la Moya, M. Madrid, C. García-López, AR Agarrabeitia, B. Verbelen, W. Dehaen, N. Boens, MJ Ortiz, J. Org. Chem.
    [0176] 2016, 81, 3700). In addition, to achieve spectral displacement towards the red zone of the visible spectrum (dye 3), a coupling reaction has also been used catalyzed by palladium, specifically a Suzuki reaction.
    [0177]
    [0178] General procedure for the Negishi reaction: In a Schlenk tube, equipped with magnetic stirring and subjected to three consecutive cycles of vacuum and argon current, the corresponding haloBODIPY (1 mol equiv) in anhydrous toluene is introduced under argon atmosphere. PdCI2 (PPh3) 2 (10% mol equiv) is added. The reaction mixture is stirred for 5 min at room temperature and then the corresponding organo-zinc (2-5 mol equiv) is added, and the period of time specified in each case is allowed to stir. After this time, the reaction crude is filtered to remove the catalyst, and the product obtained is purified by column chromatography on silica gel, using as eluent mixtures of different polarity specified in each case.
    [0179]
    [0180] 3-Chloro-5- (6-ethoxy-4-oxohexyl) -4,4-difluoro-8-mesityl-4-bora-3a, 4a-diaza-syndacene (1): Following the general Negishi reaction procedure, 3,5-dichloro-8-mes¡tilBODIPY (T. Sakida, S. Yamaguchi, H. Shinokudo, Angew. Chem. Int. Ed. 2011, 50, 2280) (50 mg, 0.132 mmol), PdCI2 are reacted (PPh 3 ) 2 (9 mg, 0.013 mmol) and 0.5 M BrZn (CH2) 5COOEt in THF (1.3 mL, 0.660 mmol) in anhydrous toluene (2 mL) for 90 min. Flash chromatography on silica gel (hexane / AcOEt, 95: 5) gives the unchanged starting compound (15 mg, 30%), and 1 (40 mg, 62%) as a brown solid. 1H NMR (700 MHz, CDCI3) <56.93 (s, 2H, 2CH), 6.62 (d, J = 4.2 Hz, 1H, CH), 6.44 (d, J = 4.2 Hz, 1H, CH), 6.36 (d, J = 4.2 Hz, 1H, CH), 6.26 (d, J - 4.2 Hz, 1H, CH), 4.12 (c, J = 7.0 Hz, 2H, CH20), 3.07 (t, J = 7.7 Hz, 2H, CH2 ), 2.34 (s, 3H, CH3), 2.32 (t, J = 7.7 Hz, 2H, CH2), 2.09 (s, 6H, 2CH3), 1.79 (q, J = 7.7 Hz, 2H, CH2), 1.70 ( q, J = 7.7 Hz, 2H, CH2), 1.49 (q, J = 7.7 Hz, 2H, CH2), 1.25 (t, J = 7.0 Hz, 3H, CH3) ppm. 13C NMR (176 MHz, CDCI3) <5173.7 (COO), 166.3, 142.9, 140.2, 138.8, 136.6, 135.6, 133.0, 131.5 (CH), 129.1, 128.2 (CH), 127.4 (CH), 120.0 (CH), 116.8 (CH), 60.3 (CH20), 34.2 (CH2), 29.1 (CH2), 29.0 (CH2), 28.1 (CH2), 24.8 (CH2), 21.1 (CH3), 20.0 (CH3), 14.3 (CH3) ppm . FTIR v2928, 2862, 1730, 1562, 1408, 1329, 1267, 1102, 1045, 970 cnr1. HRMS-EI m / z 486.2051 (486.2057 calculated for C26H3oBCIF2N202).
    [0181]
    [0182] 3- (6-Ethoxy-4-oxohexyl) -4,4-difluoro-8-mesyl-5-methyl-4-bora-3a, 4a-diaza-s-indacene (2): Following the general procedure of Negishi reaction, 1 (52 mg, 0.106 mmol), PdCI2 (PPh3) 2 (8 mg, 0.011 mmol) and 1.2 M ZnMe2 (0.22 mL, 0.265 mmol) in anhydrous toluene (2 mL) are reacted for 60 min. Flash chromatography on silica gel (hexane / AcOEt, 95: 5) yields 2 (37 mg, 75%) as a brown solid. 1H NMR (300 MHz, CDCI3) S 6.92 (s, 2H, 2CH), 6.48 (d, J = 4.2 Hz, 1H, CH), 6.46 (d, J = 4.2 Hz, 1H, CH), 6.24 (d, J ~ 4.2 Hz, 1H, CH), 6.19 (d, J = 4.2 Hz, 1H, CH), 4.12 (c, J = 7.2 Hz, 2H, CH20), 3.03 (t, J = 7.8 Hz, 2H, CH2), 2.63 (s, 3H, CH3), 2.34 (s, 3H, CH3), 2.32 (t, J = 7.5 Hz, 2H, CH2), 2.09 (s, 6H, 2CH3), 1.83-1.66 (m, 4H, 2CH2), 1.54-1.44 (m, 2H, CH2), 1.25 (t, J = 7.2 Hz, 3H, CH3) ppm. 13C NMR (75 MHz, CDCI3) 173.7 (COO), 162.0, 157.5, 142.4, 138.3, 136.6, 134.7, 134.5, 130.1, 129.0 (CH), 128.9 (CH), 128.0 (CH), 119.3 (CH), 117.6 (CH), 60.2 (CH20), 34.3 (CH2), 29.1 (CH2), 28.7 (CH2), 28.2 (CH2), 24.8 (CH2), 21.1 (CH3), 20.0 (CH3), 14.9 (CH3), 14.3 (CH3) ppm. FTIR v 2926, 2860, 1733, 1560, 1272, 1142, 1125, 1089, 999 crrr1. HRMS-EI m / z 466.2597 (466.2603 calculated for C27H33BF2N202).
    [0183]
    [0184] 3- (6-Etox¡-4-oxohexil) -4,4-difluoro-8-mesít¡l-5- (t¡en-2-il) -4-bora-3a, 4a-diaza-s-¡ ndacene (3): To a solution of BODIPY 1 (21 mg, 0.044 mmol) in anhydrous toluene (3 mL), 2-thienylboronic acid (17 mg, 0.132 mmol), Na2C03 dissolved in H20 (3 mg) is added under argon 1 mL, 1 M) and (PCy3) PdG2 (1 mg, 0.002 mmol), and the reaction mixture is maintained at 100 ° C for 4.5 hours. After this time, the reaction crude is filtered, extracted with CH2CI2 and washed with H20. The organic phase is dried over MgSO4, the desiccant is filtered off and the solvent under reduced pressure. Flash chromatography on silica gel (hexane / CH2CI2, 8: 2) yields 3 (12 mg, 50%) as a purple solid, and unchanged starting compound 1 (5 mg, 23%). 'H NMR (700 MHz, CDCI3) 8.14 (d, J = 3.5 Hz, 1H, CH), 7.45 (d, J = 4.9 Hz, 1H, CH), 7.18 (t, J = 4.2 Hz, 1H, CH ), 6.94 (s, 2H, 2CH), 6.67 (d, J = 4.2 Hz, 1H, CH), 6.53 (d, J = 4.2 Hz, 1H, CH), 6.51 (d, J = 4.9 Hz, 1H, CH), 6.31 (d, J = 4.2 Hz, 1H, CH), 4.12 (c, J = 7.0 Hz, 2H, CH20), 3.09 (t, J = 7.7 Hz, 2H, CH2), 2.36 (s, 3H , CH3), 2.35 (t, J = 7.0 Hz, 2H, CH2), 2.12 (s, 6H, 2CH3), 1.79 (q, J = 7.7 Hz, 2H, 2CH2), 1.71 (q, J = 7.7 Hz, 2H, CH2), 1.50 (q, J = 7.7 Hz, 2H, CH2), 1.25 (t, J = 7.0 Hz, 3H, CH3) ppm. 13C NMR (176 MHz, CDCI3) 173.8 (COO), 163.2, 149.3, 141.8, 138.4, 136.8, 136.3, 134.8, 134.4, 130.5 (CH), 130.2, 129.2 (CH), 128.9 (CH), 128.6 (CH ), 128.5 (CH), 128.1 (CH), 119.6 (CH), 118.7 (CH), 60.2 (CH20), 34.2 (CH2), 29.1 (CH2), 28.8 (CH2), 28.1 (CH2), 24.8 (CH2 ), 21.1 (CH3), 20.0 (CH3), 14.3 (CH3) ppm. FTIR v2925, 2855, 1734, 1570, 1465, 1372, 1269, 1125, 1039 cnr1, HRMS-EI m / z 534.2320 (534.2324 calculated for C30H33BF2N2O2S).
    [0185]
    [0186] Example 3. Synthesis of compound 11
    [0187]
    [0188] The preparation of compound 11 is carried out through the synthetic route shown in scheme 2, illustrating the procedure based on the methodology of the pre-functionalization described in this invention.
    [0189]
    [0190]
    [0191] 2,6-Diethyl-4,4-difluoro-1,3,5,7-tetramethyl-8- (5-methoxy-5 ~ oxopentyl) -4-bora-3a, 4adiaza-s-indacene ( 11): To a solution of 6-methoxy-6-oxohexanollo * chloride (175 mg, 0.97 mmol) in CH 2 CI 2 (15 ml_) is added, under argon, 3-ethyl-2,4-dimethylpyrrole (0.33 ml_, 2.44 mmol), and the reaction mixture is refluxed for 3 h. After this time, the reaction is allowed to reach room temperature and Et 3 N (0.7 mL, 4.88 mmol) is added, after 15 min, BF3Et20 (0.6 mL, 4.88 mmol) is added, maintaining stirring for 2 h. Then, the solvent is removed under reduced pressure, the reaction crude is dissolved in AcOEt and washed with an aqueous solution of HCI (10%) and H20. The organic phase is dried over Na2SC> 4, the desiccant is filtered off and the solvent under reduced pressure. Flash chromatography on silica gel (hexane / CHCl 3 , 95: 5) yields 11 (120 mg, 30%) as an orange solid. 1H NMR (300 MHz) 3.67 (s, 3H, CH30), 3.03-2.98 (m, 2H, CH2), 2.49 (s, 6H, 2CH3), 2.40 (t, J = 7.2 Hz, 2H, CH2), 2.40 (c, J = 7.5 Hz, 4H, 2CH2), 2.33 (s, 6H, 2CH3), 1.88-1.78 (m, 2H, CH2), 1.72-1.63 (m, 2H, CH2), 1.04 (t, J = 7.5 Hz, 6H, 2CH3) ppm. 13C-NMR (75 MHz, CDCI 3 ) 173.7 (COO), 152.2, 144.0, 135.6, 132.7, 130.9, 51.7 (CH30), 33.6 (CH2), 31.2 (CH2), 28.2 (CH2), 25.3 (CH2) , 17.2 (CH2), 14.9 (CHs), 13.3 (CH3), 12.4 (CH3) ppm. FTIR i / 2925, 2855, 1741, 1548, 1472, 1331, 1264, 1198, 1071, 982 CN1. HRMS-EI m / z 418.3356 (418.3358 calculated for C23H33BF2N202).
    [0192] * Synthesis of 6-methox-6-oxohexanoyl chloride: To a solution of 6-methox-6-oxohexanoic acid (0.012 mL, 0.11 mmol) in CH2CI2 (2 mL) is added, under an argon atmosphere, thionyl (0.035 mL, 0.44 mmol). The reaction mixture is refluxed for 20 min and then the excess thionyl chloride and solvent are removed under reduced pressure. The compound obtained is used without further purification.
    [0193]
    [0194] EVALUATION OF COMPOUNDS 1, 2, 3 and 11
    [0195]
    [0196] Example 3. Evaluation as efficient fluorescent markers of GL (compounds 2, 3 and 11)
    [0197]
    [0198] For evaluation, HeLa cells (from a human cervix adenocarcinoma) have been used. The cells have been grown as monolayer cultures in DMEM (Dulbecco's modified Eagle's medium) culture medium supplemented with 10% (v / v) fetal bovine serum, 50 U / mL penicillin and 50 pg / mL streptomycin. All products have been sterilized by filters with a pore size of 0.22 pm. The cells were kept in an incubator in an atmosphere with 5% CO 2 , at a temperature of 37 ° C and with a humidity of 95%. To perform the experiments, the cells were grown on glass coverslips in 24-well plates. When the cells reached a confluence level of approximately 50%, the culture medium was replaced by dilutions of compounds 2, 3 and 11 (0.1 pM for compounds 2 and 3, and 5 pM for compound 11) in medium of culture, and incubated for 24 h in the incubator. After this time, three washes were carried out with culture medium and the coverslips, still wet, were placed on slides for observation. For the observation of the cells, an Olympus BX61 epifluorescence microscope equipped with an Olympus DP50 digital camera was used.
    [0199] Results: All the compounds tested were efficiently internalized by Hela cells presenting excellent properties as fluorescent markers. In all cases, the intensity and definition of the marking was very high, as was the maintenance time of the signal under the intense excitation of the microscope. In the case of compounds 2 and 11, fluorescent signal could be detected with the green and blue excitation filters, while the marking with compound 3 was only visible under green excitation. The subcellular localization pattern of compounds 2, 3 and 11 was very similar, in the form of granules of very regular size and shape, distributed throughout the cytoplasm and absent in the cell nucleus. This morphological pattern corresponds to that described for GL, which was corroborated by staining HeLa cells with the GL marker BODIPY497 / 503 (40 pM and 10 min incubation), obtaining a distribution pattern totally similar to that obtained with compounds 2, 3 and 11. Co-location experiments could not be performed due to the superposition of the absorption / emission spectra of the commercial label with those of compounds 2, 3 and 11.
    [0200]
    [0201] Figure 1 shows the location images by using compounds 2, 3 and 11, as well as those obtained by using the commercial marker BODIPY497 / 503.
    [0202] Example 4. Evaluation as efficient photocitotoxic agents by selective accumulation in GL {compounds 2, 3 and 11).
    [0203]
    [0204] HeLa cells were used for the evaluation. The cells were seeded in 24-well plates and incubated with different concentrations of the selected compounds at different concentrations {1,2.5, 5, 7.5 and 10 pM), diluted in culture medium. After 24 h of incubation, the cells were washed three times with culture medium and irradiated with a Par 64 Short LED lamp (Showtec, Burgebrach, The Netherlands) with a light dose of 10.3 J / cm2 and a wavelength of 518 ± 10 nm After irradiation, the cells were kept in the incubator for 24 h, and cell survival was evaluated by the MTT cytotoxicity assay. For this, the cells were incubated for 3 h with a 50 pg / mL solution of the MTT compound. After this time, the culture medium was removed and dimethylsulfoxide was added to solubilize the formazan formed by the reduction of MTT. The absorbance of each well at 542 nm was measured and the percentage of cell survival in each well was obtained with respect to the viability of the control cells (considered 100%). The results are expressed as the mean cell viability of the wells of each condition ± their standard deviation. In parallel, the same experiments were performed in the absence of irradiation to assess the toxicity of the compounds in the dark.
    [0205]
    [0206] Results: All the compounds tested showed excellent phototoxic properties, leading to induce, in all cases, a decrease in viability greater than 90% in any of the selected experimental conditions. In addition, it was found that the toxicity of all compounds in the absence of irradiation (what is known in photodynamic therapy as dark toxicity) was practically nil. This is an indispensable condition for any agent for photodynamic therapy, since it avoids side effects by limiting the toxicity of the compound to the irradiation zone.
    [0207] The viability results of HeLa cells after incubation with each of the selected compounds are shown in Figure 2, both in darkness (gray bars) and in the presence of 10.3 J / cm2 of green irradiation (striped bars). These results demonstrate that the developed compounds, in the presence of an adequate dose of light, generate a high percentage of death in tumor cells of human origin, while maintaining a minimal or zero toxicity in dark conditions.
    权利要求:
    Claims (36)
    [1]
    1. Compound of general formula (I)

    [2]
    2. A compound according to claim 1, wherein R1 or R7 is not a (alkoxycarbonyl) alkyl group, is selected from the group consisting of chlorine, methyl, 2-aryletenyl, aryl and thienyl.
    [3]
    3. Compound according to any of claims 1 and 2, wherein R2, R3, R5 and R6 are hydrogen.
    [4]
    4. A compound according to any one of claims 1 to 3, wherein R4 is an optionally substituted aryl group.
    [5]
    5. A compound according to claim 4, wherein R4 is 2,4,6-trimethylphenium (mesitiium).
    [6]
    6. Compound according to claim 1, wherein the compound is selected from the compounds of formula 1 to 8.

    [7]
    7. Compound of general formula (I) wherein the substituent R2 of the boradiazaindacentric skeleton of (I) is an alkyl (alkoxycarbonyl) alkyl group comprising a Cs alkyl group and a methoxycarbonyl group and the remaining substituents are each independently selected from from hydrogen, Ci-Cie substituted or unsubstituted alkenyl, C 2 -C 18 substituted or unsubstituted, C2-cie substituted or unsubstituted, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, halogen, hydroxyl (OH) , alkoxy (OR), amino (NH2), substituted amino (NHR, NRR ', cyano (CN), carboxy (COOH), acyl (COR), alkoxycarbonyl (COOR), nitro (NO 2 ), mercapto (SH) or Mercapto substituted (SR) two of these substituents being able to be connected to each other forming a fused cycle to the boradiazaindacenic skeleton.
    [8]
    8. Compound according to claim 7, wherein R1, R3, R5 and R7 are methyl.
    [9]
    9. A compound according to claims 7 and 8, wherein R4 is an optionally substituted aryl group.
    [10]
    10. Compound according to claim 9, wherein R4 is 2,4,6-trimethylphenium (mesityl).
    [11]
    11. Compound according to claim 7 of formula 9:

    [12]
    12. Compound of general formula (I) wherein the substituent R3 of the boradlazalndacenic skeleton of (I) is a (alkoxycarbonyl) alkyl group comprising a Cs alkyl group and an ethoxycarbonyl group and the rest of the substituents are each selected, independently, from hydrogen, substituted or unsubstituted C 1 -C 18 alkyl, substituted or unsubstituted C 2 -C 8 alkenyl, substituted or unsubstituted C 2 -Cis alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, halogen , hydroxy (OH), alkoxy (OR), amino (NH 2 ), amino substituted (NHR, NRR ', damage (CN), carboxy (COOH), acyl (COR), alkoxycarbonyl (COOR), nitro (N02), mercapto (SH) or substituted mercapto (SR), two of these substituents being able to be connected to each other forming a fused cycle to the boradiazaindacentric skeleton.
    [13]
    13. Compound according to claim 12, wherein R1 R4, R5, R6 and R7 are C 1 -C 2 alkyl groups.
    [14]
    14. Compound according to claims 12 and 13, wherein R2 is a halogen.
    [15]
    15. Compound according to claim 15, wherein R2 is chlorine.
    [16]
    16. Compound according to claim 12 of formula 10.

    [17]
    17. Compound of general formula (I) wherein the substituent R 4 of the boradiazaindacenic skeleton of (I) is a group (alkoxycarbonyl) alkyl with a C 4 alkyl group and a methoxycarbonyl group, the substituents R 1 or R 7 they are 2- (3,4-dimethoxyphenyl) ethenyl and the rest of the substituents are each independently selected from hydrogen, substituted or unsubstituted C 1 -C 18 alkyl, substituted or unsubstituted C 2 -C 18 alkenyl, alkynyl , C 2 -C 18 substituted or unsubstituted aryl, substituted or unsubstituted, substituted or unsubstituted heteroaryl, halogen, hydroxyl (OH), alkoxy (oR), amino (NH2), sustitutido amino (NHR, NRR ', cyano ( CN), carboxy (COOH), acyl (COR), alkoxycarbonyl (COOR), nitro (N02), mercapto (SH) or substituted mercapto (SR) being able to connect two of these substituents to each other forming a fused cycle to the boradiazacencenic skeleton.
    [18]
    18. Compound according to claim 17, wherein the substituent R1 and R7 are methyl.
    [19]
    19. Compound according to claim 17, wherein the substituent R1 and R7 are 2- (3,4-dimethoxyphenyl) ethenyl.
    [20]
    20. Compound according to any of claims 17 to 19, wherein R2, R3, R5 and R6 are C 1 -C 2 alkyl groups.
    [21]
    21. Compounds according to claim 17 of formula 11 and 12.

    [22]
    22. Method of obtaining compounds according to any one of claims 1 to 6 and 12 to 16, comprising a Negishi coupling reaction between a compound of formula (II) and an organozine reagent, in the presence of a palladium catalyst,

    [23]
    23. Process for obtaining compounds according to any one of claims 1 to 6 and 22, comprising a Suzuki reaction between a compound of formula (I) and a boronic acid or boronic acid derivative, in the presence of a base and a palladium catalyst, where R7 is a halogen or pseudohalogen and the remaining substituents are equal to those of the final compound of formula (I).
    [24]
    24. Method of obtaining compounds according to any one of claims 7 to 11, comprising a previous Friedel-Crafts reaction between a compound of formula (II) and a halide or carboxylic acid anhydride having a (alkoxycarbonyl) alkyl group , where R2 is hydrogen, and the remaining substituents are equal to those of the final compound of formula (I), and a subsequent chemoselective reduction reaction of a compound of formula (II), where R2 is a group [(alkoxycarbonyl) alky]] carbonyl
    [25]
    25. Process for obtaining compounds according to any one of claims 17 to 21, comprising a condensation reaction between an acid chloride having an alkyl group (alkoxycarbonyl) and a pyrrole derivative, followed by complexation with boron trifluoride etherate .
    [26]
    26. Process for obtaining compounds according to any of claims 2 and 17 to 21, comprising a Knoevenagel reaction between a compound of formula (I) and an aromatic aldehyde, where R1 and / or R7 is methyl and the remaining substituents are the same as those of the final compound.
    [27]
    27. Fluorescent marker (probe) comprising the compound of general formula O)

    [28]
    28. Fluorescent marker (probe) according to claim 27, wherein the marking is cellular.
    [29]
    29. Fluorescent marker (probe) according to claim 28, wherein the cell marking is performed on lipid drops.
    [30]
    30. Fluorescent marker (probe) according to claim 29, wherein the marking is performed on living cells.
    [31]
    31. Marker, according to claim 27, covalently, or supramolecularly, bound to a chemical system selected from the group consisting of amino acid, peptide, protein, lipid, carbohydrate, nucleic acid and toxin.
    [32]
    32. Use of the marker, according to claims 27 and 31, for imaging in the diagnosis of diseases related to abnormalities in lipid drops, such as hepatitis C virus proliferation, type-2 diabetes, obesity or cancer.
    [33]
    33. Use of the marker, according to claim 27, as a photocitotoxic agent.
    [34]
    34. Use of the marker, according to claim 27, for the treatment by photodynamic therapy of human cells.
    [35]
    35. Use according to claim 34, wherein the human cells are cancerous cells.
    [36]
    36. Method for enhancing photocitotoxic activity of a photocitotoxic agent by minimizing the loss of fluorescent capacity that consists in specifically accumulating the teragnostic agent in lipid droplets.
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    ES2719000B2|2020-05-25|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US5338854A|1991-02-13|1994-08-16|Molecular Probes, Inc.|Fluorescent fatty acids derived from dipyrrometheneboron difluoride dyes|ES2800548A1|2020-06-19|2020-12-30|Univ Madrid Complutense|New BODIPY stains for photodynamic teragnosis based on accumulation in mitochondria |
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